含有芳杂环及酯基结构的1<i>H</i>-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物的合成、抑菌活性及构效关系

引用本文: 张秀君, 王兰芝, 闫婧怡, 高晨. 含有芳杂环及酯基结构的1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物的合成、抑菌活性及构效关系[J]. 有机化学, 2017, 37(2): 462-473. doi: 10.6023/cjoc201604059 shu

Citation: Zhang Xiujun, Wang Lanzhi, Yan Jingyi, Gao Chen. Synthesis, Antibacterial Activity and Structure-Activity Relationship of 1H-2, 5-Dihydro-1, 5-benzodiazepine with Ester Group and the Aromatic Heterocyclic Ring[J]. Chinese Journal of Organic Chemistry, 2017, 37(2): 462-473. doi: 10.6023/cjoc201604059 shu

含有芳杂环及酯基结构的1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物的合成、抑菌活性及构效关系

张秀君 ^a, ,
王兰芝 ^{a,*,
,} ,
闫婧怡 ^b, ,
高晨 ^b,

a.
河北师范大学化学与材料科学学院石家庄 050024)
b.
河北师范大学生命科学学院石家庄 050024

通讯作者: 王兰芝, wanglanzhi@126.com

基金项目:
河北省自然科学基金 B2016205165

国家自然科学基金 21276064

摘要: 以2-（2-噻唑基）-3-乙酯基-4-甲基-1H-2，5-二氢-1，5-苯并二氮杂䓬（A）为模型化合物，设计合成了3个系列30种1H-2，5-二氢-1，5-苯并二氮杂䓬类衍生物2~6，其结构通过核磁共振波谱、红外光谱、质谱和元素分析确证.采用常规滤纸片扩散法测试了其对真菌（新生隐球菌标准株、临床株、白色念珠菌标准株）和细菌（大肠杆菌标准株和金黄色葡萄球菌标准株）的抑菌活性.研究结果表明，大多数化合物对所测试的菌种都表现出很好的抑制作用，且抑真菌能力大于抑细菌能力；通过对活性较高的化合物2-（2-噻唑基）-3-乙酯基-4-乙基-8-甲基-1H-2，5-二氢-1，5-苯并二氮杂䓬（4b）和2-（2-噻唑基）-3-乙酯基-4-乙基-8-氟-1H-2，5-二氢-1，5-苯并二氮杂䓬（4c）的最小抑菌浓度（MIC）和最小杀菌浓度（MFC）测试，发现其对白色念珠菌的MIC和MFC都远低于标准药物氟康唑.进一步构效关系研究表明C-2位的2-噻唑基，C-4位的CH₃、CH₂CH₃和C-8位的CH₃都是该类化合物抑菌的必需基团.

关键词:

English

Synthesis, Antibacterial Activity and Structure-Activity Relationship of 1H-2, 5-Dihydro-1, 5-benzodiazepine with Ester Group and the Aromatic Heterocyclic Ring

a.
College of Chemistry & Material Science, Hebei Normal University, Shijiazhuang 050024
b.
College of Life Sciences, Hebei Normal University, Shijiazhuang 050024

Corresponding author: Wang Lanzhi, E-mail: wanglanzhi@126.com

Received Date: 29 April 2016
Revised Date: 23 May 2016
Fund Project:
the Natural Science Foundation of Hebei Province

the National Natural Science Foundation of China

Abstract: Using (ethyl 4-methyl-2-(thiazol-2-yl)-1H-2, 5-dihydro-1, 5-benzodiazepine-3-carboxylate) (A) as a model compound, thirty 1H-2, 5-dihydro-1, 5-benzodiazepine derivatives 2~6 were synthesized by nucleophilic addition reaction, elimination reaction and cyclization reaction. The structures of these new compounds were characterized by ¹H NMR, ¹³C NMR, IR, MS and elemental analysis. The antibacterial activities of those compounds were screened using the disk diffusion method against C. neoformans, C. neoformans clinical isolates, C. albicans, E. coli and S. aureus. The bioactive assay results revealed that most of the 1H-2, 5-dihydro-1, 5-benzodiazepine derivatives exhibited considerable potency against all of the tested strains. In particular, some compounds exhibited excellent antimicrobial activities against the test microorganism MS except E. coli, and exhibited better antimicrobial activity against fungi than against bacteria. Furthermore, ethyl 4-ethyl-8-methyl-2-(thiazol-2-yl)-2, 5-dihydro-1, 5-benzodiazepine-3-carboxylate (4b) and ethyl 4-ethyl-8-fluoro-2-(thiazol-2-yl)-2, 5-dihydro-1, 5-benzo-diazepine-3-carboxylate (4c), which had good antibacterial activities, were subjected to further pharmacological evaluation, including minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC) against C. neoformans, C. albicans, and S. aureus. The results showed that the MIC and MFC against C. albicans values for above compounds were much lower than those of fluconazole. A further study of the structure-activity rela-tionship revealed that the thiazole ring at C-2, the methyl, ethyl at C-4 and the methyl at C-8 are essential for antibacterial activity.

Key words:

1H-2, 5-二氢-1, 5-苯并二氮杂䓬是一类具有较高生理活性及药理活性的七元含氮杂环化合物, 具有抗抑郁、抗惊厥、抗焦虑、安眠、消炎止痛等^[1~5]作用, 在医学上常用作治疗癌症、病毒感染和心血管疾病等方面的药物^{[6, 7]}.近期有文献报道1H-2, 5-二氢-1, 5-苯并二氮杂䓬及其衍生物具有优良的抑细菌、真菌特性^{[8, 9]}.本课题组一直进行1H-2, 5-二氢-1, 5-苯并二氮杂䓬类衍生物的设计、合成及抑菌活性研究, 并且发现2-(2-噻唑基)-3-乙酯基-4-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3a) (图 1) 的抑真菌活性尤为突出, 其对新生隐球菌的抑菌活性是标准药物氟康唑的12.8倍, 而且毒性较低, 同时还确定了3位的乙酯基是其药效团之一, 而2位的噻唑基与其抑菌活性也有密切关系^[10].鉴于此, 本文在保留1H-2, 5-二氢-1, 5-苯并二氮杂䓬3位乙酯基结构特征的基础上, 以杂䓬3a为模型化合物, 设计合成了3个系列30种新型1H-2, 5-二氢-1, 5-苯并二氮杂䓬衍生物 (图 1), 考察了其抑菌效果, 研究其构效关系, 旨在筛选出抑菌活性优良的1H-2, 5-二氢-1, 5-苯并二氮杂䓬类先导化合物.

图 1 化合物2~6的结构 Figure 1. Structures of compounds 2~6

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1 结果与讨论

1.1 化合物的合成

目标化合物2~6的合成路线如Scheme 1所示, 由Scheme 1可知, 合成1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物2~6的反应分2步进行.第一步是取代的邻苯二胺在无溶剂无催化剂、室温条件下, 与羰基酸酯中的酮羰基碳发生亲核加成、消除反应, 得到的产品通过无水乙醇重结晶得到纯品N-邻氨芳基取代的β-烯胺酯1, 产率可高达96%以上.由于在反应过程中酸性催化剂的加入和高温都会促使副产物苯并咪唑^[11]的生成 (图 2), 从而使主产物的产率降低, 因此将绿色无溶剂、无催化剂、室温作为该反应的最优条件; 第二步是N-邻氨芳基取代的β-烯胺酯1与芳醛发生亲核加成、消除、环合等系列反应, 得到目标化合物2~6.本文通过反应条件优化发现, 采用二氧化硅负载的L-扁桃酸 (L-MA-SiO₂) 作催化剂, 比其他的酸性催化剂催化效果明显, 表现在反应时间可缩短0.5 h, 产率可高出其他催化剂10%, 且比消旋扁桃酸作催化剂得到目标产物的产率较高, 原因可能是L-扁桃酸更纯净, 使得目标化合物更容易从反应体系中析出.由此该反应选择二氧化硅负载的L-扁桃酸 (L-MA-SiO₂)^{[12, 13]}为催化剂, 无水乙醇为溶剂, 在-10 ℃下反应2.5 h得到目标化合物, 产率可高达92%.

图 2 副产物苯并咪唑的结构 Figure 2. Structure of by-product benzimidazole

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图图式1 1H-2, 5-二氢-1, 5-苯并二氮杂䓬2~6的合成路线 Figure 图式1. Synthetic routes of 1H-2, 3-dihydro-1, 5-benzodiaze-pines 2~6

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1.2 目标化合物的合成机理

由Scheme 2可见, 该反应的第一步是取代邻苯二胺中对位的氨基进攻羰基酸酯中的酮羰基碳, 发生亲核加成反应, 脱去一分子水生成中间体N-邻氨芳基取代的β-烯胺酯1, 第二步是N-邻氨芳基取代的β-烯胺酯1中的另外一个氨基进攻醛中的羰基碳, 发生亲核加成反应, 同样脱掉一分子水生成中间体I, 该中间体经过环合、H⁺转移到电子云密度高的N原子上得到目标化合物2~6^{[14, 15]}.

