Discovery of Strong 3-Nitro-2-Phenyl--Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite . There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. glucokinase (GlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of GlcK inhibitors that also exhibited anti- efficacy called the 3-nitro-2-phenyl-2-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited GlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds and were determined to inhibit GlcK quite well with IC values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2-chromene analogues, two of which were and (compounds and , respectively). Twelve of these compounds had a one-point change from the chemical structure of . The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the GlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl--chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.