This application claims priority to Indian patent application number 1443/CHE/2011 filed on Apr 26, 2011 the contents of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION:

The present invention relates to novel enzymatic process for the preparation of Miglitol and deoxynorjorimycin hydrochloride, a key intermediate of Miglitol.

BACKGROUND OF THE INVENTION:

Miglitol is an oral alpha-glucosidase inhibitor for use in the management of non-insulin-dependent diabetes mellitus (NIDDM). Miglitol is sold by the trade name Glyset. GLYSET tablets are available as 25 mg, 50 mg and 100 mg tablets and are made for oral use. Miglitol is a desoxynojirimycin derivative, and is chemically known as 3,4,5-piperidinetriol, l-(2-hydroxyethyl)-2-(hydroxymethyl)-, [2R-(2a,3p,4a, 50)] and its chemical structure is as follows:

It is primarily used in diabetes mellitus type 2 for establishing greater glycemic control by preventing the digestion of carbohydrates (such as disaccharides, oligosaccharides, and polysaccharides) into monosaccharides which can be absorbed by the body.

Miglitol inhibits glycoside hydrolase enzymes called alpha-glucosidases. Since miglitol works by preventing digestion of carbohydrates, it lowers the degree of postprandial hyperglycemia. It must be taken at the start of main meals to have maximal effect. Its effect will depend on the amount of non-monosaccharide carbohydrates in a person's diet.

In contrast to acarbose (another alpha-glucosidase inhibitor), miglitol is systematically absorbed; however, it is not metabolized and is excreted by the kidneys. European Patent Application 3,024,901 Al disclosed a process for the preparation of Miglitol starting from 2-hydroxymethyl-3,4,5-trihydroxypiperidines of formula V;

It is also known that compounds of the formula (I) are obtained when compounds of the formula (V) are treated with ethylene oxide in a manner known in the art.

WO2008025826 disclosed an enzymatic process for the preparation of 1-Deoxynojirimycin as depicted below:

l-L/coxyiiujirifiiycm

An aldol addition catalysed by a D-Fructose-6-Phosphate aldolase enzyme between protected amino aldehyde (N-CbZ-3-aminopropanal) and a diol (dihyroxyacetone) to form an adduct which then undergoes intramolecular reductive amination in the presence of a hydrogen source to form 1-Deozynojirimycin.

The use of gaseous carcinogenic feed materials, expensive, poorly accessible starting components are some of the disadvantages of the process known in the prior art. Also the reaction time is very long thus proving chemosynthetic process to be tedious. Thus there is a need of an economical, safe process which is industrially feasible. The inventors of the present invention have come up with an enzymatic process for the preparation of miglitol and its key intermediates using yeast and their related species and gluconobacteroxydans species which makes the reaction fast and cheap. These enzymatic reactions are safe, economical and industrially feasible.

OBJECT OF THE INVENTION.

The main object of the present invention relates to a novel enzymatic process for the preparation of Miglitol and 1-deoxynorjorimycin hydrochloride, a key intermediate of Miglitol.

SUMMARY OF THE INVENTION:

The main aspect of the present invention relates to an enzymatic process for the preparation of Miglitol and 1-deoxynorjorimycin.

Another aspect of the present invention relates to an enzymatic process for the preparation of Miglitol or its key intermediate of Miglitol (1-deoxynorjorimycin or its hydrochloride salt).

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides an enzymatic process for the preparation of Miglitol.

In another embodiment, the present invention provides an enzymatic process for the preparation of 1-deoxynorjorimycin, a key intermediate of miglitol

In another embodiment, the present invention relates to an enzymatic process for the preparation of Miglitol or 1-deoxynorjorimycin as shown in the below scheme

In another embodiment, the present invention provides an enzymatic process for the preparation of Miglitol from N-hydroxyethyl glucosamine comprising the steps of;

a) inoculating yeast into a buffer solution containing N-hydroxyethyl glucosamine,

b) adding a proton donor/acceptor,

c) stirring the mass,

d) separating the mass using resin,

e) removing the solvent,

f) optionally purifying the residue, and

g) isolating pure N-hydroxyethyl-1 -deoxynorjorimycin (Miglitol).

