Identification and immunological evaluation of novel TLR2 agonists through structural optimization of Diprovocim

As an important family member of Toll-like receptors (TLRs), TLR2 can recognize various pathogen-associated molecular patterns (PAMPs) such as bacteria and viral components. Accumulating evidence demonstrates that TLR2 agonists play a critical role in cancer immunotherapy and infectious diseases. Diprovocim is the most potent small molecule TLR2 agonist known, showing remarkably immune adjuvant activity in mice. However, the further clinical research and development of Diprovocim was hampered because of its structural complexity as well as high molecular weight. Here, we designed and synthesized 21 structurally simplified derivatives of Diprovocim, performed their TLR2 agonistic activities by HEK-Blue hTLR2 SEAP assay, and evaluated the toxicity in two human normal cell lines. Compounds B3-B4 and B9-B12 with excellent TLR2 agonistic activity were found through the structure-activity relationship study. Among them, diastereomer B10 and B12 substituted (S)-2-phenylcyclopropylamide side chain of Diprovocim with simple (R)- and (S)-n-butyl groups exhibited comparable TLR2 agonistic activities with EC₅₀ values of 35 nM and 39 nM, respectively. ELISA and western blot experiments on THP-1 cells showed that B10 and B12 displayed remarkable immunostimulatory activity in the release of various inflammatory cytokines through activating MyD88-dependent NF-κB and MAPK signaling pathways. Importantly, B10 and B12 have less structural complexity and better safety compared to Diprovocim, and the chiral center of right pyrrolidine ring has negligible influence on TLR2 activition. Our study provides simplified Diprovocim derivatives with high agonistic activity, providing a clue to further optimize Diprovocim.