苯并[<i>f</i>][1,4]氧氮杂䓬酮衍生物的合成及抗肿瘤活性研究

引用本文: 李凤琼, 穆敏婕, 杨娜, 钟凌, 胡荣, 李晋奇, 白兰, 师健友, 张梅. 苯并[f][1,4]氧氮杂䓬酮衍生物的合成及抗肿瘤活性研究[J]. 有机化学, 2016, 36(6): 1419-1425. doi: 10.6023/cjoc201512034 shu

Citation: Li Fengqiong, Mu Minjie, Yang Na, Zhong Ling, Hu Rong, Li Jinqi, Bai Lan, Bai Lan, Shi Jianyou. Synthesis and Anticancer Activity Evaluation of Benzo[f][1,4]oxazepinone Derivatives[J]. Chinese Journal of Organic Chemistry, 2016, 36(6): 1419-1425. doi: 10.6023/cjoc201512034 shu

苯并[f][1,4]氧氮杂䓬酮衍生物的合成及抗肿瘤活性研究

李凤琼 ^a,†, ,
穆敏婕 ^a,†, ,
杨娜 ^b, ,
钟凌 ^b, ,
胡荣 ^a, ,
李晋奇 ^b, ,
白兰 ^b, ,
师健友 ^{b,*,
,} ,
张梅 ^{b,*,
,}

a.
成都中医药大学药学院中药材标准化教育部重点实验室中药资源系统研究与开发利用省部共建国家重点实验室培育基地成都 611137
b.
四川省医学科学院·四川省人民医院电子科技大学附属医院个体化药物治疗四川省重点实验室成都 610072

通讯作者: 师健友, E-mail: shijianyoude@126.com; 张梅, E-mail: zhangmei63@126.com

基金项目:
中国博士后科学基金面上资助项目 No. 2014M552374

国家自然科学基金 No. 81302643
^†共同第一作者(These authors contributed equally to this work).

摘要: 端锚聚合酶与细胞的癌变和衰老密切相关, 被认为是细胞癌变机制和癌症治疗靶标研究的新热点. 为寻找新型抗肿瘤化合物, 实验采用计算机辅助的虚拟药物筛选, 设计并合成苯并[f][1,4]氧氮杂䓬酮类化合物12个, 结构经¹H NMR、¹³C NMR、HRMS确证. 对合成化合物进行初步生物活性测试和构效关系阐释, 部分化合物对肿瘤细胞显示抑制作用, 其中3h对Hep-3B具有显著的活性, IC₅₀值为3.5 μmol/L.

关键词:

English

Synthesis and Anticancer Activity Evaluation of Benzo[f][1,4]oxazepinone Derivatives

Li Fengqiong ^a,†, ,
Mu Minjie ^a,†, ,
Yang Na ^b, ,
Zhong Ling ^b, ,
Hu Rong ^a, ,
Li Jinqi ^b, ,
Bai Lan ^b, ,
Bai Lan ^{b,*,
,} ,
Shi Jianyou ^{b,*,
,}

a.
Key Laboratory of Standardization of Chinese Herbal Medicines of Ministry of Education, State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine (TCM), Chengdu 611137
b.
Sichuan Provincial Key Laboratory of Individualized Drug Therapy, Sichuan Academy of Medical Science & SichuanProvincial People's Hospital, School of Medicine, University of Electronic Science and Technology of China (UESTC), Chengdu 610072

Corresponding author: Bai Lan, shijianyoude@126.com; Shi Jianyou, zhangmei63@126.com

Received Date: 23 December 2015
Revised Date: 22 January 2016
Fund Project:
and the China Postdoctoral Science Foundation

