A method for preparing a bumen peptide

Abstract

The application relates to the field of polypeptide medicine preparation, and particularly discloses a preparation method of bumenol peptide, which adopts a solid-liquid combination mode, liquid-phase synthesis of a tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-OMe, subsequent solid-phase synthesis of a cyclic heptapeptide methyl ester, cleavage, cyclization, deprotection and hydrolysis to obtain crude bumenol peptide. The cyclization site is alpha-COOH of Trp and alpha-NH2 of Lys, the reaction yield is high, and the cyclization problem between Lys and Asp in most current patents is solved. The process operation is simple, the deprotection reagent is conducive to environmental protection requirements. The crude product is easy to purify, the finished product has high yield and purity, and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug preparation, and specifically to a method for preparing bumenopeptide. Background Technology

[0002] Hypoactive sexual desire disorder (HSDD) is one of the most common sexual dysfunctions in women, with a global prevalence of 6%-10% among women of reproductive age. It is mainly characterized by decreased libido, which also leads to psychological stress and affects women's emotional health, family harmony, interpersonal relationships, and overall quality of life.

[0003] Bremerlanotide, developed by Palatin Technologies, Inc., is a melanocortin receptor-4 (MC4R) agonist that regulates libido and sexual response by activating endogenous pathways in the brain. It is used to treat premenopausal women with acquired generalized hyposexuality. Phase III clinical trials have been completed, and the drug received FDA approval on June 12, 2019. The subcutaneous injection is marketed under the brand name [Brand Name Missing]. This makes it the second drug to receive FDA approval for the treatment of premenopausal hypoactive sexual desire disorder (HSDD) in women. Currently, no drugs for the treatment of HSDD have been approved for marketing in China; therefore, bumenotide has a broad market prospect.

[0004] Bumenotide is an analogue of the α-melanocyte-stimulating hormone (α-MSH) synthetic polypeptide. It is a cyclic polypeptide derivative of melanocortin containing seven amino acids, CAS number 1607799-13-2. It is a cyclic heptapeptide with a C-terminal carboxylic acid, containing non-natural amino acids: D-type phenylalanine and Nle leucine. The Asp side chain carboxyl group and the Lys side chain amino group form a ring via an amide bond. Nle is coupled to the N-terminus and acetylated for capping. The amino acid sequence is: Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH. Its chemical structure is as follows:

[0005]

[0006] Patent CN101092451A discloses a solid-phase synthesis method for bumenopeptides. The method involves using PAM resin to sequentially couple amino acids, obtaining an Fmoc-protected peptide resin. The Asp side chain β-COOH protecting group and the Lys side chain ε-NH2 protecting group are then removed, followed by solid-phase condensation to form a cyclization. The N-terminal Fmoc is then removed, followed by acetylation and HF cleavage to obtain the final product. This process utilizes easily corrosive HF cleavage, which is environmentally unfriendly and unsuitable for large-scale production.

[0007] Patent CN101280005A discloses a method for solid-phase synthesis of bumeno peptides. First, a peptide resin is synthesized using Wang resin in a solid phase. After removing the protecting groups, cyclization and cleavage occur on the solid phase. However, the Asp (O-2-Phipr) and Lys (Mmt) amino acid fragments are expensive, and selective deprotection using low-concentration TFA may result in incomplete removal of the protecting groups on Asp and Lys.

[0008] Patent CN110746486A discloses a solid-liquid phase 1+6 synthesis method for bumenopeptide. After solid-phase synthesis of a 6-peptide fragment, it is then synthesized in the liquid phase with Lys(Boc)-OMe to form a fully protected 7-peptide methyl ester. This is followed by cleavage to obtain a linear heptapeptide methyl ester. The β-COOH of the Asp side chain of the linear heptapeptide methyl ester is then cyclized with the ε-NH2 of the Lys side chain using a condensing agent. This process forms an amide bond after cleavage to obtain the linear heptapeptide methyl ester. Since the linear heptapeptide methyl ester is already in a deprotected state at this point, the formation of the amide bond easily generates many impurities. Furthermore, sulfur-containing reagents are preferentially used during cleavage, resulting in a pungent odor, which is detrimental to environmental protection.

[0009] Patent CN110950933A discloses a solid-liquid phase 2+5 synthesis method for bumenopeptide. The method involves synthesizing the Ac-Nle-Asp-O-2-Phipr dipeptide fragment via liquid-phase synthesis, extending the peptide sequence by coupling the main chain and side chains of Lys to obtain a fully protected peptide resin, and then forming amide bonds on the α-NH2 of His and the α-COOH of Asp in the solid phase. This method avoids end-to-end folding into rings. However, the synthesis of the Ac-Nle-Asp-O-2-Phipr dipeptide fragment is costly and expensive, requiring the use of hydrazine hydrate for deprotection, which is environmentally unfriendly. Furthermore, the dipeptide fragment is coupled to the C-terminal Lys, which is in contact with the resin. After coupling, other amino acids need to be coupled, but steric hindrance hinders the subsequent coupling, resulting in poor coupling efficiency.

