CN116370650B - 基于四嗪连接子的多肽药物偶联物及其制备方法、应用 - Google Patents
基于四嗪连接子的多肽药物偶联物及其制备方法、应用 Download PDFInfo
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- CN116370650B CN116370650B CN202310397370.2A CN202310397370A CN116370650B CN 116370650 B CN116370650 B CN 116370650B CN 202310397370 A CN202310397370 A CN 202310397370A CN 116370650 B CN116370650 B CN 116370650B
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- tetrazine
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- drug conjugate
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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Abstract
基于四嗪连接子的多肽药物偶联物及其制备方法、应用,该多肽药物偶联物采用四嗪化合物作为连接子,其释放策略为反式环辛烯与四嗪连接子发生生物正交反应断键以释放母药,由于正常生理组织中不含有反式环辛烯或具有近似结构的化合物,因此能够显著地提高药物释放的特异性和精准性,同时,该多肽药物偶联物在体内具有良好的稳定性,不易与体内的谷胱甘肽等物质发生巯基交换反应而导致脱靶毒性,易在肿瘤部位聚集。
Description
技术领域
本发明涉及生物制药技术领域,具体涉及基于四嗪连接子的多肽药物偶联物及其制备方法、应用。
背景技术
传统的抗癌药物尽管具有很强的药效,但时常伴随着药物不良反应,对正常细胞和特定器官可能具有毒副作用,例如,阿霉素的心脏毒性。这种毒副作用的存在会限制化疗药物的使用剂量,从而使暴露在肿瘤组织的药物浓度不够,最终导致癌症复发和转移。
为了在保证疗效的同时降低药物的不良反应,抗体药物偶联物(ADC)通过高亲和力抗体特异性靶向肿瘤部位,在特性的环境下释放出活性药物进行肿瘤治疗,使得药物在病灶组织高浓度富集,从而避免了药物抵达正常组织细胞,保证药效的同时提高了安全性。
近年来,越来越多的研究团体发现多肽相较小分子和大生物制品而言有许多优势,其合成路径更简单、成本更低,具有与未开发的靶标相互作用的能力,可以降低免疫原性和增强组织渗透力等。靶向肽除了具有生物活性外,它们还能出色地将药物功能分子运送到所需的靶点。因此,利用靶向肽构建的多肽药物偶联物(PDC)越来越广泛地应用到肿瘤治疗中。
专利CN111068068A公开了一种以丁二酸酐作为连接子的RGD多肽-喜树碱多肽药物偶联物,其利用RGD多肽靶头的靶向作用,可将药物特异性地运送到肿瘤组织,减少正常组织的蓄积,降低其生物毒性,通过配体-受体的相互作用增加肿瘤细胞和肿瘤新生血管对药物的摄取。
多肽药物偶联物所采用的连接子决定了药物递送策略、以及用于与连接子共同作用而释放药物的外来引入化合物,外来引入化合物在肿瘤微环境中的含量越少,多肽药物偶联物释放的特异性和精准性更高,更容易在肿瘤组织富集、减少其在非肿瘤组织的释放。目前,暂未公开有采用乙烯基四嗪作为连接子的多肽药物偶联物。
发明内容
本发明的一个目的在于提供基于四嗪连接子的多肽药物偶联物,其采用乙烯基四嗪作为连接子将带巯基的多肽和带酚羟基或氨基甲酸酯键的药物相连,不仅能够利用靶向肽的特异性靶向肿瘤相关蛋白,而且能够与四嗪母核发生生物正交反应断键释放出带酚羟基或氨基的母药的反式环辛烯(TCO)不存在于肿瘤微环境中,从而实现仅能够利用外来的TCO释放母药,显著地提高了母药释放的特异性和精准性。
