Preparation method of sitagliptin intermediate and sitagliptin isomer

Abstract

The invention relates to the technical field of medicine synthesis, in particular to a preparation method of a sitagliptin intermediate and a sitagliptin isomer. According to the preparation method provided by the invention, a chiral resolution product (S)-I is recycled at low cost, a key intermediate compound shown as a formula II is synthesized, and based on the intermediate compound shown as the formula II, not only can a sitagliptin racemate be obtained by adopting a resolution process, but also R-configuration sitagliptin can be efficiently synthesized under the catalysis of transaminase. Compared with the prior art, the preparation method provided by the invention has the advantages of mild reaction conditions, no dangerous reaction, no unacceptable taste, small dosage of key reagents, no use of rare metals, and relatively friendly cost control and production environment.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, specifically to a method for preparing sitagliptin intermediates and sitagliptin isomers. Background Technology

[0002] Sitagliptin is a drug used to treat type 2 diabetes. It belongs to the dipeptidyl peptidase-4 inhibitor (DPP-4 inhibitor) class of drugs. It works by inhibiting the activity of the DPP-4 enzyme, which slows down the inactivation of incretin hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-promoting peptide (GIP), thereby increasing the concentration of these hormones.

[0003] Sitagliptin contains one chiral carbon atom in the R configuration, and the chiral purity of the drug directly affects its absorption and efficacy. In the synthesis of sitagliptin, most methods employ a resolution route using chiral reagents to obtain the desired R configuration. However, this process results in the waste of the S configuration compound ((S)-I) and exhibits extremely poor atom economy. Therefore, recovering the S configuration isomer can significantly reduce the production cost of the resolution route.

[0004]

[0005] Existing methods for recovering the S-configuration isomer mostly suffer from problems such as being environmentally unfriendly, costly, and having low yields. Therefore, there is an urgent need to develop an environmentally friendly, low-cost, and high-yield method for preparing sitagliptin intermediates and the R-configuration sitagliptin. Summary of the Invention This invention aims to at least partially address one of the technical problems in related technologies. To this end, one object of this invention is to provide a method for synthesizing sitagliptin intermediates and isomers, which utilizes the low-cost recycling of the chiral resolution product (S)-I to synthesize the key intermediate compound of formula II. Based on the compound of formula II, not only can the racemic form of sitagliptin be obtained through resolution processes, but the R-configuration of sitagliptin can also be efficiently synthesized under transaminase catalysis. Compared with existing technologies, the preparation method provided by this invention features mild reaction conditions, no hazardous reactions, no unpleasant odor, low dosage of key reagents, and no use of rare metals, making it more cost-effective and environmentally friendly.

[0006] Therefore, the first aspect of the present invention provides a method for preparing a sitagliptin intermediate. According to an embodiment of the present invention, the preparation method includes: Will( S Compound I undergoes a first contact with an amino receptor, transaminase, and coenzyme to yield the compound shown in Formula II. Wherein, the amino receptor is pyruvate, and the coenzyme is pyridoxal 5-phosphate. During the first contact, the pH of the reaction system was 7.0-9.0.

[0007] To solve the existing ( S The high cost, environmental unfriendliness, and low yield of )-Ⅰ compound recovery methods have been addressed by the inventors through the use of special amino receptors and coenzymes, etc. S The intermediate shown in Formula II is synthesized from compound (±)-I under the action of transaminase and amino receptor. This intermediate can not only be used to obtain the racemic (±)-I by resolution, but also to efficiently synthesize sitagliptin (R)-I under the catalysis of transaminase, thereby achieving the effect of controlling (±)-I. S The invention provides a highly efficient utilization of compound I, and the method for preparing sitagliptin intermediates provided by this invention has no dangerous reactions, no unacceptable taste, requires small amounts of key reagents, does not use rare metals, has low cost, and high product yield.

[0008] According to an embodiment of the present invention, the pH of the reaction system is 7.5-8.5 when the first contact is carried out.

[0009] When the first contact is carried out, the pH of the reaction system is 7.0-9.0, preferably 7.5-8.5, which can further improve the product yield.

[0010] According to an embodiment of the present invention, the amino receptor and ( S The mass ratio of the )-Ⅰ compound is (0.26-0.63): 1, preferably (0.26-0.50): 1, and even more preferably (0.26-0.43): 1.

