WO2004067522A1

WO2004067522A1 - Azaphenylalanine derivatives and their use as antithrombotic agents

Info

Publication number: WO2004067522A1
Application number: PCT/IB2003/000403
Authority: WO
Inventors: Uros Urleb; Ales Obreza; Mojca Stegnar; Alenka Trampus Bakija
Original assignee: Lek Pharmaceuticals D.D.; University Of Ljubljana
Priority date: 2002-01-24
Filing date: 2003-01-27
Publication date: 2004-08-12
Also published as: AT503355A2; AU2003205963A8; SI21137A; DE10392134T5; CH696724A5; AU2003205963A1

Abstract

Novel azaphenylalanine derivatives of the formula I and pharmaceutically acceptable salts thereof are described wherein the substituents have the meanings as specified in the description. The compounds are useful as anticoagulants.

Description

AZAPHENYLALANINE DERIVATIVES AND THEIR USE AS ANTITHROMBOTIC AGENTS

The invention belongs to the field of pharmaceutical industry and relates to novel azaphenylalanine derivatives, procedures for their preparation and pharmaceutical compositions containing them. These novel azaphenylalanine derivatives are useful as anticoagulants.

Thrombin is a serine protease, which is one of the key enzymes in the processes of blood coagulation and in the development of thrombosis (Edit, J. F.; Allison, P.; Noble, S.; Ashton, J.; Golino, P.; McNard, J.; Buja, L M.; Willerson, J. T., J. Clin. Invest. 1989, 84, 18.).

The crystalline structure of the serine protease thrombin is known (Bode, W.; Turk,

D.; Karshikov, A. J., Protein Sci. 1992, 1, 426.

Currently used anticoagulants have many adverse effects and are of limited activity.

Low molecular weight thrombin inhibitors should have a selective activity and can be used in oral administration. There are reversible and irreversible thrombin antagonists (Kimball, S. D., Curr. Pharm. Design 1995, 1, 441, Das, J.; Kimball, S. D., Bioorg. Med. Chem. 1995, 3, 999, Kimball, S. D., Blood Coagulation and Fibrinolysis 1995, 6, 511, Breznik, M.; Pecar, S., Farm. Vestn. 1997, 48, 545, Leung, D.; Abbenante, G.; Fairlie, D.P., J. Med. Chem. 2000, 43, 305). The majority of reversible thrombin antagonists derive from peptidomimetically modified structure D-Phe-Pro-Arg. The aim of the modification is to provide chemical stability, selectivity and activity. The active substance argatroban, described in EP-A-0008746 is clinical in use. Napsagatran is a selective reversible inhibitor of thrombin described in EP-A- 0559046. Meta substituted phenylalanine derivatives with amide or amidoxime structure are selective thrombin inhibitors disclosed in WO 92/08709. Amidinophenylalanine derivatives and aminopyridylalanine derivatives, described in WO 95/13274, have selective antithrombotic activity.

Azapeptides are peptidomimetics in which the Cα atom is replaced by nitrogen. The advantage of this isoelectronic substitution is the preservation of the chemical integrity of the modified amino acid and only a minor conformational modification that is important for the process of molecular identification and stabilization of the complex ligand-receptor and metabolic stabilization (Gante, J., Synthesis 1989, 405, Gante, J., Angew. Chem. 1994, 06, 1780). Preparation of N-naphthylsulfonyl amino acids is known (Pendleton.R.G., et al. J. Pharm. Exp. Ther. 1979, 208, 24). Azaphenyalanine derivatives are described in WO 02/051824 and in Zega, A.; Mlinsek, G. Sepic, P.; et al. Bioorg. Med. Chem., 2001 ; 9, 2745-2756, Zega, A.; Trampus-Bakija, A.; Fortuna, M.; Stegnar, M.; et al. Pharmazie, 2001, 56, 683-685). A group of thrombin inhibitors with the azaphenylalanine fragment, described in Sl- 20025, showed in in vitro tests submicromolar constants.

Recently, also the inhibitors of factor Xa become research targets of the pharmaceutical industry. Structure of the active site of factor Xa, achieved with X-ray diffraction made a great impact on the synthesis of new Xa inhibitors. Despite high activity and moderate selectivity there are currently no Xa inhibitors on the market although some of them are in different stages of clinical tests. (Zega, A.; Obreza, A.; Urleb, U. Farm, vestn., 1999, 50, 53-57, Rai, R.; Sprengeler, K. C; Elrod, K. C; et al. Curr. Med. Chem. 2001 , 8, 101-119). A low-molecular weight inhibitor DX-9065a exibits high thrombin selectivity and is a bis-amidino derivative (Hara, T.; Yokoyama, A.; Ishihara, H.; et al. Thromb. Haemost. 1994, 71, 314-319. Lately, compounds with only one basic group in their structure have been reported with improved bioavailability (WO 9857951, Yee, Y. K.; Tebbe, A. L; Linebarger, J. H.; J. Med. Chem. 2000, 43, 873-882. The object of the present invention is to provide improved new compounds with anticoagulant activity, wherein these compounds are active after oral administration, highly selective and have a low toxicity, a process for the preparation thereof as well as the use of these compounds for the preparation of a pharmaceutical composition. These objects are achieved for example by the subject-matter of the independent claims 1 , 3, 4 and 11. Preferable embodiments of the invention are defined in the dependent claims.

The invention relates to novel azaphenylalanine derivatives and analogs thereof of the general formula (I)

wherein

R¹ and Z are H or a residue of the formula

with the proviso that one of R¹ and Z is H; R⁴ = H, alkyl (C C₃), OH, O-alkyl (CrC₃), NH_2;

R represents a residue of the formula

wherein R° = H, alkyl (C C₃), COOR 1^ι0^υ, R⁶ = H, alkyl (C1-C3), COOR¹⁰ ,

R⁷ = H, alkyl (CrC₃), COOR¹⁰,

R¹⁰ = H, alkyl (C C₃),

R⁸ = H, alkyl (C C₃), cycloalkyl (Cg-Cβ),

R⁹ = H, alkyl (Cι-C₃), cycloalkyl (C₃-C₆),

R¹¹ = H, alkyl (C₁-C₃), benzyl,

X = CH, 0,S,

Y= NR¹², O, S,

R¹²= H, COCH3, alkyl (C C₃);

R is a residue of the formula

ccr tsnr

or a residue of the formula

H ci^" in the case of R¹ = f The invention also relates to their pharmaceutically acceptable salts with an anticoagulant activity and to pharmaceutical compositions containing them.

The pharmaceutically acceptable salts of the compounds of formula I preferably include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, hydrobromide, hydrochloride, hydroiodide, lactate, maleate, methanesulfonate, nicitinate, nitrate, oxalate, pamoate, 3-phenylpropionate, picrate, pivalate, propionate, pectinate, succinate, sulfate, tartrate, thiocyanate, tosylate, and audecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, N-methyl-D-glucamine and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, dialkylsulfates, and diamylsulfates, long chain halides, aralkyl halides and others. The selection of the inorganic or organic acids or bases should not be restricted by these examples.

The invention also relates to a process for the preparation of azaphenylalanine derivatives and analogs of the general formula (I).

Azaphenylalanine derivatives and analogs of the general formula (I), when

R¹= ' , are preferably prepared as follows:

4-cyanobenzaldehyde of formula (II)

is converted with BOC-carbazate of the formula (III)

to the compound of the formula (IV)

which by subjecting to reduction by catalytic hydrogenation, using Pd as a catalyst, is converted to the compound (V)

which reacts with triphosgene and amine of the formula (VI)

wherein R⁵ , R⁶ and R⁷ have the same meanings as in the formula (I), or with triphosgene and amine of the formula (VII or VIII),

(vii) (VII) wherein Y has the same meaning as in the formula (I), or with triphosgene and amine of the formula (IX)

R8

HN.

^^R9 (IX) wherein R⁸ in R⁹ have the same meanings as in the formula (I), or with triphosgene and amine of the formula (X), COOR

(X) wherein R¹¹ and X have the same meanings as in the formula (I), to the compound (XI)

wherein R² has the same meaning as in the formula (I).

The BOC protecting group in the compound (XI) is removed at room temperature using the influx of gaseous hydrogen chloride in acetic acid to obtain compound (XII),

wherein R² has the same meaning as in the formula (I).

Then the compound (XII) reacts with the activated naphthylsulfonyl aminoacid or with activated arylalkylcarboxylyc acid up to the compound (XIII)

wherein R² in R³ have the same meanings as in the formula (I).

Compound (XIII) is transformed with hydroxylamine in absolute ethanol up to the compound with formula (XIV) wherein R¹=

e meanings as in the formula (I) and R⁴ is OH group.

The compound (XIII) is converted with an influx of gaseous hydrogen chloride in ethanolic solution, addition of ammonium acetate, followed by another influx of hydrogen chloride to the compound (XV),

H-N. J.R⁴ wherein R = R and R have the same meanings as in the formula (I)

and R⁴ is hydrogen.

