alsactide

Alsactide,ZYF Pharm Chemical

Abbreviations:
Boc - tert-butoxycarbonyl; TCP - 2,4,5-trichlorophenyl; Z - carbobenzyloxy.
(a) β-Ala-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys- NH-(CH2)4-NH2-acetate, aq.
Z-Lys(Boc)-NH-(CH2)4-NH-Boc: 7.0 g (31 mmols) of Boc-NH-(CH2)4-NH2-HCl and 4.2 ml (30 mmols) of triethylamine in 100 ml of dimethylformamide are stirred for 20 hours together with 16.8 g (30 mmols) of Z-Lys(Boc) OTCP. The product is filtered off the triethylammonium chloride and the filtrate is evaporated to dryness in vacuo. The residue is dissolved in ethyl acetate, thoroughly shaken at 0°C with 2 N citric acid, 1 N bicarbonate and H2O and dried over sodium sulfate. After distilling off the solvents, the residue is recrystallized two times from isopropanol/ether. Yield: 14.1 g (83%), M.P. 83°-85°C.
(b) H-Lys(Boc)-NH-(CH2)4-NH-Boc-tosylate: 11.4 g of the Z-compound prepared according to (a) are hydrogenated in the presence of Pd in 80 ml of methanol with, while adding methanolic toluene-sulfonic acid at pH 5. After the reaction is complete, the product is filtered to remove the catalyst, the methanol is distilled off in vacuo and the residue is triturated with ether. For purification purposes it is dissolved in warm isopropanol and precipitated with ether. Yield: 10.3 g (88%).
(c) Z-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Lys(Boc)-NH-(CH2)4-NH-Boc: 10.9 g (10 mmols) of Z-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-OH and 5.89 g (10 mmols) of Boc-NH-(CH2)4-NH2-tosylate in 100 ml of dimethylformamide are combined with 12.8 ml (10 mmols) of N-ethylmorpholine and 2.7 g (20 mmols) of 1-hydroxybenzotriazole. 2.2 g of dicyclohexylcarbodiimide (11 mmols) are added at -10°C. The whole is then allowed to come to room temperature. The stirring is continued for 3 hours, the solvent is distilled off in vacuo. The residue is digested with 1 N bicarbonate and water and, after drying, recrystallized from acetonitrile. Yield: 10.6 g (74.2%). M.P. 150°- 155°C (while foaming). [α]D 20: -24.0° (c=1 in dimethylformamide).
(d) H-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Lys(Boc)-NH-(CH2)4-NH-Boctosylate- dihydrate: 15.1 g (10 mmols) of the Z-compound prepared according to (c) are catalytically hydrogenated in the presence of Pd in 300 ml of methanol, a pH of 5 being maintained by simultaneously adding methanolic toluene-sulfonic acid. After complete reaction, the methanol is distilled off and the residue is reprecipitated from pyridine/ ether and methanol/water. The oil, which first precipitates becomes solid after a short time. Yield: 12.1 g (77.5%).
(e) 1.65 g (1.1 mmols) of Boc-β-Ala-Tyr-Ser-Met-Glu-(OBut)-His-Phe-Arg-Trp- Gly-OH·4H2O and 1.56 g (1 mmol) of H-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys- (Boc)-Lys(Boc)-NH-(CH2)4-NH-Boc-tosylate-dihydrate are dissolved together with 540 mg (4 mmols) of 1-hydroxybenzotriazole in 30 ml of dimethylformamide. A solution of 1.25 g (6 mmols) of dicyclohexylcarbodiimide in 4 ml of dimethylformamide is prepared and 1/3 of this solution is added to the above solution. The whole is stirred for 1 hour, then another third is added and, after a further hour, the last third is fed in. After a further 2 to 3 hours of stirring, the reaction product is precipitated with ether. Yield: 3.1 g.
Without further purification, the compound is freed from the protective groups by standing for 1 hour in 80-90% trifluoroacetic acid containing some thioglycolic acid, and is subsequently precipitated by adding 150 ml of ether. Yield: 3.06 g of crude peptide-trifluoroacetate. After purification on carboxymethyl cellulose, 1.45 g of the chromatographically pure peptide are obtained in the form of acetate. [α]D 20:-68.6°-2° (c=0.5 in 1% acetic acid). Amino acid analysis: Ser0.86Glu0.99Pro0.97Gly2.00Val1.03Met0.98Tyr0.02Phe1.00βAla1.01Lys4.00His1.02Arg0.94.

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