Preparation method of canagliflozin

Abstract

This application provides a method for preparing canagliflozin, using methods including... 20 Ser、 21 Ser's NBD pseudodipeptide and including 4 Asn- 5 Thr- 6 Solid-phase synthesis of canagliflozin using amino acid fragments of Ala can overcome the difficulty of coupling caused by α-helices in traditional solid-phase synthesis. It has the advantages of low cost, large-scale production capability, high yield, and high purity of crude peptide, which greatly reduces purification and production costs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a method for preparing canagliflozin. Background Technology

[0002] Canagliptin is a novel, long-acting acylated amylin analog in investigation. It is a non-selective dual agonist of the amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) designed for weight management. Canagliptin is a cyclic polypeptide composed of 38 amino acids, containing a single disulfide bond. It can significantly reduce weight and food intake, showing potential in obesity research. The development of canagliptin is based on amylin, a hormone secreted by pancreatic β-cells that reduces gastric emptying and induces a feeling of fullness, thereby reducing food intake. However, the clinical application of natural amylin is limited due to its poor water solubility and tendency to aggregate into amyloid protein. Canagliptin improves these physical properties by extending its half-life, enabling once-weekly dosing and enhancing its clinical feasibility.

[0003] Canagliflozin is composed of 38 amino acids and has the molecular formula C. 194 H 312 O 59 N 54 S2, with a molecular weight of 4409.01, contains a pair of disulfide bonds (Cys). 3 -Cys 8 Its sequence is: eicosanoic acid-γ-Glu-Lys-Cys-Asn-Thr 5 -Ala-Thr-Cys-Ala-Thr 10 -Gln-Arg-Leu-Ala-Glu 15 -Phe-Leu-Arg-His-Ser 20 -Ser-Asn-Asn-Phe-Gly 25 -Pro-Ile-Leu-Pro-Pro 30 -Thr-Asn-Val-Gly-Ser 35 -Asn-Thr-Pro-NH2.

[0004] Currently, the main methods for synthesizing canagliflozin include the following: Patent CN114249808A uses three pseudoproline dipeptides, which solves the problem of increased coupling difficulty caused by resin condensation, but increases production costs, and the purity and yield of the peptide prepared by this process are relatively low; Patent CN117986347A reduces the number of pseudoproline dipeptides to 1-2, but the production cost is still high, and the stepwise coupling method used may require subsequent amino acid addition, increasing time costs, and the yield is low (≤32%); Patent CN119350469A uses a large-fragment SPPS-LPPS mixed method to synthesize canagliflozin, specifically by solid-phase synthesis of four fragments, followed by fully protected cleavage of the fragment peptide resin and then liquid-phase coupling. However, liquid-phase coupling requires cleavage of all four fragment peptide resins, and post-processing requires filtration, concentration, crystallization, centrifugation, etc., which requires more equipment and incurs high time costs. Furthermore, the condensing agent HATU and solution DMSO used for fragment coupling are costly. Additionally, post-condensation processing of the liquid-phase fragments requires the addition of water for crystallization, leaving residual water that is difficult to dry. After condensation, deprotection requires further pulping and drying, making the post-processing complex and time-consuming, with a final yield of less than 45%. Therefore, there is an urgent need for an efficient method for synthesizing canagliflozin to solve the problems of high impurities, difficult coupling, and low yield in existing technologies. Thus, developing a high-yield, short-cycle, and low-impurity synthetic method for canagliflozin has significant practical implications. Summary of the Invention

[0005] In order to at least partially overcome the shortcomings of the prior art, the present invention provides a method for preparing canagliflozin.

[0006] The first aspect of this application provides a method for preparing caglitazone, comprising: S1 uses amide-type resin as a solid-phase synthesis carrier and solid-phase polypeptide synthesis technology to synthesize a fully protected cagliolipoide resin conjugate with a cagliolipoide sequence. Among them, the caglitazone sequence 20 Ser- 21 Ser is formed using intermediate 1, which has the pseudo-dipeptide structure shown in Formula I: Formula I; Among them, R A R is an amino protecting group. A Selected from C1-C16 alkoxycarbonyl groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; R B R is a hydroxyl protecting group. B The group is selected from tert-butyl, C1-C5 acyl, benzyl, TBS, and TES, with tert-butyl being preferred; R C R is a phenolic hydroxyl protecting group.C Selected from C1-C5 acyl, benzyl, TBS, TES, preferably acetyl; In the caglitazone sequence 4 Asn- 5 Thr- 6 Ala uses including 4 Asn- 5 Thr- 6 Ala is formed from protected amino acid fragments; S2. The fully protected cagliflozin resin conjugate from step S1 is cleaved using an acid hydrolysate to obtain a linear cagliflozin peptide. S3 uses a cyclizing agent to cyclize linear caglitazone peptides. 3 Cys and 8 The Cys site undergoes a disulfide bond cyclization reaction to yield canagliflozin.

[0007] According to the method of the first aspect, in step S1, the method includes... 4 Asn- 5 Thr- 6 The protective amino acid fragment of Ala was selected from R D - 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH, R D - 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala(R S )-OH, R D - 2 Lys(R S )- 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH; Wherein, the R D Each of the amino protecting groups is independently selected from C1-C16 alkoxycarbonyl protecting groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; The R SThe protecting groups are the side chain protecting groups of amino acids, each independently selected from C1-C16 alkoxycarbonyl protecting groups, C4-C21 alkyl protecting groups, and C4-C8 alkoxy protecting groups, preferably tert-butyl, triphenylmethyl, or tert-butyloxycarbonyl.

[0008] According to the method of the first aspect, in step S1, the amide resin is selected from one or more of Rink Amide resin, Rink Amide MBHA resin, Rink Amide AM resin, MBHA resin, and PAL resin, preferably Rink Amide resin; and / or The degree of substitution of the amide resin is 0.3-1.8 mmol / g, preferably 0.4-0.8 mmol / g; and / or The molar ratio of each protected amino acid to the resin is (1.2-3.5):1, preferably (1.5-3):1.

[0009] According to the method of the first aspect, in step S1, the coupling is carried out in the presence of a coupling agent selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, DIEA, and HOBT, preferably at least one of DIEA, DIC, HOBT, and EDCI.

[0010] According to the method of the first aspect, the preparation method of the intermediate 1 includes: S11 will R A -Ser(R B The condensation of )-OH with salicylaldehyde yields intermediate 1-1, which has the structure shown in Formula I-1: Formula I-1; Intermediate S12 1-1 was coupled with serine ester hydrochloride to obtain coupling product intermediate 1-2 having the structure shown in Formula I-2: Formula I-2; S13 involves acetyl protection of the salicylaldehyde phenolic hydroxyl group in intermediate 1-2 to obtain intermediate 1-3 having the structure shown in Formula I-3: Formula I-3; S14 intermediate 1-3 undergoes ester hydrolysis to obtain intermediate 1 with the structure shown in Formula I.

