HU183579B - Process for producing l-arginyl-l-lizyl-l-asparagyl-l-valyl-l-tirosine pentapeptide - Google Patents

Abstract

The present invention relates to a novel process for the preparation of L-arginyl-L-lysyl-L-aspartyl-L-valyl-L-tyrosine pentapeptide (TP-5) by stepwise chain extension in solution, in which the L-valine, L, in each coupling step is used. as the amino-terminal protecting group for aspartic acid and L-lysine, a benzyloxycarbonyl group, a t-butyloxy group for the protection of the L-aspartic acid / 1-carboxyl group, an ω-amino group of the L-lysine, and an amino-terminal protecting group for the last coupling step in L-arginine. and the tert-butyloxycarbonyl group is used and the amino-terminal protecting group is removed by catalytic hydrogenation, with the exception of the last step, after each coupling step, and after the final coupling step, all the protecting groups present are removed by treatment with trifluoroacetic acid. In this way, a very pure product is obtained without any harmful reaction. -1-

Description

The present invention relates to a novel process for the synthesis of the Arg-Lys-Asp-Val-Tyr pentapeptide.

It is known that the thymus secretes a number of hormone polypeptides that contribute to lymphocyte differentiation and thus play an important role in the body's immune system. One of these hormones, thymopoietin, which regulates selective differentiation of T-lymphocytes, was isolated in 1975 and its structure was determined [4077949]. U.S. Pat. Further studies have revealed that the probable active center of the 49 amino acid hormone is the sequence 32-36, the Arg-Lys-Asp-Val-Tyr pentapeptide [G. Goldstein, M.

P. Scheid, E. A. Boyse, D. H. Schlesinger, J. Van Wauwe (1979) Science 204: 1309] which preserve the biological effects of the whole hormone [D. H. Schlesinger, G. Goldstein, Cell, 5, 361 (1975)]. According to extensive biological studies, this pentapeptide appears to be a promising agent for the treatment of immunosuppressive conditions of various origins [S. J. Hopkins, Drugs of Future, 5, 625 (1980)].

The pentapeptide, referred to as "TP-5" in the literature, is a solid phase [No. 824121]. Belgian and 4,190,166; U.S. Pat.] And in solution [T. Abiko,

1. Tin, rí. Sekino: Chem. Pharm. Bull., 28, 2507 (1980)]. Both methods have several drawbacks, which make it difficult to scale up the reactions and to economically produce industrially. In the solid phase peptide synthesis, the coupling reagents must be used to acylate the peptide coupled to the solid support by using at least twice the coupling reagent to complete the reactions (RB Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); M. Monahan, J. Rivier: Biochem. Biophys. Gap. Commun., 48, 1100 (1972); J. Rivier et al., J. Med Chem, 16, 5 ¹ Π97)], while the whole process has a high dissolving ¹ ukséglete.?. Because of this, and because of the known problems of solid phase peptr vts (generation of defective and truncated sequences...), This method has not yet been applied to industrial synthesis.

In both solid phase and solution TP-5 syntheses, a combination of protecting groups has been used which allows for a number of undesirable side reactions. In both cases, the amino acid-sensitive protecting group was removed by trifluoroacetic acid in the syntheses, so that the peptides were repeatedly treated with acid during stepwise chain extension. At the end of the synthesis, the vehicle was cleaved and all other protecting groups removed using liquid hydrogen fluoride. Repeated acid treatments can cause a number of side effects; Thus, alkylation of the amino group [A. R. Mitchell, R. B. Merrifield: J. Org. Chem., 41, 2015 (1976)], partial cleavage of benzyl esters (E. Schnabel, H. Klostermeyer, H. Berndt: Ann. Chem., 749, 90 (1971)), partial cleavage of peptide-carrier bonds [B Gutte, RB Merrifield, J. Biol. Chem., 246, 1922 (1971); B. Gutte, J. Biol. Chem., 1975, 250, 889], tyrosine alkylation (P. Sórup, H. Braae). Villemoes, P., Christensen, T., Acta Chem. Scand., B33, 653 (1979), BF Lundt, NL, Johansen, J. Markussen, Int. J. Peptide Protein Res., 14, 344 (1979), BW Erickson. RB Merrifield, J. Am. Chem. Soc., 95, 3750 (1973);

