A synthetic method of canagliflozin

Abstract

The present invention provides a method for synthesizing canagliflozin, which belongs to the technical field of polypeptide synthesis. The method first uses Fmoc solid-phase synthesis of peptide fragments I, II, III, and IV, and then condenses each fragment in sequence. After that, the side chain protecting groups are cracked and removed to obtain a crude linear peptide of canagliflozin, which is then cyclized, separated and purified, converted to salt, concentrated, and lyophilized to obtain a canagliflozin finished product. The present 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 in the coupling reaction system of the Fmoc solid-phase synthesis peptide fragment. The method provides a method for synthesizing canagliflozin, which has the advantages of high reaction rate, low cost, ease of large-scale amplification production, easy purification of the obtained crude product, high purity and yield, low amount of waste liquid, and energy saving and environmental protection.

Description

Technical Field

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

[0002] Cagrilintide is a long-acting acylated amylin analog that acts as a non-selective amylin receptor (AMYR) and calcitonin G-protein-coupled receptor (CTR) agonist. It can reduce energy intake and regulate food choice and preference. It is co-secreted with insulin to exert glucose regulation, inhibit postprandial glucagon release, and delay gastric emptying. It has significant advantages in the treatment of diabetes, obesity, metabolic syndrome, and cardiovascular disease, and has broad application prospects.

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

[0004] The present invention aims to provide a method for synthesizing canagliflozin, which has high reaction rate, low cost, is easy to scale up production, is easy to purify the obtained crude product, has high purity and yield, greatly reduces the amount of waste liquid, and is energy-saving and environmentally friendly.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0006] The present invention discloses a method for synthesizing canagliflozin, which comprises the following steps:

[0007] Step 1, synthesizing peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV respectively using a solid phase method;

[0008] Step 2: Liquid-phase splicing of peptide fragment III and peptide fragment IV to obtain peptide fragment V, then liquid-phase splicing of peptide fragment V and peptide fragment II to obtain peptide fragment VI, and finally liquid-phase splicing of peptide fragment VI and peptide fragment I to obtain canagliflozin fully protected peptide;

[0009] Step 3: adding the fully protected canagliflozin peptide to the lysis solution, and obtaining the crude linear canagliflozin peptide through precipitation, beating and washing;

[0010] Step 4: Cyclize the crude linear peptide of canagliflozin, and then separate, purify, convert to salt, concentrate, and freeze-dry to obtain the finished canagliflozin;

[0011] 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;

[0012] The peptide fragment II is Fmoc-Thr(tBu) 10 -Gln(Trt)-Arg(Pbf)-Leu-Ala-Glu(OtBu) 15 -Phe-Leu 17 -OH;

[0013] The peptide fragment III is Fmoc-Arg (Pbf) 18 -His(Boc)-Ser(tBu) 20 -Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly 25 -OH;

[0014] 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. Canagliflozin is a 38-octadecanopyram peptide, a long peptide with a long carbon chain eicosanedioic acid modified at its N-terminus. Due to the long peptide chain, as the synthetic peptide sequence is extended on the solid-phase resin support, steric hindrance increases. Coupling difficulties, resin shrinkage, and residual reaction solution washing are all key factors that cause impurities to be generated, which will reduce the quality of the crude peptide and increase the difficulty of subsequent purification and removal. In addition, the use of a step-by-step coupling process restricts the efficiency and production capacity of large-scale production. To address the above shortcomings, the method of the present invention divides the canagliflozin peptide sequence into four small fragments, namely peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV. After synthesizing the corresponding fragment peptide resin on a solid-phase support, the fragment solid is separated and each fragment is sequentially condensed. This process can effectively avoid resin shrinkage and coupling problems at difficult sites during the peptide resin coupling process, reduce the number of impurities, improve coupling efficiency, shorten the synthesis cycle, increase the yield, purity, and content of the linear crude peptide, reduce the difficulty of purifying the linear crude peptide, and obtain a final product with high purity and yield.

[0015] In some embodiments, the above step 1 includes:

[0016] S1, using 2-CTC resin as a solid phase support, using Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin I, peptide fragment I is obtained by soft cleavage of peptide resin I;

[0017] S2, using 2-CTC resin as a solid phase support, using the Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin II, peptide resin II is softly cleaved to obtain peptide fragment II;

[0018] S3, using 2-CTC resin as a solid phase support, using the Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin III, peptide resin III is softly cleaved to obtain peptide fragment III;

[0019] S4, using Sieber amide resin as a solid phase support, using Fmoc solid phase synthesis strategy, sequentially coupled 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, namely peptide resin IV, peptide resin IV is softly cleaved to obtain peptide fragment IV.

[0020] In some embodiments, the degree of substitution of the 2-CTC resin is 0.6 to 1.0 mmol / g, preferably 0.8 to 1.0 mmol / g.

[0021] In some embodiments, the degree of substitution of the Sieber amide resin is 0.8-1.0 mmol / g, preferably 0.6-0.8 mmol / g.

[0022] In some embodiments, the lysis solution used for soft lysis in steps S1, S2, and S3 is a TFA / DCM solution with a volume ratio of 1-1.2%.

[0023] In some embodiments, the lysis solution used for the soft lysis in step S4 is a TFA / DCM solution with a volume ratio of 5-5.5%.

[0024] In some embodiments, the specific preparation method of the above-mentioned peptide resin I includes: a. swelling 2-CTC resin with DMF for 25 to 35 minutes, and then inserting 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 to 6 times, and couple it with the next protected amino acid monomer Fmoc-Cys(Trt)-OH to obtain Fmoc-Cys(Trt)-Ala 9 -CTC Resin; c. Repeat step b to sequentially add protected amino acid monomers, and finally add mono-tert-butyl eicosanedioate according to the method of step b to obtain peptide resin I.

[0025] In some embodiments, the specific preparation method of the peptide resin II comprises: a. swelling the 2-CTC resin with DMF for 25 to 35 minutes, then inserting 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 with DMF 5 to 6 times, and couple it with the next protected amino acid monomer Fmoc-Phe-OH, Fmoc-Phe-Leu 17 -CTC Resin; c. Repeat step b to sequentially add protected amino acid monomers to obtain peptide resin II.

[0026] In some embodiments, the specific preparation method of the peptide resin III includes: a. swelling 2-CTC resin with DMF for 25 to 35 minutes, and then inserting 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 with DMF 5 to 6 times, and couple it with the next protected amino acid monomer Fmoc-Phe-OH to obtain Fmoc-Phe-Gly 25 -CTC Resin; c. Repeat step b to sequentially add protected amino acid monomers to obtain peptide resin III.

[0027] In some embodiments, in the specific preparation methods of the above-mentioned peptide resin I, peptide resin II, and peptide resin III, the condensing agent used to connect the first protected amino acid monomer to the 2-CTC resin is DIEA, and the reaction solvent is DMF.

