PEGYLATED RESOLVING AGENTS FOR IMPROVED RESOLUTION OF RACEMIC MIXTURE

Abstract

The present invention is related to a product for the resolution of racemic mixtures in which various separation processes of a salt composed of a PEGylated resolving agent and an enantiomer in a racemic mixture is utilized. Separation can be caused by temperature-dependent phase transformation of the said salt pair in aqueous or organic media as well as other methods used is separation of PEG such as salting out (e.g. addition of ammonium sulfate). The first cycle of racemic resolution by this method was shown to afford 85% optically pure amino acid from a 50:50 mixture of racemic L,D-amino acids esters. An additional cycle improved optical purity up to 95%.

Claims

1. A resolving agent that can be used for the resolution of racemic mixtures and is composed of a chemically activated linear or branched polyethylene glycol (PEG) from molecular weights of 400 to 4 million in which the PEG is activated in one terminus or many termini and condensed with to a pure stereoisomer and in which the PEG termini carries an nucleophile(s) to be condensed with a pure isomer carrying an electrophile or in which the PEG termini carries an electrophile(s) to be condensed with a pure isomer carrying an nucleophile.

2. Utilizing the said PEGylated resolving agent in an aqueous or organic medium for diastereomeric salt pair formation between the said PEGylated resolving agent and a mixture of enantiomers by cooling the resulting mixture or by the addition of agents known to result in the precipitation of polyethylene glycol.

3. A resolving agent of claim 1 composed of various known polyethylene glycols such as linear or branched (PEG), attached to a pure stereoisomer (SI), including those traditionally used for resolution of racemic mixtures such as mandelic acid, brucine, strychnine, (D)- or (L)-tartaric acid, valine or any stereochemicaly pure compound capable of forming diastereomeric salts with racemic mixtures of enantiomers, via a linkage X formed between a chemically activated PEG at one terminus or many termini, and in which chemical activation depends on the nature of SI so that when SI is a nucleophile the said chemical activation of PEG involves the attachment of an electrophile to PEG terminus (or termini) and when SI is an electrophile the said chemical activation of PEG involves the attachment of a nucleophile to PEG terminus (or termini)as shown below as A:
SI-X-PEG-X-SI A

4. A resolving agent of claim 1 composed of various known polyethylene glycols such as linear or branched (PEG), attached to a pure stereoisomer (SI) carrying an electrophilic moiety. The stereoisomer is chosen, but not limited to, the traditional resolving agents such as mandelic acid, brucine, strychnine, (D)- or (L)-tartaric acid, valine or any stereochemicaly pure compound capable of forming diastereomeric salts with racemic mixtures of enantiomers, via a linkage formed between a chemically activated PEG at one terminus or many termini (Y-PEG-Y, Y=N, S, O, -carbon, etc.) used for condensation with a resolving agent carrying an electrophilic moiety as shown below as B.
SI-Y-PEG-Y-SI B

5. A resolving agent of claim 3 in which the PEG used may be substituted with group comprising of phenyl, benzyl, naphthyl, a C.sub.2-C.sub.n linear or branched alkyl group that may carry inert functional groups such as nitrile, ethers, amide, ester, olefin, alkyne, and the like.

6. A resolving agent of claim 3 in which X (Nucleophile on the resolving agent) or Y (Nucleophile on PEG) may be O, OH, SH, NH, NH.sub.2 and other nucleophiles such as -carbons that can form carbanions, etc.

7. A resolving agent of claim 1 in which a stereochemically pure amino acid, alkaloids, nucleosides, or any optically pure compound or a derivative thereof is attached to PEG and the resulting resolving agent (e.g. SI-X-PEG-X-SI or SI-Y-PEG-Y-SI vide supra) is used for the resolution of racemic amino acid esters or any other racemic mixture in aqueous or organic solvents and recovering the D-amino acid esters and L-amino acid ester or the enantiomers of the racemic mixture as shown below as 1 and 2. ##STR00004##

8. A process for the separating of enantimerically pure compounds from their racemic mixtures in which a resolving agent of claim 1 is used and by cooling the final resolution solution and filtration or centrifugation of the precipitate, followed by the release of the desired enantiomer by treatment with acid or base.

9. A process for the separation of enantimerically pure compounds from there racemic mixture in which a resolving agent of claim 1 is used and by the addition of various inorganic and organic chemicals that are known to cause precipitation of PEG.

10. A process for the separation of enantiomerically pure compounds from their racemic mixtures (compound 2) in which the SI in claim 2 is D- or L-phenyl alanine methyl ester.

