WO2021109297A1 - 一种依特卡肽中间体及依特卡肽的合成方法 - Google Patents

一种依特卡肽中间体及依特卡肽的合成方法 Download PDF

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WO2021109297A1
WO2021109297A1 PCT/CN2019/129440 CN2019129440W WO2021109297A1 WO 2021109297 A1 WO2021109297 A1 WO 2021109297A1 CN 2019129440 W CN2019129440 W CN 2019129440W WO 2021109297 A1 WO2021109297 A1 WO 2021109297A1
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cys
otbu
boc
fmoc
arg
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French (fr)
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李九远
马修•约翰逊
李常峰
荆禄涛
巴拉苏布拉马尼安•阿鲁穆加姆
雷小龙
朱自力
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凯莱英医药集团（天津）股份有限公司
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Priority to EP19955221.7A priority Critical patent/EP4071135A4/en
Priority to US17/782,200 priority patent/US20230021514A1/en
Priority to KR1020227022643A priority patent/KR20220120589A/ko
Priority to JP2022533637A priority patent/JP2023504848A/ja
Publication of WO2021109297A1 publication Critical patent/WO2021109297A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/70—Sulfur atoms
    • C07D213/71—Sulfur atoms to which a second hetero atom is attached
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C313/00—Sulfinic acids; Sulfenic acids; Halides, esters or anhydrides thereof; Amides of sulfinic or sulfenic acids, i.e. compounds having singly-bound oxygen atoms of sulfinic or sulfenic groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C313/08—Sulfenic acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/22—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/22—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides
    • C07C319/24—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides by reactions involving the formation of sulfur-to-sulfur bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the technical field of chemical synthesis, in particular, to a synthetic method of etecartide intermediates and etecartide.
  • Etecartide is a novel calcimimetic developed by Kai Pharmaceuticals, Inc., which can inhibit the secretion of parathyroid hormone. Etecartide can bind to and activate the calcium-sensitive receptors on the parathyroid glands to reduce the level of parathyroid hormone.
  • Etecartide has 3 D-configuration arginines, 2 D-configuration alanines, 1 D-configuration arginine amide, 1 L-configuration cysteine and 1 D-configuration Cysteine (the N-terminus is blocked by an acetyl group) is composed of cysteine in the D configuration and the cysteine in the L configuration by a disulfide bond (N-acetyl-D-cysteinyl-D-alanyl- D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-Argininamide, disulfide with L-cysteine).
  • Patent 2017/114238 A1 The liquid phase synthesis method adopted, the route is longer, and the total yield is only 11%; Patent WO 2017/114240 A1: According to the patent, the purity of the crude peptide is 81.0%, and there are many impurities. The total yield after purification The rate is 30.5%; Patent US 2019/0100554 A1: According to the patent, the purity of the crude peptide is low and requires multiple purifications, and the total yield is 50%; in the Chinese patent CN201811277081, the purity of the crude peptide is 88.2%, and the total yield after purification is 57.8%. In other words, the current solid-phase synthesis of 7 peptides connected to cysteine and derivatives has problems such as low purity of crude peptides, many impurities, and difficulty in purification.
  • the present invention aims to provide an etecartide intermediate and a method for synthesizing itecartide.
  • the intermediate is used to synthesize itecartide, so as to solve the technical problem of complex synthesis steps of etecartide in the prior art.
  • a method for synthesizing an intermediate of etacatide is provided.
  • the etercartide intermediate is Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and its structural formula I is shown in:
  • the synthetic method of etacatide intermediate includes the following steps: starting with N-Boc-L-Cys-OtBu, the primary product is generated through substitution reaction Wherein, R is S-Py or Cl, and the primary product is coupled with Fmoc-D-Cys-OH amino acid to obtain Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • the primary product is Py-SS-(N-Boc)-L-Cys-OtBu
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps :
  • the primary product Py-SS-(N-Boc)-L-Cys-OtBu was synthesized by substitution reaction of N-Boc-L-Cys-OtBu and disulfide dipyridine; and Py-SS-(N-Boc)- L-Cys-OtBu is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(SS-(N-Boc)-L-Cys-OtBu)-OH.
