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Chinese Journal of Organic Chemistry >

2025 , Vol. 45 >Issue 12: 4362 - 4374

DOI: https://doi.org/10.6023/cjoc202504013

ARTICLES

Design, Synthesis and Antitumor Activity of Novel Imide-β-carboline

  • Dongping Qiu ,
  • Zhaoxu Wang ,
  • Jie Zhang , * ,
  • Liang Guo , *
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  • Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, Xinjiang 832003
* E-mail:;

Received date: 2025-04-12

  Revised date: 2025-06-27

  Online published: 2025-08-27

Supported by

National Natural Science Foundation of China(22067017)

Guidance Plan Project of Xinjiang Production and Construction Corps(2022ZD019)

Guidance Plan Project of Xinjiang Production and Construction Corps(2023ZD073)

Science and Technology Research Projects of Xinjiang Production and Construction Corps(2023AB047)

Science and Technology Research Projects of Xinjiang Production and Construction Corps(2023AB054)

Xinjiang Science and Technology Major Project(2022A02006-5)

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Abstract

In order to find novel compounds with superior antitumor activity, a series of 6-(N-imide)-β-carboline derivatives with diverse substituents, totaling 30 compounds, were designed and synthesized. The inhibitory effects of these compounds on five cancer cell lines, namely lung cancer (A549), gastric cancer (BGC-823), colon cancer (CT-26), liver cancer (Bel-7402), and breast cancer (MCF-7), were evaluated using methyl thiazolyl tetrazolium (MTT) assay. The experimental results indicated that most target compounds exhibit better antitumor activity against the tested cell lines, with half-maximal inhibitory concentrations (IC50) values below 20 μmol/L. Moreover, the series of compounds showed better inhibitory activity against the A549 and Bel-7402 cell lines. In particular, compounds 5j, 5p, and 5ac exhibited excellent inhibitory activity against tumor cells, demonstrating potential value for antitumor research. Specifically, compounds 5j and 5p exhibited excellent inhibitory activity against the A549, BGC-823, and Bel-7402 cell lines, while compound 5ac showed excellent inhibitory activity against the A549, Bel-7402, and MCF-7 cell lines, all with IC50 values less than 10 μmol/L. The molecular docking results indicate that compound 5ac exhibits favorable binding interactions with multiple amino acid residues of vascular endothelial growth factor receptor-2 (VEGFR-2).

Key words： β-carboline; imide; antitumor; molecular docking

Cite this article

Dongping Qiu , Zhaoxu Wang , Jie Zhang , Liang Guo . Design, Synthesis and Antitumor Activity of Novel Imide-β-carboline[J]. Chinese Journal of Organic Chemistry, 2025 , 45(12) : 4362 -4374 . DOI: 10.6023/cjoc202504013

近年来, 全球癌症相关死亡率呈现上升趋势. 据统计, 2022年全球癌症死亡人数达到970万, 预计到2050年, 癌症确诊病例数将达到3500万[1]. 目前, 癌症治疗的主要策略涵盖外科手术、放疗和化疗, 而化疗在多数癌症治疗方案中占据着至关重要的地位. 尽管癌症治疗领域已取得了若干进展, 但实现癌症的有效治疗依旧面临严峻挑战. 治疗过程中, 系统性毒性、选择性低和肿瘤细胞的耐药性等问题构成了其限制因素[2,3]. 因此, 急需研发具有更好疗效的新型抗肿瘤药物以及更为安全的化疗药物.
天然产物及其衍生物是抗肿瘤药物的主要来源之一, 其中β-咔啉生物碱作为一种具有吡啶并[3,4-b]吲哚环系的平面三环分子, 是许多天然产物的结构母核和重要中间体[4]. 目前, 已报道β-咔啉生物碱具有广泛的生物活性, 如抗肿瘤[5-12]、抗疟疾[13-18]、抗阿尔兹海默症[19,20]、抗利什曼[21]、抗菌[22,23]、抗抑郁症[24]以及抗病毒活性[25,26]. 其中, 对β-咔啉生物碱的抗肿瘤活性的研究尤为广泛. 在已有的研究中, β-咔啉生物碱可作用于脱氧核糖核酸(DNA)[27]、微管蛋白[28]、拓扑异构酶[29]、周期蛋白依赖性激酶(CDK)[30]、驱动蛋白Eg5[31]、组蛋白脱乙酰酶(HDAC)[32]和血管内皮生长因子受体-2 (VEGFR-2)[33]等靶点, 发挥其抗肿瘤作用.
肿瘤的恶性发展和转移依赖于血管生成, 即从现有血管中生成新血管, 并为肿瘤提供营养物质和氧气[34]. VEGFR-2是调控血管生成的关键酪氨酸激酶, 参与内皮细胞增殖、迁移及血管通透性调节等过程[35]. 该受体异常激活致使多种肿瘤生成, 如肾细胞癌、肝细胞癌和胃肠道间质瘤等[36]. 目前已有多种VEGFR-2抑制剂被批准用于癌症的临床治疗, 如帕唑帕尼(Pazopanib)[37]、阿昔替尼(Axitinib)[38]、索拉非尼(Sorafenib)[39]和替沃扎尼(Tivozanib)[40] (图1). 因此, VEGFR-2成为癌症治疗的重要靶点.
图1 代表性的VEGFR-2抑制剂结构

Figure 1 Chemical structures of representative VEGFR-2 inhibitors

酰胺基团构成了蛋白质的基本结构单元, 被誉为“生命的第一物质”[41]. 在化学药物中, 许多药物分子也含有酰胺结构, 这归因于它们优异的生物活性和药理作用. 例如抗癫痫药乙琥胺(Ethosuximide)[42]、抗肿瘤药物来那度胺(Lenalidomide)[43]、镇痛抗炎药扑热息痛(Paracetamol)[44]和镇痛药利多卡因(Lidocaine)[45](图2). 因此, 酰胺结构作为关键的药效基团被广泛应用于合成具有生物活性的重要中间体.
图2 含酰胺结构的代表药物

Figure 2 Representative drugs with amide structure

目前已报道的具有酰胺结构的β-咔啉活性分子中(图3), 在3位具有酰胺结构的β-咔啉衍生物大多具有良好的抗肿瘤活性[10-12], 还具有抗疟原虫活性[16], 在9位具有酰胺结构的β-咔啉衍生物具有良好的抗疟原虫活性[14,17-18], 此外在其他位点具有酰胺结构的β-咔啉衍生物还具有镇痛[46]、抗增殖[47]以及抗阿尔兹海默症[20]等活性. 具有良好抗肿瘤活性的Canthin-6-one类生物碱同样具有环状酰胺结构, 酰胺结构在β-咔啉衍生物活性方面发挥重要作用.
图3 部分具有酰胺结构的β-咔啉衍生物

