HK1018008B - Thiol derivatives with metallopeptidase inhibitory activity - Google Patents

Description

Thiol derivatives having metallopeptidase inhibitory activity

The present invention relates to thiol derivatives having metallopeptidase inhibitory activity, and more particularly, it relates to N-mercaptoacylphenylalanine derivatives useful in the treatment of cardiovascular diseases.

Pharmacological interest in the study of metallopeptidase inhibitory molecules derives from the action exerted by the enzyme on the level of the heart circulatory system.

It is a well-known fact that compounds having Angiotensin Converting Enzyme (ACE) inhibitory activity are mainly used in the treatment of hypertension, heart failure and post-infarction, since they inhibit the formation of angiotensin ii, a substance that produces several blood pressure raising effects.

Compounds having endothelin-converting enzyme (ECE) inhibitory activity are useful as anti-vasoconstrictors because they inhibit the formation of endothelin, a 21 amino acid peptide having vasoconstrictive activity.

In contrast, compounds having Neutral Endopeptidase (NEP) (also known as enkephalinase) inhibitory activity are useful as vasodilators, since NEP enzymes cause the inactivation of not only endogenous enkephalinase, but also certain natriuretic factors such as atrial factor (ANF), a hormone secreted by the heart that increases vasodilation and, at the renal level, increases diuresis and natriuresis.

Thus, even if their action is exerted on the cardiovascular system by different mechanisms of action, compounds having metallopeptidase inhibitory activity are generally used alone or in combination for the treatment of hypertension, renal failure, congestive heart failure and ischemic heart disease.

Thioprhan among thiol derivative inhibitors of metallopeptidases

[ (DL- (3-mercapto-2-benzylpropionyl) glycine) ] (described first by Roques et al in Nature, Vol.288, pp.286-288, (1980)) and captopril (Merck index, tenth edition, 1773, 267) were identified as parent compounds for NEP-inhibitors and ACE-inhibitors, respectively.

Other molecules with metallopeptidase inhibitory activity having a thiol structure are described in the literature.

U.S. patents 4,401,677 and 4,199,512 (both in the name of e.r. squibb & Sons, inc.) describe mercaptoalkanoylamino acids having enkephalinase and ACE-inhibiting activity, respectively.

European patent application 0419327(Societe Civille Bioproject) describes amino acid derivatives having enkephalinase and ACE inhibitory activity, respectively.

European patent application 0449523(e.r. squibb & Sons, Inc.) describes mercapto or acyl thio-trifluoromethyl amides with NEP-inhibitory activity.

International patent application WO93/08162(Rhone-Poulenc Rorer S.A. -institute National de la Sante et de la Recherche medical (INSERM)) describes β, β -disubstituted α -mercaptomethylpropionamides with mixed ACE/NEP inhibitory activity.

European patent application 0524553 (Institut National de la Sante et al de la recherche mcale (INSERM)) describes acyl mercaptoalkanoyl dipeptides having neutral endopeptidase and peptidyl-dipeptidase A inhibitory activity.

European patent application 0566157(Schering Corporation) describes thiol derivatives such as N-mercaptoacylamino acids having NEP-inhibitory activity.

Alpha-mercaptoacyl dipeptides having ACE-inhibitory and NEP-inhibitory activity are also described by S.S. Bhagwat et al in Bioorganic & Medicinal Chemistry Letters,7,735-738, 1995.

In this last work the authors concluded that, although the presence of a diphenylmethyl group in a molecule with an alpha-mercaptoacyl dipeptide structure brings about an interesting mixed ACE/NEP-inhibitory activity, the substitution of the diphenyl group by groups like alpha-or beta-naphthyl gives a significant loss of activity.

We have now found N-mercaptoacylphenylalanine derivatives having a significant inhibitory activity against angiotensin converting enzymes as well as against neutral endopeptidases (mixed or dual ACE/NEP inhibitory activity), which are particularly useful in the treatment of cardiovascular diseases.

Accordingly, objects of the present invention are compounds of formula (I) and pharmaceutically acceptable salts thereofWherein

R is mercapto or R convertible in the organism to mercapto groups₄A COS group;

R₁is straight-chain or branched C₂-C₄An alkyl group or an aryl group or an aralkyl group having from l to 6 carbon atoms in the alkyl moiety, wherein the aryl group is a phenyl group or a 5-or 6-membered heteroaromatic ring having 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur, which may be substituted or unsubstituted, identically or differently, with 1 or more substituents selected from halogen atoms, hydroxyl groups, alkoxy groups, alkyl groups, alkylthio groups, alkylsulfonyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moietyGroup, C containing 1 or more fluorine atoms₁-C₃An alkyl group, an antelope group, a nitro group, an amino or aminocarbonyl group, an amido group, an aminosulfonyl group, a mono-or di-alkylamino group having from 1 to 6 carbon atoms in the alkyl moiety or a mono-or di-alkylaminocarbonyl group;

R₂is a hydrogen atom, a straight chain or a branched chain C₁-C₄An alkyl or benzyl group;

R₃is phenyl substituted by a 5-or 6-membered aromatic heterocycle having 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur, as the phenyl and heterocyclic groups, may be substituted or unsubstituted with one or more substituents, which may be the same or different, e.g. for R₁As indicated;

R₄is straight or branched C₁-C₄An alkyl group or a phenyl group;

the carbon atom marked with an asterisk is the stereostructural center.

The compounds of formula I contain two stereogenic centers and may therefore exist as stereoisomers.

The object of the present invention is therefore compounds of the formula I in the form of stereoisomeric mixtures and in the form of individual stereoisomers.

Objects of the present invention compounds of formula I have mixed ACE/NEP-inhibitory activity and are useful in the treatment of cardiovascular disease.

Although both terms of "mixed" and "dual" are used indifferently, the term "dual" is more commonly accepted in the literature for compounds having both ACE and NEP-inhibitory activity.

The terms "mixed" and "dual" are considered equivalent herein.

In this specification, unless otherwise indicated, by the term straight or branched alkyl group we mean alkyl groups 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 groups; by the term linear or branched alkoxy we mean alkoxy groups such as methoxy, ethoxy, n-propoxy and isopropoxy; by the term halogen atom we mean a fluorine, halogen, bromine or iodine atom; by the term acyl we mean acyl groups derived from aliphatic or aromatic carboxylic acids such as acetic, propionic, butyric and benzoic acids; by the term 5-or 6-membered heteroaromatic ring containing 1 or 2 heteroatoms selected from nitrogen, oxygen and sulphur we mean, for example, thiazole, isoxazole, oxazole, isothiazole, pyrazole, imidazole, thiophene, pyrrole, pyridine, pyrimidine, pyrazine pyridazine and furan, optionally benzo-fused.

Examples of pharmaceutically acceptable salts of the compounds of formula I are salts with alkali metals or alkaline earth metals and salts with pharmaceutically acceptable organic bases.

Preferred compounds of the formula I are those in which R₃Is a phenyl group substituted at the 4-position with a heterocyclic group.

More preferred compounds within this class are those wherein R is₁A compound of formula I representing an aralkyl group substituted or not with one or more substituents, which may be the same or different, selected from halogen atoms, hydroxy groups, alkyl groups or alkoxy groups.

Still further preferred compounds within this class are those wherein R is₁Compounds of formula (I) represent phenylalkyl substituted or unsubstituted by one or more substituents, identical or different, chosen from halogen atoms, hydroxy, alkyl or alkoxy groups.

