Structure–Activity\nRelationship Studies in\nBenzothiadiazoles as Novel Vaccine Adjuvants

Extracellular\nvesicles (EVs) can transfer antigens and immunomodulatory\nmolecules, and such EVs released by antigen-presenting cells equipped\nwith immunostimulatory functions have been utilized for vaccine formulations.\nA prior high-throughput screening campaign led to the identification\nof compound <b>634</b> (<b>1</b>), which enhanced EV release\nand increased intracellular Ca<sup>2+</sup> influx. Here, we performed\nsystematic structure–activity relationship (SAR) studies to\ninvestigate the scaffold for its potency as a vaccine adjuvant. Synthesized\ncompounds were analyzed in vitro for CD63 reporter activity (a marker\nfor EV biogenesis) in human THP-1 cells, induction of Ca<sup>2+</sup> influx, IL-12 production, and cell viability in murine bone-marrow-derived\ndendritic cells. The SAR studies indicated that the ester functional\ngroup was requisite, and the sulfur atom of the benzothiadiazole ring\nreplaced with a higher selenium atom (<b>9f</b>) or a bioisosteric\nethenyl group (<b>9h</b>) retained potency. Proof-of-concept\nvaccination studies validated the potency of the selected compounds\nas novel vaccine adjuvants.