PEPTIDE DERIVATIVES WITH METALLOPEPTIDASE INHIBITORY ACTIVITY

Abstract: Compounds of formula (I) wherein R is a biphenyl group optionally substituted by one or more substituents, the same or different, selected among halogen atoms, nitro groups, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C1-C3 alkyl groups containing one or more fluorine atoms, carboxy groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R1 is a hydrogen atom, a straigth or branched C1-C6 alkyl or an arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a biphenyl, a naphthyl or a 5- or 6-membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C1-C3 alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R2 is a straigth or branched C1-C6 alkyl optionally substituted by one or more substituents selected among amino, mono- or di-(C1-C6)alkylamino or aminocarbonyl groups, mono or di-(C1-C6)-alkylaminocarbonyl groups, an arylalkyl, an arylcarbonylaminoalkyl, an arylaminocarbonylalkyl or an arylalkylaminocarbonylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety, the aryl being optionally substituted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C1-C3 alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; X is a single bond or an -N(R3)- group wherein R3 is a hydrogen atom or a straight or C1-C4 alkyl group; the carbon atoms marked with an asterisk are asymmetric carbon atoms; and their pharmaceutically acceptable salts, are described. The compounds of formula (I) are endowed with a mixed ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.

Description: PEPTIDE DERIVATIVES UITH METALLOPEPTIDASE INHIBITORY ACTIVITY ***************** The present invention relates to peptide derivatives with metallo- peptidase inhibitory activity and, more particularly, it relates to carboxyalkylpeptide derivatives useful in the treatment of cardio¬ vascular diseases as metallopeptidase inhibitors. The pharmacologic interest towards the study of metallopeptidase inhibitory molecules derives from the role that said enzymes exert on the level of the cardiocirculatory system. It is well-known, in fact, that compounds with angiotensin convert¬ ing enzyme (ACE) inhibitory activity are mainly useful in the treat¬ ment of hypertension and of cardiac failure in that they inhibit the formation of angiotensin II, a substance which increases the blood pressure. Compounds with endothelin converting enzyme (ECE) inhibitory activi¬ ty are useful as anti-vasoconstrictors in that they inhibit the formation of endothelin, a 21 amino acid peptide with vasoconstric¬ tor activity. Instead, compounds with inhibitory activity of the neutral endopep- tidase enzyme (NEP), also called enkephalinase, are useful as vaso¬ dilators in that the NEP enzyme is responsible for the inactivation, not only of endogenous enkephaline, but also of atrial natriuretic factor (ANF), a vasodilator hormone secreted by heart. Therefore, even exerting their action on the cardiovascular system with different mechanisms of action, the compounds with metallopep¬ tidase inhibitory activity are generally used, alone or in combina¬ tion, in the treatment of hypertension, renal failure and congestive heart failure. Uithin the field of the metallopeptidase inhibitors, the carboxyal- kylpeptide derivatives are known since several years. For instance, N-carboxyalkyl dipeptides with ACE-inhibitory activity are described in the U.S. patent No. 4,374,829 (Merck & Co., Inc.), in the U.S. patent No. 4,487,716 (American Home Products Corp.) and in the U.S. patent No. 4,666,906 (USV Pharmaceutical Corporation); N-carboxyalkyl dipeptides with enkephalinase-inhibitory activity are described in the U.S. patent No. 4,721,726 (Schering Corporation), in the European patent application No. 54862 (Schering Corporation), in the European patent application No. 38758 (Roques et al.) and in the U.S. patent No. 4,610,816 (Schering Corporation); N-carboxyal- kylacylamino acids with ACE-inhibitory activity are described in the U.S. patent No. 4,105,789 (E.R. Squibb & Sons, Inc.). None of the aforementioned documents describes carboxyalkylpeptide derivatives wherein the C-terminal amino acid derivative is a bi¬ phenylalanine derivative. The International patent application No. 94/03481 (Schering Corpora¬ tion) describes aminoalkyl derivatives of alpha,omega-diamino acids which are synthetic intermediates for the preparation of NEP and ACE inhibitors. The European patent application No. 126974 (G.D.Searle & Co.) de¬ scribes a considerable number of N-carboxyalkyl peptides, among which also the dipeptides of formula COR "1 R 3 R 1 * I I I A 1 -A 2 -Y-(CH 3 )„-CH-NH-C-CO-N—C-COR ' " β (A) I I I R 2 1 β R 1 ~ wherein the meanings of the ~ Λ & and R 1"7 radicals, which represent the substituents of the C-terminal amino acid, are the following: R ή represents hydrogen, alkyl or aralkyl; W ~ represents hydrogen, alkyl; substituted alkyl wherein the sub- stituent can be one or more groups selected among hydroxy, alk¬ oxy, aryloxy, aralkoxy, mercapto, alkylthio, arylthio, alkyl- sulphinyl, alkylsulphonyl, carboxy, carboxamido, carboxyalkyl, carboxyaralkyl, aralkoxycarbonylamino, amino, dialkylamino, acylamino, aroylamino and trihalomethyl groups; aralkyl, substi¬ tuted aralkyl wherein the substituent in the aryl moiety can be one or more groups selected among halogen, alkyl, hydroxy, alk¬ oxy, aralkoxy, amino, aminomethyl, cyano, alkylamino, dialkylami¬ no, carboxy, sulphonamido, alkylthio, nitro and phenyl groups; or a heteroaralkyl; and R 1B represents amino, alkylamino, dialkylamino; substituted alkyl¬ amino wherein the substituent is amino, hydroxy, alkoxy, carboxy, carboxamido, carboxyalkyl, alkylthio, alkylsulphinyl or alkyl¬ sulphonyl; hydroxyamino, alkoxyamino, aralkylamino, alkoxy, aralkoxy or alkylaminoalkoxy groups. The compounds of formula (A) , among which are comprised but not exemplified the peptides wherein the C-terminal amino acid deriva¬ tive is an ester or an amide of the biphenylalanine, are endowed with collagenase inhibitory activity and are thus useful in the treatment of articular diseases such as rheumatoid arthritis and correlated diseases. As reported by the inventors in the same patent application (page 2 - last paragraph) , the compounds of formula (A) are substantially devoid of ACE-inhibitory activity. Now we have found carboxyalkylpeptide derivatives, wherein the C-terminal amino acid group is a biphenylalanine optionally substi¬ tuted in the biphenyl moiety, endowed with inhibitory activity on the angiotensin converting enzyme as well as on the neutral endopeptidase enzyme (mixed ACE/NEP-inhibitory activity) which renders them particularly useful in the cardiovascular therapy. Therefore, object of the present invention are the compounds of formula wherein R is a biphenyl group optionally substituted by one or more substi- tuents, the same or different, selected among halogen atoms, nitro groups, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxy¬ carbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C 3 alkyl groups containing one or more fluorine atoms, carboxy groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylamino¬ carbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R-, is a hydrogen atom, a straight or branched C.