CN116947963A - 一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs及其制备方法与应用 - Google Patents
一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs及其制备方法与应用,属于药物化学领域。该类化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对应异构体、水合物、或其药学上可接受的盐,具有如式Ⅰ或式Ⅱ所示的结构。本发明选用VHL的配体(S,R,S)‑AHPC作为PROTACs中与E3连接酶进行结合的部位,冠状病毒3CL蛋白酶抑制剂为靶向目标蛋白的小分子化合物,选用合适的连接链将两者相连接构建PROTACs。本发明克服了现有冠状病毒3CL蛋白酶抑制剂结构类型单一,发挥药效途径有限(仅具有抑制作用)等缺陷,对冠状病毒3CL蛋白酶具有较好的抑制和降解活性,可作为抗冠状病毒药物进行开发和研究。
Description
技术领域
本发明属于药物化学技术领域,具体涉及一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs及其制备方法与应用。
背景技术
新型冠状病毒肺炎具有高传染性和高致病性,已对人类健康、社会稳定和经济发展构成了严重威胁。冠状病毒中重要的非结构蛋白3CLpro(3C样蛋白酶,也称作主要蛋白酶Mpro),与小核糖核酸病毒的3C蛋白酶具有相似的底物特异性,在子代病毒的复制和转录过程中起到极其关键的作用。3CLpro具有三个结构域,结构域Ⅰ与Ⅱ主要由β折叠结构组成,结构域Ⅲ主要由α螺旋结构组成。活性位点位于结构域I和结构域II之间形成的凹槽中,结构域III参与二聚体的形成。相较于结构域III,不同的冠状病毒的结构域I和结构域II的序列保守性更高,这与其活性位点位于前两个结构域中相一致。3CLpro采用保守的半胱氨酸和组氨酸作为催化氨基酸,组成催化二联体,其中半胱氨酸为亲核攻击基团,组氨酸为碱性基团。冠状病毒3CLpro对病毒复制至关重要,而且保守性高,是理想的抗冠状病毒药物靶标。但是目前针对新型冠状病毒的药物仅仅对3CLpro有抑制作用,不具备降解功能。因此,寻求一种同时抑制和降解的针对新型冠状病毒3CLpro的药物迫在眉睫。
蛋白水解靶向嵌合分子(PROTACs)是近年来药物开发领域最具颠覆性的技术之一。PROTAC技术是利用泛素-蛋白酶体系统(UPS)来降解目标蛋白。PROTAC本质是一个双功能分子,其分子结构由靶蛋白配体部分、E3泛素连接酶部分以及将两部分连接起来的链状物组成。该分子的靶蛋白配体端连接靶蛋白,E3泛素连接酶配体端连接E3泛素连接酶,形成“靶蛋白-PROTAC-E3连接酶三元复合物”,驱动E3泛素连接酶复合物将泛素从泛素偶联酶(E2)转移至靶蛋白上暴露的赖氨酸,从而泛素化靶蛋白。靶蛋白被泛素化后可为蛋白酶体识别,催化降解成氨基酸与小肽。Von Hippel–Lindau(VHL)是E3泛素化连接酶中的一种,该基因于1990年代初在VHL疾病中被发现。VHL基因编码了两种VHL蛋白亚型:一种由213个氨基酸残基构成,约30kDa(pVHL30);一种由160个氨基酸残基构成,约19kDa(pVHL19),VHL的配体(S,R,S)-AHPC已成为构建PROTACs最常用的配体之一。(S,R,S)-AHPC(也称作MDK7526,VH032-NH2,VHL ligand 1)是一种VH032-based VHL ligand,可用于募集von Hippel-Lindau(VHL)蛋白。(S,R,S)-AHPC可能对androgen receptor的靶向降解有用。(S,R,S)-AHPC可以通过接头与蛋白质的配体例如BCR-ABL1连接,以形成PROTAC例如GMB-475。
利用PROTAC技术降解外源性蛋白(如病毒蛋白等)的研究目前处于起步阶段,丙型肝炎病毒(HCV)NS3/4A蛋白酶降解剂DGY-08-097,抗病毒活性和抗耐药性特征显著优于传统药物Telaprevir,证实了PROTACs可以降解病毒相关蛋白,PROTACs可能是抗病毒药物研发的新方向。
发明内容
为了克服上述现有技术的缺点,本发明的目的在于提供一种含VHL配体靶向冠状病毒3CL蛋白酶的PROTACs及其制备方法与应用,同时抑制和清除冠状病毒3CLpro,治疗冠状病毒所引起的疾病。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,为式Ⅰ或式Ⅱ所示化合物或其药学上可接受的盐,以及所述的式Ⅰ或式Ⅱ化合物或其药学上可接受的盐的溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,包括外消旋混合物;
所述式Ⅰ或式Ⅱ化合物的结构式为:
其中,n=1-6。
优选地,所述代表性化合物选自如下化合物:
优选地,所述药学上可接受的盐为靶向冠状病毒3CL蛋白酶的PROTACs与盐酸、氢溴酸、硫酸、硝酸、磷酸、醋酸、富马酸、马来酸、草酸、丙二酸、琥珀酸、柠檬酸、苹果酸、甲磺酸、乙磺酸、苯磺酸、甲苯磺酸、谷氨酸或天冬氨酸形成的盐。
本发明还公开了上述基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs的制备方法,包括以下操作步骤:
