CN118056816A - 吡唑-3-酮类衍生物及其制备方法与应用 - Google Patents
吡唑-3-酮类衍生物及其制备方法与应用 Download PDFInfo
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- CN118056816A CN118056816A CN202211455030.2A CN202211455030A CN118056816A CN 118056816 A CN118056816 A CN 118056816A CN 202211455030 A CN202211455030 A CN 202211455030A CN 118056816 A CN118056816 A CN 118056816A
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- phenyl
- pyrazol
- dihydro
- carbonyl
- dimethylamino
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Abstract
吡唑‑3‑酮类衍生物及其制备方法与应用，属于医药技术领域，具体涉及一种如通式I所示的吡唑‑3‑酮类衍生物，及其药学上可接受的盐，该衍生物的制备方法，以该衍生物为活性成分的药物组合物，以及用于治疗和/或预防各种癌症的药物的用途。本发明所述吡唑‑3‑酮类衍生物可通过活化Bax，促进Bax的线粒体转位和寡聚化，导致线粒体外膜透化作用，释放细胞色素c等线粒体蛋白至胞质中，激活caspase级联反应，直接诱导肿瘤细胞凋亡。因此，能够用于制备与Bcl‑2蛋白家族失调相关的疾病的药物中，如各种癌症。
Description
技术领域
本发明属于医药技术领域，具体涉及吡唑-3-酮类衍生物或其药学上可接受的盐，该衍生物的制备方法，以该衍生物为活性成分的药物组合物，和其作为Bax激动剂在制备预防和/或治疗肿瘤药物中的应用。
背景技术
癌细胞内部维持着Bcl-2抗凋亡信号与促凋亡信号间的动态平衡，且相较于正常细胞，癌细胞具有更高的凋亡阈值，使用小分子药物打破该平衡即可选择性诱导癌细胞发生凋亡，实现抗肿瘤效力。在过去二十年，基于抑制抗Bcl-2凋亡蛋白功能策略的凋亡诱导剂(BH3模拟物)取得了巨大的成功，其凋亡诱导活性来源于两方面，一是释放被抑制的Bax/Bak活化单体，直接激活下游凋亡通路，二是释放被抑制的BH3-only蛋白，随后激活胞质中休眠的Bax单体，进而激活下游凋亡通路。综上，基于抑制抗凋亡蛋白功能策略的凋亡诱导剂发挥诱导凋亡能力的关键是直接或间接激活凋亡执行蛋白Bax/Bak。
在线粒体凋亡通路中，凋亡执行蛋白Bax位于抗凋亡蛋白下游，且处于中心调控地位。多项研究表明Bax促凋亡功能的失调与多种肿瘤的发生发展有关，如肺癌、结肠癌、乳腺癌和白血病等。靶向激活Bax可直接激活线粒体凋亡通路，发挥与抗凋亡蛋白多靶点抑制剂相近的药理作用，可有效克服现有BH3模拟物的局限(适应症窄和复杂的耐药机制)。
本发明在参考文献的基础上，设计并合成了一系列吡唑-3-酮类衍生物。体外分子和细胞水平测试结果表明，该类衍生物表现出广谱的、强效的肿瘤细胞生长抑制活性，其通过Bax活化而非抑制Bcl-2抗凋亡蛋白，从而表现出显著优于阳性药BTSA1的凋亡诱导、诱导G0/G1期阻滞和抑制集落形成的能力。
发明内容
本发明的目的在于提供一类吡唑-3-酮类衍生物及其制备方法，和其作为Bax激动剂在制备预防和/或治疗肿瘤药物中的应用。
为了实现上述目的，本发明提供通式I所示的吡唑-3-酮类衍生物或其在药学上可接受的盐；
其中：
R1为H或
其中，R4为：
a表示在苯环上的取代位置，为3位或4位；
R2为甲基、乙基、丙基、丁基、环丙基、环丁基、环戊基、环己基、环戊基或苯基；
X为CH或N；
Y为NH或O；
或者，X、Y不存在，R3位于X的位置直接与双键相连接；
R3为
其中，R8、R9、R10、R11、R12各自独立地为氢、羟基、卤素、硝基、氨基、氰基、取代或为取代的C1-C4烷基或亚烷基、取代或为取代的C1-C4烷氧基或亚烷氧基、 所述取代或未取代的C1-C4烷基或亚烷基，或取代或未取代的C1-C4烷氧基或亚烷氧基中，所述的取代基选自卤素、羟基、氨基，所述卤素为氟、氯或溴；
R13为
进一步地，本发明所述吡唑-3-酮类衍生物为如下所述化合物中任一个：
N-(1-甲基哌啶-4-基)-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A01)
2-(4-(4-甲哌嗪-1-甲酰基)苯基)-5-苯基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A02)
N-(1-苄基哌啶-4-基)-N-甲基-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A03)
2-(4-(4-二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A04)
4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)-N-(吡啶-4-基甲基)苯甲酰胺(A05)
5-苯基-4-(2-苯基肼基)-2-(4-(哌嗪-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A06)
(R)-2-(4-(3-氨基哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A07)
2-(4-(2,7-二氮杂螺[3.5]壬烷-2-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A08)
2-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)-2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(A09)
2-(4-(4-(氧杂环丁-3-基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A10)
1-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)哌啶-4-酮(A11)
2-(4-(4-(甲基磺酰基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A12)
N-(3-(二甲氨基)丙基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A13)
5-苯基-4-(2-苯基肼基)-2-(4-(哌啶-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A14)
N,N-二甲氨基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A15)
2-(4-([1,4'-双哌啶基]-1'-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A16)
2-(3-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A17)
N-(1-甲基哌啶-4-基)-3-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A18)
2-(3-(4-甲基哌嗪-1-羰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A19)
4-(2-(4-(4-甲基哌嗪-1-基)苯基)肼基)-2,5-双苯基-2,4-二氢-3H-吡唑-3-酮(A20)
4-(2-(3,5-双(三氟甲基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A21)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-3-甲氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A22)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-2-异丙氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A23)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(3,4,5-三甲氧基苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A24)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(萘-2-基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A25)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-羟基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A26)
4-(2-(4-(苄氧基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A27)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(4-(哌啶-1-甲酰基)苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A28)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A29)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-((苯基氨基)亚甲基)-2,4-二氢-3H-吡唑-3-酮(A30)
4-((苄基氨基)亚甲基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A31)
4-环己亚基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A32)
4-(1-苄基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A33)
4-(1-(环丙基甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A34)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(1-(噻唑-4-甲酰基)哌啶-4-基亚基)-2,4-二氢-3H-吡唑-3-酮(A35)
4-(1-苯甲酰基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A36)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-羟基苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A37)
4-(1-(5-氯-1H-吲哚-2-甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A38)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-(10α-二氢青蒿素-10-基)氧基)苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A39)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-甲基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A40)
5-环丙基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A41)
除非另外指出，本发明所用的术语“卤素”是指氟、氯、溴或碘，优选氯、溴；“烷基”是指直链或支链的烷基。
通式I所示的化合物还可以以不同互变异构体形式存在，所有这些形式均包含在本发明范围内。属于“互变异构体”或“互变异构体形式”是指经有低能垒相互转化的不同能量的结构异构体。
根据本发明，药学上可接受的盐是指所述吡唑-3-酮类衍生物与下述酸形成的加成盐：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、苯甲酸、琥珀酸。
本发明的通式I所示的吡唑-3-酮类衍生物或其药学上可接受的盐作为活性成份，与药学上可接受的赋形剂混合制备成组合物，并制备成临床上可接受的剂型，上述赋形剂是指可用于药学领域的稀释剂、辅助剂或载体。上述剂型是指临床上常用的注射剂、片剂、胶囊剂。
本发明所述的吡唑-3-酮类衍生物或其药学上可接受的盐可作为唯一的抗肿瘤药物单独使用，或者与现已上市的抗肿瘤药物联合使用，用于治疗预防肿瘤等。
通过体外抗肿瘤作用机制研究，我们发现本发明所述吡唑-3-酮类衍生物可通过活化Bax，促进Bax的线粒体转位和寡聚化，导致线粒体外膜透化作用，释放细胞色素c等线粒体蛋白至胞质中，激活caspase级联反应，直接诱导肿瘤细胞凋亡。因此，本发明所述吡唑-3-酮类衍生物可用于制备与Bcl-2蛋白家族失调相关的疾病的药物中，如各种癌症。
通过体外活性筛选研究，我们发现本发明所述的吡唑-3-酮类衍生物具有抗肿瘤活性，因此本发明所述吡唑-3-酮类衍生物可以用于制备治疗和/或预防癌症的药物，所述癌症为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性粒细胞白血病、慢性白血病、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌和/或胰腺癌。
本发明化合物可作为唯一的抗癌药物使用，或者与一种或多种其它抗肿瘤药物联合使用，联合治疗通过将各个治疗组分同时、顺序或隔开给药来实现。
所述吡唑-3-酮类衍生物A01-A41的制备方法，包括如下：
目标化合物A01-A20和A40-A41的合成路线：其中，R2、R4为化合物A01-A20和A40-A41对应位置的相应基团；
目标化合物A21-A39的合成路线：其中，R3、R13为化合物A21-A39对应位置的相应基团；
附图说明
图1(A)优选化合物对Bax构象活化的影响；(B)优选化合物对Bax的线粒体转位和细胞色素C释放的影响；(C)优选化合物对凋亡相关蛋白水平的影响。
具体实施方式
实施例旨在阐述而不是限制本发明的范围。凡基于本发明上述内容所实现的技术均属于本发明的范围。
化合物的核磁共振谱用Bruker ARX-600核磁共振波谱仪在CDCl3-d或DMSO-d6中以TMS为内标测定，低分辨质谱用Agilent 1100SL型离子阱质谱仪测定，高分辨质谱用Brukermicro-TOF-Q测定。
实施例1：N-(1-甲基哌啶-4-基)-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A01)的制备
步骤A：4-(5-羟基-3-苯基-4,5-二氢-1H-吡唑-1-基)苯甲酸(1a)的制备
将苯甲酰乙酸乙酯(1.0g,5.2mmol)和4-肼基苯甲酸(0.88g,5.6mmol)溶于20mL无水乙醇，80℃反应2h。冷却至室温，抽滤，干燥，得灰白色固体1a 0.95g，收率65.2％。1HNMR(600MHz,DMSO-d6)δ12.91(s,1H),12.20(s,1H),8.05(t,J＝8.3Hz,4H),7.86–7.85(m,2H),7.43(t,J＝7.5Hz,2H),7.35(t,J＝7.4Hz,1H),6.06(s,1H).
采用上述方法，以3-肼基苯甲酸替换4-肼基苯甲酸，合成化合物1b；以乙酰乙酸乙酯或3-环丙基-3-氧代丙酸乙酯替换苯甲酰乙酸乙酯，合成化合物6a-6b。
步骤B：N-(1-甲基哌啶-4-基)-4-(5-氧代-3-苯基-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(2a)的制备
将1a(0.30g,1.1mmol)和4-氨基-1-甲基哌啶(0.13g,1.1mmol)溶于10mL干燥二氯甲烷中，随后加入HATU(0.44g,1.15mmol)和DIEA(0.97g,5.5mmol.)，室温反应1h。加入15mL饱和氯化铵溶液淬灭反应，二氯甲烷萃取(10mL*3)，收集有机相。有机相水洗、饱和氯化钠溶液洗涤，减压蒸干得粗产品，后经柱层析纯化(二氯甲烷/甲醇，V:V＝90:1)，得白色固体2a 0.19g，收率45.9％。
采用上述步骤B的方法，以中间体1a、1b、6a-6b和不同脂肪胺制得中间体2b-2r和7a-7b。
步骤C：N-(1-甲基哌啶-4-基)-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A01)的制备
将苯胺(0.084g,0.91mmol)溶于3mL 12M盐酸水溶液，冷却至0℃，逐滴加入6mL亚硝酸钠(0.069g,1.0mmol)水溶液0℃下搅拌30min。将1a(0.34g,0.91mmol)溶于5mL无水乙醇，逐滴加入5mL乙酸钠(0.075g,0.91mmol)水溶液，随后0℃下逐滴加入重氮盐溶液，滴毕，室温反应2h。反应结束后，减压蒸除有机溶剂，加入5mL水，使用6M NaOH(aq.)调节pH至7～8，乙酸乙酯萃取(15mL*3)，收集有机相，饱和氯化钠洗涤(25mL*2)，后经柱层析纯化(二氯甲烷/甲醇，V:V＝30:1)，制得橙色固体A01 0.21g，收率为47.8％。1H NMR(600MHz,CDCl3-d)δ14.00(s,1H),8.28–8.17(m,4H),7.86(s,2H),7.49(s,8H),6.03(s,1H),4.04(s,1H),2.97–2.84(m,2H),2.36(s,3H),2.24(s,2H),2.12–2.06(m,2H),1.67(s,2H).13C NMR(151MHz,CDCl3-d)δ166.25,158.46,147.23,141.02,140.66,131.01,130.17,130.07,129.85,128.65,127.85,127.58,126.99,126.40,118.12,116.24,54.53,46.07,32.20,29.71.HRMS(ESI)m/z calcd for C28H28N6O2[M-H]-:479.2201found:479.2210.
实施例2：2-(4-(4-甲哌嗪-1-甲酰基)苯基)-5-苯基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A02)的制备
按照实施例1的合成方法，以2b(0.15g,0.41mmol)和苯胺(0.038g,0.41mmol)原料，得橙色固体A02 0.095g，收率为49.7％。1H NMR(600MHz,DMSO-d6)δ13.71(s,1H),8.21(d,J＝7.3Hz,2H),8.15(d,J＝8.5Hz,2H),7.69(d,J＝8.0Hz,2H),7.64(d,J＝8.5Hz,2H),7.60(t,J＝7.4Hz,2H),7.56(d,J＝7.2Hz,1H),7.52(t,J＝7.9Hz,2H),7.30(t,J＝7.3Hz,1H),3.19–2.99(m,2H),2.78(s,3H).HRMS(ESI)m/z calcd for C27H26N6O2[M-H]-:465.2044found:465.2072.
实施例3：N-(1-苄基哌啶-4-基)-N-甲基-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A03)的合成
按照实施例1的合成方法，以2c(0.15g,0.32mmol)和苯胺(0.031g,0.32mmol)原料，得橙色固体A03 0.093g，收率为50.9％。
实施例4：2-(4-(4-二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A04)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和苯胺(0.038g,0.38mmol)原料，得橙色固体A04 0.090g，收率为48.0％。1H NMR(600MHz,DMSO-d6)δ13.07(s,1H),8.23–8.19(m,2H),8.14(d,J＝8.6Hz,2H),7.66(d,J＝7.6Hz,2H),7.57(t,J＝7.4Hz,2H),7.55–7.48(m,5H),7.27(t,J＝7.3Hz,1H),3.06(s,1H),2.84(s,1H),2.29(s,6H),1.86–1.79(m,1H),1.44–1.38(m,2H).13C NMR(151MHz,DMSO-d6)δ168.96,157.36,147.07,139.42,132.73,132.00,130.93,130.23,130.15,129.20,128.46,127.77,126.57,126.41,118.06,117.46,65.50,61.92,41.47,27.02.HRMS(ESI)m/z calcd for C29H30N6O2[M-H]-493.2357found:493.2372.
实施例5：4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)-N-(吡啶-4-基甲基)苯甲酰胺(A05)的制备
按照实施例1的合成方法，以2e(0.15g,0.40mmol)和苯胺(0.040g,0.40mmol)原料，得橙色固体D12 0.088g，收率为44.8％。1H NMR(600MHz,DMSO-d6)δ13.71(s,1H),9.18(t,J＝5.9Hz,1H),8.52(d,J＝5.1Hz,2H),8.22(d,J＝7.2Hz,2H),8.18(d,J＝8.7Hz,2H),8.08(d,J＝8.8Hz,2H),7.68(d,J＝7.9Hz,2H),7.59(t,J＝7.4Hz,2H),7.55(d,J＝7.2Hz,1H),7.52(t,J＝7.9Hz,2H),7.34(d,J＝5.5Hz,2H),7.29(t,J＝7.3Hz,1H),4.53(d,J＝5.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ166.25,157.74,150.01,149.12,147.11,141.66,140.64,130.73,130.57,130.30,130.26,129.30,128.94,127.76,126.80,126.73,122.66,117.75,117.07,42.26.HRMS(ESI)m/z calcd for C28H22N6O2[M-H]-:473.1731found:473.1755.
实施例6：5-苯基-4-(2-苯基肼基)-2-(4-(哌嗪-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A06)的制备
按照实施例1的合成方法，以2f(0.15g,0.43mmol)和苯胺(0.042g,0.43mmol)原料，得橙色固体A06 0.091g，收率为46.8％。1H NMR(600MHz,DMSO-d6)δ13.63(s,1H),8.21–8.19(m,2H),8.12(dd,J＝8.2,1.2Hz,1H),8.07(s,1H),7.67(d,J＝7.7Hz,2H),7.60–7.58(m,2H),7.57(d,J＝1.7Hz,1H),7.54(d,J＝7.2Hz,1H),7.52–7.50(m,2H),7.30–7.26(m,2H),3.66(s,2H),3.38(s,2H),2.36(d,J＝57.7Hz,4H),2.22(s,3H).13C NMR(151MHz,CDCl3-d)δ170.03,158.36,147.09,141.03,139.25,132.23,130.21,130.03,129.85,128.65,128.22,127.55,127.03,126.37,118.41,116.22,46.03,29.71.HRMS(ESI)m/zcalcd for C26H24N6O2[M+H]+:453.2024found:453.2031.
实施例7：(R)-2-(4-(3-氨基哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A07)的制备
按照实施例1的合成方法，以2g(0.15g,0.41mmol)和苯胺(0.041g,0.41mmol)原料，得橙色固体A07 0.085g，收率为44.4％。1H NMR(600MHz,CDCl3-d)δ8.23(dd,J＝8.1,1.4Hz,2H),8.20–8.17(m,2H),7.90(d,J＝8.7Hz,2H),7.52–7.49(m,2H),7.49–7.43(m,5H),7.25(ddd,J＝8.4,4.4,2.2Hz,1H),4.20(s,1H),3.12(d,J＝10.0Hz,1H),2.89–2.77(m,3H),1.87–1.82(m,1H),1.81–1.75(m,2H),1.61–1.56(m,1H).13C NMR(151MHz,CDCl3-d)δ165.92,158.34,147.05,140.94,140.46,131.11,130.09,129.95,129.75,128.55,127.88,127.48,126.93,126.27,117.99,116.14,51.34,46.52,45.69,29.91,29.62.
实施例8：2-(4-(2,7-二氮杂螺[3.5]壬烷-2-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A08)的制备
将A09(0.050g,0.084mmol)溶于10mL干燥的二氯甲烷中，冰浴下加入4mL三氟乙酸，室温反应0.5h，减压蒸馏除去有机试剂，使用饱和碳酸氢钠溶液调节pH＝10，抽滤并干燥，得橙色固体A08 0.035g，收率为84.6％。1H NMR(600MHz,DMSO-d6)δ9.61(s,1H),8.28(d,J＝8.3Hz,2H),8.20(d,J＝7.6Hz,2H),7.54(d,J＝7.7Hz,2H),7.47(t,J＝7.4Hz,2H),7.41(dd,J＝17.0,8.0Hz,5H),7.19(t,J＝7.1Hz,1H),3.79(s,4H),3.51(s,4H),1.82(s,4H).13CNMR(151MHz,DMSO-d6)δ169.64,155.39,148.11,141.46,133.81,132.00,130.75,130.12,129.71,129.52,129.14,128.57,128.13,126.20,123.81,119.81,117.54,65.50,55.14,36.92.HRMS(ESI)m/z calcd for C29H28N6O2[M+H]+:493.2347found:493.2351.
实施例9：2-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)-2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(A09)的合成
按照实施例1的合成方法，以2h(0.15g,0.31mmol)和苯胺(0.030g,0.31mmol)原料，得橙色固体A09 0.080g，收率为43.5％。1H NMR(600MHz,DMSO-d6)δ13.71(s,1H),8.21(d,J＝7.1Hz,2H),8.11(d,J＝8.6Hz,2H),7.68(d,J＝7.7Hz,2H),7.59(t,J＝7.4Hz,2H),7.55–7.50(m,5H),7.29(t,J＝7.3Hz,1H),3.64–3.57(m,4H),1.76–1.66(m,4H),1.38(s,9H),1.23(s,2H).13C NMR(151MHz,DMSO-d6)δ169.06,157.63,156.11,146.91,141.66,139.01,133.15,130.54,130.35,130.28,129.32,128.42,127.70,126.86,126.71,118.18,117.06,78.94,33.92,28.56.HRMS(ESI)m/z calcd for C34H36N6O4[M-H]-:591.2725found:591.2743.
实施例10：2-(4-(4-(氧杂环丁-3-基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A10)的制备
按照实施例1的合成方法，以2i(0.15g,0.37mmol)和苯胺(0.036g,0.37mmol)原料，得橙色固体A10 0.085g，收率为45.2％。1H NMR(600MHz,DMSO-d6)δ13.70(s,1H),8.20(d,J＝7.2Hz,2H),8.11(d,J＝8.6Hz,2H),7.67(d,J＝7.9Hz,2H),7.58(t,J＝7.4Hz,2H),7.57–7.50(m,5H),7.29(t,J＝7.3Hz,1H),4.55(t,J＝6.5Hz,2H),4.45(t,J＝6.1Hz,2H),3.73–3.56(m,2H),3.46(dt,J＝12.5,6.2Hz,3H),2.31(s,4H).13C NMR(151MHz,DMSO-d6)δ168.94,157.62,146.91,141.67,139.12,132.68,130.52,130.35,130.26,129.30,128.71,127.69,126.82,126.70,118.11,117.05,74.74,58.72,55.39,49.42.HRMS(ESI)m/z calcdfor C29H28N6O3Na[M+Na]+:531.2115found:531.2149.
实施例11：1-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)哌啶-4-酮(A11)的制备
按照实施例1的合成方法，以2j(0.15g,0.42mmol)和苯胺(0.041g,0.42mmol)原料，得橙色固体A11 0.083g，收率为42.4％。1H NMR(600MHz,DMSO-d6)δ13.71(s,1H),8.22(d,J＝7.2Hz,2H),8.15(d,J＝8.6Hz,2H),7.69(d,J＝7.9Hz,2H),7.66(d,J＝8.6Hz,2H),7.59(t,J＝7.4Hz,2H),7.55(d,J＝7.2Hz,1H),7.53–7.50(m,2H),7.29(t,J＝7.3Hz,1H),3.92–3.66(m,4H),2.47(s,4H).13C NMR(151MHz,DMSO-d6)δ207.70,169.54,157.65,146.99,139.31,132.64,130.55,130.39,130.29,130.11,129.34,128.66,127.73,126.84,126.72,118.15,117.10,40.53.HRMS(ESI)m/z calcd for C27H23N5O3[M+H]+:466.1874found:466.1880.
实施例12：2-(4-(4-(甲基磺酰基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A12)的制备
按照实施例1的合成方法，以2k(0.15g,0.35mmol)和苯胺(0.034g,0.35mmol)原料，得橙色固体A12 0.088g，收率为47.4％。1H NMR(600MHz,DMSO-d6)δ13.70(s,1H),8.22–8.20(m,2H),8.14(d,J＝8.6Hz,2H),7.69(d,J＝7.8Hz,2H),7.61–7.58(m,4H),7.55(d,J＝7.2Hz,1H),7.53–7.50(m,2H),7.29(t,J＝7.3Hz,1H),3.64(dd,J＝76.5,26.3Hz,4H),3.19(s,4H),2.92(s,3H).13C NMR(151MHz,DMSO-d6)δ169.17,157.65,146.98,141.66,139.32,132.27,130.56,130.34,130.28,129.33,128.85,127.71,126.83,126.73,118.17,117.08,45.86,34.63.HRMS(ESI)m/z calcd for C27H26N6O4S[M+H]+:531.1809found:531.1805.
实施例13：N-(3-(二甲氨基)丙基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A13)的制备
按照实施例1的合成方法，以2l(0.15g,0.41mmol)和苯胺(0.040g,0.41mmol)原料，得橙色固体A13 0.091g，收率为47.4％。1H NMR(600MHz,DMSO-d6)δ13.70(s,1H),8.66(t,J＝5.6Hz,1H),8.23–8.21(m,2H),8.17(d,J＝8.7Hz,2H),8.02(d,J＝8.8Hz,2H),7.69(d,J＝7.8Hz,2H),7.60(t,J＝7.4Hz,2H),7.56(d,J＝7.2Hz,1H),7.52(t,J＝7.9Hz,2H),7.30(t,J＝7.3Hz,1H),3.36(dd,J＝12.4,6.4Hz,2H),3.13–3.10(m,2H),2.80(s,6H),1.92–1.88(m,2H).13C NMR(151MHz,DMSO-d6)δ166.33,157.75,147.16,141.66,140.55,130.98,130.62,130.30,129.80,129.34,128.85,127.76,126.79,125.31,117.73,117.09,55.33,42.84,36.80,25.08.HRMS(ESI)m/z calcd for C27H28N6O2[M+H]+:469.2347found:469.2344.
