Semi-synthesis preparation method of canagliflozin

Abstract

The invention relates to the field of biological medicine preparation, in particular to a semi-synthesis preparation method of canagliflozin, which comprises the following steps: (1) under the condition that the pH value is 9.0-11.5, carrying out selective protection on lysine epsilon amino of a canagliflozin intermediate 36AA to obtain Boc-protected 36 peptide (BoC-36AA); (2) under the condition that the pH (Potential of Hydrogen) is 8.5 to 9.0, carrying out a reaction on side chain Glu-eicosandioic acid and the lysine alpha-amino group of the Boc-36AA to obtain 36-AA (SC-36AA) with a side chain; (3) carrying out a reaction on the SC-36AA and prolinamide, so as to obtain a canagliflozin precursor; and (4) cracking the canagliflozin precursor to obtain the canagliflozin. The preparation method is simple to operate, low in cost, high in yield and high in purity, the total reaction yield is more than 56%, and the HPLC (high performance liquid chromatography) purity is more than or equal to 99.6%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological drug preparation, and in particular to a semi-synthetic preparation method of cagrilintide. BACKGROUND

[0002] Cagrilintide is a new long-acting acylated amylin analogue, which is a non-selective amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) agonist. Cagrilintide is a cyclic polypeptide composed of 38 amino acids, containing a pair of disulfide bonds. Cagrilintide can reduce energy intake, regulate food selection and preference, and has glucose regulation effect by co-secretion with insulin, inhibit postprandial glucagon release, and delay gastric emptying. It has obvious advantages in the treatment of diabetes, obesity, metabolic syndrome and cardiovascular diseases, and has wide application prospect.

[0003] At present, the preparation method of cagrilintide is a chemical total synthesis method, including solid phase synthesis method (SPPS) or solid-liquid phase combination (SPPS-LPPS) fragment method, such as CN117986347A and CN119350469A. CN117986347A discloses a synthesis method of cagrilintide, which takes amino resin as starting resin, and sequentially couples single amino acid, and finally connects the side chain (eicosanedioic acid) on Glu. The total yield of cagrilintide is 31.87%. CN119350469A discloses a method for synthesizing cagrilintide by SPPS-LPPS mixed method of large fragments, which includes cutting cagrilintide into four polypeptide fragments, sequence 1-14 is fragment I, sequence 15-26 is fragment II, sequence 27-35 is fragment III, and sequence 36-39 is fragment IV; the protected amino acid monomers corresponding to the sequence positions are sequentially coupled by SPPS to synthesize fully protected fragments I, II and III, and fragment IV is synthesized by solid phase or liquid phase; then the fragments are connected by liquid phase synthesis method (LPPS), and the fully protected cagrilintide is obtained by repeating condensation / Fmoc removal reaction, and the total yield is about 45%. The above prior art has defects such as long reaction steps, difficult impurity control, low total yield, high preparation cost, and difficult industrial production. SUMMARY

[0004] In order to overcome the above-mentioned defects in the prior art, the present application provides a semi-synthetic preparation method of cagrilintide, which is simple in operation, low in cost, high in total yield, high in purity and easy to industrialize.

[0005] The present application provides a semi-synthetic preparation method of cagrilintide, which comprises the following steps: (1) The lysine epsilon amino group of the intermediate 36AA of carmegliptin is selectively protected under the condition of pH 9.0-11.5 to obtain Boc-36AA, the reaction formula is shown in the following reaction formula I: Reaction formula I; (2) The side chain Glu-eicosadioic acid reacts with the lysine alpha amino group of the Boc-36AA under the condition of pH 8.5-9.0 to obtain SC-36AA, the reaction formula is shown in the following reaction formula II: Reaction formula II; (3) The SC-36AA reacts with prolyl amide to obtain the precursor of carmegliptin, the reaction formula is shown in the following reaction formula III: Reaction formula III; (4) The precursor of carmegliptin is cleaved to obtain carmegliptin, the reaction formula is shown in the following reaction formula IV: Reaction formula IV.

[0006] Preferably, the 36AA is prepared by a biosynthetic method, the method comprising preparing a recombinant fusion protein comprising 36AA, and enzymatically cutting the recombinant fusion protein to obtain the 36AA, wherein the recombinant fusion protein is SEQ ID NO. 1.

[0007] Preferably, the pH of step (1) is 9.5-10.0.

