DESC:The following specification particularly describes the invention and the manner in which it is to be performed:

ENZYMATIC PROCESS FOR THE PREPARATION OF A REPAGLINIDE INTERMEDIATE, 2-(3-ETHOXY-4-(ETHOXYCARBONYL) PHENYL) ACETIC ACID.

INTRODUCTION
The present application relates to a process for the preparation of 2-(3-ethoxy-4-(ethoxycarbonyl) phenyl) acetic acid (ECPA), a key intermediate of Repaglinide by enzymatic conversion of ethyl 4-(cyanomethyl)-2-ethoxybenzoate. The present application specifically relates to a process for the preparation of ECPA by the conversion of ethyl 4-(cyanomethyl)-2-ethoxybenzoate using a Nitrilase-Amidase System.
BACKGOUND
The drug compound having the adopted name “Repaglinide” has chemical name, 2-ethoxy-4-[2-[[(1S)-3-methyl-1-(2-piperidin-1-ylphenyl)butyl]amino]-2-oxoethyl]benzoic acid and can be represented by structural formula (I) below.

Repaglinide is an oral blood glucose-lowering drug of the meglitinide class used in the management of type 2 diabetes mellitus. Repaglinide lowers blood glucose levels by stimulating the release of insulin from the pancreas. This action is dependent upon functioning beta (ß) cells in the pancreatic islets. Insulin release is glucose-dependent and diminishes at low glucose concentrations. Repaglinide is available in the market under the brand names PRANDIN® tablets containing 0.5 mg, 1 mg, or 2 mg of Repaglinide in the United States.
2-(3-ethoxy-4-(ethoxycarbonyl) phenyl) acetic acid (ECPA) is a key intermediate in the synthesis of Repaglinide. Synthesis of ECPA is disclosed in the literature but essentially through chemical means.

Prior art herein incorporated for reference such as J. Med. Chem., 1998, 41(26), page: 5219-46 (Grell et. al.,); IN 1454/MUM/2006 A (Sadashiv et al.,) and US 20040249188 A1 (Reddy et. al.,) discloses the chemical synthesis of ECPA such as scheme-1.

US 6686497 B1 / Org. Proc. Res. Dev., 2002, 6 (2), pages: 184-186, (Salman et al.,) US 6818786 B2 (Kalkote et al.,) describes the synthesis of ECPA in two steps synthesis as depicted in scheme-2.

US 8198054 B2 (Burgos et. al.,) describes a process enzymatic hydrolysis of ethyl 2-ethoxy-4-(2-ethoxy-2-oxoethyl) benzoate to produce ECPA. It involves the use of enzymes selected from a lipase, an esterase, a chirazyme and a pancreatin for the conversion of said ester to ECPA. Starting material (ethyl ester) that is used in this process may be synthesized through multiple step synthesis as reported in prior art.

Prior art describes chemical approaches for the synthesis of ECPA. These involve reagents that are difficult to handle on an industrial scale (LDA, ethyl bromide, DMSO) or low temperatures/high pressures. The prior art describes hydrolysis of a nitrile intermediate, esterification and selective monohydrolysis of the resultant diester by chemical processes. Potentially, impurities remain from unselective hydrolysis reactions.
Hence, it is an objective of the present application to provide an economic process for the commercial production of ECPA by enzymatic hydrolysis of ethyl 4-(cyanomethyl)-2-ethoxybenzoate in high yields. The enzymatic hydrolysis of the compound of formula II to yield ECPA provides an efficient and selective biotransformation which avoids the stepwise nitrile hydrolysis, esterification and regioselective monohydrolysis necessary in the prior art processes.

SUMMARY
In an aspect, the present application provides a process for the preparation of compound of formula I comprising the step of hydrolyzing compound of formula II in the presence of an enzyme.

wherein, R is C1-6 alkyl.
In another aspect, the present application provides a process for the preparation of ECPA comprising the step of hydrolyzing compound of formula IIa in the presence of an enzyme.

