RU2703991C1 - Method of producing monosodium salt of isoglutamyl-tryptophan - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to organic synthesis. Method includes steps of synthesis of carbobenzoxy-D-glutamic acid oxazolidinone, its condensation with methyl tryptophan, removal of carbobenzoxy protection with opening of oxalidinone ring by catalytic hydrogenolysis and alkaline saponification of methyl ether to obtain monosodium salt of dipeptide, characterized by that at the stage of synthesis of oxazolidinone carbobenzoxy-D-glutamic acid using washing with aqueous solution of hydroxylamine hydrochloride, and the step of removing carbobenzoxy protection with simultaneous opening of the oxalidine ring is carried out by catalytic hydrogenolysis over a palladium catalyst using formic acid as a hydrogen donor and aniline taken in amount of 2 to 4 equivalents with respect to the amount of the protected dipeptide.

EFFECT: disclosed is a method of producing D-isoglutamyl-D or L-tryptophan in form of a monosodium salt.

1 cl, 3 ex

Description

The invention relates to organic synthesis, namely to the synthesis of dipeptides, and in particular to the production of various optical isomers of isoglutamyl tryptophan in the form of a monosodium salt.

The optical isomers of isoglutamyl tryptophan (H-iGlu-Trp-OH) include synthetic dipeptides that are promising in the treatment of autoimmune diseases. Some dipeptide isomers are manufactured by the pharmaceutical industry. Disodium salt of D-isoglutamyl-D-tryptophan is available in the form of a solution for intramuscular injection or in the form of a nasal spray under the trade name Timodepressin. Disodium salt of D-isoglutamyl-L-tryptophan is available in the form of nasal drops or spray, as well as in the form of a solution for intramuscular injection or lyophilisate to obtain this solution under the trade name Bestim.

A known method for producing peptides, including D-isoglutamyl-D-tryptophan [RU 2107692, IPC C07K 5/065, A61K 38/08, 1998, analogue US 5736519], according to which the dipeptide is synthesized by sequential reactions to obtain tert-butyloxycarbonyl glutamine acid (Boc-D-Glu-OH), the opening of the internal anhydride Boc-D-Glu-OH with the corresponding tryptophan derivatives (HD-Trp-Y) to form a mixture of Boc-D-iGlu-D-Trp-OH and Boc-D- Glu-D-Trp-OH, removal of the Boc protecting group (tert-butyloxycarbonyl) under the action of formic acid and subsequent chromatographic separation of the α and γ isomers in the resulting D-glutamyl-D-tryptophan.

The known method has several disadvantages. So, in the second stage of synthesis, a mixture of Boc-D-iGlu-D-Trp-OH and Boc-D-Glu-D-Trp-OH in the ratio 1: 1 is formed, therefore, the maximum yield of the target product (Boc-D-iGlu-D -Trp-OH) cannot exceed 50%. After removal of the Boc-protection, the mixture of dipeptides must be separated using ion exchange chromatography and / or preparative high-performance reverse phase chromatography, which significantly increases the cost of synthesis and reduces the yield of the final product.

Also known is a method of producing a stable crystalline monoammonium salt of HD-iGlu-D-Trp-OH [RU 2483077, IPC C07K 5/037, A61K 38/05, 2013], according to which the target product is synthesized by sequential reactions to obtain the tert-butyloxycarbonyl glutamine ester Boc-D-Glu-OR ¹ acids, where R ^{1 is} selected from the group consisting of C ₁ -C ₄ alkyl or benzyl, condensing it with a tryptophan derivative HD-Trp-OR ² , where R ² is C ₁ -C ₄ alkyl , with the formation of Boc-D-Glu- (γ-Trp-OR ² ) -α-OR ¹ , sequentially removing the protective groups R ¹ and R ² and Boc-protection and obtaining the target pr the product is HD-iGlu-D-Trp-OH, which is then converted to a crystalline ammonium salt.

The known method allows to obtain only one isomer of D-glutamyl-D-tryptophan, and therefore does not need a chromatographic separation step of a mixture of α and γ dipeptides, but requires more expensive starting materials and an additional step — removal of the protective groups R ¹ and R ² .

The closest in technical essence and the achieved effect to the claimed method is a method for producing peptides, in particular, γ-glutamic acid when used to temporarily protect the α-carboxyl group of the reaction of formation of the oxazolidinone ring [Masumi Itoh. Chem. Pharm. Bull. 17 (8), 1679-1686 (1969)]. The method includes the step of producing oxazolidinone glutamic acid by reacting paraformaldehyde and carbobenzoxy-glutamic acid (Z-Glu-OH) in benzene in the presence of p-toluenesulfonic acid as a catalyst. The resulting oxazolidinone is introduced into a condensation reaction with a benzyl ester of another amino acid, in particular glutamic acid, isoleucine and tyrosine, followed by the opening of the oxazolidinone ring and removal of the protective groups by alkaline saponification and / or catalytic hydrogenolysis with a palladium catalyst.

The specified method has the disadvantage that at the stage of producing oxazolidinone glutamic acid, the final product is formed in the form of an oil containing a significant amount of hard to separate formaldehyde. In the case of using catalytic hydrogenolysis to remove protective groups with the simultaneous opening of the oxazolidinone ring, the presence of formaldehyde leads to poisoning of the palladium catalyst, which greatly complicates the hydrogenation process. In addition, formaldehyde is able to interact with the resulting free amino group of glutamic acid to form a Schiff base. Subsequent catalytic reduction of the Schiff base results in the formation of the N-methyl-glutamic acid derivative.

The use of alkaline saponification to open the oxazolidinone ring and / or remove protective groups, in some cases leads to the formation of a complex mixture of by-products, along with the risk of racemization during long-term processing. These disadvantages make this method unacceptable for the industrial production of a product with a high degree of purity.

The object of this invention is to develop a method for the industrial production of isoglutamyl-tryptophan dipeptides in the form of monosodium salts.

This result is achieved in that in a method for producing isoglutamyl-tryptophan, which includes the steps of synthesizing oxazolidinone carbobenzoxy-glutamic acid, condensing it with tryptophan methyl ester, removing the carbobenzoxy protection with the opening of the oxalidinone ring using catalytic hydrogenolysis and alkaline saponification of the methyl ester with the formation of the methyl ester of methoxide in order to obtain , during the synthesis of oxazolidinone carbobenzoxy-glutamic acid, washing with an aqueous solution of hydroxylamine hydrochloride is used, and with The step of removing the carbobenzoxy protection with the simultaneous opening of the oxalidinone ring is carried out by catalytic hydrogenolysis over a palladium catalyst using formic acid as a donor of hydrogen and aniline, taken in an amount of 2 to 4 equivalents relative to the amount of protected dipeptide.

Washing of oxazolidinone carbobenzoxy-glutamic acid with an aqueous solution of hydroxylamine hydrochloride allows almost completely to remove formaldehyde residues from the product. The use of aniline in the catalytic hydrogenolysis step prevents the formation of by-products.

The alkaline saponification reaction of the obtained dipeptide methyl ester is carried out with 1 N NaOH for 30 minutes.

Purification of the final product by recrystallization from aqueous isopropyl alcohol allows to obtain the crystalline monosodium salt of isoglutamyl tryptophan with a purity of> 99% according to analytical high-performance reverse phase chromatography.

The method is further illustrated by examples of a specific implementation, but is not limited to them.

Example 1

Obtaining oxazolidinone Z-D-Glu

25 g (88.9 mmol) Z-D-Gl-OH, 5.33 g (177.8 mmol) paraforma, 1.24 g (6.55 mmol) para-toluenesulfonic acid, and 150 ml benzene were placed in a 500 ml round-bottom flask. The mixture was boiled for 2 h with a Dean-Stark nozzle, removing water formed during the reaction, then cooled to room temperature and washed twice with a benzene solution of 100 ml of a saturated solution of NaCl in a separatory funnel.

The organic layer was transferred to a 500 ml round bottom flask, 100 ml of a 10% hydroxylamine hydrochloride solution was added, and stirred vigorously on a magnetic stirrer (until emulsion formed) for 30 minutes. The aqueous layer was separated in a separatory funnel and discarded, and the organic phase was re-treated with 100 ml of a 10% solution of hydroxylamine hydrochloride for 30 min.

The aqueous layer was separated; the organic phase was washed with saturated NaCl solution, dried for 3 hours with anhydrous Na ₂ SO _4, and benzene was removed on a rotary evaporator. Received 28.5 g of light oil.

Getting oxazolidinone Z-D-iGlu-D-Trp-OMe

Oxazolidinone ZD-Glu was dissolved in a 500 ml round-bottom flask in 200 ml of dry ethyl acetate, cooled to -10 ° C in an ice and salt bath, and 12.76 ml (97.6 mmol) of isobutyl chloroformate and 10.73 ml (97.6 mmol) were added with vigorous stirring on a magnetic stirrer. ) N-methylmorpholine. The mixture was stirred for 3 min, and a precipitate of N-methylmorpholine hydrochloride precipitated, the temperature of the reaction mixture rose to + 12 ° С, then 24.86 g (97.6 mmol) of H-D-Trp-OMe⋅HCl and 10.73 ml (97.6 mmol) of N-methylmorpholine were added. The reaction mass was stirred while cooling for 30 minutes until the reaction was complete according to high performance reverse phase chromatography (HPLC).

