Synthetic method of canagliptide

Abstract

The invention provides a synthesis method of canagliptin, and belongs to the technical field of polypeptide synthesizing.The method comprises the steps that firstly, a peptide fragment I, a peptide fragment II, a peptide fragment III and a peptide fragment IV are subjected to solid-phase synthesis through an Fmoc method, then all the fragments are condensed in sequence, then splitting decomposition is conducted to remove side chain protecting groups, and crude linear canagliptin peptide is obtained; the preparation method comprises the following steps: taking 7-(trifluoromethyl)-1H-[1, 2, 3] triazolo [4, 5-e] triazolo [1, 5-a] pyrazine-1-ol as a condensation system in a coupling reaction system for solid-phase synthesis of a peptide fragment by an Fmoc method, and then carrying out cyclization, separation and purification, salt conversion, concentration and freeze-drying to obtain a canagliptin finished product. The invention also uses DIC / 7-(trifluoromethyl)-1H-[1, 2, 3] triazolo [4, 5-e] triazolo [1, 5-a] pyrazine-1-ol as a condensation system. The synthesis method of the canagliflozin has the advantages that the reaction rate is high, the cost is low, large-scale production is easy, the obtained crude product is beneficial to purification, the purity and yield are high, the amount of waste liquid is small, and energy conservation and environmental protection are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing cagrilintide. Background Art

[0002] Cagrilintide is a long-acting acylated amylin analogue and acts as a non-selective amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) agonist. Cagrilintide can reduce energy intake, regulate food choice and preference, play a glucose-regulating role by co-secreting with insulin, inhibit postprandial glucagon release, and delay gastric emptying. It has obvious advantages in the treatment of diabetes, obesity, metabolic syndrome, cardiovascular diseases, etc., and has a broad application prospect.

[0003] The prior art, such as the Chinese invention patent with the publication number CN114249808A, discloses a method for synthesizing Cagrilintide, which includes the following steps: Step 1, selecting the 7th-8th amino acids of the Cagrilintide sequence as the pseudo-proline dipeptide Fmoc-Ala-Thr(pro-me-me)-OH, selecting the 10th-11th amino acids of the Cagrilintide sequence as the pseudo-proline dipeptide Fmoc-Ala-Thr(pro-me-me)-OH, and selecting the 21st-22nd amino acids of the Cagrilintide sequence as the pseudo-proline dipeptide Fmoc-Ser-Ser(pro-me-me)-OH; Step 2, performing a coupling reaction according to the amino acid sequence; Step 3, cleavage; Step 4, cyclization to obtain the crude product of Cagrilintide; The method of the present invention is simple in operation, high in reaction rate, low in cost, suitable for large-scale production, and high in purity and yield. Although this method solves the problem of increased coupling difficulty caused by resin polycondensation, it increases the production cost and is restricted by production equipment, making it difficult to further scale up production. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for synthesizing cagrilintide, which has a high reaction rate, low cost, is easy to scale up for production, the obtained crude product is conducive to purification, and has high purity and yield, and greatly reduces the amount of waste liquid, saving energy and protecting the environment.

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows: The present invention discloses a method for synthesizing cagrilintide, which includes the following steps: Step 1, respectively synthesizing peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV by a solid-phase method; Step 2: Perform liquid-phase splicing on peptide fragment III and peptide fragment IV to obtain peptide fragment V, then perform liquid-phase splicing on peptide fragment V and peptide fragment II to obtain peptide fragment VI, and finally perform liquid-phase splicing on peptide fragment VI and peptide fragment I to obtain the fully protected peptide of canagliflozin; Step 3: Add the fully protected peptide of canagliflozin to the cleavage solution, and obtain the crude linear peptide of canagliflozin through precipitation, pulping and washing; Step 4: Cyclize the crude linear peptide of canagliflozin, and then obtain the finished product of canagliflozin through separation and purification, salt conversion, concentration and freeze-drying; The peptide fragment I is Eic-(OtBu)-Glu 1 -(OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu) 5 -Ala-Thr(tBu)-Cys(Trt)-Ala 9 -OH; The peptide fragment II is Fmoc-Thr(tBu) 10 -Gln(Trt)-Arg(Pbf)-Leu-Ala-Glu(OtBu) 15 -Phe-Leu 17 -OH; The peptide fragment III is Fmoc-Arg(Pbf) 18 -His(Boc)-Ser(tBu) 20 -Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly 25 -OH; The peptide fragment IV is NH2-Pro 26 -Ile-Leu-Pro-Pro 30 -Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu) 35 -Asn(Trt)-Thr(tBu)-Pro 38-NH2. Cagrilintide is a 38-peptide, belonging to long peptides, with its N-terminus modified with a long carbon chain of eicosanedioic acid. Due to the relatively long peptide chain, as the peptide sequence extends on the solid-phase resin carrier, the steric hindrance becomes larger, and problems such as difficult coupling, resin shrinkage, and residual washing of the reaction solution are all key factors causing impurity generation, which will reduce the quality of the crude peptide and increase the difficulty of subsequent purification and removal. In addition, the stepwise coupling process restricts the efficiency and production capacity of large-scale production. In view of the above disadvantages, the method of the present invention divides the cagrilintide peptide sequence into 4 small fragments, namely peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV. After synthesizing the corresponding fragment peptide resins on the solid-phase carrier and separating to obtain the fragment solids, each fragment is then condensed in sequence. This process can well avoid the resin shrinkage and coupling problems at difficult sites during the coupling of peptide resins, reduce the number of impurities, improve the coupling efficiency, shorten the synthesis cycle, increase the yield, purity, and content of the linear crude peptide, reduce the purification difficulty of the linear peptide crude peptide, and obtain a final product with relatively high purity and yield.

[0006] In some embodiments, step one above includes: S1. Use 2-CTC resin as the solid-phase carrier, adopt the Fmoc solid-phase synthesis strategy, and sequentially couple amino acids according to the sequence of peptide fragment I to obtain Eic-(OtBu)-Glu 1 -(OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu) 5 -Ala-Thr(tBu)-Cys(Trt)-Ala 9 -CTC Resin, that is, peptide resin I. Peptide resin I is subjected to soft cleavage to obtain peptide fragment I; S2. Use 2-CTC resin as the solid-phase carrier, adopt the Fmoc solid-phase synthesis strategy, and sequentially couple amino acids according to the sequence of peptide fragment II to obtain Fmoc-Thr(tBu) 10 -Gln(Trt)-Arg(Pbf)-Leu-Ala-Glu(OtBu) 15 -Phe-Leu 17 -CTC Resin, that is, peptide resin II. Peptide resin II is subjected to soft cleavage to obtain peptide fragment II; S3. Use 2-CTC resin as the solid-phase carrier, adopt the Fmoc solid-phase synthesis strategy, and sequentially couple amino acids according to the sequence of peptide fragment III to obtain Fmoc-Arg(Pbf) 18 -His(Boc)-Ser(tBu) 20 -Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly 25 -CTC Resin, that is, peptide resin III. Peptide resin III is subjected to soft cleavage to obtain peptide fragment III; S4. Use Sieber amide resin as the solid-phase carrier, and adopt the Fmoc solid-phase synthesis strategy to sequentially couple amino acids according to the sequence of peptide fragment Ⅳ to obtain NH2-Pro 26 -Ile-Leu-Pro-Pro 30 -Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu) 35 -Asn(Trt)-Thr(tBu)-Pro 38 -Sieber Resin, that is, peptide resin Ⅳ. Peptide resin Ⅳ is subjected to mild cleavage to obtain peptide fragment Ⅳ.