[0010] Bumenotide is a cyclic peptide composed of 7 amino acids. The challenge in its synthesis lies in the construction of the intramolecular cyclic amide bond. Existing methods for preparing bumenotide primarily focus on the cyclic sites at the β-COOH of the Asp side chain and the ε-NH2 of the Lys side chain, as well as the α-COOH of Asp and the α-NH2 of His. However, existing synthetic strategies using these two cyclic sites have the following drawbacks: the use of expensive amino acid materials, such as Asp (O-2-Phipr) and Lys (Mmt), leads to high synthesis costs and complex processes; some cleavage and deprotection reagents (HF, sulfur-containing reagents, hydrazine hydrate, etc.) are environmentally unfriendly; steric hindrance results in poor coupling when dipeptide fragments are coupled to the C-terminal Lys; and the linear heptapeptide methyl ester is in a deprotected state, making it prone to producing many impurities in the crude peptide during amide bond formation, hindering purification and hindering large-scale industrial production.

[0011] Therefore, providing a method for preparing bumenotide that can reduce the generation of impurities, facilitate the purification of crude products, achieve high yield and purity of finished products, meet environmental protection requirements, and is suitable for large-scale production is of great practical significance. Summary of the Invention

[0012] To address the shortcomings of existing technologies, this invention provides a novel method for preparing bumenotide, which mainly includes the following steps:

[0013] A fully protected linear bumeno peptide resin was synthesized, which was then cleaved, cyclized, and deprotected to obtain cyclic heptapeptide ester, which was then hydrolyzed to obtain crude bumeno peptide.

[0014] The fully protected linear bumetropeptide resin is:

[0015]

[0016] Where X is selected from Trt or Boc;

[0017] Y is selected from methyl ester, ethyl ester, or benzyl ester.

[0018] Furthermore, the specific steps for obtaining crude bumenopeptide are as follows:

[0019] Step 1): Synthesize the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y;

[0020] Step 2): Fmoc-Trp(Boc)-OH is coupled to a solid-phase support resin by solid-phase synthesis to obtain Fmoc-Trp(Boc)-resin.

[0021] Step 3): Take the Fmoc-Trp(Boc)-resin obtained in step 2) and couple the corresponding protected amino acids or fragments and the tripeptide fragment synthesized in step 1) sequentially under the condensation agent to obtain a fully protected linear bumeno peptide resin.

[0022] Step 4): Take the linear bumeno peptide resin obtained in step 3), and after cleavage, cyclization and deprotection, obtain cyclic heptacapeptide methyl ester.

[0023] Step 5): Take the cyclic heptapeptide methyl ester obtained in step 4), add an alkaline reagent, and hydrolyze the methyl ester in a reaction solvent to obtain crude bumenopeptide.

[0024] The tripeptide fragment synthesized in step 1) has large steric hindrance. It is more appropriate to place it at the nitrogen end during solid-phase coupling in step 3) to effectively avoid the problem of low coupling efficiency caused by large steric hindrance.

[0025] In some embodiments, the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y in step 1) is synthesized by a liquid-phase method, specifically: H-Asp-OtBu reacts with Ac-Nle-OSu to obtain Ac-Nle-Asp-OtBu, which is then reacted with HOSu to generate the active ester Ac-Nle-Asp(OSu)-OtBu; the active ester is condensed with Fmoc-Lys-Y to obtain Fmoc-Lys(Ac-Nle-Asp-OtBu)-Y; and finally, the protecting group is removed to obtain Fmoc-Lys(Ac-Nle-Asp)-Y.

[0026] In some embodiments, the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y in step 1) can also be synthesized by liquid phase method first, followed by acetylation. Specifically, H-Asp-OtBu reacts with Boc-Nle-OSu to obtain Boc-Nle-Asp-OtBu, which is then reacted with HOSu to generate the active ester Boc-Nle-Asp(OSu)-OtBu; the active ester is condensed with Fmoc-Lys-Y to obtain Fmoc-Lys(Boc-Nle-Asp-OtBu)-Y; the protecting group is removed to obtain Fmoc-Lys(H-Nle-Asp)-Y, which is finally reacted with acetic anhydride to obtain Fmoc-Lys(Ac-Nle-Asp)-Y.

