HU211088B - Process for producing peptide derivates inhibiting coagulation of blood and pharmaceutical compositions containing them - Google Patents

Abstract

Peptide derivs. of formula A-Xaa-Arg-H (I) and their salts are new. In (I), A = a D- or L- isochroman-1-carbonyl, D- or L-isochroman-3-carbonyl gp. or an acyl gp. of formula D- or DL-A'-CH(OH)-CO; A' = phenyl, benzyl, 1-naphthyl, 1-naphthylmethyl, 2-naphthyl, 2-naphthylmethyl, 9-fluorenyl, benzhydryl, cyclohexyl, cyclohexylmethyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; Xaa = L-prolyl or L-pipecolinic acid residue; Arg = an L-arginine residue. - An effective clot inhibiting dose of (I) is 0.3-3 mg/kg s.c. and 3-20 mg/kg p.o. Specifically claimed are 15 cpds. (I) e.g. D-isochroman-1-carbonyl -L-prolyl-L-arginine aldehyde (Ia).

Description

The present invention relates to novel peptide derivatives A-Xaa-Arg-H (I)

A is D or L-isochroman-1-carbonyl, Dv or L-isochroman-3-carbonyl, and an acyl group of the formula D-A'CH (OH) -CO

A 'is phenyl, benzyl, 1-naphthyl, 2-naphthyl, benzhydryl, cyclohexyl or cyclohexylmethyl,

Xaa is an L-proline residue or an L-pipecolic acid residue, and

Arg is for the preparation of L-arginine radicals and their acid addition salts.

The invention further relates to a process for the preparation of a pharmaceutical composition containing the above compounds. Particularly valuable examples of the compounds of formula (I) obtained by the process of the invention include:

3-Cyclohexyl-D-lacto-L-prolyl-L-arginine aldehyde sulfate,

D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-L-argininaldehid hemisulfate

3,3-Diphenyl-D-laktoil-L-prolyl-L-arginine aldehidhemiszulfát,

D-2- (l-Naphthyl) -2-hydroxyacetyl-L-prolyl-L-argininaldehid hemisulfate,

D-2- (2-Naphthyl) -2-hydroxyacetyl-L-prolyl-L-argininaldehid hemisulfate.

The compounds of the invention are valuable pharmaceuticals. in particular, they have anticoagulant activity.

Aldehyde derived from the tripeptide D-phenylalanil-L-prolyl-L-arginine (U.S. Patent Nos. 169,870 and 184,368), trifluoromethyl ketone (Neises and Tarnus, Thromb Haemostas 65, 1290 (1991)) and boroarginine are known to be known. Kettner et al., J. Bioi. Chem. 265: 18289 (1990)] is an effective anticoagulant agent, having both in vitro and in vivo activity. It should be noted, however, that these compounds are less stable and are converted to an inactive product in a neutral aqueous solution [e.g. Bajusz et al., J. Med. Chem. 33, 1729 (1990). Other active tripeptide derivatives have been found to be acylated on the D-phenylalanine moiety (U.S. Pat. No. 4,478,475 and Kettner et al., J. Biol. Chem. 265, 18289 (1990)] or alkylated (Hungarian Patent No. 192,646) or analogous to amino or imino acids, e.g. With N-methylphenylglycine or 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (Shuman et al., Peptides, Proceedings of the Twelfth American Peptide Symposium, ed.

A. Smith and J.E. Rivier), ESCOM, Leiden, 1992, 801-802. page] has been replaced. Known high-potency anticoagulant peptides are thus characterized by tripeptide derivatives and the so-called L-proline moiety linked to the middle amino acid moiety of the molecule. The N-terminal group is an amino or imino acid group. [These compounds would correspond to Formula I if A is a D-phenylalanine group, an acyl or alkyl derivative thereof, or an amino or imino acid analogue thereof.]

SUMMARY OF THE INVENTION It is an object of the present invention to provide novel anticoagulant peptide derivatives which are more stable and bioavailable than those known and exert their effects upon oral administration.

Surprisingly, it has been found that the tripeptide structure is not required for anticoagulant activity, the N-terminal amino or imino acid moieties of the known compounds being preferably replaced by a position-containing carboxylic acid group (A in formula I),

Further, it has been found that the compounds of formula (I) according to the invention, wherein A, Xaa and Arg are as defined above, and their acid addition salts are stable in aqueous solution, have a strong anticoagulant activity, both in vitro and in vivo, next to.

SUMMARY OF THE INVENTION The present invention relates to novel peptide derivatives A-Xaa-Arg-H (I) wherein

A is D- or L-isochroman-1-carbonyl, Dv or L-isochroman-3-carbonyl, and an acyl group of formula D-A'CH (OH) -CO

A 'is phenyl, benzyl, 1-naphthyl, 2-naphthyl, benzhydryl, -cyclohexyl or cyclohexylmethyl,

Xaa is an L-proline residue or an L-pipecolic acid residue, and

Arg is for the preparation of L-arginine radicals and acid addition salts thereof such that an optionally O-protected oxo acid containing an A 'acid radical - wherein A' can take the meaning as defined above and DL-isochroman-1-carbonyl or Alternatively, DL-A'-CH (OH) -CO may be acyl, wherein A 'is as defined above, and an aniline derivative of formula Xaa, wherein Xaa is as defined above, is condensed and the resulting acyl-L- proline or acyl L-pipecolic acid, optionally after separation of the diastereomers, by acylation of a N ^G- protected arginine lactam in a known manner, the resulting protected acyl arginine lactam is reduced and the resulting protected acyl arginine aldehyde is protected ) and the resulting peptide derivative (I) is isolated if desired in the form of an acid addition salt.

The novel peptide derivatives of formula (I), wherein A, A ', Xaa and Arg are defined in the introduction, are preferably derived from the novel acyl L-proline or acyl L-pipecolic acid obtained in the first step of the process of the invention. may be prepared by the method disclosed in the patent.

In a preferred embodiment of the process of the invention, the DL-isochroman-1-carboxylic acid is condensed with L-proline tert-butyl ester, the resulting DL-acyl-L-proline tert-butyl ester being group was removed by acidolysis and silica2

Column with EN 211 088 Β gel of D- and L-isochroman-l-carbonyl-L-proline is separated, D-isochroman-l-carbonyl-L-proline is condensed with N ^G -benzyloxycarbonyl-L-arginine lactam, the obtained D-isochroman-1-carbonyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-argininlaktámot lithium aluminum hydride reduction of the corresponding aldehyde, hydrogenation over palladium on carbon in the presence of aqueous ethanol solution containing the N ^G -protecting group of sulfuric acid the catalyst is filtered off and the desired product is isolated by lyophilization.

In another preferred embodiment of the process of the invention, the methyl D-mandelate is reacted with dihydropyran to form the O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetic acid methyl ester after saponification with L-proline. is condensed with methyl ester, the resulting O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-proline methyl ester is saponified and then condensed with N ^G -benzyloxycarbonyl-L-arginine-lactam to give tetrahydropyran-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carboxyanhydride-arginine lactam is reduced with lithium aluminum hydride to the corresponding aldehyde, by dissolution in aqueous ethanol containing sulfuric acid, the protective groups, or in this solution, it is removed by hydrogenation in the presence of palladium on charcoal, the catalyst is filtered off and the desired product is isolated by lyophilization.

In a further preferred embodiment of the process of the invention, DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl ester isobutene is O-tert-butyl-DL-2- (2-naphthyl) -2. is converted to the methyl hydroxyacetic acid and saponified, the resulting O-tert-butyl DL-2- (2-naphthyl) -2-hydroxyacetic acid is condensed with the L-proline methyl ester to give the O-tert-butyl methyl ester. D-2- (2-Naphthyl) -2-hydroxyacetyl-L-proline methyl ester and O-tert-butyl L-2- (2-naphthyl) -2-hydroxyacetyl-L-proline methyl ester after saponification of the diastereomeric acetate anion exchange resin separated in cycles, the first effluent from the column O-tert-butyl-D-2- (2-naphthyl) -2-hydroxyacetyl-L-proline N ^G -benzyloxy-carbonyl-L-argininlaktámmal condensed the obtained O-tert-butyl-D-2- (2-naphthyl) -2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carboxyanhydride-arginine lactam after the O-tert-butyl lithium acidolysis reduced to the corresponding aldehyde with aluminum hydride, the N ^G protecting group Hydrogenated in aqueous ethanol containing sulfuric acid in the presence of palladium on charcoal, the catalyst filtered off and the desired product isolated by lyophilization.

For compounds of formula I wherein Xaa is an L-pipecolic acid radical, a preferred embodiment of the process of the present invention is that the acyl L-pipecolic acid and N0-benzyloxycarbonyl-L-arginine-lactam. the condensation is carried out with phosphorus oxychloride in pyridine solution to give Lpipekolil acyl-N ^G -benzyloxy-carbonyl-L-arginine lactam is reduced to an aldehyde, followed by the final deprotection the desired product is isolated by lyophilization.

The in vitro antithrombotic activity of the compounds of Formula I was determined as usual (Bagdy et al., Thromb Haemostas 67, 325330 (1992)), total blood clotting time (WBCT), thrombin time (TT) and activated by measuring partial thromboplastin time (APTT). Table 1 shows the in vitro efficacy of some representative compounds of the present invention in the so-called. native non-coagulated human blood (WBCT) and citrated plasma (TT and APTT).

Table J In vitro antiplatelet activity of novel peptide derivatives A-Xaa-Arg-H of formula I

A-Xaa-Arg-H ¹ Clotting parameter ² Example THE Xaa WBCT TT APTT First Ica-D (l) Pro 1.55 0,132 0.60 6th D-Man Pro 0.45 0,040 0.15 7th D-Hpl Pro 0.32 0,072 0.20 8th D-Hma Pro 0.72 0,100 0.35 12th D-DPL Pro 0.52 0.077 0.12 13th D-Nga (l) Pro 1.13 0,100 1.00 14th D-Nga (2) Pro 1.04 0,150 0.52

¹ Abbreviations: Dpi = 3,3-diphenyl-lacto-yl, Hma = 2-cyclohexyl-2-hydroxyacetyl, Hpl = 3-cyclohexyl-lactyl, Ica (I) = isochroman-1-carbonyl. Man = 2-phenyl-2-hydroxyacetyl, Nga (1) = 2- (1-naphthyl) 2-hydroxyacetyl, Nga (2) = 2- (2-naphthyl) -2-hydroxyacetyl, Pro L-proline.

² WBCT = whole blood coagulation time, TT = thrombin time and APTT = activated partial thromboplastin time parameter. The numbers refer to the concentration (pg / ml) of the compounds in which human blood or blood is not inhibited by coagulation. the coagulation time of citrate plasma is twice that of the control ηδ

The in vivo anti-thrombotic activity of the compounds of formula (I) was determined in rabbits in a known manner (Bagdy et al., 1992, Thromb Haemostas 67, 357-365). The compounds were dissolved in physiological saline at a dose of 0.5-3 mg / kg subcutaneously (s. C) or 5-15 mg / kg orally (p.). The effect of the compounds can be detected 15-30 minutes after administration, the highest value being 90-120. and the therapeutic effect is maintained in a dose-dependent manner for 3 to 5 hours.

The in vivo antithrombotic activity of 3-cyclohexyl-D-lacto-L-prolyl-L-arginine aldehyde hemisulfate (Example 7) is shown in detail in Tables 2 and 3. The compound was administered at 10 mg / kg, respectively. It was administered orally at a dose of 15 mg / kg. Blood samples were taken from the ear vein of the animals every 30 minutes. Whole blood coagulation time (WBCT) and the degree of inhibition of thrombin-induced platelet aggregation (IWBA) were determined. Plasma time (TT) and activated partial thromboplastin time (APTT) from the blood sample were also determined. The WBCT and APTT values are shown in Table 2, and the TT and IWBA values are listed in Table 3.

