A semisynthetic process for the preparation of a cagrilintide

Abstract

The application relates to the field of biological medicine preparation, in particular to a semi-synthetic preparation method of a cagrilintide, which comprises the following steps: (1) selectively protecting lysine epsilon amino groups of a cagrilintide intermediate 36AA under the condition that the pH is 9.0-11.5 to obtain a Boc-protected thirty-six peptide (Boc-36AA); (2) reacting side chain Glu-eicosanedioic acid with lysine alpha amino groups of the Boc-36AA under the condition that the pH is 8.5-9.0 to obtain a 36-AA with a side chain (SC-36AA); (3) reacting the SC-36AA with prolyl amide to obtain a cagrilintide precursor; and (4) cleaving the cagrilintide precursor to obtain the cagrilintide. The preparation method of the application has the advantages of simple operation, low cost, high yield and high purity, the total reaction yield is more than 56%, and the HPLC purity is greater than or equal to 99.6%.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical preparation, specifically to a semi-synthetic preparation method of canagliflozin. Background Technology

[0002] Canagliptin is a novel, long-acting acylated amylin analogue and a non-selective amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) agonist. Canagliptin is a cyclic polypeptide composed of 38 amino acids and contains a single disulfide bond. Canagliptin can reduce energy intake, regulate food choices and preferences, and exert glucose regulation by co-secreting with insulin. It also inhibits postprandial glucagon release and delays gastric emptying. It has significant advantages in the treatment of diabetes, obesity, metabolic syndrome, and cardiovascular diseases, and its application prospects are broad.

[0003] Currently, all methods for preparing canagliflozin are total chemical synthesis methods, including solid-phase synthesis (SPPS) or fragment synthesis methods combining solid and liquid phases (SPPS-LPPS), such as CN117986347A and CN119350469A. CN117986347A discloses a method for synthesizing canagliflozin, using an amino resin as the starting resin, sequentially coupling single amino acids, and finally attaching a side chain (eicosanoic acid) to Glu, with an overall yield of 31.87% for canagliflozin. CN119350469A discloses a method for synthesizing canagliflozin via a large-fragment SPPS-LPPS hybrid method. The method involves cleaving canagliflozin into four polypeptide fragments: fragment I (sequences 1-14), fragment II (sequences 15-26), fragment III (sequences 27-35), and fragment IV (sequences 36-39). Fully protected fragments I, II, and III are synthesized by sequentially coupling corresponding protecting amino acid monomers at their respective sequence positions using SPPS. Fragment IV is then synthesized via solid-phase or liquid-phase synthesis. Subsequently, the fragments are synthesized by repeated condensation / de-Fmoc reactions using liquid-phase synthesis (LPPS) to obtain the fully protected canagliflozin, with an overall yield of approximately 45%. The aforementioned prior art suffers from drawbacks such as long reaction steps, difficulty in impurity control, low overall yield, high preparation cost, and difficulty in industrial-scale production. Summary of the Invention

[0004] To overcome the aforementioned deficiencies in the prior art, this invention provides a semi-synthetic preparation method for canagliflozin, which is simple to operate, low in cost, high in total yield, high in purity, and easy to industrialize.

[0005] This invention provides a semi-synthetic preparation method for canagliflozin, the semi-synthetic preparation method comprising 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.

[0006] Preferably, the 36AA is prepared by a biosynthetic method, the method comprising preparing a recombinant fusion protein containing 36AA, and enzymatically digesting 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 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.

[0009] 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 prolineamide is 1.0:1.2.

[0010] Preferably, the semi-synthetic preparation method includes a reaction solvent, a base, 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.

[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, step (4) uses a pyrolysis solution prepared from trifluoroacetic acid, triisopropylsilane and water for pyrolysis, wherein the volume ratio of trifluoroacetic acid, triisopropylsilane and water is 20:2:1.

[0013] Preferably, the semi-synthetic preparation method of the present invention further includes purifying the canagliflozin by liquid chromatography, wherein the mobile phase for liquid chromatography purification is alcohol and water.

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

[0015] The beneficial effects of this invention are: This invention discloses for the first time a semi-synthetic preparation method for canagliflozin, filling a gap in the semi-synthetic preparation methods for canagliflozin. The preparation method of this invention involves fewer reaction steps, is simple to operate, greatly simplifies the process, reduces time and material costs, and minimizes the generation of waste liquid and byproducts, meeting the overall requirements of green development and facilitating industrialization.

[0016] The canagliflozin intermediate of the present invention is prepared by biosynthesis and has the advantages of strong expression targeting, safety and hygiene, wide availability of raw materials and low cost.

