A novel series of dihydrofolate reductase inhibitors was uncovered during an expansion of the SAR of 5,10-dideazatetrahydrofolic acid, and their biological activity was evaluated. These new analogs do not possess an oxygen at the 4 position and contain a monocyclic pyrimidine ring. 5,10-Dideazatetrahydrofolic acid I (DDATHF, 1) is a potent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT). The 6R diastereomer of DDATHF (Lometrexol) is currently in Phase I clinical trials. LY2315142 (3), a pyrrolo[2,3 d]pyrimidine-based antifolate which inhibits primarily at thymidylate synthase (TS) is currently undergoing Phase II clinical trials. As part of the continuing SAR study of these novel classes of deazafolates, we have examined the role of the (3H)4-oxo group on the pyrimidine ring of both DDATHF and LY231514. The replacement of the 4-oxo group of DDATHF and LY231514 with a hydrogen atom has resulted in compounds such as LY2952483 (2) and LY2887843 (4) with complete loss of inhibitory activity against GARFT and TS. Cell culture end products reversal and enzyme inhibition studies have demonstrated that these 4-deoxy analogs do not inhibit GARFT and/or TS, but instead are potent inhibitors of human dihydrofolate reductase (hDHFR). This finding has prompted us to examine the corresponding less rigid, monocyclic pyrimidine based analogs with the methylene moiety in both the reduced pyridine and pyrrole rings being removed. Herein we describe the synthesis and biological evaluations of these two monocyclic pyrimidine compounds LY3163734 (5) and LY2982074 (6).