CA2156620A1 - Chelating agents of the type xn1s1o for radioactive isotopes, metal complexes thereof, and their use in diagnosis and therapy - Google Patents
Chelating agents of the type xn1s1o for radioactive isotopes, metal complexes thereof, and their use in diagnosis and therapyInfo
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- CA2156620A1 CA2156620A1 CA002156620A CA2156620A CA2156620A1 CA 2156620 A1 CA2156620 A1 CA 2156620A1 CA 002156620 A CA002156620 A CA 002156620A CA 2156620 A CA2156620 A CA 2156620A CA 2156620 A1 CA2156620 A1 CA 2156620A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/001—Acyclic or carbocyclic compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/008—Peptides; Proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
- C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
- C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
- C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
- C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
- C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
- C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
- C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur 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
- C07D333/30—Hetero atoms other than halogen
- C07D333/34—Sulfur atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic System
- C07F13/005—Compounds without a metal-carbon linkage
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Abstract
S u m m a r y This invention relates to new bifunctional chelating agents with intermittent chalcogen atoms, pharmaceuticals containing these compounds, their use in radiodiagnostics and radiotherapy, and methods for the production of these compounds.
The compound according to the invention has the general formula (I) M - L
wherein M represents a radionuclide of Tc or Re and L a ligand of the general formula (II).
It was found, surprisingly, that these new, bifunctional chelating agents with intermittent chalcogen atoms and their coupling products with compounds that accumulate specifically are excellently suited for producing radio-pharmaceuticals for diagnostic and therapeutic purposes.
The compound according to the invention has the general formula (I) M - L
wherein M represents a radionuclide of Tc or Re and L a ligand of the general formula (II).
It was found, surprisingly, that these new, bifunctional chelating agents with intermittent chalcogen atoms and their coupling products with compounds that accumulate specifically are excellently suited for producing radio-pharmaceuticals for diagnostic and therapeutic purposes.
Description
This invention relates to new bifunctional chelating agents with intermittent chalcogen atoms, pharmaceuticals containing these compounds, their use in radiodiagnostics and radiotherapy, and methods for the production of these S compounds.
It has been known for a long time that complexing agents for radioactive isotopes or their complexes with radio-active metals can be applied in radiodiagnostics and radio-therapy. Technetium-99m is the most frequently used radio-nuclide in radiodiagnostics because it is particularly wellsuited for in-vivo applications due to its favourable physical properties (no corpuscular radiation, low half-life of 6.02 h, goo~ detectability by 140 keV y-radiation) as well as its low biological half-life and easy availabil-ity. The first step of forming complexes of technetium-99m is to gain pertechnetate from a nuclide generator; it is then converted to a lower oxidation number using appro-priate reductants (such as SnCl2, S2O42-, etc.). This oxidation number is stabilize,d by an appropriate chelating agent. As technetium may have several oxidation numbers (+7 to -1) which may vehemently alter its pharmacological properties by changing the charge of the complex, it is necessary to provide chelating agents or complex ligands for technetium-99m that are capable of binding technetium 2S in a specific oxidation number safely, firmly and stably to prevent undesirable biodistribution due to in-vivo redox processes or release of technetium from the radiodiagnostic agent which would impede the safe diagnosis of the respec-tive diseases.
For example, cyclic amines (Troutner, D.E. et al.: J. Nucl.
Med. ~1, 443 (1980)) are regarded as suitable complexing agents for technetium and rhenium isotopes but their disadvantage is that they are only capable of binding technetium-99m in sufficient quantities from a pH value ~9.
N2O2 systems (Pillai, M.R.A., Troutner, D.E. et al.: Inorg.
~ ID-13317WQ
Chem., 29, 1850 (1990)) are in clinical use. Non-cyclic N4 systems such as HMPAO have the great disadvantage of low complex stability. Tc-99m-HMPA0 has to be applied immediately after labelling due to its low stability s (Ballinger, J. R. et al., Appl. Radiat. Isot. ~, 315 (1991); Billinghurst, M. W. et al., Appl. Radiat. Isot. 42, 607 (1991)) to keep the portion of decomposition products low which have different pharmacokinetic and excretion properties. Such radiochemical impurities make detection of the diseases to be diagnosed more difficult. Any coupling of these chelates or chelating agents with other substances that accumulate selectively in centres of diseases cannot be broken by simple means so that these normally spread unspecifically in the organism.
~, N2S2 chelating agents (Bormans, G. et al.: Nucl. Med.
Biol., 17, 499 (1990)) such as ethylene dicysteine (EC;
Verbruggen, A.M. et al.; J. Nucl. Med. 33, 551 (1992)) meet the requirement of sufficient stability of their respective technetium-99m complex but form radiodiagnostic agents of a purity greater than 69~ only~at pH values ~9 of the complexing medium. N3S systems (Fritzburg, A.; EPA 0 173 424 and EPA 0 250 013) yield stable technetium-99m complexes but have to be heated up to temperatures of about 100C to insert the radionuclide.
Another disadvantage of N2S2 and N3S systems is that they are discharged partially too rapidly and without specific accumulation by the organism. Thus they are only used clinically, though to a limited extent, in renal function diagnostics. In the last few years the demand has increased for radiodiagnostics that accumulate specifically in diseased tissues. This can be accomplished if one manages to link complexing agents easily with selectively accumu-lating substances while the latter retain their favourable complexing properties. But as it happens quite frequently that a certain reduction of complex stability is observed ID-l33l7Wo 2 15 6 fi 2 ~
after coupling the complexing agent to such a molecule by means of one of its functional groups, previous approaches to coupling chelating agents with substances that accumu-late selectively are hardly satisfactory because a quantity 5 of the isotope that is not tolerable with a view to diagnostics is released in vivo from the conjugate (Brechbiel, M. W. et al.; Inorg. Chem. 1986, ~, 2772). It is therefore necessary to produce bifunctional complexing agents that have functional groups to bind the desired metallic ion and one (or several other) functional groups to bind the select~vely accumulating molecule. Such bifunc-tional ligands allow specific, chemically defined bonding of technetium or rhenium isotopes to the most various bio-logical materials even in cases in which pre-labelling is ~5 applied. Some chelating agents coupled with monoclonal antibodies (e.g. EP Appl. 0 247 866 and EP Appl. 0 188 256) or fatty acids (EP Appl. 0 200 492) have been described.
But these chelating agents were based on the N2S2 systems mentioned above which are hardly appropriate due to their low stability. As both the properties of the substances that accumulate selectively and the mechanisms of accumulation are quite varied, one should be able to vary the chelating agent meant for coupling to adapt it to the physiological requirements of its coupling partner with regard to lipophilic or hydrophilic behaviour, membrane permeability or impermeability, etc.
It is therefore an object of this invention to provide stable complex compounds coupled with or capable of cou-pling with various compounds that accumulate selectively, and to provide such linkable chelating agents or complexes whose substituents show a wider range of chemical variation to be adaptable to the above requirements. It is another object of this invention to provide such compounds and pharmaceuticals containing these compounds, as well as methods for their production.
' ID-13317wo 215662~
_ 5 This problem is solved by the invention, surprisingly, in that the new, uncommon, bifunctional chelating agents with intermittent chalcogen atoms and their coupling products with compounds that accumulate selectively are excellently suited for producing radiodiagnostic and radiotherapeutic agents.
The subject matter of this invention are compounds of the general formula (I) M~- L (I) wherein M represents a radionuclide of Tc or Re and L represents a ligand of the general formula (II) B-Co-CRlR2-A-CR3R4-CooH (II) wherein A represents an O, S, or Se chalcogen atom, Rl, R2, R3, and R4 are same or different and represent a hydrogen atom and/or a branched or unbranched Cl-C6 alkyl resldue, B represents a residue -NH-CR5R6- (CR7R8)n=1 2-S-R9, wherein R5 and R6 are same or different and represent a hydrogen atom or an unbranched, branched, cyclic, or polycyclic Cl-C60 alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, 25 aryl, alkylaryl, or arylalkyl residue which may optionally be carry an additional hydroxy, oxo, oxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or alkoxy groups containing up to 20 carbon atoms, and/or may optionally be interrupted, and/or replaced, by one or several heteroatoms from the series of O, N, S, P, As, Se, 215662~
R7 and R8 are same or different and represent a hydrogen atom and/or a branched or unbranched Cl-C6 alkyl residue, R9 represents a hydrogen atom, a branched or unbranched Cl-C6 alkyl residue, or a sulfur protecting group, with Rs and R6, together with the groups that connect them, optionally forming a 4- to 8-membered ring which may optionally carry additional hydroxy, oxo, oxy, or alkoxy groups containing up to 6 carbon atoms.