,NH cf

Azaphenylalanine derivatives of the general formula (I), wherein R¹= ' are prepared as follows:

4-cyanobenzaldehyde of (II)

is converted with ethyleneglycol in presence of 4-toluenesulfonic acid into the compound (XVI)

which is subjected to reduction with lithium aluminium hydride into the compound of formula (XVII)

which reacts with acetanhydride up to the compound (XVIII)

CH '!-"N'HCOCH-

(XVIII)

The compound (XVIII) is converted with 90% methanoic acid to the compound (XIX)

which is converted with BOC-carbazate of the formula (111)

to the compound of the formula (XX)

which by subjecting to reduction by catalytic hydrogenation is converted to the compound (XXI)

which reacts with triphosgene and amine of the formula (VI)

(Vii) (viii) wherein Y has the same meaning as in the formula (I), or with triphosgene and amine of the formula (IX)

wherein R⁸ in R⁹ have the same meanings as in the formula (I), or with triphosgene and amine of the formula (X),

(X) wherein R¹¹ and X have the same meanings as in the formula (I), to the compound (XXII)

wherein R has the same meaning as in the formula (I). The protecting BOC group in the compound (XXII) is removed with HCI (g) in AcOH at room temperature to obtain the compound (XXIII),

⁽x iii⁾

wherein R² has the same meaning as in the formula (I), which reacts with aromatic sulfonylchloride up to the compound (XXIV)

wherein R ,2 and , r R-»3 have the same meanings as in the formula (I).

The compound (XXIV) by heating to boiling with 5M HCI is converted to the compound (XXV)

,NH ci

( ³ wherein R¹= ' , R² and R³ have the same meanings as in the formula (I).

The compounds wherein R¹ is H and Z is

H-N

are prepared in analogous way as described above with the sole exception that the starting compound is 3-cyanobenzaldehyde instead of 4-cyanobenzaldehyde.

The starting compounds may be prepared, unless otherwise directed, according to the procedures described in the literature; e.g., the compound of the formula IV as described by A. Fassler, et. al., J. Med. Chem. 1996, 39, 3203-3215.

The invention further relates to the use of compounds of the formula I for the production of pharmaceutical compositions used as oral and parenteral anticoagulants e.g. mostly inhibitors of thrombine and dual inhibitors of thrombine and factor Xa. They may be useful in the treatment and prevention of a variety of thrombosis forms: (i) venous thromboembolism due to formation of a thrombus within a vein {venous thrombosis) associated with acquired (prolonged bedrest, surgery, injury, malignancy, pregnancy and postpartum states) or inherited risk factors (deficiency of natural coagulation inhibitors), obstruction or occlusion of a lung artery by a detached thrombus {pulmonary embolism), (ii) cardiogenic thromboembolism due to formation of a thrombus in the heart associated with cardiac arrhythmia, heart valve defect, prosthetic heart valves or heart disease, embolism of peripheral arteries caused by a detached thrombus, most commonly in the brain {ischemic stroke), (iii) arterial thrombosis due to underlying atherosclerotic processes in the arteries which obstructs or occludes an artery and causes myocardial ischemia {angina pectoris, acute coronary syndrome) or heart muscle cell death {myocardial infarction), obstructs or occludes a peripheral artery {ischemic peripheral artery disease) and obstructs or occludes the artery after the procedure on the blood vessel {reocclusion or restenosis after transluminal coronary angioplasty, reocclusion or restenosis after percutaneous transluminal angioplasty of peripheral arteries) and (iv) in the number of states (e.g., in complications in pregnancy, in metastasing malignant diseases, after extensive injuries, in bacterial sepsis) when thrombogenic activation causes widespread formation of thrombi within the vascular system {disseminated intravascular coagulation). The selection of the possible uses of the produced pharmaceutical compositions should not be restricted by these examples.

The compounds of the formula I may be also used as an adjunct therapy in conjunction with thrombolytic therapy in recent myocardial infarction, in combination with acetylsalicylic acid in patients with unstable angina pectoris designed to undergo percutaneous transluminal angioplasty and in the treatment of patients with thrombosis and with heparin-induced thrombocytopenia.

The anticoagulants may further be used for the prevention of blood coagulation which is in contact with nonbiological surfaces (vascular prosthesis, vascular stents, prosthetic heart valves, extracorporeal circulation systems, hemodialysis) and in vitro to prevent coagulation in biological samples for testing or storage.

Further an object of the present invention is to provide pharmaceutical compositions comprising the compounds of the formula I. They may be formulated as injectable or oral formulations. In addition to the active ingredient they may preferably contain different standard additives depending on the use. The pharmaceutical compositions are prepared according to the standard procedures. The preparation may be formulated in such a manner as to permit controlled and sustained release of the active ingredient. Dosage, frequency and mode of administration depend on a variety of factors, they also depend on individual active ingredient and its pharmacokinetic parameters and on patient's condition.

BIOLOGICAL TESTS I. Enzyme assay

1. Determination of the activity of thrombin inhibitors a) Principle

Thrombin cleaves amide bonds in a synthetic chromogenic substrate wherein yellow coloured p-nitroaniline (p-NA) was released. The amount of p-NA produced is directly proportionate to the absorbance measured at a wavelength of 405 nm using a spectrophotometer. When thrombin inhibitor is added, the amidolytic activity of the enzyme decreases. The effect of the inhibitor is expressed by the inhibition constant (Ki).

a) Reagents

Thrombin (human thrombin, 308 NIH units, Sigma): the contents of the vial are dissolved in distilled water to give a stock solution of 20 NIH units/ml. The stock solution is pipetted into 0.5 ml aliquots and stored at -70°C. Immediately before use, a working solution of thrombin of 2 NIH units/ml activity is prepared with HBSA buffer. The final concentration of thrombin in a microtiter plate is 0.5 NIH units/ml.

Chromogenic substrate for thrombin (S-2238, Chromogenix, 25 mg). 1 mM substrate solution is prepared, pipetted into 0.5 ml aliquots and stored at -20°C. Before use, 160 and 80 μM substrate solutions are prepared with distilled water. The final concentrations of the substrate in the reaction mixture are 40 and 20 μM, respectively (K_m = 2.6 μM).

HBSA buffer, pH 7.5: 10 mM Hepes buffer (HEPES, Sigma), 150 mM NaCI and 0.1% (w/v) bovine serum albumin (98% bovine serum albumin, Sigma) are dissolved in bidistilled water. The pH is adjusted with 0.1 M NaOH solution.

Inhibitors: The inhibitors are dissolved in DMSO to give a 10 mM stock solution. Working solutions (final concentrations within the range 10 to 100 μM) are prepared with distilled water. The highest concentration of DMSO in a microtiter plate does not exceed 3%. c) Procedure

Measurements are carried out in the microtiter plate. 50 μl of HBSA buffer, 50 μl of inhibitor solution of different concentrations (for control 50 μl of HBSA buffer) and 50 μl of thrombin solution are pipetted into the wells of a microtiter plate. The plate is incubated at a temperature of 25°C for 15 minutes. After incubation 50 μL of the chromogenic substrate is added and the microtiter plate is placed in the spectrophotometer (Tecan, Sunrise). The absorbance increase at 405 nM is measured at 10-second intervals for a period of 15 minutes at a temperature of 25° C.

For determination of the inhibition constant (Kj) 40 and 20 μM substrate is used. Each measurement is carried out in triplicate and the result is the averaged value of three measurements.

2. Determination of the inhibition constant ( Kj )

Kj is determined according to the principle, described by Cheng and Prusoff (Biochem Pharmacol, 1973). Initial velocities of the reaction in the presence and absence of the inhibitor are measured. The change in the absorbance in the time unit {v) is calculated from the initial, linear part of the reaction. For competitive inhibitors it holds that v,. _ K„ +S κ_M + /κ,))+s and it follows that

K_s =

((S/ + l ((v₀/v, )-l)

I = inhibitor concentration, S = substrate concentration, K_m = Michaelis constant, v₀ = initial velocity of the reaction in the absence of inhibitor, Vj = initial velocity of the reaction in the presence of inhibitor. Measurements are carried out with two concentrations of the inhibitor and two concentrations of the substrate. For each combination of the used concentrations of the substrate and the inhibitor, Kj is calculated and the result is their averaged value.

3. Determination of selectivity of the inhibitor activity against thrombin with respect to trypsin inhibition

a) Principle

Because thrombin and trypsin are closely related with respect to the specificity against the substrate due to comparable structure of the active site, the selectivity of inhibitory activity against thrombin is determined with respect to trypsin inhibition which is nonspecific serine protease. The inhibitory activity against thrombin is determined as described above. Trypsin inhibition is measured in the same manner as in determination of inhibitory activity for thrombin except that a different chromogenic substrate is used. For both enzymes Kj is calculated. Selectivity of the inhibitor is expressed as a ratio of Kj for trypsin to Kj for thrombin.

b) Reagents

Trypsin (bovine, 6000 BAEE Units/mg protein, Sigma): A stock solution of trypsin with the activity of 300 U/ml is prepared, pipetted into 0.2 ml aliquots and stored at -70°C. Immediately before use, the stock solution is thawed and a working solution of 4 mU/ml is prepared with HBSA buffer. The final trypsin activity in a microtiter plate is 1 mU/ml.

Chromogenic substrate for trypsin (S-2222, Chromogenix, 25 mg): 2 mM substrate solution is prepared, pipetted into 0.3 ml aliquots and stored at -20°C. Before use the stock solution is thawed and 400 and 200 μM substrate solutions are prepared The final concentrations of the substrate in the reaction mixture are 100 and 50 μ M, respectively (K_m = 25 μM)

Inhibitor: The inhibitors are dissolved in DMSO to give a 10 mM stock solution. Working solutions (final concentrations in the range from 10 to 600 μM) are prepared with distilled water. The highest concentration of DMSO in a microtiter plate does not exceed 10%.