[0017] Furthermore, in the semi-synthetic preparation method of the present invention, by optimizing the reaction conditions, such as by selecting and adjusting the reaction pH, reactant molar ratio, condensing agent, reaction solvent, purification method, and purification reagent, the occurrence of side reactions is reduced, thereby improving the overall yield and purity of canagliflozin. For example, the present invention reacts (Boc)₂O (Boc anhydride) with canagliflozin intermediate 36AA to obtain Boc-protected 36-peptide (Boc-36AA). Those skilled in the art know that the amino acid chain of canagliflozin intermediate 36AA has multiple active sites that can react with Boc anhydride, and the resulting byproducts not only affect the purity and yield of the final canagliflozin but also the reaction process. The present invention, through optimization of the reaction pH, enables the selective reaction of Boc anhydride with the ε-amino group of 36AA to obtain Boc-36AA with the desired amino group protected. Attached Figure Description

[0018] Figure 1 This is the liquid chromatography portion of the LC-MS spectrum of canagliflozin intermediate 36AA prepared in Example 1; Figure 2 This is the mass spectrometry portion of the LC-MS spectrum of the canagliflozin intermediate 36AA prepared in Example 1; Figure 3 This is the HPLC chromatogram of canagliflozin prepared in Example 2; Figure 4 This is the HPLC chromatogram of canagliflozin prepared in Example 3. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. However, these embodiments are exemplary and are only intended to help those skilled in the art to further understand the features of the present invention, and do not constitute any limitation on the scope of the present invention. The canagliflozin intermediate (36AA) is derived from the applicant's internal biosynthesis. Unless otherwise specified, the side chains and other reagents are all conventional commercially available products. Unless otherwise specified, all percentages appearing in this invention are mass percentages. In the following embodiments, the high-performance liquid chromatograph (HPLC) used for purity detection and the HPLC used for liquid phase separation and purification are both Hanbang Technology Newstyle® Laboratory HPLC systems, with a column of UniHybrid8-120C18.SS50:250. For specific parameters, please refer to the relevant embodiments.

[0020] In this invention, the semi-synthetic preparation method refers to using canagliflozin intermediate 36AA as a starting material, modifying the side chain at the lysine α-position and modifying the remaining peptide chain at the N-terminus to finally obtain canagliflozin. Canagliflozin intermediate 36AA is prepared using biosynthetic methods. Those skilled in the art can use conventional gene recombination techniques and bio-fermentation processes to produce recombinant fusion proteins containing canagliflozin intermediate 36AA. The vector used in this invention can be a commonly used pET series expression vector, such as pET30a or pET30b prokaryotic expression vectors. Those skilled in the art can construct recombinant expression vectors containing the DNA sequence encoding the recombinant fusion protein of this invention and suitable transcription / translation control signals using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The host cell suitable for this invention is preferably *Escherichia coli*, such as *Escherichia coli* BL21(DE3).

[0021] In this invention, the meanings of the abbreviations and their corresponding English translations are shown in Table 1: Table 1 .

[0022] This invention provides a semi-synthetic preparation method for canagliflozin. By selectively protecting the ε-amino group of lysine in a canagliflozin intermediate (36AA, biosynthesized), a Boc-protected hexapeptide (Boc-36AA) is obtained. Then, the side chain Glu-eicosanoic acid reacts with the α-amino group of lysine in Boc-36AA to obtain 36-AA with a side chain (SC-36AA). SC-36AA then reacts with proline amide to obtain a canagliflozin precursor, which is then cleaved to obtain canagliflozin. The canagliflozin intermediate 36AA has two amino groups on its lysine residue, namely an ε-amino group and an α-amino group. The inventors discovered that in step (1), adjusting the pH of the reaction solution to 9.0-11.5 makes it easier for the Boc anhydride to react with the ε-amino group of lysine in 36AA, but less likely to react with the α-amino group, thus selectively protecting the ε-amino group of lysine in 36AA to obtain Boc-36AA. The Boc-36AA obtained through the above steps has high yield and purity, and can be used directly in subsequent reactions without purification, without affecting the occurrence of subsequent reactions, with few side reactions, and the final canagliflozin has high purity. Then, the pH is adjusted to 8.5-9.0, and the α-amino group of lysine in Boc-36AA reacts with the side chain Glu-eicosanoic acid to obtain SC-36AA, avoiding the reaction between the side chain Glu-eicosanoic acid and the ε-amino group of lysine, reducing the generation of by-products, and the yield and purity of SC-36AA are high. Moreover, by adjusting the pH of the reaction solution in step (1) to 9.0-11.5, the amide bond in the hexapeptide can be prevented from breaking, preventing the generation of a large number of by-products, and improving the overall yield and purity of the final product. Preferably, the pH of the reaction solution in step (1) is 9.5-10.0.

