Understanding the cardioprotective effects of flavonols: Discovery of relaxant flavonols without antioxidant activity

Article abstract of DOI:10.1021/jm070352h

3′,4′-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that can reduce injury after myocardial ischemia and reperfusion and thus is a promising small molecule for the treatment of cardiovascular disease. Like all vasoactive flavonols reported to date, DiOHF is both relaxant and antioxidant, hindering investigation of the relative contribution of each activity for the prevention of reperfusion injury. This study investigates structure-activity relationships of variations at the 3′ and 4′ positions of the B ring of DiOHF and vasorelaxant and antioxidant activities. Relaxation of rat isolated aortic rings precontracted with KCl revealed that the most active flavonols were those with a 4′-hydroxyl group, with the opening of potassium channels as a possible contributing mechanism. For the antioxidant activity, with the exception of DiOHF, none of the flavonols investigated were able to significantly scavenge superoxide radical, and none of the three most potent vasorelaxant flavonols could prevent oxidant-induced endothelial dysfunction. The discovery of single-acting vasorelaxant flavonols without antioxidant activity, in particular 4′-hydroxy-3′-methoxyflavonol, will assist in investigating the mechanism of flavonol-induced cardioprotection.

Full text of DOI:10.1021/jm070352h

1874
J. Med. Chem. 2008, 51, 1874–1884
Understanding the Cardioprotective Effects of Flavonols: Discovery of Relaxant Flavonols
without Antioxidant Activity
Cheng Xue Qin,†,‡ Xingqiang Chen,† Richard A. Hughes,† Spencer J. Williams,‡ and Owen L. Woodman*,†
The Department of Pharmacology, UniVersity of Melbourne, ParkVille, Victoria, Australia, and School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, 30 Flemington Road, UniVersity of Melbourne, ParkVille, Victoria, Australia
ReceiVed March 27, 2007
3′,4′-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that can reduce injury after myocardial
ischemia and reperfusion and thus is a promising small molecule for the treatment of cardiovascular disease.
Like all vasoactive flavonols reported to date, DiOHF is both relaxant and antioxidant, hindering investigation
of the relative contribution of each activity for the prevention of reperfusion injury. This study investigates
structure–activity relationships of variations at the 3′ and 4′ positions of the B ring of DiOHF and vasorelaxant
and antioxidant activities. Relaxation of rat isolated aortic rings precontracted with KCl revealed that the
most active flavonols were those with a 4′-hydroxyl group, with the opening of potassium channels as a
possible contributing mechanism. For the antioxidant activity, with the exception of DiOHF, none of the
flavonols investigated were able to significantly scavenge superoxide radical, and none of the three most
potent vasorelaxant flavonols could prevent oxidant-induced endothelial dysfunction. The discovery of single-
acting vasorelaxant flavonols without antioxidant activity, in particular 4′-hydroxy-3′-methoxyflavonol, will
assist in investigating the mechanism of flavonol-induced cardioprotection.
Introduction
Cardiovascular disease, principally ischemic heart disease and
stroke, is the leading cause of death worldwide.1 The current
treatments for patients with acute myocardial infarction are
revascularization with thrombolytic drugs or interventional
procedures (e.g., balloon angioplasty or coronary artery bypass
grafting). However, the restoration of blood flow results in a
unique form of myocardial damage, i.e., reperfusion injury. The
manifestations of reperfusion injury include arrhythmias, revers-
ible contractile dysfunction (such as myocardial stunning),
endothelial dysfunction, and lethal cell damage.2–5 It has been
suggested that an overproduction of reactive oxygen species
(ROSa) and intracellular calcium overload or redistribution are
the most important mediators of myocardial ischemia/reperfu-
sion (I/R) injury.2,4,6,7 Even though there are some pharmaco-
logical interventions to limit reperfusion injury thereby pre-
venting long-term cardiac dysfunction, such as antioxidants and
calcium antagonists, none of the successful experimental treat-
ments have demonstrated clinical efficacy.5,8 Thus, there is a
need for new approaches to the prevention and/or treatment of
myocardial I/R injury.
Figure 1. Structure of 3′,4′-dihydroxyflavonol 1.
suggested that the effect is associated with the relatively high
red wine consumption in France9–12 and that the active
component of the wine may be flavonoids, a group of polyphe-
nolic compounds derived from grape skins.11 Starting from this
premise, previous studies in our laboratory have demonstrated
that a synthetic flavonol, 3′,4′-dihydroxyflavonol (DiOHF) 1
(Figure 1), significantly reduced infarct size and injury associ-
ated with myocardial I/R in anesthetized sheep, and the level
of protection by DiOHF was similar to that afforded by an
interventional procedure, ischemic preconditioning, which is the
most effective method to prevent myocardial I/R injury to date.13
The beneficial effect in this study indicates that 1 has potential
as an adjunctive therapeutic agent for reperfusion therapy in
the treatment of myocardial infarction.13 Like many flavonols,
1 improves vascular function, acting as an antioxidant and a
vasorelaxant in rat isolated aorta, properties that might explain
the ability of this compound to ameliorate reperfusion injury.14
By itself, vasorelaxation will lead to improved perfusion,
providing more effective reoxygenation of previously ischemic
tissue. However, while reperfusion is a crucial step in recovery
from acute myocardial infarction, it leads to a dramatic increase
in the formation of ROS including superoxide, hydroxyl radical,
and hydrogen peroxide, which overwhelm the body’s defenses
against oxidative stress. ROS cause lipid peroxidation and lead
to cellular damage and death, as well as form isoprostanes,
prostaglandin-like compounds resulting from the reaction of
ROS with multiple unsaturated fatty acids (e.g., arachidonic
acid) and which possess a variety of activities detrimental to
cellular and vascular function.15 In addition, ROS can react with
the endogenous vasodilator NO, depleting available NO and
leading to vasoconstriction and reduced perfusion. Thus, both
Epidemiological studies have demonstrated a much lower
morbidity and mortality due to cardiovascular disease in the
French population compared to other Western Europeans, North
Americans, and Australians, even though they consume a
comparable diet high in fats and cholesterol. This phenomenon
has been called the “French paradox”.9,10 Several studies have
* To whom correspondence should be addressed. Phone: +61-3-9925-
7305; fax, +61-3-9925-7063; e-mail, woodman@rmit.edu.au.
†
The Department of Pharmacology.
School of Chemistry and Bio21 Molecular Science and Biotechnology
‡
Institute.
a Abbreviations: DECTA, diethylthiocarbamic acid; DiOHF, 3′,4′-
dihyroxyflavonol; DPPH, 1,1-diphenyl-2-picrylhydrazyl; I/R, ischemia/
reperfusion; KPSS, high potassium physiological salt solution; NADPH,
nicotinamide adenine dinucleotide; NBT, nitroblue tetrazolium; NMR,
nuclear magnetic resonance, pEC50, potency; Rmax, maximum response;
ROS, reactive oxygen species; TLC, thin layer chromatography.
10.1021/jm070352h CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

Relaxant FlaVonols without Antioxidant ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1875
Scheme 1. Synthesis of 3′- and 4′-Monosubstituted Flavonolsa
the antioxidant and vasorelaxant activities of 1 might explain
its cardioprotective effects. However, because these effects are
simultaneously present in 1 and in all other known vasoactive
flavonols, it has not been possible to determine which of these
activities is required for effective cardioprotection. In this study
we sought to undertake a structure–activity study with the aim
to separate vasorelaxant properties from antioxidant properties,
with the ultimate goal to develop single-acting antioxidant or
vasorelaxant flavonols to investigate whether either or both of
these activates are required to prevent reperfusion injury.
Previous studies of structure–activity relationships of vaso-
active flavonols have focused on simple substituents such as
hydroxyl and methoxy groups and have shown that systematic
substitutions on the A ring do not correlate with antioxidant
activity.16 The 3-hydroxyl group on the C ring of flavonols is
essential for the endothelium-dependent component of flavonol
relaxation16–18 but plays only a small role in flavonol-induced
vasorelaxation in total.16 On the other hand, the number and
the orientation of hydroxyl groups on the B ring appear to be
crucial for both the relaxant and the antioxidant activity.
Flavonols with three contiguous hydroxyl groups (a pyrogallol
group) enhance contraction in response to a variety of stimuli.19
Flavonols with 2′,4′-dihydroxyl groups are weak vasodilators,
and a catechol group with hydroxyls at C3′ and C4′ endows
strong vasorelaxant activity.19 When the C3′ and C4′ positions
are substituted with methoxy groups, rather than hydroxyl
groups, relaxation activity is abolished, underlining the impor-
tance of substituents at the 3′ and 4′ positions for vascular
activity.20 A catechol moiety is a unifying feature of the most
potent flavonol scavengers of peroxyl, superoxide, and perox-
ynitrite radicals, as demonstrated by the enhanced inhibition of
lipid peroxidation in a total oxyradical scavenging capacity
assay.20,21
a Reagents and conditions: (a) NaOH, EtOH, room temp; (b) H2O2,
NaOH, EtO, 0 °C to room temp.
Scheme 2. (A) Synthesis of 3-tert-Butyl-4-hydroxybenzaldehyde
from 2-tert-Butylphenol and (B) Synthesis of 4′-Hydroxy-3′-
substituted Flavonols from 4-Hydroxy-3-substituted
Benzaldehydesa
Given that much of the biological activity of vasoactive
flavonols appears to reside in the substituents at the 3′ and 4′
positions, modifications at these sites offers the greatest potential
to modulate the biological activity of the parent 1. In this study,
a range of flavonols were synthesized bearing a variety of
substitutions at these two positions. Substituents were chosen
to systematically explore the effect on vasorelaxation and
antioxidant activity of electron-donating and withdrawing groups
(methoxy, halogen, and trifluoromethyl groups), steric effects
(hydrogen, methyl and tert-butyl groups), and hydrogen bond
donors and acceptors (hydroxyl and methoxy groups). Eighteen
flavonols [4′-substituted flavonols [H, OH, O CH3, Cl, CF3, CH3,
C(CH3)3], 3′-substituted flavonols (H, OH, O CH3, Cl, CF3,
CH3), and 4′-hydroxy-3′-substituted flavonols [H, OH, O CH3,
Br, CF3, CH3, C(CH3)3)] were synthesized and their vasorelaxant
and antioxidant activities were evaluated in isolated rat aorta.18,22
Surprisingly, vasorelaxant activity was found to be separable
from antioxidant activity, leading to the discovery of the first
single-acting vasorelaxant flavonols, compounds that should help
to cast light on the mechanism of cardioprotection of flavonols.
a Reagents and conditions: (a) CHCl3, NaOH, H2O, 50 °C; (b) BnBr,
K2CO3, EtOH, room temp; (c) 2-hydroxyacetophenone, dioxane, EtOH,
KOH, room temp; (d) H2O2, dioxane, EtOH, NaOH, 0 °C to room temp;
(e) HCl, AcOH, 100 °C.
the 4-hydroxy-3-substituted benzaldehydes was commercially
available except 4-hydroxy-3-tert-butylbenzaldehyde. This benz-
aldehyde was synthesized from 2-tert-butylphenol and chloro-
form as a minor product of the Reimer-Tiemann reaction using
conditions described by Birchall et al.26,27 The pure product
was isolated by steam distillation in a 2% yield (Scheme 2A).
The 4-hydroxyl group of each benzaldehyde was protected as
a benzyl ether with benzyl bromide and K2CO3, in yields of
60–80% (Scheme 2B).28 The benzyl group was chosen because
of its stability under the strong alkaline conditions of the
Claisen-Schmidt condensation and the Algar-Flynn-Oyamada
reaction and the anticipated ease of deprotection. Successful
Claisen-Schmidt condensation of 4-benzyloxybenzaldehydes
and 2-hydroxyacetophenone required more forcing conditions
than for the monosubstituted benzaldehydes.29 These conditions
involve a prolonged reaction time, use of a higher concentration
of base, and inclusion of dioxane to solubilize poorly soluble
reaction intermediates.29 The chalcones were not purified but
immediately treated with alkaline hydrogen peroxide.29 All
protected flavonols were obtained in >35% yield over these
two steps.
Results and Discussion
Monosubstituted flavonols were synthesized in two steps
(Scheme 1). Claisen-Schmidt condensation of various substi-
tuted benzaldehydes and 2-hydroxyacetophenone in the presence
of aqueous sodium hydroxide gave the corresponding chalcones
in yields of 30–60%.23 Chalcones were converted to flavonols
using the Algar-Flynn-Oyamada24,25 reaction by treatment
with alkaline hydrogen peroxide, in yields of 25–65%.23
The 4′-hydroxy-3′-substituted flavonols were synthesized by
the four- or five-step processes shown in Scheme 2. Each of
Debenzylation of protected flavonols was achieved by treat-
ment with aqueous HCl in AcOH at 100 °C,29 with partial

1876 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Qin et al.
Table 1. Comparison of the Vascular Activity (Sensitivity, pEC50; Maximum Response, Rmax) and the Antioxidant Activity of 4′-Substituted,
3′-Substituted, and 4′-Hydroxy-3′-substituted Flavonols in Rat Isolated Thoracic Aorta with Intact Endothelium
vascular activitya
antioxidant activity
X1
X2
n
pEC50
Rmax (%)
91 ( 2
n
lucigeninb
n
NBTc
n
DPPHd
2
H
H
5
5.12 ( 0.09
5
70 ( 14
3
91 ( 10
4
83 ( 9
4′-Substituted Flavonols
3
4
5
6
7
8
OH
OCH3
Cl
CF3
CH3
tBu
H
H
H
H
H
H
5
5
6
5
5
5
5.33 ( 0.11
ND
5.09 ( 0.14
ND
ND
ND
99 ( 1
16 ( 5
37 ( 4
6 ( 3
13 ( 7
10 ( 3
5
6
6
6
6
6
74 ( 9
89 ( 17
100 ( 36
85 ( 10
101 ( 21
111 ( 32
3
3
3
3
3
3
93 ( 11
91 ( 10
69 ( 8
82 ( 17
73 ( 6
81 ( 6
4
4
4
4
4
4
32 ( 9
51 ( 3
93 ( 11
90 ( 5
76 ( 5
71 ( 4
3′-Substituted Flavonols
9
H
H
H
H
H
OH
OCH3
Cl
CF3
CH3
5
6
6
5
5
5.49 ( 0.10
5.15 ( 0.05
5.17 ( 0.08
3.94 ( 0.10
4.13 ( 1.03
98 ( 2
79 ( 7
53 ( 6
23 ( 4
37 ( 12
6
6
5
6
6
78 ( 17
74 ( 16
75 ( 17
80 ( 19
85 ( 23
3
3
3
3
3
84 ( 10
97 ( 6
77 ( 11
82 ( 23
66 ( 17
4
4
4
4
4
87 ( 16
71 ( 2
87 ( 1
89 ( 1
80 ( 1
10
11
12
13
4′-Hydroxy-3′-Substituted Flavonols
1
OH
OH
OH
OH
OH
OH
OH
OCH3
Br
CF3
CH3
tBu
5
5
5
5
5
5
5.30 ( 0.07
5.35 ( 0.03
5.60 ( 0.07
4.78 ( 0.01
5.36 ( 0.14
5.33 ( 0.11
102 ( 1
98 ( 2
100 ( 1
77 ( 5
98 ( 2
38 ( 3
5
5
5
5
5
5
27 ( 4
106 ( 25
96 ( 37
109 ( 28
105 ( 34
108 ( 36
5
4
3
3
3
3
43 ( 6
88 ( 10
95 ( 10
102 ( 20
79 ( 21
84 ( 14
4
4
4
4
4
4
36 ( 10
24 ( 1
23 ( 1
26 ( 2
25 ( 1
26 ( 2
14
15
16
17
18
a ND ) not determined. b Superoxide levels in the presence of flavonols (10-5 M) expressed as percentage of control. c DPPH level in the presence of
flavonols (10-4 M) expressed as percentage of control. d NBT level in the presence of flavonols (10-5 M) expressed as percentage of control.
substitutions at the 3′ position slightly reduced the vasorelaxation
relative to the unsubstituted flavonols. The OCH3 group did not
alter the response, and the OH substitution was again the
preferred substituent that maximized vascular activity. We next
sought to investigate the effect of combinations of substituents
and chose to maintain a 4′-hydroxyl group, which provides
excellent vasorelaxant activity while investigating the effect of
variation of substituents at the 3′-position. The 4′-hydroxy-3′-
substituted flavonols were as active as the most potent compound
described to date, 1, except the tert-butyl derivative, which had
a significantly lower potency and efficacy. This suggests that
for flavonols with a 4′-hydroxy group in place, electron-
Figure 2. Examples of concentration–response curves of DiOHF 1
withdrawing or -donating groups or hydrogen bond donors and/
(O) (n ) 5), 3′-methoxy 10 (9) (n ) 5), and 4′-hydroxy-3′-methoxy
or acceptors at the 3′-position do not significantly alter the level
14 (2) (n ) 6) in endothelium-intact rat thoracic aortic rings. Aortic
of their vascular activity, but steric hindrance caused by a bulky
tert-butyl group does reduce vascular activity. These results also
suggest that a single B-ring hydroxyl group might be sufficient
for maximal vascular activity and that a catechol group may
not be as important as has been proposed previously by our
group and others.16,19 Woodman et al. proposed that a hydroxyl
group at the 3′ position of a flavonol was the main determinant
of the vascular activity.22 The results of this study suggest that
a hydroxyl group at either the 3′ or 4′ position is sufficient for
good vasorelaxant activity. Flavonols with a similar range of
substitutions at the 4′ position and with a 3′-hydroxyl could be
synthesized and investigated to test this hypothesis.
Mechanisms of Flavonol-Induced Vasorelaxation. Fla-
vonols are most commonly found to cause endothelium-
independent vasorelaxation,16,17,19,30 but Fitzpatrick et al.
demonstrated that quercetin causes endothelium-dependent
relaxation in rat thoracic aorta.31 To investigate whether the
flavonols under investigation here act in an endothelium-
dependent manner, four of the most active flavonols (1, 3, 10,
and 14) were examined. Our data for 3, 10, and 14 show that
the removal of the endothelium from rat aorta did not affect
the efficacy or potency of the compound (Figure 3, Table 2). 1
showed a reduced sensitivity in endothelium denuded rings
without affecting the maximum response. This result agrees with
Chan et al., who suggested that 1 causes vasorelaxation
rings were precontracted with isotonic high-potassium physiological
salt solution (KPSS, 30 mM KCl) to an equal level in all groups (10,
45 ( 3% of KPSS (123 mM KCl); 14, 46 ( 4%; 1, 42 ( 1%). All
values are expressed as mean with standard error of the mean indicated
by the vertical lines. The pEC50 and Rmax values determined from the
data presented are given in Table 1.
decomposition being observed for the tert-butyl derivative. All
flavonols and intermediates synthesized in this study were
purified by recrystallization.
Structure–Activity Relationships of Vascular Activity. The
vascular activities of 18 flavonols were quantified using pre-
contracted rat isolated thoracic aorta from which the potency
(pEC50) and maximum relaxation (Rmax) were determined.
Endothelium intact rings were precontracted with an isotonic,
high potassium physiological salt solution (KPSS, 30 mM KCl)
to similar levels, and the concentration–response curves to
various flavonols were generated allowing quantification of the
potency and efficacy.
The results for the 4′-monosubstituted flavonols (Table 1,
Figure 2) indicated that substitution by most groups including
Cl, OCH3, C(CH3)3, CH3, and CF3 abolished vasorelaxation
relative to the unsubstituted flavonols. By contrast, a 4′-hydroxyl
group improved the vasorelaxant activity and was the optimum
substituent among those examined. The effects of substitutions
at the 3′ position were next investigated. CH3, CF3, and Cl

Relaxant FlaVonols without Antioxidant ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1877
(KPSS, 30 mM/25 mM KCl) and thromboxane mimetic agent
U46619, were selected to investigate the possible role of
potassium channels in the vascular relaxation. To achieve the
same level of precontraction, 30 mM KCl was used in
endothelium intact aortic rings, whereas 25 mM KCl was used
in endothelium denuded aortic rings. KCl at 25 or 30 mM
partially depolarizes the aortic rings, whereas U46619 activates
thromboxane A2 receptor. Our results show that the same four
flavonols (i.e., 1, 3, 10, 14) exerted a more potent relaxing effect
in the presence of U46619 than in the presence of 30 mM KCl
(Figure 3, Table 2). This suggests that the opening of potassium
channels might play a role in flavonol-induced vasorelaxation,
which has been observed by other groups.33–39 While our results
suggest that these flavonols primarily cause endothelium-
independent relaxation through the opening of the potassium
channels, other mechanisms may also be involved, such as
inhibition of enzymes involved in vascular smooth muscle
contraction, e.g., protein kinase C and cAMP phosphodi-
esterase,19 or interference with the utilization of Ca2+ in the
contractile response.16,22
Figure 3. Role of endothelium and the effect of different contractile
agents on the responses to 4′-hydroxy-3′-methoxy 14. Comparison of
concentration–response curves to 14 after active tone is induced by
either U46619 or isotonic high-potassium physiological salt solutions
in the presence and the absence of the endothelium (endothelium intact,
U46619 induced (1); endothelium denuded and U46619 induced (O);
endothelium intact, isotonic, high-potassium physiological salt solution
(KPSS, 30 mM KCl) induced (9); endothelium denuded, isotonic, high-
potassium physiological salt solution (KPSS, 25 mM KCl) induced (2)).
Aortic rings were precontracted to a similar level in all groups. KCl-
induced precontraction was more resistant to relaxation induced by
DiOHF 1. Removal of the endothelium did not affect the response
significantly. All values are expressed as mean with standard error of
the mean indicated by the vertical lines. pEC50 and Rmax analysis results
are summarized in Table 2.
Structure–Activity Relationships of Antioxidant Activi-
ty. The antioxidant activities of the 18 flavonols were examined
next using several different in vitro assays. The effect of
flavonols on superoxide levels were first assessed using a cell-
free colorimetric nitroblue tetrazolium chloride (NBT) assay
coupled with xanthine/xanthine oxidase. The reaction of xan-
thine and xanthine oxidase results in production of superoxide
anions that is detected by the change in color of NBT.
Superoxide levels will be reduced if flavonols possess antioxi-
dant activity. None of flavonols examined in this study were
found to be as effective as the lead compound 114 for superoxide
scavenging when used at a concentration of 10-5 M (Table 1).
1 inhibited superoxide levels by 57 ( 6%, whereas most of the
other flavonols inhibited by only 10–20%.
