A kind of synthesis method of Etelcalcetide and application thereof

Abstract

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

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and specifically relates to a synthesis method of Etelcalcetide and application thereof. Background Art

[0002] Secondary hyperparathyroidism (SHPT) is a disease caused by abnormal calcium and phosphorus metabolism, vitamin D deficiency or renal damage, which leads to parathyroid hyperplasia, increased blood parathyroid hormone, and aggravated bone damage. Current treatment measures for SHPT include actively controlling the primary disease that induces SHPT, limiting dietary phosphorus intake, and using phosphorus binders and active vitamin D analogs when necessary. At the same time, calcimimetics can effectively treat SHPT.

[0003] Etelcalcetide is a calcimimetic used to treat SHPT in patients receiving hemodialysis. It is administered intravenously at the end of each dialysis period. It acts as an allosteric activator by binding to and activating the calcium-sensing receptor (CaSR) in the parathyroid glands, inhibiting and reducing the secretion of parathyroid hormone, thereby achieving the purpose of treating SHPT. This drug is also the first intravenous calcimimetic that is administered intravenously three times a week after the end of each dialysis session. PTH levels were significantly reduced in hemodialysis patients with moderate to severe hyperparathyroidism after receiving velacalcetide treatment. Etelcalcetide is a calcimimetic that can allosterically regulate the calcium-sensing receptor (CaSR). The main chain of Etelcalcetide is composed of 7 D-amino acids, including 4 D-Arg, 2 D-Ala and acetylated D-Cys. At the same time, the side chain is connected to L-cysteine ​​through a disulfide bond to obtain Etelcalcetide. The structure of Etelcalcetide is as follows Figure 1 As shown, the peptide sequence is Figure 2 shown.

[0004] The synthesis of Etelcalcetide has been reported both at home and abroad, and the patents such as publication number CN105504012 A, CN106928320, CN109280078B, CN110668984A, CN110498835A and CN110054662A all adopt solid phase synthesis Etelcalcetide. Although the solid phase coupling process operating process is simple, convenient to automated production, the product yield is higher and the product is easily separated, but resin needs to be used in the coupling process, for ensuring that the coupling is complete, the amino acid whose feeding multiple needs excessive (3-5 equivalent) usually, plus the Etelcalcetide main chain is D-type amino acid, and the price is relatively conventional amino acid, more expensive. Therefore, adopt solid phase synthesis method, cost is too high, and scale production is limited.

[0005] Patents with publication numbers CN106928321, WO2016154580A1, and CN111925418A all use liquid phase synthesis of Etelcalcetide. The liquid phase synthesis method is suitable for large-scale production and uses less amino acids, achieving the purpose of controlling costs. However, the post-reaction processing operation is complicated, the synthesis of poorly soluble peptides is difficult, the total yield is relatively low, and the formation of disulfide bonds between polypeptide chains in the liquid phase will cause disulfide bond mismatch problems, there are many types of side reactions and by-products, and the yield and purity are not ideal, further reducing the product yield. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a synthesis method of Etelcalcetide and its application. The present invention utilizes a liquid phase carrier method to synthesize Etelcalcetide and uses two dipeptide fragments to improve the synthesis efficiency and reduce defect impurities. The present invention combines the advantages of solid phase synthesis and liquid phase synthesis, and achieves the purpose of simple operation, reduced cost, reduced generation of defect impurities and suitable for large-scale production. And it has good operability, good effect of reducing impurities, and also improves the yield, reduces the cost, and is suitable for large-scale production.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A synthetic method of Etelcalcetide, characterized in that it comprises the following steps:

[0009] (1) The amino acid is placed in a solvent for coupling and recrystallization, and the Fmoc-D-Ala-D-Arg(Pbf)-OH dipeptide fragment is synthesized in liquid phase;

[0010] (2) taking the amino acid and placing it in a solvent for coupling and recrystallization to synthesize the Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH dipeptide fragment in the liquid phase;