(1)以3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮为原料,与2,4,5-三氟溴苄进行烷基化反应,得到化合物a1;然后在三嗪母核6位处引入6-氯-2-甲基-2H-吲唑单元,得到化合物a2;化合物a2的三嗪母核3位叔丁基在酸性溶剂中脱掉得到化合物a3;随后在化合物a3的三嗪母核3位引入3-丙炔基,最终得到化合物a4;
其中,a1为a2为/>a3为a4为/>
(2)以(S,R,S)-AHPC为原料,与不同长度的溴代烷酸进行酸胺缩合反应,得到相应的化合物b1-b6,然后与叠氮化钠在碘化钾的催化下,得到相应的化合物c1-c6;
其中,b1-b6为n=1-6;c1-c6为n=1-6;
或者,以(S,R,S)-AHPC为原料,与叠氮-多聚乙二醇-乙酸类化合物进行酸胺缩合反应,得到相应的化合物d1-d3;
其中,d1-d3为n=1-6;
(3)化合物a4与化合物c1-c6反应,生成如式Ⅰ结构的靶向冠状病毒3CL蛋白酶的PROTACs;
或者,化合物a4与化合物d1-d3反应,生成如式Ⅱ结构的靶向冠状病毒3CL蛋白酶的PROTACs。
优选地,步骤(1)中,3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮与2,4,5-三氟溴苄的摩尔比为1:1.1,化合物a1与6-氯-2-甲基-2H-吲唑-5-胺的摩尔比为1:1.3,化合物a3与3-溴丙炔的摩尔比为1:1.2;步骤(2)中,(S,R,S)-AHPC与不同长度的溴代烷酸的摩尔比为1:1.2,(S,R,S)-AHPC与叠氮-多聚乙二醇-乙酸类化合物的摩尔比为1:2;步骤(3)中,化合物a4与c1-c6或d1-d3的摩尔比为1:1.2。
优选地,步骤(1)中,化合物a2的合成过程中反应温度为0℃,所用催化剂为双三甲基硅基胺基锂;步骤(2)中,化合物b1-b6的合成过程中,所用缩合剂为2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯,化合物d1-d3的合成过程中,所用缩合剂为氯化亚砜,所用溶剂为四氢呋喃;步骤(3)中所用溶剂为四氢呋喃和水的混合溶剂,混合溶剂中,四氢呋喃与水的体积比为10:1。
优选地,步骤(1)中,化合物a1的合成过程中所用的溶剂为乙腈,在碳酸钾和加热回流条件下反应;化合物a2的合成过程中所用溶剂为四氢呋喃;化合物a4的合成过程所用溶剂为DMF,反应温度为60℃;步骤(2)所述化合物b1-b6的合成过程中所用溶剂为DCM;化合物c1-c6的合成所用溶剂为DMF;步骤(3)中,催化剂为五水硫酸铜和抗坏血酸钠;反应条件为氩气保护下45℃反应。
本发明还公开了上述基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs在制备抗冠状病毒药物制剂中的应用。
优选地,所述冠状病毒为新型冠状病毒SARS-CoV-2。
优选地,所述制剂为单独使用或与其他抗冠状病毒药物联合使用,或与可药用的赋形剂、稀释剂混合制成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂、预混剂或微丸剂,或者制成非口服方式给药的吸入剂、注射剂或透皮制剂。
本发明还公开了一种药物组合物,所述药物组合物包含上述基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs作为活性成分。
与现有技术相比,本发明具有以下有益效果:
本发明提供的一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,选用VHL的配体(S,R,S)-AHPC为E3连接酶配体,通过不同种类、不同链长的linker将冠状病毒3CL蛋白酶抑制剂与E3连接酶偶联,成功制备得到了靶向冠状病毒3CL蛋白酶的PROTACs,能有效靶向于目标蛋白。3CLpro抑制活性测试结果表明,本发明合成的化合物对3CLpro有较强的抑制作用,优选化合物4、5、6、8和9对3CLpro的IC50值均在100nM以下;3CLpro降解活性实验结果表明,本发明合成的化合物对3CLpro均具有降解活性,优选化合物5、6和8的对于3CLpro的DC50值均在100nM以下。本发明提供的化合物克服了现有冠状病毒3CL蛋白酶抑制剂结构类型单一,发挥药效途径有限(仅具有抑制作用)等缺陷,对冠状病毒3CL蛋白酶具有较好的抑制和降解活性,可作为抗冠状病毒药物进行开发和研究。
本发明提供的一种含VHL配体靶向冠状病毒3CL蛋白酶的PROTACs的制备方法,该方法原料简单易得,没有使用高温高压和高毒的试剂,反应条件温和,使用设备常见,产率较高,易于工业化生产。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面对本发明做进一步详细描述:
本发明提供的一种含VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,为式Ⅰ或式Ⅱ所示化合物或其药学上可接受的盐,以及所述的式Ⅰ或式Ⅱ化合物或其药学上可接受的盐的溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,包括外消旋混合物;
所述式Ⅰ或式Ⅱ化合物的结构式为:
其中,n=1-6;
所述药学上可接受的盐为靶向冠状病毒3CL蛋白酶的PROTACs与盐酸、氢溴酸、硫酸、硝酸、磷酸、醋酸、富马酸、马来酸、草酸、丙二酸、琥珀酸、柠檬酸、苹果酸、甲磺酸、乙磺酸、苯磺酸、甲苯磺酸、谷氨酸或天冬氨酸形成的盐。
