新型吡唑类衍生物合成与抗病毒和抗癌生物活性研究

【作者】 欧阳贵平；

【导师】 宋宝安；

【作者基本信息】 贵州大学 ， 农药学， 2008， 博士

【摘要】 吡唑类化合物及其衍生物因其结构的可变性和其高效广谱的生物活性，在医药和农药领域有着广泛的应用。在医药领域方面，吡唑主要表现在抗菌、抗肿瘤、抗癌、抗HIV-1和抗结核等方面；在农药领域方面，吡唑主要表现在杀虫杀螨剂，杀菌剂及除草剂等方面。多年来一直引起化学、药学和生物学领域人们的兴趣和重视，尤其是近年来，是医药、农药精细化工领域研究开发的热点课题之一。对烟草花叶病（Tobacco Mosaic Virus，简称TMV）的防治，国内外已做了大量的研究，特别是在植物抗病毒基因研究上取得了一系列进展，但是由于远缘杂交的不亲和性，使由抗性基因转移的难度很大。其次，农业栽培措施由于受耕作制度和社会经济发展的限制，在生产中有效的实施仍有一定难度，使用化学或生物制剂防治烟草花叶病不失为一种经济有效的措施。现有的药剂菌奇清、宁南霉素、病毒A、病毒必克有一定防效（防效大多在30-60％），但药剂防治效果还不理想。因此，筛选高效、低毒、高选择性、环境相容性好的农用抗病毒药剂成为当务之急。为了创制具有高效具有农用抗病毒活性的新化合物，本论文以1-取代基-3-甲基吡唑酮为起始原料，经多步反应，设计合成一系列新的含5-取代硫醚吡唑肟醚类化合物和5-取代砜吡唑肟醚类化合物（如图所示），然后进行结构表征和生物测定，希望从中筛选出较高抗TMV生物活性的化合物，并对作用机理进行研究。本论文完成工作归纳为以下几点结论：R₁=Ph，3-ClPh，4-ClPh，4-CH₃Ph：G1=H，F，Me，OMe；G₃代表的芳基杂环，等。1．目标化合物5-取代硫醚-1-取代基-3-甲基吡唑肟醚衍生物的合成（化合物编号：PS1）以1-取代芳基-3-甲基吡唑酮为原料，通过POCl₃氯化合成了5个1-取代苯基-3-甲基-5-氯-4-醛基吡唑中间体（中间体M1），取代苯基-3-甲基-5-氯-4-醛基吡唑（中间体M1）通过取代的苯硫酚醚化合成了9个1-取代苯基-3-甲基-5-取代苯硫基-4-醛基吡唑（中间体M3），1-取代苯基-3-甲基-5-取代苯硫基-4-醛基吡唑（中间体M3）和盐酸羟胺反应合成了9个1-取代苯基-3-甲基-5-取代苯硫基-4-吡唑醛肟（中间体M4），1-取代苯基-3-甲基-5-取代苯硫基-4-吡唑醛肟（中间体M4）和氯甲基芳杂环化合物（C1-G3）进行醚化反应合成了11个5-取代硫醚-1-取代基-3-甲基吡唑肟醚衍生物，其中有11个为未见报道的化合物。对合成的11个化合物进行了物理常数的测定，其结构经过了IR、¹H NMR、¹³C NMR及元素分析确证。2．目标化合物5-取代砜-1-取代基-3-甲基吡唑肟醚衍生物的合成（化合物编号：PS2）以吡唑醛肟中间体M4为原料，通过与氯甲基芳杂环化合物（C1-G3）进行醚化反应反应，在冰醋酸中用高锰酸钾室温条件下可将5-位的硫醚氧化为砜，合成了5个5-取代砜-1-取代基-3-甲基吡唑肟醚未见报道的化合物。对所合成的化合物分别进行了物理常数的测定，其结构经过了IR、¹H NMR、¹³C NMR及元素分析确证。3．目标化合物5-取代硫醚-1-取代基-3-甲基吡唑肟酯衍生物（化合物编号：PS3）、5-取代砜-1-取代基-3-甲基吡唑吡唑肟酯类衍生物的合成（化合物编号：PS4）以吡唑醛肟中间体M4为原料，通过与酰氯进行酯化反应成了14个5-取代硫醚-1-取代基-3-甲基吡唑肟酯衍生物（化合物编号：PS3），在冰醋酸中用高锰酸钾室温条件下可将5-位的硫醚氧化为砜，合成了5个5-取代砜-1-取代基-3-甲基吡唑肟酯未见报道的化合物。对所合成的化合物分别进行了物理常数的测定，其结构经过了IR、¹H NMR、¹³C NMR及元素分析确证。4．目标化合物吡唑酰胺（PS5）及吡唑酰脲类衍生物（PS6）的合成以1-取代基-3-甲基吡唑酮为原料，通过POCl₃氯化合成了5个1-取代苯基-3-甲基-5-氯-4-醛基吡唑中间体（中间体M1），通过高锰酸钾进行氧化反应合成了5个1-取代苯基-3-甲基-5-氯-4-吡唑甲酸（中间体M5），1-取代苯基-3-甲基-5-氯-4-吡唑甲酸（中间体M5）通过氯化亚砜加热回流反应合成了5个1-取代苯基-3-甲基-5-氯-4-吡唑甲酰氯中间体（中间体M6），通过26％的氨水酰胺反应合成了5个1-取代苯基-3-甲基-5-氯-4-吡唑甲酰胺中间体（中间体M7），1-取代苯基-3-甲基-5-氯-4-吡唑甲酰氯和芳胺（G5-NH₂）反应合成了9个吡唑酰胺类衍生物（PS5）目标化合物；吡唑酰胺（中间体M7）和草酰氯反应，通过含氟芳胺（G5-NH2）进行酰脲反应合成了10个吡唑酰脲类衍生物衍生物（PS6），其中有19个为未见报道的化合物。对合成的19个化合物分别进行了物理常数的测定，其结构经过了IR、¹H NMR、¹³C NMR及元素分析确证。5．目标化合物吡唑甲胺衍生物（PS8）的合成1-取代苯基-3-甲基-5-取代苯硫基-4-醛基吡唑（中间体M3）和对三氟甲基苯胺，加热回流反应，NaBH₄进行还原反应合成了5个吡唑甲胺衍生物（PS7）目标化合物，对合成的5个化合物分别进行了物理常数的测定，其结构经过了IR、¹H NMR、¹³C NMR及元素分析确证。6．部分目标化合物的抗癌活性研究采用MTT法，对目标化合物还进行了离体抗癌活性研究，结果表明部分化合物对人前列腺癌细胞（PC3）、人乳腺癌细胞Bcap37和胃癌细胞（BGC-823）具有较高的抑制活性。采用MTT法，目标化合物吡唑衍生物对癌细胞的IC₅₀值，测试化合物对PC3细胞的IC₅₀值为7．06-46．81μM，对Bcap-37细胞的IC₅₀值为1．77-206．18μM，对BGC-823细胞的IC₅₀值为7．75-38．35μM；采用TRYPANBLUE细胞活力测定和AO／EB染色荧光显微镜观察PS6-7对PC3细胞的细胞毒性，显示该化合物在该浓度下作用于PC3没有引起细胞凋亡，同时造成细胞死亡较少。7．部分目标化合物的抗TMV活性研究采用半叶法，在药剂的质量浓度均为500 mg/L时，对40个化合物进行了活体治疗抗烟草花叶病毒活性测定，测定结果表明：化合物PS3-1、PS1-1、PS3-2、PS3-6、PS3-8、PS3-7、PS3-12、PS3-13、PS5-4、PS5-5、PS5-8表现出较高的活体治疗作用，抑制率分别为47．8％，47．0％，40．4％，50．0％，46．4％，47．7％，62．0％，60．0％，44．0％，46．5％，46．0％，PS3-12，PS3-13活性较高，抑制率分别为62．0％，60．0％，略高于商品抗病毒剂宁南霉素（56-58％）。化合物PS1-1，PS3-2，PS1-7，PS3-8，PS3-12，PS3-13，PS3-14表现出较高的抗烟草花叶病毒钝化活性，抑制率分别为81．4％，74．1％，80．6％，72．1％，74．6％，73．7％，73．9％，其中PS1-1，PS1-7活性较高，抑制率为81．4％，80．6％，比商品抗病毒剂宁南霉素略低，PS3-2，PS3-8，PS3-12，PS3-13，PS3-14低于PS1-1，PS1-7活性，抑制率分别为74．1％，72．1％，74．6％，73．7％，73．