ITMI961512A1 - DERIVATIVES OF 1,2 SUBSTITUTED CYCLOALKANES AS THROMBIN INHIBITORS PROCEDURE FOR THEIR PREPARATION AND THEIR USE IN FORMULATIONS - Google Patents

Description

Description of the industrial invention having as title: "DERIVATIVES OF CYCLOALCES 1, 2 REPLACED AS THROMBIN INHIBITORS, PROCEDURE FOR THEIR PREPARATION AND THEIR EMPLOYMENT IN PHARMACEUTICAL FORMULATIONS"

The subject of the invention are new compounds containing a 1,2 disubstituted cycloalkanic or cycloalkene ring of general formula (I) and their salts, in enantio- or diastereoisorically pure form or as mixtures of stereoisomers.

The compounds are endowed with inhibitory activity towards some protected serines; in particular, they have been found to be active in inhibiting the action of the thrombin enzyme and are therefore usable, for example, as potential antithrombotic agents, as antiplatelet agents or anticoagulants.

State of art

The Phe-Pro-Arg peptide sequence is present in the fibrinogen structure and is believed to be important in recognizing the thrombin active site. Numerous examples of tranbin inhibitors based on structural modifications of the Phe-Pro-Arg sequence are known: see for example patents EP 526877, US 4478745 and the publications Bajusz et al. , J. Med. Chem. 1990, 33, 1729-1735 and Kettner et al. , Thromb. Res., 1979, 14, 969-973.

The new derivatives are characterized by the fact of presenting a 1,2 disubstituted cycloalkanic or cycloalkene ring which surprisingly is able to act as a conformational analogue of proline in a peptide sequence of the Phe-Pro-Arg type.

Detailed description of the invention

The present invention refers to new compounds having general formula (I)

in which:

A is a group in which n = 1-3 or CH = CH;

either the same or different, they are 0R4 or together they represent the residue of a diol; R4 is H, alkyl, aryl or

arylalkyl;

Y is selected from the aryl group, aryl substituted with 1-3 hydroxyl, alkoxy or halogen residues, substituted alkylaryl COTI 1-3 hydroxyl, alkoxyl or halogen residues; wherein m = 1-6, preferably 3-5 and T is H, hydroxy, alkoxy, amino amidino, imidazole, guanidino or isothioureido;

alkyl;

L is

Ar is an aromatic group, preferably selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl, indolyl, optionally substituted with 1-3 residues which are the same or different from each other selected from the alkyl, alkoxy or halogen group, or a group selected from the corresponding saturated analogs of said aromatic groups;

X is selected from the group consisting of H, alkyl, Diesyl, tosyl, benzenesulfonyl, butyloxycarbonyl, benzyloxycarbonyl, acetyl, benzoyl.

The compounds of the invention can form with various acids both inorganic and organic salts which are also the object of this invention. Salts include, for example, hydrochlorides, hydrobromides, sulphates, phosphates, maleates, fumarates.

Also included in the invention are compounds or their salts in pure enantio- or diastereomeric form or as mixtures of stereoisomers.

Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.

Examples of aryl groups are phenyl, thienyl, furyl and pyridyl; preferably phenyl.

Examples of arylalkyl groups include benzyl and phenethyl, preferably benzyl.

Examples of diol residues are ethanediol, propanediol, butanediol, pinane diol and pinacolum, preferably pinane diol or pinacolum.

Preferred compounds of formula (I) are those in which A is with n = 1-2.

Another group of preferred compounds is the one in which A is

with n = 1-2 and W is CN or COH or W is a group

Yet another group of preferred compounds is that in which A is a group

Particularly preferred compounds of general formula (I) are those in which:

A is a group in which n = 1-2 o

W is CN, the same or different, they are OR4 or together they represent the residue of a diol; R4 is H or alkyl;

Y is selected from the alkylaryl group substituted by one to three hydroxyl residues, of which m is 3 and T is alkoxy, amidino or guanidino;

Q is H;

L is

Ar is phenyl, naphthyl or cyclohexyl;

X is selected from the group H, alkyl, mesyl, benzyloxycarbonyl or acetyl.

More particularly preferred compounds are the following:

i) N-a- [cis-2- (2S) - [((R) -N-methylphenylalanyl) amino] cyclohexanecarbonyl] -arginine-aldehyde hydrochloride;

ii) N-α- [cis-2- (2R) - [((R) -N-methylphenylalanyl) amino] cyclohexanecarbonyl]

in which the terms boroglycinate and boroargininate indicate the analogues of glycine and arginine with the carboxylic group substituted with the boronic one.

