CN110668984A - 一种依特卡肽中间体及依特卡肽的合成方法 - Google Patents
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Abstract
Description
技术领域
本发明涉及化学合成技术领域,具体而言,涉及一种依特卡肽中间体及依特卡肽的合成方法。
背景技术
依特卡肽是由KaiPharmaceuticals,Inc.开发的一种新颖的拟钙剂,能够抑制甲状旁腺激素的分泌。依特卡肽可结合并激活甲状旁腺上的钙敏感受体,实现甲状旁腺激素水平的降低。
依特卡肽有3个D构型的精氨酸、2个D构型的丙氨酸、1个D构型的精氨酰胺、1个L构型半胱氨酸与1个D构型半胱氨酸(N端被乙酰基封闭)构成,其中D构型半胱氨酸与L构型半胱氨酸以二硫键连接在一起(N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl -D-alanyl-D-Argininamide ,disulfidewithL-cysteine)。
目前报道的专利中,都是固相链接至7肽,然后再选择不同的方法链接二硫键,特别是在脱除MMT过程中,需要使用1%~2%TFA/DCM反复操作15次以上,操作相当繁琐。
专利2017/114238 A1:采用的液相合成方法,路线较长,且总收率仅11%;专利WO2017/114240 A1:依照专利,粗肽纯度为81.0%, 杂质较多,纯化后总收率30.5%;专利US2019/0100554 A1:依照专利,粗肽纯度较低,需要多次纯化,总收率50%;中国专利CN201811277081中,粗肽纯度88.2%,纯化后总收率57.8%。也就是说,目前固相合成7肽连接半胱氨酸及衍生物这种方法,均存在粗肽纯度低,杂质多,难纯化等问题。
发明内容
本发明旨在提供一种依特卡肽中间体及依特卡肽的合成方法,该中间体用于合成伊特卡肽,以解决现有技术中依特卡肽合成步骤复杂的技术问题。
为了实现上述目的,根据本发明的一个方面,提供了一种依特卡肽中间体的合成方法。该依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,其结构式I所示:
结构式I
依特卡肽中间体的合成方法包括以下步骤:以N-Boc-L-Cys-OtBu为起始原料,通过取代反应生成初级产物,其中,R为S-Py或Cl,初级产物与Fmoc- D-Cys-OH氨基酸进行偶联反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
进一步地,初级产物为Py-S-S-(N-Boc)-L-Cys-OtBu,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:将N-Boc-L-Cys-OtBu与二硫二吡啶取代反应合成得到初级产物Py-S-S-(N-Boc)-L-Cys-OtBu;以及将Py-S-S-(N-Boc)-L-Cys-OtBu与Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys-(S-S- (N-Boc)-L -Cys-OtBu)-OH。
进一步地,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:室温下,将N-Boc-L-Cys-OtBu和二硫二吡啶加入溶剂A中,搅拌6~12 h,然后加入水,使用萃取剂萃取,对得到的有机相进行干燥及过滤,纯化后得到Py-S-S-(N-Boc)-L-Cys -OtBu;溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu,控温15~30oC搅拌反应0.5~2 h,对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH;优选的,溶剂A选自DMF、NMP和DMAc中的一种或多种;优选的,萃取剂选自EtOAc、MTBE和DCM中的一种或多种;优选的,溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
进一步地,N-Boc-L-Cys-OtBu与二硫二吡啶反应中,N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2~1:6.4;N-Boc-L-Cys-OtBu在溶剂A 中的浓度为0.01~0.3g/mL;溶剂B中Fmoc-D-Cys-OH的浓度为0.01~0.3 g/mL;Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1:0.8~1.4。
进一步地,初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu;以及将(N-Boc)-L-Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L- Cys(OtBu))-OH。
