"Thiol derivatives with metallopeptidase inhibitory activity"

The present invention relates to thiol derivatives with metallopeptidase inhibitory activity and, more particularly, it relates to mercaptoacylamino derivatives useful in the treatment of cardiovascular diseases.

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 converting enzyme (ACE) inhibitory activity are mainly useful in the treatment of hypertension and of heart failure in that they inhibit the formation of angiotensin D, a substance which increases the blood pressure.

Compounds with endothehn converting enzyme (ECE) inhibitory activity are useful as anti- vasoconstrictors in that they inhibit the formation of endothehn, a 21 amino acid peptide with vasoconstrictor activity.

Instead, compounds with inhibitory activity of the neutral endopeptidase enzyme (NEP), also called enkephalinase, are useful as vasodilators in that the NEP enzyme is responsible for the inactivation, not only of endogenous enkephaline, but also of atrial natriuretic factor (ANF), a hormone secreted by heart which increases the vasodilation and, on the renal level, increases diuresis and natπuresis

Therefore, even exerting their action on the cardiovascular system with different mechanisms of action, the compounds with metallopeptidase inhibitory activity are generally used, alone or in combination, in the treatment of hypertension, renal failure, congestive heart failure and ischemic cardiopathologies. Among the metallopeptidase inhibitors having a mercaptoacylamino structure, Thiorphan [ L-(3-mercapto-2-benzylpropanoyl)glycine], described for the first time by Roques et al. in Nature, Vol. 288, pages 286-288, (1980), and Captopril (The Merck Index, XI ed., No. 1773, page 267) are considered the parent compounds for NEP-inhibitors and ACE-inhibitors, respectively. Other molecules with a mercaptoacylamino structure endowed with metallopeptidase inhibitory activity are described in the literature.

The US patent No 4,401,677 (E R Squibb & Sons, Inc ) descπbes mercaptoalkanoyl amino aαds endowed with enkephahnase inhibitory activity

The European patent application No 0 566 157 (Scheπng Corporation) descπbes mercaptoalkanoyl ammo aαds endowed with neutral metalloendopeptidase inhibitory activity The European patent application No 0 419 327 (Soαete Civile Bioproject) descπbes ammo acid denvatives endowed with enkephahnase and ACE inhibitory activity The European patent application No 0 449 523 (E R Squibb & Sons, Inc ) descπbes mercapto or acylthio tnfluoromethylamides with NEP-inhibitory activity The international patent application No WO 93/08162 [Rhone-Poulenc Rorer S A - Institut National de la Sante et de la Recherche Medicale (INSERM)] descπbes ^β ,β-dιsubstituted α- mercaptomethylpropionylamides endowed with a mixed ACE/NEP inhibitory activity Among the compounds descnbed in the patent application No WO 93/08162, the compound known with the abbreviation RB 105 [N-[2-(mercaptomethyl)-3-phenylbutanoyl]-L-alanιne] is under study as mixed ACE/NEP inhibitor for the treatment of cardiovascular diseases [Fourme-Zaluski M C et al , Proc Natl Acad Sci USA, vol 91, pages 4072-4076, (1994)] The European patent application No 0 524 553 [Institut National de la Sante et de la Recherche Medicale (INSERM)] descπbes acylmercaptoalkanoyldipeptides endowed with neutral endopeptidase and peptidyldipeptidase A inhibitory activity Now we have found mercaptoacylamino denvatives which are endowed with a remarkable inhibitory activity on the angiotensin converting enzvrne as well as on the neutral endopeptidase enzyme (mixed or dual ACE/NEP inhibitory activity) which makes them particularly useful m the cardiovascular therapy Therefore, object of the present invention are the compounds of formula

wherein

R is a mercapto group or a R.COS group convertible in the organism to mercapto group,

R. is a hydrogen atom, a straight or branched C--C , alkyl group, an aryl or an arylalkyl

group having from 1 to 6 carbon atoms in the straight 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 with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C--C- alkyl groups containmg one or more fluorine atoms, carboxy groups, rutro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or dι-alkylamιno groups having from 1 to 6 carbon atoms m the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety,

R. is a hydrogen atom, a straight or branched C.-C, alkyl group or an arylalkyl group having from 1 to 6 carbon atoms m 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 with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C. -C, alkyl groups containing one or more fluoπne atoms, carboxy groups, rutro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkviammo 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~ is a biphenyl group optionally substituted with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, alkylthio or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C.-C. alkyl groups containing one or more fluoπne atoms, carboxy groups, rutro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di- alkylammo 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 is a hydrogen atom, a C.-C . alkyl group or a benzyl group, R, is a C.-C . alkyl group or a phenyl group,

m is 0 or 1,

the carbon atom marked with an asteπsk is a stereogenic centre, and pharmaceutically acceptable salts thereof

