WO2014174505A2 - Procédé de préparation de nicotine comprenant la réduction enzymatique de 4-(méthylamino)-1-(pyridine-3-yl) butan-1-one - Google Patents

Procédé de préparation de nicotine comprenant la réduction enzymatique de 4-(méthylamino)-1-(pyridine-3-yl) butan-1-one Download PDF

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WO2014174505A2
WO2014174505A2 PCT/IL2014/000019 IL2014000019W WO2014174505A2 WO 2014174505 A2 WO2014174505 A2 WO 2014174505A2 IL 2014000019 W IL2014000019 W IL 2014000019W WO 2014174505 A2 WO2014174505 A2 WO 2014174505A2
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kred
process according
nicotine
methylamino
pyridin
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PCT/IL2014/000019
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WO2014174505A3 (fr
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Roger Arthur Sheldon
Menno Jort Sorgedrager
Sander VAN PELT
Alexander Weisman
Oded Friedman
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Perrigo Api Ltd.
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    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms

Definitions

  • the present invention is directed to the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3-yl) butan-l-one .
  • Nicotine is a parasympathomimetic alkaloid found in plants of the Nicotlana genus of the Solanaceae plant family, such as tobacco, and functions as an antiherbivore chemical in the plant.
  • the naturally occurring enantiomer is (S) -nicotine, which is more physiologically active and toxic than the corresponding (R) -enantiomer .
  • Nicotine is an addictive substance and is the primary contributing factor to the dependence-forming properties of cigarette smoking.
  • the main therapeutic use of nicotine is in nicotine replacement therapy, as part of smoking cessation; such replacement therapy comprises the administration of controlled levels of nicotine through gums, dermal patches, lozenges, electronic cigarettes or nasal sprays in an effort to wean off smoking .
  • the present invention provides a process for the preparation of nicotine comprising the enzymatic reduction of 4- (methylamino) -1- (pyridin-3-yl) butan-l-one [abbreviated herein pseudooxynicotine] .
  • nicotine can be prepared by the enzymatic reduction of 4- (methylamino) -1- (pyridin-3- yl ) butan-l-one using ketoreductase enzymes.
  • the present invention provides a process for the preparation of nicotine comprising the step of reacting 4- (methylamino) -1- (pyridin-3-yl) butan-l-one, or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme.
  • provided by the invention is a process for the preparation of 4- (methylamino) -1- (pyridin-3- yl ) butan-l-ol comprising reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme, wherein the ketoreductase is capable of converting the pseudooxynicotine to the corresponding alcohol.
  • the 4- (methylamino) -1- (pyridin-3- yl ) butan-l-ol obtained is subsequently converted to nicotine .
  • the invention provides a process for the direct preparation of nicotine from pseudooxynicotine comprising the step of reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme, wherein the ketoreductase is capable of converting the pseudooxynicotine directly to nicotine.
  • nicotine can be prepared directly from pseudooxynicotine by the enzymatic reduction of pseudooxynicotine using imine reductase enzymes .
  • the invention provides a process for the direct preparation of nicotine from pseudooxynicotine comprising the step of reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with an imine reductase enzyme, wherein the imine reductase is capable of converting the pseudooxynicotine directly to nicotine.
  • the present invention provides a process for the preparation of nicotine comprising the step of reacting 4- (methylamino) -1- (pyridin-3-yl) butan-l-one
  • the invention provides a process for the preparation of nicotine comprising the step of reacting 4- (methylamino) - 1- (pyridin-3-yl) butan-l-one [pseudooxynicotine] , or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme.
  • pseudooxynicotine having the following chemical structure:
  • pseudooxynicotine refers to pseudooxynicotine per se [i.e., 4- (methylamino) -1- (pyridin-3-yl) butan-l-one] or to any of its forms which are in equilibrium in solution.
  • Ketoreductase or “KRED” refers to an enzyme that catalyzes the reduction of a ketone, optionally in a stereoselective manner.
  • Ketoreductase enzymes include, for example, those classified under EC 1.1.1. Kedoreductase enzymes are commercially available, for example, from Codexis, Inc.
  • the ketoreductase may be either a wild type or a variant enzyme, and may be either isolated from a natural source or synthesized, e.g. with recombinant technology.