进一步地,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:A.将N-Boc-L-Cys-OtBu溶解在溶剂C中,控温0~10oC,加入DIPEA,分批加入NCS,搅拌4~5 h,反应结束后,过滤、淋洗,得到滤液;B. 控温0~10oC,将Fmoc-D-Cys-OH加入滤液中,加入DIPEA,反应控温10~30oC,搅拌0.5~2 h;对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S- (N-Boc)-L-Cys(OtBu))-OH;优选的,溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
进一步地,步骤A中,N-Boc-L-Cys-OtBu溶解在溶剂C中得到浓度为0.01~0.3 g/mL的溶液,DIPEA的加入量为摩尔的2~3 eq,NCS的加入量为1.1~1.5 eq;步骤B中,Fmoc-D-Cys-OH的加入量为1.1~1.5 eq。
根据本发明的另一个方面,提供一种依特卡肽的合成方法。该合成方法包括以下步骤:S1,上述任一种的依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH;以及S2,将NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与Fmoc-D-Cys(S-S-(N-Boc)-L- Cys(OtBu))-OH反应,然后脱去Fmoc,再经过乙酰化得到依特卡肽。
进一步地,S2中,NH2-D-Ala-D-Arg-D- Arg-D-Arg-D-Ala-D-Arg为NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。
进一步地,S2包括:使用氨基树脂依照固相合成的方法链接NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽,将Fmoc-D-Cys (S-S-(N-Boc)-L-Cys( OtBu ))-OH、PyBop和DIPEA在0~5oC活化0-10min,控温20~30oC反应2~6 h,反应结束后用DMF洗涤4~6次,10%~20% 哌啶脱去Fmoc,再经过乙酰化得到依特卡肽的肽树脂。
进一步地,Fmoc-D-Cys (S-S-(N-Boc)-L-Cys( OtBu ))-OH: PyBop: DIPEA=3:3~6:3~6的比例。
应用本发明的技术方案,通过合成关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,进而再合成依特卡肽,避免了肽链构建二硫键时的副反应,简化了操作,用于合成依特卡肽可以提高纯度和收率,重要的是,合成此中间体的原料廉价易得,工艺简单。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1示出了实施例1中 Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的纯度图谱;
图2示出了实施例1的中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的 LCMS 图谱(LC-MS: m/z = 617.9 (M-1, 30 ev), 1235.7 (2M-1, 30 ev));以及
图3示出了实施例3中制备纯化后的伊特卡肽纯度图谱。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明中所涉及的缩写解释如下:
Fmoc:9-芴甲氧基羰基。
Boc:叔丁基羰基。
tBu:叔丁基。
Arg:精氨酸。
Cys:半胱氨酸。
Ala:丙氨酸。
PyBop:六氟磷酸苯并***-1-基-氧基三吡咯烷基磷。
DIPEA:N,N-二异丙基乙胺。
DCM:二氯甲烷。
NCS:N-氯代丁二酰亚胺。
DMF:N,N-二甲基甲酰胺。
NMP:N-甲基吡咯烷酮。
DMAc:N,N-二甲基乙酰胺。
THF:四氢呋喃。
OtBu:叔丁氧基。
EtOAc: 乙酸乙酯。
依特卡肽的合成中,最关键的就是二硫键的合成。该步骤的收率的好坏直接影响到最终整条路线的收率。本申请的发明人发现,关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,(N-芴甲酰基-D-半胱氨酸-S-S-(N-叔丁氧羰基-L-半胱氨酸叔丁酯))的合成预先合成出二硫键从而可以完美的避开固相反应效率低收率差的问题。
根据本发明一种典型的实施方式,提供一种依特卡肽中间体的合成方法,依特卡肽中间体为Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,依特卡肽中间体的合成方法包括以下步骤:以N-Boc-L-Cys-OtBu为起始原料,通过取代反应生成初级产物,其中,R为S-Py或Cl,初级产物与Fmoc- D-Cys-OH氨基酸进行偶联反应得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。
应用本发明的技术方案,通过合成关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,进而再合成依特卡肽,避免了肽链构建二硫键时的副反应,简化了操作,用于合成依特卡肽可以提高纯度和收率,重要的是,合成此关键中间体的原料廉价易得,工艺简单。