HU 211 088 Β

Table 2

Antithrombotic activity of 3-cyclohexyl-D-lacto-L-prolyl-L-arginine-aldehyde hemisulphate in rabbits at oral doses of 10 mg / kg and 15 mg / kg, characterized by WBCT and APTT

Time (p) 10 mg / kg 15 mg / kg WBCT (p) APTT (sec) WBCT (p) APTT (sec) 0 13.2 + 1.1 27.7 ± 1.2 14.6 ± 0.4 32.6 ± 4.6 30 17.7 ± 1.6 58.9 ± 9.0 24.2 ± 4.7 115.0 ± 36.6 60 23.6 ± 1.9 77.6 ± 14.6 29.4 ± 4.5 150.4 ± 30.9 90 21.2 ± 2.9 65.1 ± 8.4 33.0 ± 5.9 170.3 ± 18.5 120 18.8 ± 1.0 55.1 ± 11.4 34.4 ± 4.4 145.6 ± 22.7 150 19.2 ± 1.1 52.1 ± 6.6 27.2 ± 3.6 149.2 ± 22.0 180 18.4 ± 1.5 53.8 ± 8.5 29.5 ± 2.7 169.0 ± 23.0 210 17.2 ± 2.3 48.6 ± 3.8 34.0 ± 5.9 137.7 ± 25.8 240 15.8 ± 1.7 47.7 ± 7.4 33.7 ± 6.5 127.4 ± 25.7 270 16.2 ± 2.1 44.4 ± 3.7 26.0 ± 1.2 133.4 ± 24.7 300 13.5 ± 0.9 43.7 ± 5.0 23.6 ± 3.3 92.8 ± 18.4

Table 3

Antithrombotic activity of 3-cyclohexyl-D-lacto-L-prolyl-L-arginine-aldehyde hemisulphate in rabbits at oral doses of 10 mg / kg and 15 mg / kg with TT and 1WBA

Time ip) 10 mg / kg 15 mg / kg TT (p) IWBA (sec) TT (p) IWBA (sec) 0 18.4 ± 2.1 18.9 ± 2.1 30 317.1 ± 123.0 89.4 + 7.6 371.2 ± 142.3 81.4 ± 11.7 60 461.0 ± 107.2 95.8 ± 6.9 528.3 ± 72.9 97.4 ± 2.6 90 444.7 ± 108.6 81.6 ± 8.2 578.8 ± 21.5 I00,0 ± 0 120 264.6 ± 101.3 94.0 ± 3.2 > 600.0 ± 0 100.0 ± 0 150 201.1 ± 109.9 71.4 ± 13.8 549.6 ± 34.0 100.0 ± 0 180 182.7 ± 113.6 72.4 ± 19.2 380.7 ± 116.0 100.0 ± 0 210 123.0 ± 65.3 54.4 ± 21.0 342.1 ± 121.2 93.7 ± 4.9 240 141.5 ± 81.3 50.6 ± 17.7 340.9 ± 137.0 82.6 ± 17.7 270 65.4 ± 25.7 35.2 ± 17.3 376.4 ± 153.0 81.0 ± 16.9 300 41.8 ± 11.4 48.2 ± 16.9 261.0 ± 140.8 69.6 ± 20.4

The compounds of formula (I) exert their anticoagulant activity without interfering with the natural process of dissolution of the clot, the so-called. fibrinolysis. This is supported by the fact that the compounds described in Examples 6, 7 and 8 only negligibly inhibited the plasma clot fibrinolysis induced by tissue plasminogen activator in a 50 thrombelastograph [H. Hartert: Thrombelastography.

In: N. U. Bang, F. K. Beller, E. Deutsch, and E. F. Mamman, Thrombosis and Bleedings Disorders. Theory and Methods (Academic Press, London, 1971, p. 7076). 55

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

In these examples, _Rf values were determined by thin layer chromatography on silica gel (DC-Alufolien 60

Kieselgel 60 F ₂₅₄ , Merck, Darmstadt) in the following solvents:

1. ethyl acetate - pyridine - acetic acid - water (480: 20: 6: 11)

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

3. ethyl acetate - pyridine - acetic acid - water (120: 20: 6: 11)

4. ethyl acetate - pyridine - acetic acid - water (60: 20: 6: 11)

5. ethyl acetate - pyridine - acetic acid - water (45: 20: 6: 11)

6. ethyl acetate - pyridine - acetic acid - water (30: 20: 6: 11)

7. ethyl acetate - pyridine - formic acid - water (480: 20: 6: 5.5)

8. ethyl acetate - pyridine - formic acid - water (240: 20: 6: 5.5)

9. ethyl acetate - pyridine - acetic acid - water (960: 20: 6: 11)

10. Ethyl acetate-diisopropyl ether (7: 3)

11. Ethyl acetate - n-hexane (1: 1)

12. Chloroform-acetone (98: 2)

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

HU 21 1,088 Β

Column I: VYDAC C-18 reversed phase: 10 pm,

300A, 4 x 250mm ".

Buffer A: 0.1% trifluoroacetic acid in water.

Buffer B: 0.1% trifluoroacetic acid in acetonitrile.

II. Column: Q HR 5/5 (Pharmacia).

The buffer is a 2: 1 methanol-water mixture

Bpuffer: 2: 1 methanol - 0.2 mol / dm ³ solution of aqueous sodium chloride solution

Gradients used (flow rate):

1. 0-5 min: 0-15% B, 5-30 min: 15% Β (1 mL / min);

2. 0-5 min: 0-15% B, 5-30 min: 15% B (1.2 mL / min);

3. 0-30 min: 0-60% Β (1 mL / min);

4. 0-5 min: 0-6% B, 5-30 min: 6% B, 30-35 min

6-18% B (1.2 mL / min);

5. 0-5 min: 0% B, 5-30 min: 2% B (0.8 mL / min).

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

The HPLC assay conditions, columns and buffers used (I or II), and gradient data (1-5) are shown in brackets in the exemplary embodiments, e.g. HPLC (II / 5).

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

Of these, the aldehyde hydrate and one or both of the aminocyclols appear in separate peaks during HPLC. Accordingly, two or three k 'values can be used to characterize the products in the examples. An exception is the 3-cyclohexyl-D-lactyl-L-pipecolyl-L-arginyl aldehyde hemisulfate described in Example 9, where six k 'values are given after elution in six peaks, presumably the known cis-trans isomerization of acyl pipecoline. as a result.

Example 1

Preparation of D-Isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: D-Isochroman-1-carbonyl-L-prolyl-tert-benzyloxycarbonyl-L-arginine-lactam

2.73 g (7 mmol) Zerc-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] are suspended in 10 ml of chloroform, and 10 ml of hydrochloric acid ethyl acetate (concentration 0.11-0.15 g / ml) are added under stirring and ice-cooling. After 2 hours, the reaction mixture was diluted with ΙΟΙ5 mL diethyl ether, the precipitated crystalline material was filtered off, washed with 5 mL acetone and 5 mL diethyl ether, and dried overnight in a vacuum desiccator with potassium hydroxide. The resulting N-benzyloxycarbonyl-L-arginine-lactam chlorohydrate was dissolved in 10 ml of dimethylformamide, cooled to -20 ° C and added to the mixed anhydride below.

D-isochroman-1-carbonyl-L-proline cyclohexylammonium salt 2.25 g (6 mmol) (Example 1, Step D) is dissolved in 10 ml of ethyl acetate and 10 ml of 1 mol / dm ³ solution of aqueous potassium bisulfates. After drying the ethyl acetate phase over anhydrous sodium sulfate at a pressure of 20-25 millibar, the reaction mixture was dried at room temperature for 20 minutes. After evaporation from a bath at 40 DEG C., 5 ml of benzene were distilled off from the residue and dissolved in 6 ml of dimethylformamide. The DMF solution is cooled to -20 °, and was added 0.66 mL (6 mmol) of N-methylmorpholine, 0.79 ml of chloroformic acid isobutyl ester, carbon, and after 10 minutes stirring, N ^G - a suspension of the above dimethylformamide containing benzyloxycarbonyl-L-arginine lactam, and finally 2.1 ml (15 mmol) of triethylamine. The reaction mixture was stirred for 2 hours, first with cooling and then allowed to warm to room temperature. The salts are filtered off, the filtrate is diluted with 50 ml of benzene and washed 4 times with 20 ml of water, 3 times with 10 ml of 0.1 M hydrochloric acid and again with water (3 times with 20 ml) and dried over anhydrous sodium sulfate. millibar pressure approx. Evaporate from a 40 'C bath. The residue was dissolved in 1.5 mL of ethyl acetate-pyridine formic acid-water (240: 20: 6: 5.5) and applied to a column of 100 g silica gel with ethyl acetate followed by ethyl acetate-pyridine formic acid-water 480 : 20: 6: 5.5. Fractions containing pure product were combined, shaken with 1/3 portion of 1 mol / dm ³ aqueous solution of potassium bisulfate and the organic layer was washed with water and dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ Pa at a pressure of approx. Evaporate from a 40 'C bath. The residue was dissolved in benzene and evaporated again, then triturated with n-hexane. 2.3 g (70%) of the title compound are obtained.

RX7) = 0.42-0.46.

[.alpha.] D @ 20 = -28.5 DEG (c = 1 in tetrahydrofuran).

Analysis for C ₂ 9H ₃₃ N ₅ O ₆ (547.59) Calcd:

Found: C, 63.60; H, 6.07; N, 12.79; Found: C, 63.5; H, 6.1; % N, 12.5.

Step 2: D-Isochroman-1-carbonyl-L-prolyl-4-benzyloxycarbonyl-L-arginine aldehyde

1.64 g (3 mmol) of D-isochroman-1-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam (Example 1,

Step 1) dissolved in 8 mL of tetrahydrofuran and added with stirring at -20 ° C

2.25 mmol of lithium aluminum hydride in tetrahydrofuran. The progress of the reduction was monitored by layer chromatography in ethyl acetate-pyridine-acetic acid-water (240: 20: 6: 11), and additional lithium aluminum hydride was added as needed, followed by cooling and stirring with 20 mL of cooled 1 mmol / dm. Pour into aqueous ³ -potassium bisulfate. The solution was diluted with water (10 mL) and extracted with n-hexane (2 x 10 mL) and extracted with methylene chloride (3 x 10 mL). The methylene chloride solutions were combined, washed 3 times with 5 ml water, 5% after drying with aqueous sodium bicarbonate and again with water, then sodium sulfate 10 ³ 2.0-2.5 torr approx. Evaporate from a 40 'C bath. The residue was triturated with n-hexane. 1.15 g (70%) of the title product are obtained. Rf (3) = 0.50-0.52.

HU 211 088 Β

Step 3: D-Isochroman-1-Carbonyl-L-Prolyl-L-Arginine Aldehyde Hemisulfate

0.82 g (1.5 mmol) of D-isochroman-l-carbonyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 1, Step 2) was dissolved in 10 ml of 60% aqueous hydrogen ethanol containing 0.75 mmol sulfuric acid and then hydrogenated in the presence of palladium on charcoal. The progress of the reaction was monitored by layer chromatography in ethyl acetate - pyridine - acetic acid - water (45: 20: 6: 11). The reaction was carried out for ca. After 30 minutes, the catalyst is filtered off and washed with 60% aqueous ethanol and water. The filtrate was washed with ca. half concentrated under reduced pressure and diluted to 10 ml with water. The solution was first extracted with methylene chloride, then frozen and lyophilized. Yield: 0.5 g (70%).

Rf (5) = 0.48-0.52. HPLC (LZ1): k '= 3.95, 4.76 and

5.95.

[α] D = -70.86 ° (c = 0.1 mol / dm ^{3 in} aqueous hydrochloric acid).

The FAB mass spectrum (416 [M + H] ⁺ ) confirms the expected structure.

For example, the starting materials are prepared as follows:

For the preparation of D-isochroman-1-carbonyl-L-prolyl-cyclohexylammonium salt

Step A: DL-Isochroman-1-carboxylic acid (ml) (0.1 mol) in phenethyl alcohol and 10.12 g (0.11 mol) in glyoxylic acid hydrate were dissolved in 50 ml of trifluoroacetic acid. The solution is heated at reflux for 24 hours and then at a pressure of 2.0 to 2.5 bar ( ^{3 bar} ). Evaporate from a bath at 40 ° C. To the residue was added water (100 mL) and concentrated ammonium hydroxide (pH 7). The solution was extracted with diethyl ether (3 x 30 mL), saturated potassium bisulfate (ca. It was acidified to pH 3 and extracted with ethyl acetate (3 x 30 mL). The ethyl acetate layers were combined, washed with water (34 times 20 ml), and dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a bath at 40 ° C. The residue was triturated with n-hexane, filtered, washed with n-hexane and air dried. 10.7-11.6 g (60-65%) of the title product are obtained.

Rf (3) = 0.65-0.75. M.p. 92-93 ° C.

Analysis result for C ₁₀ H ₁₀ O _? With reference to formula (178.18):

Calculated: C, 67.40; H, 5.66;

Found: C, 67.45; H, 5.65.

Step B: DL-Isochroman-1-carbonyl-L-propyl-tert-butyl ester

DL-isochroman-1-carboxylic acid (4.45 g, 25 mmol) (Example 1, Step A) and N-methylmorpholine (2.75 mL, 25 mmol) were dissolved in dimethylformamide (20 mL) at -15 ° C. After cooling at this temperature, 3.3 ml of chloroformic acid isobutyl ester was added with stirring, followed by 5.2 g (25 mmol) of L-proline tert-butyl ester chlorohydrate and 3.5 ml of triethyl of a solution of amine in 25 ml of dimethylformamide; slurry, which was previously cooled to -15 ° C. After two hours stirring, the reaction mixture was filtered and 1.5 to 2.0 · 10 ³ torr approx. Evaporate from a 40 'C bath. The remainder is approx. Dissolved in 30 ml of ethyl acetate and extracted 3 times with 20 ml, respectively, 0.25 mol / dm ³ aqueous solution of sulfuric acid, water, 5% sodium bicarbonate, again with water, then dried with sodium sulfate 2.0-2.5 · 10 ³ Pa at approx. Evaporate from a bath at 40 ° C. The remaining oily product is DL-isochroman-1-carbonyl-L-propyl-tert-butyl ester [Rf (7) = 0.72 and 0.75].

Step C: DL-Isochroman-1-carbonyl-L-proline

The oily product of Example 1, Step B, was dissolved in 25 mL of trifluoroacetic acid and allowed to stand at room temperature for 2 hours. The trifluoroacetic acid solution is pressurized to about 20-25 millibar pressure. After evaporation from a bath at 40 ° C, the residue was dissolved in water (30 ml) and evaporated again. The residue thus obtained was dissolved in ethyl acetate (50 ml) and extracted with 5% sodium bicarbonate (3 x 30 ml). The sodium bicarbonate solutions were combined, acidified to pH = ^{3 with} 1 M aqueous potassium bisulfate, and extracted three times with 30 mL of ethyl acetate. The ethyl acetate solutions were evaporated after drying over sodium sulfate. The oily residue [R (<8] = 0.400.42 and 0.51-0.53] was added with 25 mmol D- L-isochroman-1-carbonyl-L-proline is considered to be the diastereomeric pair.

