Membrane-active quinoline and quinazoline antibacterials that target Gram positive pathogens

摘要 由于耐药革兰氏阳性微生物引起的感染正在上升，可能是由于多种因素，包括更长的 住院时间、侵入性手术频率增加和普遍的抗生素治疗。配混 问题是在许多革兰氏阳性病原体（MRSA，S. 表皮葡萄球菌、肠球菌和S.肺炎）。尽管有抗生素管理和感染控制， 迫切需要针对这些革兰氏阳性病原体的药剂。经过筛选， 化合物库中，我们获得了一个具有抗菌活性的4-羟基喹啉衍生物DNAC-2 抗MRSA和肠球菌我们随后合成了3个系列的类似物，涉及50多个 化合物.第二系列中的这些类似物中的两种，JRS-3-56（化合物1）和JRS-4-32（化合物2）， 对MRSA、S. epidermidis、表皮E. faecalis和E.对屎肠球菌的MIC ≤0.2 μg/ml。然而，两人1 和2具有差的预测水溶性，具有高cLogP（分别为7.7和6.0）。转化 喹啉对喹唑啉1的cLogP提高（从6.06到5.08），但导致MIC略微增加（0.25 2 μg/ml）。在最新的系列中，我们在C-4处引入了羰基，并在C-1处引入了C至N取代 位置，产生具有低cLogP和非常低的MIC（对于USA 300为0.06 μg/ml）的化合物3和4， 伴随着更紧密的SAR。使用大分子合成测定，膜特异性染料FM 4- 64和电子显微镜研究，我们有证据表明1和2靶向革兰氏+膜（3和4 也导致膜缺陷，如通过FM 4 -64染料检测到的），但不是革兰氏或真核细胞膜， 因此暗示了某种程度的特异性。然而，这些化合物的确切目标可能不同， 来自达托霉素，是未知的。在本申请中，我们试图定义这些化合物的作用机制。 喹啉/喹诺酮衍生物，并进一步探索控制体外和体内活性的SAR， 药物处置特性。因此，我们有以下具体目标：1）设计合成 通过定义控制对主要革兰氏阳性病原体的活性的SAR， 药物处置特性（MIC、溶解度、明显毒性和血清结合等）; 2)阐明机制 喹啉/喹诺酮衍生物与遗传、生物化学和生物物理工具的作用; 3） 药代动力学和功效研究，其中将评价候选化合物的药物 处置性质，以确保安全性和选择性，然后选择“先导”化合物进行全PK 两种动物模型的评价和功效研究。这些研究的目的是确定“可用药的 膜活性化合物”具有广泛的革兰氏+活性。如果成功，我们相信这些化合物将 代表了一类新的膜活性化合物，为药物开发提供了重大进展。

Abstract Infections due to resistant Gram+ organisms are on the rise, likely due to a variety of factors including longer hospital stay, increased frequency of invasive procedures and pervasive antibiotic therapy. Compounding the problem is the emergence of multi-drug resistance (MDR) among many Gram+ pathogens (MRSA, S. epidermidis, Enterococcus and S. pneumoniae). Despite antibiotic stewardship and infection control, new agents against these Gram+ pathogens are urgently needed. After screening a ~60,000 preselected compound library, we obtained DNAC-2, a 4-hydroxyquinoline derivative, that exhibited antibacterial activities against MRSA and Enterococcus. We subsequently synthesized 3 series of analogues involving over 50 compounds. Two of these analogues in the 2th series, JRS-3-56 (compound 1) and JRS-4-32 (compound 2), were cidal against MRSA, S. epidermidis, E. faecalis and E. faecium, with MIC ≤0.2 μg/ml. However, both 1 and 2 have poor predicted aqueous solubility with high cLogP (7.7 and 6.0, respectively). Conversion of quinoline to quinazoline for 1 improved the cLogP (from 6.06 to 5.08) but led to a slight increase in MIC (0.25 to 2 µg/ml). In the latest series, we introduced a carbonyl group at C-4 and a C to N substitution at the C-1 position, yielding compounds 3 and 4 with low cLogPs and very low MIC (0.06 µg/ml for USA300), accompanied by a much tighter SAR. Using macromolecular synthesis assays, membrane-specific dye FM4- 64 and electron microscopy studies, we have evidence that 1 and 2 target the Gram+ membrane (3 and 4 also resulted in membrane defect as detected by the FM4-64 dye), but not Gram- or eukaryotic membrane, thus implying some degree of specificity. However, the exact target of these compounds which likely differs from daptomycin, is not known. In this application, we seek to define the mechanism of action of these quinoline/quinolone derivatives and further explore the SAR that governs in vitro and in vivo activities and drug disposition properties. Accordingly, we have the following specific aims: 1) design and synthesize quinoline/quinolone derivates by defining the SAR that governs activity against major Gram+ pathogens and drug disposition properties (MIC, solubility, overt toxicity and serum binding etc.); 2) delineate the mechanism of action of the quinoline/quinolone derivatives with genetic, biochemical and biophysical tools; 3) pharmacokinetic and efficacy studies where candidates compounds will be evaluated for their drug disposition properties to ensure safety and selectivity followed by selection of “lead” compounds for full PK evaluation and efficacy studies with two animal models. The goal of these studies is to identify “druggable membrane-active compounds” with broad Gram+ activity. If successful, we believe these compounds will represent a new class of membrane-active compounds that offer a significant advance in drug development.