DESC:FORM 2

THE PATENTS ACT, 1970

(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION

(Section 10 and Rule 13)

A PROCESS FOR THE PREPARATION OF
OPTICALLY ACTIVE SECONDARY ALCOHOLS

AUROBINDO PHARMA LTD HAVING CORPORATE OFFICE AT
GALAXY, FLOORS: 22-24,
PLOT No.1, SURVEY No.83/1,
HYDERABAD KNOWLEDGE CITY,
RAIDURG PANMAKTHA,
RANGA REDDY DISTRICT,
HYDERABAD – 500 032,
TELANGANA, INDIA
AN INDIAN ORGANIZATION

The following specification particularly describes and ascertains the nature of this invention and the manner in which the same is to be performed:

FIELD OF THE INVENTION

The present invention is related to a process for the preparation of optically active secondary alcohols of formula I.

Formula I


wherein * is an optically active (R) or (S)- configuration; n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is a halogen or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups.

The present invention specifically provides a process for the preparation of (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol, which is an intermediate of Duloxetine.

Formula II

BACKGROUND OF THE INVENTION

Ketoreductases (KREDs, also called ‘alcohol dehydrogenases’, ADHs, or ‘carbonyl reductases’) catalyze the reduction of aldehydes and ketones to the corresponding primary and secondary alcohols, respectively. These enzymes are also capable of catalyzing the reverse reaction, i.e. the oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, respectively.

For industrial applications, the reduction of ketones to secondary alcohols is of great interest since prochiral carbonyl compounds are stereoselectively reduced to chiral alcohols. In some industrial applications, also the stereoselective conversion of secondary alcohols to ketones for chiral resolution of racemic compounds is desired, e.g. allowing the isolation of enantiomers. The enzymatic oxidation of primary alcohols to aldehydes and the enzymatic reduction of aldehydes to primary alcohols are often considered of lower relevance in industrial applications but are also catalyzed by KREDs. The use of a same KREDs for oxidation or reduction reaction, respectively, can be influenced by adjusting the chemical equilibrium of the enzyme reaction.

The reduction catalyzed by KREDs requires a reduced cofactor as the electron donor. Some KREDs use reduced nicotinamide adenine dinucleotide (NADH) as a cofactor, other KREDs use reduced nicotinamide adenine dinucleotide phosphate (NADPH) and some ketoreductases accept both, NADH and NADPH. Commercially available KREDs are derived from horse liver (HLADH), baker's yeast (YADH) and from bacteria, such as Thermoanaerobium brockii (TBADH) and Lactobacillus kefir (LKADH).

For industrial applications, it is desirable to employ KREDs with high specific activity and stereoselectivity. Another important criterion in the industrial use of KREDs is long process stability, which often correlates with high stability at elevated temperatures and high solvent stability. If the substrates are chiral already, it is further desirable to employ KREDs with high stereospecificity.

US7785847 discloses a process for stereospecifically reducing substituted alkanones using the enzymes with dehydrogenase activity, prepared from microorganisms of the genus Azoarcus, are capable of stereospecifically catalyzing the reaction with simultaneous cofactor regeneration to produce corresponding optically active alcohols.

US7888080 discloses a process for stereospecifically reducing substituted alkanones using the enzymes with dehydrogenase activity, prepared from microorganisms of the genus Lactobacillus, in particular of the species Lactobacillus brevis to produce corresponding optically active alcohols.

US 8426178 discloses a process for the preparation of optically active alcohols using engineered ketoreductase (KRED) polypeptides, wherein the engineered ketoreductase polypeptides have an improved property as compared to the naturally-occurring wild-type ketoreductase enzymes obtained from Lactobacillus kefir, Lactobacillus brevis, or Lactobacillus minor.

US 10093905 discloses new ketoreductases, particularly engineered ketoreductases exhibiting improved properties compared to the wild wild-type enzyme and their use in the carbonyl reduction to produce corresponding optically active alcohols.

However, there is a need for alternative approach to obtain the optically pure secondary alcohols with high conversion and isolated yield by utilizing an in-situ cofactor recycling system under environmentally benign conditions.

Hence, present invention cultured a cost-effective and commercially viable process for the preparation of optically active secondary alcohols of formula I.