Yet another aspect, the present application provides a process for the preparation of Repaglinide comprising the steps of
a) hydrolyzing compound of formula IIa in the presence of an enzyme to obtain ECPA
b) converting ECPA of step a) to Repaglinide.

DETAILED DESCRIPTION
In an aspect, the present application provides a process for the preparation of compound of formula I comprising the step of hydrolyzing compound of formula II in the presence of an enzyme.

Wherein, R is C1-6 alkyl.
The starting material, compound of formula II may be prepared according to any of the procedures described in the present application or by following methods known in the art.
Starting materials may be purified according any of the methods known in art such as chromatography, recrystallization, fractionation or the like before using.
In an embodiment, enzymatic hydrolysis of compound of formula II may be carried out in the presence of a suitable inert solvent. Inert solvent is selected from a group of water, an organic solvent and mixture thereof. In one embodiment, hydrolysis of compound of formula II may be carried out in water. In another embodiment, hydrolysis of compound of formula II may be carried out in a solvent mixture containing water and a miscible organic solvent.
In an embodiment, hydrolysis of compound of formula II is carried out in the presence of an enzyme. The enzyme is selected from a naturally occurring enzyme and a mutated enzyme, which is capable of hydrolysis of compound of formula II to compound of formula I.
In an embodiment, hydrolysis of compound of formula II may be carried out in the presence of a nitralase enzyme. Nitralase enzyme may be selected from the group consisting of ES-NIT-103, ES-NIT-104, ES-NIT-111, ES-NIT-119, ES-NIT-120, ES-NIT-121, ES-NIT-126, ES-NIT-129, Nitrilase 14, Zea mays, Neurospora crassa OR74A or the like.
In an embodiment, hydrolysis of compound of formula II may be carried out optionally in presence of an additional enzyme. In an embodiment, a nitralase enzyme may be co-expressed or may be combined with an enzyme such as amidase enzyme when a amide side product is formed in addition to the desired carboxylic acid, compound of formula I. Amidase enzyme may be selected from the group consisting of Rhodococcus globerulus, Aureobacterium sp. amidases or the like.
In an embodiment, hydrolysis of compound of formula II may be carried out in the presence of an enzyme, which is a mutated enzyme obtained by the mutation of naturally occurring enzyme. In an embodiment, hydrolysis may be carried out using an enzyme having atleast 50% identity or atleast 70% similarity to the protein sequence of Neurospora crassa nitralase (SEQ ID No. 1) and / or Rhodococus globerulus amidase (SEQ ID No. 2) whether genes are obtained from host strains, recombinant strains or as synthetic DNA.
In an embodiment, the enzyme may be a wild type or a recombinant enzyme and it may be used as whole cells, cell-free extract or in the isolated / semi-purified form. In preferred embodiment, the enzyme may be used at high concentration in cell free extract and the enzyme may be co-expressed in the same host strain.
In an embodiment, hydrolysis may be carried out with concentration of compound of formula II ranging from 0.1 g/L to 300 g/L. In preferred embodiment, it may range from 10 g/L to 30 g/L.
In an embodiment, hydrolysis may be carried out with enzyme loading ranging from 10 % wt. to 100 % wt. In preferred embodiment, enzyme loading may be 75 % wt. or less.
In an embodiment, hydrolysis of compound of formula II may be carried out at a suitable temperature to yield compound of formula I. Suitable temperature may be at about 10° C to about 50° C. In preferred embodiment, it may be carried out at ambient temperature of about 25°C to 40 °C.
In another embodiment, the hydrolysis of compound of formula II to yield compound of formula I is carried out at a suitable pH. In preferred embodiment, the hydrolysis of compound of formula II may be carried out at pH of about 4 to 9. In preferred embodiment, hydrolysis may be carried out in the presence of a buffer at pH of about 7-8.
In an embodiment, hydrolysis of compound of formula II may be carried out in the presence of a suitable buffer, which is a solution of a salt.
In an embodiment, hydrolysis of compound of formula II may be carried out in the presence of a buffer. The buffer added to the reaction mixture may be any buffer known in the art. Specifically, the buffer is selected from a group of potassium phosphate buffer, tris/HCl buffer and triethanolamine buffer.
In an embodiment, hydrolysis of compound of formula II the buffer may be used at concentration of about 0.01 M to 1 M. In preferred embodiment, hydrolysis may be carried out at 0.1 M.
In an embodiment metallic ions like zinc ions, magnesium ions may optionally be added to the buffer to stabilize or activate the enzyme.
In another embodiment, the enzymatic hydrolysis of compound of formula II to yield compound of formula I according to the process of the present application is carried out, for example, in a closed reaction vessel made of glass or metal. For this purpose, the components are transferred individually into the reaction vessel and stirred under an atmosphere of, e.g., nitrogen or air.
In another embodiment, the compound of formula I formed by the hydrolysis of compound of formula II may be extracted after suitable work procedures and isolated using the suitabe methods known in the art such as filtration, centrifugation, evaporation of solvent or by procedure described in the instant application.
The compound of formula I as prepared by the methods of the present aspect may be used as an intermediate for the synthesis of active ingredient such as Repaglinide.