图图式2 目标化合物2~6的合成机理 Figure 图式2. Plausible mechanism for synthesis of products 2~6

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1.3 1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物2~6的抑菌活性评价

1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物2~6的抗菌活性测试结果列于表 1中.由表 1可见, 当化合物的2位为2-噻唑基时, 大部分化合物 (2a~2e、3a(A)~3e^[10]、4a~4e) 都表现出很好的抑菌活性, 尤其是化合物4a~4e能较好地抑制真菌和细菌 (大肠杆菌除外), 其中4b的抑菌活性最高, 对真菌新生隐球菌标准株和临床株的抑菌圈值可高达28.5和23.7 mm; 当化合物的2位为苯基时, 化合物3f~3j对新生隐球菌临床株、白色念珠菌和金黄色葡萄球菌只表现出中等或者很弱的抑菌活性; 而2位为2-呋喃基的化合物 (3k~3o) 对所测试的五种菌株都没有任何抑制作用.由此看来, 2-噻唑基的加入能够有效增强该类化合物的抑菌活性 (大肠杆菌除外).

表 1 化合物2~6的抑菌圈值 (nm)^a Table 1. Zone (mm) of growth inhibition of compounds 2~6

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Compd.	R¹	R²	X	C.n.	C.n.C.	C.a.	E.c.	S.a.
2a	H	H		6.00±0.00	6.00±0.00	6.00±0.00	9.70±0.30	8.40±0.10
2b	CH₃	H		8.80±0.15	6.00±0.00	6.00±0.00	11.20±0.00	11.70±0.00
2c	F	H		6.00±0.00	6.00±0.00	6.00±0.00	10.40±0.12	9.60±0.15
2d	Cl	H		16.60±0.05	19.10±0.06	9.60±0.10	8.70±0.12	7.80±0.05
2e	Br	H		6.00±0.00	6.00±0.00	7.30±0.10	7.80±0.20	7.00±0.25
3a	H	CH₃		24.27±0.20	28.63±0.30	15.00±0.10	19.70±0.25	21.17±0.20
3b	CH₃	CH₃		21.00±0.21	24.07±0.23	14.37±0.15	15.03±0.19	17.10±0.30
3c	F	CH₃		15.83±0.32	15.90±0.25	13.33±0.15	12.67±0.20	19.00±0.32
3d	Cl	CH₃		14.23±0.21	15.75±0.22	8.10±0.12	9.50±0.20	18.60±0.10
3e	Br	CH₃		12.07±0.12	15.70±0.25	11.70±0.20	12.03±0.10	13.30±0.15
3f	H	CH₃		6.00±0.00	14.30±0.05	7.20±0.10	6.00±0.00	10.02±0.11
3g	CH₃	CH₃		6.00±0.00	15.60±0.10	16.60±0.05	6.00±0.00	11.17±0.12
3h	F	CH₃		6.00±0.00	16.70±0.11	15.25±0.03	6.00±0.00	10.95±0.11
3i	Cl	CH₃		6.00±0.00	15.70±0.15	12.23±0.04	6.00±0.00	10.83±0.13
3j	Br	CH₃		6.00±0.00	15.60±0.12	11.33±0.10	6.00±0.00	10.30±0.12
3k	H	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3l	CH₃	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3m	F	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3n	Cl	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3o	Br	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
4a	H	CH₂CH₃		22.60±0.00	17.70±0.00	18.40±0.10	6.00±0.00	21.90±0.13
4b	CH₃	CH₂CH₃		28.50±0.11	23.70±0.15	22.70±0.12	6.00±0.00	23.10±0.05
4c	F	CH₂CH₃		27.90±0.20	23.20±0.19	21.70±0.05	6.00±0.00	22.80±0.30
4d	Cl	CH₂CH₃		21.20±0.10	15.70±0.30	14.00±0.23	6.00±0.00	22.50±0.19
4e	Br	CH₂CH₃		19.40±0.05	14.30±0.12	9.60±0.15	6.00±0.00	21.10±0.19
5a	H	CH₂CH₂CH₃		20.40±0.20	16.00±0.30	9.10±0.10	6.00±0.00	23.80±0.20
5b	CH₃	CH₂CH₂CH₃		21.70±0.21	16.40±0.23	15.30±0.15	6.00±0.00	26.00±0.30
5c	F	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	25.50±0.15
5d	Cl	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	24.40±0.32
5e	Br	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	22.80±0.25
6a	H	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6b	CH₃	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6c	F	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6d	Cl	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6e	Br	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
^a Values indicate the average diameters in mm (of three trials) for the zone of growth inhibition observed after 24 h of incubation at 30 ℃. C.n. C. neoformans ATCC 32264;C.n.C.: C. neoformans clinical strain; C.a.: C. albicans ATCC 10231;E.c.: E. coli ATCC 44752; S.a.:S. aureus ATCC 25923.

表 1 化合物2~6的抑菌圈值 (nm)^a
Table 1. Zone (mm) of growth inhibition of compounds 2~6

为了研究C-4位取代基对化合物抑菌活性的影响, 通过对化合物2a~2e、3a(A)~3e^[10]、4a~4e、5a~5e和6a~6e的抑菌活性比较, 结果显示, 化合物2a~2e(C-4位为H) 对所测试的五种菌株表现出轻微的抑菌活性, 化合物3a(A)~3e^[10] (C-4位为CH₃) 不管是真菌还是细菌都表现出中等的抑菌活性, 化合物4a~4e (C-4位为CH₂CH₃)、5a~5b (C-4位为CH₂CH₂CH₃) 除了对大肠杆菌无活性以外, 对其他菌种都有很高的抑菌活性, 化合物5c~5e (C-4位为CH₂CH₂CH₃) 只对金黄色葡萄球菌有抑制作用且对细菌表现出很好的选择性, 而化合物6a~6e [C-4位为CH (CH₃)₂]对所测试的五种菌株都没有抑菌活性.

另外比较化合物4a~4e、5a~5e对各种菌的抑菌圈直径数值发现, 化合物苯环上的取代基R¹对其抑菌效果也有很大的影响, 当R¹＝CH₃时化合物4b、5b对各种菌 (大肠杆菌除外) 的抑菌圈直径值都最大, 抑菌效果最好, 当R¹＝H、F、Cl、Br时化合物的抑菌活性有所下降, 尤其是Br原子的引入, 使得化合物4e、5e的抑菌圈直径明显减小.

对于具有优良抑菌活性的1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物4b、4c, 本文进一步测试了其最小抑菌浓度 (MIC)、最小杀真菌浓度 (MFC) 和最小杀细菌浓度 (MBC) 值 (表 2和表 3), 由表 2和表 3可知, 化合物4b、4c对标准新生隐球菌的MIC值低于抗真菌药物氟康唑; 对白色念珠菌的MIC和MFC值都远低于氟康唑; 而对金黄色葡萄球菌的MIC和MBC值都高于抗细菌药物环丙沙星, 这充分说明了此类化合物的抑真菌 (白色念珠菌和标准新生隐球菌) 能力高于标准药物氟康唑, 且抑真菌能力大于抑细菌能力.由于其对白色念珠菌有较高的抑菌和杀菌效果, 因此化合物4b、4c可作为优良的抑菌药物候选分子和先导化合物.

表 2 化合物4b、4c和氟康唑的MIC及MFC值 (μg/mL)^a Table 2. MIC and MFC values for compounds 4b, 4c and fluconazole (Mg/mL)

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Fungal strain	4b		4c		Fluconazole
Fungal strain	MIC	MFC	MIC	MFC	MIC	MFC
C.n	80.0	＞128.0	60.0	＞128.0	＞128.0	＞128.0
C.a.	40.0	84.0	40.0	86.0	＞128.0	＞128.0
^aC.n.: C. neoformans ATCC 32264; C.a.: C. albicansATCC 10231.

表 2 化合物4b、4c和氟康唑的MIC及MFC值 (μg/mL)^a
Table 2. MIC and MFC values for compounds 4b, 4c and fluconazole (Mg/mL)

表 3 化合物4b、4c和环丙沙星的MIC及MBC值 (μg/mL)^a Table 3. MIC and MBC values for compounds 4b, 4c and ciprofloxacin (μg/mL)

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Bacterial strain	4b		4c		Ciprofloxacin
Bacterial strain	MIC	MBC	MIC	MBC	MIC	MBC
S.a.	56.0	＞128.0	62.0	＞128.0	28.0	36.0
^a.S.a.: S. aureus ATCC 25923.

表 3 化合物4b、4c和环丙沙星的MIC及MBC值 (μg/mL)^a
Table 3. MIC and MBC values for compounds 4b, 4c and ciprofloxacin (μg/mL)

1.4 1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物2~6的构效关系

本文从目标化合物分子C-2位芳杂环基团 (X)、C-4位烷基 (R²) 链长短及体积和C-8位取代基 (R¹) 的电负性三方面入手, 对其进行了构效关系研究 (表 1).

关于C-2位上芳杂环基团 (X) 对1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物抑菌活性的影响.结果显示, 当目标化合物分子的C-2位被呋喃基取代时 (3k~3o), 化合物对所测试的五种菌株没有任何活性; 被苯基取代时 (3f~3j), 化合物在一定程度上增强了其抑菌活性; 被噻唑基取代时 (2a~2e、3a(A)~3e、4a~4e、5a~5b), 化合物表现出中等到很强的抑菌活性而且抑真菌能力大于抑细菌能力.由此推断, C-2位上取代基X对该类化合物抑菌活性的影响顺序为: 2-噻唑基＞苯基＞呋喃基.