Project supported by the National Natural Science Foundation of China

Abstract: The telomere-associated protein tankyrase is a poly(adenosine diphosphate-ribose) polymerase and is considered to be a promising target for cancer therapy, especially selective lethality on breast cancer associated (BRCA) cell lines, including breast cancer as well as malignancies of the ovaries, pancreas, and prostate gland. A variety of drug candidates have been developed and investigated, such as XAV-939, which was identified as a tankyrase inhibitor during screening for a small-mole- cule inhibitor of the Wnt/β-catenin pathway, and it exhibits nanomolar activity in the tankyrase-2 biochemical assay. Coincidentally, almost all compounds which exhibit excellent activity in the tankyrase-2 biochemical assay showed the common structure: lactam ring or amide structure. According to the reported co-crystal structure of inhibitors with tankyrase-2 analysis, it was found that the lactam ring or amide of the compounds forms three conserved hydrogen bonds to Gly1032 and Ser1068 and a π-stacking interaction with Tyr1071. Thus the lactam ring or amide was identified to be a key structure of tankyrase inhibitors. So a new class of structure based on benzo[f][1,4]oxazepinone scaffold was designed through computer-aided virtual drug screening, and 12 new compounds were synthesized via Mannich reactions of aromatic aldehydes with aromatic amines and aromatic ketones, ring expansion, reduction and condensation. The target compound was confirmed by ¹H NMR, ¹³C NMR, HRMS. Followed by the methyl thiazolyl tetrazolium (MTT) assay [A1] to test in vitro anti-tumor activity, part compounds showed potent inhibitory effect on tumor cells. This study culminates in compound 3h, an inhibitor with potent activity against Hep-3B (IC₅₀=3.5 μmol/L). Here, the synthesis and structure-activity relationship (SAR) of this novel series were described. To explore the anti-tumor mechanism of benzo[f][1,4]oxazepinone compounds, we explored the interaction of the compound 3h complex with the Gly1032, Ser1068 and Tyr1071 binding site of tankyrase-2, which confirms the three conserved hydrogen and π-stacking binding mode, as supporting information for the in vitro anti-tumor activity.

Key words:

图2 XAV939-TANKS2 (a),DPQ-TANKS2 (b),DR-2313-TANKS2 (c),NU-1025-TANKS2 (d),P-J34-TANKS2 (e)和ABT-888-TANKS2 (f)的晶体结构

Figure 2. The co-crystal structures of XAV939-TANKS2 (a),DPQ-TANKS2 (b),DR-2313-TANKS2 (c),NU-1025-TANKS2 (d),P-J34-TANKS2 (e) and ABT-888-TANKS2 (f)

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图图式1 苯并[f]^{[1, 4]}-氧氮杂䓬酮类化合物2的合成路线

Figure 图式1. Strategies for the synthesis of benzo[f]^{[1, 4]}oxazepin-one 2

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图1 部分文献报道TANKS抑制剂

Figure 1. Part reported TANKS inhibitors

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我们课题组在查阅文献^{[8, 9]}过程中,发现目前报道的Tankyrase类抑制剂多具有酰胺、内酰胺结构(图 1). 分析各化合物与Tankyrase-2结合的晶体结构发现: 各分子的酰胺或者内酰胺结构的N、O均与Tankyrase-2的Gly1032 (G1032)及Ser1068 (S1068)形成保守的三个氢键作用,同时酰胺、内酰胺环与Tankyrase-2蛋白中Tyr1071 (Y1071)形成π键作用,且部分化合物也与Tyr1060 (Y1060)形成π键作用(图 2). 由上我们推测,内酰胺或酰胺结构可能为Tankyrase-2抑制剂的关键结构.

图图式3 化合物2f的合成路线

Figure 图式3. Strategies for the synthesis of compound 2f

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图图式2 化合物3和4的合成路线

Figure 图式2. Strategies for the synthesis of compounds 3 and 4

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端锚聚合酶(Tankyrase)属于腺苷二磷酸核糖聚合酶[poly adenosine diphosphate-ribose polymerases,PARP]家族,该家族包含17个成员,均含有高度保守的PARP催化活性中心^{[1, 2]}. Tankyrase包含两个亚型,Tankyrase-1 (TANKS1)和Tankyrase-2 (TANKS2),二者结构非常相似,后者较前者仅缺少一个HPS (region containing homopolymeric runs of His,Pro and Ser)结构域,它们的PARP活性中心序列一致性高达86%～95%^[3]. Tankyrase对端粒结合蛋白1 (telomereic repeat binding factor 1,TRF1)等的糖基化作用使其从端粒末端释放,端粒酶乘机接近端粒末端,催化端粒延长,当这种延长足以抵消细胞分裂造成的端粒缩短时,端粒保持一种稳定的长度设置,进而保证细胞的永生化增殖-癌症发生^[4]. 因此,Tankyrase是人体内端粒酶激活、端粒延长的正调控因子^[5]. 有研究指出TANKS2能够通过与Axin (Axis inhibitor,体轴发育抑制因子)相互作用,经由泛素-蛋白酶体途径促进Axin的降解而激活Wnt信号通路^[6]. Wnt信号通路的异常激活,与大量人体肿瘤的发生过程密切相关,包括结肠癌、髓母细胞瘤、黑色素瘤、肝癌、卵巢癌和子宫癌等. 因此,Tankyrase已成为最热的药物筛选靶点之一. 2009年,Nature杂志刊登了Huang等^[6]的研究: 用化学遗传筛选(Chemical Genetic Screen)得到了命名为XAV-939的化合物小分子,证明该化合物能够很好的抑制Wnt信号通路,降低核内β-catenin的数量,并成功寻找到了XAV-939的作用靶点: Tankyrase 1/2. Karlberg等^[^7]在2010年发表了XAV-939和Tankyrase-2的三维晶体结构,使人们可以更加直观的理解其相互作用模式,也为药物设计和虚拟筛选打下了基础.

