Synthesis and antioxidant properties of new chromone derivatives

Article abstract of DOI:10.1016/j.bmc.2009.08.056

Nowadays, the recognition of the benefits of antioxidants is eliciting an increasingly interest in the search for new molecules with improved activity. The aim of the present work was to search for improved reactive oxygen species (ROS) and reactive nitro

Full text of DOI:10.1016/j.bmc.2009.08.056

Bioorganic & Medicinal Chemistry 17 (2009) 7218–7226
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antioxidant properties of new chromone derivatives
Ana Gomes a, Ondrej Neuwirth a, Marisa Freitas a, Diana Couto a, Daniela Ribeiro a,
Andrea G. P. R. Figueiredo b, Artur M. S. Silva b, Raquel S. G. R. Seixas b, Diana C. G. A. Pinto b,
a
Augusto C. Tomé b, José A. S. Cavaleiro b, Eduarda Fernandes a, , José L. F. C. Lima
*
a REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal
b QOPNA, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2009
Revised 26 August 2009
Accepted 28 August 2009
Available online 3 September 2009
Nowadays, the recognition of the benefits of antioxidants is eliciting an increasingly interest in the search
for new molecules with improved activity. The aim of the present work was to search for improved reac-
tive oxygen species (ROS) and reactive nitrogen species (RNS) scavengers by testing new structures of 2-
styrylchromones (2-SC) and 3-substituted flavones, which were synthesised by the Baker–Venkataraman
approach. The new compounds were also tested for their metal chelating capacity and reducing activity.
The obtained results showed that the methylation of hydroxyl groups decreases the scavenging of ROS
and RNS by 2-SC. The decrease in the scavenging activities was, generally, more evident when the meth-
Keywords:
2-Styrylchromones
3-Substituted flavones
Reactive oxygen species
Reactive nitrogen species
Scavenging activity
Reducing activity
ylation occurred in B-ring, except for O2 and 1O2. On the other hand, the introduction of a substituent,
Åꢀ
either hydroxyl or methoxyl, in position 8 was sometimes favourable and others unfavourable to the
scavenging activities, depending on the reactive species. In conclusion, the study of the antioxidant prop-
erties of the new 2-SC and flavones allowed establishing new structure–activity relationships and
brought out, in some cases, pharmacophores with improved activity.
Metal chelating activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
molecular stabilization, increasing the compound’s antiradical
activity.
The 2-styrylchromones (2-SC) designation presently represents
a well recognized group of natural and synthetic chromones, viny-
logues of flavones (2-phenylchromones). As it occurs for several
flavones, several studies performed with 2-SC have disclosed inter-
esting activities with potential therapeutic applications, possibly in
the treatment of cancers,1–5 allergies,6 viral infections,7,8 gout,9 and
oxidative stress related damage.10 The antioxidant behaviour of
these compounds is a matter of particular interest, as previously
demonstrated by their strong protective effects against pro-oxi-
dant agents observed in cellular systems10 and in non-cellular
systems.11 Recently our group has tested several 2-SC and struc-
tural-similar flavonoids for their scavenging activities against reac-
tive oxygen species (ROS) and reactive nitrogen species (RNS),
providing outstanding results for several of the tested compounds,
considering the nanomolar to micromolar range of the IC50 values
found.12 In that study, we demonstrated that the 30,40-dihydroxy
substitution on the B-ring plays an important role in the scaveng-
ing activity of 2-SC for ROS and RNS. In addition, the number and
position of the hydroxyl groups in the A-ring also seems to contrib-
ute to the scavenging effect of these compounds. It was also
pointed out the likely contribution of the styryl moiety to the
The aim of the present work was to continue searching for im-
proved ROS and RNS scavengers by testing new structures of 2-SC
(7a–7c) and 3-substituted flavones (8a–8c, 9) (Scheme 1 and
Fig. 1). In what concerns to 2-SC we studied the effects resulting
from the methylation of the 30,40-dihydroxy group (7c), the
changes in the position of the hydroxyl groups in the A-ring as well
as their methylation (7a–7c). Regarding the flavones, we tested
new compounds with different length alkyl chains in the position
3 of the C-ring (8a–8c) and with a 3,4-dihydroxybenzoyl substitu-
ent in the same position (9).
Besides the scavenging of ROS and RNS the new compounds
were also tested for their metal chelating activity and for their
reducing capacity since these are indicators of antioxidant activity.
A compound can act as antioxidant through the chelation of tran-
sition metals (especially iron and copper), preventing their partic-
ipation in free radical generating reactions. In fact, iron and copper
have a major role in the production of the very reactive hydroxyl
radical (HOÅ), through the Fenton and Haber-Weiss reactions.13–16
In addition, through a Fenton type reaction, preformed lipid hydro-
peroxides (ROOH) are decomposed to form the alkoxyl radicals
(ROÅ), strong oxidants, which can propagate the chain reaction of
lipid peroxidation.6,17 On the other hand, the antioxidant power
of certain substances, phenolic compounds in particular, can be as-
sessed by methodologies that measure the reducing capacity
employing ferric iron as oxidant.18,19
* Corresponding author. Tel.: +351 222078968; fax: +351 222004427.
E-mail address: egracas@ff.up.pt (E. Fernandes).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.056

A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
7219
O
β
R1
R1
β
4'
2''
5''
R2
R2
CO2H
3''
4''
3'
R1 4'
5'
O
+
3'
R1
A/B
α
α
2'
R2
2
2'
6''
4
1
3
R2
OH
O
6'
O
2
1
3
a) R2 = OMe
b) R2 = OBn
a) R1 = OMe
b) R1 = OBn
C
R2
R2
3''
5'
R1
R1
R2
R1 4'
R2
4''
6'
2''
4
2
5
3
1
5''
R1
3'
2'
6''
OH
O
O
OH
O
OH
4
5
5'
R2
R2
R2
R2
D
4'
6'
β
R1
8
R1
3'
2'
R1
7
9
O
R1
O
α
E/F
2
3
6
10
4
5
O
O
7
6
For compounds 7
For compounds 3, 4, 5 and 6
a) R1 = OMe; R2 = OBn
b) R1 = OMe, R2 = OMe
c) R1 = OBn, R2 = OMe
A: Py, POCl3, room temp., N2
a) R1 = OMe; R2 = OH
b) R1 = OH, R2 = OH
c) R1 = OH, R2 = OMe
B: CH2Cl2, DCC, 4-pyrrolidinopyridine, room temp., N2
C: DMSO, KOH, 2 h, room temp.
D: p-toluenesulfonic acid, DMSO, 90ºC
E: HCl (37%)/AcOH glacial (1:10), 90ºC, 2-12 h.
F: BBr3/CH2Cl2, -78ºC to room temp. (24 h)
Scheme 1. Synthesis of the studied 2-styrylchromones.
afforded b-diketones 5a–5c in good yields (60–95%); these were
submitted to a cyclodehydration process with a p-toluenesulfonic
acid/DMSO mixture, leading to the synthesis of alkoxy-2-styrylchr-
omones 6a–6c in good yields (62–84%). The final step of our
synthetic route consisted on the demethylation of alkoxy-2-
styrylchromones 6a–6c. The cleavage of the methyl groups of
30,40,7,8-tetramethoxy-2-styrylchromones 6b was performed by
treatment with boron tribromide (2.5 equiv per methyl group) in
dichloromethane for 24 h (Scheme 1). The cleavage of the benzyl
groups of methoxybenzyloxy-2-styrylchromones 6a, 6c was car-
ried out by treatment with a mixture of chloridric acid in acetic
acid, leading to the expected methoxy-2-styrylchromones 7a, 7c.
OH
OH
OH
OH
HO
O
O
HO
O
O
OH
n
a) n = 3
b) n = 5
c) n = 9
OH
O
9
OH
8
OH
Figure 1. Chemical structures of the studied 3-substituted flavones.
