(19)
(11)EP 4 071 135 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
12.10.2022 Bulletin 2022/41

(21)Application number: 19955221.7

(22)Date of filing:  27.12.2019
(51)International Patent Classification (IPC): 
C07C 319/22(2006.01)
C07K 7/06(2006.01)
C07K 1/04(2006.01)
C07C 323/59(2006.01)
C07K 1/06(2006.01)
(52)Cooperative Patent Classification (CPC):
Y02P 20/55; C07C 2603/18; C07C 319/24; C07C 313/08; C07K 7/06
 
C-Sets:
C07C 319/24, C07C 323/59;
(86)International application number:
PCT/CN2019/129440
(87)International publication number:
WO 2021/109297 (10.06.2021 Gazette  2021/23)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 03.12.2019 CN 201911217130

(71)Applicant: Asymchem Laboratories (Tianjin) Co., Ltd.
Tianjin 300457 (CN)

(72)Inventors:
  • LI, Jiuyuan
    Tianjin 300457 (CN)
  • MATT, Johnson
    Morrisville, North Carolina 27560 (US)
  • LI, Changfeng
    Tianjin 300457 (CN)
  • JING, Lutao
    Tianjin 300457 (CN)
  • BALASUBRAMANIAN, Arumugam
    Tianjin 300457 (CN)
  • LEI, Xiaolong
    Tianjin 300457 (CN)
  • ZHU, Zili
    Tianjin 300457 (CN)

(74)Representative: Glawe, Delfs, Moll 
Partnerschaft mbB von Patent- und Rechtsanwälten Postfach 13 03 91
20103 Hamburg
20103 Hamburg (DE)

  


(54)ETELCALCETIDE INTERMEDIATE AND METHOD FOR SYNTHESIZING ETELCALCETIDE


(57) Disclosed are an etelcalcetide intermediate and a method for synthesizing etelcalcetide. The etelcalcetide intermediate is Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH. The method for synthesizing the etelcalcetide includes the following steps: using N-Boc-L-Cqs-OtBu as a starting material to generate a primary product of a formula (A) by means of a substitution reaction, herein R is S-Py or Cl; and performing a coupling reaction on the primary product and Fmoc-D-Cys-OH amino acid to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH. The key intermediate is used for synthesizing the etelcalcetide, which may improve the purity and the yield. It is important that the raw materials for synthesizing the key intermediate are cheap and readily available, and the process is simple.






Description

Technical Field



[0001] The present disclosure relates to the technical field of chemical synthesis, in particular to an etelcalcetide intermediate and a method for synthesizing etelcalcetide.

Background



[0002] Etelcalcetide is a novel calcimimetic agent developed by Kai Pharmaceuticals, Inc. that may inhibit the secretion of a parathyroid hormone. The etelcalcetide may bind and activate a calcium-sensing receptor on a parathyroid gland, and the reduction in parathyroid hormone level is achieved.

[0003] The etelcalcetide is composed of three D- arginines, two D- alanines, one D-arginine amide, one L-cysteine and one D-cysteine (N-terminal blocked by an acetyl group), herein the D-cysteine and the L-cysteine are linked together by a disulfide bond (N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-Argininamide,disulfidewithL-cysteine).

[0004] In currently disclosed patents, they are all linked to 7 peptides in solid-phase, and then choose different methods to link the disulfide bond, especially in the process of removing Methylcyclopentadienyl-Manganese-Tricarbony (MMT), it is necessary to use 1%~2% of Trifluoroacetic Acid (TFA)/Dichloromethane (DCM) to repeat an operation by more than 15 times, and the operation is quite complicated.