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH was prepared by the following steps: at room temperature, N-Boc-L-Cys-OtBu and dithiodipyridine Add to solvent A, stir for 6-12h, then add water, extract with extractant, dry and filter the obtained organic phase, and obtain Py-SS-(N-Boc)-L-Cys-OtBu after purification; solvent B Fmoc-D-Cys-OH and Py-SS-(N-Boc)-L-Cys-OtBu were added to the mixture, the temperature was controlled at 15 ⁇ 30°C, and the reaction was stirred for 0.5 ⁇ 2h.
  • reaction system was washed, concentrated and purified to obtain Fmoc- D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH; preferably, solvent A is selected from one or more of DMF, NMP and DMAc; preferably, the extractant is selected from EtOAc One or more of MTBE and DCM; preferably, solvent B is selected from one or more of DCM, DMF, THF, NMP and DMAc.
  • the molar ratio of N-Boc-L-Cys-OtBu to dithiodipyridine is 1:1.2 to 1:6.4; N-Boc- The concentration of L-Cys-OtBu in solvent A is 0.01 ⁇ 0.3g/mL; the concentration of Fmoc-D-Cys-OH in solvent B is 0.01 ⁇ 0.3g/mL; Fmoc-D-Cys-OH and Py-SS The molar ratio of -(N-Boc)-L-Cys-OtBu is 1:0.8 to 1.4.
  • the primary product is (N-Boc)-L-Cys(S-Cl)-OtBu
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps Obtained: (N-Boc)-L-Cys(S-Cl)-OtBu was synthesized by reacting N-Boc-L-Cys-OtBu with NCS; and (N-Boc)-L-Cys(S-Cl)- OtBu reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH was prepared by the following steps: A. Dissolving N-Boc-L-Cys-OtBu in solvent C, Control the temperature at 0 ⁇ 10°C, add DIPEA, add NCS in batches, stir for 4 ⁇ 5h, after the reaction, filter and rinse to obtain the filtrate; B.
  • the solvent C is selected from one or more of DCM, THF, DMF NMP and DMAc.
  • step A N-Boc-L-Cys-OtBu is dissolved in solvent C to obtain a solution with a concentration of 0.01 to 0.3 g/mL, the amount of DIPEA added is 2 to 3 eq of moles, and the amount of NCS added is 1.1 ⁇ 1.5eq;
  • step B the amount of Fmoc-D-Cys-OH added is 1.1 ⁇ 1.5eq.
  • a method for synthesizing etacatide includes the following steps: S1, the synthesis method of any one of the above-mentioned etacitide intermediates to synthesize Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH; and S2 , Combine NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg with Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu)) -OH reaction, then remove Fmoc, and then undergo acetylation to obtain etecatide.
  • NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg is NH 2 -D-Ala-D-Arg-D-Arg-D- Arg-D-Ala-D-Arg-resin hexapeptide.
  • S2 includes: using amino resin to link NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide according to the method of solid phase synthesis, and Fmoc- D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH, PyBop and DIPEA are activated at 0 ⁇ 5°C for 0-10min, and the temperature is controlled at 20 ⁇ 30°C to react for 2 ⁇ 6h. After the reaction, use DMF was washed 4-6 times, 10%-20% piperidine was removed from Fmoc, and then acetylated to obtain the peptide resin of etercartide.
  • Figure 1 shows the purity profile of Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH in Example 1;
  • FIG. 3 shows the purity profile of itercatide after preparation and purification in Example 3.
  • Fmoc 9-fluorenylmethoxycarbonyl.
  • Boc tert-butylcarbonyl.
  • Cys Cysteine
  • PyBop benzotriazol-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate.
  • DIPEA N,N-diisopropylethylamine.
  • NCS N-chlorosuccinimide
  • NMP N-methylpyrrolidone
  • DMAc N,N-dimethylacetamide.