[0011] According to the method of the first aspect, in step S11, the condensation reaction temperature is 0-25°C; and / or The condensation reaction time is 2-6 hours, preferably 2-4 hours; and / or The condensation is carried out in the presence of a condensing agent selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, HOBT, and DIEA, preferably at least one of DIEA, DIC, EDCI, and HOBT; and / or The condensation is carried out in the presence of a catalyst, preferably DMAP.

[0012] According to the method of the first aspect, in step S22, the serine ester hydrochloride is selected from at least one of serine methyl ester hydrochloride, serine ethyl ester hydrochloride, and serine benzyl ester hydrochloride, preferably serine methyl ester hydrochloride. Preferably, step S22 is carried out in a mixed system comprising an organic base and an organic acid; More preferably, the organic base is selected from at least one of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine, and is further preferably pyridine; and / or More preferably, the organic acid is selected from at least one of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid, and is even more preferably acetic acid.

[0013] According to the method of the first aspect, in step S13, the acetyl protection step includes reacting intermediates 1-2 with an acetylation agent; Preferably, the acetylation agent is selected from acetic anhydride and / or acetic halogen, more preferably acetic anhydride and / or acetyl chloride.

[0014] According to the method of the first aspect, in step S14, the ester hydrolysis step includes hydrolyzing intermediates 1-3 with a strong base; Preferably, the hydrolysis reaction temperature is 0-35℃; and / or Preferably, the hydrolysis reaction time is 1-3 hours.

[0015] According to the method of the first aspect, in step S2, the acid hydrolysant includes at least one of trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, HCl, and H2SO4, preferably trifluoroacetic acid.

[0016] According to the method of the first aspect, in step S3, the cyclizing agent is selected from at least one of I2, air, H2O2, and DMSO.

[0017] According to the method of the first aspect, step S1 includes: Protective peptide 1, containing an amino acid sequence of fragment 1, was prepared using solid-phase peptide synthesis technology. Protective peptide 1 was condensed with intermediate 1 to obtain protective peptide 2, containing an amino acid sequence of fragment 2. Protective peptide 2 was then used in a further solid-phase peptide synthesis reaction to obtain protective peptide 3, containing an amino acid sequence of fragment 3. Protective peptide 3 was then combined with... 4 Asn- 5 Thr- 6 The protective amino acid fragments of Ala are linked to form a protective peptide 4 with an amino acid sequence of fragment 4. The main chain of the protective peptide 4 is modified to obtain a fully protected cagliflozin resin conjugate with a cagliflozin sequence. Among them, including 4 Asn- 5 Thr- 6 The protected amino acid fragment of Ala is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala-OH; The first segment is: 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The second segment is: NBD ( 20 Ser- 21 Ser-) 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser-36 Asn- 37 Thr- 38 Pro-NH2; The third segment is: 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His-NBD( 20 Ser- 21 Ser)- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The fourth segment is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala- 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His- 20 Ser- 21 Ser- 22Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2.

[0018] According to the method of the first aspect, the method further includes: S4 purifies, concentrates, and freeze-dries the canagliflozin obtained in S3 to obtain canagliflozin peptide.

[0019] The method for preparing caglitazone of the present invention has, but is not limited to, the following beneficial effects: This application employs the fragment method and utilizes methods including... 20 Ser、 21 Ser's NBD pseudodipeptide and including 4 Asn- 5 Thr- 6 Solid-phase synthesis of canagliflozin using amino acid fragments of Ala can overcome the difficulty of coupling caused by α-helices in traditional solid-phase synthesis. It has the advantages of low cost, large-scale production capability, high yield, and high purity of crude peptide, which greatly reduces purification and production costs. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation of canagliflozin according to the present invention.

[0021] Figure 2 This is the HPLC chromatogram of intermediate 1 in Example 1.

[0022] Figure 3 This is the Mass spectrum of intermediate 1 in Example 1.

[0023] Figure 4 The 1H NMR spectrum of intermediate 1 in Example 1 is shown.

[0024] Figure 5 This is the HPLC chromatogram of intermediate 2 in Example 2.

[0025] Figure 6 This is the Mass spectrum of intermediate 2 in Example 2.

[0026] Figure 7 This is the HPLC chromatogram of the crude fragment 1 in Example 3.

[0027] Figure 8 The Mass spectrum of the crude fragment 1 in Example 3 is shown.

[0028] Figure 9 This is the HPLC chromatogram of the crude fragment 2 in Example 4.

[0029] Figure 10 The Mass spectrum of the crude fragment 2 in Example 4 is shown.

[0030] Figure 11 This is the HPLC chromatogram of the crude fragment 3 in Example 5.

[0031] Figure 12 The Mass spectrum of the crude fragment 3 in Example 5 is shown.

[0032] Figure 13 This is the HPLC chromatogram of the crude fragment 4 in Example 6.

[0033] Figure 14 The Mass spectrum of the crude fragment 4 in Example 6 is shown.

[0034] Figure 15 This is the HPLC chromatogram of the crude linear canagliflozin peptide from Example 8.

[0035] Figure 16 This is the Mass spectrum of the crude linear canagliflozin peptide from Example 8.

[0036] Figure 17 This is the HPLC chromatogram of crude canagliflozin from Example 9.

[0037] Figure 18 The Mass spectrum of crude canagliflozin from Example 9 is shown.

[0038] Figure 19 This is the HPLC chromatogram of canagliflozin arginine after separation and purification in Example 9. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0040] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0041] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0042] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0043] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value. In an embodiment, the term "about" indicates a range of ±1% of the following value.

[0044] In this invention, "room temperature" refers to 20-35℃.

[0045] This application provides a method for preparing caglitazone, comprising: S1 uses amide-type resin as a solid-phase synthesis carrier and solid-phase polypeptide synthesis technology to synthesize a fully protected cagliolipoide resin conjugate with a cagliolipoide sequence. The 20Ser-21Ser sequence in the calagliptin is formed using intermediate 1, which has the pseudo-dipeptide structure shown in Formula I. Formula I; Among them, R A R is an amino protecting group. A Selected from C1-C16 alkoxycarbonyl groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; R B R is a hydroxyl protecting group. B The group is selected from tert-butyl, C1-C5 acyl, benzyl, TBS, and TES, with tert-butyl being preferred; R C R is a phenolic hydroxyl protecting group. C Selected from C1-C5 acyl, benzyl, TBS, TES, preferably acetyl; In the caglitazone sequence 4 Asn- 5 Thr- 6 Ala uses including 4 Asn- 5 Thr- 6 Ala is formed from protected amino acid fragments; S2. The fully protected caglitazone resin from step S1 is cleaved using an acid hydrolysate to obtain linear caglitazone peptides. S3 uses a cyclizing agent to cyclize linear caglitazone peptides. 3 Cys and 8 The Cys site undergoes a disulfide bond cyclization reaction to yield canagliflozin.