[0007] In some embodiments, the substitution degree of the above-mentioned 2-CTC resin is 0.6 - 1.0 mmol / g, preferably 0.8 - 1.0 mmol / g.

[0008] In some embodiments, the substitution degree of the above-mentioned Sieber amide resin is 0.8 - 1.0 mmol / g, preferably 0.6 - 0.8 mmol / g.

[0009] In some embodiments, the cleavage solution used for mild cleavage in the above steps S1, S2, and S3 is a TFA / DCM solution with a volume ratio of 1 - 1.2%.

[0010] In some embodiments, the cleavage solution used for mild cleavage in the above step S4 is a TFA / DCM solution with a volume ratio of 5 - 5.5%.

[0011] In some embodiments, the specific preparation method of the above-mentioned peptide resin Ⅰ includes: a. After swelling 2-CTC resin with DMF for 25 - 35 min, introduce the first protected amino acid monomer Fmoc-Ala-OH to obtain Fmoc-Ala 9 -CTC Resin; b. Remove the Fmoc protection on the resin, wash it with DMF 5 - 6 times, and carry out a coupling reaction with the next protected amino acid monomer Fmoc-Cys(Trt)-OH to obtain Fmoc-Cys(Trt)-Ala 9 -CTC Resin; c. Sequentially introduce protected amino acid monomers in a cyclic manner according to step b, and finally introduce monoterbutyl ester of eicosanedioic acid according to the method of step b to obtain peptide resin Ⅰ.

[0012] In some embodiments, the specific preparation method of the above-mentioned peptide resin Ⅱ includes: a. After swelling 2-CTC resin with DMF for 25 - 35 min, introduce the first protected amino acid monomer Fmoc-Leu-OH to obtain Fmoc-Leu 17-CTC Resin; b. Remove the Fmoc protection on the resin, wash it 5 - 6 times with DMF, and carry out a coupling reaction with the next protected amino acid monomer Fmoc-Phe-OH to obtain Fmoc-Phe-Leu 17 -CTC Resin; c. Sequentially introduce protected amino acid monomers in a cycle according to step b to obtain peptide resin II.

[0013] In some embodiments, the specific preparation method of the above peptide resin III includes: a. After swelling 2-CTC resin with DMF for 25 - 35 min, introduce the first protected amino acid monomer Fmoc-Gly-OH to obtain Fmoc-Gly 25 -CTC Resin; b. Remove the Fmoc protection on the resin, wash it 5 - 6 times with DMF, and carry out a coupling reaction with the next protected amino acid monomer Fmoc-Phe-OH to obtain Fmoc-Phe-Gly 25 -CTC Resin; c. Sequentially introduce protected amino acid monomers in a cycle according to step b to obtain peptide resin III.

[0014] In some embodiments, in the specific preparation methods of the above peptide resin I, peptide resin II, and peptide resin III, the condensing agent used for introducing the first protected amino acid monomer to 2-CTC resin is DIEA, and the reaction solvent is DMF.

[0015] In some embodiments, the specific preparation method of the above peptide resin IV includes: a. Swell Sieber amide resin with DMF for 25 - 35 min; b. Remove the Fmoc protection on the resin, wash it 5 - 6 times with DMF, and carry out a coupling reaction with the first protected amino acid Fmoc-Pro-OH to obtain Fmoc-Pro 38 -Sieber Resin; c. Sequentially introduce protected amino acid monomers in a cycle according to step b. After the coupling is completed, remove the Fmoc protection on the resin to obtain peptide resin IV.

[0016] In some embodiments, in the specific preparation methods of the above peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, during the process of sequentially introducing amino acid monomers, the molar ratio of the resin to the protected amino acid monomer is 1:1.5 - 3.

[0017] In some embodiments, the deprotecting agent used for removing the Fmoc protection on the resin is a Pip / DMF solution with a volume ratio of 20% - 30%. Further, the deprotection time is 28 - 32 min, and the reaction temperature is 20 - 35 °C.

[0018] In some embodiments, in the specific preparation method of the above-mentioned peptide resin I, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure or DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol.

[0019] In some embodiments, in the specific preparation method of the above-mentioned peptide resin II, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure or DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol.

[0020] In some embodiments, in the specific preparation method of the above-mentioned peptide resin III, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure or DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol.

[0021] In some embodiments, in the specific preparation method of the above-mentioned peptide resin IV, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure or DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol.

[0022] In some embodiments, in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure. Preferably, in the coupling reaction system of step b in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, the molar ratio of the protected amino acid monomer:DIC:Oxyma Pure is 1:0.8-1.2:0.8-1.2.

[0023] In some embodiments, in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, the condensation system for the coupling reaction in step b is DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. The hydroxyl group of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol can form an ester with the carboxyl group. The structure of the triazole ring, the fused ring system, and the presence of the trifluoromethyl group greatly increase the activity of the activated ester, resulting in a high rate of ester aminolysis. The condensation system composed of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol as an activator and DIC has a good activation effect on amino acids, enabling a high degree of amino acid coupling reaction, with fewer by-products, and peptide fragments I, II, III, and IV with both high yield and purity can be obtained. Preferably, in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, in the coupling reaction system of step b, the molar ratio of the protected amino acid monomer:DIC:7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is 1:0.8~1.2:0.8~1.2.

[0024] In some embodiments, in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, peptide resin III, and peptide resin IV, the reaction solvent used for the coupling reaction is DMF.

[0025] In some embodiments, in the specific preparation method of the above-mentioned peptide resin I, the reaction solvent used for the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3~4:1~2:1. When 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used as an activator, a DMF / 1,1-diisopropoxymethylamine / 4-phenyl-1-butene solution with a volume ratio of 3~4:1~2:1 as the solvent can better improve the activation effect of the amino acid reagent, increase the degree of amino acid coupling reaction, and improve the purity and yield of the corresponding peptide fragment.

[0026] In some embodiments, in the specific preparation method of the above-mentioned peptide resin II, the reaction solvent used for the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3~4:1~2:1.

[0027] In some embodiments, in the specific preparation method of the above-mentioned peptide resin III, the reaction solvent used for the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3~4:1~2:1.

[0028] In some embodiments, in the specific preparation method of the above-mentioned peptide resin IV, the reaction solvent used in the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3 to 4:1 to 2:1.

[0029] In some embodiments, the above step two includes the following steps: i. After activating the C-terminal carboxyl group of peptide fragment III with a condensing agent, performing a coupling reaction with the N-terminal amino group of peptide fragment IV in a reaction solution, then removing the Fmoc protecting group and precipitating a solid to obtain peptide fragment V; ii. After activating the C-terminal carboxyl group of peptide fragment II with a condensing agent, performing a coupling reaction with the N-terminal amino group of peptide fragment V in a reaction solution, then removing the Fmoc protecting group and precipitating a solid to obtain peptide fragment VI; iii. After activating the C-terminal carboxyl group of the said peptide fragment I with a condensing agent, performing a coupling reaction with the N-terminal amino group of peptide fragment VI in a reaction solution, then removing the Fmoc protecting group and precipitating a solid to obtain the fully protected peptide of canagliflozin.

[0030] In some embodiments, the deprotecting agent used to remove the Fmoc protecting group in the above step two is Pip.

[0031] In some embodiments, the coupling reaction temperature in the above step i is 0 to 10 °C.

[0032] In some embodiments, the coupling reaction temperature in the above step ii is -10 to 0 °C.

[0033] In some embodiments, the coupling reaction temperature in the above step iii is -10 to 0 °C.