Examples of preferred pharmaceutically acceptable salts of the compounds of formula I are salts with alkali metals such as sodium, lithium and potassium.

It is clear to the person skilled in the art that: wherein R is R which is convertible into a mercapto group in an organism₄Compounds of formula I of COS and wherein R₂Compounds of the formula I which are alkyl or benzyl radicals are those in which each R is mercapto (R = SH) or R₂Is a hydrogen atom (R)₂= H) corresponding biological precursors (prodrugs) of the compounds of the formula i.

Specific examples of preferred compounds of the formula I for the purposes of the present invention are:

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-thiazolyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-pyridyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-pyridyl) -phenylalanine benzyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-furyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (5-pyrimidinyl) -phenylalanine ethyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-pyrazinyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-thienyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-thienyl) -phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-furyl) -phenylalanine methyl ester;

n- [ 3-phenylcarbonylthio-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- [ 3-acetylthio-2- (3-methoxyphenyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- [ 3-acetylthio-2- (2-fluorophenyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- [ 3-phenylcarbonylthio-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- [ 2- (2-furyl) methyl-3-phenylcarbonylthio-propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- [ 2- (3-methyl-5-isoxazolyl) methyl-3-phenylcarbonylthio-propionyl ] -4- (2-thiazolyl) -phenylalanine methyl ester;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-thiazolyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-pyridyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-pyridyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-furyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (5-pyrimidinyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-pyrazinyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-thienyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-thienyl) -phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-furyl) -phenylalanine;

n- [ 3-mercapto-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -phenylalanine;

n- [ 3-mercapto-2- (3-methoxyphenyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine;

n- [ 3-mercapto-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine;

n- [ 3-mercapto-2- (3-methyl-5-isoxazolyl) methyl-propionyl ] -4- (2-thiazolyl) -phenylalanine;

n- [ 2- (2-fluorophenyl) methyl-3-mercapto-propionyl ] -4- (2-thiazolyl) -phenylalanine;

n- [ 2- (2-furyl) methyl-3-mercapto-propionyl ] -4- (2-thiazolyl) -phenylalanine.

The object of the invention is the preparation of the compounds of formula I according to the following synthetic process, including the compounds of formula IIWherein

R and R₁Have the meaning described above;

and phenylalanine derivatives of the formula IIIWherein

R₂And R₃Have the meaning indicated above.

The condensation reaction is carried out according to conventional techniques of peptide chemistry.

Before carrying out the reaction, it is advantageous to suitably protect the selective functional groups which may interfere with the reaction.

The selective protection is performed according to conventional techniques.

In this regard, it is preferred to use a compound wherein R is R₄The compound of the COS group is used as an intermediate of a formula II, so that R = R is obtained₄The corresponding compound of formula i of COS, from which compounds of formula i wherein R = SH can be obtained by hydrolysis.

Evaluation of the effectiveness of selective protection and selection of the type of protection to be taken, in terms of the reaction to be carried out and the functional group to be protected, is within the knowledge of a person skilled in the art.

The removal of the selective protecting groups is carried out according to conventional techniques.

As a general reference for the use of protecting groups in organic chemistry, see "protecting groups in organic synthesis" by theodora w.greene and Peter g.m.wuts, john wiley & sons, inc.

The compounds of formula ii are known or readily prepared according to such conventional methods, for example as described in british patent No. 1576161 in the name of e.r.

Alternatively, the compounds of formula II may be prepared according to the synthetic procedures described by M.C. Fournie-Zalusky et al, J.Med.chem.1994,37, 1070-1083.

And the intermediate compounds of formula iii are known or readily prepared according to known methods.

For example, the compounds of formula III can be prepared according to the synthetic procedures described by W.C.Shieh et al, J.org.chem.1992,57, 379-381.

Alternatively, the compounds of the formula III can be prepared by coupling methods (cross-coupling) starting from halogenated heterocyclic compounds and stannane phenylalanine derivatives as described by D.S. Wilbur et al, Bioconjugate chem.1993,4, 574-580.

The compounds of formula i in their individual stereoisomeric forms are prepared by stereoselective synthesis or by separation of a mixture of stereoisomers according to conventional techniques.

And the preparation of the salts of the compounds of formula I, object of the present invention, is carried out according to conventional techniques.

The compounds of formula I, object of the present invention, have mixed ACE/NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.

The inhibitory activity of the compounds of formula I was evaluated by in vitro tests (example 8).

In particular, the inhibitory activity of the compounds of formula I is assessed by comparison with Thioprhan and captopril as described previously.

The in vitro inhibitory activity of the compounds of formula I is expressed as IC₅₀Values, results are in nM concentration.

The activity produced was comparable to the ACE-inhibitory activity involved in captopril and greater than the NEP-inhibitory activity involved in Thiorphan.

Furthermore, the inhibitory activity of the compounds of the formula I is also assessed by ex vivo tests in comparison with known compounds (example 9).

In particular, N- [ 3-mercapto-2- (3, 4-methylenedioxyphenyl) methyl-propionyl ] -S-phenylalanine and N- [ 3-mercapto-2- (3, 4-methylenedioxyphenyl) methyl-propionyl ] -S-tyrosine, described as ACE-inhibitors in the aforementioned European patent application 0419327, hereinafter referred to as compounds R-1 and R-2, respectively, and N- (3-mercapto-2-methylpropyl) -L-tyrosine and N- (3-mercapto-2-methylpropyl) -L-tryptophan, are described as ACE-inhibitors in U.S. Pat. No. 4,199,512 and as neprilysin inhibitors in U.S. Pat. No. 4,401,677, hereinafter referred to as compounds R-3 and R-4, respectively, as comparative compounds.

In particular, the in vivo ACE/NEP-inhibitory activity was determined by evaluating the enzymatic activity of tissue homogenates (lung and kidney, respectively, for ACE and NEP activity) of spontaneously hypertensive mice (SHR) treated with the test compounds by intravenous or oral route.

It is worth noting that: the combined activity shown in the in vitro tests (dual activity) is demonstrated by the activity shown in ex vivo tests of the compounds of formula i, also after oral administration.

But also results in which the activity is significantly higher than that of the comparative compound.

For therapeutic utility, the compounds of formula I may be formulated in solid or liquid pharmaceutical compositions suitable for oral or parenteral administration.

Accordingly, pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula I in admixture with a pharmaceutically acceptable carrier are a further object of the present invention.

Specific examples of pharmaceutical compositions according to the invention are tablets, coated tablets, capsules, granules, solutions or suspensions suitable for oral administration, solutions and suspensions suitable for parenteral administration.

The pharmaceutical compositions object of the present invention are prepared according to conventional techniques.

The daily dose of a compound of formula I or a corresponding prodrug will depend on several factors such as the severity of the disease, the individual response of the patient or the type of preparation, but will generally be between 0.1mg and 10mg per kg body weight, divided into single or multiple daily doses.

In order to illustrate the invention, the following examples are now given.

Unless otherwise indicated, flash chromatography was carried out using flash chromatography silica gel from Baker corporation (code 7024-00).

Example 1

Preparation of N-tert-butoxycarbonyl-4- (5-pyrimidinyl) -L-phenylalanine ethyl ester.