  • THF Tetrahydrofuran
  • the etecartide intermediate is Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu) )-OH
  • the synthetic method of etacitide intermediate includes the following steps: take N-Boc-L-Cys-OtBu as the starting material and generate the primary product through substitution reaction Wherein, R is S-Py or Cl, and the primary product is coupled with Fmoc-D-Cys-OH amino acid to obtain Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • the primary product is Py-SS-(N-Boc)-L-Cys-OtBu
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu)) -OH is prepared by the following steps: Py-SS-(N-Boc)-L-Cys-OtBu is synthesized by reacting N-Boc-L-Cys-OtBu with dithiodipyridine; and Py-SS-(N -Boc)-L-Cys-OtBu is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • synthetic The raw material of this intermediate is cheap and easy to obtain, and the process is simple.
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: at room temperature, (N-Boc)-L-Cys-OtBu and disulfide Dipyridine was added to solvent A, stirred for 6-12 hours, then added water, extracted with extractant, dried and filtered the obtained organic phase, and purified to obtain Py-SS-(N-Boc)-L-Cys-OtBu; Add Fmoc-D-Cys-OH and Py-SS-(N-Boc)-L-Cys-OtBu to solvent B, control the temperature at 15 ⁇ 30°C, stir and react for 0.5 ⁇ 2h, wash the reaction system with water, concentrate and purify it Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • the solvent A is selected from one or more of DMF, NMP and DMAc; preferably, the extraction agent is selected from one or more of EtOAc, MTBE and DCM; preferably, the solvent B is selected from DCM , DMF, THF, NMP and DMAc one or more.
  • the combination of N-Boc-L-Cys-OtBu and dithiodipyridine In order to improve the utilization of raw materials and ensure the rapid and effective progress of the reaction, it is further preferred that in the reaction between N-Boc-L-Cys-OtBu and dithiodipyridine, the combination of N-Boc-L-Cys-OtBu and dithiodipyridine
  • the molar ratio is 1:1.2 ⁇ 1:6.4, the concentration of (N-Boc)-L-Cys-OtBu in solvent A is 0.01 ⁇ 0.3g/mL; the concentration of Fmoc-D-Cys-OH in solvent B is 0.01 ⁇ 0.3g/mL; the molar ratio of Fmoc-D-Cys-OH to Py-SS-(N-Boc)-L-Cys-OtBu is 1:0.8 ⁇ 1.4.
  • the primary product is (N-Boc)-L-Cys(S-Cl)-OtBu
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu) )-OH is prepared by the following steps: (N-Boc)-L-Cys(S-Cl)-OtBu is synthesized by reacting N-Boc-L-Cys-OtBu with NCS; and (N-Boc)-L- Cys(S-Cl)-OtBu reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(SS-(N-Boc)-L-Cys(OtBu))-OH.
  • Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: A. Dissolving (N-Boc)-Cys-OtBu in solvent C, Control the temperature at 0 ⁇ 10°C, add DIPEA, add NCS in batches, stir for 4 ⁇ 5h, after the reaction, filter and rinse to obtain the filtrate; B.
  • the solvent C is selected from one or more of DCM, THF, DMF NMP and DMAc.
  • step A (N-Boc)-L-Cys-OtBu is dissolved in solvent C to obtain a solution with a concentration of 0.01 to 0.3 g/mL,
  • the added amount of DIPEA is 2 to 3 eq of moles, and the added amount of NCS is 1.1 to 1.5 eq;
  • step B the added amount of Fmoc-D-Cys-OH is 1.1 to 1.5 eq.
  • a method for synthesizing etacatide includes the following steps: S1, Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH is synthesized according to the synthesis method of any of the aforementioned etka peptide intermediates; and S2 , Combine NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg with the Fmoc-D-Cys(SS-(N-Boc)-L-Cys(OtBu )) -OH reaction, and then remove Fmoc, and then undergo acetylation to obtain etercartide.
  • NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg is NH 2 -D-Ala-D-Arg-D-Arg-D- Arg-D-Ala-D-Arg-resin hexapeptide.
