Omariglonide intermediate and method for preparing Omariglonide key intermediate

Abstract

The invention provides an omariglitazone intermediate and a method for preparing an omariglitazone key intermediate, and relates to the technical field of organic synthesis. The invention provides an omarignelone intermediate which has a structure as shown in a formula III. According to the present invention, the raw materials of the omariglone intermediate are easily available, the use of expensive palladium and ligand can be avoided by using the omariglone intermediate to prepare the omariglone key intermediate (the compound represented by the formula I), the production cost is low, the atom economy is high, and the method is suitable for industrial production. The invention provides a method for preparing an omarignelone key intermediate, the raw materials are easy to obtain, the preparation process is simple and easy to operate, the use of expensive palladium and ligand is avoided, and the method has the advantages of low production cost, environmental friendliness and high atom economy, and is suitable for industrial production. The omarignelone key intermediate (the compound shown in the formula I) prepared by the method provided by the invention has relatively high yield and purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and particularly relates to an orforglipron intermediate and a method for preparing a key intermediate of orforglipron. BACKGROUND

[0002] Orforglipron (CAS: 2212020-52-3), chemical name: (1R, 2R)-1-(5-((S)-2, 2-dimethyltetrahydro-2H-pyran-4-yl)-2-(3-(4-fluoro-1-methyl-2, 3-dihydro-1H-indazol-5-yl)-2-oxo-2, 3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro-3, 5-dimethylphenyl)-4, 5, 6, 7-tetrahydro-2H-pyrazolo[4, 3-c]pyridine-5-carbonyl)-1, chemical structural formula is as follows:

[0003]

[0004] Orforglipron is a glucagon-like peptide (receptor agonist 1, GLP-1) receptor agonist developed by Lilly, which can be used for studying obesity and type 2 diabetes (T2D) in adults. On April 17, 2025, Lilly announced the positive top-line results of their phase 3 clinical study, ACHIEVE-1. This study aims to evaluate the safety and efficacy of orforglipron in patients with type 2 diabetes, and the drug still shows good effect in patients with poor diet and exercise control. It is the first oral small molecule GLP-1 drug that successfully completes a phase 3 trial without restrictions on diet or water intake.

[0005] PCT patent application with publication number WO2018056453A1 discloses a preparation method of orforglipron, and the compound shown in formula I is a key intermediate for preparing orforglipron. The synthetic route for preparing the compound shown in formula I in the patent is shown in route 1. The compound shown in formula I is obtained through four steps of Negishi coupling, chiral column separation, hydrolysis and condensation, with 5-bromoindole-2-carboxylic acid ethyl ester (compound 31a) as a starting material. The problems of this route are as follows. First, the starting material is not easy to obtain, and 5-bromoindole-2-carboxylic acid ethyl ester needs to be prepared from p-bromoaniline through multi-step reactions. In addition, expensive metal palladium and ligand are used in the coupling reaction, resulting in high cost. Chiral column separation is also needed to obtain chiral intermediates, which is difficult to be used for industrial production.

[0006] Route 1:

[0007] The synthesis route of the compound shown in formula I (as shown in route 2) is disclosed in PCT patent with publication number WO2024137426A1, which uses 5-bromoindole-2-carboxylic acid (compound 1) as a starting material, and obtains the compound shown in formula I through six steps of condensation, Heck coupling and the like. This route also has the problems of difficult to obtain starting material and use of expensive metal palladium and ligand, in addition to the problems of poor atom economy and low atom utilization rate caused by difficult to recycle chiral auxiliary, which is not conducive to cost reduction and does not conform to the purpose of green chemistry.

[0008] Route 2: SUMMARY

[0009] Therefore, the purpose of the present application is to provide an alogliptin intermediate and a method for preparing a key intermediate of alogliptin. The alogliptin intermediate (the structure shown in formula III) provided by the present application is easy to obtain, and the use of expensive palladium and ligand can be avoided when preparing the key intermediate of alogliptin (the compound shown in formula I) from the alogliptin intermediate, which is low in production cost, high in atom economy, and suitable for industrialized production.

[0010] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0011] The present application provides an alogliptin intermediate, which has the structure shown in formula III:

[0012]

[0013] In formula III, R is an alkyl group or a substituted alkyl group, the substituent group of the substituted alkyl group includes an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0014] The present application provides a preparation method of the alogliptin intermediate described in the above technical solutions, which comprises the following steps:

[0015] The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain the alogliptin intermediate with the structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0016]

[0017] In formula II, R is an alkyl group or a substituted alkyl group, the substituent group of the substituted alkyl group includes an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0018] Preferably, the catalyst comprises one or more of aluminum trichloride, ferric trichloride, zinc chloride and boron trifluoride etherate; the first organic solvent comprises dichloromethane and / or 1,2-dichloroethane; the molar ratio of the compound of formula II, the acylating agent and the catalyst is 1:(1.1-3):(1.1-3); the temperature of the Friedel-Crafts acylation reaction is 0-80℃, and the time is 0.5-3h.

[0019] The present application provides an alogliptin intermediate having the structure of formula IV:

[0020]

[0021] In formula IV, R is an alkyl group or a substituted alkyl group, the substituent of the substituted alkyl group comprises an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0022] The present application provides a preparation method of the alogliptin intermediate of the above technical solution, comprising the following steps:

[0023] The compound of formula II, the acylating agent, the catalyst and the first organic solvent are mixed to perform a Friedel-Crafts acylation reaction to obtain the alogliptin intermediate having the structure of formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0024]

[0025] The alogliptin intermediate having the structure of formula III, the carbonyl reductase, the reduced coenzyme and the phosphate buffer are mixed to perform a biocatalytic reduction reaction to obtain the alogliptin intermediate having the structure of formula IV.

[0026] Preferably, the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 2, the nucleic acid sequence of the gene encoding the carbonyl reductase is shown in SEQ ID NO. 1; the reduced coenzyme is NADPH; the temperature of the biocatalytic reduction reaction is 25-35℃, the time is 12-24h, and the biocatalytic reduction reaction is performed under the condition that the pH value is 6.5-7.5.

