Preparation method of retaglutide

Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to a preparation method of retaglutide. According to the method, a fragment 1-4-methoxy salicylaldehyde ester and a fragment 2 are connected through a cysteine / penicillamine connection reaction, acidolysis and desulfurization are performed, crude retaglutide is obtained, and the purity of the crude peptide can reach 83.3%; furthermore, secondary high-pressure preparation and purification are adopted to obtain a final qualified retaglutide finished product, the secondary purification purity can reach 99.47%, and the overall yield reaches 37.2%; according to the method, a two-stage method is adopted, a CPL chemical connection method is used for chemical connection, the method has the advantages of being low in cost, capable of achieving large-scale production, high in yield, high in crude peptide purity and the like, the purification cost and the production cost are greatly reduced, all fragments 2 are natural amino acid, and biological fermentation production can be achieved. Therefore, the method has the advantages of semi-chemical and semi-biological synthesis, and the production cost is greatly reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine preparation, and particularly relates to a preparation method of Retatrutide. BACKGROUND

[0002] Retatrutide is a triple agonist of GLP-1R, GIPR and GCGR, and its peptide sequence structure is as follows: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu -Leu-Asp-Lys- Lys(icosadioic acid-rGlu-AEEA)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2.

[0003] Retatrutide is a new triple receptor agonist, mainly used for treating obesity and metabolic diseases. It can activate three kinds of incretin receptors, including GLP-1 (glucagon-like peptide-1), GIP (glucose-dependent insulin polypeptide) and glucagon receptor. This multiple mechanism makes Retatrutide have great potential in weight management, blood glucose control and overall metabolic health.

[0004] Since Retatrutide contains 39 amino acids, the sequence is long and contains more hydrophobic amino acids in the peptide sequence. If a single amino acid is used to synthesize the method, the production cycle is long, and as the synthesis process continues, the peptide chain grows, which is easy to produce missing peptides, resulting in increased impurities, and these missing peptide impurities are similar in properties to the product, making it difficult to purify the crude Retatrutide obtained in the later stage. Low yield.

[0005] Therefore, it is of great practical significance to develop a synthesis method of Retatrutide with high yield, short cycle and few impurities. SUMMARY

[0006] The first aspect of the application aims to provide a preparation method of Retatrutide.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is: The first aspect of the application provides a preparation method of Retatrutide, comprising the following steps: The fragment 1-4-methoxysalicylaldehyde ester is connected with the fragment 2 through a cysteine / penicillamine ligation (CPL) reaction, acidolysis, and then desulfurization to obtain a crude peptide of Retalipotide; The fragment 1-4-methoxysalicylaldehyde ester is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-O-4-MeO-Ph(CHO); The fragment 2 is: NH2-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2.

[0008] In some embodiments, the preparation method comprises the following steps: connecting the fragment 1-4-methoxysalicylaldehyde ester with the fragment 2 through a cysteine / penicillamine ligation reaction to obtain intermediate 1; then performing acidolysis on the intermediate 1 to obtain intermediate 2; and then performing desulfurization on the intermediate 2 to change the cysteine in the intermediate 2 back to alanine to obtain a crude peptide of Retalipotide; The fragment 1-4-methoxysalicylaldehyde ester is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-(N,C)-Benzilidine-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2; The fragment 1-4-methoxysalicylaldehyde ester is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2.

[0009] In some embodiments, the cysteine / penicillamine ligation reaction is performed in a mixture comprising: an organic base and an organic acid.

[0010] In some embodiments, the organic base in the mixture comprises at least one of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, N,N-diisopropylethylamine; further pyridine.

[0011] In some embodiments, the organic acid in the mixture comprises at least one of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, butyric acid; further acetic acid.

[0012] In some embodiments, the volume ratio of the organic base to the organic acid in the mixture is 1:(0.16-6); further 1:(1-3); still further about 1:1.

[0013] In some embodiments, the concentration of the organic base in the mixture is 10-60% v / v; further 25-55% v / v; still further about 50%.

[0014] In some embodiments, the concentration of the organic acid in the mixture is 10-80% v / v; further 45-75% v / v; still further about 50%.

[0015] In some embodiments, the time of the cysteine / penicillamine ligation reaction is 2-14 h; further 2-8 h; still further 2-6 h; yet further about 5 h.

[0016] In some embodiments, the temperature of the cysteine / penicillamine ligation reaction is room temperature.

[0017] In some embodiments, the molar ratio of Fragment 2 to Fragment 1-4-methoxy salicylaldehyde ester is (1-2): 1; further (1.3-1.7): 1; still further about 1.5: 1.

[0018] In some embodiments, the acid hydrolysis employs an acid hydrolysis agent comprising TFA, TIPS, EDT, and H2O.

[0019] In some embodiments, the volume ratio of TFA, TIPS, EDT, and H2O is (36-38):(0.8-1.2):(0.8-1.2): 1.

[0020] In some embodiments, the temperature of the acid hydrolysis is room temperature.

[0021] In some embodiments, the acidolysis is for a time of 10-30 min; further 15-25 min; still further about 20 min.

[0022] In some embodiments, the cysteine / penicillamine ligation reaction is followed by a precipitation step prior to acidolysis.

[0023] In some embodiments, the acidolysis is followed by a precipitation step prior to the desulfurization reaction.

[0024] In the present application, the precipitation is with methyl tert-butyl ether (MTBE).