Preferred compounds of the general formula (I) are characterized in that Rl, R2, R3, and R4 are hydrogen atoms, and that A is a sulfur atom.
Particularly prefer~ed compounds according to the invention of the general formula (I) are characterized in that RS and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom.
Another object of this invention is related to the new, bifunctional ligands with intermittent chalcogen atoms of the general formula (II) B-CO-CR1R2-A- CR3R4-COOH (II) wherein R1, R2, R3, R4, A and B have the meanings specified above.
Such ligands according to the invention of the general formula (II) are preferred in which A is a sulfur atom and Rl, R2, R3 and R4 are hydrogen atoms.
Particularly preferred compounds according to the invention of general formula (II) are characterized in that Rs and R6 are different, and that R6, R7 and Rs each represent a hydrogen atom.
Yet another object of this invention are conjugates containing compounds of the general formulae (I and/or II) ID-l33l7wo -and substances that accumulate selectively in diseased tissues, with a covalent bond existing between these sub-stances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups.
Particularly preferred conjugates according to the invention are characterized in that the substances that accumulate in dise~sed tissue are peptides such as endo-thelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
Other preferred embodiments of the conjugates according to the invention are characterized in that the peptides com-prise the following sequences or parts thereof:
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-~ I
phe-cys-his-leu-asp-ile-ile-trp, cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-~ I
tyr-cys-his-leu-asp-ile-ile-trp, cys-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr--phe-cys-his-leu-asp-ile-ile-trp, i rD-13317WO
-cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr--phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-i phe-cys-his-gln-asp-val-ile-trp, , ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, the partia~ sequence his-leu-asp-ile-ile-trp or the cyclic amino acid sequences Cyclo-(Dtrp-Dasp-pro-Dval-leu), Cyclo-(Dglu-ala-alloDile-leu-Dtrp).
ID-1~317Wo 9 21S6fi20 -The compounds according to the invention of the general formula (I) are produced by reacting technetium-99m in the form of pertechnetate or Re in the form of perrhenate in the presence of a reductant and, optionally, an auxiliary ligand, with a compound of the general formula (II) B-Co-CRlR2-A-CR3R4-CooH (II) wherein Rl, R2, R3, R4, A, and B, have the meanings specified above.
The ligands accordlng to the invention of the general for-mula (II) are produced by reacting compounds of the general formula (III) with compounds of the general formula (IV) according to the following reaction scheme:
CR1R2-A_CR3R4 + NH2 ~ CR5 R6 - ( CR7R8 ) n= l 2 ~ S - R9 1 5 C~- O CO
(III) (IV) B-Co-CRlR2-A-CR3R4-CooH (II) .
wherein Rl R2 R3, R4, R5, R6, R7, R8, R9, A, and B have the meanings specified above.
These reactions are carried out in polar and non-polar aprotic solvents such as dichloromethane, tetrahydrofurane, chloroform, 1,4-dioxane, DMF, or DMSO at temperatures between -30 and +100C; an auxiliary base is added to trap any acids that may be liberated. Among these bases could be, for example: tertiary amines, alkali and alkaline-earth hydroxides, alkali and alkaline-earth carbonates.
Another object of the present invention is a kit for pro-ducing radiopharmaceuticals consisting of a compound of the general formula (II) or a conjugate according to the invention containing compounds of the general formulae (I
~ ID-13317wo 21~6620 -and/or II) and substances that accumulate selectively in tissues, a reductant and, optionally, an auxiliary ligand, said agents being either dry or in solution, instructions for use including instructions for reacting the compounds described with technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.
Another object of this invention is a radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or athero-sclerotic plaques.JIt contains a compound of the generalformula (I) or a conjugate according to the invention containing compounds of the general formulae (I and/or II) and substances that accumulate selectively in tissues, optionally with the~adjuvants common in galenics; the compound is prepared in a kit using technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.
Yet another object of this invention is a method for carrying out radiodiagnostic examinations according to which the radiopharmaceutical formulation is applied at doses from 0.1 to 30 mCi, preferably from 0.5 to 10 mCi, per 70 kg of a patient's body weight, and that radiation emitted by the patient is recorded.
Many of the synthesized chelates that were labelled with Tc-99m or Re surprisingly showed a greater stability than comparable-N2S2 and N3S systems described in the litera-ture. For example, no decomposition products were found of a substance according to the invention (Examples 2a, 2b) coupled with a fatty alcohol even after 23 hours. It was also found in competition tests that the Tc-99m or-Re chelating agents complex better than comparable N2S2, N3S
and propylene aminoxium systems. The chelates and chelating agents described in the present invention are clearly bet-ter suited for diagnostic and therapeutic purposes than the systems known so far. It is a specific advantage of the 215662~
- chelating agents according to the invention that they may be synthesized without sulfur protecting groups This makes synthesis very simplei in addition, the compounds according to the invention, when radiolabelled, do not contain any other foreign molecules in the solutions used for radio-diagnostics or radiotherapy, for example, solutions to be administered intravenously. Biodistribution of the radio-pharmaceutical and thus the value of diagnostic information are frequently diminished by such foreign molecules. More-over, such ligands~or their coupling products with sub-stances that accumulate selectively in diseased tissues can be labelled very gently. The ligands according to the in-vention and their coupling products with substances that accumulate selectively in diseased tissues can be labelled at room temperature and at the physiological pH value without having to split off protecting groups using bases, acids, or other auxiliary substances known to a person skilled in the art. This guarantees that the very sensitive substances that accumulate selectively in diseased tissues are not modified chemically by such auxiliary substances, as such modification frequently reduces selective accumu-lation in diseased tissue and diminishes the value of radiodiagnostic information.
Sulfur protecting groups may be used here, of course, if the disadvantages described can be accepted. The groups are attached to sulfur atoms and split off according to methods known to a-person skilled in the art. The ways in which the substances that accumulate selectively in diseased tissues are bonded are also known to a person skilled in the art (e.g. Fritzberg et al.; J. Nucl. Med. ~, 7 (1987)), for example, by a reaction of electrophilic groups of the complex ligand with nucleophilic centres of the substances that accumulate selectively in diseased tissues. Otherwise, nucleophilic groups of the chelating agent are coupled with electrophilic groups of the substances that accumulate selectively in diseased tissues.
rD- 13317WO
21S66~0 The partners for coupling are, among others, various bio-molecules, ligands that bond to specific receptors which are capable of detecting tissue showing a modified receptor density. This includes peptides, steroid hormones, growth factors and neurotransmitters. Ways for improved diagnosis of carcinomas of the breast and the prostata were shown using ligands for steroid hormone receptors (S. J. Brandes and J. A. Katzenellenbogen, Nucl. Med. Biol. lS, S3, 1988).
Tumour cells sometimes show a modified density of receptors for peptide hormone~s or growth factors such as the epider-mal growth factor (EGF). The differences in concentration could be utilized for selective accumulation of cytostatic agents in tumour cells (E. Aboud-Pirak et al., Proc. Natl.
Acad. Sci. USA 86; 3778, 1989). Ligands for neuroreceptors labelled with positron-emitting isotopes were successfully used for the diagnosis of various brain diseases (J. J.
Forst, Trends in Pharmacol. Sci., 7, 490, 1989). Other biomolecules are metabolites that can be introduced into the metabolism of cells to make changes visible; this includes fatty acids, sacchar~des, peptides, and amino acids. Fatty acids that were coupled with the more unstable N2S2 chelating agents have been described in EPA 0 200 492.
Other metabolic products such as saccharides (desoxyglucose), lactate, pyruvate, and amino acids 2S (leucine, methylmethionine, glycine) were used in the PET
technique for visualizing changes in metabolic processes (R. Weinreich, Swiss Med., 8, 10, 1986). Likewise, non-biological substances such as misonidazol and its derivatives which bond irreversibly to cell components in tissues or parts of tissues with a reduced oxygen concentration, can be used for specific accumulation of radioactive isotopes and thus for the visualization of tumours or ischaemic regions (M. E. Shelton, J. Nucl. Med.
