氟嘧啶类药物，尤其是5-氟尿嘧啶（5-FU），仍然是多种癌症治疗方案的关键组成部分，特别是用于结直肠癌和其他消化道恶性肿瘤。为了克服影响5-FU临床效用的关键限制和药理挑战，设计了5-氟脱氧尿嘧啶的磷酸酰胺酯化合物（NUC-3373），旨在提高其疗效和安全性以及管理上的挑战。利用亚IC50剂量的NUC-3373或5-FU处理人类结直肠癌细胞株HCT116和SW480，用液相色谱-质谱分析测量细胞内激活，用Western blot确定活性抗癌代谢物FdUMP与胸腺嘧啶合成酶（TS）和DNA修复的结合情况。我们证明NUC-3373生成的FdUMP比5-FU更多，结果产生了更强的TS抑制、DNA错配、细胞周期阻滞和DNA损伤。与5-FU不同，NUC-3373引起的无胸腺嘧啶死亡可以同时添加外源脱氧胸腺嘧啶来挽救。然而，5-FU之细胞毒性只能通过补充尿苷来逆转，而用于减少5-FU引起的毒性反应的治疗方法。这与我们的发现相一致，即5-FU会生成包括在RNA中的FUTP，这是导致骨髓抑制和消化道炎症的机制。总之，这些结果强调了NUC-3373和5-FU之间由抗癌代谢物驱动的关键差异。NUC-3373是TS的有效抑制剂，也会引起DNA定向损伤。这些数据支持初步的临床证据，表明NUC-3373 在患者中具有良好的安全性。©2023年。作者（们）发布。

Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU.Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage.We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU.Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.© 2023. The Author(s).