WO2019184089A1 - 化合物及其制备方法和应用 - Google Patents

化合物及其制备方法和应用 Download PDF

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WO2019184089A1
WO2019184089A1 PCT/CN2018/090549 CN2018090549W WO2019184089A1 WO 2019184089 A1 WO2019184089 A1 WO 2019184089A1 CN 2018090549 W CN2018090549 W CN 2018090549W WO 2019184089 A1 WO2019184089 A1 WO 2019184089A1
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reaction
compound
methanol
mixture
added
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French (fr)
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袁慧星
陈学明
宓鹏程
陶安进
袁建成
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深圳翰宇药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes 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 in position 9
    • C07D311/86—Oxygen atoms, e.g. xanthones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes 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 in position 9
    • C07D311/88—Nitrogen 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
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • 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 polypeptide synthesis, in particular to compounds and preparation methods and applications thereof.
  • Lanreotide is a somatostatin octapeptide analog that is more active than native somatostatin and similar in nature to octreotide.
  • Long-acting injections are used to treat acromegaly and thyroid-stimulating hormone adenomas, and can also be used to control the symptoms of neuroendocrine tumors, especially carcinoid syndrome. It was first developed by the French Epson Pharmaceutical Group. In 1994, the powder injection for injection was first listed in France. It was approved for marketing in China in 2002, and was approved by the US FDA in 2007 for long-acting sustained-release preparations.
  • the Boc strategy is often used to synthesize lanreotide.
  • the disadvantage is that expensive diphenylmethylamine-polystyrene resin is used as a carrier, and a highly corrosive reagent (33% trifluoroacetic acid) is used as the N-terminal protecting group.
  • a highly corrosive reagent (33% trifluoroacetic acid) is used as the N-terminal protecting group.
  • it has strong acid resistance requirements for equipment and operation, and waste acid treatment does not conform to the concept of green chemistry.
  • Fmoc solid phase synthesis strategy solid phase synthesis has certain advantages in post-reaction treatment, but has the following disadvantages: a. It requires 3 to 5 times or even higher Fmoc-protected amino acid feed. b. After coupling an amino acid, a very large amount of DMF washing (about 3 to 5 washes) is required. This causes an increase in cost and difficulty in solvent treatment. c. The reaction is a heterogeneous reaction. For solid phase cyclization, coupling of hydrophobic amino acids, etc., due to large steric hindrance, the coupling effect is poor, requiring two or even three feeds. d. Due to the large volume of the resin and the large amount of solvent, the reaction efficiency is low, and the yield of one batch is low.
  • the present invention provides compounds and methods for their preparation and use.
  • the compound is synthesized by using a polypeptide, and the process operation is simplified, and the loss of the crude peptide is reduced, and the yield is improved.
  • the present invention provides the following technical solutions:
  • the present invention provides a compound having the structure shown in Formula II:
  • R is selected from the group consisting of C16 to C25 fatty chains.
  • the compound structure is as shown in Formula VIII:
  • the invention also provides a preparation method of the compound, which is prepared by using a compound having the structure of the formula IV as a raw material, by a substitution reaction, a reduction reaction, an aminolysis reaction, and then a hydrolysis reaction;
  • the reagent used in the substitution reaction is one of 1-bromohexadecane, 1-bromooctadecane, 1-iodooctadecane, and 1-bromopentacosane.
  • 1-bromooctadecane is preferred;
  • the acid binding agent of the substitution reaction is potassium carbonate, sodium carbonate, DIPEA, TEA, preferably potassium carbonate;
  • the temperature of the substitution reaction is 70-100 ° C, the substitution reaction The time is 14 to 18 hours.
  • the catalyst used in the reduction reaction is sodium borohydride, sodium cyanoborohydride or a mixture of the two, preferably sodium borohydride;
  • the solvent of the reduction reaction is tetrahydrofuran.
  • One or a mixture of two of methyltetrahydrofuran is preferably tetrahydrofuran;
  • the reagent for the reduction reaction is methanol, ethanol, isopropanol, preferably methanol, at a temperature of 50 to 65 °C.
  • the reagent for the aminolysis reaction is methanesulfonic acid and urethane; the temperature of the aminolysis reaction is 100 to 110 ° C, and the time for the aminolysis reaction is 2 to 4 minutes. .
  • the reagent for the hydrolysis reaction is one of sodium hydroxide or potassium hydroxide or a mixture of the two; the hydrolysis reaction temperature is 90 to 110 ° C, and the hydrolysis reaction The time is 14 to 17 hours.
  • the invention also provides the use of the compound or a compound prepared as described for the preparation of a polypeptide in the synthesis of a polypeptide.
  • the polypeptide is lanreotide.
  • the invention also provides a method for synthesizing lanreotide, which comprises the compound of the formula II as a raw material, sequentially coupling the amino acid, deprotecting, preparing the crude peptide of lanreotide, and cleavage.
  • the coupling solvent and the deprotection reaction are carried out using a reaction solvent of one or a mixture of two of chloroform, dichloromethane, preferably chloroform, in g/mL, the reaction.
  • the mass to volume ratio of the reaction solvent to the reaction solvent is from 8 ml/g to 15 ml/g; after the coupling and deprotection, the step further comprises the steps of mixing the reaction solution with a solvent, the solvent is methanol, ethanol, isopropanol.
  • One or a mixture of two or more of acetonitrile, preferably methanol, is used in an amount of from 8 ml/g to 15 ml/g.
  • the solvent used in the synthetic method is a fat-soluble solvent
  • the solvent used in the precipitation is a polar solvent
  • the reaction solvent in the preparation method is chloroform, DCM, etc., a fat-soluble solvent having a good dissolving power for the carrier
  • a solvent used for precipitation (a solvent for precipitating solids): methanol, acetonitrile, etc. have a strong dissolving power for protecting the amino acid.
  • Polar solvent is chloroform, DCM, etc.
  • the solvent used in the preparation process is one or both of chloroform or dichloromethane, preferably chloroform.
  • the coupling agent in the preparation process is HOBt/DIC, HOBt/EDC.HCl, EDC.HCl, preferably HOBt/EDC.HCl.
  • the synthetic process of lanreotide comprises the steps of:
  • the carrier is dissolved and clarified in a chloroform solvent system, and then the Fmoc protected amino acid is sequentially coupled to the carrier, the amino acid equivalent is 1.1-1.5 eq, the coupling reagent is EDC.HCl (1.1-1.2 eq), HOBt (1.0- 1.2 eq), reacted at room temperature for 2 to 3 hours, and the reaction was monitored by TLC. After completion of the reaction, chloroform was concentrated, and 8 to 10 eq of methanol was added to precipitate a solid, which was suction filtered, and then washed with methanol for 3 to 5 times to wash excess protective amino acid and coupling reagent. Dry it
  • the reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, acetonitrile (10 ml / g) was added to the viscous material, stirred, filtered, and the filter cake was washed twice with methanol. The filter cake was dried under vacuum at 40 ° C for 2 hours.
  • the last amino acid adopts Boc-Nal-OH without N-terminal protection.
  • the ring is first formed, and then cleavage is removed after the ring is formed.