[0025] In some embodiments, the desulfurization reaction is performed on intermediate 2 in the presence of an initiator, a reducing agent, and a sulfhydryl agent.

[0026] In some embodiments, the initiator comprises at least one of VA044 (azobisimidozolin hydrochloride), sodium tetraethylborate; further VA044.

[0027] In some embodiments, the reducing agent comprises at least one of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), DTT (dithiothreitol); further TCEP.

[0028] In some embodiments, the sulfhydryl agent comprises at least one of MESNa (sodium 2-mercaptoethanesulfonate), tBuSH (tert-butyl mercaptan), and MTG (mercaptoacetic acid monoethanolamine); further tBuSH.

[0029] In some embodiments, the molar ratio of the initiator, the reducing agent, the sulfhydryl agent, and intermediate 2 is (32-40):(145-155):(20-24): 12; further about 36:150:22:12.

[0030] In some embodiments, the desulfurization reaction system further comprises: GnHCI.

[0031] In some embodiments, the pH of the desulfurization reaction system is 6-8; further 7.6-8; still further about 8.

[0032] In some embodiments, the temperature of the desulfurization reaction is 30-40 °C; further 35-40 °C; still further about 37 °C.

[0033] In some embodiments, the time of the desulfurization reaction is 14-20 h; further 15-17 h; still further about 16 h.

[0034] In some embodiments, the method for preparing the fragment 2 comprises the following steps: synthesizing the fully-protected fragment 2 by solid phase synthesis, and deprotecting the fully-protected fragment 2; the fully-protected fragment 2 is R1NH-Cys(R4)-Phe-Ile-Glu(R2)-Tyr(R2)-Leu-Leu-Glu(R2)-Gly-Gly-Pro-Ser(R2)-Ser(R2)-Gly-Ala-Pro-Pro-Pro-Ser(R2)-CONH2; the R1 is an amino protecting group, and the R2 and R4 are side chain protecting groups of amino acids.

[0035] In some embodiments, the method for preparing the fragment 2 comprises the following steps: coupling the following protected amino acids in sequence from C-terminus to N-terminus using an amino resin as a solid phase synthesis carrier: R5-Ser(R2)-OH, R5-Pro-OH, R5-Pro-Pro-OH, R5-Ala-OH, R5-Gly-OH, R5-Ser(R2)-OH, R5-Ser(R2)-OH, R5-Pro-OH, R5-Gly-Gly-OH, R5-Glu(R2)-OH, R5-Leu-OH, R5-Leu-OH, R5-Tyr(R2)-OH, R5-Glu(R2)-OH, R5-Ile-OH, R5-Phe-OH, R1-Cys(R4)-OH, to obtain a fully-protected fragment 2 resin; performing peptide chain cleavage using an acidolysis agent and deprotecting the fully-protected fragment 2; the acidolysis agent is a strong acid, further comprising one of trifluoroacetic acid (TFA), HCl, H2SO4, and further comprising TFA, triisopropylsilane (TIPS), 1,2-ethanedithiol (EDT), and H2O; the R1 and R5 are amino protecting groups, and the R2 and R4 are side chain protecting groups of amino acids.

[0036] In some embodiments, the volume ratio of the TFA, TIPS, EDT, and H2O is (36-38):(0.8-1.2):(0.8-1.2):1; further about 37:1:1:1.

[0037] In some embodiments, the method for preparing the fragment 2 further comprises the following step: precipitation.

[0038] In some embodiments, the amino resin comprises at least one of Rink Amide resin, Rink Amide AM resin, Rink MBHA resin, and Sieber resin; further the Rink Amide resin.

[0039] In some embodiments, the degree of substitution of the amino resin is 0.2-1.2 mmol / g; further 0.5-0.7 mmol / g.

[0040] In some embodiments, each protecting amino acid is 1.2-3.5 times the total moles of the amino resin fed; further 1.5-3 times.

[0041] In the present application, the coupling is performed in the presence of a condensing agent.

[0042] In the present application, the condensing agent comprises at least one of N,N-diisopropylcarbodiimide (DIC), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-lH-benzotriazole-l-yl)-l, l,3,3-tetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), chloroacetyl chloride, azide, O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (Pybop), N,N-diisopropylethylamine (DIEA), DIC and 1-hydroxybenzotriazole (HOBT), DCC and HOBT, EDCI and HOBT, HBTU and DIEA (N,N-diisopropylethylamine), Pybop / DIEA, HATU / DIEA; further DIEA, DIC and HOBT, or EDCI and HOBT.

[0043] In the present application, the reaction solvent of the coupling comprises at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP); further DCM or DMF.

[0044] In the present application, the time of the coupling is 1-5 h.

[0045] In the present application, the treatment of removing the amino protecting group from the protecting amino acid on the peptide chain of the amino resin and / or amino resin / carboxyl resin is included before the coupling.

[0046] In the present application, the amino protecting group removing reagent is used to remove the amino protecting group.

[0047] In the present application, the amino protecting group removing reagent comprises piperidine and DMF.

[0048] In the present application, the volume ratio of the piperidine and DMF is 1:(3-5); further 1:(3.5-4.5); more further about 1:4.

[0049] In the present application, the amino protecting group is a C1-C16 alkoxycarbonyl protecting group.

[0050] In the present application, the side chain protecting group of the amino acid comprises at least one of a C1-C16 alkoxycarbonyl protecting group, a C4-C21 alkyl protecting group, and a C4-C8 alkoxy protecting group.

