KR100472754B1 - Anticoagulant Peptidyl-Arginine Aldehyde Derivatives - Google Patents

Abstract

The present invention relates to novel peptidyl-arginine aldehyde derivatives of the following general formula (I), organic or inorganic acids and their acid-addition salts formed and pharmaceutical compositions containing them.

Q-D-Xaa-Pro-Arg-H (Ⅰ)

Wherein Q is an acyl group of formula Q'-O-CO-, Q 'is an alkyl group having from 1 to 3 carbon atoms, and D-Xaa is 3-cyclobutyl-D-alanyl or 3-cyclopentyl- D-alanyl group, Pro is L-prolyl group, Arg is L-arginyl group.

Compounds of formula (I) have anticoagulant properties, especially with platelet function inhibition and thrombus progression.

Description

Anticoagulant Peptidyl-Arginine Aldehyde Derivatives

The present invention relates to novel peptidyl-arginine aldehyde derivatives of the following general formula (I), organic or inorganic acids and their acid-addition salts formed and pharmaceutical compositions containing them.

Q-D-Xaa-Pro-Arg-H (Ⅰ)

Where

Q is an acyl group of formula Q'-O-CO-, Q 'is an alkyl group having 1 to 3 carbon atoms,

D-Xaa is a 3-cyclobutyl-D-alanyl or 3-cyclopentyl-D-alanyl group,

Pro is an L-prolyl residue,

Arg is an L-arginyl residue.

The new compounds of general formula (I) are therapeutically particularly useful for anticoagulant, antiplatelet and antithrombin effects.

Particularly preferred representative examples of the compounds of general formula (I) of the present invention include the following derivatives:

Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde,

Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde,

Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine aldehyde,

Methoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine aldehyde,

And acid-addition salts of these organic and inorganic acids, preferably hemisulfate.

Justice

Amino acids, their substituents, and abbreviations for the peptides derived therefrom are known in the art, for example Biochem.J. 126 , 773 (1972); Biochemistry 14 , 449 (1975).

amino acid:

Arg = L-arginine [(2R) -2-amino-5-guanidino-pentanoic acid],

Asp = L-aspartic acid [(2S) -2-amino-3-carboxypropionic acid],

boroArg = L-boroarginine [(1R) -1-Amino-4-guanidino-butylboric acid],

DL-Cba = 3-cyclobutyl-D-alanine [(2R) -2-amino-3-cyclo-butylpropionic acid],

DL-Cbg = DL-cyclobutyl-glycine [(2RS) -2-amino-2-cyclobutylacetic acid],

D-Chg = D-2-cyclohexyl-glycine [(2R) -2-amino-cyclohexylacetic acid],

D-Cpa = 3-cyclopentyl-D-alanine [(2R) -2-amino-3-cyclopentylpropionic acid],

Gla = gamma-carboxy-L-glutamic acid [(2S) -2-amino-4,4-dicarboxybutyric acid],

Glu = L-glutamic acid [(2S) -2-amino-4-carboxybutyric acid],

Glp = L-pyroglutamic acid [(5S) -2-pyrrolidone-5-carboxylic acid],

Gly = glycine (2-aminoacetic acid),

D-MePhe = N-methyl-3-phenyl-D-alanine [(2R) -2-methyl-amino-3-phenylpropionic acid],

D-MePhg = D-N-methyl-phenylglycine [(2R) -2-amino-2-phenylacetic acid],

Nal (1) = 3- (naphth-1-yl) -L-alanine-[(2S) -2-amino-3- (naphth-1-yl) -propionic acid],

D-Phe = 3-phenyl-D-alanine [2 (R) -2-amino-3-phenyl-propionic acid],

Pro = L-proline [(2S) -pyrrolidine-2-carboxylic acid].

Substituents:

Ac = acetyl, Boc = t-butoxycarbonyl, Bz = benzoyl, Eoc = ethoxycarbonyl, Et = ethyl, iPoc = isopropoxycarbonyl, Me = methyl, 4MeP = 4-methyl-pentanoyl, Moc = Methoxycarbonyl, pNA = p-nitrophenylamino, Poc = propoxycarbonyl, Tos = p-toluene-sulfonyl, Z-benzyloxycarbonyl.

Peptides and Derivatives:

Simply abbreviated amino acid stands for each L-amino acid. D-amino acids are indicated separately, ie 3-phenyl-D-alanine = D-Phe. The hyphens (-) before and after the amino acid abbreviation are the hydrogen atoms lost in amino groups or the hydroxyl groups lost in carboxyl groups, respectively. Thus, Eoc-D-Cba-Pro-Arg-H is ethoxy-carbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde, while Bz-lle-Glu-Gly- Arg-pNA is benzoyl-L-isoloyyl-L-glutamyl-glycyl-L-arginine p-nitroanilide.

Blood clotting is part of the organism's protective mechanism. Blood clots are induced by vascular wall injury, preventing death by bleeding. In addition, pathological activation of vascular disease, congestion and blood clotting factors can also induce blood clots. In this case, the blood vessels are partially or entirely blocked by a blood clot formed in the blood vessels and progress to thrombosis. Fibrinolysis is another part of the protective mechanism. Excess blood clots are also removed here by lysis of the clots and enzymes involved in thrombolysis.

The clot formation process is a cascade of a series of catalyzed enzymatic reactions, in which the so-called coagulation factor, the plasma protein, is continuously activated. The factors are denoted by Roman numerals and the active type is denoted by the letter "a". Abbreviations are also used, ie fibrinogen = factor 1 (f1), fibrin = factor 1a (f1a), prothrombin = factor II (fII) and thrombin = factor IIa (fIIa). Serine protease (fXIIa, fVIIa, fXIa, fIXa, fXa, fXa and thrombin), several accelerating cofactors (fVa and fVIIIa) and the coagulating molecule itself (fibrin) are all blood clotting processes Is formed. fXa and thrombin are the last two of the formed proteinases. Thrombin, formed by the activity of fXa, initiates fibrinogen cleavage to form fibrin blood clots.

According to the concept of conventional blood clotting mechanisms [RG Mac Farlane, Nature 202 , 498 (1964); EW Davie and ODRatnoff, Science 145 , 1310 (1964)] fX can be activated by two methods, by endogenous and external pathways. The former is initiated by surface-activated fXII (fXIIa) with the conversion of fXI → fXIa, followed by reaction with fIX → fIXa; fX is activated by fIXa. Exogenous pathways are initiated by the appearance of cell surface receptors, tissue factor (hereinafter referred to as "TF") and the development of [fVII + TF] or [fVIIa + TF] complexes. fX is activated by the [fVIIa + TF] complex.

Recent findings suggest that blood clots in living organisms are the result of a combination of both pathways whose main steps are: [EWDavies et al., Biochemistry 43 , 10363 (1991):

1. In the case of vascular wall damage or disease, TF migrates to the surface and binds to part of Factor VII circulating in the blood. The formed [fVIIa + TF] -complex activates a small portion of plasma factors IX and X (ie, fIXa and fXa are formed) by appropriate trace proteins [fXIIa, fXa, fIXa and thrombin] Tissue Factor Pathway Inhibitor (hereinafter referred to as "TFPI", formerly called Lipoprotein-Associated Coagulation Inhibitor), which is converted to [fVIIa + TF] and is a co-inhibitor of both fXa and [fVIIa + TF] Inactivated by TJGirard et al., Narure 338 , 518-520 (1989).

2. In the presence of Ca ⁺² ions, fIXa produced with factor X and cofactor VIIIa produces a “tenase” complex with [fIXa + fVIIIa + fX + PL + Ca ^{+ 2} ] on the surface of phospholipids. FX is activated with fXa.

3. fXa produced up to this time together with prothrombin (fII) and cofactor Va produces the “prothrombinase complex” [fXa + fVa + fII + PL + Ca ⁺² ], which is the term of “tenase”. It is similar in structure. In the complex, prothrombin is converted to thrombin. fV → fVa conversion and fVIII → fVIIIa conversion may be performed by fXa or thrombin.

4. The resulting traces of thrombin convert a portion of fXI to enzyme fXIa and activate factors VIII and V, producing additional fVIIIa and fVa, respectively. To date, fXIa can convert Factor IX to the enzyme fIXa. At this stage a continuous reaction is resumed, starting with the Xase-complex and ending with thrombin formation. Repeated reactions result in increased thrombin.

5. When the thrombin concentration is appropriately high, the dissolved fibrinogen in plasma causes partial proteolysis, resulting in the fibrin-monomer first linked to the soluble fibrin polymer, which is then converted to an insoluble fibrin polymer. Thrombin also acts as fXIIIa, and the factor that performs the polymerization is generated by the activity of thrombin [L. Lorand and K. Konishi, Arch. Biochem. Biophys. 105 , 58 (1964).

Insoluble fibrin polymers are the main components of blood clots and blood clots, and others are platelet aggregates produced primarily by the action of thrombin. The thrombus or clot formed traps most of the thrombin produced during the reaction, which clings to the solution during the dissolution of the thrombus, causing a new coagulation reaction [AK Gash et al. J. Cardiol. 57 , 175 (1986); R. Kumer et al., Thromb. Haemost. 72 , 713 (1994).

This feature explains the major role of thrombin in thrombus formation. As a result, all compounds that interfere with the function and / or formation of thrombin play a very important role in the treatment of thrombosis.

The most widespread and successfully used compounds currently applied in the prevention and treatment of blood clots are heparin and vitamin K antagonists coumarins (ie syncumar and warfarin), which are indirect thrombin inhibitors.