Step D: D- and L-Isochroman-1-Carbonyl-L-Prolincyclohexylammonium Salt

The diastereomeric pair obtained in Example 1, Step C is separated by silica gel column chromatography. The column was made from 350 g of silica gel using ethyl acetate - pyridine - formic acid - water 480: 20: 6: 5.5 as eluent. The pure F containing the upper [R / 8) = 0.51-0.53] and the lower [Rf (8) = 0.40-0.42] patches were collected. Treat the resulting solutions F and A in the same manner as follows.

The solution is evaporated in the usual way (2.0-

2.5 10 ³ Pa for approx. 40 ° C bath). Water was distilled off and the residue was dissolved in methylene chloride (30 mL). The methylene chloride solution was washed with 15% aqueous saline solution and evaporated to dryness. The oily residue is dissolved in 20 ml of diethyl ether and enough cyclohexylamine is added to make the solution slightly basic. The crystalline crystalline cyclohexylammonium salt was filtered off, washed with diethyl ether and dried in vacuo in concentrated sulfuric acid.

The product from solution F was determined by NMR to be 2.75 g (58.75%) of D-isochroman-1-carbonyl-L-proline cyclohexylammonium salt.

R | (8) = 0.54-0.59. 122-125 ° C.

[α] D 20 = -41.5 ° (c = 1 in methanol).

Analysis for C ₂₁ H ₃₀ N ₂ O ₄ (374.47):

Found: C, 67.35; H, 8.07; N, 7.48. Found: C, 67.3; H, 8.15; N, 7.24.

HU 211 088 B

The product obtained from the Λ-solution, according to NMR, was the salt of L-isochroman-1-carbonyl-L-proline-cyclohexylammonium, 3.1 g (66.2%).

Rf (δ) = 0.36-0.41. 154-157 'C.

[α] D = -51.15 ° (c = 1, methanol).

Analysis for _C2] H ₃₀ N ₂ O ₄ (374.47) Calcd:

Found: C, 67.35; H, 8.07; N, 7.48. Found: C, 66.39; H, 8.45; N, 7.435.

Example 2

Preparation of L-isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

]. Step: L-Isochroman-1-carbonyl-L-prolyl-N-benzyloxycarbonyl-L-arginine lactam

2.73 g (7 mmol) of Step B: tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 2.25 g (6 mmol) of L-isochroman-1-carbonyl-L-prolyl-cyclohexylammonium salt (Example 1, Step D) and in each case, using the same amounts of solvents and reagents as in Example 1, Step 1, gave the title product (-2.3 g, 70%).

RX7) = 0.42-0.46.

[.alpha.] D @ 20 = -70.3 DEG (c = 1 in tetrahydrofuran).

Analysis for C ₂₉ H ₃₃ N ₅ O ₆ (547.59):

Found: C, 63.60; H, 6.07; N, 12.79; Found: C, 63.3; H, 6.2; N, 12.4.

Step 2: L-Isochroman-1-carbonyl-L-prolyl-N'-benzyloxycarbonyl-L-arginine aldehyde

1.64 g (3 mmol) of L-isochroman-l-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam (Example 2, Step 1) as described in Example 1, Step 2 conversion using the same amount of solvents and reagents gave 1.15 g (70%) of the title product.

Rf (3) = 0.48-0.50.

Step 3: L-Isochroman-1-Carbonyl-L-Prolyl-L-Arginine Aldehyde Hem Sulfate

0.82 g (1.5 mmol) of L-isochroman-l-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 2, Step 2) of Example 1 Step 3; Converted as described using the same amounts of solvents and reagents, 0.5 g (70%) of the title compound are obtained.

RK5) = 0.48-0.52. HPLC (I / 1): k '= 3.90,4.62 and 5.29.

[a] o = -55.2 '(c = ³ 1, 0.1 mol / dm aqueous solution of hydrochloric acid).

The FAB mass spectrum (416 [M + H] ⁺ ) confirms the expected structure.

Example 3

Preparation of D-isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: D-izokmmán-3-carbonyl-L-prolyl-N ^Q benzyloxy-carbonyl-L-arginine lactam

2.73 g (7 mmol) of tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 2.25 g (6 mmol) of D-isochroman-3-carbonyl-L-prolyl-cyclohexylammonium salt (Example 3, Step C) and In each case, using the same amounts of solvents and reagents as in Example 1, Step 1, 2.3 g (70%) of the title compound were obtained.

Rf <9) = 0.45-0.55.

[α] D = -50 ° (c = 1, in tetrahydrofuran)

Analysis for C ₂₉ H ₃₃ N ₅ O ₆ (547.59):

Found: C, 63.60; H, 6.07; N, 12.79; Found: C, 63.8; H, 6.2; % N, 12.5.

Step 2: D-Isochroman-3-carbonyl-L-prold-N ^<3 -benzyloxycarbonyl-L-arginine aldehyde

2.2 g (4 mmol) of D-isochroman-3-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam (Example 3,

Step 1), as described in Example 1, Step 2, using proportional solvents and reagents, gave 1.55 g (70%) of the title product.

Rf <2) = 0.15-0.25.

[.alpha.] D @ 20 = -28.6 DEG (c = 1 in tetrahydrofuran)

Step 3: D-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

1.1 g (2 mmol) of D-isochroman-3-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 3,

Step 2), as described in Example 1, Step 3, using proportional amounts of solvents and reagents, afforded 0.67 g (70%) of the title product.

R (<4) = 0.20-0.30. HPLC (I / 1): k '= 4.55, 5.65 and

6.95.

The FAB mass spectrum (416 [M + H] ⁺ ) confirms the expected structure.

The starting materials are prepared as follows:

Preparation of D-isochroman-3-carbonyl-L-prolyl-cyclohexylammonium salt.

Step A: D-Isochroman-3-carboxylic acid 3 g of paraformaldehyde is dissolved in 50 ml of trifluoroacetic acid and 16.6 g (0.1 mol) of 3-phenyl-lactic acid [M. Winitz et al., J. Am. Chem. Soc., 82, 2423 (1956). The solution was boiled under reflux cooling for 48 hours, and then 2.0 to 2.5 10 ³ psi max. Evaporate from a 40 'C bath. The residue was distilled off with 50 ml of water and crystallized from water. The crystals were filtered off, washed with water and dried in vacuo in a desiccator over phosphorus pentoxide. 14.2-16.0 g (80-90%) of the title product are obtained.

RK1) = 0.40-0.50. 86-88 'C.

[.alpha.] D @ 20 = + 149 DEG (c = 1, in methanol).

Analysis for C ₁₀ H ₁₀ O ₃ (178.18):

Calculated: C, 67.40; H, 5.66;

Found: C, 67.4; H, 5.65.

HU 211 088 Β

Step B: D-Isochroman-3-carbonyl-L-prolyl tert-butyl ester

D-Isochroman-3-carboxylic acid (4.45 g, 25 mmol) (Example 3, Step A) and L-prolyl-tert-butyl ester chlorohydrate (5.2 g, 25 mmol) were obtained in Example 1B. Steps are condensed using the same amounts of solvents and reagents as described in Step 1. The oily product thus obtained was considered as 25 mmol of D-isochroman-3-carbonyl-Lprolyl-tert-butyl ester.

R, (l) = 0.80-0.90.

Step C: D-Isochroman-3-carbonyl-L-prolyl-cyclohexylammonium salt

The oily product of Example 3, Step B, was dissolved in 25 mL of trifluoroacetic acid and allowed to stand at room temperature for 2 hours. The trifluoroacetic acid solution was pressurized to about 2.0-2.5 x 10 ³ Pa. After evaporation from a bath at 40 ° C, the residue was dissolved in water (30 ml) and evaporated again. Water (50 mL) was poured onto the oily residue and enough solid sodium bicarbonate was added to bring the solution to a pH above 7. The solution was extracted with ethyl acetate ( ³ x 10 mL), acidified to pH 3 with aqueous potassium bisulfate (1 mol / dm ³ ) and extracted with ethyl acetate (3 x 30 mL). The ethyl acetate solutions were evaporated after drying over anhydrous sodium sulfate. The oily residue is dissolved in 20 ml of diethyl ether and enough cyclohexylamine is added to make the solution slightly basic. The crystalline crystalline cyclohexylammonium salt was filtered off, washed with diethyl ether and dried in vacuo in concentrated sulfuric acid. 6.5 g (70%) of the title product are obtained.

Rf (l) = 0.30-0.40. Mp .: 158-160 ° C.

[α] D = -3.5 (c = 1, in methanol).

Analysis for C ₂ H ₃₀ N ₂ O ₄ (374.47):

Found: C, 67.35; H, 8.07; N, 7.48. Found: C, 67.4; H, 8.4; N, 7.3.

Example 4

Preparation of L-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: L-i-Chromane-3-carbonyl-L-prolyl-N'-benzyloxycarbonyl-L-arginine lactam

2.73 g (7 mmol) of tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 2.25 g (6 mmol) of L-isochroman-3-carbonyl-L-prolyl-cyclohexylammonium salt (Example 4, Step C). Starting with the same procedure as in Example 1, Step 1, using the same amount of solvents and reagents, 2.3 g (70%) of the title compound are obtained.

RX9) = 0.50-0.60.

Step 2: L-Isocroinan-S-carbonyl-L-prolyl-N-benzyloxycarbonyl-L-arginine aldehyde

2.2 g (4 mmol) of L-isochroman-3-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine lactam (Example 4.

Step 1), as described in Example 1, Step 2, using proportional solvents and reagents, gave 1.55 g (70%) of the title product.

Rj (2) = 0.20-0.30.

Step 3: L-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

1.1 g (2 mmol) of L-isochroman-3-carbonyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 4,

Step 2) Converted as in Example 1, Step 3 using proportional amounts of solvents and reagents to give 0.67 g (70%) of the title product.

R 4 = 0.25-0.35. HPLC (I / 1): k '= 4.65, 5.40 and 6.65. The FAB mass spectrum (416 [M + H] ⁺ ) confirms the expected structure.

The starting materials are prepared as follows:

Preparation of L-isochroman-3-carbonyl-L-prolyl-cyclohexylammonium salt.

Step A: L-Isochroman-3-carboxylic acid

1.5 g of paraformaldehyde and 8.3 g (50 mmol) of 3-phenyl L-lactic acid [M. Winitz et al., J. Am. Chem. Soc. 82, 2423 (1956)] were condensed using proportional solvents and reagents as described in Example 3, Step A. 7.1-8.0 g (80-90%) of the title product are obtained.

R (<1) = 0.40-0.50. 86-88 'C.

[α] D = -149 ° (c = 1, in methanol).

Analysis for C, ₀ H _{) 0} O ₃ (178.18):

Calculated: C, 67.40; H, 5.66;

Found: C, 67.4; H, 5.65.

Step B: L-Isochroman-3-carbonyl-L-proline tert-butyl ester

4.45 g (25 mmol) of L-isochroman-3-carboxylic acid (Example 4, Step A) and 5.2 g (25 mmol) of L-proline tert-butyl ester chlorohydrate are obtained in Example 1B. Steps are condensed using the same amounts of solvents and reagents as described in Step 1. The oily product thus obtained is considered to be 25 mmol of L-isochroman-3-carbonyl-L-propyl tert-butyl ester.

R 1) = 0.80-0.90.

Step C: L-Isochroman-3-carbonyl-L-proline-cyclohexylammonium salt

The oily product of Example 4, Step B, was converted as described in Example 3, Step C using equal amounts of solvents and reagents.

6.5 g (70%) of the title product are obtained.

RXD = 0.40-0.50. Mp 148-150 ° C.

[.alpha.] D @ 20 = -91.9 DEG (c = 1 in methanol).

Analysis for C ₂ H ₃₀ N ₂ O ₄ (374.47):

Found: C, 67.35; H, 8.07; N, 7.48. Found: C, 67.2; H, 8.0; N, 7.4.

HU 211 088 Β

Example 5

Preparation of 3-Phenyl-D-lacto-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: O-Tetrahydropyranyl-3-phenyl-D-lactoyl-L-prolyl-b-benzotoxycarbonyl-L-arginine-lactam 2.68 g (6 mmol) of O-tetrahydropyranyl-3-phenyl-D-lactoyl- The L-prolylcyclohexylammonium salt (Example 5, Step B) was dissolved in dichloromethane (30 mL) and aqueous potassium bisulfate (1 mL / dm ³ ). The dichloromethane layer was washed with water, and then dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a bath at 40 ° C. The residue and (7 mmol) of tert-buúloxi-carbonyl-N ^G -benzyloxy-carbonyl-L-arginine lactam 2.73 g [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] using the same amounts of solvents and reagents as described in Example 1, Step 1.

conversion gave 2.3 g (65%) of the title compound.

RX in ethyl acetate) = 0.55-0.65.

Analysis for C ₃₃ H ₄ iN ₅ O ₇ (619.70):

Calculated: C, 63.95; H, 6.67; N, 11.30; Found: C, 63.8; H, 6.7; % N, 11.1.