-C& alkyl or an arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a biphenyl, a naphthyl or a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substi¬ tuted by one or more substituents, the same or different, select¬ ed among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, Ci-C 3 alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; 5 R__ is a straight or branched C.-C ώ alkyl optionally substituted by one or more substituents selected among amino, mono- or di-(C.-c < _,)alkylamino or aminocarbonyl groups, mono or di-(Cι-C Λ )- alkylaminocarbonyl groups, an arylalkyl, an arylcarbonylaminoal- kyl, an arylaminocarbonylalkyl or an arylalkylaminocarbonylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety, the aryl being optionally substituted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C 3 alkyl groups C . containing one or more fluorine atoms, carboxy groups, nitro lb groups, amino or aminocarbonyl groups, acylamino groups, amino¬ sulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbon¬ yl groups having from 1 to 6 carbon atoms in the alkyl moiety; X is a single bond or a -N(R 3 )- group wherein R 3 is a hydrogen atom or a straight or branched C.-C* alkyl group; the carbon atoms marked with an asterisk are asymmetric carbon atoms; and their pharmaceutically acceptable salts. Object of the present invention are the compounds of formula I in 5 the form of stereoisomeric mixture as well as in the form of single stereoisomers. The compounds of formula I object of the present invention are endowed with a mixed ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases such as 0 hypertension, renal failure and congestive heart failure. In the present description, unless otherwise specified, with the term biphenyl group we intend a 2-biphenyl, 3-biphenyl or 4-biphenyl 5 group; with the term alkyl group we intend a straight or branched alkyl such as methyl, ethyl, n.propyl, isopropyl, n.butyl, sec-bu¬ tyl, tert-butyl, isobutyl, n.pentyl, 2-pentyl, 3-pentyl, isopentyl, tert-pentyl, n.hexyl and isohexyl group; with the term halogen atom we intend a fluorine, chlorine, bromine or iodine atom; with the term acyl we intend an acyl group deriving from an aliphatic or aromatic carboxylic acid such as acetic, propionic, butyric and benzoic acid; with the term aryl we intend an aromatic group such as phenyl, 2-biphenyl, 3-biphenyl, 4-biphenyl, 1-naphthyl and 2-naph- thyl or a 5- or 6-membered aromatic heterocyclic group containing 1 nc or 2 heteroatoms selected among nitrogen, oxygen and sulphur such as It) thiazole, isoxazole, oxazole, isothiazole, pyrazole, imidazole, thiophene, pyrrole, pyridine, pyrimidine and furan, optionally benzocondensed. Examples of pharmaceutically acceptable salts of the compounds of formula I are the salts with alkali or alkali-earth metals and the 0 salts with pharmaceutically acceptable organic bases. Preferred compounds of formula I are the compounds wherein R is a 4-biphenyl group; R-, is a straight or branched C.-C* alkyl group or an arylalkyl having from 1 to 3 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a naphthyl, or a 5-membered aromatic 5 heterocyclic group; R 2 is an arylalkyl, an arylalkylaminocarbonylal- kyl or an arylaminocarbonylalkyl group having from 1 to 4 carbon atoms in the alkyl moiety wherein the aryl is a phenyl and X is a -N(R 3 )- group wherein R 3 is a hydrogen atom. Still more preferred compounds of formula I are the compounds 0 wherein R is a 4-biphenyl group; R-, is isopropyl, isobutyl or 2-thienylmethyl and R 3 is 2-phenylethyl, 2-phenylaminocarbonylethyl or benzylaminocarbonylmethyl. Preferred examples of pharmaceutically acceptable salts of the compounds of formula I are the salts with alkali metals such as sodium, lithium and potassium. Specific examples of preferred compounds of formula I, object of the present invention, are: N-[N'-(1-carboxy-3-phenyl-propyl)-L-leucyl]-(1 ,1 '-biphenyl-4-yl)-L- -alanine; N-[N τ -(1-carboxy-3-ρhenyl-propyl)-L-valyl]-(1 ,1 '-biphenyl-4-yl)-L- -alanine; N-[N'-(1-carboxy-3-phenyl-propyl)-(2-thienyl)-L-alanyl]-(1 ,1 '-bi- phenyl-4-yl)-L-alanine; N-[N'-(1-carboxy-3-phenylaminocarbonyl-propyl)-L-leucyl]-(1 ,1 '-bi- phenyl-4-yl)-L-alanine; N-[N'-(1-carboxy-3-phenylaminocarbonyl-propyl)-L-valyl]-(1 ,1 '-bi- phenyl-4-yl)-L-alanine; N-[N 1 -(1-carboxy-3-phenylaminocarbonyl-propyl)-(2-thienyl)-L-ala¬ nyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine; N-[N'-(2-benzylaminocarbonyl-1-carboxy-ethyl)-L-leucyl]-(1 ,1 '-bi- phenyl-4-yl)-L-alanine; N-[N τ -(2-benzylaminocarbonyl-1-carboxy-ethyl)-L-valyl]-(1 ,1 '-biphen- yl-4-yl)-L-alanine; N-[N'-(2-benzylarainocarbonyl-1-carboxy-ethyl)-(2-thienyl)-L-alanyl]- -(1,1'-biphenyl-4-yl)-L-alanine. The preparation of the compounds of formula I, object of the present invention, comprises the reaction between a compound of formula R..00C-CH-X-CH-C00H ( II ) I R= 5 wherein R-, , R a and X have the above reported meanings and R__ repre¬ sents a protective group, preferably a C-,-C* alkyl, a phenyl or a phenylalkyl group having from 1 to 4 carbon atoms in the alkyl moiety; and a biphenylalanine derivative of formula NH 2 -CH-COOH (III) 10 , CH a -R wherein R has the above reported meanings. The condensation is carried out according to conventional techniques of the chemistry of peptides. Before carrying out the reaction, it can be useful to properly lb protect the optional functional groups which could interfere in the reaction. The optional protection is carried out according to conventional techniques. For instance, it can be useful to protect the free carboxy function of the compound of formula III. The evaluation of the usefulness of the optional protection as well as the selection of the kind of protection, with respect to the reaction to be carried out and to the functional groups to be pro¬ tected, are within the normal knowledge of the man skilled in the 5 art. The removal of the optional protective groups is carried out accord¬ ing to conventional techniques. For a general reference to the use of protective groups in organic chemistry see Theodora U. Greene and Peter G.M. Huts "Protective 0 Groups in Organic Synthesis", John Uiley & Sons, Inc. The compounds of formula II and III are known or easily prepared according to known methods. For instance, the compounds of formula II wherein X is a -N(R 3 )- group can be prepared through the reaction of an α-keto ester of formula 0 II R*00C-C-R 2 (IV) wherein R 2 and * have the above reported meanings; with an amino acid of formula R-, I R 3 -NH-CH-C00H (V) wherein R-, and R 3 have the above reported meanings. Alternatively, the compounds of formula II wherein X is a -N(R 3 )- group can be prepared through the reaction of a α-hydroxy ester of formula OH I R.