[0046] Currently, the main methods for synthesizing canagliflozin include the following: Patent CN114249808A uses three pseudoproline dipeptides, which solves the problem of increased coupling difficulty caused by resin condensation, but increases production costs. Furthermore, the purity and yield of the peptide prepared by this process are relatively low. Patent CN119350469A uses a large-fragment SPPS-LPPS mixed method to synthesize canagliflozin. Specifically, this method involves solid-phase synthesis of four fragments, followed by fully protected cleavage of the fragment peptide resin, and then liquid-phase coupling. However, liquid-phase coupling requires cleavage of all four fragment peptide resins, and post-processing includes filtration, concentration, crystallization, centrifugation, etc., requiring more equipment and incurring higher time costs.

[0047] The inventors of this application discovered in experiments that during the synthesis of caglitazone using solid-phase peptide synthesis (SPPS) technology, an α-helix structure is formed during the synthesis process, which causes the amino acids at positions 18 and 19 in the sequence to be affected. 18 Arg and 19 His is difficult to connect. The method in this application uses the intermediate 1NBD-(Ser-Ser)-OH of the pseudo-dipeptide structure shown in Formula I to connect the calagliptin sequence. 20 Ser、 21 The use of intermediate 1, which links serine residues at the Ser site, can disrupt the α-helix structure formed during peptide synthesis, thereby... 18 Arg and 19 His connection becomes easier, thereby improving coupling efficiency and reducing impurity generation, which is beneficial for solid-phase peptide synthesis and increases the yield of caglitazone. Furthermore, the inventors of this application have also discovered that when the peptide chain is connected to the end, the 5th amino acid in the caglitazone sequence... 5 Thr and the fourth amino acid 4 ASN connections are difficult to establish and have low efficiency. This application's method addresses these issues by designing a system that includes... 4 Asn- 5 Thr- 6 Ala's protective amino acid fragments reduced 4 Asn and 5 The increased difficulty in linking Thr improves linking efficiency and reduces impurity generation. The technical solution of this application can solve the problems of difficult coupling, easy racemization, low yield, and high cost in existing technologies, achieving efficient synthesis of canagliflozin.

[0048] In some specific embodiments, the acyl groups of C1-C5 may be at least one of formyl, acetyl, propionyl, butyryl, and valeryl.

[0049] In some implementations, in step S1, the including 4 Asn- 5 Thr- 6 The protective amino acid fragment of Ala was selected from R D - 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH, R D - 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala(R S1 )-OH, R D - 2 Lys(R S )- 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH; Wherein, the R D Each of the amino protecting groups is independently selected from C1-C16 alkoxycarbonyl protecting groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; The R S The protecting groups are the side chain protecting groups of amino acids, each independently selected from C1-C16 alkoxycarbonyl protecting groups, C4-C21 alkyl protecting groups, and C4-C8 alkoxy protecting groups, preferably tert-butyl, triphenylmethyl, or tert-butyloxycarbonyl.

[0050] In this application, the C1-C16 alkoxycarbonyl protecting group can be, for example, benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, etc.

[0051] In this application, the alkyl protecting group of C4-C21 can be, for example, triphenylmethyl, p-methoxybenzyl, benzyl, tert-butyl, 2,4-dimethoxybenzyl, etc.

[0052] In this application, the alkoxy protecting group of C4-C8 can be, for example, tert-butoxy, benzyloxy, allyloxy, etc.

[0053] In some specific embodiments, the alkoxycarbonyl protecting group of the C1-C16 is tert-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc).

[0054] In some specific embodiments, the alkyl protecting group of the C4-C21 includes at least one of tert-butyl (t-Bu) and triphenylmethyl (Trt).

[0055] In some specific embodiments, the alkoxy protecting group of the C4-C8 is tert-butoxy (t-BuO).

[0056] In some specific embodiments, the pseudo-dipeptide Fmoc-NBD-(Ser-Ser)-OH is prepared as intermediate 1 by liquid-phase synthesis.

[0057] In some specific embodiments, solid-phase organic synthesis technology is used to prepare products including... 4 Asn- 5 Thr- 6 Ala's protective amino acid fragment R D - 2 Lys(R S )- 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH is used as intermediate 2.

[0058] In some specific embodiments, the pentapeptide fragment R D - 2 Lys(R S )- 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 The preparation method of Ala-OH includes the following steps: Using carboxyl resin as a solid-phase synthetic support, the following protected amino acids were sequentially coupled from the C-terminus to the N-terminus: R D -Ala-OH, R D -Thr(R S ) -OH, R D -Asn(R S )-OH, R D -Cys(R S )-OH, R D-NH-Lys(R S The )-OH group yielded a fully protected amino acid fragment as intermediate 2; Peptide chain cleavage and deprotection of fully protected intermediate 2 were performed using an acid hydrolysant. The acid hydrolysate can be, for example, TFE / DCM, TFA / DCM, or HFIP / DCM / acetic acid, preferably TFE and DCM. Specifically, the volume ratio of TFE to DCM can be 1:(3-5); the volume ratio of TFA to DCM can be 1:(95-105), preferably 1:(98-100); and the volume ratio of HFIP to DCM can be 1:(3-5).

[0059] In some specific embodiments, the carboxyl resin includes at least one of CTC resin and Wang resin; preferably, it is CTC resin.

[0060] In some specific embodiments, the degree of substitution of the carboxyl resin is 0.3-1.8 mmol / g, more specifically 1.6-1.7 mmol / g.

[0061] In some specific embodiments, the amount of each protected amino acid is 1.2-3.5 times the total molar amount of the carboxyl resin added; further, it is 1.5-3 times.

[0062] In some specific embodiments, in step S1, the amide resin is selected from one or more of Rink Amide resin, Rink Amide MBHA resin, Rink Amide AM resin, MBHA resin, and PAL resin, preferably Rink Amide resin; and / or The degree of substitution of the amide resin is 0.3-1.8 mmol / g, preferably 0.4-0.8 mmol / g; and / or The molar ratio of each protected amino acid to the resin is (1.2-3.5):1, preferably (1.5-3):1.

[0063] In some embodiments, in step S1, the coupling is carried out in the presence of a coupling agent selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, DIEA, and HOBT, preferably at least one of DIEA, DIC, HOBT, and EDCI.

[0064] In some specific embodiments, the coupling agent may be, for example, DIEA, DIC and HOBT, EDCI and HOBT, DCC and HOBT, EDCI and HOBT, HBTU and DIEA, Pybop and DIEA, HATU and DIEA, preferably DIEA, DIC and HOBT, EDCI and HOBT.

