WO2023181046A1 - Procédé biocatalytique recyclable pour la préparation de sitagliptine - Google Patents

Procédé biocatalytique recyclable pour la préparation de sitagliptine Download PDF

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WO2023181046A1
WO2023181046A1 PCT/IN2022/050436 IN2022050436W WO2023181046A1 WO 2023181046 A1 WO2023181046 A1 WO 2023181046A1 IN 2022050436 W IN2022050436 W IN 2022050436W WO 2023181046 A1 WO2023181046 A1 WO 2023181046A1
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formula
enzyme
water
amine
sitagliptin
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PCT/IN2022/050436
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English (en)
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Dipak Kumar Roy
Kamlesh Jayantilal Ranbhan
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Iol Chemicals And Pharmaceuticals Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • Enzyme transaminase is used for the preparation of compound of Formula I comprising reuse of at least once used transaminase enzyme for stereoselective conversion of keto group of 4- Oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[l,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-one (also referred herein Ketoamide) of Formula II.
  • the said transaminase enzyme is used once only and discarded after the reaction is completed.
  • prior art discloses the reuse of the once used enzyme after immobilization process.
  • the inventors of the present invention disclose herein that the enzyme once used can be reused as such without any processing like immobilization for the future batches for the same chemical reaction step without any adverse impact on the yield and purity of the product.
  • the recycling also referred herein as reuse makes the process for preparing compound of Formula-I, which is a drug molecule Sitagliptin thereby making the said improved inventive process economical and green at industrial scale.
  • Sitagliptin is chemically known as 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]- 5,6,7,8-tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a] pyrazine represented by structural Formula-I:
  • Sitagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor and used to improve glycemic control in patients with type-2 diabetes mellitus.
  • DPP-4 dipeptidyl peptidase 4
  • Sitagliptin is sold under the trade name JANUVIA as monotherapy and JANUMET in combination with Metformin hydrochloride.
  • US6699871B2 discloses Sitagliptin and other related compounds which are inhibitors of the dipeptidyl peptidase-IV enzyme ('DP-IV inhibitors') and which are useful in the treatment or prevention of diseases in which the dipeptidyl peptidase-IV enzyme is involved, such as diabetes and particularly type 2 diabetes.
  • ‘871 discloses a process for preparation of chiral beta amino acid derivative using chiral pyrazine derivative to introduce chiral amine, which after few subsequent steps give Sitagliptin and related compounds.
  • the process involves many steps making process commercially inviable.
  • the process also uses pyrophoric reagent butyl lithium, explosive reagent diazomethane, expansive reagents silver benzoate and EDC.
  • W02004085661A2 discloses a process for the preparation of enantiomerically enriched beta amino amide including Sitagliptin by treating ketoamide compound with a chiral auxiliary S- phenyl glycine, followed by diastereoselective reduction of the enamine by hydrogenation using Pt2O and finally debenzylation by palladium catalyzed hydrogenolytic removal of benzyl group.
  • the process suffers from low diastereo- and enantioselectivity and lower yield.
  • the process uses expansive metal catalyst like Pt2O.
  • US7495123B2 discloses a process for the preparation of chiral beta amino amide by rhodium catalysed asymmetric hydrogenation of prochiral enamine in the presence of a chiral mono- or bisphosphine ligand.
  • Drawback of this process lies on high cost of catalyst and low enantiomeric purity.
  • W02006081151A1 discloses a process for preparation of chiral beta amino amide that comprises an enantioselective hydrogenation of a prochiral beta-amino acrylic acid derivative substrate in the presence of an ammonium salt and a transition metal precursor complexed with a chiral ferrocenyl bisphosphine ligand.
  • Rhodium-catalized asymmetric enamine hydrogenation originally used for the large-scale manufacture of the antidiabetic compound Sitagliptin.
  • Rhodium is replaced with a transaminase enzyme which ultimately has led to an enzymatic process that reduces waste, improves specificity, yield, safety and eliminates the need for a metal catalyst.
  • Exploration for suitable catalysts has been expanded significantly from the last few decades because of the increasing demand of environmental friendly production in the industry. In this quest, biocatalysts have much to offer because of their ease of production, substrate specificity, and green chemistry. Progress in biotechnology has paved the way for the widespread application of biocatalysis in industrial organic synthesis.
