A method for preparing purcanapeptide

Abstract

This invention belongs to the field of polypeptide drug preparation and discloses a method for preparing purcanapeptide. The method includes the following steps: sequentially inserting corresponding protecting amino acids into a sequence on Fmoc-Leu-resin using a solid-phase synthesis method to obtain a purcanapeptide peptide resin: R1-Asn(R4)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R3)-Val-Asn(R4)-Val-Ala-Cys(R2)-Thr-X-Cys(R3)-Leu-resin, wherein X is selected from (Hmb)Gly or (Dmb)Gly; R1 is selected from Fmoc, Boc, or H; R2 and R3 are selected from A or B, where A is Acm or tBu, and B is Trt or Mmt, and R2 and R3 are different; R4 is selected from H or an amino protecting group. The purcanapeptide peptide resin is then cleaved and oxidized stepwise to obtain crude purcanapeptide peptide. The process of this invention effectively solves the problems of coupling difficulties caused by peptide resin shrinkage and missing peptide impurities during solid-phase synthesis, greatly reducing the difficulty of synthesis and purification of pulcanapeptide, and significantly improving the purity and yield of crude pulcanapeptide. It is a pulcanapeptide preparation method with broad practical value and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug preparation, and particularly relates to a method for preparing the polypeptide compound purcanapeptide. Background Technology

[0002] Chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C) are two of the most common gastrointestinal disorders characterized by reduced bowel movements, tension, abdominal pain, or discomfort. The prevalence of chronic constipation in Chinese adults, including CIC, functional defecation disorders, and IBS-C, is 4%-6%. Chronic constipation can lead to secondary mental and psychological disorders, and its impact on patients' quality of life is comparable to that of chronic diseases such as diabetes and heart failure.

[0003] Plecanatide, developed by Synergy Pharmaceuticals, is a guanylate cyclase C (GC-C) receptor agonist. It was approved by the FDA on January 19, 2017, under the brand name Trulance. Plecanatide is a cyclic polypeptide containing 16 amino acids. It regulates acid-base balance in the gastrointestinal tract, induces fluid transport into the gastrointestinal tract, and increases peristalsis. It is indicated for the treatment of chronic idiopathic constipation in adults. Its structural formula is as follows:

[0004]

[0005] Currently, solid-phase synthesis methods for purcanapeptide backbones are mainly divided into fragmentation methods and stepwise coupling methods. Patent application CN103764672A uses the fragmentation method, forming the backbone through liquid-phase condensation between fragments. This method requires the pre-preparation of more than two fragments, and the post-condensation processing is cumbersome, resulting in numerous steps and high costs for industrial production. Patent application CN104211777A uses a stepwise coupling method to synthesize peptide resin, which is then cleaved to obtain a linear peptide. The linear peptide is then oxidized stepwise in the liquid phase to form two pairs of disulfide bonds to obtain purcanapeptide. For the preparation of purcanapeptide containing 16 amino acids, the stepwise coupling method has advantages over the fragmentation method, such as simpler operation and easier automation. However, in actual production, during the stepwise coupling process, when Gly 14 When no protecting base is used, in Val 8 and Glu 5 After coupling, the peptide resin shrinks significantly, making subsequent amino acid coupling difficult and resulting in a crude product with low purity and many impurities. Patent applications with publication numbers CN110981939A and CN110903350A are published on Gly... 14 Protection is provided using Dmb / Hmb, etc., while Thr 13 The side chains are protected with tBu, which significantly reduces the aforementioned peptide resin condensation phenomenon. However, through long-term experiments, the applicant of this invention unexpectedly discovered that when Gly...14 When using Fmoc-(Hmb)Gly-OH or Fmoc-(Dmb)Gly-OH, the large steric hindrance of the Hmb or Dmb groups leads to Thr 13 Coupling with the tBu protecting group is difficult, and the product contains more Thr-deficient peptide impurities, affecting the yield. Literature reports that if the N-terminal Asn side chain of the peptide is protected with Trt, this protecting group is quite stable to trifluoroacetic acid and requires a long treatment time to completely remove it (Pept. Res. 1992, Vol 5:145), which can cause other side reactions.