In another aspect, the present application provides a process for the preparation of ECPA comprising the step of hydrolyzing compound of formula IIa in the presence of an enzyme.

In an embodiment, hydrolyzing compound of formula IIa is carried out in the presence of an enzyme which is capable of hydrolysis of compound of formula IIa to ECPA.
In an embodiment, hydrolyzing compound of formula IIa may be carried out in the presence of a nitralase enzyme.
In an embodiment, hydrolyzing compound of formula IIa may be carried out in the presence of a nitralase enzyme such as Neurospora crassa OR74A.
In an embodiment, hydrolyzing compound of formula IIa may be carried out in the presence of a nitralase enzyme and an additional enzyme such as amidase enzyme.
In an embodiment, hydrolyzing compound of formula IIa may be carried out in the presence of a nitralase enzyme such as Neurospora crassa OR74A and a amidase enzyme such as Rhodococcus globerulus amidase.
In an embodiment, hydrolyzing compound of formula IIa may be carried out in the presence of Neurospora crassa OR74A and Rhodococcus globerulus amidase.
In an embodiment, hydrolyzing compound of formula IIa in the presence of an enzyme to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
In an embodiment, ECPA as prepared according to this aspect may be optionally purified according to the techniques known in the art such as re-crystallization, slurrying, chromatography or the like.
ECPA as prepared by the methods of the present aspect may be used as an intermediate for the synthesis of active ingredient, Repaglinide.

In another aspect, the present application provides a process for the preparation of Repaglinide comprising the steps of
a) hydrolyzing compound of formula IIa in the presence of an enzyme to obtain ECPA
b) converting ECPA of step a) to Repaglinide.

