基于烯胺腈结构的吡喃香豆素并嘧啶类化合物的合成及抗肿瘤活性研究

引用本文: 黄新炜, 刘建利. 基于烯胺腈结构的吡喃香豆素并嘧啶类化合物的合成及抗肿瘤活性研究[J]. 有机化学, 2018, 38(5): 1233-1241. doi: 10.6023/cjoc201708027 shu

Citation: Huang Xinwei, Liu Jianli. Synthesis and Anticancer Activities of Novel Pyranocoumarin Fused Pyrimidine Based on Cyanoenamine[J]. Chinese Journal of Organic Chemistry, 2018, 38(5): 1233-1241. doi: 10.6023/cjoc201708027 shu

基于烯胺腈结构的吡喃香豆素并嘧啶类化合物的合成及抗肿瘤活性研究

黄新炜 ^{a,
,} ,
刘建利 ^b,

a.
西安文理学院化学工程学院西安 710065
b.
西北大学生命科学学院西部资源生物与现代生物技术省部共建教育部重点实验室西安 710069

通讯作者: 黄新炜, huangxinw@sina.com

基金项目:

西安市科技计划（No.CXY1443WL13）及陕西省教育厅基金（No.15JK2145）资助项目

摘要: 吡喃香豆素是一类重要的天然产物，具有广泛的生物活性和药理作用，如抗肿瘤、抗菌、抗人类免疫缺陷病毒（HIV）、抗炎、抗氧化等.嘧啶是另一类重要的含氮杂环化合物，利用药物设计中的药效团拼合原则，如将吡喃香豆素和嘧啶结构进行拼合，有可能获得抗肿瘤活性更好的先导化合物.因此，首先以4-羟基香豆素、芳香醛、丙二腈为原料，4-二甲氨基吡啶（DMAP）为催化剂，通过多组分反应合成具有烯胺腈结构的吡喃香豆素，再与N，N-二甲基甲酰胺二甲缩醛（DMF-DMA）反应制备N，N-二甲基甲脒，最后与芳香胺通过Dimroth重排制备新型4-苯胺基取代吡喃香豆素并嘧啶类化合物，并通过熔点，IR，¹H NMR，¹³C NMR，元素分析对目标产物的结构进行了表征，所得目标产物均未见文献报道.该方法具有反应时间短、反应条件温和、操作简单、产率高、无需柱色谱分离等优点.通过四甲基偶氮唑盐微量酶反应比色法（MTT）对目标产物抑制人宫颈癌细胞Hela和人急性早幼粒白血病细胞HL-60的活性进行了体外评价，结果表明：化合物4-（4'-溴苯胺基）-5-（2'，3'-二氯苯基）-色烯[3'，4'：5，6]吡喃[2，3-d]嘧啶-6-酮（4k）和4-（4'-溴苯胺基）-5-（4'-硝基苯基）-色烯[3'，4'：5，6]吡喃[2，3-d]嘧啶-6-酮（4l）对HL-60具有较高活性，其IC₅₀分别为（11.3±0.3）和（10.8±0.2）μmol/L；化合物4-（4'-氯苯胺基）-5-（3'，4'，5'-三甲氧苯基）-色烯[3'，4'：5，6]吡喃[2，3-d]嘧啶-6-酮（4g）和4-（3'-氯-4'-氟苯胺基）-5-（3'，4'，5'-三甲氧苯基）-色烯[3'，4'：5，6]吡喃[2，3-d]嘧啶-6-酮（4h）对Hela具有较高活性，其IC₅₀分别为（9.2±0.6）和（8.5±0.2）μmol/L.

关键词:

English

Synthesis and Anticancer Activities of Novel Pyranocoumarin Fused Pyrimidine Based on Cyanoenamine

Huang Xinwei ^{a,
,} ,
Liu Jianli ^b,

a.
School of Chemical Engineering, Xi'an University, Xi'an 710065
b.
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, School of Life Science, Northwest University, Xi'an 710069

Corresponding author: Huang Xinwei, E-mail:huangxinw@sina.com

Received Date: 14 August 2017
Revised Date: 31 October 2017
Available Online: 01 May 2018
Fund Project:

Project supported by the Science and Technology Program of Xi'an City (No. CXY1443WL13) and the Foundation of the Education Department of Shaanxi Province (No. 15JK2145)

Abstract: Pyranocoumarin is an important kind of natural products, which has many biological activities and pharmacological effects, such as antitumor, anti-bacterial, anti-human immunodeficiency virus (HIV), anti-inflammatory, antioxidation, etc. Pyrimidine is another important nitrogen-containing heterocycles. Using the pharmacophore combination principle of drug design, it is possible to obtain lead compounds with better anticancer activities by puting pyranocoumarin and pyrimidine structures together. Therefore, pyranocoumarin which has cyanoenamine structure was synthesized by multicomponent reaction, taking 4-hydroxycoumarin, aromatic aldehyde, malononitrile as raw materials and 4-dimethylaminopyridine (DMAP) as catalyst. Then N, N-dimethyl formamidine derivatives were synthesized by treatment with dimethylformamide-dimethyl acetal. Finally 4-anilino substituted pyranocoumarin fused pyrimidines were synthesized by treatment with substituted anilines involving Dimroth rearrangement. The structures of target compounds were characterized by melting point, IR, ¹H NMR, ¹³C NMR and elemental analysis. This method has some advantages with short reaction time, mild reaction condition, simple operation, high yields and with no chromatographic separation procedure. All the title compounds were evaluated for anticancer activities in vitro against HL-60 cell lines and Hela human cervical cartcinoma cell lines. The results showed that 4-(4'-bromophenylamino)-5-(2', 3'-dichlorophenyl)-chromene [3', 4':5, 6]pyrano [2, 3-d]pyrimidin-6-one (4k) and 4-(4'-bromo-phenylamino)-5-(4'-nitrophenyl)-chromene [3', 4':5, 6]pyrano [2, 3-d]pyrimidin-6-one (4l) exhibited high activity against HL-60 with IC₅₀ values of (11.3±0.3) and (10.8±0.2) μmol/L, and 4-(4'-chlorophenylamino)-5-(3', 4', 5'-trimethoxyphenyl)-chromene [3', 4':5, 6]pyrano [2, 3-d]pyrimidin-6-one (4g) and 4-(3'-chloro-4'-fluorophenylamino)-5-(3', 4', 5'-trimethoxy-phenyl)-chromene [3', 4':5, 6]pyrano [2, 3-d]pyrimidin-6-one (4h) exhibited high activity against Hela with IC₅₀ value of (9.2±0.6) and (8.5±0.2) μmol/L.

