基于天然产物为导向的手性膦酸酯及查尔酮衍生物的设计、合成及生物活性研究

【作者】 张国平；

【导师】 宋宝安；

【作者基本信息】 贵州大学 ， 农药学， 2017， 博士

【摘要】 手性农药具有药效高、用量小、三废少、对环境生态安全等优点,一直引起农药化学家的关注。但限制其发展的主要因素有:（1）获得手性农药方法还不够成熟,主要存在手性催化剂昂贵、难以制备、性质不稳定、催化活性不高、底物宽泛性不够及操作条件苛刻,手性催化机理尚不清楚等诸多不足。（2）手性农药的对映体活性差异原因尚不清楚。因此针对这些挑战,本论文分别以消旋体农药和高活性天然产物为骨架,寻找有效的不对称合成方法合成高活性手性单体化合物,并通过有效途径探究其催化机理和生物选择性作用机理,为进一步不对称合成高活性手性单体化合物作好理论支持。首先,本论文以天然产物为导向的消旋体农药“毒氟磷”为骨架,采用硫脲类有机小分子不对称催化,设计合成了一系列高光学活性“毒氟磷”衍生物。通过对目标手性化合物的抗植物病毒活性筛选,研究它们的生物选择性及作用机制。现将此工作总结如下:（1）以消旋体农药“毒氟磷”为骨架,设计合成了18个含杂环2-氨-4-甲基苯并噻唑亚胺底物（A1^A18）,通过不对称膦氢化反应条件的优化,分别采用硫脲奎宁或硫脲奎尼丁小分子为手性催化剂,成功合成了18对高收率高光学活性“毒氟磷”衍生物（Ⅰa^Ⅰr）。（2）采用核磁、红外和高分辨质谱对化合物Ⅰa^Ⅰr的结构进行了表征,并通过X单晶衍射确认化合物If立体构型为R,依次类推其它手性化合物的构型。同时,采用旋光仪和含IA手性柱的高效液相色谱对手性化合物Ⅰa^Ⅰr的光学活性进行了测试。（3）采用实验和DFT计算相结合的方法进行该反应的催化机理研究,提出了亚磷酸二苯酯以五元环方式进攻催化剂与底物亚胺的复合体。（4）采用半叶枯斑法,以黄瓜花叶病毒（CMV）为测试对象,进行了手性“毒氟磷”衍生物（Ⅰa^Ⅰr）抗病毒活性筛选,其结果表明:目标化合物（R）体的抗病毒活性明显高于相对应的（S）体,其中化合物（R）-Ⅰb,Ⅰe,Ⅰf和Ⅰj在500μg/m L抗CMV的治疗活性分别为72.3%,70.2%,71.4%,和64.2%,明显优于对照药剂宁南霉素（45.3%）和毒氟磷（50.4%）;而相应地化合物（S）-Ⅰb,Ⅰe,Ⅰf和Ⅰj在500μg/m L抗CMV的治疗活性分别为25.1%,42.1%,54.5%和47.3%,明显弱于相应的（R）体,其中化合物Ⅰb有明显的生物选择性;化合物（R）-Ⅰf在500μg/m L时抗CMV的保护活性为67.4%,明显优于对照药剂宁南霉素（47.9%）和毒氟磷（54.1%）;化合物（R）-Ⅰb,Ⅰe,Ⅰf和Ⅰj在500μg/m L抗CMV的钝化活性分别为96.3%,93.4%,94.1%和90.7%,明显优于对照药剂宁南霉素（71.3%）和毒氟磷（78.2%）。其中手性化合物（R）-Ⅰf抗病毒活性在治疗、保护和钝化三方面均明显优于对照药剂,有一定的应用开发价值。（5）采用荧光滴定法对高活性化合物Ib与病毒潜在靶标蛋白CMV-CP进行了相互作用研究,其结果表明:化合物（R）-Ib,（S）-Ib,宁南霉素和毒氟磷分别与蛋白CMV-CP的结合常数为2.19×10⁵、1.17×10⁴、5.25×10⁴、2.40×10⁴ L mol^-1,该结果与化合物的活性呈正相关性,并佐证了该类手性化合物具有生物选择性。对手性化合物Ib与CMV-CP蛋白进行了分子对接,其对接结果表明:化合物Ib的两个手性体与CMV-CP蛋白的结合位点均有PHE58,THR57,VAL218,HIS55和ALA147,但两者的结合能有差别,分别为E（R）-Ib=-8.74Kcal/mo L和E（S）-Ib=-6.57Kcal/mo L,而从对接图来看存在差别的主要原因是化合物Ib与CMV-CP蛋白在三维空间的结合方式不同。其次,本论文以具有广泛生物活性天然产物查尔酮为骨架,采用硫脲类有机小分子不对称催化,设计合成了一系列高光学活性γ-硝基酮衍生物（IIa^IIm）。并通过农用活性筛选,研究它们的生物选择性。现将此工作总结如下:（1）以查尔酮为骨架,针对含杂环吡啶的查尔酮底物（AA1^AA13）,设计合成一系列不同取代基的硫脲奎宁类催化剂（Q1^Q6）,从中筛选出含3,5-三氟甲基苯基硫脲奎宁Q1的催化活性最高,建立了无溶剂1,4-迈克不对称加成合成方法,成功合成了13对高光学活性的γ-硝基酮衍生物。（2）采用核磁、红外和高分辨质谱对化合物ⅠIa^ⅠIm的结构进行了表征,并通过X单晶衍射确认化合物IIa立体构型为S,依次类推其它手性化合物的构型。同时,采用旋光仪和含手性IA柱的高效液相色谱对手性化合物的光学活性进行了表征。（3）采用实验和DFT计算相结合的方法进行该反应的催化机理研究,提出了多氢键结合模式。（4）采用浑浊度法,以水稻白叶枯为测试对象,进行了γ-硝基酮衍生物（IIa^IIm）抗细菌活性筛选,其结果表明:目标化合物（S）体的抗细菌活性高于相对应的（R）体的,其中化合物（S）-Ⅱb,Ⅱh和Ⅱm在100μg/m L时抗细菌活性分别为100.0%,100.0%,和75.0%,明显优于对照药剂叶枯唑（55.0%）;而相应地化合物（R）-Ⅱb,Ⅱh,和Ⅱm在100μg/m L抗细菌活性分别为65.0%,57.0%和35.0%,明显弱于相应的（S）体,从而表现明显的生物选择性。更多还原

