CN110746486A

CN110746486A - Method for preparing bremer langdan by solid-liquid combination

Info

Publication number: CN110746486A
Application number: CN201910974828.XA
Authority: CN
Inventors: 贾军; 徐大平; 张国庆; 白俊才; 周游
Original assignee: Shanghai Angbo Biological Technology Co Ltd
Current assignee: Shanghai Angbo Biological Technology Co Ltd
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2020-02-04

Abstract

The invention relates to the field of polypeptide drug chemical synthesis, in particular to a method for preparing bremer langdan by solid-liquid combination, which comprises the following steps: s1, connecting Fmoc-Trp (Boc) -OH to a 2-CTC Resin carrier to synthesize a hexapeptide full-protection fragment; s2, under a liquid phase system, carrying out fragment condensation on the synthesized hexapeptide full-protection fragment and amino acid through a condensing agent to obtain heptapeptide methyl ester of a side chain belt protecting group; s3, fully protecting the heptapeptide methyl ester, cracking a side chain protecting group by using a cutting fluid, and drying to obtain a methyl ester ring; s4, dissolving the methyl ester cyclic peptide in ACN/H₂In O solution, the product is precipitated and filtered, and then MTBE orAnd (4) pulping with diethyl ether, washing the solid product, and drying to obtain a crude product of the Braimeer red. The method has the advantages of mild reaction conditions, simple and convenient operation, short production period, lower cost, small environmental pollution, convenience for large-scale production, wide application prospect and better economic and social values.

Description

Method for preparing bremer langdan by solid-liquid combination

Technical Field

The invention belongs to the field of polypeptide drug chemical synthesis, and relates to a method for preparing bremer langdan by solid-liquid combination.

Background

The peptide sequence of the Braimeidan PT 141 is as follows: Ac-Nle-Cyclo (-Asp-His-D-Phe-Arg-Trp-Lys) -OH, formula: C50H68N14O10, molecular weight 1025.18, and structural formula as follows:

the bremeralangdan PT 141, also called bumenotide, is a melanocortin receptor-4 (MC4r) agonist, regulates sexual desire and response by activating endogenous pathways in the brain, helps premenopausal women with low-activity sexual desire disorder to keep normal sexual desire, is a new drug developed by Palatin Technologies pharmaceutical companies in the United states for treating female sexual dysfunction, has completed clinical phase III research at present, and is approved by FDA on 6/21 th of 2019 to be on the market. Compared with flibanserin, the bremer langdan has great advantages, does not need to be drunk, has no adverse reactions such as hypotension, dizziness and the like, has only slight nausea reaction, takes effect quickly within 30 minutes, has the curative effect lasting for 8 hours, and does not approve any medicine for treating HSDD to be on the market in China at present, so the bremer langdan has wide market prospect.

At present, the domestic situation about the synthesis of the Braimeldan is approximately as follows: as reported in patent CN106589111A, the synthesis using 5+2 fragments uses Lys (IvDde) which is expensive and not easily available in large quantity, the use of side chain protecting group IvDde in hydrazine hydrate removal has high toxicity risk, difficult operation and difficult waste liquid treatment, great environmental pollution and the like, wherein Ac-Nle-Asp-OtBu in the 5+2 fragments requires a plurality of liquid phase synthesis steps and is not easy to obtain high-purity fragments, the overall design is complicated, the efficiency is low, the large-scale production is not facilitated, expensive Asp (O-2-Phipr) is adopted in patent CN101280005B, Lys (MMt) resin is subjected to selective deprotection to form ring, the yield is only about 17%, and the large-scale production cost is high.