9％，其它化合物抗烟草花叶病毒钝化活性均低于70％；化合物PS5-7，PS5-5，PS5-2，PS3-6，PS3-10，PS1-7表现出较高的抗烟草花叶病毒保护活性，其中PS5-7活性较高，比商品抗病毒剂宁南霉素略低，PS5-5，PS5-2，PS3-6，PS3-10，PS1-7低于PS5-7活性（抑制率为59．0％），抑制率分别为50．0％，45．7％，48．4％，40．9％，43．5％。进行了化合物PS3-12处理接种TMV后的烟草植株中，抗TMV的生化研究，结果表明其PAL酶、POD酶、SOD酶等相关酶和调控物质在一定的时间内都具有相关性；对普通烟PR-5基因的研究：处理已感染TMV天的普通烟后，普通烟叶片PR-5基因表达改变不明显，说明具有诱导PR-5基因不表达上调的作用；结果表明PS3-12可诱导pathogenesis related proteins-1基因表达上调，以增加烟草抗病毒的能力，从而阻止TMV病毒的系统感染和远距离侵袭；这些结果为创制具有高效具有农用抗病毒活性的新化合物提供了先导化合物；由此，该类化合物具有较好的抗TMV活性，对该类化合物进行结构优化，筛选出抗烟草花叶病毒的药剂是很有希望的。更多还原

【Abstract】 Pyrazole and their derivatives are important class of heterocyclic compounds.They occupy important position in medicinal and pesticide chemistry with wide range of bioactivities. As medicines, many of them display antifungal, antimicrobial, anti-HIV, antitubercular, anticancer, antiinflammatory, anticonvulsant, antidepressant, hypolipidemic, antiulcer, analgesic, immunotropic activities and are also known to act as thymidyalate synthase, poly（ADP-ribose） polymerase （PARP）, and protein tyrosine kinase inhibitors. As pesticides, they are used as insecticides, fungicides, antibacterial agents, and antiviral agentssuch as TMV, CMV inhibitors. With growing application on their synthesis and bioactivity, there has been an enormous increase of interest among the biologists and chemists in recent years on the research of pyrazole derivatives.Tobacco mosaic virus （TMV） infection is very widely distributed, and can cause serious damage and large economic loss. It was found that in some fields 90-100% of the plants show mosaic by harvesting time. Regarding the unsatisfactory curatives （30-60% cure rate by common antiviral agent Ningnanmycin or Virus A） and economic loss of tobacco, it is of great necessary to develop highly efficient, novel environmental benign antiviral agent.In order to create new and high-efficient antiviral agent in agricuture, this paper hoped to use 1-substituted phenyl-3-methyl-4-formylpyrazole as the start material, designed and synthesized a series of pyrazole derivatives containing oxime ester and amide moiety （see figure）, then the structures of the compounds are characterized by elemental analysis, IR, ¹H-NMR and ¹³C-NMR spectra. The title compounds are also tested in vitro and in vivo against pathogenic virus and cancer cells. Half-leaf method was used to determine curative efficacy in vivo of title compounds. The result showed that some compounds possessed good curative effect against TMV in vivo. The mechanism of action of the title compounds PS3-13 against TMV in vivo is also studies. Some conclusions were obtained: 1. The synthesis of title compounds l-substituted-5 substitutedphenylthio-4-pyrazolaldoxime ether derivatives （PS1）Starting from 1-substituted phenyl-3-methyl-4-formylpyrazole as raw materials, the intermediate M1, 1-substituted phenyl-3-methyl-4-formyl-5-chloropyrazole, was prepared by chlorination reaction with POCl₃. Thus, treatment of 1-substituted phenyl-3-methyl-4-formyl-5-chloropyrazole with substituted thiophenol afforded 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldehyde （M3）.Reaction 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldehyde with hydroxylamine hydrochloride gave 9 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldoximes （M4）. 