WO1994017036A1 - S-lipophilic aliphatic carbonyl [n-mercaptoacyl-(amino acid or peptide)] compounds as antihypertensive agents - Google Patents

S-lipophilic aliphatic carbonyl [n-mercaptoacyl-(amino acid or peptide)] compounds as antihypertensive agents Download PDF

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WO1994017036A1
WO1994017036A1 PCT/EP1993/000147 EP9300147W WO9417036A1 WO 1994017036 A1 WO1994017036 A1 WO 1994017036A1 EP 9300147 W EP9300147 W EP 9300147W WO 9417036 A1 WO9417036 A1 WO 9417036A1
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alkyl
compound
acid
hydrogen
phenyl
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PCT/EP1993/000147
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French (fr)
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Marie-Claude Fournie-Zaluski
Bernard-Pierre Roques
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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Priority to PCT/EP1993/000147 priority Critical patent/WO1994017036A1/en
Priority to AU34940/93A priority patent/AU3494093A/en
Publication of WO1994017036A1 publication Critical patent/WO1994017036A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06078Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C327/00Thiocarboxylic acids
    • C07C327/20Esters of monothiocarboxylic acids
    • C07C327/32Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043Leu-amino acid
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052Val-amino acid
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr

Definitions

  • the present invention is directed to novel compounds useful as
  • the invention is also directed to pharmaceutical
  • compositions containing these novel compounds the preparation of these novel compounds, and methods for their pharmaceutical use.
  • U.S. Patent No. 4,053,651 discloses inhibitors of peptidyldipeptidase A (ACE) (EC3.4-15.1 ) wherein the mercapto moiety therein is substituted by hydrogen, lower alkanoyl wherein lower alkyl is defined as up to C 7 or benzoyl, or the mercapto moieties in two of the inhibitor molecules form a dimer by a disulfide linkage.
  • ACE peptidyldipeptidase A
  • German Patent No. DE 3,819,539 A1 discloses inhibitors of neutral endopeptidase (NEP) (EC 3.4-24.11) wherein the mercapto moiety therein is substituted by hydrogen, phenyl C 2-5 alkanoyl, thiophenyl C 2-5 alkanoyl, furanyl C 2-5 alkanoyl, pyridinyl C 2-5 alkanoyl or cycloalkyl C 2-5 alkanoyl.
  • NEP neutral endopeptidase
  • 4,684,660 discloses inhibitors of ACE wherein the mercapto moiety therein is substituted by hydrogen or C 2-7 alkanoyl, or the mercapto moieties in two of the inhibitor molecules form a dimer by a disulfide linkage.
  • This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • This reference also does not disclose that the ACE inhibitors also have NEP inhibitory activity.
  • French Patent FR 8320024 (2,556,721 ) discloses inhibitors of
  • U.S. Patent No. 4,248,883 discloses inhibitors of ACE wherein the mercapto moiety therein is substituted by hydrogen, G 4 CO-, G 5 S- or
  • G 4 represents a lower alkyl group, a lower alkoxy group, a phenyl group, a substituted phenyl group, a phenyl-lower alkyl group, a substituted phenyl- lower alkyl group, a phenyl-lower alkoxy group, a substituted phenyl-lower alkoxy group, a phenoxy group, or a substituted phenoxy group;
  • G5 represents a lower alkyl group, a phenyl group, a substituted phenyl group, a phenyl-lower alkyl group, a substituted phenyl-lower alkyl group,
  • G 6 represents a hydrogen atom or a lower alkyl group
  • G 7 represents a lower alkyl group, a phenyl group or a substituted phenyl group
  • X represents an oxygen or sulfur atom.
  • the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • the ACE inhibitors also have NEP inhibitory activity.
  • U.S. Patent No. 4,474,799 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted by hydrogen, C 1-4 alkanoyl or benzoyl.
  • the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • the NEP inhibitors possess antihypertensive activity.
  • U.S. Patent No. 4,798,904 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted by hydrogen, phenyl C 2-5 alkanoyl, substituted phenyl C 2-5 alkanoyl, thiophenyl C 2-5 alkanoyl, furanyl C 2-5 alkanoyl, or pyridinyl C 2-5 alkanoyl.
  • This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • This reference also does not disclose that the NEP inhibitors possess
  • U.S. Patent No. 4,513, 009 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted hydrogen, C 1-4 alkanoyl, halo substituted C 1-4 alkanoyl, benzhydrylcarbonyl, benzoyl, phenyl C 2-5 alkanoyl, halo substituted phenyl C 2-5 alkanoyl, hydroxy C 2-5 alkanoyl or amino C 2-5 alkanoyl.
  • the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • the NEP inhibitors possess antihypertensive activity.
  • U.S. Patent No. 3,246,025 discloses mercaptopropionic acid derivatives that are useful for treating drug intoxication and poisoning wherein the mercapto moiety therein is substituted by hydrogen or a radical that is readily convertible to hydrogen, e.g., benzoyl or nitrobenzoyl.
  • This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical.
  • This reference also does not disclose that the mercaptopropionic acid
  • NEP neutral endopeptidase
  • enkephalinase since the enzyme degrades the enkephalins which are endogenous opiate peptides of morphine receptors. These inhibitors are disclosed as useful analgesics. This reference does not disclose that NEP inhibitors possess antihypertensive activity. In Nature, 288, 286-288 (1980), Roques et al.
  • (R,S)-(2- mercaptomethyl-3-phenylpropionyl)glycine has an inhibitory power at a nanomolar concentration and behaves as an analgesic in potentiating the action of the enkephalins. This reference does not disclose that thiorphan possesses antihypertensive activity.
  • U.S. Patent No. 4,879,309 discloses that compounds of formulae HS- CH 2 -CH(Q 2 )-CONH-CH(Q 1 )-COOQ 3 and HS-(CH 2 ) m -CH(Q 2 )-CONH-CH(Q 1 )- CO-A-COOQ 3 are useful for augmenting natriuresis and diuresis which thereby aids in reducing blood pressure.
  • This reference does not disclose that the inhibitors possess antihypertensive activity separate from their natriuretic and diuretic effects.
  • the present invention is directed to an S-lipophilic aliphatic carbonyl [N-mercaptoacyl(amino acid or peptide)] compound that, at very low
  • the invention is directed also to the preparation of the compound, pharmaceutical compositions containing it, and methods for its pharmaceutical use.
  • FIGURE Figure 1 is a time course plot of the percent of in vivo inhibition of ACE by the mixed NEP/ACE inbitors: N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)alanine as a mixture of four stereoisomers (- ⁇ -); N-(2-adamantylthiomethyl-1-oxo-3- phenylbutyl)alanine as a mixture of four stereoisomers (- O -); and
  • Alkyl means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain.
  • “Lower alkyl” means alkyl having about 1 to about 8 carbon atoms.
  • “Higher alkyl” means alkyl having about 10 to about 20 carbon atoms.
  • the alkyl may be optionally substituted with one or more alkyl group substituents which may be the same or different, where "alkyl group substituent” includes halo, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy
  • alkoxycarbonyl, oxo and cycloalkyl "Branched" means that a lower alkyl group such as methyl, ethyl or propyl is attached to a linear alkyl chain.
  • exemplary alkyl groups include methyl, ethyl, i-propyl, n -butyl, t-butyl, n -pentyl, heptyl, octyl, decyl or dodecyl.
  • exemplary substituted alkyl groups include cyclohexylm ethyl and trifluoromethyl.
  • Preferred alkyl groups include the lower alkyl groups.
  • Alkenyl means an alkyl group containing a carbon-carbon double bond. "Lower alkenyl” means alkenyl having about 1 to about 8 carbon atoms. “Higher alkenyl” means alkenyl having about 10 to about 20 carbon atoms. The alkenyl may be optionally substituted with one or more "alkyl group substituent”.
  • alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl or tetradecadieneyl.
  • Preferred alkenyl groups include the lower alkenyl groups.
  • Alkynyl means an alkyl group containing a carbon-carbon triple bond.
  • “Lower alkynyl” means alkynyl having about 1 to about 8 carbon atoms.
  • “Higher alkynyl” means alkynyl having about 10 to about 20 carbon atoms.
  • the alkynyl may be optionally substituted with one or more "alkyl group substituent”.
  • alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl or dodecynyl.
  • Preferred alkynyl groups include the lower alkynyl groups.
  • Cycloalkyl means a non-aromatic mono or multi cyclic ring system of about 4 to about 10 carbon atoms.
  • the cycloalkyl may be optionally partially unsaturated.
  • the cycloalkyl may be also optionally substituted with an aryl group substituent, oxo or alkylene.
  • Preferred mono cyclic cycloalkyl rings include cyclopentyl, cyclohexyl or cycloheptyl.
  • Preferred multi cyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, noradamantyl, norbornane, bicyclo[2.2.2.]-oct-5-ene, cis-5-norborene, 5-norborene, (1 R)-(-)-myrtentane, norbane or anti-3-oxo-tricyclo[2.2.1.0 2,6 ]- heptane.
  • Alkyloxymethyl means an alkyl-O-CH 2 - group wherein alkyl is as previously described.
  • exemplary alkyloxymethyl groups include methoxymethyl, ethoxymethyl, n-propoxymethyl, Apropoxymethyl, n-butoxym ethyl and
  • alkyloxymethyl it is preferred that the alkyl contains about 1 to about 6 carbon atoms.
  • Aryl means aromatic carbocyclic radical containing about 6 to about 10 carbon atoms.
  • the aryl may be optionally substituted with one or more aryl group substituents which may be the same or different, where "aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano,
  • aryl include phenyl or naphthyl or substituted phenyl or substituted naphthyl
  • Heteroaryl means about a 5- to about a 10- member monocyclic or multicyclic ring system wherein one or more of the atoms in the ring system is an element other than carbon chosen amongst nitrogen, oxygen or sulfur.
  • the heteroaryl may be optionally substituted by one or more aryl group substituents.
  • Exemplary heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N- methylpyrrolyl, quinolinyl, and isoquinolinyl.
  • Heterocyclyl means an about 4 to about 10 member monocyclic or multicyclic ring system wherein one or more of the atoms in the ring system is an element other than carbon chosen amongst nitrogen, oxygen or sulfur.
  • the heterocyclyl may be optionally substituted by one or more alkyl group
  • heterocyclyl moieties include quinuclidine, and .
  • Preferred substituents of the heterocyclyl include hydroxyl, alkoxy containing about 1 to about 4 carbon atoms, trifluoromethyl, fluorine or alkylene of about 3 to about 4 carbons which alkylene when substituted on the heterocyclyl forms a saturated or unsaturated
  • acyl have an alkyl containing about 1 to about 3 carbon atoms in the alkyl group.
  • exemplary groups include acetyl, propanoyl, 2-methylpropanoyl, butanoyl or palmitoyl.
  • “Aroyl” means an aryl-CO- group wherein alkyl is as previously described.
  • Exemplary groups include benzoyl and 1- and 2-naphthoyl.
  • Alkoxy means an alkyl-O- group wherein alkyl is as previously
  • alkoxy groups include methoxy, ethoxy,
  • Aryloxy means an aryl-O- group wherein the aryl group is as previously described.
  • exemplary aryloxy groups include phenoxy and naphthoxy.
  • Alkylthio means an alkyl-S- group wherein alkyl is as previously described.
  • Exemplary alkylthio groups include methylthio, ethylthio,
  • Arylthio means an aryl-S- group wherein the aryl group is as previously described.
  • Exemplary arylthio groups include phenthio and naphththio.
  • Alkyl means an aryl-alkyl- group wherein aryl and alkyl are as previously described Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
  • Alkyloxy means an aralkyl-O- group wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxy group is benzyloxy.
  • Alkylthio means an aralkyl-S- group wherein the aralkyl group is as previously described.
  • An exemplary aralkylthio group is benzylthio.
  • Dialkylamino means an -NZZ' group wherein both Z and Z' are alkyl groups as previously described.
  • Exemplary alkylamino groups include ethylmethylamino, dimethylamino and diethylamino.
  • Alkoxycarbonyl means an alkyl-O-CO- group.
  • alkoxycarbonyl groups include methoxy- and ethoxy- carbonyl.
  • Aryloxycarbonyl means an aryl-O-CO- group.
  • aryloxycarbonyl groups include phenoxy- and naphthoxy- carbonyl.
  • Alkoxycarbonyl means an aralkyl-O-CO- group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl means an H 2 N-CO- group.
  • Alkylcarbamoyl means an Z'ZN-CO- group wherein one of Z and Z' is hydrogen and the other of Z and Z' is alkyl as defined previously.
  • Dialkylcarbamoyl means an Z'ZN-CO- group wherein both Z and Z' are alkyl as defined previously.
  • acyloxy means an acyl-O- group wherein acyl is as defined previously.
  • acylamino means an acyl-NH- group wherein acyl is as defined previously.
  • Aroylamino means an aroyl-NH- group wherein aroyl is as defined previously.
  • Alkylene means a straight or branched bivalent hydrocarbon chain group having from about 1 to about 8 carbon atoms. The alkylene group may be also optionally unsaturated. There may be optionally inserted along the alkylene group one or more oxygen or sulphur atoms, or substituted nitrogen atoms wherein the substituent is alkyl as previously described.
  • alkylene group has about 2 to about 3 carbon atoms.
  • Halo mean fluoro, chloro or bromo.
  • amino Acid may be naturally occurring or synthetic.
  • exemplary amino acids include proline, hydroxy proline, 4,4-ethylenedioxyproline, methoxyproline, thiazolidinecarboxylic acid, trytophane, glycine alanine, leucine, isoleucine, valine, tyrosine, O-benzylserine, 2-carboxylpiperidine, 1-amino-1-phenylacetic acid and 1-amino-1-indan-2-ylacetic acid.
  • Peptide may be about 2 to about 6 amino acid residues bonded by peptide linkages. Preferred are peptides having about 2 to about 3 amino acid residues.
  • Lipophilic aliphatic carbonyl radical means higher alkyl carbonyl, higher alkenyl carbonyl, higher alkynyl carbonyl, multi cycloalkyl carbonyl or multicyclic heterocyclyl carbonyl wherein the alkyl, alkenyl, alkynyl, cycloalkyl and heterocyclyl are as previously described.
  • Multi cycloalkyl carbonyl radicals are preferred, and more preferred are the multi cycloalkyl carbonyl radicals wherein the multi cycloalkyl moiety thereof has bridging carbons, for example
  • exemplary lipophilic aliphatic carbonyl radical groups include 1- or 2-adamantylacetyl, 3-methyl adamant-1-ylacetyl, 3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl, camphaneacetyl, noradamantylacetyl, norbornaneacetyl, bicyclo[2.2.2.]-oct-5- eneacetyl, 1 -methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl, cis-5-norborene-endo- 2,3-dicarbonyl, 5-norbomen-2-yl acetyl, (1 R)-(-)-myrtentaneacetyl,
  • N-mercaptoacyl(amino acid or peptide) compounds which are derivatized by an S-lipophilic aliphalic carbonyl group to form the compounds according to the invention, include the N-mercaptoacyl(amino acid or peptide) compounds disclosed in the following: U.S. Patent Nos. 4,053,651 ; 4,684,600; 4,248,883; 4,474,799; 4,798,904; 4,513,009; 4,879,309; German Patent No. DE 3,819,539 A1; French Patent No. FR 8320024 (2,556,721); European Patent Nos. EP 0038758; 0 136883; PCT/EP Application Nos.
  • N-mercaptoacyl(amino acid or peptide) compounds N-mercaptoacyl(amino acid or peptide) compounds.
  • Preferred mercaptoalkanoyl (amino acid or peptide) species which are derivatized by an S-Iipophilic aliphalic carbonyl group to form the
  • R is a lipophilic aliphatic carbonyl radical;
  • R 1 is aryl or heteroaryl;
  • R 2 is alkyl, or alkylene attached to the moiety and R 1 ;
  • R 3 is hydrogen, alkyl, aryl, alkoxy or aryloxy
  • R' is hydrogen, alkyl, aralkyl, acyl or aroyl; or a pharmaceutically acceptable salt thereof.
  • R 2 is alkylene
  • the alkylene preferably attaches at an ortho position on R 1
  • R is higher alkyl carbonyl or multi cycloalkyl carbonyl;
  • R 1 is aryl or heteroaryl;
  • R 2 lower alkyl, or alkylene attached to the moiety and R 1 ;
  • R 3 is hydrogen, lower alkyl, aryl or heteroaryl, lower alkoxy or aryloxy
  • R' is hydrogen, lower alkyl, aryl lower alkyl, acyl or aroyl.
  • R is adamantoyl or palmitoyl
  • R 1 is phenyl, 2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl radical, optionally substituted with one or more identical or different aryl group substituents chosen from halo, hydroxy, C 1-4 acyloxy, C 1-4 alkoxy, phenoxy, phenylthio and amino radicals, C 1-4 dialkylamino, methylenedioxy and ethylenedioxy;
  • R 2 is methyl or trifluoromethyl radical, or -CH 2 -Y-, -Y-CH 2 - or
  • R 1 is phenyl, 2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl
  • Y is oxygen, sulphur or nitrogen substituted by methyl, or alkylene is optionally substituted with a methyl
  • R 3 is hydrogen, trifluoromethyl, C 1-8 alkoxy, phenoxy, phenyl or thienyl or C 1-8 alkyl optionally substituted with phenyl, hydroxy, C 1-4 alkoxy, phenoxy, C 1-4 alkylthio, phenylthio, benzyloxy or benzylthio, wherein the phenyl and the phenyl portions of the phenoxy, phenylthio, benzyloxy or benzylthio radicals are optionally substituted with one or more identical or different aryl group
  • R' is hydrogen, methyl, ethyl, benzyl, cyclohexylmethyl, palmitoyl or pamoyl radical.
  • R' is hydrogen, methyl, ethyl, benzyl, cyclohexylmethyl, palmitoyl or pamoyl radical.
  • Presently preferred also is a compound of the formula (II)
  • R is a lipophilic aliphatic carbonyl ra dical
  • R 4 and R 5 are independently hydrogen, alkyl, aryl, aralkyl, alkoxy, alkyloxymethyl or aralkyloxy;
  • a 1 and A 2 are independently hydrogen, alkyl, or together with -CH- R 4 or -CH-R 5 , respectively, form phenyl, or alkylene which together with -CH- R 4 or -CH-R 5 , respectively, form benzocycloalkyl;
  • R 6 is hydrogen or alkyl
  • G 7 is cycloalkyl, aralkyl or aryloxymethyl or alkoxymethyl, or R 6 and G 7 and the carbon and nitrogen atoms through which R 6 and G 7 are attached taken together form heterocyclyl; and
  • R" is hydrogen, alkyl, aralkyl, cycloalkyl lower alkyl or palmitoyl; or a pharmaceutically acceptable salt thereof.
