HU224426B1 - Peptidyl-arginin-aldehyde derivatives of thrombin- and xa-factor-inhibiting activity and pharmaceutical compositions containing them and their use - Google Patents

Abstract

The subject of the invention is the new peptidyl-arginine-aldehyde derivatives of the general formula (I) – Q-D-Xaa-Pro-Arg-H (I) where Q is an acyl group of the formula Q'–O–CO–, in which formula Q' is 1–3 carbon atoms alkyl group, D-Xaa means D-cyclohexylglycine or D-cyclopentylglycine radical, Pro means L-proline radical and Arg means L-arginine radical - and their acid addition salts formed with organic or inorganic acids, as well as pharmaceutical preparations containing the above compounds are trained. The compounds of general formula (I) according to the invention have a valuable medicinal, more specifically anti-coagulation effect.

Description

(54) Peptidyl-arginine aldehyde derivatives having thrombin and factor Xa inhibitory activity, pharmaceutical compositions containing them and their use

HU 224 426 Β1 (57) Extracts

The present invention relates to novel peptidyl-arginine aldehyde derivatives of formula (I): Q-D-Xaa-Pro-Arg-H (I) wherein

Q is an acyl group of the formula Q'-O-CO- in which

Q 'is a C 1-3 alkyl group, D-Xaa is a D-cyclohexylglycine or D-cyclopentylglycine radical,

Pro is an L-proline residue and

Arg is the L-arginine residue and its acid addition salts with an organic or inorganic acid, and pharmaceutical compositions containing the above compounds.

The compounds of formula (I) according to the invention have valuable therapeutic properties, in particular anticoagulant activity.

The scope of the description is 16 pages

HU 224 426 Β1

The present invention relates to novel peptidyl-arginine aldehyde derivatives of formula (I): Q-D-Xaa-Pro-Arg-H (I) wherein

Q is an acyl group of the formula Q'-O-CO- in which

Q 'is C 1-3 alkyl, D-Xaa is D-cyclohexylglycine or D-cyclopentylglycine,

Pro is an L-proline residue and

Arg is the L-arginine residue and its acid addition salts with an organic or inorganic acid, and pharmaceutical compositions containing the above compounds.

The compounds of formula (I) of the present invention have valuable therapeutic properties, in particular anticoagulant activity, which is associated with a significant inhibition of platelet function and thrombosis formation.

Particularly valuable examples of the compounds of formula I according to the invention are: ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde, methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L -arginine aldehyde, ethoxycarbonyl-D-cyclopentylglycyl-L-prolyl-L-arginine aldehyde, and acid addition salts thereof (primarily hydrogen sulfate).

definitions

The abbreviations of the amino acids and substituents herein, as well as the peptides formed from them, correspond to those commonly used in the peptide chemical literature.

Amino acids: Arg = L-arginine [(2R) -2-amino-5-guanidino-pentanoic acid], Asp = L-aspartic acid [(2S) -2-amino-3-carboxypropionic acid], boroArg = L-boro-arginine [ (1R) -1-amino-4-guanidino-butylboronic acid], D-Chg = D-2-cyclohexylglycine [(2R) -2-aminocyclohexylacetic acid], D-Cpg = D-2-cyclopentylglycine [ (2R) -2-Amino-3-cyclopentylacetic acid, Gla = gamma-carboxy-L-glutamic acid [(2S) -2-amino-4,4-dicarboxy-butyric acid], Glu = L-glutamic acid [(2S) -2 -amino-4-carboxy-butyric acid], Gly = L-pyroglutamic acid [(5S) -2-pyrrolidone-5-carboxylic acid], Gly = glycine (2-aminoacetic acid), D-MePhe = N-methyl-3- phenyl-D-alanine [(2R) -2-methylamino-3-phenylpropionic acid], D-MePhg = DN-methylphenylglycine [(2R) -2-amino-2-phenylacetic acid], Nal (1) = 3- (naphthyl-1) -L-alanine [(2S) -2-amino-3- (naphthyl-1) -propionic acid], D-Phe = 3-phenyl-D-alanine [(2R) ) -2-amino-3-phenylpropionic acid], Pro = Lproline [(2S) -pyrrolidine-2-carboxylic acid].

Substituents: Ac = acetyl, Boc = tert-butoxycarbonyl, Bz = benzoyl, Eoc = ethoxycarbonyl, Et = ethyl, iPoc = isopropoxycarbonyl, Me = methyl, 4MeP = 4-methylpentanoyl, Moc = methoxy- carbonyl, pNA = p-nitrophenylamino, Poc = propoxycarbonyl, Tos = p-toluenesulfonyl, Z = benzyloxycarbonyl.

Peptides and Derivatives: Abbreviations of amino acids per se denote L-amino acid. The D-amino acid is separately designated, for example, 3-phenyl-D-alanine-D-Phe. The dash before and after the amino acid abbreviations indicates that the amino group has one H atom and the carboxyl group one OH group. Accordingly, Eoc-D-Chg-Pro-Arg-H is ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde and BzHe-Glu-Gly-Arg-pNA is benzoyl-Lizoleucyl -L-glutamyl-glycyl-L-arginine-p-nitroanilide.

Coagulation is part of the body's defense mechanism. When a blood vessel is injured, the process starts and blood clots are formed to prevent bleeding. In addition, vascular disease, congestion in the blood, and abnormal activation of coagulation factors can cause coagulation. In this case, a blood clot (thrombus) is formed within the blood vessel, which breaks it or breaks it, thrombosis. Another part of the defense mechanism is fibrinolysis. The enzymes that work in it remove excess blood clots and are involved in thrombolysis and thrombus dissolution.

The process of blood coagulation is a series of catalyzed enzymatic reactions, a cascade reaction in which plasma proteins, so-called coagulation factors, are activated sequentially. Factors are denoted by Roman numerals and the active form is indicated by the letter. In some cases the trivial name (also) is used: for example, fibrinogen = factor ll (fi), fibrin = factor ll (son), prothrombin = factor ll (phi) and thrombin = factor ll (flla). During coagulation, serine proteases (fXIIa, fVIIa, fXIIa, fIXa, fXa and thrombin), some accelerating cofactors (fVa and fVIIIa), and the coagulant substance (fibrin) are produced. Of the resulting proteases, fXa and thrombin are the last two. FXa produces thrombin which cleaves fibrinogen to form a fibrin clot.

Previous mechanism of blood coagulation [R. G. MacFarlane, Naturre 202, 498 (1964); E. W. Davie and O. D. Ratnoff, Science 145, 1310 (1964)], fX is activated by two pathways, the so-called internal or external pathways. In the former case, the fXII (fXlla) activated on some surface initiates the process by the fXI-> fXla conversion followed by the fIX-> flXa conversion; fX is activated by fIXa. Externally, the process begins with the appearance of a cell surface receptor, the tissue factor (TF), and the formation of the [fVIITF] and [fVlla-TF] complex. Activation of fX is effected by the [fVlla-TF] complex.

According to recent results, coagulation in the living body is a combination of the above [E. W. Davie et al., Biochemistry 43: 10363 (1991)], and the most important steps are as follows.

1. In the event of an injury or disease of the vascular wall, TF is exposed to the surface and binds all of Factor VII in the blood. The resulting [fVIITF] complex is converted to an active [fVIIa-TF] enzyme complex by activation of a suitable protease (such as fXIIa, fXa, fIXa and thrombin), which activates a small fraction of plasma Factors IX and X fIXa and fXa), then inactivated by TFPI (Tissue Factor Pathway Inhibitor, formerly known as LACI (Lipoprotein-Associated Coagulation Inhibitor)), a common inhibitor of plasma fXa and [fVlla-TF] complex [T. J. Girard et al., Natur. 338, 518-520 (1989).

2. The resulting fIXa with the factor X and the fVllla accelerator on the phospholipid (PL) surface, Ca ⁺⁺

In the presence of Β1, it creates the so-called “tenase complex: [flXafVlllafXPLCa ⁺⁺ ], in which fX is activated, fXa is formed.

3. The resulting fXa forms a so-called prothrombinase complex with the prothrombin (fii) and fVa accelerator structure [fXatVafllPLCa ⁺⁺ j. This is where the prothrombin to thrombin conversion takes place. The conversion of fV> fVa and fVIII -> fVllla can be accomplished by fXa or thrombin.

4. The little thrombin produced converts some of the fXI to the fXIa enzyme and activates some of the factor VIII and V to produce another fVIIIa or fVa. Now, fXIa is converting factor IX to fIXa. This can restart the chain reaction from the Xase complex to the formation of thrombin. As the process repeats itself, more and more thrombin is produced.

5. At sufficiently high thrombin concentrations, partial proteolysis of the fibrinogen dissolved in plasma occurs, the formation of the fibrin monomer, which is first associated with the so-called soluble fibrin polymer and then converted to the insoluble fibrin polymer. Thrombin also plays a role in the latter transformation, as it results in the formation of polymerization fXllla [L. Lorand and K. Konishi, Arch. Biochem. Biophys. 105, 58 (1964)].

