Abstract 1658: 5N-substituted pyrrolo[3,2-d]pyrimidines: Tumor-targeted agents with first-in-class dual inhibition of serine hydroxymethyl transferase 2 and 5-amino-4-imidazolecarboxamide ribonucleotide formyl transferase enzymes
Authors: Shah Khushbu , Dekhne Aamod , Hou Zhanjun , Matherly Larry , Gangjee Aleem 
Published: August 2018

Design, modeling and synthesis of pyrrolo[3,2-d]pyrimidine analogs were carried out based on the concept of dual mechanisms-of-action for achieving maximal antitumor efficacy. Tumor-targeted delivery via agents that are selectively transported by the folate receptor (FRα/β) and/or PCFT, overexpressed in tumor cells such as epithelial ovarian cancer and non-small cell lung cancer, over the ubiquitously expressed reduced folate carrier (RFC) would afford tumor selectivity. Gangjee, Matherly and coworkers previously reported 5-substituted pyrrolo[2,3-d]pyrimidines as inhibitors of de novo purine nucleotide biosynthesis glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). To synthesize potential dual inhibitors of de novo purine biosynthesis at cytosolic AICARFTase and mitochondrial one-carbon metabolism at serine hydroxymethyl transferase (SHMT) 2, we generated structural hybrids of these inhibitors and 5,10-methylenetetrahydrofolate (the SHMT2 cofactor), 5-substituted pyrrolo[3,2-d]pyrimidine analogs AGF291, AGF299, AGF300, AGF318, AGF320, and AGF331. Molecular modeling of these compounds utilizing X-ray crystal structures of the transporters (FRα/β) and enzyme targets (AICARFTase, SHMT2) predicted excellent activities. The influence of bridge lengths and the aromatic moieties of these novel compounds were evaluated by testing their anti-proliferative activities in Chinese hamster ovary cells expressing human FRα or PCFT, and in FRα- and/or PCFT expressing nasopharyngeal carcinoma (KB) and lung cancer (H460) cells. All the pyrrolo[3,2-d]pyrimidine analogs were inhibitory toward FR- and/or PCFT-expressing cells. Compared to its pyrrolo[2,3-d]pyrimidine analog, AGF300 increased inhibition of cells via FR uptake from IC50 8.6 nM to 2.1 nM (by 4-fold) and decreased uptake through RFC from 56.5 nM to 516 nM (by 10-fold), suggesting a FR-targeted delivery of the agent. Toward H460 xenografts in SCID mice, AGF291 was efficacious. Protection studies with thymidine, glycine, adenosine and AICA and radiotracer studies with [3-14C]serine identified the likely intracellular targets of the pyrrolo[3,2-d]pyrimidine analogs as SHMT2 and AICARFTase. Mitochondrial one-carbon metabolism originating with SHMT2 is a critical source of reducing equivalents and one-carbon units for cytosolic biosynthesis and SHMT2 has been implicated as an oncodriver in several tumor types. While inhibition of SHMT2 was the subject of exploratory studies, our novel pyrrolo[3,2-d]pyrimidine series are “first-in-class” in terms of their in vitro and in vivo antitumor efficacies attributable to dual targeting SHMT2 and AICARFTase.

Citation Format: Khushbu Shah, Aamod Dekhne, Zhanjun Hou, Larry Matherly, Aleem Gangjee. 5N-substituted pyrrolo[3,2-d]pyrimidines: Tumor-targeted agents with first-in-class dual inhibition of serine hydroxymethyl transferase 2 and 5-amino-4-imidazolecarboxamide ribonucleotide formyl transferase enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1658.