[0023] In one embodiment of the present invention, 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.

[0024] This invention adjusts the pH of the reaction solution in step (1) and regulates the condensation reactions in steps (2) and (3) using an alkali. The alkali includes inorganic 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. Preferably, the inorganic base is sodium carbonate, and the organic base is diisopropylethylamine. In step (2), an acid is used to lower the pH of the reaction solution. The acid used can be a commonly used organic or inorganic acid, as long as it does not affect the reaction, such as 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 invention, canagliflozin is further purified by liquid chromatography, wherein the mobile phase for liquid chromatography purification is alcohol and water. More preferably, the mobile phase for liquid chromatography purification is isopropanol and water.

[0030] In a preferred embodiment of the present invention, the reaction temperature of the preparation method of the present invention is 10-25°C.

[0031] Example 1: Preparation of canagliflozin intermediate 36AA

[0032] In this embodiment, the canagliflozin intermediate 36AA was used to design a recombinant fusion protein containing 36AA using gene recombination technology. The polynucleotide sequence corresponding to this recombinant fusion protein was then inserted into an expression vector to construct an expression plasmid, which was transfected into *E. coli* for expression. The expressed recombinant fusion protein was digested with enzymes to obtain 36AA. The polynucleotide sequence and plasmid construction of the recombinant fusion protein in this embodiment were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The amino acid sequence of the recombinant fusion protein containing 36AA is SEQ ID NO.1. The polynucleotide sequence corresponding to the recombinant fusion protein is SEQ ID NO.2. The polynucleotide sequence was digested with NdeI and NotI endonucleases and then inserted into the pET30b(+) prokaryotic expression vector to construct an expression plasmid. The specific sequence is shown below: SEQ ID NO.1: MQHHHHAEAEAEKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQ RLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT.

[0033] SEQ ID NO.2:

[0034] The plasmid containing the polynucleotide sequence corresponding to the recombinant fusion protein was transfected into *E. coli* BL21(DE3) competent cells (purchased from Shanghai Sangon Biotech Co., Ltd.) using the calcium phosphate precipitation method. 1 ng of plasmid aqueous solution was added to 100 µL of *E. coli* BL21(DE3) competent cells, gently mixed, and placed on ice for 30 minutes. A heat shock at 42°C for 90 seconds was performed, followed by 5 minutes on ice. Then, 700 µL of LLB liquid medium (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, 1.0 g / mL NaCl, antibiotic-free, balance water) was added. The culture was incubated at 37°C and 200 rpm for 1 hour using a shaker. After centrifugation at 5000 rpm for 2 minutes, 700 µL of supernatant was aspirated. The remaining liquid was used to resuspend the centrifuged cells. 100 µL of the resuspended culture was plated onto LB agar plates (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, NaCl...). 1.0 g / mL, 1.5 g / 100 mL agar powder, 50 ug / mL kanamycin sulfate, and the remainder water) were incubated in a 37°C incubator for 13 h.

[0035] Pick a single clone of the bacterial strain and transfer it to 5 mL of LB liquid medium (1.0 g soybean peptone / 100 mL, 0.5 g yeast extract / 100 mL, 1.0 g NaCl / mL, 50 ug kanamycin sulfate / mL, with the remainder being water). Incubate at 37°C and 200 rpm on a constant temperature shaker until OD600≈0.5. Transfer 0.5 mL of the bacterial culture to a new sterile 1.5 mL centrifuge tube and store at 4°C or proceed directly to the next step of bacterial strain preparation.

[0036] Inoculate 0.5 mL of bacterial culture into 50 mL of LB medium (1.0 g soybean peptone / 100 mL, 0.5 g yeast extract / 100 mL, 1.0 g NaCl / mL, 50 µg kanamycin sulfate / mL, with the remainder being water), and incubate at 37°C for 200 rpm until OD500. 600 The bacterial culture value is approximately between 0.4 and 0.8. Transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 5 min, collect the bacterial cells, and resuspend the bacterial cells in an appropriate amount of the above LB medium until the OD value is reached. 600 ≈2, add an equal volume of sterile 50% w / t glycerol, mix well, and dispense 1 mL / tube into cryovials and store at -80°C.

