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 (Ramdas et 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. P3 was 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 by 1H and 13C NMR spectroscopy. The proposed structure of this new inhibitor is that of a
tyrphostin 23 dimer joined at the benzylidene
carbon. P3 was 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 a Ki value of 6 microM and was less inhibitory toward the other
protein kinases with Ki values ranging from 35 to 300 microM. P3 did 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 P3 with an IC50 value of approximately 10 microM.