WO2019154151A1 - 一种中间体化合物及其制备方法，及以该中间体化合物制备多肽的固相合成方法 - Google Patents

一种中间体化合物及其制备方法，及以该中间体化合物制备多肽的固相合成方法 Download PDF

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WO2019154151A1
WO2019154151A1 PCT/CN2019/073370 CN2019073370W WO2019154151A1 WO 2019154151 A1 WO2019154151 A1 WO 2019154151A1 CN 2019073370 W CN2019073370 W CN 2019073370W WO 2019154151 A1 WO2019154151 A1 WO 2019154151A1
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compound
protecting group
resin
amino protecting
intermediate compound
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PCT/CN2019/073370
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English (en)
French (fr)
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赵明亮
李筛
田强
薛宏祥
杨燕苹
潘钧铸
蔡家强
王利春
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to CN201980006409.5A priority Critical patent/CN111479800A/zh
Publication of WO2019154151A1 publication Critical patent/WO2019154151A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/10—Formation of amino groups in compounds containing carboxyl groups with simultaneously increasing the number of carbon atoms in the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • 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
    • 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 invention relates to the field of medicinal chemistry, in particular to an intermediate compound and a preparation method thereof, and a solid phase synthesis method for preparing a polypeptide by using the intermediate compound.
  • Opioid receptors are widely present in the central nervous system and the peripheral nervous system.
  • Traditional opioid receptor agonists such as morphine and its derivatives, are the most effective drugs for the treatment of chronic arthritis, inflammatory neuralgia, postoperative pain, and moderate to severe pain caused by various cancers.
  • kappa opioid receptor agonists including spirolin and enalapril, stopped further development due to the side effects of both drugs, such as agitation and hallucination.
  • Second-generation ⁇ -opioid receptor agonists (such as acimadol) have a poor anesthetic effect at licensed doses and have abandoned their use as opioid narcotics, but instead used it to treat the digestive system. Diseases such as irritable bowel syndrome.
  • Patent PCT/CN2017/103027 uses a liquid phase method to synthesize a peptide compound with the chemical name: 4-amino-1-((R)-2-((R)-2-((R)-2-amino-) 3-phenylpropionamido)-3-phenylpropionamido)-4-methylpentanoyl)-6-((2-(2-methoxyethoxy)ethyl)amino)hexanoyl Piperidine-4-carboxylic acid, the structural formula is as follows:
  • the compound of the formula I has excellent agonistic efficacy against the ⁇ opioid receptor and can effectively attenuate the side effects of the central nervous system while maintaining peripheral analgesic effects.
  • the above patent uses the liquid phase method to synthesize the polypeptide, and the synthesis cycle is long, and the reaction process needs to be monitored in real time, and after each step of the reaction, the intermediate needs to undergo complicated purification before the next reaction can be carried out, the operation is cumbersome, the cost is increased, and it is disadvantageous for large Scale production.
  • An object of the present invention is to provide an intermediate compound represented by the formula II-1 or the formula II-2 or a salt thereof, and a method for synthesizing the compound of the formula I by a solid phase method via the intermediate compound.
  • the method for synthesizing the compound of the formula I can greatly shorten the reaction period, and the reaction operation is simple and can be industrialized and mass-produced.
  • R 1 is hydrogen or an amino protecting group
  • the amino protecting group is a basic amino protecting group or an acidic amino protecting group.
  • R 1 of intermediate compound II- 1 is hydrogen or a basic amino protecting group.
  • the basic amino protecting group is Fmoc or Tfa, more preferably Fmoc.
  • the R 1 is hydrogen, Fmoc or Tfa; preferably Fmoc.
  • the intermediate compound II-1 is:
  • R 1 is hydrogen or an amino protecting group
  • R 2 is an amino protecting group
  • the amino protecting group is a basic protecting group or an acidic protecting group
  • R 1 of intermediate compound II-2 is hydrogen or an amino protecting group as opposed to a basicity of R 2 acid.
  • R 1 is hydrogen or an acidic amino protecting group
  • R 2 is a basic amino protecting group
  • R 1 is hydrogen or a basic amino protecting group
  • R 2 is an acidic amino protecting group.
  • R 1 is hydrogen or a basic amino protecting group and R 2 is an acidic amino protecting group.
  • R 1 is hydrogen or a basic amino protecting group and R 2 is an acidic amino protecting group.
  • the basic amino protecting group is preferably Fmoc or Tfa, more preferably Fmoc.
  • the acidic amino protecting group of intermediate compound II-2 is Cbz, Boc, Trt, DMB or PMB, more preferably Boc.
  • R 1 is hydrogen, Fmoc or Tfa; and R 2 is Cbz, Boc, Trt, DMB or PMB.
  • R 1 is Fmoc and R 2 is Boc.
  • the intermediate compound II-2 is:
  • the salt of the intermediate compound II-1 or II-2 of the present invention may be a sodium salt or a potassium salt thereof, which can be produced by a method conventionally used in the art.
  • Another object of the present invention is to provide a process for the preparation of intermediate compounds II-1 and II-2 which is simple in procedure, easy to handle, and which does not require an excessive separation step, so that the reaction can be mass-produced in kilograms.
  • a method for preparing an intermediate compound II-1 comprising the steps of:
  • R 1 is as defined above; HX is trifluoroacetic acid or hydrochloric acid; preferably, HX is hydrochloric acid.
  • the compound of the formula III or its HX salt can be synthesized by a conventional method in the art.
  • the reductive amination reaction is carried out in a solvent.
  • the molar ratio of compound SM-2-1 to the HX salt of the compound of Formula III is from (1:1) to (5:1), preferably from (1:1) to (2.5:1).
  • the molar ratio of the molar amount of the HX salt of the compound of formula III to the solvent in the reductive amination reaction is (1 mol: 5 L) to (1 mol: 10 L), preferably (1 mol: 5 L) to (1 mol: 8 L).
  • 1 mol: 5 L, 1 mol: 6 L, 1 mol: 7 L or 1 mol: 8 L is 1 mol: 5 L, 1 mol: 6 L, 1 mol: 7 L or 1 mol: 8 L.
  • the solvent in the reductive amination reaction is a mixed solvent of an aprotic solvent and an alcohol solvent.
  • the aprotic solvent is selected from one or more of the group consisting of dichloromethane, tetrahydrofuran and diethyl ether;
  • the alcohol solvent is selected from one or more of methanol, ethanol and isopropanol.
  • the solvent is a mixed solvent of dichloromethane and methanol.
  • the volume ratio of the aprotic solvent to the alcohol solvent in the solvent of the reductive amination reaction is (1:5) to (10:1), preferably (1.5:1) to (5). :1).
  • the reducing agent used in the reductive amination reaction is sodium borohydride or a derivative thereof, preferably sodium triacetoxyborohydride.
  • the molar ratio of the HX salt of the compound of Formula III to the reducing agent is from (1:1) to (1:10), preferably from (1:2) to (1:8), such as 1: 2.5, 1:5 or 1:7.
  • the reductive amination reaction time is from 0.5 to 24 hours, preferably from 1 to 3 hours.
  • the reductive amination reaction temperature is from -20 °C to 25 °C, preferably from 0 °C to 15 °C.
  • compound SM-2-1 is prepared by the oxidation of diethylene glycol monomethyl ether in an oxidizing system to a compound of formula SM-2-1.
  • the product obtained by the oxidation reaction is used directly in the preparation of intermediate compound II-1 without isolation and purification.
  • the molar ratio of diethylene glycol monomethyl ether to the HX salt of the compound of formula III is from (1:1) to (5:1), preferably from (1:3) to (3:1), More preferably, it is 2.2:1.
  • the oxidizing system of the oxidation reaction includes an oxidizing agent and an organic base.
  • the molar ratio of diethylene glycol monomethyl ether to the oxidizing agent in the oxidation reaction is (1:1) to (1:5), preferably (1:1) to (1:3). More preferably, it is (1:1) - (1:2).
  • the molar ratio of diethylene glycol monomethyl ether to the organic base in the oxidation reaction is (1:2) to (1:10), preferably (1:2) to (1:6). ).
  • the oxidizing agent in the oxidation reaction is a combination of DMSO and oxalyl chloride, or a combination of DMSO and trifluoroacetic anhydride.
  • the molar ratio of oxalyl chloride to DMSO, or the molar ratio of trifluoroacetic anhydride to DSMO is (1:1) to (1:5), preferably (1:1) to (1:3). More preferably, it is (1:1) - (1: 2), More preferably (1:1) - (1: 1.5).
  • the organic base in the oxidation reaction is triethylamine.
  • the oxidation reaction is carried out in an aprotic solvent, which may be selected from one or more of the group consisting of dichloromethane, tetrahydrofuran, and diethyl ether.
  • the aprotic solvent is dichloromethane.
  • the molar ratio of the molar amount of diethylene glycol monomethyl ether to the aprotic solvent in the oxidation reaction is (1 mol: 1 L) to (1 mol: 2 L).
  • the temperature of the oxidation reaction is -30 ° C or less, preferably -60 ° C or less, more preferably -70 ° C or less.
  • compound SM-2-1 is prepared by the hydrolysis of (2-methoxyethoxy)acetaldehyde dimethylacetal in an acidic system to a compound of formula SM-2-1,
  • the acid in the acidic system includes, but is not limited to, an organic acid and a mineral acid.
  • the organic acid includes, but is not limited to, trifluoroacetic acid, acetic acid, formic acid, p-toluenesulfonic acid, fumaric acid, and tartaric acid, with trifluoroacetic acid being particularly preferred.
  • the mineral acid includes, but is not limited to, hydrochloric acid, sulfuric acid, and phosphoric acid, with sulfuric acid being particularly preferred.
  • the reaction system is an aqueous solution of sulfuric acid having a sulfuric acid aqueous solution content of from 0.1% to 50%, particularly preferably from 0.1% to 20%, still more preferably from 1% to 10%.
  • the temperature of the reaction is from 0 ° C to 50 ° C, preferably from 0 ° C to 25 ° C, more preferably from 0 ° C to 10 ° C.
  • R 1 and R 2 are as defined above.
  • intermediate compound II-1 is prepared following the methods of preparation described above. In some preferred embodiments, intermediate compound II-1 is used directly in the preparation of intermediate compound II-2 without isolation and purification.
  • the imine group of intermediate compound II-1 is protected with N,N-diisopropylethylamine and di-tert-butyl dicarbonate.