[0008] Preferably, the molar ratio of the 36AA to the (Boc)2O is 1.0:1.0-1.0:1.5, the molar ratio of the 36AA to the side chain Glu-eicosadioic acid is 1.0:1.0-1.0:1.5, and the molar ratio of the 36AA to the prolyl amide is 1.0:1.0-1.0:1.5.

[0009] More preferably, the molar ratio of the 36AA to the (Boc)2O is 1.0:1.1, the molar ratio of the 36AA to the side chain Glu-eicosadioic acid is 1.0:1.2, and the molar ratio of the 36AA to the prolyl amide is 1.0:1.2.

[0010] Preferably, the semi-synthetic preparation method comprises a reaction solvent, a base and a condensing agent; The reaction solvent comprises one or more of water, DMF, acetonitrile, dioxane, DMSO and DMAC; The base includes one or more of inorganic bases including sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium carbonate, sodium bicarbonate, disodium hydrogen phosphate and potassium bicarbonate, and organic bases including one or more of triethylamine, diisopropylethylamine, collidine and pyridine; The condensing agent includes one or more of HBTU, TCFH, HCTU, HOSU, HOBT, HOAT, COMU, HATU, PyBop, EDCI, Oxyma and PyOxim.

[0011] More preferably, the reaction solvent is water and DMF, the inorganic base is sodium carbonate, the organic base is diisopropylethylamine, and the condensing agent is COMU or HATU.

[0012] Preferably, the step (4) is carried out by using a cleavage solution prepared from trifluoroacetic acid, triisopropylsilane and water, and the volume ratio of the trifluoroacetic acid, triisopropylsilane and water is 20:2:1.

[0013] Preferably, the semi-synthetic preparation method further comprises subjecting the cagrilintide to liquid chromatography purification, and the mobile phase of the liquid chromatography purification is alcohol and water.

[0014] More preferably, the mobile phase of the liquid chromatography purification is isopropanol and water.

[0015] The present application has the following advantages: The present application discloses a semi-synthetic preparation method for synthesizing cagrilintide, which fills the blank of the semi-synthetic preparation method for cagrilintide. The preparation method has fewer reaction steps, simple operation, greatly simplified process, reduced time and material cost, reduced waste liquid and by-product generation, meets the overall requirements of green development, and is easy to industrialize.

[0016] The cagrilintide intermediate of the present application is prepared by a biosynthetic method, which has the advantages of strong expression directionality, safety and health, wide raw material sources and low cost.

[0017] In the semi-synthetic preparation method of the present application, by optimizing the reaction conditions, such as by selecting and adjusting the reaction pH, molar ratio of reactants, condensing agent, reaction solvent, purification method, purification reagent, etc., the occurrence of side reactions is reduced, and the overall yield and purity of the carmegliptin is improved. For example, in the present application, (Boc)20 (Boc anhydride) is reacted with carmegliptin intermediate 36AA to obtain Boc-protected thirty-six peptide (Boc-36AA). Those skilled in the art know that carmegliptin intermediate 36AA has multiple active sites on the amino acid chain that can react with Boc anhydride, producing by-products that not only affect the purity and yield of the final carmegliptin, but also affect the reaction process. By optimizing the reaction pH, the present application can selectively react Boc anhydride with the epsilon amino group of 36AA to obtain Boc-36AA which protects the desired amino group. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the liquid chromatography part of the LC-MS spectrum of carmegliptin intermediate 36AA prepared in Example 1; Figure 2 is the mass spectrometry part of the LC-MS spectrum of carmegliptin intermediate 36AA prepared in Example 1; Figure 3 is the HPLC spectrum of carmegliptin prepared in Example 2; Figure 4 is the HPLC spectrum of carmegliptin prepared in Example 3. DETAILED DESCRIPTION

[0019] The present application is further described below in conjunction with the accompanying drawings and examples, but these examples are exemplary and only facilitate those skilled in the art to further understand the features of the present application, and do not constitute any limitation on the scope of the present application. Carmegliptin intermediate (36AA) is derived from the applicant's internal biosynthesis, and the side chain and other reagents, if not specified, are all conventional commercially available products. In the present application, the percentages appearing therein, if not specified, are mass percentages. In the following examples, the high performance liquid chromatograph used for purity detection and the high performance liquid chromatograph used for liquid phase separation and purification are both Hanbon Science and Technology Newstyle®laboratory liquid chromatograph system, column: UniHybrid8-120 C18.SS50:250, see the relevant examples for specific parameters.