【Abstract】 It has been attention that chiral pesticides possess high efficacy,small dosage,less "three wastes",the environmental advantages of ecological security,and so on.Chiral pestcides is one of important research direction of the green pesticide.But there are two major factors in restricting the development of chiral pesticides:（1）The method about preparing chiral pesticides is not mature enough and there are someshortcomings such as expensive and unstable catalyst,low catalytic activity,narrow substrate and harsh reaction conditions,unknown chiral catalytic mechanism,and so on.（2）The mechanism of the bioselectivity of the enantiomers of chiral pesticidee is still unclear.Therefore,be faced to these challenges,we will synthesize chiral monomer with high bioactivity based on racemate pestcides and highly active natural products and investigate its catalytic mechanism and bioselective mechanism of chiral pesticides via effective methods.Firstly,based on the motif of the racemic “dufulin”,high optical activity “dufulin” derivatives were designed and synthesized via chiral thiourea catalysts.Then the agricultural biological and bioselective activity of the title chiral compounds were investigated.And the origins of bioselective activities were further investigated.The conclusions are shown as follows:（1）Based on the motif of the racemic “dufulin”,18 imines with 4-methyl-2-yl-benzothiazole（A1^A18）were designed and synthesized.Through reaction condition optimization for enantioselective hydrophosphonylation,18 pairs chiral “dufulin” derivatives（Ia^Ir）were synthesized with highly yields and ee via thiourea-quinine or thiourea-quinindium catalysts.（2）The structures of compounds（Ia^Ir）were characterized by ¹H NMR,¹³C NMR,HMRS.The absolute configuration of compound If obtained by Q1 catalyst was determined to be R by X-ray crystallography.The absolute configuration of all other products（I）was assigned by analogy.Optical activity of chiral compounds（Ia^Ir）were tested using polarimeter and HPLC with chiral IA colum.（3）The reaction mechanism was proposed that the diphenyl phosphite attacked the complex of catalyst Q1 and imine A in five-member ring via experiments and DFT.（4）The antiviral activities of chiral “dufulin” derivatives（Ia^Ir）against cucumber mosaic virus（CMV）were tested by means of half leaf.The results showed that（R）-compounds generally exhibited higher antiviral activity than the corresponding（S）-enantiomer.Among them,compounds（R）-Ⅰb,Ⅰe,Ⅰf and Ⅰj displayed the inhibitory effect of curative activity with values of 72.3,70.2,71.4 and 64.2%,respectively,which is significantly higher than that of Ningnanmycin（45.3%）and Dufulin（50.4%）at 500μg/m L.Accordingly,compounds（S）-Ⅰb,Ⅰe,Ⅰf and Ⅰj displayed the curative activity with values of 25.1,42.1,54.5 and 47.3%,respectively,which is obviously lower than that of（R）-compounds.And compound Ib exhibited the evident bioselective activities.Compound（R）-Ⅰf possess the protective activity of 67.4%,which is obviously higher than that of Ningnanmycin（47.9%）and Dufulin（54.1%） at 500μg/m L.Compounds（R）-Ⅰb,Ⅰe,Ⅰf and Ⅰj displayed the inhibitory effect of inactive activity with values of 96.3,93.4,94.1 and 90.7%,respectively,which is significantly higher than that of Ningnanmycin（71.3%）and Dufulin（78.2%）.In view of the curative,protective,inactive activity of the chiral compounds（Ia^Ir）,compound（R）-If could have great potential for further development as chiral antivirus agents.