[0027] The present application provides an alogliptin intermediate having the structure of formula V:

[0028]

[0029] In formula V, R is an alkyl group or a substituted alkyl group, the substituent of the substituted alkyl group comprises an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0030] The application provides a preparation method of the above technical scheme of an intermediate of alogliptin, and comprises the following steps:

[0031] The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0032]

[0033] The intermediate of alogliptin with the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula IV;

[0034]

[0035] The intermediate of alogliptin with the structure shown in formula IV, an alkaline compound and a second organic solvent are mixed to perform a ring-closing reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula V.

[0036] Preferably, the alkaline compound comprises one or more of potassium tert-butoxide, sodium ethoxide, sodium methoxide and sodium hydride; the second organic solvent comprises one or more of tetrahydrofuran, toluene and 2-methyltetrahydrofuran; the molar ratio of the intermediate of alogliptin with the structure shown in formula IV to the alkaline compound is 1:1.2-1:3; the ring-closing reaction is carried out at a temperature of 20-50 DEG C for 3-6 h.

[0037] The application provides a method for preparing a key intermediate of alogliptin with a structure shown in formula I, which comprises the following steps:

[0038] The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0039]

[0040] The intermediate of alogliptin with the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula IV;

[0041]

[0042] The intermediate of alogliptin with the structure shown in formula IV, an alkaline compound and a second organic solvent are mixed to perform a ring-closing reaction, so as to obtain an intermediate of alogliptin with a structure shown in formula V.

[0043]

[0044] The ogilator intermediate shown in formula V, a Lewis acid, a Grignard reagent and a third organic solvent are mixed to carry out an opening ring reaction to obtain a compound shown in formula VI; the Grignard reagent includes 2-methylallyl magnesium chloride and / or 2-methylallyl magnesium bromide;

[0045] The compound shown in formula VI, an acid and a fourth organic solvent are mixed to carry out a ring-forming reaction, and then the obtained reaction system is mixed with water to carry out an ester group hydrolysis reaction to obtain a compound shown in formula VII;

[0046] The compound shown in formula VII is activated by oxalyl chloride and then condensed with N-methyl aniline to obtain an ogilator key intermediate shown in formula I;

[0047]

[0048] The present application provides an ogilator intermediate, which has a structure shown in formula III. The ogilator intermediate (the structure shown in formula III) provided by the present application is easy to obtain, and can be prepared from an easily obtained compound shown in formula II through a one-step Friedel-Crafts acylation reaction. Furthermore, the preparation of an ogilator key intermediate (a compound shown in formula I) from the ogilator intermediate can avoid the use of expensive palladium and ligands, has low production cost, high atom economy, and is suitable for industrial production.

[0049] The present application provides a method for preparing an ogilator key intermediate, which has raw materials that are easy to obtain. The carbonyl reductase (further, the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 2, and the nucleic acid sequence of the encoding gene is shown in SEQ ID NO. 1) catalyzes the compound of formula III with high stereoselectivity and high activity. When the compound of formula III is catalyzed to prepare the compound of formula IV, the product has high concentration, high atom economy, simple operation, easy scaling-up, and the product is easy to separate and purify, and has good industrial application prospect. The present application avoids the use of expensive palladium and ligands, has the advantages of low production cost, environmental friendliness, high atom economy, and is suitable for industrial production, so that the preparation of the ogilator key intermediate is more environmentally friendly and efficient. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the compound shown in formula III-1 prepared in the examples;

[0051] Figure 2 The carbon nuclear magnetic resonance spectrum of the compound shown in formula III-1 prepared in the examples;

[0052] Figure 3A hydrogen nuclear magnetic resonance spectrum of a compound shown in Formula IV-1 prepared for an example;

[0053] Figure 4 A carbon nuclear magnetic resonance spectrum of a compound shown in Formula IV-1 prepared for an example;

[0054] Figure 5 A hydrogen nuclear magnetic resonance spectrum of a compound shown in Formula I prepared for an example;

[0055] Figure 6 A carbon nuclear magnetic resonance spectrum of a compound shown in Formula I prepared for an example. DETAILED DESCRIPTION

[0056] The present application provides an alogliptin intermediate having a structure shown in Formula III:

[0057]

[0058] In Formula III, R is an alkyl group or a substituted alkyl group, the substituent of the substituted alkyl group includes an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0059] In the present application, the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group can be 1, 2, 3, 4, 5 or 6, and the alkyl group in the alkyl group and the substituted alkyl group is preferably a straight-chain saturated alkyl group. In the present application, the aryl group is preferably a phenyl group, and the substituted aryl group is preferably a benzyl group, a phenethyl group or a halogen-substituted benzyl group (such as p-chlorobenzyl).

[0060] The alogliptin intermediate (having a structure shown in Formula III) provided by the present application is easy to obtain, and the use of expensive palladium and ligands can be avoided in the preparation of a key intermediate (a compound shown in Formula I) of alogliptin from the alogliptin intermediate, thereby reducing production cost, improving atomic economy and being suitable for industrial production.

[0061] The present application provides a preparation method of the alogliptin intermediate described in the above technical solution, which comprises the following steps:

[0062] The compound shown in Formula II, an acylating reagent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain the alogliptin intermediate having a structure shown in Formula III; the acylating reagent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0063]

[0064] In Formula II, R is an alkyl group or an aralkyl group, and the number of carbon atoms of the alkyl group in the alkyl group and the aralkyl group is 1-6.

[0065] In the present application, the raw materials involved are all commercially available products well known in the art, unless otherwise specified.

[0066] In the present application, R in the compound of formula II is consistent with R in the intermediate of olgrilone of formula III.

[0067] In the present application, the catalyst preferably comprises one or more of aluminum trichloride, ferric trichloride, zinc chloride and boron trifluoride etherate, and the aluminum trichloride is specifically anhydrous aluminum trichloride; the molar ratio of the compound of formula II, the acylating agent and the catalyst is preferably 1:(1.1-3):(1.1-3), and more preferably 1:2:2. In the present application, the first organic solvent preferably comprises dichloromethane and / or 1,2-dichloroethane, and the present application does not have a specific requirement for the amount of the first organic solvent, which can only ensure that the raw materials are dissolved and the reaction is smoothly carried out.