Key words:

吡喃香豆素是天然存在的一类具有广泛生物活性的化合物, 如抗肿瘤、抗菌、抗人类免疫缺陷病毒(HIV)、抗炎、抗氧化等.含氮杂环化合物也是一大类非常重要的有机小分子化合物, 如嘧啶、吡啶、吡唑、吲哚等, 广泛存在于自然界, 很多具有生物活性, 其已成为医药、农药和有机功能材料等的核心骨架^[1], 特别是在抗肿瘤^[2]方面尤为突出.利用药物设计中的药效团拼合原则, 如能将吡喃香豆素和嘧啶结构进行拼合有可能获得抗肿瘤活性更高、耐药性更好的先导化合物^[3].

基于以上原因, 我们设计并合成4-位含有苯胺基的吡喃香豆素并嘧啶类化合物, 并对其体外抗肿瘤活性的进行筛选, 以期发现抗肿瘤活性更好的先导化合物, 目标产物的结构如图 1所示.

图 1

图 1. 目标产物的结构

Figure 1. Structure of target compounds

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1. 结果与讨论

1.1 化合物的合成

通过查阅文献, 合成4-苯胺基取代嘧啶的方法主要有两种. (1)以氨基烯酸为原料和甲酰胺反应生成嘧啶-4-酮, 再与二氯亚砜或三氯氧磷进行氯化, 最后和苯胺类化合物反应得到4-苯胺基取代嘧啶^[4](Scheme 1). (2)以烯胺腈为原料, 首先与N, N-二甲基甲酰胺二甲基缩醛(DMF-DMA)反应得到相应的甲脒; 然后与胺发生亲核加成、环合反应生成3-氨基取代嘧啶, 再经Dimroth重排后得到4-苯胺基取代嘧啶^[5~12](Scheme 2).

图式 1

图式 1. 以氨基烯酸为原料合成4-苯胺基取代嘧啶

Scheme 1. Synthesis of 4-anilino substituted pyrimidine using amino olefin acid as raw material

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图式 2

图式 2. 以烯胺腈为原料合成4-苯胺基取代嘧啶

Scheme 2. Synthesis of 4-anilino substituted pyrimidine using cyanoenamine as raw material

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通过以上方法, 可以得到很多具有生物活性的杂环并嘧啶类化合物, 如吡唑并嘧啶^[5]、吡咯并嘧啶^[6]、三唑并嘧啶^[7]、苯(吡啶、吡嗪)并噻吩并嘧啶^[8]、苯并呋喃(噻吩)并嘧啶^[9]、吡啶并呋喃并嘧啶^{[10, 11]}、呋喃(噻吩)并嘧啶^[12]等, 其结构如图 2所示.

图 2

图 2. 杂环并嘧啶类化合物的结构

Figure 2. Structure of heterocycle fused pyrimidines

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多组分反应(Multicomponent Reactions, MCRs)因其兼具高原子经济性、高汇聚性、操作简单、快速高效、易于实现多样性导向合成等特点, 一直以来受到有机化学家的持续关注, 被广泛应用于从简单原料出发合成大量结构多样化的复杂分子^[13].

近年来, 人们以4-羟基香豆素、3-羟基香豆素、5, 7-二羟基香豆素、丙二腈及芳香醛为原料通过多组分反应合成了一系列含有烯胺腈结构的吡喃香豆素类衍生物.其结构如图 3所示.

图 3

图 3. 通过多组分反应制备具有烯胺腈结构的多取代吡喃香豆素

Figure 3. Polysubstituted pyranocoumarins with adjacent amino and nitrile functionalities synthesized by MCRs

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以上反应的产率高、后处理方便、时间短、所用催化剂价廉易得, 分别为4-二甲氨基吡啶(DMAP)^[14]、纳米氧化锌^[15]、碳酸钾^[16]及磷酸二氢铵^[17].

具有烯胺腈结构的4-取代吡喃香豆素本身也具有一定的生物活性, 如抗肿瘤及抗氧化^[18]、抗菌^[19]、抗嘌呤氧化抑制剂^[20]、乙酰胆碱酯酶抑制剂^[21]等(图 4).

图 4

图 4. 具有生物活性的含有烯胺腈结构的4-取代吡喃香豆素

Figure 4. 4-Substituted pyranocoumarins with cyanoenamine which have biological activities

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由于通过上述多组分反应可以比较容易获得含烯胺腈结构的吡喃香豆素.因此, 我们按Scheme 3路线进行吡喃香豆素并嘧啶类化合物的合成.首先通过多组分反应合成具有烯胺腈结构的吡喃香豆素(1), 再与N, N-二甲基甲酰胺二甲基缩醛(DMF-DMA)反应得到相应的甲脒; 然后与芳香胺反应得到4-苯胺基取代的吡喃香豆素并嘧啶类化合物; 并在此基础上, 对所合成目标化合物的体外抗肿瘤^[20]活性进行研究, 以期发现高效用、低成本的对肿瘤有治疗作用的此类化合物, 为进一步的临床前研究奠定一定基础.

图式 3

图式 3. 目标化合物的合成路线

Scheme 3. Synthetic route of the target compounds

4a R¹ = 3, 4-(OCH₃)₂, R² = 3-Cl-4-F; 4b R¹ = 3, 4-(OCH₃)₂, R² = 3, 4-Cl₂; 4c R1 = 3, 4-(OCH₃)₂, R² = 4-Br; 4d R¹ = 3, 4-(OCH₃)₂, R² = 4-Cl; 4e R¹ = 3, 4-(OCH₃)₂, R² = 3-Cl-4-CH₃; 4f R¹ = 3, 4, 5-(OCH₃)₃, R² = 4-Br; 4g R¹ = 3, 4, 5-(OCH₃)₃, R² = 4-Cl; 4h R¹ = 3, 4, 5-(OCH₃)₃, R² = 3-Cl-4-F; 4i R¹ = 2, 3-Cl₂, R² = 3, 4-Cl₂; 4j R¹ = 2, 3-Cl₂, R² = 4-Cl; 4k R¹ = 2, 3-Cl₂, R² = 4-Br; 4l R¹ = 4-NO₂, R² = 4-Br; 4m R¹ = 4-NO₂, R² = 4-Cl; 4n R¹ = 4-NO₂, R² = 3-Cl-4-F; 4o R¹ = 3-NO₂, R² = 4-Br; 4p R¹ = 4-Br, R² = 3-Cl-4-F; 4q R¹ = 4-Br, R² = 4-Br; 4r R¹ = 4-Br, R² = 3, 4-Cl₂; 4s R¹ = 4-Br, R² = 4-Cl

