FIELD OF THE INVENTION

The present invention relates to a process for the preparation of (S)-N-Ethyl-N-methyl-3-[l-(dimethylamino)ethyl]-phenyl-carbamate [Rivastigmine] or its pharmaceutically acceptable salts. More particularly, the present invention relates to an enzymatic process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester and further conversion of the same to Rivastigmine.

BACKGROUND OF THE INVENTION

Rivastigmine, (S)-N-Ethyl-N-methyl-3-[l -(dimethylamino)ethyl]-phenyl carbamate, marketed in the form of its tartarate salt, is a reversible cholinesterase inhibitor, prescribed for the treatment of mild to moderate Alzeimers disease.

Rivastigmine in its tartarate form, i.e (S)-N-Ethyl-N-methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate hydrogen-(2R,3R)-tartarate is marketed under the brand name of Exelon in the form of capsules and oral solution by Novartis. Exelon Capsules contain Rivastigmine tartarate, equivalent to 1.5, 3, 4.5 and 6 mg of Rivastigmine base for oral administration. Exelon Oral Solution is supplied as a solution containing Rivastigmine tartarate, equivalent to 2 mg/MmL of Rivastigmine base for oral administration.

Rivastigmine and its pharmaceutical activity is first disclosed in US 4,948,807, wherein racemic Rivastigmine is prepared by reacting a-m-hydroxyphenylethyldimethylamine with carbamoyl chloride in the presence of sodium hydride.

US 5,602,176 claims hydrogen tartarate salt of Rivastigmine and process for the preparation of Rivastigmine comprises reacting m-hydroxyphenylethyldimethyl amine with N-ethylmethylcarbamoyl chloride in presence of NaH to obtain Rivastigmine. It also describes resolution of Rivastigmine using di-O,O'-p-toluoyl tartaric acid.

Chemical Communications, 2010,46, 5500-5502; discloses chemo enzymatic asymmetric total synthesis of (S)-Rivastigmine using ©-transaminases and the reaction pathway is depicted below.

J. Org. Chem. 2009, 74, 5304-5310 discloses chemo enzymatic asymmetric total synthesis of (S)-Rivastigmine using lipase and the reaction pathway is depicted below.

J. Org. Chem. 2010, 75, 3105-3108 discloses chemo enzymatic asymmetric total synthesis of (S)-Rivastigmine using lipase and the reaction pathway is depicted below.

The present inventors have developed an enzymatic route for the preparation of Rivastigmine and its pharmaceutically acceptable salts. The enzyme used acts as a catalyst and can be recovered, reused making the process cost effective, convenient than chemical method, environmental friendly and industrially viable.

OBJECT OF THE INVENTION

The principle object of the present invention is to provide an enzymatic process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester and further conversion of the same to Rivastigmine or its pharmaceutically acceptable salts.

One object of the present invention is to provide novel enzymatic process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester.

Another object of the present invention is to provide a process for further conversion of above compound ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester to Rivastigmine or its pharmaceutically acceptable salts.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a novel enzymatic process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester comprising the steps of:
a) reducing ethyl methyl carbamic acid 3-acetyl-phenyl ester using an enzyme; and
b) isolating ethyl-methyl-carbarnic acid-3-[lS-hydroxy-ethyl)-phenyl ester.

Another aspect of the present invention is to provide a process for further conversion of above obtained compound ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester to Rivastigmine or its pharmaceutically acceptable salts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel process for the preparation of (S)-N-Ethyl-N-methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate Rivastigmine or its pharmaceutically acceptable salts.

More particularly, the present invention provides novel enzymatic process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester and further conversion of the same to Rivastigmine or its pharmaceutically acceptable salts.

Accordingly, the present invention provides a process for the preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester comprising the steps of:

a) reducing ethyl methyl carbamic acid 3-acetyl-phenyl ester using an enzyme; and
b) isolating ethyl-methyl-carbamic acid-3 - [ 1 S-hydroxy-ethyl)-phenyl ester.

The reaction pathway is schematically represented below.

