DESC:The following specification particularly describes the invention and the manner in which it is to be performed:
ENZYMATIC PROCESS FOR THE PREPARATION OF RUXOLITINIB AND ITS INTERMEDIATES

INTRODUCTION
One aspect of the present invention relates to process for preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) by stereo selective enzymatic reduction. Another aspect of the present invention provides an enzyme catalyzed resolution of 2-cyclopentyl-2-hydroxyacetate of the formula (IIa). Another aspect of the present invention provides the use of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) prepared according to the present invention for the preparation of Ruxolitinib and its pharmaceutically acceptable salts.

BACKGROUND
Ruxolitinib is useful as inhibitor of the Janus Kinase family of protein tyrosine kinases (JAKs) for treatment of inflammatory diseases, myeloproliferative disorders, and other diseases and is represented by structure of formula (I).

I
(R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) is one of the key intermediate useful in the preparation of ruxolitinib and its pharmaceutically acceptable salts.

II
wherein R=C1-C5 alkyl group.
Processes for the preparation of ruxolitinib and its intermediates have been disclosed in US7598257B2 and WO2010083283A1.

In view of the importance of JAKs inhibitors, new, cost-effective, novel methods of making such drugs and their intermediates are always of interest.

SUMMARY
The first embodiment of the present application provides a process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising a stereoselective enzymatic reduction of compound of formula (III).

III
wherein R=C1-C5 alkyl group.

The second embodiment of the present invention provides the process for preparation of ruxolitinib of formula (I) which comprises;
a) stereo selective enzymatic reduction of compound of formula (III) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib of formula (I);
d) optionally converting ruxolitinib of formula (I) to its pharmaceutically acceptable salts.

The third embodiment of the present invention provides a process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising an enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa).

IIa
wherein R=C1-C5 alkyl group.

The fourth embodiment of the present invention provides process for preparation of ruxolitinib of formula (I) which comprises;
a) enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib of formula (I);
d) optionally converting ruxolitinib of formula (I) to its pharmaceutically acceptable salts.

The fifth embodiment of the present invention provides a novel compound of formula (II);

II
wherein R=C1-C5 alkyl group.

The sixth embodiment of the present invention provides pharmaceutical compositions comprising ruxolitinib of formula (I) or its pharmaceutically acceptable salts prepared according to processes of the present application together with one or more pharmaceutically acceptable excipient, carrier and diluents.

DETAILED DESCRIPTION
The first embodiment of the present application provides a process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising a stereo selective enzymatic reduction of compound of formula (III).

III
wherein R=C1-C5 alkyl group.

Stereoselective enzymatic reduction of compound of formula (III) may be carried out comprising the use of an oxidoreductase enzyme which is capable of catalyzing the enzymatic reduction of compound of formula (III), or using isolated enzymes or purified enzymes capable of catalyzing the stereo selective enzymatic reduction of the compound of formula (III).

In an embodiment, the stereoselective enzymatic reduction of compound of formula (III) of the present application may be carried out involving the use of isolated enzymes or purified enzymes.