本发明目的通过下述技术方案实现:
基于四嗪连接子的多肽药物偶联物,所述多肽药物偶联物的结构式为:P-L-D,其中,P为具有巯基的多肽,D为具有酚羟基或者氨基的药物,L为四嗪连接子,所述四嗪连接子的结构式为:所述四嗪连接子与所述多肽的巯基连接形成C-S键,四嗪连接子与药物的酚羟基连接形成酚醚键,或者四嗪连接子与药物的氨基连接形成氨基甲酸酯键。
本技术方案中,多肽药物偶联物的结构式为P-L-D,即通过四嗪连接子L连接多肽P和药物D。其中,多肽为具有半胱氨酸的多肽,四嗪连接子能够与多肽的半胱氨酸的巯基通过C-S键进行连接。药物可以是含有酚羟基的药物例如喜树碱,四嗪连接子与这类药物的酚羟基连接构成酚醚键;药物也可以是含有氨基的药物例如阿霉素,四嗪连接子与这类药物的氨基连接后构成氨基甲酸酯键。
靶向多肽的引入使得多肽药物偶联物更容易被靶点细胞摄取,同时更难进入非靶标的正常细胞,从而在肿瘤部位发挥药效。本技术方案中,采用四嗪化合物作为连接子连接多肽和药物,在肿瘤微环境中,需要外来引入的反式环辛烯作为小分子释放药物与四嗪连接子进行生物正交断键反应,进而释放出连接的母药。
这种释放策略的一个优势在于,正常生理组织中不含有反式环辛烯,因此聚集于肿瘤部位的多肽药物偶联物必须通过外来添加的反式环辛烯方能释放药物,所以显著地提高了药物释放的特异性和精准性,药物不会在非肿瘤组织中释放。而传统的释放策略中,正常生理组织中通常含有一定量的小分子释放药物或者具有近似结构的化合物,即使其含量很低,也会造成传统的多肽药物偶联物在非肿瘤组织中释放。此外,基于四嗪连接子的多肽药物偶联物在体内的稳定性良好,不易与体内的谷胱甘肽等物质发生巯基交换反应而导致脱靶毒性,易在肿瘤部位聚集。
进一步地,所述多肽选自以下任一种:
理论上,含有半胱氨酸的靶向肽均可以通过巯基与四嗪连接子的乙烯基键反应,实现四嗪连接子和靶向肽通过C-S键进行连接的目的。作为优选的实施方案,本技术方案中,用于多肽药物偶联物合成的靶向肽选自上述六种多肽的任一种。
进一步地,所述药物选自以下任一种:
药物的选择主要有两类,一类是具有酚羟基的药物,另一类是具有氨基的药物。两类药物的合成路径虽然存在差异,但可分别通过酚醚键或氨基甲酸酯键与四嗪连接子连接得到多肽药物偶联物,并在四嗪连接子与反式环辛烯发生生物正交反应后断键。优选地,本技术方案中,用于多肽药物偶联物合成的药物选自上述十种药物中的任一种。
作为本发明中多肽药物偶联物的优选实施方案,所述多肽药物偶联物的结构式为:
本技术方案中,利用四嗪连接子分别连接了具有代表性酚羟基类药物喜树碱、氨基类药物阿霉素。药物的对位上通过C-S键连接有靶向肽c(RGDyC)。本领域技术人员应当理解,根据不同的靶向需求,四嗪连接子可以连接不同的具有巯基的靶向肽。
进一步地,若干多肽药物偶联物之间通过非共价键连接、自组装形成纳米球。在部分优选的实施例中,所述多肽为亲水性多肽,所述药物为疏水性药物。四嗪连接子结构短小的特点有利于亲水性多肽与阿霉素、喜树碱等疏水性药物以更加适当的距离相连,进而形成两亲性分子,从而在生理环境中自发地组装成球形纳米粒子。不仅能够明显地增加疏水性药物的溶解性、提高细胞摄取,还能够增加细胞富集,进一步提高疗效。
进一步地,所述纳米球的水合粒径为200~300nm。
本发明还提供了用于制备前述任一种基于四嗪连接子的多肽药物偶联物的制备方法,该制备方法包括以下步骤:
在四嗪连接子的3位或6位上形成乙烯基,之后在乙烯基的对位通过氨基甲酸酯键连接具有氨基的药物,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物;或