  • the molar ratio of intermediate compound II-1 to N,N-diisopropylethylamine is from (1:1) to (1:5).
  • the molar ratio of intermediate compound II-1 to di-tert-butyl dicarbonate is from (1:1) to (1:5).
  • the process for the preparation of the intermediate compound II-2 of the present invention further comprises the step of separating and purifying the obtained intermediate compound II-2.
  • a further object of the present invention is to provide a solid phase synthesis method for a compound of the formula I, which has a short synthesis cycle and a simple operation step, and is suitable for mass production.
  • a solid phase synthesis method for a compound of formula I comprising the steps of:
  • R 1 and R 2 are as defined above;
  • R y , R 3 , R 4 , R 5 are a basic amino protecting group or an acidic amino protecting group; and when R 3 and R 4 are both an acidic amino protecting group, R Both y and R 5 are basic amino protecting groups; when R 3 and R 4 are both basic amino protecting groups, R y and R 5 are both acidic amino protecting groups.
  • R 3 and R 4 are basic amino protecting groups
  • R y and R 5 are acidic amino protecting groups
  • R 3 and R 4 are independently selected from one or more of Fmoc and Tfa; and R y and R 5 are independently selected from one or more of Cbz, Boc, Trt, DMB, and PMB.
  • R 3 and R 4 are Fmoc; and R y and R 5 are Boc.
  • R 2 and R y , R 5 are both an acidic amino protecting group or a basic amino protecting group.
  • R 1 is an amino protecting group
  • R 3 and R 4 are the same acidic group or a basic amino-protecting an amino protecting group.
  • R 3 and R 4 are both acidic amino protecting groups, and R 2 , R y , and R 5 are both basic amino protecting groups. In some embodiments, R 3 and R 4 are both basic amino protecting groups, and R 2 , R y , and R 5 are both acidic amino protecting groups. In some embodiments, when R 1 is an amino protecting group, R 1, R 3 and R 4 is an acid with an amino protecting group, R 2, R y and R 5 are the same as the basic amino protecting group. In some embodiments, when R 1 is an amino protecting group, R 1, R 3 and R 4 are the same as the basic amino protecting group, R 2, R y and R 5 is an acid with an amino protecting group. Preferably, R 1 , R 3 and R 4 are basic amino protecting groups, and R 2 , R y and R 5 are acidic amino protecting groups.
  • step 3) optionally includes the step 4): separating and purifying the crude compound I obtained in the step 3) to obtain the pure compound I.
  • the intermediate compound II-2 is obtained by the preparation method described above.
  • the compound M-1-1 of the step 1) is prepared by immobilizing the compound SM-1 as a solid phase carrier, and then removing the protecting group Rx of the piperidine ring imine group to obtain a solid phase carrier.
  • R x is opposite to the pH of the amino-protecting group and R y;
  • R x is a basic amino protecting group; more preferably R x is or Tfa of the Fmoc; R x is particularly preferably Fmoc.
  • the molar ratio of the compound of formula SM-1 to the solid support is 1: (2 to 4), preferably 1:3.
  • the solid support is Wang resin or 2-chlorotrityl chloride resin, preferably 2-chlorotrityl chloride resin.
  • the degree of substitution of Wang resin is 0.3-1.0 mmol/g, more preferably 0.4-0.7 mmol/g; the degree of substitution of 2-chlorotrityl chloride resin is 0.2-1.6 mmol/g, preferably 0.7-1.2 mmol. /g is preferably 1.0-1.2 mmol/g.
  • the solid phase carrier has high yield of the compound, and the prepared compound has high purity, is easy to be purified, and has low cost.
  • the condensation reaction of step 1) and step 2) is carried out in a solvent selected from one or both of N,N-dimethylformamide, dichloromethane.
  • a solvent selected from one or both of N,N-dimethylformamide, dichloromethane.
  • the volume ratio of N,N-dimethylformamide to dichloromethane is (1-5):1, preferably (1-3):1.
  • the condensation of the polypeptide is carried out using a condensing agent in steps 1) and 2)
  • the condensing agent may be one or more of the following: a) HBTU, Cl-HoBt and DIEA; b) DIC and Cl-HoBt; c) PyBOP, Cl-HoBt and DIEA; d) HBTU, Oxyma, DIEA, wherein the ratio of each component of group a), group c) and group d) can be 1:1:1.1, b) The composition ratio can be 1:1.1.
  • a combination of HBTU, Cl-HoBt and DIEA, or DIC and Cl-HoBt is used as the condensing agent.
  • the condensation of the polypeptide is carried out using a condensing agent in steps 1) and 2), and the condensing agent may be a combination of DIC and HoBt as a condensing agent.
  • Protection of the amino group, imino group, and deprotection are carried out in the present invention using methods conventionally used in the art.
  • 20% piperidine/DMF solution DBLK
  • Boc protecting groups TFA, HCl, or HF can be used for removal, preferably TFA.
  • the lysate in step 3) contains 50% to 100% TFA, or further contains 0% to 10% TIS, 0% -10 One of 2H 2 O, 0%-10% TES, 2 or more, preferably 90% TFA / 5% TIS / 5% H 2 O (v / v / v) or 95% TFA / 5% H 2 O (v/v) composition to completely cleave the M-5 resin and improve the purity of the final product.
  • the ratio of the lysate in step 3) to the polypeptide compound M-5 immobilized in step 2) is (6-10) mL: 1 g, preferably 8 mL: 1 g.
  • the cleavage reaction of step 3 is first reacted at low temperature for 15-30 min, then warmed to room temperature and reacted to completion.
  • step 3) cleavage, optionally step 4), comprises the step of precipitating the crude compound of formula I using an ether solvent.
  • the ether solvent is anhydrous diethyl ether or methyl tert-butyl ether.
  • the above step of precipitating the crude compound of formula I using an ether solvent is carried out at a low temperature, such as 0 ° C, -10 ° C, and the like.
  • the separation and purification of step 4) is carried out using reverse phase high performance liquid chromatography.
  • the basic amino protecting group as used in the specification and claims refers to a protecting group on nitrogen which can be removed under basic conditions, for example, Fmoc or Tfa, etc.; an acidic amino protecting group means that it can be subjected to acidic conditions.
  • the nitrogen protecting group is removed, for example, Cbz, Boc, Trt, DMB or PMB.
  • One skilled in the art can selectively or completely remove one or more protecting groups by appropriately selecting and operating with reference to the textbooks commonly used in the art, Greene's Protective Groups in Organic Synthesis (4th Edition).
  • the synthesis method of the intermediate compound II-1 or II-2 of the invention is simple and easy to operate, and does not require excessive separation steps, so that the reaction can be mass-produced in kilograms; in particular, when When the ethylene glycol monomethyl ether and the HX salt of the compound III are used as the starting reactants, the reaction process is optimized, and mild reaction conditions and short reaction time can be used without any separation and purification steps to "one-pot method".
  • the intermediate compound II-2 of the present invention is prepared.
  • the solid phase synthesis method of the compound of the formula I of the invention has a short synthesis cycle, simple operation steps, and is suitable for large-scale production.
  • the polypeptide compound of the formula I prepared by the solid phase synthesis method is applied, and the purity of the product can be up to 99.5% or more by optimizing the reaction conditions.
  • the raw materials used in the following examples are all commercially available products.
  • Oxalyl chloride (1.65 kg, 13.0 mol) was dissolved in dichloromethane (15 L), cooled to ⁇ -70 ° C under nitrogen atmosphere, and DMSO (1.47 kg, 18.8 mol) in dichloromethane (1500 mL). ⁇ -70 ° C. After the addition was completed, the mixture was stirred at this temperature for 60 min, and a solution of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol) in dichloromethane (1500 mL) was added, and the temperature was controlled to ⁇ -70 °C. After the addition was completed, the mixture was stirred at this temperature for 60 minutes.
  • Oxalyl chloride (2.39 kg, 18.8 mol) was dissolved in dichloromethane (15 L), cooled to ⁇ -70 ° C under nitrogen atmosphere, and DMSO (1.95 kg, 25.0 mol) in dichloromethane (1500 mL) was added, and temperature was applied. ⁇ -70 ° C. After the addition was completed, the mixture was stirred at this temperature for 60 min, and a solution of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol) in dichloromethane (1500 mL) was added, and the temperature was controlled to ⁇ -70 °C. After the addition was completed, the mixture was stirred at this temperature for 60 minutes.
  • N,N-diisopropylethylamine (1.5 kg, 11.4 mol) and di-tert-butyl dicarbonate (3.75 kg, 17.1 mol) were successively added to the reaction mixture. After the addition, the temperature was raised to room temperature for 2 h. Dichloromethane and methanol were distilled off under reduced pressure, and a saturated aqueous solution of sodium carbonate was added to this crude product, and stirred for 30 min. It was extracted 3 times with methyl tert-butyl ether, and then the aqueous phase was adjusted to pH 3-4 again with 1N hydrochloric acid, and the aqueous phase was extracted twice with ethyl acetate. The ethyl acetate phase was combined and washed with 1N HCl and brine, and dried over anhydrous sodium sulfate and filtered. Compound II-2-a (1.09 kg, yield 33.74%).
  • Oxalyl chloride (3.30 kg, 25.0 mol) was dissolved in dichloromethane (15 L), cooled to ⁇ -70 ° C under nitrogen atmosphere, and DMSO (2.94 kg, 37.5 mol) in dichloromethane (1500 ml) was added, and temperature was applied. ⁇ -70 ° C. After the addition was completed, the mixture was stirred at the same temperature for 60 min, and a solution of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol) in dichloromethane (1500 ml) was added thereto, and the temperature was controlled to ⁇ -70 °C. After the addition was completed, the mixture was stirred at this temperature for 60 minutes.
  • N,N-diisopropylethylamine (3.7 kg, 28.5 mol) and di-tert-butyl dicarbonate (6.25 kg, 28.5 mol) were successively added to the reaction mixture. After the addition, the temperature was raised to room temperature for 2 h. Dichloromethane and methanol were distilled off under reduced pressure, and a saturated aqueous solution of sodium carbonate was added to this crude product, and stirred for 30 min. It was extracted 3 times with methyl tert-butyl ether, and then the aqueous phase was adjusted to pH 3-4 again with 1N hydrochloric acid, and the aqueous phase was extracted twice with ethyl acetate. The ethyl acetate phase was combined and washed with 1N HCl and brine, and dried over anhydrous sodium sulfate, and filtered and filtered. Compound II-2-a (0.95 kg, yield 29.30%).
