CN101410108B - 用于治疗特定耐药性肿瘤的激酶抑制剂的应用 - Google Patents
用于治疗特定耐药性肿瘤的激酶抑制剂的应用 Download PDFInfo
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Abstract
本发明提供低分子量化合物,即四氢吡咯并[3,4-c]吡唑,所述化合物显示与ABL酪氨酸激酶的ATP袋有高度亲和性。因此,这些化合物是ATP竞争性的酪氨酸激酶抑制剂,特别地对于耐BCR-ABL抑制剂的T315I ABL突变体也显示出显著的抑制能力。发现本发明所述化合物在治疗耐BCR-ABL抑制剂的ABL介导的疾病方面的有益应用,例如耐伊马替尼-的慢性髓性白血病。此外,本发明提供了用于鉴定化合物的筛选方法,所述化合物能够结合激酶蛋白的ATP袋,特别是T315I突变的ABL激酶的ATP袋。
Description
技术领域
本发明涉及通过应用ATP竞争性小分子激酶抑制剂的突变ABL介导的疾病的治疗。
背景技术
慢性髓性白血病(CML)是一种血液障碍,大约占成年人白血病的15%,其特征是髓细胞系恶性扩张。CML的遗传特征是在染色体9和22间相互易位,结果形成了所谓的费城(Ph)染色体。这种染色体间交换的分子结果是产生嵌合基因BCR-ABL,所述基因编码酪氨酸激酶多肽,其中所述酪氨酸激酶的结构域是组成性激活的。对于CML细胞的转化表型已经显示,此融合蛋白标记物的表达是必要的和充分的。(FADERL,S,et al.The biology of chronic myeloid leukemia.NewEngland Journal of Medicine.1999,vol.341,no.3,p.164-172.SAWYERS,C.Chronic myeloid leukemia.New England Journal ofMedicine.1999,vol.340,no.17,p.1330-1340.)。除了CML之外,在多达20%的成人成淋巴细胞性白血病(ALL)患者中检测到异常的ABL激酶活性,所述异常的ABL激酶活性由Ph染色体易位引起(OTTMANN,O.G.,et al.A phase 2 study of imatinib in patients withrelapsed or refractory Philadelphia chromosome-positive acutelymphoid leukemias.Blood.2002,vol.100,no.6,p.1965-1971.)。
ABL转换来自细胞表面生长因子受体和粘附受体的信号以调节细胞骨架结构。已经显示许多信号发送蛋白与ABL相互作用,所述ABL活化一系列信号通路。因此,抑制ABL构成了一个改善CML患者治疗的新方法。
BCR-ABL是CML的发病机制所必需的,而且ABL的酪氨酸激酶活 性是BCR-ABL介导的转化所必不可少的,上述发现使得ABL激酶成为用于治疗性干预有吸引力的靶点。伊马替尼(Gleevec,也称为STI571,EP564409A)是BCR-ABL的ATP竞争性抑制剂,所述BCR-ABL能有效抑制其酪氨酸激酶的活性。伊马替尼的高度选择性和有效性归因于它在所述酶的催化的失活构象中结合和阻断BCR-ABL的能力。临床前和临床试验已经显示伊马替尼的显著有效性和高度耐受性,所述伊马替尼现在是用于所有新近诊断为CML的患者的第一线治疗药物(DEININGER,M,et al.The development of imatinib as atherapeutic agent for chronic myeloid leukemia.Blood.2005,vol.105,no.7,p.2640-2653.)。但是,伊马替尼对疾病的早期阶段非常有效,而在CML加速和急变期的患者和那些费城染色体阳性(ph+A11)的急性淋巴细胞白血病的患者中经常形成伊马替尼耐药性(DRUKER,B.J.,et al.Activity of a specific inhibitor of theBCR-ABL tyrosine kinase in the blast crisis of chronic myeloidleukemia and acute lymphoblastic leukemia with the Philadelphiachromosome.New England Journal of Medicine.2001,vol.344,no.14,p.1038-1042.SAWYERS,C.L.,et al.Imatinib induceshematologic and cytogenetic responses in patients with chronicmyelogenous leukemia in myeloid blast crisis:results of a phaseII study.Blood.2002,vol.99,no.10,p.3530-3539.)。伊马替尼耐药性的主要机制是克隆的进化,所述克隆在BCR-ABL的酪氨酸激酶催化结构域内获得了点突变,并直接阻止或削弱了与抑制剂的相互作用。某些突变,例如苏氨酸315到异亮氨酸置换(以下称作T315I)的在ATP结合袋直接干扰伊马替尼的相互作用,而它们中的大多数诱导结构改变,所述结构改变阻止ABL激酶结构域采取伊马替尼结合所必需的闭合、失活的构象(SHAH,N.P.,et al.Mechanisms ofresistance to STI571 in Philadelphia chromosome-associatedleukemias.Oncogene.2003,vol.22,no.47,p.7389-7395.)。