在四嗪连接子的3位或6位上通过酚醚键连接具有酚羟基的药物,之后在形成的酚醚键的对位形成乙烯基,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物。
本技术方案中,对于氨基类药物的多肽药物偶联物,首先可以采用发明人于专利CN112010817A中公开的制备方法,利用腈类化合物为原料,加入催化剂硫醇化合物、肼类化合物进行反应制得四嗪化合物。之后在四嗪化合物的3位或者6位上直接或间接连接用于与氨基类药物反应连接的碳酸酯键,再在连接有碳酸酯键的对位上形成乙烯基,随后利用碳酸酯键与药物的氨基反应形成氨基甲酸酯键、连接四嗪连接子和药物,最后,利用药物对位上的乙烯基与多肽的巯基反应连接靶向肽,得到多肽药物偶联物。
对于酚羟基类药物的多肽药物偶联物,其合成四嗪化合物的方式相同,不同的是,四嗪化合物可先与酚羟基反应连接药物,之后在药物的对位形成乙烯基,最后利用乙烯基与多肽的巯基反应连接靶向肽,得到多肽药物偶联物。
靶向肽和四嗪连接子的反应既可以在水相中低浓度快速高效反应,也可以在有机相高浓度制备。在部分优选的实施例中,多肽和四嗪连接子在含有N,N-二异丙基乙胺的溶剂中常温反应制得多肽药物偶联物,所述溶剂包括乙腈和二甲基亚砜。乙腈和二甲基亚砜的体积比可以根据溶解性进行调节,在一个或多个优选的实施例中,乙腈和二甲基亚砜的体积比为3:2。
本发明还提供前述任一种基于四嗪连接子的多肽药物偶联物的用途,所述多肽药物偶联物用于与反式环辛烯发生生物正交反应,释放带有酚羟基或氨基的药物。
本发明还提供一种药物,该药物包括前述任一种基于四嗪连接子的多肽药物偶联物、以及药学上可接受的辅料。
本发明与现有技术相比,具有如下的优点和有益效果:
1、本发明提供的多肽药物偶联物采用四嗪连接子,其释放策略为反式环辛烯与四嗪连接子发生生物正交反应断键以释放母药,由于正常生理组织中不含有反式环辛烯或具有近似结构的化合物,因此能够显著地提高药物释放的特异性和精准性;
2、本发明的多肽药物偶联物在体内具有良好的稳定性,不易与体内的谷胱甘肽等物质发生巯基交换反应而导致脱靶毒性,易在肿瘤部位聚集;
3、本发明利用四嗪连接子的结构特点,通过与亲水性多肽、疏水性药物连接,能够使多肽和药物以更加适当的距离相连形成两亲性分子,不仅能够明显地增加疏水性药物的溶解性、提高细胞摄取,还能够增加细胞富集,进一步提高疗效。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解,构成本申请的一部分,并不构成对本发明实施例的限定。在附图中:
图1示出了本发明具体实施例中c(RGDyC)-Tz-DOX的HPLC-MS表征;
图2示出了本发明具体实施例中c(RGDyC)-Tz-CPT的HPLC-MS表征;
图3示出了本发明具体实施例中c(RGDyC)-Tz-DOX对U87、B16F10、SKOV3和LX2细胞的毒性实验结果;
图4示出了本发明具体实施例中c(RGDyC)-Tz-CPT对U87和SKOV3细胞的毒性实验结果;
图5示出了本发明具体实施例中两种PDC的自组装表征结果,其中(a)为c(RGDyC)-Tz-DOX,(b)为c(RGDyC)-Tz-CPT。
图6示出了本发明具体实施例中两种PDC的稳定性表征结果,其中(a):c(RGDyC)-Tz-DOX,(b):c(RGDyC)-Tz-CPT。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
在本发明中使用的术语“连接”在不进行特别说明的情况下,可以是直接相连,也可以使经由其他基团间接相连。
本发明所有原料,对其来源没有特别限制,在市场上购买的或按照本领域技术人员熟知的常规方法即可制备。本发明所有原料,对其纯度没有特别限制,本发明优选采用分析纯或生物制药领域常规的纯度要求。本发明对所述取代基的表达方式没有特别限制,均采用本领域技术人员熟知的表达方式,本领域技术人员基于常识,可根据其表达方式正确理解其含义。
本发明所有原料,其牌号和简称均属于本领域常规牌号和简称,每个牌号和简称在其相关用途的领域内均是清楚明确的,本领域技术人员根据牌号、简称以及相应的用途,能够从市售中购买得到或者通过常规方法制备得到。
一、具有氨基类药物的多肽药物偶联物的制备:
【实施例1】
1、合成(2-(6-((4-甲氧基苄基)氧基)甲基)-1,2,4,5-四嗪-3-基)乙烷-1-醇)
将2-((4-甲氧基苄基)氧基)乙腈(6.0g,0.034mol,1.0eq.)(化合物1)置于150mL圆底烧瓶中,抽换氩气三次,并于氩气保护下加入羟基丙腈(5.79mL,0.085mol,2.50eq.)和巯基丙酸(2.80mL,0.034mol,1.0eq.),然后在氩气保护和冰浴条件下,滴加水合肼(33.50mL,0.678mol,20.0eq.),滴加完毕,将反应瓶移至室温搅拌18小时。将亚硝酸钠(35.0g,0.508mol,15.0eq.)置于500mL锥形瓶中加少量水溶解,将反应液转移至锥形瓶中,冰浴搅拌下加入6mol/L盐酸调pH至2~3左右并继续搅拌20min,用二氯甲烷(150mL×3)萃取,合并有机相,经无水硫酸钠干燥后真空浓缩,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色油状产物化合物2。
2、合成3-(2-((叔丁基二甲基硅基)氧基)乙基)-6-((4-甲氧基苄基)氧基)甲基)-1,2,4,5-四嗪
将化合物2(2.234g,8.094mmol,1.0eq.)置于100mL圆底烧瓶中,加入咪唑(0.716g,10.520mmol,1.3eq.),抽换氩气三次,然后在氩气保护和冰浴条件下加入叔丁基二甲基氯硅烷(1.464g,9.713mmol,1.2eq.),滴加完毕,将反应瓶移至室温搅拌30分钟。待薄层色谱TLC监测原料消失后,加入水10mL淬灭反应,二氯甲烷(25mL×3)萃取,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色油状产物化合物3。
1H NMR(400MHz,Chloroform-d)δ7.39–7.27(m,2H),6.94–6.82(m,2H),5.08(s,2H),4.71(s,2H),4.23(t,J=6.4Hz,2H),3.81(s,3H),3.54(t,J=6.4Hz,2H),0.79(s,9H).
13C NMR(101MHz,Chloroform-d)δ169.31,166.76,159.57,129.85,129.09,113.95,73.31,69.28,61.10,55.30,38.39,25.72,18.14,-5.47.
3、合成(6-(2-((叔丁基二甲基硅基)氧基)乙基)-1,2,4,5-四嗪-3-基)甲醇
将化合物3(2.79g,7.154mmol,1.0eq.)置于100mL圆底烧瓶中,加入二氯甲烷和水于室温搅拌溶解,加入2,3-二氯-5,6-二氰基苯醌(DDQ)(3.90g,17.200mmol,2.4eq.),常温搅拌2小时,TLC监测原料消失后,加入饱和碳酸氢钠水溶液淬灭反应,二氯甲烷(50mL×3)萃取,有机相用饱和碳酸氢钠水溶液洗三次,饱和氯化钠溶液洗一次,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色油状产物化合物4。
1H NMR(400MHz,Chloroform-d)δ5.27(d,J=3.9Hz,2H),4.23(t,J=6.3Hz,2H),3.56(t,J=6.3Hz,2H),3.34(t,J=6.2Hz,1H),0.77(d,J=1.1Hz,9H),-0.02(s,6H).
13C NMR(101MHz,Chloroform-d)δ169.74,167.61,62.70,61.07,38.36,25.69,18.12,-5.50.
