CN104271557A - 去泛素化活性的抑制方法 - Google Patents
去泛素化活性的抑制方法 Download PDFInfo
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- CN104271557A CN104271557A CN201280062713.XA CN201280062713A CN104271557A CN 104271557 A CN104271557 A CN 104271557A CN 201280062713 A CN201280062713 A CN 201280062713A CN 104271557 A CN104271557 A CN 104271557A
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- DBRXOUCRJQVYJQ-CKNDUULBSA-N withaferin A Chemical compound C([C@@H]1[C@H]([C@@H]2[C@]3(CC[C@@H]4[C@@]5(C)C(=O)C=C[C@H](O)[C@@]65O[C@@H]6C[C@H]4[C@@H]3CC2)C)C)C(C)=C(CO)C(=O)O1 DBRXOUCRJQVYJQ-CKNDUULBSA-N 0.000 description 1
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Abstract
一种通式(I)的化合物,所述化合物能够消除19S RP DUB的去泛素化(DUB)活性。所述化合物可用于治疗癌症,特别是通过本领域化疗法难以治疗的癌症。还公开了相应的治疗方法和包含所述化合物的药物组合物。
Description
发明领域
本发明涉及通过抑制去泛素化活性来治疗患者癌症的方法。更具体地,本发明涉及治疗经证实用至少一种抗癌药物难以治疗的患者的癌症的方法。最具体地,本发明涉及用于所述方法的化合物和包含所述化合物的药物组合物。
发明背景
肿瘤细胞显示对于泛素-蛋白酶体系统(UPS)的破坏具有较强敏感性,这为抗癌治疗的发展提供了吸引人的靶标(1)。
经泛素标记的底物被26S蛋白酶体降解,所述26S蛋白酶体是一种多亚基复合物,包含蛋白水解性20S核心(20S CP),冠有19S调节粒(19S RP)(2,3)帽。该20S CP已发展成抗癌药物发展中的重要靶标,导致硼替佐米()被公认用于治疗骨髓性白血病(4)。
已知化合物b-AP15(NSC687852)诱导p53非依赖性和组织蛋白酶-D-依赖性的凋亡(5,6)。
发明目的
本发明的一个目的在于提供用于通过抑制去泛素化活性来治疗患者癌症的方法,所述癌症具体是用本领域化疗法难以治疗的癌症。
具体而言,本发明的一个目的在于提供一种化合物,所述化合物用于治疗用至少硼替佐米或与硼替佐米共用抑制去泛素化活性机制的药剂难以治疗的患者的癌症。
本发明的另一个目标在于提供前述种类的化合物,所述化合物相对于本领域已知的功能等同化合物而言在生理pH下具有提高的溶解度。
本发明的另一个目的在于提供对应的方法。
本发明的另一个目的在于提供包含所述化合物的药物组合物。
本发明的又另一个目的将通过了解以下发明内容、以附图形式说明的多个优选实施方式和所附权利要求书而变得显而易见。
发明内容
本发明公开了具有如下通式S的化合物:
所述化合物能够消除19S RP DUB的去泛素化(DUB)活性。
认为本发明的化合物与蛋白酶体抑制剂的新种类有关,代表性的蛋白酶体抑制剂有已知化合物b-AP15。
具体而言,如本发明所述,本发明的化合物抑制两种19S RP DUB的活性(UCHL5和USP14),但不影响非蛋白酶体DUB。更具体地,本发明化合物对于治疗用本领域化疗法难以治疗的癌症肿瘤(所述难治疗性归因于内源性凋亡抑制剂Bcl-2的过表达)有影响。
更具体地,如本发明所述,本发明的化合物在治疗用硼替佐米或与硼替佐米共用抑制去泛素化活性机制的药剂难以治疗的癌症中有效。在另一个优选实施方式中,所述化合物在治疗用本领域已知的任何抗癌药物难以治疗的癌症中有效。
在本申请中,“难以治疗”表示用单一抗癌药物剂量治疗癌症不实质性地降低在治疗前即刻观察到的所述癌症的生长率,例如使该生长率降低不超过25%/月,或不超过10%/月,或甚至不超过5%/月或更少。具体而言,本发明方法对治疗如下患者的癌症有效,所述患者在接受了一个或多次(尤其是两次或三次)标准剂量的硼替佐米或与硼替佐米或任何其它抗癌药物共用抑制去泛素化活性机制的试剂后,显示癌症生长率/月降低不超过25%或10%或甚至5%或更少,例如相较于单次治疗或者两次或三次或更多次治疗的各最末次治疗之前即刻观察到的癌症生长率而言的任何正生长率。一种公认的肿瘤生长方式是未弥散癌症的体积变化。
可受本发明方法治疗影响的癌症的示例有多发性骨髓瘤。可受治疗影响的其它癌症示例包括肺癌、前列腺癌、结肠癌、卵巢癌、胰腺癌、乳腺癌、头颈癌。
在通式S-1的本发明化合物中,
双键d1处的R1、R2和双键d2处的R3、R4可以彼此独立地具有与式S-1相反的构型,
X是CO、CS、CH2、CHC1-6-烷基、NH或NC1-6-烷基;
R1和R3彼此独立地是H或C1-6-烷基;
R2和R4彼此独立地是H;C1-6-烷基;C1-5-烷基CO;苯基,或6元杂芳基,其任选地被以下1~3种取代:C1-6-烷基、C1-6-烷氧基、CN、-COOC1-6烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8,前提条件是烷基和烷氧基中的一个或多个H可被氟取代;R5是H;C1-6-烷基;C2-6-烯基;C1-3-烷氧基-C2-6-烷基-;C1-3-烷氧基-C2-6-烯基-;芳基-C0-6-烷基-;杂芳基-C0-6-烷基-;杂环基-C0-6-烷基-;环烷基-C0-6-烷基-;-C1-6-烷基-COOC1-6-烷基;-C2-6-烷基-芳氧基;COR6;
R6选自:C1-6-烷基;C2-6-烯基;C1-6-烷氧基;C1-3-烷氧基-C1-6-烷基-;
C1-3-烷氧基-C1-6-烯基-;芳基-C0-6-烷基-;杂芳基-C0-6-烷基-;杂环基-C0-6-烷基-;环烷基-C0-6-烷基-;-C1-6-烷基-COOC1-6-烷基;NH2;-NHC1-6-烷基;-N(C1-6-烷基)2;-C0-6-烷基-芳氧基;
R7、R8彼此独立地是H或C1-3-烷基。
优选R1和R3均是或C1-3-烷基。
优选R2和R4是H;C1-6-烷基;C1-5-烷基CO;苯基或6元杂芳基,其任选地被以下1~3种取代:C1-6-烷基、C1-6-烷氧基、CN、COOC1-6-烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8,前提条件是烷基和烷氧基中的一个或多个H可被氟取代,并且其中苯基的取代优选在第3、4、5位中的一个或多个位置。
特别优选R2和R4是苯基取代的,由如下1~3个取代基,优选如下1或2个取代基在第3、4、5位的一个或多个位置取代:C1-6-烷基、C1-6-烷氧基、CN、COOC1-6-烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8,前提条件是烷基和烷氧基中的一个或多个H可被氟取代。最优选的是吸电子取代基,尤其是F、Cl、三氟甲基、NO2、CN。
R5优选选自下组:H、甲基、乙酰基、COCH=CH2、2-乙酰氧基乙基。
根据本发明的一个优选方面,X=CO。根据本发明的另一个优选方面,R1和R3均是H。根据本发明的第三个优选方面,R2和R4彼此独立地是,苯基或6元杂芳基,其任选地被以下取代基中的1~3个取代:C1-6-烷基、C1-6-烷氧基、CN、-COOC1-6-烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8,优选苯基且苯基被取代,若有取代则优选在第3、4、5位的一个或多个位置被取代。根据本发明的一个优选方面,双键d1处的R1、R2和双键d2处的R3、R4具有式S-1的构型;X是CO、CS、CH2、CHC1-6-烷基、NH或NC1-6-烷基;R1和R3彼此独立地是H或C1-6-烷基;R2和R4彼此独立地是H;C1-6-烷基;C1-5-烷基CO;苯基或6元杂芳基,其被以下取代基中的1~3个取代:CN、NO2、F、Cl、Br、I、NH2、NHC1-6-烷基、N(C1-6-烷基)2、COC1-6-烷基;R5是H、C1-6-烷基、C2-6-烯基、C1-3-烷氧基-C1-6-烷基、C1-3-烷氧基-C1-6-烯基、芳基、杂芳基、杂环基、C1-6-烷基-杂芳基、C1-6-烷基-杂环基、C1-6-烷基-环烷基、C1-6-烷基-芳基、CO-C1-6-烷基、CO-乙烯基、CO-烯丙基、CO-芳基、CO-环烷基。优选地,X独立地是CO,R2和R4彼此独立地被苯基取代,R5选自COR6,特别地选自CO-C1-6-烷基、CO-环烷基、CO-乙烯基、CO-烯丙基。
“芳基”指6~10个碳原子的包含至少一个芳族环的单环或二环烃。“芳氧基”指与氧原子结合的芳基基团。“杂芳基”代表具有5或6个环原子的单环系统,所述环原子中的一个或多个独立地选自氧、氮、硫。“烷基”指直链或支链烷基。“烯基”指指直链或支链烯基。“烷氧基”指直链或支链烷氧基。“环烷基”指3~7个碳原子的饱和单环烃。
本发明通式S-1的优选化合物公开于表1和表2。
表1.本发明优选化合物
X=CO,R1=R3=H,R5是H或烷基
*或H或H和4-硝基苯基的混合物
**或4-硝基苯基或H和4-硝基苯基的混合物
因其氨基基团的质子化作用,其中R5不是酰基的本发明氮杂环庚烷酮(azepanone)化合物以及对应取代的哌啶-4-酮在水性介质中的溶解度随pH降低而增加。然而,根据一个重要方面,其中R5不是酰基的本发明氮杂环庚烷酮化合物(即,不是-COR6)相较于对应取代的哌啶-4-酮而言在生理pH下在水性介质中具有较高溶解度。虽然这些氮杂环庚烷酮和哌啶-4-酮的溶解度在pH由高到低时增加,但氮杂环庚烷酮溶解度增加开始的pH值高于哌啶-4-酮的相应值。本申请中,“生理pH”是pH为约6~约8,尤其是7.0~7.5。
表2.本发明优选化合物
X=CO,R1=R3=H,R5=COR6
其中R5是酰基的本发明化合物(即,-COR6)在水性介质中的溶解度基本不依赖于pH。
特别优选的本发明化合物是具有如下编号的化合物:1561、1562、1567、1570、1571、1586、1591、1600、1612、1618、1622、1625、1643、1644、1647、1648、1649、1652、1653、1656、1657、1658、1662。最优选的本发明化合物是具有如下编号的化合物:1570、1571、1625、1662。
因为本发明的化合物包含1,5-未经取代的1,4-戊烯-3-酮部分,其可以四种顺/反异构体EE、ZE、ES、ZZ存在。在本发明化合物的定义中,该异构现象在上文中如此定义“双键d1处的R1、R2和双键d2处的R3、R4可彼此独立地具有与式S1相反的构型”。本发明的化合物包括任何此类异构体和此类异构体的任何混合物。
合成中的本发明化合物以异构体混合物形式获得,但有时也以在特定溶剂中溶解度最低的异构体形式获得,由此可使其沉淀或结晶。虽然由此可在控制条件下获得纯的异构体,但所有异构体均显示该化合物的药理学作用。其原因在于它们在水或其它羟基或巯基溶剂或试剂存在下的平衡,这通过酸和碱催化而加速。
因此,本文所用的术语“本发明化合物”包含前述种类的纯异构体以及两种或更多种此类异构体的混合物。本发明化合物在水性体液中的平衡速率足以在单一疗程中达到基本平衡。
本发明化合物包含氮杂环庚烷(azepane)部分,优选是氮杂环庚烷-4-酮部分。根据本发明的一个重要方面,本发明化合物显示的细胞毒活性高于含有哌啶部分(例如,4-哌啶酮部分)的结构上对应的化合物。
根据本发明的另一个重要方面,包含氮杂环庚烷部分,尤其是氮杂环庚烷-4-酮部分的本发明化合物显示,其在适合给予患者的液体运载体(例如二甲亚砜)中的溶解度高于包含哌啶部分(例如4-哌啶酮部分)的结构上对应的化合物。
“单一疗程”是给予和本发明化合物消耗之间的经历的时间过程,所述的本发明化合物消耗即为,当本发明化合物在作用位点(例如肿瘤)的浓度已减少90%或95%或99%或更多时的时间点。在药物组合物中,通过小心除湿来使本发明化合物的异构体或异构体的混合物稳定。
本发明方法包括:给予有需要的患者药理学有效量的合适药学载体中的本发明化合物,例如,溶解或悬浮在水性载体中或在包含二甲亚砜或N,N-二甲基乙酰胺的载体中。给予可通过任何合适的途径进行,例如通过静脉内、肌肉内、腹膜内或皮下注射或输注。同样预期其它给予方法,尤其是口服(per os),例如以片剂或者硬或软胶囊。
本领域技术人员已知如何确定药理学有效剂量。所述剂量可以是0.0001g/kg~0.1g/kg体重,特别是0.001g/kg~0.01g/kg kg体重,考虑该试剂是否全身性或局部给予。与DUB抑制一致,采用本发明化合物的治疗相较于硼替佐米治疗而言引起较高分子量的聚泛素化蛋白的累积,并导致较强的未折叠蛋白反应。根据本发明,已发现由本发明化合物诱导的凋亡与硼替佐米不同,其差异在于对p53肿瘤抑制剂的破坏不敏感且对凋亡抑制剂Bcl-2的过表达不敏感。
如本发明所述的采用本发明化合物的治疗抑制人肿瘤发展和小鼠乳腺癌、肺癌、结肠癌、头颈癌体内模型中的肿瘤发展,并且抑制急性骨髓性白血病(AML)模型中的浸润。因此,本文公开了用本发明化合物抑制19S RP的DUB活性是治疗人与动物中癌症的可行选择。
因此,更具体地,本发明公开了治疗用本领域化疗法难以治疗的人体内癌肿瘤的方法,所述方法包括给予药学上可接受运载体内的药理学有效量的本发明化合物。本发明方法特别有利于治疗肿瘤细胞因内源性凋亡抑制剂Bcl-2的过表达而难以治疗的肿瘤患者。
根据本发明的一个优选方面,所述19S RP DUB包含UCHL5和USP14。根据本发明的另一个优选方面,非蛋白酶体DUB的去泛素化(DUB)活性不受本发明化合物的影响。本发明化合物可溶解或悬浮在液体运载体中以任何合适的途径给予,例如通过静脉内、肌肉内和皮下给予。此外或替代性地,本发明化合物可由口给予,例如以片剂或胶囊形式给予。本发明化合物的有用的药理学有效剂量是0.0001g/kg~0.1g/kg体重,特别是0.001g/kg~0.01g/kg kg体重,考虑所述化合物是否全身性或局部给予。所述方法可包括:通过在给予硼替佐米或与硼替佐米或所述其它抗癌药物共用抑制泛素化活性机制的所述活性物质(principle)之前和之后测定癌症的生长率来选择待治疗的人,以正生长率(尤其是大于5%/月或大于10%/月或大于25%/月)定为选择标志物。如采用报告细胞系证实,本发明化合物阻滞细胞蛋白酶体功能,所述报告细胞系表达标记有黄色荧光蛋白的泛素(UbG76V-YFP),其供于蛋白酶体降解的结构性靶向(12)。免疫印迹和流式细胞术显示Ub-YFP报告子的剂量依赖性聚集(IC50=0.8μM),表明蛋白酶体功能的损伤。因为蛋白酶体功能的抑制通过泛素周转(ubiquitinturnover)(13)的缺陷来表征,用本发明化合物处理结肠癌HCT116细胞并通过免疫印迹来分析泛素连接水平。相较于采用20S CP抑制剂硼替佐米,所述处理引起较高分子量的聚泛素化蛋白质的快速时间依赖性聚集,表明本发明化合物抑制UPS的替代分支(alternative branch)。聚泛素的增加与强蛋白毒性反应相关,所述蛋白毒性反应通过HSPA6(Hsp70B')、HSPA1B和DNAJB1(Hsp40)的诱导来表征。
多种细胞周期调节蛋白的周转都受所述UPS的控制,包括细胞周期蛋白依赖性激酶p21Cip1、p27Kip1和肿瘤抑制剂p53的抑制剂(4)。采用本发明化合物的治疗使其水平以剂量依赖性的方式增加而不改变鸟氨酸脱羧酶1(DOC1)(一种泛素非依赖性蛋白酶体底物)的水平变化(8)。细胞周期调节物的增加伴随G2/M期界线处的生长停滞和亚G1DNA含量增加。观察到的细胞周期停滞与DNA损伤标志物(例如磷酸化的p53(Ser 15处)(9)或H2AX(Ser 139处)(10))水平的增加无关,表明b-AP15不是基因毒性试剂。亚G1 DNA、半胱天冬酶-3活化和聚ADP核糖聚合酶(PARP)与细胞角蛋白切割的增加与一定药物浓度下细胞活力的总体降低有关,所述药物浓度诱导与UPS抑制和凋亡有联系的聚泛素的累积。硼替佐米引起的凋亡诱导对p53肿瘤抑制剂状态和抗凋亡Bcl-2肿瘤蛋白(11,12)的过表达敏感。通过采用HCT116结肠癌细胞的等基因克隆,证明b-AP15诱导的凋亡对Bcl-2的过表达和凋亡调节物p52、BAX或PUMA的破坏不敏感。细胞毒性活性的检测显示,本发明化合物对结肠癌细胞系HTC-116的毒性大于对无限增殖化的视网膜色素上皮细胞(hTERT-RPE1)和外周血单核细胞(PBMC)的毒性。本发明化合物显示对HTC-116细胞的细胞毒性活性程度高于对正常细胞类型的细胞毒性活性。
观察到的细胞蛋白酶体活性的减少的原因不能解释为对20S CP的β亚基的蛋白水解活性的抑制。采用活性特异性底物的体外实验没有显示对以下任何一项的抑制:20S CP或26S蛋白酶体的蛋白水解活性、19S RP和20S CP的分离,以及对聚泛素与蛋白酶体的结合的抑制。
本发明化合物包含α-β二烯酮个体和两个空间上可及的β碳。早期已有描述称一类泛素异肽酶抑制剂包含结构上相似的药效团(13)。然而,当采用泛素7-氨基-4-甲基香豆素(Ub-AMC)测试经本发明化合物处理的细胞的细胞DUB活性时,没有观察到Ub-AMC切割的减少。这证明本发明化合物不是通用的DUB抑制剂。虽然不希望受理论限制,但药效团结构的相似性和显示本发明化合物不依赖20S CP而抑制蛋白酶体活性的数据指示,本发明化合物通过阻滞19S RP的去泛素化活性来抑制所述蛋白酶体。
采用Ub-AMC和纯化的19S RP或26S蛋白酶体的体外试验证实,本发明化合物抑制19S RP和26S蛋白酶体两者的去泛素化活性。重组的泛素-GFP是19S RPDUB活性的底物(15)。用b-AP15处理19S RP有效抑制Ub-GFP和泛素化HDM2的切割。聚泛素链中存在的泛素连接(ubiquitin bond)的类型决定泛素修饰的底物的命运。
K48连接的聚泛素链通常靶向接合的蛋白质以使其降解(14),然而K63连接的链涉及非蛋白水解作用,包括DNA修复(15)和有丝分裂染色体分离(16)。泛素链分解反应揭示本发明化合物抑制K48和K63连接的泛素四聚物的19S RP加工。观察到的对泛素链分解的抑制可解释用本发明化合物处理的细胞中高分子量泛素结合物的累积。
所述蛋白酶体的去泛素化活性归因于三种DUB:UCHL5、USP14和POH1的作用,这三种DUB都定位在19S RP内(17-19)。UCHL5和USP14都对N-乙基马来酰亚胺(NEM)(半胱氨酸蛋白酶的通用抑制剂)敏感,而POH1对由NEM引起的抑制不敏感但对金属螯合剂(例如N,N,N,N-四-(2-吡啶基甲基)乙二胺(TPEN))敏感(20)。抑制实验显示,甚至在用NEM和本发明化合物共同处理19S RP之后还存在残留的DUB活性。该残留DUB活性在用本发明化合物和TPEN共同处理后消除,表明本发明化合物主要抑制所述NEM敏感性半胱氨酸DUB之一或两者。本发明化合物的β-碳可作为迈克尔受体(Michael acceptor)部分,导致对靶蛋白中半胱氨酸残基的共价结合。然而,体外试验显示,本发明化合物是可逆抑制剂,并且谷胱甘肽不妨碍所述化合物的抑制活性。
为了具体鉴定哪种DUB受到本发明化合物处理的抑制,采用带血凝素标签的泛素乙烯砜(vinylsulphonone)(HA-UbVS)来进行竞争性平行实验,所述HA-UbVS是与所述半胱氨酸类的DUB不可逆反应的活性位点定向的探针(17)。用本发明化合物孵育19S RP或26S蛋白酶体消除两种DUB的Ub-VS标记,所述两种DUB的分子量对应于UCHL5和USP14。采用UbVs对于获自药物处理细胞的溶解产物获得了相似结果。免疫印迹分析显示USP14和UCHL5的分子量下移,这归因于活性损失和UbVs标记的减少。这与来自经本发明化合物处理细胞的亲和纯化的蛋白酶体显示降低的DUB活性相一致,但该现象限于蛋白酶体且在细胞溶解产物中不明显。其它体外试验显示,本发明化合物对重组非蛋白酶体半胱氨酸DUB具有最小抑制,这与该抑制并不归因于通用半胱氨酸反应性的看法一致。
本发明化合物确实使体内肿瘤生长显著减少或甚至停止,如通过将其给予负载人肿瘤或小鼠异种移植物的小鼠所示。当每天给予荷载FaDu头颈癌异种移植物的SCID小鼠本发明化合物时,在用本发明化合物每天处理之后观察到对FaDu肿瘤的显著抑制(处理/对照肿瘤体积,T/C=0.4,p=<0.001)。通过检测异种移植物源性的循环中的细胞角蛋白(CK18)分析肿瘤细胞死亡。细胞角蛋白-18是凋亡的生物标志物(21,22);观察到总人CK18血浆水平的显著升高(p=0.01)。相较于总水平,半胱天冬酶切割的CK18(CK18-Asp396)的水平适中升高,表明本发明化合物具有体内抗肿瘤细胞活性。本发明化合物还显示抑制HCT-116Bcl2+结肠癌异种移植物在裸小鼠内的肿瘤发病,这通过相较于载体处理的对照而言的肿瘤发病的显著延迟来证明。
类似地,采用较低频的给予方案,本发明化合物抑制同基因小鼠模型中的肿瘤生长。
泛素C-末端水解酶(UCH)和泛素特异性蛋白酶(USP)是大约一百组由人基因组编码的DUB的主要亚组(23)。
本发明化合物对19S RP中的UCHL5和USP14的特异性的机制可能与19SRP中这些酶的独特构象有关,或归因于药物诱导的19S RP结构变化。这些发现与本领域报道的一致,指示缺失UCHL5和USP14两者与缺失单独一个不同,导致聚泛素化蛋白质的累积和对细胞蛋白质降解的抑制(24)。
观察到的DUB抑制与高分子量的泛素-底物复合物相关联似乎具有特定的关联性。在用本发明化合物处理的细胞中观察到伴侣蛋白基因的强表达,指示诱导了蛋白毒性反应。由本发明化合物抑制DUB导致的高分子量泛素-底物复合物的累积似乎产生强细胞毒性。
将在下文中通过参考本发明的优选实施方式和包含附图编号的说明性附图来进一步详细描述本发明。
附图说明
图1a~1o的图表说明由FMCA(微培养细胞毒荧光分析)检测,通过本发明化合物的实施方式使报告细胞系HCT-116连续接触化合物72小时后诱导剂量依赖性细胞毒性,并且采用本发明范围外的结构相关化合物不产生所述诱导。将处理的细胞与未经处理的对照作比较(存活指数);
图2a~2e的图表说明本发明化合物在生理pH下在水性介质中具有较高溶解度。
图3a~3f的图表说明,通过图1a~1o的方法,本发明的氮杂环庚烷酮化合物的细胞毒性高于本发明未包含的结构上相对应的哌啶-4-酮化合物的细胞毒性。
具体实施方式
方法
采用内含人20S蛋白酶体(波士顿生物化学公司(Boston Biochem)),并且含有Suc-LLVY-AMC、Z-LLE-AMC或Boc-LRRAMC作为蛋白酶体活性底物的反应缓冲液(25mM Hepes,0.5mM EDTA,0.03%SDS)在黑96孔微量滴定板中进行体外蛋白酶体活性试验。采用人19S RP(波士顿生物化学公司),以泛素-AMC作为底物来进行去泛素化酶活性试验。将包含1×106个细胞的100-μl-细胞悬液皮下注射进入SCID的横腹部,供于FaDu异种移植物研究。在将荷瘤小鼠随机分成对照组和处理组之后,以5mg/kg给予本发明化合物或载剂。通过采用M30和M65试验(Peviva公司)检测半胱天冬酶切割的细胞角蛋白-18的水平和细胞角蛋白-18的总水平来测定凋亡和细胞死亡的体内水平。在下文中更详细地描述所述方法。
试剂试剂获自以下来源:20S蛋白酶体(E-360)、26S蛋白酶体(E-365)、19SRP蛋白酶体(E-366)、Suc-LLVY-AMC(S-280)、Z-LLE-AMC(S-230)、Boc-LRR-AMC(S-300)、泛素-AMC(U-550)、四泛素(Tetra-ubiquitin)K63(UC-310)、四泛素K48(UC-210)、去结合酶(deconjugating enzyme)组(KE10)、HA-泛素乙烯基砜(U-40212)(波士顿生物化学公司);抗-β-肌动蛋白(AC-15)、ODC-1(HPA001536)(西格玛奥德里奇公司(Sigma Aldrich));抗-LC-3(2775)、抗-GAPDH(2118)、抗-p44/42MAPK(4695)、抗-膦酸基-p44/42MAPK(9101)(细胞信号转导公司(Cell Signaling));N-乙基马来酰亚胺(34115)(EMD化学品公司(EMD Chemicals));抗-泛素K48(Apu2)、抗-泛素(MAB1510)(密理博公司(Millipore));抗-p53(DO1)、抗-UCHL5(H-110)、Hdm2(SMP14)(圣克鲁兹公司(Santa Cruz));抗-PARP(C2-10)、抗-p27(G173-524)、抗-活性半胱天冬酶3(C92-605)(BD生物科学公司);抗-USP14(A300-919A)(贝司实验室公司(BethylLaboratories));抗-HA(12CA5)(罗氏公司(Roche))。硼替佐米获自瑞典的卡珞琳斯卡医院(Karolinska Hospital),肿瘤科。
细胞培养。
使MCF7细胞保持在MEM/10%胎牛血清中。使HCT-116p53+/+、p53-/-、Bcl-2+/+、PUMA-/-和BAX-/-细胞保持在McCoy′s5A改良培养基/10%胎牛血清中。如(25)所述产生HCT-116p53+/+、p53-/-、PUMA-/-和BAX-/-。通过对亲本HCT-116p53+/+细胞转染pCEP4Bcl-2(Addgene质粒16461)(26)并分离高表达克隆来产生HCT-116 Bcl-2+/+细胞系。使FaDu和LLC3细胞保持在补充有10%胎牛血清、丙酮酸钠、Hepes和非必需氨基酸的DMEM高葡萄糖培养基中。使4T1.12B癌细胞保持在补充有10%胎牛血清的RPMI培养基中。如(12)所述产生蛋白酶体报告细胞系MelJuSo Ub-YFP。使细胞保持在杜贝克氏改良的依格培养基(Dulbecco’s Modified Eagle’s Medium)/10%胎牛血清中。如(12)所述产生视网膜上皮细胞系。使所有细胞保持在杜贝克氏改良的依格培养基(Dulbecco’sModified Eagle Medium)/10%胎牛血清中。如(28)所述产生视网膜上皮细胞系。使所有细胞保持在37℃,5%CO2中。
蛋白酶体和DUB抑制试验。
采用20S CP(2nM)(波士顿生物化学公司)的体外蛋白酶体活性试验在37℃下于100-μl反应缓冲液(25mM Hepes,0.5mM EDTA,0.03%SDS)中进行。样品用指示化合物孵育10分钟,然后添加10μM Suc-LLVY-AMC、Z-LLE-AMC或Boc-LRR-AMC以分别检测糜蛋白酶样、半胱天冬酶样和胰蛋白酶样活性。就DUB抑制试验而言,使本发明化合物与19S RP(5nM)、26S(5nM)UCH-L1(5nM)、UCH-L3(0.3nM)、USP2CD(5nM)USP7CD(5nM)USP8CD(5nM)和BAP1(5nM)孵育,然后添加泛素-AMC(1000nM)。采用Wallac Multilabel计数器或配备有360nm激发滤镜和460nm发射滤镜的Tecan Infinite M1000来监测荧光。
底物覆盖试验。
如(29)所述进行非变性凝胶电泳。简言之,使4μg纯化的26S蛋白酶体(波士顿生物化学公司)与10或50μM的本发明化合物混合,并在37℃下孵育10分钟。样品在4%非变性PAGE上显影。将凝胶浸没于试验缓冲液(20mM Tris-HCL,5mM MgCl2,1mM ATP,0.1mM Suc-LLVY-AMC)中,并在紫外线照射下目测蛋白酶体。
泛素-切割试验。
如(30)所述产生重组Ub-GFP质粒pet19b Ub-M-GFP。简言之,通过His亲和纯化法从BL21大肠杆菌细胞纯化重组Ub-GFP。对于切割试验,使19S RP(25nM)与10mM NEM、250μM TPEN或50μM本发明化合物孵育10分钟,然后添加重组Ub-GFP(200nM)。基本如上所述进行泛素链分解反应,不同之处在于,K48-或K63-连接的泛素四聚物(50ng)用Ub-GFP替代。采用抗泛素抗体,通过免疫印迹来测定Ub-GFP切割水平或泛素分解水平。按照波士顿生物化学公司的方案(K-200)来产生泛素化的Hdm2底物。对于切割试验,使19S RP(25nM)与50μM本发明化合物或DMSO孵育10分钟,然后添加泛素化的Hdm2底物(100nM)。泛素化的Hdm2底物和泛素化的Hdm2的切割通过采用抗Hdm2抗体的免疫印迹来测定。
蛋白酶体分离:HCT-116细胞用硼替佐米(100nM)或本发明化合物(1μM)处理3小时。刺激之后,使所述细胞在50mM HEPES pH7.4,250mM蔗糖,10mM MgCl2,2mM ATP,1mM DTT和0.025%洋地黄皂苷中裂解。对样品进行简短超声处理,并使其在冰上孵育15分钟。按照生产商说明来分离这些样品中的蛋白酶体。
DUB的UbVS标记。为在细胞裂解物中进行DUB标记,通过胰蛋白酶处理来收取亚培殖细胞,所述细胞用PBS洗涤三次,并以1500RPM离心5分钟。细胞沉淀用缓冲液(50mM HEPES pH7.4,250mM蔗糖,10mM MgCl2,2mMATP,1mM DTT)在冰上裂解15分钟。通过离心去除碎片,并用1μM HA-UbVS在37℃下标记μg蛋白质30分钟。样品通过SDS-PAGE显影并通过采用指示抗体的免疫印迹分析。
细胞凋亡和活力测定
为测定亲本HCT-116p53+/+的凋亡,细胞用递增剂量的本发明化合物处理24小时。处理剂量基于24小时内导致最大凋亡的药物浓度。将HCT-116细胞以10,000个细胞/孔接种于96孔微量滴定板中并孵育过夜。细胞用指示药物处理24小时。在孵育阶段的最后,向组织培养介质添加NP40至0.1%,并且25μl各孔内容物采用前述M30-ELISA(31)分析。通过检测酸性磷酸酶活性或采用FMCA方法(32)来测定细胞活力。为测酸性磷酸酶活性,将细胞以5000个细胞/孔接种在96孔培养板内并在37℃孵育12小时。向生长培养基中的细胞添加化合物并在37℃孵育72小时。细胞用200μl温PBS洗涤。每孔添加100μl内含对硝苯基磷酸盐(pNPP,2mg/ml)的乙酸钠缓冲液(pH5)(NaAc0.1M,0.1%曲通-X-100)。细胞孵育2小时,之后通过添加1N NaOH来终止反应。在405nm处检测吸光度。图1a~1o中说明了本发明化合物的多个实施方式的剂量依赖性细胞毒性。
对于FMCA试验,采用移液机械手Precision2000(佛蒙特州威努斯基的伯腾仪器公司(Bio-Tek Instruments Inc.))将细胞接种于已加药物的384孔板中。所述板孵育72小时,然后转移至整合的HTS SAIGAN核心系统,所述核心系统由以下部分组成以供自动化FMCA:带有CO2孵育箱(Cytomat2C,瑞典索伦蒂纳市的科峻公司(Kendro))的ORCA机器人(贝克曼库尔特公司(Beckman Coulter))、分配器模块(Multidrop384,亚拉巴马州亨茨维尔的泰特技术公司(Titertek))、洗涤模块(ELx405,伯腾仪器公司)、开盖台(delidding station)、置板区(plate hotel)、条形码阅读器(贝克曼库尔特公司)、液体处理器(Biomek2000,贝克曼库尔特公司)和多用阅读器(FLUOstar Optima,德国奥芬堡的BMG实验室技术公司(BMGLabtech))。存活指数(SI)定义为测试孔荧光(减去空白值)与对照孔荧光(减去空白值)的百分比。
细胞周期分析。
为测定细胞周期,HCT-116细胞用本发明化合物或DMSO处理。细胞通过胰蛋白酶消化来收取,洗涤并在70%冰冷EtOH中固定12小时。使所述细胞在含有碘化丙啶(50μg/ml)和RNA酶A(0.5μg/ml)的PBS中的染色溶液中重悬。样品在BD FACScalibur上跑动。采用ModFit软件测定各细胞周期时期的细胞百分比。
实施例1.本发明化合物优选实施方式的示例性合成
概述
所用全部溶剂为HPLC级或更高级别。需要无水条件时,在使用前至少24小时向溶剂添加过量分子筛以确保干燥。以400.1MHz在Bruker AdvanceDPX 400分光光度计上记录1H NMR核磁共振(NMR)值。采用Agilent质谱仪电离模式获得低分辨率电喷雾电离质谱。在Merck硅胶60(230-400目)上进行快速色谱。分析LCMS数据如下获得:采用Agilent质谱仪;Agilent1100系统;A:ACEC8柱(50x3.0mm,5μM);梯度:内含10-97%乙腈的水/0.1%TFA,3分钟内,1.0mL/分钟,或B:xBridge C18柱(3.5μM.50x3.0mm),梯度内含10%~97%乙腈的10mM NH4HCO3(pH10),3分钟内,1mL/分钟)。化学结构的名称采用Marvin Scech5.2.6(ChemAxon)来确定。
(3E,5E)-3,5-二(苯甲叉)氮杂环庚烷-4-酮(#1516)和(3E,5E)-3,5-二(4-甲氧基苯甲叉)-氮杂环庚烷-4-酮(#1517)
六氢-4H-氮杂卓-4-酮(0.45g,3.0mmol)与苯醛(0.70g,7.0mmol)、4-甲氧基苯醛(0.90g,7.0mmol)或4-氯苯醛(0.92g,7.0mmol)一同溶解在乙酸(10mL)中。然后逐滴添加硫酸(浓1mL)并在室温下搅拌反应24小时。添加水(30mL)并过滤沉淀物然后真空干燥过夜。不进行进一步纯化。获得99%纯度的化合物#1516(通过LCMS(系统A)MS ESI+m/z290[M+H]+测定)。也获得99%纯度的化合物#1517(通过LCMS(系统A)MS ESI+m/z350[M+H]+测定)。获得91%纯度的化合物#1518;LCMS(系统A)。MS ESI+m/z358[M]+,360[M+2]+。
(3E,5E)-3,5-二(苯甲叉)-1-(丙-2-烯酰基)-氮杂环庚烷-4-酮(#1520)
(3E,5E)-3,5-二(苯甲叉)氮杂环庚烷-4-酮(#1516)(50.0mg,0.182mmol)和丙烯酸(14.4mg,0.20mmol)、HBTU(58.4mg,0.182mmol)、三乙胺(36.7mg,0.364mmol)溶解于DMF(2mL)并搅拌过夜。添加乙酸乙酯和盐水并提取产物。合并有机层干燥并蒸发。粗产物用甲醇稀释并通过制备型HPLC纯化。获得96%纯度的化合物#1520,MS-ESI+m/z344[M+H]+。
(2R)-[(3E,5E)-3,5-二(4-硝基苯甲叉)-4-氧代-1-(吡咯烷-2-基-羰基)-氮杂环庚烷三氟乙酸酯(#1505)
使N-Boc-氮杂环庚烷酮(100mg,0.47mmol)和4-硝基苯醛(156mg,1.03mmol)溶解于乙酸(10mL)。然后逐滴添加硫酸(浓1mL)并在室温下搅拌反应三天。然后添加更多醛和硫酸并另搅拌反应24小时,隔24小时两次添加更多酸。反应通过加水淬灭,然后滤出沉淀的粗制中间体并用水洗涤。产物经真空过夜干燥后,称取2x35mg(0.09mmol)的粗制中间体置入两个烧瓶,并与琥珀酸单乙酯(14.8mg,0.10mmol)一同在DCM/DMF(2mL,4:1)中溶解。添加三乙胺(19.3μL,0.14mmol),并搅拌混合物5分钟,然后添加HATU(38.6mg,0.10mmol)。持续搅拌12小时后,添加更多的三乙胺和HATU,并持续搅拌4小时。蒸发溶剂,然后残余物通过制备型HPLC纯化。将残余物溶解于二氯甲烷/三氟乙酸(5mL,4:1),搅拌40分钟并再次浓缩。获得93%纯度的化合物#1505(通过LCMS(System A)测定)。MS ESI+m/z477[M+H]+.
实施例2.本发明化合物的优选实施方式的其它示例性合成
(2R)-2-{[(3E,5E)-3,5-二[(4-硝苯基)亚甲基]-4-氧代氮杂环庚烷-1-羰基}吡咯烷鎓三氟乙酸酯(化合物#1505)。使N-boc氮杂环庚烷-4-酮(0.10g,0.47mmol)和4-硝基苯醛(156mg,1.0mmol)溶解于乙酸(10mL)中,逐滴添加浓H2SO4(1mL)并使该反应在室温下搅拌过周末。添加更多醛(156mg)和H2SO4(1mL),并室温搅拌过夜。另加mL浓H2SO4,并使反应再次搅拌过夜。再添加一次浓H2SO4并使反应搅拌直至完全(搅拌两周)。添加水后,形成棕色沉淀,滤出,用水洗涤,并真空干燥以获得339.5mg棕色固体中间体1,其无需进一步纯化即可使用。使中间体1(35mg,0.09mmol)和N-boc脯氨酸(22mg,0.10mmol)溶解于DCM/DMF(4:1,2mL)。添加TEA(19μL,0.14mmol)并使所述混合物搅拌5分钟,然后添加HATU(38.6mg,0.10mmol)并使反应在室温下搅拌过夜。添加更多TEA(19μL,0.14mmol)和HATU(38.6mg,0.10mmol),并使该反应另搅拌4小时。使所述反应混合物浓缩然后通过制备型LC(内含40-70%ACN的0.1%TFA)纯化以获得黄色固体状的产物。使该固体溶解于DCM/TFA(4:1,5mL),并使该溶液在室温下搅拌40分钟以去除boc保护基团。回收到纯度为93%的黄色固体状的所述产物的TFA盐。LCMS A:Rt1.94/1.99,m/z[M+H]+477.1,B:Rt2.28。
(3E,5E)-1-(4-乙氧基-4-氧代丁酰基)-3,5-二[(4-硝苯基)亚甲基]-4-氧代氮杂环庚烷-1-鎓三氟乙酸酯(化合物#1507)。使中间体1(35mg,0.09mmol)和N-boc脯氨酸(22mg,0.10mmol)溶解于DCM/DMF(4:1,2mL)。添加TEA(19μL,0.14mmol)并使所述混合物搅拌5分钟,然后添加HATU(38.6mg,0.10mmol)并使反应在室温下搅拌过夜。添加更多TEA(19μL,0.14mmol)和HATU(38.6mg,0.10mmol),并使该反应另搅拌4小时。使所述反应混合物浓缩然后在制备型LC(内含40-70%ACN的0.1%TFA)上纯化以获得纯度为95%的黄色固体状的所述产物的TFA盐。LCMS A:Rt2.48/2.50m/z[M+H]+508.1.B:Rt2.48/2.52。
(3E,5E)-3,5-二[(4-氯苯基)亚甲基]氮杂环庚烷-4-酮(化合物#1518)。
使N-Boc-氮杂环庚烷酮盐酸盐(0.45g,3.0mmol)和4-氯苯醛(0.92g,6.6mmol)溶解于浓乙酸(10mL)。逐滴添加H2SO4(1mL)并使反应在室温下搅拌24小时。添加水(30mL)之后,形成沉淀物,滤出,并真空干燥以获得纯度为91%的黄色固体产物。LCMS A:Rt2.04m/z[M]+358.1。
(3E,5E)-3,5-二(苯甲叉)-1-(丙-2-烯酰基)氮杂环庚烷-4-酮(化合物#1520)。使氮杂环庚烷-4-酮盐酸盐(50mg,0.182mmol)、丙烯酸(14μL,0.20mmol)、TBTU(58mg,0.182mmol)和TEA(37mg,0.364mmol)溶解于DMF(2mL)并在室温下搅拌过夜。添加盐水和乙酸乙酯,并使相分离。过滤后干燥有机相并蒸发溶剂。将组产物溶解于乙酸(2mL)和H2SO4(0.2mL)。添加苯醛(50μL)并使该反应搅拌24小时。向该混合物添加甲醇和水,其通过制备型LC纯化。分离纯度为96%的黄色固体标题化合物。LCMS A:Rt2.68m/z[M+H]+344.1。
(3E,5E)-3,5-二(苯甲叉)-1-环丁烷羰基氮杂环庚烷-4-酮(化合物#1521)。使氮杂环庚烷-4-酮盐酸盐(50mg,0.182mmol)、环丁酸(14μL,0.20mmol)、TBTU(58mg,0.182mmol)和TEA(37mg,0.364mmol)溶解于DMF(2mL)并在室温下搅拌过夜。添加盐水和乙酸乙酯,并使相分离。过滤后干燥有机相并蒸发溶剂。将组产物溶解于乙酸(2mL)和H2SO4(0.2mL)。添加苯醛(50μL)并使反应搅拌24小时。向该混合物添加甲醇和水,其通过制备型LC纯化。分离纯度为96%的黄色固体标题化合物。LCMS A:Rt2.68m/z[M+H]+372.1。
(3E,5E)-1-(2-环丙基乙酰基)-3,5-二[(4-甲氧基苯基)亚甲基]氮杂环庚烷-4-酮(化合物1526)。使氮杂环庚烷-4-酮盐酸盐(0.45g,3.0mmol)和4-甲氧基苯-醛(0.90g,6.6mmol)溶解于浓乙酸(10mL)。逐滴添加H2SO4(1mL),并使反应在室温下搅拌24小时。添加水(30mL)。滤出沉淀物并真空干燥过夜。使该粗制物质(30mg,0.107mmol)、环丙基乙酸(12mg,0.12mmol)、TBTU(41mg,0.13mmol)和TEA(26mg,0.26mmol)溶解于DMF(2mL)并在室温下搅拌过夜。添加甲醇(1.5mL)和水(0.5mL),并使产物通过制备型LC纯化以获得纯度为95%的固体产物。LCMS A:Rt2.51m/z[M+H]+432.2。
(3E,5E)-5-[(3-硝苯基)亚甲基]-3-(苯甲叉)氮杂环庚烷-4-酮(化合物#1560)。使N-boc-氮杂环庚烷-4-酮(0.10g,0.47mmol)和3-硝基苯醛(156mg,1.0mmol)溶解于乙酸(5mL),逐滴添加浓H2SO4(0.5mL)并使该反应在室温下搅拌4天。然后,添加更多浓H2SO4(0.5mL)和醛(156mg,1.0mmol)并在室温下持续搅拌三周。获得单缩合和双缩合产物的混合物。该混合物通过柱色谱(DCM/甲醇)纯化以获得棕色油状的中间体胺中间体2(19mg).使中间体2和苯醛一起溶解于乙酸(1.5mL)。添加浓H2SO4(0.05mL)并使反应在室温下搅拌过夜。然后添加更多H2SO4并持续搅拌一周。添加更多醛(156mg,1.0mmol)和H2SO4并另持续搅拌4天。浓缩反应混合物并通过制备型LC纯化以获得纯度为98%的所述产物的黄色固体TFA盐。LCMS系统A:Rt1.78m/z[M+H]+335.1,系统B:Rt2.43/2.28。
(3E,5E)-1-甲基-3,5-二[(4-硝苯基)亚甲基]氮杂环庚烷-4-酮(化合物#1563)。使N-甲基氮杂环庚烷-4-酮·HCl(50mg,0.30mmol)和4-硝基苯醛溶解与乙酸(5mL)并搅拌10分钟,然后缓慢添加浓H2SO4(50μL)并使该混合物在室温下搅拌过夜。添加更多浓H2SO4(100μL)并在室温下持续搅拌6小时。另添加500μL浓H2SO4并使该反应搅拌过夜。另添加350μL浓H2SO4并另搅拌5小时,在此过程中再分两次添加H2SO4(500μL和250μL)。然后添加水(3x反应体积)并使该混合物搅拌直至达到室温。该反应混合物用乙酸乙酯萃取(3x反应体积)。使相分离,并浓缩有机相以获得深黄色粘性油状物。粗产物通过制备型HPLC(XBridge柱;洗脱剂pH为10的50mM碳酸铵缓冲剂和甲醇)纯化,以获得黄色固体的标题产物(26.3mg)。LCMS系统A:Rt1.87m/z[M+H]+394.1,系统B:Rt2.57。
(3E,5E)-3,5-二[(4-氟苯基)亚甲基]-1-丙基氮杂环庚烷-4-酮(化合物#1574)。使氮杂环庚烷-4-酮盐酸盐(0.25g,1.68mmol)和4-氟苯醛(0.416g,3.36mmol)溶解于乙酸(20mL)并使该溶液搅拌10分钟,然后缓慢添加浓H2SO4(200μL)并使该溶液在室温下搅拌过夜。添加更多浓H2SO4(1mL)并在室温下搅拌。6小时后再添加mL浓H2SO4,并再次搅拌该反应过夜。第二天再添加800μL浓H2SO4并持续搅拌五天,在此过程中向所述反应混合物添加两部分H2SO4(1mL和0.5mL)。然后添加水(3x反应体积)并使该混合物搅拌直至达到室温。该反应混合物用乙酸乙酯萃取(10x反应体积)。蒸发浓缩有机相。向剩余物添加水。形成沉淀物并滤出。用水洗涤该固体并真空干燥以获得黄色固体中间体3。使其中一部分(15mg,0.05mmol)溶解于DCE-丙醛(4μL,0.06mmol)添加,并使该混合物在室温下搅拌15分钟。然后添加NaBH(OAc)3(15.7mg,0.07mmol)和乙酸(2.6μL,0.05mmol)并使该反应在室温下搅拌过夜。浓缩该反应并使粗产物通过制备型LC纯化,获得纯度为90%的产物(7.2mg)。LCMS系统A:Rt2.02m/z[M+H]+368.1,系统B:Rt3.21。
(3E,5E)-3-[(4-甲氧基苯基)亚甲基]-5-[(4-硝苯基)亚甲基]氮杂环庚烷-4-酮(化合物#1575)。使氮杂环庚烷-4-酮盐酸盐(0.25g,1.68mmol)和4-硝基苯-醛(253mg,1.68mmol)溶解于乙酸(20mL)并搅拌10分钟,然后缓慢添加浓H2SO4(1mL)并使该混合物在室温下搅拌8天。在第1-3天每天添加一份浓H2SO4(0.5mL,0.75mL和0.5mL)。添加水(2x反应体积)并用乙酸乙酯(2x反应体积)萃取该混合物。有机相通过蒸发浓缩并干燥以获得粗制中间体4。使一份中间体4(100mg,0.41mmol)溶解于乙酸(6mL)并搅拌10分钟,然后缓慢添加浓H2SO4(0.6mL)并使该反应在室温下搅拌6天。添加水之后,该产物沉淀为黄色固体。滤出该沉淀物,用水洗涤并真空干燥以获得纯度为98%的黄色固体标题化合物。LCMS系统A:Rt1.82m/z[M+H]+365.1,系统B:Rt2.41.1H-NMR(400MHz,CDCl3)[ppm]=2.97-2.99(m,2H),3.41-3.44(m,2H),3.83(bs,3H),4.28(s,2H),7.06-7.08(d,2H),7.47(s,1H),7.59-7.62(d,2H),7.76(s,1H),7.78-7.80(d,2H),8.27-8.29(d,2H)。
(3E,5E)-5-[(4-氟苯基)亚甲基]-3-[(4-甲氧基苯基)亚甲基]-1-甲基氮杂环庚烷-4-酮(化合物#1577)。使N-甲基氮杂环庚烷-4-酮盐酸盐(75mg,0.46mmol)和4-氟苯醛溶解于乙酸(7mL)并搅拌10分钟,然后缓慢添加浓H2SO4(350μL)并使该混合物在室温下搅拌8天。在第2~4天中添加更多浓H2SO4(分别为0.175mL、0.35mL、0.25mL)。添加水并用乙酸乙酯(两倍反应混合物体积)萃取该溶液。浓缩有机相以获得中间体5。使一部分该中间体(35mg,0.15mmol)和4-甲氧基苯醛(17μL,0.mmol)溶解于乙酸(2.5mL)并搅拌10分钟,然后缓慢添加浓H2SO4(0.20mL)并使该反应搅拌五天。添加水(2x反应体积)并用乙酸乙酯(2x反应体积)萃取该混合物。浓缩有机相并添加水。形成并滤出沉淀物以获得纯度为91%的黄色固体标题产物(11.2mg)。LCMS系统A:Rt1.86m/z[M+H]+352.1,系统B:Rt2.79。
(3E,5E)-1-乙酰基-5-[(4-氟苯基)亚甲基]-3-[(4-乙氧基苯基)亚甲基]氮杂环庚烷-4-酮(化合物#1579)。使氮杂环庚烷-4-酮盐酸盐(0.25g,1.68mmol)和4-氟-苯醛(179μL,1.68mmol)溶解于乙酸(20mL)并搅拌分钟,然后缓慢添加浓H2SO4(1mL)并使该混合物在室温下搅拌8天,在前三天添加浓H2SO4(分别为0.5mL、0.75mL和0.5mL)。添加水(2x反应体积)并用乙酸乙酯(2x混合物体积)萃取该混合物。浓缩并干燥有机相以获得粗制中间体6。使一份该中间体(100mg,0.46mmol)溶解于乙酸(6mL)并搅拌10分钟,然后缓慢添加浓H2SO4(0.6mL)并使该反应在室温下搅拌7天。添加水(1x体积)并用饱和水性NaHCO3中和该混合物。滤出形成的沉淀物,用水洗涤并真空干燥以获得纯度为91%的黄色固体中间体7(31.5mg)。LCMS系统A:Rt1.85m/z[M+H]+338。使中间体7(10mg)溶解于DCM(1mL)并添加TEA(5.0μL,0.04mmol)。使该混合物搅拌10分钟,然后添加乙酰氯(2.3μL,0.03mmol)并使该反应在室温下搅拌30分钟。该反应用水、饱和水性NaHCO3和盐水洗涤。浓缩有机相以获得纯度为90%的黄色固体标题化合物(6.4mg)。LCMS系统A:Rt2.35m/z[M+H]+380.1,系统B:Rt2.37.1H-NMR(400MHz,CDCl3):[ppm]=1.70,1.90,1.98和1.99(4x s,3H,CH3CO-,来自两个区域异构体及其乙酸盐旋转异构体的信号),2.89-3.01(m,2H),3.68-3.77(m,2H),3.79,3.79,3.79,3.08(4x s,3H,-OMe,来自两个区域异构体及其乙酸盐旋转异构体的信号),4.65-4.68(m,2H),7.0-7.04和7.098-7.103(2x m,2H),7.22-7.30(m,3H),7.48-7.62(m,5H)。
(3E,5E)-5-[(4-氯苯基)亚甲基]-3-[(4-硝苯基)亚甲基]氮杂环庚烷-4-酮(化合物#1583)。使N-甲基氮杂环庚烷-4-酮盐酸盐(75mg,0.46mmol)和4-氯苯醛(64mg,0.46mmol)溶解于乙酸(7mL)并搅拌10分钟,然后缓慢添加浓H2SO4(350μL)并使该混合物在室温下搅拌8天。在第2~4天中添加更多浓H2SO4(分别为0.175mL、0.35mL、0.25mL)。添加水(2x反应体积)并用乙酸乙酯(2x反应体积)萃取该溶液。浓缩有机相以获得中间体8。使一部分该中间体(35mg,0.14mmol)和4-硝基苯醛(69.5mg,0.46mmol)溶解于乙酸(2.5mL)并搅拌10分钟,然后缓慢添加浓H2SO4(200μL)并使该混合物在室温下搅拌5天。添加更多浓H2SO4(0.2mL),并再持续搅拌5天。添加水(2x反应体积)并用乙酸乙酯(2x反应体积)萃取该溶液。浓缩有机相,并通过制备型LC纯化剩余物以获得纯度为94%的黄色固体标题化合物(1.8mg)。LCMS系统A:Rt1.98/2.04m/z[M+H]+383.1,系统B:Rt2.82/2.98。
缩写
Boc 叔丁基氧基羰基
ACN 乙腈
DCM 二氯甲烷
TFA 三氟乙酸
DMF 二甲基甲酰胺
TEA 三乙胺
Rt保留时间
TBTU O-(苯并三唑-1-基)-N,N,N',N'-四甲基脲四氟硼酸酯)
rt 室温
LC 液体色谱
EDC 1-乙基-3-[3-二甲基氨基丙基]碳二亚胺
HATU2-(1H-7-氮杂苯并三唑-1-基)--1,1,3,3-四甲基脲六氟磷酸酯;
DCE 1,2-二氯乙烷
实施例3.药物组合物A(水性悬浮液)。使本发明化合物(25mg)溶解于1ml二甲亚砜。向10mL剧烈搅拌的盐水逐滴添加该溶液。形成的悬液可用于肌肉内、静脉内或皮下给予,所述悬液可通过添加1%(以PVP重量计)来稳定。
实施例4.药物组合物B(片剂)。用于经口给予的片剂如下制备:掺混2.0g本发明化合物(粉末,<10mμ,90%)和微晶纤维素(1.30g)、谷物淀粉(0.50)g、硅石(0.20)g、硬脂酸镁(0.12mg)。将该混合物干燥压制成400mg片剂,其被覆糖衣。
实施例5.药物组合物C(溶液)。使本发明化合物(10mg)溶解于0.5ml的Cremophor EL(巴斯夫公司(BASF))并添加无水乙醇至1.0ml。将澄清溶液装填至玻璃小瓶以供注射。
实施例6.药物组合物D(溶液)。为了研究水性组合物在动物中的腹膜内给予,通过以下方式制备储液:在室温下将本发明化合物以2mg/mL的浓度溶解于Chremaphor EL/聚乙二醇4001:1.(v/v)中或通过超声下升温至约80℃。一等分储液用0.9%盐水1:10稀释并立即用于IP注射。
实施例7.药物组合物E(溶液)。就腹膜内给予而言,通过以下方式制备25%(以重量计)Kolliphor HS15储液:通过使整个容器的Kolliphor HS15(西格玛(Sigma)42966))升温至60℃熔化并用蒸馏水稀释至25%w/w。向内含化合物#1570(18.0mg)的10mL样品管添加10.0mL所述储液,并涡旋该管,用超声在约50℃处理2小时(有时加热至约83℃)。注射之前使获得的澄清溶液无菌过滤通过0.2μm纤维素注射器滤器。通过相同方法制备化合物#1546和#1571的溶液;然而这些化合物并未完全溶解。对未溶解的剩余物称重,并将该重量从化合物起始重量(18mg)扣除。发现所述制备的溶液(10ml)分别包含8.5mg和11.0mg的化合物#1546和#1571。
实施例8.药物组合物F(溶液)。就腹膜内给予而言,通过以下方法制备2-羟丙基-β-环糊精(奥德里奇(Aldrich)332593)储液:使环糊精以30%w/w的浓度溶解于蒸馏水。向内含化合物#1649(15.0mg)的10mL样品管添加10.0mL该储液。该管经涡旋,在约50℃用超声处理约2小时(有时加热至约83℃)。在注射前使所获溶液通过0.2μm纤维素注射器滤器无菌过滤。确定未溶解的剩余化合物#1659的重量,用于将过滤溶液的浓度校正为预计浓度的82.5%。通过相同方法制备化合物#1546的溶液。
实施例9.本发明的化合物诱导对蛋白酶体的抑制。采用报告细胞系MelJuSoUb-YFP来评价化合物,该细胞系经工程改造以使黄色荧光蛋白(YFP)在蛋白酶体抑制后累积(12)。在IncuCyte-FLR系统(英国埃森的埃森生物科学公司(EssenBioscience))内检测YFP累积48小时,该系统是自动化荧光显微镜。采用阳性细胞数量/视野作为蛋白酶体抑制的度量。