[0006] Therefore, there is a need to find a method for preparing pucanapeptide that can reduce condensation during coupling, decrease the generation of Thr-deficient peptides, avoid Asn side chain dehydration side reactions, save costs, and is suitable for industrial production. Summary of the Invention

[0007] To address the problems in existing technologies where peptide resin shrinkage occurs during the solid-phase stepwise coupling of pulcanapeptide, leading to coupling difficulties, low coupling efficiency, and the generation of missing peptide impurities, this invention provides a method for preparing pulcanapeptide, the method comprising the following steps:

[0008] 1) The resin solid support is coupled with Fmoc-Leu-OH to obtain Fmoc-Leu-resin;

[0009] 2) By solid-phase synthesis, corresponding protecting amino acids in the sequence were sequentially coupled onto Fmoc-Leu-resin to prepare purcanapeptide peptide resin: R1-Asn(R4)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R3)-Val-Asn(R4)-Val-Ala-Cys(R2)-Thr-X-Cys(R3)-Leu-resin, where X is selected from (Hmb)Gly or (Dmb)Gly; R1 is selected from Fmoc, Boc or H; R2 and R3 are selected from A or B, where A is Acm or tBu, and B is Trt or Mmt, and R2 and R3 are different; R4 is selected from H or an amino protecting group;

[0010] 3) Linear pulcanapeptide was obtained by cleaving the peptide resin;

[0011] 4) Linear purcanapeptide is cyclized stepwise to obtain purcanapeptide.

[0012] Furthermore, the four Cys side chain protecting groups R2 and R3 in the purcanapeptide sequence are preferably Trt and Acm.

[0013] Furthermore, the Asn side chain protecting group R4 in the purcanapeptide sequence is preferably H or Trt.

[0014] In a preferred embodiment of the present invention, the resin in the Fmoc-Leu- resin is selected from Wang resin, 2-chlorotriphenylmethyl chloride resin, HMP MBHA resin or HMP AM resin, and the substitution value of the Fmoc-Leu- resin is 0.20-0.60 mmol / g.

[0015] In a preferred embodiment of the present invention, step 2) specifically involves sequentially coupling Fmoc-Cys(R3)-OH, Fmoc-(Hmb)Gly-OH or Fmoc-(Dmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(R2)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(R4)-OH, Fmoc-Val-OH, Fmoc-Cys(R3)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(R4)-OH or Boc-Asn(R4)-OH to the Fmoc-Leu-resin to obtain the purcanapeptide peptide resin.

[0016] Furthermore, the condensation reagent used in step 2) of the coupling process is selected from HBTU / HOBT / DIEA, TBTU / HOBT / DIEA, or HOBt / DIC.

[0017] In a preferred embodiment of the present invention, in step 3), the pulcanapeptide peptide resin is cleaved while the resin and side chain protecting groups are removed to obtain linear pulcanapeptide: H-Asn-Asp-Glu-Cys(R2)-Glu-Leu-Cys(R3)-Val-Asn-Val-Ala-Cys(R2)-Thr-Gly-Cys(R3)-Leu-OH, wherein when the Cys side chain protecting group is Trt or Mmt, it can be cleaved during the cleavage process; if it is another protecting group, it cannot be cleaved.

[0018] Further, in step 3), the cleavage reagent is selected from TFA and reagent A. Reagent A is selected from one or more of Tis, Mpr, m-cresol, Phenol, and H2O, wherein the proportion of TFA is 80-95 (V / V) and the proportion of reagent A is 5-20 (V / V).

[0019] In a preferred embodiment of the present invention, cyclization in step 4) includes:

[0020] When the pulcanapeptide peptide resin is cleaved by a cleavage reagent to obtain a linear peptide containing a pair of Cys protected groups with Acm, the linear peptide is dissolved, an oxidant is added, and an oxidation reaction is carried out to obtain a monocyclic peptide; the monocyclic peptide is then deprotected from the Cys side chain protecting group Acm in an iodine solution, and cyclized to obtain crude pulcanapeptide peptide.

[0021] In a preferred embodiment of the present invention, cyclization in step 4) includes:

[0022] When the pulcanapeptide peptide resin is cleaved by a cleavage reagent to obtain a linear peptide containing a pair of Cys protecting groups with tBu, the linear peptide is dissolved, an oxidant is added, and an oxidation reaction is carried out to obtain a monocyclic peptide. The monocyclic peptide is then deprotected by removing the tBu side chain protecting group on Cys with trichlorophenylsilane, diphenyl sulfoxide, and anisole, and cyclization is continued in the same oxidant system to obtain crude pulcanapeptide peptide.

[0023] Furthermore, the added oxidant is selected from hydrogen peroxide, DMSO, heme chloride, or air.

[0024] In a preferred embodiment of the present invention, the crude purcanapeptide obtained in step (4) can be further purified and freeze-dried to obtain purcanapeptide.

