CA1331564C - Redox systems for brain-targeted drug delivery - Google Patents
Redox systems for brain-targeted drug deliveryInfo
- Publication number
- CA1331564C CA1331564C CA000585791A CA585791A CA1331564C CA 1331564 C CA1331564 C CA 1331564C CA 000585791 A CA000585791 A CA 000585791A CA 585791 A CA585791 A CA 585791A CA 1331564 C CA1331564 C CA 1331564C
- Authority
- CA
- Canada
- Prior art keywords
- methyl
- beta
- dihydropyridine
- dihydro
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/559—Redox delivery systems, e.g. dihydropyridine pyridinium salt redox systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C219/26—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
- C07C219/28—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton having amino groups bound to acyclic carbon atoms of the carbon skeleton
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
- C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
- C07D211/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
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- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/81—Amides; Imides
- C07D213/82—Amides; Imides in position 3
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- C—CHEMISTRY; METALLURGY
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- C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
- C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
- C07D223/18—Dibenzazepines; Hydrogenated dibenzazepines
- C07D223/22—Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines
- C07D223/24—Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines with hydrocarbon radicals, substituted by nitrogen atoms, attached to the ring nitrogen atom
- C07D223/28—Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines with hydrocarbon radicals, substituted by nitrogen atoms, attached to the ring nitrogen atom having a single bond between positions 10 and 11
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/72—Two oxygen atoms, e.g. hydantoin
- C07D233/74—Two oxygen atoms, e.g. hydantoin with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to other ring members
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/72—Two oxygen atoms, e.g. hydantoin
- C07D233/76—Two oxygen atoms, e.g. hydantoin with substituted hydrocarbon radicals attached to the third ring carbon atom
- C07D233/78—Radicals substituted by oxygen atoms
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D243/00—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms
- C07D243/06—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4
- C07D243/10—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
- C07D243/38—[b, e]- or [b, f]-condensed with six-membered rings
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
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- C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
- C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
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- C07C2601/14—The ring being saturated
Abstract
ABSTRACT OF THE DISCLOSURE
Inclusion complexes of hydroxypropyl, hydroxy-ethyl, glucosyl, maltosyl or maltotriosyl derivatives of .beta.- or .gamma.-cyclodextrin with the reduced, biooxi-dizable, blood-brain barrier penetrating, lipoidal forms of dihydropyridine ? pyridinium salt redox systems for brain-targeted drug delivery provide a means for stabilizing the redox systems, particularly against oxidation. The redox inclusion complexes also provide a means for decreasing initial drug concentra-tions in the lungs after administration of the systems, leading to decreased toxicity. In selected instances, complexation results in substantially improved water solubility of the redox systems as well.
Inclusion complexes of hydroxypropyl, hydroxy-ethyl, glucosyl, maltosyl or maltotriosyl derivatives of .beta.- or .gamma.-cyclodextrin with the reduced, biooxi-dizable, blood-brain barrier penetrating, lipoidal forms of dihydropyridine ? pyridinium salt redox systems for brain-targeted drug delivery provide a means for stabilizing the redox systems, particularly against oxidation. The redox inclusion complexes also provide a means for decreasing initial drug concentra-tions in the lungs after administration of the systems, leading to decreased toxicity. In selected instances, complexation results in substantially improved water solubility of the redox systems as well.
Description
1331~
REDOX SYSTEMS FOR
BRAIN-TARGETED DRUG DELIVERY
FIELD OF THE INVENTION:
The present invention provides a method for 5 stabilizing the reduced, dihydropyridine for~s af ~9 dihydropyridine + pyridinium salt redox systems for brain-targeted drug delivery by forming inclusion complexes of the dihydropyridine forms with selected cyclodextrins. These redox inclusion complexes also provide a means for increasing the ratio of initial brain to lung concentrations, thus leading to decreased toxicity. In selected instances, complexation results in substantially improved water solubility of the redox systems as well.
BACKGR0UND OF THE INVENTION:
Cyclodextrins are cyclic oligosaccharides. The most common cyclodextrins are ~-cyclodextrin, which is composed of a ring of six glucose residues, ~-cyclo-dextrin, which is composed of a ring of seven glucose residues, and y-cyclodextrin, which is composed of a ring of eight glucose units. The inside cavity of a cyclodextrin is lipophilic, while the outside of the cyclodextrin is hydrophilic; this combination of properties has led to widespread study of the natural cyclodextrins, particularly in connection with phar-maceuticals, and many inclusion complexes have been reported. B-Cyclodextrin has been of special interest ap 1331~3~
because of its cavity size, but its relatively low -aqueous solubility has limited its use in the ~ ~-pharmaceutical field.
Attempts to modify the properties of the natural 5 cyclodextrins have resulted in the development of ~ -heptakis (2,6-di-~-methyl)-~-cyclodextrin, heptakis (~,3,6-tri-0-methyl)-~-cyclodextrin, hydroxypropyl~
cyclodextrin, i3-cyclodextrin-epichlorohydrin polymer ~ -and others. For a comprehensive review of cyclo- ~- -dextrins and their use in pharmaceutical research, see Pitha et al, in Controlled Drug Delivery, ed. S.D.
Bruck, Vol. I, CRC Press, Boca Raton, Florida, pp. 125-148 (1983). For an even more recent overview, see Uekama et al, in CRC Critical Reviews in Therapeutic 15 Drug Carrier Systems, Vol. 3 (1), 1-40 (1987); Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. ~
.. .
Breimer and P. Speiser, Elsevier Science Publishers B.
V. (8iomedical nivision), 1987, 181-194; and Pagington, Chemistry in Britain, May 1987, 455-458.
Inclusion co~plexes of a-, ~- or Y-cyclodextrin or their ~ixtures with a variety of drugs have been de-scribed by numerous parties and various advantages have been attributed to the complexes. These descriptions include the following:
, ~
., :..
~ ,.
1 3 3 1 ~
U 5. ACTIVE
INVENTORPATEN~ NO. IN6REDlEhT USE ADV~NTA6E
Noda et al 4,024,223 menthol ~/or antiphlogistic, reduced unpleasant methyl analgesic odor, increased salicylate wet packing effect Szejtli et al 4,228,160 indomethacin ant1-inflam- reduced ulcerative matory, pro- effect tective during pregnancy Hayashi et al 4,232,009 ~halo-PGI2 hypotensive, increased stability analogs uterine con-traction :~
st1mulating, blood platelet aggregation 1nhibiting Matsumoto et al 4,351,846 3-hydroxy- and uterine contrac- increased stabil1ty 3-oxo- t10n stimulating prostaglandin analogs Yamahira et al 4,352,793 bencyclane anticonvulsant, increased stability fumarate vasodilative at strong acid pH, faster gastric emptying, h1gher blood concentrations, :
less lrritation, in~roved hemolytic act1vtty L1par1 4,383,992 stero1ds-- hormonal improved water cort1costero1ds, solubility, 1ncreased androgens, therapeut1c response anabol1c ln eye stero1ds, estrogens, progestagens N1colau 4,407,795 p-hexadecyl- ant1athero- enhanced aminobenzo1c sclerot1c b10ava11ab11ity ac1d sod1um salt ~ ",~
. .
, ~
1331~
U.S. ACTIVE :~
IINENTOR PATENT NO. INGREDIENT USE ADV~NTI~GE ~ -Tuttl el 4.424,209 3,4-diisobutyr- cardlac yloxy-N-~3-(4- contractility isobutyryloxy- agent . ::
phenyl)~
methyl-n- -propyl~- B-phenethyl amine Tuttle 4,425,336 3,4-dihydroxy- cardiac capable of oral : -N-~3-(4-hydroxy- contractility administration phenyl)-1- agent ;:
methyl-n-propyl]- B- -~
phenethyl amine l~lagu et al 4,438,106 EPA and DH~ deodorized, 7 -(fatty acids) storage stable ~ -Masuda et al2 4,474,811 2-(2-fluoro-4- anti- reduced eye biphenylyl)pro- inflammatory irritation, pionic acid ophthalmic higher concen- : - -or salt trations, no side effects, highly : :
solubl e, l ong stability, excellent ~ . .
pharmacological effects Shinoda et al4,478,995 ac1d addition anti-ulcer excellent water salt of (2'- solubility, good benzyloxycar- absorption ln diges-bonyl)phenyl t1ve tract, good trans-4-guanl- anti-ulcer activity dinomethyl cycl o-hexanecarboxylate Hayashi et al 4,479,944 PG12 analog for treatment of stabil1zation against artereosclerosis, decomposition card1ac failure or thrombosis 1331~
- 5 ~
U S. ACTIVE ~: -INVE~ITOR PA~ENT N0. INGREDIENT USE ~DVANTA6E ~ -~
Hayashi et al 4,479,966 6,9-methano- for hypertension, increased stability P612 anal ogs cerebral throm-bosis and the l~ke ::: ::::
Harada et al 4,497,803 lankacidin- ant1biotic for enhanced water group antib1Otic swine dysentery solubil~ty and stabil1ty, increased ~-rate and amount of ~:
absorption :: :
Masuda 4,499,085 prostaglandin treating anoxia :
analog of brain cells -~
Szejtli et al 4,518,588 phendil1ne, i.e.coronary dilator improved water solu-r~ -phenyl- calcium bility, accelerated ethyl)-3,3- antagonist and increased 1n diphenylpro- vivo resorption pylamine or its b dissolution at pH/ .
hydrochloride temperature of gastric ac1d Szejtli et al 4,524,068 piperonyl synergizes easily handled butoxide pesticidal effect crystalline solid;
of known insecti- improved water solu-cides and fungi- bility, increased cides absorption b veloc1ty of penetration through b1Olog1cal membranes Jones 4,555,504 a cardiac cardiac effect high aqueous solu-glycoside b11itY~ appar~elntlY
ability .
Uekama et al3 4,565,807 p1rprofen anti-lnflam- 1mproved stabil1ty matory, to ox1dation, analgesic, freedom from b1tter ant1pyret1c taste, less 1rr1ta-ting , ~ .
::~
~ .
:~:
~ .
~ ;~?~
~Xi ~
,7` j;.'.~
i ~:
- ~:
1 3 3 ~
INYENTOR PATENT N0. INGREDIENT USE ADVANTAGE
Ueda et al 4,575,548 2-nitroxymethyl- for vascular non-volatile po~der 6-chloropyridine disorders vs. volative oil Ohwaki et al4 4,598,070 tripamide anti-hyper- improved solubility tensive ~ ~ -Chiesi et al 4,6û3,123 piroxicam, i.e. anti-inflam-4-hydroxy-2- matory, analgesic methyl-N-2-pyridyl-2H-1 ,2- -benzothiazine-3-carboxamide-1,1-dioxide Hasegawa et al 4,608,366 mobenzoxamine, antiemetic, storage stability, -~
i.e. 1-t2-(4- antispasmodic better absorption methoxybenzhy- through digestive dryloxy)ethyl]- tract 4-t3-(4-fluoro-benzoyl)propyl~-piperazine Hirai et al2 4,659,696 polypeptide improving drug absorption by non-oral and non-injectlon routes Szejtli et al 4,623,641 PGt~rmethyl anti-ulcer improved storage Ninger et al 4,663,316 unsaturated antibiotic, enhanced stability ;
phosphorus- antifungal, against oxidation containing antitumor ant1biotics, includlng phosphotr1enin .,. :
i ~ ~ 3 1 r; ~
-7- ;:
U.S. ~CTIVE
INVENTORPATEIIT NO. INGREDIENT USE ADVA~ITAGE
Fukazawa et al 4,675,395 hinokitiol bactericidal; improved water odor ~ ' .
1 Tuttle also describes use of 2,6-di-0-methyl-~-cyclodextrin and 2,3,6-tri-0-methyl-~-cyclodextrin to form the inclusion complex.
REDOX SYSTEMS FOR
BRAIN-TARGETED DRUG DELIVERY
FIELD OF THE INVENTION:
The present invention provides a method for 5 stabilizing the reduced, dihydropyridine for~s af ~9 dihydropyridine + pyridinium salt redox systems for brain-targeted drug delivery by forming inclusion complexes of the dihydropyridine forms with selected cyclodextrins. These redox inclusion complexes also provide a means for increasing the ratio of initial brain to lung concentrations, thus leading to decreased toxicity. In selected instances, complexation results in substantially improved water solubility of the redox systems as well.
BACKGR0UND OF THE INVENTION:
Cyclodextrins are cyclic oligosaccharides. The most common cyclodextrins are ~-cyclodextrin, which is composed of a ring of six glucose residues, ~-cyclo-dextrin, which is composed of a ring of seven glucose residues, and y-cyclodextrin, which is composed of a ring of eight glucose units. The inside cavity of a cyclodextrin is lipophilic, while the outside of the cyclodextrin is hydrophilic; this combination of properties has led to widespread study of the natural cyclodextrins, particularly in connection with phar-maceuticals, and many inclusion complexes have been reported. B-Cyclodextrin has been of special interest ap 1331~3~
because of its cavity size, but its relatively low -aqueous solubility has limited its use in the ~ ~-pharmaceutical field.
Attempts to modify the properties of the natural 5 cyclodextrins have resulted in the development of ~ -heptakis (2,6-di-~-methyl)-~-cyclodextrin, heptakis (~,3,6-tri-0-methyl)-~-cyclodextrin, hydroxypropyl~
cyclodextrin, i3-cyclodextrin-epichlorohydrin polymer ~ -and others. For a comprehensive review of cyclo- ~- -dextrins and their use in pharmaceutical research, see Pitha et al, in Controlled Drug Delivery, ed. S.D.
Bruck, Vol. I, CRC Press, Boca Raton, Florida, pp. 125-148 (1983). For an even more recent overview, see Uekama et al, in CRC Critical Reviews in Therapeutic 15 Drug Carrier Systems, Vol. 3 (1), 1-40 (1987); Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. ~
.. .
Breimer and P. Speiser, Elsevier Science Publishers B.
V. (8iomedical nivision), 1987, 181-194; and Pagington, Chemistry in Britain, May 1987, 455-458.
Inclusion co~plexes of a-, ~- or Y-cyclodextrin or their ~ixtures with a variety of drugs have been de-scribed by numerous parties and various advantages have been attributed to the complexes. These descriptions include the following:
, ~
., :..
~ ,.
1 3 3 1 ~
U 5. ACTIVE
INVENTORPATEN~ NO. IN6REDlEhT USE ADV~NTA6E
Noda et al 4,024,223 menthol ~/or antiphlogistic, reduced unpleasant methyl analgesic odor, increased salicylate wet packing effect Szejtli et al 4,228,160 indomethacin ant1-inflam- reduced ulcerative matory, pro- effect tective during pregnancy Hayashi et al 4,232,009 ~halo-PGI2 hypotensive, increased stability analogs uterine con-traction :~
st1mulating, blood platelet aggregation 1nhibiting Matsumoto et al 4,351,846 3-hydroxy- and uterine contrac- increased stabil1ty 3-oxo- t10n stimulating prostaglandin analogs Yamahira et al 4,352,793 bencyclane anticonvulsant, increased stability fumarate vasodilative at strong acid pH, faster gastric emptying, h1gher blood concentrations, :
less lrritation, in~roved hemolytic act1vtty L1par1 4,383,992 stero1ds-- hormonal improved water cort1costero1ds, solubility, 1ncreased androgens, therapeut1c response anabol1c ln eye stero1ds, estrogens, progestagens N1colau 4,407,795 p-hexadecyl- ant1athero- enhanced aminobenzo1c sclerot1c b10ava11ab11ity ac1d sod1um salt ~ ",~
. .
, ~
1331~
U.S. ACTIVE :~
IINENTOR PATENT NO. INGREDIENT USE ADV~NTI~GE ~ -Tuttl el 4.424,209 3,4-diisobutyr- cardlac yloxy-N-~3-(4- contractility isobutyryloxy- agent . ::
phenyl)~
methyl-n- -propyl~- B-phenethyl amine Tuttle 4,425,336 3,4-dihydroxy- cardiac capable of oral : -N-~3-(4-hydroxy- contractility administration phenyl)-1- agent ;:
methyl-n-propyl]- B- -~
phenethyl amine l~lagu et al 4,438,106 EPA and DH~ deodorized, 7 -(fatty acids) storage stable ~ -Masuda et al2 4,474,811 2-(2-fluoro-4- anti- reduced eye biphenylyl)pro- inflammatory irritation, pionic acid ophthalmic higher concen- : - -or salt trations, no side effects, highly : :
solubl e, l ong stability, excellent ~ . .
pharmacological effects Shinoda et al4,478,995 ac1d addition anti-ulcer excellent water salt of (2'- solubility, good benzyloxycar- absorption ln diges-bonyl)phenyl t1ve tract, good trans-4-guanl- anti-ulcer activity dinomethyl cycl o-hexanecarboxylate Hayashi et al 4,479,944 PG12 analog for treatment of stabil1zation against artereosclerosis, decomposition card1ac failure or thrombosis 1331~
- 5 ~
U S. ACTIVE ~: -INVE~ITOR PA~ENT N0. INGREDIENT USE ~DVANTA6E ~ -~
Hayashi et al 4,479,966 6,9-methano- for hypertension, increased stability P612 anal ogs cerebral throm-bosis and the l~ke ::: ::::
Harada et al 4,497,803 lankacidin- ant1biotic for enhanced water group antib1Otic swine dysentery solubil~ty and stabil1ty, increased ~-rate and amount of ~:
absorption :: :
Masuda 4,499,085 prostaglandin treating anoxia :
analog of brain cells -~
Szejtli et al 4,518,588 phendil1ne, i.e.coronary dilator improved water solu-r~ -phenyl- calcium bility, accelerated ethyl)-3,3- antagonist and increased 1n diphenylpro- vivo resorption pylamine or its b dissolution at pH/ .
hydrochloride temperature of gastric ac1d Szejtli et al 4,524,068 piperonyl synergizes easily handled butoxide pesticidal effect crystalline solid;
of known insecti- improved water solu-cides and fungi- bility, increased cides absorption b veloc1ty of penetration through b1Olog1cal membranes Jones 4,555,504 a cardiac cardiac effect high aqueous solu-glycoside b11itY~ appar~elntlY
ability .
Uekama et al3 4,565,807 p1rprofen anti-lnflam- 1mproved stabil1ty matory, to ox1dation, analgesic, freedom from b1tter ant1pyret1c taste, less 1rr1ta-ting , ~ .
::~
~ .
:~:
~ .
~ ;~?~
~Xi ~
,7` j;.'.~
i ~:
- ~:
1 3 3 ~
INYENTOR PATENT N0. INGREDIENT USE ADVANTAGE
Ueda et al 4,575,548 2-nitroxymethyl- for vascular non-volatile po~der 6-chloropyridine disorders vs. volative oil Ohwaki et al4 4,598,070 tripamide anti-hyper- improved solubility tensive ~ ~ -Chiesi et al 4,6û3,123 piroxicam, i.e. anti-inflam-4-hydroxy-2- matory, analgesic methyl-N-2-pyridyl-2H-1 ,2- -benzothiazine-3-carboxamide-1,1-dioxide Hasegawa et al 4,608,366 mobenzoxamine, antiemetic, storage stability, -~
i.e. 1-t2-(4- antispasmodic better absorption methoxybenzhy- through digestive dryloxy)ethyl]- tract 4-t3-(4-fluoro-benzoyl)propyl~-piperazine Hirai et al2 4,659,696 polypeptide improving drug absorption by non-oral and non-injectlon routes Szejtli et al 4,623,641 PGt~rmethyl anti-ulcer improved storage Ninger et al 4,663,316 unsaturated antibiotic, enhanced stability ;
phosphorus- antifungal, against oxidation containing antitumor ant1biotics, includlng phosphotr1enin .,. :
i ~ ~ 3 1 r; ~
-7- ;:
U.S. ~CTIVE
INVENTORPATEIIT NO. INGREDIENT USE ADVA~ITAGE
Fukazawa et al 4,675,395 hinokitiol bactericidal; improved water odor ~ ' .
1 Tuttle also describes use of 2,6-di-0-methyl-~-cyclodextrin and 2,3,6-tri-0-methyl-~-cyclodextrin to form the inclusion complex.
2 This may not be an inclusion complex, but simp1y a physical mixture.
3 This is a mixture and/or an inclusion compound.
4 The inventors also mention prior known solubility improvements of cyclodextrininclusions of barbiturk acid derivatives, mefenamic acid, indomethacin and ch1oramphenicol.
5 The inventors refer to this as an ~occlusion" compound.
, , :.'", ~: :- ~
.
~331~
Inclusion complexes of 2,6-di-0-methyl~
cyclodextrin with dibenzo[bd]pyran derivatives and salts having analgesic, antemetic and narcosis-potentiating activities have been described in Nogradi et al U.S. Patent No. 4,599,327; increased water solu-bility and thus improved biological activity have been claimed for the complexes. A review of the pharma-ceutical applications of such methylated cyclodextrins has been published by Uekama, Pharm. Int., March 1985, 61-65; see also Pitha, Journal of Inclusion Phenomena 2, 477-485 (1984).
Cyclodextrin polymer has been reported by Fenyvesi et al, Chem. Pharm. 3ull. 32 (2), 665-669 (1984) to improve the dissolution of furosemide. Improvements in the dissolution and absorption of phenytoin using a water-soluble ~-cyclodextrin epichlorohydrin polymer have been described by Uekama et al, International Journal of Pharmaceutics, 23, 35-42 (1985).
Hydroxypropyl-~-cyclodextrin (HPCD) and its preparation by propylene oxide addition to ~
cyclodextrin were described in Gramera et al United States Patent No. 3,459,731 nearly 20 years ago.
Gramera et al also described the analogous preparation of hydroxyethyl-~-cyclodextrin by ethylene oxide reaction with ~-cyclodextrin. Much more recently, Pitha and co-workers have described the improved preparation of this cyclodextrin derivative and its effects on the dissolution of various drug molecules.
Pitha United States Patent No. 4,596,795, dated June ~-30 24, 1986, describes inclusion complexes of sex hormones, particularly testosterone, progesterone and .
. ,::: :. ~. .. . : , , . . , 1 3315~
g ~, estradiol, ~ith specific cyclodextrins, preferably hydroxypropyl-~-cyclodextrin and poly-B-cyclodextrin.
The co~plexes enable the sex hormones to be successfully delivered to the systemic circulation via the sublingual or buccal route; the effectiveness of this delivery is believed to be due to "the high dissolution power of hydrophilic derivatives of cyclodextrins, the non-aggregated structure of their complexes with steroids, and their low toxicity and irritancy of mouth tissue". Success with other cyclodextrins, including poly-y-cyclodextrin and hyroxypropyl-y-cyclodextrin, have also been noted in the Pitha patent. See also Pitha et al, J. Pharm.
Sci., Vol. 74, No. 9, September 1985, 987-990, concerning the same and related studies. Pitha et al also describe in the J. Pharm. Sci. article the storage stability of tablets containing a testosterone-hydroxypropyl-~-cyclodextrin complex and the lack of ~ -toxicity of the cyclodextrin itself, as well as the importance of the amorphous nature of the cyclodextrin derivatives and their complexes with drugs in improving dissolution properties.
The improved, optimized preparation and purifi-cation of hydroxypropyl-~-cyclodextrin has been recently described by Pitha et al, International Journal of Pharmaceutics, 29, 73-82 (1986). In the same publication, the authors have described increased water solubility for 32 drugs in concentrated (40 to 507O) aqueous solutions of hydroxypropyl-~-cyclodextrin.
Uekama et al, CRC Critical Reviews in Therapeutic ~rug Carrier Systems, Vol. 3 (1), 1-40 (1987), have .
. :: :
133~
described the characteristics of various cyclodextrins, including hydroxyprooyl-~-cyclo~extrin. The authors have presented data showing improved solubilization in water in the presence of 15 ~g/~L of 4PCD for the drugs carmofur, diazepam, digitoxin, digoxin, flurbiprofen, indo~ethacin, isosorbide ~initrate, phenytoin, prednisolone, progesterone and testosterone.
JANSSEN PHARMACEUTICA N.V.'s International Patent Application No. PCT/EP84/00417, published under International Publication No. ~085/n2767 on July 4, 1985, has described pharmaceutical compositions comprising inclusion compounds of drugs, which are unstable or only sparingly soluble in water, with partially etherified ~-cyclodextrin derivatives having hydroxyalkyl and optionally additional alkyl groups.
Among the cyclodextrin derivatives contemplated are hydroxypropyl-~-cyclodextrin and hydroxyethyl-~-cyclodextrin, while the drugs include non-steroidal anti-rheumatic agents, steroids, cardiac glycosides and derivatives of benzodiazepine, benzimidazole, piperi-dine, piperazine, imidazole and triazole. Preferred drugs include etomidate, ketoconazole, tubulazole itraconazole, levocabastine and flunarizine. The pharmaceutical compositions of the invention include oral, parenteral and topical formulations, with 4 to 1073 solutions of cyclodextrin derivatives being utilized to solubilize various drugs. Improved solu-bilities of indomethacin, digitoxin, progesterone, dexamethasone, hydrocortisone and diazepam using 10%
HPCD are shown, and an injectable formulation of diazepam in 7% HPCD is specifically described.
1 3 3 ~
Carpenter et al, The Journal of Pediatrics, 111, 507-512 (October 1987) describe intravenous infusion of 2-hydroxypropyl-~-cyclodextrin, prepared as a 5~ solu-tion in water, to treat severe hypervitaminosis ~. It was found that, during infusion, circulating retinyl esters increased transiently, while total vitamin A
excreted in the urine was enhanced after infusion.
Thus, intravenous infusion of 570 HPCD was found to decrease in vivo levels of the vitamin, presumably by complexing with the vitamin and removing some of the excess from the body.
The inclusion characteristics of yet other -derivatized cyclodextrins have also been described in the literature. Studies of branched cyclodextrins which are glucosyl and maltosyl derivatives of -, ~-and Y-cyclodextrin and their inclusion complexes with drugs have recently been reported. Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. D. Breimer and P. Speiser, Elsevier Science Publishers B. V.
tBiomedical Division), 1987, 181-194, has described the effects on bio-pharmaceutical properties of maltosyl and glucosyl cyclodextrin derivatives, including enhanced drug absorption. Koizumi et al, Chem. Pharm.
Bull. 35 (8~, 3413-341~ (1987), have reported on inclusion complexes of poorly water-soluble drugs with glucosyl cyclodextrins, namely 6-0-a-D-glucosyl--CD
(G1--CD), 6-0--D-glucosyl-B-Cr) (G1-~-CD) and 6A, 6D-di-O-a-D-glucosyl-~-CD (2G1-~-CD). Okada et al, Chem.
Pharm. Bull., 36 (6), 2176-2185 (1988), have reported 30 on the inclusion complexes of poorly water-soluble -drugs with maltosyl cyclodextrins, namely 6-0-a- -~
maltosyl-a-CD (G2-a-CD), 6-0-a-maltosyl-~-CD (G2-~-CD), . , . .., ~ ~
133~
, , :.'", ~: :- ~
.
~331~
Inclusion complexes of 2,6-di-0-methyl~
cyclodextrin with dibenzo[bd]pyran derivatives and salts having analgesic, antemetic and narcosis-potentiating activities have been described in Nogradi et al U.S. Patent No. 4,599,327; increased water solu-bility and thus improved biological activity have been claimed for the complexes. A review of the pharma-ceutical applications of such methylated cyclodextrins has been published by Uekama, Pharm. Int., March 1985, 61-65; see also Pitha, Journal of Inclusion Phenomena 2, 477-485 (1984).
Cyclodextrin polymer has been reported by Fenyvesi et al, Chem. Pharm. 3ull. 32 (2), 665-669 (1984) to improve the dissolution of furosemide. Improvements in the dissolution and absorption of phenytoin using a water-soluble ~-cyclodextrin epichlorohydrin polymer have been described by Uekama et al, International Journal of Pharmaceutics, 23, 35-42 (1985).
Hydroxypropyl-~-cyclodextrin (HPCD) and its preparation by propylene oxide addition to ~
cyclodextrin were described in Gramera et al United States Patent No. 3,459,731 nearly 20 years ago.
Gramera et al also described the analogous preparation of hydroxyethyl-~-cyclodextrin by ethylene oxide reaction with ~-cyclodextrin. Much more recently, Pitha and co-workers have described the improved preparation of this cyclodextrin derivative and its effects on the dissolution of various drug molecules.
Pitha United States Patent No. 4,596,795, dated June ~-30 24, 1986, describes inclusion complexes of sex hormones, particularly testosterone, progesterone and .
. ,::: :. ~. .. . : , , . . , 1 3315~
g ~, estradiol, ~ith specific cyclodextrins, preferably hydroxypropyl-~-cyclodextrin and poly-B-cyclodextrin.
The co~plexes enable the sex hormones to be successfully delivered to the systemic circulation via the sublingual or buccal route; the effectiveness of this delivery is believed to be due to "the high dissolution power of hydrophilic derivatives of cyclodextrins, the non-aggregated structure of their complexes with steroids, and their low toxicity and irritancy of mouth tissue". Success with other cyclodextrins, including poly-y-cyclodextrin and hyroxypropyl-y-cyclodextrin, have also been noted in the Pitha patent. See also Pitha et al, J. Pharm.
Sci., Vol. 74, No. 9, September 1985, 987-990, concerning the same and related studies. Pitha et al also describe in the J. Pharm. Sci. article the storage stability of tablets containing a testosterone-hydroxypropyl-~-cyclodextrin complex and the lack of ~ -toxicity of the cyclodextrin itself, as well as the importance of the amorphous nature of the cyclodextrin derivatives and their complexes with drugs in improving dissolution properties.
The improved, optimized preparation and purifi-cation of hydroxypropyl-~-cyclodextrin has been recently described by Pitha et al, International Journal of Pharmaceutics, 29, 73-82 (1986). In the same publication, the authors have described increased water solubility for 32 drugs in concentrated (40 to 507O) aqueous solutions of hydroxypropyl-~-cyclodextrin.
Uekama et al, CRC Critical Reviews in Therapeutic ~rug Carrier Systems, Vol. 3 (1), 1-40 (1987), have .
. :: :
133~
described the characteristics of various cyclodextrins, including hydroxyprooyl-~-cyclo~extrin. The authors have presented data showing improved solubilization in water in the presence of 15 ~g/~L of 4PCD for the drugs carmofur, diazepam, digitoxin, digoxin, flurbiprofen, indo~ethacin, isosorbide ~initrate, phenytoin, prednisolone, progesterone and testosterone.
JANSSEN PHARMACEUTICA N.V.'s International Patent Application No. PCT/EP84/00417, published under International Publication No. ~085/n2767 on July 4, 1985, has described pharmaceutical compositions comprising inclusion compounds of drugs, which are unstable or only sparingly soluble in water, with partially etherified ~-cyclodextrin derivatives having hydroxyalkyl and optionally additional alkyl groups.
Among the cyclodextrin derivatives contemplated are hydroxypropyl-~-cyclodextrin and hydroxyethyl-~-cyclodextrin, while the drugs include non-steroidal anti-rheumatic agents, steroids, cardiac glycosides and derivatives of benzodiazepine, benzimidazole, piperi-dine, piperazine, imidazole and triazole. Preferred drugs include etomidate, ketoconazole, tubulazole itraconazole, levocabastine and flunarizine. The pharmaceutical compositions of the invention include oral, parenteral and topical formulations, with 4 to 1073 solutions of cyclodextrin derivatives being utilized to solubilize various drugs. Improved solu-bilities of indomethacin, digitoxin, progesterone, dexamethasone, hydrocortisone and diazepam using 10%
HPCD are shown, and an injectable formulation of diazepam in 7% HPCD is specifically described.
1 3 3 ~
Carpenter et al, The Journal of Pediatrics, 111, 507-512 (October 1987) describe intravenous infusion of 2-hydroxypropyl-~-cyclodextrin, prepared as a 5~ solu-tion in water, to treat severe hypervitaminosis ~. It was found that, during infusion, circulating retinyl esters increased transiently, while total vitamin A
excreted in the urine was enhanced after infusion.
Thus, intravenous infusion of 570 HPCD was found to decrease in vivo levels of the vitamin, presumably by complexing with the vitamin and removing some of the excess from the body.
The inclusion characteristics of yet other -derivatized cyclodextrins have also been described in the literature. Studies of branched cyclodextrins which are glucosyl and maltosyl derivatives of -, ~-and Y-cyclodextrin and their inclusion complexes with drugs have recently been reported. Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. D. Breimer and P. Speiser, Elsevier Science Publishers B. V.
tBiomedical Division), 1987, 181-194, has described the effects on bio-pharmaceutical properties of maltosyl and glucosyl cyclodextrin derivatives, including enhanced drug absorption. Koizumi et al, Chem. Pharm.
Bull. 35 (8~, 3413-341~ (1987), have reported on inclusion complexes of poorly water-soluble drugs with glucosyl cyclodextrins, namely 6-0-a-D-glucosyl--CD
(G1--CD), 6-0--D-glucosyl-B-Cr) (G1-~-CD) and 6A, 6D-di-O-a-D-glucosyl-~-CD (2G1-~-CD). Okada et al, Chem.
Pharm. Bull., 36 (6), 2176-2185 (1988), have reported 30 on the inclusion complexes of poorly water-soluble -drugs with maltosyl cyclodextrins, namely 6-0-a- -~
maltosyl-a-CD (G2-a-CD), 6-0-a-maltosyl-~-CD (G2-~-CD), . , . .., ~ ~
133~
6-0--maltosyl-y-C~ (G~-y-CD), 5-0-~-maltotriosyl--CD
(G3--C~), 6-0--maltotriosyl-~-C~ (G3-~-CD) and 6-0--maltotriosyl-~-CD (G3-y-cn~.
The delivery of drugs to the brain is often seriously limited by transport and metabolism factors and, more specifically, by the functional barrier of the endothelial brain capillary wall, i.e. the blood- ;~
brain barrier or ~BB. Site-specific delivery and sustained delivery of drugs to the brain are even more difficult.
A dihydropyridine ~ pyridinium salt redox system has recently been successfully applied to delivery to the brain of a number of drugs. Generally speaking, ;~
according to this system, a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the blood-brain ~-barrier following its systemic administration. Subse-quent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
Three main approaches have been published thus far for delivering drugs to the brain using this redox system. The first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component. This approach was first applied to delivering to the brain N-methylpyridinium- -~
2-carbaldoxime chloride (2-PAM), the active nucleus of ¦ which constitutes a quaternary pyridinium salt, by way ~ of the dihydropyridine latentiated prodrug form ¦ 30 thereof. Thus, a hydrophilic compound (2-P4M) was made lipoidal (i.e. lipophilic) by making its dihydropyri-.i .
~ -.~ ~ ~ ,................................... .
c: ~'''` ~ ~`'' ~ ` ~ ` ~ ' ' ''' ': ' .:'.:
1 3 ~
dine form (Pro-2-PAM) to enahle its penetration through lipoidal barriers. This si~ple prodrug approach allowed the co~pound to get into the brain as well as other organs, but this manipulation did not and could 5 not result in any brain specificity. On the contrary, -~-such approach was delimited to relatively small mole-cule quaternary pyridinium ring-containing drug species and did not provide the overall ideal result of brain-specific, sustained release of the desired drug, with concomitant rapid elimination from the general circu-lation, enhanced drug efficacy and decreased toxi- -city. No "trapping" in the brain of the 2-PAM formed in situ resulted, and obviously no brain-specific, sustained delivery occurred as any consequence there-of: the 2-PAM was eliminated as fast from the brain as it was from the general circulation and other organs.
Co~pare U.S. Patents Nos. 3,929,813 and 3,962,447;
Bodor et al, J. Pharm. Sci, 67, No. 5, 685 (1978). See also 30dor, "Novel Approaches for the Design of Me~brane Transport Properties of Drugs", in Design of Biopharmaceutical Properties Through Prodrugs and Analogs, Roche, E.B. (ed.), APhA Academy of Pharmaceutical Sciences, Washington, D.C., 98-135 (1976). Subsequent extension of this first approach to ~-~
delivering a much larger quaternary salt, berberine, to the brain via its dihydropyridine prodrug form was, ~-~
however, found to provide site-specific sustained delivery to the brain of that anticancer agent. See Bodor et al, Science, Vol. 214, ~ecember 18, 1981, pp.
30 1370-1372.
The second approach for delivering drugs to the brain using the redox system involves the use of a :
1331!r~ ~t pyridinium carrier chemically linked to a biologically active compound. Bodor et al, Science, Vol. 214, ~ :~
~ecember 1~, 1981, pp. 137~-1372, outline a scheme for this specific and sustained delivery of drug species to the brain, as depicted in the following Scheme 1:
' ~
' ' ',"' '~;"
, ! ' . ~
1 3 3 ~
- l 'i - ' . .
' lDl ~ ~ ID~CI- ~-DHCl DEEIVER~ / 7 ~TO 80DY \ /ELIIllIUTIOH
ID-DHCI ID-DHCJ
111 THE ~RAIII IN CIRCULATORY SYSTEn . -~D O~G~S
1~ OXIDATIOII ~ oxlDAT
~ , ~ ~ , .
ID-OC1''-- ENZYI'ATtC ENZY14~TIC --ID-OCI t Ill HE SRAI~I K 111 Cl~ICI ILATORY SYSTE~
4 4 .
~'- ' . , ~ ~ ,.' 2 .~, . i.it~
IDI ~ 10Cll IDI ~1CI1 K3 ~: S 6 .. -.
B88 ~ , : I
,i 1 , ¦~ ELIYINATION
~,j.
1~ SCHE~IE 1: BBB, UOOD-~RAI~ RARRIER.
:1 '~'1 Accord1ng to the scheme ~n Science~ a drug tD] is coupled to a quaternary carrier [QC]+ and the [D-QC3+
which results is then reduced chemically to the lipoidal dihydro form [D-DHC]. After administration of ~D-DHC] in vivo, it is rapidly distributed throughout the body, includlng the brain. The dihydro form [D-DHC] is then in situ oxidized (rate constant, k1) (by the NAD ~ NADH system) to the ideally inactive original [D-QC]+ quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the -~
blood-brain barrier should prevent its elimination from the brain (k3 k2; k3 k7). Enzymatic cleavage of the [D-QC]+ that is "locked" in the brain effects a -15 sustained delivery of the drug species [D], followed by ;~
its normal elimination (k5), metabolism. A properly selected carrier [QC~ will also be rapidly eliminated from the brain (k6 k2). Because of the facile elimination of tD-QC]+ from the general circulation, only m~nor amounts of drug are released in the body ~k3 k4); [D] will be released primarily in the brain (k4>k2). The overall result ideally will be a brain-specific sustained release of the target drug spec1es. Specifically, Bodor et al worked with phenyl-ethylamine as the drug model. That compound wascoupled to nicotinic acid, then quaterni~ed to give compounds of the formula : .
[~J C51~ 32C32~ ~1 ~ CH3 or CH2 ~ )- :
;- ~: , . . ., . , :; , . . . .......... . . .
i ~
133~4 wh1ch were subsequently reduced by sodium dithion1te to the correspond~ng compounds of the formula ¢3- CONHCH2CH2~ (R = CH3 or CH ~ ).
R
Testlng of the N-methyl der1vatlve in vivo supported 5 the criteria set forth in Scheme I. Bodor et al -speculated that various types of drugs might possibly ~ -~
be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring-substituted derivatives was being studied for delivery of amino- or hydroxyl-containing drugs, including small peptldes, to the bra~n. No other possible specific carriers were disclosed. Other reports of this work with the redox carrier system have appeared in The Friday Evening Post, August 14, 1981, Health Center Communications, University of Florlda, Galnesville, Flor1da; Chemical ~ Engineerlng News, December 21, 1981, pp. 24-25; and Science News, January 2, 1982, Vol. 121, No. 1, page 7. More recently, the redox carr~er system has been substantially extended in terms of possible carriers and drugs to be delivered. See International Patent Application No. PCT/US83/00725, flled May 12, 1983 and published November 24, 1983 under International Publicat~on No. W083/03968. Also see Bodor et al, Pharmacology and Therapeutics, Vol.
19, No. 3, pp. 337-386 (1983); and Bodor United States Patent No. 4,540,564, ~ssued September 10, 1985.
~ :' . ~-, :
1~31r~/U,~
The third approach for delivering drugs to the brain using the redox system provides derivatives of centrally acting amines ln which a primary, secondary or tertiary amine funct~on has been replaced with a dihydropyridine/pyridinium salt redox system. These brain-specific analogs of centrally acting amines have been recently described in International Patent Appli-cation No. PCT/US85/00236, filed February 15, 1985 and published September 12, 1985 under International Publi-cation No. W085/03937. The dihydropyridine analogs are characterized by the structural formula . :
D - NJ
wherein D is the residue of a centrally acting primary, secondary or tertlary amlne, and -N 3 is a radical of the formula b ~ N~
( R~ ~J ( R ) q ~ o r b ) ( C ) wherein the dotted line in formula (a) indicates the presence of a double bond in either the 4 or 5 pos1t1On of the dihydropyridine ring; the dotted line in formula (b) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system; m is zero or one; n is zero~ one or two; p is 1331~
zero, one or two, prov~ded that when p is one or two, each R ~n formula (b) can be located on either of the two fused rings; q ~s zero, one, or two, provided that when q is one or two, each R in formula (c) can be located on e1ther of the two fused rings; and each R is 1ndependently selected from the group consisting of halo, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, Cl-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR"' wherein R''' is H or Cl-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different. are each H or C1-C7 alkyl. These dihydropyridine analogs act as a delivery system for the corresponding biologi-cally active quaternary compounds in vivo. Due to its lipophilic nature, the dihydropyridine analog will distribute throughout the body and has easy access to the brain through the blood-brain barrier. Oxidation in vivo w1ll then provide the quaternary form, which will be "locked" preferentially in the brain. In con-tradist~nction to the drug-carrier ent~ties described in Bodor U.S. Patent No. 4,540,564 and related publica-tlons, however, there 1s no read1ly metabol1cally cleavable bond between drug and quaternary portions, and the act1ve specles delivered is not the original drug from which the d1hydro analog was derived, but rather is the quaternary analog itself.
Each of the major d1hydropyridine ~ pyridinium redox systems for bra1n-targeted drug delivery thus has 1ts own unique characteristtcs but also has properties ln common w~th the other approaches. Common to the various approaches 1s 1ntroduction of a dihydropyri-d1ne-type nucleus into the drug molecule, wh1th renders :~ ' : :.- ' ' ' : . : :: .
133~
the dihydropyridine-containing drug derivative substantially more lipoph~lic than the parent drug from -~
which ~t is der~ved. The increased lipophilicity enables the derivative to readily penetrate biological membranes~ including the blood-brain barrier. Also common to the various approaches is the fact that the Uredoxu nature of the dihydropyridine-type moiety means that the lipophilic dihydropyridine form is oxidizable -in vivo to the hydrophilic~ ionic pyridinium salt form, thus locking ~n the brain either the active drug or i~s quaternary precursor, depending on which approach is employed.
The dihydropyridine ~ pyridinium salt redox carrier and analog systems have achieved remarkable success in targeting drugs to the brain in laboratory tests. This success is, of course, due in part to the highly lipophilic nature of the dihydropyrid~ne-containing derivatives, which allows brain penetra-tion. At the same time, the increased lipophilicity `
makes it practically imposs1ble to formulate aqueoussolutions of these derivatives for injection; moreover, even when the dihydropyridines are dissolved in organic solvents such as 4imethylsulfoxide, they have a propensity for precipitating out of solution upon injection, particularly at higher concentrations, and especially at the injection site or in the lungs.
Indeed, even in the absence of noticeable crystalli-zat10n, it has been found that the redox derivatives frequently display not only the desired concentration in the bra1n but undesired lung concentrations as well, so that while the brain to blood ratios are at appropriate high levels, the initial lung to brain ~ ~ .
~ , ' ', levels are high as well. Still further, the dihydro-pyridine-containing derivatives suffer from stability : ~
problems, since even in the dry state they are very :
sensitive to oxidation as well as to water addition.
5 These problems must be overcome so t~at the dihydro-pyridine : pyridinium salt redox systems can be fully commercialized. -SUMMARY AND OBJECTS OF THE INVENTION:
One object of the present invention is to provide a method for stabilizing the reduced, dihydropyridine form of a dihydropyridine ~ pyridinium salt redox system for brain-targeted drug delivery.
Another object of the present invention is to provide a method for increasing the ratio of brain to lung concentrations at early time points resulting from administration of the reduced, dihydropyridine form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery.
Yet another object of the present invention is to ~:
provide a method for improving the water solubility of the reduced, dihydropyridine form of selected dihydro- ::~
pyridine pyridinium salt redox systems for brain-targeted drug delivery.
Another object of the present invention is to 25 provide improved pharmaceutical formulations containing ;~ -;.
the reduced, dihydropyridine form of a dihydropyri-dine ~ pyridinium salt redox system for brain-targeted drug delivery.
The foregoing objects are achieved by means of 30 novel inclusion complexes of hydroxypropyl, ~ :
i ~ 3 ~
hydroxyethyl, glucosyl, maltosyl or ma7totriosyl derivatives of ~- or Y-cyclodextrins with the reduced, dihydropyridine form of the dihydropyridine ~ pyridi-nium salt redox system for brain-targeted drug ~:
delivery. Thus, the present inven-tion provides a novel method for stabilizing the reduced biooxidizable, blood-brain barrier penetratinq lipoidal form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery, said method comprising .
co~plexing said reduced form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of ~- or Y-cyclodextrin. The present invention further provides a novel method for improving the water-solubility of the reduced, biooxidizable, blood-brain barrier penetrating lipoidal form of dihydropyridine pyridinium salt redox systems for brain-targeted drug delivery, said method comprising complexing said reduced form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of ~- or Y-cyclodextrin. The present invention still further provides a novel method for `
increasing the ratio of brain to lung concentrations of drug at early time points resulting from administration of the reduced, biooxidizable, blood-brain penetrating - .
lipoidal form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery, said `~
method comprising administering said reduced form as its inclusion complex with a hydroxypropyl, :~
hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of B- or y-cyclodextrin.
13 ~ 3~
-~3- ... ~.
eiRIEF nEscRIpTIoN OF THE ~RAllINGS:
Other objects and advantages of the present invention will be apparent from the following detailed description and acco~panying drawings, in which:
FIG. 1 is a phase-solubility diagram illustrating the increase in solubility of an estradiol-CDS, 17~-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra- :
1,3,5(10)-trien-3-ol, hereafter referred to as E2-CDS
(-), with increasing concentrations of hydroxypropyl-~-cyclodextrin in water;
FIG. 2 is a graph comparing the brain concentra-tions of the quaternary cation, 17~ (1-methyl-3-pyridinium)carbonyloxy~estra-1,3,5(10)-trien-3-ol, hereafter referred to as E2Q+, in % dose per gram of 15 brain tissue, following systemic administration to rats :
of either 15 mg/kg E2-CDS in dimethylsulfoxide (-) or 5 mg/kg E2-CDS in aqueous hydroxypropyl-~-cyclodextrin (-), corrected for dose;
FIG. 3 is a pair of semi-logarithmic plots, the 20 first comparing the concentrations of E2-CDS in lung ~ .
tissue in ~9 per gram dose following systemic administration to rats of either 15 mg/kg E2-CDS in dimethylsulfoxide (O) or 5 mg/kg E2-CDS inclusion -~
complex with hydroxypropyl-~-cyclodextrin (~) in water, corrected for dose, and the second comparing the lung concentrations of the quaternary cation, E2Q+ or Quat, following the same E2-CDS administration;
FIG. 4 is a bar graph illustrating, at selected ~:
time points, the concentrations of the quaternary cation, E2Q+ or Quat, in the brain in n~ per gram dose, :~
. ~..-, .
':~ :}f.. ~" ' ~ ': ~ '' '.": ":' . . , ' . ` ' following syste~ic administration to rats of either 15 mg/kg E2-CDS in dimethylsulfoxide (~) or 5 mg/kg E2-CDS
inclusion co~plex with hydroxypropyl-3-cyclodextrin (~) in water, corrected for dose; and F16. 5 is a plot of first-order rate constants as a function of ferricyanide ion concentration, illus-trating the effect of added hydroxypropyl-~-cyclo-dextrin (HPCD) on the rate of E2-CDS oxidation.
DETAILED DESCRIPTION OF THE INVENTION:
.
The term "lipoidal" as used herein is intended to designate a redox moiety which is lipid-soluble or lipophilic.
The terms "redox carrier system" and "redox analog system" are intended to designate two different approaches to targeting drugs to the brain using a dihydropyridine + pyridinium salt system; compounds -~-~
representing either of these approaches are contem- -~
plated for complexation with hydroxypropyl-~-cyclodex-trin and use in accord with the present invention.
The redox carrier system provides for brain- -~
targeted drug delivery by means of carrier-drugs, which in their reduced form, which is the form intended for administration, can be represented by the formula [D-DHC]
wherein ~D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine + pyridinium salt redox carrier. In their ' .' ::
:
1 3 ~ ~ r ;~ ~ :
oxidized form, which is the form "locked" in the brain from which the active drug is ultimately released, the carrier-drugs can be represented by the formula [D-QC]+ X~
wherein X~ is the anion of a non-toxic pharmaceutically acceptable acid, [~] is a centrally acting drug species and [~C]+ is the hydrophilic, ionic pyridinium salt form of a dihydropyridine pyridinium salt redox carrier. The redox carrier approach is discussed hereinabove in the section entitled "BACKGROUND OF THE
I~VENTION"; historically, the carrier system is the second type of redox system developed for delivering drugs to the brain.
Various aspects of the redox carrier system have been described in detail in Bodor United States Patent No. 4,479,932, issued October 30, 1984; Bodor United States Patent No. 4,~40,564, issued September 10, 1985; -~ -Bodor et al United States Patent No. 4,617,298, issued October 14, 1986; and UNIVERSITY OF FLORIDA's International Application No. PCT/US83/00725, published under International Publication No. W083/03968 on November 24, 1983.
The redox analog system provides for brain~
targeted drug delivery by means of new compounds -~
containing a dihydropyridine + pyridinium salt portion which, unlike the redox carrier, is not readily meta-bolically cleavable from the original drug molecule. :
One redox analog approach, which provides deriva-tives of centrally acting amines in which a primary, `, ' ' '~ ~ '~ . ' ,, : ' '" . ~.,' . ' ' ': , . ': ' . :' '. : ' ' : ". :: ~: i ' ' " ~ ' ' r~
secondary or tertiary amine function has been replaced with a dihydropyridine ~ pyridiniu~ salt redox system, is discussed hereinabove in the section entitled "BACK-GROUND OF THE IN~ENTION"; historically, this analog systerfl is the third type of redox system developed for delivering drugs to the brain. Various aspects of this analog system are described in detail in UNIVERSITY OF
FLORIDA's International Application ~lo. PCT/US85/00236, published under International Publication No.
W085/03937 on September 12, 1985.
Another redox analog approach provides novel amino acids and peptides containing them which comprise a dihydropyridine ~ pyridinium salt portion, the redox system being appended directly or via an alkylene bridge to the carbon atom adjacent to the carboxyl carbon. These amino acids and peptides are described in detail in UNIVERSITY OF FLORIDA's copending Canadian Patent Application No. 563,439-3, filed April 6, 1988. 3riefly, the novel redox amino acids in the reduced form have the structural formula 113 ~
:::
' wherein Z is either a direct hond or C1-C6 alkylene and can be attached to the heterocyclic ring via a ring carbon atom or via the ring nitrogen atom; R1 is C1-C7 ` 25 alkyl, C1-C7 haloalkyl or C7-C12 aralkyl when Z is '~
,;
.
., . - , .
.,~
-. r ~33~
attached to a ring carbon atom; R1 is a direct bond when Z is attached to the ring nitrogen ato~; R2 and R3, which can be the same or different, are selected from the group consisting of hydrogen, halo, cyano, C
C7 alkyl, Cl-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, Cl-C7 alkylthio, C1-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR' " wherein R''' is hydrogen or Cl-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are ;~
each hydrogen or Cl-C7 alkyl; or one of R2 and R3 together with the adjacent ring carbon atom for~s a ::~
benzene ring fused to the heterocyclic ring, which benzene ring may optionally bear one or two substi-tuents, which can be the same or different, selected from the group consisting of hydroxy, protected hydroxy, halo, cyano, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, Cl-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR''' wherein R' " is hydrogen or Cl-C7 alkyl, and 20 -CONR'R'' wherein R' and R " , which can be the same or ~::
different, are each hydrogen or C1-C7 alkyl; R4 is :~
hydrogen or a carboxyl protective group; R5 is hydrogen or an amino protective group; and the dotted lines . .
indicate that the compound contains a 1,4- or 1,6- ::
25 dihydropyridine, a 1,4- or 1,2-dihydroquinoline, or a -1,2-dihydroisoquinoline ring system.
The new dihydropyridine amino~acid analogs ~ -depicted above and the corresponding oxidized forms are useful in the preparation of novel redox peptides of the partial formulas:
..
?
~ ~31~
-HI~H- CO-N-Rl (A) , :~
~reduced form) : .:
3nd R ~/N -~1 (B) :
3 X~
(oxid~2ed form) ;~
the new pePtide analogs of partial structure (A) act as a delivery system for the corresponding quaternary ~ -salts of partial structure (B) ~n vivo; the quaternary derivatives, which also are chemical intermediates to the dihydro compounds, are pharmacologically active or convertible in vivo to pharmacologically active peptides, and are characterized by site-specific and sustained delivery to the brain when administered via the corresponding dihydropyridine form. Methods for the preparation of these analog amino acids and peptides utilize methods known in the art for intro-duction of the dihydropyridine ~ pyridinium salt moiety or a precursor thereof, e.g. from the aforementioned International Publications Nos. W083/03968 and .,'''~
'. " -: . . ~ ' . ~' . . - ' ' ' : .:
W085/03937, appropriately combined with well-known -~
methods for peptide synthesis. Ultimately, the quaternary forms of the amino acids and peptides are subjected to reduction to afford the corresponding ~ -dihydropyridines, according to the methods of the Bodor U.S. patents and above-mentioned published PCT appli-cations.
In a preferred aspect of the present invention, the redox system selected for complexation with a 10 hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or ;-maltotriosyl derivative of ~- or y-cyclodextrin and use in accord with the present invention is a redox carrier system. The drug and carrier portions of the redox ~-carrier system are described in more detail below and of course in the various carrier patents and patent applications identified above. Selection of appro-priate drugs and carrier moieties need not be limited to specific drugs and specific carriers disclosed in the aforementioned patents and applications or in the present application, just so long as the selected drug and carrier meet the general requirements of the drug/carrier system as described in the aforenoted documents.
The term "drug" as used herein means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental develop- ;
ment and conditions in man or animal.
By "centrally acting" drug species, active agent or compound as utilized herein, there is of course intended any drug species or the like, a significant ; .~
1~3~
(usually, pr1ncipal) pharmacolog~cal activity of which ~s CNS and a result of d~rect action in the brain.
Exemplary such centrally acting drug species are the CNS-amines and other nervous system agents, whether sympathetic or parasympathetic, e.g., phenylethylamine (a stimulant), dopamine (a neurotransmitter and dopaminergic agent used, e.g., in the treatment of Parkinsonism or hyperprolactinemia), tyramine (a stimulant), L-DOPA (a dopamine precursor used, for example, in the treatment of Parkinsonism); muscle relaxants, tranquilizers and antidepressants, e.g., benzodiazepine tranquilizers such as diazepam and oxazepam and phenothiazine tranquilizers such as carphenazine, fluphenazine and the like; mild and strong analgesics and narcotics; sedatives and hypnotics; narcotic antagonists; vascular agents;
st~mulants; anesthet~cs; small peptides, such as the di-, tri-, tetra and pentapeptides, and other small 2-20 amino ac~d unit containing pept~des, e.g. the enkephalins (for example, Tyr-Gly-Gly-Phe-Leu), which, bes~des be1ng analges1cs, initiate epileptic activity ~n the brain at doses that are about tenfold lower than for effect1ng analges1c activity; growth-promoting substances; ant1epilept1c and anticonvulsant drugs generally, lncluding hydantoins such as phenytoln and ethoto~n, barbituates such as phenobarb~tal; hormones, such as the steroid hormones, e.g., estrad~ol, testosterone, 17 a-ethynyl testosterone (ethisterone), and the like (recent stud1es on histological mapp~ng of hormone-sensit~ve and spec~fic steroid binding cells ~n the brain have underscored the importance of the stero1d act~on 1n the bra1n on sexual behav~or);
~:
r - , -31- ;~
amphetamine-llke drugs; anticancer and anti-Parkinsonism agents; anti-hypertenslves, agents to enhance learning capac~ty and the memory processes, including treatment of dementias, such as Alzheimer's 5 disease, such as 9-amino-1,2,3,4-tetrahydroacridine; ~ ;
antibacterials; centrally acting hypotensive agents;
centrally acting prostaglandins, such as PGD2;
d1agnostic agents, such as radiopharmaceuticals;
monoamlne oxidase (MAO) inhibitor drugs; CNS or brain important/essential amino acids, such as tryptophan (which is an antidepressant as well as a nutrient); and any like centrally acting compounds. For the purposes of this invention, dopa or L-DOPA is not classified as an amino acid but rather as a CNS amine and dopa-minergic agent used, e.g. in the treatment ofParkinsonism.
Other illustrative ultimate species of centrally acting drug entities are: amphe~amine, dextroamphet-amine, levamphetamine, aletamine, cypenamine, fencam-famin, fenozolone, zylofuramine, methamphetamine, phen-metrazine and phentermine, which are sympathomimetic amines/cerebral stimulants and appetite suppressants;
etryptamine, a cerebral stimulant; codeine, oxycodone, pentazoc~ne, anileridine, hydromorphone, morph1ne and oxymorphone, which are narcotic analgesics; desipra-mine, nortriptyline, octriptyllne, maprotiline, opipramol and protriptyl1ne, which are cerebral st1mulants/tricylic ant1depressants of the dibenza-zepine type used, e.g., ~n endogenous depressions;
clon~d~ne and methyldopa, whlch are sympatholytic agents used, e.g., tn hypertension; biperiden, cycr~m~ne and procyclidine, which are centrally acting antichollnergics; tranylcypromine, a sympathomimetic cerebral stimulant/MA0 inh~bitor and antidepressant;
acetophenazine, carphenazine, fluphenazine, perphenazine and piperacetazine, which are ~ :
phenothiaz1ne-type tranquilizers; benzoctamine, a sedative/muscle relaxant which structurally is an analogue of the phenothiazine tranquilizers; chlor- - :
diazepoxide, clorazepate, nitrazepam and temazepam, which are benzodiazepine-type tranquilizers; noracy-methadol, a narcotic analgesic of the methadone type;
piminodine, a narcotic analgesic of the meperidine type; tracazolate, a sedative/hypotensive; prizidilol, a centrally acting hypotensive; sulpiride$ an anti-depressant/psychotropic; haloperidol and clopenthixol, which are tranquilizers; norepinephrine, a sympathetic stimulant/adrenergic agent; nalorphine and naloxone, narcotic antagonists; hydralazine, a hypotensive;
ethotoin, phenobarbital and aminoglutethimide, anti-convulsants; epinephrine, an adrenergic agent;
20 ethamivan, a medullary stimulant; bemegride, a ~. :
barbiturate antagonist; amiphenazole, a stimulant, iopydol, iodopyracet, iodouppurate (o-~odohippuric acid), iodamide and iopanoic acid, which are radiodiagnost~cs; ephedrine, pseudoephedrine, oxymetazoline and phenylephrine, which are sympatho-mimetic amlnes and decongestants; estradiol, estrone and estriol, the natural estrogens; amoxicillin, oxacillin, carbenicillin, benzylpenicillin, phenoxy-methylpenicillin, meth1cillin, nafcillin, ticarcillin, bacampicillin, epicillin, hetacillin, pivampacillin, the methoxymethyl ester of hetacillin, and ampicillin, which are penic1llin-type antibiotics; amobarbital, a ,, , ~ . ~
.. ~
:::
sedative; trihexyphenidyl, a centrally acting anticholinergic; hydroxyzine9 a tranquilizer; chlorte~
tracycline, demeclocycline, minocycline, doxycycline,~ :.
oxytetracycline, tetracycline and methacycline, which are tetracycl~ne-type antibiotics; flurazepam, bromazepam, demoxepam and lorazepam, benzodiazepine tranqu~lizers; phenytoin, an anticonvulsant;
glutethimide, a mild hypnotic/sedative; clindamyr-in, lincomycin, nalidix~c acid, oxolinic acid and phenazo-10 pyrldine, zntibacterials/antibiotics; bethanidine and ~:
guanethidine, hypotensives/sympatholytics; captopril, a hypotensive; methyprylon, a mild hypnotic; amedalin, bupropion, cartazolate, daledalin, difluanine, fluoxe-tine and nisoxetine, which are cerebral stimulants; ~ -15 propranolol, a B-blocker antihypertensive; cloxacillin~: :
and dicloxacillin, penicillin-type antibacterials;
butalbital, a barbiturate sedat~ve; GABA, y-vinyl GA8A, y-acetylenic GA~A, neurotransmitters for possible use in epilepsy; valproic acid and its metabolites such as S-hydroxy-2-n-propylpentanoic acid, 4-hydroxy-2-n-propylpentanoic acid, 3-hydroxy-2-n-propylpentanoic ac~d, for use as anticonvulsants; valpromide, a valproic acid derivative for use as an anticonvulsant;
apomorph1ne, a narcotic depressant/emetic which has been used in the treatment of photosensitive epilepsy;
pholcod~ne, a narcotlc antitussive; methotrexate, :
mitoxantrone, podophyllotoxin derivatives (etopside, teniposide), doxorubicin, daunamycin and cyclophosphamide, anti-cancer/antitumor agents; ~
30 methylphenidate, a stimulant; thiopental, an ~ ~ .
anesthetlc; ethinyl estradiol and mestranol, estrogens;
meptaz1nol, cyclazocine, phenazocine, profadol, $ . ~
~`
r ~
- 3 4 - ~ :
:~
metopon, drocode and myfadol, which are narcotic ~-~
analges1cs; buprenorphlne, nalbuphine, butorphanol, levallorphan, naltrexone, nalme~ene, alazocine, ~ :
oxllorphan and nalmexone, which are narcotic antagonists or igon1st-antagonists; norgestrel and norethindrone, progestins; cephalothin, cephalex1n, cefazolin, cefoxitin, moxalactam, ceforanide, cefroxadine and cephap1rin, cephalosporin ant~biotics;
atenolol, nadolol, t~molol and metoprolol, B-blockerslhypotensives; ACTH (corticotropin), a hormonewhich stimulates glucocorticoid production; LHRH, a neurotransmltter which stimulates secretion of the pituitary hormones, LH and FSH, and has been used to~:- :
1nduce ovulation as well as for fertility control/ :~
contraception; sulfadiazine and other sulfonamide antibiotics; ribavarin and acyclovir, antiviral agents;
chlorambuc11 and melphalan, nitrogen mustard-type anti-cancer/antitumor agents; methotrexate and aminopterin, which are folic acid antagonist-type anticancer/anti-tumor agents; plat1num coordination complexes, i.e.
cisplatin analogue-type anticancerlant~tumor agents; ~: .
dactinomycin and m1tomyc1n C, used in cancer chemo-therapy; th109uanine, a pur1ne/pyrimidine antagonist : used 1n cancer treatment; vincristlne and vinblastlne, ant1cancer alkaloids; hydroxyurea and DON, anticancer urea derivat1Yes; FSH, HCG and HCS, pituitary and non-pttu~tary gonadotrop1ns, used, for example, in certain reproductive disorders; N,N'-bis(dichloracetyl)-1,8-octamethylenediamlne (fertilysin), an agent for male fert11ity 1nhib1t10n; levorphanol, a narcotic anal-gesic; benzestrol and diethylst11bestrol, synthetic estrogens; ethyl B-carbol1ne-3-carboxylate, a benzo-i ~ 3 ,3 ~ ~ ~ I A
diazepine antagontst; furosemide, a diuretic/antihyper~
tensive; dipyridamole and nifedipine, coronary vaso-dilators; and progabide, a GABA-agonist and prodrug of -GABA. Yet other ultimate species include non-steroidal antiinflammatory agents/non-narcotic analgesics, e.g.
propionic acid derivatlves, acetic acid derivatives, fenamic acid derivatives and biphenylcarboxylic acid derivatives. Specific NSAID's/non-narcotic analgesics contemplated for use herein include ibuprofen, naproxen, flurbiprofen, zomepirac, sulindac, indo-methacin, fenbufen, fenoprofen, indoproxen, ketoprofen, fluprofen, bucloxic acid, tolmetin, alclofenac, fenclozic acid, ibufenac~ flufenisal, pirprofen, flufenamic acid, mefenamic acid, clonixeril, clonixin, meclofenamic acid, flunixin, diclofenac, carprofen, etodolac, fendosal, prodolic acid, sermetacin, indox-ole, tetrydamine, diflunisdl, naproxol, piroxicam, metazamide, flutiaz~n and tesicam.