[0005] Patent CN2017/114238 A1: a liquid-phase synthesis method adopted has a long route and the total yield is only 11%; Patent WO 2017/114240 A1: according to the patent, the purity of a crude peptide is 81.0%, there are many impurities, and the total yield after purification is 30.5%; Patent US 2019/0100554 A1: according to the patent, the purity of the crude peptide is relatively low, it needs to be purified by many times, and the total yield is 50%; and in Chinese Patent CN201811277081, the purity of the crude peptide is 88.2%, and the total yield after purification is 57.8%. In other words, an existing method of solid-phase synthesis of 7-peptide-linked cysteine and derivatives thereof has problems such as the low purity of the crude peptide, many impurities, and difficulty in purification.

Summary



[0006] The present disclosure aims to provide an etelcalcetide intermediate and a method for synthesizing etelcalcetide. The intermediate is used for synthesizing the etelcalcetide, as to solve a technical problem in an existing technology that the etelcalcetide synthesis process is complicated.

[0007] In order to achieve the above purpose, according to one aspect of the present disclosure, a method for synthesizing an etelcalcetide intermediate is provided. The etelcalcetide intermediate is Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and a structural formula I thereof is as follows:



[0008] The method for synthesizing the etelcalcetide intermediate includes the following steps: N-Boc-L-Cys-OtBu is taken as a starting raw material, and a primary product

is generated through a substitution reaction, herein R is S-Py or Cl, and the primary product performs a coupling reaction with Fmoc-D-Cys-OH amino acid to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH.

[0009] Further, the primary product is Py-S-S-(N-Boc)-L-Cys-OtBu, and Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: N-Boc-L-Cys-OtBu performs a substitution reaction with dithiodipyridine to synthesize and obtain a primary product Py-S-S-(N-Boc)-L-Cys-OtBu; and Py-S-S-(N-Boc)-L-Cys-OtBu is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH.

[0010] Further, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: at a room temperature, N-Boc-L-Cys-OtBu and dithiodipyridine are added to a solvent A, and stirred for 6-12 h, then water is added, an extractant is used to extract, an obtained organic phase is dried and filtered, and after purification, Py-S-S-(N-Boc)-L-Cys-OtBu is obtained; Fmoc-D-Cys-OH and Py-S-S-(N-Boc)-L-Cys-OtBu are added to a solvent B, the temperature is controlled at 15-30 °C and it is stirred and reacted for 0.5-2 h, the reaction system is water-washed, concentrated and purified to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH; preferably, the solvent A is selected from one or more of N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP) and N,N-dimethylacetamide (DMAc); preferably, the extractant is selected from one or more of ethyl acetate (EtOAc), methyl tert-butyl ether (MTBE) and dichloromethane (DCM); and preferably, the solvent B is selected from one or more of DCM, DMF, tetrahydrofuran (THF), NMP and DMAc.

[0011] Further, in the reaction between N-Boc-L-Cys-OtBu and dithiodipyridine, the molar ratio of N-Boc-L-Cys-OtBu and dithiodipyridine is 1:1.2~1:6.4; the concentration of N-Boc-L-Cys-OtBu in the solvent A is 0.01~0.3 g/mL; the concentration of Fmoc-D-Cys-OH in the solvent B is 0.01~0.3 g/mL; and the molar ratio of Fmoc-D-Cys-OH and Py-S-S-(N-Boc)-L-Cys-OtBu is 1:0.8~1.4.

[0012] Further, the primary product is (N-Boc)-L-Cys(S-Cl)-OtBu, and Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: N-Boc-L-Cys-OtBu reacts with NCS to synthesize (N-Boc)-L-Cys(S-Cl)-OtBu; and (N-Boc)-L-Cys(S-Cl)-OtBu reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH.

[0013] Further, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: A. N-Boc-L-Cys-OtBu is dissolved in a solvent C, the temperature is controlled at 0~10°C, N,N-diisopropylethylamine (DIPEA) is added, and N-chlorosuccinimide (NCS) is added in batches, it is stirred for 4-5 h, after the reaction, it is filtered, and rinsed to obtain a filtrate; and B. the temperature is controlled at 0~10°C, Fmoc-D-Cys-OH is added to the filtrate, and DIPEA is added, the reaction temperature is controlled at 10~30°C, and it is stirred for 0.5-2 h; the reaction system is water-washed, concentrated and purified to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH; and preferably, the solvent C is selected from one or more of DCM, THF, DMF NMP and DMAc.