The insoluble fibrin polymer is one of the major components of the blood clot and thrombus, and the other component is the platelet aggregate, which is also produced primarily by thrombin. The resulting thrombus or blood clot encapsulates a significant portion of the thrombin produced during the process, which initiates another clotting process when it is dissolved / dissolved in the thrombus [A. K. Gash et al., Am. J. Cardiol. 57: 175 (1986)]; R. Kumar et al., Thromb. Haemost. 72: 713 (1994)].

The foregoing highlights the key role of thrombin in thrombus formation. Therefore, substances that inhibit the function and / or production of thrombin are of particular importance in the treatment of thrombotic diseases.

Heparins and vitamin K antagonist coumarins (such as Syncumar and Warfarin), which indirectly inhibit thrombin, are currently the most widely and successfully used formulations for treating and preventing thrombosis.

Heparin catalyses the reaction between thrombin and its natural inhibitor, antithrombin-III (ΑΤ-III). However, this effect of heparin is absent if plasma concentrations of ΑΤ-III are less than 75% of normal [R. Egbring et al., Thromb. Haemost. 42, 225 (1979). Not least, this indirect mechanism cannot inhibit thrombin-bound thrombin because it is not accessible to the heparin-AT-III complex [J. I. Weitz et al., J. Clin. Invest. 86: 385 (1990)]. However, the occurrence of bleeding complications resulting from treatment and thromboembolic events due to the immune process is not negligible (see, for example, J.M. Walenga et al., Clin. Appl. Thrombosis / Hemostasis 2 (Suppl. 1), S21-S27 (1996)].

Vitamin K antagonists may also be administered orally. They act after 16 to 24 hours and are shown to inhibit the formation of reactive Gla-containing forms of some clotting factors (such as prothrombin). The therapeutic effect requires partial inhibition (60-70%) [Μ. P. Esnouf and C. V. Prowse, Biochim. Biophys. Acta 490, 471 (1977)], which can be achieved by proper administration of the drug. The use of vitamin K antagonists is complicated by the narrow therapeutic band and the high dependence on food composition (vitamin K) and variable individual sensitivity.

The first high potency synthetic substance to directly inhibit thrombin was the D-Phe-Pro-Arg-H tripeptide aldehyde, a reversible inhibitor and showed significant anticoagulant activity both in vitro and in vivo [S. Bajusz et al., In: Peptides: Chemistry, Structure and Biology (R. Walter and J. Meienhofer, Eds.), Ann Arbor Publ., Ann Arbor, Michigan, USA, 1975, p. 603-608; Int. J. Peptide Protein Res. 12, 217 (1978)]. Several related compounds of D-Phe-Pro-Arg-H have been prepared. Boc-D-Phe-Pro-Arg-H [S. Bajusz et al., Int. J. Peptide Protein Slit. 12, 217 (1978)] and the chloromethyl ketone analog (D-Phe-Pro-Arg-CH ₂ Cl), which has been shown to be an irreversible inhibitor [C. Kettner and E. Shaw, Thromb. Gap. 14, 969 (1979)]. Among these, we highlight the boro-arginine analogs of the peptide and its acyl compounds (D-Phe, Boc-D-Phe and Ac-D-Phe-Pro-boroArg), which are potent reversible inhibitors of thrombin [C. Kettner et al., J. Bioi. Chem. 265, 18289 (1990)] and other Boc-D-Phe-ProArg-H analogs, including Boc-D-Chg-ProArg-H analogue (PD Gesellchen and RT Shuman, EP 0 479 489 A2 (1992)). ].

D-Phe-Pro-Arg-H was found to be prone to spontaneous conversion in aqueous solution, but D-MePhe-Pro-Arg-H (GYKI-14,766) obtained by methylation of the terminal amino group was already sufficiently stable and similarly potent. Bajusz et al., U.S. Patent 4,703,036 (1987); J. Med. Chem. 33, 1729 (1990)]. It significantly inhibits coagulation and thrombus formation in experimental animals [D. Bagdy et al., Thromb. Haemost. 67, 357 and 68, 125 (1992); J. V. Jackson et al., J. Pharm. Ex. Ther. 261, 546 (1992)]; the inhibitory effect on fibrinolysis enzymes is insignificant, when co-administered with thrombolytics, it effectively promotes thrombus dissolution [C. V. Jackson et al., J. Cardiovasc. Pharmacol. 21, 587 (1993)], which the heparin-AT-III complex is incapable of. Syntheses of related compounds of D-MePhe-Pro-Arg-H have also been synthesized, for example, D-MePhg-Pro-Arg-H [R. T. Shuman et al., 1993, J. Med. Chem. 36, 314].

The anticoagulant effect (inhibition of the proteolytic reactions of the process) is measured in coagulation assays such as thrombin time (TT-), activated partial thromboplastin time (APTT) and prothrombin time (PT) assay (EJW Bovie et al., Mayo Clinic Laboratory Manual of Hemostasis; WB Sanuders Co., Philadelphia, 1971). Plasma inhibited by spontaneous coagulation, such as citrate plasma, is induced to coagulate and measured

EN 224 426 Β1 clotting time. The effect of anticoagulants is to prolong the coagulation time in proportion to the inhibition of the systemic reaction (s). For example, the anticoagulant effect is characterized by the concentration of the substance at which the coagulation time is doubled (IC ₅₀ ). The effect of anticoagulants on individual coagulation proteases is measured by the so-called amidolytic method [R. Lottenberg et al., Methods Enzymol. 80, 341 (1981); G. Cleason: Blood Coagulation and Fibrinolysis 5, 411 (1994)]. The isolated active factor (e.g., thrombin or fXa) and its chromogenic or fluorogenic peptide amide substrate are reacted in the absence or presence of the inhibitor. The enzyme inhibitory activity is characterized, for example, by the inhibition constant (IC ₅₀ ) as measured by amidolysis.

In the TT test, thrombin added to the citrate plasma causes coagulation. Systemic thrombin is 22 pmol / ml and its inhibition can be measured on the presence of fibrinogen (a natural substrate for thrombin) in the presence of plasma components. In the APTT and PT tests, the whole coagulation process takes place. Depending on the activator, fX is activated internally or externally. The resulting fXa activates prothrombin to thrombin, which eventually causes plasma clotting. Clotting time is prolonged when the inhibitor inhibits the enzymes in the assays or one of them. While up to 40 pmol / ml of Factor Xa can be produced in the APTT and PT assays (the amount of Factor X present in the two systems), thrombin may have a concentration of about 10 pmol / ml. 150 pmol / ml (APTT) and 350 pmol / ml (PT) are formed [cf. B. Kaiserés et al., Thromb. Gap. 65: 157 (1992)].

In the case of D-MePhe-Pro-Arg-H (C1), the double clotting time concentrations in the TT, APTT and PT tests were 87, 622 and 2915 nM, respectively. These values and the amount of thrombin present in the assays (22, 150 and 350 pmol / ml, respectively) increase similarly, suggesting that C1 acts as a thrombin inhibitor in both the APTT and PT assays and, for example, has little or no effect on the function of fXa. Consistent with this, C1 IC inhibits the amidolytic effect of thrombin on the Tos-Gly-Pro-Arg-pNA substrate at ₅₀ = 2 nM, while the amidolytic effect of fXa on the corresponding Bz-Ile-Glu-Gly-ArgpNA substrate is limited. ₅₀ = 9.1 μΜ (Bajusz S. et al., Unpublished results).

It follows from the mechanism of blood coagulation that the process is inhibited not only by direct inhibitors of thrombin, but also by inhibitors of thrombin formation, such as fXa inhibitors. An example of a fXa inhibitor with significant anticoagulant and thrombus inhibiting activity is the 60-membered polypeptide isolated from the tick, Tick Anticoagulant Peptide [L. Waxman et al., Science 248, 593 (1990); Kelly et al., Circulation 86, 1411 (1992)] and DX-9065a (C2), a synthetic non-peptide: (+) - (2S) -2- [4 [[(3S) -1-acetimidoyl-3- pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] -propionic acid hydrochloride pentahydrate [T. Hara et al., Thromb. Haemost. 71, 314 (1994); T. Yokoyama et al., Circulation 92, 485 (1995)]. These substances strongly inhibit the amidolytic activity and plasma clotting of fXa in the PT and APTT assays. That is, they inhibit both free and complex factor Xa well. However, as specific fXa inhibitors, they do not inhibit thrombin and do not work in the TT assay. Examples of fXa synthetic peptide inhibitors include 4MeP-Asp-Pro-Arg-H (C3) (WO 93/15756) and Boc-D-PheNal (1) -Arg-H (C4) (WO 95 / 13693). C3 and C4 are reported to inhibit the amidolytic effect of fXa on ZD-Arg-Gly-Arg-pNA substrate at ₅₀ = 57 and 30 nM; no data are available on their anticoagulant activity. In our own experiments the Bz-lle-Glu-Gly-Arg-pNA substrate also showed significant inhibition on C3 and C4, while their anticoagulant activity in plasma coagulation assays is extremely low. Table 1 shows the activity values reported (C2) and obtained (C3-C4) for the above synthetic fXa inhibitors as compared to C1 antiplatelet data.