对伊马替尼耐药性的分子基础的了解已经刺激寻找新的BCR-ABL 抑制剂,所述抑制剂可以有效对抗临床上可见的耐伊马替尼ABL介导的疾病。许多第二代BCR-ABL的ATP竞争性的抑制剂已经研制出来,例如AMN107(WEISBERG,E,et al.Characterization of AMN107,aselective inhibitor of native and mutant Bcr-Abl.Cancer cell.2005,vol.7,p.129-141.)(?)和BMS-354825(SHAH,N.P.,et al.Overriding imatinib resistance with a novel ABL kinaseinhibitor.Science.2004,vol.305,no.5682,p.399-401.),而且正在对这些抑制剂进行早期临床试验评估。这些化合物有效对抗大多数伊马替尼耐药性ABL突变体,但是它们无一能有效对抗所述T315I突变体,所述T315I突变体是在伊马替尼耐药性患者中出现的第二常见的突变(O′HARE,T,et al.In vitro activity of Bcr-Ablinhibitors AMN107 and BMS-354825 against clinically relevantimatinib-resistant Abl kinase domain mutants.Cancer Research.2005,vol.65,no.11,p.4500-4505.)。可以得出结论,即通过ATP竞争性化合物抑制T315I ABL的突变异常困难。实际上,直到最近,已经报道的也有效对抗T315I突变体的唯一的抑制剂是ON12380,所述ON12380实际是一个底物竞争性(即非ATP竞争性的)抑制剂(GUMIREDDY,K.,et al.A non-ATP-competitive inhibitor ofBCR-ABL overrides imatinib resistance.Proc.Natl.Acad.Sci.U.S.A..2005,vol.102,no.6,p.1992-1997.)。
最近,已经报道两个ATP-竞争性分子,VX-680(HARRINGTON,E.A.,et al.VX-680,a potent and selective small-molecule inhibitorof the Aurora kinases,suppresses tumor growth in vivo.NatureMedicine.2004,vol.10,no.3,p.262-267)和BIRB-796(PARGELLIS,C.,et al.Inhibition of p38MAP kinase by utilizing a novelallosteric binding site.Nat.Struct.Biol..2002,vol.9,vo.4,p.268-272.)能在体外以低纳摩尔Kd结合ABL的T315I突变的酪氨酸激酶结构域,但是在抑制细胞中T315I BCR-ABL方面显示较低的能力(CARTER,T.A.,et al.Inhibition of drug-resistant mutants of ABL,KIT,and EGF receptor kinases.Proc.Natl.Acad.Sci.U.S.A..2005,vol.102,no,31,p.11011-11016)。
目前,对于受耐BCR-ABL抑制剂的T315I ABL介导的疾病侵袭的患者没有有效的激酶靶点治疗,所述疾病估计占所有用伊马替尼治疗后复发患者的20%,而且预计将被选择用于用上述下一代抑制剂进行治疗。其代表了对BCR-ABL的T315I突变体的新的有效ATP竞争性抑制剂的未被满足的医药需求。本发明致力于这个问题。
发明概述
本发明提供低分子量化合物,所述化合物显示对ABL酪氨酸激酶的ATP袋有高度亲和性。因此,这些化合物是ATP竞争性的酪氨酸激酶抑制剂,显示出对于耐BCR-ABL抑制剂的T315I ABL突变体、特别是对于耐伊马替尼的T315I ABL突变体的显著抑制能力。对酪氨酸激酶活性的抑制导致由ABL和所述T315I突变体介导的信号级联发生阻塞。
显示出令人期望活性的本发明化合物是四氢吡咯并[3,4-c]吡唑,所述化合物被设计为以蛋白激酶ATP袋为靶点。这个化学类的化合物已经显示是Aurora激酶的有效ATP-竞争性抑制剂(FANCELLI,D.,etal.Potent and selective Aurora inhibitors identified by theexpansion of a novel scaffold for protein kinase inhibition.Journal of Medicinal Chemistry.2005,vol.48,no.8,p.3080-3084.PCT/WO2005005427)。意外地,已经发现同样化学骨架的化合物对ABL显示出显著的抑制能力,并且特别地,它们在体外也能抑制ABL的耐BCR-ABL抑制剂的T315I突变体形式。
考虑到它们的生物学活性,本发明的化合物提供了一条开发用于耐伊马替尼的正遭受ABL介导的疾病的患者群体治疗药物的新途径。
本发明也提供用于鉴定其它化合物的试验,所述化合物能结合靶蛋白激酶、特别是ABL酪氨酸激酶的ATP袋。该试验依赖检测结合到靶ATP袋的结果的能力,而且其在定量条件下进行以便能测定化合物 与ATP袋的结合亲和力。
发明详述
在第一方面,本发明涉及用于抑制耐BCR-ABL抑制剂的酪氨酸激酶活性的方法,所述方法包括将耐BCR-ABL抑制剂的酪氨酸激酶多肽与有效量的式(I)化合物接触,
其中R是氢或甲基,
R1是羟基或直链或支链C1-C3烷基或烷氧基,
R2是氢或卤素原子,
X是二价基团,选自亚甲基(-CH2-)或氟亚甲基(-CHF-),或它是杂原子或杂原子基团,选自氧(-O-)或氮(-NR′-),其中R′是氢原子,直链或支链C1-C4烷基或C3-C6环烷基,
或其可药用盐。
本发明式(I)化合物具有非对称的碳原子,因此可以以单独的旋光异构体、消旋混合物或任意其它混合物形式存在,包括大部分的两个旋光异构体之一,以上所有都意在本发明的范围之内。
当化合物可能以互变异构形式存在时,不论处于平衡或主要以一种形式存在,预期本发明包括每一种形式。
同样地,除非另有提供,当仅指明有下列式(Ia)或(Ib)的互变异构形式之一时,余下的另一个仍意在包含于本发明的范围之内:
在本说明书中,除非另有说明,关于术语直链或支链C1-C3或C1-C4烷基,我们意指任意的所述基团,例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基和仲丁基。
