| Abstract
| - The general class of 4-(phenylamino)quinazolines are potent(some members with IC50 values≪ 1 nM) and selective inhibitors of the tyrosine kinase activity ofthe epidermal growth factorreceptor (EGFR), via competitive binding at the ATP site of the enzyme,but many of the earlyanalogues had poor aqueous solubility (≪1 mM). A series of7-substituted 4-[(3-bromophenyl)amino]pyrido[4,3-d]pyrimidines, togetherwith selected (3-methylphenyl)amino analogues, wereprepared by reaction of the analogous 7-fluoro derivatives withappropriate amine nucleophilesin 2-BuOH or aqueous 1-PrOH. All of the compounds were evaluatedfor their ability to inhibitthe tyrosine-phosphorylating action of EGF-stimulated full-length EGFRenzyme. Selectedanalogues were also evaluated for their inhibition ofautophosphorylation of the EGF receptorin A431 human epidermoid carcinoma cells in culture and against A431tumor xenografts inmice. Analogues bearing a wide variety of polyol, cationic, andanionic solubilizing substituentsretained activity, but the most effective in terms of both increasedaqueous solubility (>40mM) and retention of overall inhibitory activity (IC50'sof 0.5−10 nM against isolated enzymeand 8−40 nM for inhibition of EGFR autophosphorylation in A431 cells)were weakly basicamine derivatives. These results are broadly consistent with aproposed model for the bindingof these compounds to EGFR, in which the 6- and 7-positions of thepyridopyrimidine ring arein a largely hydrophobic binding region of considerable steric freedom,at the entrance of theadenine binding cleft. The most active cationic analogues have aweakly basic side chain wherethe amine moiety is three or more carbon atoms away from the nucleus.Two of the compounds(bearing weakly basic morpholinopropyl and strongly basic(dimethylamino)butyl solubilizinggroups) produced in vivo tumor growth delays of 13−21 days againstadvanced stage A431epidermoid xenografts in nude mice, when administered ip twice per dayon days 7−21posttumor implant. Treated tumors did not increase in size duringtherapy and resumed growthat the termination of therapy, indicating an apparent cytostatic effectfor these compoundsunder these treatment conditions. The data suggest that continuouslong-term therapy withthese compounds may result in substantial tumor growthinhibition.
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