Discovery of Imidazoquinolines with Toll-Like Receptor 7/8 Independent Cytokine Induction

Abstract

Toll-like receptors (TLRs) are key targets in the design of immunomodulating agents for use as vaccine adjuvants and anticancer treatments. The imidazoquinolines, imiquimod and resiquimod, have been shown to activate TLR-7 and -8 which in turn induce cytokine production as part of the innate immune response. Herein, we report the synthesis and discovery of a C7-methoxycarbonyl derivative of imiquimod that stimulates cytokine production but is devoid of TLR-7/8 activity. Data is presented that shows this analog not only induces IL-12p40 and TNF production, similar to that of imiquimod and resiquimod, but greatly enhances the production of IL-1β, a key cytokine involved in activation of CD4 T cells. It is further demonstrated that TLR-7/8 activation can be recovered by the addition of a C2-alkyl substituent to this newly discovered analog. The results support the existence of an alternative mechanism of action by which imidazoquinolines can stimulate cytokine production.

The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

“…The phagosome is an efficient platform for the recognition of diverse ligands, sometimes involving co-operative interactions between TLR8 and other TLRs [56], or even other classes of PRRs [57]. Additionally, our lab has previously shown that CL075 can also activate DCs via other classes of PRRs, such as the inflammasome [17], which is supported by reports that imidazoquinolines can trigger induction of inflammatory cytokines in the absence of TLR 7 or 8 agonist activity [58]. Lastly, using molecular modeling studies, Govindaraj et al demonstrated that the binding affinity of the TLR8 agonist R848 plays a key role in species-specificity [59].…”

Toll‐Like Receptor 7 Inactive Ligands Enhanced Cytokine Induction by Conjugation to Weak Antigens

“…Clinical trials of imiquimod were discontinued owing to its serious adverse side effects. [5] Imidazoquinoline analogues such as imiquimod, [6] resiquimod, [6] gardiquimod, [7] (1), [7] and 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (2)a re all potent agonists of this pathway ( Figure 1A). Their ability to triggering remarkable levels of cytokinep roduction were revealed upon their conjugation with antigenst hat have weak immunogenicity.T his discovery demonstrated that TLR 7c an be activated by coupling an antigen to the terminal carboxyl group at N9 of the inactive ligand adenine analogues.…”

“…[1][2][3] They respondu niversally to the presence of viral genomic ssRNA within the cytoplasm. [6][7][8]16] For the guaninea nalogues, SAR studies showedt hat analogues with thiazolo[4,5-d]pyrimidine [17] or pyrazolo[3,4-d]pyrimidine [18] ring systemsa re all active. [5] Imidazoquinoline analogues such as imiquimod, [6] resiquimod, [6] gardiquimod, [7] …”

“…[8,16] SAR resultsr evealed that either a2 -hydroxy-2-methylpropyl or benzyl group at N1, and an n-butyl group at C2 are indispensable for the most potent TLR 7a gonists. [6][7][8]16] For the guaninea nalogues, SAR studies showedt hat analogues with thiazolo[4,5-d]pyrimidine [17] or pyrazolo[3,4-d]pyrimidine [18] ring systemsa re all active. [19] As hort alkyl (2-4 methylene units) or aryl alkyl chain at N7;t hio, oxo,o rs eleno substituents at C8;a nd alkyl, aryl alkyl, or morpholino groups at C2' or C3' of the sugar ring are required for,orbeneficial to, activity.T he analogues were also usually found to be active if the sugar ring was replaced with alkyl or aryl alkyl moieties at the N9 position.…”

“…[1][2][3] They respondu niversally to the presence of viral genomic ssRNA within the cytoplasm. [5] Imidazoquinoline analogues such as imiquimod, [6] resiquimod, [6] gardiquimod, [7] 4-amino-2-butyl-1-(2-hydroxy-2-methylpropyl)-7-methoxycarbonyl-1H-imidazo [4,5-c]quinolone (1), [7] and 1-benzyl-2-butyl-1H-imidazo [4,5-c]quinolin-4-amine (2)a re all potent agonists of this pathway ( Figure 1A). [5] Imidazoquinoline analogues such as imiquimod, [6] resiquimod, [6] gardiquimod, [7] 4-amino-2-butyl-1-(2-hydroxy-2-methylpropyl)-7-methoxycarbonyl-1H-imidazo [4,5-c]quinolone (1), [7] and 1-benzyl-2-butyl-1H-imidazo [4,5-c]quinolin-4-amine (2)a re all potent agonists of this pathway ( Figure 1A).…”