NO167809B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE ENZYME INHIBITIVE COMPOUNDS. - Google Patents

Description

The present invention relates to the production of new peptide analogues for use in preventing thrombus formation.

Thrombus formation

Blood clotting or coagulation depends on a complex series of actions that ultimately lead to the cleavage of florin from circulating fibrinogen by the protease thrombin and its subsequent cross-linking to form a stable structure. Thrombin production can be divided into two parts, an "intrinsic" system based on circulating blood components and an "extrinsic" system requiring tissue components. In each system there is a cascade of reactions, a series of inactive "factors" each of which is converted by a proteolytic reaction into the corresponding "a" factor which is itself a proteolytic enzyme which causes the next step.

In the intrinsic system, the effects begin when the circulating Hagemann factor (Factor XIII), which undergoes contact activation, becomes bound to damaged surfaces or collections of platelets. A peptide is cleaved from there by the circulating protease kallikrein which forms Factor Xlla. Factor XIIa in the presence of circulating high molecular weight kininogen (MHV7-K), then a) cleaves circulating plasma thromboplastin precursor (Factor XI) to yield plasma thromboplastin (Factor Xla) itself, and b) cleaves circulating pre -kallikrein (Pre-K, Fletcher factor), which gives more kallikrein and thus a self-accelerating effect. Plasma thromboplastin in the presence of calcium ions cleaves circulating Christmas factor (Factor IX) to give Factor IXa. Factor IXa with circulating antihemophilic globulin (AHG, Factor VIII) and in the presence of calcium ions and phospholipid micelles from platelets forms a lipoproteln complex with circulating Stuart factor (Factor X) and cleaves it to form Factor Xa. Finally, Factor Xa, together with circulating labile factor (Factor V) and in the presence of calcium ions and phospholipid micelles, forms a lipoproteln complex with prothrombin (Factor II) and cleaves it to form thrombin (Factor IIa) itself.

In the extrinsic system, a serum glycoprotein pro-convertin (Factor VII) is cleaved by different proteases from the intrinsic system (Factor Xla and Xlla and kallikrein) to form Factor Vila. Factor Vila together with tissue lipoprotein called tissue thromboplastin (Factor III) and in the presence of calcium ions, forms a complex with additional circulating factor X and cleaves it to form a second source of Factor Xa, which promotes thrombin production.

Finally, thrombin from both sources converts circulating fibrinogen into the soluble form of fibrin which is instantly polymerized into filaments and then cross-linked under the influence of an enzyme (Factor Xlla) which is formed by a second action of thrombin from circulating fibrin-stabilizing factor (Factor XIII).

In tabular form, the accelerating cascade of effects is as follows:

Thrombin

Besides its central action in fibrin clot formation as indicated above, thrombin is a key factor in the platelet aggregation stage of thrombus formation as described e.g. by J.H. Weiss in "Platelets: Pathophysiology and Antiplatelet Drug Therapy", ch. 1 (Liss, New York, 1982) or D. Ogsten and B. Bennett (eds.) "Haemostasis: Biochemistry, Physiology and Pathology", ch. 13 (Wiley , 1977). The details are not fully understood, but thrombin is the strongest inducer of the "platelet reaction" (change in shape, clumping, secretion of dense granule and cx granule) responsible for primary blood stasis. Prior to the action of thrombin, its interaction with a special binding protein in the platelet membrane takes place, and the inhibitors produced according to the invention also appear to interfere with this binding.

Special inhibitors for thrombin are thus expected to act as highly effective antithrombotic agents and/or anticoagulants as alternatives to existing therapeutic and prophylactic agents. Heparin is e.g. widely used against thrombosis, but it carries with it a high risk of bleeding, is ineffective in many conditions and gives a variable reaction from patient to patient and from time to time in a given patient, so that careful monitoring is necessary. Anticoagulants taken by mouth, which are used in particular for the prevention and regulation of venous thrombosis, also need a certain time to develop their effect and are exposed to the interfering effects of many other drugs. The inhibitors produced according to the present invention work immediately and due to their specificity and chemical nature, they can be expected to be without side effects or mutual influence from other drugs. Furthermore, because they act only on the thrombin that triggers platelet aggregation, they leave the collagen trigger available for maintaining blood status. The way the remedy is given is simple, e.g. intranasally.