  • reaction solution 800 mL of methanol was continuously added to block the unreacted active site, and the reaction was carried out for 45 minutes. After the reaction is completed, the solution is drained, and the resin is washed with 4 ⁇ 10 L of DMF solution. After the washing is completed, a part of the resin is taken, and deprotection is carried out using piperidine, and the amount of Fmoc in the piperidine deprotecting solution is determined by ultraviolet spectrophotometry, and M is calculated. The degree of substitution of the -1-a resin was 0.79 mmol/g.
  • the M-1-a resin obtained in the step (1) was treated with 10 L of 20% piperidine/DMF solution for 5 min, the mixture was drained, and 10 L of 20% piperidine/DMF solution was further added for 15 min to remove the Fmoc protecting group. The resin was then washed with 5 x 10 L DMF, Kaiser Test, resin reddish brown, completely deprotected to give M-1-1-a resin.
  • the M-2-a resin obtained in the step (3) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue-purple , deprotection is complete, get M-2-1-a resin.
  • the SM-3-a compound (3270 mmol, 1156.7 g) and Cl-HoBt (3270 mmol, 555.2 g) were weighed and dissolved in 7 L of a 1:1 volume ratio of DMF/DCM solution, and the solution was ice-cooled to 0 under nitrogen atmosphere. -5 ° C, then DIC (3597 mmol, 557 ml) was added and the reaction was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-3-a resin.
  • the M-3-a resin was treated with 2 ⁇ 10L 20% piperidine/DMF solution for 5 min and 15 min respectively, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue, and deprotected completely. M-3-1-a resin.
  • the SM-4-a compound (3270 mmol, 1267.8 g) and Cl-HoBt (3270 mmol, 554.9 g) were weighed and dissolved in 7 L of a 1:1 ratio of DMF/DCM solution, and the solution was ice-cooled to 0 under nitrogen atmosphere. -5 ° C, then DIC (3597 mmol, 557 ml) was added and the reaction was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L of DMF, the Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-4-a resin.
  • the M-4-a resin obtained in the step (7) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue-purple , deprotection is complete, get M-4-1-a resin.
  • the SM-5-a compound (3270 mmol, 867.8 g) and Cl-HoBt (3270 mmol, 555.1 g) were weighed and dissolved in 7 L of a 1:1 ratio of DMF/DCM solution, and the solution was ice-cooled to 0 under nitrogen atmosphere. -5 ° C, then DIC (3597 mmol, 557 ml) was added and the reaction was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was complete.
  • the resin was alternately washed with 3 ⁇ 10 L DCM and 3 ⁇ 10 L methanol. After the washing was completed, the resin was vacuum dried to a constant weight to finally obtain 1967.1 g of M-5-a resin in a yield of 96.7%.
  • reaction solution 800 mL of methanol was continuously added to block the unreacted active site, and the reaction was carried out for 45 minutes. After the reaction is completed, the solution is drained, and the resin is washed with 4 ⁇ 10 L of DMF solution. After the washing is completed, a part of the resin is taken, and deprotection is carried out using piperidine, and the amount of Fmoc in the piperidine deprotecting solution is determined by ultraviolet spectrophotometry, and M is calculated. The degree of substitution of the -1-a resin was 0.77 mmol/g.
  • the M-1-a resin was treated with 2 ⁇ 10L 20% piperidine/DMF solution for 5 min and 15 min respectively, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin reddish brown, completely deprotected. M-1-1-a resin.
  • the II-2-a intermediate compound (1917 mmol, 1093.0 g), Cl-HoBt (1917 mmol, 343.6 g), HBTU (1917 mmol, 766.3 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled under nitrogen atmosphere. To 0-5 ° C, then DIEA (2108 mmol, 367 mL) was added and the reaction was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L of DMF, the Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-2-a resin.
  • the M-2-a resin obtained in the step (3) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue, Deprotection is complete, and M-2-1-a resin is obtained.
  • SM-3-a compound (3032.4 mmol, 1071.1 g), Cl-HoBt (3032.4 mmol, 514.6 g), HBTU (3032.4 mmol, 1149.8 g) were weighed and dissolved in 7 L of DMF solution, and the solution was iced under nitrogen atmosphere. The mixture was brought to 0-5 ° C, then DIEA (3322.4 mmol, 550 mL) was added and the mixture was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-3-a resin.
  • the M-3-a resin obtained in the step (5) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue. , deprotection is complete, get M-3-1-a resin.
  • the SM-4-a compound (3032.4 mmol, 1175.7 g), Cl-HoBt (3270 mmol, 514.5 g), HBTU (3032.4 mmol, 1150.3 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled under nitrogen atmosphere. To 0-5 ° C, then DIEA (3322.4 mmol, 550 mL) was added and the reaction was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L of DMF, the Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-4-a resin.
  • the M-4-a resin obtained in the step (7) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 10 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 1 L DMF, Kaiser Test, resin blue-purple , deprotection is complete, get M-4-1-a resin.
  • the SM-5-a compound (3270 mmol, 867.8 g), Cl-HoBt (3270 mmol, 555.1 g), HBTU (3032.4 mmol, 1151.7 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled under nitrogen atmosphere. The reaction was stirred for 5 min at 0-5 ° C then DIEA (3322.4 mmol, 550 mL). After 5 min, the reaction solution was added to the resin obtained in the previous step for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-5-a resin.
  • the resin was alternately washed with 3 ⁇ 10 L of DCM and 3 ⁇ 10 L of methanol. After the completion of the washing, the resin was dried under vacuum drying to a constant weight to obtain 1941.9 g of a resin M-5-a resin in a yield of 92.1%.
  • reaction solution 800 mL of methanol was continuously added to block the unreacted active site, and the reaction was carried out for 45 minutes. After the reaction is completed, the solution is drained, and the resin is washed with 4 ⁇ 10 L of DMF solution. After the washing is completed, a part of the resin is taken, and deprotection is carried out using piperidine, and the amount of Fmoc in the piperidine deprotecting solution is determined by ultraviolet spectrophotometry, and M is calculated. The degree of substitution of the -1-a resin was 0.76 mmol/g.
  • the resin obtained in the step (1) was treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, respectively, and the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF to remove the Fmoc by-product and the residual piperidine. Kaiser Test The resin is reddish brown and completely deprotected to obtain M-1-1-a resin.
  • the II-2-a intermediate compound (1898 mmol, 1230.7 g) and Cl-HoBt (1898 mmol, 322.1 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then added. DIC (2087 mmol, 323 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L of DMF, the Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-2-a resin.
  • the M-2-a resin obtained in the step (3) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue. , deprotection is complete, get M-2-1-a resin.
  • the SM-3-a compound (3273 mmol, 1155.7 g) and Cl-HoBt (3032.4 mmol, 514.6 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then added to DIC. (2087 mmol, 323 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-3-a resin.
  • the M-3-a resin was treated with 2 ⁇ 10L 20% piperidine/DMF solution for 5 min and 15 min respectively, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue, and deprotected completely. M-3-1-a resin.
  • the SM-4-a compound (3273 mmol, 1267.4 g) and Cl-HoBt (3273 mmol, 555.5 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then DIC ( 2087 mmol, 323 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L of DMF, the Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-4-a resin.
  • the M-4-a resin obtained in the step (7) was separately treated with 2 ⁇ 10 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 10 L DMF, Kaiser Test, resin blue-purple , deprotection is complete, get M-4-1-a resin.
  • the SM-5-a compound (3273 mmol, 868.6 g) and Cl-HoBt (3273 mmol, 555.2 g) were weighed and dissolved in 7 L of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then DIC ( 2087 mmol, 323 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 10 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-5-a resin.
  • the resin was washed alternately with 3 ⁇ 10 L of DCM and 3 ⁇ 10 L of methanol. After the washing was completed, the resin was dried in a vacuum to dryness to a constant weight to obtain 1987.4 g of M-5-a resin in a yield of 98.7%.
  • M-1-a resin was treated with 2 ⁇ 100 mL of 20% piperidine/DMF solution for 5 min and 15 min, Fmoc protecting group was removed, then the resin was washed with 5 ⁇ 100 mL DMF, Kaiser Test, resin reddish brown, completely deprotected, M -1-1-a resin.
  • the II-2-a intermediate compound (66 mmol, 37.6 g) and Cl-HoBt (66 mmol, 11.2 g) were weighed and dissolved in 700 mL of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then added to DIC. (72.6 mmol, 12 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 1 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-2-a resin.
  • the M-2-a resin obtained in the step (3) was separately treated with 2 ⁇ 1 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 1 L DMF, Kaiser Test, resin blue , deprotection is complete, get M-2-1-a resin.
  • the SM-3-a compound (66 mmol, 23.3 g) and Cl-HoBt (66 mmol, 12.3 g) were weighed and dissolved in 700 ml of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then DIC ( 72.6 mmol, 12 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 1 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-3 resin.
  • the M-3 resin obtained in the step (5) was separately treated with 2 ⁇ 1 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 1 L DMF, Kaiser Test, resin blue, off The protection is complete and the M-3-1-a resin is obtained.
  • the SM-4-a compound (66 mmol, 25.6 g) and Cl-HoBt (66 mmol, 11.9 g) were weighed and dissolved in 700 mL of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then DIC ( 72.6 mmol, 12 ml) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 1 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-4-a resin.
  • the M-4-a resin obtained in the step (7) was separately treated with 2 ⁇ 1 L of 20% piperidine/DMF solution for 5 min and 15 min, the Fmoc protecting group was removed, and then the resin was washed with 5 ⁇ 1 L DMF, Kaiser Test, resin blue-purple , deprotection is complete, get M-4-1-a resin.
  • the SM-5-a compound (66 mmol, 17.5 g) and Cl-HoBt (66 mmol, 12.0 g) were weighed and dissolved in 700 mL of DMF solution, and the solution was ice-cooled to 0-5 ° C under nitrogen protection, and then DIC ( 72.6 mmol, 12 mL) was stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, reacted at room temperature for 1.5 h, the resin was drained, the resin was washed with 3 ⁇ 1 L DMF, Kaiser Test, the resin was pale yellow, and the condensation reaction was completed to obtain M-5-a resin.
  • the resin was washed alternately with 3 x 1 L of DCM and 3 x 1 L of methanol. After the washing was completed, the resin was dried under vacuum drying to constant weight to obtain 122.7 g of M-5-a resin in a yield of 95.3%.

