【作者】 张剑；

【导师】 徐文方； 李学臣；

【作者基本信息】 山东大学 ， 药物化学， 2014， 博士

【摘要】 研究背景革兰氏阴性菌耐药及耐药菌感染问题已成为公众健康的最大威胁之一,是21世纪抗感染领域面临的巨大挑战。因此,寻找具有全新抗菌机制和抗菌靶点的新型抗菌药物已迫在眉睫。在革兰氏阴性菌中,UDP-3-O-(R-羟基十四酰)-N-乙酰氨基葡萄糖脱乙酰化酶(LpxC),是一种锌离子依赖性蛋白水解酶,它催化脂质A生物合成中最关键一步,为革兰阴性菌生存和毒力所必需。此外,LpxC酶在革兰氏阴性菌不同菌株中高度保守,与其它已知锌蛋白酶相比不具有同源性。因此,LpxC作为抗菌新靶点已成为近年来抗菌药物研究中的热点领域,针对这一靶标设计合成抑制剂是当前抗菌药物研究中很有发展前景的一类。目标化合物的设计、合成及活性筛选对已有LpxC抑制剂的药效团和定量构效关系总结发现,几乎所有高活性的LpxC抑制剂都具有共同的结构特征即含有与催化活性中心Zn2+螯合的基团(如异羟肟酸等)和与酶的疏水通道之间有效结合的疏水侧链。除上述两个主要的抑制剂结合位点外,还有"UDP binding site"这一结合位点,它对于促进底物与酶的识别和结合非常重要,是当前LpxC抑制剂设计的重要方向之一。在充分调研文献的基础上,本研究以LpxC为靶标,基于LpxC与其抑制剂作用模式,借助计算机辅助药物设计,结合构象限制、电子等排等策略,设计了系列新型的LpxC抑制剂。在合成目标化合物之前,我们利用SYBYL8.0对所设计的化合物进行了对接打分,结果显示大部分化合物分值都接近于甚至超过阳性对照。这说明了我们设计思路的合理性,为我们的研究提供了理论依据。合成过程中,A系列以L-羟脯氨酸为起始原料,经过系列反应如酯化、Boc保护或磺化、SN2亲核取代、Suzuki偶联反应、缩合反应、Sonogashira Coupling、 Mitsunobu Reaction等合成带有羧酸甲酯结构的目标化合物的前体。B系列以各种L构型氨基酸为起始原料,经过系列反应如Sonogashira Coupling、酯水解、缩合反应、Glaser Coupling等合成带有羧酸甲酯结构的目标化合物的前体。C系列分别以两种光学结构的氯霉胺及D-对甲砜基苯丝氨酸乙酯为起始原料,经过系列反应如Boc保护,羟基的选择性氧化、Sonogashira Coupling、酯水解、缩合反应、Glaser Coupling等合成带有羧酸甲酯结构的目标化合物的前体。D系列由B系列的中间体(S)-2-(4-乙炔基苯甲酰胺基)-4-甲硫基-丁酸甲酯经硫醚氧化成砜或亚砜与Boc保护的乙炔苯胺进行Glaser Coupling合成带有羧酸甲酯结构的目标化合物的前体。最后这些羧酸甲酯前体化合物均通过酯交换转化成异羟肟酸而得到目标化合物。对所合成的目标化合物进行了初步的生物活性评价,包括最低抑菌浓度MIC的测定,抑菌环试验及体外抑酶活性实验三方面,以期更准确地筛选出高活性的LpxC抑制剂。结果所有目标化合物均由’HNMR、HR-MS等方法进行结构确证,经Scifinder等文献检索工具证实,所合成的目标化合物均为新型化合物,未见文献报道。四系列化合物都表现出对革兰氏阴性菌(E. coil ATCC25922和P. aeruginosa ATCC27853)的选择性抑菌作用,对两株革兰氏阳性菌(S.A. ATCC25923和MRS.A. ATCC29213)则无明显抑菌作用。通过上述两株革兰氏阴性菌株的初筛,对活性较好的化合物进行了三株耐药菌株(E. coil MDR、P. aeruginosa MDR和E. cloacae MDR)的MIC测定。部分目标化合物的抑菌活性与阳性对照LPC009相比略弱。对活性最好的化合物D1和D2还进行了大肠杆菌野生型菌株(E. coilW3110)和membrane-compromised strain (E. coil CMR300)的MIC的测定,此部分工作由Duke University完成。此外,对活性最好的两个化合物D1和D2,进行了两株细菌ATCC25922和P. aeruginosa ATCC27853)的抑菌环试验。结果显示测试化合物与阳性对照药LPC009、Levofloxacin、Claforan抑菌活性相当。LpxC抑制剂的酶活测定方法条件比较苛刻,故根据上述MIC的测定结果,选取抗菌活性最好的两个化合物D1和D2进行EcLpxC的酶活测定,此部分工作由Duke University完成,活性结果表明所测两个化合物与阳性对照LPC028的抑酶活性相当,可作为抗革兰阴性菌先导进行深入研究。结论本研究基于LpxC的晶体结构及抑制剂与LpxC的作用模式,设计、合成了四系列新型的LpxC抑制剂,在体外表现出广谱的抗革兰氏阴性菌活性及较好的抑酶活性。这些化合物是很有研究前景的先导化合物,对具有全新作用机制的新型抗生素的研发有着积极的指导意义。更多还原