[0025] This invention uses Hmb or Dmb to protect Gly, solving the coupling difficulties caused by resin shrinkage during stepwise coupling; it employs Thr without side-chain protecting groups. 13 Because of its relatively small steric hindrance, the method avoids the formation of Thr-deficient peptide impurities, thus improving the yield. Using Fmoc-Asn-OH reduces steric hindrance and production costs, while also avoiding the dehydration side reaction of the Asn side chain. The purcanapeptide synthesized using this method has a purity greater than 99.5% and a single impurity of less than 0.1%. Compared with existing technologies, this invention effectively solves the problems of coupling difficulties caused by peptide resin shrinkage and the formation of deletion peptide impurities during solid-phase synthesis. The prepared purcanapeptide has high purity, few impurities, is easy to purify, and has a high yield, possessing broad practical value and application prospects. Attached Figure Description

[0026] Figure 1 This is the crude peptide mass spectrum of purcanapeptide obtained in Example 13 of the present invention;

[0027] Figure 2 This is the mass spectrum of the crude purcanapeptide obtained in Comparative Example 3 of this invention. Detailed Implementation

[0028] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0029] Example 1: Preparation of Fmoc-Leu-Wang resin with a degree of substitution of 0.40 mmol / g

[0030] 100 g (91 mmol) of Wang resin with a substitution value of 0.91 mmol / g was weighed and added to a solid-phase reactor. The resin was washed twice with DMF and swollen with DMF for 60 minutes. Then, 32.14 g of Fmoc-Leu-OH, 13.52 g of HOBt, 17.23 g of DIC, and 1.11 g of DMAP were dissolved in 600 ml of DMF and added to the solid-phase reactor. The reaction was carried out at room temperature for 3 hours. After the reaction, the resin was washed three times with DMF and three times with DCM. Then, a capping solution was added and the resin was capped for 1.5 hours. The capping solution consisted of 92.90 g of acetic anhydride, 35.28 g of DIEA, and 2.22 g of DMAP dissolved in 600 ml of DMF. After capping, the resin was washed three times with DMF, three times with DCM, and three times with methanol. The resin was then dried under vacuum to obtain Fmoc-Leu-Wang resin with a substitution value of 0.40 mmol / g.

[0031] Example 2: Preparation of Fmoc-Leu-2-chlorotriphenylmethyl chloride resin with a degree of substitution of 0.20 mmol / g

[0032] 100 g (47 mmol) of 2-chlorotriphenylmethyl chloride resin with a substitution value of 0.47 mmol / g was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, and after swelling with DMF for 60 minutes, 24.90 g of Fmoc-Leu-OH and 36.45 g of DIEA were dissolved in 600 ml of DMF and added to the solid-phase reactor. The reaction was carried out at room temperature for 1 hour. After the reaction, the resin was washed three times with DMF and three times with DCM. Then, a capping solution was added for 1.5 h to cap the resin. The capping solution consisted of 15.04 g of methanol and 18.22 g of DIEA dissolved in 600 ml of DMF. After capping, the resin was washed three times with DMF, three times with DCM, and three times with methanol. The resin was then dried under vacuum to obtain Fmoc-Leu-2-chlorotriphenylmethyl chloride resin with a substitution value of 0.20 mmol / g.

[0033] Example 3: Preparation of Fmoc-Leu-HMP MBHA resin with a degree of substitution of 0.50 mmol / g

[0034] Weigh 100g (70mmol) of MBHA resin with a substitution value of 0.70mmol / g and add it to a solid-phase reactor. Wash twice with DMF. After swelling the resin with DMF for 60 minutes, dissolve 38.25g HMP, 31.21g HOBt, and 39.76g DIC in 600ml DMF and add it to the solid-phase reactor. React at room temperature for 3 hours. After the reaction, wash four times with DMF. Dissolve 74.17g Fmoc-Leu-OH, 31.21g HOBt, 39.76g DIC, and 2.58g DMAP in 600ml DMF and add it to the solid-phase reactor. React at room temperature for 3 hours. Then add a capping solution and cap for 1.5 hours. The capping solution is: 71.47g acetic anhydride, 27.15g DIEA, and 0.85g DMAP dissolved in 600ml DMF. After end capping, the resin was washed three times with DMF, three times with DCM, and three times with methanol, then shrunk and dried to obtain Fmoc-Leu-HMPMBHA resin, with a substitution value of 0.50 mmol / g.

[0035] Example 4: Preparation of Fmoc-Leu-HMP AM resin with a degree of substitution of 0.60 mmol / g

[0036] Weigh 100 g (80 mmol) of AM resin with a substitution value of 0.80 mmol / g and add it to a solid-phase reactor. Wash twice with DMF. After swelling the resin with DMF for 60 minutes, dissolve 43.71 g HMP, 35.66 g HOBt, and 45.44 g DIC in 600 ml of DMF and add it to the solid-phase reactor. React at room temperature for 3 hours. After the reaction, wash four times with DMF. Dissolve 84.77 g Fmoc-Leu-OH, 35.66 g HOBt, 45.44 g DIC, and 2.94 g DMAP in 600 ml of DMF and add it to the solid-phase reactor. React at room temperature for 3 hours. Then add a capping solution and cap for 1.5 hours. The capping solution is: 81.68 g acetic anhydride, 31.02 g DIEA, and 0.98 g DMAP dissolved in 600 ml of DMF. After end capping, the resin was washed three times with DMF, three times with DCM, and three times with methanol, then shrunk and dried to obtain Fmoc-Leu-HMPAM resin with a substitution value of 0.60 mmol / g.