Object of the present invention are the compounds of formula I in the form of stereoisomeπc 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- lnhibitory and NEP-mhibitory activity and are useful in the treatment of cardiovascular diseases

In the present descπption, unless otherwise specified, with the term biphenyl group we intend a 2-bιphenyl, 3-bιphenyl or 4-bιphenyl group, with the term alkyl group we intend a straight or branched alkyl such as methyl, ethyl, n propyl, isopropyl, n butyl, sec-butyl, tert-butyl, isobutyl, n.pentyl, 2-pentyl, 3-pentyl, isopentyl, tert-pentyl, n hexyl and isohexyl, with the term halogen atom we intend a fluoπne, chlorine, bromine or iodine atom, with the term acyl we intend an acyl group deπvmg from an aliphatic or aromatic carboxyhc aαd such as acetic, propionic, butync and benzoic aαd, with the term aryl we intend an aromatic group such as phenyl, 2-bιphenyl, 3-bιphenyl, 4-bιphenyl, 1 -naphthyl and 2-naphthyl or a heterocyclic group containing 1 or 2 heteroatoms selected among nitrogen, oxygen and sulphur such as thiazole, isoxazole, oxazole, isothiazole, pyrazole, imidazole, thiophene, pyrrole, pyπdine, pynmidine 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 salts with mineral aαds Preferred compounds of formula 1 are the compounds wherein R is a 4-bιphenyl group optionally substituted with from 1 to 3 substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy groups and alkyl groups, R. is a phenylalkyl group optionally substituted with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy groups and alkyl groups, R . is a hydrogen atom, m is

Still more preferred compounds of formula I are the compounds wherein R is a mercapto group, R, is a 4-bιphenyl group optionally substituted with from 1 to 3 substituents, the same

or different, selected among fluorine atoms or hydroxy groups; R. is a phenylmethyl group optionally substituted with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy groups and alkyl groups; R . is a hydrogen atom; m is

Another class of preferred compounds of formula I are the compounds wherein R is a mercapto group; R_ is a 4-biphenyl group optionally substituted with from 1 to 3 substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy groups and alkyl groups; R. is an arylalkyl group wherein the aryl group is a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted with one or more substituents, the same or different, selected among alkyl groups; R . is a hydrogen atom; m is 0 and n is i.

Preferred examples of pharmaceutically acceptable salts of the compounds of formula I are the salts with alkali metals such as sodium, lithium and potassium and the salts with mineral acids such as hydrochloric and hydrobromic acid.

It is clear that the compounds of formula I wherein R is a R^COS group convertible in the organism to mercapto group and the compounds of formula I wherein R . is a C.-C . alkyl group or a benzyl group are biologic precursors (pro-drugs) of the corresponding compounds of formula I wherein R is a mercapto group (R=SH) or R . is a hydrogen atom (R =H), respectively.

The preparation of the compounds of formula 1, object of the present invention, comprises the reaction between a compound of formula

R-(CH ₂ ) _n -CH-COOH (TJ) wherein

R, R. and n have the above reported meanings; and a biphenylalanine derivative of formula

(TH)

wherein

R_, R», R . and m have the above reported meanings

The condensation is earned out according to conventional techniques of the chemistry of peptides

Before carrying out the reaction, it can be useful to properly protect the eventual functional groups which could interfere in the reaction

The optional protection is earned out according to conventional techniques

In this respect, the compounds wherein R is a R.COS group are preferably used as intermediates of formula π, thus obtaining the corresponding compounds of formula I wherein

R=R,COS from which, by hydrolysis, the compounds of formula I wherein R=SH can be obtained

The evaluation of the usefulness of the optional protection as well as the selection of the kind of adopted protection, according to the reaction to be earned out and to the functional groups to be protected, are within the normal knowledge of the man skilled in the art

The removal of the optional protective groups is earned out according to conventional techmques

For a general reference to the use of protective groups in organic chemistry see Theodora W

Greene and Peter G M Wuts "Protective Groups in Organic Synthesis", John Wiley & Sons, Inc , π Ed , 1991

The compounds of formula II are known compounds or easily prepared according to conventional methods

For instance, the compounds of formula D can be prepared as reported in Bπtish patent No

1,576,161 (E R Squibb & Sons Inc ) or, alternatively, according to the synthetic method descnbed by M C Fourme-Zalusky et al in J Med Chem 1994, 37, 1070-1083

Also the intermediates of formula UJ are known or easily prepared according to known methods