30; 3S1, 1989). Finally, bifunctional chelating agents may be coupled with monoclonal antibodies or their fragments.
Coupling products of the chelating agents according to the rD- 133 17WO
215fi620 ~_ 13 invention or their technetium-99m or Re complexes with fatty alcohols, fatty alcohol derivatives, or fatty amines and their derivatives, or with endothelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists have proved particularly favourable for the detection of atherosclerotic vascular diseases. These derivatives were applied to WHHL rabbits that had high LDL concentrations in their blood - and thus atherosclerotic lesions - due to a genetic defect of ~heir LDL receptor. Concentration quotients from 3 to 40 were found in atheromatose plaques as compared with undamaged tissue about 4 to 5 hours after i.v. application of the derivatives to WHHL rabbits. This allowed detection of atherosclerotic areas of vessels using the common methods of radiodiagnostics (e.g. a gamma scintillation camera). Only very late stages of atherogenesis could up to now be diagnosed by using more invasive methods (e.g. arteriography). The substances according to the invention provide the decisive advantage of being able to diagnose much earlier stages of athero-sclerosis using non-invasive methods.
It is unimportant whether the chelating agent is labelled with Tc-99m or Re before or after coupling with the selec-tively accumulating molecule. But if coupling takes place after complexing, it is a prerequisite that the reaction of the radioactive complex with the accumulating compound is rapid, gentle, and nearly quantitative, requiring no subsequent purification.
The radiopharmaceuticals of the invention are produced in a generally known way by dissolving the complexing agents according to the invention in an aqueous medium and adding a reductant, preferably tin(II) salts such as chloride or tartrate, optionally adding the adjuvants common in galenics, and subsequent sterile filtration. Among the suitable additives are physiologically tolerable buffers 21~662~
(such as tromethamine), small quantities of electrolytes (e.g. sodium chloride) or stabilizers (e.g. gluconate, phosphate, or phosphonate). The pharmaceutical according to the invention is either available as a solution or as lyophilizate and is mixed shortly before application with a solution of Tc-99m pertechnetate, eluated from commercial generators, or a perrhenate solution.
For in-vivo applications in nuclear medicine, the agents according to the invention are administered at doses from 1 x 10-5 to 5 x 104~nmol/kg of body weight, preferably from 1 x 10-3 to 5 x 102 nmol/kg of body weight. The amount of radioactivity per application, based on an average body weight of 70 kg, is between 0.05 and S0 mCi, preferably between 5 and 30 mCi`, for diagnostic applications. For therapeutic applications, doses applied are between 5 and 500 mCi, preferably from 10 to 350 mCi. Normally, 0.1 to 2 ml of a solution of the agents according to the invention is applied by intravenous, intra-arterial, peritoneal or intra-tumoral injection. The intravenous injection is preferred.
The following examples shall explain the object of this invention in greater detail.
rD-13317WO
21~6620 ~_ 15 Example la N-(2-mercapto-1-(methoXycarbonyl)-ethyl)-thiodiglycolic acid monoamide 13.21 g (0.1 mol) of thiodiglycolic acid anhydride are added by dropping, and in an argon atmosphere, to a solution of 17.16 g (0.1 mol) of cysteine methyl ester hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hourf.s at room temperature and washed with aqueous 2~ citric acid. After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 18.73 g (70.1%), white powder 15 Analysis referring to the anhydrous substance:
Calc.: C 35.95 H 4.90 N 5.24 0 29.93 S 23.99 Found: C 35.72 H 5.12 N 5.03 S 23.71 Example lb Technetium-99m complex of N-(2-mercapto-1-(methoxy-carbonyl)-ethyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example la are dissolved in 1.0 ml of 0.5 M phosphate buffer, pH 7.5.
50 ~l of this ligand solution are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and lO0 ~l of a pertechnetate solution (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient elution from 100~ A to 100~ B within 7.5 minutes ~eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
16 21~66zo -acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,98~.
Example 2a S N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide 1.32 g (10 mol) of thiodiglycolic acid anhydride dissolved in 50 ml of anhydrcus-dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 2.98 g (10 mmol) of cysteine decyl ester hydrochloride and 2.02 g (20 mol) of triethyl amine in 250 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2~ citric acid.
After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 3.37 g (85.6~), white powder Analysis referring to the anhydrous substance:
Calc.: C 51.88 H 7.94 N 3.56 O 20.33 S 16.29 Found: C 51.63 H 8.07 N 3.37 S 16.02 ~D- 133 17WO
Example 2b Technetium-99m complex of N-(2-mercapto-1-(decyloxy-carbonyl)-ethyl-thiodiglycolic acid diamide 10 mg of the ligand produced according to Example 2a are dissolved in 1.0 ml of O.S M phosphate buffer, pH 7.5.
50 ~1 of this ligand solution are mixed with 250 ~1 of phosphate buffer, pH 7.5, 50 ~1 of a deoxygenated aqueous ci~rate solution (50 mg/ml), 2.5 ~1 of a deoxygenated aqueous tin(II) chlorlde solution (5 mg/ml 0.05 N HCl), and 100 ~1 of a pertechnetate solution (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ~98%.
Example 3a N-(2-oxo-1-tetrahydrothiophene-3-yl)-thiodiglycolic acid monoamide 13.21 g (0.1 mmol) of thiodiglycolic acid anhydride dissolved in 250 ml of anhydrous dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 15.36 g (0.1 mol) of homocysteine thiolactone hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2~ citric acid. After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 22.73 g (91.2~), white powder Analysis referring to the anhydrous substance:
18 21 S 6 ~ 2 0 Calc.: C 38.54 H 4.45 N 5.62 0 25.67 S 25.72 Found: C 38.37 H 4.68 N 5.41 S 25.47 Example 3b N-(3-mercapto-1-(octylaminoCarbonyl)-propyl)-thiodiglycolic mono~mide 30 ml of octyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglyco~ic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2~
citric acid and 200~ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separation, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 878 mg (23.2~), white powder Analysis referring to the anhydrous substance:
Calc.: C 50.77 H 7.99 N 7.40 0 16.91 S 16.94 Found: C 50.48 H 8.13 N 7.15 S 16.71 Example 3c Technetium 99-m complex of N-(3-mercapto-1-(octylamino-carbonyl)-propyl)-thiodiglycolic acid monsr ;de 10 mg of the ligand produced according to Example 3b are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex lg 2ls662a formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, O.oo5 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,95~ .
Example 4a N-(3-mercapto-1-(2,methoxyethylamino carbonyl)-propyl)-thiodiglycolic acid monoamide 30 ml of 2-methoxy ethyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 5 hours and evaporated under reduced pressure. The residue is mixed with 200 ml of aqueous 2~ citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separation, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 734 mg (22.6~), white powder Analysis referring to the anhydrous substance:
Calc. C 40.73 H 6.21 N 8.64 O 24.66 S 19.76 Found: C 40.47 H 6.49 N 8.38 S 19.51 ID-13317Wo 215662~
Example 4b Technetium-99m complex o~ N-(3-mercapto-l-(2-methyleth amino carbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 4a are S dissolved in 1.0 ml of ethanol. S0 ~1 of this ligand solu-tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~1 of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~1 of a deoxygenated aqueous tin(II) chloride solution (S mg/ml O.OS N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci)~ After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, S ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100% B within 7.5 minutes (eluent A:-~odium hydrogenphosphate, 0.005 M, pH
lS 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,95~ .
Example Sa N-(3-mercapto-1-(2-hydroxy-ethylamino carbonyl)-propyl)-thiodiglycolic acid monoamide 30 ml of 2-amino ethanol are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2 citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separa-tion, is dried above sodium sulfate. The solvent is evapo-rated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 435 mg (14.0~), white powder ID- 133 17WO , 2 1 5 6 6 2 0 Analysis referring to the anhydrous substance:
Calc.: C 38.70 H 5.85 N 9.03 0 25.77 S 20.66 Found: C 38.38 H 5.74 N 8.91 S 20.43 Example 5b Technetium-99m complex of N-(3-mercapto-1-(2-hydroxy-ethylaminocarbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example Sa are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 100 ~l of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, O.OOS M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ~95%.