根据本发明一种典型的实施方式,初级产物为Py-S-S-(N-Boc)-L-Cys-OtBu,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得:将N-Boc-L-Cys-OtBu与二硫二吡啶反应合成得到Py-S-S-(N-Boc)-L-Cys-OtBu;以及将Py-S-S- (N-Boc)-L-Cys-OtBu与Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys(OtBu))-OH,重要的是,合成此中间体的原料廉价易得,工艺简单。
优选的,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得:室温下,将(N-Boc)-L- Cys-OtBu和二硫二吡啶加入溶剂A中,搅拌6~12h,然后加入水,使用萃取剂萃取,对得到的有机相进行干燥及过滤,纯化后得到Py-S-S-(N-Boc)-L-Cys-OtBu;溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu,控温15~30oC搅拌反应0.5~2 h,对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH。优选的,溶剂A选自DMF、NMP和DMAc中的一种或多种;优选的,萃取剂选自EtOAc、MTBE和DCM中的一种或多种;优选的,所述溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
为了提高原料的利用率,保证反应的快速有效进行,进一步优选的,N-Boc-L-Cys-OtBu与二硫二吡啶反应中,N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2~1:6.4,(N-Boc)-L- Cys-OtBu在溶剂A中的浓度为0.01~0.3 g/mL;溶剂B中Fmoc-D-Cys-OH的浓度为0.01~0.3 g/mL;Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1: 0.8~1.4。
根据本发明一种典型的实施方式,初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得:将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu;以及将(N-Boc)-L- Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys(OtBu))-OH。
优选的,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得:A. 将(N-Boc)-Cys-OtBu溶解在溶剂C中,控温0~10oC,加入DIPEA,分批加入NCS,搅拌4~5 h,反应结束后,过滤、淋洗,得到滤液;B. 控温0~10oC,将Fmoc-D-Cys-OH加入滤液中,加入DIPEA,反应控温10~30 oC,搅拌0.5~2 h;对反应体系进行水洗、浓缩及纯化得到Fmoc-D-Cys(S-S-(N-Boc)-L-Cys (OtBu))-OH。优选的,所述溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
为了提高原料的利用率,保证反应的快速有效进行,进一步优选的,步骤A中, (N-Boc)-L-Cys -OtBu溶解在溶剂C中得到浓度为0.01~0.3 g/mL的溶液,DIPEA的加入量为摩尔的2~3 eq,NCS的加入量为1.1~1.5 eq;所述步骤B中,Fmoc-D-Cys-OH的加入量为1.1~1.5eq。
根据本发明一种典型的实施方式,提供一种依特卡肽的合成方法。该合成方法包括以下步骤:S1,按照上述任一种依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH;以及S2,将NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与所述Fmoc-D-Cys(S-S-(N-Boc)-L- Cys(OtBu))-OH反应,然后脱去Fmoc,再经过乙酰化得到依特卡肽。
由于关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH的原料廉价易得,工艺简单,也直接使得本发明中依特卡肽的合成方法原料廉价易得,工艺简单。
优选地,S2中,NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg为NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。优选的,S2包括:使用氨基树脂依照固相合成的方法链接NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽,将Fmoc-D-Cys (S-S-(N-Boc)-L-Cys( OtBu ))-OH、PyBop和DIPEA在0~10oC活化0-10 min,控温20~30oC反应2~6 h,反应结束后用DMF洗涤6次,10%~20% 哌啶脱去Fmoc,再经过乙酰化得到依特卡肽的肽树脂。更优选的,Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH:PyBop:DIPEA=3: 3~6:3~6的比例。
