Tumor Targeting with Novel Pyridyl 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of De NovoPurine 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 10showing no activity, and 6> 9> 5> 8, with 10and 7being inactive, respectively. Antiproliferative effects toward FRα- and FRβ-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6was 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, 6showed <1 nM IC50, ∼2–3-fold more potent than 1. Compound 6inhibited glycinamide ribonucleotide formyltransferase in de novopurine biosynthesis and showed potent in vivoefficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.