[0034] In some embodiments, the condensation system for the coupling reaction in the above step two consists of any one or more of HOBt, HOAT, Cl-HOBt, Oxyma Pure and DIC, or consists of any one or more of HBTU, TBTU, HATU, PyBOP, PyAOP, PyOxim and DIEA.

[0035] In some embodiments, the condensation system for the coupling reaction in the above step i is HBTU / DIEA.

[0036] In some embodiments, the condensation system for the coupling reaction in the above step ii is PyOxim / DIEA.

[0037] In some embodiments, the condensation system for the coupling reaction in the above step iii is HBTU / DIEA.

[0038] In some embodiments, the reaction solvent used in the coupling reaction in the above step two includes one or more of DCM, DMF, THF, DMSO, and NMP.

[0039] In some embodiments, the above step three specifically includes: reacting the fully protected peptide of canagliflozin with a cleavage solution at 20 - 30 °C with stirring for 2 - 3 h, filtering after reaching the reaction time, and rinsing the filter cake with TFA; pouring the obtained filtrate into MTBE at -10 - 0 °C to obtain a suspension in which a white solid precipitates, and then obtaining the crude linear peptide of canagliflozin through centrifugal sedimentation and slurry washing.

[0040] In some embodiments, the mass - to - volume ratio of the fully protected peptide of canagliflozin to the cleavage solution in the above step three is 1 g: 7 - 9 mL.

[0041] In some embodiments, the mass - to - volume ratio of the fully protected peptide of canagliflozin to MTBE in the above step three is 1 g: 62 - 65 mL.

[0042] In some embodiments, the cleavage solution in the above step three includes more than three of TFA, EDT, TIS, PhOH, and PhSMe.

[0043] In some embodiments, the cleavage solution in the above step three is composed of TFA, EDT, TIS, and H2O in a volume ratio of 83 - 86: 4 - 5: 4 - 5: 4 - 5.

[0044] Preferably, the cleavage solution in the above step three is composed of TFA, EDT, TIS, and H2O in a volume ratio of 85: 5: 5: 5.

[0045] In some embodiments, the cyclization method of the crude linear peptide of canagliflozin in the above step four is specifically: dissolving the crude linear peptide of canagliflozin in an acetonitrile / water solution with a volume ratio of 75 - 80%, and after dissolving clearly, diluting it with purified water to a concentration of 0.9 - 1.2 g / L; then dropwise adding an iodine / acetonitrile solution at 0.8 - 1.1 mmol / mL until the cyclization solution shows a light yellow color, and stirring to keep the color from fading for 25 - 32 min, and finally dropwise adding a vitamin C aqueous solution at 0.8 - 1.2 mmol / mL until the cyclization solution is clear and colorless.

[0046] Since the cleavage sites are selected according to the shrinkage phenomenon of the peptide resin during the stepwise coupling process, the present invention has the following beneficial effects: 1) It avoids multiple peptide chain foldings in the peptide sequence of canagliflozin, overcomes the problem of slow coupling rate of protected amino acids one by one, and can obtain crude peptides with much higher purity and yield than those obtained by coupling one by one; 2) During the preparation of peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV of canagliflozin of the present invention, the protected amino acids are all easy to couple. After the peptide resin is cleaved to separate the solid support, solid peptides of fully protected fragment peptides with relatively high purity are easy to precipitate, without the need to use preparative chromatography for purification, saving time and cost; 3) For peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV selected by the present invention, when they are sequentially condensed into long fragments, they can exhibit a very high coupling rate without special conditions. Moreover, due to the growth of the peptide chain, the hydrophobicity of the peptide chain is greatly increased, and it is easier to obtain solid particles with excellent properties from the reaction solvent. Through synthesis and cleavage by this method, a canagliflozin linear peptide crude peptide with fewer impurities, higher purity, and higher yield can be obtained.

[0047] Since DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used as the condensation system in the coupling reaction system for peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV of canagliflozin in solid-phase synthesis, the present invention has the following beneficial effects: It can improve the degree of the coupling reaction, obtain corresponding peptide fragments with relatively high yield and purity, and further improve the yield of the canagliflozin linear peptide crude peptide, and the prepared canagliflozin linear peptide crude peptide has relatively high purity.

[0048] The present invention provides a method for synthesizing canagliflozin. The inventive method can avoid problems such as peptide chain folding and coupling difficulties caused by solid-phase sequential coupling of amino acids, can improve the reaction rate, is easy to scale up for production, and the obtained canagliflozin crude product is easy to purify, with high purity and yield.

[0049] As used herein, the following abbreviations have the meanings given herein: "FMOC" means 9-fluorenylmethoxycarbonyl, "Trt" means triphenylmethyl, "Boc" means tert-butoxycarbonyl, "tBu" means tert-butyl, "OtBu" means tert-butyl ester group, "HOBt" means 1-hydroxybenzotriazole, "EDT" means 1,2-ethanedithiol, "DODT" means 3,6-dioxa-1,8-octanedithiol, "DTT" means dithiothreitol, "Pip" means piperidine, "DIC" means N,N'-diisopropylcarbodiimide, "TFA" means trifluoroacetic acid, "Tis" means triisopropylsilane, "PhOH" means phenol, "DMF" means N,N'-dimethylformamide, "Eic or Eicosandioic acid" means eicosanedioic acid, "Eicosandioic acid, 1-(1,1-dimethyl)ester" means eicosanedioic acid monoter-butyl ester, "Pbf" means 2,2,4,6,7-pentamethyl-2,3-dihydrobenz[B]furanyl, "PyOxim" means cyanato(hydroxyimino)acetato-O2]tri-1-pyrrolidinyl hexafluorophosphate, "PyBOP" means 1H-benzotriazol-1-yloxytrispyrrolidinyl hexafluorophosphate, "HBTU" means benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate, "DIEA" means N,N'-diisopropylethylamine, "Cl-HOBt" means 6-chloro-1-hydroxybenzotriazole, "HOAT" means 1-hydroxy-7-azabenzotriazole, "TBTU" means O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate, "HATU" means 2-(7-azabenzotriazol)-N,N,N',N'-tetramethylurea hexafluorophosphate, "PyAOP" means (3H-1,2,3-triazolo[4,5-b]pyridin-3-yloxy)tri-1-pyrrolidinyl hexafluorophosphate, "Oxyma Pure" means ethyl 2-cyano-2-hydroxyiminoacetate, "MeOH" means methanol, "PhOH" means phenol, "PhSMe" means benzyl methyl sulfide, "MTBE" means methyl tert-butyl ether, "THF" means tetrahydrofuran, "DMSO" means dimethyl sulfoxide, "NMP" means N-methylpyrrolidone, "DCM" means dichloromethane, "2-CTC Resin" means 2-chlorotrichloroethylene resin, "Sieber Resin" means Sieber amide resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the measurement result of the weight yields of peptide resin I and peptide fragment I in Test Example 1 of the present invention.

[0051] Figure 2 This is the measurement result of the purity of peptide fragment I in Test Example 1 of the present invention.

[0052] Figure 3 This is the measurement result of the purity of the crude linear peptide of calyxtide in Test Example 2 of the present invention. Detailed implementation manners

[0053] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0054] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and features in the embodiments of the present application can be combined with each other. Next, the present application will be described in detail with reference to the accompanying drawings and embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments: In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are obtained by purchasing in the market.

