RU2233289C2 - Method for preparing dipeptides - Google Patents

Abstract

FIELD: organic chemistry, chemical technology, biochemistry, medicine.

SUBSTANCE: invention relates to a method for preparing dipeptides eliciting biological activity, in particular, anti-tumor activity that provides their applying in medicinal practice. Invention proposes a method for preparing dipeptides by interaction of N-protected amino acid with esterified amino acid derivative with free amino-group in organic solvent medium in the presence of condensing agent and triethylamine. N-acetyl-L-phenylalanine or N-acetyl-4-[di-(2-chloroethyl)amino]-DL-phenylalanine is used as N-substituted amino acid; L-alanine or L-phenylalanine, or L- and DL-valine methyl or ethyl, or benzyl, or tert.-butyl ester are used as esterified derivative of amino acid; isobutylchlorocarbonate is used as a condensing agent. Triethylamine is taken with 20% excess as regards with stoichiometry value. The proposed method provides preparing dipeptides enriched with DL- or DL + LD-diasteteromers that, therefore, promotes to expanding assortment of preparing peptides used both in scientific-research aims and medicinal practice.

EFFECT: improved preparing method.

1 tbl, 2 ex

Description

The invention relates to the field of organic chemistry, and in particular to a method for producing peptides with biological activity, in particular antitumor activity, which allows their use in medical practice.

A known method of producing derivatives of peptides in solution in the presence of a condensing agent (RF patent No. 2047621, C 07 K 7/02, 1995). In the known method, derivatives of amino acids with a free carboxyl group are activated by dissolving in dimethylformamide (DMF) and adding a solution of diisopropylcarbodiimide in DMF. Then the esters of other amino acids are dissolved in N-methylpyridine and combined with a previously deblocked 20% solution of piperidine in dimethylformamide resin. Then the peptide is cleaved from the resin while the protective groups of the side chains are removed.

Thus, in the known method, it is necessary to attach the first amino acid to the resin, to which the next amino acid or a fragment with a free amino group is further attached after activation, which complicates the method by increasing its staging. The known method allows to obtain derivatives of peptides with the ability to inhibit the action of retroviral proteases. In this case, we are talking about individual stereoisomers of peptides, and in the case of obtaining a mixture of stereoisomers, in particular a mixture of diastereomers, they are separated by known methods by fractional crystallization or by chromatography. But in medical practice, the use of dipeptides enriched in DL or D, L + L, D-diastereomers is often necessary.

A known method of producing dipeptides in solution in the presence of a condensing agent and triethylamine (TEA) (Chemical-Pharmaceutical Journal, Medicine, 1970, No. 10, pp. 25-27). In the known method, di-peptides of 4-di (2-chloroethyl) amino-DL-phenylalanine (sarcolisin) are obtained by two-stage synthesis, in the first stage, when N-acetylsarcolisin is reacted with isovaleric acid chloride in the presence of TEA in chloroform medium, mixed N-acetylsarcoline anhydride is obtained isovaleric acid, in the second stage, without isolation of the anhydride, it is reacted with an ester of DL- or L-amino acids. Along with other dipeptides, N-acetylsarcolysin-DL-valine ethyl ester (asaline) was obtained, which is successfully used in medical practice in the treatment of some forms of malignant neoplasms.

In a known manner, N-acetylsarcolysin dipeptides can be prepared. To search for new drugs and conduct scientific research, peptides of various amino acid derivatives enriched with the desired diastereomer are needed.

Thus, the authors were faced with the task of developing a method to expand the range of obtained dipeptides, in particular, not only N-acetyl-sarcolisin dipeptides, but also N-acetyl-L-phenylalanine dipeptides, enriched with DL- or D, L + L, D-diastereomers.