关于术语直链或支链C1-C3烷氧基,我们意指任意的所述基团,例如甲氧基、乙氧基、正丙氧基和异丙氧基。
关于术语卤素原子,我们意指氟、氯、溴或碘原子。
关于术语C3-C6环烷基,我们意指任意基团,例如环丙基、环丁基、环戊基和环己基。
显然,取决于X基团的性质,此连接到式(I)化合物的亚苯基部分的相同的杂环可以表示哌啶基,4-氟哌啶基、哌嗪基、4-烷基-哌嗪基、4-环烷基-哌嗪基或吗啉代环。
在国际专利申请WO2005/005427中描述和要求保护的式(I)化合物,所述化合物可以根据其中公开的步骤制备。
可以在体外或体内进行本发明的方法。可以应用全长酪氨酸激酶多肽;可选择地,使用其片段、部分或类似物。
更详细地,通过凝胶激酶分析测定本发明所选化合物的抑制活性。所述分析包括,在测试化合物存在下,将通过ABL激酶放射性标记的磷酸盐部分转移到底物。一旦反应中止,将反应混合物加载在聚丙烯酰胺凝胶上,检测放射性信号。在实施例部分将更好地说明抑制分析。
此外,在细胞系统内,测定本发明所选的化合物的抑制活性。合适的细胞是那些,已经天然出现BCR-ABL染色体易位的细胞,例如人白血病细胞K-562和转染携带T315I ABL突变基因的重组构建体的细胞。用本发明的化合物处理细胞,并通过免疫印迹法检测抑制活性,正如在实施例部分更好说明的。
在本发明的优选的实施方案中,所述耐BCR-ABL抑制剂的酪氨酸激酶多肽是ABL酪氨酸激酶的T315I突变体。
在本发明的更优选的实施方案中,所述BCR-ABL抑制剂是伊马替尼。
在第二个方面,本发明涉及用于治疗耐BCR-ABL抑制剂的T315IABL介导的疾病的方法,包括给予有此需要的哺乳动物有效量的如上述定义的式(I)化合物。
在本发明中要求保护的治疗方法范围内的是所有可能的异构体和它们的混合物以及式(I)化合物的代谢物和可药用生物前体(另称作前药)的应用。根据式(I),前药在体内是任意的共价结合化合物,其释放活性母药。
本发明的上述两个方法优选通过式(I)化合物进行,其中R是氢,R1是甲氧基,R2是氢和X是亚甲基(-CH2-)或是杂原子或杂原子基团,选自氧(-O-)或氮(-NR′-),其中R′是氢原子或烷基,所述烷基选自甲基、乙基、异丙基、环丙基或叔丁基。
更为优选地,所述化合物选自:
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-异丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-环丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-哌啶-1-基苯甲酰胺;
4-(4-氟哌啶-1-基)-N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并 [3,4-c]吡唑-3-基}-4-吗啉-4-基苯甲酰胺;
N-{5-[(2R)-2-甲基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-哌啶-1-基苯甲酰胺;
4-(4-氟哌啶-1-基)-N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺;
4-吗啉-4-基-N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺。
更为优选地是由以下化合物组成的亚组:
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-异丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-环丙基哌嗪-1-基)苯甲酰胺。
应用在本发明上述两个方法中最为优选的化合物的是N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺,在下文称为“化合物1”。
式(I)化合物的可药用盐包括与无机酸或有机酸形成的酸加成盐,例如,硝酸、盐酸、氢溴酸、硫酸、高氯酸、磷酸、醋酸、三氟乙酸、丙酸、乙醇酸、乳酸、草酸、丙二酸、苹果酸、马来酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、羟乙磺酸和水杨酸。
术语“T315I ABL介导的疾病”意指包括其中需要直接或间接抑制T315I ABL酪氨酸激酶活性的疾病。
术语“BCR-ABL抑制剂”意指包括具有抑制BCR-ABL蛋白的酶活性能力的ATP-竞争性分子。这样的分子的非限制实例是伊马替尼、Desatinib和AMN107。
在上述方法的优选实施方案中,所述疾病是耐BCR-ABL抑制剂的白血病,所述白血病由ABL酪氨酸激酶的T315I突变体引起。在更为优选的实施方案中,所述白血病是慢性髓性白血病。
在上述方法的另一个优选实施方案中,所述BCR-ABL抑制剂是伊马替尼。
在确定受试者具有某种疾病或不希望的状况后,可以给受试者服用根据式(I)的化合物,所述疾病和状况可以通过用所述化合物治疗而获益。医护和临床人员可以作出决定作为受试者的疾病或状况诊断的一部分。也可应用化合物预防这样的状况,其可被视为能降低受试者出现一种或更多所述状况的概率。
将细胞与式(I)化合物接触来进行本发明的方法,所述化合物抑制一种或多种T315IABL突变体的活性。可以在体外(即用化合物培养所述细胞)或可选择地,在体内(即通过给予受试者化合物)实施本方法。本方法也可离体进行,如在来源于受试者的细胞和在其返回受试者体内后体外治疗的情况下。
在体内,在合适的动物模型中可以评价靶蛋白抑制治疗组合物的作用。
如在此处所应用的,化合物的有效量是指足以达到其预期目标的量。在完成预期效应的基础之上,确定所述有效量完全在本领域技术人员能力范围之内。有效剂量所取决的因素包括但不限于,受试者的大小和/或受试者遭受疾病或不希望的状况的进展程度。所述有效量还将取决于化合物是以单一剂量还是随时间周期的方式给予受试者。
本发明的式(I)的化合物目的是用于受试者治疗。如在此处所应用地,术语“受试者”包括哺乳动物和非哺乳动物。哺乳动物的实施例包括但不限于,哺乳动物类的任何成员:人类、非人类,例如黑猩 猩和其它猿和猴类;耕作动物例如牛、马、绵羊、山羊、猪;家畜例如兔、狗和猫;实验动物例如啮齿类动物,例如大鼠、小鼠和豚鼠等。非哺乳动物的实施例包括但不限于,鸟、鱼等。