Heparin catalyzes the reaction between thrombin and its natural inhibitor, antithrombin-III (AT-III). However, if the concentration of heparin in the plasma is lower than 75% of the normal level, this action of heparin does not exist [R.Egbring et al., Thromb. Haemost. 42 , 225 (1979). Thrombin bound to the thrombus cannot be inhibited by this indirect mechanism because it cannot access the heparin-AT-III complex [JIWeitz et al., J. Clin. Invest. 86 , 385 (1990). In addition, side effects such as progression of thromboembolism by treatment-related bleeding and immunopathological processes cannot be ignored [JMWalenga et al., Clin. Appl. Thrombosis / Haemostasis, 2 (Suppl. 1) , S21-27 (1996)].

In addition, vitamin K antagonists may be administered orally, the effect of which appears after 16 to 24 hours. This inhibits the development of the active form of some Gla-containing aggregation factors (ie prothrombin). Partial inhibition (60-70%) is required to obtain a therapeutic effect [MPEsnouf and CVProwse, Biochem. Biophys. Acta 490 , 471 (1977)] can be reached by appropriate drug dosage. However, vitamin K antagonists are difficult to use because of their narrow therapeutic range, strong dependence on dietary compositions (vitamin K), and various individual sensitivity.

The first very potent synthetic compound that directly inhibits thrombin is the reversible inhibitor tripeptide aldehyde D-Phe-Pro-Arg-H, which exhibited significant anticoagulant activity in both in vitro and in vivo [S. Bajusz et al. , in: Peptides: Chemistry, Structure and Biology (R. Walter and J. Meinhofer, Eds.), Ann Arbor Publ., Ann Arbor, Michigan, USA, 603-608 (1975); Int. J. Pepide Protein Res. 12 , 217 (1978). A series of compounds associated with D-Phe-Pro-Arg-H has been synthesized. One of the most important is Boc-D-Phe-Pro-Arg-H [S. Bausz et al., Int. J. Peptide Protein Res. 12 , 217 (1978)] and methyl ketone homologues (D-Phe-Pro-Arg-CH ₂ Cl) which have been found to be irreversible inhibitors (C. Res. 14 , 969 (1979). Additional peptides and acylpeptides to be mentioned are boroarginine homologues (Ac-D-Phe-Pro-boroArg as well as D-Phe-Pro-boroArg and Boc-D-Phe-Pro-boroArg) which are potent reversible thrombin inhibitors. Kettner et al., J. Biol. Chem. 265 , 18289 (1990) and other Boc-D-Phe-Pro-Arg homologues including Boc-D-Chg-Pro-Arg-H (PD Gesellchen and RT Shauman, European Patent Classification No. 0,479,489 A2 (1992) ].

In aqueous solution, D-Phe-Pro-Arg-H is susceptible to spontaneous conversion, while D-MePhe-Pro-Arg-H (GYKI-14766), obtained by methylation of terminal amino groups, maintains the stability of the original compound while maintaining adequate stability Have been demonstrated [S. Bajusz et al., US Pat. No. 4,703, 036 (1987); J.Med. Chem. 33 , 1729 (1990). The formation of blood clots and thrombus in animal experiments was significantly inhibited by the compound [D. Bagdy et al., Thromb. Haemost. 67 , 357 and 68 , 125 (1992); JV Jackson et al., J. Pharm. Exp. Ther. 261 , 546 (1992); Although its inhibitory activity against fibrinolytic enzymes is weak, they, when administered with thrombin solubilizers, significantly promoted the dissolution of blood clots, which could not be achieved with the heparin-AT-III complex [CV Jackson et al., J. Cardiovascular Pharmacol . 21 , 587 (1993). Various compounds associated with D-MePhe-Pro-Arg-H, such as D-MePhg-Pro-Arg-H, have been synthesized [RTShuman et al., J. Med. Chem. 36 , 314 (1993).

Anticoagulant activity (i.e. inhibiting proteolytic activity during the reaction) is, for example, thrombin time (hereinafter referred to as "TT"), active component thromboplastin time (hereinafter referred to as "APTT"). ) And anticoagulant tests, Prothrombin Time (hereinafter referred to as "PT") tests (EJW Bovies et al., Mayo Clinic Laboratory Manual of Haemostasis; WB Saunders Co., Philadelphia (1971)]. Plasma inhibited in spontaneous coagulation, for example citrate-plasma, is coagulated and the time required for coagulation is measured. After the action of the anticoagulant, the coagulation time is extended in proportion to the inhibition of the reaction in the process. The effect of the anticoagulant is characterized by the concentration of material required to double the coagulation time compared to the control (IC ₅₀ ). The effect of anticoagulants on individual coagulating proteinases is determined by the amidoletic method [R. Lottenberg et al., Method in Enzymol. 80 , 341 (1981); G. Cleanson, Blood Coagulation and Fibriolysis 5 , 411 (1994)]. Isolated active factors (eg thrombin, fXa) and their chromogen or fluorogenic peptide-amide substrates, respectively, react in the presence or absence of inhibitors. Enzyme inhibitory action is characterized by the inhibition constant (IC ₅₀ ) measured during amidolilysis.

Coagulation in the TT test is initiated by thrombin added to citrate plasma. Thrombin 22 pmole / ml in the system acts and its inhibition can be measured for fibrinogen (one of thrombin's natural substrates) in the presence of plasma components. Complete coagulation occurs in the APTT and PT tests. Depending on the activator, fX is activated by external or internal pathways. The generated fXa activates prothrombin to thrombin, which then coagulates the plasma. If the enzyme or one of the enzymes is inhibited by an inhibitor, the coagulation time is prolonged. The APTT or FT test produces thrombin 150 pmole / ml (APTT) or 350 pmole / ml (PT), while Xa can produce up to 40 pmole / ml (this is a factor of factor X present in both systems). Total amount) [B. Kaiser et al., Thromb. Res. 65 , 157 (1992).

For D-MePhe-Pro-Arg-H (Cl), the concentrations required to double the coagulation time in TT, APTT and PT tests are 87, 622 and 2915 nM, respectively. These values and the amount of thrombin acting in the test (22, 150 and 350 pmole / ml) increased similarly, indicating that C1 acts as a thrombin inhibitor in both the APTT and PT tests, with very little or no effect on the performance of fXa. Present that it does not cause. Consistent with these results, the amidolite effect of thrombin on the Tos-Gly-Pro-Arg-pNA substrate is suppressed by C1 to a value of IC ₅₀ = 2 nM, while the corresponding Bz-lle-GlU-Gly-Arg- The amidolite effect of fXa on the pNA substrate was only slightly affected, with a value of IC ₅₀ = 9.1 mM (Bajusz et al., unpublished results).

A peculiar feature of blood clot mechanisms is that the mechanism is not only directly inhibited by thrombin inhibitors, but also by a trobin formation inhibitor such as an fXa inhibitor. 60-member polypeptide isolated from tick, Tick (Tick Anticoagulant Peptide) [L. Waksman et al., Science 248 , 593 (1990); AB Kelly et al., Circulation 86 , 1411 (1992)] and DX-9065a ( C2 ), synthetic non-peptides, (+)-(2S) -2- [4 [[(3S) -1-acetimidoyl ) -3-pyrrolidinyl] oxy] phenyl] -3- [7 [amidino-2-naphthyl] -propionic acid hydrogen chloride pentahydrate [T. Hara et al., Thromb. Haemost. 71 , 314 (1994); T. Yokohama et al., Circulation 92 , 485 (1995)], are strong inhibitors of blood clots and thrombus formation. Both the amidolite effect and plasma coagulation of fXa were strongly inhibited in the PT and APTT tests by these compounds. That is, thrombin was not inhibited at all according to their properties as specific fXa inhibitors, ie did not show any activity in the TT test, while the free and complex bound Xa factors were equally well suppressed. 4-MeP-Asp-Pro-Arg-H (C3) (International Patent Application No. 93/15756) and Boc-D-Phe-Nal (1) -Arg-H ( C4 ) (International Patent Application No. 95/13693) Is a synthetic peptide inhibitor of fXa.

According to the literature, C3 and C4 inhibit the amidolite activity of fXa on the ZD-Arg-Gly-Arg-pNA-substrate at IC ₅₀ = 57 and 30 nM, respectively. There is no data on their anticoagulant activity. In our own tests, C3 and C4 showed significant inhibition on the Bz-lle-Glu-Gly-Arg-pNA substrate, while their anticoagulant activity was found to be weak in the plasma coagulation test. Table 1 shows the literature ( C2 ) and measurement ( C3 and C4 ) activity of synthetic fXa compared to antithrombin compound C1 .

The data in Table 1 indicate that for C1 the anticoagulant effect is due to thrombin inhibition, whereas for C2 it is due to the inhibition of fXa. The significant fXa inhibitory effects of C3 and C4 do not involve any significant anticoagulant effect. The fXa active center of the prothrombinase complex is likely to be inaccessible to C3 and C4 , and these peptides can only inhibit fXa released in solution.

Table 1

FXa Inhibition (A) and Anticoagulant (B) Effects of Known Synthetic Inhibitors

^a peptide concentration that doubles coagulation time when compared to control in PT, APTT and TT tests

^b Value measured by isolated human fXa on Bz-lle-Glu-Gly-Arg-pNA pigment-producing substrate

^c NA = inactive

According to the recent literature [NAPrager et al., Circulation 92 , 962 (1995)] not only thrombin but also factor Xa trapped in factor thrombus / blood clotting and released during lysis is determined by the activity of [fVII + TF] -complex or factor V and VIII, respectively. Contribute to the initiation and maintenance of new coagulation reactions. Thus, if an anticoagulant can inhibit factor Xa in addition to inhibition of thrombin, it is particularly advantageous if this inhibition extends to coagulation-bound thrombin and factor Xa.

It is an object of the present invention to prepare new peptide derivatives which have improved anticoagulant activity compared to known compounds and which exhibit anticoagulant activity even upon oral administration.