In foreign original research company patents such as US6579968, US6794489, US7176279, US7235625, US7417027 and US7473760, Lys side chains in peptide sequences are gradually coupled with queen resin by using unconventional protecting groups such as Adpoc, Alloc and Mtt, Lys side chain special protecting groups are expensive in raw material price and not easily available in large quantities in the market, Lys side chain protecting groups (Adpoc, Alloc and Mtt) are selectively cleaved without cleaving polypeptides from the queen resin by using low-concentration TFA, Asp side chain protecting groups tBu, 2-Phipr resin are cyclized, polypeptide chains are cleaved from the queen resin by using high-concentration TFA to obtain crude product of bramer erysipan (PT114), the method has the disadvantages that removal of side chain amino protecting groups mtu is not clean and repeated, the removal of side chain protecting groups tBu requires the use of expensive tetrakis (triphenylphosphine) palladium, the operation is complicated, heavy metal palladium is easily attracted to the product, and low-concentration side chain protecting groups on the resin can not be completely cleaved by selectively detecting whether tBu protecting groups, and the whole polypeptide chain is easy to be cracked from the resin, which is not beneficial to controlling the reaction, thus causing the cost to be increased and being not beneficial to mass production, so that a method for preparing the bremer-langdan by combining a solid phase and a liquid phase is needed to improve the problems.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a method for preparing the bremer-wave pill by combining solid and liquid.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing the bremer langdan by solid-liquid combination comprises the following steps:

s1, connecting Fmoc-Trp (Boc) -OH to a 2-CTC Resin carrier, and synthesizing a hexapeptide full-protection fragment Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -OH according to the Fmoc solid-phase polypeptide synthesis principle and the peptide sequence;

s2, carrying out fragment condensation on the synthesized hexapeptide full-protection fragment and H-Lys (Boc) -OMe.HCl through a condensing agent in a liquid phase system, settling by pure water, filtering and drying to obtain heptapeptide methyl ester Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Boc) -OMe with a protecting group at a side chain;

s3, cleaving the side chain protecting group by using a cleavage solution, adding the reaction solution into frozen MTBE or ether after cleavage is completed, separating out a solid, filtering and collecting the solid, drying to obtain linear heptapeptide methyl ester, dissolving the linear heptapeptide methyl ester in a DMF/THF solution, cyclizing an Asp side chain carboxyl and a Lys side chain amino in the sequence by using a condensing agent, extruding into the frozen MTBE or ether, separating out the solid, filtering and collecting the solid, and drying to obtain methyl ester cyclopeptide Ac-Nle-Cyclo (-Asp-His-D-Phe-Arg-Trp-Lys) -OMe;

s4, dissolving the methyl ester cyclic peptide in ACN/H₂And adding DBU into the O solution, introducing N2 for protection, maintaining the reaction solution at a proper temperature, hydrolyzing methyl ester, standing the reaction solution for a while, separating a lower layer solution, adding frozen MTBE or diethyl ether into the lower layer solution for pulping, separating out a product, filtering the product, pulping and washing the solid product by using the MTBE or the diethyl ether, filtering and collecting the solid product, and drying the solid product to obtain the crude product of the Brahman red.

Preferably, the S1, Fmoc-Trp (Boc) -OH is linked to the 2-CTC Resin carrier by DIPEA action, and the prepared Fmoc-Leu-CTC Resin has a degree of substitution ranging from 0.30 mmol/g to 1.00mmol/g, preferably from 0.50mmol/g to 0.90 mmol/g.

Preferably, in S1, the protected amino acids used in the synthesis of the fully protected hexapeptide fragment are as follows: Fmoc-Arg (Pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Nle-OH, wherein the molar ratio of amino acid feeding is 1.5-6 times, preferably 1.5-2.5 times when the fully-protected hexapeptide is synthesized.

Preferably, at S2, the condensing agent may be selected from any one of the following combinations: DIC/Y, EDC.HCl/Y, HBTU/Y/Z, HATU/Y/Z, HCTU/Y/Z, TBTU/Y/Z, PyBOP/Y/Z, PyAOP/Y/Z; wherein Y is HOBt or HOAt or Cl-HOBt, Z is DIPEA or NMM or TMP, preferably DIC/HOBt or HBTU/Cl-HOBt/DIPEA or HBTU/HOBt/DIPEA or HATU/HOAt/TMP or PyBOP/NMM or HATU/DIPEA.