The present invention is directed towards an enzymatic reduction using ketoreductase to produce optically active secondary alcohols of formula I from the corresponding carbonyl compounds of formula IA. In addition, glucose dehydrogenase (GDH) will be combined with the above process for cofactor (NADH/NADPH) regeneration using glucose as a co-substrate.

Formula IA


wherein n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is halogen, or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups.

OBJECTIVE OF INVENTION

The main objective of the present invention is to provide a simple, industrially feasible and cost-effective process to produce optically active secondary alcohol of formula I with high purity and good yield on a commercial scale.

SUMMARY OF THE INVENTION
The main embodiment of the present invention is to provide a process for the preparation of optically active secondary alcohols of formula I;

Formula I

wherein * is an optically active (R) or (S)- configuration; n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is a halogen or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups;
which comprises, providing the carbonyl compounds of formula (IA) and ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 or Sequence ID No. 4,

Formula IA


wherein n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is a halogen, or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups; to produce optically active alcohol of formula I.

Yet another embodiment of the present invention provides the use of ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 or Sequence ID No. 4 in the reduction of compound of formula (IA).

Formula IA

Yet another embodiment of the present invention provides a process for the preapration of (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol of formula (II)

Formula II

which comprises treating 3-(dimethylamino)-1-(thiophen-2-yl) propan-1-one of formula (IIA) or its salts

Formula IIA
with ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 or Sequence ID No. 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for the preparation of optically active secondary alcohols of formula I.

The process comprises, providing the carbonyl compounds of formula (IA) and ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 or Sequence ID No. 4 to produce the corresponding optically active secondary alcohols of formula I.
The ketoreductase enzymes (sequence ID. 1 and sequence ID. 2) used in the above reactions are derived from Serratia marcescens and Bosea caraganae respectively.

The glucose dehydrogenase enzyme (sequence ID no. 3) used in the above reaction is derived from Bacillus subtilis.

The reaction is carried out with carbonyl compound using NAD(P)H as a cofactor, glucose as a co-substrate, ketoreductase, and glucose dehydrogenase.

Yet another embodiment of the present invention provides the use of ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 or Sequence ID No. 4, in the reduction of compound of formula (IA).

The ketoreductase enzymes (sequence ID. 1 and sequence ID. 2) used in the above reactions are derived from Serratia marcescens and Bosea caraganae respectively.

The glucose dehydrogenase enzyme (sequence ID no. 3) is derived from Bacillus subtilis, while glucose dehydrogenase with sequence ID No. 4 is its modified mutant.

In a preferred embodiment the optically active secondary alcohol of formula (I) is selected from (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol of formula (II) or (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol of formula (III).

Formula II

Formula III

(S)-3-(Dimethylamino)-1-(thiophen-2-yl)propan-1-ol of formula (II) is prepared by reducing carbonyl compound of formula (IA), which is is selected from 3-(dimethylamino)-1-(thiophen-2-yl) propan-1-one (IIA).

The process is carried out by using ketoreductase enzyme having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenase having the nucleotide sequence of Sequence ID No. 3 or sequence ID No. 4.

The culture of CR-1 (SEQ ID NO 1) is grown in LB media with kanamycin antibiotic. Primary culture, LB media or TB media is inoculated with the previously prepared CR-1 glycerol stock and is grown at 37oC. The secondary culture, LB media or TB media was inoculated with 2% of primary culture and incubated at 37 oC, until the OD600 reaches 0.6 (3.0 to 4.0 h). Then, the culture is cooled down to room temperature and induced using IPTG with LB and TB media respectively. The culture with LB media is incubated at 20 oC, whereas TB media is incubated at 25 oC by following the same condition. Then, the cells are harvested by centrifugation. The harvested cells are lysed by sonication at amplitude followed by centrifugation to remove cell debris. The supernatant (cell lysate) is subjected for protein purification using Ni-NTA resin. The buffers used for CR-1 protein purification are as follows: Lysis Buffer - NaH2PO4, NaCl, pH 7.0, Wash Buffer - NaH2PO4, Imidazole, NaCl, pH 7.0 and Elution Buffer - NaH2PO4, Imidazole, NaCl, pH 7.0. After confirming the presence of proteins in the collected fractions by SDS-PAGE, dialysis was carried out to remove salts and imidazole. The purified enzymes were used as such or frozen with liquid nitrogen and stored at -80 oC for further use.

