Synthesis and studies on three-compartment flavone-containing combi-molecules designed to target EGFR, DNA, and MEK.

In order to induce a tandem targeting of EGFR, DNA, and MEK, we built complex combi-molecules containing an EGFR targeting quinazoline and an aminoethyltriazene moiety linking the entire molecule to PD98059. Two complex molecules were synthesized: one with a short aminoethyl spacer, AL232, and the other AL414 with a longer aminoethylaminoethyl spacer. AL414 was a more potent inhibitor of EGFR tyrosine kinase than AL232. Both combi-molecules blocked EGFR phosphorylation in whole cells and downregulated extracellular signaling-regulated kinases (ERK1,2). However, only AL414 was capable of inducing DNA damage. Thus, it was taken in vivo for metabolic analysis. The results showed that 3 h after injection, AL414 was hydrolyzed to an EGFR inhibitor FD105, which was further acetylated to FD105Ac, a more potent inhibitor of EGFR. The detected flavone derivative was PD98059 linked to the hydroxyalkyl moiety resulting from the decomposition of the alkyldiazonium species. Independent synthesis of the latter metabolite and further in vitro analysis showed that it was deprived of antiproliferative activity. The results in toto suggest that while AL414 is a three-compartment combi-molecule, only the EGFR and DNA targeting species can be released and the cleavage to the intact MEK inhibitor PD98059 was mitigated by the stability of the carbamate.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.