The problem is solved in the proposed method for the preparation of dipeptides by the interaction of an N-protected amino acid with an esterified amino acid derivative with a free amino group in an organic solvent in the presence of a condensing agent and triethylamine, in which N-acetyl-L-phenylalanine or N-acetyl is used as an N-protected amino acid -4 [di- (2-chloroethyl) amino] -DL-phenylalanine, as an esterified derivative of an amino acid, methyl, or ethyl, or benzyl, or tert-butyl ether of L-alanine, or L-phenyl Lanina or L- and DL-valine, and a condensing agent - isobutyl chlorocarbonate, with triethylamine present to 20% excess relative to stoichiometry.

Currently, there is no known method for the preparation of dipeptides enriched with DL- or DL + LD-diastereomers, which can be characterized by the proposed combination of distinctive features, in particular, by the initial ones, the interaction of which is carried out in the presence of isobutylchlorocarbonate and TEA taken with a 20% excess of stoichiometry.

The proposed method allows to obtain dipeptides enriched in DL or DL + LD diastereomers by reacting an N-protected amino acid in an organic solvent by the mixed anhydride method, which is accompanied by the formation of enantiomeric oxazolones. These oxazolones present in racemic mixtures show pronounced differences in reaction rates with chiral amino acid esters. The implementation of the method by using the property of mixed anhydrides to form racemic oxazolones, which not only react with amino acids at different rates, but also turn into each other, allows to obtain a wide range of dipeptides enriched in DL or DL + LD diastereomers. The use of isobutylchlorocarbonate as a condensing agent allows the use of both N-acetyl-L-phenylalanine and N-acetyl-4 [di- (2-chloroethyl) amino] -DL- as the starting N-protected amino acid. phenylalanine, thereby expanding the range of resulting peptides. Carrying out the process in the presence of a 20% excess to the stoichiometry of TEA allows one to obtain, after the initial treatment of the reaction mass, the ratio of diastereomers is much more than 50:50. However, with a lower or higher content of triethylamine, it is not possible to achieve the desired ratio.

The proposed method is as follows. N-acetyl-L-phenylalanine or its derivative N-acetyl-4 [di- (2-chloroethyl) amino] -DL-phenylalanine is dissolved in tetrahydrofuran (THF), the resulting solution is cooled to -13 ° C or to 0 ° C ( in the case of a phenylalanine derivative) and TEA is added dropwise with an excess of 20% to stoichiometry; then isobutyl chlorocarbonate is added at the same temperature. The reaction mass is maintained for 30 minutes with stirring at a temperature of -13 ° C or 0 ° C (in the case of a phenylalanine derivative) and L-alanine or L-phenylalanine or L-and DL-valine ester hydrochloride is neutralized in an THF solution, neutralized with an equimolar amount TEA and pre-chilled in the refrigerator. The reaction mass is stirred for 1 hour at a temperature of -13 ° C, 1 hour at a temperature of 0 ° C and then 3 hours at room temperature. Then left overnight without stirring. In the case of a phenylalanine derivative, the reaction mass is stirred for 2 hours at 0 ° C, then 3 hours at room temperature and left overnight without stirring. The precipitate was filtered off, the filtrate was evaporated to dryness in vacuo. The residue was dissolved in chloroform and washed with 5% NaHCO ₃ solution, water, 5% hydrochloric acid solution and again with water, then dried over Na ₂ SO ₄ . After distilling off the solvent and drying the residue in vacuo, a mixture of D, L and L, L-diastereomers is obtained, in which the D, L-diastereomer predominates (see table).

