RU2696099C1 - Chemoenzymatic synthesis of 2,5-diketomorfoline derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel method of synthesis of 2,5-diketomorpholines. Use of penicillin acylase enzyme at the stage of formation of amide bond enables high output and high enantiomeric purity of the end product.

EFFECT: novel method of producing 2,5-diketomorpholines, which involves a biocatalytic stage of formation of an amide bond between a carboxyl group of an oxyacid and an amino group of an amino acid with a subsequent chemical step of intramolecular esterification.

6 cl, 6 ex

Description

Technical field

The invention relates to the synthesis of derivatives of 2,5-diketomorpholine, which are monomers for the preparation of biodegradable polymers of polydepsipeptides (polyetheramides). Polymers of this type are used in medicine and can be used in surgical stitching of tissues, as bone plates, as well as carriers for the controlled release of drugs. Derivatives of 2,5-diketomorpholine are of interest as biologically active compounds: they have antibiotic, antioxidant, anticoagulant, as well as immunostimulatory activity, and exhibit inhibitory effects against various enzymes.

State of the art

In the scientific and patent literature, there are known methods for the preparation of 2,5-diketomorpholine derivatives by the methods of classical organic multistage synthesis, including the steps of setting and removing protective groups and using activated derivatives of amino and hydroxy acids. All known methods can be reduced to two main approaches: the initial formation of an amide bond, and then cyclization to lactone, and vice versa, esterification followed by cyclization to lactam [Smelcerovic A. et al. J. of Amino Acids. 2014, 46, P. 825.]. Most often, the authors use alpha-halogen-substituted derivatives of carboxylic acid halides [CN 108299332, 20-07-2018; Cook A. et al. J. Chem. Soc. 1949, P. 2347 .; Smelcerovic A et al. J. Mol. Struct. 2011, 985, P. 397.], N-protected derivatives of amino acids [Pedras M. et al. Org. Lett. 2004, 6, P. 4615 .; Kagamizono T. et al. J. Antibiot. 1995, 48, P. 1407.], O-protected derivatives of amino acids [Hughes A. et al. J. Org. Chem. 2005, 70, P. 3079.]. In the patent document [US 005817751, 06-10-1998], the use of the Ugi reaction for the creation of an N-substituted amide bond by condensation of four components: hydroxy acid, amino acid, aldehyde or ketone and isonitrile is used for the synthesis of a precursor of a derivative of 2,5-diketomorpholine wherein the amino acid is covalently linked via an ester bond through a carboxy group to a solid support containing hydroxyl groups. Subsequent cyclization of the precursor leads to the formation of the target product with the regeneration of the substrate.

Known methods for producing derivatives of 2,5-diketomorpholine have significant limitations. If it is necessary to obtain enantiomerically pure compounds, optically pure starting materials must be used. Activated reagents are required for regioselective conversion. Due to the multifunctionality of the starting reagents, the need arises to use protective groups, which greatly complicates the synthesis and makes it multi-stage, as a result of which the total yield of the final product does not exceed 50%, despite the rather high yields of specific stages of the synthesis. The use of biocatalysis for the synthesis of such compounds allows one to overcome these limitations and obtain the final product with a significantly higher yield and enantiomeric purity of more than 99.9%.

Closest to the claimed solution is the method described in the patent document [CN 108299332, 20-07-2018], the authors of which synthesized (3R, 6R and 3S, 6S) -3- (benzyloxycarbonyl-ethylene) -6-methyl-morpholine- 2,5-dione in two stages based on 2-bromopropionyl chloride and gamma-benzyl-glutamic acid, and depending on the purification method, the total yield of the product was small and amounted to 3.4-34.8%. In addition, the preparation of 2-bromopropionyl chloride will also require reactions involving hazardous and aggressive reagents. The method for the synthesis of 2,5-diketomorpholines specified in the patent document [US 005817751, 06-10-1998] using the Ugi reaction is described above and has similar limitations.

