Molecular networking-assisted dereplication reveals Callistenone Q, a potent antileishmanial phloroglucinol from Melaleuca viminalis.

Leishmaniasis remains a major neglected tropical disease for which current treatments are limited by toxicity, variable efficacy, and high cost. Natural products continue to represent an important source of new antiparasitic agents. In this study, a dereplication/bioactivity-guided approach was employed to investigate the antileishmanial constituents of Melaleuca viminalis leaves. The MeOH extract was partitioned into hexane, CH2Cl2, and EtOAc phases and their activities were evaluated against promastigote forms of Leishmania amazonensis. The CH2Cl2 phase showed the highest activity (EC50 = 36 ± 7 μg/mL) and was subjected to a bioactivity-guided fractionation combined with UHPLC-ESI-HRMS/MS dereplication and molecular networking (GNPS2). This approach revealed the presence of several metabolites, including the triterpenes 3-hydroxy-11-ursene-28,13-olide, betulinic acid, and uvaol, the fatty acid erucamide, and several phloroglucinol derivatives. Further purification led to the isolation of the previously undescribed phloroglucinol derivative 3,6-dihydroxy-1-isobutyryl-4-methoxy-3-methylcyclohexa-1,5-dien-2-one, named callistenone Q, whose structure was elucidated using HR-ESI-MS, NMR, IR, and ECD analysis. Evaluation of antileishmanial activity showed that callistenone Q exhibited potent activity against intracellular amastigote forms of L. amazonensis (EC50 = 5.4 ± 0.6 μg/mL), comparable to the reference drug miltefosine (EC50 = 7.6 ± 0.9 μg/mL), while displaying no cytotoxicity toward macrophages (CC50 > 100 μg/mL), resulting in a selectivity index (SI) higher than 18.5. These findings highlight M. viminalis as a promising source of bioactive phloroglucinols, with callistenone Q emerging as a potential scaffold for the development of new antileishmanial agents.