Discovery of novel selective TLR7 agonists based on chromeno[3,4-d]imidazol-4(1H)-one and 2-(trifluoromethyl)quinoline/ quinazoline-4-amine scaffold

Toll-like receptors (TLRs) are pattern-recognition receptors involved in host cell recognition and initiation of immune responses against microbes. Among 10 functional TLRs identified in human, TLR7 and TLR8, which specifically recognize single-stranded RNAs, have been considered as emerging therapeutic targets for several life-threatening diseases, i.e. viral infections, autoimmune diseases and cancers.

In our study, two different ligand-based virtual screening protocols with imiquimod as a query compound were used for identification of novel TLR7 ligands. The first hit compound with potent TLR7 antagonist activity was 1-isobutylchromeno[3,4-d]imidazol-4(1H)-one, which was used a starting point for further exploration of the chemical space around the chromenoimidazolone scaffold. The synthesis of a small library of selected analogs led to the discovery of chromenoimidazolone-based TLR7 agonists with EC₅₀ values in low micromolar range. The second in-house ligand-based virtual screening protocol LiSiCA was used for discovery of two hits TLR7 agonists that were topologically most similar to imiquimod, i.e. 2-(trifluoromethyl)quinolone and quinazoline derivatives. Synthesis of a focused library of analogs, biological evaluation on HEK293-hTLR7/8 cells, and docking studies provided systematic exploration of structure-activity relationship indicating that a secondary or tertiary amine with smaller flexible alkyl substituents or bulkier rigid aliphatic rings on the position 4 of 2-(trifluoromethyl)quinoline/ quinazoline scaffold is required for potent TLR7 agonist activity. N-Cyclopropyl-2-(trifluoromethyl)quinazolin-4-amine with EC₅₀ of 16.8 µM was able to induce increased levels of proinflammatory cytokines IL-6 and IL-8. Our study demonstrated successful in-silico discovery and further synthesis of novel TLR7 over TLR8-selective compounds as promising chemical probes for development of small-molecule immunomodulators.