Preparation method of guanylate cyclase agonist

Abstract

The invention provides the guanylate cyclase agonist preparation method which is short in reaction time, high in yield, high in purity, low in cost, simple in process, simple in post-treatment and environment-friendly. According to the preparation method provided by the invention, the generation proportion of polymer impurities is reduced by utilizing the false dilution effect of solid-phase cyclization; secondly, linear peptide resin is not cracked before cyclization, so that a large number of impurities are prevented from being generated in the cracking process, and the yield of linaclotide is increased; the iodine-containing oxidizing agent is adopted as the oxidizing agent for oxidation, the reaction can be completed only in 2-3 hours, the production period is greatly shortened, the yield, the purity and the content are remarkably improved, and industrial production is facilitated.

Description

Technical Field

[0001] The invention relates to the field of drug synthesis, and in particular to a method for preparing a guanylate cyclase agonist. Background Art

[0002] Linaclotide was launched in the United States in December 2012 for the treatment of irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC) in adults; in April 2013, the drug was launched in the United Kingdom for IBS-C; in June 2015, the product was launched in 12 European countries; in September 2018, the drug was launched in Japan for the treatment of chronic constipation; in March 2019, the drug was approved for marketing in China.

[0003] Linaclotide is a 14 amino acid peptide with the following chemical name: L-cysteinyl-L-cysteinyl-L-glutamyl-L-tyrosyl-L-cysteinyl-L-cysteinyl-L-asparagyl-L-prolyl-L-alanyl-L-cysteinyl-L-threonyl-glycyl-L-cysteinyl-tyrosine, cyclic (1-6), (2-10), (5-13)-tris(disulfide). Molecular formula C 59 H 79 N 15 O 21 S6, molecular weight 1526.8, amino acid sequence:

[0004]

[0005] Currently, there are many methods for the synthesis of linaclotide, which can be mainly divided into liquid phase synthesis and solid phase synthesis.

[0006] 1) Liquid phase synthesis: cleavage followed by oxidation

[0007] Prior art CN102875655A uses Mmt protecting group to protect cysteine ​​side chain, synthesizes crude linear peptide of linaclotide by one-by-one coupling, then cleaves and oxidizes, and finally uses GSH / GSSH oxidation system to oxidize to obtain linaclotide. However, the application uses Mmt protecting group, which is costly, uses GSH / GSSH as oxidant, has long oxidation reaction time, many mismatched impurities, and large oxidation volume; prior art CN104231051A uses DMSO and EDTA one-step oxidation method to directly oxidize crude peptide when forming three disulfide bonds, but this method is prone to mismatch, and produces more polymer impurities than solid phase synthesis, long oxidation reaction time, large oxidation volume, and high cost.

[0008] Prior art CN106008674A uses HMP-AM resin when synthesizing polypeptides, and uses an ammonia / DMSO system in the process of oxidation to form three disulfide bonds; Prior art CN106167514A uses relatively inexpensive Fmoc-Cys(Trt)-OH as a reaction raw material, and adopts a one-step oxidation method to prepare linaclotide during cyclization, using a Cystine / Cysteine ​​guanidine hydrochloride oxidation system; Prior art CN104844693A uses cysteine ​​hydrochloride / DMSO buffer solution to cyclize a linear crude peptide to obtain linaclotide; Prior art CN105884864A improves the cyclization system of linaclotide linear crude peptide, and uses ammonium carbonate / DMSO / water solution plus hydroquinone or TCEP (Chinese name: tris(2-carboxyethyl)phosphine) to cyclize the linear crude peptide to obtain linaclotide.

[0009] The above-mentioned prior arts all use liquid phase oxidation to prepare linaclotide. During the reaction, parameters such as the sample dissolution system, dissolution concentration, oxidant dosage, oxidation time, and oxidant quenching need to be controlled, and the volume of the oxidation solution is relatively large (basically more than 5 times that of solid phase oxidation). In addition, if a mild oxidant is used in the liquid phase oxidation process, the reaction time is long, and if a strong oxidant is used, the impurities increase, reducing the purity and yield. In addition, the liquid phase uses complex solvents and is difficult to operate and handle. However, this method requires a long reaction time, is prone to mismatching, produces many impurities, has a large oxidation volume, and is not easy to use in actual production

[0010] 2) Solid phase synthesis: oxidation followed by cleavage

[0011] Prior art WO2016012938 protects the process of straight chain oxidation, the oxidant is sulfur trioxide-pyridine, a polymer-bound complex of dimethyl sulfoxide (DMSO) in water, a complex of pyridine-sulfur trioxide, guanidine hydrochloride, clear-OX TM , reduced glutathione, etc., but the oxidant in this application is complex, the oxidation steps are complex, there are more oxidant solvents, the reaction time is long, and the oxidation volume is large, which is not conducive to industrial production.

