Vol. 4, 1405-1414, June 1998 Clinical Cancer Research 1405 443 ‘ -‘ hydroxylphenyl)-amino-6,7-dimethoxyquinazoline: A Novel Quinazoline Derivative with Potent Cytotoxic Activity against Human Glioblastoma Cells Rama Krishna Narla, Xing-Ping Liu, Dorothea E. Myers, and Fatih M. Uckun1 Departments of Experimental Oncology [R. K. N., F. M. U.], Biotherapy [R. K. N., D. E. M., F. M. U.], and Chemistry [X-P. L.], and Drug Discovery Program [X-P. L., D. E. M., F. M. U.], Hughes Institute, St. Paul, Minnesota 55 1 13 ABSTRACT The novel quinazoline derivative 4-(3’-bromo-4’- hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI- P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines, causing apoptotic cell death at micromolar concentrations. The in vitro antiglio- blastoma activity of WHI-P154 was amplified >200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblas- toma cells within 10-30 mm via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 ± 139 flM, whereas no cytotoxicity against EGF-R-negative leukemia cells was observed, even at concentrations as high as 100 LM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival, 19 days) and all control mice had tumors that rapidly progressed to reach an average size of > 500 mm3 by 58 days, 40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detect- able tumors for more than 58 days with a median tumor- free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size >50 mm3. Thus, targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. Received 2/4/98; revised 4/1/98; accepted 4/3/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Hughes Insti- tute, 2665 Long Lake Road, Suite 330, St. Paul, MN 551 13. Phone: (612) 697-9228; Fax: (612) 697-1042. INTRODUCTION As the most malignant primary central nervous system tumors, high-grade anaplastic astrocytoma and glioblastoma multiforme respond poorly to contemporary multimodality treatment programs using surgical resection, radiation therapy, and chemotherapy with a median survival of < 1 year after initial diagnosis (1-4). Consequently, the development of effec- tive new agents and novel treatment modalities against these very poor prognosis brain tumors remains a major focal point in translational oncology research. In a systematic effort to identify a cytotoxic agent with potent antitumor activity against glioblastoma cells, we synthe- sized several dimethoxy-substituted quinazoline derivatives and examined their in vitro and in vivo effects on human glioblas- toma cells. Here, we provide experimental evidence that the novel quinazoline derivative WHI-P1S42 exhibits potent cyto- toxic activity against human glioblastoma cells. Notably, target- ing of WHI-P154 to the surface EGF-R further enhanced its cytotoxic activity, resulting in rapid apoptotic death of glioblas- toma cells at nanomolar concentrations in vitro and significantly improved tumor-free survival in an in vivo SCID mouse glioblastoma xenograft model. MATERIALS AND METHODS Synthesis and Analysis of Quinazoline Derivatives. All chemicals were purchased from the Aldrich Chemical Com- pany (Milwaukee, WI) and were used directly for synthesis. Anhydrous solvents such as acetonitrile, methanol, ethanol, ethyl acetate, tetrahydrofuran, chloroform, and methylene chlo- ride were obtained from Aldrich as Sure Seal bottles under nitrogen and were transferred to reaction vessels by cannulation. All reactions were carried out under a nitrogen atmosphere. 2 The abbreviations used are: WHI-P154, 4-(3’-bromo-4’hydroxylphe- nyl)-amino-6,7-dimethoxyquinazoline; WHI-P79, 4-(3’-bromophenyl)- amino-6,7-dimethoxyquinazoline; WHI-P97, 4-(3’,5’-dibromo-4’-hy- droxylphenyl)-amino-6,7-dimethoxyquinazoline: WHI-PI 11 . 4-(3’- bromo-4’-methylphenyl)-amino-6,7-dimethoxyquinazoline: WHI-P131. 4-(4’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; WHI-P132, 4-(2’-hydroxylphenyl)-arnino-6,7-dimethoxyquinazoline; WHI-Pl80, 4-(3’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; WHI-P 197, 4-(3’-chloro-4’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline: WHI-P258, 4-(phenyl)-amino-6,7-dimethoxyquinazoline; SCID, severe combined immunodeficient; EGF, epidermal growth factor; rhEGF, recom- binant human EGF; EGF-R, EGF receptor; NMR, nuclear magnetic reso- nance; 5, d, t, q, m, and br, singlet, doublet, triplet, quartet. multiplet, and broad, respectively; IR, infrared; GC/MS. gas chromatography/mass spec- trometiy; m.p., melting point; HPLC, high-performance liquid chromatog- raphy; Sulfo-SANPAH, sulfosuccinimidyl 6-(4’azido-2’-nitrophenylami- no)hexanoate; MU, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyb tetrazolium bromide; GEN, genistein: TdT, terminal deoxynucleotidyl transferase: VTK, protein tyrosine kinase. 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