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Substrate structure

It is known that most of the affinity sites for thrombin are present in the N-end group (8-20) fragment of fibrinogen and that partial sequences of this region bind firmly to the enzyme and are rapidly hydrolysed by it. More specifically, thrombin acts on the A-a chain of fibrinogen

between Arg<16> and Gly<17> to remove the N-terminal hexadecapeptide fragment (fibrinopeptide A). Cleavage between the residues Arg<19> and Val<20> then takes place, again by thrombin, at a much slower rate, releasing a further tripeptide fragment. The enzyme also removes the N-terminal tetradecapeptide fragment (fibrinopeptide B) of the fibrinogen Bp chain by slow hydrolysis between Arg<14> and Gly<15>. This release of fibrinopeptides from fibrin monomers to form "soluble" fibrin. The latter is then converted to stable, "insoluble" fibrin gel by Factor Xlla, which cross-links toa or nr chains of neighboring fibrin molecules by transamidation.

The present invention is based on partial sequences of human fibrinogen, which have a high binding affinity for thrombin. These substrate fragments are modified by replacing the cleavable peptide bond -CO-NH- (i.e. the bond normally cleaved by thrombin) with a non-hydrolyzable isosteric bond (e.g. a keto-CO-CB^-, hydroxy-CH( 0H)-CH2 or reduced -CB^-NH bond.

Other positions of the substrate frequency can optionally also be modified to obtain increased binding affinity, e.g. by introducing DPhe at position 14 and/or Pro at position 15 of fibrinogen, as previously proposed by M. Pozsgay et al., Eur. J. Biochem., 115. 941 (1981), or increased metabolic stability, e.g. by isosteric substitution of further peptide bonds or protection of the N-end group and/or the C-end group with suitable protecting groups.

General reference to amino acids and aminoacyl residues and side chains in the present context shall be considered as a reference to such whether they occur naturally in proteins or not, and to both the D and L forms; and amino acid shall be deemed to include imino acid. All asymmetric centers can be of either R or S configuration unless otherwise specified.

The present invention provides new therapeutically active polypeptide analogues which show anti-thrombotic or anti-coagulant activity and have the following formula (where the numbering of the residues corresponds to that applicable to human flbrinogen):

where:

X - H or a protecting group comprising lower alkyl

(Cl-C5) or R-O-CO-, where R - t-butyl;

Y D- or L-phenylalanine;

Z - L- or D-proline;

"reduced" isostere (IV) where:

(ii) R<2> - H,

(ili) R*<*> - any of the groups X which defined above, (lv) the configuration at the asymmetric centers<*> are either R or S,

Arg may be absent;

B D- or L-valine or absent;

W = -OH or NH-(CH2)n, where n - 0-5.

According to the present invention, these peptide analogues are prepared by reacting an acid X-Y-Z-OH with an amine H-A-Pro-Arg-B-W.

Preferred compounds of formula I are:

Such peptide analogues can further exist in the form indicated above or can be modified by Isoster replacement of one or more remaining peptide bonds or by keto

-C0-CH2-, hydroxy -CH(OH)-CH2- or reduced -CH2-NH-

bonds, and can exist in the free form or in a protected form by one or more remaining functional groups, e.g. amlno, imino or amide (including peptide), nitrogen, carboxyl, hydroxyl, guanidino. More specifically, the analogous compounds may exist in the form of their physiologically acceptable acid addition salts or other derivatives which can be converted in the body to the active compound (as shown by their action). Such physiologically acceptable derivatives are included in the definition of the manufactured compounds.

The invention further extends to the use of the above-described thrombin inhibitors in the prevention or treatment of diseases associated with unwanted thrombus formation.

EXAMPLES

The following detailed examples show the invention as the text in the examples is followed by the reaction schemes shown in tabular form. Examples I to VI are given in detail followed by a more abbreviated Example VII along the same lines.

