2,3-Diarylxanthones as strong scavengers of reactive oxygen and nitrogen species: A structure-activity relationship study

Article abstract of DOI:10.1016/j.bmc.2010.07.044

Xanthones are a class of oxygen-containing heterocyclic compounds widely distributed in nature. The natural derivatives can present different substitutions in the xanthone core that include hydroxyl, methoxyl, prenyl and glycosyl groups. The inclusion of

Full text of DOI:10.1016/j.bmc.2010.07.044

Bioorganic & Medicinal Chemistry 18 (2010) 6776–6784
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,3-Diarylxanthones as strong scavengers of reactive oxygen and nitrogen
species: A structure–activity relationship study
b
b
b
c
Clementina M. M. Santos a, , Marisa Freitas , Daniela Ribeiro , Ana Gomes , Artur M. S. Silva ,
*
José A. S. Cavaleiro c, Eduarda Fernandes b,
*
a Departamento de Produção e Tecnologia Vegetal, Escola Superior Agrária de Bragança, 5301-855 Bragança, Portugal
b REQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal
c 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:
Xanthones are a class of oxygen-containing heterocyclic compounds widely distributed in nature. The
natural derivatives can present different substitutions in the xanthone core that include hydroxyl, meth-
oxyl, prenyl and glycosyl groups. The inclusion of aryl groups has only been reported for a few synthetic
derivatives, the 2,3-diaryl moiety being recently introduced by our group. Xanthones are endowed with a
broad spectrum of biological activities, many of them related to their antioxidant ability, including the
scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as metal chelat-
ing effects. Considering the interesting and promising antioxidant activities present in compounds
derived from the xanthone core, the main goal of this work was to evaluate the scavenging activity of
the new 2,3-diarylxanthones for ROS, including superoxide radical (O2ꢀꢁ), hydrogen peroxide (H2O2), sin-
glet oxygen (1O2), peroxyl radical (ROOÅ) and hypochlorous acid (HOCl), and RNS, including nitric oxide
(ÅNO) and peroxynitrite anion (ONOOꢁ). The obtained results revealed that the tested 2,3-diarylxanthones
are endowed with outstanding ROS and RNS scavenging properties, considering the nanomolar to micro-
molar range of the IC50 values found. The xanthones with two catechol rings were the most potent scav-
engers of all tested ROS and RNS. In conclusion, the new 2,3-diarylxanthones are promising molecules to
be used for their potential antioxidant properties.
Received 2 June 2010
Revised 15 July 2010
Accepted 20 July 2010
Available online 25 July 2010
Keywords:
2,3-Diarylxanthones
Reactive oxygen species
Reactive nitrogen species
Scavenging activity
Antioxidant activity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
extensively reported in the literature, namely anti-allergic,14,15
antifungal,16,17 anti-inflammatory,18,19 antimalarial,20,21 antitu-
Xanthones are a class of oxygen-containing heterocyclic com-
pounds widely distributed in nature.1 They occur in two major
plant families, Guttiferae and Gentianaceae and in some families
of fungi and lichens.2,3 These natural derivatives can present differ-
ent substitutions in the xanthone core that include hydroxyl,
methoxyl, prenyl and glycosyl groups.4,5 The inclusion of aryl
groups has only been reported for a few synthetic derivatives
and the 2,3-diaryl moiety has never been presented before our syn-
thetic work.6–10
The pharmacological properties of xanthones published in the
last decades reveal the growing interest in this type of com-
pounds.11 The pioneering work of Finnegan et al.,12 in 1968, re-
ported the diuretic action of mangiferin, a natural xanthone
glycoside. Later on, Da Re et al.13 reported the analeptic activity
of synthetic aminoalkylxanthone derivatives. Since then, the bio-
logical profiles of natural and even synthetic analogues have been
mour,22,23 hepatoprotective activity,24 inhibition of monoamine
oxidase,25 and other enzymes, like cholinesterase26 and angioten-
sin I converting-enzyme.27 In this broad spectrum of biological
activities special mention deserves also the antioxidant11 activity
demonstrated by several xanthones. The antioxidant properties
have been exemplified by their action as free radical scavengers,
inhibitors of lipid peroxidation and as metal chelators.11,28,29
The recognized participation of reactive oxygen species (ROS)
and reactive nitrogen species (RNS) in several physiopathologies
and in cellular signalling systems has attracted growing interest
from the health sector over the last few decades. In addition, the
quest for effective antioxidants for food, cosmetic and pharmaceu-
tical purposes has become a major industrial and scientific re-
search challenge. Considering the interesting and promising
studies already performed in xanthones, concerning their antioxi-
dant activities, the development of new and effective scavengers
of ROS and RNS using a xanthone scaffold seems therefore to be
an interesting approach in this area. Thus, the main goal of this
work was to evaluate the scavenging activity of the new synthetic
2,3-diarylxanthones derivatives for ROS, such as superoxide radical
* Corresponding authors. Tel.: +351 222078968; fax: +351 222004427 (E.F.); tel.:
+351 273303308; fax: +351 273325405 (C.M.M.S.).