According to the present invention, Yeast (obtained from Saccharomyces cerevasiae) and gluconobacter (obtained from Agricultural Research Service USA is inoculated in a reaction buffer containing N-hydroxyethyl glucosamine, proton donor/acceptor such as NAD, NADP, 2,3 dichloro, 5,6 dicyano 1,4 benzouinone, Phenazine methosulfate, acetoin. In the presence of the yeast, the N-hydroxyethyl glucosamine is oxidized on the 5th position and then gets cyclised by the p resence of a proton donor. The reaction mass is maintained till the completion of the reaction. The resultant mixture is separated by using a resin such as cation exchanger resin. Washing the reaction mass with water and acid and removing the solvent obtains Miglitol which is optionally purified.

Yet another embodiment, the present invention provides an enzymatic process for the preparation of 1-deoxynorjorimycin, a key intermediate of miglitol, comprising the steps of:

a. incubating 1-Amino-1-deoxy-D-glucitol taken in a buffer in presence of a proton
donor/acceptor and baker yeast,

b. maintaining the reaction mass,

c. separating the mass through a resin,

d. removing the solvent, and

e. isolating pure 1-deoxynorjorimycin.

Yet another embodiment, the present invention provides an enzymatic process for the preparation of 1-deoxynorjorimycin hydrochloride comprising the steps of:

a) inoculating 1-amino-1-deoxy-D-glucitol solution containing yeast extract and buffer,

b) maintaining the reaction medium under acidic pH,

c) separating the mass through a resin,

d) removing the solvent, and

e) isolating pure 1-deoxynorjorimycin hydrochloride.

According to the present invention, 1-Amino-1-deoxy-D-glucitol is taken in a buffer is incubated/inoculated in the presence of proton donor and baker yeast or gluconobacter species. The reaction mass is maintained under shaking and subjected to centrifugation. The filtrate is passed through a resin and washed with aq.acid. The acid fraction is distilled to obtain a residue which is precipitated by using an organic solvent such as ethanol, methanol, acetone and ethyl acetate to obtain crystals of 1-deoxynorjorimycin hydrochloride.

According to the present invention, the proton donor is selected from NAD, NADP, 2,3 dichloro, 5,6 dicyano 1,4 Benzoquinone, Phenazine methosulfate, acetoin.

According to the present invention, the buffer is selected from potassium dihydrogen phosphate, Tris HC1, HEPES, oxalic acid - sodium oxalate, acetic acid - sodium acetate.

According to the present invention, Cation exchanger resin is selected from Amberlite, Dowex and Resindion.

According to the present invention, the solvent is removed by precipitation, recovery of product from an organic solvent selected from alcohols such as methanol, isopropanol, ethanol, acetone, water or their mixtures thereof.

The following examples are merely shown as representative examples of the present invention but are not intended to be limiting.

EXAMPLES

Example 1. 1-Amino-1-deoxy-D-glucitol in buffer containing NAD, 20 g of Dextrose and 100mM phosphate buffer was incubated in presence of Locally available baker yeast at 30 -35 °C in a shaker for a period of 72 hours. Reaction mixture was centrifuged and filtrate was subjected for column chromatography. The filtrate was loaded into a column packed with resin followed by water wash and acid wash. The acid wash fraction was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin HC1.

Example 2. Gluconobacter roseus wet cells were then inoculated in autoclaved reaction media containing 1-amino-1-deoxy-D-glucitol solution containing of yeast extract, of D-sorbitol and potassium dihydrogen phosphate in Milli Q water, pH 2-10. The reaction media is stirred for 48 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-Deoxynorjorimycin HC1.

Example 3. Gluconobacter roseus inoculated in autoclaved reaction media containing 2% 1 -amino- 1-deoxy-D-glucitol solution containing yeast extract, D-sorbitol and potassium dihydrogen phosphate in water, pH 2-10 the reaction media is stirred for 48 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin.