  • the raw material Fmoc-Cys(Mmt)-OH is used in two Cys, the removal scheme is 1% TFA ⁇ 2% chloroform solution, after removal, concentrated, dried, added with methanol, washed 2 ⁇ 3 times (removed TFA), using 0.3% hydrogen peroxide to form a ring. concentrate.
  • the invention skillfully synthesizes lanreotide by means of a carrier similar to the resin connecting arm (the structure is shown in formula II, the synthesis process is shown in Figure 1), and the specific advantages are as follows:
  • the carrier and peptide are dissolved and clarified, which should belong to a homogeneous reaction, which is beneficial to the coupling and ring formation of difficult amino acids.
  • a solid precipitated in methanol, and the amino acid starting material and the coupling reagent were dissolved in methanol.
  • the ratio of amino acid raw materials is reduced from 5 times to 1.2 to 1.3 times. It is not necessary to use a large amount of DMF, but a small amount of methanol-trichloromethane or the like is used. And chloroform can be recycled.
  • the Cys (Mmt) protecting group is used to achieve the localization and removal, and the ring is formed in the environment of the carrier and the protecting group, and then the cleavage is carried out. Compared with the liquid phase of the crude peptide, the operation is simple and the yield is greatly improved.
  • Figure 1 shows the synthesis process of the compound (reaction carrier) of formula VIII
  • Example 2 is a HPLC chart showing the crude lanreotide peptide prepared in Example 2;
  • Figure 3 shows the HPLC chromatogram of the crude lanreotide peptide prepared in Example 3;
  • Figure 4 shows the HPLC chromatogram of the crude lanreotide peptide prepared in Example 4.
  • Figure 5 shows the HPLC chromatogram of the crude lanreotide peptide prepared in Comparative Example 1;
  • Figure 6 shows the HPLC chromatogram of the crude lanreotide peptide prepared in Comparative Example 2.
  • the invention discloses a compound and a preparation method and application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the invention provides a carrier (Sieber-NH 2 ):
  • the carrier (Sieber-NH 2 ), a compound of the formula VIII, is modified from the structure of the compound sieber Amide Resin of the formula III and is temporarily named Sieber-NH2.
  • the carrier introduces a long-chain alkane chain symmetrically due to side chain symmetry, and is fat-soluble and can be dissolved in a fat-soluble solvent such as chloroform.
  • the heterogeneous reaction that causes the reaction to be synthesized by the solid phase becomes a homogeneous reaction of liquid phase synthesis.
  • the carrier can be precipitated in a polar solvent such as methanol, and the protected amino acid is dissolved in a polar solvent such as methanol, and excess protective amino acid, coupling reagent, and the like are removed by filtration using such a difference in solubility.
  • the carrier draws on the advantages of solid phase synthetic resin in post-treatment, and the advantage of homogeneous reaction by means of liquid phase.
  • the compounds and reagents and reagents used in the methods and applications of the present invention are commercially available.
  • the above compound 4 was placed in a three-necked flask, toluene and ethanol were added to the reaction flask, and the mixture was stirred uniformly, and then sodium hydroxide was added.
  • the reaction solution was warmed to 90 ° C and stirring was continued for 17 hours.
  • Water, n-hexane and ethyl acetate were successively added to the reaction mixture, and the mixture was stirred for 30 minutes, and then filtered.
  • the filter cake was beaten twice with purified water, and the obtained filtrate was allowed to stand for separation, the organic phase was collected, and the organic phase was concentrated to dryness at 45 °C.
  • the filter cake was combined with the concentrated residue and beaten with a mixture of methanol and acetonitrile. After filtration, the filter cake was vacuum dried at 45 ° C for 5 hours to obtain Compound 5, the target carrier compound Sieber-NH 2 .
  • the above compound 4 was placed in a three-necked flask, toluene and ethanol were added to the reaction flask, and the mixture was stirred uniformly, and then sodium hydroxide was added.
  • the reaction solution was warmed to 100 ° C and stirring was continued for 16 hours.
  • Water, n-hexane and ethyl acetate were successively added to the reaction mixture, and the mixture was stirred for 30 minutes, and then filtered.
  • the filter cake was beaten twice with purified water, and the obtained filtrate was allowed to stand for separation, the organic phase was collected, and the organic phase was concentrated to dryness at 45 °C.
  • the filter cake was combined with the concentrated residue and beaten with a mixture of methanol and acetonitrile. After filtration, the filter cake was vacuum dried at 45 ° C for 5 hours to obtain Compound 5, the target carrier compound Sieber-NH 2 .
  • the above compound 4 was placed in a three-necked flask, toluene and ethanol were added to the reaction flask, and the mixture was stirred well, and then potassium hydroxide was added.
  • the reaction solution was warmed to 110 ° C and stirring was continued for 14 hours.
  • Water, n-hexane and ethyl acetate were successively added to the reaction mixture, and the mixture was stirred for 30 minutes, and then filtered.
  • the filter cake was beaten twice with purified water, and the obtained filtrate was allowed to stand for separation, the organic phase was collected, and the organic phase was concentrated to dryness at 45 °C.
  • the filter cake was combined with the concentrated residue and beaten with a mixture of methanol and acetonitrile. After filtration, the filter cake was vacuum dried at 45 ° C for 5 hours to obtain Compound 5, the target carrier compound Sieber-NH 2 .
  • Compound 5 The structure of the carrier (Sieber-NH 2 ) is as shown in Formula VIII:
  • reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (40 mL) was added to the viscous material and stirred for 2 hours. Filter and filter cake was washed three times with methanol (15 mL x 3). The filter cake was vacuum dried at 40 ° C for 5 hours to give Fmoc-Thr(tBu)-NH-Sieber (10.71 g, 9.62 mmol, yield 96.16%).
  • the compound Fmoc-Thr(tBu)-NH-Sieber (10.71 g, 9.62 mmol) was weighed into a 500 mL three-necked flask, and an appropriate amount of chloroform (about 100 mL of about 10 ml/g relative to the compound Fmoc-Thr(tBu)- was added to the reaction flask. NH-Sieber)), stirred and dissolved, and then added DBU (1.47 g, 9.62 mmol). The reaction solution was cooled to 5 ° C or less in an ice bath, and diethylamine (7.04 g, 96.2 mmol) was slowly added dropwise thereto, and the temperature was not more than 5 °C.
  • reaction solution was allowed to warm to room temperature and stirring was continued for 2 hours.
  • the reaction mixture was concentrated under reduced pressure at 30 ° C to a thick residue, and acetonitrile (100 mL) was added to the viscous material and stirred for 30 minutes. Filter and filter cake was rinsed twice with methanol (30 mL x 2). The filter cake was dried under vacuum at 40 °C for 2 h to give an off-white solid (8.59 g, 9.6 mmol, 100%).
  • the reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (50 mL) was added to the viscous material and stirred for 2 hours. Filter and filter cake was washed three times with methanol (20 mL x 3). The filter cake was vacuum dried at 40 ° C for 3 hours to obtain (14.03 g, yield 98.01%).
  • Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber 31.0g8.15mmol was added to 1000mL three mouths Into the flask, chloroform (200 mL) was added to the reaction flask, and the mixture was stirred and dissolved, and then a mixture of chloroform-trifluoroacetic acid (200 ml - 4 ml) (to a total concentration of trifluoroacetic acid of about 1%) was added, and the reaction was stirred for 2 hours.
  • the total yield is about 66.24%.
  • reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (50 mL) was added to the viscous material and stirred for 2 hours. Filter and filter cake was washed three times with methanol (15 mL x 3). The filter cake was vacuum dried at 40 ° C for 5 hours to give Fmoc-Thr(tBu)-NH-Sieber (10.74 g, 9.64 mmol, yield 96.44%).
  • the compound Fmoc-Thr(tBu)-NH-Sieber (10.74 g, 9.6 mmol) was weighed into a 500 mL three-necked flask, and an appropriate amount of chloroform (about 100 mL) was added to the reaction flask, and the mixture was stirred and dissolved, and then DBU (1.47 g, 9.6) was added. Mm).
  • the reaction solution was cooled to 5 ° C or less in an ice bath, and diethylamine (7.05 g, 96.4 mmol) was slowly added dropwise thereto, and the temperature was not more than 5 °C. After the dropwise addition was completed, the reaction solution was allowed to warm to room temperature and stirring was continued for 2 hours.
  • reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (50 mL) was added to the viscous material and stirred for 2 hours. Filter and filter cake was washed three times with methanol (20 mL x 3). The filter cake was vacuum dried at 40 ° C for 3 hours to obtain (14.063 g, yield 98.01%).
  • Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber 31.0g8.15mmol was added to 1000mL three mouths Into the flask, chloroform (200 mL) was added to the reaction flask, and the mixture was stirred and dissolved, and then a mixture of chloroform-trifluoroacetic acid (200 ml - 4 ml) (to a total concentration of trifluoroacetic acid of about 1%) was added, and the reaction was stirred for 2 hours.
  • the total yield was 70.28%.
  • reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (100 mL) was added to the viscous material and stirred for 3 hours. Filter and filter cake was washed three times with methanol (30 mL x 3). The filter cake was vacuum dried at 40 ° C for 5 hours to give Fmoc-Thr(tBu)-NH-Sieber (10.74 g, 9.72 mmol, yield 97.16%).
  • the compound Fmoc-Thr(tBu)-NH-Sieber (10.82g, 9.72mmol) was weighed into a 500 mL three-necked flask, and an appropriate amount of chloroform (about 150 mL) was added to the reaction flask, stirred and dissolved, and then DBU (1.48 g, 9.72) was added. Mm).
  • the reaction solution was cooled to 5 ° C or less in an ice bath, and diethylamine (7.11 g, 97.2 mmol) was slowly added dropwise thereto, and the temperature was not more than 5 ° C. After the dropwise addition was completed, the reaction solution was allowed to warm to room temperature and stirring was continued for 2 hours.
  • the reaction mixture was concentrated under reduced pressure to a viscous material at 30 ° C, and methanol (100 mL) was added to the viscous material and stirred for 2 hours. Filter and filter cake was washed three times with methanol (40 mL x 3). The filter cake was vacuum dried at 40 ° C for 3 hours to obtain (14.063 g, yield 98.01%).
  • Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber (32.5g8.38mmol) was added to 1000mL three mouths Into the flask, chloroform (100 mL) was added to the reaction flask, and the mixture was stirred and dissolved, and then a mixture of chloroform-trifluoroacetic acid (300 ml - 4 ml) (to a total concentration of trifluoroacetic acid of about 1%) was added thereto, and the reaction was stirred for 2 hours.
  • the total yield was 71.89%.
  • the diphenylmethylamine-polystyrene resin was placed in a peptide synthesizer and washed in the following order: 1) dichloromethane; 2) 33% trifluoroacetic acid in dichloromethane; 3) dichloromethane 4) Ethanol 5) Dichloromethane 6) 10% triethylamine in chloroform. Further, the neutral resin and tert-butoxycarbonyl-O-benzylthreonine and diisopropylcarbodiimide were dissolved in dichloromethane, and the reaction was stirred, and the resulting amino acidified resin was washed as above.
  • the hydrogen fluoride was purged by dry nitrogen gas, and the precipitated free peptide was collected by filtration, washed with diethyl ether, dissolved in 90% acetic acid, and added with a solution of iodine in methanol until brown. After stirring for a while, the solvent was evaporated under reduced pressure. The oil is dissolved in a minimum volume of 50% acetic acid and subjected to column chromatography to give the product. The yield was about 37% and the crude peptide was about 59.7% pure, as shown in FIG.
  • Control group 1 lanreotide crude peptide prepared in Comparative Example 1;
  • Control group 2 lanreotide crude peptide prepared in Comparative Example 2;
  • control 1 is the Boc strategy of the original research process, the yield is less than 40%, and the crude peptide purity is less than 60%;
  • control 2 is the commonly used Fmoc strategy, the yield is 62%, the purity is about 88%, and the need is 3 to 5 Double feed, and the scale of synthesis is low.
  • the two groups of data showed significant differences in both yield and crude peptide purity (P ⁇ 0.05).