For determination of Kj 100 and 50 μM substrate is used. Each measurement is carried out in triplicate and the result is the averaged value of three measurements.

c) Procedure

The same procedure as outlined above for measurement of the inhibitory activity against thrombin is used. The concentrations of the reagents described for determination of the inhibitory activity with respect to trypsin are used.

d) Determination of the inhibition constant ( Kj )

It is determined in the same manner as in determination of Kj for thrombin.

e) Determination of the selectivity

Kj for thrombin and Kj for trypsin are determined. The selectivity is defined as the ratio:

K. (trypsin) selectivity = — -

K. (thrombin) 4. Determination of selectivity of the inhibitor activity against thrombin with respect to inhibition of factor Xa

a) Principle

Because both enzymes are closely related and of comparable structure of the active site, the selectivity of inhibitory activity against thrombin is determined with respect to inhibition of factor Xa. The inhibition is of FXa is measured in the same manner as in measurement of the inhibition of thrombin with chromogenic substrate S2238. For both enzymes Kj is calculated. Selectivity of the inhibitor is expressed as a ratio of Kj for FXa to Kj for thrombin.

b) Reagents

Factor Xa (Chromogenix, 71 nkat): The contents of the vial are dissolved in distilled water to give a stock solution of 10 nkat ml. The stock solution is pipetted into 0.5 ml aliquots and stored at -20°C. Immediately before use, FXa working solution having the activity of 2 nkat/ml is prepared with HBSA buffer. The final FXa activity in a microtiter plate is 0.5 nkat ml.

Chromogenic substrate for FXa (S-2222, Chromogenix, 25 mg): 2 mM substrate solution is prepared, pipetted into 0.5 ml aliquots and stored at -20°C. Before use 800 and 400 μM substrate solutions are prepared with distilled water. The final concentrations of the substrate in the reaction mixture are 200 and 50 μM, respectively (K_m = 25 μM)

HBSA buffer, pH 7.5: the same as in the method for thrombin.

Inhibitor: The inhibitors are dissolved in DMSO to give a 10 mM stock solution. Working solutions (final concentrations in the range from 5 to 300 μM) are prepared with distilled water. The highest concentration of DMSO in a microtiter plate does not exceed 3%.

c) Procedure The same procedure as outlined bove for measurement of the inhibitory activity against thrombin is used. The concentrations of the reagents described for determination of the inhibitory activity with respect to FXa are used.

d) Determination of the inhibition constant ( Kj )

It is determined in the same manner as in determination of Kj for thrombin.

e) Determination of the selectivity

Kj for thrombin and Ki for FXa are determined. The selectivity is defined as a ratio:

II. Coagulation assay

Clotting time of normal pooled plasma is measured by the coagulation assays (thrombin time, activated partial thromboplastin time and prothrombin time) in an coagulometer after addition of different concentrations of the inhibitor. The results are presented as the inhibitor concentration that doubled the clotting time.

1. Thrombin time (TT) a) Principle

Determination of the thrombin time is used for laboratory monitoring of the treatment with nonfractionated heparin, for monitoring of thrombolytic therapy, for detecting disorders in fibrin formation and for diagnosing severe forms of fibrinogen deficiency. Thrombin time is prolonged due to reduced fibribogen concentration, the presence of fibrinogen degradation products or thrombin inhibitors in plasma. For the procedure, thrombin is added to plasma. Thrombin converts fibrinogen into fibrin and the time for the clot formation is measured. b) Reagents

Thrombin (Test Thrombin Reagent, 1.5 lU/ml): lyophilised bovine thrombin is dissolved in 5 ml of HEPES buffer ( 25 nM, pH 7.4). saline to a concentration 2

U/ml. Normal pooled plasma: Venous blood from at least 10 apparently healthy volunteers is collected in 0.11 M sodium citrate solution (1 part of sodium citrate and 9 parts of blood). Immediately after withdrawal the blood is centrifuged at 2000 x g for 30 minutes at 4°C. Plasma is removed, pipetted into 2-ml aliquots and stored at -70

°C. Inhibitors: The inhibitors are dissolved in DMSO (10 mM stock solution) and diluted with distilled water to give working solutions (the highest concentration 100 μM).

b) Procedure

Pipette 90 μl of plasma and 10 μl of the inhibitor into a cuvette of the coagulometer (Fribrintime, Dade/Behring), pre-warmed at 37°C. Incubate at 37°C for 5 minutes. Add 200 μl of the thrombin solution, pre-warmed at 37°C. After thrombin addition, start the timer and measure the time of clot formation.

2. Activated partial thromboplastin time (aPTT) a) Principle aPTT is used as a screening test for coagulation disorders of the intrinsic coagulation pathway. The method is sensitive to deficit of coagulation factors VIII and IX and contact factors. The aPTT is prolonged due to the deficit of the factors or due to the presence of inhibitors (e.g., lupus anticoagulants, heparin). Incubation of plasma with the optimal quantity of phospholipids and a contact activator leads to activation of the factors of the intrinsic coagulation pathway. The addition of calcium ions triggers the coagulation process. The time of fibrin clot formation is measured.

b) Reagents

Phospholipids with an activator (Pathrombin SL, Dade/Behring): silicon dioxide particles, vegetable phospholipids, sodium chloride (2.4 g/l), HEPES (14.3 g/l), pH 7.6 and sodium azide (<1 g/l). Before use, the reagent must have room temperature and be well agitated. Calcium chloride solution: 0.025 mol/l Normal pooled plasma: in the same as for thrombin time. Inhibitors: in the same as for thrombin time.

c) Procedure

Pipette 90 μl of plasma and 10 μl of the inhibitor into a cuvette of the coagulometer (Fribrintime, Dade/Behring), pre-warmed at 37°C. Incubate at 37°C for 5 minutes. Add 100 μl of phospholipids with an activator. Incubate at 37°C for 2 minutes. Add 100 μl of calcium chloride at 37°C. After addition of calcium chloride, measure the time of clot formation

3. Prothrombin time (PT) a) Principle

Prothrombin time is a rapid, sensitive screening test for determination of coagulation disorders of the intrinsic pathway (factors II, V, VII, and X). Owing to high sensitivity to these coagulation factors, the test is well suited for monitoring of oral anticoagulant therapy, for diagnosing genetic and acquired deficiencies in coagulation factors and checking the liver for its synthesis performance. Prothrombin time is prolonged due to the coagulation factors deficit or due to the presence of inhibitors. For the procedure, optimal amount of thromboplastin and calcium are added to plasma and the time of clot formation is measured.

b) Reagents

Thromboplastin (Thromborel S, Dade/Behring): human placental thromboplastin with calcium chloride and stabilising agents; dissolve in 4 ml of distilled water. Before use, warm the reagent to 37°C at least for 30 minutes. Normal pooled plasma: in the same as for thrombin time. Inhibitors: in the same as for thrombin time. c) Procedure

Pipette 90 μl of plasma and 10 μl of the inhibitor into a cuvette of the coagulometer

(Fribrintime, Dade/Behring), pre-warmed at 37°C. Incubate at 37°C for 5 minutes.

Add 200 μl of thromboplastin, pre-warmed at 37°C. After addition of thromboplastin, measure the time of clot formation

The invention is illustrated but in no way limited by the following examples.

EXAMPLE 1

Λ/-{2-[2-(1-azepanyIcarbonyl)-2-(4-cyanobenzyl)hydrazino]-2-oxoethyl}-2- naphthalenesulfonamide

1.00 g (3.77 mmol) 2-[(2-naphthylsulfonyl)amino]acetic acid was dissolved in 20 ml dichloromethane and while stirring 420 mg (3.87 mmol) of ethyl chloroformiate and

516 mg (4.00 mmol) of N-ethyl-N,N-diisopropylamine were added. The reaction mixture was stirred at room temperature for 1 hour, followed by the addition of powdered 2-(1-azepanyIcarbonyl)-2-(4-cyanobenzyl)hydrazine chloride (1.00 g; 3.24 mmol). After addition, the mixture was stirred at room temperature for 3 days.

The solvent was removed on a rotavapor, the residue was dissolved in 50 ml of ethylacetate, and then washed with 1M HCI, 1M NaOH and distilled water. The organic phase was dried over sodium sulphate and the solvent was evaporated in vacuo. The crude product was re-crystallised from ethanol.

Yield: 1.34 g (79%)

Melting point: 149-151 °C

IR (KBr, cm^"1): 3410, 3253, 2919, 2232, 1708, 1613, 1440, 1343, 1156, 659

¹H-NMR (DMSO-de): δ (ppm) 1.46 (m, 8H, CH₂-azepine₃',4',5'.6'); 1,65 (m, 4H, CH₂- azepine₂',7'); 3.33 (m, 2H, CO-CH₂-NH); 4.09 (m, 1H, NHCO); 4.47 (s, 2H,.Ar- Cl±.),

7.58 and 7.79 (2d, 2H each, J₂,₆= 8.27, J₃,₅=8.27, Ar-Ha.g.s.e); 7.69-8.47 (m, 7H, Ar-H

(naphthalene)); 10.29 (s, 1H NHSO₂)

Molecular mass: calculated: 519 ( ₂₇H₂₉N₅O₄S); Found: 520 ( MH ⁺)

EXAMPLE 2 4-[(1 -(1 -azepanylcarbonyl)-2-{2-[(1 -naphthylsulfonyl)amino]acetyl}hydrazino)methyl]- Λf-hydroxybenzenecarboxamide

Λ/-{2-[2-(1-azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazino]-2-oxoethyl}-2- naphthalenesulfonamide (400 mg. 0.77 mmol) was dissolved in anhydrous ethanol, and hydroxylamine (28.0 mg, 0.85 mmol) was added. The reaction mixture was heated at boiling point for 12 hours.

The solvent was removed on a rotavapor and the product was washed with ether.

Yield: 386 mg (87%)

Melting point: 104-108 °C

IR (KBr, cm^"1): 3359, 2624, 1782, 1640, 1422, 1345, 1157, 1075, 750, 661

¹H-NMR (CDCI₃):δ (ppm) 1.49-1.89 (m, 8H, CHa-azepinesV.δ'.β); 2.07 (m, 4H, CH₂- azepine_2>7'); 3.48 (m, 2H, CO-Cife-NH); 4.18 (s, 1H, NJjCO); 4.68 (m, 2H, Ar- CHh),

4.90 (s, 1H, =N-OH); 7.42 and 7.61 (2d, 2H each, J_2>6= 8.17, J_3l5=8.15. Ar-H^s.e);

7.64-8.34 (m, 7H, Ar-H (naphthalene)); 8.42 (s, 2H, Nϋ-amidoxime); 10.35 (s, 1H

NHSO₂)

Molecular mass: Calculated 552 (C₂₇H3₂N₆O₅S), Found 553 ( MH ⁺)

EXAMPLE 3

Amino{4-[(1 -(1 -azepanylcarbonyl)-2-{2-[(1 -naphthylsulfonyl)amino]acetyl} hydrazino)methyl]phenyl}methanamine hydrochloride

/V-{2-[2-(1-azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazino]-2-oxoethyl}-2- naphthalenensulfonamide (520mg, 1.00 mmol) was dissolved in 25 ml of anhydrous ethanol and the gaseous HCI was bubbled in for 20 minutes. After completed bubbling the solution was stirred at room temperature for 4 hours, followed by the addition of ammonium acetate (85.0 mg, 1.10 mmol). The reaction mixture was left at room temperature for 2 days. The gaseous HCI was re-bubbled in for 20 minutes. After 1 hour, the formed ammonium chloride was filtered off and ethanol was evaporated on a rotavapor. The product was dissolved in ether and filtered off by suction. Yield: 481 mg (84%) IR (KBr, cm^"1): 2969, 2758, 1783, 1669, 1344, 1158, 1076, 749, 660,

¹H-NMR (DMSO-de): δ (ppm) 1.57-1.76 (m, 8H,

(m, 4H,

CH2-azepine₂',₇'); 3.37 (m, 2H, CO-Chfe-NH); 4.31 (m, 1H, NHCO); 4.74 (s, 2H, Ar-

Chb), 7.62 and 7.90 (2d, 2H each, J_2>6= 8.14, J_3>5=8.19, Ar-H^s.e); 7.35-8.49 (m, 7H,

Ar-JH (naphthalene)); 9.30 (s, 1H NHS0₂); 9.42 (2s, 2H, amidine)

Molecular mass: Calculated: 537 (C₂₇H33N₆O4S); Found: 538 ( MH ⁺)

EXAMPLE 4

Ethyl 2-[2-(1 -azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazino]-1 -benzyl-2- oxoethylcarbamate

1.10 g (4.64 mmol) /V-(ethoxycarbonyl)phenylalanine was dissolved in dichloromethane (25 mL) and 0.50 mL (5.18 mmol) ethylchloroformiate was added dropwise. The mixture was stirred for 30 minutes at room temperature followed by the addition of DIEA (2 mL) and 1.35 g (4.48 mmol) 2-(1-azepanylcarbonyl)-2-(4- cyanobenzyl)hydrazinium chloride. The reaction mixture was stirred overnight, then the solvent was evaporated in vacuo and the residue dissolved in ethylacetate (50 mL). The solution was extracted with 2x25 mL 10% citric acid and 25 mL aqueous

NaHC0₃ and washed with water and brine. Organic phase was dried over Na₂S0₄ and the solvent was removed under reduced pressure. Crude product was further purified with column chromatography (dichloromethane/methanol 20:1).

Yield: 438 mg (22%)

Melting point: 133-137⁹C

IR (KBr, cm^"1) 3321 , 2950, 2225, 1666, 1531 , 1421 , 1289, 1043, 757, 559

H-NMR (CDCIs): δ (ppm) 1.21 (t, 3H, J=6.78 Hz, CH₂CH₃), 1.57 (m, 4H, Cbk), 1.75

(m, 4H, Chb), 3.01 (m, 2H, Ar-CH?), 3.43 (t, 4H, J=5.80 Hz, Ct ), 4.13 (t, 3H, J=6.73

Hz, CfcfeCHg), 4.41 (s, 2H, Ar-CHp). 4.53 (s, 1H, CH), 5.04 (s, 1H, J\}HCOOEt), 6.48

(s, 1 H, N-NHCO), 7.29 (m, 5H, Ar-H), 7.51 (d, 2H, J=8.20 Hz, Ar-H^3,5), 7.66 (d, 2H,

J=8.33 Hz, Ar-H²⁶)

Molecular mass : Calculated: 491 ( ₂₇H₃3N₅O4); Found: 492 (MH⁺)

EXAMPLE 5 Ethyl 2-[2-{4-[amino(imino)methyl]benzyl}-2-(1 -azepanylcarbonyl) hydrazino]-1 - benzyl-2-oxoethylcarbamate

Ethyl 2-[2-(1 -azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazino]-1 -benzyl-2- oxoethylcarbamate was suspended in absolute ethanol (20 mL) and the gaseous hydrogen chloride was bubbled in for half an hour. Reaction mixture was left at room temperature for four hours, then the solvent was removed in vacuo. The residue was washed with diethylether (2x20 mL) and dissolved in absolute ethanol. The solution was treated with gaseous ammonia for 10 minutes and ethanol was removed under reduced pressure.

EXAMPLE 6

[Amino(4-{[2-(2-ammonio-3-phenylpropanoyl)-1 -(1 - azepanylcarbonyl)hydrazino]methyl}phenyl)methylene]ammonium dichloride

The crude product of the previous stage was dissolved in 1 M HCI (30 mL) and was refluxed for 2 hours. The water was removed under reduced pressure and the residue was purified with column chromatography (ethylacetate/methanol 2:1).

Yield: 222 mg (49%)

Melting point: 221-223^QC

IR (KBr, cm^"1) 3232, 2940, 2229, 1784, 1716, 1538, 1403, 1257, 1054, 959, 823,

752, 701

H-NMR (CDCI3): δ (ppm) 1.24 (t, 3H, J=6.76 Hz, CH CH₃), 1.49 (m, 4H, CH_), 1.68

(m, 4H, Chfe), 3.03 (m, 2H, Ar-CHp), 3.39 (t, 4H, J=5.97 Hz, ⁽____), 4.12 (t, 3H, J=6.72

Hz, CHPCHS), 4.43 (s, 2H, Ar-CHp). 4.62 (s, 1 H, CH), 5.01 (s, 1 H, NHCOOEt), 6.45

(s, 1 H, N-NHCO), 7.26 (m, 5H, Ar-H), 7.53 (d, 2H, J=8.17 Hz, Ar-H^3,5), 7.68 (d, 2H,

J=8.29 Hz, Ar-H^2,6), 9.29 (s, 4H, HPN-C=NHP⁺)

Molecular mass : Calculated: 509 (C₂₄H₃₄N₆O₂CI₂); Found: 437 ((M-2HCI)H⁺)

EXAMPLE 7

Λ/^I-(1-azepanylcarbonyl)-Λ/'-(4-cyanobenzyl)-1-(2-naphthylsulfonyl)-2- pyrrolidinecarbohydrazide 2.20 g (7.21 mmol) 1 -(2-naphthylsulfonyl)proline was dissolved in 35 ml of dichloromethane and while stirring 815 mg (7.50 mmol) of ethyl chloroformiate and 1.20 g (9.30 mmol) of N-ethyl-N,N-diisopropylamine were added. The reaction mixture was stirred at room temperature for 1 hour, followed by the addition of powdered 2-(1-azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazine chloride (2.00 g, 6.48 mmol). After the addition, it was further stirred at room temperature for 3 days. The solvent was removed on a rotavapor and the residue was dissolved in 50 ml of ethylacetate, followed by washing with 10% citric acid, 5% NaHC0₃ solution and distilled water. The organic phase was dried over sodium sulfate and the solvent was evaporated in vacuo. The crude product was re-precipitated from acetone.

Yield: 0.82 g (23%)

Melting point: 158-159°C

IR (KBr, cm^"1) 3461 , 2234, 1708, 1614, 1527, 1428, 1342, 1156, 1082, 1015 759,

662, 548_

H-NMR (DMSO-de): δ (ppm) 1.47 (s, 4H, CH>), 1.64 (s, 4H, CH_), 1.75 (m, 4H, CHP).