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1.2 化合物结构的表征

以化合物2a、3a和4a为例说明化合物的结构表征.化合物2a的氢谱可以看到δ 7.90~6.75的芳环质子信号, δ 7.38两个氢的单峰为伯胺(NH₂)的信号, δ 4.41一个氢的单峰为次甲基的信号, δ 3.72三个氢的单峰为甲氧基的信号; 化合物2a的红外谱图可以看到氰基的特征吸收(2193 cm^-1)以及伯氨基的吸收峰(3376, 3321 cm^-1).化合物3a的氢谱可以看到δ 8.22~6.76的芳环质子信号, δ 8.56一个氢的单峰为亚胺(N＝CH)的信号, δ 4.51一个氢的单峰为次甲基的信号, δ 3.72和3.71各三个氢的单峰为两个甲氧基的信号, δ 3.25和3.05各三个氢的单峰为两个甲基的信号.在化合物3a的红外光谱图中可以看到氰基的特征吸收(2198 cm^-1), 而伯氨基的吸收峰(3376, 3321 cm^-1)消失.在化合物4a的氢谱中可以看到δ 8.98~6.83处的芳环质子信号, δ 11.99处的仲胺质子信号, δ 5.62处的次甲基信号, δ 3.70和3.66处的两个甲氧基的信号.在化合物4a的红外光谱图中可以看到仲胺质子的特征吸收(3381 cm^-1), 而2198 cm^-1的特征峰消失.

1.3 生物活性

利用CLC Drug discovery Workbench软件进行模拟, 选择PDB号为1SA0的蛋白中的秋水仙碱结合位点为靶点, 选取体外活性较好的化合物4h与该靶点进行对接, 对接结果如图 5(a)所示, 化合物4h与微管蛋白中的秋水仙碱位点具有很好的结合能力, 提示该类化合物具有潜在的抗肿瘤活性.

图 5

图 5. (a) 化合物4h与秋水仙碱结合位点的分子对接图以及(b)秋水仙碱的分子对接图

Figure 5. (a) Molecules Docking of compound 4h with colchicine-binding site and (b) molecules Docking of colchicine

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采用四甲基偶氮唑盐微量酶反应比色法(MTT)测定了吡喃香豆素并嘧啶类化合物4a~4s对人宫颈癌细胞(Hela)及人急性早幼粒白血病细胞株(HL-60)的体外抗肿瘤活性^[22], 阳性对照物为抗肿瘤药物多西紫杉醇(Docetaxel), 测试结果如表 1所示.化合物4k和4l对HL-60具有较高活性, 其IC₅₀分别为(11.3±0.3)和(10.8±0.2) μmol·L^-1; 化合物4g和4h对Hela具有较高活性, 其IC₅₀分别为(9.2±0.6)和(8.5±0.2) μmol·L^-1.

表 1

表 1 目标产物4a~4s的体外抗肿瘤活性[IC₅₀/(μmol·L^-1)]

Table 1. In vitro antitumor activities of target compounds 4a~4s against Hela and HL-60 [IC₅₀/(μmol·L^-1)]

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Compound	HL-60	Hela
4a	63.1±0.4	NA
4b	NA	NA
4c	NA	28.7±0.2
4d	NA	51.9±0.2
4e	54.6±0.2	NA
4f	16.9±0.5	47.2±0.4
4g	27.8±0.1	9.2±0.6
4h	38.9±0.6	8.5±0.2
4i	53.2±0.2	NA
4j	44.8±0.4	NA
4k	11.3±0.3	57.2±0.3
4l	10.8±0.2	52.5±0.8
4m	45.9±0.3	NA
4n	NA	42.8±0.6
4o	36.6±0.3	55.3±0.2
4p	NA	43.7±0.2
4q	20.9±0.3	38.6±0.5
4r	47.2±1.1	NA
4s	17.4±0.2	NA
Docetaxel	0.00321±0.00026	0.00716±0.00037

从表 1可以看出, 当R¹为3, 4, 5-三甲氧基时, 有利于抗肿瘤活性的提高, 原因可能与手性碳上连接有三甲氧苯基有关, 由于甲氧基上氧原子的孤对电子可以作为氢键给体, 与微管蛋白氨基上的氢原子形成氢键, 从而提高其结合能力; 当R²为吸电子基团时有利于活性提高, 吸电子基团越多, 活性越高, 详细的构效关系有待进一步深入研究.

2. 结论

以4-羟基香豆素、芳香醛、丙二腈、DMF-DMA以及芳香胺为原料, 通过三步反应合成了19个4-苯胺基取代吡喃香豆素并嘧啶类化合物, 均未见文献报道.该方法不仅具有反应时间时间短、产率高、后处理操作简便、无需柱色谱分离等优点, 而且还为以烯胺腈结构为原料制备其他类型吡喃香豆素并含氮杂环类化合物奠定了一定基础.体外抗肿瘤活性实验表明, 部分目标产物对人宫颈癌细胞Hela及人急性早幼粒白血病细胞株(HL-60)具有较好的抑制活性.

3. 实验部分

3.1 仪器与试剂

X-5显微熔点测定仪(未校准, 北京泰克仪器有限公司); 傅里叶变换红外光谱仪(KBr压片法, 美国Pekin Elmer公司); Bruker Avance-400MHz核磁共振仪(TMS作内标, CDCl₃为溶剂, 瑞士Bruker公司); Vario ELⅢ型元素分析仪(德国Elementar公司); EL311型酶标免疫检测仪(美国Bio Tek公司); 3164型水套式CO₂孵箱(美国Forma Scientific公司); IX71型倒置荧光相差显微镜(日本Olympus公司); 四氮唑蓝(MTT)购于瑞士Fluka公司; RPMI-1640培养基购于美国HyClone公司.所用醛均购于阿拉丁试剂公司, 均为分析纯.

实验所用人宫颈癌细胞株Hela由第四军医大学提供, 人急性早幼粒白血病细胞株HL-60由香港理工大学Chan Kin-Fai博士提供.

3.2 具有烯胺腈结构的吡喃香豆素2a~2f的合成

称取0.52 g (4 mmol) 4-羟基香豆素、0.26 g (4 mmol)丙二腈、4 mmol芳香醛、0.10 g (0.8 mmol) DMAP, 加入50 mL无水乙醇回流, 待反应结束后, 冷却至室温, 过滤得粗产物, 无水乙醇重结晶得产物.

2-氨基-3-氰基-4-(3', 4'-二甲氧苯基)-吡喃[3, 2-c]色烯-5-酮(2a):白色固体, 产率90%. m.p. 241~243 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.90 (d, J＝8.0 Hz, 1H), 7.72 (t, J＝8.0 Hz, 1H), 7.51 (d, J＝8.4 Hz, 1H), 7.47 (d, J＝8.0 Hz, 1H), 7.38 (s, 2H, NH₂), 6.87 (t, J＝8.0 Hz, 1H), 6.85 (s, 1H), 6.75 (dd, J＝8.4, 1.6 Hz, 1H), 4.41 (s, 1H, H-4), 3.72 (s, 6H, 2×OCH₃); IR (KBr) ν: 3376, 3321 (NH₂), 2193 (C≡N), 1729 (C＝O) cm^-1. Anal. calcd for C₂₁H₁₆N₂O₅: C 67.02, H 4.29, N 7.44; found C 67.27, H 4.31, N 7.49.