Step 2: O-tetrahydropyranyl-3-phenyl-D-prolyl-N ^Q laktoil-benzyloxy-carbonyl-L-arginine aldehyde

1 2.2 g (3.5 mmol) of O-tetrahydropyranyl-3-phenyl-D-laktoil-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam (Example 5, Step 1). EXAMPLE 1 (2) gave the title product (1.30 g, 60%), using proportional solvents and reagents.

RX2) = 0.40-0.50.

Analysis for C ₃₃ H ₄₃ N ₅ O ₇ (621.71):

Calculated: C, 63.75; H, 6.97; N, 11.26; Found: C, 63.85; H, 7.1; % N, 11.1.

Step 3: 3-Phenyl-D-lacto-L-prolyl-L-arginine aldehyde hemisulfate

(2 mmol) of O-tetrahydropyranyl-3-phenyl-D-prolyl-N ^G laktoilL-benzyloxy-carbonyl-L-arginine aldehyde (Example 5, step 2) of Example 7 1.25 3rd step (a), using proportional amounts of solvents and reagents

conversion gave 0.6 g (60%) of the title product.

Rf (5) = 0.50-0.60. HPLC (I / 1): k '= 3.96 and 4.96.

The FAB mass spectrum (404 [M + H] ⁺ ) confirms the expected structure.

The starting materials are prepared as follows:

Preparation of O-Tetrahydropyranyl-3-phenyl-D-lactoyl-L-pmylcyclohexylammonium salt

Step A: Cyclohexylammonium salt of O-tetrahydropyranyl-3-phenyl-D-lactic acid

3-Phenyl-D-lactic acid (6.64 g, 40 mmol) was reacted with dihydropyran (3.7 g, 44 mmol) in acetonitrile, Obrecht and Heimgartner, Helv. Chim. Acta, 67, 526 (1984)]. The reaction mixture was pressurized to about 2.0-2.5 x 10 ³ Pa for approx. After evaporation from a 40 ° C bath, the residue was dissolved in dichloromethane (20 ml), washed with water and then, after drying over anhydrous sodium sulfate, evaporated again. The resulting O-tetrahydropyranyl-3-phenyl-D-lactic acid was dissolved in diethyl ether and converted to the cyclohexylammonium salt. The crystals were filtered off, washed with diethyl ether and dried in a vacuum desiccator. Yield: 8.4 g (60%).

R / 9) = 0.68-0.77. Mp 148-150 ° C.

Analysis for C ₂₀ H ₃ NO ₄ (349.46):

Found: C, 68.74; H, 8.94; N, 4.01; Found: C, 68.65; H, 9.1; N, 4.1.

Step B: O-Tetrahydropyranyl-3-phenyl-D-lactoyl-Lproline-cyclohexylammonium salt

8.74 g (25 mmol) of O-tetrahydropyranyl-3-phenyl-D-lactic acid cyclohexylammonium salt (Example 5, Step A) was dissolved in 30 ml of 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and 50 ml dichloromethane. The dichloromethane layer was washed with water, and then dried over anhydrous sodium sulfate 2.0-2.5 10 ³ torr approx. Evaporate from a bath at 40 ° C. The resulting O-tetrahydropyranyl-3-phenyl-D-lactic acid and 4.15 g (25 mmol) of L-proline methyl ester chlorohydrate are described by Obrecht and Heimgartner, Helv. Chim. Acta 67, 526 (1984)], the resulting O-tetrahydropyranyl-3-phenyl-D-lactoyl-L-proline methyl ester is saponified, and the O-tetrahydropyranyl-3-phenyl-D-lactoyl thus obtained is saponified. -L-proline is converted into a crystalline cyclohexylammonium salt in diethyl ether. 6.7 g (60%) of the title product are obtained.

RX 1) = 0.3-0.4.

Analysis for C25 _3g N ₂ O ₅ (446.57) Calcd:

Found: C, 67.23; H, 8.58; N, 6.27; Found: C, 67.3; H, 8.7; N, 6.1.

Example 6

Preparation of D-2-Phenyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 7: 0-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N ^G- benzotoxycarbonyl-L-arginine lactam

From 2.6 g (6 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-proline-cyclohexylammonium salt (Example 6, step B) and 2.73 (7 mmol) of butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S.

Bajusz et al., J. Med. Chem., 33, 1729 (1990) and all of the procedures described in Example 5, Step 1, using the same amount of solvents and reagents, afforded 2.5 g (69%) of the title product. _[Rf = 0.5-0.6 (AcOEt)] as an oil which was used directly in the next step.

Step 2: 0-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N'-benzyloxycarbonyl-L-argininaldehyde

2.5 g (4.1 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-acetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginin9

The lactam (Example 6, Step 1) was converted in the same manner as in Example 5, Step 2 using proportional solvents and reagents to give the title product (1.50 g, 60%).

R (2) = 0.33-0.44.

Analysis for C ₃₂ H ₄ N ₅ O ₇ (607.69):

Found: C, 63.24; H, 6.80; N, 11.52. Found: C, 63.1; H, 6.9; N, 11.35.

Step 3: D-2-Phenyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate

1.22 g (2 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-acetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 6, Step 2) the Converted as in Example 7, Step 3 using proportional amounts of solvents and reagents, 0.6 g (60%) of the title product is obtained.

Rf <5) = 0.46-0.56. HPLC (I / 4): k '= 8.33, 9.22 and 14.20.

The FAB mass spectrum (390 [M + H] ⁺ ) confirms the expected structure.

The starting materials are prepared as follows:

Preparation of O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-propylcyclohexylammonium salt

Step A: Cyclohexylammonium salt of O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetic acid

6.1 g (40 mmol) of D-mandelic acid were prepared according to Example 5A.

Converted as described in Step 3a to the same amount of solvents and reagents to afford the title product (8.4 g, 62%).

Rf (9) = 0.68-0.77. Mp 148-150 ° C.

Analysis for C ₁₉ H ₂₉ NO ₄ (335.43):

Found: C, 68.03; H, 8.71; N, 4.18. Found: C, 67.95; H, 8.8; N, 4.1.

Step B: 0-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-proline-cyclohexylammonium salt

8.4 g (25 mmol) of cyclohexylammonium salt of O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetic acid (Example 6, Step A) and 4.15 g (25 mmol) of L-proline methyl ester chlorohydrate conversion as described in Example 5, Step B, using the same amount of solvents and reagents, afforded 6.7 g (62%) of the title product.

R t <1) = 0.3-0.4.

Analysis for C ₂₄ H ₃₆ N ₂ O ₅ (432.54):

Calculated: C, 66.64; H, 8.39; N, 6.48. found; C, 66.5; H, 8.25; N, 6.3.

Example 7

Preparation of 3-Cyclohexyl-D-Lactoyl-L-Prolyl-L-Arginine Aldehyde Hemisulfate

Step 1: O-tetrahydropyranyl-3-cyclohexyl-D-laktoil-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam 2.72 g (6 mmol) of O-tetrahydropyranyl-3-ciklohexilD- lacto-L-proline-cyclohexylammonium salt (Example 7,

Step D) was dissolved in dichloromethane (30 mL) and aqueous potassium bisulfate (1 mL / dm ^{1 x 3} ). The dichloromethane layer was washed with water, and then dried over anhydrous sodium sulfate 2.0-2.5 10 ³ torr approx. Evaporate from a bath at 40 ° C. The residue and (7 mmol) of tert-butyloxycarbonyl-N ^c benzyloxy-carbonyl-L-arginine lactam 2.73 g [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990) 1729] using the same amount of solvents and reagents as in Example 1, Step 1, afforded 2.44 g (65%) of the title product.

R, (2) = 0.73-0.80.

Step 2: O-Tetrahydropyranyl-3-cyclohexyl-D-lactoyl-L-prolyl-4-benzyloxycarbonyl-L-arginine aldehyde

O-tetrahydropyranyl-3-cyclohexyl-laktoil-L-prolyl-N ^G -benzyloxy-carbonyl-L-argininlaktámot 2.2 g (3.5 mmol) (Example 7, step 1) was dissolved in 8 ml of tetrahydrofuran and lithium aluminum hydride (2.265 mmol) in tetrahydrofuran was added with stirring at a temperature below -50 ° C. The progress of the reduction was monitored by layer chromatography in ethyl acetate-pyridine acetic acid-water (240: 20: 6.11) and additional lithium aluminum hydride was added as needed. The reaction mixture to pH 3, acidified with cooled 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate under cooling and stirring. The solution was diluted with water until slightly cloudy (about 10 mL), then extracted twice with 10 mL of 1: 1 n-hexane: ethyl acetate and extracted 3 times with 10 mL of methylene chloride. The methylene chloride solutions were combined. , washed three times with 5 ml of water, after drying in aqueous sodium bicarbonate and again with water, dried over anhydrous sodium sulfate 5% from 2.0 to 2.5 · 10 ³ torr approx. Evaporate from a bath at 40 ° C. The residue was triturated with n-hexane, filtered and dried in a vacuum desiccator. 1.30 g (59%) of the title product are obtained.

R / 2) = 0.26-0.38.

Analysis for C ₃₃ H ₄₉ N ₅ O ₇ (627.76):

Found: C, 63.13; H, 7.87; N, 11.16; found; C, 63.3; H, 8.0; N, 11.0.

Step 3: 3-Cyclohexyl-D-lacto-L-prolyl-L-argininaldehyde hemisulfate

O-tetrahydropyranyl-3-cyclohexyl-laktoil-L-prolyl-N ^G -benzyloxy-carbonyl-L-argininaldehidet 1.25 g (2 mmol) (Example 7, Step 2) was dissolved in 10 ml of 60% in aqueous ethanol containing 1.05 mmol of sulfuric acid and then hydrogenated in the presence of palladium on charcoal. The reaction was monitored by layer chromatography on ethyl acetate-pyridine-acetic acid in water (45:20: 6:11) ₍ Rf 0.45-0.54 for the benzyloxycarbonyl-free intermediate). The reaction was carried out for ca. After 60 minutes, the catalyst is filtered off and washed with 60% aqueous ethanol and water. The filtrate was washed with ca. half concentrated under reduced pressure and diluted with water to 10 ml,

Adjust the pH of the solution to 2.8 with sulfuric acid or hydroxyl ring anion exchange resin and allow to stand at room temperature for approx. For 24 hours until complete removal of the O-tetrahydropyranyl protecting group (reaction followed by layer chromatography on the above system). The aqueous solution was extracted with methylene chloride (3 x 5 mL), adjusted to pH 3.6 with an anion exchange resin on the hydroxyl cycle, then frozen and lyophilized. Yield: 0.6 g (60%).

R f (5) = 0.43-0.51. HPLC (I / 3): k '= 35.25 and 37.40.

The FAB mass spectrum (410 [M + H] ⁺ ) confirms the expected structure.

For example, the starting materials may be prepared as follows:

Preparation of O-Tetrahydropyranyl-3-cyclohexyl-D-lacto-L-prolylcyclohexylammonium salt

Step A: 3-Cyclohexyl-D-lactic acid g (30.1 mmol) 3-phenyl-D-lactic acid [Rf (2) = 0.450.55] was dissolved in 70 mL acetic acid and 0.25 g platinum (IV) oxide catalyst in the presence of hydrogen at 40 to 50 ° C. The reaction is followed by layer chromatography [final product Rj (2) = 0.6-0.7]. At the end of the reaction, the catalyst was filtered off and the solution is from 2.0 to 2.5 up to 10 ³ Torr. Evaporate from a 40 'C bath. The residue was distilled off twice with 20 ml of toluene and then crystallized from n-hexane. 4.3 g (83%) of the title compound are obtained. R / 2) = 0.60-0.70.

90-93 'C.

[α] D = +8.45 '(c = 1, in methanol).

Analysis for C ₉ H ₁₆ O ₃ (172.22):

Calculated: C, 62.76; H, 9.36;

Found: C, 62.65; H, 9.5.

Step B: 3-Cyclohexyl-D-lactic acid methyl ester

3.44 g (20 mmol) of 3-cyclohexyl-D-lactic acid (Example 7,

Step A) (R f (9) = 0.40-0.45] were dissolved in 30 ml of absolute methanol and after 4 drops of concentrated sulfuric acid is boiled for three hours under reflux cooling. The solution was 2.0-2.5 10 ³ After concentration at 40 DEG C., the residue was dissolved in methylene chloride (40 ml) and washed with water until neutral.The methylene chloride solution was dried over anhydrous sodium sulfate and then evaporated to give an oily residue (3.8 g). , Rf (9) = 0.80-0.86] 20 mmol of 3-cyclohexyl-1-D-milk are considered acid methyl 1-esters.

