CN116924987A - 4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物及其制备方法和应用 - Google Patents
4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物及其制备方法和应用 Download PDFInfo
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- CN116924987A CN116924987A CN202310958426.7A CN202310958426A CN116924987A CN 116924987 A CN116924987 A CN 116924987A CN 202310958426 A CN202310958426 A CN 202310958426A CN 116924987 A CN116924987 A CN 116924987A
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Abstract
本发明公开了一系列结构新颖的4‑哌嗪硫脲苯磺酰胺‑1,8‑萘酰亚胺衍生物及它们的制备方法。申请人的试验结果表明，本发明部分目标化合物对CA IX具有良好的抑制活性，可用于抑制碳酸酐酶IX酶活性和/或过表达的药物；部分目标化合物对多种肿瘤细胞株具有较好的抗肿瘤活性，有望用于抗肿瘤药物的制备。
Description
技术领域
本发明涉及4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物及其制备方法和应用，属于医药技术领域。
背景技术
碳酸酐酶IX(CA IX)是一种跨膜、缺氧诱导的锌金属酶，可将二氧化碳转化为碳酸氢盐和质子，这种酸碱调节功能对于维持促进恶性细胞存活和生长的碱性细胞内pH值以及放大促进其侵袭和转移的细胞外酸中毒至关重要，表达碳酸酐酶IX的癌细胞通常代表异质性肿瘤中最具临床侵袭性的成分，使碳酸酐酶IX成为关键的生物标志物和主要的治疗靶点。目前，碳酸酐酶IX抑制剂主要有磺胺类和香豆素类这两种，其中磺胺类研究居多。公布号为CN115697981A的发明专利公开了如下所示的化合物2-(3-(2-甲基-6-(对甲苯基)吡啶-3-基)脲基)苯磺酰胺：同时指出该化合物是靶向碳酸酐酶的小分子，也是碳酸酐酶活性和过表达的抑制剂，可用作药剂，特别是用于治疗和/或预防与增殖性疾病相关的病症，例如癌症。
萘酰亚胺通常含有一个共平面的、π-缺陷的芳香系统和碱性侧链，主要作为DNA和拓扑异构酶靶向抗肿瘤药物。研究发现萘酰亚胺衍生物对多种肿瘤具有潜在的抗增殖活性，如乳腺癌、肝癌、神经胶质瘤、黑色素瘤等。一些有效的抗肿瘤药物，如米托萘胺(Mitonafide)、氨萘非特(Amonafide)、Ethonafide、Elinafide等，它们正处于临床II期实验。但一些因素阻碍了萘酰亚胺衍生物的进一步发展，如：CNS神经毒性和血液毒性；恶性干细胞可能逃脱这种药物的治疗；使肿瘤细胞对治疗药物产生耐药性，并使当前的抗癌疗法效果不佳，最终导致其失败。因此，本申请人尝试对萘酰亚胺进行结构修饰，以求开发多功能、高选择性的靶向碳酸酐酶IX抗肿瘤药物。
发明内容
本发明要解决的技术问题是提供一系列结构新颖且具有较好的生物活性的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物及其制备方法和应用。
为解决上述技术问题，本发明采用以下技术方案：
本发明所述的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物为具有下述式(I)所示结构的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐：
其中：
R表示氢原子、卤原子、羟基或巯基，或者是C1～C8的烷基、烯基或炔基，或者是卤代C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基、烯基或炔基，或者是氨基取代的C1～C8的烷基、烯基或炔基，或者是酰胺基取代的C1～C8的烷基、烯基或炔基，或者是羧基取代的C1～C8的烷基、烯基或炔基，或者是C1～C8的烷氧基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0～4之间的整数。
上述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物的通式结构中，各取代基优选如下：
R表示C1～C8的烷基，或者是羟基取代的C1～C8烷基，或者是氨基取代的C1～C8的烷基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0或2。
更进一步的，各取代基优选为：R表示2-吗啉基乙基、3-吗啉丙基、苄基、4-氟苄基、4-氯苄基、4-溴苄基、4-甲基苄基、4-甲氧基苄基、3,4,5-三甲氧基苄基、4-(三氟甲基)苄基、3,4-二羟基苯乙基、环己基、丁基、辛基、2-(二甲基氨基)乙基、2-(二乙基氨基)乙基或2-羟乙基；n表示0或2。
本发明所述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物的制备方法，主要包括以下步骤：取如下式(II)所示化合物和式(III)所示化合物置于有机溶剂中反应，所得反应物料回收溶剂，得到目标化合物粗品；
其中：
R表示氢原子、卤原子、羟基或巯基，或者是C1～C8的烷基、烯基或炔基，或者是卤代C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基、烯基或炔基，或者是氨基取代的C1～C8的烷基、烯基或炔基，或者是酰胺基取代的C1～C8的烷基、烯基或炔基，或者是羧基取代的C1～C8的烷基、烯基或炔基，或者是C1～C8的烷氧基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0～4之间的整数。
上述制备方法中，所述的有机溶剂优选为选自乙腈、丙酮、甲醇和二氯甲烷中的一种或两种以上的组合。所述有机溶剂的用量以能够溶解参加反应的原料为宜，通常情况下，以1mmol的式(III)所示化合物为基准，所有参加反应的原料通常用15～20mL的有机溶剂来溶解。
上述制备方法中，反应可以在加热或不加热的条件下进行，具体可以在常温至有机溶剂沸点之间的温度条件下进行。当反应在加热条件下进行时，可以加快反应的速率，因此，本申请进一步优选反应在35～60℃的条件下进行。反应通过TLC跟踪监测直至反应完全。根据申请人的经验，当反应在常温至35～60℃条件下进行时，反应时间控制在12～48h较为适宜。
上述方法制备得到的是目标化合物的粗产物，因此，本发明所述方法还包括对制得的目标化合物粗品进行纯化的步骤。具体的，可以采用现有常规的纯化方法对其进行纯化以提高各目标化合物的纯度，如采用硅胶柱层析的方式对粗产物进行纯化。柱层析时用于洗脱的洗脱剂优选为二氯甲烷与甲醇组成的混合溶剂。所述的混合溶剂中，二氯甲烷与甲醇的体积比优选为20：1～5：1，进一步优选为15：1～10：1。
本发明所述的制备方法中，各原料的用量配比为化学计量比，在实际操作时，式(II)所示化合物和式(III)所示化合物的摩尔比通常为1：1～1：2。本发明所述制备方法中，涉及的原料式(II)所示化合物为1,8-萘酰亚胺哌嗪衍生物，其可参考现有文献(Gui-BinLiang,Jian-Hua Wei,Hong Jiang,Ri-Zhen Huang,Jing-Ting Qin,Hui-Ling Wang,Heng-Shan Wang,Ye Zhang,Design,synthesis and antitumor evaluation of new 1,8-naphthalimide derivatives targeting nuclear DNA,European Journal of MedicinalChemistry,2021,210,112951.)进行合成，也可自选设计合成路线进行合成。本申请中优选按下述合成路线(合成路线中化合物S3和化合物S4中的BOC表示叔丁氧羰基)制备式(II)所示化合物：
具体的制备式(II)所示化合物的方法包括以下步骤：
1)取化合物S1(4-溴-1,8-萘二甲酸酐)和化合物S2(叔丁氧羰基哌嗪)置于溶剂A中，于加热条件下进行反应，反应物冷却，收集沉淀，得到化合物S3；
2)取化合物S3和伯胺类化合物(R-NH2，R的选择如前所述)置于溶剂A中，于加热条件下进行反应，反应物冷却，收集沉淀，得到化合物S4；
3)脱除化合物S4中的保护基(即叔丁氧羰基)，裸露N-H，即得到式(II)所示化合物。
上述制备式(II)所示化合物方法的步骤1)和2)中，涉及的溶剂A可以是醇类溶剂和/或非质子性溶剂，其中，所述的醇类溶剂具体可以是选自甲醇、乙醇、丙醇和正丁醇中的一种或两种以上的组合；所述的非质子性溶剂具体可以是选自乙二醇甲醚、N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、甲苯、四氯化碳和丙酮中的一种或两种以上的组合。所述有机溶剂的用量可根据需要确定，通常情况下，以1mmol的4-溴-1，8-萘二甲酸酐为基准，所有反应原料用10～50mL的有机溶剂来溶解。当有机溶剂的加入量较大时，反应完全后优选先回收部分溶剂A后(通常是除去占加入量40～50％的溶剂A)再将反应物冷却。
上述制备式(II)所示化合物方法的步骤1)和2)中，反应优选是在50℃至溶剂A的沸点温度之间进行，进一步优选采用回流反应。
