The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit. In our laboratory, we have performed a cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system. Several interesting scaffolds were discovered. One of the hit compounds was a pyrimido[5,4-b]indole (1Z65) and was identified as a potent NF-κB activator. This potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. In addition, computational studies supported that active compounds in this scaffold appeared to bind primarily to MD-2 in the TLR4/MD-2 complex[1]. 1Z65 contain the fused ring structure known to bind DNA [2], and may have the potential for cell toxicity. On the other hand, we previously reported that the N-5 methylated derivative had lower toxicity in mouse cells than the non-methylated derivative. In this study, in order to reduce toxicity and maintain stimulation of TLR4, we designed and synthesized analogs of the pyrimido[5,4-b]indole scaffold (figure 1). These compounds are truncated at the benzo group, and substituted by alkyl groups for reduced DNA binding affinity. In addition, N-5 position of pyrrolopyrimidine was substituted with a methyl group. Several starting materials were used in 4-7 steps to prepare 8 final compounds which were each analyzed for TLR4 activation, cell toxicity, and DNA binding affinity. In this activity analysis, the cyclohexyl compound 3 had 74% stimulation activity, normalized to 1Z-65. The dimethyl and non-substituted derivatives had no activity (less than 1%). Therefore, it appears that bulky groups, such as cyclohexyl and benzo, are important for TLR4 stimulation potency. In addition, we present also results of studies on a small molecular TLR-7 ligand conjugated with a sugar (glucosamine). References [1] Chan, M.; Hayashi, T.; Carson, A, D.; Cottam, B, H.; et al. J. Med. Chem., 2013, 56, 4206−4223. [2] Wang, LR.; Wang, Y.; Chen, JW.; Guo, LH. Toxicology., 2009, 3, 250–7.

Modification and characterization of TLR4 and TLR7 ligands obtained by high throughput screening

251st National Meeting (2016)

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