Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Screening of a compound library for inhibitors of the fibroblast growth factor (FGFr) and platelet-derived growth factor (PDGFr) receptor tyrosine kinases led to the development of a novel series of ATP competitive pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. The initial lead, 1-[2-amino-6-(2,6-dichlorophenyl)pyrido[2,3-d]pyrimidin-7-yl]-3- tert-butylurea (4b, PD-089828), was found to be a broadly active tyrosine kinase inhibitor. Compound 4b inhibited the PDGFr, FGFr, EGFr, and c-src tyrosine kinases with IC50 values of 1.11, 0.13, 0.45, and 0.22 microM, respectively. Subsequent SAR studies led to the synthesis of new analogs with improved potency, solubility, and bioavailability relative to the initial lead. For example, the introduction of a [4-(diethylamino)butyl]amino side chain into the 2-position of 4b afforded compound 6c with enhanced potency and bioavailability. Compound 6c inhibited PDGF-stimulated vascular smooth muscle cell proliferation with an IC50 of 0.3 microM. Furthermore, replacement of the 6-(2,6-dichlorophenyl) moiety of 4b with a 6-(3',5'-dimethoxyphenyl) functionality produced a highly selective FGFr tyrosine kinase inhibitor 4e. Compound 4e inhibited the FGFr tyrosine kinase with an IC50 of 0.060 microM, whereas IC50s for the inhibition of the PDGFr, FGFr, EGFr, c-src, and InsR tyrosine kinases for this compound (4e) were all greater than 50 microM.

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity 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 formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than 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.