BIOCATALYTIC PROCESSES FOR PREPARING ESLICARBAZEPINE

INTRODUCTION

The present application relates to biocatalytic processes for the preparation of eslicarbazepine, an intermediate in the preparation of eslicarbazepine acetate.

The drug having the adopted name "eslicarbazepine" has a chemical name (S)-10,11-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide. It has the structure of formula (I).

Eslicarbazepine acetate has a chemical name (S)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide. It has the structure of formula (II).

Eslicarbazepine acetate is a voltage gated sodium channel blocker and is the active ingredient in products sold in Europe as Exalief™ and Zebinix™. Eslicarbazepine acetate is indicated as adjunctive therapy in adults with partial-onset seizures with or without secondary generalization.

IPCOM000193904D discloses reduction of 10,11-dihydro-10-oxo-5H-dibenz(b,f)azepine-5-carboxamide with a ketoreductase, such as KRED-114, 119, 130, and 101, KRED-NADH-109 and 108, and ES-KRED-144. The conversion is very low.

There remains a need to provide biocatalytic processes for the preparation of eslicarbazepine, which are cost-effective.

SUMMARY

In an aspect, the present application provides biocatalytic processes for the preparation of (S)-10,11-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide of formula (I), comprising a stereoselective enzymatic reduction of oxcarbazepine of formula (III).

DETAILED DESCRIPTION

In an aspect, the present application provides biocatalytic processes for the preparation of (S)-10,11-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide of formula (I), comprising a stereoselective enzymatic reduction of oxcarbazepine of formula (III).

Stereoselective enzymatic reduction of oxcarbazepine of formula (III) may be carried out using whole-cells of a microorganism that supplies an oxidoreductase enzyme capable of catalyzing the enzymatic reduction of oxcarbazepine 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 aspect, stereoselective enzymatic reduction of oxcarbazepine of formula (III) involves the use of whole-cells of a microorganism that supplies an oxidoreductase enzyme capable of catalyzing the enzymatic reduction of oxcarbazepine of formula (III).

The whole-cells of the microorganism may be in the form of intact wet cells or dried cells, such as lyophilized, spray-dried, or heat-dried cells, or in the form of a treated cell material such as ruptured cells or cell extracts.

Suitable microorganisms that may be used for the generation of oxidoreductase enzymes include, but are not limited to: genera from bacteria, yeasts and fungi, such as Achromobacter, Acinetobacter, Actinomyces, Alkaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Methylomonas, Mycobacterium, Nocardia, Psuedomonas, Rhodococcus, Streptomyces, Xanthomonas, Aspergillus, Candida, Fusarium, Geotrichum, Hansenula, Kloeckera, Penidllum, Pichia, Rhizopus, Rhodotorula, Saccharomyces, Trichoderma, Mortierella, Cunninghamella, Torulopsis or Rhodopseudomonas. An example of a specific microorganism is Pichia methanolica.

A co-factor and a system for re-generating the co-factor, for example glucose and glucose dehydrogenase, are used with the enzyme to drive the reaction. As suitable co-factors and reduction mechanisms are present in the whole cells, the whole cells can be used in a nutrient medium that contains a suitable carbon source, which may include one or more of the following: a sugar, e.g., maltose, sucrose, or glucose; a polyol, e.g., glycerol or sorbitol; citric acid; a suitable synthetic medium, e.g., potato dextrose broth (PDB), nutrient broth, yeast maltose peptone (YMP); or a lower alcohol, for example methanol or ethanol.

If whole cells are intended to grow during the reaction, nitrogen and phosphorus sources and trace elements should be present in the medium. These may be those normally used in culturing the organism.

Stereoselective enzymatic reduction of oxcarbazepine of formula (III) may be carried out by adding a compound of formula (III) to a culture of the growing organism, in a medium capable of supporting growth, or to a suspension of the live cells in a medium that contains a carbon source but lacks one or more nutrients necessary for growth.
Suitable pH values that may be used for stereoselective enzymatic reduction of oxcarbazepine of formula (III) may range from about 2 to about 8.5.
Suitable temperatures that may be used for stereoselective enzymatic reduction of oxcarbazepine of formula (III) may be less than about 100°C, or less than about 60°C, or less than about 40°C, or less than about 30°C, or less than about 20°C, or less than about 10°C, or less than about 0°C, or any other suitable temperatures.