[0068] In the present application, the method for mixing the compound of formula II, the acylating agent, the catalyst and the first organic solvent is preferably as follows: the catalyst and the first organic solvent are added to a reaction bottle, ice bathed under nitrogen protection, the acylating agent is then added, and after 5 minutes of incubation and stirring, the compound of formula II is added to the obtained mixture; the compound of formula II is preferably added in batches.

[0069] In the present application, the temperature of the Friedel-Crafts acylation reaction is preferably 0-80℃, and more preferably under reflux conditions; the time of the Friedel-Crafts acylation reaction is preferably 0.5-3h, which can be 1h, 2h or 3h, and the time of the Friedel-Crafts acylation reaction is calculated from the time when the addition of the compound of formula II is completed. In the present application, the reaction formula involved in the Friedel-Crafts acylation reaction is as follows:

[0070]

[0071] In the present application, after the completion of the Friedel-Crafts acylation reaction, the obtained reaction solution is preferably subjected to post-treatment, and the method for the post-treatment is preferably as follows: the obtained reaction solution is added dropwise into ice water for quenching, extracted with ethyl acetate, washed with saturated sodium bicarbonate and saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, concentrated to dryness, and the intermediate of olgrilone of formula III is obtained.

[0072] The present application provides an intermediate of olgrilone, which has a structure of formula IV:

[0073]

[0074] In formula IV, R is an alkyl group or a substituted alkyl group, and the substituent of the substituted alkyl group comprises an aryl group or a substituted aryl group; the number of carbon atoms in the alkyl group and the alkyl group of the substituted alkyl group is 1-6.

[0075] In the present application, R in the structure shown in formula IV is consistent with R in the alogliptin intermediate shown in formula III.

[0076] The present application provides a preparation method of the alogliptin intermediate shown in the above technical solution, comprising the following steps:

[0077] The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction to obtain the alogliptin intermediate shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0078]

[0079] The alogliptin intermediate shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction to obtain the alogliptin intermediate shown in formula IV.

[0080] In the present application, the Friedel-Crafts acylation reaction is consistent with the Friedel-Crafts acylation reaction described in the above technical solution, which will not be described here.

[0081] In the present application, the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 2, and the nucleic acid sequence of the gene encoding the carbonyl reductase is shown in SEQ ID NO. 1, and the SEQ ID NO. 2 and the SEQ ID NO. 1 are specifically as follows:

[0082] SEQ ID NO. 1:

[0083]

[0084] SEQ ID NO.2:

[0085] MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSFSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPESDPQKSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPALALMPPQYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA.

[0086] In the present application, the carbonyl reductase is a catalyst which can be added in the form of an enzyme or in the form of a microbial cell expressing the enzyme. In the present application, the carbonyl reductase is a carbonyl reductase with high stereoselectivity and high activity for the compound of formula III, which is screened from a large number of enzyme libraries from microbial sources, derived from Sporobolomyces salmonicolor, and used for catalyzing the preparation of the compound of formula IV from the compound of formula III, with high product concentration, high atom economy, simple operation, easy scale-up and easy separation and purification of the product, having a good industrial application prospect.

[0087] In the present application, the reduced coenzyme is preferably NADPH, which is preferably generated by a coenzyme regeneration system, i.e. the regeneration of NADPH is carried out at the same time as the biocatalytic reduction reaction; the coenzyme regeneration system is preferably any one of the following: 1) a glucose dehydrogenase regeneration system with glucose as a coenzyme regeneration substrate and glucose dehydrogenase as a coenzyme regeneration enzyme, containing NADPH or NADP + ; 2) an alcohol dehydrogenase regeneration system with isopropanol as a coenzyme regeneration substrate and alcohol dehydrogenase as a coenzyme regeneration enzyme, containing NADPH or NADP + ; 3) a formate dehydrogenase regeneration system with formate as a coenzyme regeneration substrate and formate dehydrogenase as a coenzyme regeneration enzyme, containing NADPH or NADP + . The addition of coenzyme NADP + in the reaction system can promote the reduction reaction, NADP+ The function of the coenzyme regeneration enzyme is to scavenge electrons as an oxidizing agent, and the coenzyme regeneration enzyme reduces NADP + to NADPH using a coenzyme regeneration substrate such as the above-mentioned isopropanol, glucose or formate, to generate sufficient NADPH as a reducing agent for biosynthesis, thereby facilitating the reduction reaction. The present application does not have any particular requirements for the glucose dehydrogenase, alcohol dehydrogenase and formate dehydrogenase, and corresponding enzymes well known to those skilled in the art can be used. In the examples of the present application, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO. 3, and is as follows:

[0088] MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG.

[0089] In the present application, the NADPH provides a hydrogen source and determines the stereoconfiguration of the product, and the carbonyl reductase determines the catalytic efficiency and stereoselectivity.

[0090] In the present application, the coenzyme regeneration enzyme, i.e. the glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase, can be added as an additive, or can be expressed together with the carbonyl reductase and exist simultaneously. When the carbonyl reductase and the coenzyme regeneration enzyme are co-expressed in the same microbial cell, a co-expression recombinant cell that clones the genes of the carbonyl reductase and the coenzyme regeneration enzyme is first constructed, and the two enzymes can be simultaneously and proportionally provided by simple fermentation of the microorganism, without the need for proportion adjustment according to the enzyme activities of the two enzymes, which is the advantage of co-expressing the two enzymes.

[0091] The carbonyl reductase of the present application has 343 amino acids and a clear structure, so that the coding gene, expression cassette and plasmid containing the gene, and transformant containing the plasmid can be easily obtained by those skilled in the art. These genes, expression cassettes, plasmids and transformants can be obtained by gene engineering construction methods known to those skilled in the art. For example, the nucleic acid sequence of SEQ ID NO: 1 can be connected to various commercially available conventional plasmid vectors to construct the plasmid. The plasmid is preferably a pET sequence plasmid, and more preferably a plasmid pET-28a(+). The recombinant expression plasmid can be prepared by the following method: the nucleic acid product obtained by PCR amplification and the expression vector pET-28a are digested with restriction endonuclease BamH I and Xho I respectively to form complementary cohesive ends, and then connected by T4 DNA ligase to form the recombinant expression plasmid pET-28a-SSCR (SSCR is carbonyl reductase) of the carbonyl reductase gene.