如在此处所应用的术语“治疗”包括获得治疗的益处。所谓治疗益处是指根除或改善正在治疗的潜在的障碍。例如,对于癌症患者,治疗益处包括根除或改善潜在的癌症。同样地,尽管事实上患者可能仍然受到潜在障碍的折磨,但是通过根除或改善与潜在障碍相关的一种或多种生理症状使患者获得治疗益处,从而在患者体内能观察到这种改善。
本发明的方法包括将式(I)化合物以单个药剂或可选择地,一种或多种其它分子或其它药剂的组合的方式服用,所述其它药剂适合于在此处公开的疾病和不希望的状况的治疗。适用于组合治疗的举例分子和药剂是细胞生长抑制和细胞毒性剂、抗菌剂、烷化剂、抗代谢剂、激素类药剂、免疫类药剂、干扰素类药剂、环加氧酶抑制剂(例如COX-2抑制剂)、基质金属蛋白酶抑制剂、端粒酶抑制剂、酪氨酸激酶抑制剂、抗生长因子受体药剂、抗-HER药剂、抗-EGFR药剂、抗血管生成剂(例如血管生成抑制剂)、法尼基转移酶抑制剂、ras-raf信号传导通道抑制剂、细胞周期抑制剂、其它细胞周期蛋白依赖性激酶抑制剂、微管蛋白结合剂、拓扑异构酶I抑制剂、拓扑异构酶II抑制剂等。
本发明也包括药物组合物,所述药物组合物包括与可药用的赋形剂相结合的式(I)的化合物或其可药用的盐,所述赋形剂可以是一个载体或稀释剂。
包含本发明化合物的药物组合物通常以下述的常规方法制备,并以适合的药物形式给药。
例如,固体口服形式包含与活性化合物一起的稀释剂、润滑剂、粘合剂、崩解剂、泡腾混合物、着色剂、增甜剂、湿润剂以及一般而言用于药物配方的无毒性的药理学无活性的物质,所述稀释剂例如为乳糖、右旋糖、蔗糖(saccharose)、蔗糖(sucrose)、纤维素、玉米淀粉、马铃薯淀粉;所述润滑剂例如为二氧化硅、滑石、硬脂酸、 镁或硬脂酸钙、和/或聚乙二醇;所述粘合剂例如为淀粉、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素或聚乙烯吡咯烷酮;所述崩解剂例如为淀粉、海藻酸、藻酸盐或淀粉羟乙酸钠;所述润湿剂例如为卵磷脂、聚山梨醇酯、十二烷基硫酸盐。这些药物制剂可通过公知的方法生产,例如通过包括混合、粒化、压片、包糖衣或薄膜包衣的过程生产。
用于口服给药的液体分散剂可以是,例如糖浆剂、乳剂或悬浮液。
作为载体的实施例,糖浆剂可以包含蔗糖或含甘油和/或甘露醇和山梨醇的蔗糖。
作为载体的实施例,悬浮液和乳剂可以包含天然树胶、琼脂、海藻酸钠、果胶、甲基纤维素、羧甲基纤维素或聚乙烯醇。
用于肌内注射的悬浮液或溶液可以包含与活性化合物一起的可药用载体,例如无菌水、橄榄油、油酸乙酯、二醇类,例如丙二醇,如果需要,合适量的盐酸利多卡因。
作为载体,用于静脉内注射或输注的溶液可以包含无菌水或优选地所述溶液以无菌、水性、等渗盐溶液的形式存在,或其可以包含作为载体的丙二醇。静脉输注可以按照从大约1小时到大约10小时的时间间隔进行实施,并且按照周治疗方案持续1到6周的周期。
栓剂可以包含与活性化合物一起的可药用载体,例如可可脂、聚乙二醇、聚氧乙烯脱水山梨醇脂肪酸酯表面活性剂或卵磷脂。
适用于本发明用途的药物组合物包括其中的活性成分以有效量存在的组合物,即在接受治疗的病症中可以有效获得治疗和/或预防益处的量。用于具体应用的实际有效量取决于病症或接受治疗的病症、受试者的状况、配方和给药方式、以及本领域技术人员所公知的其它因素。根据在此的公开,确定本发明化合物的有效量完全在本领域技术人员能力范围之内,而且可以通过常规优化技术确定有效量。
在治疗应用方面,本发明的化合物按照每天从大约30mg/m2-大约3000mg/m2的体表面积的剂量水平对受试者给药。特别合适的范围包括从大约100mg/m2到1000mg/m2的剂量水平。对于成年人受试者来说, 作为非限制实例的单次剂量从大约50mg到大约5000mg,更为优选地从大约150mg到大约1000mg,每天服用1到5次。根据需要,可以应用高于或低于那些在此公开的剂量。但是,这样的剂量可以根据许多变量改变,所述变量不限于所应用化合物的活性、待治疗的情况、给药方式、治疗方案、个体受试者的需求、接受治疗的病症的严重性以及医师的判断。由于就个体给药方案而言,其变量数目大而且经常相当大地偏离推荐值,所以上述范围仅仅是建议性的。
可以通过动物模型确定人用的有效量。例如,可以配制的人用剂量达到已经在动物体内证明有效的循环、肝、局部和/或胃肠道浓度。
在第三个方面,本发明涉及用于鉴定能结合激酶蛋白的ATP袋的化合物的筛选方法,其包括以下步骤:提供包含所述激酶蛋白和二氢吲哚酮衍生物的反应混合物,所述二氢吲哚酮衍生物对于所述激酶蛋白的ATP袋有亲和力且在结合所述ATP袋时能产生荧光信号;系列稀释所述测试化合物,并比较在测试化合物不存在和以不同浓度存在的情况下产生的荧光信号;由此降低的荧光水平表明测试化合物置换所述二氢吲哚酮衍生物的能力。
在所述筛选方法的优选的实施方案中,所述激酶蛋白和二氢吲哚酮衍生物是预混的。
在所述筛选方法的另一个优选的实施方案中,所述激酶蛋白和测试化合物是预混的。
在所述筛选方法进一步的优选实施方案中,所述激酶蛋白是ABL激酶。在更为优选地实施方案中,所述ABL激酶是T315I突变体ABL激酶。
术语“激酶蛋白”包括天然蛋白全长和其片断,在所述片断范围之内包括所述激酶蛋白的ATP袋。
术语“ATP袋”指在所述激酶蛋白内的ATP结合位点。
优选地,二氢吲哚酮衍生物是以下式(II)化合物
其中R1是氢或甲氨基-磺酰基或苄基-磺酰基,R2是氢或甲基,R3是甲基或4-氯苯基或2,4-二氟苯基。
更为优选地,所述二氢吲哚酮衍生物是选自下列的化合物:2,4-二甲基-5-[2-氧代-5-苯甲磺酰基-1,2-二氢-吲哚-(3Z)-亚基甲基]-1H-吡咯-3-甲酸(2-二乙氨基-乙基)-酰胺(已经在国际专利申请WO02/096361公开);4-(4-氯苯基)-N-[2-(二乙氨基)乙基]-2-甲基-5-((Z)-{5-[(甲氨基)磺酰基]-2-氧代-1,2-二氢-3H-吲哚-3-亚基}甲基)-1H-吡咯-3-甲酰胺;N-[2-(二乙氨基)乙基]-4-(2,4二氟苯基)-2-甲基-5-((Z)-{5-[(甲氨基)磺酰基]-2-氧代-1,2-二氢-3H-吲哚-3-亚基}甲基)-1H-吡咯-3-甲酰胺;4-(4-氯苯基)-N-[2-(二乙氨基)乙基]-2-甲基-5-[(Z)-(4-甲基-2-氧代-1,2-二氢-3H-吲哚-3-亚基)甲基]-1H-吡咯-3-甲酰胺。