For a bibliographic reference to the preparation of the compounds of formula HI see, for instance, Michel Sy et al , Bull Soc Chim Fr , 1276-1277, (1963) and Moses Lee et al , J Org Chem , 53(9), 1855-1859, (1988)

The compounds of formula I in the form of single stereoisomers are prepared by

stereoselective synthesis or by separation of the stereoisomeπc mixture according to conventional techniques

Also the preparation of the salts of the compounds of formula I, object of the invention, is earned 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

The inhibitory activity of the compounds of formula I was evaluated by means of in vitro tests in compaπson to the aforementioned Thiorphan, Captopπl and RB 105

The activity of the compounds of formula I, expressed as IC.- (nM) or as percentage of inhibition, is pharmacologically significant in compaπson to the NEP-inhibitory activity of Thiorphan as well as in compaπson to the ACE-mhibitory activity of Captopnl Furthermore, the mixed ACE/NEP-inhibitory activity of the compounds of formula I, object of the present mvention, is comparable or better than that of the mixed ACE/NEP inhibitor RB 105 (example 3)

The compounds of formula I, moreover, confirmed their mixed ACE/NEP-inhibitory activity also by means of ex vivo tests The ex vivo ACE/NEP-inhibitory activity of the compounds of formula I, in particular, was evaluated by consideπng the enzymatic activity in tissue homogenates (lung and kidney for the ACE- and NEP-inhibitory activity, respectively) from spontaneously hypertensive rats (SHR), 5 minutes after 1 v injection (064 μmoles/Kg) of the tested compounds For instance, in the following table are reported the percentages of inhibition of the basal enzymatic activity ex vivo after I v administration of N-(2-mercaptomethyl-3- phenyIpropιonylHl.l'-b hen l-4-yl)-L-alanιne, a compound representative of the class of compounds of formula I, object of the present invention Table 1 l v injection ACE-mhibitory activity NEP-inhibitory activity

(lung) (kidney)

064 μmoles/Kg 49% 20%

For a practical use in therapy, the compounds of formula I can be formulated in solid or liquid

pharmaceutical compositions, suitable to oral or parenteral administration Therefore, the pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I in admixture with a earner for pharmaceutical use are a further object of the present invention

Speαfic 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 techmques

The daily dose of the compound of formula I will depend on different factors such as the seπousness of the disease, the individual response of the patient or the kind of formulation but it is usually compπsed between 0 1 mg and 10 mg per Kg of body weight divided into a single dose or into more daily doses With the aim of illustrating the present invention, without limiting it, the following examples are now given

Unless otherwise specified, the flash chromatographies were earned out by using flash chromatography silica gel from Baker company (code 7024-00) and the thin layer chromatographies (TLC) were earned out by using "silica gel plates 60 F. - " from Merck company (code 1 05719)

Example 1 Preparation of N-[3-phenylcarbonylthio-2-(phenylmethyl)propionyll-( 1.1 '-bιphenyl-4-yl)-L- alanine phenylmethyl ester A solution of hydroxybenzotπazole (1 1 g, 8 15 mmoles) in tetrahydrofuran (30 ml) and, subsequently, a solution of dicyclohexylcarbodiimide (2 02 g, 9 78 mmoles) in methylene chloπde (15 ml) were added, at 0°C under stirπng, to a mixture of 3-phenylcarbonylthιo-2- (phenylmethyl)propιonιc aαd (2 5 g, 8 15 mmoles), ( l ,l'-bιphenyl-4-yl)-L-alamne methyl ester hydrochloπde (3 g, 8 15 mmoles) and tπethylamine (1 14 ml, 8 15 mmoles) in tetrahydrofuran (20 ml) and methylene chloπde (30 ml) The reaction mixture was kept under stirπng for 20 hours, then dicyclohexylurea was filtered off and the solvent was evaporated at reduced pressure

The residue was collected with ethyl acetate and the solution was washed with aqueous solutions of citric acid at 10%, sodium chloride at 20%, sodium bicarbonate at 5% and sodium chloride at 20% again. After separation of the phases and evaporation of the organic phase, the resultant white solid

(6 g) was purified by flash chromatography (silica gel, eluent ethyl acetate:hexane=25:75, pressure of nitrogen=0.1 atm) affording the title compound (4.2 g). m.p. 103-105°C

^-NMR (200 MHz, CDCy: δ (ppm): 7.98-6.53 (m, 24H, 5Ar); 5.95-5.75 (2d, IH, 2NH); 5.10-4.98 (m, 2H, COOCH ; 4.95-4.78 (m, IH, CHCOO); 3.35-2.60 (m, 7H,

SCH ₂ CHCH ₂ and CH^-biphenyl).