Preferred classes of centrally acting drugs for use herein are the central neurotransmitters, steroids, anticancer and antitumor agents, antiviral agents, tranquilizers, memory enhancers, hypotensives, seda-tives, antipsychotics and cerebral stimulants (especially tricyclic antidepressants). Among the -~
neurotransmitters, there can be mentioned amino acids, such as GABA, GABA derivatives and other omega-amino acids, as well as glycine, glutamic acid, tyrosine, aspartic ac1d and other natural amino acids; catechol-am~nes, such as dopamine, norepinephrine and epine- ~ -phrine; serotonin, histamine and tryptamine; and pep~
tldes such as neurotensin, lute~nizing hormone-releaslng hormone ~LHRH), somatostatin, enkephalins 1 3 3 ~
such as met5-enkephalin and leu5-enkephalin, endorphins such as ~-, a- and B-endorphins, oxytocin M and vasopressin. Synthetic and semi-synthetic analogues, e.g. analogues of LHRH in which one or more amino ac1d(s) has/have been eliminated and/or replaced with one or more different amino acid(s), and which may be agonists or antagonists, are also contemplated, e.g.
the primary and secondary amine LHRH analogues dis-closed in United States Patents No. 4,377,574, 3,917,825, 4,034,082 and 4,338,305. Among the stero~ds, there can be mentioned anti-inflammatory adrenal cortical steroids such as hydrocortisone, betamethasone, cortisone, dexamethasone, flumethasone, fluprednisolone, meprednisone, methyl prednisolone, prednisolone, prednisone, triamcinolone, cortodoxone, fludrocortisone, flurandrenolone acetonide (fluran-drenolide), paramethasone and the like; male sex hormones (androgens), such as testosterone and its close analogues, e.g. methyl testosterone (17-methyltestosterone); and female sex hormones, bothestrogens and progestins, e,g. progestins such as norgestrel, norethindrone, norethynodrel, ethisterone, dimeth~sterone, allylestrenol, cingestol, ethynerone, lynestrenol, norgesterone, norvinisterone, ethynodiol, oxogestone and tigestol, and estrogens such as ethinyl estradiol, mestranol, estrad~ol, estriol, estrone and qu1nestrol and the like. Among the anticancer and antitumor agents, there can be mentioned Ara-AC, pentostatln (2'-deoxycoformycin), Ara-C (cytarabine), 3-deazaguan~ne, dihydro-5-azacytidine, tiazofurîn, sangivamycin, Ara-A (v~tarabine), 6-MMPR, PCNU, FENU, HENU and other nitrosoureas, spiromustine, : ~' 13 ~1 bisbenzimidazole, L-alanosine (6-diazo-5-oxo-L-norleucine), DON, L-ICRF, trimethyl TMM, 5-methyl-tetrahydrohomofolic acid, glyoxylic acid ~
sulfonylhydrazone, DACH, SR-2555, SR-2508, desmethyl- ~ ~-misonidazole, mitoxantrone, menogarol, aclacinomycin A, phyllanthoside, bactobolin, aphidocolin, homoharring- :~
tonine, levonantradol, acivicin, streptozotocin, hydroxyurea, chlorambucil, cyclophosphamide, uracil mustard, melphalan, 5-FU (5-fluorouracil), 5-FUDR
(floxuridine), vincristine, vinblastine, cytosine arabinoside, 6-mercaptopurine, thioguanine, 5- . : :.
azacytidine, methotrexate, adriamycin (doxorubicin), ~ :
daunomycin (daunorubicin), largomycine polypeptide, :
aminopterin, dactinomycin, mitomycin C, and 1i5 podophyllotoxin derivatives, such as etoposide (VP-16) and teniposide. Among the antiviral agents, there can be mentioned ribavarin; acyclovir (ACV); amantadine (also of possible value as an anti-Parkinsonism agent); -~ :
diarylamidines such as 5-amidino-2-(5-amidino-2-benzo-furanyl)indole and 4',6-diimidazolino-2-phenylbenzo-(b)thiophene; 2-aminooxazoles such as 2-guanidino-4,5- ~:
di-n-propyloxazole and 2-guanidino-4,5-diphenyloxazole;
benzimidazole analogues such as the syn and anti ~ :
isomers of 6~(hydroxyimino)phenyl3methyl]-1-~
methylethyl)sulfonyl~-lH-benzimidazol-2-amine;
bridgehead C-nucleosides such as 5,7-dimethyl-2-3-D-ribofuranosyl-s-triazole(1,5-a)pyrimidine; glycosides such as 2-deoxy-D-glucose, glucosamine, 2-deoxy-2-fluoro-D-mannose and 6-amino-6-deoxy-D-glucose; phenyl 30 glucoside derivatives such as phenyl-6-chloro-6-deoxy- ;~
B-D-glucopyranoside; (S)-9-(2,3-dihydroxypropyl)-adenine; t~azofurin; selenazofurin; 3-deazauridine; 3- :~ ~
,"",.. ~ ,,"," ,-,"-,-: ~: :' ~' ' ' ' ' ' ' ' "., ..
-, , ~ "
- `~
~ ~ 3 ~
deazaguanosine; DHPG; 6-azauridine; idoxuridine;
trifluridine (trifluorothym~dine); BDVU (bisdihydroxy-v1nyluridine); zidovudine (AZT); dideoxycytidine; and 5,6-d~chloro-l-~-D-r1bofuranosylbenzimidazole. Among the anti-cancer/intitumor and antiviral agents, those of the nucleoside type (i.e. a purine or pyrimidine base-type structure bear~ng a singly or multiply hydroxylated substituent) are of particular lnterest.
This group ~ncludes such compounds as Arà-AC, pentostatin, Ara-C, dihydro-5-azacytidine, tiazofurin, sangivamycin, Ara-A, 6-MMPR, desmethylmisonidazole, 5-FU~R, cytosine arabinoside, 5-azacytidine, ribavirin, acyclovir, (S)-9-(2,3-dihydroxypropyl)adenine, 6-azauridine, 5,6-dichloro-1-B-D-ribofuranosyl-benzimidazole, 5,7-dimethyl-2-B-D-ribofuranosyl-s-trlazole(l,5-a)pyrimidine, zidovudine (AZT), dldeoxycytidine, dideoxyadenosine, dideoxyinosine and DHPG. Among the tranquilizers, there can be mentioned benzodiazepine tranqu~lizers, such as diazepam, oxazepam, lorazepam, chlordiazepoxide, flurazepam, bromazepam, chlorazepate, n~trazepam and temazepam;
hydantoin-type tranqu~lizers/anticonvulsants such as phenytoin, ethotoin, mephenytoin; pheno~hiazine-type tranqu111zers such as acetophenazine, carphenazlne, fluphenaz~ne, perphenaz~ne and piperacetazine; and others. Among the hypotensives, there can be mentioned clon~dine, methyldopa, bethan1dine, debr~soquin, hydralaz~ne, and guanethidine and ~ts analogues. Among the sedatives, tranquilizers and antipsychot~cs, there can be mentioned the many specific compounds of this type disclosed above, espec1ally the phenoth~azines and benzodiazep~nes and their analogues. Among the ~ /~r -.
r~
13~
cerebral stimulants, there also can be mentioned the many specific compounds set forth hereinabove, particularly the sympathomlmetic amine-type cerebral stimulants and the tricyclic antidepressants, espectally preferred tricycl~cs being the dibenzazepines and their analogues.
Also illustrative of the centrally acting drug species contemplated by this invention are centrally active metabolites of centrally acting drugs. Such metabolites are typified by hydroxylated metabolites of tricyclic antidepressants, such as the E- and ~-iscmers of 10-hydroxynortriptyline, 2-hydroxyimipramine, 2- ~-hydroxydesipramine and 8-hydroxychloripramine; hydroxy-lated metabolites of phenothiazine trarquilizers, e.g.
(G3--C~), 6-0--maltotriosyl-~-C~ (G3-~-CD) and 6-0--maltotriosyl-~-CD (G3-y-cn~.
The delivery of drugs to the brain is often seriously limited by transport and metabolism factors and, more specifically, by the functional barrier of the endothelial brain capillary wall, i.e. the blood- ;~
brain barrier or ~BB. Site-specific delivery and sustained delivery of drugs to the brain are even more difficult.
A dihydropyridine ~ pyridinium salt redox system has recently been successfully applied to delivery to the brain of a number of drugs. Generally speaking, ;~
according to this system, a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the blood-brain ~-barrier following its systemic administration. Subse-quent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
Three main approaches have been published thus far for delivering drugs to the brain using this redox system. The first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component. This approach was first applied to delivering to the brain N-methylpyridinium- -~
2-carbaldoxime chloride (2-PAM), the active nucleus of ¦ which constitutes a quaternary pyridinium salt, by way ~ of the dihydropyridine latentiated prodrug form ¦ 30 thereof. Thus, a hydrophilic compound (2-P4M) was made lipoidal (i.e. lipophilic) by making its dihydropyri-.i .
~ -.~ ~ ~ ,................................... .
c: ~'''` ~ ~`'' ~ ` ~ ` ~ ' ' ''' ': ' .:'.:
1 3 ~
dine form (Pro-2-PAM) to enahle its penetration through lipoidal barriers. This si~ple prodrug approach allowed the co~pound to get into the brain as well as other organs, but this manipulation did not and could 5 not result in any brain specificity. On the contrary, -~-such approach was delimited to relatively small mole-cule quaternary pyridinium ring-containing drug species and did not provide the overall ideal result of brain-specific, sustained release of the desired drug, with concomitant rapid elimination from the general circu-lation, enhanced drug efficacy and decreased toxi- -city. No "trapping" in the brain of the 2-PAM formed in situ resulted, and obviously no brain-specific, sustained delivery occurred as any consequence there-of: the 2-PAM was eliminated as fast from the brain as it was from the general circulation and other organs.
Co~pare U.S. Patents Nos. 3,929,813 and 3,962,447;
Bodor et al, J. Pharm. Sci, 67, No. 5, 685 (1978). See also 30dor, "Novel Approaches for the Design of Me~brane Transport Properties of Drugs", in Design of Biopharmaceutical Properties Through Prodrugs and Analogs, Roche, E.B. (ed.), APhA Academy of Pharmaceutical Sciences, Washington, D.C., 98-135 (1976). Subsequent extension of this first approach to ~-~
delivering a much larger quaternary salt, berberine, to the brain via its dihydropyridine prodrug form was, ~-~
however, found to provide site-specific sustained delivery to the brain of that anticancer agent. See Bodor et al, Science, Vol. 214, ~ecember 18, 1981, pp.
30 1370-1372.
The second approach for delivering drugs to the brain using the redox system involves the use of a :
1331!r~ ~t pyridinium carrier chemically linked to a biologically active compound. Bodor et al, Science, Vol. 214, ~ :~
~ecember 1~, 1981, pp. 137~-1372, outline a scheme for this specific and sustained delivery of drug species to the brain, as depicted in the following Scheme 1:
' ~
' ' ',"' '~;"
, ! ' . ~
1 3 3 ~
- l 'i - ' . .
' lDl ~ ~ ID~CI- ~-DHCl DEEIVER~ / 7 ~TO 80DY \ /ELIIllIUTIOH
ID-DHCI ID-DHCJ
111 THE ~RAIII IN CIRCULATORY SYSTEn . -~D O~G~S
1~ OXIDATIOII ~ oxlDAT
~ , ~ ~ , .
ID-OC1''-- ENZYI'ATtC ENZY14~TIC --ID-OCI t Ill HE SRAI~I K 111 Cl~ICI ILATORY SYSTE~
4 4 .
~'- ' . , ~ ~ ,.' 2 .~, . i.it~
IDI ~ 10Cll IDI ~1CI1 K3 ~: S 6 .. -.
B88 ~ , : I
,i 1 , ¦~ ELIYINATION
~,j.
1~ SCHE~IE 1: BBB, UOOD-~RAI~ RARRIER.
:1 '~'1 Accord1ng to the scheme ~n Science~ a drug tD] is coupled to a quaternary carrier [QC]+ and the [D-QC3+
which results is then reduced chemically to the lipoidal dihydro form [D-DHC]. After administration of ~D-DHC] in vivo, it is rapidly distributed throughout the body, includlng the brain. The dihydro form [D-DHC] is then in situ oxidized (rate constant, k1) (by the NAD ~ NADH system) to the ideally inactive original [D-QC]+ quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the -~
blood-brain barrier should prevent its elimination from the brain (k3 k2; k3 k7). Enzymatic cleavage of the [D-QC]+ that is "locked" in the brain effects a -15 sustained delivery of the drug species [D], followed by ;~
its normal elimination (k5), metabolism. A properly selected carrier [QC~ will also be rapidly eliminated from the brain (k6 k2). Because of the facile elimination of tD-QC]+ from the general circulation, only m~nor amounts of drug are released in the body ~k3 k4); [D] will be released primarily in the brain (k4>k2). The overall result ideally will be a brain-specific sustained release of the target drug spec1es. Specifically, Bodor et al worked with phenyl-ethylamine as the drug model. That compound wascoupled to nicotinic acid, then quaterni~ed to give compounds of the formula : .
[~J C51~ 32C32~ ~1 ~ CH3 or CH2 ~ )- :
;- ~: , . . ., . , :; , . . . .......... . . .
i ~
133~4 wh1ch were subsequently reduced by sodium dithion1te to the correspond~ng compounds of the formula ¢3- CONHCH2CH2~ (R = CH3 or CH ~ ).
R
Testlng of the N-methyl der1vatlve in vivo supported 5 the criteria set forth in Scheme I. Bodor et al -speculated that various types of drugs might possibly ~ -~
be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring-substituted derivatives was being studied for delivery of amino- or hydroxyl-containing drugs, including small peptldes, to the bra~n. No other possible specific carriers were disclosed. Other reports of this work with the redox carrier system have appeared in The Friday Evening Post, August 14, 1981, Health Center Communications, University of Florlda, Galnesville, Flor1da; Chemical ~ Engineerlng News, December 21, 1981, pp. 24-25; and Science News, January 2, 1982, Vol. 121, No. 1, page 7. More recently, the redox carr~er system has been substantially extended in terms of possible carriers and drugs to be delivered. See International Patent Application No. PCT/US83/00725, flled May 12, 1983 and published November 24, 1983 under International Publicat~on No. W083/03968. Also see Bodor et al, Pharmacology and Therapeutics, Vol.
19, No. 3, pp. 337-386 (1983); and Bodor United States Patent No. 4,540,564, ~ssued September 10, 1985.
~ :' . ~-, :
1~31r~/U,~
The third approach for delivering drugs to the brain using the redox system provides derivatives of centrally acting amines ln which a primary, secondary or tertiary amine funct~on has been replaced with a dihydropyridine/pyridinium salt redox system. These brain-specific analogs of centrally acting amines have been recently described in International Patent Appli-cation No. PCT/US85/00236, filed February 15, 1985 and published September 12, 1985 under International Publi-cation No. W085/03937. The dihydropyridine analogs are characterized by the structural formula . :
D - NJ
wherein D is the residue of a centrally acting primary, secondary or tertlary amlne, and -N 3 is a radical of the formula b ~ N~
( R~ ~J ( R ) q ~ o r b ) ( C ) wherein the dotted line in formula (a) indicates the presence of a double bond in either the 4 or 5 pos1t1On of the dihydropyridine ring; the dotted line in formula (b) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system; m is zero or one; n is zero~ one or two; p is 1331~
zero, one or two, prov~ded that when p is one or two, each R ~n formula (b) can be located on either of the two fused rings; q ~s zero, one, or two, provided that when q is one or two, each R in formula (c) can be located on e1ther of the two fused rings; and each R is 1ndependently selected from the group consisting of halo, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, Cl-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR"' wherein R''' is H or Cl-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different. are each H or C1-C7 alkyl. These dihydropyridine analogs act as a delivery system for the corresponding biologi-cally active quaternary compounds in vivo. Due to its lipophilic nature, the dihydropyridine analog will distribute throughout the body and has easy access to the brain through the blood-brain barrier. Oxidation in vivo w1ll then provide the quaternary form, which will be "locked" preferentially in the brain. In con-tradist~nction to the drug-carrier ent~ties described in Bodor U.S. Patent No. 4,540,564 and related publica-tlons, however, there 1s no read1ly metabol1cally cleavable bond between drug and quaternary portions, and the act1ve specles delivered is not the original drug from which the d1hydro analog was derived, but rather is the quaternary analog itself.
Each of the major d1hydropyridine ~ pyridinium redox systems for bra1n-targeted drug delivery thus has 1ts own unique characteristtcs but also has properties ln common w~th the other approaches. Common to the various approaches 1s 1ntroduction of a dihydropyri-d1ne-type nucleus into the drug molecule, wh1th renders :~ ' : :.- ' ' ' : . : :: .
133~
the dihydropyridine-containing drug derivative substantially more lipoph~lic than the parent drug from -~
which ~t is der~ved. The increased lipophilicity enables the derivative to readily penetrate biological membranes~ including the blood-brain barrier. Also common to the various approaches is the fact that the Uredoxu nature of the dihydropyridine-type moiety means that the lipophilic dihydropyridine form is oxidizable -in vivo to the hydrophilic~ ionic pyridinium salt form, thus locking ~n the brain either the active drug or i~s quaternary precursor, depending on which approach is employed.
The dihydropyridine ~ pyridinium salt redox carrier and analog systems have achieved remarkable success in targeting drugs to the brain in laboratory tests. This success is, of course, due in part to the highly lipophilic nature of the dihydropyrid~ne-containing derivatives, which allows brain penetra-tion. At the same time, the increased lipophilicity `
makes it practically imposs1ble to formulate aqueoussolutions of these derivatives for injection; moreover, even when the dihydropyridines are dissolved in organic solvents such as 4imethylsulfoxide, they have a propensity for precipitating out of solution upon injection, particularly at higher concentrations, and especially at the injection site or in the lungs.
Indeed, even in the absence of noticeable crystalli-zat10n, it has been found that the redox derivatives frequently display not only the desired concentration in the bra1n but undesired lung concentrations as well, so that while the brain to blood ratios are at appropriate high levels, the initial lung to brain ~ ~ .
~ , ' ', levels are high as well. Still further, the dihydro-pyridine-containing derivatives suffer from stability : ~
problems, since even in the dry state they are very :
sensitive to oxidation as well as to water addition.
5 These problems must be overcome so t~at the dihydro-pyridine : pyridinium salt redox systems can be fully commercialized. -SUMMARY AND OBJECTS OF THE INVENTION:
One object of the present invention is to provide a method for stabilizing the reduced, dihydropyridine form of a dihydropyridine ~ pyridinium salt redox system for brain-targeted drug delivery.
Another object of the present invention is to provide a method for increasing the ratio of brain to lung concentrations at early time points resulting from administration of the reduced, dihydropyridine form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery.
Yet another object of the present invention is to ~:
provide a method for improving the water solubility of the reduced, dihydropyridine form of selected dihydro- ::~
pyridine pyridinium salt redox systems for brain-targeted drug delivery.
Another object of the present invention is to 25 provide improved pharmaceutical formulations containing ;~ -;.
the reduced, dihydropyridine form of a dihydropyri-dine ~ pyridinium salt redox system for brain-targeted drug delivery.
The foregoing objects are achieved by means of 30 novel inclusion complexes of hydroxypropyl, ~ :
i ~ 3 ~
hydroxyethyl, glucosyl, maltosyl or ma7totriosyl derivatives of ~- or Y-cyclodextrins with the reduced, dihydropyridine form of the dihydropyridine ~ pyridi-nium salt redox system for brain-targeted drug ~:
delivery. Thus, the present inven-tion provides a novel method for stabilizing the reduced biooxidizable, blood-brain barrier penetratinq lipoidal form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery, said method comprising .
co~plexing said reduced form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of ~- or Y-cyclodextrin. The present invention further provides a novel method for improving the water-solubility of the reduced, biooxidizable, blood-brain barrier penetrating lipoidal form of dihydropyridine pyridinium salt redox systems for brain-targeted drug delivery, said method comprising complexing said reduced form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of ~- or Y-cyclodextrin. The present invention still further provides a novel method for `
increasing the ratio of brain to lung concentrations of drug at early time points resulting from administration of the reduced, biooxidizable, blood-brain penetrating - .
lipoidal form of a dihydropyridine + pyridinium salt redox system for brain-targeted drug delivery, said `~
method comprising administering said reduced form as its inclusion complex with a hydroxypropyl, :~
hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of B- or y-cyclodextrin.
13 ~ 3~
-~3- ... ~.
eiRIEF nEscRIpTIoN OF THE ~RAllINGS:
Other objects and advantages of the present invention will be apparent from the following detailed description and acco~panying drawings, in which:
FIG. 1 is a phase-solubility diagram illustrating the increase in solubility of an estradiol-CDS, 17~-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra- :
1,3,5(10)-trien-3-ol, hereafter referred to as E2-CDS
(-), with increasing concentrations of hydroxypropyl-~-cyclodextrin in water;
FIG. 2 is a graph comparing the brain concentra-tions of the quaternary cation, 17~ (1-methyl-3-pyridinium)carbonyloxy~estra-1,3,5(10)-trien-3-ol, hereafter referred to as E2Q+, in % dose per gram of 15 brain tissue, following systemic administration to rats :
of either 15 mg/kg E2-CDS in dimethylsulfoxide (-) or 5 mg/kg E2-CDS in aqueous hydroxypropyl-~-cyclodextrin (-), corrected for dose;
FIG. 3 is a pair of semi-logarithmic plots, the 20 first comparing the concentrations of E2-CDS in lung ~ .
tissue in ~9 per gram dose following systemic administration to rats of either 15 mg/kg E2-CDS in dimethylsulfoxide (O) or 5 mg/kg E2-CDS inclusion -~
complex with hydroxypropyl-~-cyclodextrin (~) in water, corrected for dose, and the second comparing the lung concentrations of the quaternary cation, E2Q+ or Quat, following the same E2-CDS administration;
FIG. 4 is a bar graph illustrating, at selected ~:
time points, the concentrations of the quaternary cation, E2Q+ or Quat, in the brain in n~ per gram dose, :~
. ~..-, .
':~ :}f.. ~" ' ~ ': ~ '' '.": ":' . . , ' . ` ' following syste~ic administration to rats of either 15 mg/kg E2-CDS in dimethylsulfoxide (~) or 5 mg/kg E2-CDS
inclusion co~plex with hydroxypropyl-3-cyclodextrin (~) in water, corrected for dose; and F16. 5 is a plot of first-order rate constants as a function of ferricyanide ion concentration, illus-trating the effect of added hydroxypropyl-~-cyclo-dextrin (HPCD) on the rate of E2-CDS oxidation.
DETAILED DESCRIPTION OF THE INVENTION:
.
The term "lipoidal" as used herein is intended to designate a redox moiety which is lipid-soluble or lipophilic.
The terms "redox carrier system" and "redox analog system" are intended to designate two different approaches to targeting drugs to the brain using a dihydropyridine + pyridinium salt system; compounds -~-~
representing either of these approaches are contem- -~
plated for complexation with hydroxypropyl-~-cyclodex-trin and use in accord with the present invention.
The redox carrier system provides for brain- -~
targeted drug delivery by means of carrier-drugs, which in their reduced form, which is the form intended for administration, can be represented by the formula [D-DHC]
wherein ~D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine + pyridinium salt redox carrier. In their ' .' ::
:
1 3 ~ ~ r ;~ ~ :
oxidized form, which is the form "locked" in the brain from which the active drug is ultimately released, the carrier-drugs can be represented by the formula [D-QC]+ X~
wherein X~ is the anion of a non-toxic pharmaceutically acceptable acid, [~] is a centrally acting drug species and [~C]+ is the hydrophilic, ionic pyridinium salt form of a dihydropyridine pyridinium salt redox carrier. The redox carrier approach is discussed hereinabove in the section entitled "BACKGROUND OF THE
I~VENTION"; historically, the carrier system is the second type of redox system developed for delivering drugs to the brain.
Various aspects of the redox carrier system have been described in detail in Bodor United States Patent No. 4,479,932, issued October 30, 1984; Bodor United States Patent No. 4,~40,564, issued September 10, 1985; -~ -Bodor et al United States Patent No. 4,617,298, issued October 14, 1986; and UNIVERSITY OF FLORIDA's International Application No. PCT/US83/00725, published under International Publication No. W083/03968 on November 24, 1983.
The redox analog system provides for brain~
targeted drug delivery by means of new compounds -~
containing a dihydropyridine + pyridinium salt portion which, unlike the redox carrier, is not readily meta-bolically cleavable from the original drug molecule. :
One redox analog approach, which provides deriva-tives of centrally acting amines in which a primary, `, ' ' '~ ~ '~ . ' ,, : ' '" . ~.,' . ' ' ': , . ': ' . :' '. : ' ' : ". :: ~: i ' ' " ~ ' ' r~
secondary or tertiary amine function has been replaced with a dihydropyridine ~ pyridiniu~ salt redox system, is discussed hereinabove in the section entitled "BACK-GROUND OF THE IN~ENTION"; historically, this analog systerfl is the third type of redox system developed for delivering drugs to the brain. Various aspects of this analog system are described in detail in UNIVERSITY OF
FLORIDA's International Application ~lo. PCT/US85/00236, published under International Publication No.
W085/03937 on September 12, 1985.
Another redox analog approach provides novel amino acids and peptides containing them which comprise a dihydropyridine ~ pyridinium salt portion, the redox system being appended directly or via an alkylene bridge to the carbon atom adjacent to the carboxyl carbon. These amino acids and peptides are described in detail in UNIVERSITY OF FLORIDA's copending Canadian Patent Application No. 563,439-3, filed April 6, 1988. 3riefly, the novel redox amino acids in the reduced form have the structural formula 113 ~
:::
' wherein Z is either a direct hond or C1-C6 alkylene and can be attached to the heterocyclic ring via a ring carbon atom or via the ring nitrogen atom; R1 is C1-C7 ` 25 alkyl, C1-C7 haloalkyl or C7-C12 aralkyl when Z is '~
,;
.
., . - , .
.,~
-. r ~33~
attached to a ring carbon atom; R1 is a direct bond when Z is attached to the ring nitrogen ato~; R2 and R3, which can be the same or different, are selected from the group consisting of hydrogen, halo, cyano, C
C7 alkyl, Cl-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, Cl-C7 alkylthio, C1-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR' " wherein R''' is hydrogen or Cl-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are ;~
each hydrogen or Cl-C7 alkyl; or one of R2 and R3 together with the adjacent ring carbon atom for~s a ::~
benzene ring fused to the heterocyclic ring, which benzene ring may optionally bear one or two substi-tuents, which can be the same or different, selected from the group consisting of hydroxy, protected hydroxy, halo, cyano, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, Cl-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, -CH=NOR''' wherein R' " is hydrogen or Cl-C7 alkyl, and 20 -CONR'R'' wherein R' and R " , which can be the same or ~::
different, are each hydrogen or C1-C7 alkyl; R4 is :~
hydrogen or a carboxyl protective group; R5 is hydrogen or an amino protective group; and the dotted lines . .
indicate that the compound contains a 1,4- or 1,6- ::
25 dihydropyridine, a 1,4- or 1,2-dihydroquinoline, or a -1,2-dihydroisoquinoline ring system.
The new dihydropyridine amino~acid analogs ~ -depicted above and the corresponding oxidized forms are useful in the preparation of novel redox peptides of the partial formulas:
..
?
~ ~31~
-HI~H- CO-N-Rl (A) , :~
~reduced form) : .:
3nd R ~/N -~1 (B) :
3 X~
(oxid~2ed form) ;~
the new pePtide analogs of partial structure (A) act as a delivery system for the corresponding quaternary ~ -salts of partial structure (B) ~n vivo; the quaternary derivatives, which also are chemical intermediates to the dihydro compounds, are pharmacologically active or convertible in vivo to pharmacologically active peptides, and are characterized by site-specific and sustained delivery to the brain when administered via the corresponding dihydropyridine form. Methods for the preparation of these analog amino acids and peptides utilize methods known in the art for intro-duction of the dihydropyridine ~ pyridinium salt moiety or a precursor thereof, e.g. from the aforementioned International Publications Nos. W083/03968 and .,'''~
'. " -: . . ~ ' . ~' . . - ' ' ' : .:
W085/03937, appropriately combined with well-known -~
methods for peptide synthesis. Ultimately, the quaternary forms of the amino acids and peptides are subjected to reduction to afford the corresponding ~ -dihydropyridines, according to the methods of the Bodor U.S. patents and above-mentioned published PCT appli-cations.
In a preferred aspect of the present invention, the redox system selected for complexation with a 10 hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or ;-maltotriosyl derivative of ~- or y-cyclodextrin and use in accord with the present invention is a redox carrier system. The drug and carrier portions of the redox ~-carrier system are described in more detail below and of course in the various carrier patents and patent applications identified above. Selection of appro-priate drugs and carrier moieties need not be limited to specific drugs and specific carriers disclosed in the aforementioned patents and applications or in the present application, just so long as the selected drug and carrier meet the general requirements of the drug/carrier system as described in the aforenoted documents.
The term "drug" as used herein means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental develop- ;
ment and conditions in man or animal.
By "centrally acting" drug species, active agent or compound as utilized herein, there is of course intended any drug species or the like, a significant ; .~
1~3~
(usually, pr1ncipal) pharmacolog~cal activity of which ~s CNS and a result of d~rect action in the brain.
Exemplary such centrally acting drug species are the CNS-amines and other nervous system agents, whether sympathetic or parasympathetic, e.g., phenylethylamine (a stimulant), dopamine (a neurotransmitter and dopaminergic agent used, e.g., in the treatment of Parkinsonism or hyperprolactinemia), tyramine (a stimulant), L-DOPA (a dopamine precursor used, for example, in the treatment of Parkinsonism); muscle relaxants, tranquilizers and antidepressants, e.g., benzodiazepine tranquilizers such as diazepam and oxazepam and phenothiazine tranquilizers such as carphenazine, fluphenazine and the like; mild and strong analgesics and narcotics; sedatives and hypnotics; narcotic antagonists; vascular agents;
st~mulants; anesthet~cs; small peptides, such as the di-, tri-, tetra and pentapeptides, and other small 2-20 amino ac~d unit containing pept~des, e.g. the enkephalins (for example, Tyr-Gly-Gly-Phe-Leu), which, bes~des be1ng analges1cs, initiate epileptic activity ~n the brain at doses that are about tenfold lower than for effect1ng analges1c activity; growth-promoting substances; ant1epilept1c and anticonvulsant drugs generally, lncluding hydantoins such as phenytoln and ethoto~n, barbituates such as phenobarb~tal; hormones, such as the steroid hormones, e.g., estrad~ol, testosterone, 17 a-ethynyl testosterone (ethisterone), and the like (recent stud1es on histological mapp~ng of hormone-sensit~ve and spec~fic steroid binding cells ~n the brain have underscored the importance of the stero1d act~on 1n the bra1n on sexual behav~or);
~:
r - , -31- ;~
amphetamine-llke drugs; anticancer and anti-Parkinsonism agents; anti-hypertenslves, agents to enhance learning capac~ty and the memory processes, including treatment of dementias, such as Alzheimer's 5 disease, such as 9-amino-1,2,3,4-tetrahydroacridine; ~ ;
antibacterials; centrally acting hypotensive agents;
centrally acting prostaglandins, such as PGD2;
d1agnostic agents, such as radiopharmaceuticals;
monoamlne oxidase (MAO) inhibitor drugs; CNS or brain important/essential amino acids, such as tryptophan (which is an antidepressant as well as a nutrient); and any like centrally acting compounds. For the purposes of this invention, dopa or L-DOPA is not classified as an amino acid but rather as a CNS amine and dopa-minergic agent used, e.g. in the treatment ofParkinsonism.
Other illustrative ultimate species of centrally acting drug entities are: amphe~amine, dextroamphet-amine, levamphetamine, aletamine, cypenamine, fencam-famin, fenozolone, zylofuramine, methamphetamine, phen-metrazine and phentermine, which are sympathomimetic amines/cerebral stimulants and appetite suppressants;
etryptamine, a cerebral stimulant; codeine, oxycodone, pentazoc~ne, anileridine, hydromorphone, morph1ne and oxymorphone, which are narcotic analgesics; desipra-mine, nortriptyline, octriptyllne, maprotiline, opipramol and protriptyl1ne, which are cerebral st1mulants/tricylic ant1depressants of the dibenza-zepine type used, e.g., ~n endogenous depressions;
clon~d~ne and methyldopa, whlch are sympatholytic agents used, e.g., tn hypertension; biperiden, cycr~m~ne and procyclidine, which are centrally acting antichollnergics; tranylcypromine, a sympathomimetic cerebral stimulant/MA0 inh~bitor and antidepressant;
acetophenazine, carphenazine, fluphenazine, perphenazine and piperacetazine, which are ~ :
phenothiaz1ne-type tranquilizers; benzoctamine, a sedative/muscle relaxant which structurally is an analogue of the phenothiazine tranquilizers; chlor- - :
diazepoxide, clorazepate, nitrazepam and temazepam, which are benzodiazepine-type tranquilizers; noracy-methadol, a narcotic analgesic of the methadone type;
piminodine, a narcotic analgesic of the meperidine type; tracazolate, a sedative/hypotensive; prizidilol, a centrally acting hypotensive; sulpiride$ an anti-depressant/psychotropic; haloperidol and clopenthixol, which are tranquilizers; norepinephrine, a sympathetic stimulant/adrenergic agent; nalorphine and naloxone, narcotic antagonists; hydralazine, a hypotensive;
ethotoin, phenobarbital and aminoglutethimide, anti-convulsants; epinephrine, an adrenergic agent;
20 ethamivan, a medullary stimulant; bemegride, a ~. :
barbiturate antagonist; amiphenazole, a stimulant, iopydol, iodopyracet, iodouppurate (o-~odohippuric acid), iodamide and iopanoic acid, which are radiodiagnost~cs; ephedrine, pseudoephedrine, oxymetazoline and phenylephrine, which are sympatho-mimetic amlnes and decongestants; estradiol, estrone and estriol, the natural estrogens; amoxicillin, oxacillin, carbenicillin, benzylpenicillin, phenoxy-methylpenicillin, meth1cillin, nafcillin, ticarcillin, bacampicillin, epicillin, hetacillin, pivampacillin, the methoxymethyl ester of hetacillin, and ampicillin, which are penic1llin-type antibiotics; amobarbital, a ,, , ~ . ~
.. ~
:::
sedative; trihexyphenidyl, a centrally acting anticholinergic; hydroxyzine9 a tranquilizer; chlorte~
tracycline, demeclocycline, minocycline, doxycycline,~ :.
oxytetracycline, tetracycline and methacycline, which are tetracycl~ne-type antibiotics; flurazepam, bromazepam, demoxepam and lorazepam, benzodiazepine tranqu~lizers; phenytoin, an anticonvulsant;
glutethimide, a mild hypnotic/sedative; clindamyr-in, lincomycin, nalidix~c acid, oxolinic acid and phenazo-10 pyrldine, zntibacterials/antibiotics; bethanidine and ~:
guanethidine, hypotensives/sympatholytics; captopril, a hypotensive; methyprylon, a mild hypnotic; amedalin, bupropion, cartazolate, daledalin, difluanine, fluoxe-tine and nisoxetine, which are cerebral stimulants; ~ -15 propranolol, a B-blocker antihypertensive; cloxacillin~: :
and dicloxacillin, penicillin-type antibacterials;
butalbital, a barbiturate sedat~ve; GABA, y-vinyl GA8A, y-acetylenic GA~A, neurotransmitters for possible use in epilepsy; valproic acid and its metabolites such as S-hydroxy-2-n-propylpentanoic acid, 4-hydroxy-2-n-propylpentanoic acid, 3-hydroxy-2-n-propylpentanoic ac~d, for use as anticonvulsants; valpromide, a valproic acid derivative for use as an anticonvulsant;
apomorph1ne, a narcotic depressant/emetic which has been used in the treatment of photosensitive epilepsy;
pholcod~ne, a narcotlc antitussive; methotrexate, :
mitoxantrone, podophyllotoxin derivatives (etopside, teniposide), doxorubicin, daunamycin and cyclophosphamide, anti-cancer/antitumor agents; ~
30 methylphenidate, a stimulant; thiopental, an ~ ~ .
anesthetlc; ethinyl estradiol and mestranol, estrogens;
meptaz1nol, cyclazocine, phenazocine, profadol, $ . ~
~`
r ~
- 3 4 - ~ :
:~
metopon, drocode and myfadol, which are narcotic ~-~
analges1cs; buprenorphlne, nalbuphine, butorphanol, levallorphan, naltrexone, nalme~ene, alazocine, ~ :
oxllorphan and nalmexone, which are narcotic antagonists or igon1st-antagonists; norgestrel and norethindrone, progestins; cephalothin, cephalex1n, cefazolin, cefoxitin, moxalactam, ceforanide, cefroxadine and cephap1rin, cephalosporin ant~biotics;
atenolol, nadolol, t~molol and metoprolol, B-blockerslhypotensives; ACTH (corticotropin), a hormonewhich stimulates glucocorticoid production; LHRH, a neurotransmltter which stimulates secretion of the pituitary hormones, LH and FSH, and has been used to~:- :
1nduce ovulation as well as for fertility control/ :~
contraception; sulfadiazine and other sulfonamide antibiotics; ribavarin and acyclovir, antiviral agents;
chlorambuc11 and melphalan, nitrogen mustard-type anti-cancer/antitumor agents; methotrexate and aminopterin, which are folic acid antagonist-type anticancer/anti-tumor agents; plat1num coordination complexes, i.e.
cisplatin analogue-type anticancerlant~tumor agents; ~: .
dactinomycin and m1tomyc1n C, used in cancer chemo-therapy; th109uanine, a pur1ne/pyrimidine antagonist : used 1n cancer treatment; vincristlne and vinblastlne, ant1cancer alkaloids; hydroxyurea and DON, anticancer urea derivat1Yes; FSH, HCG and HCS, pituitary and non-pttu~tary gonadotrop1ns, used, for example, in certain reproductive disorders; N,N'-bis(dichloracetyl)-1,8-octamethylenediamlne (fertilysin), an agent for male fert11ity 1nhib1t10n; levorphanol, a narcotic anal-gesic; benzestrol and diethylst11bestrol, synthetic estrogens; ethyl B-carbol1ne-3-carboxylate, a benzo-i ~ 3 ,3 ~ ~ ~ I A
diazepine antagontst; furosemide, a diuretic/antihyper~
tensive; dipyridamole and nifedipine, coronary vaso-dilators; and progabide, a GABA-agonist and prodrug of -GABA. Yet other ultimate species include non-steroidal antiinflammatory agents/non-narcotic analgesics, e.g.
propionic acid derivatlves, acetic acid derivatives, fenamic acid derivatives and biphenylcarboxylic acid derivatives. Specific NSAID's/non-narcotic analgesics contemplated for use herein include ibuprofen, naproxen, flurbiprofen, zomepirac, sulindac, indo-methacin, fenbufen, fenoprofen, indoproxen, ketoprofen, fluprofen, bucloxic acid, tolmetin, alclofenac, fenclozic acid, ibufenac~ flufenisal, pirprofen, flufenamic acid, mefenamic acid, clonixeril, clonixin, meclofenamic acid, flunixin, diclofenac, carprofen, etodolac, fendosal, prodolic acid, sermetacin, indox-ole, tetrydamine, diflunisdl, naproxol, piroxicam, metazamide, flutiaz~n and tesicam.
Preferred classes of centrally acting drugs for use herein are the central neurotransmitters, steroids, anticancer and antitumor agents, antiviral agents, tranquilizers, memory enhancers, hypotensives, seda-tives, antipsychotics and cerebral stimulants (especially tricyclic antidepressants). Among the -~
neurotransmitters, there can be mentioned amino acids, such as GABA, GABA derivatives and other omega-amino acids, as well as glycine, glutamic acid, tyrosine, aspartic ac1d and other natural amino acids; catechol-am~nes, such as dopamine, norepinephrine and epine- ~ -phrine; serotonin, histamine and tryptamine; and pep~
tldes such as neurotensin, lute~nizing hormone-releaslng hormone ~LHRH), somatostatin, enkephalins 1 3 3 ~
such as met5-enkephalin and leu5-enkephalin, endorphins such as ~-, a- and B-endorphins, oxytocin M and vasopressin. Synthetic and semi-synthetic analogues, e.g. analogues of LHRH in which one or more amino ac1d(s) has/have been eliminated and/or replaced with one or more different amino acid(s), and which may be agonists or antagonists, are also contemplated, e.g.
the primary and secondary amine LHRH analogues dis-closed in United States Patents No. 4,377,574, 3,917,825, 4,034,082 and 4,338,305. Among the stero~ds, there can be mentioned anti-inflammatory adrenal cortical steroids such as hydrocortisone, betamethasone, cortisone, dexamethasone, flumethasone, fluprednisolone, meprednisone, methyl prednisolone, prednisolone, prednisone, triamcinolone, cortodoxone, fludrocortisone, flurandrenolone acetonide (fluran-drenolide), paramethasone and the like; male sex hormones (androgens), such as testosterone and its close analogues, e.g. methyl testosterone (17-methyltestosterone); and female sex hormones, bothestrogens and progestins, e,g. progestins such as norgestrel, norethindrone, norethynodrel, ethisterone, dimeth~sterone, allylestrenol, cingestol, ethynerone, lynestrenol, norgesterone, norvinisterone, ethynodiol, oxogestone and tigestol, and estrogens such as ethinyl estradiol, mestranol, estrad~ol, estriol, estrone and qu1nestrol and the like. Among the anticancer and antitumor agents, there can be mentioned Ara-AC, pentostatln (2'-deoxycoformycin), Ara-C (cytarabine), 3-deazaguan~ne, dihydro-5-azacytidine, tiazofurîn, sangivamycin, Ara-A (v~tarabine), 6-MMPR, PCNU, FENU, HENU and other nitrosoureas, spiromustine, : ~' 13 ~1 bisbenzimidazole, L-alanosine (6-diazo-5-oxo-L-norleucine), DON, L-ICRF, trimethyl TMM, 5-methyl-tetrahydrohomofolic acid, glyoxylic acid ~
sulfonylhydrazone, DACH, SR-2555, SR-2508, desmethyl- ~ ~-misonidazole, mitoxantrone, menogarol, aclacinomycin A, phyllanthoside, bactobolin, aphidocolin, homoharring- :~
tonine, levonantradol, acivicin, streptozotocin, hydroxyurea, chlorambucil, cyclophosphamide, uracil mustard, melphalan, 5-FU (5-fluorouracil), 5-FUDR
(floxuridine), vincristine, vinblastine, cytosine arabinoside, 6-mercaptopurine, thioguanine, 5- . : :.
azacytidine, methotrexate, adriamycin (doxorubicin), ~ :
daunomycin (daunorubicin), largomycine polypeptide, :
aminopterin, dactinomycin, mitomycin C, and 1i5 podophyllotoxin derivatives, such as etoposide (VP-16) and teniposide. Among the antiviral agents, there can be mentioned ribavarin; acyclovir (ACV); amantadine (also of possible value as an anti-Parkinsonism agent); -~ :
diarylamidines such as 5-amidino-2-(5-amidino-2-benzo-furanyl)indole and 4',6-diimidazolino-2-phenylbenzo-(b)thiophene; 2-aminooxazoles such as 2-guanidino-4,5- ~:
di-n-propyloxazole and 2-guanidino-4,5-diphenyloxazole;
benzimidazole analogues such as the syn and anti ~ :
isomers of 6~(hydroxyimino)phenyl3methyl]-1-~
methylethyl)sulfonyl~-lH-benzimidazol-2-amine;
bridgehead C-nucleosides such as 5,7-dimethyl-2-3-D-ribofuranosyl-s-triazole(1,5-a)pyrimidine; glycosides such as 2-deoxy-D-glucose, glucosamine, 2-deoxy-2-fluoro-D-mannose and 6-amino-6-deoxy-D-glucose; phenyl 30 glucoside derivatives such as phenyl-6-chloro-6-deoxy- ;~
B-D-glucopyranoside; (S)-9-(2,3-dihydroxypropyl)-adenine; t~azofurin; selenazofurin; 3-deazauridine; 3- :~ ~
,"",.. ~ ,,"," ,-,"-,-: ~: :' ~' ' ' ' ' ' ' ' "., ..
-, , ~ "
- `~
~ ~ 3 ~
deazaguanosine; DHPG; 6-azauridine; idoxuridine;
trifluridine (trifluorothym~dine); BDVU (bisdihydroxy-v1nyluridine); zidovudine (AZT); dideoxycytidine; and 5,6-d~chloro-l-~-D-r1bofuranosylbenzimidazole. Among the anti-cancer/intitumor and antiviral agents, those of the nucleoside type (i.e. a purine or pyrimidine base-type structure bear~ng a singly or multiply hydroxylated substituent) are of particular lnterest.
This group ~ncludes such compounds as Arà-AC, pentostatin, Ara-C, dihydro-5-azacytidine, tiazofurin, sangivamycin, Ara-A, 6-MMPR, desmethylmisonidazole, 5-FU~R, cytosine arabinoside, 5-azacytidine, ribavirin, acyclovir, (S)-9-(2,3-dihydroxypropyl)adenine, 6-azauridine, 5,6-dichloro-1-B-D-ribofuranosyl-benzimidazole, 5,7-dimethyl-2-B-D-ribofuranosyl-s-trlazole(l,5-a)pyrimidine, zidovudine (AZT), dldeoxycytidine, dideoxyadenosine, dideoxyinosine and DHPG. Among the tranquilizers, there can be mentioned benzodiazepine tranqu~lizers, such as diazepam, oxazepam, lorazepam, chlordiazepoxide, flurazepam, bromazepam, chlorazepate, n~trazepam and temazepam;
hydantoin-type tranqu~lizers/anticonvulsants such as phenytoin, ethotoin, mephenytoin; pheno~hiazine-type tranqu111zers such as acetophenazine, carphenazlne, fluphenaz~ne, perphenaz~ne and piperacetazine; and others. Among the hypotensives, there can be mentioned clon~dine, methyldopa, bethan1dine, debr~soquin, hydralaz~ne, and guanethidine and ~ts analogues. Among the sedatives, tranquilizers and antipsychot~cs, there can be mentioned the many specific compounds of this type disclosed above, espec1ally the phenoth~azines and benzodiazep~nes and their analogues. Among the ~ /~r -.
r~
13~
cerebral stimulants, there also can be mentioned the many specific compounds set forth hereinabove, particularly the sympathomlmetic amine-type cerebral stimulants and the tricyclic antidepressants, espectally preferred tricycl~cs being the dibenzazepines and their analogues.
Also illustrative of the centrally acting drug species contemplated by this invention are centrally active metabolites of centrally acting drugs. Such metabolites are typified by hydroxylated metabolites of tricyclic antidepressants, such as the E- and ~-iscmers of 10-hydroxynortriptyline, 2-hydroxyimipramine, 2- ~-hydroxydesipramine and 8-hydroxychloripramine; hydroxy-lated metabolites of phenothiazine trarquilizers, e.g.
7-hydroxychlorpromazine; and desmethyl metabolites of N-methyl benzodiazepine tranquilizers, e.g. desmethyl~
diazepam. Other CNS active metabolites for use herein -~
will be apparent to those skilled in the art, e.g. SL
75102, which is an active metabolite of progabide, a GABA agonist, and hydroxy-CCNU, which is an active metabolite of CCNU, an anti-cancer nitrosourea.
Typically, these CNS active metabolites have been identified as such in the scientific literature but have not been administered as drugs themselves. In many cases, the active metabolites are believed to be comparable in CNS activity to their parent drugs;
frequently; however, the metabolites have not been -administered per se because they are not themselves able to penetrate the blood-brain barrier.
As indicated hereinabove, diagnostic agents, in-cluding radiopharmaceuticals, are encompassed by the c.";.....
~2^.', ., ~
1 3 3 ~
^40-expression "centrally acting drug" or the like as used herein. Any diagnost1c agent which can be derivatized to afford a redox carr~er system which will penetrate -the BBB and concentrate 1n the brain in its quaternary form and can be detected therein is encompassed by this invention. The diagnostic may be "cold" and be de-tected by X-ray (e.g. radiopaque agents) or other means such as mass spectrophotometry, NMR or other non- ~ I
invasive techniques (e.g. when the compound includes stable isotopes such as C13, N15, 018, S33 and S34).
The d1agnost1c alternatively may be "hot", i.e.
radiolabelled, such as with radioactive iodine (I 123, I 125, 1 131) and detected/imaged by radiation detect~on/imaging means. Typical "cold" diagnostics for derivation herein include o-iodohippuric acid, iothalamic acid, iopydol, iodamide and iopanoic acid.
Typical radiolabelled diagnostics include diohippuric ~-acid (I 125, I 131), diotyrosine (I 125, 1 131), o-iodohippuric acid (I 131), iothalamic acid (I 125, 1 131), thyroxine (I 125, I 131), iotyrosine (I 131) and iodometaraminol (I 123), which has the structural formula ;~
HO ~ CHCHNH2 ,:
In the case of diagnostics, unlike the case of drugs which are for the ~reatment of disease, the "locked in"
quaternary form will be the form that is imaged or otherw~se detected, not the original diagnostic ~ ~ 31 ~
itself. Moreover, any of the centrally acting drugs which are intended for the treatment or prevention of medical disorders but which can be radiolabelled, e.g.
with a radioisotope such as iodine, or labelled with a stable isotope, can thus be converted to a diagnostic for incorporation into the redox carrier system.
It will be apparent from the known structures of -the many drug species exemplified above, that in many cases the selected drug will possess more than one re~
10 active functional group, and, in particular, that the ~-drug may contain hydroxyl or carboxyl or amino or other functional groups in addition to the groups to which the carrier will be linked, and that these additional groups will at times benefit from being protected during synthesis and/or during administration. The nature of such protection is described in more detail in the various patents and patent applications referred to herein. Obviously, such protected drug species are encompassed by the definition of "drug" set forth hereinabove.
It too will be appreciated that by "dihydropyri~
dine carrier" or "[DHC]", there is intended any non-toxic carrier moiety comprising, containing or in~
cluding the dihydropyridine nucleus, whether or not a part of any larger basic nucleus, and whether substi-tuted or unsubstituted, the only criterion therefor being capacity for BBB penetration and in vivo oxidation thereof to the corresponding quaternary pyridinium salt carrier [QC]~. As aforesaid, the ionic pyridinium salt drug/carrier prodrug entity [D-QC]+
which results from such in vivo oxidation is prevented e~
1 3 3 ~
from efflux from the brain, while elimination from the general circulation is accelerated. Subsequently, the covalent or equivalent bond coupling the drug species [D] to the quaternary carrier ~QC]+ is metabolically cleaved, wh~ch results 1n sustained delivery of the drug [D] in the brain and facile elimination of the carrier moiety [QC]+. Such "covalent or equivalent bond" between the drug and the quaternary carrier can be a simple direct chemical bond, e.g., an amide, an ester, or any other like bond, or same can even be comprised of a linking group or function, e.g., a thiazolidine bridge or a peptide linkage, typically necessitated when the drug species is not susceptible to direct chemical coupling to either the dihydropyri-dine carrier or the quaternary carrier. Nonetheless,the bond in the formulae [D-~C]+ and [D-DHC] is intended to be, and is hereby defined as inclusive of all such alternatives. And the cleavage of the [D-QC]+
prodrug to sustainedly delivery the drug species [D] in the brain with concomitant facile elimination of the carrier moiety ~QC]~ is characteristically enzymatic cleavage, e.g., by esterase, amidase, cholinesterase, hydrolyt1c enzyme, or peptidase.
The expression Unon-toxic pharmaceutically accept-able salts" as used here1n generally includes the non-toxic salts of the reduced, d1hydropyridine forms of the redox carrier or redox analog systems, formed with nontox1c, pharmaceut1cally acceptable inorganic or organ1c acids HX. For example, the salts include those der~ved from inorganlc ac1ds such as hydrochloric, hydrobrom1c, sulfur1c, sulfamic, phosphoric, nitric and the 11ke; and the salts prepared from organlc acids 1331~
such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartar1c, citric~ ascorbic9 pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyl~c, sulfan~lic, fumaric, methanesulfonic, tolu-enesulfonic and the like. The expression "anion of anon-toxlc pharmaceutically acceptable acid" as used herein, e.g. in connection with the oxidized, pyri-dinium salt forms of the redox carrier or redox analog systems, is intended to include anions of such inorganic or organic acids HX.
In the discussion to follow, the expression "at least one reactive functional group selerted from the ;~
group consisting of amino, hydroxyl, mercapto, carboxyl, amide and imide" or portions of that expres-15 sion are used. The functional groups designated in ~ ~
that expression have ~he following meanings: ~-The word "amino" means a primary or secondary amlno function, i.e. -NH2 or -NHR. The secondary amino functlon ls also represented herein as -NH-, particu-larly since the exact ident1ty of the R portion of -NHR
is lmmaterlal, R being a part of the drug residue D
ltself whlch is left unchanged by conversion of the drug to the redox carr~er system.
The word "hydroxyl" means an -OH function.
The word "carboxyl" means a -COOH function.
The word "mercapto`' means an -SH function.
The word "amide" means a carbamoyl (-CONH2) or substltuted carbamoyl (-CONHR) or a sulfamoyl ~-502NH2) or substltuted sulfamoyl (-S02NHR) functional group.
The -CONHR and -S02NHR groups may also be represented . .
.
~3~
hereln as -CONH- and -S02NH-, respectively, since the 1dentlty of R is immaterial, R being a part of the drug : -resldue D ltself wh1c\l is left unchanged by con~ersion of the drug to the redox carrier system.
The word "imide" means a functional group having the structure -C~ ,~ ~
~ H
O . . ~:.
that is, the structure which characterizes imides (i.e.
compounds having a succinimide-type or phthalimide-type structure).
Many different dihydropyridine ~ pyridinium salt redox carrier moieties are illustrated in the carrler patents and applications referred to hereinabove.
The following i8 a list of representative :
ma~or classes of dlhydros and the correspond1ng quater-naries, but 1s not meant to be exhaustlve:
(1) For linkage to a drug having at least one hydro~yl or mercapto or primary or secondary amino functional grouping, replacing a hydrogen atom from at least one of said functional groupings with one of the following ~DHC~ groupings:
, ~ , ~..
' ,.
~',.'~.
~'",;~
1~3~
-45 ~
o .::
~lc'- ~X
R l , R3 -8-(a`) (b') ,: ~ .::
O 0 1l ~ICOC'2C- ~ , '. ;~ ~
R ~
( c ' ) ( d ) :~ ~
O O ~' '.
~X ~COC~2C-R -C-3a (e ) ~oc- ~x or I~N`R ' a (9 ) .
~ COCH2C- ~ :
`Rl ~ ::
(J') ,~ ' ' ~ ' 1~3~ ~4 wherein the dotted line ~n formulas (a'), (b') and (c') ~nd~cates the presence of a double bond in either the 4 or 5 posit~on of the d~hydropyridine ring; the dotted ~ :
line in formulas (d'), (e'~ and (f') indicates the S presence of a double bond in either the 2 or 3 posit~on of the dihydroquinollne ring; Rl is Cl-C7 alkyl, Cl-C7 : ~:
haloalkyl or C7-Clo aralkyl; R3 is Cl to C3 alkylene; X
is -CONR'R" , where~n R' and R'', which can be the same .A/~
or different, are each H or Cl-C7 alkyl, or X is -CH=NOR" ' wherein R''' is H or Cl-C7 alkyl; the carbonyl-containing groupings in formulas (a') and (c') and the X substituent in formula (b') can each be attached at the 2, 3 or 4 position of the dihydropyri- ~ ~ :
dine ring; the carbonyl-containing groupings in 15 formulas (d') and (f') and the X substituent in formula ;~ :
(e') can each be attached at the 2, 3 or 4 position of the dihydroquinol1ne ring; and the carbonyl-containing groupings in formulas (g') and (j') and the X substi- -tuent in formula (h') can each be attached at the 1, 3 or 4 posit10n of ~he d1hydro~soquinoline ring.
(2) For linkage to a drug having at least one carboxyl functional grouping, replacing a hydrogen atom from at least one of sa~d carboxyl groupings with one of the follow1ng tDHC] groupings:
(a) When there are one or more -COOH groups ~ ~.
to be derlvat~zed~
R~ R3 C Q Z
( 1 ' ) ( 11 ' ) ' ~; ~ `
o o o , OCH2C-~-Z - ~
R ~ R I
? ( l v ) X~ jCcH2c-Q-z ' -o R 1 (V' ) (vi ~ ) ~1 ~3 S az (v11~)(viii~
COCH2C-Q-Z'-1x~) whereln the dotted llne ln formulas (1'), (11') and (111') lnd1cates the presence of a double bond ln elther the 4 or 5 posltlon of the dihydropyridlne ring;
the dotted llne ln formulas (lv'), (v') and (vl') lndicates the presence of a double bond in elther the 2 or 3 posltion of the dlhydroqulnoline rlng; Z' ~s Cl-C8 stralght or branched ~lkylene, preferably Cl-C3 stralght or branched alkylene; Q ls -O- or -NH-; Rl ls Cl-C7 alkyl, Cl-C7 haloalkyl or C7-C10 aralkyl; R3 ls Cl-C3 alkylene; X ls -CONR'R'' wherein R' and R'', : 15 whlch can be the same or dlfferent, are each H or Cl-C7 ::: ~ :
~3~
-48^
alkyl, or X ~s -CH=NOR''' where~n R'" ~s H or Cl-C7 alkyl; the X substituent ln formula (1~') and the A,~
carbonyl-contaln1ng group~ng 1n formulas (i') and (~11') can each be attached at the 2, 3 or 4 position of the d~hydropyrld1ne ring; the X substituent ~n formula (v') and the carbonyl-containing groupings in formulas (~v') and (v~') can each be attached at the 2, 3 or 4 pos~t~on of the dihydroqu~noltne r~ng; and the X
substltuent ln formula (v~') and the carbonyl-conta~n~ng group~ngs in formulas (v~i') and (ix') caneach be attached at the 1, 3 or 4 position of the dihydroqu~nol~ne ring;
(b) AlternatiYely, when there ~s only one ~ --COOH group to be der1vatized:
(R~)5~ "_ ~ ~ ~ (R4)n1v ~ ~ , (x') (x1') ~
, _ , . .
O
CNH
)nV ' r (x11 ' ) , , ~, ~, .
ChH ~ I (x111'), or .- ...
~N
~A)nV ' ' ~
' ~ CNH ~ t (x (A)nV ~ ~
::::
D r ~ . ' ~ . ' ~: . : -` ~ ~ . ' . ~. : . ' . . : . ', ~, ".' -~:, ' ':' :' ': . ' ' ' : ' , . ~ , ~ ' ,- .~ :.. ' ' ' . ' ' '.:' ' ~ j, ,' . . ' ' ' , :: : . . .
j/, ~
~^~
~ 3 ~
wherein the dotted 11ne in formula (xii') 1ndicates the presence of a double bond in either the 4 or 5 position ~i of the d1hydropyr1dine ring; the dotted line in formula (x11i') 1ndicates the presence of a double bond in ~:
e1ther the 2 or 3 pos1tion of the dihydroquinoline r1ng;. ` is the skeleton of a sugar molecule; niV is a pos1tive integer equal to the total number of -OH
funct10ns 1n the sugar molecule from which sa1d skele-ton is derived; nv 1s a positive integer one less than lO the total number of -OH functions in the sugar molecule -from which said skeleton is derived; each A in each of structures (xii'), (xii1') and (xiv') can ~ndependently be hydroxy or ~', D' being the residue of a centrally acting drug containing one reactive carboxyl functional group, said residue being characterized by the absence of a hydrogen atom from said carboxyl functional group .
1n said drug; and each R4 1n each of structures (x') and (xi') can 1ndependently be hydroxy, .
o8~
R
20 oc~3 R~
oC~3 or D'~
.. ~' :
' ,:,5: ,, ` ~: ~ .~ - : : . . .
~?~
1~3~
wherein the dotted line is defined as with structures (xii') and (xiii'); D' is defined as with structures (xii ), (xiii ) and (xiv'); Rl is C1-C7 alkyl, Cl-C7 -haloalkyl or C7-C10 aralkyl; and the depicted carbonyl groupings can be attached at the 2, 3 or 4 position of the pyridinium or qu~nolinium ring or at the 1, 3 or 4 position of the isoquinolinium ring; with the proviso that at least one R4 in each of structures (x') and (xi') is 10 -C~
-OC~ or Rl . , ~ ' ' -_o = ~ ~
,N ' wherein R~, the dotted lines and the position of the carbonyl-containing groupings are def~ned as above; and wlth the further proviso that when more than one of the R4 radicals in a given compound are the aforesa~d is,, .. ., :.: . . ...
1 ~3~
carbonyl-containing groupings, then all such carbonyl-containing groupings in said compound are identical.
(3) For llnkage to a drug hav~ng at least one :
-NH- functional group which is part of an amide or 5 ~m~de structure or at least one low pKa primary or secondary amine functional group, replacing a hydrogen atom from at least one of said functional groupings with one of the follow1ng [DHC] groupings:
C O C H ~ ,X
R
~ IN /
R l R 3 - ~C1 ( k ' ) O R ( 1 O : :: :
!' COCH--, I
N
Rl (n ' ) (m' ) o O
C O C H 2 C O C H-- , : :~
~ ~ R ~: ~
R3-COCIH--. Rl ::
O R
(O') (P') .' t.~
1~31~
~COCH--, ~K
( q ' ) ( r o r ~ COCH2 COCH ~
~ R1 (s ' ) '' - ' ''-`' '"'~'~'".' wherein R is hydrogen, Cl-C7 alkyl, C3-C8 cycloalkyl, C1-C7 haloalkyl, furyl, phenyl, or phenyl substituted by one or more halo, lower alkyl, lower alkoxy, carba-moyl, lower alkoxycarbonyl, lower alkanoyloxy, lower haloalkyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, lower alkylth~o, lower alkylsulfinyl or lower alkylsulfonyl; the dotted line in formulas (k'), (1') and (m') lndicates the presence of a double bond in either the 4 or 5 position of the dihydropyri-dine ring; the dotted l~ne in formulas (n'), (o') and ~ -~: (p') indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring; Rl is ~ ~.
:
. ~".~
~ , c v;
~, ,. ~ . . . ,, ~ , ~ .. . . .
1 ~ 3 ~
C1-C7 alkyl, Cl-C7 haloalkyl or C7-C10 aralkyl; R3 is Cl to C3 alkylene; X is -CONR'R'', wherein R' and R'', which can be the same or different, are each H or Cl-C7 alkyl, or X is -CH=NOR''' wherein R'" is H or C1-C7 alkyl; the carbonyl-containing group~ngs in formulas (k') and (m') and the X substituent in formula (l') can each be attached at the 2, 3 or 4 position of the dihy-dropyridine ring; the carbonyl-containing groupings in formulas (n') and (p') and the X substltuent ln formula 10 (o') can each be attached at the 2, 3 or 4 position of .
the dihydroquinoline ring; and the carbonyl-containing groupings in formulas (q') and (s') and the X substi- .
tuent in formula (r') can each be attached at the 1, 3 or 4 position of the dihydroisoquinoline ring.
Drugs containing secondary or tertiary hydroxyl functional groups can be linked to any of the tDHC]
groupings (k') through (s') above in which the -CHO-portion is derived from an aldehyde RCH2C capable of reacting with said drug to form the corresponding hemi-acetal, e.g. chloral, acetaldehyde, formaldehyde or benzaldehyde.
The following are especially preferred reduced, dlhydropyrid1ne forms of dihydropyridine pyridinium salt redox carr~er systems, also termed "chem1cal delivery systems" or "CDS", for braln-targeted drug del1very wh~ch are contemplated for formation of ~nclus~on complexes wlth hydroxypropyl-B-cyclodextrin 1n accord with ~he present inventlon:
",,~
.
, ,~
1331 ~
~54 ~
a _ ~ c e o ~;
y ~1 'o) _ E ~ E E
L O o ~
Ç~
0_3~ -0 ~ _~
l ' ~ ~. .
X ~ y ~ ' : 0 8-o ~ _ o~8 ~=o ~
~ ~ ' 133t ~ ~
O ~
~ o .~
C
~ _ N
~~
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e~
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r ~t ,~
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,~
t- -5~
t~
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1 3 31 ~
-57~
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133~
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o~ o E C : :
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o _ _ ~ C~ _ O C O ~ ~
~ ~ _ tc ~`:
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Z o~ o r~ c c u o ~,o " ~ ~ 2 ~--' ~ o o N
~ ~ J U ~` ~
~ _ N ~ K
~J ~,N~ ~ ' L~ LJ
s~ ~- ~
'^~
I ~ L~ ::
I \r~ O
V ~0 '' 0=~ ~
~ =/ ~:
~ 3 ::::
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~... . . ~ . . ~ . . . . . .
1~ 3 1 ~::
l ~ a c - ~-_ ~ O _ ~ ~
o E ~,.. o D
O ~
!~ ~, Q : ~ :
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O _ L
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r ~, ~ y ~ ~ o ':
'~ -63~
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_ _ .. ~ ~. ,.
~ ~ U ~ .. ~ o _ _ o _ _ C o.~ g C _ C ~ ~ ~ " _ - : .
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V) ~ ~ Vo~
~, E ~o .,~
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o V~ o .~
.t ,, ~.o ~ ~ ~ ~
o o O
, Y o_ ~C~
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~ _ ~. o ~", o o Z
J __ ~ ., ~,~, , .. _ ~ .
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~ ~ o ~ O~ ~ o ' ~ '' ~C_o '.;, ~ ,:.
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uo~
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s,~
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O O ~ ~. O ~ ~ , C C c O Co O ~ O C O ~ " O " ~ .
L ~ Y ~ ~ 01 C o C
~
, o ~o o o o _ L~ C
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r O O ~ :
V~ L~
_ ~, O v v C _ C o E ~o ~ ~ o o ~ ~
n n L ~ ~ C _ .. n ~ ~ ~ G
~ o ~0 ~ ~ ~ 0 ~ ~ o ~ ~o O ~o _I n O ~ _ n ~ ~J ~ u L ~-- ~ _ _ ~ ~ ~ n O C
eE o E ' ~ ~ ~ n v, J E ~I G
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C ~ o , ~ .. el o e , -:
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The cyclodextrins contemplated for use herein are hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of ~-cyclodextrin and the ;
corresponding derivatives of y-cyclodextrin. The ~ -hydroxyalkyl groupings ~ay contain one or more hydroxyl groups, e.g. hydroxypropyl, dihydroxypropyl and the like. The glucosyl, maltosyl and maltotriosyl derivatives may contain one or more sugar residues, e.g. glucosyl or diglucosyl, maltosyl or dimaltosyl.
Various mixtures of the cyclodextrin derivatives may be used as well, e.g. a mixture of maltosyl and dimaltosyl derivatives. Specific cyclodextrin derivatives for use herein include hydroxypropyl-~-cyclodextrin (HPCD or HPBCD), hydroxyethyl-B-cyclodextrin (HEBCD), hydroxypropyl-Y-cyclodextrin (HPGCD), hydroxyethyl-y-cyclodextrin (HEGCD), dihydroxypropyl-B-cyclodextrin (2HPBCD), glucosyl-B-cyclodextrin (G1-B-CD or G1BCD), diglucosyl-B-cyclodextrin (2G1-B-CD or 2G1BC~), maltosyl-B-cyclodextrin (G2-B-CD or G2BCD), maltosyl-y-cyclodextrin (G2-y-CD or G2GCD), maltotriosyl-B-cyclodextrin (G3-B-CD or G3BCD), maltotriosyl-y-cyclodextrin (G3-Y-CD or G3GCD) and dimaltosyl-B-cyclodextrin (2G2-B-CD or 2G2BCD)s and mixtures thereof such as maltosyl-B-cyclodextrin/dimaltosyl-B-cyclodextrin.
Hydroxypropyl-B-cyclodextrin for use in the novel inclusion complexes and methods of the present inven-tion is commercially available. Alternatively, it may be prepared by known methods, especially by use of the ~`~ 30 optimized procedure of Pitha et al, International Journal of Pharmaceutics, 29, 73-82 (1986). The :: . ..
1 3 3 1 .; r r ~
-74~
. ~, : ~,:
following is a typical procedure using the Pitha et al method:
31 g of sodium hydroxide were dissolved in 250 mL
of water. Then, 100 g of ~-cyclodextrin were added and the solvent was warmed to effect solution. The flask was cooled and 50 mL of propylene oxide were added.
The flask was fitted with a dry ice/acetone condenser ~-during the addition. The solution was allowed to come to room temperature and was stirred for 72 hours. The solution was then neutralized with concentrated hydro-chloric acid and diluted with water. The solvent was removed in vacuo, leaving a syrup which was taken up in ethanol. After stirring for 30 minutes at room temperature, the sodium chloride produced was removed by filtration. The filter cake was washed with ethanol and the co~bined ethanol layers were reduced in vacuo. The residue was dissolved in water and dialyzed in cellulose acetate ($7, 38 mm 4.6 mL/cm, molecular weight cut off = 10~0, Fisher Scientific). After 5 hours at 0C, the solution was removed from the dialysis tubing and freeze-dried. The resulting solid was suspended in acetone and stirred overnight. The filtered solid was resuspended in acetone and stirred ;
for 24 hours. The solid was collected by filtration and dissolved in 200 mL of water and then lyophil-ized. 75 grams of purified hydroxypropyl-~-cyclo-dextrin were obtained. The degree of substitution was calculated by NMR and by comparison with an authentic sample.