下面将结合实施例进一步说明本发明的有益效果。
具体实施方式合成路线:
实施例1
R=S-Py
以N-Boc-L-Cys-OtBu (Cpd 1) 为原料,通过与二硫二吡啶反应合成Py-S-S-(N-Boc)-L-Cys -OtBu(Cpd 2a);将Py-S-S-(N-Boc)-L-Cys-OtBu(Cpd 2a)同Fmoc-D-Cys-OH偶联得到Fmoc-D-Cys- (S-S-(N-Boc)-L-Cys-OtBu)-OH(Cpd 3)。
步骤 1
室温下,将Cpd 1 (1.6 g, 1.0 eq)和2,2-dithiodipyridne (5.1 g, 4.0 eq)加入DMF(16 mL,10 vol.)中。反应室温搅拌6~12 h;然后体系中加入水;使用乙酸乙酯(100 mL*3)萃取;有机相合并后使用MgSO4干燥,过滤;有机相浓缩得到Cpd 2a粗品,通过柱层析纯化得到纯的Py-S-S-(N-Boc)-L -Cys-OtBu(Cpd 2a)。
步骤2
15~30oC下,DCM (30 ml, 30 vol.)中加入Fmoc-D-Cys-OH (1.0 g, 1.0 eq.),然后加入Cpd 2a(1.13 g, 1.0 eq.)至反应体系;控温15~30oC下搅拌反应0.5~2 h;体系水洗3次,浓缩得到粗品Cpd 3;通过柱层析得到纯的Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH(Cpd 3)。
图1示出了纯化后的Cpd 3纯度图谱;图2示出了纯化后的Cpd 3的LCMS图谱。
实施例2
R=Cl
以N-Boc-L-Cys-OtBu (Cpd 1)为原料,通过与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu (Cpd 2b);将(N-Boc)-L-Cys(S-Cl)-OtBu(Cpd 2b)同Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L- Cys-(OtBu))-OH(Cpd 3)。
步骤 1
(N-Boc)-Cys-OtBu(0.28 g, 0.1mmol)溶解在DCM(20 mL)中,搅拌控温0~10oC,加入DIPEA(0.19 g, 0.15 mmol);控温0~10oC分批加入NCS(0.15 g, 1.1 eq);反应保温搅拌4~5 h, HPLC 监控反应终点;反应结束后,过滤;滤饼用DCM(20 mL)淋洗;合并滤液直接用于下一步。
步骤2
控温0-10oC,将Fmoc-D-Cys-OH(0.34 g,0.1 mmol)加入上一步的滤液中。DIPEA(0.15g, 1.1 eq)滴加入反应体系;反应控温10~30oC,搅拌0.5~2 h;体系水洗3次,浓缩得到粗品Cpd 3,通过柱层析得到纯的Fmoc-D-Cys(S-S-(N-Boc)-L-Cys-(OtBu))-OH (Cpd 3)。
纯化后的Cpd 3的LCMS图谱同图2。
实施例 3
使用关键中间体合成依特卡肽:
使用氨基树脂(包括但不限于Sieber树脂,Rink Amide MBHA,Rink Amide 树脂)依照固相合成的方法链接NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽,用关键中间体按照Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu ))-OH : PyBop : DIPEA = 3:3:3的比例0-5 oC活化0~5 min,控温20-30oC反应2~6 h,kaiser检测反应终点。反应结束后用DMF洗涤6次,20% 哌啶脱去Fmoc,再经过乙酰化得到依特卡肽的肽树脂。经过切割后的粗肽纯度90.3%,制备提纯后纯度在99.51%,总收率65%。
参照新开发的依特卡肽关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu ))-OH合成工艺,100 g半胱氨酸为起始物料,最终合成关键中间体357 g。使用此中间体依照实例3合成依特卡肽,经制备纯化后得到纯品227 g(TFA 盐),纯度在99.51%(图3),总收率65%。
从以上的描述中,可以看出,本发明上述的实施例实现了如下技术效果:本申请中先合成了一种依特卡肽的关键中间体Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH,使用该中间体合成依特卡肽,制备后纯度可以达到99.51%(图3), 分离收率73%,解决了现有合成过程中存在多肽链间二硫键错配的反应,副反应和副产物的种类较多的问题。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (11)