In the TT and APTT tests, the anticoagulant effect of Boc-D-Phe-Pro-Arg-H ( C5 ) was compared to that of tripeptide aldehyde C1 . Although small, it was somewhat better in the PT test and suppressed factor Xa with IC ₅₀ , 10 times lower than C1 . Substitution of the BOC-group at the C5 position with the Eoc-group positively enhances the anticoagulant effect of the peptide since the resulting Eoc-D-Phe-Pro-Arg-H exhibits a higher effect than C5 in all three tests. It was unexpectedly found to change; In addition, its activity was higher than that of C1 in the PTTT as well as in the PT test, while its fXa inhibitory effect was similar to that of Boc-peptide C5 . The anticoagulant effect of Moc-D-Phe-Pro-Arg-H ( C7 ) was similar to that of C6 Eco-compound, but the fXa inhibitory effect was somewhat high. These findings are described in Table 2.

Table 2

Anticoagulant effect (A) of peptide aldehyde having R-D-Phe-Pro-Arg-H structure and

fXa inhibitory effect (B)

^a peptide concentration that doubles coagulation time compared to ^a control

^b Value determined by human fXa isolated against Bz-lle-Glu-Gly-Arg-pNA pigmentogenic substrate

It has also been found that the more effective anticoagulant compounds can be obtained when the N-terminal amino acid is changed from phenylalanine to 3-cyclobutylalanine, while binding homologous cyclobutyl-glycine to the N-terminus decreases the activity. In addition, apart from 3-cyclobutylalanine, substitution with 3-cyclopentylalanine produces effective peptidyl-arginine aldehydes. It has also been found that substitution of a methoxycarbonyl or propoxycarbonyl group instead of an N-terminal ethoxy-carbonyl group changes the activity spectrum, ie the activity ratio measured in the individual tests.

The present invention relates to novel peptide aldehyde derivatives of the following general formula (I), organic or inorganic acids and their acid-addition salts formed and pharmaceutical compositions containing them.

Q-D-Xaa-Pro-Arg-H (Ⅰ)

Where

Q is an acyl group of formula Q'-O-CO-, Q 'is an alkyl group having 1 to 3 carbon atoms,

D-Xaa is a 3-cyclobutyl-D-alanyl or 3-cyclopentyl-D-alanyl group,

Pro is an L-prolyl residue,

Arg is an L-arginyl residue.

Compounds of formula (I) wherein Q, Q ', Xaa, Pro and Arg have the same meaning as above, for example acyldipeptides protected with a benzyloxycarbonyl group in a guanidino group QD-Xaa-Pro is condensed with L-arginine lactam, and the resulting protected tripeptide lactam is reduced with the protected tripeptide aldehyde of formula QD-Xaa-Pro-Arg (Z) -H, and finally guanie of arginine Prepared by removing the Z groups from the dino group and separating the peptide analogs of formula (I) as organic or inorganic acids and their additional salts formed.

QD-Xaa-Pro, an acyl-dipeptide used as starting material, is prepared, for example, by acylating D-Xaa amino acids with an alkylester of Q 'of chloroformic acid in a basic medium, and thus obtained QD-Xaa acyl The amino acid is bound to L-proline.

QD-Xaa required to bind proline acylates racemic DL-Xaa-compounds, converts DL-acylamino acids to their methyl esters, and enzymatically separates Q-DL-Xaa-OMe racemic esters. Can be prepared. The Q-D-Xaa-OMe thus obtained is saponified to become the desired Q-D-Xaa acylamino acid.

In the compounds of general formula (I) of the present invention, Q, Xaa, Pro and Arg have the same meaning as above, exhibit strong anticoagulant activity in both in vitro and in vivo and have excellent bioavailability.

The in vitro anticoagulant effect of the compound of formula (I) was determined by PT, APTT and TT tests [D. Bagdy et al., Thromb. Haemost. 67 , 325 (1992). In addition, the fXa-inhibitory effect of the compounds was determined using the Bz-Ile-Glu-Gly-Arg-pNA pigment-producing substrate (see Method M6).

The results obtained are shown in Table 3. Data corresponding to C1 thrombin inhibitors, C6 Eoc-D-Phe-compounds and C8 Eoc-DL-Cba isoforms served as controls. In Table 3 the compounds are listed in order of decreasing PT activity. The data below show useful effects of terminal Cba- and Cpa- moieties compared to Phe (see 1 , 4 , and C6 compounds), and homologous cyclobutylglycines (see C8 , C9 and C10 compounds of the DL-class), for example. Is clearly shown.

TABLE 3

Anticoagulant effect (A) and fXa inhibition of novel peptide aldehydes (1-4, general QD-Xaa-Pro-Arg-H) and controls ( C1, C6 and C8- C10 ), in decreasing order of PT activity Effect (B)

^a peptide concentration that doubles thrombin-time compared to the control

^b same as the example number describing the preparation of each compound

^c Value measured by isolated human fXa on Bz-lle-Glu-Gly-Arg-pNA substrate (see methods M1 to M5)

Not only for plasmin formation induced by tissue plasminogen activator (hereinafter referred to as "tPA") and urokinase (hereinafter referred to as "UK") but also plasmin. The inhibitory effect of the novel compounds of the invention on "PL") was examined by the fibrin-plate method [D. Bagdy et al., Thromb. Haemost. 67 , 325 (1992). Compounds C1 and C5 are controls. It is already known that the antifibrinolytic effect of C1 is weak in in vivo and can be used as an adjuvant to dissolve experimental blood clots, while the antifibrinolytic activity of C5 is well detectable in in vivo [CVJackson et al. J. Cardiovasc. Pharmacol. 21 , 587 (1993). The results obtained with the novel compounds 1 and 2 as well as the control C1 to C5 are shown in Table 4 as examples.

In addition to the IC ₅₀ values (column A), the utility of the compounds related to C1 was also listed (column B). The latter data show that the antifibrin lytic activity of the novel compounds 1 and 2 is moderate, similar to compound C1 ; The activity for the three enzymes is 3.2-4.5-39 times lower than the value of compound C5 , respectively.

Table 4

Plasmin (PL) formation induced by tPA and UK, studied by fibrin-plate method, and

Novel Peptidyl-Arginine Aldehydes of the Invention for Plasmin and

Inhibitory effect of structurally similar controls (IC ₅₀ )

^a IC ₅₀ = peptide concentration (μM) in which the hydrolyzed region in the fibrin plate is reduced by 50% compared to the control

^{b The} value related to the activity of C1 (1 / IC ₅₀ = 1)

^c are the same as the numbers in the examples each illustrating the preparation of the compound

The inhibitory effects of the novel compounds of general formula (I) on bloodline-binding fXa and thrombin as well as fibrin gel-bound thrombin are shown in Table 5 together with compounds 1-3; Each value of C1 was used as a control. Plasma-coagulation was obtained by platelet-rich human plasma by calcium recalcification and fibrin gel was obtained by coagulating human fibrinogen with human thrombin. The activity of fXa and thrombin was measured using a ZD-Arg-Gly-Arg-pNA or Tos-Gly-Pro-Arg-pNA substrate in combination with methods M1 to M3.

Table 5

Novel of the invention for fibrin-gel binding thrombin as well as blood clot-binding fXa and trombone Inhibitory Effect of Peptidyl-Arginine Aldehyde (1 to 3) and C1 as a Control (IC _50, μM)

^{a In} determining the value of IC ₅₀ , the activities of fXa and thrombin were measured for Moc-D-Chg-Gly-Arg-pNA or Tos-Gly-Pro-Arg-pNA according to methods M2 and M3.

^b Same as in Example number describing novel compound or control ( C1 )

These data show that Eoc-compounds 1 and 3 inhibit plasma thrombin-bound fXa as well as thrombin at concentrations below IC ₅₀ = 1 μM.

The anticoagulant and platelet aggregation inhibitory activity of the compounds of formula (I) was investigated ex vivo in New Zealand white rabbits according to D. Bagdy et al. [Thromb. Haemost. 67 , 357 (1992). Compounds were dissolved in buffered isotonic saline solution intravenously (0.04 to 5.0 mg / kg) or infusion (0.25 to 5.0 mg / kg / h) or subcutaneously (0.5 to 6.0 mg / kg) or orally (2.5 to 20 mg / kg). ). The effect of the compound could already be detected within 30 minutes of oral administration, the peak was obtained after 60 to 180 minutes, and the therapeutic level lasted for 3 to 6 hours depending on the dose.

The in vivo effects of ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde are described in detail in Tables 6 and 7. Corresponding values of C1 are listed as controls. Compounds were administered orally at a dose of 5 mg / kg. Blood samples taken every 30 to 60 minutes were analyzed in the unmeasured veins. Whole Blood Clotting Time (hereinafter referred to as "WBCT") and inhibition of platelet aggregation (PAI) induced by thrombin were measured. In addition, APTT and TT were measured in citrate plasma obtained from blood samples. APTT and TT values are compiled in Table 6 and WBCT and PAI in Table 7.

The data in Table 6 and Table 7 show that New Compound 1 has a longer lasting anticoagulant and anticoagulant effect than C1 .

Table 6

In the APTT and TT tests, characterized by relative coagulation time ^a ,

5 mg / kg of D-MePhe-Pro-Arg-H ( C1 ) and Eoc-D-Cba-Pro-Arg-H (1) as a control

Anticoagulant Effect of Oral Administration to Rabbits

^a ratio of clotting time measured in treated / untreated animals. The therapeutic value is italicized.

TABLE 7

In the WBCT test, characterized by relative coagulation time and percentage inhibition ^a

D-MePhe-Pro-Arg-H ( C1 ) and Eoc-D-Cba-Pro-Arg-H (1) to rabbits at 5 mg / kg

Anticoagulant and PAI Effects after Oral Administration

^a Percentage of clotting measured in treated / untreated animals. The therapeutic value is italicized.

