RU1792418C - Method of somatostation analogue synthesis - Google Patents

Description

The invention relates to an improved method for the preparation of a comapostatin analog, a heptapeptide of formula I

, CH (CH3) -OH: hD-Phe-ujs-Pht-D-Trp-bjs -Thi-Cys-NH-CH

SNg-OH

which may find application in medicine.

A known method for the preparation of compound I by fragment condensation in solution.

A disadvantage of the known method is the complexity of the process, the need to isolate intermediate products per kg of each stage and the impossibility of its automation.

The purpose of the invention is to simplify the process of obtaining compound I.

The goal is achieved by the described method, which consists in the fact that the synthesis of compound 1 is carried out by the solid phase method by stepwise peptide chain growth; starting with a C-terminal cysteine linked to a polymer matrix of the formula

(CHJK1

-sng-o-

where P is aminomethylated polystyrene using the corresponding N-fluorenyloxycarbonylaminocell in the presence of a condensing agent c. subsequent processing of the obtained protected heptidylpolymer of the formula:

Fmoc-D-Phe-CystBu -Phe-D-Trp- -1y8 (Boc) -Tn-Suz (Vi1) -X (|||),

S

with

&

where Fmoc is fluorenyloxycarbonyl, Boc-tert-butyloxycarbonyl, Vi-tert-butyl, Xx-mn-sn-sn (sn5) -o.

ng-o-sn © ..

deblocking agents, an oxidizing agent and trifluoroacetic acid in an organic solvent medium.

Step 1. Preparation of threoninol-acetal p-formylphenoxyacetic acid (Na).

To 105 g (1 ml) of L-threoninol in 200 ml of methanol in a nitrogen atmosphere, 200 ml of trifluoroacetic acid methyl ester in 250 ml of methanol are pinned at 0 ° C. The reaction mixture was stirred for 1.5 hours at TO ° C and evaporated at 40 ° C. The resulting white crystalline residue was dissolved in 200 ml of ethyl acetate at 70 ° C and reprecipitated by adding 100 ml of hexane. The precipitate was filtered off at 0 ° C, washed with hexane and dried at room temperature, 50.3 g (0.25 mol) of the obtained product were dissolved in nitrogen atmosphere in 1250 ml of tetrahydrofuran, and 75 ml of tri- methylchlorosilane and 70 ml of triethylamine in 250 ml of tetrahydrofuran. After 4 hours, the precipitate formed is filtered off, the filtrate is evaporated at 40 ° C, the residue is dissolved in 1500. ml of methylene chloride. The resulting solution was mixed in portions with 90.4 g of p-formyl-phenoxyacetic acid, and then 9 ml of trifluoromethanesulfonic acid trimethylsilyl ether was added. The reaction mixture was stirred for 24 hours, the precipitate formed was filtered off, washed thoroughly with methylene chloride, the filtrate was evaporated at 40 ° C, and the residue was purified by silica gel chromatography using ethyl acetate as eluent. The purity of the obtained compound B was determined by high pressure liquid chromatography.

Step 2. Preparation of Compound II

17.2 g of aminomethylated polystyrene (0.7 wt.%, 11, which corresponds to 0.50 mmol / g) are suspended in 80 ml of a mixture of methylene chloride with dimethylformamide (4: 1). 4.17 g of Ha, 1.6 goxybenzotriazole and 4 g of dicyclohexylcarbodiimide are successively added to the resulting suspension, stirred for 2 hours at room temperature, the resin is filtered off, washed and suspended in 100 ml of a mixture of tetrahydrofuran with methanol (3: 1). The resulting suspension was mixed in portions with 10.4 g of sodium borohydrate, stirred for 6 hours at room temperature, filtered and washed.

Stage 3. Obtaining heptapeptide 1.

The resin obtained in step 2 is doped in a mixture of methylene chloride with dimethylformamide and 5.57 g of N-fluorenyloxycarbonyl, 5-tert.-butyl-L-cysteine, 1.74 goxybenzotriazole and 3.6 g of dicyclohexylcarbodiimide are successively added. After cleavage of the fluorenyloxycarbonyl protecting group by treating the resin with piperidine (2x20 min), then 1-fluorenyloxycarbonyl-1-threonine, N-fluorenoyloxy-carbonyl-1-N-benzyloxy-carbonyl-1-N-benzyloxycarbonyl-1-threonyloxy-carbonyl-1-threonyloxy-carbonyl-1-threonyloxy-carbonyl-1-threonyloxycarbonyl-1-threonyloxy-carbonyl-1-threonyloxycarbonyl protecting groups are subsequently removed by treating the resin with piperidine (2x20 min). nyl-0-tryptophan, N-fluorenyloxycarbonyl-1-phenylalanine, N-fluorenyloxycarbonyl, 3-tert, -butyl-1-cysteine, and N-fluorenyloxycarbonyl-0-phenylalanine. The result is a peptidyl polymer of the formula III.

The resulting peptidyl polymer is suspended in 100 ml of a mixture of trifluoroethanol with methylene chloride (1: 1) and mixed with 50 ml of tributylphosphine. The suspension is stirred for 70 hours at room temperature, the resin is filtered off and mixed with 100 ml of a mixture of tetrahydrofuran and a 1 M solution of ammonium acetate (1: 1). To the resulting mixture was added 1.1 ml of 30% hydrogen peroxide and stirred for 24 hours at room temperature. Then the resin is washed and mixed with a mixture of 20 ml of trifluoroacetic acid, 80 ml of methylene chloride, 10 ml of water and 2 ml of thioanisole and stirred for 2 hours. Then the resin is filtered off, washed with trifluoroacetic acid and methylene chloride, 200 ml of diethyl ether are added to the filtrate, and the precipitate is filtered off. dissolved in aqueous buffer. The resulting solution was desalted by treatment with Duolite and subjected to freeze drying.

The result is 2.1 g (70%) of compound 1; a | P -42 ° (C 0, €: 95% AcOH).

As can be seen from the above example, the proposed method for the production of heptapeptide I can significantly simplify the process of obtaining the target product by eliminating the stages of isolation and purification of intermediate compounds and due to the simple procedure of isolation of the final product by filtration and desalting. In addition, the proposed method can be automated during the process on a peptide synthesizer.

Claims (1)

SUMMARY OF THE INVENTION Method for preparing a somatostatin analog of the formula H-D-frne-Cas-Pbe-D-Trp-Lys ... CH (CH3) -OH -Thc-tijs-NH-CH Chj-oh by peptide chain extension using N-protected amino acids, characterized in that, in order to simplify the process, the synthesis is carried out by stepwise peptide chain extension, starting with the C-terminal cysteine linked to the polymer matrix of the formula HjN YISSHIN 0®. sng-sg where P is aminomethylated polystyrene using the corresponding M-fluorenyloxycarbonylamino acids in the presence of a condensing agent followed by treatment of the obtained protected peptidyl polymer of the formula Fmoc-D-Phe-CysCBu j-Pne-D-Trp-LysCBocKThr-CystBuVx. where Fmoc is fluorenyloxycarbonyl; Voe - tert.-butyloxycarbonyl; Whig-tert.-butyl; x- -NH-cH-crtcHjVo . Chsng-o-sn-O-®, successively with releasing agents 15, an oxidizing agent and trifluoroacetic acid in an organic solvent medium.