E-mail addresses: clems@ipb.pt (C.M.M. Santos), egracas@ff.up.pt (E. Fernandes).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.044

C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
6777
(O2ꢀꢁ), hydrogen peroxide (H2O2), singlet oxygen (1O2), peroxyl
radical (ROOÅ) and hypochlorous acid (HOCl), and RNS, such as ni-
tric oxide (ÅNO) and peroxynitrite anion (ONOOꢁ) (Fig. 1).
Table 1
O2ꢀꢁ, H2O2, 1O2 and HOCl scavenging activity (IC50, mean SEM) and ROOÅ scavenging
activity (concentration range 0.125–1.0
of the tested 2,3-diarylxanthones and positive controls
lM) expressed as ORAC values (mean SEM)
ꢀ
ORACROO SEM
Compound IC50 M)
(
l
2. Results
(
lM trolox equiv/
O2
H2O2
HOCl
1O2
ꢀꢁ
l
M compound)
2.1. Superoxide radical scavenging activity
2,3-Diarylxanthones
l
M
l
l
M
M
l
M
lM
1a
1b
1c
2a
2b
2c
3a
3b
3c
NA25
NA125
NA125
155 25 27%*100
72.1 8.6 80 11
NA1
Only 2,3-diarylxanthones 3a–c could prevent the O2ꢀꢁ-depen-
dent reduction of NBT in a concentration-dependent manner
(Table 1). Compound 3c was considerably the most efficient, of this
group and of all the tested compounds, presenting an IC50 of
10.4 0.8 lM. Compounds 2c, 1c and 3b are also potent scaveng-
ers, providing IC50s of 20.3 2.5, 28.1 2.3 and 31.3 3.2
NA200
1.05 0.09
0.76 0.01
l
M
28.1 2.3 9%*125
15.7 1.1 6.0 1.0
l
M
NA200
NA125
53.8 7.8 68.8 6.2 2.08 0.16
22.4 2.5 58.4 4.9 2.88 0.09
l
M
l
l
M
M
l
166 16 NA125
20.3 2.5 14%*250
14.7 1.3 3.3 0.7
10.8 0.4 4.5 0.6
7.5 0.7 6.8 0.5
1.2 0.02 2.5 0.2
0.88 0.12
0.84 0.01
0.83 0.03
0.28 0.03
M
l
M
76 11
9%*125
31.3 3.2 12%*250
10.4 0.8 17%*250
l
M
lM,
l
M
respectively.
Positive controls
2,3-Diarylxanthones 1a, 1b and 2a were completely ineffective
up to the highest tested concentration (compound 1a, 25 M and
compounds 1b and 2a, 200 M).
The IC50 values presented in Table 1 for O2 scavenging activity
vary from 10.4 to 166 M (compound 2b) and are significantly
lower than the IC50 value found for the positive control, tiron
(IC50 = 273 32 M).
Tiron
273 32
—
—
—
—
—
—
—
l
Ascorbic acid
—
602 80
—
0.22 0.03
—
l
Dihydrolipoic —
acid
2.3 0.3
ꢀꢁ
l
Quercetin
—
—
—
1.8 0.1
—
NA: no activity was found up to the highest tested concentration (in superscript).
Scavenging effect (mean%) at the highest tested concentration (in superscript).
l
*
2.2. Hydrogen peroxide scavenging activity
It was possible to determine the IC50 of 2,3-diarylxanthone 1a,
along the studied concentration range (25–200
155 25 M.
The IC50 of the positive control dihydrolipoic acid was
2.3 0.3 M.
lM) at
The results presented in Table 1 refer only to the H2O2 scaveng-
ing activity of the tested 2,3-diarylxanthones in a range of
125–250 lM, due to the precipitation that occurred in the sample
l
l
wells at higher concentrations.
Vestigial scavenging activity was observed for xanthones 3a–c.
Compounds 3b and 3c caused 17% and 12% inhibition of chemilu-
minescence, respectively, at their maximum concentration
2.4. Singlet oxygen scavenging activity
The 2,3-diarylxanthones proved to be effective scavengers of
1O2 in a concentration-dependent manner, except for compound
1a, which only reached 27% effect at the highest tested concentra-
(250
the highest tested concentration (250
and 1c presented similar effects and did not reach 10% effect at
the maximum tested concentration (125 M).
No scavenging activity was found for 2,3-diarylxanthones 1a,b
and 2a,b, at concentrations up to 125 M.