2.2. Antioxidant activity
2. Results and discussion
2.1. Chemistry
Free radical scavenging capacity of flavone derivatives, has been
primarily attributed to the high reactivity of hydroxyl substituents
that participate in the following reaction:27
Flavone-OH þ RÅ ! Flavone-OÅ þ RH
The 2-SC 7a–7c and 3-substituted flavones 8a–8c and 9 were
synthesised by the Baker–Venkataraman method.20–22 3-Substi-
tuted flavones 8a–8c and 9 have been recently published,23,24
while 2-SC 7a–7c are new compounds, reported for the first time
in the present study. The first step of this synthesis involves the
esterification of 20-hydroxyacetophenones 1a, 1b with cinnamoyl
chlorides 2a, 2b, prepared in situ from the corresponding cinnamic
acids and phosphorous oxychloride in dry pyridine (Scheme 1).25
The yield of compounds 3b and 3c is higher if the esterification
reaction is conducted with N,N-dicyclohexylcarbodiimide (DCC),
as coupling esterification agent, in the presence of a catalytic
amount of 4-pyrrolidinopyridine.26 Baker–Venkataraman rear-
rangement of esters 3a–3c with potassium hydroxide in DMSO
As reviewed by Heim et al.,27 the B-ring 30,40-catechol structure
is the most significant determinant of scavenging of ROS and RNS,
leading to the formation of fairly stable ortho-semiquinone radicals
through facilitating electron delocalization while the effects result-
ing from the hydroxylation pattern of ring A are more variable. This
was only partially confirmed in our study.
All of the tested compounds were able to prevent O2Åꢀ-depen-
dent reduction of NBT in a concentration-dependent manner. The
most effective were the 2-SC and flavone 9. The order of potencies
found was 7b > 9 > 7a > 7c > 8a > 8b > 8c (Table 1). We confirmed

7220
A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
Table 1
dent manner. The order of potencies found was 8a > 8c > 9 > 8b >
7a > 7b > 7c (Table 1). Considering what has been previously dem-
onstrated for the flavonols quercetin and rutin30 and for the iso-
flavones genistein, biochanin-A, and daidzein31 the reaction of
the tested flavones with HOCl is likely to occur via an electrophilic
aromatic substitution consisting in the chlorination of C-6 and C-8
of the A-ring, where 5-OH and 7-OH groups are the most reactive.
The presence of a substituent in C-3 is apparently favourable to the
reaction with HOCl, since all of the tested flavones where shown to
be more active than their parent compound, luteolin.12
O2Åꢀ, H2O2, 1O2 and HOCl scavenging activities (IC50, mean SE) of the tested 2-
styrylchromones, flavones and positive controls
Compound
IC50
(l
M)
1O2
Åꢀ
O2
H2O2
HOCl
2-Styrylchromones
7a
7b
7c
20.8 3.2
17.8 3.8
26.0 3.8
701.9 58.2
799.3 60.9
31%a
7.97 1.38
4.69 0.64
9.95 2.27
4.67 0.64
67.2 11.2
47%b
Flavones
All of the tested compounds were able to prevent the 1O2-
dependent oxidation of DHR. Some of them were even more effec-
tive than ascorbic acid. The order of potencies found was
7b > 8c > 7a > 7c > 9 > 8bꢁ8a (Table 1). From the analysis of the re-
sults of 2-SC, it seems that the methylation of the hydroxyl groups
is disadvantageous for the 1O2 scavenging activity, since the com-
pounds 7a and 7c where slightly less effective than compound 7b.
No meaningful differences were found between the new 2-SC and
those tested before (30,40-dihydroxy-2-styrylchromone deriva-
tives),12 which indicates that the substitution in the position 8 does
not bring important changes to the 1O2 scavenging activity. The
tested flavones where much less active then their parent com-
pound, luteolin,12 with the exception of compound 8c. The high
activity showed by this compound can be explained by the pro-
posed mechanism of the chemical reactions of flavones with 1O2.
It has been previously suggested32 that there is a cycloaddition of
1O2 to the double bond of the C-ring, leading to the formation of
a 1,2-dioxetane intermediate. This reaction is potentiated by elec-
tron-donating substituents in C-3, which activate the double bond.
Following this rationale, the higher activity of compound 8c over
8a and 8b is understandable because the alkyl chain C-3 substitu-
ent is bigger in 8c, thus more electron-donating. The higher effect
of 2-SC over flavones is probably due to the double bond in the sty-
ryl moiety, which is a possible target for 1O2.33
The results from the ORAC assay are listed in Table 2. All the
compounds were able to prevent the ROOÅ-dependent oxidation
of fluorescein. All of them were considerably more active than
ascorbic acid. The order of potencies found was 9 > 7a > 8a > 7b >
8b > 8c > 7c (Table 2). The results showed that compounds 7b
and 7a achieved similar ORAC values while compound 7c had a
lower effect. This behaviour might be caused by the methylation
of the hydroxyl substituents in B-ring. In fact, it was previously
shown that methylation reduces the antioxidant capacity of poly-
phenols, tested by the same method.34 All of the 2-SC were much
less active than the previously tested 2-SC from group 1 (30,40-
dihydroxy-2-styrylchromone derivatives).12 This can also be ex-
plained by the methylation theory, but only for compounds 7a
and 7c, not for compound 7b. Thus, it is possible that the reduced
8a
8b
8c
9
51.8 3.3
62.8 3.6
78.0 4.9
19.5 2.6
501.8 43.9
347.7 28.2
235.7 7.8
366.9 36.4
61.8 7.0
58.6 5.6
7.9 1.0
1.45 0.22
2.49 0.38
1.70 0.13
2.29 0.36
43.0 7.6
Positive controls
Tiron
Ascorbic acid
Lipoic acid
287.7 32.0
—
—
—
—
—
567.4 33.0
—
10.2 1.5
—
—
1.95 0.06
Each study corresponds to five experiments, performed in triplicate.
a
Scavenging effect (mean%) at the highest tested concentration (1 mM).
Scavenging effect (mean%) at the highest tested concentration (200 lM).
b
that the methylation of the hydroxyl groups, especially those in the
B-ring, seems to be disadvantageous to the O2Åꢀ scavenging activity
(compound 7b vs compounds 7a and 7c). However, the new 2-SC
Åꢀ
tested were shown to have improved O2 scavenging activity
when compared with those tested before, in spite of complete O-
methylation of hydroxyl groups in compound 7c.12 This corre-
sponds to a very interesting and new feature of 2-SC, implying a
greater importance of the styrylchromone backbone, under the
influence of electron-donor activation mediated by O-methoxy
groups in the B-ring. Also, compound 7b only differs from the pre-
viously tested compound 1A12 in the position of one hydroxyl in
the A-ring, that is, 5-OH in 1A became 8-OH in 7b. Thus, it seems
Åꢀ
appropriate to infer that this modification improves the O2 scav-
enging activity. In what concerns to flavones, the substitution of
the position 3 in the C-ring by an alkyl chain seems to be more
unfavourable to their scavenging activity as it becomes bigger.
From previous studies, it is known that these 3-alkylpolyhydroxyf-
lavones bearing an alkyl chain with increasing length allow the
control of oxidation processes at variable depths in organized
microstructures such as ionic or neutral micelles.28 However, the
structure–activity relationships drawn from antioxidant protection
provided by these 3-alkylflavones strongly depends on the model
studied,29 supporting our present results. On the other hand, the
C-3 3,4-dihydroxybenzoyl substituent, in compound 9, improves
Åꢀ
the O2 scavenging activity perhaps due to the catechol group in-
cluded. The IC50 of the new 2-SC was remarkably lower than the
positive control, tiron, (287.7 32.0) (Table 1).
