WO2021000543A1 - Procédé de synthèse de l'ételcalcétide - Google Patents

Procédé de synthèse de l'ételcalcétide Download PDF

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WO2021000543A1
WO2021000543A1 PCT/CN2019/127793 CN2019127793W WO2021000543A1 WO 2021000543 A1 WO2021000543 A1 WO 2021000543A1 CN 2019127793 W CN2019127793 W CN 2019127793W WO 2021000543 A1 WO2021000543 A1 WO 2021000543A1
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compound
reaction
solvent
acetonitrile
arg
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PCT/CN2019/127793
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Chinese (zh)
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陶志强
姚志军
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
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07K1/06—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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography

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  • the present invention relates to the field of polypeptide synthesis, and particularly relates to a synthetic method of Etelcalcetide.
  • Etelcalcetide is a novel calcimimetic developed by AMGEN INC. It is mainly used as a polypeptide drug for secondary hyperparathyroidism in hemodialysis treatment of adult patients with chronic kidney disease. It was launched in the United States on February 07, 2017 under the trade name Parsabiv.
  • the main chain of Etelcalcetide consists of seven D-type amino acids, and the side chain is connected to L-cysteine through a disulfide bond.
  • the peptide sequence is shown in formula I:
  • the solid phase coupling of Etelcalcetide requires the use of resins, and large-scale production is limited. Moreover, the amino acid usually needs to be excessive (3-5 equivalents) during coupling, and Etelcalcetide is mainly based on expensive D-type amino acids. Therefore, the solid-phase synthesis method is too expensive and large-scale production is restricted.
  • the coupling between patent CN106928321 and WO2016154580A1 uses traditional coupling reagents (PyBop, HATU, DIC or EDC, etc.) for activation coupling, but the coupling efficiency of this method for fragments is usually low, resulting in yield Too low.
  • the present invention provides a synthesis method of Etelcalcetide.
  • the method adopts the intramolecular migration reaction of S to N to complete the coupling, thereby improving the coupling efficiency between peptide fragments, and the total yield is 71.3%. It overcomes the defect of difficulty in coupling between fragments under the action of traditional coupling reagents, reduces production costs, and is suitable for large-scale production.
  • the present invention provides a synthetic method of Etelcalcetide, using ⁇ -S(Trt)-D-Arg(Boc)3-OH as raw material, replacing alanine in peptide sequence with cysteine, and using NCL method for coupling reaction , Etelcalcetide was prepared.
  • Step 1 Take ⁇ -S(Trt)-D-Arg(Boc) 3 -OH and thiophenol (PhSH) as raw materials, and obtain compound 1 through coupling reaction in the presence of a solvent;
  • Step 2 Ac-D-Cys(Acm)-OH and HONb are used as raw materials, activated by a coupling agent in the presence of a solvent, and dehydrated and condensed with H-Ala-OH to obtain compound 2;
  • Step 3 Take compound 2 and thiophenol (PhSH) as raw materials, and obtain compound 3 through condensation reaction in the presence of a solvent and a coupling agent;
  • Step 4 Using Boc-D-Cys(Trt)-OH and HD-Arg(Pbf)-NH 2 ⁇ HCI as raw materials, the dipeptide fragment, compound 4, is obtained by condensation reaction in the presence of solvent and coupling agent;
  • Step 5 Compound 4 undergoes deprotection reaction in the presence of lysate to obtain compound 5;
  • Step 6 Using compound 5 and compound 1 as raw materials, in the presence of solvent, buffer solution, TCEP and trifluoroethanol, undergo NCL reaction to obtain compound 6 by cracking;
  • Step 7 Using compound 6 and compound 1 as raw materials, in the presence of solvent, buffer solution, TCEP and trifluoroethanol, undergo NCL reaction to obtain compound 7 by cracking;
  • Step 8 Using compound 7 and compound 1 as raw materials, in the presence of solvent, buffer solution, TCEP and trifluoroethanol, undergo NCL reaction to obtain compound 8 by cracking;
  • Step 9 Using compound 8 and compound 3 as raw materials, reacting with NCL in the presence of solvent, buffer solution, TCEP and trifluoroethanol, and then desulfurizing and purifying in the presence of initiator and tBuSH to obtain compound 9 ;
  • Step 10 Take compound 9 and L-Cys-OH as raw materials, oxidize and purify with iodine to obtain Etecalcetide.