[0011] (3) Dpm-NH 2 The method is to use chloroform as a liquid phase synthesis carrier and sequentially couple the following amino acids: Fmoc-D-Ala-D-Arg(Pbf)-OH synthesized in the above step (1), Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH synthesized in the above step (2), Fmoc-D-Ala-D-Arg(Pbf)-OH synthesized in the above step (1), and N-Ac-D-Cys(Mmt)-OH to obtain N-Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH2 , i.e. fragment A, having the structural formula as shown in Formula 1;

[0012]

[0013] (4) Using 1% TFA / CHCl 3 The solution was used to remove the protecting group of Cys(Mmt) in fragment A 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 , i.e., fragment B, is coupled with Boc-Cys(Npys)-OH and cleaved to obtain crude Etelcalcetide.

[0014] The synthetic method of Etelcalcetide, wherein the solvent in step (1) and 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, DCC / HONb / inorganic base, and the recrystallization solvent system comprises EtOH / H 2 O、DCM / Et 2 O, THF / Et 2 O, EA / Et 2 O, MeOH / Et 2 O、CH 3 CN / Et 2 O, EA / PE, THF / PE, CH 3 CN / H 2 One or more of O.

[0015] The synthetic method of Etelcalcetide, wherein the organic base comprises at least one of DIPEA, triethylamine and N-methylmorpholine, preferably DIPEA, and the inorganic base comprises Na 2 CO 3 or NaHCO 3 At least one of the inorganic bases, preferably NaHCO 3 .

[0016] The synthetic method of Etelcalcetide, 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.

[0017] In the synthesis method of Etelcalcetide, the solvent in step (2) is DCM, the coupling system is DIC / HONb / organic base, and the recrystallization solvent system is EA / PE.

[0018] The synthesis method of Etelcalcetide, in step (3), the amino acid and Dpm-NH 2 The molar ratio of amino acid to Dpm-NH 2 The molar ratio of is 1.1:1 to 1.3:1, and the coupling agent used in the coupling includes one or more of EDCI / / HOBt, DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA and PyAop / HOAt / DIPEA. Preferably, the coupling agent used in the coupling is EDCI / / HOBt.

[0019] The synthesis method of Etelcalcetide, wherein the volume of Mmt in the protecting group of Cys(Mmt) in step (4) is TFA / CHCl 3 1%-5% of the volume of the solution, preferably the volume of Mmt in the protecting group of Cys(Mmt) is TFA / CHCl 3 1%-3% of the volume of the solution.

[0020] The synthesis method of Etelcalcetide, wherein the cleavage reagent used in the cleavage step (4) comprises TFA, PhSMe, TIS, PhOH, H 2 O, PhOMe or a mixture of the above, preferably the cleavage reagent used for cleavage is TFA:H 2 The volume ratio of O:PhSMe:PhOMe:TIS is 88:5:3:2:2.

[0021] Etelcalcetide is synthesized by any of the above-mentioned methods for synthesizing Etelcalcetide.

[0022] Application of any one of the above-mentioned methods for synthesizing Etelcalcetide in synthesizing Etelcalcetide drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention uses a liquid phase carrier Dpm-NH 2The method is a homogeneous reaction and can be applied to the direct modification of hydrophilic, hydrophobic peptides, and long-chain peptides. It can also be carried out in organic solvents, overcoming the shortcomings of the limited scale of solid-phase peptide synthesis, making it suitable for large-scale production and improving production efficiency.

[0025] 2. The amino acid equivalent used in each step of the reaction of the present invention is 1.1eqv, which improves the utilization rate of amino acids and saves costs compared with the solid phase peptide synthesis method. Each step of the reaction intermediate can be precipitated with a polar organic solvent. Compared with the traditional liquid phase peptide synthesis method, the post-processing operation is simple and the purity of each step intermediate is relatively high.

[0026] 3. The present invention uses two dipeptide fragments, Fmoc-D-Ala-D-Arg-OH and Fmoc-D-Arg-D-Arg-OH, as starting materials to further improve the coupling efficiency, greatly reduce the number of synthesis steps, reduce the generation of defective Arg impurities, and reduce the difficulty of synthesizing the peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The structural diagram of Etelcalcetide;

[0028] Figure 2 is the peptide sequence diagram of Etelcalcetide;

[0029] Figure 3 Dpm-NH 2 The structural formula of

[0030] Figure 4 The synthetic route of Etelcalcetide is shown in FIG.