The proposed method is illustrated by the following examples

Example 1. N-acetyl-L- and N-acetyl-O-phenylalanyl-L-valine methyl esters

To a solution of 0.75 g (3.62 mmol) of N-acetyl-L-phenylalanine in 17 ml of THF cooled to -13 ° C, 0.61 ml (4.34 mmol) of TEA is added dropwise with stirring, which is 20% excess to stoichiometry; and 0.48 ml (3.62 mmol) of isobutyl chlorocarbonate. The reaction mass is stirred for 30 min at a temperature of -13 ° C, a mixture cooled to a temperature of -10 ° C is added, obtained by stirring 0.61 g (3.62 mmol) of L-valine methyl ester hydrochloride and 0.51 ml (3.62 mmol) ) TEA, 3 ml DMF in 9 ml THF. The reaction mass is kept under stirring at a temperature of -13 ° C for 1 hour, then at a temperature of 0 ° C for 1 hour, then at room temperature for 3 hours, after which it is left at room temperature for 17 hours. The precipitate was filtered off, THF was distilled off in vacuo. The filtrate was dissolved in chloroform, washed with 5% NaHCO ₃ , water, 5% HCl, then water to pH 7 and dried over Na ₂ SO ₄ . The chloroform solution was evaporated to dryness in vacuo. After drying in vacuo over P ₂ O ₅ , the compound is obtained in the form of a colorless crystalline powder.

0.88 g (75.9%) of a mixture of diastereomers are obtained. According to HPLC, 23.5% of the L, L-diastereoisomer and 76.5% of the D, L-diastereoisomer. R _f 0.62 (benzene-acetic acid-methanol 11: 1: 2). Found,%: C 63.83; H 7.60; N, 8.73. C ₇ H ₂₄ N ₂ O ₄ . Calculated,%: C 63.73; H 7.55; N, 8.75. UV spectrum (λ, nm): 261.2 (benzene ring). IR spectrum (ν, cm ^-1 ): 3280 (val. Col. NH); 3070 (Val. Col. CH aromat.); 1723 (C = 0 ester); 1625 (amide lane I); 1525 (def. Col. NH, amide. Lane II).

¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.73 d (CH ₃ -5 'D, L-isomer, I _d = 6.75 Hz); 0.75 d (CH ₃ -4 'D, L, I _d = 6.5 Hz); 0.83 d and 0.87 d (2 × CH ₃ -4 ', 5', L, L-isomer, I _d = 6.8 Hz); 1.98 s (CH ₃ —CO, D, L + L, L); 2.01-2.14 m (CH3 ', D, L + L, L); 3.03 dd and 3.11 dd (CH ₂ -3, AV system D, L, I _AB = 13.7 Hz; I _3A-2 = 6.2 Hz; I _3B-2 = 8.2 Hz); 3.05 d (CH ₂ -3, L, L isom., I _3-2 = 7.2 Hz); 4.40dd (CH-2 ', D, L-isomer; I _2'-1 = 8.4 Hz, I _2'-3' = 5.0 Hz); 4.41 dd (CH-2 ', LL-isom., I _2'-1' = 8.4 Hz, I _{2'-3 '} = 5.0 Hz), 4.71 dt (CH-2, L, L -isomers, I _2-1 = 7.8 Hz; I _2-3 = 7.2 Hz); 4.76 dd (CH-2, L, L-isomer, I _2-1 = 8.1 Hz; I _2-3B -8.2 Hz; I _2-3A = 6.3 Hz); 6.23 d (NH-1, D, L-isomer; I _1-2 = 8.1 Hz); 6.28 d (NH-1, L, L-isomer, I _1-2 = 7.8 Hz); 6.37 d (NH-1 ', D, L + L, L, I _1'-2' = 8.4 Hz); 7.12-7.32 m (Ph from D, L- and L, L-isomers).

