Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure–activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5–1 μg/mL) and Acinetobacter baumannii (8–16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.


5-Bromo-2-((tert-butyldimethylsilyloxy)methyl)-4-chloro-1-methyl-1H-indole (A5).
To a solution of A4 (7.10 g, 18.94 mmol) in DMF (55 mL) at 0 o C was added NaH (60%, 0.91 g, 22.75 mmol) in portions. Upon completion of the addition, the reaction mixture was warmed to room temperature, stirred for 10 min and then cooled to 0 o C and MeI (1.8 mL, 28.91 mmol) was added. The reaction mixture was warmed to room temperature, stirred for 30 min and then cooled to 0 o C and quenched by the addition of NH 4 Cl (aq. satd.
Crude imine (0.858 g, 1.69 mmol) and trimethyl methanetricarboxylate (0.55 g, 2.89 mmol) were mixed together in Ph 2 O (4 mL). The stirred mixture was placed onto a pre-heated heat block at 230 o C and heated for 10 min after initial bubbling of MeOH was observed (occurs at ~160 o C internal reaction temperature). The reaction mixture was cooled to room temperature, loaded directly on a silica gel column, eluted with hexanes to remove Ph 2 O and followed by an EtOAc/hexanes gradient (0-70%) to yield A12 as a yellow foam (0.5425 g, 50% over 2 steps). LC−MS m / z = 633. 5
DNA gyrase mutant expression and purification. Site directed mutagenesis to generate mutant gyrase A subunit (gyrase SD-LY ) was carried out using the Invitrogen's GeneArt site directed mutagenesis PLUS kit according to the manufacturer's protocol. Primers were generated using Invitrogen's "Oligo Designer". Accuprime pfx DNA polymerase was used for increased fidelity of the pcr reactions. Plasmid DNA pET15b containing the wild-type E. coli DNA gyrase was used as the template. After sequence verification (Genewiz) of the mutation in the pcr product, the NdeI/XmaI restriction digested fragment containing the gyrase A mutant sequence was ligated into a similarly digested plasmid pET15b. The plasmid DNA containing the mutant gyrase A subunit was then transformed into competent BL21 (ƛDE3) pLysS cells for expression and purification of the mutant gyrase A subunit.
Purification of the histidine tagged gyrase A subunit (WT or mutant), was carried out by modification of a combination of methods. 3,4 Briefly, 1L cultures of the E. coli BL21(ƛDE3) pLysS containing the plasmid of interest (WT or mutant gyrase subunit A) was grown in Luria-Bertani (LB) medium containing the selective antibiotic. Induction of the protein was carried out by adding 0.5 mM IPTG to log phase culture and allowing additional growth overnight at 18 o C. The bacterial cell pellet was allowed to incubate for 30 min on ice in 40 mL of buffer containing 20mM Tris-HCl (pH 7.9), 300 mM NaCl, 0.1% triton x-100, 10% glycerol and 0.1% (weight/vol.) lysozyme for lysis. The lysate was then briefly sonicated (2 x 10 sec bursts) to reduce viscosity and then centrifuged at 20,000 x g for 30 minutes. The soluble fraction was then mixed with 5 mL of a 50% Ni-NTA slurry which was pre-equilibrated with buffer N (20 mM Tris-HCl (pH7.9), 300 mM NaCl, 0.1% triton x-100, 10% glycerol) and allowed to equilibrate for a minimum of 1 h after which the slurry was poured into a column and the unbound proteins were collected in the flow through fraction. The Ni-NTA column was then washed with 15 to 20 column volumes of buffer N with added 50 mM imidazole and washes were collected in column volume fractions to assess the protein content by a gel electrophoresis. The gyrase subunit A was then eluted with buffer N containing 200 mM imidazole in column volume fractions. The various fractions collected were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and those containing the gyrase subunit A were pooled and dialyzed against 4L of TED (50 mM Tris pH 8.0, 1 mM EDTA, and 2 mM dithiothreitol). The dialysate was then subjected to anion exchange chromatography on a Mono Q HR 10/10 column (run by a FPLC system) which was equilibrated in TED buffer. The gyrase subunit A was eluted using a 1M NaCl gradient in the same buffer. Peak fractions containing the protein were pooled and concentrated using centrifugal concentrators (Millipore), and then subjected to desalting and buffer exchange by size exclusion chromatography on a Superdex 200 column equilibrated in 10 mM Tris pH 7.5, 50 mM KCl, 0.1 mM EDTA and 2 mM DTT. Peak fractions containing the protein were pooled and concentrated and stored at −80 ºC in the presence of 25% glycerol.
The purified mutant gyrase A SD-LY was then reconstituted with WT gyrase B subunit (purchased from Topogen) in a 1:1 molar ratio at 15 micromolar concentration in a storage certified animal facilities.

Pharmacokinetic studies in mice
The pharmacokinetics of test compounds was evaluated in CD-1 mice. Compounds were dosed at 10 mg/kg IV in 10% DMSO and 1% Tween 80 in pH 8.0 phosphate buffer in CD-1 mice. Blood was collected by terminal cardiac puncture (3 mice per time point) at 0.083, 0.25, 1, 2, 4, 7, 16, and 24 hours after dosing. Plasma drug levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Briefly, the plasma samples were treated with acetonitrile-methanol mixture containing an internal standard that is a close analog of the test compounds. Pharmacokinetic parameters were calculated using a noncompartmental model using WinNonlin (Phoenix, Pharsight; St. Louis MO).

Mouse model of bacterial-induced lethality
The mice were made neutrophil-deficient with the use of cyclophosphamide IP administration at 150 mg/kg Day -4 and 100 mg/kg cyclophosphamide on Day -1. CD-1 mice were inoculated with Gram negative bacteria (E. coli, 1x10 6 CFU/mouse) mixed with 5% mucin and injected intraperitoneally. Morbidity and mortality was monitored twice per day. Moribund mice were euthanized.