为得到新一类高效Tankyrase-2抑制剂,我们课题组根据已报道的Tankyrase-2蛋白晶体结构,采用计算机辅助的虚拟筛选,设计了一类苯并[f]^{[1, 4]}-氧氮杂䓬酮母核化合物,虚拟筛选显示: 结构中的内酰胺与Tankyrase-2蛋白的Ser1068、Gly1032发生氢键作用,Tyr1071与母核苯环形成芳环堆叠作用. 因此,我们推测苯并[f]^{[1, 4]}-氧氮杂䓬酮衍生物会是一类具潜在的Tankyrase-2抑制剂. 为了验证我们的设想,实验设计并合成了12个未见文献报道的苯并[f]^{[1, 4]}-氧氮杂䓬酮类化合物. 合成路线如Scheme 1～3所示,并对所合成化合物进行了体外抗肿瘤活性测定,初步阐明构效关系. 为进一步探讨苯并[f]^{[1, 4]}-氧氮杂䓬酮类化合物的与靶标蛋白的结合作用,实验将化合物3h与Tankyrase-2蛋白进行了分子对接，初步印证其抗肿瘤活性。但，该类化合物的抗肿瘤作用机制有待进一步研究证明.

1 结果与讨论

1.1 目标化合物的合成

以2-羟基苯乙酮和取代苯甲醛为起始原料,经曼尼希缩合反应制得黄烷酮化合物1,随后经扩环反应制得苯并[f]^{[1, 4]}-氧氮杂䓬酮化合物2,其中产物2a经磺化、胺化得到磺胺化合物2f; 另产物2d和2h为右边苯环NO₂取代,经还原Fe粉还原得到3d和3h. 3d与芳香羧酸缩合制得酰胺产物4d和4e.

1.2 目标化合物的抗肿瘤活性

用噻唑蓝(MTT)比色法测定目标化合物2a～2h、3 和4抑制人肝癌细胞(Hep-3B)、人宫颈癌细胞(Hela)、人乳腺癌细胞（MDA-MB-231）等3种人癌细胞增殖活性,MTT实验结果见表 1. 从表 1中数据可知,所合成的目标化合物对3种肿瘤细胞株(Hep-3B、Hela、MDA-MB-231)均有不同程度的抑制活性,而化合物2a、2d、2f、4d、4e对肿瘤细胞的抑制活性不明显.

图3 化合物3h与Tankyrase-2结合位点作用图

Figure 3. Possible binding mode of compound 3h with Tankyrase-2 binding site

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由表 1可知,目标化合物苯环上的取代基对其生物活性具有明显的影响. 当苯环上无取代时(2a),显示无抗肿瘤活性.; 固定R²位取代不变,对R¹位取代目标化合物的活性进行探究: 例如2b抗肿瘤活性较2a明显增强,而2c抗肿瘤活性较2d明显增强,且其对抗肿瘤活性的增强作用较化合物2b对比2a的作用更强,说明R¹位的吸电子取代对化合物抗肿瘤活性贡献较大. 固定R¹取代不变,研究R²位的取代对化合物抗肿瘤活性的影响: R²引入3-NO₂时(2d)对上述3种癌细胞系增殖无明显抑制活性.; 尝试在R²引入较3-NO₂吸电子能力弱的3-Cl (2e),其对癌细胞增殖表现出明显抑制活性.; 当R²引入供电子基3-NH₂ (3d)后对上述3种癌细胞系增殖的抑制率均高于吸电子的3-NO₂ (2d)和3-Cl (2e)取代. 因此,对R²的3位取代目标化合物的探索表明: R²位的供电子取代是化合物抗肿瘤活性的关键. 于是固定化合物3d取代不变,使氨基酰胺化,得到目标化合物4d和4e,实验数据显示相对于化合物3d,其体外抗肿瘤活性明显降低,甚至显示出对肿瘤细胞增殖无抑制作用，推测氨基酰胺化后引入大基团形成位阻效应,从而使化合物与靶标位点的结合作用减弱,导致抗肿瘤活性降低.为进一步探讨R²取代对目标化合物生物活性的影响,在R²引入4-SO₂NH₂ (2f),实验显示其无体外抗肿瘤活性; 于是对R²依次引入2-NO₂ (2h)、2-Cl (2g)、2-NH₂ (3h),其体外抗肿瘤活性规律同R²的3位取代,即: NH₂ (3d、3h)＞Cl (2f、2g)＞NO₂ (2d、2h),并且,其对三种肿瘤细胞增殖的抑制作用均为R²的2位取代强于3位取代化合物,即: 3h＞3d＞2g＞2f＞2h＞2d. 表明: R²的2-NH₂(即2位供电子体)取代是目标化合物抑制肿瘤增殖活性的关键. 为了进一步了解3h活性较好的原因,我们对其进行了分子对接,发现除了设想的母核上的内酰胺与Ser1068与Gly1032有氢键作用,其2位上的氨基亦与Gly1032发生氢键作用,加强了该分子与Tankyrase -2的结合力(图 3).