11. A process for the separation of enantoimerically pure compounds from their racemic mixtures in which the SI is claim 2 is (R)- or (L)-mandelic acid.

12. A process for the separation of enantiomerically pure compounds from their racemic mixtures in which the SI in claim 2 is enantiopure-()-hydroxy acids.

13. A process for the separation of enantiomerically pure compounds from their racemic mixtures in which the SI in claim 2 is (L)- or (D)-valine.

14. A process for the separation of enantiomerically pure compounds from their racemic mixtures in according to claim 1 in which the resolving agent is used in an amount of 0.25 to 0.5 mole per mole of racemic phenyl alanine methyl ester to be resolved.

15. A process of claim 1 in which the base suitable for use in the process for synthesis of a compound of formula A is selected from the group comprising pyridine, diisopropylethyl amine, triethylamine, Na.sub.2CO.sub.3, Cs.sub.2CO.sub.3 and K.sub.2CO.sub.3.

16. The process defined in claim 2, wherein MeOH, EtOH, and H.sub.2O is used as solvent for the resolution of enantiomers.

17. Process as claimed in claim 2 wherein said resolution is carried out at from 0 C. to 25 C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0025] The process of the invention is schematically represented as follows:

##STR00003##

[0026] Preferably, the process for producing a compound of Formula 1 can be used to produce PEGylated ()-hydroxy or ()-amino acids or pharmaceutically acceptable salts thereof. Generally, PEGylated--hydroxy or amino acid may be prepared in two steps: (1) reacting the PEG-10000 with thionyl chloride and (2) reacting of activated PEG 5 with -hydroxy or amino acids. In this process PEG reacted with at least 5 eq moles of thionyl chloride.

[0027] Typically, the reaction can be conducted at a temperature comprised between 15 to 25 C. and preferably at 20 C.

[0028] The base suitable for use in the process of producing a compound of Formula 1 is selected from the group comprising pyridine, Na.sub.2CO.sub.3 and preferably Et.sub.3N or other bases used to neutralize the acid produced from the condensation reaction between 4 and activated PEG 5 for step of chlorination and K.sub.2CO.sub.3 for step of substitution.

[0029] The solvent suitable for use in the process of producing a compound of Formula 1 is selected from CH.sub.2Cl.sub.2, CHCl.sub.3, THF, CH.sub.3CN, DMF and preferably Toluene for step of chlorination and CH.sub.3CN for step of substitution reaction.

[0030] The present invention further provide a process for racemic resolution of compound Formula 2 amino acid esters was added to enantiopure PEGylated-()-hydroxy or ()-amino acids in methanol and upon cooling of the mixture to 0 C. a precipitate was formed. Diastereomeric salt formation of enantiopure PEGylated-()-hydroxy or ()-amino acids with D or L-amino acids esters forms a pair of diastereomers that possess the same chemical formula, but have different physical properties. In this process, the molecules of opposite character (amine and acid) recognize each other by various interactions on the basis of their molecular structures and functional groups.

[0031] To effect the optical resolution of racemic amino acid esters, the amino acids esters is mixed with the PEGylated-()-hydroxy or amino acids and preferably 0.5 mole of PEGylated-()-hydroxy or amino acids is used per 2 mole of DL-amino acid esters.

[0032] The most preferred starting material of Formula 4 for the process of producing a compound of Formula 1 is a-hydroxy or ()-amino acids in which R of Formula 4 is isopropyl or phenyl or other radicals known to men of art. In this embodiment, the product of Formula 1 is PEGylated-(R)-Mandelic acid or L-Valine respectively. It will of course be understood that the manner in which starting compound of Formula 3 is made is not particularly restricted as regards the process of making Formula 1 . In fact, any optically pure enantiomer with a nucleophile (to be condensed with a PEG that is activated with an electrophile) or an electrophile (to be condensed with a PEG that is activated with a nucleophile) can be used.

[0033] The most preferred racemic amino acid ester of Formula 2 is DL-amino acid ester in which R.sub.3 of Formula 2 is methyl and R.sub.4 is benzyl. In this embodiment, the DL-amino acid ester of Formula 2 is DL-phenylalanine methyl ester.

[0034] In general, it will be found that PEGylated-(R)-mandelic acid is better than of PEGylated-(L)-valine for resolving of DL-phenyl alanine methyl ester. After mixing the PEGylated-(R)-mandelic acid and the racemic phenylalanine methyl ester and stirring for 4h in room temperature, the resulting mixture is cooled to 0 C. and filtered. Optical purity in case of PEGylated-(R)-mandelic acid as a resolving agent was 85% and for PEGylated-(L)-valine was 74%. The separation of the diastereomeric complex can also be caused by precipitation of polyethylene glycol moiety of the diastereomeric salt using ammonium sulfate in water. Optical purity of e product obtained with PEGylated-(R)-mandelic acid as a resolving agent was 76%.