本发明提供的含VHL配体靶向冠状病毒3CL蛋白酶的PROTACs的制备方法如下:
(1)以3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮为原料,与2,4,5-三氟溴苄进行烷基化反应,得到化合物a1;然后在三嗪母核6位处引入6-氯-2-甲基-2H-吲唑单元,得到化合物a2;化合物a2的三嗪母核3位叔丁基在酸性溶剂中脱掉得到化合物a3;随后在化合物a3三嗪母核3位引入3-丙炔基,最终得到化合物a4,反应式如下:
其中,化合物a1的合成过程中所用的溶剂为乙腈,3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮与2,4,5-三氟溴苄的摩尔比为1:1.1,在碳酸钾和加热回流条件下反应;化合物a2的合成过程中,原料化合物a1与6-氯-2-甲基-2H-吲唑-5-胺的摩尔比为1:1.3,反应温度为0℃,所用溶剂为四氢呋喃,所用催化剂为双三甲基硅基胺基锂(LiHMDS);化合物a3的合成过程中所用的酸性溶剂为三氟乙酸(TFA);化合物a4的合成过程中,原料化合物a3与3-溴丙炔的摩尔比为1:1.2,所用溶剂为N,N-二甲基甲酰胺(DMF),反应温度为60℃;
(2)以(S,R,S)-AHPC(MDK7526,VH032-NH2,VHL LIGAND 1)(购于上海麦克林生化科技有限公司;CAS No.1448297-52-6)为原料,与不同长度的溴代烷酸进行酸胺缩合反应,得到相应的化合物b1-b6,然后与叠氮化钠在碘化钾的催化下,得到相应的化合物c1-c6;
或者,以(S,R,S)-AHPC(MDK7526,VH032-NH2,VHL LIGAND 1)为原料,与叠氮-多聚乙二醇-乙酸类化合物进行酸胺缩合反应,得到相应的化合物d1-d3;
其中,所述化合物b1-b6的合成过程中,(S,R,S)-AHPC(MDK7526,VH032-NH2,VHLLIGAND 1)与不同长度的溴代烷酸的摩尔比为1:1.2,不同长度的溴代烷酸用二氯甲烷(DCM)溶解,所用缩合剂为2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU);化合物c1-c6的合成所用溶剂为DMF,化合物b1-b6与叠氮化钠的摩尔比为1:3,所用催化剂为碘化钾;所述化合物d1-d3的合成过程中,(S,R,S)-AHPC(MDK7526,VH032-NH2,VHLLIGAND 1)与叠氮-多聚乙二醇-乙酸类化合物的摩尔比为1:2,所用缩合剂为氯化亚砜,所用溶剂为四氢呋喃;
(3)化合物a4与化合物c1-c6反应,生成如式Ⅰ结构的靶向冠状病毒3CL蛋白酶的PROTACs;
或者,化合物a4与化合物d1-d3反应,生成如式Ⅱ结构的靶向冠状病毒3CL蛋白酶的PROTACs,反应式如下:
其中,反应中化合物a4与c1-c6或d1-d3的摩尔比为1:1.2,反应所用溶剂为四氢呋喃和水的混合溶剂,体积比为四氢呋喃:水=10:1;催化剂为五水硫酸铜和抗坏血酸钠;反应条件为氩气保护下45℃反应。
1.合成化合物1-9的具体实施例
本发明代表性化合物结构式如下所示:
下面给出上述化合物合成的实施例。
实施例1
化合物1:(2S,4R)-1-((S)-2-(3-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)丙酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
(1)化合物a4的制备
步骤一:化合物a1的合成
将3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮(114.7mg,0.5mmol)、2,4,5-三氟溴苄(123.8mg,0.55mmol)和碳酸钾(79.6mg,0.48mmol)置于反应器中,使用10mL乙腈进行溶解,加热回流,搅拌反应3小时,TLC监测。反应结束后,将反应液减压浓缩除去溶剂,所得固体残留物经饱和氯化钠水溶液洗涤,乙酸乙酯萃取,收集有机相,柱层析分离纯化(正己烷:乙酸乙酯(V:V)为流动相=8:2),干燥得到a1 161.0mg,产率为86.24%。
步骤二:化合物a2的合成
将化合物a1(186.7mg,0.5mmol)和6-氯-2-甲基-2H-吲唑-5-胺(118.1mg,0.65mmol)置于反应器中,使用5mL四氢呋喃进行溶解,在0℃下向反应器中缓慢加入1mmol双三甲基硅基胺基锂(LiHMDS)(0.2mL,1mmol)的四氢呋喃溶液,搅拌反应3小时,TLC监测。反应结束后,冷却至室温,加入氯化铵水溶液进行淬灭,将反应液减压浓缩除去四氢呋喃,所得残留液经饱和氯化钠水溶液洗涤,乙酸乙酯萃取,收集有机相,柱层析分离纯化(二氯甲烷:甲醇(V:V)=10;1为流动相),干燥得到a2 62.2mg,产率为25.24%。
步骤三:化合物a3的合成
将得到的化合物a2(246.4mg,0.5mmol)置于反应器中,加入3mL三氟乙酸(TFA),室温搅拌过夜,然后与甲苯共沸减压浓缩除去溶剂,干燥得到a3 202.1mg,产率为92.52%。
步骤四:化合物a4的合成
将化合物a3(218.4mg,0.5mmol)、3-溴丙炔(43μL,0.6mmol)和碳酸钾(82.9mg,0.6mmol)置于反应器中,使用10mL N,N-二甲基甲酰胺进行溶解,60℃加热搅拌反应5小时,TLC监测。反应结束后,所得反应液经饱和氯化钠水溶液洗涤,乙酸乙酯萃取,收集有机相,柱层析分离纯化(正己烷:乙酸乙酯(V:V)=6:4为流动相),干燥得到化合物a4148.2mg,产率为62.43%。