All of the tested compounds were able to scavenge H2O2 in a
concentration-dependent manner. 2-SC were less effective than
flavones. The order of potencies found was 8c > 8b > 9 > 8a >
7a > 7b > 7c (Table 1). The structural modifications introduced in
the tested 2-SC are unfavourable for the H2O2 scavenging activity
since these compounds are much weaker scavengers than the pre-
viously tested compounds 1A–1D (30,40-dihydroxy-2-styrylchro-
mone derivatives).12 This suggests the importance of the catechol
group in the B-ring, but also a possible critical contribution of
the A-ring hydroxylation pattern. Results from the flavones 8a,
8b, 8c show that the H2O2 scavenging activity increases with the
growth of the alkyl chain. Moreover, all of the C-3 substituents
introduced in the tested flavones where shown to improve the ef-
fect of the parent compound, luteolin.12
Table 2
ROOÅ scavenging activity of the tested 2-styrylchromones, flavones, and ascorbic acid
expressed as ORAC values (mean SE)
Compound
ORACROOꢂ SE
M trolox equiv/
Concentration
range (lM)
(
l
lM compound)
2-Styrylchromones
7a
7b
7c
2.74 0.12
2.58 0.13
1.40 0.08
0.25–2
0.5–4
0.5–4
Flavones
8a
8b
8c
9
2.66 0.16
2.00 0.14
1.63 0.14
4.32 0.28
0.5–4
1–8
0.5–4
0.25–2
Positive control
Ascorbic acid
The tested flavones and compound 7a were shown to be strong
scavengers of HOCl. Compounds 7b and 7c were much weaker
scavengers although both were effective in a concentration-depen-
0.405 0.033
1–8
Each study corresponds to five experiments, performed in triplicate.

A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
7221
ꢀ
ORAC value is caused by the hydroxyl or methoxyl substitution in
position 8, which is, apparently, unfavourable to the scavenging
activity against ROOÅ. In what concerns to flavones, the analysis
of the results shows that the ORAC decreases along with the in-
crease of the length of the alkyl chain in C-3 (compounds 8a, 8b,
and 8c). Because the increase of the length of the alkyl chain im-
plies a decrease in the hydrophilicity, the obtained results are
likely due to solubility issues, since the reaction occurs in aqueous
solution. On the other hand, compound 9 was shown to be the
most active of the tested flavones, which indicates that the 3,4-di-
hydroxybenzoyl substituent in C-3 is more favourable to the scav-
enging activity against ROOÅ than the alkyl chain. Still, a hydroxyl
substituent (quercetin) or no substituent at all (luteolin) caused
stronger effects.12
in vivo are promoted by ONOOCO2 derivatives, which are more
efficient nitrating and oxidizing species than ONOOꢀ.41,42
All of the tested compounds where shown to efficiently inhibit
ÅNO-induced oxidation of DAF-2, in a concentration-dependent
manner. 2-SC showed to be better scavengers of ÅNO than flavones.
The order of potencies found was 7b > 7c > 7a > 8a > 8b > 9 > 8c
Å
(Table 3). The scavenging activity against NO in 2-SC was im-
proved by the introduction of a hydroxyl substituent in the posi-
tion 8 (compound 7b vs compounds 1A and 1B).12 On the other
hand, the methylation of the hydroxyl groups in rings A or B pro-
voked a slight decrease in the activity (compound 7b vs com-
pounds 7a and 7c). The new flavones were considerably less
active than luteolin,12 which means than none of the substituents
Å
in C-3 is favourable to the NO scavenging activity.
The ONOOꢀ-induced oxidation of DHR was efficiently prevented
by all of the tested compounds. In general, the compounds were
shown to be more effective in the absence of bicarbonate than in
its presence, with the exception of compound 7a. The order of
potencies found was 7c > 8a > 9 > 8c > 7b > 7a > 8b, without bicar-
bonate and 7a > 8a > 7b > 8b > 8c > 9 > 7c, with bicarbonate (Table
3). Compounds 7b, 7a, and 7c were all less active than the previ-
ously tested 30,40-dihydroxy-2-styrylchromone derivatives12 either
with or without the addition of bicarbonate, to mimic physiological
conditions of CO2. This means that the presence of a substituent in
position 8 and the methylation of the hydroxyl substituents are
detrimental to the scavenging effect against ONOOꢀ. The decrease
of this effect upon methylation of the hydroxyl groups has previ-
ously been shown by others for quercetin and phenol.35 The C-3
substituents in the tested flavones could not favour the ONOOꢀ
scavenging activity. In fact, all of these compounds were much less
potent than their unsubstituted counterpart, luteolin.12 It is possi-
ble that the C-3 substituents cause steric hindrance to the catechol
moiety, which is accepted to be of major importance to the scav-
enging activity of flavonoids and other phenolic compounds.36–38
All of the tested compounds, except compound 7a, were less effec-
tive scavengers of ONOOꢀ in the presence of bicarbonate. The
extremely fast reaction between ONOOꢀ and CO2 (K = 3–5.8 ꢃ
All the tested compounds were able to significantly reduce fer-
ric ion, except 8c. 2-SC showed to be better reducers than flav-
ones. The order of potencies found was 7b > 7c > 7a > 9 > 8b > 8a
(Fig. 2). The reducing capacity is an indicator of the hydrogen-
donating ability. From the results obtained one can infer that
the methylation of the hydroxyl groups in 2-SC increases the
reducing capacity (Fig. 2 7b vs 7a and 7c). Compound 9 was con-
siderably more active than the other flavones, probably because of
the presence of a catechol group in the C-3 substituent. Despite
phenolic compounds exert their antioxidant effects by redox
mechanisms, the compounds with high ferric reducing capacity
may exert pro-oxidant effects resulting from the increased pro-
duction of ferrous iron and its consequent participation in Fenton
reactions.43,44 On the other hand, several flavones are also en-
dowed with metal chelation effects, which is considered a mech-
anism of antioxidant activity. The spectral changes of the tested
2-SC upon addition of Fe(II) indicate the formation of iron com-
plexes. For compounds 7a and 7b the spectrums show bathochro-
mic shifts at 380 to about 420 nm and at 370 nm to about 390 nm,
respectively, which, according to the existent knowledge about
structural-similar flavonoids, are probably related to changes in
B-ring absorption.45 These shifts were associated with decreases
in absorbance (Fig. 3). As expected, compound 7c, gave rise to
spectral changes very different from those that occurred in com-
pounds 7a and 7b (Fig. 3). In this compound, the ‘iron binding mo-
tif’ of the B-ring, that is, the 30,40-dyhydroxy substituent, is
methylated, hindering the complexation with Fe(II). On the other
hand, the spectrum shows a small bathochromic shift (10 nm) at a
shorter wavelength (270 nm), which is likely to be related to
changes in the A-ring,45 associated with a small absorbance de-
crease. According to Guo et al.,46 the tested flavones have two
‘iron binding motifs’, the 30,40-dyhydroxy substituent and the
5,7-dihydroxy substituent. However, the substituents in the posi-
tion C-3 seem to affect the capacity of flavones to complex ferrous
iron since the expected bathochromic shifts were not observed in
the spectrum of these compounds (data not shown). This may be
due to steric hindrance to the iron binding groups caused by the
alkyl and 3,4-dihydroxybenzoyl substituents.
104 Mꢀ1 sꢀ1 39,40
originates the nitrosoperoxycarbonate anion
)
(ONOOCO2ꢀ), whose decomposition leads to the formation of
different species including the highly reactive nitrogen dioxide
(ÅNO2) and carbonate (CO3Åꢀ) radicals.41 The presence of these rad-
icals in solution affects the reactivity of the ONOOꢀ scavengers,
either increasing or decreasing their effects. Nevertheless, the re-
sults of the assays in which bicarbonate was added give us, theo-
retically, a better approach to the developments in a biological
system. In fact it is likely that many of the reactions of ONOOꢀ
Table 3
ÅNO and ONOOꢀ (with and without 25 mM NaHCO3) scavenging effects (IC50
mean SE) of the tested 2-styrylchromones, flavones and positive controls
,
Compound
IC50 (lM)
ÅNO
ONOOꢀ without
NaHCO3
ONOOꢀ with
NaHCO3
From this study it became clear that the methylation of hydro-
xyl groups decreases the scavenging of ROS and RNS by 2-SC,
which was somehow expected because it is widely known that hy-
droxyl groups are important for this type of effect. The decrease in
the scavenging activities was, generally, more evident when the
2-Styrylchromones
7a
7b
7c
0.368 0.039
0.193 0.025
0.254 0.012
1.21 0.14
0.85 0.13
0.59 0.08
0.93 0.21
1.59 0.05
2.08 0.16
methylation occurred in B-ring, except for O2 and 1O2. On the
Åꢀ
Flavones
other hand, the introduction of a substituent, either hydroxyl or
methoxyl, in position 8 was sometimes favourable and others
unfavourable to the scavenging activities, depending on the reac-
tive species. The effect of the C-3 substituents in flavones was also
variable according to the reactive species. In conclusion, the study
of the antioxidant properties of the new 2-SC and flavones allowed
establishing new structure–activity relationships and brought out,
in some cases, pharmacophores with improved activity.