2-氨基-3-氰基-4-(3', 4', 5'-三甲氧苯基)-吡喃[3, 2-c]色烯-5-酮(2b):白色固体, 产率88%. m.p. 245~247 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.90 (d, J＝8.0 Hz, 1H), 7.72 (t, J＝8.0 Hz, 1H), 7.52~7.47 (m, 2H), 7.41 (s, 2H, NH₂), 6.53 (s, 2H), 4.44 (s, 1H, H-4), 3.72 (s, 6H, 2×OCH₃), 3.64 (s, 3H, OCH₃); IR (KBr) ν: 3433, 3302 (NH₂), 2192 (C≡N), 1718 (C＝O) cm^-1; Anal. calcd for C₂₂H₁₈N₂O₆: C 65.02, H 4.46, N 6.89; found C 65.26, H 4.49, N 6.91.

2-氨基-3-氰基-4-(2', 3'-二氯苯基)-吡喃[3, 2-c]色烯-5-酮(2c):白色固体, 产率85%. m.p. 293~295 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.92 (d, J＝8.0 Hz, 1H), 7.75 (t, J＝8.0 Hz, 1H), 7.57~7.49 (m, 5H), 7.35 (d, J＝8.0 Hz, 1H), 7.31 (t, J＝8.0 Hz, 1H), 5.09 (s, 1H, H-4); IR (KBr) ν: 3406, 3304 (NH₂), 2200 (C≡N), 1712 (C＝O) cm^-1. Anal. calcd for C₁₉H₁₀Cl₂N₂O₃: C 59.24, H 2.62, N 7.27; found C 59.51, H 2.64, N 7.28.

2-氨基-3-氰基-4-(4'-硝基苯基)-吡喃[3, 2-c]色烯-5-酮(2d):黄色固体, 产率83%. m.p. 276~278 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.19 (d, J＝8.0 Hz, 2H), 7.92 (d, J＝8.0 Hz, 1H), 7.75 (t, J＝8.0 Hz, 1H), 7.61 (d, J＝8.0 Hz, 2H), 7.58 (s, 2H, NH₂), 7.54 (d, J＝8.0 Hz, 1H), 7.50 (d, J＝8.0 Hz, 1H), 4.69 (s, 1H, H-4); IR (KBr) ν: 3482, 3431 (NH₂), 2196 (C≡N), 1718 (C＝O) cm^-1. Anal. calcd for C₁₉H₁₁N₃O₅: C 63.16, H 3.07, N 11.63; found C 63.41, H 3.09, N 11.66.

2-氨基-3-氰基-4-(4'-溴苯基)-吡喃[3, 2-c]色烯-5-酮(2e):白色固体, 产率86%. m.p. 273~275 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 7.91 (d, J＝8.0 Hz, 1H), 7.73 (t, J＝8.0 Hz, 1H), 7.53~7.47 (m, 6H), 7.26 (d, J＝8.0 Hz, 2H), 4.49 (s, 1H, H-4); IR (KBr) ν: 3388, 3311 (NH₂), 2193 (C≡N), 1713 (C＝O) cm^-1; Anal. calcd for C₁₉H₁₁BrN₂O₃: C 57.74, H 2.81, N 7.09; found C 57.98, H 2.83, N 7.13.

2-氨基-3-氰基-4-(3'-硝基苯基)-吡喃[3, 2-c]色烯-5-酮(2f):白色固体, 产率82%. m.p. 261~263 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.15~8.12 (m, 2H), 7.92 (d, J＝8.0 Hz, 1H), 7.82 (d, J＝8.0 Hz, 1H), 7.74 (t, J＝8.0 Hz, 1H), 7.64 (t, J＝8.0 Hz, 1H), 7.58 (s, 2H, NH₂), 7.54~7.47 (m, 2H), 4.74 (s, 1H, H-4); IR (KBr) ν: 3405, 3321 (NH₂), 2203 (C≡N), 1705 (C＝O) cm^-1. Anal. calcd for C₁₉H₁₁N₃O₅: C 63.16, H 3.07, N 11.63; found C 63.43, H 3.09, N 11.66.

3.3 N, N-二甲基甲脒3a~3f的合成

称取1.88 g (5 mmol) 2, 加入5 mL DMF-DMA回流, 待反应结束后, 冷却至室温, 过滤得粗产物, 乙醚洗涤干燥得产物.

(E)-N'-[2-氰基-3-(3', 4'-二甲氧苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3a):白色固体, 产率84%. m.p. 280~282 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.56 (s, 1H, NCHN), 8.22 (d, J＝8.0 Hz, 1H), 7.72 (t, J＝8.0 Hz, 1H), 7.49~7.45 (m, 2H), 6.90~6.87 (m, 2H), 6.76 (dd, J＝8.0, 2.0 Hz, 1H), 4.51 (s, 1H), 3.72 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.25 (s, 3H, NCH₃), 3.05 (s, 3H, NCH₃); IR (KBr) ν: 2198 (C≡N), 1722 (C＝O), 1665 (C＝N) cm^-1. Anal. calcd for C₂₄H₂₁N₃O₅: C 66.81, H 4.91, N 9.74; found C 67.05, H 4.92, N 9.77.

(E)-N'-[2-氰基-3-(3', 4', 5'-三甲氧苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3b):灰色固体, 产率83%. m.p. 250~252 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.58 (s, 1H, NCHN), 8.25 (d, J＝8.0 Hz, 1H), 7.74 (t, J＝8.0 Hz, 1H), 7.50~7.47 (m, 2H), 6.55 (s, 2H), 4.55 (s, 1H), 3.72 (s, 6H, 2×OCH₃), 3.64 (s, 3H, OCH₃), 3.26 (s, 3H, NCH₃), 3.06 (s, 3H, NCH₃); IR (KBr) ν: 2203 (C≡N), 1720 (C＝O), 1662 (C＝N) cm^-1. Anal. calcd for C₂₅H₂₃N₃O₆: C 65.07, H 5.02, N 9.11; found C 65.33, H 5.03, N 9.14.

