KR20000016348A - Anticoagulant peptidyl-arginine aldehyde derivatives - Google Patents

Abstract

PURPOSE: A new peptidyl-arginine aldehyde derivative, an acid-addition salt thereof being formed by an organic or inorganic acid, and a pharmaceutical composition containing thereof are provided. CONSTITUTION: The invention relates to new peptidyl-arginine aldehyde derivatives of general formula (I):Q-D-Xaa-Pro-Arg-H, wherein Q represents an acyl group of formula Q'-O-CO- where Q' represents an alkyl group with 1-3 carbon atoms, D-Xaa represents 3-cyclobutyl-D-alanyl- or 3-cyclo-pentyl-D-alanyl- group, Pro stands for L-prolyl- group, and Arg stands for L-arginyl- group, and acid-addition salts thereof formed with an organic or inorganic acid and pharmaceutical compositions containing the same. The compounds of formula (I) have valuable therapeutic, particularly anticoagulant, properties together with inhibiting platelet functions and thrombosis development.

Description

Anticoagulant Peptidyl-Arginine Aldehyde Derivatives

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. Here the blood clots are also removed by the dissolution of the thrombus and the enzymes involved in thrombus lysis.

The clot formation process is a cascade of a series of catalyzed enzymatic reactions in which plasma proteins, so-called coagulation factors, are continuously activated. The argument is represented by Roman numerals and the active form is represented 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 co-factors (fVa and fVIIIa) and the clotting 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 fibr-linogen 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 as follows [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 a part of Factor VII that is 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 amounts of proteinases [fXIIa, fXa, fIXa and thrombin] Tissue Factor Pathway Inhibitor (hereinafter referred to as "TFPI"), which is converted to the complex [fVIIa + TF] and is then a co-inhibitor of both fXa and [fVIIa + TF], conventionally termed Lipoprotein-Associated Coagulation Inhibitor ) (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 with prothrombin (fII) and the cofactor Va produces the "prothrombinase complex" [fXa + fVa + fII + PL + Ca ⁺² ], which is the 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. This step resumes the continuous reaction starting with the Xase-complex and ending with thrombin formation. Repeated reactions result in increased thrombin.

5. Fibrinogen dissolved in plasma at moderately high thrombin concentrations results in partial proteolysis, in which fibrin-monomers are first linked to soluble fibrin polymers, which are then converted to insoluble fibrin polymers. Thrombin plays the role of fXIIIa, and a factor for carrying out 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 major constituents 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 causes a new coagulation reaction when it becomes in solution during the dissolution of the thrombus [AK Gash et al., Am. 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 thrombus are coumarins (ie syncumar and warfarin) and heparin, vitamin K antagonists which are direct inhibitors of thrombin.

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 is absent [R.Egbring et al., Thromb. Haemost. 42 , 225 (1979). Thrombin bound to the thrombus cannot be inhibited by this indirect mechanism because the heparin-AT-III complex is inaccessible. [JIWeitz et al., J. Clin. Invest. 86 , 385 (1990). In addition, the development 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)].

Vitamin K antagonists can also be administered orally and the effects develop after 16 to 24 hours. This inhibits the development of some active forms of 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 dosage. However, vitamin K antagonists are difficult to use because of their narrow therapeutic range, strong dependence on diet composition (vitamin K), and varying individual sensitivity.

The first very potent synthetic compound that directly inhibits thrombin is the reversible inhibitor tripeptide aldehyde D-Phe-Pro-Arg-H, which has shown significant anticoagulant effects in both in vivo and in vitro experiments [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 related to D-Phe-Pro-Arg-H has been synthesized. One of the first is Boc-D-Phe-Pro-Arg-H [S. Bausz et al., Int. J. Peptide Protein Res. 12 , 217 (1978) and C. Kettner and E. Shaw, Thromb. Res. 14 , 969 (1979)] is a methyl chloride ketone homologue. Additional peptides and acylpeptides to be mentioned are boroarginine homologues (Ac-D-Phe-Pro-boroArg as well as D-Phe and Boc-D-Phe) which are potent reversible thrombin inhibitors [C. Kettner et al., J. Biol. Chem. 265 , 18289 (1990)] and other Boc-D-Phe-Pro-Arg analogs, 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, but D-MePhe-Pro-Arg-H (GYKI-14766), obtained by methylating amino group ends, 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 experimental animals was significantly inhibited by this 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 the enzyme of fibrinolysis is weak, it significantly promotes the dissolution of blood clots when administered with thrombin solubilizers [CV Jackson et al., J. Cardiovascular Pharmacol. 21 , 587 (1993)], which cannot be reached with the heparin-AT-III complex. Various compounds related to D-MePhe-Pro-Arg-H have been synthesized, for example D-MePhg-Pro-Arg-H [RTShuman et al., J. Med. Chem. 36 , 314 (1993).

Anticoagulant activity (i.e., proteolytic activity in the process) is, for example, thrombin time (hereinafter referred to as "TT"), activated partial thromboplastin time (hereinafter referred to as "APTT"). quot;) and prothrombin time (prothrombin time; is measured by anticoagulant tests (tests anticoagulant), hereinafter referred to as the "PT") [EJWBovies etc., Mayo Clinic Laboratory Manual of Haemostasis; WB Saunders Co., Philadelphia (1971)]. For example, the time required for coagulation is determined by coagulation of plasma, which is suppressed spontaneous coagulation such as citrate-plasma. When the anticoagulant acts, the coagulation time is prolonged in proportion to the inhibition of the reaction. The effect of the anticoagulant is characterized by the concentration of material required to allow the coagulation time to be doubled compared to the control (IC ₅₀ ). The anticoagulant activity against individual coagulant protease is measured by the amidolytic method [R. Lotenberg et al., Method in Enzymol. 80 , 341 (1981); G. Cleanson, Blood Coagulation and Fibriolysis 5 , 411 (1994)]. The isolated active factors (eg thrombin, fXa) and the chromogen or flurogen peptide-amide substrates above react in the presence or absence of inhibitors, respectively. Enzyme inhibitory action is characterized by the inhibition constant (IC ₅₀ ) measured during amidolysis.