[0065] In some embodiments, the coupling reaction solvent includes at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP); more specifically, DCM or DMF.

[0066] In some implementations, the coupling time is 1-5 hours.

[0067] In some embodiments, the coupling process includes removing the amino protecting groups from the protecting amino groups on the peptide chains of amino resins and / or amino / carboxyl resins.

[0068] In some embodiments, an amino-protecting group removal reagent is used to remove the amino protecting group.

[0069] In some embodiments, the amino protecting group removal agent includes piperidine and DMF.

[0070] In some specific embodiments, the volume ratio of piperidine to DMF in the amino protecting group removal reagent can be 1:(3-5), preferably 1:(3.5-4.5), and more preferably 1:4.

[0071] In some embodiments, the coupling reaction in step S1 is carried out at room temperature.

[0072] In some embodiments, the method for preparing the intermediate 1 includes: S11 will R A -Ser(R B The condensation of )-OH with salicylaldehyde yields intermediate 1-1, which has the structure shown in Formula I-1: Formula I-1; Intermediate S12 1-1 was coupled with serine ester hydrochloride to obtain coupling product intermediate 1-2 having the structure shown in Formula I-2: Formula I-2; S13 involves acetyl protection of the salicylaldehyde phenolic hydroxyl group in intermediate 1-2 to obtain intermediate 1-3 having the structure shown in Formula I-3: Formula I-3; S14 intermediate 1-3 undergoes ester hydrolysis to obtain intermediate 1 with the structure shown in Formula I.

[0073] In some embodiments, in step S11, the condensation reaction temperature is 0-25°C; and / or The condensation reaction time is 2-6 hours, preferably 2-4 hours; and / or The condensation is carried out in the presence of a condensing agent selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, HOBT, and DIEA, preferably at least one of DIEA, DIC, EDCI, and HOBT; and / or The condensation is carried out in the presence of a catalyst, which in some embodiments is a basic catalyst, such as one or more of DMAP, DIPEA, and triethylamine, preferably DMAP.

[0074] In some specific embodiments, in step S11, the condensation reaction temperature can be, for example, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, or a range consisting of any two of the above values.

[0075] In some specific implementations, the contraction time can be, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or a range consisting of any two of the above values.

[0076] In some specific embodiments, the condensing agent may be, for example, DIEA, DIC and HOBT, EDCI and HOBT, DCC and HOBT, EDCI and HOBT, HBTU and DIEA, Pybop and DIEA, HATU and DIEA, preferably DIEA, DIC and HOBT, EDCI and HOBT.

[0077] In some embodiments, in step S22, the serine ester hydrochloride is selected from at least one of serine methyl ester hydrochloride, serine ethyl ester hydrochloride, and serine benzyl ester hydrochloride, preferably serine methyl ester hydrochloride; Preferably, step S22 is carried out in a mixed system comprising an organic base and an organic acid; More preferably, the organic base is selected from at least one of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine, and is further preferably pyridine; and / or More preferably, the organic acid is selected from at least one of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid, and is even more preferably acetic acid.

[0078] In some specific embodiments, the molar ratio of the organic base to the organic acid in the mixed system comprising organic base and organic acid is 1:6 to 6:1.

[0079] In some specific embodiments, the molar ratio of the organic base and the organic acid in the mixed system comprising the organic base and the organic acid can be, for example, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1 or any two of the above values.

[0080] In some specific embodiments, the mixed system comprising an organic base and an organic acid may, for example, consist of pyridine and acetic acid in a molar ratio of 1:1.

[0081] In some embodiments, step S13, the acetyl protection step includes reacting intermediates 1-2 with an acetylation agent; Preferably, the acetylation agent is selected from acetic anhydride and / or acetic halogen, more preferably acetic anhydride and / or acetyl chloride.

[0082] In some embodiments, step S14, the ester hydrolysis step includes hydrolyzing intermediates 1-3 with a strong base; Preferably, the hydrolysis reaction temperature is 0-35℃; and / or Preferably, the hydrolysis reaction time is 1-3 hours.

[0083] In some specific embodiments, in step S14, the hydrolysis reaction temperature can be, for example, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or a range consisting of any two of the above values.

[0084] In some specific embodiments, the hydrolysis reaction time can be, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or a range consisting of any two of the above values.

[0085] In some specific embodiments, the ester hydrolysis reaction is carried out in an isopropanol-water system.

[0086] In some specific embodiments, the hydrolysis reaction further includes the addition of calcium halides, such as one or more of CaCl2, CaBr2, and CaI2. ...

[0087] By adding calcium halide to the ester hydrolysis reaction, the concentration of OH ions in the solution can be reduced, thereby maintaining the overall alkalinity of the reaction system at a low level. This allows the ester hydrolysis reaction to proceed gently and avoids the protection of the R group. A R B R C The shedding of [something].

[0088] In some embodiments, in step S2, the acid hydrolysant may include at least one of trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, HCl, and H2SO4, preferably trifluoroacetic acid.

[0089] In some specific embodiments, the acid hydrolysate may include TFA, TIPS, EDT, and H2O.

[0090] In some specific embodiments, the acid hydrolysate is composed of TFA, TIPS, EDT and H2O; preferably, the volume ratio of TFA, TIPS, EDT and H2O is (36-38):(0.8-1.2):(0.8-1.2):1; more preferably about 37:1:1:1.

[0091] In some embodiments, in step S3, the cyclizing agent is selected from at least one of I2, air, H2O2, and DMSO.

[0092] In some implementations, step S1 includes: Protective peptide 1, containing an amino acid sequence of fragment 1, was prepared using solid-phase peptide synthesis technology. Protective peptide 1 was condensed with intermediate 1 to obtain protective peptide 2, containing an amino acid sequence of fragment 2. Protective peptide 2 was then used in a further solid-phase peptide synthesis reaction to obtain protective peptide 3, containing an amino acid sequence of fragment 3. Protective peptide 3 was then combined with... 4 Asn- 5 Thr- 6 The protective amino acid fragments of Ala are linked to form a protective peptide 4 with an amino acid sequence of fragment 4. The main chain of the protective peptide 4 is modified to obtain a fully protected cagliflozin resin conjugate with a cagliflozin sequence. Among them, including 4 Asn- 5 Thr- 6 The protected amino acid fragment of Ala is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala-OH; The first segment is: 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The second segment is: NBD ( 20 Ser- 21 Ser-) 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The third segment is: 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His-NBD( 20 Ser- 21 Ser)- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr-38 Pro-NH2; The fourth segment is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala- 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His- 20 Ser- 21 Ser- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2.

[0093] In some embodiments, the method further includes: S4 purifies, concentrates, and freeze-dries the canagliflozin obtained in S3 to obtain canagliflozin peptide.

[0094] In some specific embodiments, the purification method includes HPLC purification.

