Novel 5‑Substituted Pyrrolo[2,3‑<i>d</i>]pyrimidines as Dual Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5‑Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase and as Potential Antitumor Agents

A new series of 5-substituted thiopheneyl pyrrolo­[2,3-<i>d</i>]­pyrimidines <b>6</b>–<b>11</b> with varying chain lengths (<i>n</i> = 1–6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo­[2,3-<i>d</i>]­pyrimidines <b>2c</b> and <b>2d</b> with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds <b>6</b>–<b>11</b> inhibited KB human tumor cells in the order <b>9</b> = <b>10</b> > <b>8</b> > <b>7</b> > <b>6</b> = <b>11</b>. Compounds <b>8</b>–<b>10</b> were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of <b>8</b> and <b>9</b> appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure–activity relationship for transport and dual target inhibition