TT test coagulation is initiated by thrombin added to citrate plasma. 22 pmole / ml of thrombin in the system acts and its inhibition can be measured on 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 endogenous or exogenous pathways. The resulting fXa activates prothrombin to thrombin, which then causes plasma coagulation. If the enzyme or one of the enzymes is inhibited by an inhibitor, the coagulation time is prolonged. The APTT or PT test produces up to 150 pmoles / ml (APTT) or 350 pmoles / ml (PT) of thrombin, while up to 40 pmoles / ml of Xa can be produced (this is the total amount of factor X present in both systems). B. Kaiser et al., Thromb. Res. 65 , 157 (1992).

The concentrations of D-MePhe-Pro-Arg-H ( C1 ) required to double the coagulation time are 87, 622 and 2915 nM for TT, APTT and PT tests, 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 and had no effect on the performance of fXa in both APTT and PT tests. Suggest that you cannot. Consistent with these results, the amidolytic effect of thrombin on the Tos-Gly-Pro-Arg-pNA substrate is inhibited by C1 with a value of IC ₅₀ = 2 nM, while the corresponding Bz-lle-Glu-Gly-Arg The amidolytic effect of fXa on the -pNA substrate was only slightly affected, with an IC ₅₀ = 9.1 mM (Bajusz et al., unpublished results).

It is an inherent property that blood clotting mechanisms are inhibited not only by direct thrombin inhibitors but also by trobinogenesis inhibitors such as fXa inhibitors. Tick Anticoagulant Peptide (TAP), a 60-member polypeptide isolated from ticks [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 ( acetimidoyl) -3-pyrrolidyl] 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. Amidolytic effects on fXa and plasma coagulation were strongly inhibited in these PT and APTT tests by these compounds, ie both free and complex bound Xa factors were equally well suppressed, while their specific nature as specific fXa inhibitors It was found that thrombin was not inhibited at all, i.e. did not show any activity in the TT test. 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 amidoratic activity of fXa on the ZD-Arg-Gly-Arg-pNA-substrate with IC ₅₀ = 57 and 30nM, respectively. There is no data on their anticoagulant activity. In our own tests, C3 and C4 showed significant inhibitory action against the Bz-lle-Glu-Gly-Arg-pNA substrate, whereas their anticoagulant activity was weak in the plasma coagulation test. It turned out. The published ( C2 ) and measured ( C3 and C4 ) activities of synthetic fXa compared to antithrombin compound C1 are shown in Table 1.

The data in Table 1 indicate that for C1 , anticoagulant activity is due to inhibition of thrombin while C2 is due to inhibition of fXa. Significant fXa inhibitory activity of C3 and C4 is not accompanied by any significant anticoagulant activity. The fXa activity 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

Inhibitor A: B: IC ₅₀ , μM ^a IC ₅₀ , nM ^b 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

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

^b Value measured with isolated human Bz-lle-Glu-Gly-Arg-pNA chromogen substrate

^c NA = inactive

According to a recent publication [NAPrager et al., Circulation 92 , 962 (1995)] Thrombin, as well as factor Xa, is involved in thrombosis / blood clotting and released during lysis, and factor Xa is the [fVII + TF] -complex or the activity of factors V and VIII. Maintain new coagulation process and initiate reaction. Thus, if an anticoagulant can inhibit factor Xa in addition to the inhibition of thrombin, it is particularly beneficial 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 over known compounds and which, when orally administered, also have anticoagulant activity.

The anticoagulant activity of Boc-D-Phe-Pro-Arg-H ( C5 ) in the TT and APTT tests was smaller than that of the tripeptide aldehyde C1 , but better in the PT test and factor Xa at IC ₅₀ at 10 times lower than C1. Inhibited. Substitution of the Eoc-group to the BOC-group at the C5 position positively alters the anticoagulant effect of the peptide since Eoc-D-Phe-Pro-Arg-H thus obtained has a higher effect in three tests than C5. It was unexpectedly found; In addition, the activity was higher than C1 in the PTTT as well as the PT test, whereas the fXa inhibitory activity was similar to that of the 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)

Peptide aldehyde A: IC ₅₀ , μM ^a B No. R TT APTT PT IC ₅₀ , μM ^b 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

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

^b Value measured with isolated human Bz-lle-Glu-Gly-Arg-pNA chromogen substrate

It has also been found that the coagulation of homologous cyclochloro-glycine to the N-terminus decreases the activity, while more effective anticoagulant compounds can be obtained when the N-terminal amino acid is changed from phenylalanine to 3-cyclobutylalanine. Apart from 3-cyclobutylalanine, substitution of 3-cyclopentylaldehyde also produces effective peptidyl-arginine aldehydes. Substitution of methoxycarbonyl or propoxycarbonyl groups instead of N-terminal ethoxy-carbonyl groups has also been found to change the activity spectrum, ie the proportion of activity measured in the individual tests.

The present invention relates to novel peptidyl-arginine aldehyde derivatives of the following general formula (1), their acid-addition salts formed with organic or inorganic acids and pharmaceutical compositions containing them.

Q-D-Xaa-Pro-Arg-H (1)

In the above formula,

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 group,

Arg is an L-arginyl group.

The new compounds of formula (1) are therapeutically useful, in particular for anticoagulant, antiplatelet and antithrombin effects.

Particularly preferred representative examples of the compounds of formula (1) 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-proyl-L-arginine aldehyde,

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

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

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-cyclohexyl-acetic acid],

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

Gla = gamma-carboxyl-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-pyrrolidine-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- (naph-1-tyl) -L-alanine-[(2S) -2-amino-3- (naph-1-tyl) -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:

The abbreviation of amino acid only is L-amino acid, respectively. D-amino acids are indicated separately, ie 3-phenyl-D-alanine = D-Phe. The hyphen (-) before and after the amino acid is the hydrogen atom lost in the amino group or the hydroxyl group lost in the carboxy group, respectively. Thus, Eoc-D-Cba-Pro-Arg-H is ethoxy-carbonyl-3-cyclochloro-D-alanyl-L-prolyl-L-arginine aldehyde, whereas Bz-lle-Glu-Gly- Arg-pNA is benzoyl-L-isoleucil-L-glutamyl-glycyl-L-arginine p-nitroanilide.