表1 目标化合物抗肿瘤细胞增殖活性 Table1. Anti-tumor cell proliferative activity of target compounds

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Compd.	R¹	R²	IC₅₀^a/(μmol•L^-1)
Hep-3B	R¹	R²	Hela	MDA-MB-231
2a	H	H	NA	NA	NA
2b	4-OCH₃	H	35.1	23.8	27.9
2c	2-F	3-NO₂	10.9	9.8	15.6
2d	H	3-NO₂	NA	NA	NA
2e	H	3-Cl	35.9	26.5	NA
3d	H	3-NH₂	33.2	21.1	18.5
4d	H	3-NHCOC₆H₅	NA	NA	NA
4e	H	3-NHCOC₅NH₅	NA	NA	NA
2f	H	4-SO₂NH₂	NA	NA	NA
2g	H	2-Cl	9.7	13.3	10.9
2h	H	2-NO₂	35.2	21.1	23.4
3h	H	2-NH₂	3.5	4.7	6.2
阿霉素	—	—	5.1	2.3	5.9
^a NA: No activity.

表1 目标化合物抗肿瘤细胞增殖活性
Table1. Anti-tumor cell proliferative activity of target compounds

2 结论

根据Tankyrase-2晶体结构,采用计算机辅助的虚拟筛选,设计合成了一系列苯并[f]^{[1, 4]}-氧氮杂䓬酮化合物,其结构均经¹H NMR,¹³C NMR和HRMS确证. 采用MTT法对所合成化合物进行了体外抗肿瘤活性测试(Hep-3B、Hela、MDA-MB-231),结果表明,部分化合物对肿瘤细胞增长有一定的抑制作用,其中化合物3h对Hep-3B具有显著活性,IC₅₀值为3.5 μmol/L,对探索新一类抗肿瘤药物研究具有重要价值.

3 实验部分

3.1 仪器与试剂

Bruker Avance 400型核磁共振波谱仪(TMS为内标,DMSO-d₆为溶剂); Waters UPLC/Q-TOF-MS; SGW X-4熔点仪; Thermo Multiskan FC型酶标仪; ESCO生物安全柜; OLYMPUS CKX41生物显微镜; Thermo BB15 CO₂孵箱; MTT为Sigma Aldich 公司产品; 肝癌Hep-3B细胞、宫颈癌Hela细胞以及乳腺癌MDA-MB-231细胞均由四川省医学科学院四川省人民医院实验动物研究所提供. 其余所用试剂均为市售化学纯或分析纯.

3.2 实验方法

3.3 目标化合物的抗癌活性测试

采用标准的噻唑蓝比色法,测定目标化合物抑制Hep-3B、Hela、MDA-MB-231 等3种癌细胞系增殖的抑制活性(%). 用完全培养液调整细胞浓度为2×10⁴ mL,接种于96 孔板,每孔100 μL,培养过夜,次日分别用不同剂量的待筛选化合物配制的完全培养液(终浓度分别为40,20,10,5,2.5,1.25 μmol/L)处理细胞,同时等体积的溶剂对照组. 每个设3个复孔,37 ℃,5% CO₂培养48 h后,每孔加入5 mg/mL MTT溶液 20 μL,继续培养2～4 h,弃上清液,再加二甲基亚砜(DMSO) 150 μL,振荡混匀15 min,用酶标仪(λ＝570 nm)测定吸光度(A)值(A值与活细胞数成正比),取其平均值. 相对细胞增殖抑制率(%)＝(对照组A₅₇₀－实验组A₅₇₀)/对照组 A₅₇₀×100%. 实验至少重复3次. 阳性对照采用阿霉素.

致谢委托四川省医学科学院四川省人民医院实验动物研究所,完成了目标化合物的抗癌活性测定.