[0027] Further, the solid support resin in step 2) is selected from 2-CTC resin, with a resin substitution degree of 0.8-1.2 mmol / g.

[0028] The protecting group described in this invention is used in the field of peptide synthesis to protect amino acid backbones and side chains such as amino, carboxyl, and hydroxyl groups from interference during synthesis, preventing these groups from reacting and generating impurities during the preparation of the target product. The amino acid protected by the protecting group is called the protecting amino acid. For the amino acid whose side chain needs to be protected in this invention, those skilled in the art know that commonly used protecting groups are used to protect the amino, carboxyl, and hydroxyl groups on the amino acid side chain. Preferably, step 3) uses a stepwise coupling method to sequentially couple: Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-His(Trt)-OH, or Fmoc-His(Boc)-OH.

[0029] Further, the condensing agent in step 3) is one or more of DIC / HOBt, EDCI / HOBt, PyBOP / DIEA, TBTU / DIEA, and HBTU / DIEA. Preferably, the condensing agent is selected from TBTU / DIEA or DIC / HOBt.

[0030] Furthermore, in step 4), the pyrolysis solution required for pyrolysis is selected from a mixture of TFE and DCM, wherein TFE accounts for 20% to 50% of the volume of the mixture, preferably 20% TFE. Pyrolysis under this concentration condition has the best effect on cutting the resin.

[0031] Further, in step 4), the cyclization is performed using a condensing agent in an organic solvent; wherein the organic solvent is DMF, DCM or THF, and the condensing agent is one of DIC / HOBt, EDCI / HOBt, PyBOP / DIEA, TBTU / DIEA, or HBTU / DIEA, preferably EDCI / HOBt, PyBOP / DIEA, or HBTU / DIEA, and more preferably PyBOP / DIEA. These condensing agents are suitable for cyclization under liquid phase conditions.

[0032] Further, the deprotection reagent required in step 4) is a mixture of TFA, TIS and H2O, with a volume ratio of TFA:TIS:H2O = 90-98:1-5:1-5. Preferably, TFA:TIS:H2O = 95:2.5:2.5.

[0033] Further, in step 5), the alkaline reagent is one of LiOH, NaOH, and KOH, preferably LiOH; the reaction solvent is one of ethanol / water, methanol / water, and acetonitrile / water, wherein the organic solvent accounts for 10-50%, preferably 20%.

[0034] Further, the crude bumenotide obtained in step 5) was purified, converted to salt, concentrated, and lyophilized to obtain bumenotide acetate. Specifically, the crude product was dissolved in water, filtered through a 0.45 μm filter membrane, purified in one step with a 10 mM-100 mM sodium sulfate / acetonitrile solution, converted to salt with 10 mM-100 mM ammonium acetate, eluted with a gradient of 0.05%–0.5% acetic acid / acetonitrile system, and the fractions with a purity greater than 99% were combined, concentrated, and lyophilized to obtain the final product.

[0035] This invention provides a method for preparing bumenopeptide, employing a solid-liquid combination approach. The tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y is synthesized in the liquid phase, followed by solid-phase synthesis of cyclic heptapeptide methyl ester. After resin cleavage, cyclization, deprotection, and hydrolysis yield crude bumenopeptide. This method utilizes the cyclization sites of Trp's α-COOH and Lys's α-NH2. Due to the inherent properties of Trp, these two amino acids are less prone to racemization during cyclization, thus reducing the generation of racemic impurities. This solves the problem of numerous impurities caused by cyclization limited to Lys and Asp in most current patents, which only address cyclization between Lys and Asp. Simultaneously, it improves reaction yield. Furthermore, this process is simple to operate, avoids the use of sulfur-containing reagents for deprotection, and has no unpleasant odor, thus meeting environmental protection requirements. The crude product is easy to purify, the purification process is simple, and the finished product has high yield and purity, making it suitable for mass production. Attached Figure Description

[0036] Figure 1 The image shows the HPLC chromatogram of the bumeno peptide obtained in Example 17.

[0037] Figure 2 The image shows the HPLC chromatogram of the bumeno peptide obtained in Example 18. Detailed Implementation

[0038] 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 invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the present invention. In the specific embodiments of the present invention, all amino acids coupled with protecting groups are commercially available, and the Chinese names corresponding to the English abbreviations involved in the present invention are shown in Table 1.

[0039] Table 1. Chinese names corresponding to the English abbreviations involved in this invention.

[0040]

[0041] Unless otherwise specified, the explanations of relevant terms used in this invention shall adopt the conventional interpretations in the prior art.