The compounds of the present invention of general formula (I) are suitable for the development of seizures based on deep vein thrombosis, pulmonary embolism, arterial thrombosis, unstable angina, myocardial infarction, myocardial fibrillation and thrombosis based on thrombosis and hypercoagulability. Used to cure light prevention.

In atherosclerosis, the compounds can be used to prevent thrombosis and coronary artery disease in cerebral arteries as a surgical prophylaxis or other surgical prophylaxis for high risk patients. The compounds also include diseases with nephritis and hypercoagulation; It can be applied to the thrombus breakdown of transdermal transluminal angioplastics for adjuvant therapy of inflammation as well as diabetes (eg arthritis) and malignant tumors and to prevent re-obstruction. The compounds may be ineffective for the administration of other anticoagulants or forbidden, for example, lack of anti-thrombin-III in the case of heparin or heparin-induced thrombocytopenia (HIT), and pregnancy in the case of coumarin, for example. May be administered.

The compounds of the present invention and their pharmaceutically acceptable salts are used for themselves or in the form of pharmaceutical structures for the purpose of treatment. The invention also relates to such structural formulas.

The pharmaceutical structural formula consists of an effective amount of the general formula (I) compound or a pharmaceutically acceptable salt thereof and known pharmaceutically acceptable carriers, fillers, diluents and / or other pharmaceutical excipients.

Such carriers, diluents, or fillers may be water, alcohol, gelatin, lactose, saccharose, starch, pectin, magnesium stearate, stearic acid, talc, various animal or vegetable oils, in addition propylene, for example Glycols such as glycols or polyethylene glycols. Pharmaceutical excipients can be preservatives, various natural or synthetic emulsifiers, dispersing or wetting agents, colorants, flavoring agents, bottling accelerators, and bioavailability enhancing substances of the active ingredient.

Pharmaceutical compositions of the present invention may be used as parenteral compositions (drugs, infusions, suppositories, drugs administered without gastrointestinal tract such as warnings or ointments), as well as oral compositions (tablets, capsules, powders, pills, dragees or granules). Drug to be administered to).

The therapeutic dosage of a compound of the present invention may vary depending on the age and personal health state of the patient; Therefore, the dosage level can be delivered by the physician in charge of the treatment. In diseases in which thrombin formation or function is inhibited for the purpose of prophylaxis or treatment, the daily oral or parenteral (eg, intravenous administration) dose is 0.01 to 1000 mg / kg body weight, preferably 0.25 to 20 mg / kg body weight.

The compound of formula (I) of the present invention, when administered as a thrombolytic agent (eg, tPA or UK), actively promotes dissolution of thrombin formed in arteries or veins and effectively inhibits remodeling. In such cases, the compound of the present invention is preferably administered simultaneously with thrombolytics or immediately after thrombolytic treatment.

The following examples illustrate the invention but do not limit the scope of the invention.

The R _f values reported in the examples were determined in the following developing solvents by thin layer chromatography using silica gel as an adsorbent (DC-Alufolien Kieselgel 60 F ₂₅₄ , Merck, Darmstadt);

1. Ethyl Acetate

2. ethyl acetate-pyridine-acetic acid-water

(480: 20: 6: 11)

5. Ethyl acetate-pyridine-acetic acid-water

(45: 20: 6: 11)

6. Ethyl acetate-pyridine-acetic acid-water

(240: 20: 6: 11)

7. Ethyl acetate-pyridine-acetic acid-water

(480: 10: 3: 5.5)

9. Ethyl acetate-pyridine-acetic acid-water

(120: 20: 6: 11)

3. Ethyl acetate-n-butanol-acetic acid-water

(1: 1: 1: 1)

The capacity factor (hereinafter referred to as "k '") specified in this example was determined by the "Pharmacia LKB Analytical HPLC Syatem Two" apparatus as follows.

Column: "VYDAC C-18 Reversed Phase: 10 mm, 300 A, 4 x 250 mm"

Buffer A: 0.1% trifluoroacetic acid in water

0.1% trifluoroacetic acid in buffer B acetonitrile

Gradient was applied at 1 ml / mim.

Flow rate

I: 0-5 min, 0.25% B, then isocratic 25% B;

II: 0-30 minutes, 0-60% B

Peptide content of the eluate was detected by ultraviolet at 214 mm. Sample concentration is 1 mg / ml Buffer A, injection volume 25 μl. The slope applied for HPLC analysis (I to II) is indicated in parentheses abbreviated in the individual steps of the examples.

Acylarginine aldehydes exist in equilibrium structures, ie aldehydes, aldehyde hydrates and two aminocyclols. During HPLC analysis, the aldehyde hydrate and one or two aminocyclol forms appear as separate peaks, and eventually the acylarginine aldehydes described in the examples are characterized by 2-3 k 'values.

Mass Spectrometry (hereinafter referred to as "MS").

FAB positive ionization measurements are performed with a Finnigan MAT8430 device. The sample was dissolved in m-nitrobenzyl alcohol (hereinafter referred to as "NBA") substrate and introduced directly into the ion source. In the peptidyl-arginine aldehyde spectrum, additional molecular-ions: [M + H] ⁺ and [M + H + NBA] ⁺ of additional compounds produced with NBA can be detected. In the examples FAB spectral data is specified accordingly.

Positive ionization measurements of ESI were performed using the VG Quattro (Fisons) apparatus. The sample was dissolved in a mixture of acetonitrile-water (1: 1) containing 1% (v / v) formic acid and 10 mL of sample-loop in the ion source at an outflow rate of 15-25 mL / min. Was introduced with.

Specific rotation ([α] _D ) was determined at 20 ° C.

Example 1

Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde (Eoc-D-Cba-Pro-Arg-H) hemisulfate

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

t-butoxycarbonyl-N ^G -benzyloxy-carbonyl-L-arginine-lactam [S. Bajusz and co-workers: J. Med. Chem. 33 , 1729 (1990)] 3.28 g (8.4 mmol) were suspended in 8 ml of chloroform, and then 10 ml of ethyl acetate, saturated with hydrogen chloride gas (0.11 to 0.15 g / ml), was added with constant stirring under ice-cooling. Thin layer chromatography [R _f (9) = 0.91 (Boc-compound); 0.07 (free compound)]. Finally, the reaction mixture was diluted with 18 ml of diethyl ether and the crystalline formed was filtered off, washed with 5 ml of acetone and 5 ml of diethyl ether and then dried in a vacuum drier on potassium hydroxide pellets. The obtained N ^G -benzyloxycarbonyl-L-arginine-lactam hydrogen chloride was dissolved in 8 ml of dimethylformamide, cooled to -20 ° C and added to the mixed anhydride of the following preparation.

2.5 g (8 mmol) of ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (Example 1, Step E) was dissolved in 8 ml of dimethylformamide cooled to -20 ° C. 0.9 N (8 mmol) of N-methyl-morpholine and 1.05 mL (8 mmol) of isobutyl chloroformate were introduced under constant stirring, followed by stirring after 10 minutes, and the above N ^G -benzyloxy-carbonyl-L- Arginine-lactam hydrogen chloride solution and 2.35 mL (16.8 mmol) triethylamine were introduced. The reaction mixture was stirred at −10 ° C. for 30 minutes and at 0 ° C. for 1 hour. Salt was filtered off and the filtrate was diluted with 40 mL of benzene. The solution was washed with 3 × 10 mL of water, 10 mL of 1M KHSO ₄ ⁻ solution, again with 3 × 10 mL of water, then dried over anhydrous sodium sulfate and the solvent was evaporated under a pressure of 2.0-2.5 kPa. The product was applied to a column prepared with 70 g of Kieselgel 60 and eluted with ethyl acetate. The portion containing pure material [R _f (1) = 0.50] was collected and the solvent was evaporated at 2.0 to 2.5 kPa. The oily residue was washed with petroleum ether, filtered and dried.

Yield: 3.0 g (64%)

R _f (1) = 0.50,

[α] _D = -47.4。 (c = 1 in THF)

FAB MS data confirm the expected structure (585 [M + H] ⁺ ).

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

2.27 g (4.6 mmol) of ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Example 1, Step 1) 3.6 mmol of lithium aluminum hydrate dissolved in 15 mL of furan and dissolved in tetrahydrofuran were added at -50 ° C or lower with continuous stirring. The progress of the reaction was monitored by thin layer chromatography in solvent system 6 and further lithium aluminum hydrate was added as needed. The pre-cooled 0.5 M sulfuric acid solution was continuously added to the reaction mixture with constant stirring and cooling until the pH reached 3. The resulting solution was extracted with 2 × 15 mL of n-hexane and then extracted with 3 × 20 mL of methylene chloride. The methylene chloride extracts were collected, washed with 3 x 15 ml of water, 15 ml of cooled 5% sodium hydrogen carbonate solution, then again with 15 ml of water, dried over anhydrous sodium sulfate, and the solvent was washed with 2.0 to 2.5 kPa. Evaporated to pressure. The residue was dissolved in 10 ml of benzene and precipitated with cyclohexane. After filtration and drying, the product weighs 1.8 g (65%).

R _f (6) = 0.30,

[α] _D = -30.8。 (c = 1 in THF)

FAB MS data confirmed the expected structure (587 [M + H] ⁺ ).

Step 3: Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine-aldehyde hemisulfate

Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 1, Step 2) 1.58 g (2.7 mmol) in 14 ml of ethanol , 2.7 mL of 0.5 M sulfate, 3.2 mL of water, and then 0.17 g of Pd / C catalyst suspended in 6 mL of ethanol were added and the system was hydrogenated at 10 ° C. The reaction was monitored by thin layer chromatography and completed within 15 minutes. The catalyst was filtered off and the solution was concentrated to a volume of 7-9 mL at a pressure of 2.0-2.5 kPa. The residue was dissolved in 20 ml of water, extracted with 6 ml of methylene chloride and the aqueous layer was left at room temperature for 24 hours. Then, the pH was adjusted to 3.5 using Dowex AG 1-X8 resin in OH-cycle and the solution was lyophilized.

