Abstract 5492: Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3-d]pyrimidine scaffold.
Authors: Gangjee Aleem , Zhao Sai , Kumar Lalit , Cherian Christina , Orr Steven , Huang Jenny , Hou Zhanjun , Matherly Larry H. 
Published: August 2014

Abstract
We have been extensively involved in the elaboration of substituted pyrrolo[2,3-d]pyrimidines to obtain potential targeted agents without reduced folate carrier (RFC) activity. Lack of selectivity for RFC is a major cause of significant toxicity in the clinic associated with antifolate anticancer agents. These efforts have led to the discovery of several series of potent targeted agents, which showed specificity for folate receptors (FRs) and/or the proton coupled folate transporter (PCFT). These transport systems are two targets for selective cancer chemotherapy. One such targeted agent, AG94, was reported by us as the most efficacious targeted antifolate antitumor agent with PCFT and FR selectivity over RFC. AG94 is a GARFTase inhibitor and its targeted transport and inhibition of GARFTase was demonstrated to be responsible for its selective in vitro and in vivo antitumor activity. We were interested in carrying out a structure-activity relationship study (SAR) with AG94 as the lead compound. For this study we elected the glutamate moiety of AG94 for modification to determine the effect of this variation on transport as well as cell inhibition in culture. The glutamate moiety of AG94 was replaced with various natural and unnatural amino acids or completely deleted in an attempt to vary the chain length between the two carboxylic groups and retain one or none of the carboxylic moieties in the target compounds. The results showed that AG190 was relatively potent with an IC50 of 9.0 nM in human tumor KB cells in culture. A complete set of cell proliferation assays in CHO sublines afforded a SAR study of the transporters with defined transport as well as for GARFTase inhibitory activity. With FRα expressing RT16 CHO cells, an order of decreasing sensitivities (AG94&gt;AG189∼AG190&gt;AG213&gt;AG214&gt;AG187) was obtained. For FRβ expressing D4 CHO cells, it showed that AG94&gt;AG190&gt;&gt;AG189, whereas AG187, AG213 and AG214 are inactive. All target compounds are inactive in PCFT expressing R2/PCFT4 cells. In the binding assays towards PCFT and FRα with AG189 and AG190, we found that AG190 and AG189 still bound to PCFT although somewhat less avidly than that for AG94 towards PCFT, and AG189 along with AG190 exhibited very similar binding affinity towards FRα compared to AG94. In nucleoside protection assay, surprisingly neither adenosine nor AICA protect and suggested a change in intracellular target for AG189 and AG190 compared to AG94. Based on the profile of protection, we postulate that both AG189 and AG190 retain their targeted mechanism for transport but may not act as GARFTase inhibitors, a role previously established for AG94. Further study to elucitdate their exact role is currently underway. The ability to manipulate the intracellular target by variation in the glutamate moiety is novel and remarkable for this series.
Citation Format: Aleem Gangjee, Sai Zhao, Lalit Kumar, Christina Cherian, Steven Orr, Jenny Huang, Zhanjun Hou, Larry H. Matherly. Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3-d]pyrimidine scaffold. [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 5492. doi:10.1158/1538-7445.AM2013-5492