The present invention relates to novel peptidyl-arginine aldehyde derivatives of the following general formula (1), their acid-addition salts formed with organic or inorganic acids and pharmaceutical compositions containing them.

Q-D-Xaa-Pro-Arg-H (1)

In the above formula,

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 group,

Arg is an L-arginyl group.

The compound of formula (1), wherein Q, Q ', Xaa, Pro and Arg have the same meaning tripeptide lactam as described above, for example, by concentrating acylpeptide QD-Xaa-Pro with L-arginine lactam The guanidino group is protected with a benzyloxycarbonyl group, and the resulting protected tripeptide lactam is reduced with the protected tripeptide aldehyde of formula QD-Xaa-Pro-Arg (Z) -H, followed by organic or inorganic acid Peptide analogs of formula (1) formed together with additional salts are isolated.

QD-Xaa-Pro, an acyl peptide used as starting material, is prepared by acylating, for example, the amino acid of D-Xaa with an alkyl ester of Q 'of chloroformic acid and an acylamino acid of QD-Xaa in basic medium. Therefore, what is obtained is bound to L-proline.

QD-Xaa required to bind proline acylates racemic DL-Xaa-compounds, converts DL-acylamino acids to the methylamino acids above, and enzymatically converts Q-DL-Xaa-OMeracemic esters. It can be prepared separately. The Q-D-Xaa-OMe obtained as described above is saponified to become the required Q-D-Xaa acylamino acid.

In the compound of the formula (1) of the present invention, Q, Xaa, Pro and Arg have the same meaning as above, show strong anticoagulant activity in both in vivo and laboratory experiments and have excellent anticoagulant activity.

The anticoagulant effect of the compound of formula (1) in the laboratory was determined by PT, APTT and TT tests [D. Bagdy et al., Thromb. Haemost. 67 , 325 (1992). The fXa-inhibitory activity of the compounds is also determined using the Bz-Ile-Glu-Gly-Arg-pNA chromogen 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 are controls. In the table the compounds are listed in order of decreasing PT activity. The following data clearly show the useful effects of the Cba and Cpa end portions when compared to Phe and the homologous cyclobutylglycine (see C8 , C9 and C10 compounds of the DL-class, for example) ( 1 , 4 , and C6). See compound).

TABLE 3

Anticoagulant Effect (A) and fXa Inhibitory Effect (B) of Novel Peptide Aldehyde (1-4, Formula QD-Xaa-Pro-Arg-H) and Anticoagulant Effect of Controls ( C1, C6 and C8 to C10 ) Of PT activity of the anti- and fXa inhibitory effect

Q-Xaa-Pro-Arg-H A: IC ₅₀ , μM ^a B: IC ₅₀ Example ^b Q Xaa PT APTT TT nM ^c 2 Moc D-Cba 1.12 0.25 0.11 388 One Eoc D-Cba 1.15 0.35 0.11 40 4 Eoc D-Cpa 1.41 0.24 0.10 27 3 iPoc D-Cba 1.70 0.60 0.19 36 Control compound C8 Eoc DL-Cba 1.49 0.50 0.13 68 C9 Eoc DL-Cpa 1.90 0.48 0.26 43 C6 Eoc D-Phe 1.91 0.38 0.13 917 C1 D-MePhe 2.91 0.62 0.09 9133 C10 Eoc DL-Cbg 6.15 0.77 0.88 82

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

^{b is the} same as the number in the examples illustrating the preparation of each compound

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

Plasmin as well as for plasmin formation induced by tissue plasminogen activator (hereinafter referred to as "tPA") and urokinase (hereinafter referred to as "UK"). The inhibitory effect of the fresh compounds of the invention on the " PL " hereinafter is 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 antifibrin lytic activity of C1 is weak in vivo and can be used as an adjuvant to dissolve experimental blood clots, whereas the antifibrin lytic activity of C5 is well known in vivo [CVJackson et al. J. Cardiovasc. Pharmacol. 21 , 587 (1993). In Table 4, the results obtained with the controls C1 to C5 as well as the novel compounds 1 to 2 are shown as examples.

In addition to the IC ₅₀ values (column A), the usefulness of the compounds related to C1 is also listed (column B). The latter data indicate that, similar to compound C1 , the antifibrin lytic activity of novel compounds 1 and 2 is moderate; Activity tests for these three enzymes ranged from 3.2 to 4.5, 39 times lower than that of compound C5 .

Table 4

Inhibitory effect of the novel peptidyl-arginine aldehydes and structurally similar controls on plasmin (PL) formation and plasmin induced by tPA and UK, studied by fibrin-plate method (IC ₅₀ )

Peptidyl arginine aldehyde (example) ^c A: IC ₅₀ B: relative active ^b PL tPA UK A B A B A B Eoc-D-Cba-Pro-Arg-H (1) 39 1.4 27 4.9 120 0.7 Moc-D-Cba-Pro-Arg-H (2) 235 0.23 92 1.4 153 0.5 Eoc-D-Cpa-Pro-Arg-H (3) 98 0.55 38 3.5 46 1.8 D-MePhe-Pro-Arg-H (C1) 54 1.0 132 1.0 82 1.0 Boc-D-Phe-Pro-Arg-H (C5) 12 4.5 6 22.0 3 27.3

^a IC ₅₀ = concentration of 50% reduction in the peptide concentration (μM) hydrolyzed in the fibrin plate 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 formula (1) on fXa and thrombin that bind to coagulation as well as fibrin gel-bound thrombin are described in Table 5 with compounds 1-3; Each value of C1 was used as a control. Plasma-coagulation can be obtained by recalcification of human platelet-rich plasma, and fibrin-gel is obtained by coagulating human fibrinogen with human thrombin. ZD-Arg-Gly-Arg-pNA or Tos-Gly-Pro-Arg-pNA substrates were used in accordance with Methods M1 to M3 to determine the activity of fXa and thrombin.