  • R is higher alkyl carbonyl or multi cycloalkyl carbonyl;
  • R 4 and R 5 are independently hydrogen, lower alkyl, phenyl, aryl lower alkyl, lower alkoxy, lower alkyloxymethyl or aryl lower alkyloxy;
  • a 1 and A 2 are independently hydrogen, lower alkyl or together with -CH- R 4 or -CH-R 5 , respectively, form phenyl, or alkylene which together with -CH- R 4 or -CH-R 5 , respectively, form benzocycloalkyl;
  • R 6 is hydrogen or lower alkyl
  • G 7 is cycloalkyl, aryl lower alkyl or aryloxymethyl or lower alkoxymethyl, or R 6 and G 7 and the carbon and nitrogen atoms through which R 6 and G 7 are attached taken together form heterocyclyl; and
  • R" is hydrogen, lower alkyl, aryl lower alkyl, cycloalkylmethyl or palmitoyl.
  • R is adamantoyl or palmitoyl
  • R 4 and R 5 are independently lower alkyl, lower alkoxy, phenyl, benzyl or benzyloxy
  • a 1 and A 2 are independently hydrogen, methyl, or together with -CH- R 4 or -CH-R 5 , respectively, form phenyl, or alkylene together with -CH- R 4 or -CH-R 5 , respectively, form indanyl
  • G 7 is benzyl wherein the phenyl ring is optionally substituted by hydroxyl, or R 6 and G 7 and the carbon and nitrogen atoms through which R 6 and G 7 are attached taken together form a saturated 5 or 6 member heterocyclyl; and
  • R" is hydrogen.
  • the compounds of the present invention may be useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof. All forms are within the scope of the invention. Where the compound of the invention is substituted with an acidic moiety, base addition salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of the free acid form.
  • the bases which can be used to prepare the base addition salts preferably include those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose cations are non-toxic to the animal organism in pharmaceutical doses of the salts, so that the inhibition of NEP and/or ACE, and preferably beneficial mixed NEP and ACE inhibition, inherent in the compound of the invention is not vitiated by side effects ascribable to the cations.
  • compositions within the scope of the invention include those derived from the following bases: sodium hydroxide, potassium
  • hydroxide calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, omithine, choline, N.N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
  • Metal salts of compounds of the present invention may be obtained by contacting a hydroxide, carbonate or similar reactive compound of the chosen metal in an aqueous solvent with the free acid form of the compound.
  • the aqueous solvent employed may be water or it may be a mixture of water with an organic solvent, preferably an alcohol such as methanol or ethanol, a ketone such as acetone, an aliphatic ether such as tetrahydrofuran, or an ester such as ethyl acetate.
  • Such reactions are normally conducted at ambient temperature but they may, if desired, be conducted with heating.
  • Amine salts of compounds of the present invention may be obtained by contacting an amine in an aqueous solvent with the free acid form of the compound.
  • Suitable aqueous solvents include water and mixtures of water with alcohols such as methanol or ethanol, ethers such as tetrahydrofuran, nitriles such as acetonitrile, or ketones such as acetone.
  • Amino acid salts may be similarly prepared.
  • Preferred base addition salts have a cation selected from the group consisting of ammonium, sodium, calcium, protonated N-methyl-D-glucamine, protonated lysine, protonated arginine and protonated dicyclohexylamine.
  • acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form.
  • the acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non- toxic to the animal organism in pharmaceutical doses of the salts, so that the inhibition of NEP and/or ACE, and preferably beneficial mixed NEP and ACE inhibition, inherent in the compound of the invention is not vitiated by side effects ascribable to the anions.
  • salts within the scope of the invention are those derived from the following acids: mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like.
  • the corresponding acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, malonate,
  • methanesulfonate methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.
  • the acid addition salts of the compounds of this invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, wherein case the salt separates directly or can be obtained by concentration of the solution.
  • the compound according to the invention contains several asymmetric centers and may exist in the form of pure stereoisomers or in the form of mixtures of stereoisomers.
  • the carbon atoms marked by an asterisk (*) designate asymmetric centers.
  • configurations at the carbon atoms marked by an asterisk relate: sequentially startin from the carbon atom closest to the O-terminal side of the molecule, i.e., the amino acid residue.
  • Preferred compounds according to the invention include those wherein any of the carbon atoms marked by an asterisk (*) have the S configuration.
  • Preferred also include the compounds of formula (I) according to the invention having a stereochemical designation selected from the group consisting of R,R,S, S,R,S, S,S,S and R,S,S form, and it is more preferred having a stereochemical designation S,S,S or R,S,S.
  • Preferred compounds of formula II according to the invention are those wherein the carbon atoms of the principal chain marked by an asterisk (*) have the L configuration.
  • the S-lipophilic aliphatic carbonyl is S-lipophilic aliphatic carbonyl
  • N-mercaptoacyl(amino acid or peptide)] compound may be prepared by acylating the appropriate N-mercaptoacyl(amino acid or peptide) with the appropriate lipophilic aliphatic carbonyl halide.
  • the reaction may be carried out in the absence or presence of a base such as aqueous sodium hydroxide or pyridine, and may be carried out under an inert atmosphere such as nitrogen. It is also preferred that the reaction medium should be degassed to prevent the mercapto compound from forming a disulfide linked dimer.
  • a base such as aqueous sodium hydroxide or pyridine
  • N-(mercaptoacyl)amino acids may be prepared by hydrolyzing the product formed by the acylation of an amino acid of formula (III)
  • the hydrolysis is
  • the acylation is performed in the presence of a condensing agent such as dicyclohexylcarbodiimide, optionally in the presence of 1-hydroxybenzo-triazole.
  • a condensing agent such as dicyclohexylcarbodiimide
  • 1-hydroxybenzo-triazole optionally in the presence of 1-hydroxybenzo-triazole.
  • thioacetic acid or thiobenzoic acid is used as a sulphur derivative of formula (V).
  • the acrylic acid of formula (VI) may be obtained by saponification of a corresponding ester in a basic medium.
  • the methyl or ethyl ester is used.
  • H 2 C C- COOR 8 (VII) wherein R 1 and R 2 are defined as above and R 6 preferably represents an alkyl radical containing 1 to 4 carbon atoms, may be obtained by a Mannich reaction on the monoester of formula (VIII)
  • the Mannich reaction is performed by means of formaldehyde in the presence of a secondary amine such as diethylamine at a temperature of about 20°C.
  • the monoester of formula (VIII) is obtained by monosaponification in a basic medium of a malonic ester of formula (IX)
  • R 1 , R 2 and R 8 are defined as above.
  • the malonic ester of formula (IX) may be obtained by the action of a halide of formula (X)
  • Halo- N (X) wherein R 1 and R 2 are defined as above and Halo represents a halide atom, preferably a bromide atom, on an alkyl malonate anionised beforehand, for example by means of an alkali metal alcoholate optionally prepared in situ.
  • the malonic ester of formula (IX) may also be obtained by condensation of a ketone of formula (XI)
  • R 1 and R 2 are defined as above, with an alkyl malonate anionised beforehand as described above, to form the product of formula (XII) 1 8 8 XII
  • R 1 , R 2 and R 8 are defined as above, which is reduced catalytically, for example with hydrogen in the presence of palladium on charcoal, to the product of formula (IX).
  • the product of formula (VII) may also be obtained by a Wittig reaction with formaldehyde on a phosphonate of formula (XIII)
  • the reaction is generally performed in the presence of a strong base such as sodium hydride.
  • N-(mercaptoacyl)peptides may be prepared by hydrolyzing the product formed the acylation of a dipeptide of the formula
  • a 2 , R 2 , R 3 , R 4 and R' are as defined above, by an acid of the formula
  • R LA , A 1 and R 1 are as defined above, under the usual conditions used in peptide chemistry and described, for example, by Bodansky et al., "Peptide Synthesis", J. Wiley and Sons Edit.
  • the hydrolysis is preferably carried out in an alkaline medium, working in an inert medium so as to avoid oxidation of the mercapto group.
  • the acid reactant may be prepared from the corresponding ⁇ -amino acid by halogenating deamination according to Fischer, Ann., 357, 1-24 (1907), followed by nucleophilic substitution of the halogen atom.
  • Lipophilic aliphatic carbonyl halides may be prepared from the following lipophilic aliphatic carboxylic compounds when they are in the acid or anhydride forms: camphorcarboxylic acid, camphanecarboxylic acid, 3-methyl adamantyl carboxylic acid, noradamantyl carboxylic acid, norbornane carboxylic acid, 3- methyl 3-bromo-1-adamantyl carboxylic acid, methyl 1-methoxybicyclo[2.2.2.]- oct-5-ene-2-carboxylate, mono methyl cis-5-norborene-endo -2,3-dicarboxylate, methyl-5-norborene-2,3-dicarboxylic anhydride, (1R)-(-)-myrtentoic acid, cis-5- norbornene-endo -2,3-dicarboxylic acid, 5-norbornen-2-yl acetate, 2- norbaneacetic acid, anti-3-o
  • the resolved forms of the compounds according to the invention may be prepared by standard practices known to those skilled in the art such as fractional crystallization and column chromatography.
  • compounds of formula (IV) such as an acylthioalkanoic acid may be resolved by fractional recrystallization with optically active bases such as methylbenzylamine or 1-(1- naphthyl)ethylamine and then the resolved compounds of formula (IV) may be reacted with an optically active amino acid according to the acylation reaction described above to yield a resolved N-(mercaptoacyl)amino acid.
  • Ethyl malonate (4.1 mL) is added to a solution of sodium ethoxide prepared from sodium metal (0.75 g) and ethanol (40 mL).
  • 1-Bromo-1- phenylethane (6 g) is added at 0°C and the mixture is then stirred for 20 hours at 30°C.
  • the residue is taken up with water (100 mL) and then extracted three times with ethyl acetate (75 mL).
  • the organic phase is washed with water and then with saturated sodium chloride solution and finally dried over magnesium sulphate.
  • the product thereby obtained (2.2 g) is treated with of sodium hydroxide (1.5 eq) in an acetone/water (2:1 by volume) mixture overnight at a temperature of about 20°C.
  • the mixture is concentrated to dryness and the residue is then taken up with water (30 mL).
  • the aqueous phase is acidified to pH 2 by adding hydrochloric acid.
  • the diethyl 2-(1-phenylethyl)phosphonoacetate (40.3 g) is dissolved in 37 % formaldehyde (65 mL), and potassium carbonate (51 g) is then added.
  • N-(2-Mercaptomethyl-1-oxo-3-phenylbutyl)tyrosine 1 g of the product obtained in Example G is dissolved in an degassed acetone/water (2:1 by volume) mixture. 1M sodium hydroxide (4 eq) is added at 0°C and under a nitrogen atmosphere. The mixture is stirred for 5 hours at a temperature of about 20°C. After filtering and acidifying to pH 1 , the product is extracted three times with degassed chloroform (15 mL). The organic phase is dried over sodium sulphate.
  • EXAMPLE K N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)-O-benzylserine Benzyl Ester Using the procedure described in Example G, starting with
  • ATBA-I 2-Acetylthiomethyl-3-phenylbutanoic acid (5 g) is dissolved in ethanol (50 mL) and (R)-(+)-1-(1-naphthyl)ethylamine (3.4 g) is added to the ethanol solution. Ethyl ether (50 mL) is then added. The mixture is cooled in an ice bath and after scratching the reaction vessel a white precipitate is formed. The solution is stored at about 0°C for about 18 hours. The precipitate is filtered and washed with ethyl ether.
  • the colorless crystalline product is filtered, washed with ethyl ether and dried to yield 2 g of ATBA-III salt.
  • the product is recrystallized twice from a mixture of ethyl acetate/ethanol (2:1) (25 mL) to yield 1 g of ATBA-III salt.
  • the free acid ATBA-III is regenerated by contacting the ATBA-III salt with aqueous acid (10% hydrochloric acid) and extracting with methylene chloride. The methylene is evaporated and 0.67 g of ATBA-III is obtained as a colorless crystalline solid.
  • hydroxybenzotnazole (0.11 g) and N-methylmorpholine (0.19 g) are added to the solution and the mixture is stirred at 20°C for 18 hours.
  • the solvent is removed in vacuo, the residue is contacted with aqueous acid (10% hydrochloric acid) and extracted with ethylacetate.
  • the organic phase is separated and washed consecutively with water (20 mL) saturated sodium bicarbonate solution (20 mL) and water (20 mL).
  • the organic phase is dried and evaporated to yield a light yellow gum.
  • the gum is chromatographed on silica gel using a mixture of hexane/ethyl acetate (1 :1) to yield a colorless gum (ATBAT-IV) (0.26 g; 65%) having the following
  • Example AF(a) Using the procedure of Example AF(a), except starting with the product (ATBA-I) of Example AE(a), the product (ATBAT-I) is obtained after crystallization having the characteristics: m.p. 135-137°C.
  • the diastereomers of N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine benzyl ester are also obtained by column chromatography using Hewlett- Packard ODS Hypersil column (5 ⁇ , 200 x 4.6 mm i.d.).
  • the product of Example G is dissolved in methanol and placed on the column.
  • a mobile phase of methanol/water (62:38 by volume) with 0.2% trifluoroacetic acid is employed in effecting the separation.
  • the flow rate is 1.0 mL/min.
  • the product is detected at UV of 215 nm.
  • the retention times of the four diastereomers are 22.7, 24.75, 28.5 and 30 minutes respectively.
  • Lithium hydroxide hydrate (18 mg) is dissolved in solvent comprising tetrahydrofuran/methanol/water (1 :1:1 by volume). The solvent is purged with nitrogen gas.
  • the product (ATBAT-IV) (21 mg) of Example AF(a)is added to the solution and stirred for 75 minutes at 20°C.
  • the solution is then diluted with water (5 mL), washed twice with chloroform (3 mL), acidified with 10% hydrochloric acid (2 mL) and extracted five times with chloroform (4 mL).
  • the combined organic phases are dried over magnesium sulphate and evaporated to leave 16 mg of a colorless gum (MTBT-I) having > 95% purity.
  • MTBT-I colorless gum
  • Example AH(a) Using the procedure of Example AH(a), except starting with the product (ATBAT-II) of Example AF(b), 9.7 mg the product (MTBT-II) is obtained after crystallization having > 98% purity.
  • R f 0.25 [methylene chloride/methanol/acetic acid (9:1 :05 by volume)].
  • the residue obtained is dissolved in ethyl acetate and then washed successively with a 10% citric acid solution (w/v), with water, with a 10% sodium carbonate solution (w/v), with water, then with a saturated sodium chloride solution.
  • the organic phase is dried over sodium sulphate. After filtration and concentration to dryness, the residue is purified by
  • the product obtained above is dissolved in degassed methanol and a 1 M sodium hydroxide solution (3 equivalents) is added at 0°C.
  • the mixture is stirred for 2 hours at a temperature close to 20°C and then a partition is carried out between degassed water and degassed ethyl acetate.
  • the organic phase is washed with a saturated aqueous solution of sodium chloride, dried over sodium sulphate, filtered and then concentrated under reduced pressure.
  • the residue obtained is chromatographed on silica gel, eluting with a methylene chloride/methanol/acetic acid mixture (9:0.5:0.5 by volume).
  • N-(2- mercapto-3-phenylpropanoyl)-Val-Tyr is thus obtained with a yield of 65% in the form of a white solid having the following characteristics: high performance liquid chromatography: retention time: 8 minutes [acetonitrile/0.05%
  • N-(2-Mercapto-3-phenylpropanoyl)-lle-Tyr-OMe The coupling of 2-acetylthio-3-phenylpropanoic acid with the protected dipeptide lle-Tyr-OMe is carried out under the conditions described in Example BA.
  • N-(2-Acetylthio-3-phenylpropanoyl)-lle-Tyr-OMe is obtained with a yield of 75% in the form of an oily product.
  • R f 0.17 [hexane/ethyl acetate (5:5 by volume)].
  • Deprotection is carried out under the conditions of Example BA.
  • N-(2- Mercapto-3-phenylpropanoyl)-lle-Tyr-OMe is thus obtained with a yield of 70% in the form of a white solid melting at 185°C.
  • R f 0.28 [methylene
  • N-(2-Mercapto-3-phenylpropanoyl)-lle-Pip is thus obtained with a yield of 65% in the form of a white solid.
  • R f 0.48 [methylene chloride/methanol/acetic acid (9:0-5:0-25 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH 3 ( lle)]; 1.02, 1.50 and 2.05 [CH 2 ⁇ , ⁇ and ⁇ (Pip)]; 2.6 (HS); 2.80 and 3.05 [CH 2 ⁇ (Phe)]; 3.7 [CH 2 ⁇ (Pip)]; 4.20, 4.70 and 4.95 (CH ⁇ ; 7.20 [Ar (Phe)]; 8.20 and 8.40 (NH); 12.78 (COOH).
  • R f 0.53 [methylene chloride/methanol (9:1 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH 3 (Nle)]; 1.25 and 1.50 [CH 2 ⁇ ,g and ⁇ (Nle)]; 2.00 (HS); 2.75 and 2.95 [CH 2 ⁇ (Phe + Tyr)]; 3.70 and 4.20 [CH ⁇ (Nle, Phe, Tyr)]; 6.60 and 6.95 [Ar (Tyr)]; 7.20 [Ar (Phe)]; 8.10 and 8.20 (NH); 9.15 [OH (Tyr)]; 12.3 (COOH); high performance liquid chromatography:
  • Example BA N-(2-Acetylthic-4-phenylbutanoyl)-Val-Tyr-OCH 3 is thus obtained with a yield of 82% in the form of an oily product.
  • R f 0.49 [cyclohexane/ethyl acetate (5:5 by volume)].
  • N-(2-Mercapto-4-phenylbutanoyl)-Val-Tyr is thus obtained with a yield of 98% in the form of a white product melting at 105°C.