Abstract

本发明涉及一种式II-1或式II-2所示的中间体化合物或其盐，及其制备方法。本发明还涉及使用式II-2所示的中间体化合物以固相合成法合成式I化合物的工艺。本发明的中间体化合物式II-1或式II-2的合成方法步骤简单、易于操作，反应可以进行公斤级的量产；本发明式I化合物的固相合成方法工艺合成周期短，操作步骤简便，适于大规模生产。

Description

一种中间体化合物及其制备方法，及以该中间体化合物制备多肽的固相合成方法 技术领域
本发明涉及药物化学领域，具体涉及一种中间体化合物及其制备方法，及以该中间体化合物制备多肽的固相合成方法。
背景技术
阿片样物质受体(μ、δ和κ)广泛存在于中枢神经***和外周神经***。传统阿片样物质受体激动剂(如***及其衍生物)是治疗慢性关节炎、炎症性神经痛、术后疼痛以及各种癌症引起的中度至重度疼痛的最有效的药物。但第一代κ阿片样物质受体激动剂包括螺朵林和依那朵林，由于这两种药物可产生躁动和致幻等副作用，因此停止了对其的进一步开发。第二代κ阿片样物质受体激动剂(例如阿西马朵林)在许可剂量下的麻醉效果较差，已经放弃将其作为阿片类麻醉品的开发，而是将其用于治疗消化***疾病，例如肠易激综合征。
专利PCT/CN2017/103027采用液相法合成了一种多肽化合物，化学名为：4-氨基-1-((R)-2-((R)-2-((R)-2-氨基-3-苯基丙酰胺基)-3-苯基丙酰胺基)-4-甲基戊酰胺基)-6-((2-(2-甲氧基乙氧基)乙基)氨基)己酰基)哌啶-4-羧酸，其结构式如下：
Figure PCTCN2019073370-appb-000001
该式I化合物对于κ阿片样受体具有优异的激动效能，且在保持外周镇痛作用的同时，能够有效减弱中枢神经***的毒副作用。上述专利中采用液相法合成该多肽，合成周期长，需要实时监测反应进程，且每一步反应完成之后中间体需要经过复杂的纯化后才能进行下一步反应，操作繁琐，成本增加，不利于大规模生产。
因此需要寻求一种新的式I化合物的制备方法及该方法中使用的中间体化合物，用于解决现有技术中液相法合成周期长、反应操作复杂繁琐，不利于控制成本的问题。
发明内容
本发明的目的在于提供一种式II-1或式II-2所示的中间体化合物或其盐，以及经由所述中间体化合物以固相法合成式I化合物的方法。所述合成式I化合物的方法可大大缩短反应周期，且反应操作简便，可工业化大规模量产。
本发明提供一下技术方案实现上述目的：
一种式II-1所示的中间体化合物或其盐：
Figure PCTCN2019073370-appb-000002
其中R 1是氢或氨基保护基，所述氨基保护基是碱性氨基保护基或酸性氨基保护基。
在一些实施方案中，中间体化合物II-1的R 1为氢或碱性氨基保护基。在一些优选实施方案中，所述碱性氨基保护基为Fmoc或Tfa，更优选为Fmoc。在一些实施方案中，所述R 1为氢、Fmoc或Tfa；优选为Fmoc。在一些优选的实施方案中，中间体化合物II-1为：
Figure PCTCN2019073370-appb-000003
一种式II-2所示的中间体化合物或其盐：
Figure PCTCN2019073370-appb-000004
其中R 1是氢或氨基保护基，R 2是氨基保护基，所述的氨基保护基为碱性保护基或酸性保护基。
在一些实施方案中，中间体化合物II-2的R 1为氢或与R 2酸碱性相反的氨基保护基。 例如，R 1为氢或酸性氨基保护基，R 2为碱性氨基保护基；或者R 1为氢或碱性氨基保护基，R 2为酸性氨基保护基。在一些优选的实施方案中，R 1为氢或碱性氨基保护基，R 2为酸性氨基保护基。在一些实施方案中，R 1为氢或碱性氨基保护基，R 2为酸性氨基保护基。在一些实施方案中，所述碱性氨基保护基优选Fmoc或Tfa，更优选Fmoc。在一些优选的实施方案中，中间体化合物II-2的酸性氨基保护基为Cbz、Boc、Trt、DMB或PMB，更优选Boc。在一些实施方案中，R 1为氢、Fmoc或Tfa；R 2为Cbz、Boc、Trt、DMB或PMB。在一些实施方案中，R 1为Fmoc，R 2为Boc。在一些优选的实施方案中，中间体化合物II-2为：
Figure PCTCN2019073370-appb-000005
本发明中间体化合物II-1或II-2的盐可以是其钠盐或者钾盐，其可使用本领域常规使用的方法制备。
本发明的另一目的在于提供中间体化合物II-1和II-2的制备方法，该方法步骤简单、易于操作，无需过多的分离步骤，使反应可以进行公斤级量产。
本发明通过以下技术方案实现该目的：
一种中间体化合物II-1的制备方法，包括以下步骤：
使式SM-2-1所示化合物与化合物III的HX盐进行还原胺化反应，得到中间体化合物II-1：
Figure PCTCN2019073370-appb-000006
其中R 1如上文所定义；HX为三氟乙酸或盐酸；优选地，HX为盐酸。
本发明中，式III化合物或其HX盐可以按本领域常规方法合成。
在一些实施方案中，该还原胺化反应在溶剂中进行。
在一些实施方案中，化合物SM-2-1与式III化合物的HX盐的摩尔比为(1:1)～(5:1)，优选(1:1)～(2.5:1)。
在一些实施方案中，该还原胺化反应中式III化合物的HX盐的摩尔量与溶剂的体积 比为(1mol:5L)～(1mol:10L)，优选(1mol:5L)～(1mol:8L)，例如1mol:5L、1mol:6L、1mol:7L或1mol:8L。
在一些实施方案中，该还原胺化反应中所述溶剂是非质子性溶剂和醇溶剂的混合溶剂。在优选的实施方案中，所述非质子性溶剂选自二氯甲烷、四氢呋喃和***中的一种或多种；所述醇溶剂选自甲醇、乙醇和异丙醇中的一种或多种。在更优选的实施方案中，所述溶剂为二氯甲烷和甲醇的混合溶剂。
在一些实施方案中，所述还原胺化反应的溶剂中所述非质子性溶剂和醇溶剂的体积比为(1:5)～(10:1)，优选为(1.5:1)～(5:1)。
在一些实施方案中，该还原胺化反应中使用的还原剂为硼氢化钠或其衍生物，优选三乙酰氧基硼氢化钠。在一些实施方案中，式III化合物的HX盐与所述还原剂的摩尔比为(1:1)～(1:10)，优选为(1:2)～(1:8)，例如1:2.5、1:5或1:7。
在一些实施方案中，该还原胺化反应时间为0.5-24小时，优选为1-3小时。
在一些实施方案中，该还原胺化反应温度为-20℃-25℃,优选为0℃-15℃。
在一些实施方案中，化合物SM-2-1由以下氧化反应制备：将二乙二醇单甲醚在氧化体系中氧化为式SM-2-1所示的化合物
Figure PCTCN2019073370-appb-000007
在一些实施方案中，该氧化反应所得产物不经分离纯化直接用于中间体化合物II-1的制备。
在一些实施方案中，二乙二醇单甲醚与式III化合物的HX盐的摩尔比为(1:1)～(5:1)，优选为(1:3)～(3:1)，更优选为2.2:1。
在一些实施方案中，该氧化反应的氧化体系中包括氧化剂与有机碱。
在一些实施方案中，该氧化反应中二乙二醇单甲醚与所述氧化剂的摩尔比为(1:1)～(1:5)，优选为(1:1)～(1:3)，更优选为(1:1)～(1:2)。
在一些实施方案中，该氧化反应中二乙二醇单甲醚与所述有机碱的摩尔比为(1:2)～(1:10)，优选为(1:2)～(1:6)。
在一些实施方案中，该氧化反应中所述氧化剂为DMSO和草酰氯的组合，或DMSO和三氟乙酸酐的组合。在一些实施方案中，草酰氯和DMSO的摩尔比，或三氟乙酸酐与DSMO的摩尔比为(1:1)～(1:5)，优选为(1:1)～(1:3)，更优选为(1:1)～(1:2)，进一步优选为(1:1)～(1:1.5)。
在一些实施方案中，该氧化反应中有机碱为三乙胺。
在一些实施方案中，该氧化反应在非质子性溶剂中进行，该非质子性溶剂可以选自二氯甲烷、四氢呋喃和***中的一种或多种。在优选的实施方案中，所述非质子性溶剂为二氯甲烷。
在一些实施方案中，该氧化反应中二乙二醇单甲醚的摩尔量与所述非质子性溶剂的体积比为(1mol:1L)～(1mol:2L)。
在一些实施方案中，该氧化反应的温度为小于等于-30℃，优选小于等于-60℃，更优选小于等于-70℃。
在一些实施方案中，化合物SM-2-1由以下反应制备：将(2-甲氧基乙氧基)乙醛缩二甲醇在酸性体系中水解为式SM-2-1所示的化合物，
Figure PCTCN2019073370-appb-000008
在一些实施方案中，所述酸性体系中的酸包括但不限于有机酸与无机酸。
在一些实施方案中，所述有机酸包括但不限于三氟乙酸、乙酸、甲酸、对甲苯磺酸、富马酸和酒石酸，特别优选三氟乙酸。
在一些实施方案中，所述无机酸包括但不限于盐酸、硫酸和磷酸，特别优选硫酸。
在一些实施方案中，该反应体系为硫酸水溶液，硫酸水溶液含量为0.1％-50％，特别优选为0.1％-20％，再特别优选为1％-10％。
在一些实施方案中，该反应的温度为0℃-50℃，优选0℃-25℃，更优选0℃-10℃。
本发明进一步通过以下技术方案实现上述目的：
一种中间体化合物II-2的制备方法，该中间体化合物II-2由中间体化合物II-1进行氨基保护得到，
Figure PCTCN2019073370-appb-000009
其中R 1和R 2如上文所定义。
在一些实施方案中，中间体化合物II-1按照上文所述制备方法进行制备。在一些优选的实施方案中，中间体化合物II-1在制得后不经分离纯化直接用于中间体化合物II-2的制备。
在一些实施方案中，使用N,N-二异丙基乙胺和二碳酸二叔丁酯对中间体化合物II-1的亚胺基进行保护。在一些实施方案中，中间体化合物II-1与N,N-二异丙基乙胺的摩尔比为(1:1)～(1:5)。在一些实施方案中，中间体化合物II-1与二碳酸二叔丁酯的摩尔比为(1:1)～(1:5)。
在一些实施方案中，本发明中间体化合物II-2的制备方法进一步包括将得到的中间体化合物II-2进行分离纯化的步骤。
本发明的又一目的在于提供式I化合物的固相合成方法，整条工艺合成周期短，操作步骤简便，适于大规模生产。
本发明通过以下技术方案实现该目的：
一种式I化合物的固相合成方法，包括如下步骤：
1)将固相载体固定的化合物M-1-1与中间体化合物II-2进行缩合反应，得到固相载体固定的多肽化合物M-2，而后脱除R 1保护基，得到固相载体固定的化合物M-2-1，
