Small-molecule inhibitors targeting programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interactions can compensate for the shortcomings of antibody-based inhibitors and have attracted considerable attention, some of which have already entered clinical trials. Herein, based on our previous study on small-molecule PD-L1 inhibitors, we reported a series of 8-(o-tolyl)quinazoline derivatives by the skeleton merging strategy. Homogenous time-resolved fluorescence (HTRF) assay against PD-1/PD-L1 interaction identified compound A5, which showed the most potent inhibition with an IC₅₀ value of 23.78 nM. Meanwhile, based on the results of HTRF assay, the structure–activity relationships (SARs) of the tail were focused on. Cell-based PD-1/PD-L1 blockade assay further revealed that A5 significantly blocked the PD-1/PD-L1 interaction at 1.1 μM in the co-culture system of Jurkat-NFAT-PD-1 cells and Hep3B-OS8-hPD-L1 cells with no significant cytotoxicity on Jurkat cells. Moreover, the proposed binding mode of A5 was investigated by a docking analysis. These results indicate that compound A5 is a promising lead compound that deserves further investigation.

中文翻译：

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小分子 PD-1/PD-L1 拮抗剂 8-(邻甲苯基)喹唑啉衍生物的设计、合成和评价

靶向程序性细胞死亡-1/程序性细胞死亡-配体1（PD-1/PD-L1）相互作用的小分子抑制剂可以弥补基于抗体的抑制剂的缺点，并引起了相当大的关注，其中一些已经进入临床试验。在此，基于我们前期对小分子PD-L1抑制剂的研究，我们通过骨架合并策略报道了一系列8-(邻甲苯基)喹唑啉衍生物。针对 PD-1/PD-L1 相互作用的均相时间分辨荧光 (HTRF) 测定鉴定出化合物A5，其显示出最有效的抑制作用，IC ₅₀值为 23.78 nM。同时，根据HTRF测定的结果，重点研究了尾部的构效关系（SAR）。基于细胞的 PD-1/PD-L1 阻断测定进一步表明，在 Jurkat-NFAT-PD-1 细胞和 Hep3B-OS8 的共培养系统中，1.1 μM 的A5显着阻断 PD-1/PD-L1 相互作用。 hPD-L1细胞对Jurkat细胞没有明显的细胞毒性。此外，通过对接分析研究了A5的拟议结合模式。这些结果表明化合物A5是一种有前途的先导化合物，值得进一步研究。