  • S2 includes: using amino resin to link NH 2 -D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide according to the method of solid phase synthesis, and Fmoc- D-Cys(SS-(N-Boc)-L-Cys(OtBu))-OH, PyBop and DIPEA are activated at 0 ⁇ 10°C for 0-10min, and the temperature is controlled at 20 ⁇ 30°C to react for 2 ⁇ 6h.
  • N-Boc-L-Cys-OtBu(Cpd 1) was synthesized by reaction with disulfide dipyridine; Py-SS- (N-Boc)-L-Cys-OtBu(Cpd 2a) is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(SS-(N-Boc)-L-Cys-OtBu)-OH( Cpd 3).
  • Cpd 1 (1.6 g, 1.0 eq) and 2,2-dithiodipyridne (5.1 g, 4.0 eq) were added to DMF (16 mL, 10 vol.).
  • the reaction was stirred at room temperature for 6-12 hours; then water was added to the system; extracted with ethyl acetate (100mL*3); the organic phases were combined and dried with MgSO 4 and filtered; the organic phases were concentrated to obtain crude Cpd 2a, which was purified by column chromatography to obtain pure Py-SS-(N-Boc)-L-Cys-OtBu(Cpd 2a).
  • Figure 1 shows the purity profile of Cpd 3 after purification
  • Figure 2 shows the LCMS profile of Cpd 3 after purification.
  • N-Boc-L-Cys-OtBu(Cpd 1) as a raw material, (N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b) is synthesized by reacting with NCS; (N-Boc)- L-Cys(S-Cl)-OtBu(Cpd 2b) reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys(SS-(N-Boc)-L-Cys-(OtBu))-OH(Cpd) 3).
  • Use amino resin including but not limited to Sieber resin, Rink Amide MBHA, Rink Amide resin

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Abstract

本发明公开了一种依特卡肽中间体及依特卡肽的合成方法。其中，该依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，依特卡肽中间体的合成方法包括以下步骤：以N-Boc-L-Cys-OtBu为起始原料，通过取代反应生成初级产物式（A)，其中，R为S-Py或Cl，初级产物与Fmoc-D-Cys-OH氨基酸进行偶联反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。该关键中间体用于合成依特卡肽可以提高纯度和收率，重要的是，合成此关键中间体的原料廉价易得，工艺简单。

Description

一种依特卡肽中间体及依特卡肽的合成方法 技术领域
本发明涉及化学合成技术领域，具体而言，涉及一种依特卡肽中间体及依特卡肽的合成方法。
背景技术
依特卡肽是由KaiPharmaceuticals,Inc.开发的一种新颖的拟钙剂，能够抑制甲状旁腺激素的分泌。依特卡肽可结合并激活甲状旁腺上的钙敏感受体，实现甲状旁腺激素水平的降低。
依特卡肽有3个D构型的精氨酸、2个D构型的丙氨酸、1个D构型的精氨酰胺、1个L构型半胱氨酸与1个D构型半胱氨酸(N端被乙酰基封闭)构成，其中D构型半胱氨酸与L构型半胱氨酸以二硫键连接在一起(N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-Argininamide,disulfidewithL-cysteine)。
目前报道的专利中，都是固相链接至7肽，然后再选择不同的方法链接二硫键，特别是在脱除MMT过程中，需要使用1％～2％TFA/DCM反复操作15次以上，操作相当繁琐。
专利2017/114238 A1：采用的液相合成方法，路线较长，且总收率仅11％；专利WO 2017/114240 A1：依照专利，粗肽纯度为81.0％,杂质较多，纯化后总收率30.5％；专利US 2019/0100554 A1：依照专利，粗肽纯度较低，需要多次纯化，总收率50％；中国专利CN201811277081中，粗肽纯度88.2％，纯化后总收率57.8％。也就是说，目前固相合成7肽连接半胱氨酸及衍生物这种方法，均存在粗肽纯度低，杂质多，难纯化等问题。
发明内容
本发明旨在提供一种依特卡肽中间体及依特卡肽的合成方法，该中间体用于合成伊特卡肽，以解决现有技术中依特卡肽合成步骤复杂的技术问题。
为了实现上述目的，根据本发明的一个方面，提供了一种依特卡肽中间体的合成方法。该依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，其结构式I所示：