[0031] Figure 5 HPLC chart of refined peptide;

[0032] Figure 6 This is the mass spectrum of the refined peptide. DETAILED DESCRIPTION

[0033] The present invention will be further described below by means of drawings and embodiments.

[0034] Example 1: Synthesis of Fmoc-D-Ala-D-Arg(Pbf)-OH

[0035] Take 200mL THF to dissolve Fmoc-D-Ala-OH (15.6g, 50mmol) and HOSu (6.33g, 55mmol), add DCC (11.3g, 55mmol) under ice-water bath, remove the ice bath, and stir at room temperature for 5h. TLC shows that the raw material is basically reacted (PE:EA:HOAc=1:1:0.05), filter the reaction mixture, and use the filtrate for later use.

[0036] 200mL deionized water to dissolve Na 2 CO 3 (15.9 g, 150 mmol), HD-Arg(Pbf)-OH (21.32 g, 50 mmol), slowly add the above freshly prepared mixed solution, stir at room temperature for 18 h. The reaction solution was concentrated, 200 mL of deionized water was added to dilute the residual solution, EA was extracted (250*3 mL), the water layer was acidified to pH about 3 with 1 mol / L hydrochloric acid solution, EA was extracted, the EA layer was combined, the EA layer was washed with 200 mL of 1 mol / L hydrochloric acid solution, 200 mL*2 mL of deionized water twice, and 200 mL of saturated brine once, and quickly dried with 40 g of anhydrous sodium sulfate, filtered and concentrated, and the concentrate was washed with EtOH / H 2 O (2:1) recrystallization. The product was filtered and dried in vacuo to obtain Fmoc-D-Ala-D-Arg(Pbf)-OH: 24.9 g, HPLC: 98.2%, yield: 69.4%.

[0037] Example 2: Synthesis of Fmoc-D-Ala-D-Arg(Pbf)-OH

[0038] Dissolve Fmoc-D-Ala-OH (15.6 g, 50 mmol) and HONb (9.84 g, 55 mmol) in 200 mL DCM, add DIC (8.5 mL, 55 mmol) in an ice-water bath, remove the ice bath, and stir at room temperature for 5 h. TLC shows that the raw material is basically reacted (PE:EA:HOAc=1:1:0.05), filter the reaction mixture, and use the filtrate for later use.

[0039] HD-Arg(Pbf)-OH (21.32 g, 50 mmol) was added to the above filtrate, magnetic stirring was started, and DIPEA (10.74 mL, 65 mmol) was added dropwise with a dropping funnel, and stirred at room temperature for 10 h. The reaction solution was concentrated, 200 mL of deionized water was added to dilute the residual solution, EA was extracted (250*3 mL), the water layer was acidified to pH about 3 with 1 mol / L hydrochloric acid solution, EA was extracted, the EA layer was combined, and the EA layer was washed with 200 mL of 1 mol / L hydrochloric acid solution, 200 mL*2 mL of deionized water twice, and 200 mL of saturated brine once, and quickly dried with 40 g of anhydrous sodium sulfate, filtered and concentrated, and the concentrate was washed with EtOH / H 2 O (1:4) recrystallization. The product was filtered and dried in vacuo to obtain Fmoc-D-Ala-D-Arg(Pbf)-OH: 26.7 g, HPLC: 98.9%, yield: 73.8%.

[0040] Example 3: Synthesis of Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH

[0041] Take 200mL THF to dissolve Fmoc-D-Arg(Pbf)-OH (32.4g, 50mmol) and HOSu (6.33g, 55mmol), add DCC (11.3g, 55mmol) in an ice-water bath, remove the ice bath, and stir at room temperature for 5h. TLC shows that the raw material is basically reacted (PE:EA:HOAc=1:1:0.05), filter the reaction mixture, and use the filtrate for later use.