8a
8b
8c
9
2.72 0.46
3.29 0.39
4.85 0.87
3.62 0.70
0.60 0.13
1.48 0.36
0.83 0.09
0.66 0.09
1.24 0.19
1.80 0.29
1.82 0.22
1.97 0.21
Positive controls
Rutin
Ebselen
1.17 0.05
—
—
—
1.00 0.11
5.27 0.32
Each study corresponds to five experiments, performed in triplicate.

7222
A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
Figure 2. Reducing activity of the tested compounds and ascorbic acid. Each value represents mean SE of triplicate measurements. ***P <0.001, **P <0.01, *P <0.05,
significantly different from control.
Bruker Avance 300 spectrometer (300.13 for 1H and 70.47 MHz
0.3
for 13C), with CDCl3 as solvent, unless otherwise stated. Chemical
shifts (d) are reported in ppm, and coupling constants (J) in hertz.
The internal standard was TMS. Unequivocal 13C assignments were
made with the aid of 2D gHSQC and gHMBC (delays for one bond
and long-range J C/H couplings were optimised for 145 and 7 Hz,
respectively) experiments. Electron impact (EI, 70 eV) MS were
recorded on VG Autospec Q spectrometer. Electrospray (ESI,
3000 V, 30 V, source and desolvatation temperatures of 80 °C and
150 °C, respectively) MS were recorded on Micromass Q-Tof-2TM
spectrometer. Microanalysis were recorded on CHNS CARLO ERBA
1108 analyser (University of Vigo). Elemental analyses were
obtained with a CHNS 932 LECO analyser (University of Aveiro).
Preparative thin-layer chromatography was performed with Merck
7a
7b
7c
0.2
0.1
0
300
400
500
600
λ(nm)
0.3
0.2
0.1
0
Silica Gel 60 DGF254
.
A microplate reader (Synergy HT, BIO-TEK), for fluorescence,
absorbance in UV–vis and luminescence measurements, plus tem-
perature control capacity, was used for all the assays.
300
400
500
600
λ(nm)
3.2. Chemicals
0.2
0.1
0
All the chemicals and reagents were of analytical grade. Dihy-
drorhodamine 123 (DHR), 4,5-diaminofluorescein (DAF-2), 30%
hydrogen peroxide, ascorbic acid, sodium hypochlorite solution,
with 4% available chlorine, lipoic acid, diethylenetriaminepenta-
acetic acid (DTPA), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-
oxo-1-triazene (NOC-5), b-nicotinamide adenine dinucleotide
(NADH), phenazine methosulfate (PMS), nitroblue tetrazolium
chloride (NBT), rutin, lucigenin, tiron, and ebselen, were obtained
250
350
λ(nm)
450
Figure 3. UV–vis absorption spectrum of the tested compounds, at the concentra-
tion of 10 M, in the presence of 0, 2, 4, 6, 8, 10, 12, 14, and 16 M of Fe(II), in
20 mM phosphate buffer, pH 7.2. The direction of the arrows indicates crescent
amounts of Fe(II).
l
l
from Sigma–Aldrich (St. Louis, USA).
a,
a0-Azodiisobutyramidine
dihydrochloride (AAPH), histidine and trolox were obtained from
Fluka Chemie GmbH (Steinheim, Germany). Fluorescein sodium
salt was obtained from Aldrich (Milwaukee, USA).
3. Experimental
3.1. General methods
Column chromatography was performed with Merck Silica Gel
60, 70–230 mesh. All other chemicals and solvents used were ob-
tained from commercial sources and used as received or dried
using standard procedures.
Melting points were measured in a Büchi Melting Point B-540
apparatus and are uncorrected. NMR spectra were recorded on a

A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
7223
3.3. Synthesis of (E)-20-cinnamoyloxyacetophenones 3a–c
3.3.3. (E)-30,40-Dibenzyloxy-20-(3,4-dimethoxycinnamoyloxy)-
acetophenone (3c)
Method 1: The appropriate cinnamic acid 2a,b (9.46 mmol) and
phosphorous oxychloride (4.0 mL, 43 mmol) were added to a solu-
tion of the appropriate acetophenone 1a,b (8.6 mmol) in dry pyri-
dine (170 mL). The solution was stirred under different conditions
of time and temperature according to the substitution of the com-
pound: 3a, 2 h at room temperature; 3b, 12 h at room temperature
and 3c, initially 4 h at room temperature and then 2 h at 60 °C. After
that, thesolutionwaspouredintoice andwater(200 mL), andthepH
adjusted to 4 with hydrochloric acid. The obtained solid was filtered
and taken in chloroform (50 mL). The organic layer was washed with
water (2 ꢃ 60 mL), and dried through Na2SO4. The solvent was evap-
orated and the residue was purified by silica gel column chromatog-
raphy using a 3:7 mixture of light petroleum/dichloromethane as
eluent. The solvent was evaporated to dryness in each case, and
the residue was crystallized from ethanol to give the expected (E)-
20-cinnamoyloxyacetophenones 3a–c in moderate to good yields:
3a (86%, 3.98 g), 3b (40%, 1.33 g) and 3c (50%, 2.3 g).
Method 2: 3,4-Dimethoxycinnamic acid 2a (686.5 mg, 3.3 mmol),
4-pyrrolidinopyridine (49 mg, 0.33 mmol) and N,N0-dicyclohexyl-
carbodiimide (680 mg, 3.3 mmol) were added to a solution of the
appropriate acetophenone 1a,b (3.3 mmol) in dichloromethane
(20 mL). The solution was stirred, at room temperature, for 18 h.
After that, the solid was filteredand washed abundantly with dichlo-
romethane (2 ꢃ 20 mL). The filtrate was evaporated and the residue
was purified by silica gel column chromatography using a (3:7) mix-
ture of light petroleum/dichloromethane as eluent. The solvent was
evaporated to dryness in each case, and the residue was crystallized
from ethanol to give the expected (E)-20-cinnamoyloxyacetophe-
nones 3b,c in good yields: 3b (88%, 1.12 g), 3c (67%, 1.19 g).
Mp 92–93 °C. 1H NMR: d 7.77 (d, J 15.9 Hz, 1H, H-b), 7.65 (d, J
8.9 Hz, 1H, H-60), 7.31–7.46 (m, 7H, 300,400-OCH2C6H5), 7.20–7.23
(m, 3H, 300,400-OCH2C6H5), 7.15 (dd, J 8.3 and 1.9 Hz, 1H, H-600),
7.09 (d, J 1.9 Hz, 1H, H-200), 6.96 (d, J 8.9 Hz, 1H, H-50), 6.90 (d, J
8.3 Hz, 1H, H-500), 6.50 (d, J 15.9 Hz, 1H, H- ), 5.21 (s, 2H, 40-
a
OCH2C6H5), 5.06 (s, 2H, 30-OCH2C6H5), 3.945 (s, 3H, 400-OCH3),
3.943 (s, 3H, 300-OCH3), 2.51 (s, 3H, H-2), 13C NMR: d 196.0 (C-1),
165.0 (C@O ester), 156.3 (C-40), 151.5 (C-400), 149.2 (C-300), 147.2
(C-b), 144.7 (C-20), 140.8 (C-30), 137.0 (C-1 of 30-OCH2C6H5), 135.9
(C-1 of 40-OCH2C6H5), 128.7 (C-3,5 of 30-OCH2C6H5), 128.4 (C-3,5
of 40-OCH2C6H5), 128.3 (C-4 of 30-OCH2C6H5), 128.2 (C-2,6 of 30-
OCH2C6H5), 127.9 (C-4 of 40-OCH2C6H5), 127.4 (C-2,6 of 40-
OCH2C6H5), 127.1 (C-100), 125.9 (C-60), 125.1 (C-10), 123.2 (C-600),
114.2 (C-
a
), 110.9 (C-500), 110.5 (C-50), 109.7 (C-200), 75.1 (30-
OCH2C6H5), 71.0 (40-OCH2C6H5), 56.0 (400-OCH3), 55.9 (300-OCH3),
29.9 (C-2); MS (ESI) m/z (rel. int.): 539 ([M+H]+, 5), 561 ([M+Na]+,
100), 577 ([M+K]+, 20). Anal. Calcd for C33H30O7 (538.59): C,
73.59; H, 5.61. Found: C, 73.35; H, 5.61.