(E)-N'-[2-氰基-3-(2', 3'-二氯苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3c):白色固体, 产率81%. m.p. 270~272 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.60 (s, 1H, NCHN), 8.27 (d, J＝8.0 Hz, 1H), 7.76 (t, J＝8.0 Hz, 1H), 7.57 (dd, J＝8.0, 2.0 Hz, 1H), 7.52~7.48 (m, 2H), 7.38~7.31 (m, 2H), 5.21 (s, 1H), 3.27 (s, 3H, NCH₃), 3.06 (s, 3H, NCH₃); IR (KBr) ν: 2205 (C≡N), 1724 (C＝O), 1667 (C＝N) cm^-1. Anal. calcd for C₂₂H₁₅Cl₂N₃O₃: C 60.02, H 3.43, N 9.54; found C 60.29, H 3.44, N 9.56.

(E)-N'-[2-氰基-3-(4'-硝基苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3d):黄色固体, 产率84%. m.p. 293~295 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.60 (s, 1H, NCHN), 8.26 (d, J＝8.0 Hz, 1H), 8.19 (d, J＝8.0 Hz, 2H), 7.74 (t, J＝8.0 Hz, 1H), 7.62 (d, J＝8 Hz, 2H), 7.51~7.46 (m, 2H), 4.81 (s, 1H), 3.26 (s, 3H, NCH₃), 3.05 (s, 3H, NCH₃); IR (KBr) ν: 2202 (C≡N), 1728 (C＝O), 1660 (C＝N) cm^-1. Anal. calcd for C₂₂H₁₆N₄O₅: C 63.46, H 3.87, N 13.46; found C 63.74, H 3.89, N 13.48.

(E)-N'-[2-氰基-3-(4'-溴苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3e):亮黄色固体, 产率83%. m.p. 290~292 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.57 (s, 1H, NCHN), 8.23 (d, J＝8.0 Hz, 1H), 7.73 (t, J＝8.0 Hz, 1H), 7.53~7.45 (4H, m), 7.27 (d, J＝8.0 Hz, 2H), 4.59 (s, 1H), 3.25 (s, 3H, NCH₃), 3.05 (s, 3H, NCH₃); IR (KBr) ν: 2203 (C≡N), 1719 (C＝O), 1665 (C＝N) cm^-1. Anal. calcd for C₂₂H₁₆BrN₃O₃: C 58.68, H 3.58, N 9.33; found C 58.95, H 3.59, N 9.36.

(E)-N'-[2-氰基-3-(3'-硝基苯基)-吡喃[3, 2-c]色烯]-N, N-二甲基甲脒(3f):黄色固体, 产率80%. m.p. 289~291 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.63 (s, 1H, NCHN), 8.29 (d, J＝8.0 Hz, 1H), 8.16~8.13 (m, 2H), 7.85 (d, J＝8.0 Hz, 1H), 7.76 (t, J＝8.0 Hz, 1H), 7.66 (t, J＝8 Hz, 1H), 7.52 (d, J＝8.0 Hz, 1H), 7.48 (d, J＝8.0 Hz, 1H), 4.87 (s, 1H), 3.28 (s, 3H, NCH₃), 3.07 (s, 3H, NCH₃); IR (KBr) ν: 2201 (C≡N), 1713 (C＝O), 1667 (C＝N) cm^-1. Anal. calcd for C₂₂H₁₆N₄O₅: C 63.46, H 3.87, N 13.46; found C 63.72, H 3.89, N 13.49.

3.4 吡喃香豆素并嘧啶类化合物4a~4s的合成

在50 mL圆底烧瓶中加入3 (0.5mmol), 芳香胺(0.55 mmol)和8 mL冰醋酸回流, 薄层色谱(TLC)监测反应进程, 待反应结束后, 冷却至室温, 析出晶体, 过滤, 乙醚洗涤后, 干燥得产物4.

4-(3'-氯-4'-氟苯胺基)-5-(3', 4'-二甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4a):黄色固体, 产率85%. m.p. 246~248 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 8.98 (s, 1H), 8.50 (s, 1H), 8.04 (d, J＝8.0 Hz, 1H), 7.98 (dd, J＝8.0, 2.0 Hz, 1H), 7.75 (t, J＝8.0 Hz, 1H), 7.64~7.60 (m, 1H), 7.54 (d, J＝8.0 Hz, 1H), 7.51 (d, J＝8.0 Hz, 1H), 7.39 (t, J＝9.0 Hz, 1H), 7.28 (s, 1H), 6.83 (d, J＝8.0 Hz, 2H), 5.62 (s, 1H), 3.70 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.3, 160.4, 159.0, 156.8, 152.6, 148.7, 148.5, 134.6, 123.1, 123.0, 120.7, 117.1, 113.8, 112.4, 106.1, 99.9, 56.0, 55.8, 32.9, 21.5; IR (KBr) ν: 3381 (NH), 1728 (C＝O) cm^-1. Anal. calcd for C₂₈H₁₉ClFN₃O₅: C 63.22, H 3.60, N 7.90; found C 63.49, H 3.62, N 7.93.

4-(3', 4'-二氯苯胺基)-5-(3', 4'-二甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4b):黄色固体, 产率82%. m.p. 238~240 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.00 (s, 1H, NH), 9.06 (s, 1H), 8.54 (s, 1H), 8.10 (d, J＝2.0 Hz, 1H), 8.04 (d, J＝7.0 Hz, 1H), 7.75 (t, J＝8.0 Hz, 1H), 7.70 (dd, J＝9.0, 2.0 Hz, 1H), 7.59~7.50 (m, 2H), 7.28 (s, 1H), 6.83 (d, J＝8.0 Hz, 2H), 5.66 (s, 1H), 3.69 (s, 3H, OCH₃), 3.65 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.4, 160.4, 158.9, 156.8, 154.7, 152.6, 148.7, 148.5, 139.8, 134.6, 133.4, 131.2, 130.8, 125.1, 122.4, 120.7, 117.1, 113.8, 112.5, 106.1, 100.5, 56.0, 55.8, 32.9, 21.5; IR (KBr) ν: 3378 (NH), 1729 (C＝O) cm^-1. Anal. calcd for C₂₈H₁₉Cl₂N₃O₅: C 61.33, H 3.49, N 7.66; found C 61.58; H 3.51, N 7.67.

4-(4'-溴苯胺基)-5-(3', 4'-二甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4c):黄色固体, 产率84%. m.p. 239~241 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 8.92 (s, 1H), 8.46 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.74 (t, J＝8.0 Hz, 1H), 7.64 (d, J＝8.8 Hz, 2H), 7.53 (d, J＝8.0 Hz, 1H), 7.49 (d, J＝8.8 Hz, 2H), 7.28 (s, 1H), 6.81 (s, 2H), 5.63 (s, 1H), 3.68 (s, 3H, OCH₃), 3.64 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.2, 160.4, 159.1, 156.7, 154.7, 152.6, 148.7, 148.5, 139.0, 134.7, 131.7, 125.4, 123.6, 123.1, 120.7, 115.8, 113.9, 113.2, 112.5, 106.1, 100.0, 56.0, 55.8, 33.0, 21.5; IR (KBr) ν: 3384 (NH), 1706 (C＝O) cm^-1. Anal. calcd for C₂₈H₂₀BrN₃O₅: C 60.23, H 3.61, N 7.53; found C 60.49, H 3.63, N 7.55.