[0042] Example 1: Synthesis of the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-OMe

[0043] Ac-Nle-OSu (8.11 g) was dissolved in THF and cooled to 0–5 °C. H-Asp-OtBu (7.38 g) and sodium carbonate (4.13 g) were added to THF / water (V / V, 1 / 3), stirred until dissolved, and then added dropwise to the above reaction solution. The mixture was stirred at 0–5 °C for 1 h, then heated to room temperature for 5 h, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction was stopped, and the pH of the reaction solution was adjusted to 5 with 1 N hydrochloric acid. The mixture was extracted twice with ethyl acetate, and the organic layers were combined. The organic layers were washed three times with dilute hydrochloric acid and once with saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain 10.21 g of Ac-Nle-Asp-OtBu, with a yield of 99%.

[0044] The above solid (10.21 g) was dissolved and clarified in DCM. HOSu (4.09 g) was added, and the reaction was stirred at 0–5 °C. DCC (7.33 g) was weighed and added to the DCM solution, then added to the above solution. The reaction was stirred at 0–5 °C for 3 h, resulting in the precipitation of a white solid. The reaction progress was monitored by TLC. After the reaction was complete, the reaction was stopped, and the solid was filtered. The solid was washed twice with DCM, and the filtrates were combined. The filtrate was washed twice with water and once with saturated brine, then dried over anhydrous sodium sulfate. The organic layer was concentrated to near dryness under vacuum, and a mixed solvent (petroleum ether / isopropyl ether, V / V, 1 / 1) was added. A white solid precipitated, which was filtered, and the solid was dried under vacuum to obtain Ac-Nle-Asp(OSu)-OtBu 12.09 g, with a yield of 99%.

[0045] The above solid (12.09 g) was dissolved in THF and cooled to 0–5 °C. Fmoc-Lys-OMe (13.49 g) and sodium carbonate (3.74 g) were added to THF / water (V / V, 1 / 3), stirred until dissolved, and then added dropwise to the above reaction solution. The mixture was stirred at 0–5 °C for 1 h, then allowed to rise to room temperature for 5 h. The reaction progress was monitored by TLC. After the reaction was complete, it was stopped, and the mixture was extracted twice with 1 N hydrochloric acid at pH 5. The organic layers were combined, washed three times with dilute hydrochloric acid and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 20.24 g of Fmoc-Lys(Ac-Nle-Asp-OtBu)-OMe, with a yield of 97%.

[0046] Prepare 200 ml of frozen TFA / H2O = 90% / 10% lysis buffer and add it to a 500 ml round-bottom flask. Maintain the temperature ≤5℃. Add the solid from the previous step (20.24 g) to the lysis buffer and react at room temperature for 2 h. Filter the solution, add the filtrate to ice-cold isopropyl ether to settle, centrifuge and wash to obtain 17.36 g of white solid Fmoc-Lys(Ac-Nle-Asp)-OMe, yield 93%.

[0047] Example 2: Synthesis of the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-OMe

[0048] Following the method in Example 1, Boc-Nle-OSu (9.82 g) was used to replace Ac-Nle-OSu for the reaction, and the product was washed by centrifugation to obtain 17.24 g of white solid Fmoc-Lys(Nle-Asp)-OMe.

[0049] The above solid was dissolved in THF, and 4.29 g of acetic anhydride was added. The mixture was stirred at room temperature for 1 h, and water was added to precipitate the solid. The solid was washed with petroleum ether and dried to obtain 16.82 g of Fmoc-Lys(Ac-Nle-Asp)-OMe, with a yield of 92%.

[0050] Example 3: Synthesis of the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-OEt

[0051] Following the method of Example 1, Fmoc-Lys-OMe (13.49 g) was replaced with Fmoc-Lys-OEt (14.09 g), and finally 17.74 g of white solid Fmoc-Lys(Ac-Nle-Asp)-OEt was obtained, with a yield of 94%.

[0052] Example 4: Synthesis of the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-OBn

[0053] Following the method of Example 1, Fmoc-Lys-OMe (13.49 g) was replaced with Fmoc-Lys-OBn (16.17 g), and finally 19.37 g of white solid Fmoc-Lys(Ac-Nle-Asp)-OBn was obtained, with a yield of 92%.

[0054] Example 5: Synthesis of Fmoc-Trp(Boc)-2-CTC resin 1

[0055] Weigh 6.17 g (5 mmol) of 2-CTC resin with a substitution degree of 0.81 mmol / g, add it to the solid-phase reaction column, wash it three times with DMF, remove the waste, add DMF to swell for 30 min, and remove the waste.

[0056] Weigh 5.27 g of Fmoc-Trp(Boc)-OH, dissolve it in DMF, add 2.70 g of DIEA, stir, and control the activation solution temperature to ≤15℃. Add the activation solution to the solid-phase reaction column, stir, and control the temperature at 25±10℃ for 3.0–4.0 h. Stop the reaction, discharge the waste, and wash with DMF three times.

[0057] Methanol (3.20 g) and DIEA (1.94 g) were weighed and dissolved in DMF. The solution was then added to the reaction column and stirred. The reaction was carried out at 25 ± 10 °C for 1 h. Waste was discharged, and the resin was washed 3 times with DMF and 5 times with DCM. The resin was then removed, air-dried at room temperature, and then vacuum-dried to constant weight to obtain 8.72 g of amino acid resin with a substitution degree of 0.55 mmol / g.

[0058] Example 6: Synthesis of Fmoc-Trp(Boc)-2-CTC Resin

[0059] Weigh 4.35 g (5 mmol) of 2-CTC resin with a substitution degree of 1.15 mmol / g, add it to the solid-phase reaction column, wash three times with DMF, remove waste, add DMF to swell for 30 min, and remove waste.

[0060] Weigh 5.27 g of Fmoc-Trp(Boc)-OH, dissolve it in DMF, add 2.70 g of DIEA, stir, and control the activation solution temperature to ≤15℃. Add the activation solution to the solid-phase reaction column, stir, and react at room temperature for 3.0–4.0 h. Stop the reaction, discharge the waste, and wash with DMF three times.

[0061] Methanol (3.20 g) and DIEA (1.94 g) were weighed and dissolved in DMF. The solution was then added to the reaction column and stirred. The reaction was carried out at room temperature for 1 hour. Waste was discharged, and the resin was washed 3 times with DMF and 5 times with DCM. The resin was removed, air-dried at room temperature, and then vacuum-dried to constant weight to obtain 6.89 g of amino acid resin with a substitution degree of 0.65 mmol / g.

[0062] Example 7: Synthesis 1

[0063] The Fmoc-Trp(Boc)-2-CTC resin (7.27 g, 4 mmol) obtained in Example 5 was added to a solid-phase reaction column. The column was washed three times with DMF, and the residue was discharged. DMF was added again for swelling for 30 min, and the residue was discharged again. A 20% piperidine / DMF solution was added twice to remove the Fmoc protecting group, once for 5 min and once for 15 min. The reaction was monitored with ninhydrin; when the resin turned blue, deprotection was complete. The residue was discharged, and the resin was washed six times with DMF.

[0064] Weigh out 5.19 g (8.0 mmol) of Fmoc-Arg(Pbf)-OH and 2.59 g (8.08 mmol) of TBTU, dissolve them in DMF, and maintain the solution temperature at ≤10 °C. Add 1.55 g (12.0 mmol) of DIEA and activate for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0065] Weigh out 3.10 g (8.0 mmol) of Fmoc-D-Phe-OH and 1.30 g (9.60 mmol) of HOBt and dissolve them in DMF. Maintain the solution temperature at ≤10 °C. Add 1.51 g (12.0 mmol) of DIC and activate for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0066] Weigh out 4.96 g (8.0 mmol) of Fmoc-His(Trt)-OH and 1.30 g (9.60 mmol) of HOBt and dissolve them in DMF. Maintain the solution temperature at ≤10 °C. Add 1.51 g (12.0 mmol) of DIC and activate for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0067] Weigh out 5.22 g (8.0 mmol) of Fmoc-Lys(Ac-Nle-Asp)-OMe and 2.59 g (8.08 mmol) prepared in Example 1, dissolve them in DMF, and maintain the solution temperature at ≤10°C. Add 1.55 g (12.0 mmol) to activate the solution for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction was monitored with ninhydrin; the resin was colorless, indicating complete condensation. The solvent was removed, and the resin was washed four times with DMF. The Fmoc protecting group was removed twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turned blue with ninhydrin, indicating complete deprotection. The resin was washed six times with DMF, then three times with DCM. After washing, the peptide resin was removed and dried, yielding 11.21 g of peptide resin, with a weight gain of 95%.

[0068] Example 8: Synthesis 2

[0069] The Fmoc-Trp(Boc)-2-CTC resin (6.15 g, 4 mmol) obtained in Example 6 was added to a solid-phase reaction column. The column was washed three times with DMF, and the residue was discharged. DMF was added again to swell the resin for 30 min, and the residue was discharged again. A 20% piperidine / DMF solution was added twice to remove the Fmoc protecting group, once for 5 min and once for 15 min. The reaction was monitored with ninhydrin; when the resin turned blue, deprotection was complete. The residue was discharged, and the resin was washed six times with DMF.

