Method of producing dipeptidyl peptidase iv inhibitors and derivatives of said inhibitors

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

 

The text descriptions are given in facsimile form.

1. The method of obtaining partially purified concentrates of phenylalaninamide and/or formiatehydrogenlyase (PDH/FDH), which includes the following stages:
A. getting the culture fluid of the microorganism Escherichia coli JM110 (pBMS 2000-PPDFDH-mod), is able to produce phenylalaninamide and/or formiatehydrogenlyase;
b. exposure to the culture fluid of microfluidizer to release the enzymatic activity of the cells and obtain microfluidizer culture fluid with PDH and/or FDH activity,
c. purification of the culture fluid by processing its flocculating agent to coagulopathy cellular debris, and remove the junk DNA and proteins;
d. filtering the purified culture fluid; and
that is, the concentration of the culture fluid with obtaining partially purified enzyme concentrate with PDH/FDH activity, with PDH activity, at least about 400 IU/ml and FDH activity in the region is her least about 20 IU/ml

2. The method according to claim 1, wherein the culture fluid is subjected to microfluidizer under pressure in the range of from about 12,000 psi to about 20,000 psi.

3. The method according to claim 1, wherein the culture fluid is subjected to microfluidizer maintaining the temperature of the culture liquid below 25°C.

4. The method according to claim 3, characterized in that the temperature of the culture fluid support below 25°C.

5. The method according to claim 1, characterized in that the purified culture liquid is subjected to ultrafiltration.

6. The method of obtaining an amine of structural formula

which includes stages
A. treatment of an aqueous solution of keto-acid structural formula

by ammonium formate, dinucleotide, dithiothreitol and partially purified mixture of enzymes phenylalaninamide and formiatehydrogenlyase (PDH/FDH)obtained by the method according to claim 1 at a temperature of from 35 to 40°C;
b. maintain the pH of the reaction mixture with sodium hydroxide in the range from 7.8 to 8.2 with obtaining an amine of formula 2.

7. The method according to claim 6, characterized in that the reaction are the period of time from about 24 hours to about 48 hours to obtain an amine of formula 2.

8. The way to obtain the BOC-protected amine of structural formula

which includes stages
A. is Holocene aqueous solution of amino acids (αS)-α-amino-3-hydroxytriazine [3.3.1.1 3,7]decane-1-acetic acid structural formula

by reductive amination of a keto acid

where an aqueous solution of (αS)-α-amino-3-hydroxytriazine[3.3.1.13,7] decane-1-acetic acid, ammonium formate, dinucleotide, dithiothreitol and partially purified concentrate of enzymes
phenylalaninamide and formiatehydrogenlyase (PDH/FDH)obtained by the method according to claim 1, heated to a temperature of from 35 to 40°C and maintain the pH of the reaction mixture with sodium hydroxide in the range from 7.8 to 8.2, with the receipt of a solution of amino acids; and
b. processing the above-mentioned aqueous solution of directbuylighting.com to obtain BOC-protected amine.

9. The method according to claim 8, characterized in that the pH of the mixture aqueous solution and distritbution maintained within the range of from about 8.5 to about 12.5.

10. The method according to claim 9, characterized in that it additionally includes the stage of allocation of the BOC-protected amine from the reaction medium and its crystallization.

11. Partially purified mixture of enzymes phenylalaninamide and formiatehydrogenlyase obtained by the method according to claim 1.

12. The way to obtain the BOC-protected amine of the formula

which includes stages
A. obtain partially purified concentrate fermentatsioonitehnoloogia and formiatehydrogenlyase, by
getting the culture fluid of the microorganism Escherichia coli JM110 (pBMS 2000-PPDFDH-mod), is able to produce phenylalaninamide and/or formiatehydrogenlyase;
exposure to the culture fluid of microfluidizer to release the enzymatic activity of the cells and obtain microfluidizer culture fluid with PDH and/or FDH activity,
purification of the culture fluid by processing its flocculating agent to coagulopathy cellular debris, and remove the junk DNA and proteins;
filtering the purified culture fluid; and
concentration of the culture fluid with obtaining partially purified enzyme concentrate with PDH/FDH activity, with PDH activity, at least about 400 IU/ml and FDH activity, at least about 20 IU/ml;
b. treatment of an aqueous solution of keto-acid structural formula

by ammonium formate, dinucleotide, dithiothreitol and partially purified mixture of enzymes phenylalaninamide and formiatehydrogenlyase obtained in step (a) at a temperature of from 35 to 40°C;
c. maintain the pH of the reaction mixture with sodium hydroxide in the range from 7.8 to 8.2 with obtaining an amine of formula 2

and
d. the processing of the above-mentioned water rest the RA directbuylighting.com without isolating the intermediate amino acids to obtain the BOC-protected amine of structural formula
.

13. A method of obtaining a connection in the form of the free base of the formula

which provides for the BOC-protected compounds of formula 3

by obtaining an aqueous solution of amino acids (αS)-α-amino-3-hydroxytriazine[3.3.1.13,7]decane-1-acetic acid structural formula

by reductive amination of a keto acid

where an aqueous solution of (αS)-α-amino-3-hydroxytriazine[3.3.1.13,7]decane-1-acetic acid, ammonium formate, dinucleotide, dithiothreitol and partially purified concentrates enzymes phenylalaninamide and formiatehydrogenlyase (PDH/FDH)obtained by the method according to claim 1, heated to a temperature of from 35 to 40°C and maintain the pH of the reaction mixture with sodium hydroxide in the range from 7.8 to 8.2, with the receipt of a solution of amino acids; and
the processing of the above-mentioned aqueous solution of directbuylighting.com to obtain BOC-protected amine of formula 3;
treatment of the BOC-protected connection methylchloride and aminobutiramida-ethylamine and the compound of the formula J

and 1-hydroxybenzotriazole (NOT) to give the BOC-protected intermediate structure To a

the de-hydration of the intermediate in the presence of pyridine and triperoxonane anhydride, and subsequent hydrolysis of the reaction product in the presence of a strong base to obtain compound L

and process connection L hydrochloric acid to obtain the corresponding hydrochloric acid salt L'

processing connection L' with sodium hydroxide to obtain compound M' in the form of free base.



 

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