Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t_1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC₅₀ ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

中文翻译：

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磷酸抗原（ProPAgens）的芳氧基二酯氨基磺酸盐前药可作为Vγ9/Vδ2T细胞免疫反应的有效激活剂。

Vγ9/Vδ2T细胞被称为磷酸抗原（PAgs）的含焦磷酸盐的小分子激活。这些PAg中焦磷酸基团的存在因其不稳定性和极性性质而限制了它们的类药物性质。在这项工作中，我们报告了一种新型的短Grubbs烯烃复分解介导的PAg（E）-4-羟基-3-甲基-3-甲基-2-丁烯基焦磷酸酯（HMBP）的亚甲基和二氟亚甲基单膦酸酯衍生物的合成及其芳氧基二酯磷酸亚氨基酯前药，称为ProPAgens。这些前药在人血清中表现出出色的稳定性（t _1/2 > 12 h），并有效激活Vγ9/Vδ2T细胞（EC ₅₀范围从5 fM到73 nM），这转化为亚纳摩尔γδT细胞介导的体外消除膀胱癌细胞。另外，计算机模拟和体外酶促测定的组合证明了这些膦酰胺化物的代谢以释放未掩盖的PAg单膦酸酯类。总的来说，这项工作确立了HMBP单膦酸ProPAgens作为理想的候选药物，可以作为新的癌症免疫治疗剂进行进一步研究。