4-(4'-氯苯胺基)-5-(3', 4'-二甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4d):亮黄色固体, 产率83%. m.p. 229~231 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 8.93 (s, 1H), 8.46 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.76 (d, J＝8.0 Hz, 1H), 7.70 (d, J＝8.8 Hz, 2H), 7.54 (d, J＝8.0 Hz, 1H), 7.50 (d, J＝8.0 Hz, 1H), 7.38 (d, J＝8.8 Hz, 2H), 7.30 (s, 1H), 6.83 (s, 2H), 5.64 (s, 1H), 3.69 (s, 3H, OCH₃), 3.65 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.2, 160.4, 159.1, 155.7, 154.7, 152.6, 148.6, 148.4, 138.6, 133.4, 128.8, 123.3, 123.1, 120.7, 117.1, 113.8, 112.4, 106.1, 99.9, 56.0, 55.8, 32.9, 21.5; IR (KBr) ν: 3384 (NH), 1731 (C＝O) cm^-1. Anal. calcd for C₂₈H₂₀ClN₃O₅: C 65.44, H 3.92, N 8.18; found C 65.71, H 3.95, N 8.20.

4-(3'-氯-4'-甲基苯胺基)-5-(3', 4'-二甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4e):灰色固体, 产率80%. m.p. 246~248 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.87 (s, 1H), 8.48 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.83 (d, J＝2.0 Hz, 1H), 7.74 (t, J＝8.0 Hz, 1H), 7.54~7.49 (m, 3H), 7.29 (s, 1H), 6.81 (s, 2H), 5.62 (s, 1H), 3.69 (s, 3H, OCH₃), 3.64 (s, 3H, OCH₃), 2.29 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.1, 160.1, 159.5, 155.4, 154.9, 150.6, 148.6, 148.4, 137.6, 130.4, 128.8, 123.1, 120.7, 117.6, 113.8, 110.4, 106.1, 99.7, 55.3, 32.7, 21.5; IR (KBr) ν: 3402 (NH), 1725 (C＝O) cm^-1. Anal. calcd for C₂₉H₂₂ClN₃O₅: C 65.97, H 4.20, N 7.96; found C 66.24, H 4.24, N 7.98.

4-(4'-溴苯胺基)-5-(3', 4', 5'-三甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4f):黄色固体, 产率81%. m.p. 293~295 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.93 (s, 1H), 8.48 (s, 1H), 8.04 (d, J＝8.0 Hz, 1H), 7.74~7.71 (m, 1H), 7.67 (d, J＝8.0 Hz, 2H), 7.55~7.50 (m, 3H), 6.79 (s, 2H), 5.66 (s, 1H), 3.66 (s, 6H, 2×OCH₃), 3.57 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.3, 160.4, 159.1, 156.8, 155.1, 153.2, 152.6, 139.0, 137.6, 137.2, 131.8, 125.4, 123.6, 123.2, 117.1, 115.6, 113.9, 106.3, 105.7, 99.6, 60.3, 56.4, 33.7; IR (KBr) ν: 3386 (NH), 1736 (C＝O) cm^-1. Anal. calcd for C₂₉H₂₂BrN₃O₆: C 59.20, H 3.77, N 7.14; found C 59.49, H 3.78, N 7.17.

4-(4'-氯苯胺基)-5-(3', 4', 5'-三甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4g):黄色固体, 产率84%. m.p. 299~301 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 8.94 (s, 1H), 8.48 (s, 1H), 8.04 (d, J＝8.0 Hz, 1H), 7.77 (d, J＝8.0 Hz, 1H), 7.72 (d, J＝8.8 Hz, 2H), 7.54 (d, J＝8.0 Hz, 1H), 7.51 (d, J＝8.0 Hz, 1H), 7.39 (d, J＝8.8 Hz, 2H), 6.80 (s, 2H), 5.66 (s, 1H), 3.67 (s, 6H, 2×OCH₃), 3.58 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.3, 160.4, 159.1, 156.8, 155.1, 153.2, 152.6, 138.5, 137.6, 137.2, 133.4, 128.9, 125.4, 123.3, 123.2, 117.1, 113.9, 106.4, 105.7, 99.5, 60.3, 56.4, 33.7, 21.5; IR (KBr) ν: 3387 (NH), 1737 (C＝O) cm^-1. Anal. calcd for C₂₉H₂₂ClN₃O₆: C 64.03, H 4.08, N 7.73; found C 64.29, H 4.11, N 7.75.

4-(3'-氯-4'-氟苯胺基)-5-(3', 4', 5'-三甲氧苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4h):黄色固体, 产率82%. m.p. 280~282 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 8.96 (s, 1H), 8.50 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.99 (dd, J＝8.0, 2.0 Hz, 1H), 7.74 (t, J＝8.0 Hz, 1H), 7.64~7.60 (m, 1H), 7.53~7.48 (m, 1H), 7.39 (t, J＝8.0 Hz, 1H), 6.78 (s, 2H), 5.63 (s, 1H), 3.66 (s, 6H, 2×OCH₃), 3.59 (s, 3H, OCH₃); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.5, 160.1, 159.5, 156.2, 155.0, 153.2, 152.6, 138.1, 137.6, 137.2, 133.5, 130.9, 125.4, 123.8, 123.2, 117.0, 113.9, 106.8, 105.7, 99.8, 60.9, 56.4, 33.5, 21.5; IR (KBr) ν: 3392 (NH), 1729 (C＝O) cm^-1. Anal. calcd for C₂₉H₂₁ClFN₃O₆: C 61.98, H 3.77, N 7.48; found C 62.25, H 3.79, N 7.51.

4-(3', 4'-二氯苯胺基)-5-(2', 3'-二氯苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4i):黄色固体, 产率79%. m.p.＞320 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.73 (s, 1H), 8.56 (s, 1H), 8.06 (d, J＝8.0 Hz, 1H), 8.01~7.98 (m, 2H), 7.78 (t, J＝8.0 Hz, 1H), 7.58~7.50 (m, 4H), 7.37 (t, J＝8.0 Hz, 1H), 6.02 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.3, 159.5, 158.0, 151.4, 150.9, 145.1, 140.6, 138.3, 135.7, 130.3, 124.6, 120.2, 117.0, 115.6, 113.0, 107.9, 99.5, 33.5, 21.5; IR (KBr) ν: 3417 (NH), 1731 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₃Cl₄N₃O₃: C 56.04, H 2.35, N 7.54; found C 56.31, H 2.38, N 7.56.