[0055] Example 1: 1. Preparation of peptide resin I: 1.1 Weigh 3.0 g (scale 3.0 mmol) of 2-CTC Resin with a substitution degree of 1.0 mmol / g into a solid-phase reaction column, and swell the resin with 15 mL of DMF for 30 minutes and then evacuate the solution under vacuum. Weigh 1.87 g of Fmoc-Ala-OH and 1.4 mL of DIEA, dissolve them with 12 mL of DMF, add the mixture to the reaction column, react under nitrogen bubbling at 25 °C for 4 h, then add 3.0 mL of MeOH, and react under nitrogen bubbling for 30 min; after the reaction, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0056] 1.2 Add 15 mL of a 20% Pip / DMF solution by volume to the resin to remove Fmoc for 30 min, and wash with 15 mL of DMF 6 times. Weigh 3.53 g of Fmoc-Cys(Trt)-OH and 0.85 g of Oxyma Pure, dissolve them in 12 mL of DMF and pour them into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0057] 1.3 Repeat the steps of removing Fmoc, coupling, and washing in 1.2. Coupling Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Glu-OtBu in sequence according to the peptide sequence of peptide fragment I. Then modify Eicosandioic acid, 1-(1,1-dimethyl)ester according to this step. After the modification is completed, contract with 15 mL of MeOH three times and drain to dryness to obtain peptide resin I.

[0058] 2. Preparation of peptide fragment I: Weigh 9.0 g of peptide resin I and pour it into a reaction flask containing 90 mL of a 1% (v / v) TFA / DCM solution. Stir and react at 20 °C for 1 h. After reaching the time, filter the reaction solution. After adding 2.0 mL of DIEA to the filtrate, rotary evaporate under reduced pressure until about 30 mL remains, then add 30 mL of MeOH and continue rotary evaporation. Repeat rotary evaporation with MeOH three times to obtain about 8 mL of a thick solution containing peptide fragment I. Slowly drop it into 40 mL of purified water, stir, and white solid precipitates. After all the thick solution is added, continue to stir for 30 min, filter, wash the filter cake with 10 mL of purified water twice, and vacuum dry to obtain solid peptide fragment I.

[0059] Example 2: 1. Preparation of peptide resin II: 1.1 Weigh 3.33 g (scale 3.0 mmol) of 2-CTC Resin with a substitution degree of 0.9 mmol / g into a solid-phase reaction column, and swell the resin with 15 mL of DMF for 30 minutes, then evacuate the solution under vacuum. Weigh 2.13 g of Fmoc-Leu-OH and 1.4 mL of DIEA, dissolve them with 12 mL of DMF, add the mixture to the reaction column, react under nitrogen bubbling at 25 °C for 4 h, then add 3.0 mL of MeOH and react under nitrogen bubbling for 30 min. After the reaction is completed, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin three times.

[0060] 1.2 Add 15 mL of a 20% (v / v) Pip / DMF solution to the resin to remove Fmoc for 30 min, wash with 15 mL of DMF six times. Weigh 2.31 g of Fmoc-Phe-OH and 0.85 g of Oxyma Pure, dissolve them clearly with 12 mL of DMF and pour them into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin three times.

[0061] 1.3 Repeat the steps of removing Fmoc, coupling, and washing in 1.2, and sequentially couple Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Thr(tBu)-OH according to the peptide sequence of peptide fragment Ⅱ. Finally, contract with 150 mL of MeOH three times and drain to obtain peptide resin Ⅱ.

[0062] 2. Preparation of peptide fragment Ⅱ Weigh 8.0 g of peptide resin Ⅱ and pour it into a reaction flask containing 80 mL of a 1% (v / v) TFA / DCM solution. Stir and react at 20 °C for 1 h. After the reaction time is reached, filter the reaction solution. After adding 2.0 mL of DIEA to the filtrate, rotary evaporate under reduced pressure until about 25 mL remains. Then add 25 mL of MeOH and continue rotary evaporation. Repeat the rotary evaporation with MeOH three times to obtain about 5 mL of a thick solution containing peptide fragment Ⅱ. Slowly drop this thick solution into 25 mL of purified water, stir, and a white solid will precipitate. After all the thick solution is added, continue stirring for 30 min, filter, wash the filter cake twice with 10 mL of purified water, and vacuum dry to obtain the solid of peptide fragment Ⅱ.

[0063] Example 3: 1. Preparation of peptide resin Ⅲ: 1.1 Weigh 3.8 g (scale 3.0 mmol) of 2-CTC Resin with a substitution degree of 0.8 mmol / g into a solid-phase reaction column, and swell the resin with 15 mL of DMF for 30 minutes, then evacuate the solution under vacuum. Weigh 1.78 g of Fmoc-Gly-OH and 1.4 mL of DIEA, dissolve them in 12 mL of DMF, add the mixture to the reaction column, react under nitrogen bubbling at 25 °C for 4 h, then add 3.0 mL of MeOH and react under nitrogen bubbling for 30 min. After the reaction is completed, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin three times.

[0064] 1.2 Add 15 mL of a 20% (v / v) Pip / DMF solution to the resin to remove Fmoc for 30 min, wash with 15 mL of DMF six times. Weigh 2.33 g of Fmoc-Phe-OH and 0.86 g of Oxyma Pure, dissolve them in 12 mL of DMF until clear, then pour the solution into the reaction column, bubble nitrogen, and then add 0.9 mL of DIC. React at 25 °C for 2 h, then evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin three times.

[0065] 1.3 Repeat the steps of removing Fmoc, coupling, and washing in 1.2, and sequentially couple Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH according to the peptide sequence of peptide fragment Ⅲ. Finally, contract with 15 mL of MeOH three times and drain to dryness to obtain peptide resin Ⅲ.

[0066] 2. Preparation of peptide fragment Ⅲ Weigh 8.5 g of peptide resin Ⅲ and pour it into a reaction flask containing 85 mL of a 1% (v / v) TFA / DCM solution. Stir and react at 20 °C for 1 h. After the reaction time is reached, filter the reaction solution. After adding 2.0 mL of DIEA dropwise to the filtrate, rotary evaporate under reduced pressure until about 30 mL remains, then add 30 mL of MeOH and continue rotary evaporation. Repeat rotary evaporation with MeOH three times to obtain about 8 mL of a thick solution containing peptide fragment Ⅲ. Slowly drop it into 40 mL of purified water, stir to precipitate a white solid. After all the thick solution is added, continue stirring for 30 min, filter, wash the filter cake twice with 10 mL of purified water, and vacuum dry to obtain the solid of peptide fragment Ⅲ.

[0067] Example 4: 1. Preparation of peptide resin Ⅳ: 1.1 Weigh 3.9 g (scale 3.0 mmol) of Sieber Resin with a substitution degree of 0.77 mmol / g into a solid-phase reaction column, and swell the resin with 20 mL of DMF for 30 minutes, then evacuate the solution under vacuum.

[0068] 1.2 Add 20 mL of a 20% (v / v) Pip / DMF solution to the resin to remove Fmoc for 30 min, wash with 20 mL of DMF six times. Weigh 2.03 g of Fmoc-Pro-OH and 0.85 g of Oxyma Pure, dissolve them in 15 mL of DMF and pour them into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 20 mL of DMF to wash the resin three times.

[0069] 1.3 Repeat the steps of removing Fmoc, coupling, and washing in 1.2. Sequentially couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Pro-OH according to the peptide sequence of peptide fragment Ⅳ. After the coupling is completed, add 20 mL of a 20% Pip / DMF solution by volume to remove Fmoc for 30 min, wash 6 times with 20 mL of DMF, and finally contract 3 times with 20 mL of MeOH and drain to obtain peptide resin Ⅳ.