[0037] Example 5: Preparation of Pulcanopeptide Resin 1

[0038] 10.00 g (4.0 mmol) of Fmoc-Leu-Wang resin with a substitution value of 0.40 mol / g prepared in Example 1 of this invention was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, swollen with DMF for 30 minutes, dried, and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF for 5 min + 15 min respectively, followed by six washes with DMF. Fmoc-Cys(Acm)-OH (6.63 g, 4.0 eq.) and HOBt (2.38 g, 4.4 eq.) were weighed and dissolved in 40 ml of DMF. DIC (3.03 g, 6.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to the synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The amino group was detected using the ninhydrin method. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete, and the coupling reaction needed to continue for another 0.5–1.5 hours until the resin was colorless and transparent to the ninhydrin test. The resin was dried and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF, for 5 min and 15 min respectively, followed by six washes with DMF.

[0039] Repeat the above steps, removing the Fmoc protecting group and then sequentially coupling Fmoc-(Hmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH. The coupling of Fmoc-Thr-OH uses HBTU / HOBT / DIEA as the condensing agent, while the coupling of other amino acids uses HOBt / DIC as the condensing agent. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 0.5 hours, and the resin was colorless and transparent as determined by the ninhydrin method. After removing the Fmoc protecting group, the resin was washed four times with DMF, three times with DCM, and three times with MeOH. After drying, 21.45 g of pulcanapeptide peptide resin A: H-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr-(Hmb)Gly-Cys(Acm)-Leu-Wang resin was obtained.

[0040] Example 6: Preparation of Pulcanapeptide Resin 2

[0041] 10.00 g (2.0 mmol) of Fmoc-Leu-2-chlorotriphenylmethyl chloride resin with a substitution value of 0.20 mmol / g prepared in Example 2 of this invention was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, swollen with DMF for 30 minutes, dried, and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF at deprotection times of 5 min + 15 min, followed by six washes with DMF. Fmoc-Cys(Trt)-OH (4.69 g, 4.0 eq.) and HOBt (1.19 g, 4.4 eq.) were weighed and dissolved in 40 ml of DMF. DIC (1.51 g, 6.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to a synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The amino group was detected using the ninhydrin method. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete, and the coupling reaction needed to continue for another 0.5–1.5 hours until the resin was colorless and transparent to the ninhydrin test. The resin was dried and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF, for 5 min and 15 min respectively, followed by six washes with DMF.

[0042] Repeat the above steps, removing the Fmoc protecting group and then sequentially coupling Fmoc-(Dmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn(Trt)-OH. The coupling of Fmoc-Thr-OH uses HBTU / HOBT / DIEA as the condensing agent, while the coupling of other amino acids uses HOBt / DIC as the condensing agent. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 0.5 hours, and the resin was colorless and transparent as determined by the ninhydrin method. After washing with DMF four times, DCM three times, and MeOH three times, and drying, 14.58 g of pulcanapeptide peptide resin B was obtained: Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-Glu(OtBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr-(Dmb)Gly-Cys(Trt)-Leu-2-chlorotriphenylmethylchloro resin.

[0043] Example 7: Preparation of Pulcanapeptide Resin 3

[0044] 10.00 g (5.0 mmol) of Fmoc-Leu-HMP MBHA resin with a substitution value of 0.50 mmol / g prepared in Example 3 of this invention was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, swollen with DMF for 30 minutes, dried, and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF at deprotection times of 5 min + 15 min, followed by six washes with DMF. Fmoc-Cys(Acm)-OH (8.29 g, 4.0 eq.) and HOBt (2.97 g, 4.4 eq.) were weighed and dissolved in 40 ml of DMF. DIC (3.79 g, 6.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to the synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The amino group was detected using the ninhydrin method. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete, and the coupling reaction needed to continue for another 0.5–1.5 hours until the resin was colorless and transparent to the ninhydrin test. The resin was dried and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF, for 5 min and 15 min respectively, followed by six washes with DMF.

[0045] Repeat the above steps, removing the Fmoc protecting group and then sequentially coupling Fmoc-(Hmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn-OH. The coupling of Fmoc-Thr-OH uses TBTU / HOBT / DIEA as the condensing agent, while the coupling of other amino acids uses HOBt / DIC as the condensing agent. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 0.5 hours, and the resin was colorless and transparent as determined by the ninhydrin method. After removing the Fmoc protecting group, the resin was washed four times with DMF, three times with DCM, and three times with MeOH. After drying, 25.35 g of pulcanapeptide peptide resin C: H-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)-Thr-(Hmb)Gly-Cys(Acm)-Leu-HMP MBHA resin was obtained.

