Synthesis and Characterization of AGF94

AGF94 [(S)-2-((5-[3-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-propyl]-thio-phene-2-car-bonyl)-amino)-pentanedioic acid] was synthesized, as previously described [18 (link)]. Leucovorin [(6R,S) 5-formyl tetrahydrofolate] was obtained from the Drug Development Branch, National Cancer Institute, Bethesda, MD. [3′,5′,7-³H]MTX (20 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). 5-Aza-2′-deoxycytidine (5-Aza) was purchased from Sigma–Aldrich (St. Louis, MO). Restriction and modifying enzymes were purchased from New England Biolabs (Ipswich, MA). The mammalian expression vector pcDNA3.1/myc-His(–)A was purchased from Invitrogen (Carlsbad, CA). Reporter gene vectors pGL3-Basic and pGL4.74[hRluc/TK] were purchased from Promega (Madison, WI). Other chemicals were obtained from commercial sources in the highest available purities.