[0070] 2. Preparation of peptide fragment Ⅳ: Weigh 9.5 g of peptide resin Ⅳ and pour it into a reaction flask containing 95 mL of a 5% TFA / DCM solution by volume. Stir and react at 20 °C for 1 h; after reaching the time, filter the reaction solution. After adding 11 mL of DIEA dropwise to the filtrate, rotary evaporate under reduced pressure until about 20 mL remains, then add 20 mL of MeOH and continue rotary evaporation. Repeat rotary evaporation with MeOH 3 times to obtain about 5 mL of a thick solution of peptide fragment Ⅳ. Slowly drop it into 50 mL of MTBE, stir, and precipitate a white solid; after all the thick solution is added, continue to stir for 30 min, filter, wash the filter cake by slurrying with 10 mL of MTBE 2 times, and vacuum dry to obtain the solid of peptide fragment Ⅳ, weighing 5.58 g.

[0071] Example 5: 1. Preparation of peptide fragment Ⅴ: Weigh 4.70 g of peptide fragment Ⅳ prepared in Example 4, 5.26 g of peptide fragment Ⅲ prepared in Example 3, and 1.38 g of HBTU into a 100 mL round-bottom flask. After adding 50 mL of DMF and stirring until clear, cool the water bath to 2 °C, and gradually add 1.7 mL of DIEA dropwise, controlling the temperature at 0 - 5 °C. After dropping, maintain stirring at 5 °C for 1.0 h. Then add 10 mL of Pip to the solution and stir at 25 °C for 1.0 h. After that, add 50 mL of DMF to the reaction solution, mix well, and cool to 10 °C. Then gradually add it dropwise to 500 mL of a saturated citric acid aqueous solution, control the temperature of the water bath at 15 °C, and precipitate a white solid. After the reaction solution is dropped, maintain stirring at 10 °C for 1.0 h, filter, vacuum dry the filter cake, take out the filter cake, and add 50 mL of purified water to slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water 2 times and then drain, and then transfer it to a blast drying oven at 45 °C and dry to constant weight to obtain peptide fragment Ⅴ.

[0072] 2. Preparation of peptide fragment VI: Weigh 9.13 g of the above-prepared peptide fragment V into a 200 mL round-bottom flask, add 100 mL of THF, and dissolve it at 25 °C. Then weigh 4.55 g of the peptide fragment II prepared in Example 2 and 1.91 g of PyOxim, and pour them into the THF solution of peptide fragment V to dissolve. Cool the solution to 2 °C in a water bath, and then add 1.7 mL of DIEA dropwise while controlling the temperature at 0 - 5 °C. After the addition, stir the reaction at 5 °C for 4.0 h. Then add 20 mL of Pip to the solution and stir the reaction at 25 °C for 2.0 h. Then add the reaction solution dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate white solid. After the addition of the reaction solution is completed, stir at 10 °C for 1.0 h, filter, vacuum dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a 45 °C blast drying oven and dry it to constant weight to obtain peptide fragment VI.

[0073] 3. Preparation of canagliflozin fully protected peptide: Weigh 12.35 g of the above-prepared peptide fragment VI, 5.59 g of the peptide fragment I prepared in Example 1, and 1.31 g of HBTU into a 200 mL round-bottom flask, dissolve it with 120 mL of THF at 25 °C, cool it to -8 °C, and then add 1.6 mL of DIEA dropwise. Control the temperature at -10 - -5 °C during the addition process, stir the reaction for 2.0 h, and then add the reaction solution dropwise to 600 mL of saturated citric acid aqueous solution. Control the temperature at 15 °C in a water bath to precipitate white solid. After the addition of the reaction solution is completed, stir at 10 °C for 1.0 h, filter, vacuum dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a 45 °C blast drying oven and dry it to constant weight to obtain canagliflozin fully protected peptide.

[0074] 4. Preparation of canagliflozin linear peptide crude peptide: Weigh 15.0 g of the above-prepared canagliflozin fully protected peptide, pour it into 120 mL of a cleavage solution of TFA / EDT / TIS / H2O with a volume ratio of 85:5:5:5, stir the reaction at 25 °C for 2.0 h. After the reaction time is reached, filter and wash the filter cake with TFA; pour the obtained filtrate into 960 mL of MTBE at -5 °C to obtain a suspension with white solid precipitated. Then, after centrifugal sedimentation and slurry washing, dry it to constant weight in a vacuum drying oven at 25 °C to obtain canagliflozin linear peptide crude peptide.

[0075] Example 6: Preparation of canagliflozin finished product: Weigh 8.0 g of the crude linear peptide of canagliflozin prepared in Example 5, dissolve it in 800 mL of an acetonitrile / water solution with a volume ratio of 80%, and then dilute it with purified water to a concentration of 1 g / L. Then, dropwise add a 1 mmol / mL iodine / acetonitrile solution until the cyclization solution turns light yellow, and stir to keep the color from fading for 30 min. Finally, add a 1 mmol / mL vitamin C aqueous solution dropwise until the cyclization solution becomes clear and colorless to obtain a solution of the crude cyclic peptide of canagliflozin.

[0076] Filter the solution of the crude cyclic peptide of canagliflozin through a mixed microporous membrane with a pore size of 0.45 µm. Purification and preparation are carried out by high performance liquid chromatography, and the canagliflozin fraction is collected according to the position of the reference substance.

[0077] Purification analysis method: C18 chromatographic column:

[0078] The purified canagliflozin fraction is desalted, transferred to salt, concentrated, and freeze-dried to obtain 2.71 g of the canagliflozin finished product, with a yield of 33.88% and a purity of 99.67%.

[0079] Example 7: The difference between this example and Example 1 is that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used to replace Oxyma Pure for the activation of the protected amino acid monomer.

[0080] Preparation method of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol: Take 1.6 g of Fe(NO3)3·9H2O and 0.32 g of tetramethylpiperidine oxide, dissolve them in dichloroethane, introduce oxygen, heat to 80 °C, add 0.53 g of 6-bromo-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-A]pyrazine, react for 5 h, cool to room temperature, filter by suction, wash once with ethanol and twice with water, dry, and separate and purify by silica gel column chromatography with a petroleum ether / ethyl acetate volume ratio of 8:2 as the eluent to obtain 6-bromo-5-nitro-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine. 1 1H NMR (CDCl3, 400 MHz): δ 8.82 (1H, d). EI-MS m / z: 310.93 M peak, relative intensity 100%, 311.93 M+1 peak, relative intensity 8.4%, 312.93 M+2 peak, relative intensity 97.3%. Obtain 6-bromo-5-nitro-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine.

[0081] Add 312 mg of 6-bromo-5-nitro-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine, 200 mg of hydrazine hydrate and 280 mg of isopentanol into a 250 mL three-necked flask, heat for reaction and separate water. Heat to 100 °C and react for 2 h. After the reaction is completed, neutralize with 40 wt% NaOH solution, distill off the excessive hydrazine hydrate and isopentanol, adjust the pH value to 3.0 with hydrochloric acid, filter by suction, wash with 5 wt% ice brine, recrystallize, and dry to obtain 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. 1 1H NMR (CDCl3, 400 MHz): δ 8.82 (1H, s), δ 2.78 (1H, s). EI-MS m / z: 245.03 M peak, relative intensity 100%, 246.03 M+1 peak, relative intensity 9.2%.

[0082] Example 8: The difference between this example and Example 2 is that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used to replace Oxyma Pure for the activation of protected amino acid monomers.

[0083] Example 9: The difference between this example and Example 3 is that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used to replace Oxyma Pure for the activation of protected amino acid monomers.

[0084] Example 10: The difference between this example and Example 4 is that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used to replace Oxyma Pure for the activation of protected amino acid monomers.