实施例10.本发明化合物在水性介质中的溶解度的测定在图2a~2e的图表中,溶解度被表示为Log S(mmol/ml;软件ACD/实验室公司(ACD/Labs Inc.))。在25℃下的不同pH值的水性缓冲液中测定溶解度并预测在纯水中的溶解度。该算法采用>6,800种化合物的组作为参考。该图表显示,相较于对应取代的哌啶-4-酮而言,本发明的氮杂环庚烷酮在生理pH(例如pH6~8,特别是7.0~7.5)下的水性介质中的溶解度显著提高,例如提高至2倍或更多。
实施例11.本发明的氮杂环庚烷/氮杂环庚烷酮显示细胞毒性高于结构上对应的哌啶/哌啶-4-酮
图3b、3d、3f的图表表明带有7元环部分的本发明的编号1546、1547和1570的化合物的细胞毒性和结构上相对应的具有6元环部分的本发明以外的化合物的细胞毒性的比较。在使报告细胞系HCT-116持续接触化合物72小时后测定其诱导的剂量依赖性细胞毒性。经处理的细胞与未经处理的对照相比较。细胞毒性用存活指数(SI)进行观察,约90%~约0%之间的存活指数取决于化合物浓度。如图所示,本发明化合物比参比化合物更具细胞毒性,因为其在较低浓度产生相同水平的细胞毒性。
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Claims (26)
1.通式S-1的化合物,所述化合物能够消除19S RP DUB的去泛素化(DUB)活性,
其中:
双键d1处的R1、R2和双键d2处的R3、R4可以彼此独立地具有与式S1相反的构型,
X是CO、CS、CH2、CHC1-6-烷基、NH或NC1-6-烷基;
R1和R3彼此独立地是H或C1-6-烷基;
R2和R4彼此独立地是H;C1-6-烷基;C1-5-烷基CO;苯基或6元杂芳基,其任选地被以下1~3种基团取代:C1-6-烷基、C1-6-烷氧基、CN、-COOC1-6-烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8,前提条件是烷基和烷氧基中的一个或多个H可被氟取代;
R5是如下任何一种基团:H;C1-6-烷基;C2-6-烯基;C1-3-烷氧基-C2-6-烷基-;C1-3-烷氧基-C2-6-烯基-;芳基-C0-6-烷基-;杂芳基-C0-6-烷基-;杂环基-C0-6-烷基-;环烷基-C0-6-烷基-;-C1-6-烷基-COOC1-6-烷基;-C2-6-烷基-芳氧基;COR6;
R6是如下任何一种基团:C1-6-烷基;C2-6-烯基;C1-6-烷氧基;C1-3-烷氧基-C1-6-烷基-;C1-3-烷氧基-C1-6-烯基-;芳基-C0-6-烷基-;杂芳基-C0-6-烷基-;杂环基-C0-6-烷基-;环烷基-C0-6-烷基-;-C1-6-烷基-COOC1-6-烷基;NH2;-NHC1-6-烷基;-N(C1-6-烷基)2;-C0-6-烷基-芳氧基;
R7、R8彼此独立地是H或C1-C3-烷基。
2.如权利要求1所述的化合物,其特征在于,X=CO。
3.如权利要求1或2所述的化合物,其特征在于,R1和R3均为H。
4.如权利要求1~3中任一项所述的化合物,其特征在于,R2和R4彼此独立地是,苯基或6元杂芳基,特别是苯基,其任选地被选自下组的1~3种基团取代:C1-6-烷基、C1-6-烷氧基、CN、-COOC1-6-烷基、COOH、NO2、F、Cl、Br、I、CF3、NH2、NHC1-6-烷基、N(C1-6-烷基)2、CONR7R8。
5.如权利要求4所述的化合物,其特征在于,苯基的取代在第3、4、5位中的一个或多个位置发生。
6.如权利要求1所述的化合物,其特征在于,在双链d1处的R1、R2和在双链d2处的R3、R4具有式S-1的构型,
X是CO、CS、CH2、CHC1-6-烷基、NH或NC1-6-烷基;
R1和R3彼此独立地是H或C1-6-烷基;
R2和R4彼此独立地是H;C1-6-烷基;C1-5-烷基CO;苯基或6元杂芳基,其被选自下组的1~3种基团取代:CN、NO2、F、Cl、Br、I、NH2、NHC1-6-烷基、N(C1-6-烷基)2、COC1-6-烷基;
R5是H、C1-6-烷基、C2-6-烯基、C1-3-烷氧基-C1-6-烷基、C1-3-烷氧基-C1-6-烯基、芳基、杂芳基、杂环基、C1-6-烷基-杂芳基、C1-6-烷基-杂环基、C1-6-烷基-环烷基、C1-6-烷基-芳基、CO-C1-6-烷基、CO-乙烯基、CO-烯丙基、CO-芳基、CO-环烷基。
7.如权利要求6所述的化合物,其特征在于,X是CO。
8.如权利要求6或7所述的化合物,其特征在于,R2和R4是经取代的苯基。
9.如权利要求6~8中任一项所述的化合物,其特征在于,R5选自CO-C1-6-烷基、CO-环烷基、CO-乙烯基、CO-烯丙基。
10.一种治疗人的癌肿瘤的方法,所述癌肿瘤采用硼替佐米或与硼替佐米具有相同凋亡产生活性的试剂或任何其他抗癌药物难以治疗,所述方法包括:给予药学上可接受运载体中的药理学有效剂量的权利要求1~9中任一项所述的化合物。
11.如权利要求10所述的方法,其特征在于,所述肿瘤的细胞因内源性凋亡抑制剂Bcl-2的过表达而难以治疗。
12.如权利要求10或11所述的方法,其特征在于,19S RP DUB包含UCHL5和USP14。
13.如权利要求10~12所述的方法,其特征在于,非蛋白酶体DUB的去泛素化(DUB)活性不受影响。
14.如权利要求10~13中任一项所述的方法,其特征在于,使所述化合物溶解或悬浮于液体运载体中。
15.如权利要求10~14所述的方法,其特征在于,通过静脉内、肌肉内、腹膜内或皮下注射或输注给予。
16.如权利要求10所述的方法,其特征在于,给予是经口给予。
17.如权利要求16所述的方法,其特征在于,所述运载体是片剂或胶囊。
18.如权利要求10~17中任一项所述的方法,其特征在于,所述药理学有效剂量是0.0001g/kg~0.1g/kg体重,特别是0.001g/kg~0.01g/kg体重,鉴于所述试剂是全身性给予还是局部给予。
19.如权利要求10~18中任一项所述的方法,其特征在于,“难以治疗”表示用单一剂量的硼替佐米或与硼替佐米具有相同凋亡产生活性的试剂或任何其他抗癌药物治疗癌症不实质性降低在即将进行所述治疗前观察到的所述癌症的每月生长率,诸如降低不多于25%或不多于10%或不多于5%。
20.如权利要求10~19中任一项所述的方法,其特征在于,所述癌症选自多发性骨髓瘤、乳腺癌、卵巢癌、肺癌、结肠癌、前列腺癌、胰腺癌。
21.如权利要求10~20中任一项所述的方法,其特征在于,所述方法包括选择待治疗的人,所述选择如下进行:在给予硼替佐米或与硼替佐米具有相同去泛素化活性抑制机制的所述活性物质或所述其它抗癌药物之前或之时测定所述癌症的生长率,将正生长率,特别是每月超过5%或超过10%或超过25%的生长率设为选择标志。
22.一种包含如权利要求1~9中任一项所述的化合物和药学上可接受运载体的药物组合物。
23.如权利要求22所述的组合物,其特征在于,所述组合物是片剂或胶囊或经口给予的其它单剂量制剂形式。
24.如权利要求22所述的组合物,其特征在于,所述组合物是用于注射或输注的药学上可接受的液体运载体中的溶液或悬浮液。
25.如权利要求21所述的组合物,其特征在于,所述组合物用于静脉内、肌内、腹膜内或皮下输注或注射。
26.如权利要求1~9中任一项所述的化合物或如权利要求22~25中任一项所述的药物组合物用于治疗化疗难以治疗的人的癌症的应用。
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