[0011] According to an embodiment of the present invention, the transaminase is derived from the wet cells of microorganisms.

[0012] According to an embodiment of the present invention, the wet cell of the microorganism and ( S The mass ratio of the )-Ⅰ compound is (0.5-8.0):1, preferably (1.0-6.0):1, and even more preferably (1.0-4.0):1.

[0013] According to an embodiment of the present invention, the coenzyme and ( S The mass ratio of the )-Ⅰ compound is (0.001-0.050):1, preferably (0.001-0.040):1, and even more preferably (0.001-0.030):1.

[0014] According to an embodiment of the present invention, the first contact is performed in a buffer solution.

[0015] According to an embodiment of the present invention, the buffer solution is a phosphate buffer.

[0016] According to an embodiment of the present invention, the buffer solution is a NaH2PO4 / NaOH buffer solution.

[0017] According to an embodiment of the present invention, the concentration of the NaH2PO4 / NaOH buffer solution is 0.1 - 0.3 mol / L.

[0018] According to an embodiment of the present invention, the NaH2PO4 / NaOH buffer solution is mixed with ( S The mass ratio of the )-Ⅰ compound is (2.0-37.5): 1, preferably (2.0-30.0): 1, and even more preferably (2.95-7.0): 1.

[0019] According to an embodiment of the present invention, during the first contact, the reaction system further includes a co-solvent.

[0020] According to embodiments of the present invention, the co-solvent includes at least one selected from DMSO and isopropanol.

[0021] According to an embodiment of the present invention, the co-solvent is DMSO, and the volume ratio of the co-solvent to the phosphate buffer is (0.42-4.00):1, preferably (0.42-3.00):1, and more preferably (0.42-2.50):1.

[0022] According to an embodiment of the present invention, the temperature during the first contact is 35-70°C, and the time is 5-30 hours.

[0023] According to an embodiment of the present invention, the temperature during the first contact is 50-60°C, and the time is 10-25 hours.

[0024] According to an embodiment of the present invention, the preparation method further includes enzyme removal treatment and extraction treatment.

[0025] According to an embodiment of the present invention, the enzyme removal treatment includes using a pH adjustment method to inactivate the transaminase.

[0026] According to an embodiment of the present invention, the pH adjustment method controls the pH range to be 2-3.

[0027] According to embodiments of the present invention, the extractant used in the extraction process includes at least one selected from ethyl acetate, butyl acetate, isopropyl acetate, tetrahydrofuran, and dichloromethane.

[0028] A second aspect of the present invention provides a method for preparing a sitagliptin isomer. According to an embodiment of the present invention, the preparation method includes: S1: Sitagliptin intermediate is prepared using the preparation method described in the first aspect; S2: The sitagliptin intermediate, isopropylamine, transaminase, and coenzyme are subjected to a second contact to obtain the structure with the following formula ( R Sitagliptin isomers of α-I, The coenzyme is pyridoxal 5-phosphate.

[0029] A third aspect of this invention provides a method for preparing the racemic mixture of sitagliptin. According to an embodiment of the invention, the preparation method includes: M1: Sitagliptin intermediates are prepared using the preparation method described in the first aspect; M2: The sitagliptin intermediate is contacted with ammonium acetate in an alcoholic solution to obtain the compound shown in Formula III; M3: An addition reaction is performed on the compound shown in Formula III to obtain the racemic sitagliptin shown in Formula IV.

[0030] Sitagliptin has several synthetic routes, one of which is the resolution route, which involves chiral reagents and results in an S-configuration compound. S (I) Wasteful and extremely poor in atom economy, therefore, isomer recycling can greatly reduce the production cost of the splitting route.

[0031] This invention uses sitagliptin isomers (( S Using (±)-Ⅰ as a substrate, this invention synthesizes the key intermediate compound represented by formula II via a bioenzymatic method. The compound represented by formula II can not only be resolved to obtain the racemic (±)-Ⅰ, but can also be efficiently synthesized into sitagliptin (R)-Ⅰ under transaminase catalysis. Compared with existing technologies, this invention features mild reaction conditions, no hazardous reactions, no unpleasant odor, low dosage of key reagents, and no use of rare metals, making it more cost-effective and environmentally friendly.