辅助材料(Supporting Information) 合成化合物的结构和¹H NMR、¹³C NMR、HRMS原始图谱。这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载

3.2.3 化合物3的合成

参考文献^{[12, 13]}方法合成. 向25 mL干燥的圆底烧瓶中加入75%乙醇25 mL、还原Fe粉8 mmol,稀盐酸调节pH值3～4,80 ℃搅拌10 min后,加入化合物2d或2h 2 mmol,80 ℃回流,薄层色谱(TLC)跟踪反应进程,反应完全后,冷却,抽滤,滤液用NaHCO₃溶液调节pH值8～9,静置、抽滤,滤液用乙酸乙酯(20 mL×3)萃取. 合并有机相,无水MgSO₄干燥,浓缩有机相得目标化合物3d或3h.

2-(3-氨基苯)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(3d): 黄色固体,产率85%. m.p. 216.2～217.4 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.28～3.48 (m,2H),5.13 (s,2H),5.30 (dd,J＝6.6,3.5 Hz,1H),6.46～6.56 (m,2H),6.62 (t,J＝1.9 Hz,1H),6.97～7.08 (m,2H),7.20 (td,J＝7.5,1.2 Hz,1H),7.49 (ddd,J＝8.1,7.3,1.8 Hz,1H),7.67 (dd,J＝7.7,1.8 Hz,1H),8.34 (t,J＝5.8 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 45.09,85.73,111.60,113.48,122.22,123.21,126.87,128.85,130.37,132.76,140.10,148.70,153.96,168.86. HRMS calcd for C₁₅H₁₄N₂O₂ 255.1133,found 255.1088 .

2-(2-氨基苯)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(3h): 黄色固体,产率77%. m.p. 179.8～180.4 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.38 (t,J＝5.1 Hz,2H),5.12 (s,2H),5.56 (t,J＝4.4 Hz,1H),6.58 (t,J＝7.4 Hz,1H),6.70 (d,J＝8.0 Hz,1H),6.98～7.10 (m,2H),7.14～7.29 (m,2H),7.49 (td,J＝7.7,1.9 Hz,1H),7.69 (dd,J＝7.8,1.8 Hz,1H),8.25 (t,J＝5.7 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 43.01,80.88,115.40,115.89,122.00,122.36,123.30,126.33,127.01,128.34,130.37,132.75,145.08,154.17,168.92. HRMS calcd for C₁₅H₁₄N₂O₂ 255.1133,found 255.1137.

3.2.2 化合物2的合成

2-(3-硝基苯)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(2d): 黄色固体,产率60%. m.p. 168.3～169.7 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.45 (ddd,J＝15.7,5.9,4.8 Hz,1H),3.60 (ddd,J＝15.6,5.8,3.7 Hz,1H),5.69 (t,J＝4.2 Hz,1H),7.13 (dd,J＝8.1,1.1 Hz,1H),7.27 (td,J＝7.5,1.1 Hz,1H),7.54 (td,J＝7.7,1.8 Hz,1H),7.65～7.78 (m,2H),7.91 (dt,J＝7.8,1.4 Hz,1H),8.24 (ddd,J＝8.2,2.4,1.0 Hz,1H),8.28～8.37 (m,2H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.53,83.93,121.20,122.04,122.89,123.98,127.32,130.01,130.37,132.95,133.10,141.75,147.71,153.46,169.00. HRMS calcd for C₁₅H₁₂N₂O₄ 283.0719,found 283.0534.

参考文献^{[10, 11]}方法合成. 向25 mL干燥的圆底烧瓶中加入黄烷酮(1a) 4.5 mmol、NaN₃ 6.2 mmol、3.3 mL冰乙酸,搅拌使溶解,冰浴冷却后,缓慢滴加浓H₂SO₄ 0.66 mL. 于冰浴上搅拌,直至气泡产生并继续在冰浴完成放热反应. 转移至45～50 ℃反应45 min. 冷却,加入10% NaHCO₃冰水溶液,调pH呈中性. 乙酸乙酯萃取(20 mL×3),无水MgSO₄干燥,浓缩有机相得到粗品,柱色谱[V(环己烷)∶V(乙酸乙酯)＝2∶1]分离纯化. 得到目标化合物2-苯基-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(2a). 白色固体,产率70%. m.p. 126.3～127.6 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.36～3.47 (m,1H),3.44～3.55 (m,1H),5.45～5.52 (m,1H),7.02～7.09 (d,J＝8.1 Hz,1H),7.17～7.26 (t,J＝7.6 Hz,1H),7.32～7.44 (m,5H),7.46～7.55 (t,J＝7.7 Hz,1H),7.65～7.73 (dd,J＝7.8,1.7 Hz,1H),8.30～8.38 (t,J＝5.8 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.89,85.21,122.16,123.44,126.36,127.02,128.03,128.36,130.40,132.81,139.49,153.80,168.93. HRMS calcd for C₁₅H₁₃NO₂ 238.0868,found 238.1006.