Table 2. Comparison of the pEC50 and Rmax to Different Flavonols in
Endothelium-Intact (EI) and Endothelium-Denuded (EX) Thoracic
Aortic Rings from Rats
14
1
3
10
U46619-Induced, EI
9
na
9
7
7
pEC50
6.06 ( 0.23 5.87 ( 0.10 5.78 ( 0.09 5.52 ( 0.11
Rmax
101 ( 1
50 ( 2
101 ( 1
53 ( 2
100 ( 0
54 ( 3
101 ( 3
53 ( 4
precontractionb
U46619-Induced, EX
5
The effect of flavonols on superoxide levels was then assessed
using a tissue-based lucigenin-enhanced chemiluminescence
assay, which has greater pharmacological relevance compared
to cell-free assays because it uses the tissue itself to generate
superoxide and demands that active antioxidants scavenge
superoxide in the presence of other biological substrates.22
Isolated rat aortic rings were incubated with NADPH as the
substrate for NADPH oxidase in the vasculature, diethylthio-
carbamic acid (DETCA) to inactive endogenous superoxide
dismutase, and either vehicle or flavonols. Superoxide that is
produced by NADPH oxidase reacts with lucigenin, leading to
the emission of photons, which can be quantified to give a
measure of superoxide levels.40 1 inhibited superoxide levels
by 73 ( 4%, whereas other flavonols inhibited by up to only
30%. The findings from both of the cell-free and tissue-based
systems demonstrate that the catechol group on the B ring of 1
is important for strong antioxidant activity, as others have
reported.41–43 In addition to the presence of two contiguous
hydroxyl groups, the total number of hydroxyl groups might
also be an important determinant of the scavenging activity.22
Clearly, flavonols with three hydroxyl groups in total, such as
1, exhibit good antioxidant activity but not flavonols with one
or two hydroxyl groups. However, it is unclear whether the
antioxidant activity of flavonols is due to their direct radical
scavenging effect or inhibition of enzymes responsible for
superoxide anion production, such as NADPH oxidase.44
na
pEC50
8
5
9
5.78 ( 0.05 5.52 ( 0.10c 5.50 ( 0.06 5.43 ( 0.13
Rmax
100 ( 1
51 ( 2
100 ( 0
59 ( 2
102 ( 1
53 ( 4
93 ( 2
47 ( 5
precontractionb
KCl-Induced, EI [Isotonic, High-Potassium Physiological Salt Solution
(KPSS, 30 mM KCl)]
na
pEC50
5
6
7
6
5.32 ( 0.03c 5.26 ( 0.03d 5.29 ( 0.08d 5.11 ( 0.05c
Rmax
100 ( 1
58 ( 4
100 ( 1
57 ( 4
95 ( 4
50 ( 3
87 ( 5c
56 ( 6
precontractionb
KCl-Induced, EX [Isotonic, High-Potassium Physiological Salt Solution
(KPSS, 25 mM KCl)]
na
pEC50
4
4
4
6
5.25 ( 0.06d 5.20 ( 0.10d 5.26 ( 0.06d 5.05 ( 0.08c
Rmax
93 ( 4d
58 ( 2
100 ( 1
60 ( 5
92 ( 1
60 ( 6
86 ( 5c
59 ( 8
precontractionb
a n ) total number of rats. b Level of tone expressed as percentage of
contraction to KPSS (123 mM KCl). c Significantly different from the response
of the U46619 + EI vessel (p < 0.05, ANOVA, Dunnett’s multiple comparison
test). d Very significantly different from the response of the U46619 + EI vessel
(p < 0.001, ANOVA, Dunnett’s multiple comparison test).
predominantly in an endothelium-independent manner but that
endothelium-dependent relaxation also plays a small role.16 The
differences between 1 and other active flavonols might be due
to differences in other biological activities, such as antioxidant
activity, which may indirectly enhance the activity of endog-
enously released NO.
Several studies have demonstrated that flavonols are effective
vasodilators,16,19,31,32 but the mechanism of flavonol induced
vasodilatation remains unclear. In this study, two contractile
agents, isotonic high-potassium physiological salt solutions
The radical scavenging activity of flavonols against other free
radicals, such as the organic free radical DPPH, was also
examined. DPPH possesses an unpaired electron that is stabilized
by resonance over the whole molecule, giving rise to a deep-

1878 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Qin et al.
Table 3. Comparison of the Sensitivity (pEC50) and Maximum
Relaxation (Rmax) to acetylcholine in the Presence of 1 (10-4.5 M), 14
(10-4.5 M), or Vehicle (15% DMSO, 85% Methanol) in Endo-
thelium-Intact Rat Isolated Thoracic Aorta Exposed to Pyrogallol (10-4.5
M) or a Combination of Xanthine (10-4M)/Xanthine Oxidase (0.016
U/mL)a
pyrogallol
pEC50
xanthine/xanthine oxidase
treatment
vehicle
oxidant stress
oxidative stress
+ 1
n
Rmax
n
pEC50
Rmax
6
6
6
6.93 ( 0.08 81 ( 5
6.32 ( 0.23 47 ( 7b
7.19 ( 0.22 81 ( 8
6
7
6
7.60 ( 0.20 94 ( 2
6.96 ( 0.14 47 ( 7b
7.26 ( 0.30 84 ( 7
oxidative stress
+ 14
6
6.56 ( 0.31 42 ( 5b
7
7.05 ( 0.28 62 ( 6b
Figure 4. Effect of 4′-hydroxy-3′-methoxyflavonol 14 or DiOHF 1
on pyrogallol-induced vascular dysfunction. Concentration–responses
curves to acetylcholine in the absence (9, n ) 6) or the presence of P
(pyrogallol (2), 10-4.5 M, n ) 6), P + 1 (pyrogallol, 10-4.5 M + 1
(O), 10-4.5 M, n ) 6), or P + 14 (pyrogallol, 10-4.5 M + 14 ([),
10-4.5 M, n ) 6). Aortic rings were precontracted with U46619 to a
similar level in all groups (see Table 2). Pyrogallol significantly
inhibited the response to acetylcholine. This inhibitory effect is
markedly reduced by 1 but not by 14. All values are expressed as mean
with standard error of the mean indicated by the vertical lines.
a n ) the total number of rats. Pyrogallol: 10-4.5 M. Xanthine (10-4
M)/xanthine oxidase (0.016 U/mL). b Significantly different from the vehicle
(p < 0.05, ANOVA, Dunnett’s multiple comparison test).
violet color. When a solution of DPPH reacts with a hydrogen
atom donor, it is reduced resulting in a loss of the violet color.45
As shown in Table 1, 4′-hydroxyflavonol 3 is the most activate
flavonol among the 4′-substituted flavonols in the DPPH assay.
None of 3′-substituted flavonols demonstrated significant scav-
enging activity against the DPPH radical, and all of the
4′-hydroxy-3′-substituted flavonols could scavenge this organic
radical as effectively as quercetin, as previously reported by
Sim et al.46Owing to the delocalizaton of its free electron, DPPH
is a more stable, and less reactive, radical than superoxide ion.
These results indicate the importance of the orientation of phenolic
hydroxyl groups on the B ring in determining the anti-DPPH
efficacy, with the 4′-hydroxyl group on the B ring being required
for effective scavenging of DPPH, possibly as a result of the
resonance stabilization afforded to the resulting phenolic radial.
Figure 5. Effect of 4′-hydroxy-3′-methoxyflavonol 14 or DiOHF 1
on acetylcholine-induced endothelium-dependent relaxation in the
presence of xanthine/xanthine oxidase: concentration–response curves
to acetylcholine in the absence (9, n ) 6) or the presence of xanthine/
xanthine oxidase (2, n ) 7), xanthine/xanthine oxidase + 1 (O, n )
6), or xanthine/xanthine oxidase + 14 ([, n ) 6). Aortic rings were
precontracted with U46619 to a similar level in all groups (see Table
2). Xanthine/xanthine oxidase significantly inhibited the response to
acetylcholine. This inhibitory effect is markedly reduced by 1 but not
14. All values are expressed as mean with standard error of the mean
indicated by the vertical lines.
Effect of Flavonols on Vasorelaxation Stimulated by
Acetylcholine in the Presence of Oxidative Stress. Preserva-
tion of NO bioactivity by superoxide scavengers depends not
only on superoxide scavenging activity but also on the rate of
superoxide scavenging.47 For example, the well-known anti-
oxidant, ascorbic acid, reacts relatively slowly with superoxide
(rate ) 3.3 × 10-5 M-1 s-1 48
) and does not improve endothelial
dysfunction caused by superoxide.47 By contrast, flavonols react
almost 10 times faster with superoxide (e.g., rate ) 2.4 × 10-6
M-1 s-1 for quercetin).49 To determine if flavonols scavenge
superoxide effectively enough to increase vascular NO bio-
availability when there are high levels of superoxide, we
examined the effect of relaxant responses of acetylcholine in
rat isolated aortic rings subjected to oxidative stress induced
by two routes, pyrogallol or xanthine/xanthine oxidase. Pyro-
gallol autooxidizes and generates superoxide radical directly,
whereas xanthine is the substrate for xanthine oxidase and
generates superoxide enzymatically. Addition of pyrogallol or
xanthine/xanthine oxidase caused significant endothelial dys-
function (>50% reduction in Rmax to acetylcholine) as shown
in Figures 4 and 5 and Table 3. 1, one of the most active
flavonols reported to date,14,22 completely reversed the reduction
in relaxation caused by oxidant stress using either system,
indicating a rapid reaction between the flavonol and superoxide
anions, thereby preventing vascular dysfunction. This provides
strong evidence that this flavonol effectively protects endothe-
lium-derived NO from inactivation from endogenously and
exogenously generated superoxide. On the other hand, 14, which
is as effective a vasorelaxant as 1, did not improve endothelium-
dependent relaxation in the presence of oxidative stress in either
system, indicating that this compound cannot efficiently scav-
enge superoxide and that it lacks functional antioxidant activity.
Conclusion
Through the synthesis and pharmacological evaluation of a
series of 4′-substituted, 3′-substituted, and 4′-hydroxy-3′-
substituted flavonols, we have determined the relationship
between vasorelaxant and antioxidant activities and flavonol
structure. Surprisingly, while vasorelaxant and antioxidant
activity has been concurrent in all flavonoids studied to date,
our structure–activity relationship clearly shows that these
activities may be separated. The discovery that 4′-hydroxy-3′-
methoxyflavonol possesses only vasorelaxant activity and lacks
antioxidant properties now provides the first single-acting
flavonol that maintains one activity while being devoid of the
other. As such, it is now available as a small molecule to
determine which of these two activities is required for cardio-
protection and should cast light on the mechanisms of reper-

Relaxant FlaVonols without Antioxidant ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1879
literature procedure by Robak & Cryglewski.50 Background was
measured by a spectrophotometer (Perkin-Elmer) at 560 nm in a
96-well microplate plate containing 200 µL of 0.1 M phosphate
buffer (pH 7.4), xanthine (3 mM), EDTA (3 mM), NBT (600 µM),
and 4′-substituted flavonols, 3′-substituted flavonols, 4′-hydroxyl-
3′-substituted flavonols (10 µM, 100% DMSO), or vehicle (5%
DMSO). A total of 25 mU/mL xanthine oxidase was then added to
the reaction mixture and incubated at 25 °C after a further 25 min.
The absorbance of the reaction mixture was recounted and the level
of superoxide was calculated using the following equation: level
of superoxide (%) ) (Aflavonol+xanthine oxidase - Abackground)/(Axanthine oxidase
- Abackgroud) × 100.
fusion injury and the potential for small molecule antioxidant
therapeutics for the treatment of cardiovascular disease.
Experimental Section
Pharmacological Assays. All experimental procedures were
performed within the guidelines of National Health and Medical
Research Council of Australia and were approved by the Pharma-
cology and Physiology subcommittee of the University of Mel-
bourne Animal Experimentation Ethics Committee.
Isolation of Rat Thoracic Aortic Rings. Male Sprague–Dawley
rats (250–350 g) were euthanized by inhalation of 80% CO2/20%
O2 and subsequent cervical dislocation. The chests were opened,
and the thoracic aortae were isolated. After removal of the
superficial connective tissue, the aorta was cut into ring segments,
2 to 3 mm in width, which were then used for relaxation assays
and lucigenin chemiluminescence assays in vitro.
(2) Effects of Flavonols on Superoxide Levels in Lucigenin
Assays. Superoxide anion generation was measured in isolated
aortic segments by lucigenin-enhanced chemiluminescence. Aortic
rings were prepared as described above and then placed in
Krebs-HEPES buffer (composition (mM): NaCl 99.90, KCl 4.7,
KH2PO4 1.0, MgSO4 · 7H2O 1.2, D-glucose 11.0, NaHCO3 25.0,
CaCl2 ·2H2O 2.5, Na HEPES 20.0). These tissues were incubated
for 45 min at 37 °C at pH 7.4 in Krebs-HEPES buffer containing
diethylthiocarbamic acid (DETCA, 3 mM, to inactivate superoxide
dismutase) and ꢀ-nicotinamide adenine dinucleotide phosphate
(NADPH, 100 µM) as a substrate for NADPH oxidase and
4′-substituted flavonols, 3′-substituted flavonols, 4′-hydroxy-3′-
substituted flavonols (10 µM, 100% DMSO), or vehicle (DMSO
1%). Background photoemission was measured by a TopCount
single photocounter for 12 cycles in a 96-well Optiplate containing
(300 mL) of Krebs-HEPES buffer together with bis-N-methylacri-
dinium nitrate (lucigenin) (50 µM), NADPH (100 µM), 4′-
substituted flavonols, 3′-substituted flavonols, 4′-hydroxy-3′-
substituted flavonols (10 µM, 100% DMSO), or vehicle (DMSO
1%). After 45 min, a single ring segment of aorta was washed and
added to each well and photon-emission, as a measure of superoxide
production, was recounted. At the conclusion of the assay, aortic
tissues were removed and dried for 48 h at 65 °C before weighing.