[0042] 200mL deionized water to dissolve Na 2 CO 3 (15.9g, 150mmol), HD-Arg(Pbf)-OH(21.32g, 50mmol), slowly add the above freshly prepared mixed solution, stir at room temperature for 18h. The reaction solution is concentrated, 200mL of deionized water is added to dilute the residual solution, EA extraction (250*3mL), the water layer is acidified to pH about 3 with 1mol / L hydrochloric acid solution, EA extraction, the EA layer is combined, the EA layer is washed with 200mL 1mol / L hydrochloric acid solution, 200mL*2mL deionized water twice, and 200mL with saturated brine once, and quickly dried with 40g anhydrous sodium sulfate, filtered and concentrated, and the concentrate is recrystallized with EA:PE (3:1). The product is filtered and vacuum dried to obtain Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH: 37.1g, HPLC: 98.9%, yield: 70.2%.

[0043] Example 4: Synthesis of Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH

[0044] Dissolve Fmoc-D-Arg(Pbf)-OH (32.4 g, 50 mmol) and HONb (9.84 g, 55 mmol) in 200 mL DCM, gradually add DIC (8.5 mL, 55 mmol) under ice-water bath, then remove the ice bath and stir at room temperature for 5 h. TLC shows that the raw material is basically reacted (PE:EA:HOAc=1:1:0.05), filter the reaction mixture, and use the filtrate for later use.

[0045] HD-Arg(Pbf)-OH (21.32 g, 50 mmol) was added to the above filtrate, magnetic stirring was started, and DIPEA (10.74 mL, 65 mmol) was added dropwise with a dropping funnel, and stirred at room temperature for 10 h. The reaction solution was concentrated, 200 mL of deionized water was added to dilute the residual solution, EA was extracted (250*3 mL), the water layer was acidified to pH about 3 with 1 mol / L hydrochloric acid solution, EA was extracted, the EA layer was combined, and the EA layer was washed with 200 mL of 1 mol / L hydrochloric acid solution, 200 mL*2 mL of deionized water twice, and 200 mL of saturated brine once, and quickly dried with 40 g of anhydrous sodium sulfate, filtered and concentrated, and the concentrate was washed with EtOH / H 2 O (1:4) recrystallization. The product was filtered and dried in vacuo to obtain Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH: 39.8 g, HPLC: 99.1%, yield: 75.3%.

[0046] Example 5: Fmoc-D-Ala-D-Arg(Pbf)-Dpm-NH 2 Synthesis

[0047] Weigh the liquid phase carrier Dpm-NH 2 (8.3 g, 10.0 mmol) was added into a 250 mL three-necked flask, and CHCl 3 (80mL), then add HOBt (1.3g, 10.0mmol), and Fmoc-D-Ala-D-Arg(Pbf)-OH (8.64g, 12.0mmol) in Example 2 in sequence. Stir to dissolve. Add EDCI (2.6g, 13mmol) and continue stirring at room temperature for 3 hours. Monitor the reaction by TLC (DCM:MeOH:HAc=100:1:0.5). After the reaction is complete, the reaction solution is concentrated under reduced pressure at 30°C to a viscous substance, methanol (80mL) is added to the viscous substance, and stirred for 2 hours. Filter, and rinse the filter cake three times with methanol (30mL×3). The filter cake is vacuum dried at 40°C for 5 hours to obtain Fmoc-D-Ala-D-Arg(Pbf)-Dpm-NH 2 (14.64 g, yield 95.5%).

[0048] Example 6: Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 Synthesis

[0049] Weigh the compound Fmoc-D-Ala-D-Arg(Pbf)-Dpm-NH 2(12.27g, 8.01mmol) was added to a 1.0L three-necked flask, chloroform (400mL) was added to the reaction flask, stirred to dissolve, and then DBU (1.22g, 8.01mmol) was added. The reaction solution was cooled to below 5°C in an ice bath, and diethylamine (6.56g, 90mmol) was slowly added dropwise, and the temperature was controlled not to exceed 5°C. After the addition was completed, the reaction solution was warmed to room temperature and continued to stir for 2 hours. TLC (DCM: MeOH: HAc = 100: 1: 0.5) was used to monitor the reaction. After the reaction was complete, the reaction solution was concentrated under reduced pressure at 30°C to a viscous substance, acetonitrile (80mL) was added to the viscous substance, and stirred for 30 minutes. Filter, and rinse the filter cake twice with methanol (40mL × 2). The filter cake was vacuum dried at 40 °C for 2 h to obtain solid HD-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 =14.67g.