11 the title compounds, 1-substituted -5-substitutedphenylthio-4-pyrazolaldoxime ether derivatives （PS1） were synthesized by the thioetherication reaction of l-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes （M4） with chloromethyl-heterocyclic compounds （C1-G3）. A total of 11 compounds, of which 11 new compounds. The synthesized compounds were determied physical constants, their structures were clearly established by IR, ¹H NMR, ¹³C NMR and elemental analysis.2. The synthesis of title compounds 1-substituted-S-substitutedphenylsulfonyl-4-pyrazolaldoxime ether derivatives （PS2）. 5 1-substituted-5-substitutedphenylsulfonyl-4-pyrazolaldoxime ether derivatives （PS2） were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes （M4） with chloromethyl-heterocyclic compounds （C1-G3） through etherication reaction. Thus, Oxidiation of potassium permanganate with PS1 in HOAc solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.3. The synthesis of title compounds l-substituted-5-substitutedphenylsulfonyl-4-pyrazolaldoxime ester derivatives （PS4）.14 the title compounds, 1-substituted-5-substitutedphenylthio-4-pyrazolaldoxime ester derivatives （PS3） were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes （M4） with acyl chloride. Thus, Oxidiation of potassium permanganate with PS3 in HOAc solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.4. The synthesis of title compounds l-substituted-5-chloro-4- pyrazolecarbox-amide derivatives （PS5） and their pyrazolecarbonylurea derivatives （PS6）.Starting from 1-substituted phenyl-3-methyl-4-formyl-chloropyrazole as raw materials, the intermediate M5, 1-substituted phenyl-3-methyl-5-chloropyrazole -4-carboxylic acid, was prepared by oxidation reaction with potassium permanganate. Thus, treatment of 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carboxylic acid with SOCl₂ afforded 5 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carbonyl chloride （M6） in refluex condition. Reaction 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carbonyl chloride （M6） with 26% ammonia gave 5 1-substituted phenyl-3-methyl-5-chloro-4-pyrazolaldoximes （M7）. 9 the title compounds, 1-substituted-5-chloro-4-pyrazole-carboxamide derivatives （PS5） were synthesized by the amidiation reaction of 1-substitutedphenyl-3-methyl- 5-chloro-4-pyrazolcarbonyl chloride （M6） with aromatic amines （G5-NH2）. Reaction of 1-substituted-5-chloro-4-pyrazolecarboxamide （M7） with grass chloride, followed by carbonylurea reaction with fluoroaromatic amine （G5-NH2） to yield 10 pyrazolecarbonylurea derivatives （PS6）. A total of 19 compounds, of which 19 new compounds. The synthesized compounds were determied physical constants, their structures were clearly established by IR, ¹H NMR, ¹³C NMR and elemental analysis.5. The synthesis of title compounds l-substituted-5-substitutedphenylthio-4-pyrazolyl-methylamino derivatives （PS8）.5 the title compounds, l-substituted-5-substitutedphenylthio-4-pyrazolylmethyl-amino derivatives （PS8） were synthesized from the starting material 1-substituted phenyl-3-methyl-5-substitutedphenylthio-4-formylpyrazole （M3） with 4-trifluoro -methyaniline. Thus, reduction of NaBH₄ with 1-substitutedphenyl-3 -methyl-5-substituted phenylthio-4-pyrazoleimine （M10） in EtOH solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.6. The anticancer activities of title compounds.The antitumor activites of the title compounds were assayed by the MTT method. It was found that these compounds exhibit moderate to good activities against PC3, Bcap37 and BGC-823 cell lines in vitro. The target compounds can strongly inhibit PC3 cells, with IC₅₀ value of 7.06-46.81μM. The IC₅₀ value of title compounds to Bcap37 and BGC-823 cell lines were 1.77-206.18μM and 7.75-38.35μM, respectively. Using TRYPAN BLUE and AO/EB stained fluorescent microscope, the compound PS6-7 couldn’t induce apoptosis of PC3 cancer cells, causing significant reduction of cell death number.7. The anti-TMV activities of title compounds.Half-leaf method was used to determine curative effects in vivo of 40 title compounds. The result showed that title compounds PS3-1, PS 1-1, PS3-2, PS3-6, PS3-8, PS3-7, PS3-12, PS3-13, PS5-4, PS5-5 and PS5-8 possessed good curative effect against TMV in vivo, with values of 47.8, 47.0, 40.4, 50.0, 46.4, 47.7, 62.0, 60.0, 44.0, 46.5 and 46.0% at 500μg/mL, respectively. Among these compounds, PS3-12, PS3-13 were much more active against TMV than the other ones, with the curative rate of 62.0, 60.0%, which is equivalent to Ningnanmycin （56-58%） against TMV at 500μg/mL. Choosing fourty new compounds was used to test protective and inactivation effects.It can be observed that the title compounds PS1-1, PS3-2, PS1-7, PS3-8,PS3-12,PS3-13,PS3-14 possess potential inactivation bioactivities, with values of 81.4, 74.1, 80.6, 72.1, 74.6, 73.7, 73.9% at 500μg/mL, respectively. Among these compounds, PS1-1, PS1-7 is much more active against TMV than the other ones, with the inactivation rate of 81.0, 80.0%, which is little lower than Ningnanmycin （95-98%） against TMV at 500μg/mL. The other compounds exhibited weak to moderate inactivation bioactivities, with the inactivation rate of below to 70%. The data also indicate that title compounds PS5-7, PS5-5, PS5-2, PS3-6, PS3-10, PS1-7 show good protection activity against TMV of up to 50.0, 45.7, 48.4, 40.9, 43.5% and 59.0% at 500μg/mL. While the title compounds PS5-7, PS5-5, PS5-2, PS3-6, PS3-10 have a relatively lower protection activity than PS5-7. Primary study on the anti-TMV action mechanism of PS3-12 was inoculated TMV dealt with the tobacco plants using anti-TMV biochemical research. The results showed that their PAL enzyme, peroxidase enzyme, SOD enzyme, and control within a certain amount of time have relevance. SOD activity and chlorophyll content were increased in the tobacco plant treated by PS3-12. PS3-12 induced PAL and POD activity enhanced, different from the action of Ningnanmycin. Gene expression of Nicotiana tobacum treated by PS3-12. PS3-12 can induce up-regulation of PR-1a gene and acquire SAR and possess antiviral activity.更多还原