A Tyrphostin-Derived Inhibitor of Protein Tyrosine Kinases: Isolation and Characterization

We recently reported that tyrphostin 23 (3,4-dihydroxybenzylidene malononitrile) is unstable in solution and that some of the degradation products are better inhibitors of the tyrosine kinase activity of Src and the EGF-receptor kinase than the parent compound itself (Ramdaset al.,Cancer Res. 54, 867–868, 1994). In this study, the tyrphostin 23-derived compound designated P3, which is a more stable and potent protein tyrosine kinase inhibitor, was isolated. P3was purified from oxidized tyrphostin 23 by solvent extraction, silica-gel flash chromatography, and reverse-phase high-pressure liquid chromatography. The physical characteristics of the isolated compound were determined and its chemical structure elucidated by1H and13C NMR spectroscopy. The proposed structure of this new inhibitor is that of a tyrphostin 23 dimer joined at the benzylidene carbon. P3was evaluated in vitro as an inhibitor of four different protein tyrosine kinases (Src, Csk, EGF-receptor, and FGF-receptor) and two protein serine kinases (PK-A and PK-C). This compound exhibited the most inhibitory activity against Src with aKivalue of 6 μMand was less inhibitory toward the other protein kinases withKivalues ranging from 35 to 300 μM. P3did not inhibit other nucleotide-utilizing enzymes such as lactate dehydrogenase and hexokinase. The growth and colony formation of HT-29 colon adenocarcinoma cells that contain activated Src was inhibited by P3with an IC50value of approximately 10 μM.