[0020] In the present application, the semi-synthetic preparation method refers to using the Cagrilintide intermediate 36AA as a starting material, modifying the side chain at the alpha position of lysine and completing the modification of the remaining peptide chain at the N-terminus, to finally obtain Cagrilintide. The Cagrilintide intermediate 36AA is prepared by a biosynthetic method. Those skilled in the art can use conventional genetic recombination techniques and biological fermentation processes to produce a recombinant fusion protein containing the Cagrilintide intermediate 36AA. The vector used in the present application can be selected from the commonly used pET series of expression vectors, such as pET30a vector, pET30b vector, and other prokaryotic expression vectors. Those skilled in the art can use well-known methods to construct a recombinant expression vector containing a DNA sequence encoding the recombinant fusion protein of the present application and suitable transcription / translation control signals, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to a suitable promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The host cell suitable for the present application is preferably Escherichia coli, such as Escherichia coli BL21 (DE3).

[0021] In the present application, the abbreviations and their corresponding English meanings are shown in Table 1: Table 1 .

[0022] The application provides a semi-synthetic preparation method of carmegrelide. The method comprises the following steps: selectively protecting the lysine epsilon amino group of a carmegrelide intermediate (36AA, biosynthetic) to obtain a Boc-protected thirty-six peptide (Boc-36AA), then reacting the side chain Glu-eicosanoic acid with the Boc-36AA at the lysine alpha amino group to obtain a side chain-36-AA (SC-36AA), and then reacting the SC-36-AA with a proline amide to obtain a carmegrelide precursor, and finally cleaving the carmegrelide precursor to obtain the carmegrelide. The carmegrelide intermediate 36AA has two amino groups, i.e. the epsilon amino group and the alpha amino group of lysine. It is found that when the pH of the reaction solution is adjusted to 9.0-11.5 in step (1), the Boc anhydride can more easily react with the lysine epsilon amino group of 36AA and less easily react with the alpha amino group, thereby selectively protecting the lysine epsilon amino group of 36AA to obtain Boc-36AA. The Boc-36AA obtained through the above steps has high yield and purity, and can be directly used in subsequent reactions without purification, so that the occurrence of subsequent reactions is not affected, the side reactions are less, and the purity of the finally obtained carmegrelide is high. Then, the pH is adjusted to 8.5-9.0, the lysine alpha amino group of Boc-36AA reacts with the side chain Glu-eicosanoic acid to obtain SC-36AA, thereby avoiding the reaction of the side chain Glu-eicosanoic acid with the lysine epsilon amino group and reducing the generation of by-products, and the yield and purity of the obtained SC-36AA are high. Moreover, by adjusting the pH of the reaction solution in step (1) to 9.0-11.5, the breaking of the amide bond in the thirty-six peptide can be avoided, the generation of a large amount of by-products is prevented, and the total yield and purity of the final product are improved. Preferably, the pH of the reaction solution in step (1) is 9.5-10.0.

[0023] In an embodiment of the application, 36AA is prepared by a biosynthetic method, which comprises preparing a recombinant fusion protein comprising 36AA, and enzymatically digesting the recombinant fusion protein to obtain the 36AA, wherein the recombinant fusion protein is SEQ ID NO. 1.

[0024] In the application, the pH of the reaction solution in step (1) is adjusted by a base, and the condensation reactions in steps (2) and (3) are regulated, wherein the base comprises an inorganic base and an organic base, the inorganic base comprises one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium carbonate, sodium bicarbonate, disodium hydrogen phosphate and potassium bicarbonate, and the organic base comprises one or more of triethylamine, diisopropylethylamine, trimethylpyridine and pyridine. Preferably, the inorganic base is sodium carbonate, and the organic base is diisopropylethylamine. In step (2), the pH of the reaction solution is lowered by using an acid, and the acid used can be a commonly used organic acid and inorganic acid as long as it does not affect the reaction, for example, acetic acid.