Figure PCTCN2019073370-appb-000010
2)依次使用氨基酸衍生物SM-3、氨基酸衍生物SM-4、氨基酸衍生物SM-5为原料进行以下的反应，最终得到固相载体固定的化合物M-5，
Figure PCTCN2019073370-appb-000011
3)向步骤2)所得固相载体固定的化合物M-5中加入裂解液进行脱保护、裂解，得化合物I的粗品，
Figure PCTCN2019073370-appb-000012
其中，R 1和R 2如上文所定义；R y、R 3、R 4、R 5是碱性氨基保护基或酸性氨基保护基；且R 3和R 4同为酸性氨基保护基时，R y和R 5同为碱性氨基保护基；R 3和R 4同为碱性氨基保护基时，R y和R 5同为酸性氨基保护基。
在一些实施方案中，R 3和R 4为碱性氨基保护基，R y和R 5为酸性氨基保护基。
在一些实施方案中，R 3和R 4独立地选自Fmoc和Tfa的一种或多种；R y和R 5独立地选自Cbz、Boc、Trt、DMB和PMB的一种或多种。
在一些实施方案中，R 3和R 4为Fmoc；R y和R 5为Boc。
在一些实施方案中，R 2与R y、R 5同为酸性氨基保护基或碱性氨基保护基。在一些实施方案中，当R 1为氨基保护基时，其与R 3和R 4同为酸性氨基保护基或碱性氨基保护基。
在一些实施方案中，R 3和R 4同为酸性氨基保护基，R 2、R y和R 5同为碱性氨基保护基。在一些实施方案中，R 3和R 4同为碱性氨基保护基，R 2、R y和R 5同为酸性氨基保护基。在一些实施方案中，当R 1为氨基保护基时，R 1、R 3和R 4同为酸性氨基保护基，R 2、R y和R 5同为碱性氨基保护基。在一些实施方案中，当R 1为氨基保护基时，R 1、R 3和R 4同为碱性氨基保护基，R 2、R y和R 5同为酸性氨基保护基。优选的，R 1、R 3和R 4为碱性氨基保护基，R 2、R y和R 5为酸性氨基保护基。
在一些实施方案中，步骤3)后任选包括步骤4)：对步骤3)所得化合物I粗品进行分离提纯，得纯品化合物I。
在一些实施方案中，中间体化合物II-2通过上文所述的制备方法得到。
在一些实施方案中，步骤1)的化合物M-1-1由以下方法制备：将化合物SM-1以固相载体固定，而后脱除哌啶环亚胺基的保护基Rx，得固相载体固定的化合物M-1-1，
Figure PCTCN2019073370-appb-000013
其中，R x是酸碱性与R y相反的氨基保护基；优选地，R x是碱性氨基保护基；更优选 R x为Fmoc或Tfa；特别优选R x为Fmoc。
在一些实施方案中，式SM-1所示的化合物与固相载体的摩尔比为1:(2～4)，优选为1:3。
在一些实施方案中，所述固相载体为Wang树脂或2-氯三苯甲基氯树脂，优选2-氯三苯甲基氯树脂。优选地，Wang树脂取代度为0.3-1.0mmol/g，更优选为0.4-0.7mmol/g；2-氯三苯甲基氯树脂取代度为0.2-1.6mmol/g，优选为0.7-1.2mmol/g，优选为1.0-1.2mmol/g。采用该固相载体，化合物收率高，制备得到的化合物纯度高，容易纯化，成本低。
在一些实施方案中，步骤1)和步骤2)的缩合反应在溶剂中进行，所述溶剂选自N,N-二甲基甲酰胺、二氯甲烷中的一种或两种。当反应在混合溶剂中进行时，N,N-二甲基甲酰胺与二氯甲烷的体积比为(1-5):1，优选为(1-3):1。
在一些实施方案中，步骤1)和步骤2)中使用缩合剂进行多肽的缩合，缩合剂可以是以下组合中的一种或多种：a)HBTU、Cl-HoBt和DIEA；b)DIC和Cl-HoBt；c)PyBOP、Cl-HoBt和DIEA；d)HBTU、Oxyma、DIEA，其中a)组、c)组、d)组各成分比可以分别是1:1:1.1，b)组各成分比可以是1:1.1。在优选的实施方案中，使用HBTU、Cl-HoBt和DIEA，或DIC和Cl-HoBt的组合作为缩合剂。
在一些实施方案中，步骤1)和步骤2)中使用缩合剂进行多肽的缩合，缩合剂可以是DIC和HoBt的组合作为缩合剂。
本发明中使用本领域常规使用的方法对氨基、亚氨基进行保护基团保护和脱保护。在优选的实施方案中，对于Fmoc保护基，可使用20％哌啶/DMF溶液(DBLK)脱除；对于Boc保护基，可使用TFA、HCl、或HF脱除，优选TFA。
在一些实施方案中，当R y、R 2和R 5为酸性氨基保护基时，步骤3)中裂解液含有50％-100％TFA，或者进一步含有0％-10％TIS、0％-10％H 2O、0％-10％TES中的1种，2种或2种以上，优选由90％TFA/5％TIS/5％H 2O(v/v/v)或95％TFA/5％H 2O(v/v)组成，以使M-5树脂裂解完全，提高终产物纯度。
在一些实施方案中，步骤3)中裂解液与步骤2)所得载体固定的多肽化合物M-5的比例为(6-10)mL:1g，优选为8mL:1g。
在一些实施方案中，步骤3)的裂解反应首先在低温下反应15-30min，然后升温至室温并反应至完全。
在一些实施方案中，步骤3)裂解、任选的步骤4)前后包括使用醚类溶剂使式I化合物粗品沉淀的步骤。在优选的实施方案中，醚类溶剂是无水***或甲基叔丁基醚。
在一些实施方案中，上述使用醚类溶剂沉淀式I化合物粗品的步骤在低温下进行，例如0℃、-10℃等。
在一些的实施方案中，步骤4)的分离提纯使用反相高效液相色谱进行。
本说明书和权利要求书所使用的碱性氨基保护基是指在碱性条件下能被脱除的氮上保护基，例如：Fmoc或Tfa等；酸性氨基保护基是指在酸性条件下能被脱除的氮上保护基，例如：Cbz、Boc、Trt、DMB或PMB等。本领域技术人员可以参照本领域常用教科书Greene's Protective Groups in Organic Synthesis(4th Edition)等进行适当的选择和操作，选择性或完全脱除一个或多个保护基。
本说明书和权利要求书所使用的缩写的含义如下所示：
Figure PCTCN2019073370-appb-000014
本发明有益效果：
1、使用本发明的中间体化合物II-2以固相法制备式I化合物可大大缩短反应周期，且反应操作简便，化合物收率高，生产成本低，可工业化大规模量产。
2、通过优化工艺条件，本发明中间体化合物II-1或II-2的合成方法步骤简单、易于操作，无需过多的分离步骤，使反应可以进行公斤级的量产；特别是，当以二乙二醇单甲醚和化合物III的HX盐作为起始反应物时，通过反应流程优化，能够采用温和的反应条件以及短的反应时间，不经过任何分离提纯步骤，以“一锅法”制备得到本发明的中间体化合物II-2。
3、本发明式I化合物的固相合成方法，整条工艺合成周期短，操作步骤简便，适于大规模生产。
4、使用本发明中间体II-1及II-2，将其应用于固相合成方法制备的式I的多肽化合 物，通过优化反应条件，产物纯度可达99.5％以上。
附图说明
图1、本发明实施例5所得粗品式I化合物的HPLC图谱。
图2、本发明实施例5所得纯品式I化合物的HPLC图谱。
具体实施方式
以下实施例中使用的原料均为市售产品。
实施例中所使用仪器设备信息：
Figure PCTCN2019073370-appb-000015
实验例1化合物III-a盐酸盐的制备
Figure PCTCN2019073370-appb-000016
在300L的反应釜中加入150L乙酸乙酯，冷却至-10-0℃。向体系中通入氯化氢气体，控制体系温度为-10-0℃。将化合物Fmoc-D-Lys(Boc)-OH(14kg，29.88mol)与乙酸乙酯(50L)的混合物加入到上述反应体系中，搅拌下自然恢复至室温，3小时后，停止反应，放出反应液，离心，滤饼用乙酸乙酯淋洗3次，离心，将滤饼烘干，得化合物III-a盐酸盐(11.5kg,产率95.1％)。
实验例2中间体化合物II-1-a和II-2-a的制备
Figure PCTCN2019073370-appb-000017
将草酰氯(1.65kg,13.0mol)溶于二氯甲烷(15L)中，氮气氛围下冷却至<-70℃，加入DMSO(1.47kg,18.8mol)的二氯甲烷溶液(1500mL)，控温<-70℃。加完后，保持该温度下搅拌60min，加入二乙二醇单甲醚(1.5kg，12.5mol)的二氯甲烷溶液(1500mL)，控温<-70℃。加完后，保持该温度下搅拌60min。继续加入三乙胺(2.55kg,25.2mol)，控温<-70℃。加完后，缓慢升至室温，搅拌20min。得2-(2-甲氧基乙氧基)乙醛的二氯甲烷(25L)溶液待用。
将化合物式III-a盐酸盐(2.3kg,5.7mol)的甲醇(5L)溶液加入至上述得到的2-(2-甲氧基乙氧基)乙醛的二氯甲烷溶液(25L)，室温搅拌30min，加入三乙酰氧基硼氢化钠(3.0kg,14.2mol)，反应体系在室温反应1h。LC-MS监测反应，中间体II-1-a化合物生成后将反应液冷却至0℃。将N,N-二异丙基乙胺(2.2kg,17.1mol)和二碳酸二叔丁酯(1.36kg,6.2mol)先后加入反应液中。加毕，升温至室温反应2h。减压蒸馏除去二氯甲烷与甲醇，向此粗品中加入饱和碳酸钠的水溶液，搅拌30min后。用甲基叔丁基醚萃取3次，之后水相再用1N盐酸调节至pH 3-4，用乙酸乙酯萃取水相两次。合并乙酸乙酯相，分别用1N HCl和饱和食盐水洗涤3次，无水硫酸钠干燥，抽滤，向滤液中直接加入硅胶浓缩至干，得粗品5.0kg，直接柱层析，得中间体化合物II-2-a(1.05kg，收率32.39％)。
ESI-MS(m/z):471.2(M-Boc+H) +
1H NMR(400MHz,DMSO-d6)δ12.51(s,1H),7.90(d,J＝7.5Hz,2H),7.73(d,J＝7.4Hz,2H),7.64(d,J＝8.0Hz,1H),7.42(t,J＝7.4Hz,2H),7.33(t,J＝7.2Hz,2H),4.30–4.19(m,3H),3.92(s,1H),3.52–3.38(m,6H),3.30–3.21(m,5H),3.17-3.12(m,2H),1.78–1.55(m,2H),1.52-1.41(m,2H),1.37(s,9H),1.33-1.21(m,2H).