Suitable enzymatic catalysts that may be used in the reaction include, but are not limited to, ketoreductases such as NADH dependent ketoreductases or NADPH-dependent ketoreductases. Suitable ketoreductase enzymes include, but not limited to, for example, the Codexis Inc. products with catalog numbers KRED-EW-N106, KRED-EW-N102, KRED-EW-N107, KRED-EW-N104, KRED-EW-N103, KRED-EW-N113, KRED-119, KRED-NADH-110; the Almac Inc. products with catalog numbers Almac-CRED N151, Almac-CRED A401, Almac-CRED N131, Almac-CRED A481, Almac-CRED A121, Almac-CRED N701, Almac-CRED A571, Almac-CRED A231, Almac-CRED A641, Almac-CRED A491, Almac-CRED A651, Almac-CRED N501, Almac-CRED A671, Almac-CRED A801, Almac-CRED A201, Almac-CRED A211, Almac-CRED A181, Almac-CRED A431, Almac-CRED A681, Almac-CRED A271, Almac-CRED A301, Almac-CRED A411, Almac-CRED A421, Almac-CRED A141, Almac-CRED A391, Almac-CRED A561, Almac-CRED A631, Almac-CRED A261, Almac-CRED A281, Almac-CRED A661, Almac-CRED A241; the Prozomix Inc. products with catalog numbers Prozomix -AKR(062), Prozomix -AKR(054), Prozomix -AKR(075), Prozomix -AKR(076), Prozomix -AKR(084), Prozomix -AKR(008), Prozomix -AKR(067), Prozomix -AKR(041), Prozomix -AKR(048, Prozomix -AKR(047), Prozomix -AKR(078), Prozomix -AKR(083, Prozomix -AKR(009), Prozomix -AKR(069), Prozomix -AKR(057), Prozomix -AKR(086), Prozomix -AKR(077), Prozomix -AKR(012), Prozomix -AKR(010), Prozomix -AKR(053), Prozomix -AKR(020), Prozomix -AKR(007), Prozomix -AKR(034), Prozomix -AKR(044, Prozomix -AKR(095), Prozomix -AKR(085); the Evo Inc. products with catalog numbers Evo-ADH190, Evo-ADH250, Evo-ADH260 and Evo-ADH440; and any combinations thereof.
The reaction of first embodiment of present application may be carried out in the presence of a co-factor. The term “co-factor” refers to an organic compound that operates in combination with an enzyme, which catalyzes the reaction of interest. Co-factors include, but are not limited to, for example, nicotinamide co-factors such as, nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), any derivatives thereof, and analogs thereof.
Optionally, the reaction mixture further comprises a co-factor regeneration system. A co-factor regeneration system comprises a substrate and a dehydrogenase. The reaction between the substrate and dehydrogenase enzyme regenerates the co-factor. The co-factor regeneration system comprises a substrate/dehydrogenase pair such as: D-glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, phosphite/phosphite dehydrogenase, and 2-propanol and ketoreductase/hydrogenase; secondary alcohols such as isopropyl alcohol, secondary butanol, 2-pentanol, 3-pentanol, cyclohexanol and any other suitable co-factor regeneration system.
The reaction may be carried out in the presence of a buffer. The buffer may have pH values from about 4 to about 9, or from about 4 to about 8, or from about 5 to about 8, or from about 6 to about 8, or from about 6 to about 9, or from about to 5 about 7. In a preferred embodiment, the buffer may be a solution of a salt, such as potassium phosphate, magnesium sulfate, or mixtures thereof. Optionally, the buffer comprises a thiol, such as dithiothreitol (“DTT”).
Suitable co-solvents that may be used in the reaction include, but are not limited to, polar aprotic solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, and the like; ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dimethoxyethane, dioxane and the like; aromatic hydrocarbons such as, for example toluene, xylenes, and the like; alcohols such as, for example, methanol, ethanol, 2-propanol, n-propanol, n-butanol, 2-methoxyethanol, secondary butanol, tertiary butanol, and the like; alicyclic hydrocarbons such as, for example, hexanes, heptanes, pentanes, cyclohexane, and the like; nitrile solvents such as, for example, acetonitrile, propionitrile and the like; esters such as, for example, ethyl acetate and butyl acetate; aliphatic or alicyclic hydrocarbon solvents such as n-pentane, isopentane, neopentane, n-hexane, isohexane, 3-methylpentane, 2,3-dimethylbutane, neohexane, n-heptane, isoheptane, 3-methylhexane, neoheptane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, n-octane, isooctane, 3-methylheptane, neooctane, cyclohexane, methylcyclohexane, cycloheptane and the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, and the like; and any mixtures thereof.
Suitable temperatures that may be used for the reaction may be less than about 80°C, less than about 60°C, less than about 50°C less than about 40°C, less than about 30°C, less than about 20°C, less than about 10°C, or any other suitable temperatures based on the reaction conditions.
A representative gas chromatography (“GC”) method for determining the conversion of a compound of formula (III) to a compound of formula (II) of the present application involves the parameters shown in Table 1.
Table 1
Column Chiraldex Dex-CB (25m X 0.25mm X 0.25µm)
Oven 110 °C for 10 min
Carrier He @ 20 psi
Injector temp 200 °C
Detector temp 250°C
Injection Volume 1 ?L
Retention Times
Ethyl 2-cyclopentyl-2-oxoacetate 5.6 minutes
(R)-2-cyclopentyl-2-hydroxyacetate 8.4 minutes
(S)-2-cyclopentyl-2-hydroxyacetate 8.7 minutes