Figure 3 β-Carboline derivatives containing amide structure

另外, 本课题组曾在β-咔啉的6位引入系列N-芳基取代基[48]和芳基取代基[49], 发现该系列化合物对多种肿瘤细胞株具有较强的抑制作用. 因此, 我们设想在β-咔啉的6位引入环状酰亚胺结构, 合成系列6-酰亚胺-β-咔啉衍生物(图4), 并评价了该系列化合物的抗肿瘤活性, 得到该类化合物的初步构效关系规律. 通过分子对接技术揭示了化合物与VEGFR-2活性位点的结合模式, 初步明确化合物与靶标的相互作用特征.
图4 目标化合物设计思路

Figure 4 Design strategy of target compound

1 结果与讨论

1.1 化合物3a~3g、4a~4i及5a~5ad的合成

中间体4a~4i的合成路线如Scheme 1所示. 化合物1在硫酸和硝酸的混合溶液中冰浴条件下反应, 生成化合物2[50]. 化合物2和卤代烃在强碱氢化钠作用下, 于无水N,N-二甲基甲酰胺(DMF)溶液中反应, 生成化合物3a~3g[51]. 化合物3b、3g和氯化亚锡在甲醇溶液中反应, 加入氢氯酸后生成化合物4a和4b; 化合物3a~3g和氯化亚锡在甲醇溶液中反应, 加入氢碘酸后生成化合物4c~4i[48].
图式1 中间体4a~4i的合成路线

Scheme 1 Synthetic route of intermediate 4a~4i

化合物5a~5d的合成路线如Scheme 2所示. 化合物4a和4b与邻苯二甲酸酐在冰乙酸中反应得到目标化合物5a~5d. 所有目标化合物经1H NMR、13C NMR和HRMS确证结构.
图式2 化合物5a~5d的合成路线

Scheme 2 Synthetic route of intermediate 5a~5d

化合物5e~5ad的合成路线如Scheme 3所示. 化合物4c~4i与环状酸酐在冰乙酸中反应, 得到目标化合物5e~5ad. 所有目标化合物经1H NMR、13C NMR和HRMS确证结构.
图式3 化合物5e~5ad的合成路线

Scheme 3 Synthetic route of compounds 5e~5ad

1.2 活性研究部分

采用甲基噻唑基四唑(MTT)法[52]测试了目标化合物对肿瘤细胞株的抑制活性, 包括肺癌细胞(A549)、胃癌细胞(BGC-823)、结肠癌细胞(CT-26)、肝癌细胞(Bel-7402)和乳腺癌细胞(MCF-7), 顺铂为阳性对照. 比较9-取代-6-氨基-β-咔啉4e~4h(表1)和6-(N-酰亚胺)-β-咔啉5a~5ad(表2~4), 结果表明, 在6位引入N-邻苯二甲酰亚胺基、N-衣康酰亚胺基、N-丁二酰亚胺基可以增强对多种肿瘤细胞系的抑制活性. 下面将讨论目标化合物对五种癌细胞系的抑制活性.
表1 化合物4e~4h对肿瘤细胞的抑制活性

Table 1 Inhibitory activity of compounds 4e~4h against tumor cells

Compd. R9 (IC50±SD)a/(μmol•L－1)
A549 BGC-823 CT-26 Bel-7402 MCF-7
4e C2H5 31.8±1.5 22.1±1.1 43.2±2.1 18.8±0.8 34.2±1.6
4f n-C4H9 >50 43.9±2.2 35.2±1.6 20.3±0.9 32.7±1.5
4g CH2C6H5 37.9±2.8 >50 >50 46.6±4.4 23.2±1.1
4h CH2C6H4(p-F) 16.8±0.8 27.3±1.2 36.2±1.8 20.8±0.9 15.2±0.7
Cisplatin 18.3±0.6 10.6±0.4 5.7±0.1 14.2±0.7 10.6±0.4

a Cytotoxicity as IC50 for each cell line is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.

表2 化合物5a~5r对肿瘤细胞的抑制活性

Table 2 Inhibitory activity of compounds 5a~5r against tumor cells

Compd. R1 R5 R9 (IC50±SD)a/(μmol•L－1)
A549 BGC-823 CT-26 Bel-7402 MCF-7
5a H Cl CH3 13.1±0.6 7.1±0.3 9.9±0.5 12.6±0.6 16.1±0.8
5b Cl Cl CH3 17.3±0.8 13.3±0.6 9.6±0.5 13.4±0.6 9.9±0.5
5c H Cl CH2C6F5 14.5±0.7 8.3±0.4 11.6±0.5 9.8±0.4 17.4±0.8
5d Cl Cl CH2C6F5 12.4±0.6 13.8±0.6 8.4±0.4 10.3±0.4 11.7±0.5
5e H H H 25.3±1.2 30.6±1.5 19.4±0.9 27.7±1.2 36.6±1.8
5f H H CH3 13.3±0.6 9.6±0.4 11.7±0.5 10.4±0.5 18.9±0.8
5g H H C2H5 12.4±0.6 12.5±0.6 12.1±0.6 13.8±0.6 13.1±0.6
5h H H n-C4H9 14.2±0.6 17.3±0.8 18.2±0.9 13.1±0.6 26.9±1.3
5i H H CH2C6H5 9.9±0.4 11.6±0.5 15.1±0.7 28.7±1.3 13.2±0.6
5j H H CH2C6H4(p-F) 8.1±0.4 8.9±0.4 12.7±0.6 7.6±0.3 11.3±0.5
5k H H CH2C6F5 14.1±0.6 9.7±0.5 11.9±0.5 13.5±0.7 7.9±0.4
5l Cl H CH3 21.4±1.0 11.6±0.5 12.9±0.6 16.2±0.8 11.9±0.5
5m Cl H C2H5 12.2±0.5 22.6±1.1 28.8±1.4 10.8±0.5 26.1±1.1
5n Br H C2H5 13.9±0.6 11.7±0.5 15.7±0.7 11.3±0.5 9.7±0.4
5o CH3 H C2H5 22.1±1.1 21.3±1.1 37.4±1.8 12.8±0.6 24.7±1.1
5p Cl H CH2C6F5 6.5±0.1 9.5±0.4 12.4±0.6 7.8±0.3 11.1±0.5
5q Br H CH2C6F5 13.4±0.7 14.2±0.7 11.6±0.5 9.7±0.4 8.8±0.4
5r CH3 H CH2C6F5 23.5±1.1 15.8±0.6 20.3±1.0 12.9±0.6 14.7±0.7
Cisplatin 18.3±0.6 10.6±0.4 5.7±0.1 14.2±0.7 10.6±0.4

a Cytotoxicity as IC50 for each cell line is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.