Figure PCTCN2019129440-appb-000001
依特卡肽中间体的合成方法包括以下步骤：以N-Boc-L-Cys-OtBu为起始原料，通过取代反应生成初级产物
Figure PCTCN2019129440-appb-000002
其中，R为S-Py或Cl，初级产物与Fmoc-D-Cys-OH氨基酸进行偶联反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
进一步地，初级产物为Py-S-S-(N-Boc)-L-Cys-OtBu，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：将N-Boc-L-Cys-OtBu与二硫二吡啶取代反应合成得到初级产物Py-S-S-(N-Boc)-L-Cys-OtBu；以及将Py-S-S-(N-Boc)-L-Cys-OtBu与Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH。
进一步地，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：室温下，将N-Boc-L-Cys-OtBu和二硫二吡啶加入溶剂A中，搅拌6～12h，然后加入水，使用萃取剂萃取，对得到的有机相进行干燥及过滤，纯化后得到Py-S-S-(N-Boc)-L-Cys-OtBu；溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu，控温15～30℃搅拌反应0.5～2h，对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；优选的，溶剂A选自DMF、NMP和DMAc中的一种或多种；优选的，萃取剂选自EtOAc、MTBE和DCM中的一种或多种；优选的，溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
进一步地，N-Boc-L-Cys-OtBu与二硫二吡啶反应中，N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2～1:6.4；N-Boc-L-Cys-OtBu在溶剂A中的浓度为0.01～0.3g/mL；溶剂B中Fmoc-D-Cys-OH的浓度为0.01～0.3g/mL；Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1:0.8～1.4。
进一步地，初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu；以及将(N-Boc)-L-Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
进一步地，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：A.将N-Boc-L-Cys-OtBu溶解在溶剂C中，控温0～10℃，加入DIPEA，分批加入NCS，搅拌4～5h，反应结束后，过滤、淋洗，得到滤液；B.控温0～10℃，将Fmoc-D-Cys-OH加入滤液中，加入DIPEA，反应控温10～30℃，搅拌0.5～2h；对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；优选的，溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
进一步地，步骤A中，N-Boc-L-Cys-OtBu溶解在溶剂C中得到浓度为0.01～0.3g/mL的溶液，DIPEA的加入量为摩尔的2～3eq，NCS的加入量为1.1～1.5eq；步骤B中，Fmoc-D-Cys-OH的加入量为1.1～1.5eq。
根据本发明的另一个方面，提供一种依特卡肽的合成方法。该合成方法包括以下步骤： S1，上述任一种的依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；以及S2，将NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH反应，然后脱去Fmoc，再经过乙酰化得到依特卡肽。
进一步地，S2中，NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg为NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。
进一步地，S2包括：使用氨基树脂依照固相合成的方法链接NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽，将Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH、PyBop和DIPEA在0～5℃活化0-10min，控温20～30℃反应2～6h，反应结束后用DMF洗涤4～6次，10％～20％哌啶脱去Fmoc，再经过乙酰化得到依特卡肽的肽树脂。
进一步地，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA＝3:3～6:3～6的比例。
应用本发明的技术方案，通过合成关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，进而再合成依特卡肽，避免了肽链构建二硫键时的副反应，简化了操作，用于合成依特卡肽可以提高纯度和收率，重要的是，合成此中间体的原料廉价易得，工艺简单。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1示出了实施例1中Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的纯度图谱；
图2示出了实施例1的中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的LCMS图谱(LC-MS：m/z＝617.9(M-1,30ev),1235.7(2M-1,30ev))；以及