Step C: O-Tetrahydropyranyl-3-cyclohexyl-D-lactic acid, cyclohexylammonium salt, mmol of 3-cyclohexyl-D-lactic acid methyl ester (Example 7, Step B) was dissolved in 20 mL of methylene chloride and stirred 2 ml (22 mmol) of dihydropyran and 0.3 ml of hydrochloric acid ethyl acetate (concentration 0.1 ΙΟ, 15 g / cm ³ ) were added. The solution was allowed to stand overnight at room temperature. The solution was diluted with 20 ml of methylene chloride, washed neutral with water and 2.0-2.5 10 ³ psi max. Evaporate from a 40 'C bath. The resulting oil (4.4 g of O-tetrahydropyranyl-3-cyclohexyl-D-lactic acid methyl ester, R / 9) = 0.84-0.90] was dissolved in methanol and 20 ml of 1 mol / dm ^{3 was added.} aqueous sodium hydroxide. After standing overnight at room temperature, and after adding 20 ml of 2.0 to 2.5 10 ³ psi max. Distil from a 40 ° C bath until the solution is slightly cloudy. The resulting solution was extracted with 10 ml of methylene chloride and cooled to 5-10 C 'acidified to pH 3 with 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and extracted with 3 times 10 ml of methylene chloride. The methylene chloride solutions were combined, washed with water to neutral, dried over anhydrous sodium sulfate and evaporated. The resulting O-tetrahydropyranyl-3-cyclohexyl-lactic acid was dissolved in diethyl ether and converted to the cyclohexylammonium salt. The crystals were filtered off, washed with diethyl ether and dried in a vacuum desiccator. 5.0 g (70%) of the title product are obtained.

RK9) = 0.63-0.70.

M.p. 153-155 'C (converted to 142' C).

Analysis for C ₂₀ H ₃₇ NO ₄ (355.50):

Found: C, 67.57; H, 10.49; N, 3.98. Found: C, 68.0; H, 11.0; N, 3.85.

Step D: O-Tetrahydropyranyl-3-cyclohexyl-D-lacto-L-proline-cyclohexylammonium salt

O-tetrahydropyranyl-3-cyclohexyl-D-lactic acid cyclohexylammonium salt 4.45 g (12.5 mmol) (Example 7, Step C) was dissolved in 15 ml of 1 mol / dm ³ aqueous solution of potassium hydrogen sulphate and 25 ml of dichloromethane. The dichloromethane layer was washed with water and dried under sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a 40 'C bath. The resulting O-tetrahydropyranyl-3-cyclohexyl-D-lactic acid was dissolved in 15 ml of dichloromethane, cooled in an ice-water bath, and 2.06 g (12.5 mmol) of L-proline methyl ester chlorohydrate, 2.10 g (75 mmol) of 1-hydroxybenzotriazole hydrate, 1.75 ml of triethylamine (12.5 mmol) and 2.58 g (12.5 mmol) of dicyclohexylcarbodiimide. The reaction mixture was allowed to stand at room temperature overnight and The precipitated dicyclohexylurea was filtered off and the solution was pressurized to about 2.0-2.5 x 10 ³ Pa. Evaporate from a 40 'C bath. The residue was dissolved in methylene chloride (30 mL), washed with water (10 mL), 5% sodium bicarbonate (2 x 10 mL), water (10 mL), and dried over anhydrous sodium sulfate (2.0-2.5). ^{At a} pressure of ³ Pa millibar, approx. Evaporate from a 40 'C bath. The oily product thus obtained (O-tetrahydropyranyl-3-cyclohexyl-D-lacto-L-proline methyl ester, Rf (9) = 0.86-0.93) was dissolved in methanol (40 mL), dm ³ sodium hydroxide and stirred for 24 hours. Subsequently, 15 ml of aqueous sodium hydroxide, mol / dm ³ , and 20 ml of methylene chloride are added. The two phases were separated, the aqueous phase was acidified to pH 3 with aqueous potassium hydrogen sulfate (1M / dm ³ ) with stirring and ice-cooling, and extracted with methylene chloride (2 x 15 mL). The methylene chloride solutions were combined, washed with water to neutral and then dried over anhydrous sodium sulfate. The O-tet11 thus obtained

The dihydropyranyl-3-cyclohexyl-D-lacto-L-proline is converted into a crystalline cyclohexylammonium salt in diethyl ether. 3.2 g (56%) of the title compound are obtained. Rf (9) = 0.44-0.51. 119-120 ° C.

Analysis for C ^ HH ^ NN ^O, (452.62):

Found: C, 66.34; H, 9.80; N, 6.19; Found: C, 66.4; H, 9.9; N, 6.25.

Example 8

Preparation of D-2-Cyclohexyl-2-hydroxyacetyl-L-prolyl-L-arginine

]. Step: O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-1-benzyloxycarbonyl-L-arginine-lactam

From 2.6 g (6 mmol) of 0-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-proline-cyclohexylammonium salt (Example 8, Step C) and 2.73 g (7 mmol) of tert-butyloxy carbonyl ^G -benzyloxy-carbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem., 33, 1729 (1990)], using the same amounts of solvents and reagents as described in Step 1 of Example 5, yielded the same title compound as -3.53 g (69%).

R / 2) = 0.70-0.78.

Step 2: 0-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N'-benzyloxycarbonyl-L-arginine aldehyde

2.15 g (3.5 mmol) of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carboxyanhydride-arginine lactam (Example 8, Step 1) in Conversion as described in Example 7, Step 2, using equal amounts of solvents and reagents afforded 1.40 g (65%) of the title product.

R, (2) = 0.23-0.31.

Analysis for C32H47N5O7 (613.74):

Found: C, 62.62; H, 7.72; N, 11.41; Found: C, 62.8; H, 7.8; % N, 11.2.

Step 3: D-2-Cyclohexyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde hemisulfate

1.23 g (2 mmol) of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carboxyanhydride-arginine aldehyde (Example 8, step 2) the Conversion as described in Example 7, Step 3, using equal amounts of solvents and reagents afforded 0.6 g (60%) of the title product. Rf (5) = 0.390.46. HPLC (I / 3): k '= 5.70, 6.10 and 8.76.

The FAB mass spectrum (396 [M + H] ⁺ ) confirms the expected structure.

For example, the starting materials are prepared as follows:

Preparation of O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetylL-proline cyclohexylammonium salt

Step A: D-2-Cyclohexyl-2-hydroxy-acetic acid methyl ester

3.2 g (20 mmol) of D-2-cyclohexyl-2-hydroxyacetic acid [H. Lettré, H. Barnback and H. Staunau, Chem. Bér. 69, 1594-1598 (1936)] [R / 9] = 0.32-0.42], using the same amounts of solvents and reagents as described in Step B of Example 7, using the same amount of solvents and reagents. [Rf (9) - 0.78-0.84], which is considered to be 20 mmol of methyl D-2-cyclohexyl-2-hydroxyacetic acid.

Step B: Cyclohexylammonium salt of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acid starting from mmol D-2-cyclohexyl-2-hydroxyacetic acid methyl ester (Example 8, Step A) Example 7, Step C, Using the same amount of solvents and reagents, 4.1 g of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acid methyl ester [Rf (9) = 0.80-0.88]. as an oily intermediate, 4.1 g (60%) of the title compound are obtained.

Rf (9) = 0.55-0.70.

Mp 152-155 'C (converted to 122' C).

Analysis for C19H35NO4 (341.48):

Found: C, 66.82; H, 10.33; N, 4.10. Found: C, 66.90; H, 10.3; N, 4.20.

Step C: O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-proline-cyclohexylammonium salt

From the cyclohexylammonium salt of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acid (4.3 g, 12.5 mmol) (Example 8, Step B) and 2.1 g (12.5 mmol) of L starting from -proline methyl ester chlorohydrate and all in Example 7

In the same manner as in Step D, using the same amount of solvents and reagents, O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-proline methyl ester [Rf <9] (0.84-0.93) as oily intermediate, followed by 3.25 g (60%) of the title compound.

Rf (9) = 0.20-0.27. Mp: 140-142 ° C.

Analysis for C24H42N2O5 (438.61):

Found: C, 65.72; H, 9.65; N, 6.39. Found: C, 65.7; H, 9.7; N, 6.3.

Example 9

Preparation of 3-Cyclohexyl-D-Lactoyl-L-Pipecolyl-L-Arginine Aldehyde Hemisulfate

Step I: O-Tetrahydropyranyl-3-cyclohexyl-D-lactoyl-L-pipecolyl-1-benzyloxycarbonyl-L-arginine lactam

3.9 g (10 mmol) of tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] are suspended in 15 ml of chloroform and 14.5 ml of hydrochloric acid ethyl acetate (0.1-0.15 g / cm ³ ) are added with stirring and ice-cooling. ). After 2 hours, the reaction mixture was diluted with diethyl ether (15-20 mL), the precipitated crystalline material was filtered off, washed with acetone (7 mL) and diethyl ether (7 mL), and dried overnight in a vacuum desiccator with potassium hydroxide. The resulting N ^G -benzyloxycarbonyl-L-arginine-lactam chlorohydrate was dissolved in 10 ml of pyridine, cooled to -15 ° C, and

EN 211 088 B, while stirring and cooling

O-Tetrahydropyranyl-3-cyclohexyl-Dlactoyl-L-pipecolic acid (3.7 g, 10 mmol) (Example 9, Step A) was dissolved in 10 mL of pyridine, and 0.964 mL (10 mmol) of phosphorous oxychloride and 1.85 mL of 13.2 mmol) triethylamine. The mixture was stirred for 30 minutes at -15 ° C and one hour at 0 ° C and 2.0-2.5 torr to about 10 ^3. Evaporate from a 40 'C bath. The residue was dissolved in water (30 ml) and ethyl acetate (30 ml). The ethyl acetate layer was washed with 10-10 ml of water, after drying in 5% aqueous sodium bicarbonate, water, 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and again with water, dried over anhydrous sodium sulfate 2 02,5 10 ³ Pa for approx. Evaporate from a bath at 40 ° C. The oily product was chromatographed on a silica gel column with ethyl acetate-pyridine-acetic acid-water 960: 20: 6: 11. Fractions containing pure product were combined and extracted with 2 times 10 ml of 1 mol / dm ³ aqueous solution of potassium bisulfate, washed with water, and the above way is evaporated to dryness under reduced pressure. Obtained as an oil (2.56 g, 40%).

R (2) = 0.83-0.90.

Step 2: O-Tetrahydropyranyl-3-cyclohexyl-D-lactoyl-L-pipecolyl-N'-benzyloxycarbonyl-L-arginine aldehyde

O-Tetrahydropyranyl-3-cyclohexyl-D-lactoyl-L-pipecolyl-NG-benzyloxycarbonyl-L-arginine-lactam (2.25 g, 3.5 mmol) (Example 9, Step 1) is prepared as described in Example 7, Step 2. Converted as described in Step 3a to the same amount of solvents and reagents to give 1.46 g (65%) of the title product.

R ({2) = 0.46-0.58.

Analysis result for formula (641.79):

Found: C, 63.62; H, 8.01; N, 10.91. Found: C, 63.80; H, 8.15; N, 10.90.

Step 3: O-Cyclohexyl-D-Lactoyl-α-pplecolyl-L-Arginine Aldehyde Hemisulfate

1.3 g (2 mmol) of O-tetrahydropyranyl-3-cyclohexyl-L-Dlaktoil pipekolil-N ^G -benzyloxy-carbonyl-L-argininaldehidet (Example 9, step 2) of Example 7, step 3, Converted as described using the same amounts of solvents and reagents to give 0.6 g (60%) of the title product.

Rj (5) = 0.51-0.60. HPLC (I / 3): k '= 7.34, 7.97, 8.18, 8.45, 8.55 and 8.90.

The FAB mass spectrum (424 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material may be prepared as follows.

Step A: O-Tetrahydropyranyl-3-cyclohexyl-D-lactooyl-L-pipecolic acid

O-tetrahydropyranyl-3-cyclohexyl-lactic acid cyclohexylammonium salt 4.45 g (12.5 mmol) (Example 7, Step C) was dissolved in 15 ml of 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and 25 ml methylene chloride. The methylene chloride layer was washed with water, and then dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a bath at 40 ° C. The resulting O-tetrahydropyranyl-3-cyclohexyl-D-lactic acid was dissolved in dichloromethane (15 mL), cooled in an ice-water bath, and 2.24 g (12.5 mmol) of methyl L-pipecolic acid chlorohydrate, 2.10 g were added. (13.75 mmol) of 1-hydroxybenzotriazole hydrate, 1.75 mL of triethylamine (12.5 mol) and 2.58 g (12.5 mmol) of dicyclohexylcarbodiimide. The reaction mixture was allowed to stand at room temperature overnight, then the precipitated dicyclohexylurea is filtered off and the solution is from 2.0 to 2.5 · 10 ³ Pa was concentrated to 40 ° C on a bath. The residue was dissolved in 30 ml of methylene chloride, washed with 10 ml of water, 2 times 10 ml of 5% sodium bicarbonate and again with 10 ml of water, and then dried over anhydrous sodium sulfate 2.0 to 2.5 · 10 ³ Pa approx. Evaporate from a 40 'C bath. The oily product thus obtained (O-tetrahydropyranyl-3-cyclohexyl-D-lacto-L-pipecolic acid methyl ester, R / 9) = 0.95-0.98] was dissolved in 40 ml of methanol and 13 ml of 1 mol / dm ^{3 were added.} aqueous sodium hydroxide and stirred for 24 hours. After adding 15 ml of 1 mol / dm ³ aqueous solution of sodium hydroxide and 20 ml of methylene chloride. The two phases were separated, the aqueous phase was acidified to pH = ^{3 with} aqueous potassium bisulfate (mol / dm ³ ) with stirring and ice cooling, and extracted with methylene chloride (2 x 15 mL). The methylene chloride solutions were combined, washed with water to neutral and then evaporated after drying over anhydrous sodium sulfate. 3.75 g (82%) of O-tetrahydropyranyl-3-cyclohexyl-D-lacto-L-pipecolic acid are obtained in the form of an amorphous powder.