[0028] In some embodiments, the specific preparation method of the peptide resin IV comprises: a. swelling Sieber amide resin with DMF for 25 to 35 minutes; b. removing the Fmoc protection on the resin, washing it with DMF 5 to 6 times, and coupling it with the first protected amino acid Fmoc-Pro-OH to obtain Fmoc-Pro 38 -Sieber Resin; c. Repeat step b to sequentially add protected amino acid monomers. After the coupling is completed, remove the Fmoc protection on the resin to obtain peptide resin IV.

[0029] 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 molar ratio of resin to protected amino acid monomer during the process of sequentially accessing amino acid monomers is 1:1.5~3.

[0030] In some embodiments, the deprotection agent used to remove the Fmoc protection from the resin is a 20% to 30% by volume Pip / DMF solution. Furthermore, the deprotection time is 28 to 32 minutes, and the reaction temperature is 20 to 35°C.

[0031] In some embodiments, in the specific preparation method of the above-mentioned peptide resin I, the condensation system of 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.

[0032] In some embodiments, in the specific preparation method of the above-mentioned peptide resin II, the condensation system of 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.

[0033] 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.

[0034] 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.

[0035] In some embodiments, in the specific preparation methods of the aforementioned peptide resins I, II, III, and IV, the condensation system for the coupling reaction in step b is DIC / Oxyma Pure. Preferably, in the specific preparation methods of the aforementioned peptide resins I, II, III, and IV, in the coupling reaction system in step b, the molar ratio of protected amino acid monomer: DIC: Oxyma Pure is 1:0.8-1.2:0.8-1.2.

[0036] 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 of 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 ester aminolysis rate. 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, resulting in a high degree of amino acid coupling reaction with few by-products, and can obtain peptide fragments I, II, III, and IV with high yield and purity. 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 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.

[0037] 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 in the coupling reaction is DMF.

[0038] In some embodiments, in the specific preparation method of the above-mentioned peptide resin I, the reaction solvent used in the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution in 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 the activating agent, a DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene solution in a volume ratio of 3-4:1-2:1 can better enhance the activation effect of the amino acid reagent, increase the degree of amino acid coupling reaction, and increase the purity and yield of the corresponding peptide fragment.

[0039] In some embodiments, in the specific preparation method of the above-mentioned peptide resin II, the reaction solvent used in the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3~4:1~2:1.

[0040] In some embodiments, in the specific preparation method of the above-mentioned peptide resin III, the reaction solvent used in the coupling reaction in step b is a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3-4:1-2:1.

[0041] 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 3-4:1-2:1.

[0042] In some embodiments, the above step 2 includes the following steps:

[0043] i. Activating the C-terminal carboxyl group of peptide fragment III with a condensing agent, and then carrying out a coupling reaction with the N-terminal amino group of peptide fragment IV in a reaction solution, and then removing the Fmoc protecting group and precipitating a solid to obtain peptide fragment V;

[0044] ii. activating the C-terminal carboxyl group of peptide fragment II with a condensing agent, and then carrying out a coupling reaction with the N-terminal amino group of peptide fragment V in a reaction solution, and then removing the Fmoc protecting group and precipitating a solid to obtain peptide fragment VI;

[0045] iii. Activate the C-terminal carboxyl group of the peptide fragment I with a condensing agent, and then carry out a coupling reaction with the N-terminal amino group of the peptide fragment VI in a reaction solution, then remove the Fmoc protecting group and precipitate a solid to obtain the fully protected canagliflozin peptide.

[0046] In some embodiments, the deprotecting agent used to remove the Fmoc protecting group in the above step 2 is Pip.

[0047] In some embodiments, the coupling reaction temperature in step i is 0-10°C.

[0048] In some embodiments, the coupling reaction temperature in step ii is -10~0°C.

[0049] In some embodiments, the coupling reaction temperature in step iii is -10~0°C.

[0050] In some embodiments, the condensation system of the coupling reaction in step 2 is composed of any one or more of HOBt, HOAT, Cl-HOBt, Oxyma Pure and DIC, or any one or more of HBTU, TBTU, HATU, PyBOP, PyAOP, PyOxim and DIEA.

[0051] In some embodiments, the condensation system of the coupling reaction in step i is HBTU / DIEA.

[0052] In some embodiments, the condensation system of the coupling reaction in step ii is PyOxim / DIEA.

[0053] In some embodiments, the condensation system of the coupling reaction in step iii is HBTU / DIEA.

[0054] In some embodiments, the reaction solvent used in the coupling reaction in step 2 includes one or more of DCM, DMF, THF, DMSO, and NMP.

[0055] In some embodiments, the above step three specifically includes: reacting the fully protected canagliflozin peptide with a lysis solution at 20-30°C with stirring for 2-3 hours, filtering after the reaction time is reached, and rinsing the filter cake with TFA; pouring the obtained filtrate into MTBE at -10-0°C to obtain a suspension of precipitated white solid, and then obtaining a crude canagliflozin linear peptide after centrifugal sedimentation and pulping and washing.

[0056] In some embodiments, the mass volume ratio of canagliflozin fully protected peptide to lysate in step 3 is 1 g: 7-9 mL.

[0057] In some embodiments, the mass volume ratio of canagliflozin fully protected peptide to MTBE in step 3 is 1 g: 62-65 mL.

[0058] In some embodiments, the lysis solution in step 3 includes three or more of TFA, EDT, TIS, PhOH, and PhSMe.

[0059] In some embodiments, the lysis solution in step three above is composed of TFA, EDT, TIS, and H2O in a volume ratio of 83-86:4-5:4-5:4-5.

[0060] Preferably, the lysis solution in the above step 3 is composed of TFA, EDT, TIS, and H2O in a volume ratio of 85:5:5:5.

[0061] In some embodiments, the cyclization method of the crude linear peptide of canagliflozin in step 4 is specifically as follows: dissolving the crude linear peptide of canagliflozin in an acetonitrile / water solution with a volume ratio of 75-80%, and then diluting it with purified water to a concentration of 0.9-1.2 g / L after it is dissolved; then adding 0.8-1.1 mmol / mL of iodine / acetonitrile solution dropwise until the cyclized liquid turns light yellow, and stirring to maintain the color for 25-32 minutes without fading, and finally adding 0.8-1.2 mmol / mL of vitamin C aqueous solution dropwise until the cyclized liquid is clear and colorless.