Yield: 1.0 g (78%)

R _f (5) = 0.44,

[α] _D = -75.5。 (c = 1 in water)

HPLC (1): k '= 5.53; 6.20 and 7.17

FAB MS data confirmed the expected structure (453 [M + H] ⁺ ).

Starting materials can be prepared as follows.

Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline

(Eoc-D-Cba-Pro)

Step A: Ethoxycarbonyl-3-cyclobutyl-DL-alanine

3-cyclobutyl-DL-alanine [A. Burger et al., J. Med. Chem. 6, 221 (1963)] 10 g (70 mmol) was dissolved in 70 mL 2M sodium hydroxide, cooled to +5 ° C and added dropwise with 11.1 mL (116.2 mmol) of ethyl chloroform continuously stirring. Stirring was continued for 2 hours at this temperature and for 2 hours at room temperature. The reaction mixture was washed with 20 ml of diethyl ether and the extracted solution was acidified to pH 1 with 3 × 20 ml of ethyl acetate. The combined extracts were washed with brine, dried over anhydrous sodium sulfate, and then the solvent was evaporated at a pressure of 2.0-2.5 kPa. The resulting oil is considered pure product [yield: 13.1 g (87%), R _f (7) = 0.8] and used in the next step without further treatment.

Step B: Methyl Ethoxycarbonyl-3-cyclobutyl-DL-alanineate

11 g (51 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanine [Example 1, Step 1) 10 ml (137 mmol) of thionyl chloride while dissolving methane in 100 ml, cooling to -15 ° C and stirring continued ) Was added. Stirring was continued without further cooling. When the reaction mixture reached room temperature, it was stirred for a further 2 hours. The solvent was evaporated under a pressure of 2.0 to 2.5 kPa. The residual oil was dissolved in 40 ml of ethyl acetate, washed with 10 ml of 1N HCl, 2 × 10 ml of 5% sodium bicarbonate and 2 × 10 ml of water, dried over anhydrous sodium sulfate and dried at a pressure of 2.0 to 2.5 kPa. I was. The residue is considered a pure product [yield: 10.8 g (92%), R _f (1) = 0.85; R _f (6) = 0.65] used in the next step without further processing.

Step C: Methyl Ethoxycarbonyl-3-cyclobutyl-D-alanineate

Enzymatic hydrolysis of acyl amino acid methylester can be carried out at pH = 7.5 in an automatic titrator with two reaction vessels (one volume of 100 ml, the other 250 ml). The reagent is a 1M NaOH solution.

10.8 g (47 mmol) of methyl ethoxycarbonyl-3-cyclobutyl-DL-alanine salt (synthesized in Step B) were dissolved in 20 ml of benzene in a 250 ml titration vessel. 100 mL of water was added and the pH of the solution was adjusted to 7.5 with 1M NaOH while stirring. 40 mg of enzyme [Subtilisin Carlsberg, Protease, Type VIII (Sigma) was added, the pH was adjusted to 7.5 and the value was maintained during titration. Enzymatic hydrolysis was completed in 3 hours. The phases were separated. Pure ethoxy-carbonyl-3-cyclobutyl-L-alanine can be separated from the aqueous layer. The benzene layer was washed with 2 x 10 ml of 5% sodium hydrogen carbonate and 2 x 10 ml of water, dried over anhydrous sodium sulfate and evaporated to a pressure of 2.0 to 2.5 kPa. The resulting yellow oil is considered pure product and used in the next step without further treatment.

Step D: Ethoxycarbonyl-3-cyclobutyl-D-alanine

The oily product of Step C was dissolved in 20 mL of ethanol and 10 mL of 2.5M NaOH was added. The mixture was stirred at rt for 2 h and evaporated to a pressure of 2.0-2.5 kPa. The residue was dissolved in 30 mL of water, washed with 10 mL of diethyl ether, acidified to pH = 1 with 3M hydrochloric acid, and extracted with 3 × 10 mL of ethyl acetate. The combined organic layer extracts were washed with brine, dried over anhydrous sodium sulfate, and the solvent was then evaporated to a pressure of 2.0 to 2.5 kPa. Yield: 4.3 g (90%) of the oily product [Rf (2) = 0.8] is mostly used for the next step, the remainder being converted to crystalline salts as follows.

0.43 g (2 mmol) of oily ethoxycarbonyl-3-cyclobutyl-D-alanine (Example 1, Step D) was dissolved in 10 mL of diethylether and 0.23 mL (2.1 mmol) of cyclohexylamine was added. The separated crystals were filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 0.57 g (90%)

M.P .: 129-131 degreeC

[α] _D = -3.93。 (c = 1 in methanol)

C ₁₆ H ₃₀ N ₂ O ₄ (314.42) analysis;

Calculated value: C% = 61.12; H% = 9.62; N% = 8.91

Found: C% = 61.30; H% = 9.80; N% = 8.60

Step E: Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline

3.7 g (17.2 mmol) of ethoxycarbonyl-3-cyclobutyl-D-alanine [Example 1, Step D) were dissolved in 60 ml of tetrahydrofuran, cooled to 0 ° C., and 2,4,5-trichloro 4.85 g (24.5 mmol) of rophenol and 5.06 g (24.5 mmol) of dicyclohexyl-carbodiimide were added. The reaction mixture was stirred at rt for 2 h, filtered from the separated dicyclohexylurea and washed with tetrahydrofuran. The filtrate and washes were collected and evaporated at a pressure of 2,0 to 2.5 kPa. The residue was dissolved in 25 ml of diethylether-petroleum ether mixture (1: 1), the insoluble dicyclohexylurea was filtered off and the filtrate was evaporated again. [4.9 g (89% of dicyclohexylurea filtered) ) And 0.6 g (11%). Oily 2,4,5-trichlorophenyl-ethoxycarbonyl-3-cyclobutyl-D-alanineate was dissolved in 40 ml of pyridine and 2.12 g (18.4 mmol) of finely divided L-proline was added to 3.43 ml of triethylamine. (24.6 mmol) was added. The reaction mixture was stirred at rt for 3 h and evaporated to a pressure of 2.0-2.5 kPa. The residue was partitioned between 50 ml of ethyl acetate and 50 ml of 1M HCl. The organic layer was first washed with 10 ml of 1 M HCl, then with 2 x 10 ml of water and extracted with 3 x 100 ml of 5% sodium hydrogen carbonate. The collected hydrogen carbonate extracts were acidified to pH = 3 with 6M HCl and extracted with 3 × 20 mL of diethyl ether. The extract on ether was collected and washed with water to remove the acid, dried over anhydrous sodium sulfate and evaporated to dryness.

Yield: 3.2 g (60%) oil

R _f (6) = 0.49

FAB MS data confirmed the expected structure (313 [M + H] ⁺ ).

Example 2

Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -Benzyloxycarbonyl-L-arginine-aldehyde (MOC-D-Cba-Pro-Arg-H) hemisulfate

step 1: Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] starting with 1.64 g (4.2 mmol) and 1.25 g (4.0 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (Example 2, Step C), The components were combined and the final product was purified by chromatography according to the method described in Example 1, Step 1 using reagent and solvent proportions. The portion containing pure final product [R _f (1) = 0] was collected and evaporated at a pressure of 2.0-2.5 kPa. The residue was washed with petroleum ether and dried in a paraffin turnings and dryer of phosphorus oxyacid.

Yield: 1.15 g (50%)

R _f (2) = 0.45 to 0.47

[α] _D = -43.5。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (571 [M + H] ⁺ , 724 [M + H + NBA] ⁺ ).

Step 2: methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

1.06 g (1.8 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine lactam (Example 2, Step 1) using Example 1 using a proportional amount of reagent and solvent , According to the method described in step 2.

Yield: 0.76 g (73%)

R _f (9) = 0.47

[α] _D = -37.5。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (573 [M + H] ⁺ , 726 [M + H + NBA] ⁺ ).

Step 3: methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde hemisulfate

0.69 g (1.2 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 2, Step 2) in proportional amount of reagent And hydrogenolysis using a solvent according to the method described in Example 1, Step 3.

Yield: 0.53 g (91%)

HPLC (II): k '= 4.74; 5.47 and 5.82

[α] _D = -60.9。 (c = 1 in water)

FAB MS data confirmed the expected structure (439 [M + H] ⁺ , 592 [M + H + NBA] ⁺ ).

Starting materials can be prepared as follows.

Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (MOC-D-Cba-Pro)

Step A: Methoxycarbonyl-3-cyclobutyl-DL-alanine

3-cyclobutyl-DL-alanine [A. Burger et al., J. Med. Chem. 6 , 221 (1963)] 4.3 g (30 mmol) were dissolved in 15 ml of 2M NaOH solution, cooled to 0 ° C., and 16.5 ml (24.5 mmol) and 255 ml (33 mmol) of methyl chloroform were stirred vigorously while Added. Stirring was continued for 30 minutes at this temperature and 2 hours at room temperature. The reaction mixture was diluted with 40 mL of water, extracted with 10 mL of diethylether and acidified to pH 3 with 3M HCl. The product was extracted with ethyl acetate (4 × 20 ml), collected, washed with 20 ml of water, dried over anhydrous sodium sulfate, evaporated to 2.0-2.5 kPa and dried. Dairy residues [5.3 g (88%), Rf (7) = 0.61] were used in the next step without further purification.

Step B: Methoxycarbonyl-3-cyclobutyl-D-alanine

5.03 g (25 mmol) of methoxycarbonyl-3-cyclobutyl-DL-alanine (Example 2, Step A) were treated according to the method described in Examples 1, Steps B to D, using proportional amounts of reagent and solvent It was.