Disclosure of invention

The technical problem to which the invention is directed is the development of a simple-to-execute method for the synthesis of derivatives of 2,5-diketomorpholine, providing a high yield of the target product and its enantiomeric purity.

The claimed method of synthesis of derivatives of 2,5-diketomorpholine, General formula

where R ¹ = H, CH ₃ , aryl; R ² = H, CH ₃ , aryl, CH ₂ -aryl, isopropyl, propyl, butyl, hydroxymethyl, methaptomethyl, 1-hydroxyethyl, 1-methylpropyl, 2-methylpropyl, indol-3-yl-methyl, carbamoylmethyl, carbamoylethyl, carboxymethyl , carboxyethyl, 4-aminobutyl, 3-guanidylpropyl, 2- (methylsulfanyl) ethyl starting from amino acids and amides of hydroxyacids, which includes the step of forming an amide bond using penicillin acylase and subsequent cyclization under the action of an activating agent, for example, N, N'-dicyclohexylcarbodiimide. The method consists in preparing saturated aqueous solutions of the amino acid, which is the nucleophile, and the hydroxy acid amide, which is the acyl donor, in a molar ratio of components from 5: 1 to 1: 3, adjust the pH to 9-11, add a biocatalyst — soluble, immobilized or a mutant form of penicillin acylase to ensure the necessary conversion, the reaction mixture is maintained at 10-35 ° С until the maximum yield of N-hydroxyacyl amino acid is reached, the pH is adjusted to 2-3, the obtained N-hydroxyacyl amino acid is extracted with an organic solution Ithel added an equimolar amount of N, N'-dicyclohexylcarbodiimide, the mixture was allowed to stand until a precipitate N, N'-dicyclohexylurea precipitate is separated, the remaining solution is evaporated and the solid residue was isolated the title product. In this case, the amount of biocatalyst for optimal conversion is determined empirically when sampling the reaction mixture in a model experiment conducted in a small volume, observing similar experimental conditions and concentrations, and monitoring the accumulation of the target product. The time required to achieve the maximum yield of N-hydroxyacyl amino acids is determined empirically in a model experiment conducted in a small volume, observing similar experimental conditions and concentrations, while monitoring the accumulation of the target product. To carry out the synthesis, ethyl acetate, butyl acetate, isobutyl acetate, heptane, carbon tetrachloride, chloroform, dichloromethane, acetonitrile, cyclopentyl methyl ether or mixtures thereof are used as an organic solvent, while the solvent is removed by distillation or in vacuum. The sediment is separated by filtration through filter paper, cloth or a porous filter, as well as by decantation.