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Abstract

公开了式II所示的化合物及其制备方法和应用。使用式Ⅱ所示的化合物合成兰瑞肽，具体如下优势：1、反应时，载体和肽溶解澄清，属于均相反应，有利于困难氨基酸的偶联及成环等，反应完毕后产品在甲醇中析出，而氨基酸原料和偶联试剂在甲醇中溶解；2、通过过滤洗涤的方式分离原料和产品；3、氨基酸原料投料比由5倍降低至1.2～1.3倍，不需要使用大量DMF，而仅使用少量的甲醇、三氯甲烷等，且三氯甲烷等可以回收；4、一批次产量提高；5、利用Cys(Mmt)保护基，实现在定位脱除，在载体和保护基的环境下先成环，再进行裂解，相对于粗肽液相成环，操作简便且收率有较大提高。

Description

化合物及其制备方法和应用
本申请要求于2018年03月29日提交中国专利局、申请号为201810273684.0、发明名称为“化合物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及多肽合成领域，特别涉及化合物及其制备方法和应用。
背景技术
兰瑞肽(Lanreotide)是一种生长抑素八肽类似物，其比天然生长抑素更具活性，与奥曲肽性质相似。长效制剂注射用于治疗肢端肥大症和促甲状腺激素腺瘤，也可用于控制神经内分泌肿瘤的症状，尤其是类癌综合征。最早由法国的益普生制药集团研制年，1994以注射用粉针剂首先在法国上市，2002年在中国批准上市，2007年经美国FDA批准以长效缓释剂上市。
兰瑞肽的结构信息如下：
化学名：[3-(2-萘基)]-D-丙氨酰基-L半胱氨酰基-L-酪氨酰基-D-色氨酰基-L赖氨酰基-L-缬氨酰基-L半胱氨酰基-L-苏氨酰胺环状(3→7)-二硫化合物；
3-(2-Naphthalenyl)-D-alanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide cyclic(2-7)-disulfide；
结构如式Ⅰ所示：
Figure PCTCN2018090549-appb-000001
分子式：C 54H 69N 11O 10S 2；分子量：1096.32；CAS登记号：108736-35-2(兰瑞肽)。
目前多采用Boc策略进行合成兰瑞肽，缺点在于：采用昂贵的二苯 基甲胺-聚苯乙烯树脂作为载体，使用强腐蚀性的试剂(33％三氟乙酸)作为N端保护基的脱除试剂，对设备和操作具有较强的耐酸要求，同时废酸处理等不符合绿色化学的理念。
Fmoc固相合成策略，固相合成在反应后处理上具有一定优势，但是有以下不足：a、需要3～5倍甚至更高倍数的Fmoc-保护氨基酸投料。b、在偶联一个氨基酸后，需要极大量的DMF洗涤(约3～5次洗涤)。造成成本的增加和溶剂处理的困难。c、反应属于非均相反应。对于固相成环，偶联疏水性氨基酸等，由于位阻大，偶联效果欠佳，需要二次甚至三次投料。d、由于树脂体积较大，溶剂用量较大，导致反应釜效较低，一批次产量较低。
因此，提供一种操作简便、收率较高的兰瑞肽的合成方法具有重要的现实意义。
发明内容
有鉴于此，本发明提供了化合物及其制备方法和应用。该化合物应用多肽合成，工艺操作简化，同时减少了粗肽的损失，提高了收率。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了化合物，结构如式Ⅱ所示：
Figure PCTCN2018090549-appb-000002
R选自C16～C25脂肪链。
在本发明的一些具体实施方案中，化合物结构如式Ⅷ所示：
Figure PCTCN2018090549-appb-000003
本发明还提供了所述的化合物的制备方法，以结构如式Ⅳ所示的化合物为原料，经取代反应、还原反应、氨解反应，再经水解反应，沉淀制得；
Figure PCTCN2018090549-appb-000004
在本发明的一些具体实施方案中，所述取代反应采用的试剂为1-溴十六烷、1-溴十八烷、1-碘十八烷、1-溴二十五烷中的一种，其中优选1-溴十八烷；所述取代反应的缚酸剂为碳酸钾、碳酸钠、DIPEA，TEA，优选碳酸钾；所述取代反应的温度为70～100℃，所述取代反应的时间为14～18小时。
在本发明的一些具体实施方案中，所述还原反应采用的催化剂为硼氢化钠、氰基硼氢化钠中的一种或两者的混合物，优选硼氢化钠；所述还原反应的溶剂为四氢呋喃、甲基四氢呋喃中的一种或两者的混合物，优选四氢呋喃；所述还原反应的试剂为甲醇、乙醇、异丙醇，优选甲醇，温度为50～65℃。
在本发明的一些具体实施方案中，所述氨解反应的试剂为甲烷磺酸和乌来糖；所述氨解反应的温度为100～110℃，所述氨解反应的时间为2～4min。
在本发明的一些具体实施方案中，所述水解反应的试剂为氢氧化钠、氢氧化钾中的一种或两者的混合物；所述水解反应的温度为 90～110℃，所述水解反应的时间为14～17小时。
本发明还提供了所述的化合物或如所述的制备方法制得的化合物在多肽合成中的应用。
在本发明的一些具体实施方案中，所述多肽为兰瑞肽。
本发明还提供了兰瑞肽的合成方法，以式Ⅱ所示化合物为原料，依次偶联氨基酸，脱保护，制得兰瑞肽粗肽，裂解。
在本发明的一些具体实施方案中，所述偶联和脱保护反应采用的反应溶剂为氯仿、二氯甲烷中的一种或两种的混合物，优选氯仿，以g/mL计，所述反应物与所述反应溶剂的质量体积比为8ml/g～15ml/g；所述偶联和脱保护后还包括浓缩反应液与溶剂混合打浆的步骤，所述溶剂为甲醇、乙醇、异丙醇、乙腈中的一种或两种以上的混合物，优选甲醇，所述溶剂的使用量为8ml/g-15ml/g。
在本发明的一些具体实施方案中，所述合成方法采用的溶剂为脂溶性溶剂；所述沉淀采用的溶剂为极性溶剂。作为优选，制备方法中的反应溶剂为氯仿，DCM等对该载体具有较好溶解能力的脂溶性溶剂；沉淀采用的溶剂(析出固体的溶剂)：甲醇，乙腈等对保护氨基酸具有较强溶解能力的极性溶剂。
在本发明的一些具体实施方案中，制备方法中所用溶剂为三氯甲烷或二氯甲烷中的一种或两种，优选三氯甲烷。
在本发明的一些具体实施方案中，制备方法中偶联剂为HOBt/DIC、HOBt/EDC.HCl、EDC.HCl，优选HOBt/EDC.HCl。
在本发明的一些具体实施方案中，制备方法中裂解液为TFA和水混合溶剂，混合溶剂的配比为TFA体积比为80～95％，水的体积比5-20％，其中优选的比例是TFA：H 2O＝95：5。
在本发明的一些具体实施方案中，兰瑞肽的合成工艺包括如下步骤：
1)偶联氨基酸：
将载体在氯仿溶剂体系中，溶解澄清，然后依次将Fmoc保护氨基酸偶联至载体上，氨基酸投料当量为1.1～1.5eq，偶联试剂为EDC.HCl(1.1～1.2eq)，HOBt(1.0～1.2eq),室温反应2～3小时，TLC监控反 应。反应毕，浓缩氯仿，加入8～10eq甲醇，析出固体，抽滤，再用甲醇打浆3～5次洗涤过量的保护氨基酸和偶联试剂。烘干即可
2)脱Fmoc：
上述Fmoc-AA-NH-Sieber用氯仿(10ml/g)加入DBU(1.0～1.1eq)，将反应液冰浴冷却至5℃以下，缓慢滴加二乙胺(14～16eq)，控温不超过5℃。滴加完后，将反应液升至室温继续搅拌2小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(10ml/g)，搅拌,过滤，滤饼用甲醇冲洗两次。将滤饼在40℃条件下真空干燥2小时，即可。按照此偶联-脱Fmoc循环偶联，最后一个氨基酸采用Boc-Nal-OH，不需N端-保护。
