Discovery of Novel Oxazolo[4,3‑<i>f</i>]purine Derivatives as Antitumor Agents through PPIA Interaction

54 novel oxazolo [4,3-f]purine derivatives were designed, synthesized, and evaluated for antitumor activity, among which compound 20b exhibited potent activity against several cancer cell lines. Compound 20b inhibited cell metastasis, arrested the cell cycle in the G0/G1 phase, and induced apoptosis in HCT116 cells. Mechanistic studies revealed that 20b increased ROS levels and led to DNA damage, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction in HCT116 cells. Limited proteolysis-small molecule mapping (LiP-SMap), drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) experiments provided evidence that compound 20b bound to PPIA with a KD value of 0.52 μM. siRNA assay indicated that 20b-mediated antiproliferative and antimigration activities were abolished and that the PPIA/MAPK signaling pathway was inhibited when PPIA was silenced in HCT116 cells. Significantly, compound 20b presented significant anticolorectal cancer efficacy in vivo without obvious toxicity. These results indicate that 20b may serve as a novel anticancer agent targeting PPIA, meriting further attention in antitumor drug research.本研究设计、合成并评价了54种新型恶唑并[4,3-f]嘌呤衍生物的抗肿瘤活性，其中化合物20b对多种癌细胞系均表现出强效活性。化合物20b可抑制HCT116细胞的转移、将细胞周期阻滞于G0/G1期，并诱导细胞凋亡。机制研究显示，20b可升高HCT116细胞内活性氧（Reactive Oxygen Species, ROS）水平，进而引发DNA损伤、内质网应激（Endoplasmic Reticulum Stress, ER）与线粒体功能障碍。有限蛋白水解-小分子作图法（Limited proteolysis-small molecule mapping, LiP-SMap）、药物亲和力响应靶点稳定性（Drug Affinity Responsive Target Stability, DARTS）实验、细胞热位移分析（Cellular Thermal Shift Assay, CETSA）以及表面等离子体共振（Surface Plasmon Resonance, SPR）实验均证实，化合物20b可与亲环蛋白A（Peptidylprolyl Isomerase A, PPIA）结合，其解离常数（KD）为0.52 μM。小干扰RNA（small interfering RNA, siRNA）实验结果显示，在HCT116细胞中敲低PPIA的表达后，20b介导的抗增殖与抗迁移活性被完全消除，且PPIA/丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）信号通路的活性受到抑制。值得注意的是，化合物20b在体内展现出显著的抗结直肠癌疗效，且未表现出明显的毒性。上述研究结果表明，20b可作为一种靶向PPIA的新型抗肿瘤候选药物，值得在抗肿瘤药物研发领域获得进一步关注。