Cenobamate

Preparation Example 1: Preparation of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one

To a suspension of 2-bromo-2′-chloroacetophenone (228.3 g, 0.978 mol) and potassium carbonate (161.6 g, 1.170 mol) in acetonitrile (2000 mL) was added a 35 w/w% 1H-tetrazole dimethylformamide solution (215.1 g, 1.080 mol) at room temperature. These reactants were stirred for 2 h at 45℃ and distilled under reduced pressure to remove about 1500 mL of the solvent. The concentrate was diluted in ethyl acetate (2000 mL) and washed with 10% brine (3 x 2000 mL). The organic layer thus separated was distilled under reduced pressure to afford 216.4 g of an oily solid residue. To a solution of the solid residue in ethyl acetate (432 mL) was slowly added heptane (600 mL). The precipitate thus formed was filtered at room temperature and washed to yield 90.1 g (0.405 mol) of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one (hereinafter referred to as 1N ketone ).

¹H-NMR(CDCl ₃) 8.87(s, 1H), d7.77(d, 1H), d7.39-7.62(m, 3H), d5.98(s, 2H)

Preparation Example 2: Preparation of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one

After the filtration of Preparation Example 1, the filtrate was concentrated and dissolved in isopropanol (100 mL), and to which heptane (400 mL) was then added to complete the crystallization. Filtering and washing at 5℃ afforded 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one (hereinafter referred to as “2N ketone”) as a solid. 94.7 g (0.425 mol).

¹H-NMR(CDCl ₃) d8.62(s, 1H), d7.72(d, 1H), d7.35-7.55(m, 3H), d6.17(s, 2H)

PREPARATION EXAMPLE 3: Preparation of Alcohol Compound of (R)-Configuration by enantioselective enzymatic reduction via various oxidoreductases

The following four solutions were prepared as follows:

Competent Escherichia coli StarBL21(De3) cells (Invitrogen) were transformed with the expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 1. The Escherichia coli colonies transformed with the resulting expression constructs were then cultivated in 200 mL of LB medium (1% tryptone, 0.5 % yeast and 1% sodium chloride) with 50 micrograms/mL of ampicillin or 40 micrograms/mL of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was achieved. The expression of the desired recombinant protein was induced by the addition of isopropylthiogalactoside (IPTG) to a concentration of 0.1 mM. After 16 hours of induction at 25 ℃ and 220 rpm, the cells were harvested and frozen at -20 ℃. In the preparation of the enzyme solutions, 30 g of cells were resuspended in 150 mL of triethanolamine buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) and homogenized in a high pressure homogenizer. The resultant enzyme solution was mixed with 150 mL glycerol and stored at -20℃.

RB791 cells ( E.coli genetic stock, Yale, USA) were transformed with the expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 2. The Escherichia coli colonies transformed with the resulting expression constructs were then cultivated in 200 mL of LB medium (1% tryptone, 0.5 % yeast and 1% sodium chloride) with 50 micrograms/mL of ampicillin or 40 micrograms/mL of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was achieved. The expression of the desired recombinant protein was induced by the addition of isopropylthiogalactoside (IPTG) to a concentration of 0.1 mM. After 16 hours of induction at 25℃ and 220 rpm, the cells were harvested and frozen at -20℃. In the preparation of the enzyme solutions, 30 g of cells were resuspended in 150 mL of triethanolamine buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) and homogenized in a high pressure homogenizer. The resultant enzyme solution was mixed with 150 mL glycerol and stored at -20℃.

Enzyme solutions 3 was prepared in the same manner as described in Enzyme solution 1 except that expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 3 instead of expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 1 was used.

Enzyme solutions 4 was prepared in the same manner as described for enzyme solution 2 except that expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 4 instead of expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 2 was used.

Different oxidoreductases contained in each enzyme solutions 1 to 4 were examined as follows for the conversion of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one (1N ketone) and 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one (2N ketone) to the corresponding 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-ol (hereinafter, referred to as 1N alcohol ) and 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (hereinafter, referred to as “2N alcohol”), respectively.

160 ㎕ buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8)

100 ㎕ NADPH (40 mg/ml)

40 ㎕ 2-propanol

50 ㎕ enzyme solution 1

2 mg 1N ketone or 2N ketone

160 ㎕ buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8)

100 ㎕ NADPH (40 mg/ml)

40 ㎕ 2-propanol

50 ㎕ enzyme solution 2

2 mg 1N ketone or 2N ketone

350 ㎕ buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8)

0,05 mg NADP

50 ㎕ enzyme solution 3

10 mg 1N ketone or 2N ketone

250 ㎕ 4-methyl-2-pentanol

50 ㎕ enzyme (oxidoreductase from Thermoanerobium brockii) solution for regeneration of cofactor

350 ㎕ buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8

0,05 mg NADP

50 ㎕ enzyme solution 4

10 mg 1N ketone or 2N ketone

250 ㎕ 4-methyl-2-pentanol

50 ㎕ enzyme (oxidoreductase from Thermoanerobium brockii) solution for regeneration of cofactor