Table 5

Inhibitory effect on thrombin and blood clot-bound fXa (IC _50, μM) as well as novel peptidyl-arginine aldehydes (1 to 3) of the present invention and fibrin-gel bound thrombin of C1 as a control

Peptidyl-arginine-aldehyde (number) ^c Plasma blood clots Fibrin gel fXa Thrombin Thrombin Eoc-D-Cba-Pro-Arg-H (1) 0.70 0.39 0.32 Moc-D-Cba-Pro-Arg-H (2) 0.39 0.68 0.64 Eoc-D-Cpa-Pro-Arg-H (3) 0.28 0.49 0.67 D-MePhe-Pro-Arg-H (C1) 1.12 0.38 0.30

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

^b Same as the number in the example describing a novel compound or control ( C1 )

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

The anticoagulant and platelet aggregation inhibitory activity of the compound of formula (1) was studied ex vivo in New Zealand white rabbits according to D. Bagdy et al. [Thromb. Haemost. 67 , 357 (1992). Compounds may be 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). ). Within 30 minutes of oral administration the effect of the compound can already be detected, peak values can be obtained after 60 to 180 minutes and the therapeutic level lasts for 3 to 6 hours depending on the dosage.

The in vivo effects of ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-L-arginine aldehyde are detailed in Tables 6 and 7. Corresponding values of C1 are listed as controls. Compounds were administered orally at 5 mg / kg. Samples taken every 30 to 60 minutes were analyzed in the unmeasured veins. Whole Blood Clotting Time (hereinafter referred to as "WBCT") and platelet aggregation induced by thrombin (PAI) were determined. APTT and TT were also 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 values in Table 6 and Table 7 show that New Compound 1 had a sustained anticoagulant and anticoagulant effect than C 1.

Table 6

D-MePhe-Pro-Arg-H ( C1 ) and Eoc-D-Cba-Pro-Arg-H ( C1 ) as a control group when administered orally at 5 mg / kg in rabbits in the APTT and TT tests characterized by relative coagulation time ^a ( Anticoagulant effect of 1)

time Eoc-D-Cba-Pro-Arg-H D-MePhe-Pro-Arg-H min. APTT TT APTT TT 0 1.0 1.0 1.0 1.0 30 1.73 ± 0.47 8.30 ± 5.67 1.27 ± 0.38 1.52 ± 0.17 45 1.84 ± 0.50 9.04 ± 5.50 1.30 ± 0.02 2.50 ± 0.44 60 1.94 ± 0.51 9.28 ± 5.45 1.37 ± 0.03 4.75 ± 1.38 90 1.92 ± 0.31 13.58 ± 5.43 1.45 ± 0.09 10.50 ± 4.59 120 1.75 ± 0.26 16.33 ± 5.89 1.43 ± 0.11 5.51 ± 3.59 180 1.59 ± 0.12 6.95 ± 3.73 1.39 ± 0.09 2.05 ± 0.38 240 1.38 ± 0.08 2.03 ± 0.42 1.31 ± 0.11 1.81 ± 0.39 300 1.28 ± 0.08 1.77 ± 0.50 1.25 ± 0.22 -

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

TABLE 7

D-MePhe-Pro-Arg-H ( C1 ) and Eoc-D-Cba-Pro-Arg-H (1) when administered orally at 5 mg / kg in rabbits in a WBCT study characterized by relative coagulation time and percentage inhibition ^a Anticoagulant and PAI effects

time Eoc-D-Cba-Pro-Arg-H (1) D-MePhe-Pro-Arg-H ( C1 ) min. WBCT PAI (%) WBCT PAI (%) 0 1.0 1.0 1.0 1.0 30 1.52 ± 0.22 71.3 ± 16.6 1.07 ± 0.13 52.8 ± 14.7 45 1.58 ± 0.17 55.8 ± 17.4 1.26 ± 0.07 58.4 ± 15.6 60 1.88 ± 0.45 63.8 ± 21.2 1.55 ± 0.17 54.2 ± 11.6 90 1.73 ± 0.33 85.6 ± 10.0 1.61 ± 0.34 83.2 ± 8.9 120 1.98 ± 0.30 73.6 ± 19.1 1.45 ± 0.21 75.2 ± 12.3 180 1.43 ± 0.13 81.8 ± 11.6 1.44 ± 0.08 47.5 ± 20.9 240 1.12 ± 0.24 59.4 ± 19.1 1.22 ± 0.08 32.2 ± 16.0 300 1.18 ± 0.08 75.8 ± 11.8

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

Compounds of the present invention of formula (1) are seizures based on deep vein thrombosis, pulmonary embolism, arterial thrombosis, unstable angina, myocardial infarction, myocardial fibrillation and seizure based on thrombus and hypercoagulability Used for the treatment of light prevention.

In atherosclerosis, the compounds can be used to prevent thrombosis, coronary artery disease of cerebral arteries as a surgical prophylaxis or other surgical prophylaxis for patients at high risk. The compounds also include diseases with nephritis and hypercoagulation; It can be applied to the thrombus collapse of transdermal transluminal angioplastics for adjuvant therapy and reclosure room as well as diabetes (eg arthritis) and malignant tumors. The compound may be deficient in anti-thrombin-III if the administration of the other anticoagulant is not effective or contraindicated, for example for heparin or heparin-induced thrombocytopenia (HIT), and for example for coumarin A case is mentioned.

The compounds of the present invention and their pharmaceutically acceptable salts are used alone or in the form of pharmaceutical formulas for the purpose of treatment. The present invention also refers to these equations.

Pharmaceutical formulas are used alone or in the form of pharmaceutical formulas for the purpose of treatment. The present invention also refers to these equations.