4-(4'-氯苯胺基)-5-(2', 3'-二氯苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4j):黄色固体, 产率83%. m.p.＞320 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.53 (s, 1H), 8.49 (s, 1H), 8.06 (d, J＝8.0 Hz, 1H), 7.98 (d, J＝8.0 Hz, 1H), 7.77 (t, J＝8.0 Hz, 1H), 7.59~7.50 (m, 5H), 7.36 (t, J＝8.0 Hz, 1H), 7.35 (d, J＝8.0 Hz, 1H), 5.76 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.7, 159.5, 158.4, 150.5, 149.5, 149.0, 148.3, 140.1, 131.7, 130.6, 125.1, 123.6, 117.0, 115.4, 113.5, 104.9, 99.7, 33.0, 21.5; IR (KBr) ν: 3423 (NH), 1731 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₄Cl₃N₃O₃: C 59.74, H 2.70, N 8.04; found C 60.02, H 2.72, N 8.07.

4-(4'-溴苯胺基)-5-(2', 3'-二氯苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4k):黄色固体, 产率81%. m.p.＞320 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.54 (s, 1H), 8.49 (s, 1H), 8.06 (d, J＝8.0 Hz, 1H), 7.98 (d, J＝8.0 Hz, 1H), 7.77 (t, J＝8.0 Hz, 1H), 7.54~7.46 (m, 5H), 7.38~7.31 (m, 2H), 5.76 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.5, 159.6, 158.0, 152.4, 150.2, 145.1, 139.8, 138.3, 135.5, 130.1, 124.6, 120.5, 117.3, 115.6, 113.5, 107.9, 99.9, 33.2, 21.5; IR (KBr) ν: 3420 (NH), 1733 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₄BrCl₂N₃O₃: C 55.06, H 2.49, N 7.41; found C 55.32, H 2.51, N 7.44.

4-(4'-溴苯胺基)-5-(4'-硝基苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4l):黄色固体, 产率80%. m.p.＞320 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.10 (s, 1H), 8.49 (s, 1H), 8.15 (d, J＝8.8 Hz, 2H), 8.06 (d, J＝8.0 Hz, 1H), 7.82 (d, J＝8.8 Hz, 2H), 7.77 (d, J＝8.0 Hz, 1H), 7.58~7.48 (m, 5H), 5.93 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.3, 159.2, 157.3, 155.5, 152.7, 149.3, 147.2, 138.8, 131.7, 130.3, 124.1, 123.2, 117.1, 115.9, 113.7, 104.9, 98.9, 33.5, 21.5; IR (KBr) ν: 3432 (NH), 1718 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₅BrN₄O₅: C 57.48, H 2.78, N 10.31; found C 57.74, H 2.81; N 10.33.

4-(4'-氯苯胺基)-5-(4'-硝基苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4m):黄色固体, 产率82%. m.p. 317~319 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.97 (s, 1H, NH), 9.08 (s, 1H), 8.48 (s, 1H), 8.14 (d, J＝8.4 Hz, 2H), 8.05 (d, J＝8.0 Hz, 1H), 7.81 (d, J＝8.8 Hz, 2H), 7.76 (t, J＝8.0 Hz, 1H), 7.61 (d, J＝8.8 Hz, 2H), 7.56~7.49 (m, 2H), 7.36 (d, J＝8.4 Hz, 2H), 5.92 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.5, 160.3, 159.3, 157.3, 155.5, 152.7, 149.3, 147.2, 138.3, 133.7, 128.8, 124.1, 123.8, 123.2, 117.2, 113.7, 104.9, 98.7, 33.5, 21.5; IR (KBr) ν: 3370 (NH), 1718 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₅ClN₄O₅: C 62.60, H 3.03, N 11.23; found C 62.85, H 3.06, N 11.25.

4-(3'-氯-4'-氟苯胺基)-5-(4'-硝基苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4n): 灰色固体, 产率83%. m.p. 298~301 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.14 (s, 1H), 8.51 (s, 1H), 8.15 (d, J＝8.8 Hz, 2H), 8.05 (d, J＝8.0 Hz, 1H), 7.86 (dd, J＝8.0, 2.0 Hz, 1H), 7.80 (d, J＝8.8 Hz, 2H), 7.76 (d, J＝8.0 Hz, 1H), 7.56~7.50 (m, 3H), 7.37 (t, J＝8.0 Hz, 1H), 5.89 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.1, 159.0, 158.3, 155.4, 150.5, 149.1, 147.6, 138.2, 131.7, 130.3, 124.1, 123.1, 117.0, 115.6, 113.5, 104.9, 99.7, 33.2, 21.5; IR (KBr) ν: 3434 (NH), 1716 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₄ClFN₄O₅: C 60.42, H 2.73, N 10.84; found C 60.69, H 2.76, N 10.87.

4-(3'-氯-4'-氟苯胺基)-5-(4'-溴苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4o):黄色固体, 产率85%. m.p. 297~299 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 9.05 (s, 1H), 8.50 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.90 (dd, J＝7.0, 2.0 Hz, 1H), 7.76 (t, J＝8.0 Hz, 1H), 7.59~7.45 (m, 6H), 7.38 (t, J＝9.0 Hz, 1H), 5.72 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.8, 159.2, 157.5, 155.5, 150.8, 149.3, 147.2, 138.4, 135.7, 131.3, 125.1, 121.2, 117.6, 116.9, 113.8, 105.6, 99.0, 33.9, 21.5; IR (KBr) ν: 3375 (NH), 1734 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₄BrClFN₃O₃: C 56.70, H 2.56, N 7.63; found C 56.95, H 2.58, N 7.65.

4-(4'-溴苯胺基)-5-(4'-溴苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4p):黄色固体, 产率84%. m.p.＞320 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.97 (s, 1H, NH), 8.99 (s, 1H), 8.47 (s, 1H), 8.04 (dd, J＝8.0, 1.2 Hz, 1H), 7.76 (t, J＝8.0 Hz, 1H), 7.59 (d, J＝8.0 Hz, 2H), 7.55~7.48 (m, 7H), 5.75 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.4, 160.3, 159.1, 157.1, 155.1, 152.6, 141.4, 136.6, 133.6, 131.9, 131.0, 123.5, 122.5, 121.2, 117.2, 117.0, 113.7, 105.5, 99.3, 33.0, 21.5; IR (KBr) ν: 3374 (NH), 1693 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₅Br₂N₃O₃: C 54.10, H 2.62, N 7.28; found C 54.35, H 2.65, N 7.31.