[M-H] ⁺ =613;

By working in an analogous way the following compounds were prepared:

N-f 2-acet\dthiomethyl-3-(3-methoxyphenyl)propionyl ]-( 1.1 '-biphenyl-4-yl VL-alanine methyl ester from which the two stereoisomers were separated by flash chromatography (silica gel, eluent ethyl acetate:hexane=25:75, pressure of nitrogen=0.1 atm)

Stereoisomer A m.p. 109-110°C

TLC (ethyl acetate:hexane=30:70) R =0.18

^-NMR (200 MHz, CDC1..): δ (ppm): 7.55-6.65 (m, 13H, 3Ar); 5.78 (d, IH, NH); 4.85-

4.72 (m, IH, CHCOO); 3.73 (s, 3H, OCH,.); 3.62 (s, 3H, COOCR.); 3.20-2.71 (m, 6H,

SCH ₂ CHCH ₂ and CH biphenyi); 2.63-2.49 (m, IH, CH- _j CHCH^); 2.29 (s, 3H, CH ₃ COS)

[M-H] ⁺ =506;

N-f2-acetylthiomethyl-3-(3-methoxyphenyl)propionyl]-(l . -biphenyl-4-yl)-L-alanine methyl ester

Stereoisomer B m.p. 83-85°C

TLC (ethyl acetate:hexane=30:70) R _f =0.17

^-NMR (200 MHz, CDC1 ₃ ): δ (ppm): 7.55-6.62 (m, 13H, 3Ar); 5.83 (d, IH, NH); 4.91- 4.81 (m, IH, CHCOO); 3.77 (s, 3H, OCH ₃ ); 3.68 (s, 3H, COOCH ₃ ); 3.10-2.70 (m, 6H,

SCH ₂ CHCH ₂ and CH ₂ -biphenyl); 2.30 (s, 3H, CH ₃ COS)

[M-H] ⁺ =506,

N-[3-phenylcarbcnyl.hιo-2-(phenylmethyl)propιonyl]-(3'- cruoro-l.l'-bιphenyl-4-yl)-L-alanme methyl ester m p 111-113°C

^-NMR (200 MHz, CDCy δ (ppm) 8 00-6 62 (m, 18H. Ar), 5 89 and 5 71 (2d, IH, NH),

4 91-4 75 (m, IH, CHCOO), 3 60 (s, 3H, COOCH.,), 3 37-2 61 (m, 7H, CH ₂ CHCH ₂ and

CH.-biphenyl)

[M-H] ⁺ =572, N-[(2S)-3-phenylcarbonylthιo-2-(phenylmethyl)propιonyll-( 1 , 1 '-bιphenyl-4-yl)-L-alanιne methyl ester m p 138-140°C

TLC (ethyl acetate hexane=3 7) R =0 25

^-NM (200 MHz, CDC ) δ (ppm) 8 00-7 01 (m, 19H, Ar), 5 75 (d, IH, NH), 4 85-4 72 (m, IH, CHCOO), 3 60 (s, 3H, COOCH ₃ ), 3 35-2 61 (m, 7H, CH ₂ CHCH ₂ and CH biphenyl)

[M-H] ⁺ =538,

N-f(2RV3-phenylcarbonylthιc~2-(phenylmethyl)propιony1]- ( 1.1 '-bιphenyl-4-yl )-L-alanιne methyl ester m p 156-158°C

TLC (ethyl acetate hexane=l 1) R _f =0 5

¹ H-NMR (200 MHz, CDCI ₃ ) δ (ppm) 7 98-6 61 (m, 19H, Ar), 5 89 (d, IH, NH), 4 92-4 80

( , IH, CHCOO), 3 60 (s, 3H, COOCH-.), 3 30-2 60 (m, 7H, CH ₂ CHCH ₂ and CH ₂ - biphenyl) [M-H] ⁺ =538,

N-r2-ιsopropyl-3-(phenylcarbonylthιo)propιonyn-( 1.1 '-bιphenyl-4-yl)-L-alanιne methyl ester m p 123-125°C

^-NMR (200 MHz, CDCψ δ (ppm) 7 98-7 07 (m, 14H, Ar), 5 93 (2d, IH, NH), 5 02-

4 87 ( , IH, CHCOO), 3 71-3 65 (2s, 3H, COOCH..), 3 51-3 00 (m, 4H, S-CH ₂ and CH.,- biphenyl), 2 28-2 11 (m, IH, CH-CONH), 2 82-2 02 (m, IH, S-CH-CH-CH), 1 07 and 0 98 and 0 89 (3d, 6H, CH CH-Ciy