3.4. Synthesis of 5-aryl-3-hydroxy-1-(2-hydroxyaryl)penta-2,4-
dien-1-ones (4a–c)
Potassium hydroxide (powder, 1.4 g, 25 mmol) was added to a
solution of the appropriate 20-cinnamoyloxyacetophenone 3a–c
(5 mmol) in dimethyl sulfoxide (30 mL). The solution was stirred
under nitrogen at room temperature for 2 h. After that period,
the solution was poured into ice and water (150 mL), and the pH
adjusted to 4 with diluted hydrochloric acid. The obtained solid
was removed by filtration, taken in chloroform (80 mL) and
washed with water (2 ꢃ 80 mL). The organic layer was dried over
Na2SO4 and the solvent was evaporated to dryness in each case.
The residue was crystallized from ethanol to give the expected 5-
aryl-3-hydroxy-1-(2-hydroxyaryl)penta-2,4-dien-1-ones 4a–c: 4a
(93%, 2.5 g), 4b (62%, 1.2 g) and 4c (96%, 2.6 g).
3.3.1. (E)-20-(3,4-Dibenzyloxycinnamoyloxy)-30,40-dimethoxy-
acetophenone (3a)
Mp 111–112 °C; 1H NMR: d 7.81 (d, J 15.9 Hz, 1H, H-b), 7.66 (d, J
8.9 Hz, 1H, H-60), 7.31–7.48 (m, 10H, 300,400-OCH2C6H5), 7.21 (d, J
2.0 Hz, 1H, H-200), 7.15 (dd, J 8.4 and 2.0 Hz, 1H, H-600), 6.95
(d, J 8.4 Hz, 1H, H-500), 6.87 (d, J 8.9 Hz, 1H, H-50), 6.53 (d, J
3.4.1. 5-(3,4-Dibenzyloxyphenyl)-3-hydroxy-1-(2-hydroxy-3,4-
dimethoxyphenyl)penta-2,4-dien-1-one (4a)
15.9 Hz, 1H, H-a
), 5.23 (s, 2H, 400-OCH2C6H5), 5.21 (s, 2H,
Mp 179–180 °C; 1H NMR: d 14.56 (s, 1H, 3-OH), 12.55 (s, 1H, 20-
OH), 7.52 (d, J 15.9 Hz, 1H, H-5), 7.46 (d, J 9.2 Hz, 1H, H-60), 7.32–
7.49 (m, 10H, 300,400-OCH2C6H5), 7.15 (d, J 1.9 Hz, 1H, H-200), 7.11
(dd, J 8.3 and 1.9 Hz, 1H, H-600), 6.94 (d, J 8.3 Hz, 1H, H-500), 6.50
(d, J 9.2 Hz, 1H, H-50), 6.38 (d, J 15.9 Hz, 1H, H-4), 6.18 (s, 1H, H-
2), 5.21 (s, 2H, 400-OCH2C6H5), 5.20 (s, 2H, 300-OCH2C6H5), 3.94 (s,
3H, 40-OCH3), 3.91 (s, 3H, 30-OCH3); 13C NMR: d 194.7 (C-1),
173.8 (C-3), 158.1 (C-40), 157.2 (C-20), 150.9 (C-400), 149.0 (C-300),
139.2 (C-5), 136.9 (C-30), 136.8 and 136.7 (C-1 of 300,400-OCH2C6H5),
128.6 (C-3,5 of 300,400-OCH2C6H5), 128.0 (C-4 of 300,400-OCH2C6H5),
127.3 and 127.1 (C-2,6 of 300,400-OCH2C6H5), 127.3 (C-100), 124.6
(C-60), 122.7 (C-600), 120.2 (C-4), 114.4 (C-500), 114.3 (C-10), 113.7
(C-200), 103.0 (C-50), 96.4 (C-2), 71.4 (300-OCH2C6H5), 70.9 (400-
OCH2C6H5), 60.7 (30-OCH3), 56.1 (40-OCH3); MS (FAB+), m/z (rel.
int.): 539 [M+H]+. Anal. Calcd for C33H30O7 (538.59): C, 73.59; H,
5.61. Found: C, 73.35; H, 5.63.
300-OCH2C6H5), 3.94 (s, 3H, 40-OCH3), 3.83 (s, 3H, 30-OCH3), 2.51
(s, 3H, H-2); 13C NMR: d 196.0 (C-1), 165.0 (C@O ester), 157.1 (C-
40), 151.4 (C-400), 148.9 (C-300), 147.1 (C-b), 144.2 (C-20), 141.4 (C-
30), 136.7 and 136.5 (C-1 of 300,400-OCH2C6H5), 128.5 (C-3,5 of
300,400-OCH2C6H5), 127.9 (C-4 of 300,400-OCH2C6H5), 127.4 (C-100),
127.2 and 127.1 (C-2,6 of 300,400-OCH2C6H5), 125.8 (C-60), 124.7
(C-10), 123.5 (C-600), 114.3 (C- ), 114.0 (C-500), 113.7 (C-20), 109.0
a
(C-50), 71.2 (C-300), 70.8 (C-400), 60.8 (C-30), 56.0 (C-40), 29.8 (C-2);
MS (EI) m/z (rel. int.): 538 (M+Å, 4), 360 (3), 343 (25), 253 (3), 196
(4), 181 (10), 91 (100), 65 (7); Anal. Calcd for C33H30O7 (538.59):
C, 73.59; H, 5.61. Found: C, 73.76; H, 5.53.
3.3.2. (E)-30,40-Dimethoxy-20-(3,4-dimethoxycinnamoyloxy)-
acetophenone (3b)
Mp 124–125 C; 1H NMR: d 7.88 (d, J 15.9 Hz, 1H, H-b), 7.67 (d, J
8.9 Hz, 1H, H-60), 7.20 (dd, J 8.2 and 2.0 Hz, 1H, H-600), 7.15 (d, J
2.0 Hz, 1H, H-200), 6.91 (d, J 8.2 Hz, 1H, H-500), 6.89 (d, J 8.9 Hz,
3.4.2. 3-Hydroxy-1-(2-hydroxy-3,4-dimethoxyphenyl)-5-(3,4-
dimethoxyphenyl)penta-2,4-dien-1-one (4b)
1H, H-50), 6.62 (d, J 15.9 Hz, 1H, H-
a), 3.96 and 3.95 (2 s, 3H +
6H, 40,300,400-OCH3), 3.86 (s, 3H, 30-OCH3), 2.54 (s, 3H, H-2); 13C
NMR: d 196.0 (C-1), 165.1 (C@O ester), 157.1 (C-40), 151.5 (C-400),
149.2 (C-300), 147.3 (C-b), 144.3 (C-20), 141.5 (C-30), 127.0 (C-100),
Mp 159–160 °C; 1H NMR: d 14.58 (d, J 0.7 Hz, 1H, 3-OH), 12.58
(s, 1H, 20-OH), 7.59 (d, J 15.9 Hz, 1H, H-5), 7.48 (d, J 9.2 Hz, 1H, H-
60), 7.15 (d, J 8.3 and 1.9 Hz, 1H, H-600), 7.08 (d, J 1.9 Hz, 1H, H-200),
6.90 (d, J 8.3 Hz, 1H, H-500), 6.51 (d, J 9.2 Hz, 1H, H-50), 6.46 (d, J
15.9 Hz, 1H, H-4), 6.21 (s, 1H, H-2), 3.954 (s, 3H, 300-OCH3), 3.946
(s, 3H, 40-OCH3), 3.94 (s, 3H, 400-OCH3), 3.92 (s, 3H, 30-OCH3); 13C
NMR: d 194.7 (C-1), 173.9 (C-3), 158.0 (C-40), 157.2 (C-20), 150.9
(C-400), 149.2 (C-300), 139.4 (C-5), 136.8 (C-30), 128.1 (C-100), 124.6
125.9 (C-60), 124.8 (C-10), 123.3 (C-600), 114.2 (C- ), 110.9 (C-500),
a
109.7 (C-200), 109.0 (C-50), 60.9 (30-OCH3), 56.1, 56.0 and 55.9
(300,40,400-OCH3), 29.8 (C-2); MS (ESI+) m/z (rel. int.): 409
([M+Na]+, 100), 425 ([M+K]+, 20), 795 ([2 M+Na]+, 72). Anal. Calcd
for C21H22O7 (386.40): C, 65.28; H, 5.74. Found: C, 65.10; H, 5.68.

7224
A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
(C-60), 122.4 (C-600), 120.0 (C-4), 114.4 (C-10), 111.1 (C-5), 109.5 (C-
200), 103.0 (C-50), 96.4 (C-2), 60.7 (30-OCH3), 56.1, 56.0 and 55.9
(40,300,400-OCH3); MS (ESI) m/z (rel. int.): 387 ([M+H]+, 100), 409
([M+Na]+, 35), 425 ([M+K]+, 5), 795 ([2 M+Na]+, 40). C21H22O7
(386.40): C, 65.28; H, 5.74. Found: C, 65.45; H, 5.71.
8-OCH3), 4.01 (s, 3H, 7-OCH3), 3.98 (s, 3H, 30-OCH3), 3.94 (s, 3H,
40-OCH3); 13C NMR: d 178.0 (C-4), 161.8 (C-2), 156.6 (C-7), 150.7
(C-40), 150.3 (C-9), 149.3 (C-30), 136.7 (C-b), 136.6 (C-8), 128.1
(C-10), 122.2 (C-60), 121.0 (C-5), 118.8 (C-10), 118.2 (C-
a), 111.1
(C-50), 109.5 (C-6), 109.4 (C-3), 109.2 (C-20), 61.6 (8-OCH3), 56.4
(7-OCH3), 56.0 (30,40-OCH3); MS (ESI+) m/z (rel. int.): 396
([M+H]+, 100), 391 ([M+Na]+, 10), 759 ([2 M+Na]+, 8). Anal. Calcd
for C21H20O6 (368.38): C, 68.47; H, 5.47. Found: C, 68.74; H, 5.52.
3.4.3. 1-(3,4-Dibenzyloxy-2-hydroxyphenyl)-3-hydroxy-5-(3,4-
dimethoxyphenyl)penta-2,4-dien-1-one (4c)
Mp 147–148 °C; 1H NMR: d 14.60 (s, 1H, 3-OH), 12.59 (s, 1H, 20-
OH), 7.57 (d, J 15.7 Hz, 1H, H-5), 7.25–7.51 (m, 11H, H-60 and 30,40-
OCH2C6H5), 7.13 (d, J 8.3 Hz, 1H, H-600), 7.06 (s, 1H, H-200), 6.88 (d, J
8.3 Hz, 1H, H-500), 6.49 (d, J 9.1 Hz, 1H, H-50), 6.43 (d, J 15.7 Hz, 1H,
H-4), 6.17 (s, 1H, H-2), 5.15 (s, 2H, 40-OCH2C6H5), 5.11 (s, 2H, 30-
OCH2C6H5), 3.94 and 3.92 (s, 6H, 300,400-OCH3); 13C NMR: d 194.6
(C-1), 173.9 (C-3), 157.6 and 157.4 (C-20,40), 150.9 (C-400), 149.2
(C-300), 139.3 (C-5), 137.6 (C-1 of 30-OCH2C6H5), 136.3 (C-1 of 40-
OCH2C6H5), 136.1 (C-30), 128.6 (C-3,5 of 30,40-OCH2C6H5), 128.2
(C-2,6 of 30-OCH2C6H5), 128.0 (C-4 of 30,40-OCH2C6H5), 127.8 (C-
100), 127.2 (C-2,6 of 40-OCH2C6H5), 124.5 (C-60), 122.4 (C-600),
120.0 (C-4), 114.4 (C-10), 111.1 (C-500), 109.5 (C-200), 104.6 (C-50),
96.4 (C-2), 74.7 (30-OCH2C6H5), 70.7 (40-OCH2C6H5), 55.95 and
55.86 (300,400-OCH3); MS (ESI) m/z (rel. int.): 539 ([M+H]+, 100),
561 ([M+Na]+, 35), 577 ([M+K]+, 10). HRMS (ESI), m/z: C33H31O7:
539.2070 [M+H]+. Found: 539.2076.
3.5.3. (E)-7,8-Dibenzyloxy-30,40-dimethoxy-2-styrylchromone
(6c)
Mp 176–177 °C; 1H NMR: d 7.90 (d, J 9.0 Hz, 1H, H-5), 7.32–7.54
(m, 10H, 7,8-OCH2C6H5), 7.44 (d, J 15.9 Hz, 1H, H-b), 7.07 (d, J
9.0 Hz, 1H, H-6), 7.03 (dd, J 8.3 and 1.9 Hz, 1H, H-60), 6.98 (d, J
1.9 Hz, 1H, H-20), 6.88 (d, J 8.3 Hz, 1H, H-50), 6.60 (d, J 15.9 Hz,
1H, H-a), 6.20 (s, 1H, H-3), 5.28 (s, 2H, 7-OCH2C6H5), 5.22 (s, 2H,
8-OCH2C6H5), 3.94 (s, 3H, 30-OCH3), 3.93 (s, 3H, 40-OCH3); 13C
NMR: d 178.0 (C-4), 161.7 (C-2), 155.9 (C-7), 150.7 (C-40), 150.5
(C-9), 149.2 (C-30), 137.3 (C-8), 136.9 (C-b), 136.12 and 136.06
(C-10 of 7,8-OCH2C6H5), 128.7 and 128.4 (C-30,50 of 7,8-OCH2C6H5),
128.4 (C-20,60 of 8-OCH2C6H5), 128.3 and 128.2 (C-40 of 7,8-
OCH2C6H5), 128.0 (C-10), 127.4 (C-20,60 of 7-OCH2C6H5), 122.0
(C-60), 121.0 (C-5), 119.1 (C-10), 117.9 (C-
a), 111.2 (C-6), 111.1
(C-5’), 109.5 (C-3), 109.3 (C-20), 75.8 (8-OCH2C6H5), 71.2 (7-
OCH2C6H5), 56.0 and 55.9 (30,40-OCH3); MS (ESI) m/z (rel. int.):
521 ([M+H]+, 100), 543 ([M+Na]+, 10). Anal. Calcd for C33H28O6
(520.57): C, 76.14; H, 5.42. Found: C, 76.08; H, 5.40.
3.5. Synthesis of alkoxy-2-styrylchromones (6a–c)
p-Toluenesulfonic acid monohydrate (0.48 g, 2.5 mmol) was
added to a solution of the appropriate 5-aryl-3-hydroxy-1-(2-
hydroxyaryl)penta-2,4-dien-1-one 4a–c (5.0 mmol) in dimethyl
sulfoxide (30 mL). The solution was heated under nitrogen at
90 °C, until the complete consumption of the starting material
was observed (2–8 h). After that period, the solution was poured
into ice and water (150 mL), and the obtained solid was removed
by filtration, taken in dichloromethane (100 mL) and the organic
layer washed with water (2 ꢃ 100 mL). After evaporation of the
solvent, the residue was purified by silica gel column chromatogra-
phy using dichloromethane as eluent. The solvent was evaporated
to dryness in each case, and the residue was crystallized from eth-
anol to give the alkoxy-2-styrylchromones 6a–c: 6a (84%, 2.2 g),
6b (62%, 1.1 g) and 6c (77%, 2.0 g).