Preferably, when the S2 hexapeptide full-protection fragment is condensed with the amino acid H-Lys (Boc) -OMe.HCl, the feeding molar ratio of the full-protection hexapeptide to the amino acid is 1-3 times, and preferably 1-1.5 times; the reaction pH value is 6-10, and the pH value is preferably 7-9; the amount of pure water used in the sedimentation is 6-12 times of the volume of the reaction solution, and preferably 8-10 times.

Preferably, in S3, the cleavage agent used in the cleavage is a mixture of trifluoroacetic acid and water, a mixture of trifluoroacetic acid and thioanisole, water, EDT, triisopropylsilane, or a mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane, and when the side chain protecting group of the fully protected heptapeptide is cleaved with a cleavage solution, a mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane is preferably selected, and the ratio is 92.5%: 2.5%: 2.5%: 2.5%.

Preferably, in the S3, the concentration of DMF is 40-99%, 40% is preferred, the reaction temperature is 15-25 ℃, 25 ℃ is preferred, the reaction time is 10-45 h, and the amount of MTBE or diethyl ether used for sedimentation is 6-12 times of the volume of the filtrate, 8-10 times of the volume of the filtrate is preferred.

Preferably, the molar weight of S4, DBU and methyl cyclic peptide is 5-10: 1, preferably 5-6: 1, wherein the reaction system is ACN/H₂The concentration of ACN in the O solution is 70-99%, the concentration of ACN is preferably 80%, the reaction temperature during hydrolysis is 0-20 ℃, the reaction time is 0-5 ℃, the reaction time is 2-10 hours, and the amount of MTBE or diethyl ether used for sedimentation is 5-12 times of the volume of the filtrate, and the amount of MTBE or diethyl ether used is preferably 5-7 times.

Compared with the prior art, the invention provides a method for preparing the bremer wave pellet by combining solid and liquid, which has the following beneficial effects:

1. the invention uses a solid-liquid combined method to carry out 6+1 fragment condensation, has simple operation process, high purity and yield of crude products and little environmental pollution, is convenient to improve the downstream purification yield and the final purity of products, and has application value of large-scale production.

2. The purity of the crude product synthesized by the method is more than or equal to 70 percent, the synthesis yield is more than or equal to 70 percent, the reaction condition is mild, the operation is simple and convenient, the production period is short, the cost is lower, the environmental pollution is small, the large-scale production is convenient, and the method has wide application prospect and better economic and social values.

Drawings

FIG. 1 is a process flow diagram of a method for preparing the Braimei pellet by solid-liquid combination according to the invention;

FIG. 2 is a HPLC diagram of a crude product of the Bramerlandine (PT114) in the method for preparing the Bramerlandine by solid-liquid combination of the invention: retention time RT is 15.743min, purity 70%;

FIG. 3 is a diagram of crude ESI-MS of the Bramerlandan (PT114) in the method for preparing the Bramerlandan by solid-liquid combination of the invention: [ M + H ]]⁺＝1025.95,[M+2H]²⁺＝513.61；

Fig. 4 is an illustration of an abbreviation or full-name English method for preparing the Braimeidan by solid-liquid combination according to the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-4, a method for preparing bremer langdan by solid-liquid combination comprises the following steps: s1, connecting Fmoc-Trp (Boc) -OH to a 2-CTC Resin carrier, and synthesizing a hexapeptide full-protection fragment Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -OH according to the Fmoc solid-phase polypeptide synthesis principle and the peptide sequence;

And the S1, Fmoc-Trp (Boc) -OH is connected to a 2-CTC Resin carrier through DIPEA, and the substitution degree of the prepared Fmoc-Leu-CTC Resin ranges from 0.30 mmol/g to 1.00mmol/g, and preferably ranges from 0.50mmol/g to 0.90 mmol/g.