[0062] Since the present invention selects the cleavage site based on the shrinkage phenomenon of the peptide resin during the step-by-step coupling process, it has the following beneficial effects: 1) it avoids multiple peptide chain foldings in the canagliflozin peptide sequence, overcomes the problem of slow coupling rate of protected amino acids one by one, and can obtain a crude peptide with much higher purity and yield than that obtained by coupling one by one; 2) in the preparation process of the peptide fragments I, II, III, and IV selected in the present invention, the protected amino acids are all easy to couple, and after the peptide resin is cleaved and the solid phase carrier is separated, it is easy to precipitate a high-purity fully protected fragment peptide solid, without the need for preparative chromatography purification, saving time and cost; 3) when the peptide fragments I, II, III, and IV selected in the present invention are sequentially condensed into long fragments, they can exhibit a high coupling rate without the need for special conditions, and as the peptide chain grows, the hydrophobicity of the peptide chain is greatly increased, making it easier to obtain solid particles with excellent properties from the reaction solvent, and the synthesis and cleavage method can obtain a crude canagliflozin linear peptide with fewer impurities, higher purity, and higher yield.

[0063] Since the present invention uses DIC / 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol as a condensation system in the coupling reaction system for solid-phase synthesis of canagliflozin peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV, it has the following beneficial effects: the degree of coupling reaction can be improved, and the corresponding peptide fragments with higher yield and purity can be obtained, thereby increasing the yield of crude canagliflozin linear peptide, and the obtained crude canagliflozin linear peptide has higher purity.

[0064] The present invention provides a method for synthesizing canagliflozin. The method can avoid the problems of peptide chain folding and coupling difficulty caused by solid-phase coupling of amino acids one by one, and can increase the reaction rate, facilitate large-scale production, and the obtained crude canagliflozin is easy to purify with high purity and yield.

[0065] As used herein, the following abbreviations have the meanings as given herein: “FMOC” means 9-fluorenylmethyloxycarbonyl, “Trt” means trityl, “Boc” means tert-butyloxycarbonyl, “tBu” means tert-butyl, “OtBu” means tert-butyl carbothiol, “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 eicosandioic acid, “Eicosandioic acid, "1-(1,1-dimethyl)ester" means mono-tert-butyl eicosandioate, "Pbf" means 2,2,4,6,7-pentamethyl-2,3-dihydrobenzo[B]furanyl, "PyOxim" means cyano(hydroxyimino)ethyl acetate-O2]tris-1-pyrrolidinyl hexafluorophosphate, "PyBOP" means 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate, "HBTU" means benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and "DIEA" means N,N "-Diisopropylethylamine," "Cl-HOBt" means 6-chloro-1-hydroxybenzotriazole, "HOAT" means 1-hydroxy-7-azabenzotriazole, "TBTU" means O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, "HATU" means 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, "PyAOP" means (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinyl hexafluorophosphate, "Oxyma "Pure" means ethyl 2-oximecyanoacetate, "MeOH" means methanol, "PhOH" means phenol, "PhSMe" means thioanisole, "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, and "Sieber Resin" means Sieber amide resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 These are the results of measuring the weight yields of peptide resin I and peptide fragment I in Test Example 1 of the present invention.

[0067] Figure 2 This is the result of measuring the purity of peptide fragment I in Test Example 1 of the present invention.

[0068] Figure 3 This is the result of measuring the purity of the crude canagliflozin linear peptide in Test Example 2 of the present invention. DETAILED DESCRIPTION

[0069] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0070] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is only for the purpose of making the content of this application easier to understand, and does not limit the scope of protection of this application; at the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following application will be described in detail with reference to the accompanying drawings and in combination with the embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments:

[0071] In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are purchased from the market.

[0072] Example 1:

[0073] 1. Preparation of peptide resin I:

[0074] 1.1 Weigh 3.0 g of 2-CTC Resin (3.0 mmol scale) with a substitution degree of 1.0 mmol / g into a solid-phase reaction column. Swell the resin with 15 mL of DMF for 30 minutes, then drain the solution under vacuum. Dissolve 1.87 g of Fmoc-Ala-OH and 1.4 mL of DIEA in 12 mL of DMF. Add the mixture to the reaction column and react under nitrogen pressure at 25°C for 4 hours. Then, add 3.0 mL of MeOH and react under nitrogen pressure for 30 minutes. Upon completion of the reaction, drain the reaction mixture under vacuum, and wash the resin three times with 15 mL of DMF.

[0075] 1.2 Add 15 mL of a 20% (v / v) Pip / DMF solution to the resin to remove Fmoc residues for 30 minutes. Wash six times with 15 mL of DMF. 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 a reaction column. Purge with nitrogen and add 0.9 mL of DIC. React at 25°C for 2 hours. Drain the reaction mixture under vacuum and wash the resin three times with 15 mL of DMF.

[0076] 1.3 Repeat the Fmoc removal, coupling, and washing steps in 1.2, and sequentially couple 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 according to the peptide sequence of peptide fragment I. Then modify Eicosandioic acid, 1-(1,1-dimethyl)ester according to this procedure. After modification, shrink with 15 mL of MeOH three times and drain to obtain peptide resin I.

[0077] 2. Preparation of peptide fragment I:

[0078] Weigh 9.0 g of peptide resin I and pour it into a reaction bottle containing 90 mL of 1% by volume TFA / DCM solution. Stir and react at 20°C for 1 hour. After the time is up, filter the reaction solution, add 2.0 mL of DIEA dropwise to the filtrate, and evaporate under reduced pressure until about 30 mL remains. Then add 30 mL of MeOH and continue to evaporate. Repeat the rotary evaporation with MeOH three times to obtain about 8 mL of a thick solution containing peptide fragment I. Slowly add it dropwise to 40 mL of purified water and stir to precipitate a white solid. After all the thick solution is added, continue stirring for 30 minutes, filter, rinse the filter cake with 10 mL of purified water twice, and vacuum dry to obtain the peptide fragment I solid.

[0079] Example 2:

[0080] 1. Preparation of peptide resin II:

[0081] 1.1 Weigh 3.33 g of 2-CTC Resin (3.0 mmol scale) with a substitution degree of 0.9 mmol / g into a solid-phase reaction column. Swell the resin with 15 mL of DMF for 30 minutes, then drain the solution under vacuum. Weigh 2.13 g of Fmoc-Leu-OH and 1.4 mL of DIEA, dissolve them in 12 mL of DMF, and add the mixture to the reaction column. React at 25°C under a nitrogen atmosphere for 4 hours. Then, add 3.0 mL of MeOH and react under a nitrogen atmosphere for 30 minutes. Upon completion of the reaction, drain the reaction mixture under vacuum, and wash the resin three times with 15 mL of DMF.

[0082] 1.2 Add 15 mL of 20% by volume Pip / DMF solution to the resin to remove Fmoc for 30 min, then wash six times with 15 mL of DMF. Weigh 2.31 g of Fmoc-Phe-OH and 0.85 g of Oxyma Pure, dissolve them in 12 mL of DMF, and pour them into the reaction column. Purge with nitrogen, then add 0.9 mL of DIC, react at 25°C for 2 h, drain the reaction solution in vacuo, and wash the resin three times with 15 mL of DMF.

[0083] 1.3 Repeat the Fmoc removal, coupling, and washing steps 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, shrink with 150 mL of MeOH three times and drain to obtain peptide resin II.