  • ketoredutases which may suitably be employed in the process of the invention include, but are not limited to, Codexis Inc's products with the following catalog numbers: KRED-101, KRED-119, KRED-130, KRED-NADH- 101, KRED-NADH-110, KRED-P1-B02 , KRED-P1-B05 , KRED-P1- B10, KRED-P1-B12, KRED-Pl-COl, KRED-P1-H08 , KRED-P1-H10, KRED-P2-B02 , KRED-P2-C02 , KRED-P2-C11, RED-P2-D11 , KRED- P2-D12, KRED-P2-G03 , KRED-P3-B03, KRED-P3-G09, KRED-P3- H12 and mixtures thereof.
  • enzymes as catalysts for the reduction reaction is typically environmentally advantageous and lower in cost in comparison to metallic reducing agents used in the prior art. Moreover, enzymes are selective towards the substrate and their use generally results in less by-products.
  • the enzymatic reduction process of the invention may be carried out in a stereoselective manner, such that an optically active product is obtained.
  • the ketoreductase reduces the pseudooxynicotine to the corresponding alcohol, namely 4- (methylamino) -1- (pyridin-3-yl) butan-l- ol.
  • the 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol obtained is useful as an intermediate in the preparation of nicotine.
  • the invention provides a process for the preparation of 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol, having the following chemical structure :
  • the reduction of pseudooxynicotine to the corresponding alcohol by the ketoreductase is advantageously carried out in a stereoselective manner, such that either (S)- or (R) -4- (methylamino) -1- (pyridin-3-yl) utan-l-ol is obtained in an enantiomeric excess.
  • (S)-4- (methylamino) -1- (pyridin-3-yl) butan-l-ol is obtained in an enantiomeric excess.
  • enantiomeric excess refers to the excess of one enantiomer compared to that of the other enantiomer, and is defined as [F(R)-F(S)], wherein F(R) refers to the mole or weight fraction of the (R) -enantiomer and F(S) refers to the mole or weight fraction of the (S) -enantiomer .
  • percent enantiomeric excess or “%ee” is defined as [F (R) -F (S) ] * 100.
  • optically active refers to a chiral compound the enantiomeric excess of which differs from zero.
  • the above process for the preparation of 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol provides (S) -4- (methylamino) -1- (pyridin-3-yl) butan-l-ol, having the following formula: in an enantiomeric excess.
  • the (S) -4- (methylamino) -1- (pyridin-3-yl) butan-l-ol preferably has an enantiomeric excess greater than about 95%, and more preferably greater than 99%, as determined by HPLC.
  • the (R) -4- (methylamino) -1- (pyridin-3-yl) butan-l-ol preferably has an enantiomeric excess greater than about 95%, and more preferably greater than 99%, as determined by HPLC.
  • the starting material of the process i.e. pseudooxynicotine
  • the procedure described in Spath et al comprises reacting ethyl nicotinate with l-methyl-2-pyrrolidone in the presence of sodium ethoxide to give l-methyl-3-nicotinoylpyrrolidin- 2-one; the latter is then heated with ftiming HCl at 130°C to afford pseudooxynicotine.
  • the process for the preparation of 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol comprises reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme.
  • the ketoreductase catalyzes the reduction of the ketone to the corresponding alcohol, advantageously in a stereoselective manner.
  • the ketoreductase is one that is capable of producing the product alcohol with a yield greater than about 50% and more preferably greater than about 90%.
  • ketoredutases which may be used for the reduction of pseudooxynicotine to 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol include, but are not limited to, Codexis Inc's products with the following catalog numbers: KRED-101, KRED-119, KRED-130, KRED-NADH-110 , KRED-P1-B02, KRED-P1-B05, KRED-P1-B10, KRED-P1-B12, KRED- P1-C01, KRED-P1-H08, KRED-P1-H10, KRED-P2-B02, KRED-P2- C02, KRED-P2-C11, KRED-P2-D11, KRED-P2-D12, KRED-P2-G03 and combinations thereof.
  • the ketoreductase may be chosen from the group consisting of KRED-101, KRED-P1-B02, KRED-P1-B05, KRED-P1-B10, KRED-P1- B12, KRED-Pl-COl, KRED-P1-H10, KRED-P2-B02, KRED-P2-C02, KRED-P2-D11 and combinations thereof.
  • the ketoreductase is chosen from the group consisting of KRED-P1-B02, KRED-P1-B05, KRED-P1-B10, KRED-P1-B12 and KRED-P2-D11, with KRED-P1-B10 and KRED-P1-B12 being especially preferred.
  • KRED-P2-C11 is conveniently used as the ketoreductase.
  • the reduction of pseudooxynicotine by kedoreductase is carried out in the presence of a co-factor.
  • co-factor refers to a compound that operates in combination with the enzyme that catalyzes the reaction of interest.
  • the co-factor in its reduced form, reacts as a hydride donor.
  • nicotinamide co-factors such as reduced nicotinamide adenine dinucleotide ("NADH"), nicotinamide adenine dinucleotide ( "NAD + " ) , reduced nicotinamide adenine dinucleotide phosphate ("NADPH”), nicotinamide adenine dinucleotide phosphate ("NADP + ”) and any salts, derivatives or analogs thereof.
  • NADH reduced nicotinamide adenine dinucleotide
  • NAD + nicotinamide adenine dinucleotide
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • NADP + nicotinamide adenine dinucleotide phosphate
  • the co-factor is suitably selected from NADH, NAD + and mixtures thereof.
  • the ketoreductase is NADPH-dependent
  • the co-factor is suitably selected from NADPH, NADP + and mixtures thereof .
  • the process of the invention may advantageously be carried out, further to the co-factor, in the presence of a co-factor regenerating system, capable of regenerating the oxidized form of the co-factor to its reduced form.
  • the co-factor regenerating system comprises a dehydrogenase and a corresponding substrate.
  • suitable regenerating systems include glucose dehydrogenase and D-glucose, glucose phosphate dehydrogenase and glucose-6-phosphate, formate dehydrogenase and formate, and ketoreductase or alcohol dehydrogenase and a secondary alcohol.
  • Dehydrogenases are commercially available from e.g. Codexis Inc.
  • the ketoreductase enzyme which catalyzes the reduction of pseudooxynicotine is also capable of converting a secondary alcohol, such as isopropanol, to its corresponding ketone, thereby regenerating the oxidized form of the co-factor to its reduced form, in which case the addition of a further co- factor regenerating dehydrogenase may be omitted.
  • the process of the invention is carried out, in addition to the co-factor, in the presence of a co- factor regenerating substrate, such as a secondary alcohol, and preferably isopropanol.
  • the enzymatic reduction of pseudooxynicotine is carried out in a solvent, which is preferably water (as known in the art, an aqueous salt solution or a buffer may be used as needed in order to avoid denaturation of the reductase enzymes) .
  • a water-miscible co-solvent may be added, typically in an amount of up to about 90%.
  • the solvent consists of a mixture of isopropanol and water.
  • the reduction may be carried out in a buffer, which is preferably a solution of one or more salts, such as magnesium sulfate, potassium phosphate and mixtures thereof.
  • the buffer has a pH from about 4 to about 9, more preferably from about 6 to about 8.
  • the concentration of the pseudooxynixotine starting material in the solution is typically within the range of 1 to 1000 rtiM, and preferably 10 to 100 rtiM.
  • the ketoreductase is added in a catalytically effective amount (typically between about 0.01% and 20% weight enzyme/ weight pseudooxynicotine, and preferably between about 0.1% and 10%) .
  • the process of the invention may be carried out at a temperature between about 10°C and 50°C, more preferably between about 25°C and 30°C.
  • the pseudooxynicotine is allowed to contact the ketoreductase for a duration of from about 2 to 48 hours, and preferably from about 20 to 24 hours.
  • the obtained 4- (methylamino) -1- (pyridin- 3-yl ) butan-l-ol may be isolated and purified by means of conventional techniques such as extraction, evaporation, filtration and chromatography.
  • the 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol is useful as an intermediate in the preparation of nicotine. Accordingly, the present invention provides the use of 4- (methylamino) -1- (pyridin-3-yl ) butan-l-ol prepared by the above-described enzymatic reduction process in the preparation of nicotine. Additionally provided is a process for the preparation of nicotine comprising preparing 4- (methylamino) -1- (pyridin-3-yl ) butan-l-ol from pseudooxynicotine by the above-described enzymatic reduction process and converting it to nicotine.
  • optically active 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol is converted to (S) -nicotine [i.e. 3- [ (2S) -l-methylpyrrolidin-2- yl] pyridine] , corresponding to the following formula:
  • the (S) -nicotine obtained has an enantiomeric excess, which is preferably above 50%, 80%, 90%, 95% and most preferably above 99%, as determined by HPLC.
  • optically active 4- (methylamino) - 1- (pyridin-3-yl) butan-l-ol is converted to (R) -nicotine, [i.e. 3- [ (2R) -l-methylpyrrolidin-2-yl] pyridine] , corresponding to the following formula:
  • the (R) -nicotine obtained has an enantiomeric excess, which is preferably above 50%, 80%, 90%, 95% and most preferably above 99%, as determined by HPLC.
  • the conversion of 4- (methylamino) -1- (pyridin-3-yl ) butan- l-ol to nicotine may be carried out by reacting the 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol with a compound of formula RS0 2 X, wherein R represents an alkyl or optionally-substituted aryl, and X represents a halogen, and subsequently adding a base to the reaction mixture.
  • R is selected from methyl and toluene
  • X is chloride.
  • the reaction of the alcohol with RSO2X is thus suitably carried out under heating, wherein the reaction temperature is maintained at about 20°C to 80°C for about 2 to 96 hours.
  • a nucleophilic catalyst such as dimethylaminopyridine
  • a base is added to the reaction mixture.
  • Bases operable in the reaction are preferably inorganic, with the hydroxides of alkali metals (e.g., potassium hydroxide or sodium hydroxide) being especially preferred.
  • the obtained nicotine is isolated from the reaction mixture by means of extractive procedure, following which the solvent used for the extraction is removed by evaporation. Other conventional methods of isolation and purification may be applied in order to collect the product nicotine.
  • the 4- (methylamino) -1- (pyridin-3-yl ) butan-l-ol may conveniently be transformed to nicotine through a Mitsunobu reaction.
  • 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol is dissolved in a suitable solvent, such as dichloromethane, propylene carbonate or solvents promoting S N 2 reaction [i.e. polar aprotic solvents].
  • Triphenylphosphine is added and the mixture cooled to 0°C.
  • a basic amine such as triethylamine, may be added to prevent the formation of undesired byproducts.
  • An azodicarboxylate compound such as diethyl azodicarboxylate or diisopropyl azodicarboxylate, is dissolved in a suitable solvent such as dichloromethane, propylene carbonate or solvents promoting S N 2 reaction [i.e. polar aprotic solvents], and added dropwise to the reaction mixture.
  • a suitable solvent such as dichloromethane, propylene carbonate or solvents promoting S N 2 reaction [i.e. polar aprotic solvents]
  • the obtained nicotine is subsequently isolated from the reaction mixture by conventional means. Most conveniently, the nicotine is isolated by means of extractive procedure, following which the solvent used for the extraction is removed by evaporation. Other conventional methods of isolation and purification may be applied in order to collect the product nicotine.
  • a third suitable route for the formation of nicotine from 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol, in which route the stereochemistry of the starting material is retained, comprises chlorination of the aminoalcohol followed by cyclization, which is conveniently accomplished as follows.
  • 4- (methylamino) -1- (pyridin-3-yl ) butan-l-ol is reacted with thionylchloride [S0C1 2 ] in a suitable solvent, such as propylene carbonate.
  • a suitable solvent such as propylene carbonate.
  • the weight ratio between thionylchloride and the 4- (methylamino) -1- (pyridin-3- yl) butan-l-ol starting material in the reaction mixture is typically in the range between 1:1 and 10:1, and preferably between 1.5:1 and 4:1.
  • the reaction is suitably carried out at a temperature between about 20°C to 80°C for about 2 to 96 hours. Subsequently, a base is added to the reaction mixture.
  • Bases operable in the reaction are preferably inorganic, with the hydroxides of alkali metals (e.g., potassium hydroxide or sodium hydroxide) being especially preferred.
  • the obtained nicotine is isolated from the reaction mixture by means of extractive procedure, following which the solvent used for the extraction is removed by evaporation.
  • Other conventional methods of isolation and purification may be applied in order to collect the product nicotine.
  • the 4- (methylamino) -1- (pyridin-3- yl ) butan-l-ol starting material is optically active, such that an optically active nicotine product is obtained.
  • ketoreductase enzymes are capable of converting pseudooxynicotine directly to nicotine, thereby omitting the need for further reaction steps in the preparation of nicotine using pseudooxynicotine as starting material.
  • the present invention provides a process for the direct preparation of nicotine from pseudooxynicotine comprising the step of reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with a ketoreductase enzyme, wherein the ketoreductase is capable of converting the pseudooxynicotine directly to nicotine.
  • the conversion of pseudooxynicotine to nicotine by the ketoreductase is carried out in a stereoselective manner, such that either (R)- or (S) -nicotine is obtained in an enantiomeric excess.
  • the enantiomeric excess of the product nicotine is preferably above 50%, 80%, 90%, 95% and most preferably above 99%, as determined by HPLC.
  • KRED-P3-G09 is conveniently used as the ketoreductase.
  • the above reaction is carried out in the presence of a co-factor, such as nicotinamide co-factors (e.g. NADH, NAD + , NADPH, NADP + and mixtures thereof) , and is advantageously further carried out in the presence of a co-factor regenerating system, comprising a dehydrogenase and a corresponding substrate, such as, for example, glucose dehydrogenase and D-glucose.
  • a co-factor such as nicotinamide co-factors (e.g. NADH, NAD + , NADPH, NADP + and mixtures thereof)
  • a co-factor regenerating system comprising a dehydrogenase and a corresponding substrate, such as, for example, glucose dehydrogenase and D-glucose.
  • the ketoreductase which catalyzes conversion of pseudooxynicotine to nicotine is also capable of converting a secondary alcohol, such as isopropanol, to its corresponding ketone, thereby regenerating the oxidized form of the co-factor to its reduced form
  • a further co-factor regenerating dehydrogenase may be omitted; in such cases the reaction is carried out, in addition to the co-factor, in the presence of a co-factor regenerating substrate, such as a secondary alcohol, preferably isopropanol.
  • the above reaction is typically carried out by dissolving the pseudooxynicotine starting material in a suitable solvent, which is preferably water (as known in the art, an aqueous salt solution or a buffer may be used as needed in order to avoid denaturation of the reductase enzymes) .
  • a water-miscible co-solvent may be added, typically in an amount of up to about 90%.
  • the solvent consists of a mixture of isopropanol and water.
  • the reaction may be carried out in a buffer, which is preferably a solution of one or more salts, such as magnesium sulfate, potassium phosphate and mixtures thereof.
  • the buffer has a pH from about 4 to about 9, more preferably from about 6 to about 8.
  • the concentration of the pseudooxynixotine starting material in the solution is generally within the range of 1 to 1000. mM, and preferably 10 to 100 mM.
  • the ketoreductase is added in a catalytically effective amount (typically between about 0.01% and 20% weight enzyme/ weight pseudooxynicotine, and preferably between about 0.1% and 10%) .
  • the pseudooxynicotine is allowed to contact the ketoreductase for a duration of from about 1 to 4 days, at a temperature of, for example, between about 10°C and 50°C, and preferably between about 20°C and 30°C.
  • the obtained nicotine may be isolated and purified by means of conventional techniques such as extraction, evaporation, filtration and chromatography .
  • nicotine can be prepared directly from pseudooxynicotine by the enzymatic reduction of pseudooxynicotine using imine reductase enzymes.
  • the present invention provides a process for the direct preparation of nicotine from pseudooxynicotine comprising the step of reacting pseudooxynicotine, or any of its forms which are in equilibrium in solution, with an imine reductase enzyme, wherein the imine reductase is capable of converting the pseudooxynicotine directly to nicotine.
  • imine reductase or “IRED” refers to an enzyme that catalyzes the reduction of an imine, or of an iminium cation, optionally in a stereoselective manner.
  • Imine reductase enzymes include, for example, those classified under EC 1.5.1.
  • Some imine reductase enzymes are commercially available, for example, from Enzymicals AG (Greifswald, Germany) .
  • the imine reductase may be either a wild type or a variant enzyme .
  • imine reductase from Streptomyces sp. GF3546 or imine reductase from Streptomyces sp. GF3587 may be employed as the imine reductase in the process of the invention.
  • the imine reductase may be isolated from a natural source, such as microorganisms having the ability of reducing imines .
  • microorganisms comprising imine reductases which may be employed in the process of the invention include Streptomyces sp. GF3546 and Streptomyces sp. GF3587.
  • microorganisms having the ability of reducing imines may be employed in the process of the invention without isolation and purification of the imine reductase enzymes .
  • imine reductases which may be employed in the process of the invention may be synthesized, e.g. with recombinant technology.
  • a recombinant DNA construct encoding the imine reductase is transplanted into a host organism and expressed.
  • the production by recombinant technology of the imine reductase comprised in Streptomyces sp. GF3546 may be carried out by the process described in Leipold et al [ChemCatChem, 2013, 5(12), 3505-3508], which is incorporated herein by reference in its entirety (including its supporting information) .
  • the enzyme is produced by expression in E. coli bearing a plasmid with an inserted codon-optimized gene of the imine reductase; following cultivation, biomass is harvested by centrifugation and cell disruption is carried out using ultrasonication; the crude enzyme extract is obtained by centrifugation and subsequently purified by chromatography.
  • the isolation of the imine reductase from Streptomyces sp. GF3546, and its production by recombinant technology can be carried out, for example, according to the procedures described in US2011/0262977 , which is incorporated herein by reference in its entirety.
  • the isolation of the imine reductase from Streptomyces sp. GF3587, and its production by recombinant technology can be carried out, for example, according to the procedures described in US2011/0287494 , which is incorporated herein by reference in its entirety.
  • the conversion of pseudooxynicotine to nicotine by the imine reductase is carried out in a stereoselective manner, such that either (R)- or (S) -nicotine is obtained in an enantiomeric excess.
  • imine reductase from Streptomyces sp. GF3546 or imine reductase from Streptomyces sp. GF3587 may be used to obtain (R) -nicotine in an enantiomeric excess.
  • the enantiomeric excess of the product nicotine is preferably above 50%, 70%, 80%, 90%, 95% and most preferably above 99%, as determined by HPLC.
  • the above reaction is carried out in the presence of a co-factor, such as nicotinamide co-factors (e.g. NADH, NAD + , NADPH, NADP + and mixtures thereof) , and is advantageously further carried out in the presence of a co-factor regenerating system, comprising a dehydrogenase and a corresponding substrate, such as, for example, glucose dehydrogenase and D-glucose.
  • a co-factor such as nicotinamide co-factors (e.g. NADH, NAD + , NADPH, NADP + and mixtures thereof)
  • a co-factor regenerating system comprising a dehydrogenase and a corresponding substrate, such as, for example, glucose dehydrogenase and D-glucose.
  • the imine reductase which catalyzes conversion of pseudooxynicotine to nicotine is also capable of converting a secondary alcohol, such as isopropanol, to its corresponding ketone, thereby regenerating the oxidized form of the co-factor to its reduced form
  • a further co-factor regenerating dehydrogenase may be omitted; in such cases the reaction is carried out, in addition to the co-factor, in the presence of a co-factor regenerating substrate, such as a secondary alcohol, preferably isopropanol.
  • the above reaction is typically carried out by dissolving the pseudooxynicotine starting material in a suitable solvent, which is preferably water (as known in the art, an aqueous salt solution or a buffer may be used as needed in order to avoid denaturation of the reductase enzymes) .
  • a water-miscible co-solvent may be added, typically in an amount of up to about 90%.
  • the solvent consists of a mixture of isopropanol and water.
  • the reaction may be carried out in a buffer, which is preferably a solution of one or more salts, such as magnesium sulfate, potassium phosphate and mixtures thereof.
  • the buffer has a pH from about 4 to about 9, more preferably from about 6 to about 8.
  • the concentration of the pseudooxynixotine starting material in the solution is generally within the range of 1 to 1000 mM, and preferably 10 to 100 mM.
  • the imine reductase is added in a catalytically effective amount (typically between about 0.01% and 20% weight enzyme/ weight pseudooxynicotine, and preferably between about 0.1% and 10%) .
  • the pseudooxynicotine is allowed to contact the imine reductase for a duration of from about 2 hours to 4 days, at a temperature of, for example, between about 10°C and 50°C, and preferably between about 20°C and 30°C) .
  • the obtained nicotine may be isolated and purified by means of conventional techniques such as extraction, evaporation, filtration and chromatography .
  • room temperature refers to a temperature in the range from about 20°C to 30°C, such as, for example, 25°C.
  • HPLC analysis was performed on a Shimadzu Prominence LC20 system consisting of a LC-20AT gradient pump, DGU-20A solvent degasser, SIL-20AC autosampler, SPD-20A UV detector, RID-10A RI detector and a Jasco CO1560 column oven at 21 °C.
  • Non-chiral HPLC method (used in Examples 1-5) : Analyses were performed using two columns in series; phenomenex Gemini C18 150 x 4.6 mm x 5 micron, followed by Varian Polaris C18A 100 x 3 mm x 5 micron. The eluent consisted of pure water (+0.1 % TFA) with a flowrate of 0.6 ml/min. Detection was performed using a UV detector at 254 nm. 4- (methylamino) -1- (pyridin-3-yl) utan-l-ol elutes at 4.5 minutes, 4- (methylamino) -1- (pyridin-3-yl) butan-l-one at 8.5 minutes and nicotine at 6.1 minutes.
  • Non-chiral HPLC method (used in Example 6) : Analyses were carried out in gradient elution mode, using a Waters XBridge Shield RP18 150 x .6 mm x 5 micron column.
  • the eluent profile is presented in Table 1 below, wherein Eluent A was prepared by mixing 900 mL water, 25 mL of a 60 g/L acetic acid solution and 6 mL concentrated ammonia, adjusting the pH to 10 with dilute ammonia or dilute acetic acid, and adding water to a final volume of 1000 mL.
  • Eluent B consists of acetonitrile .
  • the eluent flow rate was 1.0 mL/minute. Detection was performed using a UV detector at 254 nm; 4- (methylamino) - 1- (pyridin-3-yl ) butan-l-ol elutes at 9 minutes, 4- (methylamino) -1- (pyridin-3-yl) butan-l-one at 10 minutes and nicotine at 20 minutes.
  • the absolute chiral configuration of the alcohol was determined by having the product of sample no. 5 of Table 1, Example 1 (see below) undergo a Mitsunobu reaction (see Example 5 below for the general procedure), which is known to cause an inversion of stereochemistry, and comparing the product obtained in a chiral HPLC with S- nicotine .
  • Racemic 4- (methylamino) -1- (pyridin-3-yl) butan-l-ol was synthesized from pseudooxynicotine for use as a reference material for the HPLC analytical analyses.
  • NaBH4 75 mg, 2eq
  • 35 additional mg of NaBH4 were added and the reaction mixture maintained for 4 additional hours at room temperature.
  • the protected alcohol obtained was isolated from the reaction mixture and purified by column chromatography using a silica column and heptane/EtOAc/EtOH in a volume ratio of 4:4:2 as eluent.
  • Reaction systems were prepared to provide for the regeneration of the enzymes. Two different systems were used, depending on the enzyme/cofactor regenerating system:
  • a stock solution was prepared, consisting of 5.5 mL water, 250 mM potassium phosphate, 2 rtiM magnesium sulfate, 1.1 mM NADP+, 1.1 mM NAD+, 10 U/ml glucose dehydrogenase and 80 mM glucose. 137 mg of pseudooxynicotine dihydrochloride was added to the solution. 1 mL potassium hydroxide solution (1 M) was added to neutralize the pH of the solution.
  • ketoreductase 5 mg was added to a vial comprising 1 mL of the above-described stock solution and the mixture was stirred at room temperature for a duration of 21 hours to give a crude product mixture.
  • Non-chiral analyses were performed by diluting 20 microliter samples of the mixture to 1 mL with water (containing 0.1% trifluoroacetic acid) and injecting the diluted samples to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analyses were performed by diluting 200 microliter samples of the mixture to 1 mL with ethanol (containing 0.1% diethyl amine) and injecting the diluted samples to the chiral HPLC (injection volume 5 microliter).
  • System B KRED/2-propanol system
  • a stock solution was prepared, consisting of 9.2 mL water, 125 mM potassium phosphate, 1.25 mM magnesium sulfate and 1.0 mM NADP+. 178 mg of pseudooxynicotine dihydrochloride was added to the solution. 1.8 mL potassium hydroxide solution (1 M) was added to neutralize the pH of the solution.
  • ketoreductase 5 mg was added to a vial comprising 1 mL of the above-described stock solution and 3.8 mL isopropanol, and the mixture was stirred at room temperature for a duration of 21 hours to give a product mixture.
  • Non-chiral analyses were performed by diluting 20 microliter samples of the mixture to 1 mL with water (containing 0.1% trifluoroacetic acid) and injecting the diluted samples to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analyses were performed by diluting 500 microliter samples of the mixture to 1 mL with ethanol (containing 0.1% diethyl amine) and injecting the diluted samples to the chiral HPLC (injection volume 5 microliter) .
  • a stock solution was prepared, consisting of 5.5 mL water, 125 mM potassium phosphate, 1.25 mM magnesium sulfate and 1.0 mM NADP+. 81 mg of pseudooxynicotine dihydrochloride was added to the solution. Potassium hydroxide solution (1 M) was added to neutralize the pH of the solution.
  • ketoreductase KRED-P3-G09 25 mg was added to a vial comprising 2.5 mL of the above-described stock solution and 9.5 mL isopropanol, and the mixture was stirred at room temperature for a duration of 4 days. Subsequently, the reaction mixture was evaporated and the residue redissolved in 3 mL of DCM/diethylether in a volume ratio of 1:2. The solution was washed with an HC1 solution (1M, 2 x 4 mL) . Sodium hydroxide solution (1 M) was added to the aqueous layer to neutralize its pH, as indicated by pH paper. The aqueous layer was then subjected to extraction using dichloromethane (3 x 10 mL) .
  • Non-chiral analysis was performed by adding 1 mL of water (containing 0.1% trifluoroacetic acid) to the residue and injecting to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analysis was performed by adding 1 mL of ethanol (containing 0.1% diethyl amine) to the residue and injecting to the chiral HPLC (injection volume 5 microliter) .
  • (S) -4- (methylamino) -1- (pyridin-3-yl) butan-l-ol was prepared according to Example 1 using KRED-P1-B12 as the ketoreductase .
  • 1 mL of the crude product mixture was evaporated to dryness under vacuum over P2O5.
  • the residue obtained was dissolved in 2 mL of propylene carbonate.
  • Mesylchloride (10 microliters) was added and the mixture maintained at a temperature of 50°C for 72 hours.
  • 2 mL of potassium hydroxide solution (10% weight/volume) was added and the reaction mixture stirred for 30 minutes. The mixture was then extracted with dichloromethane, the layers separated and the organic layer was evaporated to dryness .
  • Non-chiral analysis was performed by adding 1 mL of water (containing 0.1% trifluoroacetic acid) to the obtained residue and injecting to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analysis was performed by adding 1 mL of ethanol (containing 0.1% diethyl amine) to the obtained residue and injecting to the chiral HPLC (injection volume 5 microliter) .
  • Non-chiral analysis was performed by adding 1 mL of water (containing 0.1% trifluoroacetic acid) to the obtained residue and injecting to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analysis was performed by adding 1 mL of ethanol (containing 0.1% diethyl amine) to the obtained residue and injecting to the chiral HPLC (injection volume 5 microliter) .
  • reaction mixture was subjected to evaporation and the residue redissolved in 3 mL of dichloromethane/diethylether in a volume ratio of 1:2, following which the solution was washed with an HC1 solution (1 M, 2 x 4 mL) .
  • Non-chiral analysis was performed by adding 1 mL of water (containing 0.1% trifluoroacetic acid) to the obtained residue and injecting to the non-chiral HPLC (injection volume 5 microliter) .
  • Chiral analysis was performed by adding 1 mL of ethanol (containing 0.1% diethyl amine) to the obtained residue and injecting to the chiral HPLC (injection volume 5 microliter) .
  • Table 2 The results are presented in Table 2 below.
  • Non-chiral analysis was performed by adding 1 mL of water (containing 0.1% trifluoroacetic acid) to a 20 microliter sample of the reaction mixture and injecting the diluted sample to the non-chiral HPLC (injection volume 5 microliters) .
  • Chiral analysis was performed by adding 500 microliters of ethanol (containing 0.1% trifluoroacetic acid) to a 500 microliter sample of the reaction mixture, centrifuging and injecting the diluted sample to the chiral HPLC (injection volume 5 microliters) .
  • HPLC analysis revealed a 23% conversion of the pseudooxynicotine starting material (calculated according to HPLC peak area) . According to chiral HPLC analysis, R- nicotine was obtained in an enantiomeric excess of 70%. The formation of 4- (methylamino) -1- (pyridin-3-yl) butan-l- ol was not observed.

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Abstract

La présente invention concerne la préparation de nicotine comprenant la réduction enzymatique de 4-(méthylamino)-1-(pyridine-3-yl) butan-1-one.
PCT/IL2014/000019 2013-04-22 2014-04-06 Procédé de préparation de nicotine comprenant la réduction enzymatique de 4-(méthylamino)-1-(pyridine-3-yl) butan-1-one WO2014174505A2 (fr)

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IL225900A IL225900A0 (en) 2013-04-22 2013-04-22 A process for the preparation of nicotine that includes the enzymatic reduction of 4-(methylamino)-1-(3-pyridinyl)-1-butanone

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