[0070] Weigh out 5.19 g (8.0 mmol) of Fmoc-Arg(Pbf)-OH and 4.21 g (8.08 mmol) of PyBop, dissolve them in DMF, and maintain the solution temperature at ≤10 °C. Add 1.55 g (12.0 mmol) of DIEA and activate for 3 min. Then add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0071] Weigh out 3.10 g (8.0 mmol) of Fmoc-D-Phe-OH and 1.30 g (9.60 mmol) of HOBt and dissolve them in DMF. Maintain the solution temperature at ≤10 °C. Add 1.51 g (12.0 mmol) of DIC and activate for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0072] Weigh out 4.96 g (8.0 mmol) of Fmoc-His(Trt)-OH and 1.30 g (9.60 mmol) of HOBt and dissolve them in DMF. Maintain the solution temperature at ≤10 °C. Add 1.51 g (12.0 mmol) of DIC and activate for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction is monitored with ninhydrin; the resin is colorless, indicating complete condensation. Remove the solvent and wash the resin four times with DMF. Remove the Fmoc protecting group twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turns blue with ninhydrin, indicating complete deprotection. Wash the resin six times with DMF. Proceed to the next amino acid coupling step.

[0073] Weigh out 5.22 g (8.0 mmol) of Fmoc-Lys(Ac-Nle-Asp)-OMe and 3.06 g (8.08 mmol) of HBTU prepared in Example 2, dissolve them in DMF, and maintain the solution temperature at ≤10°C. Add 1.55 g (12.0 mmol) to activate the solution for 3 min. Then, add the activated amino acid solution to the solid-phase reactor and stir at room temperature for 2 h. The reaction was monitored with ninhydrin; the resin was colorless, indicating complete condensation. The solvent was removed, and the resin was washed four times with DMF. The Fmoc protecting group was removed twice with 20% piperidine / DMF solution, once at 5 min and once at 15 min. The resin turned blue with ninhydrin, indicating complete deprotection. The resin was washed six times with DMF, then three times with DCM. After washing, the peptide resin was removed and dried, yielding 9.75 g of peptide resin, with a weight gain of 97%.

[0074] Example 9: Synthesis 1

[0075] Prepare 110 ml of TFE / DCM = 20% / 80% lysis buffer and add it to a 250 ml round-bottom flask, maintaining the temperature at ≤5℃. Add 11.21 g of the peptide resin obtained in Example 7 to the above lysis buffer and react at room temperature for 2 h. Filter, add the filtrate to cold isopropyl ether for precipitation, centrifuge and wash to obtain 6.48 g of fully protected linear peptide methyl ester.

[0076] Example 10: Synthesis 2

[0077] Prepare 100 ml of a 50% / 50% TFE / DCM lysis buffer and add it to a 250 ml round-bottom flask, maintaining the temperature at ≤5°C. Add 9.75 g of the peptide resin obtained in Example 8 to the above lysis buffer and react at room temperature for 2 h. Filter, add the filtrate to cold isopropyl ether for precipitation, centrifuge and wash to obtain 6.60 g of fully protected linear peptide methyl ester.

[0078] Example 11: Synthesis of Ac-Nle-Cyclo[Asp-His(Trt)-D-Phe-Arg(Pbf)-Trp(Boc)-Lys]-OMe

[0079] Take 6.0 g of the fully protected linear peptide methyl ester obtained in Example 9, add 3.5 L of DCM to dissolve, control the temperature at 0-5℃, stir, add 2.83 g of PyBOP and 0.94 g of DIEA, react at 0-5℃ for 1 h, then raise to room temperature and continue the reaction for 24 h, concentrate to obtain a pale yellow oily substance, dissolve the oily substance in DMF, add 10 times the volume of purified water for crystallization, centrifuge the crystallized solution, wash three times with purified water, collect the solid, filter, dry, and obtain 5.46 g of fully protected cyclic peptide methyl ester, yield 92%.

[0080] Example 12: Synthesis of Ac-Nle-Cyclo[Asp-His(Trt)-D-Phe-Arg(Pbf)-Trp(Boc)-Lys]-OMe 2

[0081] 6.0 g of the fully protected linear peptide methyl ester obtained in Example 10 was dissolved in 3.5 L of THF, and the temperature was controlled at 0-5 °C. After stirring, 1.04 g of EDCI and 5.88 g of HOBt were added. The reaction was carried out at 0-5 °C for 1 h, then the temperature was raised to room temperature and the reaction was continued for 20 h. The mixture was concentrated to obtain a pale yellow oily substance. The oily substance was dissolved in DMF and then added to 10 times the volume of purified water for crystallization. The crystallized solution was centrifuged, washed three times with purified water, and the solid was collected, filtered, and dried to obtain 5.38 g of the fully protected cyclic peptide methyl ester, with a yield of 91%.