3.6. Synthesis of dihydroxy-2-styrylchromones (7a,c)
The appropriate benzyloxy-2-styrylchromone 6a, 6c (0.54 mmol)
was added to a solution of chloridric acid (37%, 2 mL) in glacial acetic
acid (20 mL). The mixture was refluxed at 110 °C for 2:30 h for 7a and
12 h for 7c. The solution was carefully poured into ice and water
(30 mL), and theobtainedsolidwasremovedby filtration, abundantly
washedwithwater(4 ꢃ 50 mL)andthendriedaffordingthe expected
hydroxy-2-styrylchromones in good yields: 7a (82%, 0.15 g) and 7c
(82%, 0.15 g).
3.6.1. (E)-30,40-Dihydroxy-7,8-dimethoxy-2-styrylchromone (7a)
Mpdec. 233–234 °C; 1H NMR (DMSO-d6): d 9.64 (br s, 1H, 40-OH),
9.20 (br s, 1H, 30-OH), 7.74 (d, J 8.9 Hz, 1H, H-5), 7.44 (d, J 16.0 Hz,
1H, H-b), 7.25 (d, J 8.9 Hz, 1H, H-6), 7.13 (d, J 1.6 Hz, 1H, H-20), 7.04
3.5.1. (E)-30,40-Dibenzyloxy-7,8-dimethoxy-2-styrylchromone
(6a)
(dd, J 8.2 and 1.6 Hz, 1H, H-60), 6.90 (d, J 16.0 Hz, 1H, H-
a), 6.81 (d, J
Mp 165–168 °C; 1H NMR: d 7.92 (d, J 9.0 Hz, 1H, H-5), 7.50–7.32
(m, 10H, 30,40-CH2C6H5), 7.52 (d, J 16.0 Hz, 1H, H-b), 7.19 (d, J
1.9 Hz, 1H, H-20), 7.13 (dd, J 8.3 and 1.9 Hz, 1H, H-60), 7.02 (d, J
9.0 Hz, 1H, H-6), 6.95 (d, J 8.4 Hz, 1H, H-50), 6.58 (d, J 16.0 Hz,
8.2 Hz, 1H, H-50), 6.35 (s, 1H, H-3), 3.96 (s, 6H, 7.8-OCH3); 13C NMR
(DMSO-d6): d 176.5 (C-4), 161.9 (C-2), 156.4 (C-7), 149.7 (C-9),
148.0 (C-40), 145.8 (C-30), 136.7 (C-b), 136.2 (C-8), 126.5 (C-10),
121.0 (C-60), 120.1 (C-5), 118.2 (C-10), 117.0 (C- ), 115.9 (C-50),
a
1H, H-
a
), 6.23 (s, 1H, H-3), 5.23 (s, 2H, 30-OCH2C6H5), 5.22 (s, 2H,
114.3 (C-20), 110.3 (C-6), 108.5 (C-3), 61.2 (8-OCH3), 56.5 (7-
OCH3); MS (EI) m/z (rel. int.): 340 (M+Å, 100), 323 (9), 310 (19),
255 (21), 228 (34), 214 (15), 181 (37), 167 (8), 152 (17), 126
(26), 91 (10), 69 (18). Anal. Calcd for C19H16O6 (340.33): C, 67.05;
H, 4.74. Found: C, 67.46; H, 4.76.
40-OCH2C6H5), 4.04 (s, 3H, 8-OCH3), 4.00 (s, 3H, 7-OCH3); 13C
NMR: d 178.0 (C-4), 161.8 (C-2), 156.6 (C-7), 150.7 (C-40), 150.3
(C-9), 149.1 (C-30), 136.9 and 136.6 (C-10 of 30,40-OCH2C6H5),
136.7 (C-8), 136.59 (C-b), 128.6 (C-30,50 of 30,40-OCH2C6H5), 128.4
(C-10); 127.99 and 127.96 (C-40 of 30,40-OCH2C6H5), 127.4 and
127.2 (C-20,60 of 30,40-OCH2C6H5), 122.5 (C-60), 121.0 (C-5), 118.8
3.6.2. (E)-7,8-Dihydroxy-30,40-dimethoxy-2-styrylchromone (7c)
Mpdec. 287–291 °C; 1H NMR (DMSO-d6): d 10.18 (br s, 1H, 7-OH),
9.52 (br s, 1H, 8-OH), 7.78 (d, J 15.8 Hz, 1H, H-b), 7.35 (d, J 8.7 Hz, 1H,
H-5), 7.32 (d, J 1.8 Hz, 1H, H-20), 7.22 (dd, J 8.2 and 1.8 Hz, 1H, H-60),
(C-10), 118.4 (C-a
), 114.4 (C-50), 113.4 (C-20), 109.49 and 109.44
(C-3 and C-6), 71.5 (30-OCH2C6H5), 71.0 (40-OCH2C6H5), 61.6 (8-
OCH3), 56.4 (7-OCH3); MS (ESI) m/z (rel. int.): 521 ([M+H]+, 100),
543 ([M+Na]+, 15), 559 ([M+K]+, 5). Anal. Calcd for C33H28O6
(520.57): C, 76.14; H, 5.42. Found: C, 75.83; H, 5.13.
7.07(d, J15.8 Hz, 1H, H-a
), 7.03(d, J8.2 Hz, 1H, H-50), 6.91(d, J8.7 Hz,
1H, H-6), 6.23 (s, 1H, H-3), 3.84 (s, 3H, 40-OCH3), 3.81(s, 3H, 30-OCH3);
13C NMR (DMSO-d6): d 176.8 (C-4), 161.5 (C-2), 150.41 (C-40), 150.38
(C-7), 149.1 (C-30), 146.2 (C-9), 136.4 (C-b), 132.9 (C-8), 128.1 (C-10),
3.5.2. (E)-30,40,7,8-Tetramethoxy-2-styrylchromone (6b)
Mp 171–172 °C; 1H NMR: d 7.93 (d, J 9.0 Hz, 1H, H-5), 7.59 (d, J
15.9 Hz, 1H, H-b), 7.18 (dd, J 8.3 and 2.0 Hz, 1H, H-60), 7.12 (d, J
2.0 Hz, 1H, H-20), 7.03 (d, J 9.0 Hz, 1H, H-6), 6.91 (d, J 8.3 Hz, 1H,
122.1 (C-60), 118.5 (C-
a), 117.1 (C-10), 115.0 (C-5), 113.7 (C-6), 111.7
(C-50), 109.7 (C-20), 108.7 (C-3), 55.6 (30,40-OCH3); MS (ESI+) m/z (rel.
int.): 341 ([M+H]+, 17), HRMS (ESI+), m/z: C19H17O6: calcd 341.1025
[M+H]+. Found: 341.1023.
H-50), 6.68 (d, J 15.9 Hz, 1H, H-
a), 6.26 (s, 1H, H-3), 4.06 (s, 3H,

A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
7225
3.7. Synthesis of 30,40,7,8-tetrahydroxy-2-styrylchromone (7b)
tion of DHR. Each study corresponds to five experiments, per-
formed in triplicate.
A solution of 1 M boron tribromide in dichloromethane (0.54 mL,
0.54 mmol) was slowly added to a cool solution(2-propanol cryostat
bath at ꢀ78 C) of 30,40,7,8-tetramethoxy-2-styrylchromone 6b (49.3
mg, 0.134 mmol) in freshly distilled dichloromethane (5 mL). The
solution was stirred under nitrogen at room temperature for 24 h.
After that period, ice and water (20 mL) was slowly added to the
solution and then vigorously stirred. The obtained solid was
removed by filtration and abundantly washed with water (4 ꢃ
50 mL) and then dried affording the expected 30,40,7,8-tetrahydr-
oxy-2-styrylchromone 7b (76%, 32 mg).
3.10.4. Singlet oxygen scavenging assay
The 1O2 scavenging activity was measured by monitoring the
oxidation of non-fluorescent DHR to fluorescent rhodamine by this
ROS, as previously described.12 Ascorbic acid was used as positive
control. The results were expressed as the inhibition (in percent-
age) of 1O2-induced oxidation of DHR. Each study corresponds to
five experiments, performed in triplicate.