Example I

The synthesis of H-163 was carried out according to scheme I. Arabic numerals underlined (eg 1) denote the structure in these schemes. Arabic numbers in brackets e.g. ( 1) refers to reaction steps. (1) N~-formyl-N<G>,N<G->di(benzyloxycarbonyl)-L-arginine 1 was obtained from Na<->formyl-L-arginine by the method of Smithwick and Shuman (J. Org. Chem., 1974, 39» 3441) with a yield of 6256 or by formylation of H-Arg(Z2 )-0"K+ with formic/pivalic anhydride (60SÉ). (2) Oxazolone 2. HCO-Arg(Z2)-0H (1 mmol) was dissolved in dry THF-CH 2 Cl 2 (1:5) and ring-closed by treatment with DPECI.HCl salt (1.1 mmol) at 0<*>C for 2 hours. (3) , (4) and (5). Oxazolone 2 (1 mmol 1 dry THF at 0'C was acylated with the succinic hemiester chloride 3 (1.1 mmol) 1 in the presence of Et3N (1.2 mmol). The reaction mixture was stirred at 22<*>C 1 2 hours, evaporated and the residue dissolved in pyridln and treated with DMAP (20 mg) at 22<*>C 1 90 min. AcOH (2.5 ml was added and the red solution was set aside at 22<*>C 1 16 hours After evaporation and extraction of the crude product with ethyl acetate, chromatography on silica with EtOAc-petroleum gave the pure trichloroethyl ester 6 as a colorless oil (27% from 1).(6) Trichloroethyl ester The er group was removed from 6 (1.2 mmol) by treatment at 0<*>C in THF (25 mL) with Zn powder (2.4 g) and cold IM N8LH2PO4 (6.5 mL) in 2, 5 hours. The crude product was extracted with EtOAc and crystallized from EtOAc-petroleum to give pure HClO-Arg(Z2jK-Gly-OH, 7 { 82%). (7) The protected keto isosteric compound 7 (0.189 mmol) was converted to its Pfp ester by treatment with Pfp-OH (0.2 mmol) and DCCl (0.19 mmol) in CH 2 Cl 2 (2 mL) at 0< *>C for 1.5 hours. This Pfp ester was coupled at 0<*>C to H-Pro-OMe. HCl salt (1.1 mmol) 1 DMF containing <1>Pr2 NEt (2.5 equiv). Chromatography of the crude product in EtOAc on silica afforded pure HCO-Arg(Z2)--Gly-Pro-OMe 8 (80%) as a colorless oil. (8) (9) The formyl protecting group in 8 (0.13 mmol) was removed by treatment with 1M HCl/MeOH (30 mL) at 22°C for 16 h. Evaporation gave H-Arg(Z2)^-Gly-Pro-OMe.HCl, 9, which was dissolved in DMF and coupled with Boc-DPhe-Pro-OFfp 11 (0.144 mmol) at 0<*>C in the presence of < *>Pr2 NEt (0.27 mmol). The crude methyl ester 12 thus formed was purified by chromatography on silica in EtOAc and obtained as a colorless oil (70%). (10) (11) The methyl ester 12 (0.04 mmol) 1 MeOH (0.9 mL) was treated with 1M KOH (0.1 mL) for 1.5 h at 22<*>C to remove the ester group and a of the Z-protecting groups. The crude product was purified by hplc on Lichroprep RP18 (trade mark) in a gradient of MeOH and 556 AcOH to give the pure peptidic acid 13 (26 mg). The latter was dissolved in MeOH-ACOH-H2O (5:1:1) and hydrogenated over 596 Pd/C to give pure H-163. Tic and hplc show the presence of two epimers. Tic on silica in CHCl3-MeOH-AcOH (6:2:1)RF 0.19 and 0.22. After hydrolysis at 110°C/18 hours with 6N HCl, amino acid analysis: Pro, 2.0; Phew, 0.99.

Example II

(12) The Na<->Boc-protected peptide H-163 was treated with aqueous 2M HCl for 2 h at 22°C. The resulting unprotected peptide H-173 was obtained by lyophilization. Analysis: Pro, 2.05; Phe, 0.95.

Example III

(1) The protected keto isosteric compound 7 (0.189 mmol) was converted to its Pfp ester and coupled to H-Pro-NHEt (1.1 equivalents) in the same manner as described for the methyl ester in Example I step (7) for to gl the ethylamide .15 in 9556 yield. (2) (3) The formyl protected group was removed from 15 by treatment with 1M HCl/MeOH and the resulting Na-unprotected peptidamide 16 was acylated with the Pfp ester 11 as described 1 sections (8) and (9) in Example I, to give a mixture of epimers 17a and 17b (136 mg, 7556). 11 (4) The latter were separated by chromatography on silica in 556 MeOH-EtOAc, yielding 56 mg of the faster moving epimer 17a (Rf 0.35 on a silica-tlc plate in MeOH-EtOAc 1:10) and 60 mg of the slower epimer 17b (Rp 0.30). Synthesis of 17 by an independent route (see Scheme V) afforded the slow epimer 17b, which made it possible to determine the configuration of the epimers 17a (fast moving, contains D-arginine) and 17b (slow moving, contains L-arginine) . Hydrogenolysis of 17a over 556 Pd/C afforded the Boc-protected pentapeptide ethylamine H-170. Tic on silica in CHCl3-Me0H-Ac0H (6:2:1) Rp 0.39. Amino acid analysis after hydrolysis at 110<*>C/18 hours at 6N EC1: Pro 2.04; Phe 0.96.

