虽然胸腺嘧啶酸合成酶（TYMS）抑制剂已经作为化疗方案的组成部分，但目前可以获得的抑制剂会诱导TYMS过度表达或改变叶酸运输/代谢反馈途径，肿瘤细胞利用这些途径获得耐药性，从而限制了总体疗效。在这里，我们报道了一种小分子TYMS抑制剂，它i）表现出比当前的氟嘧啶和抗叶酸更强的抗肿瘤活性，而不会诱导TYMS过度表达，ii）与传统的抗叶酸结构不同，iii）在胰腺移植瘤模型和hTS/Ink4a/Arf null基因工程小鼠肿瘤模型中可以延长生存期，iv）耐受性良好，无论是腹腔注射还是口服都具有同等疗效。在机制上，我们确认该化合物是一种多功能的非经典抗叶酸，通过一系列类似物，我们确定了直接抑制TYMS的结构特征，同时保持了抑制双氢叶酸还原酶（DHFR）的能力。总之，该研究确定了新的非经典抗叶酸抑制剂，优化了对胸腺嘧啶生物合成的抑制，并具有良好的安全性，突出了增强癌症治疗的潜力。

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibits enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in both pancreatic xenograft tumor models and hTS/Ink4a/Arf null genetically engineered mouse tumor model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non-classical antifolate, and using a series of analogues, we identify structural features allowing direct TYMS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non-classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy.