Identification of High-Potency Human TLR8 and Dual TLR7/TLR8 Agonists in Pyrimidine-2,4-diamines

The induction of toll-like receptor
7 (TLR7)-dependent type I interferons
(IFN-α/β) from plasmacytoid dendritic cells as well as
the production of TLR8-dependent type II interferon (IFN-γ),
TNF-α, and IL-12 in myeloid dendritic cells are of importance
in generating T helper-1 biased adaptive immune responses. In an effort
to identify novel dual TLR7/TLR8-active compounds, we undertook structure–activity
relationship studies in pyrimidine 2,4-diamines, focusing on substituents
at C5. Several analogues substituted with aminopropyl appendages at
C5 displayed dominant TLR8-agonistic activity. N4-Butyl-6-methyl-5-(3-morpholinopropyl)­pyrimidine-2,4-diamine
was found to be a very potent dual TLR7/TLR8 agonist. Employing novel
cytokine reporter cell assays, we verified that potency at TLR7 correlates
with IFN-α/β production in human blood, whereas IFN-γ
and TNF-α induction is largely TLR8-dependent. Dual TLR7/TLR8
agonists markedly upregulate CD80 expression in multiple dendritic
cell subsets, providing insight into the immunological basis for the
superior adjuvantic properties of such innate immune stimuli.