The compound of formula (III) may be prepared as per the processes disclosed in the literature. In one of the specific embodiment, the compound of formula (III) may be prepared by reacting cyclopentyl magnesium halide with dialkyloxalate under suitable conditions to produce a compound of formula (III).
The compound of formula (II), prepared according to the processes described in the present application may be useful as an intermediate for the manufacture of ruxolitinib of formula (I).
The R group of compound of formula (III) also can be selected from group of aryl, cycloalkyl and heterocycle.
The second embodiment of the present invention provides the process for preparation of ruxolitinib of formula (I) which comprises;
a) stereo selective enzymatic reduction of compound of formula (III) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib of formula (I);
d) optionally converting ruxolitinib of formula (I) to its pharmaceutically acceptable salts.

The enzymatic reductase, solvents and reaction conditions for step a) may be selected from one or more suitable enzymatic reductase, solvents and process conditions as described in the steps of first embodiment of the present invention.

The (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) may be converted to ruxolitinib of formula (I) by the methods known in the literature. The intermediate obtained in the present invention may be isolated or used directly in the next step obtained as such from the reaction mass.

The isolation and purification of compounds of or their pharmaceutically acceptable salt of the present invention may be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, centrifugation, extraction, acid-base treatment, crystallization, conventional isolation and refining means such as concentration, concentration under reduced pressure, solvent-extraction, crystallization, phase-transfer chromatography, column chromatography, or by a combination of these procedures.

The third embodiment of the present invention provides a process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising an enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa).

IIa
wherein R=C1-C5 alkyl group.

In the present application, the enzyme catalyzed resolution may be done by either hydrolysis or esterification. It is well known that enzymes are very specific in their functions due to the amino acids present in their active site and enzymes are chiral and have asymmetric binding sites, this asymmetric binding sites leads to enzyme stereo specificity, which results in its favor to bind one enantiomer over the other. In addition, enzymes may be recycled due to the fact that their structure does not change during the reaction, thus the use of enzymes makes the processing easier, because the isolation of the enzyme from the reaction mixture involves a simple process.
The benefit of performing the enzyme catalyzed resolution on the intermediates of present invention instead, on ruxolitinib of formula (I) may be significant, since the undesired enantiomer may be recycled while the recycling of the undesired enantiomer of ruxolitinib of formula (I) may involves a costlier process.
In one embodiment of the present invention, the enzyme catalyzed resolution process may be performed comprising the reaction of ester of formula (IIa) with hydrolase, buffer and optionally a base at a temperature of about 4°C to obtain (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II).
Suitable hydrolase that may be used in the reaction include, but are not limited to, esterase such as BC esterase, PF2 esterase, BS1 esterase, BS2 esterase, BS3 esterase, esterase RO and BS4 esterase and the like; lipase such as lipase P2, Lipase PS, Lipase RS, Lipase C2, Lipase C1, Lipase QLM, Lipase TL and the like; protease such as pepsin, trypsin, chymotrypsin, peptidase and the like.
In the present embodiment, the reaction may be carried out in the presence of a buffer. The buffer may have pH values from about 4 to about 9, or from about 4 to about 8, or from about 5 to about 8, or from about 6 to about 9, or from about to 5 about 7. The buffer frequently is a solution of a salt, such as potassium phosphate, magnesium sulfate, or mixtures thereof.
Suitable co-solvents that may be used in the reaction include, but are not limited to: polar aprotic solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, and the like; ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dimethoxyethane, dioxane and the like; aromatic hydrocarbons such as, for example toluene, xylenes, and the like; alcohols such as, for example, methanol, ethanol, 2-propanol, n-propanol, n-butanol, 2-methoxyethanol, secondary butanol, tertiary butanol, and the like; alicyclic hydrocarbons such as, for example, hexanes, heptanes, pentanes, cyclohexane, and the like; nitrile solvents such as, for example, acetonitrile, propionitrile and the like; esters such as, for example, ethyl acetate and butyl acetate; aliphatic or alicyclic hydrocarbon solvents such as n-pentane, isopentane, neopentane, n-hexane, isohexane, 3-methylpentane, 2,3-dimethylbutane, neohexane, n-heptane, isoheptane, 3-methylhexane, neoheptane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, n-octane, isooctane, 3-methylheptane, neooctane, cyclohexane, methylcyclohexane, cycloheptane and the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, and the like; and any mixtures thereof.
Suitable temperatures that may be used for the reaction may be less than about 50°C, less than about 40°C, less than about 30°C, less than about 20°C, less than about 10°C, less than about 5°C or any other suitable temperatures.
The compound of formula (IIa) may be prepared as per the processes disclosed in the literature. In one of the specific embodiment, the compound of formula (IIa) may be prepared by reacting cyclopentyl magnesium halide with dialkyloxalate under suitable conditions to produce a compound of formula (III), followed by treating with suitable reducing agent that may be selected from sodium borohydride and lithium aluminium hydride and the like, under suitable conditions to produce a compound of formula (IIa).

