Human Toll-like Receptor (TLR) 8‑Specific Agonistic Activity in Substituted Pyrimidine-2,4-diamines

Activation of human toll-like receptor-8 (TLR8) evokes a distinct cytokine profile favoring the generation of Type 1 helper T cells. A multiplexed high-throughput screen had led to the identification of N4-butyl-5-iodo-6-methylpyrimidine-2,4-diamine as a pure TLR8 agonist, and a detailed structure–activity relationship study of this chemotype was undertaken. A butyl substituent at N4 was optimal, and replacement of the 5-iodo group with chloro, bromo, or fluoro groups led to losses in potency, as did the introduction of aromatic bulk. Drawing from our previous structure-based design, several 5-alkylamino derivatives were evaluated. Significant enhancement of potency was achieved in 5-(4-aminobutyl)-N4-butyl-6-methylpyrimidine-2,4-diamine. This compound potently induced Th1-biasing IFN-γ and IL-12 in human blood, but lower levels of the proinflammatory cytokines IL-1β, IL-6, and IL-8. These results suggest that the inflammatory and reactogenic propensities of this compound could be considerably more favorable than other TLR8 agonists under evaluation.人类Toll样受体8（TLR8）的激活可诱发独特的细胞因子谱，该谱有利于1型辅助性T细胞（Type 1 helper T cells）的生成。本研究通过多重高通量筛选，鉴定出N4-正丁基-5-碘-6-甲基嘧啶-2,4-二胺为纯TLR8激动剂，并针对该化学型开展了详细的构效关系研究。N4位的正丁基取代为最优取代模式；将5位碘原子替换为氯、溴或氟原子会导致活性下降，引入芳香性大位阻基团同样会造成活性损失。我们基于此前的基于结构的设计思路，对多款5位烷基氨基衍生物开展了活性评价，其中5-(4-氨基丁基)-N4-正丁基-6-甲基嘧啶-2,4-二胺的活性得到显著提升。该化合物可在人全血中强效诱导偏向1型辅助性T细胞（Th1）的干扰素γ（IFN-γ）与白细胞介素12（IL-12）的表达，而促炎细胞因子白细胞介素1β（IL-1β）、白细胞介素6（IL-6）及白细胞介素8（IL-8）的表达水平较低。上述结果表明，相较于当前在研的其他TLR8激动剂，该化合物的炎症与反应原性倾向显著更优。