Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/001—Amines; Imines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C227/30—Preparation of optical isomers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C253/00—Preparation of carboxylic acid nitriles
  • C07C253/08—Preparation of carboxylic acid nitriles by addition of hydrogen cyanide or salts thereof to unsaturated compounds
  • C07C253/10—Preparation of carboxylic acid nitriles by addition of hydrogen cyanide or salts thereof to unsaturated compounds to compounds containing carbon-to-carbon double bonds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C253/00—Preparation of carboxylic acid nitriles
  • C07C253/30—Preparation of carboxylic acid nitriles by reactions not involving the formation of cyano groups
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/002—Nitriles (-CN)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
  • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction

Definitions

• the invention relates to a commercially viable green process for manufacturing Pregabalin (1) in high yield with high chemical and chiral purity.
• Pregabalin chemically known as 3-(S)-(aminomethyl-5-methyl hexanoic acid having structure formula (1) is known to treat several central nervous system disorders that include epilepsy, neuropathic pain, anxiety and social phobia.
• (S)-Pregabalin has been found to activate GAD (L-gl tamic acid decarboxylase) in a dose dependent manner and promote production of GABA (gamma-aminobutyric acid), one of the major inhibitory neurotransmitters of brain.
• GAD L-gl tamic acid decarboxylase
• GABA gamma-aminobutyric acid
• Pregabalin has been prepared in various ways.
• One of the common approaches involves synthesis of racemic Pregabalin typically a 50:50 mixture of R and S isomers and subsequent resolution through diastereomeric salt formation.
• Such an approach could be found in Patent publications such as WO2009122215, WO2009087674, WO2009044409, WO 2008138874, WO2009125427 and WO2009001372.
• the major difficulties associated with this approach involve the loss of R-enantiomer along with a part of S-isomer as well and this can not be effectively recycled leading to cost pressure.
• Another approach has utilized resolution in the intermediate stage as a strategy. Scheme 1 outlines the approach described in WO 9638405.
• Patent publication WO2007035789 described stereo selective ring opening of 3-isobutyl-glutaric anhydride (IBG) with (R)-PEA as described in scheme 7.
• the chiral auxiliary was replaced by amide using alkali metal amide at low temperature followed by Hoffmann degradation to give (S)- Pregabalin in overall 32.9% yield in three steps.
• the conjugated nitrile was hydrolyzed and converted to tert- butyamine salt that was stereo selectively hydrogenated to cyano acid using [(R,R)-(Me-DuPHOS)Rh(COD)].BF 4 , followed by hydrogenation of CN with Ni to give Pregabalin in 41.5% overall yield with ee 99.8% over 6 steps.
• the main object of the present invention is to provide a process for the preparation of a compound of formula (I), which is simple, economical, user- friendly and commercially viable.
• Another objective of the present invention is to provide a process for the preparation of a compound of formula (I), which would be easy to implement on commercial scale, and to avoid excessive use of reagent(s) and organic solvent(s), which makes the present invention eco-friendly as well.
• Yet another objective of the present invention is to provide a process for the preparation of a compound of formula (I) in a greater yield with higher chemical and chiral purity.
• Still another objective of the present invention is to provide a process for the preparation of a compound of formula (I), wherein the byproduct formed during the reaction can be reusable and thereby recyclable, which makes the process industrially more suitable.
• the present invention provides an improved process for the preparation of a compound of formula (I), which comprises the steps of:
• R Linear or branched lower alky
• R 2 Catatonic counter ion, hydrogen, alkali metal salt, alkaline earth metal salt, ammonium salt, alkyl ammonium salt, organic amine salt and the like
• R, Linear or branched lower alkyl C, to C 4 or C 7 to C, 0 aryl or alkyl aryl chain and the like
• substantially pure S enantiomer (s) indicates the presence of S enantiomer » R enantiomer; preferentially the ratio of S:R can be in the range of 85: 15 to 100: 0; more preferably the ratio of S:R can be 95:5 to 100:0; while most preferably the ratio of S:R can be 99: 1 to 100:0.
• R enriched enantiomer indicates the presence of R enantiomer » S enantiomer; preferentially the ratio of R:S can be in the range of 70:30 to 100:0.
• the said weak organic acid used in step (a) is preferably selected from the group consisting of benzoic acid, succinic acid maleic acid, fumaric acid, phthalic acid, acetic acid and the like more.
• the said weak base used in step (a) is preferably selected from the group consisting of triethyl amine, diisipropylethyl amine, pyridine, piperidine, l,8-diazabicyclo[5.4.0]undec-7-ene and the like more preferably diisipropylethyl amine, piperidine.
• the said salts in step (a) is preferably selected from the group consisting of sodium acetate, ammonium acetate, ammonium benzoate, ammonium succinate, alkyl ammonium acetate and the like more preferably ammonium acetate, sodium acetate.
• step (a) The crude compound of formula (IV) disclosed in step (a) can be used as such or can be purified by distillation by different techniques well understood by those skilled in the art.
• the said suitable cyanide source of step (b) and (c) is preferably selected from the group consisting of lithium cyanide, sodium cyanide potassium cyanide, trimethylsilyl cyanide and the like, more preferably sodium cyanide or potassium cyanide.
• the suitable cyanide source optionally can be used in 1-50 % excess.
• the said organic solvent in step (a) is preferably selected from the group consisting of ethyl acetate, dichloro methane, chloroform, methyl tert-butyl ether, cyclohexane, toluene and mixture thereof, more preferably cyclohexane or toluene.
• step (a) and step (c) are carried out preferably at ambient temperature to reflux temperature, more preferably at reflux temperature.
• step (b) and step (c) is preferably selected from the group consisting of water, ethyl alcohol, methyl alcohol, isopropyl alcohol, «-butyl alcohol, tetra hydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, methyl tert-butyl ether, cyclohexane and the like and more preferably solvent is methyl alcohol or ethyl alcohol or water or a mixture thereof.
• step (b) preferably carried out at a temperature range between 45°C to 120°C, more preferably 45°C to 1 10°C and the most preferably is at reflux temperature of the methanol to reflux temperature of water.
• the said genetically modified nitrilase enzyme in step (d) is Nit 9N_56_2.
• the present inventors were motivated to pursue the conversion of 2-isobutylsuccinonitrile of formula (V) to racemic 3- cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) with the said enzyme with surprising selectivity, improved conditions, higher yields, minimum waste; therefore as a result promoting the green chemistry of preparation of a compound of formula (I).
• step (d) preferably can be chosen from 30 to 300 g per liter of water or water in combination of co-solvent; more preferably 50 to 200 g per liter of water or water in combination of co-solvent whilst most preferably 60 to 150 g per liter of water or water in combination of co-solvent.
• step (d) preferably can be chosen from 4 to 25 U per g of compound (V) whilst more preferably 6 to 20 U per gram of compound (V) can be used.
• the pH of the solution be kept in the range 7.2 + 0.8 and most preferably in the range of 7.0 + 0.5 and can be maintained by a suitable buffer and are well known in the art; one of the most preferred way to achieve is to use a phosphate or acetate buffer or maintain the pH with the addition of suitable acid chosen from among acetic, citric, tartaric, hydrochloric, sulfuric, phosphoric acid and the like whilst the most preferred acid is hydrochloric acid and or a base which is selected from the group consisting of ammonia, mono, di and tri alkyl amine, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and the like, the most preferred base is sodium bicarbonate.
• the substrate can be dispersed well with micronization or using dispesion stabilizing agent well known by those skilled in the art before loading of the said enzyme in step (d).
• step (d) preferably carried out at a temperature range between 25°C to 40°C, more preferably 28°C to 38°C and the most preferably a temperature range between 30°C to 37°C.
• step (e) preferably is selected from the group consisting of methyl alcohol, ethyl alcohol, isopropyl alcohol, rc-propyl alcohol, «-butyl alcohol, cyclopentanol, cyclohexanol and the like.
• the said alkyl halide (R3X) in step (e) preferably is selected from the group C 1 -C5 alkyl halides consisting of methyl iodide, ethyl chloride, ethyl bromide, ethyl iodide, n-propyl bromide, isopropyl chloride, isopropyl bromide and the like.
• the said acid catalyst/or reagent in step (e) preferably is selected from the group consisting of hydrochloric acid, sulfuric acid, thionyl chloride, trimethylsilyl chloride, methanesulfonic acid, paratoluene sulfonic acid, benzene sulfonic acid, trifluoromethanesulfonic acid, Lewis acid or strongly acidic sulfonated resins well known in the art while most suitable catalyst and /or reagent is chosen from hydrochloric acid, sulfuric acid, paratoluene sulfonic acid, trimethyl silyl chloride and the like.
• racemic alkyl 3-cyano-5-methyl-hexanoate of formula (VII) the same can be optionally purified by distillation in step (e).
• step (f) is performed by using commercially available hydrolysis enzymes such as esterasees, lipolases, lipases and the like.
• the said hydrolysis enzymes preferably is selected from the group consisting of Candida Antarctica A, Candida Antarctica Bl, Candida Antarctica BY2, Novozymes, Novozyme 435, Rhizomucor meihei, Thermomyces lanhginosa, pseudomonas cepecia, Resinase HT, Lipex 100L, Bascillus subtillis, lipase 3.101, lipase 3.102, lipase 3.104, lipase 3.105, lipase 3.106, lipase 3.107, lipase 3.108, lipase 3.109, lipase 3.111, lipase 3.115, lipase 3.113, lipase 3.117, lipase 3.136, AYS Amino
• step (f) is in the range of > 0.1% to ⁇ 5% w/w compared to the substrate; more preferably the range is 0.5% to 4% w/w compared to the substrate; whilst most preferably the range is 1.0% to 3% w/w compared to the substrate.
• step (f) may be recovered and reused for several times till almost full enzyme activity is retained; while during recycling of enzyme if the activity is less then additional amount of fresh enzyme can be added and the additional amount can be in the range of 5% to 50 % w/w with respect to initial enzyme loading; more preferentially in the range of 5% to 25 % w/w with respect to initial enzyme loading.
• the said organic solvent in step (f) is selected from the group consisting of water, methyl alcohol, ethyl alcohol, isopropyl alcohol, rt-butyl alcohol, isobutyl alcohol, acetone, methyl isobutyl ketone, acetonitrile, methyl tert- butyl ether, tetra hydrofuran, 2-methyl tetra hydrofuran, 1 ,4-dioxane, dimethyl sulfoxide, and the like and more preferably solvent is water, 1 ,4-dioxane, dimethyl sulfoxide or a mixture thereof.
• the initial pH of the solution be kept in the range 7.5 + 0.5 and most preferably in the range of 7.2 + 0.2 by using a suitable reagent selected from the group consisting of acetic acid, citric acid, boric acid, ethylenediaminetetraacetic acid, hydrochloric acid, sulfuric acid, triethyl amine, diisopropylamine, pyridine, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, calcium hydroxide, magnesium hydroxide, magnesium oxide or its suitable combination thereof.
• the selection of the amount of this suitable reagent can be chosen in a manner so that final pH after completion of reaction does not exceed 8.5.
• the pH of the reaction mixture during the progress of the reaction preferably can be allowed to increase slowly in the range of 7 to 9 while most preferably the pH can be allowed to increase up to > 7.5 to ⁇ 8.5.
• the enzymatic step can optionally be carried out in presence of salts which can be selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like or can be generated in situ by neutralization of suitable acid and a suitable base.
• salts can be selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like or can be generated in situ by neutralization of suitable acid and a suitable base.
• the enzymatic step (f) preferably carried out at a temperature range between 20°C to 45°C, more preferably 22°C to 40°C and the most preferably a temperature range between 25°C to 35°C.
• the said organic solvent in step (g) is preferably selected from the group consisting of water, methyl alcohol, ethyl alcohol, isopropyl alcohol, ⁇ -propyl alcohol, «-butyl alcohol, isobutyl alcohol, tertiary butyl alcohol, cyclohexanol, toluene, monochlorobenzene, dichlorobenzene, tetra hydrofuran, 2-methyl tetra hydrofuran, 1 ,4-dioxane, dimethylformamide, dimethyl amine, dimethyl sulfoxide, sulfolane and the like.
• the said base used in step (g) is preferably selected from the group consisting of triethyl amine, diisipropylethyl amine, pyridine, piperidine, l,8-diazabicyclo[5.4.0]undec-7-ene, l,4-diazabicyclo[2.2.2]octane, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, alkali and alkaline earth metal, C t -C 6 alkoxide and the like.
• the said enzymatic step (g) preferably carried out at a temperature range between 25°C to 200°C for 1 to 60 hours, more preferably 50°C to 180°C for 2 to 24 hours.
• the base for hydrolysis in step (h) is selected from alkali or alkaline earth metal hydroxides selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, Ci-C 5 quaternary ammonium hydroxide and the like.
• the said preparation of pregabalin of formula (I) comprises in-situ hydrolysis of compound of structure (VIII) followed by catalytic hydrogenation while the base strength for hydrolysis can be selected from 0. IN to 5N; more preferably from 0.3 to 3N and most preferably from 0.5N to 2N.
• the said organic solvent in step (h) is selected from the group consisting of water, methyl alcohol, ethyl alcohol, isopropyl alcohol, M-propyl alcohol, rc-butyl alcohol, isobutyl alcohol, tertiary butyl alcohol, cyclohexanol, toluene, monochlorobenzene, dichlorobenzene, tetra hydrofuran, dioxane, dimethylformamide or a combination thereof.
• the said suitable hydrogenation catalyst is preferably selected from the group consisting of nickel, palladium, ruthenium, rhodium with or without support and their different chemical forms and grades optionally fresh or recovered or mixture of fresh and recovered catalyst while the most preferred catalyst is Nickel and palladium.
• step (h) preferably carried out at a temperature range between 10°C to 100°C, more preferably 15°C to 60°C and the most preferably a temperature range between 25°C to 50°C.
• catalytic hydrogenation in the step (h) is preferably carried out with the hydrogen pressure in the range of 0.5 to 25 kg/cm or equivalent unit; whilst the preferable hydrogen pressure in the range of 2 to 15 kg /cm or equivalent units and most preferred pressure range is 3 to 10 kg/cm 2 .
• the preparation of pregabalin of formula (I) comprise optional charcoalization of hydrogenation product and isolation of pregabalin by preferably isoelectric focusing in the pH range of 6.9 to 7.3, more preferably at pH 7 to 7.2 and crystallization of crude from water, C 1 -C5 alcohol or a mixture thereof.
• pregabalin of formula (I) comprise isolation of pregabalin by isoelectric focusing wherein the pH can be adjusted with any inorganic or organic acid such as hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, formic acid, trifluoroacetic acid and the like while the most preferred acid is hydrochloric acid or acetic acid.
• inorganic or organic acid such as hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, formic acid, trifluoroacetic acid and the like while the most preferred acid is hydrochloric acid or acetic acid.
• pregabalin of formula (I) comprises purification of pregabalin by crystallization of crude from water, C1 -C5 alcohol or a mixture thereof and recovering further amount of pure pregabalin of formula (I) by recrystallization of dried mother liquor.
• pregabalin of formula (I) further comprises alternative recovery of pregabalin of formula (I) from the mother liquor preferably as an amino protecting derivative such as tert-butyloxycarbonyl, carboxybenzyl, trityl and the like known in the art, more preferably tert-butyloxycarbonyl can be used and subsequent removal of tert-butyloxycarbonyl group by treatment with acid in a suitable solvent.
• an amino protecting derivative such as tert-butyloxycarbonyl, carboxybenzyl, trityl and the like known in the art, more preferably tert-butyloxycarbonyl can be used and subsequent removal of tert-butyloxycarbonyl group by treatment with acid in a suitable solvent.
• Example 2.2 In a 1 liter round bottom flask, 2-cyano-5-mefhyl-hex-2-enoic acid methyl ester (100 g) obtained in example 1.1 was dissolved in MeOH (200 ml, 2V) and a solution of sodium cyanide (29.3 g; 1.0 equiv.) in water (100 ml; 1 V) was added drop wise for ⁇ lh; an exotherm observed up to 42°C. After the addition was over, downward distillation was set up and distilled out methanol from reaction mass till the reaction mass temperature reaches to 95 °C. Continue heating at -95 °C for 3hr. Solid started to fall out after methanol distillation. The reaction mixture was cooled to 30°C.
• Example 2.3 In a 3 liter round bottom flask, 2-cyano-5-methyl-hex-2-enoic acid methyl ester (515 g) obtained in example 1.1 was dissolved in MeOH (420 ml) and a solution of sodium cyanide (147 g; 1.0 equiv.) in water (630 ml; 2.1 V) was added drop wise for -30 min; an exotherm observed up to 45-50°C. After the addition was over, downward distillation was set up and distilled out methanol from reaction mass till the reaction mass temperature reaches to 95 °C. Continue heating at -95 °C for lhr. Solid started to fall out after methanol distillation. The reaction mixture was cooled to 30°C.
• Example 2.4 In a 2 liter round bottom flask, 2-cyano-5-methyl-hex-2-enoic acid methyl ester (100 g) as obtained in example 1.1 was stirred with water (100ml; 1 V) and a solution of sodium cyanide (29.4 g; 1.0 equiv.) in water (200 ml; 2 V) was added drop wise for -15 min; an exotherm observed up to 30°C. After the addition was over, reflux the reaction mixture at 89-92°C for 10 hrs. The reaction mixture was cooled to 30°C. The organic layer was separated from the aqueous layer and washed with water (100 ml).
• Example 2.5 Arranged the reaction set up consist of 500 ml four neck RBF, mechanical stirrer and thermometer pocket over an oil bath. Methyl cyanoacetate 50g (0.505 mol) and isovaleraldehyde 43.4g (0.505 mol) were dissolved in MeOH (50 ml). A solution of sodium cyanide (24.52g; 0.505 mol) of sodium cyanide in 80 ml water was added slowly under stirring (exotherm observed up to - 25-54°C). Additional water (20 ml) was used to rinse the dropping funnel. The reaction mixture was heated at 74-76°C (reflux temperature) and maintained for 3.0 hrs.
• Example 2.6 Arranged the reaction set up consist of 1000 ml four neck RBF, mechanical stirrer and thermometer pocket over an oil bath. Methyl cyanoacetate (100.0 g) and isovaleraldehyde (86.92 g) were added to 100 ml water. A solution of sodium cyanide (49.5 g; 1.0 equiv.) in water (200 ml) was added drop wise to the above mixture under stirring for 30 min. A down ward distillation assembly was set up and started distilling out methanol from reaction mass till temperature of the reaction mass reached to >90°C Maintain the reaction mass temperature in range of 90-98°C for further 12 hrs. The reaction mixture was cooled to 30°C. The organic layer was separated from the aqueous layer and washed the organic layer with 200 ml of water. Weight of compound V was 130 g (yield 94.0%; purity 95.81% by GC analysis)
• Example 2.7 In a 2L four neck RBF, equipped with mechanical stirrer and thermometer pocket over oil bath, methyl cyanoacetate (200g; 1 eqv) and isovaleraldehyde (173.84 g; 1.0 eqv) were dissolved in 200 ml (1.0 V) MeOH at 25 °C. A solution of sodium cyanide (98.90 g; 1.0 eqv.) in 400 ml water (2.0 V) was added drop wise at 25 °C over 1.5 h. The reaction mass was heated at 80 °C for 3 h and checked the consumption of methyl cyanoacetate by GC.
• a downward distillation assembly was attached and distilled out methanol from the reaction mass till reaction mass temperature reached to 92 °C during the period of 3 h. Then reaction mass becomes solid.
• 200 ml (IV) of water was added. The reaction mixture was maintained between 92-94 °C for further 5 h.
• the organic layer formed was separated from the aqueous layer.
• the organic layer was washed with 200 ml of water (1.0 V). Weight of compound V was 240 g (87.30 % yields) with GC purity 96.89 %.
• the aqueous layer was extracted using MTBE (2 x 200 ml). The MTBE layer was dried over anhydrous sodium sulphate.
• a one liter four necked RBF was equipped with mechanical stirrer, thermometer pocket and a pH meter probe.
• Crude compound V (50.0 g) and 666 ml of water (13.3 V) were added in the flask at rt (25°C).
• the pH of the solution was adjusted at 7.5 + 0.2 using solid NaHC0 3 .
• the reaction mixture was warmed to 30°C and Nitrilase enzyme preparation (4.0 ml; sp. activity 230 U/ml) was added to the reaction mixture.
• the reaction mixture was stirred at 30°C for 24 hrs by keeping the pH at ⁇ 7.5. After 24 hrs, the reaction mixture was brought to 25°C.
• reaction mass was extracted with MTBE (150 ml) and after evaporator at 45 °C under 150 Torr gave a recovery of 0.5 g un-reacted starting material.
• the aq. layer was acidified to pH 1.0 by addition of cone. HC1 ( ⁇ 50 ml).
• the aqueous layer was extracted with MTBE (2 x 150 ml).
• Example 3.2 In a 5L four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (200.0 g; 1.0 eq) and 2660 ml of water were taken at 25 °C. The pH was adjusted at 7.5 + 0.2 using solid NaHC0 3 . The reaction mixture was warmed to 35° + 2°C and Nitrilase enzyme (16.0 ml; sp activity 230 U/ml) was added to the reaction mixture. The reaction mixture was stirred at 35 0 + 2°C for 24 h by maintaining the pH at 7.5 + 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 23 h (unreacted compound III ⁇ l%).
• the reaction mixture was cooled to 25 °C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 300 ml) and concentrate under reduced pressure at 45 °C to recover un-reacted starting material (1.0 g).
• the aqueous layer was acidified to pH 1-2 by adding cone. HC1 (200 ml).
• the aqueous layer was extracted with MTBE (3 x 400 ml).
• Example 3.3 In a 250 ml four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (10.0 g; 1.0 eq), 100 ml of water (10 V) and 5 ml MeOH (0.5 V) were taken at 30 °C. The pH was adjusted at 7.5 ⁇ 0.2 using solid NaHC0 3 . The reaction mixture was maintained at 30°C and Nitrilase enzyme (0.76 ml; sp activity 300 U/ml) was added to the reaction mixture. The reaction mixture was stirred at 30°C for 24 h by maintaining the pH at 7.5 ⁇ 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 23 h (unreacted compound V ⁇ l%).
• the reaction mixture was cooled to 25 °C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 25 ml) and concentrate under reduced pressure at 45 °C to recover un- reacted starting material (3.9 g).
• the aqueous layer was acidified to pH 1-2 by adding cone. HC1.
• the aqueous layer was extracted with MTBE (2 x 40 ml).
• Example 3.4 In a 250 ml four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (10.0 g; 1.0 eq), 50 ml of water (5 V) were taken at 30 °C. The pH was adjusted at 7.5 + 0.2 using solid NaHC0 3 . The reaction mixture was maintained at 30°C and Nitrilase enzyme (0.76 ml; sp activity 300 U/ml) was added to the reaction mixture. The reaction mixture was stirred at 30°C for 24 h by maintaining the pH at 7.5 + 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 23 h (unreacted compound V ⁇ l %).
• the reaction mixture was cooled to 25 °C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 25 ml).
• the aqueous layer was acidified to pH 1-2 by adding cone. HC1.
• the aqueous layer was extracted with MTBE (2 x 40 ml).
• Example 3.5 In a 500 ml four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (25.0 g; 1.0 eq), 332.5 ml of water (13.3 V) were taken at 30 °C. The pH was adjusted at 7.5 + 0.2 using solid NaHC0 3 (1.17g). The reaction mixture was maintained at 30°C and Nitrilase enzyme (2.7 ml; sp activity 300 U/ml) was added to the reaction mixture. The reaction mixture was stirred at 30°C for 24 h by maintaining the pH at 7.5 + 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 23 h (unreacted compound V ⁇ l%).
• the reaction mixture was cooled to 25°C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 25 ml).
• the aqueous layer was acidified to pH 1-2 by adding conc.HCl.
• the aqueous layer was extracted with MTBE (2 x 40 ml).
• Example 3.6 In a 500 ml four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (25.0 g; 1.0 eq), 332.5 ml of water (13.3 V) were taken at 30°C. The pH was adjusted at 7.5 + 0.2 using solid NaHC0 3 (1.17g). The reaction mixture was maintained at 30°C and Nitrilase enzyme (3.37 ml; sp activity 300 U/ml) was added to the reaction mixture. The reaction mixture was stirred at 30°C for 24 h by maintaining the pH at 7.5 + 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 23 h (unreacted compound V ⁇ l%).
• the reaction mixture was cooled to 25 °C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 25 ml).
• the aqueous layer was acidified to pH 1-2 by adding cone. HC1.
• the aqueous layer was extracted with MTBE (2 x 40 ml).
• Example 3.7 In a 500 ml four necked RBF, equipped with mechanical stirrer, thermometer pocket and pH meter, compound V (25.0 g; 1.0 eq), 250 ml of water (10 V) and 1.25 ml MeOH were taken at 35 °C. The pH was adjusted at 7.5 + 0.2 using solid NaHC0 3 (1.75 g). The reaction mixture was maintained at 35°C and powdered Nitrilase enzyme (1.75 g; sp activity 300 U/ml; 16.2 u/g) was added to the reaction mixture. The reaction mixture was stirred at 35°C for 24 h by maintaining the pH at 7.5 + 0.2 (if required adjust the pH by using solid NaHC0 3 or IN HC1).
• the reaction was monitored by GC after 24 h (unreacted compound V ⁇ l%).
• the reaction mixture was cooled to 25 °C and filtered the reaction mass.
• the reaction mass was extracted with MTBE (2 x 25 ml).
• the MTBE layer was evaporated to recover 4.5 g (18%) compound V.
• the aqueous layer was acidified to pH 1-2 by adding cone. HC1.
• the aqueous layer was extracted with MTBE (2 x 40 ml).
• Example 3.8 In a 30 lit reactor charged 9.0 kg of water at 25°C to 35°C. In a clean HDPE container charged 10.99 lit of water and 1.500 kg (1.0 eq) of compound V at 25°C to 35°C and micronized for 30 min. After micronization formation of fine globules observed and was transferred to the reactor. The pH of the mixture was adjusted to 7.5 ⁇ 0.2 using solid NaHC0 3 (-30.0 gm). The temperature of the reaction mass was brought to 35 ⁇ 2°C and charged nitrilase enzyme 129.26 gm (sp. activity : 0.235kU/ mL) in one portion . The container of the enzyme was rinsed with 0.2 V water and charged to reactor.
• reaction mass was adjusted to 7.5 ⁇ 0.2 by using solid NaHC0 3 .
• the reaction mixture was stirred at 35 ⁇ 2°C for 14.0 h by maintaining the pH 7.5 ⁇ 0.2 by adding sodium bicarbonate solution or with dilute hydrochloric acid.
• Continue the reaction till compound V ⁇ l%.Filter the reaction mass from reactor by using Buchner filter under vacuum.
• the filtrate was extracted with MTBE (3 x 3.0 lit). Concentrated the MTBE layer under reduced pressure at 45 °C and vacuum 400-10 tor to give compound VI in 90.3% assay based yield.
• Racemic 3-cyano-5-methyl-hexanoic acid (41 Og) was dissolved in MeOH (2050 ml;) followed by H 2 S0 4 (32.8 g; 0.12 eq.) at 25 °C. Stir the reaction mass for 1 h at reflux temperature (68 °C). After evaporation of the reaction mass under vacuum the oily residue was diluted with water (2000 ml) and extracted with toluene (300 ml x 3). The organic layer was washed with water (300 ml) followed by saturated bicarbonate solution (100 ml x 2).
• methyl cyanoacetate (l OOg; 1 eqv) and isovaleraldehyde (95.6 g; 1.0 eqv) were dissolved in 100 ml (1.0 V) methanol at 25 °C.
• a solution of sodium cyanide (98.90 g; 1.0 eqv.) in 200 ml water (2.0 V) was added drop wise at 25 °C over 1.5 h.
• the reaction mass was heated at 80 °C for 3 h and checked the consumption of methyl cyanoacetate by GC. Additional water (100 ml) was added.
• a downward distillation assembly was attached and distilled out methanol from the reaction mass till reaction mass temperature reached to 92 °C during the period of 3 h.
• the reaction mixture was maintained between 92-94 °C for further 5 h.
• the organic layer formed was separated from the aqueous layer.
• the organic layer was washed with 200 ml of water (1.0 V). Weight of compound V was 128 g (93.12 % yields) with GC purity 95.82 %.
• a one liter four necked RBF was equipped with mechanical stirrer, thermometer pocket and a pH meter probe.
• Crude compound V obtained above (25.0 g, 95.82% purity) and 333 ml of DM water (13.3 V) were added in the flask at 25°C.
• the pH of the solution was adjusted at 7.5 + 0.2 using solid NaHC0 3 ( ⁇ 2.0 g was required).
• the reaction mixture was warmed to 30°C and Nitrilase enzyme preparation (1.8 ml; 20.5 u/g substrate; sp. activity 257 U/ml) was added to the reaction mixture.
• the reaction mixture was stirred at 35°C for 24 hrs by keeping the pH at 7.5 + 0.2 (by using solid NaHC0 3 or IN HC1 as and when required).
• Table 1 optimization of racemization of R enriched acid.
• Enzyme catalyzed hydrolysis was explored with 100 mg of compound V with 20 % loading of 28 different enzymes at pH 7 phosphate buffer (1 ml, 10V) from Among these different enzymes investigated CAL B1, CAL BY2 furnished good results. However chiral purity with CAL BY2 was found to be low as compared to CAL Si. Hence only CAL B1 was considered for further investigations. Enzymes obtained from chiralvision Candida antarctica A, rhizomucor miehei, thermomyces Lanhginosa, pseudomonas cepacia, resinase HT, lipex 100L, novozymes, bacillus subtillis enzymes from Evocatal lipase 3.
• lipase 3.105, lipase 3.107, lipase 3.109 and enzymes from Amano a ano lipase PS, amano lipase AK, a ano lipase AH, amano lipase AYS did not respond to this substrate under the chosen condition
• Enzyme catalyzed hydrolysis was also explored with 100 mg of compound V with 20 % loading of different enzymes at pH 8 phosphate buffer (1 ml, 10 V) and acid formation was observed only with Candida antarctica, B1 in both buffer solution viz. pH 7 and 8 on GC.
• rate of reaction enzyme activity w.r.t reaction rate
• Example 5.6 In a 250 ml single neck RBF, equipped with magnetic needle over a magnetic stirrer, methyl 3-cyano-5-methyl-hexanoate (10 g; 1.0 eqv) was dispersed in 150 ml aqueous 7% NaHC0 3 solution (pFI 7.0) containing 10.5 g NaHC0 3 and 0.2 g of Novozyme 435 was added at 25 °C. The reaction mass was stirred for 7 h at 26 + 2 °C till hydrolysis was > 50 % monitored through chiral GC analysis. At the end of the reaction pH of the reaction was observed to be ⁇ 8.0.
• reaction mass was filtered through Buchner funnel using vacuum and the residual enzyme was washed with ethyl acetate (50 ml). The two layers were separated. The aqueous layer was extracted with ethyl acetate (3 x 20 ml). The combined organic layer was washed with saturated bicarbonate solution 60 ml and optionally dried over anhydrous sodium sulphate and concentrated under reduced pressure to give 20 g (40.1 %) of compound VIII with purity of 91.73 % by GC; and chiral purity of 99.91 % (99.82 % ee).
• reaction mass was stirred for 6 h at 26 + 2 °C till hydrolysis was > 50 % monitored through chiral GC analysis. During the maintenance no pH adjustment was done.
• the reaction mass was filtered through Buchner funnel using vacuum and the residual enzyme was washed with ethyl acetate (800 ml). The two layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 100 ml). The combined organic layer was washed with saturated bicarbonate solution optionally dried over anhydrous sodium sulphate and concentrated under reduced pressure to give 180 g (41.0 %) of compound VIII with purity of 98.74 % by GC; and chiral purity of 99.95 %.
• the catalyst was filtered through celite or any other suitable bed and washed with water (50 ml). The catalyst was recovered for reuse.
• the reaction mass was extracted with MTBE (150 ml).
• the aqueous layer was acidified to pH 1.0 with 60 - 65 ml cone. HCl. Cool the reaction mass to 0°C and adjust the pH at 7.4 by adding 10% NaOH solution (-25 ml). Racemic Pregabalin) started separating as white crystals.
• the reaction mass was maintained at 0°C for 2-3 hr.
• the solid was filtered through a Buchner funnel under vacuum. The wet cake was washed with 50 ml of water and suck dried for 30 min to get 55.0 g of wet cake.
• thermometer pocket and stopper was taken (5 -3-cyano-5-methyl hexanoic acid methyl ester 60.0 g (1 eq.) and 225.0 ml methanol and cooled to 5-10 °C.
• a solution of 32 % aq. potassium hydroxide 84.67 ml (1.16 eq.) was kept at 15 -25 °C for 1.5 h.
• the reaction mass was transferred to a autoclave containing MeOH (225 ml) and Raney Ni 18.0 g (30 % w/w).it was hydrogenated at ⁇ 40°C with 350 RPM at 10 kg/cm 2 pressure for upto 22 h. Catalyst was filtered. Optionally to the filtrate charcoal (1.5 % w / w) was added and stirred for 1 h at room temperature. The mixture was filtered through celite bed and washed with 60 ml methanol. Then the pH was adjusted within the range of 7.1 + 0.1 by adding glacial acetic acid. Methanol was removed by distillation for at ⁇ 40 °C) under vacuum followed by MeOH and water mixture at ⁇ 45°C .
• the air inside the autoclave was replaced by purging nitrogen gas twice. It was pressurized 5 kg /cm 2 of hydrogen gas and release to atmosphere twice to remove nitrogen gas.
• the stirring was initiated with 500 RPM at 70°C and hydrogen gas pressure of 5 kg /cm 2 .
• the hydrogenation was continued for 22 h.
• the catalyst was filtered through celite or any other suitable bed and washed with MeOH water (30 ml). The catalyst was recovered for reuse.
• the filtrate was adjusted to pH 7-7.4 by using glacial acetic acid ( ⁇ 10 ml).
• the methanol was recovered under reduced pressure at 40 °C.
• the reaction mass was cooled to 0 to 5 °C for 1 h.
• the solid was filtered on a Buchner funnel using vacuum.
• the stirring was initiated with 500 RPM at 30°C and hydrogen gas pressure of 10 kg /cm 2 .
• the hydrogenation was continued for 22 h.
• the catalyst was filtered through celite or any other suitable bed and washed with MeOH (30 ml).
• the catalyst was recovered for reuse.
• the filtrate was adjusted to pH 7- 7.4 by using glacial acetic acid ( ⁇ 15 ml).
• the methanol was recovered under reduced pressure at 40 °C.
• the reaction mass was cooled to 0 to 5 °C for 1 h.
• the solid was filtered on a Buchner funnel using vacuum and suck dried for 30 min to get crude 15.99 g wet Pregabalin.
• MIBK Methyl isobutyl ketone

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Wood Science & Technology (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• General Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Toxicology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (3)

Family

ID=50685262

Family Applications (1)

Country Status (5)

Cited By (5)

Families Citing this family (8)

Citations (20)

• 2013
  • 2013-11-04 JP JP2015540226A patent/JP6482465B2/ja active Active
  • 2013-11-04 EP EP13853048.0A patent/EP2916832B1/en not_active Not-in-force
  • 2013-11-04 US US14/434,658 patent/US10023885B2/en active Active
  • 2013-11-04 WO PCT/IB2013/002435 patent/WO2014072785A2/en not_active Ceased
  • 2013-11-04 CA CA2888877A patent/CA2888877C/en active Active

Patent Citations (21)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events