[0092] The recombinant expression plasmid pET-28a-SSCR can be transformed into a suitable host cell by conventional methods in the art, such as heat shock method, electroporation method, chemical method, etc., for example, into E. coli BL21 (DE3) competent cells. The preferred embodiment of the heat shock method is as follows: mix the plasmid solution with the competent cells, heat shock at 42°C for 45s, then ice bath for 2min, and then recover at 37°C for 1h, and then spread on LB agar medium containing kanamycin, and culture to obtain the recombinant expression transformant E. coli BL21 (DE3) / pET-28a-SSCR.

[0093] When the carbonyl reductase is co-expressed with a coenzyme regenerating enzyme such as glucose dehydrogenase (e.g. BmGDH), the two recombinant expression plasmids should be able to stably replicate by themselves, and the carbonyl reductase and coenzyme regenerating enzyme carried by them can be effectively expressed. Therefore, the recombinant expression plasmid pACYCDuet-1-BmGDH is also constructed, and the two recombinant plasmids pET-28a-SSCR and pACYCDuet-1-BmGDH are separately transformed into host cells, or the two recombinant plasmids pET-28a-SSCR and pACYCDuet-1-BmGDH are simultaneously transformed into E. coli BL21 (DE3) competent cells to obtain the co-expression recombinant bacteria E. coli BL21 (DE3) / pACYCDuet-1-BmGDH / pET-28a-SSCR which clones the carbonyl reductase and coenzyme regenerating enzyme genes.

[0094] When used as a biocatalyst for preparing the compound of formula IV, the carbonyl reductase of the present application can be in the form of an enzyme or in the form of a bacteria, wherein the enzyme form includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier immobilized enzyme, etc.; and the bacteria form includes viable bacteria (resting cells) and dead bacteria.

[0095] As is easily understood by those skilled in the art, the microbial cell contains coenzyme NADPH / NADP + (Nicotinamide adenine dinucleotide phosphate, coenzyme II), NADH / NAD + (Nicotinamide adenine dinucleotide, coenzyme I) is itself a natural form of enzyme immobilization and can be used as an enzyme preparation for catalyzing reactions without the need for disruption, even extraction and purification. Since the reaction substrate and reaction product can easily pass through the biological barrier of the cell membrane, the microbial cell does not need to be disrupted, which is economically advantageous. On the other hand, compared to the catalysis of isolated enzymes, the present application can provide a continuous and inexhaustible supply of enzymes or microbial cells through simple fermentation of microorganisms, without the need for further extraction, purification, and separation of enzymes, which is economically advantageous and creates conditions for industrial application.

[0096] In the present application, the concentration of the phosphate buffer is preferably 100 mM, and the pH is preferably 7.4.

[0097] In the present application, the temperature of the biocatalytic reduction reaction is preferably 25-35°C, which can be 25, 30, or 35°C, and the time is preferably 12-24 h, which can be 12, 15, 18, 20, or 24 h. The biocatalytic reduction reaction is preferably carried out at a pH of 6.5-7.5, which can be 6.5, 7, or 7.4.

[0098] In the present application, the biocatalytic reduction reaction is carried out after mixing the ogilaston intermediate of the structure shown in Formula III, the carbonyl reductase, the coenzyme regeneration system, and the phosphate buffer, and adjusting the pH of the resulting mixture to 6.5-7.5 by adding 10 wt% sodium hydroxide solution dropwise. In the present application, the mixing is preferably carried out at 25-35°C with stirring. The concentration of the ogilaston intermediate of the structure shown in Formula III in the mixture is preferably 70-140 g / L, which can be 80, 100, 110, 120, 130, or 140 g / L, and the concentration of the carbonyl reductase is preferably 14-28 g / L, which can be 15, 20, 25, or 28 g / L.

[0099] In the present application, the reaction formula involved in the biocatalytic reduction reaction is as follows:

[0100]

[0101] In the present application, after the biocatalytic reduction reaction is completed, the obtained reaction solution is preferably subjected to post-treatment, which preferably comprises the following steps: under ice-bath, hydrochloric acid is added to the obtained reaction solution to adjust the pH value to 2-3, then activated carbon is added for adsorption for 40 min, filtration is performed to obtain a filtrate; the filtrate is extracted with ethyl acetate, the organic layer is washed with saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and concentrated to dryness; then the obtained concentrate is recrystallized with toluene to obtain the alogliptin intermediate of the structure shown in formula IV.

[0102] In the present application, the carbonyl reductase is used to perform a high stereoselectivity reaction to prepare the alogliptin intermediate of the structure shown in formula IV, and chiral pure product can be obtained through simple post-treatment, and industrial production can be realized.

[0103] The present application provides an alogliptin intermediate, which has the structure shown in formula V:

[0104]

[0105] In formula V, R is an alkyl group or a substituted alkyl group, the substituent group of the substituted alkyl group comprises an aryl group or a substituted aryl group; the number of carbon atoms of the alkyl group in the alkyl group and the substituted alkyl group is 1-6.

[0106] In the present application, R in the structure shown in formula V is consistent with R in the alogliptin intermediate of the structure shown in formula III.

[0107] The present application provides a preparation method of the alogliptin intermediate as described in the above technical solution, which comprises the following steps:

[0108] The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction to obtain the alogliptin intermediate of the structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0109]

[0110] The alogliptin intermediate of the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer solution are mixed to perform a biocatalytic reduction reaction to obtain the alogliptin intermediate of the structure shown in formula IV;

[0111]

[0112] The alogliptin intermediate of the structure shown in formula IV, an alkaline compound and a second organic solvent are mixed to perform a ring-closing reaction to obtain the alogliptin intermediate of the structure shown in formula V.

[0113] In the present application, the Friedel-Crafts acylation reaction and the biocatalytic reduction reaction are consistent with the above technical solutions, and will not be described here.

[0114] In the present application, the basic compound preferably includes one or more of potassium tert-butoxide, sodium ethoxide, sodium methoxide and sodium hydride; the molar ratio of the alogliptin intermediate of formula IV to the basic compound is preferably 1:1.2-1:3, and can be 1:1.2, 1:1.5, 1:2, 1:2.5 or 1:3.