  • the solvent in step 1 includes tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane , Chloroform, dichloromethane or a mixture of two or more; preferably dichloromethane;
  • step 1 the molar ratio of ⁇ -S(Trt)-D-Arg(Boc) 3 -OH to thiophenol (PhSH) is 1:1.0 ⁇ 1.5;
  • the temperature of the coupling reaction in step 1 is 20°C to 30°C; the time of the coupling reaction is 2h-10h; preferably 5h; the coupling agent used in the coupling reaction includes a combination of DIC and compound A , A combination of DIPEA, compound A and compound B, wherein compound A is HOAt or HOBt, compound B is one or a combination of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably EDC/
  • the molar ratio of Ac-D-Cys(Acm)-OH to HONb in step 2 is 1:1.0 to 1.5;
  • the solvent selected for activation in step 2 includes tetrahydrofuran, dimethyl sulfoxide, dimethyl formamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane One or a combination of more than two; preferably dichloromethane;
  • the activation temperature in step 2 is 20°C to 30°C, and the activation time is 1h to 5h; preferably 3h;
  • the coupling agent used for activation in step 2 is a combination of DIC or DIPEA and compound B, and compound B is one or a combination of PyAOP, PyBOP, HATU, HBTU, TBTU or EDC; preferably DIC;
  • the solvents selected for the dehydration condensation in step 2 include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane , One or a mixture of two or more in water; preferably a mixed solvent of water and acetonitrile; further, the ratio of water to acetonitrile is 2:1;
  • the temperature of the dehydration and condensation in step 2 is 20°C to 30°C, and the time of the dehydration and condensation is 1h to 3h; preferably 2h;
  • the Acm protecting group in Ac-D-Cys(Acm)-OH can also be replaced with a Trt protecting group.
  • the molar ratio of compound 2 to thiophenol (PhSH) in step 3 is 1:1.0 to 1.5;
  • the solvent used in the condensation reaction includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloride One or a combination of two or more of methane; preferably methylene chloride;
  • step 3 the temperature of the condensation reaction is 20°C to 30°C, and the time of the condensation reaction is 2h-10h; preferably 5h;
  • the molar ratio of Boc-D-Cys(Trt)-OH to HD-Arg(Pbf)-NH 2 ⁇ HCI in step 4 is 1:1.0 ⁇ 1.5;
  • the temperature of the condensation reaction in step 4 is 20°C-30°C, and the time of the condensation reaction is 2h-10h; preferably 5h;
  • the solvent in step 4 includes one of tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, and dichloromethane Or a mixture of two or more; preferably dichloromethane;
  • the lysate in step 5 of TFA, H 2 O and TIS composition TFA, H 2 O and a volume ratio of the TIS 95: 2.5: 2.5;
  • step 5 the temperature of the deprotection reaction is 20°C to 30°C, and the time of the deprotection reaction is 2h-4h; preferably 3h;
  • the molar ratio of the compound 5, compound 6, compound 7, and compound 8 to the compound 1 is 1.0 to 1.1:1;
  • the buffer solution in steps 6-9 is a mixed solution of 6.0M guanidine hydrochloride (Gn ⁇ HCl) and 0.2M Na 2 HPO 4 , and the pH value ranges from 5.6 to 7.6; preferably, the pH is 6.6;
  • the solvents described in steps 6-9 include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane One or a mixture of two or more; preferably acetonitrile;
  • the temperature of the NCL reaction in steps 6-9 is 20°C-30°C, and the time of the NCL reaction is 2h-6h; preferably 4h;
  • the amount of TCEP and trifluoroethanol in steps 6-9 is 5-15 equivalents of the raw materials; preferably 10 equivalents;
  • the lysis solution used for the lysis in steps 6 to 8 is a composition of TFA, H 2 O, and TIS, and the volume ratio of TFA:H 2 O:TIS is 95:2.5:2.5;
  • the temperature of the cracking in steps 6-8 is 20°C-30°C, and the time of the cracking is 2h-4h; preferably 3h.
  • the initiator in step 9 is VA-044; the amount of the initiator is 0.01-0.03 equivalents of compound 3; preferably 0.02 equivalents; the amount of tBuSH is that of compound 3. 1 to 3 equivalents; preferably 2 equivalents;
  • the temperature of the desulfurization reaction in step 9 is 20°C to 30°C, and the time of the desulfurization reaction is 2h to 4h; preferably 3h;
  • the purification in step 9 adopts reversed-phase high pressure liquid chromatography.
  • the reversed-phase high pressure liquid chromatography includes: using reversed-phase octadecylsilane as the stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as the mobile phase, and collecting The largest peak fraction was concentrated under reduced pressure at 30°C to 1/4 volume and lyophilized.