[0085] Example 11: The difference between this example and Example 1 is in Step 1.2 of the preparation of peptide resin I: Add 15 mL of a 20% Pip / DMF solution to the resin to deprotect Fmoc for 30 min, and wash with 15 mL of DMF for 6 times. Weigh 3.53 g of Fmoc-Cys(Trt)-OH and 0.85 g of Oxyma Pure, dissolve them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1, pour the solution into the reaction column, purge with nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0086] Example 12: This example is different from Example 1 in Step 1.2 of the preparation of peptide resin Ⅰ: Add 15 mL of a 20% Pip / DMF solution to the resin to remove Fmoc for 30 min, and wash with 15 mL of DMF 6 times. Weigh 3.53 g of Fmoc-Cys(Trt)-OH and 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. After dissolving them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1 and pouring it into the reaction column, purge with nitrogen, then add 0.9 mL of DIC. After reacting at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0087] Example 13: This example is different from Example 2 in Step 1.2 of the preparation of peptide resin Ⅱ: Add 15 mL of a 20% Pip / DMF solution to the resin to remove Fmoc for 30 min, and wash with 15 mL of DMF 6 times. Weigh 2.31 g of Fmoc-Phe-OH and 0.85 g of Oxyma Pure. After dissolving them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1 and pouring it into the reaction column, purge with nitrogen, then add 0.9 mL of DIC. After reacting at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0088] Example 14: This example is different from Example 2 in Step 1.2 of the preparation of peptide resin Ⅱ: Add 15 mL of a 20% Pip / DMF solution to the resin to remove Fmoc for 30 min, and wash with 15 mL of DMF 6 times. Weigh 2.31 g of Fmoc-Phe-OH and 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. After dissolving them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1 and pouring it into the reaction column, purge with nitrogen, then add 0.9 mL of DIC. After reacting at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0089] Example 15: The difference between this example and Example 3 lies in Step 1.2 of the preparation of peptide resin III: Add 15 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 15 mL of DMF 6 times, weigh 2.33 g of Fmoc-Phe-OH, 0.86 g of Oxyma Pure, dissolve them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1, pour the solution into the reaction column, purge with nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0090] Example 16: The difference between this example and Example 3 lies in Step 1.2 of the preparation of peptide resin III: Add 15 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 15 mL of DMF 6 times, weigh 2.33 g of Fmoc-Phe-OH, 1.48 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol, dissolve them in 12 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1, pour the solution into the reaction column, purge with nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0091] Example 17: The difference between this example and Example 4 lies in Step 1.2 of the preparation of peptide resin IV: Add 20 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 20 mL of DMF 6 times, weigh 2.03 g of Fmoc-Pro-OH, 0.85 g of Oxyma Pure, dissolve them in 15 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1, pour the solution into the reaction column, purge with nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 20 mL of DMF to wash the resin 3 times.

[0092] Example 18: The difference between this example and Example 4 lies in Step 1.2 of the preparation of peptide resin IV: Add 20 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 20 mL of DMF 6 times. Weigh 2.03 g of Fmoc-Pro-OH and 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. After dissolving them in 15 mL of a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3:1:1 and clarifying, pour it into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 20 mL of DMF to wash the resin 3 times.

[0093] Example 19: The difference between this example and Example 1 lies in Step 1.2 of the preparation of peptide resin I: Add 15 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 15 mL of DMF 6 times. Weigh 3.53 g of Fmoc-Cys(Trt)-OH and 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. After dissolving them in 12 mL of a DMF / 4-isobutylacetophenone solution with a volume ratio of 3:1 and clarifying, pour it into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0094] Example 20: The difference between this example and Example 1 lies in Step 1.2 of the preparation of peptide resin I: Add 15 mL of a 20% Pip / DMF solution by volume to the resin to deprotect Fmoc for 30 min, wash with 15 mL of DMF 6 times. Weigh 3.53 g of Fmoc-Cys(Trt)-OH and 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. After dissolving them in 12 mL of a DMF / 2-pentylpyridine solution with a volume ratio of 3:1 and clarifying, pour it into the reaction column, bubble nitrogen, then add 0.9 mL of DIC, react at 25 °C for 2 h, evacuate the reaction solution under vacuum, and add 15 mL of DMF to wash the resin 3 times.

[0095] Example 21: The difference between this example and Example 5 lies in the preparation of peptide fragment V, peptide fragment VI, and the fully protected peptide of canagliflozin: 1. Preparation of peptide fragment V: Weigh 4.70 g of peptide fragment IV prepared in Example 10, 5.26 g of peptide fragment III prepared in Example 9, and 1.38 g of HBTU into a 100 mL round-bottom flask. Add 50 mL of DMF and stir until dissolved and clear. Then cool the solution to 2 °C in a water bath. Dropwise add 1.7 mL of DIEA while controlling the temperature at 0 - 5 °C. After the addition, maintain stirring at 5 °C for 1.0 h. Then add 10 mL of Pip to the solution and stir at 25 °C for 1.0 h. After that, add 50 mL of DMF to the reaction solution, mix well, and cool to 10 °C. Then dropwise add the solution to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate a white solid. After the addition of the reaction solution is complete, maintain stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it by suction. Then transfer it to a blast drying oven at 45 °C and dry to a constant weight to obtain peptide fragment V.

[0096] 2. Preparation of peptide fragment VI: Weigh 9.13 g of the above-prepared peptide fragment V into a 200 mL round-bottom flask, add 100 mL of THF and dissolve it clearly at 25 °C. Then weigh 4.55 g of peptide fragment II prepared in Example 8 and 1.91 g of PyOxim, and pour them into the THF solution of peptide fragment V to dissolve clearly. Cool the solution to 2 °C in a water bath, then dropwise add 1.7 mL of DIEA while controlling the temperature at 0 - 5 °C. After the addition, maintain stirring at 5 °C for 4.0 h. Then add 20 mL of Pip to the solution and stir at 25 °C for 2.0 h. After that, dropwise add the reaction solution to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate a white solid. After the addition of the reaction solution is complete, maintain stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it by suction. Then transfer it to a blast drying oven at 45 °C and dry to a constant weight to obtain peptide fragment VI.

[0097] 3. Preparation of canagliflozin fully protected peptide: Weigh 12.35 g of the above-prepared peptide fragment VI, 5.59 g of peptide fragment I prepared in Example 7, and 1.31 g of HBTU into a 200 mL round-bottom flask. Dissolve it clearly with 120 mL of THF at 25 °C, then cool to -8 °C, and dropwise add 1.6 mL of DIEA while controlling the temperature at -10 to -5 °C during the addition. Stir and react for 2.0 h, then dropwise add the reaction solution to 600 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate a white solid. After the addition of the reaction solution is complete, maintain stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it by suction. Then transfer it to a blast drying oven at 45 °C and dry to a constant weight to obtain canagliflozin fully protected peptide.

[0098] Example 22: The difference between this example and Example 5 lies in the preparation of peptide fragment V, peptide fragment VI and the fully protected peptide of canagliflozin: 1. Preparation of peptide fragment V: Weigh 4.70 g of peptide fragment IV prepared in Example 17, 5.26 g of peptide fragment III prepared in Example 15, and 1.38 g of HBTU into a 100 mL round-bottom flask. After adding 50 mL of DMF and stirring until clear, cool the solution to 2 °C in a water bath. Then, dropwise add 1.7 mL of DIEA while controlling the temperature at 0 - 5 °C. After the addition, maintain stirring at 5 °C for 1.0 h. Next, add 10 mL of Pip to the solution and stir at 25 °C for 1.0 h. Then, add 50 mL of DMF to the reaction solution, mix well, and cool it to 10 °C. Then, dropwise add the solution to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate a white solid. After the addition of the reaction solution is complete, maintain stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and stir overnight; filter the slurry, wash the filter cake twice with 30 mL of purified water and then dry it by suction. Then, transfer it to a blast drying oven at 45 °C and dry it to a constant weight to obtain peptide fragment V.

