Fluorine-Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>de Novo</i> Purine Nucleotide Biosynthesis

Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo­[2,3-d]­pyrimidine analogues 7–12 were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds 8, 9, 11, and 12 showed increased in vitro antiproliferative activities (∼11-fold) over the nonfluorinated analogues 2, 3, 5, and 6 toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds 8, 9, 11, and 12 also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, 9, 11, and 12 showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the de novo purine biosynthesis pathway. Molecular modeling studies validated in vitro cell-based results. NMR evidence supports the presence of an intramolecular fluorine–hydrogen bond. Potent in vivo efficacy of 11 was established with IGROV1 xenografts in severe compromised immunodeficient mice.