Elsevier

Biochemical Pharmacology

Volume 57, Issue 8, 15 April 1999, Pages 917-925
Biochemical Pharmacology

Hormones and Growth Factors
Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with In Vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785)

https://doi.org/10.1016/S0006-2952(98)00356-6Get rights and content

Abstract

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (ic50 = 370 ± 120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 ≅ 5 nM), inhibited cell proliferation (ic50 = 31–125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

Section snippets

Chemicals

Preparation of CL-387,785 began with 5-nitroanthranilonitrile. Formamidination using N,N-dimethylformamide dimethyl acetal (98% yield) followed by reduction of the nitro group with palladium on carbon and cyclohexene in refluxing methanol furnished N′-(4-amino-2-cyanophenyl)-N,N-dimethylformamidine (95% yield). Acylation with the mixed anhydride of tetrolic acid and isobutyl chloroformate gave N-[3-cyano-4-[[(dimethylamino)methylene]amino]phenyl]-2-butynamide (89% yield). In the final step,

Results

Using the homology model of EGF-R as a starting point and the observations by us and others [22] that the 6- and 7-positions of the quinazoline ring can tolerate considerable bulk, we proposed that CL-387,785 bound to EGF-R at the ATP-binding region with an orientation and conformation similar to that shown in Fig. 1. This orientation placed the bromoaniline side chain of CL-387,785 in a hydrophobic pocket surrounded by Pro770, Met769, Gln767, Ala719, Val702, and Leu820. The N1 nitrogen of the

Discussion

These data show that CL-387,785 is a specific inhibitor of EGF-R. The molecule bound covalently to EGF-R. Such binding inhibited autophosphorylation of EGF-R and blocked proliferation of tumor cells both in vitro and in vivo. It is likely that CL-387,785 inhibits c-erbB-2, since the growth of cells that overexpress the receptor was inhibited and the drug inhibited autophosphorylation of c-erbB-2 in cells. Further studies are underway to determine whether CL-387,785 directly binds to c-erbB-2 or

Acknowledgements

We thank Ms. L. Benmassoud, Ms. S. Carter, Ms. K. Collins, Mr. M. Damiani, Dr. P. Lassota, Mr. R. Jeyasselan, Ms. E. Lenox, Ms. R. Montoya, Ms. G. McLaughlin, Mr. S. Jones, and Mr. R. Yannuzzi for their excellent technical assistance, the Biotechnology Core Group at Wyeth-Ayerst for providing recombinant enzyme, and Drs. F. Loganzo, P. Frost, and J. Upeslacis for their critical review of this work.

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    Present address: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.

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