[0012] The prior art CN109311941A adopts solid phase cyclization, that is, the linaclotide linear peptide resin is directly cyclized in one step using the NX-succinimide solution oxidation system without being cracked. The patent uses the oxidizing agent NX-succinimide, and the reaction time takes up to 12 hours. In addition, NX-succinimide is difficult to handle and requires the addition of a reducing agent such as sodium sulfite. The reducing agent will also reduce the disulfide bonds, increasing the cost and being incompatible with the solid phase carrier. If the reducing agent is not added for reduction, the crude product will be dark brown, which is not convenient for subsequent treatment.

[0013] Therefore, it is necessary to provide a method for synthesizing linaclotide with short reaction time, high yield, high purity, low cost, simple process, simple post-treatment, and environmental friendliness, so that it is suitable for scale-up production. Summary of the invention

[0014] The invention provides an environmentally friendly method for synthesizing linaclotide with short reaction time, high yield, high purity, low cost, simple process, simple post-treatment.

[0015] To achieve the above object, the present invention provides the following technical solutions:

[0016] A method for preparing linaclotide comprises the following steps:

[0017] 1) Fmoc-Tyr(tBu)-resin is coupled with other amino acids with Fmoc protecting groups using a condensing agent to obtain linaclotide linear peptide resin;

[0018] 2) adding an iodine-containing oxidant and a solvent to the linaclotide linear peptide resin of step 1) for cyclization to obtain a linaclotide resin cyclized at positions Cys1-6, 2-10, and 5-13, and performing a cleavage reaction using a cleavage agent to obtain linaclotide.

[0019] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the iodine-containing oxidant in step 2) is iodine.

[0020] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:5-20.

[0021] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:10-15.

[0022] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the iodine-containing oxidant in step 2) is a mixture of iodine and perchlorate.

[0023] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:5-20.

[0024] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:10-15.

[0025] Furthermore, the perchlorate concentration in the iodine-containing oxidant is 0.2-0.6 mol / L.

[0026] Furthermore, the perchlorate concentration in the iodine-containing oxidant is 0.4 mol / L.

[0027] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the perchlorate in the iodine-containing oxidant in step 2) is sodium perchlorate.

[0028] Furthermore, the concentration of sodium perchlorate in the iodine-containing oxidant is 0.2-0.6 mol / L.

[0029] Furthermore, the concentration of sodium perchlorate in the iodine-containing oxidant is 0.4 mol / L.

[0030] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the perchlorate in the iodine-containing oxidant in step 2) is potassium perchlorate.

[0031] Furthermore, the concentration of potassium perchlorate in the iodine-containing oxidant is 0.2-0.6 mol / L.

[0032] Furthermore, the concentration of potassium perchlorate in the iodine-containing oxidant is 0.4 mol / L.

[0033] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the iodine-containing oxidant in step 2) is a mixture of iodine and urea.

[0034] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:5-20.

[0035] Furthermore, the present invention relates to a method for preparing linaclotide, wherein the molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant in step 2) is 1:10-15.

[0036] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the urea concentration in the iodine-containing oxidant in step 2) is 0.5-3.0 mol / L.

[0037] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the urea concentration in the iodine-containing oxidant in step 2) is 0.5-1.5 mol / L.

[0038] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the solvent in step 2) contains one or more of DMF, DCM, and TFE.

[0039] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the solvent in step 2) is DMF.

[0040] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the solvent in step 2) is a mixture of DMF / DCM solvents.

[0041] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the solvent in step 2) is a mixture of three solvents: DMF / DCM / TFE.

[0042] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the amino acid with a protecting group in step 1) is Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, or Fmoc-Cys(Trt)-OH.

[0043] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the amino acid with a protecting group in step 1) is Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, or Boc-Cys(Trt)-OH.

[0044] In another aspect, the present invention relates to a method for preparing linaclotide, wherein in step 1), the condensing agent is selected from DIC, HBTU, TBTU, 6-Cl-HOBt or one or more mixtures of HOBt.

[0045] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of DIC and 6-Cl-HOBt.

[0046] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of DIC and HOBt.

[0047] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of HBTU and 6-Cl-HOBt.

[0048] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of HBTU and HOBt.

[0049] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of TBTU and 6-Cl-HOBt.