表3 化合物5s~5y对肿瘤细胞的抑制活性

Table 3 Inhibitory activity of compounds 5s~5y against tumor cells

Compd. R9 (IC50±SD)a/(μmol•L－1)
A549 BGC-823 CT-26 Bel-7402 MCF-7
5s H 29.6±1.4 43.7±2.1 24.2±1.2 23.3±1.1 32.7±1.6
5t CH3 23.4±1.2 16.7±0.8 18.5±0.9 17.1±0.8 18.9±0.8
5u C2H5 25.4±1.2 11.3±0.5 17.1±0.8 16.7±0.8 24.2±1.2
5v n-C4H9 21.7±0.9 17.2±0.8 18.7±0.8 9.1±0.4 22.9±1.1
5w CH2C6H5 19.2±0.9 12.9±0.6 11.2±0.5 10.5±0.5 20.1±0.9
5x CH2C6H4(p-F) 16.6±0.6 15.1±0.7 33.7±1.6 8.3±0.4 14.9±0.6
5y CH2C6F5 18.5±0.8 16.6±0.8 18.7±0.9 24.5±1.2 21.4±1.0
Cisplatin 18.3±0.6 10.6±0.4 5.7±0.1 14.2±0.7 10.6±0.4

aCytotoxicity as IC50 for each cell line is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.

表4 化合物5z~5ad对肿瘤细胞的抑制活性

Table 4 Inhibitory activity of compounds 5z~5ad against tumor cells

Compd. R9 (IC50±SD)a/(μmol•L－1)
A549 BGC-823 CT-26 Bel-7402 MCF-7
5z CH3 22.1±1.1 22.3±1.1 16.8±0.8 15.9±0.7 11.8±0.5
5aa n-C4H9 8.5±0.4 20.3±1.0 15.2±0.7 17.2±0.8 15.7±0.7
5ab CH2C6H5 15.2±0.6 13.6±0.6 14.2±0.6 9.4±0.4 16.2±0.7
5ac CH2C6H4(p-F) 9.6±0.4 12.4±0.6 13.4±0.6 7.5±0.3 7.3±0.3
5ad CH2C6F5 10.5±0.5 6.3±0.3 8.7±0.4 15.3±0.7 7.9±0.3
Cisplatin 18.3±0.6 10.6±0.4 5.7±0.1 14.2±0.7 10.6±0.4

aCytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.

目标化合物6位为N-邻苯二甲酰亚胺基, R1为氢、R9为甲基和五氟苄基时, 探究了5位取代对目标化合物的抑制活性的影响, 结果如表2所示. 对A549细胞、BGC-823细胞、CT-26细胞和MCF-7细胞抑制活性为5a (R5=Cl)>5f (R5=H); 对A549细胞、BGC-823细胞、CT-26细胞和Bel-7402细胞抑制活性为 5c (R5=Cl)>5i (R5=H), 结果表明5位引入氯原子有助于提高其抗肿瘤活性.
目标化合物6位为N-邻苯二甲酰亚胺基, R1、R5为氢时, 探究了9位取代对目标化合物的影响, 结果如表2所示. 化合物对A549细胞系抑制活性为5j>5i>5g>5f>5k>5h, IC50值优于阳性对照顺铂[(18.3±0.6) μmol/L] (除5e以外); 化合物对Bel-7402细胞系抑制活性为5j>5f>5h>5k>5g, IC50值优于顺铂[(14.2±0.7) μmol/L] (除5e、5i以外); 化合物5f、5j、5k对BGC-823细胞系表现出较优的抑制活性, IC50值优于顺铂[(10.6±0.4) μmol/L]; 化合物5k对MCF-7细胞系表现出最优的抑制活性, IC50值低于顺铂[(10.6±0.4) μmol/L], 结果表明9位引入取代基有助于提高对多种肿瘤细胞系的抑制活性, 其影响为对氟苄基>苄基>乙基>甲基>五氟苄基>正丁基>氢.
目标化合物R5为氢、R9为乙基时, 探究了6位取代N-邻苯二甲酰亚胺基苯环上R1取代对目标化合物的影响, 结果如表2所示. 化合物对A549细胞系抑制活性为5m>5g>5n>5o, IC50值优于顺铂(除5o以外); 化合物对BGC-823细胞系抑制活性为5n>5g>5o>5m, IC50值均高于顺铂; 化合物对Bel-7402细胞系抑制活性为5m>5n>5o>5g, IC50值均优于顺铂; 化合物对MCF-7细胞系抑制活性为5n>5g>5o>5m, IC50值高于顺铂(除5n以外); 结果表明R1取代基为吸电子基团时, 抗肿瘤活性明显增加.
目标化合物6位为N-衣康酰亚胺基时, 探究了9位取代对目标化合物的影响, 结果如表3所示. 大部分化合物对4种肿瘤细胞株表现出较弱的抑制活性. 化合物5t、5u、5v、5w、5x、5y (R9=甲基、乙基、正丁基、苄基、对氟苄基、五氟苄基)对于五种细胞系表现出优于化合物5s (R9=氢)的抑制活性, 只有化合物5y (R9=五氟苄基)对Bel-7402细胞系表现出略低于化合物5s (R9=氢)的抑制活性. 其中, 化合物5x (R9=对氟苄基)对A549细胞系表现最优的抑制活性, IC50值为(16.6±0.6) μmol/L; 化合物5v、5w、5x对Bel-7402细胞系表现较优的抑制活性, IC50值优于顺铂. 结果表明9位引入取代基有助于提高对多种肿瘤细胞系的抑制活性, 其影响为对氟苄基>苄基>五氟苄基>正丁基>乙基>甲基>氢.
目标化合物6位为N-丁二酰亚胺基时, 探究了9位取代对目标化合物的影响, 结果如表4所示. 化合物5aa、5ab、5ac、5ad (R9=正丁基、苄基、对氟苄基、五氟苄基)对A549细胞系表现出较优的抑制活性, IC50值均低于顺铂; 化合物5ad (R9=五氟苄基)对BGC-823细胞系表现出较优的抑制活性, IC50值低于顺铂; 化合物5ab、5ac (R9=苄基、对氟苄基)对Bel-7402细胞系表现出较优的抑制活性, IC50值为6.3±0.3 μmol/L; 化合物5ac、5ad (R9=对氟苄基、五氟苄基)对MCF-7细胞系表现出较优的抑制活性, IC50值低于顺铂; 结果表明9位引入长链烷基和芳基取代基有助于提高对多种肿瘤细胞系的抑制活性, 其影响为对氟苄基>五氟苄基>苄基>正丁基>甲基.
综上所述, 目标化合物对测试的癌细胞系均表现出低至良好程度抗肿瘤活性, 大部分化合物的IC50值小于20 μmol/L且对Bel-7402细胞系表现出优异的抑制活性. 该系列化合物对CT-26细胞系的抑制活性相对不理想, IC50值均高于阳性对照顺铂. 基于以上数据分析, 发现6位引入不同的酰亚胺基对癌细胞系有不同程度的抑制活性, 其影响为N-(4-氯邻苯二甲酰亚胺)>N-邻苯二甲酰亚胺>N-丁二酰亚胺>N-(4-溴邻苯二甲酰亚胺)>N-衣康酰亚胺>N-(4-甲基邻苯二甲酰亚胺). 化合物5p、5ad分别对A549、BGC-823细胞系表现出最优的抑制活性, IC50值分别为(6.5±0.1)、(7.1±0.3) μmol/L; 化合物5ac对Bel-7402和MCF-7细胞系均表现出最优的抑制活性, IC50值分别为(8.4±0.4)和(8.8±0.4) μmol/L. 化合物5j、5p对A549、BGC-823和Bel-7402细胞系表现出较优的抑制活性, 化合物5ac对A549、Bel-7402和MCF-7细胞系表现出较优的抑制活性, IC50值均小于10 μmol/L, 化合物5j、5p、5ac对肿瘤细胞的抑制活性表现优异, 具有潜在的抗肿瘤研究价值.