上述制备式(II)所示化合物方法的步骤3)中，采用现有常规的方法脱化合物S4中的保护基，如在将化合物S4置于盐酸二氧六环或者是由二氯甲烷和三氟乙酸按2：1的体积比组成的混合溶剂中搅拌一定时间以脱除化合物S4上的叔丁氧羰基。为了进一步裸露N-H结构，可以再向反应体系中加入相对于化合物S4过量的二氯甲烷(作反应溶剂)和过量的碱性物质(如无机碱(如氢氧化钠、氢氧化钾等)或有机碱(如三乙胺、二乙胺等))以中和过量的酸。
上述制备式(II)所示化合物方法的步骤1)至3)所获得的都是化合物的粗产物，优选是对所得粗产物按常规进行纯化后再进行下一步骤。
本发明所述制备方法中，涉及的原料式(III)所示化合物为4-异硫氰基苯磺酰胺或其衍生物(如4-(2-异硫氰乙基)苯磺酰胺等)，其中，4-异硫氰基苯磺酰胺可直接从市场上购买得到，4-异硫氰基苯磺酰胺衍生物(如4-(2-异硫氰乙基)苯磺酰胺等)可以参考现有文献(Zhang B,Liu Z,Xia S,et al.Design,synthesis and biological evaluation ofsulfamoylphenyl-quinazoline derivatives as potential EGFR/CA IX dualinhibitors[J].Eur J Med Chem,2021,216:113300.)进行合成。
申请人通过试验发现本发明部分衍生物对CA IX具有良好的抑制活性，因此，本发明还包括上述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐在制备用于抑制碳酸酐酶IX酶活性和/或过表达的药物中的应用。另外，申请人通过实验还发现本发明部分衍生物对多种肿瘤细胞株具有较好的抗肿瘤活性，因此，本发明还包括上述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐在制备治疗肿瘤的药物中的应用，进一步是在制备治疗乳腺癌、肺癌或结肠癌的药物中的应用。
进一步的，本发明还包括一种药物组合物，其包括作为活性成分的治疗上有效剂量的上述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐，以及药学上可接受的载体。药物组合物的剂型可以是本领域惯用的剂型，例如片剂、丸剂、颗粒剂、针剂等。本发明所述药物的施用量可根据用药途径、患者的年龄、体重、所治疗的疾病的类型和严重程度等变化，其日剂量可以是0.01～10mg/kg体重，优选0.1～5mg/kg体重。可以一次或多次施用。
与现有技术相比，本发明提供了一系列结构新颖的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物及它们的制备方法。申请人的试验结果表明，本发明部分目标化合物对CA IX具有良好的抑制活性，可用于抑制碳酸酐酶IX酶活性和/或过表达的药物；部分目标化合物对多种肿瘤细胞株具有较好的抗肿瘤活性，有望用于抗肿瘤药物的制备。
附图说明
图1为透射电镜观察不同浓度的化合物I32处理MDA-MB-231细胞24h后亚细胞结构图。
图2为倒置荧光显微镜观察不同浓度的化合物I32对MDA-MB-231细胞ROS水平的影响图。
图3为倒置荧光显微镜下观察不同浓度的化合物I32对MDA-MB-231细胞JC-1染色影响图。
图4为Annexin V/PI双染法检测不同浓度的化合物I32诱导细胞MDA-MB-231凋亡率图。
图5为不同浓度的化合物I32抑制MDA-MB-231细胞的迁移能力图。
图6为化合物I32在4T1异种移植瘤模型中的体内抗肿瘤作用图(连续21天，每3天给小鼠口服载体或化合物I32(15或30mg/kg))；其中，(A)给予化合物I32连续21天后分离的肿瘤组织的照片，(B)在化合物I32治疗结束时切除的肿瘤的重量，(C)每3天测量一次不同治疗组肿瘤体积的变化，(D)化合物I32对4T1异种移植小鼠模型体重的影响。
图7为不同浓度的化合物I32抑制肺和肝的转移H&E染色图(放大倍数，×200)。
具体实施方式
为了更好的解释本发明的技术方案，下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
以下各实施例中涉及的式(II)所示化合物按下述合成路线进行制备：
R表示氢原子、卤原子、羟基或巯基，或者是C1～C8的烷基、烯基或炔基，或者是卤代C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基、烯基或炔基，或者是氨基取代的C1～C8的烷基、烯基或炔基，或者是酰胺基取代的C1～C8的烷基、烯基或炔基，或者是羧基取代的C1～C8的烷基、烯基或炔基，或者是C1～C8的烷氧基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物。
具体的制备方法包括以下步骤：
1)取化合物S1(4-溴-1,8-萘二甲酸酐，10mmol)和化合物S2(叔丁氧羰基哌嗪，mmol)置于圆底烧瓶中，加入乙二醇甲醚(50mL)，回流反应3h(TLC监测反应)，反应完成后，过滤反应液，滤饼用乙醇重结晶过夜，过滤，得到化合物S3(黄色固体)；
2)取化合物S3(1mmol)和伯胺类化合物(R-NH2，1.2mmol)置于圆底烧瓶中，加入乙醇(50mL)，回流反应3h(TLC监测反应)，反应完成后，所得物料上硅胶柱纯化(二氯甲烷/甲醇＝10/1，体积比)，得到化合物S4(黄色固体)；
3)取化合物S4(1mmol)置于圆底烧瓶中，加入盐酸二氧六环(10～20mL)搅拌1h，旋干，然后加入二氯甲烷(20mL)和三乙胺(10mL)反应(TLC监测)，反应完成后，加入适量水萃取，收集有机相并旋干，即得到式(II)所示化合物。
以下实施例中涉及的4-(2-异硫氰乙基)苯磺酰胺按下述合成路线进行制备：
在含有50mL THF(四氢呋喃)的支口瓶中加入10mmol DCC(二环己基碳二亚胺)、65mmol CS2和10mmol 4-(2-氨乙基)苯磺酰胺，室温下搅拌过夜(TLC监测反应)，反应完成后，将所得反应物料过滤并用THF洗涤，收集滤液，浓缩后经硅胶柱纯化(二氯甲烷/甲醇＝100/1，体积比)，即得到4-(2-异硫氰乙基)苯磺酰胺。
实施例1
按下述合成路线合成本发明所述的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物。
I1：R＝2-吗啉基乙基，n＝0； I18：R＝2-吗啉基乙基，n＝2；
I2：R＝3-吗啉丙基，n＝0； I19：R＝3-吗啉丙基，n＝2；
I3：R＝苄基，n＝0； I20：R＝苄基，n＝2；
I4：R＝4-氟苄基，n＝0； I21：R＝4-氟苄基，n＝2；
I5：R＝4-氯苄基，n＝0； I22：R＝4-氯苄基，n＝2；
I6：R＝4-溴苄基，n＝0； I23：R＝4-溴苄基，n＝2；
I7：R＝4-甲基苄基，n＝0； I24：R＝4-甲基苄基，n＝2；
I8：R＝4-甲氧基苄基，n＝0； I25：R＝4-甲氧基苄基，n＝2；
I9：R＝3,4,5-三甲氧基苄基，n＝0； I26：R＝3,4,5-三甲氧基苄基，n＝2；
I10：R＝4-(三氟甲基)苄基，n＝0； I27：R＝4-(三氟甲基)苄基，n＝2；
I11：R＝3,4-二羟基苯乙基，n＝0； I28：R＝3,4-二羟基苯乙基，n＝2；
I12：R＝环己基，n＝0； I29：R＝环己基，n＝2；
I13：R＝丁基，n＝0； I30：R＝丁基，n＝2；
I14：R＝辛基，n＝0； I31：R＝辛基，n＝2；
I15：R＝2-(二甲基氨基)乙基，n＝0； I32：R＝2-(二甲基氨基)乙基，n＝2；
I16：R＝2-(二乙基氨基)乙基，n＝0； I33：R＝2-(二乙基氨基)乙基，n＝2；
I17：R＝2-羟乙基，n＝0； I34：R＝2-羟乙基，n＝2。
具体的制备方法为：在圆底烧瓶中，加入1mmol式(II)所示化合物、1mmol式(III)所示化合物(4-异硫氰基苯磺酰胺或4-(2-异硫氰乙基)苯磺酰胺)和20mL乙腈，于50℃条件下搅拌反应12～18h(TLC监测反应)，反应完成后，将反应物料拌样，经硅胶柱纯化(二氯甲烷/甲醇＝10/1，体积比)，得到目标化合物I。不同的目标产物及其表征如下：
4-(2-(2-吗啉基乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I1):Yield 62％,as a yellow solid.Mp:223.1-224.5℃.1H NMR(400MHz,DMSO-d6):δ＝9.76(s,1H),8.55(d,J＝8.4Hz,1H),8.47(d,J＝7.2Hz,1H),8.42(d,J＝8.4Hz,1H),7.82(t,J＝8.0Hz,1H),7.75(d,J＝8.8Hz,2H),7.52(d,J＝8.8Hz,2H),7.37(d,J＝8.0Hz,1H),7.29(s,2H),4.24(t,J＝4.0Hz,4H),4.16(t,J＝6.8Hz,2H),3.52(t,J＝4.0Hz,4H),2.55(t,J＝6.8Hz,2H),2.45ppm(t,J＝4.8Hz,4H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.53,162.99,154.97,144.26,138.96,132.15,130.77,130.69,129.10,126.23,125.84,125.34,123.99,122.51,115.90,115.42,66.25,55.66,53.43,52.15,48.33,36.64ppm.HR-MS(m/z)(ESI):calcd for C29H32N6O5S2[M+H]+:609.1948；found:609.1941.