The compound of formula (II), prepared according to the processes described in the present application may be useful as an intermediate for the manufacture of ruxolitinib of formula (I).
The fourth embodiment of the present invention provides the process for preparation of ruxolitinib of formula (I) which comprises;
a) enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib of formula (I);
d) optionally converting ruxolitinib of formula (I) to its pharmaceutically acceptable salts.

In the fourth embodiment, the resulted acid of compound of formula (IV) may be converted to compound of formula (IIa) by subjecting to an esterification methods as disclosed in the art.

The enzyme, solvents and reaction conditions for step a) may be selected from one or more suitable enzyme, solvents and process conditions as described in the steps of third embodiment of the present invention.

The (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) may be converted to ruxolitinib of formula (I) by the methods known in the literature. The intermediate obtained in the present invention may be isolated or used directly in the next step obtained as such from the reaction mass.

The isolation and purification of compounds of or their pharmaceutically acceptable salt of the present invention can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, centrifugation, extraction, acid-base treatment, crystallization, conventional isolation and refining means such as concentration, concentration under reduced pressure, solvent-extraction, crystallization, phase-transfer chromatography, column chromatography, or by a combination of these procedures.

The fifth embodiment of the present invention provides a novel compound of formula (II);

II
wherein R=C1-C5 alkyl group.

The present invention also includes the use of novel compound as described in the fifth embodiment for the preparation of ruxolitinib of formula (I) or its pharmaceutically acceptable salts.

The sixth embodiment of the present invention provides pharmaceutical compositions comprising ruxolitinib of formula (I) or its pharmaceutically acceptable salts prepared according to processes of the present application together with one or more pharmaceutically acceptable excipient, carrier and diluents.

“Pharmaceutically acceptable salt" refers to a salt formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. Suitable pharmaceutically acceptable salts that may be used include, but are not limited to, those derived from organic and inorganic acids such as, acetic, lactic, citric, cinnamic, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, oxalic, propionic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, glycolic, pyruvic, methane sulfonic, ethane sulfonic, toluene sulfonic, salicylic, benzoic, and similarly known acceptable acids.

Certain specific aspects and embodiments of the present invention will be explained in more detail with reference to the following examples, which are provided for purposes of illustration only and should not be construed as limiting the scope of the present invention in any manner.