-CH-COOH (VI) wherein R-, has the above reported meanings; with an amino acid of formula R 2 I R 3 -NH-CH-C00R * (VII) wherein R 2 , R 3 and R * have the above reported meanings. As previously pointed out, before carrying out the reaction it can be useful to properly protect the optional functional groups which could interfere in the reaction. The compounds of formula II wherein X is a single bond, instead, are succinic acid derivatives and are preparable, for instance, accord¬ ing to the method described by Van Leeuwen et al. in Reel. Trav. Chim. Pays-Bas , 1980, 99 (3 ) , 96-99. A further process for the preparation of the compounds of formula I wherein X represents a -N(R 3 )- group, object of the present inven- 5 tion, comprises the reaction of a compound of formula OH I R 2 -CH-C00H (VIII) wherein R 2 has the above reported meanings; with a dipeptide of formula 10 j 1 Ra-NH-CH-CONH-CH-COOH (IX) I CH 2 -R wherein R, R. and R 3 have the above reported meanings. The dipeptides of formula IX, in their turn, can be easily prepared ,,_ through a condensation reaction according to conventional techniques of the chemistry of peptides. The compounds of formula I in the form of single stereoisomers are prepared by stereoselective synthesis or by separation of the ster- eoisomeric mixture according to conventional techniques. Also the preparation of the salts of the compounds of formula I, object of the invention, is carried out according to conventional techniques. The compounds of formula I object of the present invention are endowed with a mixed ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases. 5 The inhibitory activity of the compounds of formula I has been evaluated by means of in vitro tests in comparison to known mole¬ cules with ACE-inhibitory or NEP-inhibitory activity (example 11). Captopril, a drug known as the first oral active ACE-inhibitor (The Merck Index, XI ed. - No. 1773, pages 267-268), has been used as a 0 reference compound for the ACE-inhibitory activity. Thiorphan [DL-(3-mercapto-2-benzylpropanoyl)glycine] , known molecule considered the parent compound of NEP-inhibitors and described for 5 the first time by Roques et al. in Nature, Vol. 288, pages 286-288, (1980), has been used as a reference compound for the NEP-inhibitory activity. The inhibitory activity of the compounds of formula I, expressed as ICβo value, is pharmacologically significant in that it results in nM concentrations and is comparable to that of Captopril, as far as the ACE-inhibitory activity is concerned, and to that of thiorphan, as far as the NEP-inhibitory activity is concerned. For a practical use in therapy, the compounds of formula I can be formulated in solid or liquid pharmaceutical compositions, suitable C to oral or parenteral administration. It) A further object of the present invention, therefore, are the phar¬ maceutical compositions containing a therapeutically effective amount of a compound of formula I in admixture with a carrier for pharmaceutical use. Specific examples of pharmaceutical compositions according to the present invention are tablets, coated tablets, capsules, granulates, solutions and suspensions suitable to oral administration, solutions and suspensions suitable to parenteral administration. The pharmaceutical compositions object of the present invention are prepared according to conventional techniques. 5 For a practical use in therapy it can be useful to formulate the compounds of formula I in the form of pro-drugs, namely, of deriv¬ atives which enable the in. vivo release of the pharmacologically active molecule that is of the corresponding compounds of formula I. Specific examples of pro-drugs of the compounds of formula I are the 0 esters of the carboxy group present in the carboxyalkyl function. Therefore, also the pro-drugs of the compounds of formula I are within the scope of the present invention. The daily dose of the compound of formula I or of the corresponding pro-drug will depend on different factors such as the seriousness of the disease, the individual response of the patient or the kind of formulation but it is usually comprised between 0.1 mg and 50 mg per Kg of body weight divided into a single dose or into more daily doses. Uith the aim of better illustrating the present invention the fol¬ lowing examples are now given. Example 1 Preparation of N-[ (1S)-1-ethoxycarbonyl-3-phenyl-propyl1-L-leucine A suspension of L-leucine (1 g; 7.62 mmoles) and 4-phenyl-2-oxo-bu- tyric acid ethyl ester (1.89 g; 9.15 mmoles), prepared as described in Chem. Pharm. Bull., 32.(5), 1800-1807 (1984), in absolute ethanol (40 ml), was hydrogenated into a Parr apparatus in the presence of palladium on charcoal at 10 * /. (200 mg) at 40-50°C for 15 hours. After having filtered off the catalyst and evaporated to dryness, the residue was purified by silica gel column chromatography (eluent CH 2 C1 2 :CH 3 0H=95:5). The resultant solid was collected with petroleum ether and filtered. After drying under vacuum at 50°C, N-[ (1S)-ethoxycarbonyl-3-phenyl- -propyl]-L-leucine (150 mg) was thus obtained, m.p. 103-105°C ■■ H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.86-0.98 (m, 6H, CH.,-CH-CH*,) : 1.26 (t, 3H, CH 2 -CHa); 1.40-2.12 (m, 5H, Ph-CHa-CHa., NH-CH-CH^-CH) ; 2.57-2.82 (m, 2H, CH^-Ph) ; 3.13-3.38 (m, 2H, CH.-NH-CH.) ; 4.18 (q, 2H, C00CH 2 ); 7.10-7.31 (m, 5H, aryl). Example 2 Preparation of N-[N'-[ (1S)-ethoxycarbonyl-3-phenyl-propyn-L-leu- cyll-d .1 '-biphenyl-4-yl)-L-alanine methyl ester N.N'-dicyclohexylcarbodiimide (422 mg; 2.046 mmoles) was added, under stirring at room temperature, to a solution of N-[ (1S)-1-eth- oxycarbonyl-3-phenyl-propyl]-L-leucine (600 mg; 1.86 mmoles), pre¬ pared as described in example 1, and N-hydroxysuccinimide (214 mg; 1.86 mmoles) in dioxane (12 ml). After 2 hours, dicyclohexylurea was filtered off and (1 ,1 '-biphenyl- -4-yl)-L-alanine methyl ester (493 mg; 1.69 mmoles) and triethylam¬ ine (0.282 ml; 2.03 mmoles) were respectively added to the resultant solution. The reaction mixture was heated at 60°C for 3 hours. After cooling and diluting with ethyl acetate, the reaction mixture was washed 3 times with water and dried on sodium sulphate. By evaporating to dryness, a residue (1.5 g) which was purified by silica gel column chromatography (eluent 60-80°C petroleum ether:ethyl acetate=75:25) was obtained. N-[ '-[ (1S)-ethoxycarbonyl-3-phenyl-propyl]-L-leucyl]-(1 ,1 '-biphen¬ yl-4-yl)-L-alanine methyl ester (760 mg; 80% yield) was obtained as an oil. • •H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.85-0.93 (m, 6H, CHa.-CH-CHa.) ; 1.22 (t, 3H, CH 2 -CHa_); 1.28-1.98 (m, 5H, Ph-CHa-CHa., CH-CH,-CH) : 2.45- -2.70 (m, 2H, CU 2 .-Ph); 3.00-3.08 (m, 1H, NH-CH.) ; 3.12-3.19 (m, 2H, CRarbiphenyl); 3.20-3.26 (m, 1H, CH.-NH) ; 3.71 (s, 3H, C00CH 3 ); 4.11 (q, 2H, C00CH 2 ); 4.88-4.98 (m, 1H, CH.-NHC0) ; 7.02-7.50 (m, 14H, aryl); 7.58 (d, 1H, CONH) . By working as described in examples 1 and 2 and by separating the diastereoisomeric mixtures by silica gel column chromatography, as indicated below, the following compounds were prepared: N-[N τ -[ (1S)-1 -ethoxycarbonyl-4-phenyl-butyl1-L-leucyl1-(1.1 '-biphen- yl-4-yl)-L-alanine methyl ester (eluent 40-60°C petroleum ether:ethyl acetate=70:30, Rf=0.38) ^H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.85-0.92 (m, 6H, CH^-CH-CHa.) ; 1.20-1.75 [ , 10H, C00-CH 2 -CiL__, CH-CH^-CHa., CH 3L -C -CH(CH 3 ) 2 ] ; 2.48- -2.59 ( m, 2H, CH S! .-Ph); 3.00-3.25 (m, 4H, CH.-NH-CH., CHarbiphenyl) ; 3.70 (s, 3H, C00-CH 3 ); 4.07-4.19 (m, 2H, C00-CH 2 ); 4.85-4.95 (m, 1H, CH.-NHC0); 7.05-7.60 (m, 15H, CONH, aryl). N- CN ' - C ( 1 R) -1 -ethoxycarbonyl-4-phenyl-butyl 1 -L-leucyl 1 - ( 1.1 τ -biphen- yl-4-yl) -L-alanine methyl ester (eluent 40-60°C petroleum ether:ethyl acetate=70:30, Rf=0.31) ■ •H-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.80-0.89 (m, 6H, CHa.-CH-CHa_) ; 1.12-1.80 [m, 10H, C00-CH 2 -CHa_, CH-CH^-CH^., CH*.-CH.-CH(CH 3 ) 2 ] ; 2.52- -2.62 (m, 2H, CibrPh); 3.00-3.29 (m, 4H, CH.-NH-CH., CJiarbiphenyl) ; 3.71 (s, 3H, C00-CH 3 ); 4.02-4.21 (m, 2H, COO-CH 2 ); 4.80-4.91 (m, 1H, CH.