Flavone-8-acetic Acid Derivatives
411
H, H-3, 4’), 6.52 (d, J = 2, 1 H, H-5’), 3.72 (s, 3 H), 3.70 (s, 2 H).–MS (FAB);
m/z (%) = 415 (21) [M+], 371 (6), 176 (100).
[3] M. C. Bibby, J. A. Double, R. M. Phillips, P. M. Loadman, Br. J. Cancer
1987, 55, 159–163.
[4] D. J. Kerr, T. Maughan, E. Newlands, G. Rustin, N. M. Bleehen, C.
Lewis, S. B. Kaye, Br. J. Cancer 1989, 60, 104–106; M. C. Bibby, J.
A. Double, Anti-Cancer Drugs 1993, 4, 3–17.
In Vitro Chemosensitivity Studies
The activity of each analogue was evaluated in vitro in a continuous 96
hour exposure and chemosensitivity assessed using the MTT assay. The
MAC15A cell line was used for the primary evaluation. All cells were
harvested from subconfluent stocks using 0.25% trypsin, counted on a
haemocytometer (Improved Neubauer chamber, Weber UK) and diluted in
complete RPMI 1640 for use at a concentration of 1 × 104 ml–1 MAC15A.
Compounds were dissolved to the appropriate concentration in complete
RPMI tissue culture medium immediately prior to use and serially diluted.
100 µl per well of cell suspension was plated in 96 well plates (U bottomed,
tissue culture treated, Costar Cat No3799) and incubated for 3–4 hours before
addition of the test solution. To one row of eight wells, 100µl complete RPMI
1640 was added to serve as the control. Subsequent rows of eight wells
received a concentration of test solution over the range 1 mg ml–1 to 0.01 µg
ml–1. Following the addition of the test compounds, the plates were incubated
at 37 °C in an atmosphere of 5% CO2, 95% air for four days before being
assessed.
[5] R. A. Aitken, M. C. Bibby, J. A. Double, A. L. Laws, R. B. Ritchie, D.
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Archiv. Pharm. Pharm. Med. Chem., 1996, 329, 489–497.
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Gummer in Progress in Clinical and Biological Research (Eds. V.
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The tetrazolium dye reduction assay was used in these studies. 150 µl of
used medium and compound solution was removed from each of the wells
following the 4 day exposure and replaced with 150 µl fresh medium and 20
µl of 5 mg ml–1 MTT solution. After 4 hours all of the medium and MTT was
removed from all of the wells and replaced with 150 µl DMSO. The formazan
crystals produced during the assay were dissolved and mixed by reverse
pipetting and the absorbance read at a wavelength of 550 nm using an ELIZA
spectrophotometer.
The mean percentage survival of the cells at each compound concentration
was calculated relative to the control and activity expressed as an IC50 value.
A limited number of analogues were similarly evaluated against the three
human tumour cell lines DLD-1, HRT-18 and K-562.
[11] We thank Inspec Fine Chemicals Ltd., Wolverhampton, for a generous
gift of this compound.
[12] S.-O. Lawesson, Arkiv Kemi 1957, 11, 317–324.
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In Vivo Chemotherapy
[17] J. Buckingham, S. M. Donaghy (Eds.), Dictionary of Organic Com-
pounds, Fifth Ed., Chapman & Hall, London, 1982, Vol. 5, p. 5344.
For in vivo evaluation the compounds were made up immediately prior to
use at an appropriate concentration for the desired dose to be administered
in 0.1 ml per 10 g body weight. All treatments were administered intraperi-
toneally to NMRI mice. The activity of the compounds were determined
against the subcutaneous MAC15A tumour model by the measurement of
tumour growth delay. Wherever possible, activity was tested up to the
maximum tolerated dose.
[18] R. A. Hoffman, S. Gronowitz, Arkiv Kemi 1960, 16, 515–538.
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[20] J. Buckingham, S. M. Donaghy (Eds.), Dictionary of Organic Com-
pounds, Fifth Ed., Chapman & Hall, London, 1982, Vol. 3, p. 2699.
Chemotherapy was administered on day 5 after tumour implantation to
allow for vascularization to occur, as determined histologically. Tumour
growth was followed by serial calliper measurements and anti-tumour activ-
ity assessed by tumour volume. This was calculated by the formula a2 × b/2
[21] G. Pappalardo, Gazz. Chim. Ital. 1959, 89, 540–550.
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where a and b were the smaller and larger tumour diameters respectively[30]
.
Growth delay was determined as the difference in time taken for the median
tumours of the analogue treated and solvent control treated mice to reach a
relative tumour volume of two. The significance of the growth delay was
determined using a Mann-Whitney statistical analysis.
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Received: September 3, 1998 [FP329]
Arch. Pharm. Pharm. Med. Chem. 331, 405–411 (1998)