[0025] In a preferred embodiment of the present invention, the molar ratio of 36AA to (Boc)₂O is 1.0:1.0-1.0:1.5, the molar ratio of 36AA to the side-chain Glu-eicosanoic acid is 1.0:1.0-1.0:1.5, and the molar ratio of 36AA to proline is 1.0:1.0-1.0:1.5. Limiting the molar ratios of the reactants to the above ranges ensures complete reaction of the substrate 36AA while avoiding waste of raw materials such as (Boc)₂O, side-chain Glu-eicosanoic acid, and proline, as well as excessive subsequent purification steps, thus saving costs. More preferably, the molar ratio of 36AA to (Boc)₂O is 1.0:1.1, the molar ratio of 36AA to the side-chain Glu-eicosanoic acid is 1.0:1.2, and the molar ratio of 36AA to proline is 1.0:1.2.

[0026] The semi-synthetic preparation method of the present invention is a liquid-phase reaction, wherein the solvent used is a good solvent capable of effectively dissolving each reactant, and the amount used is sufficient to completely dissolve each reactant. The solvents for the compound 36AA solution, the side-chain Glu-eicosanoic acid solution, and the compound SC-36AA solution can each be independently selected from one or more combinations of water, DMF, acetonitrile, dioxane, DMSO, and DMAC. In a preferred embodiment, the solvent for the compound 36AA solution is water, the solvent for the side-chain Glu-eicosanoic acid solution is DMF, and the solvent for the SC-36AA solution is DMF. When the solvents are selected as described above, not only can each reactant be fully dissolved, but they are also miscible during the reaction, which is beneficial to the reaction of each reactant.

[0027] In a preferred embodiment of the present invention, the condensing agent used in the semi-synthetic preparation method of the present invention includes one or more of HBTU, TCFH, HCTU, HOSU, HOBT, HOAT, COMU, HATU, PyBop, EDCI, Oxyma, and PyOxim. COMU or HATU is preferred, as COMU and HATU have higher efficiency, lower side reaction rates, and better safety in peptide synthesis, and are particularly suitable for the synthesis and industrial production of complex peptide chains.

[0028] In a preferred embodiment of the present invention, the canagliflozin precursor is cleaved in a lysis buffer prepared from trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 20:2:1. This lysis buffer facilitates the removal of the Boc group on the amino group and the protecting group on the side chain without affecting other covalent bonds. Cleavage of the canagliflozin precursor with this buffer yields canagliflozin in high yield and high purity.

[0029] In a preferred embodiment of the present application, the cagrilintide is further purified by liquid chromatography, wherein the mobile phase of the liquid chromatography is an alcohol and water. More preferably, the mobile phase of the liquid chromatography is isopropanol and water.

[0030] In a preferred embodiment of the present application, the reaction temperature of the process of the present application is 10-25 °C. Example 1: Preparation of cagrilintide intermediate 36AA

[0031] The cagrilintide intermediate 36AA in this example is prepared by designing a recombinant fusion protein comprising 36AA by genetic recombination technology, and then inserting the polynucleotide sequence corresponding to the recombinant fusion protein into an expression vector to construct an expression plasmid, which is transfected into E. coli for expression, and the expressed recombinant fusion protein is digested to obtain 36AA. The polynucleotide sequence corresponding to the recombinant fusion protein in this example is synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The amino acid sequence of the recombinant fusion protein comprising 36AA is SEQ ID NO. 1. The polynucleotide sequence corresponding to the recombinant fusion protein is SEQ ID NO. 2. The polynucleotide sequence is inserted into a pET30b(+) prokaryotic expression vector after being digested by Ndel and Notl endonuclease to construct an expression plasmid. The specific sequences are as follows: SEQ ID NO. 1: MQHHHHAEAEAEAEKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT.

[0032] SEQ ID NO. 2:

[0033] The plasmid with the recombinant fusion protein corresponding polynucleotide sequence is transfected into E. coli BL21 (DE3) competent cells (purchased from Shanghai Shenguo Biotechnology Co., Ltd.) by calcium phosphate precipitation method. Add 1 ng of plasmid water solution to 100 μL of E. coli BL21 (DE3) competent cells, mix gently, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, place on ice for 5 min, add 700 μL of LB liquid medium (soybean peptone 1.0 g / 100 mL, yeast powder 0.5 g / 100 mL, NaCl 1.0 g / mL, without antibiotics, the rest is water), constant temperature shaker 37°C, 200 rpm, cultivate for 1 hour, centrifuge at 5000 rpm for 2 min, then suck out 700 μL of supernatant, resuspend the remaining liquid with the centrifuged bacteria, and 100 μL of resuspension is completely coated on LB agar plate (soybean peptone 1.0 g / 100 mL, yeast powder 0.5 g / 100 mL, NaCl 1.0 g / mL, agar powder 1.5 g / 100 mL, kanamycin sulfate 50 ug / mL, the rest is water), and place in a 37°C constant temperature incubator for 13 h.