1.3 分子对接研究

本课题组前期研究表明, 对β-咔啉化合物进行结构修饰得到系列β-咔啉衍生物, 并在鸡胚绒毛尿囊膜(CAM)实验中表现出显著的抗血管生成能力, 同时分子印迹实验进一步表明, 该类化合物可能通过VEGFR-2信号通路发挥抗肿瘤作用[53-55].
为进一步探索β-咔啉与VEGFR-2的作用机制, 选择了表现较为优异的化合物5ac与VEGFR-2的活性口袋进行了分子对接研究(图5). 化合物5ac位于由Ala113、Gly230、Leu231、Lys100、Glu117、Asp228、Cys227、Leu121、lle124、lle120、Asp46、Cys49和Ser116等氨基酸残基组成的疏水性口袋中, 在活性口袋内与周围的蛋白质氨基酸残基发生相互作用. 化合物5ac的羰基的氧和228位的天冬氨酸Asp主链上肽键的氮氢键形成氢键, 化合物5ac的β-咔啉吡啶环的氮和116位的丝氨酸Ser残基的羟基形成氢键. 该系列化合物对多个氨基酸均具有一定的相互作用, 且酰亚胺结构在发挥活性作用中具有重要贡献. 以上结果显示, 化合物5ac与VEGFR-2产生了较为稳定的结合, 可显著增强β-咔啉的抗肿瘤活性.
图5 化合物5ac与VEGFR-2 (PDB code : 4AGD)的分子对接研究

Figure 5 Molecular docking studies of compounds 5ac with VEGFR-2 (PDB code : 4AGD)

2 结论

为了寻找具有良好活性的抗肿瘤药物, 对1-甲基-β-咔啉的6位进行结构修饰共得到30个具有新结构的6-(N-酰亚胺)-β-咔啉衍生物, 采用MTT法测试了目标化合物对A549、BGC-823、CT-26、Bel-7402和MCF-7癌细胞的抗肿瘤活性. 活性数据分析表明, 该系列化合物对测试的5种肿瘤细胞系具有良好的抑制活性, 大部分化合物的IC50值小于20 μmol/L, 优于其6-氨基-β-咔啉前体, 其中化合物5j、5p和5ac对A549、BGC-823、Bel-7402和MCF-7细胞系表现出较优的抑制活性, IC50值均低于10 μmol/L. 结构活性分析表明, 在6位引入N-邻苯二甲酰亚胺基、N-衣康酰亚胺基、N-丁二酰亚胺基可以增强对多种肿瘤细胞系的抑制活性; 在9位引入长链烷基和芳基有助于提高对多种肿瘤细胞系的抑制活性; 在5位引入氯原子有助于提高其抗肿瘤活性. 分子对接结果表明, 化合物5ac与VEGFR-2的多个氨基酸残基形成了稳定的结合. 因此, 化合物5ac具有潜在的抗肿瘤研究价值, 可作为抗肿瘤药物的先导化合物.

3 实验部分

3.1 仪器与试剂

Bruker Avance III HD 400型核磁共振波谱仪(瑞士Brucker公司); LTQ Orbitrap XL型质谱仪(赛默飞世尔科技公司); FA2004型电子天平(上海舜宇恒平科学仪器有限公司); X-5型显微熔点测定仪(北京泰克仪器有限公司); WS70-1型红外快速干燥箱(巩义市予华仪器有限公司); ZF-I型三用紫外分析仪(上海宝山顾村电光仪器厂); Bio-Rad全自动酶标仪(美国伯乐公司).

3.2 实验方法

3.2.1 中间体的合成

中间体2[50]、3a~3g[51]和4a~4i[48]按参考文献方法制备.