[0051] In the present application, the C1-C16 alkoxycarbonyl protecting group is tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc).

[0052] In the present application, the C4-C21 alkyl protecting group comprises at least one of tert-butyl (t-Bu) and trityl (Trt).

[0053] In the present application, the C4-C8 alkoxy protecting group is tert-butyloxy (t-BuO).

[0054] In the present application, the R1 is tert-butyloxycarbonyl (Boc).

[0055] In the present application, the R2 is tert-butyl (t-Bu).

[0056] In the present application, the R4 is trityl (Trt).

[0057] In the present application, the R5 is 9-fluorenylmethoxycarbonyl (Fmoc).

[0058] In some embodiments, the method for preparing the fragment 1-4-methoxysalicylaldehyde ester comprises the following steps: condensing a fully protected fragment 1 with 4-methoxysalicylaldehyde comprising an aldehyde protecting group, and then deprotecting to obtain the fragment 1-4-methoxysalicylaldehyde ester. The fully protected fragment 1 is: R1NH-Tyr(R2)-Aib-Gln(R4)-Gly-Thr(R2)-Phe-Thr(R2)-Ser(R2)-Asp(R2)-Tyr(R2)-Ser(R2)-Ile-αMeLeu-Leu-Asp(R2)-Lys(R3)-Lys(AEEA-rGlu(R2)-C20 diacid(R2))-Ala-Gln(R4)-Aib-OH. The R1 is an amino protecting group, and the R2, R3, and R4 are side chain protecting groups of amino acids.

[0059] In the present application, the R3 is tert-butyloxycarbonyl (Boc).

[0060] In some embodiments, the condensation reaction is performed for 10-16 h; further for 10-14 h; and more further for about 12 h.

[0061] In some embodiments, the condensation reaction is at room temperature.

[0062] In some embodiments, the condensation reaction is in the presence of a condensing agent.

[0063] In the present application, the reaction solvent of the condensation reaction comprises at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP); further DCM or DMF.

[0064] In some embodiments, the 4-methoxysalicylaldehyde comprising an aldehyde-protecting group is acetonide-protected 4-methoxysalicylaldehyde.

[0065] In some embodiments, the molar ratio of the fully-protected fragment 1 to the 4-methoxysalicylaldehyde comprising an aldehyde-protecting group is 1:(1-2); further 1:(1.4-1.6).

[0066] In some embodiments, the deprotection in the preparation method of the fragment 1-4-methoxysalicylaldehyde ester is performed by using an acidolytic agent, and the acidolytic agent is an organic acid or an inorganic acid; further comprising TFA, H2O and phenol.

[0067] In some embodiments, the volume ratio of the TFA, H2O and phenol is (37-39):(0.9-1.1):1; further about 38:1:1.

[0068] In some embodiments, the preparation method of the fragment 1-4-methoxysalicylaldehyde ester further comprises the following step: precipitation.

[0069] In some embodiments, the fully-protected fragment 1 is synthesized by using a solid-phase synthesis method.

[0070] In some embodiments, the preparation method of the fully-protected fragment 1 comprises the following steps: using a carboxyl resin as a solid-phase synthesis carrier, sequentially coupling the following protected amino acids from C-terminal to N-terminal: R5-Aib-OH, R5-Gln(R4)-OH, R5-Ala-OH, R5-Lys(AEEA-rGlu(R2)-C 20R5-Lys(R3)-OH, R5-Asp(R2)-OH, R5-Leu-OH, R5-αMeLeu-OH, R5-Ile-OH, R5-Ser(R2)-OH, R5-Tyr(R2)-OH, R5-Asp(R2)-OH, R5-Ser(R2)-OH, R5-Thr(R2)-OH, R5-Phe-OH, R5-Thr(R2)-OH, R5-Gly-OH, R5-Gln(R4)-OH, R1-Tyr(R2)-Aib-OH, to obtain the full-protected fragment 1 resin; The acidolysis agent includes: TFA and DCM, trifluoroethanol and DCM, or hexafluoroisopropanol and dichloromethane (preferably, TFA and DCM are included). The R1 and R5 are amino protecting groups, and the R2, R3 and R4 are side chain protecting groups of amino acids.

[0071] In some embodiments, the carboxyl resin includes at least one of a CTC resin and a Wang resin; and further, the CTC resin.

[0072] In some embodiments, the degree of substitution of the carboxyl resin is 0.3-1.8 mmol / g; and further, 1.6-1.7 mmol / g.

[0073] In some embodiments, each of the protected amino acids is 1.2-3.5 times the total moles of the carboxyl resin; and further, 1.5-3 times.

[0074] In some embodiments, the volume ratio of the TFA and DCM is 1: (95-105); and further, 1: (98-100).

[0075] In some embodiments, the volume ratio of the trifluoroethanol and DCM is 1: (3-5).

[0076] In some embodiments, the volume ratio of the hexafluoroisopropanol and dichloromethane is 1: (3-5).

[0077] In some embodiments, the synthesis method further includes the following steps: purifying (preferably, HPLC purifying), concentrating and freeze-drying the crude peptide to obtain the ritate peptide.

[0078] The present application has the following beneficial effects: The present application first uses the cysteine (Cys) at the 21st position of ritate peptide as a chemical connection (coupling) site of a polypeptide, specifically connects (couples) two fragments of the polypeptide together, and then performs acidolysis and desulfurization reaction in one step to obtain the ritate peptide crude peptide.