Example IV

(4) Hydrogenolysis of the slow epimer 17b obtained in step (3) of the synthesis of H-170 gave Boc pentapeptide amide H-171. The latter was treated with 2N HCl at 22<*>C for 2 hours and lyophilized to give H-179. Tic on silica in CHCl3-Me0H-Ac0H (6:2:1) Rp 0.15. Amino acid analysis: Pro 2.00; Phew 1.00.

Example V

The synthesis of this compound was carried out according to scheme

III.

(1) H-Val-NHEt.HC1 (3 mmol) in CH2Cl2 at 0<*>C was acylated with Boc-Arg(Z2 )-0Pfp (2 mmol) in the presence of <ip>^NEt (2 mmol) for to give, after standard workup, the protected dlpeptide-ethylamide 25 (985É). (2) (3) 25 (1.87 mmol) was deprotected by treatment with HCl/EtOAc and acylated with Boc-Pro-OPfp (3 mmol) 1 DMF at 0"C 1 in the presence of <1>Pr2NEt ( 1.87 mmol).Usual work-up method gave the protected trlpeptld-ethylamld 27

(8096). (4) (5) 27 (0.587 mmol) was deprotected with BX1 /EtOAc and reacted in DMF solution at 0°C in the presence of <1>Pr2NEt with the Pfp ester 28 (prepared from 7 with DCCl) to to gel the protected pentapeptyl-ethylamide 30 (9596). (6) (7) 30 (0.17 mmol) was deformylated with 1N HCl/MeOH and the hydrochloride salt, 31 was isolated by evaporation and drying over KOH pellets. It was then reacted in DMF at 0 °C with the Pfp ester 11, in the presence of <1>Pr2NEt (2 equivalents). A mixture of epimers 32a and 32b was isolated from the reaction by standard methods. (8) (9) Epimers 32a and 32b was separated by chromatography on silica in MeOH-EtOAc (1:40) and the L-epimer 32a was hydrogenated in MeOH-AcOH-H2O (5:1:1) over 596 Pd/C to give the Boc-protected heptapeptide-ethylamide 33a. (10) 33a was treated with 2N HCl at 22°C for 2 hours. Lyophilization gave pure H-200. Tic on silica: RH 0.10 in CHCl3-MeOH-AcOH (6:2:1). Analysis: Arg 1.01, Phe 0.99, Pro 2.07, Val 0.83.

Example VI

This compound was prepared according to Scheme IV.

(1) Synthesis of Boc-Arg(Z2)-H 18 was carried out by the method according to A.Ito et al., (Chem. Pharm. Bull., 1975, 23. 3081) by reduction of Boc-Arg(Z2 )-0Me with diisobutyl aluminum hydride (<1>Bu2AlH) in toluene. The pure aldehyde 18 was obtained after purification by flash chromatography on silica with a yield of 5056. (2) Preparation of H-Gly-Pro-OBzl, 19. Boc-Gly-OH was carbonized via the mixed anhydride (prepared with isobutyl chloroformate and NMM) to proline benzyl ester. The resulting Boc-Gly-Pro-OBzl was treated with HCl/EtOAc to remove the Boc group, and the HCl salt of 19 thus formed was used in the reductive alkylation step (3). (3) Boc-Arg(Z2)HGly-Pro-0Bzl, 20. The aldehyde 18 (3 mmol) and H-Gly-Pro-OBzl 19 (3 mmol obtained from the HCl salt with NMM) in dry THF were allowed to react in the presence of a 5Å molecular sieve (10 g) at -10'C for 4 hours. The Schiff's base thus formed was reduced with NaCNBH 3 (3 mmol) in methanol at -10* C. The pure product 20 was isolated in 4456's yield by chromatography in EtOAc on silica. (4) 21. The reduced peptide 20 (0.6 mmol) in THF (40 mL) was acylated with 2,2,2-trichloroethoxycarbonyl chloride (Troc-Cl, 0.7 mmol) in the presence of NMM (0, 7 mmol) at -15'C. Silica gel chromatography of the crude product in EtOAc afforded pure 21 in 5856 yield. (5)

23

The protected reduced tripeptide 21 was treated with HCl/EtOAc to remove the Boc protecting group and the resulting N-Unprotected compound 22 (0.32 mmol) was acylated with Boc-DPhe-Pro-Opfp (0.32 mmol) in dry DMF in the presence of <1>Pr2NEt (0.32 mmol). Solid oxide chromatography of the crude product in EtOAc-benzene afforded pure 23 (4556).