Ethyl-methyl-carbamic acid

Ethyl-methyl-carbamic acid 3-[lS-hydroxy-ethyl)-phenyl ester ester 3-acetyl-phenyl ester

According to the present invention, ethyl methyl carbamic acid 3-acetyl-phenyl ester is treated with a ketoreductase [KRED] in presence of co-enzyme such as NADP, NADPH, potassium phosphate, glucose, glucose dehydrogenase or mixtures thereof. The reaction mixture is maintained for 20-40°C for 12-36 hrs. After completion of the reaction, product is isolated into organic layer using methylene dichloride to obtain ethyl-methyl-carbamic acid-3-(lS-hydroxy-ethyl)-phenyl ester.

The "KRED" or ketoreductase enzyme used in the present invention refers to an enzyme that catalyzes the reduction of a ketone to form the corresponding alcohol. Ketoreductase enzymes include, for example, those classified under the Enzyme Commission ("EC") numbers of 1.1.1. Such enzymes are given various names in addition to ketoreductase, including, but not limited to, alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, 0-hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase, and the like. NADPH-dependent ketoreductases are classified under the EC number of 1.1.1.2 and the CAS number of 9028-12-0. NADH-dependent ketoreductases are classified under the EC number of 1.1.1.1 and the CAS number of 9031 -72-5. Ketoreductases are commercially available, for example, from Syncore Laboratories (Shanghai) co., Ltd under the catalog numbers KRED- 101 to ES-KRED-180.

Suitable Ketoreductases include, but are not limited to, Syncore Laboratories products with catalog numbers labeled as KRED-101; KRED-102; KRED-103; KR.ED-104; KRED-105; KRED-106; KRED-107; KRED-108; KRED-109; KRED-110; KRED-111; KRED-112; KRED-113; KRED-114; KRED-115; KRED-116; KRED-117; KRED-118; KRED-119; KRED-120; KRED-121; KRED-122; KRED-123 ; KRED-124; KRED-125; KRED-126; KRED-127; KRED-128; KRED-129; KRED-130; KRED-131; KRED-132; KRED-133; KRED-134; KRED-135; KRED-136; KRED-137; KRED-138. KRED-139; KRED-140; KRED-141; KRED-142; KRED-143; KRED-144; KRED-145; KRED-146; KRED-147; KRED-148; KRED-149; KRED-150; KRED-151; KRED-152; KRED-153 ; KRED-154; KRED-155; KRED-156; KRED-157; KRED-158; KRED-159; KRED-160; KRED-161; KRED-162; KRED-163; KRED-164; KRED-165; KRED-166; KRED-167; KRED-168. KRED-169; KRED-170; KRED-171; KRED-172; KRED-173; KRED-174; KRED-175; KRED-176; KRED-177; KRED-178; KRED-179; KRED-180.

Crude alcohol dehydrogenase enzyme used in the present invention is prepared from commercially available baker yeast. Alcohol dehydrogenase are not limited to Sacchromyces species, but also to Rhodococus species, Lactobacillus species, Pseudomonus species. Candida species, Escherichia species and any recombinant species.

According to present invention, crude alcohol dehydrogenase from Saccharomyces cerevasiae was prepared from Sigma Aldrich or from commercially available yeast as per the processes described in prior art such as Biochem. J; (1970) 118, 375-378.

According to the present invention, reduction of compound ethyl methyl carbamic acid 3-acetyl-phenyl ester uses a co-factor with the ketoreductase enzyme. The co-factor is selected from the group consisting of NADH, NADPH, NAD+, NADP+, salts thereof or analogs thereof.

According to the present invention, reduction of ethyl methyl carbamic acid 3-acetyl-phenyl ester comprises a co-factor regeneration system. A co-factor regeneration system comprises a substrate and a "dehydrogenase enzymes". Preferably, the co-factor regeneration system comprises a substrate/dehydrogenase pair selected from the group consisting of D-glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase and phosphite/phosphite dehydrogenase. Another co-factor regeneration system comprises a alcohol selected from the group of secondary alcohol like isopropyl alcohol, 2-butanol and cyclohexonal.
Glucose dehydrogenase (GDH) includes, for example, those classified under the EC number 1.1.1.47 and the CAS number 9028-53-9, and are commercially available, for example, from Syncore Laboratories under the catalog number ES-GDH-101 to ES-GDH-104 or Codexis, Inc. under the catalogue number GDH-CDX-901, GDH 102 and GDH 103. Preferably, the glucose dehydrogenase is selected from the group consisting of the predominant enzyme in each of Syncore Laboratories products with catalogue numbers ES-GDH-101, ES-GDH-102, ES-GDH-103, ES-GDH-104 and Codexis Inc's products with catalog numbers GDH-CDX901, and mixtures thereof.