Example 2. Ethyl esters of N-acetyl-DL-sarcolysyl-DL-valine

1.5 g (4.3 mmol) of N-acetylsarcolysin is dissolved in 36 ml of THF. The solution was cooled to a temperature of -2 - 0 ° C and 0.73 ml (5.2 mmol) of TEA was added dropwise, which is 20% excess to stoichiometry, maintaining the temperature at about 0 ° C, then 0.57 ml was added dropwise ( 4.3 mmol) of isobutyl chlorocarbonate. The reaction mass is maintained for 30 minutes at a temperature of 0 ° C and with stirring, then 0.79 g (4.3 mmol) of ethyl ester hydrochloride D, L-valine, pre-cooled to a temperature of 0 ° C and neutralized with 0.61 ml, are added (4, 3 mmol) TEA. The reaction mass is stirred for 2 hours at a temperature of 0 ° C, then 3 hours at room temperature and left overnight without stirring. The precipitate was filtered off, the filtrate was evaporated to dryness in vacuo. The residue was dissolved in chloroform and washed with 5% NaHCO ₃ solution, water with 5% HCl solution, then with water to pH 7 and dried over Na ₂ SO ₄ . The chloroform solution was evaporated to dryness in vacuo. After drying in vacuo over P ₂ O ₅ , the compound is obtained as a colorless crystalline powder.

1.67 g (81.5%) of the dipeptide are obtained with the ratio of diastereomers (L, D + D, L) :( L, L + D, D) = 77: 23 according to HPLC data. After drying in vacuo over P ₂ O ₅ , a yellowish substance is obtained. R _f 0.5 (benzene-acetic acid: methanol 11: 1: 2). Found,%: C 55.26; H 6.88; N, 8.49; Cl 15.26; C ₂₂ H ₃₃ N ₃ O ₄ Cl ₂ . Calculated,%: C 55.69; H 7.01; N, 8.86; Cl 14.95. UV spectrum (λ, nm): 260. IR spectrum (ν, cm ^-1 ): 3280 (NH), 3070 (val.col. arom. Rings), 1735 (val. Col. C = 0 in ester ); 1635 (amide lane I); 1540 (amide lane II).

¹ H NMR (COCO ₃ , δ, ppm): 0.76 d and 0.78 d (6H, 2 × CH ₃ Val; I _d = 6.7 Hz); 1.25 t (3H, CH _{3 eff} , I _T = 7.2 Hz); 1.99 s (3H, CH ₃ CO); 2.03sp.d (1H, CH (3 '), I _cp = 6.7 Hz, I _d = 4.7 Hz); 2.92 d. and 3.02 d. (AV system, CH ₂ (3); I _AB = 13.9 Hz; I _B-2 = 8.1 Hz; I _A-2 = 6.0 Hz); 3.60 m and 3.7 m (8H, N (CH ₂ CH ₂ Cl ₂ )); 4.14d. and 4.18dq. (AB system, ОCH _2eff , I _AB = 10.8 Hz, I _quarter = 7.2 Hz); 4.39 dd (1H, CH (2 '), I _2'-1' = 8.6 Hz; I _{2'-3 '} = 4.7 Hz); 4.69 dd (1H, CH (2), I _2-in = 8.1 Hz, I _2-1 = 7.9 Hz, I _2-A = 6.0 Hz); 6.22d (1H, NH, I _1-2 = 7.9 Hz); 6.35d (1H, NH, I _{1'-2 '} = 8.6 Hz); 6.61d (2H, 2 × CH _arom., I _d = 8.8 Hz); 7.11d (2H, 2 × CH _arom., I _d = 8.8 Hz).

Thus, the proposed method allows to obtain dipeptides enriched in DL- or DL + LD-diastereomers, and therefore, allows to expand the range of the resulting peptides used both for research purposes and in medical practice.

Claims (1)

A method for producing dipeptides by reacting an N-protected amino acid with an esterified amino acid derivative in an organic solvent in the presence of a condensing agent and triethylamine, characterized in that N-acetyl-L-phenylalanine or N-acetyl-4 [di- ( 2-chloroethyl) amino] -DL-phenylalanine, as an esterified derivative of an amino acid, methyl, or ethyl, or benzyl, or tert-butyl ether of L-alanine, or L-phenylalanine, or L- and DL-valine, and as condensing agent and - isobutyl chlorocarbonate, with triethylamine present to 20% excess relative to stoichiometry.