4、合成6-(2-羟乙基)-1,2,4,5-四嗪-3-基)甲基(4-硝基苯)碳酸酯
将化合物4(800.0mg,2.963mmol,1.0eq.)置于200mL圆底烧瓶中抽换氩气三次,并于氩气保护下加入二氯甲烷50mL,将对硝基氯甲酸苯酯(777.5mg,3.852mmol,1.3eq.)溶于5mL二氯甲烷,滴加到上述反应液中,最后加入DIPEA(1.032mL,5.926mmol,2.0eq.)常温搅拌3小时,TLC监测原料消失后,加入水约50mL淬灭反应,二氯甲烷(30mL×3)萃取,有机相用饱和碳酸氢钠水溶液洗三次,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色油状产物化合物5。
将化合物5(184mg,0.423mmol,1.0eq.)置于50mL圆底烧瓶中,加入混合溶剂(四氢呋喃:甲醇=4:1)15mL溶解,滴加1mol/L盐酸(4.23mL,4.23mmol,10.0eq),常温搅拌20分钟,TLC监测原料消失后,加入5mL水,二氯甲烷(10mL×3)萃取,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=1:1)得红色油状产物化合物6。
1H NMR(400MHz,DMSO-d6)δ8.34(d,J=9.1Hz,2H),7.61(d,J=9.2Hz,2H),5.91(s,2H),4.86(t,J=5.6Hz,1H),4.01(q,J=6.1Hz,2H),3.45(t,J=6.3Hz,2H).
13C NMR(101MHz,DMSO-d6)δ170.08,164.66,155.52,152.40,145.83,126.01,122.96,67.37,59.65,38.66.
5、合成4-硝基苯((6-乙烯基-1,2,4,5-四氮-3-基)甲基)碳酸酯
将化合物6(60.0mg,0.187mmol,1.0eq.)置于50mL圆底烧瓶中,加入约2mL二氯甲烷溶解,加入甲基磺酰氯(26.17μL,0.281mmol,1.5eq.)和N,N-二异丙基乙胺(DIPEA)(56.6μL,0.281mmol,1.5eq.)常温搅拌30分钟,TLC监测原料消失后,加N,N-二异丙基乙胺(DIPEA)(56.6μL,0.281mmol,1.5eq.)后于室温反应,TLC监测反应结束后,加入约5mL水,二氯甲烷(10mL×3)萃取,有机相用水洗三次,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色固体粉末化合物7。
1H NMR(400MHz,DMSO-d6)δ8.38–8.30(m,2H),7.65–7.58(m,2H),7.25(dd,J=17.6,10.9Hz,1H),6.94(dd,J=17.5,1.2Hz,1H),6.19(dd,J=10.9,1.1Hz,1H),5.90(s,2H).
13C NMR(101MHz,DMSO-d6)δ164.79,164.36,155.53,152.38,145.83,130.87,129.08,126.01,122.97,67.41.
6、合成化合物8
将化合物7(100.0mg,0.330mmol,1.0eq.)置于100mL圆底烧瓶中,加入30mL二氯甲烷,DIPEA(171.2μL,0.990mmol,3.0eq.)和盐酸阿霉素(573mg,0.990mmol,3.0eq.),常温搅拌5小时,TLC监测原料消失后,加入水约50mL,二氯甲烷(50mL×3)萃取,收集有机相,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(二氯甲烷:甲醇=10:1)得红色油状产物化合物8。
1H NMR(400MHz,DMSO-d6)δ14.03(s,1H),13.27(s,1H),7.91(d,J=4.8Hz,2H),7.66(q,J=4.9,4.4Hz,1H),7.31–7.09(m,3H),6.84(dd,J=17.5,1.2Hz,1H),6.10(dd,J=11.0,1.2Hz,1H),5.55–5.49(m,2H),5.43(s,2H),5.22(d,J=3.6Hz,1H),4.94(s,1H),4.83(t,J=6.0Hz,1H),4.75(d,J=5.7Hz,1H),4.55(d,J=6.0Hz,2H),4.14(q,J=6.5Hz,1H),3.99(s,3H),3.50(s,1H),2.96(d,J=3.3Hz,2H),2.20–2.10(m,2H),1.99(q,J=7.3Hz,1H),1.86(dd,J=12.8,3.8Hz,1H),1.12(d,J=6.4Hz,3H).