-NHC0); 7.08-7.59 (m, 15H, CONH, aryl). N-rN'-r(1S)-1-ethoxvcarbonvl-butvl1-L-leucyl1-(1.T-biphenyl-4-yl)- -£.-3l9nir_e methy e~ er (eluent hexane:ethyl acetate=70:30, Rf=0.27) • "H-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.83-0.91 (m, 6H, CH_,-CH-CH,) : 1.11-1.75 tm, 10H, C00-CH 2 -CHa., CH-CH^-CHa., CH 2 .-CH_-CH(CH 3 ) a ] ; 2.95- 3.19 (m, 4H, CH.-NH-CH., CHarbiphenyl) ; 3.72 (s, 3H, C00-CH 3 ); 4.05- 4.19 (m, 2H, C00-CH 2 ); 4.85-4.98 (m, 1H, CH.-NHC0); 7.13-7.60 ( , 9H, CONH, aryl). N-[N'-_ (1R)-1 -ethoxycarbonyl-butyll-L-leucvπ-d .1 '-biphenyl-4-yl)- -L-alanine methyl ester (eluent hexane.-ethyl acetate=70:30, Rf=0.23) "•H-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.82-0.91 (m, 6H, CH^-CH-CHa.) ; 1.11-1.78 [m, 10H, C00-CH 2 -CHa_, CH-CH^-CIk., CJHa L -C -CH(CH 3 ) 2 ] ; 2.99- -3.28 (m, 4H, CH.-NH-CH., CH^.-biphenyl) ; 3.71 (s, 3H, COO-CH 3 ); 4.05- -4.19 (m, 2H, COO-CH 2 ); 4.78-4.91 (m, 1H, CJH-NHCO) ; 7.15-7.60 (m, 9H, CONH, aryl). Example 3 Preparation of N-.N'-[ (1S)-1 -ethoxycarbonyl-3-phenyl-proPyl]-L-leu- cvn-(4'-methoxy-l .1 '-biphenyl-4-yl)-L-alanine methyl ester A solution of trifluoromethanesulphonic anhydride (1.26 ml; 7.68 mmoles) in methylene chloride (10 ml) was added dropwise to a solu¬ tion of ethyl (R)-2-hydroxy-4-phenylbutyrate (1.6 g; 7.68 mmoles) and 1 ,8-bis(dimethylamino)naphthalene (1.64 g; 7.68 mmoles) in methylene chloride (16 ml) at -5°C while stirring. After 30 minutes, a solution of N-(L-leucyl)-(4'-methoxy-1 ,1 '-bi- phenyl-4-yl)-L-alanine methyl ester (2.55 g; 6.39 mmoles) and 1,8- -bis(dimethylamino)naphthalene (1.36 g; 6.39 mmoles) in acetonitrile (25 ml) was therein added at -5°C while stirring. The reaction mixture, kept at room temperature for 20 hours under stirring, was filtered and the resultant solution was evaporated to dryness at reduced pressure. The residue was collected with ethyl acetate (25 ml) and brine (25 ml) and the phases were separated. The organic phase was dried on sodium sulphate and evaporated. The residue was silica gel column chromatographed (eluent ethyl acetate:petroleum ether=30:70) affording N-[N'-[ (1S)-1-ethoxycarbon- yl-3-phenyl-propyl3-L-leucyl]-(4'-methoxy-1 ,1 '-biphenyl-4-yl)-L-ala¬ nine methyl ester (2.11 g) as a pale yellow solid. ■ •H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.85-0.92 (m, 6H, CH_,-CH-CH.,) : 1.20-2.00 [m, 8H, Ph-CH 2 -CHa_-CH, CHa.-CH 2 , CH 2 .-CH.-CH(CH 3 ) 2 ]; 2.45- -2.71 (m, 2H, CH 2 -CHa.-Ph) ; 3.00-3.28 (m, 4H, CH.-NH-CH., CHa_-biphenyl); 3.71 (s, 3H, C00-CH 3 ); 3.81 (s, 3H, biphenyl-OCHjJ ; 4.05-4.19 (m, 2H, C00-CH 2 ) ; 4.89-5.00 Cm, IH, CH.-NHCO); 6.89-7.60 Cm, 14H, CONH, aryl). 5 By working in a similar way 3 the following compounds were prepared: N-[N'-1 1S)-1-ethoxycarbonyl-3-phenyl-PΓOPV!1-L-valyl1-(1.1 '-biphen- yl-4-yl)-L-alanine methyl ester " •H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.80-0.92 (m, 6H, Ci rCH-CHi.) ; 1.21 (t, 3H, CHa_-CH 2 ); 2.40-2.70 (m, 2H, CH ^ -Ph): 2.82-3.30 (m, 4H, CH.-NH-CH.-CH_, Ci .-biphenyl); 3.72 (s, 3H, C00-CH 3 ); 4.05-4.19 (m, 2H, C00CH 2 ); 4.90-5.02 (m, 1H, CH.-NHC0) ; 7.02-7.70 (m, 15H, CONH, aryl). N-IN'-T (1S)-1-ethoxycarbonyl- -phenyl-propyl1-L-leucyl1-( τ -chloro- -1.1 '-biphenyl-4-yl)-L-alanine methyl ester ■ •H-NMR (200 MHz, CDC1 3 ) : δ(ppm): 0.85-0.95 (m, 6H, CHa.-CH-CHa.) ; ,g 1.20-2.00 [m, 8H, Ph-CHa-CH^-CH, CHa.-CH 2 , CH a .-CH.-CH(CH 3 ) 2 ]; 2.40- -2.70 (m, 2H, CHa-CU^-Ph) ; 3.00-3.30 (m, 4H, CH.-NH-CH., CHa.-biphen- yl); 3.71 (s, 3H, C00CH 3 ); 4.05-4.18 (m, 2H, C00CH 2 ); 4.89-5.00 (m, 1H, CH.-NHC0); 7.01-7.61 (m, 14H, CONH, aryl). N-[N'-T (1S)-1-ethoxycarbonyl- -phenyl-propyl1-(2-naphthyl)-L-ala- nyll-d .1 '-biphenyl-4-yl)-L-alanine methyl ester 0 " •H-NMR (200 MHz, CDC1 3 ): δ(ppm): 1.05 (t, 3H, CE___-CH 2 ); 1.60-1.90 (m, 2H, Ph-CHa-CH^-CH) ; 2.30-2.53 (m, 2H, CHa-CH^-Ph) ; 2.90-3.45 (m, 6H, CH.-NH-CH., CH ^ -biphenvl. Ci .- aphthyl) ; 3.69 (s, 3H, C00CH 3 ); 3.80-4.01 (m, 2H, COOCHa); 4.89-5.01 (m, 1H, CH.-NHC0); 6.90-7.85 (m, 22H, CONH, aryl). 5 N-TN'-r (1S)-1-ethoxycarbonyl-3-phenyl-propyl1-(1-naphthyl)-L-ala- nyll-d .1 '-biphenyl-4-yl)-L-alanine methyl ester • Η-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.80 (t, 3H, CHa.-CH 2 ); 1.60-1.88 (m, 2H, Ph-CH 2 -CH 2 .-CH) ; 2.30-2.50 (m, 2H, CHa-CH^-Ph) ; 2.90-3.70 (m, 8H, C00CH 2 , CH.-NH-CH., CH^-biphenyl, CH^-naphthyl) ; 3.71 (s, 3H, 0 C00CH 3 ); 4.88-5.00 (m, 1H, CH.-NHC0) ; 6.91-8.20 (m, 22H, CONH, aryl). N-[N'-[ (1S)-1-ethoχycarbonyl-3-phenyl-propyl]-(4-fluorophenyl)-L- -alanyll-CI .1 '-biphenyl-4-yl)-L-alanine methyl ester 5 ' 'H-NMR (200 MHz, CDC1 3 ): δ(ppm): 1.15 (t, 3H, CHa_-CH 2 ); 1.60-1.90 (m, 2H, Ph-CH 2 -Ci .-CH) ; 2.35-2.61 (m, 2H, CHa-CH^-Ph) ; 2.69-3.30 ( , 6H, CH_-NH-CH_, CHarbiphenyl, CH 2 _-fluorophenyl) ; 3.70 (s, 3H, C00CH 3 ); 3.95-4.10 (m, 2H, C00CH=); 4.88-4.90 (m, 1H, CH.-NHC0) ; 6.95-7.60 (m, 19H, CONH, aryl). ln N-CN'-[ (1S)-1-ethoxycarbonyl-3-phenyl-PΓQPV!1-(2-thienyl)-L-alanyl1- -(1.1 '-biphenyl-4-yl)-L-alanine methyl ester "• H-NMR (200 MHz, CDCl 3 ): δ(ppm): 1.20 (t, 3H, CHarCHa); 1.60-1.90 (m, 2H, Ph-CHa-CH-j_-CH) ; 2.35-2.61 (m, 2H, CHa-CH^-Ph) ; 2.98-3.31 (m, 6H, CH.-NH-CH., CΪL__.-biphenyl, CHarthienyl) ; 3.70 (s, 3H, C00CH 3 ); ,g 4.00-4.14 (m, 2H, C00CH 2 ); 4.91-5.01 Cm, IH, CH.-NHC0) ; 6.90-7.70 Cm, 18H, CONH, aryl). N-TN'-f 1S)-1-ethoχycarbonyl-3-phenyl-propyl1-L-phenylalanyl1-(1.1 '- -biphenyl-4-yl)-L-alanine methyl ester " •H-NMR (200 MHz, CDCl 3 ): δ(ppm): 1.12 (t, 3H, CHarCHa ; 1.60-1.90 (m, 2H, Ph-CHa-CHa CH) ; 2.31-2.51 Cm, 2H, CH._-CH 2 .-Ph) ; 2.71-3.35 Cm, 6H, CE-NH-CH.-CiL__.-Ph, Cϋarbiphenyl); 3.69 Cs, 3H, C00CH 3 ); 3.90-4.10 Cm, 2H, C00CH 2 ); 4.91-5.01 Cm, 1H, CH.-NHC0); 6.95-7.62 Cm, 20H, CONH, aryl). N-[N'-rciS)-1-methoxycarbonyl-2-methyl-propyl]-L-leucyl1-(1.1 '-bi- phenyl-4-yl)-L-alanine methyl ester 5 " Η-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.80-0.90 (m, 12H, 2CH*,-CH-CH.*-.) : 1.20-1.88 [m, 4H, CU 2 .-CH.-CH(CH 3 ) 2 , NH-CH-CHJ; 2.91-3.15 (m, 4H, CH.-NH-CH., Cϋarbiphenyl); 3.63, 3.72 (2s, 6H, C00CH 3 , 0CH 3 ); 4.85- -4.99 Cm, 1H, CR-NHCO); 7.12-7.54 Cm, 10H, CONH, aryl). N-CN'-r C1S)-1-methoxycarbonyl-3-methyl-butyl1-L-leucyl1-(1.1 '-bi- 0 phenyl-4-yl)-L-alanine methyl ester " •H-NMR (200 MHz, CDCl 3 ): δ(ppm) : 0.73-0.98 Cm, 12H, 2CH a -CH-CH_. ) ; 1.10-1.80 [m, 6H, CH 2 .-CH.-NH-CH-Cii 2 .-C -CHCCH 3 )a] ; 2.95-3.25 Cm, 4H, CH.-NH-CH., CH^-biphenyl); 3.65, 3.71 (2s, 6H, 2C00CH 3 ); 4.83-4.97 (m, 1H, CH.-NHC0); 7.10-7.60 (m, 10H, CONH, aryl). N-[ '-[ (1S)-1 -benzyloxycarbonyl-2-phenyl-ethyl1-L-leucyl1-(1.1 '-bi- phenyl-4-yl)-L-alanine benzyl ester 1 H-NMR ( 200 MHz, CDCl 3 ): δ(ppm): 0.75-0.83 (m, 6H, CHa.-CH-CHa.) ; 1.10-1.60 [m, 3H, CHi.-CH.-CH(CH 3 2] ; 2.61-3.20 Cm, 4H, CH -bi p henvl. Ph-CH,): 3.40-3.52 Cm, 1H, CH-NH-CH.-C0) ; 4.45-4.52 Cm, 1H, Ph-CH 2 -CH ) ; 4.69-4.80 Cm, 1H, CH.-NHC0) ; 4.98-5.19 Cm, 4H, 2Ph-CH g -0C0) : 6.95-7.51 Cm, 20H, CONH, aryl). N-fN'-T (1S)-3-benzylaminocarbonyl-1 -methoxycarbonyl-PΓOPV!1-L-leu¬ cvl.-(1.1 '-biPhenvl-4-vl)-L-alanine methyl ester 1 H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.83-0.91 (m, 6H, CHarCH-CHa.) ; 1.30-2.10 [m, 6H, CH-NH.-CH, CHa-CH^-CH, CH 2 .-CH.-CH(CH 3 ) 2 ] ; 2.21-2.30 (t, 2H, CO-CHa.); 2.95-3.14 (m, 4H, CH.-NH-CH., CH,-biphenvl) : 3.61, 3.67 (2s, 6H, 2C00CH 3 ); 4.09-4.42 (m, 2H, Ph-CH^-NH) ; 4.86-4.96 (m, 1H, Cϋ-CHa-biphenyl) ; 6.34 (bt, 1H, CO-NH-CH*) ; 7.08-7.57 (m, 15H, CH-NH.