D. Yamashiro, C. H. Li: J. Org. Chem., 38, 591 (1973)], ring closure of aspartic acid [I. Schoen, L. Kisfaludy, int. J. Peptide Protein Res., 14, 485 (1979); Peptides: Structure and Biological Function, Proc. 6th Amer. Peptide Symp. (E. Gross, J. Meienhofer ed.) Pierce Chem. Co., Rockford, 1979, 277-280. Peptides containing N-benzyl aspartic acid are also highly prone to ring closure [M. Bodanszky, J. Z. Kwei, Int. J. Peptide Protein Res., 12, 69 (1978)]. Due to the occurrence of these side reactions, TP-5 synthesized both in solid phase and in solution [No. 824121]. Belgian and 4 190166 U. S. Patent No. 5,197 to T. Abiko, I. Onode, H. Sekino, Chem. Pharm. Bull., 28, 2507 (1980)] had to be purified by ion exchange column chromatography. The solid phase synthesized product was 94% pure.

In addition to the high solvent requirements already mentioned, both the use of hydrogen fluoride deprotection and ion exchange column chromatography purification make it very difficult or uneconomic to scale up known processes.

It has been found that the above drawbacks of the known methods can be overcome and the Arg-Lys-Asp-Val-Tyr pentapeptide can be prepared in good yield without side reactions, by stepless chain extension of the solution in each step without the need for further chromatographic purification. , L-aspartic acid, or L-lysine may be deprotected by catalytic hydrogenation, namely benzyloxycarbonyl, the t-carboxyl group of L-aspartic acid and the ω-amino group of L-lysine, and in the final coupling step The amino-terminal protecting group of L-arginine is trifluoroacetic acid-deprotected and the amino-terminal protecting group is followed by catalytic hydrogenation after each coupling step, except for the last step, and all the protecting groups present after the final coupling step. the opportun is removed by treatment with trifluoroacetic acid.

Thus, for the temporary protection of the N-carboxyl group of L-aspartic acid and the ω-amino group of L-lysine, trifluoroacetic acid-deprotected protecting groups are used which are not cleaved after each coupling reaction by catalytic hydrogenation to remove the amino-terminal protecting group of the intermediate. however, after the final coupling step, they are simultaneously cleaved during removal of the amino-terminal protecting group of L-arginine with trifluoroacetic acid. Therefore, a tert-butyloxy group for the temporary protection of the N-carboxyl group of L-aspartic acid and a tert-butyloxycarbonyl group for the terminal protection of the ω-amino group of L-lysine are used.

In the final step of the process of the invention, after deprotection with trifluoroacetic acid, the Arg-Lys-Asp-Val-Tyr pentapeptide is obtained in the form of its trifluoroacetate salt. In this way, the free pentapeptide or other salt of the formula can be prepared, for example by ion exchange.

Thus, in the practice of the process according to the invention, the first step is, for example, benzyloxycarbonyl-21