实验例3中间体化合物II-1-a和II-2-a的制备
将草酰氯(2.39kg,18.8mol)溶于二氯甲烷(15L)中，氮气氛围下冷却至<-70℃，加入DMSO(1.95kg,25.0mol)的二氯甲烷溶液(1500mL)，控温<-70℃。加完后，保持该温度下搅拌60min，加入二乙二醇单甲醚(1.5kg，12.5mol)的二氯甲烷溶液(1500mL)，控温<-70℃。加完后，保持该温度下搅拌60min。继续加入三乙胺(5.10kg,50.4mol)， 控温<-70℃。加完后，缓慢升至室温，搅拌20min。得2-(2-甲氧基乙氧基)乙醛的二氯甲烷(25L)溶液待用。
将化合物式III-a盐酸盐(2.3kg,5.7mol)的甲醇(16.5L)溶液加入至上述得到的2-(2-甲氧基乙氧基)乙醛的二氯甲烷溶液(25L)，室温搅拌30min，加入三乙酰氧基硼氢化钠(3.0kg,14.2mol)，反应体系在室温反应1h。LC-MS监测反应，中间体II-1-a化合物生成后将反应液冷却至0℃。将N,N-二异丙基乙胺(1.5kg,11.4mol)和二碳酸二叔丁酯(3.75kg,17.1mol)先后加入反应液中。加毕，升温至室温反应2h。减压蒸馏除去二氯甲烷与甲醇，向此粗品中加入饱和碳酸钠的水溶液，搅拌30min后。用甲基叔丁基醚萃取3次，之后水相再用1N盐酸调节至pH 3-4，用乙酸乙酯萃取水相两次。合并乙酸乙酯相，分别用1N HCl和饱和食盐水洗涤3次，无水硫酸钠干燥，抽滤，向滤液中直接加入硅胶浓缩至干，得粗品5.0kg，直接柱层析，得中间体化合物II-2-a(1.09kg，收率33.74％)。
实验例4中间体化合物II-1-a和II-2-a的制备
将草酰氯(3.30kg,25.0mol)溶于二氯甲烷(15L)中，氮气氛围下冷却至<-70℃，加入DMSO(2.94kg,37.5mol)的二氯甲烷溶液(1500ml)，控温<-70℃。加完后，保持该温度下搅拌60min，加入二乙二醇单甲醚(1.5kg，12.5mol)的二氯甲烷溶液(1500ml)，控温<-70℃。加完后，保持该温度下搅拌60min。继续加入三乙胺(6.32kg,62.5mol)，控温<-70℃。加完后，缓慢升至室温，搅拌20min，得2-(2-甲氧基乙氧基)乙醛的二氯甲烷(25L)溶液待用。
将化合物式III-a盐酸盐(2.3kg,5.7mol)的甲醇(8.5L)溶液加入至上述得到的2-(2-甲氧基乙氧基)乙醛的二氯甲烷溶液(25L)，室温搅拌30min，加入三乙酰氧基硼氢化钠(3.0kg,14.2mol)，反应体系在室温反应1h。LC-MS监测反应，中间体II-1-a化合物生成后将反应液冷却至0℃。将N,N-二异丙基乙胺(3.7kg,28.5mol)和二碳酸二叔丁酯(6.25kg,28.5mol)先后加入反应液中。加毕，升温至室温反应2h。减压蒸馏除去二氯甲烷与甲醇，向此粗品中加入饱和碳酸钠的水溶液，搅拌30min后。用甲基叔丁基醚萃取3次，之后水相再用1N盐酸调节至pH 3-4，用乙酸乙酯萃取水相两次。合并乙酸乙酯相，分别用1N HCl和饱和食盐水洗涤3次，无水硫酸钠干燥，抽滤，向滤液中直接加入硅胶浓缩至干，得粗品5.0Kg，直接柱层析，得中间体化合物II-2-a(0.95kg，收率29.30％)。
实施例5式I化合物的制备
(1)M-1-a树脂的制备
Figure PCTCN2019073370-appb-000018
称取1000.0g 2-氯三苯甲基氯树脂(取代值：1.1mmol/g)加入至20L多肽反应器中，同时加入2L DCM洗涤并溶胀树脂45min。称取化合物4-(叔丁氧羰基氨基)-1-芴甲氧基哌啶-4-羧酸SM-1-a(1650mmol，769.7g)，加7L DCM溶解，将溶解后的反应液加入至树脂中，待树脂与反应液搅拌均匀后，向树脂反应液中加入DIEA(4950mmol，818mL)，25℃反应2h。向反应液中继续加入800mL甲醇用于封闭未反应的活性位点，反应45min。反应完成后，排干溶液，使用4×10L DMF溶液洗涤树脂，洗涤完成后，取部分树脂，使用哌啶进行脱保护，利用紫外分光度法测定哌啶脱保护液中Fmoc量，计算得到M-1-a树脂的取代度为0.79mmol/g。
(2)M-1-1-a树脂的制备
Figure PCTCN2019073370-appb-000019
使用10L 20％哌啶/DMF溶液处理步骤(1)中得到的M-1-a树脂5min，排干混合液，再加入10L 20％哌啶/DMF溶液继续处理15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂红棕色，脱保护完全,得M-1-1-a树脂。
(3)M-2-a树脂的制备
Figure PCTCN2019073370-appb-000020
称取中间体化合物II-2-a(2069mmol，1311.7g)、Cl-HoBt(2069mmol，351.3g)、HBTU(2069mmol，784.5g)溶解于7L DMF溶液中，氮气保护下将该溶液冰浴至0-5℃，然后加入DIEA(2275.9mmol，376mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-2-a树脂。
(4)M-2-1-a树脂的制备
Figure PCTCN2019073370-appb-000021
使用2×10L 20％哌啶/DMF溶液分别处理步骤(3)中所得M-2-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝紫色，脱保护完全，得M-2-1-a树脂。
(5)M-3-a树脂的制备
Figure PCTCN2019073370-appb-000022
称取SM-3-a化合物(3270mmol，1156.7g)、Cl-HoBt(3270mmol，555.2g)溶解于7L体积比1:1的DMF/DCM溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(3597mmol，557ml)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-3-a树脂。
(6)M-3-1-a树脂的制备
Figure PCTCN2019073370-appb-000023
使用2×10L 20％哌啶/DMF溶液分别处理M-3-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-3-1-a树脂。
(7)M-4-a树脂的制备
Figure PCTCN2019073370-appb-000024
称取SM-4-a化合物(3270mmol，1267.8g)、Cl-HoBt(3270mmol，554.9g)溶解于7L体积比1:1的DMF/DCM溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(3597mmol，557ml)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-4-a树脂。
(8)M-4-1-a树脂的制备
Figure PCTCN2019073370-appb-000025
使用2×10L 20％哌啶/DMF溶液分别处理步骤(7)中所得M-4-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝紫色，脱保护完全，得M-4-1-a树脂。
(9)M-5-a树脂的制备
Figure PCTCN2019073370-appb-000026
称取SM-5-a化合物(3270mmol，867.8g)、Cl-HoBt(3270mmol，555.1g)溶解于7L体积比1:1的DMF/DCM溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(3597mmol，557ml)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全。使用3×10L DCM，3×10L甲醇交替洗涤树脂，洗涤完成后，将树脂真空干燥至恒重，最终得到1967.1g M-5-a树脂，收率为96.7％。
(10)式I化合物的制备
Figure PCTCN2019073370-appb-000027
称取M-5-a树脂1900.1g加入至20L多肽裂解釜中，加入15L预先降温至0℃左右的裂解液95％TFA/5％H 2O，室温条件下搅拌反应1.5h。将反应液过滤至150L预先降温至-10℃左右的甲基叔丁基醚，有白色固体产生，-10℃下搅拌该白色淤浆物30min，随后将白色淤浆物离心，离心机参数设置为3500r/min，离心5min，离心完成后，弃掉上清液，收集白色淤浆物，加入新鲜的20L新鲜的甲基叔丁基醚，重复上述离心过程，收集白色淤浆物,真空干燥至恒重，最终获得式I化合物粗品981.9g，收率为98.1％，纯度为92.55％。该式I化合物粗品的HPLC谱见图1。
99.3g式I化合物粗品经制备型HPLC纯化，最终得到纯品42.7g，收率为43％，纯度为99.77％。该纯化后的化合物I的HPLC谱见图2。
ESI-MS(m/z):782.5(M+H) +
实验例6式I化合物的制备
(1)M-1-a树脂的制备
称取2-氯三苯甲基氯树脂(取代值：1.1mmol/g)1010.8g加入至20L多肽反应器中，同时加入2L DCM洗涤并溶胀树脂45min。称取SM-1-a化合物4-(叔丁氧羰基氨基)-1-芴甲氧基哌啶-4-羧酸(1667mmol，778.2g)，加7L DCM溶解，将溶解后的反应液加入至树脂中，待树脂与反应液搅拌均匀后，向树脂反应液DIEA(5003mmol，827mL)，25℃反应2h。向反应液中继续加入800mL甲醇用于封闭未反应的活性位点，反应45min。反应完成后，排干溶液，使用4×10L DMF溶液洗涤树脂，洗涤完成后，取部分树脂，使用哌啶进行脱保护，利用紫外分光度法测定哌啶脱保护液中Fmoc量，计算得到M-1-a树脂的取代度为0.77mmol/g。
(2)M-1-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理M-1-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂红棕色，脱保护完全，得M-1-1-a树脂。
(3)M-2-a树脂的制备
称取II-2-a中间体化合物(1917mmol，1093.0g)、Cl-HoBt(1917mmol，343.6g)、HBTU(1917mmol，766.3g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至 0-5℃，然后加入DIEA(2108mmol，367mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得的树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-2-a树脂。
(4)M-2-1-a树脂的制备
使用2×10L20％哌啶/DMF溶液分别处理步骤(3)中所得M-2-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-2-1-a树脂。
(5)M-3-a树脂的制备
称取SM-3-a化合物(3032.4mmol，1071.1g)、Cl-HoBt(3032.4mmol，514.6g)、HBTU(3032.4mmol，1149.8g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIEA(3322.4mmol，550mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-3-a树脂。
(6)M-3-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理步骤(5)中所得M-3-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-3-1-a树脂。
(7)M-4-a树脂的制备
称取SM-4-a化合物(3032.4mmol，1175.7g)、Cl-HoBt(3270mmol，514.5g)、HBTU(3032.4mmol，1150.3g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIEA(3322.4mmol，550mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-4-a树脂。