先成环，成环后再进行裂解脱除。
在两个Cys中采用原料Fmoc-Cys(Mmt)-OH，脱除方案为1％TFA～2％三氯甲烷溶液，脱除后，浓缩，至干，加入甲醇，洗涤2～3次(去除TFA)，再采用0.3％的双氧水成环。浓缩。采用配方为TFA:H2O:TIS:EDT＝91：3:3:3的裂解液裂解2小时，过滤，沉淀得粗肽。
本发明巧妙的借助一个类似于树脂连接臂的载体(结构如式Ⅱ所示，其合成工艺见图1)合成兰瑞肽，具体优势如下：
1、反应时，载体和肽溶解澄清，应属于均相反应，有利于困难氨基酸的偶联及成环等。反应完毕在甲醇中析出固体，而氨基酸原料和偶联试剂在甲醇中溶解。
2、具备固相合成树脂带来的优势，在后处理，通过过滤洗涤的方式分离原料和产品。
3、氨基酸原料投料比由5倍降低至1.2～1.3倍。不需要使用大量DMF，而使用少量的甲醇-三氯甲烷等。且三氯甲烷等可以回收。
4、因为载体在三氯甲烷体系是溶解的，需要溶剂量为正常液相反应的溶剂量(3ml/g～5ml/g)，因为在相同的反应釜中，釜效较固相合成有较大提高，一批次产量提高。
5、利用Cys(Mmt)保护基，实现在定位脱除，在载体和保护基的环 境下先成环，再进行裂解。相对于粗肽液相成环，操作简便且收率有较大提高。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示式Ⅷ所示化合物(反应载体)的合成工艺；
图2示实施例2制得的兰瑞肽粗肽的HPLC谱图；
图3示实施例3制得的兰瑞肽粗肽的HPLC谱图；
图4示实施例4制得的兰瑞肽粗肽的HPLC谱图；
图5示对比例1制得的兰瑞肽粗肽的HPLC谱图；
图6示对比例2制得的兰瑞肽粗肽的HPLC谱图。
具体实施方式
本发明公开了化合物及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供了一种载体(Sieber-NH 2)：
Figure PCTCN2018090549-appb-000005
该载体(Sieber-NH 2)——式Ⅷ所示化合物由式Ⅲ所示化合物sieber Amide Resin结构修饰而来，暂时命名为Sieber-NH2。
该载体(Sieber-NH 2)——式Ⅷ所示化合物的合成路线如图1所示。
1)该载体合成难度及成本远低于常用多肽固相合成的树脂，如Rink Resin等。
2)该载体因为侧链对称引入长链烷烃链，脂溶性增强，可以溶解于氯仿等脂溶性溶剂。使得反应由固相合成的非均相反应变成液相合成的均相反应。且该载体在甲醇等极性溶剂中可以析出，而保护氨基酸溶于甲醇等极性溶剂，利用这样的溶解度差异，通过过滤的方式除去过量的保护氨基酸和偶联试剂等。简而言之，该载体即借鉴了固相合成树脂在后处理方面的优势，由借助液相均相反应的优势。
3)由于该工艺在液相中进行，相比于固相合成常用的3～5倍投料，和大量的DMF等溶剂洗涤，该工艺采用1.1～1.5eq投料，甲醇洗涤。大幅度的节约了成本。
4)液相合成，批生产对设备要求较小，且相同反应釜，釜效能更大。
5)该工艺采用Fmoc-Cys(Mmt)-OH为原料，代替Fmoc-Cys(Trt)-OH，因为载体位阻较大，分子间成环概率较低。因此在偶联完成后，脱除Mmt，可以在正常浓度的反应体系中进行氧化成环。而不需要像固相合成那样，先裂解，再制备，再配成极稀溶液(防止分子间成环)。工艺操作简化，同时减少了粗肽的损失，提高了收率。
本发明提供的化合物及其制备方法和应用中所用原料及试剂均可由市场购得。
缩写及英文含义：
Figure PCTCN2018090549-appb-000006
Figure PCTCN2018090549-appb-000007
下面结合实施例，进一步阐述本发明：
实施例1载体(Sieber-NH 2)合成
化合物2的制备 例1：
称取22.8g(1mmol)化合物1与1-溴十八烷(2.1mmol)加入合适的三口烧瓶内，然后向反应瓶中加入DMF(适量)，搅拌均匀，再加入碳酸钾(3.0mmol)。将反应液升温至80℃继续搅拌16小时。TLC监控反应。原料消耗完后，将反应液冰浴冷却至室温。在充分搅拌条件下，向反应液中缓慢滴加1M稀盐酸，和纯化水，滴加完后继续搅拌30分钟。过滤，滤饼依次用纯化水和甲醇冲洗。在40℃条件下真空干燥8小时，得到目标化合物2。
化合物2的制备 例2：
称取22.8g(1mmol)化合物1与1-碘十八烷(2.1mmol)加入合适的三口烧瓶内，然后向反应瓶中加入DMF(适量)，搅拌均匀，再加入DIPEA(3.0mmol)。将反应液升温至70℃继续搅拌18小时。TLC监控反应。原料消耗完后，将反应液冰浴冷却至室温。在充分搅拌条件下，向反应液中缓慢滴加1M稀盐酸，和纯化水，滴加完后继续搅拌30分钟。过滤，滤饼依次用纯化水和甲醇冲洗。在40℃条件下真空干燥8小时，得到目标化合物2
化合物2的制备 例3：
称取22.8g(1mmol)化合物1与1-溴十六烷(2.1mmol)加入合适的三口烧瓶内，然后向反应瓶中加入DMF(适量)，搅拌均匀，再加入碳酸钠(3.0mmol)。将反应液升温至100℃继续搅拌14小时。TLC监控反应。原料消耗完后，将反应液冰浴冷却至室温。在充分搅拌条件下，向反应液 中缓慢滴加1M稀盐酸，和纯化水，滴加完后继续搅拌30分钟。过滤，滤饼依次用纯化水和甲醇冲洗。在40℃条件下真空干燥8小时，得到目标化合物2
化合物3的制备 例1
称取化合物2加入三口烧瓶内，然后向反应瓶中加入THF和甲醇，搅拌均匀。将反应液升温至60℃后，缓慢加入硼氢化钠，加完后保持温度继续搅拌4小时。TLC(乙酸乙酯：正己烷＝1:2，紫外灯显色)监控反应。原料消耗完后，将反应液冰浴冷却至10℃以下。在充分搅拌的条件下，缓慢滴加1mol/L稀盐酸，滴加完后，减压浓缩除去THF，向剩余反应液加入纯化水，并用1mol/L稀盐酸调pH值至5-7。过滤，滤饼依次用纯化水和甲醇冲洗。在60℃条件下真空干燥8小时，得到目标化合物3。
化合物3的制备 例2
称取化合物2加入三口烧瓶内，然后向反应瓶中加入甲基四氢呋喃和乙醇，搅拌均匀。将反应液升温至65℃后，缓慢加入氰基硼氢化钠，加完后保持温度继续搅拌4小时。TLC(乙酸乙酯：正己烷＝1:2，紫外灯显色)监控反应。原料消耗完后，将反应液冰浴冷却至10℃以下。在充分搅拌的条件下，缓慢滴加1mol/L稀盐酸，滴加完后，减压浓缩除去甲基四氢呋喃，向剩余反应液加入纯化水，并用1mol/L稀盐酸调pH值至5-7。过滤，滤饼依次用纯化水和乙醇冲洗。在60℃条件下真空干燥8小时，得到目标化合物3。
化合物3的制备 例子3
称取化合物2加入三口烧瓶内，然后向反应瓶中加入THF和异丙醇，搅拌均匀。将反应液升温至50℃后，缓慢加入硼氢化钠，加完后保持温度继续搅拌4小时。TLC(乙酸乙酯：正己烷＝1:2，紫外灯显色)监控反应。原料消耗完后，将反应液冰浴冷却至10℃以下。在充分搅拌的条件下，缓慢滴加1mol/L稀盐酸，滴加完后，减压浓缩除去THF，向剩余反应液加入纯化水，并用1mol/L稀盐酸调pH值至5-7。过滤，滤饼依次用纯化水和异丙醇冲洗。在60℃条件下真空干燥8小时，得到目标化合物3。
化合物4的制备：例1
称取化合物3加入三口烧瓶内，然后向反应瓶中加入甲苯，搅拌均匀，再依次加入甲烷磺酸、乌来糖。将反应液升温至100℃继续搅拌4小时。将反应液冷却至室温，加入碳酸钠，然后旋蒸至原体积的1/5除去溶剂。向残余物中加入甲醇，升温至70℃，回流反应30分钟，将反应液自然冷却至室温析晶。过滤。滤饼用甲醇和乙腈混合溶剂(1:1)冲洗，50℃真空干燥5小时，得到化合物4。