[0050] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the condensing agent in step 1) is a mixture of TBTU and HOBt.

[0051] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) contains one or more of TFA, TIS, Ac-Cys-OH, thioanisole and water.

[0052] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH and water.

[0053] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH and water, and the volume ratio of each component is TFA:TIS:water:Ac-Cys-OH=85-95:0-5::0-5:0-5.

[0054] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH and water, and the volume ratio of each component is TFA:TIS:water:Ac-Cys-OH=91-93:2-3:2-3:2-3.

[0055] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH, thioanisole and water.

[0056] On the other hand, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH, thioanisole and water, and the volume ratio of each component is TFA:TIS:water:Ac-Cys-OH:thioanisole=85-95:0-5:0-5:0-5:0-5:0-5.

[0057] In another aspect, the present invention relates to a method for preparing linaclotide, wherein the cleavage agent used in step 2) is TFA, TIS, Ac-Cys-OH, thioanisole and water, and the volume ratio of each component is TFA:TIS:

[0058] Water:Ac-Cys-OH:thioanisole=95:1.5:1:1:1.5.

[0059] In another aspect, the present invention relates to a method for preparing linaclotide, wherein in step 1), the carrier resin in the Fmoc-Tyr(tBu)-resin is Wang resin, and the degree of substitution is 0.3-1.2 mmol / g;

[0060] In another aspect, the present invention relates to a method for preparing linaclotide, wherein in step 1), the carrier resin in the Fmoc-Tyr(tBu)-resin is Wang resin, and the degree of substitution is 0.3-0.8 mmol / g.

[0061] The preparation method provided by the present invention utilizes the pseudo-dilution effect of solid phase cyclization to reduce the proportion of polymer impurities produced. Compared with liquid phase cyclization, solid phase oxidation can carry out cyclization reaction at a higher concentration, which can greatly improve production efficiency. Secondly, the linear peptide resin is not cracked before cyclization, which avoids the generation of a large amount of impurities during the cracking process, improves the yield of linaclotide, reduces the difficulty of purification, and also has the following advantages:

[0062] 1) Using an iodine-containing oxidant such as iodine, or a mixture of iodine and perchlorate, or a mixture of iodine and urea as an oxidant for oxidation, the reaction can be completed in only 2-3 hours, which greatly reduces the production cycle and significantly improves the yield, purity and content; in addition, using an iodine-containing oxidant for oxidation makes post-treatment simpler, reduces costs, is odor-free and more environmentally friendly, and is conducive to industrial production.

[0063] 2) The preparation method of the present invention does not use EDT with a strong odor as a lysis solution, but adopts an AC-Cys-OH lysis process to inhibit cysteine ​​alkylation, excessive oxidation of disulfide bonds, and the formation of polymers, thereby improving purity. DETAILED DESCRIPTION

[0064] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0065] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0066] Abbreviation Description:

[0067] Acm: acetylaminomethyl;

[0068] Boc: tert-butyloxycarbonyl;

[0069] DIC: N,N-diisopropylcarbodiimide;

[0070] DIEA: N,N-diisopropylhexylamine;

[0071] Fmoc: 9-fluorenylmethoxycarbonyl;

[0072] HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0073] HOBt: 1-hydroxybenzotriazole;

[0074] 6-Cl-HOBt: 6-chloro-1-hydroxybenzotriazole

[0075] TFA: trifluoroacetic acid;

[0076] TFE: 2,2,2-trifluoroethanol

[0077] TIS: triisopropylsilane;

[0078] Trt: trityl;

[0079] tBu: tert-butyl

[0080] OtBu: tert-butyloxy

[0081] AC-Cys-OH: Acetylcysteine

[0082] EDT: 1,2-ethanedithiol

[0083] DCM: dichloromethane

[0084] DMSO: dimethyl sulfoxide

[0085] DMF: N,N-dimethylformamide

[0086] NBS: N-bromosuccinimide

[0087] NCS: N-chlorosuccinimide

[0088] NIS: N-iodosuccinimide

[0089] Example 1 Preparation of Linaclotide Linear Peptide Resin

[0090] Weigh 10.5g of Fmoc-Tyr(tBu)-Wang resin (substitution degree is 0.38mmol / g, 4mmol), add 60ml of dichloromethane, and blow nitrogen for 30min at room temperature. Drain dry, wash with N,N-dimethylformamide 3 times, add deprotection solution (20% piperidine / N,N-dimethylformamide), react for 5min, drain dry, add an equal amount of deprotection solution and react for 10min. Drain the reaction solution, wash with N,N-dimethylformamide 8 times, and drain dry.