[0046] Example 8: Preparation of Pulcanapeptide Resin 4

[0047] 10.00 g (6.0 mmol) of Fmoc-Leu-AM MBHA resin with a substitution value of 0.60 mmol / g prepared in Example 4 of this invention was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, swollen with DMF for 30 minutes, dried, and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF at deprotection times of 5 min + 15 min, followed by six washes with DMF. Fmoc-Cys(tBu)-OH (9.59 g, 4.0 eq.) and HOBt (3.57 g, 4.4 eq.) were weighed and dissolved in 40 ml of DMF. DIC (4.54 g, 6.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to the synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The amino group was detected using the ninhydrin method. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete, and the coupling reaction needed to continue for another 0.5–1.5 hours until the resin was colorless and transparent to the ninhydrin test. The resin was dried and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF, for 5 min and 15 min respectively, followed by six washes with DMF.

[0048] Repeat the above steps, removing the Fmoc protecting group and then sequentially coupling Fmoc-(Hmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn-OH. The coupling of Fmoc-Thr-OH uses HBTU / HOBT / DIEA as the condensing agent, while the coupling of other amino acids uses HOBt / DIC as the condensing agent. No peptide resin volume shrinkage occurred during the coupling process, indicating easy coupling. Each reaction lasted 0.5 hours, and the resin was colorless and transparent as determined by the ninhydrin method. After washing with DMF four times, DCM three times, and MeOH three times, and drying, 28.66 g of pulcanapeptide peptide resin D: Boc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Mmt)-Glu(OtBu)-Leu-Cys(tBu)-Val-Asn-Val-Ala-Cys(Mmt)-Thr-(Hmb)Gly-Cys(tBu)-Leu-HMP-AM resin was obtained.

[0049] Example 9: Preparation of linear crude pucanatide peptide 1

[0050] 22.56 g of the purcanapeptide resin A prepared in Example 5 of this invention was placed in a pyrolysis reactor. Pyrolysis reagent was added at a ratio of 10 ml / g resin, with the ratio of TFA:Tis = 95:5 (V / V). The mixture was stirred and pyrolyzed at room temperature for 2.0 hours. The reactants were filtered through a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. The precipitate was added to cooled isopropyl ether to settle, and the solid was centrifuged. The solid was washed with isopropyl ether, centrifuged four times, and dried under vacuum to obtain the linear crude purcanapeptide peptide E: H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH. The weight of the linear crude peptide was 7.58 g, its purity was 85.4%, and the yield was 103.7%.

[0051] Example 10: Preparation of linear crude pucalatide peptide 2

[0052] Take 4.58 g of the purcanapeptide resin B1 prepared in Example 6 of this invention and place it in a pyrolysis reactor. Add pyrolysis reagent at a ratio of 10 ml / g resin. The ratio of pyrolysis reagent is as follows: TFA: Mpr: m-cresol = 95:3:2 (V / V). Pyrolysis is carried out by stirring at room temperature for 2.0 hours. Filter the reactants using a sintered glass funnel, collect the filtrate, wash the resin three times with a small amount of TFA, combine the filtrates, and concentrate under reduced pressure. Add cooled isopropyl ether to settle the precipitate, centrifuge, wash with isopropyl ether, centrifuge four times, and vacuum dry to obtain the linear crude purcanapeptide peptide F: H-Asn-Asp-Glu-Cys(Acm)-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys(Acm)-Thr-Gly-Cys-Leu-OH. The weight of the linear crude peptide is 3.12 g, its purity is 81.8%, and the yield is 85.3%.

[0053] Example 11: Preparation of linear crude pucanatide peptide 3

[0054] 25.35 g of the purcanapeptide resin C prepared in Example 7 of this invention was placed in a pyrolysis reactor. Pyrolysis reagent was added at a ratio of 10 ml / g resin, with the following ratio: TFA: Tis: Mpr: Phenol: H2O = 80:5:5:5:5 (V / V). The mixture was stirred and pyrolyzed at room temperature for 2.0 hours. The reactants were filtered through a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. The precipitate was added to cooled isopropyl ether to settle, and the solid was centrifuged. The solid was washed with isopropyl ether, centrifuged four times, and dried under vacuum to obtain the linear crude purcanapeptide peptide G: H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH. The weight of the linear crude peptide was 9.28 g, its purity was 83.6%, and the yield was 101.5%.

[0055] Example 12: Preparation of linear crude pucanatide peptide 4

[0056] 28.66 g of the purcanapeptide resin D prepared in Example 8 of this invention was placed in a pyrolysis reactor. Pyrolysis reagent was added at a ratio of 10 ml / g resin, with the following ratio: TFA: Tis: Mpr: m-cresol: H2O = 90:3:3:3:1 (V / V). The mixture was stirred and pyrolyzed at room temperature for 2.0 hours. The reactants were filtered through a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. The precipitate was added to cooled isopropyl ether to settle, and the solid was centrifuged. The solid was washed with isopropyl ether, centrifuged four times, and dried under vacuum to obtain the crude linear purcanapeptide peptide H: H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(tBu)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(tBu)-Leu-OH. The linear peptide weighed 10.03 g, with a purity of 82.9% and a yield of 91.4%.