  • a process for the preparation of Sitagliptin is reported in Science, 2010, 329, 305-309 using transaminase enzymes to reduce beta keto amide with high enantioselectivity.
  • the biotransformation is performed in a mixture of triethanol amine buffer and DMSO.
  • the process is capable for the synthesis of Sitagliptin with high enantiomeric purity.
  • One of the main drawback of the process is high cost of enzyme.
  • Another drawback of the process is use of water miscible DMSO as co-solvent, which makes work up process difficult. Further, difficulty in recovery of DMSO makes this process expensive and environmentally unsafe.
  • US8293507 discloses the use of transaminase for the biocatalytic conversion of 4-oxo-4-[3- (trifluoromethyl)-5,6-dihydro[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-one (the “ketoamide substrate”) to (2R)-4-oxo-4-[3-(trifluoromethyl)- 5,6-dihydro[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-l-(2,4,5-trifluorophenyl)butan-2-amine (the “product) in presence of an amino group donor.
  • DMSO is used in varying amount from 10-50% thereby making DMSO solvent recovery difficult, and cost is higher.
  • CN105018440 discloses transaminase from mycobacterium (Mycobacteriumvanbaalenii) PYR-lhas the function of transaminase, is a kind of new transaminase.
  • the prochirality carbonyl compound is preferably 3- carbonyls -4- (2,4,5- trifluorophenyl) -butanoic acid esters.
  • CN103014081 discloses the use of transaminase by 3- carbonyls -4- (2,4,5- trifluoros Phenylmethyl butyrate into R-3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate.
  • CN104805069B discloses immobilization of transaminase and its reuse. Restructuring transaminase is fixed on sodium alginate by the immobilization process. It discloses catalysis of prochiral carbonyl compounds using immobilised to obtain chiral amine.
  • Transaminases are a specific class of enzymes that catalyse the direct amination of ketones to chiral amines. Enantiomerically pure chiral amines are key intermediates in a number of pharmaceutical compounds that possess a wide range of biological activities. Besides, enzyme based catalysis is performed with much higher fidelity, under mild ambient environmental reaction conditions like temperature, pH, and pressure are highly efficient in terms of number of steps, giving them an edge over their chemical counter parts. The unique characteristic of enzyme makes them highly applicable for a number of chemical transformation reactions in pharmaceutical industries.
  • enzymes represent a key component in a broad range of reactions but their cost becomes a limiting factor in industries and academics. Further, despite great potential of enzymes, their industrial applications have been restricted because of the recovery of enzymes and difficulty in reusability limits their application in the industry. To overcome these limitations, enzymes have been immobilized on various solid supports. Most of the prior art cited herein above comprises the use of immobilization process which allows the enzymes to be recycled and reused, reducing cost in comparison with use of a fresh enzyme. The said immobilisation process as described herein requires a separate facility having requisite infrastructure and extra material viz solid support and the like but still cost efficient compared to process wherein a fresh enzyme is used.
  • Enzyme immobilization is defined as a process of confining the enzyme molecules to a solid support over which a substrate is passed and converted into product. It is done for protection from degradation and deactivation of enzyme with the intension of re-use of enzymes for reaction cycles, lowering the total production cost of enzyme mediated reactions. Enzyme immobilization is one of the most promising approaches for exploiting enzyme based processes in Biotransformation. Immobilized enzymes though a little cheaper than fresh unused enzyme but still are expensive as the process of immobilization process need an exclusive dedicated infrastructure which is expensive needing extra space, material and utilities. In addition, it requires specific material and additional process step which is time consuming. For immobilisation process sophisticated process equipment is needed.
  • Inventors of the present invention successfully based on exhaustive R&D efforts have evolved an efficient cost effective, green novel and inventive process comprising: developing an efficient biphasic solvent system for said biochemical reaction and the said biphasic solvent comprises water and water immiscible organic solvent; recovery of used enzyme from the biphasic solvent system; reuse of said recovered enzyme as such without immobilization; recovery of the organic solvent from the biphasic reaction mass containing compound of Formula I. no generation of effluent as both the solvents of the reaction mass are recovered ans reused.
  • the improved process disclosed herein for the preparation of compound of Formula I addresses all the issues therein in the biochemical process disclosed in the prior art and making the said improved process for industrial production of Sitagliptin and intermediates thereof green, economical and commercially viable at industrial scale.