[0084] 2. Preparation of peptide fragment II

[0085] Weigh 8.0 g of peptide resin II and pour it into a reaction bottle containing 80 mL of 1% by volume TFA / DCM solution. Stir and react at 20°C for 1 h. After the time is up, filter the reaction solution, add 2.0 mL of DIEA dropwise to the filtrate, and evaporate under reduced pressure until about 25 mL remains. Add 25 mL of MeOH and continue to evaporate. Repeat the evaporation with MeOH three times to obtain about 5 mL of a thick solution containing peptide fragment II. Slowly add it dropwise to 25 mL of purified water and stir to precipitate a white solid. After all the thick solution is added, continue stirring for 30 min, filter, rinse the filter cake with 10 mL of purified water twice, and vacuum dry to obtain the peptide fragment II solid.

[0086] Example 3:

[0087] 1. Preparation of peptide resin III:

[0088] 1.1 Weigh 3.8 g of 2-CTC Resin (3.0 mmol scale) with a substitution degree of 0.8 mmol / g into a solid-phase reaction column. Swell the resin with 15 mL of DMF for 30 minutes, then drain 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, and add the mixture to the reaction column. React at 25°C under nitrogen for 4 hours. Then, add 3.0 mL of MeOH and react under nitrogen for 30 minutes. Upon completion of the reaction, drain the reaction mixture under vacuum, and wash the resin three times with 15 mL of DMF.

[0089] 1.2 Add 15 mL of 20% by volume Pip / DMF solution to the resin to remove Fmoc for 30 min, then wash six times with 15 mL of DMF. Weigh 2.33 g of Fmoc-Phe-OH and 0.86 g of Oxyma Pure, dissolve them in 12 mL of DMF, and pour them into the reaction column. Purge with nitrogen, then add 0.9 mL of DIC, react at 25°C for 2 h, drain the reaction solution in vacuo, and wash the resin three times with 15 mL of DMF.

[0090] 1.3 Repeat the Fmoc removal, coupling, and washing steps 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, and Fmoc-Arg(Pbf)-OH according to the peptide sequence of peptide fragment III. Finally, shrink with 15 mL of MeOH three times and drain to obtain peptide resin III.

[0091] 2. Preparation of peptide fragment III

[0092] 8.5 g of peptide resin III was weighed and poured into a reaction bottle containing 85 mL of a 1% by volume TFA / DCM solution. The reaction was stirred at 20°C for 1 h. After the time was up, the reaction solution was filtered, 2.0 mL of DIEA was added dropwise to the filtrate, and the mixture was evaporated under reduced pressure until about 30 mL remained. 30 mL of MeOH was added and the evaporation was continued. After repeating the evaporation with MeOH three times, about 8 mL of a thick solution containing peptide fragment III was obtained. The solution was slowly dripped into 40 mL of purified water and stirred to precipitate a white solid. After all the thick solution was added, the mixture was stirred for 30 min, filtered, and the filter cake was rinsed twice with 10 mL of purified water and vacuum-dried to obtain a peptide fragment III solid.

[0093] Example 4:

[0094] 1. Preparation of peptide resin IV:

[0095] 1.1 Weigh 3.9 g (3.0 mmol scale) of Sieber Resin with a substitution degree of 0.77 mmol / g into a solid phase reaction column. Swell the resin with 20 mL of DMF for 30 minutes, then drain the solution under vacuum.

[0096] 1.2 Add 20 mL of 20% by volume Pip / DMF solution to the resin to remove Fmoc for 30 min, then wash six times with 20 mL of DMF. 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. Purge with nitrogen, then add 0.9 mL of DIC, react at 25°C for 2 h, drain the reaction solution in vacuo, and add 20 mL of DMF to wash the resin three times.

[0097] 1.3 Repeat the steps of 1.2 for removing Fmoc, coupling, and washing. According to the peptide sequence of peptide fragment IV, 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 were coupled in sequence. After the coupling was completed, 20 mL of 20% by volume Pip / DMF solution was added to remove Fmoc for 30 min. The mixture was washed 6 times with 20 mL of DMF. Finally, the mixture was shrunk 3 times with 20 mL of MeOH and dried to obtain peptide resin IV.

[0098] 2. Preparation of peptide fragment IV:

[0099] 9.5 g of peptide resin IV was weighed and poured into a reaction bottle containing 95 mL of a 5% by volume TFA / DCM solution. The reaction was stirred at 20°C for 1 h. After the time was up, the reaction solution was filtered, 11 mL of DIEA was added dropwise to the filtrate, and the mixture was evaporated under reduced pressure until about 20 mL remained. 20 mL of MeOH was added and the evaporation was continued. After repeating the evaporation with MeOH three times, about 5 mL of a thick solution of peptide fragment IV was obtained. The solution was slowly added dropwise to 50 mL of MTBE and stirred to precipitate a white solid. After all the thick solution was added, stirring was continued for 30 min, filtered, and the filter cake was washed twice with 10 mL of MTBE by beating. The solid of peptide fragment IV was vacuum-dried to obtain 5.58 g.

[0100] Example 5:

[0101] 1. Preparation of peptide fragment V: 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 were weighed into a 100 mL round-bottom flask, 50 mL of DMF was added and stirred to dissolve, the mixture was cooled to 2°C in a water bath, 1.7 mL of DIEA was added dropwise, and the temperature was controlled at 0-5°C. After the addition was complete, the mixture was stirred at 5°C for 1.0 h, and 10 mL of IP was added to the solution. After stirring at 25°C for 1.0 h, 50 mL of DIEA was added to the reaction solution. DMF, after mixing, cool to 10 ° C, and then add dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature in a water bath at 15 ° C, and precipitate a white solid. After the reaction solution is added dropwise, 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 beat overnight; the slurry is filtered, the filter cake is rinsed twice with 30 mL of purified water and then dried, and then transferred to a 45 ° C forced air drying oven and dried to constant weight to obtain peptide fragment V.

[0102] 2. Preparation of peptide fragment VI: Weigh 9.13 g of the peptide fragment V prepared above into a 200 mL round-bottom flask, add 100 mL of THF and dissolve at 25°C, then weigh 4.55 g of the peptide fragment II prepared in Example 2 and 1.91 g of PyOxim, pour them into the THF solution of peptide fragment V and dissolve them, cool the solution to 2°C in a water bath, then add 1.7 mL of DIEA dropwise, control the temperature at 0-5°C, maintain the reaction at 5°C with stirring for 4.0 h, and then add 20 mL of DIEA to the solution. Pip, after stirring at 25°C for 2.0h, the reaction solution was added dropwise to 500mL of saturated citric acid aqueous solution, and the temperature was controlled at 15°C in a water bath to precipitate a white solid. After the reaction solution was added dropwise, it was stirred at 10°C for 1.0h, filtered, and the filter cake was vacuum-dried. The filter cake was taken out and slurried with 50mL of purified water overnight; the slurry was filtered, and the filter cake was rinsed twice with 30mL of purified water and then dried, and then transferred to a 45°C forced air drying oven and dried to constant weight to obtain peptide fragment VI.