其次, C-4位上烷基 (R²) 链长短及体积对目标化合物分子的抑菌活性也有很大影响.通过化合物2a~2e, 3a(A)~3e, 4a~4e, 5a~5e对五种菌株的抑制作用发现, C-4位上烷基取代可增强化合物的抑菌活性, 尤其是乙基取代, 可使化合物的抑菌活性最强, 但是随着烷基体积的增大, 化合物的抑菌活性也会相应降低, 当被异丙基取代时 (化合物6a~6e), 该类化合物的抑菌活性完全消失, 这可能因为烷基体积增大导致整个化合物分子体积增大, 从而使得该类化合物难以穿过菌种的细胞并与其发生作用, 最终难以抑制菌株的生长^[16].

最后, 本文还研究了C-8位上取代基 (R¹) 的电负性对目标化合物分子抑菌活性的影响.当R¹＝CH₃时, 化合物的抑菌活性最高; 当R¹＝H时, 化合物也表现出很好的抑菌活性, 但是较R¹＝CH₃的化合物而言活性有所下降, 当R¹＝F、Cl、Br等基团时, 化合物的抑菌活性明显降低, 尤其是Br原子的引入, 使得化合物的抑菌活性会急剧下降甚至消失, 主要是因为Br不但是吸电子基团而且体积很大, 从而降低了化合物的抑菌活性^[17].因此我们推断, C-8位上取代基R¹对该类化合物的抑菌活性的影响顺序为: CH₃＞H＞F＞Cl＞Br.

2 结论

设计合成了30个新型1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物2~6, 通过考察该类化合物对真菌新生隐球菌标准株、临床株、白色念珠菌、细菌大肠杆菌和金黄色葡萄球菌等五种菌株的抑菌活性, 发现化合物4a~4e, 5a, 5b除了对大肠杆菌无活性外, 对其他四种菌株均表现出很强的抑菌活性, 其中以化合物4b、4c的活性最强, 且对白色念珠菌的抑菌活性优于标准药物氟康唑, 所以化合物4b、4c可作为抑制真菌的先导化合物, 具有进一步的研究价值.构效关系研究表明, C-2位上的噻唑环可在一定程度上增强该类化合物的抑菌活性; C-4位上的CH₃、CH₂CH₃是该类化合物的活性基团, 但是随着烷基链的增长, 化合物的活性基本消失; 同时C-8位上取代基的电性对该类化合物的抑菌活性也起到决定性作用.这一结论对于发现具有优良抑菌活性的1H-2, 5-二氢-1, 5-苯并二氮杂䓬药物分子有着重要的意义.

3 实验部分

3.1 仪器与试剂

2-噻唑甲醛、2-呋喃甲醛、无取代苯甲醛、取代的邻苯二胺、甲酰乙酸乙酯、乙酰乙酸乙酯、丙酰乙酸乙酯、丁酰乙酸乙酯、异丁酰乙酸乙酯等均未经处理直接使用; 无水乙醇、乙酸乙酯、石油醚等试剂均为国产分析析纯, 使用前都经无水处理; 真菌新生隐球菌标准株 (C. neoformans, ATCC 32264)、临床株 (C. neoformans clinical isolates, ATCC 32264)、白色念珠菌 (C. albicans, ATCC 10231)、革兰氏阴性细菌大肠杆菌 (E. coli, ATCC 44752) 和革兰氏阳性细菌金黄色葡萄球菌 (S. aureus, ATCC 25923); X-4型数字显示显微熔点测定仪、AVIII 500Q型超导核磁共振仪 (TMS为内标)、液相质谱串联质谱、VarioELⅢ型元素分析仪、Vertex70型傅里叶变换红外光谱仪、SKY-2012C型恒温培养箱摇床和722型可见分光光度计.

3.2 目标化合物的合成过程

3.3 抑菌活性测试

3.2.1 中间体N-邻氨芳基取代的β-烯胺酯 (1) 的合成

以1a'为例.在100 mL的反应瓶中依次加入0.54 g (5 mmol) 的无取代邻苯二胺和0.58 g (5 mmol) 的甲酰乙酸乙酯, 无溶剂无催化剂、室温条件下搅拌约1 h, 有固体析出, 抽滤得到粗产品, 用无水乙醇进行重结晶得到白色的纯产品1a', 产率可达到96%以上.同样的方法合成化合物1b'~1y'.

3.2.2 取代的2-芳杂环-3-乙酯基-4-烷基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬类化合物 (2~6) 的合成

以2a为例.将0.51 g (2.5 mmol) 的化合物1a'加入到100 mL的反应瓶中, 用20 mL新蒸的无水乙醇将其溶解后, 磁力搅拌下加入0.28 g (2.5 mmol) 的2-噻唑甲醛, 最后加入催化剂量的可回收利用的非均相催化剂二氧化硅负载的L-扁桃酸 (L-MA-SiO₂), 在冰水浴条件下反应大约2.5 h, 用薄层色谱 (TLC) 检测至原料点基本消失, 反应过程中有大量的固体析出, 抽滤得到粗产品2a.然后用无水乙醇进行重结晶, 得到白色纯品2a, 产率可高达86.5%.同样的方法合成其他目标化合物.

2-(2-噻唑基)-3-乙酯基-4-氢-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(2a):白色固体, 产率86.5%. m.p. 184~186 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 9.38 (m, 1H, NH), 7.59 (d, J＝3.5 Hz, 1H, CH), 7.54~6.61 (m, 6H, C₆H₄, C₃H₂NS), 6.34 (d, J＝5.5 Hz, 1H, CH), 5.68 (d, J＝5.5 Hz, 1H, NH), 4.03 (q, J＝7.0 Hz, 2H, COOCH₂), 1.16 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.39, 167.17, 142.73, 140.91, 137.68, 132.14, 128.71, 123.27, 122.25, 121.17, 119.97, 101.48, 59.53, 57.61, 14.97; IR (KBr) ν: 3308.9 (N-H), 1648.4 (C＝O), 1574.7 (C＝C) cm^-1; MS m/z: 302.1 [M+H]⁺. Anal. calcd for C₁₅H₁₅N₃O₂S: C 59.78, H 5.02, N 13.94; found C 59.79, H 5.03, N 13.92.

2-(2-噻唑基)-3-乙酯基-4-氢-8-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(2b):白色固体, 产率90%. m.p. 188~190 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 9.29 (m, 1H, NH), 7.59 (d, J＝3.5 Hz, 1H, CH), 7.53~6.43 (m, 5H, C₆H₃, C₃H₂NS), 6.17 (d, J＝5.5 Hz, 1H, CH), 5.65 (d, J＝5.5 Hz, 1H, NH), 4.02 (q, J＝7.0 Hz, 2H, COOCH₂), 2.10 (s, 3H, CH₃), 1.15 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.54, 167.19, 142.73, 140.91, 137.49, 132.14, 124.08, 122.32, 121.86, 120.23, 119.95, 101.54, 59.49, 57.68, 20.53, 14.96; IR (KBr) ν: 3276.5 (N-H), 1673.2 (C＝O), 1591.2 (C＝C) cm^-1; MS m/z: 316.1 [M+H]⁺. Anal. calcd for C₁₆H₁₇N₃O₂S: C 60.93, H 5.43, N 13.32; found C 60.95, H 5.41, N 13.32.

2-(2-噻唑基)-3-乙酯基-4-氢-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(2c):白色固体, 产率88%. m.p. 164~166 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 9.41 (m, 1H, NH), 7.62 (d, J＝3.0 Hz, 1H, CH), 7.60~6.50 (m, 5H, C₆H₃, C₃H₂NS), 6.34 (d, J＝5.5 Hz, 1H, CH), 5.66 (d, J＝5.5 Hz, 1H, NH), 4.03 (q, J＝7.0 Hz, 2H, COOCH₂), 1.16 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.98, 167.03, 159.28, 157.39, 142.81 (d, J_C-F＝7.25 Hz), 139.39 (d, J_C-F＝9.50 Hz), 128.69 (d, J_C-F＝2.13 Hz), 121.03 (d, J_C-F＝9.88 Hz), 120.04 (d, J_C-F＝8.63 Hz), 107.91, 107.50 (d, J_C-F＝22.75 Hz), 101.02, 59.57, 57.37, 14.95; IR (KBr) ν: 3308.9 (N-H), 1648.4 (C＝O), 1582.9 (C＝C) cm^-1; HRMS calcd for C₁₅H₁₅FN₃O₂S [M+H]⁺320.0864, found 320.0860.