+

[M-H] =490,

N-[2-phenylcarbonylthιomethyl-3-(3-pyridyl .propionylH 1.1 '-bιphenyl-4-yl)-L-alanιne methyl ester - Stereoisomer A m.p 158-160°C

TLC (methylene chloride:methanol=95:5) R.=0 5

^-NMR (200 MHz, CDC1 ₃ ) δ (ppm): 8 40-6.75 (m, 18H, Ar); 6.98 (d, IH, NH); 4.90-4 78 (m, IH, CHCOO), 3 60 (s, 3H, COOCR.), 3 35-2 65 (m, 7H, CH ₂ CHCH ₂ and CH ₂ - biphenyl) [M-H] ⁺ =539,

N-f 2-phenylcarbonylthιomethyl-3-(3-pyndyl)propιonyl 1— ( 1.1 '-biphenyl -4-yl)-L-alamne methyl ester - Stereoisomer B m.p 142-144°C

TLC (methylene chloride:methanol=95:5) R.=0.4 ^-NMR (200 MHz, CDCy δ (ppm): 8.30-7.02 (m, 18H, Ar); 6 65 (d, IH, NH), 4 91-4.80

(m, IH, CHCOO), 3.60 (s, 3H, COOCH ₃ ), 3.21-2 65 ( , 7H, CH ₂ CHCH ₂ and CH ₂ - biphenyl)

[M-H] ⁺ =539,

N-[3-(2-furyl 2-(phenylcarbonylthιomethyl)propιonyll-( 1. l'-bιphenyl-4-yl ,-L-alanιne methyl ester m.p 114-116°C H-NMR (200 MHz, CDC1 ₃ ): δ (ppm): 7.98-6.00 (m, 18H, Ar and NH); 4.95-4.82 (m, IH,

CHCOO), 3 69-3.62 (2s, 3H, COOCH ₃ ), 3 36-2.35 (m, 7H, CH ₂ CHCH ₂ and CH ₂ - biphenyl) [M-H] ⁺ =528,

N-f3-(3-methyl-5-ιsoxazolylV2-(phenyl.^rbonylthιomethyl )propιonyl]- ^' l. -bιphenyl-4-yl)-L- alanine methyl ester - Stereoisomer A m.p 130-132°C

TLC (ethyl acetate:hexane=l:l) R _f =0.39 ^! H-NMR (200 MHz, CDC1 ₃ ): δ (ppm): 7.92-7.00 (m, 14H, Ar), 6.34 (d, IH, NH), 5 88 (s,

IH, CH-isoxazolyl), 4.93-4 80 (m, IH, CHCOO); 3.66 (s, 3H, COOCH ₃ ), 3 40-2.80 (m, 7H,

N-[3-(3-memyl-5-isoxazolylV2-(phenylcarbonylt-tiiomethyl) propionyll-(l.r-biphenyl-4-yl ^' l-L- alanine methyl ester - Stereoisomer B m p 146-148°C

TLC (ethyl acetate hexane=l 1) R _f =0 34

'H-NMR (200 MHz, CDC1 ₃ ) δ (ppm) 7 95-7 10 (m, 14H, Ar), 6 15 (d, IH, NH), 5 91 (s,

IH, CH-isoxazolyl), 4 95-4 85 (m, IH, CHCOO), 3 70 (s, 3H, COOCH ₃ ), 3 21-2 78 (m, 7H, CH ₂ CHCH ₂ and CH ₂ -bιphenyl)

[M-H] ⁺ =543

N-f(2SV3-phenyl-2-(phenylcarbonylthιo)propιonyll-( 1 , 1 '-bιphenyl-4-yl)-L-alanιne methyl ester m p 128-130°C ^-NMR (200 MHz, CDC1 ₃ ) δ (ppm) 7 82-7 00 (m, 1 H, Ar), 6 70 (d, IH, NH), 4 90-4 80

(m, IH, CHCOO), 4 41 (t, IH, CH-S), 3 70 (s, 3H, COOCH ₃ ), 3 50-2 93 (m, 4H, CH CH-

Ar and CH.-biphenyl)

[M-H] ⁺ =524,

Example 2 Preparation of N-(2-mercaptomethyl-3-phenylpropιonyl)-(l. -bιphenyl-4-yl)-L-alanιne

(Compound 1)

N-[3-phenylcarbonylthιo-2-(phenylmethyl)propιonyl]-( 1 , 1 '-bιphenyl-4-yl)-L-alanιne phenylmethyl ester (1 84 g, 3 mmoles), prepared as descπbed in example 1, was suspended in ethanol (36 ml), degassed by nitrogen bubbling to eliminate the oxygen An aqueous degassed solution of sodium hydroxide IN (9 ml) was added dropwise at 5°C to the suspension and, at the end of the addition, further degassed ethanol was added (20 ml)