According to the invention compounds of general formula (I) can be obtained by means of coupling reactions between amino acids starting from the following intermediates:

amino acids of general formula

suitably protected on nitrogen, where Ar and X are as previously defined

2-amino cycloalkyl or alkenyl carboxylic acids

amino derivatives of general formula

where Y and W, previously defined, are possibly protected with suitable groups.

Of particular importance is the synthesis where the 2-amino cycloalkyl carboxylic acid used is one of the possible isomers of the 2-amino-cyclohexanecarboxylic acid. These were synthesized starting from the corresponding enantiraneri of 2-methoxycarbonylcyclohexanecarboxylic acid according to scheme A:

Bianchetti Giuseppe and others

DIAGRAM A

The acid 1 (by way of example the 1S, 2R isomer) is converted to azide with diphenylphosphorylazide (DPPA) and transformed into urethane by heat treatment in the presence of benzyl alcohol. The methyl ester 2 is saponified with 2M NaOH and MeOH and, after hydrogenolysis with Pd / C, the desired amino acid 4 is obtained (in the example under examination the IR, 2S isomer).

The Arg (Z) lactam residue was prepared according to the literature method (S. Bajusz et al. J. Med. Chem. 1990, 33, 1729)

The synthesis of the tripeptides with the terminal boronic group (W = was carried out according to the methods reported in the literature (-J. Wityak et al. J. Org. Chem. 1995, 60, 3717; C. Kettner et al. J. Biol . Chem. 1990, 265, 18289).

By way of example, scheme B reports the general synthesis of the tripeptides of formula (I), containing the residue Arginine aldehyde, starting from the enantiomers of 2-amino-cyclohexanecarboxylic acid 4.

DIAGRAM B

Protected amino acid 5 is condensed with 2-aminocyclohexanecarboxylic acid by 2,4,5-trichlorophenyl ester. The obtained dipeptide 7 is reacted with Arg (Z) lactam via mixed anhydride with isobutyl chloroformate and N-methyl morpholine as base, or by using Ν, Ν'-dicyclohexylcarbodiimide (DCC) and 1-hydroxy-benzotriazole (HOBT).

The lactam tripeptide 8 is reduced with at -60 ° C and finally deprotected by hydrogenolysis with Pd / C.

The compounds disclosed in the present invention act as thrombin inhibitors. An in vitro human thrombin inhibition test was used for the characterization and evaluation of their efficacy using tosyl-glycylprolyl-arginine-4-nitroaniline as a synthetic substrate.

The compounds of the invention proved to be active in the above test, showing that they have values of less than 5 nM; for example compound vii showed a 0.022 mM. The compounds of the invention can therefore be used as active ingredients of pharmaceutical compositions with arithithrombotic activity. The compositions of the invention, which can be prepared using conventional methods or excipients, will typically contain from 1 to 1000 mg of the compounds of general formula (I), and can be administered from 1 to 4 times a day by oral, parenteral, transdermal or per another convenient route of administration.

The examples given below serve to better illustrate the present invention.

Example 1

(1R, 2S) -2-amino-cyclohexanecarboxylic acid (Compound of formula 4) To a solution of monoester 1 (20 g) in toluene (200 ml) DPPA (24.5 ml, 107.5 mmoles) were added under nitrogen , NEt3 (15 ml) and benzyl alcohol (11.2 ml). The reaction mixture was refluxed for 4 hours, added with 100 ml of toluene, washed with 5% HCl, 5% NaHCO3 and H2O.

After evaporation of the solvent, the residue was purified by flashchromatography on silica gel (petroleum ether / AcOEt = 90/10) obtaining 15.8 g of the desired compound 2 as colorless oil; TLC: r.f.

0.4 (petroleum ether / ethyl acetate 9: 1); [a] D = -29.0 ° (c = 1, BtOH).

A 2M NaOH solution (76.3 ml) was added under stirring to a solution of ester 2 (11 g) in methanol (104 ml).

The suspension was stirred for 1 hour at room temperature, until a clear solution was formed. The methanol was evaporated under vacuum, the aqueous phase was washed with AcOEt, acidified with 6N HCl and extracted with

The organic phases were washed with H2O, dried and evaporated. The residue was treated with ether and filtered, obtaining 8.2 g of the desired compound 3 as a white solid; TLC r.f. 0.5 / cyclohexane / acetic acid / ethanol 45: 45: 5: 5); [a] D = -15.1 '(c = 1, BtOH).