[0099] 2. Preparation of peptide fragment VI: Weigh 9.13 g of the above-prepared peptide fragment V into a 200 mL round-bottom flask, add 100 mL of THF and dissolve it at 25 °C. Then, weigh 4.55 g of peptide fragment II prepared in Example 13 and 1.91 g of PyOxim, and pour them into the THF solution of peptide fragment V to dissolve. Cool the solution to 2 °C in a water bath, then dropwise add 1.7 mL of DIEA while controlling the temperature at 0 - 5 °C. After the addition, maintain stirring at 5 °C for 4.0 h. Next, add 20 mL of Pip to the solution and stir at 25 °C for 2.0 h. Then, dropwise add the reaction solution to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate a white solid. After the addition of the reaction solution is complete, maintain stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and stir overnight; filter the slurry, wash the filter cake twice with 30 mL of purified water and then dry it by suction. Then, transfer it to a blast drying oven at 45 °C and dry it to a constant weight to obtain peptide fragment VI.

[0100] 3. Preparation of fully protected cagrilintide peptide: Weigh 12.35 g of the peptide fragment VI prepared above, 5.59 g of the peptide fragment I prepared in Example 11, and 1.31 g of HBTU into a 200 mL round-bottom flask. Dissolve them clearly with 120 mL of THF at 25 °C, cool down to -8 °C, and then dropwise add 1.6 mL of DIEA. During the dropping process, control the water bath temperature at -10~-5 °C. After stirring and reacting for 2.0 h, dropwise add the reaction solution into 600 mL of saturated citric acid aqueous solution. Control the water bath temperature at 15 °C to precipitate white solid. After the reaction solution is added dropwise, keep stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a blast drying oven at 45 °C and dry to constant weight to obtain the fully protected cagrilintide peptide.

[0101] Example 23: The difference between this example and Example 5 lies in the preparation of peptide fragment V, peptide fragment VI and fully protected cagrilintide peptide: 1. Preparation of peptide fragment V: Weigh 4.70 g of the peptide fragment IV prepared in Example 18, 5.26 g of the peptide fragment III prepared in Example 16, and 1.38 g of HBTU into a 100 mL round-bottom flask. After adding 50 mL of DMF and stirring to dissolve clearly, cool down the water bath to 2 °C, dropwise add 1.7 mL of DIEA, control the temperature at 0~5 °C, keep stirring at 5 °C for 1.0 h after dropping, then add 10 mL of Pip to the solution, keep stirring at 25 °C for 1.0 h, then add 50 mL of DMF to the reaction solution, mix well and cool down to 10 °C, and then dropwise add it into 500 mL of saturated citric acid aqueous solution. Control the water bath temperature at 15 °C to precipitate white solid. After the reaction solution is added dropwise, keep stirring at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a blast drying oven at 45 °C and dry to constant weight to obtain peptide fragment V.

[0102] 2. Preparation of Peptide Fragment VI: Weigh 9.13 g of the above-prepared peptide fragment V into a 200 mL round-bottom flask, add 100 mL of THF and dissolve it at 25 °C. Then weigh 4.55 g of peptide fragment II prepared in Example 14 and 1.91 g of PyOxim, and pour them into the THF solution of peptide fragment V to dissolve. Cool the solution to 2 °C in a water bath, and then dropwise add 1.7 mL of DIEA while controlling the temperature at 0 - 5 °C. After the addition, stir the reaction at 5 °C for 4.0 h. Then add 20 mL of Pip to the solution and stir the reaction at 25 °C for 2.0 h. Then slowly add the reaction solution dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate white solid. After the addition of the reaction solution is completed, stir at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a 45 °C forced-air drying oven and dry it to constant weight to obtain peptide fragment VI.

[0103] 3. Preparation of Canagliflozin Fully Protected Peptide: Weigh 12.35 g of the above-prepared peptide fragment VI, 5.59 g of peptide fragment I prepared in Example 12, and 1.31 g of HBTU into a 200 mL round-bottom flask, dissolve them with 120 mL of THF at 25 °C, cool to -8 °C, and then dropwise add 1.6 mL of DIEA while controlling the temperature at -10 - -5 °C during the addition. Stir the reaction for 2.0 h, then slowly add the reaction solution dropwise to 600 mL of saturated citric acid aqueous solution, control the temperature at 15 °C in a water bath to precipitate white solid. After the addition of the reaction solution is completed, stir at 10 °C for 1.0 h, filter, vacuum-dry the filter cake, take out the filter cake, add 50 mL of purified water and slurry overnight; filter the slurry, wash the filter cake with 30 mL of purified water twice and then dry it, and then transfer it to a 45 °C forced-air drying oven and dry it to constant weight to obtain canagliflozin fully protected peptide.

[0104] Test Example 1: 1. Determine the weight yield of peptide resin I prepared in Example 1, Example 7, Example 11, Example 12, Example 19, and Example 20; after soft cleavage of peptide resin I, peptide fragment I is obtained, and determine the weight yield of peptide fragment I in the soft cleavage step. The determination results are shown in Figure 1 。

[0105] From Figure 1It can be seen that the weight yields of peptide fragment Ⅰ prepared in Example 1, Example 7, Example 11, Example 12, Example 19, and Example 20 do not differ significantly. Compared with Example 1, the weight yield of peptide resin Ⅰ prepared in Example 7 is higher, indicating that 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol with the same molar equivalent has a better amino acid activation effect than Oxyma Pure, resulting in a higher degree of amino acid coupling; compared with Example 1, the weight yield of peptide resin Ⅰ prepared in Example 11 is slightly lower, compared with Example 7, the weight yield of peptide resin Ⅰ prepared in Example 12 is higher, and compared with Example 7, the difference in the weight yields of peptide resin Ⅰ prepared in Example 19 and Example 20 is not significant, indicating that when 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used as an activator in the amino acid coupling reaction system during solid-phase synthesis, a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3-4:1-2:1 as the reaction solution can improve its activation effect on amino acids and further increase the degree of amino acid coupling.

[0106] 2. The purity of peptide fragment Ⅰ prepared in Example 1, Example 7, Example 11, Example 12, Example 19, and Example 20 was measured, and the measurement results are shown in Figure 2 .

[0107] It can be seen from Figure 2 that the purity of peptide fragment Ⅰ prepared in Example 7 is greater than that in Example 1, indicating that compared with Oxyma Pure with the same molar equivalent, the amino acid coupling reaction using 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is more complete, with fewer by-products, and the purity and yield of peptide fragment Ⅰ are higher; there is no obvious difference in the purity of peptide fragment Ⅰ prepared in Example 1 and Example 11. Compared with Example 7, Example 19, and Example 20, the purity of peptide fragment Ⅰ prepared in Example 12 is higher, and the difference between Example 19, Example 20 and Example 7 is not significant, indicating that when 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used as an activator in the amino acid coupling reaction system during solid-phase synthesis, a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3-4:1-2:1 as the reaction solution can promote the amino acid coupling reaction and improve the yield and purity of peptide fragment Ⅰ.

[0108] Test Example 2: The purity of the crude linear peptide of canagliflozin prepared in Example 5, Example 21, Example 22, and Example 23 was measured, and the measurement results are shown in Figure 3。

[0109] It can be seen from Figure 3 that, compared with Example 5, the crude linear peptide of canagliflozin prepared in Example 21 has a higher purity, indicating that compared with Oxyma Pure with the same molar equivalent, the amino acid coupling reaction using 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is more complete, with fewer by-products, resulting in a higher purity of the peptide fragment, and thus a higher purity of the crude linear peptide of canagliflozin.