[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0038] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0039] According to a specific embodiment of the present invention, the present invention provides a method for preparing a sitagliptin intermediate, comprising: Will( S Compound I undergoes a first contact with an amino receptor, transaminase, and coenzyme to yield the compound shown in Formula II. Wherein, the amino receptor is pyruvate, and the coenzyme is pyridoxal 5-phosphate. During the first contact, the pH of the reaction system was 7.0-9.0.

[0040] In the synthesis of sitagliptin, the resolution route is mostly employed using chiral reagents to obtain the desired R-configuration of sitagliptin. However, this process can lead to the formation of the S-configuration compound. SThe waste of S-configuration isomeric compounds (I) and their extremely poor atom economy mean that recycling the S-configuration isomeric compounds can significantly reduce the production cost of the resolution route. The method for preparing sitagliptin intermediates provided by this invention utilizes the S-configuration compound (I) S )-Ⅰ) Synthesizing the sitagliptin intermediate compound shown in formula II not only fully utilizes the S-configuration compound (( S Furthermore, based on the following reaction route, the compound shown in Formula II can be further utilized to obtain the racemic form of sitagliptin through a resolution process, or to efficiently synthesize the R-configuration sitagliptin under transaminase catalysis. Moreover, the method for preparing sitagliptin intermediates provided by this invention has no hazardous reactions, no unpleasant odor, requires minimal amounts of key reagents, does not use rare metals, is low-cost, and yields a high product.

[0041]

[0042] According to a preferred embodiment of the present invention, the pH of the reaction system is 7.5-8.5 during the first contact. This can further improve the product yield.

[0043] According to a specific embodiment of the present invention, the amino receptor and ( S The mass ratio of the amino receptor to compound I is (0.26-0.63):1. For example, the amino receptor and ( S The mass ratios of the amino receptor and (I) compounds are 0.26:1, 0.30:1, 0.35:1, 0.40:1, 0.45:1, 0.50:1, 0.55:1, 0.60:1, and 0.63:1. According to a preferred embodiment of the present invention, the amino receptor and (I) compounds are... S The mass ratio of the amino receptor to compound I is (0.26-0.50):1. According to a more preferred embodiment of the invention, the amino receptor and ( S The mass ratio of the )-Ⅰ compounds is (0.26-0.43): 1.

[0044] According to a specific embodiment of the present invention, the transaminase is derived from the wet cell of a microorganism.

[0045] It should be noted that the transaminase used in the preparation method provided by the present invention can be a commercially available transaminase or it can be derived from microorganisms that can produce transaminase, such as Streptomyces, Bacillus, genetically modified Escherichia coli that can produce transaminase, lactic acid bacteria, etc.

[0046] According to a specific embodiment of the present invention, the wet cell of the microorganism and ( S The mass ratio of compound I to ) is (0.5-8.0):1. For example, the wet cell mass of the microorganism and ( SThe mass ratio of compound I to the microorganism is 0.5:1, 1.0:1, 1.66:1, 2.0:1, 3.0:1, 3.33:1, 4.0:1, 5.0:1, 6.0:1, 7.0:1, or 8.0:1. According to a preferred embodiment of the present invention, the wet cell mass of the microorganism is mixed with (… S The mass ratio of compound I to () is (1.0-4.0):1. According to a preferred embodiment of the present invention, the wet cell mass of the microorganism and ( S The mass ratio of the )-Ⅰ compounds is (1.0-3.0):1.

[0047] According to a specific embodiment of the present invention, the coenzyme and ( S The mass ratio of the coenzyme to compound I is (0.001-0.050):1. For example, the coenzyme and ( S The mass ratio of the coenzyme to compound I is 0.001:1, 0.005:1, 0.010:1, 0.020:1, 0.030:1, 0.040:1, 0.050:1, etc. According to a preferred embodiment of the present invention, the coenzyme and ( S The mass ratio of the )-Ⅰ compounds is (0.001-0.040):1, and more preferably (0.001-0.030):1.

[0048] According to a specific embodiment of the present invention, the first contact is performed in a buffer solution.

[0049] It should be noted that all types of buffer solutions capable of successfully performing the synthesis reaction of the compounds shown in Formula II are covered within the scope of protection of this invention.

[0050] According to a specific embodiment of the present invention, the buffer solution is a phosphate buffer solution.

[0051] According to a preferred embodiment of the present invention, the buffer solution is a NaH2PO4 / NaOH buffer solution.

