This application claims priority to Indian patent application numbered IN 3275/CHE/2013 filed on July 22, 2013, the contents of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to an improved process for the preparation of 1-Deoxynojirimycin, an intermediate in the preparation of Miglitol.
BACK GROUND 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, 1-(2-hydroxyethyl)-2-(hydroxymethyl)-, [2R-(2a,3p,4a, 50)] and its chemical structure is as shown below:
1-deoxynojirimycin is a key intermediate in the preparation of Miglitol. It is structurally represented by formula I shown below:
US patent 4246345 discloses a process for preparing 1-deoxynojirimycins, wherein 1-aminosorbitol is microbiologically oxidized to 6-aminosorbose which is then hydrogenated to 1-deoxynojirimycin.

US4806650 discloses that D-glucose is converted to aminosorbitol of formula V which is then protected with an alkalinically detachable group to give compound of formula IV. The protected amino compound of formula IV is stable in the subsequent microbiological oxidation. The protected compound of formula IV is microbiologically oxidized to compound of the formula III and the protective groups are subsequently alkalinically split off affording the compound of the formula II. The compound of formula V is then is reduced in a manner known such as catalytically or with complex hydrides to give deoxynojirimycin of the formula I
WO2008025826 disclosed an enzymatic process for the preparation of miglitol as shown below

US2016962 patent discloses a process for preparing aminosorbitol, where in amination of saccharides by ammonia or aliphatic amine or aromatic amine having a replaceable amino hydrogen atom is carried out at an elevated pressure of more than 15 atmospheres and at a temperature between 50°C and 200°C in the presence of nickel catalyst.
Journal of the American chemical society 79, 3541-4, 1957 discloses, reductive alkylation of benzylamine with galactose in the presence of platinum catalyst gave galactamine in when hydrogenolyzed in the presence of palladium-on-charcoal.
Still there is a need for an alternate and improved process for the preparation of 1-deoxynojirimycin. The inventors of the present invention developed simple, cost effective and commercially feasible process for the preparation of 1-deoxynojirimycin with high yield.

OBJECT OF THE INVENTION
The main objective of the present invention relates to an improved process for the preparation of 1-Deoxynojirimycin, a key intermediate of Miglitol.
SUMMARY OF THE INVENTION
The main aspect of the present invention provides an improved process for the preparation of 1-Deoxynojirimycin in high yield.
Another aspect of present invention provides an improved process for the preparation of 1-Deoxynojirimycin comprising the steps of:
a) converting the compound of formula A into formula B in the presence of a metal hydrogenation catalyst,
b) reducing the compound of the formula B with a metal catalyst to give compound of formula C,
c) converting the compound of Formula C into compound of Formula D in the presence of a polar protic solvent,
d) oxidizing the compound of Formula D using enzyme to give compound of Formula E,
e) reducing the compound of the formula E to give compound of the formula I.
In yet another aspect, the present invention provides process for the preparation of deoxynojirmycin by oxidizing N-benzyl-N-formyl-1-amino-1-deoxyglucitol.
In yet another aspect, the present invention provides process for the preparation of galactonojirmycin using Gluconobacter oxydans.
In yet another aspect, the present invention provides further conversion of 1-deoxynojrimycin into Miglitol or its pharmaceutically acceptable salts with less than 0.1% impurities.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an improved process for the preparation of 1-deoxynojirimycin, a key intermediate in the preparation of Miglitol.
The present invention also relates to further conversion of intermediate 1-deoxynojirimycin into Miglitol or its pharmaceutically acceptable salts.

In one embodiment, the present invention relates to a process for the preparation of 1-deoxynojirimycin of Formula-I as represented in the scheme below:
H.
In another embodiment, the present invention relates to an improved process for the preparation of 1-deoxynojirimycin comprising the steps of:
a) converting the compound of formula A into formula B in the presence of a metal hydrogenation catalyst,
b) reducing the compound of the formula B with a metal catalyst to give compound of formula C,
c) converting the compound of Formula C into compound of Formula D in the presence of a polar protic solvent,
d) oxidizing the compound of Formula D using enzyme to give compound of Formula E,
e) reducing the compound of the formula E to give compound of the formula I which is optionally purified.
In another embodiment, the compound of formula A is treated with aryl amine, preferably benzyl amine followed by treatment with a metal hydrogenation catalyst, preferably Raney-nickel in the presence of an organic solvent preferably methanol to give compound of formula B.