[0014] Further, in the step A, N-Boc-L-Cys-OtBu is dissolved in the solvent C to obtain solution with a concentration of 0.01-0.3 g/mL, the amount of DIPEA added is 2-3 eq of moles, and the amount of NCS added is 1.1~1.5 eq; and in the step B, the amount of Fmoc-D-Cys-OH added is 1.1~1.5 eq.

[0015] According to another aspect of the present disclosure, a method for synthesizing etelcalcetide is provided. The synthesis method includes the following steps: S1, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is synthesized by any one of the methods for synthesizing the etelcalcetide intermediates; and S2, NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg reacts with Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and Fmoc is removed, and then acetylation is performed to obtain the etelcalcetide.

[0016] Further, in the S2, NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg is NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide.

[0017] Further, the S2 includes: an amino resin is used to link NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide according to a method of solid-phase synthesis, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBop) and DIPEA are activated at 0~5°C for 0-10 min, the temperature is controlled at 20~30°C and it is reacted for 2~6 h. After the reaction, it is washed by DMF for 4-6 times, Fmoc is removed by 10%~20% of piperidine, and then the acetylation is performed to obtain a peptide resin of the etelcalcetide.

[0018] Further, the ratio of Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:OIPEA is 3:3~6:3~6.

[0019] A technical scheme of the present disclosure is applied, by synthesizing the key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and then synthesizing the etelcalcetide, a side reaction while a peptide chain constructs a disulfide bond is avoided, the operation is simplified, and the purity and yield may be improved while the etelcalcetide is synthesized. It is important that raw materials for synthesizing this intermediate are cheap and easy to obtain, and the process is simple.

Brief Description of the Drawings



[0020] Drawings constituting a part of the present disclosure are used to provide further understanding of the present disclosure, and exemplary embodiments of the present disclosure and descriptions thereof are used to explain the present disclosure, and do not constitute improper limitation to the present disclosure.

Fig. 1 shows a purity map of Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH in Embodiment 1;

Fig. 2 shows an liquid chromatography mass spectrometry (LCMS) spectrum of an intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH of Embodiment 1 (LC-MS: m/z=617.9 (M-1,30ev), 1235.7(2M-1,30ev)); and

Fig. 3 shows a purity map of purified etelcalcetide prepared in Embodiment 3.


Detailed Description of the Embodiments



[0021] It should be noted that embodiments in the present disclosure and features of the embodiments may be combined with each other in the case without conflicting. The present disclosure is described in detail below with reference to the drawings and in combination with the embodiments.

[0022] Abbreviations involved in the present disclosure are explained as follows:

Fmoc: 9-fluorenemethoxycarbonyl.

Boc: tert-butylcarbonyl.

tBu: tert-butyl.

Arg: arginine.

Cys: cysteine.

Ala: alanine.

PyBop: benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate.

DIPEA: N,N-diisopropylethylamine.

DCM: dichloromethane.

NCS: N-chlorosuccinimide.

DMF: N,N-dimethylformamide.

NMP: N-methylpyrrolidone.

DMAc: N,N-dimethylacetamide.

THF: tetrahydrofuran.

OtBu: tert-butoxy.

EtOAc: ethyl acetate.



[0023] In the synthesis of etelcalcetide, the most critical is the synthesis of a disulfide bond. The yield of this step directly affects the final yield of the entire route. It is found by the inventor of the present application that in the synthesis of a key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH (N-fluorenylcarbonyl-D-cysteine-S-S-(N-tert-butoxycarbonyl-L-cysteine tert-butyl ester)), the synthesis of the disulfide bond in advance may perfectly avoid problems of low efficiency and poor yield of a solid-phase reaction.