  • R f 0.65 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]; high performance liquid chromatography: retention time: 9.10 minutes[acetonitrile/0.05% trifluoroacetic acid (45:55 by volume)]; proton nuclear magnetic resonance spectrum
  • N-(2-Acetylthio-3-methylbutanoyl)-lle-Tyr-OCH 3 is obtained with a yield of 85% in the form of an oily product.
  • R f 0.28 [n-hexane/ethyl acetate (4:6 by volume)].
  • Example N The product (1.7 g) of Example N is dissolved in degassed water (18 mL). To the solution under a nitrogen atmosphere and at 0°C is added a solution 1.3 X 10 -2 M sodium hydroxide (2.2 eq) and adamantoyl chloride (1.33 g, 1.1 eq). The mixture is stirred for four hours at ambient temperature. The mixture is then acidified to pH 3 with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with water and then a saturated NaCI solution. The organic phase is dried over anhydrous Na 2 SO 4 , filtered and evaporated to dryness. A white solid (2 g) is obtained in a yield of 74% having the following characteristics: m.p.
  • Compounds within the scope of the present invention when administered to mammals, are potent ACE and/or NEP inhibitors; preferably antihypertensives due to double inhibitory action where they inhibit both NEP and ACE.
  • the ACE inhibitors within the scope of the present invention are capable of blocking the increase in the blood pressure caused by an increase in the vascular resistance and the blood volume due to the formation of angiotensin II from angiotensin I since ACE inhibitors block the production of angiotensin II.
  • the NEP inhibitors within the scope of the present invention regulate auricular natriuretic peptide which is implicated in the regulation of arterial pressure.
  • This peptide is liberated by the heart, endowed with a vasodilatory property and capable of controlling diuresis and natriuresis.
  • Auricular natriuretic peptide is inactivated by NEP in the peripheral tissues.
  • NEP and ACE may be used to alleviate hypertension of various origins without the co-administration of other diuretics.
  • compounds according to the present invention are effective in the treatment of congestive heart failures and various types of hypertension, in particular hypertension connected with an increase in the blood volume.
  • the antihypertensive properties of the present compounds are:
  • Table III presents selected pharmacological data for the N- mercaptoacyl (amino acid or peptide) compounds having both ACE and NEP inhibitory properties. These compounds may be acylated with an S-lipophilic aliphatic acyl radical to form compounds within the scope of the present invention.
  • the results in Table III also gives the results with N-(2-mercaptomethyl-3-phenyl-1-oxopropyl)glycine (thiorphan), which is very closely related structure to the N-mercaptoacylamino acid portion of the compound of formula I, for comparison to show that thiorpan manifests activity only against NEP.
  • This invention is directed to the unexpected finding that protection of the thiol group was protected by a lipohilic aliphatic carbonyl results in compounds have an increased ACE and/or NEP inhibitory activity over the compounds that are acylated with an acylating group, e.g., acetyl, have lower lipophilic character.
  • mice administered by i.v. route to a mouse.
  • the mouse is killed 15 minutes
  • the kidney rapidly set apart, and homogenized at 4°C in a Tris HCI buffer 50 mM (pH 7.4).
  • the homogenate is filtered and the radioactivity bound to the filter is measured by liquid scintillation.
  • the non-specific binding is determined after co-injection of [3 H]HACBOGIy with 10,000 equivalents of the specific NEP inhibitor, retrothiorphan (Roques et al., PNAS, 1983, 80, 3178- 3182).
  • the inhibition of NEP in the kidney is expressed as the difference (in %) between specific [ 3 H]HACBOGIy binding in the absence (controls injected with saline) and presence of inhibitor.
  • the inhibition values in Table IV are the mean value of three determinations.
  • Example 3 The protocol in Example 3 is used also to determine NEP inhibition except for the following: the selective tritiated ACE inhibitor is
  • [ 3 H]Trandaloprilate (0.5 ⁇ Ci); and the non-specific binding is determined by the co-administration of [ 3 H]Trandaloprilate with 1000 eq. of captopril.
  • the NEP inhibition values are given in Table IV above.
  • ACE inhibition is determined at 30, 60, 120 and 240 minutes for N-(2-acetylthiomethyl-1-oxo-3- phenylbutyl)alanine as a mixture of four stereoisomers (- ⁇ -), N-(2- adamantylthiomethyl-1-oxo-3-phenylbutyl)alanine as a mixture of four stereoisomers (- O -) and the single stereoisomer (S,S,S)-N-(2-adamantyl- thiomethyl-1-oxo-3-phenylbutyl)alanine (- ⁇ -).
  • Figure 1 graphically represents the results and shows that the adamantylcarbonyl derivatized compounds exhibit unexpectedly greater inhibition than the acetylated compound.
  • the products according to the invention may generally be administered orally or parenterally for the treatment of patients suffering from hypertension.
  • the products according to the invention, in base or salt form may be presented in forms permitting administration by the most suitable route and the invention also relates to pharmaceutical compositions containing at least one product according to the invention which are suitable for use in human or veterinary medicine.
  • These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients.
  • the adjuvants comprise, inter alia, diluents, sterile aqueous media and the various non-toxic organic solvents.
  • the compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or
  • suspensions injectable solutions, elixirs or syrups, and can contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, or stabilizers in order to obtain pharmaceutically acceptable preparations.
  • excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets.
  • lactose and high molecular weight polyethylene glycols When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as ethanol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
  • suspensions or solutions of the products according to the invention in sesame oil, groundnut oil or olive oil or aqueous solutions of propylene glycol, as well as sterile aqueous solutions of the pharmaceutically acceptable salts are used.
  • the solutions of the salts of the products according to the invention are especially useful for administration by intramuscular or subcutaneous injection.
  • the aqueous solutions also be used.
  • compositions of the salts in pure distilled water may be used for intravenous administration with the proviso that their pH is suitably adjusted, that they are judiciously buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride and that they are sterilized by heating or
  • the doses used in the methods according to the invention are those which lead to a maximal therapeutic effect until an improvement is obtained.
  • the doses used are those which are therapeutically effective for lowering blood pressure during the treatment of hypertension.
  • the doses administered orally are between 0.1 and 100 mg/kg, and preferably between 1 and 10 mg/kg, and those administered intravenously are between 0.01 and 10 mg/kg, and preferably between 0.1 and 5 mg/kg, on the
  • the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health and other characteristics which can influence the efficacy of the medicinal product.
  • the products according to the invention may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the active product may be administered orally 1 to 4 times per day. It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.
  • the products according to the invention may be used in injectable form in emergency cases of acute hypertension. Such a treatment may be followed by an intravenous perfusion of the active product so as to obtain and maintain the desired therapeutic effect.

Abstract

The present invention is directed to an S-lipophilic aliphatic carbonyl [N-mercaptoacyl(amino acid or peptide)] compound of formula (I) or formula (II) wherein R is a lipophilic aliphatic carbonyl radical; R1 is aryl or heteroaryl; R2 is alkyl or alkylene attached to the (a) moiety and R1; R3 is hydrogen, alkyl, aryl, alkoxy or aryloxy; R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkoxy, alkyloxymethyl or aralkyloxy; A1 and A2 are independently hydrogen, alkyl, or toghether with -CH-R4 or -CH-R5, respectively, form phenyl, or alkylene which together with -CH-R4 or -CH-R5, respectively, form benzocycloalkyl; R6 is hydrogen or alkyl; R7 is cycloalkyl, aralkyl or aryloxymethyl or alkoxymethyl, or R6 and R7 and the carbon and nitrogen atoms through which R6 and R7 are attached taken together form heterocyclyl; and R' is hydrogen, alkyl, aralkyl, cycloalkyl, lower alkyl or palmitoyl; or a pharmaceutically acceptable salt thereof; that, at very low concentrations inhibits NEP and/or ACE, and is useful in treating hypertension; preferably by inhibiting both ACE and NEP. The invention is directed also to the preparation of the compound, pharmaceutical compositions containing it, and methods for its pharmaceutical use.

Description

S-Lipophilic aliphatic carbonyl [N-mercaρtoacyl-(amino acid or peptide)] compounds as antihypertensive agents Field of the Invention
The present invention is directed to novel compounds useful as
antihypertensives. The invention is also directed to pharmaceutical
compositions containing these novel compounds, the preparation of these novel compounds, and methods for their pharmaceutical use.
Recent Developments
U.S. Patent No. 4,053,651 discloses inhibitors of peptidyldipeptidase A (ACE) (EC3.4-15.1 ) wherein the mercapto moiety therein is substituted by hydrogen, lower alkanoyl wherein lower alkyl is defined as up to C7 or benzoyl, or the mercapto moieties in two of the inhibitor molecules form a dimer by a disulfide linkage. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the ACE inhibitors also have NEP inhibitory activity.
German Patent No. DE 3,819,539 A1 discloses inhibitors of neutral endopeptidase (NEP) (EC 3.4-24.11) wherein the mercapto moiety therein is substituted by hydrogen, phenyl C2-5 alkanoyl, thiophenyl C2-5 alkanoyl, furanyl C2-5 alkanoyl, pyridinyl C2-5 alkanoyl or cycloalkyl C2-5 alkanoyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the NEP inhibitors possess antihypertensive activity. U.S. Patent No. 4,684,660 discloses inhibitors of ACE wherein the mercapto moiety therein is substituted by hydrogen or C2-7 alkanoyl, or the mercapto moieties in two of the inhibitor molecules form a dimer by a disulfide linkage. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the ACE inhibitors also have NEP inhibitory activity. French Patent FR 8320024 (2,556,721 ) discloses inhibitors of
enkephalinase wherein the mercapto moiety therein is substituted by hydrogen, C2-6 alkanoyl or arylcarbonyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the NEP inhibitors possess
antihypertensive activity.
U.S. Patent No. 4,248,883 discloses inhibitors of ACE wherein the mercapto moiety therein is substituted by hydrogen, G4CO-, G5S- or
Figure imgf000004_0002
wherein
G4 represents a lower alkyl group, a lower alkoxy group, a phenyl group, a substituted phenyl group, a phenyl-lower alkyl group, a substituted phenyl- lower alkyl group, a phenyl-lower alkoxy group, a substituted phenyl-lower alkoxy group, a phenoxy group, or a substituted phenoxy group; G5 represents a lower alkyl group, a phenyl group, a substituted phenyl group, a phenyl-lower alkyl group, a substituted phenyl-lower alkyl group,
Figure imgf000004_0001
or an amino(-carboxy)lower alkyl group; G6 represents a hydrogen atom or a lower alkyl group; G7 represents a lower alkyl group, a phenyl group or a substituted phenyl group; X represents an oxygen or sulfur atom. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclosethat the ACE inhibitors also have NEP inhibitory activity. U.S. Patent No. 4,474,799 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted by hydrogen, C1-4 alkanoyl or benzoyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the NEP inhibitors possess antihypertensive activity.
U.S. Patent No. 4,798,904 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted by hydrogen, phenyl C2-5 alkanoyl, substituted phenyl C2-5 alkanoyl, thiophenyl C2-5 alkanoyl, furanyl C2-5 alkanoyl, or pyridinyl C2-5 alkanoyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the NEP inhibitors possess
antihypertensive activity.
U.S. Patent No. 4,513, 009 discloses inhibitors of NEP wherein the mercapto moiety therein is substituted hydrogen, C1-4 alkanoyl, halo substituted C1-4 alkanoyl, benzhydrylcarbonyl, benzoyl, phenyl C2-5 alkanoyl, halo substituted phenyl C2-5 alkanoyl, hydroxy C2-5 alkanoyl or amino C2-5 alkanoyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the NEP inhibitors possess antihypertensive activity.
U.S. Patent No. 3,246,025 discloses mercaptopropionic acid derivatives that are useful for treating drug intoxication and poisoning wherein the mercapto moiety therein is substituted by hydrogen or a radical that is readily convertible to hydrogen, e.g., benzoyl or nitrobenzoyl. This reference does not disclose that the mercapto group may be derivatized by a lipophilic aliphatic carbonyl radical. This reference also does not disclose that the mercaptopropionic acid
derivatives possess NEP inhibitory or antihypertensive activities.
European Patent EP 0,038,758 describes products which are inhibitors of neutral endopeptidase (NEP) (EC 3.4-24.11). NEP is alternatively referred to as "enkephalinase" since the enzyme degrades the enkephalins which are endogenous opiate peptides of morphine receptors. These inhibitors are disclosed as useful analgesics. This reference does not disclose that NEP inhibitors possess antihypertensive activity. In Nature, 288, 286-288 (1980), Roques et al. claim that (R,S)-(2- mercaptomethyl-3-phenylpropionyl)glycine (thiorphan) has an inhibitory power at a nanomolar concentration and behaves as an analgesic in potentiating the action of the enkephalins. This reference does not disclose that thiorphan possesses antihypertensive activity.
Other inhibitors of enkephalinase, endowed with analgesic properties, are the subject of European Patent EP 0,136,883. This reference does not disclose that the NEP inhibitors possess antihypertensive activity.
U.S. Patent No. 4,879,309 discloses that compounds of formulae HS- CH2-CH(Q2)-CONH-CH(Q1)-COOQ3 and HS-(CH2)m-CH(Q2)-CONH-CH(Q1 )- CO-A-COOQ3 are useful for augmenting natriuresis and diuresis which thereby aids in reducing blood pressure. This reference does not disclose that the inhibitors possess antihypertensive activity separate from their natriuretic and diuretic effects.
Koehn et al., J. Biol. Chem., 262, 11623-11627 (1987) and S. L
Stephenson and A. J. Renny, Biochem. J., 243, 183-187 (1987) have reported that auricular natriuretic peptide which is liberated by the heart, particularly in cardiac insufficiency, and which augments natriuretic, diuretic and vasodilator effects, is inactivated by the peripheral enzyme EP 24.11. Thus, the inhibitor of NEP, thiorphan, and certain of its derivatives are capable of augmenting the half-life of circulating auricular natriuretic peptide and thereby aids in reducing blood pressure in the rat. [G. Olins et al., Moll. Cell. Endocrinol., 61, 201-208 (1989); A.A. Seymour et al., Hypertension, 14, 87-97 (1989)]. However, none of these references disclose that the NEP inhibitors possess ACE inhibition mediated antihypertensive properties exhibited by inhibitors of ACE activity.
Furthermore, in clinical trials, the inhibitors of neutral endopeptidase, such as thiorphan, produce natriuresis and diuresis without any significant hypotensive effect, except in certain sick people showing cardiac or renal insufficiency. A.M. Richards et al., J. Gin. Endocrinol. Metab., 72, 1317-1322 (1991); A.M. Richards et al., Circulation Res., 71, 1501-1507 (1992); E.P.
Kromer et al., Am. J. Hypertens., 4, 460-463 (1991); D.B. Northridge et al., Am. J. Hypertens., 3, 682-687 (1990). B.P. Roques et al., Trends in Pharmacological Sciences, 11 (6) 1989, suggested combining in one molecule the properties of inhibition (1) of NEP, in order to potentiate the natriuretic, diuretic and vasorelaxant effects of atrial natriuretic peptide, and (2) of ACE, to block the hypertensive effects induced by angiotensin II formation. The reference does not disclose molecules capable of acting with both of these two enzymes at low concentrations.
SUMMARY OF THE INVENTION
The present invention is directed to an S-lipophilic aliphatic carbonyl [N-mercaptoacyl(amino acid or peptide)] compound that, at very low
concentrations, inhibits NEP and/or ACE and is useful in treating hypertension; preferably by inhiting both ACE and NEP. The invention is directed also to the preparation of the compound, pharmaceutical compositions containing it, and methods for its pharmaceutical use.
BRIEF DESCRIPTION OF THE FIGURE Figure 1 is a time course plot of the percent of in vivo inhibition of ACE by the mixed NEP/ACE inbitors: N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)alanine as a mixture of four stereoisomers (-□ -); N-(2-adamantylthiomethyl-1-oxo-3- phenylbutyl)alanine as a mixture of four stereoisomers (- O -); and
N-(2-adamantylthiomethyl-1-oxo-3-phenylbutyl)alanine as a single
stereoisomers (-■ -) at a dose of 2.6 x 10-5 mole/Kg p.o.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Patient" embraces both human beings and other mammals. The "*" designation on the carbons in the compound according to the invention represents that the carbons are chiral.
"Alkyl" means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain. "Lower alkyl" means alkyl having about 1 to about 8 carbon atoms. "Higher alkyl" means alkyl having about 10 to about 20 carbon atoms. The alkyl may be optionally substituted with one or more alkyl group substituents which may be the same or different, where "alkyl group substituent" includes halo, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy
alkoxycarbonyl, oxo and cycloalkyl. "Branched" means that a lower alkyl group such as methyl, ethyl or propyl is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, i-propyl, n -butyl, t-butyl, n -pentyl, heptyl, octyl, decyl or dodecyl. Exemplary substituted alkyl groups include cyclohexylm ethyl and trifluoromethyl. Preferred alkyl groups include the lower alkyl groups.
"Alkenyl" means an alkyl group containing a carbon-carbon double bond. "Lower alkenyl" means alkenyl having about 1 to about 8 carbon atoms. "Higher alkenyl" means alkenyl having about 10 to about 20 carbon atoms. The alkenyl may be optionally substituted with one or more "alkyl group substituent".
Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl or tetradecadieneyl. Preferred alkenyl groups include the lower alkenyl groups.
"Alkynyl" means an alkyl group containing a carbon-carbon triple bond. "Lower alkynyl" means alkynyl having about 1 to about 8 carbon atoms. "Higher alkynyl" means alkynyl having about 10 to about 20 carbon atoms. The alkynyl may be optionally substituted with one or more "alkyl group substituent".
Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl or dodecynyl. Preferred alkynyl groups include the lower alkynyl groups.
"Cycloalkyl" means a non-aromatic mono or multi cyclic ring system of about 4 to about 10 carbon atoms. The cycloalkyl may be optionally partially unsaturated. The cycloalkyl may be also optionally substituted with an aryl group substituent, oxo or alkylene. Preferred mono cyclic cycloalkyl rings include cyclopentyl, cyclohexyl or cycloheptyl. Preferred multi cyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, noradamantyl, norbornane, bicyclo[2.2.2.]-oct-5-ene, cis-5-norborene, 5-norborene, (1 R)-(-)-myrtentane, norbane or anti-3-oxo-tricyclo[2.2.1.02,6]- heptane.