Example 6a N-(3-mercapto-1-~carbonyl-gly-his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid monoamide 250 mg (1 mmol) of the N-(2-oxo-tetrahydrothiophene-3-yl)-thiodiglycolic acid monoamide produced in Example 3a are added in an argon atmosphere to a solution of 853 mg (1 mmol) of NH2-gly-his-leu-asp-ile-ile-trp (produced in a similar way as described by Barany and Merrifield, The Peptides: Analysis, Biology, Academic Press, New York 1980;
Stewart and Young, Solid Phase Peptides Syntheses, 2nd ed., Pierce Chemical W., Rockford, II, 1984) and 404 mg (4 mmol) _ 22 2156620 of triethyl amine in 100 ml of anhydrous dimethyl formamide. The resulting reaction mixture is stirred at room temperature for 12 hours. When the reaction is finished, the solution is filtered and the solvent removed under reduced pressure. The residual oiI is mixed three times with 50 ml of dimethyl formamide and evaporated each time. The residue is stirred up with 200 ml of anhydrous diethyl ether. A white solid material settles down which is filtered off. The material is recrystallized from mixtures of dimethyl formamide and diethyl ether for purification.
Yield: 282 mg (25.6~), white powder Analysis referring to the anhydrous substance:
Calc.: C 53.39 H 6.49 N 13.98 O 20.32 S 5.82 Found: C 53.17 H 6.63 N 13.74 S 5.61 Example 6b Technetium-99m complex of N-(3-mercapto-1-(carbonyl-gly-his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 6a are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50 ~1 of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solut-ion (5 mg/ml 0. 05 N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-l column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (7S/25)i 2.0 ml/min. Radiochemical purity is ~97%.
rD-13317WO
23 215662~
-Example 7 Accumulation of N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide, technetium-99m complex, in atherosclerotic vascular lesions of WHHL rabbit N-(2-mercapto-I-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide (produced according to Example 2a) is labelled as described in Example 3b. 99.9 GBq (2.7 mCi) of the substance label~led according to Example 3b were diluted to 1 ml with phosphate-buffered saline and administered via the ear vein to a narcotized WHHL rabbit, Rompun/Ketavet (1:2). The rabbit was killed 5 hours after the application, and an autoradiogram of the aorta as well as a Sudan(III) staining were carried out to visualize the atherosclerotic plaques (Figure 1). The accumulation factor between normal and atherosclerotic walls was between 3 and 8 depending on the thickness of the plaques (Sudan(III) staining).
It has been known for a long time that complexing agents for radioactive isotopes or their complexes with radio-active metals can be applied in radiodiagnostics and radio-therapy. Technetium-99m is the most frequently used radio-nuclide in radiodiagnostics because it is particularly wellsuited for in-vivo applications due to its favourable physical properties (no corpuscular radiation, low half-life of 6.02 h, goo~ detectability by 140 keV y-radiation) as well as its low biological half-life and easy availabil-ity. The first step of forming complexes of technetium-99m is to gain pertechnetate from a nuclide generator; it is then converted to a lower oxidation number using appro-priate reductants (such as SnCl2, S2O42-, etc.). This oxidation number is stabilize,d by an appropriate chelating agent. As technetium may have several oxidation numbers (+7 to -1) which may vehemently alter its pharmacological properties by changing the charge of the complex, it is necessary to provide chelating agents or complex ligands for technetium-99m that are capable of binding technetium 2S in a specific oxidation number safely, firmly and stably to prevent undesirable biodistribution due to in-vivo redox processes or release of technetium from the radiodiagnostic agent which would impede the safe diagnosis of the respec-tive diseases.
For example, cyclic amines (Troutner, D.E. et al.: J. Nucl.
Med. ~1, 443 (1980)) are regarded as suitable complexing agents for technetium and rhenium isotopes but their disadvantage is that they are only capable of binding technetium-99m in sufficient quantities from a pH value ~9.
N2O2 systems (Pillai, M.R.A., Troutner, D.E. et al.: Inorg.
~ ID-13317WQ
Chem., 29, 1850 (1990)) are in clinical use. Non-cyclic N4 systems such as HMPAO have the great disadvantage of low complex stability. Tc-99m-HMPA0 has to be applied immediately after labelling due to its low stability s (Ballinger, J. R. et al., Appl. Radiat. Isot. ~, 315 (1991); Billinghurst, M. W. et al., Appl. Radiat. Isot. 42, 607 (1991)) to keep the portion of decomposition products low which have different pharmacokinetic and excretion properties. Such radiochemical impurities make detection of the diseases to be diagnosed more difficult. Any coupling of these chelates or chelating agents with other substances that accumulate selectively in centres of diseases cannot be broken by simple means so that these normally spread unspecifically in the organism.
~, N2S2 chelating agents (Bormans, G. et al.: Nucl. Med.
Biol., 17, 499 (1990)) such as ethylene dicysteine (EC;
Verbruggen, A.M. et al.; J. Nucl. Med. 33, 551 (1992)) meet the requirement of sufficient stability of their respective technetium-99m complex but form radiodiagnostic agents of a purity greater than 69~ only~at pH values ~9 of the complexing medium. N3S systems (Fritzburg, A.; EPA 0 173 424 and EPA 0 250 013) yield stable technetium-99m complexes but have to be heated up to temperatures of about 100C to insert the radionuclide.
Another disadvantage of N2S2 and N3S systems is that they are discharged partially too rapidly and without specific accumulation by the organism. Thus they are only used clinically, though to a limited extent, in renal function diagnostics. In the last few years the demand has increased for radiodiagnostics that accumulate specifically in diseased tissues. This can be accomplished if one manages to link complexing agents easily with selectively accumu-lating substances while the latter retain their favourable complexing properties. But as it happens quite frequently that a certain reduction of complex stability is observed ID-l33l7Wo 2 15 6 fi 2 ~
after coupling the complexing agent to such a molecule by means of one of its functional groups, previous approaches to coupling chelating agents with substances that accumu-late selectively are hardly satisfactory because a quantity 5 of the isotope that is not tolerable with a view to diagnostics is released in vivo from the conjugate (Brechbiel, M. W. et al.; Inorg. Chem. 1986, ~, 2772). It is therefore necessary to produce bifunctional complexing agents that have functional groups to bind the desired metallic ion and one (or several other) functional groups to bind the select~vely accumulating molecule. Such bifunc-tional ligands allow specific, chemically defined bonding of technetium or rhenium isotopes to the most various bio-logical materials even in cases in which pre-labelling is ~5 applied. Some chelating agents coupled with monoclonal antibodies (e.g. EP Appl. 0 247 866 and EP Appl. 0 188 256) or fatty acids (EP Appl. 0 200 492) have been described.
But these chelating agents were based on the N2S2 systems mentioned above which are hardly appropriate due to their low stability. As both the properties of the substances that accumulate selectively and the mechanisms of accumulation are quite varied, one should be able to vary the chelating agent meant for coupling to adapt it to the physiological requirements of its coupling partner with regard to lipophilic or hydrophilic behaviour, membrane permeability or impermeability, etc.
It is therefore an object of this invention to provide stable complex compounds coupled with or capable of cou-pling with various compounds that accumulate selectively, and to provide such linkable chelating agents or complexes whose substituents show a wider range of chemical variation to be adaptable to the above requirements. It is another object of this invention to provide such compounds and pharmaceuticals containing these compounds, as well as methods for their production.
' ID-13317wo 215662~
_ 5 This problem is solved by the invention, surprisingly, in that the new, uncommon, bifunctional chelating agents with intermittent chalcogen atoms and their coupling products with compounds that accumulate selectively are excellently suited for producing radiodiagnostic and radiotherapeutic agents.