通过二氢吲哚酮衍生物与ABL的野生型和突变型的ATP袋的结合能力,与激酶蛋白的探针的亲和力,试验的可靠性并且与公知的ABL抑制剂(例如:伊马替尼)发表的数据相一致与否来选择二氢吲哚酮衍生物。
荧光信号可以通过例如一个平板读取器、如Fusion α-FP HT平板读取器(Packard)进行检测。测试化合物的置换能力与化合物对激酶蛋白的ATP袋的亲和力直接相关。如实施例部分说明,可以测定测试化合物的亲和结合常数(KD)。
本发明的试验是建立在新发现的基础之上,所述新发现是在二氢吲哚酮衍生物结合到激酶蛋白时观察到荧光强度显著增加。迄今为止,通过化学方法将基团连接到有亲和力的能结合到目标部分的探针提供了荧光信号。但是,这种方法有明显的缺点,所述缺点与对激酶袋的 低亲和力有关,其妨碍建立敏感测定的可能性。已经应用的替代方法依赖于具有固相测定典型限制(例如空间位阻和需要洗涤剂)的固定探针配体或激酶蛋白。
由于二氢吲哚酮类显示出对于靶激酶的相对较高的亲和力,而且在结合位点上产生的信号直接取决于所述结合,因此本发明克服了这些缺点,允许建立均一性测定。
尽管在这里用ABL激酶例举了本筛选试验,本领域的专家无需过多试验可以进行本筛选试验,也可以使用其它激酶蛋白,条件是激酶蛋白包含ATP袋。实际上,当应用胰岛素样生长因子(非磷酸化的和三磷酸化的)、胰岛素受体(非磷酸化的和三磷酸化的)、c-Met、Alk、Chk1和PDK1进行试验时,本发明人已经能够检测到荧光信号的变化。因此,可以预期更多的蛋白激酶可以适用于本发明测定。
最后,如上所述,本发明涉及式(I)化合物或其可药用盐在生产用于治疗耐BCR-ABL抑制剂的T315I ABL介导的疾病的药物中的应用。
为了更好地说明本发明而不造成任何限制,现在提供下列实施例。
附图简述
图1说明化合物1和伊马替尼对野生型ABL和T315I ABL突变体激酶结构域的激酶活性的抑制活性。通过凝胶激酶试验测定在增加的化合物浓度的情况下的抑制活性。用髓磷脂碱蛋白作为底物。
图2是一张免疫印迹图,其显示用化合物1(2道)或伊马替尼(3道)处理后对在K-562白血病细胞中的BCR-ABL的抑制作用。通过分析下游靶点STAT5或CRKL的磷酸化状态,监测对BCR-ABL信号活动的抑制作用。1道是未处理的K-562细胞。
图3是一张免疫印迹图,其显示化合物1在HCT-116转染细胞中对野生型和T315I ABL突变体的抑制作用。用ABL野生型(1道)或T315I ABL突变体的表达构建体(2道)转染HCT-116结直肠肿瘤细胞。通过免疫印迹法分析细胞裂解液,所述免疫印迹法应用全ABL蛋白(α-ABL)的抗体或磷酸基-特异性抗体,所述抗体在Y412(α-pY412-ABL) 位磷酸化时识别ABL。比较未处理(-)和处理组(+)细胞显示,化合物1能抑制野生型和T315I突变体ABL两者。
实施例
在下列实施例中,氨基酸残基的数目指在基因库对ABL蛋白AAB60394的同种型1a的登录号。实施例1:重组ABL激酶结构域的克隆、表达和纯化。
ABL激酶结构域(对应残基229-512)是从人类睾丸cDNA库通过聚合酶链反应(PCR)扩增得到。通过正向寡核苷酸:5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTACTGGAAGTTCTGTTCCAGGGGCCCTCCCCCAACTACGACAAGTG-3′[SEQ ID NO:1]和反向寡核苷酸:5′GGGGACCACTTTGTACAAGAAAGCTGGGTTTTATTTCCCCAGCTCCTTTTCCAC-3′完成扩增[SEQ ID NO:2]。
为了克隆目的,所述寡核苷酸包括细菌附着位点(attB)以通过 technology(Invitrogen)获得适合于克隆的attB-侧面的PCR产物。此外,为了纯化目的,正引物包括一个切割位点(Amersham Biosciences)。生成的PCR产物在杆状病毒表达载体pVL1393(Invitrogen)-modified中克隆。为了表达和纯化的目的,将GST标签加N-末端到ABL激酶结构域。根据手册所描述的方案进行克隆。
通过诱变试剂盒(Stratagene),采用定点诱变的方法产生耐伊马替尼的T315I ABL突变体。在诱变反应中应用的寡核苷酸是:5′-CCCCGTTCTATATCATCATTGAGTTCATGACCTACG-3′[SEQ IDNO:3]。
使用转染试剂盒(Pharmingen),通过用表达载体以及病毒DNA共转染Sf9昆虫细胞产生杆状病毒。5天后回收病毒上清液,然后使其接受3轮扩增以增加病毒效价。用每十亿个细胞3ml病毒上清液,以1×106个细胞/ml的密度感染High5昆虫细胞,产生重组蛋白。在感染48小时后,回收细胞、沉淀并且保存在-80℃。为了纯化重组蛋白,融化沉淀,在裂解缓冲液(Tris盐酸50mM,氯化 钠150mM,CHAPS0.2%,二硫苏糖醇20mM,甘油10%)中重悬,并且通过超声处理裂解。通过20000g离心30min使裂解液澄清,并且加载在Glutathione Sepharose(Amersham Biosciences)柱上。在充分洗涤后,通过与蛋白酶温孵,重组蛋白裂解并被洗脱出。
实施例2:激酶试验
在凝胶激酶试验中评价化合物的活性,所述试验通过重组野生型和T315I突变体的ABL激酶结构域测定髓磷脂碱蛋白的磷酸化。在一个20μl的反应混合液中进行激酶试验,所述混合液包含15nM的重组酶、10μM作为底物的的髓磷脂碱蛋白(Sigma-Aldrich)、10mM HepespH7.5、5mM氯化镁、1mM氯化锰、1mM二硫苏糖醇、15nM Na3VO4、4%DMSO、3nM ATP、0.1μ居里的γP32-ATP。测试化合物1,并且也平行测试伊马替尼作为参照。抑制剂浓度的范围从10nM到10μM。在酶和底物与抑制剂在室温预温孵15分钟后,加入ATP,在30℃进行反应30分钟,并加入凝胶加载缓冲液中止反应,煮沸5分钟。然后,将反应混合液加载在10%的SDS-聚丙烯酰胺凝胶(Polyacrilammide)上。分离后,干燥凝胶并且应用PhosphorImager系统(MolecularDynamics)显示放射性信号。
