Abstract 3239: Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution
Authors: Gangjee Aleem , Mohan Rishabh , Ravindra Manasa , Wallace-Povirk Adrianne , O’Connor Carrie , Dekhne Aamod , Hou Zhanjun , Matherly Larry H. 
Published: July 2017

Abstract
Three specialized systems exist for membrane transport of folates and antifolates across biological membranes. The reduced folate carrier (RFC) is ubiquitously expressed in tumors and normal tissues. Folate receptors (FR) α and β, and the proton-coupled folate receptor (PCFT) exhibit narrow patterns of tissue expression and serve specialized physiologic roles. FRs are expressed in a number of cancer cells (e.g., FRα in ovarian cancer and non-small cell lung cancer; FRβ in acute myelogenous leukemia), whereas PCFT is expressed in a large number of human solid tumors but not leukemias. FRs are either inaccessible to the circulation (FRα) or are nonfunctional (FRβ) in normal tissues. PCFT transport is limited in most normal tissues, given the requirement of acidic pH for optimal activity. These properties facilitate tumor targeting of cytotoxic compounds with specificities for PCFT or/and FR transport. Clinically used antifolates such as methotrexate and pemetrexed are substrates for RFC and their uptake by both normal tissues and tumors confers dose-limiting toxicity due to limited tumor selectivity. We previously reported a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate with a 4-carbon bridge and a phenyl glutamate side chain (AGF127). Previous studies of related 6-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge region plays an important role in determining tumor cell potency and transport selectivity. Thus, it was of interest to vary the length and insert heteroatoms into the side chain -(CH2)4- of AGF127, including -(CH2)n-S- (compound 1, n= 3 and compound 2, n = 4) and -(CH2)n-O- (compound 3, n= 3 and compound 4, n = 4), to determine the impact on drug potency and transport selectivity. The novel analogs were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells expressing human FRα (RT16), RFC (PC43-10), and PCFT (R2/PCFT4). AGF127 showed potent inhibition of CHO cells expressing FRα (IC50 = 8.6 nM), and reduced activity toward CHO cells expressing RFC (57 nM) or PCFT (840 nM). Compounds 1, 2, and 3, showed reduced inhibitions toward FRα-expressing RT16 cells (4-35-fold) compared to AGF127; however, compound 4 was equipotent to AGF127. Like AGF127, none of the series were effective PCFT substrates. Compound 3, like AGF127, inhibited RFC-expressing PC43-10 cells (IC50 = 37 nM); RFC activity was reduced 6- to &gt;20-fold for compounds 1, 2, and 4. Toward FRα-, RFC-, and PCFT-expressing KB human tumor cells, the order of potency was 4 = AGF127 &gt; 2 = 3 = 1. Compound 4 was also more active (15-fold) than pemetrexed with KB cells. Collectively, the potent and selective activity of compound 4 toward FRα-expressing tumor cells and limited activity toward RFC-expressing cells offers significant advantages over AGF127 and clinically used non-targeted antifolates, suggesting that further preclinical evaluation is warranted.
Citation Format: Aleem Gangjee, Rishabh Mohan, Manasa Ravindra, Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2017-3239