13C NMR(101MHz,DMSO-d6)δ214.35,186.67,186.55,166.09,164.53,161.10,156.51,155.23,136.51,135.76,134.84,134.38,130.80,128.50,120.18,119.97,119.29,111.02,110.89,100.76,75.34,70.19,68.41,67.11,64.20,63.40,56.93,55.37,47.92,36.83,32.43,30.22,17.47.
7、合成多肽偶联药物c(RGDyC)-Tz-DOX
1mM的多肽c(RGDyC)和1.1mM的化合物8在含有1mM的DIPEA的乙腈比二甲基亚砜体积比为3:2的溶液里常温反应2小时,得到多肽偶联药物c(RGDyC)-Tz-DOX,其HPLC-MS表征如图1所示。
二、具有酚羟基类药物的多肽药物偶联物的制备:
【实施例2】
本实施例中,化合物4的合成方法与实施例1相同。
1、合成3-(溴甲基)-6-(2-(叔丁基二甲基硅氧基)乙基)-1,2,4,5-四嗪
将化合物4(800mg,2.963mmol,1.0eq.)置于50mL圆底烧瓶中,加入N-溴代丁二酰亚胺(NBS)(897mg,5.037mmol,1.7eq.)抽换氩气三次,并在氩气保护下加入二氯甲烷10mL,将三苯基膦溶于5mL二氯甲烷中,缓慢滴加进反应瓶中,加毕,常温搅拌30分钟,TLC监测原料消失后,加入水10mL淬灭反应,二氯甲烷(10mL×3)萃取,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色油状物化合物9。
1H NMR(400MHz,Chloroform-d)δ4.93(s,2H),4.23(t,J=6.3Hz,2H),3.56(t,J=6.2Hz,2H),0.77(s,9H),-0.03(s,6H).
13C NMR(101MHz,Chloroform-d)δ168.82,167.54,61.00,38.33,27.54,25.70,18.10,-5.51
2、合成化合物10
将喜树碱(80mg,0.2mmol,1.0eq.)和碳酸氢钾(60mg,0.3mmol,3.0eq.)加入到100mL圆底烧瓶中,加入约10mL丙酮后于常温预搅拌20分钟,将化合物9(75mg,0.225mmol,1.1eq.)溶于约3mL丙酮,滴加到反应瓶中,将18-冠醚-6(13.2mg,0.05mmol,0.25eq.)溶于约2mL丙酮,滴加至上述反应中,加毕,将反应瓶移至40℃油浴锅中搅拌2小时,TLC监测原料消失后,加入水约5mL淬灭反应,乙酸乙酯(10mL×3)萃取,有机相用饱和碳酸氢钠水溶液洗三次,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色固体粉末化合物10。
3、合成化合物11
将化合物10(50mg,0.078mmol,1.0eq.)置于50mL圆底烧瓶中,加入混合溶剂(四氢呋喃:甲醇=4:1)5mL溶解,滴加1mol/L盐酸(0.78mL,0.78mmol,10.0eq),常温搅拌20分钟,TLC监测原料消失后,加入5mL水,乙酸乙酯(10mL×3)萃取,旋干溶剂,经硅胶柱层析分离纯化后(石油醚:乙酸乙酯=4:1)得红色固体粉末化合物11。
4、合成化合物12
将化合物11(18.0mg,0.034mmol,1.0eq.)置于50mL圆底烧瓶中,加入约2mL四氢呋喃溶解,加入甲基磺酰氯(4ul,0.05mmol,1.5eq.)和三乙胺(TEA)(7.03ul,0.05mmol,1.5eq.)常温搅拌30分钟,TLC监测原料消失后,加三乙胺(7.03ul,0.05mmol,1.5eq.)后于室温反应,TLC监测反应结束后,加入约5mL水,二氯甲烷(10mL×3)萃取,有机相用水洗三次,无水硫酸钠干燥,旋干溶剂,经硅胶柱层析分离纯化后(石油醚∶乙酸乙酯=4∶1)得红色固体粉末化合物12。
1H NMR(400MHz,Chloroform-d)δ8.21–8.13(m,1H),7.63–7.54(m,3H),7.24–7.14(m,1H),7.09(dd,J=17.5,1.3Hz,1H),6.15(dd,J=10.6,1.3Hz,1H),5.86(s,2H),5.75(d,J=16.2Hz,1H),5.35–5.20(m,3H),3.77(s,1H),3.15(q,J=7.7Hz,2H),1.98–1.83(m,2H),1.38(t,J=7.6Hz,3H),1.03(t,J=7.3Hz,3H).