[0034] Pick a single colony strain into 5 mL of LB liquid medium (soybean peptone 1.0 g / 100 mL, yeast powder 0.5 g / 100 mL, NaCl 1.0 g / mL, kanamycin sulfate 50 ug / mL, the rest is water), constant temperature shaker 37°C, 200 rpm, cultivate until OD600≈0.5, take 0.5 ml of bacterial solution into a new sterile 1.5 mL centrifuge tube, and store in a 4°C refrigerator or directly proceed to the next step of strain preparation.

[0035] Inoculate 0.5 mL of bacterial solution into 50 mL of LB medium (soybean peptone 1.0 g / 100 mL, yeast powder 0.5 g / 100 mL, NaCl 1.0 g / mL, kanamycin sulfate 50 ug / mL, the rest is water), 200 rpm, 37°C culture until OD 600 is between 0.4 and 0.8, transfer the bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 5 min, collect the bacteria, resuspend the bacteria with an appropriate amount of the above LB medium to OD 600 ≈2, add an equal volume of sterile 50% w / t glycerol, mix well, and then 1 mL / tube is divided into cryotubes and stored in a -80°C refrigerator.

[0036] Take one strain from the -80°C refrigerator, inoculate it into LB medium (soybean peptone 1.0 g / 100 mL, yeast powder 0.5 g / 100 mL, NaCl 1.0 g / mL, kanamycin sulfate 50 ug / mL, the rest is water) at a volume ratio of 1 / 100, 37°C, 200 rpm, cultivate for 5 h, and obtain the recovered strain.

[0037] High-density fermentation was carried out. The fermentation medium (soybean peptone 1%, yeast extract 1%, NaCl 0.4%, K2HPO4·3H2O 0.25%, KH2PO4 0.1%, 20% NaOH solution to adjust pH to 7.0, and the balance was water) and glucose feed (50% glucose aqueous solution) were autoclaved and then cooled to 37°C. The single colony strain was inoculated into the recovered bacterial strain at a volume ratio of 1 / 100. The OD 600 When it reached about 100, it was cooled to 30°C, IPTG (final concentration 1 mM) was added for induction, and the culture was continued until the OD 600 The fermentation was stopped when the OD reached 150-200.

[0038] The bacterial cells were resuspended in buffer A (50 mM Tris-HCl, pH 7.0) at 11 times the weight of the bacterial cells, and the bacteria were broken by high-pressure homogenization (ATS, AH-PILOT 2018) at 750 bar for 3 times. The inclusion bodies were collected by centrifugation at 8000 rpm for 60 min, and the inclusion bodies were dissolved and renatured using buffer B (1% triton x-100, 50 mM Tris-HCl, pH 8.0), which had the same volume as buffer A. 2N HCl was added to the renatured sample to adjust the pH to the isoelectric point of the fusion protein, which was 5.73. The sample was centrifuged at 8000 rpm for 15 minutes, and the white precipitate was collected. The white precipitate was dissolved using buffer C (50 mM Tris-HCl, pH 8.0), which had the same volume as buffer A. The dissolved recombinant fusion protein was added with 100X enzyme digestion buffer (200 mM CaCl2), and enterokinase (Pangolin Bio), Kex2 protease (Pangolin Bio), and carboxypeptidase B (Pangolin Bio) were added. The mass ratio of enzyme to fusion protein was Kex2 enzyme: fusion protein = 1:1000, carboxypeptidase B: fusion protein = 1:1200, and enterokinase: fusion protein = 1:1500. The enzyme digestion was carried out at 30°C for 6 hours. After enzyme digestion, the 36AA was purified using SP-FF ion exchange chromatography (Borgolon), and the purity was 93.97% after isoelectric point precipitation. The prepared 36AA was detected by LC-MS (Waters Xevo G3 QTof), and the molecular weight was (965.83-1.01) x 4 = 3859.28, which was consistent with the polypeptide molecular weight of SEQ ID NO. 1. The liquid chromatography part of the 36AA LC-MS spectrum is shown in Figure 1 , and the mass spectrum part is shown in Figure 2 . Example 2: Preparation of carmegliptin

[0039] Into a 500 mL two-neck flask, add 19.4 g (0.005 mol) of Cagrilintide intermediate 36AA prepared according to the method of Example 1, add 200 mL of purified water, stir to dissolve, add DIPEA to adjust the pH to about 10.0, dropwise add 5 mL of a DMF solution of Boc anhydride (1.2 g, 0.0055 mol), continue stirring for 2 h to obtain Boc-36AA.