-C0, aryl). N-[N'-[ (1S)-2-benzylaminocarbonyl-1-methoxycarbonyl-ethyl1-L-leu- cyll-d .1 '-biPhenyl-4-yl)-L-alanine methyl ester ■ •H-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.86 (d, 6H, CHarCH-CHa.); 1.15-1.76 [m, 3H, Cfla.-CϊL<CHa) a ]; 2.27 (bs, 1H, CH-NH.-CH) ; 2.53 (d, 2H, CO-CH -CH) : 2.95-3.60 (m, 4H, CH.-NH-CH., CH^-biphenyl) ; 3.69 ( 2s, 6H, 2C00CH 3 ); 4.21-4.44 (m, 2H, Ph-CH^-NH) ; 4.80-4.92 ( m, 1H, CH.-CHa-biphenyl); 6.26 (t, 1H, CO-NH-CH^.) ; 7.11-7.54 (m, 14H, aryl); 7.70 (d, IH, C0-N&-CH). Example 4 Preparation of N-.N'-[ (1S)-1-methoxycarbonyl-3-phenylaminocarbonyl- -propyll-L-leucyπ-d .1 '-biphenyl-4-yl)-L-alanine methyl ester 5 A solution of trifluoromethanesulphonic anhydride (2.78 ml; 16.9 mmoles) in methylene chloride (10 ml) was added dropwise to a solu¬ tion of benzyl (R)-2-hydroxy-4-methyl-valerate (3.76 g; 16.9 mmoles) and 1 ,8-bis(dimethylamino)naphthalene (3.6 g; 16.9 mmoles) in aceto- nitrile (37 ml) at -5°C while stirring. _. After 30 minutes, a solution of (S)-δ-anilide glutamic acid methyl ester (4 g; 16.9 mmoles) and 1 ,8-bis(dimethylamino)naphthalene (3 g; 13.5 mmoles) in acetonitrile (40 ml) was added dropwise at -5°C while stirring. After 18 hours the reaction mixture was evaporated and the residue , 5 was collected with ethyl acetate (25 ml) and brine (25 ml). The phases were separated and the organic phase was dried on sodium sulphate and evaporated to dryness obtaining a crude oil (7 g) which was purified by silica gel column chromatography (eluent ethyl acetate:petroleum ether=40:60) . The thus obtained oily compound (0.78 g) was dissolved in ethanol (30 ml) and hydrogenated into a Parr apparatus in the presence of palladium on charcoal at 10% (0.078 g) . After having filtered off the catalyst and evaporated to dryness, the resultant crude (0.57 g) was condensed at 0°C with (1 ,1 '-biphen- yl-4-yl)-L-alanine methyl ester hydrochloride (4.74 g; 1.62 mmoles) 5 in tetrahydrofuran (12 ml), in the presence of a solution of hy- droxybenzotriazole (0.22 g; 1.62 mmoles) and triethylamine (0.23 ml; 1.62 mmoles) in tetrahydrofuran (12 ml), by adding a solution of dicyclohexylcarbodiimide (0.335 g; 1.62 mmoles) in tetrahydrofuran (5 ml). 0 After 16 hours at room temperature, the reaction mixture was fil¬ tered and the resultant solution was evaporated. The residue was collected with ethyl acetate (15 ml) and the solu- tion was washed with 10% hydrochloric acid (2x10 ml), brine (2x10 ml), an aqueous solution of sodium bicarbonate at 5% (2x10 ml) and brine (10 ml), respectively. By drying on sodium sulphate and evaporating to dryness it was obtained an oily residue (1 g) which, purified by silica gel column chromatography (eluent ethyl acetate:petroleum ether=60:40) , fur¬ nished N-[N'-[ (1S)-1-methoxycarbonyl-3-phenylaminocarbonyl-propyl]- -L-leucyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine methyl ester (0.46 g). ■ Η-NMR (200 MHz, CDC1 3 ) : δ(ppm): 0.75-0.89 (m, 6H, CH.,-CH-CH,) : 1.10-2.05 [m, 5H, NHCO-CHa-CH^., CH*.-CH.-CH(CH 3 ) 2 ] ; 2.40-2.50 (m, 2H, NHCO-CHi ; 2.81-3.20 (m, 4H, CH.-NH-CH., CH 2 -biphenvl) : 3.68, 3.81 (2s, 6H, 2C00CH 3 ); 4.98-5.10 (m, 1H, CH.-NH-C0); 6.98-7.60 (m, 15H, CONH, aryl); 8.02 (s, 1H, Ph-NH) . By working in a similar way, the following compounds were prepared: N-CN'-[ (1 )-1-methoχycarbonyl-3-phenylaminocarbonyl-propyl1-L-leu- cyl]-(1.1 '-biphenyl-4-yl)-L-alanine methyl ester • Η-NMR (200 MHz, CDC1 3 ): δ(ppm): 0.78-0.88 (m, 6H, CH..-CH-CH*-*) : 1.20-2.20 [m, 5H, CH 2 .-CH-NH-CH-CH S -CH.-CH(CH 3 )a] ; 2.31-2.41 (m, 2H, NHCO-CJia.-CHa); 2.95-3.40 (m, 4H, CH-NH-CH., CH^-biphenyl) ; 3.60, 3.72 (2s, 6H, 2C00CH 3 ); 4.82-4.98 (m, 1H, CH.-NH-C0) ; 7.02-7.54 (m, 15H, CONH, aryl); 7.31 (s, 1H, Ph-NH) . N-TN'-[ (1S)-1-methoxycarbonyl-2-phenylaminocarbonyl-ethyl]-L-leu- cyll-d .1 '-biphenyl-4-yP-L-alanine methyl ester ^H-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.72-0.88 (m, 6H, CHa.-CH-CHa_) ; 1.20-1.80 [m, 3H, CiLa.-CH.-CH(CH 3 )a]; 2.60-3.22 ( m, 5H, CH^-CH-NH-CH., Cϋarbiphenyl) ; 3.48-3.52 (m, 1H, CONH-CH 2 -CH.-NH) ; 3.71, 3.80 (2s, 6H, 2C00CH 3 ) ; 4.87-4.99 (m , 1 H , CH.-NHC0 ) ; 7.00-7.60 (m , 1 5H , CONH , aryl ) ; 8. 00 ( s , 1 H , Ph-NH) . N-[N'-[ (1S)-1-methoxycarbonyl-5-tert-butoxycarbonylamino-pentyl]-L- -leucylT-d .1 '-biphenyl-4-yl)-L-alanine methyl ester " Η-NMR (200 MHz, CDCl 3 ): δ(ppm): 0.81-0.91 (m, 6H, CHa.-CH-CHa.) ; 1.20-1.70 [m, 18H, C00-C(CHa_) 3 , CH 2 .-Cja 2 .-CH 2 .-CH, CH 2 .-CH.-CH(CH 3 ) 2 ] ; 2.80-3.28 (m, 6H, OCO-NH-CHa., CE.-NH-CH., CH2_-biphenyl) ; 3.65, 3.75 (2s, 6H 2C00CH 3 ); 4.68 (bt, 1H, C0-NH.-CH 2 ); 4.89-5.00 (m, 1H, CH-COO); 7.12-7.60 (m, 10H, NH-CO, aryl). Example 5 Preparation of N-fN'-[ (1S)-1-carboxy-3-Phenyl-propyll-L-leucyll- -(1.1 '-biphenyl-4-yl)-L-alanine disodium salt (Compound 1 ) A solution of sodium hydroxide 1N (5 ml) was added under stirring to a solution of N-[N'-[ (1S)-1-ethoxycarbonyl-3-phenyl-propyl]-L-leu- cyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine methyl ester (700 mg; 1.25 mmoles), prepared as described in example 2, in ethanol (14 ml). After one night, the solvent was evaporated under vacuum and the residue was collected with absolute ethanol (30 ml) and kept under stirring for 1 hour. After 48 hours of rest, the precipitate was filtered washing with ethanol and ether. By drying under vacuum at 40°C, N-[N'-[ (1S)-1-carboxy-3-phenyl-pro- pyl]-L-leucyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine disodium salt (570 mg; 81% yield) was obtained as a white solid. " Η-NMR (200 MHz, D 2 0) : δ(ppm): 0.58-0.64 (m, 6H, CH-.-CH-CH-.) : 1.04- -1.11 (m, 2H, Ph-CHa-CHa.) ; 1.18-1.55 (m, 3H, CH-CH 2 .-CH) 2.16-2.42 (m, 2H, CHa-CHarPh) ; 2.65-3.08 (m, 3H, CH.-NH-CH., CH-CHarbiphenyl) ; 4.32-4.38 (m, 1H, CH-COO); 6.92-7.44 (m, 14H, aryl). Mass: 517 (M+H). By working in a similar way the following compounds were prepared: N-TN'-r (1S)-1 -carboxy-4-phenyl-butyl1-L-leucyl1-(1.1 '-biphenyl-4- -yl)-L-alanine dilithium salt (Compound 2) 5 " "H-NMR (200 MHz, D 2 0 ) : δ(ppm ) : 0.41-0.49 ( m, 6H, CHa.-CH-CH.__. ) ; 0.91- -1.44 (m, 7H, CH-CH2.-CH 2 .-CHa, NH-CH-CH2.-CE) ; 2.16-2.36 ( m, 2H, CH 2 -CU 2 _-Ph) ; 2.82-2.73 (m, 2H, CH.-NH-CH) ; 2.56-3.05 (m, 2H, CH-CH 2 _-biphenyl); 4.22-4.28 (m, 1H, CH_-CH 2 -Ph) ; 6.80-7.23 (m, 14H, aryl) . Mass: 531 (M+H) N-CN'-T (1 )-1 -carboχy-4-phenyl-butyl1-L-leucyl1-(1.1 '-biphenyl-4- -yl)-L-alanine dilithium salt (Compound 3) n H-NMR (200 MHz, D 2 0): δ(ppm): 0.49-0.54 (m, 6H, CHarCH-CHa.); 1.02- -1.47 (m, 7H, CH-CH 2 .-CH 2 .-CH, NH-CH-CH 2 .-CH.) ; 2.06-3.14 (m, 6H, ,- Ph-CU 2 .-CH 2 , CH-CHarbiphenyl); 4.31-4.38 (m, 1H, CH.-NH-C0); 6.92-7.42 lb (m, 14H, aryl). Mass: 531 (M+H) N- N'-C (1S)-1 -carboxy-butyl1-L-leucyl1-(1.1 '-biphenyl-4-yl)-L-ala¬ nine dilithium salt (Compound 4) ■ •H-NMR (200 MHz, D 2 0) : δ(ppm): 0.54-0.65 (m, 9H, CHa_-CH 2 , 0 CHarCH-CHa.); 0.88-1.31 (m, 7H, CH*.-CH**,-CH^. NH-CH-CH^-CH) ; 2.66-3.12 (m, 4H, CH.-NH-CH., CH^-biphenvl) : 4.32-4.38 (m, 1H, CH.-CH a -biphenyl) ; 7.10-7.47 (m, 9H, aryl). Mass: 455 (M+H) N-CN T -C (1R)-1 -carboxy-butyl1-L-leucyl1-(1.1 '-biphenyl-4-yl)-L-ala¬ 5 nine dilithium salt (Compound 5) "Η-NMR (200 MHz, D 2 0) : δ(ppm): 0.58-0.66 (m, 9H, CHarCH 2 , CHarCH-CHa.); 0.85-1.33 (m, 7H, CH.^-CH^-CH^. NH-CH-CH2.-CH ) ; 2.75-3.17 (m, 4H, CH.