[0050] Add the above solid to a 500mL three-necked flask, add chloroform (150mL) to the reaction flask, and then add HOBt (1.08g, 8.01mmol), and Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH (10.15g, 9.61mmol) in Example 3. Stir to dissolve. Cool the reaction solution to 0°C. Add EDCI (1.69g, 8.95mmol) and continue stirring at 0-10°C for 3 hours. Monitor the reaction by TLC (DCM:MeOH:HAc=100:1:0.5). After the reaction is complete, the reaction solution is concentrated under reduced pressure at 30°C to a viscous substance, methanol (40mL) is added to the viscous substance, and stirred for 2 hours. Filter, and rinse the filter cake with methanol (40mL×3) three times. The filter cake was dried under vacuum at 40°C for 3 hours to obtain the compound Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 (17.25 g, yield 91.6%).

[0051] Example 7: Fmoc-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 Synthesis

[0052] Weigh the compound Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2(16.45g, 7.0mmol) was added to a 1.0L three-necked flask, chloroform (200mL) was added to the reaction flask, stirred to dissolve, and then DBU (1.06g, 7.0mmol) was added. The reaction solution was cooled to below 5°C in an ice bath, and diethylamine (5.47g, 75mmol) was slowly added dropwise, and the temperature was controlled not to exceed 5°C. After the addition was completed, the reaction solution was warmed to room temperature and continued to stir for 2 hours. TLC (DCM: MeOH: HAc = 100: 1: 0.5) was used to monitor the reaction. After the reaction was complete, the reaction solution was concentrated under reduced pressure at 30°C to a viscous substance, acetonitrile (70mL) was added to the viscous substance, and stirred for 30 minutes. Filter, and rinse the filter cake twice with methanol (40mL × 2). The filter cake was dried under vacuum at 40 °C for 2 hours to obtain solid HD-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 =15.95g.

[0053] Add the above solid to a 500mL three-necked flask, add chloroform (160mL) to the reaction flask, and then add HOBt (0.94g, 7.0mmol), and Fmoc-D-Ala-D-Arg(Pbf)-OH (16.04g, 8.4mmol) in Example 3 in sequence. Stir to dissolve. Cool the reaction solution to 0°C. Add EDCI (1.45g, 7.7mmol) and continue stirring at 0-10°C for 3 hours. Monitor the reaction by TLC (DCM:MeOH:HAc=100:1:0.5). After the reaction is complete, the reaction solution is concentrated under reduced pressure at 30°C to a viscous substance, methanol (40mL) is added to the viscous substance, and stirred for 2 hours. Filter, and rinse the filter cake with methanol (40mL×3) three times. The filter cake was dried under vacuum at 40°C for 3 hours to obtain 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%).

[0054] Example 8: N-Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 Synthesis

[0055] Weigh the compound Fmoc-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2(18.4g, 6.5mmol) was added to a 1.0L three-necked flask, chloroform (200mL) was added to the reaction flask, stirred to dissolve, and then DBU (0.98g, 6.5mmol) was added. The reaction solution was cooled to below 5°C in an ice bath, and diethylamine (5.10g, 70mmol) was slowly added dropwise, and the temperature was controlled not to exceed 5°C. After the addition was completed, the reaction solution was warmed to room temperature and continued to stir for 2 hours. TLC (DCM: MeOH: HAc = 100: 1: 0.5) was used to monitor the reaction. After the reaction was complete, the reaction solution was concentrated under reduced pressure at 30°C to a viscous substance, acetonitrile (80mL) was added to the viscous substance, and stirred for 30 minutes. Filter, and rinse the filter cake twice with methanol (50mL × 2). The filter cake was dried under vacuum at 40 °C for 2 hours to obtain solid HD-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 =18.16g.