The subject matter of this invention are compounds of the general formula (I) M~- L (I) wherein M represents a radionuclide of Tc or Re and L represents a ligand of the general formula (II) B-Co-CRlR2-A-CR3R4-CooH (II) wherein A represents an O, S, or Se chalcogen atom, Rl, R2, R3, and R4 are same or different and represent a hydrogen atom and/or a branched or unbranched Cl-C6 alkyl resldue, B represents a residue -NH-CR5R6- (CR7R8)n=1 2-S-R9, wherein R5 and R6 are same or different and represent a hydrogen atom or an unbranched, branched, cyclic, or polycyclic Cl-C60 alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, 25 aryl, alkylaryl, or arylalkyl residue which may optionally be carry an additional hydroxy, oxo, oxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or alkoxy groups containing up to 20 carbon atoms, and/or may optionally be interrupted, and/or replaced, by one or several heteroatoms from the series of O, N, S, P, As, Se, 215662~
R7 and R8 are same or different and represent a hydrogen atom and/or a branched or unbranched Cl-C6 alkyl residue, R9 represents a hydrogen atom, a branched or unbranched Cl-C6 alkyl residue, or a sulfur protecting group, with Rs and R6, together with the groups that connect them, optionally forming a 4- to 8-membered ring which may optionally carry additional hydroxy, oxo, oxy, or alkoxy groups containing up to 6 carbon atoms.
Preferred compounds of the general formula (I) are characterized in that Rl, R2, R3, and R4 are hydrogen atoms, and that A is a sulfur atom.
Particularly prefer~ed compounds according to the invention of the general formula (I) are characterized in that RS and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom.
Another object of this invention is related to the new, bifunctional ligands with intermittent chalcogen atoms of the general formula (II) B-CO-CR1R2-A- CR3R4-COOH (II) wherein R1, R2, R3, R4, A and B have the meanings specified above.
Such ligands according to the invention of the general formula (II) are preferred in which A is a sulfur atom and Rl, R2, R3 and R4 are hydrogen atoms.
Particularly preferred compounds according to the invention of general formula (II) are characterized in that Rs and R6 are different, and that R6, R7 and Rs each represent a hydrogen atom.
Yet another object of this invention are conjugates containing compounds of the general formulae (I and/or II) ID-l33l7wo -and substances that accumulate selectively in diseased tissues, with a covalent bond existing between these sub-stances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups.
Particularly preferred conjugates according to the invention are characterized in that the substances that accumulate in dise~sed tissue are peptides such as endo-thelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
Other preferred embodiments of the conjugates according to the invention are characterized in that the peptides com-prise the following sequences or parts thereof:
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-~ I
phe-cys-his-leu-asp-ile-ile-trp, cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-~ I
tyr-cys-his-leu-asp-ile-ile-trp, cys-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr--phe-cys-his-leu-asp-ile-ile-trp, i rD-13317WO
-cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr--phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-i phe-cys-his-gln-asp-val-ile-trp, , ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, the partia~ sequence his-leu-asp-ile-ile-trp or the cyclic amino acid sequences Cyclo-(Dtrp-Dasp-pro-Dval-leu), Cyclo-(Dglu-ala-alloDile-leu-Dtrp).
ID-1~317Wo 9 21S6fi20 -The compounds according to the invention of the general formula (I) are produced by reacting technetium-99m in the form of pertechnetate or Re in the form of perrhenate in the presence of a reductant and, optionally, an auxiliary ligand, with a compound of the general formula (II) B-Co-CRlR2-A-CR3R4-CooH (II) wherein Rl, R2, R3, R4, A, and B, have the meanings specified above.
The ligands accordlng to the invention of the general for-mula (II) are produced by reacting compounds of the general formula (III) with compounds of the general formula (IV) according to the following reaction scheme:
CR1R2-A_CR3R4 + NH2 ~ CR5 R6 - ( CR7R8 ) n= l 2 ~ S - R9 1 5 C~- O CO
(III) (IV) B-Co-CRlR2-A-CR3R4-CooH (II) .
wherein Rl R2 R3, R4, R5, R6, R7, R8, R9, A, and B have the meanings specified above.
These reactions are carried out in polar and non-polar aprotic solvents such as dichloromethane, tetrahydrofurane, chloroform, 1,4-dioxane, DMF, or DMSO at temperatures between -30 and +100C; an auxiliary base is added to trap any acids that may be liberated. Among these bases could be, for example: tertiary amines, alkali and alkaline-earth hydroxides, alkali and alkaline-earth carbonates.
Another object of the present invention is a kit for pro-ducing radiopharmaceuticals consisting of a compound of the general formula (II) or a conjugate according to the invention containing compounds of the general formulae (I
~ ID-13317wo 21~6620 -and/or II) and substances that accumulate selectively in tissues, a reductant and, optionally, an auxiliary ligand, said agents being either dry or in solution, instructions for use including instructions for reacting the compounds described with technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.
Another object of this invention is a radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or athero-sclerotic plaques.JIt contains a compound of the generalformula (I) or a conjugate according to the invention containing compounds of the general formulae (I and/or II) and substances that accumulate selectively in tissues, optionally with the~adjuvants common in galenics; the compound is prepared in a kit using technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.
Yet another object of this invention is a method for carrying out radiodiagnostic examinations according to which the radiopharmaceutical formulation is applied at doses from 0.1 to 30 mCi, preferably from 0.5 to 10 mCi, per 70 kg of a patient's body weight, and that radiation emitted by the patient is recorded.
Many of the synthesized chelates that were labelled with Tc-99m or Re surprisingly showed a greater stability than comparable-N2S2 and N3S systems described in the litera-ture. For example, no decomposition products were found of a substance according to the invention (Examples 2a, 2b) coupled with a fatty alcohol even after 23 hours. It was also found in competition tests that the Tc-99m or-Re chelating agents complex better than comparable N2S2, N3S
and propylene aminoxium systems. The chelates and chelating agents described in the present invention are clearly bet-ter suited for diagnostic and therapeutic purposes than the systems known so far. It is a specific advantage of the 215662~
- chelating agents according to the invention that they may be synthesized without sulfur protecting groups This makes synthesis very simplei in addition, the compounds according to the invention, when radiolabelled, do not contain any other foreign molecules in the solutions used for radio-diagnostics or radiotherapy, for example, solutions to be administered intravenously. Biodistribution of the radio-pharmaceutical and thus the value of diagnostic information are frequently diminished by such foreign molecules. More-over, such ligands~or their coupling products with sub-stances that accumulate selectively in diseased tissues can be labelled very gently. The ligands according to the in-vention and their coupling products with substances that accumulate selectively in diseased tissues can be labelled at room temperature and at the physiological pH value without having to split off protecting groups using bases, acids, or other auxiliary substances known to a person skilled in the art. This guarantees that the very sensitive substances that accumulate selectively in diseased tissues are not modified chemically by such auxiliary substances, as such modification frequently reduces selective accumu-lation in diseased tissue and diminishes the value of radiodiagnostic information.
Sulfur protecting groups may be used here, of course, if the disadvantages described can be accepted. The groups are attached to sulfur atoms and split off according to methods known to a-person skilled in the art. The ways in which the substances that accumulate selectively in diseased tissues are bonded are also known to a person skilled in the art (e.g. Fritzberg et al.; J. Nucl. Med. ~, 7 (1987)), for example, by a reaction of electrophilic groups of the complex ligand with nucleophilic centres of the substances that accumulate selectively in diseased tissues. Otherwise, nucleophilic groups of the chelating agent are coupled with electrophilic groups of the substances that accumulate selectively in diseased tissues.
rD- 13317WO
21S66~0 The partners for coupling are, among others, various bio-molecules, ligands that bond to specific receptors which are capable of detecting tissue showing a modified receptor density. This includes peptides, steroid hormones, growth factors and neurotransmitters. Ways for improved diagnosis of carcinomas of the breast and the prostata were shown using ligands for steroid hormone receptors (S. J. Brandes and J. A. Katzenellenbogen, Nucl. Med. Biol. lS, S3, 1988).
Tumour cells sometimes show a modified density of receptors for peptide hormone~s or growth factors such as the epider-mal growth factor (EGF). The differences in concentration could be utilized for selective accumulation of cytostatic agents in tumour cells (E. Aboud-Pirak et al., Proc. Natl.
Acad. Sci. USA 86; 3778, 1989). Ligands for neuroreceptors labelled with positron-emitting isotopes were successfully used for the diagnosis of various brain diseases (J. J.
