DD155954A1 - PROCESS FOR PREPARING N LOW ALPHA ARYL-BZW.N LOW ALPHA HETEROARYLSULFONYLAMINOACYLATED AMIDINOPHENYLALANINAMIDES - Google Patents

Abstract

Process for the preparation of N deeply a -aryl or N-deep a-heteroarylsulfonylaminoacylated amidinophenylalanine amides. These compounds can be advantageously used as thrombin inhibitors of high specificity and thus as anticoagulants or for eliminating the activity of thrombin in the determination of other proteolytic activities. It should be achieved to produce hitherto unknown N deep a -aryl or N deep a -heteroarylsulfonylaminoacylated Amidinophenylalaninamide. This is done by reacting cyanobenzylacylaminomalonate diesters to cyanophenylalanine and carboxyphenylalanine. After isoelectric separation of the by-product, sulfonylaminoacylation of cyanophenylalanine and amide formation via an activated ester leads to cyano compounds of the desired structure. The amidino compounds are obtained by reacting the cyano compound to give imidic acid esters and treating them with alcoholic ammonia solution. The desired amidines are likewise obtained by reacting the corresponding cyano compounds to thioamides, thioimidic acid esters and reacting them with ammonium acetate. The invention can be used in pharmacy and medicine.

Description

Title of the invention

A process for the preparation of N ^ -aryl or Щ- heteroarylsulfonylaminoacylated Amidinophenylalaninamiden

Field of application of the invention

The invention relates to processes for the preparation of 1 ^ - aryl or N - heteroarylsulfonylaminoacylated Amidinophenylalaninamiden.

These compounds are thrombin inhibitors of high specificity and can thus be advantageously used as direct anticoagulants or for eliminating the enzymatic activity of thrombin in the determination of other proteolytic activities in biological materials.

C Characteristics of the Known Technical Solutions For the therapy and prophylaxis of thrombotic diseases, direct and indirect anticoagulants of the heparin and coumarin and indanedione derivatives type have hitherto been used in medicine. Both anticoagulant types have certain disadvantages: The effect of the coumarin derivatives is directed to the biosynthesis of the vitamin K dependent coagulation factors II, VII, IX and X and their effect, which amounts to a reduction of the coagulation potential in the patient's blood, occurs only some time after administration , The therapy requires a laboratory-based monitoring, as it can come in an overdose to bleeding.

Heparin, which due to its polysaccharide structure can only be used parenterally, acts as a catalyst in the body's own inactivation mechanisms. Due to its polyvalent affinity, its action can be neutralized or attenuated by reaction with other blood components (lipoproteins, platelet factor IV, etc.). Its action is dependent on a normal functioning blood antithrombin level. Small molecule enzyme inhibitors have the advantage over heparin that they act directly and are also orally administrable. Their effect occurs only after activation of the normally inactive zymogen prothrombin to the active enzyme thrombin. For the action of such inhibitors, no other components of the blood are required, e.g. Antithrombio for heparin action. Compared with coumarin-type anticoagulants, they have the particular advantage of being able to exert their effect immediately after application.

The benzimidine-derived enzyme inhibitors hitherto known, such as p-amidinophenylpyruvic acid ( Richter, P. and Wagner, G. , WP C 07C / 87029 (DDR, 1972); Pharmacie., 28, 514, 585 (1973)) show a polyvalent inhibitory Action against serine proteinases ( Arkv / ardt , Kl ., Walsmann, P. and Klöcking, H.-P., WP A 61 K / 92302 (DDR, 1972)). Next thrombin inhibiting including trypsin, plasmin, factor Xa and serum kallikrein with considerable potency (Geratz, JP, Arch Biochem 118 (1967);.. Geratz, JD, Experientia 25.4 to 83 (1969); Geratz, JD , Amer J. Physiol 216 , 1812 (1969), Markward, F , Iandar: iq, H and V / a Ismann, P. , European J. Biochem., 6, 502 (1968), Markwardt, F. and WaIsmann, P., Experientia, 4, 25 (1968), Markwardt, F. , Walsmann, P., and Drawert, J. , Acta biol.med, Germ., 24, 401 (1970), Stürzebecher, J. , Markwardt, F. and Walsmann, P. , Thrombos Res. 9 _f 637 (1976))