"Alkyloxymethyl" means an alkyl-O-CH2- group wherein alkyl is as previously described. Exemplary alkyloxymethyl groups include methoxymethyl, ethoxymethyl, n-propoxymethyl, Apropoxymethyl, n-butoxym ethyl and
heptoxymethyl. When G7 in the compound of formula (II) herein is
alkyloxymethyl, it is preferred that the alkyl contains about 1 to about 6 carbon atoms.
"Aryl" means aromatic carbocyclic radical containing about 6 to about 10 carbon atoms. The aryl may be optionally substituted with one or more aryl group substituents which may be the same or different, where "aryl group substituent" includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano,
alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene or -NZZ' where Z and Z' are independently hydrogen, alkyl, aryl, or aralkyl. Exemplary aryl include phenyl or naphthyl or substituted phenyl or substituted naphthyl
"Heteroaryl" means about a 5- to about a 10- member monocyclic or multicyclic ring system wherein one or more of the atoms in the ring system is an element other than carbon chosen amongst nitrogen, oxygen or sulfur. The heteroaryl may be optionally substituted by one or more aryl group substituents. Exemplary heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N- methylpyrrolyl, quinolinyl, and isoquinolinyl.
"Heterocyclyl" means an about 4 to about 10 member monocyclic or multicyclic ring system wherein one or more of the atoms in the ring system is an element other than carbon chosen amongst nitrogen, oxygen or sulfur. The heterocyclyl may be optionally substituted by one or more alkyl group
substituents or alkylene. Exemplary heterocyclyl moieties include quinuclidine,
Figure imgf000009_0001
and . Preferred substituents of the heterocyclyl include hydroxyl, alkoxy containing about 1 to about 4 carbon atoms, trifluoromethyl, fluorine or alkylene of about 3 to about 4 carbons which alkylene when substituted on the heterocyclyl forms a saturated or unsaturated
hydrocarbon ring having about 5 to about 6 members. "Acyl" means an alkyl-CO- group wherein alkyl is as previously described.
Preferred acyl have an alkyl containing about 1 to about 3 carbon atoms in the alkyl group. Exemplary groups include acetyl, propanoyl, 2-methylpropanoyl, butanoyl or palmitoyl. "Aroyl" means an aryl-CO- group wherein alkyl is as previously described.
Exemplary groups include benzoyl and 1- and 2-naphthoyl.
"Alkoxy" means an alkyl-O- group wherein alkyl is as previously
described. Exemplary alkoxy groups include methoxy, ethoxy,
ti-propoxy, Apropoxy, n-bustoxy an d heptoxy.
"Aryloxy" means an aryl-O- group wherein the aryl group is as previously described. Exemplary aryloxy groups include phenoxy and naphthoxy. "Alkylthio" means an alkyl-S- group wherein alkyl is as previously described. Exemplary alkylthio groups include methylthio, ethylthio,
i-propylthio and heptylthio.
"Arylthio" means an aryl-S- group wherein the aryl group is as previously described. Exemplary arylthio groups include phenthio and naphththio.
"Aralkyl" means an aryl-alkyl- group wherein aryl and alkyl are as previously described Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
"Aralkyloxy" means an aralkyl-O- group wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.
"Aralkylthio" means an aralkyl-S- group wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio. "Dialkylamino" means an -NZZ' group wherein both Z and Z' are alkyl groups as previously described. Exemplary alkylamino groups include ethylmethylamino, dimethylamino and diethylamino. "Alkoxycarbonyl" means an alkyl-O-CO- group. Exemplary
alkoxycarbonyl groups include methoxy- and ethoxy- carbonyl.
"Aryloxycarbonyl" means an aryl-O-CO- group. Exemplary
aryloxycarbonyl groups include phenoxy- and naphthoxy- carbonyl.
"Aralkoxycarbonyl" means an aralkyl-O-CO- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
"Carbamoyl" means an H2N-CO- group.
"Alkylcarbamoyl" means an Z'ZN-CO- group wherein one of Z and Z' is hydrogen and the other of Z and Z' is alkyl as defined previously.
"Dialkylcarbamoyl" means an Z'ZN-CO- group wherein both Z and Z' are alkyl as defined previously.
"Acyloxy" means an acyl-O- group wherein acyl is as defined previously.
"Acylamino" means an acyl-NH- group wherein acyl is as defined previously.
"Aroylamino" means an aroyl-NH- group wherein aroyl is as defined previously. "Alkylene" means a straight or branched bivalent hydrocarbon chain group having from about 1 to about 8 carbon atoms. The alkylene group may be also optionally unsaturated. There may be optionally inserted along the alkylene group one or more oxygen or sulphur atoms, or substituted nitrogen atoms wherein the substituent is alkyl as previously described. Exemplary alkylene groups include ethylene (-CH2CH2-), propylene (-CH2-)3, -CH=CH-CH=CH- , -CH=CH-CH2-, -CH2-NMe-CH2-, -methylene (O-CH2-O- )or ethylene
(-O-(-CH2-)2-O-). It is preferred that the alkylene group has about 2 to about 3 carbon atoms. "Halo" mean fluoro, chloro or bromo.
The symbol "φ" means phenyl.
"Amino Acid" may be naturally occurring or synthetic. Exemplary amino acids include proline, hydroxy proline, 4,4-ethylenedioxyproline, methoxyproline, thiazolidinecarboxylic acid, trytophane, glycine alanine, leucine, isoleucine, valine, tyrosine, O-benzylserine, 2-carboxylpiperidine, 1-amino-1-phenylacetic acid and 1-amino-1-indan-2-ylacetic acid.
"Peptide" may be about 2 to about 6 amino acid residues bonded by peptide linkages. Preferred are peptides having about 2 to about 3 amino acid residues.
"Lipophilic aliphatic carbonyl radical" means higher alkyl carbonyl, higher alkenyl carbonyl, higher alkynyl carbonyl, multi cycloalkyl carbonyl or multicyclic heterocyclyl carbonyl wherein the alkyl, alkenyl, alkynyl, cycloalkyl and heterocyclyl are as previously described. Multi cycloalkyl carbonyl radicals are preferred, and more preferred are the multi cycloalkyl carbonyl radicals wherein the multi cycloalkyl moiety thereof has bridging carbons, for example
adamantane, camphor and norbane. Exemplary lipophilic aliphatic carbonyl radical groups include 1- or 2-adamantylacetyl, 3-methyl adamant-1-ylacetyl, 3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl, camphaneacetyl, noradamantylacetyl, norbornaneacetyl, bicyclo[2.2.2.]-oct-5- eneacetyl, 1 -methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl, cis-5-norborene-endo- 2,3-dicarbonyl, 5-norbomen-2-yl acetyl, (1 R)-(-)-myrtentaneacetyl,
2-norbaneacetyl, anti-3-oxo-tricyclo[2.2.1.02'6]-heptane-7-carbonyl, decanoyl, dodecanoyl, dodecenoyl , tetradecadienoyl, decynoyl or dodecynoyl.
Exemplary N-mercaptoacyl(amino acid or peptide) compounds, which are derivatized by an S-lipophilic aliphalic carbonyl group to form the compounds according to the invention, include the N-mercaptoacyl(amino acid or peptide) compounds disclosed in the following: U.S. Patent Nos. 4,053,651 ; 4,684,600; 4,248,883; 4,474,799; 4,798,904; 4,513,009; 4,879,309; German Patent No. DE 3,819,539 A1; French Patent No. FR 8320024 (2,556,721); European Patent Nos. EP 0038758; 0 136883; PCT/EP Application Nos. 92/01622; 92/02412; Nature, 288, 286-288 (1980). These references are incorporated by reference herein. The precursor of compounds of formulae I and II herein, i.e., wherein the R group thereof is replaced by hydrogen are other exemplary
N-mercaptoacyl(amino acid or peptide) compounds.
Preferred mercaptoalkanoyl (amino acid or peptide) species, which are derivatized by an S-Iipophilic aliphalic carbonyl group to form the
compompounds according to the invention, include those in Tables I and II herein and the following species:
N-(2-mercaptomethyl-1-oxo-3-phenylbutyl)tyrosine; N-(2-mercaptomethyl-1-oxo- 3-phenylbutyl)glycine; N-(2-mercaptomethyl-1-oxo-3-phenylbutyl)-O-benzyl- serine;N-(2-mercaptomethyl-1-oxo-3-phenylbutyl)alanine; N-(2-mercaptomethyl1 -oxo-3-phenylbutyl)norleucine; N-[3-mercapto-2-(1-indanyl)-1-oxopropyl]- alanine; N-[2-mercaptomethyl-3-(4-hydroxyphenyl)-1-oxobutyl]alanine;
N-[2-(1-mercaptomethyl-3-(4-hydroxyphenyl)-1-oxobutyl]tyrosine; N-[N'-(2- mercapto-3-phenylpropanoyl)valyl]-tyrosine; N-[N'-(2-mercapto-3-phenylpropanoyl)isoleucinyl]tyrosine; N-[N'-(2-mercapto-3-phenylpropanoyl)- isoleucinyl]piperidin-2-oic acid; N-[N'-(2-mercapto-3-phenylpropanoyl)n-leucinyl]- tyrosine; N-[N'-(2-mercapto-3-phenylpropanoyl)leucinyl]tyrosine;
N-[N'-(2-mercapto-3-methylpentanoyl)phenalanyl]tyrosine; N-[N'-(2-mercapto-3- methylpentanoyl)valyl]tyrosine; N-[N'-(2-mercapto-4-phenylbutanoyl)valyl]- tyrosine; N-[N'-(2-mercapto-3-methylbutanoyl)isoleucinyl]tyrosine;
N-[N'-(2-mercapto-2-phenylacetyl)isoleucinyl]tyrosine; N-[N'-(2-mercapto-3- methylpentanoyl)isoleucinyl]tyrosine; N-[N'-(2-mercapto-3-methylpentanoyl)-2- amino-3-methoxybutanoyl]tyrosine; N-[N'-(2-mercapto-3-methylpentanoyl)-2- amino-3-methoxybutanoyl]proline; N-[N'-(2-mercapto-3-methylpentanoyl)-2- amino-3-methoxybutanoyl]hydroxyproline; N-[N'-(2-mercapto-3-methylpentanoyl)-2-amino-3-benzyloxybutanoyl]tyrosine; N-[N'-(2-mercapto-3- methylbutanoyl)-2-amino-3-methylbutanoyl]tyrosine; N-[N'-(2-mercapto-3- methylpentanoyl)-2-amino-3-methoxybutanoyl]tyrosine; N-[N'-(2-mercapto-3- methylbutanoyl)-2-amino-3-benzyloxybutanoyl]tyrosine; N-[N'-(2-mercapto-3- methylbutanoyl)-2-amino-3-benzyloxybutanoyl]proline; N-[N'-(2-mercapto-3- methoxybutanoyl)isoleucinyl]tyrosine; N-[N'-(2-mercapto-3-methoxybutanoyl)-3- methylphenalanyl]tyrosine; N-[N'-(2-mercapto-3-methoxybutanoyl)-2-amino-2- indan-2'-ylacetyl]tyrosine; N-[N'-(2-mercapto-3-benzyloxybutanoyl)isoleucinyl]- tyrosine; N-[N'-(2-mercapto-2-phenylacetyl)valinyl]tyrosine; N-[N'-(2-mercapto-2- indan-2'-yl)isoleucinyl]-tyrosine; N-[N'-(2-mercapto-3-methylpentanoyl)-2-amino- 2-phenylacetyl]tyrosine; N-[N'-(2-mercapto-3-methylbutanoyl)-2-amino-2- phenylacetyl]tyrosine; N-[N'-(2-mercapto-3-methylbutanoyl)-2-amino-2- phenylacetyl]piperidin-2-oic acid; N-[N'-(2-mercapto-3-methylpentanoyl)-2- amino-2-phenylacetyl]tyrosine; N-[N'-(2-mercapto-3-methylpentanoyl)alanyl]- piperidin-2-oic acid; N-[N'-(2-mercapto-3-methylbutanoyl)-2-amino-2-phenylacetyl]piperidin-2-oic acid; N-[N'-(2-mercapto-2-phenylacetyl)-2-amino-2- phenylacetyl]tyrosine; N-[N'-(2-mercapto-2-phenylacetyl)-2-amino-3-phenylbutanoyl]tyrosine; N-[N'-(2-mercapto-2-phenylacetyl)-2-amino-3-ethoxybutanoyl]tyrosine; and N-[N'-(2-mercapto-2-phenylacetyl)valinyl]tyrosine.
Figure imgf000015_0001
Figure imgf000016_0001
Description of the Preferred Embodiments
Presently preferred is a compound of formula (I)
* *
R- S - CH2 - CH- CONH- CH-COOR'
I I
* CH R3
R2 R1
Figure imgf000017_0003
(I) wherein
R is a lipophilic aliphatic carbonyl radical; R1 is aryl or heteroaryl;
R2 is alkyl, or alkylene attached to the moiety and R1 ;
Figure imgf000017_0002
R3 is hydrogen, alkyl, aryl, alkoxy or aryloxy; and
R' is hydrogen, alkyl, aralkyl, acyl or aroyl; or a pharmaceutically acceptable salt thereof.
When R2 is alkylene, the alkylene preferably attaches at an ortho position on R1
relative to the attachment of R1 to the
Figure imgf000017_0001
moiety.
More preferred compounds are described by the formula (I) above wherein
R is higher alkyl carbonyl or multi cycloalkyl carbonyl; R1 is aryl or heteroaryl; R2 lower alkyl, or alkylene attached to the
Figure imgf000018_0002
moiety and R1;
R3 is hydrogen, lower alkyl, aryl or heteroaryl, lower alkoxy or aryloxy; and
R' is hydrogen, lower alkyl, aryl lower alkyl, acyl or aroyl.
More preferred are compounds of formula (I) above wherein
Figure imgf000018_0003
R is adamantoyl or palmitoyl; R1 is phenyl, 2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl radical, optionally substituted with one or more identical or different aryl group substituents chosen from halo, hydroxy, C1-4 acyloxy, C1-4 alkoxy, phenoxy, phenylthio and amino radicals, C1-4 dialkylamino, methylenedioxy and ethylenedioxy;
R2 is methyl or trifluoromethyl radical, or -CH2-Y-, -Y-CH2- or
C2-3 alkylene attached to the
Figure imgf000018_0001
moiety and R1 wherein R1 is phenyl, 2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl, Y is oxygen, sulphur or nitrogen substituted by methyl, or alkylene is optionally substituted with a methyl; R3 is hydrogen, trifluoromethyl, C1-8 alkoxy, phenoxy, phenyl or thienyl or C1-8 alkyl optionally substituted with phenyl, hydroxy, C1-4 alkoxy, phenoxy, C1-4 alkylthio, phenylthio, benzyloxy or benzylthio, wherein the phenyl and the phenyl portions of the phenoxy, phenylthio, benzyloxy or benzylthio radicals are optionally substituted with one or more identical or different aryl group
substituents chosen among halo, hydroxy, C1-4 alkoxy, amino,
C1-4 dialkylamino, methylenedioxy or ethylenedioxy; and
R' is hydrogen, methyl, ethyl, benzyl, cyclohexylmethyl, palmitoyl or pamoyl radical. Presently preferred also is a compound of the formula (II)
R6 R7
I I
R-S-CH-CONH-CH- CON-CH-COOR"
I I
CH CH *
/ \ / \
A1 R4 A2 R5 .... wherein
R is a lipophilic aliphatic carbonyl ra
Figure imgf000019_0001
dical; R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkoxy, alkyloxymethyl or aralkyloxy;
A1 and A2 are independently hydrogen, alkyl, or together with -CH- R4 or -CH-R5, respectively, form phenyl, or alkylene which together with -CH- R4 or -CH-R5, respectively, form benzocycloalkyl;
R6 is hydrogen or alkyl; G7 is cycloalkyl, aralkyl or aryloxymethyl or alkoxymethyl, or R6 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form heterocyclyl; and
R" is hydrogen, alkyl, aralkyl, cycloalkyl lower alkyl or palmitoyl; or a pharmaceutically acceptable salt thereof.
More preferred also is a compound of the formula (II) above wherein
R is higher alkyl carbonyl or multi cycloalkyl carbonyl; R4 and R5 are independently hydrogen, lower alkyl, phenyl, aryl lower alkyl, lower alkoxy, lower alkyloxymethyl or aryl lower alkyloxy;
A1 and A2 are independently hydrogen, lower alkyl or together with -CH- R4 or -CH-R5, respectively, form phenyl, or alkylene which together with -CH- R4 or -CH-R5, respectively, form benzocycloalkyl;
R6 is hydrogen or lower alkyl; G7 is cycloalkyl, aryl lower alkyl or aryloxymethyl or lower alkoxymethyl, or R6 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form heterocyclyl; and
R" is hydrogen, lower alkyl, aryl lower alkyl, cycloalkylmethyl or palmitoyl.
More preferred compounds are described also by the formula (II) above wherein: R is adamantoyl or palmitoyl; R4 and R5 are independently lower alkyl, lower alkoxy, phenyl, benzyl or benzyloxy; A1 and A2 are independently hydrogen, methyl, or together with -CH- R4 or -CH-R5, respectively, form phenyl, or alkylene together with -CH- R4 or -CH-R5, respectively, form indanyl; G7 is benzyl wherein the phenyl ring is optionally substituted by hydroxyl, or R6 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form a saturated 5 or 6 member heterocyclyl; and
R" is hydrogen. The compounds of the present invention may be useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof. All forms are within the scope of the invention. Where the compound of the invention is substituted with an acidic moiety, base addition salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of the free acid form. The bases which can be used to prepare the base addition salts preferably include those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose cations are non-toxic to the animal organism in pharmaceutical doses of the salts, so that the inhibition of NEP and/or ACE, and preferably beneficial mixed NEP and ACE inhibition, inherent in the compound of the invention is not vitiated by side effects ascribable to the cations. Although pharmaceutically acceptable salts of said acidic compounds are preferred, all base addition salts are useful as sources of the free acid form even if the particular salt, per se, is desired only as an intermediate product as for example, when the salt is formed only for purposes of purification, and identification, or when it is used as intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures.
Pharmaceutically acceptable salts within the scope of the invention include those derived from the following bases: sodium hydroxide, potassium
hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, omithine, choline, N.N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane,
tetramethylammonium hydroxide, and the like.