A solution of acid 3 (8.1 g) in methanol (100 ml) was hydrogenated in the presence of 10% Pd / C (900 mg). After filtration of the catalyst, the solvent was evaporated under vacuum. The residue was taken up with ether and filtered, obtaining 4.1 g of compound 4 as a white solid; TLC: 0.6 (butanol / acetic acid / water 6: 2: 2); [a] D = -16.8 '

Similarly, the other enantiomers of 2-amino-cyclohexanecarboxylic acid were obtained:

The (1S, 2R) [a] D = 15.6 ° enantiomer

The enantiomer (1R, 2R) [a] D = -71.9 °

The (1S, 2S) enantiomer [a] D = 71.5 °

Example 2

N-a- [cis-2- (2S) - [((R) -N-methylphenylalanyl) amino] cyclohexanecarbonyl] -arginine aldehyde hydrochloride (Compound of formula 10)

Preparation of Z- (D) -N-methylphenylalanine-2,4,5-trichlorophenylester (6; X = CH3, L = CH2, Ar = phenyl)

2,4,5-trichlorophenol (HOTCP) (3.15 g) and DCC ( 3.29 g). The reaction mixture was kept under stirring at 0 ° C for 1 hour and at room temperature for 2 hours.

After cold filtration of the dicyclohexylurea formed, the solvent was evaporated to obtain 8.3 g of the desired compound 6 as an oily residue (HPLC titer = 86%), which was used without further purification.

Preparation of cis-2- (2S) - [((R) -N-methylphenylalanyl) amino] cyclohexanecarboxylic acid (7; X = CH3, L = CH2, Ar = phenyl)

NEt3 (1.46 ml) and Z— (D) - MePhe-OTCP 6 (6 g). The reaction mixture was left under stirring at room temperature for 24 hours and evaporated under vacuum at 35-40 ° C. The residue was taken up with AcOEt, cold washed with 2N HC1, H20, dried and evaporated. The crude product was dissolved in ether and extracted with 5% NaHC03. The combined aqueous phases were cold acidified with 1N HCl to pH 2 and extracted with AcOEt. The combined organic phases were dried and evaporated under vacuum at 30 ° C, obtaining 3.3 g of the desired compound 7 as a white solid.

TLC: r.f. 0.7 cyclohexane / acetic acid / water 45: 45: 5: 5) Preparation of N-q-rcis-2- (2S) - [((R) -N-methylphenylalanyl) amnino] cyclohexancarbonyl-Z-arqinine lactam (8; X = CH3, L = CH2, Ar = phenyl) To a solution of compound 7 (2.6 g) in CHCI3 (20 ml), cooled to -10 ° C, N-methylmorpholine (0.65 ml) was added and isobutyl chloroformate (0.78 ml). To the reaction mixture, after 5 'at -10 ° C, a solution of Arg (Z) lactam hydrochloride (2.15 g) and NEt3 (1.65 ml) in CHC13 (40 ml) was added. The reaction mixture was left under stirring at -10 ° C for 30 'and at room temperature for 3 hours, then it was diluted with CHCl3 (60 ml) and washed with 1N HCl, H20, 5% ed (60 ml). The organic phase was dried and evaporated under vacuum at 30 ° C. The residue was purified by flash chromatography, obtaining 2.4 g of the desired compound 8 as a white solid; TLC: r.f. 0.5 (ethyl acetate / hexane 8: 2).

Preparation of N-a- [cis-2- (2S) -r ((R) - (Z) -N-methylphenylalanyl) amino] cyclohexanecarbonyl] -Z-arginine aldehyde (9; X = CH3, L = CH2, Ar = phenyl ) To a solution of compound 8 (1 g) in THF (40 ml) stirred under N2 and cooled to -60 ° C, 1M in THF (1.4 ml) was added. The reaction mixture was left under stirring for 30 'at -60 ° C, added with 10% up to pH 2 and diluted with H20 (20 ml). The aqueous phase was extracted with CH2C12. The combined organic phases were washed with H2 O, dried and evaporated under vacuum, obtaining 980 mg of the desired compound 9 as a white solid.

Compound 9 (500 mg, 0) in THF (15 ml) and (5 ml), was hydrogenated in the presence of 10% Pd / C (100 mg) and IN HCl (0.63 ml) at atmospheric pressure and room temperature . At the end of the reaction the catalyst was filtered and, after addition of 1N HCl (0.77 ml), the solvent was evaporated under vacuum at 30 ° C.