[0057] Example 13, Preparation of pulcanapeptide 1

[0058] 7.85 g of the linear crude pucarnatide peptide E obtained in Example 9 of this invention was dissolved in 300 ml of acetonitrile, then diluted with 6400 ml of water. The pH was adjusted to 8.0-10.0 with ammonia. 0.45 ml of 30% hydrogen peroxide solution was added and the mixture was stirred for 1-6 hours to oxidize the first disulfide bond. Trifluoroacetic acid was then added dropwise to adjust the pH to 2.0-5.0. Iodine-acetonitrile solution was added to oxidize the second disulfide bond. After the reaction was complete, ascorbic acid aqueous solution was slowly added to consume the excess iodine. At this point, the reaction system should be colorless and transparent. The pH was then adjusted to 6.5-7.0 with ammonia. The resulting reaction solution is the crude pucarnatide peptide solution. The concentration of the crude pucarnatide peptide solution was determined by comparing it with a pucarnatide reference standard, and the yield was 0.6923 g / L, with a crude product yield of 69.0%. MS analysis of the crude pucarnatide peptide solution was performed, and the mass spectrum results are shown below. Figure 1 .

[0059] The crude peptide was prepared and purified by HPLC using a reversed-phase C18 DAC200 column at a wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA aqueous solution and acetonitrile. After purification and elution, the eluent of the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain 3.25 g of purcanapeptide peptide. The peptide yield was 48.3%, the purity was 99.6%, and the maximum single impurity was 0.05%.

[0060] Example 14, Preparation of pulcanapeptide 2

[0061] 3.72g of the linear crude pricanapeptide F obtained in Example 10 of this invention was dissolved in 150ml of acetonitrile, then diluted with 3200ml of water. 3.15g of Tris-HCl was added to dissolve and clarify the solution, and the pH was adjusted to 8.0-8.5 with hydrochloric acid. 0.26g of heme chloride was added, and 2-4 drops of DIEA were added dropwise, stirring for 1-6 hours to oxidize the first disulfide bond. Trifluoroacetic acid was then added dropwise to adjust the pH to 2.0-5.0, and iodine-acetonitrile solution was added to oxidize the second disulfide bond. After the reaction was complete, ascorbic acid aqueous solution was slowly added to consume the excess iodine. At this point, the reaction system should be colorless and transparent. The pH was then adjusted to 6.5-8.5 with ammonia water. The resulting reaction solution is the crude pricanapeptide solution. The crude pucarnatide solution was standardized to a concentration of 0.6662 g / L with pucarnatide reference standard, and the crude product yield was 66.4%. MS analysis of the crude pucarnatide solution was performed, and the mass spectrum results were consistent with... Figure 1 similar.

[0062] The crude peptide was prepared and purified by HPLC using a reversed-phase C18 DAC200 column at a wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA aqueous solution and acetonitrile. After purification and elution, the eluent of the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain 1.52 g of pricanapeptide refined peptide. The yield was 45.2%, the purity was 99.4%, and the maximum single impurity was 0.08%.

[0063] Example 15, Preparation of pulcanapeptide 3

[0064] 9.28 g of the linear crude pricanapeptide G obtained in Example 11 of this invention was dissolved in 250 ml of acetonitrile, then diluted with 4750 ml of water. The pH was adjusted to 8.0-10.0 with ammonia, and the reaction was stirred in air for 18-36 hours to oxidize the first pair of disulfide bonds. Trifluoroacetic acid was then added dropwise to adjust the pH to 2.0-5.0, and iodine-acetonitrile solution was added to oxidize the second pair of disulfide bonds. After the reaction was complete, ascorbic acid aqueous solution was slowly added to consume the excess iodine. At this point, the reaction system should be colorless and transparent. The pH was then adjusted to 6.5-8.5 with ammonia. The resulting reaction solution is the crude pricanapeptide solution. The concentration of the crude pricanapeptide solution was determined by standardization with pricanapeptide reference standard, which was 1.1523 g / L, and the crude product yield was 68.5%. MS analysis of the crude pricanapeptide solution was performed. The mass spectrum results of the crude pricanapeptide solution were consistent with... Figure 1 similar.

[0065] The crude peptide was prepared and purified by HPLC using a reversed-phase C18 DAC200 column at a wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA aqueous solution and acetonitrile. After purification and elution, the eluent of the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain 4.22 g of puricanatide peptide, with a yield of 50.2%, purity of 99.5%, and a maximum single impurity of 0.05%.