3.2.2 目标化合物5a~5ad的合成

称取中间体4a (1 mmol)、邻苯二甲酸酐(1.5 mmol), 量取冰乙酸(6~8 mL)于100 mL圆底烧瓶中, 回流反应2~4 h, 薄层色谱法(TLC)监测反应进程. 反应完毕后, 将反应液冷却至室温, 用NaHCO3调节至pH大于8, 乙酸乙酯萃取(80 mL×3), 无水Na2SO4干燥, 减压浓缩, 柱层析纯化[V(二氯甲烷)∶V(甲醇)=200∶1~50∶1], 得目标产物5a. 化合物5b~5ad的合成方法与化合物5a类似.
6-(N-邻苯二甲酰亚胺基)-5-氯-1,9-二甲基-β-咔啉(5a): 白色固体, 产率88.2%. m.p. 288.2~289.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.42 (d, J=5.4 Hz, 1H), 8.39 (d, J=5.4 Hz, 1H), 8.02 (dd, J=5.4, 3.0 Hz, 2H), 7.85 (dd, J=5.2, 3.0 Hz, 2H), 7.58 (d, J=9.2 Hz, 1H), 7.54 (d, J=8.8 Hz, 1H), 4.23 (s, 3H), 3.213 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.23, 143.41, 141.44, 136.02, 134.56, 131.96, 129.63, 129.45, 128.70, 124.01, 121.93, 119.30, 115.55, 108.78, 32.84, 22.81. HRMS (ESI) calcd for C21H15ClN3O2 [M＋H]＋ 376.0847, found 376.0847.
6-(N-3-氯邻苯二甲酰亚胺基)-5-氯-1,9-二甲基-β-咔啉(5b): 白色固体, 产率86.9%. m.p. 205.4~207.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.37 (d, J=5.4 Hz, 1H), 8.28 (d, J=5.4 Hz, 1H), 7.97 (s, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 4.15 (s, 3H), 3.10 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.36, 166.05, 142.95, 142.05, 141.28, 138.87, 136.13, 134.58, 133.61, 130.00, 128.86, 127.70, 125.21, 124.61, 124.38, 121.03, 119.46, 115.03, 108.63, 32.70, 23.81. HRMS (ESI) calcd for C21H14Cl2- N3O2 [M＋H]＋ 410.0457, found 410.0458.
6-(N-邻苯二甲酰亚胺基)-5-氯-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5c): 白色固体, 产率86.8%. m.p. 226.5~227.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.45 (d, J=5.4 Hz, 1H), 8.38 (d, J=5.4 Hz, 1H), 8.00 (dd, J=5.4, 3.0 Hz, 2H), 7.84 (dd, J=5.4, 3.0 Hz, 2H), 7.48 (d, J=8.8 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H), 5.98 (s, 2H), 3.13 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.18, 146.45 (m), 144.01 (m), 142.62 (m), 141.86, 141.52, 140.07 (m), 139.24, 136.58 (m), 135.84, 134.60, 131.90, 129.53, 129.46, 128.40, 124.03, 122.65, 120.31, 115.40, 109.75 (m), 108.38, 38.09, 23.65; 19F NMR (376 MHz, CDCl3) δ: －142.04, －152.30, －160.10. HRMS (ESI) calcd for C27H14ClF5N3O2 [M＋H]＋ 542.0689, found 542.0689.
6-(N-3-氯邻苯二甲酰亚胺基)-5-氯-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5d): 白色固体, 产率90.1%. m.p. 218.9~219.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.45 (d, J=5.4 Hz, 1H), 8.36 (d, J=5.4 Hz, 1H), 7.97 (s, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.36 (d, J=8.8 Hz, 1H), 5.97 (s, 2H), 3.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.21, 165.90, 146.46 (m), 144.02 (m), 142.66 (m), 141.91, 141.67, 141.40, 139.63, 139.13 (m), 136.60 (m), 135.96, 134.66, 133.54, 129.92, 129.39, 129.28, 128.34, 125.28, 124.47, 122.25, 120.51, 115.36, 109.69 (m), 108.39, 38.14, 24.09; 19F NMR (376 MHz, CDCl3) δ: －141.99, －152.14, －160.01. HRMS (ESI) calcd for C27H13Cl2F5N3O2 [M＋H]＋ 576.0299, found 576.0299.
6-(N-邻苯二甲酰亚胺基)-1-甲基-β-咔啉(5e): 白色固体, 产率68.7%. m.p.>290 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.69 (s, 1H), 8.35 (s, 1H), 8.23~7.64 (m, 5H), 7.53 (s, 2H), 5.30 (s, 1H), 2.83 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.95, 142.06, 139.32, 139.13, 135.06, 134.50, 131.85, 128.03, 126.97, 123.81, 122.49, 120.69, 113.04, 112.13, 20.36. HRMS (ESI) calcd for C20H14N3O2 [M＋H]＋ 328.1080, found 328.1084.
6-(N-邻苯二甲酰亚胺基)-1,9-二甲基-β-咔啉(5f): 白色固体, 产率75.7%. m.p. 265.4~267.3 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.34 (d, J=5.4 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 7.99 (dd, J=5.4, 3.0 Hz, 2H), 7.85 (d, J=5.2 Hz, 1H), 7.83 (dd, J=5.4, 3.0 Hz, 2H), 7.67 (dd, J=8.8, 1.6 Hz, 1H), 7.59 (d, J=8.8 Hz, 1H), 4.20 (s, 3H), 3.17 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.81, 141.69, 141.55, 136.19, 134.48, 131.83,129.23, 127.41, 123.93, 123.79, 121.23, 120.42, 113.47, 110.16, 32.55, 22.77. HRMS (ESI) calcd for C21H15N3O2 [M＋H]＋ 342.1237, found 342.1237.
6-(N-邻苯二甲酰亚胺基)-1-甲基-9-乙基-β-咔啉(5g): 白色固体, 产率70.5%. m.p. 260.2~261.3 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.33 (d, J=5.2 Hz, 1H), 8.16 (s, 1H), 7.98 (dd, J=5.4, 3.0 Hz, 2H), 7.87~7.74 (m, 3H), 7.61 (dd, J=8.8, 1.2 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 4.63 (q, J=7.2 Hz, 2H), 3.07 (s, 3H), 1.48 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.88, 141.48, 140.42, 138.13, 135.36, 134.41, 131.85, 128.88, 126.92, 123.73, 123.56, 121.74, 120.40, 113.21, 110.07, 39.75, 23.31, 15.73. HRMS (ESI) calcd for C22H18N3O2 [M＋H]＋ 356.1393, found 356.1393.
6-(N-邻苯二甲酰亚胺基)-1-甲基-9-正丁基-β-咔啉(5h): 白色固体, 产率60.3%. m.p. 152.3~153.2 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.35 (d, J=5.4 Hz, 1H), 8.17 (s, 1H), 7.99 (dd, J=5.4, 3.0 Hz, 2H), 7.90~7.72 (m, 3H), 7.62 (dd, J=8.8, 1.6 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 4.70~4.45 (m, 2H), 3.08 (s, 3H), 1.90~1.81 (m, 2H), 1.52~1.43 (m, 2H), 1.00 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.88, 141.41, 140.93, 137.76, 135.61, 134.44, 131.85, 129.06, 126.96, 123.76, 123.59, 121.55, 120.36, 113.34, 110.35, 44.99, 32.93, 23.03, 20.22, 13.88. HRMS (ESI) calcd for C24H22N3O2 [M＋H]＋ 384.1706, found 384.1701.
6-(N-邻苯二甲酰亚胺基)-1-甲基-9-苄基-β-咔啉(5i): 白色固体, 产率62.8%. m.p. 177.4~178.6 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.35 (d, J=5.2 Hz, 1H), 8.19 (d, J=1.6 Hz,, 1H), 7.96 (dd, J=5.4, 3.0 Hz, 2H), 7.81 (d, J=5.4 Hz, 1H), 7.85~7.75 (m, 3H), 7.54 (dd, J=8.8, 1.8 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.27 (q, J=6.8 Hz, 3H), 6.99 (d, J=6.8 Hz, 2H), 5.76 (s, 2H), 2.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.83, 141.82, 141.32, 138.22, 137.45, 136.02, 134.47, 131.82, 129.14, 127.76, 127.32, 125.42, 124.12, 123.79, 121.64, 120.36, 113.37, 110.53, 48.45, 22.82. HRMS (ESI) calcd for C27H20N3O2 [M＋H]＋ 418.1550, found 418.1552.
6-(N-邻苯二甲酰亚胺基)-1-甲基-9-(4-氟苄基)-β-咔啉(5j): 白色固体, 产率76.2%. m.p. 238.4~240.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.39 (d, J=5.4 Hz, 1H), 8.22 (d, J=1.6 Hz, 1H), 7.98 (dd, J=5.4, 3.0 Hz, 2H), 7.87 (d, J=5.4 Hz, 1H), 7.82 (dd, J=5.4, 3.0 Hz, 2H), 7.59 (dd, J=8.8, 1.6 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.09~6.90 (m, 4H), 5.77 (s, 2H), 2.91 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.81, 163.49 (d, J=245.0 Hz), 141.55, 141.22, 138.10, 135.83, 134.51, 133.10 (d, J=3.1 Hz), 131.80, 129.36, 127.50, 127.09 (d, J=8.1 Hz), 124.32, 123.82, 121.64, 120.49, 116.13 (d, J=21.6 Hz), 113.50, 110.42, 47.82, 22.42. HRMS (ESI) calcd for C27H19FN3O2 [M＋H]＋ 436.1455, found 436.1455.