The above data indicate that the anticoagulant effect observed in the PT and APTT assays is due to inhibition of thrombi in C1 and inhibition of fXa in C2. However, the significant fXa inhibitory activity of C3 and C4 is not accompanied by significant anticoagulant activity. It is likely that C3 and C4 do not have access to the active center of fXa in the prothrombinase complex, these peptides being able to inhibit only the free, soluble factor Xa.

Table 1

Effects of known synthetic inhibitors Factor Xa (A) and anticoagulant (B)

inhibitor A: IC ₅₀ , nM ^b 6: IC ₅₀ , μΜ ³ PT APTT TT C1 9133 2.91 0.62 0.09 C2 70 0.52 0.97 NA ^C C3 64 19.32 4.59 0.87 C4 86 53.62 9.96 17,24

^the peptide concentration at which the coagulation time extending the control doubling of the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) test. ^b Measured on chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA with isolated human factor Xa.

^c NA = not active.

According to another announcement, [N. A. Prager et al., Circulation 92, 962 (1995)], not only thrombin but also factor Xa, which are trapped in the thrombus / blood clot and released during dissolution, contribute to the initiation and maintenance of a new coagulation process, e.g. fVII TF] complex and activation of the V and Vili factor. Thus, it is preferred that the anticoagulants are capable of inhibiting factor Xa in addition to thrombin, and it is particularly preferred that the anticoagulant effect also extends to the thrombin and factor Xa trapped in the coagulant.

It is an object of the present invention to provide novel peptide derivatives which are known in the art for coagulation

They are more potent inhibitors, and their inhibitory effect is also apparent with oral administration.

We previously observed that Boc-D-PhePro-Arg-H (C5), although less potent in the TT and APTT assays than in the C1 tripeptide aldehyde, was slightly more active in the PT assay, and and inhibits Factor Xa with an IC 50 10-fold lower than C1. It has now surprisingly been found that replacing the Boc group of C5 with the Eoc group advantageously modifies the anticoagulant activity of the peptide, since the Eoc-D-Phe-Pro-Arg-H (C6) thus produced is more potent than C5 in all three assays. , and C1 activity is no longer present in the PT but also in the APTT assay, while its factor Xa inhibitory activity is similar to that of the C5 Boc peptide. The anticoagulant activity of Moc-D-Phe-Pro-Arg-H (C7) is similar to that of C6 Eoc, but slightly greater than that of factor Xa. Table 2 illustrates what has been said.

Table 2

R-D-Phe-Pro-Arg-H Peptide Aldehydes: Anticoagulant (A) and Factor Xa (B) Inhibition

Peptide aldehydes A- ιθ5ο> μΜ ³ Β: Woman. R TT ΑΡΤΤ PT IC ₅₀ , μΜ » C1 Me 0.09 0.62 2.93 9.13 C5 Boc 0.20 0.76 2.27 0.91 C6 EOC 0.13 0.38 1.91 0.92 C7 moc 0.20 0.29 1.96 0.13

^the concentration of peptide at which the clotting time is doubled.

^b Measured on isolated Bz-11-Glu-Gly-Arg-pNA chromogenic substrate with human factor Xa (see Method M6).

It has further been found that the beneficial influence of the Eoc group is not limited to the D-Phe-Pro-Arg-H sequence, for example the known D-Chg-Pro-Arg-H sequence [Boc-D-Chg-Pro-Arg -H (C8), PD Gesellchen and RT Shuman (EP 0479489 A2 (1992)), the replacement of the Boc group with the Eoc group enhances the anticoagulant effect. Similarly remarkable results were obtained using the D-Cpg-Pro-Arg-H sequence.

Accordingly, the present invention provides novel peptidyl-arginine aldehyde derivatives of formula (I): Q-D-Xaa-Pro-Arg-H (I) wherein

Q is an acyl group of the formula Q'-O-CO- in which

Q 'is C 1-3 alkyl, D-Xaa is D-cyclohexylglycine or D-cyclopentylglycine,

Pro is an L-proline residue and

Arg is the L-arginine residue and its acid addition salts with an organic or inorganic acid, and pharmaceutical compositions containing the above compounds.

The compounds of formula I in which Q, Q 'and D-Xaa, Pro and Arg are as defined above are prepared, for example, by condensing a acyl dipeptide QD-Xaa-Pro on the guanidino group with benzyloxy. -glycarbonyl-protected arginine lactam, the resulting protected tripeptide lactam is reduced to the protected tripeptide aldehyde QD-Xaa-Pro-Arg (Z) -H, and finally the Z group is removed from the guanidino group of arginine and (I) ) is isolated in the form of an organic or inorganic acid addition salt.

For example, the novel Q-D-XaaPro acyl dipeptide used as starting material is prepared by acylation of the corresponding D-Xaa amino acid with a Q 'alkyl ester of chloro carbonic acid in basic medium and coupling the resulting Q-D-Xaa acyl amino acid with L-proline.

Preferably, the acyl amino acid QD-Xaa required for coupling with L-proline can also be prepared by acylating the DL-Xaa racemic compound and converting it to the methyl ester, followed by the resulting Q-DL-Xaa-OMe racemic acyl amino acid methyl ester. QD-Xaa-OMe obtained after enzymatic hydrolysis is saponified to the desired QD-Xaa acyl amino acid.

The compounds of formula I according to the invention, wherein Q, D-Xaa, Pro and Arg are as defined above, exhibit potent anticoagulant activity both in vitro and in vivo, with favorable bioavailability.

The in vitro anticoagulant activity of the compounds of formula I in the Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Thrombin Time (TT) assays [D. Bagdy et al., Thromb. Haemost. 67, 325 (1992)]. We also determined the fXa inhibitory activity of the compounds on the Bz-11-Glu-Gly-Arg-pNA chromogenic substrate as well as the plasmid inhibitory activity by the so-called fibrin plate method [D. Bagdy et al., Thromb. Haemost. 67: 325 (1992)]. The results are shown in Table 3.

HU 224 426 Β1

Table 3

New peptide derivatives Q-D-Xaa-Pro-Arg-H (I) and control compound of similar structure (A), factor Xa inhibitor (B) and plasmin inhibitor (C) in descending order of PT activity

Q-D-Xaa-Pro-Arg-H A: IC ₅₀ , μΜ * β; IC ₅₀ , nM ^c C: IC ₅₀ , μΜ « Number ^b Q Xaa PT APTT TT 3 EOC CPG 1.15 0.36 0.40 44 83 1 EOC Chg 1.16 0.31 0.23 27 97 2 moc Chg 2.19 0.50 0.33 100 189 control Compound C8 Boc Chg 2.02 0.92 0.24 22 81

^the peptide concentration at which the coagulation time extending the control doubling of the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) test.

^b Identical to the number of the exemplifying embodiment of Compound a or to the Control Compound (C1, C3).

^c Measured on isolated Bz-11-Glu-Gly-Arg-pNA chromogenic substrate with human factor Xa (see Method M6).

^d Peptide concentration at which the lysis area is 50% of control.

The data indicate that the new Eoc derivative 1 exhibits a greater anticoagulant activity than the known C8 Boc compound in the PT and APTT assays, whereas the TT assay activity and factor Xa inhibitory activity are similar for the two compounds.

The plasma-clotting factor Xa and anti-thrombotic activity of the compounds of formula (I) and the fibrin-gel-bound thromboembolic activity are shown in Table 4 for Examples 1, 2 and 3; the corresponding values for C1 and C3 are shown as controls. Plasma clot was obtained by recalcination of plate-rich human citrate plasma, and fibrin gel was cleaved with human fibrinogen human thrombin, and ZD-Arg-Gly-Arg-pNA and Tos-GlyPro-Arg-pNA . (see the "Methods" section).

Table 4

Inhibitory activity (IC ₅₀ , μΜ) of the novel peptidyl arginine aldehyde and C8 as a control compound of the invention on factor Xa and thrombin and fibrin gel-bound thrombin ³

Peptidyl Arginine Aldehyde (Number ^B ) Plazmaalvadék fibrin Factor Xa thrombin thrombin Eoc-D-Chg-Pro-Arg-H (1) 0.96 0.17 0.08 Moc-D-Chg-Pro-Arg-H (2) 1.39 0.61 0.27 Eoc-D-Cpg-Pro-Arg-H (3) 0.67 0.51 0.78 control Compound Boc-D-Chg-Pro-Arg-H (C8) 1.69 0.79 0.35

The activity of factor Xa and thrombin ^on the IC ₅₀ values by determining the measured Moc-D-Chg-Gly-Arg-pNA or Tos-Gly-Pro-Arg-pNA substrate (see M1-M5. method).

^b Identified by the number of the exemplary embodiment describing the new compound and the control compound (C8).

The data show that the novel compounds closed factor Xa and human thrombin lemezkedús in plasma as well as the ability to inhibit both thrombin bound to human fibrin under micromoles (nanomolar range) _IC50 -értékkel. Both coagulated factor Xa and thrombin were inhibited by a compound with a lower IC ₅₀ than C8, indicating that the Eoc group is a more preferred substituent in this respect than the Boc group.