The reaction mixture was kept under stirnng for 4 hours at room temperature, then cooled at

0°C and acidified with hydrochloπc aαd 5% (10 ml)

The resultant solution was evaporated under reduced pressure and the residue was puπfied by flash chromatography (silica gel, eluent ethyl acetate hexane acetic aαd=30 70 5, pressure of nιtrogen=0 1 atm) affording a solid which, after treatment with hexane, furnished Compound

1 (1 2 g) m p 108-110°C

^-NMR (200 MHz, CDCy δ (ppm) 7 54-6 80 (m, 14H, Ar), 5 90 (d, IH, J _JiH =7 8 Hz, NH), 4 96-4 83 (m, IH, CHCOO), 3 30-2 42 (m, 7H, SCH ₂ CHCH ₂ and

1 62-1 53 and 1 38-1 29 (m, IH, SH)

[M-H] ⁺ =420

By working in an analogous way the following compounds were prepared

N-[2-mercaptomethyl-3-(3-methoxyphenyl)propιonyll-( 1.1 '-bιphenyl-4-yl)-L-alanιne Stereoisomer A (Compound 2) m p 122-124°C

[α] ² °=+55° (c=0 1, CHCl ₃ )

TLC (ethyl acetate hexane acetic aαd=50 50 5) R =0 37 H-NMR (200 MHz, DMSO-d.) δ (ppm) 8 31 (d, IH, J _tItr =8.1 Hz, NH), 7 62-6 69 (m, o riri 13H, Ar), 4 56-4 45 (m, IH, CHCOO), 3 69 (s, 3H, OCH ₃ ), 3 17-2 20 (m, 7H, 1 84-1 72 (bs, IH, SH)

N-12-mercaptomethyl-3-(3-methoxyphenyl)propιonyl1-(l. -bιphenyl-4-yl)-L-alan e

Stereoisomer B (Compound 3) m p 48-50°C

[α] ²⁰ =+22 7° (c=0 1 , CHC1 ₃ )

^-NMR (200 MHz, DMSO-d,) δ (ppm) 8 31 (d, IH, J _uu =8 1 Hz, NH), 7 62-6 63 (m,

13H, Ar), 4 51-4 40 ( , IH, CHCOO), 3 67 (s, 3H, OCH ₃ ), 3 05-2 32 (m, 7H, 2.26-2 08 (bs, IH, SH)

N-(2-mer(^ptomethyl-3-phenylpropιonyl.-(3'-chloro-l.l'-b ιphenyl-4-yl)-L-alamne

Stereoisomer A (Compound 4)

^-NMR (200 MHz, CDC1 ₃ ) δ (ppm) 7 49-6 80 (m, 13H, Ar), 5 87 (d, IH, J _fiH =7 6 Hz,

NH), 4 95-4 81 (m, IH, CHCOO), 3.30-3 00 (m, 2H, CH ₂ -bιphenyl), 2 94-2 43 (m, 5H, HS- CH ₂ -CH-CH ₂ ), 1 38-1 29 (m, IH, SH)

[M-H] ⁺ =454,

N-(2-mercaptomethyl-3-phenylpropιonylK3'-chloro- 1 , 1 '-bιphenyl-4-yl)-L-alamne

Stereoisomer B (Compound 5)

^! H-NMR (200 MHz, CDCL) δ (ppm) 7 49-6 68 (m, 13H, Ar), 5 93 (d, IH, J _TJI _ _I =7 8 Hz,

5 rlH

NH), 4 94-4 85 (m, IH, CHCOO), 3 12-2 45 (m, 7H- CH ₂ -CH-CH ₂ and CH biphenyl),

1 61-1 52 (m, IH, SH)

[M-H] ⁺ =454,

N-[(2SV2-mercaptomethyl-3-phenylpropionvn-( 1.1 '-bιphenyl-4-yl)-L-alanιne (Compound 6) m p 160-162°C

^] H-NMR (200 MHz, CDCL) δ (ppm) 7 54-7 08 (m, 14H, Ar), 5 84 (d, IH, J _TJT ,=7 5 Hz,

5 rlH

NH), 4 91-4 82 ( , IH, CHCOO), 3 25 and 3 07 (ABλ Jab=14 1 Hz, Jax=5 4 Hz, Jbx=7 0

Hz, CH ₂ -bιphenyl), 2 95-2 43 (m, 5H, HS-CH.,-CH-CH.,), 1 38-1 29 (m, IH, SH)

[M-H] ⁺ =420,

N-f(2RV2-mercaptomethyl-3-phenylpropιonyll-( 1.1 '-bιphenyl-4-yl)-L-alanιne (Compound 7) m p 147-149°C