In other known benzamidine-derived enzyme inhibitors, such as bis (amidinobenzyliaea) cycloallophones and bis (midinobenzylo) cycloalkanones ( Vagner, G. , Viewer H. and Horn, H. , Pharmacia 32, 141 (1977) Toxicity such that they are unsuitable for in vivo use ( Walsmann, P. , Horn, H._ , Markwardt, F. , Richter, P. , Stürzebeoher, J. , Vieweg, H. and У / agner, G. , Acta biol., Med., Germ., 22.1 ^K 1 (1976), Hauptmann, J. , Hoffmann, J., and Ivlarkwardt, F. , Acta biol., Med., Germ., 35 * ⁶³⁵ (1976)) thrombin-specific inhibitors were found by Okamoto and Mitarb, with the% arylsulfonylarginine esters and amides, in particular corresponding Dansy! derivatives ( Okamoto, S., et al . , DE-OS 2,438,851 (1975), Japanese Pat. 76 125 259 (1976), 76 125 262 (1976), 76 131 864 (1976); Okamoto, S. , Hi.jikata, A. , Ki η jo, K. , Kikumoto, R. , Onkubo, K. , Tonomura, S. and Tamao, Y. , Kobe J. Med. Sci. 21, 43 (1973); Hijikata, A. , Okamoto, S. , Mori, E. , Kietao , K. , K ikumoto, R. , Tonomura, p. , Татар, Y. and Нага, Η. , Thromb. Res., Suppl. II, 8, 83 (1976); Okamoto, S. , Hi.jikata, A. _t Ikezawa, K. , Kin jo, K. , ) R. , Tonomura, S. and П £ тао, У :. , Thromb. Res.

Suppl. II, Q, 77 (1976)). It is disadvantageous in the presentation of this compound that as a rule the guanidino grouping of arginine with a protective group which is difficult to remove must be blocked before further reactions can be carried out on the arginine. In the case of the dansyl derivatives, isolation of pure reaction products poses greater difficulties.

Thrombin-specific inhibitors can be used not only as direct anticoagulants, but also to improve laboratory diagnostic methods for the determination of coagulation factors using chromogenic peptide substrates ( H. Vinazzer , coagulation physiology and methods in the blood coagulation laboratory , G. Fischer Verlag, Stuttgart, New York 1979 ). While the specificity of these substrates for single coagulation factors is relatively high, it is not sufficient for certain methods of urine detection.

For example, the determination of the coagulation factor Xa by means of substrate S-2222 (KABI Diagnostica, Sweden) is disturbed in the presence of thrombin ( H. Vinazzer , coagulation physiology and methods in the blood coagulation laboratory , G. Fischer Verlag, Stuttgart, New York 19У9, P. Friberger and H Vinazser , Research Results of Transfusion Medicine and Imraunhaematology, Vol. 6, Ed. H. Vinazzer, Medicus Verlag GmbH, Berlin 1979). The addition of a thrombin-specific inhibitor to the assay can eliminate this source of error. The use of the natural thrombin inhibitor hirudin in the routine laboratory for this purpose does not make sense, since solutions of this protein are not stable for a long time. The thrombin inhibitors of the type of N, N -aryl or C 1-4 alkylsulfonylated O-amidiaphenyl-K-aminoalkylcarboxylic acid amides ( Wagner, G. , He, P. , Horn, H. , Voigt, B ,, Vieweg, H . , Markwardt, F « , Y / AIsmann, P. , Kazmirowski, G. , Landma η η, Η. , Falls beeher , J. , Hauptmann, J. and Lischke,!. , V / P С о7 D / 201 898 (DDR, 1977) have the disadvantage that the specificity of their He.Timwirkung is not sufficient for this use ·