In forming a complex with E2-CDS, a 50% solution (w/w) cf 2-hydroxypropyl-B-cyclodextrin (HPCD) was made ,., - 1 3 ~ 4 in distllled ~ater. An excess of E2-CDS was added and the solut~on then was purged wlth h~lium. The result-~ng suspension ~as then sonicated for 30 minutesTh f;l-tered through a glass filter (ASTM 1~-15M, Pyrex No.
36~60) and freeze-dried overnight. Best results were obtained by hard-freezing the aqueous solution of the E2-CDS/HPCD complex for at least 10 hours before lyophll1zation. The degree of complex formatlon was determined by dissolving a small amount of the dry complex in methanol and then analyzing by high pressure liquid chromatography (HPLC). The degree of complexation was found to vary between 20-40 mg/g and the solubility of the complex was determined to be 2.2x104 Ing/L.
The P1tha et al method for preparation of HPCD by condensation of propylene oxide with ~-cyclodextrin in alkaline aqueous solutlon unfortunately suffers from disadvantages, particularly in purification of the product. After comp1etlon of the condensation, the react~on m1xture is neutralized with hydrochlor~c acid, water is evaporated under vacuum and the syrupy residue ~;
1s dlssolved in ethanol to precipitate sodium chloride, the main by-product of the react1On. After filtration, ethanol is evaporated under vacuum and the residue is dissolved in water and dialyzed to remove the remaining sodium chloride and polymerization products of propylene oxide. During dialysis, part of the hydroxy-propyl-~-cyclodextrin goes through the membrane and is lost. The dialysate is then freeze-dried, twice -~ --30 stirred in acetone and washed to remove the remaining -~
polymerization products. Finally, hydroxypropyl~
cyclodextrin is freeze-dried again. The second freeze-1 ~ 3 t ~
drying is necessary because the product after washing with acetone is not homogeneous.
To overcome these difficulties with the Pitha et al process, a new method has been developed by Maciej 5Inulkowski of the University of Florida, Gainesville, Florlda for the synthesis of HPCD. This ne~ method 1nvolves removal of sodium hydroxide fro~n the reaction mixture by an ion exchange resin (H+); as a result, several time-consuming steps of Pitha et al's -~
10 purification can be avoided. Moreover, the amount of ~-sodium hydroxide used by Pitha et al t7 equivalents for one of ~ cyclodextrin) can be decreased to 2 equivalents of sodium hydroxide per cyclodextrin molecule, and still produce a product with the appropriate NMR and optical rotation.
According to the new method, ~-cyclodextrln is - ;
first condensed with propylene oxide in alkaline solu~
tion, sodiu~Thydroxide is removed on an ion exchange column (Dowex 50W-X8, H+ form), the eluate is eva- -~
porated under vacuum to one-half of the original volume, the rema~n1ng solution is freeze-drled, the resultlng whlte solld 1s washed wlth acetone and freeze-dried again, then subjected to grinding and sieving. Possible modifications of this method include: (1) use of the ionic exchange resin for neutralization ;n the reaction flask, with filtration of the resin and washing on the filter funnel; (2) use of calcium, magnesium, lithium or potassium hydroxide to dissolve the cyclodextrin; (3) removal of hydroxides 30 after the reaction by saturating the reaction mixture ;~
with carbon dioxide or neutralization with sulfuric ;~
A ~ ~
_. ~ .. .. . .. . .. . . .. . .. . .
~",, ""', ~ ,"~ . "~
~3~ ;s1~
acid in place of the ion exchange resin; (4) use of even less sodium hydroxide (between 1 and 2 equi-valents); and (5) elimination of the second freeze-drying.
The following is a typical procedure using the new, improved ~ethod:
50 9 of ~-cyclodextrin was dissolved in a solution of 3.53 9 of sodium hydroxide in 75 mL of water and treated with 29 mL of propylene oxide at 0C. The reaction mixture was maintained for 5 hours at that te~perature, then was kept at room temperature for 42 hours. At the end of that time, the reaction mixture was passed through the Dowex 50W-X8 column (H+ form), -the column was washed with water and the eluate was evaporated under vacuum to a volume of 100 mL, then freeze-dried. The resulting white solid was washed with acetone to give 51 9 of HPCD, with the same degree of substitution (4.7) and NMR as the HPCD prepared by the Pitha et al method. Riesidue on ignition was 0.0%. Optical rotation also was identical to that of the Pitha et al product. ~ ~ ~
Condensation of 25 9 of B-cyclodextrin using ~ ;
7.71 9 of sodium hydroxide gave similar results.
A further improvement in the new, improved HPCD
25 synthesis utilizes activated carbon for purification of` ;~
the solution prior to the last freeze-drying. Thus, when the aqueous solution from the Dowex 50 ionic ~-~
exchange column was treated with activated carbon, most of the polymerization products were removed without loss of HPCD, and the filtrate after only one washing with ethyl acetate was ready for final freeze-drying.
, , :, ~, ~,.
In this way, only one freeze-drying was require~.
Crystallization of the final product instead of freeze-drying is also possible, ~t least on ~ small scale.
The product from the modified new process (using 5 activated carbon) appears to he superior to that of the original new process and the Pitha et al process.
First, the product is snow white and Droduces a color-less aqueous solution, whereas solutions of the earlier products were yellow. Secondly, the product is not 10 oily, which may be due to removal of more highly sub-J stituted, less soluble, oily cyclodextrins.
HPCD can be prepared in varying degrees of sub-stitution, such as 5 or 7. Typically, the foregoing procedure is used to produce HPCD (ASDS 7). The mass 15 spectra for the isomeric misture of HPCD centers around 7 degrees of substitution. This spectra is obtained by `~
"softly" ionizing the sample using fast atom bombardment. The generated spectra is similar to those previously reported (obtained by Californium-252 plasma 20 desorption) in both the symmetry of the isomeric distribution and the numerical spread of the isomers formed.
Hydroxyethyl-~-cyclodextrin (HEBCD) can be pre-; pared analogously to HPCD, utilizing the improved 25 procedure detailed above but substituting an equivalent ; quantity of ethylene oxide for the propylene oxide there employed.
The synthesis of 2-hydroxypropyl-y-cyclodextrin (HPGCD) similarly uses the same basic procedure as for 30 HPCD, substituting y-cyclodextrin for the B-cyclo-~ dextrin starting material. However, because y-cyclo- ~
,: :
~ .
, :
1~ 3 ~ L~
-7~-dextrin contains eight glucose resi~ues compared to seven for ~-cyclodextrin, the amo~nt of propylene oxide used can be reduced in order to lower the degree of substitution. IJse of 0.75 mole of propylene oxide per 0.077 mole of Y-cyclodextrin (~ 20% excess considering
diazepam. Other CNS active metabolites for use herein -~
will be apparent to those skilled in the art, e.g. SL
75102, which is an active metabolite of progabide, a GABA agonist, and hydroxy-CCNU, which is an active metabolite of CCNU, an anti-cancer nitrosourea.
Typically, these CNS active metabolites have been identified as such in the scientific literature but have not been administered as drugs themselves. In many cases, the active metabolites are believed to be comparable in CNS activity to their parent drugs;
frequently; however, the metabolites have not been -administered per se because they are not themselves able to penetrate the blood-brain barrier.
As indicated hereinabove, diagnostic agents, in-cluding radiopharmaceuticals, are encompassed by the c.";.....
~2^.', ., ~
1 3 3 ~
^40-expression "centrally acting drug" or the like as used herein. Any diagnost1c agent which can be derivatized to afford a redox carr~er system which will penetrate -the BBB and concentrate 1n the brain in its quaternary form and can be detected therein is encompassed by this invention. The diagnostic may be "cold" and be de-tected by X-ray (e.g. radiopaque agents) or other means such as mass spectrophotometry, NMR or other non- ~ I
invasive techniques (e.g. when the compound includes stable isotopes such as C13, N15, 018, S33 and S34).
The d1agnost1c alternatively may be "hot", i.e.
radiolabelled, such as with radioactive iodine (I 123, I 125, 1 131) and detected/imaged by radiation detect~on/imaging means. Typical "cold" diagnostics for derivation herein include o-iodohippuric acid, iothalamic acid, iopydol, iodamide and iopanoic acid.
Typical radiolabelled diagnostics include diohippuric ~-acid (I 125, I 131), diotyrosine (I 125, 1 131), o-iodohippuric acid (I 131), iothalamic acid (I 125, 1 131), thyroxine (I 125, I 131), iotyrosine (I 131) and iodometaraminol (I 123), which has the structural formula ;~
HO ~ CHCHNH2 ,:
In the case of diagnostics, unlike the case of drugs which are for the ~reatment of disease, the "locked in"
quaternary form will be the form that is imaged or otherw~se detected, not the original diagnostic ~ ~ 31 ~
itself. Moreover, any of the centrally acting drugs which are intended for the treatment or prevention of medical disorders but which can be radiolabelled, e.g.
with a radioisotope such as iodine, or labelled with a stable isotope, can thus be converted to a diagnostic for incorporation into the redox carrier system.
It will be apparent from the known structures of -the many drug species exemplified above, that in many cases the selected drug will possess more than one re~
10 active functional group, and, in particular, that the ~-drug may contain hydroxyl or carboxyl or amino or other functional groups in addition to the groups to which the carrier will be linked, and that these additional groups will at times benefit from being protected during synthesis and/or during administration. The nature of such protection is described in more detail in the various patents and patent applications referred to herein. Obviously, such protected drug species are encompassed by the definition of "drug" set forth hereinabove.
It too will be appreciated that by "dihydropyri~
dine carrier" or "[DHC]", there is intended any non-toxic carrier moiety comprising, containing or in~
cluding the dihydropyridine nucleus, whether or not a part of any larger basic nucleus, and whether substi-tuted or unsubstituted, the only criterion therefor being capacity for BBB penetration and in vivo oxidation thereof to the corresponding quaternary pyridinium salt carrier [QC]~. As aforesaid, the ionic pyridinium salt drug/carrier prodrug entity [D-QC]+
which results from such in vivo oxidation is prevented e~
1 3 3 ~
from efflux from the brain, while elimination from the general circulation is accelerated. Subsequently, the covalent or equivalent bond coupling the drug species [D] to the quaternary carrier ~QC]+ is metabolically cleaved, wh~ch results 1n sustained delivery of the drug [D] in the brain and facile elimination of the carrier moiety [QC]+. Such "covalent or equivalent bond" between the drug and the quaternary carrier can be a simple direct chemical bond, e.g., an amide, an ester, or any other like bond, or same can even be comprised of a linking group or function, e.g., a thiazolidine bridge or a peptide linkage, typically necessitated when the drug species is not susceptible to direct chemical coupling to either the dihydropyri-dine carrier or the quaternary carrier. Nonetheless,the bond in the formulae [D-~C]+ and [D-DHC] is intended to be, and is hereby defined as inclusive of all such alternatives. And the cleavage of the [D-QC]+
prodrug to sustainedly delivery the drug species [D] in the brain with concomitant facile elimination of the carrier moiety ~QC]~ is characteristically enzymatic cleavage, e.g., by esterase, amidase, cholinesterase, hydrolyt1c enzyme, or peptidase.
The expression Unon-toxic pharmaceutically accept-able salts" as used here1n generally includes the non-toxic salts of the reduced, d1hydropyridine forms of the redox carrier or redox analog systems, formed with nontox1c, pharmaceut1cally acceptable inorganic or organ1c acids HX. For example, the salts include those der~ved from inorganlc ac1ds such as hydrochloric, hydrobrom1c, sulfur1c, sulfamic, phosphoric, nitric and the 11ke; and the salts prepared from organlc acids 1331~
such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartar1c, citric~ ascorbic9 pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyl~c, sulfan~lic, fumaric, methanesulfonic, tolu-enesulfonic and the like. The expression "anion of anon-toxlc pharmaceutically acceptable acid" as used herein, e.g. in connection with the oxidized, pyri-dinium salt forms of the redox carrier or redox analog systems, is intended to include anions of such inorganic or organic acids HX.
In the discussion to follow, the expression "at least one reactive functional group selerted from the ;~
group consisting of amino, hydroxyl, mercapto, carboxyl, amide and imide" or portions of that expres-15 sion are used. The functional groups designated in ~ ~
that expression have ~he following meanings: ~-The word "amino" means a primary or secondary amlno function, i.e. -NH2 or -NHR. The secondary amino functlon ls also represented herein as -NH-, particu-larly since the exact ident1ty of the R portion of -NHR
is lmmaterlal, R being a part of the drug residue D
ltself whlch is left unchanged by conversion of the drug to the redox carr~er system.
The word "hydroxyl" means an -OH function.
The word "carboxyl" means a -COOH function.
The word "mercapto`' means an -SH function.
The word "amide" means a carbamoyl (-CONH2) or substltuted carbamoyl (-CONHR) or a sulfamoyl ~-502NH2) or substltuted sulfamoyl (-S02NHR) functional group.
The -CONHR and -S02NHR groups may also be represented . .
.
~3~
hereln as -CONH- and -S02NH-, respectively, since the 1dentlty of R is immaterial, R being a part of the drug : -resldue D ltself wh1c\l is left unchanged by con~ersion of the drug to the redox carrier system.
The word "imide" means a functional group having the structure -C~ ,~ ~
~ H
O . . ~:.
that is, the structure which characterizes imides (i.e.
compounds having a succinimide-type or phthalimide-type structure).
Many different dihydropyridine ~ pyridinium salt redox carrier moieties are illustrated in the carrler patents and applications referred to hereinabove.
The following i8 a list of representative :
ma~or classes of dlhydros and the correspond1ng quater-naries, but 1s not meant to be exhaustlve:
(1) For linkage to a drug having at least one hydro~yl or mercapto or primary or secondary amino functional grouping, replacing a hydrogen atom from at least one of said functional groupings with one of the following ~DHC~ groupings:
, ~ , ~..
' ,.
~',.'~.
~'",;~
1~3~
-45 ~
o .::
~lc'- ~X
R l , R3 -8-(a`) (b') ,: ~ .::
O 0 1l ~ICOC'2C- ~ , '. ;~ ~
R ~
( c ' ) ( d ) :~ ~
O O ~' '.
~X ~COC~2C-R -C-3a (e ) ~oc- ~x or I~N`R ' a (9 ) .
~ COCH2C- ~ :
`Rl ~ ::
(J') ,~ ' ' ~ ' 1~3~ ~4 wherein the dotted line ~n formulas (a'), (b') and (c') ~nd~cates the presence of a double bond in either the 4 or 5 posit~on of the d~hydropyridine ring; the dotted ~ :
line in formulas (d'), (e'~ and (f') indicates the S presence of a double bond in either the 2 or 3 posit~on of the dihydroquinollne ring; Rl is Cl-C7 alkyl, Cl-C7 : ~:
haloalkyl or C7-Clo aralkyl; R3 is Cl to C3 alkylene; X
is -CONR'R" , where~n R' and R'', which can be the same .A/~
or different, are each H or Cl-C7 alkyl, or X is -CH=NOR" ' wherein R''' is H or Cl-C7 alkyl; the carbonyl-containing groupings in formulas (a') and (c') and the X substituent in formula (b') can each be attached at the 2, 3 or 4 position of the dihydropyri- ~ ~ :
dine ring; the carbonyl-containing groupings in 15 formulas (d') and (f') and the X substituent in formula ;~ :
(e') can each be attached at the 2, 3 or 4 position of the dihydroquinol1ne ring; and the carbonyl-containing groupings in formulas (g') and (j') and the X substi- -tuent in formula (h') can each be attached at the 1, 3 or 4 posit10n of ~he d1hydro~soquinoline ring.
(2) For linkage to a drug having at least one carboxyl functional grouping, replacing a hydrogen atom from at least one of sa~d carboxyl groupings with one of the follow1ng tDHC] groupings:
(a) When there are one or more -COOH groups ~ ~.
to be derlvat~zed~
R~ R3 C Q Z
( 1 ' ) ( 11 ' ) ' ~; ~ `
o o o , OCH2C-~-Z - ~
R ~ R I
? ( l v ) X~ jCcH2c-Q-z ' -o R 1 (V' ) (vi ~ ) ~1 ~3 S az (v11~)(viii~
COCH2C-Q-Z'-1x~) whereln the dotted llne ln formulas (1'), (11') and (111') lnd1cates the presence of a double bond ln elther the 4 or 5 posltlon of the dihydropyridlne ring;
the dotted llne ln formulas (lv'), (v') and (vl') lndicates the presence of a double bond in elther the 2 or 3 posltion of the dlhydroqulnoline rlng; Z' ~s Cl-C8 stralght or branched ~lkylene, preferably Cl-C3 stralght or branched alkylene; Q ls -O- or -NH-; Rl ls Cl-C7 alkyl, Cl-C7 haloalkyl or C7-C10 aralkyl; R3 ls Cl-C3 alkylene; X ls -CONR'R'' wherein R' and R'', : 15 whlch can be the same or dlfferent, are each H or Cl-C7 ::: ~ :
~3~
-48^
alkyl, or X ~s -CH=NOR''' where~n R'" ~s H or Cl-C7 alkyl; the X substituent ln formula (1~') and the A,~
carbonyl-contaln1ng group~ng 1n formulas (i') and (~11') can each be attached at the 2, 3 or 4 position of the d~hydropyrld1ne ring; the X substituent ~n formula (v') and the carbonyl-containing groupings in formulas (~v') and (v~') can each be attached at the 2, 3 or 4 pos~t~on of the dihydroqu~noltne r~ng; and the X
substltuent ln formula (v~') and the carbonyl-conta~n~ng group~ngs in formulas (v~i') and (ix') caneach be attached at the 1, 3 or 4 position of the dihydroqu~nol~ne ring;
(b) AlternatiYely, when there ~s only one ~ --COOH group to be der1vatized:
(R~)5~ "_ ~ ~ ~ (R4)n1v ~ ~ , (x') (x1') ~
, _ , . .
O
CNH
)nV ' r (x11 ' ) , , ~, ~, .
ChH ~ I (x111'), or .- ...
~N
~A)nV ' ' ~
' ~ CNH ~ t (x (A)nV ~ ~
::::
D r ~ . ' ~ . ' ~: . : -` ~ ~ . ' . ~. : . ' . . : . ', ~, ".' -~:, ' ':' :' ': . ' ' ' : ' , . ~ , ~ ' ,- .~ :.. ' ' ' . ' ' '.:' ' ~ j, ,' . . ' ' ' , :: : . . .
j/, ~
~^~
~ 3 ~
wherein the dotted 11ne in formula (xii') 1ndicates the presence of a double bond in either the 4 or 5 position ~i of the d1hydropyr1dine ring; the dotted line in formula (x11i') 1ndicates the presence of a double bond in ~:
e1ther the 2 or 3 pos1tion of the dihydroquinoline r1ng;. ` is the skeleton of a sugar molecule; niV is a pos1tive integer equal to the total number of -OH
funct10ns 1n the sugar molecule from which sa1d skele-ton is derived; nv 1s a positive integer one less than lO the total number of -OH functions in the sugar molecule -from which said skeleton is derived; each A in each of structures (xii'), (xii1') and (xiv') can ~ndependently be hydroxy or ~', D' being the residue of a centrally acting drug containing one reactive carboxyl functional group, said residue being characterized by the absence of a hydrogen atom from said carboxyl functional group .
1n said drug; and each R4 1n each of structures (x') and (xi') can 1ndependently be hydroxy, .
o8~
R
20 oc~3 R~
oC~3 or D'~
.. ~' :
' ,:,5: ,, ` ~: ~ .~ - : : . . .
~?~
1~3~
wherein the dotted line is defined as with structures (xii') and (xiii'); D' is defined as with structures (xii ), (xiii ) and (xiv'); Rl is C1-C7 alkyl, Cl-C7 -haloalkyl or C7-C10 aralkyl; and the depicted carbonyl groupings can be attached at the 2, 3 or 4 position of the pyridinium or qu~nolinium ring or at the 1, 3 or 4 position of the isoquinolinium ring; with the proviso that at least one R4 in each of structures (x') and (xi') is 10 -C~
-OC~ or Rl . , ~ ' ' -_o = ~ ~
,N ' wherein R~, the dotted lines and the position of the carbonyl-containing groupings are def~ned as above; and wlth the further proviso that when more than one of the R4 radicals in a given compound are the aforesa~d is,, .. ., :.: . . ...
1 ~3~
carbonyl-containing groupings, then all such carbonyl-containing groupings in said compound are identical.
(3) For llnkage to a drug hav~ng at least one :
-NH- functional group which is part of an amide or 5 ~m~de structure or at least one low pKa primary or secondary amine functional group, replacing a hydrogen atom from at least one of said functional groupings with one of the follow1ng [DHC] groupings:
C O C H ~ ,X
R
~ IN /
R l R 3 - ~C1 ( k ' ) O R ( 1 O : :: :
!' COCH--, I
N
Rl (n ' ) (m' ) o O
C O C H 2 C O C H-- , : :~
~ ~ R ~: ~
R3-COCIH--. Rl ::
O R
(O') (P') .' t.~
1~31~
~COCH--, ~K
( q ' ) ( r o r ~ COCH2 COCH ~
~ R1 (s ' ) '' - ' ''-`' '"'~'~'".' wherein R is hydrogen, Cl-C7 alkyl, C3-C8 cycloalkyl, C1-C7 haloalkyl, furyl, phenyl, or phenyl substituted by one or more halo, lower alkyl, lower alkoxy, carba-moyl, lower alkoxycarbonyl, lower alkanoyloxy, lower haloalkyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, lower alkylth~o, lower alkylsulfinyl or lower alkylsulfonyl; the dotted line in formulas (k'), (1') and (m') lndicates the presence of a double bond in either the 4 or 5 position of the dihydropyri-dine ring; the dotted l~ne in formulas (n'), (o') and ~ -~: (p') indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring; Rl is ~ ~.
:
. ~".~
~ , c v;
~, ,. ~ . . . ,, ~ , ~ .. . . .
1 ~ 3 ~
C1-C7 alkyl, Cl-C7 haloalkyl or C7-C10 aralkyl; R3 is Cl to C3 alkylene; X is -CONR'R'', wherein R' and R'', which can be the same or different, are each H or Cl-C7 alkyl, or X is -CH=NOR''' wherein R'" is H or C1-C7 alkyl; the carbonyl-containing group~ngs in formulas (k') and (m') and the X substituent in formula (l') can each be attached at the 2, 3 or 4 position of the dihy-dropyridine ring; the carbonyl-containing groupings in formulas (n') and (p') and the X substltuent ln formula 10 (o') can each be attached at the 2, 3 or 4 position of .
the dihydroquinoline ring; and the carbonyl-containing groupings in formulas (q') and (s') and the X substi- .
tuent in formula (r') can each be attached at the 1, 3 or 4 position of the dihydroisoquinoline ring.
Drugs containing secondary or tertiary hydroxyl functional groups can be linked to any of the tDHC]
groupings (k') through (s') above in which the -CHO-portion is derived from an aldehyde RCH2C capable of reacting with said drug to form the corresponding hemi-acetal, e.g. chloral, acetaldehyde, formaldehyde or benzaldehyde.
The following are especially preferred reduced, dlhydropyrid1ne forms of dihydropyridine pyridinium salt redox carr~er systems, also termed "chem1cal delivery systems" or "CDS", for braln-targeted drug del1very wh~ch are contemplated for formation of ~nclus~on complexes wlth hydroxypropyl-B-cyclodextrin 1n accord with ~he present inventlon:
",,~
.
, ,~
1331 ~
~54 ~
a _ ~ c e o ~;
y ~1 'o) _ E ~ E E
L O o ~
Ç~
0_3~ -0 ~ _~
l ' ~ ~. .
X ~ y ~ ' : 0 8-o ~ _ o~8 ~=o ~
~ ~ ' 133t ~ ~
O ~
~ o .~
C
~ _ N
~~
~n _o ~r ~? u ~1 5~
e~
~: ~
!. ' ~
~,'. ":'~'. ' .: ~:.: ~ ~ ~. .'. ~ : ' ~ ' ~ ~ 3 1 ~
-56 - ` . ~
C ~ . ~
r ~t ,~
Y ¦ X D
,~
t- -5~
t~
~ :-.
1 3 31 ~
-57~
X ' _ o o n ~J ~ o ~ ''"~
c n C n ~ ~ ~
vl ' o O
D V ~V ~V
N . ~
~ ~, O, ,~
V O o o ~r ~ s C N ~
N ~ ~ ~ U ~
C ~ C~ e ~, .
:~
~ }~
p~ .~ ~ .: ., .. .. - ~ ~ . . : . ' ' , -:
133~
e ,,, ;~
I ~ . .0 0 E .. o o ,.
C " ~i1 E ,~
_ N ~, N
C ~ C
~ K ~
C V V~ N ,, ~ ~ O ~ , ~ ~, O ' :' ~!
_ _ C _ j~ o ~ ~
~ "~ _ . 2 .. ~ ~ o ~
_ 8 K C ~ __ C ~ ~ _ C ' :, I ~ 2 _ N ~, _ N ~,_ . 3 . c ~ c c ~1 ~ L~ ~V
S~
o~
05~ ~ o=
~1 ~ o=~ o=~
~ b-~- b-s-; ~
i; ~ ~
_ _,-~ Co, ~: :
o o ac~ v-C~
~1 o~
~ C
~ o V~
~ _ C
U : .
,~ T-~s ,IT ~ v 5~ o ~1 ~o Pr ~S~ !;' .'.: ~. -: ~ ` `
~,',.,i~'.. /' . : . ': ': :.. ,' ',: , .
_ _, O .,, O
O ~ e O
_ _ o ~ o V U ~ ~ ' O ' ~ O
~ ~ a ~ o . ~ . ~
o~ o E C : :
W C K o o C V~
o _ _ ~ C~ _ O C O ~ ~
~ ~ _ tc ~`:
_ . ,, " c _ O ~ ~-- I N
E o 5 ~1 Y ~ o 3~ C o ~ N _ ~_ N ~ ~ C
Z o~ o r~ c c u o ~,o " ~ ~ 2 ~--' ~ o o N
~ ~ J U ~` ~
~ _ N ~ K
~J ~,N~ ~ ' L~ LJ
s~ ~- ~
'^~
I ~ L~ ::
I \r~ O
V ~0 '' 0=~ ~
~ =/ ~:
~ 3 ::::
~ ~
~... . . ~ . . ~ . . . . . .
1~ 3 1 ~::
l ~ a c - ~-_ ~ O _ ~ ~
o E ~,.. o D
O ~
!~ ~, Q : ~ :
_ O ~ ~':
O _ L
7 ,3~ N ~"" ~ e ~ _ _ E
~ ' O
~"~'''' 0~
Z J~' G
o .. ..
, C C ~ ~ ~
~ C
.~ .
E O
E '--_ C~
~C
r ~, ~ y ~ ~ o ':
'~ -63~
o o ~ C
_ _ .. ~ ~. ,.
~ ~ U ~ .. ~ o _ _ o _ _ C o.~ g C _ C ~ ~ ~ " _ - : .
~ O
V) ~ ~ Vo~
~, E ~o .,~
_ _ O > ~ o ~ ~
o V~ o .~
.t ,, ~.o ~ ~ ~ ~
o o O
, Y o_ ~C~
~ V o C . _ ,. .
~ ^ ,.. _ i ~ ~
_ ~ ~ C _ ~ o O o ~ O L ~ ~ L
~ _ ~. o ~", o o Z
J __ ~ ., ~,~, , .. _ ~ .
A ~
~ ~ J
i , ~:
~:
1 ~
:' ''~ `' '' ~ ' ',' ~ , ' :~; '" .'~ '~::'`" ~':1" : ' : ' ~ : ' , .
~ ~ " ~ r i~3~
'' ~o . ~
~ ~ o ~ O~ ~ o ' ~ '' ~C_o '.;, ~ ,:.
... ., . o ~ ~ :
~ o y o ,,, , ~ U ~ ~
o o ~ ~X :
-1 ~J~
uo~
2 [~
/~\ ~ ~o '' 13~,15~4 , o ~
o ", o C ~
o _ ~ ~
-I '' ~c Co ~ OO-- ~ ~
_~o~J>~ ~?c --~ '' ~ ~ ~ o ~ ~ æ
s,~
13$~
., ~
O~ o~a O ~ O,~;~,O O
. 0~ UO ~ O
O O ~ ~. O ~ ~ , C C c O Co O ~ O C O ~ " O " ~ .
L ~ Y ~ ~ 01 C o C
~
, o ~o o o o _ L~ C
~ C~ o ~ C ~ "~,, ~ O
~æ. ~, ~?ær,~
o ~ ~ ; .
,.. ~ 0 ~ ~
o ~
o o .
1 33~r~
~j l ~ C ~
C' . ;
cl~ N '~ ~ lV _ C
~ o W~ ~ W ~ C
E O ~
L~
U ~ , L~ L~' 7 . u 7~ i l -æ~ ~ æ~ ~æ~
~.--g' b ~ ; ~
r O O ~ :
V~ L~
_ ~, O v v C _ C o E ~o ~ ~ o o ~ ~
n n L ~ ~ C _ .. n ~ ~ ~ G
~ o ~0 ~ ~ ~ 0 ~ ~ o ~ ~o O ~o _I n O ~ _ n ~ ~J ~ u L ~-- ~ _ _ ~ ~ ~ n O C
eE o E ' ~ ~ ~ n v, J E ~I G
~i Z
o Y O
L _ I O _ ' , ~
O OGJ c, .
~i! ~ c a~
n >. ~
~ _ ~, L c b00 ~c~ _~c'ô :~
I~ _ ~n ~? CO ~,~ m : :
L ~ ~ o~
~ ~ ~ 2 " ' ~ ~ ~ ~ o L~rl C~
E ,o, C .. ~ o S ' c~ -- '' c 2 . c~
C ~ o , ~ .. el o e , -:
I') Y O _ I~ O 0 ~ _ N ~ :-C~
~ .
t ~
, r ~ ., U _ ~ g ~:
~ ~ ~U V
:
nl ~ æ o , O :~ :
~ t E ~
E L o ~- ~ c g ' ~' U
u ~~
O~ ~
Z
' .~ .
'..'~'`' "" "
I V V V ' ~
u I C ' ,~
~ H H H
~ ~0 _ E O ~ ol K c V E E
L u ~ U
N ~
C ~ t ~ >, _ ~ ~ :
0~
,2,~ ' 2~2 ~,~2~
U
: ~ '' .
G u -u ~, 1 3 3 ~ h~ ~
C C C
~ ~1 U ~:n _ O L o L o L
o ~,~ C~
__ .,,.,, -c ¦ ~u o N O "
I U~ ,o o V 1~ U ~ i C
Xc ~ ~
O ~ o ~ Z Z ~ ~A` '~ --, i .~.;~,~ ~
~ o ~0 _ c ~o _ C
~ _~ ~Z Z ~2Z~ ~ o .. ~CU '3U ~- ~
~ ' --o >, o N ~~ _ C ~ 4J
~ ~ u c ~ ~ , o~s 1~31~
- 7 2 - ~ ,~
U ' ~
o E ,~
' C C
UU ''~
. ~u .. ,C .'' V V) ~ ' ~u , ~ :.
.~
~ C
~ ~e -- . ~
~ ~V~I
c r 1331., 5~
The cyclodextrins contemplated for use herein are hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of ~-cyclodextrin and the ;
corresponding derivatives of y-cyclodextrin. The ~ -hydroxyalkyl groupings ~ay contain one or more hydroxyl groups, e.g. hydroxypropyl, dihydroxypropyl and the like. The glucosyl, maltosyl and maltotriosyl derivatives may contain one or more sugar residues, e.g. glucosyl or diglucosyl, maltosyl or dimaltosyl.
Various mixtures of the cyclodextrin derivatives may be used as well, e.g. a mixture of maltosyl and dimaltosyl derivatives. Specific cyclodextrin derivatives for use herein include hydroxypropyl-~-cyclodextrin (HPCD or HPBCD), hydroxyethyl-B-cyclodextrin (HEBCD), hydroxypropyl-Y-cyclodextrin (HPGCD), hydroxyethyl-y-cyclodextrin (HEGCD), dihydroxypropyl-B-cyclodextrin (2HPBCD), glucosyl-B-cyclodextrin (G1-B-CD or G1BCD), diglucosyl-B-cyclodextrin (2G1-B-CD or 2G1BC~), maltosyl-B-cyclodextrin (G2-B-CD or G2BCD), maltosyl-y-cyclodextrin (G2-y-CD or G2GCD), maltotriosyl-B-cyclodextrin (G3-B-CD or G3BCD), maltotriosyl-y-cyclodextrin (G3-Y-CD or G3GCD) and dimaltosyl-B-cyclodextrin (2G2-B-CD or 2G2BCD)s and mixtures thereof such as maltosyl-B-cyclodextrin/dimaltosyl-B-cyclodextrin.
Hydroxypropyl-B-cyclodextrin for use in the novel inclusion complexes and methods of the present inven-tion is commercially available. Alternatively, it may be prepared by known methods, especially by use of the ~`~ 30 optimized procedure of Pitha et al, International Journal of Pharmaceutics, 29, 73-82 (1986). The :: . ..
1 3 3 1 .; r r ~
-74~
. ~, : ~,:
following is a typical procedure using the Pitha et al method:
31 g of sodium hydroxide were dissolved in 250 mL
of water. Then, 100 g of ~-cyclodextrin were added and the solvent was warmed to effect solution. The flask was cooled and 50 mL of propylene oxide were added.
The flask was fitted with a dry ice/acetone condenser ~-during the addition. The solution was allowed to come to room temperature and was stirred for 72 hours. The solution was then neutralized with concentrated hydro-chloric acid and diluted with water. The solvent was removed in vacuo, leaving a syrup which was taken up in ethanol. After stirring for 30 minutes at room temperature, the sodium chloride produced was removed by filtration. The filter cake was washed with ethanol and the co~bined ethanol layers were reduced in vacuo. The residue was dissolved in water and dialyzed in cellulose acetate ($7, 38 mm 4.6 mL/cm, molecular weight cut off = 10~0, Fisher Scientific). After 5 hours at 0C, the solution was removed from the dialysis tubing and freeze-dried. The resulting solid was suspended in acetone and stirred overnight. The filtered solid was resuspended in acetone and stirred ;
for 24 hours. The solid was collected by filtration and dissolved in 200 mL of water and then lyophil-ized. 75 grams of purified hydroxypropyl-~-cyclo-dextrin were obtained. The degree of substitution was calculated by NMR and by comparison with an authentic sample.
In forming a complex with E2-CDS, a 50% solution (w/w) cf 2-hydroxypropyl-B-cyclodextrin (HPCD) was made ,., - 1 3 ~ 4 in distllled ~ater. An excess of E2-CDS was added and the solut~on then was purged wlth h~lium. The result-~ng suspension ~as then sonicated for 30 minutesTh f;l-tered through a glass filter (ASTM 1~-15M, Pyrex No.
36~60) and freeze-dried overnight. Best results were obtained by hard-freezing the aqueous solution of the E2-CDS/HPCD complex for at least 10 hours before lyophll1zation. The degree of complex formatlon was determined by dissolving a small amount of the dry complex in methanol and then analyzing by high pressure liquid chromatography (HPLC). The degree of complexation was found to vary between 20-40 mg/g and the solubility of the complex was determined to be 2.2x104 Ing/L.
The P1tha et al method for preparation of HPCD by condensation of propylene oxide with ~-cyclodextrin in alkaline aqueous solutlon unfortunately suffers from disadvantages, particularly in purification of the product. After comp1etlon of the condensation, the react~on m1xture is neutralized with hydrochlor~c acid, water is evaporated under vacuum and the syrupy residue ~;
1s dlssolved in ethanol to precipitate sodium chloride, the main by-product of the react1On. After filtration, ethanol is evaporated under vacuum and the residue is dissolved in water and dialyzed to remove the remaining sodium chloride and polymerization products of propylene oxide. During dialysis, part of the hydroxy-propyl-~-cyclodextrin goes through the membrane and is lost. The dialysate is then freeze-dried, twice -~ --30 stirred in acetone and washed to remove the remaining -~
polymerization products. Finally, hydroxypropyl~
cyclodextrin is freeze-dried again. The second freeze-1 ~ 3 t ~
drying is necessary because the product after washing with acetone is not homogeneous.
To overcome these difficulties with the Pitha et al process, a new method has been developed by Maciej 5Inulkowski of the University of Florida, Gainesville, Florlda for the synthesis of HPCD. This ne~ method 1nvolves removal of sodium hydroxide fro~n the reaction mixture by an ion exchange resin (H+); as a result, several time-consuming steps of Pitha et al's -~
10 purification can be avoided. Moreover, the amount of ~-sodium hydroxide used by Pitha et al t7 equivalents for one of ~ cyclodextrin) can be decreased to 2 equivalents of sodium hydroxide per cyclodextrin molecule, and still produce a product with the appropriate NMR and optical rotation.
According to the new method, ~-cyclodextrln is - ;
first condensed with propylene oxide in alkaline solu~
tion, sodiu~Thydroxide is removed on an ion exchange column (Dowex 50W-X8, H+ form), the eluate is eva- -~
porated under vacuum to one-half of the original volume, the rema~n1ng solution is freeze-drled, the resultlng whlte solld 1s washed wlth acetone and freeze-dried again, then subjected to grinding and sieving. Possible modifications of this method include: (1) use of the ionic exchange resin for neutralization ;n the reaction flask, with filtration of the resin and washing on the filter funnel; (2) use of calcium, magnesium, lithium or potassium hydroxide to dissolve the cyclodextrin; (3) removal of hydroxides 30 after the reaction by saturating the reaction mixture ;~
with carbon dioxide or neutralization with sulfuric ;~
A ~ ~
_. ~ .. .. . .. . .. . . .. . .. . .
~",, ""', ~ ,"~ . "~
~3~ ;s1~
acid in place of the ion exchange resin; (4) use of even less sodium hydroxide (between 1 and 2 equi-valents); and (5) elimination of the second freeze-drying.
The following is a typical procedure using the new, improved ~ethod:
50 9 of ~-cyclodextrin was dissolved in a solution of 3.53 9 of sodium hydroxide in 75 mL of water and treated with 29 mL of propylene oxide at 0C. The reaction mixture was maintained for 5 hours at that te~perature, then was kept at room temperature for 42 hours. At the end of that time, the reaction mixture was passed through the Dowex 50W-X8 column (H+ form), -the column was washed with water and the eluate was evaporated under vacuum to a volume of 100 mL, then freeze-dried. The resulting white solid was washed with acetone to give 51 9 of HPCD, with the same degree of substitution (4.7) and NMR as the HPCD prepared by the Pitha et al method. Riesidue on ignition was 0.0%. Optical rotation also was identical to that of the Pitha et al product. ~ ~ ~
Condensation of 25 9 of B-cyclodextrin using ~ ;
7.71 9 of sodium hydroxide gave similar results.
A further improvement in the new, improved HPCD
25 synthesis utilizes activated carbon for purification of` ;~
the solution prior to the last freeze-drying. Thus, when the aqueous solution from the Dowex 50 ionic ~-~
exchange column was treated with activated carbon, most of the polymerization products were removed without loss of HPCD, and the filtrate after only one washing with ethyl acetate was ready for final freeze-drying.
, , :, ~, ~,.
In this way, only one freeze-drying was require~.
Crystallization of the final product instead of freeze-drying is also possible, ~t least on ~ small scale.
The product from the modified new process (using 5 activated carbon) appears to he superior to that of the original new process and the Pitha et al process.
First, the product is snow white and Droduces a color-less aqueous solution, whereas solutions of the earlier products were yellow. Secondly, the product is not 10 oily, which may be due to removal of more highly sub-J stituted, less soluble, oily cyclodextrins.
HPCD can be prepared in varying degrees of sub-stitution, such as 5 or 7. Typically, the foregoing procedure is used to produce HPCD (ASDS 7). The mass 15 spectra for the isomeric misture of HPCD centers around 7 degrees of substitution. This spectra is obtained by `~
"softly" ionizing the sample using fast atom bombardment. The generated spectra is similar to those previously reported (obtained by Californium-252 plasma 20 desorption) in both the symmetry of the isomeric distribution and the numerical spread of the isomers formed.
Hydroxyethyl-~-cyclodextrin (HEBCD) can be pre-; pared analogously to HPCD, utilizing the improved 25 procedure detailed above but substituting an equivalent ; quantity of ethylene oxide for the propylene oxide there employed.
The synthesis of 2-hydroxypropyl-y-cyclodextrin (HPGCD) similarly uses the same basic procedure as for 30 HPCD, substituting y-cyclodextrin for the B-cyclo-~ dextrin starting material. However, because y-cyclo- ~
,: :
~ .
, :
1~ 3 ~ L~
-7~-dextrin contains eight glucose resi~ues compared to seven for ~-cyclodextrin, the amo~nt of propylene oxide used can be reduced in order to lower the degree of substitution. IJse of 0.75 mole of propylene oxide per 0.077 mole of Y-cyclodextrin (~ 20% excess considering
8 OH groups) affords HPGCD with a degree of substitu-tion of 8, while use of 0.56 mole of propylene oxide (~ 10~ less than equivalent) gives a degree of sub~
stitution of about 7.
Hydroxyethyl-y-cyclodextrin (HEGCD) can be pre-pared similarly to HPGC~ as described in the preceding paragraph, simply using an equivalent quantity of -;~b~
ethylene oxide in place of the propylene oxide.
Thus, the hydroxyalkyl cyclodextrins intended for ~
15 use herein can be prepared by procedures described by ;
Pitha et al or variations thereof. Obtained by these procedures, the cyclodextrins are intrinsically -~
amorphous mixtures. The importance of the amorphous nature of the cyclodextrins is described by Pitha et al, ~. Pharm. Sci., Vol. 74, No. 9, September 1985, 987-990. The advantages of the amorphous nature of these materials are more pronounced at higher concentrations of cyclodextrin.
The other cyclodextrins intended for use in the present invention, i.e. the glucosyl, maltosyl and maltotriosyl derivatives of ~- and y-cyclodextrin, are branched cyclodextrins which are highly soluble in water as compared to the parent cyclodextrins. These branched cyclodextrins can be produced by microbiolo-gical processes from the parent cyclodextrins.
Glucosyl-~-cyclodextrins can be obtained from the T ~
. ~. ... ~ ~ ..... . . , . : .
mother liquor of a large-scale ~-cyclodextrin synthesis with Bacillus ohbensis cyclomalto~extrin .
glucanotransferase; see Koizumi et al, Chem. Pharm.
Bull., 35 (8), 3413-341B (1987) and reference cited therein. Maltosyl and maltotriosyl ~- and y-cyclodextrins can be prepared from the parent cyclodextrin and maltose or maltotriose through the reverse action of Pseudomonas isoamylase or Klebsiella aerogenes pullulanase, while glucosyl-y-cyclodextrin can be prepared by enzymatic hydrolysis of maltosyl-y-cyclodextrin; see Okada et al, Chem. Pharm. 8ull., 36 ~ --(6), 2176-2185 (1988) and references cited therein.
The preparation of maltosyl-~-cyclodextrin by reacting maltose with ~-cyclodextrin in the presence of pullulanase is also described in Japanese Kokai 61-287902, published December 1~, 1986, and Japanese Kokai 61-197602, published September 1, 1986. A mixture of maltosyl-~-cyclodextrin and various dimaltosyl-3-cyclodextrins may be conveniently employed, e.g.
ISOELEATTM of Ensuiko Sugar Co., Ltd., Yokohama, Japan.
The develop~ent of a carrier-mediated dihydro~
pyridine ~ pyridinium salt redox system (which, in the dihydropyridine form, is also termed a chemical deli-very system or CDS) has resulted in the enhanced andtor -sustained delivery of a variety of drugs to the central nervous system. While the physiochemical properties of the CDS are optimized for brain-uptake and retention, they are often incompatible with aqueous formulations. A salient example is E2-CDS, a CDS based on estradiol. This dihydronicotinate passes the BBB
and is oxidized to the corresponding quaternary salt, E2Q+. The sustained levels of E2Q+ thus produced then ?1.'.. `: .. ~ .:. : ' ~-~ :
~'.'. '-: ~, ~ ' ' ' ' : : ' ~
1 3~3 ~ :r ~ ~
slowly release estradiol, which exerts Profound central estrogenic effects. These effects include LH-suppression in ovariectomized rats and a reversible suppression of cyclicity in intact female rats and are exerted for prolonged periods. The E2-CDS is highly lipophilic and only poorly water soluble (0.2 ~g/mL).
This requires that E2-CDS be administered in water-~iscible organic solvents such as dimethylsulfoxide (D~SO) or dimethylacetamide (DMA). While this 10 procedure is not inappropriate for laboratory animal ~
studies, it is clearly inadequate for human use. The ~ ~-development of aqueous formulation of E2-CDS was therefore investigated. Criteria for this formulation include that it have mini~al toxicity, that it be equivalent with E2-CDS in DMSO or DMA in delivering E2Q~ to the brain and that the technology developed be applicable to other dihydropyridine ~ pyridinium salt redox systems.
I EXPERIMENTAL SECTION
¦ 20 Materials 3-Hydroxy-17B-[(1-methyl 1,4_dihydropyrj_ din-3-yl)carbonyl]oxyestra-1,3,5(10)-triene (E2-CDS), ethyl-3-{{N-{~-[3,4-bis(pivalyloxy)phenyl]ethyl}-carbonyl}}-1,4-dihydropyridine (DA-CDS), 17B-[(1,4-dihydro-1-~ethyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one (T-CDS1), 1-~ethyl-3-N-[3-(benzyloxycarbonyl)- ~`
propyl]carba~oyl-1,4-dihydropyridine (GABA-CDS1), 1-methyl-3-~[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyridine (ACV-CDS) and 17~-~[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-4-en-20-yn-3-one (N-CDS) were synthesized according to published procedures. 2-Hydroxypropyl-~-cyclodextrin ,.
-~2-(statistlcal deyree of substitution = 5.1 or 7) (HPCD) was ~repared ~ccord~ng to the method of Pitha et al.
Other cyclodextrins (~,B or y) were obtained from Aldrich Che,nical Co. and other steroids (estradiol, estradiol 17-valerate, estriol 9 estrone, estradiol 3 methyl ether an~ testosterone 17-propionate), were l)urchased from Sigma Chemical Co. All prepared compounds were fully characterized by spectrosco~ic and microcolnbustlon analysis (Atlantic Microlabs) prior to study. Mass spectroscopic studies were performed on a Kratos ~1S~ORFA double-focus~ng instrument fitted with a fast ato~ gun. Cyclodextrin mixtures were analyzed by fast atom bom~ardl~ent of the sa~ples prepared in a ylycerol matrix. Degrees of substitution were deter-mined from the isomeric mass distribution. Nuclearmagnetic resonance spectra were obtalned on a Varian EM360 60 Mllz spectrometer. Values were recorded relative to an internal standard [3-(trimethylsilyl)-propionic 2,2,3,3-d4 acid, sodium salt, DDS] and all samples were run in ~2~. The degree of substltution was calculated by comparing the integrated area attributed to the anomeric hydrogen compared to that of the hydroxypropyl functionality.
Effect of Solubilizing Agents: An excess of E2-CDS was sonicated with an aqueous solution of the appropriate solubilizing agent for 30 minutes. The suspension was then centrifuged, filtered through 0.45 ~m polyvinyl-idene difluoride (Millex-HV4, lillipore~) membranes and analyzed by HPLC. For studies with 2-hydroxypropyl-~-cyclodextrin, an excess of E2-CDS was added to different concentrations (% w/v) of HPCD and the solu-~ ~:
13315~4 ;
bility (mg/~L) was determined spectroscopically (UV =
360 nm, = 6487 in methanol). An estimation of the bulk equilibriu~ constant was obtained by correlating the millimolarity of E2-CDS solubilized and the --millimolarity of the cyclodextrin added. This latter value was calculated using the average molecular weight -~
of the isomeric mixture determined by mass spectro-scopy. The solubilizing effect of a 50% w/w solution ;
of HPCD was also examined for a series of steroids and dihydropyridines (C~S). These studies were carried out in a similar manner to those previously described.
Preparation of Solid Complexes: An excess of E2-CDS or other CDS was added to a 50~ w/w solution of HPCD. The suspension was sonicated for 30 minutes, filtered through 0.45 ~m PVDF membranes and freeze-dried. The degree of incorporation was determined either spectro-photometrically or by HPLC. In some cases, the effect ;~
of solubilizing agents on the degree of incorporatlon was examined. This involved adding small amounts of polyoxyethylene 20 cetyl e~her (Brij), polyoxyethylenesorbitan monooleate (Tween 80) or ethanol to the aqueous solution prior to lyophilization.
. .
Analytical Methodology: In determining concentrations spectrophotometrically, a Cary 219 (Yarian) or an HP
8451~ Diode Array (Hewlett Packard) spectrophotometer was used. Standard curves were prepared in ~ethanol and gave correlation coefficients greater than 0.999.
; For the CDS, the wavelength monitored was 360 nm while for estrogen 220 nm was used.
' 133~
-~4-The HPLC system consisted of either an Autochrom M500 pump fitted with a Rheodyne injector or a Perkin~
Elmer Series 4 pump, a Kratos Spectroflow 757 variable wav~length detector and either a Beck~an recorder or an LCI-100 integrator (Perkin-Elmer). Separation was achieved on an Analytical Sciences, Inc. (ASI) 10 ~m particle size, C18 reversed phase ~0 cm x 3.9 mm i.d.
analytical column. The flow rate was 1 mL/min, the compounds were detected at 360 nm and in all determina~
tions the temperature was ambient. A mobile phase containing 82:1:1:16 (acetonitrile: tetrahydrofuran:
acetic acid: H20) eluted the E2-CDS at 4.4 min, the DA-CDS at 4.4 min, the T-CDS1 at 6.8 min and the N-CDS at 5.2 min. For the 6ABA-CDS1, a mobile phase consisting of 50:1:1:48 of the same components was required. The retention time was 5.2 min. Other compounds were assayed spectrophotometrically.
Animal Studies: Conscious, restrained Sprague-Dawley rats (female, BW = 200 9) were given either 15 mg~kg E2-CDS in DMSO or 5 mg/kg of E2-CDS complex in HPCD
(E2-CDS-HPCD) in water by intravenous injection (tail vein). At various times after the administration, animals were sacrificed and trunk blood and organs collected. The organs were then weighed, homogenized in water and deproteinized with cold acetonitrile. The organ homogenates were centrifuged and the supernatant analyzed for E2Q+ and E2-CDS using a precolumn enrichment technique, the details of which are given herelnbelrw.
:~
`:
.~
....,, ~
1 ~ 3 1 ~
RESULTS AND DISCUSSION
As discussed hereinabove, cyclodextrins have been used to increase the ~ater solubility of a number of drugs, including steroids. These cyclic oligomers contain various numbers of -1,4-linked glucose units. The number of these units ( = six, ~ = seven, Y - eight) determine the size of a cone-like cavity which is amenable to inclusion by many drugs. The stability of the complex formed depends on the fit of the drug into the cyclodextrin and the cyclodextrin concentration. Unfortunately, the cyclodextrin best suited for complexation with steroids, i.e. B-cyclodex-trin, is poorly water-soluble. This property is derived from the high degree of hydrogen bonding which occurs in the crystal lattice. To add to the problem, B-cyclodextrin is known to cause nephrosis in rats, a toxicity which results, at least partially, from its poor water solubility. In any case, little change in the aqueous solubility of E2-CDS was observed when it was equilibrated with various solutions of either a, B
or Y-cyclodextrin. As illustrated in Table I, concentrations of a-cyclodextrin up to 50 mm increased the aqueous solubility of E2-CDS only 25-fold while B
and y-cyclodextrin increase the solubility of the CDS
135 and 110-fold respectively. The relationship between the aqueous solubility of E2-CDS and the concentration of the unsubstituted cyclodextrins was not linear, a situation which is also observed in the case of the parent steroid. In any case, the limited water solubility and the relatively poor complexation provided by a-, B- or Y-cyclodextrin are unsuitable for :
:
~3 ~ s'~?~
-~6-pharmaceutical exploitation. The toxicity of B-cyclo~
dextrin underscores this assess~ent.
TABLE I
EFFECT OF VARIOUS CYCLODEXTRINS ON THE ~ATER SOLUBILITY
.
Conc. of Cyclo- Maximum Cyclodextrin at dextrin Conc. Ranqe Solubilitv Max_. SolubilitY n (mM or %w7v) (mg/mL) (mM or %w/v) None - 0.0D02 Alpha ()5-50 mM 0.005 50 mM 7 Beta (B)5-15 mM 0.027 10 mM 5 Gamma (y)5-50 mM 0.022 10 mM 5 HPCD0.78-62.5 (7 ASDS)%w/v 30.19 62.5 gw/v 9 HPCD 1-52.5 (5.1 ASDS)~Ow/v 35.12 62.5 ~OW/v 5 Several efforts have been made to increase the aqueous solubility and, therefore, usefulness of cyclo-dextrins. Various methylated derivatives have beendescribed but, in general, the acute toxicity of the modified compound is greater than that of the parent.
Recently, an amorphous cyclodextrin composition was obtained by hydroxypropylation of ~-cyclodextrin. The product, 2-hydroxypropyl-~-cyclodextrin (HPCD), is a mixture of isomers which can be characterized by the average statistical degree of substitution (ASDS).
Either NMR or mass spectroscopy can be used to deter-mine this value. These highly water soluble mixtures . ~
,r ~
-~7-.
were shown by Pitha et al to dra~atically increase the solubility of a nu~ber of compounds including gonadal steroi~s. In addition, preli~inary toxicity studies have shown few, if any, harmful effects after either -oral or intravenous administration.
HPCD (ASDS 5.1 or 7) was prepared according to the method of Pitha et al. The mass spectra for the isomeric mixture of HPCD centered around 7 degrees of substitution. This spectra was obtained by "softly"
ionizing the sample using fast atom bombardment. The generated spectra was similar to those previously reported ~obtained by Californium-252 plasma desorp- -tion) in both the symmetry of the isomeric distribution and the nu~erical spread of the isomers formed. In the cited example, as in the 5.1 ASDS case, no underiva-tized (toxic) ~-cyclodextrin was detected.
In applying this HPCD composition to Ez-CDS, HPCD
with low ASDS's was selected. As the degree of substitution increases, not only does the complexing propensity of the cyclodextrin decrease, presumably due to steric interactions, but the surface activity of the complex increases. This is undesirable since, in general, as the surface activity increases, so does the tendency of the material to cause hemolysis. Both the 5.1 and 7 ASDS HPCD had a profound effect on the solu-bility of E2-CDS. FIG. 1 illustrates the 7 ASDS
case. In this instance, a linear increase (r = 0.995) in the solubility of E2-CDS was evident as the concen-tration of HPCD was increased. At 62.5% w/v, 30.2 mg/mL could be solubilized. In the 5.1 ASDS material, 35 mg/ml of E2-CDS could be solubilized at 62.5% w/v.
13~
-~3~-The lower ASI)S mater;~l yave a 15qO increase in incorporation. These data reflect an increase in ~ -solubility of five orders of magnitude (150,000-fold) over the solubility of E2-CUS in water (Table I). ~
5 Plotting the data obta;ned from the 7 ASDS study as ~-millimolarlty of E2-CUS solubilized versus the millimolarity of HPCD added (based on the average molecular weight of the mixture) gave a line wlth a slope of 0.2. This is an estimation of the bulk stab~lity of the cyclodextrin complex and compares reasonably with other systems.
These solutions could be freeze-dried giving a solid complex. A 50% w/w solut~on of HPCD gave a sol;d containing 37 ~g E2-CUS/gm complex. The complex was stable as a dry pow~er and could be easily reconsti-tuted with water. In these manlpulatlons, it was important to maintain the HPCD component greater than 20% w/v. Below tll~s level, precipitation would occur. Several attempts were made to increase the 20 degree of incorporation f TtMhe complex by ad~Mng ~ -various agents such as Brij (0.7% w/w), Tween 80 (0.8g w/w) or ethanol (107~ v/v). While the addition of Brij increased the degree of incorporatlon to 189 mg/g, the complex was not stable, falling to 42 mg/g in 12 days. The other agents had only modest effects. The upper limit for a stable complex, therefore, appeared to be approximately 40 mg/g under these circumstances. ;
Since an inclusion complex is formed between E2-CDS and the various components of the cyclodextrin mixture, it is possible that some portion of the E2-CDS
would not rapidly dissociate, thus lowering the biolo-:,, ~ A
gically available concentration of E2-CDS. To investi-gate this possibility, the ability of the HPCD (5.1 ASDS) formulation of E2-CDS (E2-CDS-HPCD) to deliver E2Q+ to the brain was measured and compared with the delivery of E2Q~ when E2-CDS was administered in DMSO. This data is summarized in FIG. 2, which depicts brain concentrations of E2Q~ after systemic administra-tion of either 15 mg/kg E2-CDS in DMS0 or 5 mg/kg E2-CDS in aqueous HPCD. When the difference in dose is accounted for, i.e. the data is presented as 7O dose/g, no significant difference exists between brain levels of E2Q~ after E2-CDS administration in DMSO or E2-CDS-HPCD in an aqueous media, although the latter produce data which are strikingly more consistent and less variable. Interestingly, the levels of E2Q~ in the lung are lower after E2-CDS-HPCD administration. One explanation for this is that when E2-CDS is given in a water miscible solvent such as DMSO, there may be some tendency for the highly water insoluble E2-CDS to precipitate. After a bolus i.v. injection, the aqueous, ionic environment of the lung may provide a suitable site for this precipitation. The lower levels obtained in the lung after E2-CDS-HPCD administrations reflect not only the higher water solubility of the complex but may also indicate something of its ln vivo dissociation constant. This dissociation appears to be fast enough so not as to alter the distribution of E2-CDS in the CNS, but slow enough to allow pulmonary transit without significant precipitation. In addi-tion, the values for various organ concentrations arefar less variable after E2-CDS-HPCD administration, which may be explained by the higher water solubility ~.
::~
.~
:
1 3 ~
of the complex and its lower tendency to precipitate.
nngoing pharmacological studies corroborate the effectiveness of the E2-CDS-HPCD formulation in brain-selective delivery.
The effect of a 50% w/w solution of HPCD on the solubility of a number of steroids and other CDS is given in Table II.
TABLE II
SOLUBILIT~ OF VARIOUS STEROIDS AND VARIOUS DRUG
CHEMICAL DELIVERY SYSTEMS IN A 50S W/~ SOLUTION OF 2-HYDROXYPROPYL-~-CYCLODEXTRIN (ASDS 5.1) AND THE AMOUNT
OF DRUG INCORPORATED_IN THE FREEZE-DRIED COMPLEX
Solubility Amount of Drug (mg/mL) in in Dry Complex Drug 50% w/w HPCD (mg/g) Estradiol 25 Estriol 40 - : .
Estrone 9.52 Estradiol 3-Methyl Ether 30 - :
: Estradiol 17-Valerate 13.8 23.5 Testosterone 3~ -;, -. . .:.r~
, .
1 3 ~
stitution of about 7.
Hydroxyethyl-y-cyclodextrin (HEGCD) can be pre-pared similarly to HPGC~ as described in the preceding paragraph, simply using an equivalent quantity of -;~b~
ethylene oxide in place of the propylene oxide.
Thus, the hydroxyalkyl cyclodextrins intended for ~
15 use herein can be prepared by procedures described by ;
Pitha et al or variations thereof. Obtained by these procedures, the cyclodextrins are intrinsically -~
amorphous mixtures. The importance of the amorphous nature of the cyclodextrins is described by Pitha et al, ~. Pharm. Sci., Vol. 74, No. 9, September 1985, 987-990. The advantages of the amorphous nature of these materials are more pronounced at higher concentrations of cyclodextrin.
The other cyclodextrins intended for use in the present invention, i.e. the glucosyl, maltosyl and maltotriosyl derivatives of ~- and y-cyclodextrin, are branched cyclodextrins which are highly soluble in water as compared to the parent cyclodextrins. These branched cyclodextrins can be produced by microbiolo-gical processes from the parent cyclodextrins.
Glucosyl-~-cyclodextrins can be obtained from the T ~
. ~. ... ~ ~ ..... . . , . : .
mother liquor of a large-scale ~-cyclodextrin synthesis with Bacillus ohbensis cyclomalto~extrin .
glucanotransferase; see Koizumi et al, Chem. Pharm.
Bull., 35 (8), 3413-341B (1987) and reference cited therein. Maltosyl and maltotriosyl ~- and y-cyclodextrins can be prepared from the parent cyclodextrin and maltose or maltotriose through the reverse action of Pseudomonas isoamylase or Klebsiella aerogenes pullulanase, while glucosyl-y-cyclodextrin can be prepared by enzymatic hydrolysis of maltosyl-y-cyclodextrin; see Okada et al, Chem. Pharm. 8ull., 36 ~ --(6), 2176-2185 (1988) and references cited therein.
The preparation of maltosyl-~-cyclodextrin by reacting maltose with ~-cyclodextrin in the presence of pullulanase is also described in Japanese Kokai 61-287902, published December 1~, 1986, and Japanese Kokai 61-197602, published September 1, 1986. A mixture of maltosyl-~-cyclodextrin and various dimaltosyl-3-cyclodextrins may be conveniently employed, e.g.
ISOELEATTM of Ensuiko Sugar Co., Ltd., Yokohama, Japan.
The develop~ent of a carrier-mediated dihydro~
pyridine ~ pyridinium salt redox system (which, in the dihydropyridine form, is also termed a chemical deli-very system or CDS) has resulted in the enhanced andtor -sustained delivery of a variety of drugs to the central nervous system. While the physiochemical properties of the CDS are optimized for brain-uptake and retention, they are often incompatible with aqueous formulations. A salient example is E2-CDS, a CDS based on estradiol. This dihydronicotinate passes the BBB
and is oxidized to the corresponding quaternary salt, E2Q+. The sustained levels of E2Q+ thus produced then ?1.'.. `: .. ~ .:. : ' ~-~ :
~'.'. '-: ~, ~ ' ' ' ' : : ' ~
1 3~3 ~ :r ~ ~
slowly release estradiol, which exerts Profound central estrogenic effects. These effects include LH-suppression in ovariectomized rats and a reversible suppression of cyclicity in intact female rats and are exerted for prolonged periods. The E2-CDS is highly lipophilic and only poorly water soluble (0.2 ~g/mL).
This requires that E2-CDS be administered in water-~iscible organic solvents such as dimethylsulfoxide (D~SO) or dimethylacetamide (DMA). While this 10 procedure is not inappropriate for laboratory animal ~
studies, it is clearly inadequate for human use. The ~ ~-development of aqueous formulation of E2-CDS was therefore investigated. Criteria for this formulation include that it have mini~al toxicity, that it be equivalent with E2-CDS in DMSO or DMA in delivering E2Q~ to the brain and that the technology developed be applicable to other dihydropyridine ~ pyridinium salt redox systems.
I EXPERIMENTAL SECTION
¦ 20 Materials 3-Hydroxy-17B-[(1-methyl 1,4_dihydropyrj_ din-3-yl)carbonyl]oxyestra-1,3,5(10)-triene (E2-CDS), ethyl-3-{{N-{~-[3,4-bis(pivalyloxy)phenyl]ethyl}-carbonyl}}-1,4-dihydropyridine (DA-CDS), 17B-[(1,4-dihydro-1-~ethyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one (T-CDS1), 1-~ethyl-3-N-[3-(benzyloxycarbonyl)- ~`
propyl]carba~oyl-1,4-dihydropyridine (GABA-CDS1), 1-methyl-3-~[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyridine (ACV-CDS) and 17~-~[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-4-en-20-yn-3-one (N-CDS) were synthesized according to published procedures. 2-Hydroxypropyl-~-cyclodextrin ,.
-~2-(statistlcal deyree of substitution = 5.1 or 7) (HPCD) was ~repared ~ccord~ng to the method of Pitha et al.
Other cyclodextrins (~,B or y) were obtained from Aldrich Che,nical Co. and other steroids (estradiol, estradiol 17-valerate, estriol 9 estrone, estradiol 3 methyl ether an~ testosterone 17-propionate), were l)urchased from Sigma Chemical Co. All prepared compounds were fully characterized by spectrosco~ic and microcolnbustlon analysis (Atlantic Microlabs) prior to study. Mass spectroscopic studies were performed on a Kratos ~1S~ORFA double-focus~ng instrument fitted with a fast ato~ gun. Cyclodextrin mixtures were analyzed by fast atom bom~ardl~ent of the sa~ples prepared in a ylycerol matrix. Degrees of substitution were deter-mined from the isomeric mass distribution. Nuclearmagnetic resonance spectra were obtalned on a Varian EM360 60 Mllz spectrometer. Values were recorded relative to an internal standard [3-(trimethylsilyl)-propionic 2,2,3,3-d4 acid, sodium salt, DDS] and all samples were run in ~2~. The degree of substltution was calculated by comparing the integrated area attributed to the anomeric hydrogen compared to that of the hydroxypropyl functionality.
Effect of Solubilizing Agents: An excess of E2-CDS was sonicated with an aqueous solution of the appropriate solubilizing agent for 30 minutes. The suspension was then centrifuged, filtered through 0.45 ~m polyvinyl-idene difluoride (Millex-HV4, lillipore~) membranes and analyzed by HPLC. For studies with 2-hydroxypropyl-~-cyclodextrin, an excess of E2-CDS was added to different concentrations (% w/v) of HPCD and the solu-~ ~:
13315~4 ;
bility (mg/~L) was determined spectroscopically (UV =
360 nm, = 6487 in methanol). An estimation of the bulk equilibriu~ constant was obtained by correlating the millimolarity of E2-CDS solubilized and the --millimolarity of the cyclodextrin added. This latter value was calculated using the average molecular weight -~
of the isomeric mixture determined by mass spectro-scopy. The solubilizing effect of a 50% w/w solution ;
of HPCD was also examined for a series of steroids and dihydropyridines (C~S). These studies were carried out in a similar manner to those previously described.
Preparation of Solid Complexes: An excess of E2-CDS or other CDS was added to a 50~ w/w solution of HPCD. The suspension was sonicated for 30 minutes, filtered through 0.45 ~m PVDF membranes and freeze-dried. The degree of incorporation was determined either spectro-photometrically or by HPLC. In some cases, the effect ;~
of solubilizing agents on the degree of incorporatlon was examined. This involved adding small amounts of polyoxyethylene 20 cetyl e~her (Brij), polyoxyethylenesorbitan monooleate (Tween 80) or ethanol to the aqueous solution prior to lyophilization.
. .
Analytical Methodology: In determining concentrations spectrophotometrically, a Cary 219 (Yarian) or an HP
8451~ Diode Array (Hewlett Packard) spectrophotometer was used. Standard curves were prepared in ~ethanol and gave correlation coefficients greater than 0.999.
; For the CDS, the wavelength monitored was 360 nm while for estrogen 220 nm was used.
' 133~
-~4-The HPLC system consisted of either an Autochrom M500 pump fitted with a Rheodyne injector or a Perkin~
Elmer Series 4 pump, a Kratos Spectroflow 757 variable wav~length detector and either a Beck~an recorder or an LCI-100 integrator (Perkin-Elmer). Separation was achieved on an Analytical Sciences, Inc. (ASI) 10 ~m particle size, C18 reversed phase ~0 cm x 3.9 mm i.d.
analytical column. The flow rate was 1 mL/min, the compounds were detected at 360 nm and in all determina~
tions the temperature was ambient. A mobile phase containing 82:1:1:16 (acetonitrile: tetrahydrofuran:
acetic acid: H20) eluted the E2-CDS at 4.4 min, the DA-CDS at 4.4 min, the T-CDS1 at 6.8 min and the N-CDS at 5.2 min. For the 6ABA-CDS1, a mobile phase consisting of 50:1:1:48 of the same components was required. The retention time was 5.2 min. Other compounds were assayed spectrophotometrically.