^] H-NMR (200 MHz, CDCL) δ (ppm) 7 54-6 79 (m, 15H, Ar and COOH), 5 94 (d, IH,

J _uu =7 9 Hz, NH), 4 94-4 85 (m, IH, CHCOO), 3 13-2 43 (m, 7R HS-CFL-CH-CH., and riri 2. 1

CH ₂ -bιphenyl), 1 61-1 52 (m, IH, SH) [M-H] ⁺ =420, N-[(2S)-2-mercapto-3-phenylpropιonyl]-( 1.1 '-bιphenyl-4-yl )-L-alanιne (Compound 8) m p 93-95°C

^-NMR (200 MHz, CDC1 ₃ ) δ (ppm) 8 82 (bs, IH, COOH), 7 52-7 07 (m, 14H, Ar), 6 82 (d, IH, J _HH =7 7 Hz, NH), 4 91-4 82 (m, IH, CHCOO), 3 63-3 52 (m, IH, CH-SH), 3 26-

3 02 (m, 4H, CH.-biphenyl and CH.-Ar), 1 91 (d, IH, J,_ _πj =9 0 Hz, SH) [M-H] ⁺ =406,

N-(2-ιsopropyl-3-mercaptopropιonyl)-( 1 , 1 '-biphenyl -4-yl )-L-alamne (Compound 9) m p 182-184°C

^-NMR (200 MHz, CDC1 ₃ ) δ (ppm) 7 56-7 27 ( , 9H, Ar), 6 10-6 05 (m, IH, NH), 5 09-

4 91 (m, IH, CHCOO), 3 63-3 05 (m, 2H, 2 02-1 92 (m, IH, CH-CH ₂ -SH), 1 91-1 70 (m, IH, CH-.-CH-CH ₃ ), 1 60-1 21 (m, IH, SH),

0 91-0 70 (m, 6H, CH ₃ -CH-CH ₃ )

[M-H] ⁺ =372,

N-f2-mer<-aptomethyl-3-(3-pyπdyl)propιonyl1-(l.r-bι phenyl-4-yl)-L-alanιne hvdrochlonde

Stereoisomer A (Compound 10) ^-NMR (200 MHz, DMSO-d ) δ (ppm) 8 61-7 67 (m, 5H, pyπdyl and CONH), 7 64-7 16

(m, 9H, biphenyl), 4 47-4 37 (m, IH, CH-CH ₂ -bιphenyl), 3 05-2 43 (m, 7H, CH ₂ -CH-CH ₂ and CH ₂ -bιphenyl), 2 26 (m, IH, SH)

[M-H] ⁺ =421,

N-r2-mercaptomethyl-3-(3-pyπdyl)propιonyll-( 1.1 '-pιphenyl-4-yl)-L-alanιne hvdrochlonde Stereoisomer B (Compound 11) m p 215-217°C

¹ H-NMR (200 MHz, DMSO-d ) δ (ppm) 8 74-7 85 (m, 5H, pyπdyl and CONH), 7 61-7 28

(m, 9H, biphenyl), 4 54-4 42 (m, IH, CH-CH ₂ -bιphenyl), 3 13-2 37 (m, 7H, CH ₂ -CH-CH ₂ and CH -biphenyl), 1 86 (m, IH, SH) [M-H] ⁺ =421 ,

N-[3-(2-fuiΥlV2-(mercaptomethyl)propιonyl1-(l. -bιphenyl-4-yl)-L-alanιne (Compound 12) m p 137-140°C

^-NMR (200 MHz, CDCLJ δ (ppm) 7 56-7 08 (m, 10H, biphenyl and CH-O), 6 35 (b,

IH, COOH), 6 23-5 96 (m, 3H, CONH and O-CH-CH-CH), 5 00-4 87 (m, IH, CH-CH.,- biphenyl), 3 33-2 43 (m, 7H, CH -CH-CH and CH -biphenyl), 1 65-1 30 (m, IH, SH)

[M-H] ⁺ =410,

N-r2-mercaptomethyl-3-(3-methyl-5-ιsoxazolyl)propιonvn- (l.l'-bιphenyl-4-yl)-L-alanιne

Stereoisomer A (Compound 13)

^-NMR (200 MHz, DMSO-d^ δ (ppm) 12 82 (bs, IH, COOH), 8 51 (d, IH, 1.^=8 0 Hz, NH), 7 65-7 26 (m, 9H, biphenyl), 5 78 [s, IH, CH(ιsoxazole)], 4 53-4 42 (m, IH, CH¬ COO), 3 14-2 43 (m, 7H, CH^CH-O _, and CH ₂ -bιphenyl), 2 31 (bt, IH, SH), 1 99 (s, 3H,

