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

CHEMO-ENZYMATIC PREPARATION OF CYCLOHEXANE DICARBOXYLIC ACID DERIVATIVE

FIELD OF INVENTION
The present application relates to a chemo-enzymatic process for the preparation of pure trans-1,4-cyclohexanedicarboxylic acid monoester of formula (A) or formula (1) using hydrolase enzyme.

INTRODUCTION
WO2011149938 teaches a process for the preparation of cis-2-(methoxycarbonyl)-cyclohexane-1-carboxylic acid by enzymatic hydrolysis of cis-1,2-cyclohexanedicarboxylate using Candida antarctica type B lipase preparation (Novozym 435)
.

US8329688B2 discloses a process for the preparation of evacetrapib comprising treating (5S)-N-[3,5-bis(trifluoromethyl)benzyl]-7,9-dimethyl-N-(2-methyl-2H-tetrazol-5yl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-amine with trans-4-formylcyclohexanecarboxylate in presence of sodium triacetoxy borohydride.

Frederick et al, Organic Process Research Development, 18, 546-51 (2014) teaches hydrogenative reductive amination of (5S)-N-[3,5-bis(trifluoromethyl)benzyl]-7,9-dimethyl-N-(2-methyl-2H-tetrazol-5yl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-amine with trans-4-formyl-cyclohexanecarboxylate in presence of Pt/C for the preparation of evacetrapib.

SUMMARY
One aspect of the present application relates to a chemo-enzymatic process for the preparation of trans-1,4-cyclohexanecarboxylic acid monoester of formula (1) from 1,4-cyclohexanedicarboxylate (2) in presence of a hydrolase enzyme
;
wherein,
R1 is selected from a group of hydrogen and C1-C4 alkyl;
R2-R9 is independently selected from a group consisting of hydrogen; alkyl, optionally substituted with halogen, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; aryl, optionally having one or more heteroatoms selected from a group of N, O and S and optionally substituted with halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; alkenyl; alkynyl; hydroxyl; nitrile; trifluoromethyl; alkylaryl; alkoxy; and arylalkoxy.

Another aspect of the present application relates to a chemo-enzymatic process for the preparation of trans-1,4-cyclohexanedicarboxylic acid monoester of formula (A) from 1,4-cyclohexanedicarboxylate (B) in presence of a hydrolase enzyme
;
wherein, R1 is selected from a group of hydrogen and C1-C4 alkyl.

Still another aspect of the present application relates to a process for conversion of compound of formula (A), prepared in accordance to the process of the present application, to evacetrapib.

DETAILED DESCRIPTION
One aspect of the present application relates to a chemo-enzymatic process for the preparation of trans-1,4-cyclohexanecarboxylic acid monoester of formula (1) from 1,4-cyclohexanedicarboxylate (2) in presence of a hydrolase enzyme
;
wherein,
R1 is selected from a group of hydrogen and C1-C4 alkyl;
R2-R9 is independently selected from a group consisting of hydrogen; alkyl, optionally substituted with halogen, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; aryl, optionally having one or more heteroatoms selected from a group of N, O and S and optionally substituted with halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; alkenyl; alkynyl; hydroxyl; nitrile; trifluoromethyl; alkylaryl; alkoxy; and arylalkoxy.

Another aspect of the present application relates to a chemo-enzymatic process for the preparation of trans-1,4-cyclohexanedicarboxylic acid monoester of formula (A) from 1,4-cyclohexanedicarboxylate (B) in presence of a hydrolase enzyme
;
wherein, R1 is selected from a group of hydrogen and C1-C4 alkyl.

The enzymatic process of the present application may be performed using any hydrolase enzyme known in the art that is capable of preferentially cleaving an ester group of only one of the geometric isomers (cis or trans) of the substrate to form an acid, such as a lipase, esterase, peptidase or protease. The enzyme may be of microbial origin or from plants or animals. Such enzymes may be commercially available or prepared by methods known in the art. Specifically, the chemo-enzymatic process of the present application may be performed using any lipase enzyme known in the art.

In one embodiment, the hydrolase enzyme used for the chemo-enzymatic process of the present application may be Candida antarctica type B lipase (e.g. Novozym 435, NZL-102-LYO). In another embodiment, the hydrolase enzyme may be subtilisin from Bacillus licheniformis (e.g. Alcalase 2.5L). In yet another embodiment, the hydrolase enzyme may be lipase from Rhizomucor miehei (e.g. Novozym 388, Palatase, Lipozyme RMIM, NZL-103-LYO). In still another embodiment, the hydrolase enzyme may be AE158 (obtained from Mann Associates), CLEA 105-ST (obtained from CLEA Technologies), EL006, EL009, EL013 and EL030 (obtained from Eucodis).