图3示出了实施例3中制备纯化后的伊特卡肽纯度图谱。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明中所涉及的缩写解释如下：
Fmoc：9-芴甲氧基羰基。
Boc：叔丁基羰基。
tBu：叔丁基。
Arg：精氨酸。
Cys：半胱氨酸。
Ala：丙氨酸。
PyBop：六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷。
DIPEA：N,N-二异丙基乙胺。
DCM：二氯甲烷。
NCS：N-氯代丁二酰亚胺。
DMF：N，N-二甲基甲酰胺。
NMP：N-甲基吡咯烷酮。
DMAc：N,N-二甲基乙酰胺。
THF：四氢呋喃。
OtBu：叔丁氧基。
EtOAc:乙酸乙酯。
依特卡肽的合成中，最关键的就是二硫键的合成。该步骤的收率的好坏直接影响到最终整条路线的收率。本申请的发明人发现，关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，(N-芴甲酰基-D-半胱氨酸-S-S-(N-叔丁氧羰基-L-半胱氨酸叔丁酯))的合成预先合成出二硫键从而可以完美的避开固相反应效率低收率差的问题。
根据本发明一种典型的实施方式，提供一种依特卡肽中间体的合成方法，依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，依特卡肽中间体的合成方法包括以下步骤：以N-Boc-L-Cys-OtBu为起始原料，通过取代反应生成初级产物
Figure PCTCN2019129440-appb-000003
其中，R为S-Py或Cl，初级产物与Fmoc-D-Cys-OH氨基酸进行偶联反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
应用本发明的技术方案，通过合成关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，进而再合成依特卡肽，避免了肽链构建二硫键时的副反应，简化了操作，用于合成依特卡肽可以提高纯度和收率，重要的是，合成此关键中间体的原料廉价易得，工艺简单。
根据本发明一种典型的实施方式，初级产物为Py-S-S-(N-Boc)-L-Cys-OtBu，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：将N-Boc-L-Cys-OtBu与二硫二吡啶反应合成得到Py-S-S-(N-Boc)-L-Cys-OtBu；以及将Py-S-S-(N-Boc)-L-Cys-OtBu与 Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys(OtBu))-OH，重要的是，合成此中间体的原料廉价易得，工艺简单。
优选的，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：室温下，将(N-Boc)-L-Cys-OtBu和二硫二吡啶加入溶剂A中，搅拌6～12h，然后加入水，使用萃取剂萃取，对得到的有机相进行干燥及过滤，纯化后得到Py-S-S-(N-Boc)-L-Cys-OtBu；溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu，控温15～30℃搅拌反应0.5～2h，对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。优选的，溶剂A选自DMF、NMP和DMAc中的一种或多种；优选的，萃取剂选自EtOAc、MTBE和DCM中的一种或多种；优选的，所述溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
为了提高原料的利用率，保证反应的快速有效进行，进一步优选的，N-Boc-L-Cys-OtBu与二硫二吡啶反应中，N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2～1:6.4，(N-Boc)-L-Cys-OtBu在溶剂A中的浓度为0.01～0.3g/mL；溶剂B中Fmoc-D-Cys-OH的浓度为0.01～0.3g/mL；Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1:0.8～1.4。
根据本发明一种典型的实施方式，初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu；以及将(N-Boc)-L-Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys(OtBu))-OH。
优选的，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：A.将(N-Boc)-Cys-OtBu溶解在溶剂C中，控温0～10℃，加入DIPEA，分批加入NCS，搅拌4～5h，反应结束后，过滤、淋洗，得到滤液；B.控温0～10℃，将Fmoc-D-Cys-OH加入滤液中，加入DIPEA，反应控温10～30℃，搅拌0.5～2h；对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。优选的，所述溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
为了提高原料的利用率，保证反应的快速有效进行，进一步优选的，步骤A中，(N-Boc)-L-Cys-OtBu溶解在溶剂C中得到浓度为0.01～0.3g/mL的溶液，DIPEA的加入量为摩尔的2～3eq，NCS的加入量为1.1～1.5eq；所述步骤B中，Fmoc-D-Cys-OH的加入量为1.1～1.5eq。
根据本发明一种典型的实施方式，提供一种依特卡肽的合成方法。该合成方法包括以下步骤：S1，按照上述任一种依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；以及S2，将NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH反应，然后脱去Fmoc，再经过乙酰化得到依特卡肽。