In yet another embodiment, the compound of formula B is reduced by using a metal hydrogenation catalyst preferably palladium-carbon in the presence of an organic solvent preferably isopropanol to give compound of formula C.
In yet another embodiment, the compound of the formula C is reacted with methyl formate in the presence of a polar solvent such as methanol to give compound of formula D.
In yet another embodiment, the compound of Formula D is enzymatically oxidized by using Gluconobacter oxydans followed by pH adjustment with phosphoric acid to give compound of formula E.
In yet another embodiment, the compound of Formula E is converted to compound of formula I having impurity less than 0.05% by using a reducing agent such as sodium borohydride in presence of a base such as sodium hydroxide. Further the compound of formula I is converted to Miglitol or its pharmaceutical^ acceptable salts by conventional methods known in the art.
In yet another embodiment, the present invention relates to an improved process for the preparation of deoxynojirmycin as represented in the following scheme.

In yet another embodiment, the present invention relates to an improved process for the preparation of compound of Formula-I comprising the steps of
a) converting the compound of dextrose into N-benzyl-1-amino-1-deoxy-D-glucitol in the presence of metal catalyst,
b) converting compound of N-benzyl-1-amino-1-deoxy-D-glucitol to N-benzyl-N-formyl-1-amino-1-deoxy-D-glucitol,
c) oxidizing compound of N-benzyl-N-formyl-1-amino-1-deoxy-D-glucitol in the presence of an enzyme to give N-benzyl-N-formyl-1-amino-1-deoxy-5-keto-D-glucitol,
d) reducing the compound of N-benzyl-N-formyl-1-amino-1-deoxy-5-keto-D-glucitol in the presence of a reducing agent to give compound of formula I,
e) optionally converting compound of the formula I into its pharmaceutically acceptable salts.
According to the present invention, dextrose is reduced with metal catalyst preferably raney nickel followed by treatment with benzylamine to give N-benzyl-1-amino-1-deoxy-D-glucitol which is reacted with methylformate in the presence of polar protic solvent preferably methanol to give N-benzyl-N-formyl-1-amino-1-deoxy-D-glucitol.
N-benzyl-N-formyl-1-amino-1-deoxy-D-glucitol is oxidized using an enzyme such as Gluconobacter oxydans to give N-benzyl-N-formyl-1-amino-1-deoxy-5-keto-D-glucitol which is then reduced using a reducing agent preferably sodium borohydride in presence of a base such as sodium hydroxide, followed by hydrogenation using palladium-carbon to give compound of the formula I. The compound of the formula I optionally converted into its pharmaceutically acceptable salts.
In yet another embodiment, the present invention realtes to a process for the preparation of galactonojirmycin using Gluconobacter oxydans DSM 2003 comprising the steps of:
a) converting the compound of D-galactose into N-benzyl-1-amino-1-deoxy-D-galactitol in the presence of metal catalyst,
b) reducing the compound of N-benzyl-1-amino-1-deoxy-D-galactitol in the presence of metal catalyst to give 1-amino-1-deoxy-D- galactitol,
c) converting the compound of 1-amino-1-deoxy-D- galactitol into N-formyl-1-amino-dexoy-D-galactitol,
d) oxidizing the compound of N-formyl-1-amino-1-dexoy-D-galactitol in the presence of enzyme to give N-formyl-1-amino-1-dexoy-keto-D-galactitol,
e) reducing the compound of N-formyl-1-amino-1-dexoy-keto-D-galactitol into galactonojirmycin,

f) optionally converting galactonojirmycin to its pharmaceutical^ acceptable salts.
In yet another embodiment, the present invention relates to an improved process for the preparation of galactonojirmycin as represented in the following scheme below:
US20010019837A1, US6552176, US5916784, US5610039, US4806650, US4505714 disclosed a process for the preparation of Gluconobacter cell paste by fermentation route and used in oxidation of protected and unprotected amino compound of formula IV. US 5610039 and US5602013 disclosed immobilization of Gluconobacter whole cells on Gelrite gellan gum.
The inventors of the present invention came up with an improved process by which the productivity of cell paste and the absorbance values are increased by fermentation method.
Accordingly Gluconobacter oxydans DSM 2003 cell paste is prepared under condition like pH control at 5.5 - 6.0 using 30% dipotassium hydrogen phosphate, 0.5 - 1 wm sterile air supply and agitation at 300 - 500 RPM till absorbance value of 5.5- 6.0 has improved the cell paste production (2 folds) upto 130 g in 15 L batch in a fermentor.
The Gluconobacter oxydans whole cell paste prepared were immobilized on sodium alginate beads and recycled 3 times or till activity of cell paste reduced. The Gluconobacter oxydans DSM 2003 cell paste prepared under condition like pH at 5.5 - 6.0 using 30% dipotassium hydrogen phosphate, 0.5 - 1wm sterile air supply and agitated at 300 - 500 RPM till absorbance value of