13C NMR(101MHz,Chloroform-d)δ174.01,165.12,164.98,157.71,157.01,150.36,150.23,147.21,145.90,144.07,132.62,130.07,129.42,127.97,127.45,122.36,118.06,103.82,97.58,72.79,68.14,66.39,49.45,31.60,23.21,13.68,7.84.
5、合成多肽偶联药物c(RGDyC)-Tz-CPT
1mM的多肽c(RGDyC)和1.1mM的化合物12在含有1mM的DIPEA的乙腈比二甲基亚砜体积比为3:2的溶液里常温反应2小时,得到多肽偶联药物c(RGDyC)-Tz-CPT,其HPLC-MS表征如图2所示。
三、细胞毒性测试实验
【实施例3】
c(RGDyC)-Tz-DOX细胞毒性实验
将U87、B16F10、SKOV3和LX2细胞分别以每孔1×105数量接种于96孔板中,培养24小时后除去培养基。前药组和母药组:将连续稀释不同浓度的c(RGDyC)-Tz-DOX和DOX添加到150μL细胞培养基中,加至孔板中与细胞于37℃,5%CO2的条件下孵育48小时后,移除含多肽培养基并用PBS洗涤3次,然后每孔添加150μL的含15μL CCK-8的细胞培养基,继续温育45分钟后用酶标仪测量每个样品在450nm处的OD值。未与PDC孵育的组被视为阳性对照,单纯的CCK-8溶液被用作阴性对照。
细胞存活率的计算公式为:
IC50指半数抑制浓度,可反应药物抑制细胞生长促进凋亡的细胞毒性,该值越小药效越好。
c(RGDyC)-Tz-DOX在肿瘤环境下与反式环辛烯发生生物正交反应释放带有阿霉素的反应式如下:
实验结果如图3所示,对于所有细胞,激活后PDC(Activated Dox)药效优于前药PDC(RGD-Dox);对于高表达αvβ3的细胞系U87、SKOV3、B16F10,激活PDC优于游离Dox(FreeDox);对于低表达αvβ3的正常细胞系LX2,激活PDC次于游离Dox。说明c(RGDyC)-Tz-DOX相比传统小分子药物而言能同时提高疗效和安全性。
【实施例4】
c(RGDyC)-Tz-CPT细胞毒性实验
将U87和SKOV3细胞分别以每孔1×105数量接种于96孔板中,培养24小时后除去培养基。对于前药和母药组的将连续稀释的不同浓度梯度的c(RGDyC)-Tz-CPT和CP添加到150μL细胞培养基中,加至孔板中与细胞于37℃,5%CO2的条件下孵育48小时后,移除含多肽培养基并用PBS洗涤3次,然后每孔添加150μL的含15μL CCK-8的细胞培养基,继续温育45分钟后用酶标仪测量每个样品在450nm处的OD值。未与PDC孵育的组被视为阳性对照,单纯的CCK-8溶液被用作阴性对照。
c(RGDyC)-Tz-CPT在肿瘤环境下与反式环辛烯发生生物正交反应释放带有阿霉素的反应式如下:
实验结果如图4所示,c(RGDyC)-Tz-CPT对高表达螯合素受体的细胞U87或SKOV3具有较高的毒性且优于母药CPT。
四、自组装测试实验
【实施例5】
本实施例用于c(RGDyC)-Tz-DOX、c(RGDyC)-Tz-CPT的自组装表征。
具体地,将多肽偶联药物溶于DMSO,一边超声一边缓慢加入纯水,使得最终DMSO:H2O的比例为2:98,PDC的最终浓度为50μM。c(RGDyC)-Tz-DOX和c(RGDyC)-Tz-CPT的粒径及电镜结果如图5所示。