[0103] 3. Preparation of fully protected canagliflozin peptide: 12.35 g of the peptide fragment VI prepared above, 5.59 g of the peptide fragment I prepared in Example 1, and 1.31 g of HBTU were weighed into a 200 mL round-bottom flask, dissolved with 120 mL of THF at 25°C, cooled to -8°C, and then 1.6 mL of DIEA was added dropwise. The temperature during the addition was controlled at -10 to -5°C in a water bath. After stirring for 2.0 h, the reaction solution was added dropwise to 600 mL of a saturated aqueous citric acid solution in a water bath controlled at 15°C. A white solid precipitated. After the reaction solution was added dropwise, the mixture was stirred at 10°C for 1.0 h, filtered, and the filter cake was vacuum-dried. The filter cake was removed and slurried with 50 mL of purified water overnight. The slurry was filtered, and the filter cake was rinsed twice with 30 mL of purified water and then dried. It was then transferred to a 45°C forced air drying oven and dried to constant weight to obtain fully protected canagliflozin peptide.

[0104] 4. Preparation of crude linear peptide of canagliflozin: Weigh 15.0 g of the fully protected canagliflozin peptide prepared above, pour it into 120 mL of TFA / EDT / TIS / H2O lysis solution with a volume ratio of 85:5:5:5, and react with stirring at 25°C for 2.0 h. After the reaction time is reached, filter and rinse the filter cake with TFA; the resulting filtrate is poured into 960 mL of -5°C MTBE to obtain a suspension of precipitated white solid, which is then centrifuged, pulped and washed, and then dried in a vacuum drying oven at 25°C to constant weight to obtain crude linear peptide of canagliflozin.

[0105] Example 6:

[0106] Preparation of Canagliflozin Finished Product:

[0107] 8.0 g of the crude linear peptide of canagliflozin obtained in Example 5 was weighed and dissolved in 800 mL of 80% by volume acetonitrile / water solution, and then diluted with purified water to a concentration of 1 g / L; 1 mmol / mL iodine / acetonitrile solution was then added dropwise until the cyclized solution turned light yellow, and the color was maintained without fading for 30 minutes by stirring. Finally, 1 mmol / mL of vitamin C aqueous solution was added dropwise until the cyclized solution became clear and colorless, thereby obtaining a solution of crude canagliflozin cyclic peptide.

[0108] The crude canagliflozin cyclic peptide solution was filtered through a mixed microporous membrane with a pore size of 0.45µm. The canagliflozin cyclic peptide solution was purified by high performance liquid chromatography, and the canagliflozin fractions were collected according to the position of the reference substance.

[0109] Purification analysis method:

[0110] C18 column:

[0111]

[0112] The purified canagliflozin fraction was desalted, transsalted, concentrated, and lyophilized to obtain 2.71 g of canagliflozin finished product with a yield of 33.88% and a purity of 99.67%.

[0113] Example 7:

[0114] This example differs from Example 1 in that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol was used instead of Oxyma Pure to activate the protected amino acid monomer.

[0115] Preparation method of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol:

[0116] Take 1.6g Fe(NO3)3·9H2O and 0.32g tetramethylpiperidinyl oxide and dissolve them in dichloroethane. Bring oxygen into the mixture and heat to 80℃. Then add 0.53g 6-bromo-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine and react for 5h. After cooling to room temperature, filter, wash once with ethanol, wash twice with water, dry, and separate and purify by silica gel column chromatography with petroleum ether / ethyl acetate in a volume ratio of 8:2 as eluent to obtain 6-bromo-5-nitro-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine. 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%. 6-Bromo-5-nitro-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrazine was obtained.

[0117] To a 250 mL three-necked flask, 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 isoamyl alcohol were added. The mixture was heated to react and water was removed. The reaction was heated to 100°C for 2 h. After the reaction, the mixture was neutralized with 40 wt % NaOH solution, and the excess hydrazine hydrate and isoamyl alcohol were distilled off. The pH was adjusted to 3.0 with hydrochloric acid, and the mixture was filtered, washed with 5 wt % ice brine, recrystallized, and dried to obtain 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol. 1 H NMR (CDCl3, 400 MHz): δ8.82 (1H, s), δ2.78 (1H, s). EI-MS m / z: 245.03 M peak relative intensity 100%, 246.03 M+1 peak relative intensity 9.2%.

[0118] Example 8:

[0119] This example differs from Example 2 in that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol was used instead of Oxyma Pure to activate the protected amino acid monomer.

[0120] Example 9:

[0121] This example differs from Example 3 in that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol was used instead of Oxyma Pure to activate the protected amino acid monomer.

[0122] Example 10:

[0123] This example differs from Example 4 in that 1.47 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol was used instead of Oxyma Pure to activate the protected amino acid monomer.

[0124] Example 11:

[0125] This Example differs from Example 1 in that, in step 1.2 of the preparation of Peptide Resin I, 15 mL of a 20% (volume) Pip / DMF solution was added to the resin for Fmoc removal for 30 minutes, followed by six washes with 15 mL of DMF. 3.53 g of Fmoc-Cys(Trt)-OH and 0.85 g of Oxyma Pure were weighed and dissolved in 12 mL of a 3:1:1 (volume ratio) DMF / 4-isobutylacetophenone / 2-pentylpyridine solution. The mixture was then poured into a reaction column, purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25°C for 2 hours, the reaction solution was removed under vacuum, and the resin was washed three times with 15 mL of DMF.

[0126] Example 12:

[0127] This Example differs from Example 1 in that, in step 1.2 of the preparation of peptide resin I, 15 mL of a 20% (volume) Pip / DMF solution was added to the resin for Fmoc removal for 30 minutes, followed by six washes with 15 mL of DMF. 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 were weighed and dissolved in 12 mL of a 3:1:1 (volume ratio) DMF / 4-isobutylacetophenone / 2-pentylpyridine solution. The mixture was then poured into a reaction column, purged with nitrogen, and 0.9 mL of DIC was added. The reaction was allowed to proceed at 25°C for 2 hours, after which the reaction solution was removed in vacuo and the resin was washed three times with 15 mL of DMF.

[0128] Example 13:

[0129] The difference between this example and Example 2 lies in the preparation of peptide resin II in step 1.2: 15 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, and the resin was washed six times with 15 mL of DMF. 2.31 g of Fmoc-Phe-OH and 0.85 g of Oxyma Pure were weighed and dissolved with 12 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution, and then poured into a reaction column. The column was purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25°C for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 15 mL of DMF.