3.10.5. Peroxyl radical scavenging assay
The ROOÅ scavenging activity was measured by monitoring
the fluorescence decay resulting from ROOÅ-induced oxidation
of fluorescein and expressed as the ‘Oxygen Radical Absorbance
Capacity’ (ORAC), as previously described.12 Trolox was used as
the standard control in each assay. Ascorbic acid was used as
positive control. The results were expressed as ORAC values.
Each study corresponds to four experiments, performed in
triplicate.
3.7.1. (E)-30,40,7,8-Tetrahydroxy-2-styrylchromone (7b)
Mpdec. 254–255 °C; 1H NMR (DMSO-d6): d 10.20 (s, 1H, 7-OH),
9.57 (s, 1H, 40-OH), 9.48 (s, 1H, 8-OH), 9.20 (s, 1H, 30-OH), 7.71
(d, J 16.3 Hz, 1H, H-b), 7.35 (d, J 8.7 Hz, 1H, H-5), 7.09 (d, J
1.8 Hz, 1H, H-20), 7.01 (dd, J 7.8 and 1.8 Hz, 1H, H-60), 6.91 (d, J
8.7 Hz, 1H, H-6), 6.83 (d, J 16.3 Hz, 1H, H-a), 6.81 (d, J 7.8 Hz, 1H,
H-50), 6.25 (s, 1H, H-3); 13C NMR (DMSO-d6): d 176.8 (C-4), 161.6
(C-2), 150.3 (C-7), 147.7 (C-40), 146.2 (C-9), 145.7 (C-30), 136.9
(C-b), 132.8 (C-8), 126.9 (C-10), 120.6 (C-60), 117.0 (C-
a and C-
3.10.6. Peroxynitrite scavenging assay
10), 115.9 (C-50), 115.0 (C-5), 114.2 (C-20), 113.7 (C-6), 108.4 (C-
3); MS (EI) m/z (rel. int.): 312 (M+Å, 66), 311 (8), 295 (2), 160
(22), 153 (50), 129 (8), 115 (15), 105 (27), 101 (34), 91 (47), 83
(90), 77 (44), 69 (30), 59 (100); HRMS (ESI), m/z: C17H13O6: calcd
313.0712 [M+H]+. Found: 313.0722.
The ONOOꢀ scavenging activity was measured by monitoring
the ONOOꢀ-induced oxidation of non-fluorescent DHR to fluores-
cent rhodamine, as previously described.12 Ebselen was used as po-
sitive control. In a parallel set of experiments, the assays were
performed in the presence of 25 mM NaHCO3 in order to simulate
the physiological CO2 concentrations. This evaluation is important
because, under physiological conditions, the reaction between
ONOOꢀ and bicarbonate is predominant, with a very fast rate con-
stant (k2 = 3–5.8 ꢃ 104 Mꢀ1 sꢀ1).47 The results were expressed as
the inhibition (in percentage) of ONOOꢀ-induced oxidation of
DHR. Each study corresponds to five experiments, performed in
triplicate.
3.8. Synthesis of 3-alkyl-30,40,5,7-tetrahydroxyflavones (8a–c)
3-Alkyl-30,40,5,7-tetrahydroxyflavones 8a–c were synthesised
according to procedures previously described in the literature.23
3.9. Synthesis of 3-(3,4-dihydroxybenzoyl)-30,40,5,7-
tetrahydroxyflavone (9)
3-(3,4-Dihydroxybenzoyl)-30,40,5,7-tetrahydroxyflavone 9 was
synthesised according to procedures previously described in the
literature.24
3.10.7. Nitric oxide scavenging assay
Å
The NO scavenging activity was measured by monitoring the
ÅNO-induced oxidation of non-fluorescent DAF-2 to the fluores-
cent triazolofluorescein (DAF-2T), as previously described.12 Ru-
tin was used as positive control. The results were expressed as
3.10. ROS and RNS scavenging assays
Å
the inhibition (in percentage) of NO-induced oxidation of DAF-
2. Each study corresponds to five experiments, performed in
triplicate.
3.10.1. Superoxide radical scavenging assay
Superoxide radical was generated by the NADH/PMS system
Åꢀ
and the O2 scavenging activity was determined by monitoring
the effect of the tested compound on the O2Åꢀ-induced reduction
of NBT at 560 nm as previously described.12 The antioxidant tiron
was used as positive control. The results were expressed as the
inhibition (in percentage) of the NBT reduction to diformazan. Each
study corresponds to five experiments, performed in triplicate.
3.10.8. Reducing power assay
The reducing power was determined according to a previously
described method.48 Test compounds, in 1 mL of a ultrapure
water/DMSO 1:4 solution, were mixed with phosphate buffer
(2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6]
(2.5 mL, 1% w/v). The mixture was incubated at 50 °C for 20 min.
A portion (2.5 mL) of trichloroacetic acid (10% w/v) was added to
the mixture, which was then centrifuged for 10 min at 3000 g.
The upper layer of the solution (2.5 mL) was mixed with ultrapure
water (2.5 mL) and FeCl3 (0.5 mL, 0.1% w/v), and the absorbance
was measured at 700 nm. Higher absorbance of the reaction mix-
ture indicated greater reducing power. Ascorbic acid was used as
positive control.
3.10.2. Hydrogen peroxide scavenging assay
The H2O2 scavenging activity was measured by monitoring the
H2O2-induced oxidation of lucigenin as previously described.12 The
endogenous antioxidant ascorbic acid was used as positive control.
The results were expressed as the inhibition (in percentage) of the
H2O2-induced oxidation of lucigenin. Each study corresponds to
five experiments, performed in triplicate.
3.10.9. Determination of metal chelating activity
3.10.3. Hypochlorous acid scavenging assay
The metal chelating activity was assessed by monitoring the
changes in the UV–vis spectrum of the test compounds after addi-
tion of Fe(II).46 Experiments were performed by sequential addi-
The HOCl was measured by using a previously described fluo-
rescent methodology12 based on the HOCl-induced oxidation of
DHR to rhodamine. HOCl was prepared by adjusting the pH of a
1% (m/v) solution of NaOCl to 6.2 with dropwise addition of 10%
H2SO4. Lipoic acid was used as positive control. The results were
expressed as the inhibition (in percentage) of HOCl-induced oxida-
tions of 2
of a 10 M (final concentration) compound solution. The reactions
were performed in phosphate buffer 20 mM, pH 7.2.
lL of FeCl2 solution (300 lM stock solution) to 300 lL
l

7226
A. Gomes et al. / Bioorg. Med. Chem. 17 (2009) 7218–7226
21. Mahal, H. S.; Venkataraman, K. J. Chem. Soc. 1934, 1767.
Acknowledgements
22. Price, W. A.; Silva, A. M. S.; Cavaleiro, J. A. S. Heterocycles 1993, 36, 2601.
23. Seixas, R. S. G. R.; Pinto, D. C. G. A.; Silva, A. M. S.; Cavaleiro, J. A. S. Aust. J. Chem.
2008, 61, 718.
24. Pinto, D.; Silva, A. M. S.; Cavaleiro, J. A. S. Synlett 2007, 1897.
25. Pinto, D.; Silva, A. M. S.; Almeida, L.; Cavaleiro, J. A. S.; Elguero, J. Eur. J. Org.
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26. Pinto, D.; Silva, A. M. S.; Cavaleiro, J. A. S. New J. Chem. 2000, 24, 85.
27. Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. J. Nutr. Biochem. 2002, 13, 572.
28. Silva, A. M. S.; Filipe, P.; Seixas, R. S. G. R.; Pinto, D. C. G. A.; Patterson, L. K.; Hug,
G. L.; Cavaleiro, J. A. S.; Maziere, J. C.; Santus, R.; Morliere, P. J. Phys. Chem. B
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The authors acknowledge FCT and FEDER financial support for
the project POCI/QUI/59284/2004 and the Organic Chemistry Re-
search Unit (n° 62). Ana Gomes acknowledges FCT and FSE her
PhD grant (SFRH/BD/23299/2005). Marisa Freitas acknowledges
FCT and FSE her PhD grant (SFRH/BD/28502/2006).
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