实施例14：5-苯基-4-(2-苯基肼基)-2-(4-(哌啶-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A14)的制备
按照实施例1的合成方法，以2m(0.15g,0.43mmol)和苯胺(0.042g,0.43mmol)原料，得橙色固体A14 0.096g，收率为49.4％。1H NMR(600MHz,CDCl3-d)δ14.03(s,1H),8.25(d,J＝7.4Hz,2H),8.16(d,J＝8.2Hz,2H),7.55–7.44(m,10H),3.49(s,4H),1.70(d,J＝3.2Hz,2H),1.62(d,J＝4.6Hz,4H).13C NMR(151MHz,CDCl3-d)δ158.21,146.87,140.96,138.84,133.08,130.15,129.89,129.74,128.54,127.88,127.44,126.99,126.23,118.28,116.11,24.56.HRMS(ESI)m/z calcd for C27H26N5O2[M+H]+:452.2081found:452.2074.
实施例15：N,N-二甲氨基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A15)的制备
按照实施例1的合成方法，以2n(0.15g,0.49mmol)和苯胺(0.048g,0.49mmol)原料，得橙色固体A15 0.086g，收率为42.6％。1H NMR(600MHz,DMSO-d6)δ13.71(s,1H),8.21(d,J＝7.6Hz,2H),8.11(d,J＝8.4Hz,2H),7.68(d,J＝8.0Hz,2H),7.61–7.56(m,4H),7.56–7.50(m,3H),7.29(t,J＝7.3Hz,1H),2.99(s,6H).13C NMR(151MHz,DMSO-d6)δ169.95,157.54,146.80,141.57,138.85,133.28,130.43,130.27,130.18,129.21,128.61,127.60,126.78,126.60,117.90,116.95.
实施例16：2-(4-([1,4'-双哌啶基]-1'-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A16)的制备
按照实施例1的合成方法，以2o(0.15g,0.35mmol)和苯胺(0.034g,0.35mmol)原料，得橙色固体A16 0.081g，收率为43.3％。1H NMR(600MHz,CDCl3-d)δ14.02(s,1H),8.25(d,J＝7.2Hz,2H),8.17(d,J＝8.4Hz,2H),7.54–7.48(m,7H),7.48–7.45(m,2H),4.81(dd,J＝23.9,11.3Hz,1H),4.15–3.76(m,1H),3.14–2.95(m,1H),2.68(s,4H),2.01(s,3H),1.82–1.47(m,9H).13C NMR(151MHz,CDCl3)δ169.79,158.26,147.00,140.95,139.14,130.13,129.93,129.76,128.56,128.00,127.46,126.95,126.28,118.30,116.14,50.06,29.62.
实施例17：2-(3-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A17)的合成
按照实施例1的合成方法，以2p(0.15g,0.35mmol)和苯胺(0.034g,0.35mmol)原料，得橙色固体A17 0.095g，收率为54.9％。1H NMR(600MHz,DMSO-d6)δ13.08(s,1H),8.21(d,J＝7.5Hz,2H),8.14(d,J＝8.2Hz,1H),8.09(s,1H),7.66(d,J＝7.9Hz,2H),7.56(t,J＝7.1Hz,3H),7.51(m,3H),7.27(t,J＝8.8Hz,2H),4.50(d,J＝8.5Hz,1H),3.73–3.64(m,1H),3.10(t,J＝13.8Hz,1H),2.83(t,J＝13.2Hz,1H),2.54(s,1H),2.29(s,6H),1.94–1.86(m,1H),1.80–1.73(m,1H).13C NMR(151MHz,DMSO-d6)δ168.81,157.38,146.93,138.61,137.46,130.84,130.27,130.18,129.77,129.21,127.76,126.58,126.51,123.45,119.16,117.37,116.66,61.87,41.43,26.81.HRMS(ESI)m/z calcd for C29H30N6O2[M-H]-:493.2357found:493.2377.
实施例18：N-(1-甲基哌啶-4-基)-3-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A18)的制备
按照实施例1的合成方法，以2q(0.15g,0.40mmol)和苯胺(0.039g,0.40mmol)原料，得橙色固体A18 0.099g，收率为51.5％。Following general procedure B and C,compound 3r was obtained(0.155g,60.8％).1H NMR(600MHz,CDCl3-d)δ14.02(s,1H),8.43(s,1H),8.24(d,J＝7.1Hz,3H),7.65(d,J＝7.3Hz,1H),7.55–7.51(m,3H),7.50(d,J＝3.9Hz,2H),7.48(d,J＝5.5Hz,3H),7.46(d,J＝7.6Hz,2H),4.07–4.03(m,1H),2.93–2.88(m,2H),2.35(s,3H),2.23(s,2H),2.10(d,J＝11.3Hz,2H),1.72–1.66(m,2H).13C NMR(151MHz,CDCl3-d)δ166.57,158.24,146.90,141.01,138.29,135.81,130.16,129.99,129.82,129.29,128.62,127.54,127.00,126.32,123.98,121.33,116.81,116.21,54.59,46.76,46.20,32.29.HRMS(ESI)m/z calcd for C28H28N6O2Na[M+Na]+:503.2166found:503.2194.
实施例19：2-(3-(4-甲基哌嗪-1-羰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A19)的合成
按照实施例1的合成方法，以2r(0.15g,0.41mmol)和苯胺(0.041g,0.41mmol)原料，得橙色固体A19 0.087g，收率为45.5％。1H NMR(600MHz,DMSO-d6)δ13.63(s,1H),8.21–8.19(m,2H),8.12(dd,J＝8.2,1.2Hz,1H),8.07(s,1H),7.67(d,J＝7.7Hz,2H),7.60–7.58(m,2H),7.57(d,J＝1.7Hz,1H),7.54(d,J＝7.2Hz,1H),7.52–7.50(m,2H),7.30–7.26(m,2H),3.66(s,2H),3.38(s,2H),2.40(s,2H),2.31(s,2H),2.22(s,3H).13C NMR(151MHz,DMSO-d6)δ168.85,157.55,146.84,141.93,138.39,137.27,130.47,130.44,130.25,129.85,129.29,127.72,126.80,126.67,123.82,119.28,117.10,116.85,46.02,26.81.HRMS(ESI)m/z calcd for C27H27N6O2[M+H]+:467.2190found:467.2220.
实施例20：4-(2-(4-(4-甲基哌嗪-1-基)苯基)肼基)-2,5-双苯基-2,4-二氢-3H-吡唑-3-酮(A20)的制备
步骤A：2,5-双苯基-2,4-二氢-3H-吡唑-3-酮(3)的制备
按照实施例1中中间体1a的合成方法，以苯甲酰乙酸乙酯(1.0g,5.2mmol)和苯肼(0.61g,5.6mmol)为原料，得到白色固体3 0.78g，收率63.5％。
步骤B：4-(2-(4-(4-甲基哌嗪-1-基)苯基)肼基)-2,5-双苯基-2,4-二氢-3H-吡唑-3-酮(A20)的制备
按照实施例1的合成方法，以中间体3(0.15g,0.42mmol)和4-(4-甲基-1-哌嗪基)苯胺(0.12g,0.42mmol)原料，得橙色固体A20 0.18g，收率为65.2％。1H NMR(600MHz,DMSO-d6)δ13.93(s,1H),8.21–8.17(m,2H),8.06(d,J＝7.7Hz,2H),7.60–7.54(m,4H),7.51(t,J＝8.0Hz,3H),7.27(t,J＝7.4Hz,1H),7.07(d,J＝9.1Hz,2H),3.24–3.19(m,4H),2.49–2.45(m,4H),2.24(s,3H).13C NMR(151MHz,DMSO-d6)δ157.69,150.05,146.02,138.48,130.80,130.04,129.44,129.12,127.46,125.48,124.91,118.60,118.27,116.21,54.76,48.00,46.05.HRMS(ESI)m/z calcd for C26H27N6O[M+H]+439.2241,found:439.2279.
