CN105636612B - 抗体-药物缀合物及使用和治疗方法 - Google Patents
抗体-药物缀合物及使用和治疗方法 Download PDFInfo
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- CN105636612B CN105636612B CN201480055557.3A CN201480055557A CN105636612B CN 105636612 B CN105636612 B CN 105636612B CN 201480055557 A CN201480055557 A CN 201480055557A CN 105636612 B CN105636612 B CN 105636612B
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Abstract
本发明提供抗体‑药物缀合物及使用抗体‑药物缀合物的方法,该抗体‑药物缀合物包含经接头缀合至1‑(氯甲基)‑2,3‑二氢‑1H‑苯并[e]吲哚(CBI)二聚体药物模块的抗体。
Description
对相关申请的交叉引用
依据37CFR§1.53(b)提交的此非临时申请依据35USC§119(e)要求2013 年8月12日提交的流水号61/864,889的美国临时申请,2013年12月16日提交的流水号61/916,388的美国临时申请,和2014年3月24日提交的流水号 61/969,499的美国临时申请的权益,据此通过援引完整收录它们全部。
发明领域
一般而言,本发明涉及缀合至1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚(CBI) 二聚体药物模块以形成具有治疗或诊断应用的抗体-药物缀合物的抗体。可以用对于与CBI二聚体药物-接头中间体缀合而言反应性的游离半胱氨酸氨基酸工程化改造抗体。本发明还涉及使用CBI二聚体抗体-药物缀合物化合物进行哺乳动物细胞或相关病理疾患的体外,原位,和体内诊断或治疗的方法。
发明背景
抗体-药物缀合物(ADC)是靶向性化疗分子,组合抗体和细胞毒性药物二者的特性,这通过将有力的细胞毒性药物靶向表达抗原的肿瘤细胞,内在化,并释放药物,由此增强它们的抗肿瘤活性来实现(Carter,P.and Senter, P.(2008)The Cancer Jour.14(3):154-169)。针对给定靶抗原的成功ADC开发依赖于抗体选择的优化,接头设计和稳定性,细胞毒性药物效力和药物和接头与抗体缀合的模式(Polakis,P.(2005)Current Opinion inPharmacology 5:382-387)。
5-氨基-1-(氯甲基)-1,2-二氢-3H-苯并[e]吲哚(氨基CBI)类的DNA小沟烷化剂是有力的细胞毒素(Atwell et al.(1999)J.Med.Chem.,42:3400),而且已经作为效应器单元用于为癌症疗法设计的多类前体药物。这些包括抗体缀合物(Jeffrey et al.(2005)J.Med.Chem.,48:1344),基于氨基甲酸硝基苄酯的用于基因疗法的前体药物(Hay et al.(2003)J.Med.Chem.46:2456)和作为缺氧活化的前体药物的相应硝基CBI衍生物(Tercelet al.(2011)Angew. Chem.,Int.Ed.,50:2606-2609)。已经通过烷基链将CBI和吡咯并[2,1-c][1,4] 苯并二氮杂卓(PBD)药效团连接到一起(Tercel et al.(2003)J.Med.Chem 46:2132-2151)。PBD二聚体(其中通过亚烷基或亚烷基-亚芳基链拴系两个吡咯并[2,1-c][1,4]苯并二氮杂卓单元)是高度有效的链间交联剂,与DNA小沟中的鸟嘌呤反应(Rahman etal.(2009)Jour.Amer.Chem.Soc. 131(38):13756-13766;Thurston et al.(1994)Chem.Rev.,94:433-465;Bose et al. (1992)J.Am.Chem.Soc.114:4939-4941;Gregson etal.(2004)Jour.Med. Chem.47(5):1161-1174;US 7511032;US 7528126;US 7557099;US7049311; US 7067511;US 7265105),而且具有针对革兰氏阳性细菌(Doyle et al.(2009)Jour.Antimicrob.Chemo.65(5):949-959;Hadjivassileva et al.(2005)Jour.Antimicrob.Chemo.56(3):513-518),人B细胞慢性淋巴细胞性白血病(CLL) 细胞(Pepperet al.(2004)Cancer Res.64(18):6750-6755),和实体瘤 (Hochhauser et al.(2009)Clin.Cancer Res.15(6):2140-2147;Alley et al.(2004) 64(18):6700-6706;Hartleyet al.(2004)Cancer Res.64(18):6693-6699)的活性。已经将二聚体形式的PBD连接至抗体以形成ADC(US 2009/304710;US 2010/047257;US 2009/036431;WO 2011/130598;WO2011/130616;US 2013/0028919)。
发明概述
本发明包括通过接头共价附着以形成具有治疗或诊断应用的抗体-药物缀合物(ADC)化合物的1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚(CBI)二聚体药物模块。
本发明的一个方面是具有下式的抗体-药物缀合物化合物:
Ab-(L-D)p,
其中:
Ab为抗体;
L为具有下式的接头:
-Str-(Pep)m-(Sp)n-,
其中Str为共价附着至抗体的延伸物单元,Pep为任选的2-12个氨基酸残基的肽单元,Sp为任选的共价附着至二聚体药物模块的间隔物单元,且m和n独立选自0和1;
p为1至8的整数;
D为具有下式的二聚体药物模块:
其中
R1选自H,P(O)3H2,C(O)NRaRb,或与L的键;
R2选自H,P(O)3H2,C(O)NRaRb,或与L的键;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D′为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,或与L的键,其中R为C1-C6烷基或苄基;且
R5为H或C1-C6烷基。
本发明的一个方面是抗体-药物缀合物化合物和药学可接受载剂,助流剂,稀释剂,或赋形剂的药物组合物。
本发明的一个方面是治疗癌症的方法,其包括对患者施用治疗有效量的抗体-药物缀合物化合物。
本发明的一个方面是用于治疗癌症的试剂盒,其包含:
a)药物组合物;和
b)使用说明书。
本发明的一个方面是选自下述的接头-药物中间体:
X-L-D,
其中:
X为选自马来酰亚胺,硫醇,氨基,溴化物,溴乙酰胺基,碘乙酰胺基,对甲苯磺酸根,碘化物,羟基,羧基,吡啶基二硫化物,和N-羟基琥珀酰亚胺的反应性官能团;
L为具有下式的接头:
-Str-(Pep)m-(Sp)n-,
其中Str为共价附着至X的延伸物单元,Pep为任选的2-12个氨基酸残基的肽单元,Sp为任选的共价附着至二聚体药物模块的间隔物单元,且m和n独立选自0和1;
D为具有下式的二聚体药物模块:
其中
R1选自H,P(O)3H2,C(O)NRaRb,或与L的键;
R2选自H,P(O)3H2,C(O)NRaRb,或与L的键;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D′为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,或与L的键,其中R为C1-C6烷基或苄基;且
R5为H或C1-C6烷基。
本发明的一个方面是通过将抗体缀合至接头-药物中间体来生成抗体-药物缀合物的方法。
本发明的一个方面是具有下式的CBI二聚体药物模块化合物:
其中
R1选自H,P(O)3H2,C(O)NRaRb;
R2选自H,P(O)3H2,C(O)NRaRb;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D'为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,其中R为C1-C6烷基或苄基;且
R5为H或C1-C6烷基。
附图简述
图1显示自1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a 合成2-(2-溴-N-甲基乙酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3- 二氢-1H-苯并[e]吲哚-5-基酯51。
图2显示自1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯53a合成N-((R)-1-(氯甲基)-3-(5-((R)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基)-6-(2,5-二氧 -2,5-二氢-1H-吡咯-1-基)己酰胺53。
图3显示自(S)-5-(5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸57c合成1-((S)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)- 基)-5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮53j。
图4显示自1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯 53k合成非天然对映异构体,1-((R)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)- 基)-5-((R)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮53p。
图5显示自(S)-(2-氨基-4-羟基-5-甲氧基苯基)(2-(羟基甲基)吡咯烷-1-基) 甲酮54a合成N-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧-2,3,5,11a-四氢 -1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e] 吲哚-5-基氧)甲基)苯基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺54。
图6显示自1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a 合成N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并 [e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H- 吡咯-1-基)己酰胺55。
图7显示自8-(6-((S)-1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并 [1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯54g合成 8-(6-((S)-5-(4-((S)-2-((S)-2-氨基-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5- 氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯56d。
图8显示自8-(6-((S)-5-(4-((S)-2-((S)-2-氨基-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7- 甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)- 羧酸(S)-叔丁酯56d合成N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧-2,3,5,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺56。
图9显示自1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a 合成(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)-基)-5- 氧戊酸57d。
图10显示自6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸合成2-(6-(2,5-二氧 -2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57i。
图11显示自2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57i合成 2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸 (S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)- 基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57。
图12显示自1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯 51a合成4-甲基哌嗪-1-羧酸(S)-3-(5-((S)-5-(4-氨基苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58e。
图13显示自4-甲基哌嗪-1-羧酸(S)-3-(5-((S)-5-(4-氨基苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e] 吲哚-5-基酯58e合成4-甲基哌嗪-1-羧酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58。
图14显示自5-(4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)- 叔丁酯55e合成二氢磷酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯59。
图15显示自5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯 61a合成(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)乙酯61。
图16显示自51a合成(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)丙酯62。
图17显示自6-(5-(叔丁氧羰基氨基)-4-(2-(羟基甲基)吡咯烷-1-羰基)-2-甲氧基苯氧基)己酸(S)-2,2,2-三氯乙酯54c合成4-甲基哌嗪-1-羧酸 (S)-3-(6-(4-((S)-2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-氨基-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯65d。
图18显示自(S)-2-(烯丙基氧羰基氨基)-6-(叔丁氧羰基氨基)己酸65e合成苯甲醇赖氨酸65g。
图19显示自4-甲基哌嗪-1-羧酸(S)-3-(6-(4-((S)-2-(乙酰氧基甲基)吡咯烷 -1-羰基)-5-氨基-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯65d合成8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e] 吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并 [e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-4-((S)-2-((S)-2-(((9H-芴-9-基) 甲氧基)羰基氨基)-3-甲基丁酰胺基)-6-(叔丁氧羰基氨基)己酰胺基)苄酯 65k。
图20显示自65k合成8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H- 苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-4-((S)-6-氨基 -2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)己酰胺基)苄酯65,为双三氟乙酸盐。
图21显示自5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯57a(自51a制备)合成磷酸(S)-二叔丁基1-(氯甲基)-2,3-二氢-1H-苯并[e] 吲哚-5-基酯66d。
图22显示自3,3′-(2-硝基-1,4-亚苯基)二丙烯酸(2E,2′E)-叔丁酯66e合成 N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-膦酰氧基-1H-苯并[e]吲哚-3(2H)-基)-3- 氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺 66。
图23显示自3,3′-(2-(3-(((9H-芴-9-基)甲氧基)羰基氨基)丙酰胺基)-1,4-亚苯基)二丙烯酸(2E,2′E)-叔丁酯66g合成N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5- 羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5- 二氧-2,5-二氢-1H-吡咯-1-基)己酰胺67。
图24显示自66d,67c,67d合成二氢磷酸(S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)-2-(3-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯68。
图25以3天时SK-BR-3体外细胞存活力对Thio hu抗CD22HC A121C-MC-vc-PAB-(CBI二聚体)101和Thio hu抗Her2HC A121C-MC-vc-PAB-(CBI二聚体)102浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图26以3天时SK-BR-3体外细胞存活力对Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI-PBD)103和Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI-PBD)104浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图27以3天时SK-BR-3体外细胞存活力对Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI二聚体)116和Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体)117浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图28以3天时EOL-1体外细胞存活力对Thio Hu抗CD33 15G15.33HC A118C-MC-MMED-(CBI二聚体phos)125浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图29以3天时EOL-1体外细胞存活力对Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126和Thio Hu抗CD33 15G15.33 HC A118C-MC-ED-(CBI二聚体DVBdiphos)127浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图30以3天时EOL-1体外细胞存活力对Thio Hu抗CD33 15G15.33HC A118C-MC-ED-(CBI二聚体DVB diphos)127,Thio Hu抗CD33 15G15.33HC A118C-MC-ED-(CBI二聚体DVBphos)129,和Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB phos)130浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。
图31以接种入CRL nu/nu小鼠乳房脂肪垫中的MMTV-HER2Fo5转基因乳房肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯, pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD22 10F4v3HC A118C-MC-MMED-(CBI二聚体phos)110,(3)Thio Hu抗CD22 10F4v3HCA118C-MC-vc-PAB-(CBI二聚体MePip)108,(4)Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI二聚体phos)111,(5)Thio Hu抗Her2 4D5HC A118C-MC-MMED-(CBI二聚体phos)109,(6)Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体MePip)107,(7)Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体phos)112。ADC以10mg/kg给药。
图32以接种入CRL nu/nu小鼠乳房脂肪垫中的MMTV-HER2Fo5转基因乳房肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯, pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD22 10F4v3HC A118C-DSE-(CBI二聚体phos)120,10mg/kg,(3)Thio Hu抗CD2210F4v3HC A118C-DSE-(CBI二聚体phos)122,10mg/kg,(4)Thio Hu抗CD22 10F4v3HCA118C-MC-vc-PAB-(N10,PBD-CBI MePip)124,10mg/kg,(5)Thio Hu抗 Her2 4D5HC A118C-DSE-(CBI二聚体phos)119,3mg/kg,(6)Thio Hu抗Her2 4D5HC A118C-DSE-(CBI二聚体phos)119,10mg/kg,(7)Thio Hu抗Her2 4D5HC A118C-DSP-(CBI二聚体phos)121,3mg/kg,(8)Thio Hu抗Her2 4D5 HC A118C-DSP-(CBI二聚体phos)121,10mg/kg,(9)Thio Hu抗Her24D5HC A118C-MC-vc-PAB-(N10,PBD-CBI MePip)123,3mg/kg,(10)Thio Hu抗 Her2 4D5HCA118C-MC-vc-PAB-(N10,PBD-CBI MePip)123,10mg/kg。
图33以接种入C.B-17SCID小鼠中的OVCAR3X2.1人卵巢肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯,pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD3315G15.33HC A118C-MC-ED-(CBI二聚体DVB diphos)127,3mg/kg,(3)Thio Hu抗MUC163A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126,3mg/kg,(4)Thio Hu抗MUC16 3A5HCA118C-MC-ED-(CBI二聚体DVB diphos)126,1mg/kg。
图34以接种入C.B-17SCID小鼠中的HL-60人急性髓样白血病在下述以 20μg/m2一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯,pH 5.5, 240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD33 15G15.33HC A118C-MC-MMED-(CBI二聚体phos)125,(3)Thio Hu抗CD33 15G15.33HCA118C-MC-ED-(CBI二聚体DVB diphos)127,(4)Thio Hu抗MUC16 3A5HC A118C-MC-MMED-(CBI二聚体phos)128,(5)Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126。
图35a显示ADC138在具有HL-60人急性髓样白血病肿瘤的SCID小鼠中的功效。与媒介组相比,ADC138展现出对肿瘤生长的剂量依赖性抑制。非靶向性对照ADC135对肿瘤生长没有影响。
图35b显示ADC139在具有HL-60人急性髓样白血病肿瘤的SCID小鼠中的功效。与媒介组相比,ADC139展现出清楚的肿瘤生长抑制。非靶向性对照ADC136在1mg/kg对肿瘤生长具有适度影响;然而,ADC139在匹配剂量实质性更加有效,导致完全肿瘤消退。
图36显示ADC134在具有Igrov-1人卵巢肿瘤的SCID-米色小鼠中的功效。与媒介组相比,ADC134展现出对肿瘤生长的剂量依赖性抑制。非靶向性对照ADC137对肿瘤生长没有影响。
图37显示N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物编号69,表4)的合成。
图38显示2,5-二((E)-3-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚 -3(2H)-基)-3-氧丙-1-烯基)苯基氨基甲酸2-(吡啶-2-基二硫基)丙酯(表4,化合物编号72,图38)的合成。
图39显示二氢磷酸[(1S)-1-(氯甲基)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(氯甲基)-5-膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-3-氧-丙-1-烯基]-2-[2-[2-(2,5-二氧吡咯-1-基)乙氧基]乙氧基]苯基]丙-2-烯酰基]-1,2-二氢苯并[e]吲哚-5-基]酯 (化合物编号78,表4,图39)的合成。
图40显示二氢磷酸[(1S)-1-(氯甲基)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(氯甲基)-5-膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-3-氧-丙-1-烯基]-2-[2-(2,5-二氧吡咯-1-基)乙氧基]苯基]丙-2-烯酰基]-1,2-二氢苯并[e]吲哚-5-基]酯(化合物编号 79,表4,图40)的合成。
图41显示N-[1-(氯甲基)-3-[5-[1-(氯甲基)-5-羟基-1,2-二氢苯并[e]吲哚-3-基]-5-氧-戊酰基]-1,2-二氢苯并[e]吲哚-5-基]氨基甲酸2-(2-吡啶基二硫基)丙酯(化合物编号80,表4,图41)的合成。
图42-43显示3-[6-[1-(氯甲基)-5-(4-甲基哌嗪-1-羰基)氧-1,2-二氢苯并[e]吲哚-3-基]-6-氧-己氧基]-6-羟基-2-甲氧基-11-氧-6a,7,8,9-四氢-6H-吡咯并 [2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基)丙酯,3-[6-[1-(氯甲基)-5- 膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-6-氧-己氧基]-6-羟基-2-甲氧基-11-氧 -6a,7,8,9-四氢-6H-吡咯并[2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基) 丙酯,和3-[6-[1-(氯甲基)-5-羟基-1,2-二氢苯并[e]吲哚-3-基]-6-氧-己氧基]-6- 羟基-2-甲氧基-11-氧-6a,7,8,9-四氢-6H-吡咯并[2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基)丙酯(化合物编号81-83,表4,图42-43)的合成。
图44显示甲基β-D-吡喃葡萄糖苷酸(1S)-1-(氯甲基)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(氯甲基)-5-[(6-甲基-β-D-吡喃葡萄糖酸苷基) 氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)-2-[(3-{[6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰基]氨基}丙酰基)氨基]苯基}-2-丙烯酰基)-1,2-二氢-3H- 苯并[e]吲哚-5-基酯(化合物编号84,表4,图44)的合成。
图45显示(S)-(1-甲基-1H-吡咯-2,5-二基)二(((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)甲酮)(化合物编号15,表1,图45)的合成。
图46显示N-(2,5-二((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基)乙酰胺(化合物编号16,表1,图46)的合成。
图47显示(S,2E,2'E)-3,3'-(2-甲氧基-1,4-亚苯基)二(1-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)丙-2-烯-1-酮)(化合物编号17,表1,图47)的合成。
图48显示(S,2E,2′E)-3,3'-(1-甲基-1H-吡咯-2,5-二基)二(1-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)丙-2-烯-1-酮)(化合物编号18,表1,图 48)的合成。
图49显示(S)-3,3'-(2-甲氧基-1,4-亚苯基)二(1-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)丙-2-炔-1-酮)(化合物编号19,表1,图49)的合成。
发明详述
现在要详细描述本发明的某些实施方案,其实例以所附结构和通式例示。尽管会结合例示实施方案描述本发明,但是会理解它们并非旨在将本发明限于那些实施方案。相反,本发明旨在涵盖所有备选方案,变更,和等同方案,它们可以包括在权利要求限定的本发明范围内。
本领域技术人员会认识到可以用于实施本发明的与本文中所描述的那些相似或等同的许多方法和材料。本发明绝不限于所描述的方法和材料。
除非另有定义,本文中所使用的技术和科学术语具有与本发明所属领域普通技术人员的通常理解相同的含义,并且与下述一致:Singleton et al.(1994) Dictionary ofMicrobiology and Molecular Biology,2nd Ed.,J.Wiley&Sons, New York,NY;和Janeway,C.,Travers,P.,Walport,M.,Shlomchik(2001) Immunobiology,5th Ed.,Garland Publishing,New York。
定义
除非另有陈述,本文中所使用的下列术语和短语旨在具有如下含义:
当本文中使用商品名时,申请人旨在独立包括商品名产品配制剂,仿制药,和商品名产品的活性药物组分。
术语“抗体”在本文中以最广义使用,而且具体涵盖单克隆抗体,多克隆抗体,二聚体,多聚体,多特异性抗体(例如双特异性抗体),和抗体片段,只要它们展现期望的生物学活性(Miller et al(2003)Jour.of Immunology 170:4854-4861)。抗体可以是鼠的,人的,人源化的,嵌合的的,或衍生自其它物种。抗体是由免疫系统产生的能够识别和结合特定抗原的蛋白质 (Janeway,C.,Travers,P.,Walport,M.,Shlomchik(2001)Immuno Biology,5th Ed.,Garland Publishing,New York)。靶抗原一般具有众多结合位点,也称作表位,受到多种抗体上的CDR识别。特异性结合不同表位的每种抗体具有不同结构。因此,一种抗原可以具有超过一种相应抗体。抗体包括全长免疫球蛋白分子或全长免疫球蛋白分子的免疫学活性部分,即含有免疫特异性结合感兴趣靶物的抗原或其部分的分子,此类靶物包括但不限于癌细胞或产生与自身免疫性疾病相关的自身免疫性抗体的细胞。本文中公开的免疫球蛋白可以是任何型(例如IgG,IgE,IgM,IgD,和IgA),类(例如IgG1,IgG2,IgG3, IgG4,IgA1和IgA2)或亚类的免疫球蛋白分子。免疫球蛋白可以衍生自任何物种。然而,在一个方面,免疫球蛋白是人,鼠,或家兔起源的。
“抗体片段”包含全长抗体的一部分,一般是其抗原结合或可变区。抗体片段的例子包括Fab,Fab′,F(ab')2,和Fv片段;双抗体;线性抗体;微抗体(minibody)(Olafsen etal(2004)Protein Eng.Design&Sel.17(4):315-323);通过Fab表达文库生成的片段;抗独特型(抗Id)抗体;CDR(互补决定区);和免疫特异性结合癌细胞抗原,病毒抗原或微生物抗原的任何上述的表位结合片段;单链抗体分子;和自抗体片段形成的多特异性抗体。
如本文中使用的,术语“单克隆抗体”指从基本上同质的抗体群体获得的抗体,即除了可能少量存在的可能的天然发生突变,构成该群体的抗体个体是相同的。单克隆抗体是高度特异性的,针对单个抗原位点。而且,与包括针对不同决定簇(表位)的不同抗体的多克隆抗体制备物对比,每种单克隆抗体针对抗原上的单一决定簇。在它们的特异性之外,单克隆抗体的优点还在于它们可以在不受其它抗体污染的情况下合成。修饰语“单克隆”指示抗体自基本上同质的抗体群体获得的特征,不应解释为需要通过任何特定方法来生成抗体。例如,要依照本发明使用的单克隆抗体可以通过首先由Kohler et al(1975)Nature,256:495描述的杂交瘤方法来生成,或者可以通过重组 DNA方法来生成(例如参见US4816567;US 5807715)。还可以使用例如 Clackson et al(1991)Nature,352:624-628;Marks et al(1991)J.Mol.Biol., 222:581-597中描述的技术自噬菌体抗体文库分离单克隆抗体。
本文中的单克隆抗体具体包括“嵌合”抗体,其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类或亚类的抗体中的相应序列相同或同源,而链的剩余部分与衍生自另一物种或属于另一抗体类或亚类的抗体中的相应序列相同或同源,以及此类抗体的片段,只要它们展现期望的生物学活性(US 4816567;及Morrison et al(1984)Proc.Natl.Acad.Sci.USA, 81:6851-6855)。本文中感兴趣的嵌合抗体包括“灵长化”抗体,其包含衍生自非人灵长动物(例如旧世界猴,猿,等)的可变域抗原结合序列和人恒定区序列。
本文中的“完整抗体”为包含VL和VH域,以及轻链恒定域(CL)和重链恒定域(CH1,CH2和CH3)的抗体。恒定域可以为天然序列恒定域(例如人天然序列恒定域)或其氨基酸序列变体。完整抗体可以具有一种或多种“效应器功能”,指那些可归于抗体Fc恒定区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性。抗体效应器功能的例子包括C1q结合;补体依赖性细胞毒性;Fc受体结合;抗体依赖性细胞介导的细胞毒性(ADCC);吞噬;和细胞表面受体(诸如B细胞受体和BCR)的下调。
术语“Fc区”在本文中用于定义免疫球蛋白重链中至少含有恒定区一部分的C端区。该术语包括天然序列Fc区和变体Fc区。在一个实施方案中,人 IgG重链Fc区自Cys226或Pro230延伸至重链的羧基端。然而,Fc区的C端赖氨酸(Lys447)可以存在或不存在。除非本文中另有规定,Fc区或恒定区中的氨基酸残基的编号方式依照EU编号系统,也称作EU索引,如记载于Kabat et al.,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,Bethesda,MD,1991。
“框架”或“FR”指除高变区(HVR)残基以外的可变域残基。可变域的FR一般由4个FR域组成:FR1,FR2,FR3,和FR4。因而,HVR和FR 序列在VH(或VL)中一般以如下顺序出现:FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。
根据其重链恒定域的氨基酸序列,可以将完整抗体指派至不同的“类”。存在五大类完整免疫球蛋白抗体:IgA,IgD,IgE,IgG,和IgM,而且其中数项可以进一步分成“亚类”(同种型),例如IgG1,IgG2,IgG3,IgG4,IgA1,和IgA2。与不同类的抗体对应的重链恒定域分别称作α,δ,ε,γ,和μ。不同类的免疫球蛋白的亚基结构和三维构造是公知的。Ig形式包括铰链修饰或无铰链形式(Roux et al(1998)J.Immunol.161:4083-4090;Lund et al(2000)Eur.J.Biochem.267:7246-7256;US 2005/0048572;US 2004/0229310)。
“人抗体”为拥有与由人或人细胞生成的或自利用人抗体全集或其它人抗体编码序列的非人来源衍生的抗体的氨基酸序列对应的氨基酸序列的抗体。人抗体的此定义具体排除包含非人抗原结合残基的人源化抗体。
“人共有框架”为代表人免疫球蛋白VL或VH框架序列的选集中最常发生的氨基酸残基的框架。通常,人免疫球蛋白VL或VH序列的选集来自可变域序列的亚组。通常,序列的亚组为如Kabat et al.,Sequences of Proteins of Immunological Interest,FifthEdition,NIH Publication 91-3242,Bethesda MD (1991),vols.1-3中的亚组。在一个实施方案中,对于VL,亚组是如Kabat等人,见上文中的亚组卡帕I。在一个实施方案中,对于VH,亚组是如Kabat 等人,见上文中的亚组III。
“人源化”抗体指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中,人源化抗体会包含至少一个,通常两个基本上整个如下的可变域,其中所有或基本上所有HVR(例如CDR)对应于非人抗体的那些,且所有或基本上所有FR对应于人抗体的那些。任选地,人源化抗体可以至少包含自人抗体衍生的抗体恒定区的一部分。“人源化形式”的抗体(例如非人抗体)指已经经历人源化的抗体。
如本文中使用的,术语“高变区”或“HVR”指抗体可变域中在序列上高变和/或形成结构上定义的环(“高变环”)的每个区。一般地,天然四链抗体包含六个HVR;三个在VH中(H1,H2,H3),三个在VL中(L1,L2, L3)。HVR一般包含来自高变环和/或“互补决定区”(CDR)的氨基酸残基,后者具有最高序列可变性和/或牵涉抗原识别。例示性高变环存在于氨基酸残基26-32(L1),50-52(L2),91-96(L3),26-32(H1),53-55(H2),和96-101 (H3)(Chothiaand Lesk,J.Mol.Biol.196:901-917(1987))。例示性CDR (CDR-L1,CDR-L2,CDR-L3,CDR-H1,CDR-H2,和CDR-H3)存在于氨基酸残基24-34(L1),50-56(L2),89-97(L3),31-35B(H1),50-65(H2),和95-102(H3)(Kabat et al.,Sequences of Proteins of ImmunologicalInterest, 5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD (1991))。除VH中的CDR1以外,CDR一般包含形成高变环的氨基酸残基。 CDR还包含“特异性决定残基”或“SDR”,它们是接触抗原的残基。SDR 包含在称作缩短的CDR或a-CDR的CDR区内。例示性a-CDR(a-CDR-L1, a-CDR-L2,a-CDR-L3,a-CDR-H1,a-CDR-H2,和a-CDR-H3)存在于氨基酸残基31-34(L1),50-55(L2),89-96(L3),31-35B(H1),50-58(H2),和95-102(H3)(参见Almagro and Fransson,Front.Biosci.13:1619-1633 (2008))。除非另有指示,HVR残基和可变域中的其它残基(例如FR残基) 在本文中依照Kabat等人,见上文编号。
术语“可变区”或“可变域”指抗体重链或轻链中牵涉抗体结合抗原的域。天然抗体的重链和轻链可变域(分别为VH和VL)一般具有相似的结构,其中每个域包含四个保守的框架区(FR)和三个高变区(HVR)(参见例如 Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,page 91(2007))。单个VH或VL域可以足以赋予抗原结合特异性。此外,可以分别使用来自结合抗原的抗体的VH或VL域筛选互补VL或VH域的文库来分离结合特定抗原的抗体。参见例如Portolano et al.,J.Immunol.150:880-887(1993);Clarkson etal.,Nature 352:624-628(1991)。
如本文中使用的,术语“载体”指能够增殖与其连接的另一核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸表达。此类载体在本文中称作“表达载体”。
“游离半胱氨酸”指已经改造入亲本抗体中,具有硫醇官能团(-SH),且未作为分子内或分子间二硫桥配对的半胱氨酸氨基酸残基。
“接头”,“接头单元”,或“连接”意指包含将抗体共价附着至药物模块的原子链的化学模块。在各个实施方案中,接头为二价基,以L表示。
在指出取代基的数目时,术语“一个或多个”指自一个取代基至最高可能数目的取代的范围,即用取代基替换一个氢直至替换所有氢。术语“取代基”表示替换亲本分子上的一个氢原子的一个或一组原子。术语“取代的”表示指定基团携带一个或多个取代基。在任何基团可携带多个取代基且提供多种可能取代基的情况中,取代基是独立选择的且不需要是相同的。术语“未取代的”意味着指定基团不携带取代基。术语“任选取代的”意味着指定基团是未取代的或用一个或多个独立选自可能取代基组的取代基取代的。在指出取代基的数目时,术语“一个或多个”意指一个取代基至最高可能数目的取代,即用取代基替换一个氢直至替换所有氢。
如本文中使用的,术语“烷基”指1至12个碳原子(C1-C12)的任何长度的饱和的线性或分支链的单价的烃根,其中烷基根可以是任选用一个或多个下文所述取代基独立取代的。在另一个实施方案中,烷基根是1至8个碳原子 (C1-C8),或1至6个碳原子(C1-C6)。烷基基团的例子包括但不限于甲基(Me, -CH3),乙基(Et,-CH2CH3),1-丙基(n-Pr,正丙基,-CH2CH2CH3),2- 丙基(i-Pr,异丙基,-CH(CH3)2),1-丁基(n-Bu,正丁基,-CH2CH2CH2CH3), 2-甲基-1-丙基(i-Bu,异丁基,-CH2CH(CH3)2),2-丁基(s-Bu,仲丁基, -CH(CH3)CH2CH3),2-甲基-2-丙基(t-Bu,叔丁基,-C(CH3)3),1-戊基(正戊基,-CH2CH2CH2CH2CH3),2-戊基(-CH(CH3)CH2CH2CH3),3-戊基 (-CH(CH2CH3)2),2-甲基-2-丁基(-C(CH3)2CH2CH3),3-甲基-2-丁基 (-CH(CH3)CH(CH3)2),3-甲基-1-丁基(-CH2CH2CH(CH3)2),2-甲基-1-丁基 (-CH2CH(CH3)CH2CH3),1-己基(-CH2CH2CH2CH2CH2CH3),2-己基 (-CH(CH3)CH2CH2CH2CH3),3-己基(-CH(CH2CH3)(CH2CH2CH3)),2-甲基 -2-戊基(-C(CH3)2CH2CH2CH3),3-甲基-2-戊基(-CH(CH3)CH(CH3)CH2CH3), 4-甲基-2-戊基(-CH(CH3)CH2CH(CH3)2),3-甲基-3-戊基(-C(CH3)(CH2CH3)2),2-甲基-3-戊基(-CH(CH2CH3)CH(CH3)2),2,3-二甲基-2-丁基 (-C(CH3)2CH(CH3)2),3,3-二甲基-2-丁基(-CH(CH3)C(CH3)3),1-庚基,1- 辛基,等等。
如本文中使用的,术语“亚烷基”指1至12个碳原子(C1-C12)的任何长度的饱和的线性或分支链的二价的烃根,其中亚烷基根可以是任选用一个或多个下文所述取代基独立取代的。在另一个实施方案中,亚烷基根是1至8个碳原子(C1-C8),或1至6个碳原子(C1-C6)。亚烷基基团的例子包括但不限于亚甲基(-CH2-),亚乙基(-CH2CH2-),亚丙基(-CH2CH2CH2-),等等。
术语“烯基”指2至8个碳原子(C2-C8)的任何长度的,有至少一个不饱和位点(即碳-碳,sp2双键)的,线性或分支链的单价的烃根,其中烯基根可以是任选用一个或多个本文所述取代基独立取代的,而且包括具有“顺式”和“反式”取向或者“E”和“Z”取向的根。例子包括但不限于乙烯基 (-CH=CH2),烯丙基(-CH2CH=CH2),等等。
术语“亚烯基”指2至8个碳原子(C2-C8)的任何长度的,有至少一个不饱和位点(即碳-碳,sp2双键)的,线性或分支链的二价的烃根,其中亚烯基根可以是任选用一个或多个本文所述取代基独立取代的,而且包括具有“顺式”和“反式”取向或者“E”和“Z”取向的根。例子包括但不限于亚乙烯基(-CH=CH-),亚烯丙基(-CH2CH=CH-),等等。
术语“炔基”指2至8个碳原子(C2-C8)的任何长度的,有至少一个不饱和位点(即碳-碳,sp三键)的,线性或分支的单价的烃根,其中炔基根可以是任选用一个或多个本文所述取代基独立取代的。例子包括但不限于乙炔基(-C≡CH),丙炔基或炔丙基(-CH2C≡CH),等等。
术语“亚炔基”指2至8个碳原子(C2-C8)的任何长度的,有至少一个不饱和位点(即碳-碳,sp三键)的,线性或分支的二价的烃根,其中亚炔基根可以是任选用一个或多个本文所述取代基独立取代的。例子包括但不限于亚乙炔基(-C≡C-),亚丙炔基或亚炔丙基(-CH2C≡C-),等等。
术语“碳环”,“碳环基”,“碳环环”和“环烃基”指具有作为单环环的 3至12个碳原子(C3-C12)或作为双环环的7至12个碳原子的,单价的,非芳香族的,饱和或部分未饱和的环。具有7至12个原子的双环碳环可以排列成例如双环[4,5],[5,5],[5,6]或[6,6]体系,而具有9或10个环原子的双环碳环可以排列成双环[5,6]或[6,6]体系,或桥连体系诸如双环[2.2.1]庚烷,双环[2.2.2] 辛烷和双环[3.2.2]壬烷。螺模块也包括在此定义的范围内。单环碳环的例子包括但不限于环丙基,环丁基,环戊基,1-环戊-1-烯基,1-环戊-2-烯基,1-环戊-3-烯基,环己基,1-环己-1-烯基,1-环己-2-烯基,1-环己-3-烯基,环己二烯基,环庚基,环辛基,环壬基,环癸基,环十一烷基,环十二烷基,等等。碳环基基团任选是用一个或多个本文所述取代基独立取代的。
“芳基”意指6至20个碳原子(C6-C20)的,通过自亲本芳香族环体系的一个碳原子去除一个氢原子而衍生的,单价的芳香族的烃根。一些芳基基团在例示结构中以“Ar”表示。芳基包括包含与饱和,部分未饱和的环或芳香族碳环环稠合的芳香环的双环根。典型的芳基基团包括但不限于自苯(苯基),取代的苯,萘,蒽,联苯,茚基,茚满基,1,2-二氢萘,1,2,3,4-四氢萘基,等等衍生的根。芳基基团任选是用一个或多个本文所述取代基独立取代的。
“亚芳基”指6至20个碳原子(C6-C20)的,通过自亲本芳香族环体系的两个碳原子去除两个氢原子而衍生的,二价的芳香族的烃根。一些亚芳基基团在例示结构中以“Ar”表示。亚芳基包括包含与饱和,部分未饱和的环或芳香族碳环环稠合的芳香环的双环根。典型的亚芳基基团包括但不限于自苯 (亚苯基),取代的苯,萘,蒽,亚联苯,亚茚基,亚茚满基,1,2-二氢亚萘基,1,2,3,4-四氢亚萘基,等等衍生的根。亚芳基基团任选是用一个或多个本文所述取代基取代的。
术语“杂环”,“杂环基”和“杂环环”在本文中可互换使用,指3至约 20个环原子的,饱和或部分未饱和的(即在环内具有一个或多个双键和/或三键)碳环根,其中至少一个环原子是选自氮,氧,磷和硫的杂原子,其余环原子是C,其中一个或多个环原子是任选用一个或多个下文所述取代基独立取代的。杂环可以是具有3至7个环成员(2至6个碳原子和1至4个选自N,O, P,和S的杂原子)的单环或具有7至10个环成员(4至9个碳原子和1至6个选自N,O,P,和S的杂原子)的双环,例如双环[4,5],[5,5],[5,6],或[6,6] 体系。杂环记载于Paquette,Leo A.;“Principles of Modern Heterocyclic Chemistry”(W.A.Benjamin,New York,1968),特别是第1,3,4,6,7,和9 章;“The Chemistry of HeterocyclicCompounds,A series of Monographs”(John Wiley&Sons,New York,1950to present),特别是第13,14,16,19,和28 卷;及J.Am.Chem.Soc.(1960)82:5566。“杂环基”还包括杂环根与饱和,部分未饱和的环或芳香族碳环或杂环环稠合的根。杂环环的例子包括但不限于吗啉-4-基(morpholin-4-yl),哌啶-1-基(piperidin-1-yl),哌嗪基(piperazinyl),哌嗪-4-基-2-酮(piperazin-4-yl-2-one),哌嗪-4-基-3-酮(piperazin-4-yl-3-one),吡咯烷-1-基(pyrrolidin-1-yl),硫代吗啉-4-基(thiomorpholin-4-yl),S-二氧硫代吗啉-4-基(S-dioxothiomorpholin-4-yl),氮杂环辛烷-1-基(azocan-1-yl),氮杂环丁烷-1-基(azetidin-1-yl),八氢吡啶并[1,2-a]吡嗪-2-基 (octahydropyrido[1,2-a]pyrazin-2-yl),[1,4]二氮杂环庚烷-1-基 ([1,4]diazepan-1-yl),吡咯烷基(pyrrolidinyl),四氢呋喃基(tetrahydrofuranyl),二氢呋喃基(dihydrofuranyl),四氢噻吩基(tetrahydrothienyl),四氢吡喃基 (tetrahydropyranyl),二氢吡喃基(dihydropyranyl),四氢硫代吡喃基 (tetrahydrothiopyranyl),哌啶并(piperidino),吗啉并(morpholino),硫代吗啉并(thiomorpholino),噻烷基(thioxanyl),哌嗪基(piperazinyl),高哌嗪基 (homopiperazinyl),氮杂环丁烷基(azetidinyl),氧杂环丁烷基(oxetanyl),硫杂环丁烷基(thietanyl),高哌啶基(homopiperidinyl),氧杂环庚烷基(oxepanyl),硫杂环庚烷基(thiepanyl),氧氮杂基(oxazepinyl),二氮杂基(diazepinyl),硫杂基(thiazepinyl),2-吡咯啉基(2-pyrrolinyl),3-吡咯啉基(3-pyrrolinyl),二氢吲哚基(indolinyl),2H-吡喃基(2H-pyranyl),4H-吡喃基(4H-pyranyl),二烷基(dioxanyl),1,3-二氧戊环基(1,3-dioxolanyl),吡唑啉基(pyrazolinyl),二噻烷基(dithianyl),二硫戊环基(dithiolanyl),二氢吡喃基(dihydropyranyl),二氢噻吩基(dihydrothienyl),二氢呋喃基(dihydrofuranyl),吡唑烷基 (pyrazolidinyl)咪唑啉基(imidazolinyl),咪唑烷基(imidazolidinyl),3-氮双环[3.1.0]己烷基(3-azabicyco[3.1.0]hexanyl),3-氮双环[4.1.0]庚烷基 (3-azabicyclo[4.1.0]heptanyl),氮双环[2.2.2]己烷基(azabicyclo[2.2.2]hexanyl), 3H-吲哚基(3H-indolyl)喹嗪基(quinolizinyl)和N-吡啶基脲(N-pyridyl urea)。螺模块也包括在此定义的范围内。其中2个环原子被氧(=O)模块取代的杂环基团的例子有嘧啶酮基(pyrimidinonyl)和1,1-二氧-硫代吗啉基 (1,1-dioxo-thiomorpholinyl)。本文中的杂环基团任选是用一个或多个本文所述取代基独立取代的。
术语“杂芳基”指5,6,或7元环的单价的芳香族的根,而且包括5-20 个原子(含有一个或多个独立选自氮,氧,和硫的杂原子)的稠环体系(其中至少一个是芳香族的)。杂芳基基团的例子有吡啶基(pyridinyl)(包括例如 2-羟基吡啶基),咪唑基(imidazolyl),咪唑并吡啶基(imidazopyridinyl),1-甲基-1H-苯并[d]咪唑,[1,2,4]三唑并[1,5-a]吡啶,嘧啶基(pyrimidinyl)(包括例如4-羟基嘧啶基),吡唑基(pyrazolyl),三唑基(triazolyl),吡嗪基(pyrazinyl),四唑基(tetrazolyl),呋喃基(furyl),噻吩基(thienyl),异唑基(isoxazolyl),噻唑基(thiazolyl),二唑基(oxadiazolyl),唑基(oxazolyl),异噻唑基 (isothiazolyl),吡咯基(pyrrolyl),喹啉基(quinolinyl),异喹啉基(isoquinolinyl),四氢异喹啉基(tetrahydroisoquinolinyl),吲哚基(indolyl),苯并咪唑基 (benzimidazolyl),苯并呋喃基(benzofuranyl),噌啉基(cinnolinyl),吲唑基(indazolyl),吲嗪基(indolizinyl),酞嗪基(phthalazinyl),哒嗪基(pyridazinyl),三嗪基(triazinyl),异吲哚基(isoindolyl),喋啶基(pteridinyl),嘌呤基(purinyl),二唑基(oxadiazolyl),噻二唑基(thiadiazolyl),噻二唑基(thiadiazolyl),呋咱基(furazanyl),苯并呋咱基(benzofurazanyl),苯并噻吩基(benzothiophenyl),苯并噻唑基(benzothiazolyl),苯并唑基(benzoxazolyl),喹唑啉基 (quinazolinyl),喹啉基(quinoxalinyl),二氮杂萘基(naphthyridinyl),和呋喃并吡啶基(furopyridinyl)。杂芳基基团任选是用一个或多个本文所述取代基独立取代的。