R, (9) = 0.51-0.63.

Analysis for C ₂₀ H ₃₃ NO ₅ (367.47):

Found: C, 65.36; H, 9.05; N, 3.81; Found: C, 63.35; H, 9.2; N, 4.0.

Example 10

Preparation of D-2-Cyclohexyl-2-hydroxyacetyl-L-pipecoline-L-arginine aldehyde hemisulfate

Step 1: O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecoline-N ^G -benzyloxy-carbonyl-L-arginine lactam

From 3.55 g (10 mmol) of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolic acid (Example 10, Step A) and 3.9 g (10 mmol) of tert-butyloxycarbonyl-N ^{G from} benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem., 33, 1729 (1990), 171, and using the same amounts of solvents and reagents as in Example 9, Step 1, gave 2.8 g (45%) of the title compound.

R / 2) = 0.80-0.88.

Step 2: O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecoline-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecoline-N ^G -benzyloxy-karbo13 2.2 g (3.5 mmol)

Conversion of nyl-L-arginine lactam (Example 10, Step 1) using the same amount of solvents and reagents as described in Example 9, Step 2 gave 1.45 g (66%) of the title product. .

Rf <2) = 0.42-0.53.

Analysis for C33 ₄₉ N ₅ O ₇ (627.76) Calcd:

Found: C, 63.13; H, 7.87; N, 11.16; Found: C, 63.3; H, 7.95; % N ll 0th

Step 3: D-2-Cyclohexyl-2-hydroxyacetyl-L-pipecoline-L-arginine aldehyde hemisulfate

O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecoline-NG-benzyloxycarbonyl-L-arginine aldehyde (1.26 g, 2 mmol) (Example 10, Step 2) Example

Converted as described in Step 3 using the same amount of solvents and reagents to afford 0.62 g (65%) of the title product.

R (<5) = 0.50-0.58. HPLC (I / 3): k '= 6.16,7,17 and 7.67.

The FAB mass spectrum (410 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

Step A: Preparation of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolic acid

4.27 g (12.5 moles) of the cyclohexylammonium salt of O-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acid (Example 8).

Step B) and starting from 2.47 g (13.75 mmol) of L-pipecolic acid methyl ester chlorohydrate and using the same amount of solvents and reagents as described in Example 9, Step A, using the same amount of solvents and reagents. ) to give the title product as an amorphous powder.

R (<9) = 0.50-0.60.

Analysis for C C^H jNNOS (353.45):

Found: C, 64.56; H, 8.84; N, 3.96. Found: C, 66.65; H, 8.85; N, 4.05.

Example 77

Preparation of D-2-phenyl-2-hydroxyacetyl-L-pipecoline-L-argininaldehyde hemisulfate

Step 1: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecoline-NG-benzyloxycarbonyl-L-arginine-lactam

3.47 g (10 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolic acid (Example 11, Step A) and 3.9 g (10 mmol) of tert-butyloxycarbonyl-NG benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Meg. Chem., 33, 1729 (1990)], using the same amounts of solvents and reagents as described in Example 9, Step 1, to give the title product (2.54 g, 41%).

Rf <2) = 0.80-0.85.

Step 2: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecoline-NG-benzotoxycarbonyl-L-arginine aldehyde

2.2 g (3.5 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-acetyl-L-pipecoline-N ^G -benzyloxy-carbonyl-L-arginine lactam (Example 11, step 1) in the ninth The same procedure as in Example 2, Step 2, using the same amount of solvents and reagents gave 1.3 g (65%) of the title compound.

R (<2) = 0.40-0.45.

Step 3: D-2-Phenyl-2-hydroxyacetyl-L-pipecoline-Larginine aldehyde hemisulfate

1.24 g (2 mmol) of O-tetrahydropyranyl-D-2-phenyl-2-acetyl-L-pipecoline-N ^G -benzyloxy-carbonyl-L-arginine aldehyde (Example 11, step 2) the 7th The same procedure as in Example 3, Step 3, using the same amount of solvents and reagents gave 0.56 g (60%) of the title product.

R (<5) = 0.48-0.53. HPLC (I / 2): k '= 4.55, 5.78 and 10.78.

The FAB mass spectrum (404 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material may be prepared as follows:

Step A: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolic acid

O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetoxyacetic acid cyclohexylammonium salt (4.2 g, 12.5 mmol) (Example 6, Step A) and 2.47 g (13.75 mmol) of L-pipecolic acid Starting from methyl ester chlorohydrate and using the same amount of solvents and reagents as described in Example 9, Step A, 3.65 g (84%) of the title product are obtained in the form of an amorphous powder.

R / 9) = 0.43-0.53.

Analysis for C _I9 5NO ₂ H ₄ (347.40) Calcd:

Calculated: C, 65.69; H, 7.25; N, 4.03; Found: C, 65.8; H, 7.4; N, 4.3.

Example 72

Preparation of 3,3-Diphenyl-D-lacto-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: O-Tetrahydropyranyl-3,3-diphenyl-D-lactoyl-L-propyl-b-benzotoxycarbonyl-L-arginine-lactam

2.73 g (7 mmol) phen? Butyloxy-carbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Meg. Chem. 33, 1729 (1990)], suspended in 10 ml of chloroform, and 10 ml of hydrochloric acid ethyl acetate (concentration 0.11 to 0.15 g / cm ² ) are added with stirring and ice-cooling. After 2 hours, the reaction mixture was diluted with diethyl ether (10 mL, 15 mL), the precipitated crystalline material was filtered off, washed with acetone (5 mL) and diethyl ether (5 mL), and dried in vacuo overnight with potassium hydroxide. The resulting N ^G -benzyloxycarbonyl-L-arginine lactam chlorohydrate was dissolved in 10 ml of dichloromethane, cooled to -20 ° C and added to the mixed anhydride below.

O-Tetrahydropyranyl-3,3-diphenyl-DL-lactoyl-L-propyl hemihydrate (2.60 g, 6 mmol) (Example 12, Step E) was dissolved in 6 mL of dimethylformamide and cooled to -20 'C. and 0.66 mL (6 mmol) was added with stirring.

HU 211 088 Β

N-methylmorpholine, 0.79 ml of chlorosulphonic acid isobutyl ester and the N ^G -benzyloxy-carbonyl-L-arginine lactam After 10 minutes of stirring the above slurry in dichloromethane and finally 2.1 ml (15 mmol) of triethylamine. The reaction mixture was stirred for 2 hours, first with cooling and then allowed to warm to room temperature. The salts were filtered off and the filtrate was diluted with 40 ml of methylene chloride. The resulting solution was washed with 10 ml of 1 mol / dm ^3, after drying in a concentrated aqueous solution of potassium hydrogen sulfate and 3 times with 10 ml water, dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a 30 ”C bath. The product obtained, which is a mixture of 3.8 g (90%) and D-L-3,3-diphenyl lactic acid, is subjected to silica gel column chromatography using ethyl acetate / diisopropyl ether 7: 3 (R). _f = 0.23-0.27 and 0.28-0.32 in the given system). The fractions containing the pure higher _Rf moving the D value of the product and the smaller _Rf value of L product were combined separately, 2.0 to 2.5 - 10 ³ torr approx. Concentrate from a bath at 30 ° C and triturate with diisopropyl ether. 1.0 g (48%) of D and 0.98 g (47%) of product L are obtained. Of these, product D is the peptide lactam containing the D-3,3-diphenyl lactic acid moiety, whereupon the aldehyde derivative with high thrombin inhibiting activity is obtained as described in steps 2 and 3 of this example.

Rf (10) = 0.28-0.32.

156 ° C (shrinks at 150 ° C).

Analysis for C C^H^NNjOt (695.79):

Calculated: C, 67.32; H, 6.52; N, 10.07; Found: C, 67.4; H, 6.65; N, 10.0.

Step 2: O-Tetrahydropyranyl-3,3-diferul-D-lactoyl-L-propyl-N'-benzoxytoxycarbonyl-L-arginine aldehyde 0.95 g (1.36 mmol) of O-tetrahydropyranyl-3,3- laktoil diphenyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-argininlaktámot (Example 12, step 1) was dissolved in 8 ml of tetrahydrofuran and under stirring, was added at -20 ° C at less than 1.02 mmol of lithium aluminum hydride in tetrahydrofuran. The progress of the reduction was monitored by layer chromatography on ethyl acetate-pyridine acetic acid-water (240: 20: 6: 11) and, if necessary, additional lithium aluminum hydride was added, followed by cooling and stirring at a volume of 0.5 mol / dm. Sulfuric acid ( ^{3) was added} dropwise to pH 3. The resulting solution was partitioned between diethyl ether (5 mL). To the lower phase is added 5% sodium bicarbonate to a pH of ca. 6.5 and extracted 3 times with 5 ml of methylene chloride. The methylene chloride layers were combined and dried over anhydrous sodium sulfate 2.0-2.5 10 ³ torr approx. Evaporate from a 40 'C bath. The residue was triturated with diisopropyl ether, filtered and dried in vacuo. 0.86 g (90%) of the title product is obtained. Rj (2) = 0.38-0.42.

Step 3: 3,3-Diphenyl-D-lactoyl-L-pmlll-L-argininaldehyde bemisulfate

0.8 g (1.15 mmol) of O-tetrahydropyranyl-3,3-diphenyl-laktoil-L-prolyl-N ^G -benzyloxy-carbonyl-L-argininaldehidet (Example 12, Step 2) [Rf ( 4) = 0.80 to 0.85], was dissolved in 6 ml of dimethylformamide and 1.2 ml of 0.5 mol / dm ³ aqueous solution of sulfuric acid was added to 0.12 g of palladium on carbon and approx. Hydrogenate at 10 ° C. The progress of the reaction is monitored by layer chromatography (Rf (4) 0.43-0.47 for the intermediate benzoyloxycarbonyl). Takes place for 30 minutes, then the catalyst is filtered, washed with 1 ml of 50% aqueous solution of DMF and 1 mL of water and the combined solutions were 2.0-2.5 · 10 ³ torr approx. Evaporate from a 40 'C bath. The oily residue was dissolved in water (10 mL), adjusted to pH 2.8 with sulfuric acid or hydroxyl ring anion exchange resin and allowed to stand at room temperature for ca. For 24 hours until complete deprotection of the O-tetrahydropyranyl [R / 4] = 0.40-0.50]. The aqueous solution was extracted with methylene chloride (3 x 5 mL), adjusted to pH 3.6 with an anion exchange resin on the hydroxyl cycle, then frozen and lyophilized. Yield: 0.5 g (70%).

R f 5) = 0.48-0.52. HPLC (I / 3): k '= 7.80 and 8.23.

The FAB mass spectrum (480 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of O-tetrahydropyranyl-3,3-diphenyl-DL-lactoyl-L-proline

Step A: 3,3-Diphenyl-DL-lactic acid methyl ester

22.33 g (0.1 mol) of 3,3-diphenyl-DL-lactic acid nitrile [Wiese, Ann. 248: 39-41 (1988)] was dissolved in 100 ml of diethyl ether and 4.04 ml (0.1 mol) of methanol were added. The solution was saturated with dry hydrogen chloride gas under ice-cooling and allowed to stand for 1 hour and 10 ³ 2.0-2.5 torr approx. Evaporate from a 40 'C bath. The remainder is approx. It is crystallized with 200 ml of diethyl ether, the crystals are filtered off and washed with diethyl ether and dried in a vacuum desiccator. The product thus obtained [20.55 g (70%), 3,3-diphenyl-DL-lactiminomethyl ether hydrochloride, m.p. 82-83 ° C] is suspended in 140 ml of water and at 50-60 ° C. heat for 1-2 hours. Meanwhile, it is converted into an oily product which solidifies on cooling and stirring. The solid product is filtered off, washed first with water and then with n-hexane and dried in a vacuum desiccator. 12.8 g (50% based on nitrile) of the title compound are obtained.

Rf (12) = 0.4. Op .: 49-50 'C.

Analysis result for C | ₆ H _{) 6} O ₃ (256.29):

Calculated: C, 74.98; H, 6.29;

Found: C, 75.0; H, 6.3.

Step B: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methyl ester

Methyl 3,3-diphenyl-DL-lactic acid 7.83 g (30.5 mmol) (Example 12, Step A) was dissolved in methylene chloride (75 mL) and dihydropyran (40 mmol) was added under stirring and ice-cooling. and 0.8 ml of hydrochloric acid ethyl acetate (concentration 0.11-0.15 g / ml) and then the solution

EN 211 088 Β leave to stand overnight. The reaction mixture was diluted with 50 ml of methylene chloride, washed neutral with water, and dried over anhydrous calcium chloride from 2.0 to 2.5 · 10 ³ torr approx. Evaporate from a bath at 30 ° C. The residue was considered to be 30.5 mmol of O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methyl ester and was used in Step C.

Step C: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid hydrate

30.5 mmol of O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methyl ester (Example 12, Step B) was dissolved

30.5 ml of acetone and 30.5 ml of 1 mol / dm ³ solution of ethanolic potassium hydroxide solution. The progress of the reaction is monitored by layer chromatography. The solution was pressurized to about 2025 millibar for approx. Evaporate from a bath at 40 ° C. The residue was dissolved in water (40 mL), washed with diethyl ether (2 x 10 mL), and acidified to pH 3 with ice-cold potassium hydrogen sulfate (1 mol / dm ³ ) under ice-cooling. The separated solid product was filtered off, washed with water and dried in a vacuum desiccator. 7.9 g (75%) of the title product are obtained.