The anticoagulant and platelet aggregation inhibitory activity of the compounds of formula (I) was tested ex vivo on New Zealand white rabbits [D. Bagdy

HU 224 426 Β1 et al., Thromb. Haemost. 67: 357 (1992)].

The compounds were dissolved in buffered saline by intravenous injection (0.04-5.0 mg / kg) or infusion (0.25-5.0 mg / kg / h) or 0.5-6.0 mg / kg. subcutaneously or at a dose of 2.5 to 20 mg / kg orally. The effect of the compounds can be demonstrated <30 minutes after oral administration, the maximum value being 60-180. and the therapeutic effect is maintained in a dose-dependent manner for 3-> 6 hours.

In particular, the in vivo effect of ethoxycarbonyl-D-cyclohexylglycyl-10-cil-L-prolyl-L-arginine aldehyde (1) at oral doses of 10 mg / kg and 5 mg / kg is demonstrated.

5-6 and 7-8. table; wherein the corresponding values found for C1 are shown as controls. After administration of the compounds, blood samples were taken from the ear vein of the animals every 30-60 minutes. Whole blood coagulation time (WBCT) and the degree of inhibition of thrombin-induced platelet aggregation (PAI) were determined. The activated partial thromboplastin time (APTT) and the thrombin time (TT) of the citrate plasma obtained from the blood sample were also determined. The APTT and TT values are shown in Tables 5 and 7, while the WBCT and PAI values are shown in Tables 6 and 8.

Table 5

The anticoagulant effect of Eoc-D-Chg-Pro-Arg-H and D-MePhe-Pro-Arg-H in rabbits at oral doses of 10 mg / kg, characterized by relative coagulation times in the APTT and TT test ³

Time (p) Eoc-D-Chg-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (C1) APTT TT APTT TT 0 1.0 1.0 1.0 1.0 30 2.24 ± 0.61 15.46 ± 4.84 2.07 ± 0.38 7.89 ± 4.13 60 2.59 ± 0.53 20.18 ± 3.44 2.30 ± 0.31 19.27 ± 3.60 120 3.11 ± 0.73 24.31 ± 2.25 1.93 ± 0.14 17.21 ± 4.73 180 2.72 ± 0.68 27.31 ± 2.32 1.75 ± 0.17 7.23 ± 2.00 240 2.26 ± 0.48 14.92 ± 4.12 1.61 ± 0.15 3.77 ± 1.25 300 2.09 ± 0.39 11.67 + 3.70 1.25 + 0.22 2.36 ± 1.21 360 1.81 ± 0.45 8.30 ± 4.86 - -

^θ Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

Table 6

Eoc-D-Chg-Pro-Arg-H and D-MePhe-Pro-Arg-H inhibit the anticoagulant and platelet aggregation (PAI) effects in rabbits at a relative dose of 10 mg / kg in the WBCT assay and characterized by% inhibition ³

Time (p) Eoc-D-Chg-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (R1) WBCT PAI (%) WBCT PAI (%) 0 1.0 0 1.0 0 30 1.61 ± 0.18 81.5 ± 14.8 1.38 ± 0.16 89.0 ± 7.5 60 1.77 ± 0.20 77.0 ± 17.0 1.49 + 0.13 96.5 ± 2.2 120 1.96 ± 0.19 99.0 ± 1.0 1.45 ± 0.09 100.0 ± 0.0 180 1.69 ± 0.14 98.0 ± 2.0 1.25 ± 0.08 94.0 ± 3.7 240 1.65 ± 0.19 88.5 ± 11.5 1.00 ± 0.18 81.8 ± 14.0 300 1.71 ± 0.18 93.0 ± 7.0 1.09 + 0.06 55.0 ± 29.3 360 1.22 ± 0.17 72.0 ± 25.0 - -

^a Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

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Table 7

Anticoagulant activity of Eoc-D-Chg-Pro-Arg-H and D-MePhe-Pro-Arg-H in rabbits at 5 mg / kg oral dose, characterized by relative coagulation times in the APTT and TT test ³

Time (p) Eoc-D-Chg-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (C1) APTT TT APTT TT 0 1.0 1.0 1.0 1.0 30 1.38 ± 0.27 6.21 ± 4.36 1.27 ± 0.38 1.52 & 0.17 45 1.53 ± 0.33 9.83 ± 5.48 1.30 + 0.02 2.50 ± 0.44 60 1.59 ± 0.38 10.55 ± 5.54 1.37 ± 0.03 4.75 ± 1.38 120 1.85 ± 0.25 14.10 ± 5.46 1.43 ± 0.11 5.51 ± 3.59 180 1.78 + 0.18 10.77 + 4.47 1.39 ± 0.09 2.05 ± 0.38 240 1.72 ± 0.20 6.32 ± 2.30 1.31 ± 0.11 1.81 ± 0.39

^a Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

Table 8

Eoc-D-Chg-Pro-Arg-H and D-MePhe-Pro-Arg-H inhibit the blood coagulation and platelet aggregation (PAI) activity in rabbits at 5 mg / kg oral dose with the relative coagulation time found in the WBCT test. characterized by% inhibition ³

Time (p) Eoc-D-Chg-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (C1) WBCT PAI (%) WBCT PAI (%) 0 1.0 0 1.0 0 30 1.18 ± 0.02 63.2 + 13.2 1.07 ± 0.13 52.8 ± 14.7 45 1.23 ± 0.09 58.2 ± 12.0 1.26 ± 0.07 58.4 ± 15.6 60 1.42 ± 0.09 73.8 + 16.5 1.55 & 0.17 54.2 ± 11.6 120 1.69 + 0.10 78.4 ± 13.5 1.45 ± 0.21 75.2 ± 12.3 180 1.45 ± 0.19 88.6 ± 17.4 1.44 ± 0.08 47.5 ± 20.9 240 1.13 ± 0.08 91.8 ± 7.6 1.22 + 0.08 32.2 ± 16.0

^a Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

The data in the tables show that anticoagulant 1 is preferred over C1 in terms of both magnitude and duration of action. 45

The compounds of the invention (I) are used for the treatment and prevention of the following conditions associated with thrombosis and / or increased blood coagulation: deep vein thrombosis, pulmonary embolism, arterial thrombosis, unstable angina, myocardial death, atrial fibrillation and thrombosis. They can also be used in the prevention of thrombotic diseases of the coronary artery and cerebral artery in the case of atherosclerosis, for the surgical prophylaxis of high-risk patients and for other surgical prophylaxis. In addition, they are useful in the prevention of thrombolysis or percutaneous transluminal angioplasty, and in the treatment of hemodialysis and adjunctive therapy in nephrosis syndrome, and in the treatment of diseases associated with hypercoagulability, such as cancer, inflammation, They may also be used in cases where the administration of other anticoagulants is ineffective or contraindicated: for example, in the case of heparin, lack of antithrombin-III or HIT (for heparin-induced thrombocytopenia) or, for example, in the case of coumarins, gravity.

For therapeutic purposes, the compounds of the present invention and their pharmaceutically acceptable salts may be used alone or conveniently in the form of a pharmaceutical composition. Such compositions are also included within the scope of the present invention.

These pharmaceutical compositions contain as an active ingredient an amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in order to exert its effect in a pharmaceutically acceptable carrier known in the art.

EN 224 426 Β1 with excipients, diluents and / or other pharmaceutical excipients.

Suitable carriers, diluents or fillers as described above include water, alcohols, gelatin, lactose, sucrose, starch, pectin, magnesium stearate, stearic acid, talc, various animal or vegetable oils, and glycols such as propylene glycol or polyethylene glycol. The pharmaceutical excipients include preservatives, various natural or synthetic emulsifying, dispersing or wetting agents, coloring agents, flavoring agents, buffering agents, disintegrating agents, and other agents that promote the bioavailability of the active ingredient.

The pharmaceutical compositions of the invention may be in conventional forms. Examples of such common dosage forms include oral (oral) formulations (tablets, capsules, powders, pills, dragees or granules) and parenteral (gastro-intestinal by-products), suppository, patch or ointment).

Although the dose of the compounds of the present invention required to exert a therapeutic effect will vary depending upon the individual condition and age of the patients being treated, it may vary and will ultimately be determined by the attending physician; for the prevention and / or treatment of diseases in which inhibition of thrombin function and / or production is desirable, generally from about 0.01 mg to about 1000 mg, and preferably from 0.25 mg to 20 mg, per kg of body weight of these compounds. daily or parenteral, for example, intravenous doses.

When administered in combination with thrombolytics (e.g., tPA or urokinase), the compounds of formula (I) of the present invention can effectively promote dissolution of thrombi formed in arteries or veins or effectively inhibit thrombus re-formation. In such cases, the compounds of the invention may be administered concurrently with the thrombolytics or following thrombolytic therapy.

The invention is illustrated by the following non-limiting examples.

