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CN114524860B - A kind of synthesis method of Etelcalcetide and application thereof - Google Patents

A kind of synthesis method of Etelcalcetide and application thereof Download PDF

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CN114524860B
CN114524860B CN202111644861.XA CN202111644861A CN114524860B CN 114524860 B CN114524860 B CN 114524860B CN 202111644861 A CN202111644861 A CN 202111644861A CN 114524860 B CN114524860 B CN 114524860B
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arg
pbf
etelcalcetide
ala
fmoc
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CN114524860A (en
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黄志颖
陶志强
尹传龙
唐洋明
余品香
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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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
    • 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

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Abstract

一种Etelcalcetide的合成方法及其应用，属于药物合成技术领域。本发明包括以下步骤：(1)液相合成Fmoc‑D‑Ala‑D‑Arg(Pbf)‑OH二肽片段1；(2)液相合成Fmoc‑D‑Arg(Pbf)‑D‑Arg(Pbf)‑OH二肽片段2；(3)依次偶联氨基酸：二肽片段1、二肽片段2、二肽片段1、N‑Ac‑D‑Cys(Mmt)‑OH，获得片段A；(4)脱除片段A中Cys(Mmt)的保护基，获得片段B，再与Boc‑Cys(Npys)‑OH偶联后，裂解获得Etelcalcetide粗品。本发明可以应用于亲水性、疏水性多肽、长链多肽的直接修饰同时可以在有机溶剂中进行。

A synthesis method and application of Etelcalcetide, belonging to the technical field of drug synthesis. The present invention comprises the following steps: (1) liquid phase synthesis of Fmoc-D-Ala-D-Arg(Pbf)-OH dipeptide fragment 1; (2) liquid phase synthesis of Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH dipeptide fragment 2; (3) sequentially coupling amino acids: dipeptide fragment 1, dipeptide fragment 2, dipeptide fragment 1, N-Ac-D-Cys(Mmt)-OH, to obtain fragment A; (4) removing the protecting group of Cys(Mmt) in fragment A, obtaining fragment B, and then coupling with Boc-Cys(Npys)-OH, and cleaving to obtain Etelcalcetide crude product. The present invention can be applied to the direct modification of hydrophilic, hydrophobic polypeptides and long-chain polypeptides and can be carried out in an organic solvent.

Description

ETELCALCETIDE synthesis method and application thereof
Technical Field
The invention belongs to the technical field of medicine synthesis, and particularly relates to a method for synthesizing ETELCALCETIDE and application thereof.
Background
Secondary Hyperparathyroidism (SHPT) is a disease in which parathyroid hyperplasia, elevated blood parathyroid hormone, and thus increased skeletal damage are caused by abnormal calcium-phosphorus metabolism, vitamin D deficiency, or kidney function impairment. Current measures for treating SHPT include active control of SHPT-inducing primary morbidity, limiting dietary phosphorus intake, and optionally phosphorus binders, active vitamin D analogues. Meanwhile, the calcimimetic can effectively treat SHPT.
Eptic peptides (ETELCALCETIDE) are calcimimetics for use in the treatment of SHPT in hemodialysis patients. Intravenous administration was performed at the end of each dialysis session. It acts as an allosteric activator by binding to and activating the calcium sensitive receptor (CaSR) in parathyroid glands, inhibiting and reducing secretion of parathyroid hormone, thereby achieving the goal of being able to treat SHPT. The drug is also an intravenous calcimimetic for administration by intravenous injection three times a week after the end of each dialysis of the patient. After moderately severe hyperparathyroid hemodialysis patients receive veracapeptide treatment, PTH levels are significantly reduced. ETELCALCETIDE is a calcium-mimicking agent that allosterically modulates a calcium sensitive receptor (CaSR). The ETELCALCETIDE backbone consists of 7D-amino acids of 4D-Arg, 2D-Ala and acetylated D-Cys, while the side chain is linked to L-cysteine by disulfide bonds, yielding ETELCALCETIDE. ETELCALCETIDE has a structure shown in figure 1, and a peptide sequence shown in figure 2.