Suitable solvents that may be used for the reaction include, but are not limited to: alcohols such as, for example, methanol, ethanol, 2-propanol, n-propanol, n- butanol, 2-methoxyethanol, secondary butanol, tertiary butanol, and the like; ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dimethoxyethane, and the like; esters such as, for example, ethyl acetate and butyl acetate; aliphatic or alicyclic hydrocarbons such as, for example, n-hexane, n-heptane, pentanes, cyclohexane, and the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, and the like; aromatic hydrocarbons such as, for example toluene, xylenes, and the like; polar aprotic solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, and the like; including any mixtures thereof.

The processes of the present application may be carried out under aerobic conditions. The agitation and aeration of the reaction mixture affects the amount of oxygen available during the stereoselective reduction process, which may be conducted, for example, in shake-flask cultures or fermenter tanks during growth of microorganisms. The suitable agitation may range from 0 to about 500 RPM, but any rotation speed is acceptable. An aeration rate equivalent to about 0.01 to about 10 volumes of oxygen, measured at standard temperature and pressure, per volume of the culture medium, per minute, is suitably employed at the aforesaid conditions of pH and temperature.

Purified enzymes may be isolated by any means, such as by centrifuging a suspension of disintegrated cells and separating a clear solution from debris, separating the desired enzyme from the solution, for example by ion exchange chromatography, in embodiments with elution from the column using liquids of increasing ionic strength, and/or by selective precipitation from the addition of an ionic material, for example ammonium sulphate. Such operations may be repeated, if desired, to enhance purity.
Stereoselective enzymatic reduction of oxcarbazepine of formula (III) of the present application may be carried out using 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 ketoreductases enzymes include, for example, the Codexis Inc. products with catalog numbers KRED-101, KRED-166, KRED-172, KRED-147, KRED-175, KRED-NADH-107, KRED-NADH-108, KRED-NADH-109, KRED-NADH-129; the Enzysource Inc. products with catalog numbers ES-KRED--139, ES-KRED-113, ES-KRED-112; the Almac Sciences product with catalog number CRED-A401; the Daicel Chemical Industries products with catalog numbers Chiralscreen E007 or Chiralscreen E008; Juelich RS1; and any combinations thereof.

The reaction 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 secondary butanol, 2-pentanol, or 3-pentanol; and any other suitable co-factor regeneration system.

The reaction may be carried out in the presence of a buffer. The buffer can 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 to 5 about 7. The buffer frequently is 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: alcohols such as, for example, methanol, ethanol, 2-propanol, n-propanol, n-butanol, 2-methoxyethanol, secondary butanol, tertiary butanol, and the like; ethers such as, for example, diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dimethoxyethane, and the like; esters such as, for example, ethyl acetate and butyl acetate; aliphatic or alicyclic hydrocarbons such as, for example, hexanes, heptanes, pentanes, cyclohexane, and the like; halogenated hydrocarbons such as, for example, dichloromethane, chloroform, and the like; aromatic ~ 'hydrocarbons such; as; for example toluene, xylenes, and the like; polar aprotic : solvents such as, for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide, 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 40°C, less than about 30°C, less than about 20°C, less than about 10°C, or any other suitable temperatures.

A representative supercritical fluid chromatography ("SFC") 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 3.

Table 3

The compound of formula (I), prepared according to the processes described in the present application is useful as an intermediate for the manufacture of eslicarbazepine acetate of formula (II).

Certain specific aspects and embodiments of the present application 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 application in any manner.

EXAMPLES Example 1: Preparation of (S)-(+)-10,i1-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide.

Inoculum of Pichia methanolica 103660 is grown in a medium containing potato dextrose broth, at 30°C for 16 hours at 150 rpm. The inoculum is transferred into a production medium containing PDB (potato dextrose broth) or molasses and grown at 30°C and 150 rpm for 48 hours. All of the media components are autoclaved at 15 psi, 121 °C for 15 minutes before inoculation. Cells are harvested by centrifugation at 6000 rpm for 3 minutes and then washed with water.