3.24 (m, 4H, _____), 3.24 (m,2H, ⁽____), 4.12 (m, 1H, CH), 4.47 (s, 2H, Ar-Chfe), 7.57 (d,

2H, J=8.29 Hz, Ar-H³'⁵), 7.71 (dqu, 2H, J₁₌7.16 Hz, J₂=1.50 Hz, Ar-H), 7.80 (d, 2H,

J=8.29 Hz, Ar-H²'⁶), 7.84 (dd, 1 H, Jι=8.67 Hz, J₂=1.89 Hz, Ar-H), 8.07 (d, 1H, J=7.91

Hz, Ar-H), 8.15 (m, 2H, Ar-H), 8.47 (s, 1H, Ar-H), 10.29 (s,1 H, J\MH)

Molecular mass: Calculated: 559 (C₃oH₃₃N₅θ4S); Found: 560 ( MH ⁺)

EXAMPLE 8

Amino{4-[(1 -(1 -azepanylcarbonyl)-2-{[1 -(2-naphthylsulfonyl)-2- pirrolidinyl]carbonyl}hydrazino)methyl]phenyl}methanamine hydrochloride

/V-(1 -azepanylcarbonyl)-V-(4-cyanobenzyl)-1-(2-naphthylsulfonyl)-2- pirrolidinecarbohydrazide (754 mg, 1.35 mmol) was dissolved in 25 ml of anhydrous ethanol and the gaseous HCI was bubbled in for 20 minutes. After completed bubbling, the solution was stirred at room temperature for 4 hours, followed by the addition of ammonium acetate (115.0 mg, 1.50 mmol). The reaction mixture was aged at room temperature for 2 days. The gaseous HCI was re-bubbled for 20 minutes. After 1 hour, ammonium chloride formed was filtered and ethanol evaporated on a rotavapor. The product was dissolved in ether and filtered off by suction.

Yield: 320 mg (40%)

Melting point: 123-127°C

IR (KBr, cm^"1): 3034, 1781 , 1681 , 1602, 1402, 1347, 1158, 1076, 1010, 823, 748,

660

¹H-NMR (DMSO-de): δ (ppm) 1.57 (m, 4H, Chb-azepine^s'); 1.76 (m, 4H, Chfe- azepinejt'.e'); 2.03 (m, 4H, CJhfe-proline); 3.02 (m, 4H, Cϋ-azepine^.r); 3.32 (m, 2H,

CjHg-proline); 3.63 (q, 1H, J=3.63 Hz, CH^-proline); 4.96 (s, 2H, Ar- Cji), 7.52 (d, 2H,

J=8.18 Hz, Ar-H³'⁵), 7.68 (dqu, 2H, J₁₌7.14 Hz, J₂=1.67 Hz, Ar-H), 7.81 (d, 2H,

J=8.18 Hz, Ar-H²'⁶), 7.93 (dd, 1H, ^=8.59 Hz, J₂=2.11 Hz, Ar-H), 8.12 (m, 3H, Ar-H),

8.49 (s, 1H, Ar-H), 9.41 (s, 4H, NH_.-C=NH₂ ⁺) 9.59 (s,1H, NH)

Molecular mass: Calculated: 599 (C₃oH₃7N₅0₄SCI); Found: 577 (( M-HCI)H⁺)

EXAMPLE 9 /V-(1-azepanylcarbonyl)-/V-(4-cyanobenzyl)-2-(2-naphthyloxy)acetohydrazide

750 mg (3.71 mmol) of 2-(2-naphthyloxy)acetic acid and 1.00 g (3.24 mmol) of 2-(1- azepanylcarbonyl)-2-(4-cyanobenzyl)hydrazine chloride were dissolved in 15 ml of dimethylformamide. While stirring 500 mg (3.70 mmol) of 1-hydroxy-benzotriazole and 748 mg (3.90 mmol) of EDC were added. The reaction mixture was stirred at room temperature for 1 hour.

The solvent was removed on a rotavapor and the residue was dissolved in 50 ml of ethylacetate, followed by washing with 10% of citric acid, 5% NaHC0₃ solution and distilled water. The organic phase was dried over sodium sulfate and the solvent was evaporated in vacuo. The crude product was purified by column chromatography (mobile phase: dichloromethane:methanol 9:1).

Yield: 963 mg (63%) Melting point: 142-144°C

IR (KBr, cm^"1) 3228, 2925, 2854, 2227, 1630, 1509, 1426, 1216, 1120, 960, 838,

748, 547_

H-NMR (CDCI₃): δ (ppm) 1.55 (m, 4H,

1.75 (m, 4H, C_λ_- azepine₃',6'); 3.43 (t, 4H, J=5.86 Hz, CH₂-azepine₂-,₇'); 4.54 (s, 2H, CjHg); 4.68 (s, 2H,

Ar-Chfe); 7.06 (s,1H, NH); 7.34 (d, 2H, J=8.40 Hz, Ar-H^3,5); 7.39-7.58 (m, 3H, Ar-H);

7.47 (d, 2H, J=8.40 Hz, Ar-H²'⁶); 7.68-7.84 (m, 3H, Ar-H); 8.14 (s, 1H, Ar-H)

Molecular mass: Calculated: 456 (C₂7H₂₈N₄0₃); Found: 457 ( MH ⁺)

EXAMPLE 10: fe/f-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-[(4-methyl-1 -piperidinyl)carbonyl]-1 - hydrazinecarboxylate

l.26g (4.39 mmol) of bis-trichloromethylcarbonate was dissolved in 20 ml of dichloromethane and the solution was degassed with argon. 2.50 g (8.53 mmol) of fe/f-butyl 2-{4-[(acetylamino)methyl]benzyl}-1 -hydrazinecarboxylate was dissolved in 30 ml dichloromethane, 1.65 g (12.79 mmol) of N-diisopropyl-N-ethylamine was added and the mixture was added dropwise to the solution of bis- trichloromethylcarbonate. While adding dropwise, the reaction mixture was cooled on an icewater bath. The mixture was stirred for additional half an hour at room temperature. 2.04 ml (25.60 mmol) of 4-methylpiperidine was added and stirred for another hour at room temperature. The reaction mixture was then extracted with 4 x 25 ml of 10% citric acid solution and 30 ml saturated NaHC0₃ solution, followed by washing with 30 ml of purified water and 20 ml of saturated brine. The organic phase was dried over Na₂SO₄. The solvent was evaporated in vacuo and the residue was dissolved in 10 ml of diethylether. A white precipitate was formed and filtered off by suction.

Yield: 2.10 g (59%)

Melting point: 124-126°C

IR (KBr, cm^"1) 3282, 2923, 2858, 1727, 1638, 1443, 1253, 1158, 1020, 978, 757, 611 H-NMR (CDCI₃): δ (ppm) 0.95 (d, 2H, J=6.40 Hz, pip-CH₃); 1.10-1.25 (m, 2H, pip- Chfe); 1.44 (s, 9H, C(CH₃)₃); 1.60- 1.73 (m, 3H, pip-CHp, pip-CH); 2.03 (s, 3H, CO- CH₃), 2.82 (m, 2H, pip-CHp): 3,92 (d, 2H, J=13.19 Hz, pip-CHp): 4.42 (d, 4H, J=5.65 Hz, CHp-Ar-CHp): 5.89 (s, 1 H, NH-CO); 6.27 (s, 1 H, J-COO); 7.27 (s, 4H, Ar-H) Molecular mass: Calculated: 418 (C₂₂H₃4N₄0₄); Found: 419 ( MH ⁺)

EXAMPLE 11 :

2-{4-[(acetylamino)methyl]benzyl}-2-[(4-methyl-1-piperidinyl)carbonyl]hydrazinium chloride

2.65 g (6.33 mmol) terf-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-[(4-methyl-1- piperidinyl) carbonyl]-1 -hydrazinecarboxylate was dissolved in acetic acid (20 ml) and the gaseous HCI was bubbled in for half an hour. The acid was evaporated in vacuo and diethylether was added to the residue. The flask wall was rubbed with a glass rod until a white powder was formed; meantime washing with diethylether was repeated twice.

Yield: 1.98 g (88%)

Melting point: 196-198°C

IR (KBr, cm^"1) 3419, 3256, 2926, 2685, 1689, 1552, 1434, 1235, 972, 728

H-NMR (CDCI₃): δ (ppm) 0.97 (d, 2H, J=6.03 Hz, pip-CH₃); 1.19 (m, 2H, pip-CHp):

1.52-1.70 (m, 3H, pip-CHp. pip-CH); 2.07 (s, 3H, CO-CH₃); 2.91 (t, 2H, J=12.43 Hz, pip-CHp): 3.96 (d, 2H, J=12,43 Hz, pip-Chk); 4.31 (d, 2H, J=4.15 Hz, Ar-CHp): 4.61

(s, 2H, Ar-CHp): 7.26 (d, 2H, J=7.91 Hz, Ar-H); 7.32 (d, 2H, J=7.92 Hz, Ar-H); 7.99 (s,

1H, JN1H-C0)

Molecular mass: Calculated: 354.5 (Ci₇H₂₇N₄0₂CI); Found: 319 ((M-HCI)H⁺, 100)

EXAMPLE 12:

N-(4-{[1 -[(4-methyl-1 -piperidinyl)carbonyl]-2-(1 -naphthylsulfonyl)hydrazino] methyl}benzyl)acetamide 1.98 g (5.59 mmol) of 2-{4-[(acetylamino)methyl]benzyl}-2-[(4-methyl-1- piperidinyl)carbonyl]hydrazine chloride and 1.39 g (6.13 mmol) of naphthalen-2- sulfonylchloride were dissolved in dichloromethane (25 ml), 2.16 g (16.7 mmol) of N- diisopropyl-N-ethylamine was added and stirred at room temperature for 3 days. The solution extracted with 4 x 25 ml of 10% citric acid and 30 ml of saturated NaHCO₃ solution. The organic phase was washed with 30 ml of purified water and 30 ml of brine, and dried over Na₂Sθ₄. Dichloromethane was evaporated in vacuo and a pale brown-yellow foamy solid was formed.