S1, the protected amino acids used in the synthesis of the fully protected hexapeptide fragment were as follows: Fmoc-Arg (Pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Nle-OH, wherein the molar ratio of amino acid feeding is 1.5-6 times, preferably 1.5-2.5 times when the fully-protected hexapeptide is synthesized.

The S2, the condensing agent, may be any one of the following combinations: DIC/Y, EDC.HCl/Y, HBTU/Y/Z, HATU/Y/Z, HCTU/Y/Z, TBTU/Y/Z, PyBOP/Y/Z, PyAOP/Y/Z; wherein Y is HOBt or HOAt or Cl-HOBt, Z is DIPEA or NMM or TMP, preferably DIC/HOBt or HBTU/Cl-HOBt/DIPEA or HBTU/HOBt/DIPEA or HATU/HOAt/TMP or PyBOP/NMM or HATU/DIPEA.

When the S2 hexapeptide full-protection fragment is condensed with amino acid H-Lys (Boc) -OMe.HCl, the feeding molar ratio of the full-protection hexapeptide to the amino acid is 1-3 times, and 1-1.5 times is preferred; the reaction pH value is 6-10, and the pH value is preferably 7-9; the amount of pure water used in the sedimentation is 6-12 times of the volume of the reaction solution, and preferably 8-10 times.

In S3, the cleavage agent used in the cleavage is a mixture of trifluoroacetic acid and water, a mixture of trifluoroacetic acid and thioanisole, water, EDT, triisopropylsilane, or a mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane, and when the side chain protecting group of the fully protected heptapeptide is cleaved with a cleavage solution, a mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane is preferably selected, and the ratio is 92.5%: 2.5%: 2.5%: 2.5%.

And in the S3, the concentration of DMF is 40-99%, 40% is preferred, the reaction temperature is 15-25 ℃, 25 ℃ is preferred, the reaction time is 10-45 h, and the amount of MTBE or diethyl ether used for sedimentation is 6-12 times of the volume of the filtrate, 8-10 times of the volume of the filtrate is preferred.

And the molar weight of the DBU and the methyl ester cyclic peptide of S4 is 5-10: 1, preferably 5-6: 1, wherein the reaction system is ACN/H₂The concentration of ACN in the O solution is 70-99%, the concentration of ACN is preferably 80%, the reaction temperature during hydrolysis is 0-20 ℃, the reaction time is 0-5 ℃, the reaction time is 2-10 hours, and the amount of MTBE or diethyl ether used for sedimentation is 5-12 times of the volume of the filtrate, and the amount of MTBE or diethyl ether used is preferably 5-7 times.

The specific implementation mode is as follows:

example 1

1. Synthesis of fully protected hexapeptide Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -OH

30.0g of 2-CTC Resin (1.04mmol/g) is weighed in a solid phase polypeptide synthesis reactor, adding DCM to swell for 30min, pumping out, weighing 19.716g of Fmoc-Trp (Boc) -OH, adding about 120ml of DMF to dissolve, cooling in ice water bath, adding 5.434ml of IPEA to activate for 10min, adding the amino acid activation solution into a reaction column to react, adding 3.804ml of IPEA into the reactor after 5min, continuing to react for about 1h, after the reaction is finished, sequentially adding DMF, MTBE and DMF, alternately washing for 2 times, pouring prepared blocking solution DCM/MeOH/DIPEA (17/2/1 (V/V/V)) into resin to block unreacted functional groups, blocking twice for 10min each time, adding DMF after blocking is finished, washing, the resin was then shrunk with MTBE and transferred to a vacuum oven to dry to constant weight, and the degree of substitution was determined to be 0.54 mmol/g.

