Heliyon · 8 authors, 7 centres
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Seventeen synthetic 1,4-benzothiazine derivatives were tested for the first time against Acanthamoeba castellanii. Compounds IX, X, and XVI showed the most potent anti-amoebic activity, significantly reducing trophozoite viability and inhibiting encystation and excystation. These findings suggest that 1,4-benzothiazine derivatives, especially X and XVI with low cytotoxicity, are promising candidates for developing new treatments against Acanthamoeba infections.
**Background:** Acanthamoeba species are free-living protists that can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE), a CNS infection with >90% mortality. Current treatments are limited by the double-walled cyst stage, which resists many drugs and allows recurrence. There is an urgent need for new therapeutic agents effective against both trophozoites and cysts, with low human cell toxicity. 1,4-Benzothiazine derivatives are known for diverse biological activities, including CNS effects, but have not been tested against free-living amoebae. This study synthesized seventeen 1,4-benzothiazine derivatives and evaluated their anti-amoebic potential against Acanthamoeba castellanii.
**Methods:** Seventeen 1,4-benzothiazine derivatives (I–XVII) were synthesized via a base-catalyzed one-pot reaction of 2-amino thiophenol with substituted bromoacetophenones. Four compounds (VII, IX, XI, XVI) were new; the rest were known. Structures were confirmed by EI-MS, 1H NMR, and 13C NMR. Anti-amoebic activity was tested against A. castellanii (T4 genotype, ATCC 50492) trophozoites at 50 and 100 μM for 24 h. Encystation and excystation assays were performed at the same concentrations for 72 h. Cytotoxicity was assessed against human keratinocyte (HaCaT) cells using an LDH assay kit. Statistical analysis used Student's t-test (two-tailed, p<0.05).
**Key Results:** All 17 compounds showed significant anti-amoebic activity against trophozoites in a dose-dependent manner. The most potent were compounds IX, X, and XVI, reducing viability to 5.37×10^4 cells/mL (p<0.0003), 2.00×10^5 cells/mL (p<0.006), and 2.48×10^5 cells/mL (p<0.002), respectively, at 100 μM. In encystation assays, compounds IX, XVI, and X reduced cyst numbers to 5.62×10^4, 9.87×10^4, and 1.15×10^4, respectively, at 100 μM. In excystation assays, compound IX was most effective, followed by XI and XVI, reducing emerging trophozoites to 1.87×10^4, 1.15×10^4, and 7.37×10^4, respectively; compound XI showed results equivalent to chlorhexidine. Cytotoxicity against HaCaT cells at 100 μM was low to moderate: compound X showed only 2% toxicity, compound XVI 21%, and compound IX 36%. Most other compounds showed <10% toxicity.
**Clinical Implications:** This study identifies 1,4-benzothiazine derivatives as a new class of anti-amoebic agents effective against both trophozoites and cysts of A. castellanii. Compounds X and XVI combine potent anti-amoebic activity with low cytotoxicity, making them promising leads for developing treatments for Acanthamoeba keratitis and GAE. Compound IX, despite higher cytotoxicity, may be suitable as a disinfectant for contact lenses or surfaces. These findings address the critical need for drugs that can prevent recurrence by inhibiting encystation and excystation. Further studies are needed to elucidate mechanisms of action, evaluate in vivo efficacy, and optimize structure-activity relationships.