The chemo-enzymatic conversion of the present application may be carried out in a solvent selected from a group of water, an organic solvent and mixture thereof. Specifically, the chemo-enzymatic conversion of the present application may be carried out in water. Alternatively, the chemo-enzymatic conversion of the present application may be carried out in an aqueous system comprising water and water-miscible organic co-solvent. The organic solvent may include but not limited to ethers such as tetrahydrofuran and the like; polar-protic solvents such as dimethyl sulfoxide, dimethyl formamide and the like; ketones such as acetone and the like, nitrile such as acetonitrile and the like; alcohols such as methanol, ethanol and the like. Alternatively, the chemo-enzymatic conversion of the present application may be carried out in a bi-phasic system containing water and an organic solvent. The organic solvent may be selected from a group of ethers such as diethyl ether, tertiary butyl methyl ether, diisopropyl ether and the like; hydrocarbons like heptanes, hexane, cyclohexane, toluene, benzene and the like.

The chemo-enzymatic conversion of the present application may be carried out in presence of a buffer. A salt selected from a group of potassium phosphate, magnesium sulfate, tris(hydroxymethyl)aminomethane and mixture thereof may be used as buffer. Optionally, the buffer comprises a thiol.

The chemo-enzymatic conversion of the present application may be carried out at a suitable pH. Specifically, the pH of the above chemo-enzymatic conversion of the present application may be from about 4 to about 9; more specifically from about 5 to about 8 and most specifically from about 5.5 to about 7.5.

The chemo-enzymatic conversion of the present application may be carried out at a suitable temperature. Specifically, the temperature may be of about 10 °C to about 50 °C; more specifically of about 20 °C to about 40 °C and more specifically of about 25 °C to about 30 °C.

The concentration of the substrate for the chemo-enzymatic conversion of the present application may be from about 0.1 g/L to about 1000 g/L. The loading of the enzyme, for the chemo-enzymatic conversion of the present application, may be from about 0.1 wt% to about 100 wt% with respect to substrate. Specifically, the loading of the enzyme, for the chemo-enzymatic conversion of the present application, may be less than 10 wt% with respect to substrate.
Pure

The chemo-enzymatic conversion of the present application may be carried out in presence of a stabilizer. Any stabilizer known in the art may be used for the above chemo-enzymatic conversion of the present application.

The chemo-enzymatic conversion of the present application may be carried out, for example, in a closed reaction vessel made of glass or metal. The chemo-enzymatic conversion of the present application may be carried out under an atmosphere of nitrogen or air.

The present application also relates to a process for the preparation and isolation of pure cis-isomer of compound of formula (3) or compound of formula (C)
;
Wherein, R1 to R9 is defined above.

The pure cis-isomer of compound of formula (3) or compound of formula (C) may be isolated from the reaction mixture during the process for preparation of pure trans-isomer of compound of formula (1) or compound of formula (A) starting from a compound of formula (2) or compound of formula (B).

One specific aspect of the present process for isolation of pure cis-isomer of compound of formula (3) or compound of formula (C) relates to basification of the reaction mixture to pH more than about 8 to about 11 and extraction with a suitable organic solvent.

The process of the present application relates to a simple yet efficient process for the preparation of pure trans-1,4-cyclohexanecarboxylic acid monoester of formula (A) and/or pure cis-1,4-cyclohexanecarboxylic acid diester of formula (C) starting from 1,4-cyclohexanedicarboxylate (B) using hydrolase enzyme. The process is environmentally friendly and cost-effective.

DEFINITIONS
The following definitions are used in connection with the present disclosure unless the context indicates otherwise.

“Chemo-enzymatic” is defined as a chemical reaction wherein an enzyme is used as a catalyst.

“Halogen” is defined as non-metallic elements found in group VII of the periodic table and is selected from fluorine, bromine, chlorine and iodine.

“Hydroxy” is defined as the group –OH.

“Nitrile” is defined as the group –CN.

“Trifluoromethyl” is defined as the group –CF3.

“Alkyl” is defined as straight or branched chain alkyl having 1-8 carbon atoms. Examples of alkyl chain are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.

“Alkenyl” is defined as an alkyl group, as defined above, containing one or more double bonds.

“Alkynyl” is defined as an alkyl group, as defined above, containing one or more triple bonds.