As a biocatalyst, various forms (soluble, immobilized, mutant) of the bacterial enzyme penicillin acylase from Escherichia coli, Alcaligenes faecalis, Kluyvera citrophila, Providencia rettgeri, Arthrobacter viscosus, Bacillus megaterium and other sources of amide amino acids and the carboxy group of hydroxy acids (Scheme 1). It is known that numerous methods of immobilization of penicillin acylases, as well as other enzymes, allow preserving the catalytic properties of the enzyme, and also make it possible to reuse the biocatalyst, often lead to its stabilization, and also improve the efficiency of the process and its economic indicators [R.A. Sheldon et al. Chem. Soc. Rev. 2013, 42, p. 6223; A.I. Kallenberg et al. Adv. Synth. Catal. 2005, 347, p. 905; N.A. Pchelintsev et al. J. Mol. Catal. B: Enzymatic 2009, 56, P. 202]. The biocatalytic acyl transfer reaction proceeds stereoselectively: the amino acid L-enantiomer is predominantly acylated, which allows the racemic mixture of amino acids to be included in the reaction, rather than the individual enantiomer. In addition, the biocatalytic reaction proceeds in the temperature range from 10 to 35 ° C under mild conditions in the aquatic environment, without the use of organic solvents and the formation of toxic waste. The hydroxy acid amide in the form of a racemic mixture or an individual enantiomer is used as the acyl donor, the amino acid in the form of a racemic mixture or an individual enantiomer as the nucleophile, the molar ratio of the acyl donor and nucleophile varies from 1: 5 to 3: 1, respectively. The pH of the reaction mixture varies from 9 to 11. The reaction time is proportional to the enzyme content in the reaction mixture and is ensured by using a certain amount of biocatalyst, which is selected empirically for each pair of reagents based on the need to conduct the reaction for the required period of time for technological, economic or other reasons, for example, according to the results of a preliminary kinetic experiment conducted under the same conditions as preparative synthesis, but not in pain on a large scale, for example, in 1-2 ml of a reaction mixture with tracking of the kinetics of the reaction, which consists in sampling and determining the concentration of components at each time point, for example, using high performance liquid chromatography. The choice of optimal conditions, concentrations of reagents and the amount of biocatalyst is also possible using other approaches, for example, microreactors or calculations based on a fundamental understanding of the kinetic laws of the process [R. Wohlgemuth et al. Trends Biotechnol. 2015, 33, P. 302; J.L. van Roon et al. Biotechnol. Adv. 2007, 25, R. 137]. The use of biocatalysis allows you to achieve a very high yield of N-hydroxyacyl amino acids. The reaction product is isolated from the reaction mixture by extraction with an organic solvent, for example, ethyl acetate in an acidic medium, for example, at pH 2-3. If it is necessary to separate an unreacted acyl donor after a biocatalytic conversion, for example, in the case of a process with an acyl donor to nucleophile ratio greater than 1: 1, the pH of the reaction mixture is adjusted to 12 ± 1 and the unreacted acyl donor is extracted by extraction with an organic solvent.

In the second stage, the N-hydroxyacyl amino acid undergoes an intramolecular esterification reaction to form a 2,5-diketomorpholine derivative. The cyclization takes place in an organic solvent with the addition of an activating reagent, for example, N, N'-dicyclohexylcarbodiimide in an amount of an equimolar N-hydroxyacyl amino acid. During the reaction, N, N'-dicyclohexylurea is formed, which precipitates. The yield of the cyclization reaction is also very high, since a thermodynamically stable six-membered cycle is formed. Purification of the target product consists in separating the precipitate of N, N'-dicyclohexylurea, for example, by decantation, centrifugation or filtration through filter paper, cloth or a porous filter and subsequent removal (distillation) of the solvent, for example, in vacuum or using a rotary evaporator.

The structure and composition of all synthesized compounds were confirmed by ¹ H NMR and mass spectrometry, chemical purity was confirmed by high performance liquid chromatography.

The technical result of the claimed method is a derivative of 2,5-diketomorpholine, synthesized with the participation of penicillin acylase as a highly effective biocatalyst that allows stereoselectively creating an amide bond in one stage. Subsequent cyclization leads to the desired product with a total yield of at least 80 mass. % in relation to the starting component (nucleophile or acyl donor), taken in smaller quantities. An applied aspect of the present invention is that 2,5-diketomorpholine derivatives are monomers for producing polydepsipeptides, promising biodegradable biomedical polymers [Feng Y. et al. Int. J. Mol. Sci. 2009, 10, P. 589], and also have biological activity.

The implementation of the invention

Below is a more detailed description of the proposed method, which does not limit the scope of claims of the claimed invention, but demonstrates the possibility of carrying out the invention with the achievement of the claimed technical result.

All reagents used are commercially available, all procedures, unless otherwise specified, were carried out in the temperature range from 10 to 35 ° C.

Example 1. Synthesis of (3S, 6S) -3-benzyl-6-phenyl-morpholine-2,5-dione.

