Tumor Targeting with Novel Pyridyl 6‑Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>De Novo</i> Purine Nucleotide Biosynthesis

Tumor-targeted specificities of 6-substituted pyrrolo­[2,3-d]­pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3′,6′ (5, 8), 2′,5′ (6, 9), and 2′,6′ (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and β were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRβ-expressing cells were reflected in competitive binding with [3H]­folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (∼4-fold more potent than 1) and inhibited [3H]­methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, ∼2–3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.