“Aryl” is defined as monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" also refers to heteroaryl ring systems. The term “heteroaryl” refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members.

“Alkoxy” is defined as an alkyl group or an alkenyl group or an alkynyl group, as previously defined, attached to the principal carbon chain through oxygen.

“Aralkyl” is defined as an aryl group, as previously defined, attached to the principal carbon chain through an alkyl group.

“Aralkoxy” is defined as is defined as an aryl group, as previously defined, attached to the principal carbon chain through oxygen.

“Aryloxyalkyl” is defined as an aryl group, as previously defined, attached to an oxygen atom which in turn is attached to the principal carbon chain through an alkyl group or an alkenyl group or an alkylnyl group, as defined above.

“Mixture of cis/trans isomer” indicates from about 10% by weight of cis-isomer to about 90% by weight of cis-isomer and the remaining is the trans-isomer or 10% by weight of trans-isomer to about 90% by weight of trans-isomer and the remaining is the cis-isomer.

The term “pure” indicates that one of the geometrical isomers (cis or trans) is present in excess amount with respect to the other isomer. Specifically, the “pure trans-isomer” means more than about 55% of trans-isomer present in the compound and the remaining is the cis-isomer. Similarly, the “pure cis-isomer” means more than about 55% of cis-isomer present in the compound and the remaining is the trans-isomer.

EXAMPLES
Example 1: Preparation of trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (A1) and cis-4-cyclohexane-1-carboxylic acid dimethyl ester (C1) using Novozym 435

Dimethyl 1,4-cyclohexyldicarboxylate (B1, 2.0g, cis:trans::3:1) was added to the reactor containing phosphate buffer (20 mL, pH 7.0) and MTBE (20 mL) at 30 ºC. Novozym 435 (100 mg) was added. The reaction was carried out with constant temperature and constant pH; pH was maintained by using aqueous potassium carbonate solution (20%). The reaction progress was monitored by NMR and the base uptake. After 4 hours, the reaction pH was adjusted to 9.3 and extracted with methyl tert-butyl ether (2 × 50 mL). The solvent was evaporated to give 1.35 g of cis-diester (C1) as transparent oil. The aqueous layer was acidified to pH 2 using dilute hydrochloric acid (1M) and extracted with ethyl acetate (3 × 50 mL) to provide the trans-compound (A1).
Cis-Compound:
Yield: 1.35 g
Purity: 98:2 (cis:trans)
Trans-Compound:
Yield: 0.5 g
Purity: 1:1.3 (cis:trans)

Example 2: Preparation of trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (A1) using EL030 lipase

Dimethyl 1,4-cyclohexyldicarboxylate (B1, 1.0 g, cis:trans::3.3:1) was added to the reactor containing phosphate buffer (30 mL, pH 7.0) at 30 ºC. EL030 (10 mg) was added and the reaction was carried out with constant temperature and pH; pH was maintained by using aqueous potassium carbonate solution (20%). The reaction progress was monitored by NMR and the base uptake. After 36 hours, the pH of the reaction mixture was adjusted to 12 and extracted with methyl tert-butyl ether (2 × 50 mL). The solvent was evaporated to give cis-diester (C1) as transparent oil (cis:trans::64:1). The aqueous layer was acidified to pH 2 using dilute hydrochloric acid (1M) and extracted with ethyl acetate (3 × 50 mL) to provide the trans-compound.
Cis-Compound:
Yield: 0.6 g
Purity: 64:1 (cis:trans)
Trans-Compound:
Yield: 0.20 g
Purity: 1:8.2 (cis:trans)

Example 3: Preparation of trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (A1) using Novozym 388

Dimethyl trans-1,4-cyclohexyldicarboxylate (B2, 200 mg) was added to the reactor containing phosphate buffer (30 mL, pH 7.0) and methyl tert-butyl ether (2 mL) at 30 ºC. Novozym solution (150 µL) was added and the reaction was stirred vigorously with constant temperature and pH; pH was maintained by using aqueous potassium carbonate solution (20%). The reaction progress was monitored by NMR and the base uptake. After 36 hours, the reaction pH was adjusted to 12 and extracted with methyl tert-butyl ether (2 × 30 mL). The aqueous layer was acidified to pH 2 using dilute hydrochloric acid (1M) and extracted with methyl tert-butyl ether (2 × 30 mL). The organic layer was evaporated to provide the desired compound as white solid.
Yield: 180 mg
1H NMR (400MHz, CDCl3) d (ppm): 1.30-1.50 (m, 4H), 1.90-2.10 (m, 4H), 2.15-2.25 (m, 2H), 3.70 (s, 3H).
Chemical Purity: 97.3% (2.7% diacid)

