WO1996005189A1

WO1996005189A1 - Inhibitors of the benzamidine type

Info

Publication number: WO1996005189A1
Application number: PCT/CH1995/000174
Authority: WO
Inventors: Jörg STÜRZEBECHER; Helmut Vieweg; Peter Wikstroem
Original assignee: Pentapharm Ag
Priority date: 1994-08-09
Filing date: 1995-08-09
Publication date: 1996-02-22
Also published as: AU3107795A

Abstract

Disclosed are thrombin inhibitors of the amidinophenylalanine type. The compounds are particular suitable for oral and rectal resorption.

Description

New inhibitors of the benzamidine type

The invention relates to new thrombin inhibitors of the amidinophenylalanine type, their preparation and their use for combating diseases. They are distinguished in particular by the fact that they carry a substituted piperazine residue in different positions of the molecule.

Among the trypsin-like serine proteinases, such enzymes are of particular physiological importance that trigger coagulation and fibrinolysis in the blood. Since the pathologically increased activity of the enzymes or the lack of natural inhibitors can lead to diseases, attempts are made to control these diseases by increasing the inhibitor potential. Accordingly, natural inhibitors - endogenous to the body such as antithrombin III or those from other natural sources such as hirudin - are used or prepared for the therapy.

The development of inhibitors of the coagulation enzymes is being worked on particularly intensively. Extensive biochemical and (animal) experimental pharmacological studies have shown that inhibitors of throbine in particular are effective anticoagulants and antithrombotics.

The search for anticoagulant active ingredients from natural sources led to the characterization of the thrombin inhibitor hirudin from the leech. Although potency and selectivity make hirudin the 'ideal' anticoagulant, it has the disadvantage that only parenteral administration is possible due to the protein structure. The development to a therapeutic agent, which is absorbed after oral application, is therefore only possible with a synthetic tables, small molecular, selective inhibitor possible.

Three classes of compounds have so far been described as low molecular weight thrombin inhibitors, namely a) N-ci-arylsulfonylated argininamides

[Okamoto et al. (Mitsubishi), U.S. Pat. 4,125,604] b) Compounds of the (D) -Phe-Pro-Arg type [Kettner et al. (DuPont), U.S. Pat. 178368] c) Benzamidine derivatives

[Wagner et al. WP C 07 D / 201 898,

Lintel cup, Wikstroe, Vieweg, WO 92 08709, WO 94 18185

Delabassee et al. (Sanofi), FR 8601398 and FR 8601399, Stüber et al. (Behring-Werke), DE 4115486, DE 4111394 and

DE 4206858].

Highly effective inhibitors were found in all three classes, but with only a few exceptions they are not absorbed after oral administration. In the case of benzamidines, 3-amidinophenylalanine derivatives had, for the first time, achieved a certain, but not sufficient oral availability (WO 92 08709).

Oral availability was further increased by introducing a C-terminal piperazide (WO 94 18185). However, only a few derivatives with strong antithrombin activity (K ^ <IOμmol / 1) could be found.

It has now surprisingly been found derivatives of (L) -3-amidino-phenylalanine which contain piperazine groups at the C-terminus and are protected N-terminally with a straight-chain alkylsulfonyl radical and have strong antithrombin activity. The introduction of a (L) -P2-aminodicarboxylic acid, such as (L) -asparaginic acid or (L) -glutamic acid in (D) -4-amidinophenyl- alanine piperidides, the second carboxyl group (in the β or Y position) carrying a substituted piperazine radical. Very effective thrombin inhibitors have been found, in which an increased selectivity towards thrombin is to be emphasized.

The present invention relates to new thrombin inhibitors having a benzidine structure of the general formulas I and II

wherein R ¹ in the general formula I is a straight-chain alkylsulfonyl radical having 1 to 16 carbon atoms, preferably 6 to 10 carbon atoms, R * ^{2 is} a straight-chain Alkylsulfonyl radical having 1 to 8 carbon atoms and R ^ in the general formula II is a straight-chain alkylsulfonyl radical having 1 to 4 carbon atoms, an alkanoyl radical having 1 to 4 carbon atoms and n is an integer of 1 or 2.