[0091] Weigh 4.68g (8mmol) Fmoc-Cys (Trt) -OH and 1.36g (8mmol) 6-Cl-HOBt, dissolve in N, N-dimethylformamide / dichloromethane (volume ratio 3:1) mixed solvent, after the system is dissolved, control the temperature at 0℃~5℃, slowly add 1.5ml (8mmol) N, N'-diisopropylcarbodiimide to activate for 5min. Add to the reactor, blow nitrogen at room temperature for 2h. After the reaction is completed, remove the reaction solution and wash with N, N-dimethylformamide 4 times.

[0092] The above operation was repeated to condense the protected amino acids in the following order: Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, and dried to obtain 19.85 g of linaclotide linear peptide resin with a yield of 99.27%.

[0093] Example 2 Preparation of Linaclotide Linear Peptide Resin

[0094] Weigh 3 portions of Fmoc-Tyr(tBu)-Wang resin, labeled as 1), 2), and 3), each about 1.5 g (substitution degree 0.64 mmol / g, 1 mmol), add 60 ml of dichloromethane, and blow nitrogen for 30 min at room temperature. Drain dry, wash with N,N-dimethylformamide 3 times, add deprotection solution 20% piperidine / N,N-dimethylformamide, react for 5 min, drain dry, add an equal amount of deprotection solution, and react for 10 min. Drain the reaction solution, wash with N,N-dimethylformamide 8 times, and drain dry.

[0095] Weigh 1.76g (3mmol) Fmoc-Cys (Trt) -OH, 0.51g (3mmol) 6-Cl-HOBt, dissolve in N, N-dimethylformamide / dichloromethane (volume ratio 3: 1) mixed solvent, after the system is dissolved, control the temperature at 0 ℃ ~ 5 ℃, slowly add 0.5ml (3mmol) N, N'-diisopropylcarbodiimide to activate for 5min, add to reactor 1), and react for 2h at room temperature with nitrogen blowing; Weigh 1.76g (3mmol) Fmoc-Cys (Trt) -OH, 0.51g (3mmol) 6-Cl-HOBt, dissolve in DMF / DCM (volume ratio 3: 1) Mixed solvent, after the system is dissolved, control the temperature at 0℃~5℃, slowly add 0.5ml (3mmol) N, N'-diisopropylcarbodiimide to activate for 5min, add to reactor 2), and react for 2h at room temperature with nitrogen blowing; weigh 1.24g (3mmol) Fmoc-Cys (Acm) -OH and 0.51g (3mmol) 6-Cl-HOBt, dissolve in DMF / DCM (volume ratio 3:1) mixed solvent, after the system is dissolved, control the temperature at 0℃~5℃, slowly add 0.5ml (3mmol) N, N'-diisopropylcarbodiimide to activate for 5min, add to reactor 3), and react for 2h at room temperature with nitrogen blowing. After the reaction is completed, the reaction liquid is pumped out and washed with N, N-dimethylformamide 4 times.

[0096] Reactors 1), 2), and 3) are used to condense protected amino acids in the following order: 1) Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH H, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH; 2) Fmoc-Gly-OH, Fmoc-Thr(tBu)-O H, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-C ys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH; 3) Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt) -OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Cys(Trt)-OH were dried to obtain 4.10 g, 3.32 g, and 2.95 g of linaclotide linear peptide resin, respectively, with yields of 112%, 90.95%, and 80.82%, respectively.

[0097] Example 3 Preparation of Linaclotide Linear Peptide Resin

[0098] Add 22.73g Fmoc-Tyr(tBu)-Wang resin (substitution degree 0.67mmol / g, 15mmol) and 200ml dichloromethane to a 500ml peptide solid phase reactor, and blow nitrogen for 30min at room temperature. Drain dry, wash with N,N-dimethylformamide 3 times, add deprotection solution, react for 5min, drain dry, add an equal amount of deprotection solution, react for 10min. Drain the reaction solution, wash with N,N-dimethylformamide 8 times, and drain dry.

[0099] Weigh 17.57g (30mmol) Fmoc-Cys (Trt) -OH, 5.10g (30mmol) 6-Cl-HOBt, add 200ml N, N-dimethylformamide / dichloromethane (volume ratio 3:1) mixed solvent, after the system is dissolved, control the temperature at 0℃~5℃, slowly add 4.7 (30mmol) ml N, N'-diisopropylcarbodiimide to activate for 5min. Add to the reactor, blow nitrogen at room temperature for 2h. After the reaction is completed, remove the reaction solution and wash it with N, N-dimethylformamide 4 times.