Oxcarbazepine (190 mg) and hexane (400 ml_) are combined in a flask and stirred at 25-35°C for 15 minutes. Water washed cells of the Pichia methanolica (46 g) are suspended in glucose solution (5 g in 90 ml_ of water) and charged into the mixture at 25-35°C. The mixture is incubated in an orbital shaker at 30°C and 150 rpm, then centrifuged for 5 minutes at 7000 rpm, and hexane is separated from the mixture. The mixture is filtered, ethyl acetate (400 ml_) is added to the filtrate, and the mixture is stirred for 1 hour with a magnetic stirrer at 1400 rpm. Solvent from the mixture is evaporated at 40°C. A mixture of dichloromethane (3 ml_) and heptane (1.5 mL) is added to the residue and stirred at 25-35°C for 4- 5 hours. The obtained solid is filtered and dried, to afford 130 mg of the title compound.

Chiral purity by HPLC: 93%.
Example 2: Preparation of (S)-(+)-10,11-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide.

Screening is carried out in 96-well plate format. Each well is rehydrated with a buffer mixture (500 uL). A 40 mg/mL solution of oxcarbazepine in DMSO (25 uL) is added to each well, then the plates are sealed and shaken at 750 rpm and 25°C overnight. The reactions are diluted with 500 ul_ methanol, and then samples are prepared for assay by diluting a 120 uL aliquot of this mixture with methanol (480 uL) containing MgS04 (2 uM). The supernatant is analyzed by SFC for conversion and product e.e. The results are shown in Table 2.

Buffer mixtures that may be used for enzymatic reduction of the present application, to provide for the regeneration and recovery of the enzymes, are summarized in Table 1.
Table 1

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

We claim:

1. A process for the preparation of (S)-10,11-dihydro-10-hydroxy-5H-dibenz[b,f]azepine-5-carboxamide of formula (I), (I) comprising a stereoselective enzymatic reduction of oxcarbazepine of formula (III).

2. The process of claim 1, wherein stereoselective enzymatic reduction of oxcarbazepine of formula (III) carried out using whole-cells of a microorganism that supplies an oxidoreductase enzyme capable of catalyzing the enzymatic reduction of oxcarbazepine of formula (III).

3. The process of claim 1, wherein the oxidoreductase is obtainable from Achromobacter, Acinetobacter, Actinomyces, Alkaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Methylomonas, Mycobacterium, Nocardia, Psuedomonas, Rhodococcus, Streptomyces, Xanthomonas, Aspergillus, Candida, Fusarium, Geotrichum, Hansenula, Kloeckera, Penidllum, Pichia, Pichia methanolica, Rhizopus, Rhodotorula, Saccharomyces, Trichoderma, Mortierella, Cunninghamella, Torulopsis or Rhodopseudomonas.

4. The process of claim 1, wherein stereoselective enzymatic reduction of oxcarbazepine of formula (III) carried out using isolated enzymes or purified enzymes capable of catalyzing the stereo selective enzymatic reduction of the compound of formula (III) and in the presence of a co-factor.

5. The process of claims 1 & 4, wherein isolated or purified enzyme is selected from the group consisting of at least one of KRED-101, KRED-166, KRED-172, KRED-147, KRED-175, KRED-NADH-107, KRED-NADH-108, KRED-NADH- 109, KRED-NADH-129, ES-KRED-139, ES-KRED-113, ES-KRED-112, CRED-A401, E007, E008orRS1.

6. The process of claims 1 & 4, wherein the co-factor is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and any mixtures thereof.

7. The process according to any preceding claim, wherein the reaction mixture further comprises a co-factor regeneration system that comprises a substrate and a dehydrogenase.

8. The process of claim 7, wherein the substrate/dehydrogenase pair selected from the group consisting of D-glucose/glucose dehydrogenase, sodium formate/formate dehydrogenase, phosphite/phosphite dehydrogenase, and 2-propanol and ketoreductase/hydrogenase; secondary alcohols such as secondary butanol, 2-pentanol, or 3-pentanol; or any other suitable co-factor regeneration system.