[0079] Specifically, the fragment 1-4-methoxysalicylaldehyde ester is connected with the fragment 2 by a cysteine / penicillamine ligation (CPL) reaction, acidolysis and desulfurization to obtain a crude retaspumycin peptide, and the purity of the crude peptide can reach 83.3%; further purification by secondary high-pressure preparation can obtain a final qualified retaspumycin peptide product, and the purity of the secondary purified product can be as high as 99.47%, and the overall yield can reach 37.2%; the method uses a two-stage method and uses a CPL chemical ligation method for chemical ligation, has the characteristics of low cost, large-scale production, high yield and high purity of the crude peptide, and greatly reduces the purification cost and production cost, wherein the fragment 2 is all natural amino acids, and can be produced by biological fermentation. Therefore, the method has the advantages of semi-chemical and semi-biological synthesis, and greatly reduces the production cost.

[0080] Further, the fragment 1-4-methoxysalicylaldehyde ester is obtained by condensing the fully protected fragment 1 with 4-methoxysalicylaldehyde containing an aldehyde group protecting group; and the fully protected fragment 1 and the fragment 2 are synthesized by a solid-phase synthesis method.

[0081] Further, two dipeptide structures (for example, Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-OH) are used in the synthesis process of the fragment 2 to reduce the generation of missing peptides due to coupling difficulties in the coupling process, greatly improving the crude peptide purity of the fragment 2 and the purification efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 A flowchart for preparing the retaspumycin peptide of the present application.

[0083] Figure 2 An HPLC spectrum of the fully protected fragment 1 in Example 1.

[0084] Figure 3 A Mass spectrum of the fully protected fragment 1 in Example 1.

[0085] Figure 4 An HPLC spectrum of the fragment 1-4-methoxysalicylaldehyde ester in Example 2.

[0086] Figure 5 A Mass spectrum of the fragment 1-4-methoxysalicylaldehyde ester in Example 2.

[0087] Figure 6 An HPLC spectrum of the fragment 2 crude product in Example 3.

[0088] Figure 7 A Mass spectrum of the fragment 2 crude product in Example 3.

[0089] Figure 8The image shows the HPLC chromatogram of intermediate 1 of rataglutide in Example 4, “1. Synthesis of intermediate 1 of rataglutide”.

[0090] Figure 9 The Mass spectrum of intermediate 1 of retalistatin 1 in “1. Synthesis of intermediate 1 of retalistatin 1” of Example 4 is shown in Mass spectrum.

[0091] Figure 10 The HPLC chromatogram of intermediate 1 of rataglutide in Example 4, “1. Synthesis of intermediate 1 of rataglutide” (2).

[0092] Figure 11 The HPLC chromatogram of intermediate 1 of rataglutide in Example 4, “1. Synthesis of intermediate 1 of rataglutide” (3) is shown.

[0093] Figure 12 The HPLC chromatogram of intermediate 2 of retalidinide in Example 4 is shown.

[0094] Figure 13 This is the HPLC chromatogram of the crude peptide of retalistatin in Example 4.

[0095] Figure 14 This is the Mass spectrum of the crude peptide of retalistatin in Example 4.

[0096] Figure 15 This is the HPLC chromatogram of the purified and isolated retaliptide peptide from Example 4. Detailed Implementation

[0097] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0098] 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.

[0099] 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.

[0100] "room temperature" in the present application means 20-35°C.

[0101] The following examples and drawings are provided to aid the understanding of the present application, but are not intended to limit the present application in any way. The actual scope of the application is set forth in the appended claims. It is understood that any modifications and changes can be made thereto without departing from the spirit of the present application. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0102] It should be noted that, in the examples, the specific conditions are not specified, and the routine conditions, the manufacturer's recommendations or the reported experimental conditions are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market. The reagents used are specified by the manufacturer, and similar products from other manufacturers have substitutability.

[0103] In the present application, the 1st to 20th amino acids, and the 21st to 39th amino acids refer to each amino acid of the raltitrexed from the N-terminal to the CONH2 terminal of the C-terminal.

[0104] The flow chart for preparing raltitrexed according to the present application is shown in Figure 1 .

[0105] Example 1. Synthesis of fully protected fragment 1 Fully protected fragment 1: BocNH-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(tBu)-Lys(Boc)-Lys(AEEA-rGlu(tBu)-C 20 diacid(tBu))-Ala-Gln(Trt)-Aib-OH CTC resin 60.6 g (substitution degree 1.65 mmol / g, total moles of resin 100 mmol) was weighed into a solid phase synthesis reactor, and 200 ml of dichloromethane solution was added to swell the resin for 10 min. Fmoc-Aib-OH (48.8 g, 150 mmol) was weighed into 300 ml of dichloromethane, and after mixing with DIEA (38.7 g, 300 mmol), it was added to the above-mentioned solid phase reactor, and the reaction was carried out at room temperature for 3 h. After drying, it was washed with DMF 4 times. After adding 300 ml of DMF, methanol (16 g, 500 mmol) and DIEA (38.7 g, 300 mmol) were added, and the reaction was continued at room temperature for 1.0 h. After drying, it was washed with DMF 5 times. The peptide resin in the solid phase reactor was deprotected with 20% piperidine / DMF (V / V) solution once (25 min), and after completion, it was washed with DMF 5 times, and the ninhydrin test result was positive.