After 24h of incubating each reaction batch A, B, C and D, 1 mL of acetonitrile was added to each reaction batch which was centrifuged and transferred into a HPLC analysis vessel for enantiomeric excess and conversion. Conversion and ee-value of products are listed in Table 1 below calculated using the following equations:

Conversion Rate (%) = [(Area of Product)/(Area of Reactant + Area of Product)]x100

ee-value(%) = [(Area of R-Configuration – Area of S-Configuration)/(Area of R-Configuration + Area of S-Configuration)] x 100

Table 1 [Table 1] 

PREPARATION EXAMPLE 4: Enzymatic reduction via oxidoreductase SEQ NO: 2

For the conversion of 1N/2N ketone to R-1N/R-2N alcohol, 30㎕ of the enzyme solution 2 containing the oxidoreductase SEQ NO: 2 were added to a mixture of 300㎕ of a buffer (100 mM TEA, pH 8, 1mM MgCl2, 10% glycerol), 100mg of a mixture of 1N ketone and 2N ketone (1N:2N=14%:86%), 0.04mg NADP and 300㎕ 2-butanol. The reaction mixture was incubated at room temperature under constant thorough mixing. After 48 hours, more than 98% of the ketones were reduced to an alcohol mixture of the following composition(R-2N alcohol 80%; S-2N alcohol 0%; R-1N alcohol 20%, S-1N alcohol 0%; 1N ketone 0%; 2N ketone 0%).

After general work up and recrystallization with ethyl acetate/hexane, optically pure alcohols were obtained as below:

(R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-ol (1N alcohol

¹H-NMR(CDCl ₃) d8.74(s, 1H), d7.21-7.63(m, 4H), d5.57(m, 1H), d4.90(d, 1H), d4.50(d, 1H), d3.18(d, 1H);

(R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (2N alcohol)

¹H-NMR(CDCl ₃) d8.55(s, 1H), d7.28-7.66(m, 4H), d5.73(d, 1H), d4.98(d, 1H), d4.83(d, 1H), d3.38(br, 1H).

Preparation of Carbamate

Preparation Example 5: Preparation of Carbamic Acid (R)-1-(2-Chlorophenyl)-2-(tetrazol-2-yl)ethyl ester

50ml of the enzyme solution 2 containing the oxidoreductase SEQ NO: 2 were added to a mixture of 250ml of a buffer (100 mM TEA, pH 8, 1mM MgCl2, 10% glycerol), 50g (225mmol) of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one(2N ketone), 4mg NAD, 300 ml of 2-propanol and 150mL of butyl acetate. The reaction mixture was stirred at room temperature. After 48 hours more than 98% of 2N ketone was reduced to corresponding (R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2N alcohol) with >99%ee values. To this resulting mixture, 500mL of ethyl acetate was added. After being separated, the organic layer thus formed was washed with 10% brine (3 x 500mL). The organic layer thus formed was dried over magnesium sulfate and filtered and the filtrate was distilled under reduced pressure to give 50.4g (224 mmol) of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2N alcohol, optical purity 99.9%) as an oily residue. To this resulting crude product, 450mL of tetrahydrofuran was added. After cooling to -15℃, 38g (267mmol) of chlorosulfonyl isocyanate was slowly added and stirred at -10℃ for 2 h. The slow addition of water induced termination of the reaction. The resulting solution was concentrated under reduced pressure until about 300 mL of the solvent was removed. The concentrate was diluted with 600mL of ethyl acetate and washed with 10% brine (3 x 500 mL). The organic layer was concentrated under reduced pressure and the concentrate was dissolved in isopropanol (90 mL) to which heptane (180 mL) was slowly added, leading to the completion of crystallization. The precipitate thus obtained was filtered and washed to afford 51.8 g (194 mmol) of carbamic acid (R)-1-(2-chlorophenyl)-2-(tetrazol-2-yl)ethyl ester (optical purity 99.9%).

¹H-NMR(Acetone-d ₆) d8.74(s, 1H), d7.38-7.54(m, 4H), d6.59(m, 1H), d6.16(Br, 2H), d4.90(d, 1H), d5.09(m, 2H)

As described hitherto, carbamate compounds with high optical and chemical purity can be produced with an economical benefit in accordance with the present invention.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

SEQ ID NO 1: Oryctolagus cuniculus from rabbit DSMZ 22167

SEQ ID NO 2: Candida magnoliae DSMZ 22052 protein sequence carbonyl reductase

SEQ ID NO 3: Candida vaccinii CBS7318 protein sequence carbonyl reductase

SEQ ID NO 4: Candida magnoliae CBS6396 protein sequence carbonyl reductase

SEQ ID NO 5: Oryctolagus cuniculus from rabbit DSMZ 22167

SEQ ID NO 6: Candida magnoliae DSMZ 22052 nucleic acid sequence carbonyl reductase

SEQ ID NO 7: Candida vaccinii CBS7318 nucleic acid sequence carbonyl reductase

SEQ ID NO 8: Candida magnoliae CBS6396 nucleic acid sequence carbonyl reductase