[0110] Compared with Example 21, the crude linear peptide of canagliflozin prepared in Example 23 has a higher purity, and the purity of the crude linear peptide of canagliflozin prepared in Example 5 is slightly higher than that of the crude linear peptide of canagliflozin prepared in Example 22, indicating that when 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is used as an activator in the amino acid coupling reaction system during solid-phase synthesis, the DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3-4:1-2:1 as the reaction solution can promote the amino acid coupling reaction, improve the purity of the corresponding peptide fragment, and thus improve the purity of the crude linear peptide of canagliflozin.

[0111] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any form of limitation on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0112] Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of written expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A synthetic method of canagliflozin, characterized in that, It includes the following steps: Step 1: Peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV are respectively synthesized by solid-phase methods; Step 2: Peptide fragment III and peptide fragment IV are ligated in liquid phase to obtain peptide fragment V, then peptide fragment V is ligated with peptide fragment II in liquid phase to obtain peptide fragment VI, and finally peptide fragment VI is ligated with peptide fragment I in liquid phase to obtain the fully protected canagliflozin peptide; Step 3: The fully protected canagliflozin peptide is added to the cleavage solution, and after precipitation, pulping and washing, the crude canagliflozin linear peptide is obtained; Step 4: The crude canagliflozin linear peptide is cyclized, and then through separation and purification, salt conversion, concentration, and freeze-drying, the canagliflozin finished product is obtained; The peptide fragment Ⅰ is Eic-(OtBu)-Glu 1 -(OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu) 5 -Ala-Thr(tBu)-Cys(Trt)-Ala 9 -OH; The peptide fragment II is Fmoc-Thr(tBu) 10 -Gln(Trt)-Arg(Pbf)-Leu-Ala-Glu(OtBu) 15 -Phe-Leu 17 -OH; The peptide fragment Ⅲ is Fmoc-Arg(Pbf) 18 -His(Boc)-Ser(tBu) 20 -Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly 25 -OH; The peptide fragment Ⅳ is NH2-Pro 26 -Ile-Leu-Pro-Pro 30 -Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu) 35 -Asn(Trt)-Thr(tBu)-Pro 38 -NH2。 2. The synthesis method according to claim 1, characterized in that, The said Step 1 includes: S1. Use 2-CTC resin as the solid-phase carrier, adopt the Fmoc solid-phase synthesis strategy, and sequentially couple amino acids according to the sequence of peptide fragment I to obtain Eic-(OtBu)-Glu 1 -(OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu) 5 -Ala-Thr(tBu)-Cys(Trt)-Ala 9 -CTC Resin, namely peptide resin I, and peptide resin I is obtained by soft cleavage to obtain peptide fragment I; S2. Using 2-CTC resin as the solid-phase carrier and the Fmoc solid-phase synthesis strategy, amino acids are sequentially coupled according to the sequence of peptide fragment Ⅱ to obtain Fmoc-Thr(tBu) 10 -Gln(Trt)-Arg(Pbf)-Leu-Ala-Glu(OtBu) 15 -Phe-Leu 17 -CTC Resin, that is, peptide resin Ⅱ. Peptide resin Ⅱ is obtained by soft cleavage to obtain peptide fragment Ⅱ; S3. Use 2-CTC resin as the solid-phase carrier, adopt the Fmoc solid-phase synthesis strategy, and sequentially couple amino acids according to the sequence of peptide fragment III to obtain Fmoc-Arg(Pbf) 18 -His(Boc)-Ser(tBu) 20 -Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly 25 -CTC Resin, namely peptide resin III, and peptide resin III is obtained by soft cleavage to obtain peptide fragment III; S4. Using Sieber amide resin as the solid-phase carrier and the Fmoc solid-phase synthesis strategy, amino acids are sequentially coupled according to the sequence of peptide fragment Ⅳ to obtain NH2-Pro 26 -Ile-Leu-Pro-Pro 30 -Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu) 35 -Asn(Trt)-Thr(tBu)-Pro 38 -Sieber Resin, namely peptide resin Ⅳ. Peptide resin Ⅳ is subjected to mild cleavage to obtain peptide fragment Ⅳ.

3. The synthesis method according to claim 2, characterized in that, The specific preparation method of the said peptide resin I includes: a. After swelling 2-CTC resin with DMF for 25 - 35 min, the first protected amino acid monomer Fmoc-Ala-OH was introduced to obtain Fmoc-Ala 9 -CTC Resin; b. Deprotect the Fmoc group on the resin, wash it 5 - 6 times with DMF, and perform a coupling reaction with the next protected amino acid monomer Fmoc-Cys(Trt)-OH to obtain Fmoc-Cys(Trt)-Ala 9 -CTC Resin; c. Sequentially and repeatedly connect protected amino acid monomers according to Step b, and finally connect monoterbutyl docosanedioate according to the method of Step b to obtain peptide resin I.

4. The synthesis method according to claim 3, characterized in that: In the said Step a, the condensing agent used when the 2-CTC resin connects the first protected amino acid monomer is DIEA, and the reaction solvent is DMF.

5. The synthesis method according to claim 3, characterized in that: In the said Step b, the condensation system for the coupling reaction is DIC / Oxyma Pure or DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol.

6. The synthesis method according to claim 1, wherein The said Step 2 includes: i. After activating the C-terminal carboxyl group of peptide fragment III with a condensing agent, perform a coupling reaction with the N-terminal amino group of peptide fragment IV in the reaction solution, then remove the Fmoc protecting group and precipitate a solid to obtain peptide fragment V; ii. After activating the C-terminal carboxyl group of peptide fragment II with a condensing agent, perform a coupling reaction with the N-terminal amino group of peptide fragment V in the reaction solution, then remove the Fmoc protecting group and precipitate a solid to obtain peptide fragment VI; iii. After activating the C-terminal carboxyl group of the said peptide fragment I with a condensing agent, perform a coupling reaction with the N-terminal amino group of peptide fragment VI in the reaction solution, then remove the Fmoc protecting group and precipitate a solid to obtain the fully protected canagliflozin peptide.

7. The synthesis method according to claim 6, characterized in that, The condensation systems for the coupling reactions in the said Step i and Step iii are both HBTU / DIEA.

8. The synthesis method according to claim 6, characterized in that, The condensation system for the coupling reaction in the said Step ii is PyOxim / DIEA.

9. The synthesis method according to claim 1, wherein The said Step 3 specifically includes: Stir and react the fully protected canagliflozin peptide with the cleavage solution at 20~30°C for 2~3h. After reaching the reaction time, filter, and rinse the filter cake with TFA; Pour the obtained filtrate into MTBE at -10~0°C to obtain a suspension in which a white solid precipitates, and then obtain the crude canagliflozin linear peptide after centrifugal sedimentation, pulping and washing.

10. The synthesis method according to claim 1, characterized in that, The cyclization method of the crude canagliflozin linear peptide in the said Step 4 is specifically: Dissolve the crude canagliflozin linear peptide in an acetonitrile / water solution with a volume ratio of 75~80%. After dissolving clearly, dilute it with purified water to a concentration of 0.9~1.2 g / L; Then dropwise add an iodine / acetonitrile solution with a concentration of 0.8~1.1 mmol / mL until the cyclization solution shows a light yellow color, and stir to keep the color from fading for 25~32 min. Finally, dropwise add a vitamin C aqueous solution with a concentration of 0.8~1.2 mmol / mL until the cyclization solution is clear and colorless.

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