Example 4. Baker yeast inoculated in autoclaved reaction media containing 1-amino-1-deoxy-D-glucitol solution containing yeast extract, D-sorbitol and potassium dihydrogen phosphate in water, pH 2-10. The reaction media is stirred for 72 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin HC1.

Example 5. Yeast from Saccharomyces cerevasiae inoculated in a reaction buffer containing 1-amino-1-deoxy-D-glucitol, NAD, D-Glucose in Milli Q water, 100mM potassium phosphate. The reaction media is stirred for 72 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin.

Example 6. Yeast from Saccharomyces cerevasiae inoculated in a reaction buffer containing 1-amino-1-deoxy-D-glucitol, Nicotinic acid, D-Glucose in water, 100mM potassium phosphate. The reaction media is stirred for 72 hours and monitored by HPLC. The reaction media is stirred for 138 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin.

Example 7. Yeast from Saccharomyces cerevasiae inoculated in a reaction buffer containing 1-amino-1-deoxy-D-glucitol, and of D-Glucose in water, 100mM potassium phosphate. The reaction media is stirred for 72 hours and monitored by HPLC. The reaction media is stirred for 138 hours, centrifuged and separated by column chromatography using resin. The acid washed was distilled off and the residue obtained was precipitated with ethanol to obtain white crystals of 1-deoxynorjorimycin.

Example 8. Yeast from Saccharomyces cerevasiae inoculated in a reaction buffer containing 1% N-hydroxyethyl glucosamine, NAD, D-Glucose in water, 100mM potassium phosphate. The reaction media is stirred for 168 hours, centrifuged and separated by column chromatography using Amberlyst-15 H+ resin. The water wash was distilled off and the residue obtained was further purified using n-PrOH: water: Ammonia through Silica gel to obtain pure N-hydroxyethyl- l-deoxynorjorimycin (Miglitol)

We Claim:

1. An enzymatic conversion of wherein the enzyme is selected from Saccharomyces cerevasiae, gluconobacter, or baker yeast.

2. An enzymatic process for the preparation of Miglitol comprising the steps of:

a) inoculating yeast into a buffer solution containing N-hydroxyethyl glucosamine, proton donor/acceptor, D-Glucose in aqueous medium,

b) separating the mass using resin,

c) removing the solvent,

d) optionally purifying the residue, and

e) isolating pure Miglitol.

3. An enzymatic process for the preparation of 1-deoxynorjorimycin comprising the steps of:

a. incubating 1-Amino-1-deoxy-D-glucitol taken in a buffer in presence of a proton
donor/acceptor and yeast,

b. separating the mass through a resin,

c. removing the solvent, and

d. isolating pure 1-deoxynorjorimycin.

4. An enzymatic process for the preparation of 1-deoxynorjorimycin hydrochloride
comprising the steps of:

a) inoculating 1 -amino- 1-deoxy-D-glucitol solution containing yeast extract and buffer,

b) maintaining the reaction medium under acidic pH,

c) separating the mass through a resin,

d) removing the solvent, and

e) isolating pure 1-deoxynorjorimycin hydrochloride.

5. The process according to the 2,3 or 4, wherein yeast is selected from Saccharomyces cerevasiae, gluconobacter, or baker yeast.

6. The process according to claim 2,3 or 4 wherein proton donors/acceptor is selected from NAD, NADP, 2,3 dichloro, 5,6 dicyano 1,4 benzouinone, Phenazine methosuifate, or acetoin.

7. The process according to claim 2,3 or 4, wherein resin is selected from Amberlyst-15 H+, Amberlite, Dowex or Resindion.

8. The process according to claim 2, 3 or 4, wherein the solvent is removed by filtration, distillation or vaccum distillation.

9. The process according to claim 3 or 4, wherein buffer is selecting from dihydrogen phosphate, Tris HC1, HEPES, oxalic acid - sodium oxalate, acetic acid - sodium acetate.

10. An enzymatic process comprising the steps of;
Converting to Miglitol by using Saccharomyces cerevasiae, gluconobacter, or baker yeast Or converting to 1-deoxynorjorimycin using Saccharomyces cerevasiae, gluconobacter, or baker yeast.