[0130] Example 14:

[0131] The difference between this example and Example 2 is that in step 1.2 of the preparation of peptide resin II, 15 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, and the mixture was washed six times with 15 mL of DMF. 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 were weighed and dissolved with 12 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution, and then poured into a reaction column. The mixture was purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25° C. for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 15 mL of DMF.

[0132] Example 15:

[0133] The difference between this example and Example 3 lies in the preparation of peptide resin III in step 1.2: 15 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, followed by washing with 15 mL of DMF six times. 2.33 g of Fmoc-Phe-OH and 0.86 g of Oxyma Pure were weighed and dissolved with 12 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution. The mixture was then poured into a reaction column, purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25°C for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 15 mL of DMF.

[0134] Example 16:

[0135] The difference between this example and Example 3 lies in the preparation step 1.2 of peptide resin III: 15 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, and the mixture was washed six times with 15 mL of DMF. 2.33 g of Fmoc-Phe-OH and 1.48 g of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol were weighed and dissolved with 12 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution, and then poured into a reaction column. The mixture was purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25° C. for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 15 mL of DMF.

[0136] Example 17:

[0137] The difference between this example and Example 4 lies in the preparation step 1.2 of peptide resin IV: 20 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, and the resin was washed six times with 20 mL of DMF. 2.03 g of Fmoc-Pro-OH and 0.85 g of Oxyma Pure were weighed and dissolved with 15 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution, and then poured into a reaction column. The column was purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25°C for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 20 mL of DMF.

[0138] Example 18:

[0139] The difference between this example and Example 4 is that in step 1.2 of the preparation of peptide resin IV, 20 mL of a 20% by volume Pip / DMF solution was added to the resin to remove Fmoc for 30 minutes, and the mixture was washed six times with 20 mL of DMF. 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 were weighed and dissolved with 15 mL of a 3:1:1 by volume DMF / 4-isobutylacetophenone / 2-pentylpyridine solution, and then poured into a reaction column. The mixture was purged with nitrogen, and 0.9 mL of DIC was added. After reacting at 25° C. for 2 hours, the reaction solution was drained in vacuo, and the resin was washed three times with 20 mL of DMF.

[0140] Example 19:

[0141] This Example differs from Example 1 in that, in step 1.2 of the preparation of Peptide Resin I, 15 mL of a 20% (volume) Pip / DMF solution was added to the resin for Fmoc removal for 30 minutes, followed by six washes with 15 mL of DMF. 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 were weighed and dissolved in 12 mL of a 3:1 (volume) DMF / 4-isobutylacetophenone solution. The mixture was then poured into a reaction column, purged with nitrogen, and 0.9 mL of DIC was added. The reaction was allowed to proceed at 25°C for 2 hours, after which the reaction solution was removed in vacuo and the resin was washed three times with 15 mL of DMF.

[0142] Example 20:

[0143] This Example differs from Example 1 in that, in step 1.2 of the preparation of Peptide Resin I, 15 mL of a 20% (volume) Pip / DMF solution was added to the resin for Fmoc removal for 30 minutes, followed by six washes with 15 mL of DMF. 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 were weighed and dissolved in 12 mL of a 3:1 (volume) DMF / 2-pentylpyridine solution. The mixture was then poured into a reaction column, purged with nitrogen, and 0.9 mL of DIC was added. The reaction was allowed to proceed at 25°C for 2 hours, after which the reaction solution was removed in vacuo and the resin was washed three times with 15 mL of DMF.

[0144] Example 21:

[0145] The difference between this example and example 5 lies in the preparation of peptide fragment V, peptide fragment VI and canagliflozin fully protected peptide:

[0146] 1. Preparation of peptide fragment V: 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 were weighed into a 100 mL round-bottom flask, 50 mL of DMF was added and stirred to dissolve, the mixture was cooled to 2°C in a water bath, 1.7 mL of DIEA was added dropwise, and the temperature was controlled at 0-5°C. After the addition was complete, the mixture was stirred at 5°C for 1.0 h, and then 10 mL of IP was added to the solution. After stirring at 25°C for 1.0 h, 50 mL of DIEA was added to the reaction solution. DMF, after mixing, cool to 10 ° C, and then add dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature in a water bath at 15 ° C, and precipitate a white solid. After the reaction solution is added dropwise, 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 beat overnight; the slurry is filtered, the filter cake is rinsed twice with 30 mL of purified water and then dried, and then transferred to a 45 ° C forced air drying oven and dried to constant weight to obtain peptide fragment V.

[0147] 2. Preparation of peptide fragment VI: 9.13 g of the peptide fragment V prepared above was weighed into a 200 mL round-bottom flask, 100 mL of THF was added and dissolved at 25°C, 4.55 g of the peptide fragment II prepared in Example 8 and 1.91 g of PyOxim were weighed and poured into the THF solution of peptide fragment V to dissolve, the solution was cooled to 2°C in a water bath, and 1.7 mL of DIEA was added dropwise, the temperature was controlled at 0-5°C, and after the addition was completed, the reaction was maintained at 5°C with stirring for 4.0 h, and 20 mL of DIEA was added to the solution. Pip, after stirring at 25°C for 2.0h, the reaction solution was added dropwise to 500mL of saturated citric acid aqueous solution, and the temperature was controlled at 15°C in a water bath to precipitate a white solid. After the reaction solution was added dropwise, it was stirred at 10°C for 1.0h, filtered, and the filter cake was vacuum-dried. The filter cake was taken out and slurried with 50mL of purified water overnight; the slurry was filtered, and the filter cake was rinsed twice with 30mL of purified water and then dried, and then transferred to a 45°C forced air drying oven and dried to constant weight to obtain peptide fragment VI.

[0148] 3. Preparation of fully protected canagliflozin peptide: 12.35 g of the peptide fragment VI prepared above, 5.59 g of the peptide fragment I prepared in Example 7, and 1.31 g of HBTU were weighed into a 200 mL round-bottom flask, dissolved with 120 mL of THF at 25°C, cooled to -8°C, and then 1.6 mL of DIEA was added dropwise. The temperature during the addition was controlled at -10 to -5°C in a water bath. After stirring for 2.0 h, the reaction solution was added dropwise to 600 mL of a saturated aqueous solution of citric acid. The temperature in a water bath was controlled at 15°C. A white solid precipitated. After the reaction solution was added dropwise, the mixture was stirred at 10°C for 1.0 h, filtered, and the filter cake was vacuum-dried. The filter cake was removed and slurried with 50 mL of purified water overnight. The slurry was filtered, and the filter cake was rinsed twice with 30 mL of purified water and then dried. It was then transferred to a 45°C forced air drying oven and dried to constant weight to obtain fully protected canagliflozin peptide.

