5106
S. Yap et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5102–5106
In summary, DiOHF–NIm 2 lacks vascular activity compared to
Supplementary data
the parent DiOHF. Both DiOHF and 2 possess functional antioxidant
activity and can significantly reduce NADPH-activated superoxide
(O2ꢀꢁ) levels in vitro to similar extents. Studies in normoxic and
hypoxic cells indicate selective consumption of 2 only under
hypoxic conditions, consistent with the proposed mechanism
of hypoxia-targeting by the 2-nitroimidazole group. In rat
hindquarters I/R, where tissue injury was quantified by assaying
serum LDH and muscle MPO levels, it was found that while both
DiOHF and 2 were able to significantly reduce serum LDH levels,
only 2 provided a significant reduction at the early time points
of 30 min and 1 h. Finally, muscle MPO levels were significantly
increased after hindquarters I/R in rats. Only DiOHF–NIm 2, and
not DiOHF, was able to significantly reduce muscle MPO levels
after I/R. Taken together these results indicates that while both
of these flavonols are protective against I/R-induced injury, the
protective effect is enhanced in the hypoxia-targeted derivative
2. A possible explanatory mechanism is that the antioxidant activ-
ity of 2 reduces the amount of ROS produced upon reperfusion,
limiting the damage produced and the resultant inflammatory
response, thereby protecting against neutrophil-mediated reperfu-
sion injury.
Several aspects of this study deserve further comment. Firstly,
the fact that DiOHF–NIm 2 lacks vasorelaxant activity yet exhibits
improved efficacy relative to DiOHF in limitation of I/R injury sug-
gests that the antioxidant activity of flavonols is the most impor-
tant contributor to the pharmacological mechanism of action for
protection against I/R injury. In some circumstances the co-occur-
rence of antioxidant and vasorelaxant activities in a cardioprotec-
tive agent may be a liability for clinical usage, such as in patients
suffering a myocardial infarction where impaired cardiac output
can threaten adequate organ perfusion and any further hypoten-
sive effect in response to an administered therapy may exacerbate
that situation. Secondly, these studies investigated the administra-
tion of flavonols with clinically relevant timing as a bolus dose five
minutes before reperfusion. As this is late in the period of ischae-
mia, and as perfusion is incomplete due to the no reflow phenom-
enon, the potential for accumulation of 2 may well be limited.
Alternative timings for the administration of flavonols in the
course of the I/R program may be useful for other therapeutic
applications such as organ preservation. Thirdly, I/R injury arises
from a range of phenomena in addition to ROS stress. At the single
dose at which these flavonols were administered, the antioxidant
effects may already be maximized and so there may be no possibil-
ity for further benefit to be provided. The ability of DiOHF–NIm 2
to reduce I/R injury indicates that it deserves further investigation
as an adjunctive therapy for the prevention and treatment of I/R in-
jury, for example in acute myocardial infarction.
Supplementary data (complete experimental details for the
synthesis of all new compounds and biological analyses) associated
with this article can be found, in the online version, at doi:10.1016/
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This work was supported by grants from the Australian Re-
search Council and the National Health and Medical Research
Council ofAustralia. S.Y. acknowledges support from the Australian
Government through an Endeavour Scholarship and from the Uni-
versity of Melbourne through the Albert Shimmins Fund.