An Efficient and Novel Synthesis of Pregabalin with Enantioselective Enzymatic Hydrolysis using CAL-B Enzyme

 

Kishore Gokavarapu1*, Rahul Uttamrao Devkar1, Sarita Gokavarapu3, Shiva Rama Krishna Samala2, Durga Prasad Rao3

1Production Department-Helios Lifesciences Limited, 79 and 100, Industrial Growth Center, Malanpur,

District: Bhind, MP–477117

2 Research and Development Department–Helios Lifesciences Limited, 79 & 100, Industrial Growth Center, Malanpur, District: Bhind, MP-477117

3QC and ADL Department–Helios Lifesciences Limited, 79 & 100, Industrial Growth Center, Malanpur, District: Bhind, MP-477117

*Corresponding Author E-mail: g.kishore@live.com

 

ABSTRACT:

The present paper describes the CAL-B (Enzymatic) desmmetrization of Prochiral Dimethyl 3-Isobutylpentanedioate was performed to prepare optically active (S)-3-(2-methoxy2-oxoethyl)-5-methylhexanoic acid. Based on this reaction, the synthesis of (S)-(+)-3-aminomethyl-5-methylhexanoic acid (S-Pregabalin) was achieved with a 70-75% overall yield.

 

KEYWORDS: Pregabalin, CAL-B, Enantioselective, Enzyme, Hydrolysis.

 

 

INTRODUCTION:

(S)-(+)-3-aminomethyl-5-methylhexanoic acid (Pregabalin) is a lipophilic γ-amino butyric acid (GABA) analogue that was developed for the treatment of central nervous system disorders including epilepsy, neuropathic pain, and anxiety. During the initial process development of Pregabalin, several routes were examined in detail. The first-generation manufacturing process (Scheme I) was selected on the basis of overall yield, cost, and high throughput and executed as a racemic synthesis followed by resolution with (S)-(+)-mandelic acid.

 

  

Scheme-I: First Generation Manufacturing process:

 

 

Scheme-II

Another latest industrial commercial Manufacturing process of racemic synthesis followed by resolution with (R)-(+)-Phenyl ethylamine (MBA)

 

Obtaining an optically pure isomer that is free from other stereoisomers is strongly demanded, particularly in the pharmaceutical industry, 1 which drives the development of various catalysts including enzymes. Among many industrially applicable enzymes, lipases are the most useful to catalyze diverse reactions based on the carbonyl functionality of carboxylic acids and their derivatives.2 Many different types of lipases are available from various sources including mammalian cells and diverse microorganisms. Furthermore, most lipases are tolerant of a wide spectrum of substrates and reaction media. However, there is a significant limitation in obtaining optically pure compounds from lipase-mediated resolution of racemates, and only 50% reaction yields can be obtained. To overcome this limitation, racemization of the unreacted isomer with a strong base or a metal complex is a possible alternative.3 Desmmetrization is another way to produce an optically pure reaction product with a 100% theoretical yield in the absence of a racemizer.4 various chemical and enzymatic methods have been reported for desmmetrization to yield optically pure products. However, few studies have. Reported the chemical and enzymatic desmmetrization of 3-alkylglutaric acid diesters to obtain chiral 3-alkylglutaric acid monoesters, which are good starting materials for biologically important molecules.5 In this study, enzymatic desmmetrization of 3-alkylglutaric acid diesters was undertaken to achieve a practical synthetic route to prepare Pregabalin. We describe one of the most efficient synthetic routes for Pregabalin through lipase mediated desmmetrization of 3-alkylglutaric acid diesters. In addition, a systematic study of the enzymatic desmmetrization of the 3-alkylglutaric acid diesters provides valuable information regarding the active site binding mode, which is dependent not only on the alkyl group at C3 but also the alkyl group at the ester. Many structurally diverse organic molecules have been utilized as lipase substrates for reactions, including hydrolysis, esterification and ammoniolysis, some of which provide quite clear pictures of the binding mode near the active site residues combined with their crystalline structures. However, there are still many uncertainties predicting the reactivity and stereo selectivity of certain substrates due to enzyme mobility and remote interactions. Throughout this study, we found methyl ester showed better stereo selectivity with possible remote interactions between the substrate and the enzyme active site.

 

RESULTS AND DISCUSSION:

The enzymatic route of synthesis of S-Pregabalin key starting material is Isovelardehyde, reacted with cyanoacetamide in the presence of potassium hydroxide water as medium followed by acidic hydrolysis gave the diacid (CMMHA), obtained diacid converted as diester in the presence of methanol under catalytic acidic condition, diester converted as monoester in the presence of CAL-B enzyme as R isomer with 95.5% of optical purity which was converted as amide in the presence of Aqueous ammonia and finally with Hoffman reaction gave the desired isomer of S-Pregabalin.

 

Route of synthesis:

Experimental protocols

Using CAL-B Enzyme we have conducted few experiments on Diester (3) to get optically pure isomer of Monoester (4), same mentioned in table. Out of all reaction conditions Aq. Ammonia reaction gave the desired and enantioselective pure product.

 

 

 

Diester (3) to Monoester (4) with CAL-B Enzyme and Chiral purity study

S.No

Diester (3)

Solvent

Temperature

Base

Chiral purity of (4)

1

Dimethyl 3-Isobutylpentanedioate (3)

Water

15-20oC

K2CO3

85:15

2

Na2CO3

87:13

3

Cs2CO3

83:17

4

NHCO3

88:12

5

KHCO3

86:14

6

KOH

79:21

7

NaOH

80:20

8

Aq. NH3

95.5:4.5

 

 

CONCLUSION:

We have been developed a new, facile and efficient process for preparation of (S)-Pregabalin enantioselective enzymatic hydrolysis with CAL-B enzyme, also explained and shown the detailed experimental study with all the identified observations. Chiral selectivity enhanced in experiment aqueous base with CAL-B enzyme, water as medium at 15-20oC.

 

ACKNOWLEDGMENT:

The authors sincerely acknowledge the constant support and encouragement received from Mr. Sunil Gandhi Chairman, Helios Lifesciences Limited

 

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Received on 24.12.2018                    Modified on 22.02.2019

Accepted on 04.04.2019                   ©AJRC All right reserved

Asian J. Research Chem. 2019; 12(2):55-57.

DOI:10.5958/0974-4150.2019.00012.9