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

PROCESS FOR THE PREPARATION OF CHIRAL N-BOC-3-HYDROXYPIPERIDINE

INTRODUCTION
The present invention provides a process for the enzymatic reduction of 3-ketopiperidines to 3-hydroxypiperidines.

BACKGROUND OF THE INVENTION
Reduction of cyclic amino ketones to the corresponding alcohols using conventional methods such as NaBH4, aluminium isopropoxide or the like is known in the literature. However, such reduction results in a mixture of stereoisomers.
Organic Letters, 11(6), 1245-1248; 2009 described the enzymatic reduction of cyclic amino ketones using plant enzymes (carrot). The yields and enantiomeric excess reported by Lacheretz et al. are low, potentially due to the action of multiple enzymes in the biotransformation.

CN103131734A described the enantioselective reduction of N-Boc-3-piperidone to the R- or S-alcohol using either Thermoanaerobacter ethanolicus alcohol dehydrogenase or a Rhodococcus sp. ST-1O alcohol dehydrogenase.

WO2013/190341 A1 described the enantioselective reduction of N-Boc-3-piperidone to N-Boc-(3S)-hydroxypiperidine using a ketoreductase from yeast Pichia capsulata.

CN103571908A described an enantioselective reduction of N-Boc-3-piperidone to N-Boc-(3S)-hydroxypiperidine using a commercial ketoreductase (KRED 198) of unspecified sequence as the last step in the synthetic route.

Organic Process Research & Development, 18(6), 827-830; 2014 described an enzymatic process for the production of N-Boc-(3S)-hydroxypiperidine. In the process, enantioselective conversion was achieved using 5 wt % of an (unspecified) NAD-dependent ketoreductase. The cofactor was regenerated by the oxidation of IPA. The reaction was performed at 25 °C due to poor enzyme stability. The substrate (100 g/L) was added in two 50 g batches to achieve 99.8 % conversion in 24 hrs.
Chiral 3-hydroxypiperidines are the key intermediates in the synthesis of many biologically active compounds. One such compound is Ibrutinib, a kinase inhibitor indicated for the treatment of patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).
Though several processes are available for the enzymatic conversion of ketones to the corresponding alcohols, there is still a need for an improved process, which achieves high conversion of keto compounds to the corresponding alcohols.
The inventors of the present invention have found enzymatic processes that achieve a high conversion rate and high enantiomeric excess at high substrate loading (?100 g/L), using low enzyme and NAD(P) loading, where the substrate was added in a single batch.
Summary
The present invention provides a process for the stereo selective reduction of 3-ketopiperidines to the corresponding 3-hydroxypiperidines in high enantiomeric purity of (R)- and (S)-alcohols using alcohol dehydrogenases (ADHs).

Detailed Description
In an embodiment, the present invention provides a process for the stereo selective reduction of 3-keto piperidines to the corresponding 3-hydroxy piperidines in high enantiomeric purity of (R)- and (S)-alcohols using alcohol dehydrogenases (ADHs).
In a preferred embodiment, the 3-ketopiperidine is N-protected; preferably, the N-group is Boc-protected and yields the corresponding Boc-protected alcohol after reduction.
The substrate concentration can be in the range of 50-250 g/L, preferably 100-200 g/L.
The ADH enzyme can be a wild type or a recombinant enzyme, used as whole cells, cell-free extract or in the isolated/semi-purified form.
A number of ADHs were screened for the reduction of 3-ketopiperidines. The following table shows the percentage of conversion and the enantiomeric excess obtained using different enzymes.
At 0.4 mg/mL enzyme loading At 1.6 mg/mL enzyme loading
Enzyme Conversion ee Conversion ee Configuration
AKR031 80% 94% 89% 96% (S)
AKR049 94% 96% 94% 96% (S)
AKR054 90% 96% 89% 96% (S)
AKR075 91% 96% 89% 96% (S)
AKR046 62% 93% 82% 95% (S)
AKR001 44% 85% 87% 93% (S)
AKR067 89% 93% 91% 93% (S)
AKR076 93% 94% 90% 93% (S)
AKR038 33% 83% 62% 92% (S)
AKR068 36% 80% 80% 90% (S)
AKR007 93% 100% 94% 100% (R)
AKR009 72% 4% 91% 3% (R)
AKR074 8% 21% 87% 95% (S)
Chiralscreen E007 33% 87% 67% 94% (R)
Chiralscreen E008 30% 86% 79% 91% (R)
ES-KRED-119 63% 89% 90% 88% (R)
KRED-NADH-110 94% 83% 98% 86% (R)
KRED-136 81% 73% 91% 86% (R)
CRED A401 38% 85% 45% 89% (R)
CRED A161 67% 84% 47% 84% (R)
KRED-113 54% 73% 98% 73% (R)
KRED-112 56% 71% 98% 70% (R)
KRED-101 81% 73% 98% 68% (R)
KRED-118 91% 99% 98% 94% (S)
KRED-NADH-118 98% 98% 99% 98% (S)
KRED-126 100% 99% 99% 99% (S)
KRED-146 97% 99% 99% 99% (S)
KRED-NADH-102 98% 99% 98% 99% (S)
JM-ADH-A4 100% 99% 95% 99% (S)
KRED-NADH-101 90% 98% 99% 99% (S)