37.04g (20mmol) of Fmoc-Trp (Boc) -CTC Resin is weighed into a polypeptide synthesis reactor, DCM is added for swelling for 30min, the solution is pumped out, and a proper amount of 5% PIP/2% DBU/1% HOBt/DMF solution is added for deprotection twice, wherein the deprotection time is 10min and 20min respectively. Adding DMF, MeOH and DMF for alternately washing for 2 rounds, weighing 18.74g of Fmoc-Arg (Pbf) -OH and 4.06g of HOBt in a beaker, adding DMF for dissolving, then adding 5.24 g of LDIPEA, cooling to 0-5 ℃ under the condition of ice-water bath, finally adding 11.38g of HBTU for activation for about 10min, pouring the activation solution into a reactor for reaction, detecting and judging whether condensation is complete by ninhydrin, after the reaction is finished, carrying out next condensation of amino acid according to deprotection, washing and condensation cycles, carrying out acetylation on the condensed protected amino acid sequentially comprising Fmoc-D-Phe-OH, Fmoc-His Trt) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Nle-OH, removing the Fmoc protecting group at the N terminal after the coupling is finished, detecting and judging whether condensation is complete by ninhydrin, adding DMF, MeOH and DMF for alternately washing for 2 rounds, the resin was washed 4 more times with DCM and dried to give 59.76g, yield: 95.07 percent

59.76g of the fragment (1-6) peptide resin obtained above was added to 600mL of 1% TFA/DCM (V/V) solution and stirred for 10min, the filtrate was taken up in a suction flask containing an appropriate amount of pyridine and repeated three times, the filtrates were combined and concentrated, the filtrate was added to an appropriate amount of pure water, a white solid precipitated, the solid was filtered off and dried under vacuum to constant weight to give 26.98g of a solid, and ESI-MS was determined: [ M + H ]]⁺＝1565.90。

2.6+1 fragment condensation

Weighing 5.22g of amino acid H-Lys (Boc) -OMe.HCl, dissolving in 20ml of DMF, stirring until the amino acid H-Lys (Boc) is dissolved, adding DIPEA to adjust the pH value to 7, weighing 25.08g of hexapeptide full-protection peptide, 2.85gCl-HOBt, dissolving in a beaker by using 100ml of DMF, adding 8.36ml of LDIPEA, cooling to-5-0 ℃ by using a salt-freezing bath, adding 6.67g of HBTU for activation for about 10min, adding the mixture into the amino acid solution, reacting at-5-0 ℃ for 3-4H, controlling the reaction pH value to 7, monitoring the reaction process by using HPLC, after the reaction is finished, adding the reaction solution into 1.2L of ice water cooled in advance, precipitating white solid, filtering, washing the solid for 3-4 times by using pure water, drying the obtained solid to constant weight in vacuum, weighing 27.78g of the obtained solid, and measuring ESI-MS: [ M + H ]]⁺＝1808.21。

3. Removing side chain protection to obtain linear heptapeptide methyl ester, and performing side chain cyclization by using a condensing agent liquid phase system

25g of the fully protected heptapeptide Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Boc) -OMe obtained above was dissolved in 250mL of 92.5% TFA/2.5% TIS/2.5% H2O/2.5% MPS solution, stirred to be fully dissolved and the temperature of the solution was controlled atAnd (2) splitting at 25 ℃, after the reaction is finished, adding the reaction solution into 1.95L of frozen MTBE, separating out a solid, filtering, washing the solid for 3-4 times by using the MTBE, transferring the solid into a vacuum drier, drying to constant weight, weighing the obtained linear heptapeptide methyl ester solid to be 14.1g, wherein the molecular weight of the solid containing 4 TFA salts is as follows: 1513.22, ESI-MS: [ M + H ]]⁺＝1057.22。

Dissolving 12g of the linear heptapeptide methyl ester Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-OMe obtained above in 360mL of 40% DMF/THF solution, adding 8.754mL of NMM to adjust the pH and stirring for 10min, weighing 16.525g of PyBOP in a round-bottomed flask and dissolving with 1800mL of 40% DMF/THF solution, adding 5.574mLNMM to stir uniformly, slowly dripping the linear heptapeptide methyl ester solution into the round-bottomed flask solution of PyBOP/NMM by using a constant pressure funnel, dripping for 4h, controlling the reaction by HPLC, forming a loop bundle after reacting for 45h, evaporating THF in reduced pressure at the temperature of 32 ℃ or less, adding into 6.8L of frozen MTBE, precipitating and filtering the solid, washing the solid for 3-4 times by using MTBE, drying to constant weight in vacuum, weighing 9.83g of the obtained solid, measuring ESI-MS: [ M + H ]]⁺＝1039.21

