Systematic Optimization of Potent and Orally Bioavailable Purine Scaffold as a Dual Inhibitor of Toll-Like Receptors 7 and 9

Abstract

Several toll-like receptors (TLRs) reside inside endosomes of specific immune cellsamong them, aberrant activation of TLR7 and TLR9 is implicated in myriad contexts of autoimmune diseases, making them promising therapeutic targets. However, small-molecule TLR7 and TLR9 antagonists are not yet available for clinical use. We illustrate here the importance of C2, C6, and N9 substitutions in the purine scaffold for antagonism to TLR7 and TLR9 through structure–activity relationship studies using cellular reporter assays and functional studies on primary human immune cells. Further in vitro and in vivo pharmacokinetic studies identified an orally bioavailable lead compound 29, with IC50 values of 0.08 and 2.66 μM against TLR9 and TLR7, respectively. Isothermal titration calorimetry excluded direct TLR ligand–antagonist interactions. In vivo antagonism efficacy against mouse TLR9 and therapeutic efficacy in a preclinical murine model of psoriasis highlighted the potential of compound 29 as a therapeutic candidate in relevant autoimmune contexts.

Development, Optimization, and In Vivo Validation of New Imidazopyridine Chemotypes as Dual TLR7/TLR9 Antagonists through Activity-Directed Sequential Incorporation of Relevant Structural Subunits

“…The conceptual design of the compounds was influenced by our previous experience with the essential structural features and geometrical arrangements in different chemotypes such as benzoxazole, , oxoadenine, − imidazoquinoline, quinazoline, − and other cores. − To identify the minimal substitutions required to attain TLR7/9 antagonism, initially, 7–11 were prepared with small substituents at the C4′ and C2′ positions. The C2′ substitution was to impart obvious geometrical restrictions in the molecule.…”

“…Sequential incorporation of relevant structural subunits in a basic molecular framework of imidazopyridine at C7, C3, C2, and C2′ positions provided compounds 13 , 16 , 22 , 25 , 26 , 36 , 37 , and 41 as the prospective lead candidate. To further optimize these lead candidates we evaluated them in a panel of in vitro ADME assays (Tables and ). ,,, These compounds have different substitutions at C7, C3, and C2′ positions. Thus, this would provide an understanding of the correlation between in vitro pharmacokinetic properties and different structural modifications in the imidazopyridine scaffold for lead selection.…”

“…TLR7 and TLR9 activation with their respective ligands lead to identical downstream signaling pathways initiated by the association with their adaptor protein Myd88 and culminating in the transcriptional activation of several inflammatory genes such as type I interferons or pro-inflammatory cytokines depending on the cell type and the endosomal localization of the ligands. , Dual antagonists targeting TLR7/9 serve as important tools to prevent undesirable activation and production of type I interferons in various inflammatory disease contexts. Antimalarial drugs chloroquine and hydroxychloroquine, with established inhibitory action on endosomal TLR activation, have been in clinical use against several major autoimmune diseases such as rheumatoid arthritis. , Several oligonucleotide TLR7/TLR9 antagonists such as IMO-8400, IMO-3100, and IMO-9200 have undergone evaluation in clinical trials to monitor their safety and efficacy in patients diagnosed with autoimmune disorders. − Pfizer initiated a clinical trial of a small molecule (CpG-52364) with a quinazoline core, but it failed to pass the phase I stage. ,, Several groups along with us have also previously demonstrated systematic development of potent TLR7 and TLR9 small-molecule antagonists based on benzoxazole, , quinazoline, − imidazoquinoline, and purine − scaffolds by employing different medicinal chemistry strategies. − Through agonist-to-antagonist strategy, we identified a chemical switch in the purine scaffold leading to a switch from TLR7 agonism to antagonism . An activity-guided strategy was employed for the development of selective TLR9 and dual TLR7/TLR9 antagonists in the quinazoline scaffold. , …”

Application and prospect of targeting innate immune sensors in the treatment of autoimmune diseases

“…For example, ( +)-naltrexone [( +)-NTX], an antagonist of TLR2 and TLR4, was effective at treating MS-related memory deficits with both lower- and higher-dose in EAE mice but failed to alleviate EAE-induced motor deficits [ 148 ]. TLR7/9 antagonists, compound 29 and IRS-954 (immunoregulatory DNA sequence-954), showed therapeutic efficacy in a preclinical murine model of psoriasis and lupus, respectively [ 149 , 150 ]. Wang et al found that total coumarins from Urtica dentata Hand could prevent murine autoimmune diabetes by suppressing the TLR4 signaling pathways in DCs [ 151 ].…”

Integration of Ligand-Based and Structure-Based Methods for the Design of Small-Molecule TLR7 Antagonists

“…The antimalarial agent hydroxychloroquine (HCQ), which operates as a nonselective endosomal TLR inhibitor, is widely utilized in the treatment of various autoimmune disorders [28]. Various researchers have reported molecules with quinazoline [29,30], benzoxazole [31][32][33], imidazopyridine [34,35], purine [36][37][38], and other varied scaffolds [39,40] that have been significantly investigated for the development of selective small-molecule TLR7 antagonists in several recent studies through empirical screenings and activity-guided strategies.…”