Yield: 2.22 g (11 mmol) oily product

[R _f (7) = 0.61]

Step C: Methoxycarbonyl-3-cyclobutyl-D-alanine-L-proline

1.21 g (6 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanine (Example 2, Step B) were treated according to the method described in Example 1, Step E using proportional amounts of reagent and solvent. Yield: 1.6 g (5.4 mmol, 90%) of most of the oily product [R _f (9) = 0.48] was used directly in step 1 and the residue was converted to crystalline salt as follows.

0.30 g (1 mmol) of oily methoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline was dissolved in 5 ml of diethether and 0.21 ml (1.05 mmol) of dicyclohexyl amine was added. The crystals were filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 0.43 g (90%)

M.P .: 149.5-164 degreeC

C ₂₄ H ₄₅ N ₃ O ₅ (479.64) analysis

Calc .: C% = 65.10; H% = 9.46; N% = 8.76

Found: C% = 60.4; H% = 8.98; N% = 10.47

Example 3

Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine-aldehyde (iPoc-D-Cba-Pro-Arg-H) hemisulfate

Step 1: Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] starting with 0.43 g (1.1 mmol) and 0.33 g (1 mmol) of isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (Example 3, Step C) The components were combined according to the method described in Example 1, Step 1 using a proportional amount of reagent and solvent, and the product was chromatographically purified. The portion containing pure final product [R _f (1) = 0.55] was collected, evaporated at a pressure of 2.0-2.5 kPa, the residue was washed with petroleum ether, filtered and dried in a drier.

Yield: 0.3 g (50%)

R _f (1) = 0.55

FAB MS data confirmed the expected structure (599 [M + H] ⁺ , 752 [M + H + NBA] ⁺ ).

Step 2: Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

0.26 g (0.43 mmol) of isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Example 2, Step 1) in proportional amounts Reagents and solvents were used to hydrogenate according to the method described in Example 1, Step 2.

Yield: 0.15 g

R _f (6) = 0.30

FAB MS data confirmed the expected structure (601 [M + H] ⁺ , 754 [M + H + NBA] ⁺ ).

Step 3: Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde hemisulfate

Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 2, Step 2) 0.13 g (0.2 mmol) in proportional amount Reagents and solvents were used following the method described in Example 1, Step 3.

Yield: 0.07 g (75%)

HPLC (I): k '= 3.69; 4.15 and 5.04

FAB MS data confirmed the expected structure (467 [M + H] ⁺ , 620 [M + H + NBA] ⁺ ).

Starting materials may be made of the following materials.

Isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (iPoc-D-Cba-Pro)

Step A: 3-cyclobutyl-D-alanine

To 1.0 g (5 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanine (Example 2, Step 2) was added 10 ml of 6M HCl and refluxed for 3 hours. The solution was evaporated at a pressure of 2.0-2.5 kPa, the residue was taken up in 5 ml of water, evaporated to dryness and the procedure repeated. Traces of water can be removed with the ethanol-benzene mixture by azeotropic distillation. The residual oil was dissolved in 10 ml of ethanol and 1,2-epoxy-propane was added. After cooling the precipitate was filtered off and the solid was washed with diethyl ether and dried.

Yield: 0.65 g (90%) of 3-cyclobutyl-D-alanine

R _f (13) = 0.65

[α] _D = -34.7。 (c = 1 in 1M HCl)

C ₇ H ₁₃ NO ₂ analysis:

Calc .: C% = 58.71; H% = 9.15; N% = 9.78

Found: C% = 58.70; H% = 9.30; N% = 9.50

Step B: Isopropoxycarbonyl-3-cyclobutyl-D-alanine

0.57 g (4 mmol) of 3-cyclobutyl-D-alanine was dissolved in 8 ml of a 1: 1 mixture of 1 M NaOH solution and dioxane. The mixture was cooled to 0 ° C. and 4 ml of 1 M NaOH solution and 4 ml of isopropyl chloroform (1 M concentrated in toluene) were added simultaneously with vigorous stirring. Stirring was continued for 1 hour at this temperature and for 5 hours at room temperature. Dioxane and toluene were distilled to a pressure of 2.0 to 2.5 kPa. The remaining aqueous solution was washed with 5 ml of diethyl ether and acidified to pH = 3 with 1 M potassium hydrogen sulfate. The emulsifier was extracted with 3 x 5 ml of ethyl ester and the combined extracts were washed with water and evaporated over anhydrous sodium sulfate. The yield is 0.83 g (90%) at a pressure of 2.0 to 2.5 kPa after evaporation.

R _f (6) = 0.60

[α] _D = +9.5。 (c = 0.5 in methanol)

Step C: Isopropoxycarbonyl-3-cyclobutyl-D-alanine-L-proline

0.46 g (2 mmol) of isopropoxycarbonyl-3-cyclobutyl-D-alanine (Example 3, Step B) were treated according to the method described in Example 1, Step E using proportional amounts of reagent and solvent. .

Yield: 1.3 g (4.35 mmol, 87%) oil.

R _f (2) = 0.40

FAB MS data confirmed the expected structure (327 [M + H] ⁺ ).

Example 4

Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine-aldehyde (Eoc-D-Cpa-Pro-Arg-H) hemisulfate

Step 1: Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-N ^G -benzyl-oxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] 2.93 g (7.5 mmol) and 2.3 g (7.1 mmol) of ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-proline (Example 4, Step C) in proportional amounts of reagents And a solvent was used in accordance with the method described in Example 1, Step 1. The portion containing pure final product [R _f (1) = 0.55] was collected, evaporated to a pressure of 2.0 to 2.5 kPa, the remaining oil was washed with n-hexane, filtered, washed with n-hexane and drier Dried over.

Yield: 2.5 g (59%)

R _f (1) = 0.55

[α] _D = -48.6。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (599 [M + H] ⁺ , 752 [M + H + NBA] ⁺ ).

Step 2: ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

2.34 g (3.9 mmol) of ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Example 4, step 1) in proportional amount of reagent And using a solvent according to the method described in Example 1, Step 2.

Yield: 1.75 g (74.7%)

R _f (6) = 0.42

[α] _D = -34.4。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (601 [M + H] ⁺ , 754 [M + H + NBA] ⁺ ).

Step 3: Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine aldehyde hemisulfate

1.59 g (2.65 mmol) of ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 4, Step 2) in proportional amount of reagent And a solvent was used following the method described in Example 1, Step 3.

Yield: 1.13 g (83%)

R _f (3) = 0.37

[α] _D = -34.4。 (c = 1 in water)

HPLC (I): k '= 4.76; 5.40 and 7.32

FAB MS data confirmed the expected structure (467 [M + H] ⁺ , 620 [M + H + NBA] ⁺ ).

Starting materials can be made of the following materials:

Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-proline (Eoc-D-Cpa-Pro)

Step A: Ethoxycarbonyl-3-cyclopentyl-DL-alanine

3-cyclopentyl-DL-alanine [PRPal et al., J. Am. Chem. Soc., 78 , 5116 (1956)] 7.87 g (50 mmol) was extracted with 2 x 50 mL of the product from the reaction mixture, dried over anhydrous sodium sulfate and evaporated at a pressure of 2.0 to 2.5 kPa. Except for the proportional amount of reagents and solvents, the process was carried out according to the method described in Example 1, Step A. Yield: 10.55 g (98%) of amorphous white powder, [R _f (2) = 0.84], used for next step without further purification.

Step B: Ethoxycarbonyl-3-cyclopentyl-D-alanine

Ethoxycarbonyl-3-cyclopentyl-DL-alanine (Example 4, Step A) 7.8 g (34 mmol) was added to the method described in Examples 1, Steps B, C, D using proportional amounts of reagent and solvent. Treated accordingly. Yield: 3.45 g (88.6%) oil, [R _f (6) = 0.062]. Most are used directly in step C. The residue is converted to crystalline salt as follows:

0.46 g (2 mmol) of oily ethoxycarbonyl-3-cyclopentyl-D-alanine was dissolved in 10 mL of diethyl ether and 0.42 mL (2.1 mmol) of dicyclohexylamine was added. The precipitate was filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 0.74 g (90%) crystalline salt.

M.P .: 138-140 degreeC

[α] _D = -1.5。 (c = 1 in methanol)

C ₂₃ H ₄₂ N ₂ O ₄ Assay:

Calc .: C% = 67.28; H% = 10.31; N% = 6.81

Found: C% = 67.40; H% = 10.40; N% = 6.80

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

2.43 g (10.5 mmol) of ethoxycarbonyl-3-cyclopentyl-D-alanine (Example 4, Step B) were treated according to the method described in Example 1, Step E, using a proportional amount of reagent and solvent It was. The resulting 2,4,5-trichlorophenylester was combined with 1.15 g (10 mmol) of L-proline to yield 2.3 g (71%) of an oily product.

R _f (6) = 0.53

FAB MS data confirmed the expected structure (327 [M + H] ⁺ ).

Synthesis of Control Compound

Collage 1

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

Step 1: Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] starting from 2.3 g (5.8 mmol) and 1.67 g (5 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-proline (synthesis 1, step C), The proportions of reagents and solvents were used to combine the components and purify the product according to the method described in Example 1, Step 1. Portions containing pure product [R _f (1) = 0.37] were collected and evaporated to a pressure of 2.0-2.5 kPa. The residue was dried in a vacuum drier.

Yield: 1.18 g (60%) oil

FAB MS data confirmed the expected structure (607 [M + H] ⁺ ).

Step 2: Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Synthesis 1, Step 1) 0.9 g (1.5 mmol) in proportional amount of reagent and solvent Using to reduce according to the method described in Example 1, Step 2.

Yield: 0.33 g (36%)

R _f (1) = 0.16

Step 3: Ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-L-arginine aldehyde hemiacetate

0.33 g (0.54 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (synthesis 1, step 2) in proportional amounts of reagent and solvent Was processed according to the method described in Example 1, Step 3.