【Abstract】 BackgroundThe rapid increase of infections by Gram-negative pathogens along with the emergence of drug-resistant bacterial strains, posing the serious threat to the public health, is the great challenge in21century and demands the development of novel antibiotics directed against the previously unexploited targets.One of the promising targets in Gram-negative bacteria is the zinc-dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC catalyzes the first committed, second overall step in the biosynthetic pathway of lipid A, which is essential for the bacterial viability and toxicity. Additionally, LpxC is highly conserved among Gram-negative bacteria and shares no sequence homology with any other known zinc-metalloenzymes. Thus LpxC has become an attractive target for the structure-based drug design, and research on LpxC inhibitors is a very promising strategy in the development of current antibiotic therapy for Gram-negative bacteria.Design、synthesis and activity evaluation of target compoundsCurrently identified LpxC shared the same structural characters and almost all of the potent inhibitors contain a hydroxamate group for chelating the catalytic Zn2+and a side chain for effectively interacting with the hydrophobic tunnel. Based on the LpxC crystal structures and the binding mode of the known LpxC inhibitors in complex with LpxC, we designed4series of novel compounds as LpxC inhibitors with the strategies of CADD, conformational restriction and biosiostere. Before synthesizing the designed compounds, we docked our compounds with EcLpxC via FlexX of sybyl8.0. The result showed that almost all of the compounds had similar or higher score than the positive control, which, to some extent, ensured the rationality of our design strategy and supplied basement for our study.During the synthesis of series A, all the compounds designed were synthesized using L-hydroxyproline as starting material through a reaction sequence including esterification, Boc-protection or sulfonylation, SN2nucleophilic substitution, Suzuki couplingcon, condensation, Sonogashira coupling, mitsunobu reaction to obtain the key intermediate. During the synthesis of series B, all the compounds designed were synthesized using various L amino acids as starting material through a reaction sequence including Sonogashira coupling, hydrolysis, condensation, Galaser coupling to obtain the key intermediate. During the synthesis of series C, all the compounds designed were synthesized using (S)-methyl2-(4-ethynylbenzamido)-4-(methylthio)-butanoate as starting material through a reaction sequence including oxidation, Galaser coupling to obtain the key intermediate. The methyl ester groups of all the former compounds were converted to hydroxymate group to obtain the target compounds.Additionally, aiming for high activity compounds, preliminary activity assay was also carried out in vitro including MIC assay, antibacterial annulus and anti-LpxC assay.ResultsIn this research, all target compounds were obtained and identified by1H-NMR and HRMS spectra. Literature retrieval proved that all the compounds were new and not reported.In MIC assay, the results showed that four series of these compounds exhibited highly selective antibacterial activity against Gram-negative bacteria (E.coil ATCC 25922and P. aeruginosa ATCC27853) as compared with Gram-positive bacteria (S.A. ATCC25923和MRS.A. ATCC29213). Some potent compounds were also assayed for their antibacterial activities against E.coil (MDR), P. aeruginosa (MDR) and E.cloacae (MDR). Among them, a few target compounds showed slightly lower activity compared with positive control. While, the most potent compounds D1and D2were chosen to be assayed for their antibacterial activities against E. coil W3110(wild-type strain) and E. coil CMR300(membrane-compromised strain), which was finished in Duke University.Compounds D1and D2were also assayed for their antibacterial annulus against Ecoil ATCC25922and P. aeruginosa ATCC27853. The results showed that these two compounds was comparable to the positive control LPC009, Levofloxacin, Claforan towards these two strains.Additionally, the most potent compounds Dl and D2were also chosen to be carried out anti-LpxC assay. The results showed that these two compounds was comparable to the positive control LPC028towards EcLpxC and they can be researched deeply in the future work.ConclusionsIn conclusion, based on the crystal structure of LpxC and the binding mode of the known LpxC inhibitors with the enzyme, we designed and synthesized four series of the target compounds. Preliminary antibacterial activities assay and enzyme activity assay showed most compounds possess potential activity. These compounds are promising lead compounds for developing new LpxC inhibitors as novel antibiotic.更多还原