6-(N-邻苯二甲酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5k): 白色固体, 产率78.3%. m.p.>290 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (d, J=5.2 Hz, 1H), 8.17 (s, 1H), 7.99 (dd, J=5.4, 3.0 Hz, 2H), 7.92~7.69 (m, 3H), 7.58 (dd, J=8.8, 1.8 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 5.98 (s, 2H), 3.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.72, 146.50 (m), 144.07 (m), 142.51 (m), 141.63, 140.18, 139.05, 136.54 (m), 136.12, 134.52, 131.77, 129.27, 127.27, 124.65, 123.83, 122.44, 120.43, 113.42, 110.11 (m), 109.89, 43.45, 38.02, 23.84. HRMS (ESI) calcd for C27H15F5N3O2 [M＋H]＋ 508.1078, found 508.1079.
6-(N-3-氯邻苯二甲酰亚胺基)-1,9-二甲基-β-咔啉(5l): 白色固体, 产率73.6%. m.p. 278.9~279.4 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.33 (d, J=5.2 Hz, 1H), 8.14 (s, 1H), 7.95 (s, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.79 (t, J=6.4 Hz, 2H), 7.61 (d, J=7.8 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 4.18 (s, 3H), 3.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.90, 166.58, 142.04, 141.46, 141.17, 138.25 136.36, 134.51, 133.51, 129.89, 128.60, 126.77, 125.00, 124.17, 123.28, 121.45, 120.22, 113.18, 110.08, 32.51, 23.54. HRMS (ESI) calcd for C21H15ClN3O2 [M＋H]＋ 376.0847, found 376.0848.
6-(N-3-氯邻苯二甲酰亚胺基)-1-甲基-9-乙基-β-咔啉(5m): 白色固体, 产率71.1%. m.p. 227.6~228.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.34 (d, J=5.4 Hz, 1H), 8.15 (s, 1H), 7.95 (d, J=1.4 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.81 (d, J=5.2 Hz, 1H), 7.78 (dd, J=8.0, 1.6 Hz, 1H), 7.58 (s, 2H), 4.65 (q, J=7.2 Hz, 2H), 3.08 (s, 3H), 1.49 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.92, 166.60, 141.55, 141.16, 140.49, 138.22, 135.41, 134.50, 133.51, 129.89, 128.86, 126.74, 125.00, 124.17, 123.26, 121.78, 120.33, 113.22, 110.14, 39.79, 23.32, 15.73. HRMS (ESI) calcd for C22H17ClN3O2 [M＋H]＋ 390.1003, found 390.1006.
6-(N-3-溴邻苯二甲酰亚胺基)-1-甲基-9-乙基-β-咔啉(5n): 白色固体, 产率79.0%. m.p. 225.7~226.4 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.36 (d, J=5.4 Hz, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.82 (dd, J=9.2, 6.8 Hz, 2H), 7.58 (t, J=6.2 Hz, 2H), 4.63 (q, J=7.2 Hz, 2H), 3.09 (s, 3H), 1.48 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.04, 166.51, 141.28, 140.63, 137.61, 137.48, 135.31, 133.44, 130.33, 129.39, 129.13, 127.08, 126.97, 125.12, 123.38, 121.63, 120.41, 113.37, 110.19, 39.82, 22.78, 15.75. HRMS (ESI) calcd for C22H17BrN3O2 [M＋H]＋ 434.0498, found 434.0499.
6-(N-3-甲基邻苯二甲酰亚胺基)-1-甲基-9-乙基-β-咔啉(5o): 白色固体, 产率80.6%. m.p. 255.6~256.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.35 (d, J=5.4 Hz, 1H), 8.16 (s, 1H), 7.87 (d, J=7.6 Hz, 1H), 7.83 (d, J=5.2 Hz, 1H), 7.79(s, 1H), 7.66~7.54 (m, 3H), 4.66 (q, J=7.2 Hz, 2H), 3.09 (s, 3H), 2.57 (s, 3H), 1.49 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 168.09, 167.97, 145.80, 141.45, 140.45, 138.04, 135.40, 135.00, 132.24, 129.26, 129.01, 127.00, 124.27, 123.74, 123.68, 121.75, 120.42, 113.29, 110.07, 39.79, 23.23, 22.11, 15.74. HRMS (ESI) calcd for C23H20N3O2 [M＋H]＋ 370.1550, found 370.1551.
6-(N-3-氯邻苯二甲酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5p): 白色固体, 产率79.3%. m.p. 274.3~275.6 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (d, J=5.2 Hz, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.85 (d, J=5.2 Hz, 1H), 7.79 (dd, J=8.0, 1.6 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.45(d, J=8.8 Hz, 1H), 5.99 (s, 2H), 3.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.75, 166.44, 146.54 (m), 141.65, 141.30, 140.29, 139.05 (m), 136.60 (m), 136.14, 134.61, 133.42, 129.79, 129.29, 127.15, 125.09, 124.38, 124.26, 122.45, 120.36, 113.45, 109.97, 38.03, 23.79; 19F NMR (376 MHz, CDCl3) δ: －142.12, －152.54, －160.43. HRMS (ESI) calcd for C27H14ClF5N3O2 [M＋H]＋ 542.0689, found 542.0688.
6-(N-3-溴邻苯二甲酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5q): 白色固体, 产率68.2%. m.p. 289.4~290.3 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (d, J=5.4 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 7.94~7.88 (m, 1H), 7.85 (d, J=5.2 Hz, 1H), 7.74 (d, J=3.8 Hz, 2H), 7.53 (dd, J=8.8, 1.6 Hz, 1H), 7.45 (d, J=8.8 Hz, 1H), 5.98 (s, 2H), 3.14 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 166.90, 166.37, 146.49 (m), 144.05 (m), 142.46 (m), 141.72, 140.20, 139.23, 137.55, 136.61 (m), 136.13, 133.37, 130.26, 129.48, 129.13, 127.16, 127.04, 125.17, 124.28, 122.48, 120.33, 113.37, 110.10 (m), 109.93, 38.01, 23.93; 19F NMR (376 MHz, CDCl3) δ: －142.13, －152.61, －160.32. HRMS (ESI) calcd for C27H14BrF5N3O2 [M＋H]＋ 587.0262, no found.
6-(N-3-甲基邻苯二甲酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5r): 白色固体, 产率70.6%. m.p. 284.4~285.2 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.42 (d, J=5.4 Hz, 1H), 8.19 (s, 1H), 7.86 (d, J=7.8 Hz, 2H), 7.79 (s, 1H), 7.61 (d, J=7.6 Hz, 2H), 7.45 (d, J=8.8 Hz, 1H), 5.99 (s, 2H), 3.16 (s, 3H), 2.57 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 167.91, 167.79, 146.53 (m), 145.90, 144.05 (m), 142.43 (m), 141.60, 140.06, 139.05, 139.04 (m), 136.63 (m), 136.05, 135.06, 132.14, 129.16, 127.26, 124.76, 124.29, 123.71, 122.38, 120.40, 113.36, 110.18 (m), 109.82, 37.95, 23.82, 22.10; 19F NMR (376 MHz, CDCl3) δ: －142.15, －152.81, －160.43. HRMS (ESI) calcd for C28H17F5N3O2 [M＋H]＋ 446.1299, no found.
6-(N-衣康酰亚胺基)-1-甲基-β-咔啉(5s): 白色固体, 产率51.4%. m.p. >290 ℃; 1H NMR (400 MHz, CDCl3) δ: 11.79 (s, 1H), 8.24 (d, J=5.2 Hz, 1H), 8.15 (s, 1H), 7.94 (d, J=5.2 Hz, 1H), 7.68 (d, J=8.6 Hz, 1H), 7.45 (dd, J=8.8, 1.6 Hz, 1H), 6.84 (d, J=1.2 Hz, 1H), 2.79 (s, 3H), 2.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.27, 170.26, 145.96, 141.06, 139.97, 136.41, 134.95, 128.56, 127.52, 127.18, 123.96, 121.80, 120.43, 113.15, 112.45, 19.02, 11.29. HRMS (ESI) calcd for C17H14N3O2 [M＋H]＋ 292.1080, found 292.1088.