Superoxide production was normalized to dry tissue weight.
(3) Effects of Flavonols on DPPH Levels. The free radical
scavenging activity of flavonols was determined using the stable
free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and measured
according to the method of Sim et al.46,51 All 4′-substituted
flavonols, 3′-substituted flavonols, 4′-hydroxy-3′-substituted fla-
vonols (30 µM) were dissolved in 0.1 mL of DMSO and then added
to 0.1 mL of 0.1 mM DPPH in methanol. The mixture was allowed
to stand for 10 min at room temperature in the dark, and the
absorbance was measured using a microplate reader (Perkin-Elmer)
at 560 nm. The level of DPPH was calculated using the following
equation: level of superoxide (%) ) (Aflavonol/ADPPH) × 100.
B. Effect of Flavonols on Vasorelaxation to Acetylcholine
in the Presence of Oxidative Stress. To test the ability of the
compounds to preserve endothelial function in the presence of
oxidative stress, responses to acetylcholine were examined in the
presence of pyrogallol, which auto-oxidizes to release superoxide,
or xanthine/xanthine oxidase, which produces superoxide via an
enzymatic pathway.14 The two systems were chosen to indicate
whether flavonols acts by enzyme inhibition or radical scavenging.
(1) Pyrogallol. Aortic rings were isolated and mounted in
standard 10 mL organ baths containing Krebs bicarbonate solution
as described above. After aortic rings were equilibrated, the
maximum responses were established and the endothelium integrity
was tested. Rings were incubated for 20 min with pyrogallol (30
µM), pyrogallol (30 µM) with 14 (30 µM, 15% DMSO, 85%
methanol), pyrogallol (30 µM) with the positive control 1 (30 µM,
15% DMSO, 85% methanol), or vehicle (15% DMSO, 85%
methanol). The L-type Ca2+ channel blocker nifedipine (30 nM)
was also added to all aortic rings to prevent spontaneous activity.16
After a stable precontraction tone [50% of KPSS (123 mM KCl)-
induced contraction] with thromboxane mimetic U46619 (1–10 nM)
was established, cumulative concentration–response curves to
acetylcholine were determined and the effects of vehicle, 1, and
14 in the presence of oxidative stress were compared.
Relaxation by Flavonols. Aortic rings were mounted in standard
10 mL organ baths containing Krebs’ bicarbonate solution (com-
position (mM): NaCl 118.0, KCl 4.7, MgSO4 ·7H2O 1.2, KH2PO4
1.2, NaHCO3 25.0, D-glucose 11.0, CaCl2 · 2H2O 2.5). The bath
medium was maintained at 37 °C with a pH of 7.4 and continuously
bubbled with carbogen (5% CO2 in O2). When required, the
endothelium was mechanically removed by gently rubbing the
lumen with the tips of fine forceps. Aortic rings were equilibrated
for 60 min at a resting tension of 1 g. The rings were precontracted
with an isotonic, high-potassium physiological salt solution (Krebs’
potassium salt solution, KPSS, 123 mM KCl) to achieve the
maximum tension. After a washout of 30 min, the integrity of the
endothelium was tested. Rings were precontracted with phenyle-
phrine (PE, 10-100 nM), and only rings showing greater than 80%
relaxation in response to acetylcholine (10 µM) were accepted as
endothelium intact. Endothelium-denuded rings that did not relax
to acetylcholine showed 100% relaxation to sodium nitroprusside
(SNP, 10-6 M). Following testing of endothelial integrity, rings
were repeatedly washed and re-equilibrated to the resting tension.
(1) Effect on KCl-Induced Precontraction. Isotonic, high-
potassium physiological salt solution (KPSS, 30 mM KCl) was then
used to establish a stable active force in the range of 40-60% of
KPSS (123 mM KCl) induced contraction. Cumulative concentra-
tion–response curves were conducted for all 4′-substituted, 3′-
substituted, 4′-hydroxyl-3′-substituted flavonols (10 nM to 30 µM)
or vehicle (15% DMSO, 85% methanol). Only one concentration–
response curve was obtained from each thoracic aortic ring.
(2) Effect on U4661-Induced Precontraction. To investigate
the mechanism of flavonol-induced relaxation, a different contractile
agent, the thromboxane mimetic 9,11-dideoxy-9R,11R-epoxymetha-
noprostaglandin F2R (U46619, 1–10 nM) was used. Before precon-
traction, rings were incubated with the L-type Ca2+ channel blocker
nifedipine (30 nM) to prevent spontaneous activity.16 Cumulative
concentration–response curves were obtained for the 1, 3, 10, and
14.
(3) Effect of the Removal of Endothelium on the Re-
sponses to Flavonols. In order to examine the role of endothelium
in the vasorelaxation caused by flavonols, cumulative response
curves to 1, 3, 10, and 14 were determined in the absence and the
presence of endothelium for both KPSS (25 mM for endothelium
denuded rings or 30 mM KCl for endothelium intact rings) and
U46619 precontracted vessels.
Antioxidant Activities of Flavonols. A. Effect of Flavonols
on Free Radicals in in Vitro Assays. Several in vitro test systems
were used to assess the antioxidant activities of the 18 flavonols.
Effects of flavonols on superoxide levels were assessed using
lucigenin-enhanced chemiluminescence assay and the colorimetric
NBT (nitroblue tetrazolium chloride) assay coupled with xanthine/
xanthine oxidase system. The effect on a less reactive free radical
was assessed using the DPPH assay.
(1) Effects of Flavonols on Superoxide Levels in NBT
Assays. The scavenging potential for superoxide radicals was also
analyzed via xanthine/xanthine oxidase generating system, coupled
with NBT (nitroblue tetrazolium) reduction using a modified

1880 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Qin et al.
(2) Xanthine/Xanthine Oxidase. Isolated aortic rings were
incubated with xanthine oxidase (0.016 U/mL) for 30 min to allow
it to permeate the tissue, 20 min with compound 1 (30 µM) or 14
(30 µM), and 10 min with xanthine (100 µM) before precontraction.
Cumulative concentration–response curves to acetylcholine were
determined, and the effect of vehicle, 1, and 14 in the presence of
oxidative stress was compared.
at 1 mL/min with a 25 min gradient of 0–100% solvent B, where
solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in
acetonitrile.
4-Hydroxy-3-tert-butylbenzaldehyde. Chloroform (64 mL, 799
mmol) was added dropwise with stirring to a mixture of NaOH
(120 g, 3.0 mol), water (120 mL), methanol (50 mL), and 2-tert-
butylphenol (666 mmol, 100 g) over 4 h at 50 °C. The mixture
was stirred at 50 °C for another 30 min and then poured into ice/
water (600 mL) and acidified to pH 5 with concentrated hydro-
chloric acid. The dark mixture was extracted with dichloromethane,
dried (MgSO4), and concentrated, and the residue was steam-
distilled for 18 h until the distillate ran clear. The remaining residue
was dissolved in chloroform (400 mL) and extracted with sodium
hydroxide (2 M, 400 mL). The aqueous extracts were acidified with
concentrated hydrochloric acid to pH 2 and extracted with
chloroform (3 × 300 mL). The combined organic extracts were
dried (MgSO4), filtered, and evaporated. The dark-red residue was
triturated with hot petroleum spirit (10 × 100 mL), and the
combined petroleum spirit extract evaporated to give a bright-red
residue. The residue was recrystallized from dichloromethane and
petroleum spirit to give 4-hydroxy-3-tert-butylbenzaldehyde as
colorless crystals (1.70 g, 2%), mp 146–150 °C (lit.27 142 °C). 1H
NMR (400 MHz, CDCl3) δ ppm 1.45 (s, 9H, C(CH3)3), 6.10 (s,
1H, OH), 6.84 (d, 1H, J5,6 ) 8.4 Hz, H5), 7.66 (dd, 1H, J5,6 ) 8.4
Hz, J2,6 ) 2.0 Hz, H6), 7.84 (d, 1H, J2,6 ) 2.0 Hz, H2), 9.86 (s,
1H, COH). 13C NMR (100 MHz, CDCl3) δ 29.5 (C(CH3)3), 34.9
(C(CH3)3), 117.3, 129.4, 130.0, 130.6, 137.4, 161.3 (Ar,Ph), 192.5
(CdO).
General Procedure for Benzylation of 4-Benzyloxy-3-
substituted Benzaldehydes. Benzyl bromide (1.05–1.40 mmol) was
added to a stirred solution of 3-substituted-4-hydroxybenzaldehyde
(1 mmol) and potassium carbonate (1.1–1.8 mmol) in absolute
ethanol (1 mL). The reaction mixture was stirred under nitrogen
for 1 or 2 days until TLC indicated consumption of the benzalde-
hyde. The mixture was filtered through Celite, washed with
dichloromethane (3 × 1 mL), and concentrated under vacuum. The
yellow residue was dissolved in dichloromethane (2 mL), washed
with sodium hydroxide (1 M, 1 mL), dried (MgSO4), concentrated,
and recrystallized from the solvents indicated.
Drugs and Chemicals Used. Unless stated otherwise, all
chemical reagents were purchased from Aldrich and were dissolved
in buffer solution. 4-Hydroxy-3-trifluoromethylbenzaldehyde was
supplied by Fluorochem (Derbyshire, U.K.). Acetylcholine per-
chlorate was obtained from BDH Chemicals (Poole, Dorset,
England). 9,11-Dideoxy-9R,11R-epoxymethanoprostaglandin F2R
(U46619) was from Cayman Chemical Company (Ann Arbor, MI).
DiOHF 1, flavonol 2, 4′-hydroxyflavonol 3, and 3′-hydroxyflavonol
9 were from Indofine Chemical Co., Inc. (Belle Mead, NJ), and
p-nitroblue tetrazolium chloride (NBT) was from Calbiochem
(Darmstadt, Germany). U46619 was dissolved in 1 M NaHCO3,
and nifedipine was dissolved in absolute ethanol and subsequently
in Krebs bicarbonate solution. For use in organ bath experiments,
all flavonols were dissolved in 15% DMSO, 85% methanol (0.01
M stock), subsequent dilutions were made in 100% methanol (1
µM) and deionized water (0.1 and 0.01 µM). The maximum
concentrations of DMSO and methanol in the organ bath were
0.15% and 0.85%, respectively. For use in the lucigenin, NBT, and
DPPH assays, all flavonols were dissolved in 100% DMSO, and
the maximum concentration of DMSO was 1%. DPPH was
dissolved in methanol.
Data Presentation and Statistical Analysis. The results are
expressed as the mean ( standard error of the mean, and n indicates
the number of experiments (i.e., number of rats). Relaxation
responses to flavonols were expressed as a percentage of the KCl
or U46619 induced precontraction. Relaxation responses to ace-
tylcholine were expressed as a percentage of U46619 induced
precontraction. Concentration–response data were fitted to a sig-
moidal curve using GraphPad Prism, version 4 (GraphPad Software
Inc., San Diego, CA) to provide estimates of the pEC50 value. The
calculated pEC50 and observed maximum response (Rmax) were
compared using the one-way analysis of variance test (ANOVA)
with post hoc multiple comparisons using Newman-Keuls or
Dunnett’s test (GraphPad Software Inc., San Diego, CA). P < 0.05
was considered statistically significant.