杂环或杂芳基基团可以是成碳(碳连的)或氮(氮连的)键的,在那里可能的情况中。举例而非限制,成碳键的杂环或杂芳基在吡啶的2,3,4,5,或6位,哒嗪的3,4,5,或6位,嘧啶的2,4,5,或6位,吡嗪的2,3,5,或6位,呋喃,四氢呋喃,硫代呋喃(thiofuran),噻吩,吡咯或四氢吡咯的2, 3,4,或5位,唑,咪唑或噻唑的2,4,或5位,异唑,吡唑或异噻唑的3,4,或5位,氮杂环丙烷的2或3位,氮杂环丁烷的2,3,或4位,喹啉的2, 3,4,5,6,7,或8位,或异喹啉的1,3,4,5,6,7,或8位处成键。
举例而非限制,成氮键的杂环或杂芳基在氮杂环丙烷,氮杂环丁烷,吡咯,吡咯烷,2-吡咯啉,3-吡咯啉,咪唑,咪唑烷,2-咪唑啉,3-咪唑啉,吡唑,吡唑啉,2-吡唑啉,3-吡唑啉,哌啶,哌嗪,吲哚,二氢吲哚,1H- 吲唑的1位,异吲唑或异二氢吲哚的2位,吗啉的4位,和咔唑或β-咔啉的9位处成键。
术语“手性”指分子具有镜像对映体的不可重叠的特性,而术语“非手性”指分子在其镜像对映体上可重叠。
术语“立体异构体”指具有相同的化学结构,但是在原子或基团的空间排列方面有所不同的化合物。
“非对映异构体”指具有两个或更多个手性中心且其分子彼此不为镜像的立体异构体。非对映异构体具有不同的物理特性,例如熔点,沸点,光谱特性,和反应性。非对映异构体的混合物可以在高分辨率的分析规程下分开,诸如电泳和层析。
“对映异构体”指化合物的如下两种立体异构体,它们彼此为不可重叠的镜像。
本文中使用的立体化学定义和规则一般遵循S.P.Parker,Ed., McGraw-HillDictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York;及Eliel,E.and Wilen,S.,Stereochemistry of Organic Compounds(1994)John Wiley&Sons,Inc.,New York。许多有机化合物以旋光形式存在,即它们具有旋转平面偏振光的平面的能力。在描述旋光化合物时,前缀D和L或R和S用于表示分子关于其手性中心的绝对构型。前缀d和l或(+) 和(-)用于表示化合物对平面偏振光的旋转的标记,其中(-)或1意味着化合物是左旋的。以(+)或d为前缀的化合物是右旋的。对于给定的化学结构,这些立体异构体是相同的,只是它们互为镜像。特定的立体异构体还可以称作对映体,而且此类异构体的混合物常常称作对映混合物。对映体的50:50混合物称作外消旋混合物或外消旋物,它们可以在没有立体选择性或立体特异性的化学反应或过程中存在。术语“外消旋混合物”和“外消旋物”指两种对映体种类的等摩尔混合物,没有旋光性。
如本文中使用的,短语“药学可接受盐”指抗体-药物缀合物(ADC) 的药学可接受的有机或无机盐。例示性的盐包括但不限于硫酸盐,柠檬酸盐,乙酸盐,草酸盐,氯化物,溴化物,碘化物,硝酸盐,硫酸氢盐,磷酸盐,酸式磷酸盐,异烟酸盐,乳酸盐,水杨酸盐,酸式柠檬酸盐,酒石酸盐,油酸盐,丹宁酸盐,泛酸盐,酒石酸氢盐,抗坏血酸盐,琥珀酸盐,马来酸盐,龙胆酸盐,富马酸盐,葡糖酸盐,葡糖醛酸盐,糖酸盐,甲酸盐,苯甲酸盐,谷氨酸盐,甲磺酸盐,乙磺酸盐,苯磺酸盐,对甲苯磺酸盐,和扑酸盐(即 1,1’-亚甲基-双-(2-羟基-3-萘甲酸盐))。药学可接受盐可能牵涉包含另一种分子,诸如乙酸根离子,琥珀酸根离子或其它抗衡离子。抗衡离子可以是稳定母体化合物上的电荷的任何有机或无机模块。另外,药学可接受盐可以在其结构中具有超过一个带电荷的原子。在多个带电荷的原子是药学可接受盐的组成部分的情况中可以具有多个抗衡离子。因此,药学可接受盐可以具有一个或多个带电荷的原子和/或一个或多个抗衡离子。
本文中使用下述缩写,它们具有指定定义:BME为β-巯基乙醇,Boc为 N-(叔丁氧羰基),cit为瓜氨酸(2-氨基-5-脲基戊酸),DCC为1,3-二环己基碳二亚胺,DCM为二氯甲烷,DEA为二乙胺,DEAD为偶氮二羧酸二乙酯, DEPC为氰基磷酸二乙酯(diethylphosphorylcyanidate),DIAD为偶氮二羧酸二异丙酯,DIEA为N,N-二异丙基乙胺,DMA为二甲基乙酰胺,DMAP为4-二甲基氨基吡啶,DME为乙二醇二甲醚(或1,2-二甲氧基乙烷),DMF为N,N- 二甲基甲酰胺,DMSO为二甲亚砜,DTT为二硫苏糖醇,EDCI为盐酸1-(3- 二甲基氨基丙基)-3-乙基碳二亚胺,EEDQ为2-乙氧基-1-乙氧羰基-1,2-二氢喹啉,ES-MS为电喷射质谱术,EtOAc为乙酸乙酯,Fmoc为N-(9-芴基甲氧羰基), gly为甘氨酸,HATU为O-(7-氮杂苯并三唑-1-基)-N,N,N’,N’-四甲基脲鎓六氟磷酸酯(O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate),HOBt为1-羟基苯并三唑,HPLC为高压液相层析术,ile 为异亮氨酸,lys为赖氨酸,MeCN(CH3CN)为乙腈,MeOH为甲醇,Mtr为4- 茴香基二苯甲基(或4-甲氧基三苯甲基),NHS为N-羟基琥珀酰亚胺,PBS 为磷酸盐缓冲盐水(pH 7),PEG为聚乙二醇或乙二醇单元(-OCH2CH2-), Ph为苯基,Pnp为对硝基苯基,MC为6-马来酰亚胺基己酰基,phe为L-苯丙氨酸,PyBrop为溴三吡咯烷膦六氟磷酸酯(bromotris-pyrrolidino phosphonium hexafluorophosphate),SEC为大小排阻层析术,Su为琥珀酰亚胺,TFA为三氟乙酸,TLC为薄层层析术,UV为紫外线,而val为缬氨酸。
半胱氨酸改造的抗体
本发明的化合物包括抗体-药物缀合物,其包含半胱氨酸改造的抗体,其中野生型或亲本抗体的一个或多个氨基酸用半胱氨酸替换。可以这样改造 (即突变)任何形式的抗体。例如,可以改造亲本Fab抗体片段以形成半胱氨酸改造的Fab,在本文中称作“ThioFab”。类似地,可以改造亲本单克隆抗体以形成“ThioMab”。应当注意,单位点突变在ThioFab中产生一个改造的半胱氨酸残基,而单位点突变在ThioMab中产生两个改造的半胱氨酸残基,这是IgG抗体的二聚体本质所致。对具有替换的(“改造的”)半胱氨酸(Cys) 残基的突变体评估新引入的改造的半胱氨酸硫醇基团的反应性。硫醇反应性值为0至1.0的范围中的一个相对数值术语,而且可以为任何半胱氨酸改造的抗体进行测量。本发明的半胱氨酸改造的抗体的硫醇反应性值在0.6至1.0; 0.7至1.0;或0.8至1.0的范围中。
可以在抗体的反应性位点处改造半胱氨酸氨基酸,它们不形成链内或链间二硫化物连接(Junutula et al.,2008b Nature Biotech.,26(8):925-932;Dornan et al(2009)Blood 114(13):2721-2729;US 7521541;US 7723485; WO2009/052249;Shen et al(2012)Nature Biotech.,30(2):184-191;Junutula et al(2008)Jour of Immun.Methods 332:41-52)。改造的半胱氨酸硫醇可以与本发明的具有硫醇反应性亲电子基团(诸如马来酰亚胺或α-卤代酰胺)的接头试剂或接头-药物中间体反应以形成具有半胱氨酸改造的抗体(ThioMab)和药物(D)模块的ADC。药物模块的定位可以如此设计,控制,和知晓。可以控制药物载荷,因为改造的半胱氨酸硫醇基团通常以高产率与硫醇反应性接头试剂或接头-药物中间体反应。改造抗体,通过替代重链或轻链上的一个位点来引入半胱氨酸氨基酸在对称的抗体上给出两个新的半胱氨酸。能实现接近2的药物载荷,而且缀合产物ADC接近均质。
本发明的半胱氨酸改造的抗体优选保留其野生型亲本抗体对应物的抗原结合能力。如此,半胱氨酸改造的抗体能够结合(优选特异性地)抗原。此类抗原包括例如肿瘤相关抗原(TAA),细胞表面受体蛋白和其它细胞表面分子,跨膜蛋白,信号传导蛋白,细胞存活调节因子,细胞增殖调节因子,与组织发育或分化相关的(例如,已知或怀疑在功能上有贡献的)分子,淋巴因子,细胞因子,牵涉细胞周期调节的分子,牵涉维管发生(vasculogenesis)的分子和与血管发生(angiogenesis)相关的(例如,已知或怀疑在功能上有贡献的)分子。肿瘤相关抗原可以为簇分化因子(即CD蛋白)。半胱氨酸改造的抗体能够结合的抗原可以为上文所述范畴之一的一个子集的成员,其中所述范畴的其它子集包含具有不同特征(就感兴趣抗原而言)的其它分子/抗原。
制备半胱氨酸改造的抗体,用于通过链内二硫化物基团的还原和再氧化来与接头-药物中间体缀合(实施例19)。
在癌症治疗中的本发明抗体-药物缀合物中可能有用的半胱氨酸改造的抗体包括但不限于针对细胞表面受体和肿瘤相关抗原(TAA)的抗体。肿瘤相关抗原是本领域已知的,而且可以制备它们以用于使用本领域公知的方法和信息生成抗体。在发现用于癌症诊断和治疗的有效细胞靶物的尝试中,研究人员寻求鉴定与一种或多种正常非癌性细胞相比在一种或多种特定类型的癌细胞的表面上特异性表达的跨膜或其它肿瘤相关多肽。通常,与在非癌性细胞的表面上相比,此类肿瘤相关多肽在癌细胞的表面上更加丰富地表达。对此类肿瘤相关细胞表面抗原多肽的鉴定已经提高经由基于抗体的疗法特异性靶向癌细胞进行破坏的能力。
肿瘤相关抗原TAA的例子包括但不限于下文所列TAA(1)-(51)。为方便起见,下文列出涉及这些抗原(它们均是本领域已知的)的信息,包括名称,备选名称,Genbank登录号和主要参考文献,遵循National Center for Biotechnology Information(NCBI)的核酸和蛋白质序列鉴定规则。与TAA (1)-(51)对应的核酸和蛋白质序列在公共数据库中可得,诸如GenBank。抗体靶向的肿瘤相关抗原包括相对于所引用的参考文献中鉴定的序列拥有至少约70%,80%,85%,90%,或95%序列同一性,或展现与具有在所引用的参考文献中找到的序列的TAA基本上相同的生物学特性或特征的所有氨基酸序列变体和同等型。例如,具有变体序列的TAA通常能够特异性结合如下抗体,该抗体特异性结合具有所列相应序列的TAA。通过援引明确收录本文中具体引用的参考文献中的序列和公开内容。
肿瘤相关抗原:
(1)BMPR1B(IB型骨形态发生蛋白受体,Genbank登录号 NM_001203);ten Dijke,P.et al.,Science 264(5155):101-104(1994);Oncogene 14(11):1377-1382(1997);WO2004063362(Claim 2);WO2003042661(Claim 12);US2003134790-A1(Page 38-39);WO2002102235(Claim 13;Page 296); WO2003055443(Page 91-92);WO200299122(Example2;Page 528-530); WO2003029421(Claim 6);WO2003024392(Claim 2;Fig 112);WO200298358 (Claim 1;Page 183);WO200254940(Page 100-101);WO200259377(Page349-350);WO200230268(Claim 27;Page 376);WO200148204(Example;Fig 4);NP_001194骨形态发生蛋白受体,IB型/pid=NP_001194.1-交叉参考: MIM:603248;NP_001194.1;AY065994;
(2)E16(LAT1,SLC7A5,Genbank登录号NM_003486);Biochem.Biophys.Res.Commun.255(2):283-288(1999);Nature 395(6699):288-291 (1998);Gaugitsch,H.W.et al.(1992)J.Biol.Chem.267(16):11267-11273; WO2004048938(Example 2);WO2004032842(Example IV);WO2003042661 (Claim 12);WO2003016475(Claim 1);WO200278524(Example 2); WO200299074(Claim 19;Page 127-129);WO200286443(Claim 27;Pages 222, 393);WO2003003906(Claim 10;Page 293);WO200264798(Claim 33;Page 93-95);WO200014228(Claim 5;Page 133-136);US2003224454(Fig 3); WO2003025138(Claim 12;Page 150);NP_003477溶质载体家族7(阳离子氨基酸转运蛋白,y+系统),成员5/pid=NP_003477.3-人;交叉参考: MIM:600182;NP_003477.3;NM_015923;NM_003486_1;
(3)STEAP1(前列腺六次跨膜上皮抗原,Genbank登录号NM_012449); CancerRes.61(15):5857-5860(2001);Hubert,R.S.et al.(1999)Proc.Natl.Acad.Sci.U.S.A.96(25):14523-14528;WO2004065577(Claim 6); WO2004027049(Fig1L);EP1394274(Example 11);WO2004016225(Claim 2); WO2003042661(Claim 12);US2003157089(Example 5);US2003185830 (Example 5);US2003064397(Fig 2);WO200289747(Example 5;Page 618-619); WO2003022995(Example 9;Fig 13A,Example53;Page 173,Example 2;Fig 2A);NP_036581前列腺六次跨膜上皮抗原;交叉参考:MIM:604415; NP_036581.1;NM_012449_1;
(4)0772P(CA125,MUC16,Genbank登录号AF361486);J.Biol.Chem. 276(29):27371-27375(2001);WO2004045553(Claim 14);WO200292836 (Claim 6;Fig 12);WO200283866(Claim 15;Page 116-121);US2003124140 (Example 16);交叉参考:GI:34501467;AAK74120.3;AF361486_1;
(5)MPF(MPF,MSLN,SMR,巨核细胞强化因子,间皮素,Genbank 登录号NM_005823);Yamaguchi,N.et al.,Biol.Chem.269(2):805-808(1994);Proc.Natl.Acad.Sci.U.S.A.96(20):11531-11536(1999);Proc.Natl.Acad.Sci.U.S.A.93(1):136-140(1996);J.Biol.Chem.270(37):21984-21990(1995); WO2003101283(Claim 14);WO2002102235(Claim 13;Page 287-288); WO2002101075(Claim 4;Page308-309);WO200271928(Page 320-321); WO9410312(Page 52-57);交叉参考:MIM:601051;NP_005814.2; NM_005823_1;
(6)Napi3b(NAPI-3B,NPTIIb,SLC34A2,溶质载体家族34(磷酸钠),成员2,II型钠依赖性磷酸转运蛋白3b,Genbank登录号NM_006424); J.Biol.Chem.277(22):19665-19672(2002);Genomics 62(2):281-284(1999); Feild,J.A.et al.(1999)Biochem.Biophys.Res.Commun.258(3):578-582; WO2004022778(Claim 2);EP1394274(Example 11);WO2002102235(Claim 13;Page 326);EP875569(Claim 1;Page 17-19);WO200157188(Claim 20; Page 329);WO2004032842(Example IV);WO200175177(Claim24;Page 139-140);交叉参考:MIM:604217;NP_006415.1;NM_006424_1;
(7)Sema 5b(FLJ10372,KIAA1445,Mm.42015,SEMA5B, SEMAG,脑信号蛋白5bHlog,sema域,七次血小板反应蛋白重复(1型和类1型),跨膜域(TM)和短胞质域,(脑信号蛋白)5B,Genbank登录号AB040878);Nagase T.et al.(2000)DNA Res.7(2):143-150;WO2004000997(Claim 1);WO2003003984(Claim 1);WO200206339(Claim 1; Page 50);WO200188133(Claim 1;Page 41-43,48-58);WO2003054152(Claim 20);WO2003101400(Claim 11);登录号:Q9P283;EMBL;AB040878; BAA95969.1.Genew;HGNC:10737;
(8)PSCA hlg(2700050C12Rik,C530008O16Rik,RIKEN cDNA 2700050C12,RIKENcDNA 2700050C12基因,Genbank登录号AY358628); Ross et al.(2002)Cancer Res.62:2546-2553;US2003129192(Claim 2); US2004044180(Claim 12);US2004044179(Claim11);US2003096961(Claim 11);US2003232056(Example 5);WO2003105758(Claim 12);US2003206918 (Example 5);EP1347046(Claim 1);WO2003025148(Claim 20);交叉参考:GI:37182378;AAQ88991.1;AY358628_1;
(9)ETBR(内皮缩血管肽B型受体,Genbank登录号AY275463);Nakamuta, M.etal.,Biochem.Biophys.Res.Commun.177:34-39,1991;Ogawa,Y.et al.,Biochem.Biophys.Res.Commun.178:248-255,1991;Arai,H.et al.,Jpn.Circ.J. 56:1303-1307,1992;Arai,H.et al.,J.Biol.Chem.268:3463-3470,1993; Sakamoto,A.,Yanagisawa,M.et al.,Biochem.Biophys.Res.Commun. 178:656-663,1991;Elshourbagy,N.A.et al.,J.Biol.Chem.268:3873-3879, 1993;Haendler,B.et al.,J.Cardiovasc.Pharmacol.20:S1-S4,1992;Tsutsumi,M. et al.,Gene 228:43-49,1999;Strausberg,R.L.et al.,Proc.Natl.Acad.Sci.U.S.A. 99:16899-16903,2002;Bourgeois,C.et al.,J.Clin.Endocrinol.Metab. 82:3116-3123,1997;Okamoto,Y.etal.,Biol.Chem.272:21589-21596,1997; Verheij,J.B.et al.,Am.J.Med.Genet.108:223-225,2002;Hofstra,R.M.W.et al., Eur.J.Hum.Genet.5:180-185,1997;Puffenberger,E.G.et al.,Cell 79:1257-1266,1994;Attie,T.et al.,Hum.Mol.Genet.4:2407-2409,1995; Auricchio,A.et al.,Hum.Mol.Genet.5:351-354,1996;Amiel,J.et al.,Hum. Mol.Genet.5:355-357,1996;Hofstra,R.M.W.et al.,Nat.Genet.12:445-447, 1996;Svensson,P.J.et al.,Hum.Genet.103:145-148,1998;Fuchs,S.et al.,Mol. Med.7:115-124,2001;Pingault,V.et al.(2002)Hum.Genet.111:198-206; WO2004045516(Claim 1);WO2004048938(Example 2);WO2004040000 (Claim151);WO2003087768(Claim 1);WO2003016475(Claim 1); WO2003016475(Claim 1);WO200261087(Fig 1);WO2003016494(Fig 6); WO2003025138(Claim 12;Page 144);WO200198351(Claim 1;Page 124-125); EP522868(Claim 8;Fig 2);WO200177172(Claim1;Page 297-299); US2003109676;US6518404(Fig 3);US5773223(Claim 1a;Col 31-34);WO2004001004;
(10)MSG783(RNF124,推定的蛋白质FLJ20315,Genbank登录号 NM_017763);WO2003104275(Claim 1);WO2004046342(Example 2); WO2003042661(Claim 12);WO2003083074(Claim 14;Page 61); WO2003018621(Claim 1);WO2003024392(Claim 2;Fig 93);WO200166689 (Example 6);交叉参考:LocusID:54894;NP_060233.2;NM_017763_1;
(11)STEAP2(HGNC_8639,IPCA-1,PCANAP1,STAMP1,STEAP2, STMP,前列腺癌相关基因1,前列腺癌相关蛋白1,前列腺六次跨膜上皮抗原2,六次跨膜前列腺蛋白,Genbank登录号AF455138);Lab.Invest.82 (11):1573-1582(2002);WO2003087306;US2003064397(Claim 1;Fig 1); WO200272596(Claim 13;Page 54-55);WO200172962(Claim 1;Fig4B); WO2003104270(Claim 11);WO2003104270(Claim 16);US2004005598 (Claim 22);WO2003042661(Claim 12);US2003060612(Claim 12;Fig 10); WO200226822(Claim 23;Fig 2);WO200216429(Claim 12;Fig 10);交叉参考: GI:22655488;AAN04080.1;AF455138_1;
(12)TrpM4(BR22450,FLJ20041,TRPM4,TRPM4B,瞬时受体电位阳离子通道,亚家族M,成员4,Genbank登录号NM_017636);Xu,X.Z.et al., Proc.Natl.Acad.Sci.U.S.A.98(19):10692-10697(2001);Cell 109(3):397-407 (2002);J.Biol.Chem.278(33):30813-30820(2003);US2003143557(Claim 4); WO200040614(Claim 14;Page 100-103);WO200210382(Claim 1;Fig 9A); WO2003042661(Claim 12);WO200230268(Claim 27;Page391); US2003219806(Claim 4);WO200162794(Claim 14;Fig 1A-D);交叉参考: MIM:606936;NP_060106.2;NM_017636_1;
(13)CRIPTO(CR,CR1,CRGF,CRIPTO,TDGF1,畸胎瘤衍生的生长因子,Genbank登录号NP_003203或NM_003212);Ciccodicola,A.et al., EMBO J.8(7):1987-1991(1989);Am.J.Hum.Genet.49(3):555-565(1991); US2003224411(Claim 1);WO2003083041(Example 1);WO2003034984 (Claim 12);WO200288170(Claim 2;Page 52-53);WO2003024392(Claim 2; Fig 58);WO200216413(Claim 1;Page 94-95,105);WO200222808(Claim 2; Fig 1);US5854399(Example 2;Col 17-18);US5792616(Fig 2);交叉参考:MIM:187395;NP_003203.1;NM_003212_1;
(14)CD21(CR2(补体受体2)或C3DR(C3d/EB病毒受体)或Hs.73792, Genbank登录号M26004);Fujisaku et al.(1989)J.Biol.Chem.264 (4):2118-2125;Weis,J.J.et al.,J.Exp.Med.167:1047-1066,1988;Moore,M.et al.,Proc.Natl.Acad.Sci.U.S.A.84:9194-9198,1987;Barel,M.et al.,Mol. Immunol.35:1025-1031,1998;Weis,J.J.et al.,Proc.Natl.Acad.Sci.U.S.A. 83:5639-5643,1986;Sinha,S.K.et al.(1993)J.Immunol.150:5311-5320; WO2004045520(Example 4);US2004005538(Example 1);WO2003062401 (Claim 9);WO2004045520(Example 4);WO9102536(Fig 9.1-9.9);WO2004020595(Claim 1);登录号:P20023;Q13866;Q14212;EMBL; M26004;AAA35786.1;
(15)CD79b(CD79B,CD79β,IGb(免疫球蛋白相关β),B29,Genbank 登录号NM_000626或11038674);Proc.Natl.Acad.Sci.U.S.A.(2003)100 (7):4126-4131;Blood(2002)100(9):3068-3076;Muller et al.(1992)Eur.J. Immunol.22(6):1621-1625;WO2004016225(claim 2,Fig 140); WO2003087768;US2004101874(claim 1,page 102);WO2003062401(claim 9); WO200278524(Example 2);US2002150573(claim 5,page 15);US5644033; WO2003048202(claim 1,pages 306and 309);WO 99/558658;US6534482(claim 13,Fig 17A/B);WO200055351(claim 11,pages 1145-1146);交叉参考: MIM:147245;NP_000617.1;NM_000626_1;
(16)FcRH2(IFGP4,IRTA4,SPAP1A(含SH2域的磷酸酶锚定蛋白1a), SPAP1B,SPAP1C,Genbank登录号NM_030764,AY358130);Genome Res. 13(10):2265-2270(2003);Immunogenetics 54(2):87-95(2002);Blood 99 (8):2662-2669(2002);Proc.Natl.Acad.Sci.U.S.A.98(17):9772-9777(2001); Xu,M.J.et al.(2001)Biochem.Biophys.Res.Commun.280(3):768-775; WO2004016225(Claim 2);WO2003077836;WO200138490(Claim 5;Fig 18D-1-18D-2);WO2003097803(Claim 12);WO2003089624(Claim 25);交叉参考:MIM:606509;NP_110391.2;NM_030764_1;
(17)HER2(ErbB2,Genbank登录号M11730);Coussens,L.et al.,Science (1985)230(4730):1132-1139;Yamamoto,T.et al.,Nature 319:230-234,1986; Semba,K.etal.,Proc.Natl.Acad.Sci.U.S.A.82:6497-6501,1985;Swiercz,J.M. et al.,J.CellBiol.165:869-880,2004;Kuhns,J.J.et al.,J.Biol.Chem. 274:36422-36427,1999;Cho,H.-S.et al.,Nature 421:756-760,2003;Ehsani,A. et al.(1993)Genomics 15:426-429;WO2004048938(Example 2); WO2004027049(Fig 1I);WO2004009622;WO2003081210;WO2003089904 (Claim 9);WO2003016475(Claim 1);US2003118592;WO2003008537(Claim1);WO2003055439(Claim 29;Fig 1A-B);WO2003025228(Claim 37;Fig 5C); WO200222636(Example 13;Page 95-107);WO200212341(Claim 68;Fig 7); WO200213847(Page 71-74);WO200214503(Page 114-117);WO200153463 (Claim 2;Page 41-46);WO200141787(Page 15);WO200044899(Claim 52;Fig 7);WO200020579(Claim 3;Fig 2);US5869445(Claim 3;Col 31-38); WO9630514(Claim 2;Page 56-61);EP1439393(Claim 7);WO2004043361 (Claim 7);WO2004022709;WO200100244(Example 3;Fig 4);登录号:P04626; EMBL;M11767;AAA35808.1.EMBL;M11761;AAA35808.1;
(18)NCA(CEACAM6,Genbank登录号M18728);Barnett,T.et al., Genomics 3:59-66,1988;Tawaragi,Y.et al.,Biochem.Biophys.Res.Commun. 150:89-96,1988;Strausberg,R.L.et al.,Proc.Natl.Acad.Sci.U.S.A. 99:16899-16903,2002;WO2004063709;EP1439393(Claim 7);WO2004044178 (Example 4);WO2004031238;WO2003042661(Claim 12);WO200278524 (Example 2);WO200286443(Claim 27;Page427);WO200260317(Claim 2);登录号:P40199;Q14920;EMBL;M29541;AAA59915.1.EMBL;M18728;
(19)MDP(DPEP1,Genbank登录号BC017023);Proc.Natl.Acad.Sci. U.S.A.99(26):16899-16903(2002);WO2003016475(Claim 1);WO200264798 (Claim 33;Page 85-87);JP05003790(Fig 6-8);WO9946284(Fig 9);交叉参考: MIM:179780;AAH17023.1;BC017023_1;
(20)IL20Rα(IL20Ra,ZCYTOR7,Genbank登录号AF184971);Clark,H.F. et al.,Genome Res.13:2265-2270,2003;Mungall,A.J.et al.,Nature 425:805-811,2003;Blumberg,H.et al.,Cell 104:9-19,2001;Dumoutier,L.et al., J.Immunol.167:3545-3549,2001;Parrish-Novak,J.et al.,J.Biol.Chem. 277:47517-47523,2002;Pletnev,S.et al.(2003)Biochemistry 42:12617-12624; Sheikh,F.et al.(2004)J.Immunol.172:2006-2010;EP1394274(Example 11); US2004005320(Example 5);WO2003029262(Page 74-75);WO2003002717 (Claim 2;Page 63);WO200222153(Page 45-47);US2002042366(Page 20-21); WO200146261(Page 57-59);WO200146232(Page 63-65);WO9837193(Claim 1;Page 55-59);登录号:Q9UHF4;Q6UWA9;Q96SH8;EMBL;AF184971;AAF01320.1;
(21)短蛋白聚糖(BCAN,BEHAB,Genbank登录号AF229053);Gary, S.C.et al.,Gene 256:139-147,2000;Clark,H.F.et al.,Genome Res. 13:2265-2270,2003;Strausberg,R.L.et al.,Proc.Natl.Acad.Sci.U.S.A. 99:16899-16903,2002;US2003186372(Claim 11);US2003186373(Claim 11); US2003119131(Claim 1;Fig 52);US2003119122(Claim 1;Fig 52); US2003119126(Claim 1);US2003119121(Claim 1;Fig52);US2003119129 (Claim 1);US2003119130(Claim 1);US2003119128(Claim 1;Fig52); US2003119125(Claim 1);WO2003016475(Claim 1);WO200202634(Claim 1);
(22)EphB2R(DRT,ERK,Hek5,EPHT3,Tyro5,Genbank登录号 NM_004442);Chan,J.and Watt,V.M.,Oncogene 6(6):1057-1061(1991); Oncogene 10(5):897-905(1995);Annu.Rev.Neurosci.21:309-345(1998);Int. Rev.Cytol.196:177-244(2000);WO2003042661(Claim 12);WO200053216 (Claim 1;Page 41);WO2004065576(Claim 1);WO2004020583(Claim 9); WO2003004529(Page 128-132);WO200053216(Claim 1;Page42);交叉参考: MIM:600997;NP_004433.2;NM_004442_1;
(23)ASLG659(B7h,Genbank登录号AX092328);US20040101899(Claim 2);WO2003104399(Claim 11);WO2004000221(Fig 3);US2003165504(Claim 1);US2003124140(Example 2);US2003065143(Fig 60);WO2002102235 (Claim 13;Page 299);US2003091580(Example 2);WO200210187(Claim 6; Fig 10);WO200194641(Claim 12;Fig7b);WO200202624(Claim 13;Fig 1A-1B);US2002034749(Claim 54;Page 45-46);WO200206317(Example 2; Page 320-321,Claim 34;Page 321-322);WO200271928(Page468-469); WO200202587(Example 1;Fig 1);WO200140269(Example 3;Pages 190-192);WO200036107(Example 2;Page 205-207);WO2004053079(Claim 12); WO2003004989(Claim 1);WO200271928(Page 233-234,452-453);WO 0116318;
(24)PSCA(前列腺干细胞抗原前体,Genbank登录号AJ297436);Reiter, R.E.etal.,Proc.Natl.Acad.Sci.U.S.A.95:1735-1740,1998;Gu,Z.et al., Oncogene 19:1288-1296,2000;Biochem.Biophys.Res.Commun.(2000) 275(3):783-788;WO2004022709;EP1394274(Example 11);US2004018553 (Claim 17);WO2003008537(Claim 1);WO200281646(Claim 1;Page 164); WO2003003906(Claim 10;Page 288);WO200140309(Example 1;Fig 17); US2001055751(Example 1;Fig 1b);WO200032752(Claim 18;Fig1); WO9851805(Claim 17;Page 97);WO9851824(Claim 10;Page 94);WO9840403 (Claim2;Fig 1B);登录号:O43653;EMBL;AF043498;AAC39607.1;
(25)GEDA(Genbank登录号AY260763);AAP14954脂肪瘤HMGIC融合配偶样蛋白/pid=AAP14954.1-人;物种:人;WO2003054152(Claim 20); WO2003000842(Claim 1);WO2003023013(Example 3,Claim 20); US2003194704(Claim 45);交叉参考:GI:30102449;AAP14954.1; AY260763_1;
(26)BAFF-R(B细胞活化因子受体,BLyS受体3,BR3,Genbank登录号AF116456);BAFF受体/pid=NP_443177.1-人;Thompson,J.S.et al., Science 293(5537):2108-2111(2001);WO2004058309;WO2004011611; WO2003045422(Example;Page 32-33);WO2003014294(Claim 35;Fig 6B); WO2003035846(Claim 70;Page 615-616);WO200294852(Col 136-137); WO200238766(Claim 3;Page 133);WO200224909(Example3;Fig 3);交叉参考:MIM:606269;NP_443177.1;NM_052945_1;AF132600;
(27)CD22(B细胞受体CD22-B同等型,BL-CAM,Lyb-8,Lyb8, SIGLEC-2,FLJ22814,Genbank登录号AK026467);Wilson et al.(1991)J. Exp.Med.173:137-146;WO2003072036(Claim 1;Fig 1);交叉参考: MIM:107266;NP_001762.1;NM_001771_1;
(28)CD79a(CD79A,CD79α,免疫球蛋白相关α,一种B细胞特异性蛋白,其与Igβ(CD79B)共价相互作用且在表面上与Ig M分子形成复合物,转导牵涉B细胞分化的信号),pI:4.84,MW:25028,TM:2[P]基因染色体:19q13.2,Genbank登录号NP_001774.10);WO2003088808; US20030228319;WO2003062401(claim 9);US2002150573(claim 4,pages13-14);WO9958658(claim 13,Fig 16);WO9207574(Fig 1);US5644033;Ha et al.(1992)J.Immunol.148(5):1526-1531;Mueller et al.(1992)Eur.J.Biochem. 22:1621-1625;Hashimoto et al.(1994)Immunogenetics 40(4):287-295; Preud’homme et al.(1992)Clin.Exp.Immunol.90(1):141-146;Yu et al.(1992)J. Immunol.148(2)633-637;Sakaguchi et al.(1988)EMBO J.7(11):3457-3464;
(29)CXCR5(伯基特氏淋巴瘤受体1,一种G蛋白偶联受体,由CXCL13 趋化因子活化,在淋巴细胞迁移和体液防御中发挥功能,在HIV-2感染中及可能在AIDS,淋巴瘤,黑素瘤,和白血病发生中起作用);372aa,pI:8.54, MW:41959,TM:7[P]基因染色体:11q23.3,Genbank登录号NP_001707.1); WO2004040000;WO2004015426;US2003105292(Example 2);US6555339 (Example 2);WO200261087(Fig 1);WO200157188(Claim 20,page 269);WO200172830(pages 12-13);WO200022129(Example 1,pages 152-153, Example 2,pages254-256);WO9928468(claim 1,page 38);US5440021 (Example 2,col 49-52);WO9428931(pages 56-58);WO9217497(claim 7,Fig 5); Dobner et al.(1992)Eur.J.Immunol.22:2795-2799;Barella et al.(1995)Biochem. J.309:773-779;
(30)HLA-DOB(MHC II类分子的β亚基(Ia抗原),其结合肽并将它们呈递给CD4+T淋巴细胞);273aa,pI:6.56,MW:30820,TM:1[P] 基因染色体:6p21.3,Genbank登录号NP_002111.1);Tonnelle et al.(1985) EMBO J.4(11):2839-2847;Jonsson et al.(1989)Immunogenetics 29(6):411-413; Beck et al.(1992)J.Mol.Biol.228:433-441;Strausberg et al.(2002)Proc.Natl. Acad.Sci USA 99:16899-16903;Servenius etal.(1987)J.Biol.Chem. 262:8759-8766;Beck et al.(1996)J.Mol.Biol.255:1-13;Naruse et al.(2002) Tissue Antigens 59:512-519;WO9958658(claim 13,Fig 15);US6153408(Col 35-38);US5976551(col 168-170);US6011146(col 145-146);Kasaharaet al. (1989)Immunogenetics 30(1):66-68;Larhammar et al.(1985)J.Biol.Chem.260(26):14111-14119;
(31)P2X5(嘌呤能受体P2X配体门控离子通道5,一种由胞外ATP门控的离子通道,可能牵涉突触传递和神经发生,其缺陷可能有助于特发性逼尿肌不稳定性的病理生理学);422aa,pI:7.63,MW:47206,TM:1[P] 基因染色体:17p13.3,Genbank登录号NP_002552.2);Le et al.(1997)FEBS Lett.418(1-2):195-199;WO2004047749;WO2003072035(claim10);Touchman et al.(2000)Genome Res.10:165-173;WO200222660(claim 20);WO2003093444(claim 1);WO2003087768(claim 1);WO2003029277(page 82);
(32)CD72(B细胞分化抗原CD72,Lyb-2)完整蛋白质序列 maeaity...tafrfpd(1..359;359aa),pI:8.66,MW:40225,TM:1[P]基因染色体:9p13.3,Genbank登录号NP_001773.1);WO2004042346(claim 65); WO2003026493(pages 51-52,57-58);WO200075655(pages 105-106);Von Hoegen et al.(1990)J.Immunol.144(12):4870-4877;Strausberget al.(2002)Proc. Natl.Acad.Sci USA 99:16899-16903;
(33)LY64(淋巴细胞抗原64(RP105),富亮氨酸重复(LRR)家族的 I型膜蛋白,调节B细胞活化和凋亡,在患有系统性红斑狼疮的患者中其功能丧失与疾病活性升高有关);661aa,pI:6.20,MW:74147,TM:1[P] 基因染色体:5q12,Genbank登录号NP_005573.1);US2002193567;WO9707198(claim 11,pages 39-42);Miura et al.(1996)Genomics 38(3):299-304;Miura et al.(1998)Blood 92:2815-2822;WO2003083047; WO9744452(claim 8,pages 57-61);WO200012130(pages 24-26);
(34)FcRH1(Fc受体样蛋白1,一种含有C2型Ig样域和ITAM域的免疫球蛋白Fc域的假定受体,可能在B淋巴细胞分化中起作用);429aa, pI:5.28,MW:46925,TM:1[P]基因染色体:1q21-1q22,Genbank登录号NP_443170.1);WO2003077836;WO200138490(claim 6,Fig18E-1-18-E-2); Davis et al.(2001)Proc.Natl.Acad.Sci USA 98(17):9772-9777;WO2003089624 (claim 8);EP1347046(claim 1);WO2003089624(claim 7);
(35)IRTA2(免疫球蛋白超家族受体易位相关的2,一种可能在B细胞发育和淋巴瘤发生中起作用的假定免疫受体;在一些B细胞恶性肿瘤中发生易位所致基因失调);977aa,pI:6.88,MW:106468,TM:1[P]基因染色体:1q21,Genbank登录号,人:AF343662,AF343663,AF343664, AF343665,AF369794,AF397453,AK090423,AK090475,AL834187, AY358085;小鼠:AK089756,AY158090,AY506558;NP_112571.1; WO2003024392(claim 2,Fig 97);Nakayama et al.(2000)Biochem.Biophys.Res. Commun.277(1):124-127;WO2003077836;WO200138490(claim 3,Fig 18B-1-18B-2);
(36)TENB2(TMEFF2,tomoregulin,TPEF,HPP1,TR,推定的跨膜蛋白聚糖,与EGF/调蛋白家族的生长因子和卵泡抑素有关);374aa,NCBI 登录号:AAD55776,AAF91397,AAG49451,NCBI RefSeq:NP_057276; NCBI基因:23671;OMIM:605734;SwissProt Q9UIK5;Genbank登录号 AF179274;AY358907;CAF85723;CQ782436;WO2004074320(SEQ ID NO810);JP2004113151(SEQ ID NOS 2,4,8);WO2003042661(SEQ ID NO 580); WO2003009814(SEQ ID NO 411);EP1295944(pages 69-70);WO200230268 (page 329);WO200190304(SEQID NO 2706);US2004249130;US2004022727; WO2004063355;US2004197325;US2003232350;US2004005563; US2003124579;Horie et al.(2000)Genomics 67:146-152;Uchida et al.(1999) Biochem.Biophys.Res.Commun.266:593-602;Liang et al.(2000)Cancer Res. 60:4907-12;Glynne-Jones et al.(2001)Int J Cancer.Oct 15;94(2):178-84;
(37)PMEL17(银同系物;SILV;D12S53E;PMEL17;(SI);(SIL); ME20;gp100)BC001414;BT007202;M32295;M77348;NM_006928; McGlinchey,R.P.et al.(2009)Proc.Natl.Acad.Sci.U.S.A.106 (33):13731-13736;Kummer,M.P.et al.(2009)J.Biol.Chem.284(4):2296-2306;
(38)TMEFF1(具有EGF样和两个卵泡抑素样域的跨膜蛋白1; Tomoregulin-1;H7365;C9orf2;C9ORF2;U19878;X83961)NM_080655; NM_003692;Harms,P.W.(2003)GenesDev.17(21):2624-2629;Gery,S.et al. (2003)Oncogene 22(18):2723-2727;
(39)GDNF-Ra1(GDNF家族受体α1;GFRA1;GDNFR;GDNFRA; RETL1;TRNR1;RET1L;GDNFR-alpha1;GFR-ALPHA-1;U95847; BC014962;NM_145793)NM_005264;Kim,M.H.et al.(2009)Mol.Cell.Biol. 29(8):2264-2277;Treanor,J.J.et al.(1996)Nature 382(6586):80-83;
(40)Ly6E(淋巴细胞抗原6复合物,基因座E;Ly67,RIG-E,SCA-2, TSA-1)NP_002337.1;NM_002346.2;de Nooij-van Dalen,A.G.et al.(2003)Int. J.Cancer 103(6):768-774;Zammit,D.J.et al.(2002)Mol.Cell.Biol.22 (3):946-952;
(41)TMEM46(shisa同系物2(非洲爪蟾);SHISA2)NP_001007539.1; NM_001007538.1;Furushima,K.et al.(2007)Dev.Biol.306(2):480-492;Clark, H.F.et al.(2003)Genome Res.13(10):2265-2270;
(42)Ly6G6D(淋巴细胞抗原6复合物,基因座G6D;Ly6-D,MEGT1) NP_067079.2;NM_021246.2;Mallya,M.et al.(2002)Genomics 80(1):113-123; Ribas,G.et al.(1999)J.Immunol.163(1):278-287;
(43)LGR5(含富亮氨酸重复的G蛋白偶联受体5;GPR49,GPR67) NP_003658.1;NM_003667.2;Salanti,G.et al.(2009)Am.J.Epidemiol.170 (5):537-545;Yamamoto,Y.etal.(2003)Hepatology 37(3):528-533;
(44)RET(ret原癌基因;MEN2A;HSCR1;MEN2B;MTC1;(PTC); CDHF12;Hs.168114;RET51;RET-ELE1)NP_066124.1;NM_020975.4; Tsukamoto,H.et al.(2009)CancerSci.100(10):1895-1901;Narita,N.et al. (2009)Oncogene 28(34):3058-3068;
(45)LY6K(淋巴细胞抗原6复合物,基因座K;LY6K;HSJ001348; FLJ35226)NP_059997.3;NM_017527.3;Ishikawa,N.et al.(2007)Cancer Res. 67(24):11601-11611;deNooij-van Dalen,A.G.et al.(2003)Int.J.Cancer 103 (6):768-774;
(46)GPR19(G蛋白偶联受体19;Mm.4787)NP_006134.1;NM_006143.2; Montpetit,A.and Sinnett,D.(1999)Hum.Genet.105(1-2):162-164;O'Dowd, B.F.et al.(1996)FEBSLett.394(3):325-329;
(47)GPR54(KISS1受体;KISS1R;GPR54;HOT7T175;AXOR12) NP_115940.2;NM_032551.4;Navenot,J.M.et al.(2009)Mol.Pharmacol.75 (6):1300-1306;Hata,K.et al.(2009)Anticancer Res.29(2):617-623;
(48)ASPHD1(含天冬氨酸β-羟化酶域的1;LOC253982)NP_859069.2; NM_181718.3;Gerhard,D.S.et al.(2004)Genome Res.14(10B):2121-2127;
(49)酪氨酸酶(TYR;OCAIA;OCA1A;酪氨酸酶;SHEP3)NP_000363.1; NM_000372.4;Bishop,D.T.et al.(2009)Nat.Genet.41(8):920-925;Nan,H.et al.(2009)Int.J.Cancer125(4):909-917;
