Abstract 5493: Tumor-targeting with novel non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis.
Authors: Cherian Christina , Wang Yiqiang , Mitchell-Ryan Shermaine , Orr Steven , Hou Zhanjun , Gangjee Aleem , Matherly Larry H. 
Published: August 2014

Abstract
Interest has focused on folate receptor (FR) α for delivering cytotoxic folate-based therapeutics to solid tumors, as FRα is highly expressed in certain tumors, including ovarian and non-small cell lung cancers. While FRα is present in normal epithelial tissues, this is restricted to apical membranes and is inaccessible to the circulation. Selectivity of FR-targeted agents would be further enhanced if these were poor substrates for the ubiquitously expressed reduced folate carrier (RFC). Both 5- and 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolates with 3 (AG19, AG126, respectively) and 4 (AG23, AG127, respectively) carbon bridge lengths potently inhibited proliferation of KB human tumor cells via uptake by FRα and inhibition of de novo purine nucleotide biosynthesis at β-glycinamide ribonucleotide formyltransferase (GARFTase) (all drugs) and 5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase (AG126, AG127). Thienoyl-for-benzoyl B ring replacement in AG19 and AG23 increased inhibitory potencies, reflecting FRα uptake and increased GARFTase inhibition. To further examine the role in biological activities of the side chain benzoyl ring and terminal glutamate for this series, non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine analogs were synthesized without a phenyl ring but with 5-8 carbon substituents with (AG143, AG145, AG147, AG149, respectively) and without a terminal glutamate. 5-Substituted analogs of AG145, AG147, and AG149 were also synthesized. Compounds were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC (PC43-10) or FRα (RT16). Additional testing was performed with KB human tumor cells. Analogs without a terminal glutamate and 5-substituted non-benzoyl pyrrolopyrimidine analogs were all inactive. AG143, AG145, AG147, and AG149 were also inactive with RFC- expressing CHO cells. For FRα-expressing RT16 and KB cells, growth inhibition was detectable in the order: AG147 ∼ AG149 &gt; AG145 &gt;&gt; AG143. For AG147 and AG149, IC50s toward KB cells were ∼1 nM. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in binding affinities to FRα, measured by competition with [3H]folic acid. By protection experiments with excess thymidine or adenosine, de novo purine nucleotide biosynthesis was confirmed as the targeted pathway for the active analogs. 5-Amino-4-imidazolecarboxamide also reversed the growth inhibitory effects of this series, establishing GARFTase as the principal intracellular target. Collectively, our studies establish an important structure-activity relationship for FRα-targeted antifolates, namely that a side chain benzoyl group is not essential for tumor selective drug uptake by FRα and for inhibition of GARFTase.
Citation Format: Christina Cherian, Yiqiang Wang, Shermaine Mitchell-Ryan, Steven Orr, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Tumor-targeting with novel non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5493. doi:10.1158/1538-7445.AM2013-5493