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

Abstract

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. Electronic supplementary material The online version of this article (10.1186/s12936-019-2873-0) contains supplementary material, which is available to authorized users.

Adsorption to the Surface of Hemozoin Crystals: Structure‐Based Design and Synthesis of Amino‐Phenoxazine β‐Hematin Inhibitors

“…A follow‐up study showed that this compound inhibited growth of all stages, but affected ring‐stage parasites in particular at low concentration [40] . More recently, a series of acylphenoxazine compounds with low cytotoxicity against HEK293T and HepG2 cells, but potent selectivity for Plasmodium strains has been reported [41] . While the literature certainly points to the antimalarial potential of phenoxazine derivatives, this scaffold has not been considered specifically with respect to HZ inhibition.…”

“…[40] More recently, a series of acylphenoxazine compounds with low cytotoxicity against HEK293T and HepG2 cells, but potent selectivity for Plasmodium strains has been reported. [41] While the literature certainly points to the antimalarial potential of phenoxazine derivatives, this scaffold has not been considered specifically with respect to HZ inhibition. Thus given its highest overall ranking (3 rd place) for a nonantimalarial scaffold with respect to adsorption to the crystal surface, phenoxazine (T9) was our scaffold of choice moving forward to experimentally validate the adsorption hypothesis.…”

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“…The absorbance was measured using a microplate reader at 570 nm and 630 nm wavelengths. The percentage growth inhibition was calculated after subtracting the background and the blank followed by the determination of the IC 50 value from the dose–response curve (Davis et al, 2012; Riss et al, 2016; Tougan et al, 2019). The selectivity index is a measure of relative effectiveness and safety of an investigational drug which can be determined as the ratio of the cytotoxic concentration to the effective bioactive concentration.…”

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“…Since Guttmann and Ehrlich reported the clinical cure of two patients after oral administration of a synthetic dye, methylene blue which contains a phenothiazine moiety, in 1891, numerous synthetic dyes were investigated for their antimalarial properties [ 16 , 80 ]. The only adverse effect they reported was ‘periodic irritation of the bladder’ which was somewhat alleviated by taking nutmeg powder.…”