Novel 1,5-Benzodiazepines
9
The numbering of atoms and schematic structure of the title compounds are
presented in Figure 1 and Figure 2.
[8] M. Di Braccio, G. Roma, G.C. Grossi, G. Leoncini, M. Maresca,
Farmaco, 1992, 47, 77.
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Tokkyo Koho J.P. 04 74, 181 [92 74, 181]; Chem. Abstr. 1992, 117,
69891t.
Antiproliferative Assay In Vitro
Compounds
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Naylor-Olsen, Eur. J. Med. Chem. 1998, 33, 471.
The compounds coded 1–6 were examined in in vitro screening assay. Test
solutions of the compounds tested (1 mg/ml) were prepared ex tempore by
dissolving the substance in 100 µl of DMSO completed with 900 µl of tissue
culture medium. Afterwards, the tested compounds were diluted in culture
medium (described below) to reach the final concentrations of 100, 10, 1,
and 0.1 µg/ml. The solvent (DMSO) in the highest concentration used in test
did not reveal any cytotoxic activity.
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Jenkins, S. Nadie, L.H. Hurley, D.E. Thurson, J. Am. Chem. Soc., 1992,
114, 4939.
Cells
The following established in vitro human cancer cell lines were applied:
SW707 (rectal adenocarcinoma), A549 (non-small cell lung carcinoma) and
MCF-7 (breast cancer). All lines were obtained from the American Type
Culture Collection (Rockville, Maryland, U.S.A.) and are maintained in the
Cell Culture Collection of the Institute of Immunology and Experimental
Therapy, Wroclaw, Poland. Human uroepithelial cell line HCV29T estab-
lished in the Fibiger Institute, Copenhagen, Denmark, was obtained from
Dr. J. Kieler in 1982 and maintained at the Institute of Immunology and
Experimental Therapy, Wroclaw, Poland.
Twenty-four hours before addition of the tested agents, the cells were
plated in 96-well plates (Sarstedt, U.S.A.) at a density of 104 cells per well.
The cells were cultured in the opti-MEM medium supplemented with 2mM
glutamine (Gibco, Warsaw, Poland), streptomycin (50 µg/ml), penicillin
(50 U/ml) (both antibiotics from Polfa, Tarchomin, Poland) and 5% fetal calf
serum (Gibco, Grand Island, U.S.A.). The cell cultures were maintained at
37 °C in humid atmosphere saturated with 5% CO2.
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EP 462, 522; Chem. Abstr . 1992, 116, 1289978f.
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194363c.
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214539v.
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Pharm. Pharm. Med. Chem. 1997, 330, 29.
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Chem. 1991, 26, 489.
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Gigler, I. Gacsalyi, I. Gyertyan, K. Reiter, Eur. Pat. Appl. 425, 282;
Chem. Abstr. 1991, 115, 136129z.
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SRB Assay
Chem. Pharm. Bull. 1991, 39, 81.
The details of this technique were described by Skehan et al. [44]. The
cytotoxicity assay was performed after 72-hour exposure of the cultured cells
to varying concentrations (from 0.1 to 100 µg/ml) of the tested agents. The
cells attached to the plastic were fixed by gently layering cold 50% TCA
(trichloroacetic acid, Aldrich-Chemie, Germany) on the top of the culture
medium in each well. The plates were incubated at 4 °C for 1 h and then
washed five times with tap water. The background optical density was
measured in the wells filled with culture medium, without the cells. The
cellular material fixed with TCA was stained with 0.4% sulforhodamine B
(SRB, Sigma, Germany) dissolved in 1% acetic acid (POCh, Gliwice,
Poland) for 30 minutes. Unbound dye was removed by rinsing (4×) with 1%
acetic acid. The protein-bound dye was extracted with 10mM unbuffered Tris
base (POCh, Gliwice, Poland) for determination of optical density (at
540 nm) in a computer-interfaced, 96-well microtiter plate reader Multiskan
RC photometer (Labsystems, Helsinki, Finland). Each compound in given
concentration was tested in triplicates in each experiment, which was re-
peated 3–5 times.
[21] M.G. Bock, B.E. Evans, R.M. Freidinger, Eur. Pat. Appl. EP 421, 802;
Chem. Abstr. 1992, 115, 208026p.
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