Object of the invention

The aim of the invention is the provision of highly specific thrombin inhibitors, which overcome the disadvantages of the anticoagulants of the types of heparin, coumarin, indandiones and all their derivatives, previously known benzamidine-derived kleinmoiekularer enzyme inhibitors and the N ^ -Arylsulfonylargininester and amides and surpass the antithrombin activity of 1-alkyl or γ-arylsulfonylated W-amidinophenyl K-aminoalkylcarboxamides. Due to their potent inhibitor activity directed specifically against thrombin, they should be able to increase the accuracy of laboratory diagnostic procedures using chromogenic peptide substrates by eliminating thrombin.

Statement of the invention

The invention has the object of finding as specific as possible Thrombininhüfitoren. Des'., Eiteren synthesis methods are found v / earth, which allow the production of these inhibitors in a technically relatively simple way.

The invention is based on the scientific knowledge that compounds of structure I NH

and their salts are thrombin inhibitors of high specificity. In the formula, the AmidinogrupOe in m- or

-j p position, η = 1 to 5; R = aryl or heteroaryl,

R ² = H, alkyl, aryl or aralkyl and R ⁵ = alkyl, aryl or aralkyl. R and R, with the N atom to which they are attached, form a heteroaliphatic ring which may contain other heteroatoms and may be substituted. The object is achieved in that compounds of structure I are synthesized, including the method described below. The procedure consists in that a Cyanbenzylhalogenid with a Acylaminomalonsäurediester to a Cyanbenzylacy! Aminomalonsäurediester of the general formula II

^C00R

I TC-NH-CO-R-yy-

COOR "~

in which the cyano group can be in the m- or p-position and R = alkyl.

The compounds thus obtained are reacted in a mixture of 3N HCl and glacial acetic acid under reflux conditions. In this case, compounds of structure III and IV

HOOC

_ / ^w Z

CH, -CH-COOH

IU

NC

CH, -CH-COOH

IV

receive. By titration with alkalis to the isoelectric point of the mixture of III and IV largely pure cyanophenylalanine (IV) is obtained.

By acylation with a sulfonylated amino acid halide in the presence of a base compounds of the formula V

NH-CO- (CH ₂ ) -NH-SO ₁ -R

in which η = 1 to 5, R = aryl or heteroaryl and the cyano group can be in the m- or p-position. The representation of the amides of formula VI

CRpCH-CO-N

12 3

wherein n, R, R and H are the meaning of the structure I,

takes place after the reaction of V to an activated ester, preferably nitrophenol ester, and its treatment with corresponding amines.

In a first embodiment, the cyano compounds obtained in this way are reacted with an anhydrous lower alcohol and anhydrous hydrogen halide in imidare

ester salts of the structure Ia NH

NH-CO- (CHJ-NH-

12 3

where n, R, R, R are the above-mentioned

have meanings, R = alkyl and X = halogen. In the following synthesis step, amide salts of the following structure (Ib) are obtained by the action of ammonia in alcoholic solution on the imidic acid ester salts Ia, NH

_r CH-CO-N _n ^ ₃

NH-СО- (снД-NH-SO ₁ -

wherein the amidino group can be in the m- or p-position

12 3

and o, R, R, R and X have the meanings given above.

Other amidine salts with physiologically compatible inorganic or organic acids are obtained by converting the imidic acid ester hydrohalides into other salts after the imidic acid ester bases have been obtained, which are reacted with alcoholic ammonia solution in the manner described above to give corresponding amide salts.

Furthermore, amidine salts can be obtained by reacting the imidazoline ester bases with the ammonium salt of physiologically compatible inorganic or organic acids to give corresponding amidine salts. A third possibility is to obtain other amidine salts from amidine salts after liberation of the amidine base and reaction with physiologically compatible inorganic or organic acids.