[0100] The above operation was repeated to sequentially condense Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, and Boc-Cys(Trt)-OH OH. The condensation agent was a 6-Cl-HOBt / DIC system. After the condensation was completed, 57.37 g of linaclotide linear peptide resin was obtained by drying with a yield of 106%.

[0101] Example 4 Preparation of Linaclotide Linear Peptide Resin

[0102] Add 106g Fmoc-Tyr(tBu)-Wang resin (substitution degree 0.66mmol / g, 70mmol) and 1.5L dichloromethane to a 3L peptide solid phase reactor, and blow nitrogen for 30min at room temperature. Drain dry, wash with N,N-dimethylformamide 3 times, add deprotection solution, react for 5min, drain dry, add an equal amount of deprotection solution, react for 10min. Drain the reaction solution, wash with N,N-dimethylformamide 8 times, and drain dry.

[0103] Weigh 82.03g (140mmol) Fmoc-Cys (Trt) -OH, 23.72g (140mmol) 6-Cl-HOBt, add 600ml N, N-dimethylformamide / dichloromethane (volume ratio 3:1) mixed solvent, after the system is dissolved, control the temperature at 0℃~5℃, slowly add 20ml (140mmol) N, N'-diisopropylcarbodiimide to activate for 5min. Add to the reactor, blow nitrogen at room temperature for 2h. After the reaction is completed, remove the reaction solution and wash it with N, N-dimethylformamide 4 times.

[0104] The above operation was repeated to condense Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, and Boc-Cys(Trt)-OH OH in sequence. The condensation agent was 6-Cl-HOBt / DIC system. 283.27 g of linaclotide linear peptide resin was obtained with a yield of 92.31%.

[0105] Example 5 Preparation of Linaclotide Linear Peptide Resin

[0106] Add 1.2mol Fmoc-Tyr(tBu)-Wang resin (substitution degree 0.66mmol / g, 1.2mol) and 26.5kg dichloromethane to a 50L peptide solid phase synthesizer, start stirring, control the temperature at 10℃~30℃, and stir for 30min. Press filter, wash the filter cake with N,N-dimethylformamide (industrial grade) 3 times, each time with 20L (19kg), each time with 3min, press filter until almost no liquid flows out, and obtain the filter cake. Add 20L deprotection solution to the above filter cake, stir for 5min, press filter until almost no liquid flows out, then add 20L deprotection solution, stir for 10min, press filter until almost no liquid flows out. Wash the filter cake with N,N-dimethylformamide 10 times, each time with 20L (19kg), each time with 3min, and the last N,N-dimethylformamide is left in the kettle.

[0107] Add 1406g (2.4mol) Fmoc-Cys (Trt) -OH, 407g (2.4mol) 6-Cl-HOBt and 21kg N, N-dimethylformamide / dichloromethane (volume ratio 3: 1) mixed solvent to a 30L double-layer glass reactor, start stirring, and after the system is dissolved, control the temperature to 0℃~5℃, slowly add 303g (2.4mol) N, N-diisopropylcarbodiimide, stir for 5min-10min, stop stirring, and set aside. Press filter the last washing liquid in a 50L polypeptide reactor, add the above reaction liquid, start stirring, control the temperature to 20℃±5℃, keep warm and stir for 2h, press filter, wash the filter cake with N, N-dimethylformamide 4 times, each time with 20L (19kg), each time washing for 3min, press filter until there is basically no liquid outflow, and the last N, N-dimethylformamide remains in the reactor.

[0108] The above operation was repeated to sequentially condense Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, and Boc-Cys(Trt)-OH OH, and the condensation agent was a 6-Cl-HOBt / DIC system. After final drying, 5.03 kg of linaclotide linear peptide resin was obtained with a yield of 96.73%.

[0109] Example 6 Preparation of Crude Linaclotide

[0110] The linaclotide linear peptide resin of Example 1 was equally divided into two parts: 1) 10 times the molar amount of iodine and N,N-dimethylformamide were added to oxidize for 3 hours; 2) 4 times the molar amount of NBS and N,N-dimethylformamide were added to oxidize for 3 hours. After the oxidation was completed, it was washed and dried, and a cleavage solution (TFA: TIS: water: Ac-Cys-OH = 91: 3: 3: 3) was added to cleave the resin, and the crude product was obtained by filtration and centrifugation.