[0052] According to a specific embodiment of the present invention, the concentration of the NaH2PO4 / NaOH buffer solution is 0.1-0.3 mol / L.

[0053] According to a specific embodiment of the present invention, the NaH2PO4 / NaOH buffer solution is mixed with ( S The mass ratio of compound )-Ⅰ is (2.0-37.5): 1. For example, the NaH2PO4 / NaOH buffer solution is mixed with ( SThe mass ratio of the NaH2PO4 / NaOH buffer to the compound is 2.0:1, 2.95:1, 4.0:1, 5.0:1, 6.0:1, 7.0:1, 7.5:1, 8.0:1, 10.0:1, 15.0:1, 20.0:1, 25.0:1, 30.0:1, 37.5:1, etc. According to a preferred embodiment of the present invention, the NaH2PO4 / NaOH buffer solution is mixed with (… S The mass ratio of the )-Ⅰ compound is (2.0-30.0): 1, and more preferably (2.95-7.0): 1.

[0054] According to a specific embodiment of the present invention, when the first contact is carried out, the reaction system further includes a co-solvent.

[0055] There are no particular limitations on the type of co-solvent, including but not limited to DMSO, isopropanol, etc., all of which are covered within the scope of protection of this invention. According to a preferred embodiment of the invention, the co-solvent is DMSO.

[0056] According to a specific embodiment of the present invention, the co-solvent is DMSO, and the volume ratio of the co-solvent to the phosphate buffer is (0.42-4.00):1. For example, the volume ratio of DMSO to NaH2PO4 / NaOH buffer is 0.42:1, 0.50:1, 1.00:1, 1.50:1, 2.00:1, 2.50:1, 3.00:1, etc. According to a preferred embodiment of the present invention, the volume ratio of DMSO to NaH2PO4 / NaOH buffer is (0.42-3.00):1, and more preferably (0.42-2.50):1.

[0057] According to a specific embodiment of the present invention, the temperature during the first contact is 35-70°C, and the time is 5-30 hours. According to a preferred embodiment of the present invention, the temperature during the first contact is 50-60°C, and the time is 10-25 hours. For example, the temperature during the first contact is 50-60°C, and the time is 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, or 25 hours.

[0058] According to a specific embodiment of the present invention, the preparation method further includes enzyme removal treatment and extraction treatment.

[0059] The enzyme removal treatment can be any physical or chemical method that inactivates the enzyme, such as heat treatment or acid / alkali treatment. Preferably, the enzyme removal treatment includes using pH adjustment to inactivate the transaminase.

[0060] According to a specific embodiment of the present invention, the pH adjustment method controls the pH range to be 2-3.

[0061] According to a specific embodiment of the present invention, the extractant used in the extraction process includes at least one selected from ethyl acetate, butyl acetate, isopropyl acetate, tetrahydrofuran, and dichloromethane.

[0062] According to a specific embodiment of the present invention, the method for preparing the sitagliptin intermediate provided by the present invention adopts the following synthetic route:

[0063] With sitagliptin isomers (( S Using pyruvate (Formula II) as the substrate, the key intermediate compound is synthesized via a bioenzymatic method. The mass ratio of pyruvate to the starting material is 0.26:1 to 0.63:1, preferably 0.26:1 to 0.50:1, and more preferably 0.26:1 to 0.43:1. The mass ratio of NaH₂PO₄ / NaOH buffer solution (0.10 mol / L, pH = 6.5 to 7.5) to the starting material is 2.0:1 to 37.5:1, preferably 2.0:1 to 30.0:1, and more preferably 2.95:1 to 7.0:1. The volume ratio of the co-solvent (DMSO) to the NaH₂PO₄ / NaOH buffer solution (0.10 mol / L, pH = 6.5 to 7.5) is 0.42:1 to 4.00:1, preferably 0.42:1 to 3.00:1, and more preferably 0.42:1 to 2.50:1. The mass ratio of PLP (pyridoxal 5-phosphate) to starting material is 0.001:1 to 0.050:1, preferably 0.001:1 to 0.040:1, and more preferably 0.001:1 to 0.030:1. The mass ratio of bioenzyme (wet bacterial cells) to starting material is 0.5:1 to 8.0:1, preferably 1.0:1 to 6.0:1, and more preferably 1.0:1 to 4.0:1. The reaction temperature is 35 to 70°C, preferably 50 to 60°C. The reaction pH is 7.0 to 9.0, preferably 7.5 to 8.5. The post-treatment extractant can be ethyl acetate, butyl acetate, isopropyl acetate, tetrahydrofuran, dichloromethane, etc.