[0024] According to a typical embodiment of the present disclosure, a method for synthesizing an etelcalcetide intermediate is provided. The etelcalcetide intermediate is Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and the method for synthesizing the etelcalcetide intermediate includes the following steps: N-Boc-L-Cys-OtBu is taken as a starting raw material, and a primary product

is generated through a substitution reaction, herein R is S-Py or Cl, and the primary product performs a coupling reaction with Fmoc-D-Cys-OH amino acid to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH.

[0025] A technical scheme of the present disclosure is applied, by synthesizing the key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and then synthesizing the etelcalcetide, a side reaction while a peptide chain constructs a disulfide bond is avoided, the operation is simplified, and the purity and yield may be improved while the etelcalcetide is synthesized. It is important that raw materials for synthesizing this intermediate are cheap and easy to obtain, and the process is simple.

[0026] According to a typical embodiment of the present disclosure, the primary product is Py-S-S-(N-Boc)-L-Cys-OtBu, and Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: N-Boc-L-Cys-OtBu performs a substitution reaction with dithiodipyridine to synthesize and obtain a primary product Py-S-S-(N-Boc)-L-Cys-OtBu; and Py-S-S-(N-Boc)-L-Cys-OtBu is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH. It is important that the raw materials for synthesizing this intermediate are cheap and easy to obtain, and the process is simple.

[0027] Preferably, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: at a room temperature, N-Boc-L-Cys-OtBu and dithiodipyridine are added to a solvent A, and stirred for 6~12 h, then water is added, an extractant is used to extract, an obtained organic phase is dried and filtered, and after purification, Py-S-S-(N-Boc)-L-Cys-OtBu is obtained; Fmoc-D-Cys-OH and Py-S-S-(N-Boc)-L-Cys-OtBu are added to a solvent B, the temperature is controlled at 15-30 °C and it is stirred and reacted for 0.5~2 h, a reaction system is water-washed, concentrated and purified to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH; preferably, the solvent A is selected from one or more of DMF, NMP and DMAc; preferably, the extractant is selected from one or more of eEtOAc, MTBE and DCM; and preferably, the solvent B is selected from one or more of DCM, DMF, THF, NMP and DMAc.

[0028] In order to improve the utilization rate of the raw materials and guarantee the rapid and effective progress of the reaction, further preferably, in the reaction between N-Boc-L-Cys-OtBu and dithiodipyridine, the molar ratio of N-Boc-L-Cys-OtBu and dithiodipyridine is 1:1.2~1:6.4; the concentration of N-Boc-L-Cys-OtBu in the solvent A is 0.01~0.3 g/mL; the concentration of Fmoc-D-Cys-OH in the solvent B is 0.01~0.3 g/mL; and the molar ratio of Fmoc-D-Cys-OH and Py-S-S-(N-Boc)-L-Cys-OtBu is 1:0.8~1.4.

[0029] According to a typical embodiment of the present disclosure, the primary product is (N-Boc)-L-Cys(S-Cl)-OtBu, and Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: N-Boc-L-Cys-OtBu reacts with NCS to synthesize (N-Boc)-L-Cys(S-Cl)-OtBu; and (N-Boc)-L-Cys(S-Cl)-OtBu reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH.

[0030] Preferably, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is prepared by the following steps: A. N-Boc-L-Cys-OtBu is dissolved in a solvent C, the temperature is controlled at 0~10°C, DIPEA is added, and NCS is added in batches, it is stirred for 4~5 h, after the reaction, it is filtered, and rinsed to obtain a filtrate; and B. the temperature is controlled at 0~10°C, Fmoc-D-Cys-OH is added to the filtrate, and DIPEA is added, the reaction temperature is controlled at 10~30°C, and it is stirred for 0.5~2 h; the reaction system is water-washed, concentrated and purified to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH; and preferably, the solvent C is selected from one or more of DCM, THF, DMF NMP and DMAc.