The protein purifications are carried out on AKTA protein explorer system.

The buffers used for CR-1 protein purification comprises, Lysis Buffer - 50 mM NaH2PO4, 300 mM NaCl, pH 7.0, Wash Buffer - 50 mM NaH2PO4, 30 mM Imidazole, 300 mM NaCl, pH 7.0 and Elution Buffer - 50 mM NaH2PO4, 300 mM Imidazole, 300 mM NaCl, pH 7.0.

The purified enzymes are used as such or frozen with liquid nitrogen and stored at -80 ºC for further use.

The salts as mentioned in the present invention prefereably selected from H2SO4, HCl, NH4HSO4, (NH4)2SO4, acetic acid, NH4Cl, H3PO4, (NH4)H2PO4, HClO4 or (NH4)ClO4.

The compound (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol (II) prepared according to the present invention is converted to Duloxetine or its salts as reported in the literature.

The invention is illustrated with the following examples, which are provided by way of illustration only and should not be construed to limit the scope of the invention in any manner whatsoever.

EXAMPLE 1: Synthesis of (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol (II):

Biotransformation was performed with cell-free extracts obtained from CR-1 and GDH cell cultures on a 20 mL scale. The reaction was carried out with 5 mL each of CR-1 and GDH cell-free extracts, 25 mM substrate 3-(dimethylamino)-1-(thiophen-2-yl)propan-1-one (IIA), 0.5 mM NADP+ and 50 mM glucose in 100 mM sodium phosphate buffer. The reaction mixture was incubated at 28ºC and 100 rpm. After 12 h, the pH of the reaction mixture was adjusted to 11, extracted with ethyl acetate, concentrated under reduced pressure. Similar protocol was adopted for biotransformation with purified enzymes where the final concentration of enzymes CR-1 and GDH is 1 mg/mL and 0.5 mg/mL respectively.

The enantiopurity of the product was determined as > 95% on HPLC using the chiral column (Chiralpak IA3).

EXAMPLE 2: Whole Cell biotransformations for the synthesis of (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol (II):

In whole cell biotransformations, 1.2 g of the pellet of CR-1 cells cultured in TB media and 0.3 g of GDH cells were used. The final substrate concentration is 25 mM, NADP+ is 0.5 mM and concentration of glucose is 500 mM. The final reaction volume is 10 mL in sodium phosphate buffer (100 mM NaH2PO4, pH 7.0). The reactions were proceeded at 30ºC and 150 rpm. The pH of the reaction mixture was adjusted to 7.0 for every two hours with 6 M NaOH. After the respective reaction times, the pH of the reaction mixtures was bought to 11, extracted with HPLC grade ethyl acetate, dried over magnesium sulphate

The enantiopurity of the product was determined as > 95% on HPLC using the chiral column (Chiralpak IA3).

Sequence Listing

<110> Applicant name: Aurobindo Pharma Limited
<120> Title of invention: A process for the preparation of optically active secondary alcohols
<160> Number of SEQ ID NOs: 4
<210> SEQ ID NO: 1
<211> Length: 777
<212> Type: Artificial DNA
<213> Organism: Serratia marcescens
<400> Sequence: 1

1 ATGGGCAGCA GCCATCACCA CCATCATCAC ACCACCGCGC ACCCGCTGCA GGGCAAGGTG
61 GCGTTCGTTC AAGGTGGCAG CCGTGGTATT GGTGCGGCGA TTGTGAAACG TCTGGCGAGC
121 GAGGGTGCGG CGGTTGCGTT TACCTACGCG GCGAGCGCGG ATCGTGCGGA GGCGGTGGCG
181 AGCGCGGTTA CCACCGCGGG TGGCAAGGTG CTGGCGATCA AAGCGGACAG CGCGGATGCT
241 GCGGCGCTGC AGCAAGCGGT GCGTCAGGCG GTTAGCCACT TCGGCAACCT GGATATTCTG
301 GTGAACAACG CGGGTGTTTT TACCCTGGGT GGCACCGAGG AACTGGCGCT GGACGATCTG
361 GACCGTATGC TGGCGGTGAA CGTTCGTAGC GTGTTCGTTG CGAGCCAAGA GGCGGCGCGT
421 CACATGAACG ATGGTGGCCG TATCATTCAC ATCGGCAGCA CCAACGCGGA ACGTGTGCCG
481 TTTGGTGGCG CGGCGGTTTA CGCGATGAGC AAGAGCGCGC TGGTTGGTCT GACCAAAGGC
541 ATGGCGCGTG ATCTGGGTCC GCGTAGCATT ACCGTGAACA ACGTTCAGCC GGGCCCGGTT
601 GACACCGAGA TGAACCCGGA TGCGGGCGAG TTCGCGGACC AGCTGAAGCA ACTGATGGCG
661 ATCGGTCGTT ATGGCAAAGA CGAGGAAATT GCGGGTTTTG TGGCGTACCT GGCGGGTCCG
721 CAAGCGGGTT ATATCACCGG TGCGAGCCTG AGCATTGATG GTGGCTTCAG CGCGTAA