2.根据权利要求1所述的合成方法,其特征在于,所述初级产物为Py-S-S-(N-Boc)-L-Cys- OtBu,所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH通过以下步骤制得:
将N-Boc-L-Cys-OtBu与二硫二吡啶取代反应合成得到初级产物Py-S-S-(N-Boc)-L-Cys-OtBu;以及
将所述Py-S-S-(N-Boc)-L-Cys-OtBu与Fmoc-D-Cys-OH偶联得到所述Fmoc-D-Cys-(S-S-(N-Boc)-L-Cys-OtBu)-OH。
3.根据权利要求2所述的合成方法,其特征在于,所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:
室温下,将N-Boc-L-Cys-OtBu和二硫二吡啶加入溶剂A中,搅拌6~12 h,然后加入水,使用萃取剂萃取,对得到的有机相进行干燥及过滤,纯化后得到Py-S-S-(N-Boc)-L-Cys -OtBu;
溶剂B中加入Fmoc-D-Cys-OH和Py-S-S-(N-Boc)-L-Cys-OtBu,控温15~30oC搅拌反应0.5~2 h,对反应体系进行水洗、浓缩及纯化得到所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH;
所述溶剂A选自DMF、NMP和DMAc中的一种或多种;
所述萃取剂选自EtOAc、MTBE和DCM中的一种或多种;
所述溶剂B选自DCM、DMF、THF、NMP和DMAc中的一种或多种。
4.根据权利要求3所述的合成方法,其特征在于,所述N-Boc-L-Cys-OtBu与二硫二吡啶反应中,N-Boc-L-Cys-OtBu和二硫二吡啶的摩尔比为1:1.2~1:6.4;N-Boc-L-Cys-OtBu在所述溶剂A 中的浓度为0.01~0.3g/mL;所述溶剂B中Fmoc-D-Cys-OH的浓度为0.01~0.3 g/mL;Fmoc-D-Cys-OH与Py-S-S-(N-Boc)-L-Cys-OtBu的摩尔比为1:0.8~1.4。
5.根据权利要求1所述的合成方法,其特征在于,所述初级产物为(N-Boc)-L-Cys(S-Cl)-OtBu,所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:
将N-Boc-L-Cys-OtBu与NCS反应合成(N-Boc)-L-Cys(S-Cl)-OtBu;以及
将(N-Boc)-L-Cys(S-Cl)-OtBu与Fmoc-D-Cys-OH反应得到Fmoc-D-Cys(S-S-(N-Boc)-L- Cys(OtBu))-OH。
6.根据权利要求5所述的合成方法,其特征在于,所述Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))- OH通过以下步骤制得:
A. 将N-Boc-L-Cys-OtBu溶解在溶剂C中,控温0~10oC,加入DIPEA,分批加入NCS,搅拌4~5 h,反应结束后,过滤、淋洗,得到滤液;
B. 控温0~10oC,将Fmoc-D-Cys-OH加入所述滤液中,加入DIPEA,反应控温10~30oC,搅拌0.5~2 h;对反应体系进行水洗、浓缩及纯化得到所述Fmoc-D-Cys(S-S- (N-Boc)-L-Cys(OtBu))-OH;
所述溶剂C选自DCM、THF、DMF NMP和DMAc中的一种或多种。
7.根据权利要求6所述的合成方法,其特征在于,所述步骤A中,N-Boc-L-Cys-OtBu溶解在所述溶剂C中得到浓度为0.01~0.3 g/mL的溶液,DIPEA的加入量为摩尔的2~3 eq,NCS的加入量为1.1~1.5 eq;所述步骤B中,Fmoc-D-Cys-OH的加入量为1.1~1.5 eq。
8.一种依特卡肽的合成方法,其特征在于,包括以下步骤:
S1,按照如权利要求1至7中任一项所述的依特卡肽中间体的合成方法合成Fmoc-D-Cys(S-S-(N-Boc)-L-Cys(OtBu))-OH;以及
S2,将NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg与所述Fmoc-D-Cys(S-S-(N-Boc)-L- Cys(OtBu))-OH反应,然后脱去Fmoc,再经过乙酰化得到依特卡肽。
9.根据权利要求8所述的合成方法,其特征在于,所述S2中,所述NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg为NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽。
10.根据权利要求8所述的合成方法,其特征在于,所述S2包括:使用氨基树脂依照固相合成的方法链接NH2-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-树脂六肽,将Fmoc-D-Cys(S-S-(N-Boc)-L-Cys( OtBu ))-OH、PyBop和DIPEA在0~5oC活化0-10min,控温20~30oC反应2~6 h,反应结束后用DMF洗涤4~6次,10%~20% 哌啶脱去Fmoc,再经过乙酰化得到依特卡肽的肽树脂。
11.根据权利要求10所述的合成方法,其特征在于,Fmoc-D-Cys (S-S-(N-Boc)-L-Cys( OtBu ))-OH: PyBop: DIPEA=3:3~6:3~6的比例。
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CN116947966A (zh) * | 2023-09-18 | 2023-10-27 | 哈药集团生物工程有限公司 | 一种伊特卡肽中间体及伊特卡肽的制备方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016154580A1 (en) * | 2015-03-26 | 2016-09-29 | Amgen Inc. | Solution phase method for preparing etelcalcetide |
CN106928320A (zh) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | 一种合成Etelcalcetide的方法 |
CN106928321A (zh) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | 一种合成Etelcalcetide的方法 |
CN107434820A (zh) * | 2017-08-07 | 2017-12-05 | 南京工业大学 | 一种维拉卡肽的合成方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1769000B1 (en) * | 2004-07-16 | 2014-12-24 | Amgen Research (Munich) GmbH | Expression-enhanced polypeptides |
WO2009069959A2 (en) * | 2007-11-30 | 2009-06-04 | Korea University Industrial & Academic Collaboration Foundation | A nanoparticle for separating peptide, method for preparing the same, and method for separating peptide using the same |
CN201714238U (zh) | 2010-07-19 | 2011-01-19 | 董昭 | 一种便于开关的推拉窗 |
TW201915009A (zh) | 2017-10-03 | 2019-04-16 | 中化合成生技股份有限公司 | 合成依特卡肽(Etelcalcetide)或其鹽類之方法 |
CN110498835B (zh) * | 2018-05-17 | 2021-06-08 | 深圳翰宇药业股份有限公司 | 一种合成etelcalcetide的方法 |
-
2019
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016154580A1 (en) * | 2015-03-26 | 2016-09-29 | Amgen Inc. | Solution phase method for preparing etelcalcetide |
CN106928320A (zh) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | 一种合成Etelcalcetide的方法 |
CN106928321A (zh) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | 一种合成Etelcalcetide的方法 |
CN107434820A (zh) * | 2017-08-07 | 2017-12-05 | 南京工业大学 | 一种维拉卡肽的合成方法 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111925418A (zh) * | 2020-09-27 | 2020-11-13 | 凯莱英生命科学技术(天津)有限公司 | 伊特卡肽的液相合成方法 |
CN116947966A (zh) * | 2023-09-18 | 2023-10-27 | 哈药集团生物工程有限公司 | 一种伊特卡肽中间体及伊特卡肽的制备方法 |
CN116947966B (zh) * | 2023-09-18 | 2023-12-26 | 哈药集团生物工程有限公司 | 一种伊特卡肽中间体及伊特卡肽的制备方法 |
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EP4071135A4 (en) | 2023-02-01 |
CN110668984B (zh) | 2020-04-10 |
KR20220120589A (ko) | 2022-08-30 |
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