[0031] In order to improve the utilization rate of the raw materials and guarantee the rapid and effective progress of the reaction, further preferably, in the step A, N-Boc-L-Cys-OtBu is dissolved in the solvent C to obtain solution with a concentration of 0.01~0.3 g/mL, the amount of DIPEA added is 2~3 eq of moles, and the amount of NCS added is 1.1~1.5 eq; and in the step B, the amount of Fmoc-D-Cys-OH added is 1.1~1.5 eq.

[0032] According to a typical embodiment of the present disclosure, a method for synthesizing etelcalcetide is provided. The synthesis method includes the following steps: S1, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH is synthesized by any one of the methods for synthesizing the etelcalcetide intermediates; and S2, NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg reacts with Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, and Fmoc is removed, and then acetylation is performed to obtain the etelcalcetide.

[0033] Because the raw materials of the key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH are cheap and easy to obtain, and the process is simple, it also directly makes the raw materials for the synthesis method of the etelcalcetide in the present disclosure cheap and easy to obtain, and the process is simple.

[0034] Preferably, in the S2, NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg is NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide. Preferably, the S2 includes: an amino resin is used to link NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide according to a method of solid-phase synthesis, Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, PyBop and DIPEA are activated at 0~5°C for 0-10 min, the temperature is controlled at 20~30°C and it is reacted for 2~6 h. After the reaction, it is washed with DMF for 4~6 times, Fmoc is removed by 10%~20% of piperidine, and then the acetylation is performed to obtain a peptide resin of the etelcalcetide. More preferably, the ratio of Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA is 3:3~6:3~6.

[0035] The beneficial effects of the present disclosure are further described below in combination with the embodiments.

[0036] Specific synthetic routes of the embodiments:


Embodiment 1


R=S-Py



[0037] N-Boc-L-Cys-OtBu(Cpd 1) is used as a raw material, and Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a) is synthesized by reacting N-Boc-L-Cys-OtBu(Cpd 1) with dithiodipyridine; and Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a) is coupled with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH(Cpd 3).

Step 1



[0038] At a room temperature, Cpd 1 (1.6 g, 1.0 eq) and 2,2-dithiodipyridne (5.1 g, 4.0 eq) are added to DMF (16 mL, 10 vol.). It is reacted and stirred at the room temperature for 6~12 h; then water is added to a system; ethyl acetate (100 mL*3) is used to extract; organic phases are dried by using MgSO4 after being combined, and filtered; and the organic phase is concentrated to obtain a crude product Cpd 2a, and it is purified by a column chromatography to obtain pure Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a).

Step 2



[0039] At 15~30°C, Fmoc-D-Cys-OH (1.0 g, 1.0 eq.) is added to DCM (30 ml, 30 vol.), then Cpd 2a (1.13 g, 1.0 eq.) is added to a reaction system; the temperature is controlled at 15~30°C, it is stirred and reacted for 0.5~2 h; the system is washed with water for 3 times, and concentrated to obtain a crude product Cpd 3; and pure Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd3) is obtained by the column chromatography.

[0040] Fig. 1 shows a purity map of purified Cpd 3; and Fig. 2 shows an LCMS spectrum of purified Cpd 3.

Embodiment 2


R=Cl



[0041] N-Boc-L-Cys-OtBu(Cpd 1) is used as a raw material, and (N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b) is synthesized by reacting with NCS; and (N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b) reacts with Fmoc-D-Cys-OH to obtain Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd 3).

Step 1



[0042] (N-Boc)-Cys-OtBu (0.28 g, 0.1 mmol) is dissolved in DCM (20 mL), it is stirred and the temperature is controlled at 0~10°C, and DIPEA (0.19 g, 0.15 mmol) is added; the temperature is controlled at 0~10°C and NCS (0.15 g, 1.1 eq) is added in batches; the reaction temperature is kept and it is stirred for 4~5 h, and an end point of the reaction is monitored by a high performance liquid chromatography (HPLC); after the reaction, it is filtered; a filter cake is rinsed with DCM (20 mL); and a filtrate is directly used for the next step after being combined.