183,579

L-valine (Z-Val-OH) is reacted with L-tyrosine methyl ester (H-Tyr-OMe) in the presence of dicyclohexylcarbodiimide. The resulting (Z-Val-Tyr-OMe) protected dipeptide ester [J. Ramachadran, C. H. Li: J. Org. Chem., 28, 173 (1963); H. Schwarz, F. M. Bumpus, I. H. Page: J. Am. Chem. Soc., 79, 5697 (1957); S. Sakakibara, M. Itoh: Bull. Chem. Soc. Japan, 40, 656 (1967)] and from a protected dipeptide Z-Val-Tyr-OH [R. Schwyzer et al; Helv. Chim. Acta, 41, 1273 (1958)] to remove the benzyloxycarbonyl protecting group by catalytic hydrogenation. The free dipeptide [R. Schwyzer et al., Helv. Chim. Acta, 41, 1273 (1958)] acylated with benzyloxycarbonyl-L-aspartic acid (N-tert-butyl ester) -α-N-hydroxy-succinimide ester (Z-Asp (O'Bu) -OSu). Again, the protected tripeptide is deprotected by catalytic hydrogenation and the free tripeptide is acylated with a-benzyloxycarbonyl- (e-tert-butyloxycarbonyl) -L-lysine-N-hydroxysuccinimide ester (Z-Lys (Boc) Osu). The protected tctrapctide is then deprotected by catalytic hydrogenation and the free tetrapeptide is mixed with the acetic anhydride of t-butyloxycarbonyl-L-arginine (Boc-Arg-OH) with pivaloyl chloride, chloro-carbonyl ethyl or isobutyl ester. In one step, all the protecting groups are removed from the protected pentapeptide with trifluoroacetic acid and the free peptide is treated with a strongly basic ion exchange resin introduced into the acetate ring. The product was found to be 99.3% pure by thin layer chromatography, electrophoresis, and high performance liquid chromatography.

The advantage of the process is that said side reactions are either not at all possible or can be minimized. Thus, for example, thin layer chromatography did not show acylation of the hydroxyl group of tyrosine [S. K. Girin, J. P. Svacskin: Zsur. Zh. Him., 49, 451 (1979)], the ring closure of aspartic acid to a succinimide derivative. The particular protecting group combination precludes the partial acidolysis of benzyl ester bonds and the formation of by-products resulting from the non-selective removal of the nitro group of arginine. The coupling methods employed and the protecting group combination make it possible to obtain a final product which does not need to be subjected to high solvent and time consuming column chromatography purification.

Further benefits are in the line of incremental growth and industrial production. The operations used in the synthesis can be magnified indefinitely and can be performed in devices commonly used in the pharmaceutical industry. This results in economical production of TP-5, taking into account the productivity of the process.

The Arg-Lys-Asp-Val-Tyr pentapeptide is obtained as an acetate in the process of the invention. From this the base can be liberated by methods known per se. The free base, on the other hand, can be converted into pharmaceutically acceptable acid addition salts by reaction with acids. Inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acids, organic acids such as formic acid, longer chain fatty acids, lactic acid, tartaric acid, citric acid, maleic acid and amino acids such as aspartic acid, glutamic acid and the like can be used.

The pentapeptide prepared by the process of the present invention may be used in the form of standard pharmaceutical compositions, but is not included in the invention. Such pharmaceutical compositions contain the pentapeptide of the invention in an organic or inorganic carrier suitable for enteral or parenteral administration. Thus, the pharmaceutical compositions may be in the form of solid lyophilisates such as, for example, non-peptide-reactive compounds such as mannitol, milk sugar, starch, solutions and suspensions, and emulsions containing various neutral, preservative, stabilizing excipients. Pharmaceutical compositions containing the pentapeptide in the form of complexes are preferred because of their prolonged action.

Practical embodiments of the process of the invention are illustrated by the following example.

Abbreviations used in this example to denote amino acid and peptide derivatives are recommended by IJPAC-IUB [J. Biol. Chem., 1977, 247, 977]. All amino acids are L-antipodes. Other abbreviations are: DCC = dicyclohexylcarbodiimide, DCU = dicyclohexylurea, Z = benzyloxycarbonyl, Boc = tert-butyloxycarbonyl, B-tert-butyl, OSu = N-succinimidoxy.