[0035] It has been suggested that the underlying principle for the precipitation of PEG as a result of addition of salts is due to interference in H-bond formation between the numerous oxygens of the polyether and water (Duong-Ly et. al. Methods Enzymol. 2014, 541, 85-94; Nathaniel et. al. J. Mol. Liq. 2008, 143-170).

[0036] The resolution medium can be water or alcohols or mixture thereof and suitable alcohols are methanol, ethanol, isopropanol, butanol and the like. In general, methanol is preferred resolution medium. The temperature ranges at which resolution is carried out are from 0 C. to 25 C.

[0037] Aspects of the present invention will be described with reference to the following examples which should not be considered to limit the scope of the invention.

EXAMPLE 1

[0038] Preparation of Activated PEG (Chlorination of PEG)

[0039] PEG (10000) (50 g, 5 mmol) was dissolved in 500 ml of toluene and 100 ml of toluene was distilled from the solution to remove traces of moisture. After cooling to 35 C., freshly distilled anhydrous triethylamine (3.75 ml, 27 mmol) was added. Within 1 h freshly distilled thionyl chloride (1.5 ml, 21 mmol) was dissolved in 20 ml of dry toluene, and added dropwise at 35 C. to the mixture with continuous stirring under a dry nitrogen atmosphere. The mixture was refluxed for 1 h and triethylammonium chloride was removed by passing the hot solution through Celite. After 4h incubation at room temperature, the solution was heated to 50 C. and treated with 5 g of decolorizing carbon which was then removed by filtration over Celite. The filtrate was stored at 4 C. overnight, affording activated PEG which was filtered at 4 C. The solid product was further purified by dissolving in 2,5 1 of absolute ethanol at 60 C. and treating with 30 g of decolorizing carbon, followed by filtration over Celite. The ethanolic filtrate was stored overnight at 4 C. to recrystallize the product. The solid material was separated by filtration and washed with cold ethanol and then ether. After drying in a vacuum desiccator 49 g of a pale yellow product was obtained.

EXAMPLE 2

[0040] Preparation of PEGylated-(R)-Mandelic acid

[0041] Mandelic acid (0.6 g, 4 mmol) was dissolved in 200 ml of acetonitrile, followed by to addition of K.sub.2CO.sub.3 (1.1 g, 8 mmol). Activated PEG (10000) (20 g, 2 mmol), prepared from example 1 was dissolved in 50 ml of acetonitrile and added dropwise with continuous stirring. Thereafter, the resulting pale yellow solution was stirred at reflux for 24 h. Then the reaction mixture was cooled to ambient temperature and adjusted to pH 2-3 using concentrated hydrochloric acid. The resulting solution treated with decolorizing charcoal and filtered over a layer of Celite. The resulting filtrate was evaporated under reduced pressure to obtain a solid which dissolved in methanol and the solution was stored at 4 C. overnight to crystallize the product. The solid material was separated by filtration and washed with cold methanol and then ether. After drying in a vacuum desiccator 15 g of a pale yellow product was obtained. The product was characterized by UV, NMR and IR spectra. .sup.1H-NMR (500 MHz, CDCl.sub.3): 3.57 (s, n CH.sub.2), 7.21-7.40 (m, 10H of Ar), 9.22 (bs, OH), 5.12 (s, CH). MS (EI) m/z: 152 (15), 107 (80), 79 (100).

EXAMPLE 3

[0042] Preparation of PEGylated-(L)-Valine

[0043] L-valine (0.469 g, 4 mmol) was dissolved in 200 ml of acetonitrile. Trietylamine (1.11 ml, 8 mmol) was added to the reaction mixture. Activated PEG (10000) (20 g of, 2 mmol), prepared in example 1, was dissolved in 50 ml of acetonitrile and added dropwise to the reaction mixture with continuous stirring. Thereafter, the resulting yellow solution was stirred at room temperature for 24 h and then treated with decolorizing charcoal and filtered over a layer of Celite. The resulting filtrate was evaporated under reduced pressure to obtain a solid, which was then dissolved in methanol and the solution was stored at 4 C. overnight to crystallize the product. The solid material was separated by filtration and washed with cold methanol and then ether. After drying in a vacuum desiccator 18 g of a pale yellow product was obtained. The product was characterized by UV, NMR and IR spectra. .sup.1H-NMR (500 MHz, CDCl.sub.3): 0.98 (m, 2CH.sub.3), 2.63 (m, CH (CH.sub.3).sub.2), 3.59 (s, n CH.sub.2), 3.59 (m, CH(NH)). MS (EI) m/z: 116 (15), 72 (100), 55 (76), 43 (62).

