CTP synthetase inhibitors as new potential agents for the treatment of African Trypanosomiasis : synthesis and in vitro/in vivo evaluation of trypanocidal activity of 3-Br-Acivicin

CTP synthetase (CTPS), a glutamine amidotransferase responsible for the de novo synthesis of cytidine triphosphate (CTP), was suggested to be a potential drug target for the treatment of Human African Trypanosomiasis. In fact, despite CTP being essential for cell survival, T. brucei has low pools of CTP compared to mammalian cells and completely lacks the ability to salvage cytosine or cytidine. Acivicin, an antibiotic isolated from the fermentation broths of Streptomyces sviceus and previously tested as an anti-tumor agent, was shown to behave as a CTPS inhibitor acting at the glutamine site; its inhibitory activity has been correlated to the observed trypanocidal activity in bloodstream T. brucei cell cultures. Since the proposed mechanism of action involves a nucleophilic attack of a thiol group to the C-3 of the isoxazoline nucleus, with displacement of the chlorine atom, we decided to compare the biological activity of Acivicin with that of its 3-bromo analogue. For this reason, we developed the first convenient synthesis of enantiomerically pure (aS,5S)-a-amino-3-bromo-4,5-dihydroisoxazol-5-yl acetic acid (3-Br-Acivicin) and tested it in parallel with Acivicin. Br-Acivicin was about 12 times more effective than Acivicin against trypanosomes, with an IC50 in the nanomolar range. Very interestingly, toxicity against HEK (s293) cell lines was moderate, and similar to that of Acivicin. Thus, Br-Acivicin seemed to have a better therapeutic window than Acivicin. Therefore, we decided to test Br-Acivicin in vivo for acute toxicity and efficacy against T. b. brucei. The pharmacological results obtained as well as preliminary structure-activity relationship studies will be presented and discussed.