2-(2-噻唑基)-3-乙酯基-4-氢-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(2d):白色固体, 产率87%. m.p. 182~184 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 9.44 (m, 1H, NH), 7.62 (d, J＝3.0 Hz, 1H, CH), 7.52~6.60 (m, 5H, C₆H₃, C₃H₂NS), 6.49 (d, J＝5.5 Hz, 1H, CH), 5.68 (d, J＝5.5 Hz, 1H, NH), 4.04 (q, J＝7.0 Hz, 2H, COOCH₂), 1.16 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.74, 166.99, 144.71, 142.93, 139.17, 133.51, 126.47, 121.30, 121.02, 120.71, 120.17, 101.87, 59.68, 57.31, 14.93; IR (KBr) ν: 3268.3 (N-H), 1681.5 (C＝O), 1582.9 (C＝C) cm^-1; MS m/z: 336.1 [M+H]⁺. Anal. calcd for C₁₅H₁₄ClN₃O₂S: C 53.65, H 4.20, N 12.51; found C 53.67, H 4.21, N 12.52.

2-(2-噻唑基)-3-乙酯基-4-氢-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(2e):白色固体, 产率88.2%. m.p. 152~154 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 9.43 (m, 1H, NH), 7.60 (d, J＝3.0 Hz, 1H, CH), 7.51~6.57 (m, 5H, C₆H₃, C₃H₂NS), 6.54 (d, J＝5.5 Hz, 1H, CH), 5.67 (d, J＝5.5 Hz, 1H, NH), 4.04 (q, J＝7.0 Hz, 2H, COOCH₂), 1.16 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.74, 166.98, 142.84, 140.42, 137.22, 133.84, 125.49, 123.85, 121.82, 120.13, 112.07, 102.60, 59.73, 57.29, 14.91; IR (KBr) ν: 3301.3 (N-H), 1681.4 (C＝O), 1607.7 (C＝C) cm^-1; MS m/z: 380.0 [M+H]⁺. Anal. calcd for C₁₅H₁₄BrN₃O₂S: C 47.38, H 3.71, N 11.05; found C 47.36, H 3.70, N 11.07.

2-(2-噻唑基)-3-乙酯基-4-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3a):白色固体, 产率83.8%. m.p. 166~168 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 7.64~6.58 (m, 5H, C₆H₃, C₃H₂NS), 6.11 (s, 1H, NH), 6.15 (s, 1H, CH), 4.86 (s, 1H, NH), 4.13 (q, J＝7.0 Hz, 2H, COOCH₂), 2.60 (s, 3H, CH₃), 1.18 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz)δ: 175.08, 167.59, 152.17, 142.97, 136.71, 131.37, 123.53, 121.73, 121.64, 119.63, 118.59, 100.83, 59.85, 58.88, 25.38, 14.41; IR (KBr) ν: 3321.2 (N-H), 1680.3 (C＝O), 1632.8 (C＝C) cm^-1; MS m/z: 316.1 [M+H]⁺. Anal. calcd for C₁₆H₁₇N₃O₂S: C 60.93, H 5.43, N 13.32; found C 60.95, H 5.44, N 13.34.

2-(2-噻唑基)-3-乙酯基-4, 8-二甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3b):白色固体, 产率91.8%. m.p. 162~164 ℃; ¹H NMR (CDCl₃, 500 MHz)δ: 7.65~6.40 (m, 5H, C₆H₃, C₃H₂NS), 6.16 (s, 1H, NH), 6.12 (s, 1H, CH), 4.81 (s, 1H, NH), 4.12 (q, J＝7.0 Hz, 2H, COOCH₂), 2.57 (s, 3H, CH₃), 2.12 (s, 3H, CH₃), 1.17 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 175.34, 167.71, 152.63, 142.89, 136.56, 133.20, 128.84, 122.36, 121.86, 119.65, 118.65, 100.16, 59.73, 58.68, 25.21, 20.44, 14.44; IR (KBr) ν: 3317.3 (N-H), 1674.7 (C＝O), 1621.4 (C＝C) cm^-1; MS m/z: 330.1 [M+H]⁺. Anal. calcd for C₁₇H₁₉N₃O₂S: C 61.98, H 5.81, N 12.76; found C 61.96, H 5.83, N 12.78.

2-(2-噻唑基)-3-乙酯基-4-甲基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3c):白色固体, 产率89.7%. m.p. 168~169 ℃; ¹H NMR (CDCl₃, 500 MHz)δ: 7.66~6.33 (m, 5H, C₆H₃, C₄H₂NS), 6.16 (s, 1H, CH), 6.12 (d, J＝6.5 Hz, 1H, NH), 5.01 (d, J＝6.0 Hz, 1H, NH), 4.12 (q, J＝5.0 Hz, 2H, COOCH₂), 2.57 (s, 3H, CH₃), 1.17 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.76, 167.55, 153.66, 152.86, 142.89 (d, J_C-F＝10.13 Hz), 139.84 (d, J_C-F＝9.88 Hz), 128.13 (d, J_C-F＝1.63 Hz), 121.39 (d, J_C-F＝9.50 Hz), 120.18 (d, J_C-F＝15.50 Hz), 106.61, 106.42 (d, J_C-F＝13.13 Hz), 100.61, 59.36, 57.39, 23.84, 14.87; IR (KBr) ν: 3356.2 (N-H), 1670.5 (C＝O), 1616.1 (C＝C) cm^-1; HRMS calcd for C₁₆H₁₇FN₃O₂S [M+H]⁺ 334.1020, found 334.1017.

2-(2-噻唑基)-3-乙酯基-4-甲基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3d):黄色固体, 产率88.9%. m.p. 168~170 ℃; ¹H NMR (CDCl₃, 500 MHz)δ: 7.67~6.57 (m, 5H, C₆H₃, C₃H₂NS), 6.22 (s, 1H, NH), 6.13 (s, 1H, CH), 5.81 (s, 1H, NH), 4.10 (q, J＝7.0 Hz, 2H, COOCH₂), 2.57 (s, 3H, CH₃), 1.17 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.46, 167.52, 153.49, 142.96, 139.60, 130.61, 126.13, 121.62, 120.26, 119.77, 119.54, 101.48, 59.48, 57.29, 23.74, 14.86; IR (KBr) ν: 3317.7 (N-H), 1664.9 (C＝O), 1607.2 (C＝C) cm^-1; MS m/z: 350.1 [M+H]⁺. Anal. calcd for C₁₆H₁₆ClN₃O₂S: C 54.93, H 4.61, N 12.01; found C 54.95, H 4.62, N 12.03.

2-(2-噻唑基)-3-乙酯基-4-甲基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3e):黄色固体, 产率89.4%. m.p. 168~170 ℃; ¹H NMR (CDCl₃, 500 MHz)δ: 7.67~6.48 (m, 5H, C₆H₃, C₄H₂NS), 6.12 (s, 1H, CH), 6.11 (s, 1H, NH), 4.91 (s, 1H, NH), 4.13 (q, J＝7.0 Hz, 2H, COOCH₂), 2.57 (s, 3H, CH₃), 1.18 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125 MHz) δ: 174.44, 167.34, 151.44, 143.04, 135.95, 132.65, 126.00, 122.81, 121.99, 118.64, 113.34, 102.27, 59.96, 58.70, 24.90, 14.30; IR (KBr) ν: 3340.3 (N-H), 1670.5 (C＝O), 1625.7 (C＝C) cm^-1; MS m/z: 394.0 [M+H]⁺. Anal. calcd for C₁₆H₁₆BrN₃O₂S: C 48.74, H 4.09, N 10.66; found C 48.76, H 4.10, N 10.68.

2-苯基-3-乙酯基-4-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3f):黄色固体, 产率91%. m.p. 130~132 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.28 (s, 1H, NH), 7.12~6.47 (m, 9H, C₆H₄, C₆H₅), 6.19 (d, J＝6.5 Hz, 1H, NH), 5.62 (d, J＝6.5 Hz, 1H, CH), 4.03 (q, J＝7.0 Hz, 2H, CH₂), 2.45 (s, 3H, CH₃), 1.12 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.01, 152.67, 144.56, 140.59, 138.43, 136.53, 136.30, 131.09, 132.14, 130.09, 122.62, 120.45, 120.08, 119.46, 100.35, 59.17, 49.07, 23.76, 14.89; IR (KBr) ν: 3345.3 (N-H), 1667.5 (C＝O), 1630.1 (C＝C) cm^-1; MS m/z: 309.2 [M+H]⁺. Anal. calcd for C₁₉H₂₀N₂O₂: C 74.00, H 6.54, N 9.08; found C 74.02, H 6.52, N 9.11.

2-苯基-3-乙酯基-4, 8-二甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3g):白色固体, 产率91%. m.p. 132~134 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.23 (s, 1H, NH), 7.13~6.30 (m, 8H, C₆H₃, C₆H₅), 6.13 (d, J＝6.5 Hz, 1H, NH), 5.60 (d, J＝6.5 Hz, 1H, CH), 4.23 (q, J＝7.0 Hz, 2H, CH₂), 2.46 (s, 3H, CH₃), 1.29 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.18, 152.49, 145.21, 138.45, 131.96, 131.13, 129.58, 128.21, 127.83, 126.24, 120.55, 120.44, 119.96, 100.46, 59.25, 59.02, 49.08, 23.89, 20.64, 14.92; IR (KBr) ν: 3306.3 (N-H), 1673.2 (C＝O), 1630.5 (C＝C) cm^-1; MS m/z: 323.2 [M+H]⁺. Anal. calcd for C₂₀H₂₂N₂O₂: C 74.51, H 6.88, N 8.69; found C 74.48, H 6.87, N 8.72.

