TYROSINE KINASE INHIBITORS - 14 - STRUCTURE-ACTIVITY-RELATIONSHIPS FOR METHYLAMINO-SUBSTITUTED DERIVATIVES OF ENYL)AMINO]-6-(METHYLAMINO)PYRIDO[3,4-D]PYRIMIDINE (PD-158780), A POTENT AND SPECIFIC INHIBITOR OF THE TYROSINE KINASE-ACTIVITY OF RECEPTORS FOR THE EGF FAMILY OF GROWTH-FACTORS

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a ver y potent in vitro inhibitor of the tyrosine kinase activity of the epi dermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these s ignal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepare d by reaction of the 6-fluoro derivatives with appropriate amine nucle ophiles. These were evaluated for their ability to inhibit the tyrosin e phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epide rmoid carcinoma cells in culture. The most effective analogues were th ose bearing weakly basic substituents through a secondary amine linkag e, which proved water-soluble (>10 mM) and potent (IC(50)s generally < 1 nM). No clear SAR could be discerned for these compounds with respec t to amine base strength or the distance of the cationic center from t he chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerg ed for the ability of the compounds to inhibit EGFR. autophosphorylati on in A431 cells, where analogues bearing lipophilic weak bases were p referred. Representative analogues were evaluated for antitumor effect iveness against four in vivo tumor models. Significant in vitro activi ty was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model , suggesting that while this cell line requires EGF for clone formatio n in soft agar, other growth factors may be able to replace EGF in viv o. Also, no activity was seen against the SK-OV-3 ovarian cancer model , which is known to express other EGF receptor family members (althoug h it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tum or models, the treated tumors remained approximately the same size thr oughout therapy, suggesting that the compounds are cytostatic rather t han cytotoxic under these test conditions. it remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.