Yield: 1.89 g (67%)

Melting point: 80-83°C

IR (KBr, cm^"1) 3285, 2925, 1656, 1546, 1430, 1338, 1165, 970, 750, 554

H-NMR (CDCI₃): δ (ppm) 0.52 (d, 3H, J=6.03 Hz, pip-CH₃); 1.28 (m, 2H, pip-CHp):

1.64 (m, 3H, pip-CHp. pip-CH ); 2.05 (s, 3H, CO-CH₃); 2.32-2.71 (m, 2H, pip-CHp):

3.72 (m, 2H, pip-CHp): 4.32 (m, 2H, Ar-CHp): 4.41 (d, 2H, J=5.66 Hz, Ar-CH₉): 5.72

(s, 1 H, NH-CO); 7.16 (d, 2H, J=8.29 Hz, Ar-H); 7.22 (d, 2H, J=7.91 Hz, Ar-H); 7.46

(s, 1 H, naphth-H); 7.62 (m, 2H, 2xnaphth-H); 7.81 (dd, 1 H, J₁₌8.67 Hz, J₂=1.88 Hz, naphth-H); 7.94 (q, 3H, J=7.66 Hz, 3xnaphth-H); 8.45 (s, 1H, NH-S0₂)

Molecular mass: Calculated: 508 (C₂7H₃₂N₄O₄S); Found: 509( MH ⁺)

EXAMPLE 13:

(4-{[1 -[(4-methyl-1 -piperidinyl)carbonyl]-2-(1 -naphthylsulfonyl)hydrazino] methyl}phenyl)methanamine hydrochloride

1.89 g (3.72 mmol) of N-(4-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(1- naphthylsulfonyl)hydrazino]methyl}benzyl)acetamide was dissolved in 30 ml pre- warmed isopropyl alcohol (60°C). 30 ml of 4M HCI was added and the mixture was refluxed for 5 hours. The solvent was evaporated in vacuo. The product was purified by column chromatography; stationary phase: silicagel, mobile phase: dichloromethane : methanol (9:1).

Yield: 550 mg (29%) Melting point: 156-160°C

IR (KBr, cm^"1) 3285, 2925, 1656, 1546, 1430, 1338, 1165, 970, 750, 554

H-NMR (CDCI₃): δ (ppm) 0.17 (m, 2H, pip-CHp). 0.44 (d, 3H, J=6.03 Hz, pip-CH₃),

1.22 (d, 2H, J=10.54 Hz, pip-CHp. pip-CH), 2.27-2.74 (m, 2H, pip-CHp). 3.53 (s, 2H, pip-CHp). 3.97 (s, 2H, Ar-CHp), 4.32 (m, 2H, Ar-CHp). 7.22 (d, 2H, J=8.29 Hz, Ar-H),

7.42 (d, 2H, J=8.29 Hz, Ar-H), 7.64-7,75 (m, 3H, 3xnaphth-H), 7.77 (dd, 1H, J₁₌8.67

Hz, J₂=1,88 Hz, naphth-H), 7.81-8.00 (m, 1H, naphth-H), 8.02-8.18 (m, 2H,

2xnaphth-H), 8.46 (d, 1H, J=1.88 Hz, NH-S0₂)

Molecular mass: Calculated: 502.5 (C₂₅H₃ιN₄0₃SCI); Found: 467 ((M-HCI)H⁺)

EXAMPLE 14: fetf-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-(4-morpholinylcarbonyl)-1- hydrazinecarboxylate

1.26 g (4.39 mmol) of bis-trichloromethylcarbonate was dissolved in 20 ml of dichloromethane and the solution was degassed with argon. 2.50 g (8.53 mmol) of terf-butyl 2-{4-[(acetylamino)methyl]benzyl}-1 -hydrazinecarboxylate was dissolved in 30 ml of dichloromethane, 1.65 g (12.79 mmol) of N-diisopropyl-N-ethylamine was added and the mixture was added dropwise to the bis-trichloromethylcarbonate solution. While adding dropwise, the mixture was cooled on an icewater bath. The mixture was stirred for additional half an hour at room temperature. 2.23 ml (25.60 mmol) of morpholine was added and stirred for another hour at room temperature. The reaction mixture was then extracted with 4 x 25 ml of 10% citric acid solution, 30 ml of saturated NaHC0₃ solution, followed by washing with 30 ml of purified water and 20 ml of saturated brine. The organic phase was dried over Na₂S0₄. The solvent was evaporated in vacuo, and the residue was dissolved in 10 ml of diethylether. A white precipitate was formed which was filtered off by suction.

Yield: 1.81 g (52%)

Melting point: 125-128°C

IR (KBr, cm^"1) 3308, 2980, 2857, 1728, 1641 , 1430, 1289, 1114, 1025, 873, 746, 604 H-NMR (CDCI3): δ (ppm) 1.46 (s, 9H, C(CH₃)₃), 2.03 (s, 3H, CO-CH₃), 3.44 (t, 4H, J= 4.71 Hz, 2xmorph-CHp), 3.68 (t, 4H, J=4.71 Hz, 2xmorph-CHp). 4.43 (d, 2H, J=5.65 Hz, Ar-CHp). 4.49 (s, 2H, Ar-CH₂), 5.84 (d, 1 H, NJH-CO), 6.27 (s, 1 H, NH-COO), 7.27 (s, 4H, Ar-H) Molecular mass: Calculated: 406 (C₂oH₃₀N₄θ5); Found: 407 ( MH ⁺)

EXAMPLE 15: 2-{4-[(acetylamino)methyl]benzyl}-2-(4-morpholinylcarbonyl)hydrazinium chloride

1.81 g (4.46 mmol) of fe/f-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-(4- morpholinylcarbonyl)-1 -hydrazinecarboxylate was dissolved in acetic acid (20 ml) and the gaseous HCI was bubbled in for half an hour. The acid was evaporated in vacuo and the residue was dissolved in diethylether. The flask wall was rubbed with a glass rod until a white powder was formed; meantime washing with diethylether was repeated twice.

Yield: 0.92 g (60%)

Melting point: 209-211°C

IR (KBr, cm^"1) 3427, 1685, 1560, 1432, 1274, 1112, 1022, 892, 571

H-NMR (CDCI₃): δ (ppm) 1.88 (s, 3H, CO-CH₃), 3.46 (d, 4H, J=4.90 Hz, 2xmorph-

CHP). 3.61 (d, 4H, J=4.90 Hz, 2xmoph-CHp). 4.25 (d, 2H, J=6.03 Hz, Ar-CHp). 4.50

(s, 2H, Ar-CHp). 7.28 (s, 4H, Ar-H), 8.43 (s, NH-CO)

Molecular mass: Calculated: 342.5 (Cι₅H₂₃N₄0₃CI); Found: 307 ((M-HCI)H⁺, 100)

EXAMPLE 16:

N-(4-{[1-(4-morpholinylcarbonyl)-2-(1-naphthylsulfonyl)hydrazino] methyl}benzyl)acetamide

0.92 g (2.69 mmol) of 2-{4-[(acetylamino)methyl]benzyl}-2-(4- morpholinylcarbonyl)hydrazine chloride) and 0.67 g (2.95 mmol) naphthalen-2- sulfonylchloride were dissolved in dichloromethane (25 ml), 1.04 g (8.06 mmol) of diisoropylethylamine was added and stirred at room temperature for 3 days. The mixture was extracted with 4 x 25 ml of 10% citric acid and 30 ml of saturated NaHCO₃ solution. The organic phase was washed with 30 ml of purified water and 30 ml of saturated brine and dried over Na₂S0 . Dichloromethane was evaporated in vacuo and a pale brown-yellow foamy solid was formed.

Yield: 0.66 g (50%)

Melting point: 84-88°C

IR (KBr, cm^"1) 3426, 2856, 1655, 1420, 1340, 1274, 1166, 1114, 1024, 752, 547

H-NMR (CDCIs): δ (ppm) 2.05 (s, 3H, CH₃), 3.14 (s, 4H, 2xmorph-CHp). 3.33-3.40

(m, 2H, morph-CHp), 3.58-3.81 (m, 2H, morph-CHp., 4.30 (m, 2H, Ar-CHp). 4.41 (d,

2H, J=5.65 Hz, Ar-CHp). 5.77 (s, 1H, NH-CO), 7.19 (m, 4H, Ar-H), 7.38 (s, 1 H, naphth-H), 7.68 (m, 2H, 2xnaphth-H), 7.81 (dd, Jι= 8.67 Hz, J₂=1.86 Hz, naphth-H),

7.95 (m, 3H, 3xnaphth-H), 8.44 (s, NH-S0₂)

Molecular mass: Calculated: 496 (C₂₅H₂₈N₄O₅S); Found: 497 ( MH ⁺)

EXAMPLE 17:

(4-{[1-(4-morpholinylcarbonyl)-2-(1-naphthylsulfonyl)hydrazino] methyl}phenyl)methanamine hydrochloride

0.66 g (1.33 mmol) of N-(4-{[1 -(4-morpholinylcarbonyl)-2-(1 - naphthylsulfonyl)hydrazino]methyl}benzyl)acetamide was dissolved in 30 ml of pre- warned isopropyl alcohol (60°C). 30 ml of 4M HCI was added and the mixture was refluxed for 5 hours. The solvent was evaporated in vacuo. The product was purified by column chromatography; stationary phase: silicagel, mobile phase: dichloromethane : methanol (9:1).