[0095] In some specific embodiments, the preparation method of canagliflozin of this application may include: Fragment 1 and the NBD pseudo-dipeptide molecule of intermediate 1 were condensed using solid-phase peptide synthesis technology to obtain fragment 2. Fragment 2 was then used to continue solid-phase peptide synthesis to obtain fragment 3. Fragment 3 was then linked with intermediate 2, which has a pentapeptide structure and protects the amino acid fragment, to form fragment 4. Fragment 4 was then modified in the main chain and acid-hydrolyzed to obtain linear peptide 5. After disulfide cyclization, crude canagliflozin peptide was obtained. The intermediate 1 is: Fmoc-NBD-(Ser-Ser)-OH, with the following structure: The intermediate 2 is: 2 Lys(BOC)- 3 Cys(Trt- 4 Asn(Trt)- 5 Thr(tBu)- 6 Ala-OH The first segment is: 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2 The second segment is: NBD ( 20 Ser- 21 Ser-) 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2 The third segment is: 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His-NBD( 20 Ser- 21 Ser)- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2 The fourth segment is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala- 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His- 20 Ser- 21 Ser- 22 Asn- 23 Asn- 24 Phe- 25 Gly-26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2 Fragment 5 is: {Eicosanedioic acid-γ-Glu}- 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala- 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His- 20 Ser- 21 Ser- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2 The fragment canagliflozin is: {Eicosanedioic acid-γ-Glu}-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Glu-Phe-Leu-Arg-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Pro-NH2 (Disulfide bridge:Cys3-Cys8).

[0096] The method described in this application involves condensing intermediate 1, which has an NBD pseudo-dipeptide molecular structure, to obtain fragment 2 using solid-phase organic synthesis technology. Fragment 2 is then reacted and linked to obtain fragment 3, which is then reacted with intermediate 2 to form fragment 4. Fragment 4 is then modified in the main chain, and after acid hydrolysis, a linear peptide is obtained. After cyclization, crude canagliflozin peptide is obtained. Further purification using a two-stage high-pressure preparation yields the final qualified canagliflozin product. The purity of the first purification can reach up to 99.66%, and the overall yield is 47.2%.

[0097] In some specific embodiments, the preparation method of the intermediate 2 includes the following steps: Using CTC resin as a solid-phase synthesis support, the following protected amino acids were sequentially coupled from the C-terminus to the N-terminus: R1-Ala-OH, R1-Thr(R2)-OH, R1-Asn(R3)-OH, R1-Cys(R3)-OH, and R1-NH-Lys(R4)-OH to obtain the fully protected intermediate 2. Peptide chain cleavage and deprotection of fully protected intermediate 2 are performed using an acid hydrolysate; the acid hydrolysate is a weak acid, further comprising one of TFE / DCM, TFA / DCM, 1% TFA / DOM, HFIP / DCM acetic acid, and even further comprising TFE / DCM; R1 is an amino protecting group, and R2, R3, R4, and R5 are side chain protecting groups of amino acids.

[0098] In some specific embodiments, the acid hydrolysate may include, for example, TFA and DCM, trifluoroethanol and DCM, or hexafluoroisopropanol and dichloromethane.

[0099] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0100] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0101] The present application will be further described in detail below through embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0102] The Chinese meanings of the English abbreviations used in this application are shown in Table 1.

[0103] Table 1

[0104] In this invention, the first to the 20th amino acids and the 21st to the 39th amino acids refer to the amino acids of canagliflozin sequentially from the N-terminus to the CONH2-terminus of the C-terminus.

[0105] The flowchart for the preparation of caglitazone in this invention is attached. Figure 1 As shown.

[0106] like Figure 1 As shown, using an amide-type resin as a solid-phase synthesis carrier, fully protected fragment 1 resin was obtained by sequential coupling from the C-terminus to the N-terminus through solid-phase peptide synthesis (SPPS). Fragment 1 was then condensed with the NBD pseudo-dipeptide molecule of intermediate 1 using SPPS to obtain fragment 2. Fragment 2 was then used in a further SPPS reaction to obtain fragment 3. Fragment 3 was then linked with intermediate 2, which has a pentapeptide structure, to form fragment 4. Fragment 4 was then modified in the backbone to obtain fragment 5. Fragment 5 was acid-hydrolyzed to obtain a linear canagliflozin peptide. The linear canagliflozin peptide was then subjected to a disulfide cyclization reaction to obtain a crude canagliflozin peptide. Here, PG stands for protecting group.

[0107] Example 1. Synthesis of Intermediate 1 This embodiment illustrates the synthesis of intermediate 1 Fmoc-NBD-(Ser-Ser)-OH.

[0108] Step 1. Fmoc-Ser(tBu)-OH (33.9 g, 100.0 mmol), salicylaldehyde (1.0 eq, 12.2 g, 100.0 mmol), DMAP (0.1 eq, 13 mg, 0.1 mmol), and EDCI (~20.1 g, 1.05 eq, 105 mmol) were added to DCM (500 mL) and stirred at room temperature for 2.0–3.0 h (2 h in this example). The reaction was monitored by TLC. After the reaction was complete, the mixture was washed with 0.5 M HCl / H2O (3…). The organic phase was dried over anhydrous Na2SO4 (100 mL), concentrated and dried, and no further purification was required to obtain crude salicylaldehyde ester 1-1 (46.2 g) in a pale yellow oil.

[0109] Step 2. The crude salicylaldehyde ester 1-1 (46.2 g) obtained in Step 1 was dissolved by sonication in a pyridine-acetic acid solution (final volume 5-10%, pyridine:acetic acid molar ratio 1:1, final volume 460 mL in this example) with HCl·H2N-Ser-OMe (1.2-1.5 eq., 1.2 eq in this example). Then it was added dropwise to a Py:AcOH solution (final concentration 100-200 mM, 100 mM in this example). The reaction mixture was stirred at room temperature for 3 h to overnight, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with ethyl acetate (500 mL), followed by 0.2 M HCl / H2O (100 mL). 3) Saturated saline solution (100 mL) 2) Wash, dry the organic phase with anhydrous Na2SO4, concentrate and dry, and obtain 51.2 g of pale yellow oily crude product 1-2 without further purification.

[0110] Step 3. Dissolve 51.2 g of intermediate 1-2 obtained in Step 2 in 300 mL of dichloromethane. Then, slowly add acetic anhydride (2.0 eq.) and DIPEA (3.0-4.0 eq., 4.0 eq. in this example) dropwise to the above solution, stirring the reaction mixture for several hours to overnight to ensure complete conversion. Monitor the reaction process by TLC. After the reaction is complete, the resulting dichloromethane solution is sequentially treated with 0.2 M HCl / H2O (100 mL). 3) NaHCO3 (100mL) 3) Saturated saline solution (100mL) 2) Wash, concentrate and dry the obtained organic phase, and obtain 56.4 g of pale yellow oily crude product 1-3 without further purification.