4-(2-(3-吗啉丙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I2):Yield 70％,as a yellow solid.Mp:211.8-213.2℃.1HNMR(400MHz,DMSO-d6):δ＝9.79(s,1H),8.52(d,J＝8.4Hz,1H),8.44(d,J＝7.2Hz,1H),8.36(d,J＝8.4Hz,1H),7.29(t,J＝8.0Hz,1H),7.75(d,J＝8.4Hz,2H),7.53(d,J＝8.4Hz,2H),7.35(d,J＝8.0Hz,1H),7.30(s,2H),4.25(t,J＝4.8Hz,4H),4.06(t,J＝7.2Hz,2H),3.34(t,J＝4.8Hz 4H),2.35(t,J＝6.4Hz,4H),2.29(s,4H),1.83-1.71ppm(m,2H).13C NMR(100MHz,DMSO-d6):δ＝181.69,163.62,163.09,154.86,144.25,138.99,132.03,130.65,130.54,129.09,126.18,125.85,125.31,124.03,122.64,116.09,115.36,66.11,55.98,53.18,52.19,48.38,38.17,24.09ppm.HR-MS(m/z)(ESI):calcd for C30H34N6O5S2[M+H]+:623.2105；found:623.2104.
4-(2-苄基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I3):Yield 77％,as a yellow solid.Mp:262.8-263.3℃.1HNMR(400MHz,DMSO-d6):δ＝9.77,(s,1H),8.54(d,J＝8.4Hz,1H),8.47(d,J＝6.8Hz,1H),8.38(d,J＝8.4Hz,1H),7.84-7.74(m,3H),7.54(d,J＝8.8Hz,2H),7.37-7.20(m,8H),5.22(s,2H),4.25(s,4H),3.36ppm(s,4H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.56,163.01,155.12,144.20,138.98,137.52,132.36,130.97,130.86,129.15,128.36,127.51,127.02,126.20,125.84,125.29,124.00,122.36,115.65,115.38,52.13,48.32,42.74ppm.HR-MS(m/z)(ESI):calcd for C30H27N5O4S2[M+H]+:586.1577；found:586.1580.
4-(2-(4-氟苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I4):Yield 52％,as a yellow solid.Mp:204.1-206.5℃.1HNMR(400MHz,DMSO-d6):δ＝9.78(s,1H),8.56(d,J＝8.4Hz,1H),8.49(d,J＝7.2Hz,1H),8.41(d,J＝8.0Hz,1H),7.83(t,J＝8.0Hz,1H),7.75(d,J＝8.8Hz,2H),7.52(d,J＝8.4Hz,2H),7.45–7.35(m,3H),7.29(s,2H),7.11(t,J＝8.0Hz,2H),5.20(s,2H),4.25ppm(s,4H).13C NMR(100MHz,DMSO-d6):δ＝181.60,163.62,163.06,162.47,160.06,155.23,144.40,138.91,133.74,132.44,131.03,130.97,129.84,129.76,129.22,126.25,125.84,125.34,124.00,122.40,115.67,115.43,115.22,115.01,52.16,48.31,42.10ppm.HR-MS(m/z)(ESI):calcd for C30H26FN5O4S2[M+H]+:604.1483；found:604.1471.
4-(2-(4-氯苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I5):Yield 77％,as a yellow solid.Mp:233.5-234.5℃.1HNMR(400MHz,DMSO-d6):δ＝9.76(s,1H),8.58(d,J＝8.4Hz,1H),8.50(d,J＝7.2Hz,1H),8.42(d,J＝8.0Hz,1H),7.84(t,J＝8.0Hz,1H),7.75(d,J＝8.8Hz,2H),7.52(d,J＝8.8Hz,2H),7.42-7.33(m,5H),7.30(s,2H),5.21(s,2H),4.25(t,J＝4.9Hz,4H),3.37ppm(t,J＝4.5Hz,4H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.62,163.06,155.25,144.20,138.98,136.57,132.46,131.62,131.06,131.00,129.49,129.26,128.34,126.27,125.84,125.35,123.99,122.40,115.67,115.45,52.14,48.32,42.19ppm.HR-MS(m/z)(ESI):calcdfor C30H26ClN5O4S2[M+H]+:620.1187；found:620.1168.
4-(2-(4-溴苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I6):Yield 47％,as a yellow solid.Mp:267.9-270.2℃.1HNMR(400MHz,DMSO-d6):δ＝9.76(s,1H),8.58(d,J＝8.4Hz,1H),8.50(d,J＝7.2Hz,1H),8.42(d,J＝8.4Hz,1H),7.84(t,J＝7.2Hz,1H),7.75(d,J＝8.4Hz,2H),7.52(d,J＝8.8Hz,2H),7.49(d,J＝8.4Hz,2H),7.39(d,J＝8.0Hz,1H),7.31(d,J＝8.4Hz,2H),7.30(s.2H),5.20(s,2H),4.25(t,J＝4.4Hz,4H),3.36ppm(t,J＝4.8Hz,4H).13C NMR(100MHz,DMSO-d6):δ＝181.63,163.60,163.04,155.23,144.18,138.97,136.98,132.45,131.25,131.04,131.00,129.82,129.24,126.25,125.83,125.34,123.98,122.38,120.10,115.65,115.43,52.13,48.30,42.24ppm.HR-MS(m/z)(ESI):calcd for C30H26BrN5O4S2[M+H]+:664.0682；found:664.0672.
4-(2-(4-甲基苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I7):Yield 66％,as a yellow solid.Mp:256.1-257.8℃.1HNMR(400MHz,DMSO-d6):δ＝9.76,(s,1H),8.55(d,J＝8.4Hz,1H),8.48(d,J＝6.8Hz,1H),8.40(d,J＝8.4Hz,1H),7.81(t,J＝8.0Hz,1H),7.76(d,J＝8.8Hz,2H),7.53(d,J＝8.4Hz,2H),7.36(d,J＝8.4Hz,1H),7.30(s,2H),7.23(d,J＝8.0Hz,2H),7.09(d,J＝8.0Hz,2H),5.18(s,2H),4.25(t,J＝4.8Hz,4H),3.40-3.35(m,4H),2.23ppm(s,3H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.55,163.00,155.11,144.20,138.99,136.15,134.54,132.35,130.96,130.86,129.14,128.88,127.60,126.22,125.84,125.32,124.00,122.41,115.71,115.41,52.12,48.32,42.47,20.66ppm.HR-MS(m/z)(ESI):calcd for C31H29N5O4S2[M+H]+:600.1734；found:600.1729.
4-(2-(4-甲氧基苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I8):Yield 32％,as a yellow solid.Mp:205.1-206.7℃.1H NMR(400MHz,DMSO-d6):δ＝9.76(s,1H),8.57(d,J＝8.4Hz,1H),8.50(d,J＝7.2Hz,1H),8.42(d,J＝8.0Hz,1H),7.84(t,J＝8.0Hz,1H),7.75(d,J＝8.4Hz,2H),7.52(d,J＝8.4Hz,2H),7.39(d,J＝8.0Hz,1H),7.31(d,J＝8.4Hz,2H),7.30(s,2H),6.85(d,J＝8.8Hz,2H),5.16(s,2H),4.24(s,4H),3.69ppm(s,3H).13C NMR(150MHz,DMSO-d6):δ＝181.63,163.60,163.06,158.38,155.16,144.21,138.99,132.40,131.01,130.92,129.59,129.29,129.18,126.29,125.85,125.36,124.02,122.49,115.79,115.47,113.74,55.06,52.15,48.33,42.17ppm.HR-MS(m/z)(ESI):calcd for C31H29N5O5S2[M+H]+:616.1683；found:616.1667.
4-(1,3-二氧代-2-(3,4,5-三甲氧基苄基)-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I9):Yield 76％,as a yellow solid.Mp:250.0-250.2℃.1H NMR(400MHz,DMSO-d6):δ＝9.76,(s,1H),8.54(t,J＝7.6Hz,1H),8.50(d,J＝7.6Hz,1H),8.41(t,J＝8.0Hz,1H),7.83(t,J＝7.6Hz,1H),7.76(d,J＝8.4Hz,2H),7.53(d,J＝8.4Hz,2H),7.36(t,J＝8.4Hz,1H),7.30(s,2H),6.68(s,2H),5.15(s,2H),4.25(s,4H),3.71(s,6H),3.60ppm(s,3H).13C NMR(100MHz,DMSO-d6):δ＝181.65,163.69,163.13,155.09,152.76,144.20,138.99,136.68,133.29,132.37,130.99,130.84,129.20,126.23,125.85,125.34,124.00,122.48,115.80,115.41,105.30,59.96,55.86,52.13,48.33,43.08ppm.HR-MS(m/z)(ESI):calcd for C33H33N5O7S2[M+H]+:676.1894；found:676.1892.