The synthesis of ETELCALCETIDE at home and abroad has been reported, for example, the solid phase synthesis ETELCALCETIDE is adopted in the patents with publication numbers of CN 105504012A, CN106928320, CN109280078B, CN110668984A, CN110498835A and CN 110054662A. Although the solid phase coupling process has the advantages of simple operation process, convenient automatic production, higher product yield and easy separation of products, resin is needed in the coupling process, the feeding multiple of amino acid is usually required to be excessive (3-5 equivalents) in order to ensure the complete coupling, and the main chain of ETELCALCETIDE is D-type amino acid, so that the price is more expensive than that of conventional amino acid. Therefore, the solid phase synthesis method is too costly and is limited in production scale.
The liquid phase synthesis ETELCALCETIDE is used in the patents of publication numbers CN106928321, WO2016154580A1 and CN 111925418A. The liquid phase synthesis method is suitable for large-scale production, has less amino acid consumption, and achieves the purpose of controlling cost. However, the post-reaction treatment is complicated, the synthesis of insoluble peptide is difficult, the total yield is relatively low, disulfide bond mismatch problem can occur due to the formation of disulfide bonds between polypeptide chains in a liquid phase, the types of side reactions and byproducts are more, the yield and purity are not ideal, and the product yield is further reduced.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a ETELCALCETIDE synthesis method and application thereof. The invention synthesizes ETELCALCETIDE by utilizing a liquid phase carrier method, and uses two dipeptide fragments to improve the synthesis efficiency and reduce defective impurities. The invention combines the advantages of solid phase synthesis and liquid phase synthesis, and achieves the purposes of simple operation, reduced cost, reduced generation of defective impurities and suitability for large-scale production. And the operability is good, the effect of reducing impurities is good, the yield is improved, the cost is reduced, and the method is suitable for large-scale production.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method of ETELCALCETIDE synthesis, comprising the steps of:
(1) Amino acid is taken and placed in a solvent for coupling and recrystallization, and Fmoc-D-Ala-D-Arg (Pbf) -OH dipeptide fragment is synthesized in liquid phase;
(2) Amino acid is taken and placed in a solvent for coupling and recrystallization, and Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH dipeptide fragment is synthesized in liquid phase;
(3) Sequentially coupling the Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the step (1), the Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH synthesized in the step (2) and the Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the step (1) with chloroform as a reaction solvent to obtain N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-Arg (Pbf) -Dpm-NH 2, namely a fragment A, with the structural formula shown in the following formula 1;
(4) The protecting group of Cys (Mmt) in the fragment A is removed by 1% TFA/CHCl 3 solution to obtain N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2, namely fragment B, and the fragment B is coupled with Boc-Cys (Npys) -OH, and then cleaved to obtain ETELCALCETIDE crude product.
The synthesis method of ETELCALCETIDE, wherein the solvent in the step (1) and the step (2) comprises one or more of NMP, THF, DCM, ACN and DMF, the coupling system comprises one or more of DIC/HONb/organic base, DCC/HONb/organic base, EDCI/HOSu/organic base, DCC/HOSu/organic base, DIC/HONb/inorganic base and DCC/HONb/inorganic base, and the recrystallisation solvent system comprises one or more of EtOH/H2O、DCM/Et2O、THF/Et2O、EA/Et2O、MeOH/Et2O、CH3CN/Et2O、EA/PE、THF/PE、CH3CN/H2O.
The synthetic method of ETELCALCETIDE comprises the steps that the organic base comprises at least one of DIPEA, triethylamine and N-methyl morpholine, preferably the organic base is DIPEA, the inorganic base comprises at least one of Na 2CO3 or NaHCO 3, and preferably the inorganic base is NaHCO 3.
In the synthetic method of ETELCALCETIDE, the solvent in the step (1) is DCM, the coupling system is DIC/HONb/organic base, and the recrystallized solvent system is EtOH/H 2 O.
In the synthetic method of ETELCALCETIDE, the solvent in the step (2) is DCM, the coupled system is DIC/HONb/organic base, and the recrystallized solvent system is EA/PE.
According to the ETELCALCETIDE synthesis method, in the step (3), the molar ratio of the amino acid to the Dpm-NH 2 is 1:1-3:1, preferably the molar ratio of the amino acid to the Dpm-NH 2 is 1.1:1-1.3:1, and the coupling agent adopted by the coupling comprises one or more of EDCI// HOBt, DIPCDI/HOBt, pyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/HOAt/DIPEA and PyAop/HOAt/DIPEA, and preferably the coupling agent adopted by the coupling is EDCI// HOBt.