Rf <9) = 0.65-0.75.

115-117 'C (shrinks at 76' C).

Analysis calculated for C ₂₀ H ₂₂ O ₄ H ₂ O (344.39): C, 69.75; H, 7.02; Found: C, 69.7; H, 6.55.

Step D: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid 2,4,5-trichlorophenyl Ester

O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid hydrate (4.5 g, 13 mmol) (Example 12, Step C) and 2,4,5-trichlorophenol (2.8 g, 14.2 mmol) were dissolved in water. dichlorohexylcarbodiimide (2.8 g, 13.6 mmol) was added under stirring and ice-cooling. The reaction mixture was allowed to stand at room temperature for 3 hours. then the precipitated dicyclohexylurea is filtered off and washed with tetrahydrofuran. The tetrahydrofuran solutions were combined and 2.0 to 2.5 · 10 ³ torr approx. Evaporate from a 40 'C bath. The residue was triturated with diisopropyl ether (15 mL), cooled in ice water, added with n-hexane (5 mL) and filtered. The solid product was washed with a 1: 1 mixture of diisopropyl ether and n-hexane and dried in a vacuum desiccator. The product thus obtained [4.6 g Rf (11) = 0.750.85] is treated with 10 mmol O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid 2,4,5-trichlorophenyl ester.

Step E: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactoyl-Lp-rolirt hemihydrate O2-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid 2,4,4-trichlorophenyl mmol (Example 12) (Step D) dissolved in pyridine (10 mL), L-propin (1.15 g, 10 mmol) and triethylamine (1.4 mL, 10 mmol) were added. After stirring at room temperature for 6 hours and 10 ³ 2.0-2.5 torr approx. Evaporate from a 40 'C bath. The residue was dissolved in water (20 ml) and diethyl ether (10 ml). The aqueous layer was washed with 10 ml of diethyl ether, then under ice-cooling to pH 3, extracted with acidified mol / dm ³ aqueous solution of potassium hydrogen sulfate and diethyl ether 3 times with 10 ml. The combined diethyl ether solutions were washed with water, dried over anhydrous sodium sulfate, and then evaporated. The residue was triturated with diisopropyl ether (10 mL), cooled with ice-water and then filtered, washed with cold diisopropyl ether and dried in a vacuum desiccator. 3.05 g (70%) of the title product are obtained.

RX2) = 0.45-0.55. 132-135 ° C.

Analysis for C ₂₅ H 9 NO _{2 5} 1 / 2H ₂ O (432.50) Calcd:

Found: C, 69.42; H, 6.99; N, 3.24; Found: C, 69.4; H, 6.85; N, 3.2.

Example 13

Preparation of D-2- (2-Naphthyl) -2-hydroxyacetyl-L-proline-L-arginaldehyde hemihydrate

Step 1: O-tert-Butyl-D-2- (2-naphthyl) -2-hydroxyacetyl-L-prohl-N-benzyloxycarbonyl-L-arginine-lactam

3.12 g (8 mmol) of? Tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] are suspended in 12 ml of chloroform and 12 ml of hydrochloric acid ethyl acetate (concentration 0.11-0.15 g / cm ³ ) are added under stirring and ice-cooling. After 2 hours, the reaction mixture was diluted with diethyl ether (15 mL), the precipitated crystalline material was filtered off, washed with acetone (6 mL) and diethyl ether (6 mL) and dried overnight in a vacuum desiccator with potassium hydroxide. The resulting N ^c benzyloxy-carbonyl-L-arginine lactam hydrochloride are dissolved in 12 ml of dimethylformamide, cooled to -20 ° C and added to the following mixed anhydride.

O-tert-Butyl-D-2- (2-naphthyl) -2-hydroxyacetyl-L-proline (2.8 g, 7.8 mmol) (Example 13, Step F) was dissolved in 8 mL dimethylformamide and added at -20 ° C with stirring. N-methylmorpholine (0.87 mL, 7.8 mmol), isobutyl ester of chlorocarbon (1.03 mL) followed by dimethyl-N- ³ benzyloxycarbonyl-L-arginine lactam (10 min). formamide suspension, and finally 2.8 ml (20 mmol) of triethylamine. The reaction mixture was stirred for 2 hours, first with cooling and then allowed to warm to room temperature. The salts are filtered off and the filtrate is diluted with 50 ml of benzene. The resulting solution was washed with 15 ml of 1 mol / dm ^3, after drying in a concentrated aqueous solution of potassium hydrogen sulfate and 3 times with 15 ml water, dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from a 40 'C bath. The residue was triturated with n-hexane, filtered, washed with n-hexane and dried in a vacuum desiccator. Yield: 4.14 g (85%) Rf (9) = 0.72-0.75.

Step 2: D-2- (2-naphthyl) ^{-2-hydroxyacetyl-L-prolyl-benzyl-G-L-OXT-lactam} argiuin

4.08 g (6.5 mmol) O? Tert-butyl-D-2- (2-naphthyl) -2-acetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam ( Example 13, Step 1) was dissolved in 20 ml of a 1: 1 mixture of trifluoroacetic acid and methylene chloride and allowed to stand at room temperature for 30 minutes. Areak16

Dilute the mixture with 40 ml of methylene chloride, wash three times with 20 ml of water and 3-4 times with 20 ml of 5% sodium bicarbonate each time to anhydrous and then dry over anhydrous sodium sulphate to 2,0-2. At 5 · 10 ³ Pa for approx. Evaporate from a 40 'C bath. The residue was chromatographed on a 100 g silica gel column with ethyl acetate-pyridine-acetic acid-water (480: 20: 6: 11). The fractions containing pure migratory Rf {7) = 0.44 to 0.46 values are combined, washed with 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and water, and then dried over anhydrous sodium sulfate, 2.0 to 2 At 5 · 10 ³ Pa for approx. Evaporate from a 40 'C bath. The residue was triturated with n-hexane, filtered, washed with n-hexane and dried in a vacuum desiccator. 1.8 g (48%) of the title compound are obtained.

R (<7) = 0.44-0.46.

Analysis for C _3] H ₃₃ N ₅ O ₆ (571.14) Calcd:

Calculated: C, 65.12; H, 5.82; N, 12.25; Found: C, 65.20; H, 5.90; N, 12.20.

Step 3: D-2- (2-naphthyl) -2-hydroxyacetyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde

D-2- (2-naphthyl) -2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam 1.71 g (3 mmol) (Example 13. Step 2) was dissolved Lithium aluminum hydride (2.25 mmol) in tetrahydrofuran (2.25 mmol) was added under stirring at -20 ° C in 15 mL of tetrahydrofuran. The progress of the reduction was monitored by layer chromatography in ethyl acetate-pyridine-acetic acid-water (30: 20: 6: 11) and further lithium aluminum hydride added as necessary. and then added dropwise with cooling and stirring as much chilled 0.5 mol / dm ³ solution of sulfuric acid to be between pH 3 and 4. The resulting solution was diluted with water (15 mL), washed with ethyl acetate (10 mL) and extracted with butanol (3x10 mL) saturated with water. The butanol solutions are combined, washed with 10 ml of 5% sodium bicarbonate and 2 times with 10 ml of butanol-saturated water, and then at a pressure of 20-25 millibar for approx. Evaporate from a 40 'C bath. The residue was triturated with n-hexane, filtered, washed with n-hexane and dried in a vacuum desiccator. 1.13 g (65.7%) of the title product are obtained. Rf (6) = 0.62-0.66.

Step 4: D-2- (2-Naphthyl) -2-hydroxyacetyl-L-prolyl-Larginine aldehyde hemisulfate

D-2- (2-naphthyl) -2-hydroxy-L-prolyl-N ^c benzyloxy-carbonyl-L-arginine aldehyde (Example 13, Step 3 (1.0 g, 1.75 mmol) ) dissolved in 20 ml of ethanol was added 1.75 ml of 0.5 mol / dm ³ solution of sulfuric acid and 2 ml water was hydrogenated in the presence of palladium on carbon. The reaction was followed by layer chromatography in ethyl acetate - pyridine - acetic acid - water (30: 20: 6: 11). The reaction was carried out for ca. Playback takes 4 hours. The catalyst is filtered off and washed with 5 ml of 60% aqueous ethanol and 5 ml of water. Ethyl alcohol was distilled off from the filtrate under reduced pressure, water (15 mL) was added, washed with methylene chloride (10 mL), and the solution was adjusted to pH 3.3 with sulfuric acid or a hydroxyl ring anion exchange resin. If the resulting solution is not completely clear (cloudy with precipitation), the precipitate is taken up in 0.5-1.5 ml of t-butanol (max. 10%) and the already clear solution is frozen and lyophilized. Yield: 0.78 g (88%).

R / 5) = 0.29-0.32. HPLC (I / 1): k '= 8.25, 9.25 and 11.3.

The FAB mass spectrum (440 [M + H] ⁺ ) confirms the expected structure.

The starting materials are prepared, for example, as follows: Preparation of O-tert-Butyl-D-2- (2-naphthyl) -2-hydroxyacetyl-L-proline

Step A: DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl ester

20.2 g (100 mmol) of DL-2- (2-naphthyl) -2-hydroxyacetic acid [I. M. Panaiotov: Compt. order. acad. bulgare sci. 10, No. 2, 137-140 (1957); Chem. Abstr. 52, 5336e (1958). M.p. 158-160 ° C, Rf (8) = 0.28-0.30], and proceeding as described in Example 7, Step B, using proportional solvents and reagents, to give 22 g of an oily product [Rf <2) = 0.79-0.81], which is considered to be 100 mmol DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl esters.

Step B: Methyl O-tert-butyl-DL-2- (2-naphthyl) -2-hydroxyacetic acid

DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl ester (Example 13, Step A) (100 mmol) was dissolved in methylene chloride (100 mL), cooled to -25 ° C, and treated with 0.88 mL (10 mL). Trifluoromethanesulfonic acid (11 mmol) and isobutylene (116120 mL) and allowed to stand at -20 ° C for 30 minutes. Pyridine (1.0 ml) was then added to the reaction mixture, which was left at -20 ° C for 30 minutes. Subsequently, 1.0 ml of pyridine were added and the reaction was allowed to warm to room temperature and 2.0 to 2.5 · 10 ³ torr approx. Evaporate from a 40 'C bath. The residue was dissolved in 100 ml of benzene and washed with 20-20 mL of water, 5% sodium bicarbonate, water, 1 mol / dm ³ aqueous solution of potassium hydrogen sulfate and water, followed by drying over sodium sulfate anhydrous at a pressure of 2,02,5 10 ³ Pa for approx. Evaporate from a 40 'C bath. The residue was treated with 100 mmol of O-tert-butyl DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl ester and used in Step C. Rf (12) = 0.50-0.55.

Step C: O-tert-Butyl-DL-2- (2-naphthyl) -2-hydroxy-acetic acid

100 mmol copper-zinc-O-butyl-DL-2- (2-naphthyl) -2-hydroxyacetic acid methyl ester (Example 13, Step B) is dissolved in 100 ml of methanol was added 50 ml of 2 mol / dm ³ aqueous solution of sodium hydroxide and stir overnight at room temperature. The reaction mixture was saturated with carbon dioxide (with addition of dry ice), extracted twice with 20 ml of n-hexane, acidified with solid potassium bisulfate and extracted 3 times with 40 ml of ethyl acetate. The ethyl acetate solutions were combined, water17

EN 211 088 Β and neutralized and after drying over anhydrous sodium sulfate at a pressure of 2,0-2,5 · 10 ³ Pa for approx. Evaporate from a 40 'C bath. The oily residue is stirred with 100 ml of n-hexane, the crystalline product formed is filtered off, washed with n-hexane and dried in a vacuum desiccator. 13.2 g (51%) of the title product are obtained.

Rf (9) = 0.34-0.38. 114-116 ° C.

Analysis for _{C) 6} (258.30) Calcd _Ig H O _3:

Found: C, 74.39; H, 7.02;

Found: C, 74.0; H, 7.0.

Step D: O-tert-Butyl DL-2- (2-naphthyl) -2-hydroxyacetic acid 2,4,5-trichlorophenyl ester

12.9 g (50 mmol) of O- tert -butyl-DL-2- (2-naphthyl) -2-hydroxyacetic acid (Example 13, Step C) and 10.24 g (52 mmol) of 2.4, 5-Trichlorophenol was dissolved in 30 ml of dimethylformamide, cooled to 0 ° C, dicyclohexylcarbodiimide (10.3 g, 50 mmol) was added and stirred for half an hour at 0 ° C and then for 2 hours at room temperature. The precipitated dicyclohexylurea was filtered off, washed with dimethylformamide (2 x 5 mL), and the filtrate and washings were combined. The resulting solution was used in the next reaction as O-tert-butyl-DL-2- (2-naphthyl) -2-hydroxyacetic acid 2,4,5-trichlorophenyl ester [Rf (9) - 0, 50 mmol]. 82-0.85] in dimethylformamide.