Amide of (S) -indical acid and D, L-phenylalanine (to final concentrations of 0.2 and 0.1 M, respectively) were dissolved in distilled water, the pH was adjusted to 11 with a 5 M potassium hydroxide solution, and the reaction was started by adding an aliquot of the initial concentrated penicillin acylase solution from Alcaligenes faecalis, creating a concentration of active sites of the enzyme in the reaction mixture of 1 μM. The reaction was carried out in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 15 ° С. After 25 minutes, the pH of the reaction mixture was adjusted to 12 with a 5 M KOH solution, the unreacted (S) -mindal acid amide was extracted three times with ethyl acetate, and the volume of ethyl acetate at each extraction was equal to the volume of the reaction mixture. To extract N- (S) -mandelil- (S) -phenylalanine with concentrated HCl, the pH was adjusted to 2, the product was extracted three times with ethyl acetate, and the volume of ethyl acetate at each extraction was equal to the volume of the reaction mixture. Ethyl acetate was evaporated on a rotary evaporator to form a 0.5 M solution, N, N'-dicyclohexylcarbodiimide was added to a final concentration of 0.5 M. The precipitate of N, N'-dicyclohexylurea was filtered through a porous filter with a pore diameter of 40 μm, and the filtrate was evaporated on a rotary evaporator , the solid residue was dried in vacuo to constant weight. The yield of (3S, 6S) -3-benzyl-6-phenyl-morpholine-2,5-dione was 94%, calculated on the L-enantiomer of phenylalanine. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry; chemical purity by analysis by high performance liquid chromatography was 99.1%.

Example 2. Synthesis of (3S, 6S) -3-benzyl-6-phenyl-morpholine-2,5-dione.

Amide of (S) -indic acid and D, L-phenylalanine (to final concentrations of 0.1 and 0.3 M, respectively) were dissolved in distilled water, the pH was adjusted to 10 with a 5 M potassium hydroxide solution and the reaction was started by adding an aliquot of the initial concentrated penicillin acylase solution from Alcaligenes faecalis, creating a concentration of active sites of the enzyme in the reaction mixture of 1 μM. The reaction was carried out in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 20 ° С. After 30 minutes, the pH of the reaction mixture was brought to 2, the product was extracted with butyl acetate, the volume of butyl acetate being equal to one third of the volume of the reaction mixture, an equimolar N- (S) -mandelyl- (S) -phenylalanine was added, the amount of N, N'- dicyclohexylcarbodiimide, after the formation of a precipitate of N, N'-dicyclohexylurea, it was separated using a porous filter, the filtrate was evaporated on a rotary evaporator, the solid residue was dried in vacuum to constant weight. The yield of (3S, 6S) -3-benzyl-6-phenyl-morpholine-2,5-dione was 91%, calculated on the basis of the amide of (S) -mindal acid. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry; chemical purity according to the analysis by high performance liquid chromatography was 99%.

Example 3. Synthesis of (3S, 6R) -3-methyl-6-phenyl-morpholine-2,5-dione

Amide of (R) -mindal acid and D, L-alanine (to final concentrations of 0.2 and 0.5 M, respectively) were dissolved in distilled water, the pH was adjusted to 9.0 with a 5 M potassium hydroxide solution and the reaction was started by adding an aliquot of the initial concentrated a solution of the mutant form of beta D484N penicillin acylase from Escherichia coli, creating a concentration of active sites of the enzyme in the reaction mixture of 1 μM. The reaction was carried out in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 10 ° С. After 40 minutes, the pH of the reaction mixture with concentrated HCl was adjusted to 2 and the product was extracted twice with ethyl acetate, while the volume of ethyl acetate at each extraction was equal to the volume of the reaction mixture. Ethyl acetate was evaporated on a rotary evaporator to form a 0.5 M solution, N, N'-dicyclohexylcarbodiimide was added to a final concentration of 0.5 M. The precipitate of N, N'-dicyclohexylurea was filtered through a porous glass filter with a pore diameter of 40 μm, and the filtrate was evaporated on rotary evaporator, the solid residue was dried in vacuum to constant weight. The yield of (3S, 6R) -3-methyl-6-phenyl-morpholine-2,5-dione was 86%, calculated on the basis of the amide of (R) -indamic acid. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry, chemical purity according to the analysis by high performance liquid chromatography was 99.5%.

