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Article

Use of a Pharmacophore Model for the Design of EGF-R Tyrosine Kinase Inhibitors: 4-(Phenylamino)pyrazolo[3,4-d]pyrimidines

Peter Traxler,* Guido Bold, Joerg Frei, Marc Lang, Nicholas Lydon, Helmut Mett, Elisabeth Buchdunger, Thomas Meyer, Marcel Mueller, and Pascal Furet

Novartis Pharmaceuticals, Therapeutic Area Oncology, Novartis Limited, CH-4002 Basel, Switzerland

J. Med. Chem., 1997, 40 (22), pp 3601–3616

DOI: 10.1021/jm970124v

Publication Date (Web): October 24, 1997

To whom correspondence should be addressed: Dr. Peter Traxler, Novartis Pharmaceuticals, Therapeutic Area Oncology, K136.4.94, Novartis Limited, CH-4002 Basel, Switzerland. Phone: +41 61/696 52 86. Fax: +41 61/696 34 29.

Abstract

In the course of the random screening of a pool of CIBA chemicals, the two pyrazolopyrimidines 1 and 2 have been identified as fairly potent inhibitors of the EGF-R tyrosine kinase. Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), the class of the pyrazolo[3,4-d]pyrimidines was then optimized in an interactive process leading to a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines as highly potent inhibitors of the EGF-R tyrosine kinase. The most potent compounds 13, 14, 15, 17, 19, 22, 26, 28, and 30 of this series inhibited the EGF-R PTK with IC₅₀ values below 10 nM. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC α, CDK1) was observed. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 15, 19, 22, and 23 at IC₅₀ values below 50 nM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 μM, thus indicating high selectivity for the inhibition of the ligand-activated EGF-R signal transduction pathway. Compounds 15 and 19 inhibited proliferation of the EGF-dependent MK cell line with IC₅₀ values below 0.5 μM. In addition, two compounds, 9 and 11, showing satisfactory oral bioavailability in mice after oral administration, exhibited good in vivo efficacy at doses of 12.5 and 50 mg/kg in a nude mouse tumor model using xenografts of the EGF-R overexpressing A431 cell line. From SAR studies, a binding mode for 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines, especially for compound 15, at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)-1H-pyrazolo[3,4-d]pyrimidines represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.