[0149] Example 22:

[0150] The difference between this example and example 5 lies in the preparation of peptide fragment V, peptide fragment VI and canagliflozin fully protected peptide:

[0151] 1. Preparation of peptide fragment V: 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 were weighed into a 100 mL round-bottom flask, 50 mL of DMF was added and stirred to dissolve, the mixture was cooled to 2°C in a water bath, 1.7 mL of DIEA was added dropwise, and the temperature was controlled at 0-5°C. After the addition was complete, the mixture was stirred at 5°C for 1.0 h, and then 10 mL of IP was added to the solution. After stirring at 25°C for 1.0 h, 50 mL of DIEA was added to the reaction solution. DMF, after mixing, cool to 10 ° C, and then add dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature in a water bath at 15 ° C, and precipitate a white solid. After the reaction solution is added dropwise, 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 beat overnight; the slurry is filtered, the filter cake is rinsed twice with 30 mL of purified water and then dried, and then transferred to a 45 ° C forced air drying oven and dried to constant weight to obtain peptide fragment V.

[0152] 2. Preparation of peptide fragment VI: 9.13 g of the peptide fragment V prepared above was weighed into a 200 mL round-bottom flask, 100 mL of THF was added and dissolved at 25°C, 4.55 g of the peptide fragment II prepared in Example 13 and 1.91 g of PyOxim were weighed and poured into the THF solution of peptide fragment V to dissolve, the solution was cooled to 2°C in a water bath, and 1.7 mL of DIEA was added dropwise, the temperature was controlled at 0-5°C, and after the addition was completed, the reaction was maintained at 5°C with stirring for 4.0 h, and 20 mL of DIEA was added to the solution. Pip, after stirring at 25°C for 2.0h, the reaction solution was added dropwise to 500mL of saturated citric acid aqueous solution, and the temperature was controlled at 15°C in a water bath to precipitate a white solid. After the reaction solution was added dropwise, it was stirred at 10°C for 1.0h, filtered, and the filter cake was vacuum-dried. The filter cake was taken out and slurried with 50mL of purified water overnight; the slurry was filtered, and the filter cake was rinsed twice with 30mL of purified water and then dried, and then transferred to a 45°C forced air drying oven and dried to constant weight to obtain peptide fragment VI.

[0153] 3. Preparation of fully protected canagliflozin peptide: 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 were weighed into a 200 mL round-bottom flask, dissolved with 120 mL of THF at 25°C, cooled to -8°C, and then 1.6 mL of DIEA was added dropwise. The temperature during the addition was controlled at -10 to -5°C in a water bath. After stirring for 2.0 h, the reaction solution was added dropwise to 600 mL of a saturated aqueous citric acid solution in a water bath controlled at 15°C. A white solid precipitated. After the reaction solution was added dropwise, the mixture was stirred at 10°C for 1.0 h, filtered, and the filter cake was vacuum-dried. The filter cake was removed and slurried with 50 mL of purified water overnight. The slurry was filtered, and the filter cake was rinsed twice with 30 mL of purified water and then dried. It was then transferred to a 45°C forced air drying oven and dried to constant weight to obtain fully protected canagliflozin peptide.

[0154] Example 23:

[0155] The difference between this example and example 5 lies in the preparation of peptide fragment V, peptide fragment VI and canagliflozin fully protected peptide:

[0156] 1. Preparation of peptide fragment V: 4.70 g of peptide fragment IV prepared in Example 18, 5.26 g of peptide fragment III prepared in Example 16, and 1.38 g of HBTU were weighed into a 100 mL round-bottom flask, 50 mL of DMF was added and stirred to dissolve, the mixture was cooled to 2°C in a water bath, 1.7 mL of DIEA was added dropwise, and the temperature was controlled at 0-5°C. After the addition was complete, the mixture was stirred at 5°C for 1.0 h, and then 10 mL of IP was added to the solution. After stirring at 25°C for 1.0 h, 50 mL of DIEA was added to the reaction solution. DMF, after mixing, cool to 10 ° C, and then add dropwise to 500 mL of saturated citric acid aqueous solution, control the temperature in a water bath at 15 ° C, and precipitate a white solid. After the reaction solution is added dropwise, 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 beat overnight; the slurry is filtered, the filter cake is rinsed twice with 30 mL of purified water and then dried, and then transferred to a 45 ° C forced air drying oven and dried to constant weight to obtain peptide fragment V.

[0157] 2. Preparation of peptide fragment VI: 9.13 g of the peptide fragment V prepared above was weighed into a 200 mL round-bottom flask, 100 mL of THF was added and dissolved at 25°C, 4.55 g of the peptide fragment II prepared in Example 14 and 1.91 g of PyOxim were weighed and poured into the THF solution of peptide fragment V to dissolve, the solution was cooled to 2°C in a water bath, and 1.7 mL of DIEA was added dropwise, the temperature was controlled at 0-5°C, and after the addition was completed, the reaction was maintained at 5°C with stirring for 4.0 h, and 20 mL of DIEA was added to the solution. Pip, after stirring at 25°C for 2.0h, the reaction solution was added dropwise to 500mL of saturated citric acid aqueous solution, and the temperature was controlled at 15°C in a water bath to precipitate a white solid. After the reaction solution was added dropwise, it was stirred at 10°C for 1.0h, filtered, and the filter cake was vacuum-dried. The filter cake was taken out and slurried with 50mL of purified water overnight; the slurry was filtered, and the filter cake was rinsed twice with 30mL of purified water and then dried, and then transferred to a 45°C forced air drying oven and dried to constant weight to obtain peptide fragment VI.

[0158] 3. Preparation of fully protected canagliflozin peptide: 12.35 g of the peptide fragment VI prepared above, 5.59 g of the peptide fragment I prepared in Example 12, and 1.31 g of HBTU were weighed into a 200 mL round-bottom flask, dissolved with 120 mL of THF at 25°C, cooled to -8°C, and then 1.6 mL of DIEA was added dropwise. The temperature during the addition was controlled at -10 to -5°C in a water bath. After stirring for 2.0 h, the reaction solution was added dropwise to 600 mL of a saturated aqueous citric acid solution in a water bath controlled at 15°C. A white solid precipitated. After the reaction solution was added dropwise, the mixture was stirred at 10°C for 1.0 h, filtered, and the filter cake was vacuum-dried. The filter cake was removed and slurried with 50 mL of purified water overnight. The slurry was filtered, and the filter cake was rinsed twice with 30 mL of purified water and then dried. It was then transferred to a 45°C forced air drying oven and dried to constant weight to obtain fully protected canagliflozin peptide.

[0159] Test Example 1:

[0160] 1. The weight yield of the peptide resin I obtained in Example 1, Example 7, Example 11, Example 12, Example 19, and Example 20 was determined; the peptide resin I was subjected to soft cleavage to obtain peptide fragment I, and the weight yield of the peptide fragment I in the soft cleavage step was determined. The results are shown in FIG. Figure 1 .