[0064] According to a specific embodiment of the present invention, the present invention provides a method for preparing sitagliptin isomers, comprising: S1: Sitagliptin intermediate was prepared using the preparation method described above; S2: The sitagliptin intermediate, isopropylamine, transaminase, and coenzyme are subjected to a second contact to obtain the structure with the following formula ( R Sitagliptin isomers of α-I, The coenzyme is pyridoxal 5-phosphate.

[0065] According to a specific embodiment of the present invention, the present invention provides a method for preparing the racemic mixture of sitagliptin, comprising: M1: Sitagliptin intermediate was prepared using the preparation method described above; M2: The sitagliptin intermediate is contacted with ammonium acetate in an alcoholic solution to obtain the compound shown in Formula III; M3: An addition reaction is performed on the compound shown in Formula III to obtain the racemic sitagliptin shown in Formula IV.

[0066] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0067] In the following examples, "eq" refers to molar equivalent.

[0068] Example 1

[0069] Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 6.0 mL) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (pyridoxal 5-phosphate, 15 mg) and biological enzyme (wet bacterial cells, 1.0 g), stir the system thoroughly after addition, and then add the compound ( S The reaction mixture was prepared by adding 600 mg of DMSO (6.0 mL) and 1.47 mmol (1.0 equiv) to DMSO. The mixture was stirred at 50–60 °C for 16 h. Five drops of the mixture were taken and acetonitrile was added to 1.0 mL. The mixture was centrifuged and the supernatant was sent as a sample. The reaction was stopped when the starting material was less than 1.5%. The pH was adjusted to 2–3 with concentrated hydrochloric acid, and the mixture was stirred at 50 °C for 1 h. The mixture was filtered to remove the enzyme and extracted with isopropyl acetate (10 mL * 2). The organic phases were combined, washed with brine, and distilled under reduced pressure to obtain 580 mg of solid, with a yield of 96.9%. ¹H NMR (599 MHz, DMSO-d6) δ 7.55–7.46 (m, 1H), 7.45–7.36 (m, 1H), 4.93 (d, J = 6.0 Hz, 2H), 4.28–4.11 (m, 2H), 4.04–3.89 (m, 6H). LC-MS: [M+H] = 407.1.

[0070] Example 2 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet bacterial cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO, then stir the mixture at 50–60 °C for 16 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill the organic phase under reduced pressure to obtain 580 mg of solid, with a yield of 96.8%.

[0071] Example 3 Add pyruvate (0.17 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet bacterial cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO, then stir the mixture at 50–60 °C for 16 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill the organic phase under reduced pressure to obtain 550 mg of solid, with a yield of 91.8%.

[0072] Example 4

[0073] Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet bacterial cells, 1.0 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO. Stir the mixture at 50–60 °C for 16 h. Take 5 drops of the sample, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill under reduced pressure to obtain 575 mg of solid, with a yield of 96.0%.

[0074] Example 5 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (0.6 mg) and biological enzyme (wet bacterial cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S The reaction mixture was prepared with 600 mg (1.47 mmol, 1.0 equiv) of α-I and 3.0 ml of DMSO. The mixture was stirred at 50–60 °C for 16 h. Five drops of the mixture were taken and acetonitrile was added to 1.0 mL. The mixture was shaken and centrifuged. The supernatant was sent as a sample. The reaction was stopped when the starting material was less than 1.5%. The pH was adjusted to 2–3 with concentrated hydrochloric acid, and the mixture was stirred at 50 °C for 1 h. The mixture was filtered to remove the enzyme, and extracted with isopropyl acetate (10 mL * 2). The organic phases were combined, washed with brine, and distilled under reduced pressure to obtain 577 mg of solid, with a yield of 96.4%.

[0075] Example 6 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 0.6 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO. Stir the mixture at 50–60 °C for 24 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge. Send the supernatant as a sample. If the starting material is less than 1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill under reduced pressure to obtain 571 mg of solid, with a yield of 95.4%.