A mixture of 5-pyrimidinylboronic acid (850 mg; 2 mmol), N-tert-butoxycarbonyl-4-trifluoromethylsulfonyl-L-phenylalanine ethyl ester (450 mg; 2.2 mmol), sodium carbonate (530mg) in water (2.59ml) and a mixture of toluene: ethanol = 10: 4.5(20ml) was degassed with nitrogen for 30 minutes.

Subsequently, tetrakis (triphenylphosphine) palladium (0) (120 mg; 0.06 mmol) was added thereto, and the reaction mixture was heated at 90 ℃ and kept under stirring for 3 hours.

The mixture was then kept at room temperature and N-tert-butoxycarbonyl-4-trifluoromethylsulfonyl-L-phenylalanine ethyl ester (112mg) and tetrakis (triphenylphosphine) palladium (0) (30mg) were added thereto.

The mixture was further heated at 90 ℃ and held under stirring for a further 18 hours.

After the reaction mixture was cooled at room temperature, ethyl acetate (100ml) and water (40ml) were added.

The phases were separated and the organic phase was dried over sodium sulphate and evaporated under vacuum. The resulting residue was purified by flash chromatography (silica gel, eluent hexane: ethyl acetate = 7: 3, nitrogen pressure 0.1 atm) to give N-tert-butoxycarbonyl-4- (5-pyrimidinyl) -L-phenylalanine ethyl ester (130 mg; 14% yield) as a colorless oil.

¹H-NMR(200MHz,CDCl₃)；δ(ppm):1.24(t,3H,CH₂-CH₃)；1.40[s,9H,C(CH₃)₃]；3.01-3.25(m,2H,CH₂-CH)；4.19(q,2H,CH₂-CH₃) (ii) a 452-4.65(m,1H, CH-COO); 5.06(d,1H, NH); 7.25-7.52(m,4H, phenylene); 8.91(s,2H, N-CH-C-CH-N); 9.19(s,1H, N-CH-N).

Example 2

Preparation of N-tert-butyloxycarbonyl-4- (2-thiazolyl) -L-phenylalanine methyl ester

N-tert-Butoxycarbonyl-4- (trifluoromethylsulfonyl) -L-phenylalanine methyl ester (8 g; 32.3 mmol) and bis (triphenylphosphine) palladium chloride (2.3g) were added to a solution of 2-trimethylstannyl-thiazole (13.8 g; 32.3 mmol) in a mixture of tetrahydrofuran: toluene = 10: 1 (50ml) previously degassed with nitrogen.

The mixture was refluxed for 24 hours, then 2-trimethylstannyl-thiazole (2g) was added.

After refluxing for 6 hours, N-tert-butoxycarbonyl-4- (trifluoromethylsulfonyl) -L-phenylalanine methyl ester (2g) and bis (triphenylphosphine) palladium chloride (700mg) were added.

The resulting reaction mixture was kept at 70 ℃ for 16 hours with stirring and subsequently cooled at room temperature.

Water (200ml) was then added to the mixture, which was extracted with dichloromethane (4X 200 ml).

The collected organic phases were dried over sodium sulfate and evaporated under vacuum.

The resulting residue was purified by flash chromatography (silica gel, eluent dichloromethane: ethyl acetate = 9: 1, nitrogen pressure =0.1 atm), which gave N-tert-butoxycarbonyl-4- (2-thiazolyl) -L-phenylalanine methyl ester (2.3 g; 20% yield).

¹H-NMR(200MHz,CDCl₃)；δ(ppm):1.40[s,9H,C(CH₃)₃]；3.00-3.21(m,2H,CH₂)；3.70(s,3H,COOCH₃) (ii) a 4.42-4.65(m,1H, CH-COO); 5.02(bd,1H, NH); 7.30(d,1H, S-CH-N); 7.81(d,1H, S-CH-N); 7.15-7.90(m,4H, phenylene).

Example 3

Preparation of N-tert-butoxycarbonyl-4- (3-pyridyl) -L-phenylalanine methyl ester

3-bromopyridine (1.67 g; 10 mmol) and tetrakis (triphenylphosphine) palladium (0) (370 mg; 0.219 mmol) were added to a solution of N-tert-butoxycarbonyl-4- (tributylstannyl) -L-phenylalanine methyl ester (4 g; 7.03 mmol) prepared as described by D.S. Wilbur et al, bioconjugatcchem.,1973,4,574-580 in anhydrous dimethylformamide (30ml) which had previously been degassed with nitrogen.

The reaction mixture was kept at room temperature for 10 minutes with stirring and subsequently heated at 105 ℃ for 6 hours.

Tetrakis (triphenylphosphine) palladium (0) (0.0035 mmol) was added and the reaction mixture was maintained at 105 ℃ for 8 hours with stirring and then cooled at room temperature.

Water (100ml) was added to the reaction solution, and the reaction mixture was extracted with hexane (3X 150 ml).

The collected organic phases were washed with saturated aqueous potassium fluoride solution, dried over sodium sulfate and evaporated in vacuo.

The resulting residue was collected with ethyl acetate and filtered.

The resulting solution was evaporated in vacuo and the residue was purified by flash chromatography (silica gel, eluent hexane: ethyl acetate = 8: 2, nitrogen pressure 0.1 atm) to give N-tert-butoxycarbonyl-4- (3-pyridyl) -L-phenylalanine methyl ester (1.5 g; 60% yield) as a colorless oil.

Mass Spectrometry (C.I.) (M + H)⁺=357

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.40[s,9H,C(CH₃)₃]；3.00-3.20(m,2H,CH₂)；3.71(s,3H,COOCH₃) (ii) a 4.55(m,1H, CH-COO); 5.05(bd,1H, NH); 7.19-7.51(m,4H, phenylene); 7.30-8.82(bm,4H, pyridyl)

The following compounds were prepared by similar procedures:

N-Boc-4- (2-pyridyl) -L-phenylalanine methyl ester

Mass Spectrometry (C.I.) (M + H)⁺=357

¹H-NMR(200MHz,CDCl₃):δ(ppm)1.40[s,9H,C(CH₃)₃]；3.03-3.21(m,2H,CH₂-CH)；3.70(s,3H,COOCH₃) (ii) a 4.54-4.65(m,1H, CH-COO); 4.98(d,1H, CONH); 7.16-7.21(m,1H, N-C-CH-CH); 7.65-7.77(m,2H, N-CH-CH-CH); 7.19-7.93(m,4H, phenylene); 8.63-8.68(m,1H, N-CH).

Boc-4- (2-pyrazinyl) -L-phenylalanine methyl ester

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.40[s,9H,C(CH₃)₃]；3.02(m,2H,CH₂)；3.70(s,3H,COOCH₃) (ii) a 4.53-4.70(m,1H, CH-COO); 5.03(bd,1H, NH); 7.21-7.98(m,4H, phenylene); 8.49 and 8.62[2(bs,2H, N-CH-CH-N)]；9.00(s,1H,CH-N-CH-CH)；

N-Boc-4- (2-thienyl) -L-phenylalanine methyl ester

¹H-NMR(200MHz,CDCl₃):δ(ppm)1.41[s,9H,C(CH₃)₃]；2.98-3.18(m,2H,CH₂)；3.71(s,3H,COOCH₃) (ii) a 4.53-4.64(m,1H, CH-COO); 4.98(bd,1H, NH); 7.02-7.28(m,3H, thienyl); 7.10-7.54(m,4H, phenylene).