[0111] Step 4. Dissolve the yellow oily substance 1-3 (56.4 g) obtained in Step 3 in iPrOH (200 mL). Weigh CaCl2 and add it to the dissolved iPrOH (200 mL). The concentration of CaCl2 is 0.8 mol / L. Under ice bath conditions, slowly add NaOH (4.0 eq) aqueous solution (concentration 1 mol / L). The reaction is carried out for 0.5-1 h, monitored by TLC. After the reaction is complete as monitored by TLC, add DCM (100 mL). 2) Wash and collect the aqueous phase. Slowly adjust the pH to approximately 5.6 with 1 mol / L hydrochloric acid aqueous solution, causing a white solid to slowly precipitate. Filter to obtain the white solid, then recrystallize using a toluene / dioxane = 1:1 (v / v) mixed solution to obtain 48.6 g of intermediate 1 white crystals. The HPLC chromatogram is shown below. Figure 2 As shown: HPLC purity: 96.48%, Mass spectrum as follows. Figure 3 As shown, the 1H NMR spectrum is as follows: Figure 4 As shown.

[0112] Example 2. Synthesis of Intermediate 2 This embodiment is used to illustrate the synthesis of intermediate 2.

[0113] Weigh 2Cl-CTC resin (1.5 mmol / g, 100 mmol) and add it to the reaction vessel. Weigh Fmoc-Ala-OH (62.20 g, 200 mmol) and DIEA (51.6 g, 400 mmol) and dissolve them in 500 ml of DCM. Then add the solution to the solid-phase reactor and react at room temperature for 2.0-4 h (3 h in this example). Add MeOH (12.8 g, 400 mmol), end-cap for 30 min, dry under vacuum, and wash 4 times with DMF.

[0114] Prepare the Fmoc / Boc-protected amino acids corresponding to the amino acids at positions 2-5 of caglitazone (the corresponding Fmoc / Boc-protected amino acids and their corresponding resin equivalents are shown in Table 2). Repeat the above steps using DIC / HOBt as a condensing agent, and couple the remaining amino acids according to the peptide sequence to obtain fully protected intermediate 2. Perform small-scale resin lysis detection on fully protected intermediate 2, and the HPLC chromatogram is shown below. Figure 5 As shown: HPLC purity: 81.58%, Mass spectrum as follows. Figure 6 As shown.

[0115] Table 2. Fmoc / Boc amino acids and corresponding resin equivalents for amino acids.

[0116] Example 3. Synthesis of Fragment 1 This embodiment is used to illustrate the synthesis of fragment 1.

[0117] Weigh Rink AM resin (0.61 mmol / g, 20 mmol, degree of substitution 0.61 mmol / g) and swell it in DMF for 30 min. Add 300 mL of 20% piperidine / DMF solution to remove Fmoc for 20 min. Dry the solution and wash it with DMF 6 times. Weigh Fmoc-Pro-OH (19.14 g, 60 mmol) and HOBT (8.10 g, 72 mmol) and dissolve them in 300 mL of DMF at 0 °C. Add DIC (9.0 g, 72 mmol) and activate for 5 min. Add the solution to the solid-phase reactor and react at room temperature for 2.0 h. The ninhydrin test result is negative. Dry the solution and wash it with DMF 4 times.

[0118] Prepare the Fmoc / Boc-protected amino acids corresponding to positions 22-37 of the caglitazone (the corresponding Fmoc / Boc-protected amino acids and their corresponding resin equivalents are shown in Table 3). Repeat the above steps to couple the remaining amino acids according to the peptide sequence to obtain the fully protected fragment 1: (R5) 22 Asn(R4)- 23 Asn(R4)- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr(R2)- 32 Asn(R4)- 33 Val- 34 Gly- 35 Ser(R2)- 36 Asn(R4)- 37 Thr(R2)- 38 Pro-NH2 Where R2 is tBu, R4 is TTrt, and R5 is Fmoc.

[0119] Fragment 1 was subjected to small-scale resin pyrolysis detection, and the HPLC chromatogram is shown below. Figure 7 As shown: HPLC purity: 96.32%, Mass spectrum as follows. Figure 8 As shown.

[0120] Table 3. Fmoc / Boc amino acids and corresponding resin equivalents for amino acids.

[0121] Example 4. Synthesis of canagliflozin fragment 2 This embodiment is used to illustrate the synthesis of fragment 2.

[0122] Fragment 1 prepared in Example 3 was added to 300 mL of 20% piperidine / DMF solution to remove Fmoc for 20 min, dried, and washed 6 times with DMF. Intermediate 1 (39.98 g, 60 mmol) and HOBT (8.10 g, 72 mmol) were weighed and dissolved in 300 mL of DMF at 0 °C. After activation with DIC (9.0 g, 72 mmol) for 5 min, the solution was added to a solid-phase reactor and reacted at room temperature for 2.0 h. The ninhydrin test result was negative. The fragment was dried, washed 4 times with DMF, and canagliflozin fragment 2 was obtained. Fragment 2 was subjected to small-scale resin cleavage detection. The HPLC chromatogram is shown below. Figure 9 As shown: HPLC purity: 89.60%, Mass spectrum as follows. Figure 10 As shown.

[0123] Example 5. Synthesis of canagliflozin fragment 3 This embodiment is used to illustrate the synthesis of fragment 3.

[0124] Fragment 2 prepared in Example 4 was added to 300 mL of 20% piperidine / DMF solution to remove Fmoc for 20 min, dried, and washed 6 times with DMF. Fmoc-His(Trt)-OH (36.96 g, 60 mmol) and HOBT (8.10 g, 72 mmol) were weighed and dissolved in 300 mL of DMF at 0 °C. DIC (9.0 g, 72 mmol) was added and activated for 5 min. The solution was then added to a solid-phase reactor and reacted at room temperature for 2.0 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0125] Prepare the Fmoc / Boc-protected amino acids corresponding to positions 7-18 of the caglitazone (the Fmoc / Boc-protected amino acids and their corresponding resin equivalents are shown in Table 4). Repeat the above steps to couple the remaining amino acids according to the peptide sequence to obtain the fully protected fragment 3: (R5) 7 Thr(R2)- 8 Cys(R4)- 9 Ala- 10 Thr(R2)-11 Gln(R4)- 12 Arg(R3)- 13 Leu- 14 Ala- 15 Glu(R2)- 16 Phe- 17 Leu- 18 Arg(R3)- 19 His(R4)- 20 Ser- 21 Ser(DNB)- 22 Asn(R4)- 23 Asn(R4)- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr(R2)- 32 Asn(R4)- 33 Val- 34 Gly- 35 Ser(R2)- 36 Asn(R4)- 37 Thr(R2)- 38 Pro-NH2 Where R2 is tBu, R3 is pbf, R4 is Trt, and R5 is Fmoc.