According to the synthetic method of ETELCALCETIDE, in the step (4), the volume of Mmt in the protecting group of Cys (Mmt) is 1% -5% of the volume of the TFA/CHCl 3 solution, and preferably the volume of Mmt in the protecting group of Cys (Mmt) is 1% -3% of the volume of the TFA/CHCl 3 solution.
According to the synthetic method of ETELCALCETIDE, the cracking reagent adopted in the step (4) comprises one or a mixture of more than one of TFA, phSMe, TIS, phOH, H 2 O, phOMe, preferably the cracking reagent adopted in the cracking is TFA (methyl methacrylate) and H 2 O (PhSMe) and Phome (Phome) and TIS (TIS) in a volume ratio of 88:5:3:2:2.
ETELCALCETIDE is synthesized by any one of the methods of ETELCALCETIDE.
The application of any one of the synthetic methods ETELCALCETIDE in synthesizing ETELCALCETIDE medicaments.
Compared with the prior art, the invention has the following beneficial effects:
1. the method adopts the liquid phase carrier Dpm-NH 2, is a homogeneous reaction, can be applied to direct modification of hydrophilic, hydrophobic and long-chain polypeptides and can be carried out in an organic solvent, overcomes the defect of limited solid-phase polypeptide synthesis scale, is suitable for large-scale production, and improves the production efficiency.
2. The equivalent weight of the amino acid used in each step of reaction is 1.1eqv, and compared with the solid-phase polypeptide synthesis method, the method improves the utilization rate of the amino acid and saves the cost. The reaction intermediates of each step can be precipitated by using a polar organic solvent, and compared with the traditional liquid-phase polypeptide synthesis method, the post-treatment operation is simple, and the purity of the intermediates of each step is relatively high.
3. According to the invention, two dipeptide fragments of Fmoc-D-Ala-D-Arg-OH and Fmoc-D-Arg-D-Arg-OH are used as starting materials, so that the coupling efficiency is further improved. Greatly reduces the synthesis steps, simultaneously reduces the generation of defective Arg impurities and reduces the synthesis difficulty of the peptide.
Drawings
FIG. 1 is a block diagram of ETELCALCETIDE;
FIG. 2 is a peptide sequence diagram of ETELCALCETIDE;
FIG. 3 is a structural formula of Dpm-NH 2;
FIG. 4 is a synthetic route diagram of ETELCALCETIDE;
FIG. 5 is an HPLC plot of the refined peptide;
FIG. 6 is a mass spectrum of refined peptide.
Detailed Description
The invention will be further illustrated by the following figures and examples.
EXAMPLE 1 Synthesis of Fmoc-D-Ala-D-Arg (Pbf) -OH
200ML of THF was used to dissolve Fmoc-D-Ala-OH (15.6 g,50 mmol), HOSu (6.33 g,55 mmol), DCC (11.3 g,55 mmol) was added in an ice-water bath, the ice-bath was removed, and the mixture was stirred at room temperature for 5h. TLC showed the reaction mixture was filtered and the filtrate was ready for use, indicating the starting material was substantially complete (PE: EA: hoac=1:1:0.05).
200ML of deionized water was used to dissolve Na 2CO3 (15.9 g,150 mmol) and H-D-Arg (Pbf) -OH (21.32 g,50 mmol), and the freshly prepared mixture was slowly added and stirred at room temperature for 18H. The reaction was concentrated, the residue was diluted with 200mL of deionized water, extracted with EA (250 x 3 mL), the aqueous layer was acidified to pH about 3 with 1mol/L hydrochloric acid solution, the EA layers were combined, the EA layers were washed 2 times with 200mL of 1mol/L hydrochloric acid solution, 200mL x 2mL of deionized water, 200mL of saturated brine, dried rapidly over 40g anhydrous sodium sulfate, filtered, concentrated, and the concentrate recrystallized from EtOH/H 2 O (2:1). The product was filtered and dried under vacuum to give Fmoc-D-Ala-D-Arg (Pbf) -OH in 24.9g, HPLC in 98.2% yield in 69.4%.
EXAMPLE 2 Synthesis of Fmoc-D-Ala-D-Arg (Pbf) -OH
200ML of DCM was taken to dissolve Fmoc-D-Ala-OH (15.6 g,50 mmol) and HONb (9.84 g,55 mmol), DIC (8.5 mL,55 mmol) was added in an ice-water bath, the ice-bath was removed, and stirring was performed at room temperature for 5h. TLC showed the reaction mixture was filtered and the filtrate was ready for use, indicating the starting material was substantially complete (PE: EA: hoac=1:1:0.05).