Step a) of this aspect may be carried out by hydrolyzing the compound of formula IIa in the presence of an enzyme to obtain ECPA.
In an embodiment, hydrolyzing compound of formula IIa in the presence of an enzyme to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
In an embodiment, hydrolyzing compound of formula IIa in the presence of a nitralase enzyme to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
In an embodiment, hydrolyzing compound of formula IIa in the presence of a Neurospora crassa OR74A to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
In an embodiment, hydrolyzing compound of formula IIa in the presence of a nitralase enzyme and an amidase enzyme to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
In an embodiment, hydrolyzing compound of formula IIa in the presence of a Neurospora crassa OR74A and Rhodococcus globerulus amidase to obtain ECPA may be carried out according to the procedures described in any aspects of the instant application or methods known in the art.
Step b) of this aspect involves converting ECPA of step a) to Repaglinide.
In an embodiment, ECPA as prepared in step a) may be converted to Repaglinide according to the methods known in the art or by following procedures described in the instant application.
In an embodiment, ECPA as prepared in step a) may be isolated or used directly for the preparation of Repaglinide.
In an embodiment, ECPA as prepared according to step a) may be optionally purified before using according to the techniques known in the art such as re-crystallization, slurrying, chromatography or the like.
In an embodiment, ECPA as prepared according to step a) may be reacted with S(+)-3-methyl-1-(2-piperidinophenyl)-1-butyl amine or a salt thereof to yield S(+)-Ethyl- 2-ethoxy-4(2((3-methyl-1-(2-(1-piperidinyl) phenyl)butyl) amino)-2-oxoethyl) benzoate followed by its hydrolysis under basic / acidic conditions to produce Repaglinide.
In an embodiment, ECPA as prepared according to step a) may be reacted with L-N-acetly glutamic acid salt of S(+)-3-methyl-1-(2-piperidinophenyl)-1-butyl amine to yield S(+)-Ethyl- 2-ethoxy-4(2((3-methyl-1-(2-(1-piperidinyl) phenyl)butyl) amino)-2-oxoethyl) benzoate followed by its hydrolysis under basic / acidic conditions to produce Repaglinide.
ECPA obtained according to the procedures described in any aspects of the instant application can be used as an intermediate for the preparation of various drugs, particularly for the preparation of Repaglinide.
ECPA has a purity of more than 99.8% as measured by HPLC and preferably more than 99.9%.
Repaglinide as prepared by the methods of the any aspects of the present application has a purity of more than 99 % as measured by HPLC and preferably more than 99.5%.
Certain specific aspects and embodiments are further described by the following examples, being provided only for purposes of illustration, and the scope of the disclosure is not intended to be limited by the examples.
EXAMPLES
Example-1: Preparation of ethyl 4-(cyanomethyl)-2-ethoxybenzoate
A solution of ethyl 4-(bromomethyl)-2-ethoxybenzoate (100 g) in dichloromethane (700 mL) was slowly added to a mixture of water (60 mL), sodium cyanide (25.94 g) and tetra butyl ammonium bromide (5.05 g) at 30°C. The reaction mass was stirred at the same temperature for 36 hours. After the completion of the reaction, aqueous layer was separated and quenched. The organic layer was washed with water (4x500 mL). The organic layer was separated and evaporated under reduced pressure at 40°C. Isopropyl alcohol (400 mL) was added to the reaction mass and stirred for 10 minutes at the same temperature and concentrated the reaction mass by evaporating half of the Isopropyl alcohol and cooled the reaction mass to 30°C. Further cooled the reaction mass to 10°C and stirred at same temperature for 3 hours. Filtered the solid and washed with Isopropyl alcohol (100 mL) and water (200 mL). Product was dried under vacuum to obtain title compound. Yield: 62.6 g; Purity by HPLC: 99.5%.

Example-2: Preparation of ethyl 4-(cyanomethyl)-2-ethoxybenzoate
A solution of ethyl 4-(bromomethyl)-2-ethoxybenzoate (100 g) in dichloromethane (700 mL) was slowly added to a mixture of water (60 mL), sodium cyanide (25.94 g) and tetra butyl ammonium bromide (5.05 g) at 30°C. The reaction mass was stirred at the same temperature for 36 hours. After the completion of the reaction, aqueous layer was separated and quenched. The organic layer was washed with water (4x500 mL). The organic layer was separated and evaporated under reduced pressure at 40°C. Isopropyl alcohol (400 mL) was added to the reaction mass and stirred for 10 minutes at the same temperature and concentrated the reaction mass by evaporating half of the Isopropyl alcohol and cooled the reaction mass to 30°C. Further cooled the reaction mass to 10°C and stirred at same temperature for 3 hours. Filtered the solid and washed with Isopropyl alcohol (100 mL) and water (200 mL). Product was dried under vacuum to obtain title compound. Yield: 61.5 g; Purity by HPLC: 99.0%.