[0115] In the present application, the method for mixing the alogliptin intermediate of formula IV, the basic compound and the second organic solvent is preferably as follows: under nitrogen protection, the alogliptin intermediate of formula IV and the basic compound are sequentially added into a reaction bottle containing the second organic solvent.

[0116] In the present application, the temperature of the ring-closing reaction is preferably 20-50℃, and can be 30, 40 or 50℃, and the time is preferably 3-6h, and can be 3, 4, 5 or 6h; the ring-closing reaction is preferably carried out under stirring.

[0117] In the present application, the reaction formula involved in the ring-closing reaction is as follows:

[0118]

[0119] After the ring-closing reaction is completed, the present application preferably carries out post-treatment on the obtained reaction liquid, and the method for post-treatment is preferably as follows: methanol is added into the reaction liquid for quenching, the obtained reaction system is concentrated to dryness, dichloromethane is added for slurry, filtration is carried out, the filtrate is concentrated to dryness, and the alogliptin intermediate of formula V is obtained.

[0120] The present application provides a method for preparing an alogliptin key intermediate, the alogliptin key intermediate having a structure shown in formula I, comprising the following steps:

[0121] The compound of formula II, the acylation reagent, the catalyst and the first organic solvent are mixed to carry out Friedel-Crafts acylation reaction, and the alogliptin intermediate of formula III is obtained; the acylation reagent is 3-chloropropionyl chloride or 3-chloropropionic anhydride;

[0122]

[0123] The ogilator intermediate of the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to carry out a biocatalytic reduction reaction, so as to obtain an ogilator intermediate of the structure shown in formula IV;

[0124]

[0125] The ogilator intermediate of the structure shown in formula IV, a basic compound and a second organic solvent are mixed to carry out a ring closure reaction, so as to obtain an ogilator intermediate of the structure shown in formula V;

[0126]

[0127] The ogilator intermediate of the structure shown in formula V, a Lewis acid, a Grignard reagent and a third organic solvent are mixed to carry out an opening ring reaction, so as to obtain a compound shown in formula VI;

[0128] The compound shown in formula VI, an acid and a fourth organic solvent are mixed to carry out a ring formation reaction, and then the obtained reaction system is mixed with water to carry out an ester hydrolysis reaction, so as to obtain a compound shown in formula VII;

[0129] The compound shown in formula VII is activated by oxalyl chloride, and then condensed with N-methylaniline, so as to obtain an ogilator key intermediate of the structure shown in formula I;

[0130]

[0131] In the present application, the Friedel-Crafts acylation reaction, the biocatalytic reduction reaction and the ring closure reaction are the same as those in the technical scheme, and will not be repeated here.

[0132] The ogilator intermediate of the structure shown in formula V, a Lewis acid, a Grignard reagent and a third organic solvent are mixed to carry out an opening ring reaction, so as to obtain a compound shown in formula VI.

[0133] In the present application, the Lewis acid preferably includes one or more of aluminum chloride, boron trifluoride ether, tin chloride, zinc chloride, cuprous bromide and ferric chloride; the Grignard reagent includes 2-methylallyl magnesium chloride and / or 2-methylallyl magnesium bromide; the substance amount ratio of the ogilator intermediate of the structure shown in formula V, the Lewis acid and the Grignard reagent is preferably 1:0.1:(1.5-3), and more preferably 1:0.1:(1.5-2). In the present application, the third organic solvent preferably includes one or more of tetrahydrofuran, toluene and 2-methyltetrahydrofuran, and the present application does not have special requirements for the amount of the third organic solvent, which can only ensure that the raw materials are dissolved and the reaction is carried out smoothly.

[0134] In the present application, the method for mixing the alogliptin intermediate of the structure shown in formula V, the Lewis acid, the Grignard reagent and the third organic solvent is preferably as follows:

[0135] Under nitrogen protection, the alogliptin intermediate of the structure shown in formula V and the Lewis acid are sequentially added into a reaction bottle containing the third organic solvent, and the obtained system is stirred at 0-10℃ for 30 min; then the solution of the Grignard reagent is added dropwise thereinto; the solution of the Grignard reagent is obtained by dissolving the Grignard reagent in the third organic solvent.

[0136] In the present application, the temperature of the ring-opening reaction is preferably 0-20℃, more preferably 0-10℃, and the time is preferably 1.5-3h, which can be 2, 2.5 or 3h; the ring-opening reaction is preferably carried out under stirring.

[0137] In the present application, the reaction formula involved in the ring-opening reaction is as follows:

[0138]

[0139] After the ring-opening reaction is completed, the obtained reaction liquid is preferably subjected to post-treatment in the present application, and the method for the post-treatment is preferably as follows: ice water is added into the obtained reaction liquid, most of the organic solvent is recovered by concentration under reduced pressure to obtain a concentrated liquid; the concentrated liquid is diluted and extracted with ethyl acetate, the organic layer is washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, concentrated to dryness to obtain the compound shown in formula VI.

[0140] After the compound shown in formula VI is obtained, the compound shown in formula VI, an acid and a fourth organic solvent are mixed for ring-closing reaction in the present application, and the obtained reaction system is mixed with water for ester hydrolysis to obtain the compound shown in formula VII.

[0141] In the present application, the acid preferably includes one or more of p-toluenesulfonic acid, methanesulfonic acid, sulfuric acid, hydrochloric acid and trifluoroacetic acid; and the ratio of the amount of substance of the compound shown in formula VI to the acid is preferably 1:2-1:3, wherein the sulfuric acid is calculated as H2SO4 and the hydrochloric acid is calculated as HCl. In the present application, the fourth organic solvent preferably includes one or more of cyclopentyl methyl ether, toluene, methyl tert-butyl ether, tetrahydrofuran, 2-methyl tetrahydrofuran, dichloromethane and 1,4-dioxane; and the amount of the fourth organic solvent is not particularly required in the present application, as long as the raw material can be dissolved and the reaction can be smoothly carried out.

[0142] In the present application, the method for mixing the compound shown in formula VI, the acid and the fourth organic solvent is preferably as follows: the compound shown in formula VI and the acid are sequentially added into a reaction bottle containing the fourth organic solvent.

