# US 9,434,970 B2 · Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester

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- Number: US 9,434,970 B2
- Document ID: US9434970B2
- Status: Granted
- Kind: B2
- Application number: 14/719,728
- Filed: 2015-05-22
- Published: 2015-09-10
- Granted: 2016-09-06
- Earliest claimed: 2009-10-15
- Assignees: SK Biopharmaceuticals Co., Ltd.
- Applicants: SK Biopharmaceuticals Co., Ltd.
- Inventors: Sang Chul Lim; Moo Yong Uhm; Dae Won Lee; Hui Ho Kim; Dong Ho Lee; Hyun Seok Lee
- Web page: https://patentgrep.com/patent/US9434970B2

## CPC

- A61P 25/00: Drugs for disorders of the nervous system
- C12P 17/10: Nitrogen as only ring hetero atom
- C07D 257/04: Five-membered rings
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)

## Abstract

Disclosed is a method for the preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl esters, comprising the enantioselective enzyme reduction of a 1-aryl-2-tetrazolyl-ethyl ketone to form a (R)-1-aryl-2-tetrazolyl-ethyl alcohol and the carbamation of said alcohol.

## Claims

### Claim 1

A method for preparing carbamic acid aryl-2-tetrazolyl ethyl ester of Chemical Formula 1, comprising:
subjecting an arylketone of Chemical Formula 2 to enantioselective enzymatic reduction in a reaction mixture comprising said compound, an oxidoreductase having at least 90% homology with amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, NADH or NADPH as a cofactor that is oxidized during the reduction process and continuously regenerated, a second substrate comprising a secondary alcohol having the Formula RR′CHOH, wherein R and R′ are independent from one another and have from 1 to 10 carbon atoms, and a suitable buffer to form an alcohol compound of (R)-configuration of Chemical Formula 3; and
carbamating said alcohol,
wherein R1 and R2 are independently selected from the group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; and
one of A1 and A2 is CH with the other being N.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the oxidoreductase is encoded by nucleic acid sequence SEQ ID NO: 5 or SEQ ID NO: 7.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the oxidoreductase is isolated from Candida magnolia, Candida vaccinii or Oryctolagus cuniculus.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the oxidoreductase is purified or in a microbial cell.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the oxidoreductase is partially purified.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the oxidoreductase is a microbial cell lysate.

### Claim 7 (depends on 4)

The method according to claim 4, wherein the oxidoreductase is present in the microbial cells and is permeabilized.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the microbial cells are transformed Escherichia coli cells.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the oxidized cofactor is reduced to the cofactor by the oxidation of said second substrate.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said second substrate is a secondary alcohol selected from the group consisting of 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and 2-octanol.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said oxidoreductase affects both the reduction of the arylketone of Chemical Formula 2 and the oxidation of the second substrate.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the carbamating step comprises reacting the alcohol compound of (R)-configuration of Chemical Formula 3 with inorganic cyanate and an organic acid.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the carbamating step comprises hydrolyzing a product resulting from reaction between the alcohol compound of (R)-configuration of Chemical Formula 3 and an isocyanate compound selected from the group consisting of chlorosulfonic isocyanate, trichloroacetyl isocyanate and trimethylsilyl isocyanate.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the carbamating step comprises introducing ammonia into a product resulting from reaction between the alcohol compound of (R)-configuration of Chemical Formula 3 and a carbonyl compound comprising 1,1′-carbonyldiimidazole, carbamoylhalide, N,N-disuccinimidyl carbonate, phosgene, triphosgene or chloroformate.

### Claim 15 (depends on 1)

The method according to claim 1, further comprising crystallizing the compound of Chemical Formula 1.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the crystallizing step comprises:
adding to a reaction product a solubilizing agent selected from the group consisting of acetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, an alcohol of 1 to 4 carbon atoms and mixtures thereof; and
adding a precipitating agent thereto selected from the group consisting of water, an alcohol of 1 to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptane and mixtures thereof.

### Claim 17 (depends on 1)

The method according to claim 1, further comprising: preparing the arylketone of Chemical Formula 2 by a substitution reaction between an arylketone of Chemical Formula 4 with a tetrazole of Chemical Formula 5:
wherein R1 and R2 are as defined in claim 1; and X is a leaving group selected from the group consisting of halides and sulfonates.

### Claim 18 (depends on 17)

A method according to claim 17, further comprising a crystallizing step comprising:
adding a solubilizing agent selected from the group consisting of acetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, an alcohol of 1 to 4 carbon atoms and mixtures thereof to a product obtained by the substitution reaction; and
adding a precipitating agent selected from the group consisting of water, an alcohol of 1 to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptanes and mixtures thereof.

### Claim 19

A method for preparing an alcohol compound of Chemical Formula 3, through the enantioselective enzymatic reduction of an arylketone of Chemical Formula 2:
wherein
R1 and R2 are independently selected from the group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms;
one of A1 and A2 is CH with the other being N; and
said reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 90% homology with amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, NADH or NADPH as a cofactor that is oxidized during the reduction process and continuously regenerated, a second substrate comprising a secondary alcohol having the Formula RR′CHOH, wherein R and R′ are independent from one another and have from 1 to 10 carbon atoms, and a suitable buffer.

### Claim 20 (depends on 19)

The method according to claim 19, wherein the oxidoreductase is encoded by nucleic acid sequence SEQ ID NO: 5 or SEQ ID NO: 7.

### Claim 21 (depends on 19)

The method according to claim 19, wherein the oxidoreductase is isolated from Candida magnolia, Candida vaccinii or Oryctolagus cuniculus.