In a second embodiment of the invention, hydrogen sulfide is added to the cyano compounds of structure VI in pyridine / triethylamine to form the thiocarboxaaido compounds. These are reacted with an alkyl halide to give thioimidic acid ester hydrohalides of structure VII

NH

СИ · "СО- N

NH-CO- (CH ₂ ) - NH- SO ₂ - Pf

12 3

implemented, wherein η, R, R, R and X have the abovementioned meanings and R = alkyl.

From the thioimidic acid ester salts, the corresponding amidine salts are obtained by reaction with ammonium nitrate.

The Cyanbenzylacy! Aminomalonsäurediester is expediently reacted in a mixture of glacial acetic acid and 3 N hydrochloric acid in the Volurnenverhältnis 1: 2 to Cyanphenylalanin and Carboxyphenylal ~: iin, which we largely separated by isoelectric precipitation.

The invention will be explained below in two embodiments.

Example 1:

10.0 g of 3- or 4-Cyanbenzylbrornid, 11.0 g Acetaminomalonsäurediethylester and 2.5 g of KI are abs in 10ü ml. Dioxane at room temperature while stirring with one of 1.15 g of sodium and 50 ml of abs. Ethanol produced ethylate solution implemented. After five hours of stirring on a boiling water bath, the Fiitrat the suspension thus obtained is treated with plenty of water, the precipitate is filtered off with suction and recrystallized from methanol (m.p. 143-145 ⁰ C (m-compound);. 168-170 ⁰ C (p-compound) ).

12.0 g of diethyl 3- or 4-cyanobenzylacetaminomalonate are refluxed in a mixture of 32 ml of glacial acetic acid and 64 ml of 3N HCl for 6 hours. I is distilled. Vak. most of the solvent. The residue is dried on the clay plate and washed with ether. For the substantial separation of the 3- or 4-Carboxyphenylalanins from the hydrolysis product for the recovery of 3-Cyanphenylalanin 6.0 g of the hydrolysis in 20proz. Dissolved methanol and titrated potentiometrically to pH 6.8 - 7.0 with 1 N NaOH. It crystallizes out 3-cyanophenylalanine (R ^ value 0.50-0.70, DC in n-propanol / 25% NH, (v / v) 7/3), leaving only one trace of the 3-carboxy compound (R "). Value 0.30-0.50). The precipitate is dissolved in methanolic HCl, filtered and 3-Cyanphenylalaninhydrochlorid precipitated with ether. In order to obtain 4-cyanophenylalanine, 6.0 g of hydrolysis product are dissolved in 1: 1 ethanol and titrated potentiometrically to pH 3.8-4.0 with 1 N NaOH. The resulting precipitate of 4-carboxyphenylalanine (R _f -V / ert 0.30 - 0.50) v / ird sucked off immediately and the fitrate i. Vak. concentrated to a volume of about 20 ml. The precipitate of 4-Cyanphenylalanin (R ~ - \ tert 0,50 - 0,70) is pressed on the clay plate, dissolved in methanolic HCl, the solution filtered and precipitated 4-Cyanphenylalaninhydrochlorid with ether.

2.3 g of 3- or 4-cyanophenylalanine hydrochloride are dissolved in 30 ml of 1 N NaOH and shaken for 10 hours with a solution of 2.5 g of tosylglycyl chloride in 50 ml of ether. After the ethereal phase has ended, the aqueous alkaline phase is extracted by shaking with ether. After acidification of the aqueous phase, N - (tosylglycyl) -3- or -4-cyanophenylalanine precipitate. The precipitate is filtered off and recrystallized from aqueous methanol to (m.p. 147-148 C (m-compound);. 158-160 ⁰ C (p-compound)).