(2)化合物c1的制备
步骤一:化合物b1的合成
将3-溴丙酸(91.8mg,0.6mmol)溶于3mL二氯甲烷后置于反应器中,向其中加入HATU(380.2mg,1mmol)和DIPEA(260μL,1.5mmol),室温下搅拌活化1小时后备用。将(S,R,S)-AHPC(MDK7526,VH032-NH2,VHL LIGAND 1)(215.3mg,0.5mmol)溶于3mL的二氯甲烷,滴加加入到上述反应器中,后在室温下搅拌反应5小时,TLC监测。反应结束后,将所得反应液减压浓缩除去二氯甲烷,所得固体残留物经饱和氯化钠水溶液洗涤,乙酸乙酯萃取,收集有机相,柱层析分离纯化b1 230.6mg(二氯甲烷:甲醇(V:V)=10:1为流动相),产率为81.55%。
步骤二:化合物c1的合成
将化合物b1(282.8mg,0.5mmol)、叠氮化钠(97.5mg,1.5mmol)和碘化钾(8.3mg,0.05mmol)置于反应器中,使用10mL N,N-二甲基甲酰胺进行溶解,70℃加热搅拌反应5小时,TLC监测。反应结束后,所得反应液经饱和氯化钠水溶液洗涤,乙酸乙酯萃取,收集有机相,干燥得到化合物c1 218.3mg,产率为82.74%。
(4)化合物1的制备
将化合物a4(142.4mg,0.3mmol)、化合物c1(190.0mg,0.36mmol)、五水硫酸铜(30.0mg,0.12mmol)和抗坏血酸钠(23.8mg,0.12mmol)置于反应器中,使用10mL四氢呋喃和1mL水的混合溶液进行溶解,氩气保护,45℃加热搅拌过夜,TLC监测。反应结束后,所得反应液减压浓缩除去溶剂,柱层析分离纯化(二氯甲烷:甲醇(V:V)=15:1为流动相),干燥得到化合物1 106.1mg,产率为35.27%。
1H NMR(400MHz,DMSO-d6)δ10.55(s,1H),9.17(s,1H),9.02(s,1H),8.59(t,J=8.2Hz,1H),8.41(s,1H),7.89(d,J=9.0Hz,1H),7.73(s,1H),7.69-7.58(m,2H),7.50–7.40(m,4H),7.32(m,1H),5.24(s,2H),5.13(s,2H),5.06(s,1H),4.62(d,J=8.8Hz,1H),4.54–4.43(m,2H),4.36(s,1H),4.23(t,J=6.5Hz,2H),4.15(dd,J=15.6,5.6Hz,1H),4.08(s,3H),3.41–3.30(m,2H),2.73-2.62(m,2H),2.54(s,3H),1.92–1.79(m,2H),0.98(s,9H)
13C NMR(101MHz,DMSO-d6)δ178.88,175.24,164.81,156.92,155.33,153.89,151.38,150.91,150.52,149.76,148.14,147.87,145.78,144.45,142.15,136.67,133.28,129.44,128.61,126.99,125.52,122.12,121.03,117.89,116.56,116.04,114.89,107.54,72.33,66.80,57.05,53.78,51.62,49.37,44.24,41.87,37.65,34.82,31.94,30.93,27.93,16.12.
实施例2
化合物2:(2S,4R)-1-((S)-2-(4-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)丁酰胺基)-3,3-二甲基丁酰)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法参照实施例1,只需更换相应的原料即可。所得化合物2的产率:32.62%。
1H NMR(400MHz,DMSO-d6)δ10.58(s,1H),9.21(s,1H),9.00(s,1H),8.61(t,J=8.4Hz,1H),8.39(s,1H),7.92(d,J=9.3Hz,1H),7.75(s,1H),7.71-7.60(m,2H),7.52–7.40(m,4H),7.35(m,1H),5.28(s,2H),5.17(s,2H),5.08(s,1H),4.58(d,J=9.0Hz,1H),4.57–4.45(m,2H),4.42(s,1H),4.38(t,J=6.5Hz,2H),4.26(dd,J=15.6,5.6Hz,1H),4.20(s,3H),3.42–3.30(m,2H),2.58(s,3H),2.34-2.23(m,2H),2.10–1.99(m,2H),1.89–1.77(m,2H),1.01(s,9H)
13C NMR(101MHz,DMSO-d6)δ179.04,176.32,165.56,156.02,155.23,154.33,151.27,150.89,150.43,149.46,148.32,147.12,145.89,144.62,141.11,136.48,133.12,129.24,128.58,126.71,126.57,121.34,120.89,117.65,116.62,116.21,114.63,106.43,71.34,68.69,56.71,54.38,52.59,48.34,42.31,40.65,38.45,35.78,32.54,31.73,26.23,24.22,16.01.