4-(1,3-二氧代-2-(4-(三氟甲基)苄基)-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I10):Yield 83％,as a yellow solid.Mp:265.3-266.5℃.1H NMR(600MHz,DMSO-d6):δ＝9.77(s,1H),8.59(d,J＝8.4Hz,1H),8.51(d,J＝6.6Hz,1H),8.42(d,J＝7.8Hz,1H),7.85(t,J＝8.4Hz,1H),7.76(d,J＝8.4Hz,2H),7.66(d,J＝7.8Hz,2H),7.55(d,J＝7.8Hz,2H),7.53(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,1H),7.31(s,2H),5.31(s,2H),4.25(t,J＝4.8Hz,4H),3.37ppm(t,J＝4.8Hz,4H).13C NMR(150MHz,DMSO-d6):δ＝181.65,163.69,163.12,155.32,144.22,142.36,139.01,132.52,131.12,131.08,129.33,128.11,128.00,127.79,127.58,127.00,126.30,125.87,125.37,125.34,125.31,125.29,125.19,124.02,123.39,122.39,115.64,115.46,52.16,48.32,42.56ppm.HR-MS(m/z)(ESI):calcd for C31H26F3N5O4S2[M+Na]+:676.1271；found:676.1282.
4-(2-(3,4-二羟基苯乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I11):Yield 45％,as a yellow solid.Mp:191.4-192.5℃.1H NMR(400MHz,DMSO-d6):δ＝9.77(brs,1H),8.92-8.63(m,2H),8.55(d,J＝8.4Hz,1H),8.47(d,J＝7.2Hz,1H),8.39(d,J＝8.0Hz,1H),7.83(t,J＝8.0Hz,1H),7.75(d,J＝8.4Hz,2H),7.53(d,J＝8.8Hz,2H),7.38(d,J＝8.0Hz 1H),7.30(s,2H),6.68(d,J＝2.0Hz,1H),6.63(d,J＝7.6Hz,1H),6.49(dd,J＝8.0,1.6Hz,1H),4.29(s,4H),4.14(t,J＝8.0Hz,2H),3.34(s,4H),2.71ppm(t,J＝7.6Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.40,162.87,154.95,145.20,144.24,143.73,138.98,132.12,130.74,130.67,129.55,129.10,126.24,125.85,125.36,124.02,122.56,119.27,115.99,115.97,115.63,115.45,52.17,48.36,41.29,33.05ppm.HR-MS(m/z)(ESI):calcd for C31H29N5O6S2[M+H]+:632.1632；found:632.1637.
4-(2-环己基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I12):Yield 46％,as a yellow solid.Mp:260.7-262.9℃.1H NMR(400MHz,DMSO-d6):δ＝9.91,(s,1H),8.59(d,J＝8.4Hz,1H),8.51(d,J＝7.2Hz,1H),8.42(d,J＝8.0Hz,1H),7.85(t,J＝8.0Hz,1H),7.75(d,J＝8.4Hz,2H),7.55(d,J＝8.4Hz,2H),7.40(d,J＝8.0Hz 1H),7.31(s,2H),4.88(t,J＝6.4Hz,1H),4.24(s,4H),2.48-2.35(m,2H),1.83(d,J＝10.7Hz,2H),1.70-1.58(m,2H),1.41-1.06ppm(m,4H).13C NMR(101MHz,DMSO-d6):δ＝181.63,163.89,163.40,154.67,144.20,138.96,132.06,130.66,130.34,129.09,126.22,125.83,125.18,123.97,123.07,116.48,115.39,52.59,52.14,48.33,28.69,26.14,25.23ppm.HR-MS(m/z)(ESI):calcd for C29H31N5O4S2[M+H]+:578.1890；found:578.1891.
4-(2-丁基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I13):Yields 46％,as a yellow solid.Mp:192.5-193.7℃.1H NMR(400MHz,DMSO-d6):δ＝9.75,(s,1H),8.56(dd,J＝7.6,5.2Hz,1H),8.51-8.45(m,1H),8.44-8.37(m,1H),7.87-7.79(m,1H),7.75(d,J＝8.8Hz,2H),7.52(d,J＝8.8Hz,2H),7.39(dd,J＝7.7,4.8Hz 1H),7.30(s,2H),4.24(t,J＝4.6Hz,4H),4.02(t,J＝5.2Hz,2H),1.65-1.54(m,2H),1.39-1.28(m,2H),0.92ppm(t,J＝7.2Hz,3H).13C NMR(150MHz,DMSO-d6):δ＝181.64,163.54,163.02,154.94,144.21,138.99,132.13,130.76,130.65,129.10,126.25,125.86,125.37,124.02,122.59,116.02,115.45,52.18,48.35,40.06,29.74,19.85,13.78ppm.HR-MS(m/z)(ESI):calcd for C27H29N5O4S2[M+H]+:552.1734；found:552.1763.
4-(2-辛基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I14):Yield 42％,as a yellow solid.Mp:147.9-149.5℃.1HNMR(600MHz,DMSO-d6):δ＝9.78,(s,1H),8.53(d,J＝9.6Hz,1H),8.46(d,J＝7.2Hz,1H),8.38(d,J＝7.8Hz,1H),7.81(t,J＝7.8Hz,1H),7.75(d,J＝9.0Hz,2H),7.53(d,J＝9.0Hz,2H),7.36(d,J＝7.8Hz,1H),7.30(s,2H),4.24(s,4H),3.99(t,J＝7.2Hz,2H),3.34(t,J＝3.6Hz,4H),1.62-1.57(m,2H),1.26-1.16(m,10H),0.83ppm(t,J＝6.6Hz,3H).13C NMR(100MHz,DMSO-d6):δ＝181.65,163.46,162.96,154.88,144.21,138.97,132.08,130.71,130.57,129.04,126.17,125.83,125.34,123.98,122.53,115.96,115.37,52.16,48.34,31.23,28.71,28.59,27.50,26.53,22.08,13.94ppm.HR-MS(m/z)(ESI):calcd forC31H37N5O4S2[M+H]+:608.2360；found:608.2361.
4-(2-(2-(二甲基氨基)乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I15):Yield 62％,as a yellow solid.Mp:232.2-234.5℃.1H NMR(400MHz,DMSO-d6):δ＝9.90(s,1H),8.57(d,J＝8.4Hz,1H),8.49(d,J＝7.2Hz,1H),8.41(d,J＝8.0Hz,1H),7.85(t,J＝8.0Hz,1H),7.76(d,J＝8.4Hz,2H),7.56(d,J＝8.6Hz,2H),7.39(d,J＝8.0Hz,1H),7.31(s,2H),4.30-4.20(m,6H),3.35(t,J＝4.2Hz,4H),2.93(s,2H),2.52ppm(s,6H).13C NMR(150MHz,DMSO-d6):δ＝181.63,163.80,163.24,155.08,144.28,138.92,132.22,130.82,130.79,129.21,126.24,125.80,125.29,124.00,122.60,115.94,115.38,55.73,52.21,48.38,44.18,36.31ppm.HR-MS(m/z)(ESI):calcd for C27H30N6O4S2[M+H]+:567.1843；found:567.1844.
4-(2-(2-(二乙基氨基)乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I16):Yield 44％,as a yellow solid.Mp:214.9-215.3℃.1H NMR(400MHz,DMSO-d6):δ＝9.94(s,1H),8.58(d,J＝8.4Hz,1H),8.50(d,J＝6.8Hz,1H),8.42(d,J＝8.0Hz,1H),7.85(t,J＝7.9Hz,1H),7.74(d,J＝8.8Hz,2H),7.55(d,J＝8.8Hz,2H),7.40(d,J＝8.4Hz 1H),7.31(s,2H),4.31(s,2H),4.27(s,4H),3.16(s,4H),1.26ppm(t,J＝6.8Hz,6H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.91,163.32,155.24,144.27,138.93,132.30,130.98,130.89,129.33,126.24,125.79,125.28,123.97,122.59,115.85,115.36,52.21,48.35,47.58,46.59,34.18,8.45ppm.HR-MS(m/z)(ESI):calcd for C29H36N6O4S2[M+H]+:595.2156；found:595.2147.
4-(2-(2-羟乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯基)哌嗪-1-甲硫酰胺(I17):Yield 42％,as a yellow solid.Mp:238.7-241.9℃.1HNMR(400MHz,DMSO-d6):δ＝9.77(s,1H),8.56(d,J＝8.0Hz,1H),8.50(d,J＝7.2Hz,1H),8.42(d,J＝8.0Hz,1H),7.84(t,J＝8.0Hz,1H),7.75(d,J＝8.8Hz,2H),7.52(d,J＝8.8Hz,2H),7.40(d,J＝4.4Hz,1H),7.30(s,2H),4.81(t,J＝6.0Hz,1H),4.24(t,J＝4.4Hz,4H),4.14(t,J＝6.4Hz,2H),3.61ppm(dt,J＝12.4,6.4Hz,2H).13C NMR(150MHz,DMSO-d6):δ＝181.64,163.73,163.20,154.91,144.23,138.98,132.09,130.73,130.61,129.21,126.26,125.85,125.38,124.02,122.77,116.21,115.45,57.86,52.20,48.36,41.70ppm.HR-MS(m/z)(ESI):calcd for C25H25N5O5S2[M+H]+:540.1370；found:540.1364.