  • Disclosed herein is a novel and innovative process particularly for the preparation of Sitagliptin of Formula I with high optical purity comprising reuse of at least once used transaminase enzyme as such without having any kind of immobilization or the like.
  • Used enzyme is collected from the reaction mass of the biochemical enzymatic process for the preparation of Sitagliptin, wherein the said used transaminase enzyme is obtained just by separation of aqueous phase of the biphasic solvent system which contains said enzyme which is reused as such for the same biocatalytic amination of ketoamide of formula II into Sitagliptin of formula I using buffer and a water immiscible organic solvent selected from the group comprising isopropyl acetate, n-butanol, isobutanol, Methyl tert-butyl ether, 2-methyl tetrahydrofuran, cyclohexanol and the like or mixture thereof.
  • the process is also characterized by the fact that use of water immiscible solvent ensures good conversion due to non-precipitation of the ketoamide substrate of Formula II as in the single phase solvent system, substrate of Formula II being insoluble in water precipitates from a single phase solvent system and substantial quantity remains unreacted.
  • One of the objective of the present invention is to make the process green avoiding generation of waste effluent and commercially viable at industrial scale for the production of Sitagliptin of Formula I comprising recycle and reuse of at least once used transaminase enzyme as such without immobilization or any other additional processing act and the said used enzyme is isolated directly from the reaction mass of amination used for the preparation of Sitagliptin.
  • the process for recovery or isolation of at least once used enzyme from the reaction mass comprises separation of organic solvent layer from the reaction mass and separated aqueous layer contains used transaminase enzyme and the separated aqueous layer containing said used enzyme is reused as such without immobilization by adding substrate in a water immiscible organic solvent into the said separated aqueous layer containing used enzyme separated from the early production batch and proceeding for the biocatalytic reaction to obtain substantially pure Sitagliptin of Formula I in high yield.
  • biphasic solvent system comprising water and water immiscible organic solvent enables the separation of enzyme from the reaction mass and the said separated enzyme is reused for the same biochemical reaction without having any other extra efforts like immobilization. Moreover, the novel process also enables the recovery of organic solvent. There is no teaching or motivation to a person skilled in the art to use biphasic solvent system for the biochemical reaction and to recover and reuse the enzyme as such without immobilization thus making the process green and economical at industrial scale.
  • One of the objectives of the present invention is to reuse of at least once used transaminase enzyme without having any kind of processing such as immobilization and the like.
  • One of the objectives of the present invention is to reduce the overall cost of the process for the preparation of Sitagliptin.
  • One of the objectives of the present invention is to use water immiscible solvent to simplify the workup procedure characterized by the feature that enzyme used remains in aqueous layer and easily isolated/recovered for reuse in the same water solvent for next batch for the preparation of same product.
  • One of the objectives of the present invention is to provide the robust process comprising using water immiscible solvent which otherwise may lead to incomplete conversion in water miscible solvent due to precipitation of the substrate.
  • One of the objectives of the present invention is to avoid the use of immobilized enzymes, thereby avoiding the expenses required for the process of immobilization.
  • Another objective is to recover the organic solvent which can be reused avoiding the generation of effluent making the process economical and green at industrial scale.
  • Disclosed herein is a novel and innovative process to prepare Sitagliptin of Formula I with high optical purity comprising reuse of at least once used transaminase enzyme as such without any kind of processing like immobilization and the said used enzyme is recovered/isolated from the reaction mass of early batch.
  • the process of recovery/isolation of used enzyme comprises separating aqueous layer of biphasic solvent system of reaction mass containing used enzyme and the same aqueous mother liquor containing used enzyme is used as such again and substrate of Formula II in a water immiscible organic solvent is added to the said aqueous mother liquor containing enzyme.
  • Recycled and reused transaminase under the consideration of the present invention is used for the amination of ketoamide compound of formula II into Sitagliptin of Formula I.
  • the separated organic solvent layer containing product (Sitagliptin of Formula I) is also separated and product isolated and solvent recovered which too can be used again avoiding the generation of effluent making the process economical and green at industrial scale.