[0037] Take one bacterial culture from the -80℃ freezer and inoculate it into LB medium (1.0g / 100mL soybean peptone, 0.5g / 100mL yeast extract, 1.0g / mL NaCl, 50µg / mL kanamycin sulfate, and the remainder water) at a volume ratio of 1 / 100. Incubate at 37℃ and 200rpm for 5 hours to obtain a well-recovered bacterial culture.

[0038] High-density fermentation was carried out by autoclaving the fermentation medium (1% soybean peptone, 1% yeast extract, 0.4% NaCl, 0.25% K₂HPO₄·3H₂O, 0.1% KH₂PO₄, pH adjusted to 7.0 with 20% NaOH solution, the remainder being water) and glucose feed (50% glucose aqueous solution), followed by cooling to 37°C. Single-clonal strains were picked and inoculated with the revived inoculum at a volume ratio of 1 / 100. The OD of the fermentation broth was measured. 600 When the concentration reaches approximately 100, cool the temperature to 30°C, add IPTG (final concentration 1 mM) for induction, and continue culturing until the OD reaches 100. 600 Stop fermentation when the temperature reaches 150-200°C.

[0039] The bacterial cells were resuspended in buffer A (50 mM Tris-HCl, pH 7.0) at 11 times their weight. The cells were homogenized three times using an autoclave (ATS, AH-PILOT 2018) at 750 bar. Inclusion bodies were collected by centrifugation at 8000 rpm for 60 min. The inclusion bodies were dissolved and renatured using buffer B (1% Triton X-100, 50 mM Tris-HCl, pH 8.0), with the volume of buffer B being the same as that of buffer A. 2N HCl was added to the renatured sample to adjust the pH to the isoelectric point of the fusion protein, 5.73. The sample was centrifuged at 8000 rpm for 15 min, and the white precipitate was collected. The white precipitate was dissolved in buffer C (50 mM Tris-HCl, pH 8.0), with the same volume as buffer A. The dissolved recombinant fusion protein was then added to 100X restriction enzyme buffer (200 mM CaCl2), along with enterokinase (Pakin Bio), Kex2 protease (Pakin Bio), and carboxypeptidase B (Pakin Bio). The enzyme-to-fusion protein ratio was 1:1000 for Kex2, 1:1200 for carboxypeptidase B, and 1:1500 for enterokinase. The digestion temperature was 30°C, and the digestion time was 6 hours. After digestion, 36AA was purified using SP-FF ion exchange chromatography (BorgLoughlin), and after isoelectric point precipitation, the purity was 93.97%. The molecular weight of the prepared 36AA was confirmed by LC-MS (Waters Xevo G3 QTof) to be (965.83-1.01)×4=3859.28, which matches the molecular weight of the target peptide in SEQ ID NO.1. The liquid chromatographic portion of the 36AA LC-MS chromatogram is shown below. Figure 1 As shown, the mass spectrometry section is as follows Figure 2 As shown.

[0040] Example 2: Preparation of canagliflozin

[0041] At 25°C, 19.4 g (0.005 mol) of canagliflozin intermediate 36AA prepared according to the method of Example 1 was added to a 500 mL two-necked flask, 200 mL of purified water was added, and the mixture was stirred to dissolve. DIPEA was added to adjust the pH to about 10.0, and 5 mL of DMF solution of Boc anhydride (1.2 g, 0.0055 mol) was added dropwise. The mixture was stirred for 2 h to obtain Boc-36AA.

[0042] In a 100 mL three-necked flask, add 3.5 g (0.006 mol) of side-chain Glu-eicosanoic acid and 30 mL of DMF and stir to dissolve. Then add 2.6 g of COMU (0.006 mol) and 2 mL of DIPEA and continue stirring for 2 h to obtain a side-chain activated ester solution for later use.

[0043] Acetic acid was added to the Boc-36AA system in the 500mL two-necked flask to adjust the pH to 8.8. The side-chain activated ester solution was then added dropwise to the solution, stirred, and reacted at room temperature for 3 hours. After that, the pH was adjusted to about 5.0, and a solid precipitated. The solid was filtered and dried under vacuum to obtain SC-36AA.

[0044] SC-36AA was placed in a 500mL three-necked flask, 200mL of DMF was added, and the mixture was stirred until completely dissolved. Then, 2.6g COMU (0.006mol) and 2mL of DIPEA were added, and the mixture was stirred for 30min. Then, 0.68g of proline (0.006mol) was added, and the mixture was stirred for 2h. The solution was then added to a beaker containing 400mL of water, and the pH was adjusted to about 5.0. The solid precipitated out, was filtered, and dried under vacuum to obtain canagliflozin precursor.