4-(2-(2-吗啉基乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I18):Yield 65％,as a yellow solid.Mp:239.7-240.9℃.1H NMR(400MHz,DMSO-d6):δ＝8.54(d,J＝8.4Hz,1H),8.49(d,J＝6.4Hz,1H),8.41(d,J＝8.0Hz,1H),8.01(s,1H),7.83(t,J＝8.0Hz,1H),7.77(d,J＝8.0Hz,2H),7.43(d,J＝8.4Hz,2H),7.37(d,J＝8.0Hz,1H),7.30(s,2H),4.17(t,J＝6.8Hz,2H),4.09(t,J＝5.2Hz,4H),3.76(dt,J＝11.2,7.2Hz,2H),3.52(t,J＝4.4Hz,4H),3.26(t,J＝4.8Hz,4H),2.99(t,J＝8.0Hz,2H),2.55(t,J＝6.8Hz,2H),2.46ppm(t,J＝4.4Hz,4H).13C NMR(100MHz,DMSO-d6):δ＝181.74,163.74,163.20,155.28,143.93,142.13,132.38,130.95,130.88,129.31,129.25,126.38,125.92,125.47,122.62,115.94,115.53,66.36,55.78,53.54,52.26,47.32,46.57,36.76,34.58ppm.HR-MS(m/z)(ESI):calcd for C31H36N6O5S2[M+H]+:637.2261；found:637.2254.
4-(2-(3-吗啉丙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I19):Yield 72％,as a yellow solid.Mp:189.7-194.7℃.1H NMR(400MHz,DMSO-d6):δ＝8.51(d,J＝8.4Hz,1H),8.46(d,J＝6.8Hz,1H),8.37(d,J＝8.4Hz,1H),8.04(t,J＝5.2Hz,1H),7.80(t,J＝8.0Hz,1H),7.77(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.34(d,J＝8.0Hz,1H),7.31(s,2H),4.17-3.98(m,6H),3.76(dt,J＝12.8,6.3Hz,2H),3.25(t,J＝6.0Hz,4H),2.99(t,J＝7.2Hz,2H),2.36(t,J＝6.8Hz,2H),2.29(s,4H),1.81-1.73ppm(m,2H).13C NMR(100MHz,DMSO-d6):δ＝181.67,163.66,163.13,155.03,143.80,142.07,132.06,130.66,130.59,129.17,129.17,126.19,125.80,125.34,122.67,116.04,115.35,66.11,55.98,53.19,52.17,47.23,46.47,38.17,34.49,24.09ppm.HR-MS(m/z)(ESI):calcd for C32H38N6O5S2[M+H]+:651.2418；found:651.2418.
4-(2-苄基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I20):Yield 48％,as a yellow solid.Mp:254.2-256.1℃.1H NMR(400MHz,DMSO-d6):δ＝8.57-8.46(m,2H),8.46-8.36(m,1H),8.04(s,1H),7.87-7.71(m,3H),7.43(d,J＝8.0Hz,2H),7.38-7.14(m,8H),5.23(s,2H),4.10(s,4H),3.76(dt,J＝12.4,7.6Hz,2H),3.26(s,4H),2.99ppm(t,J＝6.8Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.62,163.60,163.04,155.29,143.79,142.08,137.55,132.40,130.98,130.91,129.17,128.39,127.51,127.49,127.04,126.20,125.80,125.31,122.37,115.60,115.37,52.14,47.18,46.48,42.74,34.49ppm.HR-MS(m/z)(ESI):calcd for C32H31N5O4S2[M+H]+:614.1890；found:614.1897.
4-(2-(4-氟苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I21):Yield 59％,as a yellow solid.Mp:204.1-206.5℃.1H NMR(400MHz,DMSO-d6):δ＝8.53(d,J＝8.4Hz,1H),8.48(d,J＝6.8Hz,1H),8.39(d,J＝8.4Hz,1H),8.01(t,J＝4.8Hz,1H),7.81(t,J＝8.0Hz,1H),7.77(d,J＝8.4Hz,2H),7.47-7.37(m,4H),7.35(d,J＝8.0Hz,1H),7.31(s,2H),7.11(d,J＝8.4Hz,2H),5.20(s,2H),4.09(t,J＝4.4Hz,4H),3.76(dt,J＝13.4,6.3Hz,2H),3.26(t,J＝4.8Hz,4H),2.99ppm(t,J＝7.6Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.65,163.59,163.03,162.45,160.04,155.32,143.76,142.07,133.75,133.72,132.40,130.98,130.93,129.82,129.74,129.19,129.15,126.19,125.78,125.31,122.37,115.58,115.36,115.20,114.99,52.11,47.16,46.45,42.07,34.47ppm.HR-MS(m/z)(ESI):calcd for C32H30FN5O4S2[M+H]+:646.1789；found:632.1782.
4-(2-(4-氯苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I22):Yield 69％,as a yellow solid.Mp:176.7-178.2℃.1H NMR(400MHz,DMSO-d6):δ＝8.55(d,J＝8.4Hz,1H),8.49(d,J＝7.2Hz,1H),8.41(d,J＝8.0Hz,1H),8.02(t,J＝5.6Hz,1H),7.83(t,J＝7.2Hz,1H),7.77(d,J＝8.0Hz,2H),7.43(d,J＝8.0Hz,2H),7.39–7.33(m,5H),7.31(s,2H),5.21(s,2H),4.09(t,J＝4.4Hz,4H),3.76(dt,J＝12.8,6.4Hz,2H),3.26(t,J＝4.8Hz,4H),2.99ppm(t,J＝7.2Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.61,163.05,155.37,143.76,142.07,136.56,132.45,131.62,131.02,130.99,129.48,129.24,129.15,128.33,126.22,125.78,125.34,122.37,115.57,115.39,52.12,47.16,46.45,42.18,34.47ppm.HR-MS(m/z)(ESI):calcd forC31H30ClN5O4S2[M+H]+:648.1501；found:648.1478.
4-(2-(4-溴苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I23):Yield 45％,as a yellow solid.Mp:190.9-191.5℃.1H NMR(400MHz,DMSO-d6):δ＝8.55(d,J＝8.4Hz,1H),8.49(d,J＝6.8Hz,1H),8.41(d,J＝8.4Hz,1H),8.02(t,J＝5.2Hz,1H),7.83(t,J＝8.0Hz,1H),7.77(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),7.36(d,J＝8.0Hz,1H),7.33-7.28(m,4H),5.19(s,2H),4.09(t,J＝4.6Hz,4H),3.75(dt,J＝14.0,6.0Hz,2H),3.27(t,J＝4.8Hz,4H),2.99ppm(t,J＝7.2Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.63,163.61,163.05,155.37,143.76,142.07,136.99,132.45,131.25,131.02,131.00,129.82,129.24,129.15,126.23,125.78,125.34,122.37,120.10,115.57,115.39,52.12,47.16,46.45,42.24,34.47ppm.HR-MS(m/z)(ESI):calcd for C32H30BrN5O4S2[M+H]+:692.0995；found:692.0997.
4-(2-(4-甲基苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I24):Yield 55％,as a yellow solid.Mp:220.1-221.2℃.1H NMR(400MHz,DMSO-d6):δ＝8.52(d,J＝8.4Hz,1H),8.48(d,J＝7.2Hz,1H),8.42-8.36(m,1H),8.02(t,J＝4.8Hz,1H),7.84-7.74(m,3H),7.43(d,J＝8.0Hz,2H),7.37-7.32(m,1H),7.31(s,2H),7.23(d,J＝8.0Hz,2H),7.09(d,J＝8.0Hz,2H),5.18(s,2H),4.09(t,J＝3.6Hz,4H),3.76(dt,J＝13.2,6.4Hz,2H),3.26(t,J＝4.8Hz,4H),2.99(t,J＝6.4Hz,2H),2.23ppm(s,3H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.55,163.01,155.25,143.76,142.07,136.13,134.54,132.35,130.94,130.87,129.14,128.87,127.59,126.19,125.78,125.32,122.40,115.64,115.36,52.11,47.16,46.45,42.46,34.47,20.65ppm.HR-MS(m/z)(ESI):calcd for C33H33N5O4S2[M+H]+:628.2047；found:628.2049.
4-(2-(4-甲氧基苄基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I25):Yield 82％,as a yellow solid.Mp:167.9-169.8℃.1H NMR(400MHz,DMSO-d6):δ＝8.52(d,J＝8.4Hz,1H),8.48(d,J＝6.8Hz,1H),8.40(d,J＝8.0Hz,1H),8.01(t,J＝4.8Hz,1H),7.81(t,J＝7.6Hz,1H),7.77(d,J＝8.0Hz,2H),7.43(d,J＝8.4Hz,2H),7.37–7.26(m,5H),6.86-6.82(m,2H),5.15(s,2H),4.09(t,J＝4.6Hz,4H),3.76(dt,J＝14.0,6.0Hz,2H),,3.69(s,3H),3.26(t,J＝5.2Hz,4H),2.99ppm(t,J＝7.6Hz,2H).13CNMR(100MHz,DMSO-d6):δ＝181.65,163.54,163.00,158.35,155.24,143.75,142.06,132.33,130.92,130.85,129.57,129.26,129.14,129.12,126.19,125.77,125.31,122.42,115.66,115.36,113.70,55.03,52.10,47.16,46.44,42.13,34.46ppm.HR-MS(m/z)(ESI):calcd for C33H33N5O5S2[M+H]+:644.1996；found:644.1982.
4-(1,3-二氧代-2-(3,4,5-三甲氧基苄基)-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I26):Yield 48％,as a yellow solid.Mp:270.9-272.9℃.1H NMR(400MHz,DMSO-d6):δ＝8.56(d,J＝8.8Hz,1H),8.52(d,J＝7.2Hz,1H),8.44(d,J＝8.0Hz,1H),8.01(t,J＝5.2Hz,1H),7.84(t,J＝8.0Hz,1H),7.77(d,J＝8.4Hz,2H),7.43(d,J＝8.0Hz,2H),7.37(d,J＝8.4,1H),7.31(s,2H),6.67(s,2H),5.16(s,2H),4.09(t,J＝5.2Hz,4H),3.76(dt,J＝12.4,6.0Hz,2H),3.70(s,6H),3.60(s,3H),3.28(t,J＝4.8Hz,4H),2.99ppm(t,J＝7.2Hz,2H).13C NMR(150MHz,DMSO-d6):δ＝181.63,163.76,163.21,155.29,152.77,143.78,142.08,136.64,133.32,132.44,131.03,130.92,129.27,129.18,126.27,125.80,125.39,122.55,115.79,115.45,105.23,59.98,55.87,52.14,47.19,46.47,43.10,34.49ppm.HR-MS(m/z)(ESI):calcd for C35H37N5O7S2[M+H]+:704.2207；found:704.2202.
4-(1,3-二氧代-2-(4-(三氟甲基)苄基)-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I27):Yield 63％,as a yellow solid.Mp:206.5-207.4℃.1H NMR(400MHz,DMSO-d6):δ＝8.55(d,J＝8.4Hz,1H),8.49(d,J＝7.2Hz,1H),8.40(d,J＝8.4Hz,1H),8.03(t,J＝5.2Hz,1H),7.86-7.80(m,1H),7.78(d,J＝8.4Hz,2H),7.66(d,J＝8.0Hz,2H),7.55(d,J＝8.0Hz,2H),7.44(d,J＝8.0Hz,2H),7.36(d,J＝8.0Hz,1H),7.32(s,2H),5.31(s,2H),4.10(s,4H),3.76(dt,J＝13.2,6.4Hz,,2H),3.27(s,4H),2.99ppm(t,J＝6.8Hz,2H).13C NMR(150MHz,DMSO-d6):δ＝181.63,163.66,163.09,155.43,143.79,142.35,142.08,132.50,131.07,129.29,129.18,128.10,127.99,127.78,127.57,127.36,126.99,126.24,125.81,125.34,125.32,125.30,125.27,125.18,123.38,122.34,121.58,115.52,115.39,52.14,47.17,46.48,42.55,34.49,30.73ppm.HR-MS(m/z)(ESI):calcd for C33H30F3N5O4S2[M+H]+:682.1764；found:682.1787.
4-(2-(3,4-二羟基苯乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I28):Yield 48％,as a yellow solid.Mp:254.1-255.9℃.1H NMR(400MHz,DMSO-d6):δ＝8.81(s,1H),8.74(s,1H),8.53(d,J＝8.4Hz,1H),8.48(d,J＝7.2Hz,1H),8.39(d,J＝8.4Hz,1H),8.01(s,1H),7.82(t,J＝8.0Hz,1H),7.76(d,J＝8.4Hz,2H),7.43(t,J＝8.4Hz,2H),7.36(d,J＝8.0Hz,1H),7.31(s,2H),6.67(d,J＝2.0Hz,1H),6.63(d,J＝8.0Hz,1H),6.49(dd,J＝8.0,1.6Hz,1H),4.14(t,J＝8.0Hz,2H),4.08(s,4H),3.78-3.72(m,2H),3.25(s,4H),2.98(t,J＝7.6Hz,2H),2.71ppm(t,J＝8.0Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.72,163.55,163.02,155.20,145.24,143.89,143.78,142.11,132.27,130.85,130.79,129.65,129.26,129.19,126.32,125.88,125.45,122.63,119.37,116.08,115.95,115.70,115.49,52.22,47.29,46.54,41.35,34.55,33.11ppm.HR-MS(m/z)(ESI):calcd for C33H33N5O6S2[M+H]+:660.1945；found:660.1946.
4-(2-环己基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I29):Yield 49％,as a yellow solid.Mp:287.3-288.9℃.1HNMR(400MHz,DMSO-d6):δ＝8.50(d,J＝8.4Hz,1H),8.45(d,J＝7.2Hz,1H),8.37(d,J＝8.0Hz,1H),8.02(t,J＝5.6Hz,1H),7.84-7.74(m,3H),7.43(d,J＝8.0Hz,2H),7.38–7.28(m,3H),4.88(t,J＝11.8Hz,1H),4.08(s,4H),3.76(dt,J＝12.4,7.2Hz,2H),3.23(s,4H),2.99(t,J＝6.8Hz,2H),2.47-2.37(m,2H),1.83(d,J＝12.0Hz,2H),1.71-1.57(m,2H),1.42-1.13ppm(m,4H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.90,163.40,154.80,143.76,142.07,132.06,130.64,130.34,129.14,129.08,126.18,125.78,125.17,123.05,116.40,115.35,52.58,52.11,47.18,46.45,34.47,28.69,26.14,25.23ppm.HR-MS(m/z)(ESI):calcd for C31H35N5O4S2[M+H]+:606.2203；found:606.2203.
4-(2-丁基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I30):Yield 38％,as a yellow solid.Mp:268.4-269.7℃.1H NMR(400MHz,DMSO-d6):δ＝8.50(d,J＝8.4Hz,1H),8.45(d,J＝7.2Hz,1H),8.36(d,J＝8.0Hz,1H),8.01(t,J＝5.2Hz,1H),7.83-7.75(m,3H),7.43(d,J＝8.0Hz,2H),7.36–7.28(m,3H),4.09(s,4H),4.01(t,J＝6.8Hz,2H),3.76(dt,J＝13.6,6.0Hz,2H),3.24(s,4H),2.99(t,J＝7.2Hz,2H),1.65-1.62(m,2H),1.40-1.27(m,2H),0.91ppm(t,J＝7.2Hz,3H).13C NMR(100MHz,DMSO-d6):δ＝181.65,163.46,162.94,154.99,143.77,142.07,132.04,130.65,130.54,129.16,129.01,126.12,125.79,125.29,122.49,115.86,115.31,52.13,47.19,46.46,39.20,34.48,29.71,19.83,13.75ppm.HR-MS(m/z)(ESI):calcd for C29H33N5O4S2[M+H]+:580.2047；found:580.2050.
4-(2-辛基-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I31):Yield 38％,as a yellow solid.Mp:142.3-144.5℃.1H NMR(600MHz,DMSO-d6):δ＝8.53(d,J＝8.4Hz,1H),8.47(d,J＝6.6Hz,1H),8.39(d,J＝8.4Hz,1H),8.02(t,J＝5.4Hz,1H),7.82(t,J＝7.8Hz,1H),7.43(d,J＝8.4Hz,2H),7.43(t,J＝8.4Hz,2H),7.36(d,J＝8.4Hz,1H),7.31(s,2H),4.08(t,J＝4.8Hz,4H),4.01(t,J＝7.2Hz,2H),3.76(dt,J＝13.2,6.0Hz,,2H),3.25(t,J＝4.8Hz,4H),2.98(t,J＝7.2Hz,2H),1.63-1.57(m,2H),1.36-1.20(m,10H),0.83ppm(t,J＝7.2Hz,3H).13C NMR(150MHz,DMSO-d6):δ＝181.63,163.53,163.00,155.07,143.77,142.08,132.12,130.73,130.64,129.17,129.09,126.21,125.79,125.36,122.57,115.93,115.39,52.15,47.19,46.47,34.48,31.25,28.73,28.60,27.52,26.54,22.09,13.97ppm.HR-MS(m/z)(ESI):calcd for C33H41N5O4S2[M+H]+:636.2673；found:636.2674.
4-(2-(2-(二甲基氨基)乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I32):Yield 76％,as a yellow solid.Mp:263.9-264.6℃.1H NMR(400MHz,DMSO-d6):δ＝8.53(d,J＝8.4Hz,1H),8.47(d,J＝7.2Hz,1H),8.38(d,J＝8.0Hz,1H),8.10(t,J＝5.2Hz,1H),7.82(t,J＝8.0Hz,1H),7.77(d,J＝8.2Hz,2H),7.43(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,1H),7.32(s,2H),4.23(t,J＝6.4Hz,2H),4.10(t,J＝4.8Hz,4H),3.76(dt,J＝13.2,6.4Hz,2H),3.25(t,J＝4.8Hz,4H),2.99(t,J＝7.2Hz,2H),2.87(t,J＝7.2Hz 2H),2.48ppm(s,6H).13C NMR(100MHz,DMSO-d6):δ＝181.60,163.91,163.34,155.23,143.79,142.06,132.21,130.79,129.25,129.14,126.18,125.78,125.27,122.61,115.88,115.32,55.28,52.17,47.22,46.45,43.52,35.75,34.45ppm.HR-MS(m/z)(ESI):calcd for C29H34N6O4S2[M+H]+:595.2156；found:595.2150.