Animal Studies: Conscious, restrained Sprague-Dawley rats (female, BW = 200 9) were given either 15 mg~kg E2-CDS in DMSO or 5 mg/kg of E2-CDS complex in HPCD
(E2-CDS-HPCD) in water by intravenous injection (tail vein). At various times after the administration, animals were sacrificed and trunk blood and organs collected. The organs were then weighed, homogenized in water and deproteinized with cold acetonitrile. The organ homogenates were centrifuged and the supernatant analyzed for E2Q+ and E2-CDS using a precolumn enrichment technique, the details of which are given herelnbelrw.
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RESULTS AND DISCUSSION
As discussed hereinabove, cyclodextrins have been used to increase the ~ater solubility of a number of drugs, including steroids. These cyclic oligomers contain various numbers of -1,4-linked glucose units. The number of these units ( = six, ~ = seven, Y - eight) determine the size of a cone-like cavity which is amenable to inclusion by many drugs. The stability of the complex formed depends on the fit of the drug into the cyclodextrin and the cyclodextrin concentration. Unfortunately, the cyclodextrin best suited for complexation with steroids, i.e. B-cyclodex-trin, is poorly water-soluble. This property is derived from the high degree of hydrogen bonding which occurs in the crystal lattice. To add to the problem, B-cyclodextrin is known to cause nephrosis in rats, a toxicity which results, at least partially, from its poor water solubility. In any case, little change in the aqueous solubility of E2-CDS was observed when it was equilibrated with various solutions of either a, B
or Y-cyclodextrin. As illustrated in Table I, concentrations of a-cyclodextrin up to 50 mm increased the aqueous solubility of E2-CDS only 25-fold while B
and y-cyclodextrin increase the solubility of the CDS
135 and 110-fold respectively. The relationship between the aqueous solubility of E2-CDS and the concentration of the unsubstituted cyclodextrins was not linear, a situation which is also observed in the case of the parent steroid. In any case, the limited water solubility and the relatively poor complexation provided by a-, B- or Y-cyclodextrin are unsuitable for :
:
~3 ~ s'~?~
-~6-pharmaceutical exploitation. The toxicity of B-cyclo~
dextrin underscores this assess~ent.
TABLE I
EFFECT OF VARIOUS CYCLODEXTRINS ON THE ~ATER SOLUBILITY
.
Conc. of Cyclo- Maximum Cyclodextrin at dextrin Conc. Ranqe Solubilitv Max_. SolubilitY n (mM or %w7v) (mg/mL) (mM or %w/v) None - 0.0D02 Alpha ()5-50 mM 0.005 50 mM 7 Beta (B)5-15 mM 0.027 10 mM 5 Gamma (y)5-50 mM 0.022 10 mM 5 HPCD0.78-62.5 (7 ASDS)%w/v 30.19 62.5 gw/v 9 HPCD 1-52.5 (5.1 ASDS)~Ow/v 35.12 62.5 ~OW/v 5 Several efforts have been made to increase the aqueous solubility and, therefore, usefulness of cyclo-dextrins. Various methylated derivatives have beendescribed but, in general, the acute toxicity of the modified compound is greater than that of the parent.
Recently, an amorphous cyclodextrin composition was obtained by hydroxypropylation of ~-cyclodextrin. The product, 2-hydroxypropyl-~-cyclodextrin (HPCD), is a mixture of isomers which can be characterized by the average statistical degree of substitution (ASDS).
Either NMR or mass spectroscopy can be used to deter-mine this value. These highly water soluble mixtures . ~
,r ~
-~7-.
were shown by Pitha et al to dra~atically increase the solubility of a nu~ber of compounds including gonadal steroi~s. In addition, preli~inary toxicity studies have shown few, if any, harmful effects after either -oral or intravenous administration.
HPCD (ASDS 5.1 or 7) was prepared according to the method of Pitha et al. The mass spectra for the isomeric mixture of HPCD centered around 7 degrees of substitution. This spectra was obtained by "softly"
ionizing the sample using fast atom bombardment. The generated spectra was similar to those previously reported ~obtained by Californium-252 plasma desorp- -tion) in both the symmetry of the isomeric distribution and the nu~erical spread of the isomers formed. In the cited example, as in the 5.1 ASDS case, no underiva-tized (toxic) ~-cyclodextrin was detected.
In applying this HPCD composition to Ez-CDS, HPCD
with low ASDS's was selected. As the degree of substitution increases, not only does the complexing propensity of the cyclodextrin decrease, presumably due to steric interactions, but the surface activity of the complex increases. This is undesirable since, in general, as the surface activity increases, so does the tendency of the material to cause hemolysis. Both the 5.1 and 7 ASDS HPCD had a profound effect on the solu-bility of E2-CDS. FIG. 1 illustrates the 7 ASDS
case. In this instance, a linear increase (r = 0.995) in the solubility of E2-CDS was evident as the concen-tration of HPCD was increased. At 62.5% w/v, 30.2 mg/mL could be solubilized. In the 5.1 ASDS material, 35 mg/ml of E2-CDS could be solubilized at 62.5% w/v.
13~
-~3~-The lower ASI)S mater;~l yave a 15qO increase in incorporation. These data reflect an increase in ~ -solubility of five orders of magnitude (150,000-fold) over the solubility of E2-CUS in water (Table I). ~
5 Plotting the data obta;ned from the 7 ASDS study as ~-millimolarlty of E2-CUS solubilized versus the millimolarity of HPCD added (based on the average molecular weight of the mixture) gave a line wlth a slope of 0.2. This is an estimation of the bulk stab~lity of the cyclodextrin complex and compares reasonably with other systems.
These solutions could be freeze-dried giving a solid complex. A 50% w/w solut~on of HPCD gave a sol;d containing 37 ~g E2-CUS/gm complex. The complex was stable as a dry pow~er and could be easily reconsti-tuted with water. In these manlpulatlons, it was important to maintain the HPCD component greater than 20% w/v. Below tll~s level, precipitation would occur. Several attempts were made to increase the 20 degree of incorporation f TtMhe complex by ad~Mng ~ -various agents such as Brij (0.7% w/w), Tween 80 (0.8g w/w) or ethanol (107~ v/v). While the addition of Brij increased the degree of incorporatlon to 189 mg/g, the complex was not stable, falling to 42 mg/g in 12 days. The other agents had only modest effects. The upper limit for a stable complex, therefore, appeared to be approximately 40 mg/g under these circumstances. ;
Since an inclusion complex is formed between E2-CDS and the various components of the cyclodextrin mixture, it is possible that some portion of the E2-CDS
would not rapidly dissociate, thus lowering the biolo-:,, ~ A
gically available concentration of E2-CDS. To investi-gate this possibility, the ability of the HPCD (5.1 ASDS) formulation of E2-CDS (E2-CDS-HPCD) to deliver E2Q+ to the brain was measured and compared with the delivery of E2Q~ when E2-CDS was administered in DMSO. This data is summarized in FIG. 2, which depicts brain concentrations of E2Q~ after systemic administra-tion of either 15 mg/kg E2-CDS in DMS0 or 5 mg/kg E2-CDS in aqueous HPCD. When the difference in dose is accounted for, i.e. the data is presented as 7O dose/g, no significant difference exists between brain levels of E2Q~ after E2-CDS administration in DMSO or E2-CDS-HPCD in an aqueous media, although the latter produce data which are strikingly more consistent and less variable. Interestingly, the levels of E2Q~ in the lung are lower after E2-CDS-HPCD administration. One explanation for this is that when E2-CDS is given in a water miscible solvent such as DMSO, there may be some tendency for the highly water insoluble E2-CDS to precipitate. After a bolus i.v. injection, the aqueous, ionic environment of the lung may provide a suitable site for this precipitation. The lower levels obtained in the lung after E2-CDS-HPCD administrations reflect not only the higher water solubility of the complex but may also indicate something of its ln vivo dissociation constant. This dissociation appears to be fast enough so not as to alter the distribution of E2-CDS in the CNS, but slow enough to allow pulmonary transit without significant precipitation. In addi-tion, the values for various organ concentrations arefar less variable after E2-CDS-HPCD administration, which may be explained by the higher water solubility ~.
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1 3 ~
of the complex and its lower tendency to precipitate.
nngoing pharmacological studies corroborate the effectiveness of the E2-CDS-HPCD formulation in brain-selective delivery.
The effect of a 50% w/w solution of HPCD on the solubility of a number of steroids and other CDS is given in Table II.
TABLE II
SOLUBILIT~ OF VARIOUS STEROIDS AND VARIOUS DRUG
CHEMICAL DELIVERY SYSTEMS IN A 50S W/~ SOLUTION OF 2-HYDROXYPROPYL-~-CYCLODEXTRIN (ASDS 5.1) AND THE AMOUNT
OF DRUG INCORPORATED_IN THE FREEZE-DRIED COMPLEX
Solubility Amount of Drug (mg/mL) in in Dry Complex Drug 50% w/w HPCD (mg/g) Estradiol 25 Estriol 40 - : .
Estrone 9.52 Estradiol 3-Methyl Ether 30 - :
: Estradiol 17-Valerate 13.8 23.5 Testosterone 3~ -;, -. . .:.r~
, .
1 3 ~
-9 1 -SolubilityAmount of Drug (mg/mL) inin Dry Complex Drug 50X w/w HPCD(mg/g) --Testosterone 17-Propionate 38 65.6 Testosterone-CDS
~T-CDS1) 17.1 29.0 Norethindrone 68 -Norethindrone-CDS
(~-CDS) 0.3s 0.6 ACV-CDS 14.9 25 Thus, E2-CDS and several other CDS were success-fully solubilized with HPCD, although this is not universal; norethindrone-CDS, for example, was not readily solubilized. The best solubilization of E2-CDS
occurred in an aqueous solution of HPCD, ASDS 5.1 or 7. These complexes could be freeze-dried and were -20 stable. They were easily reconstituted in water so ~-long as the cyclodextrin co~ponent was at least 20X
w/v. Thls formulation was equivalent with E2-CDS
adm~nistered in DMS0 in delivering E2-~ to the brain ~ ~-of rats. In addition, the formulation significantly 25 reduced the lung concentrations of E2Q~. Data ava~lable at present indicates this excipient is less ~ ;
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13~
-92~
toxic, easily compressed into tahlets, rapidly dis-solved and readily and reproducibly synthesized.
E2-CDS and several other CDS have also been successfully complexed with other cyclodextrin deriva-tives as defined herein. The other cyclodextrin/CDS
complexes have the improved characteristics as noted -before for the HPCD complexes.
In one example of the for~ulation of a solid complex, an excess of a representative CDS, E2-CDS, was added to a 40% aqueous solution of a mixture of maltosyl-~-cyclodextrin (71Z) and various dimaltosyl-~-cyclodextrins (29%) obtained from Ensuiko Sugar Co., Ltd., Lot No. 8~190, and mixed for several hours. The suspension was then filtered and freeze-dried, and the amount of complexed drug was determined analytically by UV spectraphotometry. The amount of incorporation was determined to be 81.88 mg/g. The comparable figure for ~ ;
HPC~ is approximately 36.2.
In further testing, maxi~um concentrations of selected CDS in varying concentrations of selected ; cyclodextrins were determined. The concentration of E2-CDS (mg/mL) was as follows: ~
Cyclodextrin 20% 40% -HPCD 5.73 11.9 - - .`.
HEBCD 3.09 6.07 HPGCD -- 4.0 The concentration (mg/g) of the norethindrone-CDS was l~ 7.13 in 40Z HPCD and 15.9 in 40Z HPGCD.
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-As noted herein above, use of a representativedihydropyridine redox system drug, i.e. E2-CDS, complexed with a representative cyclodextrin deriva~
tive, HPCD, led to lower initial lung concentrations (and thus increased initial brain to lung concentra-tions) of the quaternary form as compared to ad~inis-tration of the redox system drug in DMSO. In studies of another representative redox system drug, namely a testosterone-CDS, T-CDS1, similar observations were made, as detailed below.
:::
EXPERIMENTAL SECTION -Materials: 2-Hydroxypropyl-~-cyclodextrin (HPCD, degree of substitution 5.1) was prepared and purified according to the method of Pitha et al. The cyclodex-15 trin inclusion complexes were prepared by equilibrating ~:
an excess of either testosterone propionate or T-CDS1 -with a 50% w/v aqueous solution of 2-hydroxypropyl-~
cyclodextrin. The solution was degassed and the ;~
suspension was sonicated for 30 minutes, after which it was filtered and the filtrate was lyophilized. The dried filtrate contained 65.6 mg testosterone propionate or 29.6 mg T-CDS1 per gram of cyclodextrin complex. Compounds were analyzed for decomposition by thin-layer chromatography and ultraviolet absorption.
: '~
:
Animals: ~1ale Sprague-Dawley rats, weighing 250-275 9, were purchased from Charles River Breeding Laboratories (Wilmington, MA) and were housed in an animal roo~
which was light (14 hours; lights on at 0500 hours) and temperature (23 i 1 C) controlled. To elevate serum luteinizing hormone (LH) and to reduce the source of endogenous testosterone, animals were bilaterally orchidectomized via a mid-ventral incision under light ether anesthesia. All experiments were initiated 2 IO weeks after orchidectomy.
Experiment 1: On day 15 after orchidectomy, rats were ether-anesthetized and the right external jugular vein exposed. Animals were then administered one of the following: testosterone-chemical delivery system (T-CDSl or T-CDS2), testosterone (Steraloids Inc., Wilton, NH) or the vehicle, dimethyl sulfoxide (DMSO; Fisher Scientific, Fair Lawn, NJ). The testosterone-chemical delivery systems were given at doses equimolar to testosterone (25 mg/kg) so that T-CDSl was administered at 35.5 mg/kg and rats received T-CDS2 at a dose of 45.1 mg/kg. DMSO was injected at a volume of 1 mL/kg. All compounds were administered by infusion over a 2 minute period. One milliliter of blood was withdrawn from the external jugular vein immediately before giving the drugs (1000 hours) and blood was sampled by cardiac puncture after 6, 12, 24 hours and on days 4 and 7. The sera were separated by centrifu-gation at 500 x 9 for 20 min at 4C and stored at -20C.
1 3 ~ ~ ri ~1 ~
Experiment 2: Two weeks after orchidectomy, rats were administered either T-CDS1, testosterone propionate (TP; Steraloids Inc.) or DMSO by ~eans o~ intravenous infusion into the right external jugular vein in an effort to more effectively enhance the brain-delivery of testosterone. It has been shown that slow infusion improves brain delivery of drugs attached to the che~ical-delivery systems. TP was selected for com-parison since it, like both of the T-CDS compounds, has an ester grouping (propionate) attached at carbon-17 (C17). Gonadally-intact animals received the drug vehicle only. Two Harvard Apparatus reciprocal infusion/withdrawal pumps (model 944) were used so that 4 animals could be simultaneously infusedO Rate of infusion was 15 ~L/min and animals were infused for 17 to 25 minutes. TP was given at 25 mg/kg and T-CDS was infused at a dose equimolar to TP (29.7 mg T-CDS1 per kg body weight). The drug vehicle, DMSO, was adminis- ;~
tered at a dose of 1 mL/kg. One mL of blood was removed from the external jugular vein prior to drug infusion and from the sub-orbital sinus at 1, 3, 5, and 7 days. The sera were separated and stored as pre-viously described.
.
Experiment 3: Orchidectomized rats were administered either testosterone-chemical delivery system (T-CDS1) in HPCD (T-CDS1-HPCD), testosterone propionate in cyclodextrin (TP-HPCD) or the vehicle, cyclodextrin (HPCD), via a single tail vein injection. T-CDS1-HPCD
(11.9 mg/kg) was given so that animals received T-CDS
at a dose equimolar to TP-HPCD (10 mg TP/kg body weight). Control rats received 25% HPCD (w/v) at 3.0 mL/kg. 8lood was removed by cardiac puncture on days 0, 1, 3, 5 and 7, and separated and stored as pre- ~
viously described. -To evaluate peripheral effects of the drugs, the right seminal vesicle, vas deferens and ventral pros-tate gland were removed, cleaned, expressed of fluid and weighed to 0.1 mg. Data are reported as mg per 100 g body weight.
Radioim~unoassay of LH: Serum LH concentrations were determined in duplicate in our laboratory with a radio-immunoassay kit (reference preparation LH-RP-2) pro vided through the Pituitary Hor~one Distribution Program of the NIADDK. The intra- and interassay coefficients of variation were 2.9 and 15.6, respec~
tively.
Radioimmunoassay of Testosterone: Serum testosterone concentrations were determined in duplicate with a Coat-A-Count radioimmunoassay kit (Diagnostic Products;
Los Angeles, CA).
Statistical treatment: The significance of difference among mean values for LH and peripheral tissues was determined by analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) tests. The level of significance for both tests was 0.05.
RESULTS AND DISCUSSION
In Experiments 1 and 2, in which DMSO served as the drug vehicle, indications of drug insolubility upon 1331~
injecti on were observed, i.e. respiratory distress accompanied by lesioning of the lungs, regardless of the rate of injection or i~fusion. In an effort to increase water solubility of the steroids, T-CDS1 and TP were solubilized in a HPCD in Experiment 3. The improvement in solubility for the T-CDS1 suggests that a lower dose (10 mg/kg vs. 25 mg/kg) could be administered with, presu~ably, a diminished risk of toxicity to the animal. A 2.5-fold decrease in T-CDS
dosage resulted in a similar suppression of serum LH
levels observed in the previous two experiments. An injection of T-CDS1-HPCD resulted in a 50% decrease in serum LH by 24 hours and this suppression was observed -~-through 3 days. Suppression of LH occurred in animals treated with TP-HPCD at day 1 only.
Mild stimulation of the seminal vesicles by T-CDS1-HPCD and of the ventral prostate gland by T-CDS1- ~
HPCD and TP-HPCD was observed at 7 days post- -injection. As observed previously, the extent of stimulation by T-CDS1-HPCD or TP-HPCD was minor relative to tissue weights observed in control (gonadally-intact) rats.
A 5.5-fold increase in serum testosterone was observed 1 day after rats were administered T-CDS1-HPCD
and serum testosterone remained elevated at day 3.
However, testosterone levels returned to pre-injection levels 5 days after injection. At no time did TP-HPCD
or HPCD induce an increase in serum testosterone.
These experiments offer support for the improved delivery of testosterone to the brain when the repre-sentative redox carrier drug, T-CDS1, is complexed to .
;
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.
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9~
the representative cyclodextrin, HPC~. Our data show an equivalent suppression of LH by complexing T-C~S1 to ~PCD and lowering the effective single dose of T-CDS
by 2.5-fold. This finding implies that the dihydro-pyridine form of T-CDSl remains in solution in an aqueous medium (e.g. blood) for a longer time, thereby permitting improved passage of the drug through the blood-brain barrier. Earlier studies revealed that, when administered in a DMSO vehicle, T-CDSl probably precipitated in the blood (and lungs), causing respira-tory distress and/or death in rats. No respiratory distress or animal loss occurred when T-CDSl was complexed with HPCD.
To quantitate the improvement provided by the representative cyclodextrin, HPCD, in lowering initial lung concentrations of redox carrier compounds compared to brain concentrations, another series of experiments was undertaken investigating the HPCD complex of E2-CDS. These studies, which are detailed below, utilize a reversed-phase-high-performance liquid chromato-graphic method for the analysis of E2-CDS and its oxidized quaternary metabolite E2-Quat in biological fluids or tissues. The assay utilizes a precolumn enrichment technique and detects plasma levels down to
~T-CDS1) 17.1 29.0 Norethindrone 68 -Norethindrone-CDS
(~-CDS) 0.3s 0.6 ACV-CDS 14.9 25 Thus, E2-CDS and several other CDS were success-fully solubilized with HPCD, although this is not universal; norethindrone-CDS, for example, was not readily solubilized. The best solubilization of E2-CDS
occurred in an aqueous solution of HPCD, ASDS 5.1 or 7. These complexes could be freeze-dried and were -20 stable. They were easily reconstituted in water so ~-long as the cyclodextrin co~ponent was at least 20X
w/v. Thls formulation was equivalent with E2-CDS
adm~nistered in DMS0 in delivering E2-~ to the brain ~ ~-of rats. In addition, the formulation significantly 25 reduced the lung concentrations of E2Q~. Data ava~lable at present indicates this excipient is less ~ ;
~' ~
,.~ . . . ... .. . .
13~
-92~
toxic, easily compressed into tahlets, rapidly dis-solved and readily and reproducibly synthesized.
E2-CDS and several other CDS have also been successfully complexed with other cyclodextrin deriva-tives as defined herein. The other cyclodextrin/CDS
complexes have the improved characteristics as noted -before for the HPCD complexes.
In one example of the for~ulation of a solid complex, an excess of a representative CDS, E2-CDS, was added to a 40% aqueous solution of a mixture of maltosyl-~-cyclodextrin (71Z) and various dimaltosyl-~-cyclodextrins (29%) obtained from Ensuiko Sugar Co., Ltd., Lot No. 8~190, and mixed for several hours. The suspension was then filtered and freeze-dried, and the amount of complexed drug was determined analytically by UV spectraphotometry. The amount of incorporation was determined to be 81.88 mg/g. The comparable figure for ~ ;
HPC~ is approximately 36.2.
In further testing, maxi~um concentrations of selected CDS in varying concentrations of selected ; cyclodextrins were determined. The concentration of E2-CDS (mg/mL) was as follows: ~
Cyclodextrin 20% 40% -HPCD 5.73 11.9 - - .`.
HEBCD 3.09 6.07 HPGCD -- 4.0 The concentration (mg/g) of the norethindrone-CDS was l~ 7.13 in 40Z HPCD and 15.9 in 40Z HPGCD.
! ~ fi~
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,. -.
~" ~,,",, 1.. ~ "~ "~ ,.. "'- " ," ~,, "
~ 3 3 ~
-93- ~;
:"
-As noted herein above, use of a representativedihydropyridine redox system drug, i.e. E2-CDS, complexed with a representative cyclodextrin deriva~
tive, HPCD, led to lower initial lung concentrations (and thus increased initial brain to lung concentra-tions) of the quaternary form as compared to ad~inis-tration of the redox system drug in DMSO. In studies of another representative redox system drug, namely a testosterone-CDS, T-CDS1, similar observations were made, as detailed below.
:::
EXPERIMENTAL SECTION -Materials: 2-Hydroxypropyl-~-cyclodextrin (HPCD, degree of substitution 5.1) was prepared and purified according to the method of Pitha et al. The cyclodex-15 trin inclusion complexes were prepared by equilibrating ~:
an excess of either testosterone propionate or T-CDS1 -with a 50% w/v aqueous solution of 2-hydroxypropyl-~
cyclodextrin. The solution was degassed and the ;~
suspension was sonicated for 30 minutes, after which it was filtered and the filtrate was lyophilized. The dried filtrate contained 65.6 mg testosterone propionate or 29.6 mg T-CDS1 per gram of cyclodextrin complex. Compounds were analyzed for decomposition by thin-layer chromatography and ultraviolet absorption.
: '~
:
Animals: ~1ale Sprague-Dawley rats, weighing 250-275 9, were purchased from Charles River Breeding Laboratories (Wilmington, MA) and were housed in an animal roo~
which was light (14 hours; lights on at 0500 hours) and temperature (23 i 1 C) controlled. To elevate serum luteinizing hormone (LH) and to reduce the source of endogenous testosterone, animals were bilaterally orchidectomized via a mid-ventral incision under light ether anesthesia. All experiments were initiated 2 IO weeks after orchidectomy.
Experiment 1: On day 15 after orchidectomy, rats were ether-anesthetized and the right external jugular vein exposed. Animals were then administered one of the following: testosterone-chemical delivery system (T-CDSl or T-CDS2), testosterone (Steraloids Inc., Wilton, NH) or the vehicle, dimethyl sulfoxide (DMSO; Fisher Scientific, Fair Lawn, NJ). The testosterone-chemical delivery systems were given at doses equimolar to testosterone (25 mg/kg) so that T-CDSl was administered at 35.5 mg/kg and rats received T-CDS2 at a dose of 45.1 mg/kg. DMSO was injected at a volume of 1 mL/kg. All compounds were administered by infusion over a 2 minute period. One milliliter of blood was withdrawn from the external jugular vein immediately before giving the drugs (1000 hours) and blood was sampled by cardiac puncture after 6, 12, 24 hours and on days 4 and 7. The sera were separated by centrifu-gation at 500 x 9 for 20 min at 4C and stored at -20C.
1 3 ~ ~ ri ~1 ~
Experiment 2: Two weeks after orchidectomy, rats were administered either T-CDS1, testosterone propionate (TP; Steraloids Inc.) or DMSO by ~eans o~ intravenous infusion into the right external jugular vein in an effort to more effectively enhance the brain-delivery of testosterone. It has been shown that slow infusion improves brain delivery of drugs attached to the che~ical-delivery systems. TP was selected for com-parison since it, like both of the T-CDS compounds, has an ester grouping (propionate) attached at carbon-17 (C17). Gonadally-intact animals received the drug vehicle only. Two Harvard Apparatus reciprocal infusion/withdrawal pumps (model 944) were used so that 4 animals could be simultaneously infusedO Rate of infusion was 15 ~L/min and animals were infused for 17 to 25 minutes. TP was given at 25 mg/kg and T-CDS was infused at a dose equimolar to TP (29.7 mg T-CDS1 per kg body weight). The drug vehicle, DMSO, was adminis- ;~
tered at a dose of 1 mL/kg. One mL of blood was removed from the external jugular vein prior to drug infusion and from the sub-orbital sinus at 1, 3, 5, and 7 days. The sera were separated and stored as pre-viously described.
.
Experiment 3: Orchidectomized rats were administered either testosterone-chemical delivery system (T-CDS1) in HPCD (T-CDS1-HPCD), testosterone propionate in cyclodextrin (TP-HPCD) or the vehicle, cyclodextrin (HPCD), via a single tail vein injection. T-CDS1-HPCD
(11.9 mg/kg) was given so that animals received T-CDS
at a dose equimolar to TP-HPCD (10 mg TP/kg body weight). Control rats received 25% HPCD (w/v) at 3.0 mL/kg. 8lood was removed by cardiac puncture on days 0, 1, 3, 5 and 7, and separated and stored as pre- ~
viously described. -To evaluate peripheral effects of the drugs, the right seminal vesicle, vas deferens and ventral pros-tate gland were removed, cleaned, expressed of fluid and weighed to 0.1 mg. Data are reported as mg per 100 g body weight.
Radioim~unoassay of LH: Serum LH concentrations were determined in duplicate in our laboratory with a radio-immunoassay kit (reference preparation LH-RP-2) pro vided through the Pituitary Hor~one Distribution Program of the NIADDK. The intra- and interassay coefficients of variation were 2.9 and 15.6, respec~
tively.
Radioimmunoassay of Testosterone: Serum testosterone concentrations were determined in duplicate with a Coat-A-Count radioimmunoassay kit (Diagnostic Products;
Los Angeles, CA).
Statistical treatment: The significance of difference among mean values for LH and peripheral tissues was determined by analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) tests. The level of significance for both tests was 0.05.
RESULTS AND DISCUSSION
In Experiments 1 and 2, in which DMSO served as the drug vehicle, indications of drug insolubility upon 1331~
injecti on were observed, i.e. respiratory distress accompanied by lesioning of the lungs, regardless of the rate of injection or i~fusion. In an effort to increase water solubility of the steroids, T-CDS1 and TP were solubilized in a HPCD in Experiment 3. The improvement in solubility for the T-CDS1 suggests that a lower dose (10 mg/kg vs. 25 mg/kg) could be administered with, presu~ably, a diminished risk of toxicity to the animal. A 2.5-fold decrease in T-CDS
dosage resulted in a similar suppression of serum LH
levels observed in the previous two experiments. An injection of T-CDS1-HPCD resulted in a 50% decrease in serum LH by 24 hours and this suppression was observed -~-through 3 days. Suppression of LH occurred in animals treated with TP-HPCD at day 1 only.
Mild stimulation of the seminal vesicles by T-CDS1-HPCD and of the ventral prostate gland by T-CDS1- ~
HPCD and TP-HPCD was observed at 7 days post- -injection. As observed previously, the extent of stimulation by T-CDS1-HPCD or TP-HPCD was minor relative to tissue weights observed in control (gonadally-intact) rats.
A 5.5-fold increase in serum testosterone was observed 1 day after rats were administered T-CDS1-HPCD
and serum testosterone remained elevated at day 3.
However, testosterone levels returned to pre-injection levels 5 days after injection. At no time did TP-HPCD
or HPCD induce an increase in serum testosterone.
These experiments offer support for the improved delivery of testosterone to the brain when the repre-sentative redox carrier drug, T-CDS1, is complexed to .
;
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.
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9~
the representative cyclodextrin, HPC~. Our data show an equivalent suppression of LH by complexing T-C~S1 to ~PCD and lowering the effective single dose of T-CDS
by 2.5-fold. This finding implies that the dihydro-pyridine form of T-CDSl remains in solution in an aqueous medium (e.g. blood) for a longer time, thereby permitting improved passage of the drug through the blood-brain barrier. Earlier studies revealed that, when administered in a DMSO vehicle, T-CDSl probably precipitated in the blood (and lungs), causing respira-tory distress and/or death in rats. No respiratory distress or animal loss occurred when T-CDSl was complexed with HPCD.
To quantitate the improvement provided by the representative cyclodextrin, HPCD, in lowering initial lung concentrations of redox carrier compounds compared to brain concentrations, another series of experiments was undertaken investigating the HPCD complex of E2-CDS. These studies, which are detailed below, utilize a reversed-phase-high-performance liquid chromato-graphic method for the analysis of E2-CDS and its oxidized quaternary metabolite E2-Quat in biological fluids or tissues. The assay utilizes a precolumn enrichment technique and detects plasma levels down to
10 ng/mL E2-Quat and 20 ng/mL E2-CDS. Sample preparation is rapid and simple. Samples are homo-genized with acetonitrile, centrifuged, and the super-natant is directly injected into the HPLC-system. A
water-delivering pump injects the sample on a pre-column where the drug is concentrated. Mobile phase backflushes the retained compound onto the analytical ~;
column. At the same time, another sample can be r~
injected onto a second pre-colu~n. This alternating pre-column sample enrich~ent technique allows the injection of large volu~es up to 1800 ~L.
EXPERIMENTAL SECTION
Materials: E2-CDS, E2-Quat and E2-CDS-HPCD were synthesized as described previously. Steroids (estradiol and ethinyl estradiol) were obtained from Sigma Chemical Co. HPLC grade acetonitrile and dis-tilled, deionized water were used for the preparation of mobile phases. All other reagents used were of analytical grade. ~
: ~. ' Instrumentation: The HPLC system consisted of a LDC/Milton Roy Constametric III high-pressure pump, a LDC/Milton Roy variable wavelength UV detector, a Perkin Elmer ISS-100 automatic ,injector equipped with a 2000 ~L loop and a DuPont Zorb`ax ODS column, 15 cm x 4.6 mm I.D. (6 ~m particle size). Vydac guard columns (5 cm x 3.2 mm I.D.~, dry-packed with DuPont Zorbax ODS
material, were used. Chromatograms were recorded on a Hewlett-Packard Model 3390A computing integrator at a chart speed of 0.2 cm/min. In addition, in the pre-column enrichment system, an enrichment injector (Rheodyne Model 7067-005) with two high pressure switching valves, pneumatically turned by a tandem actuator (Rheodyne Model 7163), was inserted between the autoinjector and the analytical column. Switching of the valves was controlled via the autoinjector.
This system also contained a Bodine Electric Co. RR/035 HPLC Solvent Pump for flushing the samples onto the enrichment columns.
~ethods: ~-Assay Conditions: direct on-line HPLC
Chromatographic conditions for the analysis of E2- ;
CDS, E2-Quat and estradiol were developed. The optimal wavelength for all compounds was 224 nm, but E2-CDS can also be detected at 360 nm due to t,he dihydropyridine structure. Although the absorptivity at this wave-length is only about half as high as it is at 224 nm, 360 nm was chosen as the analytical wavelength for E2CDS because of the increased selectivity. Different analytical columns were tested and mobile phases for a reversed phase chromatography of all three compounds were varied widely with respect to the ratio of aqueous and organic phase as well as buffer concentration and pH. No isocratic system could be found that would detect all three compounds within a reasonable reten-tion time and with satisfying compactness and separa-tion of peaks. Therefore, two different syste~s wereused for analysis.
E2-Quat and E2: The optimal mobile phase was found to consist of acetonitrile/water 40:60 containing 0.03 M/L sodium salt of octanesulfonic acid and 0.003 M/L tetrabutylammonium phosphate. The pH was adjusted to pH 5-5.5. The flow rate was 1.5 mL/minute and the ~ ~ -peaks were recorded at 224 nm.
E2-CDS: The mobile phase used for E2-CDS analysis was acetonitrile/water 70:30 at a flow rate of 1.5 mL/minute. Absorbance was monitored at 360 nm.
~ ,," ,, ~ "
Analysis of E2, E~-CDS and Ej-Quat by pre-column enrichment technique:
- . .
The loss of sensitivity resulting from the -~
dilution step in the procedure optimal for pre-5 treatment of biological samples ~see sample preparation ~ -without extraction) could be compensated for by developing an HPLC system that allows injection of large volumes. A suitable HPLC-method which has been described in the literature ~Roth et al~ J. Chromatogr.
222: 13-22 (1981)] is based on alternating pre-column sample enrichment. The procedure used herein was as follows: The sample containing the drug is injected with a first pump A, delivering pure water, onto one of two pre-columns, which are alternatingly connected with the injection system by two pneumatically driven valves. Provided a certain lipophilicity, the drug is retained and concentrated on the pre-column, while accompanying water soluhle co-products like proteins are being washed out as long as water is pumped through the pre-column. This allows the direct injection of body fluids. After a certain enrichment time (6 and 8 minutes), simultaneous rotation of the two valves is ;
induced, causing pre-column 1, where the injected drug has been absorbed, to be switched to the solvent stream of the second pump, B. Also, at this point, the recording integrator is started. Pump B delivers the mobile phase, necessary for separation and chromatography, and backflushes the sample from pre-column 1 onto the analytical column. Parallel to this process, pre-column 2 is switched to the water stream of pump A so that a sample can be injected and enriched 3 ~
f~
:
while the previous one is ~eing eluted (alternating mode). Volumes up to 13~ ~L can be injected due to .. ;:P.iYr.
the concentration effect of the enrichment phase.
Chro~atographic conditions: This system was applicable to the quantification of E2, E2-CDS and E2-Quat. The mobile phase for E2-CDS was: Acetoni- -trile/water 80:20 at a flow rate of 1.8 mL/minute.
Optimal peak shape and retention time for E2 and E2-Quat were obtained with pump ~ delivering a mixture of acetonitrile/water 42:58 which contained 0.025 M/L
sodium salt of 1-octanesulfonic acid and ~.003 M/L
tetrabutylammonium phosphate. The pH was adjusted to -pH 5, and the flow rate was 1.5 mL/minute.
Standard solutions and stability Sample stock solutions of E2-CDS, E2-Quat, E2 and ethinyl-E2 containing each 50 ~g/mL were prepared in acetonitrile. All solutions were stored at 6C. For E2-CDS, the stock solution was prepared freshly every 2 ;-weeks. All other solutions were stable over a period --of at least six months. Spiked plasma samples con-taining all four compounds were frozen at -20C and analyzed repeatedly at different time intervals. No loss of drug was found under these storage conditions during two months.
Dihydropyridine derivatives like E2-CDS are known ~ ~-to be easily oxidized and very labile in acidic solu-tions. The stability of E2-CDS was investigated under different conditions at room temperature. These studies were performed by diluting the E2-CDS stock solution 1:2 with different solvents or solutions at -- ~ :
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~. : . : . ~ - . : . .
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-1~3-different pH values and monitoring eventual peak height loss for 24 hours by use of a modification of the direct on-line H~LC method described above: If water in the E2-CDS mobile phase is replaced by 0.05 M
phosphate buffer at pH 7 and if the detection wave-length is set to 224 nm, E2-Quat can be detected simul-taneously at 6.33 min. However, the peak is relatively broad. These conditions were used to determine the degree of E2-CDS oxidation under the tested conditions.
Sa~ple preparation Extraction of E2-Quat and E2: Various extraction procedures from plasma were investigated under dif-ferent conditions and with several solvents and solvent mixtures. Estrone could be used as an internal standard, but is known to be a potential metabolite of estradiol. Therefore, 17-~-ethinyl estradiol was ;
chosen as internal standard. Its peak did not inter-fere with E2, E2-Quat or estrone. Without addition of an anion reagent, E2-Quat could not be extracted from 20 aqueous solutions. Optimal results were obtained after -~
a single-step extraction of the drugs with potassium iodide as an ion-pairing reagent to facilitate quaternary salt extraction. The method applied was as follows: 200 ~L of a saturated potassium iodide solution were added to 1 mL of spiked plasma. After vortexing for a few seconds, 10 mL of a mixture of chloroform/ethyl acetate 9:1 was added. The tubes were shaken for 10 minutes and then centrifuged for 10 minutes at 2000 rpm. The upper aqueous phase was discarded and the organic layer transferred to a clean tube to achieve complete separation from proteins and -' .
, ; ..
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~ 33~ ~$
traces of aqueous phase. The organic layer was eva-porated to dryness under nitrogen at 40C and recon-stituted in 150 ~L of mobile phase. 40 ~L were injected into the HPLC system. Appropriate blanks were prepared accordingly.
Extraction of E2-CDS: Plasma and water containing E2-CDS were repeatedly extracted with different organic solvents like chloroform, hexane, toluene, benzene and ethyl acetate. It was impossible to extract E2-CDS
reproducibly from aqueous phases, since the compound was shown to deteriorate unreproducibly during evapora-tion, even at room temperature and in the presence of oxygen-free nitrogen. Therefore, the compound had to be analyzed from biological fluids without an extrac-tion procedure.
Preparation of plas~a and tissues for analysis ofE2-CDS and E2-Quat without extraction: Using the HPLC
technique with pre-column enrichment described above, drugs can be detected from directlv injected plasma without sa~ple preparation. However, when large volumes are injected, in order to obtain maximum sensi-tivity, it is desirable to remove proteins to a large extent prior to injection in order to prevent frequent pre-column packing. The procedure of deproteinization was chosen to be applicable for subsequent analysis of both E2-CDS and E2-Quat so that only one preparation step had to be performed. -~
Acidic precipitating agents, which remove proteins when only small volumes are added to biological fluids, could not be used because they induce degradative loss of E2-CDS. Neutral or slightly basic aqueous reagents ' , 7;' ' 1 3 3 ~
used efficiently for deproteinization like ZnS04/NaOH, CuS04/Na2S04 or saturated (NH4)2S04 would be more ideal to be injected onto the enrichment columns than organic solvents. But all of these reagents were shown to absorb the water-insoluble E2-CDS on the precipitate.
Thus, the method of choice to avoid instability prob-lems and at the same time keep all compounds in solu-tion was deproteinization with acetonitrile.
To obtain these results, the following sample ~-preparation procedures were used for plasma and tissues: Plasma: 0.6 mL plasma was added to 1.2 mL ;-acetonitrile. The mixture was vortexed for 5 seconds and allowed to stand for 10 minutes at room tempera-ture, vortexed again and centrifuged for 10 minutes at 15 2000 rpm. 1000-1500 ~L of the supernatant was injected into the pre-column enrichment system. Tissue (e.g.
brain): 1 mL of water was added to one rat brain and the organ was thorcughly meshed. After sonication for 2 minutes and centrifugation at 2000 rpm for 10 20 minutes, the supernatant (1000-1500 ~L) was injected into the enrichment system.
Animal Stud~es~
~ ,, In a first experiment, 15 mg/kg E2-CDS dissolved in dimethylsulfoxide (DMSO) were administered intra-venously to conscious, restrained male Sprague-Dawley rats weighing 190-300 9 each. Animals were sacrificed in groups of 4 at 5, 15 and 30 minutes and at 1, 2, 4, 8, 24 and 48 hours after drug injection. Trunk blood ~-~
was collected into heparinized tubes and plasma obtained and immediately frozen at -20C until analy-;~ sis. Organs were dissected and placed on dry ice ~' :
:~.. ' `' - ~' : ' ' '~ :
~331~
-1~6-within 2 minutes of death and stored at -20C for later analysis by the HPLC method described above.
~ n a second experiment, the same procedure as above was followed, except that 5 mg/kg of E2-CDS were administered as a complex with hydroxypropyl-~-cyclo-dextrin (HPCD) in water.
Results and Discussion The results are depicted in FIG. 3 and FlG. 4.
FIG. 3 consists of a pair of semi-logarithmic plots comparing the concentrations in lung tissue in ~9 per 9 dose (CB/D) of E2-CDS in the first graph and of E2-Quat in the second, corrected for dose. It can be seen from FIG. 3 that when E2-CDS was administered in DMS0, -~
initial lung concentrations (i.e. concentrations within the first hour after drug injection) of both E2-CDS and E2-Quat were significantly higher than the initial lung concentrations observed when E2-CDS was administered as a complex with HPCD in water. The corresponding levels of E2-Quat in brain tissue, also corrected for dose, are given in FIG. 4 in the form of a bar graph depict-ing the brain levels in ng per g dose (CB/D) at selected time points. It can be seen that the brain levels after 1 hour are not significantly different for administration as HPCD complex in water as compared to administration in DMS0. Clearly, then, the carrier~
drug can be administered as a complex with a selected cyclodextrin as defined herein, such as HPCD, in water and still achieve the brain levels needed to produce the desired biological effect, while avoiding the high ,~ ~
133~
-1~7-initial lung concentrations responsible for respiratory distress and dysnia.
Complexation with 2-hydroxypropyl-~-cyclodextrin (HPCD) or other selected cyclodextrin derivative as defined herein has been found to be particularly advantageous in that it stabilizes the dihydropyridine redox syste~s. A direct comparison of stabilities in aqueous solution is, of course, not possible because of the low solubility of the dihydropyridine redox system drugs in water; for example, the solubility of E2-CDS
in water is only 0.0002 mg/mL. The E2-CDS-HPCD complex contains about 40 mg of E2-CDS/g and easily gives aqueous solutions containing 5 mg E2-CDS/mL at 20% w/v -~
cyclodextrin. Thus, complexation affords a 25,000-fold increase in aqueous solubility of E2-CDS. The half-life of E2-CDS in such a solution at room temperature in the dark is about 12.5 days (rate: 0.0554 n .0047d~
Since the dihydropyridine redox system drugs are especially prone to oxidative degradation, a study was undertaken to quantitate the effect of a cyclodextrin derivative intended for use herein, e.g. HPCD, on the rate of oxidation of these drugs. A representative carrier-drug, E2-CDS, was selected for this study.
The rate of ferricyanide-mediated oxidation of E2-CDS was determined using a previously published method (Okamoto et al, J. Chem. Soc. Chem. Comm., 1977, 1i31). In this procedure, 27.5 ~L of a 5 x 10-3 M
solution of E2-CDS in acetonitrile was added to 2.75 mL
of a solution containing 1 x 10-4 M Fe(CN)6~4, 0.06 M
K+, O.001 M Fe(CN)6~3. All solutions were made using ~ :
1 3 ~
- 1 o ~
water whicll ha~ been boile~ for 30 m~nutes and cooled while a strealn of pyrogallol-scrubbed nitrogen passed through it. The E2-C~S was introduced via a syringe to -~
the solution which was maintained a~ 37C in a thermo~
stated cell holder and contained in aTM anaerobic screw-top cuvette. The cuvette had a Teflon-lined septum -~
through wh1ch the compound was injected. For a given --concentration of ferr~cyanide ions (6 x 10-4 to 8 x 10-3 M), the rate of d~sappearance of the ~
I0 E2-CDS was determined. This was done by calculating ~- -the decrease in the absorbance band at 360 nm (i I0 nm) subtracted fro~ base 11ne absorbance (S00 ~ 10 nm). A
plot of the ln ~Abs] versus time gave a slope for the pseudo-first-order rate constant. This was done at several dlfferent ferricyanide ion concentrat~ons. The obta~ned first-order rate constants were then plotted ;~
as a function of ferricyanide ion concentration generating a slope from which the second order rate constant (ko s~I M-I) was obta1ned. In examlning the effect of 2-hydroxypropyl-~-cyclodextrin on the rate of E2-CDS oxidation, solutions containing the HPCD as well as those ions present in the first phase of the experiment were prepared. The second order constant was derived for each cyclodextrin concentration and a plot developed. The results, depicted in FIG. 5, show tha-t the cyclodextrin dramatically slowed the rate of oxidation. There appears to be a saturation effect in that after 2% w/v, not much change in rate is evident. The second order rate of oxidation is inhibited by 42% at 0.5% w/v cyclodextrin, 60% at 1.0%
` w/v, 81% at 2% cyclodextrin and at 5-20% a value of approximately 90% reduction in the rate was obtained.
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The following Examples illustrate the preparation of preferred reduced, dihydropyridine . pyridinium salt ~.
redox carrier systems for brain-targeted drug delivery which are contemplated for formation of cyclodextrin (e.g. HPCD) complexes in accord with this invention and which have not been specifically described in :~
publications to date.
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1 1 o -EXAMPLE 1 ~ -Preparation of N-~icotinoyldopamine~
To a pyridine solution containing 11.7 9 (0.05 ~ -mol) dopamine hydrobromide and 6.15 9 (0.05 mol) --~
nicotinic acid at 0C were added 10.3 9 (0.05 mol) -~
dicyclohexylcarbodiimide (DCC). The reaction mixture was stirred at room temperature for 24 hours and the formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo and the residue was trystallized from water at 0C. The product was isolated by filtration and dried over phosphorous pentoxide. Recrystallization from isopropanol gave 9.0 -~
9 (0.035 mol), 70X N-nicotinoyldopamine, m.p. 159- ~ -162C; aqueous solution of the compound gave a green -color with Fe~3 and reduced AgN03; IR (KBr) 3300, 2960, 1725, 1630, 1590, 1520, 1430, 1290, 1190, 1115, 720 and 710 cm~1; NMR (d6-DMS0) ~ 9.25-6.25 (m, 7H), 3.3 (m, 2H) and 2.65 (m, 2H) ppm. Anal. (C14H14N203) C, H, N-Preparation of 1-Methyl-3-¦N-[B-(3,4-dihydroxyphenyl)-ethyl]~carbamoylpyridinium iodide:
To a solution of 2 9 (7.7 mmol) of N-nicotinoyl-dopamine ~n 40 mL of dry methanol were added 2.5 9 (17.6 mmol) of methyl iodide. The reaction mixture was refluxed with stirring for 6 hours. Methyl iodide (1.5 9, 1.05 mmol) was added and refluxing was continued overnight. Methanol was removed and ethyl acetate was added, affording yellowish crystals of the desired -~
product. Yield 2.4 9 (77X), m.p. 173-174C.
-''~`' ~ ' , 1 3 3 1 ~ 6 4 ~ -Preparation of l-Methyl-3-~N-t[B-t3,4-bis(isobutyryl-oxy)phenyl]ethyl]]~carbamoylpyridinium trifluoroace-tate-~. ~
To an ice-cold solution of the product of Example 2 (3 9. 7.5 mmol) in 30 mL of trifluoroacetic acid, isobutyryl chloride (2.4 9, 22.5 mmol) was added ;~
slowly, with stirring. Stirring was continued over-night at room temperature. Trifluoroacetic acid was evaporated under vacuum and the residue was crystal-lized from ethyl ether:hexane (3:1). Yield 1.2 9 ~
(30.4X), m.p. 87-91C. ;
EXA~PLE 4 .
Preparation of 1-Methyl-3-~N-[tB-[3,4-bis(isobutyryl- ~
15 oxy)phenyl]ethyl]~carbamoyl-1,4-dihydropyridine: ~;
A solution of O.SS 9 (1 mmol) of 1-methyl-3-{N-[tB-[3,4-bis(isobutyryloxy)phenyl]ethyl]]~carbamoyl-pyridinium trifluoroacetate in S0 mL of deaerated water containing 10 mL of methanol was extracted three times with 3n mL portions of ether. To the resultant aqueous solution were added NaHC03 (0.25 9, 3 mmol) and 50 mL
of ethyl ether and the mixture was kept under ~ -;
nitrogen. To this ice-cold mixture was added sodium dithionite (0.52 9, 3 mmol) and the mixture was stirred vigorously for 30 minutes. The ether layer was sepa-rated and the aqueous layer was extracted twice with ether. The combined ether extracts were washed with ~ . .:
water and dried over sodium sulfate. Ether was removed under vacuum, leaving an oily product. NMR analysis ~ ~ -30 confirmed that the product had the structural formula: ~
~" .
~.
~' ..
~ ~,, ;~
~ I '; ' ~ ': . ' . ~ ' . ' . ' ' ~, .,,, ' ' : : ' . ~ : : , . , - 1 3 3 1 5 ~ 4 ~NHCH2CH2~o~c (c 3) Preparation of 5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidinedione: -Phenytoin (5 9, 0.02 mol) was suspended in 180 mL
of water; 20 mL of formaldehyde (37X solution) and 0.25 g K2C03 were added and the mixture was stirred at 25-30C for 24 hours. The white solid which formed was removed by filtration and washed repeatedly with a 3X
10 solution of formaldehyde, then air-dried for 3 to 4 hours and over P205 in a vacuum dessicator. Yield 91-93X, m.p. 185-189C. Anal. calc. for C16H14N203: C, 68.07; H, 5.00; N, 9.93. Found: C, 67.97; H, 5.05; N, 9.93. The product had the formula:
N
@~
H20H -:
.~
; ~ ;
: . i ,~ ~`; ::, .,~., . :,: --113- 1331~4 Preparation of 5,5-Diphenyl-3-[(3'-pyridyl)carbonyloxy-methyl]-2,4-imidazolidinedione:
The product of Example 5 (3.00 9, 0.011 mol) was dissolved in 150 mL of dry pyridine, then nicotinic anhydride (4.25 9, 0.019 mol) was added. The resultant solution was stirred at room temperature (25-30C), under dry conditions, for 40 hours. The solution was :
poured into 2.5 L of water and the resultant white solid was removed by filtration, washed well with water and dried over P205 in a vacuum dessicator. 95X yield, m.p. 178-182C. Anal calc. for C22H17N304: C, 68-21;
~, 4.42; N, 10.85. Found: C, 68.12; H, 4.43; N, 10.83. The product had the formula:
~ N
~ ~ ~2~
EXA~PLE 7 Preparation of 5,5-Diphenyl-3-[(1'-methyl-3'-pyri-dinium)carbonyloxymethyl]-2,4-imidazolidinedione iodide:
The product of Example 6 (0.5 9, 0.0013 mol) was dissolved in 50 mL of acetonitrile, then û.3 mL of methyl iodide was added and the reaction mixture was maintained at room temperature for 6 days. The solvent .
,~ ,,"
133151~
~ -:
was removed by vacuum distillation and ethyl ether was added to the residue. The ether solution was refrig-erated for 2 hours, then the yellow, hygroscopic crys-tals which formed were dried over P205 in a vacuum dessicator, giving the desired product in 85X yield.
UV and HlNMR spectra confirmed that the product had the structure:
': ~
~H ~ ~
~ ~DH20~SCH 3 :
Repeating the above procedure in nitromethane at a 10 50-70C bath temperature using excess methyl iodide, ~m added gradually, for 5 to 6 hours, afforded the same product in nearly quantitative yield.
EXA~PLE 8 Preparation of 5,5-Diphenyl-3-[(1'-methyl-1',4'-d~hydropyridin-3'-yl)carbonyloxymethyl]-2,4-imidazolidinedione:
The quaternary salt obtained in Example 7 t0.4 9.
0.0008 mol) was dissolved in 40 mL of water, 3 mL of methanol and 15 mL of ethyl acetate. The reaction mixture was cooled to 0 to 5C and deaerated, then sodium bicarbonate (0.39 9, 0.0046 mol) and sodium dithionite (0.54 9, 0.0032 mol) were added. The mixture was stirred under nitrogen at 0-5C for 35 minutes. The organic layer was removed and the aqueous .
-115- 1~315~4 layer was extracted twice with 15 mL portions of ethyl acetate and the organic solutions were extracted with 10 mL of cold deaerated water. After drying over Na2S04, the solvent was removed by vacuum distillation and the oily yellow solid was crystallized by addition of ether. Yield 70~. UV and HlNMR analyses confirmed that the product had the formula:
~CO ~CH3 Preparation of 3-Bromoacetyloxymethyl-5,5-diphenyl-2,4-imidazolidinedione:
.
5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (2 9, 0.0071 mol) was dissolved in bromoacetyl-chloride (15 9, 8 mL, o.ng6 mol) by heating in an oil bath (70-80C bath temperature) for about 15 minutes, until the formation of HCl ceased. The mixture was cooled and 30 mL of ethyl ether were added. White crystals formed. The mixture was cooled to 0C, then the crystals were removed by filtration and dried over P205. Yield: 2.15 9 (75X), m.p. 179-183C. Anal.
calc. for C18H15N204Br: C, 53.61, H, 3.75; N, 6.95;
Br, 19.82. Found: C, 53.60; H, 3.79; N, 6.92; Br, 19.90. The product had the formula:
: :
~ ~ 3~iJ~I~
N
CH20~CH2Br ' EXA~PLE 10 ::
Preparation of 3-t3'-Bromopropionyl)oxymethyl-5,5-diphenyl-2,4-imidazolidinedione: -5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (5 9, 0.018 mol) was reacted according to the procedure of Example 9 with 3-bromopropionyl chloride ~ -(6.~ 9, 0.04 mol, 4 mL) using d bath temperature of 100C. A white crystalline product was obtained in 65%
yield (4.9 9), m.p. 133-134C. Anal. calc. for ~ -C1gH17N204Br: C, 54.69; H, 4.11; N, 6.72; Br, 19.15.
Found: C, 54.79; H, 4.12; N, 6.69; Br, 19.25. The product had the formula:
~: .
~S~ :
`CH20~CH2CH2Br ~: ' -EXA~PLE 11 Preparation of 3-(2'-Bromopropionyl)oxymethyl-5,5-diphenyl-2,4-imidazolidinedione: -S,S-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (2 9, 0.0071 mol) was dissolved in 2-bromopro-. ~.. :
:, :
133~r5'~ 1 pionyl chloride (8.5 9, 5 mL, n.o5 mol) by heating for 30 minutes on a 100~110C Oi 1 bath. The reaction mixture was cooled, 20 mL of ethyl ether were added, and the resultant solution was extracted wi~h aqueous potassium carbonate, dried and then crystallized. The product was obtained as a solid white substance (1 9, 34X), m.p. 112-115C. Anal. calc. for ClgH17N204Br:
C, 54 69; H, 4.11; N, 6.72; Br, 19.15. Found: C, 54.77; H, 4.15; N, 6.69; Br, 19.25. The product had the formula:
:
H
CH20i~HCH3 Preparation of 3-(3'-Carbamoyl-l'-pyridinium)acetyl-oxymethyl-5,5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example 9 (2.0~ 9, O.On5 mol) dissolved in lS mL of nitromethane was mixed with nicotinamide (0.61 9, 0.005 mol). The solution was stirred on a 90-100C temperature oil bath for 2 hours. The mixture was cooled to 60-70C and the white crystals which had formed were removed by filtration and washed with nitromethane. Yield filX (1.65 9), m.p.
193-197C (dec). Anal. calc. for C24H~1N405Br: C, 54.87; H, 4.03; N, 10.67; Br, 15.21. Found: C, 54.70;
H, 4.05; N, 10.64; Br, 15.25. The p oduct had the formula:
1 3 3 1 ~
H2OflC~2-N ~ ~r .
EXA~PLE 13 Preparation of 3-[3'-(3''-Carbamoyl-l''-pyridinium)- ~ ;
propionyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example lO (2.09 9, 0.005 mol) was dissolved in 15 mL acetonitrile, then nicotinamide (0.61 9, 0.005 mol) was added. The solution was refluxed for 6 days, then the solvent was removed. To the gum-like residue, 30 mL of ethyl ether was added and the mixture was stirred for 2 hours. The white substance which formed was removed by filtration and washed with ether. Yield 78~ (2.1 9); m,p, 98-100C
(dec.); UV and HlNMR as expected. The product had the formula:
N~O ONH2 ~/o~ ~ClI2o~cH2cH2-N~Br : .
. ~ .
' -:-., .~. . - ~ : ~ , . .. ,. ::: ~ : ~ , . . .
'.` ' ' ' ! ' : ' , -:: , ' ; :: . I
Preparation of 3-[2'-(3''-Carbamoyl-1''-pyridinium)pro-pionyloxymethyl]-5?5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example 11 (0.69 9, 0.00165 mol) was dissolved in 8 mL of acetonitrile, then nicotin-amide (0.2 9, n.ool65 mol) was added and the solution was refluxed for 22 hours. The solvent was removed from the resultant brown noncrystalline substance at 50C, then ethyl ether (15 mE) was added and the mix-ture was stirred for 2 hours. The light brown sub-stance was removed by filtration and washed with ether. Yield 56~ (0.5 9), m.p. 158C (dec.). The product had the formula:
~V N
~ 2 ~ C 3 N~ _ Br EXA~PLE lS
Preparation of 3-[(3'-Carbamoyl-1',4'-dihydropyridin-1'-yl)acetyloxymethyl]-5,5-diphenyl-2,4-imidazolidine-dione:
The product of Example 12 (0.52 9, 0.001 mol) was dissolved in a mixture of 60 mL of water and 30 mL of ethyl acetate. The mixture was cooled at 5C and deaerated, then sodium bicarbonate (0.5 9, 0.006 mol) and sodium dithionite (0.7 9, 0.004 mol) were added and the resultant mixture was stirred, with deaeration and .~ .
.
.
,, F~
i 3 3 ~
cooling, for 30 minutes. The laye s were separated and the aqueous layer was extracted with 30 mL of ethyl acetate. The organic solution was extracted with 20 mL
of cooled, deaerated water. After drying over sodium sulfate, the solvent ~as removed. Yield 55% (0.25 9) of yellow crystals, melting at 155-160C (dec.). The product reduced alcoholic silver nitrate solution and had the formula:
~: :
Ny~ CNH2 ~N~CH 20~CH 2-N~
EXA~PLE 16 Pre aration of 3-[3'-(3''-Carbamovl-1'',4''-dihvdro~
p - -: ~
pyridin-1''-yl)propionyloxymethyl]-5,5-diphenyl-2,4 imidazolidinedione~
Substitution of the product of Example 13 in the general procedure of Example 15 and substantial repeti-tion of the sodium dithionite reduction there detailed afforded the desired product in 85% yield. The product melted at 100~C (dec.) and had the formula:
: .
N2olcicN2cN2-~ ~
~331~ 3~
The product of Example 14 can be similarly reduced to the corresponding dihydro derivative, melting at 105C (dec.).
Preparation of 4-Aminobutanoic acid cyclohexyl ester hydrochloride:
~ ABA (8 9, 77.6 mmol) was suspended in 100 mL
~0.96 mol) of cyclohexanol. Thionyl chloride (40 mL) was added dropwise to the mixture at 0C. The mixture was then refluxed for 4 hours, cooled and crystallized from ethyl ether. The white crystals obtained in this manner were filtered and dried. NMR analysis confirmed the identity of the product.
EXA~PLE 18 Preparation of 3-~-[(3'-Cyclohexyloxycarbonyl)-propyl]~carbamoylpyridine:
Nicotinic acid (2.2 9, 18 mmol) was suspended in 50 mL of dry pyridine. Dicyclohexylcarbodiimide (3.68 9, 17.9 mmol) was dissolved in the solution, with stir-ring. 4-Aminobutanoic acid cyclohexyl ester hydro-; chloride (4 9, 18 mmol) was added and the mixture was stirred for 48 hours. Precipitated dicyclohexylurea was removed by filtration and the filtrate was evapo-rated to dryness. The residue was washed with 25 mL of ice cold water and extracted into ethyl acetate. The layers were separated and the organic layer was evapo-rated to dryness. ~MR analysis confirmed the structure ;~ of the product.
1 ~ 3 ~
EXAMPLE l9 Preparation of l-Methyl-3-fN'-[(3'-Cyclohexyloxycar-bonyl)propyl]~carbamoylpyridinium iodide:
The product of Example 18 (1.74 9, 6 mmol) was dissolved in a minimum amount of acetone and the re-sulting white precipitate was filtered. ~ethyl iodide (1.5 mL, 24 mmol) was added in one portion to the~ ~ ;
solution, with stirring, at 0C. The mixture was allowed to gently reflux overnight. Filtration of a ;~
white precipitate and evaporation of the yellow filtrate produced a reddish oil, which was dissolved in acetone, filtered and evaporated to dryness. Anal.
calc- for C22H233N21: C, 47-26; H~ 5.79; N~ 6-48; 1, 29.38. Found: C, 47.03; H, 5.85; N, 6.44; 1, 29.26.
Preparation of l-Methyl-3-~N-[(3'-cyclohexylcarbonyl)-propyl]~carbdmoyl-1,4-dihydropyridine:
The product of Example 19 (0.11 9, 0.26 mmol) was dissolved in 25 mL of ice cold deaerated water. NaHC03 20 (0.09 9, 4-fold excess) was added, followed by Na2S204 (0.14 9, 3-fold excess). Ethyl acetate (25 mL) was added and the mixture was stirred under nitrogen for 30 minutes. The organic layer was extracted and dried to give an orange oil that reduced methanolic silver nitrate immediately. NMR analysis confi med that the product had the structure:
¢~NIlC~2C~2C~2Qo{) .
133~
EXA~PLE 21 Preparation of Valproic acid chloride (2-Propyl-pentanoyl chloride):
To 4.32 9 (30 mmol) of valproic acid in an ice bath, thionyl chloride (3.6n 9, 30 mmol) was slowly added, with stirring. The neat mixture was allowed to -come to room temperature and then heated in a water hath at 50C for 30 minutes. 50 mL portions of dry benzene were twice added and removed under reduced pressure. The resultant product was used in subse-quent reactions without further purification.
: ~ .
EXA~PLE 22 Preparation of Valproic acid 2-iodoethyl ester (2'-Iodoethyl 2-propylpentanoate):
To the product of Example 21 (4.87 9, 3~ mmol), 2-iodoethanol (5.16 9, 30 mmol) was added with stirring and cooling in an ice bath. The neat mixture was then '.~ J~
heated to 100C in a water bath for 10 minutes, then removed from the heat and stirred for an additional 10 minutes. The reaction mixture was then dissolved in 50 mL of ether, washed with water (1 x 30 mL), 5% NaOH (2 x 30 mL), and again with water (2 x 30 mL). The ether layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. A light yellow liquid product was obtained in 67X yield from valproic acid (6.0 9). Silver nitrate gave a bright yellow precipitate. N~R analysis confirmed the identity of the product.
1 3 ~
Preparation of 1-[2'-(2''-Propyl)pentanoyloxy]ethyl-3-carbamoylpyridinium iodide~
The product of Example 22 (3.28 9, 11 mmol) and 50 mL of dimethylformamide were added to nicotinamide (1.22 9, 10 mmol). The mixture was heated to reflux for 3 hours, then was cooled. Removal of solvent under reduced pressure afforded a brown oily residue, which was stirred with ether (60 mL) for 30 m~nutes, giving a yellow powder. The ether was decanted and a fresh portion of ether (50 mL) was added. The crude product was vacuum filtered under N2, then wdS recrystallized from isopropanoltether to give 3.5 9 of the desired product (84% yield), m.p. 111-112C. The product had the formula:
~ NH2 ~OJ I ~ ~
R ~ ~:
CH2CH20~CH2CIHcH2cH3 EXA~PLE 24 Preparation of 1-[2'-(2''-Propyl)pentanoyloxy]ethyl-3-carbamoyl-1,4-dihydropyridine:
To S0 mL of ice-cold degassed deionized water, the product of Example 23 (420 mg, 1 mmol) was added. To that solution, NaH003 (366 mg, 4 mmol) and Na2S204 (696 mg, 4 mmol) were added, with stirring. Nitrogen gas was bubbled through the solution for 30 minutes. The aqueous solution was then extracted with ether (6 x 25 .
1~31~
mL) until the ether layer was no longer yellow. The combined ether extracts were washed with water (1 x S0 mL) and dried over MgS04. The ether laye was decanted from the drying agent and the solvent was removed under reduced pressure. To the oily residue, ether was added and then removed (10 x 5 mL) on a vacuum pump. A foam was formed, which returned to an oil upon exposure to the atmosphere. Structure was confirmed by NMR `
analysis.
Preparation of N-Nicotinoyltyrosine eth~l ester~
Nicotinic acid (12.3 9, 0.1 mol) was dissolved in ^'~
dry pyridine (300 mL). The solution was cooled and dicyclohexylcarbodiimide (20.6 9, 0.1 mol) was added.
~fter dissolution, tyrosine ethyl ester hydrochloride (24.6 9, 0.1 mol) was added and the solution was stirred overnight. The precipitated dicyclohexylurea (DCU) was removed by filtration. Additional ~CU was removed by triturating the oil with hot water. The product was purified with acetone. Calculated for C17Hl~N204-1/2H20: C, 63.16; H, 5.88; N, 8.66.
Found: C, 63.10; H, 5.96; N, 8.59. The product can also be named N-[l-ethoxycarbonyl-2-(4'-hydroxyphenyl)-ethyl]nicotinamide.
Preparation of N-[(l-Methyl-3-pyridinium)carbonyl3-tyrosine ethyl ester iodide:
N-Nicotinoyltyrosine ethyl ester (20 9, 0.06 mol) was dissolved in 200 mL of acetone. A two molar excess ':~'" " ~'' , . ..
of methyl iodine (25.6 9, 0.18 mol) was added and the mixture was refluxed for 6 hours. The solvent was removed under reduced pressure to yield the desired product as a solid form. NMR analysis confirmed the identity of the product, which had the structural formula: ~ -~ ~--NH~ COOCH2cH3 CH ~ I
and can also be named l-methyl-3-~N-t(1'-ethoxy-carbonyl)-2'-(4''-hydroxyphenyl)ethyl}carbamoyl-pyridinium iodide.
EXA~PLE 27 -Prepa,ation of l-Methyl-3-~N-[(1'-ethoxycarbonyl)-2'-(4''-pivaloyloxyphenyl)ethyl]~carbamoylpyridinium trifluoroacetate:
,_ The product of Example 26 (6 9, n.013 mol) was dissolved in 50 mL of cold trifluoroacetic acid at 0C
in an ice bath. Pivaloyl chloride (3.14 9, 0.026 mol) was slowly added and the solution was wa,med to room temperature. After 24 hours, the solvent was removed under reduced pressure. The resulting dark oil was triturated with petroleum ether but no solidification occur,ed. Identity of the product was confirmed by NMR
analysis. The product was dissolved in aqueous methanol (1OX) and extracted with ethyl ether to remove a highly colored contaminant before using as the starting material in Example 29 below.
