A-315 Synthesis and biological evaluation of orally active anti-Trypanosoma agents

Abstract Background Human African Trypanosomiasis (HAT), or sleeping sickness, is a life-threatening disease endemic to sub-Saharan Africa, caused by the protozoan parasite Trypanosoma brucei. The disease progresses from hemolymphatic to neurological stages, often leading to death if untreated. Current treatments are limited by severe side effects and the necessity for parenteral administration, underscoring the urgent need for safe, effective, and orally bioavailable therapies. Previous studies have identified tubulin inhibitors with promising in vitro activity against T. brucei; however, their clinical application is hindered by poor oral bioavailability. This study aims to optimize these inhibitors to enhance their oral efficacy and therapeutic potential. Methods A series of 30 novel tubulin inhibitor analogs were synthesized using combinatorial chemistry techniques, focusing on reducing molecular weight and hydrophobicity to improve solubility and oral bioavailability. The anti-trypanosomal activity of these compounds was evaluated in vitro using T. brucei brucei cultures, while cytotoxicity assays were conducted on human kidney (HEK293) and mouse macrophage (RAW264.7) cell lines to assess selectivity. Compounds demonstrating low micromolar inhibitory concentrations (IC50) against T. brucei and minimal cytotoxicity to mammalian cells. Structure-activity relationship (SAR) analyses and molecular docking studies were performed to elucidate binding affinities to the trypanosome tubulin homolog. The most promising compound, referred to as compound 7, was selected for in vivo evaluation. In vivo efficacy was assessed using an acute mouse infection model. Mice were intraperitoneally injected with 1×106 T. brucei cells to induce infection. Compound 7 was administered orally for three consecutive days at dosages up to 50 mg/kg/day. Daily diagnostic microscopic examinations of tail blood samples were conducted to monitor parasitemia levels. Body weights of the mice were measured before and after the treatment period to evaluate the compound*s impact. Results Several synthesized analogs exhibited potent in vitro anti-trypanosomal activity, with IC50 values in the low micromolar range, effectively inhibiting T. brucei proliferation without significant cytotoxic effects on mammalian cells. SAR analysis revealed that reducing aromatic moieties and molecular weight correlated with enhanced activity and selectivity. Molecular docking studies indicated a strong correlation between binding energies to trypanosome tubulin and observed anti-parasitic activity. Compound 7, in particular, demonstrated a favorable binding profile and was advanced to in vivo studies. In the acute mouse infection model, the control group exhibited a steady rise in parasitemia, peaking at an average of 40 million parasites/mL in the blood by day 5. In contrast, mice treated with compound 7 experienced a significant reduction in parasite numbers, averaging 10 million parasites/mL on day 5. These findings strongly suggest a potential protective effect of compound 7. Conclusion The optimization of tubulin inhibitors has led to the identification of compound 7, which exhibits potent oral activity against T. brucei in vitro and in vivo. This compound represents a promising development of orally administered therapies for HAT, addressing the limitations of current treatment regimens. Evaluating the pharmacokinetics, and efficacy of compound 7 in clinical settings is essential to determine its potential as a more accessible and effective treatment option for populations affected by HAT.