[0045] At 25°C, canagliflozin precursor was added to a 500 mL three-necked flask. A lysis buffer prepared with 100 mL trifluoroacetic acid, 10 mL triisopropylsilane, and 5 mL water was added to the reaction system. The mixture was stirred for 2 h. Then, the reaction system was added dropwise to 500 mL methyl tert-butyl ether, and a solid precipitated. The solid was filtered and dried under vacuum to obtain crude canagliflozin.

[0046] Crude canagliflozin was purified by liquid chromatography to obtain 25.26 g of canagliflozin, with a yield of 57.3%. The HPLC purity was greater than 99.6%. The HPLC chromatogram of canagliflozin is shown below. Figure 3 As shown.

[0047] The instruments and parameters for liquid chromatography purification are as follows: Newstyle® Laboratory Liquid Chromatography System (Hanbang Technology). Pillar: UniHybrid8-120C18.SS50:250; Elution buffer: Mobile phase A is water:isopropanol = 95:5 (v / v); mobile phase B is water:isopropanol = 70:30 (v / v). The elution gradient is:

[0048] Example 3: Preparation of canagliflozin

[0049] At 25°C, 19.4 g (0.005 mol) of canagliflozin intermediate 36AA prepared according to the method of Example 1 was added to a 500 mL two-necked flask, 200 mL of purified water was added, and the mixture was stirred to dissolve. Sodium carbonate solution was added to adjust the pH to about 10.0, and 5 mL of DMF solution of Boc anhydride (1.2 g, 0.0055 mol) was added dropwise. The mixture was stirred for 2 h to obtain Boc-36AA.

[0050] In a 100 mL three-necked flask, add 3.5 g (0.006 mol) of side-chain Glu-eicosanoic acid and 30 mL of DMF and stir to dissolve. Then add 2.28 g of HATU (0.006 mol) and 2 mL of DIPEA and continue stirring for 3 h to obtain a side-chain activated ester solution for later use.

[0051] Acetic acid was added to the Boc-36AA system in the 500mL two-necked flask to adjust the pH to 8.8. The side-chain activated ester solution was then added dropwise to the solution, stirred, and reacted at room temperature for 3 hours. After that, the pH was adjusted to about 5.0, and a solid precipitated. The solid was filtered and dried under vacuum to obtain SC-36AA.

[0052] SC-36AA was placed in a 500mL three-necked flask, 200mL of DMF was added, and the mixture was stirred until completely dissolved. Then, 2.28g of HATU (0.006mol) and 2mL of DIPEA were added, and the mixture was stirred for 30min. Then, 0.68g of proline (0.006mol) was added, and the mixture was stirred for 2h. After that, the solution was added to a beaker containing 400mL of water, the pH was adjusted to about 5.0, the solid precipitated, filtered, and dried under vacuum to obtain canagliflozin precursor.

[0053] At 25°C, canagliflozin precursor was added to a 500 mL three-necked flask. A lysis buffer prepared with 100 mL trifluoroacetic acid, 10 mL triisopropylsilane, and 5 mL water was added to the reaction system. The mixture was stirred for 2 h. Then, the reaction system was added dropwise to 500 mL methyl tert-butyl ether, and a solid precipitated. The solid was filtered and dried under vacuum to obtain crude canagliflozin.

[0054] The crude canagliflozin was purified by liquid chromatography using the same purification method as in Example 2, yielding 25.03 g of canagliflozin, with a yield of 56.8% and an HPLC purity of 99.8%. The HPLC chromatogram of canagliflozin is shown below. Figure 4As shown.

[0055] As can be seen from the above embodiments, the preparation method of the present invention has fewer reaction steps and is simple to operate, which greatly simplifies the process, reduces time and material costs, reduces the generation of waste liquid and by-products, and the prepared canagliflozin has a high total yield, high purity, and is easy for large-scale industrial production of canagliflozin.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

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; The 36AA is prepared by a biosynthetic method, which includes preparing a recombinant fusion protein containing 36AA, and digesting the recombinant fusion protein with enterokinase, Kex2 enzyme and carboxypeptidase B to obtain the 36AA, wherein the recombinant fusion protein is SEQ ID NO.1; 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.

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

0.

3. 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.

4. The semi-synthetic preparation method according to claim 3, 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.

5. 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.

6. The semi-synthetic preparation method according to claim 5, 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.

7. The semi-synthetic preparation method according to any one of claims 1-6, characterized in that, It also includes purifying the canagliflozin by liquid chromatography, wherein the mobile phase for liquid chromatography purification is alcohol and water.

8. The semi-synthetic preparation method according to claim 7, characterized in that, The mobile phase for the liquid chromatography purification is isopropanol and water.

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