实施例21：4-(2-(3,5-双(三氟甲基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A21)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和3,5-双(三氟甲基)苯胺(0.087g,0.38mmol)原料，得橙色固体A21 0.16g，收率为66.8％。1H NMR(600MHz,CDCl3-d)δ14.02(s,1H),8.20(dd,J＝7.4,2.0Hz,2H),8.17(d,J＝8.6Hz,2H),7.87(s,2H),7.71(s,1H),7.56(d,J＝8.4Hz,3H),7.54(d,J＝1.1Hz,2H),3.31–3.22(m,1H),2.75(s,6H),2.26(d,J＝8.9Hz,2H),1.75(dd,J＝4.2,2.7Hz,2H).13C NMR(151MHz,DMSO-d6)δ169.09,156.18,147.76,139.67,132.11,131.90,131.68,131.46,130.85,130.35,128.99,128.55,128.00,126.37,124.56,122.75,120.95,118.12,117.92,62.49,30.89.HRMS(ESI)m/zcalcd for C31H29F6N6O2[M+H]+:631.2251found:631.2285.
实施例22：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-3-甲氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A22)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和4-氟-3-甲氧基苯胺(0.054g,0.38mmol)原料，得橙色固体A22 0.14g，收率为67.9％。1H NMR(600MHz,DMSO-d6)δ12.51(s,1H),8.19(d,J＝7.3Hz,2H),8.15(d,J＝8.5Hz,2H),7.55–7.51(m,4H),7.48(dd,J＝9.2,4.6Hz,2H),7.30(dd,J＝10.8,8.8Hz,1H),7.23–7.19(m,1H),3.90(s,3H),3.06(d,J＝8.2Hz,1H),2.83(d,J＝10.9Hz,1H),2.67(s,1H),2.36(s,6H),1.85(s,2H),1.44(dt,J＝11.7,8.1Hz,2H).13CNMR(151MHz,DMSO-d6)δ169.07,156.83,150.83,149.22,148.29,147.27,139.89,132.18,131.49,129.88,128.99,128.40,128.08,127.88,125.63,117.85,116.98,109.91,103.43,62.05,56.56,56.35,41.24.HRMS(ESI)m/z calcd for C30H32FN6O3[M+H]+:543.2514found:543.2547.
实施例23：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-2-异丙氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A23)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和4-氟-2-异丙氧基苯胺(0.064g,0.38mmol)原料，得橙色固体A23 0.11g，收率为50.7％。1H NMR(600MHz,DMSO-d6)δ13.98(s,1H),8.21(d,J＝7.3Hz,2H),8.10(d,J＝8.3Hz,2H),7.77(dd,J＝8.7,6.1Hz,1H),7.59–7.52(m,6H),,7.28–7.25(m,1H),6.99(td,J＝8.5,1.8Hz,1H),4.85(dd,J＝11.9,6.0Hz,1H),3.10(d,J＝19.0Hz,2H),2.79(dd,J＝49.4,9.3Hz,2H),2.57(dd,J＝6.3,2.6Hz,1H),2.37(s,6H),1.93–1.82(m,2H),1.45(d,J＝8.6Hz,2H),1.40(d,J＝5.9Hz,6H).13C NMR(151MHz,DMSO-d6)δ168.90,158.26,147.97,147.90,146.50,139.02,133.06,132.00,130.51,130.40,129.32,129.13,128.53,127.65,127.45,118.37,116.29,108.99,108.83,103.43,103.25,72.90,62.02,41.08,22.23.HRMS(ESI)m/z calcd for C32H35FN6O3[M+H]+:571.2827found:571.2820.
实施例24：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(3,4,5-三甲氧基苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A24)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和3,4,5-三甲氧基苯胺(0.070g,0.38mmol)原料，得橙色固体A24 0.15g，收率为67.5％。1H NMR(600MHz,DMSO-d6)δ12.76(s,1H),8.18(dd,J＝33.0,7.9Hz,4H),7.53(dd,J＝12.4,7.8Hz,4H),7.50–7.45(m,1H),7.03(s,2H),3.84(s,6H),3.68(s,3H),3.46–3.22(m,4H),2.56(dd,J＝10.7,6.3Hz,1H),2.31(s,6H),1.83(s,2H),1.46–1.39(m,2H).13C NMR(151MHz,DMSO-d6)δ169.06,156.87,154.02,147.24,139.89,136.17,132.27,130.11,129.90,128.99,128.39,127.89,125.23,117.86,95.46,61.95,60.68,56.26,49.07,41.45.HRMS(ESI)m/z calcd forC32H36N6O5[M+H]+:585.2820found:585.2825
实施例25：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(萘-2-基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A25)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和2-萘胺(0.054g,0.38mmol)原料，得橙色固体A25 0.11g，收率为53.1％。1H NMR(600MHz,DMSO-d6)δ13.07(s,1H),8.25(d,J＝7.4Hz,2H),8.16(d,J＝8.5Hz,2H),8.09(s,1H),8.05(d,J＝8.9Hz,1H),7.97(d,J＝8.2Hz,1H),7.94(d,J＝8.1Hz,1H),7.90(dd,J＝8.8,1.9Hz,1H),7.60(t,J＝7.6Hz,2H),7.57–7.52(m,4H),7.49(t,J＝7.5Hz,1H),4.60–4.34(m,1H),3.86–3.61(m,1H),3.14–2.99(m,1H),2.92–2.76(m,1H),2.61(d,J＝1.6Hz,1H),2.33(s,6H),1.91–1.76(m,2H),1.46–1.41(m,2H).13CNMR(151MHz,DMSO-d6)δ169.00,157.33,147.09,139.58,133.82,132.54,131.85,131.16,130.23,130.11,129.18,129.14,128.45,128.35,128.33,127.85,127.55,126.43,126.23,118.03,116.54,115.42,61.98,41.34.HRMS(ESI)m/z calcd forC33H32N6O2[M+H]+:545.2660found:545.2656.
实施例26：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-羟基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A26)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和4-羟基苯胺(0.054g,0.38mmol)原料，得橙色固体A26 0.14g，收率为72.2％。1H NMR(600MHz,DMSO-d6)δ9.86(s,1H),8.16(dd,J＝36.7,7.9Hz,4H),7.59–7.50(m,7H),6.91(d,J＝8.8Hz,2H),3.83–3.65(m,1H),3.16–3.03(m,1H),2.93–2.74(m,2H),2.42(s,6H),1.98–1.82(m,2H),1.51–1.44(m,2H).13CNMR(151MHz,DMSO-d6)δ168.88,157.74,157.03,146.60,139.30,132.57,130.73,130.14,129.14,128.41,127.55,124.81,118.92,117.97,116.73,62.00,40.80.HRMS(ESI)m/z calcd for C29H31N56O3[M+H]+511.2452,found:511.2443.
实施例27：4-(2-(4-(苄氧基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A27)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和4-(苄氧基)苯胺(0.076g,0.38mmol)原料，得橙色固体A27 0.13g，收率为56.9％。1H NMR(600MHz,DMSO-d6)δ13.83(s,1H),8.20(d,J＝7.4Hz,2H),8.14(d,J＝8.5Hz,2H),7.65(d,J＝8.9Hz,2H),7.58(t,J＝7.7Hz,4H),7.55–7.52(m,1H),7.47(d,J＝7.4Hz,2H),7.41(t,J＝7.5Hz,2H),7.35(t,J＝7.2Hz,1H),7.17(d,J＝8.9Hz,2H),5.17(s,2H),3.43(t,J＝10.8Hz,1H),3.06(d,J＝6.9Hz,4H),2.71(s,6H),2.04–1.94(m,2H),1.69–1.63(m,2H).13C NMR(151MHz,DMSO-d6)δ169.00,157.77,157.54,146.80,139.35,137.27,132.49,130.56,130.41,130.11,129.29,128.95,128.62,128.41,128.24,127.67,125.62,118.74,118.08,116.47,70.07,62.40,45.80,40.54.HRMS(ESI)m/z calcd for C36H36FN6O3[M+H]+:601.2922found:601.2997.
实施例28：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(4-(哌啶-1-甲酰基)苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A28)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和4-(哌啶-1-甲酰基)苯胺(0.076g,0.38mmol)原料，得橙色固体A28 0.098g，收率为42.6％。
实施例29：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A29)的制备
按照实施例1的合成方法，以2d(0.15g,0.38mmol)和3-氨基吡啶(0.036g,0.38mmol)原料，得橙色固体A29 0.12g，收率为63.7％。1H NMR(600MHz,DMSO-d6)δ12.00(s,1H),8.88(d,J＝2.0Hz,1H),8.42(d,J＝4.5Hz,1H),8.20(d,J＝7.5Hz,2H),8.16(d,J＝8.5Hz,2H),8.02(d,J＝8.2Hz,1H),7.56(t,J＝7.7Hz,4H),7.52–7.48(m,2H),3.96–3.65(m,1H),3.44(dt,J＝10.6,6.9Hz,1H),3.21–3.04(m,2H),2.94–2.75(m,1H),2.58(s,6H),2.05–1.89(m,2H),1.56(qd,J＝12.1,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ169.11,156.74,147.34,146.84,140.56,139.86,132.05,131.31,130.01,129.09,128.50,127.90,127.24,124.72,124.37,117.92,62.34,56.49,55.40.HRMS(ESI)m/z calcd for C28H30N7O2[M+H]+:496.2455found:496.2478.