Forst, Trends in Pharmacol. Sci., 7, 490, 1989). Other biomolecules are metabolites that can be introduced into the metabolism of cells to make changes visible; this includes fatty acids, sacchar~des, peptides, and amino acids. Fatty acids that were coupled with the more unstable N2S2 chelating agents have been described in EPA 0 200 492.
Other metabolic products such as saccharides (desoxyglucose), lactate, pyruvate, and amino acids 2S (leucine, methylmethionine, glycine) were used in the PET
technique for visualizing changes in metabolic processes (R. Weinreich, Swiss Med., 8, 10, 1986). Likewise, non-biological substances such as misonidazol and its derivatives which bond irreversibly to cell components in tissues or parts of tissues with a reduced oxygen concentration, can be used for specific accumulation of radioactive isotopes and thus for the visualization of tumours or ischaemic regions (M. E. Shelton, J. Nucl. Med.
30; 3S1, 1989). Finally, bifunctional chelating agents may be coupled with monoclonal antibodies or their fragments.
Coupling products of the chelating agents according to the rD- 133 17WO
215fi620 ~_ 13 invention or their technetium-99m or Re complexes with fatty alcohols, fatty alcohol derivatives, or fatty amines and their derivatives, or with endothelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists have proved particularly favourable for the detection of atherosclerotic vascular diseases. These derivatives were applied to WHHL rabbits that had high LDL concentrations in their blood - and thus atherosclerotic lesions - due to a genetic defect of ~heir LDL receptor. Concentration quotients from 3 to 40 were found in atheromatose plaques as compared with undamaged tissue about 4 to 5 hours after i.v. application of the derivatives to WHHL rabbits. This allowed detection of atherosclerotic areas of vessels using the common methods of radiodiagnostics (e.g. a gamma scintillation camera). Only very late stages of atherogenesis could up to now be diagnosed by using more invasive methods (e.g. arteriography). The substances according to the invention provide the decisive advantage of being able to diagnose much earlier stages of athero-sclerosis using non-invasive methods.
It is unimportant whether the chelating agent is labelled with Tc-99m or Re before or after coupling with the selec-tively accumulating molecule. But if coupling takes place after complexing, it is a prerequisite that the reaction of the radioactive complex with the accumulating compound is rapid, gentle, and nearly quantitative, requiring no subsequent purification.
The radiopharmaceuticals of the invention are produced in a generally known way by dissolving the complexing agents according to the invention in an aqueous medium and adding a reductant, preferably tin(II) salts such as chloride or tartrate, optionally adding the adjuvants common in galenics, and subsequent sterile filtration. Among the suitable additives are physiologically tolerable buffers 21~662~
(such as tromethamine), small quantities of electrolytes (e.g. sodium chloride) or stabilizers (e.g. gluconate, phosphate, or phosphonate). The pharmaceutical according to the invention is either available as a solution or as lyophilizate and is mixed shortly before application with a solution of Tc-99m pertechnetate, eluated from commercial generators, or a perrhenate solution.
For in-vivo applications in nuclear medicine, the agents according to the invention are administered at doses from 1 x 10-5 to 5 x 104~nmol/kg of body weight, preferably from 1 x 10-3 to 5 x 102 nmol/kg of body weight. The amount of radioactivity per application, based on an average body weight of 70 kg, is between 0.05 and S0 mCi, preferably between 5 and 30 mCi`, for diagnostic applications. For therapeutic applications, doses applied are between 5 and 500 mCi, preferably from 10 to 350 mCi. Normally, 0.1 to 2 ml of a solution of the agents according to the invention is applied by intravenous, intra-arterial, peritoneal or intra-tumoral injection. The intravenous injection is preferred.
The following examples shall explain the object of this invention in greater detail.
rD-13317WO
21~6620 ~_ 15 Example la N-(2-mercapto-1-(methoXycarbonyl)-ethyl)-thiodiglycolic acid monoamide 13.21 g (0.1 mol) of thiodiglycolic acid anhydride are added by dropping, and in an argon atmosphere, to a solution of 17.16 g (0.1 mol) of cysteine methyl ester hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hourf.s at room temperature and washed with aqueous 2~ citric acid. After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 18.73 g (70.1%), white powder 15 Analysis referring to the anhydrous substance:
Calc.: C 35.95 H 4.90 N 5.24 0 29.93 S 23.99 Found: C 35.72 H 5.12 N 5.03 S 23.71 Example lb Technetium-99m complex of N-(2-mercapto-1-(methoxy-carbonyl)-ethyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example la are dissolved in 1.0 ml of 0.5 M phosphate buffer, pH 7.5.
50 ~l of this ligand solution are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and lO0 ~l of a pertechnetate solution (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient elution from 100~ A to 100~ B within 7.5 minutes ~eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
16 21~66zo -acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,98~.
Example 2a S N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide 1.32 g (10 mol) of thiodiglycolic acid anhydride dissolved in 50 ml of anhydrcus-dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 2.98 g (10 mmol) of cysteine decyl ester hydrochloride and 2.02 g (20 mol) of triethyl amine in 250 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2~ citric acid.
After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 3.37 g (85.6~), white powder Analysis referring to the anhydrous substance:
Calc.: C 51.88 H 7.94 N 3.56 O 20.33 S 16.29 Found: C 51.63 H 8.07 N 3.37 S 16.02 ~D- 133 17WO
Example 2b Technetium-99m complex of N-(2-mercapto-1-(decyloxy-carbonyl)-ethyl-thiodiglycolic acid diamide 10 mg of the ligand produced according to Example 2a are dissolved in 1.0 ml of O.S M phosphate buffer, pH 7.5.
50 ~1 of this ligand solution are mixed with 250 ~1 of phosphate buffer, pH 7.5, 50 ~1 of a deoxygenated aqueous ci~rate solution (50 mg/ml), 2.5 ~1 of a deoxygenated aqueous tin(II) chlorlde solution (5 mg/ml 0.05 N HCl), and 100 ~1 of a pertechnetate solution (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ~98%.
Example 3a N-(2-oxo-1-tetrahydrothiophene-3-yl)-thiodiglycolic acid monoamide 13.21 g (0.1 mmol) of thiodiglycolic acid anhydride dissolved in 250 ml of anhydrous dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 15.36 g (0.1 mol) of homocysteine thiolactone hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2~ citric acid. After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether.
Yield: 22.73 g (91.2~), white powder Analysis referring to the anhydrous substance:
18 21 S 6 ~ 2 0 Calc.: C 38.54 H 4.45 N 5.62 0 25.67 S 25.72 Found: C 38.37 H 4.68 N 5.41 S 25.47 Example 3b N-(3-mercapto-1-(octylaminoCarbonyl)-propyl)-thiodiglycolic mono~mide 30 ml of octyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglyco~ic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2~
citric acid and 200~ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separation, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 878 mg (23.2~), white powder Analysis referring to the anhydrous substance:
Calc.: C 50.77 H 7.99 N 7.40 0 16.91 S 16.94 Found: C 50.48 H 8.13 N 7.15 S 16.71 Example 3c Technetium 99-m complex of N-(3-mercapto-1-(octylamino-carbonyl)-propyl)-thiodiglycolic acid monsr ;de 10 mg of the ligand produced according to Example 3b are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex lg 2ls662a formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, O.oo5 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,95~ .
Example 4a N-(3-mercapto-1-(2,methoxyethylamino carbonyl)-propyl)-thiodiglycolic acid monoamide 30 ml of 2-methoxy ethyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 5 hours and evaporated under reduced pressure. The residue is mixed with 200 ml of aqueous 2~ citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separation, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 734 mg (22.6~), white powder Analysis referring to the anhydrous substance:
Calc. C 40.73 H 6.21 N 8.64 O 24.66 S 19.76 Found: C 40.47 H 6.49 N 8.38 S 19.51 ID-13317Wo 215662~
Example 4b Technetium-99m complex o~ N-(3-mercapto-l-(2-methyleth amino carbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 4a are S dissolved in 1.0 ml of ethanol. S0 ~1 of this ligand solu-tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50 ~1 of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~1 of a deoxygenated aqueous tin(II) chloride solution (S mg/ml O.OS N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci)~ After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, S ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100% B within 7.5 minutes (eluent A:-~odium hydrogenphosphate, 0.005 M, pH
lS 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ,95~ .