5.5- 6.0 has improved the cell paste production (2 folds) upto 130 g in 15 L batch in a fermentor. The Gluconobacter oxydans whole cell paste prepared were immobilized on sodium alginate beads and recycled 3 times or till activity of cell paste reduced.
The following examples are provided to illustrate the process of the present invention. They, are however, not intended to limiting the scope of the present invention in any way and several variants of these examples would be evident to person ordinarily skilled in the art.
Experimental procedure:
Example 1: Preparation of 1-amino-1-deoxy-D-glucitol
Anhydrous dextrose (100g) and benzyl amine (72 mL) were added to a pressure reactor containing (2000 mL) of methanol under nitrogen atmosphere. To this mixture (15g) of activated Raney-Nickel with moisture content less than 1% was added maintaining under nitrogen atmosphere and hydrogenated at 10-12 kg/cm2 for 60 - 90 hours at 50 °C under stirring. After completion of reaction with the formation of N-benzyl-1-amino-1-deoxy-D-glucitol, the hydrogen pressure was released under nitrogen atmosphere. The reaction mixture was cooled to 45 °C. and (2000 mL) of D.M. water was added. The reaction mixture was again cooled to 35 °C and filtered through hiflow and 5 micron filter under nitrogen atmosphere. The filtrate mLs were collected and added once again to a pressure reactor. To this mixture (15 g) of 10% palladium on Carbon (50% wet) was added and hydrogenated at 10-12 kg/cm2 for 24 - 30 hours at 50 °C under stirring. After the completion of reaction, the hydrogen pressure was released under nitrogen atmosphere. The reaction mixture was cooled to ambient temperature and filtered through hiflow and 5 micron filter under nitrogen atmosphere. The filtrate mLs were collected and the reaction mixture was concentrated under high vacuum at temperature below 40 °C. To the concentrated oily reaction mass (600 mL) of isopropyl alcohol was added, stirred at 50 °C for 30 minutes. The reaction was concentrated under reduced vacuum at temperature below 40 °C till moisture content reaches below 5%. To the concentrated reaction mass (750 mL) of isopropyl alcohol was added and stirred for 30 - 40 minutes. The reaction mixture was further stirred at 3 °C for 60-90 minutes and filtered under nitrogen atmosphere. The solid obtained was dried under vacuum till moisture content reaches below 3%. Yield: 98 %; HPLC purity: > 98% which contained dimer and sorbitol impurities less than 2%.
Example 2. Preparation of N-formyl-1-amino-1-deoxy-D-glucitol:

1-amino-1-deoxy-D-glucitol (100g) was charged into a reactor containing (64 mL) methyl formate and (700 mL) of methanol. The reaction mixture was refluxed for 3 hours at 60 °C. After the completion of reaction, the reaction mixture was cooled to ambient temperature and filtered off. The solid obtained was dried under vacuum at temperature below 35 °C to obtain N-formyl-1-amino-1-deoxy-D-glucitol with dimer impurity less than 0.2% . Yield : 85% ; HPLC purity > 99%
Example 3. Preparation of Gluconobacter oxydans DSM 2003 glycerol stock
Gluconobacter oxydans lyophilized cells were used in the preparation of cell paste. Lyophilized cells of Gluconobacter oxydans DSM 2003 procured from Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH were first cultivated in a (50 mL) of sterile liquid medium containing (100 mL/L Sorbitol 70% solution, (10g/L) peptone, (7.5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 6.0 and incubating at 250RPM for 24 hours The above growth medium was used in preparation of 10% glycerol stocks of Gluconobacter oxydans and stored at -70 °C.
Example 4. Preparation of Seed I of Gluconobacter oxydans DSM 2003
A 1 mL of (10%) glycerol stocks of Gluconobacter oxydans was made to room temperature and inoculated into a (50 mL) of liquid medium containing (100 mL/L Sorbitol 70% solution, (10g/L) peptone, (7.5g/L) yeast extract 1.5g potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 250RPM at 28 °C for 24 hours.
Example 5. Preparation of Seed II of Gluconobacter oxydans DSM 2003
A 5 - 10% of Seed I was inoculated into a sterile liquid medium containing (100 mL/L) Sorbitol 70% solution, (10g/L) peptone, (7.5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 6.0 and incubating at 250RPM at 28 °C for 24 hours.
Example 6. Preparation of Gluconobacter oxydans DSM 2003 cell paste.
A 1% of Seed II of Gluconobacter oxydans DSM 2003 was inoculated into a (20L) fermentor containing (15 L) sterile liquid medium with composition viz. (100 mL/L) sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 300 RPM at 28 °C for 24 hours. The fermentation was carried out without adjustment of pH. The growth medium was harvested at time of early stationary phase by centrifugation at 9600 RPM at 10- 15 °C for 20 min. The cell paste obtained was washed with (0.02M) magnesium sulfate hepta hydrate solution and centrifuged at 9600 RPM at 10-15 °C for 20 min to obtain a 60-90 g of cell paste with moisture content 75-80%.

Example 7. Preparation of Gluconobacter oxydans DSM 2003 cell paste with pH
adjustment with 10% NaOH
A 1% of Seed II of Gluconobacter oxydans DSM 2003 was inoculated into a (20L) fermentor containing (15 L) sterile liquid medium with composition viz. (100 mL/L) sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 6.0 and incubating at 400 RPM at 28 °C for 30- 40 hours. The fermentation was carried out with adjustment of pH at 5.8 to 6.0 using 10% NaOH and 0.5WM air purging. The growth medium was harvested at time of early stationary phase by centrifugation at 9600 RPM at 10-15 °C for 20 min. The cell paste obtained was washed with 0.02M magnesium sulfate hepta hydrate solution and centrifuged at 9600 RPM at 10- 15 °C for 20 min to obtain a 100 - 110 g of cell paste with LOD 75-80%.
Example 8. Preparation of Gluconobacter oxydans DSM 2003 cell paste with pH
adjustment with 30% dipotassium hydrogen phosphate.
A 1% of Seed II of Gluconobacter oxydans DSM 2003 was inoculated into a (20L) fermentor containing e 15 L sterile liquid medium with composition viz. (100 mL/L) sorbitol (70%) solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 500 RPM at 28 °C for 30- 40 hours. The fermentation was carried out with adjustment of pH at 5.8 to 6.0 using 30% dipotassium hydrogen phosphate and 0.5-1 WM air purging. The growth medium was harvested at time of early stationary phase by centrifugation at 9600 RPM at 10-15 °C for 20 min. The cell paste obtained was washed with (0.02M) Magnesium sulfate hepta hydrate solution and centrifuged at 9600 RPM at 10- 15 °C for 20 min to obtain a 120-130 g of cell paste with LOD 70-80%.
Example 9. Preparation of Gluconobacter oxydans DSM 2003 cell paste at 40% dissolved
oxygen maintence with pure oxygen
A 1% of Seed II of Gluconobacter oxydans DSM 2003 was inoculated into a (20L) fermentor containing (15 L) sterile liquid medium with composition viz. (100 mL/L) Sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 500 RPM at 28 °C for 24- 40 hours. The fermentation was carried out with adjustment of pH at 5.8 to 6.0 using 30% dipotassium hydrogen phosphate and 0.5WM air purging. The DO was maintained above 40% by injecting pure oxygen at intervals. The growth medium was harvested at time of early stationary phase by centrifugation at 9600 RPM at 10-15 °C for 20 min. The cell paste obtained was washed with 0.02M magnesium sulfate hepta hydrate

solution and centrifuged at 9600 RPM at 10-15 °C for 20 min to obtain a 100-120 g of cell paste with LOD 70-80%
Example 10. Preparation of Gluconobacter oxydans DSM 50049 glycerol stock
Gluconobacter oxydans lyophilized cells were used in the preparation of cell paste. Lyophilized cells of Gluconobacter oxydans DSM 50049 procured from Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH was first cultivated in a (50 mL) of sterile liquid medium containing (50 mL/L) Sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 250RPM for 24 hours The above growth medium was used in preparation of 10% glycerol stocks of Gluconobacter oxydans and stored at -20 °C.
Example 11. Preparation of Seed I of Gluconobacter oxydans DSM 50049
A 10% glycerol stocks of Gluconobacter oxydans DSM 50049 was thawed to room temperature and inoculated into a (30 mL) of sterile liquid medium containing (50 mL/L) Sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 250RPM at 28 °C for 24 hours.
Example 12. Preparation of Seed II of Gluconobacter oxydans DSM 50049
A 10% of Seed I was inoculated into a sterile liquid medium containing (50 mL/L) Sorbitol (70%) solution, (10g/L) peptone, (5g/L) yeast extract (1.5g) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 250RPM at 28 °C for 24 hours.
Example 13. Preparation of Gluconobacter oxydans DSM 50049 cell paste by shake flask batch
A 1% of Seed II was inoculated into a 2L sterile liquid medium with composition viz. (50 mL/L) Sorbitol 70% solution, (10g/L) peptone, (5g/L) yeast extract (1.5g ) potassium dihydrogen phosphate in DM water pH 5.9 and incubating at 250RPM at 28 °C for 24 hours. The growth medium was harvested by centrifugation at 8500g at 6 °C for 10 min. The cell paste obtained was washed with 0.02M magnesium sulfate.hepta hydrate solution at 5-10 °C and centrifuged at 8500g to obtain a 4-5 g of cell paste.

Example 14. Preparation of immobilized Gluconobacter oxydans DSM 2003 cells
A 3% sodium alginate solution prepared in (100 uM) potassium phosphate buffer pH 7.0 was mixed with thawed whole cells of Gluconobacter oxydans DSM 2003. The mixture was then sonicated and dripped into (100 mL) of (0.05 M) calcium chloride drop wise using syringe needle. The beads were incubated for 15 minutes and filtered under vacuum. The beads were then washed with 0.1 M phosphate buffer saline solution and filtered under vacuum. The immobilized Gluconobacter oxydans DSM 2003 cells were stored at temperature below -20 °C for further use.
Example 15. Preparation of Deoxynojirmycin using Gluconobacter oxydans DSM 2003
whole cells
N-formyl-1-amino-1-deoxy-D-glucitol (100g) solution was dissolved in (1000mL) of DM water and pH was adjusted to 4.5 using dil. phosphoric acid. To this 5% of Gluconobacter oxydans DSM 2003 cell paste was added and stirred at 200 RPM for 15-30 hours at 25- 30°C along with oxygen purging. The progress of reaction was monitored by TLC and HPLC analysis. A 70-80% N-formyl-1-amino-1-deoxy-5-keto-D-glucitol formation was observed by HPLC. After the completion of reaction, the whole cells were first filtered through hiflow, followed by 0.22 micron filter. To filtrate mLs, was slowly added , (210 mL) of 10% sodium hydroxide solution at 5°C and stirred at 20 °C for 2- 5 hours. The reaction mixture was again cooled to 5°C and a (120 mL) of sodium borohydride solution (12.5 g sodium borohydride in 10% NaOH) was added slowly at 5°C. The temperature of reaction mixture was raised to 20°C and stirred at 20°C for 2- 5 hours. The reaction mixture was again cooled and adjusted pH to 7.0 using dil.HCI. The cooled neutral reaction mixture was loaded into a column containing pre-activated Indion 225 H+ resin. The column was washed with water till NaCI removed completely. The desired product was eluted with 6% chilled ammonia solution. The eluant containing desired product was collected, degasified under nitrogen and concentrated under high reduced vacuum at 40 °C till ~ 5 volumes with respect to batch size. The reaction was then cooled to 5 °C and pH was adjusted to 2 with 1:1 HCI solution. To the reaction mixture is added CECA Carbon/ carbon and stirred for 30 minutes. The reaction mixture was stirred at 5 °C for 30 minutes and filtered through Hi-flo bed followed by 0.2 micron filter at ambient temperature. The filtrate mLs was concentrated under reduced vacuum at 40 °C till moisture content below 1%. The reaction was then stirred in ethanol for 20- 30 minute at 40 °C and concentrated under reduced vacuum at 40 °C. A thick residue is obtained. To the thick residue, was added, (70 mL) of water and (700 mL) of ethanol at 40 °C and stirred for 3 hours. The reaction mixture was filtered. The residue obtained was washed with (200 mL) of ethanol and dried under vacuum till LOD reaches below 2%. A 60- 75% yield with purity > 98% of 1- deoxynojirmycin hydrochloride with 1-deoxygalactonjirimycin impurity (0.25 to 0.6%) was obtained. Deoxynojirimycin HCI is further purified in 60 volumes of 9:1 IPA:water to obtain

compound with purity in excess of 99.5 % and galactonojirimycin impurity less than 0.25 % with a yield of 50-60%
Example 16. Preparation of Deoxynojirmycin/ / 1-deoxynojirimycin HCI or its salts containing
1-deoxygalactonojirimycin imputity <0.05% using Gluconobacter oxydans DSM 2003
whole cells
N-formyl-1-amino-1-deoxy-D-glucitol solution (100g) was dissolved in (1000mL) of DM water and pH was adjusted to 4.5 using dil. phosphoric acid. To this 5% of Gluconobacter oxydans DSM 2003 cell paste was added and stirred at 200 RPM for 15-30 hours at 25- 30°C along with oxygen purging. The progress of reaction was monitored by TLC and HPLC analysis. A 70-80% N-formyl-1-amino-1-deoxy-5-keto-D-glucitol formation was observed by HPLC. After the completion of reaction, the whole cells were first filtered through hiflow, followed by 0.22 micron filter was slowly added , (275 mL) of 10% sodium hydroxide solution at 5 °C and stirred at 20 °C for 2- 5 hours. The reaction mixture was again cooled to 5 °C and a (120 mL) of sodium borohydride solution (12.5 g sodium borohydride in 10% NaOH) was added slowly at 5 °C. The temperature of reaction mixture was raised to 20 °C and stirred at 20 °C for 1- 2 hours. The reaction mixture was again cooled to 5 °C and adjusted pH to 7.0 using Cone. HCI. The neutral reaction mixture was loaded into a column containing pre-activated Indion 225 H+ resin. The column was washed with water till NaCI removed completely. The desired product was eluted with (6000mL) of ~6% chilled ammonia solution (5-10 °C). The eluant containing desired product was collected, degasified under nitrogen and concentrated under high reduced vacuum at 40 °C till dryness. Ethanol (200 mL) was added to residue and distilled of completely till moisture content of the residue reaches 0.5%. DM water (80 mL) was added and stirred for 30-40 minute at 50 °C. To the clear reaction mixture, (1200 mL) of 2-methoxy ethanol was added. The reaction mixture was heated to 50 °C and stirred at room temperature for 12-16 hours. Then it is cooled slowly to 5 °C. The precipitate obtained was filtered off and dried under vacuum to obtain a light brown crystalline product, Deoxynojirimycin base (50 - 70 g). To the above product DM water (360 mL) was added and stirred to obtain clear solution at 27 °C. The pH of the solution was slowly adjusted to 2.0 using Cone. HCI solution. To this reaction mixture was added, 5 g of Ceca carbon and stirred for 15- 30 minutes at 10 °C. The temperature of the mixture was raised to 25 °C and filtered through Hi-flow bed followed by 0.2 micron filter. The filtrate mLs was concentrated under high vacuum till almost complete water get removed. A thick residue was obtained. To this thick residue, (400 mL) of ethanol was added and stirred at 40°C , filtered off and dried under vacuum to obtain a white crystalline solid of 1- deoxynojirmycin hydrochloride with 1-deoxygalactonojirmycin impurity less than 0.05 % was obtained. Yield: 50- 65% ; purity > 99.5%
IS

Example 17. Preparation of 1-Deoxynojirmycin using immobilized Gluconobacter oxydans
DSM 2003 whole cells beads
N-formyl-1-amino-1-deoxy-D-glucitol (10%) solution was prepared and pH was adjusted to 4.5 using dil. phosphoric acid. To this mixture, (20g) immobilized Gluconobacter oxydans DSM 2003 whole cells beads was added and incubated over an orbital shaker at 200 RPM for 24-30 hours. The progress of reaction was monitored by TLC and DIP-MS analysis. N-formyl-1-amino-1-deoxy-5-keto-D-glucitol (60-70%) formation was observed by HPLC. After the completion of reaction, the beads were filtered off and reaction mixture was taken for the preparation of Deoxynojirmycin synthesis as explained in example No. 15. The immobilized Gluconobacter oxydans DSM 2003 beads were reused upto 3-5 cycles.
Example 18. Preparation of Deoxynojirmycin using Gluconobacter oxydans DSM 50049
whole cells
N-formyl-1-amino-1-deoxy-D-glucitol (100g) solution was dissolved in 1000mL of DM water and pH was adjusted to 4.5 using dil. phosphoric acid. The reaction mixture was filtered through 0.22 micron filter to remove undissolved particles and microbial contaminants. To this mixture 10% of Gluconobacter oxydans DSM 50049 cell paste was added and stirred at 200 RPM for 15-30 hours at 25- 30 °C along with oxygen purging. The progress of reaction was monitored by TLC and HPLC analysis. N-formyl-1-amino-1-deoxy-5-keto-D-glucitol (50-60%) formation was observed by HPLC. After the completion of reaction, the whole cells were first filtered through hiflow, followed by 0.22 micron filter and reaction mixture was taken for the preparation of 1-Deoxynojirmycin as explained in Example No. 15.
Example 19. Preparation of 1-amino-1-deoxy-D-galactitol
Anhydrous D-galactose (100g) and benzyl amine (73 mL) were added to a pressure reactor containing (2000 mL) of methanol under nitrogen atmosphere. To this, 15g of activated Raney-Nickel was added with moisture content less than 1% under nitrogen atmosphere and hydrogenated at 10-12 kg/cm2 for 65 - 70 hours at 50 °C under stirring. After completion of reaction with the formation of N-benzyl-1-amino-1-deoxy-D-galactitol, the hydrogen pressure was released under nitrogen atmosphere. The reaction mixture was cooled to 45 °C and D.M. water (2000 mL) was added. The reaction mixture was again cooled to 35 °C and filtered through hiflow and 5 micron filter under nitrogen atmosphere. The filtrate mLs were collected and added once again to a pressure reactor. To this (15 g) 10% palladium on carbon (50% wet) was added and hydrogenated at 10-12 kg/cm2 for 24 - 30 hours at 50 °C under stirring. After the completion of

reaction, the hydrogen pressure was released under nitrogen atmosphere. The reaction mixture was cooled to ambient temperature and filtered through hiflow and 5 micron filter under nitrogen atmosphere. The filtrate mLs were collected and the reaction mixture was concentrated under high vacuum at temperature below 40 °C. To the concentrated oily reaction mass, a 600 mL of isopropyl alcohol was added, stirred at 50 °C for 30 minutes. The reaction was concentrated under reduced vacuum at temperature below 40 °C till moisture content reaches below 5%. To the concentrated reaction mass, 750 mL of isopropyl alcohol was added, stirred for 30 - 40 minutes. The reaction mixture was again stirred at 3 °C for 60-90 minutes and filtered under nitrogen atmosphere. The solid obtained was dried under vacuum till moisture content reaches below 3%. A 80-95% yield with of 1-amino-1-deoxy-D-galactitol was obtained. Yield: 80-95%; purity > 95 %
Example 20. Preparation of N-formyl-1-amino-1-deoxy-D-galactitol:
amino-1-deoxy-D-galactitol (100g) was charged into a reactor containing (64 mL) methyl formate and (700 mL) methanol. The reaction mixture was refluxed for 6 hours at 60 °C. After the completion of reaction, the reaction mixture was cooled to ambient temperature and filtered off. The solid obtained was dried under vacuum at temperature below 35 °C. to give N-formyl-1-amino-1 -deoxy-D-galactitol. Yield : 90 -95% ; purity > 95%
Example 21. Preparation of 1-Galactonojirmycin using Gluconobacter oxydans DSM 2003 whole cells
N-formyl-1-amino-1 -deoxy-D-galactitol (100g) solution was dissolved in (1000mL) of DM water and pH was adjusted to 4.5 using dil. phosphoric acid. The reaction mixture was filtered through 0.22 micron filter to remove undissolved particles and microbial contaminants. To this 20% of Gluconobacter oxydans DSM 2003 cell paste was added and stirred at 200 RPM for 20 days at 25- 30 °C along with oxygen purging. The progress of reaction was monitored by TLC and HPLC analysis. After 20 days, a 50% conversion was observed by HPLC. The reaction mixture was first filtered through hiflow followed by 0.22 micron filter. To filtrate mLs, was slowly added , (210 mL) of 10% sodium hydroxide solution at 5 °C and stirred at 20 °C for 2- 5 hours. The reaction mixture was again cooled to 5 °C and a (120 mL) of sodium borohydride solution (12.5 g sodium borohydride in 10% NaOH) was added slowly at 5 °C. The temperature of reaction mixture was raised to 20 °C and stirred at 20 °C for 2- 5 hours. The reaction mixture was again cooled and adjusted pH to 7.0 using dil. HCI. The cooled neutral reaction mixture was loaded into a column containing pre-activated Indion 225 H+ resin. The column was washed with water till NaCI removed completely. The desired product was eluted with 6% chilled ammonia solution. The eluant containing desired product was collected, degasified under nitrogen and concentrated

under high reduced vacuum at 40 °C till ~ 5 volumes with respect to batch size. The reaction was then cooled to 5 °C and pH was adjusted to 2 with 1:1 HCI solution. To the reaction mixture is added Ceca C and stirred for 30 minutes. The reaction mixture was stirred at 5 °C for 30 minutes and filtered through Hi-flo bed followed by 0.2 micron filter at ambient temperature. The filtrate mLs was concentrated under reduced vacuum at 40 °C till moisture content below 1%. The reaction was then stirred in ethanol for 20- 30 minute at 40 °C and concentrated under reduced vacuum at 40 °C. A thick residue is obtained. To the thick residue, was added, 70 mL of water and (700 mL) of ethanol at 40 °C and stirred for 3 hours. The reaction mixture was filtered. The residue obtained was washed with (200 mL) of ethanol and dried under vacuum till LOD reaches below 2%. A 50-70 % yield of 1-deoxygalactonijirimycin hydrochloride with purity of 65 -75% was obtained. The crude was purified by preparative chromatography to obtain a pure 1-deoxygalactonijirimycin hydrochloride with yield of 40 -50% and purity more than 99%.
Example 22. Preparation of N-benzyl-1-amino-1-deoxy-D-glucitol
Dextrose (100 g) was added to (7.3) volume of MeOH: water (70:3). To this mixture (68 mL) of Benzyl amine was added. The reaction mixture was stirred at 50°C for 30 - 60 minutes. Raney-Nickel (15 g) was added and hydrogenated at 11 kg/cm2 at 60 °C for 24 hours. The reaction mixture was filtered and distilled off under reduced pressure. The product obtained was washed in MeOH and dried under reduced pressure to obtain N-benzyl-1-amino-1-deoxy-D-glucitol. Yield: 90 - 95%; purity of >99%
Example 23. Preparation of N-formyl-N-benzyl-1-amino-1-deoxy-D-glucitol
N-benzyl-1-amino-1-deoxy-D-glucitol (5 g) was taken in (10) volume of MeOH, to this mixture (10 mL) of Methyl formate was added, the reaction mixture was refluxed for 2 hours. The reaction mixture was distilled off under reduced pressure. N-formyl- N-benzyl-1-amino-1-deoxy-D-glucitol was obtained with yield of 80 -950% and purity more than 95%.
Example 24. Preparation of Deoxynojirmycin using Gluconobacter oxydans DSM 2003 whole cells.
N-fomyl-N-benzyl-1-amino-1-deoxy-D-glucitol (5 g) was taken in (20 volume) of demineralized water. The pH of the reaction was adjusted to 4.5 using dil. Phosphoric acid. To this reaction mass 1 g of Gluconobacter oxydans DSM 2003 cell paste was added. The reaction mixture was stirred at room temperature with oxygen purging for 6-10 hours. The formation of N-benzyl-1-amino-1-deoxy-5-keto-D-glucitol was monitored by TLC and Mass spectroscopy. After the completion of reaction, the whole cells were first filtered through hiflow, followed by 0.22 micron filter and reaction mixture was taken for the preparation of N-benyl-1 -Deoxynojirmycin as explained in Example No. 15 and further converted to Deoxynojirmycin by debenzylating the

compound by hydrogenating at 10 -12 kg/cm2 in presence of 10% palladium on carbon (50% wet). A 50 - 60% yield of Deoxynojirmycin was obtained. 1-deoxynojirimycin / 1-deoxynojirimycin HCI or its salts with 1-deoxygalactonojirimycin impurity <0.25% is a key intermediate in the preparation of Miglitol with 1-deoxygalactomiglitol impurity<0.1%

We claim:
1. A process for the preparation of compound of the formula I comprising the steps of:
a) converting the compound of formula A into formula B in the presence of a metal hydrogenation catalyst,
b) reducing the compound of the formula B with a metal catalyst to give compound of formula C,
c) converting the compound of Fonnula C into compound of Formula D in the presence of a polar protic solvent,

d) oxidizing the compound of Formula D using enzyme to give compound of Formula E,
e) reducing the compound of the formula E to give compound of the formula I.
2. The process according to claim 1, wherein
i. the metal hydrogenation catalyst in step (a) is Raney-nickel
ii. the reduction in step (b) is carried out using palladium carbon iii. the polar protic solvent in step (c) is methanol iv. the oxidation in step (d) is carried out using Gluconobacter oxydans. v. the reduction in step (e) is carried out using sodium borohydride in presence of sodium hydroxide.
3. A process for the preparation of compound of the formula I comprising the steps of:

a) converting dextrose into N-benzyl-1-amino-1-deoxy-D-glucitol in the presence of metal catalyst,
b) converting compound of N-benzyl-1-amino-1-deoxy-D-glucitol to N-benzyl-N-formyl-1-amino-1 -deoxy-D-glucitol,
c) oxidizing compound of N-benzyl-N-formyl-1-amino-1 -deoxy-D-glucitol in the presence of an enzyme to give N-benzyl-N-fonnyl-1-amino-1-deoxy-5-keto-D-glucitol,
d) reducing the compound of N-benzyl-N-formyl-1-amino-1-deoxy-5-keto-D-glucitol in the presence of a reducing agent to give compound of formula I,

e) optionally converting compound of the formula I into its pharmaceutical^ acceptable salts.
4. The process according to claim 3, wherein
i. the metal hydrogenation catalyst in step (a) is Raney-nickel
ii. the oxidation in step (c) is carried out using Gluconobacter oxydans
iii. the reduction in step (d) is carried out using sodium borohydride in presence of sodium hydroxide followed by debenzylation using palladium carbon.
iv. Optionally purifiying the Deoxynojirmycin.
5. A process for the preparation of galactonojirmycin using Gluconobacter oxydans DSM 2003
comprising the steps of:
a) converting the compound of D-galactose into N-benzyl-1-amino-1-deoxy-D-galactitol in the presence of metal catalyst,
b) reducing the compound of N-benzyl-1-amino-1-deoxy-D-galactitol in the presence of metal catalyst to give 1-amino-1-deoxy-D- galactitol,

c) converting the compound of 1-amino-1-deoxy-D- galactitol into N-formyl-1-amino-dexoy-D-galactitol,
d) oxidizing the compound of N-formyl-1-amino-1-dexoy-D-galactitol in the presence of enzyme to give N-formyl-1-amino-1-dexoy-keto-D-galactitol,
e) reducing the compound of N-formyl-1-amino-1-dexoy-keto-D-galactitol into galactonojirmycin,
f) optionally converting galactonojirmycin to its pharmaceutical^ acceptable salts.

6. The process according to claim 5, wherein
i. the metal hydrogenation catalyst in step (a) is Raney-nickel
ii. the reduction in step (b) is carried out using palladium carbon
iii. the polar protic solvent in step (c) is methanol
iv. the oxidation in step (d) is carried out using Gluconobacter oxydans.
v. the reduction in step (e) is carried out using sodium borohydride in presence of sodium hydroxide.
7. The process according to any of the preceding claims, wherein the gluconaobacter oxydans
cell paste is prepared at a pH of 5.5 to6 using potassium hydrogen phosphate.
8. The process according to claim 7, wherein the cell paste is immobilized on sodium alginate beads and beads and recycled.