EXAMPLES:
Example 1: Preparation of (R)-Ethyl-2-cyclopentyl-2-hydroxyacetate (II; where R is Ethyl).
Racemic ethyl-2-cyclopentyl-2-hydroxyacetate (30 g) in 0.05 M potassium phosphate buffer (20 mL, pH 7.5) was added to the reactor and stirred at 4°C. A solution of BC esterase (3g in 10 mL 0.05 M potassium phosphate buffer) was added to the reactor and maintained the pH at 7.5 by using 2M NaOH. After maintaining the reaction mixture for 8-10 hours, the unreacted optical pure ester was extracted with ethyl acetate (2 X 100 mL) at pH 9-9.5 and the aqueous layer pH was adjusted to 1-2 by using 5 M HCl. At this pH, the acid product was extracted with ethyl acetate and concentrated in vacuo to give undesired (S)-2-cyclopentyl-2-hydroxyacetic acid as a white solid. The organic layer was filtered through celite and dried with MgSO4. The organic layer was evaporated to provide the desired compound as pale yellow oil.
Yield: 13.3 g (89%)
Chiral Purity: 99% e.e.
Example 2: Preparation of (R)-Ethyl-2-cyclopentyl-2-hydroxyacetate (II; where R is Ethyl).
A solution of Prozomix-AKR054 (510 mg), GDH-CDX901 (3.3 mg) and NADP+ (10.3 mg) in 50 mM potassium phosphate buffer (20 mL, pH 7.0) were added to the reactor containing D-glucose (2.08 g) and maintained at 25°C until a solution was obtained. Ethyl 2-cyclopentyl-2-oxoacetate (1.0 g) in DMSO (1 mL) was added and the reaction was stirred with constant temperature and pH; the pH was maintained by using 1M NaOH. After maintaining the reaction mass for 19 hours, the reaction mass was extracted with methyl tert-butyl ether (4 × 25 mL). The combined organic layers were filtered through celite and dried with MgSO4. The organic layer was evaporated to provide the desired compound as pale yellow oil.
Yield: 0.70 g (70%)
Chiral Purity: 94% e.e.

Example 3: Preparation of (R)-Ethyl-2-cyclopentyl-2-hydroxyacetate (II; where R is Ethyl).
Screening conditions:
Screening is carried out in 96-well plate format. Ketoreductase enzyme (KRED, 1 mg) was dissolved in 50 mM potassium phosphate buffer (0.5 mL, pH 7.0) containing GDH (0.1 mg/mL), NAD+ (0.5 mg/mL) and NADP+(0.5 mg/mL) then the plates were sealed and shaken at 750 rpm and 25°C temperature for 30 minutes. Ethyl 2-cyclopentyl-2-oxoacetate (25 ?L of a 200 mg/mL in DMSO) was added then the plates were sealed and shaken at 750 rpm at 25°C temperature for 18 hours. The reaction mixture was diluted with acetonitrile (900 mL), and passed through a MgSO4 plug and analyzed by GC for conversion to know the content of product in enantiomeric excess.
Stereoselective enzymatic reduction of a compound of formula (III) with different enzymatic catalysts, using the above procedures, provides a compound of formula (II) and the results are summarized in Table 2.
Table 2
S.No Enzyme Conversion (%) e.e. (%)
1) Almac-CRED N151 96% 92%
2) KRED-EW-N106 98% 91%
3) Prozomix -AKR(062) 99% 91%
4) Prozomix -AKR(054) 94% 89%
5) Prozomix -AKR(075) 99% 89%
6) KRED-EW-N102 100% 89%
7) Prozomix -AKR(076) 99% 88%
8) Prozomix -AKR(084) 99% 88%
9) Prozomix -AKR(008) 96% 86%
10) Prozomix -AKR(067) 100% 86%
11) KRED-EW-N107 97% 86%
12) Almac-CRED A401 98% 85%
13) Prozomix -AKR(041) 85% 85%
14) Almac-CRED N131 94% 85%
15) Prozomix -AKR(048) 75% 71%
16) Almac-CRED A481 49% 69%
17) Prozomix -AKR(047) 81% 69%
18) Almac-CRED A121 86% 68%
19) Almac-CRED N701 64% 67%
20) Prozomix -AKR(078) 65% 67%
21) Almac-CRED A571 57% 65%
22) Almac-CRED A231 100% 64%
23) Prozomix -AKR(083) 88% 64%
24) Almac-CRED A641 98% 63%
25) Almac-CRED A491 55% 62%
26) Almac-CRED A651 52% 62%
27) Prozomix -AKR(009) 78% 61%
28) Almac-CRED N501 42% 60%
29) Almac-CRED A671 100% 85%
30) Prozomix -AKR(069) 100% 84%
31) Evo-ADH190 94% 82%
32) KRED-EW-N104 100% 82%
33) Prozomix -AKR(057) 92% 81%
34) Almac-CRED A801 92% 80%
35) Almac-CRED A201 93% 79%
36) Evo-ADH250 99% 79%
37) KRED-EW-N103 95% 79%
38) Prozomix -AKR(086) 83% 77%
39) KRED-EW-N113 100% 76%
40) Prozomix -AKR(077) 82% 76%
41) Prozomix -AKR(012) 91% 76%
42) Almac-CRED A211 91% 76%
43) Almac-CRED A181 98% 74%
44) Almac-CRED A431 77% 74%
45) Almac-CRED A681 100% 74%
46) Almac-CRED A271 60% 74%
47) Prozomix -AKR(010) 100% 72%
48) Almac-CRED A301 76% 72%
49) Almac-CRED A411 99% 72%
50) Almac-CRED A421 44% 60%
51) Almac-CRED A141 88% 60%
52) Prozomix -AKR(053) 71% 59%
53) Prozomix -AKR(020) 92% 59%
54) Almac-CRED A391 63% 58%
55) KRED-119 100% 58%
56) Prozomix -AKR(007) 99% 58%
57) Prozomix -AKR(034) 37% 57%
58) Almac-CRED A561 52% 56%
59) Almac-CRED A631 99% 56%
60) KRED-NADH-110 100% 56%
61) Evo-ADH260 57% 55%
62) Prozomix -AKR(044) 54% 55%
63) Almac-CRED A261 57% 55%
64) Almac-CRED A281 54% 54%
65) Evo-ADH440 99% 53%
66) Almac-CRED A661 50% 52%
67) Almac-CRED A241 82% 52%
68) Prozomix -AKR(095) 54% 51%
69) Prozomix -AKR(085) 54% 51%
,CLAIMS:We Claim:
1. A process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising a stereoselective enzymatic reduction of compound of formula (III).