实施例30：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-((苯基氨基)亚甲基)-2,4-二氢-3H-吡唑-3-酮(A30)的制备
步骤A：4-((二甲氨基)亚甲基)-2-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(4)的制备
将2d(0.3g,0.77mmol)和N,N-二甲基甲酰胺二甲基缩醛(0.11g,0.92mmol,DMF-DMA)溶于10mL 1,4-二氧六环，回流反应12h。反应结束后，将反应液倒入150mL水中，乙酸乙酯萃取(60mL*3)，减压蒸馏，后经柱层析纯化(二氯甲烷/甲醇，V:V＝30:1)得白色固体40.15g，收率为43.7％。
步骤B:2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-((苯基氨基)亚甲基)-2,4-二氢-3H-吡唑-3-酮(A30)的制备
将4(0.1g,0.22mmol)和苯胺(0.022g,0.22mmol)溶于5mL无水乙醇，随后加入1滴哌啶，回流反应12h。反应结束后，将反应液倒入30mL冰水中，乙酸乙酯萃取(15mL*3)，收集有机相、使用饱和氯化钠溶液洗涤(10mL*3)，后经柱层析分离纯化(二氯甲烷/甲醇，V:V＝30:1)，得黄绿色固体A30 0.065g，收率47.8％。1H NMR(600MHz,DMSO-d6)δ11.97(s,1H),8.60(s,1H),8.17(d,J＝8.6Hz,2H),7.90(d,J＝6.7Hz,2H),7.61(d,J＝7.8Hz,2H),7.57–7.53(m,2H),7.53–7.49(m,3H),7.46(t,J＝7.9Hz,2H),7.27(t,J＝7.3Hz,1H),4.62–4.21(m,1H),3.89–3.54(m,1H),3.13–2.96(m,1H),2.92–2.74(m,1H),2.44–2.36(m,1H),2.21(s,6H),1.89–1.71(m,2H),1.37(ddd,J＝22.6,10.9,2.4Hz,2H).13C NMR(151MHz,DMSO-d6)δ169.06,165.92,150.48,148.19,140.05,138.99,132.26,132.00,130.12,129.79,129.40,128.37,128.28,126.40,119.06,118.08,99.63,61.81,41.78.HRMS(ESI)m/zcalcd for C30H32N5O2[M+H]+494.2551,found:494.2599.
实施例31：4-((苄基氨基)亚甲基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A31)的制备
按照实施例31的合成方法，以苄胺(0.024g,0.22mmol)替换苯胺，得黄绿色固体A310.073g，收率为65.4％。1H NMR(600MHz,DMSO-d6)δ10.47–10.38(m,1H),8.39(d,J＝13.6Hz,1H),8.14(d,J＝8.3Hz,2H),7.77(d,J＝7.1Hz,2H),7.56–7.48(m,3H),7.46(d,J＝8.3Hz,2H),7.41(s,4H),7.33(d,J＝5.4Hz,1H),4.78(d,J＝4.9Hz,2H),4.64–4.29(m,1H),3.84–3.62(m,1H),3.09–2.94(m,1H),2.90–2.74(m,1H),2.71–2.58(m,1H),2.33(s,6H),1.90–1.70(m,2H),1.41(d,J＝10.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ169.17,166.01,155.48,150.21,140.52,137.96,132.35,131.63,130.12,129.55,129.31,129.20,128.31,128.26,128.23,128.11,117.83,97.12,62.02,52.89,49.07,41.21.HRMS(ESI)m/z calcdfor C31H34N5O2[M+H]+508.2707,found:508.2765.
实施例32：4-环己亚基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A32)
将2d(0.3g,0.77mmol)和环己酮(0.090g,0.92mmol)溶于10mL 1,4-二氧六环，回流反应12h。反应结束后，将反应液倒入150mL水中，乙酸乙酯萃取(60mL*3)，减压蒸馏，后经柱层析纯化(二氯甲烷/甲醇，V:V＝30:1)，得白色固体A39 0.22g，收率为50.8％。HRMS(ESI)m/z calcd for C29H35N4O2 +[M+H]+471.2755,found:471.1363.
实施例33：4-(1-苄基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A33)
按照实施例32的合成方法，以N-苄基哌啶酮(0.17g,0.92mmol)为原料，得白色固体A33 0.32g，收率为55.5％。HRMS(ESI)m/z calcd for C35H40N5O2 +[M+H]+562.3177,found:562.2904.
实施例34：4-(1-(环丙基甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A34)的制备
步骤A：4-(1-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-氧代-3-苯基-1,5-二氢-4H-吡唑-4-亚基)哌啶-1-甲酸叔丁酯(5)的制备
按照实施例32的合成方法，以N-叔丁氧羰基-4-哌啶酮(0.18g,0.92mmol)为原料，得白色固体5 0.23g，收率为52.2％。1H NMR(600MHz,CDCl3-d)δ8.04(d,J＝8.7Hz,2H),7.79(d,J＝7.0Hz,2H),7.52–7.48(m,3H),7.46(dd,J＝11.4,4.6Hz,2H),4.81(d,J＝76.6Hz,1H),4.04–3.76(m,1H),3.64(dd,J＝18.7,8.7Hz,1H),3.56–3.52(m,1H),3.46–3.41(m,1H),3.39–3.33(m,1H),2.94(d,J＝102.5Hz,2H),2.55(s,1H),2.43–2.39(m,1H),2.38(s,6H),2.18–2.13(m,1H),1.97(ddd,J＝35.1,19.1,11.8Hz,2H),1.66(dd,J＝9.7,4.9Hz,2H),1.53(dd,J＝30.1,17.7Hz,2H),1.42(s,9H).
步骤B：4-(1-(环丙基甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A34)的制备
将中间体5(0.15g,0.26mmol)溶于10mL干燥的二氯甲烷中，冰浴下加入4mL三氟乙酸，室温反应0.5h，减压蒸馏除去有机试剂，得白色固体，不需纯化即可用于下一步反应。将制得的白色固体和环丙甲酸(0.022g,0.26mmol)溶于10mL二氯甲烷中，依次加入HATU(0.10g,0.27mmol)和DIEA (0.17g,1.3mmol)，室温反应1h。加入15mL饱和氯化铵溶液淬灭反应，二氯甲烷萃取(10mL*3)，收集有机相。有机相水洗、饱和氯化钠溶液洗涤，减压蒸干溶剂，经柱层析分离(二氯甲烷/甲醇，V:V＝30:1)得白色固体A34 0.081g，收率70.8％。
实施例35：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(1-(噻唑-4-甲酰基)哌啶-4-基亚基)-2,4-二氢-3H-吡唑-3-酮(A35)的制备
按照实施例34的合成方法，以噻唑-4-甲酸(0.034g,0.26mmol)为原料，得白色固体A350.075g，收率为49.5％。1H NMR(600MHz,DMSO-d6)δ9.12(d,J＝49.4Hz,1H),8.14(s,1H),7.98–7.94(m,2H),7.75(d,J＝15.3Hz,2H),7.57–7.51(m,5H),3.79–3.70(m,2H),3.64–3.54(m,2H),2.34(dd,J＝29.8,11.7Hz,2H),2.26(s,6H),2.22(d,J＝13.4Hz,2H),2.03–1.96(m,1H),1.78–1.56(m,4H),1.37(dd,J＝26.1,10.2Hz,4H).13C NMR(151MHz,DMSO-d6)δ168.72,167.42,162.45,154.97,154.50,150.76,139.05,133.21,132.08,131.00,130.02,129.28,128.28,128.13,127.70,124.95,118.53,73.07,66.42,61.76,41.42,40.44,26.94.HRMS(ESI)m/z calcd for C32H34N6O3S+[M+H]+:583.2436found,583.3265.