4-(2-(2-(二乙基氨基)乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I33):Yield 74％,as a yellow solid.Mp:214.1-216.4℃.1H NMR(400MHz,DMSO-d6):δ＝8.51(dd,J＝8.4,2.8Hz,1H),8.46(dd,J＝7.2,3.2Hz,1H),8.37(dd,J＝8.0,3.2Hz,1H),8.02(t,J＝5.1Hz,1H),7.81(dd,J＝8.1,2.5Hz,1H),7.77(d,J＝8.0Hz,2H),7.43(d,J＝8.4Hz,2H),7.34(dd,J＝8.4,2.4Hz,1H),7.31(s,2H),4.13-4.05(m,6H),3.76(dt,J＝14.4,6.0Hz,2H),3.24(t,J＝4.8Hz,4H),2.99(t,J＝8.0Hz,2H),2.64(t,J＝7.2Hz,2H),2.53(q,J＝7.2Hz,2H),0.95ppm(t,J＝7.2Hz,6H).13C NMR(100MHz,DMSO-d6):δ＝181.64,163.52,162.99,155.04,143.77,142.08,132.07,130.68,130.61,129.16,129.07,126.18,125.79,125.34,122.54,115.90,115.37,52.14,49.33,47.19,47.01,46.47,37.42,34.48,12.14ppm.HR-MS(m/z)(ESI):calcd forC31H38N6O4S2[M+H]+:623.2469；found:623.2471.
4-(2-(2-羟乙基)-1,3-二氧代-2,3-二氢-1H-苯并[de]异喹啉-6-基)-N-(4-氨磺基苯乙基)哌嗪-1-甲硫酰胺(I34):Yield 50％,as a yellow solid.Mp:239.6-241.7℃.1H NMR(400MHz,DMSO-d6):δ＝8.51(d,J＝8.4Hz,1H),8.46(d,J＝7.2Hz,1H),8.37(d,J＝8.0Hz,1H),8.02(t,J＝5.0Hz,1H),7.81(t,J＝8.0Hz,1H),7.77(d,J＝8.0Hz,2H),7.43(d,J＝8.0Hz,2H),7.35(d,J＝8.0Hz,1H),7.31(s,2H),4.81(t,J＝6.0Hz,1H),4.13(t,J＝6.4Hz,2H),4.09(t,J＝5.2Hz,4H),3.76(dt,J＝13.6,6.4Hz,,2H),3.60(dt,J＝12.4,6.0Hz,,2H),3.24(t,J＝4.8Hz,4H),2.99ppm(t,J＝7.2Hz,2H).13C NMR(100MHz,DMSO-d6):δ＝181.65,163.66,163.14,154.98,143.77,142.07,132.03,130.64,130.53,129.16,129.13,126.15,125.79,125.32,122.67,116.06,115.33,57.85,52.15,47.20,46.45,41.66,34.47ppm.HR-MS(m/z)(ESI):calcd for C27H29N5O5S2[M+H]+:568.1683；found:568.1678.
本实施例所得目标化合物I1～I34的结构式分别如下所示：
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实施例2：化合物I15、I32、I33的制备
化合物I15：重复实施例1，不同的是，反应全程在室温下进行(反应至完全约3天)。最后得到黄色固体，产率为44％。经核磁氢谱、碳谱及质谱表征，确定为化合物I15。
化合物I32：重复实施例1，不同的是，用丙酮代替乙腈，反应在55℃条件下进行(反应至完全约12h)。最后得到黄色固体，产率为76％。经核磁氢谱、碳谱及质谱表征，确定为化合物I32。
化合物I33：重复实施例1，不同的是，用甲醇代替乙腈，反应在70℃条件下进行(反应至完全约8h)。最后得到黄色固体，产率为74％。经核磁氢谱、碳谱及质谱表征，确定为化合物I33。
实验例1：体外抗肿瘤活性试验
以临床使用的药物氨萘非特、CA抑制剂SLC-0111为阳性对照药，以相应的溶剂作阴性对照，以人乳腺癌细胞MDA-MB-231、MCF-7，人非小细胞肺癌细胞A549，人结肠癌细胞HCT-116、SW480为受试细胞株；MTT法对本发明所述目标化合物进行体外抗肿瘤活性测试。
取生长状态良好的细胞以2×104～4×104个/孔接种于96孔板中，37℃培养箱中培养24h后加入20μL待测化合物(起始浓度为40μM，按照2倍法梯度稀释，每个浓度设置5个复孔)，置于细胞培养箱培养24h。向96孔板中加入5mg/mL MTT工作液10μL，于培养箱孵育4h，小心弃去上清液，加入150μL DMSO溶解还原产生的甲瓒紫色晶体，用酶标仪测定每孔吸光度。计算每个给药孔细胞增值抑制率，结果如下表1所示。
表1.本发明目标化合物对不同细胞株的抑制活性
aIC50值是平均值±S.D.3个独立的实验。
由表1可以看出，部分化合物显示较好的抗肿瘤活性，对于人乳腺癌细胞MDA-MB-231，化合物I15、I32和I33具有较好的细胞毒性，IC50分别为8.89±1.80μM、4.31±0.46μM和13.51±0.93μM，优于氨萘非特(22.37±0.60μM)，其中化合物I32比阳性药氨萘非特强约5倍；对于细胞MCF-7仅有化合物I32存在细胞毒性，IC50＝10.84±0.45μM；对于细胞HCT-116，化合物I10和I13具有中等的细胞毒性，IC50值分别为15.78±0.17μM和14.83±0.21μM优于阳性药氨萘非特；对于人结肠癌细胞SW480，化合物I15和I32具有明显抑制作用，IC50分别为16.15±0.68μM、8.53±0.95μM，化合物I32对SW480毒性约是氨萘非特(13.80±0.27μM)的两倍。
上述体外抗肿瘤活性测试表明，本发明所述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物有望用于抗肿瘤药物的制备。
实验例2：CA II、CA IX酶活性测试
将CA抑制剂SLC-0111作为阳性对照进行CA II和CA IX酶活性测试，按下面步骤进行：
于96孔板中每个孔各加18μL CA溶液(CA II浓度3.33ng/μL、CAIX浓度为11.1ng/μL)，加入2μL待测药物(100μM为初试浓度加入待测药物，按3倍法梯度稀释的受试药物，每个浓度设置5个复孔)，加完药物后放入25℃的恒温培养箱中培养15min；取出后加入20μL 1mM的4-NPA溶液，将检测CA II亲和力的放于25℃恒温培养箱培养60min(检测CA IX的放于25℃恒温培养箱培养90min)，后在酶标仪下405nm测得取出测得吸光值。
通过酯酶法评估了生理相关的CA II和CA IX酶抑制活性，结果如表2所示。
表2.本发明部分目标化合物对于CA II和CA IX的抑制活性
aIC50值是平均值±S.D.3个独立的实验。
由表2可知，所检测的化合物对CA IX存在较好的酶抑制活性和选择性都优于阳性对照药SLC-0111(IC50＝0.25±0.053μM、SI(CA II/IX)＝32)。其中化合物I32的半抑制浓度(IC50)为0.0025±0.0005μM，比SLC-0111好100倍，选择性SI(CA II/IX)＝296；化合物I33对CAII存在明显的脱靶作用，SI(CA II/IX)＝665。
实验例3：细胞缺氧环境下增殖实验
将CA抑制剂SLC-0111作为阳性对照模拟缺氧环境下对细胞进行活性测试，按下面步骤进行：
将MDA-MB-231细胞以2×104～4×104/孔的密度接种到96孔板中，并放置于37℃培养箱中培养24h。去除培养基并添加100μM CoCl2的新鲜培养基于37℃培养箱中培养24h，后将CoCl2暴露维持48h。之后加入待测后加入20μL待测化合物(起始浓度为40μM，2倍法稀释，每个浓度设置5个复孔)，置于细胞培养箱37℃培养24h。最后加入5mg/mL MTT工作液10μL，于培养箱孵育4h，小心弃去上清液，加入150μL DMSO溶解还原产生的甲瓒紫色晶体，用酶标仪测定每孔吸光度。
实验结果以IC50表示(如下述表3所示)，其中SLC-0111(>20μM)为阳性参照。化合物I15、I32、I33缺氧条件下IC50分别为7.68±0.31、3.45±0.06、7.51±0.48μM均高于正常氧环境下的细胞毒性8.89±1.80、4.31±0.46、13.51±0.93μM。化合物在缺氧条件下对肿瘤细胞MDA-MB-231有更高的细胞毒性，说明化合物通过抑制CA IX的活性进一步抑制细胞的活性。
表3.本发明部分化合物正常含氧量和缺氧条件下对MDA-MB-231细胞系抗增殖活性
aIC50值是平均值±S.D.3个独立的实验
实验例4：透射电子显微镜成像
化合物I32透射电子显微镜成像实验过程如下：