The inhibitor derivatives of the general formulas I and II according to the invention can be treated with a mineral acid, e.g. HC1, HBr, H2SO4 or H3PO4, or with an organic acid, e.g. Formic, acetic, oxalic or tartaric acid may be protonized.

The compounds of the general formula I are prepared by the known production process described below.

(L) -3-Cyanphenylalaninalkylester are reacted in a suitable solvent with a straight-chain alkylsulfonyl chloride and then subjected to ester hydrolysis, whereby the free carboxyl groups are obtained. The N -alkylsulfonated 3- (L) -cyanophenylalanine derivatives are reacted with piperazine derivatives of the formula III, in which R ^ has the meanings given above:

The corresponding amidine derivatives of the formula I are prepared from these cyanphenylalanine derivatives obtained by known processes (via thioamide, thioimido ester or imido ester route).

The amidino derivatives of the general formula II are built up in stages in accordance with the production process known per se and described below. (D) -4-cyanophenylalanine is first N-acylated with an acid-labile protective group and then the σ (carboxyl group is converted to a piperidide. After the o-amino protective group has been cleaved off, the (D) - -4-cyanophenylalanine piperidide is also converted a Noχ- -2-naphthylsulfonyl derivative of the formula IV,

C = 0

wherein R ^ is an acid labile carboxyl protecting group, e.g. t-butyl, and n represents the number 1 or 2, implemented. After the acid-labile carboxyl protecting group has been split off, these derivatives with a free β or V ′ carboxyl group are reacted with a piperazine derivative of the formula V

in which R *** has the meanings given above, converted and then the 4-cyano group of (D) -phenylalanine converted into the corresponding 4-amidino derivative by known processes.

The compounds according to the invention can be used per se or - in combination with carriers and auxiliaries - as medicaments, and they can be administered subcutaneously, intravenously, but especially orally or rectally. In particular, tablets, coated tablets, capsules, pellets, suppositories, solvents, solutions, injections or transdermal systems such as plaster. In diagnostics, the compounds according to the invention can be used in particular in test kits for determining thrombin.

The invention is explained in more detail in the following examples.

example 1

N <Λ- (2-Naphthylsulfonyl) -ß-4-methylsulfonyl-piperazino- (L) - asρartyl- (D) -4-amidino-phenylalanine-piperidide hydrochloride

Connection 1

No ⁽ - (2-naphthylsulfonyl) -ß-t-butoxycarbonyl- (L) -aspartic acid

6.5 g of β-t-butoxycarbonyl- (L) -aspartic acid were dissolved in a solution of 8 g of sodium carbonate in 120 ml of water, mixed with 9.34 g of naphthalene-2-sulfochloride in 120 ml of diethyl ether and stirred for 16 hours, during which an oily product failed. So much water was added until 2 clear phases were obtained. The aqueous phase was separated, washed with 2 × 60 ml of diethyl ether, then acidified with 1N HCl and extracted 3 × with 100 ml of ethyl acetate. After washing the ethyl acetate solution with saturated sodium chloride solution and drying over sodium sulfate, the solvent was distilled off. 8.3 g (64%) of a crystalline product were obtained.

Connection 2

(D) -4-cyanophenylalanine piperidide ^• Hydrochloride

4.9 g of Boc- (D, L) -3-cyanophenylalanine were dissolved in 50 ml of DMF, mixed with 3.3 g of HOBt, cooled to 0 ° C. and 4.2 g of DCC were added. After stirring for one hour, 2.9 g of piperidine added and stirred overnight. The precipitated urea derivative was then filtered off with suction and the solvent was distilled off under vacuum. The product obtained was purified over KG60 (chloroform). Yield: 5.5g (90%).

This product was dissolved in 50 ml of 2N HCl in ethyl acetate and stirred for 2 days at room temperature. The solvent was then distilled off, the product obtained was triturated with ether, the precipitate was filtered off with suction and dried. Yield: 4.1g (90%).