[0126] Canagliflozin fragment 3 was obtained, and a small amount was analyzed by resin lysis. The HPLC chromatogram is shown below. Figure 11 As shown: HPLC purity: 76.88%, Mass spectrum as follows. Figure 12 As shown.

[0127] Table 4. Fmoc / Boc amino acids and corresponding resin equivalents for amino acids.

[0128] Example 6. Synthesis of canagliflozin fragment 4 This embodiment is used to illustrate the synthesis of fragment 4.

[0129] Fragment 3 prepared in Example 5 was added to 300 mL of 20% piperidine / DMF solution to remove Fmoc for 20 min, dried, and washed 6 times with DMF. Intermediate 2 (86.74 g, 60 mmol) and HOBT (8.10 g, 72 mmol) were weighed, dissolved in 300 mL of DMF at 0 °C, and activated with DIC (9.0 g, 72 mmol) for 5 min. The solution was then added to a solid-phase reactor and reacted at room temperature for 4.0 h. The ninhydrin test result was negative. The fragment was dried, washed 4 times with DMF, and the fully protected canagliflozin fragment 4 was obtained. (R5) 2 Lys(R3)- 3 Cys(R4)- 4 Asn(R4)- 5 Thr(R2)- 6 Ala- 7 Th(R2)- 8 Cys(R4)- 9 Ala- 10 Thr(R2)- 11 Gln(R4)- 12 Arg(R3)- 13 Leu- 14 Ala- 15 Glu(R2)- 16 Phe- 17 Leu- 18 Arg(R3)- 19 His(R4)- 20 Ser- 21 Ser(DNB)- 22 Asn(R4)- 23 Asn(R4)- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr(R2)- 32 Asn(R4)- 33 Val- 34 Gly- 35 Ser(R2)- 36 Asn(R4)- 37 Thr(R2)- 38 Pro-NH2 Where R2 is tBu, R3 is pbf, R4 is Trt, and R5 is Fmoc.

[0130] The obtained canagliflozin fragment 4 was analyzed by small-scale resin lysis, and the HPLC chromatogram is shown below. Figure 13 As shown: HPLC purity: 72.76%, Mass spectrum as follows. Figure 14 As shown.

[0131] Example 7. Synthesis of canagliflozin fragment 5 This embodiment is used to illustrate the synthesis of fragment 5.

[0132] Fragment 4 prepared in Example 6 was added to 300 mL of 20% piperidine / DMF solution to remove Fmoc for 20 min. The solution was dried, washed 6 times with DMF, and Fmoc-Glu-OtBu (25.5 g, 60 mmol) and HOBT (8.10 g, 72 mmol) were dissolved in 300 mL of DMF at 0 °C. DIC (9.0 g, 72 mmol) was added and activated for 5 min. The solution was then added to a solid-phase reactor and reacted at room temperature for 4.0 h. The ninhydrin test result was negative. The solution was dried, washed 4 times with DMF, and then eicosanoic acid monotert-butyl ester (23.92 g, 60 mmol) and HOBT (8.10 g, 72 mmol) were dissolved in 300 mL of DMF at 0 °C. DIC (9.0 g, 72 mmol) was added and activated for 5 min. The solution was then added to a solid-phase reactor and reacted at room temperature for 4.0 h. h, the ninhydrin test result was negative, the sample was dried, washed four times with DMF, and the fully protected fragment of canagliflozin was obtained: {Eicosanedioicacid-γ-Glu}-(R5) 2 Lys(R3)- 3 Cys(R4)- 4 Asn(R4)- 5 Thr(R2)- 6 Ala- 7 Th(R2)- 8 Cys(R4)- 9 Ala- 10 Thr(R2)- 11 Gln(R4)- 12 Arg(R3)- 13 Leu- 14 Ala- 15 Glu(R2)- 16 Phe- 17 Leu- 18 Arg(R3)- 19 His(R4)- 20 Ser- 21 Ser(DNB)- 22Asn(R4)- 23 Asn(R4)- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr(R2)- 32 Asn(R4)- 33 Val- 34 Gly- 35 Ser(R2)- 36 Asn(R4)- 37 Thr(R2)- 38 Pro-NH2 Where R2 is tBu, R3 is pbf, R4 is Trt, and R5 is Fmoc.

[0133] Example 8. Synthesis of linear canagliflozin peptide This example illustrates the synthesis of caglitazone linear peptide.

[0134] Fragment 5 prepared in Example 7 was added to 600 mL of cutting buffer TFA / TIPS / EDT / H2O (92.5% / 2.5% / 2.5% / 2.5%, V / V / V / V). After reacting at room temperature for 120 min, 5 L of frozen methyl tert-butyl ether was added to precipitate 87.05 g of crude white solid canagliflozin linear peptide. The HPLC chromatogram of the crude canagliflozin linear peptide is shown below. Figure 15 As shown: HPLC purity: 67.97%, Mass spectrum as follows. Figure 16 As shown.

[0135] Example 9. Synthesis of crude canagliflozin peptide The linear peptide prepared in Example 8 was dissolved in an aqueous solution to obtain a linear peptide aqueous solution (linear peptide concentration 2-5 g / L). A 0.1-1 mol / L iodine methanol solution (0.5 mol / L was used in this example) was slowly added dropwise to oxidize the solution until it changed from transparent to yellow. Then, a 0.1-1 mol / L vitamin C aqueous solution (0.5 mol / L was used in this example) was added to reduce excess iodine until the solution changed from yellow to transparent, yielding the crude canagliflozin peptide solution. The HPLC chromatogram of the crude canagliflozin peptide is shown below. Figure 17 As shown, the purity detected by HPLC was 78.60%, and the Mass spectrum is as follows. Figure 18 As shown.

[0136] The crude canagliflozin solution from Example 8 was filtered. HPLC gradient elution was performed using octadecyl silica gel as the stationary phase and 0.2% sodium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B. The purification conditions are shown in Table 5 below. Table 5

[0137] The qualified fraction of canagliflozin was obtained, acetonitrile was removed, and the product was lyophilized to obtain 41.62 g of refined canagliflozin peptide with an HPLC purity of 99.66% and an overall yield of 47.2%. The chromatogram of the purified product after separation is shown below. Figure 19 As shown.

