Abstract 4765: Synthesis and evaluation of nonclassical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates: Role of terminal amino acid moiety in membrane transport and antitumor activity.
Authors: Gangjee Aleem , Kumar Lalit , Cherian Christina , Orr Steven , Etnyre Erika , Matherly Larry H. 
Published: August 2012

Abstract
Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) ≤ and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates is due to their lack of selectivity for tumor cells over normal cells, since these drugs are transported by RFC. Antifolates with selective substrate activity for FRs and/or PCFT over RFC can potentially target tumors that express these non-RFC transporters relative to normal tissues. Thus, antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four carbon bridge length and with L-aspartic acid (AG170), aminomalonic acid (AG173) and α-glutamic acid (AG172) were designed and synthesized in place of the normal L-glutamic acid (AG118) of this series to evaluate the importance of L-glutamic acid for RFC, FRα, and PCFT transport and inhibition of cell proliferation. AG170 was slightly less active than AG118 in inhibiting growth of a CHO cell line (RT16) expressing human FRα (IC50s of 18.7 and 2.6 nM, respectively) but similarly active in inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 81.7 and 63.8, nM, respectively). AG173 and AG172 were inactive toward both RT16 and R2/PCFT4 cells (IC50&gt;1000nM). All four analogues were inactive toward a CHO cell line (PC43-10) expressing human RFC but not FRα or PCFT. These analogues were further evaluated in KB and IGROV-1 human tumor cell lines in culture. Whereas AG118 was potently inhibitory toward KB and IGROV1 tumor cells (IC50s of 0.27 and 1.4 nM, respectively), AG170, AG173, and AG172 all were inert toward these tumor cells at concentrations up to 1000 nM. Collectively, these results establish an important role for the terminal amino acid in the anti-proliferative effects of this series of 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The disparate inhibitions of AG170 toward FRα-expressing CHO versus human tumor cells implies that there may be differences in metabolic pathways and/or target enzymes in CHO cell lines compared to human cancer cell lines that are responsible.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4765. doi:1538-7445.AM2012-4765