化合物4的制备：例2
称取化合物3加入三口烧瓶内，然后向反应瓶中加入甲苯，搅拌均匀，再依次加入甲烷磺酸、乌来糖。将反应液升温至110℃继续搅拌2小时。将反应液冷却至室温，加入碳酸钠，然后旋蒸至原体积的1/5除去溶剂。向残余物中加入甲醇，升温至70℃，回流反应30分钟，将反应液自然冷却至室温析晶。过滤。滤饼用甲醇和乙腈混合溶剂(1:1)冲洗，50℃真空干燥5小时，得到化合物4。
化合物4的制备：例3
称取化合物3加入三口烧瓶内，然后向反应瓶中加入甲苯，搅拌均匀，再依次加入甲烷磺酸、乌来糖。将反应液升温至105℃继续搅拌3小时。将反应液冷却至室温，加入碳酸钠，然后旋蒸至原体积的1/5除去溶剂。向残余物中加入甲醇，升温至70℃，回流反应30分钟，将反应液自然冷却至室温析晶。过滤。滤饼用甲醇和乙腈混合溶剂(1:1)冲洗，50℃真空干燥5小时，得到化合物4。
化合物5的制备(Sieber-NH2的合成)例1：
将上述化合物4加入三口烧瓶内，向反应瓶中加入甲苯和乙醇，搅拌均匀，再加入氢氧化钠。将反应液升温至90℃继续搅拌17小时。向反应液中依次加水、正己烷、乙酸乙酯，搅拌30分钟后，过滤。滤饼用纯化水打浆两次，得到的滤液静置分层后收集有机相，将有机相在45℃条件下浓缩至干。将滤饼与浓缩后残余物合并，用甲醇与乙腈混合溶剂打浆。过滤，滤饼在45℃条件下真空干燥5小时，得到化合物5即目标载体化合物Sieber-NH2。
化合物5的制备(Sieber-NH2的合成)例2：
将上述化合物4加入三口烧瓶内，向反应瓶中加入甲苯和乙醇，搅拌均匀，再加入氢氧化钠。将反应液升温至100℃继续搅拌16小时。向反应液中依次加水、正己烷、乙酸乙酯，搅拌30分钟后，过滤。滤饼用纯化水打浆两次，得到的滤液静置分层后收集有机相，将有机相在45℃条件下浓缩至干。将滤饼与浓缩后残余物合并，用甲醇与乙腈混合溶剂打浆。过滤，滤饼在45℃条件下真空干燥5小时，得到化合物5即目标载体化合物Sieber-NH2。
化合物5的制备(Sieber-NH2的合成)例3
将上述化合物4加入三口烧瓶内，向反应瓶中加入甲苯和乙醇，搅拌均匀，再加入氢氧化钾。将反应液升温至110℃继续搅拌14小时。向反应液中依次加水、正己烷、乙酸乙酯，搅拌30分钟后，过滤。滤饼用纯化水打浆两次，得到的滤液静置分层后收集有机相，将有机相在45℃条件下浓缩至干。将滤饼与浓缩后残余物合并，用甲醇与乙腈混合溶剂打浆。过滤，滤饼在45℃条件下真空干燥5小时，得到化合物5即目标载体化合物Sieber-NH2。
上述Sieber-NH2的合成路线如图1所示。
其中，化合物1的结构如式Ⅳ所示：
Figure PCTCN2018090549-appb-000008
化合物2的结构如式Ⅴ所示：
Figure PCTCN2018090549-appb-000009
化合物3的结构如式Ⅵ所示：
Figure PCTCN2018090549-appb-000010
化合物4的结构如式Ⅶ所示：
Figure PCTCN2018090549-appb-000011
化合物5：载体(Sieber-NH 2)的结构如式Ⅷ所示：
Figure PCTCN2018090549-appb-000012
实施例2
1、Fmoc-Thr(tBu)-NH-Sieber(第一个氨基酸偶联)：
称取化合物Sieber-NH 2(7.34g,10mmol)加入500mL三口烧瓶内，向反应瓶中加入氯仿(800mL约8ml/g相对于固体底物)，再依次加入HOBt(1.36g.10.05mmol)、Fmoc-Thr(tBu)-OH(3.99g,10.05mmol)，搅拌，溶解，加入EDC·HCl(1.93g,10.05mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(40mL)，搅拌2小时。过滤，滤饼用甲醇(15mL×3)冲洗三次。将滤饼在40℃条件 下真空干燥5小时，得到Fmoc-Thr(tBu)-NH-Sieber(10.71g,9.62mmol,收率96.16％)。
2、H-Thr(tBu)-NH-Sieber(脱Fmoc)：
称取化合物Fmoc-Thr(tBu)-NH-Sieber(10.71g,9.62mmol)加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约100mL约10ml/g相对于化合物Fmoc-Thr(tBu)-NH-Sieber))，搅拌溶清，再加入DBU(1.47g，9.62mmol)。将反应液冰浴冷却至5℃以下，缓慢滴加二乙胺(7.04g,96.2mmol)，控温不超过5℃。滴加完后，将反应液升至室温继续搅拌2小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(100mL)，搅拌30分钟。过滤，滤饼用甲醇(30mL×2)冲洗两次。将滤饼在40℃条件下真空干燥2小时，得到类白色固体(8.59g,9.6mmol,100％)。
3、Fmoc-Cys(Mmt)-Thr(tBu)-NH-Sieber(偶联第二个氨基酸)：
将上述固体加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约100mL)，再依次加入HOBt(1.36g,10.1mmol)、Fmoc-Cys(Mmt)-OH(6.22g,10.1mmol)。搅拌溶清。将反应液冷却至0℃。加入EDC·HCl(1.93g,10.1mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(50mL)，搅拌2小时。过滤，滤饼用甲醇(20mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(14.03g,收率98.01％)。
4、重复2～3进行第3、4、5、6、7个基酸的偶联和脱保护至最后一个Boc-Nal-OH的偶联：
将Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(30.48g,8.50mmol)加入到500ml三口烧瓶，向反应瓶中加入适量氯仿(约200ml),再依次加入HOBt(1.21g,8.92mmol)、Boc-Nal-OH(2.96g,32,8.92mmol)。搅拌溶解。将反应液冷却至0℃。加入 EDC·HCl(1.71g,8.92mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(100mL)，搅拌2小时。过滤，滤饼用甲醇(30mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(31.35g,收率95.01％，总收率79～82％)。
5、兰瑞肽粗肽的合成：
Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(31.0g8.15mmol)加入1000mL三口烧瓶内，向反应瓶中加入氯仿(200mL)，搅拌溶清，再加入氯仿-三氟乙酸混合液(200ml-4ml)(三氟乙酸总浓度约1％),搅拌反应2小时。小样裂解，色谱监控Mmt是否脱除完全监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(110mL)，搅拌30分钟。过滤，滤饼用甲醇(60mL×2)冲洗两次,再将滤饼溶解在氯仿300ml中，加入0.3％双氧水(约1ml)搅拌2小时，得到全保护的目标肽。
