CS235151B1 - Method of separating Dal isomers of oxytocin analogs - Google Patents

Abstract

The subject of the invention is a method for separating D and L isomers of oxytocin analogues containing an unnatural amino acid in position 2. Analogs of this type are the subject of several applications. copyright certificates OA number 216 722, AO number 226 923) and have a significant natriuretic effect if they contain an amino acid of configuration L in position 2, or they are effective inhibitors of the uterotonic effect of oxytocin if the amino acid in position 2 is of configuration D. These analogues are currently prepared by gradual construction of the peptide chain followed by cyclization, either through the stage of the active ester of the ω-carboxyl of the modified cysteine residue (in carba-analogs), which reacts with the α-amino group of the amino acid in position 2 to form a peptide bond, or (in the case of analogues containing a disulfide bridge) by oxidation of sulfhydryl groups formed after removal of the protecting groups from the sulfur of cysteine. So far, the preparation of these analogues has been carried out with an optically pure unnatural amino acid, which had to be obtained by resolution, e.g. using enzymes [see e.g. Zhuze A. L. et al.: Collect. Czech. Chem. Comtnun. 29, 2648 (1964), AO No. 228,431], This disadvantage is eliminated by the method according to the invention, the essence of which is that the D and L isomers of the oxytocin analogues of the general formula I

Description

The present invention provides a method for separating the D and L isomers of oxytocin analogs containing an unnatural amino acid at position 2.

Analogs of this type are the subject of several aces. No. 216,722, AO No. 226,923) and have a significant natriuretic effect if they contain an L-configuration amino acid at position 2, or are potent inhibitors of the uterotonic effect of oxytocin if the amino acid is at position 2 of the D configuration.

Meanwhile, these analogues are prepared by sequential construction of the peptide chain by subsequent cyclization, either through the active ω-carboxy ester stage of the modified cysteine residue (for the carba-analogues), which reaR * ^1.

R-C 1

R

-. 1 + I? - CH - CO - HH - CH - CO - H & - Gpi - n - NH - CH - CO - Pro - L au - Gly - NH

It reacts with the α-amino group of the amino acid at position 2 to form a peptide bond, or (in the case of disulfide-containing analogs) by oxidation of the sulfhydryl groups formed after deprotection of cysteine sulfur. obtained by resolution, eg using enzymes [see, eg, Zhuze AL et al .: Collect. Czech. Chem. Comtnun. 29, 2648 (1964), AO No. 228 431],

This disadvantage is overcome by the process according to the invention, which is based on the fact that the D and L isomers of oxytocin analogs of the formula I

CH, (I)

where

R ¹ and R ² is H or CH 3, R ³ is H or NH ² , R ⁴ is H, OH, alkyl of 1 to 3 carbons or alkoxy of one to two carbons, R ⁵ is S-S, S-CH 2 or CH2-S and the amino group configuration at the 2 ^- position marked ⁺ is D or L, separated by reversed-phase high-pressure silica gel column chromatography with an octadecyl chain anchors in methanol or tetrahydrofuran as organic solvent and 0,1% by volume trifluoroacetic acid or 0, 05 M ammonium acetate pH 7 as aqueous buffer.

The separation of diastereoisomers is further illustrated in the following examples. The composition of the mobile phase is expressed in percent by volume.

Example 1 (2-D- and L-phenylalanine) amino-6-carbo-oxytocin o-Nitrobenzenesulfenyl-D, L-phenylalanyl-isoleucyl-glutaminyl-asparaginyl-S- (ω-carboxyethyl) homocysteinyl-prolyl-leucyl-glycinamide (200 A portion of the diastereoisomeric peptide mixture (100 mg) was dissolved in 4 mL of methanol and 7 mL of water, filtered and loaded onto a column of octadecyl chain-anchored silica gel. (Partisil ODS-2) 50 cm 0.9 cm eluted with 56% methanol and 44% 0.1% trifluoroacetic acid to give two fractions containing the peptide material, which were concentrated in vacuo and lyophilized. 12.3 mg of mesh ' ⁼ 14.9, which was identical to the analogue standard containing L-phenylalanine (OA No. 216 722) and 13 mg of mesh' = 6.5, which was identical to the standard of D- phenylalanine (AO No. 226 923).

Example 2 (2-D- and LO-ethyltyrosine) deamino-6-carbaoxytocin o-Nitrobenzylsulfenyl-D, 10-ethyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (β-carboxyethyl) homocysteinyl-propyl-leucyl-glycinamide (100 mg) was cyclized as in Example 1 and the diastereoisomers were separated on the same column using a mobile phase containing 55% methanol and 45% 0.1% trifluoroacetic acid. After concentration and lyophilization, 10.7 mg of a substance containing L-O-ethyltyrosine (k ‘= 8.0) and 11.6 mg of a substance containing D-O-ethyltyrosine (k‘ = 10.1) were obtained. Both analogs were consistent with standards.