由于关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的原料廉价易得，工艺简单，也直接使得本发明中依特卡肽的合成方法原料廉价易得，工艺简单。
优选地，S2中，NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg为NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。优选的，S2包括：使用氨基树脂依照固相合成的方法链接NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽，将Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH、PyBop和DIPEA在0～10℃活化0-10min，控温20～30℃反应2～6h，反应结束后用DMF洗涤6次，10％～20％哌啶脱去Fmoc，再经过乙酰化得到依特卡肽的肽树脂。更优选的，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA＝3:3～6:3～6的比例。
下面将结合实施例进一步说明本发明的有益效果。
具体实施方式合成路线：
Figure PCTCN2019129440-appb-000004
实施例1
R＝S-Py
以N-Boc-L-Cys-OtBu(Cpd 1)为原料，通过与二硫二吡啶反应合成Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a)；将Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a)同Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH(Cpd 3)。
步骤1
室温下，将Cpd 1(1.6g,1.0eq)和2,2-dithiodipyridne(5.1g,4.0eq)加入DMF(16mL，10vol.)中。反应室温搅拌6～12h；然后体系中加入水；使用乙酸乙酯(100mL*3)萃取；有机相合并后使用MgSO 4干燥，过滤；有机相浓缩得到Cpd 2a粗品，通过柱层析纯化得到纯的Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a)。
步骤2
15～30℃下，DCM(30ml,30vol.)中加入Fmoc-D-Cys-OH(1.0g,1.0eq.)，然后加入Cpd 2a(1.13g,1.0eq.)至反应体系；控温15～30℃下搅拌反应0.5～2h；体系水洗3次，浓缩得到粗品Cpd 3；通过柱层析得到纯的Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd 3)。
图1示出了纯化后的Cpd 3纯度图谱；图2示出了纯化后的Cpd 3的LCMS图谱。
实施例2
R＝Cl
以N-Boc-L-Cys-OtBu(Cpd 1)为原料，通过与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b)；将(N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b)同Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd 3)。
步骤1
(N-Boc)-Cys-OtBu(0.28g,0.1mmol)溶解在DCM(20mL)中，搅拌控温0～10℃，加入DIPEA(0.19g,0.15mmol)；控温0～10℃分批加入NCS(0.15g,1.1eq)；反应保温搅拌4～5h,HPLC监控反应终点；反应结束后，过滤；滤饼用DCM(20mL)淋洗；合并滤液直接用于下一步。
步骤2
控温0-10℃，将Fmoc-D-Cys-OH(0.34g，0.1mmol)加入上一步的滤液中。DIPEA(0.15g,1.1eq)滴加入反应体系；反应控温10～30℃，搅拌0.5～2h；体系水洗3次,浓缩得到粗品Cpd 3，通过柱层析得到纯的Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd 3)。
纯化后的Cpd 3的LCMS图谱同图2。
实施例3
使用关键中间体合成依特卡肽：
使用氨基树脂(包括但不限于Sieber树脂，Rink Amide MBHA，Rink Amide树脂)依照固相合成的方法链接NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽，用关键中间体按照Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA＝3:3:3的比例0-5℃活化0～5min，控温20-30℃反应2～6h，kaiser检测反应终点。反应结束后用DMF洗涤6次，20％哌啶脱去Fmoc，再经过乙酰化得到依特卡肽的肽树脂。经过切割后的粗肽纯度90.3％，制备提纯后纯度在99.51％，总收率65％。
参照新开发的依特卡肽关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH合成工艺，100g半胱氨酸为起始物料，最终合成关键中间体357g。使用此中间体依照实例3合成依特卡肽，经制备纯化后得到纯品227g(TFA盐)，纯度在99.51％(图3)，总收率65％。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本申请中先合成了一种依特卡肽的关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，使用该中间体合成依特卡肽，制备后纯度可以达到99.51％(图3),分离收率73％，解决了现有合成过程中存在多肽链间二硫键错配的反应，副反应和副产物的种类较多的问题。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 一种依特卡肽中间体的合成方法，其特征在于，所述依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH，所述依特卡肽中间体的合成方法包括以下步骤：以N-Boc-L-Cys-OtBu为起始原料，通过取代反应生成初级产物
