A series of azasterol derivatives, designed as potential inhibitors of the ⌬ 24 -sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.

Graphical abstract

In this article, the design and synthesis of some novel azasterols is described, followed by their evaluation against Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum, the causative agents of human African trypanosomiasis, Chagas disease, leishmaniasis, and malaria, respectively. Some of the compounds showed anti-parasitic activity. In particular, a number of compounds appeared to very potently inhibit the growth of the blood stream form T. b. rhodesiense, with one compound giving an IC50 value of 12 nM. Clear structure activity relationships could be discerned. These compounds represent important leads for further optimization. Azasterols have previously been shown to inhibit sterol biosynthesis in T. cruzi and L. donovani by the inhibition of the enzyme sterol 24-methyltransferase. However, in this case, none of the compounds showed inhibition of the enzyme. Therefore, these compounds have an unknown mode of action.