[0138] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing canagliflozin, comprising: S1 uses amide-type resin as a solid-phase synthesis carrier and solid-phase polypeptide synthesis technology to synthesize a fully protected cagliolipoide resin conjugate with a cagliolipoide sequence. Among them, the caglitazone sequence 20 Ser- 21 Ser is formed using intermediate 1, which has the pseudo-dipeptide structure shown in Formula I: Equation I; Among them, R A R is an amino protecting group. A Selected from C1-C16 alkoxycarbonyl groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; R B R is a hydroxyl protecting group. B Selected from tert-butyl, C1-C5 acyl, benzyl, TBS, TES, preferably tert-butyl; R C R is a phenolic hydroxyl protecting group. C Selected from C1-C5 acyl, benzyl, TBS, TES, preferably acetyl; In the caglitazone sequence 4 Asn- 5 Thr- 6 Ala uses including 4 Asn- 5 Thr- 6 Ala is formed by protecting amino acid fragments; S2. The fully protected cagliflozin resin conjugate from step S1 is cleaved using an acid hydrolysate to obtain a linear cagliflozin peptide. S3 uses a cyclizing agent to cyclize linear caglitazone peptides. 3 Cys and 8 The Cys site undergoes a disulfide bond cyclization reaction to yield canagliflozin.

2. The method according to claim 1, wherein, In step S1, the included 4 Asn- 5 Thr- 6 The protective amino acid fragment of Ala was selected from R D - 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH, R D - 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala(R S )-OH, R D - 2 Lys(R S )- 3 Cys(R S )- 4 Asn(R S )- 5 Thr(R S )- 6 Ala-OH; Wherein, the R D Each of the amino protecting groups is independently selected from C1-C16 alkoxycarbonyl protecting groups, preferably tert-butoxycarbonyl or 9-fluorenemethyloxycarbonyl; The R S The protecting groups are the side chain protecting groups of amino acids, each independently selected from C1-C16 alkoxycarbonyl protecting groups, C4-C21 alkyl protecting groups, and C4-C8 alkoxy protecting groups, preferably tert-butyl, triphenylmethyl, or tert-butyloxycarbonyl.

3. The method according to claim 1, wherein, In step S1, the amide resin is selected from one or more of Rink Amide resin, Rink Amide MBHA resin, Rink Amide AM resin, MBHA resin, and PAL resin, preferably Rink Amide resin; and / or The degree of substitution of the amide resin is 0.3-1.8 mmol / g, preferably 0.4-0.8 mmol / g; and / or The molar ratio of each protected amino acid to the resin is (1.2-3.5):1, preferably (1.5-3):

1.

4. The method according to claim 1, wherein, In step S1, the coupling is carried out in the presence of a coupling agent, which is selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, DIEA, and HOBT, preferably at least one of DIEA, DIC, HOBT, and EDCI.

5. The method according to claim 1, wherein, The preparation method of the intermediate 1 includes: S11 will R A -Ser(R B The condensation of )-OH with salicylaldehyde yields intermediate 1-1, which has the structure shown in Formula I-1: Equation I-1; Intermediate S12 1-1 was coupled with serine ester hydrochloride to obtain coupling product intermediate 1-2 having the structure shown in Formula I-2: Equation I-2; S13 involves acetyl protection of the salicylaldehyde phenolic hydroxyl group in intermediate 1-2 to obtain intermediate 1-3 having the structure shown in Formula I-3: Formula I-3; S14 intermediate 1-3 undergoes ester hydrolysis to obtain intermediate 1 with the structure shown in Formula I.

6. The method according to claim 5, wherein, In step S11, the condensation reaction temperature is 0-25℃; and / or The condensation reaction time is 2-6 hours, preferably 2-4 hours; and / or The condensation is carried out in the presence of a condensing agent selected from at least one of DIC, HBTU, HATU, DCC, EDCI, chloroacetyl chloride, azide, TBTU, Pybop, HOBT, and DIEA, preferably at least one of DIEA, DIC, EDCI, and HOBT; and / or The condensation is carried out in the presence of a catalyst, preferably DMAP.

7. The method according to claim 5, wherein, In step S22, the serine ester hydrochloride is selected from at least one of serine methyl ester hydrochloride, serine ethyl ester hydrochloride, and serine benzyl ester hydrochloride, preferably serine methyl ester hydrochloride; Preferably, step S22 is carried out in a mixed system comprising an organic base and an organic acid; More preferably, the organic base is selected from at least one of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine, and is further preferably pyridine; and / or More preferably, the organic acid is selected from at least one of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid, and is even more preferably acetic acid.

8. The method according to claim 5, wherein, In step S13, the acetyl protection step includes reacting intermediates 1-2 with an acetylation reagent; Preferably, the acetylation agent is selected from acetic anhydride and / or acetic halogen, more preferably acetic anhydride and / or acetyl chloride.

9. The method according to claim 5, wherein, In step S14, the ester hydrolysis step includes hydrolyzing intermediates 1-3 with a strong base; Preferably, the hydrolysis reaction temperature is 0-35℃; and / or Preferably, the hydrolysis reaction time is 1-3 hours.

10. The method according to claim 1, wherein, In step S2, the acid hydrolysant includes at least one of trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, HCl, and H2SO4, preferably trifluoroacetic acid.

11. The method according to claim 1, wherein, In step S3, the cyclizing agent is selected from at least one of I2, air, H2O2, and DMSO.

12. The method according to claim 1, wherein, Step S1 includes: Protective peptide 1, containing an amino acid sequence of fragment 1, was prepared using solid-phase peptide synthesis technology. Protective peptide 1 was condensed with intermediate 1 to obtain protective peptide 2, containing an amino acid sequence of fragment 2. Protective peptide 2 was then used in a further solid-phase peptide synthesis reaction to obtain protective peptide 3, containing an amino acid sequence of fragment 3. Protective peptide 3 was then combined with... 4 Asn- 5 Thr- 6 The protective amino acid fragments of Ala are linked to form a protective peptide 4 with an amino acid sequence of fragment 4. The main chain of the protective peptide 4 is modified to obtain a fully protected calagliptin resin conjugate with a calagliptin sequence. Among them, including 4 Asn- 5 Thr- 6 The protected amino acid fragment of Ala is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala-OH; The first segment is: 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The second segment is: NBD ( 20 Ser- 21 Ser-) 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The third segment is: 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His-NBD( 20 Ser- 21 Ser)- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2; The fourth segment is: 2 Lys- 3 Cys- 4 Asn- 5 Thr- 6 Ala- 7 Thr- 8 Cys- 9 Ala- 10 Thr- 11 Gln- 12 Arg- 13 Leu- 14 Ala- 15 Glu- 16 Phe- 17 Leu- 18 Arg- 19 His- 20 Ser- 21 Ser- 22 Asn- 23 Asn- 24 Phe- 25 Gly- 26 Pro- 27 lle- 28 Leu- 29 Pro- 30 Pro- 31 Thr- 32 Asn- 33 Val- 34 Gly- 35 Ser- 36 Asn- 37 Thr- 38 Pro-NH2.

13. The method according to any one of claims 1 to 12, wherein, The method further includes: S4 purifies, concentrates, and freeze-dries the canagliflozin obtained in S3 to obtain canagliflozin peptide.

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