WO2019113872A1 - 一种合成利那洛肽的方法 - Google Patents

一种合成利那洛肽的方法 Download PDF

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WO2019113872A1
WO2019113872A1 PCT/CN2017/116095 CN2017116095W WO2019113872A1 WO 2019113872 A1 WO2019113872 A1 WO 2019113872A1 CN 2017116095 W CN2017116095 W CN 2017116095W WO 2019113872 A1 WO2019113872 A1 WO 2019113872A1
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fmoc
cys
trt
tbu
resin
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PCT/CN2017/116095
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English (en)
French (fr)
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姚林
李新宇
支钦
张利香
吴丽芬
朱亮平
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深圳市健元医药科技有限公司
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Priority to CN201780018911.9A priority Critical patent/CN109311941B/zh
Priority to PCT/CN2017/116095 priority patent/WO2019113872A1/zh
Publication of WO2019113872A1 publication Critical patent/WO2019113872A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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 drug synthesis, in particular to a method for synthesizing linaclotide.
  • Linaclotide is a novel GC-C (intestinal epithelial cell uridine cyclase C) receptor agonist developed by Ironwood that activates the GC-C receptor on the apical surface of intestinal epithelial cells, resulting in Increased intracellular and extracellular cyclic guanosine monophosphate.
  • the net effect is an increase in the secretion of chlorine and bicarbonate into the intestinal lumen, which in turn leads to increased fluid secretion and accelerated stools for the treatment of adult patients with slow transit constipation and constipation-type irritable bowel syndrome (IBS-C).
  • IBS-C immediate transit constipation and constipation-type irritable bowel syndrome
  • Linaclotide consists of 14 amino acids and contains 3 pairs of disulfide bonds.
  • the specific structural sequence is as follows:
  • the disulfide bond in the structure of linaclotide is the main action point of its pharmacodynamics. Moreover, the synthesis of disulfide bonds is the most difficult in its synthetic route, which becomes the key point in the synthesis of peptide sequences.
  • nalopeptide (1) All the peptide sequences were synthesized by using Trt as a Cys protecting group for solid phase synthesis of linear crude peptide, and then lining in the liquid phase at room temperature to obtain linaclotide; (2) using five different peptide sequences Cysteine protecting group, using 2StBu, 2pMeOBzl, 2Trt or 2Mmt, 2Acm, 2Trt or 2Acm, 2Trt, 2pMeOBzl three orthogonal protection methods for solid phase synthesis of linear crude peptide, and then using step-by-step cyclization strategy Synthesis of disulfide bonds; (3) Two different cysteine protecting groups are used in the peptide sequence, that is, two StBu and four Trts are used for solid phase synthesis of linear crude peptide, and then a stepwise cyclization strategy is used to complete disulfide The synthesis of the keys.
  • Chinese Patent No. CN102875655B discloses a method for synthesizing linaclotide, which uses a Mmt protecting group to protect a cysteine side chain, and synthesizes a linaclotide resin coupled with a resin solid phase carrier at the C-terminus by coupling one by one.
  • the cleavage removal protecting group and the resin solid phase carrier are used to obtain the linear crude peptide of linaclotide, and finally the oxidation reaction is carried out by using the GSH/GSSH oxidation system to obtain crude linaclotide.
  • Chinese patent CN104231051A discloses a preparation method of linaclotide, which adopts Fmoc-Cys(Trt)-OH and Fmoc-Cys(Mmt)-OH are used as reaction raw materials, and linaclotide resin is obtained by coupling one by one, and linear linaclotide is obtained by cleavage reaction, followed by phosphate buffer system, DMSO, The EDTA is oxidized to obtain crude linaclotide.
  • liquid phase cyclization method is adopted in the above literature reports, that is, the linaclotide linear peptide resin coupled with the resin solid phase carrier at the C-terminus is first synthesized, and then the protective group and the resin solid phase carrier are cleaved to obtain the Lina. Lopeptide linear crude peptide, and finally cyclization reaction using an oxidation system to obtain crude linaclotide.
  • such methods include Chinese patents CN104628826A, CN103626849A, CN104974229A, CN105884864A, CN104844693A, CN106008674A, CN106167514A.
  • liquid phase cyclization can only be carried out at a lower concentration, and the reaction efficiency is lower.
  • the reaction concentration of Miriam et al. is only 0.5 mg/mL, otherwise the peptide chain will be absent.
  • Sequence polymerization which produces a large amount of impurities, is very unfavorable for industrial scale production; the multi-step removal of cracking and protecting groups before cyclization is easy to cause a large amount of impurities, and the intermediates need to be carried out in a multi-step cyclization process.
  • the purification step is complicated, and the difficulty of purification is increased; the use of some special amino acids, such as Fmoc-Cys(Mmt)-OH and Fmoc-Cys(Hqm)-OH, makes the production cost high, which is not conducive to popularization.
  • the object of the present invention is to provide a method for synthesizing linaclotide, which can increase the reaction concentration, improve the reaction efficiency, reduce the generation of impurities, avoid the use of special raw materials, simplify the production process, and improve the profit.
  • the total yield of naloxide, while reducing the volume of the oxidation system, reducing production costs, is suitable for scale-up production.
  • the present invention provides the following technical solutions:
  • a method of synthesizing linaclotide comprising the steps of:
  • Fmoc-Tyr(tBu)-resin is coupled to other amino acids having an N-terminal Fmoc protecting group and a side chain linked to a specific protecting group by one-by-one coupling or stepwise coupling to obtain a linaclotide linear peptide resin;
  • the resin is subjected to a cleavage reaction, purified and lyophilized to obtain linaclotide.
  • the activation system is selected from one or more of HOBt, DIC, DCC, HBTU, TBTU, PyBop, DMAP, DIPEA, DIEA.
  • the activation system is DIEA, HOBt/DIC/DMAP, HOBt/DCC/DMAP, HBTU/HOBt/DIPEA, TBTU/HOBt/DIPEA, PyBOP/HOBt/DIPEA.
  • the activation system is more preferably DIEA, HOBt/DIC/DMAP or HBTU/HOBt/DIPEA.
  • the activation system is dissolved using any one or two of DMF, DCM, NMP and DMSO.
  • the carrier resin is selected from the group consisting of Wang resin, CTC resin or hydroxy resin.
  • the carrier resin is Wang resin, and the degree of substitution is preferably 0.4 to 1.2 mmol/g.
  • the carrier resin is Wang resin, and the degree of substitution is preferably from 0.5 to 1.0 mmol/g.
  • the protecting group of the present invention is a protecting group for protecting the main chain amino group of the amino acid and the amino group, the carboxyl group, the hydroxyl group, the sulfhydryl group and the like which interfere with the synthesis in the field of polypeptide synthesis, and prevents the above reactive group from preparing the target product. The reaction takes place and impurities are formed.
  • the N-terminal protecting group in the present invention is Fmoc.
  • the side chain structure is well known to those skilled in the art, and it is known to use a common protecting group to protect the amino group and carboxyl group in the amino acid side chain. a group such as a hydroxyl group or a thiol group.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt -OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(Trt)-OH.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Cys(Trt -OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(StBu)-OH.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc- Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)- OH.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm - OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH and Fmoc-Cys(Trt)-OH.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(tBu -OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(tBu)-OH and Fmoc-Cys(Trt)-OH.