According to the present invention chiral selective reduction of ethyl methyl carbamic acid 3-acetyl-phenyl ester is carried out using Microorganisms like baker yeast, Rhodococus species, Lactobacillus species, Pseudomonus species. Saccharomyces species, Candida species, Escherichia species and any recombinant species.

According to the present invention, chiral selective reduction of ethyl methyl carbamic acid 3-acetyl-phenyl ester to the desired product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester is cost effective, less cumbersome than chemical method, environment friendly and enantiomeric excess of 99% is obtained.

According to the present invention, hydroxy acetophenone is reacted with N-ethyl-N-methyl carbamoyl chloride using a base such as triethylamine to obtain ethyl methyl carbamic acid 3-acetyl-phenyl ester.

In another embodiment, the present invention provides a process for further conversion of the above obtained compound ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester to Rivastigmine or its pharmaceutically acceptable salts.

According to the present invention, Rivastigmine is prepared by a process described in prior art such as WO 2005/061446 A2, WO 2005/058804 and J. Org. Chem. 2010, 75, 3105-3108.

The following examples will further illustrate the invention, without, however, limiting it thereto. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.

Examples:

Example 1: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl
Ester

ES-KRED-101 to 180 ES-KREDs (5 mg, Syncore Laboratories (Shanghai) co., Ltd) each was dissolved in 5ml buffer (containing 100mM potassium phosphate, 5mg NADP-',
330mM D-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (10 mg in 0.1 ml) was added.

The mixture was stirred at 30 °C for 24 hrs and monitored by HPLC. Methylene dichloride (5 ml) was added the phases were separated. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, cc: 99%).

Example 2: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester

ES-KRED-119 (200 mg, Syncore Laboratories (Shanghai) co., Ltd) was dissolved in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NADP'., 330mM l)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added, the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, ee: 99%).

Example 3:

Ketoreductases were screened for effective reduction of Ethyl methyl carbamic acid-3-acetyl phenyl ester and the results are tabulated below.

Example 3: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using alcohol dehydrogenase from Saccharomyces cerevasieae

Alcohol dehydrogenase from Saccharomyces cerevasieae (Sigma Aldrich) was dissolved in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NAD'., 330mM 1)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added; the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, ee: 99%).

Example 4: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using alcohol dehydrogenase from Lactobacillus kefir

Alcohol dehydrogenase from Lactobacillus kefir (Sigma Aldrich) was dissolved in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NAD'., 330mM l)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added, the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, ee: 99%).

Example 5: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using Saccharomyces cerevasieae Type II

Saccharomyces cerevasieae Type I (Sigma Aldrich) was mixed in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NAD'., 330mM l)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%), ee: 99%).

Example 6: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using Saccharomyces cerevasieae Type II

Saccharomyces cerevasieae Type II (Sigma Aldrich) was mixed in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NAD'., 330mM l)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, ee: 99%).

Example 7: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using commercially available baker yeast

Commercially available baker yeast was mixed in 150 ml buffer (containing 0.25 mM potassium phosphate, 5mg NAD'., 330mM l)-glucose, 2 U/ml glucose dehydrogenase, pH 7.0). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (2 g in 15 ml) was added. pH 7.0 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 24 hrs and monitored by HPLC. Methylene dichloride (200 ml) was added the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 60-99%, ee: 99%).

Example 8: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using crude alcohol dehydrogenase from Saccharomyces cerevasieae Type I (Sigma aldrich)

A 20 g of yeast was first grinded in a mixer and stirred in 40 mL of toluene at 40 °C for 1 hour. It was then cooled to room temperature and mixed with 60 mL 1 mM EDTA solution and stirred slowly for 1 hour at 5-10 °C. The mixture was filtered through filter cloth, followed by hyflow under gentle suction. The result clear yellowish liquid (crude alcohol dehydrogenase extract) is then mixed with 200 mL of buffer (containing 0.25 mM potassium phosphate, lg NAD'., 50g glucose, lg glucose dehydrogenase, pH 6.8). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (10 g in 30 ml) was added. pH 6.8 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 16-24 hrs and monitored by HPLC. Methanol (500 mL) was added to reaction mixture and filtered through celite, Methanol was distilled off. Methylene dichloride (200 ml) was added, the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 75-99%, ee: >99%).