化合物2f按文献方法^[9]合成. 取2-苯基-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬-5-(2H)-酮(2a) 2.5 mmol置于25 mL三口瓶中,于冰浴上加入ClSO₃H 2 mL,连接尾气处理装置,搅拌直至无气泡产生. 缓慢将反应液倾入干净冰水中、不断搅拌,有固体析出,抽滤,清水洗涤滤饼. 将滤饼置于反应瓶中,冰浴上缓慢加入浓氨水3 mL,转移至100 ℃搅拌20～40 min,加入15 mL水,继续搅拌5～10 min,静置、抽滤,滤饼干燥得目标化合物4-(5-氧亚基-2,3,4,5-四氢苯并[f]^{[1, 4]}氧氮杂卓-2-基)苯磺酰胺(2f),黄色固体,产率63%. m.p. 226.1～227.2 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.75 (dddd,J＝12.8,7.5,5.2,2.3 Hz,1H),4.45 (dt,J＝11.2,5.5 Hz,1H),6.43～6.53 (m,1H),6.82 (dd,J＝8.4,1.1 Hz,1H),7.28～7.44 (m,4H),7.70～7.81 (m,2H),8.48 (d,J＝2.9 Hz,1H),12.87 (s,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 45.30,115.70,124.99,125.86,125.93,128.63,134.51,141.32,142.70,145.42,169.28. HRMS calcd for C₁₅H₁₄N₂O₄S 319.0752,found 319.0740.

2-(2-硝基苯)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬-5- (2H)-酮(2h): 黄色固体,产率67%. m.p. 205.1～205.9 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.50 (ddd,J＝15.5,6.0,4.4 Hz,1H),3.56～3.74 (m,1H),5.88 (t,J＝4.0 Hz,1H),7.08 (dd,J＝8.2,1.1 Hz,1H),7.26 (td,J＝7.5,1.1 Hz,1H),7.53 (td,J＝7.7,1.8 Hz,1H),7.60～7.74 (m,2H),7.77～7.88 (m,2H),8.07～8.14 (m,1H),8.37 (t,J＝5.9 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.58,81.33,122.34,124.48,125.11,127.49,129.26,129.92,130.89,133.50,134.14,134.52,147.89,154.07,169.40. HRMS calcd for C₁₅H₁₂N₂O₄ 285.0875,found 285.0872.

8-甲氧基-2-苯基-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(2b): 白色固体,产率65%. m.p. 127.5～129.4 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.43～3.52 (m,2H),3.79 (s,3H),5.47 (dd,J＝5.3,3.9 Hz,1H),6.58 (d,J＝2.5 Hz,1H),6.77 (dd,J＝8.8,2.5 Hz,1H),7.31～7.47 (m,5H,),7.68 (d,J＝8.7 Hz,1H),8.17 (t,J＝5.6 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 45.44,55.47,84.82,106.00,109.75,118.14,126.31,128.00,128.38,132.24,139.50,162.82,168.39. HRMS calcd for C₁₆H₁₅NO₃ 268.0974,found 268.0890.

2-(3-氯苯基)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(2e): 白色固体,产率69%. m.p. 79.2～81.3 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.41(dt,J＝15.6,5.6 Hz,1H),3.52 (ddd,J＝15.6,5.8,3.7 Hz,1H),5.51 (dd,J＝5.3,3.7 Hz,1H),7.08 (dd,J＝8.1,1.1 Hz,1H),7.24 (td,J＝7.5,1.1 Hz,1H),7.36～7.50 (m,3H),7.46～7.56 (m,2H),7.68 (dd,J＝7.7,1.8 Hz,1H),8.35 (t,J＝5.8 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.59,84.26,122.05,123.73,125.11,126.30,127.15,127.94,130.29,130.37,132.87 133.01,141.95,153.57,168.94. HRMS calcd for C₁₅H₁₂ClNO₂ 272.0479,found 272.0371.

化合物2b～2e,2g～2h按化合物2a的方法合成.