Example 4

Preparation of 4- (3-pyridyl) -L-phenylalanine methyl ester dihydrochloride

Thionyl chloride (0.85 ml; 4.78 mmol) was added dropwise to a solution of N-tert-butoxycarbonyl-4- (3-pyridyl) -L-phenylalanine methyl ester (1.4 g; 3.93 mmol) prepared as described in example 3 in methanol (30 ml). The temperature was maintained at 0 ℃.

At the end of the dropwise addition, the reaction mixture was brought to room temperature and kept under stirring for 8 hours.

The solvent was then evaporated under vacuum to give 4- (3-pyridyl) -L-phenylalanine methyl ester dihydrochloride (820 mg; 71% yield), which was used as such in the following reaction.

Mass Spectrometry (C.I.) (M + H)⁺=257 (free base)

¹H-NMR(200MHz,D₂O):δ(ppm):3.11-3.32(m,2H,CH₂)；3.67(s,3H,CH₃) (ii) a 4.31-4.37(m,1H, CH); 7.30-7.63(m,4H, phenylene); 7.97(dd,1H, CH-N-CH-CH-CH); 8.59(d,1H, CH-N-CH-CH-CH); 8.65-8.71(m,1H, CH-N-CH-CH); 8.89(d,1H, CH-N-CH-CH-CH).

The following compounds were prepared by similar procedures:

4- (2-pyridinyl) -L-phenylalanine methyl ester dihydrochloride

Mass Spectrometry (C.I.) (M + H)⁺=257 (free base)

¹H-NMR(200MHz,D₂O):δ(ppm):3.12-3.33(m,2H,CH₂)；3.64(s,3H,CH₃) (ii) a 4.30-4.37(m,1H, CH); 7.36-7.73(m,4H, phenylene); 7.78-8.58(m,4H, pyridyl)

4- (5-pyrimidinyl) -L-phenylalanine ethyl ester dihydrochloride

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.11(t,3H,CH₂-CH₃)；3.10-3.35(m,2H,CH₂-CH)；4.04-4.20(m,2H,CH₂-CH₃) (ii) a 4.22-4.35(m,1H, CH); 7.40-7.84(m,4H, phenylene); 9.15(s,2H, N-CH-C-CH-N); 9.19(s,1H, N-CH-N);

4- (2-pyrazinyl) -L-phenylalanine methyl ester dihydrochloride

Mass Spectrometry (C.I.) (M + H)⁺=258 (free base)

¹H-NMR(200MHz,DCl 1N):δ(ppm):3.45-3.67(m,2H,CH₂)；3.98(s,3H,CH₃) (ii) a 4.65-4.72(m,1H, CH); 7.68-8.26(m,4H, phenylene); 9.02(d,1H, CH-N-CH-CH); 9.44(dd,1H, CH-N-CH-CH); 9.54(d,1H, CH-N-CH-CH);

4- (2-thienyl) -L-phenylalanine methyl ester hydrochloride

Mass Spectrometry (C.I.) (M + H)⁺=262 (free base)

¹H-NMR(200MHz,D₂O):δ(ppm):2.98-3.19(m,2H,CH₂)；3.65(s,3H,CH₃) (ii) a 4.21-4.28(m,1H, CH); 6.96-7.28(m,3H, thienyl); 7.08-7.52(m,4H, phenylene);

4- (2-thiazolyl) -L-phenylalanine methyl ester dihydrochloride

¹H-NMR(200MHz D₂O):δ(ppm):3.10-3.32(m,2H,CH₂-CH)；3.68(s,3H,CH₃) (ii) a 4.30-4.38(m,1H, CH); 7.30-7.80(m,4H, phenylene); 7.70-7.91(m,2H, thiazolyl).

Example 5

Preparation of 2-isobutyl-3-phenylcarbonylthio-propionic acid

A mixture of 2-isobutyl-acrylic acid (6.34 g; 49 mmol) and thiobenzoic acid (5.96 ml; 51 mmol) was heated at 100 ℃ for 2 hours with stirring.

The reaction mixture was then treated with petroleum ether 40-60 deg.C (100ml) and cooled in a dry ice/acetone bath at-70 deg.C.

After filtration and washing with petroleum ether at-70 ℃ a residue was collected which was dried under reduced pressure to give 2-isobutyl-3-phenylcarbonylthio-propionic acid (11.12 g; 85% yield) as a white solid.

¹H-NMR(200MHz,CDCl₃):δ(ppm):0.90-1.00(m,6H)；1.40-1.90(m,3H)；2.70-2.90(m,1H)；3.10-3.40(m,2H)；7.35-7.62(m,3H)；7.90-8.00(d,2H).

The following compounds were prepared by similar procedures:

2- (3-methoxyphenyl) methyl-3-phenylcarbonylthio-propionic acid

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.32(s,3H)；2.80-3.15(m,5H)；3.77(s,3H)；6.70-6.80(m,3H)；7.14-7.25(m,1H).

3-acetylthio-2- (2-fluorophenyl) methyl-propionic acid

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.31(s,3H)；2.90-3.20(m,5H)；6.95-7.30(m,4H).

3-Phenylcarbonylthio-2- (2-thienyl) methyl-propionic acid benzylamine salt

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.45-3.25(m,5H,s-CH₂-CH-CH₂)；3.90(s,2H,CH₂-phenyl); 6.85-7.91(m,13H, aryl).

Example 6

Preparation of N- [ (2S) -3-Phenylcarbonylthio-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (Compound 1)

A solution of hydroxybenzotriazole (0.54 g; 4 mmol) in tetrahydrofuran (30ml) and subsequently a solution of dicyclohexylcarbodiimide (0.825 g; 4 mmol) in dichloromethane (15ml) were added with stirring at 0 ℃ to a mixture of (2S) -3-phenylcarbonylthio-2-phenylmethylpropionic acid (1.2 g; 4 mmol), methyl 4- (2-thiazolyl) -L-phenylalanine dihydrochloride (1.34 g; 4 mmol), prepared as described in example 4, triethylamine (1.11 ml; 8 mmol) in tetrahydrofuran (20ml) and dichloromethane (30 ml).

The reaction mixture was kept under stirring for 20 hours, then the dicyclohexylurea was filtered off and the solvent was evaporated under reduced pressure.

The residue was collected with ethyl acetate and the solution was washed with 20% sodium chloride, 5% sodium bicarbonate and again 20% aqueous sodium chloride solution.

After separation of the phases and evaporation of the organic phase, the resulting white solid was purified by flash chromatography (silica gel, eluent ethyl acetate: hexane = 40: 60, nitrogen pressure 0.1 atm), thus giving N- [ (2S) -3-phenylcarbonylthio-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (1.5 g).

m.p.98-1002

Mass Spectrometry (C.I.) (M + H)⁺=545

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.63-3.35(m,7H,CH₂-CH-CH₂,CH₂-C₆H₄-thiazolyl); 3.68(s,3H, COOCH)₃) (ii) a 4.75-4.85(m,1H, CH-COO); 5.78(d,1H, NH); 7.10-8.00(m,16H, aryl).

By similar operating procedures, starting from compounds known or prepared as described in examples 4 and 5, the following compounds are obtained:

n- [ (2S) -3-Phenylcarbonylthio-2-phenylmethylpropionyl ] -4- (2-furyl) -L-phenylalanine methyl ester (Compound 2).

m.p.132-134℃

Mass spectrum (C.I.) (M + H)⁺=528

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.60-3.35(m,7H,CH₂-CH-CH₂,CH₂-C₆H₄-furyl); 3.58(s,3H, COOCH)₃) (ii) a 4.71-4.81(m,1H, CH-COO); 5.73(d,1H, NH); 6.40-8.00(m,17H, aryl).

N- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (5-pyrimidinyl) -L-phenylalanine ethyl ester (Compound 3)

m.p.117-119℃

Mass spectrum (C.I.) (M + H)⁺=554

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.18(t,3H,CH₃-CH₂)；2.65-3.35(m,7H,CH₂-CH-CH₂,CH₂-C₆H₄-pyrimidinyl); 3.95-4.20(m,2H, COOCH)₂) (ii) a 4.70-4.80(m,1H, CH-COO); 5.78(d,1H, NH); 7.05-8.00(m,14H, phenyl, phenylene); 8.68(s,2H, CH-N-CH-N-CH); 9.11(s,1H, N-CH-N).

N- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (2-pyrazinyl) -L-phenylalanine methyl ester (Compound 4)

m.p.145-147℃

Mass spectrum (C.I.) (M + H)⁺=540

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.65-3.35(m,7H,CH₂-CH-CH₂,CH₂-C₆H₄-a pyrazinyl group); 3.61(s,3H, COOCH)₃) (ii) a 4.75-4.85(m,1H, CH-COO); 5.78(d,1H, NH); 7.10-8.00(m,14H, phenyl, phenylene); 8.48-8.75(m,3H, CH-N-CH-CH-N);

n- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (3-pyridyl) -L-phenylalanine methyl ester (Compound 5)

m.p.132-134℃

Mass spectrum (C.I.) (M + H)⁺=539

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.66-2.79(m,1H,CH₂-CH-CH₂)；2.88-3.35(m,6H,CH₂-CH-CH₂,CH₂-C₆H₄-a pyridyl group); 3.61(s,3H, COOCH)₃) (ii) a 4.75-4.85(m,1H, CHCOO); 5.77(d,1H, NH); 7.07-7.99(m,16H, CH-CH-CH-N, phenyl, phenylene); 8.51-8.64(m,2H, CH-N-CH);

n- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (2-pyridyl) -L-phenylalanine methyl ester (Compound 6)

m.p.123-125℃

Mass spectrum (C.I.) (M + H)⁺=539

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.63-2.77(m,1H,CH₂-CH-CH₂)；2.85-3.35(m,6H,CH₂-CH-CH₂,CH₂-C₆H₄-a pyridyl group); 3.56(s,3H, COOCH)₃) (ii) a 4.75-4.85(m,1H, CHCOO); 5.75(d,1H, NH); 7.09-7.99(m,16H, CH-CH-CH-N, phenyl, phenylene); 8.61-8.65(m,1H, N-CH-CH);

n- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (2-thienyl) -L-phenylalanine methyl ester (Compound 7)

Mass spectrum (C.I.) (M + H)⁺=544

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.64-3.36(m,7H,CH₂-CH-CH₂,CONH-CH-CH₂)；3.58(s,3H,COOCH₃) (ii) a 4.74-4.83(m,1H, CH-COO); 5.74(d,1H, NH); 6.97-7.99(m,17H, aryl);

n- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (3-thienyl) -L-phenylalanine methyl ester (Compound 8)

Mass spectrum (C.I.) (M + H)⁺=544

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.63-3.35(m,7H,CH₂-CH-CH₂,NH-H-CH₂)；3.58(s,3H,COOCH₃) (ii) a 4.73-4.85(m,1H, CH-COO); 5.70-5.76(bd,1H, NH); 7.00-7.62(m,15H, phenyl, phenylene, CH-CH-S); 7.93-8.00(m,2H, CH-S-CH);

n- [ (2S) -3-Phenylcarbonylthio-2-Phenylmethylpropionyl ] -4- (3-furyl) -L-phenylalanine methyl ester (Compound 9)

m.p.115-117℃

Mass spectrum (C.I.) (M + H)⁺=528

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.62-3.36(m,7H,CH₂-CH-CH₂,NH-CH-CH₂)；358(s,3H,COOCH₃) (ii) a 4.71-4.82(m,1H, CH-COO); 5.72(bd,1H, NH); 6.50-8.00(m,17H, aryl);

n- [ 3-Phenylcarbonylthio-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester stereoisomer A (Compound 10)

Mass spectrum (C.I.) (M + H)⁺=546

TCL (Ethyl acetate: Petroleum Ether = 95: 5), R_f=0.33

¹H-NMR(200MHz,CDCl₃)；δ(ppm):2.60-3.38(m,7H,CH₂-CH-CH₂,NH-CH-CH₂)；3.60(s,3H,COOCH₃) (ii) a 4.79-4.90(m,1H, CH-COO); 6.21(bd,1H, NH); 7.29-7.81(m,2H, thiazolyl); 6.75-8.48(m,13H, phenyl, phenylene, pyridyl)

N- [ 3-Phenylcarbonylthio-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester stereoisomer B (Compound 11)

Mass spectrum (C.I.) (M + H)⁺=546

TLC (ethyl acetate: petroleum ether = 95: 5), R_f=0.24

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.61-3.25(m,7H,CH₂-CH-CH₂,NH-CH-CH₂)；3.60(s,3H,COOCH₃) (ii) a 4.80-4.91(m,1H, CH-COO); 6.09(bd,1H, NH); 7.27-7.80(m,2H, thiazolyl); 7.10-8.40(m,13H, phenyl, phenylene, pyridyl);

n- [ 2-isobutyl-3-phenylcarbonylthio-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (Compound 12)

¹H-NMR(200MHz,CDCl₃):δ(ppm):0.80-0.95(m,6H)；1.30-1.80(m,3H)；2.40-2.60(m,1H)；3.00-3.30(m,4H)；3.70(d,3H)；4.90-5.05(m,1H)；6.00-6.15(bt,1H)；7.10-8.00(m,11H)；

N- [ 3-Acetylthio-2- (3-methoxyphenyl) methyl-propionyl ] 4- (2-thiazolyl) -L-phenylalanine methyl ester (Compound 13)

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.30(d,3H)；2.45-3.20(m,7H)；3.63(d,3H)；3.75(d,3H)；4.70-4.93(m,1H)；5.70-5.90(dd,1H)；6.60-6.85(m,4H)；7.17-7.32(m,3H)；7.68-7.90(m,3H)；

N- [ 3-Acetylthio-2- (3-fluorophenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (Compound 14)

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.29(s,3H)；2.55-3.20(m,7H)；3.65(2s,3H)；4.70-4.90(m,1H)；5.80-6.00(2d,1H)；6.70-7.32(m,3H)；

N- [ 3-Phenylcarbonylthio-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (Compound 15)

¹H-NMR(200MHz,CDCl₃):δ(ppm):2.68-3.37(m,7H)；3.60-3.61(2s,3H,COOCH₃)；4.31-4.45(m,1H,CH-COOCH₃)；6.01-6.10(2d,1H,NH)；6.80-8.00(m,14H)；

Example 7

Preparation of N- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 16)

N- [ (2S) -3-Phenylcarbonylthio-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester (1.4 g; 2.57 mmol) prepared as described in example 6 was suspended in ethanol (30ml) purged of oxygen with nitrogen.