实施例3
化合物3:(2S,4R)-1-((S)-2-(5-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)戊酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例1,只需更换相应的原料即可。所得化合物3的产率:28.28%。
1H NMR(400MHz,DMSO-d6)δ10.65(s,1H),9.25(s,1H),9.02(s,1H),8.58(t,J=8.5Hz,1H),8.41(s,1H),7.89(d,J=9.4Hz,1H),7.72(s,1H),7.69-7.58(m,2H),7.50–7.39(m,4H),7.33(m,1H),5.32(s,2H),5.19(s,2H),5.12(s,1H),4.62(d,J=9.2Hz,1H),4.54–4.42(m,2H),4.38(s,1H),4.30(t,J=6.8Hz,2H),4.24(dd,J=15.8,6.0Hz,1H),4.12(s,3H),3.38–3.27(m,2H),2.65(s,3H),2.48-2.38(m,2H),2.06–1.94(m,2H),1.90–1.79(m,2H),1.71-1.60(m,2H),1.02(s,9H)
13C NMR(101MHz,DMSO-d6)δ178.89,175.17,164.45,155.92,156.23,154.23,151.12,150.62,150.37,149.83,148.12,146.85,145.91,144.64,141.06,136.45,133.02,129.14,128.55,126.68,126.45,121.24,120.71,117.45,116.74,116.31,114.58,106.23,72.27,69.35,57.76,55.57,51.73,45.52,41.45,40.38,39.02,36.45,34.74,29.23,27.21,25.89,23.72,15.44.
实施例4
化合物4:(2S,4R)-1-((S)-2-(6-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)己酰胺基)-3,3-二甲基丁酰)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例1,只需更换相应的原料即可。所得化合物4的产率:24.29%。
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.19(s,1H),9.03(s,1H),8.60(t,J=8.6Hz,1H),8.39(s,1H),7.91(d,J=9.8Hz,1H),7.76(s,1H),7.71-7.60(m,2H),7.52–7.41(m,4H),7.37(m,1H),5.29(s,2H),5.15(s,2H),5.13(s,1H),4.62(d,J=9.4Hz,1H),4.58–4.46(m,2H),4.42(s,1H),4.34(t,J=7.2Hz,2H),4.28(dd,J=16.0,6.1Hz,1H),4.16(s,3H),3.42–3.30(m,2H),2.67(s,3H),2.52-2.38(m,2H),1.98–1.87(m,2H),1.85–1.71(m,4H),1.54-1.42(m,2H),0.96(s,9H)
13C NMR(101MHz,DMSO-d6)δ179.23,175.22,164.33,155.90,156.12,154.12,150.98,150.54,150.48,149.78,148.04,146.76,145.89,144.56,141.12,136.27,132.89,128.97,128.45,126.62,126.37,121.13,120.68,117.34,116.67,116.24,114.45,106.31,72.18,68.45,58.36,57.27,53.83,44.36,40.71,40.44,38.29,36.19,35.64,28.91,28.18,27.62,26.25,23.42,16.38.
实施例5
化合物5:(2S,4R)-1-((S)-2-(7-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)庚酰胺基)-3,3-二甲基丁酰)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例1,只需更换相应的原料即可。所得化合物5的产率:20.97%。
1H NMR(400MHz,DMSO-d6)δ10.58(s,1H),9.27(s,1H),9.00(s,1H),8.58(t,J=8.3Hz,1H),8.39(s,1H),7.92(d,J=9.4Hz,1H),7.76(s,1H),7.65-7.53(m,2H),7.50–7.39(m,4H),7.35(m,1H),5.29(s,2H),5.12(s,2H),5.11(s,1H),4.62(d,J=10.2Hz,1H),4.51–4.40(m,2H),4.37(s,1H),4.34(t,J=6.6Hz,2H),4.22(dd,J=16.0,5.8Hz,1H),4.09(s,3H),3.27–3.15(m,2H),2.52(s,3H),2.44-2.32(m,2H),1.93–1.81(m,2H),1.78–1.66(m,4H),1.44(s,4H),0.98(s,9H)
13C NMR(101MHz,DMSO-d6)δ177.92,173.64,164.12,155.94,155.82,153.43,151.29,150.71,150.78,148.90,148.11,146.78,146.25,144.43,141.21,136.27,132.76,129.34,128.46,126.62,126.38,121.44,120.98,117.27,117.08,116.63,114.34,106.42,72.26,68.56,58.21,57.36,52.83,44.42,40.71,40.34,38.28,36.19,35.72,33.26,28.93,28.17,27.63,26.12,25.13,16.32.