Step E: O-tert-Butyl-DL-2- (2-naphthyl) -2-hydroxyacetyl-L-proline

To a solution of O-tert-butyl-DL-2 (2-naphthyl) -2-hydroxyacetic acid 2,4,5-trichlorophenyl ester (50 mmol) obtained in Example 13, Step D, was 5.8 g. L-proline (50 mmol) and triethylamine (7.0 mL, 50 mmol) were added and stirred overnight at room temperature. The reaction mixture was filtered, and from 2.0 to 2.5 · 10 ³ torr approx. Evaporate from a bath at 40 ° C. To the oily residue was added 60 ml of 5% sodium bicarbonate. The resulting cloudy solution was washed with diethyl ether (3 x 40 mL), acidified to pH 3 with solid potassium bisulfate and extracted with ethyl acetate (3 x 40 mL). The combined ethyl acetate solutions are washed neutral with water, and then dried over anhydrous sodium sulfate 2.0-2.5 · 10 ³ torr approx. Evaporate from 40 'Cos bath. 18 g of an oily residue are obtained, which is obtained in 25 mmol of 0-tert-butyl-DL-2- (2-naphthyl) -2-hydroxyacetyl-L-proline [R 1 (0.38-0.40)] and A diastereomeric mixture of Ozert-butyl DL-2- (2-naphthyl) -2-hydroxyacetyl-L-proline [Rf (1) = 0.35-0.37] mmol was used and used as such (oil). up in the next step (F).

Step E: O-tert-Butyl-DL-2- (2-naphthyl) -2-hydroxyacetyl-L-proline

Anion exchange resin (1200 meq anion capacity) of BIO-RAD AG 1-X2 (50-100 mesh) in 2000 ml acetate cycle was suspended in distilled water, passed through a 6 cm diameter and 80 cm long glass column at 15 ml / min. 4000 ml

A 2: 1 methanol / water mixture was then added to a solution of 18 g of the oily product from Example 13, Step E in 2000 ml of a 2: 1 methanol / water mixture and the column washed with 4,000 ml of the above methanol / water mixture. Elution with methanol - 1 mol / dm ³ aqueous solution of acetic acid 2: 1 is carried out with a mixture of 200 ml fractions were collected. Material The fractions were monitored by thin layer chromatography using ethyl acetate - pyridine - acetic acid - water 480: 20: 6: 11 mixture of [the spots exposed to UV light (254 nm) or _KMnO4 in aqueous call visible]. The pure isomeric fractions were combined and ca. Concentrate to 1/3 volume at a pressure of 2.0-2.5 10 ³ Pa for approx. 40 ° C bath. The precipitated material was redissolved by the addition of acetonitrile, then the solutions were frozen and lyophilized. The products thus obtained:

5.93 g of the previously eluting isomer [Rf <1] = 0.38-0.40] /

5.63 g of the latter eluting isomer [Rf (l) = 0.35-0.37] Π,

1.0 g of product containing the mixed isomers 7 and II.

The isomer is O-tert-butyl-DL-2- (2-naphthyl) -2-hydroxyacetyl-L-proline based on the chromatographic behavior of the isomers and the enzymatic inhibitory activity of the aldehyde derivatives obtained therefrom. g (16.68 mmol, 66.7% based on O-tert-butyl-DL-2- (2-naphthyl) -2-hydroxyacetic acid intermediate used in Step D of this Example). 90-93 'C.

Analysis for C ₂₁ H ₂₅ NO ₄ (355.42):

Calculated: C, 70.96; H, 7.09; N, 3.94. Found: C, 71.0; H, 7.15; N, 3.85.

Example 14

Preparation of D-2- (1-Naphthyl) -2-hydroxyacetyl-L-prolyl-L-argininaldehyde hemisulfate

Step 1: O-tert-Butyl-D-2- (1-naphthyl) -2-hydroxyacetyl-L-prolyl-1, 4-benzyloxycarbonyl-L-arginine-lactam

3.12 g (8 mmol) of tert-butyloxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 2.8 g (7.8 mmol) of O-tert-butyl-D-2- (1-naphthyl) -2-hydroxyacetyl. Starting from L-proline (Example 14, Step F) and using the same amount of solvents and reagents as described in Example 13, Step 1, 4.14 g (85%) of the title compound are obtained.

R (<9) = 0.71-0.77.

Step 2: D-2- (N-Naphthyl) -2-hydroxyacetyl-L-prolyl-N-benzyloxycarbonyl-L-arginine lactam

4.08 g (6.5 mmol) O / tert-butyl-D-2- (l-naphthyl) -2-acetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine laktámol ( Example 14, Step 1) Converted as described in Example 13, Step 2 using the same amount of solvents and reagents to give 1.85 g (50%) of the title product.

Rf (9) = 0.44-0.46.

HU 211 088 B

Analysis for C _3I H ₃₃ N ₅ O ₆ (571.61) Calcd:

Found: C, 65.13; H, 5.82; N, 12.25; Found: C, 65.0; H, 5.95; % N, 12.0.

Step 3: D-2 - (] - naphthalenyl) -2-hydroxyacetyl-L-prolyl ^G -benzyloxy-carbonyl-L-arginine aldehyde

D-2- (1-naphthyl) -2-hydroxyacetyl-L-propyl-N-benzyloxycarbonyl-L-arginine-lactam (1.71 g, 3 mmol) (Example 14, Step 2) Converted as described in Example 13, Step 3, using the same amount of solvents and reagents, 1.2 g (70%) of the title product are obtained.

Rf (3) = 0.40-0.44.

Step 4: D-2- (1-Naphthyl) -2-hydroxyacetyl-L-prolyl-Larginine aldehyde hemisulfate

D-2- (l-naphthyl) -2-hydroxy-L-prolyl-N ^c benzyloxy-carbonyl-L-arginine aldehyde (Example 14, Step 3 (1.0 g, 1.75 mmol) ), as described in Example 13, Step 4, using the same amounts of solvents and reagents, afforded 0.8 g (90%) of the title compound. R f (5) = 0.28-0.30. HPLC (I / 3): k '= 5.99, 6.39 and 7.17.

The FAB mass spectrum (440 [M + H] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of O-tert-Butyl-D-2- (1-naphthyl) -2-hydroxyacetyl-L-proline

Step A: DL-2-yl-naphthyl) -2-hydroxy-acetic acid methyl ester

20.2 g (100 mmol) of DL-2- (1-naphthyl) -2-hydroxyacetic acid [I. M. Panaiotov: Compt. order. acad. bulgare sci. 70, No. 2, 137-140 (1957); Chem. Abstr. 52, 5336e (1958). M.p. 92-94 ° C, Rf (2) = 0.28-0.30], starting from the reaction and proceeding in the same manner as in Example 7, Step B, using a proportional amount of solvents and reagents (R / 2). = 0.69-0.71] 100 mmol DL-2 (1-naphthyl) -2-hydroxyacetic acid methyl esters are considered.

Step B: Methyl O-tert-butyl DL-2- (7-naphthyl) -2-hydroxyacetic acid

Starting from 100 mmol of DL-2- (1-naphthyl) -2-hydroxyacetic acid methyl ester (Example 14, Step A) and proceeding as described in Example 13, Step B, using the same amount of solvents and reagents. the product obtained is considered to be 100 mmol of methyl O-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetic acid and is used in Step C

Rj (12) = 0.66-0.70.

Step C: O-tert-Butyl-DL-2- (1-Naphthyl) -2-hydroxyacetic acid

Starting from 100 moles of methyl O-Z-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetic acid (Example 14, Step B) and proceeding as described in Example 13, Step C above.

14.6 g (56.5%) of the title product are obtained using the same amounts of solvents and reagents.

R / 9) = 0.73-0.76. 132-135 ° C.

Analysis result for C | ₆ H, _{g of} O ₃ (258.30):

Found: C, 74.39; H, 7.02;

Found: C, 7.3; H, 7.05.

Step D: O-tert-Butyl-DL-2- (7-Naphthyl) -2-hydroxy-acetic acid 2,4,5-trichlorophenyl Ester

12.9 g (50 mmol) of O-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetic acid (Example 14, Step C) and 10.24 g (52 mmol) of 2.4.5 starting from trichlorophenol and using the same amount of solvents and reagents as described in Step D of Example 13, the resulting solution was treated with 50 mmol of O-tert-butyl DL-2 (1-naphthyl) -2-hydroxyacetic acid. A solution of 2,4,5-trichlorophenyl ester (Rf (9) = 0.84-0.86) in dimethylformamide is used as such in the next reaction.

Step E: O-tert-Butyl-DL-2- (7-naphthyl) -2-hydroxyacetyl-L-proline

From O-tert-butyl DL-2 (1-naphthyl) -2-hydroxyacetic acid 2,4,5-trichlorophenyl ester (50 mmol) obtained in Step D of Example 14 and 5.8 g (50 mmol) ) Starting from L-proline and proceeding as described in Example 13, Step E above

yielding 15.65 g of an oily residue using the same amount of solvents and reagents (Rf (1) = 0.530.55). HPLC (H / 5): k '= 12.85 and 16.95], which was 22 mmol O-tert-butyl DL-2- (1-naphthyl) -2-hydroxyacetyl-L-proline and 22 mmol It is considered to be a mixture of O-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetyl-L-proline and is used as such (oil) in the next step (F).

Step F: O-tert-Butyl-DL-2- (1-naphthyl) -2-hydroxyacetyl-L-proline

15.65 g of the oily product from Example 14, Step E are chromatographed as described in Example 13, Step F on 2000 ml of an acetate cycle BIORAD AG 1-X2 anion exchange resin, except that the fractions are determined by HPLC. (II / 5). In this way, the following products are obtained.

4.4 g of the previously eluting (k '= 12.84) 7 isomers,

4.3 g of the last eluted (k '= 16.95) 77 isomers,

4.5 g of the product, which contains 7 and 77 isomers mixed together.

The isomer is O-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetyl-L-proline based on the chromatographic behavior of the isomers and the aldehyde derivatives obtained therefrom, yielding 4.4 g. [12.4 mmol, 49.6%, relative to the O-tert-butyl-DL-2- (1-naphthyl) -2-hydroxyacetic acid intermediate used in Step D of this Example]. M.p. 88-90 'C.

Analysis for C21H25NO4 (355.42):

Calculated: C, 70.96; H, 7.09; N, 3.94. Found: C, 70.95; H, 7.15; N, 3.8.

Claims (4)

PATENT CLAIMS A process for the preparation of novel peptide derivatives A-Xaa-Arg-H (I) A is D- or L-isochroman-1-carbonyl, Dv or L-isochroman-3-carbonyl, and an acyl group of formula D-A'CH (OH) -CO A is phenyl, benzyl, 1-naphthyl, 2-naphthyl, benzhydryl, cyclohexyl or cyclohexylmethyl, Xaa is an L-proline residue or an L-pipecolic acid residue, and Arg is for the preparation of L-arginine radicals and their acid addition salts, characterized in that an optionally O-protected oxo acid containing an A 'acid radical - wherein A' can take the meaning given in A, DL-isochroman-1 carbonyl group or DL-A'-CH (OH) -CO may also represent an acyl group wherein A 'is as defined in the parent ring, and an amino acid of the formula Xaa, where Xaa is as defined in the parent ring, then condensed to form an acyl group. After the separation of the diastereomers, an N ^G- protected arginine lactam is known to be acylated with L-proline and acyl L-pipecolic acid, if appropriate, and the resulting protected acyl arginine lactam is reduced and the resulting protected acyl arginine aldehyde is protected. (s) is removed and the resulting peptide derivative of formula (I) is isolated if desired in the form of an acid addition salt. (Priority: 19/05/1992) 2. A process for the preparation of the novel peptide derivatives A-Xaa-Arg-H (I) A is D- or L-isochroman-1-carbonyl, Dv or L-isochroman-3-carbonyl, and an acyl group of formula D-A'CH (OH) -CO A 'is phenyl, benzyl, benzhydryl, cyclohexyl or cyclohexylmethyl, Xaa is an L-proline residue and Arg is for the preparation of L-arginine radicals and their acid addition salts, characterized in that an optionally O-protected oxo acid containing an A 'acid radical - wherein A' can take the meaning given in A, DL-isochroman-1-carbonyl or DL -A'-CH (OH) -CO may also represent an acyl group wherein A 'is as defined in the parent ring - and an amino acid of the formula Xaa - wherein Xaa is as defined in the parent ring, then the resulting acyl-L- proline, optionally, in a known manner with an N-protected arginine lactam acylation ^c after the separation of diastereomers of the resulting protected acyl-arginine lactam is reduced, and the resulting protected acyl-arginine aldehidről the protecting group (s) are removed and the resulting The peptide derivative of formula (I) is optionally isolated in the form of an acid addition salt. (Priority 03/04/1992) 3. A process for the preparation of a pharmaceutical composition comprising the compound of formula (I) according to claim 1, wherein A, Xaa and Arg are as defined in claim 1, or an acid addition salt thereof, a solvent, diluent conventionally used in pharmaceutical preparation. , carriers and excipients are conventionally formulated in pharmaceutical formulations. (Priority: 19/05/1992) A process for the preparation of a pharmaceutical composition comprising the compound of formula (I) according to claim 2, wherein A, Xaa and Arg are as defined in claim 2, or an acid addition salt thereof, a solvent, diluent conventionally used in pharmaceutical preparation. , carriers and excipients are conventionally formulated in pharmaceutical formulations. (Priority 03/04/1992)