[0066] Example 16, Preparation of pulcanapeptide 4

[0067] 1.21 g of the linear crude purcanapeptide H1 obtained in Example 12 of this invention was dissolved in 250 ml of DMSO (dimethyl sulfoxide), then diluted with 4750 ml of water. 1.0 g of NaH2PO4 and 1.2 g of guanidine hydrochloride were added to adjust the pH to 7.5 ± 1.0. The reaction was carried out with stirring for 12-24 hours to oxidize the first pair of disulfide bonds. The Cys side chain tBu protecting group was then removed using trifluoroacetic acid, trichlorophenylsilane, diphenyl sulfoxide, and anisole. Finally, the second pair of disulfide bonds was oxidized by air oxidation under conditions of pH 8.0 adjusted with 0.1 M Tris buffer (tris(hydroxymethyl)aminomethane). The resulting reaction solution was the crude purcanapeptide solution. The concentration of the crude purcanapeptide solution was determined by calibrating with purcanapeptide standard, and the yield was 1.3699 g / L, with a crude product yield of 67.9%. MS analysis of the crude purcanapeptide solution was performed. The mass spectrum results of the crude purcanapeptide were consistent with... Figure 1 similar.

[0068] The crude peptide was purified using an HPLC preparative column packed with a reversed-phase C18 DAC200 column at a wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA aqueous solution and acetonitrile. After purification and elution, the eluent of the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain 5.45 g of purcanapeptide refined peptide. The yield was 54.0%, the purity was 99.3%, and the maximum single impurity was 0.08%.

[0069] Comparative Example 1: Preparation of Pulcanapeptide Resin

[0070] 10 g (4.0 mmol) of Fmoc-Leu-Wang resin with a substitution value of 0.40 mol / g prepared in Example 1 of this invention was weighed and added to a solid-phase reactor. The resin was washed twice with DMF, swollen with DMF for 30 minutes, dried, and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF at deprotection times of 5 min and 15 min respectively, followed by six washes with DMF. Fmoc-Cys(Acm)-OH (6.63 g, 4.0 eq.) and HOBt (2.38 g, 4.4 eq.) were weighed and dissolved in 40 ml of DMF. DIC (3.03 g, 6.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to the synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The amino group was detected using the ninhydrin method. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete, and the coupling reaction needed to continue for another 0.5–1.5 hours until the resin was colorless and transparent to the ninhydrin test. The resin was dried and washed four times with DMF. Fmoc protection was removed twice with 20% PIPE / DMF, for 5 min and 15 min respectively, followed by six washes with DMF.

[0071] Repeat the above steps, removing the Fmoc protecting group and then sequentially coupling Fmoc-(Hmb)Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH. HOBt / DIC is used as the condensing agent for the amino acid coupling process. No peptide resin volume shrinkage occurred during the coupling process. However, when coupling Fmoc-Thr(tBu)-OH at the S13 site, coupling difficulties occurred due to steric hindrance. After 2.5 hours of reaction, the resin was light blue as detected by the ninhydrin method. Repeated addition of the resin did not significantly improve the situation. After removing the Fmoc protecting group, the resin was washed 4 times with DMF, 3 times with DCM, and 3 times with MeOH. After drying, 20.32 g of pulcanapeptide peptide resin I was obtained: H-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-(Hmb)Gly-Cys(Acm)-Leu-Wang resin.

[0072] Comparative Example 2: Preparation of linear crude pucanatide peptide

[0073] 20.32 g of the purcanapeptide resin I prepared in Comparative Example 1 of this invention was placed in a pyrolysis reactor. Pyrolysis reagent was added at a ratio of 10 ml / g resin, with the ratio of TFA:Tis = 95:5 (V / V). The mixture was stirred and pyrolyzed at room temperature for 2.0 hours. The reactants were filtered through a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. The precipitate was settled in cooled isopropyl ether, and the solid was centrifuged. The solid was washed with isopropyl ether, centrifuged four times, and dried under vacuum to obtain the linear crude purcanapeptide peptide J: H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH. The weight of the linear crude peptide was 6.95 g, the yield was 95.0%, and its purity was 55.3%.

[0074] Comparative Example 3: Preparation of Pulcanapeptide

[0075] 6.95 g of the linear crude pucarnatide J obtained in Comparative Example 2 of this invention was dissolved in 300 ml of acetonitrile, then diluted with 6400 ml of water. The pH was adjusted to 8.0-10.0 with ammonia. 0.45 ml of 30% hydrogen peroxide solution was added and the mixture was stirred for 1-6 hours to oxidize the first disulfide bond. Trifluoroacetic acid was then added dropwise to adjust the pH to 2.0-5.0. Iodine-acetonitrile solution was added to oxidize the second disulfide bond. After the reaction was complete, ascorbic acid aqueous solution was slowly added to consume the excess iodine. At this point, the reaction system should be colorless and transparent. The pH was then adjusted to 6.5-8.5 with ammonia. The resulting reaction solution is the crude pucarnatide solution. The concentration of the crude pucarnatide solution was determined by standardization with pucarnatide reference standard, which was 0.5123 g / L. The crude product yield was 51.1%. MS analysis of the crude pucarnatide solution was performed. The mass spectrum results of the crude pucarnatide solution are shown below. Figure 2 .