Connection 3

No <- (2-naphthylsulfonyl) -ß-t-butoxycarbonyl- (L) -aspartyl-

(D) -4-cyanophenylalanine piperidide

1.11 g of compound 1 were dissolved in 20 ml of DMF, 0.6 g of HOBt was added, the mixture was cooled to 0 ° C., 0.8 g of DCC was added and the mixture was stirred for one hour. Then 1.0 g of compound 2 and 0.37 ml of NMM were added and the mixture was stirred for 2 days. The urea derivative obtained was then filtered off, the solvent was distilled off under vacuum and the crude product obtained was purified over KG60 (chloroform). Yield: 1.5g (77%).

Connection 4

Nc, - (2-naphthylsulfonyl) -ß-carboxy- (L) -aspartyl- (D) -4-cyanophenylalanine piperidide

1.1 g of compound 3 were dissolved in 20 ml dichloromethane and 8 ml TFA, stirred for 2 hours at room temperature, the solvent was distilled off and codistilled twice with toluene. Yield: 0.95g (97%).

Connection 5

No (- (2-naphthylsulfonyl) -ß-4-methylsulfonyl-piperazino- (L) - aspartyl- (D) -4-cyanophenylalanine piperidide 0.92 g of compound 4 were dissolved in 20 ml of DMF, 1.4 g of complex F were added and the mixture was stirred for 2 hours. Then 0.39 g of 1-methylsulfonyl-piperazine hydrochloride and 0.2 ml of NMM in 15 ml of DMF were added and the mixture was stirred overnight. The urea derivative obtained was then filtered off, the solvent was distilled off under vacuum and the crude product obtained was purified on silica gel 60 (chloroform / methanol, 9: 1). Yield: 0.36g (31%).

Connection 6

Not- (2-naphthylsulfonyl) -ß-4-methylsulfonyl-piperazino- (L) - aspartyl- (D) -4-amidino-phenylalanine-piperidide ^• hydrochloride

0.36 g of compound 5 were dissolved in 20 ml of pyridine and 20 drops of TEA, in the solution for 10 minutes. Hydrogen sulfide was introduced and the mixture was left to stand for 2 days at room temperature. After the solvent had been distilled off, the thioamide obtained was taken up in ethyl acetate, shaken out with 1N HCl and, after drying over sodium sulfate, the solvent was distilled off. The residue was dissolved in 30 ml of acetone, 2 g of methyl iodide was added to the solution, the mixture was left to stand under light protection for 20 hours at room temperature, then the solvent was distilled off and the thioimido ester hydroiodide in the form obtained was used for the formation of amine. For this purpose, 0.27 g of this product was dissolved in 15 ml of methanol, 0.1 g of ammonium acetate was added and the mixture was heated in a water bath at 60 ° C. for 4 hours. The solvent was then distilled off and the residue was purified over KG60 (a: chloroform, b: chloroform / methanol, 9: 1). The resulting hydroiodide was converted into the hydrochloride via Amberlite IRA-420 ion exchanger. 85 mg (37% of theory) were obtained.

Example 2

Ne -octylsulfonyl-3-amidino- (L) -phenylalanine-4-methylsulfo- nyl-pyperazide • Hydrochloride

Compound 1 N-o <'- octylsulfonyl-3-cyan- (L) -phenylalanine

1.7 g of (L) -3-cyanophenylalanine methyl ester hydrochloride were suspended in 50 ml of ethyl acetate, then 2.0 ml of NMM and 2.4 ml of n-octylsulfonyl chloride solution were added and the mixture was stirred overnight. After filtering off, the solvent was distilled off. The crude product was purified over KG60 (chloroform). Yield: 2.1g (79%). The ester obtained was heated in 30 ml of acetic acid and 1N hydrochloric acid under reflux for 2 hours. After extraction with ethyl acetate and distilling off the solvent, 2.4 g of product were obtained (74%).

Connection 2

Netf-octylsulfonyl-3-cyan- (L) -phenylalanine-4-methylsulfonyl-piperazide

2.3 g of compound 1 were dissolved in 20 ml of DMF, 1.22 g of HOBt were added, the mixture was cooled to 0 ° C., 1.56 g of DCC were added and the mixture was stirred for one hour. Then 1.51 g of 1-methylsulfonylpiperazine hydrochloride and 0.82 ml of NMM were added and the mixture was stirred overnight. The urea derivative obtained was then filtered off, the solvent was distilled off and the crude product obtained was purified over KG60 (chloroform: methanol, 9: 1). Yield: 2.56g (79%).

Connection 3

No (-Octylsulfonyl-3-amidino- (L) -phenylalanine-4-methylsulfonyl-piperazide ^■ hydrochloride

2.47 g of compound 2 were dissolved in 40 ml of pyridine and 20 drops of TEA, in the solution for 10 minutes. Sulfur hydrogen initiated and the mixture left for 2 days at room temperature. After distilling off the solvent the thioamide obtained was taken up in ethyl acetate, shaken out with 1N HCl and, after drying over sodium sulfate, the solvent was distilled off. The residue was dissolved in 50 ml of acetone, the solution was mixed with 8 g of methyl iodide, left to stand under light protection for 20 hours at room temperature, then 200 ml of ether were added, and after one hour the precipitated product was filtered off, washed with fresh ether and dried. 3.13 g of this thioi idoester hydroiodide were used to form the amidine. It was dissolved in 50 ml of methanol, 0.7 g of ammonium acetate was added and the mixture was heated in a water bath at 60 ° C. for 4 hours. The solvent was then distilled off and the residue was purified on KG60 (a: chloroform, b: chloroform / methanol, 9: 1). The hydroiodide obtained was converted into the hydrochloride via Amberlite IRA-420 ion exchanger. 0.72 g (28% of theory) were obtained.

Abbreviations

Boc t-butoxycarbonyl

DCC dicyclohexylcarbodiimide

DMF dimethylformamide

HOBt hydroxybenzotriazole

KG60 silica gel 60

NMM N-methylmorpholine

TFA trifluoroacetic acid

Complex F (pentafluorophenol) 3 X DCC

Table 1

K ^ values and inhibitor concentration for doubling the clotting time of the compounds with the general formula I

Table 2

K ^ values and inhibitor concentration for doubling the clotting times (human plasma) of the compounds with the general formula II

I.

Legend for en ta e en un

TT - thrombin time aPTT - thromboplastin time

PT - prothrombin time

FX _a - factor X _a

Ki values according to Dixon, M., Bioche. J. 55, pp. 170-171 (1953). Coagulation data according to Lechner, K., 'Blood coagulation disorders', pp. 157-165, Springer Verlag, Berlin, (1982).

Claims

PATENT CLAIMS

Compounds of general formulas I and II

wherein R 'in the general formula I is a straight-chain alkylsulfonyl radical with 1 to 16 carbon atoms, preferably 6 to 10 carbon atoms, R ** is a straight-chain alkylsulfonyl radical with 1 to 8 carbon atoms and R * - * ¹ in the general formula II is a straight-chain alkylsulfonyl ¬ radical with 1 to 4 carbon atoms, an alkanoyl radical with 1 to 4 carbon atoms and n is an integer of 1 or 2, or their salts.

2. Use of the compounds according to claim 1 al thrombin inhibitors for diagnostic and pharmaceutical purposes.

3. Use of the compounds according to claim 1 for the production of subcutaneously, intravenously, in particular orally or rectally administrable antithrombotic drugs.

4. Subcutaneously, intravenously, in particular orally or rectally administrable antithrombotic medicinal product characterized by an effective amount of a compound according to claim 1 and optionally suitable carriers and / or excipients.

5. Medicament according to claim 4 in the form of tablets, coated tablets, capsules, pellets, suppositories, solutions, injections or transdermal systems, such as plaster.

6. Procedure for blood coagulation resp. Thrombin inhibition in living beings, in particular in humans, characterized by administration of an effective amount of a compound according to claim 1 or a medicament according to one of claims 4 or 5.

7. Test kit for determining thrombin, containing an effective amount of a compound according to claim 1.