  • the solvent used for the iodine oxidation in step 10 is tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, One or a mixture of two or more of toluene, hexane, chloroform, dichloromethane, methanol, ethanol, and water; preferably a mixed solvent of methanol and water; further, the ratio of methanol to water is 1:5;
  • step 10 the pH value of the iodine oxidation is adjusted to 2 to 4 using glacial acetic acid; preferably, the pH value is 3;
  • the amount of iodine used is 1 to 3 equivalents of the raw materials; preferably 2 equivalents;
  • step 10 the temperature of the iodine oxidation is 20°C to 30°C, and the time of the iodine oxidation is 1h to 5h; preferably 3h;
  • the purification adopts reversed-phase high pressure liquid chromatography;
  • the reversed-phase high pressure liquid chromatography includes: using reversed-phase octadecylsilane as a stationary phase, and using a 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a flow Phase, collect the largest peak fraction, concentrate under reduced pressure at 30°C to 1/4 volume, and freeze-dry.
  • the solid phase coupling of Etelcalcetide requires the use of resins, and large-scale production is limited. Moreover, the amino acid usually needs to be excessive (3-5 equivalents) during coupling, and Etelcalcetide is mainly based on expensive D-type amino acids. Therefore, the solid-phase synthesis method is too expensive and large-scale production is restricted.
  • the traditional fragment method is used for the large steric hindrance between the fragments, resulting in generally low coupling efficiency, resulting in too low yield.
  • the coupling yield between the two fragments in the comparative example is only 43.2%, plus The loss of the synthesis of the fragment itself, the yield is not higher than 5%.
  • the NCL (Natural Chemical Linking) method is an efficient method for assembling two or more unprotected peptides to construct a protein.
  • ⁇ -S(Trt)-D-Arg(Boc)3-OH is used as the raw material, and cysteine is substituted for the alanine in the peptide sequence, and the NCL (Natural Chemical Linking) method is adopted for coupling reaction.
  • the present invention improves upon the shortcomings of the existing liquid phase method. According to the unique characteristics of this peptide sequence, the S to N intramolecular migration reaction is used to complete the coupling, thereby improving the coupling efficiency between peptide fragments. This patent The total yield of the method is 71.3%. Therefore, the present invention overcomes the defect of difficulty in coupling between fragments under the action of traditional coupling reagents, reduces production costs, and is suitable for large-scale production.
  • Figure 1 shows a flow chart of the preparation method provided by the present invention
  • FIG. 1 shows the HPLC spectrum of compound 6
  • Figure 3 shows the mass spectrum of compound 6
  • FIG. 4 shows the HPLC spectrum of compound 8.
  • Figure 5 shows the mass spectrum of compound 8.
  • FIG. 6 shows the HPLC spectrum of compound 9
  • Figure 7 shows the mass spectrum of compound 9
  • FIG. 8 shows the HPLC spectrum of Etelcalcetide
  • Figure 9 shows the mass spectrum of the refined peptide of Etelcalcetide
  • Figure 10 shows the HPLC spectrum of the comparative example Etelcalcetide
  • Figure 11 shows the mass spectrum of the comparative example Etelcalcetide.
  • the invention discloses a synthetic method of Etelcalcetide, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it.
  • all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the NCL (Natural Chemical Linking) method is an efficient method for assembling two or more unprotected peptides to construct a protein. Therefore, in order to reduce the cost and overcome the difficulty in coupling between fragments, according to the characteristics of the peptide sequence of Etelcalcetide, the present invention uses ⁇ -S(Trt)-D-Arg(Boc) 3 -OH as the raw material and uses cysteamine The acid replaces the alanine in the peptide sequence, and the NCL (Natural Chemical Ligation) method is used for coupling reaction. The details are shown in Figure 1.
  • the present invention uses ⁇ -S(Trt)-D-Arg(Boc) 3 -OH and thiophenol as raw materials to synthesize thioester compound 1 under the action of a coupling reagent.
  • thioester compound 3 is synthesized with thiophenol under the action of coupling reagent.
  • the dipeptide fragment compound 4 was synthesized under the action of coupling reagent, and compound 5 was obtained after deprotection.
  • Compound 5 and thioester compound 1 are in a buffer solution through NCL reaction through the action of TCEP and trifluoroethanol to obtain compound 6. Continue to couple two thioester compounds 1 sequentially by NCL method to obtain compound 8.
