A series of 6-substituted classical pyrrolo [2,3-d]pyrimidine antifolates with a 3-to 6-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2, 3, 4 and 5, respectively) was synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the α-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the α-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 FRα or FRβ, and toward FRα-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 analogs as antitumor agents.

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Purpose: To determine the possibility of synergistic antileukemic activity and the underlying molecular mechanisms associated with cytarabine combined with valproic acid (VPA; a histone deacetylase inhibitor and a Food and Drug Administration-licensed drug for treating both children and adults with epilepsy) in pediatric acute myeloid leukemia (AML).Experimental Design: The type and extent of antileukemic interactions between cytarabine and VPA in clinically relevant pediatric AML cell lines and diagnostic blasts from children with AML were determined by MTT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis and cell cycle distributions were determined by flow cytometry analysis and caspase enzymatic assays. The effects of the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting.Results: We showed synergistic antileukemic activities between cytarabine and VPA in four pediatric AML cell lines and nine diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensitive to VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AML cases. Cytarabine and VPA cooperatively induced DNA double-strand breaks, reflected in induction of γH2AX and apoptosis, accompanied by activation of caspase-9 and caspase-3. Further, VPA induced Bim expression and short hairpin RNA knockdown of Bim resulted in significantly decreased apoptosis induced by cytarabine and by cytarabine plus VPA.Conclusions: Our results establish global synergistic antileukemic activity of combined VPA and cytarabine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment of children with this deadly disease. Clin Cancer Res; 16(22); 5499-510. ©2010 AACR.