Abstract 3823: 2-Methyl-6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates as selective folate receptor substrates, glycinamide ribonucleotide formyltransferase inhibitors and antitumor agents

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The important role of reduced folates in one-carbon transfer reactions has rendered folate metabolism an attractive target in cancer chemotherapy for decades. Clinically used antifolates enter cells via folate uptake systems, such as the reduced folate carrier (RFC), folate receptors (FRs), and the proton-coupled folate transporter (PCFT). Toxicity of clinically used antifolates is due to their lack of selectivity for tumor cells, since these antifolates are transported by RFC which occurs in normal as well as tumor cells. FRs are not usually expressed in normal cells. However, the ≤ isoform of FR is abundantly expressed in certain tumor cells, such as ovarian, endometrial, etc. Thus, selective FR-targeted antifolates that also possess cytotoxic activity without transport by RFC would likely circumvent the major toxicities of currently used antifolates. 2-Methyl-6-substituted pyrrolo[2,3-d]pyrimidine antifolates were designed to evaluate the importance of the 2-amino group for transport by RFC, FR, and /or PCFT, as well as for inhibition of α-glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis, compared with the 2-amino analogs. The most active compound in our series, AG179, showed an IC50 of 1.6 nM in inhibiting proliferation of KB human tumor cells, and was ∼3-fold less potent than the corresponding 2-amino compound AG71 (0.55 nM). In Chinese hamster ovary (CHO) cells engineered to individually express the major folate transporters, AG179 also inhibited growth of FRα-expressing RT16 cells but was inactive toward RFC-expressing PC43-10 and PCFT-expressing R2/PCFT4 CHO cells. The 2-amino analog, AG71, is inert toward RFC but is avidly transported by PCFT and is cytotoxic toward PCFT-expressing cells. In KB cells, growth inhibition by AG179 like AG71 is protected by excess adenosine and 5-amino-4-imidazole carboxamide, establishing GARFTase and de novo purine biosynthesis as the likely intracellular targets. These results establish that replacement of the 2-amino group of AG71 with a methyl group has a limited impact on FRα uptake and anti-proliferative activity, however, this replacement has a profound impact on cellular uptake by PCFT. This report will present the details of the design, synthesis, biological evaluation and molecular modeling of the title compounds. 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 3823. doi:1538-7445.AM2012-3823