4> 0 g of N _t ^ - (tosylglccyl) -4-cyanophen _< 7lalanine and 1.6 g of 4-nitrophenol are dissolved in 20 ml of abs. Pyridine dissolved. After addition of 2.0 g of dicyclohexylcarbodiimide (DGC), the mixture is kept for 24 hours at room temperature. Subsequently, the precipitated dicyclohexylurea is filtered off with suction and the pyridinic solution poured onto ice / HCl. % - (Tosylglycyl) -4-cyanphenylalanine-4-nitrophenyl ester is filtered off and recrystallized from ethanol (mp. 261 - 263 ⁰ C). 1.5 g of the nitrophenyl ester thus obtained are dissolved in 20 ml of abs. Dissolved pyridine, treated with 2 ml of piperidine and the approach stored for 12 hours at tree temperature. The solution is then poured onto ice / HCl, filtered off with suction N - (tosylglycyl) -4-cyanphenylalaninpiperidid and recrystallized from methanol (mp 188-191 ⁰ C).

Isolation of the nitrophenyl ester is omitted in the preparation of the corresponding m compound, followed by reaction of% - (tosylglycyl) -3-cyanophenylalanine with 4-nitrophenol and DCC after separation of the precipitated dicyclohexylurea. The filtrate with 2 ml Piperidine added and worked up the approach as above. N, - (tosylglycyl) -3-cyanophenylalanine piperidide is uracolitized after aspiration from methanol (mp 184-185 C). 2.0 g of the thus obtained N ^ - (tosylglycyl) cyanphenylalaninpiperidide be in abs. Pyridine, containing a few drops of triethylamine, dissolved. The mixtures are saturated with H ₂ S and stored for several days at room temperature. The solutions are then stirred into ice / HCl, the thiocLrnide incurred in solid form. Ss is sucked off,

Washed a lot of water and dried. For the v / urther implementation of a recrystallization is not required (Sohmp 152-153 ⁰ C.) (M-compound); 191-194 ⁰ C (p compound)),

1.5 g of the thioamides are dissolved in acetone and, after addition of 1 ml of methyl iodide, stored under light protection at room temperature. . After 24 hours the Thioimidsäureesterhydroiodide (C (ra-compound) mp 1 84 187 ^0., 118-122 ⁰ C (p-compound)) are precipitated with ether and filtered with suction. 1.0 g of thioimidic acid ester salts are dissolved in abs. Dissolved methanol and after addition of an equimolar amount of ammonium acetate 3 hrs. At 60 ⁰ C heated. Thereafter, it is filtered and i. Vak. the solution is largely concentrated. The residue is in little abs. Methanol and N ^ - (Tos; ylglyc; yl) -3-amidinophenylalaninpiperididhydroiodid (mp. From 134 ⁰ C) or ^ - (TosylgLycyO ^ -amidinophenylalanine piperididhydroiodid (mp 188-1902 ⁰ C) precipitated with ether.

Example 2;

1.5 g of N 2 - (tosylglycyl) -4-cyanophenylalanine 4-nitrophenyl ester (cf., Example 1) are dissolved in 20 ml of abs. Pyridine dissolved, mixed with 2 ml of morpholine and the mixture stored for several hours at room temperature. The solution is then poured onto ice / HCl and filtered with suction from N ^ - (tosylglycyl) -4-cyanophenylalaninmorpholid and uricristaliisiert from methanol (mp 244 - 248 ⁰ C).

1.0 g of the cyano compound is in abs. Dioxane solved. After addition of 2 ml of abs. Methanol is introduced into the cooled to 0 - 5 ⁰ C solution 1.0 g of dried HCl gas. The batch remains at 5 ⁰ C for a few days. Subsequently, the Imidsäureestersalz is precipitated by addition of a lot of ether, filtered off with suction, washed with ether and Vak.-Sxsikkator about K0H / H ₂ S0 _4, dried (mp. 128 - 132 ⁰ C).

0.9 g of Imidsäuremethylesterhydrochlorids v / earth in 40 ml of abs. Dissolved methanol and treated with 2 ml of ammonia-saturated methanol. The batch is held for 3 hours at 60 ⁰ G. Subsequently, the major part of the solvent i. Vak. distilled off, the residue in a little abs. Methanol was added, N ^ - (Tos _<7lglycyl) -4-like amidinophenylalaninmorpholidhydrochlorid with ether and purification from methanol / ether reprecipitated (mp. 118 - 121 ⁰ C)

According to Examples 1 and 2, the following compounds were obtained NH

HX-RN

position

the Ami- X η

dino group

CO

R ¹

schrap,

t ^B cJ ^P

I 1

I 1

I 1

(Η)

148-152

188-192

123-127

I 1

Cl 1

I 1

I 1

I 2

158-160

118-121

from 127

from 134

135-147 (Zers.)