The R _r values in the examples were determined by layer chromatography on silica gel (DC-Alufolien Kieselgel 60 F ₂₅ 4, Merck, Darmstadt) in the following solvents:

1. Ethyl acetate

2. Ethyl acetate-pyridine-acetic acid-water (480: 20: 6: 11)

4. Chloroform-methanol (9: 1)

6. Ethyl acetate-pyridine-acetic acid-water (240: 20: 6: 11)

9. Ethyl acetate-pyridine-acetic acid-water (120: 20: 6: 11)

10. Ethyl acetate-hexane (1: 1)

12. Ethyl acetate-cyclohexane (9: 1)

The capacity factor values (k ') in the examples were determined using a Pharmacia LKB analytical HPLC System Two as follows.

Column: "VYDAC C-18 reversed phase: 10 pm, 300,, 4 250 250 mm."

Buffer A: 0.1% trifluoroacetic acid in water.

Buffer B: 0.1% trifluoroacetic acid in acetonitrile.

The gradient used was a flow rate of 1 ml / min

I: 0-5 min 0-25% B, then isocratic 25% B;

II: 0-30 min. 0-60% B.

The peptide content of the eluate was detected by UV light at 214 nm. Sample Concentration 1 mg / ml Buffer, injection volume 25 µl.

For HPLC analysis, the gradient used (I or II) is given in brackets after each truncation in each step of the example.

Acyl-arginine aldehydes exist in equilibrium forms: aldehyde and aldehyde hydrate, as well as two aminocyclic forms.

Of these, the aldehyde hydrate and one or both of the aminocyclols appear in separate peaks during HPLC. Accordingly, the acyl-arginine aldehydes are characterized by the two or three k'-values found in the examples.

For FAB mass spectra, materials were dissolved in m-nitrobenzyl alcohol or glycerol. In addition to the molecular ion in the spectrum of peptidyl-arginine lactams and peptidyl-arginine aldehydes, there is also the molecular ion of the addition compound formed with m-nitrobenzyl alcohol (NBA): [M + H] ⁺ and [M + H + NBAj®]. Accordingly, FAB mass spectral data are given in the examples. ESI positive ionization measurements were performed on a VG Quattro (Fisons). The samples were dissolved in a 1: 1 mixture of acetonitrile / water (1% v / v formic acid) and flowed to the ion source using a 10 mL sample loop at a flow rate of 15-25 mL / min.

The specific rotations (ja] _D) were determined at 20 ° C.

Example 1

Preparation of ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde (Eoc-D-Chg-Pro-Arg-H) hemisulfate

Step 1: Ethoxycarbonyl-D-cyclohexylglycyl-L-prolylNG-benzyloxycarbonyl-L-arginine-lactam

2.73 g (7 mmol) of tert-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] are suspended in 7 ml of chloroform and then 7 ml of hydrochloric acid ethyl acetate (concentration 0.11-0.15 g / ml) are added under stirring and ice-cooling. After 2 hours, the reaction mixture was diluted with diethyl ether (13-15 ml), the precipitated crystalline material was filtered off, washed with acetone (4 ml) and diethyl ether (4 ml) and dried overnight in a vacuum desiccator with potassium hydroxide. The resulting N ^G -benzyloxy-carbonyl-L-arginine lactam hydrochloride is dissolved in 7 ml of dimethylformamide, cooled to -20 ° C and added to the following mixed anhydride.

Ethoxycarbonyl-D-cyclohexylglycyl-L-proline (1.96 g, 6 mmol) [1. (Example C, Step C)] was dissolved in 7 mL of dimethylformamide, cooled to -20 ° C, and 0.68 mL (6 mmol) of N-methylmorpholine, 0.79 mL (6 mmol) of chloro-carbonic acid were added with stirring. isobutyl ester and the N ^G -benzyloxy-carbonyl-L-arginine lactam after 10 minutes of stirring the above dimethylformamide solution, and finally

HU 224 426 Β1

1.76 mL (12.6 mmol) of triethylamine. The reaction mixture was stirred for 30 minutes at -10 ° C and then for 1 hour at 0 ° C. The salts are filtered off and the filtrate is diluted with 30 ml of benzene. The resulting solution was washed with water (3 x 10 mL), 1M potassium bisulfate (10 mL) and water (3 x 10 mL), and after drying over anhydrous sodium sulfate, concentrated to a pressure of 2.0-2.5 kPa. The product was chromatographed on a silica gel column (55 g, Kieselgel 60) using ethyl acetate as eluent. After combining and evaporating the pure fractions containing the product [Rf (1) = 0.52], the oily residue was triturated with n-hexane. Yield: 2.62 g (73%). [α] _D = -40.7 ° (c = 0.5, in tetrahydrofuran). The FAB mass spectrum (599 [M + H] ⁺ ) confirms the expected structure.

Step 2: Ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

Ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (2.3 g, 3.8 mmol) (Example 1, step 1) was dissolved in 13 ml in tetrahydrofuran and 3.0 mmol of lithium aluminum hydride dissolved in tetrahydrofuran was added with stirring at -50 ° C. The progress of the reduction is monitored by layer chromatography in ethyl acetate-pyridine-acetic acid-water (240: 20: 6: 11) and additional lithium aluminum hydride is added as needed. Cooled 0.5 M sulfuric acid was added dropwise to the reaction mixture under cooling and stirring to a pH of 3, then diluted with water (30 mL). The resulting solution was extracted with 2 x 15 mL of n-hexane and extracted with 3 ^x 20 mL of methylene chloride. The methylene chloride solutions were combined, washed with water (3.times.15 mL), cold 5% sodium bicarbonate (15 mL) and water (15 mL), and then, after drying over anhydrous sodium sulfate, concentrated under a pressure of 2.0-2.5 kPa. The residue was triturated with n-hexane. After filtration and drying in a vacuum desiccator, 1.8 g (76%) of the title product are obtained. R _f (2) = 0.20. [α] _D = -31 ° (c = 0.5; in tetrahydrofuran). FAB mass spectrum (601 [M + H] ⁺ ,

754 [M + H + NBA] ⁺ ) confirms the expected structure.

Step 3: Ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde hemisulfate

Ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde 1.6 g (2.7 mmol) (Example 1, Step 2) was dissolved in 30 ml ethanol, 3.0 ml of water and 2.7 ml of 0.5 M sulfuric acid were added 0.20 g of palladium on charcoal, suspended in 10 ml of ethanol, and hydrogenated at about 10 ° C. The reaction, followed by layer chromatography, is complete within about 30 minutes. The catalyst was filtered off and the solution was pressurized to about 2.0-2.5 kPa. Concentrate to a volume of 5-6 ml. The residue was diluted with water (20 mL), extracted with methylene chloride (7 mL), and adjusted to pH 3.5 with Dowex AG 1-X8 hydroxyl ring ion exchange resin. The solution was then allowed to stand for 24 hours at 20-22 ° C, then frozen and lyophilized. 1.27 g (90%) of the title product are obtained. HPLC (I): k '= 4.13; 4.65 and 6.17. [α] _D = -65 ° (c = 1; in water). The FAB mass spectrum (467 [M + H] ⁺ , 620 [M + H + NBA] *) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of Ethoxycarbonyl-D-cyclohexylglycyl-L-Proline (Eoc-DChg-Pro)

Step A: Ethoxycarbonyl-D-cyclohexylglycine dicyclohexylammonium salt

D-Cyclohexylglycine (2.41 g, 16 mmol) was dissolved in 8 mL of 2 M sodium hydroxide, cooled to 0 ° C, and stirred with 9 mL of 2 M sodium hydroxide and 1.73 mL (18 mL). mmol) ethoxycarbonyl chloride. After stirring for one hour, the reaction mixture was diluted with water (20 mL), extracted with diethyl ether (5 mL), acidified to pH 3 with 3M hydrochloric acid, and extracted with ethyl acetate (4 x 10 mL). The combined ethyl acetate solutions were washed with water (10 mL) and then, after drying over anhydrous sodium sulfate, concentrated under a pressure of 2.0-2.5 kPa. The residue was dissolved in diisopropyl ether (30 mL) and dicyclohexylamine (3.2 mL, 16 mmol) was added. The precipitated crystalline material is filtered off, washed with diisopropyl ether and air dried. Yield: 4.7 g (71.5%). R _f (2) = 0.73. M.p. 149-153 ° C. [α] _D = -12.8 ° (c = 1, in ethanol).

Analysis for C23H42N2O4 (410.58):

Calculated: C, 67.28; H = 10.31%; % N, 6.82. Found: C, 67.5; H% = 10.6; % N, 6.45.

Step B: Ethoxycarbonyl-D-cyclohexylglycine-2,4,5-trichlorophenyl Ester

4.5 g (11 mmol) of ethoxycarbonyl-D-cyclohexylglycine-dicyclohexylammonium salt [1. Example 1, Step A) was dissolved in 30 mL of ethyl acetate and 12 mL of 1 M potassium bisulfate. The phases are separated, the ethyl acetate phase is washed with water to neutral and then dried over anhydrous sodium sulfate and concentrated to a volume of about 15 ml at 2.0-2.5 kPa. The residual solution was cooled to 0 ° C, and 2.16 g (11 mmol) of 2,4,5-trichlorophenol and 2.26 g (11 mmol) of dicyclohexylcarbodiimide were added with stirring, and the reaction mixture was stirred at ca. leave for an hour. The precipitated dicyclohexylurea was filtered off and the filtrate was concentrated under a pressure of 2.0-2.5 kPa. 3.8 g of oil which was purified by _[Rf (4) = 0.79] considered by 9 mmol product of the title compound.