[0111] Serial number Mass yield (%) purity(%) Product Appearance 1) 40.9 50.6 White 2) 20.3 43.7 Dark brown

[0112] It can be seen from the above table that when NBS is used as the oxidant, its yield and purity are lower than when iodine is used as the oxidant, and the use of NBS causes the crude resin to turn black-brown, which is not convenient for post-processing and causes the sample properties to be unqualified. At the same time, large-scale use of NBS is difficult to quench and is relatively dangerous. After replacing NBS with NCS and NIS, the same operation is repeated, and the results are similar to those using NBS.

[0113] Example 7 Preparation of Linaclotide Linear Peptide Resin

[0114] Add iodine and N,N-dimethylformamide equivalent to 10 times the molar amount of linaclotide linear peptide resin to Example 2 for oxidation for 3 hours, wash and dry after oxidation, add lysis solution (TFA: TIS: water: Ac-Cys-OH = 91: 3: 3: 3) to cleave the resin, and filter and centrifuge to obtain a crude product. The data are shown in the following table:

[0115] Sequence number (corresponding to the condensation sequence of Example 2) Mass yield (%) purity(%) 1) 40.8 60.0 2) 10.4 56.4 3) 13.5 43.7

[0116] As can be seen from the table above, when the condensation sequence 1) is selected, that is, when the cysteine ​​protecting groups at positions 2 and 10 are Acm and the rest are Trt protected, the yield and purity are higher than those of other protecting groups at the same time.

[0117] Example 8 Preparation of Crude Linaclotide

[0118] The linear peptide resin in Example 3 was weighed into 7 portions, each with 2 g (0.5 mmol), and 20 ml of dichloromethane (DCM) was added to swell for 30 min, then washed twice with 20 ml of N, N-dimethylformamide and dried. Iodine was used as an oxidant, and different moles of iodine were dissolved in different types of solvents with different contents according to the table below to react for 2 h. After the reaction, N, N-dimethylformamide (DMF) was used to wash the filtrate until the filtrate was colorless, and then washed with dichloromethane 3 times and dried to obtain the peptide resin. 20 ml of pre-cooled lysis solution was added to the oxidized peptide resin and reacted at room temperature for 2 h, wherein the ratio of the lysis solution was TFA: TIS: water: AC-Cys-OH: anisyl thioether = 95: 1.5: 1: 1: 1.5. After filtration, tertiary methyl ether was added for precipitation, and then centrifuged and dried to obtain a white solid. The yield and purity are shown in the table.

[0119]

[0120] It can be seen from the above table that the target product cannot be obtained by using 3 times the amount of iodine as the linear peptide resin of linaclotide; when the amount of iodine is increased to 5 times, the mass yield is improved, but the mass yield is increased due to the presence of incompletely reacted linear peptides, and the peptide resin generated after complete oxidation is difficult to cleave from the resin, so the sample content is relatively low; when the amount of iodine is increased to 10 times, the content of incompletely reacted linear peptides decreases, the mass yield decreases, but the sample content and purity are improved; when the amount of iodine is increased to 15 times, the mass yield and purity are slightly reduced, which is similar to the 10-fold amount; in addition, it can be seen from the above table that when different solvents are used, the yield and purity change, and the comprehensive purity, mass yield and content, among which N,N-dimethylformamide is selected as the solvent or mixed with other solvents, whether it is from the purity, mass yield and content alone, or the comprehensive mass yield and content product, are relatively high.

[0121] Example 9 Preparation of Crude Linaclotide

[0122] Weigh 2g (0.5mmol) of the linear peptide resin in Example 3, add 20ml of dichloromethane (DCM) to swell for 30min, wash twice with 20ml of N, N-dimethylformamide (DMF), and drain. Use iodine and a mixture of iodine and sodium perchlorate as oxidants, dissolve different amounts of iodine in the solvent according to the table below, then add or not add 0.4mol / L sodium perchlorate, add the reactor to react for 2h after it is fully dissolved, wash with N, N-dimethylformamide after the reaction until the filtrate is colorless, then wash with dichloromethane 3 times and drain to obtain the peptide resin. Add 20ml of pre-cooled cleavage solution to the oxidized peptide resin and react at room temperature for 2h, wherein the ratio of the cleavage solution is TFA: TIS: water: Ac-Cys-OH = 93: 2: 2: 3. After filtration, add tertiary methyl ether to settle, and centrifuge and dry to obtain a white solid. The yield and purity are shown in the table.

[0123]

[0124] It can be seen from the above table that when iodine and sodium perchlorate are used as oxidants, the purity and mass yield are relatively high.