4-Benzyloxy-3-methoxybenzaldehyde. Vanillin (5.00 g, 32.9
mmol), potassium carbonate (5.00 g, 36.2 mmol), and benzyl
bromide (4.2 mL, 34 mmol) were reacted for 24 h. Workup as for
the general procedure gave a crude residue that was recrystallized
to yield 4-benzyloxy-3-methoxybenzaldehyde as a colorless fine
solid (4.63 g, 58%), mp 69–70 °C (EtOH, lit.28 62–63 °C). 1H NMR
(400 MHz, CDCl3) δ ppm 3.93 (s, 3H, CH3), 5.23 (s, 2H, CH2),
6.97 (d, 1H, J5,6 ) 8.4 Hz, H5), 7.31–7.43 (m, 7H, Ph,H2,6), 9.81
(s, 1H, COH). The 1H NMR spectrum was in agreement with that
previously reported.28
Superoxide levels from rat aortic rings in the lucigenin assay
were expressed as average counts per second normalized to dry
tissue weight and calculated as a percentage of the counts in the
presence of vehicle (0.1% Krebs-HEPES buffer).The superoxide
levels in NBT assays was expressed as a percentage of the counts
in the presence of vehicle (0.1 M phosphate buffer) minus the
background, and the level of DPPH was expressed as percentage
of vehicle (0.1 mM DPPH in methanol).
Synthetic Procedures and Characterization. Nuclear magnetic
resonance (NMR) spectra were obtained on a Varian Unity 400 in
solutions of CDCl3 or DMSO-d6. Multiplicity abbreviations used
are as follows: s ) singlet, d ) doublet, m ) multiplet, dd )
doublet of doublet, ddd ) doublet of doublet of doublet, t ) triplet.
Thin layer chromatography (TLC) was performed with aluminum
sheets precoated with Merck silica gel 60 F254, using mixtures of
ethyl acetate-petroleum spirits. Detection was effected by visual-
ization in UV light. Solvents were evaporated under reduced
pressure using a rotary evaporator. Melting points were obtained
using a Riechert-Jung Hotstage melting point apparatus. Low-mass
spectral data were obtained by triple quad electrospray using a
Quattro II instrument (Melbourne University), and HRMS experi-
ments were performed by Sally Duck at the Chemistry Department
(Monash University). Elemental analyses were performed by
C.M.A.S (Belmont, Victoria). HPLC analysis was performed by
John Karas (University of Melbourne) using an Agilent 1200 series
instrument, with UV detector set at 254 nm, and a Zorbax Eclipse
XDB-C18, 4.6 mm × 150 mm, 5 µm particle size column (eluting
4-Benzyloxy-3-bromobenzaldehyde. 3-Bromo-4-hydroxybenz-
aldehyde (2.00 g, 9.95 mmol), potassium carbonate (2.50 g, 18.1
mmol), and benzyl bromide (1.80 mL, 15.1 mmol) were reacted
for 2 days. Workup as for the general procedure gave a crude
residue that was recrystallized to yield 4-benzyloxy-3-bromobenz-
aldehyde as a colorless fine solid (2.39 g, 83%), mp 97–98 °C (Et2O/
1
petroleum spirit, lit.52 78–80 °C). H NMR (400 MHz, CDCl3) δ
ppm 5.27 (s, 2H, CH2Ph), 7.05 (d, 1H, J5,6 ) 8.8 Hz, H5), 7.26–7.48
(m, 5H, Ph), 7.64 (dd, 1H, J5,6 ) 8.8 Hz, J2,6 )1.6 Hz, H6), 8.11
(d, 1H, J2,6 ) 1.6 Hz, H2), 9.84 (s, 1H, CHO). The 1H NMR
spectrum was in agreement with that previously reported.52
4-Benzyloxy-3-trifluoromethylbenzaldehyde. 4-Hydroxy-3-tri-
fluoromethylbenzaldehyde (0.25 g, 1.31 mmol), potassium carbonate
(0.68 g, 4.92 mmol), and benzyl bromide (0.40 mL, 3.34 mmol)
were reacted for 2 days. Workup as for the general procedure gave
a crude residue that was recrystallized to yield 4-benzyloxy-3-
trifluorobenzaldehyde as a colorless solid (0.18 g, 72%), mp 83–85
°C (Et2O/petroleum spirit, lit.28 81.5–82.5 °C). 1H NMR (400 MHz,
DMSO-d6) δ ppm 5.31 (s, 2H, CH2), 7.17 (d, 1H, J5,6 ) 8.4 Hz,
H5), 7.35–7.43 (m, 5H, Ph), 8.01 (dd, 1H, J5,6 ) 8.4 Hz, J2,6 ) 2.0
Hz, H6), 8.14 (d, 1H, J2,6 ) 2.0 Hz, H2), 9.92 (s, 1H, CHO). 13C
NMR (100 MHz, DMSO-d6) δ 70.4 (CH2Ph), 114.6 (Ar,Ph), 117.8

Relaxant FlaVonols without Antioxidant ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1881
(q, 1C, J ) 30.7 Hz, CF3), 121.8, 124.6, 127.3, 128.2, 128.6, 128.9,
129.1, 135.6, 135.9, 160.4 (Ar,Ph), 190.9 (CdO).
H3,5), 7.00 (d, 1H, J3′,4′ ) 7.9 Hz, H3′), 7.47 (t, 1H, J4′,5′ ) J5′,6′
) 7.4 Hz, H5′), 7.53 (d, 1H, Jtrans )15.5 Hz, CdCH), 7.62 (d, 2H,
J ) 7.9 Hz, H2,6), 7.88 (d, 1H, Jtrans ) 15.5 Hz, CdCH), 7.89–7.92
(m, 1H, H6′). 13C NMR (100 MHz, CDCl3) δ 56.5 (OCH3), 115.6,
118.6, 119.6, 119.8, 121.2, 128.4, 130.6, 131.6, 137.2, 146.4, 163.1,
164.6 (Ar, Ph), 194.7 (CdO). Treatment of 2′-hydroxy-4-meth-
oxychalcone (1.00 g, 3.93 mmol) according to method A afforded
the flavonol as pale-yellow needles (536 mg, 51%), mp 230–231.5
°C (EtOH, lit.54 231–232 °C). 1H NMR (CDCl3, 400 MHz) δ ppm
3.88 (s, 3H, Me), 6.93 (br s, OH), 7.04 (d, 2H, J ) 6.9 Hz, H3′,5′),
7.39 (ddd, 1H, J5,6 ) 5.8 Hz, J6,7 ) 5.4 Hz, J6,8 ) 0.6 Hz, H6),
7.57 (br d, 1H, J7,8 ) 6.2 Hz, H8), 7.68 (ddd, 1H, J5,7 ) 1.2 Hz,
J6,7 ) 5.4 Hz, J7,8 ) 6.2 Hz, H7), 8.22 (d, 2H, J ) 6.9 Hz, H2′,6′),
8.23 (d, 1H, J5,6 ) 5.4 Hz, H5). 13C NMR (100 MHz, CDCl3) δ
56.0 (CH3), 114.5, 118.8, 122.1, 124.5, 124.9, 125.7, 130.2, 133.9,
139.0, 155.5, 161.3 (Ar,Ph), 173.7 (CdO). LRMS (ESI-) m/z: 268.1
[C16H12O4 (M + H)+ requires 268.1]. Anal. RP-HPLC tR ) 16.6
min (purity 96%).
4-Benzyloxy-3-methylbenzaldehyde. 4-Hydroxy-3-methylben-
zaldehyde (0.93 g, 6.20 mmol), potassium carbonate (1.54 g, 11.1
mmol), and benzyl bromide (1.1 mL, 9.2 mmol) were reacted for
40 h. Workup as for the general procedure gave a crude residue
that was recrystallized to yield 4-benzyloxy-3-methylbenzaldehyde
as a brown fine solid (0.84 g, 56%), mp 51–53 °C (THF/petroleum
spirit). 1H NMR (400 MHz, CDCl3) δ ppm 2.33 (s, 3H, CH3), 5.18
(s, 2H, CH2), 6.99 (d, 1H, J5,6 ) 8.4 Hz, H5), 7.35–7.45 (m, 5H,
Ph), 7.73–7.70 (m, 2H, H2,H6), 9.87 (s, 1H, CHO). 13C NMR (100
MHz, CDCl3) δ 16.6 (CH3), 70.2 (CH2), 111.1, 127.3, 128.2, 128.3,
128.8, 129.8, 130.7, 131.8, 136.5, 162.1 (Ar,Ph), 191.3 (CdO).
4-Benzyloxy-3-tert-butylbenzaldehyde. 4-Hydroxy-3-tert-bu-
tylbenzaldehyde (0.75 g, 4.21 mmol), potassium carbonate (1.05
g, 7.57 mmol), and benzyl bromide (0.8 mL, 6.3 mmol) were
reacted for 24 h. Workup as for the general procedure gave a crude
residue that was recrystallized to yield 4-benzyloxy-3-tert-butyl-
benzaldehyde as a yellow fine solid (0.76 g, 68%), mp 87–88 °C
4′-Chloroflavonol (5). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 4-chlorobenzaldehyde (2.11 g, 15.0 mmol) were reacted
for 5 h according to method A to afford 4-chloro-2′-hydroxychal-
cone as yellow needles (1.37 g, 65%), mp 149–150 °C (EtOH, lit.53
1
(Et2O/petroleum spirit). H NMR (400 MHz, CDCl3) δ ppm 1.42
(s, 9H, C(CH3)3), 5.21 (s, 2H, CH2), 7.04 (d, 1H, J5,6 ) 8.0 Hz,
H5), 7.35–7.46 (m, 5H, Ph), 7.71 (dd, 1H, J5,6 ) 8.0 Hz, J2,6 ) 2.0
Hz, H6), 7.97 (d, 1H, J2,6 ) 2.0 Hz, H2), 9.88 (s, 1H, CHO). 13C
NMR (100 MHz, CDCl3) δ 29.7 (C(CH3)3), 35.3 (C(CH3)3), 70.7
(CH2), 112.4, 127.6, 128.4, 128.5, 128.9, 129.7, 130.8, 136.3, 139.3,
162.8 (Ar,Ph), 191.6 (CdO).
1
149–150 °C). H NMR (CDCl3, 300 MHz) δ ppm 6.94 (dd, 1H,
J4′,5′ ) J5′,6′ ) 7.7 Hz, H5′), 7.03 (d, 1H, J3′,4′ ) 7.7 Hz, H3′), 7.40
(d, 2H, J ) 8.5 Hz, H3,5), 7.50 (dd, J3′,4′ ) J4′,5′ ) 7.7 Hz, H4′),
7.58 (d, 2H, J ) 8.5 Hz, H2,6), 7.61 (d, 1H, Jtrans ) 15.5 Hz,
CdCH), 7.85 (d, 1H, Jtrans ) 15.5 Hz, CdCH), 7.09 (d, 1H, J5′,6′
) 7.7 Hz, H6′), 12.81 (s, OH). 13C NMR (125 MHz, CDCl3) δ
118.9, 119.2, 120.1, 120.8, 129.6, 129.9, 130.0, 133.3, 136.8, 137.1,
144.2, 163.9 (Ar,Ph), 193.7 (CdO). Treatment of the crude 4′-
chloro-2′-hydroxychalcone (1.64 g) according to method A afforded
the flavonol as pale-yellow needles (597 mg, 22%), mp 198.5–200
°C (EtOH, lit.54 203–205 °C). 1H NMR (CDCl3, 400 MHz) δ ppm
7.09 (br s, OH), 7.42 (dd, 1H, J5,6 ) J6,7 ) 8.0 Hz, H6), 7.49 (d,
2H, J ) 8.8 Hz, H3′,5′), 7.57 (d, 1H, J7,8 ) 8.8 Hz, H8), 7.71
(ddd, 1H, J5,7 ) 1.6 Hz, J6,7 ) 8.0, J7,8 ) 8.8 Hz, H7), 8.21 (d, 2H,
J ) 8.8 Hz, H2′,6′), 8.24 (dd, 1H, J5,7 ) 1.6, J5,6 ) 8.0 Hz, H5).