采用配方为TFA:PhSMe:PhOMe:EDT＝90:5:3:2的裂解液裂解2小时，过滤，沉淀得粗肽7.26g，收率约82％，粗肽纯度92.07％(HPLC谱图如图2所示)。
总收率约：66.24％。
实施例3兰瑞肽线性肽树脂(Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber)的合成
1、Fmoc-Thr(tBu)-NH-Sieber(第一个氨基酸偶联)：
称取化合物Sieber-NH 2(7.34g,10mmol)加入500mL三口烧瓶内，向反应瓶中加入氯仿(100mL)，再依次加入HOBt(1.49g.11mmol)、Fmoc-Thr(tBu)-OH(4.37g,11mmol)，搅拌，溶解，加入EDC·HCl(2.1g,11mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(50mL)，搅拌2 小时。过滤，滤饼用甲醇(15mL×3)冲洗三次。将滤饼在40℃条件下真空干燥5小时，得到Fmoc-Thr(tBu)-NH-Sieber(10.74g,9.64mmol,收率96.44％)。
2、H-Thr(tBu)-NH-Sieber(脱Fmoc)：
称取化合物Fmoc-Thr(tBu)-NH-Sieber(10.74g,9.6mmol)加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约100mL)，搅拌溶清，再加入DBU(1.47g，9.6mmol)。将反应液冰浴冷却至5℃以下，缓慢滴加二乙胺(7.05g,96.4mmol)，控温不超过5℃。滴加完后，将反应液升至室温继续搅拌2小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(100mL)，搅拌30分钟。过滤，滤饼用甲醇(30mL×2)冲洗两次。将滤饼在40℃条件下真空干燥2小时，得到类白色固体(8.59g,9.6mmol,100％)。
3、Fmoc-Cys(Mmt)-Thr(tBu)-NH-Sieber(偶联第二个氨基酸)：
将上述固体加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约100mL)，再依次加入HOBt(1.44g,10.604mmol)、Fmoc-Cys(Mmt)-OH(6.53g,10.604mmol)。搅拌溶清。将反应液冷却至0℃。加入EDC·HCl(2.033g,10.604mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(50mL)，搅拌2小时。过滤，滤饼用甲醇(20mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(14.063g,收率98.01％)。
4、重复2～3进行第3、4、5、6、7个基酸的偶联和脱保护至最后一个Boc-Nal-OH的偶联：
将Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(30.56g,8.53mmol)加入到500ml三口烧瓶，向反应瓶中加入适量氯仿(约200ml),再依次加入HOBt(1.26g,9.38mmol)、 Boc-Nal-OH(2.96g,32,9.38mmol)。搅拌溶解。将反应液冷却至0℃。加入EDC·HCl(1.80g,9.38mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(100mL)，搅拌2小时。过滤，滤饼用甲醇(30mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(31.64g,收率95.62％，总收率70～81％)。
5、兰瑞肽粗肽的合成：
Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(31.0g8.15mmol)加入1000mL三口烧瓶内，向反应瓶中加入氯仿(200mL)，搅拌溶清，再加入氯仿-三氟乙酸混合液(200ml-4ml)(三氟乙酸总浓度约1％),搅拌反应2小时。小样裂解，色谱监控Mmt是否脱除完全监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(110mL)，搅拌30分钟。过滤，滤饼用甲醇(60mL×2)冲洗两次,再将滤饼溶解在氯仿300ml中，加入0.3％双氧水(约1ml)搅拌2小时，得到全保护的目标肽。
采用配方为TFA:PhSMe:PhOMe:EDT＝90:5:3:2的裂解液裂解2小时，过滤，沉淀得粗肽7.77g，收率约87％,粗肽纯度约93.71％(HPLC谱图如图3所示)。
总收率70.28％。
实施例4
1、Fmoc-Thr(tBu)-NH-Sieber(第一个氨基酸偶联)：
称取化合物Sieber-NH 2(7.34g,10mmol)加入500mL三口烧瓶内，向反应瓶中加入氯仿(150mL)，再依次加入HOBt(1.62g.12mmol)、Fmoc-Thr(tBu)-OH(4.77g,12mmol)，搅拌，溶解，加入EDC·HCl(2.3g,12mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(100mL)，搅拌3小时。过滤，滤饼用甲醇(30mL×3)冲洗三次。将滤饼在40℃条件下 真空干燥5小时，得到Fmoc-Thr(tBu)-NH-Sieber(10.74g,9.72mmol,收率97.16％)。
2、H-Thr(tBu)-NH-Sieber(脱Fmoc)：
称取化合物Fmoc-Thr(tBu)-NH-Sieber(10.82g,9.72mmol)加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约150mL)，搅拌溶清，再加入DBU(1.48g，9.72mmol)。将反应液冰浴冷却至5℃以下，缓慢滴加二乙胺(7.11g,97.2mmol)，控温不超过5℃。滴加完后，将反应液升至室温继续搅拌2小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(150mL)，搅拌30分钟。过滤，滤饼用甲醇(60mL×2)冲洗两次。将滤饼在40℃条件下真空干燥2小时，得到类白色固体(8.65g,9.72mmol,100％)。
3、Fmoc-Cys(Mmt)-Thr(tBu)-NH-Sieber(偶联第二个氨基酸)：
将上述固体加入500mL三口烧瓶内，向反应瓶中加入适量氯仿(约150mL)，再依次加入HOBt(1.57,11.66mmol)、Fmoc-Cys(Mmt)-OH(7.18g,11.66mmol)。搅拌溶清。将反应液冷却至0℃。加入EDC·HCl(2.23g,11.66mmol)，在室温条件下继续搅拌3小时。TLC(DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(100mL)，搅拌2小时。过滤，滤饼用甲醇(40mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(14.063g,收率98.01％)。
