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Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.

A series of new 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitrile derivatives that function as irreversible inhibitors of human epidermal growth factor receptor-2 (HER-2) and epidermal growth factor receptor (EGFR) kinases have been prepared. These compounds demonstrated enhanced activities for inhibiting HER-2 kinase and the growth of HER-2 positive cells compared to our EGFR kinase inhibitor 86 (EKB-569). Three synthetic routes were used to prepare these compounds. They were prepared mostly by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles with unsaturated acid chlorides or by amination of 4-chloro-6-(crotonamido)quinoline-3-carbonitriles with monocyclic or bicyclic anilines. The third route was developed to prepare a key intermediate, 6-acetamido-4-chloroquinoline-3-carbonitrile, that involved a safer cyclization step. We show that attaching a large lipophilic group at the para position of the 4-(arylamino) ring results in improved potency for inhibiting HER-2 kinase. We also show the importance of a basic dialkylamino group at the end of the Michael acceptor for activity, due to intramolecular catalysis of the Michael addition. This, along with improved water solubility, resulted in compounds with enhanced biological properties. We present molecular modeling results consistent with the proposed mechanism of inhibition. Binding studies of one compound, 25o (C-14 radiolabeled), showed that it binds irreversibly to HER-2 protein in BT474 cells. Furthermore, it demonstrated excellent oral activity, especially in HER-2 overexpressing xenografts. Compound 25o (HKI-272) was selected for further studies and is currently in phase I clinical trials for the treatment of cancer.

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25-30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G(1)-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.