4. Hydrolysis of cyclic heptapeptide methyl ester

9.5g of the above-obtained methyl cyclopentadienylester Ac-Nle-Cyclo (-Asp-His-D-Phe-Arg-Trp-Lys) -OMe were dissolved in 285mL of 80% ACN/H₂Stirring the mixture in an O solution until the mixture is completely dissolved, cooling the mixture to 0-5 ℃ in an ice water bath, adding 5.7mL of DBU into a cyclic heptapeptide methyl ester solution, protecting the mixture with N2 for reaction, maintaining the reaction temperature at 0-5 ℃, reacting for 4-6h, monitoring the reaction process through HPLC, standing the solution for 10min after the reaction is finished, layering, adding 720mL of frozen MTBE into a lower layer solution to separate out a solid, pulping the MTBE for 3-4 times, washing the solid for 3-4 times, filtering, collecting the solid, drying the solid in a vacuum drier to constant weight, weighing the obtained solid to 8.585g, and measuring ESI-MS: [ M + H ]]⁺The crude HPLC purity was 72% with 91.4% yield as 1025.95.

Example 2

30.0g of 2-CTC Resin (1.04mmol/g) is weighed in a solid phase polypeptide synthesis reactor, adding DCM to swell for 30min, pumping out, weighing 24.64g of Fmoc-Trp (Boc) -OH, adding about 120ml of DMF to dissolve, cooling in ice-water bath, adding 9.238ml of IPEA to activate for 10min, adding the amino acid activation solution into a reaction column to react, adding 7.065ml of IPEA into the reactor after 5min, continuing to react for about 1h, after the reaction is finished, sequentially adding DMF, MTBE and DMF, alternately washing for 2 times, pouring prepared blocking solution DCM/MeOH/DIPEA (17/2/1 (V/V/V)) into resin to block unreacted functional groups, blocking twice for 10min each time, adding DMF after blocking is finished, washing, the resin was then shrunk with MTBE and transferred to a vacuum oven to dry to constant weight, and the degree of substitution was determined to be 0.76 mmol/g.

26.315g (20mmol) of Fmoc-Trp (Boc) -CTC Resin was weighed into a polypeptide synthesis reactor, added with DCM to swell for 30min, pumped out, added with an appropriate amount of 5% PIP/2% DBU/1% HOBt/DMF solution to deprotect twice, 10min and 20min respectively. Adding DMF, MeOH and DMF for washing for 2 rounds alternately, weighing 25.92g of Fmoc-Arg (Pbf) -OH and 5.41g of HOBt in a beaker, adding DMF for dissolving, then adding 6.194 g of LDIC for cooling to 0-5 ℃ under the condition of ice-water bath for activation for about 3min, pouring the activation solution into a reactor for reaction, detecting whether condensation is complete by ninhydrin, after the reaction is finished, carrying out condensation of amino acid in the next step according to deprotection, washing and condensation cycles, wherein the condensed protected amino acids are Fmoc-D-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Nle-OH, removing the Fmoc protecting group at the N terminal after coupling is finished, acetylating by 20.7mL of acetic anhydride, 17.26mL of pyridine and DMFF172.56mL, detecting whether condensation is complete by ninhydrin, adding DMF, washing for 2 rounds alternately, washing for 4 times by MeOH, the resin was dried to give 48.55g, yield: 93 percent.