Melting point determinations were made by dr. They were made in a Toltoli apparatus (Biichi, Switzerland). Thin layer chromatography was performed on a pre-fabricated silica gel adsorbent (DC-Fertigplatten, Merck) in the following solvent mixtures:

1. ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 9: 1

2. ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 4: 1

3. ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 7: 3

4. ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 1: 1

5. ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 2: 3

6. Ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11) = 3: 7 Chromatograms were developed with ninhydrin and after chlorination with KJ-tolidine. Specific optical rotation was measured on a Perkin Elmer 141-digit photoelectric polarimeter. The tyrosine content of the peptides was determined in 0.1 M NaOH at 294 nm using a Pye-Unicam SP 1800 UV spectrophotometer.

High Performance Liquid Chromatographic Testing of Boc-Arg (HCL) -Lys (Boc) -Asp (OBu) -Val-Tyr-OH from a Hewlett-Packard 1081A Pump, Schoeffel SF 770 Variable Wavelength Detector, Rheodyne 7010 Loop Injector and KUTESZ A 185 "writing apparatus was performed on a 250 x 4.6 mm Nucleosil 10 Ci ₈ column (Chrompack BU, The Netherlands) at a flow rate of 1.08 ml / min at 210 nm. Eluting with methanol, acetonitrile, and pH 2.2 with Mc Ilvaine buffer 5: 4 was used as eluent, the mixture contained 0.1 mole / 1 NaCl cent _4: 1st

High-performance liquid chromatography testing of Arg-Lys-Asp-Val-Tyr and its acid addition salts was performed on a Liquopump 312 high pressure pump, Liquodet 308 variable wavelength detector (Labor MIM, Budapest), 20 μΐ loop injector and Varian Type A-25 recorder. was performed on a 250 x 4.6 mm Lichrosorb RP-18 10 μηι column (Applied Science Lab., USA) at a flow rate of 1.0 ml / min at 275 nm. Elution was carried out with a 1: 9 mixture of acetonitrile and Mc Ilvaine buffer, pH 2.2. A 1% aqueous solution of the test substance was prepared.

183,579

1st step

N-Benzyloxycarbonyl-L-valyl-L-tyrosine methyl ester 12.55 g (50 mmol) of Z-Val-OH and 11.60 g (50 mmol)

H-Tyr-OMe-HCl was suspended in 170 ml of chloroform in the presence of 7.0 ml (50 mmol) of triethylamine. The suspension was cooled to 0 ° C and dicyclohexylcarbodiimide (10.3 g, 50 mmol) was added. After stirring at room temperature overnight, the precipitated dicyclohexylurea was filtered off and washed with 50 ml of 1M chloroform each time. The chloroform solution was washed with 1M hydrochloric acid (2 x 100 mL), water (100 mL), 5% sodium bicarbonate (2 x 100 mL), water (100 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in 200 mL of ethyl acetate while warm. After half an hour the precipitated dicyclohexylurea was filtered off, the filtrate was concentrated in vacuo and the residue was solidified with petroleum ether. 18.37 g (86%) of Z-Val-Tyr-OMe are obtained. M.p. 145-147 ° C, Rf 0.70, [α] 20 D = + 8.38 ° (c = 1.0 pyridine).

Step 2

N-Benzyloxycarbonyl-L-valyl-L-tyrosine

Z-Val-Tyr-OMe (26.4 g, 61.6 mmol) (Step 1) was treated with 6.0 g (150 mmol) of sodium hydroxide in 150 mL of methanol and 50 mL of water. After one hour, water (100 mL) was added, the methanol was evaporated in vacuo and the aqueous solution was acidified to pH 3 with concentrated hydrochloric acid. The precipitate was filtered off and washed thoroughly with water. 24.0 g (94%) of Z-Val-Tyr-OH are obtained. Melting point: 158 to 160 ° C, Rf 0.50, [a] f> ^-l-p = 32.0 ° (c = 4, pyridine).