[0082] Example 13: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OMe

[0083] Prepare 50 ml of frozen TFA / TIS / H2O = 95% / 2.5% / 2.5% lysis buffer and add it to a 100 ml round-bottom flask. Maintain the temperature ≤5℃. Weigh 5.0 g of the fully protected cyclic peptide methyl ester from Example 11 and add it to the above lysis buffer. React at room temperature for 2 h. Filter, add the filtrate to 500 ml of cold isopropyl ether for precipitation, centrifuge and wash to obtain 3.46 g of white solid, yield 109%.

[0084] Example 14: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OMe 2

[0085] Prepare 50 ml of frozen TFA / TIS / H2O = 90% / 5% / 5% lysis buffer and add it to a 100 ml round-bottom flask. Maintain the temperature ≤5°C. Weigh 5.0 g of the fully protected cyclic peptide methyl ester from Example 12 and add it to the above lysis buffer. React at room temperature for 2 h. Filter, add the filtrate to 500 ml of cold isopropyl ether for precipitation, centrifuge and wash to obtain 3.52 g of white solid, yield 111%.

[0086] Example 15: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH

[0087] Take 3.4g of cyclic peptide methyl ester from Example 13 and add it to 70ml of 20% acetonitrile / water. Stir to dissolve and clarify. Add 0.39g of LiOH and react at room temperature for 1h. The reaction is complete and proceed directly to the next step of purification.

[0088] Example 16: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH 2

[0089] Take 3.5g of cyclic peptide methyl ester from Example 14 and add it to 70ml of 20% ethanol / water. Stir to dissolve and clarify. Add 0.65g of NaOH and react at room temperature for 1h. The reaction is complete and proceed directly to the next step of purification.

[0090] Example 17: Purification of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH

[0091] The cyclic peptide solution from Example 15 was diluted 2-fold with water, filtered through a 0.45 μm microporous membrane, and purified in one step with a 50 mM sodium sulfate / acetonitrile solution. It was then converted to 50 mM ammonium acetate and eluted with a gradient of 0.1% acetic acid / acetonitrile. Fractions with a purity greater than 99% were combined, concentrated, and lyophilized to obtain 1.68 g of bumenopeptide acetate, with a total yield of 50.9% and a purity of 99.8%. The HPLC chromatogram of the obtained bumenopeptide is shown below. Figure 1 .

[0092] Example 18: Purification of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH

[0093] The cyclic peptide solution from Example 16 was diluted 2-fold with water, filtered through a 0.45 μm microporous membrane, and purified in one step with 80 mM sodium sulfate / acetonitrile solution. It was then converted to 80 mM ammonium acetate and eluted with a gradient of 0.5% acetic acid / acetonitrile. Fractions with a purity greater than 99% were combined, concentrated, and lyophilized to obtain 1.76 g of bumenopeptide acetate, with a total yield of 51% and a purity of 99.5%. The HPLC chromatogram of the obtained bumenopeptide is shown below. Figure 2 .

[0094] Example 19: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OEt 3

[0095] Following the steps of Examples 5, 7, 9, 11, and 13, Fmoc-Lys(Ac-Nle-Asp)-OEt (5.33 g, 8.0 mmol) from Example 3 was used to replace Fmoc-Lys(Ac-Nle-Asp)-OMe for reaction. After cleavage, cyclization, and deprotection, Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OBn 3.83 g was obtained.

[0096] Example 20: Purification of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH 3

[0097] Following the methods and steps of Examples 15 and 17, the product of Example 19 was hydrolyzed and purified, and then lyophilized to obtain 1.71 g of bumenotide acetate, with a total yield of 49% and a purity of 99.2%. The HPLC chromatogram of the obtained bumenotide peptide is consistent with... Figure 2 similar.

[0098] Example 21: Synthesis of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OBn 4

[0099] Following the steps of Examples 5, 7, 9, 11, and 13, Fmoc-Lys(Ac-Nle-Asp)-OBn (5.83 g, 8.0 mmol) from Example 4 was used to replace Fmoc-Lys(Ac-Nle-Asp)-OMe for reaction. After cleavage, cyclization, and deprotection, Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OBn 3.96 g was obtained.

[0100] Example 22: Purification of Ac-Nle-Cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH 4

[0101] Following the methods and steps of Examples 15 and 17, the product of Example 21 was hydrolyzed, purified, and lyophilized to obtain 1.62 g of bumenotide acetate, with a total yield of 47% and a purity of 99.1%. The HPLC chromatogram of the obtained bumenotide peptide is consistent with... Figure 2 similar.