-NH-CH., CH rbiphenyl) ; 4.36-4.43 (m, 1H, CHrCH 2 -biphenyl) ; 7.19-7.57 (m, 9H, aryl ) . 0 Mass : 455 (M+H) N-CN'-C (1S)-1-carboxy-2-methyl-propyll-L-leucyl]-(1.1 '-biphenyl-4- -yl)-L-alanine dilithium salt (Compound 6) n H-NMR (200 MHz, D 2 0): δ(ppm): 0.58-0.69 (m, 12H, 2CHarCH-CHa.) ; 1.00-1.56 [m, 4H, CH-CH_CCH 3 ) 2 , CH 2 .-CH_CCH 3 ) 2 ] ; 2.40 [d, 1H, NH-CH_-CH(CH 3 ) 2 ]; 2.75-3.13 (m, 3H, CH 2 .-biphenyl, NH-CH,-C0NH); 4.34- -4.41 (m, 1H, CH_-CH-biphenyl); 7.17-7.56 (m, 9H, aryl). Mass: 455 (M+H) N-CN'- (1S)-1-carboxy- -methyl-butyl1-L-leucyl1-(1.1 '-biphenyl-4- -vl)-L-alanine dilithium salt (Compound 7) ■ •H-NMR (200 MHz, D 2 0) : δ(ppm): 0.55-0.70 (m, 12H, 2CH-.-CH-CH-.) : 1.00-1.50 [m, 6H, 2CH 2 .-CH.-CHCCH 3 ) 2 ]; 2.71-3.13 (m, 4H, CH.-NH-CH., CHarbiphenyl) ; 4.32-4.38 ( , 1H, CH.-NHC0); 7.15-7.55 (m, 9H, aryl). Mass: 469 (M+H) N-CN'-C (1R)-1-carboχy-3-phenylaminocarbonyl-propyl]-L-leucyl]-(1.1 '- -biphenyl-4-yl)-L-alanine dilithium salt CCompound 8) ■ •H-NMR C200 MHz, D 2 0) : δCppm): 0.56-0.62 Cm, 6H, CH.-_--CH-CH.-__) : 1.08- -1.28 [m, 3H, CH 2 .-CH.-CHCCH 3 ) 2 ]; 1.46-1.57 Cm, 2H, C0-CH 3 -CH 2 _-CH) ; 1.79-2.15 Cm, 2H, CO-CHa.) J 2.67-3.15 Cm, 4H, CH.-NH-CH., CH 2 .-biph.en- yl); 4.45-5.38 Cm, 1H, CHrCOO) ; 6.97-7.43 Cm, 14H, aryl). Mass: 558 CM+H) Example 6 Preparation of N-CN'-CC1S)-1-carboxy-3-phenyl-propyl]-L-leucyl]-C4'- -methoxy-1.1 '-biphenyl-4-yl)-L-alanine CCompound 9) A solution of sodium hydroxide 1N C4.14 ml; 4.1 mmoles) was added dropwise, at 0-5°C and under stirring, to a solution of N-[N'-[C1S)- -1-ethoxycarbonyl-3-phenyl-propyl]-L-leucyl]-C4'-methoxy-1 ,1 '-bi¬ phenyl-4-yl)-L-alanine methyl ester C1.11 g; 1.88 mmoles), prepared as described in example 3, in ethanol C22 ml). After one hour at 5°C, the reaction mixture was kept at room temper¬ ature for 20 hours and the solvent was evaporated at reduced pres¬ sure. 5 The residue was collected with water (10 ml) and acidified with an aqueous solution of hydrochloric acid at 10% (10 ml). Tetrahydrofuran (10 ml) was therein added and the organic phase was separated, dried on sodium sulphate and evaporated to dryness. The resultant crude was purified by silica gel column chromatography ( eluent methylene chloride:methanoi:acetic acid=90:10:1 ) affording N-[N'-[ (1S)-1-carboxy- -phenyl-propyl]-L-leucyl]-(4'-methoxy-1,1'- -biphenyl-4-yl)-L-alanine (0.57 g). m.p. 214-217°C ■• H-NMR (200 MHz, D 3 0+Na0D): δ(ppm): 0.40-0.48 (m, 6H, CHa-CH-CHa.) ; C 0.89-1.60 (m, 5H, CH 2 .-CH-NH-CH-CH 2 .-CH) ; 2.12-2.38 (m, 2H, lb CH 2 .-CH 2 -CH) ; 2.62-2.89 (m, 4H, CH.-NH-CH., CHarbiphenyl) ; 3.14 (s, 3H, 0CH 3 ); 4.20-4.26 (m, 1H, CH.-C00) ; 6.30-6.95 (m, 13H, aryl). Mass: 547 (M+H) By working in a similar way the following compounds were prepared: N- N'-C (1S)-1-carboχy-3-phenyl-propyl1-L-valyl1-(1.1 '-biPhenyl-4- 0 -yl)-L-alanine (Compound 10) m.p. 228-231°C 1 H-NMR (200 MHz, DMS0-d Λ ) : δ(ppm): 0.79 (t, 6H, CHarCH-CHa.); 1.64-1.85 (m, 3H, CH 2 -CH 2 .-CH, CH 3 -CHrCH 3 ) ; 2.31-2.61 (m, 2H, CH-_.-Ph) : 2.73-3.18 (m, 4H, CH.-NH-CH, CHarbiphenyl) ; 4.53-4.64 (m, 5 1H, CH-COO); 7.00-7.58 (m, 14H, aryl); 7.95 (d, 1H, NH-CO). Mass: 503 (M+H) N-CN'-C (1S)-1-carboxy-3-phenyl-propyl]-L-leucyl]-( '-chloro-1.1 '- -biphenyl-4-vl)-L-alanine (Compound 11) m. p. 21 4-216°C 0 " Η-NMR ( 200 MHz , DMS0-d Λ ) : δ (ppm) : 0. 80 ( t , 6H , CHarCH-CHa. ) ; 1 .20- 1 . 29 [m, 2H, CH 2 _-CH(CH 3 ) 2 ] ; 1 . 53-1 . 77 (m, 3H, CH 2 .-CH 2 -Ph, CH 3 -CH_-CH 3 ); 2.32-2.63 (m, 2H, CHa h ; 2.88-3.20 Cm, 4H, CE-NH-CH_, 5 CHarbiphenyl); 4.52-4.63 Cm, 1H, CH-COO); 7.00-7.63 Cm, 13H, aryl); 7.95 Cd, 1H, NH-CO). Mass: 551 CM+H) N-CN'-C C1S)-1-carboxy-3-phenv1-propyl1- 2-naphthyl)-L-alanyl]-(1.1 '- -biphenyl-4-yl)-L-alanine (Compound 12) - 0 m.p. 217-219°C " Η-NMR (200 MHz, DMS0-d Λ ) : δ(ppm): 1.68-1.79 (m, 2H, CH 2 _-CH 2 -Ph) ; 2.39-2.47 (m, 2H, CHarP ) ; 2.87-3.11 (m, 5H, Cibrbiphenyl, CHrCHarnaphthyl) ; 3.65-3.72 (m, 1H, CH_-CH 2 -CH 2 ) ; 4.55-4.62 (m, 1H, CO-NH-CH-CO) ; 6.95-7.85 (m, 21H, aryl). 5 Mass: 601 (M+H) N-CN'-C(1S)-1-carboxy-3-phenyl-proρyl]-(1-naphthyl)-L-alanyl1-(1.1 '- -biPhenyl-4-yl)-L-alanine (Compound 13) m.p. 219-220°C " -NMR (200 MHz, DMSO-d*) : δ(ppm): 1.62-1.75 (m, 2H, CH 2 .-CH 2 -Ph) ; Q 2.34-2.62 Cm, 2H, CH 2 .-Ph ; 2.96-3.39 Cm, 6H, CHarbiphenyl, CH.-NH-CHrCH___-.naphthyl); 4.50-4.60 Cm, 1H, CH-COO); 7.02-8.09 Cm, 22H, NH-CO, aryl). N-CN'-C C1S)-1-carboxy-3-Phenyl-propyl1-(4-fluorophenyl)-L-alanyl]- -(1.1 '-biphenyl-4-yP-L-alanine (Compound 14) m.p. 229-231°C 5 " -NMR (200 MHz, DMS0-d A ) : δ(ppm): 1.55-1.75 (m, 2H, CH ^ -CH ^ -Ph) : 2.33-2.52 (m, 2H, CH,-Ph) : 2.68-2.86 (m, 2H, CHarfluorophenyl) ; 2.96-3.04 (m, 3H, CH.-CH 2 -CHa-Ph, CHarbiphenyl) ; 3.21-3.27 (m, 1H, CH_-CH 2 -Ph); 4.51-4.62 (m, 1H, CHrCH 2 -biphenyl) ; 6.98-7.57 (m, 18H, aryl); 7.95 (d, 1H, NH-CO). 0 Mass : 569 CM+H) N- CN' - C ( 1 S) -1 -carboxy-3-phenyl-propyl ] - ( 2-thienyl ) -L-alanyl 1 - ( 1 . 1 ' - -biphenyl-4-yl ) -L-alanine (Compound 1 5) 5 m. p. 209-21 1 °C " Η-NMR (200 MHz, DMS0-d A ) : δ (ppm) : 1 . 58-1 .78 (m, 2H, CH 2 .-CH 2 -Ph) ; 2.36-2. 51 (m , 2H, CHarPh) ; 2.99-3.09 (m, 5H , CE-CH 2 -CH 2 , CHarbiphenyl, CH 2 _-thienyl) ; 3.24-3.30 (m, 1H, CH_-CH 2 -thienyl) ; 4.49-4.59 (m, 1H, CH_-CH 2 -biphenyl) ; 6.84-7.57 (m, 17H, aryl); 8.00 (d, 1H, NH-CO). Mass: 557 (M+H) N-CN'-C (1S)-1 -carboxy- -phenyl-propyl]-L-Phenylalanyl1-(1.1 '-biphen- yl-4-yl)-L-alanine (Compound 16) m.p. 221-222°C - 5 " -NMR (200 MHz, DMSO-d Λ ) : δ(ppm): 1.55-1.75 (m, 2H, CH 2 .-CH_.-Ph) ; 2.30-2.51 (m, 2H, CH_.-CH 2 .-Ph) ; 2.69-2.89 (m, 2H, CH.-CH 2 -Ph) ; 2.97- -3.03 (m, 3H, CH.-CHa-CH 2 , CHarbiphenyl) ; 3.24-3.30 (m, 1H, CHrCHa-Ph); 4.51-4.61 (m, 1H, CHrCH 3 -biphenyl) ; 6.98-7.57 (m, 19H, aryl); 7.93 (d, 1H, NH-CO). Mass: 551 (M+H) N-CN'-C (1S)-1 -carboxy-5-tert-butoχycarbonylamino-pentyl1-L-leucyl]- -(1.1 '-biphenyl-4-yl)-L-alanine " Η-NMR (200 MHz, DMS0-d Λ ) : δ(ppm): 0.75-0.88 (m, 6H, CHarCH-CHa) ; 1.10-1.70 [m, 18H, C00-C(CH 3 ) 3 , CH 2 rCH 2 .-CH 2 .-CH, CH-_--CH-CH(CH*_.),] : 2.70-3.20 (m, 6H, OCO-NH-CH 2 ., CH 3 -CHrCH 3 , CH.-C0-NH, CH 2 .-biphenyl) ; 5 4.45-4.60 (m, 1H, CH-COO ) ; 6.70 (t, 1H, C0-NH_-CHa ) ; 7.20-7.75 (m, 9H, aryl); 7.88 (d, 1H, CO-NH-CH). N-CN'-C (1S)-1-carboxy-3-phenylaminocarbonyl-propyl]-L-leucyl1-(1.1 '- -biphenyl-4-yl)-L-alanine (Compound 17) " Η-NMR (200 MHz, DMS0-d__) : δ(ppm): 0.72-0.78 (m, 6H, CH,-CH-CK, ) : 0 1.09-1.17 Cm, 2H, CH-CH 2 .-CH); 1.44-1.60 Cm, 1H, CH 3 -CHrCH 3 ) ; 1.69- 1.98 Cm, 2H, C0-CHa-CU 2 .-CH) ; 2.30-2.54 Cm, 2H, CO-CH2. ) ; 2.84-3.21 Cm, 4H, CU 2 .-biphenyl, CHrNH-CH.) ; 4.30-4.39 Cm, 1H, CH-COO); 6.93- 5 7.63 Cm, 14H, aryl); 7.85 (d, 1H, CO-NH-CH); 10.30 Cs, 1H, NH-Ph) . N-CN'-f C1S)-1-carboxy-2-Phenylaminocarbonyl-ethyl1-L-leucyl1-C1.1 '- -biphenyl-4-yl)-L-alanine (Compound 18) " Η-NMR (200 MHz, DMSO-d Λ ) : δ(ppm): 0.84-0.89 (m, 6H, CHarCH-CHa.); 1.15-1.22 [m, 2H, CH 2 .-CH(CH 3 ) 3 ]; 1.51-1.72 (m, 1H, CH 3 -CH-CH 3 ) ; 2.58-2.78 (m, 2H, CO-CH 2 .-CH-NH) ; 2.96-3.23 (m, 3H, CH.-CO-NH, CH rbiphenyl); 3.47-3.53 (m, 1H, CO-CH 2 -CH.-NH) ; 4.38-4.49 (m, 1H, CH_-CH 2 -biphenyl); 6.99-7.63 (m, 14H, aryl); 8.25 (d, 1H, CO-NH.-CH); 10.12 (s, 1H, N -Ph). Mass: 546 (M+H) , c N-CN'-C (1S)-3-benzylaminocarbonyl-1-carboxy-propyl]-L-leucyl]-(1.1 '- It) -biPhenvl-4-vl)-L-alanine (Compound 19) " Η-NMR C200 MHz, DMSO-d β ): δCppm): 0.75-0.81 Cm, 6H, CH-.-CH-CH*.) : 1.12-1.22 Cm, 2H, CHa-CHzrCH-NH) ; 1.46-1.87 Cm, 3H, CH 2 .