4-(3', 4'-二氯苯胺基)-5-(4'-溴苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4q):黄色固体, 产率81%. m.p. 310~312 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.94 (s, 1H, NH), 9.11 (s, 1H), 8.53 (s, 1H), 8.04 (s, 1H), 8.02 (d, J＝2.0 Hz, 1H), 7.75 (t, J＝8.0 Hz, 1H), 7.63 (dd, J＝9.0, 2.0 Hz, 1H), 7.57~7.43 (m, 6H), 5.75 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.4, 160.3, 159.1, 157.0, 155.1, 152.6, 141.5, 138.9, 133.5, 131.9, 131.7, 131.0, 123.9, 123.1, 121.1, 117.1, 115.8, 113.8, 105.5, 99.3, 33.0, 21.5; IR (KBr) ν: 3370 (NH), 1733 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₄BrCl₂N₃O₃: C 55.06, H 2.49, N 7.41; found C 55.32, H 2.52, N 7.44.

4-(4'-氯苯胺基)-5-(4'-溴苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4r):黄色固体, 产率83%. m.p. 300~302 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.99 (s, 1H, NH), 9.01 (s, 1H), 8.47 (s, 1H), 8.04 (d, J＝8.0 Hz, 1H), 7.76 (t, J＝8.0 Hz, 1H), 7.64 (d, J＝8.0 Hz, 2H), 7.38 (d, J＝8.0 Hz, 2H), 7.56~7.48 (m, 5H), 5.74 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 172.5, 161.4, 160.3, 159.3, 157.3, 155.5, 152.7, 149.3, 147.2, 138.3, 133.7, 128.8, 124.1, 123.8, 123.2, 117.1, 113.7, 104.9, 98.7, 33.5, 21.5; IR (KBr) ν: 3376 (NH), 1695 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₅BrClN₃O₃: C 58.61, H 2.84, N 7.89; found C 58.87, H 2.86, N 7.91.

4-(4'-溴苯胺基)-5-(3'-硝基苯基)-色烯[3', 4':5, 6]吡喃[2, 3-d]嘧啶-6-酮(4s):黄色固体, 产率78%. m.p. 277~279 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.96 (s, 1H), 8.48 (s, 1H), 8.03 (d, J＝8.0 Hz, 1H), 7.77~7.73 (m, 7H), 7.55~7.45 (m, 2H), 7.28 (d, J＝8.0 Hz, 1H), 5.72 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 161.4, 160.3, 159.2, 157.0, 155.1, 152.6, 141.5, 138.4, 128.8, 125.5, 123.6, 123.2, 121.1, 117.1, 113.8, 105.5, 99.3, 33.0, 21.5; IR (KBr) ν: 3409 (NH), 1731 (C＝O) cm^-1. Anal. calcd for C₂₆H₁₅BrN₄O₅: C 57.48, H 2.78, N 10.31; found C 57.76, H 2.80, N 10.33.

3.5 MTT法体外抗肿瘤活性实验

取肿瘤细胞株, 用RPMI-1640培养液于CO₂孵箱中37 ℃, 5% CO₂饱和湿度下培养, 细胞贴壁后用胰蛋白酶消化传代.将处于对数生长期肿瘤细胞以5×10⁴个/ mL的密度接种于96孔培养板中, 每孔100 μL.次日, 每孔加入不同浓度受试化合物, 每药设4个剂量组, 每组设3个平行孔, 另设空白组和对照组(以紫杉醇为阳性对照).加药完毕后于CO₂孵箱中在37 ℃, 5% CO₂饱和湿度下培养48 h, 然后每孔再加入5 mg/mL MTT溶液20 μL, 继续培养4 h后, 吸弃上清液, 每孔加入150 μL二甲基亚砜(DMSO), 振荡10 min (60次/min)后, 用自动酶标仪于490 nm波长处测定吸光度(OD)值, 取各组平均值, 重复实验3次.以浓度为横轴, 细胞存活率为纵轴绘制曲线, 用两点法计算出每个样品的IC₅₀值, 结果见表 1.

$ \begin{align} &\text{细胞存活率}\left( \% \right)=\left( \text{样品组平均OD值}-\text{空白孔} \right)/ \\ &\left( \text{对照组平均OD值}-\text{空白孔} \right)\times 100\% \\ \end{align} $

辅助材料(Supporting Information) 化合物2a~2f, 3a~3f和4a~4s的¹H NMR和¹³C NMR图谱.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

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图 1 目标产物的结构

Figure 1 Structure of target compounds

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图式 1 以氨基烯酸为原料合成4-苯胺基取代嘧啶

Scheme 1 Synthesis of 4-anilino substituted pyrimidine using amino olefin acid as raw material

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图式 2 以烯胺腈为原料合成4-苯胺基取代嘧啶

Scheme 2 Synthesis of 4-anilino substituted pyrimidine using cyanoenamine as raw material

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图 2 杂环并嘧啶类化合物的结构

Figure 2 Structure of heterocycle fused pyrimidines

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图 3 通过多组分反应制备具有烯胺腈结构的多取代吡喃香豆素

Figure 3 Polysubstituted pyranocoumarins with adjacent amino and nitrile functionalities synthesized by MCRs

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图 4 具有生物活性的含有烯胺腈结构的4-取代吡喃香豆素

Figure 4 4-Substituted pyranocoumarins with cyanoenamine which have biological activities

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图式 3 目标化合物的合成路线

Scheme 3 Synthetic route of the target compounds

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图 5 (a) 化合物4h与秋水仙碱结合位点的分子对接图以及(b)秋水仙碱的分子对接图

Figure 5 (a) Molecules Docking of compound 4h with colchicine-binding site and (b) molecules Docking of colchicine

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表 1 目标产物4a~4s的体外抗肿瘤活性[IC₅₀/(μmol·L^-1)]

Table 1. In vitro antitumor activities of target compounds 4a~4s against Hela and HL-60 [IC₅₀/(μmol·L^-1)]

Compound	HL-60	Hela
4a	63.1±0.4	NA
4b	NA	NA
4c	NA	28.7±0.2
4d	NA	51.9±0.2
4e	54.6±0.2	NA
4f	16.9±0.5	47.2±0.4
4g	27.8±0.1	9.2±0.6
4h	38.9±0.6	8.5±0.2
4i	53.2±0.2	NA
4j	44.8±0.4	NA
4k	11.3±0.3	57.2±0.3
4l	10.8±0.2	52.5±0.8
4m	45.9±0.3	NA
4n	NA	42.8±0.6
4o	36.6±0.3	55.3±0.2
4p	NA	43.7±0.2
4q	20.9±0.3	38.6±0.5
4r	47.2±1.1	NA
4s	17.4±0.2	NA
Docetaxel	0.00321±0.00026	0.00716±0.00037

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沈阳化工大学材料科学与工程学院沈阳 110142