[0040] Into a 100 mL three-neck flask, add 3.5 g (0.006 mol) of side chain Glu-eicosanedioic acid and 30 mL of DMF, stir to dissolve, then add 2.6 g of COMU (0.006 mol), DIPEA 2 mL, continue stirring for 2 h to obtain a side chain activated ester solution for later use.

[0041] To the Boc-36AA system in the above 500 mL two-neck flask, add acetic acid to adjust the pH to 8.8, dropwise add the side chain activated ester solution to the above solution, stir, and react at room temperature for 3 h, then adjust the pH to about 5.0, precipitate the solid, suction filter, and vacuum dry to obtain SC-36AA.

[0042] Place SC-36AA in a 500 mL three-neck flask, add 200 mL of DMF, stir, and after complete dissolution, add 2.6 g of COMU (0.006 mol), DIPEA 2 mL, stir for 30 min, add proline amide 0.68 g (0.006 mol), continue stirring for 2 h, then add the above solution to a beaker containing 400 mL of water, adjust the pH to about 5.0, precipitate the solid, suction filter, and vacuum dry to obtain a Cagrilintide precursor.

[0043] At 25°C, add the Cagrilintide precursor to a 500 mL three-neck flask, add 100 mL of a cleavage solution of trifluoroacetic acid, 10 mL of triisopropylsilane, and 5 mL of water to the reaction system, stir for 2 h, then dropwise add the reaction system to 500 mL of methyl tert-butyl ether, precipitate the solid, suction filter, and vacuum dry to obtain a Cagrilintide crude product.

[0044] Purify the Cagrilintide crude product by liquid chromatography to obtain 25.26 g of Cagrilintide with a yield of 57.3%. The HPLC purity is greater than 99.6%, and the HPLC chromatogram of Cagrilintide is shown in Figure 3

[0045] The instrument and parameters for liquid chromatography purification are as follows: Newstyle® laboratory liquid chromatography system (Hanbon Science and Technology); Column: UniHybrid8-120 C18.SS50:250; Eluent: ​Mobile phase A is water: isopropanol = 95:5 (v / v); mobile phase B is water: isopropanol = 70:30 (v / v); The elution gradient is: Example 3: Preparation of carbaglu tides

[0046] Into a 500 mL two-neck flask, carbaglu tide intermediate 36AA 19.4 g (0.005 mol) prepared according to the method of Example 1 was added, 200 mL of purified water was added, stirred and dissolved, sodium carbonate solution was added to adjust the pH to about 10.0, 5 mL of Boc anhydride (1.2 g, 0.0055 mol) DMF solution was added dropwise, and stirring was continued for 2 h to obtain Boc-36AA.

[0047] Into a 100 mL three-neck flask, side chain Glu-eicosanedioic acid 3.5 g (0.006 mol) and 30 mL DMF were added and stirred and dissolved, then 2.28 g HATU (0.006 mol), DIPEA 2 mL were added, and stirring was continued for 3 h to obtain a side chain activated ester solution for standby.

[0048] To the Boc-36AA system in the above 500 mL two-neck flask, acetic acid was added to adjust the pH to 8.8, the side chain activated ester solution was added dropwise to the above solution, stirred, and reacted at room temperature for 3 h, then the pH was adjusted to about 5.0, the solid was precipitated, suction filtered, and vacuum dried to obtain SC-36AA.

[0049] SC-36AA was placed in a 500 mL three-neck flask, 200 mL DMF was added, stirred, and after complete dissolution, 2.28 g HATU (0.006 mol), DIPEA 2 mL were added, stirred for 30 min, proline amide 0.68 g (0.006 mol) was added, and stirring was continued for 2 h, then the above solution was added to a beaker containing 400 mL water, the pH was adjusted to about 5.0, the solid was precipitated, suction filtered, and vacuum dried to obtain a carbaglu tide precursor.

