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

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by <b>4</b>–<b>9</b> over the CH₂ analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations of <b>4</b> required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidines related to <b>1</b> provide targeted antifolates that warrant further evaluation as anticancer agents

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

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by <b>4</b>–<b>9</b> over the CH₂ analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations of <b>4</b> required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidines related to <b>1</b> provide targeted antifolates that warrant further evaluation as anticancer agents