Example Sa N-(3-mercapto-1-(2-hydroxy-ethylamino carbonyl)-propyl)-thiodiglycolic acid monoamide 30 ml of 2-amino ethanol are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2 citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separa-tion, is dried above sodium sulfate. The solvent is evapo-rated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether.
Yield: 435 mg (14.0~), white powder ID- 133 17WO , 2 1 5 6 6 2 0 Analysis referring to the anhydrous substance:
Calc.: C 38.70 H 5.85 N 9.03 0 25.77 S 20.66 Found: C 38.38 H 5.74 N 8.91 S 20.43 Example 5b Technetium-99m complex of N-(3-mercapto-1-(2-hydroxy-ethylaminocarbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example Sa are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50~l of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 100 ~l of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, O.OOS M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is ~95%.
Example 6a N-(3-mercapto-1-~carbonyl-gly-his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid monoamide 250 mg (1 mmol) of the N-(2-oxo-tetrahydrothiophene-3-yl)-thiodiglycolic acid monoamide produced in Example 3a are added in an argon atmosphere to a solution of 853 mg (1 mmol) of NH2-gly-his-leu-asp-ile-ile-trp (produced in a similar way as described by Barany and Merrifield, The Peptides: Analysis, Biology, Academic Press, New York 1980;
Stewart and Young, Solid Phase Peptides Syntheses, 2nd ed., Pierce Chemical W., Rockford, II, 1984) and 404 mg (4 mmol) _ 22 2156620 of triethyl amine in 100 ml of anhydrous dimethyl formamide. The resulting reaction mixture is stirred at room temperature for 12 hours. When the reaction is finished, the solution is filtered and the solvent removed under reduced pressure. The residual oiI is mixed three times with 50 ml of dimethyl formamide and evaporated each time. The residue is stirred up with 200 ml of anhydrous diethyl ether. A white solid material settles down which is filtered off. The material is recrystallized from mixtures of dimethyl formamide and diethyl ether for purification.
Yield: 282 mg (25.6~), white powder Analysis referring to the anhydrous substance:
Calc.: C 53.39 H 6.49 N 13.98 O 20.32 S 5.82 Found: C 53.17 H 6.63 N 13.74 S 5.61 Example 6b Technetium-99m complex of N-(3-mercapto-1-(carbonyl-gly-his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 6a are dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50 ~1 of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated aqueous tin(II) chloride solut-ion (5 mg/ml 0. 05 N HCl), and 100 ~1 of a pertechnetate solu-tion (400-900 ~Ci). After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-l column, 5 ~m, 125 x 4.6 mm; gradient eluation from 100~ A to 100~ B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (7S/25)i 2.0 ml/min. Radiochemical purity is ~97%.
rD-13317WO
23 215662~
-Example 7 Accumulation of N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide, technetium-99m complex, in atherosclerotic vascular lesions of WHHL rabbit N-(2-mercapto-I-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide (produced according to Example 2a) is labelled as described in Example 3b. 99.9 GBq (2.7 mCi) of the substance label~led according to Example 3b were diluted to 1 ml with phosphate-buffered saline and administered via the ear vein to a narcotized WHHL rabbit, Rompun/Ketavet (1:2). The rabbit was killed 5 hours after the application, and an autoradiogram of the aorta as well as a Sudan(III) staining were carried out to visualize the atherosclerotic plaques (Figure 1). The accumulation factor between normal and atherosclerotic walls was between 3 and 8 depending on the thickness of the plaques (Sudan(III) staining).
Claims
C l a i m s 1. Compounds of the general formula (I) B-CO-CR1R2-A-CR3R4-COOH (I) wherein A represents an O, S, or Se chalcogen atom, R1, R2, R3, and R4 are same or different and repre-sent a hydrogen atom and/or a branched or unbranched C1-C6 alkyl residue, B represents a residue -NH-CR5R6-(CR7R8)n=1,2-S-R9, wherein R5 and R6 are same or different and represent a hydrogen atom or an unbranched, branched, cyclic, or polycyclic C1-C60 alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, aryl, alkylaryl, or arylalkyl residue which may optionally carry additional hydroxy, oxo, oxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or alkoxy groups containing up to 20 carbon atoms, and/or may optionally be interrupted, and/or replaced, by one or several heteroatoms from the series of O, N, S, P, As, Se, R7 and R8 are same or different and represent a hydrogen atom and/or a branched or unbranched C1-C6 alkyl residue, R9 represents a hydrogen atom, a branched or unbranched C1-C6 alkyl residue, or a sulfur protecting group, with R9 and R5, together with the groups that connect them, optionally forming a 4- to 8-membered ring which may optionally carry additional hydroxy, oxo, oxy, or alkoxy groups containing up to 6 carbon atoms.
their conjugates with substances that accumulate selectively in diseased tissues or tumours, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups and their complexes with radioisotopes of Tc or Re.
2. Compounds according to Claim 1, characterized in that R1, R2, R3 and R4 are hydrogen atoms and A is a sulfur atom.
3. Compounds according to Claims 1 or 2, characterized in that R5 and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom.
Compounds according to Claims 1 to 3, characterized in that the substances that accumulate in diseased tissue are peptides such as endothelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
5. Compounds according to Claims 1 to 4, characterized in that the peptides comprise the following sequences or parts thereof:
Method for the production of compounds according to Claim 1, characterized in that compounds of the general formula (II) are reacted with compounds of the general formula (III) according to the following reaction scheme:
+ NH2-CR5R6-(CR7R8)n=1,2-S-R9 (II) (III) --> B-CO-CR1R2-A-CR3R4-COOH (I) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, A and B have the meanings specified in Claim 1 and that optionally the compounds produced in this way are conjugated with substances that accumulate selectively in deseased tissues or tumours, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, anti-bodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups and that optionally the compounds and conjugates produced in this way are reacted with technetium -99m or Re in the form of pertechnetate or perrhenate in the presence of a reductant and, optionally, an auxiliary ligand.
7. Radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or atherosclerotic plaques characte-rized in that it contains a compound or a conjugate according to one of Claims 1 to 5.
their conjugates with substances that accumulate selectively in diseased tissues or tumours, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups and their complexes with radioisotopes of Tc or Re.
2. Compounds according to Claim 1, characterized in that R1, R2, R3 and R4 are hydrogen atoms and A is a sulfur atom.
3. Compounds according to Claims 1 or 2, characterized in that R5 and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom.
Compounds according to Claims 1 to 3, characterized in that the substances that accumulate in diseased tissue are peptides such as endothelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
5. Compounds according to Claims 1 to 4, characterized in that the peptides comprise the following sequences or parts thereof:
Method for the production of compounds according to Claim 1, characterized in that compounds of the general formula (II) are reacted with compounds of the general formula (III) according to the following reaction scheme:
+ NH2-CR5R6-(CR7R8)n=1,2-S-R9 (II) (III) --> B-CO-CR1R2-A-CR3R4-COOH (I) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, A and B have the meanings specified in Claim 1 and that optionally the compounds produced in this way are conjugated with substances that accumulate selectively in deseased tissues or tumours, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, anti-bodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups and that optionally the compounds and conjugates produced in this way are reacted with technetium -99m or Re in the form of pertechnetate or perrhenate in the presence of a reductant and, optionally, an auxiliary ligand.
7. Radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or atherosclerotic plaques characte-rized in that it contains a compound or a conjugate according to one of Claims 1 to 5.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4311023A DE4311023C2 (en) | 1993-03-31 | 1993-03-31 | Bifunctional chalcogen atom-interrupted chelating agents of type XN¶1¶S¶1¶O¶1¶ for radioactive isotopes, their metal complexes, processes for their preparation and pharmaceutical compositions containing them |
| DEP4311023.1 | 1993-03-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2156620A1 true CA2156620A1 (en) | 1994-10-13 |
Family
ID=6484687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002156620A Abandoned CA2156620A1 (en) | 1993-03-31 | 1994-03-29 | Chelating agents of the type xn1s1o for radioactive isotopes, metal complexes thereof, and their use in diagnosis and therapy |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0692976B1 (en) |
| JP (1) | JPH08508263A (en) |
| KR (1) | KR960700777A (en) |
| AT (1) | ATE179076T1 (en) |
| AU (1) | AU691806B2 (en) |
| CA (1) | CA2156620A1 (en) |
| DE (2) | DE4311023C2 (en) |
| HU (1) | HUT73665A (en) |
| NO (1) | NO953867L (en) |
| NZ (1) | NZ263794A (en) |
| WO (1) | WO1994022497A1 (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6051207A (en) * | 1996-10-28 | 2000-04-18 | Nycomed Imaging As | Contrast agents |
| US6322770B1 (en) | 1998-03-31 | 2001-11-27 | Dupont Pharmaceuticals Company | Indazole vitronectin receptor antagonist pharmaceuticals |
| US6511649B1 (en) | 1998-12-18 | 2003-01-28 | Thomas D. Harris | Vitronectin receptor antagonist pharmaceuticals |
| US6511648B2 (en) | 1998-12-18 | 2003-01-28 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6524553B2 (en) | 1998-03-31 | 2003-02-25 | Bristol-Myers Squibb Pharma Company | Quinolone vitronectin receptor antagonist pharmaceuticals |
| US6537520B1 (en) | 1998-03-31 | 2003-03-25 | Bristol-Myers Squibb Pharma Company | Pharmaceuticals for the imaging of angiogenic disorders |
| US6548663B1 (en) | 1998-03-31 | 2003-04-15 | Bristol-Myers Squibb Pharma Company | Benzodiazepine vitronectin receptor antagonist pharmaceuticals |
| US6558649B1 (en) | 1998-12-18 | 2003-05-06 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6569402B1 (en) | 1998-12-18 | 2003-05-27 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6794518B1 (en) | 1998-12-18 | 2004-09-21 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4311022C2 (en) * | 1993-03-31 | 1996-07-11 | Diagnostikforschung Inst | Bifunctional chalcogen atom-interrupted chelating agents of the type S¶3¶N¶2¶ for radioactive isotopes and their metal complexes, processes for their preparation and pharmaceutical compositions containing them |
| DE4310999C2 (en) * | 1993-03-31 | 1996-07-18 | Diagnostikforschung Inst | Bifunctional chalkogen atom-interrupted chelating agents of the type XN¶1¶S¶1¶X 'for radioactive isotopes and their metal complexes, processes for their preparation and pharmaceutical compositions containing them |
| DE4425778A1 (en) * | 1994-07-13 | 1996-01-18 | Diagnostikforschung Inst | Complex compounds for the diagnosis of vascular diseases |
| DE19652374A1 (en) * | 1996-12-04 | 1998-06-10 | Schering Ag | Use of endothelin conjugates in therapy, new endothelin conjugates, agents containing them, and processes for their preparation |
| PT2151250E (en) * | 2002-05-06 | 2013-12-09 | Endocyte Inc | Vitamin-targeted imaging agents |
| SE526214C2 (en) | 2003-02-28 | 2005-07-26 | Amersham Biosciences Ab | One way to generate metal chelating affinity ligands |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57188527A (en) * | 1981-04-21 | 1982-11-19 | Amersham Int Plc | Diagnosis of kidney function |
| US4980147A (en) * | 1984-06-25 | 1990-12-25 | University Of Utah Research Foundation | Radiolabeled technetium chelates for use in renal function determinations |
| EP0188256B1 (en) * | 1985-01-14 | 1991-08-21 | NeoRx | Metal radionuclide labeled proteins for diagnosis and therapy |
| ATE52516T1 (en) * | 1986-05-28 | 1990-05-15 | Mallinckrodt Inc | TECHNETIUM CHELATE FOR DETERMINING KIDNEY FUNCTION. |
| US4861869A (en) * | 1986-05-29 | 1989-08-29 | Mallinckrodt, Inc. | Coupling agents for joining radionuclide metal ions with biologically useful proteins |
| ES2033433T3 (en) * | 1987-07-16 | 1993-03-16 | Nycomed As | PROCEDURE TO PREPARE AMINOPOLICARBOXILIC ACIDS AND THEIR DERIVATIVES. |
| JPH0570481A (en) * | 1991-09-12 | 1993-03-23 | Hitachi Chem Co Ltd | Peptide and its salt |
-
1993
- 1993-03-31 DE DE4311023A patent/DE4311023C2/en not_active Expired - Lifetime
-
1994
- 1994-03-29 AT AT94912441T patent/ATE179076T1/en active
- 1994-03-29 EP EP94912441A patent/EP0692976B1/en not_active Expired - Lifetime
- 1994-03-29 CA CA002156620A patent/CA2156620A1/en not_active Abandoned
- 1994-03-29 AU AU65017/94A patent/AU691806B2/en not_active Ceased
- 1994-03-29 KR KR1019950703669A patent/KR960700777A/en not_active Application Discontinuation
- 1994-03-29 WO PCT/DE1994/000371 patent/WO1994022497A1/en active IP Right Grant
- 1994-03-29 HU HU9502868A patent/HUT73665A/en unknown
- 1994-03-29 JP JP6521542A patent/JPH08508263A/en active Pending
- 1994-03-29 NZ NZ263794A patent/NZ263794A/en unknown
- 1994-03-29 DE DE59408142T patent/DE59408142D1/en not_active Expired - Lifetime
-
1995
- 1995-09-29 NO NO953867A patent/NO953867L/en unknown
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6051207A (en) * | 1996-10-28 | 2000-04-18 | Nycomed Imaging As | Contrast agents |
| US6548663B1 (en) | 1998-03-31 | 2003-04-15 | Bristol-Myers Squibb Pharma Company | Benzodiazepine vitronectin receptor antagonist pharmaceuticals |
| US6322770B1 (en) | 1998-03-31 | 2001-11-27 | Dupont Pharmaceuticals Company | Indazole vitronectin receptor antagonist pharmaceuticals |
| US7052673B2 (en) | 1998-03-31 | 2006-05-30 | Bristol-Myers Squibb Pharma Company | Pharmaceuticals for the imaging of angiogenic disorders |
| US6524553B2 (en) | 1998-03-31 | 2003-02-25 | Bristol-Myers Squibb Pharma Company | Quinolone vitronectin receptor antagonist pharmaceuticals |
| US6537520B1 (en) | 1998-03-31 | 2003-03-25 | Bristol-Myers Squibb Pharma Company | Pharmaceuticals for the imaging of angiogenic disorders |
| US6569402B1 (en) | 1998-12-18 | 2003-05-27 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6558649B1 (en) | 1998-12-18 | 2003-05-06 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6511648B2 (en) | 1998-12-18 | 2003-01-28 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6683163B2 (en) | 1998-12-18 | 2004-01-27 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6689337B2 (en) | 1998-12-18 | 2004-02-10 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6743412B2 (en) | 1998-12-18 | 2004-06-01 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6794518B1 (en) | 1998-12-18 | 2004-09-21 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6818201B2 (en) | 1998-12-18 | 2004-11-16 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US7018611B2 (en) | 1998-12-18 | 2006-03-28 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US6511649B1 (en) | 1998-12-18 | 2003-01-28 | Thomas D. Harris | Vitronectin receptor antagonist pharmaceuticals |
| US7090828B2 (en) | 1998-12-18 | 2006-08-15 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US7321045B2 (en) | 1998-12-18 | 2008-01-22 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
| US7332149B1 (en) | 1998-12-18 | 2008-02-19 | Bristol-Myers Squibb Pharma Company | Vitronectin receptor antagonist pharmaceuticals |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994022497A1 (en) | 1994-10-13 |
| NZ263794A (en) | 1996-10-28 |
| HU9502868D0 (en) | 1995-11-28 |
| AU6501794A (en) | 1994-10-24 |
| KR960700777A (en) | 1996-02-24 |
| HUT73665A (en) | 1996-09-30 |
| NO953867L (en) | 1995-11-23 |
| EP0692976B1 (en) | 1999-04-21 |
| AU691806B2 (en) | 1998-05-28 |
| DE59408142D1 (en) | 1999-05-27 |
| EP0692976A1 (en) | 1996-01-24 |
| DE4311023A1 (en) | 1994-10-06 |
| JPH08508263A (en) | 1996-09-03 |
| DE4311023C2 (en) | 1996-05-02 |
| NO953867D0 (en) | 1995-09-29 |
| ATE179076T1 (en) | 1999-05-15 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |