含苯并1,1-二氧代噻嗪结构的芳氨基嘧啶类ALK抑制剂的合成及抗肿瘤活性
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摘要
目的设计并合成结构新颖的含苯并1,1-二氧代噻嗪结构的芳氨基嘧啶类ALK抑制剂。方法基于色瑞替尼与ALK蛋白的共晶模型及其构效关系,设计了含苯并1,1-二氧代噻嗪结构的芳氨基嘧啶类新化合物;以2-氟硝基苯为原料,经取代、氯代、氧化、还原、环合、N-烃化反应得到关键中间体4-(2-氨基-5-氯嘧啶-4-基)-3,4-二氢-2H-苯并[1,4-b]1,1-二氧代噻嗪,该中间体与取代芳胺进行氨解反应得目标化合物Ⅰ_1~Ⅰ_(11);化合物Ⅰ_(11)与环胺进行Mannich反应得到目标物Ⅰ_(12)~Ⅰ_(15)。采用MTT法及HTRF激酶测试法评价目标化合物的体外抗肿瘤活性。结果与结论合成了15个未见文献报道的新化合物,其结构经MS、~1H-NMR谱确证;部分目标化合物显示出较好的活性,其中,4-(5-氯-2-((2-(4-乙基哌嗪-1-基)-6-甲氧基吡啶-4-基)氨基)嘧啶-4-基)-3,4-二氢-2H-苯并[1,4-b]噻嗪-1,1-二氧化物(Ⅰ_1)活性最为突出,其抑制人间变性大细胞淋巴瘤细胞株Karpas299和人肺腺癌细胞株H2228的IC_(50)值分别为0.024、0.14μmol·L~(-1),化合物Ⅰ_1抑制ALK激酶的IC_(50)值为14.2 nmol·L~(-1),与色瑞替尼接近。
The rate of anaplastic lymphoma kinase(ALK) gene rearrangements in non-small cell lung cancer(NSCLC) tissues is 4%-6%. Aiming to explore novel anaplastic lymphoma kinase(ALK) inhibitors,a series of novel arylaminopyrimidine derivatives bearing benzothiazine~(-1),1-dioxide moiety were designed and synthesized based upon the cocrystal structure of ceritinib with ALKWT(PDB: 4 M KC) as well as the reported structure-activity relationships(SARs). Taking 2-fluoronitrobenzene as starting material,the critical intermediate 4-(2,5-dichloropyrimidin-4-yl)-3,4-dihydro-2 H-benzo [1,4-b]thiazine~(-1),1-dioxide was prepared through S-alkylation,chlorination,oxidation,reduction,intermolecular cyclization and N-alkylation reactions. Subsequently,ammonolysis reaction of 4-(2,5-dichloropyrimidin-4-yl)-3,4-dihydro-2 H-benzo[1,4-b]thiazine~(-1),1-dioxide with a variety of arylamine obtained newcompounds Ⅰ_1~Ⅰ_(11). Compounds Ⅰ_(12)~Ⅰ_(15) were synthesized from compound Ⅰ11 by M annich reaction. The cellular and enzymatic assays validated compound Ⅰ1 as a most promising ALK(IC50= 14. 2 nmol·L~(-1)) inhibitor. CompoundⅠ1 showed significant inhibitory activities on ALK-dependent cell lines Karpas299(IC50= 0. 024 μmol·L~(-1)) and H2228(IC50=0. 14 μmol·L~(-1)) superior to ceritinib(IC50= 0. 048 μmol·L~(-1) and 0. 22 μmol·L~(-1)). The preliminary SARs studies suggested that the introduction of benzothiazine~(-1),1-dioxide moiety did improve the potency; diverse arylamine(R) and a variety of water-soluble groups(R1) had dramatic impacts on cellular activity; overall,SARs identified molecules bearing the 2-methoxypyridine and 4-ethylpiperazine moieties with more favorable potency.
The rate of anaplastic lymphoma kinase(ALK) gene rearrangements in non-small cell lung cancer(NSCLC) tissues is 4%-6%. Aiming to explore novel anaplastic lymphoma kinase(ALK) inhibitors,a series of novel arylaminopyrimidine derivatives bearing benzothiazine~(-1),1-dioxide moiety were designed and synthesized based upon the cocrystal structure of ceritinib with ALKWT(PDB: 4 M KC) as well as the reported structure-activity relationships(SARs). Taking 2-fluoronitrobenzene as starting material,the critical intermediate 4-(2,5-dichloropyrimidin-4-yl)-3,4-dihydro-2 H-benzo [1,4-b]thiazine~(-1),1-dioxide was prepared through S-alkylation,chlorination,oxidation,reduction,intermolecular cyclization and N-alkylation reactions. Subsequently,ammonolysis reaction of 4-(2,5-dichloropyrimidin-4-yl)-3,4-dihydro-2 H-benzo[1,4-b]thiazine~(-1),1-dioxide with a variety of arylamine obtained newcompounds Ⅰ_1~Ⅰ_(11). Compounds Ⅰ_(12)~Ⅰ_(15) were synthesized from compound Ⅰ11 by M annich reaction. The cellular and enzymatic assays validated compound Ⅰ1 as a most promising ALK(IC50= 14. 2 nmol·L~(-1)) inhibitor. CompoundⅠ1 showed significant inhibitory activities on ALK-dependent cell lines Karpas299(IC50= 0. 024 μmol·L~(-1)) and H2228(IC50=0. 14 μmol·L~(-1)) superior to ceritinib(IC50= 0. 048 μmol·L~(-1) and 0. 22 μmol·L~(-1)). The preliminary SARs studies suggested that the introduction of benzothiazine~(-1),1-dioxide moiety did improve the potency; diverse arylamine(R) and a variety of water-soluble groups(R1) had dramatic impacts on cellular activity; overall,SARs identified molecules bearing the 2-methoxypyridine and 4-ethylpiperazine moieties with more favorable potency.
引文
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[15]吴永谦.二并环类间变性淋巴瘤激酶抑制剂:中国,106118029[P]. 2016-12-07.
[16] HUANG Y,ZHANG J,YU Z T,et al. Discovery of First-in-Class,potent,and orally bioavailable embryonic ectoderm development(EED)inhibitor w ith robust anticancer efficacy[J]. J M ed Chem,2017,60(6):2215-2226.
[17]赵嘉惠,张华屏,王春芳. MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3):262-263.
[18]杨燕,胡万福. MTT法检测复方大果木姜子软胶囊抗肿瘤作用[J].三峡大学学报(自然科学版),2017(S1):15-17.
[19] MATHIS G. HTRF technology[J]. J Biomol Screen,1999,4(6):309-313.
[20]王慧洁,周子涵,徐嘉辰,等.基于均相时间分辨荧光技术(HTRF)的HSP90-HOP相互作用抑制剂活性测试方法的构建及其应用[J].药学学报,2017,52(4):592-597.
[2] SIEGEL R L,MILLER K D,FEDEWA S A,et al.Colorectal cancer statistics,2017[J]. CA Cancer J Clin,2017,67(3):177-193.
[3] SHIOTA M,FUJIMOTO J,SEMBA T,et al. Hyperphosphorylation of a novel 80 k Da protein-tyrosine kinase similar to Ltk in a human Ki-1 lymphoma cell line,AM S3[J]. Oncogene,1994,9(6):1567-1574.
[4] HATCHER J M,BAHCALL M,CHOI H G,et al.Discovery of inhibitors that overcome the G1202R anaplastic lymphoma kinase resistance mutation[J].J M ed Chem,2015,58(23):9296-9308.
[5] SODA M,CHOI Y L,ENOMOTO M,et al. Identification of the transforming EM IA-ALK fusion gene in non-small-cell lung cancer[J]. Nature,2007,448(7153):561-566.
[6] SAOIRSE O D,DEARBHAILE C C,RAGHAV S,et al. Advances in the development of molecularly targeted agents in non-small-cell lung cancer[J].Drugs,2017,77(8):813-827.
[7] MARSILJE T H,PEI W,CHEN B,et al. Synthesis,structure-activity relationships,and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase(ALK)inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)-phenyl)pyrimidine-2, 4-diamine(LDK378)currently in phase 1 and phase 2 clinical trials[J]. J M ed Chem,2013,56(14):5675-5690.
[8] FRIBOULET L,LI N,KATAYAMA R,et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer[J]. Cancer Discov,2014,4(6):662-673.
[9] HUANG W S,LIU S Y,ZOU D,et al. Discovery of brigatinib(AP26113),a phosphine oxide-containing,potent,orally active inhibitor of anaplastic lymphoma kinase[J]. J M ed Chem,2016,59(10):4948-4964.
[10] SOLOMON B J. Ceritinib in ALK-rearranged nonsmall cell lung cancer[J]. N Engl J M ed,2015,371(21):1963-1971.
[11] CHEN J Y,JIANG C,WANG S M. LDK378:apromising anaplastic lymphoma kinase(ALK)inhibitor[J]. J Med Chem,2013,56(14):5673-5674.
[12] LIU Z Q,AI J,PENG X,et al. Novel 2,4-diarylaminopyrimidine analogues(DAAP alogues)showing potent c-M et/ALK multikinase inhibitory activities[J]. M ed Chem Lett,2014,5(4):304-308.
[13] SONG Z L,YANG Y H,LIN Z Q,et al. Discovery of novel 2, 4-diarylaminopyrimidine analogues(DAAP alogues)showing potent inhibitory activities against both w ild-type and mutant ALK kinases[J].J M ed Chem,2015,58(1):197-211.
[14] LEWIS D P,DEMETRI T M. The necessary nitrogen atom:a versatile high-impact design element for multiparameter optimization[J]. J M ed Chem,2017,60(9):3552-3579.
[15]吴永谦.二并环类间变性淋巴瘤激酶抑制剂:中国,106118029[P]. 2016-12-07.
[16] HUANG Y,ZHANG J,YU Z T,et al. Discovery of First-in-Class,potent,and orally bioavailable embryonic ectoderm development(EED)inhibitor w ith robust anticancer efficacy[J]. J M ed Chem,2017,60(6):2215-2226.
[17]赵嘉惠,张华屏,王春芳. MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3):262-263.
[18]杨燕,胡万福. MTT法检测复方大果木姜子软胶囊抗肿瘤作用[J].三峡大学学报(自然科学版),2017(S1):15-17.
[19] MATHIS G. HTRF technology[J]. J Biomol Screen,1999,4(6):309-313.
[20]王慧洁,周子涵,徐嘉辰,等.基于均相时间分辨荧光技术(HTRF)的HSP90-HOP相互作用抑制剂活性测试方法的构建及其应用[J].药学学报,2017,52(4):592-597.