CH.-ιsoxazolyl),

N-r2-mercaptomethyl-3-(3-methyl-5-ιsoxazolvI)propιonyl1 -(l. -bιphenyl-4-yl)-L-alamne

Stereoisomer B (Compound 14) m p 215-217°C

^] H-NMR (200 MHz, DMSO-d..) δ (ppm) 12 81 (bs, IH, COOH), 8 49 (d, IH, J _HH =8 3

Hz, NH), 7 63-7 30 (m, 9H, biphenyl), 6 04 [s, IH, CH(ιsoxazole)], 4 59-4 48 (m, IH, CH¬ COO), 3 18-2 33 (m, 7H, 2 12 (s, 3H, 1 86 (t, IH, J _τ _ _rτJ =8 5 Hz, SH) Example 3

In vitro evaluation of the pharmacologic activitv a) NEP-inhibitory activity

The NEP-inhibitory activity was evaluated in membranes from rat kidney cortex prepared according to the procedure descnbed by T Maeda et al in Biochim Biophys Acta 1983, 731(1 ), 1 15-120

By working at 0-4°C, kidneys were removed from male Sprague-Dawley rats weighing approximately 300 g

Cortex was carefully dissected, finely minced and suspended in a homogenization buffer ( 10 mM sodium phosphate pH 7 4 containing 1 mM MgCL, 30 mM NaCl, 0 02% NaN ) 1 15 weight/ vol ume

The tissue was then homogenized for 30 seconds using an Ultra-Turrax homogemzer Approximately 10 ml of homogenate were layered over 10 ml of sucrose (41% weight volume) and centnfuged 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 according to the method descnbed by C Llorens et al , in Eur J Pharmacol , 69, (1981 ), 1 13-1 16, as reported hereinafter Aliquots of the membrane suspension prepared as above descnbed (concentration 5 μg ml of proteins) were premcubated in the presence of an aminopeptidase inhibitor (Bestatin - 1 mM) for 10 minutes at 30°C

[ H][Leu ⁵ ]-enkephahne (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)

3 TThhee ffoorrmmaattiioonn ooff tthhee mmeettaabboolliittee [ H]Tyr-Gly-Gly was quantified by chromatography on polystyrene columns (Porapak Q)

The inhibition of the metabolite formation in the membrane preparations treated with the compounds of formula I and with the comparative compounds with respect to the untreated membrane preparations was expressed as IC .. (nM) value or as percentage of inhibition at a

-7 concentration corresponding to 10 M b) ACE-inhibitory activity

The ACE-inhibitory activity was evaluated according to the method reported in the literature by B Holmquist et al , m Analytical Biochemistry 95, 540-548 (1979)

50 μM of ACE (250 mU/ml puπfied by lung rabbit, EC 3 4 15 1 SIGMA) were premcubated with 50 μl of the compound of formula I or of the reference compound in thermostated cuvettes at 37°C

The reaction was started by adding furylacryloylphenylalanylglycylglyαne 0 8 mM (FAPGG-

SIGMA)

Contemporaneously, by using a Beckman DU-50 spectrophotometer provided with a program for calculating delta A minutes and regression coeffiαents of the enzyme kinetics curves, the absorbance 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 or with the comparative compounds with respect to the untreated preparations was expressed as IC,. (nM) value As an example, we report in the following table 2 the IC,.. (nM) values or the percentages of inhibition (10 M) related to the ACE-inhibitory activity and NEP-inhibitory activity of the compounds 1, 2, 6, 7, 9-14 and of Thiorphan, Captopnl and RB 105 as comparative compounds

Table 2

ACE-inhibitory and NEP-inhibitory activity of compounds 1-2, 6-7, 9-14, Thiorphan, Captopπl and RB 105

Compound ACE-inhibitory activity NEP-inhibitory activity IC _<0 (nM) IC (nM) % inhibition ( lθ ^"7 M)

1 5 5

2 75 16

6 26 18

7 253 61

9 38 12

10 13 29

11 2 13

12 6 37

13 13 94%

14 11 76%

RB 105 5 24

Thiorphan 99 11

Captopnl 3 not active

The data reported in table 2 show that the compounds of formula I, object of the present invention, are endowed with a significant mixed ACE/NEP inhibitory activity Said activity is comparable to the ACE-inhibitory activity of Captopnl as well as to the NEP- lnhibitory activity of Thiorphan

Furthermore, the mixed ACE/NEP-inhibitory activity of the compounds of formula I is comparable or better than that of the mixed ACE/NEP-inhibitor RB 105