Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, , produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo--quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a sponge species, and the first description of distinct chemotypes within a species of the family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.