[0056] Add the above solid to a 500mL three-necked flask, add chloroform (200mL) to the reaction flask, and then add HOBt (0.87g, 6.5mmol) and N-Ac-D-Cys(Mmt)-OH (3.39g, 7.8mmol) in sequence. Stir to dissolve. Cool the reaction solution to 0℃. Add EDCI (1.35g, 7.2mmol) and continue stirring at 0-10℃ for 3 hours. Monitor the reaction by TLC (DCM:MeOH:HAc=100:1:0.5). After the reaction is complete, the reaction solution is concentrated under reduced pressure at 30℃ to a viscous substance, methanol (50mL) is added to the viscous substance, and stirred for 2 hours. Filter, and rinse the filter cake with methanol (50mL×3) three times. The filter cake was dried under vacuum at 40°C for 3 hours to obtain 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%).

[0057] Example 9: N-Ac-D-Cys(SH)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH 2 Synthesis

[0058] Weigh 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 (18.0 g, 5.95 mmol) was added into a 1.0 L three-necked flask, and 300 mL of 1% TFA / CHCl was added 3 After 5 minutes of magnetic stirring at room temperature, the reaction solution was concentrated under reduced pressure at 30°C to a viscous substance. Acetonitrile (80 mL) was added to the viscous substance and stirred for 30 minutes. Filter, rinse the filter cake twice with methanol (50 mL × 2); repeat this process 2 times, a total of 3 times. 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%)

[0059] Example 10: C 152 H 243 N 21 O 26 S 6 Synthesis

[0060] Weigh 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 (17.65g, 6.41mmol) was added to a 1.0L three-necked flask, and chloroform (250mL) was added to the reaction flask, followed by Boc-Cys(Npys)-OH (2.64g, 7.05mmol). Stir until dissolved. The reaction solution was cooled to 0°C. DIPEA (2.0mL, 10mmol) was added and continued to stir at 20-30°C for 24 hours. TLC (DCM:MeOH:HAc=100:1:0.5) was used to monitor the reaction. After the reaction was complete, the reaction solution was concentrated to a viscous substance at 30°C under reduced pressure, and methanol (50mL) was added to the viscous substance and stirred for 2 hours. Filter, and rinse the filter cake three times with methanol (50mL×3). The filter cake was vacuum dried at 40°C for 3 hours to obtain compound C 152 H 243 N 21 O 26 S 6 16.19 g, yield 85%, its structural formula is as follows.

[0061]

[0062] Example 11: C 38 H 73 N 21 O 10 S 2 Synthesis

[0063] 16.0 g of the compound in Example 10 was placed in a 1.0 L reaction bottle, and 160 mL of the cleavage reagent TFA:H was added at a ratio of 10 mL / g of compound. 2 O:PhSMe:Anisole:TIS=88:5:3:2:2, and the cleavage reaction was carried out under magnetic stirring at room temperature for 2.5 hours. Then the reaction solution was slowly poured into frozen MTBE (1600mL), stirred for 30 minutes, and then placed in the refrigerator for 1 hour. Centrifuged and washed three times with ether (100mL×3). The obtained precipitate was dried at 30°C for 2 hours, then slurried with methanol (100mL) for 2 hours, filtered, the filter cake was discarded, and the filtrate was dried at 40°C to obtain a crude peptide (10.03g, 136.8%), which is Etelcalcetide (chemical formula C 38 H 73 N 21 O 10 S 2 ). The HPLC purity of the crude peptide was about 89.34%.

[0064] After purification, 6.08 g of Etelcalcetide peptide was obtained, with a total yield of 82.95% and a purity of 99.37%. Its chemical structure is as follows:

[0065]

[0066] The abbreviations and their English meanings involved in the above Examples 1-11 are shown in Table 1 below.