Step 3

N-Benzyloxycarbonyl-L-aspartic acid-.beta.-t-biitiiészter-L-valyl-L-tyrosine

Z-Val-Tyr-OH (step 2) (32.0 g, 77.2 mmol) was dissolved in acetic acid (300 mL) and hydrogenated with bubbling under stirring in the presence of 3.5 g of 10% palladium on activated carbon. The reaction was completed within three hours. The catalyst is filtered off, the filtrate is concentrated in vacuo and the residue is crystallized from 150 ml of methanol and filtered. 19.8 g (90.7%) of H-Val-Tyr-OH are obtained, Rf 0.45, [α] D 20 = + 47.7 ^° (c = 1.01 pyridine).

The free dipeptide obtained above was suspended in 150 ml of anhydrous dioxane and reacted with 9.85 ml (70.5 mmol) of Z-Asp (O'Bu) -OSu. The next day, the clear solution was concentrated in vacuo to 100 mL and the residue quenched with 300 mL of 10% aqueous acetic acid. The resulting oil solidifies by scraping. The crude product is filtered off, washed well with water and then boiled with 250 ml of methanol. The next day, the suspension was filtered and the precipitate was washed with 100 mL of 80% methanol. 35.7 g (79% of Z-Val-Tyr-OH) of Z-Asp-Buj-Val-Tyr-OH are obtained, m.p. 210-212 ° C, Rf 0.65, [α] = 0 (c = 1, dimethylformamide), [α] 25 D = - 3.0 ° (c = 1.06, dimethylsulfoxide).

Step 4

N-a-benzyloxycarbonyl-N-e-tert-butyloxycarbonyl-L-lysyl-L-aspartic acid P-tert-butyl ester-L-valyl-L-tyrosine

Z-Asp (O'Bu) -ValI-Tyr-OH (step 3) (35.4 g, 60.3 mmol) was suspended in acetic acid (500 mL) in the presence of 3.6 g of 10% palladium on activated carbon catalyst, stirring while hydrogenation is bubbled through. After two hours, the precipitate dissolves and the reaction is complete. The catalyst was filtered off and the filtrate was concentrated in vacuo. The residual oil was crystallized with 100 mL of water and the suspension was refrigerated the next day. The product is filtered off and washed with ice-cold water. 25.4 g of H-Asp (O'Bu) -Val-Tyr-OH are obtained. Mp: 168-170 ° C, Rf 0.30, [α] ?? = - 2.7 ° (c = 0.74, dimethylformamide, [a] ^D: -1-21,7 (c? = 1, acetic acid).

The above free tripeptide was treated with Z-Lys (Boc) -OSu (26.8 g, 56.1 mmol) in anhydrous dioxane (200 mL) and triethylamine (7.87 mL, 56.1 mmol). The next day, the reaction mixture was concentrated in vacuo and the residue was dissolved in chloroform (500 mL) and washed with 1M HCl (3 x 100 mL) and water (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was solidified with anhydrous ether. 35.2 g (71.3%) of Z-Lys (Boc) -Asp (O'Bu) -Val-Tyr-OH are obtained. Mp 152-157 ° C, Rf 0.70, [α] 25 D -10.3 ° (c = 0.99 acetic acid).

Step 5

N-tert-Butyloxycarbonyl-L-arginyl-6-hydrochloride-N-E-tert-butyloxycarbonyl-L-lysyl-L-asparagil-β-tert-butyl ester-L-vaid-L-tyrosine

30.5 g (37.5 mmol) of Z-Lys (Boc) -Asp (O'Bu) -Val-Tyr-OH (Step 4) in 300 mL of acetic acid were treated with 3.0 g of 10% palladium on activated carbon. hydrogenation with bubbling in the presence of a catalyst. After 2 hours the reaction was complete, the catalyst was filtered off, the filtrate was concentrated in vacuo and the residue was crystallized with 100 ml of water. The suspension was kept in the refrigerator for the next day and then filtered.