13C NMR (100 MHz, CDCl3 + DMSO-d6) δ 118.9, 122.0, 125.0,
125.8, 129.2, 129.9, 130.8, 134.2, 135.8, 140.1, 144.8, 155.5
(Ar,Ph), 174.0 (CdO). LRMS (ESI-) m/z: 272.0 [C15H9ClO3 (M
+ H)+ requires 272.0]. Anal. RP-HPLC tR ) 18.4 min (purity 99%).
4′-Trifluoromethylflavonol (6). 2-Hydroxyacetophenone (1.10
mL, 9.0 mmol) and 4-trifluoromethylbenzaldehyde (0.80 mL, 5.97
mmol) according to method A afforded the crude chalcone as yellow
needles. Treatment of the crude 4-trifluoromethyl-2′-hydroxychal-
cone (561 mg) according to method A afforded the flavonol as pale-
yellow needles (293 mg, 43% over two steps), mp 190–191 °C
(ethyl acetate/petroleum spirit, lit.55 181 °C). 1H NMR (CDCl3,
400 MHz): δ ppm 7.12 (br s, OH), 7.46 (t, 1H, J6,7 ) J5,6 ) 8.0
Hz, H6), 7.63 (d, 2H, J2′,3′ ) J5′,6′ ) 8.2 Hz, H3′,5′), 7.81–7.74 (m,
General Procedure for Sequential Claisen-Schmidt Con-
densation of 2-Hydroxyacetophenone and Various Sub-
stituted Benzaldehydes and Algar-Flynn-Oyamada Reac-
tion. Method A. 2-Hydroxyacetophenone (1 mmol) was added to
a suspension of the aldehyde (1.0–1.5 mmol) in ethanol (2 mL)
and aqueous NaOH (1 mL of 25.2 g/100 mL). The mixture was
stirred at room temperature for 5 h or overnight. The reaction
mixture was cooled on ice, and aqueous AcOH (30%) was added
until the mixture was acidic. The mixture was stirred for an
additional 30 min at 0 °C, and the solid precipitate was collected
by filtration. Recrystallization from the indicated solvent afforded
the pure chalcone. Hydrogen peroxide (30%, 0.25 mL) was then
added to an ice-cold suspension of the chalcone (1 mmol) in ethanol
(5 mL) and 1 M NaOH (2 mL). The mixture was allowed to warm
to room temperature and was stirred overnight. The mixture was
acidified with 1 M HCl until slightly acidic, and the precipitate
formed was collected by filtration. Recrystallization from the
indicated solvent afforded the flavonol.
Method B. Method B is as for method A except for the workup
of the Claisen-Schmidt condensation reaction. The reaction mixture
was extracted with ethyl acetate and washed with saturated
NaHCO3, saturated NaCl and water. The organic layer was dried
(MgSO4) and evaporated to dryness to afford the crude chalcone.
Method C. KOH solution (40% w/v, 0.8 mL) was added
dropwise to a suspension of 4-benzyloxy-3-substituted benzaldehyde
(1 mmol) and 2-hydroxyacetophenone (1 mmol) in ethanol (2 mL)
and dioxane (2 mL) cooled on ice. The reaction mixture was
allowed to warm to room temperature, and stirring was continued
for 70 h. Dichloromethane (13 mL) was added, and the organic
layer was washed with water (3 × 2 mL), dried (MgSO4) and
concentrated in vacuo. The yellow oily residue was dissolved in
dioxane (4 mL), ethanol (10 mL), and NaOH solution (5.4% w/v,
3 mL) and was treated dropwise with H2O2 solution (30%, 0.4 mL).
The reaction mixture was stirred in an ice bath for 2 h and
subsequently at room temperature overnight, resulting in a yellow
suspension. The suspension was acidified with 2 M HCl (3 mL),
and the resultant precipitate was collected by filtration and washed
with water (16 mL). The crude product was recrystallized from
the solvents indicated.
2H, H7,8), 8.28 (d, 1H, J5,6 ) 8.0 Hz, H5), 8.40 (d, 2H, J2′,3′
)
J 5′,6′ ) 8.2 Hz, H2′,6′). 13C NMR (100 MHz, DMSO-d6) δ 118.5,
121.3, 122.7, 124.7, 124.9, 125.4, 128.2 (Ar,Ph), 129.2 (q, 1C, J
31.9 Hz, CF3), 134.1, 135.3, 140.1, 143.3, 154.6 (Ar), 173.2 (CdO).
LRMS (ESI-) m/z: 306.1 [C16H9F3O3 (M + H)+ requires 306.1].
Anal. RP-HPLC tR ) 18.8 min (purity 99%).
4′-Methylflavonol (7). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 4-methylbenzaldehyde (1.77 mL, 15.0 mmol) were
reacted for 5 h according to method A to afford 2′-hydroxy-4-
methylchalcone as yellow needles (1.54 g, 65%), mp 115–117 °C
1
(EtOH, lit.53 114–115 °C). H NMR (CDCl3, 400 MHz) δ ppm
2.39 (s, 3H, CH3), 6.93 (dd, 1H, J4′,5′ ) 7.2 Hz, J5′,6′ ) 8.0 Hz,
H5′), 7.01 (d, 1H, J3′,4′ ) 8.4 Hz, H3′), 7.23 (d, 1H, J ) 8.0
Hz, H3,5), 7.48 (ddd, 1H, J3′,4′ ) 8.8 Hz, J4′,5′ ) 7.2, J5′,6′ ) 8.0
Hz, H4′), 7.55 (d, 2H, J ) 8.0 Hz, H2,6), 7.61 (d, 1H, Jtrans ) 15.6
Hz, CdCH), 7.89 (d, 1H, Jtrans ) 15.6 Hz, CdCH), 7.92 (dd, 1H,
J4′,6′ ) 1.6 Hz, J5′,6′ ) 8.0 Hz, H6′). 13C NMR (100 MHz, CDCl3)
δ 22.7 (CH3), 119.6, 119.9, 120.0, 121.1, 129.8, 130.7, 130.9, 132.9,
137.3, 142.7, 146.6, 164.6 (Ar,Ph), 194.8 (CdO). Treatment of 2′-
hydroxy-4-methylchalcone (1.00 g, 4.20 mmol) according to method
A afforded the flavonol as pale-yellow needles (475 mg, 45%),
4′-Methoxyflavonol (4). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 4-methoxybenzaldehyde (1.72 mL, 15.0 mmol) were
reacted for 5 h according to method A to afford 2′-hydroxy-4-
methoxychalcone as yellow needles (1.42 g, 56%), mp 93–94 °C
(EtOH, lit.53 89–91 °C). 1H NMR (CDCl3, 400 MHz) δ ppm 3.85
(s, 3H, CH3), 6.90–6.95 (m, 1H, H4′), 6.94 (d, 2H, J ) 7.9 Hz,

1882 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Qin et al.
mp 190–192 °C (EtOH, lit.54 196–198 °C). 1H NMR (CDCl3, 400
MHz) δ ppm 2.42 (s, 3H, CH3), 7.95 (br s, OH), 7.33 (d, 2H, J )
8.0 Hz, H3′,5′), 7.40 (dd, 1H, J5,6 ) J6,7 ) 6.7 Hz, H6), 7.77 (d,
1H, J7,8 ) 8.3 Hz, H8), 7.68 (ddd, 1H, J5,7 ) 1.8 Hz, J6,7 ) 6.7,
J7,8 ) 8.3 Hz, H7), 8.24 (d, 2H, J ) 8.0 Hz, H2′,6′), 8.23 (dd, 1H,
J5,6 ) 7.8 Hz, J5,7 ) 1.8 Hz, H5). 13C NMR (100 MHz, CDCl3 +
DMSO-d6) δ 21.3 (CH3), 118.0, 121.2, 124.1, 125.0, 127.6, 128.5,
129.0, 133.1, 138.7, 139.8, 145.4, 154.8 (Ar), 173.1 (CdO). LRMS
(ESI-) m/z: 252.1 [C16H12O3 (M + H)+ requires 252.1]. Anal. RP-
HPLC tR ) 17.8 min (purity 99%).
7.11 (br s, 1H, OH), 7.41–7.51 (m, 3H, H4′,5′,6), 7.62 (d, 1H, J7,8
) 8.8 Hz, H8), 7.75 (ddd, 1H, J5,7 ) 1.6 Hz, J6,7 ) J 7,8 ) 8.0 Hz,
H7), 8.19–8.28 (m, 3H, H2′,5,6′). 13C NMR (100 MHz, CDCl3) δ
118.5, 121.2, 124.7, 124.8, 126.0, 127.1, 129.5, 130.5, 133.3, 133.9,
139.6, 143.3, 154.6 (Ar,Ph), 173.1 (CdO). LRMS (ESI+) m/z: 272.0
[C15H9ClO4 (M + H)+ requires 272.0]. Anal. RP-HPLC tR ) 18.3
min (purity 98%).
3′-Trifluoromethylflavonol (12). 2-Hydroxyacetophenone (0.50
mL, 4.15 mmol) and 3-trifluoromethylbenzaldehyde (0.40 mL, 2.99
mmol) according to method A afforded the crude 3-trifluoromethyl-
2′-hydroxychalcone. Treatment of the chalcone (461 mg, 1.58
mmol) according to method A afforded the flavonol as pale-yellow
needles (203 mg, 22% over two steps), mp 171–172 °C (ethyl
4′-tert-Butylflavonol (8). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 4-tert-butylbenzaldehyde (2.51 mL, 15.0 mmol) were
reacted for 5 h according to method A to afford 4′-tert-butyl-2′-
hydroxychalcone as yellow needles (2.07 g, 74%), mp 87.5–88 °C
(EtOH). 1H NMR (CDCl3, 400 MHz) δ ppm 1.37 (s, 9H, C(CH3)3),
6.96 (dd, 1H, J4′,5′ ) 7.0 Hz, J5′,6′ ) 7.0 Hz, H5′), 7.04 (d, 1H, J3′,4′
1
acetate/petroleum spirit). H NMR (DMSO-d6, 400 MHz) δ ppm
7.45–7.51 (m, 1H, H4′), 7.79–7.89 (m, 5H, OH, H5′,7,6, 6′), 8.11
(d, 1H, J7,8 ) 8.0 Hz, H8), 8.49 (d, 1H, J5,6 ) 8.0 Hz, H5), 8.56 (s,
H2′). 13C NMR (100 MHz, DMSO-d6) δ 119.3, 122.0, 123.4, 124.7,
125.4, 125.5, 126.1, 126.9 (Ar,Ph), 130.0 (q, 1C, J ) 34.3 Hz,
CF3), 131.9, 133.1, 134.7, 140.5, 144.0, 155.3 (Ar,Ph), 173.8
(CdO). LRMS (ESI-) m/z: 306.1 [C16H9F3O3(M + H)+ requires
306.1]. Anal. RP-HPLC tR ) 18.5 min (purity 99%).