Step C: Ethoxycarbonyl-D-cyclohexylglycyl-L-proline mmol Ethoxycarbonyl-D-cyclohexylglycine-2,4,5-trichlorophenyl Ester [1. Example B, Step B) is dissolved in 10 mL of pyridine, added 1.15 g (10 mmol) of L-proline and

Triethylamine (1.4 mL, 10 mmol) was added and the reaction stirred for about 16 hours. The reaction mixture was then concentrated under a pressure of 2.0-2.5 kPa. The residue was dissolved in 30 ml of 5% sodium bicarbonate and the solution was extracted with 3 x 5 ml of diethyl ether. The diethyl ether phases were combined and extracted with 5 ml of 5% sodium bicarbonate. The sodium bicarbonate solutions were combined, acidified to pH 3 with 3M hydrochloric acid, and extracted with ethyl acetate (3 x 10 mL). The combined ethyl acetate solutions, after drying over anhydrous sodium sulfate, are evaporated under a pressure of 2.0-2.5 kPa and the residue is crystallized with isopropyl ether. 1.9 (65%) of the title compound are obtained.

HU 224 426 Β1

R _f (4) = 0.49. Melting point: 116-119 ° C. [α] _D = -22.5 ° (c = 1, in ethanol). The FAB mass spectrum (327 [M + H] *) confirms the expected structure.

Example 2

Preparation of methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde (Moc-D-Chg-Pro-Arg-H) hemisulfate

Step 1: Methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam

1.95 g (5 mmol) tert-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 1.4 g (4.5 mmol) of methoxycarbonyl-D-cyclohexylglycyl-L-proline [2]. Example 1, Step C] Starting as described in Example 1, Step 1, using proportional amounts of reagents and solvents, coupling of the components and chromatographic purification of the final product were performed. The pure fractions containing the final product [Rf (1) = 0.38] were combined and, after evaporation, the resulting oily residue was triturated with n-hexane. 2.1 g (79%) of the title compound are obtained. Rf (2) = 0.61. [α] _D = -41.6 ° (c = 0.5, in tetrahydrofuran). The FAB mass spectrum (585 [M + H] ⁺ , 738 [M + H + NBA] *) confirms the expected structure.

Step 2: Methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

1.97 g (3.37 mmol) of methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Example 2, Step 1) of Example 1, 2 The reaction mixture is reduced as described in Step (a), using proportional amounts of reagents and solvents, except that at the end of the step, the oily final product is rubbed with cyclohexane. After filtration and drying in a vacuum desiccator, 1.25 g (63.5%) of the title product are obtained. R _f (6) = 0.57. [α] _D = -29.8 ° (c = 0.5, in tetrahydrofuran). The FAB mass spectrum (587 [M + H] ⁺ , 740 [M + H + NBA] *) confirms the expected structure.

Step 3: Methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde hemisulfate

Methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde 1.1 g (1.8 mmol) (Example 2, step 2), the first Example 3 was converted using proportional amounts of reagents and solvents. 0.81 g (90%) of the title compound is obtained. HPLC (I):? -3.57; 3.87 and 4.74. [α] _D = -74.8 ° (c = 1; in water). The FAB mass spectrum (453 [M + H] ⁺ , 606 [M + H + NBA] *) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of methoxycarbonyl-D-cyclohexylglycyl-L-proline (Moc-DChg-Pro)

Step A: Methoxycarbonyl-D-cyclohexylglycine dicyclohexylammonium salt

D-Cyclohexylglycine (1.73 g, 11 mmol) was dissolved in 2.75 mL of 2 M sodium hydroxide, cooled to 0 ° C, and 7.65 mL of 2 M sodium hydroxide was added simultaneously with stirring. 1 mL (14.3 mmol) of methoxycarbonyl chloride. After stirring for one hour, the reaction mixture was diluted with water (20 mL), extracted with diethyl ether (2 x 10 mL), acidified to pH = 3 with solid potassium bisulfate, and extracted with ethyl acetate (4 x 10 mL). The combined ethyl acetate solutions were washed with water to neutral and then dried over anhydrous sodium sulfate and concentrated at 2.0-2.5 kPa. The residue was dissolved in diisopropyl ether (30 mL) and dicyclohexylamine (2.4 mL, 12 mmol) was added. The precipitated crystalline material is filtered off, washed with diisopropyl ether and air dried. 3.4 g (78%) of the title compound are obtained. R (2) = 0.81.

Analysis for C ₂₂ H ₄₀ N ₂ O ₄ (396.56):

Found: C, 66.63; H = 10.17%; % N, 7.06. Found: C, 66.7; H = 10.15%; % N, 7.1.

Step B: Methoxycarbonyl-D-cyclohexylglycine-2,4,5-trichlorophenyl ester

Methoxycarbonyl-D-cyclohexylglycine-dicyclohexylammonium salt (3.3 g, 8.3 mmol) [Example 2]. Example 1, Step A] was converted as described in Example 1, Step B using proportional amounts of reagents and solvents. The oil (2.8 g) [R _f (12) = 0.29] was taken as the title product (6.7 mmol).

Step C: Methoxycarbonyl-D-cyclohexylglycyl-L-proline

2.8 g (6.7 mmol) of methoxycarbonyl-D-cyclohexylglycine-2,4,5-trichlorophenyl ester. Example 1, Step B] and 0.86 g (7.5 mmol) of L-proline were coupled as described in Example 1, Step C using proportional amounts of reagents and solvents. 1.6 g (76%) of the title compound are obtained. R _f (2) = 0.19. Mp 147.5-148 ° C. [α] _D = -23.3 ° (c = 1, in ethanol).

Example 3

Preparation of ethoxycarbonyl-D-cyclopentylglycyl-L-prolyl-L-arginine aldehyde (Eoc-D-Cpg-Pro-Arg-H) hemisulfate

Step 1: Ethoxycarbonyl-D-ciklopentilglicil-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam

S. 0.43 g (1.1 mmol) tert-butoxycarbonyl-N ^G benzyloxy-carbonyl-L-arginine lactam [ Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 0.31 g (1.0 mmol) of ethoxycarbonyl-D-cyclopentylglycyl-L-proline [3]. Example 1, Step C] Starting as described in Example 1, Step 1, using proportional amounts of reagents and solvents, coupling of the components and chromatographic purification of the final product were performed. The pure fractions containing the final product [R _f (1) = 0.40] were combined and evaporated under a pressure of 2.0-2.5 kPa. The residue was triturated with n-hexane and dried in a desiccator. 0.38 g (65%) of the expected product are obtained. Rf (1) = 0.40. [α] _D = -41.2 ° (c = 1, in tetrahydrofuran). The ESI mass spectrum (584 [M + H] ⁺ ) confirms the expected structure.

Step 2: Ethoxycarbonyl-D-ciklopentilglicil-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

1 0.36 g (0.6 mmol) of ethoxycarbonyl-D-cyclopentyl-glycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Example 3, Step 1). using a proportional amount of reagents and solvents, except that

At the end of the operation, the oily product obtained after evaporation of the methylene chloride solution was crystallized with n-hexane. Filtration, washing with n-hexane and drying in a vacuum desiccator afforded 0.2 g (55%) of the title product. Rf (6) = 0.21. The ESI mass spectrum (587 [M + H] ⁺ ) confirms the expected structure.

Step 3: Ethoxycarbonyl-D-cyclopentylglycyl-L-prolyl-L-arginine aldehyde hemisulfate

0.18 g (0.3 mmol) of ethoxycarbonyl-D-cyclopentyl-glycyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 3, step 2), the first Example 3 was converted using proportional amounts of reagents and solvents. Yield: 0.145 g (96%). HPLC (I): k '= 3.39; 3.75 and 4.6. The ESI mass spectrum (453 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of ethoxycarbonyl-D-cyclopentylglycyl-L-proline (Eoc-D-CpgPro)

Step A: Ethoxycarbonyl-D-cyclopentylglycine

0.26 g (1.8 mmol) of D-cyclopentylglycine [J. T. Hill and F. W. Dunn, J. Org. Chem. 30, 1321 (1965)]

The reaction mixture is acylated as described in Step A using proportional amounts of reagents and solvents except that the oil obtained by evaporation of the reaction mixture by evaporation of the ethyl acetate solution containing the final product is crystallized with n-hexane, washed with n-hexane and dried in a vacuum desiccator. 0.29 g (76%) of the title product are obtained. Melting point: 89.5-93.6 ° C. R _f (6) = 0.69. [α] _D = + 11.5 ° (c = 1, in methanol).