[0143] In the present application, the temperature of the ring formation reaction is preferably 60-70°C, and can be 60, 65 or 70°C, and the time is preferably 10-12h, and can be 10, 11 or 12h. After the ring formation reaction is completed, water is added to the obtained reaction system to perform an ester group hydrolysis reaction. In the present application, the temperature of the ester group hydrolysis reaction is preferably 60-70°C, and can be 60, 65 or 70°C, and the time is preferably 6-10h. In the present application, the ring formation and ester group hydrolysis reactions are preferably performed under stirring. This step is a process in which the hydroxyl group on the compound shown in formula VI attacks the olefinic bond to form a six-membered ring (nucleophilic addition ring closure reaction), and then the ester group is hydrolyzed.

[0144] In the present application, the reaction formula involved in the ring formation and ester group hydrolysis reactions is as follows:

[0145]

[0146] In the present application, after the ring formation and ester group hydrolysis reactions are completed, the obtained reaction solution is preferably subjected to post-treatment. The method of the post-treatment is preferably as follows: the pH value of the obtained reaction solution is adjusted to 8-9 with liquid alkali, the solution is separated, the aqueous phase is adjusted to a pH value of 2-3 with hydrochloric acid, and then extracted with dichloromethane, the organic layer is washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, concentrated to dryness, and the compound shown in formula VII is obtained.

[0147] After the compound shown in formula VII is obtained, the compound shown in formula VII is activated with oxalyl chloride in the present application, and then subjected to condensation reaction with N-methylaniline to obtain the key intermediate of olendron shown in formula I.

[0148] In the present application, the molar ratio of the compound shown in formula VII, oxalyl chloride and N-methylaniline is preferably 1:(1.1-1.5):(1.1-1.5), and can be 1:1.25:1.25.

[0149] In the present application, the specific operation of the activation is preferably as follows: the compound shown in formula VII, the fifth organic solvent and oxalyl chloride are sequentially added to a reaction bottle, stirred at room temperature, and then a solution of N,N-dimethylformamide is added dropwise, and after the dropping is completed, stirring is continued for 45 min. In the present application, the fifth organic solvent preferably includes one or more of acetonitrile, tetrahydrofuran and dichloromethane; the solution of N,N-dimethylformamide is a solution of N,N-dimethylformamide in the fifth organic solvent, and N,N-dimethylformamide is an aprotic solvent, which can reduce the hydrolytic damage of trace water in the system or the environment to acyl chloride.

[0150] In the present application, the temperature of the condensation reaction is preferably 0-40°C, more preferably 0-10°C, and the time is preferably 2-4h, which can be 2, 3 or 4h. The temperature of the activated system is preferably adjusted to 0-40°C, and N-methylaniline is added to the system for the condensation reaction.

[0151] In the present application, the reaction formula involved in the condensation reaction is as follows:

[0152]

[0153] In the present application, after the condensation reaction is completed, the obtained reaction solution is preferably subjected to post-treatment, which is preferably as follows: triethylamine is added to the obtained reaction solution, stirring is performed for 2h, water is added to the obtained system, stirring is performed at room temperature for 2h, and then filtration is performed, the filter cake is sequentially washed with acetonitrile and water, and drying is performed in an oven at 50-80°C to obtain the key intermediate of alogliptin with the structure shown in formula I.

[0154] The overall reaction route for preparing the key intermediate of alogliptin with the structure shown in formula I according to the present application is as follows:

[0155]

[0156] The method for preparing the key intermediate of alogliptin provided by the present application uses indole-2-carboxylate compounds as starting materials, which are easy to obtain and low in cost, and the preparation process is simple and easy to operate, the reaction conditions are mild, the atom economy is high, the use of expensive palladium and ligands is avoided, it is green, economical and environmentally friendly, the cost is greatly reduced, heavy metal pollution is avoided, and it is suitable for industrial production. The preparation method provided by the present application has the advantages of easy-to-obtain raw materials, low cost and environmental friendliness, and the key intermediate of alogliptin prepared by the method provided by the present application has high yield and purity.

[0157] In order to further illustrate the present application, the key intermediate of alogliptin and the method for preparing the key intermediate of alogliptin provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0158] Materials and methods in the examples:

[0159] The whole gene synthesis, primer synthesis and sequencing in the examples are completed by GenScript Biotech Corporation.

[0160] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, preparation of competent cells, transformation and the like, are mainly performed according to the Molecular Cloning Laboratory Manual (3rd Edition, J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translated, Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined through simple tests.

[0161] PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple test.

[0162] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium plus 20 g / L agar powder).

[0163] In the examples, the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 2, and the nucleic acid sequence of the encoding gene is shown in SEQ ID NO. 1; the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO. 3.

[0164] Preparation of the compound of formula III-1 in Example 1

[0165]

[0166] A reaction bottle was added with 53.2 g of anhydrous aluminum chloride (0.4 mol) and 500 mL of 1,2-dichloroethane, and ice bathed under nitrogen protection, 50.4 g of 3-chloropropionyl chloride (0.4 mol) was added. After 5 min of continued stirring under heating, 37.8 g of the compound of formula II-1 (0.2 mol) was added in batches, and the reaction was carried out for 3 h after heating to reflux. The reaction liquid was added dropwise into ice water for quenching, extracted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the compound of formula III-1 (36.2 g, yield 65%).

[0167] 1 H-NMR (400M, DMSO-d6): δ 12.25 (s, 1H), 8.39 (ddd, 1H), 7.79-7.88 (dd, 1H), 7.45-7.53 (m, 1H), 7.24-7.28 (m, 1H), 4.25-4.36 (q, 2H), 3.83-3.94 (t, 2H), 3.55-3.47 (t, 2H), 1.26-1.33 (t, 3H); 13 C-NMR (101M, DMSO-d6): δ 196.6, 161.47, 140.27, 129.80, 129.75, 126.74, 125.15, 124.36, 113.22, 110.01, 61.25, 40.92, 40.34, 14.75.

[0168] Figure 1 and Figure 2 are respectively the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum of the compound of formula III-1.