The residue was taken up with AcOEt and filtered to obtain 280 mg of the desired compound 10 as a white solid: m.p. 190 -4 ° C; TLC r.f. 0.4 (butanol / acetic acid / water 6: 2: 2).

With the procedure described in Example 2, the following were obtained: ii) N-a- [cis-2- (2R) - [((R) -N-methylphenylalanyl) amino] cyclohexancarbonyl] -argininaldehyde hydrochloride;

iii) N-a- [trans-2- (2S) - [((R) -N-methylphenylalanyl) amino] cyclohexanecarbon 1] -argininaldehyde hydrochloride;

iv) N-α- [trans-2- (2R) - [((R) -N-methylphenylalanyl) amino] cyclohexancar

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Claims (4)

CLAIMS Compounds of formilia general (I) in which: A is a group (CH2) n in which n = 1-3 or CH = CH; W is CN, where H, 0R4, or equal 0 different, are 0R4 or together represent the residue of a diol; R4 is H, alkyl, aryl or arylalkyl; Y is selected from the aryl group, aryl substituted with 1-3 hydroxyl, alkoxy or halogen residues, alkylaryl substituted with 1-3 hydroxyl residues, C1-C3 alkoxy or halogens; wherein m = 1-6, preferably 3-5 and T is H, hydroxy, alkoxy, amino, amidino, imidazole, guanidino or isothioureido; Q is H or alkyl; Ar is an aromatic group, preferably selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl, indolyl, optionally substituted with 1-3 residues which are the same or different from each other selected from the alkyl group, C1-C3 alkoxy or halogen, or a group selected from the corresponding saturated analogues of said aromatic groups; X is selected from the group consisting of H, alkyl, mesyl, tosyl, benzenesulfonyl, butyloxycarbonyl, benzyloxycarbonyl, acetyl, benzoyl, their stereoisomeric forms and their salts with pharmaceutically acceptable acids. 2. Compounds according to claim 1 wherein A is with n = 1-2. 3. Compounds according to claim 2 wherein wherein is H. 4. Compounds according to claim 3, which are: Ν-α- [cis-2- (2S) - [((R) -β-cyclohexylalanyl) amino] cyclohexanecarbonyl] - (4-amidinophenyl methyl) boroglycinate of pinanediol hydrochloride; N-a- [cis-2- (2S) - [((R) -N-methyl-β- (2-naphthyl) alanyl) amino] cyclopentancarbonyl] -borargininate of methyl hydrochloride. 7. Compounds according to claim 1 wherein A is CH = CH. 8. Compounds according to claim 7, which are: N-a- [cis-2- (2S) - [((R) -N-methyl-β- 2-naphthyl) alanyl) amino] cyclohex-4-encarbonyl] -arginine; N-a- [cis-2- (2S) - [((R) -N-methyl-β- (2-naphthyl) alanyl) amino] cyclohex-4-encarbonyl] - (4-amidinophenylmethyl) boroglycinate of pinacolum hydrochloride; N-a- [cis-2- (2S) - [((R) -N-methyl-β- (2-naphthyl) alanyl) amino] cyclohex-4-encarbonyl] - (4-hydroxyphenylmethyl) glycinate of ethyl. 9. A process for the preparation of compounds of general formula (I) obtainable by coupling, according to the methods known in the literature, of amino acids of general formula suitably protected on nitrogen, with 2-amino cycloalkyl or alkenyl carboxylic acids and with amino derivatives of general formula suitably protected on the functional groups of Y and W. 10. A process for the preparation of compounds of general formula (I), according to claim 9, wherein the 2-amino cycloalkyl carboxylic acid used is one of the isomers of the 2-amino cyclohexane carboxylic acid obtained in an enantiomerically pure form, which comprises : a) the treatment of the suitable monoester of the enantiomerically pure 1,2-cyclohexandicarboxylic acid with diphenylphosphorylazide; b) transformation of the acyl azide obtained into urethane by heat treatment in the presence of benzyl alcohol; c) saponification of the methyl ester and deprotection of the amino group by hydrogenolysis with Pd / C. 11. The optically active synthetic intermediate (1R, 2S) -2-aminocyclohexanecarboxylic acid. 12. The optically active synthetic intermediate (1S, 2R) -2-amninocyclohexanecarboxylic acid. 13. Compounds of claims 1-8, such as antithrombotic, anticoagulant and anti-aggregating therapeutic agents. 14. Pharmaceutical compositions containing cooked active ingredient an effective amount of a compound, of claims 1-8, in association with suitable excipients.