  • Step 1 Synthesis of compound 1 ( ⁇ -S(Trt)-D-Arg(Boc) 3 -SPh)
  • Step 3 Synthesis of compound 3 (Ac-D-Cys(Acm)-D-Ala-SPh)
  • Step 4 Synthesis of compound 4 (Boc-D-Cys(Trt)-D-Arg(Pbf)-NH 2 )
  • Step 5 Synthesis of compound 5 (HD-Cys-D-Arg-NH 2 )
  • Step 6 Synthesis of compound 6 (HD-Arg( ⁇ -SH)-D-Cys-D-Arg-NH 2 )
  • Step 7 Synthesis of compound 7 (HD-Arg( ⁇ -SH)-D-Arg( ⁇ -SH)-D-Cys-D-Arg-NH 2 )
  • step 1 ⁇ -S(Trt)-D-Arg(Boc) 3 -OH and thiophenol (PhSH) are mainly used as raw materials, and the molar ratio is 1:1.0 to 1.5.
  • the coupling reaction was carried out at room temperature (25°C ⁇ 5°C) to obtain compound 1.
  • the selected solvents include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and other solvents, preferably dichloromethane .
  • the coupling reaction time is 2-10 hours, preferably 5 hours.
  • the coupling agent is a combination of DIC and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is HOAt or HOBt, and compound B is PyAOP or PyBOP , HATU, HBTU or TBTU, EDC.
  • compound A is HOAt or HOBt
  • compound B is PyAOP or PyBOP , HATU, HBTU or TBTU, EDC.
  • EDC EDC/HOBT/DIPEA composition
  • step 2 Ac-D-Cys(Acm)-OH and HONb are used as raw materials, and the molar ratio is 1:1.0 ⁇ 1.5. It is activated by coupling agent at room temperature (25°C ⁇ 5°C). After activation, it is activated in DIPEA Under the action of, it is dehydrated and condensed with H-Ala-OH to obtain compound 2.
  • the solvents selected for the activation reaction include tetrahydrofuran, dimethyl sulfoxide, dimethyl formamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and other solvents, preferably two Methyl chloride.
  • the activation reaction is carried out at room temperature (25°C ⁇ 5°C) for 1-5 hours, preferably 3 hours.
  • the activated coupling agent is a combination of DIC or DIPEA and compound B, compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, and EDC is preferably DIC.
  • the solvent selected for the dehydration condensation reaction with H-Ala-OH includes tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, Solvents such as dichloromethane and water are preferably mixed solvents of water and acetonitrile, and the ratio of water to acetonitrile is further 2:1.
  • the coupling reaction is carried out at room temperature (25°C ⁇ 5°C) for 1-3 hours, preferably 2 hours.
  • the Acm protecting group in Ac-D-Cys(Acm)-OH can also be replaced with a Trt protecting group.
  • step 3 compound 2 and thiophenol (PhSH) are used as raw materials with a molar ratio of 1:1.0 to 1.5, and the coupling agent undergoes condensation reaction at room temperature (25°C ⁇ 5°C).
  • the selected solvents include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and other solvents, preferably dichloromethane .
  • the reaction time is 2-10 hours, preferably 5 hours.
  • the coupling agent is a combination of DIC and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU , HBTU or TBTU, EDC.
  • compound A is HOAt or HOBt
  • compound B is PyAOP, PyBOP, HATU , HBTU or TBTU
  • EDC is an EDC/HOBT/DIPEA composition.
  • step 4 Boc-D-Cys(Trt)-OH, HD-Arg(Pbf)-NH 2 ⁇ HCI are used as raw materials, and the molar ratio is 1:1.0 ⁇ 1.5.
  • the coupling reagent undergoes condensation reaction to obtain the dipeptide fragment compound 4.
  • the selected solvents include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and other solvents, preferably dichloromethane .
  • the reaction time is 2-10 hours, preferably 5 hours.
  • the coupling agent is a combination of DIC and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU , HBTU or TBTU, EDC.
  • it is an EDC/HOBT/DIPEA composition.
  • step 5 the compound undergoes deprotection reaction through the lysate.
  • the volume ratio of lysis buffer TFA:H 2 O:TIS was 95:2.5:2.5 for lysis.
  • the lysis time at room temperature is 2 to 4 hours, preferably 3 hours.
  • step 6 compound 5 and compound 1 are in a buffer solution at a molar ratio of 1.0-1.1:1, and NCL reaction is performed through the action of TCEP and trifluoroethanol, and then compound 6 is obtained by cleavage.
  • the buffer solution is a mixed solution of 6.0M guanidine hydrochloride (Gn ⁇ HCl) and 0.2M Na2HPO4, and its pH value ranges from 5.6 to 7.6, preferably pH 6.6.
  • the solubilizing solvents selected for the reaction are tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane and other solvents. Acetonitrile is preferred.
  • the reaction time at room temperature (25°C ⁇ 5°C) is 2-6 hours, preferably 4 hours.