2-苯基-3-乙酯基-4-甲基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3h):棕色固体, 产率89%. m.p. 131~132 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.32 (s, 1H, NH), 7.17~6.32 (m, 8H, C₆H₃, C₆H₅), 6.29 (d, J＝6.5 Hz, 1H, NH), 5.60 (d, J＝6.5 Hz, 1H, CH), 3.94 (q, J＝7.0 Hz, 2H, CH₂), 2.45 (s, 3H, CH₃), 1.09 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.09, 157.06, 152.41, 144.73, 140.35 (d, J_C-F＝9.88 Hz), 128.70, 128.26 (d, J_C-F＝8.13 Hz), 127.80, 127.72, 126.41 (d, J_C-F＝5.50 Hz), 120.84 (d, J_C-F＝9.75 Hz), 106.09 (d, J_C-F＝17.13 Hz), 105.92 (d, J_C-F＝15.63 Hz), 100.62, 59.26, 59.15, 49.07, 23.74, 14.88; IR (KBr) ν: 3317.2 (N-H), 1648.4 (C＝O), 1542.3 (C＝C) cm^-1; HRMS calcd for C₁₉H₂₀FN₂O₂ [M+H]⁺ 327.1503, found 327.1500.

2-苯基-3-乙酯基-4-甲基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3i):棕色固体, 产率89%. m.p. 133~134 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.39 (s, 1H, NH), 7.17~6.52 (m, 8H, C₆H₃, C₆H₅), 6.48 (d, J＝7.5 Hz, 1H, NH), 5.61 (d, J＝6.5 Hz, 1H, CH), 3.95 (q, J＝7.5 Hz, 2H, CH₂), 2.46 (s, 3H, CH₃), 1.09 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.07, 152.19, 144.54, 137.75, 131.22, 128.42, 127.69, 126.55, 125.66, 121.14, 119.30, 119.16, 118.95, 101.51, 59.27, 59.23, 49.08, 23.63, 14.86; IR (KBr) ν: 3301.4 (N-H), 1624.3 (C＝O), 1534.1 (C＝C) cm^-1; MS m/z: 343.1 [M+H]⁺. Anal. calcd for C₁₉H₁₉ClN₂O₂: C 66.59, H 5.57, N 8.15; found C 66.57, H 5.59, N 8.17.

2-苯基-3-乙酯基-4-甲基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3j):棕色固体, 产率88%. m.p. 134~135 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.39 (s, 1H, NH), 7.17~6.64 (m, 8H, C₆H₃, C₆H₅), 6.48 (d, J＝6.5 Hz, 1H, NH), 5.59 (d, J＝6.5 Hz, 1H, CH), 3.96 (q, J＝7.0 Hz, 2H, CH₂), 2.45 (s, 3H, CH₃), 1.11 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.05, 152.19, 144.51, 140.43, 138.14, 133.78, 131.62, 128.43, 127.69, 124.53, 122.15, 121.95, 121.71, 121.51, 101.60, 59.24, 49.08, 23.63, 14.86; IR (KBr) ν: 3303.2 (N-H), 1682.4 (C＝O), 1627.1 (C＝C) cm^-1; MS m/z: 387.1 [M+H]⁺. Anal. calcd for C₁₉H₁₉BrN₂O₂: C 58.93, H 4.95, N 7.23; found C 58.89, H 4.90, N 7.28.

2-(2-呋喃基)-3-乙酯基-4-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3k):白色固体, 产率90.1%. m.p. 110~112 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.34 (s, 1H, NH), 7.34~6.10 (m, 7H, C₆H₄, C₄H₃O), 5.74 (d, J＝3.0 Hz, 1H, NH), 5.59 (d, J＝6.5 Hz, 1H, CH), 3.98 (q, J＝7.0 Hz, 2H, COOCH₂), 2.42 (s, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 167.80, 157.19, 152.91, 141.96, 138.63, 131.91, 122.55, 120.15, 120.05, 120.03, 110.22, 106.63, 99.77, 59.19, 53.82, 23.84, 14.92; IR (KBr) ν: 3350.2 (N-H), 1632.6 (C＝O), 1542.3 (C＝C) cm^-1; MS m/z: 299.1 [M+H]⁺. Anal. calcd for C₁₇H₁₈N₂O₃: C 68.47, H 6.08, N 9.35; found C 68.44, H 6.08, N 9.39.

2-(2-呋喃基)-3-乙酯基-4, 8-二甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3l):白色固体, 产率92%. m.p. 113~115 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.29 (s, 1H, NH), 7.35~6.05 (m, 6H, C₆H₃, C₄H₃O), 5.75 (d, J＝3.0 Hz, 1H, NH), 5.59 (d, J＝6.0 Hz, 1H, CH), 3.97 (q, J＝7.0 Hz, 2H, COOCH₂), 2.42 (s, 3H, CH₃), 2.05 (s, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 167.79, 157.31, 152.97, 141.93, 138.43, 131.31, 129.35, 120.69, 120.31, 120.06, 110.27, 106.55, 99.30, 59.11, 53.54, 23.91, 20.65, 14.95; IR (KBr) ν: 3350.2 (N-H), 1624.3(C＝O) cm^-1, 1582.9 (C＝C); MS m/z: 313.1 [M+H]⁺. Anal. calcd for C₁₈H₂₀N₂O₃: C 69.20, H 6.48, N 8.94; found C 69.21, H 6.45, N 8.97.

2-(2-呋喃基)-3-乙酯基-4-甲基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3m):白色固体, 产率89%. m.p. 117~118 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.39 (s, 1H, NH), 7.39~6.13 (m, 6H, C₆H₃, C₄H₃O), 5.79 (d, J＝3.0 Hz, 1H, NH), 5.59 (d, J＝6.5 Hz, 1H, CH), 3.99 (q, J＝7.0 Hz, 2H, COOCH₂), 2.42 (s, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 167.68, 157.13, 156.82, 152.88, 142.21 (d, J_C-F＝6.00 Hz), 140.29 (d, J_C-F＝9.75 Hz), 128.52 (d, J_C-F＝1.38 Hz), 120.99 (d, J_C-F＝9.63 Hz), 110.29, 106.79, 106.21 (d, J_C-F＝22.50 Hz), 105.84 (d, J_C-F＝23.88 Hz), 99.32, 59.23, 53.58, 23.77, 14.91; IR (KBr) ν: 3350.2 (N-H), 1624.3 (C＝O), 1534.1 (C＝C) cm^-1; HRMS calcd for C₁₇H₁₇FN₂O₃ [M+H]⁺ 317.1296, found 317.1298.

2-(2-呋喃基)-3-乙酯基-4-甲基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3n):白色固体, 产率88.7%. m.p. 120~122 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.45 (s, 1H, NH), 7.40~6.14 (m, 6H, C₆H₃, C₄H₃O), 5.80 (d, J＝3.0 Hz, 1H, NH), 5.58 (d, J＝6.5 Hz, 1H, CH), 3.99 (q, J＝7.0 Hz, 2H, COOCH₂), 2.42 (s, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 167.64, 156.56, 152.73, 142.35, 140.09, 131.03, 125.80, 121.28, 119.57, 118.96, 110.33, 106.84, 100.21, 59.35, 53.53, 23.67, 14.89; IR (KBr) ν: 3375.1 (N-H), 1632.6 (C＝O), 1582.9 (C＝C) cm^-1; MS m/z: 333.1 [M+H]⁺. Anal. calcd for C₁₇H₁₇ClN₂O₃: C 61.38, H 5.17, N 8.46; found C 61.36, H 5.15, N 8.42.

2-(2-呋喃基)-3-乙酯基-4-甲基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(3o):白色固体, 产率90%. m.p. 138~140 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.45 (s, 1H, NH), 7.41~6.15 (m, 6H, C₆H₃, C₄H₃O), 5.80 (d, J＝3.0 Hz, 1H, NH), 5.59 (d, J＝6.5 Hz, 1H, CH), 3.96 (q, J＝7.0 Hz, 2H, COOCH₂), 2.42 (s, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 167.64, 156.58, 152.73, 142.35, 140.38, 131.04, 122.41, 121.18, 119.57, 118.96, 110.29, 106.83, 100.22, 59.43, 53.57, 23.72, 14.90; IR (KBr) ν: 3358.5 (N-H), 1624.3 (C＝O), 1542.3 (C＝C) cm^-1; MS m/z: 377.0 [M+H]⁺. Anal. calcd for C₁₇H₁₇BrN₂O₃: C 54.16, H 4.58, N 7.50; found C 54.13, H 4.54, N 7.43.

2-(2-噻唑基)-3-乙酯基-4-乙基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(4a):白色固体, 产率85.2%. m.p. 166~168 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.46 (s, 1H, NH), 7.57~6.55 (m, 6H, C₆H₄, C₃H₂NS), 6.36 (d, J＝6.5 Hz, 1H, CH), 5.86 (d, J＝6.5 Hz, 1H, NH), 4.01 (q, J＝7.0 Hz, 2H, COOCH₂), 2.82 (q, J＝7.0 Hz, 2H, CH₂), 1.22 (t, J＝7.0 Hz, 3H, CH₃), 1.12 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 175.15, 167.25, 159.29, 142.82, 138.06, 131.70, 122.76, 120.65, 120.36, 120.18, 119.99, 100.35, 59.36, 57.63, 29.41, 14.83, 14.59; IR (KBr) ν: 3259.9 (N-H), 1664.9 (C＝O), 1624.3 (C＝C) cm^-1; MS m/z: 330.1 [M+H]⁺. Anal. calcd for C₁₇H₁₉N₃-O₂S: C 61.98, H 5.81, N 12.76; found C 61.97, H 5.83, N 12.74.