) 8.5 Hz, H3′), 7.47 (d, 2H, J ) 8.0 Hz, H3,5), 7.51 (ddd, J3′,4′
)
8.5 Hz, J4′,5′ ) 7.0 Hz, J4′,6′ ) 1.5 Hz, H4′), 7.62 (d, 2H, J ) 8.0
Hz, H2,6), 7.65 (d, 1H, Jtrans ) 16.0 Hz, CdCH), 7.94 (d, 1H,
Jtrans ) 16.0 Hz, CdCH), 7.95 (dd, 1H, J4′,6′ ) 1.5 Hz, J5′,6′ ) 7.0
Hz, H6′). 13C NMR (100 MHz, CDCl3) δ 32.2 (CH3), 36.1
(C(CH3)3), 119.9, 120.3, 121.1, 127.1, 129.7, 130.7, 132.9, 137.3,
146.6, 155.8, 164.7 (Ar, CdC), 194.9 (CdO). Treatment of 4-tert-
butyl-2′-hydroxychalcone (561 mg, 2.00 mmol) according to method
A afforded the flavonol as pale-yellow needles (365 mg, 62%),
mp 175–177 °C (EtOH). 1H NMR (CDCl3, 400 MHz) δ ppm 1.38
(s, 9H, C(CH3)3), 6.96 (br s, OH), 7.40 (dd, 1H, J5,6 ) 5.7 Hz, J6,7
) 5.4 Hz, H6), 7.55 (d, 2H, J ) 6.4 Hz, H3′,5′), 7.57 (d, 1H, J7,8
) 6.6 Hz, H8), 7.68 (ddd, J5,7 )1.2 Hz, J6,7 ) 5.4 Hz, J7,8 ) 6.6
Hz, H7), 8.18 (d, 2H, J ) 6.4 Hz, H2′,6′), 8.24 (dd, 1H, J5,6 ) 5.7
Hz, J5,7 ) 1.2 Hz, H5). 13C NMR (100 MHz, CDCl3) δ 32.2 (CH3),
36.0 (C(CH3)3), 119.3, 121.8, 125.5, 126.5, 126.6, 128.7, 129.3,
134.5, 139.3, 146.4, 154.7, 156.4 (Ar), 174.4 (CdO). LRMS (ESI-)
m/z: 294.1 [C19H18O3 (M + H)+ requires 294.1]. Anal. RP-HPLC
tR ) 21.0 min (purity 96%).
3′-Methylflavonol (13). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 3-methylbenzaldehyde (1.41 mL, 12.0 mmol) were
reacted overnight according to method B to afford the crude
3-methoxy-2′-hydroxychalcone as orange needles. 1H NMR (CDCl3,
400 MHz) δ ppm 2.42 (s, 3H, CH3), 6.96 (dd, 1H, J4′,5′ ) J5′,6′
)
8.4 Hz, H5′), 7.04 (d, 1H, J3′,4′ ) 8.4 Hz, H3′), 7.25–7.27 (m, 1H,
H4), 7.34 (dd, 1H, J4,5 ) J5,6 ) 8.4 Hz, H5), 7.47–7.51 (m, 3H,
H2,4′,6), 7.66 (d, 1H, Jtrans ) 15.6 Hz, CdCH), 7.91 (d, 1H, Jtrans
) 15.6 Hz, CdCH), 7.95 (dd, 1H, J4′,6′ ) 1.6, J5′,6′ ) 8.4 Hz, H6′).
Treatment of 3-methyl-2′-hydroxychalcone (1.89 g, 7.50 mmol)
according to method A afforded the flavonol as pale-yellow needles
(1.18 g, 47% over two steps), mp 146–148 °C (EtOH, lit.59 146–148
1
°C). H NMR (CDCl3, 400 MHz) δ ppm 2.48 (s, 3H, CH3), 6.99
(br s, 1H, OH), 7.29–7.31 (m, 1H, H6′), 7.41–7.45 (m, 2H, H4′,5′),
7.62 (ddd, 1H, J7,8 ) 8.5 Hz, J6,8 ) 2.0 Hz, J5,8 ) 0.5 Hz, H8),
7.72 (ddd, 1H, J6,7 ) J7,8 ) 8.5 Hz, J5,7 ) 2.0 Hz, H7), 8.07–8.08
3′-Methoxyflavonol (10). 2-Hydroxyacetophenone (1.20 mL,
10.0 mmol) and 3-methoxybenzaldehyde (1.82 mL, 15.0 mmol)
were reacted overnight according to method A to afford 3-methoxy-
2′-hydroxychalcone as yellow needles (1.49 g, 59%), mp 92–94
(m, 2H, H2′,6′), 8.26 (ddd, 1H, J5,8 ) 0.5 Hz, J5,7 ) 2.0 Hz, J5,6
)
8.0 Hz, H5). 13C NMR (100 MHz, CDCl3) δ 21.9 (CH3), 119.1,
122.0, 125.2, 125.5, 125.7, 128.6, 129.1, 131.2, 131.9, 134.4, 138.3,
139.7, 146.0, 155.27 (Ar,Ph), 173.6 (CdO). LRMS (ESI-) m/z:
252.1 [C16H12O3 (M + H)+ requires 252.1]. Anal. RP-HPLC tR )
17.7 min (purity 99%).
1
°C (EtOH, lit.56 94–96 °C). H NMR (CDCl3, 400 MHz) δ ppm
3.87 (s, 3H, CH3), 6.95 (dd, 1H, J4′,5′ ) J5′,6′ ) 8.0 Hz, H5′), 6.99
(dd, 1H, J4,6 ) 2.4 Hz, J5,6 ) 8.0 Hz, H6), 7.03 (d, 1H, J3′,4′ ) 8.4
Hz, H3′), 7.17 (s, 1H, H2), 7.27 (d, 1H, J4,5 ) 8.0 Hz, H4), 7.36
(dd, 1H, J4,5 ) J5,6 ) 8.0 Hz, H5), 7.51 (m, 1H, H4′), 7.64 (d, 1H,
Jtrans ) 15.6 Hz, CdCH), 7.89 (d, 1H, Jtrans ) 15.6 Hz, CdCH),
7.92 (dd, 1H, J4′,6′ ) 1.2, J5′,6′ ) 8.0 Hz, H6′). Treatment of
3-methoxy-2′-hydroxychalcone (1.03 g, 4.00 mmol) according to
method A afforded the flavonol as pale-yellow needles (530 mg,
4′-Benzyloxy-3′-methoxyflavonol. 4-Benzyloxy-3-methoxyben-
zaldehyde (5.74 g, 23.7 mmol) and 2-hydroxyacetophenone (2.85
mL, 23.7 mmol) according to method C gave 4′-benzyloxy-3′-
methoxyflavonol as bright-yellow crystals (3.91 g, 44%), mp
187–190 °C (EtOH). 1H NMR (400 MHz, CDCl3) δ ppm 3.99 (s,
3H, CH3), 5.24 (s, 2H, CH2), 7.01 (d, 1H, J5′,6′ ) 8.4 Hz, H5′),
7.32 (t, 1H, J ) 7.2 Hz, H4′′), 7.37–7.41 (m, 3H, H6,3′′,5′′), 7.46
(d, 2H, J ) 7.2 Hz, H2′′,6′′), 7.55 (d, 1H, J7,8 ) 8.4 Hz, H8), 7.67
(ddd, 1H, J6,7 ≈ J7,8 ) 8.4, J5,7 ) 1.6 Hz, H7), 7.81 (dd, 1H, J2′,6′
) 2.0, J5′,6′ ) 8.4 Hz, H6′), 7.87 (d, 1H, J2′,6′ ) 2.0 Hz, H2′), 8.23
(dd, 1H, J5,6 ) 8.0, J5,7 ) 1.6 Hz, H5). 13C NMR (100 MHz,
DMSO-d6) δ 56.1 (CH3), 70.7 (CH2), 111.2, 113.2, 118.1, 120.6,
121.3, 123.9, 124.4, 125.3, 127.2, 128.0, 128.6, 133.3, 136.5, 137.7,
145.1, 149.3, 149.8, 155.1 (Ar,Ph), 173.0 (CdO). LRMS (ESI+)
m/z: 375.1 [C23H18O5 (M + H)+ requires 375.1]. Anal. Calcd for
C23H18O5: C, 73.79; H, 4.85. Found: C, 73.79; H, 4.79%.
4′-Benzyloxy-3′-bromoflavonol. 4-Benzyloxy-3-bromobenzal-
dehyde (1.00 g, 3.43 mmol) and 2-hydroxyacetophenone (0.41 mL,
3.43 mmol) according to method C gave 4′-benzyloxy-3′-meth-
oxyflavonol as bright-yellow crystals (0.50 g, 35%), mp 204–208
°C (THF/petroleum spirit). 1H NMR (400 MHz, DMSO-d6) δ ppm
5.25 (s, 2H, CH2), 6.90 (br s, OH), 7.06 (d, 1H, J5′,6′ ) 8.8 Hz,
H5′), 7.34 (t, 1H, J ) 7.6 Hz, H4′′), 7.38–7.42 (m, 3H, H6,3′′,5′′),
7.48 (d, 2H, J ) 8.8 Hz, H2′′,6′′), 7.58 (d, 1H, J7,8 ) 8.0 Hz, H8),
7.70 (ddd, 1H, J6,7 ≈ J7,8 ) 8.0 Hz, J5,7 ) 1.6 Hz, H7), 8.20–8.24
(m, 2H, H5,6′), 8.47 (d, 1H, J2′,6′ ) 2.0 Hz, H2′). 13C NMR (400
MHz, DMSO-d6) δ 70.63 (CH2), 114.0, 114.0, 118.6, 121.7, 124.7,
125.2, 125.7, 127.7, 128.3, 128.7, 128.8, 132.4, 133.8, 136.5, 139.2,
1
49%), mp 131 °C (EtOH, lit.57 131 °C). H NMR (CDCl3, 500
MHz) δ ppm 3.92 (s, 3H, CH3), 7.03–7.06 (m, 2H, OH, H4′),
7.42–7.49 (m, 2H, H5′,6), 7.61 (d, 1H, J7,8 ) 8.0 Hz, H8), 7.73
(ddd, 1H, J6,7 ) J7,8 ) 8.2 Hz, J5,7 ) 1.6 Hz, H7), 7.84 (dd, 1H,
J2′,4′ ) J2′,6′ ) 2.4 Hz, H2′), 7.88 (d, 1H, J5′,6′ ) 8.0 Hz, H6′), 8.27
(dd, 1H, J5,7 ) 1.6 Hz, J5,6 ) 8.4 Hz, H5). 13C NMR (100 MHz,
CDCl3) δ 55.2 (CH3), 113.4, 115.2, 118.5, 120.0, 121.2, 124.6,
124.8, 129.6, 132.5, 133.7, 139.2, 146.8, 154.5, 159.1 (Ar,Ph), 173.0
(CdO). LRMS (ESI-) m/z: 268.1 [C16H12O4 (M + H)+ requires
268.0]. Anal. RP-HPLC tR ) 16.7 min (purity 99%).
3′-Chloroflavonol (11). 2-Hydroxyacetophenone (1.20 mL, 10.0
mmol) and 3-chlorobenzaldehyde (1.70 mL, 15.0 mmol) were
reacted overnight according to method A to afford 3-chloro-2′-
hydroxychalcone as yellow needles (0.76 g, 29%), mp 106 °C
1
(EtOH, lit.56 105–106 °C). H NMR (CDCl3, 400 MHz) δ ppm
6.97 (dd, 1H, J4′,5′ ) J5′,6′ ) 8.0 Hz, H4′), 7.06 (dd, 1H, J3′,5′ ) 1.0
Hz, J3′,4′ ) 8.0 Hz, H3′), 7.38–7.41 (m, 2H, H4,5), 7.52–7.54 (m,
2H, H5′,6), 7.66 (d, 1H, Jtrans ) 15.6 Hz, CdCH), 7.67 (s, 1H,
H2), 7.85 (d, 1H, Jtrans ) 15.6 Hz, CdCH), 7.93 (dd, 1H,J4′,6′
)
1.6 Hz, J5′,6′ ) 8.0 Hz, H6′). Treatment of 3-chloro-2′-hydroxy-
chalcone (1.03 g, 4.00 mmol) according to method A afforded the
flavonol as pale-yellow needles (0.37 g, 34%), mp 158–160 °C
1
(EtOH, lit.58 158–160 °C). H NMR (CDCl3, 400 MHz) δ ppm

Relaxant FlaVonols without Antioxidant ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1883
144.1, 154.8, 155.7 (Ar,Ph), 173.2 (CdO). LRMS (ESI+) m/z: 423.3
[C22H15BrO4 (M + H)+ requires 423.0]. Anal. Calcd for
C22H15BrO4: C, 62.43; H, 3.57. Found: C, 62.50; H, 3.41%.