  • the amount of the catalyst TCEP/trifluoroethanol is 5-15 equivalents of the substrate, preferably 10 equivalents. Compound 6 undergoes deprotection reaction through the lysate.
  • the volume ratio of lysis buffer TFA:H 2 O:TIS was 95:2.5:2.5 for lysis.
  • the lysis time at room temperature (25°C ⁇ 5°C) is 2 to 4 hours, preferably 3 hours.
  • Step 7 is the same as step 6, and compound 1 is coupled in sequence to obtain compound 7.
  • Step 8 is the same as steps 6 and 7, and compound 1 is sequentially coupled to obtain compound 8.
  • step 9 The description of the NCL reaction conditions in step 9 is the same as that in step 6.
  • VA-044 is used as an initiator for desulfurization reaction.
  • compound 9 was prepared and purified by reverse phase HPLC.
  • the amount of initiator used is 0.01-0.03 equivalents of compound 3, preferably 0.02 equivalents.
  • TBuSH is used to provide protons, and the amount is 1-3 equivalents of compound 3, preferably 2 equivalents.
  • the desulfurization reaction is carried out at room temperature, and the desulfurization reaction time is 2-4 hours, preferably 3 hours.
  • the purification step can be reversed-phase high pressure liquid chromatography.
  • the reverse-phase high-pressure liquid chromatography method includes: using reverse-phase octadecylsilane as a stationary phase, using a 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, collecting the largest peak fraction, and concentrating under reduced pressure at 30°C to 1/4 volume, freeze-dried.
  • step 10 compound 9 and L-Cys-OH are oxidized by iodine to obtain Etecalcetide.
  • the selected solvents for iodine oxidation are tetrahydrofuran, dimethyl sulfoxide, dimethyl formamide, dioxane, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, methanol, ethanol , Water, etc., preferably a mixed solvent of methanol and water, and further, the ratio of methanol to water is 1:5.
  • Use glacial acetic acid to adjust the pH to the range of 2-4, preferably pH 3.
  • the amount of iodine used is 1 to 3 equivalents of the substrate, preferably 2 equivalents. Oxidation at room temperature, the oxidation time is 1 to 5 hours, preferably 3 hours.
  • the purification step can be reversed-phase high pressure liquid chromatography. Further, the reverse-phase high-pressure liquid chromatography method includes: using reverse-phase octadecylsilane as a stationary phase, using a 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, collecting the largest peak fraction, and concentrating under reduced pressure at 30°C to 1/4 volume, freeze-dried.
  • the raw materials and reagents used in the synthetic method of Etelcalcetide provided by the present invention can all be purchased from the market.
  • the organic phase was concentrated under reduced pressure at 30°C, the residue was stirred and dissolved with ethyl acetate (300 mL), and washed with purified water (300 mL), saturated sodium bicarbonate solution (300 mL), and saturated sodium chloride solution (300 mL) in turn.
  • the organic phase was collected and dried overnight with anhydrous sodium sulfate. After filtration, the filtrate was concentrated to 60 mL, and n-hexane (240 mL) was slowly added dropwise. A white solid precipitated out, and after continuing to stir for 1 hour, it was allowed to stand and crystallize overnight at 0°C. After filtration, it was vacuum dried at 40°C to obtain 30.6 g of white solid with a yield of 91.2%.
  • Example 8 Synthesis of Compound 8 (HD-Arg( ⁇ -SH)--D-Arg( ⁇ -SH)-D-Arg( ⁇ -SH)-D-Cys-D-Arg-NH 2 )

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Abstract

La présente invention relève du domaine de la synthèse des polypeptides et concerne un procédé de synthèse de l'ételcalcétide. Le procédé utilise du β-S(Trt)-D-Arg(Boc)3-OH en tant que matière première, remplace l'alanine dans une séquence peptidique avec de la cystéine, et utilise un procédé de ligature chimique native (NCL) pour la réaction de couplage. Le procédé utilise une réaction de migration intramoléculaire de S à N pour achever le couplage, surmonte le problème de difficulté de couplage entre des fragments sous l'action d'un réactif de couplage classique, améliore l'efficacité de couplage entre des fragments peptidiques, et a un rendement total de 71,3 %. La présente invention réduit les coûts de production et est appropriée pour une production à grande échelle.
PCT/CN2019/127793 2019-07-03 2019-12-24 Procédé de synthèse de l'ételcalcétide WO2021000543A1 (fr)

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CN201910595277.6A CN112175042B (zh) 2019-07-03 2019-07-03 Etelcalcetide的合成方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117486968A (zh) * 2024-01-03 2024-02-02 深圳创元生物医药科技有限公司 一种类蛇毒肽的制备方法

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