The reaction mixture was transferred to a separatory funnel, 100 ml of water was added - the precipitate dissolved; the aqueous layer was discarded, the organic phase was washed 2 more times with 100 ml of water and 2 times with 100 ml of a saturated NaCl solution, dried for 3 hours with anhydrous Na ₂ SO ₄ and the ethyl acetate was removed on a rotary evaporator. Received 52 g of a light brown oil.

Obtaining H-D-iGlu-D-Trp-OMe

To the oxazolidinone Z-D-iGlu-D-Trp-OMe, 35 ml (392 mmol, 4 equiv.) Of aniline was added and dissolved in 400 ml of distilled isopropyl alcohol. The resulting solution was transferred into a 1000 ml round-bottom flask, and with vigorous stirring on a magnetic stirrer, 3.2 g of 5% Pd / C (palladium on charcoal) in 80 ml of water and 37 ml of 90% formic acid were added. According to HPLC, the reaction is complete in 1 hour.

The catalyst was filtered off, isopropyl alcohol was removed on a rotary evaporator, the residue was diluted with water to 250 ml and extracted with ethyl acetate 1 × 200 ml and 2 × 100 ml to remove aniline. The aqueous layer was evaporated to dryness on a rotary evaporator at 40 ° C - a crystalline precipitate formed. 250 ml of isopropyl alcohol was added to the precipitate and stirred for 1 h on a rotary evaporator, then left in the refrigerator overnight. The precipitate was filtered off, washed 3 times with 50 ml of isopropyl alcohol and dried in vacuum.

Received 18.5 g of a crystalline powder of light cream color. Yield 60% based on the starting Z-D-Glu-OH.

Obtaining H-D-iGlu-D-Trp-ONa

To a suspension of 17.3 g (50 mM) of H-D-iGlu-D-Trp-OMe in 50 ml of water was added with stirring on a magnetic stirrer 150 ml of 1 n NaOH - the precipitate dissolved, the solution changed color from light pink to yellow. After 30 minutes, the reaction was complete according to HPLC. The pH of the solution was adjusted to 6.8 by the addition of approximately 2.5 ml of concentrated acetic acid. The solution was evaporated on a rotary evaporator to a volume of 80 ml and 280 ml of isopropyl alcohol was added - a white precipitate formed. The flask was left overnight in the refrigerator, then the precipitate was filtered off, washed 2 times with 80 ml of 80% aqueous isopropyl alcohol and 1 time with pure isopropyl alcohol. The precipitate was dissolved in 70 ml of water (on a rotary evaporator to remove residual alcohol) and 170 ml of isopropyl alcohol was added. The precipitate formed was left in the refrigerator overnight, then it was filtered off, washed twice with 80 ml of 80% aqueous isopropyl alcohol, once with pure isopropyl alcohol, and dried at 30 ° C in vacuo. Received 11 g of product (yield 37% based on the original ZD-Glu-OH and 62% on HD-iGlu-D-Trp-OMe. Evaporation of the mother liquor, followed by dissolution of the residue in water and addition of isopropyl alcohol, allows 2 g of product to be additionally isolated (total yield 43% based on the starting ZD-Glu-OH).

Example 2

The synthesis was carried out as in example 1, but to 40 g (81.3 mmol) of oxazolidinone ZD-iGlu-D-Trp-OMe, prepared as described previously, 15 ml (162.6 mmol, 2 equiv.) Of aniline were added and dissolved in 160 ml of isopropyl alcohol . The resulting solution was transferred into a 1000 ml round-bottom flask, and with vigorous stirring on a magnetic stirrer, 1.7 g of palladium black in 40 ml of water and 31 ml of 90% formic acid were added. According to HPLC, the reaction is complete in 1 hour.

The catalyst was filtered off, isopropyl alcohol was removed on a rotary evaporator, the residue was diluted with water to 250 ml and extracted with ethyl acetate 1 × 200 ml and 2 × 100 ml to remove aniline. The aqueous layer was evaporated to dryness on a rotary evaporator at 40 ° C - a crystalline precipitate formed. 250 ml of isopropyl alcohol was added to the precipitate and stirred for 1 h on a rotary evaporator, then left in the refrigerator overnight. The precipitate was filtered off, washed 3 times with 50 ml of isopropyl alcohol and dried in vacuum.

Received 19.35 g of a crystalline powder of a light cream color. Yield 62% based on the starting Z-D-Glu-OH.