To this suspension was added dropwise a degassed aqueous solution of sodium hydroxide 1N (7.7ml) at 5 ℃ and, after the addition was complete, degassed ethanol (20ml) was added dropwise.

The reaction mixture was kept at room temperature for 4 hours with stirring, then cooled at 0 ℃ and acidified with 5% (10ml) hydrochloric acid.

The reaction mixture was evaporated to dryness and the residue was collected with acetonitrile and water, followed by filtration, whereby N- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine (1g) was given.

m.p.180-182℃

Mass spectrum (C.I.) (M + H)⁺=427

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.80-1.88(m,1H,SH)；2.22-2.84(m,5H,CH₂-CH-CH₂)；2.86-3.18(m,2H,CH₂-CH-COO); 4.46-4.57(m,1H, CH-COO); 7.10-7.25(m,5H, phenyl); 7.32-7.84(m,4H, phenylene); 7.74(d,1H, N-CH = CH-S); 7.89(d,1H, N-CH = CH-S); 8.35(d,1H, NH); 12.76(s,1H, COOH);

the following compounds were prepared by similar procedures:

n- [ (2R) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 17)

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.15-2.23(m,1H,SH)；2.31-2.74(m,5H,CH₂-CH-CH₂)；2.78-3.07(m,2H,CH₂-CH-COO); 4.42-4.53(m,1H, CH-COO); 7.02-7.80(m,9H, phenyl and phenylene); 7.75(d,1H, N-CH = CH-S); 7.90(d,1H, N-CH = CH-S); 8.36(d,1H, NH);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-furyl) -L-phenylalanine (Compound 18)

m.p.153-155℃

Mass spectrum (C.I.) (M + H)⁺=410

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.40(t,1H,SH)；2.45-3.25(m,7H,CH₂-CH-CH₂,CH₂-CH-COO); 4.80-4.90(m,1H, CH-COO); 5.86(d,1H, NH); 6.42-7.42(m,3H, furyl) 7.07-7.57(m,9H, phenyl, phenylene);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (5-pyrimidinyl) -L-phenylalanine (Compound 19)

m.p.193-195℃

Mass spectrum (C.I.) (M + H)⁺=422

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.81(bm,1H,SH)；2.21-3.20(m,7H,CH₂-CH-CH₂,CH₂-C₆H₄-pyrimidinyl); 4.46-4.57(m,1H, CH-COO); 7.06-7.29(m,5H, phenyl); 7.36-7.72(m,4H, phenylene); 8.33(d,1H, NHCO); 9.08(s,2H, N-CH-C-CH-N); 9.15(s,1H, N-CH-N);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-pyrazinyl) -L-phenylalanine (Compound 20)

m.p.176-178℃

Mass spectrum (C.I.) (M + H)⁺=422

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.84(bt,1H,SH)；2.21-3.21(m,7H,CH₂-CH-CH₂,CH₂-CH-COO); 4.48-4.59(m,1H, CH-COO); 7.10-7.26(m,5H, phenyl); 7.37-8.05(m,4H, phenylene); 8.37(d,1H, NHCO); 8.58(d,1H, CH-N-CH-CH-N); 8.68-8.70(m,1H, CH-N-CH-CH-N); 9.21(d,1H, C-CH-N); 12.78(b,1H, COOH);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (3-pyridyl) -L-phenylalanine (Compound 21)

m.p.146-148℃

Mass spectrum (C.I.) (M + H)⁺=421

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.69-1.89(b,1H,SH)；2.23-3.18(m,7H,CH₂-CH-CH₂,CH₂-phenylene radicals); 4.45-4.56(m,1H, CH-COO); 7.09-7.26(m,5H, phenyl); 7.42-7.48(m,1H, CH-N-CH-CH-CH); 7.62-7.33(m,4H, phenylene); 7.98-8.04(m,1H, CH-N-CH-CH-CH); 8.30(d,1H, CONH); 8.53(dd,1H, CH-N-CH-CH-CH); 8.83(d,1H, CH-N-CH-CH-CH);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-pyridyl) -L-phenylalanine (Compound 22)

m.p.157-159℃

Mass spectrum (C.I.) (M + H)⁺=421

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.87(b,1H,SH)；2.23-3.19(m,7H,S-CH₂-CH-CH₂,CONH-CH-CH₂) (ii) a 4.44-4.45(m,1H, CH-COO); 7.09-7.93(m,8H, N-CH-CH-CH-CH-phenyl); 7.30-7.99(m,4H, phenylene); 8.29(d,1H, CONH); 8.61-8.65(m,1H, C-N-CH);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (2-thienyl) -L-phenylalanine (Compound 23)

m.p.152-154℃

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.34-1.43(m,1H,SH)；2.44-3.26(m,7H,S-CH₂-CH-CH₂,CONH-CH-CH₂) (ii) a 4.80-4.89(m,1H, NH-CH-COO); 5.81(d,1H, NH); 7.02-7.52(m,12H, aryl);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (3-thienyl) -L-phenylalanine (Compound 24)

m.p.169-171℃

Mass spectrum (C.I.) (M + H)⁺=426

TLC (ethyl acetate: hexane: acetic acid = 50: 5), R_f=0.44

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.84(bs,1H,SH)；2.23-3.13(m,7H,S-CH₂-CH-CH₂,NH-CH-CH₂)；4.42-4.53(m,1H,NH-CH-COO)；7.12-7.80(m,12H, aryl); 8.30(d,1H, JHH =8.2Hz, NH);

n- [ (2S) -3-mercapto-2-phenylmethylpropionyl ] -4- (3-furyl) -L-phenylalanine (Compound 25)

m.p.140-142℃

Mass spectrum (C.I.) (M + H)⁺=410

TLC (ethyl acetate: hexane: acetic acid = 50: 5), R_f=0.42

¹H-NMR(200MHz,CDCl₃):δ(ppm):1.79-1.90(m,1H,SH)；2.22-3.11(m,7H,S-CH₂-CH-CH₂,NH-CH-CH₂) (ii) a 4.41-4.53(m,1H, NH-CH-COO); 7.11-7.50(m,9H, phenyl, phenylene); 6.91-8.12(m,3H, furyl); 8.30(d,1H, JHH =8.2Hz, NH);

n- [ 3-mercapto-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine-stereoisomer A (Compound 26)

m.p.193-196℃

Mass spectrum (C.I.) (M + H)⁺=428

TLC (dichloromethane: methanol: acetic acid = 85: 15: 1.5), R_f=0.53

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.37-3.05(m,7H,S-CH₂-CH-CH₂,NH-CH-CH₂) (ii) a 4.36-4.47(m,1H, NH-CH-COO); 7.76-7.90(m,2H, thiazolyl); 7.10-8.33(m,9H, NH, pyridyl, phenylene);

n- [ 3-mercapto-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine-stereoisomer B (Compound 27)

Mass spectrum (C.I.) (M + H)⁺=428

TLC (dichloromethane: methanol: acetic acid = 85: 15: 1.5), R_f=0.47

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.28-3.20(m,7H,S-CH₂-CH-CH₂,NH-CH-CH₂) (ii) a 4.20-4.35(m,1H, NH-CH-COO); 7.70-7.90(m,2H, thiazolyl); 7.17-8.40(m,9H, NH, pyridyl, phenylene);

n- (2-isobutyl-3-mercapto-propionyl) -4- (2-thiazolyl) -L-phenylalanine (Compound 28)