  • Major advantage of the present invention is the reuse of at least once used transaminase enzyme as such without involving extra efforts like immobilization and the like for the commercially economical production of Sitagliptin at of Formula I at industrial level with minimization of waste.
  • biphasic solvent system makes work up and enzyme and solvent recovery simpler and cost effective.
  • the process with water immiscible solvent makes the process robust which otherwise may lead to incomplete conversion in water miscible solvent as the ketoamide substrate of Formula II being insoluble in water precipitate out from the reaction mass. Separation of water layer containing enzyme and the organic layer containing desired products is separated at great ease and the used enzyme in water layer is reused as such for the next batch for the preparation of desired compounds.
  • the product is isolated from the organic solvent layer and the solvent is recovered avoiding generation of effluent making the process economical and green.
  • Transaminases are one of the most promising biocatalysts in organic synthesis for the preparation of chiral amino compounds.
  • the concise reaction, excellent enantioselectivity, environmental friendliness and compatibility with other enzymatic or chemical systems have brought transaminase to the attention of scientists working in the area of biocatalysis.
  • transaminase attempts are made to optimize their performance using recycled and reuse of transaminase enzymes as such without involving any extra enzyme activation process like immobilization and the like.
  • biocatalysts including aspects such as shorter routes without functional group protecting steps, lower cost of equipment and energy, less environmental waste, easier procedures for work-up and even lower cost of catalysts considering the recycling of enzymes as disclosed herein, has made them one of the preferred options for industrial organic preparation.
  • biphasic solvent system comprising water and water immiscible organic solvent for the stereoselective amination of selectively a ketoamide of Formula II comprising reuse of at least once used enzyme as such without immobilisation.
  • the substrate of Formula II being soluble in organic solvent phase doesn’t precipitate whilst reaction is in progress, therefore maximum substrate of Formula II gets converted into corresponding compound of Formula I.
  • Water phase containing used enzyme, amine donor and buffer is separated and is reused as such for the next production batch for the production of compound of compound of Formula I comprising addition of the substrate ketoamide of Formula II in a water immiscible organic solvent into the said water solvent phase containing used enzyme from earlier batch.
  • Prior art discloses the said biocatalytic process for the preparation of Sitagliptin of Formula I wherein the fresh enzyme is used once and no provision of isolating the used enzyme from a single phase reaction mass.
  • the used enzyme can be reused only after immobilization for the preparation of Sitagliptin.
  • the immobilization process is expensive and time consuming as it comprises additional process steps.
  • Inventors of the present invention disclose herein the reuse of recovered enzyme as such without any additional process step like immobilization.
  • the used enzyme therein in the aqueous mother liquor separated from the biphasic solvent system is reused as such by the inventors of the present invention for the same purpose viz. amination of a ketoamide of compound of Formula II into amino group to prepare Sitagliptin of Formula I at industrial scale making the process economical and green.
  • biphasic solvent system comprising water and water immiscible solvent which has following advantages.
  • the amino product of Formula I obtained remains in the organic solvent and the enzyme, cofactor and buffer remains in the aqueous layer.
  • the organic layer is separated and product isolated and solvent is recovered making the process economical and green as due to solvent recovery there is no generation of effluent.
  • the aqueous layer containing used enzyme, cofactor and buffer is reused as such as an aqueous part of the biphasic solvent system containing enzyme, cofactor and buffer to which substrate of Formula II in water immiscible organic solvent is added for same biocatalytic process.
  • a process for the preparation of compound of Formula I comprising: a) an aqueous layer is separated from the biphasic solvent system of the previous production lot of production of compound of Formula I and the said aqueous layer contains cofactor, buffer, water, alkyl amine and at least once used transaminase enzyme; b) pH of the said aqueous solvent phase is adjusted to about 7 to about 10 preferably about 8.5 using an acid; c) a solution containing ketoamide of Formula II in a water immiscible organic solvent is added slowly to the aqueous mixture of step b) and the temperature of the reaction mass is maintained at about 35°C to 70°C preferably about 45-50°C;adjusting pH between about 8.2 to about 9.5.