  • the other amino acids having an Fmoc protecting group at the N-terminus and a specific protecting group at the side chain in the step 2) are: Fmoc-Cys(StBu)-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(StBu -OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(StBu)-OH and Fmoc-Cys(Trt)-OH.
  • the coupling of the present invention may be coupled one by one or stepwise.
  • the one-by-one coupling means that after the first amino acid is coupled with the solid phase carrier, the remaining amino acids are coupled to the previous amino acid one by one in the order of the respective sequences.
  • the condensation reaction condensation reaction of the main chain amino group and the carboxyl group
  • the coupling one by one since each amino acid has a protecting group at the N-terminus, it is necessary to first remove the N-terminal protecting group and then couple it, which is common knowledge to those skilled in the art.
  • amino acid sequence of the N-terminus to the C-terminus of the linear backbone of linaclotide is numbered as follows:
  • the stepwise coupling means an "8+6" or "5+9" mode.
  • the "8+6" mode refers to the first synthesis of the 1-8 octapeptide fragment I in the linaclotide peptide sequence and the 9-14 hexapeptide fragment II resin in the peptide sequence, respectively, in the coupling system. In the presence of fragment I, fragment I is ligated to fragment II to obtain a linaclotide linear peptide resin.
  • the model firstly synthesizes the 1-5 pentapeptide fragment I in the linaclotide peptide sequence and the 6-14 nonapeptide fragment II resin in the peptide sequence, and then connects the fragment I with the fragment II in the presence of a coupling system. Linaclotide linear peptide resin.
  • Purification according to the present invention may employ conventional purification methods in the art, such as HPLC purification methods.
  • the NX succinimide described in the step 3) of the present invention is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, N-hydroxythiosuccinyl One of the imines.
  • the solvent of the N-X succinimide solution described in the step 3) of the present invention is DMF or DCM.
  • the N-X generation succinimide solution described in the step 3) of the present invention is used in an amount of from 1 to 5 eq.
  • the method for synthesizing linaclotide according to the present invention after synthesizing the linear peptide resin of linaclotide, is cyclized by an oxidation system of NX succinimide solution, and 1, 6, 2, 10, 5 in the cyclization process.
  • the side chain protecting group of the 13th Cys is removed and forms a Cys 1-6, 2-10, 5-13 disulfide bond, while the remaining amino acid side chain protecting groups and the resin are not removed, forming Cys 1-6.
  • the linaclotide resin having a 2-10, 5-13 position cyclized while the other amino acid side chain is still attached to a specific protecting group.
  • the cleavage reagent described in the step 4) of the present invention is: TFA/Mpr/Tis, TFA/EDT/Tis/H 2 O.
  • the amount of the cleavage reagent described in the step 4) of the present invention is: TFA/Mpr/Tis: (92 to 95) / (2 to 5) / (2 to 5), TFA / EDT / Tis / H 2 O : (90 to 96) / (1 to 5) / (1 to 5) / (1 to 5).
  • the crude peptide of linaclotide synthesized by the method of the invention has a weight yield of 99.3-108.9%, and the purity is between 72.1-80.5% by HPLC, and the purity of the linaclotide sperm peptide obtained by HPLC is 99.34%.
  • the total yield is 45.8-52.6%.
  • the existing synthetic linaclotide technology firstly synthesizes a linaclotide linear peptide resin coupled with a resin solid phase carrier at the C-terminus, and then cleaves the deprotection group and the resin solid phase carrier.
  • the linaclotide linear crude peptide is obtained, and finally cyclized by an oxidation system to obtain a crude linaclotide, that is, a liquid phase cyclization method.
  • the synthesis of linaclotide is carried out by solid phase cyclization, that is, linaclotide linear peptide resin is directly cyclized by NX succinimide solution oxidation system without cleavage to obtain linaclotide resin.
  • the resin is cleaved, purified and lyophilized to obtain linaclotide.
  • Liquid phase cyclization can only be carried out at a lower concentration, and the reaction efficiency is lower.
  • the reaction concentration of Miriam et al. is only 0.5 mg/mL, otherwise the disordered polymerization of the peptide chain will occur, and a large amount of impurities will be generated.
  • Solid phase cyclization plays a role of false dilution, avoids repeated folding of peptide chains, can carry out cyclization reaction at higher concentration, can greatly improve production efficiency; secondly, linear peptide resin before cyclization The cleavage is carried out to avoid the generation of a large amount of impurities, and the efficiency of cyclization of linaclotide is improved.
  • One-step cyclization using N-X succinimide can avoid multi-step purification of the intermediate, reduce the cost of the intermediate purification step, and increase the total yield of linaclotide.
  • FIG. 1 Schematic diagram of linalopeptide linear peptide resin cyclization
  • the mixture was sealed with 70 ml of acetic anhydride and 62 ml of pyridine for 24 h.
  • the mixture was washed 3 times with DMF, 3 times with DCM, and the methanol was subjected to shrink-drying to obtain Fmoc-Tyr(tBu)-Wang resin, and the detection degree of substitution was 0.48 mmol/g.
  • Trt -OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH.
  • reaction column was added and reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. The above procedure was repeated, and Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn were sequentially coupled according to the peptide sequence.
  • Trt -OH, Fmoc-Cys(StBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(StBu)-OH.
  • reaction column was added and reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. The above procedure was repeated, and Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn were sequentially coupled according to the peptide sequence.
  • Trt -OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)-OH.
  • Fmoc-Cys(StBu)-OH 129.5g (300mmol), HOBt 48.6g (360mmol), HBTU 114.1g (300mmol) was dissolved in DMF, 77ml DIPEA (360mmol) was added in 0°C ice water bath, activated for 5 minutes, added The reaction column was reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK.
  • the mixture was washed three times with DMF, washed three times with DCM, and sealed with anhydrous methanol for 30 minutes, and the methanol was dried under reduced pressure to obtain Fmoc-Pro-CTC resin, and the detection degree of substitution was 0.52 mmol/g.
  • the mixture was washed three times with DMF, washed three times with DCM, and sealed with anhydrous methanol for 30 minutes, and the methanol was shrink-dried to obtain Fmoc-Pro-CTC resin, and the detection degree of substitution was 0.54 mmol/g.
  • Fmoc-Cys(Trt)-OH175.7g (300mmol), HOBt 45.2g (300mmol), HBTU 114.1g (300mmol) was dissolved in NMP, 77ml DIPEA (360mmol) was added in 0°C ice water bath, activated for 5 minutes, added The reaction column was reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. The above procedure was repeated, and Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH and Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH were sequentially coupled in the order of the peptide.
  • the Fmoc-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC resin obtained in Example 13 was placed in a reaction column, washed 3 times with DCM, and then swollen with DMF 30. minute. The Fmoc protecting group was then removed with DBLK and then washed 6 times with DMF. The Fmoc-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-OH, HOBt obtained in Example 11 was weighed.
  • the mixture was washed three times with DMF, washed three times with DCM, and sealed with anhydrous methanol for 30 minutes, and the methanol was dried under reduced pressure to obtain Fmoc-Cys(Trt)-CTC resin, and the detection degree of substitution was 0.80 mmol/g.
  • the resin was added to a 5000 mL three-neck round bottom flask, and 3200 ml of a lysate was placed at a volume ratio of 1:4 TFE and DCM.
  • the lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and washed with a small amount of TFA.
