Bisarylthiourea comprising cycloalkyl-thiophene-3-carboxylate derivatives as potential Human Toll-Like Receptor (TLR)2 Agonists: Design, Synthesis, and Structure-Activity Relationship Enabling the Switch from TLR2/1 to TLR2/6 Activation

Abstract

Toll-like receptor 2 (TLR2) acts as a pivotal family of pattern-recognition receptors that bridges innate and adaptive immunity through the detection of PAMPs and DAMPs. In pursuit of novel immunomodulators, a series of bisarylthiourea (BAT) comprising cycloalkyl-thiophene-3-carboxylate derivatives was rationally designed, synthesized, and evaluated as potential human TLR2 agonists. Systematic SAR studies were performed through strategic modifications at five distinct regions of the scaffold. The structural integrity of the synthesized derivatives was confirmed by 1 H and 13 C NMR, HRMS, and FT-IR analysis, with two analogues further confirmed by single-crystal XRD. The agonistic activity was assessed using various HEK-Blue human pan-TLR-2, -2/1 and -2/6 reporter assays. Among the series, compounds 10f and 12d exhibited the strongest agonistic activity with an EC 50 of 6.6 μM and 1.3 μM on pan-hTLR2, respectively, with selectivity for the TLR2/1 heterodimer, and no activity for the TLR2/6 heterodimer. In contrast, compound 10a displayed pan-hTLR2 potency with an EC 50 value of 6.6 μM, but selectively stimulated the TLR2/6 heterodimer (EC 50 = 8.8 μM), with no activity for the TLR2/1 heterodimer. Notably, 10f induced a dose-dependent cytokine response in human peripheral blood mononuclear cells, inducing IL-6, IL-8, IL-10, IL-1β and TNF-α secretion. Furthermore, all potent compounds, including 10f, were non-toxic toward HEK-Blue hTLR2 cells (IC 50 >100 μM) and exhibited a favourable ADMET profile, highlighting their potential as promising therapeutic candidates. Collectively, these results identify 10f as a novel small-molecule hTLR2 agonist scaffold and provide a foundation for the rational design of TLR2 heterodimer-specific modulators for future immunotherapeutic applications.