Example 9: Preparation of ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester using crude alcohol dehydrogenase from commercially available baker yeast

A 20 g of yeast was first grinded in a mixer and stirred in 40 mL of toluene at 40 °C for 1 hour. It was then cooled to room temperature and mixed with 60 mL 1 mM EDTA solution and stirred slowly for 1 hour at 5-10 °C. The mixture was filtered through filter cloth, followed by hyflow under gentle suction. The result clear yellowish liquid (crude alcohol dehydrogenase extract) is then mixed with 200 mL of buffer (containing 0.25 mM potassium phosphate, lg NAD'., lg glucose, 2 U/ml glucose dehydrogenase, pH 6.8). A solution of Ethyl methyl carbamic acid-3-acetyl phenyl ester in dimethyl sulfoxide (10 g in 30 ml) was added. pH 6.8 was maintained by adding 1 M NaOH. The mixture was stirred at 30 °C. for 16-24 hrs and monitored by HPLC. Methanol (500 mL) was added to reaction mixture and filtered through celite, Methanol was distilled off. Methylene dichloride (200 ml) was added, the phases were separated, dried over sodium sulfate and distilled off. The obtained alcohol product ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester in the organic phase was analyzed (Yield: 75-99%, ee: >99%).

We Claim:

1. A process for preparing ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl
ester comprising the steps of:

a) reducing ethyl methyl carbamic acid 3-acetyl-phenyl ester using an enzyme; and

b) isolating ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester.

2. The process of claim 1, wherein the enzyme is selected from ketoreductase enzymes, alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, P-hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase.

3. The process of claim 2, wherein the enzyme is ketoreductase.

4. The process of claim 3, wherein the ketoreductase enzyme is selected from KRED-101; KRED-102; KRED-103; KR.ED-104; KRED-105; KRED-106; KRED-107; KRED-108; KRED-109; KRED-110; KRED-111; KRED-112; KRED-113; KRED-114; KRED-115; KRED-116; KRED-117; KRED-118; KRED-119; KRED-120; KRED-121; KRED-122; KRED-123 ; KRED-124; KRED-125; KRED-126; KRED-127; KRED-128; KRED-129; KRED-130; KRED-131; KRED-132; KRED-133; KRED-134; KRED-135; KRED-136; KRED-137; KRED-138. KRED-139; KRED-140; KRED-141; KRED-142; KRED-143; KRED-144; KRED-145; KRED-146; KRED-147; KRED-148; KRED-149; KRED-150; KRED-151; KRED-152; KRED-153 ; KRED-154; KRED-155; KRED-156; KRED-157; KRED-158; KRED-159; KRED-160; KRED-161; KRED-162; KRED-163; KRED-164; KRED-165; KRED-166; KRED-167; KRED-168. KRED-169; KRED-170; KRED-171; KRED-172; KRED-173; KRED-174; KRED-175; KRED-176; KRED-177; KRED-178; KRED-179;KRED-180.

5. The process of claim 2, wherein the ketoreductase enzyme is used in presence of a co-enzyme.

6. The process of claim 5, wherein the co-enzyme is selected from NADP, NADPH, potassium phosphate, glucose, glucose dehydrogenase or mixtures thereof.

7. A process for preparing ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester comprising the steps of:

a) reducing ethyl methyl carbamic acid 3-acetyl-phenyl ester using a microorganism; and

b) isolating ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester.

8. The process of claim 7, wherein the microorganism is selected from Rhodococus species, Lactobacillus species, Pseudomonus species. Saccharomyces species, Candida species, Escherichia species and any recombinant species.

9. The process of the proceeding claims, wherein the obtained ethyl-methyl-carbamic acid-3-[lS-hydroxy-ethyl)-phenyl ester is further converted into Rivastigmine.