H-D-Arg (Pbf) -OH (21.32 g,50 mmol) was added to the above filtrate, magnetic stirring was started, DIPEA (10.74 mL,65 mmol) was added dropwise with a dropping funnel, and stirring was continued at room temperature for 10H. The reaction was concentrated, the residue was diluted with 200mL of deionized water, extracted with EA (250 x3 mL), the aqueous layer was acidified to pH about 3 with 1mol/L hydrochloric acid solution, the EA layers were combined, the EA layers were washed 2 times with 200mL of 1mol/L hydrochloric acid solution, 200mL x 2mL of deionized water, 200mL of saturated brine, dried rapidly over 40g anhydrous sodium sulfate, filtered, concentrated, and the concentrate recrystallized from EtOH/H 2 O (1:4). The product was filtered and dried under vacuum to give Fmoc-D-Ala-D-Arg (Pbf) -OH 26.7g, HPLC 98.9% yield 73.8%.
EXAMPLE 3 Synthesis of Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH
200ML of THF was used to dissolve Fmoc-D-Arg (Pbf) -OH (32.4 g,50 mmol) and HOSu (6.33 g,55 mmol), DCC (11.3 g,55 mmol) was added under ice-water bath conditions, the ice-bath was removed, and the mixture was stirred at room temperature for 5h. TLC showed the reaction mixture was filtered and the filtrate was ready for use, indicating the starting material was substantially complete (PE: EA: hoac=1:1:0.05).
200ML of deionized water was used to dissolve Na 2CO3 (15.9 g,150 mmol) and H-D-Arg (Pbf) -OH (21.32 g,50 mmol), and the freshly prepared mixture was slowly added and stirred at room temperature for 18H. The reaction was concentrated, the residue was diluted with 200mL of deionized water, extracted with EA (250 x 3 mL), the aqueous layer was acidified to pH about 3 with 1mol/L hydrochloric acid solution, the EA layers were combined, the EA layers were washed 2 times with 200mL of 1mol/L hydrochloric acid solution, 200mL x 2mL of deionized water, 200mL of saturated brine, and dried rapidly with 40g of anhydrous sodium sulfate, filtered, concentrated, and the concentrate recrystallized from EA: PE (3:1). The product was filtered and dried under vacuum to give Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH 37.1g, HPLC 98.9% yield 70.2%.
EXAMPLE 4 Synthesis of Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH
200ML of DCM was taken to dissolve Fmoc-D-Arg (Pbf) -OH (32.4 g,50 mmol) and HONb (9.84 g,55 mmol) and DIC (8.5 mL,55 mmol) was gradually added dropwise with ice-water bath, then the ice-bath was removed and stirred at room temperature for 5h. TLC showed the reaction mixture was filtered and the filtrate was ready for use, indicating the starting material was substantially complete (PE: EA: hoac=1:1:0.05).
H-D-Arg (Pbf) -OH (21.32 g,50 mmol) was added to the above filtrate, magnetic stirring was started, DIPEA (10.74 mL,65 mmol) was added dropwise with a dropping funnel, and stirring was continued at room temperature for 10H. The reaction was concentrated, the residue was diluted with 200mL of deionized water, extracted with EA (250 x 3 mL), the aqueous layer was acidified to pH about 3 with 1mol/L hydrochloric acid solution, the EA layers were combined, the EA layers were washed 2 times with 200mL of 1mol/L hydrochloric acid solution, 200mL x 2mL of deionized water, 200mL of saturated brine, dried rapidly over 40g anhydrous sodium sulfate, filtered, concentrated, and the concentrate recrystallized from EtOH/H 2 O (1:4). The product was filtered and dried under vacuum to give Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH 39.8g, HPLC 99.1% yield 75.3%.
EXAMPLE 5 Synthesis of Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH 2
Liquid carrier Dpm-NH 2 (8.3 g,10.0 mmol) was weighed into a 250mL three-necked flask, CHCl 3 (80 mL) was added to the flask, followed by HOBt (1.3 g,10.0 mmol) and Fmoc-D-Ala-D-Arg (Pbf) -OH (8.64 g,12.0 mmol) as in example 2. Stirring and dissolving. EDCI (2.6 g,13 mmol) was added and stirring continued for 3 hours at room temperature. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30℃to give a viscous material, and methanol (80 mL) was added to the viscous material and stirred for 2 hours. The mixture was filtered and the filter cake was rinsed three times with methanol (30 mL. Times.3). The filter cake was dried under vacuum at 40℃for 5 hours to give Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH 2 (14.64 g, yield 95.5%).