正如所料,伊马替尼仅能抑制野生型ABL激酶结构域的激酶活性,而对T315I突变体完全无效。与之相反,测试化合物以同样的效力对野生型和T315I突变体均有效。
实施例3ATP位点依赖的取代试验的建立和验证
建立ATP位点依赖的取代试验用于测试化合物的定量亲和性评价。
试验利用显著增加的荧光强度,所述荧光强度可以在某种二氢吲哚酮类结合到激酶蛋白时观察到。如下所示,我们显示使用两个不同的二氢吲哚酮类化合物获得的结果,所述二氢吲哚酮基于结合到ABL 时荧光强度增加的量级选作潜在的探针。
3.1二氢吲哚酮探针2,4-二甲基-5-[2-氧代-5-苯甲磺酰基-1,2-二氢-吲哚-(3Z)-亚基甲基]-1H-吡咯-3-羧酸(2-二乙氨基-乙基)-酰胺。
在滴定实验中,评价二氢吲哚酮2,4-二甲基-5-[2-氧代-5-苯甲磺酰基-1,2-二氢-吲哚-(3Z)-亚基甲基]-1H-吡咯-3-甲酸(2-二乙氨基-乙基)-酰胺与野生型ABL激酶结构域和T315I ABL突变体的结合能力。通过ATPγS和伊马替尼评价试验完成(Z′因子)和探针的取代情况。在所有的实验中,通过融合α-FP HT读盘器(Packard)检测荧光信号。应用Dynafit软件完成数据分析。
特别地,滴定数据符合下列平衡:酶+探针<==>酶-探针复合物,而取代数据符合下列平衡:酶+探针<==>酶-探针复合物,酶+化合物<==>酶-化合物复合物,由此探针与化合物在酶上的结合是相互排斥的(单纯竞争性机制)。
如下进行滴定实验:10nM ABL激酶结构域(野生型或T315I突变体),二氢吲哚酮浓度从3μM到0,稀释梯度:1:1.5,在50mMTris盐酸,pH7.5,150mM氯化钠,5mM氯化镁,1mM氯化锰,1mM二硫苏糖醇,1%DMSO,3μM Na3VO4(缓冲液1)。所得结果(在下表1中显示)表明所述探针能结合ABL激酶结构域的所有测试形式。如下测定Z′因子(Z′=1-(3*(SD探针+蛋白+SD蛋白)/(Mean探针+蛋白-Mean探针)):在存在(探针+蛋白)或不存在(探针)10和5nM蛋白的情况下,测定饱和探针浓度(0.15、0.12和0.42μM,分别用于ABL非磷酸化野生型(0P),野生型和T315I)。在所有情况下,Z′值均大于0.8,表明该试验是可靠的。
在置换试验中,应用ATPγS和伊马替尼验证试验,如下进行:系列稀释的测试化合物(稀释梯度1:2)首先在7%DMSO中配制,在试验缓冲液1中进一步稀释以使DMSO的终浓度为1%。在250μM时检测ATPγS作为最高浓度,而伊马替尼的最高浓度是10μM。酶的终浓度为10nM。对于ABL非磷酸化野生型(0P)、野生型和T315I,探针的 终浓度分别是0.15、0.12和0.42μM。将酶和探针的混合物加到先前稀释的化合物中。结果(表1)表明,探针能从所有的测试的ABL构建体中完全被ATPγS置换,其表明二氢吲哚酮在ATP袋内结合。一致地,通过符合单纯竞争性机制测定亲和性结合常数(KD)。KD是三次独立试验的平均值。
正如所料,伊马替尼显示出与ABL非磷酸化形式的亲和力高于与ABL磷酸化形式的亲和力,而在T315I ABL突变体中没有观察到置换。
表1
野生型ABL0P | 野生型ABL | T315I ABL | |
探针滴定,KD(μM) | 0.05±0.03 | 0.04±0.02 | 0.14±0.07 |
ATPγS,KD′(μM) | 9.5±3.4(100%置换) | 9±1.9(100%置换) | 2±0.21(100%置换) |
伊马替尼,KD′(μM) | 0.022±0.003(100%置换) | 0.17±0.02(100%置换) | (0%置换) |
总的来说,这些结果显示,所述置换试验是ATP位点依赖性的,而且所测的亲和力与伊马替尼发表的数据一致(CARTER,T.A.,et al.Inhibition of drug-resistant mutants of ABL,KIT,and EGFreceptor kinases..Proc.Natl.Acad.Sci.U.S.A..2005,vol.102,no.31,p.11011-11016.FABIAN,Miles A.,et al.A smallmolecule-kinase interaction map for clinical kinase inhibitors.Nat.biotechnol..2005,vol.23,no.3,p.329-336)。
用重组ABL激酶结构域的ATP位点依赖性取代试验。
在取代试验中,评价化合物与重组野生型和T315I突变体ABL激酶结构域的结合亲和力。测试了吡咯并吡唑类化合物,并与包括的伊马替尼进行比较。
表2显示了不同ABL构建体的亲和结合常数(KD)的测定。VX-680是来源于Vertex的ATP-竞争性的酪氨酸激酶抑制剂,最近已经报道其有体外结合T315I ABL突变体的能力;化合物1是本发明的实施例化合物。
表2
化合物 | 野生型ABL0PKD(μM) | 野生型ABLKD(μM) | T315I ABLKD(μM) |
VX680(Vertex) | 0.032±0.007 | 0.031±0.007 | 0.027±0.001 |
化合物1 | 0.008±0.002 | 0.008±0.001 | 0.001±0.0001 |
伊马替尼 | 0.022±0.003 | 0.117±0.02 | 0%置换 |
测定了本发明中其它的实例化合物对于T315I ABL突变体的亲和结合常数(KD)。结果在如下表3中报道,其中化合物2是N-[5-((R)-2-甲氧基-2-苯基-乙酰基)-1,4,5,6-四氢-吡咯并[3,4-c]吡唑-3-基]-4-哌啶-1-基-苯甲酰胺;化合物3是4-(4-乙基-哌嗪-1-基)-N-[5-((R)-2-甲氧基-2-苯基-乙酰基)-1,4,5,6-四氢-吡咯并[3,4-c]吡唑-3-基]-苯甲酰胺;化合物4是4-(4-环丙基-哌嗪-1-基)-N-[5-((R)-2-甲氧基-2-苯基-乙酰基)-1,4,5,6-四氢-吡咯并[3,4-c]吡唑-3-基]-苯甲酰胺;化合物5是4-(4-异丙基-哌嗪-1-基)-N-[5-((R)-2-甲氧基-2-苯基-乙酰基)-1,4,5,6-四氢-吡咯并[3,4-c]吡唑-3-基]-苯甲酰胺。