  • the resin was combined 3 times, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, washed with diethyl ether for 6 times, and dried under vacuum to give Fmoc-Cys(StBu)-Cys(Trt)-Glu. (OtBu)-Tyr(tBu)-Cys(Trt)-OH 165.37g
  • the StBu protecting group was removed by using 20% ⁇ -mercaptoethanol, 0.1 M N-methylmorpholine DMF lysate, and then washed 6 times with DMF to obtain Fmoc-Cys-Asn(Trt)-Pro-Ala- Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang resin, which was deprotected by Fmoc with a mixed solution of DMF: pyridine in a volume ratio of 4:1, and then washed 6 times with DMF.
  • Example 18 H-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly Synthesis of -Cys(Trt)-Tyr(tBu)-Wang Resin
  • the linaclotide peptide resin prepared in Example 19 was placed in a cleavage reactor, and a lysis reagent (TFA/Mpr/Tis: 94/3/3) (V/V) was added at a ratio of 15 ml/g resin. Stir at room temperature for 2.5 h. The reaction was filtered through a sep. funnel, and the filtrate was collected. The solvent was washed three times with a small portion of TFA. The precipitate was precipitated by adding ice-cold diethyl ether.
  • the precipitate was filtered or centrifuged, and the obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, which was 140.32 g of linalo peptide.
  • the crude peptide weight yield was 108.9% and the HPLC purity was 72.1%, as shown in FIG.
  • the linaclotide peptide resin prepared in Example 20 was placed in a cleavage reactor, and a lysis reagent (TFA/Mpr/Tis: 92/4/4) (V/V) was added at a ratio of 15 ml/g resin. Stir at room temperature for 2.5 h. The reaction was filtered through a sep. funnel, and the filtrate was collected. The solvent was washed three times with a small portion of TFA. The mixture was poured into ice-cold anhydrous diethyl ether, and the precipitate was filtered or centrifuged. The obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 147. The crude peptide weight yield was 99.9%, the HPLC purity was 75.4%, and the HPLC pattern was similar to that of Example 26.
  • a lysis reagent (TFA/Mpr/Tis: 92/4/4) (V/V) was added at a
  • the linaclotide peptide resin prepared in Example 21 was placed in a cleavage reactor, and a lysis reagent (TFA/EDTA/Tis/H2O: 90/3/3/4) was added at a ratio of 15 ml/g resin (V/ V)), stirring at room temperature for 2.5 h.
  • the reaction was filtered through a sep. funnel, and the filtrate was collected.
  • the solvent was washed three times with a small portion of TFA.
  • the precipitate was precipitated by adding ice-cold diethyl ether.
  • the precipitate was filtered or centrifuged.
  • the obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 147.
  • Crude peptide weight The yield was 98.9%, the HPLC purity was 80.3%, and the HPLC chart was similar to that of Example 26.
  • the linaclotide peptide resin prepared in Example 22 was placed in a cleavage reactor, and a lysis reagent (TFA/Mpr/Tis: 94/2/4) (V/V) was added at a ratio of 15 ml/g resin. Stir at room temperature for 2.5 h. The reaction was filtered through a sep. funnel, and the filtrate was collected. The solvent was washed three times with a small portion of TFA. The precipitate was precipitated by adding ice-cold diethyl ether. The precipitate was filtered or centrifuged. The obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 147. The crude peptide weight yield was 98.9%, the HPLC purity was 80.5%, and the HPLC chart was similar to that of Example 26.
  • a lysis reagent (TFA/Mpr/Tis: 94/2/4) (V/V) was added at a ratio
  • the linaclotide peptide resin prepared in Example 23 was placed in a cleavage reactor, and a lysis reagent (TFA/EDTA/Tis/H2O: 92/1/5/2) was added at a ratio of 15 ml/g resin (V/ V)), stirring at room temperature for 2.5 h.
  • the reaction was filtered through a sep. funnel, and the filtrate was collected.
  • the solvent was washed three times with a small portion of TFA.
  • the mixture was poured into ice-cold anhydrous diethyl ether, and the precipitate was filtered or centrifuged.
  • the obtained filter cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 143.
  • the crude peptide weight yield was 100.9%, the HPLC purity was 76.3%, and the HPLC pattern was similar to that of Example 26.
  • the linaclotide peptide resin prepared in Example 24 was placed in a cleavage reactor, and a lysis reagent (TFA/EDTA/Tis/H2O: 94/2/3/1) was added at a ratio of 15 ml/g resin (V/ V)), stirring at room temperature for 2.5 h.
  • the reaction was filtered through a sep. funnel, and the filtrate was collected.
  • the solvent was washed three times with a small portion of TFA.
  • the precipitate was precipitated by adding ice-cold diethyl ether.
  • the precipitate was filtered or centrifuged.
  • the obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 147.
  • the weight yield of the crude peptide was 100.9%, the HPLC purity was 78.2%, and the HPLC chart was similar to that of Example 26.
  • the linaclotide peptide resin prepared in Example 24 was placed in a cleavage reactor, and a lysis reagent (TFA/EDTA/Tis/H2O: 94/2/3/1) was added at a ratio of 15 ml/g resin (V/ V)), stirring at room temperature for 2.5 h.
  • the reaction was filtered through a sep. funnel, and the filtrate was collected.
  • the solvent was washed three times with a small portion of TFA.
  • the precipitate was precipitated by adding ice-cold diethyl ether.
  • the precipitate was filtered or centrifuged.
  • the obtained cake was washed three times with anhydrous diethyl ether and dried in vacuo to give a white powder solid, 143.
  • the weight yield of the crude peptide was 99.3%, the HPLC purity was 77.6%, and the HPLC chart was similar to that of Example 26.
  • the crude peptide of linaclotide prepared in Example 26 was prepared by using NOVASEPRP-HPLC system, wave
  • the column is 220nm long, the column is reversed phase C18 column, purified by conventional 0.1% TFA/water and acetonitrile mobile phase system.
  • the salt is removed, the peak fraction is collected, concentrated by rotary evaporation, and lyophilized to obtain 116.45g of linaclotide. 99.23%, the total yield was 45.8%.
  • the spermatic peptide spectrum is shown in Figure 3.
  • the crude peptide of linaclotide prepared in Example 27 was prepared by NOVASEPRP-HPLC system at a wavelength of 220 nm, and the column was a reverse phase C18 column, purified by a conventional 0.1% TFA/water, acetonitrile mobile phase system, and desalted to collect a target peak.
  • the fraction was concentrated by rotary evaporation, and lyophilized to obtain 114.53 g of linaclotide phage peptide, the HPLC purity was 99.32%, and the total yield was 50.3%.
  • the spermatic peptide spectrum was similar to that of Example 31.
  • the crude peptide of linaclotide prepared in Example 28 was prepared by NOVASEPRP-HPLC system at a wavelength of 220 nm, and the column was a reverse phase C18 column, purified by a conventional 0.1% TFA/water, acetonitrile mobile phase system, and desalted to collect a target peak. The fraction was concentrated by rotary evaporation and lyophilized to obtain 115.32 g of linaclotide phlopeptide. The HPLC purity was 99.21%, and the total yield was 46.8%. The spermatic peptide spectrum was similar to that of Example 31.