将生长状态良好的MDA-MB-231细胞接种在6孔板，每孔约5×105个细胞，于37℃恒温培养箱中培养24h。去除培养基，然后加入不同浓度的化合物I32(每个浓度设置3个复孔)，培养24h后用PBS缓冲液洗涤3次，再用2.5％戊二醛溶液固定12h，最后通过透射电子显微镜观察细胞形态。
在化合物I32以不同的浓度处理MDA-MB-231细胞24h，透射电镜下细胞线粒体结构发生明显的铁死亡。在以5μM化合物I32处理细胞时，细胞线粒体结构相对空白组明显缩小，结构变的紧密；在10μM化合物I32能使线粒体皱缩的更加明显(图1)。上述形态特征表明化合物I32可以有效的诱导铁死亡。
实验例5：活性氧(ROS)实验
化合物I32活性氧(ROS)实验过程如下：
将生长状态良好的MDA-MB-231细胞接种在6孔板中，每孔约5×105个细胞，37℃的恒温培养箱中培养待用。待细胞生长密度到60～70％时，加入不同浓度的化合物I32，孵育24h后，去除细胞培养基，用PBS溶液洗涤细胞。在每个孔中补充10μΜDCFH-DA，避光37℃培养箱中培养20～60min后用无血清培养基洗涤细胞3次，最后使用倒置荧光显微镜观察拍照。
在倒置荧光显微镜观察结果如图2所示。由图2可以看出：对照组中的绿色荧光信号缺失或微弱，实验组绿色荧光随着化合物I32加药浓度增大变强，说明细胞内ROS的水平相对空白组显著提高了。说明化合物I32提高了细胞内ROS的水平，从而导致细胞铁死亡。
实验例6：JC-1染色实验
化合物I32 JC-1染色实验过程如下：
取生长状态良好的MDA-MB-231细胞接种到6孔板中，每孔约5×105个细胞，37℃的恒温培养箱中培养待用。待细胞贴壁完全且密度达60-70％，更换含有不同浓度化合物11o或载体的新鲜培养基，于培养箱培养24h。去除培养液，用PBS缓冲液洗涤2次、胰蛋白酶消化，将细胞收集于15mL离心管中，离心后弃去上清液，用无血清培养基洗涤细胞2次。加入500μL JC-1染色工作液，避光孵育20min。孵育结束后离心、弃去上清液，JC-1染色缓冲液洗涤两次。加500μL无血清培养基悬浮细胞，用荧光倒置显微镜观察拍照。
使用不同浓度的化合物I32处理MDA-MB-231细胞，JC-1染色，荧光倒置显微镜拍照(如图3所示)。结果表明：随着化合物I32浓度的增加红色的荧光在变淡，即聚合物的比例在减少，绿色荧光物质明显增加，说明线粒体膜电位下降，进一步说明了线粒体受损。
实验例7：化合物I32细胞凋亡测试
化合物I32细胞凋亡测试试验过程如下：
将MDA-MB-231细胞以5×105个/孔接种到6孔板中，孵育过夜至密度到60-70％。将培养基更换为含不同浓度的I32的培养基于培养箱中孵育24h，接着通过消化、PBS洗涤将细胞转移至15mL离心管中，1000r/min离心，加入含有5μL的Annexin V-FITC的200μL缓冲液37℃避光孵育20min，随后加入300μL的缓冲液和5μL的PI染色剂，混合均匀后将转移到1.5mL的离心管中，最后使用流式细胞仪进行数据分析和收集。
使用不同浓度的化合物I32孵育MDA-MB-231细胞24h。结果如图4所示，对照组中细胞Q2期所占比例为6.2％，化合物I32的浓度为5μM和10μM处理的Q2期所占比例分别为66.2％、92.9％，表明化合物I32可以通过浓度依赖性的方式促进细胞晚期凋亡。
实验例8：化合物I32细胞细胞划痕实验
化合物I32细胞划痕实验过程如下：
将MDA-MB-231细胞接种在6孔细胞培养板培养过夜，观察细胞刚好铺满板孔。用无菌微量移液枪枪头刮擦单层细胞，用不完全培养基洗涤细胞去除分离的细胞，拍照后补充含有不同浓度的化合物I32的完全新鲜培养基孵育24h。然后用4％多聚甲醛固定细胞，并通过相差倒置显微镜拍照。
基于I32能有效诱导细胞铁死亡和细胞凋亡，申请人对化合物I32影响细胞迁移运动与修复能力进行评估。在不同浓度化合物I32孵育MDA-MB-231细胞24h后，分析MDA-MB-231细胞的迁移潜力。加药前后测量划痕的宽度(如图5所示)，与I32孵育过的细胞相比，未处理的对照组的细胞划痕区域明显变窄了，表明化合物I32能有效抑制MDA-MB-231细胞的迁移。
实验例9：化合物I32动物实验
化合物I32动物实验过程如下：
通过皮下移植乳腺癌4T1细胞建立小鼠模型，当小鼠平均肿瘤体积达到约100mm3时，将小鼠随机分为3组，每组5只，即载体组、化合物I32 15mg/kg剂量组、化合物I32 30mg/kg剂量组。每3天经静脉注射给予化合物I32，药物处理后每隔一天记录肿瘤体积和体重。治疗21天后，颈椎脱臼处死动物，剥离瘤块称重。观察期未采血进行生化参数检测，取脏器组织(肺、肝)，4％多聚甲醛固定、苏木精-伊红染色。
由于体外抗肿瘤实验存在一定的局限性，不能完全模拟体内的生存环境，所以将化合物I32进行体内实验进一步评估抗肿瘤活性能力。申请人使用具有高度转移性的乳腺癌4T1细胞的异种移植模型研究化合物I32的体内抗肿瘤活性。接种24h后，将小鼠随机分为三组(n＝5)，每3天经静脉注射给予化合物I32(15或30mg/kg)或载体，连续21天。结果如图6所示，与对照组相比，化合物I32能有效抑制肿瘤生长(图6A)，在以15mg/kg和30mg/kg剂量下的肿瘤生长抑制率分别为45.5％和51.2％(图6B)。与空白组相比，加入了化合物I32的实验组明显减少了肿瘤体积(图6C)，但是每组小鼠的体重逐渐增加，进一步证明化合物11o的安全性(图6D)。小鼠体内抗肿瘤活性测试结果表明化合物I32能有效地抑制乳腺癌4T1肿瘤生长。
据报道，在乳腺癌导致的死亡中，超过90％归因于转移相关的并发症。研究表明，肺和肝脏是乳腺癌最常见的转移部位。因此，申请人通过对肝脏进行切片并用苏木精-伊红(H&E)染色，评估肿瘤细胞在肺和肝脏中的浸润情况。结果如图7所示，4T1细胞植入21天后发生了肺和肝脏的转移，化合物I32治疗显著抑制4T1细胞的肺和肝转移，并呈剂量依赖性。这些结果表明化合物I32具有良好的治疗潜力。
Claims (10)
1.下述式(I)所示结构的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐：
其中：
R表示氢原子、卤原子、羟基或巯基，或者是C1～C8的烷基、烯基或炔基，或者是卤代C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基、烯基或炔基，或者是氨基取代的C1～C8的烷基、烯基或炔基，或者是酰胺基取代的C1～C8的烷基、烯基或炔基，或者是羧基取代的C1～C8的烷基、烯基或炔基，或者是C1～C8的烷氧基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0～4之间的整数。
2.根据权利要求1所述的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物，其特征是，
R表示C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基，或者是氨基取代的C1～C8的烷基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0或2。
3.权利要求1所述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物的制备方法，其特征是，取如下式(II)所示化合物和式(III)所示化合物置于有机溶剂中反应，所得反应物料回收溶剂，得到目标化合物粗品；
其中：
R表示氢原子、卤原子、羟基或巯基，或者是C1～C8的烷基、烯基或炔基，或者是卤代C1～C8的烷基、烯基或炔基，或者是羟基取代的C1～C8烷基、烯基或炔基，或者是氨基取代的C1～C8的烷基、烯基或炔基，或者是酰胺基取代的C1～C8的烷基、烯基或炔基，或者是羧基取代的C1～C8的烷基、烯基或炔基，或者是C1～C8的烷氧基，或者是苄基或其取代的衍生物，或者是苯基或其取代的衍生物，或者是五元或六元杂环甲基或其取代的衍生物；
n表示0～4之间的整数。
4.根据权利要求3所述的制备方法，其特征是，所述的有机溶剂为选自乙腈、丙酮、甲醇和二氯甲烷中的一种或两种以上的组合。
5.根据权利要求3所述的制备方法，其特征是，反应在加热或不加热的条件下进行。
6.根据权利要求3所述的制备方法，其特征是，反应在常温至有机溶剂沸点之间的温度条件下进行。
7.根据权利要求3～6中任一项所述的制备方法，其特征是，还包括对制得的目标化合物粗品进行纯化的步骤。
8.权利要求1所述的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐在制备用于抑制碳酸酐酶IX酶活性和/或过表达的药物中的应用。
9.权利要求1所述的4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐在制备治疗肿瘤的药物中的应用。
10.一种药物组合物，其包括作为活性成分的治疗上有效剂量的权利要求1所述4-哌嗪硫脲苯磺酰胺-1,8-萘酰亚胺衍生物或其药学上可接受的盐，以及药学上可接受的载体。
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