2-(2-氯苯基)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬-5- (2H)-酮(2g): 白色固体,产率61%. m.p. 158.8～160.0 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.41 (ddd,J＝15.6,6.0,4.7 Hz,1H),3.55 (ddd,J＝15.6,5.8,3.6 Hz,1H),5.72 (dd,J＝4.6,3.5 Hz,1H),7.14 (dd,J＝8.2,1.1 Hz,1H),7.25 (td,J＝7.4,1.1 Hz,1H),7.37～7.48 (m,2H),7.48～7.64 (m,3H),7.70 (dd,J＝7.7,1.8 Hz,1H),8.33 (t,J＝5.8 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 43.26,82.10,122.01,123.78,126.90,127.28,128.26,129.46,129.73,130.43,130.70,132.96,136.50,153.70,168.87. HRMS calcd for C₁₅H₁₂ClNO₂ 272.0479,found 272.0370.

6-氟-2-(3-硝基苯)-3,4-二氢苯并[f]^{[1, 4]}-氧氮杂䓬- 5-(2H)-酮(2c): 黄色固体,产率63%. m.p. 178.4～179.3 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.52 (dt,J＝15.8,5.6 Hz,1H),3.64 (ddd,J＝15.8,5.9,3.7 Hz,1H),5.78 (t,J＝4.4 Hz,1H),7.26 (td,J＝8.0,4.8 Hz,1H),7.45～7.56 (m,2H),7.74 (t,J＝8.0 Hz,1H),7.86～7.94 (m,1H),8.25 (ddd,J＝8.2,2.4,1.0 Hz,1H),8.33 (s,1H),8.47 (t,J＝5.8 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.28,84.49,119.43,121.24,123.10,124.42,125.59,129.93,133.09,141.19,147.72,154.05,167.94. HRMS calcd for C₁₅H₁₁FN₂O₄ 303.0781,found 303.0781.

3.2.4 化合物4的合成

向25 mL干燥的圆底烧瓶中加入羧酸2 mmol,适量SOCl₂,催化量N,N-二甲基甲酰胺(DMF),80 ℃回流,薄层色谱(TLC)跟踪反应进程,反应完全后,旋蒸除去多余SOCl₂,溶加CH₂Cl₂溶解,缓慢滴加至冰浴冷却的含化合物3d、DIEA的CH₂Cl₂溶液中. 薄层色谱(TLC)跟踪反应进程,反应结束后,浓缩有机相得到粗品,用[V(环己烷)︰V(乙酸乙酯)＝2︰1]柱色谱分离纯化. 得到目标化合物4d或4e.

N-[3-(5-氧亚基-2,3,4,5-四氢苯并[f]^{[1, 4]}氧氮杂䓬- 2-基)-苯基]吡啶酰胺(4e): 白色固体,产率71%. m.p. 169.2～1 71.0 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.42 (dt,J＝15.5,5.9 Hz),3.54 (ddd,J＝15.5,6.0,3.6 Hz,1H),5.50 (dd,J＝6.3,3.6 Hz,1H),7.10～7.28 (m,3H),7.40 (t,J＝7.9 Hz,1H),7.52 (td,J＝7.8,1.8 Hz,1H),7.69 (ddd,J＝7.6,4.3,1.4 Hz,2H),7.90 (dd,J＝8.2,2.0 Hz,1H),8.01～8.13 (m,2H),8.17 (dt,J＝7.8,1.2 Hz,1H),8.37 (t,J＝5.8 Hz,1H),8.75 (dt,J＝4.7,1.2 Hz,1H),10.70 (s,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 44.88,85.18,118.25,119.82,121.83,122.30,122.3,123.49,126.94,127.09,128.72,130.34,132.83,138.13,138.38,140.07,148.40,149.79,153.77,162.49,168.94. HRMS calcd for C₂₁H₁₇N₃O₃ 358.1192,found 358.0386.

N-[3-(5-氧亚基-2,3,4,5-四氢苯并[f]^{[1, 4]}氧氮杂䓬- 2-基)-苯基]苯甲酰胺(4d): 白色固体,产率74%. m.p. 105.5～106.0 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 3.38～3.63 (m,2H),5.52 (t,J＝4.5 Hz,1H),7.16 (dd,J＝8.2,4.0 Hz,2H),7.24 (t,J＝7.5 Hz,1H),7.40 (t,J＝7.8 Hz,1H),7.55 (tt,J＝14.0,7.2 Hz,3H),7.73 (dd,J＝7.9,3.9 Hz,1H),7.87 (t,J＝6.6 Hz,1H),7.93～8.04 (m,3H),8.43 (q,J＝5.7 Hz,1H),10.40 (s,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 48.63,85.28,118.31,119.95,121.49,122.29,123.53,127.15, 127.65,128.33,128.65,130.37,131.55,132.86,134.85,139.28,140.04,153.83,165.62,169.09. HRMS calcd for C₂₂H₁₈N₂O₃ 359.1395,found 359.1397.