Example 3

Getting oxazolidinone Z-D-iGlu-L-Trp-OMe

Oxazolidinone Z-D-Glu obtained from 2.06 g of Z-D-Glu-OH was dissolved in a 100 ml round bottom flask in 20 ml of dry ethyl acetate; cooled to -10 ° C in an ice and salt bath and 0.96 ml (7.38 mmol) of isobutyl chloroformate and 0.81 ml (7.38 mmol) of N-methylmorpholine were added with vigorous stirring on a magnetic stirrer. The mixture was stirred for 3 min, a precipitate of N-methylmorpholine hydrochloride precipitated, the temperature of the reaction mixture rose to + 12 ° C, then 1.88 g (7.38 mmol) of H-L-Trp-OMe HCl and 0.81 ml (7.38 mmol) of N-methylmorpholine were added. The reaction mass was stirred while cooling for 30 minutes until complete reaction according to HPLC.

The reaction mixture was transferred to a separatory funnel, 10 ml of water was added - the precipitate dissolved; the aqueous layer was discarded, the organic phase was washed 2 more times with 10 ml of water and 2 times with 10 ml of a saturated NaCl solution, dried for 3 hours with anhydrous Na ₂ SO ₄ and the ethyl acetate was removed on a rotary evaporator.

Received 4.25 g of a light yellow oil.

Obtaining H-D-iGlu-L-Trp-OMe

To oxazolidinone Z-D-iGlu-L-Trp-OMe, 1.57 ml (17.28 mmol, 2 equiv.) Of aniline was added and dissolved in 34 ml of isopropyl alcohol. The resulting solution was transferred into a 100 ml round bottom flask, and with vigorous stirring on a magnetic stirrer, 0.28 g of 5% Pd / C in 8 ml of water and 3.31 ml (86.4 mmol) of 90% formic acid were added. According to HPLC, the reaction is complete in 1 hour.

The catalyst was filtered off, isopropyl alcohol was removed on a rotary evaporator, the residue was diluted with water to 20 ml and extracted with ethyl acetate 2 × 10 ml to remove aniline. The aqueous layer was evaporated to dryness on a rotary evaporator at 40 ° C - a crystalline precipitate formed. 10 ml of isopropyl alcohol was added to the precipitate and left in the refrigerator overnight. The precipitate was filtered off, washed 3 times with 3 ml of isopropyl alcohol and dried in vacuum.

Obtained 1.53 g of a crystalline powder of a light cream color. Yield 60% based on the starting Z-D-Glu-OH.

Obtaining H-D-iGlu-L-Trp-ONa

To a suspension of 1.53 g (4.43 mmol) of H-D-iGlu-L-Trp-OMe in 4 ml of water, 13.28 ml of 1 N NaOH was added with stirring on a magnetic stirrer. After 30 minutes, the reaction was complete according to HPLC. The pH of the solution was adjusted to 6.8 by the addition of concentrated acetic acid. The solution was evaporated on a rotary evaporator to a volume of 5 ml and 30 ml of isopropyl alcohol was added - a white precipitate formed. The flask was left overnight in the refrigerator, then the precipitate was filtered off, washed 2 times with 8 ml of 80% aqueous isopropyl alcohol and 1 time with pure isopropyl alcohol. The precipitate was dissolved in 5 ml of water and 30 ml of isopropyl alcohol was added. The precipitate formed was left in the refrigerator overnight, then it was filtered off, washed twice with 8 ml of 80% aqueous isopropyl alcohol, once with pure isopropyl alcohol, and dried at 30 ° C in vacuo.

1.13 g of product is obtained (yield 44% based on the starting Z-D-Glu-OH).

The inventive method allows to obtain a product with a high degree of purity (more than 99% according to analytical HPLC), without requiring chromatographic purification methods. However, there is no need to use expensive starting compounds such as benzyl ether Boe D-glutamic acid or benzyl ether D-tryptophan. Taking into account the yield of the final product, the cost of industrial synthesis of D-isoglutamyl-D-tryptophan can be reduced by a factor of 2–3 compared with the known analogues.

The relatively high yield, high purity of the final product and its relative cheapness make it possible to organize, on the basis of the proposed method, the industrial production of isoglutamyl-tryptophan monosodium salt.

Claims (1)

A method for producing a monosodium salt of D-isoglutamyl-D or L-tryptophan, comprising the steps of synthesizing oxazolidinone carbobenzoxy-D-glutamic acid, condensing it with tryptophan methyl ester, removing the carbobenzoxy protection by opening the oxalidinone ring using catalytic hydrogenolysis and alkaline saponification methyl dipeptide monosodium salt, characterized in that at the stage of the synthesis of oxazolidinone carbobenzoxy-D-glutamic acid, washing with an aqueous solution of hydroxy hydrochloride is used amine, and the step of removing the carbobenzoxy protecting oksalidinonovogo with simultaneous opening of the ring is performed by catalytic hydrogenolysis over palladium catalyst using formic acid as hydrogen donor and aniline, taken in an amount of from 2 to 4 equivalents relative to the amount of the protected dipeptide.