EXAMPLE 6 Synthesis of Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2
The compound Fmoc-D-Ala-D-Arg (Pbf) -Dpm-NH 2 (12.27 g,8.01 mmol) of example 5 was weighed into a 1.0L three-necked flask, chloroform (400 mL) was added to the flask, the solution was stirred, and DBU (1.22 g,8.01 mmol) was added. The reaction solution was cooled to 5 ℃ or below in an ice bath, and diethylamine (6.56 g,90 mmol) was slowly added dropwise, with the temperature controlled to not more than 5 ℃. After the completion of the dropwise addition, the reaction solution was warmed to room temperature and stirred for 2 hours. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction solution was concentrated under reduced pressure at 30 ℃ to form a viscous substance, acetonitrile (80 mL) was added to the viscous substance, and stirring was carried out for 30 minutes. The mixture was filtered and the filter cake was rinsed twice with methanol (40 mL. Times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give solid H-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 = 14.67g.
The above solid was charged into a 500mL three-necked flask, chloroform (150 mL) was added to the reaction flask, and HOBt (1.08 g,8.01 mmol) and Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH (10.15 g,9.61 mmol) of example 3 were added sequentially. Stirring and dissolving. The reaction solution was cooled to 0 ℃. EDCI (1.69 g,8.95 mmol) was added and stirring was continued for 3 hours at 0-10 ℃. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30℃to give a viscous material, and methanol (40 mL) was added to the viscous material and stirred for 2 hours. The mixture was filtered and the filter cake was rinsed three times with methanol (40 mL. Times.3). The filter cake was dried under vacuum at 40℃for 3 hours to give the compound Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (17.25 g, yield 91.6%).
EXAMPLE 7 Synthesis of Fmoc-D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2
The compound Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (16.45 g,7.0 mmol) of example 6 was weighed into a 1.0L three-necked flask, chloroform (200 mL) was added to the flask, the solution was stirred, and DBU (1.06 g,7.0 mmol) was further added. The reaction solution was cooled to below 5 ℃ in an ice bath, and diethylamine (5.47 g,75 mmol) was slowly added dropwise, with the temperature controlled to not exceed 5 ℃. After the completion of the dropwise addition, the reaction solution was warmed to room temperature and stirred for 2 hours. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction solution was concentrated under reduced pressure at 30 ℃ to form a viscous substance, acetonitrile (70 mL) was added to the viscous substance, and stirring was carried out for 30 minutes. The mixture was filtered and the filter cake was rinsed twice with methanol (40 mL. Times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give solid H-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 = 15.95g.
The above solid was charged into a 500mL three-necked flask, chloroform (160 mL) was added to the reaction flask, and HOBt (0.94 g,7.0 mmol), and Fmoc-D-Ala-D-Arg (Pbf) -OH (16.04 g,8.4 mmol) in this order were added. Stirring and dissolving. The reaction solution was cooled to 0 ℃. EDCI (1.45 g,7.7 mmol) was added and stirring continued for 3 hours at 0-10 ℃. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30℃to give a viscous material, and methanol (40 mL) was added to the viscous material and stirred for 2 hours. The mixture was filtered and the filter cake was rinsed three times with methanol (40 mL. Times.3). The filter cake was dried under vacuum at 40℃for 3 hours to give the compound Fmoc-D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (18.52 g, yield 93.5%).
EXAMPLE 8 Synthesis of N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2
The compound Fmoc-D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (18.4 g,6.5 mmol) of example 7 was weighed into a 1.0L three-necked flask, chloroform (200 mL) was added to the flask, the solution was stirred, and DBU (0.98 g,6.5 mmol) was further added. The reaction solution was cooled to below 5 ℃ in an ice bath, diethylamine (5.10 g,70 mmol) was slowly added dropwise, and the temperature was controlled to not exceed 5 ℃. After the completion of the dropwise addition, the reaction solution was warmed to room temperature and stirred for 2 hours. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction solution was concentrated under reduced pressure at 30 ℃ to form a viscous substance, acetonitrile (80 mL) was added to the viscous substance, and stirring was carried out for 30 minutes. The mixture was filtered and the filter cake was rinsed twice with methanol (50 mL. Times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give solid H-D-Ala-D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 = 18.16g.
