In vitro and in vivo characterization of anti-malarial acylphenoxazine derivatives prepared from basic blue 3

Background Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Plasmodium falciparum , Trypanosoma cruzi , Trypanosoma brucei rhodesiense , and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated. Methods Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model. Results All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, Plasmodium berghei NK65 strain. Conclusions The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.