2-(2-噻唑基)-3-乙酯基-4-乙基-8-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(4b):白色固体, 产率86.4%. m.p. 142~144 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.42 (s, 1H, NH), 7.58~6.37 (m, 5H, C₆H₃, C₃H₂NS), 6.29 (d, J＝7.0 Hz, 1H, CH), 5.85 (d, J＝6.5 Hz, 1H, NH), 3.99 (q, J＝7.0 Hz, 2H, COOCH₂), 2.81 (q, J＝7.0 Hz, 2H, CH₂), 2.05(s, 3H, CH₃), 1.21 (t, J＝7.5 Hz, 3H, CH₃), 1.12 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 175.23, 167.24, 159.35, 142.81, 137.87, 131.54, 129.14, 120.87, 120.74, 120.39, 120.03, 99.91, 59.28, 57.41, 29.43, 20.66, 14.85, 14.58; IR (KBr) ν: 3268.3 (N-H), 1664.9 (C＝O), 1616.0 (C＝C) cm^-1; MS m/z: 344.1 [M+H]⁺. Anal. calcd for C₁₈H₂₁N₃O₂S: C 62.95, H 6.16, N 12.23; found C 62.94, H 6.14, N 12.25.

2-(2-噻唑基)-3-乙酯基-4-乙基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(4c):黄色固体, 产率85%. m.p. 146~148 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.55 (s, 1H, NH), 7.62~6.72 (m, 5H, C₆H₃, C₃H₂NS), 6.68 (d, J＝7.0 Hz, 1H, CH), 5.84 (d, J＝6.5 Hz, 1H, NH), 4.02 (q, J＝7.0 Hz, 2H, COOCH₂), 2.80 (q, J＝7.0 Hz, 2H, CH₂), 1.21 (t, J＝7.0 Hz, 3H, CH₃), 1.13 (t, J＝7.0 HZ, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.82, 167.14, 159.36, 157.23, 142.91 (d, J_C-F＝6.50 Hz), 139.81 (d, J_C-F＝10.00 Hz), 128.21 (d, J_C-F＝2.25 Hz), 121.37 (d, J_C-F＝9.63 Hz), 120.05 (d, J_C-F＝17.13 Hz), 106.46, 106.27 (d, J_C-F＝14.00 Hz), 100.17, 59.42, 57.29, 29.36, 14.81, 14.51; IR (KBr) ν: 3259.9 (N-H), 1664.9 (C＝O), 1624.3 (C＝C) cm^-1; HRMS calcd for C₁₇H₁₈FN₃O₂S [M+H]⁺ 348.1177, found 348.1180.

2-(2-噻唑基)-3-乙酯基-4-乙基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(4d):黄色固体, 产率84.8%. m.p. 148~150 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.54 (s, 1H, NH), 7.61~6.65 (m, 5H, C₆H₃, C₃H₂NS), 6.56 (d, J＝6.5 Hz, 1H, CH), 5.85 (d, J＝6.5 Hz, 1H, NH), 4.02 (q, J＝7.0 Hz, 2H, COOCH₂), 2.80 (q, J＝7.0 Hz, 2H, CH₂), 1.21 (t, J＝7.0 Hz, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.54, 167.13, 159.18, 143.02, 139.59, 130.72, 126.05, 121.61, 120.25, 119.66, 119.41, 101.07, 59.54, 57.24, 29.31, 14.79, 14.48; IR (KBr) ν: 3268.3 (N-H), 1664.9 (C＝O), 1632.6 (C＝C) cm^-1; MS m/z: 364.1 [M+H]⁺. Anal. calcd for C₁₇H₁₈ClN₃O₂S: C 56.12, H 4.99, N 11.55; found C 56.14, H 4.98, N 11.56.

2-(2-噻唑基)-3-乙酯基-4-乙基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(4e):黄色固体, 产率83.9%. m.p. 150~152 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.55 (s, 1H, NH), 7.62~6.72 (m, 5H, C₆H₃, C₃H₂NS), 6.68 (d, J＝7.0 Hz, 1H, CH), 5.84 (d, J＝6.5 Hz, 1H, NH), 4.02 (q, J＝7.0 Hz, 2H, COOCH₂), 2.80 (q, J＝7.0 Hz, 2H, CH₂), 1.21 (t, J＝7.0 Hz, 3H, CH₃), 1.13 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.47, 167.12, 159.19, 143.03, 139.89, 131.12, 122.50, 122.21, 121.96, 120.27, 114.01, 101.17, 59.55, 57.24, 29.30, 14.79, 14.47; IR (KBr) ν: 3325.4 (N-H), 1640.1 (C＝O), 1599.5 (C＝C) cm^-1; MS m/z: 408.0 [M+H]⁺. Anal. calcd for C₁₇H₁₈BrN₃O₂S: C 50.01, H 4.44, N 10.29; found C 50.02, H 4.46, N 10.27.

2-(2-噻唑基)-3-乙酯基-4-丙基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(5a):白色固体, 产率83.7%. m.p. 146~148 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.44 (s, 1H, NH), 7.57~6.57 (m, 6H, C₆H₄, C₃H₂NS), 6.36 (d, J＝6.5 Hz, 1H, CH), 5.87 (d, J＝6.5 Hz, 1H, NH), 4.00 (q, J＝7.0 Hz, 2H, COOCH₂), 2.79 (t, J＝5.5 Hz, 2H, CH₂), 1.62~1.67 (m, 2H, CH₂), 1.12 (t, J＝7.0 Hz, 3H, CH₃), 0.98 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 175.19, 167.37, 157.96, 142.79, 138.11, 131.69, 122.75, 120.67, 120.36, 120.19, 119.96, 100.61, 59.37, 57.76, 37.92, 23.22, 14.84, 14.44; IR (KBr) ν: 3251.7 (N-H), 1681.4 (C＝O), 1607.8 (C＝C) cm^-1; MS m/z: 344.1 [M+H]⁺. Anal. calcd for C₁₈H₂₁N₃O₂S: C 62.95, H 6.16, N 12.23; found C 62.93, H 6.17, N 12.20.

2-(2-噻唑基)-3-乙酯基-4-丙基-8-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(5b):白色固体, 产率85.6%. m.p. 148~150 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.44 (s, 1H, NH), 7.57~6.57 (m, 6H, C₆H₄, C₃H₂NS), 6.36 (d, J＝6.5 Hz, 1H, CH), 5.87 (d, J＝6.5 HZ, 1H, NH), 4.00 (q, J＝7.0 Hz, 2H, COOCH₂), 2.75 (t, J＝6.0 Hz, 2H, CH₂), 2.04 (s, 3H, CH₃), 1.63~1.69 (m, 2H, CH₂), 1.12 (t, J＝7.0 Hz, 3H, CH₃), 0.97 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 175.27, 167.37, 158.02, 142.78, 137.91, 131.54, 129.13, 120.88, 120.75, 120.39, 119.99, 100.18, 59.29, 57.53, 37.96, 23.20, 20.65, 14.86, 14.43; IR (KBr) ν: 3235.9 (N-H), 1656.6 (C＝O), 1624.3 (C＝C) cm^-1; MS m/z: 358.2 [M+H]⁺. Anal. calcd for C₁₉H₂₃-N₃O₂S: C 63.84, H 6.49, N 11.75; found C 63.85, H 6.47, N 11.75.

2-(2-噻唑基)-3-乙酯基-4-丙基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(5c):黄色固体, 产率82.8%. m.p. 134~136 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.47 (s, 1H, NH), 7.60~6.38 (m, 5H, C₆H₃, C₃H₂NS), 6.65 (d, J＝6.5 Hz, 1H, CH), 5.85 (d, J＝6.5 Hz, 1H, NH), 4.01 (q, J＝7.0 Hz, 2H, COOCH₂), 2.72 (t, J＝6.5 Hz, 2H, CH₂), 1.62~1.69 (m, 2H, CH₂), 1.13 (t, J＝7.0 Hz, 3H, CH₃), 0.97 (t, J＝7.5 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.86, 167.27, 159.13, 157.24, 142.92 (d, J_C-F＝6.50 Hz), 139.86 (d, J_C-F＝9.88 Hz), 128.21 (d, J_C-F＝1.75 Hz), 121.37 (d, J_C-F＝9.37 Hz), 120.08 (d, J_C-F＝7.13 Hz), 106.48, 106.29 (d, J_C-F＝11.75 Hz), 100.44, 59.42, 57.42, 37.88, 23.15, 14.82, 14.41; IR (KBr) ν: 3251.7 (N-H), 1664.9 (C＝O), 1624.3 (C＝C) cm^-1; HRMS calcd for C₁₈H₂₀FN₃O₂S [M+H]⁺ 362.1333, found 362.1331.