Metal salts of compounds of the present invention may be obtained by contacting a hydroxide, carbonate or similar reactive compound of the chosen metal in an aqueous solvent with the free acid form of the compound. The aqueous solvent employed may be water or it may be a mixture of water with an organic solvent, preferably an alcohol such as methanol or ethanol, a ketone such as acetone, an aliphatic ether such as tetrahydrofuran, or an ester such as ethyl acetate. Such reactions are normally conducted at ambient temperature but they may, if desired, be conducted with heating. Amine salts of compounds of the present invention may be obtained by contacting an amine in an aqueous solvent with the free acid form of the compound. Suitable aqueous solvents include water and mixtures of water with alcohols such as methanol or ethanol, ethers such as tetrahydrofuran, nitriles such as acetonitrile, or ketones such as acetone. Amino acid salts may be similarly prepared. Preferred base addition salts have a cation selected from the group consisting of ammonium, sodium, calcium, protonated N-methyl-D-glucamine, protonated lysine, protonated arginine and protonated dicyclohexylamine.
Where the compound of the present invention is substituted with a basic moiety, acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form. The acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non- toxic to the animal organism in pharmaceutical doses of the salts, so that the inhibition of NEP and/or ACE, and preferably beneficial mixed NEP and ACE inhibition, inherent in the compound of the invention is not vitiated by side effects ascribable to the anions. Pharmaceutically acceptable salts within the scope of the invention are those derived from the following acids: mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like. The corresponding acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, malonate,
methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.
The acid addition salts of the compounds of this invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, wherein case the salt separates directly or can be obtained by concentration of the solution. The compound according to the invention contains several asymmetric centers and may exist in the form of pure stereoisomers or in the form of mixtures of stereoisomers. The carbon atoms marked by an asterisk (*) designate asymmetric centers. In the compound according to the invention, unless stated otherwise, designations regarding the stereochemical
configurations at the carbon atoms marked by an asterisk relate: sequentially startin from the carbon atom closest to the O-terminal side of the molecule, i.e., the amino acid residue. Preferred compounds according to the invention include those wherein any of the carbon atoms marked by an asterisk (*) have the S configuration. Preferred also include the compounds of formula (I) according to the invention having a stereochemical designation selected from the group consisting of R,R,S, S,R,S, S,S,S and R,S,S form, and it is more preferred having a stereochemical designation S,S,S or R,S,S. Preferred compounds of formula II according to the invention are those wherein the carbon atoms of the principal chain marked by an asterisk (*) have the L configuration.
According to the invention, the S-lipophilic aliphatic carbonyl
[N-mercaptoacyl(amino acid or peptide)] compound may be prepared by acylating the appropriate N-mercaptoacyl(amino acid or peptide) with the appropriate lipophilic aliphatic carbonyl halide. The reaction may be carried out in the absence or presence of a base such as aqueous sodium hydroxide or pyridine, and may be carried out under an inert atmosphere such as nitrogen. It is also preferred that the reaction medium should be degassed to prevent the mercapto compound from forming a disulfide linked dimer. "Preparative Organic Chemistry" edited by G. Hilgetag and A. Martini at page 642, J. Wiley & Sons (1972).
N-(mercaptoacyl)amino acids may be prepared by hydrolyzing the product formed by the acylation of an amino acid of formula (III)
H2N- CH- COOR'
I
R3 (III) wherein R3 and R' are defined as above, by means of an acid of formula (IV)
RLA- S - CH2- CH-COOH
Figure imgf000024_0002
1 (IV) wherein R1 and R2 are defined as above and RLA is a lower acyl group, under the customary conditions used in peptide chemistry, described, for example, by Bodansky et al., "Peptide Synthesis", J. Wiley and Sons Edit. The hydrolysis is
Figure imgf000024_0003
preferably carried out in an alkaline medium, working in an inert medium so as to avoid oxidation of the mercapto group.
More especially, the acylation is performed in the presence of a condensing agent such as dicyclohexylcarbodiimide, optionally in the presence of 1-hydroxybenzo-triazole. The products of general formula (IV) may be obtained by the Michael addition of a sulphur derivative of formula (V)
RLA- SH
(V) wherein RLA is defined as above, to an acrylic acid of formula (VI)
H2C= C- COOH
I CH
Figure imgf000024_0001
2 (V.) wherein R1 and R2 are defined as above.
Preferably, thioacetic acid or thiobenzoic acid is used as a sulphur derivative of formula (V). The acrylic acid of formula (VI) may be obtained by saponification of a corresponding ester in a basic medium. Preferably, the methyl or ethyl ester is used. The acrylic ester of formula (VII)
H2C=C- COOR8 (VII)
Figure imgf000025_0001
wherein R1 and R2 are defined as above and R6 preferably represents an alkyl radical containing 1 to 4 carbon atoms, may be obtained by a Mannich reaction on the monoester of formula (VIII)
R8OCO- CH-COOH
I
Figure imgf000025_0002
(VIII) wherein R1, R2 and R8 are defined as above.
Generally, the Mannich reaction is performed by means of formaldehyde in the presence of a secondary amine such as diethylamine at a temperature of about 20°C.
The monoester of formula (VIII) is obtained by monosaponification in a basic medium of a malonic ester of formula (IX)
(R8OCO)2- CH
1 (IX)
Figure imgf000025_0003
wherein R1, R2 and R8 are defined as above.
The malonic ester of formula (IX) may be obtained by the action of a halide of formula (X)
Halo-
Figure imgf000025_0004
N (X) wherein R1 and R2 are defined as above and Halo represents a halide atom, preferably a bromide atom, on an alkyl malonate anionised beforehand, for example by means of an alkali metal alcoholate optionally prepared in situ. The malonic ester of formula (IX) may also be obtained by condensation of a ketone of formula (XI)
O
Figure imgf000026_0003
wherein R1 and R2 are defined as above, with an alkyl malonate anionised beforehand as described above, to form the product of formula (XII) 1 8 8 XII
Figure imgf000026_0002
wherein R1, R2 and R8 are defined as above, which is reduced catalytically, for example with hydrogen in the presence of palladium on charcoal, to the product of formula (IX).
The product of formula (VII) may also be obtained by a Wittig reaction with formaldehyde on a phosphonate of formula (XIII)
Figure imgf000026_0004
(R8O)2P- CH- COOR8 1
Figure imgf000026_0001
(XIII) wherein R1, R2 and R8 are defined as above. The product of formula (XIII) may be obtained by the action of a product of formula (IX) on an alkyl phosphonoacetate of formula (XIV)
Figure imgf000027_0004
(R8O)2P- CH2-COOR8
(XIV) wherein R8 is defined as above.
The reaction is generally performed in the presence of a strong base such as sodium hydride.
N-(mercaptoacyl)peptides may be prepared by hydrolyzing the product formed the acylation of a dipeptide of the formula
R3 R4
Figure imgf000027_0003
I I
H2N- CH- CON-CH-COOR'
I *
Figure imgf000027_0002
wherein
A2, R2, R3, R4 and R' are as defined above, by an acid of the formula
RLA-S-CH-COOH
I 1 1
Figure imgf000027_0001
wherein RLA, A1 and R1 are as defined above, under the usual conditions used in peptide chemistry and described, for example, by Bodansky et al., "Peptide Synthesis", J. Wiley and Sons Edit. The hydrolysis is preferably carried out in an alkaline medium, working in an inert medium so as to avoid oxidation of the mercapto group. The acid reactant may be prepared from the corresponding α-amino acid by halogenating deamination according to Fischer, Ann., 357, 1-24 (1907), followed by nucleophilic substitution of the halogen atom.
Lipophilic aliphatic carbonyl halides may be prepared from the following lipophilic aliphatic carboxylic compounds when they are in the acid or anhydride forms: camphorcarboxylic acid, camphanecarboxylic acid, 3-methyl adamantyl carboxylic acid, noradamantyl carboxylic acid, norbornane carboxylic acid, 3- methyl 3-bromo-1-adamantyl carboxylic acid, methyl 1-methoxybicyclo[2.2.2.]- oct-5-ene-2-carboxylate, mono methyl cis-5-norborene-endo -2,3-dicarboxylate, methyl-5-norborene-2,3-dicarboxylic anhydride, (1R)-(-)-myrtentoic acid, cis-5- norbornene-endo -2,3-dicarboxylic acid, 5-norbornen-2-yl acetate, 2- norbaneacetic acid, anti-3-oxo-tricyclo[2.2.1.02, 6]heptane-7-carboxylic acid, 2- decalincarboxylic acid or 2-quinuclidinecarboxlic acid. "Preparative Organic Chemistry" edited by G. Hilgetag and A. Martini at pages 242-251 , J. Wiley & Sons (1972).
The resolved forms of the compounds according to the invention may be prepared by standard practices known to those skilled in the art such as fractional crystallization and column chromatography. For example, compounds of formula (IV) such as an acylthioalkanoic acid may be resolved by fractional recrystallization with optically active bases such as methylbenzylamine or 1-(1- naphthyl)ethylamine and then the resolved compounds of formula (IV) may be reacted with an optically active amino acid according to the acylation reaction described above to yield a resolved N-(mercaptoacyl)amino acid.
The present invention is further exemplified but not limited by the following illustrative examples. 1 - PREPARATION OF THE INTERMEDIATES
EXAMPLE A 2-Acetylthiomethyl-3-phenylbutanoic Acid
Ethyl malonate (4.1 mL) is added to a solution of sodium ethoxide prepared from sodium metal (0.75 g) and ethanol (40 mL). 1-Bromo-1- phenylethane (6 g) is added at 0°C and the mixture is then stirred for 20 hours at 30°C. After concentrating to dryness, the residue is taken up with water (100 mL) and then extracted three times with ethyl acetate (75 mL). The organic phase is washed with water and then with saturated sodium chloride solution and finally dried over magnesium sulphate. After filtering and concentrating to dryness, ethyl (1-phenylethyl)malonate (6 g) is obtained in an 84 % yield in the form of a pale yellow oil, having the following characteristics: - Rf = 0.67
[hexane/ethyl acetate (65:35 by volume)].
The ethyl (1-phenylethyl)malonate is stirred overnight with sodium hydroxide (1.3 eq) in an acetone/water (3:1 by volume) mixture. After
concentrating to dryness, the residue is taken up with water (40 mL) and the mixture is then extracted three times with ethyl acetate (25 mL). The organic phase is washed, dried, filtered and then concentrated to dryness to yield 3.5 g of 2-ethoxycarbonyl-3-phenylbutanoic acid in a 65 % yield in the form of an oil, having the following characteristics: - Rf = 0.50 [methylene chloride/methanol (9:1 by volume)].
To the 2-ethoxycarbonyl-3-phenylbutanoic acid (3.5 g) is added diethylamine (1.53 mL, 1 eq) and 30 % formaldehyde (1.78 mL) at 0°C. The mixture is stirred overnight at a temperature of about 20°C and is then taken up with diethyl ether (100 mL). The organic phase is separated after settling takes place, washed with 10 % aqueous citric acid solution (40 mL), with water (40 mL) and with saturated sodium chloride solution (40 mL) and finally dried over sodium sulphate. After filtering and concentrating to dryness, 2.2 g of ethyl 2 ~ phenylethyl)acrylate is obtained in a 71 % yield in the form of a yellow oil, having the following characteristics: - Rf = 0.86 [methylene chloride/methanol (9:1 by volume)]. The product thereby obtained (2.2 g) is treated with of sodium hydroxide (1.5 eq) in an acetone/water (2:1 by volume) mixture overnight at a temperature of about 20°C. The mixture is concentrated to dryness and the residue is then taken up with water (30 mL). The aqueous phase is acidified to pH 2 by adding hydrochloric acid.
The product is then extracted three times with ethyl acetate (20 mL). The organic phase is washed with water (15 mL) and then with saturated sodium chloride solution (15 mL) and finally dried over sodium sulphate. After filtering and concentrating to dryness, 1.8 g of 2-(1-phenylethyl)acrylic acid is obtained in a 95 % yield in the form of a white solid, having the following characteristics: - melting point (m.p.); 115°C - Rf = 0.61 [methylene chloride/methanol (9:1 by volume)]. The acid thereby obtained (1.5 g) is heated to 80°C with thioacetic acid (6 mL). After concentration to dryness, 0.21 g of 2-acetylthiomethyl-3- phenylbutanoic acid is obtained in a 98 % yield in the form of a yellow oil, having the following characteristics: - Rf = 0.73 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE B
2-Acetylthiomethyl-3-phenylbutanoic Acid Sodium hydride (4.15 g) in 80 % suspension in oil is added to a solution of triethyl phosphonoacetate (25 mL) in anhydrous dimethylformamide (36 mL). After 15 minutes at 0°C, l-bromo-1-phenylethane (18 mL, 1.05 eq) is added. The mixture is stirred overnight at a temperature of about 20°C under a nitrogen atmosphere. After evaporation of dimethylformamide, the residue is taken up with ethyl acetate (200 mL). The organic phase is washed three times with water (60 mL) and then with saturated sodium chloride solution (60 mL) and finally dried over sodium sulphate. After filtering and concentrating to dryness, 40.3 g of diethyl 2-(1-phenylethyl)phosphonoacetate is obtained in a 97 % yield in the form of an oil, having the following characteristics: - Rf = 0.57 [cyclohexane/ethyl acetate/acetic acid(5:5:0.5 by volume)]. The diethyl 2-(1-phenylethyl)phosphonoacetate (40.3 g) is dissolved in 37 % formaldehyde (65 mL), and potassium carbonate (51 g) is then added. The mixture is heated to reflux for 3 hours 30 minutes. After cooling, the mixture is taken up with hexane (400 mL). The organic phase is washed with water twice 100 mL) and then with saturated sodium chloride solution (100 mL) and finally dried over sodium sulphate. After filtering and concentrating to dryness, 24.2 g of ethyl 2-(1-phenylethyl)acrylate are obtained in a 96 % yield in the form of an oil, the characteristics of which are identical to those of the product obtained in Example A.
EXAMPLE C 3-Acetylthio-2-(1-indanyl)propanoic Acid Using the procedure described in Example B, but starting with 1- bromoindane and triethyl phosphonoacetate, ethyl 2-(1-indanyl)acrylate is obtained, which product, by the action of thioacetic acid under the conditions described in Example A, yields 3-acetylthio-2-(1-indanyl)propanoic acid in the form of a pale yellow oily product, having the following characteristics:
Rf = 0.60 [hexane/ethyl acetate/acetic acid (5:5:0.5 by volume)].
EXAMPLE D
2-Acetylthiomethyl-3-(4-hydroxyphenyl)butanoic Acid
Using the procedure described in Example A, but starting with 1-bromo-1- (4-hydroxyphenyl)ethane, 2-acetylthiomethyl-3-(4-hydroxyphenyl)butanoic acid is obtained, having the following characteristics: - m.p.: 48°C - Rf = 0.58
[methylene chloride/methanol (9:1 by volume)].
EXAMPLE E
2-Acetylthiomethyl-3-f(4-fluoro)phenyl]butanoic Acid Working as in Example A, but starting with 1-bromo-1-[(4- fluoro)phenyl]ethane, 2-acetylthiomethyl-3-[(4-fluoro)phenyl]butanoic acid is obtained in a 98% yield as a thick oil having the following characteristics: - Rf = 0.38 [n-hexane/ethyl acetate/acetic acid (7:3:0.5 by volume)].
EXAMPLE F 2-Acetylthiomethyl-3-f(3,4-difluoro)phenyl]butanoic Acid
Working as in Example A, but starting with 1-bromo-1-[(3,4- difluoro)phenyl]ethane, 2-acetylthiomethyl-3-[(3,4-difluoro)phenyl]-butanoic acid is obtained in a 95% yield as a thick oil having the following characteristics: Rf = 0.33 [hexane/ethyl acetate (1:1 by volume)].
EXAMPLE G N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine Benzyl Ester
2-acetylthiomethyl-3-phenylbutanoic acid (5 g) are dissolved in dry tetrahydrofuran (25 mL). A solution of (S)-tyrosine benzyl ester p-tosylate (1 equivalent) and triethylamine (1 eq) in chloroform (25 mL), a solution of 1- hydroxybenzotriazole (1 eq) in tetrahydrofuran (30 mL) and a solution of dicyclohexylcarbodiimide (1.2 eq) in chloroform (25 mL) are added successively at 0°C. The mixture is stirred for 1 hour at 0°C and then overnight at a
temperature in the region of 20°C. After filtering, the mixture is concentrated to dryness and the residue is then taken up with ethyl acetate (80 mL). The organic phase is washed successively with 10 % citric acid solution (20 mL), with water (20 mL), with saturated sodium bicarbonate solution (20 mL), with water (20 mL) and then with saturated sodium chloride solution (20 mL). After drying over sodium sulphate, filtering and concentrating to dryness, 8 g of N-(2- acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine benzyl ester is obtained in an 80% yield in the form of a white solid, having the following characteristics: - Rf = 0.65 [chloroform/methanol (9.5:0.5 by volume)].
The product is purified by chromatography on silica. EXAMPLE H
N-(2-Mercaptomethyl-1-oxo-3-phenylbutyl)tyrosine 1 g of the product obtained in Example G is dissolved in an degassed acetone/water (2:1 by volume) mixture. 1M sodium hydroxide (4 eq) is added at 0°C and under a nitrogen atmosphere. The mixture is stirred for 5 hours at a temperature of about 20°C. After filtering and acidifying to pH 1 , the product is extracted three times with degassed chloroform (15 mL). The organic phase is dried over sodium sulphate. After filtering and concentrating to dryness, 0.73 g of N-(2-mercaptomethyl-l-oxo-3-phenylbutyl)tyrosine is obtained in an 81 % yield, the characteristics of which product are as follows: - m.p.: 80°C - Rf = 0.43 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]. EXAMPLE I
N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)glycine Benzyl Ester
Using the procedure described in Example G, starting with 2- acetylthiomethyl-3-phenylbutanoic acid and glycine benzyl ester, and after purifying by chromatography, eluting with a cyclohexane/ethyl acetate (6:4 by volume) mixture, N-(acetylthiomethyl-1-ox-3-phenylbutyl)glycine benzyl ester is obtained in a 61 % yield, the characteristics of which product are as follows: - Rf = 0.39 [cyclohexane/ethyl acetate(6:4 by volume)].
