N4-Substituted Piperazinyl Norfloxacin Derivatives with Broad-Spectrum Activity and Multiple Mechanisms on Gyrase, Topoisomerase IV, and Bacterial Cell Wall Synthesis.

Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.

Rational design, synthesis, molecular modeling, biological activity, and mechanism of action of polypharmacological norfloxacin hydroxamic acid derivatives.

Fluoroquinolones are broad-spectrum antibiotics that target gyrase and topoisomerase IV, involved in DNA compaction and segregation. We synthesized 28 novel norfloxacin hydroxamic acid derivatives with additional metal-chelating and hydrophobic pharmacophores, designed to enable interactions with additional drug targets. Several compounds showed equal or better activity than norfloxacin against Gram-positive, Gram-negative, and mycobacteria, with MICs as low as 0.18 µM. The most interesting derivatives were selected for in silico, in vitro, and in vivo mode of action studies. Molecular docking, enzyme inhibition, and bacterial cytological profiling confirmed inhibition of gyrase and topoisomerase IV for all except two tested derivatives (10f and 11f). Further phenotypic analysis revealed polypharmacological effects on peptidoglycan synthesis for four derivatives (16a, 17a, 17b, 20b). Interestingly, compounds 17a, 17b, and 20b, showed never seen before effects on cell wall synthetic enzymes, including MreB, MurG, and PonA, suggesting a novel mechanism of action, possibly impairing the lipid II cycle.

Design, Synthesis, Molecular Modeling, Biological Activity, and Mechanism of Action of Novel Amino Acid Derivatives of Norfloxacin.

Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.

A diselenide additive enables photocatalytic hydroalkoxylation of gem-difluoroalkenes.

A photocatalytic hydroalkoxylation reaction enables the coupling of aliphatic alcohols with gem-difluoroalkenes, expanding the scope of accessible α,α-difluorinated ethers, a desirable substructure for medicinal and agricultural chemists. This reaction exploits an uncommon diselenide co-catalyst to facilitate the net hydrofunctionalization process, which contrasts alternate single-electron reactions that deliver dioxidation products. Future use of this co-catalyst might enable other currently unknown photocatalytic reactions.

General Co-catalytic Hydrothiolation of gem-Difluoroalkenes.

Regioselective functionalization of gem-difluoroalkenes enables convergent late-stage access to fluorinated functional groups, though most functionalization reactions proceed through defluorinative functionalization processes that deliver mono-fluorovinyl products. In contrast, fewer reactions undergo net hydrofunctionalization to generate difluorinated products. Herein, we report a photocatalytic hydrothiolation of gem-difluoroalkenes that enables access to a broad spectrum of α,α-difluoroalkylthioethers. Notably, the reaction successfully couples nonactivated substrates, which expands the scope of accessible molecules relative to previously reported reactions involving organo- or photocatalytic strategies. Further, this reaction successfully couples biologically relevant molecules under aqueous conditions, highlighting potential applications in both late-stage and biorthogonal functionalizations.

Design, Synthesis, Molecular Modeling, and Anticancer Evaluation of New VEGFR-2 Inhibitors Based on the Indolin-2-One Scaffold.

A new series of indoline-2-one derivatives was designed and synthesized based on the essential pharmacophoric features of VEGFR-2 inhibitors. Anti-proliferative activities were assessed for all derivatives against breast (MCF-7) and liver (HepG2) cancer cell lines, using sunitinib as a reference agent. The most potent anti-proliferative derivatives were evaluated for their VEGFR-2 inhibition activity. The effects of the most potent inhibitor, 17a, on cell cycle, apoptosis, and expression of apoptotic markers (caspase-3&-9, BAX, and Bcl-2) were studied. Molecular modeling studies, such as docking simulations, physicochemical properties prediction, and pharmacokinetic profiling were performed. The results revealed that derivatives 5b, 10e, 10g, 15a, and 17a exhibited potent anticancer activities with IC50 values from 0.74-4.62 µM against MCF-7 cell line (sunitinib IC50 = 4.77 µM) and from 1.13-8.81 µM against HepG2 cell line (sunitinib IC50 = 2.23 µM). Furthermore, these compounds displayed potent VEGFR-2 inhibitory activities with IC50 values of 0.160, 0.358, 0.087, 0.180, and 0.078 µM, respectively (sunitinib IC50 = 0.139 µM). Cell cycle analysis demonstrated the ability of 17a to induce a cell cycle arrest of the HepG2 cells at the S phase and increase the total apoptosis by 3.5-fold. Moreover, 17a upregulated the expression levels of apoptotic markers caspase-3 and -9 by 6.9-fold and 3.7-fold, respectively. In addition, 17a increased the expression level of BAX by 2.7-fold while decreasing the expression level of Bcl-2 by 1.9-fold. The molecular docking simulations displayed enhanced binding interactions and similar placement as sunitinib inside the active pocket of VEGFR-2. The molecular modeling calculations showed that all the test compounds were in accordance with Lipinski and Veber rules for oral bioavailability and had promising drug-likeness behavior.

Enantioselective Total Synthesis of (+)-Sieboldine A.

The first total synthesis of (+)-sieboldine A was completed starting from 5-(p-methoxybenzyloxy)pentyne in 19 steps. The enantioselective Keck allylation provided the dienyne derivative, which was exposed to the Pauson-Khand conditions to afford the bicyclo[4.3.0]nonenone derivative with high stereoselectivity with an ee value of 93%. The following Ueno-Stork reaction formed the cis-hydrindane core with a quaternary carbon center. The late-stage Schmidt glycosylation led to the formation of the N-hydroxyazacyclononane ring.

Synthesis, biological investigation and molecular docking study of N-malonyl-1,2-dihydroisoquinoline derivatives as brain specific and shelf-stable MAO inhibitors.

A group of N-malonyl-1,2-dihydroisoquinoline derivatives were synthesized and investigated as brain specific and shelf-stable MAO inhibitors. N-malonyl-1,2-dihydroisoquinoline redox carrier system was linked through amidic bond to 4-chloro and 4-nitrobenzylidenehydrazines (9a-b), as monoamine oxidase inhibitors (MAOIs), and ß-phenethylamine (14), as a model drug, to afford a novel group of N-malonyl-1,2-dihydroisoquinoline chemical delivery systems (DHIQCDSs) (13a-b and 18). These systems are expected to be stable against air oxidation due to the presence of the carbonyl group close to nitrogen of the dihydroisoquinoline. The synthesized DHIQCDS (18) was subjected to various chemical and biological investigations to evaluate its stability and prove its ability to cross the blood brain barrier and "lock-in" the brain. The in vitro chemical and enzymatic oxidation studies showed reasonable stability and adequate rate of conversion of DHIQCDS (18) to its corresponding quaternary metabolites. In vivo distribution study in rats revealed preferential concentration of the active moiety in the brain. Moreover, compounds (9a-b, 12a-b and 17) were screened for their in vitro MAO inhibitory activity compared to clorgyline as a reference compound. The inhibition profile was found to be competitive for both MAO-A and MAO-B isozymes with more selectivity toward MAO-A. Molecular docking study of compounds (9a-b, 12a-b and 17) and the suggested metabolites was carried out on both MAO-A and MAO-B isozymes. Observation of the docked poses revealed many interactions with many residues previously reported to have an effect on the inhibition of MAO enzyme.