[0076] Example 7 Add pyruvate (0.156 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 2.0 g), stir the system thoroughly after addition, and then add the compound ( SAdd 600 mg (1.47 mmol, 1.0 equiv) of α-I and 3.0 mL of DMSO, then stir the mixture at 50–60 °C for 24 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill the organic phase under reduced pressure to obtain 568 mg of solid, with a yield of 94.9%.

[0077] Example 8 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 1.77 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 2.0 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg (1.47 mmol, 1.0 equiv) of α-I and 4.22 ml of DMSO, then stir the mixture at 50-60 °C for 24 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2-3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL*2), combine the organic phases, wash with brine, and distill the organic phase under reduced pressure to obtain 570 mg of solid, with a yield of 95.2%.

[0078] Example 9 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 4.22 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 2.0 g), stir the system thoroughly after addition, and then add the compound ( S The reaction mixture was prepared with 600 mg (1.47 mmol, 1.0 equiv) of α-I and 1.77 mL of DMSO. The mixture was stirred at 50–60 °C for 24 h. Five drops of the mixture were taken and acetonitrile was added to 1.0 mL. The mixture was shaken and centrifuged. The supernatant was sent as a sample. The reaction was stopped when the starting material was less than 1.5%. The pH was adjusted to 2–3 with concentrated hydrochloric acid, and the mixture was stirred at 50 °C for 1 h. The mixture was filtered to remove the enzyme, and extracted with isopropyl acetate (10 mL * 2). The organic phases were combined, washed with brine, and distilled under reduced pressure to obtain 565 mg of solid, with a yield of 94.4%.

[0079] Example 10 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.3 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet bacterial cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO, then stir the mixture at 50–60 °C for 16 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. If the starting material is <1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with isopropyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill the organic phase under reduced pressure to obtain 571 mg of solid, with a yield of 95.4%.

[0080] Example 11 Add pyruvate (0.24 g) and phosphate buffer solution (PBS, 0.1 M, 3.0 ml) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet bacterial cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 3.0 mL of DMSO. Stir the mixture at 50–60 °C for 16 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge. Send the supernatant as a sample. If the starting material is less than 1.5%, stop the reaction. Adjust the pH to 2–3 with concentrated hydrochloric acid, stir at 50 °C for 1 h, filter to remove enzymes, extract with ethyl acetate (10 mL * 2), combine the organic phases, wash with brine, and distill under reduced pressure to obtain 582 mg of solid, with a yield of 97.3%.

[0081] Comparative Example 1

[0082] Add methyl acetoacetate (376 mg) and phosphate buffer solution (6.0 mL) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 1.0 g), stir the system thoroughly after addition, and then add the compound ( S Add 600 mg of α-I (1.47 mmol, 1.0 equiv) and 6.0 mL of DMSO, then stir the mixture at 50-60 °C for 16 h. Take 5 drops of the mixture, add acetonitrile to 1.0 mL, shake and centrifuge, and send the supernatant as a sample. Only a trace amount of product was obtained.

[0083] Comparative Example 2

[0084] Add acetone (188 mg) and phosphate buffer solution (6.0 mL) to the reaction flask, adjust the pH to 7.5-8.5, add PLP (15 mg) and biological enzyme (wet cells, 1.0 g), stir the system thoroughly after addition, and then add the compound ( S )-Ⅰ (600mg, 1.47mmol, 1.0 equiv) and DMSO (6.0 mL) were added, and the system was sealed and reacted at 50~60℃ for 16h. 5 drops of the sample were taken, acetonitrile was added to 1.0 mL, the mixture was shaken and centrifuged, and the supernatant was sent as a sample. The residue of the raw material was 85.3%.

[0085] The results of Comparative Examples 1 and 2 show that the yield of intermediate product II obtained by using methyl acetoacetate or acetone as amino acceptors is low, while only by using pyruvate of the present invention as amino acceptor can a product yield of more than 90% be obtained.

