Abstract 2706: Tumor-targeting with novel pyridyl 6-substituted pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor (FR) α and the proton-coupled folate transporter (PCFT) and inhibition of de novo purine nucleotide biosynthesis
Authors: Cherian Christina , Wang Lei , Wallace Adrianne , Orr Steven , Hou Zhanjun , Gangjee Aleem , Matherly Larry H. 
Published: May 2015

Abstract
Recent interest has focused on exploiting non-reduced folate carrier (RFC) uptake processes including FRα and PCFT for delivering cytotoxic folate-based therapeutics to solid tumors. FRα is highly expressed in certain tumors, including ovarian and non-small cell lung cancers, whereas PCFT is expressed in most solid tumors and is active at pHs characterizing the tumor microenvironment. Although FRα is present in normal epithelial tissues, this is restricted to apical membranes and is inaccessible to the circulation. PCFT is essentially inactive at neutral pH characterizing most normal tissues. Selectivity of FR-targeted agents would be enhanced if these were also poor substrates for RFC. We previously demonstrated potent antiproliferative activities for a novel 6-substituted-pyrrolo[2,3-d]pyrimidine benzoyl antifolate with a 4-carbon bridge length (AG23). With KB human tumor cells, AG23 inhibited cell proliferation via FRα uptake and inhibition of de novo purine biosynthesis at β-glycinamide ribonucleotide formyltransferase (GARFTase), resulting in ATP depletion. We replaced the side-chain benzoyl ring of AG23 with a pyridine to generate region-isomeric pyridyl 6-substituted-pyrrolo[2,3-d]pyrimidine antifolates, AG105 (3N, 6N), AG107 (2N, 5N), and AG109 (2N, 6N). AG105, AG107, and AG109 were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC (PC43-10), FRα (RT16), or PCFT (R2/PCFT4) and the results compared to those for AG23. Additional testing was performed with KB cells. All analogs were inactive with RFC-expressing CHO cells. For FRα-expressing RT16 and KB cells, growth inhibition was in the order, AG107&gt;AG23&gt;AG105&gt;&gt;AG109. For AG107, the IC50 toward KB cells was ∼0.6 nM, slightly exceeding that for AG23. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in high affinity binding to FRα, measured by competition with [3H]folic acid. AG107, but not AG105 or AG109, inhibited proliferation of R2/PCFT4 CHO cells expressing PCFT without FRα (IC50∼58 nM; ∼4-fold lower than for AG23). AG107 bound to PCFT, as reflected in inhibition of [3H]methotrexate uptake at pH 5.5 and 6.8; AG105 and AG109 were also inhibitory, albeit less than AG107. By protection experiments with excess thymidine or adenosine, de novo purine biosynthesis was confirmed as the targeted pathway. GARFTase was inhibited in KB cells in situ at concentrations approximating those for AG23 and which inhibited cell proliferation. Our results establish an interesting structure-activity relationship for inhibitory pyridyl 6-substituted-pyrrolo[2,3-d]pyrimidine antifolates which promotes efficacy toward FRα- and PCFT-expressing cells.
Citation Format: Christina Cherian, Lei Wang, Adrianne Wallace, Steven Orr, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Tumor-targeting with novel pyridyl 6-substituted pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor (FR) α and the proton-coupled folate transporter (PCFT) and inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2706. doi:10.1158/1538-7445.AM2014-2706