The positive control, ascorbic acid, provided an IC50 of
602 80 M.
lM). From 2a–c, only 2c presented a slight effect of 14% at
lM). The derivatives 3a
tion (100
lM) (Table 1). For compounds 1a–c, 1c (IC50 = 6.0 1.0
l
lM) was noticeably more active than compound 1b (IC50 = 80
11
l
M).
For compounds 2a–c, 2c was the most potent (IC50 = 3.3
M) and the obtained IC50 for 2b (IC50 = 58.4 4.9 M) was
M).
l
0.7
l
l
l
slightly lower than that of 2a (IC50 = 68.8 6.2
l
2,3-Diarylxanthones 3a–c revealed to be very active com-
pounds, the scavenging activity order being 3c > 3a > 3b, with
2.3. Hypochlorous acid scavenging activity
IC50s of 2.5 0.2, 4.5 0.6 and 6.8 0.5
Quercetin (positive control) efficiently scavenge 1O2
(IC50 = 1.8 0.1 M).
lM, respectively.
All the tested compounds were able to scavenge HOCl in a con-
centration-dependent manner. The HOCl-induced oxidation of
dihydrorhodamine 123 (DHR) was efficiently prevented by hydro-
xy-2,3-diarylxanthones 3a–c, being 3c and 3b the most effective
l
2.5. Peroxyl radical scavenging activity
derivatives, providing IC50 values of 1.2 0.02 and 7.5 0.7
respectively (Table 1).
lM,
As seen in Table 1, all the assayed compounds, except 1a, were
able to delay the loss of fluorescence, due to ROOÅ-dependent fluo-
rescein oxidation, in a concentration-dependent way. Compound
1a did not show any scavenging activity along the studied concen-
From compounds 1a–c and 2a–c, 1c and 2c proved to be the
most active ones (IC50 = 15.7 1.1 and 14.7 1.3 M, respectively).
However, the IC50 values were slightly higher than the obtained for
l
the compound 3a (IC50 = 10.8 0.4
lM).
5''
E
OH
OH
4''
3''
6''
5
4
O
O
O
3
O
O
6
OH
R2
1''
1'
2''
2'
B
8
A
C
2
R2
R2
R1
3'
7
9
1
D
5'
4'
O
6'
R1
R1
1a-c
2a-c
3a-c
a) R1 = R2 = H
b) R1 = OH, R2 = H
c) R1 = R2 = OH
a) R1 = R2 = H
b) R1 = OH, R2 = H
c) R1 = R2 = OH
a) R1 = R2 = H
b) R1 = OH, R2 = H
c) R1 = R2 = OH
Figure 1. Chemical structures of the studied 2,3-diarylxanthones.

6778
C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
tration range (0.125–1
considerably more active than the endogenous antioxidant, ascor-
bic acid, which presented an ORAC (oxygen radical absorbance
l
M) and the other tested compounds were
Similar results were obtained for compounds 1b and 2b, being
the former more efficient in the absence of NaHCO3 and the latest
more effective in the presence of this salt.
capacity) value of 0.22 0.03
lM.
Compound 1a was also able to scavenge ONOOꢁ although its ef-
fect could only reach 29% and 48%, at the maximum concentration
2,3-Diarylxanthones 1b, 2a and 2b were the most powerful
scavengers of ROOÅ, providing ORAC values of 1.05 0.09,
tested (50
respectively.
Ebselen, a well known ONOOꢁ scavenger, presented an IC50 of
0.50 0.03 M in the absence of NaHCO3 and 2.01 0.22 M in
its presence.
lM), in the absence or in the presence of NaHCO3,
2.08 0.16 and 2.88 0.09
In contrast, compounds 3a–c along with compound 1c were the
less active ones, presenting ORAC values from 0.28 to 0.84
lM, respectively.
l
M.
l
l
2.6. Nitric oxide scavenging activity
3. Discussion
Å
All the tested compounds were able to scavenge NO-induced
oxidation of 4,5-diaminofluorescein (DAF-2) in a concentration-
dependent manner (Table 2).
Compounds 2c and 3c were the most effective ones, presenting
a very similar activity with IC50 rounding 0.4 lM, followed by 1c,
The present study indicates that the tested 2,3-diarylxanthones
1–3 are promising molecules to be used for their potential antiox-
idant properties. The scavenging activity for ROS/RNS of these xan-
thone derivatives is reported for the first time, with outstanding
results, considering the nanomolar to micromolar range of the
3a and 3b. The order of potencies found for these derivatives
was: 3c > 2c > 3b > 3a > 1c.
ꢀꢁ
IC50 values found. In our first approach, O2 scavenging activity
was assayed using a non enzymatic (NADH/phenazine methosul-
fate) generating system. The results seem to be largely affected
by the number and position of hydroxyl groups in D and E rings
of the xanthone core. Several authors had already referred the
importance of structure–activity relationship for the ability of sev-
eral hydroxyxanthones to scavenge O2ꢀꢁ, using different methodol-
ogies.30–32 Compound 3c, bearing four hydroxyl groups, is the most
2,3-Diarylxanthones 1b and 2b revealed to be less potent and
provided IC50s of 175 36 and 108 18
es 1a and 2a presented only a slight effect of 39% and 41%, respec-
tively, at the maximum tested concentration (200 M).