Step 2



[0043] The temperature is controlled at 0-10 °C, and Fmoc-D-Cys-OH (0.34 g, 0.1 mmol) is added to the filtrate of the previous step. DIPEA (0.15 g, 1.1 eq) is dropwise added to a reaction system; the reaction temperature is controlled at 10~30°C, and it is stirred for 0.5~2 h; the system is washed with water for 3 times, and concentrated to obtain a crude product Cpd 3, and pure Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd3) is obtained by a column chromatography.

[0044] An LCMS spectrum of purified Cpd 3 is the same as that in Fig. 2.

Embodiment 3



[0045] Synthesis of etelcalcetide using key intermediate:
An amino resin (including but not limited to a Sieber resin, Rink Amide MBHA, and a Rink Amide resin) is used to link NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide according to a method of solid-phase synthesis, a key intermediate is used to activate at 0-5°C for 0~5 min according to the ratio of Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA=3:3:3, the temperature is controlled at 20-30°C for 2~6 h, and a reaction end point is detected by kaiser. After the reaction, it is washed with DMF for 6 times, Fmoc is removed by 20% of piperidine, and then acetylation is performed to obtain a peptide resin of the etelcalcetide. The purity of a crude peptide after cleavage is 90.3%, the purity after preparation and purification is 99.51%, and the total yield is 65%.

[0046] Referring to the newly developed synthesis process of the etelcalcetide key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH, 100 g of a cysteine is used as a starting material, and the finally synthesized key intermediate is 357 g. This intermediate is used to synthesize the etelcalcetide according to Embodiment 3. After preparation and purification, 227 g of a pure product (TFA salt) is obtained, the purity is 99.51% (Fig. 3), and the total yield is 65%.

[0047] From the above description, it may be seen that the above embodiments of the present disclosure achieve the following technical effects: in the present application, a key intermediate Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH of the etelcalcetide is firstly synthesized, this intermediate is used to synthesize the etelcalcetide, the purity may reach 99.51% after preparation (Fig. 3), and the separation yield is 73%, it solves a problem in an existing synthesis process that a reaction of disulfide bond mismatch exists between polypeptide chains, and there are many types of side reactions and by-products.

[0048] The above are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent replacements, improvements and the like made within the spirit and principle of the present disclosure shall be included within a scope of protection of the present disclosure.


Claims

1. A synthetic method for an etelcalcetide intermediate, wherein the etelcalcetide intermediate is Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH, and the synthetic method for the etelcalcetide intermediate comprises the following steps: using N-(Boc)-L-Cys-OtBu as a raw material, and generating a primary product

through a substitution reaction, wherein R is S-Py or Cl, enabling the primary product to perform a coupling reaction with a Fmoc-D-Cys-OH amino acid, as to obtain the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH.
 
2. The synthetic method of claim 1, wherein the primary product is Py-S-S-Boc-L-Cys-OtBu, and the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH is prepared by the following steps:

enabling the N-(Boc)-L-Cys-OtBu to perform the substitution reaction with dithiodipyridine to obtain a primary product Py-S-S-Boc-L-Cys-OtBu; and

enabling the Py-S-S-Boc-L-Cys-OtBu to be coupled with Fmoc-D-Cys-OH to obtain the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu)-OH.