  • the crude peptide of linaclotide prepared in Example 29 was prepared by NOVASEPRP-HPLC system with a wavelength of 220 nm, the column was reversed C18 column, purified by conventional 0.1% TFA/water and acetonitrile mobile phase system, and desalted to collect the target peak. The fraction was concentrated by rotary evaporation, and lyophilized to obtain 114.26 g of linaclotide sperm. The HPLC purity was 99.16%, and the total yield was 52.6%. The spermatic peptide spectrum was similar to that of Example 31.
  • the crude peptide of linaclotide prepared in Example 30 was prepared by NOVASEPRP-HPLC system at a wavelength of 220 nm, and the column was a reverse phase C18 column, purified by a conventional 0.1% TFA/water, acetonitrile mobile phase system, and desalted to collect a target peak. The fraction was concentrated by rotary evaporation, and lyophilized to obtain 112.34 g of linalotide peptide, HPLC purity 99.34%, total yield 49.5%. The spermatic peptide spectrum was similar to that of Example 31.

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Abstract

涉及医药合成领域，公开了一种合成利那洛肽的方法。所述方法采用固相一步环化方法制备利那洛肽，利那洛肽线性树脂未经裂解直接采用N-X代琥珀酰亚胺溶液氧化体系进行一步环化得到利那洛肽树脂，树脂经裂解，纯化和冻干得到利那洛肽。所述的N-X代琥珀酰亚胺为N-氯代琥珀酰亚胺、N-溴代琥珀酰亚胺、N-碘代琥珀酰亚胺、N-羟基硫代琥珀酰亚胺中的一种。所述方法具有以下优点：1、采用固相环化，首先起到了假稀释效应，避免肽链的重复折叠，能在较高的浓度下进行环化反应，可极大的提高生产效率；其次环化之前线性肽树脂未进行裂解，避免了大量杂质的产生，提高了利那洛肽环化的效率。2、采用N-X代琥珀酰亚胺进行一步环化，可避免中间体的多步纯化，降低了中间纯化步骤的成分，提高了利那洛肽的总收率。3、采用特定的氨基酸侧链保护基，在环化过程中定位了1对二硫键，减少了错配副产物的生成，提高了利那洛肽的纯度，可极大的提高生产效率，降低生产成本。

Description

一种合成利那洛肽的方法 技术领域
本发明涉及药物合成领域，具体涉及一种合成利那洛肽的方法。
技术背景
利那洛肽(Linaclotide)为Ironwood公司开发的一种新型GC-C(肠上皮细胞尿苷酸环化酶C)受体激动剂，可激活肠上皮细胞顶端表面的GC-C受体，导致细胞内和细胞外环鸟苷酸增多。其净效应是氯和碳酸氢盐分泌进入肠腔增加，进而导致液体分泌增多以及大便通过加速，用于治疗成人慢传输型便秘和便秘型肠易激综合征(IBS-C)患者。该药物于2012年12月17日首次获批在美国上市，商品名为LINZESS。
利那洛肽由14个氨基酸组成，并且含有3对二硫键，具体结构序列如下：
Figure PCTCN2017116095-appb-000001
利那洛肽的结构中二硫键是其发挥药效的主要作用点，不仅如此，在其合成路线中二硫键的合成难度最大，成为整个肽序列合成研究中的关键点。
关于利那洛肽的合成技术，最早见于Miriam等人的文献报道，(“OptimizedFmocSolid-PhaseSynthesisoftheCysteine-RichPeptideLinaclotide”，PeptideScience,2011,Volume96,Issue1,pages69-80)其分别采用了三种不同的策略进行利那洛肽的合成：(1)肽序中全部采用Trt为Cys保护基固相合成线性粗肽，然后在液相中室温一步氧化得到利那洛肽；(2)肽序中采用五种不同半胱氨酸保护基，即采用2StBu、2pMeOBzl、2Trt或2Mmt、2Acm、2Trt或2Acm、2Trt、2pMeOBzl共三种正交保护方式，进行固相合成线性粗肽，然后采用分步环化策略完成二硫键的合成；(3)肽序中采用两种不同半胱氨酸保护基，即采用2个StBu和4个Trt进行固相合成线性粗肽，然后采用分步环化策略完成二硫键的合成。
中国专利CN102875655B公开了一种合成利那洛肽的方法，其采用Mmt保护基保护半胱氨酸侧链，用逐一偶联方式合成在C端偶联有树脂固相载体的利那洛肽树脂，裂解脱除保护基和树脂固相载体得到利那洛肽线性粗肽，最后采用GSH/GSSH氧化体系进行氧化反应，得到利那洛肽粗品。
中国专利CN104231051A公开了一种利那洛肽的制备方法，其采用 Fmoc-Cys(Trt)-OH、Fmoc-Cys(Mmt)-OH作为反应原料，逐一偶联获得利那洛肽树脂，经裂解反应获得线性利那洛肽，之后采用磷酸盐缓冲体系、DMSO、EDTA进行氧化获得利那洛肽粗品。
以上文献报道中均采用了液相环化的方法，即首先合成在C端偶联有树脂固相载体的利那洛肽线形肽树脂，然后裂解脱除保护基和树脂固相载体得到利那洛肽线性粗肽，最后采用氧化体系进行环化反应，得到利那洛肽粗品。此外采用此类方法的还包括中国专利CN104628826A、CN103626849A、CN104974229A、CN105884864A、CN104844693A、CN106008674A、CN106167514A。然而，此类方法存在诸多不足：液相环化只有在较低的浓度下才能进行反应，反应效率较低，如Miriam等的方法反应浓度仅为0.5mg/mL，否则会造成肽链的无序聚合，产生大量杂质，十分不利于工业放大化生产；在环化之前进行裂解和保护基的多步脱除，容易造成大量杂质的产生，多步环化的过程中需要对中间体进行多步纯化，操作复杂且增加纯化的难度；一些特殊氨基酸的使用，如Fmoc-Cys(Mmt)-OH、Fmoc-Cys(Hqm)-OH，使得生产成本较高，不利于推广使用。
有鉴于此，本发明的目的在于提供一种合成利那洛肽的方法，本发明所述方法能够加大反应浓度，提高反应效率，减少杂质产生，避免使用特殊原料，简化生产工艺，提高利那洛肽的总收率，同时减少氧化体系的体积，降低生产成本，适合于放大生产。
发明内容
为实现上述目的，本发明提供如下技术方案：
一种合成利那洛肽的方法，包括以下步骤：
1)在活化体系存在下，将Fmoc-Tyr(tBu)-OH与树脂载体偶联，获得Fmoc-Tyr(tBu)-树脂；
2)Fmoc-Tyr(tBu)-树脂采用逐一偶联或分步偶联的方式偶联N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸，获得利那洛肽线性肽树脂；
3)采用N-X代琥珀酰亚胺溶液氧化体系进行环化，获得Cys 1-6,2-10,5-13位环化而其他氨基酸侧链仍连有特定保护基的利那洛肽树脂；
4)树脂经裂解反应，纯化和冻干，获得利那洛肽。
在合成Fmoc-Tyr(tBu)-树脂载体中，活化体系选自由HOBt、DIC、DCC、HBTU、TBTU、PyBop、DMAP、DIPEA、DIEA中的一种或多种组成。
在合成Fmoc-Tyr(tBu)-树脂载体中，进一步优选，所述活化体系为DIEA、 HOBt/DIC/DMAP，HOBt/DCC/DMAP，HBTU/HOBt/DIPEA，TBTU/HOBt/DIPEA，PyBOP/HOBt/DIPEA。
一些实施例中，所述活化体系更优选为DIEA、HOBt/DIC/DMAP或HBTU/HOBt/DIPEA。
在上述合成Fmoc-Tyr(tBu)-树脂载体中，所述活化体系采用DMF、DCM、NMP和DMSO中任意一种或两种溶剂溶解。
优选地，采用DMF或体积比DMF:DCM为1:1的溶剂溶解。
一些实施例中，在上述合成Fmoc-Tyr(tBu)-树脂载体中，进一步优选，采用体积比DMF:DCM为1:1的混合溶剂溶解。
在合成Fmoc-Tyr(tBu)-树脂载体中，载体树脂选自Wang树脂、CTC树脂或羟基类树脂。
优选地，载体树脂为Wang树脂，替代度优选为0.4～1.2mmol/g，
更优选地，载体树脂为Wang树脂，替代度优选为0.5～1.0mmol/g。