[0106] Fmoc-Gln(Trt)-OH (122.14 g, 200 mmol) and HOBT (27.0 g, 200 mmol) were weighed into 300 ml of DMF at 0°C, and after activation for 5 min by adding DIC (25.2 g, 200 mmol), the solution was added to the solid phase reactor, and the reaction was carried out for 2.0 h. The ninhydrin test result was negative, and after drying, it was washed with DMF 4 times.

[0107] Fmoc / Boc-protected amino acids corresponding to the 3rd to 20th amino acids were prepared in advance (the Fmoc / Boc-protected amino acids corresponding to the amino acids and the equivalent number of the corresponding resin are shown in Table 1). The above-mentioned steps were repeated to couple the remaining amino acids according to the peptide sequence, and the weight of the obtained fully protected fragment 1 peptide resin was 425.8 g. The peptide resin was reacted with 300 ml of TFA / DCM (V / V, the amount of TFA:DCM = 1:99) solution for 2.0 h, filtered, and the filtrate was collected. After adding an appropriate amount of purified water, it was extracted twice to obtain the organic layer filtrate, and after adding an appropriate amount of anhydrous sodium sulfate, it was dried, and after filtering the filtrate, it was concentrated to dryness to obtain the fully protected fragment 1: Boc-NH-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(tBu)-Lys(Boc)-Lys(AEEA-rGlu(tBu)-C 20 diacid(tBu))-Ala-Gln(Trt)-Aib-OH, and the fully protected fragment 1 was 365.2 g. The fully protected fragment 1 was subjected to small-scale resin cleavage detection, and the HPLC spectrum is shown in FIG. 1.Figure 2 Purity of crude peptide: 88.3%, HPLC profile as shown in Figure 3 Purity of crude peptide: 88.3%, HPLC profile as shown in

[0108] Table 1 Fmoc / Boc amino acids and corresponding resin equivalent numbers corresponding to amino acids

[0109] Example 2. Synthesis of Fragment 1-4-methoxysalicylaldehyde ester The fully protected Fragment 1 (365.2 g, 88.8 mmol) obtained from Example 1 was dissolved in DCM (2 L) with the acetone protected 4-methoxysalicylaldehyde compound 2 (26.37 g, 133.2 mmol), followed by the addition of HOBt (35.99 g, 266.4 mmol) and EDCI (51.0 g, 266.4 mmol). The reaction was stirred at room temperature overnight (12 h), after the reaction was monitored to be complete by LCMS, the reaction was washed with 1 mol / L aqueous hydrochloric acid solution for 3 times to obtain the organic layer filtrate, and then dried by adding an appropriate amount of anhydrous sodium sulfate, and the filtrate was filtered and concentrated to dryness to obtain the crude product of the fully protected Fragment 1-4-methoxysalicylaldehyde ester. The crude product was cut with 1 L of TFA / H2O / phenol (v / v / v, wherein the amount of TFA:H2O:phenol was 95:2.5:2.5 by volume) at room temperature for 2 h, and after the reaction was detected to be complete by LCMS, 7 L of ice-cold methyl tert-butyl ether was added to precipitate white solids, which were dried by centrifugation for 3 times to obtain the crude product of the white Fragment 1-4-methoxysalicylaldehyde ester, and the purity of the crude product was determined by high performance liquid chromatography to be 83%. After one purification by high performance liquid chromatography, the white Fragment 1-4-methoxysalicylaldehyde ester was obtained in the form of a pure powder (145.6 g, purity of the pure product: 92.3%, yield: 54.8%) after freeze-drying. The HPLC profile is shown in Figure 4 Purity of crude peptide: 88.3%, HPLC profile as shown in Figure 5 Purity of crude peptide: 88.3%, HPLC profile as shown in

[0110] Example 3. Synthesis of Fragment 2 166.7 g of Fmoc-Rink Amide AM resin (degree of substitution 0.6 mmol / g, total moles of resin used 100 mmol) was first swelled with 1 L of DMF for 5 min, and then filtered, and the resin was deprotected with 1 L of 20% piperidine / DMF for 25 min, and the result was positive by ninhydrin detection, and then filtered, and washed with DMF for 5 times.

[0111] Take 150 mmol of Fmoc-Ser(tBu)-OH and 200 mmol HOBt, dissolve in 600 ml of DMF; take another 200 mmol of DIC, slowly add to the above DMF solution under stirring, stir for 5 min at room temperature, then slowly add the above Fmoc-deprotected Rink Amide AM resin, and couple for 2 h. After the ninhydrin test result is negative, the coupling is complete. After filtration, the resin is washed with 500 ml of DMF for 5 times to obtain Fmoc-Ser(tBu)-amino resin. Then use 600 ml of 20% piperidine / DMF to deprotect for 20 min, and the ninhydrin test result is positive. After filtration, wash with DMF for 5 times to obtain NH2-Ser(tBu)-amino resin.

[0112] First, prepare the Fmoc-protected amino acids corresponding to the 2nd-19th amino acid pairs, and the structure is shown in Table 2.

[0113] The activation method of the Fmoc-protected amino acid corresponding to the 2nd amino acid pair is as follows: Take 200 mmol of Fmoc-Pro-OH amino acid and 200 mmol HOBt, dissolve in 600 ml of DMF; take another 200 mmol of DIC, slowly add to the above DMF solution under stirring, stir for 5 min at room temperature, then slowly add the above Fmoc-deprotected NH2-Ser(tBu)-amino resin, and couple for 2 h. After the ninhydrin test result is negative, the coupling is complete. After filtration, the resin is washed with 500 ml of DMF for 5 times to obtain Fmoc-Pro-Ser(tBu)-amino resin. Then use 600 ml of 20% piperidine / DMF to deprotect for 20 min, and the ninhydrin test result is positive. After filtration, wash with DMF for 5 times to obtain NH2-Pro-Ser(tBu)-amino resin.