Example 4: Preparation of trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (A1) using Alcalase 2.5L

Dimethyl trans-1,4-cyclohexyldicarboxylate (B2, 200 mg) was added to the reactor containing phosphate buffer (30 mL, pH 7.0) and methyl tert-butyl ether (2 mL) at 30 ºC. Alcalase 2.5L solution (150 µL) was added and the reaction was stirred vigorously with constant temperature and pH; pH was maintained by using aqueous potassium carbonate solution (20%). The reaction progress was monitored by NMR and the base uptake. After 36 hours, the reaction pH was adjusted to 12 and extracted with methyl tert-butyl ether (2 × 30 mL). The aqueous layer was acidified to pH 2 using dilute hydrochloric acid (1M) and extracted with methyl tert-butyl ether (2 × 30 mL). The organic layer was evaporated to provide the desired compound as white solid.
Yield: 190 mg
Chemical Purity: 99.5% (0.5% diacid)

Example 5: Preparation of trans-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid (A1)
Dimethyl 1,4-cyclohexyldicarboxylate (B1 or B2, 2.0 g) was added to the reactor containing phosphate buffer (20 mL, pH 7.0) and methyl tert-butyl ether (20 mL) at 30 ºC. Adequate amount of enzyme was added (Table 1) and the reaction was carried out with constant temperature and pH; pH was maintained by using aqueous potassium carbonate solution (20%). The reaction progress was monitored by NMR and the base uptake. The reaction pH was adjusted to 9.3, the reaction mixture was extracted with methyl tert-butyl ether (2 × 50 mL). The organic solvent was evaporated to give diester as transparent solid. The aqueous layer was acidified to pH 2 using dilute hydrochloric acid (1M) and extracted with ethyl acetate (3 × 50 mL) to provide the title compound. The purity of monoesters was determined by GC using ZB-1 column. The yields and purities are presented in Table 1.

Table 1
Sl. No. Substrate
(Weight in g) Enzyme
(Quantity) Time Yield of
diester Yield of monoester Chemical purity of monoester
(GC)
1 B2
(trans)
(2.0) Alcalase 2.5
(300 µL) 24 hrs 1.75 g (88%) 0.17 g
(9%) 93.7
2 B2
(trans)
(2.0) Novozym 388
(300 µL) 24 hrs 0.50 g (25%) 1.40 g
(75%) 95.7
3 B2
(trans)
(2.0) Palatase
(300 µL) 24 hrs 0.50 g (25%) 1.40 g
(75%) 99.6
4 B1
(cis:trans::3.2:1)
(2.0) Novozym 435
(100 mg) 24 hrs 1.35 g (68%)
(cis:trans::>98:2) 0.50 g
(21%)
(cis:trans::1:1.4) 95.8
5 B2
(trans)
(2.0) Alcalase 2.5
(1000 µl) 24 hrs 0.95 g (43%) 0.58 g
(28%) 97.6
6 B2
(trans)
(2.0) Novozym 435
(150 mg) 4 hrs 0.25 g (13%) 1.50 g
(81%) 99
7 B1
(cis:trans::2.7:1)
(2.5) Novozym 435
(420 mg) 3 days 0 g
(0%) 2.20 g
(94%)
(cis:trans::9:1) 82.5
,CLAIMS:1. A chemo-enzymatic process for the preparation of trans-1,4-cyclohexanecarboxylic acid monoester of formula (1) from 1,4-cyclohexanedicarboxylate (2) in presence of a hydrolase enzyme
;
wherein,
R1 is selected from a group of hydrogen and C1-C4 alkyl;
R2-R9 is independently selected from a group consisting of hydrogen; alkyl, optionally substituted with halogen, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; aryl, optionally having one or more heteroatoms selected from a group of N, O and S and optionally substituted with halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkylaryl, alkoxy, arylalkoxy, nitrile, trifluoromethyl; alkenyl; alkynyl; hydroxyl; nitrile; trifluoromethyl; alkylaryl; alkoxy; and arylalkoxy.

2. A chemo-enzymatic process for the preparation of trans-1,4-cyclohexanedicarboxylic acid monoester of formula (A) from 1,4-cyclohexanedicarboxylate (B) in presence of a hydrolase enzyme
;
wherein, R1 is selected from a group of hydrogen and C1-C4 alkyl.