Yield: 340 mg (52%)

Melting point: 124-126°C

IR (KBr, cm^"1) 3411 , 16662, 1504, 1459, 1419, 1335, 12272, 1211 , 1166, 1113,

1067, 1023, 830, 754, 688, 544_

H-NMR (CDCIs): δ (ppm) 2.96 (m, 2H, morph-CHp). 3.11 (m, 6H, 3xmorph-CHp). 3.97

(d, 2H, J=5.66 Hz, Ar-CHp). 4.17-4.52 (m, 2H, Ar-CHp). 7.22 (d, 2H, J=7.53 Hz, Ar- H), 7.41 (d, 2H, J=7.92 Hz, Ar-H), 7.82-7.66 (m, 3H, 3xnaphth-H), 7.99-7.84 (m, 1 H, naphth-H), 8.03-8.21 (m, 3H, 3xnaphth-H), 8.47 (s, 1H, NH-S0₂)

Molecular mass: Calculated : 490.5 (C₂₅H₃₁N₄O₃SCI); Found: 455 ((M-HCI)H ⁺)

EXAMPLE 18:

Te/f-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-{[cyclopentyl(methyl)amino] carbonyl}-

1 -hydrazinecarboxylate

1.26 g (4.39 mmol) of bis-trichloromethylcarbonate was dissolved in 20 ml of dichloromethane and cooled to 0°C. The solution of 2.00 g (6.83 mmol) of ferf-butyl 2-{4-[(acetylamino)methyl]benzyl}-1 -hydrazinecarboxylate and 1.65 g (12.79 mmol) of N-diisopropyl-N-ethylamine in 30 ml dichloromethane was added dropwise allowing the temperature to increase slowly The resulting mixture was stirred at room temperature for half an hour, followed by the addition of 2.14 g (21.60 mmol) N- cyclopentyl-/V-methylamine. After another hour of stirring, the solvent was evaporated in vacuo and the residue was dissolved in 50 ml of ethylacetate. The solution was extracted with 4x 25 ml of 10% aqueous solution of citric acid and 30 ml of aqueous sodium hydrogencarbonate. The organic phase was washed with water and brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the residue dissolved in 10 ml of diethylether. After 2 days the precipitate was collected and further purified by column chromatography (dichloromethane/methanol 20:1). Yield: 1.31 g (46%) Melting point: 124-127°C

IR (KBr, cm^"1) 3274, 2974, 1726, 1640, 1550, 1369, 1285, 1160, 1067, 794 H-NMR (CDCI3): δ (ppm) 1.44 (s, 9H, C(CH₃)₃), 1.46-1.85 (m, 8H, CH,), 2.03 (s, 3H, CO-CH3), 2.32 (m, 1 H, CH), 2.80 (s, 3H, N-CH₃), 4.38 (m, 2H, Ar-Cfcfe), 4.42 (d, 2H, J=5.64 Hz, Ar-CHp). 5.93 (s, 1H, NH-COO), 6.54 (s, 1H, ISIH-CO), 7.28 (m, Ar-H) Molecular mass : Calculated: 418 (C22H34N4O4); Found: 419 ( MH⁺)

EXAMPLE 19: 2-{4-[(Acetylamino)methyl]benzyl}-2- {[cyclopentyl(methyl)amino]carbonyl}hydrazinium chloride

1.20 g (2.87 mmol) of tetf-butyl 2-{4-[(acetylamino)methyl]benzyl}-2-

{[cyclopentyl(methyl)amino] carbonyl}-1 -hydrazinecarboxylate was dissolved in 20 ml of acetic acid and the gaseous hydrogen chloride was bubbled in for 30 minutes. The acid was evaporated in vacuo and diethylether was added to the residue. After vigorous stirring and numerous washings with diethylether, a white solid was obtained.

Yield: 765 mg (77%)

Melting point: 165-169°C

IR (KBr, cm^"1) 3254, 2987, 1782, 1653, 1548, 1425, 1234, 1022, 798, 744, 602

H-NMR (CDCI₃): δ (ppm) 1.51-1.89 (m, 8H, CHP.), 2.02 (s, 3H, CO-CH₃), 2.49 (m, 1H,

CH), 2.87 (s, 3H, N-CHs), 4.24 (m, 2H, Ar-CHp). 4.52 (s, 2H, AΓ-CHP). 7.22 (m, Ar-H),

10.12 (s, I H, JNΠH-CO)

Molecular mass : Calculated: 445 (C₁₇H₂₇N₄O₂CI); Found: 319 ((M-HCI)H⁺

EXAMPLE 20:

1-{4-[(Acetylamino)methyl]benzyl}-Λ/-cyclopentyl-/V-methyl-2-(2-naphthylsulfonyl)-1- hydrazinecarboxamide

733 mg (2.07 mmol) of 2-{4-[(acetylamino)methyl]benzyl}-2- {[cyclopentyl(methyl)amino]carbonyl}hydrazine chloride, 481 mg (2.12 mmol) of naphthalene-2- sulfonylchloride and 843 mg (6.54 mmol) of N-diisopropyl-N- ethylamine were dissolved in dichloromethane (25 ml), and stirred at room temperature for 3 days. The solvent was removed in vacuo and the residue was dissolved in 30 ml of ethylacetate and extracted with 4x 25 ml 10% of citric acid and 30 ml aqueous of NaHC0₃. The organic phase was further washed with water (30 ml) and brine (30 ml), dried over Na₂SU₄and the solvent was removed to yield a pale yellow solid which was further purified by recrystallisation from ethanol. Yield: 211 mg (20%) Melting point: 91 -93^SC IR (KBr, cm^"1) 3448, 2962, 1772, 1651 , 1558, 1395, 1262, 1166, 1025, 859, 803, 669, 549

H-NMR (CDCI₃): δ (ppm) 1.27-1.73 (m, 8H, _____), 2.06 (s, 3H, CO-CH₃), 2.29 (m, 1H, CH), 2.60 (s, 3H, N-CΗg), 4.32 (m, 2H, Ar-Chb), 4.44 (d, 2H, J=6.13 Hz, Ar-CHp). 7.15 (d, 2H, J=8.13 Hz, Ar-H³'⁵), 7.21 (d, 2H, J=8.11 Hz, Ar-H²'⁶), 7.42 (s, NHS0₂), 7.67 (dqu, 2H, Jι=7.51 Hz, J₂=1.79 Hz, Ar-H), 7.79 (dd, 1 H, ^=8.70 Hz, J₂=1.76 Hz, Ar-H), 7.90 (s, 1 H, NH-CO), 7.97 (m, 3H, Ar-H), 8.43 (s, 1 H, Ar-H) Molecular mass : Calculated: 508 (C₂₇H₃₂N₄O₄S); Found: 509 ( MH ⁺)

EXAMPLE 21 :

(4-{[1-{[Cyclopentyl(methyl)amino]carbonyl}-2-(2-naphthylsulfonyl) hydrazino]methyl}phenyl)methanaminium chloride

The solution of 183 mg (0.36 mmol) of 1-{4-[(acetylamino)methyl]benzyl}-/V- cyclopentyl-Λ/-methyl-2-(2-naphthylsulfonyl)-1-hydrazinecarboxamide in 30 ml of isopropanol at 60°C (30 ml), was treated with 4M HCI (30 ml) and refluxed for 5 hours. The solvents were removed in vacuo and the product was purified by column cromatography (dichloromethan/methanol 9:1).

Yield: 81 mg (45%)

Melting point: 170-173^aC

IR (KBr, cm^"1) 3425, 3137, 2968, 1744, 1591 , 1275, 1122, 973, 815, 637

H-NMR (CDCI₃): δ (ppm) 1.18-1.75 (m, 8H, _____), 2.34 (m, 1H, CH), 2.57 (s, 3H, N-

CH₃), 4.22 (m, 2H, Ar-CHp). 4.43 (m, 2H, Ar-CHp). 7.18 (d, 2H, J=8.35 Hz, Ar-H^3,5),

7.24 (d, 2H, J=8.27 Hz, Ar-H²'⁶), 7.37 (s, NHS0₂), 7.64 (dqu, 2H, Jι=8.29 Hz, J₂=1.84

Hz, Ar-H), 7.82 (dd, 1 H, ^=8.56 Hz, J₂=1.72 Hz, Ar-H), 8.04 (m, 3H, Ar-H), 8.41 (s,

1H, Ar-H)

Molecular mass : Calculated: 503 (C₂₅H₃ιN₄θ₃SCI); Found: 467 ((M-HCI)H⁺)

EXAMPLE 22: /V-(3-Cyanobenzyl)-/V-(2-naphthoyl)-1-azepanecarbohydrazide 2-(1-azepanylcarbonyl)-2-(3-cyanobenzyl)hydrazinium chloride (312 mg, 1.03 mmol), N-diisopropyl-N-ethylamine (2 ml) and benzoylchloride (210 mg, 1.11 mmol) were dissolved in dichloromethane (30 ml). The reaction mixture was stirred at room temperature for 2 days. The solvent was removed in vacuo and the remainder was dissolved in 30 ml ethylacetate and extracted with 4x 25 ml 10% of citric acid and 30 ml of aqueous NaHCO₃. The organic phase was further washed with water (30 ml) and brine (30 ml), dried over Na₂S0₄ and the solvent was removed. The yield was washed with diethylether and further purified by recrystallisation from ethanol. Yield: 378 mg (86%) Melting point: 139-141 ^eC

IR (KBr, cm^"1) 3281 , 2922, 2236, 1637, 1524, 1289, 1210, 1010, 912, 822, 759, 698, 595