实施例36：4-(1-苯甲酰基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A36)的制备
按照实施例34的合成方法，以苯甲酸(0.032g,0.26mmol)为原料，得白色固体A360.069g，收率为46.1％。HRMS(ESI)m/z calcd for C35H38N5O3 +[M+H]+:576.2969,found:576.4133.
实施例37：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-羟基苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A37)的制备
按照实施例34的合成方法，以4-羟基苯甲酸(0.036g,0.26mmol)为原料，得白色固体A37 0.071g，收率为46.1％。1H NMR(600MHz,CDCl3-d)δ8.03(d,J＝8.1Hz,3H),7.78(t,J＝5.7Hz,3H),7.52–7.47(m,7H),4.75(s,1H),4.15(d,J＝14.1Hz,1H),3.94(d,J＝13.0Hz,1H),3.87(d,J＝14.2Hz,1H),3.72–3.54(m,3H),3.41(t,J＝11.6Hz,1H),2.65(dd,J＝21.8,13.3Hz,3H),2.42(s,6H),2.19(dd,J＝26.6,12.3Hz,2H),1.84–1.69(m,4H).HRMS(ESI)m/z calcd for C35H38N5O4 +[M+H]+:592.2918,found:592.2919.
实施例38：4-(1-(5-氯-1H-吲哚-2-甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A38)的制备
按照实施例34的合成方法，以5-氯吲哚-2-羧酸(0.051g,0.26mmol)为原料，得白色固体A38 0.082g，收率为48.6％。1H NMR(600MHz,DMSO-d6)δ11.75(s,1H),7.97(d,J＝8.4Hz,2H),7.78(d,J＝5.3Hz,2H),7.61(s,1H),7.54(d,J＝8.4Hz,5H),7.40(d,J＝8.6Hz,1H),7.17(d,J＝8.5Hz,1H),6.77(s,1H),3.89–3.74(m,2H),3.71–3.60(m,2H),2.41(d,J＝15.4Hz,4H),2.26(s,6H),1.99(dt,J＝18.3,6.1Hz,1H),1.82–1.66(m,4H),1.43–1.31(m,4H).13C NMR(151MHz,DMSO-d6)δ174.65,168.72,167.44,161.97,154.95,139.06,134.73,133.23,132.05,131.48,131.03,130.02,129.29,128.30,128.21,127.69,124.52,123.74,120.77,118.52,114.02,104.13,73.09,66.48,61.76,41.44,40.44,26.98.
实施例39：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-(10α-二氢青蒿素-10-基)氧基)苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A39)的制备
按照实施例34的合成方法，以4-(10α-二氢青蒿素-10-基)氧基)苯甲酸(0.1g,0.26mmol)为原料，得白色固体A39 0.093g，收率为41.7％。1H NMR(600MHz,DMSO-d6)δ7.96(d,J＝8.2Hz,2H),7.74(s,2H),7.53(d,J＝8.1Hz,5H),7.34(d,J＝6.3Hz,2H),7.07(d,J＝6.6Hz,2H),5.59(s,1H),5.40(s,1H),3.71–3.51(m,4H),2.61–2.59(m,1H),2.47–2.42(m,1H),2.37–2.29(m,2H),2.24(s,6H),2.21–2.16(m,2H),2.00(d,J＝14.6Hz,2H),1.92–1.85(m,2H),1.85–1.77(m,4H),1.74–1.68(m,2H),1.62(d,J＝11.2Hz,2H),1.54–1.51(m,1H),1.40–1.36(m,4H),1.34(s,1H),1.28(s,3H),1.21–1.19(m,1H),0.95(d,J＝7.0Hz,3H),0.90(d,J＝6.1Hz,3H).13CNMR(151MHz,DMSO-d6)δ169.36,168.70,167.40,158.22,154.99,139.03,133.27,132.03,131.00,129.29,129.28,129.11,128.29,127.66,118.53,116.87,116.83,116.80,103.95,99.54,99.49,87.83,80.72,73.18,66.37,61.72,44.00,41.54,40.44,36.84,36.33,34.48,30.69,26.72,25.96,24.52,24.29,20.54,12.90.
实施例40：2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-甲基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A40)的制备
按照实施例1的制备方法，以7a(0.15g,0.46mmol)和苯胺(0.045g,0.46mmol)为原料，得橙色固体A40 0.13g，收率为65.3％。1H NMR(600MHz,CDCl3-d)δ13.56(s,1H),8.04(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.44(q,J＝8.3Hz,4H),7.23(t,J＝6.6Hz,1H),4.85–4.54(m,1H),4.00–3.69(m,1H),3.07–2.79(m,2H),2.42(s,1H),2.38(s,3H),2.32(s,6H),1.95–1.80(m,2H),1.56–1.44(m,2H).13C NMR(151MHz,CDCl3-d)δ169.80,157.90,148.99,140.95,139.02,132.31,129.65,128.17,127.92,125.95,117.92,115.83,62.09,41.62,29.62,11.75.HRMS(ESI)m/z calcd for C24H29N6O2[M+H]+433.2347,found:433.2394.
实施例41：5-环丙基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A41)的制备
按照实施例1的制备方法，以7b(0.15g,0.42mmol)和苯胺(0.042g,0.42mmol)为原料，得橙色固体A41 0.15g，收率为77.9％。1H NMR(600MHz,DMSO-d6)δ7.99(d,J＝8.4Hz,2H),7.61(d,J＝7.9Hz,2H),7.50(d,J＝8.5Hz,2H),7.47(t,J＝7.7Hz,2H),7.25(t,J＝7.3Hz,1H),4.65–4.37(m,1H),3.83–3.60(m,1H),2.91–2.70(m,2H),2.43(s,6H),2.19–2.15(m,1H),1.95–1.78(m,2H),1.47(dd,J＝20.9,10.0Hz,2H),1.09(d,J＝5.8Hz,4H),0.85(t,J＝5.8Hz,1H).13C NMR(151MHz,DMSO-d6)δ168.98,157.23,153.55,142.02,139.24,132.33,130.12,128.48,128.35,127.30,126.29,117.57,116.80,62.10,40.73,8.71.HRMS(ESI)m/z calcd for C24H29N6O2[M+H]+459.2503,found:459.2501.