  • step (c) Byproduct like acetone generated during the reaction is removed under vacuum or bubbling or both from the reaction mass to enable the reaction to proceed further and the stirring of the said reaction mass is continued until achievement of maximum conversion; d) the reaction mass of step (c) is cooled to room temperature; e) organic solvent layer is separated from the aqueous layer of the biphasic solvent reaction mass and the said aqueous layer is further extracted with same water immiscible organic used in step (c); f) combined organic solvent is concentrated by removing solvent by standard process like vacuum distillation and the like to obtain substantially pure desired amino product of Formula I and recovering the organic solvent; g) aqueous layer as separated in step e) from the water immiscible organic solvent layer and the said aqueous layer contains the residual used enzyme, buffer, alkyl amine and cofactor used for the biocatalytic amination to obtain compound of Formula I and the said aqueous layer containing used enzyme is used as a water solvent phase of the biphasic solvent system as such for the
  • Used enzyme is isolated after its first use for its further reaction as disclosed in the present invention simply by the separation of reaction mass into water immiscible organic solvent containing product of Formula I and aqueous layer containing used enzyme.
  • aqueous layer herein above containing used enzyme, buffer, alkyl amine and cofactor is reused in subsequent cycle/batch for the preparation of compound of Formula I and likewise the aqueous layer containing reused enzyme is again reused for subsequent cycle/batch for the same purpose of converting compound of Formula II into compound of Formula I.
  • the process of the present invention is further characterized by the feature that use of biphasic solvent medium comprising water and water immiscible organic solvent makes work up, isolation of product simple and effective. Furthermore, said biphasic solvent medium also enables the recovery of enzyme and organic solvent from the reaction mass just by separation of each phase of the said biphasic solvent medium also refereed as system.
  • the process with water immiscible solvent make the process robust which otherwise may lead to incomplete conversion in water miscible solvent due to precipitation of the substrate of molecule of Formula II.
  • Cofactor in step a) is Pyridoxal phosphate.
  • the alkyl amine is used as an amine donor moiety.
  • the said alkyl amine is also acts as a base for pH adjustment.
  • the alkyl amine used in herein above is selected from the group comprising isopropyl amine, isobutyl amine, isopropyl amine, 2-methyl benzyl amine, diphenyl methyl amine or any C-substitutes isopropyl amine and the mixture thereof and the likes.
  • the water immiscible solvent used herein above step c) is selected from alkyl ester, ether, water immiscible alcohols and 2-methyl tetrahydrofuran or mixture thereof.
  • Water immiscible alkyl ester is selected from the group comprising ethyl acetate, isopropyl acetate, isobutyl acetate, ter. Butyl acetate or mixture thereof and the likes.
  • Water immiscible ether is selected from the group comprising diisopropyl ether, methyl, methyl isopropyl ether, methyl ter-butyl ether or mixture thereof and the likes.
  • Water immiscible alcohol is selected from the group comprising n-butanol, ter.-butanol, cyclohexanol, or mixture thereof and the likes.
  • the aq. Buffer used herein is selected from the group comprising triethanol amine buffer, phosphate buffer, Tris buffer, or any other buffer in which the said enzyme used herein is stable or mixture thereof.
  • an aqueous phase comprising addition of cofactor Pyridoxal phosphate, buffer triethanolamine and isopropyl amine as an amine donor and transaminase enzyme, pH of said aq. Mixture is adjusted to about 8.5 using hydrochloric acid. The said buffer aq. mixture is stirred at room temperature to get the clear solution. Temperature of the said aq. Mixture is raised to about 45°C. A water immiscible organic solvent isopropyl acetate solution containing ketoamide of Formula II is added slowly to the said aq. mixture resulting into a biphasic solvent system also referred herein as reaction mass.
  • reaction mass Temperature of said reaction mass is maintained at about 45-50°C and pH is adjusted between about 8.2-9.5 using isopropyl amine. Acetone generated as a by-product therein is removed under the reduced pressure to enable the reaction to proceed to completion. Stirring is continued until achievement of maximum conversion.
  • the reaction mass is cooled to room temperature.
  • Organic solvent Isopropyl acetate layer is separated from aqueous layer of the biphasic solvent reaction mass and aqueous layer is further extracted with isopropyl acetate.
  • Combined isopropyl acetate is distilled under vacuum to afford substantially pure Sitagliptin of Formula I as a product as oily mass which turns to solid on standing.
  • Aqueous layer which contains used transaminase enzyme is reused as such for next cycle/batch for amination of a ketoamide of Formula II for the next production batch of Sitagliptin of Formula I as described herein before in earlier embodiment.