Example-3: Preparation of ethyl 4-(bromomethyl)-2-ethoxybenzoate
A mixture of 4-methyl salicylic acid (50 g) in cyclohexane (575 mL) was taken in a flask and potassium carbonate (113.4 g), tetra butyl ammonium bromide (5.29 g) and diethylsulphate (126.5 g) were added at 30°C. Heated the reaction mixture to 80°C and stirred for 6 hours at the same temperature while removing water azeotropically. After completion of the reaction, the reaction mixture was cooled to 30°C. Water (250 mL) was added to the reaction mass and stirred for 10 minutes at 40°C. The organic layer was separated and the aqueous layer was taken into a flask. Water (100 mL) and cyclohexane (100 mL) were added to the aqueous layer and stirred for 20 minutes at 40°C and separated the organic layer. The combined organic layer was taken into a flask and water (3x200 mL) was added and stirred at reflux temperature for 1 hour. Separated the organic layer and repeated washing with water (2x200 mL). The organic layer was heated at reflux for 2 hours to remove water azeotropically. Azobisisobutyronitrile, AIBN (3.23 g) in cyclohexane (0.3 mL) and 1,3-Dibromo-5,5-dimethylhydantoin, DDH (46.99 g) in cyclohexane (4.6 mL) were added at 80°C sequentially in small lots and stirred for 2.5 hours at the same temperature. The reaction mixture was heated to reflux and washed with water (2x340 mL) at reflux and cooled to 30°C. The cyclohexane layer was washed with saturated solution of sodium bicarbonate (340 mL). Organic layer was separated and concentrated under vacuum. Isopropyl alcohol (200 mL) was added to the concentrate and stirred at 50°C for 30 minutes and filtered the solution and washed the filter with Isopropyl alcohol (50 mL). Cooled the solution to -5°C and stirred for 3 hours at same temperature. Filtered the solid and washed with Isopropyl alcohol (50 mL). Wet solid was combined with Isopropyl alcohol (117 mL) and cyclohexane (39 mL) and heated to 50°C. Cooled the reaction mixture to -5°C and stirred for 3 hours at same temperature. Filtered the solid and washed with cyclohexane (39 mL). The solid was dried at 45°C under vacuum for 10 hours to obtain the title compound. Yield: 34.8 g; Purity by HPLC: 98.6%

Example-4: Preparation of ethyl 4-(bromomethyl)-2-ethoxybenzoate
A mixture of 4-methyl salicylic acid (50 g) in cyclohexane (575 mL) was taken in a flask and potassium carbonate (113.4 g), tetra butyl ammonium bromide (5.29 g) and diethylsulphate (126.5 g) were added at 30°C. Heated the reaction mixture to 80°C and stirred for 6 hours at the same temperature while removing water azeotropically. After completion of the reaction, the reaction mixture was cooled to 30°C. Water (250 mL) was added to the reaction mass and stirred for 10 minutes at 40°C. The organic layer was separated and the aqueous layer was taken into a flask. Water (100 mL) and cyclohexane (100 mL) were added to the aqueous layer and stirred for 20 minutes at 40°C and separated the organic layer. The combined organic layer was taken into a flask and water (3x200 mL) was added and stirred at reflux temperature for 1 hour. Separated the organic layer and repeated washing with water (2x200 mL). The organic layer was heated at reflux for 2 hours to remove water azeotropically. Azobisisobutyronitrile, AIBN (3.23 g) in cyclohexane (0.3 mL) and 1,3-Dibromo-5,5-dimethylhydantoin, DDH (46.99 g) in cyclohexane (4.6 mL) were added at 80°C sequentially in small lots and stirred for 2.5 hours at the same temperature. The reaction mixture was heated to reflux and washed with water (2x340 mL) at reflux and cooled to 30°C. The cyclohexane layer was washed with saturated solution of sodium bicarbonate (340 mL). Organic layer was separated and concentrated under vacuum. Isopropyl alcohol (200 mL) was added to the concentrate and stirred at 50°C for 30 minutes and filtered the solution and washed the filter with Isopropyl alcohol (50 mL). Cooled the solution to -5°C and stirred for 3 hours at same temperature. Filtered the solid and washed with Isopropyl alcohol (50 mL). Wet solid was combined with Isopropyl alcohol (115.5 mL) and cyclohexane (38.5 mL) and heated to 50°C. Cooled the reaction mixture to -5°C and stirred for 3 hours at same temperature. Filtered the solid and washed with cyclohexane (38.5 mL). The solid was dried at 45°C under vacuum for 10 hours to obtain the title compound. Yield: 34.0 g; Purity by HPLC: 98.9%