（5）The interaction between chiral compound Ib and potential target protein（CMV-CP）was investigated via fluorescence titration.The results showed that the binding constant（K_a）of（R）-Ib,（S）-Ib,Ningnanmycin and Dufulin with CMV-CP is 2.19 × 10⁵,1.17 × 10⁴,5.25 × 10⁴,2.40 × 10⁴ L/mo L,respectively.Results of the binding studies are consistent with our experimental observations that compounds exhibited the antivirus activity.Molecular docking between chiral Ib and CMV-CP was performed.The interactions between both enantiomers of 3b and CMV-CP can likely occur in the binding pocket defined by five residues（Phe58,Thr57,Val218,His55 and Ala147）.The binding energy of（R）-Ib with CMV-CP（-8.74 Kcal/mol）is lower than that of（S）-Ib（-6.57 Kcal/mol）.The difference in the selective bioactivity could be affected by the combination mode of the three-dimensional space.Secondly,based on the motif of the diverse activities chalcones,high optical activity chalcones derivatives were designed and synthesized via chiral thiourea catalysts.Then the agricultural biological and bioselective activity of the title chiral compounds were investigated.The conclusions are shown as follows:（1）Based on the motif of the diverse chalcones,a series of chiral thioureas（Q1^Q6）derived from quinine have been tested as catalysts in the enantioselective Michael additions of nitromethane to chalcones containing pyridine（AA1^AA13）.The best results are obtained with the bifunctional catalyst prepared from 3,5-di（trifluoromethyl）-aniline under solvent-free conditions.13 pairs chiral γ-nitroketones（IIa^IIm）were synthesized with highly yields and ee via thiourea-quinine or thiourea-quinindium.（2）The structures of compounds（IIa^IIm）were characterized by ¹H NMR,¹³C NMR,HMRS.The absolute configuration of compound IIa obtained by Q1 catalyst was determined to be S by X-ray crystallography.The absolute configuration of all other products（II）was assigned by analogy.Optical activity of chiral compounds（IIa^IIm）were tested using polarimeter and HPLC with chiral IA colum.（3）The reaction mechanism was proposed that nitromethane attacked the complex of catalyst Q1 and chalcone AA1 in more hydrogen bonding interaction via experiments and DFT.（4）The antibacterial activities of chiral γ-nitroketones derivatives（IIa^IIm）against rice bacterial leaf blight were tested by turbidity method.The result showed that（S）-compounds generally exhibited higher antibacterial activity than the corresponding（R）-enantiomer.Among them,compounds（S）-Ⅱb,Ⅱh and Ⅱm displayed showed good antibacterial activity（inhibition,100,100 and 57% respectively）,which is significantly higher than that of Bismerthiazol（inhibition,55%）.at 100 μg/m L.Accordingly,compounds（R）-Ⅱb,Ⅱh and Ⅱm displayed the activity with values of 65.0,57.0 and 35.0%,respectively,which is obviously lower than that of（S）-compounds.Therefore,chiral γ-nitroketones derivatives（IIa^IIm）also exhibited the evident bioselective activities.更多还原