TYROSINE KINASE INHIBITORS .13. STRUCTURE-ACTIVITY-RELATIONSHIPS FOR SOLUBLE 7-SUBSTITUTED 4-[(3-BROMOPHENYL)AMINO]PYRIDO[4,3-D]PYRIMIDINESDESIGNED AS INHIBITORS OF THE TYROSINE KINASE-ACTIVITY OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR

The general class of 4-(phenylamino)quinazolines are potent (some memb ers with IC50 values much less than 1 nM) and selective inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor ( EGFR), via competitive binding at the ATP site of the enzyme, but many of the early analogues had poor aqueous solubility (much less than 1 mM). A series of 7-substituted 4-[(3-bromophenyl)amino]pyrido[4,3-d]py rimidines, together with selected (3-methylphenyl)amino analogues, wer e prepared by reaction of the analogous 7-fluoro derivatives with appr opriate amine nucleophiles in 2-BuOH or aqueous 1-PrOH. All of the com pounds were evaluated for their ability to inhibit the tyrosine-phosph orylating action of EGF-stimulated full-length EGFR enzyme. Selected a nalogues were also evaluated for their inhibition of autophosphorylati on of the EGF receptor in A431 human epidermoid carcinoma cells in cul ture and against A431 tumor xenografts in mice. Analogues bearing a wi de variety of polyol, cationic, and anionic solubilizing substituents retained activity, but the most effective in terms of both increased a queous solubility (>40 mM) and retention of overall inhibitory activit y (IC50's of 0.5-10 nM against isolated enzyme and 8-40 nM for inhibit ion of EGFR autophosphorylation in A431 cells) were weakly basic amine derivatives. These results are broadly consistent with a proposed mod el for the binding of these compounds to EGFR, in which the 6- and 7-p ositions of the pyridopyrimidine ring are in a largely hydrophobic bin ding region of considerable steric freedom, at the entrance of the ade nine binding cleft. The most active cationic analogues have a weakly b asic side chain where the amine moiety is three or more carbon atoms a way from the nucleus. Two of the compounds (bearing weakly basic morph olinopropyl and strongly basic (dimethylamino)butyl solubilizing group s) produced in vivo tumor growth delays of 13-21 days against advanced stage A431 epidermoid xenografts in nude mice, when administered ip t wice per day on days 7-21 posttumor implant. Treated tumors did not in crease in size during therapy and resumed growth at the termination of therapy, indicating an apparent cytostatic effect for these compounds under these treatment conditions. The data suggest that continuous lo ng-term therapy with these compounds may result in substantial tumor g rowth inhibition.