[0114] Using the same method as described above, sequentially couple the Fmoc / Boc-protected amino acids corresponding to the 3rd-19th amino acid pairs, i.e. after deprotecting the Fmoc of the Fmoc-[1~(n-1)] amino acid-amino resin obtained in the previous step, couple with the activated Fmoc / Boc-protected amino acid (the nth one) for 60-300 min, n=3~17. After coupling all the protected amino acids, wash with DMF for 3 times, methanol for 3 times, dry, and vacuum dry to obtain 445.3 g of polypeptide resin.

[0115] Take the above 445.3 grams of polypeptide resin, add 5 L lysis reagent [TFA / water / 1,2-ethanedithiol / TIPS = 92.5% / 2.5% / 2.5% / 2.5% (V / V / V / V)], stir evenly, stir at room temperature for 3 h, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the resin with a small amount of TFA for 3 times, after combining the filtrate, add at least 5 times the volume of ice-cold methyl tert-butyl ether to precipitate, centrifuge to obtain white solid, then wash with methyl tert-butyl ether for 2 times, and dry to obtain white powder, which is polypeptide fragment 2 crude product 178.6 g, the purity is 90.8% detected by high performance liquid chromatography, the crude product spectrum is shown in Figure 6 , and the Mass spectrum is shown in Figure 7 .

[0116] Table 2 Fmoc / Boc amino acids corresponding to amino acids and corresponding resin equivalent numbers

[0117] Example 4. Synthesis of Retalutide 1. Synthesis of Retalutide Intermediate 1 1) Dissolve fragment 1-4-methoxysalicylaldehyde ester (299.4 g, 100.0 mmol) obtained in Example 2 and fragment 2 (288.2 g, 150.0 mmol) obtained in Example 3 in 3 L pyridine / acetic acid = 1:1 (V / V) solution at room temperature, react for 5 h, after HPLC detection that fragment 1-4-methoxysalicylaldehyde ester is consumed, add ice-cold methyl tert-butyl ether to precipitate 576.7 g of white solid Retalutide Intermediate 1, the purity is 85.08% detected by HPLC, the HPLC spectrum is shown in Figure 8 , (after CPL, two coupled product peaks will appear, and after acidolysis, it will return to one peak), and the Mass spectrum of Intermediate 1 is shown in Figure 9 .

[0118] 2) Dissolve fragment 1-4-methoxysalicylaldehyde ester (299.4 g, 100.0 mmol) obtained in Example 2 and fragment 2 (288.2 g, 150.0 mmol) obtained in Example 3 in 3 L pyridine / acetic acid = 1:3 (V / V) solution at room temperature, react for 5 h, HPLC detection that fragment 1-4-methoxysalicylaldehyde ester is not completely reacted. After 8 h of reaction, HPLC detection that fragment 1-4-methoxysalicylaldehyde ester is consumed, add ice-cold methyl tert-butyl ether to precipitate 572.3 grams of white solid Retalutide Intermediate 1, the purity is 73.8% detected by HPLC, as Figure 10 shown (after CPL, two coupled product peaks will appear, and after acidolysis, it will return to one peak).

[0119] 3) The fragment 1-4-methoxysalicylaldehyde ester (299.4 g, 100.0 mmol) obtained from Example 2 and the fragment 2 obtained from Example 3 (288.2 g, 150.0 mmol) were dissolved in 3 L of pyridine / acetic acid / DMF = 1:1:2 (V / V) solution at room temperature, and reacted for 6 h. HPLC detection showed that the fragment 1-4-methoxysalicylaldehyde ester was not completely reacted. After the reaction was allowed to proceed overnight (12 h), HPLC detection showed that the fragment 1-4-methoxysalicylaldehyde ester was completely consumed. Then, 572.3 g of white solid Retaspumycin intermediate 1 was precipitated by adding chilled methyl tert-butyl ether. HPLC detection showed that the purity was 69.5%. As shown in Figure 11 (CPL will appear two coupled product peaks after acid hydrolysis, and will return to one peak again).

[0120] 2. Synthesis of Retaspumycin intermediate 2 The 576.7 g of white solid Retaspumycin intermediate 1 crude product obtained from 1) of “1. Synthesis of Retaspumycin intermediate 1” was dissolved in 3 L of cleavage solution TFA / TIPS / EDT / H2O (92.5% / 2.5% / 2.5% / 2.5%, V / V / V / V). After reaction at room temperature for 20 min, HPLC detection showed that the reaction was complete. Then, 568.2 g of white solid Retaspumycin intermediate 2 crude product was precipitated by adding chilled methyl tert-butyl ether. HPLC detection showed that the purity was 82.13%. The HPLC detection spectrum is shown in Figure 12 .