EXAMPLE J
N-(2-Mercaptomethyl-1-oxo-3-phenylbutyl)glycine Using the procedure described in Example H, starting with the product obtained in Example I, N-(2-mercaptomethyl-1-oxo-3-phenylbutyl)glycine is obtained in an 89 % yield, the characteristics of which product are as follows: - m.p.: 129°C - Rf = 0.62 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]. EXAMPLE K N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)-O-benzylserine Benzyl Ester Using the procedure described in Example G, starting with
2-acetylthiomethyl-3-phenylbutanoic acid and O-benzylserine benzyl ester, and after purification by chromatography, eluting with a hexane/ethyl acetate (8:2 by volume) mixture, N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)-O-benzylserine benzyl ester is obtained in a 62 % yield, the characteristics of which product are as follows: - Rf = 0.16 [hexane/ethyl acetate (8:2 by volume)].
EXAMPLE L
N-(2-Mercaptomethyl-1-oxo-3-phenylbutvB-O-benzylserine
Using the procedure described in Example H, starting with the product obtained in Example K, N-(mercaptomethyl-l-oxo-3-phenylbutyl)-O-benzylserine is obtained in a 72 % yield in the form of an oil, having the following
characteristics: - Rf = 0.29 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE M
N-92-Acetylthiomethyl-1-oxo-3-phenylbuty)alanine Benzyl Ester Using the procedure described in Example G, starting with 2- acetylthiomethyl-3-phenylbutanoic acid and alanine benzyl ester, and after purification by chromatography, eluting with a cyclohexane/ethyl acetate (75:25 by volume) mixture, N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)alanine benzyl ester is obtained in a 68 % yield, the characteristics of which product are as follows: - Rf = 0.31 [hexane/ethyl acetate (75:25 by volume)].
EXAMPLE N N-(2-Mercaptomethyl-1-oxo-3-phenylbutyl)alanine
Using the procedure described in Example H, starting with the product obtained in Example M, N-(2-mercaptomethyl-1-oxo-3-phenylbutyl)alanine is obtained in a 75 % yield in the form of a colorless oil, having the following characteristics: - Rf = 0.27 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE O
N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)norleucine Benzyl Ester
Using the procedure described in Example G, but starting with 2- acetylthiomethyl-3-phenylbutanoic acid and norleucine benzyl ester, and after purifying by chromatography, eluting with a hexane/ethyl acetate (75:25 by volume) mixture, N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)norleucine benzyl ester is obtained in a 75 % yield in the form of an oil, having the following characteristics: - Rf = 0.46 [hexane/ethyl acetate (6.5:3.5 by volume)]. EXAMPLE P
N-(2-Mercaptomethyl-1-oxo-3-phenylbutyl)norleucine
Using the procedure described in Example H, but starting with the product obtained in Example O, N-(2-mercaptomethyl-1-oxo- phenylbutyl)norleucine is obtained in a 90 % yield, the characteristics of which product are as follows: - m.p.: 55°C - Rf = 0.47 [methylene chloride/methanol (9:1 by volume)]. EXAMPLE Q
N-[3-Acetylthio-2-(1-indanyh-1-oxopropyl]alanine Benzyl Ester
Using the procedure described in Example G, but starting with 3- acetylthio-2-(1-indanyl)propanoic acid and alanine benzyl ester, N-[3-acetylthio- 2-(1-indanyl)-1-oxopropyl)alanine benzyl ester is obtained in a 72 % yield in the form of a white solid, having the following characteristics: - m.p.: 95°C - Rf = 0.17 [cyclohexane/ethyl acetate (4:1 by volume)]. EXAMPLE R
N-[3-Mercapto-2-(1-indanyl)-1-oxopropyl]alanine Using the procedure described in Example H, but starting with the product obtained in Example Q, N-[3-mercapto-2-(1-indanyl)-1-oxopropyl]- alanine is obtained in a 96 % yield, the characteristics of which product are as follows: - m.p.: 121 °C - Rf = 0.22 [methylene chloride/methanol (9:1 by volume). EXAMPLE S
N-[2-Acetylthiomethyl-3-(4-hydroxyphenyl)- 1-oxobutyl]alanine Benzyl Ester
Using the procedure described in Example G, but starting with 2- acetylthiomethyl-3-(4-hydroxyphenyl)butanoic acid and alanine benzyl ester, N- [2-acetylthiomethyl-3-(4-hydroxyphenyl)-1-oxobutyl]alanine benzyl ester is obtained in a 69 % yield, the characteristics of which product are as follows: Rf = 0.22 [hexane/ethyl acetate (65:35 by volume)]. EXAMPLE T
N-[2-Mercaptomethyl-3-(4-hvdroxyphenyl)-1-oxobutyl]alanine
Using the procedure described in Example H, but starting with the product obtained in Example S, N-[2-mercaptomethyl-3-(4-hydroxyphenyl)-1- oxobutyl]alanine is obtained in an 85 % yield in the form of a white solid, having the following characteristics: - m.p.: 72°C - Rf = 0.18 [methylene
chloride/methanol (9:1 by volume)]. EXAMPLE U
N-[2-Acetylthiomethyl-3-(4-hydroxyphenyl)-1-oxobutyl]tyrosine Benzyl Ester
Using the procedure described in Example G, but starting with 2- acetylthiomethyl-3-(4-hydroxyphenyl)butanoic acid and tyrosine benzyl ester, N- [2-acetylthiomethyl-3-(4-hydroxyphenyl)-1-oxobutyl]tyrosine benzyl ester is obtained in a 78 % yield in the form of a white solid, having the following characteristics: - Rf = 0.16 [hexane/ethyl acetate (65:35 by volume)].
EXAMPLE V N-[2-Mercaptomethyl-3-(4-hydroxyphenyl)- 1-oxobutyl]tyrosine
Using the procedure described in Example H, but starting with the product obtained in Example U, N-[2-mercaptomethyl-3-(4-hydroxyphenyl)-1- oxobutyl]tyrosine is obtained in a 70 % yield in the form of a white solid, having the following characteristics: - m.p.: 115°C - Rf = 0.27 [methylene
chloride/methanol (8:2 by volume)].
EXAMPLE W N-[2-Acetylthiomethy-3-(4-fluorophenyl)-1-oxobutyl]alanine Benzyl Ester
Working as in Example G, but starting with 2-acetylthiomethyl-3-(4- fluorophenyl)butanoic acid and benzyl esters of alanine, the titled compound is obtained in a yield of 73% as an oil having the characteristics: Rf = 0.23
[cyclohexane/ethyl acetate (7:3 by volume)].
EXAMPLE X N-[2-Acetylthiomethyl-3-C4-fluorophenyl)-1-oxobutyl]alanine
Working as in Example H, but starting with the product of Example W, the titled compound is obtained in a yield of 78% as a white solid having the following characteristics: m.p. 93°C; Rf = 0.5 [methylene
chloride/methanol/acetic acid (9: 1 :0.5 by volume)]
EXAMPLE Y
N-[2-Acetylthio-3-(4-fluorophenyl) -1-oxobutyl]tyrosine Benzyl Ester
Working as in Example G but starting with 2-acetylthio-3-(4- fluorophenyl)butanoic acid and benzyl ester of lysine, the titled compound is obtained in a yield of 89% as a pale yellow oil having the following
characteristics: Rf = 0.14 [cyclohexane/ethylacetate (2:3 by volume)].
EXAMPLE Z N-[2-Acetylthio-3-(4-fluorophenyl)- 1-oxobutyl]tyrosine
Working as in Example H, but starting with the product of Example Y, the titled compound is obtained in a 74% yield as an oily composition having the following characteristic: Rf = 0.45 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)].
EXAMPLE AA N-[2-Acetylthiomethyl-3-(3,4-difluorophenyl)-1-oxobutyl]alanine Benzyl Ester
Working as in Example G but starting with 2-acetylthiomethyl-3-(3,4- difluoro)phenyl butanoic acid and benzyl ester of alanine the title compound is formed as an oil having the following characteristic: Rf = 0.2 [hexane/ethyl acetate (3:1 by volume)].
EXAMPLE AB
N-[2-Mercaptomethyl-3-(3,4-difluorophenyl)-1-oxobutyl]alanine
Working as in Example H, but using the product of Example AA, the titled compound is obtained in a yield of 72% as a white solid having the following characteristics: m.p. 61°C; Rf = 0.17 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE AC
N-[2-Mercaptomethyl-3-(3,4-difluorophenyl)-1-oxobutyl]tyrosine Benzyl Ester Working as in Example G, but using the benzyl ester of tyrosine and 2- acetylthiomethyl-3-(3,4-difluoro)phenyl butanoic acid, the title compound is obtained in a yield of 78% as an oil having the following characteristic: Rf = 0.26 [cyclohexane/ethyl acetate (6:4 by volume)].
EXAMPLE AD
N-[2-Mercaptomethyl-3-(3,4-difluorophenyl)-1-oxobutyl]tyrosine
Working as in Example H, but using the product of Example AC, the title compound is obtained in a yield of 80% as a pale yellow oil having the following characteristic: Rf = 0.10 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE AE
Resolution of 2-Acetylthiomethyl-3-phenylbutanoic
(ATBA) Acid to Four Diastereomers (l-IV)
(a) Resolution Using (R)-(+)-1-(1-Naphthyl)ethylamine to Yield
ATBA-I 2-Acetylthiomethyl-3-phenylbutanoic acid (5 g) is dissolved in ethanol (50 mL) and (R)-(+)-1-(1-naphthyl)ethylamine (3.4 g) is added to the ethanol solution. Ethyl ether (50 mL) is then added. The mixture is cooled in an ice bath and after scratching the reaction vessel a white precipitate is formed. The solution is stored at about 0°C for about 18 hours. The precipitate is filtered and washed with ethyl ether.
1.47 g of a white precipitate is obtained. The precipitate is recrystallized from absolute ethanol to yield 0.42 g of off-white crystals of a diastereomer, (R)-(+)-1-(1-naphthyl)ethylamonium 2-acetylthiomethyl-3- phenylbutanoate salt (ATBA-I salt).
The free acid ATBA-I is regenerated by contacting the ATBA-I salt (50 mg) aqueous acid (10% hydrochloric) and extracting with methylene chloride. The methylene chloride is evaporated and 32.1 mg of ATBA-I is obtained as an oil having the following characteristic: [α]
Figure imgf000039_0001
= +78.25 [28.5 mg in methanol (1 mL)]. (b) Resolution Using (S)-(-)-1-(1-Naphthyl)ethylamine to Yield
ATBA-II
The procedure is as in Example AE(a) above, except that (S)-(-)-1- (1-naphthyl)ethylamine is used as the amine. 1.2 g of a white precipitate
(ATBA-II salt) is obtained and following recrystallization 0.31 g is obtained.
The free acid (ATBA-II) is regenerated from the salt (50 mg) in a yield of 28.7 mg as an oil having the following characteristic [α] = -79.38
Figure imgf000040_0001
[29.1 mg in methanol (1 mL)].
(c) Resolution Using (S)-Methylbenzylamine to Yield ATBA-III
2-Acetylthiomethyl-3-methylbutanoic acid (5 g) is dissolved in ethanol (35 mL) and a solution of (S)-methylbenzylamine (2.4 g) in methanol (10 mL) is added. The mixture is diluted to 200 mL with ethyl ether. A precipitate forms and the solution is cooled at about 0°C for about 18 hours.
The colorless crystalline product is filtered, washed with ethyl ether and dried to yield 2 g of ATBA-III salt. The product is recrystallized twice from a mixture of ethyl acetate/ethanol (2:1) (25 mL) to yield 1 g of ATBA-III salt.
The free acid ATBA-III is regenerated by contacting the ATBA-III salt with aqueous acid (10% hydrochloric acid) and extracting with methylene chloride. The methylene is evaporated and 0.67 g of ATBA-III is obtained as a colorless crystalline solid.
(d) Resolution Using (R)-Methylbenzylamine to Yield ATBA-IV The procedure in Example AE(c) above is used except that (R)- methylbenzylamine is used to obtain 1.1 g of ATBA-IV is obtained as a colorless crystalline solid. EXAMPLE AF
Preparation of Four Diastereomers of N-(2-Acetylthiomethyl-1-oxo-3- phenylbutyl)-tyrosine Benzyl Ester (ATBAT-(I-IV)) by Synthetic Coupling of
Resolved Reactants
(a) N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine Benzyl Ester (ATBAT-IV) The product (ATBA-IV) (0.2 g) of Example AE(d) is dissolved in methylene chloride (5 mL). (S)-Tyrosine benzyl ester p-tosylate (0.35 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.15 g),
hydroxybenzotnazole (0.11 g) and N-methylmorpholine (0.19 g) are added to the solution and the mixture is stirred at 20°C for 18 hours.
The solvent is removed in vacuo, the residue is contacted with aqueous acid (10% hydrochloric acid) and extracted with ethylacetate. The organic phase is separated and washed consecutively with water (20 mL) saturated sodium bicarbonate solution (20 mL) and water (20 mL). The organic phase is dried and evaporated to yield a light yellow gum. The gum is chromatographed on silica gel using a mixture of hexane/ethyl acetate (1 :1) to yield a colorless gum (ATBAT-IV) (0.26 g; 65%) having the following
characteristic: [α] = +98.90.
Figure imgf000041_0001
(b) N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine
Benzyl Ester (ATBAT-III)
Using the procedure of Example AF(a), except starting with the product (ATBA-III) of Example AE(c), the product (ATBAT-III) is obtained after crystallization having the characteristics: m.p. 151-153°C; [α]
Figure imgf000041_0002
= -89.7°C.
(c) N-(2-Acetylthiomethyl-1 -oxo-3-phenylbutyl)tyrosine
Benzyl Ester (ATBAT-II) Using the procedure of Example AF(a), except starting with the product (ATBA-II) of Example AE(b), the product (ATBAT-II) is obtained. (d) N-(2-Acetylthiomethyl-1-oxo-3-phenylbutyI)tyrosine
Benzyl Ester (ATBAT-I)
Using the procedure of Example AF(a), except starting with the product (ATBA-I) of Example AE(a), the product (ATBAT-I) is obtained after crystallization having the characteristics: m.p. 135-137°C.
EXAMPLE AG
Preparation of Diastereomers of N-(2-Acetylthiomethyl-1-oxo-3- phenylbutyl)tyrosine Benzyl Ester (ATBAT-(I-IV)) by Column Chromatography
The diastereomers of N-(2-acetylthiomethyl-1-oxo-3-phenylbutyl)tyrosine benzyl ester are also obtained by column chromatography using Hewlett- Packard ODS Hypersil column (5 μ, 200 x 4.6 mm i.d.). The product of Example G is dissolved in methanol and placed on the column. A mobile phase of methanol/water (62:38 by volume) with 0.2% trifluoroacetic acid is employed in effecting the separation. The flow rate is 1.0 mL/min. The product is detected at UV of 215 nm. The retention times of the four diastereomers are 22.7, 24.75, 28.5 and 30 minutes respectively.
EXAMPLE AH Two Diastereomers of N-(2-mercaptothiomethyl-1- oxo-3-phenylbutyl)tyrosine (MTBT-(l-H))
(a) N-(2-Mercaptothiomethyl-1-oxo-3-phenylbutyl)tyrosine
(MTBT-(I))
Lithium hydroxide hydrate (18 mg) is dissolved in solvent comprising tetrahydrofuran/methanol/water (1 :1:1 by volume). The solvent is purged with nitrogen gas. The product (ATBAT-IV) (21 mg) of Example AF(a)is added to the solution and stirred for 75 minutes at 20°C. The solution is then diluted with water (5 mL), washed twice with chloroform (3 mL), acidified with 10% hydrochloric acid (2 mL) and extracted five times with chloroform (4 mL). The combined organic phases are dried over magnesium sulphate and evaporated to leave 16 mg of a colorless gum (MTBT-I) having > 95% purity.
(b) N-(2-mercaptothiomethyl-1-oxo-3-phenylbutyl)tyrosine
(MTBT-(II))
Using the procedure of Example AH(a), except starting with the product (ATBAT-II) of Example AF(b), 9.7 mg the product (MTBT-II) is obtained after crystallization having > 98% purity.
EXAMPLE Al (R)-2-Bromo-3-phenylpropanoic Acid Sodium nitrite (27 g in water) is added at 0°C to a solution of D- phenylalanine (40 g) in a mixture of 48% hydrobromic acid/water (1 :1 by volume). The mixture is stirred for 30 minutes at 0°C and then for 2 hours 30 minutes at a temperature close to 20°C. The reaction mixture is extracted with ether. The organic extracts are washed with water and then with a saturated sodium chloride solution and then dried over sodium sulphate. After filtration and concentration to dryness, the obtained residue is purified by distillation. (R)-2- Bromo-3-phenylpropanoic acid (33 g) is obtained which has the following characteristics: B.p.1 kPa = 154°C; Rf = 0.47 (methylene chloride/methanol). The yield is 60%.
EXAMPLE AJ
(S)-2-Acetylthio-3-phenylpropanoic Acid Thioacetic acid (9.3 cc) and potassium carbonate (7.5 g) in water (150 cc) is added under nitrogen atmosphere to a solution of (R)-2-bromo-3- phenylpropanoic acid (25 g) in 1M sodium hydroxide (110 cc). The solution is stirred for 15 hours at a temperature close to 20°C. After evaporation under reduced pressure, the residue is taken up with water and ethyl acetate. The aqueous phase is separated, acidified to pH = 2 and then extracted with ethyl acetate. After washing, the organic phase is dried over sodium sulphate. After filtration and concentration under reduced pressure, (S)-2-acetylthio-3- phenylpropanoic acid (18.5 g) is obtained with a yield of 75% in the form of an oil having the following characteristics: Rf = 0.49 [hexane/ethyl acetate/acetic acid (6:4:0.5 by volume)]. EXAMPLE AK
(R)-2-Bromo-3-phenylpentanoic Acid
Working as in Example Al, but starting from D-isoleucine, (R)-2-bromo-3- phenylpentanoic acid having the following characteristics is obtained with a yield of 74%: Rf = 0.38 [methylene chloride/methanol (1:1 by volume)].
