Carotti Andrea, Ianni Federica, Sabatini Stefano, Di Michele Alessandro, Sardella Roccaldo, Kaatz Glenn W, Lindner Wolfgang, Cecchetti Violetta, Natalini Benedetto
Department of Pharmaceutical Sciences, Section of Chemistry and Technology of Drugs, University of Perugia, via del Liceo 1, 06123 Perugia, Italy.
Department of Physics and Geology, University of Perugia, Via Pascoli 1, 06123 Perugia, Italy.
J Pharm Biomed Anal. 2016 Sep 10;129:182-189. doi: 10.1016/j.jpba.2016.07.003. Epub 2016 Jul 4.
Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional structures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1-4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H2O-49/49/2 (v/v/v)+25mM formic acid, 12.5mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6mg and 2.8mg, for (R)-1 and (S)-1, respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the (R)-1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B (norA+/A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order.
在细菌采用的机制中，外排泵（EPs）被认为在导致对常用抗菌剂产生耐药性方面发挥了重要作用。然而，关于它们的三维结构或抑制的空间要求却知之甚少。缺乏此类知识的情况包括对金黄色葡萄球菌外排泵中研究最多的NorA之一。在本研究中，使用两种商业化的基于金鸡纳生物碱的两性离子手性固定相，实现了四种2-((2-(4-丙氧基苯基)喹啉-4-基)氧基)烷基胺1-4的对映体分离，这四种化合物在作为外消旋体测试时，先前被发现是有效的金黄色葡萄球菌NorA外排泵抑制剂。在确定的最佳极性离子条件（甲醇/四氢呋喃/水-49/49/2（v/v/v）+25 mM甲酸，12.5 mM二乙胺）下，对1进行连续重复进样，得以分离出足够量的对映体（(R)-1为2.6 mg，(S)-1为2.8 mg），进而评估它们抑制金黄色葡萄球菌NorA外排泵的能力。生物学评估突出了(R)-1对映体在溴化乙锭外排抑制以及与针对SA-1199B（norA+/A116E GrlA）的协同效应方面相对于外消旋体活性的主要贡献。对两种分离出的对映体馏分的实验电子圆二色光谱和含时密度泛函理论计算光谱进行比较，使得所有化合物的对映体洗脱顺序都能清晰且容易地确定。