[0161] Depend on Figure 1 It can be seen that the weight yields of the peptide fragment I prepared in Example 1, Example 7, Example 11, Example 12, Example 19, and Example 20 are not significantly different. Compared with Example 1, the weight yield of the peptide resin I prepared in Example 7 is higher, indicating that the same molar equivalent of 7-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol is higher than that of Oxyma Pure has a better amino acid activation effect, 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 weight yields of peptide resin I prepared in Examples 19 and 20 are not significantly different, 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 is used as a reaction solution to improve its activation effect on amino acids and further improve the degree of amino acid coupling.

[0162] 2. The purity of the peptide fragments I obtained in Example 1, Example 7, Example 11, Example 12, Example 19 and Example 20 was determined. The results are shown in FIG. Figure 2 .

[0163] Depend on Figure 2 It can be seen that the purity of peptide fragment I prepared in Example 7 is greater than that in Example 1, indicating that the purity of peptide fragment I prepared in Example 7 is greater than that of peptide fragment I prepared in Example 1, which indicates ... Compared with Example 1, Example 19 and Example 20, the purity of the peptide fragment I prepared in Example 12 was higher than that in Example 7, Example 19 and Example 20, and there was no significant difference between Example 19 and Example 20 and Example 7. This shows 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 coupling reaction of the amino acid and improve the yield and purity of the peptide fragment I.

[0164] Test Example 2:

[0165] The purity of crude canagliflozin linear peptides obtained in Examples 5, 21, 22 and 23 was determined. The results are shown in Table 2. Figure 3 .

[0166] Depend on Figure 3 It can be seen that compared with Example 5, the purity of the crude linear peptide of canagliflozin obtained in Example 21 is higher, indicating that compared with the same molar equivalent of Oxyma Pure, 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 obtained peptide fragment is of higher purity, thereby obtaining a crude linear peptide of canagliflozin.

[0167] Compared with Example 21, the purity of the crude linear peptide of canagliflozin prepared in Example 23 is higher, and the purity of the crude linear peptide of canagliflozin prepared in Example 5 is slightly higher than the purity 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, a DMF / 4-isobutylacetophenone / 2-pentylpyridine solution with a volume ratio of 3-4:1-2:1 is used as a reaction solution to promote the coupling reaction of amino acids, improve the purity of the corresponding peptide fragment, and thereby improve the purity of the crude linear peptide of canagliflozin.

[0168] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0169] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for synthesizing canagliflozin, characterized in that: The steps include: Step 1, synthesizing peptide fragment I, peptide fragment II, peptide fragment III, and peptide fragment IV respectively using a solid phase method; Step 2: Liquid-phase splicing of peptide fragment III and peptide fragment IV to obtain peptide fragment V, then liquid-phase splicing of peptide fragment V and peptide fragment II to obtain peptide fragment VI, and finally liquid-phase splicing of peptide fragment VI and peptide fragment I to obtain canagliflozin fully protected peptide; Step 3: adding the fully protected canagliflozin peptide to the lysis solution, and obtaining the crude linear canagliflozin peptide through precipitation, beating and washing; Step 4: Cyclize the crude linear peptide of canagliflozin, and then separate, purify, convert to salt, concentrate, and freeze-dry to obtain the finished canagliflozin; 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; The step one comprises: S1, using 2-CTC resin as a solid phase support, using Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin I, peptide fragment I is obtained by soft cleavage of peptide resin I; S2, using 2-CTC resin as a solid phase support, using the Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin II, peptide resin II is softly cleaved to obtain peptide fragment II; S3, using 2-CTC resin as a solid phase support, using the Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin III, peptide resin III is softly cleaved to obtain peptide fragment III; S4, using Sieber amide resin as a solid phase support, using Fmoc solid phase synthesis strategy, sequentially coupled 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, i.e., peptide resin IV, peptide resin IV is softly cleaved to obtain peptide fragment IV; 7-(Trifluoromethyl)-1H-[1,2,3]triazolo[4,5-e]triazolo[1,5-a]pyrazin-1-ol was used as an activating agent in the amino acid coupling reaction system during solid phase synthesis.

2. The synthesis method according to claim 1, wherein The specific preparation method of the peptide resin I includes: a. After swelling 2-CTC resin with DMF for 25-35 minutes, insert the first protected amino acid monomer Fmoc-Ala-OH to obtain Fmoc-Ala 9 -CTC Resin; b. Remove the Fmoc protection from the resin, wash with DMF 5 to 6 times, and couple with the next protected amino acid monomer Fmoc-Cys(Trt)-OH to obtain Fmoc-Cys(Trt)-Ala 9 -CTC Resin; c. Repeat step b to sequentially add protected amino acid monomers, and finally add mono-tert-butyl eicosanedioate according to the method of step b to obtain peptide resin I.

3. The synthesis method according to claim 2, wherein: In step a, the condensing agent used when the 2-CTC resin is connected to the first protected amino acid monomer is DIEA, and the reaction solvent is DMF.

4. The synthesis method according to claim 1, characterized in that The second step includes: i. Activating the C-terminal carboxyl group of peptide fragment III with a condensing agent, and then carrying out 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. activating the C-terminal carboxyl group of peptide fragment II with a condensing agent, and then carrying out a coupling reaction with the N-terminal amino group of peptide fragment V in a reaction solution, and then removing the Fmoc protecting group and precipitating a solid to obtain peptide fragment VI; iii. Activate the C-terminal carboxyl group of the peptide fragment I with a condensing agent, and then carry out a coupling reaction with the N-terminal amino group of the peptide fragment VI in a reaction solution, then remove the Fmoc protecting group and precipitate a solid to obtain the fully protected canagliflozin peptide.

5. The synthesis method according to claim 4, characterized in that The condensation system for the coupling reaction in step i and step iii is HBTU / DIEA.

6. The synthesis method according to claim 4, characterized in that The condensation system of the coupling reaction in step ii is PyOxim / DIEA.

7. The synthesis method according to claim 1, characterized in that The step three specifically includes: stirring the fully protected canagliflozin peptide with a lysis solution at 20-30°C for 2-3h, filtering after the reaction time is reached, and rinsing the filter cake with TFA; pouring the obtained filtrate into MTBE at -10-0°C to obtain a suspension of precipitated white solid, and then centrifuging, beating and washing to obtain a crude canagliflozin linear peptide.

8. The synthesis method according to claim 1, characterized in that The cyclization method of the crude linear peptide of canagliflozin in step 4 is specifically as follows: dissolving the crude linear peptide of canagliflozin in an acetonitrile / water solution with a volume ratio of 75-80%, and then diluting it with purified water to a concentration of 0.9-1.2 g / L after it is dissolved; then adding 0.8-1.1 mmol / mL of iodine / acetonitrile solution dropwise until the cyclized liquid turns light yellow, and stirring to maintain the color for 25-32 minutes without fading, and finally adding 0.8-1.2 mmol / mL of vitamin C aqueous solution dropwise until the cyclized liquid is clear and colorless.

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