2-(2-噻唑基)-3-乙酯基-4-丙基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(5d):棕色固体, 产率83%. m.p. 146~148 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.52 (s, 1H, NH), 7.61~6.65 (m, 5H, C₆H₃, C₃H₂NS), 6.59 (d, J＝6.5 Hz, 1H, CH), 5.86 (d, J＝6.5 Hz, 1H, NH), 4.02 (q, J＝7.0 Hz, 2H, COOCH₂), 2.75 (t, J＝6.0 Hz, 2H, CH₂), 1.64~1.69 (m, 2H, CH₂), 1.13 (t, J＝7.0 Hz, 3H, CH₃), 0.97 (t, J＝7.5 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.56, 167.25, 157.86, 142.99, 139.64, 130.69, 126.06, 121.61, 120.21, 119.67, 119.42, 101.34, 59.55, 57.35, 37.79, 23.14, 14.80, 14.40; IR (KBr) ν: 3366.7 (N-H), 1640.1 (C＝O), 1599.5 (C＝C) cm^-1; MS m/z: 378.1 [M+H]⁺. Anal. calcd for C₁₈H₂₀ClN₃O₂S: C 57.21, H 5.33, N 11.12; found C 57.22, H 5.35, N 11.14.

2-(2-噻唑基)-3-乙酯基-4-丙基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(5e):棕色固体, 产率84.2%. m.p. 146~148 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 8.52 (s, 1H, NH), 7.62~6.70 (m, 5H, C₆H₃, C₃H₂NS), 6.67 (d, J＝7.0 Hz, 1H, CH), 5.85 (d, J＝6.5 Hz, 1H, NH), 4.01 (q, J＝7.0 Hz, 2H, COOCH₂), 2.75 (t, J＝6.5 Hz, 2H, CH₂), 1.63~1.70 (m, 2H, CH₂), 1.13 (t, J＝7.0 Hz, 3H, CH₃), 0.97 (t, J＝7.0 Hz, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 174.50, 167.25, 157.86, 142.99, 139.94, 131.09, 129.13, 122.52, 122.22, 121.96, 120.22, 101.46, 59.56, 57.36, 37.79, 23.12, 14.80, 14.39; IR (KBr) ν: 3333.6 (N-H), 1640.1 (C＝O), 1599.4 (C＝C) cm^-1; MS m/z: 422.3 [M+H]⁺. Anal. calcd for C₁₈H₂₀BrN₃O₂S: C 51.19, H 4.77, N 9.95; found C 51.17, H 4.76, N 9.97.

2-(2-噻唑基)-3-乙酯基-4-异丙基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(6a):白色固体, 产率82.1%. m.p. 138~140 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 11.25 (s, 1H, NH), 7.74~6.80 (m, 6H, C₆H₄, C₃H₂NS), 6.68 (d, J＝7.0 Hz, 1H, CH), 5.49 (m, 1H, CH), 4.98 (d, J＝4.5 Hz, 1H, NH), 3.92 (q, J＝7.0 Hz, 2H, COOCH₂), 1.39 (s, 3H, CH₃), 0.96 (t, J＝7.0 Hz, 3H, CH₃), 0.73 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.98, 168.32, 166.76, 142.86, 140.64, 133.34, 132.99, 127.98, 120.83, 118.50, 118.27, 86.72, 61.02, 60.08, 53.55, 24.68, 23.56, 14.18; IR (KBr) ν: 3382.6 (N-H), 1713.8 (C＝O), 1640.1 (C＝C) cm^-1; MS m/z: 344.4 [M+H]⁺. Anal. calcd for C₁₈H₂₁N₃-O₂S: C 62.95, H 6.16, N 12.23; found C 62.96, H 6.17, N 12.24.

2-(2-噻唑基)-3-乙酯基-4-异丙基-8-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(6b):白色固体, 产率84.3%. m.p. 136~138 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 11.24 (s, 1H, NH), 7.72~6.59 (m, 5H, C₆H₃, C₃H₂NS), 6.49 (d, J＝8.0 Hz, 1H, CH), 5.42 (m, 1H, CH), 4.98 (d, J＝4.0 Hz, 1H, NH), 3.94 (q, J＝7.0 Hz, 2H, COOCH₂), 2.20 (s, 3H, CH₃), 1.38 (s, 3H, CH₃), 0.99 (t, J＝7.0 Hz, 3H, CH₃), 0.74 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.82, 168.32, 164.98, 142.70, 140.46, 137.35, 133.79, 130.54, 120.84, 119.25, 118.38, 86.76, 60.98, 58.99, 53.68, 24.65, 23.59, 21.14, 14.25; IR (KBr) ν: 3399.1 (N-H), 1713.8 (C＝O), 1640.1 (C＝C) cm^-1; MS m/z: 358.5 [M+H]⁺. Anal. calcd for C₁₉H₂₃N₃O₂S: C 63.80, H 6.49, N 11.73; found C 63.84, H 6.48, N 11.76.

2-(2-噻唑基)-3-乙酯基-4-异丙基-8-氟-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(6c):黄色固体, 产率82%. m.p. 140~142 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 11.28 (s, 1H, NH), 7.74~7.13 (m, 5H, C₆H₃, C₃H₂NS), 6.46 (d, J＝6.5 HZ, 1H, CH), 5.43~5.45 (m, 1H, CH), 5.03 (d, J＝4.0 Hz, 1H, NH), 3.95 (q, J＝7.0 Hz, 2H, COOCH₂), 1.39 (s, 3H, CH₃), 1.00 (t, J＝7.0 Hz, 3H, CH₃), 0.71 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.11, 168.13, 165.49, 162.69, 160.67, 142.49 (d, J_C-F＝11.75 Hz), 135.93 (d, J_C-F＝10.38 Hz), 129.39 (d, J_C-F＝2.13 Hz), 120.98 (d, J_C-F＝14.50 Hz), 104.78 (d, J_C-F＝22.25 Hz), 103.28 (d, J_C-F＝25.38 Hz), 86.80, 61.06, 58.48, 53.96, 24.63, 23.56, 14.23; IR (KBr) ν: 3399.1 (N-H), 1706.3 (C＝O), 1648.4 (C＝C) cm^-1; HRMS calcd for C₁₈H₂₀FN₃O₂S [M+H]⁺ 362.1333, found 362.1327.

2-(2-噻唑基)-3-乙酯基-4-异丙基-8-氯-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(6d):棕色固体, 产率81.8%. m.p. 132~134 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 11.30 (s, 1H, NH), 7.74~6.85 (m, 5H, C₆H₃, C₃H₂NS), 6.68 (d, J＝7.0 Hz, 1H, CH), 5.45~5.47 (m, 1H, CH), 5.04 (d, J＝4.5 Hz, 1H, NH), 3.96 (q, J＝7.0 Hz, 2H, COOCH₂), 1.39 (s, 3H, CH₃), 0.99 (t, J＝7.0 Hz, 3H, CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.82, 168.32, 166.99, 143.03, 139.78, 134.25, 131.95, 121.11, 121.02, 120.68, 120.07, 74.57, 61.39, 57.28, 52.22, 31.15, 28.50, 14.20; IR (KBr) ν: 3382.6 (N-H), 1706.3 (C＝O), 1640.1 (C＝C) cm^-1; MS m/z: 378.8 [M+H]⁺. Anal. calcd for C₁₈H₂₀Cl-N₃O₂S: C 57.21, H 5.33, N 11.12; found C 57.20, H 5.34, N 11.14.

2-(2-噻唑基)-3-乙酯基-4-异丙基-8-溴-1H-2, 5-二氢-1, 5-苯并二氮杂䓬(6e):棕色固体, 产率83.1%. m.p. 138~140 ℃; ¹H NMR (DMSO-d₆, 500 MHz)δ: 11.31 (s, 1H, NH), 7.74~7.00 (m, 5H, C₆H₃, C₃H₂NS), 6.81 (d, J＝6.5 HZ, 1H, CH), 5.45~5.47 (m, 1H, CH), 5.03 (d, J＝4.5 Hz, 1H, NH), 3.96 (q, J＝7.0 Hz, 2H, COOCH₂), 1.39 (s, 3H, CH₃), 1.00 (t, J＝7.0 Hz, 3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 173.08, 167.95, 167.49, 142.99, 142.36, 135.60, 131.89, 121.01, 120.63, 120.39, 119.96, 86.84, 61.13, 58.85, 54.04, 24.65, 23.47, 14.22; IR (KBr) ν: 3375.1 (N-H), 1730.4 (C＝O), 1648.4 (C＝C) cm^-1; MS m/z: 422.3 [M+H]⁺. Anal. calcd for C₁₈H₂₀Br-N₃O₂S: C 51.19, H 4.77, N 9.95; found C 51.18, H 4.75, N 9.93.