[0169] Preparation of recombinant expression transformant of carbonyl reductase

[0170] The recombinant plasmid pET-28a-SSCR (SSCR is carbonyl reductase) of carbonyl reductase obtained by biosynthesis of GenScript was transformed into E. coli DH5α competent cells, spread on LB medium plates containing 50 μg / mL kanamycin, and incubated at 37°C for 8 h. Single colonies were picked for colony PCR verification, and after sequencing verification, the corresponding plasmid was extracted and further transformed into E. coli BL21. Positive clones were picked to obtain the recombinant expression transformant E. coli BL21(DE3) / pET-28a-SSCR.

[0171] According to the same method as described above, the recombinant expression plasmid E. coli BL21(DE3) / pACYCDuet-1-BmGDH of glucose dehydrogenase was constructed using the plasmid pACYCDuet-1-BmGDH (BmGDH is glucose dehydrogenase).

[0172] Preparation of carbonyl reductase and glucose dehydrogenase co-expression cells

[0173] After 100 μL of competent E. coli BL21 was placed in an ice box for 15 min, 1 μL of recombinant plasmid pET-28a-SSCR and 1 μL of recombinant plasmid pACYC Duet-1-BmGDH were added to the E. coli BL21 in a sterile operation table. After continuing to stand in the ice box for 30 min, it was immediately placed in an ice box for 2 min after heat shock at 42°C for 45 s in a constant temperature water tank. Then, 500 μL of LB medium was added in a sterile operation table, and then incubated at 37°C for 1 h. After centrifugation for 2 min, a small amount of supernatant was retained, mixed by a pipette gun, and then spread on solid medium. Then, it was incubated in a 37°C biochemical incubator for 12 h. Positive clones were picked to obtain carbonyl reductase SSCR and glucose dehydrogenase BmGDH co-expression cells.

[0174] Induced expression of carbonyl reductase SSCR

[0175] The recombinant expression transformant obtained by construction in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin, and incubated at 37°C, 220 rpm on a shaking table. When the OD 600 of the culture solution reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM for induction. After 18 h of induction at 18°C, the culture solution was centrifuged at a speed of 8500 rpm, the cells were collected, and washed with physiological saline to obtain resting cells.

[0176] Preparation of compound of structural formula IV-1

[0177]

[0178] The co-expression cell of carbonyl reductase SSCR and glucose dehydrogenase BmGDH obtained in Example 3 was cultured and wet bacteria were collected according to the method of Example 4.

[0179] 5.6 g of wet bacteria were suspended in 200 mL of phosphate buffer (100 mM, pH 7.4), 28 g (0.1 mol) of compound of structural formula III-1, 0.4 g of coenzyme NADP + , 1.5 M of glucose, and 10 wt% sodium hydroxide solution were added in sequence under stirring at 30°C, the pH value of the reaction solution was maintained at 7.4, the reaction progress was monitored by HPLC, and the reaction was completed after 18 h. The pH value was adjusted to 2.5 by adding hydrochloric acid under ice bath, activated carbon was added for adsorption for 40 min, filtration was performed, the filtrate was extracted with ethyl acetate, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated to dryness, recrystallized from toluene, and compound of structural formula IV-1 (26.6 g, yield 95%) was obtained.

[0180] 1 H-NMR (400M, CDCl3): δ 9.32 (s, 1H), 8.38 (ddd, 1H), 7.95-8.05 (m, 1H), 7.43-7.49 (m, 1H), 7.30-7.34 (m, 1H), 3.91-3.96 (m, 1H), 3.72-3.78 (m, 2H), 3.03-3.12 (q, 2H), 1.83-1.87 (m, 2H), 1.22-1.28 (t, 3H); 13 C-NMR (101M, CDCl3): δ 162.15, 139.16, 130.67, 128.74, 127.02, 125.20, 124.52, 112.04, 110.30, 68.08, 52.37, 31.72, 25.70, 8.68.

[0181] Figure 3 and Figure 4 are respectively the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum of compound of structural formula IV-1.

[0182] Preparation of compound of structural formula V-1

[0183]

[0184] Into a three-necked flask containing 100 mL of tetrahydrofuran, under nitrogen protection, 26.6 g of compound shown in formula IV-1 (94 mmol) and 12.6 g of potassium tert-butoxide (113 mmol) were sequentially added, and the system was stirred at 50°C for 6 h after 10 mL of methanol was added to quench the reaction. The reaction system was concentrated to dryness, slurried with dichloromethane, filtered, and the filtrate was concentrated to dryness to obtain the compound of structural formula V-1 (20.3 g, yield 88%).

[0185] Example 7 Preparation of compound of structural formula VI-1

[0186]

[0187] Into a three-necked flask containing 250 mL of tetrahydrofuran, under nitrogen protection, 20.3 g of compound shown in formula V-1 (83 mmol) and 1.2 g of cuprous bromide (8.3 mmol) were sequentially added, and the system was stirred at 0°C for 30 min. Then 250 mL of 0.5M 2-methylallyl magnesium chloride in tetrahydrofuran (125 mmol) was added dropwise through an addition funnel, and after the addition was completed, the reaction system was continuously stirred for 3 h, and then ice water was added to the reaction system, most of the tetrahydrofuran was recovered by concentration under reduced pressure, and the concentrated solution was diluted with ethyl acetate for extraction, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the compound of structural formula VI-1 (18.2 g, yield 72%).

[0188] Example 8 Preparation of compound of structural formula VII

[0189]

[0190] Into a three-necked flask containing 50 mL of cyclopentyl methyl ether, 18.2 g of compound shown in formula VI-1 (60 mmol) and 25.8 g of p-toluenesulfonic acid (150 mmol) were sequentially added, and the system was stirred at 70°C for 12 h. Then 10 mL of water was added, and the stirring was continued for 6 h. Then the pH value of the system was adjusted to 8.5 with liquid alkali, and the mixture was separated, the aqueous phase was adjusted to pH 2.5 with hydrochloric acid, and extracted with dichloromethane, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the compound of structural formula VII (15.2 g, yield 93%).