(23.1 g H-Lys (Boc) -Asp (Bu ^l) -Val-Tyr-OH was obtained mp: 195-197 ° C, Rf 0.10, [a] r - + I.? 8 ⁰ (c = l, acetic acid), [0 ^ = - 7.6 ° (c = 0.99, dimethylformamide).

Boc-Arg (OH-HClH ₂ O, 11.15 g, 34 mmol) was dissolved in dimethylformamide (100 mL), triethylamine (4.76 mL, 34 mmol) was added and the mixture was brought to -10 ° C. cool and add 4.13 ml (34 mmol) of pivaloyl chloride at -10 [deg.] C. The mixed anhydride was stirred for 10 minutes at -10 [deg.] C. and then 20.4 g (30 mmol) of H- was added dropwise at this temperature. A solution of Lys (Boc) -Asp (O'Bu) -Val-Tyr-OH in 100 ml of dimethylformamide was added, the reaction mixture was stirred for 30 minutes at 0 ° C and allowed to stand for 4 hours at room temperature. of chloroform / methanol (100 ml) and washed three times with 50 ml of cold 0.1M hydrochloric acid.

183,579 turns The hydrochloric acid phases were combined and extracted three times with 50 ml of chloroform each time. The latter chloroform phases were combined and washed with 20 ml of cold 0.1 M hydrochloric acid. The combined chloroform layers were washed with 100 mL of saturated water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was dissolved in 100 ml of chloroform / 100 ml of methanol and then precipitated with 300 ml of ether. 17.0 g (52.5%) of Boc-Arg (HCl) -Lys (Boc) -Asp (O'Bu) -Val-Tyr-OH are obtained. M.p no amorphous substance, Rf 0.30, [^ $ - 17.0 ⁰ (c '0.99, acetic acid). It has a content of 101% by UV-spectrophotometric determination of tyrosine.

Step 6

L-Arginyl-L-lysyl-L-asparagyl-L-valyl-L-lyrosine acetate 5.0 g (5.132 mmol) of Boc-Arg (HCL) -Lys (Boc) -Asp (O'Bu) -Val- Tyr-OH (step 5) was treated with 60 mL of trifluoroacetic acid. After 30 minutes, the solution was diluted with 500 mL of cold anhydrous ether. The slurry was cooled to 0 ° C, filtered, and the precipitate washed thoroughly with anhydrous ether and dried in a desiccator over phosphorus pentoxide and sodium hydroxide in vacuo. The pentapeptide trifluoroacetate was dissolved in 100 mL of water and ion-exchanged with Dowex 2x8 resin in acetate acetate. The resinous suspension was filtered, the filtrate was concentrated in vacuo, the residue was solidified with ethanol, and the precipitate was filtered off and washed thoroughly with ethanol. 3.56 gf (93.2%) of Arg-Lys-Asp-Val-Tyr are obtained in the form of monoacetate. Amorphous material, Rf = 0.10, Rf = 0, [α] r> ⁰ = 28, r (c = 0.99, 10% aqueous acetic acid). Amino acid analysis: Lys 1.02 (1), Arg 1.02 (1), Asp 1.00 (1), Val 0.99 (1), Tyr 0.83 (1). The content determined by tyrosine by UV spectrophotometry is 100.2%. HPLC showed 99.3%.

Claims (1)

A process for the stepwise preparation of L-arginyl-L-lysyl-L-asparagyl-L-valyl-L-tyrosine pentapeptide in solution! chain extension, characterized in that in each coupling step, benzyloxycarbonyl as the amino protecting group of L-valine, L-as20 paraginic acid and L-lysine, the t-butyloxy group of L-aspartic acid, L-lysine ω and in the final coupling step the amino terminus of L-arginine The tert-butyloxycarbonyl group is used as its protecting group and the amino protecting group is removed after each coupling step except for the final step by catalytic hydrogenation and after the final coupling step all the protecting groups present are removed by treatment with trifluoroacetic acid.