从图中可以看出,两种PDC均自组装形成了纳米球,c(RGDyC)-Tz-DOX的水合粒径为293.9±2.86nm,c(RGDyC)-Tz-CPT的水合粒径为211±0.35nm。该纳米球结构不仅能够明显地增加疏水性药物的溶解性、提高细胞摄取,还能够增加细胞富集,进一步提高疗效。
五、GSH稳定性测试
【实施例5】
本实施例用于c(RGDyC)-Tz-DOX、c(RGDyC)-Tz-CPT的稳定性表征。
为了探索两种多肽偶联药物在体内还原性物质GSH(谷胱甘肽)的作用下是否仍能稳定存在,将50μM的PDC在高浓度还原性条件:10mM GSH,PBS(含10%DMSO),pH 7.4,37℃下孵育,HPLC-MS检测其520nm处的峰面积。实验结果如图6所示,表明72小时内两种PDC在高浓度还原性条件下均能保持80%以上的物质稳定存在。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.基于四嗪连接子的多肽药物偶联物,其特征在于,所述多肽药物偶联物的结构式为:P-L-D,其中,P为具有巯基的多肽,D为具有酚羟基或者氨基的药物,L为四嗪连接子,所述四嗪连接子的结构式为:所述四嗪连接子与所述多肽的巯基连接形成C-S键,四嗪连接子与药物的酚羟基连接形成酚醚键,或者四嗪连接子与药物的氨基连接形成氨基甲酸酯键;
所述多肽药物偶联物的制备方法包括以下步骤:
在四嗪连接子的3位或6位上形成乙烯基,之后在乙烯基的对位通过氨基甲酸酯键连接具有氨基的药物,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物;或
在四嗪连接子的3位或6位上通过酚醚键连接具有酚羟基的药物,之后在形成的酚醚键的对位形成乙烯基,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物。
2.根据权利要求1所述的基于四嗪连接子的多肽药物偶联物,其特征在于,所述多肽选自以下任一种:
3.根据权利要求1所述的基于四嗪连接子的多肽药物偶联物,其特征在于,所述药物选自以下任一种:
4.根据权利要求1~3中任一项所述的基于四嗪连接子的多肽药物偶联物,其特征在于,所述多肽药物偶联物的结构式为:
5.根据权利要求4所述的基于四嗪连接子的多肽药物偶联物,其特征在于,若干多肽药物偶联物之间通过非共价键连接、自组装形成纳米球。
6.根据权利要求5所述的基于四嗪连接子的多肽药物偶联物,其特征在于,所述纳米球的水合粒径为200~300nm。
7.如权利要求1~6中任一项所述的基于四嗪连接子的多肽药物偶联物的制备方法,其特征在于,包括以下步骤:
在四嗪连接子的3位或6位上形成乙烯基,之后在乙烯基的对位通过氨基甲酸酯键连接具有氨基的药物,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物;或
在四嗪连接子的3位或6位上通过酚醚键连接具有酚羟基的药物,之后在形成的酚醚键的对位形成乙烯基,最后四嗪连接子的乙烯基与多肽的巯基反应连接制得所述多肽药物偶联物。
8.根据权利要求7所述的基于四嗪连接子的多肽药物偶联物的制备方法,其特征在于,多肽和四嗪连接子在含有N,N-二异丙基乙胺的溶剂中常温反应制得多肽药物偶联物,所述溶剂包括乙腈和二甲基亚砜。
9.一种药物,其特征在于,包括权利要求1~6中任一项所述的基于四嗪连接子的多肽药物偶联物、以及药学上可接受的辅料。
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