The (R)-selective ADHs that may be used include Chiralscreen E007, Chiralscreen E008 (each commercially available from Daicel), ES-KRED-119 (commercially available from Syncore), KRED-NADH-110, KRED-113, KRED-112, KRED-136 (each commercially available from Codexis Inc.), CRED A161, CRED A401 (each commercially available from Almac), AKR (007), AKR (009) (each commercially available from Prozomix).
The (S)-selective ADHs that may be used include KRED-118, KRED-NADH-101, KRED-NADH-102, KRED-NADH-118, KRED-126, KRED-146, (each commercially available from Codexis Inc.), ADH A4 (commercially available from Johnson Matthey), AKR001, AKR(031), AKR(038), AKR(046), AKR(049), AKR(054), AKR(067), AKR(068), AKR(074), AKR(075), AKR(076) (each commercially available from Prozomix), or a Kluyveromyces lactis NRRL Y-1140 ADH
The enzymes may have = 50 % identity or = 70 % similarity to the protein sequence of Kluyveromyces lactis NRRL Y-1140 reductase whether genes are obtained from host strains, recombinant strains or as synthetic DNA.
A commercial glucose dehydrogenase is employed for regeneration of the NAD(P)H cofactor.
Enzyme loading can be in the range 0.01% to 100% wt/wt with respect of the substrate, preferably less than 0.01 to 10% wt/wt.
The process of the invention may be carried out at a temperature of about 10 °C to about 50 °C; preferably, the process is carried out at ambient temperature of about 25 to 40 °C. Most preferably, the process is carried out at a temperature of about 25 to 30 °C, such as at a temperature of about 30 °C.
The process of the invention may be carried out in a buffer. Preferably the buffer has a pH from about 4 to 9, more preferably from 5.5 to 7.0, or more preferably from 6.0 to 6.8. Preferably the buffer is a solution of a salt. Preferably the salt is selected from the group consisting of potassium phosphate, sodium phosphate or Tris-HCl. Most preferably, the buffer is potassium phosphate at 0.1 M.
Analytical method:
Column DexCB
Column Dimensions 25 m x 0.25 mm, 0.25µm, 7 inch cage
Method BOC 3HP.M
Column Temperature
Hold
Temperature Gradient 135 ° C
20 minutes
None
Injection volume
Run time
Retention times (of compounds in the reaction mixture)
N-Boc-3-piperidinone
N-Boc-(3R)-hydroxypiperidine
N-Boc-(3S)-hydroxypiperidine 1 µL
16 min

9.9 min
14.25 min
14.75 min

Examples
Example 1: Synthesis of N-Boc-(3R)-hydroxypiperidine (using KRED-NADH-110 from Codexis)
10 mg of KRED-NADH-110 enzyme (1% w/w), glucose (13.5 mg/mL), GDH-102 (Codexis, 2.7 mg), NAD+ (2.6 mg) was dissolved in 0.1 M phosphate buffer (8 mL). N-Boc-3-piperidinone (1 g) was dissolved in butyl acetate as co-solvent (2 mL, 20% v/v). The bi-phasic mixture was stirred at 400 rpm at 25 ºC and the pH was maintained during the reaction using 10% NaOH. After 22h the reaction mixture was extracted into ethyl acetate. The organic layer was dried over MgSO4 and evaporated to dryness to give the crude product in 93% ee. The N-Boc-(3R)-hydroxypiperidinewas short-path distilled at 88 °C/0.6 mm to give pure product as oil in 80% yield.