The pharmaceutical formula consists of an effective amount of the compound of formula (1) or a pharmaceutically acceptable salt thereof and known pharmaceutically acceptable carriers, fillers, diluents and / or other pharmaceutical excipients.

The carrier, diluent, or filler material 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 or glycols of polyethylene glycol. Pharmaceutical excipients can be preservatives, various natural or synthetic emulsifiers, dispersing or wetting agents, coloring materials, flavoring agents, facilitation agents, and substances that improve the bioavailability 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 stage can be fixed by the doctor who devised the treatment. In diseases where thrombin formation or inhibition of function is required for the purpose of prophylaxis or treatment, daily oral or parenteral administration (eg, intravenous administration) may range from 0.01 to 1000 mg / kg body weight, preferably 0.25. To 20 mg / kg body weight may be administered.

The compound of formula (1) of the present invention actively promotes dissolution of thrombin formed in arteries or veins and inhibits remodeling when administered as a thrombolytic agent (eg, tPA or UK). In this case, the compound of the present invention is preferably administered simultaneously with the thrombin solubilizer or immediately after the thrombin solubilizer treatment.

The following examples illustrate, but do not limit the scope of the invention. The R _f values reported in the examples were determined in the following developing solvents using silica gel as 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 is determined by the instrument of "High Performance Liquid Chromatography (HPLC) System 2 of Pharmacia ELKB assay" as follows. It became.

Column : "VYDAC C-18 Reverse Phase: 10mm, 300A, 4X250mm"

Buffer A : trifluoroacetic acid in 0.1% water

Trifluoroacetic acid in buffer B 0.1% acetonitrile

Gradient was applied at 1 ml / mim. Flow rate

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

II: 0 to 30 minutes 0 to 60% B

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

Acyl arginine is shown in equilibrium structure: aldehyde, aldehyde hydrate and two acylcyclo forms. During HPLC analysis the aldehyde hydrate and one or two aminocyclones are shown as separate peaks, and eventually the acylarginine aldehydes described in the examples are specified with 2-3 k 'values.

Mass Spectrometry (hereinafter referred to as "MS"). FAB positive ionization measurements are performed with a Finnigan MAT8430 instrument. Samples were 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 molecule-ions produced with NBA: [M + H] and [M + H + NBA] can be detected. In the examples FAB spectrum data are specified accordingly.

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

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) hemi sulfate

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

3.28 g (8.4 mmol) of t-butoxycarbonyl-N ^G -benzyloxy-carbonyl-L-arginine-lactam [S.Bajusz and colleagues: J.Med.Chem. 33 , 1729 (1990)] was suspended in 8 ml of chloroform, then 10 ml of ethyl acetate and saturated with hydrogen chloride gas (0.11 to 0.15 g / ml) was added with continuous stirring and ice cooling. The reaction was thin layer chromatography [R _f (9) = 0.91 (Boc-compound); 0.07 (free compound)]. At the end of the reaction the mixture was diluted with 18 ml of diethyl ether, the crystalline formed was filtered off, washed with 5 ml of acetone and 5 ml of diethyl ether and dried in a vacuum dryer on potassium hydroxide pellets. The resulting hydrogen chloride N ^G -benzyloxycarbonyl-L-arginine-lactam was dissolved in 8 ml of dimethylformamide, cooled to -20 ° C and added to the mixed anhydride prepared below. 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. Continuous stirring introduces 0.9 mL (8 mmol) of N-methylmorpholine and 1.05 mL (8 mmol) of isobutyl chloroformate, which is then stirred for 10 minutes and the aforementioned 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. The salt was filtered off and the filtrate was diluted with 40 ml of benzene. The solution was washed with 3 × 10 water, 10 ml of 1M potassium hydrogen sulfate, again with 3 × 10 ml of water, dried over anhydrous sodium sulfate and the solvent was evaporated to a pressure of 2.0 to 2.5 kPa. The resulting product was applied to a column made of 70 g Kigelgel 60 product and eluted with ethyl acetate. The portions containing pure material [R _f (1) = 0.50] are combined and the solvent is evaporated at 2.0 to 2.5 kPa. The oily portion was rubbed 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) was 15 Dissolved in ml of tetrahydrofuran and 3.6 mmol of lithium aluminum hydrate, and dissolved in tetrahydrofuran was added at -50 ° C or lower with continuous stirring. The evolution of the reaction was measured by thin layer chromatography in solvent system 6 and further lithium aluminum hydrate was added if necessary. With constant stirring and cooling, a precooled 0.5 M sulfuric acid solution was added to the reaction mixture until the pH reached 3. The resulting solution was extracted with 2 × 15 mL of n-hexane followed by 3 × 20 mL of methylene chloride. The methylene chloride extracts are combined and washed with 3 x 15 ml of water, 15 ml of cold 5% sodium bicarbonate solution, then again with 15 ml of water, dried over anhydrous sodium sulfate, and then the solvent is pressured from 2.0 to 2.5 kPa. Evaporated. The residue was dissolved in 10 ml of benzene and precipitated with cyclohexane. The product weighs 1.8 g (65%) after filtration and drying.

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

1.58 g (2.7 mmol) of ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 1, Step 2) is 14 Dissolved in ethanol, 2.7 mL 0.5 M sulfate, 3.2 mL water and then 0.17 g Pd / C catalyst, suspended in 6 mL ethanol and the system hydrogenated at 10 ° C. The development of the reaction was completed in 15 minutes and measured by thin layer chromatography. 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. pH was adjusted to 3.5 with Dowex AG 1-X8 resin in the 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] ⁺ ).

The starting material can be prepared as follows.