Example 4. Synthesis of (3S, 6S) -3- (indol-3-yl-methyl) -6-phenyl-morpholin-2,5-dione.

Amide of (S) -mindal acid and D, L-tryptophan (to final concentrations of 0.2 and 0.1 M, respectively) were dissolved in distilled water, the pH was adjusted to 10.6 with a 5 M potassium hydroxide solution and the reaction was started by adding an aliquot of the initial concentrated a solution of penicillin acylase from Alcaligenes faecalis, creating a concentration of active sites of the enzyme in the reaction mixture of 1 μM. The reaction was carried out in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 25 ° С. After 25 minutes, the pH of the reaction mixture was adjusted to 12 with a 5 M KOH solution, the unreacted (S) -amndamic acid amide was extracted twice with butyl acetate, and the volume of butyl acetate at each extraction was equal to the volume of the reaction mixture. To extract N- (S) -mandelil- (S)-tryptophan with concentrated HCl, the pH was adjusted to 2, the precipitated oil was centrifuged for 5 min at 5000 rpm, decanted, dissolved in acetonitrile to form a 0.5 M solution, N, N was added 'β-dicyclohexylcarbodiimide to a final concentration of 0.5 M. The precipitate of N, N'-dicyclohexylurea was filtered through a glass porous filter with a pore diameter of 40 μm, the filtrate was evaporated on a rotary evaporator, and the solid residue was dried in vacuum to constant weight. The yield of (3S, 6S) -3- (indol-3-yl-methyl) -6-phenyl-morpholin-2,5-dione was 91% in terms of the L-enantiomer of tryptophan. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry, chemical purity according to the analysis by high performance liquid chromatography was 99.2%.

Example 5. Synthesis of (3S, 6S) -3-carbamoylmethyl-6-phenyl-morpholine-2,5-dione.

Amide of (S) -mindal acid and D, L-asparagine monohydrate (to final concentrations of 0.2 and 0.1 M, respectively) were dissolved in distilled water, the pH was adjusted to 10 with a 5 M potassium hydroxide solution, and the reaction was started by adding an aliquot of the original immobilized preparation penicillin acylases from Alcaligenes faecalis to provide the necessary reaction rate, creating a concentration of active sites of the enzyme in the reaction mixture of 1 μM. The reaction was carried out in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 30 ° С. After 30 minutes, the preparation of the immobilized enzyme was separated by filtration, the pH of the reaction mixture (supernatant) was adjusted with a 5 M KOH solution to 12, the unreacted (S) -amidic acid amide was extracted three times with ethyl acetate, and the volume of ethyl acetate at each extraction was equal to the volume of the reaction mixture . To extract N- (S) -mandelil- (S) -asparagine with concentrated HCl, the pH was adjusted to 7, the aqueous phase was evaporated in vacuo to a dry residue, which was washed 2 times with ethyl acetate. The product was removed from the solid residue with acetonitrile to form a 0.5 M solution, N, N'-dicyclohexylcarbodiimide was added to a final concentration of 0.5 M. The precipitate of N, N'-dicyclohexylurea was filtered through a porous glass filter with a pore diameter of 40 μm, and the filtrate was evaporated on rotary evaporator, the solid residue was dried in vacuum to constant weight. The yield of (3S, 6S) -3-carbamoylmethyl-6-phenyl-morpholine-2,5-dione was 82%, calculated on the L-enantiomer of asparagine. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry, chemical purity according to the analysis by high performance liquid chromatography was 99.0%.

Example 6. Synthesis of (6S) -phenyl-morpholine-2,5-dione.