实施例6
化合物6:(2S,4R)-1-((S)-2-(8-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)辛酰胺基)-3,3-二甲基丁酰)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例1,只需更换相应的原料即可。所得化合物6的产率:17.83%。
1H NMR(400MHz,DMSO-d6)δ10.54(s,1H),9.22(s,1H),9.00(s,1H),8.54(t,J=8.2Hz,1H),8.41(s,1H),7.87(d,J=9.5Hz,1H),7.74(s,1H),7.67-7.54(m,2H),7.48–7.38(m,4H),7.33(m,1H),5.31(s,2H),5.14(s,2H),5.10(s,1H),4.57(d,J=9.5Hz,1H),4.53–4.41(m,2H),4.39(s,1H),4.30(t,J=6.7Hz,2H),4.25(dd,J=15.9,5.6Hz,1H),4.12(s,3H),3.23–3.11(m,2H),2.47(s,3H),2.32-2.21(m,2H),1.89–1.75(m,2H),1.72–1.57(m,4H),1.36(s,6H),0.97(s,9H)
13C NMR(101MHz,DMSO-d6)δ178.21,173.24,165.35,156.20,155.78,153.42,151.27,150.67,150.53,149.13,147.68,146.42,146.03,144.23,140.81,136.27,132.56,129.28,128.44,126.58,126.37,120.98,120.70,117.22,116.89,116.49,114.21,106.21,72.19,68.45,58.36,57.25,52.48,44.33,40.67,40.20,38.14,36.13,35.68,34.91,33.28,28.92,28.14,27.66,25.90,24.91,15.48.
实施例7
化合物7:(2S,4R)-1-((S)-2-(2-(2-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)乙氧基)乙酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
(1)化合物a4的制备
制备方法同实施例1。
(2)化合物d1的制备
将2-(2-叠氮基乙氧基)乙酸(290.2mg,2mmol)溶解于5mL氯化亚砜中,加热回流2小时,反应结束后,减压浓缩去除溶剂,随后加入(S,R,S)-AHPC(MDK7526,VH032-NH2,VHLLIGAND 1)(1mmol,430.6mg),10mL四氢呋喃作溶剂,加热回流反应5小时,TLC监测。反应结束,冷却至室温,加入2mL甲醇后继续搅拌1小时。减压浓缩除去溶剂,柱层析分离得到d1407.0mg(洗脱剂为二氯甲烷:甲醇(V:V)=10:1),产率为72.98%。
(3)化合物7的制备
将化合物a4(142.4mg,0.3mmol)、化合物d1(200.7mg,0.36mmol)、五水硫酸铜(30.0mg,0.12mmol)和抗坏血酸钠(23.8mg,0.12mmol)置于反应器中,使用10mL四氢呋喃和1mL水的混合溶液进行溶解,氩气保护,45℃加热搅拌过夜,TLC监测。反应结束后,所得反应液减压浓缩除去溶剂,柱层析分离纯化(二氯甲烷:甲醇(V:V)=20:1为流动相),干燥得到化合物7 82.5mg,产率为26.63%。
1H NMR(400MHz,DMSO-d6)δ10.44(s,1H),9.15(s,1H),8.92(s,1H),8.51(t,J=8.0Hz,1H),8.28(s,1H),7.87(d,J=9.2Hz,1H),7.71(s,1H),7.65-7.54(m,2H),7.48–7.38(m,4H),7.29(m,1H),5.17(s,2H),5.13(s,2H),5.05(s,1H),4.62(d,J=9.0Hz,1H),4.54–4.43(m,2H),4.38(s,1H),4.30(t,J=6.6Hz,2H),4.24(dd,J=15.6,5.6Hz,1H),4.16(s,3H),4.02-3.89(m,2H),3.58(s,2H),3.38–3.26(m,2H),2.61(s,3H),1.94–1.83(m,2H),0.95(s,9H)
13C NMR(101MHz,DMSO-d6)δ178.99,175.44,164.23,156.18,155.55,154.23,151.17,150.78,150.22,149.58,148.67,147.55,145.78,144.34,142.33,135.52,132.14,129.58,128.76,126.90,125.67,122.12,121.15,117.45,116.56,116.33,114.56,108.58,72.41,71.12,69.16,67.32,57.51,55.42,52.59,47.24,44.61,42.47,39.48,36.12,32.84,27.45,16,23.
实施例8
化合物8:(2S,4R)-1-((S)-2-(2-(2-(2-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)乙氧基)乙氧基)乙酰胺基)-3,3-二甲基丁酰基)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例7,只需更换相应的原料即可。所得化合物8的产率:21.89%。
1H NMR(400MHz,DMSO-d6)δ10.52(s,1H),9.18(s,1H),8.95(s,1H),8.50(t,J=8.2Hz,1H),8.32(s,1H),7.91(d,J=9.2Hz,1H),7.73(s,1H),7.66-7.56(m,2H),7.52–7.40(m,4H),7.31(m,1H),5.21(s,2H),5.10(s,2H),5.03(s,1H),4.58(d,J=9.2Hz,1H),4.50–4.41(m,2H),4.34(s,1H),4.28(t,J=6.6Hz,2H),4.22(dd,J=15.8,5.6Hz,1H),4.17(s,3H),4.09-3.98(m,2H),3.63(s,2H),3.45(s,4H),3.36–3.24(m,2H),2.59(s,3H),1.97–1.87(m,2H),0.98(s,9H)
13C NMR(101MHz,DMSO-d6)δ179.07,175.78,166.45,156.56,155.24,154.76,152.27,151.16,150.78,149.35,148.76,147.63,146.32,144.46,142.89,136.91,131.36,129.07,127.86,126.53,125.72,122.45,121.63,117.83,116.95,116.35,114.67,109.45,73.78,71.48,70.23,68.71,68.32,66.62,58.31,55.34,51.60,48.64,45.33,43.28,39.79,36.55,33.21,27.28,15,90.
实施例9
化合物9:(2S,4R)-1-((S)-2-(叔丁基)-14-(4-(((E)-4-((6-氯-2-甲基-2H-吲唑-5-基)亚氨基)-2,6-二氧基-3-(2,4,5-三氟苄基)-1,3,5-三嗪-1-基)甲基)-1H-1,2,3-三唑-1-基)-4-氧代-6,9,12-三唑-3-氮杂十四烷基)-4-羟基-N-(4-(4-甲基噻唑-5-基)苄基)吡咯烷-2-甲酰胺的制备
制备方法同实施例7,只需更换相应的原料即可。所得化合物9的产率:17.42%。
1H NMR(400MHz,DMSO-d6)δ10.56(s,1H),9.22(s,1H),9.01(s,1H),8.56(t,J=8.4Hz,1H),8.36(s,1H),7.89(d,J=9.0Hz,1H),7.72(s,1H),7.64-7.54(m,2H),7.48–7.36(m,4H),7.28(m,1H),5.28(s,2H),5.15(s,2H),5.09(s,1H),4.62(d,J=9.4Hz,1H),4.52–4.40(m,2H),4.32(s,1H),4.26(t,J=6.5Hz,2H),4.20(dd,J=16.0,6.2Hz,1H),4.14(s,3H),4.06-3.95(m,2H),3.67(s,2H),3.52(s,8H),3.38–3.30(m,2H),2.62(s,3H),1.94–1.83(m,2H),0.96(s,9H)
13C NMR(101MHz,DMSO-d6)δ178.77,176.23,167.41,156.78,155.45,154.32,153.03,152.36,151.90,149.83,148.19,147.45,146.90,145.42,143.56,138.26,132.34,129.56,126.90,126.13,125.48,123.75,122.83,118.81,116.67,116.05,113.89,110.42,74.28,72.04,71.67,70.45,69.67,68.85,68.42,66.34,59.68,56.90,52.67,49.54,46.54,44.12,39.77,35.53,32.81,26.88,15,78.
2.生物活性测定
(1)3CLpro抑制活性测试
使用荧光共振能量转移技术测定化合物对SARS-CoV-2 3CLpro的抑制活性。
将10μL不同浓度的制备的上述化合物溶液(终浓度为1000,500,250,125,62.5,31.25,15.63,7.81,3.90,1.95nM,DMSO配制)和40μL SARS-CoV-2 3CLpro(购于上海碧云天生物技术有限公司,终浓度为0.5μM,Tris-HCl缓冲液稀释(20mM Tris-HCl,100mM NaCl,1mM EDTA,pH值7.4))混合,加入黑色96孔板中,在37℃下孵育10min。加入50μL荧光底物Dabcyl-KTSAVLQSGFRKME-Edans(购于上海碧云天生物技术有限公司,终浓度为20μM)引发反应,孵育10min后在多功能酶标仪(赛默飞世尔科技有限公司,Varioskan Flash)上进行荧光检测,激发波长为340nm,发射波长为490nm,记录荧光值计算样品的抑制百分率。以不含化合物的DMSO为酶活性对照,不含SARS-CoV-2 3CLpro的Tris-HCl缓冲液为空白对照,其余处理方法相同。使用GraphPad Prism软件进行非线性回归分析计算得出样品(本发明合成的化合物1-9)的IC50值。
实验结果如表1所示(表1中,IC50所在列,A:IC50<100nM,B:IC50=100-1000nM),实施例化合物对3CLpro均具有抑制活性,其中化合物4、5、6、8和9对3CLpro的抑制作用较强,IC50值均在100nM以下。
(2)Western Blot测定3CLpro降解活性
将对数生长期的HEK293E细胞(购于中国科学院细胞库)以6.0×105细胞/孔密度接种于6孔板中,在含5%CO2的37℃温箱中孵育8-24h,待细胞长至密度为70%融合时,换入2mL预热的无血清培养基(购于上海奥浦迈生物科技股份有限公司)。用10μM PEI(聚乙烯亚胺,购于上海麦克林生化科技有限公司)溶液按质量体积比为3:4转染SARS-CoV-23CLpro表达质粒(2μg/孔,1×HBS配制,购于北京义翘神州科技股份有限公司),将PEI-质粒混合液逐滴加入上述无血清培养基中,轻摇混匀,置于含5%CO2的37℃温箱中孵育10h,后除去含转染试剂的培养基,并加入含有梯度浓度待测样品的培养基(样品终浓度为1000,500,250,125,62.5,31.25,15.63,7.81,3.90,1.95nM)。37℃、5%CO2条件下培养24h后弃去上清,收集细胞,加入RIPA细胞裂解液(购于上海麦克林生化科技有限公司)冰上裂解细胞30min,用Western Blot检测3CLpro表达,Image J分析3CLpro相对表达量,计算蛋白降解率。以不含待测样品的培养基为对照组,其余处理方法相同。使用GraphPad Prism软件进行非线性回归分析计算得出样品的蛋白降解活性(DC50)。