[0191] Example 9 Preparation of compound of structural formula I

[0192]

[0193] The reaction bottle was added with 15.2 g of the compound shown in formula VII (56 mmol), 120 mL of acetonitrile and 8.9 g of oxalyl chloride (70 mmol) successively, and stirred at room temperature. 2 g of N,N-dimethylformamide was added dropwise in 10 mL of acetonitrile, and after the dropping was completed, the stirring was continued for 45 min. The system was cooled to 0°C, 7.5 g of N-methylaniline (70 mmol) was added, and the reaction was continued at 0°C for 2 h. 14.6 g of triethylamine (145 mmol) was added, and stirred for 2 h. 20 mL of water was added to the system, and after stirring at room temperature for 2 h, filtration was performed. The filter cake was washed successively with acetonitrile and water, and dried in an oven at 50°C to obtain the compound shown in formula I (18 g, yield 89%).

[0194] 1 H-NMR (400M, DMSO-d6): δ 11.50 (br s, 1H), 7.40-7.55 (3H, m), 7.27-7.39 (m, 3H), 7.09 (m, 1H), 7.00-7.07 (m, 1H), 5.20 (br s, 1H), 3.38 (s, 3H), 2.78-2.93 (m, 1H), 0.7-1.78 (m, 12H); 13 C-NMR (101M, DMSO-d6): δ 162.04, 144.89, 137.97, 134.91, 130.67, 130.22, 128.47, 128.33, 127.40, 123.95, 118.93, 112.55, 106.03, 71.78, 61.37, 44.98, 38.96, 37.09, 34.24, 32.13.

[0195] Figure 5 and Figure 6 are respectively the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum of the compound shown in formula I.

[0196] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. It should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An intermediate of ostarolrøn, characterized by, has a structure shown in formula III: In formula III, R is alkyl or substituted alkyl, and the substituent of the substituted alkyl includes aryl or substituted aryl; the number of carbon atoms in the alkyl and the alkyl of the substituted alkyl is 1-6.

2. The process for the preparation of an intermediate of Origluron according to claim 1, characterized in that, The method comprises the following steps: a compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain an alogliptin intermediate with a structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride; In formula II, R is alkyl or substituted alkyl, and the substituent of the substituted alkyl includes aryl or substituted aryl; the number of carbon atoms in the alkyl and the alkyl of the substituted alkyl is 1-6.

3. The preparation method according to claim 2, characterized in that, The catalyst includes one or more of aluminum trichloride, iron trichloride, zinc chloride and boron trifluoride etherate; the first organic solvent includes dichloromethane and / or 1,2-dichloroethane; the substance amount ratio of the compound shown in formula II, the acylating agent and the catalyst is 1:(1.1-3):(1.1-3); the temperature of the Friedel-Crafts acylation reaction is 0-80℃, and the time is 0.5-3h.

4. An intermediate of ostarolrøn, characterized by, has a structure shown in formula IV: In formula IV, R is alkyl or substituted alkyl, and the substituent of the substituted alkyl includes aryl or substituted aryl; the number of carbon atoms in the alkyl and the alkyl of the substituted alkyl is 1-6.

5. The process for the preparation of an intermediate of olgrifedone according to claim 4, characterized by, The method comprises the following steps: a compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain an alogliptin intermediate with a structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride; The alogliptin intermediate with the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction, so as to obtain an alogliptin intermediate with a structure shown in formula IV.

6. The production method according to claim 5, wherein The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 2, and the nucleic acid sequence of the gene encoding the carbonyl reductase is shown in SEQ ID NO. 1; the reduced coenzyme is NADPH; the temperature of the biocatalytic reduction reaction is 25-35℃, and the time is 12-24h; the biocatalytic reduction reaction is performed under the condition that the pH value is 6.5-7.

5.

7. An intermediate of ostarolrøn, characterized by, has a structure shown in formula V: In formula V, R is alkyl or substituted alkyl, and the substituent of the substituted alkyl includes aryl or substituted aryl; the number of carbon atoms in the alkyl and the alkyl of the substituted alkyl is 1-6.

8. A process for the preparation of an intermediate of olgrifedone according to claim 7, characterized by, The method comprises the following steps: a compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction, so as to obtain an alogliptin intermediate with a structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride; The alogliptin intermediate with the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction, so as to obtain an alogliptin intermediate with a structure shown in formula IV; The alogliptin intermediate with the structure shown in formula IV, an alkaline compound and a second organic solvent are mixed to perform a ring-closing reaction, so as to obtain an alogliptin intermediate with a structure shown in formula V.

9. The production method according to claim 8, characterized by, The basic compound includes one or more of potassium tert-butoxide, sodium ethoxide, sodium methoxide and sodium hydride; the second organic solvent includes one or more of tetrahydrofuran, toluene and 2-methyltetrahydrofuran; the molar ratio of the alogliptin intermediate of the structure shown in formula IV to the basic compound is 1:1.2-1:3; the temperature of the ring-closing reaction is 20-50°C, and the time is 3-6h.

10. A process for the preparation of a key intermediate of ostarol, said key intermediate of ostarol having the structure shown in Formula I, characterized in that, ###00003### Formula I The method comprises the following steps: The compound shown in formula II, an acylating agent, a catalyst and a first organic solvent are mixed to perform a Friedel-Crafts acylation reaction to obtain an alogliptin intermediate of the structure shown in formula III; the acylating agent is 3-chloropropionyl chloride or 3-chloropropionic anhydride; The alogliptin intermediate of the structure shown in formula III, a carbonyl reductase, a reduced coenzyme and a phosphate buffer are mixed to perform a biocatalytic reduction reaction to obtain an alogliptin intermediate of the structure shown in formula IV; The alogliptin intermediate of the structure shown in formula IV, a basic compound and a second organic solvent are mixed to perform a ring-closing reaction to obtain an alogliptin intermediate of the structure shown in formula V; The alogliptin intermediate of the structure shown in formula V, a Lewis acid, a Grignard reagent and a third organic solvent are mixed to perform an opening reaction to obtain a compound shown in formula VI; the Grignard reagent includes 2-methylallyl magnesium chloride and / or 2-methylallyl magnesium bromide; The compound shown in formula VI, an acid and a fourth organic solvent are mixed to perform a ring-forming reaction, and then the obtained reaction system is mixed with water to perform an ester hydrolysis reaction to obtain a compound shown in formula VII; The compound shown in formula VII is activated by oxalyl chloride and then condensed with N-methylaniline to obtain an alogliptin key intermediate of the structure shown in formula I;

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