Step B: Ethoxycarbonyl-D-cyclopentylglycine-2,4,5-trichlorophenyl Ester

0.25 g (1.16 mmol) of ethoxycarbonyl-D-cyclopentylglycine [3. Example 2, Step A) was dissolved in 2.5 mL dimethylformamide, cooled to 0 ° C, and treated with 0.26 g (1.3 mmol) of 2,4,5-trichlorophenol and 0 25 g (1.2 mmol) of dicyclohexylcarbodiimide. The reaction mixture was further stirred at 0 ° C for 30 minutes and at room temperature for 3 hours. The precipitated dicyclohexylurea was filtered off and washed with 0.5 ml of dimethylformamide, and the filtrate and washings were evaporated at 2.0-2.5 kPa. The residue was treated with 1.1 mmol of ethoxycarbonyl-D-cyclopentylglycine-2,4,5-trichlorophenyl ester (Rf (6) = 0.94) and used directly in Step C of this Example.

Step C: Ethoxycarbonyl-D-cyclopentylglycyl-L-proline

Ethoxycarbonyl-D-cyclopentylglycine-2,4,5-trichlorophenyl ester (1.1 mmol) obtained in Step B above is dissolved in 3 ml of dimethylformamide, and 0.14 g (1.2 mmol) of L are added. -proline and 0.17 mL (1.2 mmol) of triethylamine and the reaction mixture was stirred for 18 hours at room temperature. Subsequently, 5 ml of 5% sodium bicarbonate are added and the mixture is extracted with 2 x 3 ml of diethyl ether. The lower layer was acidified to pH 3 with 3M hydrochloric acid and extracted with ethyl acetate (3 x 5 mL). The combined ethyl acetate solutions were washed with water to anhydrous and then dried over anhydrous sodium sulfate at 2.0-2.5 kPa. The residue was triturated with n-hexane, the precipitate was filtered off, washed with n-hexane and dried in a vacuum desiccator. 0.32 g (90%) of the title product are obtained. Rf (6) = 0.47.

Synthesis of control compounds

Synthesis 1

Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-arginine aldehyde (Eoc-D-Phe-Pro-Arg-H) Hemisulfate

Step 1: Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam

S. 2.3 g (5.8 mmol) tert-butoxycarbonyl-N ^G benzyloxy-carbonyl-L-arginine lactam [ Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 1.67 g (5 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-proline [1]. Synthesis, Step C), starting from the coupling of the components and chromatographic purification of the final product as described in Example 1, Step 1, using proportional amounts of solvents and reagents. The pure fractions containing the final product [Rf (1) = 0.37] were combined and evaporated under a pressure of 2.0 to 2.5 kPa, and the residue was dried in a desiccator. Obtained as an oil (1.18 g, 60%). The FAB mass spectrum (607 [M + H] ⁺ ) confirms the expected structure.

Step 2: Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde

Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Synthesis 1, Step 1) 1 0.9 g (1.5 mmol). Example 2 is reduced using proportional amounts of reagents and solvents. Yield 0.33 g (36%). R _f (1) = 0.16.

Step 3: Ethoxycarbonyl-3-phenyl-Dalanyl-L-prolyl-L-arginine aldehyde hemisulfate

0.33 g (0.54 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Synthesis 1, Step 2) for Example 1, Step 3 was converted using proportional amounts of reagents and solvents. 0.26 g (92%) of the title product are obtained. HPLC (I): k-4.0; 4.45 and 5.45. The FAB mass spectrum (475 [M + H] ⁺ , 628 [M + H + NBA] *) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of Ethoxycarbonyl-3-phenyl-D-alanyl-L-proline (Eoc-D-Phe-Pro)

Step A: Ethoxycarbonyl-3-phenyl-D-alanine

16.5 g (0.1 mol) of 3-phenyl-D-alanine were acylated as described in Example 1, Step A, using proportional reagents and solvents, except that the oil obtained by working up the reaction mixture by evaporation of the ethyl acetate containing the final product. dried in a vacuum desiccator. 14.6 g (55%) of the title compound are obtained. R _f (9) = 0.70.

Step B: Ethoxycarbonyl-3-phenyl-Dalanine-N-hydroxysuccinimide ester

14.5 g (55.4 mmol) of ethoxycarbonyl-3-phenyl-D-alanine [1. Synthesis, Step A) and 6.4 g (56 mmol) of N-hydroxysuccin

The titanium dioxide is dissolved in 60 ml of tetrahydrofuran. The solution was cooled to 0 ° C and dicyclohexylcarbodiimide (11.5 g, 56 mmol) was added with stirring. The reaction mixture was further stirred under cooling for 10 hours, then filtered and evaporated at 2.0-2.5 kPa. The residue was crystallized with benzene. The crystals were filtered off, washed with benzene and diethyl ether and dried in a vacuum desiccator. 11.3 g (60%) of the title product are obtained. Rf (10) = 0.53. [.alpha.] D @ 20 = + 58.1 DEG (c = 1; dimethylformamide). Analysis for C ₁₆ H ₁₈ N ₂ O ₆ (334.33):

Found: C, 57.48; % H, 5.42; % N, 8.30;

Found: C, 57.4; % H, 5.4; % N, 8.3.

The FAB mass spectrum (335 [M + H] ⁺ ) confirms the expected structure.

Step C: Ethoxycarbonyl-3-phenyl-D-alanyl-L-proline

3.34 g (10 mmol) of ethoxycarbonyl-3-phenyl-D-alanine-N-hydroxysuccinimide ester [1. Synthesis, Step B) is dissolved in 15 ml of pyridine, 1.15 g (10 mmol) of L-proline and 1.4 ml (10 mmol) of triethylamine are added. The reaction mixture was stirred at room temperature for 10 hours and then concentrated at 2.0-2.5 kPa. The residue was dissolved in 30 ml of 5% sodium bicarbonate and 10 ml of diethyl ether. The aqueous phase was extracted with 3 x 5 mL of diethyl ether. The diethyl ether phases were combined and extracted with 5 ml of 5% sodium bicarbonate. The sodium bicarbonate solutions were combined, acidified to pH 3 with 1M potassium hydrogen sulfate, and extracted with ethyl acetate (3 x 10 mL). The combined ethyl acetate solutions were dried over anhydrous sodium sulfate and concentrated under a pressure of 2.0-2.5 kPa. The oily residue was crystallized with diethyl ether. The crystals were filtered off, washed with diethyl ether and dried in a vacuum desiccator. 2.6 g (77%) of the title compound are obtained. Melting point: 141-143 ° C. Rf (6) = 0.40.

Analysis for C ₁₇ H ₂₂ N ₂ O ₅ (334.33) Calcd:

Calculated: C, 61.06; H, 6.63%; % N, 8.38;

Found: C, 61.1; % H, 6.7; % N, 8.1.

The FAB mass spectrum (335) M + H] ⁺ confirms the expected structure.

Synthesis 2

Methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-L-arginine aldehyde (Moc-D-Phe-Pro-Arg-H) hemisulfate

Step 1: Methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam

4.1 g (10.5 mmol) tert-butoxycarbonyl-N ^G benzyloxy-carbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)], and 2.88 g (9 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-proline [2]. Synthesis, Step C), starting from the procedure described in Step 1 of Example 1, using proportional reagents and solvents, coupling the components and isolating the crude end product. The crude final product is purified by crystallization from cyclohexane. Yield: 4.65 g (86%). Melting point: 72-82.7 ° C. R _f (2) = 0.56.

[α] _D = + 72.9 ° (c = 1, tetrahydrofuran). The FAB mass spectrum (593 [M + H] ⁺ , 746 [M + H + NBA] *) confirms the expected structure.

Step 2: Methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde

4.13 g (7 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Synthesis 2, Step 1) the first Example 2 was reduced using proportional amounts of reagents and solvents except that at the end of the step, the oily product obtained after evaporation of the methylene chloride solution was crystallized with diethyl ether. After filtration, washing with diethyl ether and drying in a vacuum desiccator, 3.06 g (74%) of the title product are obtained. Melting point: 103-107 ° C. R _f (6) = 0.44. [α] _D = -69 ° (c = 1, tetrahydrofuran). The FAB mass spectrum (595 [M + H] ⁺ , 748 [M + H + NBA] *) confirms the expected structure.

Step 3: Methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-L-arginine aldehyde hemisulfate

2.68 g (4.5 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Synthesis 2, Step 2) for Example 1, Step 3, was converted using proportional amounts of reagents and solvents. 2.15 g (94%) of the title compound are obtained. HPLC (I): k '= 3.3; 3.57 and 4.09. [α] _D = -87.6 ° (c = 1, water). The FAB mass spectrum (461 [M + H] ⁺ , 614 [M + H + NBA] *) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of methoxycarbonyl-3-phenyl-D-alanyl-L-proline (Moc-D-PhePro)

Step A: Methoxycarbonyl-3-phenyl-D-alanine dicyclohexylammonium salt (30 mmol) D-phenylalanine was dissolved in 15 mL 2M sodium hydroxide, cooled to 0 ° C and stirred. 16.5 mL of 2M sodium hydroxide and 2.55 mL (33 mmol) of methoxycarbonyl chloride were added in parallel. The reaction mixture was stirred for half an hour under cooling and for 2 hours at room temperature. The reaction mixture was diluted with water (40 mL), extracted with diethyl ether (10 mL), acidified to pH 3 with 3M hydrochloric acid, and extracted with ethyl acetate (4 x 20 mL). The combined ethyl acetate solutions were washed with water (20 mL) and then, after drying over anhydrous sodium sulfate, concentrated under a pressure of 2.0-2.5 kPa. The residue was dissolved in diisopropyl ether (60 mL) and dicyclohexylamine (6 mL, 30 mmol) was added. The precipitated crystals are filtered off, washed with diisopropyl ether and air dried. 11.5 g (28.5%) of the title product are obtained. R _f (2) = 0.56. Melting point: 167-170 ° C. [α] _D = -37.9 ° (c = 0.5 in ethanol).