Preparation of 1-Methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4''-isobutyryloxyphenyl)ethyl]~carbamoylpyridinium trifluoroacetate:
The product of Example 26 (6 9, 0.013 mol) was dissolved in 50 mL of trifluoroacetic acid cooled to 0C in an ice bath. To that solution, with stirring, 10 was slowly added isobutyryl chloride (2.77 9, 2.76 mL). The solution was stirred overnight at ambient temperature and the solvent was removed unde, reduced pressure. The oil was stirred overnight with petroleum -ether and then dried in vacuo, but no $olidification occurred. Identity of the product was confirmed by NMR
analysis. The product was dissolved in aqueous metha-nol (lOX) and extracted with ethyl ether to remove a highly colored contaminant before using in Example 30 ;~ below.
~:. . .: ..
~',' . ' ,'- -:
- ~:
,- ' ,:
~ $ ~ 1 ~r~
Preparation of l-Methyl-3-{N-t(l'-ethoxycarbonyl)-2'-(4''-pivaloyloxyphenyl)ethyl]~ca bamoyl-1,4-dihydro-pyridine:
The product of Example 27 (4.07 9, n.oo79 mol) was dissolved in lO0 mL of 25% aqueous methanol. Nitrogen gas was bubbled through the solution. To the solution, stirring in an ice bath, was then added NaHC03 (2.02 9, 0.024 mol). Ethyl ether (lO0 mL) was added, followed 10 by the addition of Na2S204 (4.12 9, t).024 mol). The yellow biphasic solution was stirred for 30 minutes, then the layers were separated and the aqueous layer was extracted twice with 75 mL portions of ethyl ether. The combined organic fractions were dried ove 15 lla2so4 and the solvent was ,emoved under reduced pressure to afford a solid foam which was oxidi~ed by ethanolic silver nitrate. Anal. calc. for C23H20N20 l/2H20: C, 65.23; H, 7.33. Found: C, 65.76; H, 7.28; N, 6.95.
. .
3 ~
Preparation of 1-Methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4''-isobutyryloxyphenyl)ethyll~calbamoyl-1,4-dihydropyridine:
The product of Example 28 (2.20 9, 0.0044 mol) was dissolved in 100 mL of aqueous methanol. The solution WdS cooled in an ice bath with a stream of N2 passing through it. To this solution, NaHC03 (1.11 9, 0.0132 mol) and ether (100 mL) were added. Then, sodium dithionite (2.30 9, 0.0132 mol) was added and the solution was stirred for 30 minutes. The layers were separated and the aqueous phase was washed with ethyl ether. The com~ined organic layers were dried over ¦ anhydrous Na2S04 and reduced in volume. The resultant orange oil was oxidized by ethanolic silver nitrate.
Identity of the product was confirmed by NMR analysis.
EXA~PLE 31 Preparation of Chloromethyl [2S-(2,5,6~)]-3,3-dimethyl-7-oxo-6-t(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclot3.2.0]heptane-2-carboxylate~
To a solution of 4.02 9 (0.01 mol) methicillin sodium salt in 10 mL water and 10 mL CH2Cl2, 2.4 9 sodium bicarbonate and 0.34 9 tetrabutylammonium ~ -hydrogen sulfate were added. Then, 1.9 9 (0.0115 mol) 25 chloromethyl chlorosulfate dissolved in 3 mL CH2Cl2 ~--were added with stirring, over a 5 minute period, keeping the temperature below 30C. After an additional 30 minutes of stirring, the organic phase was separated, washed twice with water and dried over ~gS04. 8y removing the solvent in vacuo, 4.24 9 of the : , . ~ ~ :
. ~ ~ " ~ .. . . . ' . . ~ ' ~ ' .
desined product were ohtaine~ dS a yellow solid, melting at 88-90C.
Preparation of Chloromethyl [2S-(2a,5,6~)]-3,3-5 dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7^oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
Substantial repetition of the procedure of Example 31 using 2.12 9 (O.i)05 mol) oxacillin sodium salt with 1.2 9 NaHC03, 0.17 9 tetrabutylammonium hydrogen 10 sulfate and 0.95 9 chloromethylchlorosulfate afforded 1.87 9 of the desired product melting at 78-80C ~dec.).
EXA~PLE 33 Preparation of Chloromethyl t2S-(2,5,6B)]-6-~3-(2-15 chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
:
Using the same procedure as in Example 31, but substituting 2.38 9 (0.005 mol) cloxacillin sodium salt ~ ~-20 (1 rnol water), 1.2 9 NaHC03, 0.17 9 Bu4NHS04 and 0.95 9 chloromethyl chlorosulfate gave 2.27 9 of the desired product melting at 97-100C (dec.).
. , .
i . ~ .. ., ~ . .. . . ~ . , ~ ......
~ ~ 3 ~ r ~ ,~
Preparation of Chloromethyl [2S-(2~,5,6B)]-6-[3-(2,fi-dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicycloC3.2.0]heptane-2-carboxylate:
Similarly, following the procedure of Example 31 but using 2.55 9 (0.005 mol) dicloxacillin Na salt (1 mol water) with 1.7 9 NaHC03, 0.17 9 Bu4NHS04 and 0.95 g chloromethyl chlorosulfate, 2.43 9 of product were obtained melting at 98-101C (dec.).
EXA~PLE 35 Prepa ation of [(3-Pyridinylcarbonyl)oxy]methyl [2S-(2~,5,~B)]-3,3-dimethyl-7-oxo-6-[(2,6-dimethoxy)-benzamido]-4-thia-1-azabicyclo[3.2.0]heptane-~-carboxy-late~
Three and eight-tenth grams (0.0089 mol) of the methicillin chloromethyl ester produced in Example 31 ;
and 1.6 9 (0.01 mL) potassium nicotinate in 70 mL nMF
were stirred 6 days at room temperature (20-25C). 300 ~
mL ethyl acetate were added, the resultant solid was ~ `
removed by filtration and the solution was extracted 4 times with 50 mL concentrated aqueous NaCl and dried over MgS04. The sol vent was removed in vacuo and the resultant residue was purified by chromatography (silica gel). Obtained as a white solid were 3 9 of the desired product melting at 151-157C.
.
~ 3 ~
Preparation of [(3-Pyridinylcarbonyl)oxy~metnyl [2S-(2,5,fiB)]-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxa-zolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.n]-heptane-2-carboxylate:
Following the procedure of Example 35, but utiliz-ing 1.81 9 (0.004 mol) of the oxacillin chloromethyl ester produced in Example 32 and 0.75 9 (0.0046 mol) K
nicotinate, afforded, after purification by chromato-graphy, 0.75 9 of the desired product as a white solid melting at 79-82C (dec.).
Preparation of [(3-Pyridinylcarbonyl)oxy~methyl [2S-(2a,5a,6B)]-6-[3-(2-chlorophenyl)-5-methyl-4-isoXazole-15 carboxamido]-3,3 dimethyl-7-oxo-4-thia-1-azabicyclo-t3.2.0]heptane-2-carboxylate:
Using the procedure of Example 35, but substi-tuting 2.1 9 (0.0043 mol) of the cloxacillin chloro- 1 methyl ester produced in Example 33 and 0.8 9 (0.005 20 mol) K nicotinate, gave 1.2 9 of product melting at 83-85C (dec.).
Preparation of [(3-Pyridinylcarbonyl)oxy]methyl ~2S-(2a,5a,6B)]-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxa-25 zolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabi-cyclo[3.2.0]heptane-2-carboxylate:
Similarly, following the procedure of Example 35 but using 2.27 9 (0.0047 mol) of the dicloxacillin chloromethyl ester produced in Example 34 and 0.87 9 :~ :
`','~
. ~ . ~ , . . ... . .
6 ~
(0.0054 mol) K nicotinate afforded 1.1 9 of the product as a white solid melting at ~7-90C tdec.).
EXA~PLE 39 Preparation of [2S-(2a,5a,6~)]-3-~t[t[3,3-Dimethyl-7-oxo-6-[t2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo-[3.2.0]hept-2-yl~carbonyl]oxy~methoxy]carbonyl]-1-methylpyridinium iodide:
One and one-fourth grams (0.0024 mol) of the methicillin derivative produced in Example 35 in 35 mL
nitromethane and 1.14 9 (0.5 mL) (0.008 mol) methyl -iodide were reacted in a closed system at room -temperature (20-25C) for 7 days. The solvent was removed in vacuo, the resultant residue was stirred ~-~
~ith ether, filtered off, washed with ether and - -dried. There were thus obtained 1.6 9 of yellow hygroscopic product melting at 95-100C and being further characterized by the structural formula~
' ~ONH~ ~CH3 \=<~ ~ N-- CH3 C 3 o~
CO H2CC~ ~
~' ~ . .
~, ' - ` :, , ' : ' ' ` ~ :, . ` ' ` : ~ ': : : ' ` ' ~:,"''. ' "' .' ` ~ ~ ~ ` ' ` ` ' ~ ~ : . ,' - \
1331~
EXA~PLE 40 Preparation of C2S-(2a,5.6R)]-3-[[[[[3.3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]oxy]-methoxy]carbonyl]-1-methylpyridinium iodide: ;
Using the procedure of Example 39, but substitu-ting 0.5 9 (0.0009 mol) of the oxacillin derivative produced in Example 36 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH3I produced, after 6 days, 0.6 9 10 of the desired product melting at 75-80C and having the formula:
COCH2iC~ 1 Preparation of [2S-(2,5,6B)]-3-[[~C[6-[3-(2-chloro-15 phenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl_ 7-oxo-4-thia-1-azabicyclo~3.2.0]hept-2-yl]carbonyl]-oxy]methoxy]carbonyl]-1-methylpyridinium iodide:
S~milarly, using the procedure of Example 39, but substituting 0.44 9 (0.0008 mol) of the cloxacillin 20 derivitive produced in Example 37 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH3I, gave 0.45 9 of product melting at 90-95C (dec.) and having the formula:
~3~4 ~[~CH
COCH~OC~
Preparation of [2S-(2~,5a,6~)-3-t[[[[6-t3-(2,6- -dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]-carbonyl]oxy]methoxy]carbon~l]-1-methylpyridinium iodide~
In a similar manner, using the procedure of Example 39, but substituting 0.5 9 (0.007 mol) of the dicloxacillin derivative produced in Example 38 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH31 gave 0.55 9 of product melting at 95-100C (dec.) and having the formula: ~
cl :
~C1~3~ ~Cll3 2 ~
~ ' CH3 :
::
i~.. :" i., ::. - :: :`
. ~, . ~ . . : ,.: , :
?~
1 3 ~
EXA~PLE 43 Preparation of t[(l,4-Dihydro-l-methyl-3-pyridinyl)-carbonyl]oxy]methyl [2S-(2a,5,6 ~)]-3,3-dimethyl-7-oxo- ~ i~
6-[(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo-t3.2.0]heptane-2-carboxylate~
0.45 9 (0.0007 mol) of the product of Example 39 dissolved in a mixture of deaerated 25 mL ethyl acetate and 70 mL water were reduced with a mixture of 0.34 9 (0.004 mol) NaHC03 and 0.48 9 (0.0028 mol) sodium dithionite at 0-5C over 70 minutes. The disappearance of the 268 nm maxima and increase of 366 nm maxima in the U.V. spectra were followed. The layers were separated and the aqueous layer was extracted with 2 x 25 mL ethyl acetate, then the organic layers were extracted with 2 x 20 mL cold, deaerated water. After drying over Na2S04, the solvent was removed in vacuo. -~
0.25 9 of the product was obtained as a yellow solid melting at 88-90C (dec.). The product had the formula: - ~
, :
~COMH~ ~CH3 . i \=< ,L -- CH3 0CH3 o~
Z 11--¢3 ., ~ ,", `; ~
,~
,,`~ ,.. ,, .~ ~ . .
; ~X'. . .` ~
133~
Preparation of ~[(1,4-Dihydro-l-methyl-3-pyridinyl)- ;-carbonyl]oxy]methyl [2S-(2a,5~,6B)]-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1- ~-azabicyclo[3.2.0]heptane-2-carboxylate~
Similarly, repetition of the general procedure of Example 43 using 0.17 9 (0.00025 mol) of the product of Example 40, 0.08 9 (0.0001 mol) NaHC03 and 0.51 9 (0.001 mol) Na2S204, in 15 mL water and 15 mL ethyl acetate, afforded 0.1 9 of product melting at 93-100C
(dec.). The product had the formula:
S,~CH3 ~CONH T1~CH3 I~ O CH3 o~ , .
COCH201CI~ ;
Preparation of t~(l,4-~ihydro-l~methyl-3-pyridinyl)-carbonyl]oxy]methyl ~2S-(2,5~,6~)~-6-~3-(2-chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo~3.2.0]heptane-2-carboxylate:
In a similar manner, following the procedure of Example 43, but substituting 0.18 9 (0.00025 mol) of the product of Example 41, 0.089 NaHC03 and 0.17 9 Na2S204. gave 0~13 9 of product as a yellow solid, ,: :
~ ,, . ;.
~ .~
5.
~y,~
13315~4 :
melting at 80-85C (dec.) and having the structural f o r m u l a ~
Cl ~<CR3 CoCH20 '~:
Preparation of [[(1,4-Dihydro-1-methyl-3-pyridinyl)-carbonyl]oxy]methyl [2S-(2,5,6~)]-6-[3-t2,6-dichloro- ~-phenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
In like manner, repetition of the procedure of Example 43 using 0.19 9 (0.00025 mol) of the product of Example 42, 0.08 9 NaHC03, 0.17 9 Na2S204 yielded 0.14 g of desired product melting at 98-102C (dec.) and ; having the formula:
Cl CH3 H F~<
Cl CH3 o N
ICIOCH201CI~ ; .
cL -:
:'~ 3 b .''. - .
~æ~
Preparation of [(3-Pyridinylcarbonyl)oxy]methyl [2S-(2~,5,6B)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]- -4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
~ suspension of 3.83 9 (o.nl mol) of the chloro~
methyl ester of benzylpenicillin, namely chloromethyl [2S-(2,5,6B)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)-amino]-4-thia-1-azabicyclot3.2.0]heptane-2-carboxylate, and 1.93 9 (0.012 mol) potassium pyridine-3-carboxylate in 100 mL of dimethylformamide was stirred at 20-25C
for 6 days. Then, 300 mL of ethyl acetate were added and the solid was removed by filtration. The solution was extracted 4 times with concentrated aqueous sodium chloride solution, then dried over ~gS04. The solvent was removed in vacuo to give 4.5 9 of foamy solid.
Purification by chromatography over silica gel using ethyl acetate as eluent afforded 2.5 9 of product melting at 127-130C.
EXA~PLE 48 Preparatlon of [2S-(2,5,6B)]-3-~[[[t3,3-Dimethyl-7-oxo-6-[(phenylacetyl)am1no]-4-thia-1-azabicyclo[3.2.0]-hept-2-yl]carbonyl]oxy]methoxy]carbonyl]-1-methyl-pyridiniùm iodide:
Two and one-half grams (0.053 mol) of the product of Example 47 dissolved in 100 mL of dry nitromethane were reacted with 2.25 9 (1 mL, 0.016 mol) of methyl iodide in a closed system at 20-25C for 6 days, at the end of which time thin layer chromatography showed complete reaction. The solvent was removed in vacuo and the solid residue was slurried with ether, filtered :
~ " ~ . ~ . . ~ ,: . . , 7 ~
.';-~
~ ~ 3 1 ~
and dried in vacuo over P205. The product, ~elting at ~ ;~
90-95C (dec.~, was obtained as a yellow solid (2.91 9). It was assigned the structural for~ula:
.:
C6H~CH2CNH~/ \<CH3 ~ :
N \1/ CH3 CoCH20 O I ' ' , ~
5EXA~PLE 49 Preparation of ~t(1,4-Dihydro-1-methyl-3-pyridinyl)car-bonyl]oxy]methyl [2S-(2,5a,6~)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]hep-tane-2-carboxylate:
The quaternary salt prepared in Example 48 (3.25 ~
9, 0.0053 mol) was dissolved in a mixture of 350 mL of ~-water and 150 mL of ethyl acetate. The resultant mlxture was cooled at 0-5C and deaerated with nitrogen, then a mixture of 2.67 9 (0.032 mol) of sodium bicarbonate and 3.69 9 (0.021 mol) of sodium dithionite was added over a 2-3 minute period. The reaction mixture was st~rred for 1 hour under the same conditions as before, then the layers were separated, the aqueous layer was extracted twice with 50 mL
portIons of ethyl acetate, and the combined organic extracts were washed twice with 30 mL portions of cold, deaerated water. Dr~ing over sodium sulfate and removal of the solvent in vacuo afforded 1.7 9 of yellow solld melting 98-100C and having the formula:
.
- :
~"
.
'.'~'`` . . .. ',' 'i ~ ' ' ,' :
1~31~
o 5 C6H5CH2CNH ~/ \/ CH3 N ~/\ CH3 ;
COCH20C--¢3 EXA~PLE 50 Preparation of Chloromethyl N-[3-(10,11-dihydro)-5H-dibenztb,f~azepin-5-yl)]propyl-N-methylcarbamate:
~ethod A:
Desipramine hydrochloride (1.5 9, 0.005 mol) was dissolved in 20 mL of methylene chloride, cooled at 0-5C. Then I g NaHC03 was added, followed by 0.92 9 (0.007 mol) chloromethyl chloroformate. The reaction mixture was st~rred for 1 hour, then the salts were removed by filtration and the solution was extracted twice with 10 mL portions of SX HCl. The organic layer was dried over ~gS04 and the solvent was removed in vacuo to give the desired compound as a colorless oily substance in 76X yield (1.35 9).
~ethod B:
Imipramine hydrochloride (1.59 9, O.OOS mol) was dissolved in 15 m~ of water, then 5 mL of 4X sodium hydroxide solution was added, with cooling. The resultant imipramine base was extracted twice with 10 mL portions of benzene. The solution was concentrated ~ .
1~31~6~
to 10 mL, then a solution of 0.7 9 (0.0054 mol) chloro-methyl chloroformate in 5 mL benzene was added with cooling at 10C. The reaction mixture was stirred at 20-25C for 30 minutes, then was refluxed for 1 hour.
A small amount of imipramine hydrochloride resulted and was filtered off. The solution was extracted twice with 20 mL portions of 4X HCl and dried over MgS04.
Removal of solvent in vacuo afforded 1.2 9 (66%) of product having the same characteristics as that obtained by Method ~.
Preparation of l-Chloroethyl N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-S-yl)]propyl-N-methylcarbamate:
Following the general procedure of Example 50, but using 1.5 9 (O.nO5 mol) of desipramine hydrochloride and 0.86 9 (0.006 mol) chloroethyl chloroformate and carrying out the reaction at 5-10C for 2 hours, gave 1.6 9 (86X) of the title compound as a colorless oil.
Preparation of ~N-t3-(10,11-Dihydro-5H-dibenz[b,f]-azepin-S-yl)]propyl-N-methylamino~carbonyloxy]methyl 3-pyridinecarboxylate:
The product of Example 50 (1.35 g, 0.0037 mol) dissolved in 5 mL dimethylformamide was added to a solution prepared from 0.57 9 (0.046 mol) nicotinic acid and 0.45 9 triethylamine in 5 mL dimethyl-formamide. The mixture was stirred for 24 hours at 25-30C, then 30 mL of ethyl acetate were added. The precipitated salts were removed by filtration and the .
' ; , .. ~ . " . ...
~3~
solution was extracted 4 times with 15 ~L portions of saturated aqueous sodium chloride solution. Drying over ~gS04 and removal of the solvent in vacuo afforded 1 9 (61X) of pure product as an oil.
PreQaration of ~ N-~3-(10,11-Dihydro-5H-dibenz~b~f~-azepin-5-yl)]propyl-N-methylamino~carbonyloxy]ethyl 3-pyridinecarboxylate:
Following the general procedure of Example 52, but using 1.05 9 (0.0028 mol) of the product of Exa~ple 51, 0.45 9 (0.036 mol) of nicotinic acid and 0.36 9 of tri-ethylamine in 10 mL dimethylformamide and carrying out the reaction at 25-30C for 48 hours, gave 0.5 9 of the title compound as a yellow oil.
EXA~PLE 54 Preparation of 3-C~N-~3-(10,11-Dihydro-5H-dibenz[b,f]-azepin-5-yl)]prop~l-N-methylamino~carbonyloxy]methoxy-carbonyl-1-methylpyridin1um iodide:
Eight-tenths gram (0.0018 mol) of the product of Example 52 in ~0 mL of nitromethane was methylated with 0.8 mL of methyl iodide at 25-30C for 48 hours. The solvent was removed in vacuo and the residue was slurried with ethyl ether, filtered and dried over P205. The quaternary salt was obtained in 83X yield (0.88 9) as a light yellow solid melting at 172-175C
(dec.) and having the structural formula:
~".,~, ~...~.,.,, . ~ ..~ , .~. ., . ' :
1331 ~4 ~o ~1OCH
CH2CN2 2 \CH
tH3 Preparation of 3-[1-~N-[3-(10,11-Oihyd,o-SH-dibenz-~b,f]azepin-5-yl)]propyl-N-methylamino~carbonyloxy]-S ethoxycarbonyl-l-methylpyridinium iodide:
Following the general alkylation procedure of Example 54, but using 0.5 9 (0.0011 mol) of the product of Example 53 in lS mL nitro~ethane with 0.5 mL methyl iodide, and carrying out the reaction at 20-25C for 6 days, afforded 0.33 9 (50~) of the desired quaternary salt as a dark yellow solid melting at 101-103C (dec.) ~-and having the structural formula:
,~, H3 ll CH2CH2 2 <CH ~ 1- :
L : ~
~,:
: ' ~: ' r.~ ~
~3i~
`_reparation of [~N-[3-(10,11-Dihydro-5H-dibenz[b,f~
azepin-5-yl)~propyl-N-methylamino~carbonyloxy]methyl 1,4-dihydro-1-methyl-3-py,idinecarboxylate: -Three-tenths gram (O.û005 mol) of the product of Example 54 in 30 mL water and 15 mL ethyl acetate was reduced with 0.25 9 (0.003 mol) NaHC03 and 0.35 9 (0.002 mol) sodium dithionite at 0-5C, with deaera-tion, for a 60 minute period. The layers were separated and the aqueous layer was extracted twice with 30 mL portions of ethyl acetate. The combined organic layers were then extracted twice with 20 mL
portions of cool deaerated water. Drying over Na2S04 and removal of the solvent in vacuo afforded 0.22 9 (9S%) of the title compound, melting at 59-63C (dec.) and having the structural formula:
~q ~'~ ' ~N ~ O O
~YOCH20C
CH2CH2CH2 \ ~N J
EXAltPLE 57 Preparation of ~ N-~3-(10,11-Dihydro-SH-dibenz[btf]-azepin-5-yl)]propyl-N-methylamino~carbonyloxy]ethyl ~ ~;
1,4~-dlhydro-1-methyl-3~pyridinecarboxylate~
Following the procedure of Example 56, but us1ng 0.1 9 (0.0017 mol) of the product of Example 55 in 10 ~ i mL water and 6 m~ ethyl acetate, 0.11 9 NaHC03 and 0.15 ~
~" . . ;.. ~ . ;~.. . - ~ . . ; . , ,: . .
r ~ ~ :
g Na2S204 and carring out the react~on for a 60 minute per~od, gave 0.07 9 (88X) of the title compound as a yellow sol~d, melt1ng at 60-65C (dec.) and having the structural formula:
~ 8 IH3 ~COCH
CH2CH2CH2N~CH3 ~
EX~#PLE 58 Preparat1On of N-(2-~ydroxyethyl)-3-pyridinecarboxa-mide:
A solutlon of 49.2 9 (0.32525 mol) ethyl n~co-t~nate and 72 9 (1.17 mol) ethanolamine was heated at 70C for 60 hours. The excess ethanolam1ne was removed under reduced pressure and the result~ng v1scous cream o11 was stlrred w1th ether for 48 hours. The result~ng wh1te solld was removed by f11trat~on, afford1ng 46 9 (85.1X) of the t1tle compound melting at 75-78C.
EXA#PLE 59 Preparatlon of 3-t2~-(2U-Propyl)pentanoyloxy]ethylcar bamoylpyrldine:
To a st~rred solut~on of 1.0 9 (0.006021 mol) of ~ ~i the product of Example 58 and 0.61 9 (0.00598 mol) tri-ethylam~ne in 40 mL dry dlchloromethane, 1.96 9 (0.012047 mol) of (2-propyl)pentanoyl chlor1de were , :
added and the mixture was refluxed for 4 hours. The resultant solution was washed sequentially with 30 mL
SX NaHC03, 30 mL 5X HCl and 30 mL water. The organic layer was dried over ~gS04 and the solvent was removed under reduced pressure to give 0.6 9 (34.3X) of the product as a pale brown oil.
EXA~PLE 60 Preparation of 1-Methyl-3-t2'-(2"-propy7)pentanoylox~]-ethylcarbamoylpyridinium lodide:
To a solution of 1 9 (0.00342 mol) of the product of Example 59 in 20 mL of dry ethyl acetate, 0.73 9 (0.00513 mol) of methyl iodide was added. The reaction mixture was stirred overnight at room temperature. The pale yellow solid which formed was removed by filtra-tion and recrystallized from ethyl acetate to give 1.35 9 (90.9X) of the quaternary salt as a yellow crystal-line solid. The product htd the formula:
o ,, `NHCH2CH20C-cH(cH2cH2cH3)2 as confirmed by IR, NMR and UV analyses.
-:
EXA~PLE 61 Preparation of 1-Methyl-3-~2'-~2"-propyl)pentanoyloxy]-ethylcarbamoyl-1,4-dihydropyridine:
To 50 mL of vigorously stirred, degassed, ice-cold deionized water, a solutlon of 3.0 9 (0.006909 mol) of the quaternary product of Example 60 in 50 mL of ethyl acetate was added. Throughout the reaction, the temperature and pH were maintained at 0C and 8 respectively, while nitrogen was bubbled through the reaction m~xture. A mixture of 3.5 9 (0.04145 mol) of sodium bicarbonate and 4.8 9 (0.02763 mol) of sodium dithionite was added portionwise. After 45 minutes, the organic layer was separated and the aqueous layer was extracted with 100 mL of ice-cold ethyl acetate.
The combined organic extracts were washed with ice-cold water and dried over ~gS04. Solvent was removed under reduced pressure to give 2.1 9 (98.8X) of the product as a pale yellow solid having the structural formula:
cN2c2oc-C(C 2C 2C 3)2 ' 20 dS confirr3ed by IR. NMR and UV anaI~ses.
~, .
: .
, ., .
1 3 ~
Preparat~on of 3-Pyridinecarboxylic acid (2-hydroxy)-ethyl ester hydrochlorlde:
To 120 mL cold (-10C) ethylene glycol, 16 mL of thionyl chloride were added dropwise. Upon completion o~ the addition, 24.6 9 (0.2 mo1) of nicotinic acid were added portlonw~se and the reaction m~xture was heated overn19ht at 60C. rhen, 700 mL of hot tetra-hydrofuran were added and the mixture was cooled. The sol1d which formed was removed by filtration and washed with ether to give 28.5 9 of the title compound as white crystals.
EX~PLE 63 Preparation of 3-[2'-(2"-Propyl)pentanoyloxy]ethoxy-carbonylpyr1dine To a solut1On of 10.0 9 (0.0491 mol) of the product of Example 62 ln 150 mL of dry CH2C12, 10.7 9 (0.09819 mol) of triethylamine were added. After all of the solld was dlssolved, 11.92 9 (0.07364 mol) of 2-propylpentanoyl chlorlde were added and the reactionm~xture was st1rred at room temperature for 36 hours.
Sequent1al washing w1th SX NaHC03, 5X HCl and water afforded an organlc layer ~hich was then dried over -anhydrous ~gS04. Solvent was removed under reduced pressure to g1ve a yellow-brown o11 that was tr~turated w1th a 40:60 mixture of ether and petroleum ether to y1eld 9.7 9 of the product as an orange o~l.
1 3 3 1 ~
EXA~PLE 64 Preparation of l-Methyl-3-t2'~ -Propyl)pentanoyloxy]-ethoxycarbonyl~yridinium iodide:
To a solut~on of 2.0 9 (0.006816 mol) of the S product of Example 63 in 10 mL of dry acetone, 1.45 9 (0.01022 mol) of methyl iodide were added and the mixture was refluxed overnight. Removal of solvent under reduced pressure afforded 1.84 9 of the quater-nary salt as a brown oil. The product had the struc-tural formula~
~ : -~OCH2CH20~-cH(cH2cH2cH3)2 "; ~';
EXA~PLE 65 .~ ,., Preparation of 1-Methyl-3-~2'-(2U-propyl)pentanoyloxy]-ethoxycarbonyl-1,4-dihydropyridine:
~o S0 mL of vlgorously stirred, degassed, ice-cold deionlzed water, a solution of 1.84 9 (0.004226 mo1) of the quaternary product of Example 64 in 50 mL of ethyl acetate was added. Throughout the reaction, the temperature and pH were malntained at 0C and 8, respectively, wh11e argon was bubbled through the reaction mixture. A m1xture of 2.13 9 (0.02536 mol) of HaHC03 and 2.94 9 (0.0169 mol) of ~a2S204 was added portionwlse. After 55 minutes, the organic layer was separated and the aqueous layer was extracted with 100 mL of ~ce-cold ethyl acetate. The combined organ~c ..,.,:
. ~ .
1331~B~
layers were washed with ice-cold water and dried over MgS04O ~he solvent was re~oved under reduced pressure to give 0.9 9 of the title compound as a yellow oil. h ~he product had the formula~
¢~/ ~ocH2cH2o~ cH(cH2cH2cH3)2 CH3 :
'`~"'; ;' Preparation of 3,17~-Bis[(3-pyridinylcarbonyl)oxy]-19-nor-17-pregna-1,3,5(10)-trien-20-yne:
To 2.0 9 (6.7 mmol) of ethinyl estradiol dissolved in 50 mL of dry pyridine were added 6.16 9 (0.027 mol) of nicotinic anhydride and a catalytic quantity of 4-(d1methylamino)pyr~dine (DMAP). The solut~on was warmed gently to 50C to effect solution. After 2 weeks, the pyridlne solut1On was poured over ice and the solid produced was collected by filtration. The solid was dried over P205 in vacuo to give 3 9 (85X) of an off-white powder.
EXA~PLE 67 Preparation of 3-Hydroxy-17B-~(3-pyridinylcarbonyl)-oxy]-19-nor-17a-pregna-1,3,5(10)-trien-20-yne:
~ o 200 mL of O.St methanolic KHC03, 2.0 9 (3.9 mmol) of the product of Example 66 were added. After 6 `
~' .
-~.. . . .
1 3 3 ~
hours, the slurry was dlluted with 200 mL of water and the mlxture was extracted with chloroform. The organic layers were combined, dr~ed over MgS04 and concentrated in vacuo. The resulting oil was triturated with hexane to g~ve 1.48 9 (94X) of a wh~te solid. NMR and UV
spectra and elemental analysis confirmed the identity of the title compound.
EXA~PLE 68 Preparat~on of 1-Methyl-3-{~(19-nor-17-pregna-1,3,5(10)-tr~en-20-yn-17B-yl)oxy]carbonyl~pyridinium iodide:
To 50 mL of acetone, 1.0 9 (2.5 mmol) of the ~
product of Example 67 was added, followed by 2 mL of '.`I.!.~i*~`
methyl ~odide. The reaction mixture was refluxed for 12 hours. The solid which formed was collected by flltrat1On, y1elding 1.15 9 (85X) of the quaternary salt as a yellow solid havlng the structural formula f ~ CH3 ~-C~CH
HO~ '' ~
The asslgned structure was conflrmed by UV and NMR
spectral analyses and by elemental analysls.
1 3 ~
Preparation of 3-Hydroxy-17B-~ methyl^1,4 dihydropyridin-3-yl)carbonyl]oxy~-19-nor-17~-pregna~
1 ,3,5[10)-trien-20-yne~
To a cooled suspension of 1.0 9 (1.8 mmol) of the product of Example 68 in 100 mL of SO:S0 water: tert-butanol, 0.77 9 of ~aHC03 and 0.96 9 of Na2S204 were added. The react~on m~xture was stirred at 0C for 1 hour, then was extracted twice with 100 mL portions of CH2C12. The organic extracts were combined, dried over ~gS04 and concentrated under reduced pressure to sive 520 mg (69%) of the t~tle compound as a yellow foam. : -The product was assigned the structure : -:
o ~ CH3 OC~ ~ `
CH3 l ~-C--CH
~ ' HO
lS wh~ch was ~n accord wlth UV and N~R values as well as elemental analysis.
,: : :: . : ~ , . . .
; ~ , , ~ . .
1 ~ 3 ~
' Preparation of 3,17~-Bis[(3-pyridinylcarbonyl)oxy]-estra-1,3,5(10)-triene~
To 5.3 9 (0.03 mol) of nicotinoyl chloride in 30 mL of dry pyridine at 0C were added 2.0 9 (0.0073 mol) of ~-estradiol. The reaction mixture was refluxed for 1 hour, then was poured over 100 ~L of ice water, and the resulting precipitate was collected by filtration. The precipitate was dried in vacuo over P205, affording 3.18 9 (90X) of the title compound melting at 148-150C.
Preparation of 1,1'-Dimethyl-3,3'-{[(estra-1,3,5(10)-triene-3,17~-diyl~dioxy]dicarbonyl~dip~ridinium diiodide:
To 50 mL of acetone and 2 mL (0.032 mol) of methyl iodide, 2.0 9 (0.004 mol) of the product of Example 70 were added. The solution was heated at reflux overnight. The precipitate which formed was filtered, washed with acetone and dried to give 2.75 9 (88%) of the quaternary salt melting at 251-252C and having the structural formula - ~C~13 CH O~J~ -as confirmed by UV, NMR and elemental analyses.
''' ~,':,''. ~
-:
13~1 -155~
EXA~PLE 72 Preparation of 3,17B-Bis~t(l-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy~estra-1,3,5(10)-triene:
One gram (1.31 mmol) of the product of Example 71 was dissolved in 100 mL of dry acetonitrile. To that solut10n, which was flushed with nitrogen, 0.28 9 (1.31 mmol) of l-(phenylmethyl)-4-(aminocarbonyl)-1,2-dihy-dropyr~dine was added, and the reaction mixture was st~rred at 0C for 1 hour. Removal of the solvent under reduced pressure afforded a solid, which was suspended in methylene chloride and removed by filtration. The filtrate was chromatographed several times on a neutral alumina column prepared with methylene chloride. Purification and evaporation of the solvent ln vacuo gave a solid foam. The product had the formula ~3 ~ ' dS confirned by UV, NMR and elemental analyses.
1333 ~
-156- ~ `
Preparation of 3-(Phenylcarbonyloxy)-17B-[(3-pyridinyl- ,.~ tr~.
carbonyl~oxy]estra-1,3,5(10)-triene:
Estradiol benzoate (2.5 9, 6.6 mmol) was dissolved in 50 mL of dry pyridine, then 1.66 9 of nicotinic anhydride and a catalytic amount of 4-(dimethylamino)-pyridine (DMAP) were added. The reaction mixture was -st~rred for 5 days at room temperature, then was poured into ice water. The solid which formed was collected by filtration and dried in vacuo, yielding 3.01 9 (94~) of the product as a white solid melting at 151-154C.
EXA~PLE ~4 Preparation of l-Methyl-3-(~3-(phenylcarbonyloxy)-estra-1.3,5(10)-trien-17g-yl]oxy~carbonyl)pynidinium iodide:
The product of Example 73 (1.5 9, 3.1 mmol) was suspended in 2.5 mL of acetone. Then, 2 mL of methyl iodide were added and the reaction mixture was refluxed overnight. The yellow solid (1.8 9, 93X) was collected by filtration and drled in vacuo. UV, NMR and elemental analyses confirmed that the product had the ass~gned structure:
C~3 .
OC~ 1~
~~
!~
;?~' ::' : ' : :~ ~ ~ : . - ' :
1~31~
EXA~PLE 75 Preparation of 3-(Phenylcarbonyloxy)-17B-~[1-methyl- -~
1,4-dihydropyridin-3-yl)carbonyl~oxy~estra-1,3,5(10)~
triene:
The quaternary salt prepared in Example 74 (1.2 9, 1.93 mmol) was suspended in 100 mL of 50:50 tert-butyl alcohol/water. Then, 0.81 9 of NaHC03 and 1.0 9 of Na2S204 were added and the reaction was allowed to cont~nue for 1.5 hours. The resultant solution was extracted with CH2Cl2~ and the organic phase was dried over MgS04 and concentrated in vacuo to afford 650 mg of title compound as a yellow foam. The identity of the product was confirmed by UV, NMR and elemental analyses. It was assigned the structure:
~3 ''~' , ~ H3 ~Co~J ''' ~ "
EXA~PLE 76 Preparation of_H-(2-~4-tBis(2-chloroethyl)amino]-butanoyloxy}ethyl)-3-pyridinecarboxam~de~
Chlorambucil (2n g, 0.0657 mol) was dissolved in 800 mL of dry acetonitrile, then 13.1 g (0.079 mol) of N-(2-hydroxyethyl)-3-pyr~dinecarboxamide were added.
Acetonitrile was added until the solution was clear.
The total volume of aceton~trile used at this stage was ..,'~
,, ", '~
' a '~
~ ~ 3 ~
850 mL. To the stirred solution, maintained over argon, there were added 1.492 9 (0.0723 mol) of di-cyclohexylcarbodiimide and 0.802 9 (0.0066 mol) of 4-(dimethylamino)pyridine (DMAP). The reaction mixture was stirred overnight at room temperature under dry conditions, and the progress of the reaction was followed by thin layer chromatography. At the end of the reaction period, the solid which formed was removed and washed with 50 mL of cold acetonitrile. The filtrate was evaporated in vacuo at 30C, and the yellow solid thus obta~ned was dissolved in a minimum amount (15 mL) of 8:2 chloroform/tetrahydrofuran and appl~ed to a column packed with 900 9 of sllica gel.
The column was eluted with 8:2 chloroform/tetrahydro-furan. The adduct and chlorambucil were eluted withinthe first 500 mL, and the desired ester was then collected upon evaporation of the eluent under ~ ~-vacuum. The title compound was obtained in 82.7~ yield as a yellow solid melt~ng at 73-75C. It had the formula (Clt~2c ~)2~(c`2)3-c-oc zCrz~
~ ' ~'~ ' ~ ' '' ~ ' ' i' 1 'I `
_ 1 33 ~
EXAMPLE ~7 Preparation of 1-Methyl-3-[(N- ~ t4-(~4-bis(2-chloro- -ethyl)amino]~phenyl)butanoyloxy]ethyl~)carbamoyl]-pyridinium methanosulfate:
The product of Example 76 t2 9, 0.04 mol) was dissolved in 200 mL dry acetonitrile. Dimethyl sulfate ~0.613 9, 0.0486 mol) was added and the mixture was refluxed overnight. The reaction was followed by thin layer chromatography (8:2 chloroform/tetrahydrofuran) until no more unquaternlzed ester remained. Evapora-tion of the solvent in vacuo gave a residue which was washed several times with dry ether. The red viscous liquid which remained was stored o~er argon for further use. Yield 97.35X. The product was identified as the desired quaternary salt by NMR analysis. It was assigned the structural formula~
(ClCH2CH2)zN~(CH2)3-C-OCH2C112~HC~ CH3504 CH3 ;
EXA~PLE 78 Preparation of l-Methyl~3-~(N-~2-L4-(~4-[bis(2-chloro-ethyl)]amino~phenyl)butanoyloxy]ethyl~)carbamoyl]-1,4-d~hydropyridine:
The quaternary salt prepared in Example 77 (2.49 9, 0.0043 mol) was dissolved in 350 mL of water.
~1trogen was bubbled through the solution throughout the reactlon period. The aqueous solut~on was cooled :
.
'~: - ~ - ': ~ ,. .
133~`~6~
over 1ce to 5C, then 2.17 9 (0.026 mol) of NaHC03 were added over a 5 mi nute per~od. followed by 2.997 9 (0.017 mol) of sodium dithionite over a 10 minute period. The reactlon mixture was mainta~ned at 5C for 120 minutes, then the layers were separated. The aqueous layer was extracted 4 times with ethyl acetate. The ethyl acetate extracts were combined, dried over MgS04 and evaporated to dryness in vacuo.
~he sem~-solid thus obtained was washed several times wlth dry ether to give the title compound as a yellow solid melting at 90-g2C and having the structural formula~
(clcH2cH2)2N~3(cH2)3-c-ocH2cH2NHc~
CH3 ~
EXAMPLE ~9 Preparation of N-(4-Hydroxycyclohexyl)-3-pyridinecar-boxam~de:
Trans-4-aminocyclohexanol hydrochloride (5.05 9, 0.033 mol) was suspended ~n 50 mL of ethanol, then 33 mL of 1~ NaOH were added slowly while cooling the reactlon mixture to 10C. The homogeneous mixture was evaporated to dryness, then three port~ons of a 50:50 m~xture of benzene and acetone were added and evaporated to dryness in vacuo each time. The dry -solld was extracted w~th 100 mL of chloroform and f~ltered, and the filtrate was evaporated to dryness.
The residue was tr~turated with ether and dr~ed to give ~, . . .
13 3 ~
3.50 9 (91.23X) of the free aminocyclohexanol ~elt~ng at 111-112C.
Nicotin~c ac~d (2.14 ~. 0.017 mol) was suspended ~n 75 mL of dry tetrahydrofuran, then 1.76 9 of freshly distilled triethylam~ne were added. The clear solution thus obta~ned was cooled to -4C ~n an 1ce bath under argon, then ethyl chloroformate ~1.88 9, 0.014 mol) in 10 mL of tetrahydrofuran were added such that the temperature did not go above 0C. The free ~ -am1nocyclohexanol (2.0 9, 0.017 mol) was added as a powder to the cold reaction mixture, which was allowed to come to room temperature and stirred for 2 hours.
~he precipitate which formed was collected by filtrat1On, dissolved in 28 mL of hot water and recrystall~zed as 3.25 9 (85X) of fine colorless needles melt1ng at 208-210C and having the formula ~,o~
~ .
';
as confirmed by elemental analysis.
Preparation of N-(4-~4-(~4-[Bis(2-chloroethyl)~amino~
phenyl)butanoyloxy]cyclohexyl~-3-pyrid~necarboxam~de:
Chlorambucll (1.38 9, 0.0045 mol) and N-(4-hydroxycyclohexyl3-3-pyr~dinecarboxam~de (1.1 9, 0.0049 mol) were mixed together w~th 1.03 9 (0.00459 mol) of dlcyclohexylcarbodilmide and 55 mg (0.00045 mol) of 4-(d~methylamino)pyrid~ne (DMAP) in 50 mL of freshly distilled acetonitrlle. The reaction mixture was stirred at room temperature in the presence of argon 5 for 2 days. The progress of the react~on was followed -by thin layer chromatography using 8:2 chloro~
form/tetrahydrofuran. At the end of the reaction per~od, the precip1tate was removed by filtrat~on and the filtrate was evaporated to dryness in vacuo at low ~ -10 temperature. The res~due was appl~ed to a silica ~ ~ -column and eluted with 8:2 chloroform/tetrahydrofuran.
The appropriate eluting portlons were combined and evaporated to dryness in vacuo. The product (1.86 9, 81X) was obtained as a light cream-colored powder melting at 120-122C and having the formula o ' (ClCH2CH2)2N ~ (CH2)3-C'C~ :
I~N~
: ::
The ident~ty of the product was confirmed by elemental analysis.
.~
XA~PLE 81 ~
P~ ration of l-Methyl-3-(N-~4-t4-(4-l[bis(2-chloro- ' '' ethyl)]amino~phenyl)butanoyloxy]cyclohexyl~carbamoyl)-pyridinium methanosulfate:
. ~
The product of Example 80 (1 9, 0.0019 mol) was d~ssolved in 30 mL of dry acetonitr~le and 0.249 9 (0.0019 mol) of dimethyl sulfate was added. The '~
mixture was refluxed under argon until thin layer '~
chromatography (8:2 chloroform/tetrahydrofuran on -,;~
silica) indicated quaternization was complete (about one and one-half days). The solvent was evaporated in '~r~
vacuo, leav1ng an orange residue which was washed several times with anhydrous ether and evaporated in vacuo. The quaternary salt (1.04 9, 80.6X) was obtained as a sticky yellow mass. It had the structural formula~
(clcH2cH2)2ll ~ (cH2)3~
H3 ::
EXA~PLE 82 Preparation of l-Methyl-3-(N-¦4-t4-(4-~bis~2-chloro-ethyl)]am1no~phenyl)butanoyloxy]cyclohexyl karbamoYl)-1,4-dihydropyridine:
The quaternar~ salt prepared in Example 81 (0.34 9, 0.0005 mol) was dissolved in 0.5 mL of acetonitrile and taken up in 20 mL of degassed water (bubbling N2) 25 cooled to 0C. To the ,stirr1ng solution, sod~um r-. .
:, -, ~ . - . .,, . - :
,r ~
Y~
~ :. ........................... r bicarbonate (0.27 9~ 0.003 mol) was added, followed ~ `
f~rst by 0.37 9 (0.002 mol) of sodium dithionite and then by 20 mL of ethyl acetate. After 90 mi nutes, the organic phase was removed and the aqueous phase was 5 extracted 3 to 4 times with ethyl acetate. The ethyl -~
acetate extracts were comb1ned, dried over sodium sulfate and evaporated 1n vacuo. The residual solid ~-was washed several times w~th anhydrous ether and dr1ed. The res~due thus obta~ned was applied to a neutral alum~na column and eluted with chloroform under pressure. Evaporation of chloroform left 0.18 9 (65X) of a hygroscopic yellow solid of the formula C1CH2CH2 ~(CH2)3-C-O ~
C1CH2CH2 ::
I--~HC~ ~:
The ~dentlty of the product was conf~rmed by UV
ana1ys1s.
Preparatlon of N-(2-Hydroxy)propyl-3-pyr~dinecarboxa-m~de: -To 4.29 9 (0.039 mol) of nicot~nic acid suspended ln 1~0 mL of dry tetrahydrofuran, 4.04 9 (0.039 mol) of freshly dlst~lled tr~ethylam~ne was added ~n one port1On. The resultant clear solutlon was cooled to -4C ~n an lce bath under argon. Ethyl chloroformate (4.33 9, 0.039 mol) ~n 25 mL of tetrahydrofuran was l ~
r' ~ ~
added at such a rate that the temperature of the solu~
t1On d1d not exceed 0C. Then, 3 9 (0.039 mol) of 1-am1no-2-propanol were added directly to the cold ~
react1On mixture. The react10n m1xture was allowed to ~ -come to room temperature and stirred for 2 hours. The preclpitate was removed by filtration and the filtrate was evaporated 1n vacuo. The o11y residue was washed several t~mes with anhydrous ether and allowed to stand. The t~tle compound was obtained as a white, ;
hygroscop~c, low-melting, waxy solid melting at 40C
(6.11 9, 85X) and hav~ng the formula ~-NHCH2CHOH
N
EXA~PLE 84 Preparat~on of N-12-t4-(~4-tB~s(2-chloroethyl)]amlno~-phenyl)butanoyloxy~propyll-3-pyrid1necarboxamlde:
Chlorambucil (1.0 9, 0.003 mol) and ~-(2-hydroxy)-propyl-3-pyr~d1necarboxam1de ~0.065 9, 0.0036 mol) were comb1ned w1th 0.68 9 (0.003 mol) of dicyclohexylcarbo-d11m1de and 41 mg (0.0003 mol) of 4-(d1methylam1no)-pyr1d1ne (DMAP) 1n 40 mL of freshly distilledaceton1tr~1e. The react1On m~xture was st~rred at room temperature ln the presence of argon for 2 days, the progress of the react~on be1ng followed by thin layer chromatography on s~l~ca us~ng 8:2 methylene chlo-r~de/ethyl acetate. At the end of the react1On period, `:;
,~:
~D`~
1 3 3 1 ~
the preclp~tate was removed by filtration and the filtrate was evaporated to dryness ~n vacuo at 30C.
The residue was applied to a s11ica column and eluted with 8:2 methylene chloride/ethyl acetate. The appropriate elut~ng portions were combined and evaporated to dryness ~n vacuo. The t~tle compound was obta1ned as a sticky material in 84X yield (1.53 9).
It had the structural formula:
' (ClCH2CH2)2N ~ (CH2)3-COcHcH2~Hc ~ ; ~
10EXA~PLE 85 ;~
Preparation of l-Me_hyl-3-t(N-~?-~4-(~4-~bis(2-chloro-ethyl)]amlno~phenyl)butanoyloxy]propyl~)carbamoyl]-pyr~d~n~um methanosulfate:
:
The product of Example 84 (2.2 9, ~.0047 mol) was dissolved ~n 45 mL of dry aceton~trile, dimethyl sul-fate (0.59 9, 0.0047 mol) was added and the mixture was refluxed under argon. The progress of the reaction was followed by th1n layer chromatography us~ng 8:2 methy-lene chlor~de/ethyl acetate. After one and one-half days, the solvent was removed by evaporation 1n vacuo, leav1ng an orange res~due. The residue was washed thoroughly with anhydrous ether and was dr~ed in vacuo. The product, obtained as a yellow stlcky mass In 92.47S yleld. had the structural formula:
, "~
i ~
.
. . A
., 'i' i~ ~ '. ' ''~ '. ~ ~ ~ ' ' . ' . " ' ~ ~ ' ' ' ' .
-167~
(clcH2cH2)2H~(cH2)3-cocHcHzNH~
C~3 ~ N~ CH3S04 Z'H3 ~ `
EXAMP~E 86 Preparation of l-Methyl-3-[(N-~2-1{4-({4-~bis~2-chloro-ethyl)~am1no~phenyl)butanoyloxy]propyl~)carbamoyl]-1,4-dthydropyrtZdtne:
The quaternary salt prepared in Example 85 (2.399, 0.004 mol) was dissolved in 1 mL of acetonitrile and then taken up tn 100 mL of degassed water (bubbling N2) and cooled to 0C in an tce-water bath. Sodtum btcarbonate (2.03 9, 0.024 mol) was added to the sttrrtng solutton, followed by 2.81 9 (0.016 mol) of sodtum dithton~te. To the resultant mixture, 60 mL of ethyl acetate were added. The reactton was allowed to continue for 90 mtnutes, then the phases were separated and the aqueous phase was extracted 3 or 4 ttmes with 30 mL porttons of ethyl acetate. The combtned ethyl acetate extracts were drted over sodium sulfate and evaporated tn vacuo. The restdue was applted to a neutral alumtna column and eluted with chloroform under pressure. The approprtate fracttons were evaporated to gtve a hygroscoptc yellow solid tn 60X yield. The product had the formula:
'~
.
:'~
. :.
:
; r. , ~
. ' i' ' '~
~ "' ~ "~
~3.~
;
~CIC 2C 2)2N~C 2)3~ CI C 21 as conf~rmed by UV analysis.
EXA~PLE 87 Preparat~on of ~-(2-Hydroxy-2-phenyl)ethyl-3-pyridine-carboxam1de:
Nicot~nic acid (1.79 9, 0.014 mol) was suspended ~n 60 mL of dry tetrahydrofuran and 1.48 g (0.014 mol) of freshly dist~lled triethylam~ne were added. The clear solutlon wh1ch resulted was cooled to -4C in an lce bath and argon was bubbled through it cont~nuously.
Ethyl chloroformate (1.58 9, 0.014 mol) in 10 mL of tetrahydrofuran was added at such a rate that the -temperature d~d not exceed 0C. Then, 2.0 g (0.014 mol) of 2-am~no-1-phenylethanol were added as a solutlon 1n 5 mL of tetrahydrofuran. The reaction m1xture was allowed to warm to room temperature and was st~rred for 2 hours. The prec~p1tate which formed was removed by f11trat1On and the f11trate was evaporated in vacuo to g1ve 3.22 9 (91.lS) of a wh1te crystall1ne sol1d melt~ng at 122-124C and havlng the formula : :`
13 31~ ~ ~
Elemental analys~s confirmed the identity of the product.
EXA~PLE 88 Preparation of n-(~2-phenyl-2-~4-(l4-~bis(2-chlor 5 ethyl)~am~no~phenyl)butanoyloxy]~ethyl)-3-pyridinecar- -~
boxamide:
Chlorambucil (1.0 9, n.oo3 mol) and N-(2-hydroxy-2-phenyl)ethyl-3-pyridinecarboxamide (0.88 9, 0.003 mol) were combined w~th 0.68 9 (0.003 mol) of dicyclo-hexylcarbodiimide and 41 mg (0.0003 mol) of 4-(dimethylamino)pyridine (DMAP) in 35 mL of freshly distilled acetonitrile. The reaction mixture was stlrred at room temperature under argon for 3 days.
Th1n layer chromatography using 8:2 methylene chloride/ethyl acetate was used to follow the progress of the reaction. The precipitate which formed was removed by filtrat10n and the acetonitrile was eva-porated in vacuo. The residue thus obtained was applied to a s11ica column and eluted with 8:2 methylene chloride/ethyl aceta~e. The appropriate fract10ns were collected and evaporated in vacuo to g1ve a 119ht tan powder (1.21 9, 70X) melting at 99-101C and hav~ng the formula ~.
~ClC 2cH2)2K~(cN2)~2NN ~) ::
~`
1 3 ~ 1 ~ 6 4 Preparation of 1-Methyl-3-~(N-j2-phenyl-2-~4-(~4-tbis-(2-chloroethyl)]aminolphenyl)butanoyloxy]~ethyl)carba-moyl]pyrid~nlum methanosulfate:
S The product of Example 88 (0.5 9, 0.00094 mol) was dissolved in 20 mL of dry acetonitrile and 0.12 9 (0.00094 mol) of dimethyl sulfate was added. The mixture was ref1uxed under argon for 2 days, then the solvent was removed by evaporation in vacuo. The res~due which was obtained was washed several times with anhydrous ether and dried to give 0.54 9 (9lX) of a sticky l~ght yellow product having the formula (CI~N~Nz)zN~(~N2)3~Z ~ CK350~
EXA~PLE 90 Preparat~on of l-~ethyl-3-~(N-~2-phenyl-2-~4-(~4-tbls(2-chloroethyl)~am~nolPhenyl)butanoyloxy]leth carbamoyl-l,4-d~hydropyr~d1ne:
The quaternary salt prepared in Example 89 (0.53 9, 0.0008 mol) was dissolved in 0.5 mL of acetonitrile and taken up in 20 mL of degassed, deion1zed water cooled to 0C. Sodium b~carbonate was added to the st1rring solutlon at 0C, followed by 0.56 9 (0.0032 ol) of sodium dithionite. Then, 20 mL of ethyl acetate were added and the reaction was allowed to continue for 2 hours. The organic phase was removed and the aqueous phase was extracted several tlmes with ethyl acetate (total volume 70 mL), until color was no S longer observed in the organic phase. The ethyl acetate extracts were comb~ned and dried over sodium sulfate and evaporated ~n vacuo. The residue was applied to a neutral alum~na column and eluted with chloroform. Evaporation of chloroform gave 0.2 9 (45X) of a hygroscop~c orang~sh yellow compound of the formula (C~2cNi!)2~ 2~3~32~
The ~dentity of the product was conf1rmed by UY
spectral analysis.
EX~PLE 91 Preparat10n of N-(2-Hydroxyethyl)-3-pyridinecarboxa-m~de:
A neat m1xture of 2-amlnoethanol (6.1 9, 0.10 mol) and ethyl nicot~nate (15.1 9, 0.10 mol) was refluxed overn~ght. As the m1xture was cooled to room tempera-ture, the product prec~p1tated as a crystall1ne sol~d. It was ftltered, washed wlth ether and then recrystalllzed from 2-propanol/ether. The f~nal product was collected by vacuum f~ltrat10n and washed w~th ether. The drled, wh~te compound weighed 10.7 9, j,",",, .. , ~ .. . . ~ ; , .. . .. . . . . . ..
", ~ , ~,, " "~ , " ~ ",~
1331~6A
result~ng 1n a 64.5X yield; mp 88.5-89.5C (l~t. value 92C).
EXA~PLE 92 Preparation_of ~+)N-t2-(6-Methoxy-~-methyl-2-naph-thalenylacetoxy)ethyl]-3-pyridinecarboxamide:
10Naproxen (2.30 9, l~.0 mmol) was coupled with the product of Example 91 (1.71 9, 10.0 mmol) using di-cyclohexylcarbod~m~de (2.30 9, 11.0 mmol) and 4-~dl-methylamino)pyridine (122 mg, 1.00 mmol) ~n aceton~-trile (150 mL). The react~on was stirred at room temperature for 48 hours. The precipitate was fil-tered, rinsed w~th acetonitrile and dried to a weight of 2.3 9. The solvent was removed under reduced pressure and the residual clear oil was stirred with anhydrous ether. The resulting white solid was vacuum ;
20 f~ltered, washed w~th ether and air-dried. The crude -product we~ghed 2.80 9. The compound was recrystal-l~zed from 2-propanol. The final product was fil~ered, washed w1th 0.5X aqueous sodium bicarbonate, water, and f1nally w~th ether. The compound was dr~ed ~n a 25 deslccator over P205. The recrystalllzed ma~er~al ~i welghed 2.40 9 result~ng ~n an overall y~eld of 63.4X;
mp 79-82C.
EXA~PLE 93 - ~ -Preparat1On of N-(2-~1-(p-Chlorobenzoyl)-5-methoxy-2-methyl-3-lndoly!]acetoxy~ethyl)-3-pyr~dinecarboxamtde~
A react~on of ~ndomethacln (1.79 9, 5.00 mmol) and the product of Example 91 (0.830 9, 5.00 mmol) was carr1ed out, uslng d~cyclohexylcarbodi~m~de (1.10 9, .,, . ~ ~ , .. . . . . . .
1331~4 5.50 mmol) as the coupling agent and acetonitrile as the solvent. The first two reactants were dissolved completely and the solution was then cooled to 0C.
The dicyclohexylcarbodiimide was added and the mixture was stirred overnight. The reaction was allowed to continue for 48 hours. The precip~tate (1.2 gl was removed by vacuum filtration. The solvent was removed from the filtrate under reduced pressure leaving an oily residue. The product was solidified by stirring w~th anhydrous ether. It was filtered, air-dried and recrystall~zed from ethanol/ether. The final product was vacuum filtered, washed with ether, and air dried. The product weighed 1.65 9, giving a 65.2X
yield; mp 123-125C. ~ -EXA~PLE 94 Preparation of 1-Methyl-3-~N-~2-(6-methoxy--methyl-2-naphthalenylacetoxy)ethyl]carbamoyl~pyridinium iodide:
The quaternizat~on of the naproxen ester prepared ~n Example 92 (1.0 9, 2.6 mmol) was carried out using methyl iodide (2.3 9, 16 mmol) in acetone (45 mL). The solution was heated to reflux for 20 hours. Methyl ~odlde (I.l 9, 8.0 mmol) was again added to the react~on flask. The prec~p~tated product was filtered after an add~tlonal 4 hours of reaction time. The off-wh~te powder was dried. The mater1al weighed 2.2 9 and was found to be analytically pure without recrystalli- ~
zatlon. The solvent was removed from the acetone - - -filtrate and the residue was sol~dlfied with anhydrous ~ --ether. The result~ng dark yellow powder was dissolved in water and washed w~th ether (4 x 30 mL). The water was then removed under vacuum g~ving 0.2 9 of a lighter 1 3 ~
:.~
yellow powder. The overall yield of the reaction was 93X; mp 169-170C. The product had the structural formula O o ~ OCH3 : ~:
~CNH(CH2)20CCH~-- ~ ~
5 as further conf1rmed by UV, NMR and elemental analyses. ~ ;
EXAMPLE 95 ~
::
Preparation of l-~ethyl-3-[N-(2-~[1-(p-chlorobenzoyl~
5-methoxy-2-methyl-3-indolyl]acetoxy~ethyl)carbamoyl]-pyr~d1n~um ~odlde~
The quaternizat~on of the indomethacin ester pre-pared in Example 93 (0.50 9, 1.0 mmol) was carried out 1n acetone, using methyl ~od~de (1.7 9, 12 mmol). The react10n was refluxed overnlght. The solvent was re-moved under reduced pressure and a yellow solid was ~ ;
15 obta1ned. The product was recrystall~zed us~ng ethanol~ ~
and a very small amount of ether. Small mold-11ke -~ -crystals were ~bta1ned whlch were 119ht yellow ~n color. The react10n gave 0.43 9 or a 66X y~eld of the purlf~ed mater~al; mp 178-179C. UY, NMR and elemental 20 analyses conf~rmed that the product had the structural ;
forl~ula:
~ ~ ' .
r,~
H3CO~ 2cO~cH2)2NH~
C=O 3 (~ `
EXA~PLE 96 Preparation of l-Methyl-3-jN-~2-(6-methDxy-~-methyl-2-naphthalenylacetoxy~ethyl]carbamoyl~-1,4-dihydro-pyridine:
The quaternary salt prepared in Example 94 (780 mg~ 1.5 mmol) was dissolved in degassed, deionized water (200 mL) and acetonitr11e (10 mL~. Sodium d~thionite (780 mg~ 4.5 mmol) and sodium bicarbonate (630 mg, 7.5 mmol) were combined and added to the solution at room tempera~ure. The reaction was ~ ~
cont~nued for 1 hour, while nitrogen gas was slowly ~`
bubbled through the solution. The partially pre- ;
c~pitated product was extracted repeatedly w~th ether (8 x 30 mL). The extracts were comb~ned, washed with water (25 mL) and dr1ed over magnesium sulfate. The ;~
dry~ng agent was f11tered and the solvent was removed from the filtrate under reduced pressure. The oily res~due was tissolved ln methylene chloride (3 x 5 mL) and removed under reduced pressure. The resulting foam was rinsed w~th anhydrous ether (3 mL) and the solvent was removed under vacuum. The final product weighed -~; 390 mg, g~v1ng a 66X yield. The hygroscopic solid foam was stored under n~trogen at -100C. It had the struc-tural formula:
, ~
''~
. ~ ~
1~3~
¢~ INH(CH2)2O~C ~ ~
as conf~rmed by UV. NMR and elemental analyses.
EXA~PLE 97 Preparatlon of 1-~ethyl-3-~N-(2-~[1-(p-chlorobenzoyl)~
5-methoxy-2-methyl-3-~ndolyl]acetoxy~ethyl)carbamoyl]-1,4-dihydropyrid~ne The indomethac~n quaternary salt prepared in Example 95 (140 mg, 0.2Z mmol), was dissolved in a ~inimum amount of water:acetonitrile (8:2). The water had been bubbled with nitrogen for 20 minutes prior to its use. Sodium b~carbonate (91 mg, 1.1 mmol) and sodium dithionite (110 mg, 0.6~ mmol) were added to th~e ~ ;
solut~on while stirr~ng at 0C. The solutton was then allowed to warm to room temperature. The reactlon was cont1nued for about 1 hour. Some of the product had preclp~tated dur~ng the react1On. Th~s was dlssolved ln ethyl ether. The water layer was extracted several t~mes w~th ether unt~l no more yellow color transferred to the organic layer. The ether portions were combined and dr~ed with magnesium sulfate, flltered and the ether was removed under reduced pressure. The result-1ng o~l was d~ssolved 1n acetone and the solvent was removed (2 x 10 mL) under reduced pressure to form a dry foam. The f~nal product we~ghed 92 mg. The y~eld was 82X; mp 60-65C. The product had the formula:
13315~
H3Co_~cH2~0(cH2)2HH
Cl as confirmed by UV. NMR and elemental analyses. ;~
Preparation of N~ Hydroxyc~clohexyl)methyl~-3-S pyridinecarboxam~de:
To 1.48 9 (0.012 mol) of nicot~nic acid suspended ~'n 50 mL of dry tetrahydrofuran, 2.44 9 (0~014 mol) of freshly dist~lled triethylamine were added, with ~ -st~rr~ng. The resultant clear solution was cooled to -4C in an ~ce bath, under argon. Then, 1.3 9 (n.012 mol) of ethyl chloroformate in 10 mL of tetrahydrofuran were added at such a rate that the temperature of the reactlon m~xture did not exceed 0C. To the cold react~on m1xture, 2.0 9 (0.012 mol) of 1-am~nomethyl-1-lS cyclohexanol hydrochloride were added directly as apowder. The reaction mlxture was allowed to warm to room temperature and st1rred for 2 hours, then the tr7ethylam1ne hydrochlor~de wh~ch formed was removed by f11trat~on and the f~ltrate was evaporated tn vacuo to afford a white sol~d. The sol~d was recrystall~zed from water, washed w~th acetone and ether and dried.
The t~le compound, obtained 1n ,35S y~eld (2.4 9), melted at around 110C, and was further character~zed by the structural formula i~' , ~ :
~ ' .. ...
133~
OH
CH2HH~
N
as confirmed by elemental analysis. ;
EXA~PLE 99 Preparation of N-(~1-[4-(~4-~Bis(2-chloroethyl)]amino~
phenyl)butanoyloxy]cyclohexyl~methyl~-3-pyridine-carboxamide Chlorambuc~l (1.18 9, 0.0038 mol) and hydroxycyclohexyl)methyl]-3-pyridinecarboxamide (0.99 9, 0.004 mol3 were combined with 0.8 9 (0.0038 mol) of d~cyclohexylcarbodiimide and 47 mg (0.00038 mol) of 4-(dimethylam~no)pyridine (DMAP) ~n 60 mL of freshly distilled acetonitrile. The reaction mixture was stirred at room temperature under argon for 7 days. At the end of that t1me, the prec1p1tate which formed was separated by filtratlon and the filtrate was evaporated to dryness at low temperature ~n vacuo. The res~due was applied to a silica column and eluted, f1rst w1th 8:2 methylene chlor1de/ethyl acetate, then with 8:2 chloroform/tetrahydrofuran. The appropriate elut~ng port~ons were comb1ned and evaporated to dryness in vacuo. The t~tle compound was obta~ned ~n 26X yield as a light yellow solid melt1ng at 92-94C.
~ It had the structural formula :~ :
~L3~
O-C-(CH2)3~ ~C1i2CH2 ~y \CH2CH2Cl .
C1~2NH~
as confirmed by elemental analysis.