The solid was placed in a 500mL three-necked flask, chloroform (200 mL) was added to the flask, and HOBt (0.87 g,6.5 mmol), N-Ac-D-Cys (Mmt) -OH (3.39 g,7.8 mmol) were added sequentially. Stirring and dissolving. The reaction solution was cooled to 0 ℃. EDCI (1.35 g,7.2 mmol) was added and stirring was continued for 3 hours at 0-10 ℃. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30℃to give a viscous material, and methanol (50 mL) was added to the viscous material and stirred for 2 hours. The mixture was filtered and the filter cake was rinsed three times with methanol (50 mL. Times.3). The filter cake was dried under vacuum at 40℃for 3 hours to give the compound N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (18.15 g, yield 92.3%).
EXAMPLE 9 Synthesis of N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2
The compound N-Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH2(18.0g,5.95mmol) of example 8 was weighed into a 1.0L three-necked flask, 300mL of 1% TFA/CHCl 3 was added, and after magnetically stirring at room temperature for 5 minutes, the reaction solution was concentrated under reduced pressure at 30℃to a viscous material, acetonitrile (80 mL) was added to the viscous material, and stirring was carried out for 30 minutes. The filtration was carried out and the filter cake was rinsed twice with methanol (50 mL. Times.2) and the procedure was repeated 2 times for a total of 3 times. Obtaining the compound N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2 (17.65 g, yield 98.05%)
EXAMPLE 10 Synthesis of C 152H243N21O26S6
The compound N-Ac-D-Cys(SH)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH2(17.65g,6.41mmol) of example 9 was weighed into a 1.0L three-necked flask, chloroform (250 mL) was added to the flask, and Boc-Cys (Npys) -OH (2.64 g,7.05 mmol) was added. Stirring and dissolving. The reaction solution was cooled to 0 ℃. DIPEA (2.0 mL,10 mmol) was added and stirring continued for 24 hours at 20-30 ℃. TLC (DCM: meOH: hac=100:1:0.5) monitored the reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30℃to give a viscous material, and methanol (50 mL) was added to the viscous material and stirred for 2 hours. The mixture was filtered and the filter cake was rinsed three times with methanol (50 mL. Times.3). The filter cake was dried under vacuum at 40 ℃ for 3 hours to give compound C 152H243N21O26S6 16.19 g in 85% yield, having the following structural formula.
EXAMPLE 11 Synthesis of C 38H73N21O10S2
The compound of example 10, 16.0g, was placed in a 1.0L reaction flask, and 160mL of cleavage reagent TFA: H 2 O: phSMe: anisole: TIS=88:5:3:2, was added in a ratio of 10mL/g of the compound, and the cleavage reaction was carried out with magnetic stirring at room temperature for 2.5 hours. The reaction was then slowly poured into frozen MTBE (1600 mL) and allowed to stand in a refrigerator for 1 hour after stirring for 30 minutes. Centrifugation was performed and washed three times with diethyl ether (100 mL. Times.3). The resulting precipitate was dried at 30℃for 2 hours, slurried with methanol (100 mL) for 2 hours, filtered, and the cake discarded, and the filtrate was dried by spinning at 40℃to give a crude peptide (10.03 g, 136.8%), which was ETELCALCETIDE (chemical formula C 38H73N21O10S2). The crude peptide HPLC purity was approximately 89.34%.
After purification, ETELCALCETIDE g of refined peptide was obtained, the total yield was 82.95%, and the purity of the refined peptide was 99.37%. The chemical structural formula is as follows:
Abbreviations and English meanings referred to in examples 1-11 above are shown in Table 1 below.