3.3.2 最小抑菌浓度和最小杀菌浓度的测定

MIC的测定:将目标化合物和氟康唑 (环丙沙星) 分别用无菌酵母浸出粉胨葡萄糖液体培养基 (YPD) 倍比稀释, 接入等体积的菌浓度为 (2~3)×10³/mL CFU的新生隐球菌、白色念珠菌 (金黄色葡萄球菌) 菌悬液, 使目标化合物和氟康唑 (环丙沙星) 的浓度分别为128, 64, 32, 16, 8, 4, 2, 1, 0.5和0.25 μg/mL, 以不加任何试样的处理为生长对照, 所有处理的菌体终浓度为 (1~1.5)×10³/mL CFU.于30 ℃ (37 ℃) 下恒温培养36 h, 用可见分光光度计在530 nm波长下测定光密度 (OD) 值, 所测OD值和空白培养基OD值无显著差异时对应的最小试样浓度为最小抑菌浓度.

MFC (MBC) 的测定:在MIC基础上, 所测OD值和空白培养基OD值无显著差异时, 将相应不同浓度的试样处理的培养液涂平板上, 无菌落生长对应的最小试样浓度为最小杀菌浓度

辅助材料 (Supporting Information) 目标化合物2a~6e的¹H NMR、¹³C NMR和质谱谱图.这些材料可以免费从本刊网站 (http://sioc-journal.cn/) 上下载.

3.3.1 抑菌圈的测定

采用滤纸片法测定目标化合物对新生隐球菌标准株、临床株、白色念珠菌、大肠杆菌和金黄色葡萄球菌的抑菌活性, 将上述菌种接种到Mueller-Hinton (MH) 液体培养基中, 于30 ℃(真菌)、37 ℃(细菌) 下振荡培养24~28 h至对数生长期, 用新鲜的MH液体培养基将对数生长期的菌液稀释到10⁶/mL CFU (CFU:菌落形成单位), 取0.1 mL该溶液, 加在MH琼脂平板上, 用涂棒将其涂布均匀, 然后将直径为6 mm的滤纸片放在培养基上.将样品分别用二甲基亚砜 (DMSO) 配制成一定浓度的溶液, 用微量加样器取5 μL加到滤纸片上, 使待测物的含量为200 μg/片, 于30 ℃(真菌)、37 ℃(细菌) 下培养48 h观察结果, 测量抑菌圈的直径, 平行测试3次, 结果取平均值.以2-(2-噻唑基)-3-乙酯基-4-甲基-1H-2, 5-二氢-1, 5-苯并二氮杂䓬为阳性对照, 以溶剂DMSO为阴性对照.

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图 1 化合物2~6的结构

Figure 1 Structures of compounds 2~6

2a~2e: X＝thiazol-2-yl: R²＝H; 2a: R¹＝H, 2b: R¹＝CH₃, 2c: R¹＝F, 2d: R¹＝Cl, 2e: R¹＝Br; 3a~3e: X＝thiazol-2-yl: R²＝CH₃; 3a: R¹＝H, 3b: R¹＝CH₃, 3c: R¹＝F, 3d: R¹＝Cl, 3e: R¹＝Br; 3f~3j: X＝phenyl: R²＝CH₃, 3f: R¹＝H, 3g: R¹＝CH₃, 3h: R¹＝F, 3i: R¹＝Cl, 3j: R¹＝Br; 3k~3o: X＝furan-2-yl: R²＝CH₃, 3k: R¹＝H, 3l: R¹＝CH₃, 3m: R¹＝F, 3n: R¹＝Cl, 3o: R¹＝Br; 4a~4e: X＝thiazol-2-yl: R²＝CH₂CH₃, 4a: R¹＝H, 4b: R¹＝CH₃, 4c: R¹＝F, 4d: R¹＝Cl, 4e: R¹＝Br; 5a~5e: X＝thiazol-2-yl: R²＝CH₂CH₂CH₃, 5a: R¹＝H, 5b: R¹＝CH₃, 5c: R¹＝F, 5d: R¹＝Cl, 5e: R¹＝Br; 6a~6e: X＝thiazol-2-yl: R²＝CH (CH₃)₂, 6a: R¹＝H, 6b: R¹＝CH₃, 6c: R¹＝F, 6d: R¹＝Cl

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图 2 副产物苯并咪唑的结构

Figure 2 Structure of by-product benzimidazole

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图式1 1H-2, 5-二氢-1, 5-苯并二氮杂䓬2~6的合成路线

Scheme 1 Synthetic routes of 1H-2, 3-dihydro-1, 5-benzodiaze-pines 2~6

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图式2 目标化合物2~6的合成机理

Scheme 2 Plausible mechanism for synthesis of products 2~6

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表 1 化合物2~6的抑菌圈值 (nm)^a

Table 1. Zone (mm) of growth inhibition of compounds 2~6


Compd.	R¹	R²	X	C.n.	C.n.C.	C.a.	E.c.	S.a.
2a	H	H		6.00±0.00	6.00±0.00	6.00±0.00	9.70±0.30	8.40±0.10
2b	CH₃	H		8.80±0.15	6.00±0.00	6.00±0.00	11.20±0.00	11.70±0.00
2c	F	H		6.00±0.00	6.00±0.00	6.00±0.00	10.40±0.12	9.60±0.15
2d	Cl	H		16.60±0.05	19.10±0.06	9.60±0.10	8.70±0.12	7.80±0.05
2e	Br	H		6.00±0.00	6.00±0.00	7.30±0.10	7.80±0.20	7.00±0.25
3a	H	CH₃		24.27±0.20	28.63±0.30	15.00±0.10	19.70±0.25	21.17±0.20
3b	CH₃	CH₃		21.00±0.21	24.07±0.23	14.37±0.15	15.03±0.19	17.10±0.30
3c	F	CH₃		15.83±0.32	15.90±0.25	13.33±0.15	12.67±0.20	19.00±0.32
3d	Cl	CH₃		14.23±0.21	15.75±0.22	8.10±0.12	9.50±0.20	18.60±0.10
3e	Br	CH₃		12.07±0.12	15.70±0.25	11.70±0.20	12.03±0.10	13.30±0.15
3f	H	CH₃		6.00±0.00	14.30±0.05	7.20±0.10	6.00±0.00	10.02±0.11
3g	CH₃	CH₃		6.00±0.00	15.60±0.10	16.60±0.05	6.00±0.00	11.17±0.12
3h	F	CH₃		6.00±0.00	16.70±0.11	15.25±0.03	6.00±0.00	10.95±0.11
3i	Cl	CH₃		6.00±0.00	15.70±0.15	12.23±0.04	6.00±0.00	10.83±0.13
3j	Br	CH₃		6.00±0.00	15.60±0.12	11.33±0.10	6.00±0.00	10.30±0.12
3k	H	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3l	CH₃	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3m	F	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3n	Cl	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
3o	Br	CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
4a	H	CH₂CH₃		22.60±0.00	17.70±0.00	18.40±0.10	6.00±0.00	21.90±0.13
4b	CH₃	CH₂CH₃		28.50±0.11	23.70±0.15	22.70±0.12	6.00±0.00	23.10±0.05
4c	F	CH₂CH₃		27.90±0.20	23.20±0.19	21.70±0.05	6.00±0.00	22.80±0.30
4d	Cl	CH₂CH₃		21.20±0.10	15.70±0.30	14.00±0.23	6.00±0.00	22.50±0.19
4e	Br	CH₂CH₃		19.40±0.05	14.30±0.12	9.60±0.15	6.00±0.00	21.10±0.19
5a	H	CH₂CH₂CH₃		20.40±0.20	16.00±0.30	9.10±0.10	6.00±0.00	23.80±0.20
5b	CH₃	CH₂CH₂CH₃		21.70±0.21	16.40±0.23	15.30±0.15	6.00±0.00	26.00±0.30
5c	F	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	25.50±0.15
5d	Cl	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	24.40±0.32
5e	Br	CH₂CH₂CH₃		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	22.80±0.25
6a	H	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6b	CH₃	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6c	F	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6d	Cl	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
6e	Br	CH (CH₃)₂		6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00	6.00±0.00
^a Values indicate the average diameters in mm (of three trials) for the zone of growth inhibition observed after 24 h of incubation at 30 ℃. C.n. C. neoformans ATCC 32264;C.n.C.: C. neoformans clinical strain; C.a.: C. albicans ATCC 10231;E.c.: E. coli ATCC 44752; S.a.:S. aureus ATCC 25923.

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表 2 化合物4b、4c和氟康唑的MIC及MFC值 (μg/mL)^a

Table 2. MIC and MFC values for compounds 4b, 4c and fluconazole (Mg/mL)

Fungal strain	4b		4c		Fluconazole
Fungal strain	MIC	MFC	MIC	MFC	MIC	MFC
C.n	80.0	＞128.0	60.0	＞128.0	＞128.0	＞128.0
C.a.	40.0	84.0	40.0	86.0	＞128.0	＞128.0
^aC.n.: C. neoformans ATCC 32264; C.a.: C. albicansATCC 10231.

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表 3 化合物4b、4c和环丙沙星的MIC及MBC值 (μg/mL)^a

Table 3. MIC and MBC values for compounds 4b, 4c and ciprofloxacin (μg/mL)

Bacterial strain	4b		4c		Ciprofloxacin
Bacterial strain	MIC	MBC	MIC	MBC	MIC	MBC
S.a.	56.0	＞128.0	62.0	＞128.0	28.0	36.0
^a.S.a.: S. aureus ATCC 25923.

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沈阳化工大学材料科学与工程学院沈阳 110142