New Derivatives of Flavone-8-acetic Acid
187
2-(3-Allyl-2-methoxyphenyl)-6-phenylpyran-4-one (32)
activity was tested up to the maximum tolerated dose. At the dose levels used
there was no significant body weight loss.
Sodium hydride (0.24 g, 9.8 mmol) was washed with light petroleum (40
ml) which was decanted off and replaced by THF (200 ml). The suspension
was then heated under reflux for 10 min and 31 (3.0 g, 9.8 mmol) was added
portionwise over 10 min followed by dimethyl sulfate (3.7 g, 39 mmol). After
heating for a further 2 h, the mixture was allowed to cool and concentrated
ammonia (40 ml) was added. After stirring for 12 h, water (200 ml) was added
and the mixture extracted with methylene chloride which was dried and
evaporated to yield a yellow oil. Column chromatography (silica, ethyl
acetate/petroleum 2:1) gave the product.–1H NMR: δ = 7.88–7.81 (m, 2 H),
7.63 (dd, J = 8, 2, 1 H), 7.52–7.49 (m, 3 H), 7.37 (dd, J = 8, 2, 1 H), 7.20 (t,
J = 8, 1 H), 7.00 (d, J = 2, 1 H), 6.83 (d, J = 2, 1 H), 6.10–5.90 (m, 1 H),
5.20–5.03 (m, 2 H), 3.69 (s, 3 H), 3.50 (d, J = 7, 2 H).–13C NMR: δ = 180.5,
163.7, 162.0, 156.8, 136.6, 134.8, 133.5, 131.4, 129.1, 129.0, 127.7, 125.9,
125.3, 124.4, 116.5, 115.6, 111.1, 61.3, 33.8.
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
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.
References
✩
Dedicated to Professor Richard Neidlein, Heidelberg on the occasion
of his 70th birthday.
2-(3-Carboxymethyl-2-methoxyphenyl)-6-phenylpyran-4-one (33)
[1] G. Atassi, P. Briet, J.-J. Berthelon, F. Collonges, Eur. J. Med. Chem.
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1H NMR (CD3SOCD3): δ = 8.06 (m, 2 H), 7.79 (d, J = 8, 1 H), 7.63 (m, 3
H), 7.59 (d, J = 8, 1 H), 7.36 (t, J = 8, 1 H), 7.11 (s, 1 H), 6.83 (s, 1 H), 3.75
(s, 2 H), 3.69 (s, 3 H) (acid proton not apparent).–13C NMR: δ = 184.7, 182.3,
166.7, 164.6, 159.1, 138.3, 135.2, 134.6, 131.9, 131.5, 129.9, 128.0, 127.3,
125.6, 116.2, 111.6, 63.2, 41.4.
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1H NMR (D2O): δ = 7.51–7.20 (m, 5 H), 7.16–7.01 (m, 3 H), 6.82 (s, 1 H),
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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, 100 µl 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.
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.
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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.
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In Vivo Chemotherapy
[14] R. A. Aitken, M. C. Bibby, F. Bielefeldt, J. A. Double, A. L. Laws,
A.-L. Mathieu, R. B. Ritchie, D. W. J. Wilson, Archiv. Pharm. Pharm.
Med. Chem. 1998, 331, 405–411.
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. The vehicles used were Arachis oil (19, 20,
21), 0.9% saline (18d, 22, 23, 24) and 0.9% saline/DMSO (9:1) (29a, 29b,
34). All treatments were administered intraperitoneally to NMRI mice. A
minimum of 5 mice were used for each compound. The activity of the
compounds were determined against the subcutaneous MAC15A tumour
model by the measurement of tumour growth delay. Wherever possible,
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