Synthesis and antitumor activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidines as potential nonclassical antifolates targeting both thymidylate and purine nucleotide biosynthesis

• Published in: European journal of medicinal chemistry 2015-03, Vol.93, p.142-155
• Main Authors: Liu, Yi, Zhang, Chuang, Zhang, Hongying, Li, Meng, Yuan, Jiangsong, Zhang, Yurui, Zhou, Jiaqi, Guo, Huicai, Zhao, Lijuan, Du, Yumin, Wang, Lei, Ren, Leiming
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - chemical synthesis; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Antiproliferation; Cell Culture Techniques; Cell Cycle - drug effects; Cell Line, Tumor; Cell Proliferation - drug effects; Cell Survival - drug effects; Drug discovery; Folic Acid Antagonists - chemical synthesis; Folic Acid Antagonists - chemistry; Folic Acid Antagonists - pharmacology; Humans; Molecular Docking Simulation; Molecular Structure; Purine biosynthesis; Purine Nucleotides - antagonists & inhibitors; Pyrimidines - chemical synthesis; Pyrimidines - chemistry; Pyrimidines - pharmacology; Pyrroles - chemical synthesis; Pyrroles - chemistry; Pyrroles - pharmacology; Pyrrolo[2,3-d]pyrimidines; Structure-Activity Relationship; Thymidylate biosynthesis; Thymidylate Synthase - antagonists & inhibitors
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/j.ejmech.2015.01.055

A novel series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential nonclassical antifolates targeting both thymidylate and purine nucleotide biosynthesis. Condensation of 2,4-diamino-6-hydroxypyrimidine with ethyl-4-chloroacetoacetate and subsequent hydrolysis afforded the key intermediate, 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid. Coupling with various amino acid methyl esters followed by saponification and condensation with 3-(aminomethyl)pyridine provided target compounds 1–9. The new compounds exhibited micromolar to submicromolar antiproliferative potencies against a panel of tumor cell lines including KB, A549 and HepG2. Growth inhibition of compound 2 toward KB cells resulted in cytotoxicity and G1/G2-phase accumulation, and was partially protected by excess thymidine and adenosine, but was completely reversed in the combination of thymidine and adenosine, indicating both thymidylate and de novo purine nucleotide synthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both glycinamide ribonucleotide formyltransferase (GARFTase) and AICA ribonucleotide formyltransferase (AICARFTase). The results of the docking studies show that 2 could bind and inhibit both thymidylate synthase (TS) and the two folate-dependent purine biosynthetic enzymes (GARFTase and AICARFTase), which is consistent with the results of in vitro metabolic assays. Our studies establish that compound 2 is an excellent lead analog as a multitargeted antifolate for further structure optimization. [Display omitted] •Pyrrolo[2,3-d]pyrimidines were synthesized as potential nonclassical antifolates.•Compounds exhibited antiproliferative potencies against several tumor cell lines.•Growth inhibition resulted in cytotoxicity and G1/G2-phase accumulation.•Thymidylate and purine nucleotide synthesis were indicated as the targeted pathway.•The docking results are consistent with the results of in vitro metabolic assays.