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increasing its reactivity towards electrophiles. On the other
hand, methyl groups are weak activators, they can only donate
References and notes
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Jing, Y.; Zhao, G. J. Med. Chem. 2010, Publication Date (Web): January 7, 2010.
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electron density into the aromatic system through the
r
-bond,
resulting in only
electrophiles.
a slight increase in reactivity towards
It can be speculated that the strong increase of electron density
in the aromatic system is responsible for the anti-migratory prop-
erties of compounds IIIa-4, IIIa-5, and IIIa-6. In further investiga-
tions, other electron donating substituents will be attached to
the phenyl ring. These new compounds will subsequently be eval-
uated for their effect on the migratory capacity of human MCF-7/
AZ breast cancer cells.
Due to the relative lack of potency and its diuretic properties,
EA is not suitable as a drug in cancer treatment. Several of the ana-
logues mentioned above demonstrate anti-migratory activity to-
wards the migration of human MCF-7/AZ breast cancer cells at
non-toxic concentrations. Due to the considerable difference in
structure of the new analogues versus EA, it can be speculated that
these analogues do not possess a diuretic nature. These trends are
therefore worth to be studied further with an eye to drug
development.
9. Christofori, G. Nature 2006, 25, 444.
10. Fidler, I. J. Nat. Rev. Cancer 2003, 3, 453.
11. The cytotoxicities of ethacrynic acid and its analogues, lacking the
a,b-
unsaturated carbonyl unit, were tested in accordance to Sigstedt et al.12
Mitochondrial dehydrogenase activities were measured by an MTT-reagent.
Cells were seeded in 96-well plates at an initial density of 1.5 Â 104 cells in
200 lL of the appropriate culture medium, supplemented with 5% fetal bovine
serum (FBS), 100 IU/mL penicillin, 100
incubation, at 37 °C in a humidified atmosphere containing 5% CO2, cells were
treated with eight different concentrations (1, 5, 10, 20, 40, 60, 80, and 100 M)
of EA and various EA analogues in culture medium. After 24 h of incubation,
100 L medium was removed prior to the addition of the MTT reagent. Three
lg/mL streptomycin. After 24 h of
l
l
independent experiments were carried out for each concentration, to
determine the mean optical density (OD) referring to cell viability. OD80/
IC20 values were determined from the respective graphs. These values
represent the concentrations of the analogues, required for 20% inhibition
in vitro or leaving approximately 80% of the cells viable.
In summary, we have synthesized EA analogues, lacking the
,b-unsaturated carbonyl unit, with enhanced potency, relative
a
to EA, towards the inhibition of migration of human MCF-7/AZ
breast cancer cells (Table 1 and Fig. 3). Differences in potency
among the various analogues may simply be due to the presence
of different substituents at the phenyl ring of the EA analogues.
Further studies with other EA analogues and various cancer cell
lines are underway.
12. Sigstedt, S. C.; Hooten, C. J.; Callewaert, M. C.; Jenkins, A. R.; Romero, A. E.;
Pullin, M. J.; Kornienko, A.; Lowrey, T. K.; Van Slambrouck, S.; Steelant, W. F. Int.
J. Oncol. 2008, 32, 1085.
13. Cells were grown in 6-well plates in the appropriate medium until confluence
and then washed twice with phosphate buffered saline (PBS).
A gap of
approximately 1500 m (the wound) was created in the cell monolayer and
l
images were captured. Subsequently, 3 mL of medium, in the presence or
absence of ethacrynic acid or EA analogues, was added. The respective
concentrations of these solutions were determined in the 24 h MTT assay.
After 24 h of cell migration in order to close the wound, images were once
again captured, and compared to the respective previous images in order to
Acknowledgments
We thank New Mexico Tech for supplying the start-up funds for
Dr. I.J. The biomedical evaluation of this work was supported by
the US National Institutes of Health (RR-16480) under the INBRE
program of the National Center for Research Resources.
quantify the migration rate, which is expressed as migratory velocity (
lm/h).
At least three independent experiments were performed.
14. Van slambrouck, S.; Hilkens, J.; Bisoffi, M.; Steelant, W. F. A. Int. J. Oncol. 2009,
35, 693.