[0050] At 25°C, the carbaglu tide precursor was added to a 500 mL three-neck flask, and a cleavage solution prepared from 100 mL trifluoroacetic acid, 10 mL triisopropylsilane, and 5 mL water was added to the reaction system, stirred for 2 h, then the reaction system was added dropwise to 500 mL methyl tert-butyl ether, the solid was precipitated, suction filtered, and vacuum dried to obtain carbaglu tide crude product.

[0051] The carbaglu tide crude product was purified by liquid chromatography in the same way as Example 2 to obtain carbaglu tide 25.03 g, with a yield of 56.8%, a HPLC purity of 99.8%, and a HPLC chromatogram of carbaglu tide as shown in Figure 4 .

[0052] As can be seen from the above examples, the preparation method of the present application has fewer reaction steps, simple operation, greatly simplifies the process, reduces the time and material cost, reduces the generation of waste liquid and by-products, and the total yield of the prepared carmegiline is high, the purity is high, and the large-scale industrial production of carmegiline is easy.

[0053] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application.

Claims

1. A semi-synthetic preparation method for canagliflozin, characterized in that, The preparation method includes the following steps: (1) Under pH conditions of 9.0-11.5, the lysine ε-amino group of canagliflozin intermediate 36AA was selectively protected to obtain Boc-36AA, as shown in the following reaction formula I: Reaction I; (2) Under pH conditions of 8.5-9.0, the side chain Glu-eicosanoic acid reacts with the α-amino group of lysine in the Boc-36AA to obtain SC-36AA, as shown in the following reaction formula II: Reaction II; (3) The SC-36AA reacts with proline to obtain the canagliflozin precursor, as shown in the following reaction formula III: Reaction III; (4) The canagliflozin precursor is cleaved to obtain canagliflozin, as shown in the following reaction formula IV: Reaction IV.

2. The semi-synthetic preparation method according to claim 1, characterized in that, The 36AA is prepared by a biosynthetic method, the method comprising preparing a recombinant fusion protein containing 36AA, and enzymatically cleaving the recombinant fusion protein to obtain the 36AA, wherein the recombinant fusion protein is SEQ ID NO.

1.

3. The semi-synthetic preparation method according to claim 1, characterized in that, The pH of step (1) is 9.5-10.

0.

4. The semi-synthetic preparation method according to claim 1, characterized in that, The molar ratio of 36AA to (Boc)₂O is 1.0:1.0-1.0:1.5, the molar ratio of 36AA to the side-chain Glu-eicosanoic acid is 1.0:1.0-1.0:1.5, and the molar ratio of 36AA to proline is 1.0:1.0-1.0:1.

5.

5. The semi-synthetic preparation method according to claim 4, characterized in that, The molar ratio of 36AA to (Boc)2O is 1.0:1.1, the molar ratio of 36AA to the side-chain Glu-eicosanoic acid is 1.0:1.2, and the molar ratio of 36AA to proline is 1.0:1.

2.

6. The semi-synthetic preparation method according to claim 1, characterized in that, The preparation method includes a reaction solvent, an alkali, and a condensing agent; The reaction solvent includes one or more of water, DMF, acetonitrile, dioxane, DMSO, and DMAC; The base includes inorganic bases and organic bases. The inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium carbonate, sodium bicarbonate, disodium hydrogen phosphate, and potassium bicarbonate. The organic base includes one or more of triethylamine, diisopropylethylamine, trimethylpyridine, and pyridine. The condensing agent includes one or more of HBTU, TCFH, HCTU, HOSU, HOBT, HOAT, COMU, HATU, PyBop, EDCI, Oxyma, and PyOxim.

7. The semi-synthetic preparation method according to claim 6, characterized in that, The reaction solvent is water and DMF, the inorganic base is sodium carbonate, the organic base is diisopropylethylamine, and the condensing agent is COMU or HATU.

8. The semi-synthetic preparation method according to claim 1, characterized in that, Step (4) involves pyrolysis using a pyrolysis solution prepared from trifluoroacetic acid, triisopropylsilane and water, wherein the volume ratio of trifluoroacetic acid, triisopropylsilane and water is 20:2:

1.

9. The semi-synthetic preparation method according to any one of claims 1-8, characterized in that, It also includes purifying the canagliflozin by liquid chromatography, wherein the mobile phase for liquid chromatography purification is alcohol and water.

10. The semi-synthetic preparation method according to claim 9, characterized in that, The mobile phase for the liquid chromatography purification is isopropanol and water.

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