Example-5: Preparation of 2-(3-ethoxy-4-(ethoxycarbonyl) phenyl) acetic acid (ECPA)
The ethyl 4-(cyanomethyl)-2-ethoxybenzoate substrate (0.4 g, 20g/L) was added to potassium phosphate buffer (18.5 mL, 100 mM, pH 7.8) and stirred at 600 rpm in a MultiMax vessel at 37 °C. The reaction was initiated with addition of cell-free extract containing the Rhodococcus globerulus amidase and Neurospora crassa OR74A nitrilase enzymes (750 µl, 37.5 wt % each) and the pH maintained in the 20 mL reaction by addition of 0.5 M NaOH. At 18 hours, an additional dose of enzyme (750 µl, 37.5 wt % each) was added to the reactions. After 42 hours, the reaction was sampled by removal of ~ 25 µl reaction mixture into 1 mL MeCN:dH2O (1:1) for LC-MS analysis which confirmed that conversion is > 99 %. The base addition was stopped and the pH adjusted to ~ 3.0 with 1M HCl. The product was extracted with ethyl acetate (3 x 30 mL) and the combined organic layer was dried over magnesium sulfate. The organic layer was filtered and concentrated under vacuum to yield ECPA (0.406g, 94.5 % isolated yield) as a yellow solid which was analysed by 1H NMR (CDCl3) and LC-MS to confirm the nature of the title compound.
,CLAIMS:We Claim:
1. A process for the preparation of compound of formula I comprising the step of hydrolyzing compound of formula II in the presence of an enzyme, wherein R is C1-6 alkyl.

2. A process of claim 1, wherein the enzyme is a mutated enzyme capable of hydrolysis of compound of formula II to compound of formula I.
3. A process of claim 1, wherein enzyme is a nitralase enzyme selected from a group comprising of ES-NIT-103, ES-NIT-104, ES-NIT-111, ES-NIT-119, ES-NIT-120, ES-NIT-121, ES-NIT-126, ES-NIT-129, Nitrilase 14, Zea mays, Neurospora crassa OR74A.
4. A process of claim 1, wherein the hydrolysis of compound of formula II is carried out in presence of an additional enzyme.
5. A process of claim 4, wherein the additional enzyme is an amidase selected from the group comprising of Rhodococcus globerulus and Aureobacterium sp. amidases.
6. A process for the preparation of ECPA comprising the step of hydrolyzing compound of formula IIa in the presence of an enzyme.

7. A process of claim 6, wherein the enzyme is a mutated enzyme capable of hydrolysis of compound of formula IIa to ECPA.
8. A process of claim 6, wherein the enzyme is a nitralase such as Neurospora crassa OR74A.
9. A process of claim 6, wherein the hydrolysis of compound of formula IIa is carried out in presence of an additional enzyme.
10. A process of claim 9, wherein the additional enzyme is an amidase selected from the group comprising of Rhodococcus globerulus amidase.