Yield: 0.26 g (92%)

HPLC (I): k '= 4.00; 4.45 and 5.45

FAB MS data confirmed the expected structure (475 [M + H] ⁺ , 628 [M + H + NBA] ⁺ ).

Starting materials can be prepared as follows.

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

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

16.5 g (0.1 mmol) of 3-phenyl-D-alanine was evaporated in an ethyl acetate solution of the final product, followed by drying of the resulting oil in a vacuum dryer, using a proportional amount of reagent and solvent. , According to the method described in step A.

Yield: 14.6 g (55%)

R _f (9) = 0.70

Step B: Ethoxycarbonyl-3-phenyl-D-alanine N-hydroxy-succinimidate

14.5 g (55.4 mmol) of ethoxycarbonyl-3-phenyl-D-alanine (synthesis 1, step A) and 6.4 g (56 mmol) of N-hydroxy-succinimide were dissolved in 60 ml of tetrahydrofuran. The solution was cooled to 0 ° C. and 11.5 g (56 mmol) of dicyclohexyl-carbodimide were added with stirring. Stirring was continued for 10 hours at this temperature, and the mixture was filtered and the filtrate was evaporated to a pressure of 2.0 to 2.5 kPa. The residue was crystallized from benzene. The crystals were filtered off, washed with benzene and diethyl ether and dried in a vacuum bath.

Yield: 11.3 g (60%)

R _f (10) = 0.53

[α] _D = + 58.1。 (c = 1 in dimethylformamide)

FAB MS data confirmed the expected structure (335 [M + H] ⁺ ).

C ₁₆ H ₁₈ N ₂ O ₆ (334.33) Assay:

Calc .: C% = 57.48; H% = 5.42; N% = 8.30

Found: C% = 57.40; H% = 5.40; N% = 8.30

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

3.34 g (10 mmol) of ethoxycarbonyl-3-phenyl-D-alanine hydroxysuccinimidate (synthesis 1, step B) was dissolved in 15 ml of pyridine, and 15 ml of L-proline and 1.4 ml (10 mmol) of triethylamine were added. Added. The reaction mixture was stirred at rt for 10 h and evaporated to a pressure of 2.0-2.5 kPa. The residue was partitioned between 30 ml of 5% sodium hydrogen carbonate and 10 ml of diethyl ether. The aqueous layer was washed with 3 x 5 ml of diethyl ether, and the collected organic layers were extracted with 5 ml of 5% sodium hydrogen carbonate solution. The bicarbonate layer was collected, acidified to pH = 3 with 1 M potassium hydrogen sulfate solution and extracted with 3 × 10 ml of ethyl acetate. The collected organic extracts were dried over anhydrous sodium sulfate and evaporated at a pressure of 2.0 to 2.5 kPa. The oily residue was crystallized with diethyl ether and the crystalline was filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 2.6 g (77%)

M.P .: 141 to 143 ° C

R _f (6) = 0.40.

FAB MS data confirmed the expected structure (335 [M + H] ⁺ ).

C ₁₇ H ₂₂ N ₂ O ₅ (334.33) Assay:

Calc .: C% = 61.06; H% = 6.63; N% = 8.38

Found: C% = 61.10; H% = 6.70; N% = 8.1

Synthetic 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-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-argininelactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] A proportional amount of 4.1 g (10.5 mmol) and 2.88 g (9 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-proline (synthesis 1, step C) as starting materials. Coupling was carried out according to the method described in Example 1, Step 1 using reagents and solvents of. The crude product was purified by crystallization from cyclohexane.

Yield: 4.65 g (86%)

M.P .: 72 to 82.7 ° C

R _f (2) = 0.56

[α] _D = + 72.9。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (593 [M + H] ⁺ , 746 {M + H + NBA]).

Step 2: methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

4.13 g (7 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Synthesis 2, step 1) was added to a proportional amount of reagent and solvent. Was reduced according to the method described in Example 1, Step 2, except that the final product of the oily phase was crystallized with diethyl ether after evaporation of the methylene chloride solvent. The crystals were filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 3.06 g (74%)

M.P .: 103-107 degreeC

R _f (6) = 0.44

[α] _D = -69。 (c = 1 in tetrahydrofuran)

FAB MS data confirmed the expected structure (595 [M + H] ⁺ , 748 {M + H + NBA]).

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 -benzyloxycarbonyl-L-arginine aldehyde (synthesis 2, step 2) was added in proportional amounts to reagents and solvents. Was processed according to the method described in Example 1, Step 2.

Yield: 2.15 g (94%)

[α] _D = -87.6。 (c = 1 in water)

HPLC (I): k '= 3.3; 3.57 and 4.09

FAB MS data confirmed the expected structure (461 [M + H] ⁺ , 614 [M + H + NBA] ⁺ ).

Starting materials may be prepared from the following materials.

Methoxycarbonyl-3-phenyl-D-alanyl-L-proline (Moc-D-Phe-Pro)

Step A: Methoxycarbonyl-3-phenyl-D-alanine dicyclohexylamine salt

5 g (30 mmol) of D-phenylalanine was dissolved in 15 mL of 2M NaOH, cooled to 0 ° C., and 16.5 mL of 2M NaOH solution and 2.55 mL (33 mmol) of methyl chloroformate were added simultaneously. The mixture was stirred at this temperature for 30 minutes and then at room temperature for 2 hours. After diluting with 40 mL of water, the mixture was washed with 10 mL of diethyl ether, acidified to pH = 3 with 3M HCl and extracted with 4 × 20 mL of telacetate. The collected organic extracts were washed with water (20 mL), dried over anhydrous sodium sulfate and evaporated to a pressure of 2.0-2.5 kPa. The residue was dissolved in 60 mL of isopropyl ether and 6 mL (30 mmol) of dicyclohexylamine were added. The separated crystals were filtered off, washed with diisopropyl ether and dried in air.

Yield: 11.5 g (28.5%)

R _f (2) = 0.56

M.P.:167-170 degreeC

[α] _D = -37.9。 (c = 0.5 in ethanol)

C ₂₃ H ₃₆ N ₂ O ₄ (404.53) analysis:

Calc .: C% = 68.28; H% = 8.97; N% = 6.92

Found: C% = 68.30; 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 dicyclohexyl ammonium salt (Synthesis 2, Step A) was partitioned between 75 ml of ethyl acetate and 30.5 ml of 1 M potassium hydrogen sulfate. The layers were separated, the organic layer was washed with water to neutralize, dried over anhydrous sodium sulfate and concentrated to a volume of 15 ml at a pressure of 2.0 to 2.5 kPa. To the residue cooled to about 0 ° C., 5.5 g (28 mmol) of 2,4,5-trichlorophenol and 5.75 g (28 mmol) of dicyclohexyl carbodiimide were added and the reaction mixture was left overnight. The separated cyclohexylurea was filtered off and the filtrate was evaporated. The oily residue was crystallized from ethanol. The crystalline was filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 6.86 g (61%)

M.P.:109-110 degreeC

R _f (4) = 0.84

C ₁₇ H ₁₄ NO ₄ Cl ₃ (402.66) Assay:

Calc .: C% = 50.70; H% = 3.50; N% = 3.48 Cl% = 26.42

Found: C% = 50.80; H% = 3.20; N% = 3.40 Cl% = 26.60

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

4.83 g (12 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-2,4,5-trichlorophenyl ester (Synthesis 2, step B) was dissolved in 12 ml of pyridine and 1.38 g of finely divided L-proline (12 mmol) and 1.68 mL (12 mmol) of triethylamine were added with stirring. The reaction mixture was stirred for 16 h and then evaporated to a pressure of 2.0-2.5 kPa. The residue was taken up in 40 ml of 5% sodium hydrogen carbonate and the solution was extracted with 3 × 7 ml of diethyl ether. The collected ether layers were washed with 7 ml of 5% sodium hydrogen carbonate. The bicarbonate washes were collected, acidified to pH = 3 with 3M hydrochloric acid solution and extracted with 3 × 10 mL of ethyl acetate. The extract was dried over anhydrous sodium sulfate and evaporated to a pressure of 2.0 to 2.5 kPa. The residue was crystallized with diethyl ether, the crystals were filtered off, washed with diethyl ether and dried in a vacuum drier.

Yield: 3.05 g (79%)

M.P.:154-157 degreeC

R _f (2) = 0.47

FAB MS data confirmed the expected structure (321 [M + H] ⁺ ).

Synthetic 3

Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-L-arginine-aldehyde (Eoc-DL-Cba-Pro-Arg-H) hemisulfate

Step 1: Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] 1.35 g (3.46 mmol) and 1.03 g (3.3 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-proline (Synthesis 3, Step A) in proportional amounts of reagents and The solvents were used to combine the components according to the method described in Example 1, Step 1 and the final product was purified by chromatography. The portion containing pure final product [R _f (1) = 0.50] was collected and evaporated to a pressure of 2.0 to 2.5 kPa, the residue triturated with n-hexane, filtered, washed with n-hexane and vacuum Dried in a dryer.

Yield: 1.0 g (51%)

R _f (2) = 0.50

ESI MS data confirmed the expected structure (585 [M + H] ⁺ ).

Step 2: Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

0.94 g (1.6 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Synthesis 3, step 1) was added in proportional amounts to the reagents and The solvent was reduced according to the method described in Example 1, Step 2.

Yield: 0.64 g (68%)

R _f (6) = 0.30

ESI MS data confirmed the expected structure (587 [M + H] ⁺ ).

Step 3: Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-L-arginine aldehyde hemisulfate

0.59 g (1.0 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (synthesis 3, step 2) was added in proportional amounts to the reagents and The solvent was used in accordance with the method described in Example 1, Step 3.

Yield: 0.39 g (78%)

HPLC (I): k '= 5.55; 6.27 and 7.09

FAB MS data confirmed the expected structure (453 [M + H] ⁺ , 606 [M + H + NBA] ⁺ ).

Starting materials can be prepared as follows.

Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-proline (Eoc-DL-Cba-Pro)

Step A: Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-proline

1.13 g (5.25 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanine (Example 1, Step A) was treated according to the method described in Example 1, Step E using a proportional amount of reagent and solvent An oily 2,4,5-trichlorophenyl ester was produced, which was combined with 0.58 g (5.0 mmol) of finely divided L-proline.

Yield: 1.1 (71%) of oil.

R _f (6) = 0.53

ESI MS data confirmed the expected structure (313 [M + H] ⁺ ).

Synthetic 4

Ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-L-arginine-aldehyde (Eoc-DL-Cpa-Pro-Arg-H) hemisulfate

Step 1: Ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] 3.7 g (9.5 mmol) and 2.94 g (9.0 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-proline (Synthesis 4, step A) were used as starting materials. The compounds were treated according to the method described in Example 1, Step 1 using proportional amounts of reagents and solvents. The portion containing pure product was collected, evaporated to a pressure of 2.0-2.5 kPa and the residue triturated with n-hexane. The solid product was filtered off, washed with n-hexane and dried in a vacuum drier.

Yield: 2.87 g (54%)

R _f (1) = 0.55

ESI MS data confirmed the expected structure (599 [M + H] ⁺ ).

Step 2: Ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

2.75 g (4.6 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Synthesis 4, step 1) were added in proportional amounts to reagents and The solvent was reduced according to the method described in Example 1, Step 2.

Yield: 1.74 g (64%)

R _f (6) = 0.42

ESI MS data confirmed the expected structure (601 [M + H] ⁺ ).

Step 3: ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-L-arginine aldehyde hemisulfate

1.2 g (2.0 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Synthesis 4, step 2) were added in proportional amounts to reagents and Solvent was used according to the method described in Example 1, Step 3.

Yield: 0.8 g (78%)

HPLC (I): k '= 3.96; 4.4 and 4.92

FAB MS data confirmed the expected structure (467 [M + H] ⁺ , 620 [M + H + NBA] ⁺ ).

Starting materials can be prepared as follows.

Ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-proline (Eoc-DL-Cpa-Pro)

Step A: Ethoxycarbonyl-3-cyclobutyl-DL-alanyl-L-proline

3.1 g (13.5 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanine (Example 4, Step A) were treated according to the method described in Example 1, Step E using proportional amounts of reagent and solvent An oily 2,4,5-trichlorophenyl ester was produced, which was combined with L-proline.

Yield: 3.1 (74%).

R _f (6) = 0.50

ESI MS data confirmed the expected structure (327 [M + H] ⁺ ).

Synthetic 5

Ethoxycarbonyl-DL-cyclobutyl-glysil-L-prolyl-L-arginine-aldehyde (Eoc-DL-Cbg-Pro-Arg-H) Hemisulfate

t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] A proportional amount of 1.2 g (3.0 mmol) and 0.69 g (2.3 mmol) of ethoxycarbonyl-DL-cyclobutyl-glycyl-L-proline (synthesis 5, step A) as starting materials Using the reagents and solvents of the above, as described in Example 1, steps 1,2 and 3, each bound to the blocked tripeptide lactam [R _f (1) = 0.32], and the blocked tripeptide aldehyde [ R _f (1) = 0.53] was reduced and deblocked.

Yield: 0.22 g

HPLC (I): k '= 2.74; 2.96 and 3.41

FAB MS data confirmed the expected structure (439 [M + H] ⁺ , 592 [M + H + NBA] ⁺ ). . The starting material can be made of the following materials.

Ethoxycarbonyl-DL-cyclobutyl-glysil-L-proline (Eoc-DL-Cbg-Pro)

Step A: Ethoxycarbonyl-DL-cyclobutyl-glycyl-L-proline

DL-cyclobutyl-glycine [THPorter et al., Arch. Biochem. Biophys, 179 , 266 (1977)] 0.78 g (6.0 mmol) was acylated using a proportional amount of reagent and solvent, according to the method described in Examples 1, Steps A and E [R _f (7) = 0.60 ], 2,4,5-trichlorophenol [R _f (4) = 0.84] and bound with L-proline.

Yield: 3.68 g (50%) of oil.

R _f (2) = 0.17 and 0.22

FAB MS data confirmed the expected structure (299 [M + H] ⁺ ).

Way

Method M1

Preparation of blood clots in human plasma

a) Platelet-rich plasma was prepared by centrifuging human plasma and a 3.8% sodium citrate solution 9: 1 mixture at 240 × g for 5 minutes.

b) 200 μl of platelet-rich plasma and 80 μl of 40 mM CaCl ₂ solution were left at room temperature for 1 hour. The clots formed as above were washed with 6 × ₂ mL of 0.9% NaCl solution with gentle stirring to remove residual enzymes (thrombin and factor X _a ). Wash media was analyzed for thrombin-like activity as follows;

c) 100 µl of 1 mM Tos-Gly-Pro-Arg-pNA substrate solution was added to 400 µl of washing medium. The mixture was incubated at 37 ° C. for 30 minutes, and then 100 μl of 50% acetic acid was added to stop the reaction. 150 μl aliquots from this mixture were applied to 96-well microtitre plates and extinction coefficients were measured at 405 nm (ELISA READER SLT Laborinstrument GmbH, Australia). . If washing was efficient, the extinction coefficient was 5% smaller than the control.

Method M2

Plasma binding factor X _a Inhibition Measurement of

a) 0.1, 1.0, 10 and 100 μl / ml solutions were prepared from peptide inhibitors in 0.1 M Tris-buffer containing 0.02% human albumin (pH = 8.5).

b) After draining the wash medium (Method 1), 400 μl of peptide solution (three parallel samples for each concentration) were added to the blood clots and incubated at 37 ° C. with 400 μl of buffer as a control for 5 minutes. It was. Then 100 μl of a 2mM Moc-D-Chg-Gly-Arg-pNA substrate solution was added to each vessel and incubated again at 37 ° C. for 30 minutes. The reaction was stopped with 100 μl of 50% acetic acid.

c) 150 μl was applied to a 96-well microtiter plate from each reaction vessel, and the extinction coefficient was measured at 405 nm (ELISA READER SLT Laborinstrument GmbH, Australia). IC ₅₀ (peptide concentration required for 50% inhibition) was determined graphically on average absorbance compared to control.

Method 3

Determination of Inhibition of Blood clot-bound Thrombin

a) A solution was prepared from the peptide inhibitor according to Method M2 / a.

b) After draining the wash medium, the blood clots (method M1) are mixed with 400 μl of peptide solution (3 parallel samples for each concentration), and the sample is 400 μl of buffer serving as a control for 5 minutes. Incubated at 37 ° C with. Then 100 μl of 1 mM Tos-Gly-Pro-Arg-pNA substrate solution was added to each vessel and incubated at 37 ° C. for 30 minutes.

The experiment was continued according to method M2 / c.

Method M4

Preparation of Fibrin Blood Clots

a) 200 μl of human fibrinogen solution (SIGMA), 25 μl of 25 NIH / E / ml human thrombin (SIGMA) and 40 μl of 100 mM potassium chloride solution were placed in each reaction vessel, and the mixtures were kept at 20 to 22 ° C. for 1 hour. Left for a while. Fibrin blood clots were washed with isotonic saline (3 × 2 mL) with gentle stirring for 5 minutes to remove residual thrombin and precipitate. The effectiveness of the wash can be checked by method M1 / c.

Method 5

Determination of thrombin inhibition bound to fibrin-blood clots

a) 0.1, 1.0, 10 and 100 μg / ml solutions were prepared from peptide inhibitors in Hepes / NaCl buffer (0.001 M Hepes and 0.1 M NaCl, pH = 7.4).

b) Measurement was continued according to method M3 / b.

Method M6

Factor X in solution on 96-well microtiter plate _a Inhibition Measurement of

a) 1.3 E / mL solution was prepared from human factor X _a (SIGMA) with phosphate buffer (0.1 M sodium phosphate and 0.05 M sodium chloride, pH = 7.4) and 0.1, 1.0, 10 and 100 μg from peptide inhibitor A / ml solution was prepared. The Bz-lle-Glu-Gly-Arg-pNA substrate was used in distilled aqueous solution (0.33 nM).

b) 30 μl aliquots from control and peptide inhibition solutions (3 parallel samples for each concentration) were placed in the wells. 30 μL of Factor X _a , 90 μL of buffer and 150 μL of substrate were added to the peptide solution and the absorbance was determined at 405 nm after 10 minutes. The step is then the same as that of M2 / c.

Claims (7)

Peptidyl-arginine aldehyde derivatives of the general formula (I) below, and their acid-addition salts formed with organic or inorganic acids: Q-D-Xaa-Pro-Arg-H (Ⅰ) (here, Q is an acyl group of formula Q'-O-CO-, wherein Q 'is an alkyl group having 1 to 3 carbon atoms, D-Xaa is a 3-cyclobutyl-D-alanyl or 3-cyclopentyl-D-alanyl group, Pro is an L-prolyl group, Arg is an L-arginyl group). Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. Methoxycarbonyl-3-cyclopentyl-D-alanyl-L-prolyl-L-arginine aldehyde and acid addition salts thereof. Use in the pharmaceutical field with at least one compound of the general formula (I) wherein Q, D-Xaa, Pro and Arg are as defined in claim 1 or as pharmaceutically acceptable acid addition salts thereof as the active ingredient A pharmaceutical composition comprising a mixture of possible carriers or additives and used as a medicament for the treatment and prevention of thrombus and accelerated blood clots. Compounds of formula (I) used as medicaments for the treatment and prophylaxis of thrombus or accelerated blood clots, wherein Q, D-Xaa, Pro and Arg are as defined in claim 1.