-CH_(CH 3 ) 3 ] ; 2.23-2.30 Cm, 2H, NH-CO-CHa.) ; 2.83-3.18 Cm, 4H, CU 2 .-biphenyl, CHrNH-CH..; 4.01-4.35 Cm, 3H, Ph-CH_ 2 .-NH, NH-CH.-CH 2 -biphenyl) ; 7.13- -7.59 Cm, 16H, 2C00H, aryl); 7.81 Cbd, IH, CO-NHrCH) ; 8.63 (bt, 1H, NH.-CH 2 ). Mass: 574 C +H) N-CN'- C1S)-2-benzylaminocarbonyl-l-carboxy-ethyl]-L-leucyl]-(1.1 '- -biPhenyl-4-yl)-L-alanine (Compound 20) 5 " Η-NMR (200 MHz, DMSO-d Λ ) : δ(ppm): 0.74-0.79 (m, 6H, CH_,-CH-CH.,) : 1.15-1.22 [m, 2H, CH 2 .-CH(CH 3 ) 2 ] ; 1.50-1.70 (m, 1H, CH 3 -CHrCH 3 ); 2.42-2.65 (m, 2H, CO-CH^-CH) : 2.94-3.19 [m, 3H, C -CH 2 -CH(CH 3 ) 2 , CHarbiphenyl]; 3.47-3.54 (m, IH, CHrCH 3 -CO) ; 4.19-4.43 (m, 3H, CH 2 .-NH, C -CH a -biphenyl); 7.22-7.60 (m, 14H, aryl); 8.18 (d, 1H, 0 NH_-CH-CH a -biphenyl ) ; 8.60 ( t , 1 H , NH_-CH 3 -Ph) . Mass : 558 (M-H) Example 7 5 Preparation of N-CN T -[ (1S)-5-amino-1-carboxy-pentyl1-L-leucyl1- -(1.1 '-biPhenyl-4-yl)-L-alanine dihvdrochloride (Compound 21 ) A saturated solution of hydrochloric acid in ethyl ether (5 ml; 11% weight/volume) was added at room temperature and under stirring to a solution of N-[N'-[ (1S)-1-carboxy-5-tert-butoxycarbonylamino-pent- 1Q yl]-L-leucyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine (1 g; 1.71 mmoles), prepared as described in example 6, in an ethyl ether:methylene chloride=1 :1 mixture (30 ml). After 16 hours the reaction mixture was evaporated and the residue was triturated with ethyl ether and filtered affording N-[N'-[(1S)- ^5 -5-amino-1-carboxy-pentyl]-L-leucyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine dihydrochloride (0.83 g) as a white powder. " Η-NMR (200 MHz, D 3 0) : δ(ppm): 0.69-0.74 (m, 6H, CH„-CH-CH„) : 0.79- -1.52 [m, 9H, NH-CH 3 -CH(CHa_)__. CH 2 .-CH.(CH 3 ) 2 ] ; 2.26-2.34 Cm, 2H, CH a -N); 2.64-3.32 Cm, 2H, CH 2 _-biphenyl) ; 2.38-2.43, 2.64-3.71 C2m, 0 2H, CHrNH-CH); 4.72-4.80 Cm, 1H, CH-C0); 7.18-7.61 Cm, 9H, aryl). Mass: 484 (M+H) Example 8 Preparation of N-CN'-C (1S)-1-carboχy-2-phenyl-ethyl]-L-leucyll- -(1.1 '-biPhenyl-4-yl)-L-alanine disodium salt (Compound 22) A solution of N-[N'-[ (1S)-1-benzyloxycarbonyl-2-phenyl-ethyl]-L-leu- 5 cyl]-d ,1 '-biphenyl-4-yl)-L-alanine benzyl ester (3.5 g; 5.13 mmo¬ les), prepared as described in example 3, in ethanol (114 ml) was hydrogenated into a Parr apparatus in the presence of palladium on charcoal at 10% (0.6 g) . After having filtered off the catalyst, the reaction mixture was 0 evaporated to dryness and the resultant crude was purified by silica gel column chromatography (eluent methylene chloride:methanoi:acetic acid=90:10:1). 5 The thus obtained compound (1.65 g) was treated with a solution of sodium hydroxide 1N (5.9 ml) at room temperature and the solution was evaporated to dryness. The residue, triturated with acetonitrile and with ethanol and filtered, furnished N-[N'-[ 1S)-1-carboxy-2-phenyl-ethyl]-L-leucyl]- Q C1 ,1 '-biphenyl-4-yl)-L-alanine disodium salt C1.3 g) as a white powder. " Η-NMR C200 MHz, D a 0) : δCppm): 0.39 Cd, 3H, CHarCH) ; 0.45 Cd, 3H, CHarCH); 0.79-0.86 Cm, 2H, CH-CH 2 .-CH); 0.97-1.16 Cm, 1H, CH 3 -CH.-CH 3 ); 2.29-3.10 Cm, 6H, CH-NH-CH-CH^-Ph. CHarbiphenyl) ; 5 4.12-4.19 Cm, 1H, CH.-C00H) ; 6.91-7.36 Cm, 14H, aryl). Mass: 503 CM+H) Example 9 Preparation of N- 2.3-dimethyl-succinyl)-(l .1 '-biphenyl-4-yl)-L-ala¬ nine methyl ester n Meso-2,3-dimethyl-succinic acid (1.5 g; 10.27 mmoles) was suspended in trifluoroacetic anhydride (15 ml) and kept under stirring up to complete dissolution (10 minutes). The reaction mixture was stirred overnight at room temperature and the excess of anhydride was removed under reduced pressure. The resultant oil was dissolved in methylene chloride (10 ml) and added dropwise to a solution of (1 ,1 '-biphenyl-4-yl)-L-alanine methyl ester (3.6 g; 12.32 mmoles) in methylene chloride (20 ml) at 0°C and under stirring. After 15 minutes the reaction mixture was kept at room temperature and the solution was stirred overnight. The organic phase was washed with hydrochloric acid 1N (30 ml) and water (30 ml), dried on sodium sulphate and evaporated to dryness under reduced pressure. 5 The crude product was purified by flash chromatography [flash chro¬ matography silica gel, (Baker - code 7024-00), eluent methylene chloride:methanol:acetic acid=90:10:1 , pressure of nitrogen=0.2 atmospheres) furnishing N-(2,3-dimethyl-succinyl)-(1 ,1 '-biphenyl-4- -yl)-L-alanine methyl ester (3.4 g; 86% yield) as a mixture of two Q diastereoisomers. m.p. 187-189°C " Η-NMR (200 MHz, CDC1 3 ): δ(ppm): 1.01-1.20 (m, 6H, 2CHa_-CH) ; 2.40- -2.81 (m, 2H, CH-CH); 3.00-3.30 (m, 2H, CH rbiphenyl) ; 3.75, 3.76 (2s, 3H, OCHa); 4.87-5.00 (m, IH, CH-COO); 6.30-6.40 (m, 1H, CON ; 5 7.10-7.60 (m, 9H, aryl). Example 10 Preparation of N-(2.3-dimethyl-succinyl)-(1.1 '-biphenyl-4-yl)-L-ala¬ nine (Compound 23) Lithium hydroxide monohydrate C409 mg; 9.73 mmoles) was added to a Q suspension of N-C2,3-dimethyl-succinyl)-C1 ,1 '-biphenyl-4-yl)-L-ala¬ nine methyl ester C1.5 g; 3.9 mmoles), prepared as described in example 9, in a tetrahydrofuran:water=2:1 mixture (15 ml). The reaction mixture was kept under stirring at room temperature for one night. After that, tetrahydrofuran was evaporated under reduced pressure 5 and water (20 ml) was therein added. The solution was acidified with hydrochloric acid 1N and the thus obtained precipitated was extracted with ethyl acetate (3x20 ml). The organic phase was dried on sodium sulphate and evaporated at reduced pressure affording N-(2,3-dimethyl-succinyl)-(1 ,1 '-biphenyl- -4-yl)-L-alanine (1.2 g; 83% yield) as a mixture of two diastereo- isomers. m.p. 170-172°C 5 " Η-NMR (200 MHz, DMSO-d.*) : δ(ppm): 0.61-0.99 (m, 6H, 2CHa_-CH) ; 2.14-2.45 (m, 2H, CH-CH); 2.79-3.18 (m, 2H, CH-_,-biphenvl) : 4.39-4.54 (m, 1H, CH-C00); 7.29-7.64 (m, 9H, aryl); 8.30-8.38 (m, 1H, CONE); 12.04 (bs, 2H, 2C00H). Mass: 370 (M+H) 0 Example 11 "In vitro" evaluation of the pharmacologic activity a) NEP-inhibitory activity The NEP-inhibitory activity was evaluated according to the method reported in the literature by C. Llorens et al., in Eur. J. 5 Pharmacol., £2., (1981), 113-116. Membranes from kidney cortex were prepared according to the following procedure. By working at 0-4°C, kidneys were removed from male Sprague-Daw- ley rats weighing approximately 300 g. Q Cortex was carefully dissected, finely minced and suspended in a homogenization buffer (10 mM sodium phosphate pH 7.4 containing 1 mM MgCla, 30 mM NaCl, 0.02% NaN 3 ) 1:15 weight/volume. The tissue was then homogenized for 30 seconds using an Ultra- -Turrax homogenizer. Approximately 10 ml of homogenate were layered over 10 ml of 5 sucrose (41% weight/volume) and centrifuged at 31200 rpm for 30 minutes at 4°C in a fixed angle rotor. The membranes were collected from the buffer/sucrose interface, washed twice with 50 mM TRIS/HCl buffer (pH 7.4) and resuspended into the same buffer for storage. The membranes were stored in small aliquots at -80°C until use. The NEP-inhibitory activity was evaluated by using the following method. Aliquots of the membrane suspension prepared as above described (concentration 5 μg/ml of proteins) were preincubated in the presence of an aminopeptidase inhibitor (Bestatin - 1 mM) for 10 minutes at 30°C. [*H][Leu β ]-enkephaline (15 nM) and buffer TRIS/HCl pH 7.4 (50 mM) were added in order to obtain a final volume of 100 μl. Incubation (20 minutes at 30°C) was stopped by adding HC1 0.1M (100 μl). The formation of the metabolite [ 3 H]Tyr-Gly-Gly was quantified by chromatography on polystyrene columns (Porapak Q) . The percentage of inhibition of the metabolite formation in the membrane preparations treated with the compounds of formula I and the reference compounds, in comparison to the untreated membrane preparations, was expressed as IC β o value (nM) . b) ACE-inhibitory activity The ACE-inhibitory activity was evaluated according to the method reported in the literature by B. Holmquist et al., in Analytical Biochemistry 95. 