(50)TMEM118(环指蛋白,跨膜2;RNFT2;FLJ14627)NP_001103373.1; NM_001109903.1;Clark,H.F.et al.(2003)Genome Res.13(10):2265-2270; Scherer,S.E.etal.(2006)Nature 440(7082):346-351;
(51)GPR172A(G蛋白偶联受体172A;GPCR41;FLJ11856; D15Ertd747e)NP_078807.1;NM_024531.3;Ericsson,T.A.et al.(2003)Proc. Natl.Acad.Sci.U.S.A.100(11):6759-6764;Takeda,S.et al.(2002)FEBS Lett. 520(1-3):97-101。
亲本抗体还可以是包含清蛋白结合肽(ABP)序列的融合蛋白(Dennis et al.(2002)“Albumin Binding As A General Strategy For Improving ThePharmacokinetics Of Proteins”J Biol Chem.277:35035-35043;WO 01/45746)。本发明的抗体包括具有下列文献教导的ABP序列的融合蛋白:(i)Dennis et al. (2002)J BiolChem.277:35035-35043,表III和IV,第35038页;(ii)US 20040001827,[0076];和(iii)WO01/45746,第12-13页,通过援引将它们都收入本文。
为了通过诱变来制备半胱氨酸改造的抗体,通过本领域已知的多种方法制备编码起始多肽的氨基酸序列变体的DNA。这些方法包括但不限于通过对较早制备的编码多肽的DNA的定点(或寡核苷酸介导的)诱变,PCR诱变,和盒式诱变的制备。也可以通过限制性片段操作或通过使用合成寡核苷酸的交叠延伸PCR来构建重组抗体的变体。诱变引物编码半胱氨酸密码子替换。可采用标准诱变技术来生成编码此类突变体半胱氨酸改造的抗体的DNA。一般性指导方针可见于:Sambrook et al.,Molecular Cloning,A LaboratoryManual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.,1989;及Ausubel et al.,Current Protocols in Molecular Biology,Greene Publishing andWiley-Interscience,New York,N.Y.,1993。
可以使用重组方法和组合物来生成抗体,例如如US 4,816,567中记载的。在一个实施方案中,提供了编码包含抗体的VL的氨基酸序列和/或包含VH的氨基酸序列(例如抗体的轻链和/或重链)的分离的核酸。在又一个实施方案中,提供了包含此类核酸的一种或多种载体(例如表达载体)。在又一个实施方案中,提供了包含此类核酸的宿主细胞。在一个此类实施方案中,宿主细胞包含(例如已经用其转化):(1)包含如下核酸的载体,该核酸编码包含抗体的VL的氨基酸序列和包含抗体的VH的氨基酸序列,或(2)第一载体和第二载体,该第一载体包含编码包含抗体的VL的氨基酸序列的核酸,该第二载体包含编码包含抗体的VH的氨基酸序列的核酸。在一个实施方案中,宿主细胞是真核的,例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如Y0, NS0,Sp20细胞)。在一个实施方案中,一种方法包括在适合于抗体表达的条件下培养上文提供的包含编码抗体的核酸的宿主细胞,并任选地自宿主细胞(或宿主细胞培养液)回收抗体。
为了重组生成,分离编码抗体的核酸(例如如上文所述),并插入一种或多种载体中,以在宿主细胞中进一步克隆和/或表达。可以使用常规规程容易地对此类核酸进行分离和测序(例如通过使用如下寡核苷酸探针,该寡核苷酸探针能够特异性结合编码抗体的重链和轻链的基因)。对于克隆或表达抗体编码载体合适的宿主细胞包括本文中描述的原核或真核细胞。例如,可以在细菌中生成抗体,特别是在不需要糖基化和Fc效应器功能时。对于在细菌中表达抗体片段和多肽,参见例如US 5,648,237;US 5,789,199;及US 5,840,523(还可参见Charlton,Methods in Molecular Biology,Vol.248(B.K.C. Lo,ed.,HumanaPress,Totowa,NJ,2003),pp.245-254,其描述在大肠杆菌中表达抗体片段)。表达后,可以在可溶性级分中自细菌细胞糊分离抗体,而且可以进一步纯化。在原核生物以外,真核微生物(诸如丝状真菌或酵母)也是对于抗体编码载体合适的克隆或表达宿主,包括如下的真菌和酵母株,其糖基化途径已经“人源化”,导致生成具有部分或完全人的糖基化样式的抗体。参见Gerngross,Nat.Biotech.22:1409-1414(2004);及Li et al.,Nat.Biotech. 24:210-215(2006)。对于表达糖基化抗体合适的宿主细胞也是自多细胞生物体(无脊椎动物和脊椎动物)衍生的。无脊椎动物细胞的例子包括植物和昆虫细胞。已经鉴定出众多杆状病毒株,它们可以与昆虫细胞联合使用,特别是用于转染草地夜蛾(Spodoptera frugiperda)细胞。也可以利用植物细胞培养物作为宿主,诸如US 5,959,177;US 6,040,498;US 6,420,548;US 7,125,978;及US 6,417,429(描述用于在转基因植物中生成抗体的PLANTIBODIESTM技术)中描述的那些。也可以使用脊椎动物细胞作为宿主。例如,适应悬浮生长的哺乳动物细胞系可能是有用的。有用的哺乳动物宿主细胞系的其它例子有经SV40转化的猴肾CV1系(COS-7);人胚肾系(293或例如Graham et al.,J. Gen Virol.36:59(1977)中记载的293细胞);幼仓鼠肾细胞(BHK);小鼠塞托利(Sertoli)细胞(TM4细胞,如记载于例如Mather,Biol.Reprod.23:243-251 (1980));猴肾细胞(CV1);非洲绿猴肾细胞(VERO-76);人宫颈癌细胞 (HELA);犬肾细胞(MDCK);牛鼠(buffalo rat)肝细胞(BRL 3A);人肺细胞(W138);人肝细胞(Hep G2);小鼠乳房肿瘤(MMT 060562);TRI 细胞,如记载于例如Mather et al.,Annals N.Y.Acad.Sci.383:44-68(1982); MRC 5细胞;和FS4细胞。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞,包括DHFR-CHO细胞(Urlaub et al.,Proc.Natl.Acad.Sci.USA 77:4216(1980));和骨髓瘤细胞系,诸如Y0,NS0和Sp2/0。关于适合于抗体生成的某些哺乳动物宿主细胞系的综述,参见例如Yazaki and Wu,Methods inMolecular Biology,Vol.248(B.K.C.Lo,ed.,Humana Press,Totowa,NJ),pp. 255-268(2003)。
定点诱变是一种用于制备替代变体(即突变蛋白)的方法(Carter et al. (1985)Nucleic Acids Res.13:4431-4443;Ho et al.(1989)Gene(Amst.) 77:51-59;及Kunkelet al.(1987)Proc.Natl.Acad.Sci.USA 82:488)。如下改变起始DNA,即首先使编码期望突变的寡核苷酸与此类起始DNA的单链杂交。杂交后,使用DNA聚合酶来合成完整的第二链,其中使用杂交的寡核苷酸作为引物且使用起始DNA的单链作为模板。如此,将编码期望突变的寡核苷酸掺入所得双链DNA中。可以在表达质粒中在表达要诱变的蛋白质的基因内进行定点诱变,而且可以对所得质粒测序以确认期望半胱氨酸替换突变的引入(Liu et al.(1998)J.Biol.Chem.273:20252-20260)。定点诱变方案和型式是广泛可得的,例如多重定点诱变试剂盒(Stratagene, La Jolla,CA)。
PCR诱变也适合于生成起始多肽的氨基酸序列变体。参见Higuchi(1990) in PCRProtocols,pp.177-183,Academic Press;Ito et al.(1991)Gene 102:67-70;Bernhardet al.(1994)Bioconjugate Chem.,5:126-132;及Vallette et al.(1989)Nuc.AcidsRes.,17:723-733。简言之,在PCR中使用少量模板DNA 作为起始材料时,可以使用在序列上略微不同于模板DNA中的相应区域的引物来生成相对大量的特定DNA片段,它们仅在引物不同于模板的位置处不同于模板序列。
用于制备变体的另一种方法,即盒式诱变基于Wells et al.(1985)Gene, 34:315-323描述的技术。起始材料为包含要突变的起始多肽DNA的质粒(或其它载体)。鉴定起始DNA中要突变的密码子。在鉴定出的突变位点的每侧上必须有独特的限制性内切核酸酶位点。如果不存在此类限制性位点,那么可以产生它们,即使用上文所述寡核苷酸介导的诱变方法在起始多肽DNA 中的适宜位置处引入它们。在这些位点处切割质粒DNA以使它线性化。使用标准规程合成编码在限制性位点之间的DNA的序列但含有期望突变的双链寡核苷酸,其中使用标准技术分开合成寡核苷酸的两条链,然后杂交到一起。这种双链寡核苷酸称作盒。将这种盒设计成具有与线性化的质粒的末端相容的5′和3′末端,使得它能直接连接至质粒。这种质粒现在含有突变的DNA序列。可以通过DNA测序来确认含有所编码的半胱氨酸替换的突变DNA。
还通过基于PCR的诱变使用双链质粒DNA作为模板通过寡核苷酸指导的诱变产生单突变(Sambrook and Russel(2001)Molecular Cloning:A Laboratory Manual,3rdedition;Zoller et al.(1983)Methods Enzymol. 100:468-500;Zoller,M.J.and Smith,M.(1982)Nucl.Acids Res. 10:6487-6500)。
在某些实施方案中,可以将一处或多处氨基酸修饰引入本文中提供的抗体的Fc区中,由此生成Fc区变体。Fc区变体可以包含在一个或多个氨基酸位置处包含氨基酸修饰(例如替代)的人Fc区序列(例如人IgG1,IgG2,IgG3 或IgG4Fc区)。
在某些实施方案中,本发明涵盖拥有一些但非所有效应器功能的抗体变体,使其成为如下应用的期望候选,其中抗体的体内半衰期是重要的,但某些效应器功能(诸如补体和ADCC)是不必要的或有害的。可以进行体外和/ 或体内细胞毒性测定法以确认CDC和/或ADCC活性的降低/消减。例如,可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此很可能缺乏ADCC活性),但保留FcRn结合能力。
CBI二聚体药物模块
本发明的抗体-药物缀合物化合物包含CBI二聚体药物模块D。CBI二聚体药物模块可以由两个CBI药物单元或一个CBI药物单元和一个PBD药物单元构成。
CBI二聚体药物模块D具有式:
其中|
R1选自H,P(O)3H2,C(O)NRaRb;
R2选自H,P(O)3H2,C(O)NRaRb;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D′为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,其中R为C1-C6烷基或苄基;且
R5为H或C1-C6烷基。
在某些实施方案中,Y为苯基,吡啶基,1-甲基-1H-苯并[d]咪唑,或[1,2,4] 三唑并[1,5-a]吡啶。
CBI二聚体药物模块D化合物包括表1中的那些,它们对于制备表3的接头-CBI药物中间体是有用的。
表1:CBI二聚体药物模块化合物
对于制备CBI二聚体药物模块D化合物有用的拴系试剂包括表2中的那些。
表2:拴系试剂
接头
本发明的抗体-药物缀合物(ADC)化合物包含具有下式的接头L:
-Str-(Pep)m-(Sp)n-,
其中Str为共价附着至抗体的延伸物单元,Pep为任选的2-12个氨基酸残基的肽单元,Sp为任选的共价附着至二聚体药物模块的间隔物单元,且m和n独立选自0和1。
在一个例示性实施方案中,Str具有1,3-双取代的吡咯烷-2,5-二酮(琥珀酰亚胺)式:
其中R6选自下组:C1-C10亚烷基,C3-C8碳环基,O-(C1-C8烷基),亚芳基, C1-C10亚烷基-亚芳基,亚芳基-C1-C10亚烷基,C1-C10亚烷基-(C3-C8碳环基), (C3-C8碳环基)-C1-C10亚烷基,C3-C8杂环基,C1-C10亚烷基-(C3-C8杂环基), (C3-C8杂环基)-C1-C10亚烷基,C1-C10亚烷基-C(O)N(R8)-C2-C6亚烷基 -N(R8),N(R8)-(C2-C6亚烷基),和(CH2CH2O)r-CH2;其中R8为H或C1-C6烷基,且r为范围为1至10的整数。
Str延伸物单元的1,3-双取代的吡咯烷-2,5-二酮实施方案可以自抗体的半胱氨酸硫醇与接头-药物中间体(诸如表4中的那些)的马来酰亚胺基团缀合而形成。
在另一个例示性实施方案中,Str具有式:
其中R7选自C1-C10亚烷基,C1-C10亚烷基-O,N(R8)-(C2-C6亚烷基)-N(R8), N(R8)-(C2-C6亚烷基),和(CH2CH2O)r-CH2;其中R8为H或C1-C6烷基,且 r为范围为1至10的整数。
在另一个例示性实施方案中,Str具有式:
其中R9选自C1-C10亚烷基,C1-C10亚烷基-O,(C2-C6亚烷基)-N(R8),和 (CH2CH2O)r-CH2;其中R8为H或C1-C6烷基,且r为范围为1至10的整数。
在另一个例示性实施方案中,L与抗体的半胱氨酸氨基酸形成二硫键,且R9为C2-C6亚烷基-O,其中亚烷基任选是用F,OH,O(C1-C6烷基),NH2, NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的。
接头试剂和接头-药物中间体可以具有2-12个或更多个氨基酸残基的肽单元。
在一个例示性实施方案中,m为1且Pep包含2-12个独立选自甘氨酸,丙氨酸,苯丙氨酸,赖氨酸,精氨酸,缬氨酸,和瓜氨酸的氨基酸残基。
在一个例示性实施方案中,Pep为缬氨酸-瓜氨酸。
肽-接头试剂如记载的那样制备(WO 2012113847;US 7659241;US 7498298;US2009/0111756;US 2009/0018086;US 6214345;Dubowchik et al. (2002)BioconjugateChem.13(4):855-869)。
在一个例示性实施方案中,Sp包含对氨基苄基或对氨基苄氧羰基。
表3显示对于制备表4的接头-CBI药物中间体有用的例示性接头试剂。
表3:肽-接头试剂
对于ADC有用的接头-药物中间体
对于缀合至抗体以制备抗体-药物缀合物有用的接头-药物中间体具有式:
X-L-D,
其中:
X为选自马来酰亚胺,硫醇,氨基,溴化物,溴乙酰胺基,碘乙酰胺基,对甲苯磺酸根,碘化物,羟基,羧基,吡啶基二硫化物,和N-羟基琥珀酰亚胺的反应性官能团;
L为具有下式的接头:
-Str-(Pep)m-(Sp)n-,
其中Str为共价附着至反应性官能团X的延伸物单元,Pep为任选的2-12个氨基酸残基的肽单元,Sp为任选的共价附着至二聚体药物模块的间隔物单元,且 m和n独立选自0和1;
D为具有下式的二聚体药物模块:
其中
R1选自H,P(O)3H2,C(O)NRaRb,或与L的键;
R2选自H,P(O)3H2,C(O)NRaRb,或与L的键;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D'为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,或与L的键,其中R为C1-C6烷基或苄基;且
R5为H或C1-C6烷基。
如实施例1-18和图1-24中例示的,表4的接头-药物中间体通过将CBI二聚体药物模块与接头试剂偶联来制备。
表4:接头-CBI药物中间体
抗体-药物缀合物(ADC)
本发明的抗体-药物缀合物(ADC)化合物包含连接至有力的CBI二聚体药物模块的对肿瘤相关抗原特异性的抗体,而且包括那些具有治疗活性,针对一些过度增殖性病症(包括癌症)有效的。通过缀合至抗体来调控药物模块的生物学活性。本发明的ADC将有效剂量的CBI二聚体药物或毒素选择性投递至肿瘤细胞或部位,由此在提高治疗指数(“治疗窗”)的同时可实现更大的选择性(即更低的有效剂量)。在一个例示性实施方案中,ADC化合物包括缀合(即通过接头共价附着)至CBI二聚体药物模块的半胱氨酸改造的抗体。
本发明的抗体-药物缀合物化合物具有式:
Ab-(L-D)p,
其中:
Ab为抗体;
L为具有下式的接头:
-Str-(Pep)m-(Sp)n-,
其中Str为共价附着至抗体的延伸物单元,Pep为任选的2-12个氨基酸残基的肽单元,Sp为任选的共价附着至二聚体药物模块的间隔物单元,且m和n独立选自0和1;
p为1至8的整数;
D为具有下式的二聚体药物模块:
其中
R1选自H,P(O)3H2,C(O)NRaRb,或与L的键;
R2选自H,P(O)3H2,C(O)NRaRb,或与L的键;
Ra和Rb独立选自H和任选用一个或多个F取代的C1-C6烷基,或者Ra和Rb形成五或六元杂环基基团;
T为选自C3-C12亚烷基,Y,(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C1-C6亚烷基)-Y-(C1-C6亚烷基)-Y-(C1-C6亚烷基),(C2-C6亚烯基)-Y-(C2-C6亚烯基),和(C2-C6亚炔基)-Y-(C2-C6亚炔基)的拴系基团;
其中Y独立选自O,S,NR1,芳基,和杂芳基;
其中亚烷基,亚烯基,芳基,和杂芳基是独立且任选用F,OH,O(C1-C6烷基),NH2,NHCH3,N(CH3)2,OP(O)3H2,和C1-C6烷基取代的,其中烷基任选是用一个或多个F取代的;
或者亚烷基,亚烯基,芳基,和杂芳基是独立且任选用与L的键取代的;
D′为选自下述的药物模块:
其中波浪线指示附着至T的位点;
X1和X2独立选自O和NR3,其中R3选自H和任选用一个或多个F取代的C1-C6烷基;
R4为H,CO2R,其中R为C1-C6烷基或苄基;且
R5为H。
在一个实施方案中,Ra和Rb形成选自N-甲基哌嗪基,吗啉基,哌啶基,和吡咯烷基的五或六元杂环基基团。
在一个实施方案中,可以经组织蛋白酶B(在大多数哺乳动物细胞类型中找到的一种溶酶体蛋白酶)可切割的蛋白酶可切割肽接头将药物模块连接至抗体(US 6214345;Dubowchik et al(2002)Bioconj.Chem.13:855-869)。虽然本发明不受任何特定作用机制限制或限定,但是ADC可以作为前体药物起作用,即药物是无活性的,直至接头受到切割。ADC能够在疾病通过常规化疗可能得到较差治疗的肿瘤细胞部位中特异性地集中活性药物。在其它实施方案中,药物模块经非肽,非蛋白酶可切割接头(可以包括诸如二硫化物或琥珀酰亚胺基基团等官能度)附着至抗体。
可以经反应性接头模块缀合至抗体分子的药物模块的数目可以受到通过本文所述方法引入的游离半胱氨酸残基的数目限制。因此,例示性ADC 包含具有1,2,3,或4个工程化改造的半胱氨酸氨基酸的抗体(Lyon,R.et al(2012)Methods in Enzym.502:123-138)。
在一个实施方案中,抗体-药物缀合物化合物具有式:
其中AA1和AA2独立选自氨基酸侧链。氨基酸侧链独立选自H,-CH3, -CH2(C6H5),-CH2CH2CH2CH2NH2,-CH2CH2CH2NHC(NH)NH2,-CHCH(CH3)CH3,和-CH2CH2CH2NHC(O)NH2。
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
在一个实施方案中,抗体-药物缀合物化合物具有式:
其中R7独立选自H和C1-C12烷基。
在一个实施方案中,p为1,2,3或4。
在一个实施方案中,p为2。
在一个实施方案中,D为选自表1所列化合物或其衍生物的模块。
在一个实施方案中,L-D为选自表4所列化合物或其衍生物的模块。
在一个实施方案中,本发明涉及选自表5所列分子的缀合物。
ADC的药物载荷
药物载荷为每个抗体的CBI药物模块平均数。药物载荷的范围可以为每个抗体(Ab)1至8个药物(D),即其中1,2,3,4,5,6,7,和8个药物模块共价附着至抗体。ADC的组合物包括缀合有一定范围(1至8个)药物的抗体的集合。来自缀合反应的ADC制备物中每个抗体的药物平均数可通过常规手段来表征,诸如质谱术,ELISA,电泳,和HPLC。还可以测定ADC在p 方面的定量分布。通过ELISA,可以测定ADC的特定制备物中p的平均值 (Hamblett et al(2004)Clin.Cancer Res.10:7063-7070;Sanderson et al(2005) Clin.Cancer Res.11:843-852)。然而,p(药物)值的分布无法通过ELISA的抗体-抗原结合和检测限制来辨别。还有,用于检测抗体-药物缀合物的ELISA 不测定药物模块在哪里附着于抗体,诸如重链或轻链片段,或特定氨基酸残基。在一些情况中,p为某数值的同质ADC自具有其它药物载荷的ADC的分开,纯化,和表征可通过诸如反相HPLC或电泳等手段来实现。
对于一些抗体-药物缀合物,p可能受到抗体上附着位点的数目限制。例如,抗体可能只有一个或数个半胱氨酸硫醇基团,或者可能只有一个或数个有足够反应性的硫醇基团,可附着接头。较高的药物载荷(例如p>5)可引起某些抗体-药物缀合物的聚集,不溶性,毒性,或丧失细胞通透性。
通常,在缀合反应期间少于理论最大值的药物模块缀合至抗体。抗体可以包含例如许多赖氨酸残基,它们不与接头-药物中间物(X-L-D)或接头试剂反应。只有反应性最高的赖氨酸基团可以与胺反应性接头试剂反应。还有,只有反应性最高的半胱氨酸硫醇基团可以与硫醇反应性接头试剂或接头-药物中间体反应。一般地,如果有的话,抗体不包含许多游离的且反应性的半胱氨酸硫醇基团,它们可以连接至药物模块。化合物的抗体中的大多数半胱氨酸硫醇残基作为二硫桥存在,必须在部分或完全还原条件下用还原剂(诸如二硫苏糖醇(DTT)或TCEP还原。ADC的载荷(药物/抗体比,“DAR”) 可以以数种不同方式来控制,包括:(i)限制接头-药物中间体或接头试剂相对于抗体的摩尔过量,(ii)限制缀合反应时间或温度,和(iii)用于半胱氨酸硫醇修饰的部分或限制性还原性条件。
在抗体的多于一个亲核或亲电子基团与接头-药物中间体或接头试剂,接着是CBI二聚体药物模块试剂反应的情况中,所得产物是ADC化合物的混合物,具有例如1,2,3,等个药物模块附着于抗体的分布。液体层析方法 (诸如聚合物反相(PLRP)和疏水相互作用(HIC))可以根据药物载荷值将混合物中的化合物分开。可以分离具有单一药物载荷值(p)的ADC制备物,然而,这些单一载荷值ADC可能仍然是异质混合物,因为药物模块可以经接头附着于抗体上的不同位点。如此,本发明的抗体-药物缀合物组合物包括抗体-药物缀合物化合物的混合物,其中抗体具有一个或多个药物模块且其中药物模块可以在各个氨基酸残基处附着于抗体。
制备抗体-药物缀合物的方法
依照实施例20及如表4所示自接头-药物中间体51-68制备本发明的例示性抗体-药物缀合物(ADC)化合物101-139。
表5:抗体-药物缀合物(ADC)
*DAR=药物/抗体比平均
**A118C(EU编号方式)=A121C(连续编号方式)=A114C(Kabat编号方式)
抗CD22抗体
表5中的ADC的抗CD22抗体包含依照US 8226945的三个轻链高变区 (HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和 HVR-H3):
抗MUC16抗体
表5中的ADC的抗MUC16抗体包含依照US 7989595的三个轻链高变区 (HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和 HVR-H3):
表5中的ADC的抗MUC16抗体包含SEQ ID NO:13的可变重链序列和 SEQ ID NO:14的可变轻链序列。
在一个实施方案中,本发明的ADC的抗MUC16抗体是半胱氨酸改造的 ThioMab,包含一个或多个位于选自SEQ ID NO:15-32的轻链序列或选自 SEQ ID NO:33-46的重链序列中的游离半胱氨酸氨基酸残基:
在一个实施方案中,抗MUC16半胱氨酸改造的ThioMab为人源化抗体,包含SEQ IDNO:47的重链序列。
EVQLVESGGGLVQPGGSLRLSCAASGYSITNDYAWNWVRQAPGKGLEWVGYISYSGYTTY NPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARWTSGLDYWGQGTLVTVSSCSTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO:47)
在一个实施方案中,抗MUC16半胱氨酸改造的ThioMab为人源化抗体,包含SEQ IDNO:48的轻链序列。
DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAPKLLIYGATSLETGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO:48)
在一个实施方案中,抗MUC16半胱氨酸改造的ThioMab为嵌合抗体,包含SEQ IDNO:49的重链序列。
DVQLQESGPGLVNPSQSLSLTCTVTGYSITNDYAWNWIRQFPGNKLEWMGYINYSGYTTY NPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCARWDGGLTYWGQGTLVTVSACSTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO:49)
在一个实施方案中,抗MUC16半胱氨酸改造的ThioMab为嵌合抗体,包含SEQ IDNO:50的轻链序列。
DIQMTQSSSFLSVSLGGRVTITCKASDLIHNWLAWYQQKPGNAPRLLISGATSLETGVPS RFSGSGSGNDYTLSIASLQTEDAATYYCQQYWTTPFTFGSGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO:50)
抗HER2抗体
在某些实施方案中,表5的ADC包含抗HER2抗体。在本发明的一个实施方案中,本发明的ADC的抗HER2抗体包含人源化的抗HER2抗体,例如huMAb4D5-1,huMAb4D5-2,huMAb4D5-3,huMAb4D5-4, huMAb4D5-5,huMAb4D5-6,huMAb4D5-7和huMAb4D5-8,如US 5821337表3中描述的。那些抗体含有人框架区及结合HER2的鼠抗体 (4D5)的互补决定区。人源化抗体huMAb4D5-8也称作曲妥珠单抗 (trastuzumab),以商品名可购得。在本发明的另一个实施方案中,本发明的ADC的抗HER2抗体包含人源化抗HER2抗体,例如人源化2C4,如US 7862817中描述的。一种例示性人源化2C4抗体为帕妥珠单抗(pertuzumab),以商品名可购得。
用于制备表5的ADC的半胱氨酸改造的Thiomab抗体具有在重链118- 丙氨酸位点(EU编号方式)处引入的半胱氨酸残基。此位点根据连续编号方式编号为121或根据Kabat编号方式编号为114。
抗CD33抗体
表5中的ADC的抗CD33抗体15G15.33包含三个轻链高变区(HVR-L1, HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和HVR-H3)。
表5中的ADC的抗CD33抗体15G15.33包含SEQ ID NO:57的轻链可变区和/或SEQ IDNO:58的重链可变区。
EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSV SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK
(SEQ ID NO:57)
QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAREWADVFDIWGQGTMVTVSS
(SEQ ID NO:58)
抗CD33抗体9C3和其它实施方案
9C3 VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK
(SEQ ID NO:65)
9C3 VH
EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS
(SEQ ID NO:66)
9C3.2 VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK
(SEQ ID NO:67)
9C3.2 VH
EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS
(SEQ ID NO:68)
9C3.3 VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK
(SEQ ID NO:69)
9C3.3 VH
EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS
(SEQ ID NO:70)
9C3.4 VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK
(SEQ ID NO:71)
9C3.4 VH
EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS
(SEQ ID NO:72)
在一些实施方案中,本发明提供抗CD33抗体,其包含至少1,2,3,4, 5,或6种选自下述的HVR:(a)包含SEQ ID NO:62的氨基酸序列的HVR-H1; (b)包含SEQ ID NO:63的氨基酸序列的HVR-H2;(c)包含SEQ ID NO:64 的氨基酸序列的HVR-H3;(d)包含SEQ ID NO:59的氨基酸序列的HVR-L1; (e)包含SEQ ID NO:60的氨基酸序列的HVR-L2;和(f)包含SEQ IDNO: 61的氨基酸序列的HVR-L3。
在一个方面,本发明提供一种抗体,其包含至少一种,至少两种,或所有三种选自下述的VH HVR序列:(a)包含SEQ ID NO:62的氨基酸序列的 HVR-H1;(b)包含SEQ ID NO:63的氨基酸序列的HVR-H2;和(c)包含SEQ ID NO:64的氨基酸序列的HVR-H3。在一个实施方案中,该抗体包含包含 SEQ ID NO:64的氨基酸序列的HVR-H3。在另一个实施方案中,该抗体包含包含SEQ ID NO:64的氨基酸序列的HVR-H3和包含SEQ ID NO:61的氨基酸序列的HVR-L3。在又一个实施方案中,该抗体包含包含SEQ ID NO: 64的氨基酸序列的HVR-H3,包含SEQID NO:61的氨基酸序列的HVR-L3,和包含SEQ ID NO:63的氨基酸序列的HVR-H2。在又一个实施方案中,该抗体包含(a)包含SEQ ID NO:62的氨基酸序列的HVR-H1;(b)包含SEQ IDNO:63的氨基酸序列的HVR-H2;和(c)包含SEQ ID NO:64的氨基酸序列的HVR-H3。
在另一个方面,本发明提供一种抗体,其包含至少一种,至少两种,或所有三种选自下述的VL HVR序列:(a)包含SEQ ID NO:59的氨基酸序列的 HVR-L1;(b)包含SEQ ID NO:60的氨基酸序列的HVR-L2;和(c)包含SEQ ID NO:61的氨基酸序列的HVR-L3。在一个实施方案中,该抗体包含(a)包含SEQ ID NO:59的氨基酸序列的HVR-L1;(b)包含SEQ ID NO:60的氨基酸序列的HVR-L2;和(c)包含SEQ ID NO:61的氨基酸序列的HVR-L3。
在另一个方面,本发明的抗体包含(a)VH域,其包含至少一种,至少两种,或所有三种选自下述的VH HVR序列:(i)包含SEQ ID NO:62的氨基酸序列的HVR-H1,(ii)包含SEQ IDNO:63的氨基酸序列的HVR-H2,和(iii)包含SEQ ID NO:64的氨基酸序列的HVR-H3;和(b)VL域,其包含至少一种,至少两种,或所有三种选自下述的VL HVR序列:(i)包含SEQ ID NO:59的氨基酸序列的HVR-L1,(ii)包含SEQ ID NO:60的氨基酸序列的HVR-L2,和(c)包含SEQ IDNO:61的氨基酸序列的HVR-L3。
在另一个方面,本发明提供一种抗体,其包含(a)包含SEQ ID NO:62 的氨基酸序列的HVR-H1;(b)包含SEQ ID NO:63的氨基酸序列的HVR-H2; (c)包含SEQ ID NO:64的氨基酸序列的HVR-H3;(d)包含SEQ ID NO:59 的氨基酸序列的HVR-L1;(e)包含SEQ ID NO:60的氨基酸序列的HVR-L2;和(f)包含SEQ ID NO:61的氨基酸序列的HVR-L3。
在任何上述实施方案中,抗CD33抗体是人源化的。在一个实施方案中,抗CD33抗体包含任何上述实施方案中的HVR,且进一步包含人受体框架,例如人免疫球蛋白框架或人共有框架。在某些实施方案中,人受体框架为人 VL卡帕I共有(VLKI)框架和/或VH框架VH1。在某些实施方案中,人受体框架为包含任一下述突变的人VL卡帕I共有(VLKI)框架和/或VH框架VH1。
在另一个方面,抗CD33抗体包含与SEQ ID NO:66,SEQ ID NO:68, SEQ ID NO:70,和/或SEQ ID NO:72的氨基酸序列具有至少90%,91%, 92%,93%,94%,95%,96%,97%,98%,99%,或100%序列同一性的重链可变域(VH)序列。在某些实施方案中,与SEQID NO:66,SEQ ID NO: 68,SEQ ID NO:70,和/或SEQ ID NO:72的氨基酸序列具有至少90%,91%, 92%,93%,94%,95%,96%,97%,98%,或99%同一性的VH序列相对于参照序列含有替代(例如保守替代),插入,或删除,但是包含该序列的抗 CD33抗体保留结合CD33的能力。在某些实施方案中,已经在SEQ ID NO: 66,SEQ ID NO:68,SEQ ID NO:70,和/或SEQ ID NO:72中替代,插入和/或删除总共1至10个氨基酸。在某些实施方案中,已经在SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70,和/或SEQ ID NO:72中替代,插入和/ 或删除总共1至5个氨基酸。在某些实施方案中,替代,插入,或删除发生在 HVR以外的区域(即,在FR中)。任选地,抗CD33抗体包含SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70,和/或SEQID NO:72的VH序列,包括该序列的翻译后修饰。在一个特定实施方案中,VH包含1,2或3种选自下述的HVR:(a)包含SEQ ID NO:62的氨基酸序列的HVR-H1,(b)包含SEQ ID NO:63的氨基酸序列的HVR-H2,和(c)包含SEQ ID NO:64的氨基酸序列的HVR-H3。
在另一个方面,提供抗CD33抗体,其中该抗体包含与SEQ ID NO:65, SEQ ID NO:67,SEQ ID NO:69,和/或SEQ ID NO:71的氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,或100%序列同一性的轻链可变域(VL)序列。在某些实施方案中,与SEQ ID NO: 65,SEQ ID NO:67,SEQ ID NO:69,和/或SEQ ID NO:71的氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,或99%同一性的VL序列相对于参照序列含有替代(例如保守替代),插入,或删除,但是包含该序列的抗CD33抗体保留结合CD33的能力。在某些实施方案中,已经在SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,和/或SEQ ID NO: 71中替代,插入和/或删除总共1至10个氨基酸。在某些实施方案中,已经在 SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,和/或SEQ ID NO:71 中替代,插入和/或删除总共1至5个氨基酸。在某些实施方案中,替代,插入,或删除发生在HVR以外的区域(即,在FR中)。任选地,抗CD33抗体包含SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,和/或SEQ ID NO:71的VL 序列,包括该序列的翻译后修饰。在一个特定实施方案中,VL包含一种,两种或三种选自下述的HVR:(a)包含SEQ ID NO:59的氨基酸序列的 HVR-L1,(b)包含SEQ ID NO:60的氨基酸序列的HVR-L2,和(c)包含SEQ ID NO:61的氨基酸序列的HVR-L3。
在另一个方面,提供抗CD33抗体,其中该抗体包含上文提供的任何实施方案中的VH和上文提供的任何实施方案中的VL。
在一个实施方案中,抗体包含分别在SEQ ID NO:66和SEQ ID NO:65 中的VH和VL序列,包括那些序列的翻译后修饰。在一个实施方案中,抗体包含分别在SEQ ID NO:68和SEQ ID NO:67中的VH和VL序列,包括那些序列的翻译后修饰。在一个实施方案中,抗体包含分别在SEQ ID NO:70和 SEQ ID NO:69中的VH和VL序列,包括那些序列的翻译后修饰。在一个实施方案中,抗体包含分别在SEQ ID NO:72和SEQ ID NO:71中的VH和VL 序列,包括那些序列的翻译后修饰。
在又一个方面,本文中提供的是与本文中提供的抗CD33抗体结合相同表位的抗体。例如,在某些实施方案中,提供与包含SEQ ID NO:66,SEQ ID NO:68,SEQ ID NO:70,和/或SEQ ID NO:72的VH序列和SEQ ID NO: 65,SEQ ID NO:67,SEQ ID NO:69,和/或SEQ IDNO:71的VL序列的抗 CD33抗体结合相同表位的抗体。
在本发明的又一个方面,依照任何上述实施方案的抗CD33抗体是单克隆抗体,包括人抗体。在一个实施方案中,抗CD33抗体是抗体片段,例如 Fv,Fab,Fab’,scFv,双抗体,或F(ab’)2片段。在另一个实施方案中,抗体是基本上全长抗体,例如IgG1抗体,IgG2a抗体或本文中定义的其它抗体类或同种型。
在又一个方面,依照任何上述实施方案的抗CD33抗体可以单一地或组合地并入下文描述的任何特征。
体外细胞增殖测定法
一般地,如下测量抗体-药物缀合物(ADC)的细胞毒性或细胞抑制性活性:在细胞培养基中使具有受体蛋白(例如HER2)的哺乳动物细胞暴露于ADC的抗体;将细胞培养约6小时至约5天的时段;并测量细胞存活力。使用基于细胞的体外测定法来测量存活力(增殖),细胞毒性,和本发明ADC 的凋亡诱导(胱天蛋白酶活化)。
通过细胞增殖测定法测量抗体-药物缀合物(ADC)的体外效力(实施例21)。ADC在抑制肿瘤细胞增殖方面显示出令人惊讶且出乎意料的效力。将ADC的效力与细胞的靶抗原表达关联起来。图25-30的数据证明所测试的缀合物在体外能够结合细胞表面上表达的特定抗原并引起那些细胞死亡。
发光细胞存活力测定法是可商购的(Promega Corp., Madison,WI),基于鞘翅目(Coleoptera)萤光素酶的重组表达的同质测定法方法(US 5583024;US5674713;US 5700670)。此细胞增殖测定法基于对存在的ATP(代谢活性细胞的一种指示剂)进行定量来测定培养物中存活细胞的数目(Crouch et al(1993)J.Immunol.Meth.160:81-88;US 6602677)。以96孔型式进行测定法,使之易于进行自动化高通量筛选 (HTS)(Cree et al(1995)AntiCancer Drugs 6:398-404)。同质测定法规程牵涉将单一试剂(试剂)直接添加至在补充有血清的培养基中培养的细胞。无需细胞清洗,去除培养基和多次吸移的步骤。该系统在添加试剂并混合后10分钟里检测384孔型式低至15个细胞/孔。可以用ADC连续处理细胞,或者可以处理它们并与ADC分开。一般地,短暂(即3小时)处理的细胞显示出与连续处理的细胞相同的效力作用。
同质“添加-混合-测量”型式导致细胞裂解及产生与存在的ATP的量成比例的发光信号。ATP的量与培养物中存在的细胞的数目成正比。测定法产生“辉光型”发光信号,它是由萤光素酶反应产生的,具有一般大于5小时的半衰期,这取决于所使用的细胞类型和培养基。以相对发光单位(RLU)反映存活细胞。底物甲虫萤光素被重组萤火虫萤光素酶氧化脱羧基,同时ATP转化成AMP并产生光子。
使用基于细胞的体外测定法来测量存活力(增殖),细胞毒性,和本发明ADC的凋亡诱导(胱天蛋白酶活化)。一般地,如下测量抗体-药物缀合物 (ADC)的细胞毒性或细胞抑制性活性:在细胞培养培养基中使表达抗原(诸如Her2或MUC16多肽)的哺乳动物细胞暴露于ADC;将细胞培养约6小时至约5天的时段;并测量细胞存活力。对于抗MUC16ADC的细胞增殖测定法有用的哺乳动物细胞包括:(1)表达MUC16多肽的细胞系OVCAR-3;(2)工程化改造成在其细胞表面上稳定表达MUC16多肽一部分的PC3衍生细胞系 (PC3/MUC16);(3)不表达MUC16多肽的亲本PC3细胞系;和(4)不表达 MUC16多肽但携带用于驱动外源MUC16表达的载体的PC3细胞系 (PC3/neo)。
图25以3天时SK-BR-3体外细胞存活力对Thio hu抗CD22HC A121C-MC-vc-PAB-(CBI二聚体)101和Thio hu抗Her2HC A121C-MC-vc-PAB-(CBI二聚体)102浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。Her2抗原在SK-BR-3细胞中高度表达。抗Her2ADC 102显示线性的非剂量响应性的细胞杀伤活性,其中作为对照的脱靶抗CD22ADC 101 显示更少的活性。
图26以3天时SK-BR-3体外细胞存活力对Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI-PBD)103和Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI-PBD)104浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。抗Her2ADC 103显示线性的非剂量响应性的细胞杀伤活性,而对照脱靶抗CD22ADC 104显示更少的活性。
图27以3天时SK-BR-3体外细胞存活力对Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI二聚体)116和Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体)117浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。抗Her2ADC 117显示线性的非剂量响应性的细胞杀伤活性,而对照脱靶抗CD22ADC 116显示更少的活性。
图28以3天时EOL-1体外细胞存活力对Thio Hu抗CD33 15G15.33HC A118C-MC-MMED-(CBI二聚体phos)125浓度(μg/ml)的曲线显示抗体-药物缀合物的功效,其显示适度的剂量响应性的细胞杀伤活性。
图29以3天时EOL-1体外细胞存活力对Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126和Thio Hu抗CD33 15G15.33 HC A118C-MC-ED-(CBI二聚体DVBdiphos)127浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。抗CD33ADC 127显示有力的剂量响应性的细胞杀伤活性,而对照脱靶抗MUC16ADC 126显示更少的活性。
图30以3天时EOL-1体外细胞存活力对Thio Hu抗CD33 15G15.33HC A118C-MC-ED-(CBI二聚体DVB diphos)127,Thio Hu抗CD33 15G15.33HC A118C-MC-ED-(CBI二聚体DVBphos)129,和Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB phos)130浓度(μg/ml)的曲线显示抗体-药物缀合物的功效。抗CD33ADC 127和129显示有力的剂量响应性的细胞杀伤活性,而对照脱靶抗MUC16ADC 130显示更少的活性。
体内功效
本发明的抗体-药物缀合物(ADC)的体内功效可以通过小鼠中的肿瘤异种移植物研究(实施例22)来测量。以小鼠中的肿瘤生长抑制(实施例21) 测量抗体-药物缀合物(ADC)的体内功效。ADC在抑制肿瘤生长方面显示出令人惊讶且出乎意料的效力。将ADC的功效与肿瘤细胞的靶抗原表达关联起来。
如下在体内测量抗体-药物偶联物的功效,即在啮齿动物中植入癌细胞的同种异体移植物或异种移植物并用ADC处理肿瘤。预期会有可变的结果,这取决于细胞系,ADC对癌细胞上存在的受体的抗体结合的特异性,剂量给药方案,和其它因素。使用表达中等至高水平的肿瘤相关抗原(诸如Her2, MUC16,和CD33)的转基因外植体小鼠模型测量ADC的体内功效。将受试者用ADC处理一次并监测3-6周以测量肿瘤倍增时间,细胞杀伤对数,和肿瘤收缩。进行跟踪剂量-响应和多剂量实验。
例如,可以通过高表达HER2转基因外植体小鼠模型(Phillips et al(2008)Cancer Res.68:9280-90)测量本发明的抗HER2ADC的体内功效。自不响应或较差响应疗法的Fo5mmtv转基因小鼠扩充同种异体移植物。将受试者用ADC于某个剂量水平(mg/kg)和安慰剂缓冲液对照(媒介) 处理一次并监测2周或更久以测量肿瘤倍增时间,细胞杀伤对数,和肿瘤收缩。
图31以接种入CRL nu/nu小鼠乳房脂肪垫中的MMTV-HER2Fo5转基因乳房肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯, pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD22 10F4v3HC A118C-MC-MMED-(CBI二聚体phos)110,(3)Thio Hu抗CD22 10F4v3HCA118C-MC-vc-PAB-(CBI二聚体MePip)108,(4)Thio Hu抗CD22 10F4v3HC A118C-MC-vc-PAB-(CBI二聚体phos)111,(5)Thio Hu抗Her2 4D5HC A118C-MC-MMED-(CBI二聚体phos)109,(6)Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体MePip)107,(7)Thio Hu抗Her2 4D5HC A118C-MC-vc-PAB-(CBI二聚体phos)112。ADC以10mg/kg给药。
图32以接种入CRL nu/nu小鼠乳房脂肪垫中的MMTV-HER2Fo5转基因乳房肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯, pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD22 10F4v3HC A118C-DSE-(CBI二聚体phos)120,10mg/kg,(3)Thio Hu抗CD2210F4v3HC A118C-DSE-(CBI二聚体phos)122,10mg/kg,(4)Thio Hu抗CD22 10F4v3HCA118C-MC-vc-PAB-(N10,PBD-CBI MePip)124,10mg/kg,(5)Thio Hu抗 Her2 4D5HC A118C-DSE-(CBI二聚体phos)119,3mg/kg,(6)Thio Hu抗Her2 4D5HC A118C-DSE-(CBI二聚体phos)119,10mg/kg,(7)Thio Hu抗Her2 4D5HC A118C-DSP-(CBI二聚体phos)121,3mg/kg,(8)Thio Hu抗Her2 4D5 HC A118C-DSP-(CBI二聚体phos)121,10mg/kg,(9)Thio Hu抗Her24D5HC A118C-MC-vc-PAB-(N10,PBD-CBI MePip)123,3mg/kg,(10)Thio Hu抗 Her2 4D5HCA118C-MC-vc-PAB-(N10,PBD-CBI MePip)123,10mg/kg。
图33以接种入C.B-17SCID小鼠中的OVCAR3X2.1人卵巢肿瘤在下述一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯,pH 5.5,240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD3315G15.33HC A118C-MC-ED-(CBI二聚体DVB diphos)127,3mg/kg,(3)Thio Hu抗MUC163A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126,3mg/kg,(4)Thio Hu抗MUC16 3A5HCA118C-MC-ED-(CBI二聚体DVB diphos)126,1mg/kg。
ADC 126的抗MUC16抗体,而且包括3A5和11D10变体,已经记载于WO 2007/001851;US 7989595;US 8449883,通过援引收录其内容。根据OVCAR-3 Scatchard分析,3A5单克隆抗体以433pM亲和力结合MUC16多肽的多个位点 (Chen et al(2007)Cancer Res.67(10):4924-4932)。
图34以接种入C.B-17SCID小鼠中的HL-60人急性髓样白血病在下述以 20μg/m2一次IV给药后随时间的体内拟合肿瘤体积变化的曲线显示抗体-药物缀合物的功效:(1)媒介:组氨酸缓冲液#8:20mM组氨酸乙酸酯,pH 5.5, 240mM蔗糖,0.02%PS 20,(2)Thio Hu抗CD33 15G15.33HC A118C-MC-MMED-(CBI二聚体phos)125,(3)Thio Hu抗CD33 15G15.33HCA118C-MC-ED-(CBI二聚体DVB diphos)127,(4)Thio Hu抗MUC16 3A5HC A118C-MC-MMED-(CBI二聚体phos)128,(5)Thio Hu抗MUC16 3A5HC A118C-MC-ED-(CBI二聚体DVB diphos)126。
抗体-药物缀合物的施用
可以通过对于要治疗的疾患适宜的任何路径来施用本发明的抗体-药物缀合物(ADC)。通常会胃肠外施用ADC,即输注,皮下,肌肉内,静脉内,真皮内,鞘内和硬膜外。
药物配制剂
本发明的治疗性抗体-药物缀合物(ADC)的药物配制剂通常与药学可接受胃肠外媒介一起以单位剂量可注射形式配制,供胃肠外施用,即推注,静脉内,肿瘤内注射。任选地,以冻干配制剂或水溶液的形式将具有期望纯度的抗体-药物缀合物(ADC)与药学可接受稀释剂,载剂,赋形剂或稳定剂混合(Remington's Pharmaceutical Sciences(1980)16thedition,Osol,A. Ed.)。
抗体-药物缀合物治疗
涵盖本发明的抗体-药物缀合物(ADC)可以用于治疗各种疾病或病症,例如特征在于肿瘤抗原过表达的。例示性疾患或过度增殖性病症包括良性或恶性实体瘤和血液学病症(诸如白血病和淋巴样恶性肿瘤)。其它包括神经元,胶质,星形细胞,下丘脑,腺体,巨噬细胞,上皮,基质,囊胚腔,炎性,血管发生性和免疫学病症,包括自身免疫性病症。
一般地,要治疗的疾病或病症为过度增殖性疾病,诸如癌症。本文中要治疗的癌症的例子包括但不限于癌瘤,淋巴瘤,母细胞瘤,肉瘤,和白血病或淋巴样恶性肿瘤。此类癌症的更特别例子包括鳞状细胞癌(例如上皮鳞状细胞癌),肺癌,包括小细胞肺癌,非小细胞肺癌,肺的腺癌和肺的鳞癌,腹膜癌,肝细胞癌,胃癌,包括胃肠癌,胰腺癌,成胶质细胞瘤,宫颈癌,卵巢癌,肝癌,膀胱癌,肝瘤,乳腺癌,结肠癌,直肠癌,结直肠癌,子宫内膜癌或子宫癌,唾液腺癌,肾癌,前列腺癌,外阴癌,甲状腺癌,肝癌,肛门癌,阴茎癌,以及头和颈癌。
ADC化合物可用于治疗的自身免疫性疾病包括风湿病学病症(诸如例如类风湿性关节炎,斯耶格仑氏综合征,硬皮病,狼疮,诸如系统性红斑狼疮(SLE)和狼疮性肾炎,多肌炎/皮肌炎,冷球蛋白血症,抗磷脂抗体综合征,和银屑病性关节炎),骨关节炎,自身免疫性胃肠和肝病症(诸如例如炎性肠病(例如溃疡性结肠炎和克罗恩(Crohn)氏病),自身免疫性胃炎和恶性贫血,自身免疫性肝炎,原发性胆汁性肝硬化,原发性硬化性胆管炎,和乳糜泻(celiac disease)),血管炎(诸如例如ANCA相关血管炎,包括丘-施(Churg-Strauss)血管炎,韦格纳(Wegener)氏肉芽肿病和多动脉炎),自身免疫性神经学病症(诸如例如多发性硬化,眼阵挛肌阵挛综合征,重症肌无力,视神经脊髓炎,帕金森(Parkinson)氏病,阿尔茨海默(Alzheimer) 氏病,和自身免疫性多神经病),肾病症(诸如例如肾小球肾炎,古德帕斯丘(Goodpasture)氏综合征,和贝格尔(Berger)氏病),自身免疫性皮肤学病症(诸如例如银屑病,荨麻疹(urticaria),荨麻疹(hives),寻常型天疱疮,大疱性类天疱疮,和皮肤红斑狼疮),血液病学病症(诸如例如血小板减少性紫癜,血栓性血小板减少性紫癜,输血后紫癜,和自身免疫性溶血性贫血),动脉粥样硬化,葡萄膜炎,自身免疫性听觉疾病(诸如例如内耳疾病和听力丧失),贝切特(Behcet)氏病,雷诺(Raynaud)氏综合征,器官移植,和自身免疫性内分泌病症(诸如例如糖尿病相关自身免疫性疾病,诸如胰岛素依赖性糖尿病(IDDM),阿狄森(Addison)氏病,和自身免疫性甲状腺病 (例如格雷夫斯(Graves)氏病和甲状腺炎))。更优选的此类疾病包括例如类风湿性关节炎,溃疡性结肠炎,ANCA相关血管炎,狼疮,多发性硬化,斯耶格仑氏综合征,格雷夫斯氏病,IDDM,恶性贫血,甲状腺炎,和肾小球肾炎。
对于预防或治疗疾病,ADC的适宜剂量会取决于如上文定义的要治疗的疾病的类型,疾病的严重程度和进程,施用该分子是为了预防还是治疗目的,先前的疗法,患者的临床史和对抗体的响应,及主治医师的斟酌。一次或在一系列治疗里恰当地将该分子施用于患者。根据疾病的类型和严重程度,约 1μg/kg至15mg/kg(例如0.1-20mg/kg)的分子是对患者施用的初始候选剂量,无论是例如通过一次或多次分开的施用,还是通过连续输注。典型的日剂量的范围可以是约1μg/kg至100mg/kg或更多,取决于上文所述因素。要施用于患者的ADC的一种例示性剂量在约0.1至约10mg/kg患者体重的范围中。
制品
在本发明的另一个实施方案中,提供装有对于治疗上文所述病症有用的材料的制品或“试剂盒”。制品包括容器和在容器上或与容器相关的标签或包装插页。合适的容器包括例如瓶,管形瓶,注射器,泡罩包,等。容器可以自各种材料(诸如玻璃或塑料)形成。容器装有对于治疗疾患有效的抗体 -药物缀合物(ADC)组合物,而且可以具有无菌存取口(例如,容器可以是具有皮下注射针可刺穿的塞子的管形瓶或静脉内溶液袋)。组合物中的至少一种活性剂是ADC。标签或包装插页指示组合物用于治疗所选择的疾患,诸如癌症。或者/另外,制品可以进一步包括第二(或第三)容器,其装有药学可接受缓冲液,诸如抑菌性注射用水(BWFI),磷酸盐缓冲盐水,林格 (Ringer)氏溶液和右旋糖溶液。它可以进一步包括从商业和使用者立场看想要的其它材料,包括其它缓冲剂,稀释剂,滤器,针,和注射器。
实施例
实施例1:2-(2-溴-N-甲基乙酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3- 二氢-1H-苯并[e]吲哚-5-基酯51
遵循L.F.Tietze,J.M.von Hof,M.Müller,B.Krewer,I.Schuberth,Angew.Chem.Int.Ed.(2010)49:7336-7339的规程,将1-(氯甲基)-5-羟基-1H-苯并[e] 吲哚-3(2H)-羧酸(S)-叔丁酯51a用HCl去保护,并用戊二酰基二氯酰化(图1)。代替制备性HPLC,在真空下去除反应溶剂,并与甲醇一起研磨所得残留物,给出1,5-二((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮 51b,为灰白色固体(产率51%)。Rf=0.50(乙酸乙酯/石油醚=2:1)。NMR 和MS数据与报告数值相同。[α]D 26=-46.3°(c=0.41,DMA)。
对在冰浴中冷却的51b(650mg,1.15mmol)在DMA(5mL)中的溶液添加DIPEA(0.40mL,2.31mmol)和氯甲酸4-硝基苯酯(302mg,1.50 mmol)。容许混合物升温至室温并搅动过夜之后,添加甲基(2-(甲基氨基)乙基)氨基甲酸叔丁酯(652mg,3.00mmol)。于室温将混合物搅动7小时,然后在乙酸乙酯和冷的稀NaHCO3水溶液之间重分配。用乙酸乙酯提取水相三次。用水,接着用盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过柱层析使用乙酸乙酯和石油醚的梯度混合物(v/v 1:1至4:1),然后是仅乙酸乙酯作为洗脱液进一步纯化残留物,给出甲基(2-(甲基氨基)乙基)氨基甲酸Boc(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5- 羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯51c,为灰白色固体(359mg,40%);mp 157℃(dec.)。[α]D 26=-45.4° (c=1.08,乙酸乙酯)。1H NMR(DMSO)(旋转异构体的混合物)δ10.35 (s,1H),8.25(s,1H),8.08(d,J=8.2Hz,1H),8.02(s,1H),7.87-7.77 (m,2H),7.58(t,J=7.6Hz,1H),7.50-7.42(m,2H),7.31(t,J=7.9 Hz,1H),4.42(t,J=9.7Hz,1H),4.36-4.31(m,2H),4.25(d,J=10.3 Hz,1H),4.19-4.13(m,2H),4.05(dd,J=2.9,11.0Hz,1H),3.98(dd, J=2.6,10.9Hz,1H),3.92(dd,J=7.2,10.9Hz,1H),3.79(dd,J=8.5,10.2Hz,1H),3.69(br s,1H),3.52-3.42(m,3H),3.21-2.79(m,6H, 2NMe),2.75-2.67(m,2H),2.64-2.58(m,2H),2.00-1.93(m,2H),1.44-1.35 (m,9H,But)ppm。HRMS(ESI)发现m/z777.2801(M+H)。C41H47Cl2N4O7要求777.2816。
还自同一层析分开获得二Boc保护的51d和无保护的回收的起始材料 51b,为白色固体(15mg,2%)。获得51d(429mg,37%),为灰白色固体。 [α]D 26=-32.0°(c=1.00,乙酸乙酯)。1H NMR(DMSO)(旋转异构体的混合物)δ8.25(s,2H),7.95(d,J=8.4Hz,2H),7.87-7.76(m,2H),7.58 (t,J=7.6Hz,2H),7.44(t,J=7.6Hz,2H),4.41(t,J=9.8Hz,2H), 4.32(brs,2H),4.24(d,J=10.6Hz,2H),4.06-4.03(m,2H),3.98(dd, J=7.3,10.8Hz,2H),3.69(brs,2H),3.52-3.43(m,6H),3.21-2.79(m, 12H,4NMe),2.75-2.69(m,2H),2.66-2.58(m,2H),1.99-1.95(m,2H), 1.44-1.35(m,18H,2But)ppm。HRMS(ESI)发现m/z 1013.3991(M+Na)。C51H64Cl2N6NaO10要求1013.3991。
对在冰浴中冷却的51c(200mg,0.26mmol)在DCM(3mL)中的溶液逐滴添加三氟乙酸,TFA(1.5mL)。容许混合物升温至室温,并搅动2小时。去除所有挥发性成分,给出粗制的甲基(2-(甲基氨基)乙基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯51e的三氟乙酸盐,为灰白色固体,直接使用。1H NMR(DMSO)(旋转异构体的混合物)δ10.36(s,1H),8.64(br s,1H),8.48(br s,1H),8.35(s,1H),8.08(d,J=8.1Hz,1H), 8.01(s,1H),7.97(d,J=8.4Hz,1H),7.94-7.88(m,1H),7.78(d,J =8.4Hz,1H),7.61-7.57(m,2H),7.51-7.45(m,2H),7.34-7.30(m, 1H),4.43(t,J=9.8Hz,1H),4.36-4.31(m,2H),4.26(d,J=10.4Hz,1H),4.18-4.14(m,2H),4.06(dd,J=2.9,11.0Hz,1H),4.00(dd,J= 2.7,10.8Hz,1H),3.94(dd,J=7.5,11.0Hz,1H),3.89-3.84(m,1H), 3.79(dd,J=8.1,10.8Hz,1H),3.63(t,J=5.7Hz,1H),3.33-3.30(m, 6H,2NMe),2.78-2.55(m,6H),2.00-1.94(m,2H)ppm。HRMS(ESI)发现m/z 677.2306(M+H)。C36H39Cl2N4O5要求677.2292。
于-5℃,对51e在THF(4mL)中的溶液添加一滴DIPEA,接着缓慢添加溴乙酰基溴化物(34μL,0.39mmol),然后是剩余的DIPEA(总共448μL, 2.57mmol)。容许混合物升温至室温,并搅动1小时。通过旋转蒸发器,然后是高真空泵去除所有挥发性成分。与乙酸乙酯一起搅动所得残留物,并过滤掉不溶性固体,之后将滤出液加载到层析柱上。使用乙酸乙酯和石油醚的梯度混合物(v/v 1:4至1:1)作为洗脱液,给出2-(2-溴-N-甲基乙酰胺基)乙基 (甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯51,为灰白色固体 (80mg,39%);mp167-170℃。[α]D 26=-64.0°(c=0.25,乙酸乙酯)。1H NMR (DMSO)(旋转异构体的混合物)δ10.39(s,1H),8.25-8.20(m,1H), 8.08(d,J=8.3Hz,1H),8.01(s,1H),7.96(d,J=8.3Hz,1H),7.89 (d,J=7.4Hz,0.34H),7.82(d,J=7.4Hz,0.66H),7.78(d,J=8.4Hz, 1H),7.58(t,J=7.6Hz,1H),7.51-7.45(m,2H),7.31(t,J=7.6Hz, 1H),4.42(t,J=9.6Hz,1H),4.36-4.31(m,1H),4.24(d,J=10.7Hz, 1H),4.18-4.09(m,3H,包括CH2Br),4.07-4.03(dd,J=2.8,10.9Hz, 1H),4.00-3.97(dd,J=2.6,10.8Hz,1H),3.96-3.91(m,1H),3.82-3.75(m,1H),3.70-3.68(m,1H),3.58-3.43(m,3H),3.26-2.89(m,6H, 2NMe),2.78-2.67(m,2H),2.65-2.55(m,2H),1.99-1.92(m,2H)ppm。 HRMS(ESI)发现m/z 819.1316(M+Na)。C38H38BrCl2N4O6要求819.1322。
实施例2:8-(6-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H- 苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(11S,11aS)-叔丁酯52
遵循实施例6的实验规程,制备接头-药物中间体52(图7和8)。HPLC: 96.7%纯;mp210℃(dec.);1H NMR[(CD3)2SO]δ10.03(s,与D2O可交换, 1H),8.23-8.11(m,2H),8.09(d,J=7.3Hz,与D2O可交换,1H),7.85 (d,J=8.5Hz,1H),7.80(d,J=8.3Hz,与D2O可交换,1H),7.66(d, J=8.6Hz,2H),7.59-7.44(m,3H),7.38(br t,J=7.6Hz,1H),7.04 (s,1H),6.99(s,2H),6.69(s,1H),6.38(br s,与D2O可交换,1H), 5.99(t,J=5.5Hz,与D2O可交换,1H),5.49-5.34(m,3H,D2O后降至1 H成为d,J=9.5Hz),5.20(s,2H),4.44-4.30(m,2H),4.26-4.13(m, 3H),4.10-3.91(m,3H),3.88-3.76(m,1H),3.79(s,3H),3.53-3.44 (m,1H),3.41-3.20(m,被水峰部分模糊,4H),3.09-2.88(m,2H), 2.66-2.42(m,被DMSO峰部分模糊,2H),2.25-1.24(m,21H),1.31(s, 9H),1.24-1.11(m,2H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.7Hz, 3H)。分析物(C55H71ClN8O11·H2O)计算:C,61.53;H,6.85;N,10.44。发现:C,61.39;H,7.11;N,10.15。
实施例3:N-((R)-1-(氯甲基)-3-(5-((R)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基)-6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺53
于室温(r.t.)将乙酸(50mL)添加至1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯53a(1.00g,2.01mmol)在 THF-H2O(150mL/75mL)中的搅动溶液,并将混合物搅动过夜(图2)。19.5 小时后在真空下去除THF,用EtOAc稀释混合物,充分摇动各层,然后分开。用饱和NaHCO3水溶液(4次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:EtOAc 100:0至90:10的纯化给出5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯53b(382mg,57%),为橙色凝胶样固体。1HNMRδ(400MHz,DMSO-d6)8.01(d,J=8.4Hz, 1H),7.64(d,J=8.0Hz,1H),7.42-7.38(m,1H),7.37(br s,1H),7.22-7.18 (m,1H),5.91(s,2H),4.08-3.91(m,4H),3.66(dd,J=10.6,8.2Hz, 1H),1.53(s,9H)。
在氮下于室温将53b(380mg,1.14mmol),6-(2,5-二氧-2,5-二氢-1H- 吡咯-1-基)己酸(361mg,1.71mmol),盐酸1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDCI.HCl,765mg,3.99mmol)和对甲苯磺酸(TsOH,49mg, 0.285mmol)在干的DMA(10mL)中的混合物搅动过夜。17小时后在真空下去除溶剂。在硅胶上通过柱层析使用DCM:己烷75:25至100:0,然后是DCM:MeOH 99:1至97:3纯化粗制的产物,并蒸发含有产物的级分至干燥。然后在EtOAc中溶解所得材料,用H2O(2次)清洗有机层,干燥(Na2SO4),并去除溶剂,给出1-(氯甲基)-5-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯53c(282mg,47%),为黄色固体。1H NMRδ(400MHz,DMSO-d6)9.86(s,1H),8.24(br s,1H),7.99 (d,J=8.4Hz,1H),7.88(d,J=8.3Hz,1H),7.55-7.51(m,1H),7.43-7.39 (m,1H),7.01(s,2H),4.21-4.11(m,2H),4.08-4.00(m,2H),3.87 (dd,J=10.9,6.9Hz,1H),3.42(t,J=7.0Hz,2H),2.45(t,J=7.1Hz, 2H),1.69-1.62(m,2H),1.56-1.54(m,2H),1.54(s,9H),1.35-1.28 (m,2H)。
于0℃将三氟乙酸(3.9mL)和H2O(0.1mL)添加至53c(66mg,0.125 mmol)在DCM(4mL)中的溶液。于0℃将混合物搅动1小时20分钟,然后添加冰和H2O。于0℃将混合物用饱和NaHCO3水溶液碱化至pH 8。分开有机层和水层,用H2O(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂,给出(R)-N-(1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺53d(50mg,94%),为黄色固体,无需纯化在下一步中使用。1H NMRδ(400MHz,DMSO-d6)9.70(s,1H),7.87(d,J= 8.5Hz,1H),7.64(d,J=8.2Hz,1H),7.39(ddd,J=8.1,6.9,1.0Hz, 1H),7.21-7.15(m,2H),7.01(s,2H),5.92(s,1H),4.02-3.96(m, 1H),3.85(dd,J=10.8,3.5Hz,1H),3.69(t,J=9.3Hz,1H),3.63-3.55 (m,2H),3.42(t,J=7.0Hz,2H),2.42(t,J=7.1Hz,2H),1.64(td, J=15.2,7.6Hz,2H),1.55(td,J=14.5,7.2Hz,2H),1.35-1.26(m, 2H)。
在氮下于室温将53d(50mg,0.117mmol),(R)-5-(1-(氯甲基)-5-羟基-1H- 苯并[e]吲哚-3(2H)-基)-5-氧戊酸53e(56mg,0.161mmol),盐酸1-(3-二甲基氨基丙基)-3-乙基碳二亚胺EDCI.HCl(58mg,0.303mmol)和TsOH(8mg,0.0465mmol)在干的DMA(2mL)中的混合物搅动过夜。19小时后用H2O 稀释混合物,并自溶液沉淀出固体。用EtOAc(1次),DCM(1次),DCM:MeOH 95:5(1次)提取含水悬浮液,干燥(Na2SO4)合并的有机物,并在真空下去除溶剂。在硅胶上通过柱层析使用DCM:MeOH 100:0至94:6纯化粗制的产物,给出N-((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基)-6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺53(15mg,17%,HPLC纯度:82.3%),为黄色固体。1H NMRδ(400MHz,DMSO-d6)10.35(s,1H),9.88(s,1H),8.58(s, 1H),8.08(d,J=8.2Hz,1H),8.02(s,1H),7.96(d,J=8.3Hz,1H), 7.92(d,J=8.4Hz,1H),7.78(d,J=8.3Hz,1H),7.55(t,J=7.4Hz, 1H),7.51-7.47(m,1H),7.46-7.42(m,1H),7.33-7.30(m,1H),7.01 (s,2H),4.42-4.28(m,3H),4.25-4.13(m,3H),4.01(ddd,J=19.6, 10.9,2.6Hz,2H),3.90(dd,J=10.9,7.5Hz,1H),3.79(dd,J=10.8,8.1Hz,1H),3.43(t,J=6.7Hz,2H),2.77-2.57(m,4H),2.46-2.43(m, 2H),2.01-1.96(m,2H),1.70-1.62(m,2H),1.60-1.53(m,2H),1.36-1.28 (m,2H)。HRMS m/z 777.2186[为C41H40Cl2N4NaO6计算的(M+Na)+为 777.2217]。
实施例3a:1-((S)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)- 基)-5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮53j
在氮下于-20℃将10%Pd/C(1.5g)添加至(S)-5-(5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸57c(2.00g,4.57mmol)在THF-25% NH4HCO2水溶液(60mL/23mL)中的搅动溶液(图3)。于-15至-10℃将反应混合物搅动3.5小时。然后将反应混合物于-20℃保持过夜。17.5小时后将混合物升温至-10℃,并于-10至-5℃搅动5小时。然后容许混合物升温至0℃,并于此温度搅动30分钟,然后用MeOH稀释,穿过硅藻土过滤,用MeOH(3次)清洗硅藻土栓,并在真空下浓缩溶剂直至自溶液沉淀出固体。然后将这用H2O(150mL)和己烷(150mL)稀释,并于室温在用浓HCl酸化至pH 1 时搅动。将混合物再搅动30分钟,然后通过过滤来收集固体,用H2O和己烷清洗,并干燥,给出(S)-5-(1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸53h(1.43g,90%),为米色固体。1H NMRδ(400MHz,DMSO-d6)12.07(br s,1H),10.35(s,1H),8.08(d,J=8.0Hz,1H),7.98(s,1H), 7.77(d,J=8.3Hz,1H),7.50-7.46(m,1H),7.33-7.29(m,1H),4.30 (t,J=10.4Hz,1H),4.14-4.12(m,2H),3.98(dd,J=10.9,2.8Hz, 1H),3.78(dd,J=10.8,7.8Hz,1H),2.63-2.45(m,2H),2.35(t,J=7.4 Hz,2H),1.89-1.78(m,2H)。
于室温将氯化氢气体(HCl)鼓泡穿过1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯53f(425mg,0.855mmol)在干的二烷(12mL)中的溶液(在3A分子筛上)(图3)。自溶液沉淀出固体,并在15分钟后在真空下去除溶剂。无需纯化在下一步中使用粗制的固体, (S)-1-(氯甲基)-N-(二苯基亚甲基)-2,3-二氢-1H-苯并[e]吲哚-5-胺53g。在氮下于室温将干的DMA(10mL)添加至53g,(S)-5-(1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸53h(327mg,0.941mmol),EDCI.HCl(573mg, 2.99mmol)和3A(埃)分子筛的混合物。2天19.5小时后在真空下去除溶剂。在硅胶上通过柱层析使用DCM:MeOH 100:0至90:10纯化粗制的材料,然后使用己烷:DCM 100:0至50:50至0:100,然后是DCM:MeOH 99:1至98:2再次对材料进行层析,给出1-((S)-1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚 -3(2H)-基)-5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮 53i(193mg,31%,在2个步骤上,自53f)。1H NMRδ(400MHz,DMSO-d6) 10.35(s,1H),8.08(d,J=8.1Hz,1H),8.00(s,1H),7.84(t,J=9.0 Hz,2H),7.79-7.74(m,3H),7.62-7.57(m,2H),7.54-7.46(m,4H), 7.40-7.36(m,1H),7.33-7.29(m,1H),7.27-7.22(m,3H),7.09-7.08(m, 2H),4.34-4.26(m,2H),4.22-4.12(m,4H),4.01-3.97(m,2H),3.83-3.76 (m,2H),2.69-2.50(m,4H),1.93-1.86(m,2H)。HRMS m/z 726.2264[为C44H38Cl2N3O3计算的(M+H)+为726.2285]。
于室温将乙酸(HOAc,8mL)添加至53i(190mg,0.261mmol)在 THF-H2O(24mL/12mL)中的搅动溶液,并将混合物搅动过夜。19小时后用H2O稀释混合物,并沉淀出固体。在真空下去除THF,并用DIPEA处理含水的悬浮液直至中性。通过过滤来收集固体,用H2O清洗,并干燥。在DMF (1.5mL)中溶解固体,并用MeOH稀释,引起固体沉淀。倒掉溶剂,并将己烷:DCM90:10添加至固体,摇动悬浮液,并倒掉溶剂。使用己烷:EtOAc 90:10,接着是单独的己烷重复此过程。然后在DMF/THF中溶解固体,吸收到硅胶上,并使用DCM:MeOH 100:0至90:10洗脱产物。通过制备性HPLC(柱: Synergi-MAX RP 4μ,21.20x 250mm;流速:13mL/min;流动相:溶剂A: H2O/TFA pH 2.5,溶剂B:MeCN/H2O 90:10;方法:等度,溶剂A:溶剂B 20:80, 15分钟;波长:254nm,330nm)来进一步纯化材料,给出1-((S)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)戊烷-1,5-二酮53j(30mg,22%,HPLC纯度:87.9%),为白色固体。1H NMRδ(400MHz,DMSO-d6)10.36(s,1H),8.08(d,J=8.1Hz, 1H),8.04-8.02(m,2H),7.79-7.77(m,2H),7.69(d,J=8.3Hz,1H),7.51-7.47(m,1H),7.42(t,J=7.5Hz,1H),7.34-7.30(m,1H),7.23 (t,J=7.6Hz,1H),5.91(s,2H),4.36-4.26(m,2H),4.19-4.13(m, 3H),4.08-4.04(m,1H),4.01-3.93(m,2H),3.79(dd,J=10.7,8.2Hz, 1H),3.70(dd,J=10.5,8.9Hz,1H),2.75-2.66(m,2H),2.63-2.54(m,2H),2.00-1.93(m,2H)。HRMS m/z 584.1456[为C31H29Cl2N3NaO3计算的 (M+Na)+为584.1478]。[α]D 28=-37.6°(c=0.559,DMSO)。
实施例3b:1-((R)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)- 基)-5-((R)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮53p
于0℃将三乙胺(Et3N,0.54mL,3.89mmol)和三氟甲磺酸酐(0.60mL, 3.60mmol)添加至1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯 53k(1.00g,3.00mmol)在DCM(100mL)中的搅动溶液(图4)。于0℃将反应搅动20分钟,然后用H2O稀释,分出各层,并用DCM(1次)提取水层。干燥(Na2SO4)合并的有机层,并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:EtOAc 100:0至96:4的纯化给出1-(氯甲基)-5-(三氟甲基磺酰基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯531(1.30g,93%),为橙色泡沫状固体。1H NMRδ(400MHz,CDCl3)8.30(br s,1H),8.03(d,J=8.5Hz, 1H),7.76(d,J=8.4Hz,1H),7.62-7.58(m,1H),7.53-7.49(m,1H), 4.32(br s,1H),4.20-4.15(m,1H),4.09-4.03(m,1H),3.92(dd,J=11.2, 2.8Hz,1H),3.54-3.49(m,1H),1.61(s,9H)。
在氮下将53l(1.30g,2.79mmol)在干的THF(60mL,脱气的)中的溶液添加至Cs2CO3(1.27g,3.91mmol),BINAP(209mg,0.336mmol)和 Pd(OAc)2(63mg,0.281mmol)的混合物。然后添加二苯甲酮亚胺(0.56mL, 3.34mmol),并在氮下将混合物回流过夜。20小时后将反应温度降至60-65 ℃,并在氮下于此温度将反应搅动1天。添加另外的THF(10mL),并于相同温度将混合物再搅动1天,之后再次将更多的THF(25mL)添加至混合物。再过1天后添加另外部分的Pd(OAc)2(19mg,0.0846mmol),BINAP(52mg, 0.0835mmol)和THF(30mL),并在氮下将混合物于70℃加热过夜。再过 28小时后再次添加另外部分的Pd(OAc)2(31mg,0.138mmol),BINAP(104 mg,0.167mmol),并继续反应22小时。然后将反应混合物冷却至室温,用 DCM稀释,穿过硅藻土过滤,用DCM清洗硅藻土栓直至清洗液中不再有颜色,并在真空下蒸发滤出液。在硅胶上通过柱层析使用己烷:DCM 100:0至 50:50的纯化给出1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(R)-叔丁酯53a(1.19g,85%),为黄色泡沫状固体。1H NMRδ(400MHz, DMSO-d6)7.85(d,J=8.3Hz,1H),7.80(d,J=8.4Hz,1H),7.75(d, J=7.2Hz,2H),7.61-7.57(m,1H),7.54-7.49(m,3H),7.37-7.33(m, 1H),7.30-7.23(m,3H),7.06(d,J=6.8Hz,2H),4.13-4.02(m,2H), 4.00-3.94(m,2H),3.77(dd,J=10.9,7.5Hz,1H),1.46(s,9H),1H 未观察到。[α]D 27=+101°(c=1.04,DCM)。
于室温将氯化氢气体(HCl(g))鼓泡通过53a(300mg,0.604mmol) 在干的二烷(10mL)中的溶液(在3A分子筛上)。10分钟后自溶液沉淀出固体,并在20分钟后在真空下去除溶剂。无需纯化在下一步中使用粗制的固体,(R)-1-(氯甲基)-N-(二苯基亚甲基)-2,3-二氢-1H-苯并[e]吲哚-5-胺53m。
在氮下于-10℃将碳上的钯,10%Pd/C(690mg)添加至(R)-5-(5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸53n(1.38g,3.15mmol) 在THF-25%NH4HCO2水溶液(40mL/16mL)中的搅动溶液。于-10至-5℃将反应混合物搅动3小时。然后将反应混合物于-20℃保持过夜。15小时后于 -20℃用MeOH稀释混合物,穿过硅藻土过滤,用MeOH清洗硅藻土栓,并在真空下浓缩溶剂直至自溶液沉淀出固体。然后用H2O(130mL)和己烷(100 mL)稀释悬浮液,并于室温在用浓HCl酸化至pH 1时搅动。将混合物搅动30 分钟,沉降,并倒掉己烷。添加另外的己烷(120mL),并将混合物再搅动 30分钟,再次倒掉己烷,然后通过过滤来收集固体,用H2O和己烷清洗,并干燥,给出(R)-5-(1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸53e (925mg,84%),为米色固体。1H NMRδ(400MHz,DMSO-d6)12.06(br s,1H),10.35(s,1H),8.08(d,J=8.0Hz,1H),7.98(s,1H),7.77 (d,J=8.3Hz,1H),7.50-7.46(m,1H),7.33-7.29(m,1H),4.30(t, J=10.5Hz,1H),4.14-4.12(m,2H),3.98(dd,J=10.9,2.8Hz,1H), 3.78(dd,J=10.8,7.9Hz,1H),2.63-2.45(m,2H),2.35(t,J=7.4Hz, 2H),1.89-1.78(m,2H)。
在氮下于室温将干的DMA(8mL)添加至来自先前反应的53m,53e(241 mg,0.693mmol),EDCI.HCl(404mg,2.11mmol)和3A分子筛的混合物。将反应混合物搅动过夜。19.5小时后用H2O稀释混合物,并过滤所得悬浮液。用EtOAc(3次)提取含水的滤出液,在EtOAc/MeOH中溶解过滤出的固体,并与EtOAc提取液合并。将合并的有机溶液吸收到硅胶上,并使用己烷:DCM 50:50至0:100,然后是DCM:MeOH 99.5:0.5至97:3洗脱产物,给出1-((R)-1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-基)-5-((R)-1-(氯甲基)-5- 羟基-1H-苯并[e]吲哚-3(2H)-基)戊烷-1,5-二酮53o(155mg,35%,在2个步骤上,自53a),为深黄色固体。1H NMRδ(400MHz,DMSO-d6)10.35(s, 1H),8.08(d,J=8.0Hz,1H),8.00(s,1H),7.85(t,J=8.9Hz,2H), 7.79-7.74(m,3H),7.62-7.57(m,2H),7.54-7.46(m,4H),7.40-7.36(m, 1H),7.33-7.29(m,1H),7.27-7.22(m,3H),7.09-7.08(m,2H),4.34-4.27 (m,2H),4.22-4.11(m,4H),4.02-3.97(m,2H),3.83-3.76(m,2H), 2.69-2.51(m,4H),1.93-1.85(m,2H),NMR谱匹配53i的。HRMS m/z 748.2077 [为C44H37Cl2N3NaO3计算的(M+Na)+为748.2104]。
于室温将乙酸(HOAc,4mL)添加至53o(80mg,0.110mmol)在THF-H2O (12mL/6mL)中的搅动溶液,并将混合物搅动过夜。18小时后用H2O稀释混合物,并沉淀出固体。在真空下去除THF,并用DIPEA处理含水的悬浮液直至pH 8,导致更多固体沉淀。通过过滤来收集固体,干燥,并在硅胶上通过柱层析使用DCM:MeOH 100:0至97:3纯化,给出1-((R)-5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-((R)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)戊烷-1,5-二酮53p(20mg,32%),为棕色固体。1H NMRδ(400 MHz,DMSO-d6)10.36(s,1H),8.08(d,J=8.2Hz,1H),8.04-8.02(m, 2H),7.79-7.77(m,2H),7.69(d,J=8.3Hz,1H),7.51-7.47(m,1H), 7.42(t,J=7.5Hz,1H),7.34-7.30(m,1H),7.23(t,J=7.6Hz,1H),5.91(s,2H),4.36-4.26(m,2H),4.19-4.13(m,3H),4.08-4.04(m,1H), 4.01-3.93(m,2H),3.79(dd,J=10.7,8.3Hz,1H),3.71(dd,J=10.5, 8.9Hz,1H),2.75-2.66(m,2H),2.63-2.53(m,2H),2.00-1.93(m,2H), NMR谱匹配53j的。
实施例4:N-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧-2,3,5,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e] 吲哚-5-基氧)甲基)苯基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺54
在氮气氛中在回流下将通过Tercel et al.(2003)J.Med.Chem 46:2132-2151的规程制备的(S)-(2-氨基-4-羟基-5-甲氧基苯基)(2-(羟基甲基) 吡咯烷-1-基)甲酮54a(7.6g,28.6mmol)和二碳酸二叔丁酯(12.48g,57.2 mmol)在无水THF(140mL)中的混合物搅动18小时。将反应混合物冷却至室温,并添加2N NaOH(57.2mL,114mmol)和MeOH(70mL)。于室温将混合物搅动6小时。于35-40℃(浴温度)在减压下蒸发挥发物。添加冰水(250mL),并于0℃将pH调节至8-9。于室温将混合物与石油醚-乙酸乙酯 (20:1)(2x400mL)一起搅动15分钟。分出有机层,并丢弃。用DCM(4x300 mL)提取水层,干燥(MgSO4)合并的提取液,并在减压下蒸发,给出5- 羟基-2-(2-(羟基甲基)吡咯烷-1-羰基)-4-甲氧基苯基氨基甲酸(S)-叔丁酯54b,为粉色-白色固体(9.36g,89%);mp 154-156℃;1H NMR[(CD3)2SO]δ9.51 (s,1H),8.90(s,1H),7.27(s,1H),6.91(s,1H),4.73(t,J=5.8 Hz,1H),4.16-4.02(m,1H),3.73(s,3H),3.64-3.34(m,4H),1.99-1.60 (m,4H),1.43(s,9H)。分析物(C18H26N2O6)计算:C,59.00;H,7.15; N,7.65。发现:C,58.94;H,7.31;N,7.39。
对54b(2.88g,7.87mmol)和通过Tercel et al.(2003)J.Med.Chem 46:2132-2151的规程制备的6-溴己酸2,2,2-三氯乙酯(3.86g,11.8mmol)在干的DMA(7mL)中的溶液添加无水K2CO3(2.61g,18.9mmol)。于室温将所得混合物搅动68小时。将它倒入冰-水(600mL)中,并将产物提取入乙酸乙酯(600mL)中。用冷的(0℃)2N Na2CO3水溶液(2x400mL)和水(400mL)连续清洗提取液,然后干燥(MgSO4)。蒸发溶剂给出褐色油,通过SiO2柱层析(DCM-乙酸乙酯=2:1)纯化,给出纯的6-(5-(叔丁氧羰基氨基)-4-(2-(羟基甲基)吡咯烷-1-羰基)-2-甲氧基苯氧基)己酸(S)-2,2,2-三氯乙酯54c(3.62g,76%),为浅黄色泡沫;mp 36-39℃;1H NMR[(CD3)2SO]δ 9.90(s,1H),7.33(s,1H),6.93(s,1H),4.89(s,2H),4.74(t,J =5.8Hz,1H),4.17-4.02(m,1H),3.94(t,J=6.4Hz,2H),3.73(s, 3H),3.63-3.26(m,4H),2.55-2.46(m,2H,被DMSO峰部分模糊),2.00-1.55 (m,8H),1.53-1.36(m,11H)。分析物(C26H37N2O8)计算:C,51.03; H,6.09;N,4.58。发现:C,51.33;H,6.21;N,4.35。
于室温在15分钟里对54c(3.62g,5.92mmol)在干的DCM(12mL)中的溶液分批添加戴斯-马丁高碘烷(DMP,1,1,1-三乙酰氧基-1,1-二氢-1,2-苯碘酰-3(1H)-酮,CAS登录号87413-09-0)(3.27g,7.70mmol)。于室温将反应混合物搅动45分钟。用DCM(800mL)稀释,并用10%Na2S2O3(100mL),冷的(0℃)NaHCO3溶液(400mL),和水(300mL)连续清洗,然后干燥(MgSO4)。蒸发溶剂给出琥珀色固体,通过SiO2柱层析(石油醚-乙酸乙酯= 3:4)纯化,给出11-羟基-7-甲氧基-5-氧-8-(6-氧-6-(2,2,2-三氯乙氧基)己基氧)-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)- 叔丁酯54d(2.61g,72%),为粘性泡沫;1H NMR[(CD3)2SO]δ7.03(s,1 H),6.67(s,1H),6.38(s,1H),5.41(s,1H),4.89(s,2H),4.06-3.87 (m,2H),3.79(s,3H),3.52-3.43(m,1H),3.42-3.28(m,1H,被水峰部分模糊),3.27-3.20(m,1H),2.08-1.82(m,5H),1.81-1.71(m, 2H),1.71-1.61(m,2H),1.53-1.40(m,3H),1.31(s,9H)。HRMS (ESI)m/z为C26H35Cl3N2NaO8计算:631.1351,发现:631.1361[MNa+]。
在氮下对54d(1.80g,2.95mmol)在丙酮-水(3:2)(100mL)中的搅动溶液添加Zn(7.72g,118mmol)和NH4Cl(6.32g,118mmol)。于室温将混合物搅动28小时。倒掉上清液,并用NaHCO3水溶液(3x100mL)清洗 Zn残留物。合并清洗液和上清液,并与DCM(300mL,然后是2x100mL) 一起搅动。分出DCM层,并丢弃。于0℃将水层用浓HCl酸化至pH<1。将产物提取入DCM(400mL;2x200mL)中,干燥(MgSO4),并蒸发,给出8-(5- 过氧羟基己-5-烯基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯54e(1.34g,95%),为无色泡沫;mp 65-67℃;1H NMR[(CD3)2SO]δ11.99(brs,1H),7.04(s,1 H),6.67(s,1H),6.37(br s,1H),5.41(d,J=9.4Hz,1H),4.06-3.87 (m,2H),3.79(s,3H),3.53-3.19(m,3H,被水峰部分模糊),2.22 (t,J=7.2Hz,2H),2.09-1.81(m,4H),1.80-1.67(m,2H),1.62-1.49 (m,2H),1.49-1.37(m,2H),1.31(s,9H)。分析物(C24H34N2O8·1/4H2O) 计算:C,59.68;H,7.20;N,5.80。发现:C,59.37;H,7.20;N,5.62。
在氮气氛下于室温将54e(1.16g,2.42mmol),(S)-1-(氯甲基)-5-(4-硝基苄基氧)-2,3-二氢-1H-苯并[e]吲哚54f(893mg,2.42mmol),EDCI.HCl(1.39 g,7.26mmol),和无水TsOH(83mg,0.48mmol)在DMA(7mL)中的混合物搅动4小时。添加水(120mL),并于室温将混合物搅动15分钟。过滤出沉淀的固体,用水(4x40mL),0.01%NH4OH(4x40mL),和石油醚(4x40 mL)连续清洗,然后干燥,给出N-Boc-(S)-8-(6-((S)-1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5(10H)-酮54g(1.71g,85%),为浅黄色固体;mp130-133℃;1H NMR[(CD3)2SO]δ8.30(d,J=8.8Hz,2H), 8.23(d,J=8.1Hz,1H),8.18(s,1H),7.92-7.82(m,3H),7.57(td,J=8.2,1.1Hz,1H),7.43(t,J=8.0Hz,1H),7.03(s,1H),6.69(s, 1H),6.39(br s,1H),5.51-5.35(m,3H),4.36(t,J=9.5Hz,1H), 4.28-4.15(m,2H),4.10-3.90(m,3H),3.85(dd,J=11.1,7.8Hz,1H), 3.79(s,3H),3.52-3.42(m,1H),3.42-3.20(m,2H,被水峰部分模糊), 2.68-2.50(m,2H,被DMSO峰部分模糊),2.11-1.95(m,1H),1.95-1.75 (m,5H),1.75-1.61(m,2H),1.59-1.45(m,2H),1.31(s,9H)。分析物(C44H49ClN4O10)计算:C,63.72;H,5.96;N,6.76。发现:C,63.33; H,5.97;N,6.92。
在氮下对54g(1.70g,2.05mmol)在THF(90mL),丙酮(70mL),和水(40mL)的混合物中的搅动溶液添加Zn(2.68g,41.0mmol)和NH4Cl (4.39g,82.0mmol)。于室温将混合物搅动45分钟,然后穿过硅藻土过滤,用THF清洗数次。于室温在减压下将滤出液浓缩至约60mL。添加0.01% NH4OH的溶液(400mL),并于室温将混合物搅动15分钟。收集固体,并用 0.01%NH4OH(3x100mL),水(3x100mL),和石油醚(3x100mL)连续清洗。干燥固体,给出54g的苯胺基衍生物(1.16g,100%),用干的DMA (2mL)中的6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸(253mg,1.20mmol), EDCI.HCl(576mg,3.00mmol)和无水TsOH(34.4mg,0.20mmol)处理。在氮下于室温将混合物搅动17小时。添加NaHCO3溶液(50mL),并于室温将混合物搅动30分钟。收集固体,用水清洗,干燥,并通过硅土柱层析(DCM- 乙酸乙酯=1:1)纯化,给出纯的8-(6-((S)-1-(氯甲基)-5-(4-(6-(2,5-二氧-2,5- 二氢-1H-吡咯-1-基)己酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯54h(194mg,33%),为黄色固体,mp 128-132 ℃;1H NMR[(CD3)2SO]δ9.91(s,1H),8.20-8.11(m,2H),7.84(d,J= 8.3Hz,1H),7.62(d,J=8.5Hz,2H),7.54(br t,J=8.1Hz,1H),7.47 (d,J=8.5Hz,2H),7.38(brt,J=8.0Hz,1H),7.04(s,1H),7.00 (s,2H),6.69(s,1H),6.38(br s,1H),5.45-5.37(m,1H),5.20(s, 2H),4.36(t,J=10.2Hz,1H),4.26-4.13(m,2H),4.10-3.92(m,3H), 3.88-3.79(m,1H),3.79(s,3H),3.52-3.30(m,4H),3.24(br t,J=8.9 Hz,1H),2.66-2.50(m,2H,被DMSO峰部分模糊),2.28(t,J=7.3Hz, 2H),2.08-1.95(m,1H),1.95-1.76(m,5H),1.76-1.64(m,2H),1.64-1.20 (m,8H),1.31(s,9H)。分析物(C54H62ClN5O11)计算:C,65.35;H,6.30;N,7.06。发现:C,65.08;H,6.39;N,6.67。
对在氮气氛下于-10至-11℃搅动的N-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧-2,3,5,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺54h(204mg,0.21mmol)在DCM(15mL)中的溶液逐滴添加(在30分钟里)含有2.5%水的TFA(15mL)。添加后于此温度将混合物进一步搅动4小时。于0℃将混合物倒入冰,DCM,和足以给出pH 7-8的饱和NaHCO3溶液的混合物中。于室温将混合物搅动15分钟。分出DCM层,并用更多的NaHCO3水溶液和水清洗,然后干燥(MgSO4)。于25℃(浴温度) 蒸发溶剂,给出黄色固体(172mg,回收94%材料)。通过制备性HPLC (Synergi-Max RP柱)(用30%甲酸铵缓冲液pH=3.5;70%(10%)乙腈水溶液洗脱;流速:13mL/min)来纯化此粗制产物,给出54(30mg,16%), HPLC:98.8%纯;mp 190℃(dec.);[α20 D]+320°(c 0.100,DCM);1H NMR [CDCl3]δ8.29(d,J=8.3Hz,1H),8.18(s,1H),7.69-7.63(m,2H), 7.61-7.44(m,6H),7.37(br t,J=7.3Hz,2H),6.82(s,1H),6.66(s, 2H),5.24(s,2H),4.34-4.19(m,2H),4.19-4.00(m,3H),3.99-3.92 (m,1H),3.89(s,3H),3.86-3.78(m,1H),3.76-3.70(m,1H),3.63-3.49 (m,3H),3.42(t,J=10.8Hz,1H),2.68-2.47(m,2H),2.40-2.27(m, 3H),2.12-1.92(m,5H),1.92-1.82(m,2H),1.82-1.71(m,2H),1.71-1.54 (m,4H,被水峰部分模糊),1.43-1.32(m,2H)。HRMS(ESI)m/z为 C49H52ClN5NaO8计算:896.3397,发现:896.3375[MNa+]。
实施例5:N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基 -1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5- 二氢-1H-吡咯-1-基)己酰胺55
于室温将碳酸钾,K2CO3(2.50g,18.1mmol)添加至1-(氯甲基)-5-羟基 -1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a(2.01g,6.02mmol)和1-(溴甲基)-4-硝基苯(5.20g,24.1mmol)在DMF(12mL)中的混合物(图6)。于室温将反应混合物搅动2小时,然后用EtOAc和H2O稀释,并分出各层。用H2O (3x),盐水(1x)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。两次在硅胶上通过柱层析使用己烷:EtOAc 100:0至96:4的纯化给出1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯55a(2.17g, 77%),为亮黄色固体。1HNMRδ(400MHz,CDCl3)8.31-8.27(m,3H), 7.85(br s,1H),7.72(d,J=8.4Hz,2H),7.67(d,J=8.3Hz,1H),7.54 (ddd,J=8.2,6.8,1.2Hz,1H),7.38(ddd,J=8.2,6.8,1.1Hz,1H),4.28-4.25(m,1H),4.16-4.10(m,1H),4.02-3.92(m,2H),3.45(t,J= 10.6Hz,1H),1.60(s,9H)。HRMS m/z 491.1338[为C25H25ClN2NaO5计算的(M+Na)+为491.1344]。
还原方法A:在THF-丙酮(75mL/60mL)中溶解55a(1.53g,3.26mmol)。一旦55a溶解就添加H2O(30mL)。添加NH4Cl(10.5g,196mmol)和Zn粉 (6.40g,97.9mmol),并在氮下于室温将所得混合物搅动1小时。然后将反应混合物穿过硅藻土过滤,用DCM清洗硅藻土栓,并用H2O(1次)清洗合并的滤出液,干燥(Na2SO4),并在真空下去除溶剂,给出化合物55b,为橙色固体。无需纯化在下一步中使用粗制的产物。
还原方法B:将汞-铝汞齐添加至55a在THF-MeOH-H2O(150mL/50 mL/20mL)中的溶液。15分钟后用DCM稀释反应混合物,穿过硅藻土过滤,并用DCM清洗硅藻土栓。用H2O清洗有机物,干燥(Na2SO4),并在真空下去除溶剂,给出5-(4-氨基苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)- 叔丁酯55b,为橙色固体。无需纯化在下一步中使用产物。1H NMRδ(400 MHz,DMSO-d6)8.06(d,J=8.6Hz,1H),7.80(d,J=8.3Hz,1H), 7.53-7.49(m,1H),7.34-7.30(m,1H),7.19(d,J=8.2Hz,2H),6.60 (d,J=8.4Hz,2H),5.16(s,2H),5.04(d,J=1.3Hz,2H),4.18-4.05 (m,3H),4.00-3.97(m,1H),3.81(dd,J=10.9,6.9Hz,1H),1.56(s, 9H),1H未观察到。
在氮下于室温将(S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5-脲基戊酸 (Fmoc-L-瓜氨酸,3.26g,8.20mmol)和2-乙氧基-1-乙氧基羰基-1,2-二氢喹啉(EEDQ,CAS登录号16357-59-8,3.12g,12.6mmol)在DMA(15mL) 中的混合物搅动20分钟。然后添加55b(2.77g,6.31mmol)在DMA(15mL) 中的溶液,用氮冲洗所得混合物,并搅动过夜。16小时后将反应混合物倒在冰上,并用H2O稀释。过滤出所得沉淀物,用H2O清洗,在DCM/MeOH中溶解,干燥(Na2SO4),并在真空下去除溶剂。通过研磨来纯化粗制的产物,其中用己烷:EtOAc 94:6沉淀产物,倒掉溶剂,并使用己烷:EtOAc 90:10,然后是己烷:EtOAc 95:5重复该过程。然后在硅胶上使用DCM:MeOH 100:0至 95:5对材料进行过柱,给出5-(4-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯55c (4.23g,79%,在两个步骤里,自55a,HPLC纯度:95.3%),为黄色固体。1HNMRδ(400MHz,DMSO-d6)10.12(s,1H),8.12(d,J=8.4Hz,1H), 7.89(d,J=7.5Hz,2H),7.82(d,J=8.3Hz,1H),7.77-7.74(m,2H), 7.70-7.67(m,3H),7.55-7.49(m,3H),7.42(t,J=7.4Hz,2H),7.37-7.31 (m,3H),6.00(t,J=5.7Hz,1H),5.43(s,2H),5.22(s,2H),4.29-4.27 (m,2H),4.24-4.05(m,6H),4.01-3.98(m,1H),3.82(dd,J=10.9, 7.0Hz,1H),3.09-2.92(m,2H),1.74-1.35(m,4H),1.55(s,9H)。HRMS m/z 840.3101[为C46H48ClN5NaO7计算的(M+Na)+为840.3134]。
于室温将哌啶(1.5mL,10%v/v)添加至55c(4.18g,5.11mmol)在 DMF(15mL)中的搅动溶液。将反应混合物搅动1小时。用己烷:EtOAc 90:10 (100mL)稀释所得悬浮液,并搅动10分钟。形成两层,并倒掉顶层。在真空下去除保留的底层中的溶剂。在硅胶上通过柱层析使用DCM:MeOH 100:0 至85:15的纯化给出5-(4-((S)-2-氨基-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H- 苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯55d(2.97g,97%,HPLC纯度:99.1%),为黄色粉末。1H NMRδ(400MHz,DMSO-d6)9.93(br s,1H),8.12(d, J=8.3Hz,1H),7.82(d,J=8.3Hz,1H),7.70(d,J=8.6Hz,2H),7.55-7.48 (m,3H),7.37-7.33(m,1H),5.94(t,J=5.7Hz,1H),5.37(s,2H), 5.22(s,2H),4.19-4.05(m,4H),4.01-3.98(m,1H),3.82(dd,J=10.9, 7.0Hz,1H),3.04-2.91(m,2H),1.71-1.36(m,4H),1.55(s,9H),3H 未观察到。HRMS m/z 596.2627[为C31H39ClN5O5计算的(M+H)+为596.2634]。
在氮下于室温将2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酸(S)-2,5- 二氧吡咯烷-1-基酯(Fmoc-Val-OSu,3.19g,7.31mmol)和55d(2.91g,4.87 mmol)在DMA(15mL)中的混合物搅动过夜。20小时后添加己烷:EtOAc 80:20(150mL),并将悬浮液搅动30分钟。然后倒掉溶剂,剩下固体。使用己烷:EtOAc 75:25重复数次。然后在DCM:MeOH 75:25中悬浮固体,并超声处理悬浮液。用己烷(200mL)稀释悬浮液,过滤出固体,用己烷:EtOAc 65:35清洗,并干燥,给出5-(4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯55e(3.79g,85%,HPLC纯度:92.4%),为橙色粉末。1H NMR δ(400MHz,DMSO-d6)10.12(s,1H),8.14-8.12(m,2H),7.89(d,J= 7.5Hz,2H),7.82(d,J=8.4Hz,1H),7.75(t,J=7.6Hz,2H),7.66 (d,J=8.5Hz,2H),7.55-7.48(m,3H),7.45-7.30(m,6H),5.98(t, J=5.7Hz,1H),5.41(s,2H),5.22(s,2H),4.45(dd,J=13.6,7.7Hz, 1H),4.36-4.04(m,6H),4.02-3.90(m,2H),3.82(dd,J=10.8,6.9Hz, 1H),3.08-2.91(m,2H),2.05-1.96(m,1H),1.76-1.34(m,4H),1.55 (s,9H),0.89(d,J=6.8Hz,3H),0.86(d,J=6.8Hz,3H)。HRMS m/z939.3808[为C51H57ClN6NaO8计算的(M+Na)+为939.3819]。
Boc去除方法A:于0℃将TFA(9.5mL)分批添加至55e(685mg,0.747 mmol)在DCM(19mL)中的悬浮液。于0℃将反应混合物搅动1小时45分钟。然后于0℃将氨水(0.25%,100mL)分批添加至混合物,接着添加浓氨水直至pH 9-10。然后添加己烷:EtOAc 90:10,并于0℃将混合物搅动40分钟,超声处理悬浮液,并通过过滤来收集沉淀物,用H2O,H2O:MeOH80:20,己烷:EtOAc 60:40,己烷:Et2O 50:50,己烷清洗,并干燥,给出 (S)-1-((S)-1-(4-(((S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基氨基甲酸(9H-芴-9-基)甲酯 55f,为褐色固体(364mg,60%)。
Boc去除方法B:于0℃将BF3.Et2O(0.07mL,0.552mmol)添加至55e (0.106g,0.116mmol)在DCM(40mL)中的悬浮液。1小时50分钟后于室温在真空下浓缩悬浮液直至只保留很少的DCM。添加很少的MeOH直至沉淀物溶解,然后用H2O稀释溶液。沉淀出固体,并倒掉H2O。添加己烷:EtOAc 90:10(20mL),并超声处理悬浮液,之后通过过滤来收集固体。用H2O和己烷清洗固体,并干燥,给出(S)-1-((S)-1-(4-(((S)-1-(氯甲基)-2,3-二氢-1H-苯并 [e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基氨基甲酸(9H-芴-9-基)甲酯55f(66mg,70%),为褐色固体。1H NMRδ(400 MHz,DMSO-d6)10.10(s,1H),8.12(d,J=7.6Hz,1H),8.00(d,J= 8.3Hz,1H),7.89(d,J=7.4Hz,2H),7.74(t,J=7.7Hz,2H),7.64 (d,J=8.5Hz,2H),7.58(d,J=8.3Hz,1H),7.46-7.37(m,5H),7.34-7.30 (m,2H),7.13-7.09(m,1H),6.55(s,1H),5.97(t,J=5.7Hz,1H), 5.41(s,2H),5.17(s,2H),4.43(dd,J=13.0,7.5Hz,1H),4.34-4.21 (m,3H),3.95-3.90(m,2H),3.83(dd,J=10.7,3.4Hz,1H),3.70-3.66 (m,1H),3.61-3.51(m,2H),3.08-2.90(m,2H),2.04-1.96(m,1H),1.76-1.33(m,4H),0.89(d,J=6.8Hz,3H),0.86(d,J=6.8Hz,3H), 2H未观察到。HRMS m/z839.3266[为C46H49ClN6NaO6计算的(M+Na)+为 839.3294]。
用氮冲洗55f(485mg,0.593mmol),53h(206mg,0.593mmol), EDCI.HCl(293mg,1.48mmol)和TsOH(26mg,0.151mmol)在DMA(10 mL)中的混合物,并于室温搅动过夜。19.5小时后用H2O稀释反应混合物,并通过过滤来收集所得固体,用H2O和己烷:EtOAc 50:50清洗。硅胶栓上使用DCM:MeOH 100:0至90:10的过滤柱层析给出(S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基氨基甲酸(9H-芴-9-基)甲酯55g(391mg),在下一步中使用粗制品。HRMS m/z1144.4169[为C64H64Cl2N7O9计算的(M-H)+为1144.4148]。
于室温将哌啶(0.39mL,3.95mmol)添加至55g(910mg,0.793mmol) 在DMF(6mL)中的悬浮液。10分钟后在真空下浓缩混合物。在硅胶上通过柱层析使用DCM:MeOH 95:5至85:15的纯化,接着是通过制备性HPLC(柱: Synergi-MAX RP 4μ,21.20x 250mm;流速:12mL/min;流动相:溶剂A: H2O/TFA pH 2.47,溶剂B:MeCN/H2O 90:10;方法:梯度,溶剂A:溶剂B60:40 至22:78至60:40,24分钟;波长:254nm,330nm)的进一步纯化给出三氟乙酸(S)-2-((S)-2-氨基-3-甲基丁酰胺基)-N-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基)-5-脲基戊酰胺55h(75mg,9%),为乳脂状固体。1H NMRδ(400MHz,DMSO-d6)10.35(s,1H),10.24(s,1H),8.69(d, J=7.7Hz,1H),8.20(s,1H),8.15(d,J=8.4Hz,1H),8.09-8.02(m, 4H),7.86(d,J=8.4Hz,1H),7.78(d,J=8.4Hz,1H),7.65(d,J=8.5 Hz,2H),7.57-7.47(m,3H),7.40-7.37(m,1H),7.34-7.30(m,1H), 6.04(t,J=5.8Hz,1H),5.48(br s,2H),5.22(s,2H),4.57-4.51(m, 1H),4.40-4.33(m,2H),4.25-4.14(m,4H),4.03-3.98(m,2H),3.85 (dd,J=10.7,7.6Hz,1H),3.79(dd,J=10.3,8.6Hz,1H),3.66(t, J=5.2Hz,2H),3.09-2.97(m,2H),2.75-2.57(m,4H),2.11-2.06(m, 1H),2.01-1.96(m,2H),1.78-1.70(m,1H),1.68-1.58(m,1H),1.53-1.40 (m,2H),0.95(d,J=6.8Hz,3H),0.94(d,J=6.8Hz,3H)。HRMS m/z 924.3672[为C49H56Cl2N7O7计算的(M+H)+为924.3613]。
将DMF(3mL)中的二异丙基乙基胺,DIPEA(10mg,0.0774mmol) 添加至55h(74mg,0.0712mmol)和6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(33mg,0.107mmol),并在氮下于室温搅动所得混合物。5.5小时后添加另外部分的DIPEA(0.9mg,0.00693mmol)和6-(2,5- 二氧-2,5-二氢-1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(4.4mg,0.0143 mmol)。再过1小时后添加另外部分的6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(8mg,0.0259mmol),并将混合物于-20℃保持过夜。15小时后将混合物升温至室温,并添加另外部分的6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(4.4mg,0.0143mmol)。再过1 小时后添加另外部分的6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(7.5mg,0.0243mmol)。1小时后添加己烷:EtOAc 95:5(25mL),接着是DCM(5mL),并将混合物搅动20分钟。在此时段里自溶液沉淀出固体。让混合物沉降,然后倒掉溶剂。然后添加己烷:DCM 95:5,搅动悬浮液,沉降,倒掉溶剂,并干燥固体。通过制备性HPLC(柱:Synergi-MAX RP 4μ, 21.20x250mm;流速:13mL/min;流动相:溶剂A:H2O/TFA pH 2.47,溶剂B:MeCN/H2O 90:10;方法:等度,溶剂A:溶剂B 35:65,35分钟;波长: 254nm,330nm)的纯化给出N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3- 二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺55(13.7mg, 17%,HPLC纯度:92.5%),为浅黄色粉末。1H NMRδ(400MHz,DMSO-d6) 10.35(s,1H),10.02(s,1H),8.19(s,1H),8.15(d,J=8.3Hz,1H), 8.08(d,J=8.1Hz,2H),8.02(s,1H),7.86(s,1H),7.81-7.77(m, 2H),7.66(d,J=8.5Hz,2H),7.57-7.53(m,1H),7.50-7.47(m,3H), 7.40-7.36(m,1H),7.33-7.30(m,1H),6.99(s,2H),5.97(t,J=5.5Hz, 1H),5.40(br s,2H),5.21(s,2H),4.42-4.32(m,3H),4.22-4.14(m, 4H),4.03-3.98(m,2H),3.87-3.77(m,2H),3.34(t,J=7.0Hz,2H), 3.07-3.05(m,2H),2.76-2.59(m,4H),2.22-2.08(m,2H),2.02-1.92(m,3H),1.74-1.57(m,2H),1.51-1.33(m,6H),1.23-1.14(m,3H),0.85 (d,J=6.7Hz,3H),0.82(d,J=6.8Hz,3H)。HRMS m/z 1115.4170[为 C59H65Cl2N8O10计算的(M-H)+为1115.4206]。
实施例6:N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧 -2,3,5,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基 -1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺56
将8-(6-((S)-1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4] 二氮杂卓-10(5H)-羧酸(S)-叔丁酯54g(829mg,1.00mmol)还原(Zn/NH4Cl) 成相应的苯胺(通过上文为54h的合成而报告的方法),并在干的DMA(3mL) 中溶解。对此溶液添加通过于室温将(S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5- 脲基戊酸(Fmoc-L-瓜氨酸,1.19g,3.00mmol)和2-乙氧基-1-乙氧基羰基-1,2- 二氢喹啉,EEDQ(0.99g,4.00mmol)在干的DMA(4mL)中搅动40分钟而形成的混合物。在氮气氛下于室温将最终的反应混合物搅动19小时。将混合物倒入水中,并于室温搅动5小时。收集固体,用水清洗数次,干燥,并通过硅土柱层析(DCM-MeOH梯度0-5%)来纯化,给出 8-(6-((S)-5-(4-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5- 氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯56a(0.91g,77%),为纯的米色固体,mp 172℃;1H NMR[(CD3)2SO] δ10.10(s,1H),8.22-8.11(m,2H),7.94-7.62(m,8H),7.59-7.46(m, 3H),7.46-7.27(m,5H),7.04(s,1H),6.69(s,1H),6.39(br s,1H), 5.99(t,J=5.6Hz,1H),5.51-5.32(m,3H),5.22(s,2H),4.42-3.90 (m,10H),3.88-3.75(m,1H),3.79(s,3H),3.53-3.18(m,3H),3.15-2.87(m,2H),2.66-2.44(m,2H,被DMSO峰部分模糊),2.11-1.20 (m,14H),1.31(s,9H)。分析物(C65H72ClN7O12·1/2H2O)计算:C,65.73; H,6.20;N,8.26。发现:C,65.64;H,6.19;N,8.27。
在氮气氛下于0℃对N-Fmoc 56a(0.91g,0.77mmol)在干的DMA(9mL) 中的搅动溶液添加哌啶在DMA中的溶液(1.0mmol每mL溶液)(3.85mL, 3.85mmol)。添加后于此温度将混合物再搅动2小时,然后倒入乙酸乙酯-石油醚(1:10)(150mL)的混合物中,并于0℃搅动30分钟。自不溶性材料倒出溶剂,并丢弃。于室温用更多的乙酸乙酯-石油醚(1:3)(2x150mL)重复清洗步骤。收集固体,用乙酸乙酯-石油醚(1:3)清洗,并干燥,给出 8-(6-((S)-5-(4-((S)-2-氨基-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e] 吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯56b,为无色固体(0.73g, 99%);mp 223℃(dec.);1H NMR[(CD3)2SO]δ10.60-9.30(br s,3H),8.24-8.12 (m,2H),7.84(d,J=8.2Hz,1H),7.70(d,J=8.5Hz,2H),7.54 (br t,J=7.5Hz,1H),7.50(d,J=8.5Hz,2H),7.39(br t,J=7.6Hz,1H),7.04(s,1H),6.69(s,1H),6.39(br s,1H),5.93(t,J=5.7Hz, 1H),5.47-5.28(m,3H),5.21(s,2H),4.36(t,J=10.8Hz,1H), 4.28-4.13(m,2H),4.10-3.92(m,3H),3.90-3.77(m,1H),3.79(s, 3H),3.54-3.20(m,3H,被水峰部分模糊),3.06-2.89(m,2H),2.70-2.49 (m,3H,被DMSO峰部分模糊),2.10-1.25(m,14H),1.31(s,9H)。分析物(C50H62ClN7O10·3/4H2O)计算:C,61.91;H,6.60;N,10.11。发现: C,62.05;H,6.96;N,10.08。
在氮气氛下于室温将胺56b(0.73g,0.763mmol)和2-(((9H-芴-9-基) 甲氧基)羰基氨基)-3-甲基丁酸(S)-2,5-二氧吡咯烷-1-基酯(Fmoc-Val-Osu,0.50 g,1.15mmol)在干的DMA(7mL)中的混合物搅动18小时。添加乙酸乙酯-石油醚(1:2)(100mL),并于室温将混合物搅动30分钟。自不溶性材料倒出溶剂,并用更多的乙酸乙酯-石油醚(1:1)(2x100mL)重复清洗步骤。干燥无色固体,给出8-(6-((S)-5-(4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚 -3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯56c(0.89g,91%);mp 191 ℃(dec.);1HNMR[(CD3)2SO]δ10.11(s,1H),8.22-8.08(m,3H),7.92-7.81 (m,3H),7.74(t,J=7.4Hz,2H),7.65(d,J=8.4Hz,2H),7.54 (br t,J=7.2Hz,1H),7.48(d,J=8.4Hz,2H),7.46-7.35(m,4H), 7.32(br t,J=7.4Hz,2H),7.04(s,1H),6.69(s,1H),6.39(br s,1 H),5.97(brs,1H),5.47-5.34(m,3H),5.21(s,2H),4.53-4.13(m, 7H),4.10-3.75(m,5H),3.79(s,3H),3.53-3.20(m,3H,被水峰部分模糊),3.10-2.87(m,2H),2.69-2.45(m,2H,被DMSO峰部分模糊), 2.10-1.25(m,15H),1.31(s,9H),0.88(d,J=6.8Hz,3H),0.85(d, J=6.7Hz,3H)。分析物(C70H81ClN8O13·3/4H2O)计算:C,65.10;H,6.44; N,8.68。发现:C,64.85;H,6.48;N,8.67。
在氮气氛下于0℃对N-Fmoc化合物56c(0.89g,0.70mmol)在干的DMA (6mL)中的搅动溶液添加哌啶在DMA中的溶液(1.0mmol每mL溶液)(3.48 mL,3.48mmol)。添加后于此温度将混合物再搅动1.5小时。添加乙酸乙酯- 石油醚的混合物(1:2)(90mL),并于0℃将混合物搅动10分钟。自不溶性材料倒出溶剂层,并丢弃。于室温用更多的乙酸乙酯-石油醚(1:2)(2x90mL) 重复清洗步骤。干燥剩下的无色固体,给出8-(6-((S)-5-(4-((S)-2-((S)-2-氨基-3- 甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并 [1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯56d(0.68g,93%);mp 225℃ (dec.);1H NMR[(CD3)2SO]δ10.16(s,与D2O可交换,1H),8.23-8.07(m, 3H,D2O后降至2H),7.84(d,J=8.2Hz,1H),7.65(d,J=8.5Hz,2H), 7.59-7.45(m,3H),7.37(br t,J=7.5Hz,1H),7.04(s,1H),6.69(s, 1H),6.38(br s,与D2O可交换,1H),5.96(t,J=5.8Hz,与D2O可交换, 1H),5.45-5.30(m,3H,D2O后降至1H成为d,J=9.6Hz),5.21(s,2H), 4.41(br s,D2O后变成dd,J=8.4,5.4Hz,1H),4.36(br t,J=10.7Hz, 1H),4.26-4.13(m,2H),4.10-3.91(m,3H),3.88-3.76(m,1H),3.79 (s,3H),3.53-3.43(m,1H),3.41-3.20(m,2H),3.09-2.88(m,3H),2.70-2.50(m,2H,被DMSO峰部分模糊),2.10-1.20(m,15H),1.31(s, 9H),0.88(d,J=6.9Hz,3H),0.79(d,J=6.8Hz,3H),2H未观察到。分析物(C55H71ClN8O11·H2O)计算:C,61.53;H,6.85;N,10.44。发现: C,61.39;H,7.11;N,10.15。
在氮气氛下于0℃将胺56d(0.68g,0.64mmol)和6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(马来酰亚胺基-Osu,0.50g,1.61 mmol)在干的DMA(6mL)中的混合物搅动1小时。添加乙酸乙酯-石油醚的混合物(1:2)(90mL),并于0℃将混合物搅动15分钟。自不溶性材料倒出溶剂层,并丢弃。于室温用更多的乙酸乙酯-石油醚(1:1)(90mL),然后是纯的乙酸乙酯(50mL)重复清洗步骤。干燥剩下的米色固体,给出 8-(6-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)- 基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并 [1,2-a][1,4]二氮杂卓-10(5H)-羧酸(S)-叔丁酯52(0.72g,90%);HPLC:96.7%纯;mp 210℃(dec.);1H NMR[(CD3)2SO]δ10.03(s,与D2O可交换,1H), 8.23-8.11(m,2H),8.09(d,J=7.3Hz,与D2O可交换,1H),7.85(d, J=8.5Hz,1H),7.80(d,J=8.3Hz,与D2O可交换,1H),7.66(d,J= 8.6Hz,2H),7.59-7.44(m,3H),7.38(br t,J=7.6Hz,1H),7.04(s, 1H),6.99(s,2H),6.69(s,1H),6.38(br s,与D2O可交换,1H),5.99 (t,J=5.5Hz,与D2O可交换,1H),5.49-5.34(m,3H,D2O后降至1H 成为d,J=9.5Hz),5.20(s,2H),4.44-4.30(m,2H),4.26-4.13(m,3 H),4.10-3.91(m,3H),3.88-3.76(m,1H),3.79(s,3H),3.53-3.44 (m,1H),3.41-3.20(m,被水峰部分模糊,4H),3.09-2.88(m,2H), 2.66-2.42(m,被DMSO峰部分模糊,2H),2.25-1.24(m,21H),1.31(s, 9H),1.24-1.11(m,2H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.7Hz, 3H)。分析物(C55H71ClN8O11·H2O)计算:C,61.53;H,6.85;N,10.44。发现:C,61.39;H,7.11;N,10.15。
于-10至-12℃(浴温度)对N-tBoc衍生物52(125mg,0.10mmol)在DCM(10mL)中的搅动溶液在10分钟里逐滴添加2.5%水在TFA中的溶液(10 mL)。添加后于此温度将混合物再搅动3小时。添加冷的(-25℃)乙酸乙酯 -石油醚(1:10)(300mL),接着于-10℃(浴温度)缓慢添加饱和NaHCO3水溶液以给出pH 6-7。去除有机层,并用更多的乙酸乙酯-石油醚(1:1)(300 mL)重复清洗步骤。收集固体,用水和乙酸乙酯连续清洗数次,并干燥,给出粗制的产物,为浅黄色固体(102mg)。通过制备性HPLC[Genentech] 来纯化,给出N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(6-((S)-7-甲氧基-5-氧 -2,3,5,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-8-基氧)己酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基 -1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺56(4.1mg,3.6%); HRMS(ESI)m/z为C60H72ClN9NaO11计算:1152.4932,发现:1152.4906[MNa+]。为C60H73ClN9O11计算:1130.5113,发现:1130.5077[MH+]。
实施例7:2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基 (甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e] 吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57
于室温对1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a(2.00g,5.99mmol)在DMF(5mL)中的溶液添加苄基溴(7.13mL,59.90 mmol),碘化钾KI(50mg,0.30mmol)和碳酸钾K2CO3(4.14g,30.00mmol)。见图9。将混合物搅动2小时,然后用乙酸乙酯稀释。过滤掉沉淀物。在乙酸乙酯和水之间重分配滤出液。用乙酸乙酯提取水相三次。用水和盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。通过旋转蒸发器去除溶剂,并抽走过量的苄基溴。通过柱层析使用乙酸乙酯和石油醚的混合物(v/v1:10)作为洗脱液纯化所得残留物,给出5-(苄基氧)-1-(氯甲基)-1H- 苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯57a,为白色固体(1.97g,78%);mp 186-188℃。1H NMR(CDCl3)δ8.29(d,J=8.3Hz,1H),7.86(br s,1H), 7.65(d,J=8.29Hz,1H),7.55-7.49(m,3H),7.45-7.41(m,2H),7.38-7.31 (m,2H),5.26(s,2H),4.26(br s,1H),4.13(t,J=10.8Hz,1H), 4.00-3.92(m,2H),3.44(t,J=10.5Hz,1H),1.61(s,9H)ppm。LRMS (APCI)发现m/z 424.8(M+H)。C25H27ClNO3要求424.2(Boger D.,Ishizakilb T.,Kitos P.and Suntornwat O.,(1990)J.Org.Chem.,55,5823-5832)。
用乙酸乙酯和石油醚的混合物(v/v 1:1)的进一步洗脱给出图9中所示环丙基产物,为黄色油(345mg,19%)(Lajiness J.and Boger D.,(2011)J.Org. Chem.,76,583-587)。
对在冰浴中冷却的57a(1.60g,3.77mmol)在DCM(15mL)中的溶液添加二烷(40mL)中的4N HCl。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分,给出(S)-5-(苄基氧)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚57b,为氢氯化物盐。在THF(15mL)中溶解该盐,并在冰浴中冷却下来。添加戊二酸酐(646mg,5.66mmol),DMAP(46mg,0.38mmol)和吡啶(5mL),并于室温将所得混合物搅动4小时。抽走所有挥发性成分后,在稀NaHCO3水溶液中溶解残留物,并用乙酸乙酯清洗3次。使用1N HCl将水相酸化至pH 2,并用乙酸乙酯提取三次。用水和盐水清洗合并的乙酸乙酯提取液,在无水Na2SO4上干燥,并穿过硅胶垫过滤,用MeOH和乙酸乙酯的混合物(v/v 1:10)清洗。去除溶剂,给出(S)-5-(5-(苄基氧)-1-(氯甲基)-1H-苯并 [e]吲哚-3(2H)-基)-5-氧戊酸57c,为灰白色固体(978mg,59%)。
于-10℃对57c(978mg,2.23mmol)在THF(20mL)中的溶液添加25%甲酸铵水溶液(20mL),接着是Pd-C催化剂(10%,湿的,500mg),并于 -10℃将混合物搅动7小时。添加更多的Pd-C催化剂(500mg),并于相同温度将混合物搅动过夜。穿过硅藻土过滤掉催化剂,并用THF清洗硅藻土。自滤出液抽走THF,并用乙酸乙酯提取剩余的水溶液三次。用水和盐水清洗合并的提取液,在无水Na2SO4上干燥,并过滤。去除溶剂给出(S)-5-(1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸53h,为灰白色固体(487mg, 63%);1H NMR(DMSO)δ12.08(br s,1H),10.35(br s,1H),8.08(d, J=8.0Hz,1H),7.98(s,1H),7.77(d,J=8.3Hz,1H),7.50-7.46(m, 1H),7.33-7.29(m,1H),4.30(t,J=10.5Hz,1H),4.14-4.12(m,2H), 3.98(dd,J=2.8,10.9Hz,1H),3.78(dd,J=7.8,10.8Hz,1H),2.63-2.54 (m,2H),2.34(t,J=7.4Hz,2H),1.99-1.83(m,2H)ppm。LRMS(APCI) 发现m/z 348.6(M+H)。C18H19ClNO4要求348.1。
对53h(500mg,1.44mmol)在THF(15mL)中的溶液添加四唑(3%,在乙腈中,51mL,17.25mmol),接着是N,N-二异丙基亚磷酰胺二叔丁酯(5.73 mL,17.25mmol)。于室温将混合物搅动过夜,然后在冰浴中冷却,并逐滴添加H2O2(30%水溶液,3.53mL,34.5mmol)。容许所得混合物升温至室温,并搅动5小时。通过在冰浴中冷却时添加10%亚硫酸钠水溶液来淬灭反应。通过旋转蒸发器去除有机挥发物,给出含有悬浮的油的水相。添加石油醚,并将混合物搅动半小时。通过过滤来收集形成的沉淀物,用水和石油醚清洗,并在真空下干燥,给出(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e] 吲哚-3(2H)-基)-5-氧戊酸57d(660mg,85%),为灰白色泡沫;1H NMR (DMSO)δ12.07(br s,1H),8.56(s,1H),8.04(d,J=8.2Hz,1H), 7.93(d,J=8.4Hz,1H),7.60-7.56(m,1H),7.50-7.46(m,1H),4.38 (t,J=9.8Hz,1H),4.32-4.26(m,1H),4.20-4.18(m,1H),4.02(dd, J=2.9,11.0Hz,1H),3.90(dd,J=7.1,11.0Hz,1H),2.67-2.53(m, 2H),2.34(t,J=7.4Hz,2H),1.87-1.78(m,2H),1.481和1.476(2×s, 18H)ppm。31P NMR(DMSO)δ-15.46ppm。HRMS(ESI)发现m/z 562.1719 (M+Na)。C26H35ClNNaO7P要求562.1732。
对在冰浴中冷却的6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸(1.00g,4.73mmol)在DCM(20mL)中的悬浮液添加一滴DMF,然后逐滴添加草酰氯 (2.03mL,23.67mmol)。见图10。容许混合物升温至室温,并搅动过夜,给出深褐色溶液。通过旋转蒸发器,然后是高真空泵去除所有挥发性成分。在DCM(5mL)中溶解所得残留物,并通过旋转蒸发器,然后是高真空泵去除溶剂。将上述溶解和去除规程再重复一次,给出粗制的6-马来酰亚胺基己酰氯,为深褐色油。对在冰浴中冷却的甲基(2-(甲基氨基)乙基)氨基甲酸叔丁酯(891mg,4.73mmol)在DCM(5mL)中的溶液逐滴添加上文生成的 6-马来酰亚胺基己酰氯在DCM中的溶液(20mL)。容许所得混合物升温至室温,并搅动过夜。去除DCM,并在乙酸乙酯中溶解残留物。用NaHCO3水溶液,冷的5%柠檬酸水溶液,和盐水清洗溶液,然后在无水Na2SO4上干燥,并穿过硅胶垫过滤,用乙酸乙酯清洗。去除溶剂,给出2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸叔丁酯57e,为褐色油(1.33g,74%);1H NMR(DMSO)(旋转异构体的混合物)δ7.006和7.005 (2×s,1H),3.39-3.36(m,4H),3.29-3.25(m,2H),2.92-2.75(m,6H, 2NMe),2.21(t,J=7.38Hz,2H),1.50-1.44(m,4H),1.37(s,9H),1.24-1.16(m,2H)ppm。HRMS(ESI)发现m/z 382.2338(M+H)。C19H32N3O5要求382.2336。
对在冰浴中冷却的57e(274mg,0.72mmol)在DCM(5mL)中的溶液逐滴添加TFA(5mL)。于相同温度将混合物搅动2小时,之后通过旋转蒸发器,然后是高真空泵去除所有挥发性成分。就这样使用所得残留物,三氟乙酸6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基-N-(2-(甲基氨基)乙基)己酰胺 57f。
于室温在DCM(5mL)中溶解51a(200mg,0.60mmol),添加DIPEA (0.3mL,1.72mmol),接着是氯甲酸4-硝基苯酯(145mg,0.72mmol),形成1-(氯甲基)-5-((4-硝基苯氧基)羰基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯57g。将混合物搅动5小时后,添加粗制的57f在DCM(5mL)和DIPEA (0.7mL,4.02mmol)中的溶液,给出亮黄色溶液,搅动过夜。去除所有挥发性成分。在乙酸乙酯中溶解残留物,并用5%氨水和盐水清洗。通过柱层析使用乙酸乙酯,DCM,和石油醚的混合物(v/v/v 1:2:1),接着是的乙酸乙酯和DCM的混合物(v/v 1:2)作为洗脱液进一步纯化获得的粗制的材料,给出1-(氯甲基)-5-((2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基)(甲基)氨基甲酰基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯57h(223 mg,58%),为灰白色固体;mp 53-56℃。1H NMR(CDCl3)(旋转异构体的混合物)δ8.04(br s,1H),7.86-7.78(m,1H),7.72-7.68(m,1H), 7.53-7.48(m,1H),7.40-7.34(m,1H),6.67(s,2H),4.25(br s,1H), 4.46-4.10(m,1H),4.06-3.98(m,1H),3.92-3.72(表观d,J=11.2Hz, 1H),3.72-3.42(m,7H),3.28,3.10,3.09,2.99(4×s,6H,2NMe),2.38-2.21 (m,2H),1.67-1.54(m,4H),1.57(s,9H),1.33-1.25(m,2H)ppm。 HRMS(ESI)发现m/z 641.2728(M+H)。C33H42ClN4O7要求641.2737。
对在冰浴中冷却的57h(110mg,0.17mmol)在DCM(2mL)中的溶液逐滴添加TFA(2mL)。于相同温度将混合物搅动2小时,然后去除所有挥发性成分。在乙酸乙酯和冷的稀NaHCO3水溶液之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水 Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,给出2-(6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-2,3-二氢 -1H-苯并[e]吲哚-5-基酯57i,为黄色固体(86mg,92%),无需进一步纯化就这样使用;1H NMR(CDCl3)(旋转异构体的混合物)δ7.76(d,J=8.4Hz, 1H),7.63-7.59(m,1H),7.48-7.41(m,1H),7.25-7.20(m,1H),6.79 (s,1H),6.68(s,2H),4.01-3.94(m,1H),3.88-3.78(m,3H),3.74-3.68 (m,2H),3.62-3.47(m,5H),3.28,3.10,3.06,3.00(4×s,6H,2NMe), 2.38-2.21(m,2H),1.69-1.50(m,4H),1.33-1.25(m,2H)ppm。HRMS (ESI)发现m/z541.2217(M+H)。C28H34ClN4O5要求541.2212。
对在冰浴中冷却的57i(83mg,0.15mmol)在DMA(3mL)中的溶液添加(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)-基)-5- 氧戊酸57d(99mg,0.18mmol),接着是EDCI氢氯化物(88mg,0.46mmol),然后是对甲苯磺酸(2.6mg,0.015mmol)。见图11。容许混合物升温至室温,并搅动过夜。在乙酸乙酯和冷的稀NaHCO3水溶液之间重分配混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水 Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并与石油醚一起研磨所得残留物。自DCM和异丙醇重沉淀获得的固体,给出2-(6-(2,5-二氧-2,5-二氢-1H- 吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)- 基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57j,为黄色固体(116mg, 71%);mp 81℃(dec.)。1H NMR(CDCl3)(旋转异构体的混合物)δ8.63 (s,1H),8.36(s,1H),8.23(d,J=8.5Hz,1H),7.89-7.82(m,1H), 7.72-7.67(m,2H),7.54-7.50(m,2H),7.43-7.39(m,2H),6.66(s,2H), 4.34-4.25(m,4H),4.10-4.05(m,2H),3.98-3.93(m,2H),3.72-3.69(m, 2H),3.50-3.46(m,5H),3.28,3.10,3.09,2.99(4×s,6H,2NMe),2.79-2.73 (m,2H),2.70-2.62(m,2H),2.38-2.32(m,1H),2.27-2.20(m,3H),1.67-1.54(m,3H),1.56(s,9H),1.55(s,9H),1.33-1.25(m,4H)ppm。31P NMR(CDCl3)δ-15.71ppm。HRMS(ESI)发现m/z 1084.3755(M+Na)。 C54H66Cl2N5NaO11P要求1084.3766。
对在冰浴中冷却的57j(55mg,0.052mmol)在DCM(1mL)中的溶液逐滴添加TFA(1mL)。于相同温度将混合物搅动1.5小时,然后去除所有挥发性成分。自DCM和乙酸乙酯重沉淀所得残留物,给出2-(6-(2,5-二氧-2,5- 二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57,为灰色固体(33mg,67%,HPLC 纯度:89%);mp 191-194℃(dec.)。1H NMR(DMSO)(旋转异构体的混合物)δ8.49(s,1H),8.23-8.21(m,1H),8.10(d,J=8.4Hz,1H),7.95-7.76 (m,3H),7.59-7.53(m,2H),7.45-7.39(m,2H),6.97,6.96,6.94, 6.90(4×s,总计2H,马来酰亚胺基基团),4.34-4.21(m,4H),4.06-4.01 (m,2H),3.95-3.85(m,2H),3.71-3.61(m,2H),3.54-3.30(m,5H),3.23,3.18,3.04,3.00,2.97,2.95,2.89,2.85(8×s,总计6H,2NMe), 2.37-2.28(m,2H),2.19(d,J=7.4Hz,1H),2.00-1.95(m,2H),1.50-1.40 (m,5H),1.25-1.15(m,5H)ppm。31PNMR(DMSO)δ-5.79ppm。HRMS (ESI)发现m/z 972.2488(M+Na)。C46H50Cl2N5NaO11P要求972.2514。
实施例8:4-甲基哌嗪-1-羧酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58
于室温将1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a(3.338g,10mmol),4-甲基哌嗪-1-羰基氯氢氯化物(5.98g,30mmol), Et3N(3.5g,35mmol)和DMAP(1.34g,11mmol)在CH2Cl2(80mL)中的混合物搅动2天。见图12。用水清洗混合物,在真空下干燥并去除溶剂,以定量产率给出1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)- 羧酸(S)-叔丁酯58a(Boger D.L.et al.,Synthesis(1999)1505-1509):mp 98 ℃;1H NMR(CDCl3)δ8.11(br,1H),7.84(d,J=8.4Hz,1H),7.70 (d,J=8.4Hz,1H),7.50(ddd,J=8.2,6.9,1.1Hz,1H),7.37(ddd, J=8.1,6.9,1.0Hz,1H),4.34-4.20(m,1H),4.17-4.10(m,1H),4.01-3.98 (m,1H),3.94(dd,J=9.6,2.4Hz,1H),3.87-3.80(br,2H),3.68-3.60 (br,2H),3.47(t,J=10.8Hz,1H),2.57-2.48(m,4H),2.83(s,3H), 1.58(s,9H);MS(APCI+)m/z 461.2MH+。分析为C24H30ClN3O4计算:C, 62.7;H,6.6;N,9.1。发现:C,62.5;H,6.8;N,9.2%。
于0℃将58a(2.30g,5mmol)在CH2Cl2(50mL)中的溶液用过量的三氟乙酸(TFA)处理4小时,并用冷的氨水中和混合物。用己烷稀释导致固体沉淀,通过过滤来收集,用水和己烷清洗,并干燥,给出4-甲基哌嗪-1- 羧酸(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58b(1.60g,89%): mp 144-147℃;1H NMR(CDCl3)δ7.69(d,J=8.4Hz,1H),7.63(d,J =8.4Hz,1H),7.45(ddd,J=8.3,6.9,1.2Hz,1H),7.25(ddd,J=8.4, 6.8,1.2Hz,1H),6.82(s,1H),5.30(s,1H),4.17-4.05(m,2H),4.03-3.96 (m,2H),3.89-3.77(m,4H),3.54(t,J=10.9Hz,1H),3.20-2.90(m, 4H),2.76(s,3H)。分析为C19H22ClN3O2计算:C,63.4;H,6.2;N,11.7。发现:C,63.2;H,6.2;N,11.5%。
用HCl(4M,在二烷中,10mL)处理55a(4.689g,10mmol)在二烷(30mL)中的溶液,并于室温将混合物搅动过夜。添加氢氧化铵,去除溶剂,给出(S)-1-(氯甲基)-5-(4-硝基苄基氧)-2,3-二氢-1H-苯并[e]吲哚54f,将它与戊二酸酐(3.4g,30mmol)在CH2Cl2(50mL)中混合。冷却至0℃后添加Et3N(5.05g,50mmol),并容许混合物缓慢升温,并于室温搅动过夜。添加稀HCl,给出固体,通过过滤来收集,用水和CH2Cl2清洗,并干燥,给出(S)-5-(1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸 58c(2.95g,61%):1HNMR(DMSO-d6)δ12.07(br s,1H),8.30(br d, J=8.8Hz,2H),8.24(d,J=8.1Hz,1H),8.17(s,1H),7.89-7.85(m, 3H),7.57(ddd,J=8.2,6.9,1.1Hz,1H),7.43(ddd,J=8.1,7.1,1.0Hz,1H),5.46(s,2H),4.34(t,J=9.7Hz,1H),4.25-4.13(m,2H), 4.01(dd,J=11.0,2.8Hz,1H),3.85(dd,J=10.9,7.4Hz,1H),2.66-2.57 (m,1H),2.55-2.46(m,1H),2.35(t,J=7.3Hz,2H),1.87-1.79(m, 2H)。
于室温将58b(1.33g,3.7mmol)和58c(1.63g,3.38mmol)在含1-(3- 二甲基氨基丙基)-3-乙基碳二亚胺氢氯化物(EDCI.HCl,3.26g,17.0mmol) 的DMA(25mL)中的混合物搅动过夜。用NaHCO3水溶液稀释混合物,并用水和甲醇连续清洗所得沉淀物,并干燥。在硅土上层析,首先用EtOAc/MeOH 9:1,然后是EtOAc/MeOH 4:1洗脱给出4-甲基哌嗪-1-羧酸 (S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-硝基苄基氧)-1H-苯并[e]吲哚-3(2H)- 基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58d(0.98g,35%):1H NMR(CDCl3)δ8.35(s,1H),8.33-8.27(m,3H),8.17(s,1H),7.85 (d,J=8.3Hz,1H),7.73-7.68(m,4H),7.58-7.49(m,2H),7.45-7.38 (m,2H),5.33(q,J=13.0Hz,2H),4.39-4.27(m,4H),4.14-4.06(m,2H),4.01-3.95(m,2H),3.83-3.77(m,2H),3.64-3.59(m,2H),3.48 (dt,J=10.5,7.3Hz,2H),2.85-2.65(m,4H),2.55-2.47(m,4H),2.38 (s,3H),2.28-2.21(m,2H)。
于0℃用铝汞齐(2g)处理58d(0.41g,5mmol)在THF(35mL), MeOH(15mL),和水(5mL)的混合物中的悬浮液,并容许搅动混合物在 3小时里升温至室温。用MeOH稀释后穿过硅藻土过滤混合物,并蒸发滤出液至干燥,给出4-甲基哌嗪-1-羧酸(S)-3-(5-((S)-5-(4-氨基苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e] 吲哚-5-基酯58e(0.31g,78%),在下一步中直接使用:1H NMR(CDCl3) δ8.37(s,1H),8.26(d,J=8.2Hz,1H),8.18(s,1H),7.86(d,J=8.3 Hz,1H),7.67(d,J=8.3Hz,1H),7.60(d,J=8.3Hz,1H),7.49-7.43 (m,2H),7.39(br t,J=7.6Hz,1H),7.34-7.26(m,3H),6.68(d,J= 8.3Hz,2H),5.10(q,J=10.9Hz,2H),4.32-4.15(m,4H),4.07-3.97 (m,2H),3.94-3.90(m,2H),3.86-3.79(m,2H),3.66-3.60(m,2H), 3.50-3.40(m,2H),2.77-2.58(m,4H),2.56-2.48(m,4H),2.27(s,3H), 2.22-2.14(m,2H)。
在氮下将2-乙氧基-1-乙氧基羰基-1,2-二氢喹啉(EEDQ,0.58g,2.3 mmol)和(S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5-脲基戊酸(Fmoc-L-瓜氨酸, 0.69g,1.7mmol)的混合物在干的DMA(10mL)中搅动10分钟直至所有固体溶解。见图13。添加粗制的58e(0.31g,0.39mmol),并继续搅动过夜。用EtOAc稀释混合物,并添加水以沉淀产物,通过过滤来收集,并与沸腾的 MeOH一起研磨,给出粗制的4-甲基哌嗪-1-羧酸 (S)-3-(5-((S)-5-(4-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢 -1H-苯并[e]吲哚-5-基酯58f(0.62g,>100%),于室温用干的DMF(20mL) 中的哌啶(50mg,0.59mmol)处理30分钟。用EtOAc,己烷和水稀释给出沉淀物,通过过滤来收集,并用己烷和水清洗,给出粗制的4-甲基哌嗪-1- 羧酸(S)-3-(5-((S)-5-(4-((S)-2-氨基-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58g(0.33g,89%,85%纯度,通过HPLC),用干的DMA(10mL)中的 1.5个当量的Fmoc-Val-OSu(N-α-Fmoc-L-缬氨酸N-羟基琥珀酰亚胺酯,0.23g, 0.53mmol)处理过夜。用EtOAc和水稀释给出固体,通过过滤来收集,并干燥,给出粗制的4-甲基哌嗪-1-羧酸(S)-3-(5-((S)-5-(4-((S)-2-((S)-2-(((9H-芴-9- 基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e] 吲哚-5-基酯58h(0.38g,85%):1H NMR(DMSO-d6)δ10.11(s,1H), 8.24-8.11(m,3H),7.96(d,J=8.4Hz,1H),7.90-7.80(m,4H),7.74 (t,J=7.4Hz,2H),7.65(d,J=8.4Hz,2H),7.61-7.53(m,2H),7.50-7.36 (m,6H),7.32(t,J=7.5Hz,2H),5.97(t,J=5.4Hz,1H),5.40(s, 2H),5.20(s,2H),4.44-4.19(m,9H),4.08-3.81(m,5H),3.74-3.70(br,2H),3.50-3.42(br,2H),3.07-2.89(m,2H),2.77-2.55(m,4H), 2.52-2.36(m,4H),2.25(s,3H),2.03-1.94(m,3H),1.76-1.53(m,2H), 1.49-1.30(m,2H),0.87(dd,J=11.2,6.8Hz,6H)。
于室温使粗制的58h(0.38g,0.3mmol)与DMA中的哌啶反应1小时,并用EtOAc和水稀释混合物,给出固体,收集,并干燥,给出4-甲基哌嗪-1- 羧酸(S)-3-(5-((S)-5-(4-((S)-2-((S)-2-氨基-3-甲基丁酰胺基)-5-脲基戊酰胺基) 苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58i(0.23g,73%,83%纯度,通过HPLC):1H NMR (CDCl3)δ9.34(br,1H),8.35(br s,1H),8.28(d,J=8.1Hz,1H), 8.14(s,1H),8.02(s,1H),7.86(d,J=8.2Hz,1H),7.75(d,J=7.5 Hz,1H),7.70(d,J=7.6Hz,1H),7.65-7.60(m,2H),7.54-7.47(m, 2H),7.46-7.33(m,4H),7.29(td,J=7.4,1.3Hz,1H),5.20(br s,2H), 5.17-5.09(m,1H),4.82-4.73(m,1H),4.59-4.50(m,1H),4.33-4.15(m, 3H),4.07-4.01(m,1H),3.98-3.90(m,2H),3.86-3.76(m,3H),3.65-3.57 (m,2H),3.55-3.39(m,3H),3.25(br s,1H),2.81-2.60(m,3H),2.59-2.41 (m,6H),2.37(s,3H),2.27-2.16(m,3H),1.55-1.48(m,2H),0.98 (d,J=6.7Hz,3H),0.83(d,J=6.6Hz,3H)。
于室温将粗制的58i(0.105g,0.1mmol)和6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(62mg,0.2mmol)在干的DMF(10mL) 中的混合物搅动过夜,并用EtOAc和水稀释混合物,给出固体,收集,并干燥,给出粗制的材料(70mg,71%纯度,通过HPLC),通过制备性HPLC来纯化,给出4-甲基哌嗪-1-羧酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58。MS m/z 1243.4[为C65H76Cl2N10O11计算的(M+H)+为1243.5]。
实施例9:二氢磷酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯59
对5-(4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯55e(1.00g,1.09mmol)在DMF(10mL)中的溶液添加哌啶(1.08mL, 10.90mmol)。见图14。于室温将混合物搅动2小时,然后抽走所有挥发性成分。与醚一起研磨所得残留物,给出5-(4-((S)-2-((S)-2-氨基-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯59a,为白色固体(700mg,92%)。1H NMR(DMSO)δ10.17(s,1H), 8.17(d,J=8.0Hz,1H),8.12(d,J=8.4Hz,1H),7.82(d,J=8.4Hz, 1H),7.66(d,J=8.6Hz,2H),7.55-7.49(m,3H),7.37-7.33(m,1H), 5.98(t,J=5.7Hz,1H),5.41(s,2H),5.22(s,2H),4.51-4.48(m,1H), 4.19-3.98(m,4H),3.84-3.80(m,1H),3.08-2.90(m,3H),1.99-1.91(m, 2H),1.75-1.65(m,2H),1.55(s,9H),1.49-1.33(m,2H),0.89(d,J =6.8Hz,3H),0.80(d,J=6.8Hz,3H)ppm。
于室温将59a(688mg,0.99mmol),6-(2,5-二氧-2,5-二氢-1H-吡咯-1- 基)己酸2,5-二氧吡咯烷-1-基酯(SuOMC,320mg,1.04mmol),和DIPEA (190μL,1.09mmol)在DMSO(10mL)中的混合物搅动过夜。抽走所有挥发性成分。与乙酸乙酯一起研磨所得残留物,给出1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯 59b,为灰白色固体(832mg,95%)。1H NMR(DMSO)δ10.03(s,1H), 8.13-8.08(m,2H),7.83-7.79(m,2H),7.66(d,J=8.4Hz,2H),7.55-7.48 (m,3H),7.35(t,J=7.5Hz,1H),6.99(s,2H),5.97(t,J=5.4Hz, 1H),5.41(s,2H),5.21(s,2H),4.43-4.38(m,1H),4.22-3.98(m, 4H),3.84-3.80(m,1H),3.38-3.33(m,3H),3.08-2.90(m,2H),2.24-2.06 (m,2H),2.01-1.91(m,1H),1.74-1.14(m,10H),1.55(s,9H),0.86 (d,J=6.8Hz,3H),0.83(d,J=6.7Hz,3H)ppm。HRMS(ESI)发现 m/z 910.3897(M+Na)。C46H58ClN7NaO9要求910.3877。
对在冰浴中冷却的59b(100mg,0.11mmol)在DCM(2mL)中的悬浮液添加TFA(2mL)。在冰浴中将混合物搅动3小时。抽走所有挥发性成分。在THF中溶解所得残留物,并在乙酸乙酯和冷的5%氨水之间重分配。用乙酸乙酯提取水相三次。用水和盐水清洗合并的有机提取液,在无水Na2SO4上干燥,穿过硅藻土过滤,并去除溶剂,给出N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-2,3- 二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺59c,为带绿色的褐色固体(80mg,90%),直接使用。1H NMR(DMSO)δ10.02(s,1H), 8.09-8.07(m,1H),7.99(d,J=8.3Hz,1H),7.80(d,J=8.5Hz,1H), 7.65(d,J=8.3Hz,2H),7.56(d,J=8.3Hz,1H),7.45(d,J=8.4Hz, 2H),7.38(t,J=7.1Hz,1H),7.09(t,J=7.4Hz,1H),6.99(s,2H),5.97(br s,1H),5.41(s,2H),5.16(s,2H),4.46-4.35(m,1H),4.21-4.17 (m,1H),3.96-3.87(m,1H),3.84-3.80(m,1H),3.70-3.65(m,1H), 3.60-3.49(m,1H),3.38-3.33(m,3H),3.06-2.92(m,2H),2.22-2.08(m, 2H),2.02-1.92(m,1H),1.75-1.14(m,10H),0.86(d,J=6.7Hz,3H), 0.82(d,J=6.7Hz,3H)ppm。HRMS(ESI)发现m/z 810.3332(M+Na)。C41H50ClN7NaO7要求810.3352。
于室温对59c(75mg,0.095mmol)和(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酸57d(62mg,0.11mmol)在 DMA(3mL)中的溶液添加EDCI氢氯化物(40mg,0.21mmol),然后是对甲苯磺酸(1.6mg,0.0095mmol)。将混合物搅动5小时后,添加更多的EDCI 氢氯化物(35mg,0.18mmol),并将混合物搅动过夜。抽走所有挥发性成分。在THF中溶解所得残留物,并在乙酸乙酯和冷的稀NaHCO3水溶液之间重分配。用乙酸乙酯提取水相三次。用水和盐水清洗合并的有机提取液,在无水Na2SO4上干燥,穿过硅藻土过滤,并去除溶剂,给出磷酸二叔丁基酯·(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯59d,为褐色固体(104mg,83%),直接使用。1H NMR(DMSO)δ10.03(s,1H), 8.60(s,1H),8.19-7.38(m,14H),6.87(s,2H),5.97(br s,1H),5.40 (s,2H),5.20(brs,2H),4.45-3.82(m,10H),3.38-3.33(m,3H),3.07-2.90 (m,2H),2.75-2.50(m,2H),2.20-2.08(m,4H),2.02-1.92(m,2H), 1.75-1.10(m,12H),1.48(s,18H),0.86-0.82(m,6H)ppm。31P NMR (DMSO)δ-15.46ppm。HRMS(ESI)发现m/z 1331.5071(M+Na)。 C67H83Cl2N8NaO13P要求1331.5086。
对在冰浴中冷却的59d(95mg,0.073mmol)在DCM(2mL)中的悬浮液添加TFA(1mL)。在冰浴中将混合物搅动1.5小时。抽走所有挥发性成分。与乙酸乙酯一起研磨所得残留物,给出带蓝色的灰色固体(77mg,90%),通过制备性HPLC(柱:Synergi-Max RP 4μ,250×21.20mm;流动相:A/B =30:70(A:H2O-TFA pH 2.56,B:90%乙腈,在水中);流速13mL/min)进一步纯化,给出二氢磷酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯59,为灰白色固体(5mg,HPLC纯度 97%)。1H NMR(DMSO)δ10.04(s,1H),8.49(s,1H),8.19-8.09(m, 4H),7.87-7.81(m,2H),7.66(d,J=8.5Hz,2H),7.56-7.48(m,4H), 7.40-7.36(m,2H),6.98(s,2H),6.01(br s,1H),5.43(br s,2H),5.22 (s,2H),4.42-3.34(m,2H),4.25-4.12(m,4H),4.03-3.98(m,2H), 3.88-3.81(m,2H),3.38-3.33(m,3H),3.10-2.90(m,2H),2.75-2.55(m, 4H),2.20-2.08(m,2H),2.00-1.92(m,2H),1.75-1.10(m,12H),0.88-0.81 (m,6H)ppm。31P NMR(DMSO)δ-5.60ppm。HRMS(ESI)发现m/z 1219.3794 (M+Na)。C59H67Cl2N8NaO13P要求1219.3834。
实施例10:N-((S)-1-((S)-1-(4-(((S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基 -1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氧)甲基)苯基氨基)-1-氧-5-脲基戊-2-基氨基)-3-甲基-1-氧丁-2-基)-6-(2,5-二氧-2,5- 二氢-1H-吡咯-1-基)己酰胺60
将二氢磷酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(4-((S)-2-((S)-2-(6-(2,5- 二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)-5-脲基戊酰胺基)苄基氧)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯59酶促去磷酸化,给出60。
实施例11:(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶 -2-基二硫基)乙酯61
于室温将三光气(136mg,0.458mmol)在干的DCM(10mL)中的溶液添加至5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯61a(150 mg,0.451mmol)和DMAP(386mg,3.16mmol)在干的DCM(30mL)中的混合物。见图15。45分钟后添加2-(吡啶-2-基二硫基)乙醇(Chem.Eur.J. (2006)12:3655-3671)(350mg,1.87mmol)在干的DCM(10mL)中的溶液,并将反应混合物搅动过夜。20小时后用MeOH(30mL)稀释混合物,并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:DCM 100:0至0:100,然后是DCM:EtOAc 100:0至95:5的纯化给出化合物61b和起始材料2-(吡啶 -2-基二硫基)乙醇的混合物(389mg)。在下一步中使用此混合物。于0℃将 TBDMSCl(262mg,1.74mmol)在DMF(1.5mL)中的溶液添加至产物61b, 2-(吡啶-2-基二硫基)乙醇,和咪唑(118mg,1.74mmol)在DMF(4mL)中的搅动混合物。将混合物升温至室温,搅动45分钟,然后用EtOAc和H2O稀释。分出各层,并用H2O(3次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:DCM 50:50至0:100,然后是 DCM:EtOAc 98:2至94:6的纯化给出1-(氯甲基)-5-((2-(吡啶-2-基二硫基)乙氧基)羰基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯61b(190mg,77%,在两个步骤里,自61a),为浅黄色泡沫状固体。1H NMRδ(400MHz,CDCl3) 8.49(br s,1H),8.48-8.47(m,1H),7.84(d,J=8.4Hz,1H),7.71(t, J=7.0Hz,2H),7.63(t,J=7.3Hz,1H),7.54-7.50(m,1H),7.40(ddd, J=8.2,6.8,1.1Hz,1H),7.09(ddd,J=7.3,4.9,1.0Hz,1H),6.93(br s,1H),4.48(t,J=6.3Hz,2H),4.31-4.27(m,1H),4.15-4.10(m,1H), 4.04-3.98(m,1H),3.91(dd,J=11.1,2.4Hz,1H),3.45(t,J=10.7Hz, 1H),3.13(t,J=6.3Hz,2H),1.60(s,9H)。
于0℃将TFA(4.8mL)缓慢添加至61b(180mg,0.330mmol)在DCM (9.5mL)中的溶液,并于此温度将混合物搅动1小时。然后用DCM和H2O 稀释反应混合物,并用饱和NaHCO3水溶液中和直至pH 7-8。分出各层,并用H2O(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂,给出1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸(S)-2-(吡啶-2-基二硫基)乙酯 61c(113mg,77%),为黄色固体,无需纯化在下一步中使用。1H NMRδ (400MHz,DMSO-d6)9.52(s,1H),8.48-8.46(m,1H),7.88(d,J=8.4 Hz,1H),7.83-7.78(m,2H),7.64(d,J=8.2Hz,1H),7.39(ddd,J= 8.1,6.9,1.0Hz,1H),7.25(ddd,J=6.5,4.8,2.2Hz,1H),7.14(ddd, J=8.2,6.8,1.0Hz,1H),7.09(s,1H),5.94(br s,1H),4.33(t,J=6.2 Hz,2H),4.02-3.96(m,1H),3.85(dd,J=10.8,3.5Hz,1H),3.70(t, J=9.3Hz,1H),3.63-3.55(m,2H),3.18(t,J=6.1Hz,2H)。
在氮下于室温将(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e] 吲哚-3(2H)-基)-5-氧戊酸57d(135mg,0.250mmol)在DMA(6mL)中的溶液添加至61c(110mg,0.247mmol)和EDCI.HCl(116mg,0.605mmol) 的混合物,并将混合物搅动过夜。18.5小时后添加另外的57d(40mg,0.0741 mmol)在DMA(0.5mL)中的溶液和固体EDCI.HCl(28.4mg,0.148mmol),并在氮下于室温搅动混合物。再过6小时后添加另外部分的DMA(0.5mL) 中的57d(7mg,0.0130mmol)和固体EDCI.HCl(24mg,0.125mmol),并将混合物再搅动2天16.5小时。然后用H2O稀释混合物,并沉淀出固体。通过过滤来收集固体,用H2O,饱和NaHCO3水溶液,H2O和己烷清洗。在EtOAc 中溶解固体,用饱和NaHCO3水溶液(3次)和H2O(1次)清洗溶液,然后干燥(Na2SO4),并在真空下去除溶剂。然后在DCM中重溶解固体,用更多的饱和NaHCO3水溶液(3次)清洗,干燥(Na2SO4),并在真空下去除溶剂。然后与己烷:EtOAc 95:5至90:10一起研磨固体,给出(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)- 基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基) 乙酯61d(117mg,HPLC纯度:86.1%),为米色固体,无需进一步纯化在下一步中使用。HRMS m/z 989.2289[为C47H53Cl2N4NaO8PS2计算的(M+Na)+为989.2312]。
于0℃将TFA(1mL)逐滴添加至61d在DCM(2mL)中的搅动溶液,并于此温度将混合物搅动70分钟。然后于25℃在真空下去除溶剂。在DCM 中溶解所得黑色残留物,并用EtOAc稀释溶液。在真空下去除DCM,给出在 EtOAc中的悬浮液。通过过滤来收集固体,与EtOAc和己烷一起研磨,并干燥,给出绿色固体。通过制备性HPLC(柱:Synergi-MAX RP 4μ,21.20x 250mm;流速:12mL/min;流动相:溶剂A:H2O/TFA pH 2.6,溶剂B:MeCN/H2O 90:10;方法:梯度,溶剂A:溶剂B 60:40至2:98,35分钟;波长:254nm,330 nm)进一步纯化,给出61(16mg,8%,在两个步骤里,自61c,HPLC纯度: 97.2%)。1H NMRδ(400MHz,DMSO-d6)9.69(s,1H),8.57-8.46(m, 3H),8.09(d,J=8.3Hz,1H),8.01(d,J=8.5Hz,1H),7.91(dd,J= 8.3,2.4Hz,2H),7.82(d,J=3.3Hz,2H),7.59-7.52(m,2H),7.47-7.40 (m,2H),7.27-7.23(m,1H),4.42-4.22(m,8H),4.06-4.01(m,2H), 3.90(td,J=11.2,7.4Hz,2H),3.19(t,J=6.0Hz,2H),2.77-2.59(m, 4H),2.01-1.94(m,2H)。未观察到2个质子。31P NMRδ(400MHz,DMSO-d6) -6.01。HRMS m/z 877.1053[为C39H37Cl2N4NaO8PS2计算的(M+Na)+为 877.1060]。[α]D 24=-42.3°(c=0.213,DMSO)。
实施例12:(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶 -2-基二硫基)丙酯62
于0℃将三乙胺,Et3N(0.92mL,6.62mmol)和三氟甲磺酸酐(1.03mL, 6.11mmol)添加至51a(1.70g,5.09mmol)在DCM(160mL)中的搅动溶液。见图16。于0℃将反应搅动20分钟,然后用H2O稀释,分出各层,并用 DCM(1次)提取水层。干燥(Na2SO4)合并的有机层,并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:EtOAc 100:0至95:5的纯化给出1-(氯甲基)-5-(三氟甲基磺酰基氧)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯62a(2.20 g,93%),为米色泡沫状固体。1H NMRδ(400MHz,CDCl3)8.30(br s, 1H),8.03(d,J=8.5Hz,1H),7.76(d,J=8.4Hz,1H),7.62-7.59(m, 1H),7.53-7.49(m,1H),4.32(br s,1H),4.21-4.15(m,1H),4.09-4.03 (m,1H),3.92(dd,J=11.2,2.8Hz,1H),3.54-3.49(m,1H),1.61(s, 9H)。
在氮下在密封管中将62a(2.15g,4.61mmol)在干的THF(60mL)中的溶液添加至Cs2CO3(2.10g,6.44mmol),BINAP(430mg,0.690mmol) 和Pd(OAc)2(155mg,0.690mmol)的混合物。然后将二苯基甲酮亚胺(1.0 mL,5.98mmol)添加至反应混合物,并将氮鼓泡穿过混合物10分钟。将密封管于60-65℃加热4天。然后将反应混合物冷却至室温,用DCM稀释,穿过硅藻土过滤,用DCM清洗硅藻土栓直至清洗液中不再有颜色,并在真空下蒸发滤出液。残留物在硅胶上通过柱层析使用己烷:DCM 100:0至50:50的纯化给出1-(氯甲基)-5-(二苯基亚甲基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯53f(2.09g,91%),为黄色泡沫状固体。1H NMRδ(400MHz,DMSO-d6) 7.85(d,J=8.4Hz,1H),7.80(d,J=8.4Hz,1H),7.75(d,J=7.3Hz, 2H),7.61-7.57(m,1H),7.54-7.49(m,3H),7.37-7.33(m,1H),7.30-7.23 (m,3H),7.06(d,J=6.7Hz,2H),4.14-4.02(m,2H),3.99-3.94(m, 2H),3.77(dd,J=10.8,7.4Hz,1H),1.46(s,9H),1H未观察到。HRMS m/z 497.1984[为C31H30ClN2O2计算的(M+H)+为497.1990]。[α]D 28=-101.5°(c =0.995,DCM)。
于室温将冰乙酸,HOAc(65mL)添加至53f(1.30g,2.62mmol)在 THF和H2O(195mL/98mL)中的搅动溶液,并将混合物搅动过夜。18小时后在真空下浓缩反应混合物以去除大多数THF,没有加热到30℃以上。然后用EtOAc(200mL)稀释混合物,分出有机层,用饱和NaHCO3水溶液(4次) 清洗(直至清洗液为pH 8),干燥(Na2SO4),并在真空下去除溶剂。残留物在硅胶上通过柱层析使用己烷:EtOAc 100:0至90:10的纯化给出5-氨基-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯61a(503mg,58%)。1H NMR δ(400MHz,DMSO-d6)8.01(d,J=8.4Hz,1H),7.64(d,J=8.0Hz, 1H),7.40(ddd,J=8.1,6.8,0.9Hz,1H),7.36(br s,1H),7.20(ddd, J=8.1,6.8,1.1Hz,1H),5.91(s,2H),4.11-3.91(m,4H),3.66(dd, J=10.6,8.2Hz,1H),1.53(s,9H)。
于0℃将2-巯基丙酸(3.02g,28.5mmol)在干的THF(10mL)中的溶液逐滴添加至LiAlH4(1.29g,34.0mmol)在干的THF(40mL)中的搅动悬浮液。将反应混合物升温至室温,并搅动3小时。然后将混合物冷却至0℃,并用H2O(5mL)和5%NaOH水溶液(3mL)淬灭。于0℃将混合物搅动20 分钟,穿过硅藻土过滤,用Et2O(3次)清洗硅藻土栓,干燥(Na2SO4)合并的有机物,过滤,并去除溶剂,给出2-巯基丙-1-醇(944mg),无需纯化在下一步中使用。于室温将1,2-二(吡啶-2-基)二硫烷(Bioorg.Med.Chem.Lett. (2011)21:4985-4988)(470mg,5.10mmol)在MeOH(7mL)中的溶液添加至2-巯基丙-1-醇在MeOH(4mL)中的溶液,并将混合物搅动过夜。17.5小时后在真空下去除溶剂。在矾土(中性)上通过柱层析使用己烷:DCM 50:50 至0:100,然后是DCM:EtOAc 99:1至75:25的纯化给出2-(吡啶-2-基二硫基)丙 -1-醇(528mg,18%,在两个步骤里,自2-巯基丙酸),为黄色油。1H NMR δ(400MHz,CDCl3)8.50(ddd,J=5.0,1.8,0.9Hz,1H),7.57(ddd, J=8.0,7.4,1.8Hz,1H),7.39(td,J=8.1,1.0Hz,1H),7.15(ddd,J= 7.4,5.0,1.1Hz,1H),5.93(dd,J=8.8,5.8Hz,1H),3.68(ddd,J=12.5, 8.8,3.8Hz,1H),3.40(ddd,J=12.4,7.8,5.8Hz,1H),3.14-3.06(m, 1H),1.31(d,J=6.9Hz,3H)。
于室温将三光气(127mg,0.428mmol)在干的DCM(10mL)中的溶液缓慢添加至61a(250mg,0.751mmol)和DMAP(551mg,4.51mmol) 在干的DCM(40mL)中的混合物。黄色固体立即沉淀。30分钟后添加2-(吡啶-2-基二硫基)丙-1-醇(420mg,2.09mmol)在干的DCM(6mL)中的溶液,并溶解沉淀物。将反应混合物搅动过夜。18小时后添加1M NaOH(30 mL),并搅动混合物。然后分出各层,并干燥(Na2SO4)有机层,用MeOH (15mL)稀释,并吸收到硅胶上。使用己烷:DCM 100:0至50:50至0:100,然后是DCM:EtOAc 99:1至92:8洗脱产物。然后再次将材料在硅胶上使用己烷:DCM 100:0至50:50至0:100,然后是DCM:EtOAc 98:2至95:5进行层析。这给出62b和2-(吡啶-2-基二硫基)丙-1-醇的混合物(385mg)。在下一步中使用此混合物。于0℃将TBDMSCl(81mg,0.535mmol)在DMF(1mL)中的溶液添加至62b和2-(吡啶-2-基二硫基)丙-1-醇,和DMF(2mL)中的咪唑(36 mg,0.535mmol)的搅动混合物。将混合物升温至室温,搅动50分钟,然后用EtOAc和H2O稀释。充分搅动混合物,分出各层,并用H2O(3次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用己烷:DCM 50:50至0:100,然后是DCM:EtOAc 95:5的纯化给出1-(氯甲基)-5-((2-(吡啶-2-基二硫基)丙氧基)羰基氨基)-1H-苯并[e]吲哚-3(2H)-羧酸 (1S)-叔丁酯62b(295mg,70%,在两个步骤里,自化合物61a),为浅黄色泡沫状固体。1H NMRδ(400MHz,CDCl3)8.49(br s,1H),8.46(ddd, J=4.8,1.7,0.8Hz,1H),7.86(d,J=8.4Hz,1H),7.75-7.73(m,2H), 7.64-7.60(m,1H),7.54(ddd,J=8.1,6.9,1.0Hz,1H),7.42(ddd,J= 8.2,6.8,1.1Hz,1H),7.08(ddd,J=7.4,4.9,0.8Hz,1H),6.97(br s, 1H),4.37-4.28(m,3H),4.17-4.11(m,1H),4.05-3.99(m,1H),3.92(dd,J=11.1,2.5Hz,1H),3.47(t,J=10.7Hz,1H),3.38-3.30(m, 1H),1.62(s,9H),1.41(d,J=6.9Hz,3H)。HRMS m/z 582.1265[为 C27H30ClN3NaO4S2计算的(M+Na)+为582.1258]。
于0℃将TFA(7mL)缓慢添加至62b(285mg,509mmol)在DCM(14 mL)中的溶液,并于此温度将混合物搅动1小时。然后用DCM和H2O稀释反应混合物,并用饱和NaHCO3水溶液中和直至pH 7。分出各层,并用H2O(1 次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂,给出(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)丙酯62c(220mg,94%),为黄色固体,无需纯化在下一步中使用。1H NMRδ(400 MHz,DMSO-d6)9.52(s,1H),8.45(ddd,J=4.8,1.7,0.8Hz,1H), 7.88(d,J=8.5Hz,1H),7.85-7.83(m,1H),7.79-7.75(m,1H),7.64 (d,J=8.2Hz,1H),7.39(ddd,J=8.1,6.8,1.0Hz,1H),7.23(ddd, J=7.3,4.8,1.0Hz,1H),7.15(ddd,J=8.1,6.8,1.0Hz,1H),7.08(s, 1H),5.93(br s,1H),4.24-4.13(m,2H),4.02-3.96(m,1H),3.85(dd, J=10.8,3.5Hz,1H),3.70(t,J=9.3Hz,1H),3.63-3.55(m,2H),3.43-3.36 (m,1H),1.34(d,J=6.9Hz,3H)。
于室温将(S)-5-(1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酸57d(365mg,0.676mmol)在DMA(10mL)中的溶液添加至62c(215mg,0.467mmol)和EDCI.HCl(268mg,1.40mmol)的混合物。添加TsOH(16mg,0.0929mmol),并在氮下(并在3A分子筛上) 将混合物搅动过夜。27.5小时后用EtOAc和H2O稀释混合物,充分摇动,并分出各层。用饱和NaHCO3水溶液(3次),盐水(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在DCM中溶解所得残留物,并用己烷稀释直至自溶液沉淀出固体。在真空下去除溶剂,并与己烷:EtOAc 95:5一起研磨固体,给出 (S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)丙酯62d(372mg),为棕色固体,无需进一步纯化在下一步中使用。 HRMS m/z 1003.2445[为C48H55Cl2N4NaO8PS2计算的(M+Na)+为1003.2468]。
于0℃将TFA(3.5mL)缓慢添加至62d(如上制备)在DCM(7mL) 中的搅动溶液。于此温度将混合物搅动1小时。然后于25℃在真空下去除溶剂。在DCM中溶解所得深绿色残留物,并用EtOAc稀释溶液,引起自溶液沉淀出固体。于30℃在真空下去除溶剂,并再次在DCM中溶解残留物,并用 EtOAc稀释。在真空下去除DCM,给出在EtOAc中的悬浮液。通过过滤来收集固体,然后用EtOAc和己烷清洗固体,并干燥,给出绿色固体。通过制备性HPLC(柱:Synergi-MAX RP 4μ,21.20 x 250mm;流速:12mL/min;流动相:溶剂A:H2O/甲酸铵缓冲液pH 3.5,溶剂B:MeCN/H2O 90:10;方法:梯度,溶剂A:溶剂B 25:75至0:100,在19分钟里)进一步纯化,给出(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚-3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)丙酯62(78 mg,19%,在两个步骤里,自62c,HPLC纯度:87.4%),为乳脂状粉末。1H NMRδ(400MHz,DMSO-d6)9.69(s,1H),8.56(s,1H),8.48(s,1H), 8.44(d,J=4.5Hz,1H),8.15(d,J=8.3Hz,1H),8.00(d,J=8.5Hz, 1H),7.90(d,J=8.4Hz,1H),7.85-7.76(m,3H),7.55-7.48(m,2H), 7.44-7.40(m,1H),7.36-7.32(m,1H),7.24-7.21(m,1H),4.41-4.14(m, 8H),4.05-3.99(m,2H),3.91(dd,J=10.8,7.2Hz,1H),3.85-3.81(m, 1H),3.37-3.28(m,1H),2.74-2.57(m,4H),1.98-1.95(m,2H),1.34 (d,J=6.7Hz,3H),未观察到2个质子。31P NMRδ(400MHz,DMSO-d6) -5.09。HRMS m/z 891.1221[为C40H39Cl2N4NaO8PS2计算的(M+Na)+为891.1216]。
实施例13:2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯63
将2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺基)乙基(甲基)氨基甲酸(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯57酶促去磷酸化,给出63。
实施例14:(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)乙酯64
将(S)-1-(氯甲基)-3-(5-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚 -3(2H)-基)-5-氧戊酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基氨基甲酸2-(吡啶-2-基二硫基)乙酯61酶促去磷酸化,给出64。
实施例15:8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸(11aS)-4-((S)-6-氨基-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3-甲基丁酰胺基)己酰胺基)苄酯65
于室温对6-(5-(叔丁氧羰基氨基)-4-(2-(羟基甲基)吡咯烷-1-羰基)-2-甲氧基苯氧基)己酸(S)-2,2,2-三氯乙酯54c(1.66g,2.71mmol)在干的DCM(10 mL)中的搅动溶液添加乙酸酐(1.29mL,13.6mmol)和三乙胺(2.27mL, 16.3mmol)。见图17。将反应混合物再搅动4小时。添加干的MeOH(1.5mL),并将混合物搅动30分钟。添加乙酸乙酯(200mL),并分出乙酸乙酯层,然后用水清洗数次。干燥(MgSO4)乙酸乙酯溶液,并蒸发,给出6-(4-(2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-(叔丁氧羰基氨基)-2-甲氧基苯氧基)己酸 (S)-2,2,2-三氯乙酯65a(1.8g,100%),为浅黄色胶水;1H NMR[(CD3)2SO] δ8.82(br s,1H),7.27(s,1H),6.86(s,1H),4.89(s,2H),4.39-4.20 (m,3H),3.93(t,J=6.4Hz,2H),3.74(s,3H),3.50-3.33(m,2H), 2.10-1.94(m,4H),1.92-1.61(m,7H),1.53-1.42(m,2H),1.43(s, 9H),2H被DMSO峰模糊。HRMS(ESI)m/z为C28H39Cl3N2NaO9计算: 675.1613,发现:675.1603[MNa+]。为C28H40Cl3N2O9计算:653.1794,发现: 653.1778[MH+]。
在氮下对65a(1.76g,2.69mmol)在丙酮(30mL),水(20mL),和 THF(12mL)的混合物中的搅动溶液添加Zn(7.06g,108mmol)和NH4Cl (11.6g,216mmol)。于室温将混合物搅动23小时。添加乙酸乙酯(100mL),并将混合物搅动15分钟。倒掉有机层。用更多的乙酸乙酯(2x100mL)重复提取。用水(2x100mL)清洗合并的有机溶液,干燥(MgSO4),穿过硅藻土过滤,并蒸发,给出(S)-6-(4-(2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-(叔丁氧羰基氨基)-2-甲氧基苯氧基)己酸65b(1.36g,96%),为粘性无色泡沫;1H NMR[(CD3)2SO]δ11.49(非常br s,1H),8.83(s,1H),7.27(s,1H), 6.86(br s,1H),4.39-4.02(m,3H),3.93(t,J=6.4Hz,2H),3.74(s, 3H),3.51-3.33(m,2H,被水峰部分模糊),2.21(t,J=7.1Hz,2H), 2.11-1.93(m,4H),1.90-1.66(m,5H),1.62-1.50(m,2H),1.50-1.35 (m,2H),1.43(s,9H)。分析物(C26H38N2O9)计算:C,59.76;H,7.33; N,5.36。发现:C,59.66;H,7.49;N,5.29。
在氮气氛下于0℃对65b(0.87g,2.41mmol)和4-甲基哌嗪-1-羧酸 (S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯58b(1.26g,2.41mmol)在干的DMA(5mL)中的搅动溶液添加对二烷(1.21mL,4.82mmol)中的 4M HCl,接着是EDCI.HCl(1.39g,7.23mmol),和无水TsOH(83mg,0.48 mmol)。在氮下于0℃将反应混合物搅动21小时,然后在乙酸乙酯(500mL) 和水(500mL)之间分配。分出乙酸乙酯层,并用更多的乙酸乙酯(200mL) 进一步提取水层。用水(200mL),饱和NaHCO3溶液(2x200mL)和水(200 mL)连续清洗合并的乙酸乙酯提取液。干燥并蒸发乙酸乙酯层,给出4-甲基哌嗪-1-羧酸(S)-3-(6-(4-((S)-2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-(叔丁氧羰基氨基)-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯 65c(1.66g,80%),为米色固体泡沫;mp 84-87℃;1H NMR[(CD3)2SO]δ8.84 (br s,1H),8.21(s,1H),7.95(d,J=8.3Hz,1H),7.81(d,J=8.3 Hz,1H),7.58(br t,J=7.7,1H),7.46(br t,J=8.1Hz,1H),7.29(s, 1H),6.86(s,1H),4.40(t,J=10.0Hz,1H),4.36-3.86(m,10H), 3.83-3.74(m,1H),3.73(s,3H),3.54-3.36(m,4H),2.67-2.34(m, 6H,被DMSO峰部分模糊),2.26(s,3H),2.02(br s,3H),1.93-1.62(m, 8H),1.60-1.47(m,2H),1.42(s,9H)。分析物(C45H58ClN5O10·11/2H2O) 计算:C,60.63;H,6.90;N,7.86。发现:C,60.39;H,6.66;N,8.08。
在氮气氛下于0℃对65c(2.17g,2.51mmol)在DCM(20mL)中的搅动溶液添加TFA(20mL)。添加后于此温度将混合物再搅动2.5小时。将混合物倒入冷的(0℃)NaHCO3(50g),水(700mL),和DCM(500mL)的混合物中,并搅动15分钟(pH约为8)。分出DCM层,并用更多的NaHCO3水溶液(200mL)和水(200mL)清洗,然后干燥(MgSO4)。蒸发溶剂,给出4-甲基哌嗪-1-羧酸(S)-3-(6-(4-((S)-2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-氨基-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯 65d,为浅褐色固体-泡沫(1.76g,92%);mp 62℃;1H NMR[(CD3)2SO]δ8.21 (s,1H),7.95(d,J=8.3Hz,1H),7.81(d,J=8.3Hz,1H),7.57(br t,J=7.6Hz,1H),7.46(br t,J=7.2Hz,1H),6.67(s,1H),6.37(s,1H),5.09(s,2H),4.41(t,J=9.7Hz,1H),4.36-4.20(m,3H),4.17-4.00 (m,3H),3.97-3.86(m,3H),3.81-3.70(m,2H),3.63(s,3H),3.54-3.32 (m,5H),2.66-2.34(m,6H,被DMSO峰部分模糊),2.26(s,3H), 2.08-1.96(m,1H),2.10(s,3H),1.93-1.63(m,7H),1.57-1.45(m,2H)。分析物(C40H50ClN5O8·1/2H2O)计算:C,62.13;H,6.65;N,9.06。发现:C,62.12;H,6.76;N,8.77。
在氮下于室温将(S)-2-(烯丙基氧羰基氨基)-6-(叔丁氧羰基氨基)己酸 65e(3.30g,10.0mmol)和EEDQ(3.71g,15.0mmol)在干的DMA(10mL) 中的混合物搅动15分钟。见图18。对此预先形成的混合物添加4-((叔丁基二甲基甲硅烷基氧)甲基)苯胺(在DMF中自相应的对硝基苯甲醇和TBDMSCl 制备;接着使用Zn/NH4Cl还原)(2.37g,10.0mmol)在干的DMA(3mL) 中的溶液。在氮气氛下于室温将最终的反应混合物再搅动23小时。在乙酸乙酯(500mL)和水(500mL)之间分配混合物。分出乙酸乙酯层,并用饱和 NaHCO3(2x300mL)和水(300mL)连续清洗,然后干燥(MgSO4)。蒸发溶剂给出橙色油,通过硅土柱层析(石油醚-乙酸乙酯梯度10-35%)纯化以提供受到TBDMS保护的赖氨酸65f(4.87g,89%),为粘性米色固体-泡沫;1H NMR[(CD3)2SO]δ9.97(s,1H),7.55(d,J=8.50Hz,2H),7.44 (d,J=7.8Hz,1H),7.21(d,J=8.5Hz,2H),6.75(t,J=5.3Hz,1H), 5.99-5.82(m,1H),5.28(br d,J=17.2Hz,1H),5.17(br d,J=10.5Hz, 1H),4.64(s,2H),4.46(d,J=5.2Hz,2H),4.12-4.02(m,1H),2.93-2.83 (m,2H),1.70-1.52(m,2H),1.46-1.20(m,4H),1.35(s,9H),0.89 (s,9H),0.06(s,6H)。HRMS(ESI)m/z为C28H47N3NaO6Si计算:572.3126,发现:572.3136[MNa+]。
于室温对65f(4.81g,8.75mmol)在THF(30mL)中的搅动溶液添加四丁基氟化铵在THF中的1M溶液(17.5mL,17.5mmol)。添加后于此温度将混合物再搅动2.5小时。添加NH4Cl水溶液(300mL),并将产物提取入乙酸乙酯(500mL)中。用水(2x100mL)清洗乙酸乙酯,并干燥(MgSO4)。蒸发溶剂,给出苯甲醇赖氨酸65g(3.81g,100%),为米色固体;mp 101-103 ℃;1H NMR[(CD3)2SO]δ9.94(s,1H),7.52(d,J=8.4Hz,2H),7.44 (d,J=7.8Hz,1H),7.23(d,J=8.4Hz,2H),6.76(t,J=5.4Hz,1H), 5.97-5.84(m,1H),5.29(br d,J=17.2Hz,1H),5.17(br d,J=10.4Hz, 1H),5.08(t,J=5.7Hz,1H),4.47(d,J=5.3Hz,2H),4.43(d,J=5.7Hz,2H),4.13-4.03(m,1H),2.96-2.82(m,2H),1.72-1.52(m,2 H),1.46-1.20(m,4H),1.36(s,9H)。HRMS(ESI)m/z为C22H33N3NaO6计算:458.2262,发现:458.2272[MNa+];为C22H33N3KO6计算:474.2001,发现:474.1998[MK+]。
在氮下于室温对65d(764mg,1.00mmol)和DMAP(367mg,3.00mmol) 在干的DCM(15mL)中的搅动溶液添加双光气在干的DCM中的溶液(0.05 mmol每mL,12mL,0.60mmol),并将混合物再搅动20分钟。见图19。对此混合物添加65g(3.97g,9.13mmol)在干的DCM(80mL)中的溶液。在氮气氛下于室温将最终的反应混合物再搅动48小时。在乙酸乙酯(500mL)和水(300mL)之间分配混合物。分出乙酸乙酯层,并用乙酸乙酯(2x200mL) 进一步提取水层。用更多的水(2x200mL)清洗合并的乙酸乙酯溶液,并干燥(MgSO4)。于30℃(浴温度)蒸发溶剂给出橙色油,通过硅土柱层析(乙酸乙酯-MeOH=10:1)纯化以提供4-甲基哌嗪-1-羧酸(S)-3-(6-(4-((S)-2-(乙酰氧基甲基)吡咯烷-1-羰基)-5-((4-((S)-2-(烯丙基氧羰基氨基)-6-(叔丁氧羰基氨基)己酰胺基)苄基氧)羰基氨基)-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯65h(1.04g,85%),为浅橙色固体;mp 90-93℃;1HNMR[(CD3)2SO]δ10.04(s,1H),9.10(br s,1H),8.21(s,1H), 7.95(d,J=8.3Hz,1H),7.81(d,J=8.3Hz,1H),7.63-7.53(m,3H), 7.51-7.42(m,2H),7.32(d,J=8.5Hz,2H),7.21(brs,1H),6.85(br s,1H),6.79-6.72(m,1H),5.97-5.83(m,1H),5.29(br d,J=17.2Hz, 1H),5.17(br d,J=10.4Hz,1H),5.08-4.96(m,2H),4.52-4.37(m, 3H),4.37-3.85(m,10H),3.83-3.66(m,2H),3.74(s,3H),3.54-3.41 (m,2H),3.41-3.23(m,2H,被水峰部分模糊),2.95-2.83(m,2H), 2.66-2.34(m,6H,被DMSO峰部分模糊),2.25(s,3H),2.07-1.92(m,4H),1.87-1.45(m,11H),1.45-1.20(m,4H),1.35(s,9H)。HRMS (ESI)m/z为C63H82ClN8O15计算:1225.5583,发现:1225.5557[MH+];为 C63H81ClN8NaO15计算:1247.5402,发现:1247.5401[MNa+];为C63H81ClKN8O15计算:1263.5142,发现:1263.5141[MK+]。
于室温将65h(1.01g,0.824mmol)和K2CO3(1.14g,8.24mmol)在 DCM(20mL)和MeOH(10mL)中的混合物搅动1小时40分钟。用DCM(200 mL)稀释混合物,并与冰-水(200mL)一起搅动10分钟。分出DCM层,并用DCM(2x100mL)进一步提取水层。用更多的水(200mL)清洗合并的 DCM溶液,并干燥(MgSO4)。于25℃(浴温度)蒸发溶剂给出4-甲基哌嗪 -1-羧酸(S)-3-(6-(5-((4-((S)-2-(烯丙基氧羰基氨基)-6-(叔丁氧羰基氨基)己酰胺基)苄基氧)羰基氨基)-4-((S)-2-(羟基甲基)吡咯烷-1-羰基)-2-甲氧基苯氧基)己酰基)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯65i(0.94g,96%),为米色固体;mp 104-107℃;1H NMR[(CD3)2SO]δ10.04(s,1H),9.17(br s, 1H),8.21(s,1H),7.95(d,J=8.4Hz,1H),7.80(d,J=8.3Hz,1H), 7.63-7.53(m,3H),7.51-7.42(m,2H),7.38-7.21(m,3H),6.93(s, 1H),5.32(t,J=5.4Hz,1H),5.98-5.83(m,1H),5.30(br d,J=17.2 Hz,1H),5.17(br d,J=11.7Hz,1H),5.03(s,2H),4.73(t,J=5.7 Hz,1H),4.52-4.36(m,3H),4.36-4.17(m,2H),4.17-3.85(m,6H), 3.83-3.66(m,2H),3.73(s,3H),3.61-3.40(m,4H),3.40-3.20(m, 2H,被水峰部分模糊),2.94-2.83(m,2H),2.67-2.34(m,6H,被DMSO 峰部分模糊),2.25(s,3H),1.96-1.45(m,12H),1.45-1.20(m,4H), 1.35(s,9H)。HRMS(ESI)m/z为C61H80ClN8O14计算:1183.5477,发现: 1183.5445[MH+];为C61H79ClN8NaO14计算:1205.5296,发现:1205.5256 [MNa+];为C61H79ClKN8O14计算:1221.5036,发现:1221.5026[MK+]。
于0℃对65i(0.92g,0.78mmol)在干的DCM(20mL)中的搅动溶液逐滴(在8分钟里)添加戴斯-马丁高碘烷(DMP,1,1,1-三乙酰氧基-1,1-二氢-1,2-苯碘酰-3(1H)-酮,CAS登录号87413-09-0,492mg,1.16mmol)。添加完成后,将反应混合物于0℃再搅动2小时,然后于室温45小时。用DCM(100 mL)稀释混合物,并于室温与10%Na2S2O3(100mL)一起搅动10分钟。在DCM(400mL)和饱和NaHCO3溶液(400mL)之间分配所得混合物。分出 DCM层,并用DCM(2x100mL)进一步提取水层。用饱和NaHCO3溶液(200 mL)和水(200mL)进一步清洗合并的DCM溶液,然后干燥(MgSO4)。于 25℃(浴温度)蒸发溶剂给出浅褐色固体,通过SiO2柱层析(DCM-乙酸乙酯-MeOH=15:15:1至15:15:3梯度)纯化,给出8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸 (S)-4-((S)-2-(烯丙基氧羰基氨基)-6-(叔丁氧羰基氨基)己酰胺基)苄酯65j (0.64g,70%),为浅黄色固体;mp 137℃(dec.);1H NMR[(CD3)2SO]δ10.02 (s,1H),8.21(s,1H),7.95(d,J=8.4Hz,1H),7.80(d,J=8.3Hz, 1H),7.65-7.38(m,5H),7.18(d,J=7.0Hz,2H),7.03(s,1H),6.82-6.63 (m,2H),6.49(解析较差的d,J=4.7Hz,与D2O可交换,1H),5.96-5.82 (m,1H),5.46(解析较差的dd,J=9.8,4.7Hz,D2O后变成d,J=10.1Hz, 1H),5.27(br d,J=17.1Hz,1H),5.21-5.10(m,2H),4.81(br d,J= 12.3Hz,1H),4.51-4.17(m,5H),4.13-3.84(m,4H),3.84-3.67(m, 2H),3.77(s,3H),3.55-3.20(m,6H,被水峰部分模糊),2.66-2.30(m, 6H,被DMSO峰部分模糊),2.26(s,3H),2.10-1.20(m,16H),1.35(s, 9H)。HRMS(ESI)m/z为C61H78ClN8O14计算:1181.5321,发现:1181.5286 [MH+];为C61H77ClN8NaO14计算:1203.5140,发现:1203.5130[MNa+];为 C61H77ClKN8O14计算:1219.4879,发现:1219.4861[MK+]。
在氮气氛下于0℃对65j(623mg,0.53mmol)在干的DCM(15mL) 中的搅动溶液添加吡咯烷(0.86mL,10.5mmol),接着是Pd(Ph3P)4(30mg, 9.8%Pd)。添加后于室温将反应混合物再搅动5小时。用石油醚(100mL) 稀释混合物,并于室温搅动30分钟。自不溶性材料倒掉溶剂,并用DCM-石油醚(1:10)(100mL)重复清洗步骤。在DCM(200mL)中溶解留下的粘性固体,并用水(3x100mL)清洗,然后干燥(MgSO4),并通过短SiO2柱以去除基线材料。使用DCM-MeOH梯度(2至5%)洗脱所需化合物。于25℃ (浴温度)蒸发溶剂给出浅黄色固体-泡沫(472mg,82%),在下一步中直接使用。在氮气氛下于室温用干的DMA(8mL)中的Fmoc-val-Osu (N-α-Fmoc-L-缬氨酸N-羟基琥珀酰亚胺酯,550mg,1.26mmol)处理此粗制的胺,并将混合物搅动5小时。添加乙酸乙酯-石油醚(1:10,100mL),并于室温将混合物搅动20分钟。自不溶性材料倒掉溶剂,并用更多的乙酸乙酯 -石油醚(1:10,2x50mL)重复清洗步骤。干燥留下的粘性固体,并通过SiO2柱层析(DCM-乙酸乙酯-MeOH=20:10:3)纯化,给出8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓 -10(5H)-羧酸(S)-4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基氨基)-3-甲基丁酰胺基)-6-(叔丁氧羰基氨基)己酰胺基)苄酯65k(246mg,47%),为无色固体; mp 143℃(dec.);1H NMR[(CD3)2SO]δ10.03(s,与D2O可交换,1H),8.21 (s,1H),8.05(br d,J=7.5Hz,与D2O可交换,1H),7.95(d,J=8.5Hz, 1H),7.87(d,J=7.4Hz,2H),7.81(d,J=8.3Hz,1H),7.72(t,J=7.0Hz,2H),7.62-7.25(m,10H,D2O后降至9H),7.18(解析较差的d, J=5.8Hz,2H),7.04(s,1H),6.70(br s,2H,D2O后降至1H),6.50 (br s,与D2O可交换,1H),5.53-5.40(m,D2O后变成d,J=9.9Hz,1H),5.15(br d,J=12.4Hz,1H),4.82(br d,J=12.3Hz,1H),4.46-4.16(m,7H),4.07-3.85(m,4H),3.83-3.67(m,2H),3.76(s,3H),3.56-3.23 (m,5H,被水峰部分模糊),2.93-2.79(m,2H),2.64-2.32(m,6H,被DMSO峰部分模糊),2.25(s,3H),2.09-1.20(m,17H),1.35(s,9H), 0.87(d,J=7.0Hz,3H),0.83(d,J=6.8Hz,3H)。HRMS(ESI)m/z 为C77H93ClN9O15计算:1418.6474,发现:1418.6420[MH+];为C77H92ClN9NaO15计算:1440.6294,发现:1440.6231[MNa+];为C77H92ClKN9O15计算: 1456.6033,发现:1456.6021[MK+]。
在氮气氛下于0℃对65k(224mg,0.158mmol)在干的DMA(2mL) 中的搅动溶液添加哌啶在N,N-二甲基乙酰胺(DMA)中的溶液(1.0mmol 每mL,1.58mL,1.58mmol)。见图20。添加后于此温度将混合物再搅动1 小时45分钟。添加乙酸乙酯-石油醚的混合物(1:5,50mL),并于0℃将混合物搅动20分钟。自不溶性材料倒掉溶剂层,并丢弃。于室温用更多的乙酸乙酯-石油醚(1:5,2x30mL)重复清洗步骤。干燥留下的浅黄色固体,给出 8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4] 二氮杂卓-10(5H)-羧酸(S)-4-((S)-2-((S)-2-氨基-3-甲基丁酰胺基)-6-(叔丁氧羰基氨基)己酰胺基)苄酯651(173mg,99%);mp 74℃(dec.);1H NMR [(CD3)2SO]δ10.09(s,与D2O可交换,1H),8.21(s,1H),8.08(br s,与D2O可交换,1H),7.96(d,J=8.4Hz,1H),7.81(d,J=8.2Hz,1H), 7.66-7.42(m,4H),7.19(解析较差的d,J=7.5Hz,2H),7.04(s,1H), 6.71(br s,2H,D2O后降至1H),6.49(br s,与D2O可交换,1H),5.52-5.40 (m,D2O后变成d,J=10.6Hz,1H),5.16(br d,J=12.1Hz,1H),4.81 (br d,J=11.7Hz,1H),4.49-4.18(m,4H),4.10-3.85(m,3H),3.85-3.67 (m,2H),3.77(s,3H),3.59-3.22(m,5H,被水峰部分模糊),3.03-2.97 (m,D2O后变成d,J=4.8Hz,1H),2.91-2.81(m,2H),2.71-2.33(m, 7H,被DMSO峰部分模糊),2.25(s,3H),2.11-1.20(m,17H),1.34(s, 9H),0.86(d,J=6.5Hz,3H),0.76(d,J=6.7Hz,3H),2H未观察到。 HRMS(ESI)m/z为C62H83ClN9O13计算:1196.5793,发现:1196.5804[MH+];为C62H82ClN9NaO13计算:1218.5613,发现:1218.5612[MNa+];为 C62H82ClKN9O13计算:1234.5352,发现:1234.5359[MK+]。
在氮气氛下于0℃将651(173mg,0.158mmol)和6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酸2,5-二氧吡咯烷-1-基酯(马来酰亚胺基-Osu,122mg, 0.395mmol)在干的DMA(2mL)中的混合物搅动1小时45分钟。添加乙酸乙酯-石油醚的混合物(1:5,50mL),并于0℃将所得混合物搅动15分钟。自不溶性材料倒掉溶剂层,并丢弃。于室温用更多的乙酸乙酯-石油醚(1:5, 2x30mL)重复清洗步骤。干燥留下的固体,并通过硅土柱层析(DCM:乙酸乙酯:MeOH=20:10:3)纯化,给出8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5-氧 -2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸 (S)-4-((S)-6-(叔丁氧羰基氨基)-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基) 己酰胺基)-3-甲基丁酰胺基)己酰胺基)苄酯65m(152mg,69%),为浅黄色固体;HPLC:90.6%纯;mp 120℃;1H NMR[(CD3)2SO]δ9.95(s,与D2O 可交换,1H),8.21(s,1H),8.01(d,J=7.4Hz,与D2O可交换,1H), 7.95(d,J=8.4Hz,1H),7.85-7.73(m,2H,D2O交换后降至1H),7.63-7.50 (m,3H),7.46(t,J=7.7Hz,1H),7.17(解析较差的d,J=8.4Hz,2 H),7.04(s,1H),6.98(s,2H),6.81-6.66(m,2H,D2O交换后降至1H), 6.49(解析较差的d,J=5.3Hz,与D2O可交换,1H),5.51-5.41(m,D2O 后变成d,J=9.9Hz,1H),5.15(d,J=12.6Hz,1H),4.81(br d,J=11.3 Hz,1H),4.41(t,J=9.7Hz,1H),4.37-4.27(m,2H),4.27-4.13(m, 2H),4.10-3.85(m,3H),3.85-3.63(m,2H),3.77(s,3H),3.60-3.21 (m,8H,被水峰部分模糊,1H),2.92-2.81(m,2H),2.67-2.33(m,6 H,被DMSO峰部分模糊),2.26(s,3H),2.23-1.11(m,25H),1.30(s, 9H),0.84(d,J=6.8Hz,3H),0.81(d,J=6.7Hz,3H)。HRMS(ESI) m/z为C72H94ClN10O16计算:1389.6532,发现:1389.6478[MH+];为 C72H94ClN10NaO16计算:706.3212,发现:706.3244[MH+Na+];为 C72H93ClN10Na2O16计算:717.3122,发现:717.3121[MNa+Na+]。
在氮下于0℃(浴温度)对65m(32.2mg,0.023mmol)在DCM(0.4mL) 中的搅动溶液添加TFA(0.8mL),接着是2%水在TFA中的溶液(0.8mL)。添加后于此温度将混合物再搅动8小时。添加乙酸乙酯-石油醚(1:5,25mL),并于0℃将混合物搅动15分钟。收集沉淀的固体,用乙酸乙酯-石油醚(1:5, 2x30ml)清洗,并干燥,给出粗制的产物(30mg),通过制备性HPLC[SynergiMaxRP柱;水-TFA(pH=2.56;95%至55%)/10%H2O,在CH3CN 中(5%至45%);流速:12mL/min]纯化,给出8-(6-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-6-氧己基氧)-11-羟基-7-甲氧基-5- 氧-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-10(5H)-羧酸 (S)-4-((S)-6-氨基-2-((S)-2-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)-3- 甲基丁酰胺基)己酰胺基)苄酯65,为双三氟乙酸盐(22.2mg,64%),为玻璃样固体;HPLC:97.1%纯;mp 114℃;[α]D+43.6°(c 0.275,MeOH);1H NMR[(CD3)2SO]δ9.97(s,1H),9.90(br s,1H),8.73(br s,2H),8.27 (s,1H),8.09(d,J=7.6Hz,1H),8.04-7.93(m,2H),7.89(d,J= 8.4Hz,1H),7.82(d,J=7.9Hz,1H),7.74-7.50(m,5H),7.46(br t, J=7.6Hz,1H),7.22(m,2H),7.04(s,1H),7.00(s,2H),6.75(s, 1H),6.51(br s,1H),5.52-5.40(m,1H),5.18-5.04(m,1H),4.94-4.82 (m,1H),4.48-3.70(m,14H),3.43-3.20(m,4H,被水峰部分模糊), 3.17-3.05(m,3H),2.89(s,3H),2.82-2.70(m,2H),2.66-2.48(m, 2H,被DMSO峰部分模糊),2.25-1.08(m,25H),0.91-0.77(m,6H),2 H未观察到。HRMS(ESI)m/z为C67H86ClN10O14计算:1289.6008,发现: 1289.5975[MH+];为C67H85ClN10NaO14计算:1311.5827,发现:1311.5772 [MNa+];为C67H85ClN10Na2O14计算:667.2860,发现:667.2874[MNa+Na+];为C67H86ClN10NaO14计算:656.2950,发现:656.2963[MH+Na+];为 C67H87ClN10O14计算:645.3040,发现:645.3052[MH+H+]。
实施例16:N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-膦酰氧基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺66
对在冰浴中冷却的实施例7中自51a制备的5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯57a(1.595g,3.76mmol)在DCM(15mL) 中的溶液添加二烷(40mL)中的4N HCl。见图21。容许混合物升温至室温,并搅动2小时。抽走所有挥发性成分。在乙酸乙酯和冷的5%氨水之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,给出(S)-5-(苄基氧)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚57b,为褐色胶质,直接使用;1HNMR(DMSO)δ8.04(d,J=8.2Hz,1H),7.61(d,J=8.3Hz, 1H),7.53(d,J=7.2Hz,2H),7.45-7.34(m,4H),7.14(t,J=7.3Hz, 1H),6.60(s,1H),5.24(s,2H),3.96-3.92(m,1H),3.84(dd,J=3.4, 10.7Hz,1H),3.70(t,J=9.3Hz,1H),3.60(dd,J=2.4,10.0Hz,1H), 3.55(t,J=10.3Hz,1H)ppm。HRMS(ESI)发现m/z 324.1150(M+H)。 C20H19ClNO要求324.1150。
在冰浴中冷却中间体57b,并添加吡啶(15mL),接着是三氟乙酸酐 (3.14mL,22.57mmol)。将所得混合物搅动10分钟,并添加冰。在乙酸乙酯和水之间重分配混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:10)作为洗脱液纯化所得残留物,给出(S)-1-(5-(苄基氧)-1-(氯甲基)-1H-苯并[e]吲哚-3(2H)-基)-2,2,2-三氟乙酮66a,为白色固体(1.11g,70%);mp 167-170℃。1H NMR (CDCl3)δ8.37(d,J=8.3Hz,1H),8.05(s,1H),7.72(d,J=8.2Hz, 1H),7.61-7.54(m,3H),7.49-7.42(m,3H),7.39-7.35(m,1H),5.30 (AB q,J=11.7,15.7Hz,2H),4.63-4.59(m,1H),4.43-4.38(m,1H), 4.15-4.09(m,1H),3.97-3.93(m,1H),3.49(dd,J=9.9,11.3Hz,1H) ppm。HRMS(ESI)发现m/z 442.0799(M+Na)。C22H17ClF3NNaO2要求 442.0795。
于-10℃对66a(1.10g,2.62mmol)在THF(20mL)中的溶液添加25%甲酸铵水溶液(20mL),接着是Pd-C催化剂(10%,湿的,550mg),并将混合物搅动2小时,之后添加更多的Pd-C催化剂(550mg)。于-10℃将所得混合物搅动过夜,并穿过硅藻土过滤掉催化剂。自滤出液去除THF,并在乙酸乙酯和水之间重分配残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:5)作为洗脱液纯化所得残留物,给出(S)-1-(1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)- 基)-2,2,2-三氟乙酮66b,为灰白色固体(758mg,88%);mp 209-212℃。1H NMR(CDCl3)δ8.33(d,J=8.2Hz,1H),8.10(s,1H),7.85(s,1H), 7.64(d,J=8.2Hz,1H),7.60-7.56(m,1H),7.51-7.47(m,1H),4.60-4.56 (m,1H),4.41-4.36(m,1H),4.00-3.95(m,1H),3.93-3.90(m,1H), 3.44(dd,J=9.8,11.3Hz,1H)ppm。HRMS(ESI)发现m/z 352.0331(M +Na)。C15H11ClF3NNaO2要求352.0323。
对66b(250mg,0.76mmol)在THF(15mL)中的溶液添加四唑(3%,在乙腈中,13.5mL,4.55mmol),接着是N,N-二异丙基亚磷酰胺二叔丁酯 (1.51mL,4.55mmol)。于室温将混合物搅动过夜,然后在冰浴中冷却,并逐滴添加H2O2(30%水溶液,0.78mL,7.58mmol)。容许所得混合物升温至室温,并搅动5小时。通过在冰浴中冷却时添加10%亚硫酸钠水溶液来淬灭反应。通过旋转蒸发器去除有机挥发物。在乙酸乙酯和水之间重分配所得混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过(US 硅土)柱层析使用乙酸乙酯和石油醚的梯度混合物(v/v 1:6至1:3)作为洗脱液纯化所得残留物,给出磷酸(S)-二叔丁酯·1-(氯甲基)-3-(2,2,2-三氟乙酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯66c,为无色油(367mg,93%);1HNMR (DMSO)δ8.44(d,J=1.0Hz,1H),8.11(d,J=8.1Hz,1H),8.06(d, J=8.2Hz,1H),7.69-7.65(m,1H),7.63-7.59(m,1H),4.61-4.56(m, 1H),4.46-4.41(m,1H),4.15-4.12(m,1H),4.06-4.00(m,1H),1.50 (s,9H),1.48(s,9H)ppm。31P NMR(DMSO)δ-15.54ppm。HRMS(ESI) 发现m/z 544.1236(M+Na)。C23H28ClF3NNaO5P要求544.1238。
对在冰浴中冷却的66c(239mg,0.46mmol)在MeOH(2mL)中的溶液添加CsCO3(298mg,0.92mmol)和数滴水。将混合物在冰浴中搅动1小时,然后在乙酸乙酯和水之间重分配。用乙酸乙酯提取水相三次。用水和盐水清洗合并的有机提取液,在无水Na2SO4上干燥,穿过硅藻土过滤,并去除溶剂。在乙酸乙酯中溶解所得残留物,并穿过(US硅土)垫过滤柱层析,给出磷酸(S)-二叔丁酯·1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯66d,为灰白色胶质(183mg,94%),无需进一步纯化直接使用;1H NMR (DMSO)δ8.08(d,J=8.4Hz,1H),7.58(d,J=8.3Hz,1H),7.46-7.42 (m,1H),7.25-7.21(m,1H),7.13(d,J=0.8Hz,1H),4.00-3.93(m, 1H),3.87-3.78(m,2H),3.54-3.42(m,2H),1.50(s,9H),1.49(s,9H)ppm。31P NMR(DMSO)δ-15.58ppm。HRMS(ESI)发现m/z 426.1587 (M+H)。C21H30ClNO4P要求426.1595。
在三乙胺(50mL)中溶解2,5-二溴硝基苯(5.0g,17.8mmol)和丙烯酸叔丁酯(7.75mL,53.40mmol)。见图22。通过将氮气鼓泡穿过溶液来冲洗烧瓶,然后在氮流下添加三对甲苯基膦(433mg,1.42mmol)和乙酸钯 (80mg,0.36mmol)。在氮下将混合物回流搅动过夜。抽走三乙胺。在乙酸乙酯中溶解所得残留物,并穿过硅胶垫过滤。浓缩滤出液,并在层析柱上加载。使用乙酸乙酯和石油醚的混合物(1:10)作为洗脱液,给出3,3'-(2-硝基-1,4-亚苯基)二丙烯酸(2E,2'E)-叔丁酯66e,为白色固体(5.85g,88%); mp 123-124℃。1H NMR(CDCl3)δ8.14(d,J=1.7Hz,1H),7.99(d,J =15.8Hz,1H),7.74(dd,J=1.7,8.2Hz,1H),7.66(d,J=8.1Hz,1H), 7.58(d,J=16.0Hz,1H),6.50(d,J=16.0Hz,1H),6.36(d,J=15.8Hz, 1H),1.58(s,9H),1.56(s,9H)ppm。HRMS(ESI)发现m/z 398.1574 (M+Na)。C20H25NNaO6要求398.1574。
对在冰浴中冷却的66e(5.85g,15.58mmol)在丙酮(40mL)中的溶液添加锌粉(8.15g,125.0mmol),接着是NH4Cl(3.33g,62.30mmol)在水(20mL)中的溶液。于室温将混合物搅动1小时。添加更多的锌粉(4.00 g)和更多的水(10mL)中的NH4Cl(1.7g)。1小时后添加乙酸乙酯(100mL),并通过倾倒来收集上层清澈溶液。将清洗和倾倒步骤再重复两次。用水,接着是盐水清洗合并的有机溶液,在无水Na2SO4上干燥,并穿过硅胶垫过滤。去除溶剂,给出3,3′-(2-氨基-1,4-亚苯基)二丙烯酸(2E,2'E)-叔丁酯66f,为黄色胶质(5.46g,100%);mp73-75℃。1H NMR(CDCl3)δ7.68(d,J=15.8 Hz,1H),7.46(d,J=16.0Hz,1H),7.37(d,J=8.1Hz,1H),6.92(dd, J=1.5,8.1Hz,1H),6.81(d,J=1.4Hz,1H),6.33(d,J=16.0Hz,1H),6.31(d,J=15.8Hz,1H),3.99(br s,2H),1.53(s,9H),1.51(s,9H) ppm。HRMS(ESI)发现m/z368.1830(M+Na)。C20H27NNaO4要求368.1832。
于室温将66f(2.73g,7.90mmol),3-(((9H-芴-9-基)甲氧基)羰基氨基) 丙酸(Fmoc-β-丙氨酸,3.69g,11.85mmol),EDCI氢氯化物(7.58g,39.50 mmol)和对甲苯磺酸(136mg,0.79mmol)在DMA(25mL)中的混合物搅动过夜。在乙酸乙酯和水之间重分配混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,给出3,3′-(2-(3-(((9H-芴-9-基)甲氧基)羰基氨基)丙酰胺基)-1,4-亚苯基)二丙烯酸(2E,2'E)-叔丁酯66g,为白色固体(4.89g,97%); mp 102-105℃。1H NMR(CDCl3)δ7.87(s,1H),7.70-7.66(m,2H),7.62-7.52 (m,4H),7.44(br s,1H),7.38-7.34(m,3H),7.29-7.25(m,2H),6.41 (d,J=16.0Hz,1H),6.34(d,J=15.7Hz,1H),4.42(br s,2H),4.19 (t,J=6.4Hz,1H),3.59(br s,2H),2.67(br s,2H),1.53(s,9H),1.51(s,9H)ppm。HRMS(ESI)发现m/z 661.2878(M+Na)。C38H42N2NaO7要求661.2884。
对在冰浴中冷却的66g(530mg,0.83mmol)在DCM(4mL)中的溶液添加TFA(1mL,12.98mmol)。容许混合物升温至室温,并搅动过夜,给出白色悬浮液。添加乙酸乙酯以沉淀出更多的固体,通过过滤来收集,并用乙酸乙酯和石油醚清洗,给出(2E,2′E)-3,3'-(2-(3-(((9H-芴-9-基)甲氧基)羰基氨基) 丙酰胺基)-1,4-亚苯基)二丙烯酸66h,为白色固体(404mg,92%)。mp 282 ℃(dec.)。1H NMR(DMSO)δ12.46(br s,1H),9.92(s,1H),7.89-7.84(m,3H),7.74-7.68(m,4H),7.58-7.54(m,2H),7.44-7.38(m,3H), 7.31-7.28(m,2H),6.54(dd,J=3.2,16.0Hz,2H),4.30(d,J=6.5Hz, 2H),4.22(t,J=6.8Hz,1H),3.30(br s,2H),2.83-2.79(m,2H)ppm。 HRMS(ESI)发现m/z 549.1643(M+Na)。C30H26N2NaO7要求549.1632。
于室温将66d(178mg,粗制的,约0.42mmol),66h(55mg,0.10mmol), EDCI氢氯化物(160mg,0.84mmol)和对甲苯磺酸(1.8mg,0.01mmol) 在DMA(2mL)中的混合物搅动过夜。在乙酸乙酯和冷的稀NaHCO3水溶液之间重分配混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过(US硅土)柱层析使用MeOH和乙酸乙酯的梯度混合物(v/v 0.25-5%)进一步纯化残留物,给出3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-(二叔丁氧基磷酰基氧)-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基氨基甲酸(9H-芴-9-基)甲酯66i,为黄色固体(62mg,44%);1H NMR (DMSO)δ10.04(s,1H),8.67(s,2H),8.11-8.05(m,3H),7.97(d, J=8.2Hz,2H),7.88-7.82(m,4H),7.78(d,J=8.4Hz,1H),7.73-7.67 (m,3H),7.63-7.58(m,2H),7.53-7.47(m,3H),7.38(t,J=7.4Hz, 2H),7.30-7.24(m,4H),4.60-4.50(m,4H),4.42-4.35(m,2H),4.30 (d,J=6.7Hz,2H),4.22(t,J=6.6Hz,1H),4.06-3.93(m,4H),3.40-3.33 (m,2H),2.63-2.59(m,2H),1.50(2×s,18H),1.47(2×s,18H)ppm。31P NMR(DMSO)δ-15.45ppm。HRMS(ESI)发现m/z 1341.4677(M+H)。 C72H81Cl2N4O13P2要求1341.4647。
对66i(60mg,0.045mmol)在DMF(1mL)中的溶液添加哌啶(44μL, 0.45mmol)。于室温将混合物搅动3小时,然后抽走所有挥发性成分。与醚和石油醚的混合物(v/v 1:1)一起研磨所得残留物,给出游离胺66j,为白色固体(44mg,96%)。1H NMR(DMSO)δ8.68(s,2H),8.11-8.05(m, 3H),7.98(d,J=8.4Hz,2H),7.91-7.86(m,2H),7.78-7.85(m,1H), 7.70(d,J=15.3Hz,1H),7.63-7.58(m,2H),7.51(t,J=7.9Hz,2H), 7.26(d,J=15.3Hz,2H),4.66-4.52(m,4H),4.44-4.35(m,2H),4.07-3.95 (m,4H),2.95(t,J=6.5Hz,2H),2.56-2.50(m,2H),1.50-1.49(m, 36H)ppm。31P NMR(DMSO)δ-15.42,15.45ppm。HRMS(ESI)发现m/z1119.3981(M+H)。C57H71Cl2N4O11P2要求1119.3966。
于室温将66j(40mg,0.036mmol),6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基) 己酸2,5-二氧吡咯烷-1-基酯(SuOMC,12mg,0.037mmol),和DIPEA(6.8 μL,0.039mmol)在DMSO(1mL)中的混合物搅动过夜,之后抽走所有挥发性成分。与醚一起研磨所得残留物,给出二(磷酸二叔丁酯)·N-(3-(2,5-二 ((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺66k,为黄色固体 (32mg,67%)。1H NMR(DMSO)δ10.02(s,1H),8.67(s,2H),8.11-8.05 (m,3H),7.98-7.92(m,3H),7.85-7.75(m,2H),7.70(d,J=15.3Hz, 1H),7.63-7.58(m,2H),7.51(t,J=7.9Hz,2H),7.26(dd,J=4.4, 15.4Hz,2H),6.96(s,2H),4.66-4.52(m,4H),4.44-4.35(m,2H), 4.07-3.95(m,4H),3.45-3.30(m,4H),2.52-2.50(m,2H),2.08(t,J= 7.2Hz,2H),1.50-1.40(m,40H),1.33-1.25(m,2H)ppm。31P NMR(DMSO) δ-15.42,15.45ppm。HRMS(ESI)发现m/z 1334.4515(M+Na)。 C67H81Cl2N5NaO14P2要求1334.4525。
对在冰浴中冷却的66k(30mg,0.02mmol)在DCM(1mL)中的溶液添加TFA(1mL,12.98mmol)。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分,并与乙酸乙酯一起研磨所得残留物,给出N-(3-(2,5-二 ((E)-3-((S)-1-(氯甲基)-5-膦酰氧基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基) 苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺66,为黄色固体(20mg,80%,HPLC纯度95%);1H NMR(DMSO)δ10.01(s,1H), 8.59(s,2H),8.16-8.08(m,3H),7.97-7.90(m,3H),7.88-7.73(m,2H), 7.69(d,J=14.5Hz,1H),7.61-7.55(m,2H),7.46(t,J=7.3Hz,2H), 7.29-7.24(d,J=14.0Hz,2H),6.97(s,2H),4.62-4.52(m,3H),4.40-4.30 (m,3H),4.05-3.90(m,4H),3.43-3.37(m,2H),3.24-3.18(m,2H), 2.61-2.55(m,2H),2.08(br s,2H),1.54-1.44(m,4H),1.27-1.15(m,2H)ppm。31P NMR(DMSO)δ-5.81ppm。HRMS(ESI负)发现m/z 1086.2067 (M-H)。C51H48Cl2N5O14P2要求1086.2056。
实施例17:N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯 -1-基)己酰胺67
对3,3′-(2-(3-(((9H-芴-9-基)甲氧基)羰基氨基)丙酰胺基)-1,4-亚苯基)二丙烯酸(2E,2′E)-叔丁酯66g(4.89g,7.66mmol)在DCM(30mL)中的溶液添加哌啶(4.5mL,45.50mmol)。见图23。于室温将混合物搅动5小时,之后通过旋转蒸发器去除所有挥发性成分。使用乙酸乙酯作为洗脱液,接着是 TEA,MeOH,和乙酸乙酯的混合物(v/v 1:10:100)的柱层析给出3,3'-(2-(3- 氨基丙酰胺基)-1,4-亚苯基)二丙烯酸(2E,2'E)-叔丁酯67a,为白色固体(2.33 g,73%)。mp 62-63℃。1H NMR(CDCl3)δ11.04(s,1H),8.32(d,J=1.4 Hz,1H),7.91(d,J=15.7Hz,1H),7.55(d,J=16.1Hz,1H),7.54(d, J=8.0Hz,1H),7.24(dd,J=1.5,8.2Hz,1H),6.42(d,J=16.0Hz, 1H),6.34(d,J=15.7Hz,1H),3.18(d,J=5.6Hz,2H),2.52(t,J=5.6 Hz,2H),1.53(s,18H)ppm。HRMS(ESI)发现m/z 417.2394(M+H)。C23H33N2O5要求417.2384。
于室温将67a(1.00g,2.40mmol),6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基) 己酸2,5-二氧吡咯烷-1-基酯(SuOMC,740mg,2.40mmol),和DIPEA(460 μL,2.64mmol)在DMF(10mL)中的混合物搅动过夜。抽走所有挥发性成分。通过柱层析使用乙酸乙酯和石油醚的混合物(v/v 2:1)作为洗脱液,接着是单独的乙酸乙酯纯化所得残留物,给出3,3′-(2-(3-(6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺基)丙酰胺基)-1,4-亚苯基)二丙烯酸(2E,2'E)-叔丁酯 67b,为白色固体(1.00g,68%)。mp 69-72℃。1H NMR(CDCl3)δ7.95 (s,1H),7.73(s,1H),7.65(d,J=15.9Hz,1H),7.61(d,J=8.2Hz, 1H),7.53(d,J=16.0Hz,1H),7.37(d,J=8.2Hz,1H),6.69(br s, 1H),6.64(s,2H),6.39(d,J=16.0Hz,1H),6.38(d,J=15.8Hz,1H), 3.68-3.63(m,2H),3.43(d,J=7.0Hz,2H),2.69(d,J=5.5Hz,2H), 2.21(t,J=7.3Hz,2H),1.67-1.50(m,4H),1.53(s,9H),1.52(s,9H), 1.31-1.24(m,2H)ppm。HRMS(ESI)发现m/z 632.2931(M+Na)。 C33H43N3NaO8要求632.2942。
对在冰浴中冷却的67b(500mg,0.82mmol)在DCM(4mL)中的溶液逐滴添加TFA(2mL)。容许混合物升温至室温,并搅动4小时。抽走所有挥发性成分后,与乙酸乙酯一起研磨所得残留物,给出 (2E,2′E)-3,3'-(2-(3-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)-1,4-亚苯基)二丙烯酸67c,为白色固体(380mg,93%)。mp 257-261℃。1HNMR(DMSO)δ12.47(s,2H),9.90(s,1H),7.89-7.83(m,2H), 7.71-7.67(m,2H),7.57-7.53(m,2H),6.99(s,2H),6.53(d,J=15.9 Hz,2H),3.36-3.30(m,4H),2.54-2.50(m,2H),2.06(t,J=7.3Hz, 2H),1.52-1.42(m,4H),1.22-1.17(m,2H)ppm。HRMS(ESI)发现m/z 520.1683(M+Na)。C25H27N3NaO8要求520.1690。
对在冰浴中冷却的1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a(298mg,0.89mmol)在DCM(5mL)中的溶液添加二烷(10mL) 中的4N HCl。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分,给出(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-醇67d,为氢氯化物盐,添加 67c(185mg,0.37mmol),EDCI氢氯化物(428mg,2.23mmol),对甲苯磺酸(6mg,0.037mmol)和DMA(5mL)。于室温将混合物搅动6小时后,添加更多的EDCI氢氯化物(285mg,1.49mmol)和甲苯磺酸(6mg,0.037 mmol)。将混合物搅动过夜,然后在乙酸乙酯和水之间重分配。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土过滤。去除溶剂,并通过柱层析使用MeOH和乙酸乙酯的梯度混合物(v/v 1-15%)作为洗脱液进一步纯化所得残留物,给出67,为黄色固体(160mg,46%,HPLC纯度96%);mp 230-234℃(dec)。1H NMR (DMSO)δ10.43(s,1H),10.40(s,1H),10.00(s,1H),8.14-8.08(m, 5H),7.95(br s,1H),7.83-7.75(m,5H),7.68(d,J=15.2Hz,1H), 7.52(t,J=7.4Hz,2H),7.35(t,J=7.6Hz,2H),7.28-7.23(dd,J=5.2, 15.2Hz,2H),6.95(s,2H),4.58-4.45(m,4H),4.29-4.20(m,2H), 4.04-3.97(m,2H),3.88-3.80(m,2H),3.43-3.37(m,2H),3.23(d,J= 6.9Hz,2H),2.60-2.56(m,2H),2.09(d,J=7.1Hz,2H),1.50-1.40(m, 2H),1.40-1.30(m,2H),1.24-1.14(m,2H)ppm。HRMS(ESI)发现 m/z 950.2672(M+Na)。C51H47Cl2N5NaO8要求950.2694。
实施例18:二氢磷酸(S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)-2-(3-(6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺基)丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚 -5-基酯68
对在冰浴中冷却的磷酸(S)-二叔丁酯·1-(氯甲基)-3-(2,2,2-三氟乙酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯66c(125mg,0.24mmol)在MeOH(1 mL)中的溶液添加CsCO3(298mg,0.92mmol)和数滴水。在冰浴中将混合物搅动1小时,然后在乙酸乙酯和水之间重分配。用乙酸乙酯提取水相三次。用水和盐水清洗合并的有机提取液,在无水Na2SO4上干燥,穿过硅藻土过滤,并去除溶剂。在乙酸乙酯中溶解所得残留物,并穿过(US硅土)垫过滤。去除溶剂给出磷酸(S)-二叔丁酯·1-(氯甲基)-2,3-二氢-1H-苯并[e] 吲哚-5-基酯66d,为灰白色胶质,无需进一步纯化直接使用。
对在冰浴中冷却的1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-羧酸(S)-叔丁酯51a(80mg,0.24mmol)在DCM(2mL)中的溶液添加二烷(4mL) 中的4N HCl。容许混合物升温至室温,并搅动2小时。抽走所有挥发性成分,并直接使用(S)-1-(氯甲基)-2,3-二氢-1H-苯并[e]吲哚-5-醇67d。
对在盐-冰浴(-10℃)中冷却的66d和67d(均如上制备)在DMA(2mL) 中的溶液添加(2E,2'E)-3,3′-(2-(3-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)-1,4-亚苯基)二丙烯酸67c(119mg,0.24mmol),接着是EDCI 氢氯化物(276mg,1.44mmol)和对甲苯磺酸(4mg,0.024mmol)。容许混合物升温至室温,搅动过夜,然后倒在冰上。通过过滤来收集所得沉淀物,用水清洗,并在真空下干燥。通过(US硅土)层析柱使用MeOH 和DCM的梯度混合物(v/v 2-10%)纯化给出黄色固体(50mg),通过LC-MS 鉴定为磷酸二叔丁酯·((S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯-1-基)-2-(3-(6-(2,5-二氧-2,5-二氢 -1H-吡咯-1-基)己酰胺基)丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚 -5-基)酯68a和磷酸二叔丁酯·((S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯-1-基)-3-(3-(6-(2,5-二氧 -2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基)酯68b(分别为20%和23%,通过HPLC),67(44%,通过 HPLC),和66(8%,通过HPLC)的混合物。通过制备性HPLC(柱:RP 4μ,250×21.20mm;流动相:A/B=25%至0%(A:甲酸铵pH 3.45,B:90%乙腈,在水中);流速12mL/min,梯度方法;波长:254 nm,325nm)进一步纯化混合物,给出68a和68b的混合物,为黄色固体(18 mg,8%)。HRMS(ESI)发现m/z 1142.3577(M+Na)。C59H64Cl2N5NaO11P 要求1142.3609。
对在冰浴中冷却的68a和68b(17mg,0.015mmol)在DCM(0.5mL) 中的溶液逐滴添加TFA(0.5mL)。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分。与乙酸乙酯一起研磨所得残留物,给出二氢磷酸 (S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-3-氧丙-1-烯基)-2-(3-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基) 丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯68和磷酸酯区域异构体,二氢磷酸(S)-1-(氯甲基)-3-((E)-3-(4-((E)-3-((S)-1-(氯甲基)-5-羟基 -1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)-3-(3-(6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)苯基)丙烯酰基)-2,3-二氢-1H-苯并[e]吲哚-5-基酯68c的混合物,为橙色固体(11mg,72%,HPLC纯度:95%,异构体比率1:1)。1H NMR(DMSO)δ10.45(s,1H),10.01(s,1H),8.58(s,1H), 8.15-7.23(m,17H),6.95(s,2H),4.60-3.80(m,10H),3.43-3.37(m, 2H),3.27-3.18(m,2H),2.60-2.50(m,2H),2.10-2.00(m,2H),1.55-1.35 (m,4H),1.20-1.10(m,2H)ppm。31P NMR(DMSO)δ-5.81ppm。HRMS (ESI负)发现m/z1006.2394(M-H)。C51H47Cl2N5O11P要求1006.2392。
实施例23:N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e]吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰胺(化合物编号69,表4,图37)
对DMA(2mL)中192mg(0.53mmol)的1(通过上文所述规程新鲜制备)添加2(100mg,0.20mmol),EDCI氢氯化物(231mg,1.21mmol) 和甲苯磺酸(3.5mg,0.020mmol)。将混合物搅动过夜,然后倒入氨水和冰的混合物中。通过过滤来收集所得沉淀物,用水清洗,干燥,并通过硅胶柱层析进行纯化。使用MeOH和DCM的梯度混合物(v/v 1-15%)作为洗脱液来提供N-(3-(2,5-二((E)-3-((S)-1-(氯甲基)-5-(4-甲基哌嗪-1-羰基氧)-1H-苯并[e] 吲哚-3(2H)-基)-3-氧丙-1-烯基)苯基氨基)-3-氧丙基)-6-(2,5-二氧-2,5-二氢-1H- 吡咯-1-基)己酰胺,为黄色固体(30mg,13%,HPLC纯度96%)。mp 268℃ (dec)。1H NMR(CDCl3)δ8.60(s,1H),8.46(s,1H),8.42(s,1H), 7.87-7.84(m,3H),7.75-7.60(m,5H),7.40-7.33(m,5H),7.22(br s, 1H),6.77(t,J=15.3Hz,2H),6.56(s,2H),4.46-4.40(m,2H),4.30-4.25(m,2H),4.11-4.00(m,2H),3.95-3.90(m,7H),3.68(表观s,6H), 3.53-3.46(m,3H),3.38(t,J=7.0Hz,2H),2.73(表观s,2H),2.59-2.55 (m,8H),2.42(s,6H),2.28(t,J=7.0Hz,2H),1.56-1.49(m,2H), 1.31-1.23(m,2H)ppm。HRMS(ESI)发现m/z 1180.4416(M+H)。C63H68Cl2N9O10要求1180.4416。
实施例24:2,5-二((E)-3-((S)-1-(氯甲基)-5-(膦酰氧基)-1H-苯并[e]吲哚 -3(2H)-基)-3-氧丙-1-烯基)苯基氨基甲酸2-(吡啶-2-基二硫基)丙酯(化合物编号72,表4,图38)
对207mg(0.49mmol)的1(通过上文所述规程新鲜制备)添加2(70mg, 0.15mmol),EDCI氢氯化物(233mg,1.22mmol),甲苯磺酸(3mg,0.015 mmol)和DMA(0.5mL)。将混合物搅动过夜后,在真空下去除大部分DMA,并在乙酸乙酯和水之间重分配残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,在最小限度的DCM中溶解所得残留物,并通过添加庚烷来沉淀,给出粗制的产物(156mg),通过制备性HPLC(柱:Synergi-Max RP 4μ, 250×21.20mm;流动相:A/B=10%至1%(A:甲酸铵pH 3.45,B:90%乙腈,在水中);流速12mL/min,梯度方法;波长:254nm,325nm)进一步纯化,给出3(78mg,40%),为黄色固体。1H NMR(DMSO)δ9.67(s, 1H),8.67(s,2H),8.43(d,J=4.5Hz,1H),8.11-8.06(m,3H),7.97 (表观d,J=8.5Hz,2H),7.92(d,J=15.3Hz,1H),7.84-7.76(m,3H), 7.70(d,J=15.3Hz,2H),7.61(t,J=7.6Hz,2H),7.51(t,J=7.7Hz, 2H),7.28(d,J=15.3Hz,1H),7.27(d,J=15.3Hz,1H),7.23-7.19(m,1H),4.62-4.53(m,4H),4.43-4.37(m,2H),4.23-4.13(m,2H),4.05-3.95 (m,4H),3.42-3.37(m,1H),1.51(s,9H),1.50(s,9H),1.49(s, 9H),1.48(s,9H),1.34(d,J=6.5Hz,3H)ppm。31P NMR(DMSO)δ-15.46(s)和-15.48(s)ppm。HRMS(ESI)发现m/z 1297.3471(M+Na)。C63H74Cl2N4NaO12P2S2要求1297.3489。
对在冰浴中冷却的3(60mg,0.047mmol)在DCM(2mL)中的溶液添加TFA(1mL,6.49mmol)。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分,并与乙酸乙酯一起研磨所得残留物,给出4,为黄色固体(49 mg,99%,HPLC纯度100%);1H NMR(DMSO)δ9.65(s,1H),8.59(s, 2H),8.46-8.44(m,1H),8.15-8.08(m,3H),7.96-7.90(m,3H),7.82-7.70(m,3H),7.70(d,J=15.6Hz,1H),7.59(t,J=6.8Hz,2H),7.48(t, J=7.5Hz,2H),7.31-7.22(m,3H),5.75(s,1H),4.64-4.53(m,4H), 4.40-4.33(m,3H),4.25-4.15(m,3H),4.06-3.93(m,3H),1.35-1.32(m, 3H)ppm。31P NMR(DMSO)δ-5.95(s)ppm。HRMS(ESI)发现m/z1073.0949 (M+Na)。C47H42Cl2N4NaO12P2S2要求1073.0985。
实施例25:二氢磷酸[(1S)-1-(氯甲基)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(氯甲基)-5-膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-3-氧-丙-1-烯基]-2-[2-[2-(2,5-二氧吡咯-1-基)乙氧基]乙氧基]苯基]丙-2-烯酰基]-1,2-二氢苯并[e]吲哚-5-基]酯(化合物编号78,表4,图39)
中间体3
对76mg(0.18mmol)的1(通过上文所述规程新鲜制备)添加2(18mg, 0.045mmol),EDCI氢氯化物(69mg,0.36mmol),甲苯磺酸(0.8mg,0.005 mmol)和DMA(0.25mL)。将混合物搅动过夜后,在真空下去除大部分DMA,并在乙酸乙酯和水之间重分配残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,在最小限度的DCM中溶解所得残留物,并通过添加庚烷来沉淀,给出粗制的产物(54mg),通过制备性HPLC(柱:Synergi-Max RP 4μ, 250×21.20mm;流动相:A/B=20%至1%(A:甲酸铵pH 3.45,B:90%乙腈,在水中);流速12mL/min,梯度方法;波长:254nm,325nm)进一步纯化,给出3(17mg,30%),为黄色固体。1H NMR(CDCl3)δ8.72(br s,2H),8.23(d,J=8.4Hz,2H),7.96(d,J=15.2Hz,1H),7.83(d,J= 15.3Hz,1H),7.71(d,J=8.2Hz,2H),7.54-7.50(m,3H),7.42-7.39 (m,2H),7.26-7.12(m,3H),6.95-6.88(m,1H),6.67(s,2H),4.57-4.52 (m,2H),4.47-4.38(m,2H),4.28-4.24(m,2H),4.16-4.09(m,2H),4.00-3.94(m,4H),3.78(表观s,4H),3.55-.348(m,2H),1.57(s,36H) ppm。31P NMR(CDCl3)δ-15.64(s)ppm。HRMS(ESI)发现m/z 1238.3862 (M+Na)。C62H73Cl2N3NaO14P2要求1238.3837。
对在冰浴中冷却的3(16mg,0.013mmol)在DCM(1mL)中的溶液添加TFA(0.5mL,3.24mmol)。容许混合物升温至室温,并搅动3小时。抽走所有挥发性成分,并与乙酸乙酯一起研磨所得残留物,给出化合物78,为黄色固体(13mg,100%,HPLC纯度100%)。1H NMR(DMSO)δ8.60(s, 2H),8.12(d,J=8.4Hz,2H),7.95-7.87(m,4H),7.72(d,J=15.1Hz, 1H),7.61-7.57(m,2H),7.53-7.45(m,4H),7.38-7.32(m,2H),6.97 (s,2H),4.60-4.48(m,4H),4.30-4.28(m,4H),4.08-3.88(m,6H), 3.68-3.58(m,4H)。31P NMR(DMSO)δ-5.94(s)ppm。HRMS(ESI)发现m/z 1014.1301(M+Na)。C46H41Cl2N3NaO14P2要求1014.1333。
实施例26:二氢磷酸[(1S)-1-(氯甲基)-3-[(E)-3-[4-[(E)-3-[(1S)-1-(氯甲基)-5-膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-3-氧-丙-1-烯基]-2-[2-(2,5-二氧吡咯-1-基)乙氧基]苯基]丙-2-烯酰基]-1,2-二氢苯并[e]吲哚-5-基]酯(化合物编号 79,表4,图40)
中间体3
对52mg(0.12mmol)的1(通过上文所述规程新鲜制备)添加2(11mg, 0.031mmol),EDCI氢氯化物(35mg,0.18mmol),甲苯磺酸(0.5mg,0.003 mmol)和DMA(0.25mL)。将混合物搅动过夜后,在真空下去除大部分DMA,并在乙酸乙酯和水之间重分配残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,在最小限度的DCM中溶解所得残留物,并通过添加庚烷来沉淀,给出粗制的产物(71mg),通过制备性HPLC(柱:Synergi-Max RP 4μ, 250×21.20mm;流动相:A/B=20%至1%(A:甲酸铵pH 3.45,B:90%乙腈,在水中);流速12mL/min,梯度方法;波长:254nm,300nm)进一步纯化,给出3(15mg,42%),为黄色固体。1H NMR(CDCl3)δ8.71(br s,2H),8.25(d,J=8.4Hz,2H),7.95(d,J=15.5Hz,1H),7.83(d,J= 15.3Hz,1H),7.72(d,J=8.2Hz,2H),7.60-7.52(m,3H),7.46-7.40 (m,2H),7.28-7.20(m,2H),7.13-6.99(m,2H),6.78(s,2H),4.64-4.60 (m,2H),4.51-4.45(m,2H),4.38-4.32(m,2H),4.15-4.05(m,4H),4.00-3.95(m,2H),3.57-3.49(m,2H),1.58(s,36H)ppm。31P NMR(CDCl3) δ-15.67(s)ppm。HRMS(ESI)发现m/z 1194.3606(M+Na)。 C60H69Cl2N3NaO13P2要求1194.3575。
化合物编号79
对在冰浴中冷却的3(13mg,0.011mmol)在DCM(0.5mL)中的溶液添加TFA(0.2mL,1.30mmol)。容许混合物升温至室温,并搅动0.5小时。添加二乙醚,给出沉淀物,过滤,并用乙酸乙酯清洗,给出4(化合物编号 79),为黄色固体(8.4mg,80%,HPLC纯度90%)。1H NMR(DMSO)δ8.59 (s,2H),8.13(d,J=8.3Hz,2H),7.97-7.82(m,4H),7.71(d,J=15.1 Hz,1H),7.60-7.52(m,3H),7.49-7.44(m,3H),7.35-7.24(m,2H), 7.08(s,2H),4.65-4.50(m,4H),4.40-4.30(m,4H),4.05-3.90(m,6H)。31P NMR(DMSO)δ-5.81(s)ppm。HRMS(ESI)发现m/z970.1036(M+ Na)。C44H37Cl2N3NaO13P2要求970.1071。
实施例27:N-[1-(氯甲基)-3-[5-[1-(氯甲基)-5-羟基-1,2-二氢苯并[e]吲哚 -3-基]-5-氧-戊酰基]-1,2-二氢苯并[e]吲哚-5-基]氨基甲酸2-(2-吡啶基二硫基) 丙酯(化合物编号80,表4,图41)
在氮下于室温将62c(31.0mg,0.0674mmol,通过上文所述规程新鲜制备),53h(23.0mg,0.0674mmol,通过上文所述规程新鲜制备),EDCI.HCl (38.7mg,0.202mmol)和TsOH(2.3mg,0.0135mmol)在干的DMA(2mL) 中的混合物搅动过夜。18小时后用EtOAc和H2O稀释反应混合物,并充分混合。分出各层,并用H2O(3次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用DCM:MeOH 100:0至98:2纯化粗制的产物,接着与二异丙醚一起研磨,给出化合物1(化合物编号80,26.0mg,49%, HPLC纯度:95.2%),为乳脂状固体。1H NMRδ(400MHz,DMSO-d6)10.36 (s,1H),9.69(s,1H),8.56(s,1H),8.44(d,J=4.3Hz,1H),8.08 (d,J=8.0Hz,1H),8.02-8.00(m,2H),7.92(d,J=8.3Hz,1H),7.85-7.77(m,3H),7.57-7.53(m,1H),7.51-7.47(m,1H),7.45-7.41(m,1H), 7.34-7.30(m,1H),7.24-7.21(m,1H),4.42-4.29(m,3H),4.25-4.13(m, 5H),4.05-3.97(m,2H),3.91(dd,J=11,7.2Hz,1H),3.79(dd,J=11, 8.3Hz,1H),3.43-3.36(m,1H),2.77-2.56(m,4H),2.01-1.93(m,2H), 1.34(d,J=6.7Hz,3H)。HRMS m/z 811.1542[为C40H38Cl2N4NaO5S2计算的 (M+Na)+为811.1553]。
实施例28:3-[6-[1-(氯甲基)-5-(4-甲基哌嗪-1-羰基)氧-1,2-二氢苯并[e] 吲哚-3-基]-6-氧-己氧基]-6-羟基-2-甲氧基-11-氧-6a,7,8,9-四氢-6H-吡咯并 [2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基)丙酯,3-[6-[1-(氯甲基)-5-膦酰氧基-1,2-二氢苯并[e]吲哚-3-基]-6-氧-己氧基]-6-羟基-2-甲氧基 -11-氧-6a,7,8,9-四氢-6H-吡咯并[2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基)丙酯,和3-[6-[1-(氯甲基)-5-羟基-1,2-二氢苯并[e]吲哚-3-基]-6-氧-己氧基]-6-羟基-2-甲氧基-11-氧-6a,7,8,9-四氢-6H-吡咯并[2,1-c][1,4]苯并二氮杂卓-5-羧酸2-(2-吡啶基二硫基)丙酯(化合物编号81-83,表4,图42-43)
于室温将1(1.40g,4.00mmol,遵循文献规程制备:J.Med.Chem.2003, 46,2132-2151),2(1.31g,5.22mmol,遵循文献规程制备:WO2004065491 A1)和K2CO3(829mg,6.00mmol)在干的DMA(15mL)中的混合物搅动43小时。然后用EtOAc和H2O稀释混合物,充分混合,并分出各层。用H2O (3次),盐水(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用EtOAc:Hex 50:50至67:33至100:0纯化粗制的产物,给出化合物3(1.74g,84%),为黄色油。1H NMRδ(400MHz,CDCl3)8.77 (br s,1H),7.79(s,1H),6.82(s,1H),6.02-5.92(m,1H),5.36(dq, J=17.2,1.5Hz,1H),5.26(dq,J=10.4,1.2Hz,1H),4.69-4.60(m, 2H),4.47-4.39(m,1H),4.28(br s,1H),4.09-4.05(m,2H),3.83(s, 3H),3.90-3.80(m,1H),3.74-3.70(m,1H),3.65-3.59(m,1H),3.54-3.47 (m,1H),2.25(t,J=7.4Hz,2H),2.20-2.15(m,1H),1.93-1.84(m, 3H),1.81-1.72(m,1H),1.70-1.63(m,3H),1.53-1.47(m,2H),1.44 (s,9H)。HRMS m/z 543.2666[为C27H40N2NaO8计算的(M+Na)+为543.2677]。
于室温将Et3N(1.32mL,9.47mmol)添加至3(821mg,1.58mmol) 在干的DCM(6mL)中的溶液。然后添加乙酸酐(0.75mL,7.93mmol),并于室温将混合物搅动4.5小时。将反应混合物冷却至0℃,添加干的MeOH (1mL),并于0℃将混合物搅动15分钟。然后添加EtOAc(120mL),并用 H2O(2次),盐水(1次)清洗混合物,干燥(Na2SO4),并在真空下去除溶剂,给出化合物4(891mg,定量的),无需纯化在下一步中使用。1H NMRδ (400MHz,CDCl3)8.88(br s,1H),7.82(s,1H),6.81(s,1H),6.01-5.91 (m,1H),5.36(dq,J=17.2,1.5Hz,1H),5.25(dq,J=10.4,1.3Hz, 1H),4.65-4.62(m,2H),4.61-4.54(m,1H),4.32-4.22(m,2H),4.09-4.06(m,2H),3.83(s,3H),3.55-3.47(m,2H),2.26-2.23(m,2H),2.18-2.12 (m,1H),2.07(s,3H),1.97-1.77(m,5H),1.70-1.63(m,2H),1.54-1.47 (m,2H),1.44(s,9H)。HRMS m/z585.2774[为C29H42N2NaO9计算的(M+Na)+为585.2783]。
于室温将吡咯烷(1.6mL,19.2mmol)添加至4(1.06g,1.88mmol) 在干的DCM(20mL)中的溶液。然后添加Pd(PPh3)4(109mg,0.0943mmol),并于室温将反应混合物搅动40分钟。用0.25M HCl溶液(2x 75mL)清洗反应混合物,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用EtOAc:Hex 50:50至100:0纯化粗制的产物,给出化合物5(726mg,81%),为黄色油。1H NMRδ(400MHz,DMSO-d6)6.67(s,1H),6.35(s,1H), 5.08(s,2H),4.35-4.30(m,1H),4.13-4.06(m,2H),3.87(t,J=6.4Hz, 2H),3.63(s,3H),3.50-3.44(m,1H),3.42-3.35(m,1H),2.21(t,J= 7.2Hz,2H),2.07-2.00(m,1H),2.01(s,3H),1.89-1.82(m,1H),1.77-1.67 (m,4H),1.59-1.51(m,2H),1.44-1.36(m,2H),1.39(s,9H)。HRMS m/z 501.2573[为C25H38N2NaO7计算的(M+Na)+为501.2571]。
在氮下于室温将双光气(0.22mL,1.82mmol)添加至5(726mg,1.52 mmol)和DMAP(557mg,4.56mmol)在干的DCM(25mL)中的混合物。 30分钟后添加6(2.60g,12.9mmol;通过上文所述规程新鲜制备-先前未给醇指派代码)在干的DCM(25mL)中的溶液,并于室温将混合物搅动过夜。18小时后用H2O(1次)清洗反应混合物,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用DCM:EtOAc 100:0至95:5至94:6(直至过量的6洗脱),然后是EtOAc:Hex 70:30纯化粗制的产物,给出化合物7(920 mg,86%),为浅黄色油。1H NMRδ(400MHz,DMSO-d6)9.16(br s,1H), 8.45-8.43(m,1H),7.83-7.78(m,2H),7.25-7.21(m,1H),7.15(d,J= 2.8Hz,1H),6.87(s,1H),4.29(br s,1H),4.17-3.99(m,4H),3.92 (t,J=6.4Hz,2H),3.75(s,3H),3.42-3.30(m,3H),2.20(t,J=7.2 Hz,2H),2.06-1.95(m,4H),1.83(br s,1H),1.77-1.68(m,4H),1.58-1.49 (m,2H),1.43-1.36(m,2H),1.39(s,9H),1.29(d,J=6.8Hz,3H)。 HRMS m/z 706.2832[为C34H48N3O9S2计算的(M+H)+为706.2826]。
于室温将7(949mg,1.34mmol)和K2CO3(1.85g,13.4mmol)在 DCM-MeOH(34mL/17mL)中的混合物搅动45分钟。用DCM稀释混合物,倒入冰H2O(200mL)中,充分混合,并分出各层。用DCM(1次)提取水层,干燥(Na2SO4)合并的有机层,并在真空下去除溶剂。在硅胶上通过柱层析使用DCM:EtOAc 100:0至50:50纯化粗制的产物,给出化合物8(808mg, 91%),为浅黄色油。1H NMRδ(400MHz,DMSO-d6)9.20(br s,1H), 8.44(d,J=4.7Hz,1H),7.81-7.80(m,2H),7.25-7.20(m,2H),6.94 (s,1H),4.75(t,J=5.6Hz,1H),4.17-3.99(m,3H),3.92(t,J=6.4 Hz,2H),3.74(s,3H),3.60-3.46(m,2H),3.37-3.20(m,3H),2.20 (t,J=7.2Hz,2H),1.93-1.76(m,3H),1.75-1.68(m,3H),1.58-1.51 (m,2H),1.44-1.36(m,2H),1.39(s,9H),1.29(d,J=6.9Hz,3H)。 HRMS m/z 664.2721[为C32H46N3O8S2计算的(M+H)+为664.2724]。
于室温将(二乙酰氧基碘)苯(259mg,0.804mmol)添加至8(349mg, 0.526mmol)和TEMPO(82.2mg,0.526mmol)在干的DCM(10mL)中的混合物,并将反应混合物搅动过夜。24小时后用DCM和饱和Na2S2O3水溶液稀释混合物,并充分混合。分出各层,并用饱和Na2S2O3水溶液(1次),饱和NaHCO3水溶液(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用EtOAc:Hex 70:30至100:0纯化粗制的产物,给出化合物9(248mg,71%),为白色泡沫。1H NMRδ(400MHz,DMSO-d6) 8.45-8.43(m,1H),7.79-7.69(m,1H),7.51-7.48(m,1H),7.24-7.20(m, 1H),7.10(s,1H),6.96和6.91(2s,1H),6.55(t,J=5.9Hz,1H),5.46(dd,J=8.9,6.1Hz,1H),4.31-4.21(m,1H),4.02-3.84(m,3H),3.80 和3.79(2s,3H),3.52-3.46(m,1H),3.40-3.18(m,3H),2.19-2.13(m, 2H),2.09-2.00(m,1H),1.96-1.85(m,3H),1.70-1.67(m,2H),1.56-1.45 (m,2H),1.40-1.34(m,2H),1.38和1.37(2s,9H),1.15-1.10(m,3H)。 HRMS m/z 662.2592[为C32H44N3O8S2计算的(M+H)+为662.2564]。
于室温将9(254mg,0.384mmol)和二烷(11mL)中的4M HCl的混合物搅动1小时15分钟。于25-30℃在真空下去除溶剂,给出化合物10(162 mg,70%),无需纯化在下一步中使用。