6-(N-衣康酰亚胺基)-1,9-二甲基-β-咔啉(5t): 白色固体, 产率58.7%. m.p. 239.2~240.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.32 (d, J=5.2 Hz, 1H), 8.04 (s, 1H), 7.77 (d, J=5.2 Hz, 1H), 7.53~7.48 (m, 2H), 6.53 (d, J=1.6 Hz, 1H), 4.15 (s, 3H), 3.10 (s, 3H), 2.22 (d, J=1.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.20, 170.17, 145.85, 142.01, 141.15, 138.25, 136.32, 128.51, 127.46, 126.52, 123.54, 121.39, 119.77, 113.12, 109.96, 32.44, 23.60, 11.26. HRMS (ESI) calcd for C21H15ClN3O2 [M＋H]＋ 306.1237, found 306.1231.
6-(N-衣康酰亚胺基)-1-甲基-9-乙基-β-咔啉(5u): 白色固体, 产率55.1%. m.p. 195.6~196.8 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.33 (d, J=5.2 Hz, 1H), 8.05 (s, 1H), 7.79 (d, J=5.2 Hz, 1H), 7.52 (d, J=8.8 Hz, 1H), 7.49(d, J=8.8 Hz, 1H), 6.57~6.46 (m, 1H), 4.61 (q, J=7.2 Hz, 2H), 3.06 (s, 3H), 2.21 (s, 3H), 1.45 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.22, 170.21, 145.85, 141.56, 140.18, 138.29, 135.39, 128.83, 127.46, 126.49, 123.50, 121.75, 119.91, 113.15, 110.02, 39.72, 23.42, 15.69, 11.26. HRMS (ESI) calcd for C19H18N3O2 [M＋H]＋ 320.1393, found 320.1392.
6-(N-衣康酰亚胺基)-1-甲基-9-正丁基-β-咔啉(5v): 白色固体, 产率44.8%. m.p. 129.3~130.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.33 (d, J=5.2 Hz, 1H), 8.04 (s, 1H), 7.79 (d, J=5.2 Hz, 1H), 7.56~7.44 (m, 2H), 6.61~6.44 (m, 1H), 4.60~4.46 (m, 2H), 3.05 (s, 3H), 2.28~2.14 (m, 3H), 1.86~1.78 (m, 2H), 1.50~1.40 (m, 2H), 0.98 (t, J=7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 170.19, 169.18, 144.82, 140.54, 139.56, 137.15, 134.58, 127.71, 126.44, 125.40, 122.44, 120.55, 118.78, 112.11, 109.24, 43.88, 31.88, 22.47, 19.17, 12.84, 10.23. HRMS (ESI) calcd for C21H22N3O2 [M＋H]＋ 348.1706, found 348.1706.
6-(N-衣康酰亚胺基)-1-甲基-9-苄基-β-咔啉(5w): 白色固体, 产率52.7%. m.p. 119.3~120.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.13 (d, J=1.6 Hz, 1H), 7.90 (d, J=5.4 Hz, 1H), 7.51 (dd, J=8.8, 1.6 Hz, 1H), 7.45 (d, J=8.8 Hz, 1H), 7.30~7.23 (m, 4H), 6.96 (d, J=6.4 Hz, 2H), 6.54 (d, J=1.6 Hz, 1H), 5.77 (s, 2H), 2.96 (s, 3H), 2.22 (d, J=1.6Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.06, 170.03, 145.95, 141.60, 137.09, 135.83, 129.94, 129.58, 129.19, 127.85, 127.52, 125.34, 124.55, 121.15, 120.06, 113.73, 110.67, 48.49, 21.91, 11.28. HRMS (ESI) calcd for C24H20N3O2 [M＋H]＋ 382.1550, found 382.1557.
6-(N-衣康酰亚胺基)-1-甲基-9-(4-氟苄基)-β-咔啉(5x): 白色固体, 产率50.4%. m.p. 205.2~206.3 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.38 (d, J=5.2 Hz, 1H), 8.11 (s, 1H), 7.87 (d, J=5.2 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.03~6.90 (m, 4H), 6.54 (s, 1H), 5.76 (s, 2H), 2.90 (s, 3H), 2.22 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.12, 170.10, 162.26 (d, J=246.7 Hz), 145.94, 141.56, 141.01, 138.07, 135.82, 133.10 (d, J=3.0 Hz), 129.32, 127.52, 127.12, 127.02 (d, J=8.1 Hz), 124.32, 121.60, 119.95, 116.12 (d, J=21.7 Hz), 113.47, 110.40, 47.81, 22.48, 11.28. HRMS (ESI) calcd for C27H19FN3O2 [M＋H]＋ 436.1455, found 436.1455.
6-(N-衣康酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5y): 白色固体, 产率66.1%. m.p.>290 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.39 (d, J=5.2 Hz, 1H), 8.06 (s, 1H), 7.81 (d, J=5.2 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.38 (d, J=8.8 Hz, 1H), 6.52 (d, J=1.2Hz, 1H), 5.95 (s, 2H), 3.10 (s, 3H), 2.21 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.04, 170.01, 146.52 (m), 145.94, 143.99 (m), 142.50 (m), 141.64, 139.94, 139.07, 136.53 (m), 136.11, 129.19, 127.51, 126.84, 124.63, 122.41, 119.89, 113.36, 110.15 (m), 109.85, 37.99, 23.87, 11.27. HRMS (ESI) calcd for C24H15F5N3O2 [M＋H]＋ 472.1078, found 472.1076.
6-(N-丁二酰亚胺基)-1,9-二甲基-β-咔啉(5z): 白色固体, 产率64.5%. m.p. 88.7~89.6 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.32 (d, J=5.2 Hz, 1H), 8.02 (s, 1H), 7.77 (d, J=5.2 Hz, 1H), 7.53 (d, J=8.8 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H), 4.16 (s, 3H), 3.11 (s, 3H), 2.96 (s, 4H); 13C NMR (100 MHz, CDCl3) δ: 176.72, 141.98, 141.60, 138.12, 136.31, 128.62, 126.53, 123.83, 121.42, 120.08, 113.18, 110.13, 32.48, 28.49, 23.43. HRMS (ESI) calcd for C17H16N3O2 [M＋H]＋ 294.1237, found 294.1238.
6-(N-丁二酰亚胺基)-1-甲基-9-正丁基-β-咔啉(5aa): 白色固体, 产率58.9%. m.p. 184.6~185.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.34 (d, J=5.2 Hz, 1H), 8.03 (d, J=1.6 Hz, 1H), 7.80 (d, J=5.2 Hz, 1H), 7.54 (d, J=8.8 Hz, 1H), 7.45 (dd, J=8.8, 1.8 Hz, 1H), 4.63~4.47 (m, 2H), 3.06 (s, 3H), 2.97 (s, 4H), 1.87~1.79 (m, 2H), 1.51~1.41 (m, 2H), 0.99 (t, J=7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 176.77, 141.60, 141.02, 138.17, 135.63, 128.79, 126.38, 123.73, 121.65, 120.09, 113.19, 110.43, 44.97, 32.90, 28.48, 23.45, 20.20, 13.86. HRMS (ESI) calcd for C20H22N3O2 [M＋H]＋ 336.1706, found 336.1707.
6-(N-丁二酰亚胺基)-1-甲基-9-苄基-β-咔啉(5ab): 白色固体, 产率54.0%. m.p. 196.2~197.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.36 (d, J=5.2 Hz, 1H), 8.07 (s, 1H), 7.83 (d, J=5.2 Hz, 1H), 7.43 (d, J=8.6 Hz, 1H), 7.39 (d, J=8.6 Hz, 1H), 7.28~7.26 (m, 3H), 6.99 (d, J=7.2Hz, 2H), 5.78 (s, 2H), 2.95 (s, 4H), 2.88 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 176.76, 142.04, 141.36, 138.76, 137.49, 136.05, 128.95, 127.71, 126.69, 125.42, 124.20, 121.74, 120.10, 113.22, 110.58, 48.39, 28.49, 23.12. HRMS (ESI) calcd for C23H20N3O2 [M＋H]＋ 370.1550, found 370.1551.
6-(N-丁二酰亚胺基)-1-甲基-9-(4-氟苄基)-β-咔啉(5ac): 白色固体, 产率69.2%. m.p. 277.0~278.4 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.38 (d, J=5.2 Hz, 1H), 8.09 (s, 1H), 7.85 (d, J=5.2 Hz, 1H), 7.43 (s, 2H), 7.06~6.89 (m, 4H), 5.76 (s, 2H), 2.97 (s, 4H), 2.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 176.70, 162.25 (d, J=245 Hz), 141.67, 141.36, 138.33, 135.84, 133.03 (d, J=3.1 Hz), 129.17, 127.10 (d, J=8.1 Hz), 127.02, 124.46, 121.68, 120.25, 116.11 (d, J=21.8 Hz), 113.41, 110.50, 47.80, 28.48, 22.58. HRMS (ESI) calcd for C23H19FN3O2 [M＋H]＋ 388.1455, found 388.1455.
6-(N-丁二酰亚胺基)-1-甲基-9-(2,3,4,5,6-五氟苄基)-β-咔啉(5ad): 白色固体, 产率71.8%. m.p. 257.6~259.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.39 (d, J=5.6 Hz, 1H), 8.03 (s, 1H), 7.80 (d, J=4.4 Hz, 1H), 7.47~7.36 (m, 2H), 5.94 (s, 2H), 3.09 (s, 3H), 3.00~2.92 (m, 4H); 13C NMR (100 MHz, CDCl3) δ: 176.60, 146.48 (m), 144.05(m), 142.58 (m), 141.73, 140.32, 139.26, 136.54 (m), 136.10, 129.05, 126.80, 124.80, 122.49, 120.22, 113.33, 110.22 (m), 109.96, 37.96, 28.47, 23.96. HRMS (ESI) calcd for C23H15F5N3O2 [M＋H]＋ 460.1078, found 460.1076.