 
3. The synthetic method of claim 2, wherein the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH is prepared by the following steps:

at a room temperature, adding the N-(Boc)-L-Cys-OtBu and the dithiodipyridine to a solvent A, stirring for 6-12 h, and adding water, extracting by using an extraction agent, drying and filtering an obtained organic phase, to obtain the Py-S-S-Boc-L-Cys-OtBu after purifying;

adding the Fmoc-D-Cys-OH and the Py-S-S-Boc-L-Cys-OtBu to a solvent B, controlling a temperature at 15-30 °C, stirring and reacting for 0.5-2 h, washing, concentrating and purifying a reaction system, as to obtain the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH;

preferably, the solvent A is selected from one or more of DMF, NMP or DMAc;

preferably, the extraction agent is selected from one or more of EtOAc, MTBE or DCM; and

preferably, the solvent B is selected from one or more of DCM, DMF, THF, NMP or DMAc.


 
4. The synthetic method of claim 3, wherein in the reaction of the N-(Boc)-L-Cys-OtBu and the dithiodipyridine, a mole ratio of the N-(Boc)-L-Cys-OtBu and the dithiodipyridine is 1:1.2-1:6.4; a concentration of the N-(Boc)-L-Cys-OtBu in the solvent A is 0.01-0.3 g/mL; a concentration of the Fmoc-D-Cys-OH in the solvent B is 0.01-0.3 g/mL; and a mole ratio of the Fmoc-D-Cys-OH and the Py-S-S-Boc-L-Cys-OtBu is 1:0.8-1.4.
 
5. The synthetic method of claim 1, wherein the primary product is N-(Boc)-L-Cys(S-Cl)-OtBu, and the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH is prepared by the following steps:

enabling N-(Boc)-L-Cys-OtBu to react with NCS to synthesize N-(Boc)-L-Cys(S-Cl)-OtBu; and

enabling the N-(Boc)-L-Cys(S-Cl)-OtBu to react with the Fmoc-D-Cys-OH to obtain the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH.


 
6. The synthetic method of claim 5, wherein the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH is prepared by the following steps:

A. dissolving Boc-L-Cys-OtBu in a solvent C, controlling a temperature at 0-10 °C, adding DIPEA, and adding NCS in batches, stirring for 4-5 h, after ending a reaction, filtering, and eluting, to obtain filtrate;

B. controlling a temperature at 0-10 °C, adding the Boc-L-Cys-OtBu to the filtrate, adding DIPEA, reacting and controlling the temperature at 10-30 °C, stirring for 0.5-2 h; and washing, concentrating and purifying a reaction system to obtain the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH; and

preferably, the solvent C is selected from one or more of DCM, THF, DMF, NMP or DMAc.
 
7. The synthetic method of claim 6, wherein in the step A, dissolving the Boc-L-Cys-OtBu in the solvent C to obtain a solution with a concentration of 0.01-0.3 g/mL, and an addition amount of the DIPEA is 2-3 eq of moles, and an addition amount of the NCS is 1.1-1.5 eq; and in the step B, an addition amount of the Fmoc-D-Cys-OH is 1.1-1.5 eq.
 
8. A synthetic method for an etelcalcetide, comprising the following steps:

S1, synthesizing Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH by the synthetic method for the etelcalcetide intermediate of any one of claims 1 to 7; and

S2, enabling NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg to react with the Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH, and removing Fmoc, to obtain the etelcalcetide after acetylation.


 
9. The synthetic method of claim 8, wherein in the S2, the NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg is NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide.
 
10. The synthetic method of claim 8, wherein the S2 comprises the following steps: linking the NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-resin hexapeptide by using an amino resin according to a method of solid-phase synthesis, activating the Fmoc-D-Cys (S-S-Boc-L-Cys(OtBu))-OH, PyBop and DIPEA for 0-10 min at 0-5 °C, controlling a temperature at 20-30 °C and reacting for 2-6 h, after the reaction, washing for 4-6 times by DMF, removing the Fmoc by 10%-20% of piperidine, to obtain peptide resin of the etelcalcetide after the acetylation.
 
11. The synthetic method of claim 10, wherein a ratio of Fmoc-D-Cys(S-S-Boc-L-Cys(OtBu))-OH: PyBop: DIPEA is 3:3-6:3-6.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description