<210> SEQ ID NO: 2
<211> Length: 696
<212> Type: Artificial DNA
<213> Organism: Bosea caraganae
<400> Sequence: 2

1 ATGGGTCCGC AGACCTGGAT GATCACCGGT GCGAACCGTG GTATTGGCCT GGCGCTGACC
61 ACCGTGCTGC TGGCGCGTGG TGATCACGTT ATTGCGGCGG CGCGTGACCC GAACAGCGGT
121 CCGCTGCGTG CGCTGGCGGA GAAACATCCG GGTCAAGTGA CCCCGCTGGC GCTGGATGTT
181 ACCAGCGACG CGAGCGTGGC GGAAGCGAAA GTTGCGCTGG GTAACCGTCC GATCGATGTG
241 CTGGTTAACA ACGCGGGTAT TTACGGCAAC CGTGACCGTC AGAGCGCGGT GGACATGGAT
301 TTCGACGCGT GGCGTGAGGT TTTTGAAGTG AACGTTTATG CGCCGCTGCG TGTGGCGCAA
361 GCGTTCCTGC CGAACGTTGA GAGCAGCAAG AGCCGTAAAA TCGCGACCAT TAGCAGCCGT
421 ATGGGTAGCA TCGGCAACAA CCCGAGCGGT AGCATTGCGT ACCGTAGCAG CAAGAGCGCG
481 GTGAACATGA CGATGGTGGC GTTCGGCAAC GAAGTGCGTG CGCGTGGTGT TGGCATCTAC
541 CTGTTCCACC CGGGTTGGGT GCAGACCGAT ATGGGTGGCC TGGGTGCGGA CATTGCGCCG
601 AGCCAAAGCG CGACCGGTCT GATCAAAACC ATTGATGCGA GCGGCACCGC GGAGAGCGGC
661 AGCTTTCGTA ACTGGGACGG TGCGCCGATT GCGTGG

<210> SEQ ID NO: 3
<211> Length: 810
<212> Type: Artificial DNA
<213> Organism: Bacillus subtilis
<400> Sequence: 3

1 ATGGGTAGCA GCCACCACCA CCACCACCAC TACCCGGACC TGAAGGGTAA AGTGGTTGCG
61 ATTACCGGTG CGGCGAGCGG TCTGGGCAAG GCGATGGCGA TCCGTTTCGG CAAGGAGCAG
121 GCGAAGGTGG TTATCAACTA CTACAGCAAC AAGCAAGATC CGAACGAGGT TAAGGAAGAG
181 GTGATCAAAG CGGGTGGCGA AGCGGTGGTT GTGCAGGGTG ACGTTACCAA GGAAGAGGAC
241 GTTAAAAACA TCGTGCAAAC CGCGATTAAG GAATTTGGCA CCCTGGACAT CATGATTAAC
301 AACGCGGGCC TGGAGAACCC GGTTCCGAGC CACGAAATGC CGCTGAAGGA CTGGGATAAA
361 GTGATCGGCA CCAACCTGAC CGGTGCGTTC CTGGGCAGCC GTGAGGCGAT CAAGTACTTC
421 GTTGAAAACG ACATCAAGGG CAACGTTATT AACATGAGCA GCGTGCATGC GTTCCCGTGG
481 CCGCTGTTTG TGCACTACGC GGCGAGCAAG GGTGGCATCA AACTGATGAC CGAGACCCTG
541 GCGCTGGAAT ATGCGCCGAA GGGTATCCGT GTTAACAACA TTGGTCCGGG CGCGATCAAC
601 ACCCCGATTA ACGCGGAGAA ATTCGCGGAC CCGAAGCAGA AAGCGGATGT GGAGAGCATG
661 ATCCCGATGG GTTATATTGG CGAACCGGAA GAAATTGCGG CGGTTGCGGC GTGGCTGGCG
721 AGCAAAGAAG CGAGCTACGT GACCGGCATT ACCCTGTTCG CGGACGGTGG CATGACCCAG
781 TATCCGAGCT TTCAAGCGGG TCGTGGCTAA

<210> SEQ ID NO: 4
<211> Length: 846
<212> Type: Artificial DNA
<213> Organism: Bacillus subtilis
<400> Sequence: 4

1 ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT
61 ATGTACCCGG ATCTGAAAGG TAAAGTGGTG GCAATTACCG GCGCAGCCAG CGGCCTGGGT
121 AAAGCAATGG CCATTCGTTT TGGCAAAGAA CAGGCCAAAG TGGTTATTAA TTATTATAGT
181 AACAAGCAGG ACCCGAATGA AGTGAAAGAA GAAGTGATTA AGGCAGGTGG TGAAGCCGTG
241 GTTGTTCAGG GCGATGTGAC CAAAGAAGAA GATGTTAAAA ATATCGTGCA GACCGCAATT
301 AAGGAATTTG GCACCCTGGA TATTATGATT AATAATGCAG GTCTGGAAAA CCCGGTTCCG
361 AGTCATGAAA TGCCGCTGAA AGATTGGGAT AAAGTGATTG GTACCAATCT GACCGGTGCC
421 TTTCTGGGTA GTCGTGAAGC AATTAAGTAT TTTGTGGAAA ATGACATCAA GGGTAATGTT
481 ATTAACATGA GTAGTGTGCA TGAAGTGATT CCGTGGCCGC TGTTTGTTCA TTATGCCGCA
541 AGCAAAGGTG GTATTAAGCT GATGACCCGT ACCCTGGCCC TGGAATATGC CCCGAAAGGC
601 ATTCGCGTTA ATAATATTGG TCCGGGTGCC ATTAATACCC CGATTAATGC AGAAAAATTC
661 GCCGATCCGA AACAGAAAGC CGATGTGGAA AGTATGATTC CGATGGGCTA TATTGGTGAA
721 CCGGAAGAAA TTGCCGCAGT TGCCGCCTGG CTGGCAAGCA AAGAAGCCAG TTATGTTACC
781 GGTATTACCC TGTTTGCAGA TGGCGGTATG ACCCTGTATC CGAGTTTTCA GGCAGGCCGT
841 GGCTAA ,CLAIMS:WE CLAIM:
1. A novel series of synthetic gene having comprising of nucleotide SEQ ID NO: 1, 2, 3 and 4.

2. A process for the preparation of optically active secondary alcohols of formula I;

Formula I

wherein * is an optically active (R) or (S)- configuration; n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is a halogen or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups;
which comprises, Providing the carbonyl compounds of formula (IA) or its salts and ketoreductase enzymes having nucleotide sequences of Sequence ID No. 1 or Sequence ID No. 2 and glucose dehydrogenases having the nucleotide sequence of Sequence ID No. 3 and Sequence ID No. 4,

Formula IA


wherein n is 0 to 5; R1 is saturated or unsaturated, carbocyclic or heterocyclic ring; and R2 is a halogen, or NR’R’’, wherein R’ and R’’ independently are hydrogens or lower alkyl groups;
to produce optically active alcohol of formula I.

3. The process as claimed in claim 2, wherein, compound of formula I is preferably (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol of formula (II).

Formula II
4. The process as claimed in claim 2, wherein, compound of formula IA is preferably 3-(dimethylamino)-1-(thiophen-2-yl)propan-1-one of formula (IIA) or its salts.

Formula IIA

5. The process as claimed in claim 3, wherein (S)-3-(dimethylamino)-1-(thiophen-2-yl)propan-1-ol of formula (II) is converted to Duloxetine or its salts.