Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region

Marine-derived heterocycles : structural, synthetic and biological investigations

This dissertation describes synthetic, structural and biological studies on three different groups of heterocyclic marine natural products. Chapter 2 describes the assignment of the absolute configuration of phorbasides A and B, cytotoxic macrolide glycosides from the marine sponge Phorbas sp. The synthesis of a series of ene-yne chlorocyclopropane model compounds is described; the circular dichroism spectrum for each model is presented and compared to the spectra obtained for the natural products. The relationship between configuration and structure of the extended ene-yne chromophore and the observed Cotton effect is discussed. Chapter 3 describes i) The characterization of three new antazirine analogues from the marine sponge Dysidea fragilis; ii) The total synthesis of (Z)-dysidazirine, a related 2H-azirine natural product isolated in 1988 from Dysidea fragilis, and iii) The synthesis of a series of dysidazirine analogues. The antifungal activity of all compounds against a panel of clinically-relevant pathogenic fungi is reported. The data is used to develop a basic Structure- Activity-Relationship for antifungal activity of long- chain 2H-azirine carboxylates. Implications for the mechanism of action of (Z)-dysidazirine are discussed. Chapter 4 details the first total synthesis of the cytotoxic polyketide macrololide enigmazole A, isolated from the marine sponge Cynachyrella enigmatica. Enigmazole A was synthesized in 22 steps (longest linear sequence) and 0.41% overall yield from known compounds. The development of a method for preparation of functionalized oxazol-2-yl zinc reagents by direct zinc insertion is described. Oxazol-2-yl zincates were found to undergo palladium catalyzed Negishi coupling and copper catalyzed acylation reactions. The application of this methodology to the preparation of the oxazole-containing side chain of enigmazole A is presente

Marine-derived heterocycles : structural, synthetic and biological investigations

This dissertation describes synthetic, structural and biological studies on three different groups of heterocyclic marine natural products. Chapter 2 describes the assignment of the absolute configuration of phorbasides A and B, cytotoxic macrolide glycosides from the marine sponge Phorbas sp. The synthesis of a series of ene-yne chlorocyclopropane model compounds is described; the circular dichroism spectrum for each model is presented and compared to the spectra obtained for the natural products. The relationship between configuration and structure of the extended ene-yne chromophore and the observed Cotton effect is discussed. Chapter 3 describes i) The characterization of three new antazirine analogues from the marine sponge Dysidea fragilis; ii) The total synthesis of (Z)-dysidazirine, a related 2H-azirine natural product isolated in 1988 from Dysidea fragilis, and iii) The synthesis of a series of dysidazirine analogues. The antifungal activity of all compounds against a panel of clinically-relevant pathogenic fungi is reported. The data is used to develop a basic Structure- Activity-Relationship for antifungal activity of long- chain 2H-azirine carboxylates. Implications for the mechanism of action of (Z)-dysidazirine are discussed. Chapter 4 details the first total synthesis of the cytotoxic polyketide macrololide enigmazole A, isolated from the marine sponge Cynachyrella enigmatica. Enigmazole A was synthesized in 22 steps (longest linear sequence) and 0.41% overall yield from known compounds. The development of a method for preparation of functionalized oxazol-2-yl zinc reagents by direct zinc insertion is described. Oxazol-2-yl zincates were found to undergo palladium catalyzed Negishi coupling and copper catalyzed acylation reactions. The application of this methodology to the preparation of the oxazole-containing side chain of enigmazole A is presente

Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade

Transannular Diels–Alder/1,3-Dipolar Cycloaddition Cascade of 1,3,4-Oxadiazoles: Total Synthesis of a Unique Set of Vinblastine Analogues

A powerful tandem [4 + 2]/[3 + 2] cycloaddition cascade of 1,3,4-oxadiazoles initiated by a transannular [4 + 2] cycloaddition is detailed. An impressive four rings, four carbon–carbon bonds, and six stereocenters are set on each site of the newly formed central six-membered ring in a cascade thermal reaction that proceeds at temperatures as low as 80 °C. The resulting cycloadducts provide the basis for the synthesis of unique analogues of vinblastine containing metabolically benign deep-seated cyclic modifications at the C3/C4 centers of the vindoline-derived subunit of the natural product

Optimization of CoaD Inhibitors against Gram-Negative Organisms through Targeted Metabolomics

Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure-activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type E. coli

Discovery and Optimization of Phosphopantetheine Adenylyltransferase Inhibitors with Gram-Negative Antibacterial Activity

In the preceding manuscript [Moreau et al. 2018, 10.1021/acs.jmedchem.7b01691] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli

Optimization of CoaD Inhibitors against Gram-Negative Organisms through Targeted Metabolomics

Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure–activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type <i>E. coli</i>