(6) Removal of the Troc group from 23 to give the intermediate 24. The fully protected reduced penta-peptide 23 (0.03 mmol) was dissolved in glacial acetic acid (0.8 mL) and treated with zinc powder (0 .6 mmol) in an atmosphere of nitrogen for 3 hours. Chromatography of the crude product on silica gave pure 24 in a yield of 4796. (7) Preparation of H-172. 24 obtained in step (6) was dissolved in a mixture of MeOH-AcOH-B^O (5:1:1) and hydrogenated over 556 Pd/C for 3 hours. HPLC of the crude product on Partisil (trademark) 10 ODS II in 6496 MeOH- 2 O containing 0.296 formic acid gave pure H-172. Tic in EtOAc-Py-AcOH-^O (30:20:6:11) RH - 0.25 on silica. After hydrolysis at 110°C/18 hours with 6N HCl: Found Phe 0.96; Pro 2.04.

Synthetic. 1 euro. aer

The synthesis schemes referred to above are as follows:

Biological activity

Compounds were tested in vitro for the following activities using standard procedures: (a) Inhibition of human thrombin hydrolyzing the chromogenic substrate S-2238 (for method details, see M.F. Scully and V.V. Kakkar, Cl in. Chim Acta, 1977, 79, 595). Series of measurements were performed using a number of different inhibitor concentrations and at least two different substrate concentrations. The inhibition constant K was determined graphically using a Dixon plot (M. Dixon, Biochem. J., 1953, 5j>, 170). (b) Prolongation of kaolin/cephalin clumping time (KCCT; for procedure see D.E.G. Austen and I.L. Phymes: "Laboratory Manual of Blood Coagulation", Blackwell, Oxford 1975). The results are expressed as the molar concentration of inhibitor required to double the KCCT. (c) Inhibition of thrombin-induced platelet aggregation (for

the procedure see G.V.R. Born, Nature, 1962, 194. 927).

Representative results for Examples I-VI are shown in

the table.

Some of the compounds described herein have been tested in vivo for their ability to prolong clot formation time and have shown marked activity. These experiments were performed on rabbits using 0.1-4 mg/kg of the inhibitor and comparing KCCT or thrombin clot formation time taken before and at various intervals after administration of the inhibitors. H 179, for example, prolongs the clot formation time from 25 to 220 sec. at 2.35 mg/kg, and from 25 to 280 sec. at 4.7 mg/kg, and is comparable to heparin both in terms of efficacy and its in vivo half-life 1 plasma (18 min.; heparin 15 min.).

Application in treatment

As indicated earlier herein, the compounds of formula I can be used in the prevention or treatment of diseases accompanied by the unwanted formation of thrombi instead of e.g. the well-known use of heparin. Doses vary according to the potency of the inhibitor and the degree of efficacy desired, and their frequency with the lifetime of the inhibitor in the body, but are likely to be in the range of 0.01-10 mg/kg, eg, as unit doses of 0.75 mg - 0.75 g. Specifically, e.g. the compound H179 is given in 1 mg doses either parenterally or orally.

Claims (7)

1. Analogy method for the preparation of therapeutically active polypeptide analogues of the formula: where: X - H or a protecting group comprising lower alkyl (C1-C5) or R-O-CO-, where R - t-butyl; Y - D- or L-phenylalanine; Z - L- or D-proline; A - "keto" isostere (II) "hydroxy" isostere (III) "reduced" isostere (IV) where: with n - 2 - 4 (ii) R<2> - H, (lii) R<3> - any of the groups X which defined above, (iv) the configuration at the asymmetric centers<*> are either R or S, Arg may be absent; B - D- or L-valine or absent; W - -OH or NH-(CH2)n, where n - 0-5; characterized by that an acid X-Y-Z-OH reacts with an amine H-A-Pro-Arg-B-W. 2. Analogy method according to claim 1 for production of characterized by using correspondingly substituted starting materials. 3. Analogy method according to claim 1 for production of characterized by using correspondingly substituted starting materials. 4. Analogy method according to claim 1 for production of characterized by using correspondingly substituted starting materials. 5. Analogy method according to claim 1 for production of characterized by using correspondingly substituted starting materials. 6. Analogy method according to claim 1 for production of k a-rect, rice serted by using correspondingly substituted starting materials. 7. Analogy method according to claim 1 for production of character cert by using correspondingly substituted starting materials.