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-<i>d</i>]­pyrimidine analogues <b>7</b>–<b>12</b> 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 <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> showed increased <i>in vitro</i> antiproliferative activities (∼11-fold) over the nonfluorinated analogues <b>2</b>, <b>3</b>, <b>5</b>, and <b>6</b> toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, <b>9</b>, <b>11</b>, and <b>12</b> showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the <i>de novo</i> purine biosynthesis pathway. Molecular modeling studies validated <i>in vitro</i> cell-based results. NMR evidence supports the presence of an intramolecular fluorine–hydrogen bond. Potent <i>in vivo</i> efficacy of <b>11</b> was established with IGROV1 xenografts in severe compromised immunodeficient mice