Preparation of l-Methyl-3-[N-(~ 4-(4-{~bis(2 chloroethyl)]amino~phenyl)butanoyloxy]cyclohexyl~
methyl~carbamoyl]pyridinium methanosulfate:
.:
To 0.69 9 (0.0013 mol) of the product of Example ';!~
99, dissolved in 25 mL of dry acetonitrile, was added 0.17 9 (0.0013 mol) of dimethyl sulfate. The mixture was refluxed unt~l the reaction was complete (approxi-mately 2 days), as evidenced by thin layer chromato-graphy us~ng 8:2 chloroform/tetrahydrofuran. The solvent was removed by evaporation ~n vacuo to afford an orange res~due, whlch was washed several t~mes w~th anhydrous ether and drled. The product was obtained as a st~cky yellow mass (85X, 0.72 9) having the formula:
g ~ /CH2CH2Cl O-C-(CH2)3~ ~CH2CH2Cl ~ :
Z~tU3SO~- ~
~ ' ~ '`' ~ ~ "
133~
- 1 ~ 0 - . :
Preparation of 1-Methyl-3-[N-(~ 4-(4-~[bis(2 chloroethyl)]am~no~phenyl)butanoyloxy]CyClOhe methyl)carbamoyl-1,4-dihydropyrid~ne~
The quaternary salt prepared in Example 100 (0.78 ~ -9, 0.0012 mol) was dissolved in 0.5 mL of acetonitrile and taken up ln 20 mL of water degassed with bubbling N2, cooled to 0C. To the stirring solutlon, 0.61 9 (0.0072 mol) of sodium blcarbonate was added, followed by 0.84 9 (0.0048 mol) of sodium dithionite and 20 mL
of ethyl acetate. The reaction was allowed to proceed for 75 minutes, then the layers were separated and the aqueous layer was extracted 3 to 4 times with 20 mL of ethyl acetate. The organic extracts were combined, dr~ed over sodium sulfate and evaporated in vacuo. The residue was applied to a neutral alumina column and eluted with chloroform under pressure. Evaporation afforded the product as a sticky yellow mass (0.31 9, 49X) havlng the structural formula:
O-C-~CH2)3~ ~CH2CH2Cl :
~ \cH2CH2Cl -\--I
CH2NH~
EX~MPLE 102 Preparatlon of 1-Methyl-3-{~2-(9-guanylmethoxy)ethoxy]
carbonyllpyridin~um lodlde:
~rigonelline anhydr1de dllodide (l-methylpyrl-25 dlnlum-3-carboxyl~c acld anhydride dllodlde) was ~:
.
1 33.t5~4 prepared as described by Brewster et al, Synthetic Commun~cations, 17(4), 451-455 (1987).
To a solution of 1.0 9 (4.4 mmol) of acyclovir in 25 mL of freshly dlstilled dry pyridine were added 2.27 5 g (4.4 mmol) of tr~gonell~ne anhydride diiodide and a - ~:
catalytic amount (5.4 mg, 4 mmol) of 4-(dimethyl-am1no)pyridine (DMAP). The resultant suspension was stirred for 4 days under argon at room temperature. As the react~on proceeded, the orange color of the anhydride was replaced with a yellow color. When all of the acyclovir had been consumed, the react~on was stopped, the precipitate (containing the product ester ~: :
plus the tr19Onell~ne formed as a by-product) was removed by filtration and washed with acetone and ether to remove DMAP. The yellow solid was then stirred in dry methanol at room temperature to remove tr~gonelllne, unreacted anhydride and acyclovir. The t~tle compound was obta~ned ~n 87X yield (1.82 9), melt~ng at 201-202~C. NMR and UV analyses conf~rmed that the product had the formula:
o ~Z~ 2'(Cr2'2~
~H3 :- `.. :.
31S~
, .
EXA~PLE 103 Preparation of 1-Methyl-3-~C2-(9-guanylmethoxy)ethoxy]-carbonyl~-1,4-dihydropyrid~ne~
To a solution of 1.58 9 (3.3 mmol) of the product S of Example 102 ln 120 mL of degassed water were added 1.69 9 (20.1 mmol) of NaHC03 in one portion. The m~xture was stirred at 0C wh~le 2.33 9 (13.18 mmol) of sodium dithlon~te were added over a S minute period.
The flask was flushed wlth nitrogen throughout the reaction process. The dihydropyridine product was insoluble ~n water and formed cream-colored crystals on top of the water layer. The crystals were separated by f~ltration and washed, first with ice-cold water and then w~th anhydrous ether. Drying over P205 in a dessicator maintained at -15C afforded 0.626 9 (54X) ~-of the title compound melting at 163-165C. NMR and UV
analyses confirmed that the product had the formula: -z~
H I O
CH2oCH2cH2 ~H3 .
Preparation of 5'-Pivaloyltrifluorothymidine~
To a stirr~ng solution of 150 mg of trifluoro~
thymidine tn S mL of pyridine was added a solution of ~' ~:
6`~
90 mg of pivaloyl chloride in 1 mL of pyridine, with cooling. Stirring was continued at room temperature for 10 hours. then the reaction mixture was poured into 20 mL of ice water and extracted with 50 mL of ethyl acetate. The extract was washed with water and dried over sodium sulfate. The ethyl acetate was removed and the residue was purified by silica gel column chroma-tography us~ng 20:1 chloroform/methanol as eluent. The title compound melted at 130-132C after recrystalliza-t~on from a m~xture of ether and n-hexane.
' EXA~PLE 105 Preparation of 3'-t3-Pyr1dylcarbonyl)-5'-pivaloyltri-fluorothymidine:
To a stirring solution of 450 mg of 5'-pivaloyl-trifluorothymidine ~n 10 mL of pyridine was added 1.0 9 of nicot1noyl chloride hydrochlor1de under ice-cooling. The reaction mixture was stirred at room temperature for 3 days, then was poured into 100 mL of 1ce water and extracted with 100 mL of ethyl acetate.
The extract was washed w1th water, dried over anhydrous sodlum sulfate and then evaporated in vacuo to g~ve an o11. Crystall~zatlon from n-hexane afforded S00 mg (87X) of colorless needles melt~ng at 175-177C. The product had the structure '~.' -," '~', -' .
1 3 3 1 ~
: -HN~CF3 OJ~ N
( CH3 ) 3C -~ -O CL~ O ~¦ . , '`Ir'l ' OC~
N ~ . ~
as further confirmed by NMR spectral analysis.
EXA~PLE 106 Preparation of 3'-(1-Methyl-3-pyridiniumcarbonyl)-5'-S pivaloyltrifluorothymidine iodide: :
To 440 mg of the product of Example lOS dissolved ~n 10 mL of acetone, 1.0 9 of methyl iodide was added. The mixture was refluxed for 10 hours, then the precip~tate which formed was collected by suct~on filtration to give 550 mg of the desired product as yellow leaves melting at 188-190C with decomposition.
NMR analysis confirmed that the product had the structural formula~
t 3 ~ r - 185- .
~3~CF3 (CH3)3C~ o ~ '~
~H
.. ..
Preparation of 3'-(1,4-D~hydro-l-methyl-3-pyridinyl- -carbonyl)-5'-pivaloyltrifluorothymidine~
To a stirring solution of 100 mg of the product of Example 106 1n a mixture of 20 mL of water and 20 mb of ethyl acetate were added 64 mg of NaHC03 and llS mg of Na2S204 under N2 gas. The resultant mixture was -stlrred at room temperature for 1 hour, then the organlc layer was separated and washed with water. The extract was dr~ed over anhydrous Na2S04 and evaporated 1n vacuo. The residue was trlturated wlth a mixture of - -ether and n-hexane and the yellow needles which formed were collected by suction filtration (50 mg, h2X). The product melted at 168-170C. NMR analysls confirmed that the product had the structural formula~
i' : .-:
~ ' , , ~ ' .
~ r. ~
~s 13~ $~
., o ,~F3 (CH3)3C--C--O CH~o ~
1`~'1 , :. .
' ; , -Preparation of 3'-Azido-3'-deoxy-5'-(3-pyridyl-carbonyl)thymidine A m1xture of 1.18 9 (4.42 mmol) of azidothymidine, 5 1.11 9 (4.86 mmol) of nicotinic anhydride and 0.15 9 (1.22 mmol) of N-(dimethylamino)pyridine was combined in 50 mL of pyrid~ne. The reaction mixture was st1rred at room temperature overn19ht. The clear, colorless reaction mixture was concentrated in vacuo to a semisol1d opaque mass which was triturated with ether overnight. ~he suspension was filtered and drled to give 1.97 9 of solid. Then, 1.0 9 of the solid was chromatographed over 20 9 of sil~ca gel using 10X ~-ethanol/chloroform as eluent. The desired fraction was 1solated as 0.53 9 of a white foam, wh1ch was crystal~
lized from a solvent mixture of ethanol, diethyl ether and hexane. The product melted at 138.5-141.5C and had the structural formula -t~
::
~ ~r.. ~
:1 3 ~
~3~ CH3 k~CH20C~
~13 '' ` ~' ''.
as confirmed by NMR and IR.
EXA~PLE 109 Preparation of 3'-Azido-3'-deoxy-5'-~(1-methyl-3- ~ -pyridinium)carbonyl~thymidine iodide~
A m1xture of 0.53 9 (2.0 mmol) of azidothymidine, 1.02 9 (2.2 mmol) of trigonelline anhydride d~iodide and 67 mg (0.5 mmol) of N-(dimethylamino)p~ridine was comblned 1n 25 mL of pyrid~ne. The reaction mixture was stirred at room temperature for 5 days, then was f11tered. The f~ltrate was concentrated in vacuo to a residue which was triturated w~th acetone overnight.
The result~ng suspens1On was filtered and the filtrate was concentrated 1n vacuo to a foam, which was treated 15 with water and filtered to remove i small amount of -insoluble material. The filtrate was concentrated in vacuo to a solid yellow glass (0.50 9, 49X). N~R and -UV analys~s conf~rmed tha~ the product had the formula:
. . , ;' , ~ : . ' ' ' ~ . ~ ~, ~ . ~ ' . ' .: ; - :
~31~
HNJ~/ CH3 k~CH20~) :
3 : :~
EXA~PLE 110 Preparat1On of 3'-Azido-3'-deoxy-5'-[~1-methyl-1,4-dihydro-3-pyridinyl)carbonyl]thymidine~
The crude azidothymid~ne quaternary derivative prepared according to the procedure of Example 109 -~
(1.45 9, 2.82 mmol) was dissolved in 50 mL of water and filtered. The filtrate was cooled in an ice bath and saturated w~th argon. Then, 100 mL of ethyl acetate and 2.90 9 of NaHC03 were added, followed by 1.45 9 of Na2S204 after 5 m~nutes. The reaction was allowed to proceed for 1 hour, then the ethyl acetate layer was removed and fresh ethyl acetate was added. This procedure was repeated to g~ve three organ~c extracts and a react~on t~me of 3 hours. The extracts were pooled and concentrated ~n vacuo to a foam we19h1ng 1.01 9 (92S). The foam was crystallized from methanol to g~ve the tltle compound of the formula . .
~ 3 ~
O
H20C ¢3 ' melting at 138-140C. Its structure was confirmed by ele0ental analysis as well as NMR and UV.
EXA~PLE 1 1 1 - ~ ~:
Preparation of Dopamine dipivalate, oxalate salt To a st~rred mixture of 28.1 9 of pivaloyl chloride and lsn mL of trifluoroacetic actd, 18.01 9 of dopamine hydrobromide were added. The mixture was stirred for 2 hours, then 14 mL of water were added and the mixture was concentrated in vacuo. The residual oil was dissolved in chloroform and washed with cold 10X KHC03 solution until C02 evolution ceased. The layers were separated and washed with water and the chloroform layer was dr~ed over MgS04, filtered and evaporated to dryness. The residue was taken up in 100 mL of ethyl acetate and 7 9 of oxalic acid were added together with 100 mL of ethyl acetate. The resultant solution was f~ltered to remove insoluble materials and 1.6 9 oxalic acid in 25 mL of ethyl acetate were added. The mixture was concentrated in vacuo and , .- . ., 1331~6~
.
cooled. The crystals which formed were isolated by filtration, giving 13 9 of the title compound. Cooling of the mother liquor afforded a second crop of crystals -(5.9 9). The product had the formula~
(CH3~3cco~cH2cH2hH2 . COOH
( CH3 ) 3CCO
EXA~PLE 112 Preparation of Chloromethyl N-~B-t3,4-bis(pivalyloxy)~
phenyl~ethyl~aminocarboxylate: ;
Dopamine dipivalate, oxalate salt (822 mg, 2 mmol) - 10 was suspended in 15 mL of dry tetrahydrofuran. Tri-ethylamine (278 mL, 1 mmol) was added, the mixture was stirred for 15 minutes and a further 278 mL (1 mmol) of tr~ethylamine were then added. Addition of ClC02CH2Cl (390 mg, 6 mmol) resulted in immediate formation of a heavy white precipitate and the evolution of gas. The reaction mixture was st~rred overnight at room tempera-ture, then the precipitate was removed by filtration and the filtrate was washed with 10 mL of 0.1 M hydro-chloric ac~d. Drying over magnesium sulfate and evaporat~on to dryness afforded 1.1 9 of a golden o~l of the structural formula .~ .
.~ .
,. . ~."~
~ i"~
... ~. ,.. , -~ -~ . .
~ 3~
- 1 9 1 ~
~''' '~''' -.
3)3CCO~CH2CH2NHCOCH2C~
(CH3)3CCO -~
O . ~'''.
:: .
The identity of the product was confirmed by elemental analysis.
EXA~PLE 113 s Preparation of N-~B~[3,4-Bis(pivalyloxy)ph_nyl]ethyl~-am~nocarbonyloxymethyl 3-pyridinecarboxylate: - -The chloromethyl carbamate prepared in Example 112 (1.26 9, 3.04 mmol) was combined with 10 mL of dry dimethylformamide and that mixture was added to a pre-mixed solution of nicotinic acid (375 mg, 3.04 mmol)and triethylamine (445 mL, 5X excess) in 15 mL of dry dimethylformamide at room temperature. The reaction mixture was stirred for 4 days, then the precipitate whlch formed was removed by filtration. The filtrate was evaporated to dryness and the residue was taken up in 2n mL of methylene chloride. ~hat solution was washed twice with 10 mL portions of water. Removal of ~ the solvent in vacuo afforded the t~tle compound of the - formula:
., -c~
:
.
.
:
o o o (c 3~3CC0~ 2CH2NHCOCH20C~
The identity of the product was confirmed by NMR.
EXA~PLE 114 Preparation of N-~B-[3,4-Bis(pivalyloxy)phenyl]ethyl~-aminocarbonyloxymethyl 1-methyl-3-pyridinium carboxylate iodide:
The product of Example 113 (860 mg, 1.78 mmol) was combined with 15 mL of dry acetonitrile and that mixture was treated with 223 mL (3.56 mmol) of methyl 10 iodide. The resultant mixture was stirred for 6 hours -at room temperature, then an additional 223 mL (3.56 mmol) of methyl iodide was added and the mixture was stirred overnight. Evaporation to dryness afforded, as an orange-red oi l, the ti tl e compound of the formul a `:
~ .
~ 3 ~
o o O
(C )3CCO~f H2CH2HHCOCH2 The identity of the product was confirmed by NMR ~ ~
analysis. ;~ ~-Preparation of N~ 3,4-Bis(pivalyloxy)phenyl~ethyl~-am1nocarbonyloxymethyl 1~4-dihydro-1-methyl-3-pyridine- -~
carboxylate The quaternary salt prepared in Example 114 (54 mg, n.o84 mmol) in 10 mL of water was treated at 0C
under nitrogen with NaHC03 (30 mg, 4 equivalents), Na2S204 (60 mg, 4 equivalents) and ethyl acetate (20 -mL). The reaction was allowed to proceed for 1 hour 20 minutes, then the aqueous and organic layers were separated and the aqueous layer was re-extracted with 20 mL of ethyl acetate. The combined organics were dried over magnesium sulfate. Removal of the solvent ~n vacu_ gave a red-orange oil which was taken up in chloroform and partially purified by elut~on with chloroform from a short neutral alumina column. The desired fract~on was subjected to preparative thin layer chromatography on silica using 80:20 chloro-form/acetone. The highest band was taken as the title compound of the structural formula~
o o o Il 11 11 ~
(CH3)3Cc0~3/CH2cll2NHcocH2 CH3)3CCO CH3 ~
O : ':
The identity of the product was confirmed by HPLC (high S pressure liquid chromatography) determinations for its ability to release dopamine from plasma and brain homogenate.
EXA~PLE 116 Preparation of 9-Fluoro-llB,17-dihydroxy-16 methyl-21-~(3-pyridinylcarbonyl)oxy]pregna-1,4-diene-3,20-dione:
nexamethasone (l 9, 2.5 mmol) was dissolved in 50 mL of dry pyridlne. To that solution were added 680 mg (3.0 mmol) of nlcotinic anhydride and a trace of 4-(dimethylamlno)pyr~dine (DMAP). The reaction was allowed to proceed for 4 hours, then the reaction mixture was poured over ice water and refrigerated overnlght. The sol1d was collected by filtration and dried to give l.n8 9 (87X) of product melting at 262-265C and hav1ng the structural formul~
~ .
~ .
~.. - ,... ~ ~ ~ .~ . . - .
, ?
.:,, ~, . ,:~. ~: ~': ~'` : ' ~: ' . ~ ' ,' ' ' ' ' . ,' ` '~ ' ~ ' ~ : ~ :
133~4 :
..
CH
HO~ 3 , .
~ ~ :~
0~ ; ~' ' as conf1rmed by elemental analysis.
EXA~PLE 117 Preparation of 1-Methyl-3-~(9-fluoro-llB,17-dihydroxy- ~ -16~-methylpregna-1,4-diene-3,20-dion-21-yl)oxy]-carbonyl ~yridinium iodide:
The product of Example 116 (0.74 9, 1.5 mmol) was d1ssolved in 50 mL of acetone to wh~ch 2 mL of methyl ~ :~
1Od1de was added. A small amount (10 mL) of CH3N03 was subsequently added to lncrease solubi7ity. The react~on was allowed to proceed for 2 days, then the sol1d was collected to g1ve 0.54 9 ~56X y~eld) of the t1tle compound melt1ng at 218-221C and hav1ng the formula ~CH3 C--O
H3C~ CH3 :
0~ :
~ ~ 3 1 ~
- 196- :
The structure of the product was confirmed by elemental an al y s i s . . - ~ .
.
EXA~PLE 118 Preparation of g-Fluoro~ ,17-dihydroxy-16~-methyl-21-~(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy~
pregna-1,4-diene-3,20-dione:
The general reduction procedure of Example 11 of U.S. Patent No. 4,617,298 was followed, using 0.78 mmol of the steroidal quaternary salt prepared in Example 117, 0.33 9 of NaHC03 and 0.41 9 of Na2S204 in 50X
aqueous methanol at 0C, with a nitrogen purge. The product had the structural formula~
l /CH3 \_y jC ::
~ - tH3 o . '~, EXAI~PLE 119 :
Preparatlon of 11~,17-D~hydroxy-21-[(3-pyridinyl-carbonyl)oxy]pregn-4-ene-3,20-dione:
- Hydrocort~sone (2 9, 5.5 mmol) was dissolved in 50 mL of dry pyridine. Then, 1.38 9 of nicotinoyl anhy-dride (6.05 mmol) and a trace of 4-(dimethylamino)pyri-d~ne (D~AP) were added and the react~on was allowed to proceed for 4 ho~rs at room temperature. The pyridine solut~on was poured into ~ce water and the resulting solld was collected by filtration. The solid was dried over P205 in vacuo to give 2.4 g (93~) of the t1tle ~ -compound of the formula ~-o ~11 ICH20C~) C=O .
~H
': ~
~' as conf1rmed by elemental analys~s and UV spectral analysis.
,:
~ "
13~1~64 EXA~PLE 120 Preparatlon of l-~ethyl-3-f[(llB~17-dihydroxypregn-4-ene-3,20-d10n-2l-yl)oxy]carbonyl~eyridinium iodide:
The product of Example 119 (1 9, 2.1 mmol) was dissolved in S0 mL of acetone and 4 mL of methyl iodide were added. The solution was st1rred at the reflux temperature overnight. Removal of the solvent gave the t~tle compound as a yellow powder in 98X yield. Ele-mental analysis conf~rmed that the product had the formula:
O r~N~ 3 C-O ~) H3C~ j 0~
EXA~PLE 121 Preparat~on of 11 e,17-D1hydroxy-21-~(1-methyl-1,4-d~hydropyr1dln-3-yl)carbonyl~oxy~pregn-4-ene-3,20-d~one:
The general reduction procedure of Example 11 ofU.S. Patent No. 4,617,298 was followed, using 0.8 mmol of the stero~dal quaternary salt prepared in Example 120, 0.34 9 of NaHC03 and 0.42 9 of Na2S204 ~n 50X
aqueous methanol at 0C, w~th a nitrogen purge. The product had the structural formula:
~-, : ,-........ ,., .. , . . , . - - .
~r~
!1 3 3 1 ~
..
o ~/ CH3 :
H3C I ;`~
~- OH
Preparation of N-(2-Chloroethyl)-N'-[2-(3-pyridine- ::~
carbonyloxy)ethyl]-N-nitrosourea: :
S A solution of 2-am1noethyl nicotinate dihydro-chlor1de (1.25 9, 52 mmol) and 2,4,5-trichlorophenyl-N-(2-chloroethyl)-N-n1trosocarbamate (2 9, 6 mmol) in 40 :
mL of pyr1d1ne was st1rred under nitrogen at room temperature for 24 hours. The react1on was monitored 10 by th1n layer chromatography (sil1ca, 1:1 chloro- ~n :~:
form/ethyl acetate, Rf 0.26). The solvent was removed 1n vacuo and the res1due was chromatographed on a :::~
s111ca gel column by eluting, f1rst with benzene to remove unreacted n1trosocarbamate and trichlorophenol by-product, and then w1th chloroform, to 91ve the des1red product. The resultant o11 solidifled in the freezer. It melted at 63-64C and had the formula :~
:
~3~
-OCH2CH2RHC=O
CH2 : :
Cl EXA~PLE 123 Preparat~on of N-(2-Chloroethyl)-N'-[2-~1-methyl-3-pyridiniumcarbonyloxy)ethyl]-N-nitrosourea iodide:
A solution of the product of Example 122 (1.5 9, 5 mmol) in 40 m~ of tetrahydrofuran was treated with excess methyl 1Odide. The mixture was stirred at 50C
for 4 hours. The finely crystalline, yellow solid thus obtained (1.8 9, 82X) melted at 120-121C and had the structure .
CNzCH
as confirmed by elemental analysis.
'' ~ ~.
~331~6~
Preparation of N-(2-Chloroethyl)-N'-[2-(1,4-dihydro-l-methyl-3-pyrid~necarbonyloxy)ethyl~-N-nitrosourea:
A solution of the quaternary nltrosourea prepared in Example 123 (0.48 9, 1.1 mmol) and 1-benzyl-1,2-dihydroisonicot1namide (0.23 9, 1 mmol) in 25 mL of anhydrous methanol was stirred at 0C for 4 hours under nitrogen. The solid which separated was filtered and washed with methanol and ether. The solid was identi-fied as 1-benzyl-4-carbamoylpyridinium iodide by NMR.
The filtrate was evaporated in vacuo at about 30C and the residue was suspended in methylene chloride. The sol~d that separated was filtered and washed with - -methylene chloride. The filtrate was evaporated in vacuo and the residue was dissolved in chloroform.
flash chromatography on a short column of neutral alumina, using chloroform as eluent, gave a chloroform solution which was evaporated in vacuo to afford 0.2 9 (63X) of a gummy res~due, identified by NMR as the -des~red product of the formula ¢~tl~2t~2~1~N2tl~2t~
An alcoholic solut~on of s11ver nitrate was readily ; reduced by the compound thus obtained. -~z,, - ., , .. . . .. :, ~ , ., . .. , . , .......... , : . . ::
13315~
Preparation of N-(2-Fluoroethyl)-N -[2-(3-pyr1d~ne-carbonyloxy)ethyl]-N-n~trosourea:
A solut~on of 2-aminoethyl n~cot~nate dihydro-chlor~de (1.5 9, 6.3 mmol) and 2,4,5-trichlorophenyl-N-(2-fluoroethyl)-N-n~trosocarbamate (2.18 9, 6.9 mmol) ~n 50 mL of pyridine was stirred under nitrogen at room temperature for 24 hours. The reaction was mon~tored by th~n layer chromatography (silica, 1:1 chloro-form/ethyl acetate, Rf 0.25). The solvent was removed1n vacuo and the residue was chromatographed on a sil~ca gel column by elut~ng, first wlth benzene to remove unreacted n1trosocarbamate and trichlorophenol, and then with chloroform to elute the desired -~
product. The compound (1.56 9, 87.4X) melted at 75- -~
77C and was character~zed by the structural formula:
~; ~
[~C-OCH2CH2NHC O ~
F2 -~
:' -13~fi~
EXA~PLE 126 Preparation of N-(2-Fluoroethyl)-N'-[2-(1-methyl-3-pyr1d~n1umcarbonyloxy)ethyl]-N-nitrosourea iodide:
A solution of the product of Example 125 (1.56 9, 5.4 mmol) 1n 40 mL of tetrahydrofuran was treated with excess methyl iodide. The mixture was st~rred at 50C
for 4 hours. The f1nely crystall~ne, yellow solid thus obta~ned ~2.20 9, 94.1X) melted at 123-125C and had the structural formula [~ CH2ch2 EXA~PLE 127 Preparat10n of N-(2-Fluoroethy!)-N'-~2-(1,4-dihydro-1-methyl-3-pyr1d1necarbonyloxy)ethyl]-N-n1trosourea:
A solut10n of the quaternary nitrosourea prepared 1n Example 126 (0.426 9, 1 mmol) and 1-benzyl-1,2-d1hydro1son1cotlnam1de (0.21 9, 1 mmol) 1n 25 mL of anhydrous methanol was st1rred at 0C for 4 hours under n1trogen. The solvent was evaporated in vacuo at about 30C and the res1due was suspended ~n chloroform, f~ltered and flash chromatographed on a short column of neutral alum~n2. The title compound was obtained in 13315~4 55X y~eld after elution with chloroform and was assigned the structure ¢;~,, ~OCH2CH2NHC-NCH2cH2 ~ -~H3 consistent with UV analysis.
EXA~PLE 128 Preparation of Chloromethyl nicotinate~
To a suspension of nicotinic acid (1.23 9, 0.01 mol) 1n a mixture of 10 mL of water and 20 mL of tetrahydrofuran were added tetrabutylammonium hydro- -gensulfate (0.34 9, 1 mmol) and sod~um bicarbonate (3.19 9, 0.038 mol), w~th v~gorous st1rr1ng. Chloro- .t~
methyl chlorosulfate (1.81 9, 0~011 mol) in 5 mL of tetrahydrofuran was added dropw1se, keep1ng the tem-perature below 30C. The react1On mixture was st1rred for 1 hour, then the layers were separated and the organ1c layer was dr1ed by azeotroplng w~th 1~
aceton1tr1le/benzene. The res~due was passed through a ~ column of neutral alum1na, elut1ng w1th chloroform.
; The chloroform layer was evaporated to give 1.28 9 (74.8X) of an o11y res1due, which was confirmed by NMR
analys1s to have the structural formula:
.
~ . .. .
1~3~5~
~-O-CH2-EXA~PLE 129 Preparat~on of l-(Pyr~dine-3-carbonyloxymethyl)-S~
fluorouracil~
S S-FU (1.31 9, 0.01 mol) was dissolved in 5 mL of dimethylacetamide and treated wlth triethylamine (2.78 mL, 0.02 mol). Chloromethyl n~cotinate (2.95 9, 0.012 mol) in 5 mL of dimethylacetamide was added in one port~on, and the mlxture was stirred for 24 hours, f11tered, washed with ethyl acetate and evaporated.
The resldue was chromatographed on a column of sil~ca gel, us1ng as eluent f~rst benzene, then 3:1 ben-zene/chloroform, then 1:1 benzene/chloroform, then 3:1 chloroform/benzene, then chloroform and finally 99:1 chloroform/methanol. Unreacted nicot~nate was eluted 1n~t~ally, followed by the 1,3-bis-isomer and finally the l-lsomer. The l-lsomer (1.3 9, SOX) melted at 190-192C and had the formula COCH2-N ~ F ~:
OJ~ N ~ O
Il N
.
~331~
as confirmed by NMR analysis.
EXA~PLE 130 Preparation of 1,3-Bis(pyridine-3-carbonyloxymethyl)-5-fluorouracil:
S-FU (1.31 9, 0.01 mol) was dissolved in 5 mL of dimethylacetamide and ereated w~th triethylamine (5.6 mL, 0.04 mol). Chloromethyl nicotinate (6.8 9, 0.04 mol) in 15 mL of d~methylacetamide was added in one portion, then the mixture was stirred for 48 hours and filtered, and the filter cake was washed with ethyl acetate. The filtrate was evaporated in vacuo and the residue was diluted with 100 mL of water and extracted with chloroform. The organic layer was evaporated and the residue was dried by azeotroping with 1~ r aceton~trile/benzene. The res~due was chromatographed on a column of neutral alum~na, eluting successively wlth benzene, 1:1 benzene/chloroforml and 50:50:1 benzene/chloroform/methanol, to g~ve 3.2 9 (80X) of the b~s-~somer of the formula ~OCH2~N ~
~ 0 1 N
as conf1rmed by N~R analysls.
1331~6~
EXA~PLE 131 Preparation of 3-(Pyridine-3-carbonyloxymethyl)-5 fluorouracil~
:: :
The 1,3-bis-~somer prepared in Example 130 was dissolved in 10 mL of methanol and mixed with 20 mL of potassium carbonate:sodium bicarbonate buffer (O.lM), pH 10.00. The mixture was stirred at room temperature ;~
for 2 hours, by which time thin layer chromatography indicated that the bis-isomer had disappeared 10 completely. The mixture was evaporated and the residue -was chromatographed on a column of alumina, eluting successively with chloroform, 99:1 chloroform/methanol and 96:4 chloroform/methanol. The fractions were collected and those containing the 3-isomer were pooled and evaporated to give the compound of the formula (~ F :
as a solid (0.2 9, 30X) melting at 179-180C. The ~ -identity of the product was confirmed by N~R analysis.
'.;, -~
:
:
1~31~
EXA~PLE 132 Preparat~on of 1-(1-Methyl-3-pyridiniumcarbonyloxy-methyl)-5-fluorouracil iodide:
The l-isomer prepared in Example 129 (1.93 9) was combined w~th sufficient quantities of methyl iodide and acetonitr~le, and the mixture was refluxed for 4 hours, then cooled and f~ltered to give 2.5 9 of a -. ::
light yellow, fluffy solid. The filtrate was eva-porated, triturated with acetonitrile and filtered to g~ve an addit~onal 0.23 9; total yield 2.73 9 (92.25~). UV and NMR analyses confirmed that the product had the structural formula: -CocH2-N ~ F
~- oJ` N O
EXA~PLE 133 Preparation of 1-(1,4-Dihydro-1-methyl-3-pyridinyl-c~rbonyloxymethyl)-5-fluorourac11:
The product of Example 132 (1 9) was d~ssolved in 20 mL of delonized water (degassed w~th argon) and cooled in an i.ce-bat.h, then 20 mL of ethyl acetate were 20 added. To the stirred solution, 1.24 9 of sodium :~
bicarbonate were added, followed after about one minute ~ :
with 1.75 9 of sodium dithionite. The reaction was -.
allowed to proceed under argon and was monitored by UV. After approximately 75 minutes, ethanol was added .
1 3 ~ 1 5 ~
and the solid was filtered, washed with water and methylene chloride and dried under argon to give 400 mg of the title compound. UV and N~R analyses confirmed : ~-that the product had the structural formula~
~OCH2--N~
¢~ 0~ N O
N H
The aqueous layer was repeatedly extracted with chloro-form and combined with the ethyl acetate layer used in the reaction. The solid obtained after removal of the organic solvent was suspended in acetonitrile and filtered to give an additional 250 mg of product melting at 173-174C.
EX~PLE 13~
Preparation of 3-(1-Methyl-3-pyr1dinlumcarbonyloxy-methyl)-5-fluorouracil tod1de:
lS The 3-~somer prepared ~n Example 131 can be sub-~ected to the general procedure of Example 132 to a~ford the correspond1ng quaternary salt of the forrluld~
;,., . `--~
~3156~
-21()- ~ ~ .
COCH2-N J~, F
~r J~ Jl . .
_ O
CH3 1 . :~
EXAMPLE 135 ~ :
Preparation of 3-(1,4-Dihydro-1-methyl-3-pyridinyl-carbonyloxymethyl)-5-fluorouracil:
The product of Example 134 can be subjected to the general procedure of Example 133 to afford the corres-ponding dihydropyridine of the formula: ~: :
COCH2--NJ~ . ~ . ;.
l l 1 ~J ; .
The cyclodextrin complexes of this invention can 10 be administered to warm-blooded animals to treat a -~
variety of conditions, depending upon the nature of the carrier-drug, specifically of the parent drug itself . .:.
from which the carrier-drug is derived. Thus, in one -~
preferred embodiment, the redox system is derived from 15 dopamine or L-DOPA or a protected counterpart thereof, :~
, a~ ... ;,.. , .. , , , , , , " ~"~"", "~
133~64 and the redox derivative/cyclodextrin complex is thus designed to elicit a sustained and brain-specific dopaminergic (e.g. anti-Parkinsonism or anti-hyperprolactinemia) response in the animal to which the complex is administered. In analogous fashion, the redox derivative/cyclodextrin complex derived from any other centrally acting drug as defined herein is designed to elicit the kind of pharmacological response ;~
which would be obtained by delivery of the drug itself to the brain, i.e. when the centrally acting parent drug is an antitumor/anticancer agent, the instant complex is employed to elicit an antitumor/anticancer response; when the parent drug is a sympathetic stimulant, the instant complex is used to elicit a sympathetic stimulant or amphetamine-like response;
when the parent drug is an anticonvulsant compound, the instant complex is used to elicit an anticonvulsant response; when the parent drug is a tranquilizer, the instant complex is used to elicit a tranquilizing response; when the parent drug is an antidepressant, the instant complex is used to elicit an antidepressant response; and so forth.
The cyclodextrin complexes of the invention are preferably administered in the form of a pharmaceutical composition comprising the selected complex and a nontoxic pharmaceutically acceptable carrier therefor. Suitable nontoxic pharmaceutically acceptable carriers for use with the topic complexes, e.g., those less toxic than the target drug species themselves, will be apparent to those skilled in this art. Compare, for example, Remington's Pharmaceutical Science_, seventeenth edition, ed. Alfonso R. Gennaro, ::
`,j~
13~1~64L
-212- r ~ack Publishing Company, Easton, PA (19~5). Obviously, the choice of suitable carriers will depend upon the route of administration and the exact nature of the particular dosage form selected, as well as upon the identity of the active drug species, the redox derivative and the complex to be administered.
Contemplated routes of administration for the complexes of the invention include oral, buccal, sublingual, topical (including ophthalmic), rectal, vaginal, nasal and parenteral (including intravenous, intramuscular and subcutaneous).
The therapeutic dosage ranges for administration of the complexes according to this invention will generally be equimolar to, or less than (in some instances, substantially less than), those which would characteristically be used in this art for adminis- ;
tration of the parent drug species, per se. Naturally, such therapeutic dosage ranges will vary with the size and species of the patient, the condition for which the -~
20 complex is administered, the particular dosage form ~ 9 employed, the route of administration and the like.
The quantity of given dosage form needed to deliver the desired dose of active ingredient will of course depend upon the concentration of the redox derivative in the complex and in any given pharmaceutical composi~
tion/dosage form thereof. Obviously, in the case of ~ ;
diagnostic agents, the dosage used will be a quantity sufficient to deliver to the target body area a quantity sufficient to deliver an amount of radio- -~
isotope, stable isotope or the like which can be effectively detected by radioimaging or other detection means. The amount of radioisotope, stable isotope or ~ ~
. .
- -' ::
~ .
''~,'": " "': ' '. ' ': ::: . ' ' l 331 ~
the like present in the dosage form will be within or below the ranges conventionally used for diagnostic ~ -~
purposes.
While the invention has been described in terms of : ;
various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended :. :.
that the scope of the present invention be limited solely by the scope of the following claims.
:.
.- . ~
. . ~
water-delivering pump injects the sample on a pre-column where the drug is concentrated. Mobile phase backflushes the retained compound onto the analytical ~;
column. At the same time, another sample can be r~
injected onto a second pre-colu~n. This alternating pre-column sample enrich~ent technique allows the injection of large volu~es up to 1800 ~L.
EXPERIMENTAL SECTION
Materials: E2-CDS, E2-Quat and E2-CDS-HPCD were synthesized as described previously. Steroids (estradiol and ethinyl estradiol) were obtained from Sigma Chemical Co. HPLC grade acetonitrile and dis-tilled, deionized water were used for the preparation of mobile phases. All other reagents used were of analytical grade. ~
: ~. ' Instrumentation: The HPLC system consisted of a LDC/Milton Roy Constametric III high-pressure pump, a LDC/Milton Roy variable wavelength UV detector, a Perkin Elmer ISS-100 automatic ,injector equipped with a 2000 ~L loop and a DuPont Zorb`ax ODS column, 15 cm x 4.6 mm I.D. (6 ~m particle size). Vydac guard columns (5 cm x 3.2 mm I.D.~, dry-packed with DuPont Zorbax ODS
material, were used. Chromatograms were recorded on a Hewlett-Packard Model 3390A computing integrator at a chart speed of 0.2 cm/min. In addition, in the pre-column enrichment system, an enrichment injector (Rheodyne Model 7067-005) with two high pressure switching valves, pneumatically turned by a tandem actuator (Rheodyne Model 7163), was inserted between the autoinjector and the analytical column. Switching of the valves was controlled via the autoinjector.
This system also contained a Bodine Electric Co. RR/035 HPLC Solvent Pump for flushing the samples onto the enrichment columns.
~ethods: ~-Assay Conditions: direct on-line HPLC
Chromatographic conditions for the analysis of E2- ;
CDS, E2-Quat and estradiol were developed. The optimal wavelength for all compounds was 224 nm, but E2-CDS can also be detected at 360 nm due to t,he dihydropyridine structure. Although the absorptivity at this wave-length is only about half as high as it is at 224 nm, 360 nm was chosen as the analytical wavelength for E2CDS because of the increased selectivity. Different analytical columns were tested and mobile phases for a reversed phase chromatography of all three compounds were varied widely with respect to the ratio of aqueous and organic phase as well as buffer concentration and pH. No isocratic system could be found that would detect all three compounds within a reasonable reten-tion time and with satisfying compactness and separa-tion of peaks. Therefore, two different syste~s wereused for analysis.
E2-Quat and E2: The optimal mobile phase was found to consist of acetonitrile/water 40:60 containing 0.03 M/L sodium salt of octanesulfonic acid and 0.003 M/L tetrabutylammonium phosphate. The pH was adjusted to pH 5-5.5. The flow rate was 1.5 mL/minute and the ~ ~ -peaks were recorded at 224 nm.
E2-CDS: The mobile phase used for E2-CDS analysis was acetonitrile/water 70:30 at a flow rate of 1.5 mL/minute. Absorbance was monitored at 360 nm.
~ ,," ,, ~ "
Analysis of E2, E~-CDS and Ej-Quat by pre-column enrichment technique:
- . .
The loss of sensitivity resulting from the -~
dilution step in the procedure optimal for pre-5 treatment of biological samples ~see sample preparation ~ -without extraction) could be compensated for by developing an HPLC system that allows injection of large volumes. A suitable HPLC-method which has been described in the literature ~Roth et al~ J. Chromatogr.
222: 13-22 (1981)] is based on alternating pre-column sample enrichment. The procedure used herein was as follows: The sample containing the drug is injected with a first pump A, delivering pure water, onto one of two pre-columns, which are alternatingly connected with the injection system by two pneumatically driven valves. Provided a certain lipophilicity, the drug is retained and concentrated on the pre-column, while accompanying water soluhle co-products like proteins are being washed out as long as water is pumped through the pre-column. This allows the direct injection of body fluids. After a certain enrichment time (6 and 8 minutes), simultaneous rotation of the two valves is ;
induced, causing pre-column 1, where the injected drug has been absorbed, to be switched to the solvent stream of the second pump, B. Also, at this point, the recording integrator is started. Pump B delivers the mobile phase, necessary for separation and chromatography, and backflushes the sample from pre-column 1 onto the analytical column. Parallel to this process, pre-column 2 is switched to the water stream of pump A so that a sample can be injected and enriched 3 ~
f~
:
while the previous one is ~eing eluted (alternating mode). Volumes up to 13~ ~L can be injected due to .. ;:P.iYr.
the concentration effect of the enrichment phase.
Chro~atographic conditions: This system was applicable to the quantification of E2, E2-CDS and E2-Quat. The mobile phase for E2-CDS was: Acetoni- -trile/water 80:20 at a flow rate of 1.8 mL/minute.
Optimal peak shape and retention time for E2 and E2-Quat were obtained with pump ~ delivering a mixture of acetonitrile/water 42:58 which contained 0.025 M/L
sodium salt of 1-octanesulfonic acid and ~.003 M/L
tetrabutylammonium phosphate. The pH was adjusted to -pH 5, and the flow rate was 1.5 mL/minute.
Standard solutions and stability Sample stock solutions of E2-CDS, E2-Quat, E2 and ethinyl-E2 containing each 50 ~g/mL were prepared in acetonitrile. All solutions were stored at 6C. For E2-CDS, the stock solution was prepared freshly every 2 ;-weeks. All other solutions were stable over a period --of at least six months. Spiked plasma samples con-taining all four compounds were frozen at -20C and analyzed repeatedly at different time intervals. No loss of drug was found under these storage conditions during two months.
Dihydropyridine derivatives like E2-CDS are known ~ ~-to be easily oxidized and very labile in acidic solu-tions. The stability of E2-CDS was investigated under different conditions at room temperature. These studies were performed by diluting the E2-CDS stock solution 1:2 with different solvents or solutions at -- ~ :
: . :: . : ~ :. ~ . - . . .. . . . .
. ~ ,. ", ~ " , " , ,, ~ , , , , . . .
~. : . : . ~ - . : . .
1 ~ 3 ~
-1~3-different pH values and monitoring eventual peak height loss for 24 hours by use of a modification of the direct on-line H~LC method described above: If water in the E2-CDS mobile phase is replaced by 0.05 M
phosphate buffer at pH 7 and if the detection wave-length is set to 224 nm, E2-Quat can be detected simul-taneously at 6.33 min. However, the peak is relatively broad. These conditions were used to determine the degree of E2-CDS oxidation under the tested conditions.
Sa~ple preparation Extraction of E2-Quat and E2: Various extraction procedures from plasma were investigated under dif-ferent conditions and with several solvents and solvent mixtures. Estrone could be used as an internal standard, but is known to be a potential metabolite of estradiol. Therefore, 17-~-ethinyl estradiol was ;
chosen as internal standard. Its peak did not inter-fere with E2, E2-Quat or estrone. Without addition of an anion reagent, E2-Quat could not be extracted from 20 aqueous solutions. Optimal results were obtained after -~
a single-step extraction of the drugs with potassium iodide as an ion-pairing reagent to facilitate quaternary salt extraction. The method applied was as follows: 200 ~L of a saturated potassium iodide solution were added to 1 mL of spiked plasma. After vortexing for a few seconds, 10 mL of a mixture of chloroform/ethyl acetate 9:1 was added. The tubes were shaken for 10 minutes and then centrifuged for 10 minutes at 2000 rpm. The upper aqueous phase was discarded and the organic layer transferred to a clean tube to achieve complete separation from proteins and -' .
, ; ..
., . ~
~ 33~ ~$
traces of aqueous phase. The organic layer was eva-porated to dryness under nitrogen at 40C and recon-stituted in 150 ~L of mobile phase. 40 ~L were injected into the HPLC system. Appropriate blanks were prepared accordingly.
Extraction of E2-CDS: Plasma and water containing E2-CDS were repeatedly extracted with different organic solvents like chloroform, hexane, toluene, benzene and ethyl acetate. It was impossible to extract E2-CDS
reproducibly from aqueous phases, since the compound was shown to deteriorate unreproducibly during evapora-tion, even at room temperature and in the presence of oxygen-free nitrogen. Therefore, the compound had to be analyzed from biological fluids without an extrac-tion procedure.
Preparation of plas~a and tissues for analysis ofE2-CDS and E2-Quat without extraction: Using the HPLC
technique with pre-column enrichment described above, drugs can be detected from directlv injected plasma without sa~ple preparation. However, when large volumes are injected, in order to obtain maximum sensi-tivity, it is desirable to remove proteins to a large extent prior to injection in order to prevent frequent pre-column packing. The procedure of deproteinization was chosen to be applicable for subsequent analysis of both E2-CDS and E2-Quat so that only one preparation step had to be performed. -~
Acidic precipitating agents, which remove proteins when only small volumes are added to biological fluids, could not be used because they induce degradative loss of E2-CDS. Neutral or slightly basic aqueous reagents ' , 7;' ' 1 3 3 ~
used efficiently for deproteinization like ZnS04/NaOH, CuS04/Na2S04 or saturated (NH4)2S04 would be more ideal to be injected onto the enrichment columns than organic solvents. But all of these reagents were shown to absorb the water-insoluble E2-CDS on the precipitate.
Thus, the method of choice to avoid instability prob-lems and at the same time keep all compounds in solu-tion was deproteinization with acetonitrile.
To obtain these results, the following sample ~-preparation procedures were used for plasma and tissues: Plasma: 0.6 mL plasma was added to 1.2 mL ;-acetonitrile. The mixture was vortexed for 5 seconds and allowed to stand for 10 minutes at room tempera-ture, vortexed again and centrifuged for 10 minutes at 15 2000 rpm. 1000-1500 ~L of the supernatant was injected into the pre-column enrichment system. Tissue (e.g.
brain): 1 mL of water was added to one rat brain and the organ was thorcughly meshed. After sonication for 2 minutes and centrifugation at 2000 rpm for 10 20 minutes, the supernatant (1000-1500 ~L) was injected into the enrichment system.
Animal Stud~es~
~ ,, In a first experiment, 15 mg/kg E2-CDS dissolved in dimethylsulfoxide (DMSO) were administered intra-venously to conscious, restrained male Sprague-Dawley rats weighing 190-300 9 each. Animals were sacrificed in groups of 4 at 5, 15 and 30 minutes and at 1, 2, 4, 8, 24 and 48 hours after drug injection. Trunk blood ~-~
was collected into heparinized tubes and plasma obtained and immediately frozen at -20C until analy-;~ sis. Organs were dissected and placed on dry ice ~' :
:~.. ' `' - ~' : ' ' '~ :
~331~
-1~6-within 2 minutes of death and stored at -20C for later analysis by the HPLC method described above.
~ n a second experiment, the same procedure as above was followed, except that 5 mg/kg of E2-CDS were administered as a complex with hydroxypropyl-~-cyclo-dextrin (HPCD) in water.
Results and Discussion The results are depicted in FIG. 3 and FlG. 4.
FIG. 3 consists of a pair of semi-logarithmic plots comparing the concentrations in lung tissue in ~9 per 9 dose (CB/D) of E2-CDS in the first graph and of E2-Quat in the second, corrected for dose. It can be seen from FIG. 3 that when E2-CDS was administered in DMS0, -~
initial lung concentrations (i.e. concentrations within the first hour after drug injection) of both E2-CDS and E2-Quat were significantly higher than the initial lung concentrations observed when E2-CDS was administered as a complex with HPCD in water. The corresponding levels of E2-Quat in brain tissue, also corrected for dose, are given in FIG. 4 in the form of a bar graph depict-ing the brain levels in ng per g dose (CB/D) at selected time points. It can be seen that the brain levels after 1 hour are not significantly different for administration as HPCD complex in water as compared to administration in DMS0. Clearly, then, the carrier~
drug can be administered as a complex with a selected cyclodextrin as defined herein, such as HPCD, in water and still achieve the brain levels needed to produce the desired biological effect, while avoiding the high ,~ ~
133~
-1~7-initial lung concentrations responsible for respiratory distress and dysnia.
Complexation with 2-hydroxypropyl-~-cyclodextrin (HPCD) or other selected cyclodextrin derivative as defined herein has been found to be particularly advantageous in that it stabilizes the dihydropyridine redox syste~s. A direct comparison of stabilities in aqueous solution is, of course, not possible because of the low solubility of the dihydropyridine redox system drugs in water; for example, the solubility of E2-CDS
in water is only 0.0002 mg/mL. The E2-CDS-HPCD complex contains about 40 mg of E2-CDS/g and easily gives aqueous solutions containing 5 mg E2-CDS/mL at 20% w/v -~
cyclodextrin. Thus, complexation affords a 25,000-fold increase in aqueous solubility of E2-CDS. The half-life of E2-CDS in such a solution at room temperature in the dark is about 12.5 days (rate: 0.0554 n .0047d~
Since the dihydropyridine redox system drugs are especially prone to oxidative degradation, a study was undertaken to quantitate the effect of a cyclodextrin derivative intended for use herein, e.g. HPCD, on the rate of oxidation of these drugs. A representative carrier-drug, E2-CDS, was selected for this study.
The rate of ferricyanide-mediated oxidation of E2-CDS was determined using a previously published method (Okamoto et al, J. Chem. Soc. Chem. Comm., 1977, 1i31). In this procedure, 27.5 ~L of a 5 x 10-3 M
solution of E2-CDS in acetonitrile was added to 2.75 mL
of a solution containing 1 x 10-4 M Fe(CN)6~4, 0.06 M
K+, O.001 M Fe(CN)6~3. All solutions were made using ~ :
1 3 ~
- 1 o ~
water whicll ha~ been boile~ for 30 m~nutes and cooled while a strealn of pyrogallol-scrubbed nitrogen passed through it. The E2-C~S was introduced via a syringe to -~
the solution which was maintained a~ 37C in a thermo~
stated cell holder and contained in aTM anaerobic screw-top cuvette. The cuvette had a Teflon-lined septum -~
through wh1ch the compound was injected. For a given --concentration of ferr~cyanide ions (6 x 10-4 to 8 x 10-3 M), the rate of d~sappearance of the ~
I0 E2-CDS was determined. This was done by calculating ~- -the decrease in the absorbance band at 360 nm (i I0 nm) subtracted fro~ base 11ne absorbance (S00 ~ 10 nm). A
plot of the ln ~Abs] versus time gave a slope for the pseudo-first-order rate constant. This was done at several dlfferent ferricyanide ion concentrat~ons. The obta~ned first-order rate constants were then plotted ;~
as a function of ferricyanide ion concentration generating a slope from which the second order rate constant (ko s~I M-I) was obta1ned. In examlning the effect of 2-hydroxypropyl-~-cyclodextrin on the rate of E2-CDS oxidation, solutions containing the HPCD as well as those ions present in the first phase of the experiment were prepared. The second order constant was derived for each cyclodextrin concentration and a plot developed. The results, depicted in FIG. 5, show tha-t the cyclodextrin dramatically slowed the rate of oxidation. There appears to be a saturation effect in that after 2% w/v, not much change in rate is evident. The second order rate of oxidation is inhibited by 42% at 0.5% w/v cyclodextrin, 60% at 1.0%
` w/v, 81% at 2% cyclodextrin and at 5-20% a value of approximately 90% reduction in the rate was obtained.
. ' :, ~
:
,~
,~ " ,;; ;~
$ ~ . .
The following Examples illustrate the preparation of preferred reduced, dihydropyridine . pyridinium salt ~.
redox carrier systems for brain-targeted drug delivery which are contemplated for formation of cyclodextrin (e.g. HPCD) complexes in accord with this invention and which have not been specifically described in :~
publications to date.
. ~ , .~ : .
, -~ : ?
.7'.',3.;~
i 3 3 1 ~ 6 ~
1 1 o -EXAMPLE 1 ~ -Preparation of N-~icotinoyldopamine~
To a pyridine solution containing 11.7 9 (0.05 ~ -mol) dopamine hydrobromide and 6.15 9 (0.05 mol) --~
nicotinic acid at 0C were added 10.3 9 (0.05 mol) -~
dicyclohexylcarbodiimide (DCC). The reaction mixture was stirred at room temperature for 24 hours and the formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo and the residue was trystallized from water at 0C. The product was isolated by filtration and dried over phosphorous pentoxide. Recrystallization from isopropanol gave 9.0 -~
9 (0.035 mol), 70X N-nicotinoyldopamine, m.p. 159- ~ -162C; aqueous solution of the compound gave a green -color with Fe~3 and reduced AgN03; IR (KBr) 3300, 2960, 1725, 1630, 1590, 1520, 1430, 1290, 1190, 1115, 720 and 710 cm~1; NMR (d6-DMS0) ~ 9.25-6.25 (m, 7H), 3.3 (m, 2H) and 2.65 (m, 2H) ppm. Anal. (C14H14N203) C, H, N-Preparation of 1-Methyl-3-¦N-[B-(3,4-dihydroxyphenyl)-ethyl]~carbamoylpyridinium iodide:
To a solution of 2 9 (7.7 mmol) of N-nicotinoyl-dopamine ~n 40 mL of dry methanol were added 2.5 9 (17.6 mmol) of methyl iodide. The reaction mixture was refluxed with stirring for 6 hours. Methyl iodide (1.5 9, 1.05 mmol) was added and refluxing was continued overnight. Methanol was removed and ethyl acetate was added, affording yellowish crystals of the desired -~
product. Yield 2.4 9 (77X), m.p. 173-174C.
-''~`' ~ ' , 1 3 3 1 ~ 6 4 ~ -Preparation of l-Methyl-3-~N-t[B-t3,4-bis(isobutyryl-oxy)phenyl]ethyl]]~carbamoylpyridinium trifluoroace-tate-~. ~
To an ice-cold solution of the product of Example 2 (3 9. 7.5 mmol) in 30 mL of trifluoroacetic acid, isobutyryl chloride (2.4 9, 22.5 mmol) was added ;~
slowly, with stirring. Stirring was continued over-night at room temperature. Trifluoroacetic acid was evaporated under vacuum and the residue was crystal-lized from ethyl ether:hexane (3:1). Yield 1.2 9 ~
(30.4X), m.p. 87-91C. ;
EXA~PLE 4 .
Preparation of 1-Methyl-3-~N-[tB-[3,4-bis(isobutyryl- ~
15 oxy)phenyl]ethyl]~carbamoyl-1,4-dihydropyridine: ~;
A solution of O.SS 9 (1 mmol) of 1-methyl-3-{N-[tB-[3,4-bis(isobutyryloxy)phenyl]ethyl]]~carbamoyl-pyridinium trifluoroacetate in S0 mL of deaerated water containing 10 mL of methanol was extracted three times with 3n mL portions of ether. To the resultant aqueous solution were added NaHC03 (0.25 9, 3 mmol) and 50 mL
of ethyl ether and the mixture was kept under ~ -;
nitrogen. To this ice-cold mixture was added sodium dithionite (0.52 9, 3 mmol) and the mixture was stirred vigorously for 30 minutes. The ether layer was sepa-rated and the aqueous layer was extracted twice with ether. The combined ether extracts were washed with ~ . .:
water and dried over sodium sulfate. Ether was removed under vacuum, leaving an oily product. NMR analysis ~ ~ -30 confirmed that the product had the structural formula: ~
~" .
~.
~' ..
~ ~,, ;~
~ I '; ' ~ ': . ' . ~ ' . ' . ' ' ~, .,,, ' ' : : ' . ~ : : , . , - 1 3 3 1 5 ~ 4 ~NHCH2CH2~o~c (c 3) Preparation of 5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidinedione: -Phenytoin (5 9, 0.02 mol) was suspended in 180 mL
of water; 20 mL of formaldehyde (37X solution) and 0.25 g K2C03 were added and the mixture was stirred at 25-30C for 24 hours. The white solid which formed was removed by filtration and washed repeatedly with a 3X
10 solution of formaldehyde, then air-dried for 3 to 4 hours and over P205 in a vacuum dessicator. Yield 91-93X, m.p. 185-189C. Anal. calc. for C16H14N203: C, 68.07; H, 5.00; N, 9.93. Found: C, 67.97; H, 5.05; N, 9.93. The product had the formula:
N
@~
H20H -:
.~
; ~ ;
: . i ,~ ~`; ::, .,~., . :,: --113- 1331~4 Preparation of 5,5-Diphenyl-3-[(3'-pyridyl)carbonyloxy-methyl]-2,4-imidazolidinedione:
The product of Example 5 (3.00 9, 0.011 mol) was dissolved in 150 mL of dry pyridine, then nicotinic anhydride (4.25 9, 0.019 mol) was added. The resultant solution was stirred at room temperature (25-30C), under dry conditions, for 40 hours. The solution was :
poured into 2.5 L of water and the resultant white solid was removed by filtration, washed well with water and dried over P205 in a vacuum dessicator. 95X yield, m.p. 178-182C. Anal calc. for C22H17N304: C, 68-21;
~, 4.42; N, 10.85. Found: C, 68.12; H, 4.43; N, 10.83. The product had the formula:
~ N
~ ~ ~2~
EXA~PLE 7 Preparation of 5,5-Diphenyl-3-[(1'-methyl-3'-pyri-dinium)carbonyloxymethyl]-2,4-imidazolidinedione iodide:
The product of Example 6 (0.5 9, 0.0013 mol) was dissolved in 50 mL of acetonitrile, then û.3 mL of methyl iodide was added and the reaction mixture was maintained at room temperature for 6 days. The solvent .
,~ ,,"
133151~
~ -:
was removed by vacuum distillation and ethyl ether was added to the residue. The ether solution was refrig-erated for 2 hours, then the yellow, hygroscopic crys-tals which formed were dried over P205 in a vacuum dessicator, giving the desired product in 85X yield.
UV and HlNMR spectra confirmed that the product had the structure:
': ~
~H ~ ~
~ ~DH20~SCH 3 :
Repeating the above procedure in nitromethane at a 10 50-70C bath temperature using excess methyl iodide, ~m added gradually, for 5 to 6 hours, afforded the same product in nearly quantitative yield.
EXA~PLE 8 Preparation of 5,5-Diphenyl-3-[(1'-methyl-1',4'-d~hydropyridin-3'-yl)carbonyloxymethyl]-2,4-imidazolidinedione:
The quaternary salt obtained in Example 7 t0.4 9.
0.0008 mol) was dissolved in 40 mL of water, 3 mL of methanol and 15 mL of ethyl acetate. The reaction mixture was cooled to 0 to 5C and deaerated, then sodium bicarbonate (0.39 9, 0.0046 mol) and sodium dithionite (0.54 9, 0.0032 mol) were added. The mixture was stirred under nitrogen at 0-5C for 35 minutes. The organic layer was removed and the aqueous .
-115- 1~315~4 layer was extracted twice with 15 mL portions of ethyl acetate and the organic solutions were extracted with 10 mL of cold deaerated water. After drying over Na2S04, the solvent was removed by vacuum distillation and the oily yellow solid was crystallized by addition of ether. Yield 70~. UV and HlNMR analyses confirmed that the product had the formula:
~CO ~CH3 Preparation of 3-Bromoacetyloxymethyl-5,5-diphenyl-2,4-imidazolidinedione:
.
5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (2 9, 0.0071 mol) was dissolved in bromoacetyl-chloride (15 9, 8 mL, o.ng6 mol) by heating in an oil bath (70-80C bath temperature) for about 15 minutes, until the formation of HCl ceased. The mixture was cooled and 30 mL of ethyl ether were added. White crystals formed. The mixture was cooled to 0C, then the crystals were removed by filtration and dried over P205. Yield: 2.15 9 (75X), m.p. 179-183C. Anal.
calc. for C18H15N204Br: C, 53.61, H, 3.75; N, 6.95;
Br, 19.82. Found: C, 53.60; H, 3.79; N, 6.92; Br, 19.90. The product had the formula:
: :
~ ~ 3~iJ~I~
N
CH20~CH2Br ' EXA~PLE 10 ::
Preparation of 3-t3'-Bromopropionyl)oxymethyl-5,5-diphenyl-2,4-imidazolidinedione: -5,5-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (5 9, 0.018 mol) was reacted according to the procedure of Example 9 with 3-bromopropionyl chloride ~ -(6.~ 9, 0.04 mol, 4 mL) using d bath temperature of 100C. A white crystalline product was obtained in 65%
yield (4.9 9), m.p. 133-134C. Anal. calc. for ~ -C1gH17N204Br: C, 54.69; H, 4.11; N, 6.72; Br, 19.15.
Found: C, 54.79; H, 4.12; N, 6.69; Br, 19.25. The product had the formula:
~: .
~S~ :
`CH20~CH2CH2Br ~: ' -EXA~PLE 11 Preparation of 3-(2'-Bromopropionyl)oxymethyl-5,5-diphenyl-2,4-imidazolidinedione: -S,S-Diphenyl-3-hydroxymethyl-2,4-imidazolidine-dione (2 9, 0.0071 mol) was dissolved in 2-bromopro-. ~.. :
:, :
133~r5'~ 1 pionyl chloride (8.5 9, 5 mL, n.o5 mol) by heating for 30 minutes on a 100~110C Oi 1 bath. The reaction mixture was cooled, 20 mL of ethyl ether were added, and the resultant solution was extracted wi~h aqueous potassium carbonate, dried and then crystallized. The product was obtained as a solid white substance (1 9, 34X), m.p. 112-115C. Anal. calc. for ClgH17N204Br:
C, 54 69; H, 4.11; N, 6.72; Br, 19.15. Found: C, 54.77; H, 4.15; N, 6.69; Br, 19.25. The product had the formula:
:
H
CH20i~HCH3 Preparation of 3-(3'-Carbamoyl-l'-pyridinium)acetyl-oxymethyl-5,5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example 9 (2.0~ 9, O.On5 mol) dissolved in lS mL of nitromethane was mixed with nicotinamide (0.61 9, 0.005 mol). The solution was stirred on a 90-100C temperature oil bath for 2 hours. The mixture was cooled to 60-70C and the white crystals which had formed were removed by filtration and washed with nitromethane. Yield filX (1.65 9), m.p.
193-197C (dec). Anal. calc. for C24H~1N405Br: C, 54.87; H, 4.03; N, 10.67; Br, 15.21. Found: C, 54.70;
H, 4.05; N, 10.64; Br, 15.25. The p oduct had the formula:
1 3 3 1 ~
H2OflC~2-N ~ ~r .
EXA~PLE 13 Preparation of 3-[3'-(3''-Carbamoyl-l''-pyridinium)- ~ ;
propionyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example lO (2.09 9, 0.005 mol) was dissolved in 15 mL acetonitrile, then nicotinamide (0.61 9, 0.005 mol) was added. The solution was refluxed for 6 days, then the solvent was removed. To the gum-like residue, 30 mL of ethyl ether was added and the mixture was stirred for 2 hours. The white substance which formed was removed by filtration and washed with ether. Yield 78~ (2.1 9); m,p, 98-100C
(dec.); UV and HlNMR as expected. The product had the formula:
N~O ONH2 ~/o~ ~ClI2o~cH2cH2-N~Br : .
. ~ .
' -:-., .~. . - ~ : ~ , . .. ,. ::: ~ : ~ , . . .
'.` ' ' ' ! ' : ' , -:: , ' ; :: . I
Preparation of 3-[2'-(3''-Carbamoyl-1''-pyridinium)pro-pionyloxymethyl]-5?5-diphenyl-2,4-imidazolidinedione bromide:
The product of Example 11 (0.69 9, 0.00165 mol) was dissolved in 8 mL of acetonitrile, then nicotin-amide (0.2 9, n.ool65 mol) was added and the solution was refluxed for 22 hours. The solvent was removed from the resultant brown noncrystalline substance at 50C, then ethyl ether (15 mE) was added and the mix-ture was stirred for 2 hours. The light brown sub-stance was removed by filtration and washed with ether. Yield 56~ (0.5 9), m.p. 158C (dec.). The product had the formula:
~V N
~ 2 ~ C 3 N~ _ Br EXA~PLE lS
Preparation of 3-[(3'-Carbamoyl-1',4'-dihydropyridin-1'-yl)acetyloxymethyl]-5,5-diphenyl-2,4-imidazolidine-dione:
The product of Example 12 (0.52 9, 0.001 mol) was dissolved in a mixture of 60 mL of water and 30 mL of ethyl acetate. The mixture was cooled at 5C and deaerated, then sodium bicarbonate (0.5 9, 0.006 mol) and sodium dithionite (0.7 9, 0.004 mol) were added and the resultant mixture was stirred, with deaeration and .~ .
.
.
,, F~
i 3 3 ~
cooling, for 30 minutes. The laye s were separated and the aqueous layer was extracted with 30 mL of ethyl acetate. The organic solution was extracted with 20 mL
of cooled, deaerated water. After drying over sodium sulfate, the solvent ~as removed. Yield 55% (0.25 9) of yellow crystals, melting at 155-160C (dec.). The product reduced alcoholic silver nitrate solution and had the formula:
~: :
Ny~ CNH2 ~N~CH 20~CH 2-N~
EXA~PLE 16 Pre aration of 3-[3'-(3''-Carbamovl-1'',4''-dihvdro~
p - -: ~
pyridin-1''-yl)propionyloxymethyl]-5,5-diphenyl-2,4 imidazolidinedione~
Substitution of the product of Example 13 in the general procedure of Example 15 and substantial repeti-tion of the sodium dithionite reduction there detailed afforded the desired product in 85% yield. The product melted at 100~C (dec.) and had the formula:
: .
N2olcicN2cN2-~ ~
~331~ 3~
The product of Example 14 can be similarly reduced to the corresponding dihydro derivative, melting at 105C (dec.).
Preparation of 4-Aminobutanoic acid cyclohexyl ester hydrochloride:
~ ABA (8 9, 77.6 mmol) was suspended in 100 mL
~0.96 mol) of cyclohexanol. Thionyl chloride (40 mL) was added dropwise to the mixture at 0C. The mixture was then refluxed for 4 hours, cooled and crystallized from ethyl ether. The white crystals obtained in this manner were filtered and dried. NMR analysis confirmed the identity of the product.
EXA~PLE 18 Preparation of 3-~-[(3'-Cyclohexyloxycarbonyl)-propyl]~carbamoylpyridine:
Nicotinic acid (2.2 9, 18 mmol) was suspended in 50 mL of dry pyridine. Dicyclohexylcarbodiimide (3.68 9, 17.9 mmol) was dissolved in the solution, with stir-ring. 4-Aminobutanoic acid cyclohexyl ester hydro-; chloride (4 9, 18 mmol) was added and the mixture was stirred for 48 hours. Precipitated dicyclohexylurea was removed by filtration and the filtrate was evapo-rated to dryness. The residue was washed with 25 mL of ice cold water and extracted into ethyl acetate. The layers were separated and the organic layer was evapo-rated to dryness. ~MR analysis confirmed the structure ;~ of the product.
1 ~ 3 ~
EXAMPLE l9 Preparation of l-Methyl-3-fN'-[(3'-Cyclohexyloxycar-bonyl)propyl]~carbamoylpyridinium iodide:
The product of Example 18 (1.74 9, 6 mmol) was dissolved in a minimum amount of acetone and the re-sulting white precipitate was filtered. ~ethyl iodide (1.5 mL, 24 mmol) was added in one portion to the~ ~ ;
solution, with stirring, at 0C. The mixture was allowed to gently reflux overnight. Filtration of a ;~
white precipitate and evaporation of the yellow filtrate produced a reddish oil, which was dissolved in acetone, filtered and evaporated to dryness. Anal.
calc- for C22H233N21: C, 47-26; H~ 5.79; N~ 6-48; 1, 29.38. Found: C, 47.03; H, 5.85; N, 6.44; 1, 29.26.
Preparation of l-Methyl-3-~N-[(3'-cyclohexylcarbonyl)-propyl]~carbdmoyl-1,4-dihydropyridine:
The product of Example 19 (0.11 9, 0.26 mmol) was dissolved in 25 mL of ice cold deaerated water. NaHC03 20 (0.09 9, 4-fold excess) was added, followed by Na2S204 (0.14 9, 3-fold excess). Ethyl acetate (25 mL) was added and the mixture was stirred under nitrogen for 30 minutes. The organic layer was extracted and dried to give an orange oil that reduced methanolic silver nitrate immediately. NMR analysis confi med that the product had the structure:
¢~NIlC~2C~2C~2Qo{) .
133~
EXA~PLE 21 Preparation of Valproic acid chloride (2-Propyl-pentanoyl chloride):
To 4.32 9 (30 mmol) of valproic acid in an ice bath, thionyl chloride (3.6n 9, 30 mmol) was slowly added, with stirring. The neat mixture was allowed to -come to room temperature and then heated in a water hath at 50C for 30 minutes. 50 mL portions of dry benzene were twice added and removed under reduced pressure. The resultant product was used in subse-quent reactions without further purification.
: ~ .
EXA~PLE 22 Preparation of Valproic acid 2-iodoethyl ester (2'-Iodoethyl 2-propylpentanoate):
To the product of Example 21 (4.87 9, 3~ mmol), 2-iodoethanol (5.16 9, 30 mmol) was added with stirring and cooling in an ice bath. The neat mixture was then '.~ J~
heated to 100C in a water bath for 10 minutes, then removed from the heat and stirred for an additional 10 minutes. The reaction mixture was then dissolved in 50 mL of ether, washed with water (1 x 30 mL), 5% NaOH (2 x 30 mL), and again with water (2 x 30 mL). The ether layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. A light yellow liquid product was obtained in 67X yield from valproic acid (6.0 9). Silver nitrate gave a bright yellow precipitate. N~R analysis confirmed the identity of the product.
1 3 ~
Preparation of 1-[2'-(2''-Propyl)pentanoyloxy]ethyl-3-carbamoylpyridinium iodide~
The product of Example 22 (3.28 9, 11 mmol) and 50 mL of dimethylformamide were added to nicotinamide (1.22 9, 10 mmol). The mixture was heated to reflux for 3 hours, then was cooled. Removal of solvent under reduced pressure afforded a brown oily residue, which was stirred with ether (60 mL) for 30 m~nutes, giving a yellow powder. The ether was decanted and a fresh portion of ether (50 mL) was added. The crude product was vacuum filtered under N2, then wdS recrystallized from isopropanoltether to give 3.5 9 of the desired product (84% yield), m.p. 111-112C. The product had the formula:
~ NH2 ~OJ I ~ ~
R ~ ~:
CH2CH20~CH2CIHcH2cH3 EXA~PLE 24 Preparation of 1-[2'-(2''-Propyl)pentanoyloxy]ethyl-3-carbamoyl-1,4-dihydropyridine:
To S0 mL of ice-cold degassed deionized water, the product of Example 23 (420 mg, 1 mmol) was added. To that solution, NaH003 (366 mg, 4 mmol) and Na2S204 (696 mg, 4 mmol) were added, with stirring. Nitrogen gas was bubbled through the solution for 30 minutes. The aqueous solution was then extracted with ether (6 x 25 .
1~31~
mL) until the ether layer was no longer yellow. The combined ether extracts were washed with water (1 x S0 mL) and dried over MgS04. The ether laye was decanted from the drying agent and the solvent was removed under reduced pressure. To the oily residue, ether was added and then removed (10 x 5 mL) on a vacuum pump. A foam was formed, which returned to an oil upon exposure to the atmosphere. Structure was confirmed by NMR `
analysis.
Preparation of N-Nicotinoyltyrosine eth~l ester~
Nicotinic acid (12.3 9, 0.1 mol) was dissolved in ^'~
dry pyridine (300 mL). The solution was cooled and dicyclohexylcarbodiimide (20.6 9, 0.1 mol) was added.
~fter dissolution, tyrosine ethyl ester hydrochloride (24.6 9, 0.1 mol) was added and the solution was stirred overnight. The precipitated dicyclohexylurea (DCU) was removed by filtration. Additional ~CU was removed by triturating the oil with hot water. The product was purified with acetone. Calculated for C17Hl~N204-1/2H20: C, 63.16; H, 5.88; N, 8.66.
Found: C, 63.10; H, 5.96; N, 8.59. The product can also be named N-[l-ethoxycarbonyl-2-(4'-hydroxyphenyl)-ethyl]nicotinamide.
Preparation of N-[(l-Methyl-3-pyridinium)carbonyl3-tyrosine ethyl ester iodide:
N-Nicotinoyltyrosine ethyl ester (20 9, 0.06 mol) was dissolved in 200 mL of acetone. A two molar excess ':~'" " ~'' , . ..
of methyl iodine (25.6 9, 0.18 mol) was added and the mixture was refluxed for 6 hours. The solvent was removed under reduced pressure to yield the desired product as a solid form. NMR analysis confirmed the identity of the product, which had the structural formula: ~ -~ ~--NH~ COOCH2cH3 CH ~ I
and can also be named l-methyl-3-~N-t(1'-ethoxy-carbonyl)-2'-(4''-hydroxyphenyl)ethyl}carbamoyl-pyridinium iodide.
EXA~PLE 27 -Prepa,ation of l-Methyl-3-~N-[(1'-ethoxycarbonyl)-2'-(4''-pivaloyloxyphenyl)ethyl]~carbamoylpyridinium trifluoroacetate:
,_ The product of Example 26 (6 9, n.013 mol) was dissolved in 50 mL of cold trifluoroacetic acid at 0C
in an ice bath. Pivaloyl chloride (3.14 9, 0.026 mol) was slowly added and the solution was wa,med to room temperature. After 24 hours, the solvent was removed under reduced pressure. The resulting dark oil was triturated with petroleum ether but no solidification occur,ed. Identity of the product was confirmed by NMR
analysis. The product was dissolved in aqueous methanol (1OX) and extracted with ethyl ether to remove a highly colored contaminant before using as the starting material in Example 29 below.
Preparation of 1-Methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4''-isobutyryloxyphenyl)ethyl]~carbamoylpyridinium trifluoroacetate:
The product of Example 26 (6 9, 0.013 mol) was dissolved in 50 mL of trifluoroacetic acid cooled to 0C in an ice bath. To that solution, with stirring, 10 was slowly added isobutyryl chloride (2.77 9, 2.76 mL). The solution was stirred overnight at ambient temperature and the solvent was removed unde, reduced pressure. The oil was stirred overnight with petroleum -ether and then dried in vacuo, but no $olidification occurred. Identity of the product was confirmed by NMR
analysis. The product was dissolved in aqueous metha-nol (lOX) and extracted with ethyl ether to remove a highly colored contaminant before using in Example 30 ;~ below.
~:. . .: ..
~',' . ' ,'- -:
- ~:
,- ' ,:
~ $ ~ 1 ~r~
Preparation of l-Methyl-3-{N-t(l'-ethoxycarbonyl)-2'-(4''-pivaloyloxyphenyl)ethyl]~ca bamoyl-1,4-dihydro-pyridine:
The product of Example 27 (4.07 9, n.oo79 mol) was dissolved in lO0 mL of 25% aqueous methanol. Nitrogen gas was bubbled through the solution. To the solution, stirring in an ice bath, was then added NaHC03 (2.02 9, 0.024 mol). Ethyl ether (lO0 mL) was added, followed 10 by the addition of Na2S204 (4.12 9, t).024 mol). The yellow biphasic solution was stirred for 30 minutes, then the layers were separated and the aqueous layer was extracted twice with 75 mL portions of ethyl ether. The combined organic fractions were dried ove 15 lla2so4 and the solvent was ,emoved under reduced pressure to afford a solid foam which was oxidi~ed by ethanolic silver nitrate. Anal. calc. for C23H20N20 l/2H20: C, 65.23; H, 7.33. Found: C, 65.76; H, 7.28; N, 6.95.
. .
3 ~
Preparation of 1-Methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4''-isobutyryloxyphenyl)ethyll~calbamoyl-1,4-dihydropyridine:
The product of Example 28 (2.20 9, 0.0044 mol) was dissolved in 100 mL of aqueous methanol. The solution WdS cooled in an ice bath with a stream of N2 passing through it. To this solution, NaHC03 (1.11 9, 0.0132 mol) and ether (100 mL) were added. Then, sodium dithionite (2.30 9, 0.0132 mol) was added and the solution was stirred for 30 minutes. The layers were separated and the aqueous phase was washed with ethyl ether. The com~ined organic layers were dried over ¦ anhydrous Na2S04 and reduced in volume. The resultant orange oil was oxidized by ethanolic silver nitrate.
Identity of the product was confirmed by NMR analysis.
EXA~PLE 31 Preparation of Chloromethyl [2S-(2,5,6~)]-3,3-dimethyl-7-oxo-6-t(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclot3.2.0]heptane-2-carboxylate~
To a solution of 4.02 9 (0.01 mol) methicillin sodium salt in 10 mL water and 10 mL CH2Cl2, 2.4 9 sodium bicarbonate and 0.34 9 tetrabutylammonium ~ -hydrogen sulfate were added. Then, 1.9 9 (0.0115 mol) 25 chloromethyl chlorosulfate dissolved in 3 mL CH2Cl2 ~--were added with stirring, over a 5 minute period, keeping the temperature below 30C. After an additional 30 minutes of stirring, the organic phase was separated, washed twice with water and dried over ~gS04. 8y removing the solvent in vacuo, 4.24 9 of the : , . ~ ~ :
. ~ ~ " ~ .. . . . ' . . ~ ' ~ ' .
desined product were ohtaine~ dS a yellow solid, melting at 88-90C.
Preparation of Chloromethyl [2S-(2a,5,6~)]-3,3-5 dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7^oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
Substantial repetition of the procedure of Example 31 using 2.12 9 (O.i)05 mol) oxacillin sodium salt with 1.2 9 NaHC03, 0.17 9 tetrabutylammonium hydrogen 10 sulfate and 0.95 9 chloromethylchlorosulfate afforded 1.87 9 of the desired product melting at 78-80C ~dec.).
EXA~PLE 33 Preparation of Chloromethyl t2S-(2,5,6B)]-6-~3-(2-15 chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
:
Using the same procedure as in Example 31, but substituting 2.38 9 (0.005 mol) cloxacillin sodium salt ~ ~-20 (1 rnol water), 1.2 9 NaHC03, 0.17 9 Bu4NHS04 and 0.95 9 chloromethyl chlorosulfate gave 2.27 9 of the desired product melting at 97-100C (dec.).
. , .
i . ~ .. ., ~ . .. . . ~ . , ~ ......
~ ~ 3 ~ r ~ ,~
Preparation of Chloromethyl [2S-(2~,5,6B)]-6-[3-(2,fi-dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicycloC3.2.0]heptane-2-carboxylate:
Similarly, following the procedure of Example 31 but using 2.55 9 (0.005 mol) dicloxacillin Na salt (1 mol water) with 1.7 9 NaHC03, 0.17 9 Bu4NHS04 and 0.95 g chloromethyl chlorosulfate, 2.43 9 of product were obtained melting at 98-101C (dec.).
EXA~PLE 35 Prepa ation of [(3-Pyridinylcarbonyl)oxy]methyl [2S-(2~,5,~B)]-3,3-dimethyl-7-oxo-6-[(2,6-dimethoxy)-benzamido]-4-thia-1-azabicyclo[3.2.0]heptane-~-carboxy-late~
Three and eight-tenth grams (0.0089 mol) of the methicillin chloromethyl ester produced in Example 31 ;
and 1.6 9 (0.01 mL) potassium nicotinate in 70 mL nMF
were stirred 6 days at room temperature (20-25C). 300 ~
mL ethyl acetate were added, the resultant solid was ~ `
removed by filtration and the solution was extracted 4 times with 50 mL concentrated aqueous NaCl and dried over MgS04. The sol vent was removed in vacuo and the resultant residue was purified by chromatography (silica gel). Obtained as a white solid were 3 9 of the desired product melting at 151-157C.
.
~ 3 ~
Preparation of [(3-Pyridinylcarbonyl)oxy~metnyl [2S-(2,5,fiB)]-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxa-zolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.n]-heptane-2-carboxylate:
Following the procedure of Example 35, but utiliz-ing 1.81 9 (0.004 mol) of the oxacillin chloromethyl ester produced in Example 32 and 0.75 9 (0.0046 mol) K
nicotinate, afforded, after purification by chromato-graphy, 0.75 9 of the desired product as a white solid melting at 79-82C (dec.).
Preparation of [(3-Pyridinylcarbonyl)oxy~methyl [2S-(2a,5a,6B)]-6-[3-(2-chlorophenyl)-5-methyl-4-isoXazole-15 carboxamido]-3,3 dimethyl-7-oxo-4-thia-1-azabicyclo-t3.2.0]heptane-2-carboxylate:
Using the procedure of Example 35, but substi-tuting 2.1 9 (0.0043 mol) of the cloxacillin chloro- 1 methyl ester produced in Example 33 and 0.8 9 (0.005 20 mol) K nicotinate, gave 1.2 9 of product melting at 83-85C (dec.).
Preparation of [(3-Pyridinylcarbonyl)oxy]methyl ~2S-(2a,5a,6B)]-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxa-25 zolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabi-cyclo[3.2.0]heptane-2-carboxylate:
Similarly, following the procedure of Example 35 but using 2.27 9 (0.0047 mol) of the dicloxacillin chloromethyl ester produced in Example 34 and 0.87 9 :~ :
`','~
. ~ . ~ , . . ... . .
6 ~
(0.0054 mol) K nicotinate afforded 1.1 9 of the product as a white solid melting at ~7-90C tdec.).
EXA~PLE 39 Preparation of [2S-(2a,5a,6~)]-3-~t[t[3,3-Dimethyl-7-oxo-6-[t2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo-[3.2.0]hept-2-yl~carbonyl]oxy~methoxy]carbonyl]-1-methylpyridinium iodide:
One and one-fourth grams (0.0024 mol) of the methicillin derivative produced in Example 35 in 35 mL
nitromethane and 1.14 9 (0.5 mL) (0.008 mol) methyl -iodide were reacted in a closed system at room -temperature (20-25C) for 7 days. The solvent was removed in vacuo, the resultant residue was stirred ~-~
~ith ether, filtered off, washed with ether and - -dried. There were thus obtained 1.6 9 of yellow hygroscopic product melting at 95-100C and being further characterized by the structural formula~
' ~ONH~ ~CH3 \=<~ ~ N-- CH3 C 3 o~
CO H2CC~ ~
~' ~ . .
~, ' - ` :, , ' : ' ' ` ~ :, . ` ' ` : ~ ': : : ' ` ' ~:,"''. ' "' .' ` ~ ~ ~ ` ' ` ` ' ~ ~ : . ,' - \
1331~
EXA~PLE 40 Preparation of C2S-(2a,5.6R)]-3-[[[[[3.3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]oxy]-methoxy]carbonyl]-1-methylpyridinium iodide: ;
Using the procedure of Example 39, but substitu-ting 0.5 9 (0.0009 mol) of the oxacillin derivative produced in Example 36 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH3I produced, after 6 days, 0.6 9 10 of the desired product melting at 75-80C and having the formula:
COCH2iC~ 1 Preparation of [2S-(2,5,6B)]-3-[[~C[6-[3-(2-chloro-15 phenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl_ 7-oxo-4-thia-1-azabicyclo~3.2.0]hept-2-yl]carbonyl]-oxy]methoxy]carbonyl]-1-methylpyridinium iodide:
S~milarly, using the procedure of Example 39, but substituting 0.44 9 (0.0008 mol) of the cloxacillin 20 derivitive produced in Example 37 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH3I, gave 0.45 9 of product melting at 90-95C (dec.) and having the formula:
~3~4 ~[~CH
COCH~OC~
Preparation of [2S-(2~,5a,6~)-3-t[[[[6-t3-(2,6- -dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]-carbonyl]oxy]methoxy]carbon~l]-1-methylpyridinium iodide~
In a similar manner, using the procedure of Example 39, but substituting 0.5 9 (0.007 mol) of the dicloxacillin derivative produced in Example 38 in 25 mL nitromethane and 0.45 9 (0.2 mL) (0.003 mol) CH31 gave 0.55 9 of product melting at 95-100C (dec.) and having the formula: ~
cl :
~C1~3~ ~Cll3 2 ~
~ ' CH3 :
::
i~.. :" i., ::. - :: :`
. ~, . ~ . . : ,.: , :
?~
1 3 ~
EXA~PLE 43 Preparation of t[(l,4-Dihydro-l-methyl-3-pyridinyl)-carbonyl]oxy]methyl [2S-(2a,5,6 ~)]-3,3-dimethyl-7-oxo- ~ i~
6-[(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo-t3.2.0]heptane-2-carboxylate~
0.45 9 (0.0007 mol) of the product of Example 39 dissolved in a mixture of deaerated 25 mL ethyl acetate and 70 mL water were reduced with a mixture of 0.34 9 (0.004 mol) NaHC03 and 0.48 9 (0.0028 mol) sodium dithionite at 0-5C over 70 minutes. The disappearance of the 268 nm maxima and increase of 366 nm maxima in the U.V. spectra were followed. The layers were separated and the aqueous layer was extracted with 2 x 25 mL ethyl acetate, then the organic layers were extracted with 2 x 20 mL cold, deaerated water. After drying over Na2S04, the solvent was removed in vacuo. -~
0.25 9 of the product was obtained as a yellow solid melting at 88-90C (dec.). The product had the formula: - ~
, :
~COMH~ ~CH3 . i \=< ,L -- CH3 0CH3 o~
Z 11--¢3 ., ~ ,", `; ~
,~
,,`~ ,.. ,, .~ ~ . .
; ~X'. . .` ~
133~
Preparation of ~[(1,4-Dihydro-l-methyl-3-pyridinyl)- ;-carbonyl]oxy]methyl [2S-(2a,5~,6B)]-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1- ~-azabicyclo[3.2.0]heptane-2-carboxylate~
Similarly, repetition of the general procedure of Example 43 using 0.17 9 (0.00025 mol) of the product of Example 40, 0.08 9 (0.0001 mol) NaHC03 and 0.51 9 (0.001 mol) Na2S204, in 15 mL water and 15 mL ethyl acetate, afforded 0.1 9 of product melting at 93-100C
(dec.). The product had the formula:
S,~CH3 ~CONH T1~CH3 I~ O CH3 o~ , .
COCH201CI~ ;
Preparation of t~(l,4-~ihydro-l~methyl-3-pyridinyl)-carbonyl]oxy]methyl ~2S-(2,5~,6~)~-6-~3-(2-chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo~3.2.0]heptane-2-carboxylate:
In a similar manner, following the procedure of Example 43, but substituting 0.18 9 (0.00025 mol) of the product of Example 41, 0.089 NaHC03 and 0.17 9 Na2S204. gave 0~13 9 of product as a yellow solid, ,: :
~ ,, . ;.
~ .~
5.
~y,~
13315~4 :
melting at 80-85C (dec.) and having the structural f o r m u l a ~
Cl ~<CR3 CoCH20 '~:
Preparation of [[(1,4-Dihydro-1-methyl-3-pyridinyl)-carbonyl]oxy]methyl [2S-(2,5,6~)]-6-[3-t2,6-dichloro- ~-phenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
In like manner, repetition of the procedure of Example 43 using 0.19 9 (0.00025 mol) of the product of Example 42, 0.08 9 NaHC03, 0.17 9 Na2S204 yielded 0.14 g of desired product melting at 98-102C (dec.) and ; having the formula:
Cl CH3 H F~<
Cl CH3 o N
ICIOCH201CI~ ; .
cL -:
:'~ 3 b .''. - .
~æ~
Preparation of [(3-Pyridinylcarbonyl)oxy]methyl [2S-(2~,5,6B)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]- -4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate:
~ suspension of 3.83 9 (o.nl mol) of the chloro~
methyl ester of benzylpenicillin, namely chloromethyl [2S-(2,5,6B)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)-amino]-4-thia-1-azabicyclot3.2.0]heptane-2-carboxylate, and 1.93 9 (0.012 mol) potassium pyridine-3-carboxylate in 100 mL of dimethylformamide was stirred at 20-25C
for 6 days. Then, 300 mL of ethyl acetate were added and the solid was removed by filtration. The solution was extracted 4 times with concentrated aqueous sodium chloride solution, then dried over ~gS04. The solvent was removed in vacuo to give 4.5 9 of foamy solid.
Purification by chromatography over silica gel using ethyl acetate as eluent afforded 2.5 9 of product melting at 127-130C.
EXA~PLE 48 Preparatlon of [2S-(2,5,6B)]-3-~[[[t3,3-Dimethyl-7-oxo-6-[(phenylacetyl)am1no]-4-thia-1-azabicyclo[3.2.0]-hept-2-yl]carbonyl]oxy]methoxy]carbonyl]-1-methyl-pyridiniùm iodide:
Two and one-half grams (0.053 mol) of the product of Example 47 dissolved in 100 mL of dry nitromethane were reacted with 2.25 9 (1 mL, 0.016 mol) of methyl iodide in a closed system at 20-25C for 6 days, at the end of which time thin layer chromatography showed complete reaction. The solvent was removed in vacuo and the solid residue was slurried with ether, filtered :
~ " ~ . ~ . . ~ ,: . . , 7 ~
.';-~
~ ~ 3 1 ~
and dried in vacuo over P205. The product, ~elting at ~ ;~
90-95C (dec.~, was obtained as a yellow solid (2.91 9). It was assigned the structural for~ula:
.:
C6H~CH2CNH~/ \<CH3 ~ :
N \1/ CH3 CoCH20 O I ' ' , ~
5EXA~PLE 49 Preparation of ~t(1,4-Dihydro-1-methyl-3-pyridinyl)car-bonyl]oxy]methyl [2S-(2,5a,6~)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]hep-tane-2-carboxylate:
The quaternary salt prepared in Example 48 (3.25 ~
9, 0.0053 mol) was dissolved in a mixture of 350 mL of ~-water and 150 mL of ethyl acetate. The resultant mlxture was cooled at 0-5C and deaerated with nitrogen, then a mixture of 2.67 9 (0.032 mol) of sodium bicarbonate and 3.69 9 (0.021 mol) of sodium dithionite was added over a 2-3 minute period. The reaction mixture was st~rred for 1 hour under the same conditions as before, then the layers were separated, the aqueous layer was extracted twice with 50 mL
portIons of ethyl acetate, and the combined organic extracts were washed twice with 30 mL portions of cold, deaerated water. Dr~ing over sodium sulfate and removal of the solvent in vacuo afforded 1.7 9 of yellow solld melting 98-100C and having the formula:
.
- :
~"
.
'.'~'`` . . .. ',' 'i ~ ' ' ,' :
1~31~
o 5 C6H5CH2CNH ~/ \/ CH3 N ~/\ CH3 ;
COCH20C--¢3 EXA~PLE 50 Preparation of Chloromethyl N-[3-(10,11-dihydro)-5H-dibenztb,f~azepin-5-yl)]propyl-N-methylcarbamate:
~ethod A:
Desipramine hydrochloride (1.5 9, 0.005 mol) was dissolved in 20 mL of methylene chloride, cooled at 0-5C. Then I g NaHC03 was added, followed by 0.92 9 (0.007 mol) chloromethyl chloroformate. The reaction mixture was st~rred for 1 hour, then the salts were removed by filtration and the solution was extracted twice with 10 mL portions of SX HCl. The organic layer was dried over ~gS04 and the solvent was removed in vacuo to give the desired compound as a colorless oily substance in 76X yield (1.35 9).
~ethod B:
Imipramine hydrochloride (1.59 9, O.OOS mol) was dissolved in 15 m~ of water, then 5 mL of 4X sodium hydroxide solution was added, with cooling. The resultant imipramine base was extracted twice with 10 mL portions of benzene. The solution was concentrated ~ .
1~31~6~
to 10 mL, then a solution of 0.7 9 (0.0054 mol) chloro-methyl chloroformate in 5 mL benzene was added with cooling at 10C. The reaction mixture was stirred at 20-25C for 30 minutes, then was refluxed for 1 hour.
A small amount of imipramine hydrochloride resulted and was filtered off. The solution was extracted twice with 20 mL portions of 4X HCl and dried over MgS04.
Removal of solvent in vacuo afforded 1.2 9 (66%) of product having the same characteristics as that obtained by Method ~.
Preparation of l-Chloroethyl N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-S-yl)]propyl-N-methylcarbamate:
Following the general procedure of Example 50, but using 1.5 9 (O.nO5 mol) of desipramine hydrochloride and 0.86 9 (0.006 mol) chloroethyl chloroformate and carrying out the reaction at 5-10C for 2 hours, gave 1.6 9 (86X) of the title compound as a colorless oil.
Preparation of ~N-t3-(10,11-Dihydro-5H-dibenz[b,f]-azepin-S-yl)]propyl-N-methylamino~carbonyloxy]methyl 3-pyridinecarboxylate:
The product of Example 50 (1.35 g, 0.0037 mol) dissolved in 5 mL dimethylformamide was added to a solution prepared from 0.57 9 (0.046 mol) nicotinic acid and 0.45 9 triethylamine in 5 mL dimethyl-formamide. The mixture was stirred for 24 hours at 25-30C, then 30 mL of ethyl acetate were added. The precipitated salts were removed by filtration and the .
' ; , .. ~ . " . ...
~3~
solution was extracted 4 times with 15 ~L portions of saturated aqueous sodium chloride solution. Drying over ~gS04 and removal of the solvent in vacuo afforded 1 9 (61X) of pure product as an oil.
PreQaration of ~ N-~3-(10,11-Dihydro-5H-dibenz~b~f~-azepin-5-yl)]propyl-N-methylamino~carbonyloxy]ethyl 3-pyridinecarboxylate:
Following the general procedure of Example 52, but using 1.05 9 (0.0028 mol) of the product of Exa~ple 51, 0.45 9 (0.036 mol) of nicotinic acid and 0.36 9 of tri-ethylamine in 10 mL dimethylformamide and carrying out the reaction at 25-30C for 48 hours, gave 0.5 9 of the title compound as a yellow oil.
EXA~PLE 54 Preparation of 3-C~N-~3-(10,11-Dihydro-5H-dibenz[b,f]-azepin-5-yl)]prop~l-N-methylamino~carbonyloxy]methoxy-carbonyl-1-methylpyridin1um iodide:
Eight-tenths gram (0.0018 mol) of the product of Example 52 in ~0 mL of nitromethane was methylated with 0.8 mL of methyl iodide at 25-30C for 48 hours. The solvent was removed in vacuo and the residue was slurried with ethyl ether, filtered and dried over P205. The quaternary salt was obtained in 83X yield (0.88 9) as a light yellow solid melting at 172-175C
(dec.) and having the structural formula:
~".,~, ~...~.,.,, . ~ ..~ , .~. ., . ' :
1331 ~4 ~o ~1OCH
CH2CN2 2 \CH
tH3 Preparation of 3-[1-~N-[3-(10,11-Oihyd,o-SH-dibenz-~b,f]azepin-5-yl)]propyl-N-methylamino~carbonyloxy]-S ethoxycarbonyl-l-methylpyridinium iodide:
Following the general alkylation procedure of Example 54, but using 0.5 9 (0.0011 mol) of the product of Example 53 in lS mL nitro~ethane with 0.5 mL methyl iodide, and carrying out the reaction at 20-25C for 6 days, afforded 0.33 9 (50~) of the desired quaternary salt as a dark yellow solid melting at 101-103C (dec.) ~-and having the structural formula:
,~, H3 ll CH2CH2 2 <CH ~ 1- :
L : ~
~,:
: ' ~: ' r.~ ~
~3i~
`_reparation of [~N-[3-(10,11-Dihydro-5H-dibenz[b,f~
azepin-5-yl)~propyl-N-methylamino~carbonyloxy]methyl 1,4-dihydro-1-methyl-3-py,idinecarboxylate: -Three-tenths gram (O.û005 mol) of the product of Example 54 in 30 mL water and 15 mL ethyl acetate was reduced with 0.25 9 (0.003 mol) NaHC03 and 0.35 9 (0.002 mol) sodium dithionite at 0-5C, with deaera-tion, for a 60 minute period. The layers were separated and the aqueous layer was extracted twice with 30 mL portions of ethyl acetate. The combined organic layers were then extracted twice with 20 mL
portions of cool deaerated water. Drying over Na2S04 and removal of the solvent in vacuo afforded 0.22 9 (9S%) of the title compound, melting at 59-63C (dec.) and having the structural formula:
~q ~'~ ' ~N ~ O O
~YOCH20C
CH2CH2CH2 \ ~N J
EXAltPLE 57 Preparation of ~ N-~3-(10,11-Dihydro-SH-dibenz[btf]-azepin-5-yl)]propyl-N-methylamino~carbonyloxy]ethyl ~ ~;
1,4~-dlhydro-1-methyl-3~pyridinecarboxylate~
Following the procedure of Example 56, but us1ng 0.1 9 (0.0017 mol) of the product of Example 55 in 10 ~ i mL water and 6 m~ ethyl acetate, 0.11 9 NaHC03 and 0.15 ~
~" . . ;.. ~ . ;~.. . - ~ . . ; . , ,: . .
r ~ ~ :
g Na2S204 and carring out the react~on for a 60 minute per~od, gave 0.07 9 (88X) of the title compound as a yellow sol~d, melt1ng at 60-65C (dec.) and having the structural formula:
~ 8 IH3 ~COCH
CH2CH2CH2N~CH3 ~
EX~#PLE 58 Preparat1On of N-(2-~ydroxyethyl)-3-pyridinecarboxa-mide:
A solutlon of 49.2 9 (0.32525 mol) ethyl n~co-t~nate and 72 9 (1.17 mol) ethanolamine was heated at 70C for 60 hours. The excess ethanolam1ne was removed under reduced pressure and the result~ng v1scous cream o11 was stlrred w1th ether for 48 hours. The result~ng wh1te solld was removed by f11trat~on, afford1ng 46 9 (85.1X) of the t1tle compound melting at 75-78C.
EXA#PLE 59 Preparatlon of 3-t2~-(2U-Propyl)pentanoyloxy]ethylcar bamoylpyrldine:
To a st~rred solut~on of 1.0 9 (0.006021 mol) of ~ ~i the product of Example 58 and 0.61 9 (0.00598 mol) tri-ethylam~ne in 40 mL dry dlchloromethane, 1.96 9 (0.012047 mol) of (2-propyl)pentanoyl chlor1de were , :
added and the mixture was refluxed for 4 hours. The resultant solution was washed sequentially with 30 mL
SX NaHC03, 30 mL 5X HCl and 30 mL water. The organic layer was dried over ~gS04 and the solvent was removed under reduced pressure to give 0.6 9 (34.3X) of the product as a pale brown oil.
EXA~PLE 60 Preparation of 1-Methyl-3-t2'-(2"-propy7)pentanoylox~]-ethylcarbamoylpyridinium lodide:
To a solution of 1 9 (0.00342 mol) of the product of Example 59 in 20 mL of dry ethyl acetate, 0.73 9 (0.00513 mol) of methyl iodide was added. The reaction mixture was stirred overnight at room temperature. The pale yellow solid which formed was removed by filtra-tion and recrystallized from ethyl acetate to give 1.35 9 (90.9X) of the quaternary salt as a yellow crystal-line solid. The product htd the formula:
o ,, `NHCH2CH20C-cH(cH2cH2cH3)2 as confirmed by IR, NMR and UV analyses.
-:
EXA~PLE 61 Preparation of 1-Methyl-3-~2'-~2"-propyl)pentanoyloxy]-ethylcarbamoyl-1,4-dihydropyridine:
To 50 mL of vigorously stirred, degassed, ice-cold deionized water, a solutlon of 3.0 9 (0.006909 mol) of the quaternary product of Example 60 in 50 mL of ethyl acetate was added. Throughout the reaction, the temperature and pH were maintained at 0C and 8 respectively, while nitrogen was bubbled through the reaction m~xture. A mixture of 3.5 9 (0.04145 mol) of sodium bicarbonate and 4.8 9 (0.02763 mol) of sodium dithionite was added portionwise. After 45 minutes, the organic layer was separated and the aqueous layer was extracted with 100 mL of ice-cold ethyl acetate.
The combined organic extracts were washed with ice-cold water and dried over ~gS04. Solvent was removed under reduced pressure to give 2.1 9 (98.8X) of the product as a pale yellow solid having the structural formula:
cN2c2oc-C(C 2C 2C 3)2 ' 20 dS confirr3ed by IR. NMR and UV anaI~ses.
~, .
: .
, ., .
1 3 ~
Preparat~on of 3-Pyridinecarboxylic acid (2-hydroxy)-ethyl ester hydrochlorlde:
To 120 mL cold (-10C) ethylene glycol, 16 mL of thionyl chloride were added dropwise. Upon completion o~ the addition, 24.6 9 (0.2 mo1) of nicotinic acid were added portlonw~se and the reaction m~xture was heated overn19ht at 60C. rhen, 700 mL of hot tetra-hydrofuran were added and the mixture was cooled. The sol1d which formed was removed by filtration and washed with ether to give 28.5 9 of the title compound as white crystals.
EX~PLE 63 Preparation of 3-[2'-(2"-Propyl)pentanoyloxy]ethoxy-carbonylpyr1dine To a solut1On of 10.0 9 (0.0491 mol) of the product of Example 62 ln 150 mL of dry CH2C12, 10.7 9 (0.09819 mol) of triethylamine were added. After all of the solld was dlssolved, 11.92 9 (0.07364 mol) of 2-propylpentanoyl chlorlde were added and the reactionm~xture was st1rred at room temperature for 36 hours.
Sequent1al washing w1th SX NaHC03, 5X HCl and water afforded an organlc layer ~hich was then dried over -anhydrous ~gS04. Solvent was removed under reduced pressure to g1ve a yellow-brown o11 that was tr~turated w1th a 40:60 mixture of ether and petroleum ether to y1eld 9.7 9 of the product as an orange o~l.
1 3 3 1 ~
EXA~PLE 64 Preparation of l-Methyl-3-t2'~ -Propyl)pentanoyloxy]-ethoxycarbonyl~yridinium iodide:
To a solut~on of 2.0 9 (0.006816 mol) of the S product of Example 63 in 10 mL of dry acetone, 1.45 9 (0.01022 mol) of methyl iodide were added and the mixture was refluxed overnight. Removal of solvent under reduced pressure afforded 1.84 9 of the quater-nary salt as a brown oil. The product had the struc-tural formula~
~ : -~OCH2CH20~-cH(cH2cH2cH3)2 "; ~';
EXA~PLE 65 .~ ,., Preparation of 1-Methyl-3-~2'-(2U-propyl)pentanoyloxy]-ethoxycarbonyl-1,4-dihydropyridine:
~o S0 mL of vlgorously stirred, degassed, ice-cold deionlzed water, a solution of 1.84 9 (0.004226 mo1) of the quaternary product of Example 64 in 50 mL of ethyl acetate was added. Throughout the reaction, the temperature and pH were malntained at 0C and 8, respectively, wh11e argon was bubbled through the reaction mixture. A m1xture of 2.13 9 (0.02536 mol) of HaHC03 and 2.94 9 (0.0169 mol) of ~a2S204 was added portionwlse. After 55 minutes, the organic layer was separated and the aqueous layer was extracted with 100 mL of ~ce-cold ethyl acetate. The combined organ~c ..,.,:
. ~ .
1331~B~
layers were washed with ice-cold water and dried over MgS04O ~he solvent was re~oved under reduced pressure to give 0.9 9 of the title compound as a yellow oil. h ~he product had the formula~
¢~/ ~ocH2cH2o~ cH(cH2cH2cH3)2 CH3 :
'`~"'; ;' Preparation of 3,17~-Bis[(3-pyridinylcarbonyl)oxy]-19-nor-17-pregna-1,3,5(10)-trien-20-yne:
To 2.0 9 (6.7 mmol) of ethinyl estradiol dissolved in 50 mL of dry pyridine were added 6.16 9 (0.027 mol) of nicotinic anhydride and a catalytic quantity of 4-(d1methylamino)pyr~dine (DMAP). The solut~on was warmed gently to 50C to effect solution. After 2 weeks, the pyridlne solut1On was poured over ice and the solid produced was collected by filtration. The solid was dried over P205 in vacuo to give 3 9 (85X) of an off-white powder.
EXA~PLE 67 Preparation of 3-Hydroxy-17B-~(3-pyridinylcarbonyl)-oxy]-19-nor-17a-pregna-1,3,5(10)-trien-20-yne:
~ o 200 mL of O.St methanolic KHC03, 2.0 9 (3.9 mmol) of the product of Example 66 were added. After 6 `
~' .
-~.. . . .
1 3 3 ~
hours, the slurry was dlluted with 200 mL of water and the mlxture was extracted with chloroform. The organic layers were combined, dr~ed over MgS04 and concentrated in vacuo. The resulting oil was triturated with hexane to g~ve 1.48 9 (94X) of a wh~te solid. NMR and UV
spectra and elemental analysis confirmed the identity of the title compound.
EXA~PLE 68 Preparat~on of 1-Methyl-3-{~(19-nor-17-pregna-1,3,5(10)-tr~en-20-yn-17B-yl)oxy]carbonyl~pyridinium iodide:
To 50 mL of acetone, 1.0 9 (2.5 mmol) of the ~
product of Example 67 was added, followed by 2 mL of '.`I.!.~i*~`
methyl ~odide. The reaction mixture was refluxed for 12 hours. The solid which formed was collected by flltrat1On, y1elding 1.15 9 (85X) of the quaternary salt as a yellow solid havlng the structural formula f ~ CH3 ~-C~CH
HO~ '' ~
The asslgned structure was conflrmed by UV and NMR
spectral analyses and by elemental analysls.
1 3 ~
Preparation of 3-Hydroxy-17B-~ methyl^1,4 dihydropyridin-3-yl)carbonyl]oxy~-19-nor-17~-pregna~
1 ,3,5[10)-trien-20-yne~
To a cooled suspension of 1.0 9 (1.8 mmol) of the product of Example 68 in 100 mL of SO:S0 water: tert-butanol, 0.77 9 of ~aHC03 and 0.96 9 of Na2S204 were added. The react~on m~xture was stirred at 0C for 1 hour, then was extracted twice with 100 mL portions of CH2C12. The organic extracts were combined, dried over ~gS04 and concentrated under reduced pressure to sive 520 mg (69%) of the t~tle compound as a yellow foam. : -The product was assigned the structure : -:
o ~ CH3 OC~ ~ `
CH3 l ~-C--CH
~ ' HO
lS wh~ch was ~n accord wlth UV and N~R values as well as elemental analysis.
,: : :: . : ~ , . . .
; ~ , , ~ . .
1 ~ 3 ~
' Preparation of 3,17~-Bis[(3-pyridinylcarbonyl)oxy]-estra-1,3,5(10)-triene~
To 5.3 9 (0.03 mol) of nicotinoyl chloride in 30 mL of dry pyridine at 0C were added 2.0 9 (0.0073 mol) of ~-estradiol. The reaction mixture was refluxed for 1 hour, then was poured over 100 ~L of ice water, and the resulting precipitate was collected by filtration. The precipitate was dried in vacuo over P205, affording 3.18 9 (90X) of the title compound melting at 148-150C.
Preparation of 1,1'-Dimethyl-3,3'-{[(estra-1,3,5(10)-triene-3,17~-diyl~dioxy]dicarbonyl~dip~ridinium diiodide:
To 50 mL of acetone and 2 mL (0.032 mol) of methyl iodide, 2.0 9 (0.004 mol) of the product of Example 70 were added. The solution was heated at reflux overnight. The precipitate which formed was filtered, washed with acetone and dried to give 2.75 9 (88%) of the quaternary salt melting at 251-252C and having the structural formula - ~C~13 CH O~J~ -as confirmed by UV, NMR and elemental analyses.
''' ~,':,''. ~
-:
13~1 -155~
EXA~PLE 72 Preparation of 3,17B-Bis~t(l-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy~estra-1,3,5(10)-triene:
One gram (1.31 mmol) of the product of Example 71 was dissolved in 100 mL of dry acetonitrile. To that solut10n, which was flushed with nitrogen, 0.28 9 (1.31 mmol) of l-(phenylmethyl)-4-(aminocarbonyl)-1,2-dihy-dropyr~dine was added, and the reaction mixture was st~rred at 0C for 1 hour. Removal of the solvent under reduced pressure afforded a solid, which was suspended in methylene chloride and removed by filtration. The filtrate was chromatographed several times on a neutral alumina column prepared with methylene chloride. Purification and evaporation of the solvent ln vacuo gave a solid foam. The product had the formula ~3 ~ ' dS confirned by UV, NMR and elemental analyses.
1333 ~
-156- ~ `
Preparation of 3-(Phenylcarbonyloxy)-17B-[(3-pyridinyl- ,.~ tr~.
carbonyl~oxy]estra-1,3,5(10)-triene:
Estradiol benzoate (2.5 9, 6.6 mmol) was dissolved in 50 mL of dry pyridine, then 1.66 9 of nicotinic anhydride and a catalytic amount of 4-(dimethylamino)-pyridine (DMAP) were added. The reaction mixture was -st~rred for 5 days at room temperature, then was poured into ice water. The solid which formed was collected by filtration and dried in vacuo, yielding 3.01 9 (94~) of the product as a white solid melting at 151-154C.
EXA~PLE ~4 Preparation of l-Methyl-3-(~3-(phenylcarbonyloxy)-estra-1.3,5(10)-trien-17g-yl]oxy~carbonyl)pynidinium iodide:
The product of Example 73 (1.5 9, 3.1 mmol) was suspended in 2.5 mL of acetone. Then, 2 mL of methyl iodide were added and the reaction mixture was refluxed overnight. The yellow solid (1.8 9, 93X) was collected by filtration and drled in vacuo. UV, NMR and elemental analyses confirmed that the product had the ass~gned structure:
C~3 .
OC~ 1~
~~
!~
;?~' ::' : ' : :~ ~ ~ : . - ' :
1~31~
EXA~PLE 75 Preparation of 3-(Phenylcarbonyloxy)-17B-~[1-methyl- -~
1,4-dihydropyridin-3-yl)carbonyl~oxy~estra-1,3,5(10)~
triene:
The quaternary salt prepared in Example 74 (1.2 9, 1.93 mmol) was suspended in 100 mL of 50:50 tert-butyl alcohol/water. Then, 0.81 9 of NaHC03 and 1.0 9 of Na2S204 were added and the reaction was allowed to cont~nue for 1.5 hours. The resultant solution was extracted with CH2Cl2~ and the organic phase was dried over MgS04 and concentrated in vacuo to afford 650 mg of title compound as a yellow foam. The identity of the product was confirmed by UV, NMR and elemental analyses. It was assigned the structure:
~3 ''~' , ~ H3 ~Co~J ''' ~ "
EXA~PLE 76 Preparation of_H-(2-~4-tBis(2-chloroethyl)amino]-butanoyloxy}ethyl)-3-pyridinecarboxam~de~
Chlorambucil (2n g, 0.0657 mol) was dissolved in 800 mL of dry acetonitrile, then 13.1 g (0.079 mol) of N-(2-hydroxyethyl)-3-pyr~dinecarboxamide were added.
Acetonitrile was added until the solution was clear.
The total volume of aceton~trile used at this stage was ..,'~
,, ", '~
' a '~
~ ~ 3 ~
850 mL. To the stirred solution, maintained over argon, there were added 1.492 9 (0.0723 mol) of di-cyclohexylcarbodiimide and 0.802 9 (0.0066 mol) of 4-(dimethylamino)pyridine (DMAP). The reaction mixture was stirred overnight at room temperature under dry conditions, and the progress of the reaction was followed by thin layer chromatography. At the end of the reaction period, the solid which formed was removed and washed with 50 mL of cold acetonitrile. The filtrate was evaporated in vacuo at 30C, and the yellow solid thus obta~ned was dissolved in a minimum amount (15 mL) of 8:2 chloroform/tetrahydrofuran and appl~ed to a column packed with 900 9 of sllica gel.
The column was eluted with 8:2 chloroform/tetrahydro-furan. The adduct and chlorambucil were eluted withinthe first 500 mL, and the desired ester was then collected upon evaporation of the eluent under ~ ~-vacuum. The title compound was obtained in 82.7~ yield as a yellow solid melt~ng at 73-75C. It had the formula (Clt~2c ~)2~(c`2)3-c-oc zCrz~
~ ' ~'~ ' ~ ' '' ~ ' ' i' 1 'I `
_ 1 33 ~
EXAMPLE ~7 Preparation of 1-Methyl-3-[(N- ~ t4-(~4-bis(2-chloro- -ethyl)amino]~phenyl)butanoyloxy]ethyl~)carbamoyl]-pyridinium methanosulfate:
The product of Example 76 t2 9, 0.04 mol) was dissolved in 200 mL dry acetonitrile. Dimethyl sulfate ~0.613 9, 0.0486 mol) was added and the mixture was refluxed overnight. The reaction was followed by thin layer chromatography (8:2 chloroform/tetrahydrofuran) until no more unquaternlzed ester remained. Evapora-tion of the solvent in vacuo gave a residue which was washed several times with dry ether. The red viscous liquid which remained was stored o~er argon for further use. Yield 97.35X. The product was identified as the desired quaternary salt by NMR analysis. It was assigned the structural formula~
(ClCH2CH2)zN~(CH2)3-C-OCH2C112~HC~ CH3504 CH3 ;
EXA~PLE 78 Preparation of l-Methyl~3-~(N-~2-L4-(~4-[bis(2-chloro-ethyl)]amino~phenyl)butanoyloxy]ethyl~)carbamoyl]-1,4-d~hydropyridine:
The quaternary salt prepared in Example 77 (2.49 9, 0.0043 mol) was dissolved in 350 mL of water.
~1trogen was bubbled through the solution throughout the reactlon period. The aqueous solut~on was cooled :
.
'~: - ~ - ': ~ ,. .
133~`~6~
over 1ce to 5C, then 2.17 9 (0.026 mol) of NaHC03 were added over a 5 mi nute per~od. followed by 2.997 9 (0.017 mol) of sodium dithionite over a 10 minute period. The reactlon mixture was mainta~ned at 5C for 120 minutes, then the layers were separated. The aqueous layer was extracted 4 times with ethyl acetate. The ethyl acetate extracts were combined, dried over MgS04 and evaporated to dryness in vacuo.
~he sem~-solid thus obtained was washed several times wlth dry ether to give the title compound as a yellow solid melting at 90-g2C and having the structural formula~
(clcH2cH2)2N~3(cH2)3-c-ocH2cH2NHc~
CH3 ~
EXAMPLE ~9 Preparation of N-(4-Hydroxycyclohexyl)-3-pyridinecar-boxam~de:
Trans-4-aminocyclohexanol hydrochloride (5.05 9, 0.033 mol) was suspended ~n 50 mL of ethanol, then 33 mL of 1~ NaOH were added slowly while cooling the reactlon mixture to 10C. The homogeneous mixture was evaporated to dryness, then three port~ons of a 50:50 m~xture of benzene and acetone were added and evaporated to dryness in vacuo each time. The dry -solld was extracted w~th 100 mL of chloroform and f~ltered, and the filtrate was evaporated to dryness.
The residue was tr~turated with ether and dr~ed to give ~, . . .
13 3 ~
3.50 9 (91.23X) of the free aminocyclohexanol ~elt~ng at 111-112C.
Nicotin~c ac~d (2.14 ~. 0.017 mol) was suspended ~n 75 mL of dry tetrahydrofuran, then 1.76 9 of freshly distilled triethylam~ne were added. The clear solution thus obta~ned was cooled to -4C ~n an 1ce bath under argon, then ethyl chloroformate ~1.88 9, 0.014 mol) in 10 mL of tetrahydrofuran were added such that the temperature did not go above 0C. The free ~ -am1nocyclohexanol (2.0 9, 0.017 mol) was added as a powder to the cold reaction mixture, which was allowed to come to room temperature and stirred for 2 hours.
~he precipitate which formed was collected by filtrat1On, dissolved in 28 mL of hot water and recrystall~zed as 3.25 9 (85X) of fine colorless needles melt1ng at 208-210C and having the formula ~,o~
~ .
';
as confirmed by elemental analysis.
Preparation of N-(4-~4-(~4-[Bis(2-chloroethyl)~amino~
phenyl)butanoyloxy]cyclohexyl~-3-pyrid~necarboxam~de:
Chlorambucll (1.38 9, 0.0045 mol) and N-(4-hydroxycyclohexyl3-3-pyr~dinecarboxam~de (1.1 9, 0.0049 mol) were mixed together w~th 1.03 9 (0.00459 mol) of dlcyclohexylcarbodilmide and 55 mg (0.00045 mol) of 4-(d~methylamino)pyrid~ne (DMAP) in 50 mL of freshly distilled acetonitrlle. The reaction mixture was stirred at room temperature in the presence of argon 5 for 2 days. The progress of the react~on was followed -by thin layer chromatography using 8:2 chloro~
form/tetrahydrofuran. At the end of the reaction per~od, the precip1tate was removed by filtrat~on and the filtrate was evaporated to dryness in vacuo at low ~ -10 temperature. The res~due was appl~ed to a silica ~ ~ -column and eluted with 8:2 chloroform/tetrahydrofuran.
The appropriate eluting portlons were combined and evaporated to dryness in vacuo. The product (1.86 9, 81X) was obtained as a light cream-colored powder melting at 120-122C and having the formula o ' (ClCH2CH2)2N ~ (CH2)3-C'C~ :
I~N~
: ::
The ident~ty of the product was confirmed by elemental analysis.
.~
XA~PLE 81 ~
P~ ration of l-Methyl-3-(N-~4-t4-(4-l[bis(2-chloro- ' '' ethyl)]amino~phenyl)butanoyloxy]cyclohexyl~carbamoyl)-pyridinium methanosulfate:
. ~
The product of Example 80 (1 9, 0.0019 mol) was d~ssolved in 30 mL of dry acetonitr~le and 0.249 9 (0.0019 mol) of dimethyl sulfate was added. The '~
mixture was refluxed under argon until thin layer '~
chromatography (8:2 chloroform/tetrahydrofuran on -,;~
silica) indicated quaternization was complete (about one and one-half days). The solvent was evaporated in '~r~
vacuo, leav1ng an orange residue which was washed several times with anhydrous ether and evaporated in vacuo. The quaternary salt (1.04 9, 80.6X) was obtained as a sticky yellow mass. It had the structural formula~
(clcH2cH2)2ll ~ (cH2)3~
H3 ::
EXA~PLE 82 Preparation of l-Methyl-3-(N-¦4-t4-(4-~bis~2-chloro-ethyl)]am1no~phenyl)butanoyloxy]cyclohexyl karbamoYl)-1,4-dihydropyridine:
The quaternar~ salt prepared in Example 81 (0.34 9, 0.0005 mol) was dissolved in 0.5 mL of acetonitrile and taken up in 20 mL of degassed water (bubbling N2) 25 cooled to 0C. To the ,stirr1ng solution, sod~um r-. .
:, -, ~ . - . .,, . - :
,r ~
Y~
~ :. ........................... r bicarbonate (0.27 9~ 0.003 mol) was added, followed ~ `
f~rst by 0.37 9 (0.002 mol) of sodium dithionite and then by 20 mL of ethyl acetate. After 90 mi nutes, the organic phase was removed and the aqueous phase was 5 extracted 3 to 4 times with ethyl acetate. The ethyl -~
acetate extracts were comb1ned, dried over sodium sulfate and evaporated 1n vacuo. The residual solid ~-was washed several times w~th anhydrous ether and dr1ed. The res~due thus obta~ned was applied to a neutral alum~na column and eluted with chloroform under pressure. Evaporation of chloroform left 0.18 9 (65X) of a hygroscopic yellow solid of the formula C1CH2CH2 ~(CH2)3-C-O ~
C1CH2CH2 ::
I--~HC~ ~:
The ~dentlty of the product was conf~rmed by UV
ana1ys1s.
Preparatlon of N-(2-Hydroxy)propyl-3-pyr~dinecarboxa-m~de: -To 4.29 9 (0.039 mol) of nicot~nic acid suspended ln 1~0 mL of dry tetrahydrofuran, 4.04 9 (0.039 mol) of freshly dlst~lled tr~ethylam~ne was added ~n one port1On. The resultant clear solutlon was cooled to -4C ~n an lce bath under argon. Ethyl chloroformate (4.33 9, 0.039 mol) ~n 25 mL of tetrahydrofuran was l ~
r' ~ ~
added at such a rate that the temperature of the solu~
t1On d1d not exceed 0C. Then, 3 9 (0.039 mol) of 1-am1no-2-propanol were added directly to the cold ~
react1On mixture. The react10n m1xture was allowed to ~ -come to room temperature and stirred for 2 hours. The preclpitate was removed by filtration and the filtrate was evaporated 1n vacuo. The o11y residue was washed several t~mes with anhydrous ether and allowed to stand. The t~tle compound was obtained as a white, ;
hygroscop~c, low-melting, waxy solid melting at 40C
(6.11 9, 85X) and hav~ng the formula ~-NHCH2CHOH
N
EXA~PLE 84 Preparat~on of N-12-t4-(~4-tB~s(2-chloroethyl)]amlno~-phenyl)butanoyloxy~propyll-3-pyrid1necarboxamlde:
Chlorambucil (1.0 9, 0.003 mol) and ~-(2-hydroxy)-propyl-3-pyr~d1necarboxam1de ~0.065 9, 0.0036 mol) were comb1ned w1th 0.68 9 (0.003 mol) of dicyclohexylcarbo-d11m1de and 41 mg (0.0003 mol) of 4-(d1methylam1no)-pyr1d1ne (DMAP) 1n 40 mL of freshly distilledaceton1tr~1e. The react1On m~xture was st~rred at room temperature ln the presence of argon for 2 days, the progress of the react~on be1ng followed by thin layer chromatography on s~l~ca us~ng 8:2 methylene chlo-r~de/ethyl acetate. At the end of the react1On period, `:;
,~:
~D`~
1 3 3 1 ~
the preclp~tate was removed by filtration and the filtrate was evaporated to dryness ~n vacuo at 30C.
The residue was applied to a s11ica column and eluted with 8:2 methylene chloride/ethyl acetate. The appropriate elut~ng portions were combined and evaporated to dryness ~n vacuo. The t~tle compound was obta1ned as a sticky material in 84X yield (1.53 9).
It had the structural formula:
' (ClCH2CH2)2N ~ (CH2)3-COcHcH2~Hc ~ ; ~
10EXA~PLE 85 ;~
Preparation of l-Me_hyl-3-t(N-~?-~4-(~4-~bis(2-chloro-ethyl)]amlno~phenyl)butanoyloxy]propyl~)carbamoyl]-pyr~d~n~um methanosulfate:
:
The product of Example 84 (2.2 9, ~.0047 mol) was dissolved ~n 45 mL of dry aceton~trile, dimethyl sul-fate (0.59 9, 0.0047 mol) was added and the mixture was refluxed under argon. The progress of the reaction was followed by th1n layer chromatography us~ng 8:2 methy-lene chlor~de/ethyl acetate. After one and one-half days, the solvent was removed by evaporation 1n vacuo, leav1ng an orange res~due. The residue was washed thoroughly with anhydrous ether and was dr~ed in vacuo. The product, obtained as a yellow stlcky mass In 92.47S yleld. had the structural formula:
, "~
i ~
.
. . A
., 'i' i~ ~ '. ' ''~ '. ~ ~ ~ ' ' . ' . " ' ~ ~ ' ' ' ' .
-167~
(clcH2cH2)2H~(cH2)3-cocHcHzNH~
C~3 ~ N~ CH3S04 Z'H3 ~ `
EXAMP~E 86 Preparation of l-Methyl-3-[(N-~2-1{4-({4-~bis~2-chloro-ethyl)~am1no~phenyl)butanoyloxy]propyl~)carbamoyl]-1,4-dthydropyrtZdtne:
The quaternary salt prepared in Example 85 (2.399, 0.004 mol) was dissolved in 1 mL of acetonitrile and then taken up tn 100 mL of degassed water (bubbling N2) and cooled to 0C in an tce-water bath. Sodtum btcarbonate (2.03 9, 0.024 mol) was added to the sttrrtng solutton, followed by 2.81 9 (0.016 mol) of sodtum dithton~te. To the resultant mixture, 60 mL of ethyl acetate were added. The reactton was allowed to continue for 90 mtnutes, then the phases were separated and the aqueous phase was extracted 3 or 4 ttmes with 30 mL porttons of ethyl acetate. The combtned ethyl acetate extracts were drted over sodium sulfate and evaporated tn vacuo. The restdue was applted to a neutral alumtna column and eluted with chloroform under pressure. The approprtate fracttons were evaporated to gtve a hygroscoptc yellow solid tn 60X yield. The product had the formula:
'~
.
:'~
. :.
:
; r. , ~
. ' i' ' '~
~ "' ~ "~
~3.~
;
~CIC 2C 2)2N~C 2)3~ CI C 21 as conf~rmed by UV analysis.
EXA~PLE 87 Preparat~on of ~-(2-Hydroxy-2-phenyl)ethyl-3-pyridine-carboxam1de:
Nicot~nic acid (1.79 9, 0.014 mol) was suspended ~n 60 mL of dry tetrahydrofuran and 1.48 g (0.014 mol) of freshly dist~lled triethylam~ne were added. The clear solutlon wh1ch resulted was cooled to -4C in an lce bath and argon was bubbled through it cont~nuously.
Ethyl chloroformate (1.58 9, 0.014 mol) in 10 mL of tetrahydrofuran was added at such a rate that the -temperature d~d not exceed 0C. Then, 2.0 g (0.014 mol) of 2-am~no-1-phenylethanol were added as a solutlon 1n 5 mL of tetrahydrofuran. The reaction m1xture was allowed to warm to room temperature and was st~rred for 2 hours. The prec~p1tate which formed was removed by f11trat1On and the f11trate was evaporated in vacuo to g1ve 3.22 9 (91.lS) of a wh1te crystall1ne sol1d melt~ng at 122-124C and havlng the formula : :`
13 31~ ~ ~
Elemental analys~s confirmed the identity of the product.
EXA~PLE 88 Preparation of n-(~2-phenyl-2-~4-(l4-~bis(2-chlor 5 ethyl)~am~no~phenyl)butanoyloxy]~ethyl)-3-pyridinecar- -~
boxamide:
Chlorambucil (1.0 9, n.oo3 mol) and N-(2-hydroxy-2-phenyl)ethyl-3-pyridinecarboxamide (0.88 9, 0.003 mol) were combined w~th 0.68 9 (0.003 mol) of dicyclo-hexylcarbodiimide and 41 mg (0.0003 mol) of 4-(dimethylamino)pyridine (DMAP) in 35 mL of freshly distilled acetonitrile. The reaction mixture was stlrred at room temperature under argon for 3 days.
Th1n layer chromatography using 8:2 methylene chloride/ethyl acetate was used to follow the progress of the reaction. The precipitate which formed was removed by filtrat10n and the acetonitrile was eva-porated in vacuo. The residue thus obtained was applied to a s11ica column and eluted with 8:2 methylene chloride/ethyl aceta~e. The appropriate fract10ns were collected and evaporated in vacuo to g1ve a 119ht tan powder (1.21 9, 70X) melting at 99-101C and hav~ng the formula ~.
~ClC 2cH2)2K~(cN2)~2NN ~) ::
~`
1 3 ~ 1 ~ 6 4 Preparation of 1-Methyl-3-~(N-j2-phenyl-2-~4-(~4-tbis-(2-chloroethyl)]aminolphenyl)butanoyloxy]~ethyl)carba-moyl]pyrid~nlum methanosulfate:
S The product of Example 88 (0.5 9, 0.00094 mol) was dissolved in 20 mL of dry acetonitrile and 0.12 9 (0.00094 mol) of dimethyl sulfate was added. The mixture was ref1uxed under argon for 2 days, then the solvent was removed by evaporation in vacuo. The res~due which was obtained was washed several times with anhydrous ether and dried to give 0.54 9 (9lX) of a sticky l~ght yellow product having the formula (CI~N~Nz)zN~(~N2)3~Z ~ CK350~
EXA~PLE 90 Preparat~on of l-~ethyl-3-~(N-~2-phenyl-2-~4-(~4-tbls(2-chloroethyl)~am~nolPhenyl)butanoyloxy]leth carbamoyl-l,4-d~hydropyr~d1ne:
The quaternary salt prepared in Example 89 (0.53 9, 0.0008 mol) was dissolved in 0.5 mL of acetonitrile and taken up in 20 mL of degassed, deion1zed water cooled to 0C. Sodium b~carbonate was added to the st1rring solutlon at 0C, followed by 0.56 9 (0.0032 ol) of sodium dithionite. Then, 20 mL of ethyl acetate were added and the reaction was allowed to continue for 2 hours. The organic phase was removed and the aqueous phase was extracted several tlmes with ethyl acetate (total volume 70 mL), until color was no S longer observed in the organic phase. The ethyl acetate extracts were comb~ned and dried over sodium sulfate and evaporated ~n vacuo. The residue was applied to a neutral alum~na column and eluted with chloroform. Evaporation of chloroform gave 0.2 9 (45X) of a hygroscop~c orang~sh yellow compound of the formula (C~2cNi!)2~ 2~3~32~
The ~dentity of the product was conf1rmed by UY
spectral analysis.
EX~PLE 91 Preparat10n of N-(2-Hydroxyethyl)-3-pyridinecarboxa-m~de:
A neat m1xture of 2-amlnoethanol (6.1 9, 0.10 mol) and ethyl nicot~nate (15.1 9, 0.10 mol) was refluxed overn~ght. As the m1xture was cooled to room tempera-ture, the product prec~p1tated as a crystall1ne sol~d. It was ftltered, washed wlth ether and then recrystalllzed from 2-propanol/ether. The f~nal product was collected by vacuum f~ltrat10n and washed w~th ether. The drled, wh~te compound weighed 10.7 9, j,",",, .. , ~ .. . . ~ ; , .. . .. . . . . . ..
", ~ , ~,, " "~ , " ~ ",~
1331~6A
result~ng 1n a 64.5X yield; mp 88.5-89.5C (l~t. value 92C).
EXA~PLE 92 Preparation_of ~+)N-t2-(6-Methoxy-~-methyl-2-naph-thalenylacetoxy)ethyl]-3-pyridinecarboxamide:
10Naproxen (2.30 9, l~.0 mmol) was coupled with the product of Example 91 (1.71 9, 10.0 mmol) using di-cyclohexylcarbod~m~de (2.30 9, 11.0 mmol) and 4-~dl-methylamino)pyridine (122 mg, 1.00 mmol) ~n aceton~-trile (150 mL). The react~on was stirred at room temperature for 48 hours. The precipitate was fil-tered, rinsed w~th acetonitrile and dried to a weight of 2.3 9. The solvent was removed under reduced pressure and the residual clear oil was stirred with anhydrous ether. The resulting white solid was vacuum ;
20 f~ltered, washed w~th ether and air-dried. The crude -product we~ghed 2.80 9. The compound was recrystal-l~zed from 2-propanol. The final product was fil~ered, washed w1th 0.5X aqueous sodium bicarbonate, water, and f1nally w~th ether. The compound was dr~ed ~n a 25 deslccator over P205. The recrystalllzed ma~er~al ~i welghed 2.40 9 result~ng ~n an overall y~eld of 63.4X;
mp 79-82C.
EXA~PLE 93 - ~ -Preparat1On of N-(2-~1-(p-Chlorobenzoyl)-5-methoxy-2-methyl-3-lndoly!]acetoxy~ethyl)-3-pyr~dinecarboxamtde~
A react~on of ~ndomethacln (1.79 9, 5.00 mmol) and the product of Example 91 (0.830 9, 5.00 mmol) was carr1ed out, uslng d~cyclohexylcarbodi~m~de (1.10 9, .,, . ~ ~ , .. . . . . . .
1331~4 5.50 mmol) as the coupling agent and acetonitrile as the solvent. The first two reactants were dissolved completely and the solution was then cooled to 0C.
The dicyclohexylcarbodiimide was added and the mixture was stirred overnight. The reaction was allowed to continue for 48 hours. The precip~tate (1.2 gl was removed by vacuum filtration. The solvent was removed from the filtrate under reduced pressure leaving an oily residue. The product was solidified by stirring w~th anhydrous ether. It was filtered, air-dried and recrystall~zed from ethanol/ether. The final product was vacuum filtered, washed with ether, and air dried. The product weighed 1.65 9, giving a 65.2X
yield; mp 123-125C. ~ -EXA~PLE 94 Preparation of 1-Methyl-3-~N-~2-(6-methoxy--methyl-2-naphthalenylacetoxy)ethyl]carbamoyl~pyridinium iodide:
The quaternizat~on of the naproxen ester prepared ~n Example 92 (1.0 9, 2.6 mmol) was carried out using methyl iodide (2.3 9, 16 mmol) in acetone (45 mL). The solution was heated to reflux for 20 hours. Methyl ~odlde (I.l 9, 8.0 mmol) was again added to the react~on flask. The prec~p~tated product was filtered after an add~tlonal 4 hours of reaction time. The off-wh~te powder was dried. The mater1al weighed 2.2 9 and was found to be analytically pure without recrystalli- ~
zatlon. The solvent was removed from the acetone - - -filtrate and the residue was sol~dlfied with anhydrous ~ --ether. The result~ng dark yellow powder was dissolved in water and washed w~th ether (4 x 30 mL). The water was then removed under vacuum g~ving 0.2 9 of a lighter 1 3 ~
:.~
yellow powder. The overall yield of the reaction was 93X; mp 169-170C. The product had the structural formula O o ~ OCH3 : ~:
~CNH(CH2)20CCH~-- ~ ~
5 as further conf1rmed by UV, NMR and elemental analyses. ~ ;
EXAMPLE 95 ~
::
Preparation of l-~ethyl-3-[N-(2-~[1-(p-chlorobenzoyl~
5-methoxy-2-methyl-3-indolyl]acetoxy~ethyl)carbamoyl]-pyr~d1n~um ~odlde~
The quaternizat~on of the indomethacin ester pre-pared in Example 93 (0.50 9, 1.0 mmol) was carried out 1n acetone, using methyl ~od~de (1.7 9, 12 mmol). The react10n was refluxed overnlght. The solvent was re-moved under reduced pressure and a yellow solid was ~ ;
15 obta1ned. The product was recrystall~zed us~ng ethanol~ ~
and a very small amount of ether. Small mold-11ke -~ -crystals were ~bta1ned whlch were 119ht yellow ~n color. The react10n gave 0.43 9 or a 66X y~eld of the purlf~ed mater~al; mp 178-179C. UY, NMR and elemental 20 analyses conf~rmed that the product had the structural ;
forl~ula:
~ ~ ' .
r,~
H3CO~ 2cO~cH2)2NH~
C=O 3 (~ `
EXA~PLE 96 Preparation of l-Methyl-3-jN-~2-(6-methDxy-~-methyl-2-naphthalenylacetoxy~ethyl]carbamoyl~-1,4-dihydro-pyridine:
The quaternary salt prepared in Example 94 (780 mg~ 1.5 mmol) was dissolved in degassed, deionized water (200 mL) and acetonitr11e (10 mL~. Sodium d~thionite (780 mg~ 4.5 mmol) and sodium bicarbonate (630 mg, 7.5 mmol) were combined and added to the solution at room tempera~ure. The reaction was ~ ~
cont~nued for 1 hour, while nitrogen gas was slowly ~`
bubbled through the solution. The partially pre- ;
c~pitated product was extracted repeatedly w~th ether (8 x 30 mL). The extracts were comb~ned, washed with water (25 mL) and dr1ed over magnesium sulfate. The ;~
dry~ng agent was f11tered and the solvent was removed from the filtrate under reduced pressure. The oily res~due was tissolved ln methylene chloride (3 x 5 mL) and removed under reduced pressure. The resulting foam was rinsed w~th anhydrous ether (3 mL) and the solvent was removed under vacuum. The final product weighed -~; 390 mg, g~v1ng a 66X yield. The hygroscopic solid foam was stored under n~trogen at -100C. It had the struc-tural formula:
, ~
''~
. ~ ~
1~3~
¢~ INH(CH2)2O~C ~ ~
as conf~rmed by UV. NMR and elemental analyses.