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

(Eoc-D-Cba-Pro)

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

10 g (70 mmol) of 3-cyclobutyl-DL-alanine [A. Burger et al., J. Med. Chem. 6 , 221 (1963)] was dissolved in 70 mL of sodium hydroxide, cooled to + 5 ° C. and added dropwise with 11.1 mL (116.2 mmol) of ethyl chloroform stirring continued. 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 solution was oxidized until pH was 1 and then extracted with 3 × 20 ml of ethyl acetate. The combined 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. The resulting oil was 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-alanine Salt

11 g (51 mmol) of ethoxycarbonyl-3-cyclobutyl-DL-alanine [Example 1, Step 1] was dissolved in 100 mL of methanol, cooled to -15 [deg.] C. and 10 mL (137 mmol) of thionyl chloride Was added with continued stirring. Stirring continued without further cooling. When the reaction mixture reached room temperature, stirring was performed for an additional 2 hours. The solvent was evaporated to a pressure of 2.0 to 2.5 kPa. The remaining 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 water, dried over anhydrous sodium sulfate, and at a pressure of 2.0 to 2.5 kPa. Dried. 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 treatment.

Step C : Methyl Ethoxycarbonyl-3-cyclobutyl-D-alanine Salt

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

10.8 g (47 mmol) of methyl ethoxycarbonyl-3-cyclobutyl-DL-alanine salt (synthesized in step B) was 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 and the pH was readjusted to 7.5 and maintained at this value during titration. Enzymatic hydrolysis was completed in 3 hours. 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 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 ethanol and 10 mL 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, oxidized to pH = 1 with 3M hydrochloric acid and extracted with 3 × 10 ml of ethyl acetate. The combined oil 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 [R _f (2) = 0.8] was mostly used for the next step and the residue was converted to crystalline salt as follows.

0.43 g (2 mmol) of oily ethoxycarbonyl-3-cyclobutyl-D-alanine (Example 1, Step D) was dissolved in 10 ml of diethan ether and 0.23 ml (2.1 mmol) of cycloamine was QNRK. It was. The separated crystals were filtered, 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;

Calc .: 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) was dissolved in 60 mL of tetrahydrofuran, cooled to 0 ° C., and 4.85 g (24.5 mmol) 2,4,5-trichlorophenol and 5.06 g (24.5 mmol) of dicyclohexyl-carbodimide were added. The reaction was stirred at rt for 2 h, filtered from the separated dicyclohexylurea and washed with tetrahydrofuran. The filtrate and washes were combined and evaporated at a pressure of 2.0 to 2.5 kPa. The residue was dissolved in 25 mL diethyl ether-petroleum mixture (1: 1), the insoluble dicyclohexyl urea was filtered off and the filtrate was evaporated again [4.9 g (89%) respectively And 0.6 g (11%). The oily 2,4,5-trichlorophenyl-ethoxycarbonyl-3-cyclochloro-D-alanine salt is dissolved in 40 ml of pyridine and 2.12 g (18.4 mmol) of finely ground L-proline is 3.43. Add with mL (24.6 mmol) triethylamine. 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 oil 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 combined hydrogen carbonate extracts were acidified to pH = 3 with 6M HCl and extracted with 3 × 20 mL of diethyl ether. The extracts on ether were combined, the acid was removed with water, 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) hemi sulfate

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)] and 1.25 g (4.0 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (Example 2, Step C), and Chromatographic purification of the final product was carried out according to the method described in Example 1, Step 1 using proportional amounts of reagents and solvents. The portions containing pure final product [R _f (1) = 0] were combined and evaporated to a pressure of 2.0 to 2.5 kPa. The residue was rubbed with petroleum ether and dried in a drier of paraffin turnings and phosphorus pentoxide.

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) was prepared using a proportional amount of reagents and solvents. 1, was reduced 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 half sulfate

0.69 g (1.2 mmol) of methoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 2, Step 2) is a proportional amount Hydrogenolysis was carried out according to the method described in Example 1, Step 3 using reagents and solvents.

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] ⁺ ).

The starting material can be made of the following materials.

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

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

3-cyclochloro-DL-alanine [A. Burger et al., J. Med. Chem. 6 , 221 (1963)] was dissolved in 15 mL of 2M NaOH, cooled to 0 ° C., and 16.5 mL of 2M NaOH solution (24.5 mmol) and 2.55 mL (33 mmol) of methyl chloroform were added simultaneously with vigorous stirring. Stirred continuously at this temperature for 30 minutes and at room temperature for 2 hours. The reaction mixture was diluted with 40 mL of water, extracted with 10 mL diethyl ether and oxidized with 3M HCl. The product is extracted with ethyl acetate (4x20), washed with 20 ml of water, the extracts are combined, the acid is removed with water, dried over anhydrous sodium sulfate, dried over anhydrous sodium sulfate, and pressure of 2.0-2.5 kPa Evaporated to dryness. An oily residue [5.3 g (88%), Rf (7) = 0.61] was used for 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) was prepared according to the method described in Examples 1, Steps B to D using proportional amounts of reagents and solvents. Was processed.

Yield: 2.22 g (11 mmol) oily product

[R _f (7) = 0.61]

Step C : Methoxycarbonyl-3-cyclochloro-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 the majority 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 diethan ether and 0.21 ml (1.05 mmol) of dicyclohexyl amine was added. . The separated crystals were filtered, 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

Calculated: 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) hemisulphate

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)] and 0.33 g (1 mmol) of isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-proline (Example 3, Step C), binding of these compounds Chromatographic purification of the final product was carried out according to the method described in Example 1, Step 1 using proportional amounts of reagents and solvents. The portions containing pure final product [R _f (1) = 0.55] were combined and evaporated to a pressure of 2.0 to 2.5 kPa. The residue was rubbed 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-cyclochloro-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) is proportional Hydrogenated according to the method described in Example 1, Step 2 using a reagent and a solvent.

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

0.13 g (0.2 mmol) of isopropoxycarbonyl-3-cyclobutyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Example 2, step 2) Was treated according to the method described in Example 1, Step 3 using a reagent and a solvent.

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] ⁺ ).