540-548 (1979). 50 μM of ACE (250 mU/ml purified by lung rabbit, EC 3.4.15.1 SIGMA) with 50 μl of the compound of formula I or of the refer¬ ence compounds, were preincubated in thermostated cuvettes at 37°C for 5 minutes. The reaction was started by adding furylacryloylphenylalanylgly- cylglycine 0.8 mM (FAPGG-SIGMA) . Contemporaneously, by using a Beckman DU-50 spectrophotometer provided with a program for calculating delta A/minutes and regression coefficients of the enzyme kinetics curves, the ab- sorbance at 340 nm was recorded in continuo for 5 minutes. The percentage of the enzyme inhibition in the preparations treated with the compounds of formula I and with the reference compounds with respect to the untreated preparations was expres¬ sed as ICβo value (nM). The compounds of formula I were tested in the form of sodium salts. Ue report in the following table 1 the ACE-inhibitory and NEP-inhib- itory activity of compounds 1, 10, 15, 17 and 20 in comparison to thiorphan and Captopril. Table 1 ACE-inhibitory activity and NEP-inhibitory activity, expressed as ICβo (nM), of compound 1, compound 10, compound 15, compound 17, compound 20, thiorphan and Captopril. Compound ACE-inhibitory Activity NEP-inhibitory activity 27.7 41 10 27 53 11.3 not active The compounds of formula I, object of the present invention, are endowed with a significant mixed ACE/NEP-inhibitory activity. Moreover, the inhibitory activity of the compounds of formula I is comparable to the ACE-inhibitory activity of Captopril as well as to the NEP-inhibitory activity of thiorphan.

Claims: Claims 1 ) A compound of formula wherein R is a biphenyl group optionally substituted by one or more sub¬ stituents, the same or different, selected among halogen atoms, nitro groups, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxy- carbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C 3 alkyl groups containing one or more fluorine atoms, carboxy groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylamino¬ carbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R. is a hydrogen atom, a straight or branched C-,-C Λ alkyl or an arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a biphenyl, a naphthyl or a 5- or 6-membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substit¬ uted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C 3 alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-al¬ kylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R 3 is a straight or branched C-,-C_s alkyl optionally substituted by one or more substituents selected among amino, mono- or di-CC.-C Λ )alkylamino or aminocarbonyl groups, mono or di-Cd-d)- alkylaminocarbonyl groups, an arylalkyl, an arylcarbonylaminoal- kyl, an arylaminocarbonylalkyl or an arylalkylaminocarbonylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety, the aryl being optionally substituted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C 3 alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, amino- sulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbon¬ yl groups having from 1 to 6 carbon atoms in the alkyl moiety; X is a single bond or a -NCR 3 )- group wherein R 3 is a hydrogen atom or a straight or C-,-C_w alkyl group; the carbon atoms marked with an asterisk are asymmetric carbon atoms; and its pharmaceutically acceptable salts. 2) A compound according to claim 1 wherein R is a 4-biphenyl group; R-, is a straight or branched C.-C__ alkyl group or an arylalkyl having from 1 to 3 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a naphthyl or a 5 membered aromatic heterocyclic group; R a is an arylalkyl, an arylalkylaminocarbonylalkyl or an arylamino¬ carbonylalkyl group having from 1 to 4 carbon atoms in the alkyl moiety wherein the aryl is a phenyl and X is a -NCR 3 )- group wherein R 3 is a hydrogen atom. 3) A compound according to claim 1 wherein R is a 4-biphenyl group; R-i is isopropyl, isobutyl or 2-thienylmethyl and R 3 is 2-phenyleth- yl, 2-phenylaminocarbonylethyl or benzylaminocarbonylmethyl. 5 4) A compound according to claim 1 in the form of a salt with an alkali metal selected among sodium, lithium and potassium. 5) A process for the preparation of a compound of formula I accord¬ ing to claim 1 which comprises the reaction between a compound of formula 10 *- ■ I R__00C-CH-X-CH-C00H (II) I Ra wherein Ri , R 3 and X have the meanings reported in claim 1 and R__ represents a protective group selected among a Ci-C* alkyl, a phenyl 5 or a phenylalkyl having from 1 to 4 carbon atoms in the alkyl moi¬ ety; and a biphenylalanine derivative of formula NH 2 -CH-C00H (III) I CH 2 -R 0 wherein R has the meanings reported in claim 1. 6) A process for the preparation of a compound of formula I accord¬ ing to claim 1 wherein X is a -N(R 3 )- group which comprises the reaction between a compound of formula OH I c Ra-CH-COOH (VIII) wherein R 3 has the meanings reported in claim 1 ; and a dipeptide of formula R*. R 3 -NH-C IH-C0NH-CH-C00H (IX) I CHa-R wherein R, R-, and R 3 have the meanings reported in claim 1. 7) A pharmaceutical composition containing a therapeutically effec¬ tive amount of a compound of formula I according to claim 1 in 5 admixture with a carrier for pharmaceutical use. 8) A pharmaceutical composition according to claim 7 for the treat¬ ment of cardiovascular diseases. 9) N-[N'-(1-carboxy-3-phenyl-propyl)-L-leucyl]-(1 ,1 '-biphenyl-4-yl)- -L-alanine. 10 10) N-[N'-(1-carboxy-3-phenyl-propyl)-L-valyl]-(1 ,1 '-biphenyl-4-yD- -L-alanine. 11 ) N-[N'-(1-carboxy-3-phenyl-propyl)-(2-thienyl)-L-alanyl]-(1 ,1 '- -biphenyl-4-yl)-L-alanine. 12) N-[N'-(1-carboxy-3-phenylaminocarbonyl-propyl)-L-leucyl]-(1 ,1 '- 15 -biphenyl-4-yl)-L-alanine. 13) N-[N'-(1-carboxy-3-phenylaminocarbonyl-propyl)-L-valyl]-(1 ,1 '- -biphenyl-4-yl)-L-alanine. 14) N-[N'-(1-carboxy-3-phenylaminocarbonyl-propyl)-(2-thienyl)-L- -alanyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine. 20 15) N-[N'-(2-benzylaminocarbonyl-1-carboxy-ethyl)-L-leucyl]-(1 ,1 '- -biphenyl-4-yl)-L-alanine. 16) N-[N'-(2-benzylaminocarbonyl-1-carboxy-ethyl)-L-valyl]-(1 ,1 '- -biphenyl-4-yl)-L-alanine. 17) N-[N'-(2-benzylaminocarbonyl-1-carboxy-ethyl)-(2-thienyl)-L-ala- _ nyl]-(1 ,1 '-biphenyl-4-yl)-L-alanine. 18) A pharmaceutically acceptable salt of a compound according to anyone of claim 9 to 17. 0

Inventors: BOTTA DANIELA, NORCINI GABRIELE, PELLACINI FRANCO, ROMAGNANO STEFANO, SANTANGELO FRANCESCO, BOTTA, DANIELA, NORCINI, GABRIELE, PELLACINI, FRANCO, ROMAGNANO, STEFANO, SANTANGELO, FRANCESCO
Applicants: BOTTA DANIELA, NORCINI GABRIELE, PELLACINI FRANCO, ROMAGNANO STEFANO, SANTANGELO FRANCESCO, ZAMBON SPA, BOTTA, DANIELA, NORCINI, GABRIELE, PELLACINI, FRANCO, ROMAGNANO, STEFANO, SANTANGELO, FRANCESCO, ZAMBON GROUP S.P.A.
Application year: 1995
Main classification: C07K5
IPC codes: A61K38, C07K5