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):0.50-1.44(m,9H)；1.68-2.25(m,4H)；2.79-3.22(m,2H)；4.50-4.63(m,1H)；7.34-7.85(m,4H)；7.73-7.90(m,2H)；8.27-8.39(2d,1H)；

Mass spectrum (C.I.) (M + H)⁺=393

N- [ 3-mercapto-2- (3-methoxyphenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 29)

Mass spectrum (C.I.) (M + H)⁺=457

¹H-NMR(200MHz,DMSO-d₆+D₂O):δ(ppm):2.20-3.21(m,7H)；3.70(d,3H)；4.48(m,1H)；6.55-6.86(m,3H)；7.00-7.40(m,3H)；7.65-7.95(m,4H)；8.27-8.45(bt,CONH)；

N- [ 3-mercapto-2- (2-fluorophenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 30)

Mass spectrum (C.I.) (M + H)⁺=445

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.20-3.18(m,8H)；4.35-4.55(m,1H)；6.85-7.35(m,6H)；7.65-7.90(m,4H)；8.35(d,1H)；

N- [ 3-mercapto-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 31)

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.82-3.19(m,8H,CH₂-CH₂-CH₂；NH-CH-CH₂) (ii) a 4.44-4.59(m,1H, CH-COO); 6.62-7.91(m,9H, aryl); 8.42(d,1H, NH);

n- [ 3-mercapto-2- (2-furyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 32)

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):1.79-3.18(m,8H,S-CH₂-CH₂-CH₂；NH-CH-CH₂) (ii) a 4.43-4.58(m,4H, CH-NH); 5.79-7.47(m,3H, furyl); 7.30-7.85(m,4H, phenylene); 7.73-7.91(m,2H, thiazolyl); 8.40-8.46(2d,1H, NH);

n- [ 3-mercapto-2- (3-methyl-5-isoxazolyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine (Compound 33)

¹H-NMR(200MHz,DMSO-d₆):δ(ppm):2.00-2.12(2S,3H,CH₃-isoxazolyl); 2.28-3.19(m,8H, S-CH)₂-CH₂-CH₂；CH₂-phenylene radicals); 4.43-4.59(m,1H, CH-NH); 5.75-6.03(2s,1H, isoxazolyl); 7.29-7.86(m,4H, phenylene); 7.74-7.90(m,2H, thiazolyl); 8.46-8.53(2d,1H, NH);

example 8

In vitro evaluation of pharmacological Activity

a) NEP-inhibiting Activity

NEP-inhibitory activity was assessed in murine kidney cortical membranes prepared as described by T Maeda et al in Biochim. Biophys. acta 1983,731(1), 115-120.

Kidneys were removed from male Sprague-Dawley rats weighing approximately 300g and maintained at 4 ℃.

The renal cortex was carefully dissected, finely divided and suspended in 1: 15 w/v homogenization buffer (containing 1mM MgCl₂、30mMNaCl、0.02％NaN₃10mM sodium phosphate, pH 7.4).

The tissues were then cold homogenized for 30 seconds using an Ultra-Turrax homogenizer. Approximately 10ml of homogenate was layered in 10ml of sucrose (41% w/v) and centrifuged at 31200rpm for 30 minutes at 4 ℃ on a fixed angle rotor. Membranes were collected from the buffer/sucrose interface, washed twice with 50mM Tris/HCl buffer (pH7.4) and resuspended in the same buffer and stored in small aliquots at-80 ℃ until use.

NEP-inhibitory activity was evaluated by using the method described by c.lrorens et al in eur.j.pharmacol, 69, (1981),113-116 as reported below.

Aliquots of membrane suspensions (protein at a concentration of 5. mu.g/ml) prepared as described above were preincubated in the presence of aminopeptidase inhibitor (bestatin-1 mM) for 10 minutes at 30 ℃.

Add [ ()³H〕〔Leu⁵Enkephalinase (15nM) and buffer TRIS/HClpH7.4(50mM) to reach a final volume of 100. mu.l.

The incubation was stopped by adding HCl0.1M (100. mu.l) (20 min at 30 ℃).

Metabolite [ (l)³H ] Tyr-Gly-Gly was quantified by chromatography on a polystyrene column (Porapak Q).

The percent inhibition of metabolite production in membrane preparations treated with the compound of formula I and with the comparative compound relative to untreated membrane preparations was expressed as IC₅₀(nM) values.

b) ACE-inhibitory Activity

ACE-inhibitory activity was evaluated according to the literature reported by B.Holmquist et al in analytical biochemistry 95,540-548 (1979).

50 μ M of ACE (250mU/ml purified from rabbit lungs, EC3.4.15.1SIGMA) was preincubated with 50 μ l of a compound of formula I or a reference compound or related vector in a thermostatted cuvette at 37 ℃.

The reaction was started by adding 0.8mM furylacrylphenylalanylglycylglycine (FAPGG-SIGMA).

At the same time, the absorbance at 340nm was recorded continuously for 5 minutes by using a Beckman DU-50 spectrophotometer with a regression coefficient program to calculate the delta A/min and enzyme kinetic curves.

The percent enzyme inhibition in the preparations treated with the compound of formula I and with the comparative compound relative to the untreated preparation is expressed as IC₅₀(nM) values.

IC's relating to ACE-inhibitory and NEP-inhibitory Activity of Compounds 16, 18-25, 27-33 and comparative Compounds Thiopran and Captopril₅₀The (nM) values are reported in table 1 below.

TABLE 1

ACE-inhibitory and NEP-inhibitory activity of Compounds 16, 18-25, 27-33 and Thioprhan and Captopril, expressed as IC₅₀(nM values)

Compound (I)	ACE-inhibitory Activity IC50(nM)	NEP-inhibiting Activity IC50(nM)
			16	3.2	1.8
18	1.8	1.8
			19	1.9	1.8
20	1.5	2.5
			21	1.7	2.6
22	1.8	2.0
			23	1.6	0.6
24	2.5	1.3
			25	2.4	1
27	9.1	17.3
			28	5.8	1.8
29	4.6	9.0
			30	10.7	11.2
31	8.6	1.2
			32	8.6	4.0
33	7.9	4.5
			Thiorphan	99	18
Captopril	4.6	Is inactive

The data reported in table 1 show: the compounds of formula I, object of the present invention, have a significant mixed ACE/NEP inhibitory activity.

The activity is comparable to that of captopril, which is involved in ACE inhibitory activity, but greater than that of Thiorphan, which is involved in NEP inhibitory activity.

Example 9

Evaluation of pharmacological Activity "ex vivo

a) NEP-inhibiting Activity

The in vivo NEP-inhibitory activity was evaluated as reported in the literature by M.Orlowsky et al, Biochemistry 1981,20, 4942-4950.

The inhibitory activity of the compounds of formula I was evaluated in the kidney 5 minutes after intravenous injection (0.6 and 21. mu. mol/kg) and 30 minutes, 60 minutes and 4 hours after oral administration (30. mu. mol/kg) of the test compound in spontaneously hypertensive mice (SHR).

After removal of the kidney from the SHR, the kidney tissue was homogenized and incubated for 15 minutes at 37 ℃ in the presence of glutaryl-alanyl-phenylalanyl-2-naphthylamide (GAAP) as substrate and aminopeptidase M at ph 7.6.

The reaction was terminated by the addition of 10% aqueous trichloroacetic acid. The released 2-naphthylamine was determined by addition of fast purple paste dye (2 ml).