The starting material can 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 to a pressure of 2.0 to 2.5 kPa, the residue was dissolved in 5 ml of water and evaporated to dryness, and the procedure was repeated. Residual water can be removed by boiling distillation with ethanol-benzene mixture. The remaining oil was dissolved in 10 ml of ethanol and 5 mmol of 1,2-epoxy-propane were added. After cooling the precipitate was filtered off, 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-cyclochloro-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 1M NaOH solution and 4 ml of isopropyl chloroform (1M concentrated in toluene) were added simultaneously with vigorous stirring. Stirring was continued at this temperature for 1 hour and at room temperature for 5 hours. Dioxane and toluene were distilled at a pressure of 2.0 to 2.5 kPa. The remaining aqueous solution was washed with 5 ml diethyl ether and oxidized to pH = 3 with 1 M potassium hydrogen sulfate. The emulsion was extracted with 3 x 5 ml of ethyl acetate, 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) was treated according to the method described in Example 1, Step E using proportional amounts of reagent and solvent It became.

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) hemi sulfate

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

2.93 g (7.5 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] and 2.3 g (7.1 mmol) of ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-proline (Example 4, Step C) were prepared using proportional amounts of reagents and solvents. Processing was carried out according to the method described in Example 1, Step 1. The portions containing pure final product [R _f (1) = 0.55] were combined and evaporated to a pressure of 2.0 to 2.5 kPa, the residual oil was rubbed with n-hexane, filtered and dried in a drier.

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) were in proportional amounts. Reagents and solvents were used following 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) Reagents and solvents were 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

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

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

7.8 g (34 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanine (Example 4, Step A) was prepared using the method described in Example 1, Steps B, C, D using proportional amounts of reagents and solvent. Treated according to. Yield: 3.45 g (88.6%), most of the oil [R _f (6) = 0.062] was used directly for step C. The residue is converted to crystalline 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-ala (Example 4, Step B) was treated according to the method described in Example 1, Step E using proportional amounts of reagent and solvent It became. The resulting 2,4,5-trichlorophenyl ester was combined with 1.15 g (10 mmol) of L-proline to yield 2.3 g (71%) of 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) hemi sulfate

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

2.3 g (5.8 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine-lactam [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] and 1.67 g (5 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-proline (Synthesis 1, Step C) were prepared using proportional amounts of reagents and solvents. The constructs were combined and the product was purified according to the method described in 1, step 1. The portions containing pure final product [R _f (1) = 0.37] were combined and evaporated to a pressure of 2.0 to 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

0.9 g (1.5 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (Synthesis 1, Step 1) is a proportional amount of reagent and The solvent was reduced using 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 half sulfate

0.33 g (0.54 mmol) of ethoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Synthesis 1, step 2) is a proportional amount of reagent and The solvent was used in accordance with 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] ⁺ ).

The starting material can be made of the following materials.

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 were treated according to the method described in Example 1, Step A using proportional amounts of reagents and solvents, but the resulting oil was evaporated from the final material after evaporation. Dried in a vacuum dryer.

Yield: 14.6 g (55%)

R _f (9) = 0.70

Step B : Ethoxycarbonyl-3-phenyl-D-alanine N-hydroxy-lysinimide salt

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 salt were added to 60 ml of tetrahydrofuran. Dissolved. The solution was cooled to 0 ° C. and 11.5 g (56 mmol) of dicyclohexyl-carbodimide were added with stirring. Stirring was continued at this temperature for 10 hours, the mixture was filtered and the filtrate was evaporated to a pressure of 2.0 to 2.5 kPa. The residue was crystallized in 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 hydroxy-succinimide salt (synthesis 1, step B) was dissolved in 15 ml of pyridine and 1.15 g (10 mmol) of L-proline and 1.4 ML (10 mmol) triethylamine was added. The reaction mixture was stirred for 10 hours at room temperature and evaporated to a pressure of 2.0-2.5 kPa. The residue was partitioned between 10 mL diethyl ether and 30 mL 5% sodium hydrogen carbonate. The aqueous layer was washed with 3 x 5 ml of diethyl ether, and the collected oil layer was extracted with 5 ml of 5% sodium hydrogen carbonate. The carbonate layer was collected, acidified to pH = 3 with 1 M potassium hydrogen sulfate solution and extracted with 3 × 10 mL of ethyl acetate. The combined oil layer was dried over anhydrous sodium sulfate and evaporated to a pressure of 2.0-2.5 kPa. The oily residue was crystallized with diethyl ether and the crystalline was filtered off, washed with diethyl ether and dried on 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) hemi sulfate

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

4.1 g (10.5 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] and 2.88 g (9 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-proline (Synthesis 1, Step C) were prepared using Example 1 using proportional amounts of reagents and solvents. Purification was carried out according to the method described in step 1, but the crude product was purified by crystallization from cyclohexane. The portions containing [R _f (1) = 0.37] were combined to a pressure of 2.0 to 2.5 kPa. Evaporated. The residue was dried in a vacuum drier.

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) is a proportional amount of reagent and solvent Reduction was carried out according to the method described in Example 1, Step 2, but after evaporation of the methylene chloride solvent, the final product of the oily phase was crystallized with diethyl ether. The crystalline was filtered off, washed with diethyl ether and dried on 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 half sulfate

2.68 g (4.5 mmol) of methoxycarbonyl-3-phenyl-D-alanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (Synthesis 2, step 2) is a proportional amount of reagent and The solvent was used in accordance with 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] ⁺ ).

The starting material can be made of 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 simultaneously added 16.5 mL of 2M NaOH solution and 2.55 mL (33 mmol) of methyl chloroform. 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, oxidized to pH = 3 with 3M HCl and extracted with 4 x 20 mL of ethyl acetate. The combined 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 isolated crystalline was 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 salt (Synthesis 2, Step A) was dispersed between 75 ml of ethyl acetate and 30.5 ml of 1M potassium hydrogen sulfate. The layers were separated, the oil layer was washed with water, 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 carbodimide were added and the reaction mixture was left overnight. The separated cyclohexyl urea was filtered 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) is dissolved in 12 ml of pyridine and 1.38 g (12 mmol) Powdered L-proline and 1.68 mL (12 mmol) triethylamine were added with stirring. The reaction mixture was stirred for 16 h and 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 extracted with 3 × 7 ml diethyl ether. The collected ether layers were extracted with 7 ml of 5% sodium hydrogen carbonate. The carbonic acid wash was 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 2.0-2.5 kPa. The residue was crystallized with diethyl ether, the crystalline was 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) hemi sulfate

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

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

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) is a proportional amount of reagent And using a solvent 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) is a proportional amount of reagent And a solvent was used following 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] ⁺ ).