本发明所述保护基是在多肽合成领域常用的保护氨基酸主链氨基以及侧链上氨基、羧基、羟基、巯基等干扰合成的基团的保护基团，防止上述活性基团在制备目标产物过程中发生反应，生成杂质。本发明中的N端保护基为Fmoc，对于本发明中需要保护侧链的氨基酸来说，本领域技术人员公知其侧链结构以及知晓采用常用保护基来保护氨基酸侧链上的氨基、羧基、羟基、巯基等基团。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Trt)-OH。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(StBu)-OH。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc- Cys(Acm)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Acm)-OH。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Acm)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Acm)-OH和Fmoc-Cys(Trt)-OH。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(tBu)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(tBu)-OH和Fmoc-Cys(Trt)-OH。
在一个实施例中，步骤2)中所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(StBu)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(StBu)-OH和Fmoc-Cys(Trt)-OH。
本发明所述偶联可以采用逐一偶联或分步偶联。
在上述固相合成利那洛肽树脂的优选方案中，所述逐一偶联是指在第一个氨基酸与固相载体偶联后，剩余氨基酸按照各自序列的顺序逐个与前一个偶联的氨基酸发生缩合反应(主链氨基和羧基的缩合反应)进行偶联。在逐一偶联中，由于每个氨基酸N端都有保护基，因此需要先脱除N端保护基再偶联，这对本领域技术人员来说是公知常识。
在本发明所述方法中，以利那洛肽线性主链N端到C端的氨基酸顺序编号，如下式：
1 2 3 4 5 6 7 8 9 10 11 12 13 14
H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(3对二硫键为1-6、2-10、5-13)。
在上述固相合成利那洛肽树脂的优选方案中，进一步优选，所述分步偶联是指“8+6”或“5+9”模式。本领域技术人员应当知晓，“8+6”模式是指首先分别合成利那洛肽肽序中1-8八肽片段Ⅰ和肽序中9-14六肽片段Ⅱ树脂，再在偶联体系的存在下，将片段Ⅰ与片段Ⅱ连接得到利那洛肽线性肽树脂。同样地，“5+9” 模式是指首先分别合成利那洛肽肽序中1-5五肽片段Ⅰ和肽序中6-14九肽片段Ⅱ树脂，再在偶联体系的存在下，将片段Ⅰ与片段Ⅱ连接得到利那洛肽线性肽树脂。
本发明所述纯化可以采用本技术领域常规纯化方法，如HPLC纯化方法。
本发明步骤3)中所述的N-X代琥珀酰亚胺为N-氯代琥珀酰亚胺、N-溴代琥珀酰亚胺、N-碘代琥珀酰亚胺、N-羟基硫代琥珀酰亚胺中的一种。
本发明步骤3)中所述的N-X代琥珀酰亚胺溶液的溶剂为DMF、DCM。
本发明步骤3)中所述的N-X代琥珀酰亚胺溶液的用量为：1～5eq。
本发明所述合成利那洛肽的方法，在合成利那洛肽线性肽树脂后，采用N-X代琥珀酰亚胺溶液氧化体系进行环化，环化过程中1，6，2，10，5，13位Cys的侧链保护基脱除并形成Cys 1-6,2-10,5-13二硫键，而其余氨基酸侧链保护基及树脂并未脱除，形成了Cys 1-6,2-10,5-13位环化而其他氨基酸侧链仍连有特定保护基的利那洛肽树脂。
本发明步骤4)中所述的裂解试剂为：TFA/Mpr/Tis、TFA/EDT/Tis/H2O。
作为优选，本发明步骤4)中所述的裂解试剂用量为：TFA/Mpr/Tis：(92～95)/(2～5)/(2～5)，TFA/EDT/Tis/H2O：(90～96)/(1～5)/(1～5)/(1～5)。
由本发明所述方法合成的利那洛肽粗肽，重量收率99.3-108.9％，经HPLC检测纯度介于72.1-80.5％，由此得到的利那洛肽精肽经HPLC检测纯度达99.34％，总收率达45.8-52.6％。
需要说明的是，现有的合成利那洛肽的技术中均是首先合成在C端偶联有树脂固相载体的利那洛肽线形肽树脂，然后裂解脱除保护基和树脂固相载体得到利那洛肽线性粗肽，最后采用氧化体系进行环化，得到利那洛肽粗品，即液相环化的方法。本发明中利那洛肽的合成采用的是固相环化，即利那洛肽线性肽树脂未经裂解而直接采用N-X代琥珀酰亚胺溶液氧化体系进行一步环化得到利那洛肽树脂，树脂经裂解，纯化和冻干得到利那洛肽。
液相环化只有在较低的浓度下才能进行反应，反应效率较低，如Miriam等的方法反应浓度仅为0.5mg/mL，否则会造成肽链的无序聚合，产生大量杂质，十分不利于工业放大化生产；在环化之前进行裂解和保护基的多步脱除，容易造成大量杂质的产生，多步环化的过程中需要对中间体进行多步纯化，操作复杂且增加纯化的难度；一些特殊氨基酸的使用，如Fmoc-Cys(Mmt)-OH、Fmoc-Cys(Hqm)-OH，使得生产成本较高，不利于推广使用。
本发明合成利那洛肽的方法，具有以下优点：
1、采用固相环化，首先起到了假稀释效应，避免肽链的重复折叠，能在较高的浓度下进行环化反应，可极大的提高生产效率；其次环化之前线性肽树脂未进行裂解，避免了大量杂质的产生，提高了利那洛肽环化的效率。
2、采用N-X代琥珀酰亚胺进行一步环化，可避免中间体的多步纯化，降低了中间纯化步骤的成本，提高了利那洛肽的总收率。
3、采用特定的氨基酸侧链保护基，在环化过程中定位了1对二硫键，减少了错配副产物的生成，提高了利那洛肽的纯度，可极大的提高生产效率，降低生产成本。
附图说明
图1利那洛肽线性肽树脂环化示意图
图2实施例26利那洛肽粗肽HPLC图
图3实施例31利那洛肽精肽HPLC图
具体实施例
下面结合具体实施例对本发明的利那洛肽的合成方法作进一步详细的说明以便于本领域的技术人员进一步地理解本发明，而不构成对其权利的限制。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内。
说明书和权利要求书中所使用的英文缩写具体含义如表1所示。
表1说明书和权利要求书中所使用的英文缩写具体含义
Figure PCTCN2017116095-appb-000002
Figure PCTCN2017116095-appb-000003
实施例1：替代度为0.53mmol/g的Fmoc-Tyr(tBu)-Wang树脂的制备
称取替代度为1.0mmol/g的Wang树脂100g于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Tyr(tBu)-OH 45.9g(100mmol)，HOBt 16.2g(120mmol)，DMAP 1.2g(10mmol)，用DMF溶解，0℃冰水浴下加入20.3ml DIC(120mmol)，活化5分钟，加入反应柱，反应2小时后，加入70ml醋酸酐和60ml吡啶，混合封闭24h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Tyr(tBu)-Wang树脂，检测替代度为0.53mmol/g。
实施例2：替代度为0.48mmol/g的Fmoc-Tyr(tBu)-Wang树脂的制备
称取替代度为1.0mmol/g的Wang树脂100g，加入到固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，将Fmoc-Tyr(tBu)-OH 45.9g(100mmol)、HOBt 16.2g(120mmol)，DMAP 1.2g(10mmol)，用DMF/DCM＝1:1(V/V)混合液溶解，冰水浴下加入20.3ml DIC(120mmol)活化5min后，加入上述装有树脂的反应柱中，反应2小时后。加入70ml乙酸酐和62ml吡啶混合液封闭24h。用DMF洗涤3次，DCM洗3次，甲醇收缩抽干，得到Fmoc-Tyr(tBu)-Wang树脂，检测替代度为0.48mmol/g。
实施例3：替代度为0.55mmol/g的Fmoc-Tyr(tBu)-Wang树脂的制备
称取替代度为1.0mmol/g的Wang树脂100g于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Tyr(tBu)-OH 45.9g(100mmol)，HOBt 16.2g(120mmol)，HBTU 38.0g(100mmol)，DMAP 2.4g(20mmol)，用DMF溶解，0℃冰水浴下加入32.0ml DIPEA(150mmol)，活化5分钟，加入反应柱，反应2小时后，加入70ml醋酸酐和60ml吡啶，混合封闭24h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Tyr(tBu)-Wang树脂，检测替代度为0.55mmol/g。
实施例4：利那洛肽线性肽树脂的制备
称取实施例1中得到的替代度为0.53mmol/g的Fmoc-Tyr(tBu)-Wang树脂188.7g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。 然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(Trt)-OH175.7g(300mmol)，HOBt 48.6g(360mmol)，用DMF溶解，0℃冰水浴下加入61ml DIC(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Trt)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂487.5g。
实施例5：利那洛肽线性肽树脂的制备
