Structural Optimization and Biological Evaluation of Isoxazolo[5,4-d]pyrimidines as Selective Toll-Like Receptor 7 Agonists

Toll-like receptors (TLRs) are components of innate immunity that play a crucial role in several diseases, including chronic inflammatory and infectious diseases, autoimmune diseases, and cancer. In particular, TLR7 has been identified as a key player in the innate immune response against viral infections and small-molecule TLR7 agonists have shown potential for vaccine therapy, for treatment of asthma and allergies, and as anticancer drugs. Inspired by our previous discovery of selective TLR7 agonists, our goal was to develop and introduce a new chemotype of TLR7 agonists by replacing the quinazoline ring with a new heterocycle isoxazolo-[5,4-d]-pyrimidine. Here, we report design, optimized synthesis, and structure-activity relationship studies of a novel class of TLR7 agonists based on the 6-(trifluoromethyl)-isoxazolo-[5,4-d]-pyrimidine-4-amine scaffold that demonstrate high selectivity and low micromolar potencies. The best-in-class agonist 21a, with an EC50 value of 7.8 mu M, also proved to be noncytotoxic and induced secretion of cytokines, including IL-1 beta, IL-12p70, IL-8, and TNF-alpha, indicating its potential to modulate the immune response.