Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors.

New antibacterial agents with novel target and mechanism of action are urgently needed to combat problematic bacterial infections and mounting antibiotic resistances. Topoisomerase IA represents an attractive and underexplored antibacterial target, as such, there is a growing interest in developing selective and potent topoisomerase I inhibitors for antibacterial therapy. Based on our initial biological screening, fluoroquinophenoxazine 1 was discovered as a low micromolar inhibitor against E. coli topoisomerase IA. In the literature, fluoroquinophenoxazine analogs have been investigated as antibacterial and anticancer agents, however, their topoisomerase I inhibition was relatively underexplored and there is little structure-activity relationship (SAR) available. The good topoisomerase I inhibitory activity of 1 and the lack of SAR prompted us to design and synthesize a series of fluoroquinophenoxazine analogs to systematically evaluate the SAR and to probe the structural elements of the fluoroquinophenoxazine core toward topoisomerase I enzyme target recognition. In this study, a series of fluoroquinophenoxazine analogs was designed, synthesized, and evaluated as topoisomerase I inhibitors and antibacterial agents. Target-based assays revealed that the fluoroquinophenoxazine derivatives with 9-NH2 and/or 6-substituted amine functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with IC50s ranging from 0.24 to 3.9 µM. Notably, 11a bearing the 6-methylpiperazinyl and 9-amino motifs was identified as one of the most potent topoisomerase I inhibitors (IC50 = 0.48 µM), and showed broad spectrum antibacterial activity (MICs = 0.78-7.6 µM) against all the bacteria strains tested. Compound 11g with the 6-bipiperidinyl lipophilic side chain exhibited the most potent antituberculosis activity (MIC = 2.5 µM, SI = 9.8). In addition, CoMFA analysis was performed to investigate the 3D-QSAR of this class of fluoroquinophenoxazine derivatives. The constructed CoMFA model produced reasonable statistics (q2 = 0.688 and r2 = 0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive correlation coefficient r2pred of 0.767. Collectively, these promising data demonstrated that fluoroquinophenoxazine derivatives have the potential to be developed as a new chemotype of potent topoisomerase IA inhibitors with antibacterial therapeutic potential.

Inhibition of Zn(II) binding type IA topoisomerases by organomercury compounds and Hg(II).

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3ß may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.

Synthesis and antibacterial evaluation of anziaic acid and analogues as topoisomerase I inhibitors.

Naturally occurring anziaic acid was very recently reported as a topoisomerase I inhibitor with antibacterial activity. Herein total synthesis of anziaic acid and structural analogues is described and the preliminary structure-activity relationship (SAR) has been developed based on topoisomerase inhibition and whole cell antibacterial activity.