[0067] Table 1 Abbreviations and English meanings

[0068]

[0069]

[0070]

Claims

1. A method for synthesizing Etelcalcetide, characterized in that The following steps are involved: (1) Fmoc-D-Ala-OH and HOSu are reacted, and after the reaction is completed, HD-Arg(Pbf)-OH is added in an alkaline solution, and a solvent is added to form a coupling system, and then recrystallization is performed to synthesize Fmoc-D-Ala-D-Arg(Pbf)-OH in a liquid phase; Among them, the molar ratio of Fmo cD-Ala-OH to HOSu was 50:55; (2) Fmoc-D-Arg(Pbf)-OH is reacted with HONb, and after the reaction is completed, HD-Arg(Pbf)-OH is added in an alkaline solution, and a solvent is added to form a coupling system, and then recrystallized to synthesize Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH in a liquid phase; Among them, the molar ratio of Fmoc-D-Arg(Pbf)-OH to HONb is 50:55; (3) Using Dpm-NH2 as a liquid phase synthesis carrier and chloroform as a reaction solvent, sequentially couple the following amino acids: Fmoc-D-Ala-D-Arg(Pbf)-OH synthesized in the above step (1), Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OH synthesized in the above step (2), Fmoc-D-Ala-D-Arg(Pbf)-OH synthesized in the above step (1), and N-Ac-D-Cys(Mmt)-OH to obtain N-Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH2, i.e., fragment A. The structural formula of Dpm-NH2 is as follows: Among them, the first coupling of Fmoc-D-Ala-D-Arg(Pbf)-OH and Dpm-NH 2 The molar ratio is 1:1 to 3:1; (4) Using 1% TFA / CHCl3 solution to remove the protecting group of Cys(Mmt) in fragment A, N-Ac-D-Cys(SH)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Dpm-NH2, i.e., fragment B, was then coupled with Boc-Cys(Npys)-OH, and then cleaved to obtain crude Etelcalcetide, which was purified to obtain refined Etelcalcetide peptide; The lysis reagent consists of TFA, H2O, PhSMe, PhOMe and TIS.

2. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that The solvent in step (1) and step (2) includes one or more of NMP, THF, DCM, ACN, and DMF; the coupling system includes 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 solvent system for recrystallization includes one or more of EtOH / H2O, DCM / Et2O, THF / Et2O, EA / Et2O, MeOH / Et2O, CH3CN / Et2O, EA / PE, THF / PE, and CH3CN / H2O.

3. A method for synthesizing Etelcalcetide as claimed in claim 2, characterized in that The organic base includes at least one of DIPEA, triethylamine, and N-methylmorpholine, and the inorganic base includes at least one of Na2CO3 or NaHCO3.

4. A method for synthesizing Etelcalcetide as claimed in claim 2, characterized in that The organic base is DIPEA, and the inorganic base is NaHCO3.

5. A method for synthesizing Etelcalcetide as claimed in claim 2, characterized in that In the step (1), the solvent is DCM, the coupling system is DIC / HONb / organic base, and the solvent system for recrystallization is EtOH / H2O.

6. A method for synthesizing Etelcalcetide as claimed in claim 2, characterized in that The solvent in step (2) is DCM, the coupling system is DIC / HONb / organic base, and the solvent system for recrystallization is EA / PE.

7. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that The coupling agent used in the coupling in the step (3) includes one or more of EDCI / / HOBt, DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA, and PyAop / HOAt / DIPEA.

8. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that The molar ratio of Fmoc-D-Ala-D-Arg(Pbf)-OH to Dpm-NH2 in the first coupling is 1.1:1 to 1.3:1, and the coupling agent used in the coupling is EDCI / / HOBt.

9. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that In the step (4), the volume of Mmt in the protecting group of Cys(Mmt) is 1%-5% of the volume of the TFA / CHCl3 solution.

10. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that The volume of Mmt in the protecting group of Cys(Mmt) is 1%-3% of the volume of the TFA / CHCl3 solution.

11. A method for synthesizing Etelcalcetide as claimed in claim 1, characterized in that The lysis reagent is TFA:H2O:PhSMe:PhOMe:TIS in a volume ratio of 88:5:3:2:

2.

12. Use of the synthesis method of Etelcalcetide according to any one of claims 1 to 11 in synthesizing Etelcalcetide drugs.

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