[0125] Example 10 Preparation of crude linaclotide

[0126] Weigh 2g (0.5mmol) of each linear peptide resin in Example 3, add 20ml of dichloromethane (DCM) to swell for 30min, wash twice with 20ml of N, N-dimethylformamide (DMF), and drain. Use iodine as an oxidant, dissolve different amounts of iodine in the solvent according to the table below, then add the corresponding amount of urea, add it to the reactor to react for 2h after it is fully dissolved, wash with N, N-dimethylformamide after the reaction until the filtrate is colorless, then wash with dichloromethane 3 times and drain to obtain the peptide resin. Add 20ml of pre-cooled cleavage solution to the oxidized peptide resin and react at room temperature for 2h, wherein the ratio of the cleavage solution is TFA: TIS: water: Ac-Cys-OH = 93: 2: 2: 3. After filtration, add tertiary methyl ether to precipitate and centrifuge to obtain a white solid. The yield and purity are shown in the table.

[0127]

[0128]

[0129] Example 11 Preparation of Crude Linaclotide

[0130] Weigh 2g (0.5mmol) of the linear peptide resin in Example 4 and put it into the reactor. Add 20ml of dichloromethane to swell for 30min, then wash it twice with 15ml of N,N-dimethylformamide and drain it. Add 1.5g (1.5mol / L) urea and 1.27g (5mmol) iodine and dissolve them in 15ml of N,N-dimethylformamide. After fully dissolving, add them to the reactor and react for 2h. After the reaction is completed, wash with N,N-dimethylformamide until the filtrate is colorless, then wash it with DCM 3 times and drain it to obtain the peptide resin. Add 36ml of pre-cooled lysis solution to the peptide resin and react at 27℃ for 2h, wherein the ratio of the lysis solution is TFA:TIS:water:Ac-Cys-OH:thiophenyl ether=95:1.5:1:1:1.5. After filtration, adding tertiary methyl ether to settle, centrifugal drying is obtained to obtain 585mg of white solid with a purity of 60.89% and a crude mass yield of 76.67%.

[0131] Example 12 Preparation of Crude Linaclotide

[0132] Weigh 150.06g (about 37mmol) of the linear peptide resin in Example 4 and put it into the reactor. Add 1.5L DCM to swell for 30min, then wash twice with N,N-dimethylformamide and drain. Add 90g (1.5mol / L) urea and 94g (370mmol) iodine and dissolve them in 1L N,N-dimethylformamide. After fully dissolved, add them to the reactor and react for 2h. After the reaction is completed, wash with N,N-dimethylformamide until the filtrate is colorless. A small amount of DIEA is added during the fifth N,N-dimethylformamide wash, and then wash with DCM 4 times and drain. Determine linaclotide peptide resin. Weigh 5g (1.2mmol) of peptide resin and add it to 80ml pre-cooled cleavage solution, then add 20ml dichloromethane and react at 27°C for 2h, wherein the ratio of the cleavage solution is TFA: TIS: water: Ac-Cys-OH: anisyl thioether = 95: 1.5: 1: 1: 1.5. After the reaction was completed, the mixture was filtered, methyl tert-ether was added for precipitation, and then centrifuged and dried to obtain 1.85 g of a white solid with a purity of 40.25% and a crude product mass yield of 98.56%.

[0133] Example 13 Preparation of Crude Linaclotide

[0134] Weigh 1.8 kg (1.5 mol / L) of urea and fully dissolve it in 20 L N, N-dimethylformamide, then add 2.44 kg (9.6 mol) of iodine, and after the dissolution is complete, add 4 kg (0.96 mol) of peptide resin (prepared in Example 5 above) and blow nitrogen at room temperature for 2 hours. After the reaction is completed, remove the reaction solution and wash it with N, N-dimethylformamide 4 times, each time with 20 L, and each time for 1 minute. For the fifth wash, add 2 ml of DIEA to 20 L N, N-dimethylformamide, and then wash it with N, N-dimethylformamide 3 times. Finally, wash it with dichloromethane four times and then dry it to obtain the peptide resin. Add the cleavage solution (TFA: TIS: water: Ac-Cys-OH = 93: 2: 2: 3) to the above peptide resin and stir it at 25 ° C for 2 hours. The mixture was filtered, methyl tert-ether was added for precipitation and then centrifuged and dried to obtain 1.14 kg of white solid with a purity of 60.21% and a crude product mass yield of 77.82%.