(8)M-4-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理步骤(7)中所得M-4-a树脂5min和10min，脱除Fmoc保护基，然后使用5×1L DMF洗涤树脂，Kaiser Test，树脂蓝紫色，脱保护完全，得M-4-1-a树脂。
(9)M-5-a树脂的制备
称取SM-5-a化合物(3270mmol，867.8g)、Cl-HoBt(3270mmol，555.1g)、HBTU(3032.4mmol，1151.7g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIEA(3322.4mmol，550mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-5-a树脂。使用3×10L DCM，3×10L甲醇交替洗涤树脂，洗涤完成 后，将树脂置于真空干燥中干燥至恒重，最终得到1941.9g树脂M-5-a树脂，收率为92.1％。
(10)式I化合物的制备
称取上述所得M-5-a树脂1905.4g加入至20L多肽裂解釜中，加入15L预先降温至0℃左右的裂解液95％TFA/2.5％H 2O/2.5％TIS，室温条件下搅拌反应1.5h。将反应液过滤至150L预先降温至-10℃左右的甲基叔丁基醚，有白色固体产生，-10℃下搅拌该白色淤浆物30min，随后将白色淤浆物离心，离心机参数设置为3500r/min，离心5min，离心完成后，弃掉上清液，收集白色淤浆物，加入新鲜的20L新鲜的甲基叔丁基醚，重复上述离心过程，收集白色淤浆物，真空干燥至恒重，最终获得式I化合物粗品984.1g，收率为98.1％，纯度为92.54％。
将103.2g式I化合物粗品经制备型HPLC纯化，最终得到纯品42.31g，收率41％，纯度为99.75％。
实验例7式I化合物的制备
(1)M-1-a树脂的制备
称取2-氯三苯甲基氯树脂(取代值：1.1mmol/g)1000.1g加入至20L多肽反应器中，同时加入2L DCM洗涤并溶胀树脂45min。称取SM-1-a化合物4-(叔丁氧羰基氨基)-1-芴甲氧基哌啶-4-羧酸(1651mmol，771.0g)，加7L DCM溶解，将溶解后的反应液加入至树脂中，待树脂与反应液搅拌均匀后，向树脂反应液DIEA(4954.8mmol，818mL)，25℃反应2h。向反应液中继续加入800mL甲醇用于封闭未反应的活性位点，反应45min。反应完成后，排干溶液，使用4×10L DMF溶液洗涤树脂，洗涤完成后，取部分树脂，使用哌啶进行脱保护，利用紫外分光度法测定哌啶脱保护液中Fmoc量，计算得M-1-a树脂的取代度为0.76mmol/g。
(2)M-1-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理步骤(1)中所得树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，去除Fmoc副产物以及残余哌啶，Kaiser Test，树脂红棕色，脱保护完全，得M-1-1-a树脂。
(3)M-2-a树脂的制备
称取II-2-a中间体化合物(1898mmol，1230.7g)、Cl-HoBt(1898mmol，322.1g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(2087mmol，323mL)搅拌反应5min。5min后，将反应溶液加入上一步所得的树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-2-a树脂。
(4)M-2-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理步骤(3)中所得M-2-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全,得M-2-1-a树脂。
(5)M-3-a树脂的制备
称取SM-3-a化合物(3273mmol，1155.7g)、Cl-HoBt(3032.4mmol，514.6g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(2087mmol，323mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-3-a树脂。
(6)M-3-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理M-3-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-3-1-a树脂。
(7)M-4-a树脂的制备
称取SM-4-a化合物(3273mmol，1267.4g)、Cl-HoBt(3273mmol，555.5g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(2087mmol，323mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-4-a树脂。
(8)M-4-1-a树脂的制备
使用2×10L 20％哌啶/DMF溶液分别处理步骤(7)中所得M-4-a树脂5min和15min，脱除Fmoc保护基，然后使用5×10L DMF洗涤树脂，Kaiser Test，树脂蓝紫色，脱保护完全，得M-4-1-a树脂。
(9)M-5-a树脂的制备
称取SM-5-a化合物(3273mmol，868.6g)、Cl-HoBt(3273mmol，555.2g)溶解于7L DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(2087mmol，323mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×10L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-5-a树脂。使用3×10L DCM，3×10L甲醇交替洗涤树脂，洗涤完成后，将树脂置于真空干燥中干燥至恒重，最终得到1987.4g M-5-a树脂，收率为98.7％。
(10)式I化合物的制备
称取上述所得M-5-a树脂1911.3g加入至20L多肽裂解釜中，加入15L预先降温至0℃左右的裂解液95％TFA/5％H 2O，室温条件下搅拌反应1.5h。将反应液过滤至150L预先 降温至-10℃左右的甲基叔丁基醚，有白色固体产生，-10℃下搅拌该白色淤浆物30min，随后将白色淤浆物离心，离心机参数设置为3500r/min，离心5min，离心完成后，弃掉上清液，收集白色淤浆物，加入新鲜的20L新鲜的甲基叔丁基醚，重复上述离心过程，收集白色淤浆物，真空干燥至恒重，最终获得式I化合物粗品971.2g，收率为97.7％，纯度为92.71％。
将105.1g式I化合物粗品经制备型HPLC纯化，最终得到纯品43.1g，收率41％，纯度为99.70％。
实验例8式I化合物的制备
(1)M-1-a树脂的制备
称取Wang树脂(取代值：0.45mmol/g)100.1g加入至5L多肽反应器中，同时加入1L DCM洗涤并溶胀树脂45min。称取SM-1-a化合物4-(叔丁氧羰基氨基)-1-芴甲氧基哌啶-4-羧酸(135mmol，63.1g)，Cl-HoBt(135mmol，22.9g)溶解于700mLDMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(148.5mmol，23mL)将溶解后的反应液加入至树脂中，待树脂与反应液搅拌均匀后，向树脂反应液加入4-二甲氨基吡啶(27mmol，2.44g)，25℃反应4h。向反应液中加入封闭液封闭未反应的活性位点，反应60min。反应完成后，排干溶液，使用4×1L DMF溶液洗涤树脂，洗涤完成后，取部分树脂，利用紫外分光度法测定哌啶脱保护液中Fmoc量，计算得M-1-a树脂的取代度为0.22mmol/g。
(2)M-1-1-a树脂的制备
使用2×100mL20％哌啶/DMF溶液分别处理M-1-a树脂5min和15min，脱除Fmoc保护基，然后使用5×100mL DMF洗涤树脂，Kaiser Test，树脂红棕色，脱保护完全，得M-1-1-a树脂。
(3)M-2-a树脂的制备
称取II-2-a中间体化合物(66mmol，37.6g)、Cl-HoBt(66mmol，11.2g)溶解于700mLDMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(72.6mmol，12mL)搅拌反应5min。5min后，将反应溶液加入上一步所得的树脂中，室温反应1.5h，抽干树脂，使用3×1L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-2-a树脂。
(4)M-2-1-a树脂的制备
使用2×1L 20％哌啶/DMF溶液分别处理步骤(3)中所得M-2-a树脂5min和15min，脱除Fmoc保护基，然后使用5×1L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-2-1-a树脂。
(5)M-3-a树脂的制备
称取SM-3-a化合物(66mmol，23.3g)、Cl-HoBt(66mmol，12.3g)溶解于700ml DMF 溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(72.6mmol，12mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×1L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-3树脂。
(6)M-3-1-a树脂的制备
使用2×1L 20％哌啶/DMF溶液分别处理步骤(5)中所得M-3树脂5min和15min，脱除Fmoc保护基，然后使用5×1L DMF洗涤树脂，Kaiser Test，树脂蓝色，脱保护完全，得M-3-1-a树脂。
(7)M-4-a树脂的制备
称取SM-4-a化合物(66mmol，25.6g)、Cl-HoBt(66mmol，11.9g)溶解于700mL DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(72.6mmol，12ml)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×1L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-4-a树脂。
(8)M-4-1-a树脂的制备
使用2×1L 20％哌啶/DMF溶液分别处理步骤(7)中所得M-4-a树脂5min和15min，脱除Fmoc保护基，然后使用5×1L DMF洗涤树脂，Kaiser Test，树脂蓝紫色，脱保护完全，得M-4-1-a树脂。
(9)M-5-a树脂的制备
称取SM-5-a化合物(66mmol，17.5g)、Cl-HoBt(66mmol，12.0g)溶解于700mL DMF溶液中，氮气保护条件下将该溶液冰浴至0-5℃，然后加入DIC(72.6mmol，12mL)搅拌反应5min。5min后，将反应溶液加入至上一步所得树脂中，室温反应1.5h，抽干树脂，使用3×1L DMF洗涤树脂，Kaiser Test，树脂淡黄色，缩合反应完全，得M-5-a树脂。使用3×1L DCM，3×1L甲醇交替洗涤树脂，洗涤完成后，将树脂置于真空干燥中干燥至恒重，最终得到122.7g M-5-a树脂，收率为95.3％。
(10)式I化合物的制备
称取上述所得M-5-a树脂100.1g加入至5L多肽裂解釜中，加入800ml预先降温至0℃左右的裂解液95％TFA/5％H 2O，室温条件下搅拌反应1.5h。将反应液过滤至10L预先降温至-10℃左右的甲基叔丁基醚，有白色固体产生，-10℃下搅拌该白色淤浆物30min，随后将白色淤浆物离心，离心机参数设置为3500r/min，离心5min，离心完成后，弃掉上清液，收集白色淤浆物，加入新鲜的2L新鲜的甲基叔丁基醚，重复上述离心过程，收集白色淤浆物，真空干燥至恒重，最终获得式I化合物18.7g，收率为79.1％.