4′-Benzyloxy-3′-trifluoromethylflavonol. 4-Benzyloxy-3-trif-
luoromethylbenzaldehyde (0.42 g, 1.49 mmol) and 2-hydroxyac-
etophenone (0.18 mL, 1.49 mmol) according to method C gave
4′-benzyloxy-3′-trifluoromethylflavonol as bright-yellow crystals
144.3, 154.4, 155.6 (Ar,Ph), 172.6 (CdO). Anal. Calcd for
C15H9BrO4: C, 54.08; H, 2.72. Found: C, 54.04; H, 2.80%. LRMS
(ESI+) m/z: 333.1 [C15H9BrO4 (M + H)+ requires 333.1]. Anal.
RP-HPLC tR ) 15.2 min (purity 97%).
4′-Hydroxy-3′-trifluoromethylflavonol (16). 4′-Benzyloxy-3′-
trifluoromethylflavonol (0.20 g, 0.49 mmol) gave a brown solid
after recrystallization (100 mg, 64%), mp 205–206 °C (THF/
petroleum spirit). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.20 (d,
1H, J5′,6′ ) 8.8 Hz, H5′), 7.46 (t, 1H, J6,7 ≈ J5,6 ) 7.6 Hz, H6),
7.76–7.77 (m, 2H, H7,8), 8.08 (d, 1H, J5′,6′ ) 8.8 Hz, H6′), 8.29
1
(0.25 g, 41%), mp 165–166 °C (THF/petroleum spirit). H NMR
(500 MHz, CDCl3) δ ppm 5.31 (s, 2H, CH2), 6.91 (br s, OH), 7.17
(d, 1H, J5′,6′ ) 7.8 Hz, H5′), 7.34 (d, 1H, J5,6 ) 6.3 Hz, H6),
7.35–7.43 (m, 5H, H2′′,3′′,4′′,5′′,6′′), 7.60 (d, 1H, J7,8 ) 7.8 Hz,
H8), 7.71 (d, 1H, J7,8 ) 7.8 Hz, H7), 8.24 (d, 1H, J5′,6′ ) 7.8 Hz,
H6′), 8.57 (d, 1H, J5,6 ) 6.3 Hz, H5), 8.51 (s, 1H, H2′). 13C NMR
(100 MHz, DMSO-d6) δ 70.8 (CH2), 115.1 (Ar,Ph), 118.0 (q, 1C,
J ) 30.0 Hz, CF3), 119.1, 122.0, 124.3, 125.4, 125.4, 125.6, 126.9,
127.0, 127.8, 128.9, 129.3, 129.3, 133.9, 134.4, 136.7, 139.4, 144.6,
155.1, 157.4 (Ar,Ph), 173.5 (CdO).
(d, 1H, J5,6 ) 7.6 Hz, H5), 8.43 (s, 1H, H2′), 9.62 (br s, OH). 13
C
NMR (100 MHz, DMSO-d6) δ 115.6 (CF3, 1C, J ) 29.2 Hz), 117.3,
118.4, 121.3, 121.8, 122.5, 124.6, 124.7, 126.4, 132.9, 133.6, 138.3,
144.5, 154.4, 157.2 (Ar, Ph), 172.7 (CdO). LRMS (ESI+) m/z:
322.2 [C16H9F3O4 (M + H)+ requires 322.2]. Anal. RP-HPLC tR
) 15.8 min (purity 99%).
4′-Hydroxy-3′-methylflavonol (17). 4′-Benzyloxy-3′-methylfla-
vonol (0.39 g, 1.08 mmol) gave a brown solid after recrystallization
(0.15 g, 52%), mp 255–257 °C (THF/petroleum spirit). 1H NMR (400
MHz, DMSO-d6) δ ppm 2.22 (s, 3H, CH3), 6.95 (d, 1H, J5′,6′ ) 8.4
Hz, H5′), 7.45 (t, 1H, J6,7 ≈ J5,6 ) 8.1 Hz, H6), 7.73–7.80 (m, 2H,
H7,8), 7.96 (d, 1H, J5′,6′ ) 8.4 Hz, H6′), 8.00 (s, 1H, H2′), 8.09 (d,
1H, J5,6 ) 8.1 Hz, H5), 9.28 (br s, OH), 10.02 (br s, OH). 13C NMR
(100 MHz, DMSO-d6) δ 16.8 (CH3), 115.3, 118.9, 122.0, 122.5, 124.8,
125.1, 125.4, 127.8, 130.9, 134.0, 138.5, 146.9, 155.1, 158.0 (Ar,Ph),
173.1 (CdO). Anal. Calcd for C16H12O4: C, 71.64; H, 4.51. Found:
C, 71.59; H, 4.58%. LRMS (ESI+) m/z: 268.2 [C16H12O4 (M + H)+
requires 268.2]. Anal. RP-HPLC tR ) 14.6 min (purity 98%).
4′-Hydroxy-3′-tert-butylflavonol (18). 4′-Benzyloxy-3′-tert-bu-
tylflavonol (0.39 g, 0.97 mmol) gave a brown solid after recrys-
tallization (0.052 g, 17%), mp 203–207 °C (THF/petroleum spirit).
1H NMR (400 MHz, DMSO-d6) δ ppm 1.40 (s, 9H, C(CH3)3), 6.96
(d, 1H, J5′,6′ ) 8.4 Hz, H5′), 7.45 (t, 1H, J6,7 ≈ J5,6 ) 8.0 Hz, H6),
7.73 (d, 1H, J7,8 ) 8.0 Hz, H8), 7.78 (ddd, 1H, J7,8 ) 8.0 Hz, J5,7
) 1.6 Hz, H7), 7.83 (d, 1H, J5′,6′ ) 8.4 Hz, J2′,6′ ) 2.4 Hz, H6′),
8.00 (dd, 1H, J5,6 ) 8.0 Hz, J5,7 ) 1.6 Hz, H5), 8.12 (dd, 1H, J2′,6′
) 2.4 Hz, H2′), 9.27 (br s, OH), 10.11 (br s, OH). 13C NMR (100
MHz, DMSO-d6) δ 29.3 (C(CH3)3), 34.6 (C(CH3)3), 116.3, 118.3,
121.4, 121.6, 124.5, 124.7, 126.6, 133.4, 135.3, 137.7, 146.7, 154.4,
157.8 (Ar,Ph), 172.5 (CdO). HRMS (ESI+) m/z: 333.1095
[C19H18NaO4 (M + Na)+ requires 333.1103]. Anal. RP-HPLC tR
) 17.9 min (purity 97%).
4′-Benzyloxy-3′-methylflavonol. 4-Benzyloxy-3-methylbenzal-
dehyde (0.75 g, 3.32 mmol) and 2-hydroxyacetophenone (0.40 mL,
3.32 mmol) according to method C gave 4′-benzyloxy-3′-meth-
ylflavonol as bright-yellow crystals (0.50 g, 42%), mp 199–202
°C (THF/petroleum spirit). 1H NMR (400 MHz, DMSO-d6) δ ppm
2.29 (s, 3H, CH3), 5.23 (s, 2H, CH2), 7.20 (d, 1H, J5′,6′ ) 8.8 Hz,
H5′), 7.32–7.50 (m, 6H, H6,2′′,3′′,4′′,5′′,6′′), 7.74–7.80 (m, 2H,
H7,8), 8.05–8.10 (m, 3H, H5,2′,6′), 9.40 (br s, OH). 13C NMR (100
MHz, DMSO-d6) δ 16.4 (CH3), 69.4 (CH2), 111.8, 118.4, 120.4,
123.4, 124.6, 124.8, 126.2, 127.4, 127.5, 128.0, 128.6, 129.8, 133.6,
137.1, 138.3, 145.8, 154.5, 157.7 (Ar,Ph), 172.7 (CdO). LRMS
(ESI+) m/z: 358.4 [C23H18O5 (M + H)+ requires 358.4]. Anal. Calcd
for C23H18O4: C, 77.08; H, 5.06. Found: C, 77.05; H, 5.06%.
4′-Benzyloxy-3′-tert-butylflavonol. 4-Benzyloxy-3-tert-butyl-
benzaldehyde (0.57 g, 2.11 mmol) and 2-hydroxyacetophenone
(0.21 mL, 2.11 mmol) according to method C gave 4′-benzyloxy-
3′-tert-butylflavonol as brown crystals (0.47 g, 55%), mp 163–164
°C (THF/petroleum spirit). 1H NMR (400 MHz, DMSO-d6) δ ppm
1.41 (s, 9H, C(CH3)3), 5.26 (s, 2H, CH2), 7.28 (d, 1H, J5′,6′ ) 8.8
Hz, H5′), 7.36 (t, 1H, J ) 7.2 Hz, H4′′), 7.42–7.48 (m, 3H,
H6,3′′,5′′), 7.53 (d, 2H, J ) 7.6 Hz, H2′′,6′′), 7.75 (d, 1H, J7,8
)
8.4 Hz, H8), 7.79 (ddd, 1H, J6,7 ≈ J7,8 ) 8.4 Hz, J5,7 ) 1.2 Hz,
H7), 8.07–8.11 (m, 2H, H5,6′), 8.22 (d, 1H, J2′,6′ ) 2.4 Hz, H2′).
13C NMR (100 MHz, DMSO-d6) δ 29.5 (C(CH3)3), 34.8 (C(CH3)3),
69.8 (CH2), 112.8, 118.4, 121.4, 123.2, 124.5, 124.8, 126.2, 127.4,
127.8, 128.0, 128.6, 133.5, 136.8, 137.2, 138.2, 146.1, 154.4, 158.3
(Ar, Ph), 172.6 (CdO). Anal. Calcd for C26H24O4: C, 77.98; H,
6.04. Found: C, 78.01; H, 6.09%.
Acknowledgment. This work was supported by the Shep-
herd Foundation, the Australian Research Council, and by
University of Melbourne MIFRS and MIRS Scholarships to
C.X.Q.
General Procedure for Debenzylation of 4′-Benzyloxy-
3′-substituted Flavonols. A suspension of protected flavonol (1
mmol) in HCl (36%, 25 mL) and acetic acid (100%, 25 mL) was
heated at 100 °C for 2 h. The solvent was evaporated under reduced
pressure, and the product was recrystallized from the solvents indicated.
4′-Hydroxy-3′-methoxyflavonol (14). 4′-Benzyloxy-3′-methoxy-
flavonol (1.00 g, 2.67 mmol) gave a yellow solid after recrystallization
(0.48 g, 63%), mp 249–251 °C (EtOH). 1H NMR (400 MHz, DMSO-
d6) δ ppm 3.79 (s, 3H, CH3), 6.84 (d, 1H, J5′,6′ ) 8.8 Hz, H5′), 7.22
(t, 1H, J6,7 ≈ J5,6 ) 8.4 Hz, H6), 7.41 (d, 1H, J7,8 ) 8.4 Hz, H8), 7.51
(t, 1H, J6,7 ≈ J7,8 ) 8.4 Hz, H7), 7.63 (dd, 1H, J2′,6′ ) 2.0, J5′,6′ ) 8.8
Hz, H6′), 7.68 (d, 1H, J2′,6′ ) 2.0 Hz, H2′), 8.03 (d, 1H, J5,6 ) 8.4 Hz,
H5), 8.40 (br s, OH). 13C NMR (100 MHz, DMSO-d6 + CDCl3) δ
55.8 (CH3), 111.6, 115.4, 118.0, 121.3, 121.8, 122.4, 124.0, 124.8,
132.9, 138.0, 145.8, 147.3, 148.7, 154.5 (Ar,Ph), 172.6 (CdO). Anal.
Calcd for C16H12O5: C, 67.60; H, 4.25. Found: C, 67.62; H, 4.23%.
LRMS (ESI +) m/z: 284.3 [C16H12O5 (M + H)+ requires 284.3]. Anal.
RP-HPLC tR ) 13.5 min (purity 96%).
Supporting Information Available: Table indicating results
from HPLC purity analysis and/or combustion analysis of com-
pounds 1-18, HPLC traces for compounds 1-18, and 1H and 13
C
NMR spectra for compounds 4–8 and 10–18. This material is
available free of charge via the Internet at http://pubs.acs.org.
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