[0135] Example 14 Preparation of Linaclotide

[0136] 585 mg of crude linaclotide peptide of Example 11 was used, and a high-efficiency preparative liquid phase, SP-100-10-C18-P filler, a trifluoroacetic acid-acetonitrile purification system was used for primary purification, and an ammonium acetate-acetonitrile system was used for secondary purification. The acetic acid-acetonitrile system was used for salt conversion, and the crude peptide was refined to obtain 134 mg of refined linaclotide peptide, with a purification yield of 76.5%, a total yield of 22.9%, a purity of 99.5%, and a maximum single impurity of 0.04%.

[0137] Example 15 Preparation of Linaclotide

[0138] 1.85 g of the crude linaclotide peptide of Example 12 was used, and a high-efficiency preparative liquid phase, SP-100-10-C18-P filler, a trifluoroacetic acid-acetonitrile purification system was used for primary purification, and an ammonium acetate / acetic acid-acetonitrile system was used for secondary purification. The water-acetonitrile system was used for salt conversion, and the crude peptide was refined to obtain 425.8 mg of linaclotide refined peptide, with a purification yield of 76.7%, a total yield of 23.0%, a purity of 99.0%, and a maximum single impurity of 0.10%.

[0139] Example 16 Preparation of Linaclotide

[0140] 1072 g of the crude linaclotide peptide of Example 13 was used, and a high-efficiency preparative liquid phase, SP-100-10-C18-P filler, a trifluoroacetic acid-acetonitrile purification system was used for primary purification, and an ammonium acetate / acetic acid-acetonitrile system was used for secondary purification. The water-acetonitrile system was used for salt transfer, and the crude peptide was refined to obtain 238 g of linaclotide refined peptide, with a purification yield of 79.3%, a total yield of 22.2%, a purity of 99.5%, and a maximum single impurity of 0.03%.

Claims

1. A method for preparing linaclotide, characterized in that The following steps are involved: 1) Fmoc-Tyr(tBu)-resin is coupled with other amino acids with Fmoc protecting groups using a condensing agent to obtain linaclotide linear peptide resin; 2) adding an iodine-containing oxidant and a solvent to the linaclotide linear peptide resin of step 1) for cyclization to obtain a linaclotide resin cyclized at positions Cys1-6, 2-10, and 5-13, and performing a cleavage reaction using a cleavage agent to obtain linaclotide.

2. The preparation method according to claim 1, characterized in that The iodine-containing oxidant is selected from iodine, a mixture of iodine and perchlorate, or a mixture of iodine and urea.

3. The preparation method according to claim 2, characterized in that The molar ratio of iodine to linaclotide linear peptide resin in the iodine-containing oxidant is 1:5-20, preferably 1:10-15.

4. The preparation method according to claim 2, characterized in that The perchlorate in the iodine-containing oxidant is selected from sodium perchlorate and potassium perchlorate, and the concentration is 0.2-0.6 mol / L, preferably 0.4 mol / L.

5. The preparation method according to claim 2, characterized in that The urea concentration in the iodine-containing oxidant is 0.5-3.0 mol / L, preferably 0.5-1.5 mol / L.

6. The preparation method according to claim 1, characterized in that The solvent contains one or more of DMF, DCM and TFE; preferably DMF, a DMF / DCM mixed solvent, or a DMF / DCM / TFE mixed solvent; more preferably DMF.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The amino acid with a protecting group in step 1) is Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH; or Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Al a-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Boc-Cys(Trt)-OH.

8. The preparation method according to claim 1, characterized in that: In step 1), the condensing agent contains one or more of DIC, HBTU, TBTU, 6-Cl-HOBt or a mixture of HOBt.

9. The preparation method according to claim 1, characterized in that: The cleavage agent used in step 2) contains one or more of TFA, TIS, Ac-Cys-OH, thioanisole and water, preferably TFA, TIS, Ac-Cys-OH and water, the volume ratio of each component is TFA:TIS:water:Ac-Cys-OH=85-95:0-5:0-5:0-5, preferably TFA:TIS:water:Ac-Cys-OH=91-93:2-3:2-3:2-3; or preferably TFA, TIS, Ac-Cys-OH, thioanisole and water, the volume ratio of each component is TFA:TIS:water:Ac-Cys-OH:thioanisole=85-95:0-5:0-5:0-5:0-5, preferably TFA:TIS:water:Ac- Cys-OH: thioanisole = 95:1.5:1:1:1.

5.

10. The preparation method according to claim 1, characterized in that: In step 1), the carrier resin in the Fmoc-Tyr(tBu)-resin is Wang resin, and the degree of substitution is 0.3-1.2 mmol / g, preferably 0.3-0.8 mmol / g.

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