EXAMPLE 4

[0044] Resolution of Racemic Phenyl Alanine Methyl Ester with PEGylated-(R)-Mandelic Acid

[0045] PEGylated-(R)-mandelic acid (10 g, 1 mmol) was dissolved in 50 ml methanol followed by the addition of racemic mixture of phenyl alanine methyl ester (0.36 g, 2 mmol) and the mixture was stirred at room temperature for 12 h. It was then cooled to 0-5 C. and stirred for 1 h. A voluminous precipitate of white solids was formed, followed by the addition of 20 ml cold methanol. The slurry was filtered and the cake was washed with 10 ml cold methanol, resulting in white solids, consisting of optically impure PEGylated-(R)-mandelic acid.(L)-phenyl alanine methyl ester. The cake was then dissolved in 50 ml methanol and acidified with concentrated hydrochloric acid and cooled to 0-5 C. The white solid was filtered and washed with 10 ml cold methanol. The filtrate was evaporated under reduced pressure to dryness at 50 C. to yield 0.19 g (L)-phenyl alanine methyl ester hydrochloride (90%). Specific optical rotation was performed using sodium lamp D-line wavelength and shown +28 (EtOH, c=2) corresponding to 85% of theory.

EXAMPLE 5

[0046] Resolution of Racemic Phenyl Alanine Methyl Ester with PEGylated-(L)-Valine

[0047] Using the method of example 4, PEGylated-(L)-Valine was used for resolving of racemic phenyl alanine methyl ester. In this example specific optical rotation shown +24 (EtOH, c=2) corresponding to 75% of theory.

[0048] It will be understood that the specification and example are illustrative, but not limiting to the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

EXAMPLE 6

[0049] Ammonium Sulfate-Assisted Resolution of Racemic Phenyl Alanine Methyl Ester with PEGylated-(L)-Mandelic Acid in Water

[0050] PEGylated-(R)-mandelic acid (10 g, 1 mmol), racemic phenyl alanine methyl ester (0.36 g, 2 mmol) were added to 50 ml water and the mixture was stirred at room temperature for 12 h. The resulting mixture was cooled to 0-5 C. and stirred for 1 h. Unlike Experiment 3.5, no precipitation occurred. Solid ammonium sulfate was added to the mixture. After 1 h a voluminous precipitate of a white solid occurred. The slurry was filtered and the cake was washed with 10 ml saturated ammonium sulfate, resulting in white solids consisting of optically impure PEGylated-(R)-mandelic acid.(L)-phenyl alanine methyl ester. The cake was dissolved in 50 ml water and acidified with concentrated hydrochloric acid and stirred for 1 h to liberate (L)-phenyl alanine methyl ester hydrochloride. Dry ammonium sulfate was then added to the resulting solution to precipitate PEGylated (R)-mandelic acid which was filtered and the cake was washed with 10 ml saturated ammonium sulfate. The filtrate was adjusted to pH 7 using sodium bicarbonate (1M) to neutralize (L)-phenyl alanine methyl ester. The resulting white precipitate was filtered, washed by 5 ml water and dried to afford 0.20 g of optically impure (D)-phenyl alanine methyl ester (yield: 93%). Specific optical rotation performed using sodium lamp D-line wavelength and shown +24 (EtOH, c=2) corresponding to 75% of theory.

EXAMPLE 7

[0051] Improvement of Enantiomeric Excess by Additional Cycles of Resolution

[0052] A mixture of PEGylated-(R)-mandelic acid (5 g, 0.5 mmol), optically impure (L)-phenyl alanine methyl ester obtained from Experiment 3.5 (0.16 g, 0.9 mmol, optical purity 85%), and 25 ml methanol was stirred at room temperature for 12 h. The mixture was cooled to 0-5 C. and stirred for 1 h. A voluminous white precipitate formed, followed by the addition of 20 ml cold methanol. The slurry was filtered, and washed with 10 mL cold methanol. The resulting white solid was dissolved in 50 ml methanol and acidified with concentrated hydrochloric acid pH=2-3 and cooled to 0-5. The white solid was filtered and washed with 10 ml cold methanol. The filtrate was evaporated to dryness at 50 C. under vacuum to yield 0.18 g (L)-phenyl alanine methyl ester hydrochloride (yield: 92%). Specific optical rotation performed using sodium lamp D-line wavelength and shown +32 (EtOH, c=2) corresponding to 95% of theory.