Abstract 3250: Design, synthesis and biological evaluation of side chain pemetrexed (PMX) analogues as potent inhibitors of tumors expressing folate receptors via GARFTase inhibition
Authors: Wang Yiqiang , Mitchell Shermaine , Cherian Christina , Desmoulin Sita Kugel , Hou Zhanjun , Kisliuk Roy L. , Matherly Larry H. , Gangjee Aleem 
Published: June 2012

Two major hurdles in cancer therapy are the inability of chemotherapy agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The most active of this series (AG71) included a 4 carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) (but not RFC) whereupon it inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 was highly active toward both KB and IGROV1 tumors; in SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine structure of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogs with 1 to 6 carbon atoms in the bridge (AG133-AG138) as hybrid molecules of PMX and AG71. The compounds were synthesized via a Heck coupling of the thiophene iodide with the alkene alcohols followed by α-bromonation and cyclization with the 2,4-diamino-6-oxo-pyrimidine to afford the 5-substituted pyrrolo[2,3-d]pyrimidine and elaborated to the L-glutamate. These analogs could be envisaged to afford the multi-targeted attributes of PMX, while preserving FRα and/or PCFT specificity of our 6-substituted analogs. Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). Analogs with 4 and 5-carbon atom chains (AG136 and AG137) showed IC50 values of 17.1 nM and 18.1 nM, respectively, against KB tumor cells in culture; other analogs of this series were 13- to &gt;58-fold less active. In the CHO sublines, AG136 and AG137 were growth inhibitory in the order RT-16 = PC43-10 &gt; R2/PCFT4 and RT-16 &gt;&gt; PC43-10 = R2/PCFT4, respectively. Thus, at least for AG136 unlike AG71, significant RFC activity is preserved. Whereas PMX is a multi-targeted agent with inhibitory effects on both de novo thymidylate and purine nucleotide biosynthetic pathways, the anti-proliferative effects of AG136 and AG137 were abolished by adenosine but not thymidine, establishing exclusive inhibition of purine biosynthesis. Our results suggest that 6-subtitution of the pyrrolo[2,3-d]pyrimidine scaffold is more effective in conferring selective transport of FRα over RFC, although inhibition of de novo purine biosynthesis is preserved in the 5-substituted compounds.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3250. doi:10.1158/1538-7445.AM2011-3250