[0086] Comparative Example 3 Add pyruvate (0.24 g) and phosphate buffer solution (3.0 ml) to the reaction flask, adjust the pH to 9.3-9.5, add PLP (15 mg) and biological enzyme (wet cells, 2 g), stir the system thoroughly after addition, and then add the compound ( S The reaction mixture was prepared with 600 mg (1.47 mmol, 1.0 equiv) of α-I and 3.0 mL of DMSO. The mixture was stirred at 50–60 °C for 16 h. Five drops of the mixture were taken and acetonitrile was added to 1.0 mL. The mixture was shaken and centrifuged. The supernatant was sent as a sample. A large amount of raw material remained, so the reaction was stopped. The pH was adjusted to 2–3 with concentrated hydrochloric acid, and the mixture was stirred at 50 °C for 1 h. The mixture was filtered to remove the enzyme, and extracted with isopropyl acetate (10 mL * 2). The organic phases were combined, washed with brine, and distilled under reduced pressure to obtain 412 mg of solid, with a yield of 68.8%.

[0087] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0088] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a sitagliptin intermediate, characterized in that, include: Will( S Compound I undergoes a first contact with an amino receptor, transaminase, and coenzyme to yield the compound shown in Formula II. Wherein, the amino receptor is pyruvate, and the coenzyme is pyridoxal 5-phosphate. During the first contact, the pH of the reaction system was 7.0-9.

0. 。 2. The preparation method according to claim 1, characterized in that, During the first contact, the pH of the reaction system is 7.5-8.

5.

3. The preparation method according to claim 1, characterized in that, The amino receptor and ( S The mass ratio of the )-Ⅰ compound is (0.26-0.63): 1, preferably (0.26-0.50): 1, and even more preferably (0.26-0.43):

1.

4. The preparation method according to claim 1, characterized in that, The transaminase is derived from the wet cells of microorganisms; Optionally, the wet cell mass of the microorganism and ( S The mass ratio of the )-Ⅰ compound is (0.5-8.0):1, preferably (1.0-6.0):1, and even more preferably (1.0-4.0):

1.

5. The preparation method according to claim 1, characterized in that, The coenzyme and ( S The mass ratio of the )-Ⅰ compound is (0.001-0.050):1, preferably (0.001-0.040):1, and even more preferably (0.001-0.030):

1.

6. The preparation method according to claim 1, characterized in that, The first contact takes place in a buffer solution; Optionally, the buffer solution is a phosphate buffer; Optionally, the buffer solution is a NaH2PO4 / NaOH buffer solution; Optionally, the concentration of the NaH2PO4 / NaOH buffer solution is 0.1-0.3 mol / L; Optionally, the NaH2PO4 / NaOH buffer solution is mixed with ( S The mass ratio of the )-Ⅰ compound is (2.0-37.5): 1, preferably (2.0-30.0): 1, and even more preferably (2.95-7.0):

1.

7. The preparation method according to claim 6, characterized in that, During the first contact, the reaction system further includes a co-solvent; Optionally, the co-solvent includes at least one selected from DMSO and isopropanol; Optionally, the co-solvent is DMSO, and the volume ratio of the co-solvent to the phosphate buffer is (0.42-4.00):1, preferably (0.42-3.00):1, and more preferably (0.42-2.50):

1.

8. The preparation method according to claim 1, characterized in that, The temperature during the first contact is 35-70℃, and the time is 5-30 hours. Preferably, the temperature during the first contact is 50-60°C, and the time is 10-25 hours.

9. The preparation method according to claim 1, characterized in that, The preparation method further includes enzyme removal treatment and extraction treatment; Optionally, the enzyme removal treatment includes using a pH adjustment method to inactivate the transaminase; Optionally, the pH adjustment method controls the pH range to 2-3; Optionally, the extractant used in the extraction process includes at least one selected from ethyl acetate, butyl acetate, isopropyl acetate, tetrahydrofuran, and dichloromethane.

10. A method for preparing a sitagliptin isomer, characterized in that, include: S1: Sitagliptin intermediate is prepared by the preparation method according to any one of claims 1-9; S2: The sitagliptin intermediate, isopropylamine, transaminase, and coenzyme are subjected to a second contact to obtain the structure with the following formula ( R Sitagliptin isomers of α-I, The coenzyme is pyridoxal 5-phosphate. 。 11. A method for preparing a racemic mixture of sitagliptin, characterized in that, include: M1: Sitagliptin intermediate is prepared by the preparation method according to any one of claims 1-9; M2: The sitagliptin intermediate is contacted with ammonium acetate in an alcoholic solution to obtain the compound shown in Formula III; M3: An addition reaction is performed on the compound shown in Formula III to obtain the racemic sitagliptin shown in Formula IV. 。

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