3-氨基苯并[d]异噁唑类大环ALK抑制剂的设计、合成及活性评价  

Design,synthesis and activity evaluation of 3-aminobenzo[d]isoxazole macrocyclic ALK inhibitors

作　　者：周振 刘耀阳 黎瑶 华炫智 刘洋[1] 杨华丽[1] 程卯生[1] ZHOU Zhen;LIU Yaoyang;LI Yao;HUA Xuanzhi;LIU Yang;YANG Huali;CHENG Maosheng(Key Laboratory of Structure-Based Drug Design&Discovery,Ministry of Education,Shenyang Pharmaceutical University,Shenyang 110016,China)

机构地区：[1]沈阳药科大学基于靶点的药物设计与研究教育部重点实验室,辽宁沈阳110016

出　　处：《沈阳药科大学学报》2025年第1期52-66,共15页Journal of Shenyang Pharmaceutical University

基　　金：辽宁省大学生创新创业项目(S202410163089);沈阳药科大学“优青”托举计划项目(YQ202306)。

摘　　要：目的间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)的活性失调与多种癌症的发生密切相关。以恩曲替尼为先导化合物,设计并合成一系列3-氨基苯并[d]异噁唑类ALK抑制剂,测试其对ALK阳性的人肺腺癌H2228细胞的抑制活性。方法以邻甲氧基苯甲醛为起始原料,经过还原性C-C偶联、杂环合成和酰胺缩合得到目标化合物A1。以间羟基苯甲酸甲酯和水杨酸甲酯为起始原料,经亲核取代、水解酸化得到中间体5a、5b、13a和13b。以水杨醛及取代水杨醛为起始原料,经过还原性C-C偶联、亲核取代、杂环合成、酰胺缩合、Boc保护与脱保护,以及分子内烯烃复分解反应最终合成得到B、C、D系列大环化合物B1-B4、C1-C4和D1-D7。以恩曲替尼为阳性对照药,使用ALK阳性人肺腺癌H2228细胞株和ALK阴性的人非小细胞肺癌A549细胞株,采用CCK-8法测试目标化合物对细胞株的抑制活性。采用Annexin V-FITC/PI双染流式细胞实验测试化合物B1对H2228肿瘤细胞的凋亡诱导情况。对目标化合物B1和D1进行了ALK酶抑制活性测试。结果与结论合成了16个3-氨基苯并[d]异噁唑类化合物,其中B、C、D系列化合物为大环结构,其结构经核磁共振氢谱和碳谱及高分辨质谱确证。体外H2228肿瘤细胞增殖抑制实验结果表明,化合物B1的抑制活性最佳,IC50=(16.55±3.79)μmol·L^(-1)。细胞凋亡实验表明B1能够浓度依赖性地诱导H2228细胞凋亡。ALK体外酶抑制活性实验中B1和D1的IC50分别为303.7 nmol·L^(-1)和288.0 nmol·L^(-1)。The dysregulation of anaplastic lymphoma kinase is closely associated with the development of various cancers.Using Entrectinib as the lead compound,sixteen 3-aminobenzo[d]isoxazole ALK inhibitors were designed and synthesized.The target compound A1 was synthesized from 2-methoxybenzaldehyde by reductive C-C coupling,heterocyclic synthesis,and amide condensation.Intermediates 5a、5b、13a,and 13b were synthesized from methyl 3-hydroxybenzoate and methyl salicylate via nucleophilic substitution,hydrolysis,and acidification.Target compounds B1-B4、C1-C4,and D1-D7 belonging to macrocyclic compounds respectively were synthesized from salicylaldehyde or substituted salicylaldehyde via reductive C-C coupling,nucleophilic substitution,heterocyclic synthesis,amide condensation,Boc protection,deprotection,and intramolecular olefin metathesis.Target compounds were confirmed by means of 1 H-NMR,13 C-NMR,and HRMS.Entrectinib was employed as a positive control drug to evaluate the inhibitory activity of the target compounds on ALK-positive human lung adenocarcinoma H2228 cell line and ALK-negative human non-small cell lung cancer A549 cell line by the CCK-8 method.Among them,compound B1 exhibited superior inhibitory activity against the H2228 cell line,with an IC50 value of(16.55±3.79)μmol·L^(-1).The apoptosis induction of compound B1 on H2228 tumor cells was assessed using Annexin V-FITC/PI double staining flow cytometry.The results demonstrated that B1 effectively induced concentration-dependent apoptosis in H2228 cells.The ALK enzyme activity of the target compounds B1 and D1 was detected.In vitro ALK inhibition test results showed that IC50 of B1 and D1 were 303.7 mol·L^(-1)and 288.0 mol·L^(-1),respectively.

关 键 词：3-氨基苯并[d]异噁唑 ALK抑制剂 大环化合物 

分 类 号：R914[医药卫生—药物化学]