Enzymatic reaction rates were measured at 524nm (OD) relative to a standard obtained by complexing 2-naphthylamine with fast purplish paste₅₂₄) Is determined.

The NEP-inhibitory activity of the test compounds was expressed as percent NEP-inhibition in SHR kidneys.

b) ACE-inhibitory Activity

The ACE-inhibitory activity ex vivo was assessed using a radio assay as reported by J.W.Ryan et al in biochem.J. (1977),167,501-504 literature.

The inhibitory activity of the compounds of formula I was evaluated in the lungs after 5 minutes of intravenous injection (0.6 and 21. mu. mol/kg) and after 30 minutes, 60 minutes and 4 hours of oral administration (30. mu. mol/kg) of the test compounds in spontaneously hypertensive mice (SHR).

After removal of the lung from the SHR, lung tissue was homogenized and³h ] Hyp-glycyl-glycine substrate at 37 ℃ for 2 hours.

The reaction was terminated by addition of hydrochloric acid.

The liberated radiolabeled hyppuric acid was extracted with ethyl acetate and counted by liquid scintillation spectrometry according to conventional methods.

The ACE-inhibitory activity of the test compounds was expressed as a percentage of ACE-inhibition in the lungs of SHR.

As an example, the percentage of basal enzyme activity obtained from ex vivo assays following intravenous and oral administration of Compound 16 and comparative compounds R-1, R-2, R-3 and R-4 is reported in Table 2 below.

TABLE 2

Percentage of ACE-inhibition and NEP-inhibition ex vivo of Compound 16 and Compounds R-1, R-2, R-3 and R-4

Compound (I)	Treatment of	ACE-inhibition (lung)	NEP-INHIBITION (Kidney)
						5 minutes and 60 minutes	5 minutes and 60 minutes
16	Intravenous injection (0.6 mu mol/kg)	15％	34％
				16	Intravenous injection (21 mu mol/kg)	72％	49％
16	Orally administered (30. mu. mol/kg)	36％	39％
						30 minutes and 4 hours	30 minutes and 4 hours
16	Orally administered (30. mu. mol/kg)	60％ 45％	55％ 40％
				R-1	Orally administered (30. mu. mol/kg)	25％ 20％	5% inactive
R-2	Orally administered (30. mu. mol/kg)	30％ 25％	30% inactive
				R-3	Orally administered (30. mu. mol/kg)	25％ 20％	10％ 5％
R-4	Orally administered (30. mu. mol/kg)	25％ 10％	No activity and no activity

The data reported in table 2 confirm that: the compounds of formula I, object of the present invention, have significant ACE/NEP-inhibitory activity after intravenous as well as oral administration.

Furthermore, the ACE/NEP-inhibitory activity of the compounds of formula I ex vivo gave significantly higher results than the comparative compounds.

Claims (10)

1. A compound of formula I:wherein R is a mercapto group or R convertible in an organism to a mercapto group₄COS；R₁Is straight or branched C₂-C₄Alkyl or aryl or an aralkyl group having from 1 to 6 carbon atoms in the alkyl moiety, wherein aryl is phenyl or a 5 or 6 membered heteroaryl having 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur, which may or may not be substituted, identical or different, by 1 or more substituents selected from the group consisting of pro-halogensA hydroxy group, an alkoxy group, an alkyl group, an alkylthio group, an alkylsulfonyl or alkoxycarbonyl group having from 1 to 6 carbon atoms in the anti-basic moiety, C containing 1 or more fluorine atoms₁-C₃An alkyl group, a carboxyl group, a nitro group, an amino or aminocarbonyl group, an amido group, an aminosulfonyl group, a mono-or di-alkylamino group having from 1 to 6 carbon atoms in the alkyl moiety or a mono-or di-alkylaminocarbonyl group; r₂Is a hydrogen atom, straight or branched C₁-C₄An alkyl or benzyl group; r₃Is phenyl substituted by a 5-or 6-membered aromatic heterocycle having 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur, as the phenyl and heterocyclic groups, may be substituted or unsubstituted with one or more substituents, which may be the same or different, e.g. for R₁As indicated; r₄Is straight or branched C₁-C₄An alkyl group or a phenyl group;

the carbon atom marked with an asterisk is the stereostructural center;

2. a compound of formula I according to claim 1, wherein R₃Is a phenyl group substituted in the 4-position by a heterocyclic group.

3. A compound of formula I according to claim 2, wherein R₁Represents an aralkyl group which may be substituted by one or more substituents, which may be the same or different, selected from a halogen atom, a hydroxyl group, an alkyl group or an alkoxy group.

4. A compound of formula I according to claim 3, wherein R₁Represents phenylalkyl which is optionally substituted by 1 or more substituents, which may be the same or different, selected from halogen atoms, hydroxy, alkyl or alkoxy groups.

5. A compound of formula i according to claim 1, in the form of a salt with an alkali metal selected from sodium, lithium and potassium.

6. A compound according to claim 1 which is a compound selected from the group consisting of the following and stereoisomers (2S) or (2R) thereof;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-pyridyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-pyridyl) -L-phenylalanine benzyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-furyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (5-pyrimidinyl) -L-phenylalanine ethyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-pyrazinyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (2-thienyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-thienyl) -L-phenylalanine methyl ester;

n- (3-phenylcarbonylthio-2-phenylmethylpropionyl) -4- (3-furyl) -L-phenylalanine methyl ester;

n- [ 3-phenylcarbonylthio-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-thiazolyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-pyridyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-pyridyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-furyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (5-pyrimidinyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-pyrazinyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (2-thienyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-thienyl) -L-phenylalanine;

n- (3-mercapto-2-phenylmethylpropionyl) -4- (3-furyl) -L-phenylalanine;

n- [ 3-mercapto-2- (3-pyridylmethyl) propionyl ] -4- (2-thiazolyl) -L-phenylalanine;

n- [ 3-acetylthio-2- (3-methoxyphenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- [ 3-acetylthio-2- (2-fluorophenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- [ 3-phenylcarbonylthio-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- [ 2- (2-furyl) methyl-3-phenylcarbonylthio-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- [ 2- (3-methyl-5-isoxazolyl) methyl-3-phenylcarbonylthio-propionyl ] -4- (2-thiazolyl) -L-phenylalanine methyl ester;

n- [ 3-mercapto-2- (3-methoxyphenyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine;

n- [ 3-mercapto-2- (2-thienyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine;

n- [ 3-mercapto-2- (3-methyl-5-isoxazolyl) methyl-propionyl ] -4- (2-thiazolyl) -L-phenylalanine;

n- [ 2- (2-fluorophenyl) methyl-3-mercapto-propionyl ] -4- (2-thiazolyl) -L-phenylalanine;

n- [ 2- (2-furyl) methyl-3-mercapto-propionyl ] -4- (2-thiazolyl) -L-phenylalanine.

7. A process for the preparation of a compound of formula i according to claim 1, which comprises reacting a compound of formula iiWherein

R and R₁Having the meanings reported above;

reaction with a phenylalanine derivative of formula iii:wherein

R₂And R₃Have the meaning stated in claim 1.

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula i according to claim 1 in a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 7, which is a medicament for the treatment of cardiovascular diseases.

10. Use of a compound according to claim 1 for the preparation of a medicament for the treatment of cardiovascular diseases.