EXA~PLE 97 Preparatlon of 1-~ethyl-3-~N-(2-~[1-(p-chlorobenzoyl)~
5-methoxy-2-methyl-3-~ndolyl]acetoxy~ethyl)carbamoyl]-1,4-dihydropyrid~ne The indomethac~n quaternary salt prepared in Example 95 (140 mg, 0.2Z mmol), was dissolved in a ~inimum amount of water:acetonitrile (8:2). The water had been bubbled with nitrogen for 20 minutes prior to its use. Sodium b~carbonate (91 mg, 1.1 mmol) and sodium dithionite (110 mg, 0.6~ mmol) were added to th~e ~ ;
solut~on while stirr~ng at 0C. The solutton was then allowed to warm to room temperature. The reactlon was cont1nued for about 1 hour. Some of the product had preclp~tated dur~ng the react1On. Th~s was dlssolved ln ethyl ether. The water layer was extracted several t~mes w~th ether unt~l no more yellow color transferred to the organic layer. The ether portions were combined and dr~ed with magnesium sulfate, flltered and the ether was removed under reduced pressure. The result-1ng o~l was d~ssolved 1n acetone and the solvent was removed (2 x 10 mL) under reduced pressure to form a dry foam. The f~nal product we~ghed 92 mg. The y~eld was 82X; mp 60-65C. The product had the formula:
13315~
H3Co_~cH2~0(cH2)2HH
Cl as confirmed by UV. NMR and elemental analyses. ;~
Preparation of N~ Hydroxyc~clohexyl)methyl~-3-S pyridinecarboxam~de:
To 1.48 9 (0.012 mol) of nicot~nic acid suspended ~'n 50 mL of dry tetrahydrofuran, 2.44 9 (0~014 mol) of freshly dist~lled triethylamine were added, with ~ -st~rr~ng. The resultant clear solution was cooled to -4C in an ~ce bath, under argon. Then, 1.3 9 (n.012 mol) of ethyl chloroformate in 10 mL of tetrahydrofuran were added at such a rate that the temperature of the reactlon m~xture did not exceed 0C. To the cold react~on m1xture, 2.0 9 (0.012 mol) of 1-am~nomethyl-1-lS cyclohexanol hydrochloride were added directly as apowder. The reaction mlxture was allowed to warm to room temperature and st1rred for 2 hours, then the tr7ethylam1ne hydrochlor~de wh~ch formed was removed by f11trat~on and the f~ltrate was evaporated tn vacuo to afford a white sol~d. The sol~d was recrystall~zed from water, washed w~th acetone and ether and dried.
The t~le compound, obtained 1n ,35S y~eld (2.4 9), melted at around 110C, and was further character~zed by the structural formula i~' , ~ :
~ ' .. ...
133~
OH
CH2HH~
N
as confirmed by elemental analysis. ;
EXA~PLE 99 Preparation of N-(~1-[4-(~4-~Bis(2-chloroethyl)]amino~
phenyl)butanoyloxy]cyclohexyl~methyl~-3-pyridine-carboxamide Chlorambuc~l (1.18 9, 0.0038 mol) and hydroxycyclohexyl)methyl]-3-pyridinecarboxamide (0.99 9, 0.004 mol3 were combined with 0.8 9 (0.0038 mol) of d~cyclohexylcarbodiimide and 47 mg (0.00038 mol) of 4-(dimethylam~no)pyridine (DMAP) ~n 60 mL of freshly distilled acetonitrile. The reaction mixture was stirred at room temperature under argon for 7 days. At the end of that t1me, the prec1p1tate which formed was separated by filtratlon and the filtrate was evaporated to dryness at low temperature ~n vacuo. The res~due was applied to a silica column and eluted, f1rst w1th 8:2 methylene chlor1de/ethyl acetate, then with 8:2 chloroform/tetrahydrofuran. The appropriate elut~ng port~ons were comb1ned and evaporated to dryness in vacuo. The t~tle compound was obta~ned ~n 26X yield as a light yellow solid melt1ng at 92-94C.
~ It had the structural formula :~ :
~L3~
O-C-(CH2)3~ ~C1i2CH2 ~y \CH2CH2Cl .
C1~2NH~
as confirmed by elemental analysis.
Preparation of l-Methyl-3-[N-(~ 4-(4-{~bis(2 chloroethyl)]amino~phenyl)butanoyloxy]cyclohexyl~
methyl~carbamoyl]pyridinium methanosulfate:
.:
To 0.69 9 (0.0013 mol) of the product of Example ';!~
99, dissolved in 25 mL of dry acetonitrile, was added 0.17 9 (0.0013 mol) of dimethyl sulfate. The mixture was refluxed unt~l the reaction was complete (approxi-mately 2 days), as evidenced by thin layer chromato-graphy us~ng 8:2 chloroform/tetrahydrofuran. The solvent was removed by evaporation ~n vacuo to afford an orange res~due, whlch was washed several t~mes w~th anhydrous ether and drled. The product was obtained as a st~cky yellow mass (85X, 0.72 9) having the formula:
g ~ /CH2CH2Cl O-C-(CH2)3~ ~CH2CH2Cl ~ :
Z~tU3SO~- ~
~ ' ~ '`' ~ ~ "
133~
- 1 ~ 0 - . :
Preparation of 1-Methyl-3-[N-(~ 4-(4-~[bis(2 chloroethyl)]am~no~phenyl)butanoyloxy]CyClOhe methyl)carbamoyl-1,4-dihydropyrid~ne~
The quaternary salt prepared in Example 100 (0.78 ~ -9, 0.0012 mol) was dissolved in 0.5 mL of acetonitrile and taken up ln 20 mL of water degassed with bubbling N2, cooled to 0C. To the stirring solutlon, 0.61 9 (0.0072 mol) of sodium blcarbonate was added, followed by 0.84 9 (0.0048 mol) of sodium dithionite and 20 mL
of ethyl acetate. The reaction was allowed to proceed for 75 minutes, then the layers were separated and the aqueous layer was extracted 3 to 4 times with 20 mL of ethyl acetate. The organic extracts were combined, dr~ed over sodium sulfate and evaporated in vacuo. The residue was applied to a neutral alumina column and eluted with chloroform under pressure. Evaporation afforded the product as a sticky yellow mass (0.31 9, 49X) havlng the structural formula:
O-C-~CH2)3~ ~CH2CH2Cl :
~ \cH2CH2Cl -\--I
CH2NH~
EX~MPLE 102 Preparatlon of 1-Methyl-3-{~2-(9-guanylmethoxy)ethoxy]
carbonyllpyridin~um lodlde:
~rigonelline anhydr1de dllodide (l-methylpyrl-25 dlnlum-3-carboxyl~c acld anhydride dllodlde) was ~:
.
1 33.t5~4 prepared as described by Brewster et al, Synthetic Commun~cations, 17(4), 451-455 (1987).
To a solution of 1.0 9 (4.4 mmol) of acyclovir in 25 mL of freshly dlstilled dry pyridine were added 2.27 5 g (4.4 mmol) of tr~gonell~ne anhydride diiodide and a - ~:
catalytic amount (5.4 mg, 4 mmol) of 4-(dimethyl-am1no)pyridine (DMAP). The resultant suspension was stirred for 4 days under argon at room temperature. As the react~on proceeded, the orange color of the anhydride was replaced with a yellow color. When all of the acyclovir had been consumed, the react~on was stopped, the precipitate (containing the product ester ~: :
plus the tr19Onell~ne formed as a by-product) was removed by filtration and washed with acetone and ether to remove DMAP. The yellow solid was then stirred in dry methanol at room temperature to remove tr~gonelllne, unreacted anhydride and acyclovir. The t~tle compound was obta~ned ~n 87X yield (1.82 9), melt~ng at 201-202~C. NMR and UV analyses conf~rmed that the product had the formula:
o ~Z~ 2'(Cr2'2~
~H3 :- `.. :.
31S~
, .
EXA~PLE 103 Preparation of 1-Methyl-3-~C2-(9-guanylmethoxy)ethoxy]-carbonyl~-1,4-dihydropyrid~ne~
To a solution of 1.58 9 (3.3 mmol) of the product S of Example 102 ln 120 mL of degassed water were added 1.69 9 (20.1 mmol) of NaHC03 in one portion. The m~xture was stirred at 0C wh~le 2.33 9 (13.18 mmol) of sodium dithlon~te were added over a S minute period.
The flask was flushed wlth nitrogen throughout the reaction process. The dihydropyridine product was insoluble ~n water and formed cream-colored crystals on top of the water layer. The crystals were separated by f~ltration and washed, first with ice-cold water and then w~th anhydrous ether. Drying over P205 in a dessicator maintained at -15C afforded 0.626 9 (54X) ~-of the title compound melting at 163-165C. NMR and UV
analyses confirmed that the product had the formula: -z~
H I O
CH2oCH2cH2 ~H3 .
Preparation of 5'-Pivaloyltrifluorothymidine~
To a stirr~ng solution of 150 mg of trifluoro~
thymidine tn S mL of pyridine was added a solution of ~' ~:
6`~
90 mg of pivaloyl chloride in 1 mL of pyridine, with cooling. Stirring was continued at room temperature for 10 hours. then the reaction mixture was poured into 20 mL of ice water and extracted with 50 mL of ethyl acetate. The extract was washed with water and dried over sodium sulfate. The ethyl acetate was removed and the residue was purified by silica gel column chroma-tography us~ng 20:1 chloroform/methanol as eluent. The title compound melted at 130-132C after recrystalliza-t~on from a m~xture of ether and n-hexane.
' EXA~PLE 105 Preparation of 3'-t3-Pyr1dylcarbonyl)-5'-pivaloyltri-fluorothymidine:
To a stirring solution of 450 mg of 5'-pivaloyl-trifluorothymidine ~n 10 mL of pyridine was added 1.0 9 of nicot1noyl chloride hydrochlor1de under ice-cooling. The reaction mixture was stirred at room temperature for 3 days, then was poured into 100 mL of 1ce water and extracted with 100 mL of ethyl acetate.
The extract was washed w1th water, dried over anhydrous sodlum sulfate and then evaporated in vacuo to g~ve an o11. Crystall~zatlon from n-hexane afforded S00 mg (87X) of colorless needles melt~ng at 175-177C. The product had the structure '~.' -," '~', -' .
1 3 3 1 ~
: -HN~CF3 OJ~ N
( CH3 ) 3C -~ -O CL~ O ~¦ . , '`Ir'l ' OC~
N ~ . ~
as further confirmed by NMR spectral analysis.
EXA~PLE 106 Preparation of 3'-(1-Methyl-3-pyridiniumcarbonyl)-5'-S pivaloyltrifluorothymidine iodide: :
To 440 mg of the product of Example lOS dissolved ~n 10 mL of acetone, 1.0 9 of methyl iodide was added. The mixture was refluxed for 10 hours, then the precip~tate which formed was collected by suct~on filtration to give 550 mg of the desired product as yellow leaves melting at 188-190C with decomposition.
NMR analysis confirmed that the product had the structural formula~
t 3 ~ r - 185- .
~3~CF3 (CH3)3C~ o ~ '~
~H
.. ..
Preparation of 3'-(1,4-D~hydro-l-methyl-3-pyridinyl- -carbonyl)-5'-pivaloyltrifluorothymidine~
To a stirring solution of 100 mg of the product of Example 106 1n a mixture of 20 mL of water and 20 mb of ethyl acetate were added 64 mg of NaHC03 and llS mg of Na2S204 under N2 gas. The resultant mixture was -stlrred at room temperature for 1 hour, then the organlc layer was separated and washed with water. The extract was dr~ed over anhydrous Na2S04 and evaporated 1n vacuo. The residue was trlturated wlth a mixture of - -ether and n-hexane and the yellow needles which formed were collected by suction filtration (50 mg, h2X). The product melted at 168-170C. NMR analysls confirmed that the product had the structural formula~
i' : .-:
~ ' , , ~ ' .
~ r. ~
~s 13~ $~
., o ,~F3 (CH3)3C--C--O CH~o ~
1`~'1 , :. .
' ; , -Preparation of 3'-Azido-3'-deoxy-5'-(3-pyridyl-carbonyl)thymidine A m1xture of 1.18 9 (4.42 mmol) of azidothymidine, 5 1.11 9 (4.86 mmol) of nicotinic anhydride and 0.15 9 (1.22 mmol) of N-(dimethylamino)pyridine was combined in 50 mL of pyrid~ne. The reaction mixture was st1rred at room temperature overn19ht. The clear, colorless reaction mixture was concentrated in vacuo to a semisol1d opaque mass which was triturated with ether overnight. ~he suspension was filtered and drled to give 1.97 9 of solid. Then, 1.0 9 of the solid was chromatographed over 20 9 of sil~ca gel using 10X ~-ethanol/chloroform as eluent. The desired fraction was 1solated as 0.53 9 of a white foam, wh1ch was crystal~
lized from a solvent mixture of ethanol, diethyl ether and hexane. The product melted at 138.5-141.5C and had the structural formula -t~
::
~ ~r.. ~
:1 3 ~
~3~ CH3 k~CH20C~
~13 '' ` ~' ''.
as confirmed by NMR and IR.
EXA~PLE 109 Preparation of 3'-Azido-3'-deoxy-5'-~(1-methyl-3- ~ -pyridinium)carbonyl~thymidine iodide~
A m1xture of 0.53 9 (2.0 mmol) of azidothymidine, 1.02 9 (2.2 mmol) of trigonelline anhydride d~iodide and 67 mg (0.5 mmol) of N-(dimethylamino)p~ridine was comblned 1n 25 mL of pyrid~ne. The reaction mixture was stirred at room temperature for 5 days, then was f11tered. The f~ltrate was concentrated in vacuo to a residue which was triturated w~th acetone overnight.
The result~ng suspens1On was filtered and the filtrate was concentrated 1n vacuo to a foam, which was treated 15 with water and filtered to remove i small amount of -insoluble material. The filtrate was concentrated in vacuo to a solid yellow glass (0.50 9, 49X). N~R and -UV analys~s conf~rmed tha~ the product had the formula:
. . , ;' , ~ : . ' ' ' ~ . ~ ~, ~ . ~ ' . ' .: ; - :
~31~
HNJ~/ CH3 k~CH20~) :
3 : :~
EXA~PLE 110 Preparat1On of 3'-Azido-3'-deoxy-5'-[~1-methyl-1,4-dihydro-3-pyridinyl)carbonyl]thymidine~
The crude azidothymid~ne quaternary derivative prepared according to the procedure of Example 109 -~
(1.45 9, 2.82 mmol) was dissolved in 50 mL of water and filtered. The filtrate was cooled in an ice bath and saturated w~th argon. Then, 100 mL of ethyl acetate and 2.90 9 of NaHC03 were added, followed by 1.45 9 of Na2S204 after 5 m~nutes. The reaction was allowed to proceed for 1 hour, then the ethyl acetate layer was removed and fresh ethyl acetate was added. This procedure was repeated to g~ve three organ~c extracts and a react~on t~me of 3 hours. The extracts were pooled and concentrated ~n vacuo to a foam we19h1ng 1.01 9 (92S). The foam was crystallized from methanol to g~ve the tltle compound of the formula . .
~ 3 ~
O
H20C ¢3 ' melting at 138-140C. Its structure was confirmed by ele0ental analysis as well as NMR and UV.
EXA~PLE 1 1 1 - ~ ~:
Preparation of Dopamine dipivalate, oxalate salt To a st~rred mixture of 28.1 9 of pivaloyl chloride and lsn mL of trifluoroacetic actd, 18.01 9 of dopamine hydrobromide were added. The mixture was stirred for 2 hours, then 14 mL of water were added and the mixture was concentrated in vacuo. The residual oil was dissolved in chloroform and washed with cold 10X KHC03 solution until C02 evolution ceased. The layers were separated and washed with water and the chloroform layer was dr~ed over MgS04, filtered and evaporated to dryness. The residue was taken up in 100 mL of ethyl acetate and 7 9 of oxalic acid were added together with 100 mL of ethyl acetate. The resultant solution was f~ltered to remove insoluble materials and 1.6 9 oxalic acid in 25 mL of ethyl acetate were added. The mixture was concentrated in vacuo and , .- . ., 1331~6~
.
cooled. The crystals which formed were isolated by filtration, giving 13 9 of the title compound. Cooling of the mother liquor afforded a second crop of crystals -(5.9 9). The product had the formula~
(CH3~3cco~cH2cH2hH2 . COOH
( CH3 ) 3CCO
EXA~PLE 112 Preparation of Chloromethyl N-~B-t3,4-bis(pivalyloxy)~
phenyl~ethyl~aminocarboxylate: ;
Dopamine dipivalate, oxalate salt (822 mg, 2 mmol) - 10 was suspended in 15 mL of dry tetrahydrofuran. Tri-ethylamine (278 mL, 1 mmol) was added, the mixture was stirred for 15 minutes and a further 278 mL (1 mmol) of tr~ethylamine were then added. Addition of ClC02CH2Cl (390 mg, 6 mmol) resulted in immediate formation of a heavy white precipitate and the evolution of gas. The reaction mixture was st~rred overnight at room tempera-ture, then the precipitate was removed by filtration and the filtrate was washed with 10 mL of 0.1 M hydro-chloric ac~d. Drying over magnesium sulfate and evaporat~on to dryness afforded 1.1 9 of a golden o~l of the structural formula .~ .
.~ .
,. . ~."~
~ i"~
... ~. ,.. , -~ -~ . .
~ 3~
- 1 9 1 ~
~''' '~''' -.
3)3CCO~CH2CH2NHCOCH2C~
(CH3)3CCO -~
O . ~'''.
:: .
The identity of the product was confirmed by elemental analysis.
EXA~PLE 113 s Preparation of N-~B~[3,4-Bis(pivalyloxy)ph_nyl]ethyl~-am~nocarbonyloxymethyl 3-pyridinecarboxylate: - -The chloromethyl carbamate prepared in Example 112 (1.26 9, 3.04 mmol) was combined with 10 mL of dry dimethylformamide and that mixture was added to a pre-mixed solution of nicotinic acid (375 mg, 3.04 mmol)and triethylamine (445 mL, 5X excess) in 15 mL of dry dimethylformamide at room temperature. The reaction mixture was stirred for 4 days, then the precipitate whlch formed was removed by filtration. The filtrate was evaporated to dryness and the residue was taken up in 2n mL of methylene chloride. ~hat solution was washed twice with 10 mL portions of water. Removal of ~ the solvent in vacuo afforded the t~tle compound of the - formula:
., -c~
:
.
.
:
o o o (c 3~3CC0~ 2CH2NHCOCH20C~
The identity of the product was confirmed by NMR.
EXA~PLE 114 Preparation of N-~B-[3,4-Bis(pivalyloxy)phenyl]ethyl~-aminocarbonyloxymethyl 1-methyl-3-pyridinium carboxylate iodide:
The product of Example 113 (860 mg, 1.78 mmol) was combined with 15 mL of dry acetonitrile and that mixture was treated with 223 mL (3.56 mmol) of methyl 10 iodide. The resultant mixture was stirred for 6 hours -at room temperature, then an additional 223 mL (3.56 mmol) of methyl iodide was added and the mixture was stirred overnight. Evaporation to dryness afforded, as an orange-red oi l, the ti tl e compound of the formul a `:
~ .
~ 3 ~
o o O
(C )3CCO~f H2CH2HHCOCH2 The identity of the product was confirmed by NMR ~ ~
analysis. ;~ ~-Preparation of N~ 3,4-Bis(pivalyloxy)phenyl~ethyl~-am1nocarbonyloxymethyl 1~4-dihydro-1-methyl-3-pyridine- -~
carboxylate The quaternary salt prepared in Example 114 (54 mg, n.o84 mmol) in 10 mL of water was treated at 0C
under nitrogen with NaHC03 (30 mg, 4 equivalents), Na2S204 (60 mg, 4 equivalents) and ethyl acetate (20 -mL). The reaction was allowed to proceed for 1 hour 20 minutes, then the aqueous and organic layers were separated and the aqueous layer was re-extracted with 20 mL of ethyl acetate. The combined organics were dried over magnesium sulfate. Removal of the solvent ~n vacu_ gave a red-orange oil which was taken up in chloroform and partially purified by elut~on with chloroform from a short neutral alumina column. The desired fract~on was subjected to preparative thin layer chromatography on silica using 80:20 chloro-form/acetone. The highest band was taken as the title compound of the structural formula~
o o o Il 11 11 ~
(CH3)3Cc0~3/CH2cll2NHcocH2 CH3)3CCO CH3 ~
O : ':
The identity of the product was confirmed by HPLC (high S pressure liquid chromatography) determinations for its ability to release dopamine from plasma and brain homogenate.
EXA~PLE 116 Preparation of 9-Fluoro-llB,17-dihydroxy-16 methyl-21-~(3-pyridinylcarbonyl)oxy]pregna-1,4-diene-3,20-dione:
nexamethasone (l 9, 2.5 mmol) was dissolved in 50 mL of dry pyridlne. To that solution were added 680 mg (3.0 mmol) of nlcotinic anhydride and a trace of 4-(dimethylamlno)pyr~dine (DMAP). The reaction was allowed to proceed for 4 hours, then the reaction mixture was poured over ice water and refrigerated overnlght. The sol1d was collected by filtration and dried to give l.n8 9 (87X) of product melting at 262-265C and hav1ng the structural formul~
~ .
~ .
~.. - ,... ~ ~ ~ .~ . . - .
, ?
.:,, ~, . ,:~. ~: ~': ~'` : ' ~: ' . ~ ' ,' ' ' ' ' . ,' ` '~ ' ~ ' ~ : ~ :
133~4 :
..
CH
HO~ 3 , .
~ ~ :~
0~ ; ~' ' as conf1rmed by elemental analysis.
EXA~PLE 117 Preparation of 1-Methyl-3-~(9-fluoro-llB,17-dihydroxy- ~ -16~-methylpregna-1,4-diene-3,20-dion-21-yl)oxy]-carbonyl ~yridinium iodide:
The product of Example 116 (0.74 9, 1.5 mmol) was d1ssolved in 50 mL of acetone to wh~ch 2 mL of methyl ~ :~
1Od1de was added. A small amount (10 mL) of CH3N03 was subsequently added to lncrease solubi7ity. The react~on was allowed to proceed for 2 days, then the sol1d was collected to g1ve 0.54 9 ~56X y~eld) of the t1tle compound melt1ng at 218-221C and hav1ng the formula ~CH3 C--O
H3C~ CH3 :
0~ :
~ ~ 3 1 ~
- 196- :
The structure of the product was confirmed by elemental an al y s i s . . - ~ .
.
EXA~PLE 118 Preparation of g-Fluoro~ ,17-dihydroxy-16~-methyl-21-~(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy~
pregna-1,4-diene-3,20-dione:
The general reduction procedure of Example 11 of U.S. Patent No. 4,617,298 was followed, using 0.78 mmol of the steroidal quaternary salt prepared in Example 117, 0.33 9 of NaHC03 and 0.41 9 of Na2S204 in 50X
aqueous methanol at 0C, with a nitrogen purge. The product had the structural formula~
l /CH3 \_y jC ::
~ - tH3 o . '~, EXAI~PLE 119 :
Preparatlon of 11~,17-D~hydroxy-21-[(3-pyridinyl-carbonyl)oxy]pregn-4-ene-3,20-dione:
- Hydrocort~sone (2 9, 5.5 mmol) was dissolved in 50 mL of dry pyridine. Then, 1.38 9 of nicotinoyl anhy-dride (6.05 mmol) and a trace of 4-(dimethylamino)pyri-d~ne (D~AP) were added and the react~on was allowed to proceed for 4 ho~rs at room temperature. The pyridine solut~on was poured into ~ce water and the resulting solld was collected by filtration. The solid was dried over P205 in vacuo to give 2.4 g (93~) of the t1tle ~ -compound of the formula ~-o ~11 ICH20C~) C=O .
~H
': ~
~' as conf1rmed by elemental analys~s and UV spectral analysis.
,:
~ "
13~1~64 EXA~PLE 120 Preparatlon of l-~ethyl-3-f[(llB~17-dihydroxypregn-4-ene-3,20-d10n-2l-yl)oxy]carbonyl~eyridinium iodide:
The product of Example 119 (1 9, 2.1 mmol) was dissolved in S0 mL of acetone and 4 mL of methyl iodide were added. The solution was st1rred at the reflux temperature overnight. Removal of the solvent gave the t~tle compound as a yellow powder in 98X yield. Ele-mental analysis conf~rmed that the product had the formula:
O r~N~ 3 C-O ~) H3C~ j 0~
EXA~PLE 121 Preparat~on of 11 e,17-D1hydroxy-21-~(1-methyl-1,4-d~hydropyr1dln-3-yl)carbonyl~oxy~pregn-4-ene-3,20-d~one:
The general reduction procedure of Example 11 ofU.S. Patent No. 4,617,298 was followed, using 0.8 mmol of the stero~dal quaternary salt prepared in Example 120, 0.34 9 of NaHC03 and 0.42 9 of Na2S204 ~n 50X
aqueous methanol at 0C, w~th a nitrogen purge. The product had the structural formula:
~-, : ,-........ ,., .. , . . , . - - .
~r~
!1 3 3 1 ~
..
o ~/ CH3 :
H3C I ;`~
~- OH
Preparation of N-(2-Chloroethyl)-N'-[2-(3-pyridine- ::~
carbonyloxy)ethyl]-N-nitrosourea: :
S A solution of 2-am1noethyl nicotinate dihydro-chlor1de (1.25 9, 52 mmol) and 2,4,5-trichlorophenyl-N-(2-chloroethyl)-N-n1trosocarbamate (2 9, 6 mmol) in 40 :
mL of pyr1d1ne was st1rred under nitrogen at room temperature for 24 hours. The react1on was monitored 10 by th1n layer chromatography (sil1ca, 1:1 chloro- ~n :~:
form/ethyl acetate, Rf 0.26). The solvent was removed 1n vacuo and the res1due was chromatographed on a :::~
s111ca gel column by eluting, f1rst with benzene to remove unreacted n1trosocarbamate and trichlorophenol by-product, and then w1th chloroform, to 91ve the des1red product. The resultant o11 solidifled in the freezer. It melted at 63-64C and had the formula :~
:
~3~
-OCH2CH2RHC=O
CH2 : :
Cl EXA~PLE 123 Preparat~on of N-(2-Chloroethyl)-N'-[2-~1-methyl-3-pyridiniumcarbonyloxy)ethyl]-N-nitrosourea iodide:
A solution of the product of Example 122 (1.5 9, 5 mmol) in 40 m~ of tetrahydrofuran was treated with excess methyl 1Odide. The mixture was stirred at 50C
for 4 hours. The finely crystalline, yellow solid thus obtained (1.8 9, 82X) melted at 120-121C and had the structure .
CNzCH
as confirmed by elemental analysis.
'' ~ ~.
~331~6~
Preparation of N-(2-Chloroethyl)-N'-[2-(1,4-dihydro-l-methyl-3-pyrid~necarbonyloxy)ethyl~-N-nitrosourea:
A solution of the quaternary nltrosourea prepared in Example 123 (0.48 9, 1.1 mmol) and 1-benzyl-1,2-dihydroisonicot1namide (0.23 9, 1 mmol) in 25 mL of anhydrous methanol was stirred at 0C for 4 hours under nitrogen. The solid which separated was filtered and washed with methanol and ether. The solid was identi-fied as 1-benzyl-4-carbamoylpyridinium iodide by NMR.
The filtrate was evaporated in vacuo at about 30C and the residue was suspended in methylene chloride. The sol~d that separated was filtered and washed with - -methylene chloride. The filtrate was evaporated in vacuo and the residue was dissolved in chloroform.
flash chromatography on a short column of neutral alumina, using chloroform as eluent, gave a chloroform solution which was evaporated in vacuo to afford 0.2 9 (63X) of a gummy res~due, identified by NMR as the -des~red product of the formula ¢~tl~2t~2~1~N2tl~2t~
An alcoholic solut~on of s11ver nitrate was readily ; reduced by the compound thus obtained. -~z,, - ., , .. . . .. :, ~ , ., . .. , . , .......... , : . . ::
13315~
Preparation of N-(2-Fluoroethyl)-N -[2-(3-pyr1d~ne-carbonyloxy)ethyl]-N-n~trosourea:
A solut~on of 2-aminoethyl n~cot~nate dihydro-chlor~de (1.5 9, 6.3 mmol) and 2,4,5-trichlorophenyl-N-(2-fluoroethyl)-N-n~trosocarbamate (2.18 9, 6.9 mmol) ~n 50 mL of pyridine was stirred under nitrogen at room temperature for 24 hours. The reaction was mon~tored by th~n layer chromatography (silica, 1:1 chloro-form/ethyl acetate, Rf 0.25). The solvent was removed1n vacuo and the residue was chromatographed on a sil~ca gel column by elut~ng, first wlth benzene to remove unreacted n1trosocarbamate and trichlorophenol, and then with chloroform to elute the desired -~
product. The compound (1.56 9, 87.4X) melted at 75- -~
77C and was character~zed by the structural formula:
~; ~
[~C-OCH2CH2NHC O ~
F2 -~
:' -13~fi~
EXA~PLE 126 Preparation of N-(2-Fluoroethyl)-N'-[2-(1-methyl-3-pyr1d~n1umcarbonyloxy)ethyl]-N-nitrosourea iodide:
A solution of the product of Example 125 (1.56 9, 5.4 mmol) 1n 40 mL of tetrahydrofuran was treated with excess methyl iodide. The mixture was st~rred at 50C
for 4 hours. The f1nely crystall~ne, yellow solid thus obta~ned ~2.20 9, 94.1X) melted at 123-125C and had the structural formula [~ CH2ch2 EXA~PLE 127 Preparat10n of N-(2-Fluoroethy!)-N'-~2-(1,4-dihydro-1-methyl-3-pyr1d1necarbonyloxy)ethyl]-N-n1trosourea:
A solut10n of the quaternary nitrosourea prepared 1n Example 126 (0.426 9, 1 mmol) and 1-benzyl-1,2-d1hydro1son1cotlnam1de (0.21 9, 1 mmol) 1n 25 mL of anhydrous methanol was st1rred at 0C for 4 hours under n1trogen. The solvent was evaporated in vacuo at about 30C and the res1due was suspended ~n chloroform, f~ltered and flash chromatographed on a short column of neutral alum~n2. The title compound was obtained in 13315~4 55X y~eld after elution with chloroform and was assigned the structure ¢;~,, ~OCH2CH2NHC-NCH2cH2 ~ -~H3 consistent with UV analysis.
EXA~PLE 128 Preparation of Chloromethyl nicotinate~
To a suspension of nicotinic acid (1.23 9, 0.01 mol) 1n a mixture of 10 mL of water and 20 mL of tetrahydrofuran were added tetrabutylammonium hydro- -gensulfate (0.34 9, 1 mmol) and sod~um bicarbonate (3.19 9, 0.038 mol), w~th v~gorous st1rr1ng. Chloro- .t~
methyl chlorosulfate (1.81 9, 0~011 mol) in 5 mL of tetrahydrofuran was added dropw1se, keep1ng the tem-perature below 30C. The react1On mixture was st1rred for 1 hour, then the layers were separated and the organ1c layer was dr1ed by azeotroplng w~th 1~
aceton1tr1le/benzene. The res~due was passed through a ~ column of neutral alum1na, elut1ng w1th chloroform.
; The chloroform layer was evaporated to give 1.28 9 (74.8X) of an o11y res1due, which was confirmed by NMR
analys1s to have the structural formula:
.
~ . .. .
1~3~5~
~-O-CH2-EXA~PLE 129 Preparat~on of l-(Pyr~dine-3-carbonyloxymethyl)-S~
fluorouracil~
S S-FU (1.31 9, 0.01 mol) was dissolved in 5 mL of dimethylacetamide and treated wlth triethylamine (2.78 mL, 0.02 mol). Chloromethyl n~cotinate (2.95 9, 0.012 mol) in 5 mL of dimethylacetamide was added in one port~on, and the mlxture was stirred for 24 hours, f11tered, washed with ethyl acetate and evaporated.
The resldue was chromatographed on a column of sil~ca gel, us1ng as eluent f~rst benzene, then 3:1 ben-zene/chloroform, then 1:1 benzene/chloroform, then 3:1 chloroform/benzene, then chloroform and finally 99:1 chloroform/methanol. Unreacted nicot~nate was eluted 1n~t~ally, followed by the 1,3-bis-isomer and finally the l-lsomer. The l-lsomer (1.3 9, SOX) melted at 190-192C and had the formula COCH2-N ~ F ~:
OJ~ N ~ O
Il N
.
~331~
as confirmed by NMR analysis.
EXA~PLE 130 Preparation of 1,3-Bis(pyridine-3-carbonyloxymethyl)-5-fluorouracil:
S-FU (1.31 9, 0.01 mol) was dissolved in 5 mL of dimethylacetamide and ereated w~th triethylamine (5.6 mL, 0.04 mol). Chloromethyl nicotinate (6.8 9, 0.04 mol) in 15 mL of d~methylacetamide was added in one portion, then the mixture was stirred for 48 hours and filtered, and the filter cake was washed with ethyl acetate. The filtrate was evaporated in vacuo and the residue was diluted with 100 mL of water and extracted with chloroform. The organic layer was evaporated and the residue was dried by azeotroping with 1~ r aceton~trile/benzene. The res~due was chromatographed on a column of neutral alum~na, eluting successively wlth benzene, 1:1 benzene/chloroforml and 50:50:1 benzene/chloroform/methanol, to g~ve 3.2 9 (80X) of the b~s-~somer of the formula ~OCH2~N ~
~ 0 1 N
as conf1rmed by N~R analysls.
1331~6~
EXA~PLE 131 Preparation of 3-(Pyridine-3-carbonyloxymethyl)-5 fluorouracil~
:: :
The 1,3-bis-~somer prepared in Example 130 was dissolved in 10 mL of methanol and mixed with 20 mL of potassium carbonate:sodium bicarbonate buffer (O.lM), pH 10.00. The mixture was stirred at room temperature ;~
for 2 hours, by which time thin layer chromatography indicated that the bis-isomer had disappeared 10 completely. The mixture was evaporated and the residue -was chromatographed on a column of alumina, eluting successively with chloroform, 99:1 chloroform/methanol and 96:4 chloroform/methanol. The fractions were collected and those containing the 3-isomer were pooled and evaporated to give the compound of the formula (~ F :
as a solid (0.2 9, 30X) melting at 179-180C. The ~ -identity of the product was confirmed by N~R analysis.
'.;, -~
:
:
1~31~
EXA~PLE 132 Preparat~on of 1-(1-Methyl-3-pyridiniumcarbonyloxy-methyl)-5-fluorouracil iodide:
The l-isomer prepared in Example 129 (1.93 9) was combined w~th sufficient quantities of methyl iodide and acetonitr~le, and the mixture was refluxed for 4 hours, then cooled and f~ltered to give 2.5 9 of a -. ::
light yellow, fluffy solid. The filtrate was eva-porated, triturated with acetonitrile and filtered to g~ve an addit~onal 0.23 9; total yield 2.73 9 (92.25~). UV and NMR analyses confirmed that the product had the structural formula: -CocH2-N ~ F
~- oJ` N O
EXA~PLE 133 Preparation of 1-(1,4-Dihydro-1-methyl-3-pyridinyl-c~rbonyloxymethyl)-5-fluorourac11:
The product of Example 132 (1 9) was d~ssolved in 20 mL of delonized water (degassed w~th argon) and cooled in an i.ce-bat.h, then 20 mL of ethyl acetate were 20 added. To the stirred solution, 1.24 9 of sodium :~
bicarbonate were added, followed after about one minute ~ :
with 1.75 9 of sodium dithionite. The reaction was -.
allowed to proceed under argon and was monitored by UV. After approximately 75 minutes, ethanol was added .
1 3 ~ 1 5 ~
and the solid was filtered, washed with water and methylene chloride and dried under argon to give 400 mg of the title compound. UV and N~R analyses confirmed : ~-that the product had the structural formula~
~OCH2--N~
¢~ 0~ N O
N H
The aqueous layer was repeatedly extracted with chloro-form and combined with the ethyl acetate layer used in the reaction. The solid obtained after removal of the organic solvent was suspended in acetonitrile and filtered to give an additional 250 mg of product melting at 173-174C.
EX~PLE 13~
Preparation of 3-(1-Methyl-3-pyr1dinlumcarbonyloxy-methyl)-5-fluorouracil tod1de:
lS The 3-~somer prepared ~n Example 131 can be sub-~ected to the general procedure of Example 132 to a~ford the correspond1ng quaternary salt of the forrluld~
;,., . `--~
~3156~
-21()- ~ ~ .
COCH2-N J~, F
~r J~ Jl . .
_ O
CH3 1 . :~
EXAMPLE 135 ~ :
Preparation of 3-(1,4-Dihydro-1-methyl-3-pyridinyl-carbonyloxymethyl)-5-fluorouracil:
The product of Example 134 can be subjected to the general procedure of Example 133 to afford the corres-ponding dihydropyridine of the formula: ~: :
COCH2--NJ~ . ~ . ;.
l l 1 ~J ; .
The cyclodextrin complexes of this invention can 10 be administered to warm-blooded animals to treat a -~
variety of conditions, depending upon the nature of the carrier-drug, specifically of the parent drug itself . .:.
from which the carrier-drug is derived. Thus, in one -~
preferred embodiment, the redox system is derived from 15 dopamine or L-DOPA or a protected counterpart thereof, :~
, a~ ... ;,.. , .. , , , , , , " ~"~"", "~
133~64 and the redox derivative/cyclodextrin complex is thus designed to elicit a sustained and brain-specific dopaminergic (e.g. anti-Parkinsonism or anti-hyperprolactinemia) response in the animal to which the complex is administered. In analogous fashion, the redox derivative/cyclodextrin complex derived from any other centrally acting drug as defined herein is designed to elicit the kind of pharmacological response ;~
which would be obtained by delivery of the drug itself to the brain, i.e. when the centrally acting parent drug is an antitumor/anticancer agent, the instant complex is employed to elicit an antitumor/anticancer response; when the parent drug is a sympathetic stimulant, the instant complex is used to elicit a sympathetic stimulant or amphetamine-like response;
when the parent drug is an anticonvulsant compound, the instant complex is used to elicit an anticonvulsant response; when the parent drug is a tranquilizer, the instant complex is used to elicit a tranquilizing response; when the parent drug is an antidepressant, the instant complex is used to elicit an antidepressant response; and so forth.
The cyclodextrin complexes of the invention are preferably administered in the form of a pharmaceutical composition comprising the selected complex and a nontoxic pharmaceutically acceptable carrier therefor. Suitable nontoxic pharmaceutically acceptable carriers for use with the topic complexes, e.g., those less toxic than the target drug species themselves, will be apparent to those skilled in this art. Compare, for example, Remington's Pharmaceutical Science_, seventeenth edition, ed. Alfonso R. Gennaro, ::
`,j~
13~1~64L
-212- r ~ack Publishing Company, Easton, PA (19~5). Obviously, the choice of suitable carriers will depend upon the route of administration and the exact nature of the particular dosage form selected, as well as upon the identity of the active drug species, the redox derivative and the complex to be administered.
Contemplated routes of administration for the complexes of the invention include oral, buccal, sublingual, topical (including ophthalmic), rectal, vaginal, nasal and parenteral (including intravenous, intramuscular and subcutaneous).
The therapeutic dosage ranges for administration of the complexes according to this invention will generally be equimolar to, or less than (in some instances, substantially less than), those which would characteristically be used in this art for adminis- ;
tration of the parent drug species, per se. Naturally, such therapeutic dosage ranges will vary with the size and species of the patient, the condition for which the -~
20 complex is administered, the particular dosage form ~ 9 employed, the route of administration and the like.
The quantity of given dosage form needed to deliver the desired dose of active ingredient will of course depend upon the concentration of the redox derivative in the complex and in any given pharmaceutical composi~
tion/dosage form thereof. Obviously, in the case of ~ ;
diagnostic agents, the dosage used will be a quantity sufficient to deliver to the target body area a quantity sufficient to deliver an amount of radio- -~
isotope, stable isotope or the like which can be effectively detected by radioimaging or other detection means. The amount of radioisotope, stable isotope or ~ ~
. .
- -' ::
~ .
''~,'": " "': ' '. ' ': ::: . ' ' l 331 ~
the like present in the dosage form will be within or below the ranges conventionally used for diagnostic ~ -~
purposes.
While the invention has been described in terms of : ;
various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended :. :.
that the scope of the present invention be limited solely by the scope of the following claims.
:.
.- . ~
. . ~
Claims (74)
1. An inclusion complex of a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of .beta.- or .gamma.-cyclodextrin with the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal dihydropyridine. form of a dihydropyridine ? pyridinium salt redox system for brain-targeted drug delivery.
2. An inclusion complex according to Claim 1, wherein said dihydropyridine form is a compound of the formula [D-DHC]
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
3. An inclusion complex according to Claim 2, wherein the centrally acting drug species is a dopa-minergic agent, an androgenic agent, an anticonvulsant, an anxiolytic agent, a neurotransmitter, an antibiotic or antibacterial agent, an antidepressant, an antiviral agent, an anticancer or antitumor agent, an antiinflam-matory agent, an estrogen or a progestin.
4. An inclusion complex according to Claim 3, wherein the centrally acting drug species is dopamine, testosterone, phenytoin, GABA, valproic acid, tyrosine, methicillin, oxacillin, benzylpenicillin, cloxacillin, dicloxacillin, desipramine, acyclovir, trifluorothymi-dine, zidovudine, hydroxy-CCNU, chlorambucil, trypta-mine, dexamethasone, hydrocortisone, ethinyl estradiol, norethindrone, estradiol, ethisterone, norgestrel, estrone, estradiol 3-methyl ether, estradiol benzoate, norethynodrel, mestranol, indomethacin9 naproxen, FENU, HENU or 5-FU.
5. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-{{N-{.beta.-[3,4-bis(pivalyloxy)phenyl]ethyl}-carbamoyl}}-1,4-dihydropyridine, 1-methyl-3-{N-[[.beta.-[3,4-bis(isobutyryloxy)phenyl]ethyl]]}carbamoyl-1,4-dihydropyridine or N-{.beta.-[3,4-bis(pivalyloxy)phenyl]-ethyl}aminocarbonyloxymethyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate.
6. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 17.beta.-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one or 17.beta.-{[(3"-carbamoyl-1',4'-dihydropyri-dinyl)acetyl]oxy}androst-4-en-3-one.
7. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 5,5-diphenyl-3-[(1'-methyl-1',4'-dihydropyridin-3'-yl)-carbonyloxymethyl]-2,4-imidazolidinedione, 3-[(3'-carbamoyl-1',4'-dihydropyridin-1'-yl)acetyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione or 3-[3'-(3"-carbamoyl-1",4"-dihydropyridin-1"-yl)propionyloxy-methyl]-5,5-diphenyl-2,4-imidazolidinedione.
8. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[3-(benzyloxycarbonyl)propyl]carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(3'-cyclohexyl-carbonyl)propyl]}carbamoyl-1,4-dihydropyridine.
9. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-[2'-(2"-propyl)pentanoyloxy]ethylcarbamoyl-1,4-dihydropyridine, 1-methyl-3-[2'-(2"-propyl)-pentanoyloxy]ethoxycarbonyl-1,4-dihydropyridine or 1-[2'-(2"-propyl)pentanoyloxy]ethyl-3-carboxamide-1,4-dihydropyridine.
10. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4"-pivaloyloxy-phenyl)ethyl]}carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4"-isobutyryl-oxyphenyl)ethyl]}carbamoyl-1,4-dihydropyridine.
11. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(2,6-dimethoxy)-benzamido]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxy-late, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]-oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, C[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]meth [2S-(2.alpha.,5.alpha.,6.beta.)]-6-[3-(2-chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate or [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.
12. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is [{N-[3-(10,11-dihydro-SH-dibenz(b,f]azepin-S-yl)]propyl-N-methylamino}carbonyloxy]methyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate or [1-{N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)]propyl-N-methylamino}-carbonyloxy]ethyl 1,4-dihydro-1-methyl 3-pyridine-carboxylate.
13. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-f[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyridine.
14. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 3'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-5'-pivaloyltri-fluorothymidine.
15. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 3'-azido-3'-deoxy-5'-(1-methyl-1,4-dihydro-3-pyridinyl)-carbonyl]thymidine.
16. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is N-(2-chloroethyl)-N'-[4-(1,4-dihydro-1-methyl-3-pyridine-carbonyloxy)cyclohexyl]-N-nitrosourea, N-(2-fluoro-ethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyl-oxy)ethyl]-N-nitrosourea or N-(2-chloroethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)ethyl]-N-nitrosourea.
17. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-[(N-{2-[4-({4-[bis(2-chloroethyl]amino}-phenyl)butanoyloxy]ethyl})carbamoyl]-1,4-dihydropyri-dine, 1-methyl-3-(N-{4-[4-(4-{[bis(2-chloroethyl)]-amino}phenyl)butanoyloxy]cyclohexyl}carbamoyl)-1,4-dihydropyridine, 1-methyl-3-[(N-{2-[4-({4-bis(2-chloro-ethyl)]amino}phenyl)butanoyloxy]propyl})carbamoyl]-1,4-dihydropyridine, 1-methyl-3-[(N-{2-phenyl-2-({4-[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]}ethyl)carba-moyl]-1,4-dihydropyridine or 1-methyl-3-[N-({1-[4-(4-{[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]cyclo-hexyl}methyl)carbamoyl]-1,4-dihydropyridine.
18. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[2-(3-indolyl)ethyl]carbamoyl-1,4-dihydro-pyridine.
19. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 9-fluoro-11.beta.,17-dihydroxy-16.alpha.-methyl-21-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}pregna-1,4-diene-3,20-dione or 11.beta.,17-dihydroxy-21-{[(1-methyl-1,4-dihydro-pyridin-3-yl)carbonyl]oxy}pregn-4-ene-3,20-dione.
20. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 3-hydroxy-17.beta.-[(1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxyestra-1,3,5(10)-triene.
21. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 3-hydroxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxy}-19-nor-17.alpha.-pregna-1,3,5(10)-trien-20-yne, 3-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra-1,3,5(10)-trien-17-one, 17.beta.-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra-1,3,5(10)-trien-3-ol 3-methyl ether, 3,17.beta.-bis-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}estra-1,3,5(10)-triene, 3-(phenylcar-bonyloxy)-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxy}estra-1,3,5(10-triene or 3-methoxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-nor-17.alpha.-pregna-1,3,5(10)-trien-20-yne.
22. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-4-en-20-yn-3-one, 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}pregn-4-en-20-yn-3-one, 13-ethyl-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-18,19-dinorpregn-4-ene-20-yn-3-one or 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-5(10)-en-20-yn-3-one.
23. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 1-methyl-3-[N-(2-{1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolyl]acetoxy}ethyl)carbamoyl]-1,4-dihydropyridine or 1-methyl-3-{N-[2-(6-methoxy-a-methyl-2-naphthalenyl-acetoxy)ethyl]carbamoyl-1,4-dihydropyridine.
24. An inclusion complex according to Claim 4, wherein the compound of the formula [D-DHC] is 3-(1,4-dihydro-1-methyl-3-pyridinylcarbonyloxymethyl)-5-fluorouracil or 1-(1,4-dihydro-1-methyl-3-pyridine-carbonyloxymethyl-5-fluorouracil.
25. A method for stabilizing the reduced, biooxi-dizable, blood-brain barrier penetrating, lipoidal dihydropyridine form of d dihydropyridine ? pyridinium salt redox system for brain-targeted drug delivery, said method comprising complexing said dihydropyridine form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of .beta.- or .gamma.-cyclodextrin.
26. A method according to Claim 25, wherein said dihydropyridine form is a compound of the formula [D-DHC]
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
27. A method according to Claim 26, wherein the centrally acting drug species is a dopaminergic agent, an androgenic agent, an anticonvulsant, an anxiolytic agent, a neurotransmitter, an antibiotic or anti-bacterial agent, an antidepressant, an antiviral agent, an anticancer or antitumor agent, an antiinflammatory agent, an estrogen or a progestin.
28. A method according to Claim 27, wherein the centrally acting drug species is dopamine, testos-terone, phenytoin, GABA, valproic acid, tyrosine, methicillin, oxacillin, benzylpenicillin, cloxacillin, dicloxacillin, desipramine, acyclovir, trifluorothy-midine, zidovudine, hydroxy-CCNU, chlorambucil, trypta-mine, dexamethasone, hydrocortisone, ethinyl estradiol, norethindrone, estradiol, ethisterone, norgestrel, estrone, estradiol 3-methyl ether, estradiol benzoate, norethynodrel, mestranol, indomethacin, naproxen, FENU, HENU or 5-FU.
29. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-{{N-[.beta.-[3,4-bis(pivalyloxy)phenyl]ethyl}carbamoyl}}-1,4-dihydropyridine, 1-methyl-3-{N-[[.beta.-[3,4-bis(isobutyryl-oxy)phenyl]ethyl]]}carbamoyl-1,4-dihydropyridine or N-{.beta.-[3,4-bis(pivalyloxy)phenyl]ethyl}aminocarbonyloxy-methyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate.
30. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 17.beta.-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one or 17.beta.-{[(3"-carbamoyl-1',4'-dihydropyridinyl)acetyl]-oxy}androst-4-en-3-one.
31. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 5,5-diphenyl-3-[(1'-methyl-1',4'-dihydropyridin-3'-yl)carbonyloxymethyl]-2,4-imidazolidinedione, 3-[(3'-carbamoyl-1',4'-dihydropyridin-1'-yl)acetyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione or 3-[3'-(3"-carbamoyl-1",4"-dihy-dropyridin-1"-yl)propionyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione.
32. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[3-(benzyloxycarbonyl)propyl]carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(3'-cyclohexylcarbonyl)propyl]}-carbamoyl-1,4-dihydropyridine.
33. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-[2'-(2"-propyl)pentanoyloxy]ethylcarbamoyl-1,4-dihydropyridine, 1-methyl-3-[2'-(2"-propyl)pentanoyloxy]ethoxycarbonyl-1,4-dihydropyridine or 1-[2'-(2"-propyl)pentanoyloxy]-ethyl-3-carboxamide-1,4-dihydropyridine.
34. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-{N-[1'-ethoxycarbonyl)-2'-(4"-pivaloyloxyphenyl)ethyl]}-carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4"-isobutyryloxyphenyl)ethyl]}-carbamoyl-1,4-dihydropyridine.
35. A method according to Claim 28, wherein the compound of the formula [D-DHC] is [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-5-[3-(2-chlorophenyl)-5-methyl-4-isoxazole-carboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylate or [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazole-carboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylate.
36. A method according to Claim 28, wherein the compound of the formula [D-DHC] is [{N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)]propyl-N-methylamino}carbonyloxy]methyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate or [1-{N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)]propyl-N-methylamino}-carbonyloxy]ethyl 1,4-dihydro-1-methyl-3-pyridine-carboxylate.
37. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-{[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyridine.
38. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 3'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-5'-pivaloyltrifluorothy-midine.
39. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 3'-azido-3'-deoxy-5'-(1-methyl-1,4-dihydro-3-pyridinyl)carbonyl]thymi-dine.
40. A method according to Claim 28, wherein the compound of the formula [D-DHC] is N-(2-chloroethyl)-N'-[4-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)-cyclohexyl]-N-nitrosourea, N-(2-fluoroethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)ethyl]-N-nitrosourea or N-(2-chloroethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)ethyl]-N-nitrosourea.
41. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-[(N-{2-[4-({4-[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]-ethyl})carbamoyl]-1,4-dihydropyridine, 1-methyl-3-(N-{4-[4-(4-{[bis(2-chloroethyl)]amino}phenyl)butanoyl-oxy]cyclohexyl}carbamoyl)-1,4-dihydropyridine, 1-methyl-3-[(N-{2-[4-({4-bis(2-chloroethyl)]amino}-phenyl)butanoyloxy]propyl})carbamoyl]-1,4-dihydropyri-dine, 1-methyl-3-[(N-{2-phenyl-2-({4-[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]}ethyl)carba-moyl]-1,4-dihydropyridine or 1-methyl-3-[N-({1-[4-(4-{[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]cyclo-hexyl}methyl)carbamoyl]-1,4-dihydropyridine.
42. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[2-(3-indolyl)ethyl]carbamoyl-1,4-dihydropyridine.
43. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 9-fluoro-11.beta.,17-dihydroxy-16.alpha.-methyl-21-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}pregna-1,4-diene-3,20-dione or 11.beta.,17-dihydroxy-21-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}pregn-4-ene-3,20-dione.
44. A method according to Claim 28, wherein the compound of the formula [D-OHC] is 3-hydroxy-17.beta.-[(1-methyl-1,4-dihydropyridin-3-yl)carhonyl]oxyestra-1,3,5(10)-triene.
45. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 3-hydroxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-nor-17a-pregna-1,3,5(10-trien-20-yne, 3-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra-1,3,5(10)-trien-17-one, 17.beta.-[(1-methyl-1,4-dihydro-3-pyridinyl)-carbonyloxy]estra-1,3,5(10)-trien-3-ol 3-methyl ether, 3,17.beta.-bis-{[(1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxy}estra-1,3,5(10)-triene, 3-(phenylcarbonyl-oxy)-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]-oxy}estra-1,3,5(10)-triene or 3-methoxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-nor-17.alpha.-pregna-1,3,5(10)-trien-20-yne.
46. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-4-en-20-yn-3-one, 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)car-bonyl]oxy}pregn-4-en-20-yn-3-one, 13-ethyl-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-18,19-dinorpregn-4-en-20-yn-3-one or 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-5(10)-en-20-yn-3-one.
47. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 1-methyl-3-[N-(2-{1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolyl]-acetoxy}ethyl)carbamoyl]-1,4-dihydropyridine or 1-methyl-3-{N-[2-(5-methoxy-.alpha.-methyl-2-naphthalenyl-acetoxy)ethyl]carbamoyl-1,4-dihydropyridine.
48. A method according to Claim 28, wherein the compound of the formula [D-DHC] is 3-(1,4-dihydro-1-methyl-3-pyridinylcarbonyloxymethyl)-5-fluorouracil or 1-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxymethyl-5-fluorouracil.
49. A method for decreasing the tendency of the reduced, biooxidizable, blood-brain barrier pene-trating, lipoidal dihydropyridine form of a dihydro-pyridine ? pyridinium salt redox system for brain-targeted drug delivery to provide high initial concentrations of drug in the lungs following administration, said method comprising complexing said dihydropyridine form with a hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivative of .beta.- or .gamma.-cyclodextrin prior to administration.
50. A method according to Claim 49, wherein said dihydropyridine form is a compound of the formula [D-DHC]
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
wherein [D] is a centrally acting drug species and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyri-dine ? pyridinium salt redox carrier.
51. A method according to Claim 50, wherein the centrally acting drug species is a dopaminergic agent, an androgenic agent, an anticonvulsant, an anxiolytic agent, a neurotransmitter, an antibiotic or antibac-terial agent, an antidepressant, an antiviral agent, an anticancer or antitumor agent, an antiinflammatory agent, an estrogen or a progestin.
52. A method according to Claim 51, wherein the centrally acting drug species is dopamine, testos-terone, phenytoin, GABA, valproic acid, tyrosine, methicillin, oxacillin, benzylpenicillin, cloxacillin, dicloxacillin, desipramine, acyclovir, trifluorothy-midine, zidovudine, hydroxy-CCNU, chlorambucil, tryp-tamine, dexamethasone, hydrocortisone, ethinyl estradiol, norethindrone, estradiol, ethisterone, norgestrel, estrone, estradiol 3-methyl ether, estradiol benzoate, norethynodrel, mestranol, indo-methacin, naproxen, FENU, HENU or 5-FU.
53. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-{{N-{.beta.-[3,4-bis(dipivalyloxy)phenyl]ethyl}carbamoyl}}-1,4-dihydropyridine, 1-methyl-3-{N-[[.beta.-[3,4-bis(isobutyryloxy)phenyl]ethyl]]}carbamoyl-1,4-dihydropyridine or N-{.beta.-[3,4-bis(pivalyloxy)phenyl]-ethyl}aminocarbonyloxymethyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate.
54. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 17.beta.-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one or 17.beta.-{[(3"-carbamoyl-1',4"-dihydropyridinyl)acetyl]-oxy}androst-4-en-3-one.
55. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 5,5-diphenyl-3-[(1'-methyl-1',4'-dihydropyridin-3'-yl)carbonyloxymethyl]-2,4-imidazolidinedione, 3-[(3'-carbamoyl-1',4'-dihydro-pyridin-1'-yl)acetyloxymethyl]-5,5-diphenyl-2,4-imida-zolidinedione or 3-[3'-(3"-carbamoyl-1",4"-dihydro-pyridin-1"-yl)propionyloxymethyl]-5,5-diphenyl-2,4-imidazolidinedione.
56. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[3-(benzyloxycarbonyl)propyl]carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(3'-cyclohexylcarbonyl)propyl]}-carbamoyl-1,4-dihydropyridine.
57. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-[2'-(2"-propyl)pentanoyloxy]ethylcarbamoyl-1,4-dihydropyridine, 1-methyl-3-[2'-(2"-propyl)pentanoyloxy]ethoxycarbonyl-1,4-dihydropyridine or 1-[2'-(2"-propyl)pentanoyloxy]-ethyl-3-carboxamide-1,4-dihydropyridine.
58. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4"-pivaloyloxyphenyl)ethyl]}-carbamoyl-1,4-dihydropyridine or 1-methyl-3-{N-[(1'-ethoxycarbonyl)-2'-(4"-isobutyryloxyphenyl)ethyl]}-carbamoyl-1,4-dihydropyridine.
59. A method according to Claim 52, wherein the compound of the formula [D-DHC] is [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(2,6-dimethoxy)benzamido]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolecarboxamido)-7-oxo-4-thia-1-azabicyclo[3.2.0]-heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-6-[3-(2-chlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate or [[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]oxy]methyl [2S-(2.alpha.,5.alpha.,6.beta.)]-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.
60. A method according to Claim 52, wherein the compound of the formula [D-DHC] is [{N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)]propyl-N-methyl-amino}carbonyloxy]methyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate or [1-{N-[3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)]propyl-N-methylamino}carbonyl-oxy]ethyl 1,4-dihydro-1-methyl-3-pyridinecarboxylate.
51. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-{[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyridine.
62. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 3'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-5'-pivaloyltrifluorothymi-dine.
63. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 3'-azido-3'-deoxy-5'-(1-methyl-1,4-dihydro-3-pyridinyl)carbonyl]thymi-dine.
64. A method according to Claim 52, wherein the compound of the formula [D-DHC] is N-(2-chloroethyl)-N'-[4-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)-cyclohexyl]-N-nitrosourea, N-(2-fluoroethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)ethyl]-N-nitrosourea or N-(2-chloroethyl)-N'-[2-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)ethyl]-N-nitrosourea.
65. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-[(N-{2-[4-({4-[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]-ethyl})carbamoyl]-1,4-dihydropyridine, 1-methyl-3-(N-{4-[4-(4-{[bis(2-chloroethyl)]amino}phenyl)butanoyl-oxy]cyclohexyl}carbamoyl)-1,4-dihydropyridine, 1-methyl-3-[(N-{2-[4-({4-bis(2-chloroethyl)]amino}-phenyl)butanoyloxy]propyl})carbamoyl]-1,4-dihydro-pyridine, 1-methyl-3-[(N-{2-phenyl-2-({4-[bis(2-chloro-ethyl)]amino}phenyl)butanoyloxy]}ethyl)carbamoyl]-1,4-dihydropyridine or 1-methyl-3-[N-({1-[4-(4-{[bis(2-chloroethyl)]amino}phenyl)butanoyloxy]cyclohexyl}-methyl)carbamoyl]-1,4-dihydropyridine.
5fi. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-N-[2-(3-indolyl)ethyl]carbamoyl-1,4-dihydropyridine.
67. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 9-fluoro-11.beta.,17-dihydroxy-16.alpha.-methyl-21-{[(1-methyl-1,4-dihydropyri-dine-3-yl)carbonyl]oxy}pregna-1,4-diene-3,20-dione or 11.beta.,17-dihydroxy-21-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}pregn-4-ene-3,20-dione.
68. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 3-hydroxy-17.beta.-[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxyestra-1,3,5(10)-triene.
69. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 3-hydroxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-nor-17.alpha.-pregna-1,3,5(10)-trien-20-yne, 3-[(1-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra-1,3,5(10)-trien-17-one, 17.beta.-[(1-methyl-1,4-methyl-1,4-dihydro-3-pyridinyl)carbonyloxy]estra-1,3,5(10)-trien-3-ol 3-methyl ether, 3,17.beta.-bis-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}estra-1,3,5(10)-triene, 3-(phenyl-carbonyloxy)-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxy}estra-1,3,5(10)-triene or 3-methoxy-17.beta.-{[1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-nor-17.alpha.-pregna-1,3,5(10)-trien-20-yne.
70. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-4-en-20-yn-3-one, 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)-carbonyl]oxy}pregn-4-en-20-yn-3-one, 13-ethyl-17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-18,19-dinorpregn-4-en-20-yn-3-one or 17.beta.-{[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy}-19-norpregn-5(10)-en-20-yn-3-one.
71. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 1-methyl-3-[N-(2-{1-(p-chlorobenzoyl)-5-methoxy-2-methyl-3-indolyl]-acetoxy}ethyl)carbamoyl]-1,4-dihydropyridine or 1-methyl-3-{N-[2-(6-methoxy-.alpha.-methyl-2-naphthalenyl-acetoxy)ethyl]carbamoyl-1,4-dihydropyridine.
72. A method according to Claim 52, wherein the compound of the formula [D-DHC] is 3-(1,4-dihydro-1-methyl-3-pyridinylcarbonyloxymethyl)-5-fluorouracil or 1-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxymethyl)-5-fluorouracil.
73. A method for improving the water solubility of the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal dihydropyridine form of a dihydropyridine ? pyridinium salt redox system for brain-targeted drug delivery, said method comprising complexing said dihydropyridine form with a hydroxy-propyl, hydroxyethyl, glucosyl, maltosyl or malto-triosyl derivative of .beta.- or .gamma.-cyclodextrin.
74. A method for improving the water solubility of the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal dihydropyridine form of a dihydropyridine ? pyridinium salt redox system for brain-targeted drug delivery, said method comprising complexing hydroxypropyl-.beta.-cyclodextrin with 3-hydroxy-17.beta.-[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy-estra-1,3,5(10)-triene, 17.beta.-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3-one, 1-methyl-3-N-[3-(benzyloxycarbonyl)propyl]carbamoyl-1,4-dihydropyri-dine, 1-methyl-3-{{N-{.beta.-[3,4-bis(pivalyloxy)phenyl]-ethyl}carbamoyl}}-1,4-dihydropyridine or 1-methyl-3-{[2-(9-guanylmethoxy)ethoxy]carbonyl}-1,4-dihydropyri-dine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/139,755 US5002935A (en) | 1987-12-30 | 1987-12-30 | Improvements in redox systems for brain-targeted drug delivery |
| US139,755 | 1987-12-30 |
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| Publication Number | Publication Date |
|---|---|
| CA1331564C true CA1331564C (en) | 1994-08-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000585791A Expired - Fee Related CA1331564C (en) | 1987-12-30 | 1988-12-13 | Redox systems for brain-targeted drug delivery |
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|---|---|
| US (3) | US5002935A (en) |
| EP (1) | EP0327766B1 (en) |
| JP (1) | JP3038715B2 (en) |
| AT (1) | ATE164855T1 (en) |
| AU (1) | AU618995B2 (en) |
| CA (1) | CA1331564C (en) |
| DE (1) | DE3856160T2 (en) |
| ES (1) | ES2118707T3 (en) |
| IE (1) | IE64886B1 (en) |
| ZA (1) | ZA889679B (en) |
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-
1987
- 1987-12-30 US US07/139,755 patent/US5002935A/en not_active Expired - Lifetime
-
1988
- 1988-03-29 US US07/174,945 patent/US4983586A/en not_active Expired - Lifetime
- 1988-12-13 CA CA000585791A patent/CA1331564C/en not_active Expired - Fee Related
- 1988-12-13 IE IE371788A patent/IE64886B1/en not_active IP Right Cessation
- 1988-12-19 EP EP88312016A patent/EP0327766B1/en not_active Expired - Lifetime
- 1988-12-19 AT AT88312016T patent/ATE164855T1/en not_active IP Right Cessation
- 1988-12-19 ES ES88312016T patent/ES2118707T3/en not_active Expired - Lifetime
- 1988-12-19 DE DE3856160T patent/DE3856160T2/en not_active Expired - Lifetime
- 1988-12-28 ZA ZA889679A patent/ZA889679B/en unknown
-
1989
- 1989-01-04 JP JP64000037A patent/JP3038715B2/en not_active Expired - Lifetime
- 1989-03-28 AU AU31762/89A patent/AU618995B2/en not_active Expired
- 1989-12-11 US US07/448,655 patent/US5024998A/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10035784B2 (en) | 2010-11-16 | 2018-07-31 | Texas Heart Institute | Agonists that enhanced binding of integrin-expressing cells to integrin receptors |
| US10071980B2 (en) | 2010-11-16 | 2018-09-11 | Texas Heart Institute | Agonists that enhanced binding of integrin-expressing cells to integrin receptors |
| US10287264B2 (en) | 2010-11-16 | 2019-05-14 | Texas Heart Institute | Agonists that enhance binding of integrin-expressing cells to integrin receptors |
Also Published As
| Publication number | Publication date |
|---|---|
| US4983586A (en) | 1991-01-08 |
| DE3856160T2 (en) | 1998-11-12 |
| AU619788B2 (en) | 1992-02-06 |
| AU618995B2 (en) | 1992-01-16 |
| DE3856160D1 (en) | 1998-05-14 |
| AU3176289A (en) | 1989-07-27 |
| AU2733988A (en) | 1989-07-06 |
| EP0327766A2 (en) | 1989-08-16 |
| IE64886B1 (en) | 1995-09-20 |
| JPH01294663A (en) | 1989-11-28 |
| EP0327766B1 (en) | 1998-04-08 |
| EP0327766A3 (en) | 1990-09-26 |
| US5024998A (en) | 1991-06-18 |
| US5002935A (en) | 1991-03-26 |
| IE883717L (en) | 1989-06-30 |
| ES2118707T3 (en) | 1998-10-01 |
| ATE164855T1 (en) | 1998-04-15 |
| ZA889679B (en) | 1990-08-29 |
| JP3038715B2 (en) | 2000-05-08 |
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