本发明产物药理作用研究
实施例42：荧光偏振分析法测定化合物对Bcl-2、Mcl-1、Bcl-xL的抑制常数
以维奈妥拉、A-1210477和BTSA1为对照药，采用荧光偏振(FP)方法考察目标化合物竞争抑制Bcl-2、Mcl-1、Bcl-xL蛋白与促凋亡蛋白Bim或Bid的BH3肽段(荧光素标记)结合的能力来评价其与靶蛋白的亲和力和选择性。荧光偏振信号由荧光分光光度计在激发光波长485nm和发射光波长为535nm的条件下检测。将系列浓度目标化合物与荧光素标记的Bim或Bid的BH3肽段与Bcl-2、Mcl-1、Bcl-xL蛋白一起在室温下培养20分钟后，检测其荧光偏振信号、计算该化合物的IC50，并根据测量中所使用的蛋白总浓度、荧光多肽的总浓度、蛋白-多肽复合物的解离常数以及检测化合物的IC50，计算检测化合物的竞争性抑制常数Ki。
实施例43：体外细胞抗肿瘤活性测试
1)细胞培养
人肺癌细胞(A549、H1581)、结肠癌细胞(HCT-116)和白血病细胞(THP-1、K562、MV4-11)培养于RMPI-1640培养基，人乳腺癌细胞MDA-MB-231培养基。培养基中加入10％(V/V)加热灭活的胎牛血清、100μg/mL的链霉素和100U/mL的青霉素。实验所用细胞均于37℃、5％ CO2饱和湿度的培养箱中培养
2)生长抑制活性测试
化合物对贴壁细胞的生长抑制活性采用MTT法进行测试。取对数生长期细胞进行实验，将100μL具有一定密度的(0.5-3.0×104)细胞悬液埋入96孔板中，培养24h使其贴壁。加入100μL特定浓度的待测药物，培养96h。再向每孔加入50μL的MTT溶液(2mg/mL)，继续培养3.5h。甩板，使96孔板中的液体全部弃去，并倒扣在滤纸上充分吸干。随后在每孔加入200mL DMSO溶液，于微型振荡器振荡10min。用酶标仪测定每孔于570nm波长下的吸光度，根据以下公式算出抑制率，并计算出半数生长抑制浓度GI50。
抑制率＝(1–A加药/A对照)×100％，A加药为加药孔吸光度，A对照为对照孔吸光度。
化合物对悬浮细胞的生长抑制活性采用台盼蓝染色法进行测试。取对数生长期细胞进行实验，将2mL具有一定密度的(5-10×104)细胞悬液埋入24孔板中，随即加入低于40μL特定浓度的待测药物，培养72h。将细胞液混匀，取50μL细胞悬液与等体积的台盼蓝溶液(4％)轻轻吹打混合，于显微镜下计数。活细胞是圆形、透亮的，死细胞则呈蓝色，总细胞数为活细胞和死细胞的总和。根据加药组和对照组总细胞数的比值可计算抑制率，通过相应浓度的抑制率求得半数生长抑制浓度GI50。
下表1、2、3为化合物分子水平以及细胞水平活性(实验数值为3次独立实验平均值)：
表1：优选化合物的细胞水平活性
表2：优选化合物的细胞水平活性
表3：优选化合物的分子水平活性
aN.A.代表无抑制活性(IC50>200μM)
实施例44：初步作用机制研究
Western Blot实验
A549细胞与特定浓度的药物孵育一定时间后，离心收集细胞，经RIPA裂解液冰上裂解30-60min，低温高速离心得到蛋白样品。BCA法测定蛋白浓度，取40μg蛋白利用非连续SDS聚丙烯酰胺凝胶电泳对蛋白样品进行分离。电泳结束后通过伯乐电转移装置将胶上蛋白转移到硝酸纤维素滤膜上。用含有0.2％的丽春红染料充分染色，得到不同分子量的蛋白条带。膜冲洗干净后加入5％脱脂奶粉室温封闭1h，特异性抗体与目标蛋白条带封闭于杂交袋中，4℃孵育过夜。TBST清洗膜上一抗后加入二抗，室温孵育1h。洗掉二抗后应用ECL发光试剂盒中A液和B液等体积混合涂于膜上，反应2min，与X光片置于暗盒中反应一定时间，经显影液和定影液处理。
如图1所示，优选实施例4化合物与阳性药BTSA1作用机制相同，均通过诱导Bax活化，促进Bax的线粒体转位和细胞色素c的释放，以诱导肿瘤细胞发生凋亡。
尽管已经通过特定实施方案描述了本发明，但修改和等价变化对于精通此领域的技术人员而言是显见的，且它们都包含在本发明范围内。
Claims (9)
1.一种吡唑-3-酮类衍生物或其药学上可接受的盐，其特征在于，所述吡唑-3-酮类衍生物通式为：
其中：
R1为H或
其中，R4为：
a表示在苯环上的取代位置，为3位或4位；
R2为甲基、乙基、丙基、丁基、环丙基、环丁基、环戊基、环己基、环戊基或苯基；
X为CH或N；
Y为NH或O；
或者，X、Y不存在，R3位于X的位置直接与双键相连接；
R3为
其中，R8、R9、R10、R11、R12各自独立地为氢、羟基、卤素、硝基、氨基、氰基、取代或为取代的C1-C4烷基或亚烷基、取代或为取代的C1-C4烷氧基或亚烷氧基、 所述取代或未取代的C1-C4烷基或亚烷基，或取代或未取代的C1-C4烷氧基或亚烷氧基中，所述的取代基选自卤素、羟基、氨基；
R13为
2.根据权利要求1所述的吡唑-3-酮类衍生物或其药学上可接受的盐，其特征在于，其为如下所述化合物中任一个：
N-(1-甲基哌啶-4-基)-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A01)
2-(4-(4-甲哌嗪-1-甲酰基)苯基)-5-苯基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A02)
N-(1-苄基哌啶-4-基)-N-甲基-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A03)
2-(4-(4-二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A04)
4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)-N-(吡啶-4-基甲基)苯甲酰胺(A05)
5-苯基-4-(2-苯基肼基)-2-(4-(哌嗪-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A06)
(R)-2-(4-(3-氨基哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A07)
2-(4-(2,7-二氮杂螺[3.5]壬烷-2-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A08)
2-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)-2,7-二氮杂螺[3.5]壬烷-7-甲酸叔丁酯(A09)
2-(4-(4-(氧杂环丁-3-基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A10)
1-(4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰基)哌啶-4-酮(A11)
2-(4-(4-(甲基磺酰基)哌嗪-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A12)
N-(3-(二甲氨基)丙基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A13)
5-苯基-4-(2-苯基肼基)-2-(4-(哌啶-1-甲酰基)苯基)-2,4-二氢-3H-吡唑-3-酮(A14)
N,N-二甲氨基-4-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A15)
2-(4-([1,4'-双哌啶基]-1'-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A16)
2-(3-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A17)
N-(1-甲基哌啶-4-基)-3-(5-氧代-3-苯基-4-(2-苯基肼基)-4,5-二氢-1H-吡唑-1-基)苯甲酰胺(A18)
2-(3-(4-甲基哌嗪-1-羰基)苯基)-5-苯基-4-(2-苯基肼基)-2,4-二氢-3H-吡唑-3-酮(A19)
4-(2-(4-(4-甲基哌嗪-1-基)苯基)肼基)-2,5-双苯基-2,4-二氢-3H-吡唑-3-酮(A20)
4-(2-(3,5-双(三氟甲基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A21)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-3-甲氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A22)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-氟-2-异丙氧基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A23)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(3,4,5-三甲氧基苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A24)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(萘-2-基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A25)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(4-羟基苯基)肼基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A26)
4-(2-(4-(苄氧基)苯基)肼基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A27)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(4-(哌啶-1-甲酰基)苯基)肼基)-2,4-二氢-3H-吡唑-3-酮(A28)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A29)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-((苯基氨基)亚甲基)-2,4-二氢-3H-吡唑-3-酮(A30)
4-((苄基氨基)亚甲基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A31)
4-环己亚基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A32)
4-(1-苄基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A33)
4-(1-(环丙基甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A34)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-4-(1-(噻唑-4-甲酰基)哌啶-4-基亚基)-2,4-二氢-3H-吡唑-3-酮(A35)
4-(1-苯甲酰基哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A36)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-羟基苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A37)
4-(1-(5-氯-1H-吲哚-2-甲酰基)哌啶-4-基亚基)-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A38)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(1-(4-(10α-二氢青蒿素-10-基)氧基)苯甲酰基)哌啶-4-基亚基)-5-苯基-2,4-二氢-3H-吡唑-3-酮(A39)
2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-5-甲基-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A40)
5-环丙基-2-(4-(4-(二甲氨基)哌啶-1-甲酰基)苯基)-4-(2-(吡啶-3-基)肼基)-2,4-二氢-3H-吡唑-3-酮(A41)。
3.根据权利要求1所述的吡唑-3-酮类衍生物或其药学上可接受的盐，其特征在于，所述的药学上可接受的盐是指所述吡唑-3-酮类衍生物与盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、苯甲酸或琥珀酸形成的加成盐。
4.一种权利要求2所述的吡唑-3-酮类衍生物或其药学上可接受的盐的制备方法，其特征在于，包括如下步骤：
制备化合物A01-A20和A40-A41：其中，R2、R4为化合物A01-A20和A40-A41对应位置的相应基团；
制备化合物A21-A39：其中，R3、R13为化合物A21-A39对应位置的相应基团；
5.一种药物组合物，其特征在于，其为由权利要求1-3任一项所述的吡唑-3-酮类衍生物或其药学上可接受的盐作为活性成分，与药学上可接受的赋形剂混合制备成的组合物，并制备成临床上可接受的剂型，所述赋形剂是指能够用于药学领域的稀释剂、辅助剂或载体，所述剂型为注射剂、片剂或胶囊剂。
6.权利要求1-3任一项所述的吡唑-3-酮类衍生物或其药学上可接受的盐的应用，其特征在于，所述吡唑-3-酮类衍生物或其药学上可接受的盐作为活性成分，或单独使用，或与其他抗肿瘤药物联合使用，用于制备治疗和/或预防肿瘤的药物。
7.权利要求1-3任一项所述的吡唑-3-酮类衍生物或其药学上可接受的盐的应用，其特征在于，所述吡唑-3-酮类衍生物或其药学上可接受的盐作为活性成分，用于制备与Bcl-2蛋白家族失调相关疾病的药物。
8.根据权利要求7所述的应用，其特征在于，所述与Bcl-2蛋白家族失调相关的疾病为癌症，所述癌症为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性粒细胞白血病、慢性白血病、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌和/或胰腺癌。
9.权利要求5所述的药物组合物的应用，其特征在于，所述药物组合物用于制备与Bcl-2蛋白家族失调相关疾病的药物。
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