称取将实施例2中得到的替代度为0.48mmol/g的Fmoc-Tyr(tBu)-Wang树脂208.2g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(Trt)-OH175.7g(300mmol)，HOBt 48.6g(360mmol)，HBTU 114.1g(300mmol)用DMF溶解，0℃冰水浴下加入77.5ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(StBu)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(StBu)-Asn(Trt)-Pro-Ala-Cy s(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂482.9g。
实施例6：利那洛肽线性肽树脂的制备
称取将实施例1中得到的替代度为0.53mmol/g的Fmoc-Tyr(tBu)-Wang树脂189.2g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(Acm)-OH 127.8g(300mmol)，HOBt 48.6g(360mmol)，DMAP 36g(300mmol)，用DMF溶解，0℃冰水浴下加入61ml DIC(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、 Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Acm)-OH和Fmoc-Cys(Trt)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Tyr(tBu)-Cys(Acm)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Acm)-Tyr(tBu)-Wang树脂481.5g。
实施例7：利那洛肽线性肽树脂的制备
称取将实施例3中得到的替代度为0.55mmol/g的Fmoc-Tyr(tBu)-Wang树脂181.8g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(tBu)-OH 119.9g(300mmol)，HOBt 48.6g(360mmol)，HBTU 114.1g(300mmol)用DMF溶解，0℃冰水浴下加入77ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(tBu)-OH和Fmoc-Cys(Trt)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Trt)-Cys(tBu)-Glu(OtBu)-Tyr(tBu)-Cys(tBu)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(tBu)-Thr(tBu)-Gly-Cys(tBu)-Tyr(tBu)-Wang树脂484.3g。
实施例8：利那洛肽线性肽树脂的制备
称取将实施例2中得到的替代度为0.48mmol/g的Fmoc-Tyr(tBu)-Wang树脂208.3g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(Trt)-OH175.7克(300mmol)，HOBt 48.6g(360mmol)，DMAP 36g(300mmol)，用DMF溶解，0℃冰水浴下加入61ml DIC(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Acm)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂475.3g。
实施例9：利那洛肽线性肽树脂的制备
称取将实施例3中得到的替代度为0.55mmol/g的Fmoc-Tyr(tBu)-Wang树脂181.8g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(StBu)-OH 129.5g(300mmol)，HOBt 48.6g(360mmol)，HBTU 114.1g(300mmol)用DMF溶解，0℃冰水浴下加入77ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(StBu)-OH和Fmoc-Cys(Trt)-OH。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Trt)-Cys(StBu)-Glu(OtBu)-Tyr(tBu)-Cys(StBu)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(StBu)-Thr(tBu)-Gly-Cys(StBu)-Tyr(tBu)-Wang树脂494.6g。
实施例10：替代度为0.52mmol/g的Fmoc-Pro-CTC树脂的合成
称取替代度为1.00mmol/g的2-CTC树脂200.00g，加入到固相反应柱中，加入到固相反应柱中，用DMF洗涤1次，用DMF溶胀树脂30分钟后，取33.84g Fmoc-Pro-OH(100mmol)用DMF溶解，冰水浴下加入16.5ml DIEA(100mmol)活化后，加入上述装有树脂的反应柱中，反应2小时后，加入200ml无水甲醇封闭1小时。用DMF洗涤3次，DCM洗涤3次，用无水甲醇封闭30分钟，甲醇收缩干燥，得到Fmoc-Pro-CTC树脂，检测替代度为0.52mmol/g。
实施例11：Fmoc-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-OH的制备
称取将实施例10中得到的替代度为0.52mmol/g的Fmoc-Pro-CTC树脂192.3g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Asn(Trt)-OH 113.8g(300mmol)，HOBt 45.2g(300mmol)，HBTU 114.1g(300mmol)用DMF溶解，0℃冰水浴下加入77ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Cys(Acm)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Acm)-OH。反应结束后，用甲醇收缩，得到肽树脂359.6g，真空干燥过夜，加入至25L 玻璃反应器中。
配置裂解液85％TFA/H2O 5L，将裂解试剂倒入烧瓶中，室温反应2h。反应结束，过滤树脂，收集滤液。将滤液浓缩后加至10L异丙醚中，析出白色固体，过滤收集沉淀，异丙醚洗涤，并且真空干燥，得到Fmoc-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-OH白色固体276.2g。
实施例12：替代度为0.54mmol/g的Fmoc-Tyr(tBu)-CTC树脂的制备
称取替代度为1.00mmol/g的2-CTC树脂200.00g，加入到固相反应柱中，加入到固相反应柱中，用DMF洗涤1次，用DMF溶胀树脂30分钟后，取45.95g Fmoc-Tyr(tBu)-OH(100mmol)用DMF溶解，冰水浴下加入16.5ml DIEA(100mmol)活化后，加入上述装有树脂的反应柱中，反应2小时后，加入200ml无水甲醇封闭1小时。用DMF洗涤3次，DCM洗涤3次，用无水甲醇封闭30分钟，甲醇收缩干燥，得到Fmoc-Pro-CTC树脂，检测替代度为0.54mmol/g。
实施例13：Fmoc-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC树脂的制备
称取将实施例12中得到的替代度为0.54mmol/g的Fmoc-Tyr(tBu)-CTC树脂185.3g(100mmol)于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取Fmoc-Cys(Trt)-OH175.7g(300mmol)，HOBt 45.2g(300mmol)，HBTU 114.1g(300mmol)用NMP溶解，0℃冰水浴下加入77ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。重复上述操作，按照肽序依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH和Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH。偶联完毕，用DMF洗涤3次，DCM洗涤3次，抽干得到275.6g Fmoc-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC树脂。
实施例14：利那洛肽线性肽树脂的制备
取实施例13中得到Fmoc-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC树脂于反应柱中，用DCM清洗3次，再用DMF溶胀30分钟。然后用DBLK脱除Fmoc保护基团，然后用DMF洗涤6次。称取实施例11中得到的Fmoc-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-OH，HOBt 48.6g(360mmol)，HBTU 114.1g(300mmol)用DMSO溶解，0℃冰水浴下加入77ml DIPEA(360mmol)，活化5分钟，加入反应柱，反应2小时，然后 用DBLK脱除Fmoc保护基团。反应结束后，用甲醇收缩，得到利那洛肽线性肽树脂：H-Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC树脂480.3g。
实施例15：取代度为0.80mmol/g的Fmoc-Cys(Trt)-CTC树脂的合成
称取取代度为1.20mmol/g的2-CTC树脂50.00g，加入到固相反应柱中，加入到固相反应柱中，用DMF洗涤1次，用DMF溶胀树脂30分钟后，取43.16g Fmoc-Cys(Trt)-OH(100mmol)用DMF溶解，冰水浴下加入17ml DIEA(100mmol)活化后，加入上述装有树脂的反应柱中，反应2小时后，加入500ml无水甲醇封闭1小时。用DMF洗涤3次，DCM洗涤3次，用无水甲醇封闭30分钟，甲醇收缩干燥，得到Fmoc-Cys(Trt)-CTC树脂，检测替代度为0.80mmol/g。
实施例16：Fmoc-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-OH的合成
称取200.00g取代度为0.80mmol/g的Fmoc-Cys(Trt)-CTC树脂125g(100mmol)，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-Cys(Trt)-CTC树脂30分钟后，用DMF:吡啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，称取137.91gFmoc-Tyr(tBu)-OH(300mmol)、40.52gHOBt(300mmol)加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入46mlDIC(300mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照利那洛肽主链肽序，依次完成Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(StBu)-OH的偶联。
将树脂加入到5000mL的三口圆底烧瓶中，按体积比为1:4的TFE和DCM配置裂解液3200ml，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到Fmoc-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-OH 165.37g
实施例17：H-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂的合成
称取将实施例1中得到的替代度为0.53mmol/g的Fmoc-Tyr(tBu)-Wang树脂189.2g(100mmol)于反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-Tyr(tBu)-Wang树脂30分钟后，用DMF:吡啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，称取175.7gFmoc-Cys(Trt)-OH(300mmol)、40.5gHOBt(300mmol)加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入46mlDIC(300mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照利那洛肽主链肽序，依次Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(StBu)-OH的偶联。
偶联完毕，采用20％β-巯基乙醇，0.1M N-甲基吗啉的DMF裂解液去StBu保护基，然后用DMF洗涤6次，得到Fmoc-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂，用DMF:吡啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到354.8g H-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂。
实施例18：H-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂的合成
称取实施例16制得的165.37g Fmoc-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-OH(100mmol)，15.00gHSBzl(120mmol)加入2000mlTHF中，冰水浴下加入19mlDIC(120mmol)，反应1小时，升温到室温反应3小时，反应液过滤，母液旋干，加DCM溶解，过滤，饱和碳酸氢钠洗3遍，纯水2遍，反萃2遍，合并有机相，无水碳酸钠干燥，旋干，冰乙醇重结晶3次，过滤，固体油泵拉干的到158.62g Fmoc-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-SBzl。
称取实施例17制得的354.8g H-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂(100mmol)，加入固相反应柱中，用DMF洗涤 1次，用DMF溶胀30分钟后，称取158.62g Fmoc-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-SBzl(100mmol)加入DMF溶液，加入上述装有树脂的反应柱中，25℃下反应4小时后，得到Fmoc-Cys(StBu)Cys(Trt)Glu(OtBu)Tyr(tBu)Cys-CysAsn(Trt)Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang王树脂。用DMF:吡啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到425.4g H-Cys(StBu)Cys(Trt)Glu(OtBu)Tyr(tBu)Cys-CysAsn(Trt)Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂。
实施例19：利那洛肽肽树脂的制备
以60mg/ml的浓度将487.5g实施例4制得的利那洛肽线性肽树脂(100mmol)溶于8.13L DMF中。在溶液中加入1eq的N-溴代琥珀酰亚胺DMF溶液，25℃搅拌反应12小时，得到利那洛肽肽树脂292.85g。反应如图1所示。
实施例20：利那洛肽肽树脂的制备
以45mg/ml的浓度将482.9g实施例5制得的利那洛肽线性肽树脂(100mmol)溶于10.73LDMF中。在溶液中加入2eq的N-氯代琥珀酰亚胺DMF溶液，30℃搅拌反应12小时，得到利那洛肽肽树脂289.74g。
实施例21：利那洛肽肽树脂的制备
以50mg/ml的浓度将481.5g实施例6制得的利那洛肽线性肽树脂(100mmol)溶于9.63LDMF中。在溶液中加入3eq的N-碘代琥珀酰亚胺DCM溶液，30℃搅拌反应12小时，得到利那洛肽肽树脂289.37g。
实施例22：利那洛肽肽树脂的制备
以50mg/ml的浓度将484.3g实施例7制得的利那洛肽线性肽树脂(100mmol)溶于9.69LDMF中。在溶液中加入3eq的N-羟基硫代琥珀酰亚胺DMF溶液，25℃搅拌反应12小时，得到利那洛肽肽树脂285.61g。
实施例23：利那洛肽肽树脂的制备
以55mg/ml的浓度将475.3g实施例8制得的利那洛肽线性肽树脂(100mmol)溶于8.64LDMF中。在溶液中加入4eq的N-羟基硫代琥珀酰亚胺DMF溶液，28℃搅拌反应12小时，得到利那洛肽肽树脂294.31g。
实施例24：利那洛肽肽树脂的制备
以48mg/ml的浓度将494.6g实施例9制得的利那洛肽线性肽树脂(100mmol) 溶于10.30LDMF中。在溶液中加入2.5eq的N-氯代琥珀酰亚胺DMF溶液，25℃搅拌反应12小时，得到利那洛肽肽树脂287.34g。
实施例25：利那洛肽肽树脂的制备
以52mg/ml的浓度将480.3g实施例14制得的利那洛肽线性肽树脂(100mmol)溶于9.24LDMF中。在溶液中加入2.5eq的N-溴代琥珀酰亚胺DMF溶液，25℃搅拌反应12小时，得到利那洛肽肽树脂297.36g。
实施例26：利那洛肽肽树脂的制备
以42mg/ml的浓度将425.4g实施例18制得的H-Cys(StBu)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys-Cys-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang树脂(100mmol)溶于10.13LDMF中。在溶液中加入2.5eq的N-溴代琥珀酰亚胺DMF溶液，25℃搅拌反应12小时，得到利那洛肽肽树脂259.46g。
实施例27：利那洛肽粗肽的制备
将实施例19制备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/Mpr/Tis：94/3/3)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽140.32g。粗肽重量收率为108.9％，HPLC纯度为72.1％，如图2所示。
实施例28：利那洛肽粗肽的制备
将实施例20制备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/Mpr/Tis：92/4/4)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽145.21g。粗肽重量收率为99.9％，HPLC纯度为75.4％，HPLC图与实施例26的相似。