将10g式I化合物粗品经制备型HPLC纯化，最终得到纯品3.51g，收率35.1％，纯度为99.50％

Claims (21)

  1. 一种式II-1所示的中间体化合物或其盐：
    Figure PCTCN2019073370-appb-100001
    其中，R 1是氢或氨基保护基，所述氨基保护基是碱性氨基保护基或酸性氨基保护基；
    优选地，所述R 1为氢或碱性氨基保护基；
    优选地，所述碱性氨基保护基为Fmoc或Tfa；更优选地，所述碱性氨基保护基为Fmoc。
  2. 一种式II-2所示的中间体化合物或其盐：
    Figure PCTCN2019073370-appb-100002
    其中，R 1是氢或氨基保护基，R 2是氨基保护基，所述氨基保护基是碱性氨基保护基或酸性氨基保护基；
    优选地，所述R 1为氢或与R 2酸碱性相反的氨基保护基；
    更优选地，所述R 1为氢或碱性氨基保护基，R 2为酸性氨基保护基；
    更优选地，所述碱性氨基保护基为Fmoc或Tfa，所述酸性氨基保护基为Cbz、Boc、Trt、DMB或PMB；更优选地，所述碱性氨基保护基为Fmoc，所述酸性氨基保护基为Boc。
  3. 一种如权利要求1所述的中间体化合物II-1的制备方法，其特征在于，包括以下步骤：使式SM-2-1所示化合物与式III·HX所示化合物III的盐进行还原胺化反应，得到中间体化合物II-1：
    Figure PCTCN2019073370-appb-100003
    其中，R 1如权利要求1所定义；优选地，HX选自三氟乙酸和盐酸。
  4. 根据权利要求3所述的中间体化合物II-1的制备方法，其中所述还原胺化反应在溶剂中进行，并且所述化合物III的HX盐的摩尔量与所述溶剂的体积比优选为(1mol:4L)～(1mol:10L)，更优选为(1mol:4L)～(1mol:8L)；
    优选地，所述溶剂是非质子性溶剂和醇类溶剂的混合溶剂；更优选地，所述非质子性溶剂选自二氯甲烷、四氢呋喃或***中的一种或多种，所述醇类溶剂选自甲醇、乙醇和异丙醇中的一种或多种；更优选地，所述溶剂为二氯甲烷和甲醇的混合溶剂；更优选地，所述非质子性溶剂和醇溶剂的体积比为(1:5)～(10:1)，更优选为(1.5:1)～(5:1)。
  5. 根据权利要求3或4所述的中间体化合物II-1的制备方法，其中所述还原胺化反应中使用硼氢化钠或其衍生物为还原剂；优选地，使用三乙酰氧基硼氢化钠为还原剂；
    更优选地，所述化合物III·HX与还原剂的摩尔比为(1:1)～(1:10)，更优选(1:2)～(1:8)。
  6. 根据权利要求3～5中任一项所述的中间体化合物II-1的制备方法，其中所述化合物SM-2-1由以下氧化反应制备：将二乙二醇单甲醚在氧化体系中氧化为式SM-2-1所示的化合物，
    Figure PCTCN2019073370-appb-100004
  7. 根据权利要求6所述的中间体化合物II-1的制备方法，其中所述氧化反应产物不经分离纯化直接用于中间体化合物II-1的制备。
  8. 根据权利要求6或7所述的中间体化合物II-1的制备方法，其中所述氧化反应中的氧化体系包括氧化剂与有机碱；
    优选地，所述二乙二醇单甲醚与所述氧化剂的摩尔比为(1:1)～(1:5)，更优选(1:1)～(1:3)；
    优选地，所述二乙二醇单甲醚与所述有机碱的摩尔比为(1:2)～(1:10)，更优选(1:2)～(1:6)；
    优选地，所述氧化剂为DMSO和草酰氯的组合或DMSO和三氟乙酸酐的组合，进一 步优选地，所述草酰氯和DMSO的摩尔比或三氟乙酸酐与DSMO的摩尔比为(1:1)～(1:5)，更优选(1:1)～(1:3)；
    优选地，所述有机碱为三乙胺。
  9. 根据权利要求6～8中任一项所述的中间体化合物II-1的制备方法，其中所述氧化反应在非质子性溶剂中进行；优选地，所述非质子性溶剂选自二氯甲烷、四氢呋喃和***中的一种或多种；更优选二氯甲烷；
    优选地，所述二乙二醇单甲醚的摩尔量与所述非质子性溶剂的体积比为(1mol:1L)～(1mol:2L)。
  10. 根据权利要求6～9中任一项所述的中间体化合物II-1的制备方法，其中所述氧化反应的温度为小于等于-30℃，优选小于等于-60℃，更优选小于等于-70℃。
  11. 一种如权利要求2所述的中间体化合物II-2的制备方法，其中所述中间体化合物II-2由如权利要求1所述的中间体化合物II-1进行氨基保护得到，
    Figure PCTCN2019073370-appb-100005
    其中R 1和R 2如权利要求2所定义。
  12. 根据权利要求11所述的中间体化合物II-2的制备方法，其中所述中间体化合物II-1按照权利要求3～10中任一项所述的制备方法制备；优选地，所述中间体化合物II-1在制得后不经分离纯化直接用于中间体化合物II-2的制备。
  13. 一种下式所示的化合物I的固相合成方法，
    Figure PCTCN2019073370-appb-100006
    包括如下步骤：
    1)将固相载体固定的化合物M-1-1与中间体化合物II-2进行缩合反应，得到固相载体固定的多肽化合物M-2，而后脱除R 1保护基，得到固相载体固定的化合物M-2-1，
    Figure PCTCN2019073370-appb-100007
    2)依次使用氨基酸衍生物SM-3、氨基酸衍生物SM-4、氨基酸衍生物SM-5为原料进行以下的反应，最终得到固相载体固定的化合物M-5，
    Figure PCTCN2019073370-appb-100008
    3)向步骤2)所得固相载体固定的化合物M-5中加入裂解液进行脱保护、裂解，得化合物I的粗品，
    Figure PCTCN2019073370-appb-100009
    其中，R 1和R 2如权利要求2所定义；R y、R 3、R 4、R 5是碱性氨基保护基或酸性氨基保护基；且R 3和R 4同为酸性氨基保护基时，R y和R 5同为碱性氨基保护基；R 3和R 4同为碱性氨基保护基时，R y和R 5同为酸性氨基保护基；优选地，R 3和R 4为碱性氨基保护基，R y和R 5为酸性氨基保护基；
    步骤3)后任选地包括步骤4)：对步骤3)所得化合物I粗品进行分离提纯，得纯品化合物I。
  14. 根据权利要求13所述的化合物I的固相合成方法，其中步骤1)所述固相载体固定的化合物M-1-1由以下方法制备：将化合物SM-1以固相载体固定，而后脱除哌啶环亚胺基的保护基Rx，得所述固相载体固定的化合物M-1-1，
    Figure PCTCN2019073370-appb-100010
    其中，R x是酸碱性与R y相反的氨基保护基；优选地，R x是碱性氨基保护基。
  15. 根据权利要求13或14所述的化合物I的固相合成方法，其中所述中间体化合物II-2根据权利要求11或12所述的制备方法得到。
  16. 根据权利要求13～15中任一项所述的化合物I的固相合成方法，其中所述固相载体为Wang树脂或2-氯三苯甲基氯树脂，优选2-氯三苯甲基氯树脂；
    优选地，所述Wang树脂取代度为0.3-1.0mmol/g；所述2-氯三苯甲基氯树脂取代度为0.2-1.6mmol/g。
  17. 根据权利要求13～16中任一项所述的化合物I的固相合成方法，其中步骤1)和步骤2)中使用缩合剂进行多肽的缩合；优选地，所述缩合剂为以下组合中的一种或多种：a)HBTU、Cl-HoBt和DIEA；b)DIC和Cl-HoBt；c)PyBOP、Cl-HoBt和DIEA；d)HBTU、Oxyma、DIEA；e)DIC和HoBt；优选地，所述缩合剂为HBTU、Cl-HoBt和DIEA的组合，DIC和Cl-HoBt的组合，或者DIC和HoBt的组合。
  18. 根据权利要求13～17中任一项所述的化合物I的固相合成方法，其中当R y、R 2和R 5为酸性氨基保护基时，步骤3)中裂解液含有50％-100％TFA；优选地，所述裂解液中进一步含有0％-10％TIS、0％-10％H 2O和0％-10％TES中的1中、2种或2种以上；优选地，所述裂解液由90％TFA/5％TIS/5％H 2O(v/v/v)或95％TFA/5％H 2O(v/v)组成。
  19. 根据权利要求13-18中任一项所述的化合物I的固相合成方法，其中步骤3)中所述裂解液与步骤2)所得载体固定的多肽化合物M-5的比例为(6-10)ml:1g。
  20. 根据权利要求13-19中任一项所述的化合物I的固相合成方法，在步骤3)中所述裂解后、步骤4)所述分离提纯前还包括使用醚类溶剂使化合物I粗品沉淀的步骤；优选地，所述醚类溶剂是无水***或甲基叔丁基醚。
  21. 根据权利要求13-20中任一项所述的化合物I的固相合成方法，其中步骤4)中所述分离提纯在反相高效液相色谱中进行。
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CN101341118A (zh) * 2004-12-22 2009-01-07 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN101627049A (zh) * 2006-11-10 2010-01-13 卡拉治疗学股份有限公司 合成酞酰胺
CN106459150A (zh) * 2014-06-26 2017-02-22 丸石制药株式会社 合成五肽的制造方法

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CN101341118A (zh) * 2004-12-22 2009-01-07 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN101627049A (zh) * 2006-11-10 2010-01-13 卡拉治疗学股份有限公司 合成酞酰胺
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