Claims

1. A method for preparing bumenopeptide, characterized in that, The main steps include the following: A fully protected linear bumeno peptide resin was synthesized, which was then cleaved, cyclized, and deprotected to obtain cyclic heptapeptide ester, which was then hydrolyzed to obtain crude bumeno peptide. The specific steps are as follows: Step 1): Synthesize the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y; Step 2): Fmoc-Trp(Boc)-OH is coupled to a solid-phase support resin by solid-phase synthesis to obtain Fmoc-Trp(Boc)-resin; Step 3): Take the Fmoc-Trp(Boc)-resin obtained in Step 2), and sequentially couple the corresponding protected amino acids or fragments and the tripeptide fragment synthesized in Step 1) under the presence of a condensing agent to obtain a fully protected linear bumeno peptide resin: Where X is selected from Trt or Boc; Y is selected from methyl ester, ethyl ester, or benzyl ester; Step 4): Take the linear bumeno peptide resin obtained in Step 3), and after cleavage, cyclization, and deprotection, obtain cyclic heptacapeptide methyl ester; Step 5): Take the cyclic heptapeptide methyl ester obtained in step 4), add an alkaline reagent, and hydrolyze the methyl ester in a reaction solvent to obtain crude bumenopeptide. Specifically, the tripeptide fragment Fmoc-Lys(Ac-Nle-Asp)-Y in step 1) is synthesized by a liquid-phase method, as follows: H-Asp-OtBu reacts with Ac-Nle-OSu to give Ac-Nle-Asp-OtBu, which is then reacted with HOSu to generate the active ester Ac-Nle-Asp(OSu)-OtBu; the active ester is condensed with Fmoc-Lys-Y to give Fmoc-Lys(Ac-Nle-Asp-OtBu)-Y; finally, the protecting group is removed to give Fmoc-Lys(Ac-Nle-Asp)-Y; or H-Asp-OtBu reacts with Boc-Nle-OSu to give Boc-Nle-Asp-OtBu, which is then reacted with HOSu to generate the active ester Boc-Nle-Asp(OSu)-OtBu. The active ester is condensed with Fmoc-Lys-Y to give Fmoc-Lys(Boc-Nle-Asp-OtBu)-Y. The protecting group is removed to give Fmoc-Lys(H-Nle-Asp)-Y, which is then reacted with acetic anhydride to give Fmoc-Lys(Ac-Nle-Asp)-Y.

2. The preparation method according to claim 1, characterized in that, The solid support resin in step 2) is selected from 2-CTC resin, and the resin substitution degree is 0.8-1.2 mmol / g.

3. The preparation method according to claim 1, characterized in that, The condensing agent in step 3) is one or more of DIC / HOBt, EDCI / HOBt, PyBOP / DIEA, TBTU / DIEA, and HBTU / DIEA.

4. The preparation method according to claim 1, characterized in that, The pyrolysis solution required for the pyrolysis is selected from a mixture of TFE and DCM, wherein TFE accounts for 20% to 50% of the volume of the mixture.

5. The preparation method according to claim 1, characterized in that, The cyclization conditions are as follows: cyclization is performed in an organic solvent using a condensing agent; wherein the organic solvent is DMF, DCM or THF, and the condensing agent is one of DIC / HOBt, EDCI / HOBt, PyBOP / DIEA, TBTU / DIEA, or HBTU / DIEA.

6. The preparation method according to claim 1, characterized in that, The deprotection reagent required is a mixture of TFA, TIS and H2O, with a volume ratio of TFA:TIS:H2O = 90~98:1~5:1~5.

7. The preparation method according to claim 6, characterized in that, The volume ratio of the deprotection reagent is TFA:TIS:H2O = 95:2.5:2.

5.

8. The preparation method according to claim 1, characterized in that, In step 5), the alkaline reagent is one of LiOH, NaOH, and KOH, and the reaction solvent is one of ethanol / water, methanol / water, and acetonitrile / water.

9. The preparation method according to claim 8, characterized in that, In step 5), the alkaline reagent is LiOH, and the reaction solvent is acetonitrile / water.

10. The preparation method according to any one of claims 1-9, characterized in that, The crude bumenotide obtained was further purified, converted to salt, concentrated, and freeze-dried to obtain bumenotide acetate.

11. The preparation method according to claim 10, characterized in that, The crude bumenotide was further purified by the following steps to obtain bumenotide acetate: the crude product was dissolved in water, filtered through a 0.45 μm filter membrane, purified in one step with a 10 mM-100 mM sodium sulfate / acetonitrile solution, then converted to salt with 10 mM-100 mM ammonium acetate, and eluted with a gradient of 0.05%~0.5% acetic acid / acetonitrile system. After combining the fractions with a purity greater than 99%, the product was concentrated and lyophilized to obtain bumenotide acetate.

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