Example 3 (2-D- and Lp-methylphenylaniline) deamino-6-carbo-oxytocin o-Nitrobenzylsulfenyl-D, Lp-methylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S- (β-carboxyethyl) homocysteinyl-prolyl-leucyl- glycinamide (200 mg) was cyclized as in Example 1 and a portion of the product was separated on the same column using a mobile phase containing 60% methanol and 40% water. After concentration and lyophilization, 12.6 mg of a substance containing L-p-methylphenylalanine (k ‘5.7) and 13.7 mg of an analogue containing D-p-methylphenylalanine (k‘ = 7.6) were obtained. Both substances were identical to standards.

EXAMPLE 4 (2-D- and Lp-ethylphenylalanine) Deamino-6-Carboxytocin o-Nitrobenzenesulfenyl-D, Lp-ethylphenylalanyl-isoleucyl-glutaminylasparaginyl-S- (N / 3-carboxyethyl) homocysteinyl-prolyl- leucylglycinamide (200 mg) was cyclized as in Example 1 and part of the diastereoisomers (100 mg) were separated on the same column using a mobile phase containing 70% methanol and 30% 0.1% trifluoroacetic acid. After concentration and lyophilization, 18.3 mg of a substance containing L-p-ethylphenylalanine (k ‘= 6.18) and 17.6 mg of a substance containing D-p-ethylphenylalanine (k‘ = 8.48) were obtained. Both analogs were identical to standards.

Example 5 (2-D- and Lp-methylphenylalanine) deamino-1-carbo-oxytocin o-Nitrobenzenesulfenyl-D, L-tyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (χ-carboxypropyl) cysteinyl-prolyl-leucyl-glycinamide ( 100 mg) was cyclized as in Example 1 and the diastereoisomers were separated on the same column using a mobile phase containing 12% tetrahydrofuran and 88% 0.5 M ammonium acetate pH 7. After concentration and lyophilization, 12.1 mg was obtained. containing L-tyrosine (k '= 17.2) and 11 milligrams of D-tyrosine-containing substance. Both substances were identical to standards [Jošt K .: Collect. Czech. Chem. Commun. 38, 218 (1971) and Lehl M .: unpublished results).

Example 6 (2-D- and L-p-ethylphenylalanine) deamino-oxytocin

From S-benzylmercaptopropionyl-D, Lp-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (100 mg), the protecting groups were cleaved with sodium in liquid ammonia and the oxidation cyclization was carried out in the usual manner (A0). 923). The diastereoisomers were separated on the same column as in Example 1 using a mobile phase containing 65% methanol and 35% 0.1% trifluoroacetic acid. After concentrating and freeze-drying afforded 16.2 mg of material containing the Lp-ethylphenylalanine (k '= 10.2) and 15.3 mg of material containing Dp-ethylphenylalanine (k' = ^'1 = 15.9). Both compounds were identical to analogs prepared using optically pure amino acid.

Example 7 (2-D- and L-p-ethylphenylalanine) oxytocin

N -benzyloxycarbonyl-S-benzylcysteinyl-D, Lp-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (100 mg) was cyclized as in Example 6 and the diastereoisomers were separated on the same column using mobile phase containing 60 1% methanol / 40% 0.05 M ammonium acetate, pH 7. After concentration and lyophilization, 8.2 mg of a substance containing Dp-ethylphenylalanine (k '= 11.6) and 7.9 mg were obtained compounds containing Dp-ethylphenylalanine (k '= 23.3 µ). Both were identical to standards. [Zhuze AL et al .: Collect. Czech. Chem. Commun. 29, (1964)].

Example 8 (1-Penicillamine-, 2-D and L-p-ethylphenylalanine joxytocin)

N-Benzyloxycarbonyl-S-benzylpenicilaminyl-D, Lp-ethylphenylalaninosolcucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (200 mg) was cyclized as in Example 6 and part (100 mg) of the diastereoisomers was separated on the same column using a mobile phase containing 65 percent methanol and 35% 0.05 M ammonium acetate pH 7. After concentration and lyophilization, 7.2 mg of a substance containing Lp-ethylphenylalanine (k '= 8.05 J and 7.4 mg of a substance containing Dp-ethylphenylalanine (k '= 13.8) Both were identical to analogs prepared using optically pure amino acids.

Claims (3)

OBJECT OF THE INVENTION A process for separating the D and L isomers of oxytocin analogs of formula I R < 2, * ^{R R} ~~ C --— --_____ _CH 3 * + | * R · - CH-CO-NH-CH-CO-lis, -Gto-Asn-NH-CH-CO-PrO-L & u-Glv-NH R ¹ * (I) wherein R ¹ and R ² is H or CH 3, R ³ is H or NH2, ^R4 is H, OH, alkyl of 1-3 carbons, or alkoxy of one to two carbons, R ⁵ is S-S, S-CH2 or CH2 —S and the amino group configuration at position 2 is designated + is D or L, wherein the mixture of analogs of formula I having a racemic amino acid at position 2 is separated by reverse-phase high-pressure chromatography on an octadecyl chain-anchored silica gel column in methanol or tetrahydrofuran as an organic solvent and 0.1 vol% trifluoroacetic acid or 0.05 M ammonium acetate pH 7 as an aqueous buffer.