Chloroacetic acid amide and D, L-phenylalanine (to final concentrations of 0.3 and 0.1 M, respectively) were dissolved in distilled water, the pH was adjusted to 10 with a 5 M potassium hydroxide solution and the reaction started by adding an aliquot of the initial concentrated solution of penicillin acylase from Alcaligenes faecalis, creating a concentration of active sites of the enzyme in the reaction mixture of 8 μm. The reaction was carried out in a thermostatic cell of the pH stat Titrino 719 (Metrohm, Switzerland) at 35 ° С. After 40 minutes, the pH of the reaction mixture was adjusted to 12 with a 5 M KOH solution, the unreacted chloroacetic acid amide was extracted three times with butyl acetate, and the volume of butyl acetate at each extraction was equal to the volume of the reaction mixture. To extract N-chloroacetyl- (S) -phenylalanine with concentrated HCl, the pH was adjusted to 2 and the product was extracted three times with butyl acetate, and the volume of butyl acetate at each extraction was equal to the volume of the reaction mixture. Butyl acetate was evaporated on a rotary evaporator, the residue was dissolved in 5 M KOH and heated for 1 hour at 80 ° C. Concentrated HCl adjusted the pH to 2, extracted three times with butyl acetate N-hydroxyacetyl- (S) -phenylalanine. Butyl acetate was evaporated on a rotary evaporator to form a 0.5 M solution, N, N'-dicyclohexylcarbodiimide was added to a final concentration of 0.5 M. The precipitate of N, N'-dicyclohexylurea was filtered through a porous glass filter with a pore diameter of 40 μm, and the filtrate was evaporated on a rotary evaporator, the solid residue was dried in vacuum to constant weight. The yield of (6S) -phenyl-morpholine-2,5-dione was 87% in terms of the L-enantiomer of phenylalanine. The structure and composition of the synthesized compound were confirmed by ¹ H NMR and mass spectrometry; chemical purity according to the results of analysis by high performance liquid chromatography was 99.6%.

Claims (8)

1. The method of synthesis of derivatives of 2,5-diketomorpholine of the General formula

where R ¹ = H, CH ₃ , aryl; R ² = H, CH ₃ , aryl, CH ₂ -aryl, isopropyl, propyl, butyl, hydroxymethyl, methaptomethyl, 1-hydroxyethyl, 1-methylpropyl, 2-methylpropyl, indol-3-yl-methyl, carbamoylmethyl, carbamoylethyl, carboxymethyl , carboxyethyl, 4-aminobutyl, 3-guanidylpropyl, 2- (methylsulfanyl) ethyl, characterized in that saturated aqueous solutions of the nucleophile amino acid and the hydroxy acid amide, an acyl donor, are prepared in a molar ratio of components from 5: 1 to 1 : 3, adjust the pH to 9-11, add a biocatalyst - a soluble, immobilized or mutant form of bact ryal penicillin acylase to ensure the necessary conversion, maintain the reaction mixture at 10-35 ° С until the maximum yield of N-hydroxyacyl amino acid is reached, adjust the pH to 2-3, the resulting N-hydroxyacyl amino acid is extracted with an organic solvent, an equimolar amount of N, N 'is added β-dicyclohexylcarbodiimide, the mixture is allowed to precipitate N, N'-dicyclohexylurea, the precipitate is separated, the remaining solution is evaporated and a solid residue of the target product is isolated. 2. The method according to p. 1, characterized in that the amount of biocatalyst for optimal conversion is determined empirically when sampling the reaction mixture in a model experiment conducted in a small volume with the same experimental conditions and concentrations, and monitoring the accumulation of the target product. 3. The method according to p. 1, characterized in that the time required to achieve the maximum yield of N-hydroxyacyl amino acids is determined empirically in a model experiment conducted in a small volume with the same experimental conditions and concentrations, while monitoring the accumulation of the target product. 4. The method according to p. 1, characterized in that the organic solvent used is ethyl acetate, butyl acetate, isobutyl acetate, heptane, carbon tetrachloride, chloroform, dichloromethane, acetonitrile, cyclopentyl methyl ether or mixtures thereof. 5. The method according to p. 1, characterized in that the separation of the precipitate is carried out by filtration through filter paper, cloth or a porous filter, as well as by decantation. 6. The method according to p. 1, characterized in that the removal of the solvent is carried out by distillation or in vacuum.