Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Evaluation of the metabolism, bioactivation and pharmacokinetics of triaminopyrimidine analogs toward selection of a potential candidate for antimalarial therapy.

1. During the course of metabolic profiling of lead Compound 1, glutathione (GSH) conjugates were detected in rat bile, suggesting the formation of reactive intermediate precursor(s). This was confirmed by the identification of GSH and N-acetylcysteine (NAC) conjugates in microsomal incubations. 2. It was proposed that bioactivation of Compound 1 occurs via the formation of a di-iminoquinone reactive intermediate through the involvement of the C-2 and C-5 nitrogens of the pyrimidine core. 3. To further investigate this hypothesis, structural analogs with modifications at the C-5 nitrogen were studied for metabolic activation in human liver microsomes supplemented with GSH/NAC. 4. Compounds 1 and 2, which bear secondary nitrogens at the C-5 of the pyrimidine core, were observed to form significant amounts of GSH/NAC-conjugates in vitro, whereas compounds with tertiary nitrogens at C-5 (Compound 3 and 4) formed no such conjugates. 5. These observations provide evidence that electron/hydrogen abstraction is required for the bioactivation of the triaminopyrimidines, potentially via a di-iminoquinone intermediate. The lack of a hydrogen and/or steric hindrance rendered Compound 3 and 4 incapable of forming thiol conjugates. 6. This finding enabled advancement of compound 4, with a desirable potency, safety and PK profile, as a lead candidate for further development in the treatment of malaria.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

4-aminoquinolone piperidine amides: noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Rat thalamic α4ß2 neuronal nicotinic acetylcholine receptor occupancy assay using LC-MS/MS.

INTRODUCTION: In vivo brain receptor occupancy has been the key assay in driving preclinical drug discovery program and there is a need to hasten this screening step. Radiolabeled methods, which are time consuming and expensive, are most widely employed to measure receptor occupancy. Thus we sought to develop and validate an alternative novel approach for measuring rat brain α4ß2 neuronal nicotinic acetylcholine receptor occupancy using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS). METHODS: Tracer optimization studies like in vivo dose and time dependent brain regional distribution; saturation binding and blocking study with nicotine and atropine were carried out for ZW-104 in rats. Assay validity was tested by pretreatment with potent α4ß2 ligands; TC-1734, cytisine, ABT-089, ABT-594 and A-366833. Receptor occupancy along with plasma and brain exposure levels of α4ß2 ligand was measured in the same set of animals. RESULTS: The regional distribution of ZW-104 in rat was found to be, thalamus>frontal cortex>striatum>hippocampus>cerebellum, and is in accordance with the distribution and regional densities of α4ß2 nAChRs measured using [¹8F]ZW-104 in mice and baboons. Pretreatment with nicotine and α4ß2 ligands dose dependently reduced the binding of ZW-104 in the thalamus. Non-nicotinic antagonist atropine did not alter the binding of ZW-104 in the thalamus, indicating the tracer specificity. The ED50 values calculated for occupancy were found to be 3.01, 0.83, 14.81, 0.001 and 0.11 mg/kg for TC-1734, cytisine, ABT-089, ABT-594, and A-366833, respectively. DISCUSSION: These findings demonstrate that non-radiolabeled ZW-104 is suitable for determining the α4ß2 receptor occupancy in rat brain. The LC-MS/MS based receptor occupancy assay is a rapid method and allows the generation of occupancy data along with the brain and plasma concentration in the same group of animals.