在氮下于室温将10(161mg,0.266mmol),58b(195mg,0.542mmol,通过上文所述规程新鲜制备),EDCI.HCl(253mg,1.32mmol)和TsOH(19.5 mg,0.113mmol)在干的DMA(5mL)中的混合物搅动过夜。23小时后用 EtOAc和饱和NaHCO3水溶液稀释反应混合物,并充分混合。分出各层,并用EtOAc(1次)提取水层。用H2O(1次),盐水(1次)清洗合并的有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用DCM:MeOH 100:0至93:7纯化粗制的产物,并将材料使用DCM:MeOH 99:1至94:6再次柱层析,给出11(化合物编号81,118mg,47%,HPLC纯度:98.0%),为浅黄色泡沫。1H NMRδ(400MHz,DMSO-d6)8.43-8.41(m,1H),8.22(s, 1H),7.96(d,J=8.4Hz,1H),7.83(d,J=8.4Hz,1H),7.73-7.66(m, 1H),7.61-7.56(m,1H),7.51-7.45(m,2H),7.22-7.17(m,1H),7.10 (s,1H),6.97和6.92(2s,1H),6.56(t,J=6.0Hz,1H),5.46(dd,J= 9.1,6.2Hz,1H),4.42-4.20(m,4H),4.05-3.76(m,7H),3.80和3.79(2s, 3H),3.52-3.47(m,1H),3.38-3.11(m,4H),2.09-1.99(m,1H),1.94-1.88 (m,3H),1.77-1.74(m,2H),1.65-1.62(m,2H),1.48-1.42(m,2H), 1.35-1.23(m,1H),1.15-1.10(m,3H),9H被DMSO部分模糊。HRMS m/z 969.3070[为C47H55ClN6NaO9S2计算的(M+Na)+为969.3053]。
在氮下于室温将10(162mg,0.267mmol),66d(178mg,0.418mmol,通过上文所述规程新鲜制备),EDCI.HCl(184mg,0.960mmol)和TsOH(11 mg,0.0639mmol)在干的DMA(5mL)中的混合物搅动过夜。18.5小时后用EtOAc和H2O稀释反应混合物,并充分混合。分出各层,并用饱和NaHCO3水溶液(1次),H2O(1次),盐水(1次)清洗有机层,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用EtOAc:MeOH 100:0至95:5纯化粗制的产物,给出黄色残留物。通过制备性HPLC(柱:Synergi-MAX RP 4μ, 21.20x 250mm;流速:12mL/min;流动相:溶剂A:H2O/甲酸铵缓冲液pH 3.45,溶剂B:MeCN/H2O 90:10;方法:梯度,溶剂A:溶剂B 90:10至10:90 至0:100,在30分钟里)进一步纯化,给出化合物12(89.3mg,33%,HPLC 纯度:99.5%),为白色泡沫。1H NMRδ(400MHz,DMSO-d6)8.56(s, 1H),8.43-8.41(m,1H),8.04(d,J=8.2Hz,1H),7.92(d,J=8.4Hz, 1H),7.73-7.67(m,1H),7.60-7.56(m,1H),7.51-7.45(m,2H),7.22-7.17 (m,1H),7.10(s,1H),6.98和6.92(2s,1H),6.55(t,J=5.6Hz,1H), 5.47-5.44(m,1H),4.40-4.36(m,1H),4.30-4.19(m,3H),4.04-3.86(m, 4H),3.86-3.75(m,1H),3.80和3.79(2s,3H),3.52-3.46(m,1H),3.38-3.22 (m,3H),3.21-3.15(m,1H),2.09-1.99(m,1H),1.94-1.85(m,3H), 1.78-1.74(m,2H),1.69-1.60(m,2H),1.55-1.40(m,2H),1.47和1.47 (2s,18H),1.28-1.23(m,1H),1.15-1.10(m,3H)。HRMS m/z 1035.3162 [为C49H62ClN4NaO11PS2计算的(M+Na)+为1035.3175]。
于室温将12(84.0mg,0.0829mmol)和TFA(1mL)在干的DCM(2mL) 中的混合物搅动40分钟。然后于25℃在真空下去除溶剂,给出绿色残留物。在DCM中溶解残留物,用EtOAc稀释溶液,并在真空下去除DCM,给出白色固体,倒掉剩余溶剂。重复这个过程,与EtOAc一起研磨所得固体,并干燥,给出化合物13(化合物编号82,43.8mg,59%,HPLC纯度:93.8%),为白色固体。1H NMRδ(400MHz,DMSO-d6)8.47(s,1H),8.44-8.42(m, 1H),8.08(d,J=8.3Hz,1H),7.90(d,J=8.3Hz,1H),7.74-7.68(m, 1H),7.58-7.54(m,1H),7.51-7.48(m,1H),7.47-7.43(m,1H),7.22-7.18 (m,1H),7.10(s,1H),6.98和6.93(2s,1H),5.46(d,J=9.5Hz,1H),4.39-4.18(m,4H),4.04-3.95(m,3H),3.90-3.85(m,1H),3.84-3.76(m, 1H),3.80和3.80(2s,3H),3.52-3.47(m,1H),3.40-3.27(m,3H),3.21-3.15 (m,1H),2.10-2.02(m,1H),1.94-1.88(m,3H),1.78-1.74(m,2H), 1.69-1.60(m,2H),1.48-1.42(m,2H),1.35-1.23(m,1H),1.16-1.10(m, 3H),3H未观察到。HRMS m/z 923.1938[为C41H46ClN4NaO11PS2计算的 (M+Na)+为923.1923]。
在氮下于室温将10(45.0mg,0.0743mmol),67d(24.3mg,0.0899mmol,通过上文所述规程新鲜制备),EDCI.HCl(42.7mg,0.223mmol)和TsOH (3mg,0.0174mmol)在干的DMA(3mL)中的混合物搅动5小时。将另外部分的67d(24.3mg,0.0899mmol)和EDCI.HCl(16.0mg,0.0835mmol) 添加至混合物,并于室温将反应搅动过夜。22小时后用EtOAc稀释反应混合物,并用H2O(2次),盐水(1次)清洗,干燥(Na2SO4),并在真空下去除溶剂。在硅胶上通过柱层析使用EtOAc纯化粗制的产物,给出绿色粉末。第二次在硅胶上通过柱层析使用EtOAc进一步纯化,给出化合物14(化合物编号83,8.3mg,13.5%,HPLC纯度:81.2%),为米色固体。1HNMRδ(400MHz, DMSO-d6)10.33(s,1H),8.43-8.42(m,1H),8.07(d,J=8.1Hz,1H), 7.98(s,1H),7.77(d,J=8.4Hz,1H),7.74-7.67(m,1H),7.50-7.46 (m,2H),7.33-7.29(m,1H),7.24-7.18(m,1H),7.10(s,1H),6.98 和6.92(2s,1H),6.56(t,J=6.0Hz,1H),5.47-5.44(m,1H),4.33-4.21 (m,2H),4.15-4.13(m,2H),4.05-3.93(m,3H),3.90-3.75(m,2H), 3.80和3.79(2s,3H),3.52-3.47(m,1H),3.38-3.13(m,4H),2.10-1.99 (m,1H),1.94-1.89(m,3H),1.77-1.74(m,2H),1.66-1.62(m,2H), 1.52-1.41(m,2H),1.32-1.24(m,1H),1.15-1.10(m,3H)。HRMS m/z 843.2258 [为C41H45ClN4NaO8S2计算的(M+Na)+为843.2260]。
实施例29:β-D-吡喃葡萄糖苷酸(1S)-1-(氯甲基)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(氯甲基)-5-[(6-甲基-β-D-吡喃葡萄糖酸苷基) 氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)-2-[(3-{[6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰基]氨基}丙酰基)氨基]苯基}-2-丙烯酰基)-1,2-二氢-3H- 苯并[e]吲哚-5-基甲基酯(化合物编号84,表4,图44)
依照文献规程制备三氯亚氨基乙酸酯(1):L.Lázár,E.M. Herczeg,A.Lipták,S.Antus,A.Borbás,Tetrahedron 2012,68,7386-7399; L.Tietze,H.Schuster,K.Schmuck,I.Schuberth,F.Alves,Bioorg.&Med.Chem. 2008,16,6312-6318。
于室温将三氯亚氨基乙酸酯(1)(360mg,0.75mmol),酚-CBI(2,化合物51a,第一次专利提交)(200mg,0.60mmol)和活化的分子筛(1g) 在无水CH2Cl2(20mL)中的悬浮液搅动1小时。将混合物冷却至-10℃,然后逐滴添加BF3OEt2(40μl,0.3mmol)。将温度在-10℃和-5℃之间保持1小时,然后于0℃搅动30分钟。随后于0℃逐滴添加BF3OEt2(0.24mL,1.8mmol),容许温度升高至室温,并搅动2小时。然后在硅藻土上过滤悬浮液,并蒸发溶剂,给出粗制的CBI-葡萄糖苷酸(3),无需进一步纯化在下一步中使用。将胺(3)和二酸(4,化合物66h,第一次专利提交中)(126mg,0.24 mmol)在无水DMA(4mL)中的溶液冷却至0℃。然后添加pTsOH(17mg, 0.096mmol)和EDCI·HCl(276mg,1.44mmol),容许温度升高至室温,并搅动16小时。在减压下去除溶剂,并通过柱层析(SiO2,CH2Cl2/MeOH 0-2%) 然后是(SiO2,CH2Cl2/MeOH 1-2%)纯化残留物,给出2,3,4-三-O-乙酰基-β-D- 吡喃葡萄糖苷酸(1S)-1-(氯甲基)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(氯甲基)-5-[(2,3,4-三-O-乙酰基-6-甲基-β-D-吡喃葡萄糖酸苷基)氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)-2-[(3-{[(9H-芴-9-基甲氧基)羰基]氨基}丙酰基)氨基]苯基}-2-丙烯酰基)-1,2-二氢-3H-苯并[e]吲哚-5-基甲基酯(5)(127 mg,27%),为黄色固体。1H NMR(300MHz,[(CD3)2SO])δ10.04(s,1H, NH),8.38(br s,2H),8.11(d,J=8.1Hz,1H),7.97(d,J=8.5Hz, 2H),7.93(d,J=8.2Hz,2H),7.83-7.88(m,4H),7.78(d,J=7.6Hz, 1H),7.68-7.72(m,3H),7.59(t,J=7.5Hz,2H),7.45-7.48(m,3H), 7.38(t,J=7.4Hz,2H),7.25-7.29(m,4H),5.85(d,J=7.7Hz,1H), 5.83(d,J=7.8Hz,1H),5.62-5.65(m,2H),5.32(t,J=8.7Hz,2H), 5.14(t,J=9.6Hz,2H),4.78(t,J=8.2Hz,2H),4.55(m,4H),4.31-4.38 (m,4H),4.22(t,J=6.9Hz,1H),4.01-4.03(m,2H),3.91-3.96(m, 2H),3.65(s,3H),3.63(s,3H),3.36-3.38(m,2H),2.59-2.64(m, 2H),1.98-2.03(m,18H);LC-MS(ESI)为C82H79Cl2N4O25(M+H)+计算 m/z1591.4,发现m/z 1591.4;为C82H78Cl2N4O25Na(M+Na)+计算m/z 1613.4,发现m/z 1613.4。
于室温将衍生物(5)(110mg,0.07mmol)和哌啶(468μL,0.7mmol) 在无水DMF(5mL)中的溶液搅动30分钟。于室温在减压下去除溶剂,并与冷的Et2O一起研磨残留物,给出粗制的2,3,4-三-O-乙酰基-β-D-吡喃葡萄糖苷酸(1S)-3-{(2E)-3-[2-[(3-氨基丙酰基)氨基]-4-((1E)-3-{(1S)-1-(氯甲基)-5-[(2,3,4-三-O-乙酰基-6-甲基-β-D-吡喃葡萄糖酸苷基)氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)苯基]-2-丙烯酰基}-1-(氯甲基)-1,2-二氢-3H- 苯并[e]吲哚-5-基甲基酯(6)(86mg,90%),无需进一步纯化在下一步中使用。1H NMR(300MHz,[(CD3)2SO]δ8.38(br s,2H),8.10(d,J=8.4Hz, 1H),7.98(d,J=8.4Hz,2H),7.94(d,J=8.4Hz,2H),7.90(s,1H), 7.88(d,J=15.1Hz,1H),7.76(d,J=7.6Hz,1H),7.70(d,J=15.1Hz, 1H),7.59(t,J=7.9Hz,2H),7.47(t,J=7.9Hz,2H),7.27(dd,J= 3.4,15.3Hz,2H),5.85(d,J=7.8Hz,1H),5.84(d,J=7.8Hz,1H), 5.63-5.68(m,2H),5.32(dd,J=7.8,9.6Hz,2H),5.14(dt,J=1.5, 9.6Hz,2H),4.79(d,J=9.6Hz,2H),4.56-4.60(m,4H),4.29-4.41 (m,2H),4.02-4.04(m,2H),3.92-3.97(m,2H),3.67(s,6H),2.93 (t,J=6.4Hz,2H),2.03-2.04(m,18H),2H在DMSO峰下,NH和NH2未观察到。
于0℃对胺(6)(110mg,0.08mmol)在MeOH/CH2Cl2的(1:1)混合物(10mL)中的搅动溶液逐滴添加NaOMe(8.7mg,0.16mmol)在MeOH (1mL)中的溶液,并于0℃将反应混合物搅动2小时。然后添加AcOH(8 滴),于室温在减压下去除溶剂,并在高真空下干燥,给出β-D-吡喃葡萄糖苷酸(1S)-3-{(2E)-3-[2-[(3-氨基丙酰基)氨基]-4-((1E)-3-{(1S)-1-(氯甲基)-5-[(6- 甲基-β-D-吡喃葡萄糖酸苷基)氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)苯基]-2-丙烯酰基}-1-(氯甲基)-1,2-二氢-3H-苯并[e]吲哚-5-基甲基酯(7),为橙色固体,无需进一步纯化在下一步中使用。对胺(7)和6-马来酰亚胺基己酸N-琥珀酰亚胺酯(24mg,0.077mmol)在无水DMF(5mL)中的搅动溶液添加DIEA(78μL,0.1mmol),并于室温将混合物搅动过夜。然后于室温在减压下去除溶剂,并通过柱层析(SiO2,EtOAc/MeOH 10-20%),然后是两次(SiO2,EtOAc/MeOH 15%)纯化残留物,给出β-D-吡喃葡萄糖苷酸(1S)-1-(氯甲基)-3-((2E)-3-{4-((1E)-3-{(1S)-1-(氯甲基)-5-[(6-甲基-β-D-吡喃葡萄糖酸苷基)氧]-1,2-二氢-3H-苯并[e]吲哚-3-基}-3-氧-1-丙烯基)-2-[(3-{[6-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)己酰基]氨基}丙酰基)氨基]苯基}-2-丙烯酰基)-1,2-二氢-3H-苯并[e]吲哚-5-基甲基酯8(化合物编号84)(31 mg,34%),为黄色固体。HPLC纯度95.9%;1HNMR(300MHz,[(CD3)2SO] δ10.01(s,1H),8.32(s,2H),8.31(d,J=8.6Hz,2H),8.09(d,J=8.2Hz,1H),7.89-7.95(m,3H),7.84(t,J=7.6Hz,2H),7.77(d,J =8.0Hz,1H),7.70(d,J=15.1Hz,1H),7.58(t,J=7.9Hz,2H), 7.43(t,J=8.0Hz,2H),7.27(d,J=15.6Hz,2H),6.96(s,2H),5.67 (d,J=5.3Hz,1H),5.66(d,J=5.3Hz,1H),5.45(d,J=5.6Hz,2 H),5.35(d,J=4.6Hz,2H),5.16(d,J=7.5Hz,2H),4.52-4.62(m, 4H),4.33(br s,2H),3.99-4.04(m,4H),3.93(dd,J=7.4,10.5Hz, 2H),3.68(s,3H),3.67(s,3H),3.37-3.48(m,8H),2.57-2.61(m, 2H),2.09(t,J=7.3Hz,2H),1.41-1.54(m,4H),1.14-1.23(m,4H); HRMS(ESI)为C65H67Cl2N5NaO20(M+Na+)计算m/z 1330.3649;发现m/z 1330.3600。
实施例30:(S)-(1-甲基-1H-吡咯-2,5-二基)二(((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)甲酮)(化合物编号15,表1,图45)
将1(500mg,3.22mmol)和N,N-二甲基甲酰胺二叔丁基乙缩醛(2, 5.24g,25.77mmol)在NMP(10mL)中的混合物加热至100℃,并搅动过夜。在减压下去除大部分挥发性成分,并在乙酸乙酯和水之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过垫硅胶过滤。去除溶剂,获得3,为灰白晶体固体(331mg,38%);1H NMR(DMSO)δ12.26(s,1H),6.68(d,J=2.0 Hz,2H),1.51(s,18H)ppm。HRMS(ESI)发现m/z 290.1362(M+Na)。 C14H21NNaO4要求290.1363。
于35-40℃加热3(50mg,0.18mmol),K2CO3(52mg,0.37mmol), MeI(0.12mL,1.87mmol)和四丁基碘化铵(3.5mg,0.0094mmol)在MeCN (1mL)和水(0.01mL)中的混合物,并搅动3天。在乙酸乙酯和水之间重分配反应混合物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过垫硅胶过滤。去除溶剂,获得4,为灰白油,在几小时里变成无水晶体固体(45mg,86%);1H NMR(CDCl3) δ6.77(s,2H),4.21(s,3H),1.56(s,18H)ppm。HRMS(ESI)发现 m/z 304.1512(M+Na)。C15H23NNaO4要求304.1519。
于室温对4(45mg,0.16mmol)在DCM(1mL)中的溶液添加TFA(0.5 mL,6.49mmol)。将混合物搅动3小时,给出粉色溶液。抽走所有挥发性成分,并与石油醚一起研磨所得残留物,给出5,为粉色固体(25mg,93%)。1H NMR(DMSO)δ12.81(s,2H),6.80(s,2H),4.15(s,3H)ppm。 HRMS(ESI负)发现m/z 168.0306(M-H)。C7H6NO4要求168.0302。
于室温对Boc-CBI-OH(化合物51a,130mg,0.39mmol)在DCM(2mL) 中的溶液添加二烷(2mL)中的4N HCl。将混合物搅动2.5小时。抽走所有挥发性成分,就这样直接使用所得残留物(6)。
于室温将6(上文制备),5(22mg,0.13mmol),EDCI氢氯化物(150mg, 0.78mmol)和甲苯磺酸(2.2mg,0.013mmol)在DMA(2mL)中的混合物搅动过夜。抽走大部分溶剂,并在乙酸乙酯和水之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,并通过硅胶柱层析使用MeOH和DCM的混合物(v/v 2%)作为洗脱液纯化所得残留物,给出7(化合物编号15,表1),为灰白色固体(60mg,77%)。1H NMR(DMSO)δ10.43 (s,2H),8.12(d,J=8.3Hz,2H),7.83(d,J=8.3Hz,2H),7.73(br s,2H),7.52(dd,J=1.0,8.0Hz,2H),7.37(dd,J=0.4,8.0Hz,2H),6.77(s,2H),4.62-4.57(m,2H),4.31-4.27(m,2H),4.10-4.07(m, 2H),4.02-4.00(m,2H),3.88-3.85(m,5H)ppm。HRMS(ESI)发现m/z 622.1255(M+Na)。C33H27Cl2N3NaO4要求622.1271。
实施例31:N-(2,5-二((E)-3-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚 -3(2H)-基)-3-氧丙-1-烯基)苯基)乙酰胺(化合物编号16,表1,图46)
在冰浴中对1(66f,500mg,1.45mmol)在THF(5mL)和吡啶(5mL) 中的溶液添加乙酰氯(0.50mL,7.03mmol)。容许混合物升温至室温,并搅动过夜。抽走所有挥发性成分,并在乙酸乙酯和碳酸氢钠水溶液之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,并通过硅胶柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:2)作为洗脱液纯化所得残留物,给出2,为灰白色固体(475mg,85%);1H NMR(CDCl3)δ8.00(s, 1H),7.70(d,J=15.8Hz,1H),7.55-7.51(m,2H),7.31(表观d,J=8.6 Hz,2H),6.40(d,J=16.0Hz,1H),6.37(d,J=15.8Hz,1H),2.25(s, 3H),1.54(s,9H),1.53(s,9H)ppm。HRMS(ESI)发现m/z 410.1921 (M+Na)。C22H29NNaO5要求410.1938。
于室温对2(470mg,1.21mmol)在DCM(5mL)中的溶液添加TFA(2.5 mL,32.40mmol)。将混合物搅动3小时,给出白色悬浮液。添加更多的DCM 以沉淀出更多的固体,通过过滤来收集,并用乙酸乙酯和石油醚清洗。获得 3,为白色固体(290mg,87%)。1H NMR(DMSO)δ12.40(br s,2H), 9.89(s,1H),7.85(d,J=8.3Hz,1H),7.70(表观d,J=16.0Hz,2H),7.57-7.53(m,2H),6.54(d,J=15.9Hz,1H),6.53(d,J=16.0Hz,1H), 2.09(s,3H)ppm。HRMS(ESI)发现m/z 298.0672(M+Na)。C14H13NNaO5要求298.0686。
于室温对Boc-CBI-OH(化合物51a,291mg,0.87mmol)在DCM(3mL) 中的溶液添加二烷(3mL)中的4N HCl。将混合物搅动2.5小时。抽走所有挥发性成分,就这样直接使用所得残留物(4)。
于室温将4(上文制备),3(80mg,0.29mmol),EDCI氢氯化物(334mg, 1.74mmol)和甲苯磺酸(5mg,0.029mmol)在DMA(3mL)中的混合物搅动过夜。抽走所有挥发性成分,并数次与甲醇一起研磨所得残留物,给出粗制的产物(123mg),在THF中溶解,并通过添加MeOH来沉淀,给出5(化合物编号16,表1),为黄色固体(96mg,47%,HPLC纯度97%);1H NMR (DMSO)δ10.43(s,2H),9.97(s,1H),8.12-8.07(m,5H),7.85-7.81 (m,4H),7.74(表观d,J=6.7Hz,1H),7.68(d,J=15.4Hz,1H),7.73 (t,J=7.4Hz,2H),7.35(t,J=7.5Hz,2H),7.26(dd,J=3.2,15.3Hz, 2H),4.58-4.46(m,4H),4.28-4.22(m,2H),4.05-3.98(m,2H),3.88-3.82 (m,2H),2.15(s,3H)ppm。HRMS(ESI)发现m/z 728.1662(M+Na)。 C40H33Cl2N3NaO5要求728.1689。
实施例32:(S,2E,2′E)-3,3'-(2-甲氧基-1,4-亚苯基)二(1-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)丙-2-烯-1-酮)(化合物编号17,表1,图47)
在N2下在干冰-MeCN浴中于-30至-40℃对1(2.50g,13.30mmol)在干的THF(12mL)中的溶液逐滴添加三氟化硼二乙醚合物(BF3·Et2O,4.92mL, 39.90mmol)。于-30℃将混合物搅动10分钟后,逐滴添加tBuONO(2.39mL, 19.94mmol)。容许反应混合物升温至室温,并搅动1.5小时,给出悬浮液。添加石油醚(50mL)以给出更多的沉淀物。通过倾倒去除上清液,并用石油醚清洗留下的固体以获得白色固体。在干的MeCN(20mL)中溶解此固体,并在冰浴中冷却。添加KI(11.00g,66.26mmol)和I2(6.00g,23.64mmol)。于室温将反应混合物搅动4小时,之后添加饱和Na2S2O3溶液(50mL)以淬灭反应。用乙酸乙酯提取混合物三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,并通过硅胶柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:9)作为洗脱液纯化所得残留物,给出2,为灰白色固体(2.83g,71%);1H NMR(CDCl3)δ7.50(d,J =8.5Hz,1H),7.16(d,J=2.2Hz,1H),6.84(dd,J=2.2,8.5Hz,1H), 5.39(s,1H)ppm。
在N2下将2(500mg,1.67mmol),丙烯酸叔丁酯(0.728mL,5.02mmol),乙酸钯(II)(7.5mg,0.033mmol)和三-邻-甲苯基膦(41mg,0.13mmol) 在重蒸馏三乙胺(5mL)中的混合物回流加热过夜,给出深灰色悬浮液。抽走所有挥发性成分。在乙酸乙酯中溶解所得残留物,并过滤掉沉淀物。蒸发滤出液,并通过柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:6)作为洗脱液纯化获得的残留物,给出3(160mg,28%),为灰白色固体。1H NMR (DMSO)δ10.43(s,1H),7.75(d,J=16.4Hz,1H),7.63(d,J=8.4Hz, 1H),7.45(d,J=16.0Hz,1H),7.18(d,J=8.0Hz,1H),7.07(d,J= 0.8Hz,1H),6.56(d,J=16.4Hz,1H),6.41(d,J=15.6Hz,1H),1.483 (s,9H),1.478(s,9H)ppm。HRMS(ESI)发现m/z 369.1687(M+Na)。C20H26NaO5要求369.1672。
对3(160mg,0.46mmol)在DMF(2mL)中的溶液添加K2CO3(254mg, 1.84mmol)和MeI(0.28mL,4.50mmol)。于室温将混合物搅动过夜,并过滤掉沉淀物。用水,接着是盐水清洗所得滤出液,在无水Na2SO4上干燥,并穿过硅藻土垫过滤。去除溶剂,并通过硅胶柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:10)作为洗脱液纯化所得残留物,给出4,为无色油(72mg,43%);1H NMR(CDCl3)δ7.87(d,J=16.2Hz,1H),7.54(d,J=15.9Hz, 1H),7.49(d,J=8.0Hz,1H),7.10(dd,J=1.3,8.0Hz,1H),7.00(d, J=1.2Hz,1H),6.47(d,J=16.1Hz,1H),6.38(d,J=15.9Hz,1H), 3.91(s,3H),1.539(s,9H),1.533(s,9H)ppm。HRMS(ESI)发现 m/z383.1838(M+Na)。C21H28NaO5要求383.1829。
于室温对4(70mg,0.19mmol)在DCM(2mL)中的溶液添加TFA(1 mL,12.98mmol)。将混合物搅动2.5小时,给出白色悬浮液。抽走所有挥发性成分,并与DCM和乙酸乙酯一起研磨所得残留物,给出5,为白色固体(41 mg,85%)。1H NMR(DMSO)δ12.41(br s,2H),7.80(d,J=16.2Hz, 1H),7.72(d,J=8.0Hz,1H),7.58(d,J=16.0Hz,1H),7.41(d,J= 1.2Hz,1H),7.30(dd,J=1.0,8.1Hz,1H),6.47(d,J=16.0Hz,1H), 6.38(d,J=16.1Hz,1H),3.92(s,3H)ppm。HRMS(ESI)发现m/z 271.0573 (M+Na)。C13H12NaO5要求271.0577。
于室温对Boc-CBI-OH(化合物51a,161mg,0.48mmol)在DCM(2mL) 中的溶液添加二烷(2mL)中的4N HCl。将混合物搅动2.5小时。抽走所有挥发性成分,就这样直接使用所得残留物(6)。
于室温将6(上文制备),5(40mg,0.16mmol),EDCI氢氯化物(185mg, 0.97mmol)和甲苯磺酸(2.8mg,0.016mmol)在DMA(1mL)中的混合物搅动过夜。抽走所有挥发性成分,并与甲醇一起研磨所得残留物,给出黄色固体,在THF中溶解,并通过添加甲醇来沉淀,提供7(化合物编号17,表1),为黄色固体(45mg,41%,HPLC纯度98%);1H NMR(DMSO)δ10.43 (s,2H),8.12-8.10(m,4H),8.00-7.95(m,2H),7.85-7.80(m,2H), 7.72(d,J=15.4Hz,1H),7.54-7.50(m,4H),7.37-7.26(m,4H),4.57-4.45 (m,4H),4.28-4.22(m,2H),4.00-3.99(m,5H),3.90-3.83(m,2H) ppm。HRMS(ESI)发现m/z 701.1596(M+Na)。C39H32Cl2N2NaO5要求701.1580。
实施例33:(S,2E,2'E)-3,3'-(1-甲基-1H-吡咯-2,5-二基)二(1-((S)-1-(氯甲基)-5-羟基-1H-苯并[e]吲哚-3(2H)-基)丙-2-烯-1-酮)(化合物编号18,表1,图 48)
1使用文献方法制备(参考文献Russ J Org Chem,2007,43,855-860)。
2
于40℃加热1(50mg,0.41mmol),K2CO3(112mg,0.81mmol),MeI (0.25mL,4.06mmol)和四丁基碘化铵(7.5mg,0.020mmol)在MeCN(2 mL)和水(0.02mL)中的混合物,并搅动3小时。在减压下去除大部分挥发性成分,并在乙酸乙酯和水之间重分配所得残留物。用乙酸乙酯提取水相三次。用水,接着是盐水清洗合并的有机提取液,在无水Na2SO4上干燥,并穿过垫硅胶过滤。去除溶剂,获得2,为白色固体(47mg,84%);1H NMR谱与报告的相同(参考文献C.E.Loader,G.H.Barnett and H.J.Anderson,Can. J.Chem.,1982,60,383)
4
将2(40mg,0.29mmol)和(三苯基亚膦基)乙酸甲酯(3,400mg,1.20 mmol)在DCM(3mL)中的混合物搅动2天,给出黄色溶液。通过硅胶柱层析使用乙酸乙酯和石油醚的梯度混合物(v/v=1:5,1:4和1:3)作为洗脱液纯化混合物,给出4,为黄色固体(63mg,87%);1HNMR(CDCl3)δ7.61 (d,J=15.6Hz,2H),6.70(s,2H),6.26(d,J=15.6Hz,2H),3.79 (s,6H),3.73(s,3H)ppm。HRMS(ESI)发现m/z 272.0898(M+Na)。 C13H15NNaO4要求272.0893。
5
于70℃将4(60mg,0.24mmol)和KOH(100mg,1.78mmol)在EtOH (2mL)和THF(1mL)中的混合物搅动2小时。在减压下蒸发混合物至干燥。在水中溶解所得残留物,并用1N HCl酸化直至pH 5,给出黄色沉淀物,通过过滤来收集,然后用水和石油醚清洗,给出5,为黄色固体(51mg,96%);1H NMR(DMSO)δ12.20(s,2H),7.54(d,J=15.6Hz,2H),6.89(s, 2H),6.30(d,J=15.6Hz,2H),3.70(s,3H)ppm。HRMS(ESI)发现 m/z 244.0590(M+Na)。C11H11NNaO4要求244.0580。
7
于室温对Boc-CBI-OH(化合物51a,200mg,0.60mmol)在DCM(2mL) 中的溶液添加二烷(10mL)中的4N HCl。将混合物搅动2小时。抽走所有挥发性成分,就这样直接使用所得残留物(6,化合物67d)。
于室温将6(上文制备),5(49mg,0.22mmol),EDCI氢氯化物(255mg, 1.33mmol)和甲苯磺酸(3.8mg,0.022mmol)在DMA(2mL)中的混合物搅动过夜。去除溶剂,并通过硅胶柱层析使用MeOH和乙酸乙酯的混合物 (v/v 3%)纯化所得残留物,与乙酸乙酯一起研磨粗制的产物,给出7,为橙色固体(60mg,41%,HPLC 100%);1H NMR(DMSO)δ10.41(s,2H), 8.11(d,J=8.2Hz,4H),7.81(d,J=8.3Hz,2H),7.70(d,J=14.9Hz, 2H),7.50(t,J=7.4Hz,2H),7.33(t,J=7.5Hz,2H),7.12(s,2H), 7.01(d,J=14.9Hz,2H),4.49-4.42(m,4H),4.24-4.19(m,2H),4.01-3.97 (m,2H),3.85-3.82(m,5H)ppm。HRMS(ESI负)发现m/z 650.1600(M-H)。C37H30Cl2N3O4要求650.1619。
实施例34:(S)-3,3′-(2-甲氧基-1,4-亚苯基)二(1-((S)-1-(氯甲基)-5-羟基 -1H-苯并[e]吲哚-3(2H)-基)丙-2-炔-1-酮)(化合物19,表1,图49)
2
基于US2007/49758所述通过改良规程合成化合物2。对1(5.50g,25.00 mmol)和乙酸银(I)(6.26g,37.5mmol)在DCM(100mL)中的悬浮液逐滴添加碘(6.98g,27.50mmol)在DCM(150mL)中的溶液。于室温将所得混合物搅动过夜,然后穿过硅藻土垫过滤。蒸发滤出液,并通过硅胶柱层析使用乙酸乙酯和石油醚的混合物(v/v 1:10)作为洗脱液纯化所得残留物,给出2,为白色固体(966mg,11%);mp 97-99℃。1H NMR(CDCl3) δ7.342(d,J=8.3Hz,1H),7.339(d,J=2.0Hz,1H),7.00(dd,J=2.0,8.3Hz,1H),5.32(s,1H)ppm。13C NMR(CDCl3,100.6MHz)δ155.70, 139.46(CH),131.80(CH),124.44(CH),94.65,85.52ppm。
3
对2(270mg,0.78mmol)在DMF(4mL)中的溶液添加K2CO3(162mg, 1.17mmol)和MeI(0.24mL,3.90mmol)。于室温将混合物搅动2小时。穿过硅胶垫过滤混合物,并蒸发滤出液,给出3,为无色油(277mg,99%);1H NMR(CDCl3)δ7.45(d,J=8.1Hz,1H),7.09(d,J=1.8Hz,1H), 7.04(dd,J=1.8,8.1Hz,1H),3.87(s,3H)ppm。
4
在N2下于60℃将3(274mg,0.76mmol),丙炔酸叔丁酯(0.314mL, 2.28mmol),碘化酮(I)(5.8mg,0.030mmol),乙酸钯(II)(3.4mg,0.015 mmol)和三苯基膦(12mg,0.046mmol)在重蒸馏三乙胺(5mL)中的混合物加热过夜,给出深色悬浮液。抽走所有挥发性成分。将所得残留物与DCM 一起搅动,并过滤掉沉淀物。蒸发滤出液,并通过硅胶柱层析使用DCM和石油醚的混合物(v/v=1:1)作为洗脱液纯化获得的残留物,给出4(195mg, 72%),为灰白色固体。1H NMR(CDCl3)δ7.48(d,J=7.9Hz,1H),7.13 (d,J=7.9Hz,1H),7.06(s,1H),3.89(s,3H),1.545(s,9H)和1.514 (s,9H)ppm。HRMS(ESI)发现m/z 379.1510(M+Na)。C21H24NaO5要求379.1516。
5
于室温对4(100mg,0.28mmol)在DCM(2mL)中的溶液添加TFA(1 mL,12.98mmol)。将混合物搅动2.5小时,给出白色悬浮液。抽走所有挥发性成分,并与DCM和石油醚的混合物(v/v=1:1)一起研磨所得残留物,给出5,为白色固体(66mg,96%)。1H NMR(DMSO)δ13.92(br s,2H), 7.61(d,J=7.9Hz,1H),7.37(表观s,1H),7.25(dd,J=1.1,7.9Hz, 1H),3.90(s,3H)ppm。
7
于室温对Boc-CBI-OH(化合物51a,267mg,0.80mmol)在DCM(3mL) 中的溶液添加二烷(3mL)中的4N HCl。将混合物搅动2.5小时。抽走所有挥发性成分,就这样直接使用所得残留物(6)。
于室温将6(上文制备),5(65mg,0.27mmol),EDCI氢氯化物(306mg, 1.60mmol)和甲苯磺酸(4.6mg,0.027mmol)在DMA(1mL)中的混合物搅动过夜。抽走所有挥发性成分,并与甲醇一起研磨所得残留物,给出黄色固体,在THF中溶解,并通过添加甲醇来沉淀,提供7,为黄色固体(140 mg,78%,HPLC纯度99%);1H NMR(DMSO)δ10.52(s,2H),8.12(d, J=8.4Hz,2H),7.90-7.85(m,4H),7.76(d,J=7.9Hz,1H),7.56-7.50 (m,3H),7.41-7.37(m,3H),4.63-4.49(m,4H),4.29-4.21(m,2H), 4.09-4.06(m,2H),4.01(s,3H),3.99-3.94(m,1H),3.91-3.86(m,1H) ppm。HRMS(ESI)发现m/z 697.1267(M+Na)。C39H28Cl2N2NaO5要求697.1267。
实施例19:用于通过还原和再氧化来偶联的半胱氨酸改造的抗体的制备
在某些条件下,通过用还原剂(诸如DTT(Cleland氏试剂,二硫苏糖醇) 或TCEP(盐酸三(2-羧乙基)膦;Getz et al(1999)Anal.Biochem.Vol 273:73-80; Soltec Ventures,Beverly,MA)处理,可以使得半胱氨酸改造的抗体对于与本发明的接头-药物中间体(诸如表4中的那些)偶联成为反应性的。例如,将在CHO细胞中表达的全长半胱氨酸改造的单克隆抗体(ThioMab)(Gomez et al(2010)Biotechnology and Bioeng.105(4):748-760;Gomezet al(2010) Biotechnol.Prog.26:1438-1445)于室温用约50倍过量的DTT还原过夜以还原可以在新引入的半胱氨酸残基与培养基中存在的半胱氨酸之间形成的二硫键。
轻链氨基酸依照Kabat(Kabat et al.,Sequences of proteins ofimmunological interest,(1991)5th Ed.,US Dept of Health and Human Service,National Institutes of Health,Bethesda,MD)编号。除了像Kabat系统注释的地方,重链氨基酸依照EU编号系统(Edelman et al(1969)Proc.Natl.Acad.of Sci. 63(1):78-85)编号。使用单字母氨基酸缩写。
由于细胞培养条件,在CHO细胞中表达的全长半胱氨酸改造的单克隆抗体(ThioMab)携带半胱氨酸加合物(胱氨酸)或在工程化改造的半胱氨酸上谷胱甘肽化。为了解放工程化改造的半胱氨酸的反应性硫醇基团,在约pH 8.0的500mM硼酸钠和500mM氯化钠中溶解ThioMab,并用约50-100倍过量的1mM TCEP(盐酸三(2-羧乙基)膦)(Getz et al(1999)Anal.Biochem.Vol 273:73-80;Soltec Ventures,Beverly,MA)于37℃还原约1-2小时。或者,可使用DTT作为还原剂。或是通过非还原性SDS-PAGE或是通过变性反相 HPLCPLRP柱层析来监测链间二硫键的形成。在pH 5的10mM乙酸钠中稀释还原的ThioMab,加载到HiTrap SP FF柱上,并用含有0.3M氯化钠的PBS或含有150mM氯化钠的50mM Tris-Cl pH7.5洗脱。
通过进行再氧化,在亲本单抗中存在的半胱氨酸残基之间重新建立二硫键。将洗脱的还原的ThioMab用pH 7的15X或2mM脱氢抗坏血酸(dhAA)处理3小时,或在50mM Tris-ClpH 7.5中处理3小时,或用2mM硫酸铜(CuSO4) 水溶液于室温处理过夜。可以使用本领域已知的其它氧化剂(即氧化性试剂) 和氧化性条件。环境空气氧化也可能是有效的。这种温和的部分再氧化步骤以高保真度有效形成链内二硫化物。通过在Sephadex G25树脂上洗脱来更换缓冲液,并用含1mM DTPA的PBS洗脱。如此检查硫醇/抗体值,即自溶液在 280nm处的吸光度确定还原的抗体的浓度及通过与DTNB(Aldrich, Milwaukee,WI)反应并测定412nm处的吸光度来确定硫醇浓度。
在具有扩大的质量范围的TSQ量子三联四极TM质谱仪(Thermo Electron, SanJose,California)上实施液体层析/质谱分析。将样品在加热至75℃的1000A,Microbore柱(50mm x 2.1mm,Polymer Laboratories, Shropshire,UK)上进行层析。使用30-40%B的线性梯度(溶剂A:含0.05% TFA的水,溶剂B:含0.04%TFA的乙腈),并使用电喷射源直接电离洗提液。通过数据系统收集数据,并使用(Novatia,LLC,New Jersey)实施解卷积。在LC/MS分析之前,将抗体或药物缀合物(50微克) 用PNG酶F(2U/ml;PROzyme,San Leandro,CA)于37℃处理2小时以去除N连接的碳水化合物。
将疏水相互作用层析(HIC)样品注射到丁基HIC NPR柱(2.5微米粒度, 4.6mm x3.5cm)(Tosoh Bioscience)上,并以0.8ml/min用0-70%B的线性梯度洗脱(A:含1.5M硫酸铵的50mM磷酸钾pH 7,B:50mM磷酸钾pH 7, 20%异丙醇)。使用配备有多波长检测仪和Chemstation软件的Agilent 1100系列HPLC系统来解析和定量具有不同药物对抗体比的抗体种类。
实施例20:接头-药物中间体对抗体的缀合
在实施例19的还原和再氧化规程之后,在PBS(磷酸盐缓冲盐水)中溶解抗体,并在冰上冷却。在DMSO中溶解过量(约1.5摩尔至20个当量)的具有硫醇反应性官能团(诸如马来酰亚胺基或溴乙酰胺)的接头-药物中间体 (包括但不限于表4中的51-68),在乙腈和水中稀释,并添加至PBS中的冷却的还原,再氧化的抗体。约1小时后,添加过量的马来酰亚胺以淬灭反应,并给任何未反应的抗体硫醇基团加帽。可以在HiTrap SP FF柱上加载并洗脱缀合混合物以去除过量的药物-接头中间体和其它杂质。通过离心超滤来浓缩反应混合物,并将半胱氨酸改造的抗体药物缀合物通过在PBS中穿过G25 树脂洗脱来纯化和脱盐,在无菌条件下穿过0.2μm滤器过滤,并冷冻,供贮存。
通过上文规程,制备了表3的半胱氨酸改造的抗体药物缀合物101-133。
实施例21:体外细胞增殖测定法
通过采用下述方案的细胞增殖测定法来测量ADC的功效(CELLTITER GLOTM发光细胞存活力测定法,Promega Corp.Technical Bulletin TB288; Mendoza et al(2002)Cancer Res.62:5485-5488):
1.在壁不透明的96孔板的每个孔中分配在培养基中含有约104个细胞 (SKBR-3,BT474,MCF7或MDA-MB-468)的100μl细胞培养物等分试样。
2.制备含有培养基且没有细胞的对照孔。
3.将ADC添加至实验孔并温育3-5天。
4.将板平衡至室温达大约30分钟。
5.添加与每个孔中存在的细胞培养液的体积相等体积的CELLTITER GLOTM试剂。
6.在轨道摇床上将内容物混合2分钟以诱导细胞裂解。
7.将板于室温温育10分钟以稳定发光信号。
8.以RLU=相对发光单位记录发光并以图报告。
作为每套复制品的发光均值及标准偏差误差棒将数据绘图。方案是 CELLTITERGLOTM发光细胞的一种改良
培养基:SK-BR-3在50/50/10%FBS/谷氨酰胺/250μg/mL G-418中培养, OVCAR-3在RPMI/20%FBS/谷氨酰胺中培养
实施例22:高表达HER2的转基因外植体小鼠和其它肿瘤模型中的体内功效,肿瘤生长抑制
建立肿瘤,并容许生长至体积为150-200mm3(使用测径器测量),之后进行第0天的单次处理。依照公式使用测径器测量肿瘤体积:V(mm3)=0.5 A X B2,其中A和B分别是长和短直径。在肿瘤体积达到3000mm3之前或当肿瘤显示溃疡迫近的迹象时对小鼠处以安乐死。自每个实验组(10只小鼠每组)收集的数据表述成均值±SE。
如先前所述(Phillips GDL,Li GM,Dugger DL,et al.Targeting HER2-PositiveBreast Cancer with Trastuzumab-DM1,an Antibody-Cytotoxic Drug Conjugate.(2008)Cancer Res.68:9280-90,通过援引收入本文),采用Fo5 小鼠乳房肿瘤模型来评估本发明的抗体-药物缀合物在单剂静脉内注射后的体内功效。用Fo5模型测试抗Her2ADC,Fo5模型是一种转基因小鼠模型,其中在乳房上皮中在鼠乳房肿瘤病毒启动子的转录调节下过表达人HER2基因(MMTV-HER2),如图31和32中所示。HER2过表达引起乳房肿瘤的自发发生。通过连续移植肿瘤碎片(约2x 2mm尺寸),在FVB小鼠的后续世代中扩充这些建立者动物之一(建立者#5[Fo5])的乳房肿瘤。所有研究依照实验动物护理和使用指南进行。在研究开始时及在移植后14天给九只动物静脉内服用每种抗体-药物缀合物(单剂)。初始肿瘤尺寸为约200mm3体积。本发明抗体-药物缀合物和对照随时间的肿瘤生长抑制的测量结果显示于图 31-34。
如记载的那样(Chen et al.(2007)Cancer Res 67:4924-4932)采用 OVCAR-3乳房脂肪垫移植物功效模型,在静脉内单剂之后评估肿瘤体积,并使用自携带腹膜内肿瘤的小鼠切除的肿瘤,然后在接受者小鼠的乳房脂肪垫中连续传代(图33)。
在Igrov-1小鼠异种移植物模型(人卵巢癌)中调查抗Napi2B抗体-药物缀合物(ADC)的功效。
给每只雌性C.B-17SCID-米色小鼠(Charles River Laboratories;San Diego,CA)在胸部乳房脂肪垫区域中接种5百万个Igrov-1细胞。当异种移植物肿瘤达到100-300mm3的平均肿瘤体积时(称作第0天),将动物随机化入各组中(每组7-10只小鼠),并接受ADC的单次静脉内注射。贯穿研究一周1-2次测量小鼠的肿瘤和体重。当体重损失超过它们起始重量的20%时,迅速对小鼠处以安乐死。在肿瘤达到3000mm3或显示溃疡迫近的迹象之前对所有动物除以安乐死。
在HL-60或EOL-1小鼠异种移植物模型(人急性髓样白血病)中调查抗 CD33抗体-药物缀合物(ADC)的功效。HL-60细胞系得自ATCC(美国典型培养物保藏中心;Manassas,VA),而EOL-1细胞系源自DSMZ(德国微生物和细胞培养物保藏中心;Braunschweig,Germany)。
给每只雌性C.B-17SCID小鼠(Charles River Laboratories;Hollister,CA) 在体侧区域中皮下接种5百万个HL-60或EOL-1细胞。当异种移植物肿瘤达到 100-300mm3的平均肿瘤体积时(称作第0天),将动物随机化入各组中(每组7-10只小鼠),并接受ADC的单次静脉内注射。在施用ADC之前大约4小时,给动物腹膜内服用过量(30mg/kg)的抗gD对照抗体以封闭肿瘤细胞上可能的非特异性抗体结合位点。贯穿研究一周1-2次测量小鼠的肿瘤和体重。当体重损失超过它们起始重量的20%时,迅速对小鼠处以安乐死。在肿瘤达到3000mm3或显示溃疡迫近的迹象之前对所有动物除以安乐死。
虽然出于清楚理解的目的已经通过举例说明较为详细地描述了前述发明,但是说明书和实施例不应解释为限制本发明的范围。通过援引明确收录贯穿说明书引用的所有专利,专利申请,和参考文献。
Claims (36)
2.权利要求1的抗体-药物缀合物化合物,其中该抗体为抗CD22抗体。
3.权利要求2的抗体-药物缀合物化合物,其中该抗CD22抗体包含三个轻链高变区(HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和HVR-H3),其中: HVR-L1包含SEQ ID NO:1的氨基酸序列; HVR-L2包含SEQ ID NO:2的氨基酸序列; HVR-L3包含SEQID NO:3的氨基酸序列; HVR-H1包含SEQ ID NO:4的氨基酸序列; HVR-H2包含SEQ ID NO:5的氨基酸序列;且 HVR-H3包含SEQ ID NO:6的氨基酸序列。
4.权利要求1的抗体-药物缀合物化合物,其中该抗体为抗MUC16抗体。
5.权利要求4的抗体-药物缀合物化合物,其中该抗MUC16抗体包含三个轻链高变区(HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和HVR-H3),其中: HVR-L1包含SEQ ID NO:7的氨基酸序列; HVR-L2包含SEQ ID NO:8的氨基酸序列; HVR-L3包含SEQID NO:9的氨基酸序列; HVR-H1包含SEQ ID NO:10的氨基酸序列; HVR-H2包含SEQ ID NO:11的氨基酸序列;且 HVR-H3包含SEQ ID NO:12的氨基酸序列。
6.权利要求4的抗体-药物缀合物化合物,其中该抗MUC16抗体为半胱氨酸改造抗体,其包含一个或多个位于选自SEQ ID NO:15-32的轻链序列或选自SEQ ID NO:33-46的重链序列中的游离半胱氨酸氨基酸残基。
7.权利要求1的抗体-药物缀合物化合物,其中该抗体为抗HER2抗体。
8.权利要求1的抗体-药物缀合物化合物,其中该抗HER2抗体为曲妥珠单抗(trastuzumab)。
9.权利要求1的抗体-药物缀合物化合物,其中该抗体为抗CD33抗体。
10.权利要求9的抗体-药物缀合物化合物,其中该抗CD33抗体包含三个轻链高变区(HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和HVR-H3),其中: HVR-L1包含SEQ ID NO:51的氨基酸序列; HVR-L2包含SEQ ID NO:52的氨基酸序列; HVR-L3包含SEQID NO:53的氨基酸序列; HVR-H1包含SEQ ID NO:54的氨基酸序列; HVR-H2包含SEQ IDNO:55的氨基酸序列;且 HVR-H3包含SEQ ID NO:56的氨基酸序列。
11.权利要求9的抗体-药物缀合物化合物,其中该抗CD33抗体包含SEQ ID NO:57的轻链可变区或SEQ ID NO:58的重链可变区。
12.权利要求9的抗体-药物缀合物化合物,其中该抗CD33抗体包含三个轻链高变区(HVR-L1,HVR-L2和HVR-L3)和三个重链高变区(HVR-H1,HVR-H2和HVR-H3),其中: HVR-L1包含SEQ ID NO:59的氨基酸序列; HVR-L2包含SEQ ID NO:60的氨基酸序列; HVR-L3包含SEQID NO:61的氨基酸序列; HVR-H1包含SEQ ID NO:62的氨基酸序列; HVR-H2包含SEQ IDNO:63的氨基酸序列;且 HVR-H3包含SEQ ID NO:64的氨基酸序列。
13.权利要求9的抗体-药物缀合物化合物,其中该抗CD33抗体包含选自SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,和SEQ ID NO:71的轻链可变区;或选自SEQ ID NO:66,SEQID NO:68,SEQ ID NO:70,和SEQ ID NO:72的重链可变区。
14.一种药物组合物,其包含权利要求1至13任一项的抗体-药物缀合物化合物,和药学可接受赋形剂。
15.权利要求14的药物组合物在制备用于治疗癌症的药物中的用途,其中所述治疗包括对患者施用治疗有效量的权利要求14的药物组合物。
16.一种用于治疗癌症的试剂盒,其包含: a)权利要求14的药物组合物;和 b)使用说明书。
18.一种用于生成权利要求1的抗体-药物缀合物化合物的方法,该方法包括将该抗体缀合至权利要求17的接头-药物中间体。
19.依照权利要求1的缀合物,其中p为1,2,3或4。
20.依照权利要求1的缀合物,其中p为1或2。
24.权利要求21-23任一项的缀合物,其中Ab为抗HER2抗体。
25.权利要求21-23任一项的缀合物,其中Ab为抗CD22抗体。
30.一种药物组合物,其包含权利要求27至29任一项的抗体-药物缀合物和药学可接受赋形剂。
31.权利要求27至29任一项的抗体-药物缀合物在制备用于治疗血液学病症的药物中的用途,其中所述治疗包括对患者施用治疗有效量的权利要求27至29任一项的抗体-药物缀合物。
32.权利要求27至29任一项的抗体-药物缀合物在制备用于治疗白血病的药物中的用途,其中所述治疗包括对患者施用治疗有效量的权利要求27至29任一项的抗体-药物缀合物。
33.权利要求27至29任一项的抗体-药物缀合物在制备用于治疗淋巴瘤的药物中的用途,其中所述治疗包括对患者施用治疗有效量的权利要求27至29任一项的抗体-药物缀合物。
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CN105636612A (zh) | 2016-06-01 |
JP2016530254A (ja) | 2016-09-29 |
US10442836B2 (en) | 2019-10-15 |
AU2014307080A1 (en) | 2016-02-11 |
CA2918139A1 (en) | 2015-02-19 |
HK1222566A1 (zh) | 2017-07-07 |
WO2015023355A1 (en) | 2015-02-19 |
JP2020040954A (ja) | 2020-03-19 |
TW201534327A (zh) | 2015-09-16 |
JP6993084B2 (ja) | 2022-02-03 |
KR20160042080A (ko) | 2016-04-18 |
CR20160125A (es) | 2016-05-04 |
TWI636792B (zh) | 2018-10-01 |
HK1222544A1 (zh) | 2017-07-07 |
EP3033111A1 (en) | 2016-06-22 |
CL2016000318A1 (es) | 2016-08-19 |
SG11201601005XA (en) | 2016-03-30 |
US20150165063A1 (en) | 2015-06-18 |
EA201690195A1 (ru) | 2016-05-31 |
MX2016001862A (es) | 2016-08-03 |
BR112016002829A2 (pt) | 2017-09-19 |
EP3033111B1 (en) | 2019-03-13 |
PH12016500246A1 (en) | 2016-05-16 |
AU2014307080B2 (en) | 2018-06-07 |
IL243586A0 (en) | 2016-02-29 |
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