Table 1 abbreviations and English meanings

Claims (12)

1. A method of ETELCALCETIDE synthesis, comprising the steps of:
(1) Fmoc-D-Ala-OH and HOSu are taken for reaction, H-D-Arg (Pbf) -OH is added into alkaline solution after the reaction is finished, a solvent is added to form a coupling system for coupling reaction, then recrystallization is carried out, and Fmoc-D-Ala-D-Arg (Pbf) -OH is synthesized in a liquid phase;
wherein the molar ratio of Fmo c-D-Ala-OH to HOSu is 50:55;
(2) Fmoc-D-Arg (Pbf) -OH is taken to react with HONb, H-D-Arg (Pbf) -OH is added into alkaline solution after the reaction is finished, a solvent is added to form a coupling system to perform coupling reaction, then recrystallization is performed, and Fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH is synthesized in a liquid phase;
Wherein, the molar ratio of Fmoc-D-Arg (Pbf) -OH to HONb is 50:55;
(3) Sequentially coupling the following amino acids, namely Fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the step (1), fmoc-D-Arg (Pbf) -D-Arg (Pbf) -OH synthesized in the step (2), fmoc-D-Ala-D-Arg (Pbf) -OH synthesized in the step (1) and N-Ac-D-Cys (Mmt) -OH with chloroform as a reaction solvent to obtain N-Ac-D-Cys (Mmt) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2, namely a fragment A, dpm-NH 2, wherein the structural formula of the fragment A is shown in the formula 1;
wherein the mole ratio of Fmoc-D-Ala-D-Arg (Pbf) -OH to Dpm-NH 2 of the first coupling is 1:1-3:1;
(4) Removing a protective group of Cys (Mmt) in the fragment A by using 1% TFA/CHCl 3 solution to obtain N-Ac-D-Cys (SH) -D-Ala-D-Arg (Pbf) -D-Arg (Pbf) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -Dpm-NH 2, namely fragment B, coupling with Boc-Cys (Npys) -OH, then cracking to obtain a ETELCALCETIDE crude product, and purifying to obtain ETELCALCETIDE refined peptide;
wherein the cleavage reagent consists of TFA, H 2 O, phSMe, phOMe and TIS.
2. The method of synthesis of ETELCALCETIDE according to claim 1, wherein the solvent in step (1) and step (2) comprises one or more of NMP, THF, DCM, ACN, DMF, the coupling system comprises one or more of DIC/HONb/organic base, DCC/HONb/organic base, EDCI/HOSu/organic base, DCC/HOSu/organic base, DIC/HONb/inorganic base, DCC/HONb/inorganic base, and the recrystallized solvent system comprises one or more of EtOH/H2O、DCM/Et2O、THF/Et2O、EA/Et2O、MeOH/Et2O、CH3CN/Et2O、EA/PE、THF/PE、CH3CN/H2O.
3. A method of synthesizing ETELCALCETIDE as in claim 2 wherein said organic base comprises at least one of DIPEA, triethylamine, N-methyl morpholine and said inorganic base comprises at least one of Na 2CO3 or NaHCO 3.
4. A method of synthesizing ETELCALCETIDE as claimed in claim 2 wherein said organic base is DIPEA and said inorganic base is NaHCO 3.
5. A method of synthesizing ETELCALCETIDE as claimed in claim 2 wherein the solvent in step (1) is DCM, the coupling system is DIC/HONb/organic base and the solvent system for recrystallization is EtOH/H 2 O.
6. A method of synthesizing ETELCALCETIDE as in claim 2 wherein in step (2) the solvent is DCM, the coupling system is DIC/HONb/organic base and the recrystallised solvent system is EA/PE.
7. A method of synthesizing ETELCALCETIDE according to claim 1, wherein said coupling agent employed in said coupling of step (3) comprises one or more of EDCI//HOBt、DIPCDI/HOBt、PyBop/HOBt/DIPEA、HBTU/HOBt/DIPEA、DIPCDI/HOAt、HATU/HOAt/DIPEA、PyAop/HOAt/DIPEA.
8. The method of claim 1, wherein the first coupling comprises Fmoc-D-Ala-D-Arg (Pbf) -OH and Dpm-NH 2 in a molar ratio of 1.1:1 to 1.3:1, and the coupling agent is EDCI// HOBt.
9. A method of synthesizing ETELCALCETIDE according to claim 1, wherein the volume of the protecting group of Cys (mt) in step (4) is 1% to 5% of the volume of the TFA/CHCl 3 solution.
10. A method of synthesizing ETELCALCETIDE according to claim 1, wherein the volume of Mmt in the Cys (Mmt) protecting group is 1% to 3% of the volume of TFA/CHCl 3 solution.
11. A method of synthesizing ETELCALCETIDE as claimed in claim 1 wherein the cleavage reagent is TFA: H 2 O: phSMe: phOMe: TIS in a ratio of 88:5:3:2:2 by volume.
12. Use of a method of synthesis of ETELCALCETIDE according to any one of claims 1 to 11 for the synthesis of a ETELCALCETIDE medicament.
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