3.2.3 体外抗肿瘤活性实验

采用MTT法测试了目标化合物对肿瘤细胞株的抑制活性, 包括肺癌细胞(A549)、胃癌细胞(BGC-823)、结肠癌细胞(CT-26)、肝癌细胞(Bel-7402)和乳腺癌细胞(MCF-7). 分别将细胞株以1×104个/mL的浓度接种于96孔板, 置于37 ℃的CO2培养箱中培养24 h, 使其处于对数生长期. 换新鲜培养液, 加入灭菌处理的不同浓度梯度的待测样品, 继续培养48 h, 每个梯度平行测试三次. 以等体积的溶样品试剂作为空白对照. 量取20 μL含5 mg•mL－1 MTT的RPMI1640培养液, 按批加入至每个孔板中, 加毕, 继续进行为期4 h的培养. 除去上层清液, 继续加入100 μL二甲基亚砜对沉淀进行溶解, 使用酶标仪在波长490 nm下检测OD值. 重复实验, 计算出各浓度测试样品的细胞存活率, 以细胞存活率对药物浓度对数作图, 按作图法求出每个样品的IC50值, 细胞存活率的计算公式如下:
细胞存活率(%)=(样品组平均OD值/对照组平均OD值)×100%
辅助材料(Supporting Information) 化合物5a~5ad的1H NMR和13C NMR谱图. 这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.
(Lu, Y.)

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