Synthesis and discovery of high affinity folate receptor-specific glycinamide ribonucleotide formyltransferase inhibitors with antitumor activity

6-Substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a three- to six-carbon bridge between the heterocycle and the benxoyl-L-glutamate (compounds 2-5, respectively) were synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the a-bromomethyl ketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the alpha-bromoketones, coupling with diethyl-L-glutamate, and saponification afforded 2-5. Compounds 2-5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FR alpha or FR beta and toward FR alpha-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogues as antitumor agents.