表3
化合物 | T315I ABL突变体KD(μM) |
化合物2 | 0.002±0.0008 |
化合物3 | 0.0017±0.0006 |
化合物4 | 0.007±0.003 |
化合物5 | 0.0024±0.0007 |
伊马替尼 | 0%置换 |
如前所述,用3μM作为最高抑制剂浓度进行置换试验,所述最高抑制剂浓度是用稀释梯度1:1.5测试的。结果如表2和3所示,其表明所有的测试化合物对T315I ABL突变体均有效。
3.2二氢吲哚酮探针N-[2-(二乙氨基)乙基]-4-(2,4-二氟苯基)-2-甲基-5-((Z)-{5-[(甲氨基)磺酰基]-2-氧代-1,2-二氢-3H-吲哚-3-亚基}甲基)-1H-吡咯-3-甲酰胺。
如上所述,在滴定实验中评价二氢吲哚酮N-[2-(二乙氨基)乙基]-4-(2,4-二氟苯基)-2-甲基-5-((Z)-{5-[(甲氨基)磺酰基]-2- 氧代-1,2-二氢-3H-吲哚-3-亚基}甲基)-1H-吡咯-3-甲酰胺与野生型ABL激酶结构域和T315I ABL突变体的结合能力。除了饱和探针浓度(2、3.3和0.2μM分别用于ABL非磷酸化野生型(0P),野生型和T315I)以及探针的终浓度(2、3.3和0.2μM分别用于ABL非磷酸化野生型(0P),野生型和T315I)外,其它应用条件均一样。
仍在此情况下,结果(表4)表明,探针能从所有测试的ABL构建体中完全被ATPγS置换,其表明二氢吲哚酮在ATP袋内结合。正如所料,伊马替尼显示出与ABL非磷酸化形式的亲和力高于与ABL磷酸化形式的亲和力,而在T315I ABL突变体中没有观察到置换。
表4
野生型ABL0P | 野生型ABL | T315I ABL | |
探针滴定,KD(μM) | 0.2±0.1 | 0.33±0.12 | 0.02±0.008 |
ATPγS,KD′(μM) | 6.6±1.6(100%置换) | 4.8±1(100%置换) | 2.61±0.9(100%置换) |
伊马替尼,KD′(μM) | 0.021±0.008(100%置换) | 0.23±0.09(100%置换) | (0%置换) |
总的来说,这些结果显示,置换试验是ATP位点依赖性的,而且所测的亲和力与伊马替尼发表的数据一致(CARTER,T.A.,et al.,ibid.;FABIAN,Miles A.,et al.,ibid.)。
重组ABL激酶结构域的ATP位点依赖取代试验。
如3.1中所报道的,在所述置换试验中,评价了化合物与重组野生型和T315I突变体ABL激酶结构域的亲和力,并且测试了同样的吡咯并吡唑类化合物。表5显示不同ABL构建体的亲和结合常数的测定(KD)。
表5
野生型ABL0PKD(μM) | 野生型ABLKD(μM) | T315I ABLKD(μM) | |
VX680(Vertex) | 0.058±0.02 | 0.080±0.01 | 0.043±0.014 |
化合物1 | 0.014±0.003 | 0.018±0.007 | 0.004±0.001 |
伊马替尼 | 0.020±0.006 | 0.122±0.022 | 0%置换 |
测定了本发明中其它实施例化合物(如上述定义的化合物2到5) 对于T315I ABL突变体的亲和结合常数(KD)。
结果在下表6中报道。
表6
T315I ABL突变体KD(μM) | |
化合物2 | 0.0043±0.001 |
化合物3 | 0.0028±0.001 |
化合物4 | 0.0040±0.001 |
化合物5 | 0.0026±0.0005 |
伊马替尼 | 0%置换 |
如前所述,用3μM作为最高抑制剂浓度进行置换试验,如表5和6所示,结果是三次独立试验的平均值。它们表明所有的测试化合物对T315I ABL突变体均有效。
实施例4在细胞中的ABL活性抑制
材料和方法
ABL多肽的克隆
ABL cDNA(对应残基27-1130)是从人类睾丸cDNA库通过聚合酶链反应(PCR)扩增得到。使用正向寡核苷酸:5′GGGACAAGTTTGTACAAAAAAGCAGGCTTACTGGAAGTTCTGTTCCAGGGGCCCGAAGCCCTTCAGCGGCCAGTAG-3′[SEQ ID NO:4]和反向寡核苷酸:5′GGGGACCACTTTGTACAAGAAAGCTGGGTTTTACCTCTGCACTATGTCACTG-3′完成扩增[SEQ ID NO:5]。
为了克隆目的,所述寡核苷酸包括细菌附着位点(attB)以通过使用technology(Invitrogen)获得适合于克隆的attB-侧面的PCR产物。生成的PCR产物在哺乳动物表达载体pcDNA3.1/nV5DEST(Invitrogen)中克隆。根据手册所描述的方案进行相应的克隆。
使用诱变试剂盒(Stratagene),通过定点诱变的方法产生耐伊马替尼的突变体T315I。在诱变反应中应用的寡核苷酸 是:5′-CCCCGTTCTATATCATCATTGAGTTCATGACCTACG-3′[SEQ ID NO:6]。
免疫印迹法
根据标准方法进行免疫印迹法。将细胞在125mM Tris盐酸,pH6.8和2%SDS中配制。超声样品,然后在95℃加热5min。将如BCATM蛋白试验(Pierce,Rockford,IL)测定的10μg蛋白质提取物加载在SDS-聚丙烯酰胺凝胶上。用Super signal chemiluminescenece试剂盒(Pierce,Rockford,IL)进行检测。应用下列抗体进行免疫印迹分析:抗-ABL(SIGMA,目录号A5844);抗-pY412-ABL(CELL SIGNALING,目录号2865);抗-Stat-5(CELL SIGNALING,目录号9352);抗-pStat-5、抗-CrkI、抗p-CrkI、抗-EIF4e(BCR/ABL活性测定,CELLSIGNALING,目录号7130)。
结果