Example 2: Synthesis of N-Boc-(3S)-hydroxypiperidine (using JM ADH-A4)
A solution of Na2HPO4 (5.3 g; 37.3 mmol) in water (375 mL) was prepared (pH ~8.4). The pH was adjusted to 7.0 using 2M HCl and then an aliquot (15 mL) was removed. Glucose (77 g; 0.43 mol; 1.13 eq) and crude ketone (75 g; contains ~8% EtOAc; 0.38mol uncorrected) were added and the pH was adjusted to 7.0. A solution of 18 wt% Na2CO3 in water was prepared and connected to a pH stat pump. The biphasic mixture was stirred (magnetic stirrer, 500 rpm) and warmed to 30°C. Separately, ADH A4 (JM, 375 mg; 0.5 wt %), GDH-CDX-901 (Codexis, 188 mg; 0.25 wt%) and NAD+ (Europa Bioproducts; 375 mg; 0.5 wt%) were charged to a vial and then dissolved in 10 mL of the pH 7 buffer prepared previously. Once the desired temperature was achieved, the solution of enzymes and NAD was charged to the reaction mixture, washing with the remaining buffer (5 mL) and then with water (5 mL). The reaction was left to stir at 30°C, whilst maintaining the pH at 6.8 using the pH stat previously connected to the reaction mixture. After 18h, the pH had increased to 6.85. GC analysis of a sample indicated 98.8% conversion to the desired (S)-alcohol and the base consumption was 104 g (= 18.72 g Na2CO3; 176.6mmol, approx. 0.5 equivalents). Ethyl acetate (300 mL) was added and after stirring for 15 min the mixture was filtered through Celite, washing with both water (200 mL) and ethyl acetate (200 mL). The filtrate was transferred to a separating funnel and the phases were separated. The aqueous was extracted with ethyl acetate (300 mL) and then discarded. The combined organic phase was washed with saturated brine (200 mL). The organic solution was filtered through filter paper and then concentrated to the product as thick orange oil, 69.5 g, 92% yield, >99% ee. 1H NMR analysis showed very pure product N-Boc-(3S)-hydroxypiperidine and a trace of residual solvent, GC analysis showed ~1% ketone.

Example 3: Synthesis of N-Boc-(3S)-hydroxypiperidine at 200 g/L (using Kluyveromyces lactis NRRL Y-1140 ADH)
A reactor was charged with 4.0 g of N-Boc-3-piperidone in phosphate buffer (0.1M, 20 mL, pH 6.5,), containing 2 mM NADP+, glucose dehydrogenase (10 mg, 0.25 wt%, Codexis, CDX901) and 1.25 eq. glucose. The reactions were equilibrated at 30 °C with stirring at 400 rpm and the pH was set to 6.5. The reaction was initiated with addition of 120 mg enzyme (3% wt/wt, 40 U, 10U/gram substrate) produced by in-house fermentation. The pH during reaction was maintained at 6.5 using 18 wt % aqueous sodium carbonate. The reaction progress was monitored by the base consumption and by chiral GC (25 µl sample mixture was diluted with 1 mL MeCN for GC). After 20h the reaction achieved > 99% conversion to the product N-Boc-(3S)-hydroxypiperidine in > 99% ee.
,CLAIMS:1. A process for the stereo selective reduction of 3-ketopiperidines to the corresponding 3-hydroxypiperidines using alcohol dehydrogenases (ADHs).
2. The process of claim 1, wherein (R)-selective ADHs are selected from Chiralscreen E007, Chiralscreen E008, ES-KRED-119, KRED-NADH-110, KRED-113, KRED-112, KRED-136, CRED A161, CRED A401, AKR (007), AKR (009).
3. The process of claim 1, wherein the (S)-selective ADHs are selected from KRED-118, KRED-NADH-101, KRED-NADH-102, KRED-NADH-118, KRED-126, KRED-146, ADH A4, AKR001, AKR(031), AKR(038), AKR(046), AKR(049), AKR(054), AKR(067), AKR(068), AKR(074), AKR(075), AKR(076), or a Kluyveromyces lactis NRRL Y-1140 ADH.
4. The process of claim 1, wherein 3-ketopiperidine is N-Boc-3-piperidinone.
5. The process of claim 1, wherein 3-hydroxypiperidines are N-Boc-(3R)-hydroxypiperidine and N-Boc-(3S)-hydroxypiperidine.
6. A process according to claim 3 for the synthesis of N-Boc-(3S)-hydroxypiperidine using Kluyveromyces lactis NRRL Y-1140 ADH.