实验结果如表1所示(表1中,DC50所在列,A:DC50<100nM,B:DC50=100-1000nM),实施例化合物对3CLpro均具有降解活性,其中化合物5、6和8的对于3CLpro的降解活性较强,DC50值均在100nM以下。
表1化合物1-9对3CLpro的抑制活性和降解活性
表1数据显示,化合物1-9对3CLpro均具有不同程度抑制作用和降解作用。其中化合物5、6和8对3CLpro的IC50值和DC50值均小于100nM。表明本发明中靶向冠状病毒3CL蛋白酶的PROTACs对3CLpro既具有的抑制活性,又具有良好的降解活性,可作为抗冠状病毒药物进行开发和研究。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
Claims (10)
1.一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,其特征在于,为式Ⅰ或式Ⅱ所示化合物或其药学上可接受的盐,以及所述的式Ⅰ或式Ⅱ化合物或其药学上可接受的盐的溶剂化合物、对映异构体、非对映异构体、互变异构体或其任意比例的混合物,包括外消旋混合物;
所述式Ⅰ或式Ⅱ化合物的结构式为:
其中,n=1-6。
2.根据权利要求1所述的一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,其特征在于,所述代表性化合物选自如下化合物:
3.根据权利要求1所述的一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs,其特征在于,所述药学上可接受的盐为靶向冠状病毒3CL蛋白酶的PROTACs与盐酸、氢溴酸、硫酸、硝酸、磷酸、醋酸、富马酸、马来酸、草酸、丙二酸、琥珀酸、柠檬酸、苹果酸、甲磺酸、乙磺酸、苯磺酸、甲苯磺酸、谷氨酸或天冬氨酸形成的盐。
4.权利要求1-3任意一项所述的一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs的制备方法,其特征在于,包括以下操作步骤:
(1)以3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮为原料,与2,4,5-三氟溴苄进行烷基化反应,得到化合物a1;然后在三嗪母核6位处引入6-氯-2-甲基-2H-吲唑单元,得到化合物a2;化合物a2的三嗪母核3位叔丁基在酸性溶剂中脱掉得到化合物a3;随后在化合物a3的三嗪母核3位引入3-丙炔基,最终得到化合物a4;
其中,a1为a4为/>
(2)以(S,R,S)-AHPC为原料,与不同长度的溴代烷酸进行酸胺缩合反应,得到相应的化合物b1-b6,然后与叠氮化钠在碘化钾的催化下,得到相应的化合物c1-c6;
其中,b1-b6为n=1-6;c1-c6为n=1-6;
或者,以(S,R,S)-AHPC为原料,与叠氮-多聚乙二醇-乙酸类化合物进行酸胺缩合反应,得到相应的化合物d1-d3;
其中,d1-d3为n=1-6;
(3)化合物a4与化合物c1-c6反应,生成如式Ⅰ结构的靶向冠状病毒3CL蛋白酶的PROTACs;
或者,化合物a4与化合物d1-d3反应,生成如式Ⅱ结构的靶向冠状病毒3CL蛋白酶的PROTACs。
5.根据权利要求4所述的方法,其特征在于,步骤(1)中,3-叔丁基-6-(乙硫基)-1,3,5-三嗪-2,4(1H,3H)-二酮与2,4,5-三氟溴苄的摩尔比为1:1.1,化合物a1与6-氯-2-甲基-2H-吲唑-5-胺的摩尔比为1:1.3,化合物a3与3-溴丙炔的摩尔比为1:1.2;步骤(2)中,(S,R,S)-AHPC与不同长度的溴代烷酸的摩尔比为1:1.2,(S,R,S)-AHPC与叠氮-多聚乙二醇-乙酸类化合物的摩尔比为1:2;步骤(3)中,化合物a4与c1-c6或d1-d3的摩尔比为1:1.2。
6.根据权利要求4所述的方法,其特征在于,步骤(1)中,化合物a2的合成过程中反应温度为0℃,所用催化剂为双三甲基硅基胺基锂;步骤(2)中,化合物b1-b6的合成过程中,所用缩合剂为2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯,化合物d1-d3的合成过程中,所用缩合剂为氯化亚砜,所用溶剂为四氢呋喃;步骤(3)中所用溶剂为四氢呋喃和水的混合溶剂,混合溶剂中,四氢呋喃与水的体积比为10:1。
7.权利要求1-3中任意一项所述的一种基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs在制备抗冠状病毒药物制剂中的应用。
8.根据权利要求7所述的应用,其特征在于,所述冠状病毒为新型冠状病毒SARS-CoV-2。
9.根据权利要求7所述的应用,其特征在于,所述制剂为单独使用或与其他抗冠状病毒药物联合使用,或与可药用的赋形剂、稀释剂混合制成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂、预混剂或微丸剂,或者制成非口服方式给药的吸入剂、注射剂或透皮制剂。
10.一种药物组合物,其特征在于,所述药物组合物包含权利要求1-3任意一项所述的基于VHL配体靶向冠状病毒3CL蛋白酶的PROTACs作为活性成分。
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