为了证实本发明的一个实施例化合物对内源性表达BCR-ABL细胞的抑制活性,在K-562白血病细胞中测试化合物1,所述K-562白血病细胞具有与BCR-ABL易位有关的费城染色体。用化合物1或伊马替尼处理细胞1个小时。然后收集细胞,并且通过免疫印迹法分析在412(Y412)位点的酪氨酸残基的ABL自身磷酸化的抑制作用,所述位点位于ABL的激酶活化环。同样对公知的下游信号蛋白例如Stat-5或CrkI的磷酸化状态进行分析。化合物1和伊马替尼(处于临床相关浓度下)抑制Y412的磷酸化以及BCR-ABL的下游介体Stat-5和CrkI的磷酸化(图2),其证实了预期的作用机制,而除去对蛋白磷酸化的一般作用,没有观察到对Src的作用(数据没有显示)。
为了检测伊马替尼和化合物1在细胞内对T315I ABL突变体的细胞活性,用野生型或突变体的表达构建体转染HCT-116细胞(请参考上述材料和方法),并且在5μM时测试伊马替尼和化合物1的抑制活性。正如所料,伊马替尼仅对野生型ABL构建体显示出抑制活性,而T315I ABL突变体是耐药的。与之相反,化合物1对野生型和T315I ABL突变体两者均显示出强的抑制活性(图3)。
序列表
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Claims (6)
3.根据前述权利要求2的用途,其使用式(I)化合物或其可药用盐进行,其中R是氢,R1是甲氧基,R2是氢且X是亚甲基(-CH2-),或是杂原子或杂原子基团,选自氧(-O-)或氮(-NR′-),其中R′是氢原子,或是选自甲基、乙基、异丙基、环丙基或叔丁基的烷基。
4.根据权利要求2的用途,其中所述式(I)化合物选自:
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-异丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-环丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-哌啶-1-基苯甲酰胺;
4-(4-氟哌啶-1-基)-N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-吗啉-4-基苯甲酰胺;
N-{5-[(2R)-2-甲基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-哌啶-1-基苯甲酰胺;
4-(4-氟哌啶-1-基)-N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺;
4-吗啉-4-基-N-{5-[(2R)-2-苯基丙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}苯甲酰胺。
5.根据权利要求3的用途,其中所述式(I)化合物选自:
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-乙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-异丙基哌嗪-1-基)苯甲酰胺;
N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-环丙基哌嗪-1-基)苯甲酰胺。
6.根据权利要求5的用途,其中所述式(I)化合物是N-{5-[(2R)-2-甲氧基-2-苯基乙酰基]-1,4,5,6-四氢吡咯并[3,4-c]吡唑-3-基}-4-(4-甲基哌嗪-1-基)苯甲酰胺。
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BRPI0717998A2 (pt) * | 2006-11-03 | 2013-12-03 | Nerviano Medical Sciences Srl | Método para administrar um composto antitumor. |
WO2010009985A2 (en) * | 2008-07-24 | 2010-01-28 | Nerviano Medical Sciences S.R.L. | Therapeutic combination comprising an aurora kinase inhibitor and antiproliferative agents |
CA2747326C (en) | 2008-12-22 | 2017-05-16 | Millennium Pharmaceuticals, Inc. | Combination of aurora kinase inhibitors and anti-cd20 antibodies |
CN103626777B (zh) * | 2009-07-29 | 2015-10-21 | 内尔维阿诺医学科学有限公司 | Plk抑制剂的盐类 |
GB2508652A (en) * | 2012-12-07 | 2014-06-11 | Agency Science Tech & Res | Heterocyclic piperazine derivatives |
CN103113355B (zh) * | 2013-02-27 | 2014-08-13 | 无锡爱内特生物科技有限公司 | 一种Bcr/Abl酪氨酸激酶抑制剂及其制备方法和在治疗慢性粒细胞白血病中的应用 |
CN104072498B (zh) * | 2013-03-26 | 2016-12-28 | 沈阳药科大学 | (R)‑N‑[5‑(2‑甲氧基‑2‑苯基乙酰基)‑1,4,5,6‑四氢吡咯并[3,4‑c]吡唑‑3‑基]‑4‑(4‑甲基哌嗪‑1‑基)苯甲酰胺的合成方法 |
WO2015085289A1 (en) | 2013-12-06 | 2015-06-11 | Millennium Pharmaceuticals, Inc. | Combination of aurora kinase inhibitors and anti-cd30 antibodies |
CN105037399B (zh) * | 2014-04-17 | 2017-04-26 | 深圳永泽医药股份有限公司 | 一类Bcr‑Abl双倍体抑制剂及制备方法与用途 |
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