3.2.1 黄烷酮(1a)的合成

向25 mL干燥的圆底烧瓶中加入20 mL甲醇,依次加入苯甲醛(1.0 mmol)、苯胺(1.5 mmol)、2-羟基苯乙酮(1.2 mmol)、I₂ (0.3 mmol)于45 ℃搅拌,经曼尼希缩合反应,薄层色谱(TLC)跟踪反应进程. 反应结束后,除去溶剂,加入冰的饱和Na₂S₂O₃溶液20 mL,用CH₂Cl₂ (20 mL×3)萃取. 合并有机相,依次用饱和NaCl溶液、冰水洗,无水Na₂SO₄干燥. 浓缩有机相得到粗品,柱色谱[V(环己烷)∶V(乙酸乙酯)＝6∶1]分离纯化,得到黄烷酮(1a). 白色固体,产率72%. m.p. 66.7～67.1 ℃; ¹H NMR (400 MHz,DMSO-d₆) δ: 2.85 (dt,J＝16.9,2.3 Hz,1H),3.27 (ddd,J＝16.6,12.8,2.4 Hz,1H),5.68 (dt,J＝13.0,2.6 Hz,1H),7.06～7.16 (m,2H),7.31～7.49 (m,3H),7.52～7.66 (m,3H),7.81 (dt,J＝8.3,1.6 Hz,1H); ¹³C NMR (101 MHz,DMSO-d₆) δ: 43.98,79.28,118.51,121.13,121.94,126.79,127.09,128.98,129.02,136.75,139.38,161.54,192.02.

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图 1 部分文献报道TANKS抑制剂

Figure 1 Part reported TANKS inhibitors

(a)XAV-939 is a inhibitor of TNKS1 and TNKS2 with IC₅₀ of 11 and 4 nmol/L,respectively; (b) DPQ is a inhibitor of TNKS1 and TNKS2 with IC₅₀ of 0.033 and 2.8 μmol/L,respectively; (c) DR-2313 is a novel inhibitor of TNKS2 with IC₅₀ of 3.0 μmol/L; (d) NU-1025 is a potent inhibitor of TNKS2 with IC₅₀ of 1.4 μmol/L; (e) P-J34 is a specific inhibitor of TNKS2 with IC₅₀ of 0.963 μmol/L; (f) ABT-888 is a potent inhibitor of TNKS1 and TNKS2 with IC₅₀ of 14.97 and 6.52 μmol/L,respectively.

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图 2 XAV939-TANKS2 (a),DPQ-TANKS2 (b),DR-2313-TANKS2 (c),NU-1025-TANKS2 (d),P-J34-TANKS2 (e)和ABT-888-TANKS2 (f)的晶体结构

Figure 2 The co-crystal structures of XAV939-TANKS2 (a),DPQ-TANKS2 (b),DR-2313-TANKS2 (c),NU-1025-TANKS2 (d),P-J34-TANKS2 (e) and ABT-888-TANKS2 (f)

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图式1 苯并[f]^{[1, 4]}-氧氮杂䓬酮类化合物2的合成路线

Scheme 1 Strategies for the synthesis of benzo[f]^{[1, 4]}oxazepin-one 2

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图式2 化合物3和4的合成路线

Scheme 2 Strategies for the synthesis of compounds 3 and 4

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图式3 化合物2f的合成路线

Scheme 3 Strategies for the synthesis of compound 2f

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图 3 化合物3h与Tankyrase-2结合位点作用图

Figure 3 Possible binding mode of compound 3h with Tankyrase-2 binding site

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表 1 目标化合物抗肿瘤细胞增殖活性

Table 1. Anti-tumor cell proliferative activity of target compounds


Compd.	R¹	R²	IC₅₀^a/(μmol•L^-1)
Hep-3B	R¹	R²	Hela	MDA-MB-231
2a	H	H	NA	NA	NA
2b	4-OCH₃	H	35.1	23.8	27.9
2c	2-F	3-NO₂	10.9	9.8	15.6
2d	H	3-NO₂	NA	NA	NA
2e	H	3-Cl	35.9	26.5	NA
3d	H	3-NH₂	33.2	21.1	18.5
4d	H	3-NHCOC₆H₅	NA	NA	NA
4e	H	3-NHCOC₅NH₅	NA	NA	NA
2f	H	4-SO₂NH₂	NA	NA	NA
2g	H	2-Cl	9.7	13.3	10.9
2h	H	2-NO₂	35.2	21.1	23.4
3h	H	2-NH₂	3.5	4.7	6.2
阿霉素	—	—	5.1	2.3	5.9
^a NA: No activity.

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