    Figure PCTCN2019129440-appb-100001
    其中，R为S-Py或Cl，所述初级产物与Fmoc-D-Cys-OH氨基酸进行偶联反应得到所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
  2. 根据权利要求1所述的合成方法，其特征在于，所述初级产物为Py-S-S-(N-Boc)-L-Cys-OtBu，所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：
    将N-Boc-L-Cys-OtBu与二硫二吡啶取代反应合成得到初级产物Py-S-S-(N-Boc)-L-Cys-OtBu；以及
    将所述Py-S-S-(N-Boc)-L-Cys-OtBu与Fmoc-D-Cys-OH偶联得到所述Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH。
  3. 根据权利要求2所述的合成方法，其特征在于，所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：
    室温下，将N-Boc-L-Cys-OtBu和二硫二吡啶加入溶剂A中，搅拌6～12h，然后加入水，使用萃取剂萃取，对得到的有机相进行干燥及过滤，纯化后得到Py-S-S-(N-Boc)-L-Cys-OtBu；
    溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu，控温15～30℃搅拌反应0.5～2h，对反应体系进行水洗、浓缩及纯化得到所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；
    所述溶剂A选自DMF、NMP和DMAc中的一种或多种；
    所述萃取剂选自EtOAc、MTBE和DCM中的一种或多种；
    所述溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
  4. 根据权利要求3所述的合成方法，其特征在于，所述N-Boc-L-Cys-OtBu与二硫二吡啶反应中，N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2～1:6.4；N-Boc-L-Cys-OtBu在所述溶剂A中的浓度为0.01～0.3g/mL；所述溶剂B中Fmoc-D-Cys-OH的浓度为0.01～0.3g/mL；Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1:0.8～1.4。
  5. 根据权利要求1所述的合成方法，其特征在于，所述初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu，所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：
    将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu；以及
    将(N-Boc)-L-Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
  6. 根据权利要求5所述的合成方法，其特征在于，所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得：
    A.将N-Boc-L-Cys-OtBu溶解在溶剂C中，控温0～10℃，加入DIPEA，分批加入NCS，搅拌4～5h，反应结束后，过滤、淋洗，得到滤液；
    B.控温0～10℃，将Fmoc-D-Cys-OH加入所述滤液中，加入DIPEA，反应控温10～30℃，搅拌0.5～2h；对反应体系进行水洗、浓缩及纯化得到所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；
    所述溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
  7. 根据权利要求6所述的合成方法，其特征在于，所述步骤A中，N-Boc-L-Cys-OtBu溶解在所述溶剂C中得到浓度为0.01～0.3g/mL的溶液，DIPEA的加入量为摩尔的2～3eq，NCS的加入量为1.1～1.5eq；所述步骤B中，Fmoc-D-Cys-OH的加入量为1.1～1.5eq。
  8. 一种依特卡肽的合成方法，其特征在于，包括以下步骤：
    S1，按照如权利要求1至7中任一项所述的依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH；以及
    S2，将NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH反应，然后脱去Fmoc，再经过乙酰化得到依特卡肽。
  9. 根据权利要求8所述的合成方法，其特征在于，所述S2中，所述NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg为NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。
  10. 根据权利要求8所述的合成方法，其特征在于，所述S2包括：使用氨基树脂依照固相合成的方法链接NH 2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽，将Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH、PyBop和DIPEA在0～5℃活化0-10min，控温20～30℃反应2～6h，反应结束后用DMF洗涤4～6次，10％～20％哌啶脱去Fmoc，再经过乙酰化得到依特卡肽的肽树脂。
  11. 根据权利要求10所述的合成方法，其特征在于，Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA＝3:3～6:3～6的比例。
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