实施例29：利那洛肽粗肽的制备
将实施例21制备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/EDTA/Tis/H2O：90/3/3/4)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽144.21g。粗肽重量 收率为98.9％，HPLC纯度为80.3％，HPLC图与实施例26的相似。
实施例30：利那洛肽粗肽的制备
将实施例22制备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/Mpr/Tis：94/2/4)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽144.21g。粗肽重量收率为98.9％，HPLC纯度为80.5％，HPLC图与实施例26的相似。
实施例31：利那洛肽粗肽的制备
将实施例23制备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/EDTA/Tis/H2O：92/1/5/2)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽143.21g。粗肽重量收率为100.9％，HPLC纯度为76.3％，HPLC图与实施例26的相似。
实施例32：利那洛肽粗肽的制备
将实施例24备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/EDTA/Tis/H2O：94/2/3/1)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽146.34g。粗肽重量收率为100.9％，HPLC纯度为78.2％，HPLC图与实施例26的相似。
实施例33：利那洛肽粗肽的制备
将实施例24备得到的利那洛肽肽树脂置于裂解反应器中，以15ml/g树脂的比例加入裂解试剂(TFA/EDTA/Tis/H2O：94/2/3/1)(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉降，沉降液经过滤或离心，所得滤饼用无水乙醚洗涤3次，真空干燥得到白色粉末固体，即利那洛肽粗肽143.34g。粗肽重量收率为99.3％，HPLC纯度为77.6％，HPLC图与实施例26的相似。
实施例34：利那洛肽精肽的制备
取实施例26制备得到的利那洛肽粗肽，采用NOVASEPRP-HPLC系统，波 长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化，除盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到利那洛肽精肽116.45g，HPLC纯度99.23％，总收率45.8％。精肽谱图如图3所示。
实施例35：利那洛肽精肽的制备
取实施例27制备得到的利那洛肽粗肽，采用NOVASEPRP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化，除盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到利那洛肽精肽114.53g，HPLC纯度99.32％，总收率50.3％。精肽谱图如与实施例31相似。
实施例36：利那洛肽精肽的制备
取实施例28制备得到的利那洛肽粗肽，采用NOVASEPRP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化，除盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到利那洛肽精肽115.32g，HPLC纯度99.21％，总收率46.8％。精肽谱图如与实施例31相似。
实施例37：利那洛肽精肽的制备
取实施例29制备得到的利那洛肽粗肽，采用NOVASEPRP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化，除盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到利那洛肽精肽114.26g，HPLC纯度99.16％，总收率52.6％。精肽谱图如与实施例31相似。
实施例38：利那洛肽精肽的制备
取实施例30制备得到的利那洛肽粗肽，采用NOVASEPRP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化，除盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到利那洛肽精肽112.34g，HPLC纯度99.34％，总收率49.5％。精肽谱图如与实施例31相似。

Claims (20)

  1. 一种合成利那洛肽的方法，其特征在于，包括以下步骤：
    1)在活化体系存在下，将Fmoc-Tyr(tBu)-OH与树脂载体偶联，获得Fmoc-Tyr(tBu)-树脂；
    2)Fmoc-Tyr(tBu)-树脂采用逐一偶联或分步偶联的方式偶联N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸，获得利那洛肽线性肽树脂；
    3)采用N-X代琥珀酰亚胺溶液氧化体系进行环化，获得Cys 1-6,2-10,5-13位环化而其他氨基酸侧链仍连有特定保护基的利那洛肽树脂；
    4)肽树脂经裂解反应，纯化和冻干，获得利那洛肽。
  2. 根据权利要求1所述的方法，其特征在于，所述的活化体系为HOBt、DIC、DCC、HBTU、TBTU、PyBop、DMAP、DIPEA、DIEA中的一种或多种组成。
  3. 根据权利要求2所述的方法，其特征在于，所述的活化体系为DIEA、HOBt/DIC/DMAP，HOBt/DCC/DMAP，HBTU/HOBt/DIPEA，TBTU/HOBt/DIPEA，PyBOP/HOBt/DIPEA；
  4. 根据权利要求2或3任一所述的方法，其特征在于，所述的活化体系更优选为DIEA、HOBt/DIC/DMAP或HBTU/HOBt/DIPEA。
  5. 根据权利要求1所述方法，其特征在于，所述活化体系采用DMF、DCM、NMP和DMSO中任意一种或两种溶剂溶解。
  6. 根据权利要求1所述的方法，其特征在于，所述的载体树脂选自Wang树脂、CTC树脂或羟基类树脂。
  7. 根据权利要求5或6任一所述的方法，其特征在于，所述的载体树脂为Wang树脂，替代度为0.4～1.2mmol/g，
  8. 根据权利要求7所述的方法，其特征在于，所述的载体Wang树脂替代度为0.5～1.0mmol/g。
  9. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Trt)-OH。
  10. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、 Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(StBu)-OH。
  11. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Cys(Acm)-OH。
  12. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(Acm)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Acm)-OH和Fmoc-Cys(Trt)-OH。
  13. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(tBu)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(tBu)-OH和Fmoc-Cys(Trt)-OH。
  14. 根据权利要求1所述的方法，其特征在于，所述的N端有Fmoc保护基以及侧链连有特定保护基的其他氨基酸为：Fmoc-Cys(StBu)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(StBu)-OH和Fmoc-Cys(Trt)-OH。
  15. 根据权利要求1所述的方法，其特征在于，步骤2)中所述的分步偶联为8+6模式或5+9模式。
  16. 根据权利要求1所述的方法，其特征在于，步骤3)中所述的N-X代琥珀酰亚胺为N-氯代琥珀酰亚胺、N-溴代琥珀酰亚胺、N-碘代琥珀酰亚胺、N-羟基硫代琥珀酰亚胺中的一种。
  17. 根据权利要求1所述的方法，其特征在于，步骤3)中所述的N-X代琥珀酰亚胺溶液的溶剂为DMF、DCM。
  18. 根据权利要求1所述的方法，特征在于，步骤3)中所述的N-X代琥珀 酰亚胺溶液的用量为：1～5eq。
  19. 根据权利要求1所述的方法，特征在于，步骤4)中所述的裂解试剂为：TFA/Mpr/Tis、TFA/EDT/Tis/H2O，
  20. 根据权利要求1所述的方法，特征在于，步骤4)中所述的裂解试剂用量为：TFA/Mpr/Tis：(92～95)/(2～5)/(2～5)，TFA/EDT/Tis/H2O：(90～96)/(1～5)/(1～5)/(1～5)。
PCT/CN2017/116095 2017-12-14 2017-12-14 一种合成利那洛肽的方法 WO2019113872A1 (zh)

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