H-NMR (CDCI₃): δ (ppm) 1.55 (m, 4H, azep-CH?), 1.73 (m, 4H, azep-CHp). 3.49 (t, 4H, J=5.84 Hz, azep-CHp). 4.69 (s, 2H, Ar-CHp). 7.42 (t, 1H, J=7.89 Hz, Ar-H), 7.59 (m, 3H, Ar-H), 7.69 (d, 1 H, J=7.91 Hz, Ar-H), 7.77 (dd, 1 H, Jι=8.29 Hz, J₂=1.88 Hz, Ar-H), 7.81 (s, 1 H, Ar-H), 7.89 (m, 3H, Ar-H), 8.24 (s, 1 H, Ar-H), 8.47 (s, 1 H, NH-CO) Molecular mass : Calculated: 426 (C₂6H₂₆N₄O₂); Found: 427 (MH⁺)

EXAMPLE 23:

Amino(3-{[1-(1-azepanylcarbonyl)-2-(2-naphthoyl)hydrazino]methyl} phenyl)methaniminium chloride

Λ/-(3-cyanobenzyl)-/V-(2-naphthoyl)-1-azepanecarbohydrazide (212 mg, 0.50 mmol) was suspended in absolute ethanol (20 ml) and the gaseous hydrogen chloride was bubbled in for half an hour. The reaction mixture was left at room temperature for four hours, then the solvent was removed in vacuo. The residue was washed with diethylether (2x20 ml) and dissolved in absolute ethanol. The solution was treated with gaseous ammonia for 10 minutes and ethanol was removed under reduced pressure.

Yield: 193 mg (81%)

Melting point: 247-251⁹C

IR (KBr, cm^"1) 3410, 1773, 1652, 1559, 1394, 955, 753 H-NMR (CDCI3): δ (ppm) 1.58 (m, 4H, azep-CHp). 1.76 (m, 4H, azep-CHp). 3.02 (t, 4H, J=5.15 Hz, azep-CHp). 3.82 (s, 2H, Ar-CHp). 7.61-7.67 (m, 5H, Ar-H), 7.84 (m, 2H, Ar-H), 7.98-8.12 (m, 3H, Ar-H), 8.42 (s, 1 H, Ar-H), 9.39 (s, 1 H, NH-CO), 9.43 in

Molecular mass : Calculated: 479 (C₂₆H₃oN₅O₂CI); Found: 444 ((M-HCI)H⁺)

EXAMPLE 24:

3-{[1-(1-Azepanylcarbonyl)-2-(2-naphthoyl)hydrazino]methyl}-/V- hydroxybenzenecarboximidamide

Λ/-(3-cyanobenzyl)-/V-(2-naphthoyl)-1-azepanecarbohydrazide (133 mg, 0.31 mmol) and hydroxylamine (12 mg, 0.36 mmol) were dissolved in absolute ethanol (10 ml) and refluxed overnight. The solvent was evaporated and the product was purified by column chromatography (dichloromethan/methanol 9:1).

Yield: 107 mg (75%)

Melting point: 129-131⁹C

IR (KBr, cm^"1) 3334, 2924, 1772, 1652, 1506, 1374, 1301 , 1129, 954, 749

H-NMR (CDCI₃): δ (ppm) 1.43 (m, 4H, azep-CHp). 1.57 (m, 4H, azep-CH≥), 3.36 (t,

4H, J=5.61 Hz, azep-CHp). 4.53 (s, 2H, Ar-CHp). 5.69 (s, 1H, NOH), 7.31 (t, 1 H,

J=7.76 Hz, Ar-H), 7.49 (m, 3H, Ar-H), 7.57 (m, 2H, Ar-H), 7.78 (dd, 1 H, J₁₌8.69 Hz,

J₂=1.83 Hz, Ar-H), 7.89 (m, 3H, Ar-H), 8.38 (s, 1 H, Ar-H), 9.57 (s, 2H, JNHg), 10.64(s,

1 H, NH-CO)

Molecular mass : Calculated: 459 (C₂₆H₂9N₅O₃); Found: 460 (MH⁺)

EXAMPLE 25

Results of the biological assays:

In the following table the results of the biological assays for compounds according to the above examples are identified. The other compounds represent further embodiments not particularly illustrated by an example.

Novel azaphenylalanine derivatives of the formula

and pharmaceutically acceptable salts thereof are described wherein the substituents have the meanings as specified in the description. The compounds are useful as anticoagulants.

Claims

1. A compound of the formula I

wherein

R¹ and Z are H or a residue of the formula,

H, .^ ^N' R NH ci^"

with the proviso that one of R¹ and Z is H; R⁴ = H, alkyl (C C₃), OH, O-alkyl (C C₃), NH_2;

R² represents a residue of the formula

wherein R⁵ = H, alkyl (C C₃), COOR¹⁰, R⁶ = H, alkyl (C C₃), COOR¹⁰ , R⁷ = H, alkyl (Cι-C₃), COOR¹⁰, R¹⁰ = H, alkyl (C C₃), R⁸ = H, alkyl (C C₃), cycloalkyl (C₃-C₆), R⁹ = H, alkyl (C C₃), cycloalkyl (C₃-C₆), R¹¹ = H, alkyl (d-C₃), benzyl,

X = CH, 0, S,

Y= NR¹², O, S,

R¹²= H, COCHg, alkyl (CrC₃)

R -3 is a residue of the formula

or a residue of the formula

NH ci^" in the case of R = r

and pharmaceutically acceptable salts thereof.

2. The compound according to claim 1 , wherein the compound is (4-{[1 ■ {[Cyclopentyl(methyl)amino]carbonyl}-2-(2-naphthylsulfonyl) hydrazino]methyl}phenyl)methanaminium chloride

3. A process for the preparation of the compound according to Claim 1 , wherein Z is H, comprising the following steps:

a) in the case R¹= '

4-cyanobenzaldehyde of formula (II)

is converted with BOC-carbazate of the formula (III)

to the compound of the formula (IV)

which reacts with triphosgene and amine of the formula (VI)

wherein R⁵ , R⁶ and R⁷ have the same meanings as in Claim 1 , or with triphosgene and amine of the formula (VII or VIII),

(VII) (^vι"> wherein Y has the same meaning as in Claim 1 , or with triphosgene and amine of the formula (IX) ⁸ ^VR9 (IX) wherein R⁸ in R⁹ have the same meanings as in Claim 1 , or with triphosgene and amine of the formula (X),

(X) wherein R¹¹ and X have the same meanings as in Claim 1 , to the compound (XI)

wherein R² has the same meaning as in Claim 1 , and from which the BOC protecting group is removed at room temperature using the influx of gaseous hydrogen chloride in acetic acid to obtain compound (XII),

wherein R² has the same meaning as in Claim 1 ; which then reacts with the activated naphthylsulfonyl aminoacid or with activated arylalkylcarboxylyc acid up to the compound (XIII)

wherein R .2 a „.nd r R.3 have the same meanings as in Claim 1 ; which is then transformed with hydroxylamine in absolute ethanol up to the compound with formula (XIV)

wherein R¹=

meanings as in Claim 1 and R⁴ is an OH group; and which is then converted with an influx of gaseous hydrogen chloride in ethanolic solution, addition of ammonium acetate, followed by another influx of hydrogen chloride to the compound (XV),

wherein R i1 =

R •,2 and i r R,3 have the same meanings as in the formula (I) and R⁴ is hydrogen;

NH ci b) in the case R¹= ' 4-cyanobenzaldehyde of formula (II)

which reacts with acetanhydride up to the compound (XVIII)

which is then converted with 90% methanoic acid to the compound (XIX)

H-NHCOCH-

CHO (XIX)

which is converted with BOC-carbazate of the formula (III)

to the compound of the formula (XX)

which reacts with triphosgene and amine of the formula (VI)

R^β

HN I-

^R9 (IX) wherein R⁸ in R⁹ have the same meanings as in the formula (I); or with triphosgene and amine of the formula (X),

(X) wherein R¹¹ and X have the same meanings as in the formula (I); to the compound (XXII)

wherein R ^■>2 has the same meanings as in the formula (I); from which the protecting BOC group in the compound is removed with HCI (g) in AcOH at room temperature in order to obtain the compound (XXIII),

(XXIII)

wherein R² has the same meanings as in the formula (I); which reacts with aromatic sulfonylchloride up to the compound (XXIV)

wherein R² and R³ have the same meanings as in Claim 1 , which is then converted by heating to boiling with 5M HCI to the compound (XXV)

,-NH cf wherein R¹= ' , R ,2 and , R p->^ύ3 have the same meanings as in the formula (I).

4. The use of the compound according to Claim 1 for the production of a pharmaceutical composition having therapeutic activity.

5. The process according to claim 3, NH ci^" wherein Z is

, '

characterized in that that the starting compound (II) is 3-cyanobenzaldehyde.

6. The use according to Claim 4, wherein the pharmaceutical composition is a medicament with anticoagulant activity.

7. The use according to Claim 4, wherein the pharmaceutical composition is a inhibitor of thrombine.

8. The use according to Claim 4, wherein the pharmaceutical composition is a dual inhibitor of thrombine and factor Xa.

9. The use according to Claim 4, wherein the pharmaceutical composition inhibits thrombin in the blood of humans and other mammals.

10. The use according to Claim 4, wherein the pharmaceutical composition inhibits fibrin and thrombus formation in the blood of humans and other mammals.

1. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to Claim 1 and pharmaceutically acceptable auxiliary substances.