In our continued efforts to develop targeted prodrugs activated by prostate-specific antigen (PSA), we designed and synthesized novel phosphoramide mustard peptide conjugates using previously optimized PSA substrates. Initial Nu/Nu mouse PK studies indicated that prodrug I (glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) exhibits high clearance with significant extrahepatic metabolism in vivo. Substrate optimization studies were thus carried out to further improve PSA specificity and enable the design of prodrugs with reduced in vivo clearance and enhanced tumor selectivity. To assess the utility of the newly optimized sequences as promoieties, they were coupled to phosphoramide mustard using a 4-amino-2-fluorobenzyl alcohol linker akin to prodrug I. In the presence of human PSA, prodrug I was rapidly cleaved with a half-life (t_1/2) of 35 min. Prodrugs II (glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) and III (GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) were hydrolyzed at slower rates with t_1/2 values of 80 and 107 min, respectively. These results we observed here are different from our previously reported data but may be explained by the fact that PSA-activated release of phosphoramide mustard and reactive quinonimine methides resulted in mechanism-based inhibition of PSA, thereby preventing further hydrolysis of prodrugs I–III. Prodrug I was cytotoxic to PSA-producing LNCaP cells with an IC₅₀ value of 7.3 μM and demonstrated 14-fold selectivity over the non-PSA-producing DU145. Despite its poor in vitro antiproliferative activity (IC₅₀ = 30 µM), prodrug III was found to be more stable against non-PSA-mediated hydrolysis compared with prodrug I as revealed by metabolite profiling studies, which was in agreement with its improved stability in human hepatocyte cultures. These results suggested that a combination of the peptide sequence GABA ← mGly-Ala-Ser-Chg-Gln with optimal linkers and/or other cytotoxic agents can help achieve an adequate balance between PSA cleavage rate and enhanced resistance to non-PSA-mediated hydrolysis.

中文翻译：

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作为前列腺特异性抗原激活的前药的新型4-肽亚氨基-2-氟苄基磷酰胺芥末共轭物的合成和评价

在我们继续努力开发被前列腺特异性抗原（PSA）激活的靶向前药的过程中，我们使用先前优化的PSA底物设计和合成了新型磷酰胺芥末肽结合物。最初的Nu / Nu小鼠PK研究表明，前药I（戊二酰-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-磷酰胺芥末）表现出很高的清除率，并且在体内具有明显的肝外新陈代谢。因此，进行了底物优化研究，以进一步提高PSA特异性，并使设计的前药具有降低的体内清除率和增强的肿瘤选择性。为了评估新优化序列作为原基的实用性，使用类似于前药I的4-氨基-2-氟苄基醇接头将其与磷酰胺芥末偶联。在人PSA的存在下，前药I的半衰期（t _1/2）为35分钟，迅速裂解。前药II（戊二酰-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-磷酰胺芥末）和III（GABA←mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-磷酰胺（芥末）的水解速度较慢，t _1/2值分别为80和107分钟。我们在这里观察到的这些结果与以前报道的数据不同，但可以用以下事实解释：PSA活化的磷酰胺芥子油和反应性奎宁胺甲基化物的释放导致基于机理的PSA抑制，从而防止前药I–III进一步水解。前药我对产生PSA的LNCaP细胞具有细胞毒性，IC ₅₀值为7.3μM，与未产生PSA的DU145相比，具有14倍的选择性。尽管其体外抗增殖活性差（IC ₅₀  = 30 µM），但代谢物谱分析研究表明，前药III与前药I相比对非PSA介导的水解更稳定，这与前者的稳定性有所提高相一致。人肝细胞培养。这些结果表明，肽序列GABA←mGly-Ala-Ser-Chg-Gln与最佳连接子和/或其他细胞毒剂的组合可帮助在PSA裂解率和增强的抗非PSA介导的水解抗性之间实现适当的平衡。