EXAMPLE AL (S)-2-Acetylthio-3-phenylpentanoic Acid
A solution of (R)-2-bromo-3-phenylpentanoic acid in dimethylformamide is added to a solution of thioacetic acid (1.5 equivalents) and sodium hydride (2.5 equivalents) in anhydrous dimethylformamide. The mixture is stirred for 3 hours at a temperature close to 20°C. After customary treatment, (S)-2-acetylthio-3- phenylpentanoic acid is obtained with a yield of 78% in the form of a yellow oil having the following characteristics: Rf = 0.44 [methylene chloride/methanol (9:1 by volume)]. EXAMPLE AM
(R)-2-Bromo-4-phenylbutanoic Acid
Working as in Example Al, but starting from D-2-amino-4-phenylbutanoic acid, (R)-2-bromo-4-phenylbutanoic acid having the following characteristics is obtained with a yield of 72%: Rf = 0.30 [methylene chloride/methanol/acetic acid (10:0.1:0.2 by volume)]. EXAMPLE AN
(S)-2-Acetylthio-4-phenylbutanoic Acid Working as in Example AL, but starting from (R)-2-bromo-4- phenylbutanoic acid, (S)-2-acetylthio-4-phenylbutanoic acid having the following characteristics is obtained with a yield of 80%: Rf = 0.35 [methylene
chloride/methanol/acetic acid (9:0.3:0.1 by volume)]. EXAMPLE AO
(R)-2-Bromo-3-methylbutanoic Acid
Working as in Example Al, but starting from D-valine, (R)-2-bromo-3- methylbutanoic acid is obtained with a yield of 69% in the form of an oil which crystallizes slowly having the following characteristics: Rf = 0.52 [methylene chloride/methanol/acetic acid (3:0.5:0.5 by volume)].
EXAMPLE AP (S)- 2-Acetylthio-3-methylbutanoic Acid
Working as in Example AJ, but starting from (R)-2-bromo-3- methylbutanoic acid, (S)-2-acetylthio-3-methylbutanoic acid is obtained with a yield of 94% in the form of an oil having the following characteristics: Rf = 0.28 [n-hexane/ethyl acetate (4:6 by volume)].
EXAMPLE AQ (R)- 2-Bromo-2-phenylacetic Acid
Working as in Example Al, but starting from D-phenylglycine, (R)-2- bromo-2-phenylacetic acid is obtained with a yield of 72% in the form of an oil which crystallizes slowly having the following characteristics: Rf = 0.63
[methylene chloride/methanol/acetic acid (9:1 :0.5 by volume)]. EXAMPLE AR
(S)-2-Acetylthio-2-phenylacetic Acid Working as in Example AJ, but starting from (R)-2-bromo-2-phenylacetic acid, (S)-2-acetylthio-2-phenylacetic acid is obtained with a yield of 74% in the form of an oil which crystallizes slowly having the following characteristics: Rf = 0.42 [n-hexane/ethyl acetate (4:6 by volume)]. EXAMPLE AS
L-lle-Tyr-OMe
1) A solution of methyl tyrosinate hydrochloride (1 equivalent) and triethylamine (1 equivalent) in chloroform, a solution of hydroxybenzotnazole (1 equivalent) in tetrahydrofuran and a solution of dicyclohexylcarbodiimide in chloroform are added successively at 0°C to a solution of t-butoxycarbonyl-L- isoleucine in tetrahydrofuran. The mixture is stirred for 1 hour at 0°C and then for 16 hours at a temperature close to 20°C. After customary treatment, the protected dipeptide (Boc-L-lle-Tyr-OMe) having the following characteristics is obtained with a yield of 98%: Rf = 0.47 [methylene chloride/methanol (9:1 by volume)].
2) The protected dipeptide is dissolved in methylene chloride and then treated with 20 equivalents of trifluoroacetic acid. After 1 hour at 0°C and 2 hours at a temperature close to 20°C, the mixture is concentrated under reduced pressure. The residue is triturated with ether and then dried under reduced pressure. The dipeptide L-lle-Tyr-OMe is thus obtained with a yield of 98% in the form of a white solid having the following characteristics: Rf = 0.23 [methylene chloride/methanol (9:1 by volume)].
EXAMPLE AT
L-Val-Tyr-OMe
1) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L-valine and methyl tyrosinate hydrochloride, the protected dipeptide (Boc-L-Val-Tyr- OMe) is obtained with a yield of 98% in the form of an oil having the following characteristics: Rf = 0.53 [methylene chloride/methanol (9:1 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-Val-Tyr-OMe is thus obtained with a yield of 98% in the form of a white solid having the following characteristics: Rf = 0.40 [methylene chloride/methanol/acetic acid (8:2:0-5 by volume)].
EXAMPLE AU L-Nle-Tyr-OMe
1 ) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L- norleucine and methyl tyrosinate hydrochloride, the protected dipeptide Boc-L- Nle-Tyr-OMe is obtained with a yield of 99% in the form of an oil having the following characteristics: Rf = 0.67 [methylene chloride/methanol (9:1 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-Nle-Tyr-OMe is thus obtained with a yield of 70% in the form of a white solid having the following characteristics: Rf = 0.10 [methylene chloride/methanol (25:5 by volume)].
EXAMPLE AV L-Leu-Tyr-OMe
1 ) Working as in Example AS-1, but starting from t-butoxycarbonyl-L-leucine and methyl tyrosinate hydrochloride, the protected dipeptide Boc-L-Leu-Tyr- OMe is obtained with a yield of 99% in the form of an oil having the following characteristics: Rf = 0.42 [methylene chloride/methanol (9:1 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-Leu-Tyr-OMe is thus obtained with a yield of 82% in the form of a white solid having the following characteristics: Rf = 0.11 [methylene chloride/methanol (9:1 by volume)]. EXAMPLE AW L-Phe-L-Tyr-OMe 1) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L- phenylalanine and methyl L-tyrosinate hydrochloride, the protected dipeptide Boc-L-Phe-L-Tyr-OMe is obtained with a yield of 97% in the form of an oil having the following characteristics: Rf = 0.5 [methylene chloride/methanol (9:1 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-Phe-L-Tyr-OMe is thus obtained with a yield of 85% in the form of a white solid having the following
characteristics: Rf = 0.25 [methylene chloride/methanol/acetic acid (9:1 :05 by volume)].
EXAMPLE AX
L-lle-L-Pro-OMe
1) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L- isoleucine and methyl L-prolinate, the protected dipeptide Boc-L-lle-L-Pro-OMe is obtained with a yield of 92% in the form of an oil having the following characteristics: Rf = 0.44 [methylene chloride/methanol (9:0.5 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-lle-L-Pro-OMe is thus obtained with a yield of 96% in the form of a white solid having the following characteristics: Rf = 0.37 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)].
EXAMPLE AY
L-Val-L-Pro-OMe 1) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L-valine and methyl L-prolinate, the protected dipeptide Boc-L-Val-L-Pro-OMe is obtained with a yield of 98% in the form of an oil having the following
characteristics: Rf = 0.42 [methylene chloride/methanol (9:0.5 by volume)].
2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-Val-L-Pro-OMe is thus obtained with a yield of 86% in the form of a white solid having the following
characteristics: Rf = 0.50 [methylene chloride/methanol/acetic acid (9:1 :0.5 by volume)]. EXAMPLE AZ
L-lle-L-Pip-OMe
1 ) Working as in Example AS-1 , but starting from t-butoxycarbonyl-L- isoleucine and L-2-methoxycarbonylpiperidine, the protected dipeptide Boc-L- lle-L-Pip-OMe is obtained with a yield of 89% in the form of an oil having the following characteristics: Rf = 0.70 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]. 2) The elimination of the protective group is carried out under the conditions described in Example AS-2. The dipeptide L-lle-L-Pip-OMe is thus obtained with a yield of 79% in the form of a white solid having the following characteristics: Rf = 0.42 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]. EXAMPLE BA
N-(2-mercapto-3-phenylpropanoyh-Val-Tyr
Val-Tyr-OMe trifluoroacetate (1 equivalent) and triethylamine (1 equivalent) in chloroform and then a solution of 1-hydroxybenzotnazole (1 equivalent) in tetrahydrofuran and a solution of dicyclohexylcarbodiimide (1 equivalent) in chloroform are added successively at 0°C to a solution of 2- acetylthio-3-phenyipropanoic acid (1 equivalent) in tetrahydrofuran. The mixture is stirred for 1 hour at 0°C and for 16 hours at a temperature close to 20°C. The precipitate is separated by filtration and the filtrate is concentrated to dryness under reduced pressure. The residue obtained is dissolved in ethyl acetate and then washed successively with a 10% citric acid solution (w/v), with water, with a 10% sodium carbonate solution (w/v), with water, then with a saturated sodium chloride solution. The organic phase is dried over sodium sulphate. After filtration and concentration to dryness, the residue is purified by
chromatography. N-(2-Acetylthio-3-phenylpropanoyl)-Val-Tyr-OMe is thus obtained with a yield of 70%, in the form of an oily product. Rf = 0.54
[methylene chloride/methanol (9:0.5 by volume)].
The product obtained above is dissolved in degassed methanol and a 1 M sodium hydroxide solution (3 equivalents) is added at 0°C. The mixture is stirred for 2 hours at a temperature close to 20°C and then a partition is carried out between degassed water and degassed ethyl acetate. The aqueous phase is acidified to pH = 2 and is then extracted with degassed ethyl acetate. The organic phase is washed with a saturated aqueous solution of sodium chloride, dried over sodium sulphate, filtered and then concentrated under reduced pressure. The residue obtained is chromatographed on silica gel, eluting with a methylene chloride/methanol/acetic acid mixture (9:0.5:0.5 by volume). N-(2- mercapto-3-phenylpropanoyl)-Val-Tyr is thus obtained with a yield of 65% in the form of a white solid having the following characteristics: high performance liquid chromatography: retention time: 8 minutes [acetonitrile/0.05%
trifluoroacetic acid (45:55 by volume)]: proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.8
[CH3(Val)]; 1.90 [CHβ (Val)]; 2.60 (HS); 2.70, 2.80 and 2.05 [CH2β (Tyr + Phe)]; 3.70 [CH α (Val)]; 4.20 (CH α (Phe + Tyr)]; 6.6 and 6.95 [Ar (Tyr)]; 7.8 [Ar (Phe)]; 8.05 and 8.20 (NH); 9.15 [OH (Tyr)]; 12.45 (COOH).
EXAMPLE BB
N-(2-Mercapto-3-phenylpropanoyl)-lle-Tyr-OMe The coupling of 2-acetylthio-3-phenylpropanoic acid with the protected dipeptide lle-Tyr-OMe is carried out under the conditions described in Example BA. N-(2-Acetylthio-3-phenylpropanoyl)-lle-Tyr-OMe is obtained with a yield of 75% in the form of an oily product. Rf = 0.17 [hexane/ethyl acetate (5:5 by volume)]. Deprotection is carried out under the conditions of Example BA. N-(2- Mercapto-3-phenylpropanoyl)-lle-Tyr-OMe is thus obtained with a yield of 70% in the form of a white solid melting at 185°C. Rf = 0.28 [methylene
chloride/methanol/acetic acid (9:1 :0.5 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.75 [CH3 (lle)]; 1.1 , 1.5 and 1.7 [CHβ CH2γ (lle)]; 2.70 (HS); 2.80, 2.90 and 3.15 [CH2β (Phe + Tyr)]; 3.65, 4.15 and 4.30 [CH2 α (Phe + lle + Tyr)]; 6.60 and 7.00 [Ar (Tyr)]; 7.20 [Ar (Phe)]; 8.00 and 8.20 (NH); 12.20 (COOH). EXAMPLE BC
N-(2-Mercapto-3-phenylpropanoyl)-lle-Pip
The coupling of 2-acetylthio-3-phenylpropanoic acid with the protected dipeptide lle-Pip-OMe is carried out under the conditions described in Example BA. N-(2-Mercapto-3-phenylpropanoyl)-lle-Pip-OMe is obtained with a yield of 51% in the form of an oily product. Rf = 0.30 [cyclohexane/ethyl acetate (5:5 by volume)]. Deprotection is carried out under the conditions described in Example
BA. N-(2-Mercapto-3-phenylpropanoyl)-lle-Pip is thus obtained with a yield of 65% in the form of a white solid. Rf = 0.48 [methylene chloride/methanol/acetic acid (9:0-5:0-25 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH3 ( lle)]; 1.02, 1.50 and 2.05 [CH2β,γ and γ(Pip)]; 2.6 (HS); 2.80 and 3.05 [CH2β (Phe)]; 3.7 [CH2ε (Pip)]; 4.20, 4.70 and 4.95 (CH α; 7.20 [Ar (Phe)]; 8.20 and 8.40 (NH); 12.78 (COOH).
EXAMPLE BD N-(2-Mercapto-3-phenylpropanoyl) -Nle-Tyr
The coupling of 2-acetylthio-3-phenylpropanoic acid with the protected dipeptide Nle-Tyr-OMe is carried out under the conditions described in Example BA. N-(2-Acetylthio-3-phenylpropanoyl)-Nle-Tyr-OMe is thus obtained with a yield of 64% in the form of an oily product. Rf = 0.21 [hexane/ethyl acetate (6:4 by volume)]. Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-3-phenylpropanoyl)-Nle-Tyr is thus obtained with a yield of 61% in the form of a white solid. Rf = 0.53 [methylene chloride/methanol (9:1 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH3 (Nle)]; 1.25 and 1.50 [CH2 β,g and δ (Nle)]; 2.00 (HS); 2.75 and 2.95 [CH2β (Phe + Tyr)]; 3.70 and 4.20 [CH α (Nle, Phe, Tyr)]; 6.60 and 6.95 [Ar (Tyr)]; 7.20 [Ar (Phe)]; 8.10 and 8.20 (NH); 9.15 [OH (Tyr)]; 12.3 (COOH); high performance liquid chromatography:
retention time: 9.3 minutes [acetonitrile/0.05% trifluoroacetic acid (46:54 by volume)].
EXAMPLE BE N-(2-Mercapto-3-phenylpropanoyl)-Leu-Tyr
The coupling of 2-acetylthio-3-phenylpropanoic acid with the protected dipeptide Leu-Tyr-OMe is carried out under the conditions described in Example BA. N-(2-Acetylthio-3-phenylpropanoyl)-Leu-Tyr-OMe is thus obtained in the form of an oily product. Rf = 0.18 [cyclohexane/ethyl acetate (5:5 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-3-phenylpropionyl)-Leu-Tyr is thus obtained with a yield of 70% in the form of a white solid. Rf = 0.45 [methylene chloride/methanol/acetic acid (9:1 :0.5 by volume)]; proton nuclear magnetic resonance spectrum
(deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH3 (Leu)]; 1.30 [CH2β (Leu)]; 1.5 (CH2γ (Leu)]; 2.60 (HS); 2.70, 2.80 and 3.00 [CH2β (Phe + Tyr)]; 3.50 and 4.20 [CH2 α (Phe, Leu, Tyr)]; 6.60 and 6.90 [Ar (Tyr)]; 7.20 [Ar (Phe)]; 7.90 and 8.05 (NH); 9.10 [OH (Tyr)]; 12.50 (COOH).
EXAMPLE BF
N-(2-Mercapto-3-methylpentanovyl)Phe-Tyr The coupling of 2-acetylthio-3-methylpentanoic acid with the protected dipeptide Phe-Tyr-OMe is carried out under the conditions described in Example BA. N-(2-Acetylthio-3-methylpentanoyl)-Phe-Tyr-OMe is thus obtained with a yield of 78% in the form of an oily product. Rf = 0.14 [cyclohexane/ethyl acetate (6:4 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-3-methylpentanoyl)-Phe-Tyr melting at 120°C is thus obtained with a yield of 70%. Rf = 0.40 [methylene chloride/methanol/acetic acid (9:1:0.2 by volume)]; proton nuclear magnetic resonance spectrum
(deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.75 [CH3 (lle)]; 1.00, 1.20 and 1.60 [CH2β, CH2γ (lle)]; 2.65 (HS); 2.70 and 2.90 [CH2β (Phe + Tyr)]; 6.60 and 6.95 [Ar (Tyr)]; 7.13 [Ar (Phe)]; 8.00 and 8.05 (NH); 9.10 [OH (Tyr)]; 12.6 (COOH).
EXAMPLE BG N-(2-Mercapto-3-methylpentanoyl)-Val-Tyr
The coupling of 2-acetylthio-3-methylpentanoic acid with the protected dipeptide Val-Tyr-OMe is carried out under the conditions described in Example BA. N-(2-Acetylthio-3-methylpentanoy!)-Val-Tyr-OMe is thus obtained with a yield of 78% in the form of an oily product. Rf = 0.41 [methylene
chloride/methanol (9:5:0.5 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-3-methylpentanoyl)-Val-Tyr is thus obtained with a yield of 72% in the form of a white solid melting at 150°C. Rf = 0.20 [methylene chloride/methanol/acetic acid (9:0.5:0.25 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.80 [CH3 (Val + lle)]; 1.00 and 1.25 [CH2γ (lle)]; 1.65 [CHβ (lle)]; 1.90 [CHβ (Val)]; 2.30 (HS); 2.70 and 2.85 [CH2β (Tyr)]; 3.30 [CH α (lle)]; 4.20 and 4.32 [CH α (Val and Tyr)]; 6.68 and 6.95 [Ar (Tyr)]; 7.90 and 8.10 [NH (Val and Tyr)]; 9.20 [OH (Tyr)]; 12.56 (COOH). EXAMPLE BH N-(2-Mercapto-4-phenylbutanoyl-Val -Tyr The coupling of 2-acetylthio-4-phenylbutanoic acid with the protected dipeptide Val-Tyr-OCH3 is carried out under the conditions described in
Example BA. N-(2-Acetylthic-4-phenylbutanoyl)-Val-Tyr-OCH3 is thus obtained with a yield of 82% in the form of an oily product. Rf = 0.49 [cyclohexane/ethyl acetate (5:5 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-4-phenylbutanoyl)-Val-Tyr is thus obtained with a yield of 98% in the form of a white product melting at 105°C. Rf = 0.65 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]; high performance liquid chromatography: retention time: 9.10 minutes[acetonitrile/0.05% trifluoroacetic acid (45:55 by volume)]; proton nuclear magnetic resonance spectrum
(deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.75 [CH3 (Val)]; 1.75 [CHβ (Val)]; 1.95 [CH2γ (Phe)]; 2.50 [CH2β (Phe)]; 2.72 and 2.90 [CH2β (Tyr)]; 3.45 [CH α (Phe)]; 4.20 and 4.30 [CH α (Tyr and Val)]; 6.60 and 7.00 [Ar (Tyr)]; 7.12 and 7.20 [Ar (Phe)]; 7.20 and 8.10 (NH); 9.15 [OH (Tyr)]; 12.35 (COOH).
EXAMPLE Bl
N-(2-Mercapto-3-Methylbutanoyl) -lle-Tyr
The coupling of (S)-2-acetylthio-3-methylbutanoic acid with the protected dipeptide lle-Tyr-OCH3 is carried out under the conditions described in Example BA.
N-(2-Acetylthio-3-methylbutanoyl)-lle-Tyr-OCH3 is obtained with a yield of 85% in the form of an oily product. Rf = 0.28 [n-hexane/ethyl acetate (4:6 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-3-methylbutanoyl)-lle-Tyr is obtained with a yield of 89% in the form of a white product melting at 199°C. Rf = 0.42 [methylene
chloride/methanol/acetic acid (9:1 :0.5 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.75 and 0.90 [CH3 (Val and lle)]; 1 [CHβ (lle)]; 1.40 and 1.65 [CH2γ (lle)]; 1.80 [CHβ (Val)]; 2.30 (HS); 2.75 and 2.89 [CH2β (Tyr)]; 3.15 [CH (SH)]; 4.15 [CH cc (lle)]; 4.32 [CH cc (Tyr); 6.55 and 6.95 [Ar (Tyr)]; 7.9 and 8.10 (NH); 9.12 [OH (Tyr)]; 12.10 (COOH); high performance liquid chromatography: retention time: 5.84 minutes [acetonitrile/0.05% trifluoroacetic acid (5:5 by volume)].
EXAMPLE BJ
N-(2-Mercapto-2-phenylacetyl) -lle-Tyr
The coupling of (S)-2-acetylthio-2-phenylacetic acid with the protected dipeptide lle-Tyr-OCH3 is carried out under the conditions described in Example BA. N-(2-Acetylthio-2-phenylacetyl)-lle-Tyr-OCH3 is obtained with a yield of 46% in the form on an oily product. Rf = 0.42 [n-hexane/ethylacetate (4:6 by volume)].
Deprotection is carried out under the conditions described in Example BA. N-(2-Mercapto-2-phenylacetyl)-lle-Tyr is obtained with a yield of 73% in the form of an oil. Rf = 0.42 [methylene chloride/methanol/acetic acid (9:1:0.5 by volume)]; proton nuclear magnetic resonance spectrum (deuterated dimethyl sulphoxide; chemical shifts in ppm): 0.61 and 0.72 [CH3 (lle)]; 1.1 [CHβ (lle)]; 1.4 and 1.7 [CH2γ (lle)]; 2.32 (HS); centered on 2.80 [CH2β (Tyr)]; 3.3 [CH (SH)]; 4.20 [CH2 α (Tyr)]; 4.82 [CH α (lle)]; 6.55 and 6.95 [Ar (Tyr)]; centered on 7.30 [Ar (Phe)]; 8.15 [NH ( lle and Tyr)]; 9.10 [OH (Tyr)]; 12.45 (COOH).
EXAMPLES BK TO Cl
Working as in Example BA, the products which are collected in Table II are obtained. * *
- * 1 1
Figure imgf000056_0001
* *
-C H- - *
Figure imgf000057_0001
* *
- 1 1
Figure imgf000058_0001
2. PREPARATION OF THE PRODUCT ACCORDING TO THE INVENTION EXAMPLE 1 - N-[2-adamantoylthiomethyl-1-oxo-3-phenylbutyl]alanine
The product (1.7 g) of Example N is dissolved in degassed water (18 mL). To the solution under a nitrogen atmosphere and at 0°C is added a solution 1.3 X 10-2 M sodium hydroxide (2.2 eq) and adamantoyl chloride (1.33 g, 1.1 eq). The mixture is stirred for four hours at ambient temperature. The mixture is then acidified to pH 3 with 1 M hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with water and then a saturated NaCI solution. The organic phase is dried over anhydrous Na2SO4, filtered and evaporated to dryness. A white solid (2 g) is obtained in a yield of 74% having the following characteristics: m.p. 80°C; Rf = 0.45 [chloroform/methanol/ acetic acid (7:3:0.3 by volume)]. EXAMPLE 2 - (S,S,S)-N-[2-adamantoylthiomethyl-1-oxo-3-phenylbutyl]alanine
The title compound is prepared as in Example 1, except starting with the product of Example AH(a)
Compounds within the scope of the present invention, when administered to mammals, are potent ACE and/or NEP inhibitors; preferably antihypertensives due to double inhibitory action where they inhibit both NEP and ACE. The ACE inhibitors within the scope of the present invention are capable of blocking the increase in the blood pressure caused by an increase in the vascular resistance and the blood volume due to the formation of angiotensin II from angiotensin I since ACE inhibitors block the production of angiotensin II. The NEP inhibitors within the scope of the present invention regulate auricular natriuretic peptide which is implicated in the regulation of arterial pressure. This peptide is liberated by the heart, endowed with a vasodilatory property and capable of controlling diuresis and natriuresis. Auricular natriuretic peptide is inactivated by NEP in the peripheral tissues. By inhibiting NEP with the compound according to the invention, a significant augmentation in diuresis and natriuresis in man is induced without causing the appearance of an increase in the amounts of renin and aldosterone that is usually found with the diuretics generally used in association with ACE inhibitors. The compounds according to the invention that are mixed inhibitors of
NEP and ACE may be used to alleviate hypertension of various origins without the co-administration of other diuretics.
Thus compounds according to the present invention are effective in the treatment of congestive heart failures and various types of hypertension, in particular hypertension connected with an increase in the blood volume.
Accordingly, the antihypertensive properties exhibited by the present
compounds that are inhibitors of both ACE and NEP are preferred to the effects obtained with the concomitant administration of either or both of NEP and ACE inhibitors. The enzymatic inhibitory properties of compounds according to the present invention are measured using [3H]D-Ala2-Leu-enkephalin as a substrate for neutral endopeptidase as described previously by Llorens et al., Biochem. Biophys. Res. Commun., 96 , 1710 (1980) and Z-Phe-His-Leu in the case of peptidyldipeptidase A according to the process described in Biochem. Biophys. Acta, 206, 136-142 (1970).
The antihypertensive properties of the present compounds are
determined in rat models including that of rat hypertension induced by DOCA salt and the spontaneously hypertensive male rat (SHR) according to Trapani et al., J. Cardiovasc. Pharmacol., 14, 419-424 (1989).
Table III below presents selected pharmacological data for the N- mercaptoacyl (amino acid or peptide) compounds having both ACE and NEP inhibitory properties. These compounds may be acylated with an S-lipophilic aliphatic acyl radical to form compounds within the scope of the present invention. The results in Table III also gives the results with N-(2-mercaptomethyl-3-phenyl-1-oxopropyl)glycine (thiorphan), which is very closely related structure to the N-mercaptoacylamino acid portion of the compound of formula I, for comparison to show that thiorpan manifests activity only against NEP.
Figure imgf000060_0001
Figure imgf000061_0001
This invention is directed to the unexpected finding that protection of the thiol group was protected by a lipohilic aliphatic carbonyl results in compounds have an increased ACE and/or NEP inhibitory activity over the compounds that are acylated with an acylating group, e.g., acetyl, have lower lipophilic character.
Example 3 - Comparison of In Vivo Inhibition of NEP in Mouse by Orally
Administered [S,S,S]-N-[2-adamantoylthiomethyl-1-oxo-3- phenylbutyl]alanine (I') and diastereomeric mixture of N-[2-acetyl- thiomethyl-1-oxo-3-phenylbutyl]alanine (I")
[S,S,S]-N-[2-adamantoylthiomethyl-1-oxo-3-phenylbutyl]alanine, diastereomeric mixture of N-[2-acetyl-thiomethyl-1-oxo-3-phenylbutyl]alanine (four diasteromers) and a control solution of saline are orally administered at a concentration of 2.6 x 10-5 mole/Kg) in mice at t=o. At 30 and 480 minutes following their administration, a highly selective and tritiated, NEP inhibitor,
[3H]HACBOGIy (1μCi) (Waksman et al., PNAS, 1986, 1523-1527) is
administered by i.v. route to a mouse. The mouse is killed 15 minutes
thereafter, the kidney rapidly set apart, and homogenized at 4°C in a Tris HCI buffer 50 mM (pH 7.4). The homogenate is filtered and the radioactivity bound to the filter is measured by liquid scintillation. The non-specific binding is determined after co-injection of [3H]HACBOGIy with 10,000 equivalents of the specific NEP inhibitor, retrothiorphan (Roques et al., PNAS, 1983, 80, 3178- 3182).
The inhibition of NEP in the kidney is expressed as the difference (in %) between specific [3H]HACBOGIy binding in the absence (controls injected with saline) and presence of inhibitor. The inhibition values in Table IV are the mean value of three determinations.
Table IV
Time % Inhibition
ACE NEP
|, ||, I"
30 min. 75 30 85 65 240 min. 30 15 70 45
Example 4 - Comparison of In Vivo Inhibition of ACE in Mouse by Orally
Administered (S,S,S)-N-[2-adamantoylthiomethyl-1-oxo-3-phenyl- butyl]alanine (I') and diastereomeric mixture of N-[2-acetyl- thiomethyl-1-oxo-3-phenylbutyl]alanine (I")
The protocol in Example 3 is used also to determine NEP inhibition except for the following: the selective tritiated ACE inhibitor is
[3H]Trandaloprilate (0.5μ Ci); and the non-specific binding is determined by the co-administration of [3H]Trandaloprilate with 1000 eq. of captopril. The NEP inhibition values are given in Table IV above. Under the same reaction parameters as in Example 4, ACE inhibition is determined at 30, 60, 120 and 240 minutes for N-(2-acetylthiomethyl-1-oxo-3- phenylbutyl)alanine as a mixture of four stereoisomers (-□ -), N-(2- adamantylthiomethyl-1-oxo-3-phenylbutyl)alanine as a mixture of four stereoisomers (- O -) and the single stereoisomer (S,S,S)-N-(2-adamantyl- thiomethyl-1-oxo-3-phenylbutyl)alanine (-■ -). Figure 1 graphically represents the results and shows that the adamantylcarbonyl derivatized compounds exhibit unexpectedly greater inhibition than the acetylated compound. The products according to the invention may generally be administered orally or parenterally for the treatment of patients suffering from hypertension. The products according to the invention, in base or salt form, may be presented in forms permitting administration by the most suitable route and the invention also relates to pharmaceutical compositions containing at least one product according to the invention which are suitable for use in human or veterinary medicine. These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants comprise, inter alia, diluents, sterile aqueous media and the various non-toxic organic solvents. The compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or
suspensions, injectable solutions, elixirs or syrups, and can contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, or stabilizers in order to obtain pharmaceutically acceptable preparations.
The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the product, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as ethanol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
For parenteral administration, suspensions or solutions of the products according to the invention in sesame oil, groundnut oil or olive oil or aqueous solutions of propylene glycol, as well as sterile aqueous solutions of the pharmaceutically acceptable salts, are used. The solutions of the salts of the products according to the invention are especially useful for administration by intramuscular or subcutaneous injection. The aqueous solutions, also
comprising solutions of the salts in pure distilled water, may be used for intravenous administration with the proviso that their pH is suitably adjusted, that they are judiciously buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride and that they are sterilized by heating or
microfiltration.
The doses used in the methods according to the invention are those which lead to a maximal therapeutic effect until an improvement is obtained. In general, the doses used are those which are therapeutically effective for lowering blood pressure during the treatment of hypertension. In general, the doses administered orally are between 0.1 and 100 mg/kg, and preferably between 1 and 10 mg/kg, and those administered intravenously are between 0.01 and 10 mg/kg, and preferably between 0.1 and 5 mg/kg, on the
understanding that, in each particular case, the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health and other characteristics which can influence the efficacy of the medicinal product.
The products according to the invention may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the active product may be administered orally 1 to 4 times per day. It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.
The products according to the invention may be used in injectable form in emergency cases of acute hypertension. Such a treatment may be followed by an intravenous perfusion of the active product so as to obtain and maintain the desired therapeutic effect.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than the specification, as indicating the scope of the invention.

Claims

WHAT IS CLAIMED IS:
1. An S-lipophilic aliphatic carbonyl [N-mercaptoacyl(amino acid or peptide)] compound.
2. The compound of claim 1 of formula (I)
* *
R- S - CH2 - CH- CONH-CH-COOR'
| |
* R3
1
Figure imgf000065_0002
(I) wherein
R is a lipophilic aliphatic carbonyl radical; R1 is aryl or heteroaryl;
I
R2 is alkyl, or alkylene attached to the
Figure imgf000065_0001
moiety and R1;
R3 is hydrogen, alkyl, aryl, alkoxy or aryloxy; and R' is hydrogen, alkyl, aralkyl, acyl or aroyl; or a pharmaceutically acceptable salt thereof.
3. The compound of claim 2 wherein
R is higher alkyl carbonyl or multi cycloalkyl carbonyl; R1 is aryl or heteroaryl; R2 lower alkyl, or alkylene attached to the
Figure imgf000066_0002
moiety and R1;
R3 is hydrogen, lower alkyl, aryl or heteroaryl, lower alkoxy or aryloxy; and
R' is hydrogen, lower alkyl, aryl lower alkyl, acyl or aroyl.
4. The compound of claim 3 wherein
R is adamantoyl or palmitoyl; R1 is phenyl, 2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl radical, optionally substituted with one or more identical or different aryl group substituents chosen from halo, hydroxy, C1-4 acyloxy, C1-4 alkoxy, phenoxy, phenylthio and amino radicals, C1-4 dialkylamino, methylenedioxy and ethylenedioxy; R2 is methyl or trifluoromethyl radical, or -CH2-Y-, -Y-CH2- or
|
C2-3 alkylene attached to the
Figure imgf000066_0001
moiety and R1, wherein R1 is phenyl,
2-, 3- or 4-pyridyl, N-methyl-2- or -3-pyrrolyl, 2- or 3-furyl or 2- or 3-thienyl, Y is oxygen, sulphur or nitrogen substituted by methyl, or alkylene is optionally substituted with a methyl;
R3 is hydrogen, trifluoromethyl, C1-8 alkoxy, phenoxy, phenyl or thienyl or C1-8 alkyl optionally substituted with phenyl, hydroxy, C1-4 alkoxy, phenoxy, C1-4 alkylthio, phenylthio, benzyloxy or benzylthio, wherein the phenyl and the phenyl portions of the phenoxy, phenylthio, benzyloxy or benzylthio radicals are optionally substituted with one or more identical or different aryl group
substituents chosen among halo, hydroxy, C1-4 alkoxy, amino,
C1-4 dialkylamino, methylenedioxy or ethylenedioxy; and
5. The compound of claim 1 having a configuration for the asterisked carbons selected from the group consisting of R,R,S, S,R,S, S,S,S and R,S,S.
6. The compound of claim 5 wherein the configuration is selected from the group consisting of S,S,S and R,S,S.
7. The compound of claim 6 having a configuration of R,S,S.
8. The compound of claim 1 of formula (II) R6 R7
* *
R-S-CH-CONH-CH-CON-CH-COOR"
I I
CH CH *
/ \ / \
A1 R4 A2 R5
(II) wherein
R is a lipophilic aliphatic carbonyl radical; R4 and R5 are independently hydrogen, alkyl, aryl, aralkyl, alkoxy, alkyloxymethyl or aralkyloxy;
A1 and A2 are independently hydrogen, alkyl, or together with -CH-R4 or -CH-R5, respectively, form phenyl, or alkylene which together with -CH- R4 or -CH-R5, respectively, form benzocycloalkyl; R8 is hydrogen or alkyl; G7 is cycloalkyl, aralkyl or aryloxymethyl or alkoxymethyl, or R8 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form heterocyclyl; and
R" is hydrogen, alkyl, aralkyl, cycloalkyl lower alkyl or palmitoyl; or a pharmaceutically acceptable salt thereof. or a pharmaceutically acceptable salt thereof.
9. The compound of claim 8 wherein
R is higher alkyl carbonyl or multi cycloalkyl carbonyl; R4 and R5 are independently hydrogen, lower alkyl, phenyl, aryl lower alkyl, lower alkoxy, lower alkyloxymethyl or aryl lower alkyloxy;
A1 and A2 are independently hydrogen, lower alkyl or together with -CH- R4 or -CH-R5, respectively, form phenyl, or alkylene which together with -CH- R4 or -CH-R5, respectively, form benzocycloalkyl;
R6 is hydrogen or lower alkyl; G7 is cycloalkyl, aryl lower alkyl or aryloxymethyl or lower alkoxymethyl, or R6 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form heterocyclyl; and
R" is hydrogen, lower alkyl, aryl lower alkyl, cycloalkylmethyl or palmitoyl.
10. The compound of claim 9 wherein
R is adamantoyl or palmitoyl; R4 and R5 are independently lower alkyl, lower alkoxy, phenyl, benzyl or benzyloxy;
A1 and A2 are independently hydrogen, methyl, or together with -CH- R4 or -CH-R5, respectively, form phenyl, or alkylene together with -CH- R4 or -CHHR5, respectively, form indanyl; G7 is benzyl wherein the phenyl ring is optionally substituted by hydroxyl, or R6 and G7 and the carbon and nitrogen atoms through which R6 and G7 are attached taken together form a saturated 5 or 6 member heterocyclyl; and R" is hydrogen.
11. The compound of claim 8 wherein the configuration at each of the asterisked carbons is L
12. Process for preparing the compound of claim 1 comprising acylating an N-mercaptoacyl(amino acid or peptide) with a lipophilic aliphatic carbonyl halide.
13. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
14. A method of treating hypertension in a mammal comprising administering an therapeutic effective antihypertensive amount of a compound of claim 1.
15. The method of claim 14 wherein the compound is an inhibitor of peptidyldipeptidase A and neutral endopeptidase.
16. The method of claim 14 wherein the compound is an inhibitor of peptidyldipeptidase A.
17. The method of claim 14 wherein the compound is an inhibitor of neutral endopeptidase.
PCT/EP1993/000147 1993-01-22 1993-01-22 S-lipophilic aliphatic carbonyl [n-mercaptoacyl-(amino acid or peptide)] compounds as antihypertensive agents WO1994017036A1 (en)

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