  • aqueous layer obtained herein before from a reaction mass where reused enzyme is used and said reused enzyme is reused again for the preparation of Sitagliptin of Formula I contains reused enzyme, cofactor pyridoxal phosphate, buffer triethanolamine and amine donor isopropyl amine and the said aqueous layer is heated to about 45 °C and a solution ketoamide of Formula II in isopropyl acetate is added slowly.
  • the biphasic reaction mass is stirred at about 45-50°C by adjusting pH between about 8.2-9.5 using isopropyl amine. Acetone generated as by-product is removed under the reduced pressure to enable the reaction to proceed further to completion.
  • a specific embodiment disclosed herein is an economically efficient and green enzymatic process for preparation of Sitagliptin of formula I as presented in SCHEME - 1, comprising at least once used transaminase enzyme is reused for the enzymatic conversion of compound of formula II in a biphasic solvent medium comprising aqueous buffer and water immiscible organic solvent as described herein in earlier embodiments.
  • Example 1 Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine (or Sitagliptin)
  • Triethanol amine (2.75g), water (50 mL) & Isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1. Pyridoxal phosphate (56 mg) and transaminase enzyme (1g) was added to the above solution. Temperature was raised to 45°C. A solution of ketoamide of formula I (25g) in isopropyl acetate was added to the above solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).
  • Aqueous layer from the above process which contains enzyme was used for second cycle of biotransformation.
  • Example 2 pH of the aqueous layer from Example 1 was adjusted to 8.2-9.5 using con. HC1 and added pyridoxal phosphate (56 mg). Temperature was raised to 45°C. A solution of ketoamide (25g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).
  • Triethanol amine (1.1g), water (20 mL) & isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1. Pyridoxal phosphate (59 mg) and transaminase enzyme (170 mg) was added to the above solution. Temperature was raised to 45°C. A solution of ketoamide of formula I (10g) in isopropyl acetate was added to the above solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.8-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).
  • Example 4 pH of the aqueous layer from Example 3 was adjusted to 8.2-9.5 using cone. HC1 and temperature was raised to 45°C. A solution of ketoamide (10g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).
  • Triethanol amine (11g), water (200 mL) & isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1.
  • Pyridoxal phosphate (600 mg) and transaminase enzyme (2.5 g) was added to the above solution.
  • Temperature was raised to 45°C.
  • a solution of ketoamide of formula I (100g) in isopropyl acetate was added to the above solution.
  • the reaction mass was stirred at 45-50°C by adjusting pH between 8.8-9.5 using isopropyl amine while removing acetone formed in the reaction.
  • the reaction mass was further cooled to room temperature.
  • Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (91% yield).
  • Example 6 pH of the aqueous layer (510 mL) from Example 5 was adjusted to 8.5-9.5 using cone. HC1. Added pyridoxal phosphate (608 mg) and transaminase enzyme (500 mg). Temperature was raised to 45°C. A solution of ketoamide (100g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (89 % yield).
  • Example 7 pH of the aqueous layer from Example 7 was adjusted to 8.5-9.5. Added pyridoxal phosphate (608 mg) and transaminase enzyme (500 mg). Temperature was raised to 45 °C. A solution of ketoamide (100g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (89 % yield).
  • Example 8 Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine phosphate.

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Abstract

L'invention concerne un procédé biocatalytique recyclable pour la préparation de sitagliptine comprenant la réutilisation d'au moins une fois d'une enzyme utilisée pour la préparation d'un composé de formule I dans un système de solvant biphasique comprenant de l'eau et un solvant organique non miscible dans l'eau pour une conversion stéréosélective de groupe céto de 4-Oxo-4-[3-(trifluorométhyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5-trifluorophényl)butan-2-one (ou cétoamide) de formule II pour obtenir un composé de sitagliptine de formule I.
PCT/IN2022/050436 2022-03-23 2022-05-06 Procédé biocatalytique recyclable pour la préparation de sitagliptine WO2023181046A1 (fr)

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WO2021135886A1 (fr) * 2019-12-31 2021-07-08 弈柯莱生物科技(上海)股份有限公司 Mutant de transaminase, transaminase immobilisée et utilisation dans une préparation de sitagliptine

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