46.78g of the fragment (1-6) peptide resin obtained above was added to 470mL of 1% TFA/DCM (V/V) solution and stirred for 10min, the filtrate was taken up in a filtration flask containing an appropriate amount of pyridine, repeated three times, the filtrates were combined and concentrated, the filtrate was added to an appropriate amount of pure water, a white solid precipitated, the solid was filtered off and dried to constant weight in vacuo to give 25.8g of a solid, and ESI-MS: [ M + H ]]⁺＝1565.90。

2.6+1 fragment condensation

2.61g of the amino acid H-Lys (Boc) -OMe.HCl are weighed out and dissolved in 12ml DMF and stirred until dissolvedDissolving, adding TMP to adjust the pH value to 7, weighing 12.525g of hexapeptide full-protection peptide, dissolving 1.15g of HOAt in a beaker by 88mL of DMF, adding 3.19mL of TMP, cooling to-5-0 ℃ by using a ice salt bath, adding 3.495g of HATU for activating for about 10min, adding the mixture into an amino acid solution, reacting for 3-4 h at-5-0 ℃, controlling the pH value of the reaction to be 7, monitoring the reaction process by using HPLC (high performance liquid chromatography), adding the reaction solution into 800mL of ice water cooled in advance after the reaction is finished, separating out white solid, filtering, washing the solid by using pure water for 3-4 times, drying the obtained solid to constant weight in vacuum, weighing the obtained solid to be 14.29g, and measuring ESI-MS: [ M + H ]]⁺＝1808.21。

3. Removing side chain protection to obtain linear heptapeptide methyl ester, dissolving 14g of the obtained fully-protected heptapeptide Ac-Nle-Asp (OtBu) -His (Trt) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Boc) -OMe in 140mL of 92.5% TFA/2.5% TIS/2.5% H2O/2.5% MPS solution by using a condensing agent liquid phase system for side chain cyclization, stirring until the solution is completely dissolved, controlling the solution temperature to crack at 25 ℃, adding the reaction solution into 1.95L of frozen MTBE after the reaction is finished, precipitating solid and filtering, washing the solid for 3-4 times by using the MTBE, transferring the solid to a vacuum drier for drying until the solid reaches a constant weight, weighing the obtained linear heptapeptide methyl ester to be 7.78g, and weighing the molecular weight of 4 TFA salts: 1513.22, ESI-MS: [ M + H ]]⁺＝1057.22。

7.5g of the linear heptapeptide methyl ester Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-OMe obtained above were dissolved in 225mL of 40% DMF/THF solution, adding 8.625mL of DIPEA to adjust the pH, stirring for 5-10 min, weighing 11.25g of HATU in a round-bottomed flask, dissolving with 1125mL of 40% DMF/THF solution, adding 5.175mL of DIPEA, stirring uniformly, slowly dripping a linear heptapeptide methyl ester solution into the HATU/DIPEA round-bottomed flask solution by using a constant pressure dropping funnel, dropwise adding for 4h, performing HPLC (high performance liquid chromatography) central control on the reaction, performing cyclization after 12h, performing rotary evaporation on THF (tetrahydrofuran) in the reaction solution at the temperature of less than or equal to 32 ℃ under reduced pressure, adding into 5.5L of frozen MTBE, separating out a solid, filtering, washing the solid with MTBE for 3-4 times, drying in vacuum to constant weight, weighing the obtained solid to be 5.96g, and measuring ESI-MS: [ M + H ]]⁺＝1039.21。

4. Hydrolysis of cyclic heptapeptide methyl ester

5.9g of the above-obtained cyclic heptapeptide methyl ester Ac-Nle-Cyclo (-Asp-His-D)-Phe-Arg-Trp-Lys) -OMe dissolved in 120mL 80% ACN/H₂Stirring to completely dissolve O solution, cooling to 0-5 ℃ in ice water bath, adding 3.54mL of DBU into a cyclic heptapeptide methyl ester solution, protecting the reaction with N2, maintaining the reaction temperature at 0-5 ℃, reacting for 4-6h, monitoring the reaction process through HPLC, standing the solution for 10min after the reaction is finished, layering, adding 1.7L of frozen MTBE into a lower layer solution to separate out a solid, pulping with MTBE, washing the solid for 3-4 times, filtering, collecting the solid, drying the solid in a vacuum drier to constant weight, weighing the obtained solid to 5.25g, and measuring ESI-MS: [ M + H ]]⁺The crude HPLC purity was 71.3% with a yield of 90.1% as 1025.95.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The method for preparing the bremer langdan by solid-liquid combination is characterized by comprising the following steps:

2. The method for preparing the bremer-wave pill by solid-liquid combination according to claim 1, wherein the S1, Fmoc-Trp (Boc) -OH is connected to the 2-CTCResin carrier by DIPEA, and the substitution degree of the prepared Fmoc-Leu-CTC resin is in the range of 0.30-1.00 mmol/g, preferably 0.50-0.90 mmol/g.

3. The method for preparing the bremer langdan according to claim 1, wherein the protected amino acids used for synthesizing the fully protected hexapeptide fragment of S1 are as follows: Fmoc-Arg (Pbf) -OH, Fmoc-D-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Nle-OH, wherein the molar ratio of amino acid feeding is 1.5-6 times, preferably 1.5-2.5 times when the fully-protected hexapeptide is synthesized.

4. The method for preparing the brameral in a solid-liquid combination manner according to claim 1, wherein the S2 condensing agent can be any one of the following combinations: DIC/Y, EDC.HCl/Y, HBTU/Y/Z, HATU/Y/Z, HCTU/Y/Z, TBTU/Y/Z, PyBOP/Y/Z, PyAOP/Y/Z; wherein Y is HOBt or HOAt or Cl-HOBt, Z is DIPEA or NMM or TMP, preferably DIC/HOBt or HBTU/Cl-HOBt/DIPEA or HBTU/HOBt/DIPEA or HATU/HOAt/TMP or PyBOP/NMM or HATU/DIPEA.

5. The method for preparing the bremer-wave pill by solid-liquid combination according to claim 1, wherein when the S2 hexapeptide full-protection fragment is condensed with the amino acid H-Lys (Boc) -OMe.HCl, the feeding molar ratio of the full-protection hexapeptide to the amino acid is 1-3 times, preferably 1-1.5 times; the reaction pH value is 6-10, and the pH value is preferably 7-9; the amount of pure water used in the sedimentation is 6-12 times of the volume of the reaction solution, and preferably 8-10 times.

6. The method of claim 1, wherein the cleavage agent used in the cleavage at S3 is a mixture of trifluoroacetic acid and water, a mixture of trifluoroacetic acid and thioanisole, water, EDT, triisopropylsilane, or a mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane, and when the side chain protecting group of the fully protected heptapeptide is cleaved with the cleavage solution, the mixture of trifluoroacetic acid and thioanisole, water, DODT, triisopropylsilane is preferably selected, and the ratio is 92.5%: 2.5%: 2.5%: 2.5%.

7. The method for preparing the bremerrand by solid-liquid combination according to claim 1, wherein the concentration of DMF in S3 is 40-99%, 40% is preferred, the reaction temperature is 15-25 ℃, 25 ℃ is preferred, the reaction time is 10-45 h, and the amount of MTBE or diethyl ether used for sedimentation is 6-12 times, 8-10 times of the volume of the filtrate.

8. The method for preparing the brameran in a solid-liquid combination manner according to claim 1, wherein the molar amount of S4, DBU and methyl cyclic peptide is 5-10: 1, preferably 5-6: 1, wherein the reaction system is ACN/H₂The concentration of ACN in the O solution is 70-99%, the concentration of ACN is preferably 80%, the reaction temperature during hydrolysis is 0-20 ℃, the reaction time is 0-5 ℃, the reaction time is 2-10 hours, and the amount of MTBE or diethyl ether used for sedimentation is 5-12 times of the volume of the filtrate, and the amount of MTBE or diethyl ether used is preferably 5-7 times.