III
wherein R=C1-C5 alkyl group.

2. The enzyme according to claim 1 can be selected from the group of ketoreductases such as NADH dependent ketoreductases or NADPH-dependent ketoreductases.

3. A process for preparation of ruxolitinib which comprises;
a) stereo selective enzymatic reduction of compound of formula (III) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib;
d) optionally converting ruxolitinib to its pharmaceutically acceptable salts.

4. A process for the preparation of (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II),

II
wherein R=C1-C5 alkyl group
comprising an enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa).

IIa
wherein R=C1-C5 alkyl group.

5. The enzyme catalyzed resolution according to claim 4 is carried out in presence of hydrolase, buffer and optionally a base.

6. Suitable hydrolase according to claim 4 can be selected from BC esterase, PF2 esterase, BS1 esterase, BS2 esterase, BS3 esterase, esterase RO, BS4 esterase; lipase such as lipase P2, Lipase PS, Lipase RS, Lipase C2, Lipase C1, Lipase QLM, Lipase TL; protease such as pepsin, trypsin, chymotrypsin, peptidase.

7. The buffer according to claim 4 is a solution of a salt, such as potassium phosphate, magnesium sulfate, or mixtures thereof.

8. A process for preparation of ruxolitinib which comprises;
a) enzyme catalyzed resolution of racemic 2-cyclopentyl-2-hydroxyacetate of the formula (IIa) to (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II);

wherein R=C1-C5 alkyl group
b) converting (R)-2-cyclopentyl-2-hydroxyacetate of the formula (II) to ruxolitinib;
c) optionally purifying ruxolitinib;
d) optionally converting ruxolitinib to its pharmaceutically acceptable salts.

9. A novel compound of formula (II);

II
wherein R=C1-C5 alkyl group.

10. A pharmaceutical compositions comprising ruxolitinib or its pharmaceutically acceptable salts prepared according to processes of the present application together with one or more pharmaceutically acceptable excipient, carrier and diluents.