[0121] 3. Synthesis of Retaspumycin crude peptide The 568.2 g of Retaspumycin intermediate 2 (568.2 g, 0.12 mol) crude product obtained from “2. Synthesis of Retaspumycin intermediate 2” was dissolved in 5 L of pure water. Ammonia water was added to assist dissolution, and the pH was adjusted to 7.8. Then, an aqueous solution (3 L) containing 8 M GnHCl and 0.5 M TCEP was added. VA044 (98.2 g, 0.36 mol) was slowly added to the above solution under stirring at room temperature. After the dropwise addition was completed, 20 mL of tBuSH (20.0 g, 0.22 mol) was finally added. The above solution was stirred at 37°C for 16 h. After LCMS detection showed that the reaction was complete, the pH was adjusted to 5.3 with AcOH. White solid gradually precipitated, and centrifugation gave 522.3 g of Retaspumycin crude peptide. HPLC detection showed that the purity was 83.3%. The HPLC detection spectrum is shown in Figure 13 , and the Mass spectrum is shown in Figure 14 .

[0122] 4. Preparation of Retaspumycin fine peptide The 522.3 g of crude Retalipotide obtained in "3. Synthesis of crude Retalipotide" was dissolved in 4 L of water at pH = 8.5 and clarified, and filtered. Gradient elution was performed by HPLC with octadecylsilica gel as the stationary phase and sodium acetate aqueous solution and acetonitrile as the mobile phase to obtain a qualified fraction of Retalipotide. The acetonitrile was removed, and freeze-drying was performed to obtain 43.2 g of Retalipotide fine peptide, with a HPLC purity of 99.47%. The yield of this step was 82.3%, and the overall yield was 37.2%. The separation of the qualified pure product is shown in the following figure Figure 15

[0123] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the scope of protection of the present application.​

Claims

1. A method for preparing retaglutide, comprising the following steps: Fragment 1, 4-methoxysalicylaldehyde ester and fragment 2 were linked by a cysteine / penicillamine linkage reaction, followed by acid hydrolysis and desulfurization to obtain crude retaliptide peptide. The fragment 1-4-methoxysalicylaldehyde ester is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-O-4-MeO-Ph(CHO); Fragment 2 is NH2-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2.

2. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: linking fragment 1-4-methoxysalicylaldehyde ester and fragment 2 via a cysteine / penicillamine linkage reaction to obtain intermediate 1; then acid hydrolyzing intermediate 1 to obtain intermediate 2; and then desulfurizing intermediate 2 to obtain crude retaliptide peptide. The intermediate 1 is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-(N,C)-Benzilidine-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2; The intermediate 2 is: NH2-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-rGlu-C 20 diacid)-Ala-Gln-Aib-Cys-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2; Preferably, the cysteine / penicillamine linkage reaction is carried out in a mixture comprising an organic base and an organic acid; Preferably, the organic base in the mixture includes at least one selected from pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine; more preferably, pyridine. Preferably, the organic acid in the mixture includes at least one selected from acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid; more preferably, acetic acid. Preferably, the acid hydrolysis uses an acid hydrolysate, which includes TFA, TIPS, EDT and H2O.

3. The preparation method according to claim 2, characterized in that, The volume ratio of organic base to organic acid in the mixture is 1:(0.16-6); more specifically, it is 1:(1-3). Preferably, the concentration of the organic base in the mixture is 10-60% v / v; more preferably, it is 25-55% v / v. Preferably, the concentration of the organic acid in the mixture is 10-80% v / v; more preferably, it is 45-75% v / v. Preferably, the cysteine / penicillamine linkage reaction takes 2-14 hours; more preferably 2-8 hours; and even more preferably 2-6 hours. Preferably, intermediate 2 is subjected to a desulfurization reaction in the presence of an initiator, a reducing agent, and a mercapto reagent; Preferably, the initiator includes at least one of VAO44 (azobisisobutyrazoline hydrochloride) and sodium tetraethylborate; more preferably, it is VAO44. Preferably, the reducing agent includes at least one of TCEP (tris(2-carbonylethyl)phosphohydrochloride) and DTT (dithiothreitol); more preferably, TCEP. Preferably, the thiol reagent includes at least one of MESNa (sodium 2-mercaptoethanesulfonate), tBuSH (tert-butylthiol), and MTG (mercaptoacetic acid monoethanolamine); more preferably, tBuSH.

4. The preparation method according to any one of claims 1-3, characterized in that, The preparation method of the fragment 1-4-methoxysalicylaldehyde ester includes the following steps: condensing the fully protected fragment 1 with 4-methoxysalicylaldehyde containing an aldehyde protecting group, and then deprotecting it to obtain fragment 1-4-methoxysalicylaldehyde ester. The fully protected fragment 1 is: R1NH-Tyr(R2)-Aib-Gln(R4)-Gly-Thr(R2)-Phe-Thr(R2)-Ser(R2)-Asp(R2)-Tyr(R2)-Ser(R2)-Ile-αMeLeu-Leu-Asp(R2)-Lys(R3)-Lys(AEEA-rGlu(R2)-C 20 diacid(R2))-Ala-Gln(R4)-Aib-OH; R1 is an amino protecting group, and R2, R3, and R4 are side chain protecting groups of amino acids; Preferably, the 4-methoxysalicylaldehyde containing the aldehyde protecting group is acetone-protected 4-methoxysalicylaldehyde. Preferably, the condensation reaction takes 10-16 hours; more preferably, 10-14 hours. Preferably, the condensation reaction is carried out in the presence of a condensing agent; Preferably, the preparation method of the 1-4-methoxysalicylaldehyde fragment uses an acid hydrolysate for deprotection. The acid hydrolysate is an organic or inorganic acid, and further includes TFA, H2O and phenol.