Analysis for C ₂₃ H ₃₆ N ₂ O ₄ (404.53):

Found: C, 68.28; % H, 8.97; % N, 6.92.

Found: C, 68.3; % H, 9.15; % N, 6.75.

Step B: Methoxycarbonyl-3-phenyl-D-alanine-2,4,5-trichlorophenyl ester

11.3 g (28 mmol) of methoxycarbonyl-3-phenyl-D-alanine [2. Synthesis, Step A) was dissolved in ethyl acetate (75 mL) and treated

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30.5 ml of 1 M potassium hydrogen sulfate. The phases are separated, the ethyl acetate phase is washed with water to neutral and then dried over anhydrous sodium sulfate and concentrated to a volume of about 15 ml at 2.0-2.5 kPa. The remaining solution was cooled to 0 ° C and 5.5 g (28 mmol) of 2,4,5-trichlorophenol and 5.75 g (28 mmol) of dicyclohexylcarbodiimide were added and the reaction mixture was stirred at ca. leave for an hour. The precipitated dicyclohexylurea was filtered off and the filtrate was concentrated under a pressure of 2.0-2.5 kPa. The residual oily product was crystallized from ethanol. Filtration, washing with diethyl ether and drying in a vacuum desiccator gave 6.86 g (61%) of the title product. Melting point: 109-110'C. Rf (4) = 0.84.

Analysis for C ₁₇ H ₁₄ NO ₄ Cl ₃ (402.66):

Calculated: C, 50.70; % H, 3.50; % N, 3.48;

0 = 26.42;

Found: C, 50.8; % H, 3.2; % N, 3.4;

0 = 26.6.

Step C: Methoxycarbonyl-3-phenyl-D-alanyl-L-proline 4.83 g (12 mmol) of methoxycarbonyl-3-phenyl-D-alanine-2,4,5-trichlorophenyl ester [2nd Synthesis, Step B) is dissolved in 12 ml of pyridine, 1.38 g (12 mmol) of L-proline and 1.68 ml (12 mmol) of triethylamine are added and the reaction mixture is stirred for about 16 hours. The reaction mixture was then concentrated under a pressure of 2.0-2.5 kPa. The residue was dissolved in 40 ml of 5% sodium bicarbonate and the solution was extracted with 3 x 7 ml of diethyl ether. The diethyl ether phases were combined and extracted with 7 ml of 5% sodium bicarbonate. The sodium bicarbonate solutions were combined, acidified to pH 3 with 3M hydrochloric acid, and extracted with ethyl acetate (3 x 10 mL). After drying over anhydrous sodium sulfate, the combined ethyl acetate solutions are concentrated under a pressure of 2.0 to 2.5 kPa, and the residue is crystallized with diethyl ether, the crystals are filtered off, washed with diethyl ether and dried in a vacuum desiccator. 3.05 g (79%) of the title compound are obtained. Melting point: 154-157 ° C. R _f (2) = 0.47. The FAB mass spectrum (321 [M + H] ⁺ ) confirms the expected structure.

Methods

M1. method Plasma clot preparation

a) Plate-like plasma (PRP) is prepared by centrifugation of a 9: 1 mixture of human blood and 3.8% sodium citrate-water (1/2) at 240 x g for 5 minutes.

b) Add 200 μΙ PRP to each reaction vessel, add 80 μΙ 40 mM calcium chloride, and allow to stand at room temperature for 1 hour. The resulting plasma clot was washed with 6 x 2 mL of 0.9% sodium chloride with gentle agitation and 5 minutes of sedimentation to remove enzymes remaining in solution (factor Xa and thrombin). The thrombin content of the wash was determined as follows.

c) To 400 μΙ wash solution, add 100 μΙ 1 mM Tos-Gly-Pro-Arg-pNA substrate solution, incubate for 30 min at 37 ° C and stop the reaction with 100 μΙ 50% acetic acid. 150-150 μΙ of the mixture is placed in the wells of a 96-well microtitre plate and the extinction values are determined at 405 nm (ELISA READER SLT Laborinstrument GmbH, Austria). With effective washing, the extinction value is less than 5% of the control.

M2. method

Determination of Plasma Clot Factor Xa Inhibition

a) A solution of 0.1-1.0-10 and 100 μg / ml of peptide inhibitor is prepared in 0.1 M Tris buffer containing 0.02% human albumin pH 8.5.

(b) After aspiration of the washings, place 400 μΙ peptide solution (3 replicate samples at each concentration) in 400 μΙ buffer (Method M1) or 400 μΙ buffer as a control and incubate for 5 minutes at 37 ° C. Subsequently, 100 μΙ of 2 mM Moc-D-Chg-Gly-Arg-pNA substrate solution was added, incubated for another 30 minutes at 37 ° C, and then quenched with 100 μΙ of 50% acetic acid.

c) 150-150 μΙ of each reaction mixture was placed in wells of a 96-well microtitre plate and the extinction values were determined at 405 nm (ELISA READER SLT Laborinstrument GmbH, Austria). The peptide concentration (IC50) required for 50% inhibition is plotted against the mean extinction values relative to the control.

M3. method

Determination of inhibition of plasma clot thrombin

a) Prepare solutions of the peptide inhibitor according to M2. Method A (a).

(b) After aspiration of the washings, place 400 μΙ peptide solution (3 replicate samples at each concentration) in 400 μΙ buffer (Method M1) and 400 μΙ buffer as a control and incubate for 5 minutes at 37 ° C. Subsequently, 100 μΙ of 1 mM Tos-Gly-Pro-Arg-pNA substrate solution was added, incubated for another 30 min at 37 ° C, and then quenched with 100 μΙ of 50% acetic acid. Hereinafter referred to as M2. (c).

M4. Method Fibringel

(a) Add 200 μΙ 6 mg / ml human fibrinogen (SIGMA), 25 μΙ 25 NIH U / ml human thrombin (SIGMA) and 40 μΙ 100 mM calcium chloride to each reaction vessel and allow to stand for 1 h. hours at 20-22 ° C. The resulting fibrin gel is washed with 3 x 2 ml of physiological saline solution with gentle stirring and 5 minutes of sedimentation to remove any remaining thrombin in the solution. The washing performance is determined by M1. (c).

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M5. method

Determination of inhibition of fibrin gel bound thrombin

a) Peptide inhibitors are prepared in 0.1-1.0-10 and 100 μρ / ηιΙ-θβ solutions with Hepes / NaCl buffer (0.01 M Hepes and 0.1 M sodium chloride, pH 7.4).

(b) hereinafter referred to as "M3",. (b).

M6. method

Measurement of Factor Xa Inhibition in 96-well Microtitre Plate

a) Solutions of factor AXa (human, SIGMA) at 1.3 U / ml, peptide inhibitors at 0.1-1.0-10 and 100 pg / ml were prepared with phosphate buffer (0.1 M sodium phosphate and 0, 05 M sodium chloride, pH 7.4). A 0.33 nM distilled aqueous solution of Bz-II-GluGly-Arg-pNA substrate was used.

b) Prepare 3-3 reaction mixtures of control and each peptide solution. 30 μΙ peptide was added to the wells of the plate as well as buffer, 30 μΙ factor Xa, 90 μΙ buffer and 150 μΙ substrate as control, and after 10 minutes read the extinction values at 405 nm. Hereinafter referred to as M2. method

(c).

Claims (7)

PATIENT INDIVIDUAL POINTS 1. New peptidyl arginine aldehyde derivatives of formula (I) Q-D-Xaa-Pro-Arg-H (I) wherein Q is an acyl group of the formula Q'-O-CO- Q 'is C 1-3 alkyl, D-Xaa is D-cyclohexylglycine or D-cyclopentylglycine, Pro is L-proline radical and Arg is an acid addition salt of L-arginine radical and its organic or inorganic acid. 2. Ethoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. 3. Methoxycarbonyl-D-cyclohexylglycyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. 4. Ethoxycarbonyl-D-cyclopentylglycyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. 5. A pharmaceutical composition comprising as active ingredient a compound of formula (I) wherein Q, D-Xaa, Pro and Arg are as defined in claim 1, or a pharmaceutically acceptable salt thereof; with diluents, carriers and fillers. 6. Compounds of formula I wherein Q, D-Xaa, Pro and Arg are for use as medicaments as defined in claim 1. Use of a compound of formula (I), wherein Q, D-Xaa, Pro and Arg are as defined in claim 1, for the manufacture of a medicament for the treatment and prevention of thrombosis and / or increased blood coagulation conditions.