4、重复2～3进行第3、4、5、6、7个基酸的偶联和脱保护至最后一个Boc-Nal-OH的偶联：
将Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(32.21g,8.67mmol)加入到500ml三口烧瓶，向反应瓶中加入适量氯仿(约200ml),再依次加入HOBt(1.41g,10.4mmol)、Boc-Nal-OH(3.28g,10.4mmol)。搅拌溶解。将反应液冷却至0℃。加入EDC·HCl(2.0g,10.4mmol)，在室温条件下继续搅拌3小时。TLC (DCM:MeOH:HAc＝100:1:0.5)监控反应。反应完全后，反应液在30℃减条件下，减压浓缩至成粘稠物，向粘稠物中加入甲醇(200mL)，搅拌2小时。过滤，滤饼用甲醇(60mL×3)冲洗三次。将滤饼在40℃条件下真空干燥3小时，得到(32.54g,收率96.61％，总收率70～81％)。
5、兰瑞肽粗肽的合成：
Boc-Nal-Cys(Mmt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Val-Cys(Mmt)-Thr(tBu)-NH-Sieber(32.5g8.38mmol)加入1000mL三口烧瓶内，向反应瓶中加入氯仿(100mL)，搅拌溶清，再加入氯仿-三氟乙酸混合液(300ml-4ml)(三氟乙酸总浓度约1％),搅拌反应2小时。小样裂解，色谱监控Mmt是否脱除完全监控反应。反应完全后，将反应液在30℃条件下减压浓缩至成粘稠物，向粘稠物中加入乙腈(150mL)，搅拌30分钟。过滤，滤饼用甲醇(80mL×2)冲洗两次,再将滤饼溶解在氯仿300ml中，加入0.3％双氧水(约1ml)搅拌2小时，得到全保护的目标肽。
采用配方为TFA:PhSMe:PhOMe:EDT＝90:5:3:2的裂解液裂解2小时，过滤，沉淀得粗肽8.17g，收率约87％,粗肽纯度94.44％(HPLC谱图如图4所示)。
总收率71.89％。
对比例1
将二苯基甲胺-聚苯乙烯树脂置于肽合成器中，按下列顺序进行洗涤：1)二氯甲烷；2)33％三氟乙酸的二氯甲烷溶液；3)二氯甲烷4)乙醇5)二氯甲烷6)10％三乙胺的氯仿溶液。再该中性的树脂和叔丁氧羰基-O-苄基苏氨酸和二异丙基碳化二亚胺溶于二氯甲烷，搅拌反应，生成的氨基酸化的树脂按上面过程洗涤。依次接保护氨基酸：叔丁氧羰基-3-甲基苄基-Cys、叔丁氧羰基-Val、叔丁氧羰基-Ne-苄氧羰基-Lysine、叔丁氧羰基-D-Trp、叔丁氧羰基-Tyr、叔丁氧羰基-5-甲基苄基-Cys和叔丁氧羰基-D-β-萘胺。该树脂水洗后干燥，和苯甲醚及无水氟化氢在0℃搅拌。通入干燥氮气吹走氟化氢，过滤收集沉淀的游离的肽，用乙醚洗，溶于90％乙酸，加入碘的甲醇溶液至呈棕色。搅拌片刻后，减压蒸出溶剂。油状物溶于最 少体积的50％乙酸，进行柱层析分离，即得产物。收率约37％，粗肽纯度约59.7％，如图5所示。
对比例2
将Rink酰胺树脂DCM和DMF洗涤溶胀后，脱除Fmoc保护基，再依次偶联Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Val-OH、Fmoc-Lys(Boc)-OH、Fmoc-D-Trp(Boc)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-D-3-(2-萘基)-Ala-OH，偶联体系均采用HOBt/DIC，DMF为溶剂，20％哌啶的DMF溶液脱除Fmoc保护基。偶联毕，DMF洗涤3次，DCM洗涤3次，抽干，甲醇收缩，再采用TFA-水-捕获剂体系裂解，乙醚沉淀得到粗肽，粗肽再进行氧化成环，最后采用C18反相高效液相色谱柱纯化得到醋酸兰瑞肽。收率约62％，粗肽纯度约88.19％，如图6所示。
实施例5
实验组：实施例2～4制得的兰瑞肽粗肽；
对照组1：对比例1制得的兰瑞肽粗肽；
对照组2：对比例2制得的兰瑞肽粗肽；
比较结果见表1。
表1
Figure PCTCN2018090549-appb-000013
Figure PCTCN2018090549-appb-000014
注： *示与对照组相比，具有显著差异(P＜0.05)； #示与对照组相比，具有极显著差异(P＜0.01)。
表1结果表明，实验组三组数据显示粗肽的平均收率约86％，其中实验4的收率最高，粗肽纯度也最高(即采用1.2倍氨基酸原料和1.2倍偶联试剂进行投料)，但是三者无显著性差异。
而对照组，对照1即原研工艺Boc策略，收率不足40％，且粗肽纯度不到60％；对照2即常用的Fmoc策略，收率62％，纯度约88％，且需要3～5倍投料，并且合成规模较低。该两组数据与实验组相比，无论是收率还是粗肽纯度均存在显著性差异(P＜0.05)。
以上对本发明所提供的化合物及其制备方法和应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 化合物，其特征在于，结构如式Ⅱ所示：
    Figure PCTCN2018090549-appb-100001
    R选自C16～C25脂肪链。
  2. 根据权利要求1所述的化合物的制备方法，其特征在于，以结构如式Ⅳ所示的化合物为原料，经取代反应、还原反应、氨解反应，再经水解反应，沉淀制得；
    Figure PCTCN2018090549-appb-100002
  3. 根据权利要求2所述的制备方法，其特征在于，所述取代反应采用的试剂为1-溴十六烷、1-溴十八烷、1-碘十八烷、1-溴二十五烷中的一种或两者以上的混合物；所述取代反应的缚酸剂为碳酸钾、碳酸钠、DIPEA，TEA中的一种或两者以上的混合物；所述取代反应的温度为70～100℃，所述取代反应的时间为14～18小时。
  4. 根据权利要求2或3所述的制备方法，其特征在于，所述还原反应采用的催化剂为硼氢化钠、氰基硼氢化钠中的一种或两者的混合物；所述还原反应的溶剂为四氢呋喃、甲基四氢呋喃中的一种或两者的混合物；所述还原反应的试剂为甲醇、乙醇、异丙醇；所述还原反应的温度为50～65℃。
  5. 根据权利要求2至4任一项所述的制备方法，其特征在于，所述氨解反应的试剂为甲烷磺酸和乌来糖；所述氨解反应的温度为100～110℃，所述氨解反应的时间为2～4min。
  6. 根据权利要求2至5任一项所述的制备方法，其特征在于，所述水解反应的试剂为氢氧化钠、氢氧化钾中的一种或两者的混合物；所述水解反应的温度为90～110℃，所述水解反应的时间为14～17小时。
  7. 如权利要求1所述的化合物或如权利要求2至6任一项所述的制备方法制得的化合物在多肽合成中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述多肽为兰瑞肽。
  9. 兰瑞肽的合成方法，其特征在于，以式Ⅱ所示化合物为原料，依次偶联氨基酸，脱保护，制得兰瑞肽粗肽，裂解。
  10. 根据权利要求9所述的合成方法，其特征在于，所述偶联和脱保护反应采用的反应溶剂为氯仿、二氯甲烷中的一种或两种的混合物，以g/mL计，所述反应物与所述反应溶剂的质量体积比为:8ml/g～15ml/g；所述偶联和脱保护后还包括浓缩反应液与溶剂混合打浆的步骤，所述溶剂为甲醇、乙醇、异丙醇、乙腈中的一种或两种以上的混合物，所述溶剂的使用量为8ml/g-15ml/g。
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