5. The preparation method according to claim 4, characterized in that, The fully protected segment 1 was synthesized using a solid-state synthesis method. Preferably, the method for preparing the fully protected fragment 1 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: R5-Aib-OH, R5-Gln(R4)-OH, R5-Ala-OH, and R5-Lys(AEEA-rGlu(R2)-C 20 The following compounds were added: diacid(R2)-OH, R5-Lys(R3)-OH, R5-Asp(R2)-OH, R5-Leu-OH, R5-αMeLeu-OH, R5-Ile-OH, R5-Ser(R2)-OH, R5-Tyr(R2)-OH, R5-Asp(R2)-OH, R5-Ser(R2)-OH, R5-Thr(R2)-OH, R5-Phe-OH, R5-Thr(R2)-OH, R5-Gly-OH, R5-Gln(R4)-OH, and R1-Tyr(R2)-Aib-OH, to obtain resin with fully protected fragment 1. The peptide chain was cleaved using an acid hydrolysant to obtain a fully protected fragment 1; the acid hydrolysant included: TFA and DCM, trifluoroethanol and DCM, or hexafluoroisopropanol and dichloromethane; R1 and R5 are amino protecting groups, and R2, R3, and R4 are side chain protecting groups of amino acids.

6. The preparation method according to claim 5, characterized in that, The carboxyl resin includes at least one of CTC resin and Wang resin; more specifically, it is CTC resin. Preferably, the degree of substitution of the carboxyl resin is 0.3-1.8 mmol / g; more preferably, it is 1.6-1.7 mmol / g. Preferably, the amount of each protected amino acid is 1.2-3.5 times the total molar amount of the carboxyl resin added; more preferably, it is 1.5-3 times.

7. The preparation method according to any one of claims 1-6, characterized in that, The preparation method of fragment 2 includes the following steps: Fully protected fragment 2 was synthesized using a solid-state synthesis method, and then deprotected from fully protected fragment 2. The fully protected fragment 2 is: R1NH-Cys(R4)-Phe-Ile-Glu(R2)-Tyr(R2)-Leu-Leu-Glu(R2)-Gly-Gly-Pro-Ser(R2)-Ser(R2)-Gly-Ala-Pro-Pro-Pro-Ser(R2)-CONH2; R1 is an amino protecting group, and R2 and R4 are side chain protecting groups of amino acids; Preferably, the method for preparing fragment 2 includes the following steps: Using amino resin as a solid-phase synthesis support, the following protected amino acids were sequentially coupled from the C-terminus to the N-terminus: R5-Ser(R2)-OH, R5-Pro-OH, R5-Pro-Pro-OH, R5-Ala-OH, R5-Gly-OH, R5-Ser(R2)-OH, R5-Ser(R2)-OH, R5-Pro-OH, R5-Gly-Gly-OH, R5-Glu(R2)-OH, R5-Leu-OH, R5-Leu-OH, R5-Tyr(R2)-OH, R5-Glu(R2)-OH, R5-Ile-OH, R5-Phe-OH, and R1-Cys(R4)-OH, to obtain resin with fully protected fragment 2. Peptide chain cleavage and deprotection of fully protected fragment 2 are performed using an acid hydrolysate; the acid hydrolysate is a strong acid, further including one of TFA, HCl, and H2SO4, and even further including TFA, TIPS, EDT, and H2O; R1 and R5 are amino protecting groups, and R2 and R4 are side chain protecting groups of amino acids.

8. The preparation method according to claim 7, characterized in that, The amino resin includes at least one of Rink Amide resin, Rink Amide AM resin, Rink MBHA resin, and Sieber resin; more specifically, it is Rink Amide resin. Preferably, the degree of substitution of the amino resin is 0.2-1.2 mmol / g; more preferably 0.5-0.7 mmol / g; Preferably, the amount of each protected amino acid is 1.2-3.5 times the total molar amount of the amino resin added; more preferably, it is 1.5-3 times.

9. The preparation method according to any one of claims 1-8, characterized in that, The coupling is carried out in the presence of a condensing agent; Preferably, the condensing agent includes N,N-diisopropylcarbodiimide (DIC), benzotriazole-N,N,N,N'-tetramethylurea hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), chloroacetyl chloride, azide, and O-benzotriazole-N,N,N',N' -At least one of the following: tetramethylurea tetrafluoroborate (TBTU), benzotriazine-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (Pybop), N,N-diisopropylethylamine (DIEA), DIC and 1-hydroxybenzotriazole (HOBT), DCC and HOBT, EDCI and HOBT, HBTU and DIEA (N,N-diisopropylethylamine), Pybop / DIEA, HATU / DIEA; further comprising DIEA, DIC and HOBT, EDCI and HOBT. Preferably, the side chain protecting group of the amino acid includes at least one of C1-C16 alkoxycarbonyl protecting groups, C4-C21 alkyl protecting groups, and C4-C8 alkoxy protecting groups; Preferably, the amino protecting group is a C1-C16 alkoxycarbonyl protecting group; Preferably, the C1-C16 alkoxycarbonyl protecting group is tert-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc). Preferably, the C4-C21 alkyl protecting group comprises at least one of tert-butyl (t-Bu) and triphenylmethyl (Trt); Preferably, the alkoxy protecting group of the C4-C8 is tert-butoxy (t-BuO).

10. The preparation method according to any one of claims 1-9, characterized in that, The preparation method further includes the following steps: purifying, concentrating, and freeze-drying the crude peptide to obtain the refined retaliptide peptide.

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