I 2

I 2

124-140 (Zers.)

118-128 (Zers.)

Claims (4)

Invention claim. 1 2 can, η = 1 to 5, R = aryl or heteroaryl, R = H, alkyl, Aryl, aralkyl and R = alkyl, aryl, aralkyl, and R and R with the N-atom to which they are attached, form a heteroaliphatic ring containing further heteroatoms and may be substituted; b) according to a second embodiment of the invention, a N ^ -aryl or Ify-heteroarylsulfonylaminoacyliertes Cyanphenylalaninamid prepared in the first embodiment in pyridine / triethylamine with H ₂ S to Thiocarboxamidoverbindung v / ird who after reaction with an alkyl halide a Thioimidsäureesterhydrohalogenid from which is obtained by heating with ammonium acetate in alcoholic solution a! ^ - substituted Amidinophenylalaninamidsalz. 1. "A process for the preparation of N ^ -aryl bzv /. N ^ -heteroarylsulfonylaminoacylated Amidinophenylalaninaraiden the general formula I characterized in that a) according to a first Ausfcührungs form of the invention, a cyanobenzyl halide is converted to a cyanobenzylacy! Aminomalonsäurediester this after reaction in a mixture of glacial acetic acid and HCl in the Y / poor Cyanphenylalaninhydrochlorid and Carboxyphenylalaninhydrochlorid results, which are separated by isoelectric precipitation, the further reaction of the thus purified Cyanphenylalanins with a sulfonylated amino acid halide to a N ^ -aryl- or, N ^ -heteroarylsulfonylaminoacyliertem Cyanpheny! alanine , which is converted via an activated ester, preferably 4-nitrophenyl ester, to the acid amide, which is converted by reaction with a lower alcohol and hydrogen halide in a Tnidsäureestersalz, from which by Amnoniakeinwirkung in alcoholic solution, an N ^ -substituierte amidinophenylal Aninamidhydrohalogenid arises, which is the general formula
NH NH-CO- (CH-U KIH-SO-. has, in which the A ^ idinogruüpe in m- or p-Steliung stand 2. The method according to item 1, characterized in that a Cyanphenylalanin shown first according to the first embodiment with a sulfonylated glycyl halide or ß-Alanylhalogenid to an N-aryl or N-heteroarylsulfonyliertem% -Glycyl- or N ^ - (ß- & lanyl ) -4-Cyanphenylalanin reacted via an activated ester, preferably 4-nitrophenyl ester, with amines to the acid amide, the a) is converted into an acidic ester salt by reaction with a lower alcohol and hydrogen halide, resulting in an aminonia-effect in alcoholic solution, resulting in a! -substituted amidinophenylalanine amide hydrohalide of the general formula I, or that b) is reacted in pyridine / triethylamine with hydrogen sulfide to Thiocarboxamidoverbindung that after reaction with an alkyl halide provides a Thioimidsäureestersalz, from which by heating with Am ^ oniumacetat in alcoholic solution a! ^ - substituted Aoiidinophenylalaninamidhydrohalogenid is obtained. 3 · Procedure after. Item 1 characterized in that the% -substituted amidinophenylalanine amide salt is converted into another salt of a physiologically acceptable inorganic or organic acid, e.g. HCl, HpSO., Lactic acid is converted · 4. The method according to item 1, characterized in that Cyanbenzylacy! Aminomalonsäurediester in a ... isocyanate of glacial acetic acid and 3 N hydrochloric acid in a volume ratio 1: 2 under reflux zn cyanophenylalanine and carboxyphenylalanine, which is largely separated by isoelectric precipitation, reacted.