The starting material can be made of the following materials.

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

Step A : Ethoxycarbonyl-3-cyclochloro-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 proportional amounts of reagent and solvent To give an oily 2,4,5-trichlorophenyl ester, which was then combined with 0.58 g (5.0 mmol) of finely ground 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) hemi sulfate

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

3.7 g (9.5 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] and 2.94 g (9.0 mmol) of ethoxycarbonyl-3-cyclopentyl-DL-alanyl-L-proline (Synthesis 4, Step A) were carried out using proportional amounts of reagents and solvents. Processing was performed according to the method described in Example 1, Step 1. Compounds containing pure portions were combined and evaporated to a pressure of 2.0-2.5 kPa and the residue was 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) is a proportional amount of reagent And using a solvent 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) is a proportional amount of reagent And a solvent was used following 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] ⁺ ).

The starting material can be made of the following materials.

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

Step A : Ethoxycarbonyl-3-cyclochloro-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 reagents and solvent To give an oily 2,4,5-trichlorophenyl ester, which was then combined with finely ground 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) hemi sulfate

1.2 g (3.0 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine [S. Bajusz et al., J. Med. Chem. 33 , 1729 (1990)] and 0.69 g (2.3 mmol) of ethoxycarbonyl-DL-cyclobutyl-glycyl-L-proline (Synthesis 5, Step A) were prepared using Example 1 using proportional amounts of reagents and solvents. Bind to blocked tripeptide lactam [R _f (1) = 0.32], respectively, according to the method described in steps 1,2 and 3, and reduce blocked tripeptide aldehyde [R _f (1) = 0.53], Unblocked.

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-glysyl-L-proline (Eoc-DL-Cbg-Pro)

Step A : Ethoxycarbonyl-DL-Cyclobutyl-Glysyl-L-Proline

0.78 g (6.0 mmol) of DL-cyclobutyl-glycine [THPorter et al., Arch. Biochem. Biophys, 179 , 266 (1977), acylation using a proportional amount of reagent and solvent according to the method described in Example 1, steps A and E [R _f (7) = 0.60], 2,4,5 -phenol trichloromethyl _{[R f (4) = 0.84} ] and the reaction and was combined 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 a 9: 1 mixture of human plasma and 3.8% sodium citrate 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 ). The wash medium 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 wash medium, the mixture was incubated at 37 ° C. for 30 minutes, and then the reaction was stopped by adding 100 μl of 50% acetic acid. I was. 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 is efficient, the extinction coefficient is 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 of solution 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 (3 parallel samples for each concentration) were added to the blood clots and at 37 ° C. with 400 μl of buffer as a control for 5 minutes. Incubated. 100 μl of 2mM Moc-D-Chg-Gly-Arg-pNA substrate was then added to each vessel and incubated again at 37 ° C. for 30 minutes. The reaction was stopped by adding 100 μl of 50% acetic acid.

c) 150 μL from this reaction vessel was applied to a 96-well microtitre plate 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 M3

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 every concentration) and the sample is mixed with 400 μl of buffer as a control for 5 minutes. Incubated at ° C. 100 μl of 1 mM Tos-Gly-Pro-Arg-pNA substrate was then added to each vessel and incubated again at 37 ° C. for 30 minutes.

The reaction continued according to M2 / c.

Method M4

Preparation of Fibrin Blood Clots

a) 200 μl human fibrinogen solution (SIGMA), 25 μl 25NIH / E / ml of human thrombin (SIGMA) and 40 μl of 100 mM potassium chloride solution are mixed in each vessel for 1 hour at 20-22 ° C. 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 M5

Determination of Inhibition of Thrombin Binding to Fibrin-Blood

a) 0.1 to 1.0 to 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 a 96-well microtiter plate _a Inhibition Measurement of

a) With phosphate buffer (0.1 M sodium phosphate and 0.05 M sodium chloride, pH = 7.4), 1.3 E / mL solution was prepared from human factor X _a (SIGMA) 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 of control and peptide inhibition solutions (three 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 after 10 minutes the extinction was determined at 405 nm. The step is then the same as that of M2 / c.

Claims (8)

The peptidyl-arginine aldehyde analogs of formula (1) and their acid-addition salts formed with organic or inorganic acids. Q-D-Xaa-Pro-Arg-H (1) In the above formula, 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 group, Arg is an L-arginyl group. Ethoxycarbonyl-3-cyclobutyl-D-alanyl-L-proyl-L-arginine aldehyde and acid addition salts thereof. Methoxycarbonyl-3-cyclobutyl-D-alanyl-L-proyl-L-arginine aldehyde and acid addition salts thereof. Ethoxycarbonyl-3-cyclopentyl-D-alanyl-L-proyl-L-arginine aldehyde and acid addition salts thereof. Methoxycarbonyl-3-cyclopentyl-D-alanyl-L-proyl-L-arginine aldehyde and acid addition salts thereof. As active ingredients one or more compounds of formula (1), wherein Q, D-Xaa, Pro and Arg are as defined in claim 1, or pharmaceutically acceptable salts thereof, are commonly used in the pharmaceutical industry. Pharmaceutical compositions consisting of additives and / or carriers. A medicament comprising a compound of formula (1), wherein Q, D-Xaa, Pro and Arg are as defined in claim 1. A medicament for treating and preventing blood clots and / or accelerated blood clots made of a compound of formula (1), wherein Q, D-Xaa, Pro and Arg are as defined in claim 1.