[0076] The crude peptide was purified using an HPLC preparative column packed with a reversed-phase C18 DAC200 column at a wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA aqueous solution and acetonitrile. After purification and elution, the eluent of the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain 2.52 g of purcanapeptide refined peptide. The yield was 37.5%, the purity was 98.2%, and the maximum single impurity was 0.32%.

Claims

1. A method for preparing pulcanapeptide, characterized in that, The main steps include: 1) The resin solid support is coupled with Fmoc-Leu-OH to obtain Fmoc-Leu-resin; 2) By solid-phase synthesis, corresponding protecting amino acids or fragments in the sequence are sequentially coupled to the Fmoc-Leu-resin obtained in step 1) to obtain purcanapeptide peptide resin: R1-Asn(R4)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R3)-Val-Asn(R4)-Val-Ala-Cys(R2)-Thr-X-Cys(R3)-Leu-resin, wherein X is selected from (Hmb)Gly or (Dmb)Gly; R1 is selected from Fmoc, Boc or H; R2 and R3 are selected from A or B, where A is Acm or tBu, and B is Trt or Mmt, and R2 and R3 are different; R4 is selected from H or an amino protecting group; 3) The pulcanapeptide resin obtained in step (2) is cleaved to obtain linear pulcanapeptide; 4) Cyclate the linear purcanapeptide obtained in step (3) to obtain crude purcanapeptide. Specifically, when the pulcanapeptide peptide resin is cleaved by a cleavage reagent to obtain a linear peptide containing a pair of Cys with Acm protecting groups in the peptide sequence, the linear peptide is dissolved, an oxidant is added, and an oxidation reaction is carried out to obtain a monocyclic peptide; the monocyclic peptide is then de-protected by the side chain protecting group Acm on Cys in an iodine solution, and cyclized to obtain crude pulcanapeptide peptide. or When the pulcanapeptide peptide resin is cleaved by a cleavage reagent to obtain a linear peptide containing a pair of Cys protecting groups with tBu, the linear peptide is dissolved, an oxidant is added, and an oxidation reaction is carried out to obtain a monocyclic peptide; the monocyclic peptide is then deprotected by the tBu side chain protecting group on Cys with trichlorophenylsilane, diphenyl sulfoxide, and anisole, and cyclization is continued in the same oxidant system to obtain crude pulcanapeptide peptide. The added oxidant is selected from hydrogen peroxide, DMSO, heme chloride, or air.

2. The method for preparing pulcanapeptide according to claim 1, characterized in that: In step 2), R2 and R3 are selected from Trt and Acm; R4 is selected from H or Trt.

3. The method for preparing pulcanapeptide according to claim 1, characterized in that, In step 1), the resin solid support is selected from Wang resin, 2-chlorotriphenylmethyl chloride resin, HMP MBHA resin, or HMP AM resin.

4. The method for preparing pulcanapeptide according to claim 1, characterized in that, In step 1), the degree of substitution of Fmoc-Leu-resin is 0.20-0.60 mmol / g.

5. The method for preparing pulcanapeptide according to claim 1, characterized in that, Step 2) specifically involves sequentially coupling Fmoc-Cys(R3)-OH, Fmoc-(Hmb)Gly-OH or Fmoc-(Dmb)Gly-OH, Fmoc-Thr-OH, Fmoc-Cys(R2)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(R4)-OH, Fmoc-Val-OH, Fmoc-Cys(R3)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(R4)-OH or Boc-Asn(R4)-OH onto Fmoc-Leu-resin to obtain purcanapeptide peptide resin.

6. The method for preparing pulcanapeptide according to claim 1, characterized in that: The condensation reagent used in step 2) of the coupling process is selected from HBTU / HOBT / DIEA, TBTU / HOBT / DIEA or HOBt / DIC.

7. The method for preparing pulcanapeptide according to claim 1, characterized in that: The lysis reagent is selected from TFA and reagent A. Reagent A is selected from one or more of Tis, Mpr, m-cresol, Phenol, and H2O, wherein the proportion of TFA is 80-95 (V / V) and the proportion of reagent A is 5-20 (V / V).

8. The method for preparing pulcanapeptide according to claim 1, characterized in that: The crude purcanapeptide obtained in step 4) can be further purified and freeze-dried to obtain refined purcanapeptide.

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