The IC50 obtained for the flavonoid rutin (positive control) was
2.53 0.37 M.
lM, respectively. Xanthon-
l
l
ꢀꢁ
effective scavenger of O2 (Fig. 2) followed by compounds 3b and
2.7. Peroxynitrite scavenging activity
2c, which possess 3 hydroxyl groups. In this case, the scavenging
activity of compound 2c is slightly higher than compound 3b, sug-
gesting that the presence of a catechol group in D-ring is more rel-
evant than the presence of a catechol group in E-ring. The same
effect was observed in compounds 1c and 3a, possessing an
ortho-dihydroxyl substitution in D-ring and E-ring, respectively.
Compound 2b that contains two hydroxyl groups, but no catechol
moiety, was noticeably less potent than the compounds described
before; the remaining phenol derivatives showed no activity.
Poor H2O2 scavenging activity was observed for all the tested
compounds. However, it was not possible to test beyond the con-
The 2,3-diarylxanthones tested were shown to efficiently inhi-
bit ONOOꢁ-induced oxidation of DHR, in a concentration-depen-
dent manner. However, the concentration–effect relationship on
scavenging activity was different in the absence or in the presence
of 25 mM NaHCO3. Generally, the compounds were shown to be
more effective in the absence of NaHCO3 than in its presence, with
the exception of compounds 2a and 2b.
Compound 3c proved to be the most active derivative, with
IC50s of 0.17 0.01 and 0.33 0.06 lM, in the absence and in the
presence of NaHCO3, respectively (Table 2).
centration of 250
hones 1–3 in the tested system. Nevertheless, the scavenging
values of derivatives 2c, 3b and 3c (12–17% at a 250 M concentra-
tion) also suggest that the number and the position of hydroxyl
groups are responsible for the H2O2-scavenging effects (Table 1).
This hypothesis is only partially confirmed in the literature. Sun
et al.30 monitored the H2O2-induced oxidation of luminol (a similar
methodology to the described in this paper) and showed that
lM due to the low solubility of 2,3-diarylxant-
Concerning other results in the absence of NaHCO3, compounds
2c and 3b (IC50 = 0.26 0.05 and 0.22 0.03
were visibly more efficient than compounds 1c and 3a
(IC50 = 0.40 0.03 and 0.37 0.09 M, respectively). However, in
lM, respectively)
l
l
the presence of NaHCO3, the relative order of potencies for these
compounds is the opposite. The scavenging activity order was: 1c
> 3a > 2c > 3b, with IC50 values of 0.54 0.08, 0.67 0.09,
0.78 0.26 and 0.89 0.18 lM, respectively.
a
-mangostin (a trihydroxyxanthone) presented evident inhibitory
effect on H2O2 when compared with -mangostin (a dihydroxy-
xanthone). On the other hand, Pedraza-Chaverrí et al.,33 using a
different procedure, showed that -mangostin was unable to scav-
c
Table 2
ÅNO and ONOOꢁ (with and without 25 mM NaHCO3) scavenging effects (IC50
mean SEM) of the tested 2,3-diarylxanthones and positive controls
,
a
enge H2O2. From the analysis of our results, the tetrahydroxy-2,3-
diarylxanthone 3c is more active than the trihydroxyxanthones, 2c
and 3b. Therefore comparing the results of both trihydroxyxant-
hones one can conclude that the presence of a catechol unit in
the D-ring of compound 2c can contribute for their slight higher
effect relatively to the corresponding isomer 3b.
The HOCl-induced oxidation of DHR was efficiently prevented
by all the tested 2,3-diarylxanthones. The results from the HOCl-
scavenging assay show, once again, the importance of the ortho-
dihydroxyl substitution for the antioxidant activity of 2,3-diarylx-
anthones (Fig. 3). In addition, the number of the hydroxyl groups
also seems to contribute to the scavenging effect of these com-
pounds. Indeed, compound 2b, possessing two hydroxyl groups
but not a catechol moiety, presents almost 100% inhibition of
Compound
IC50
(lM)
ÅNO
ONOOꢁ without NaHCO3 ONOOꢁ with NaHCO3
2,3-Diarylxanthones
l
M
l
M
lM
1a
1b
1c
2a
2b
2c
3a
3b
3c
39%*200
175 36
29%*50
48%*50
1.55 0.14
1.80 0.39
0.54 0.08
2.00 0.15
0.97 0.25
0.78 0.26
0.67 0.09
0.89 0.18
0.33 0.06
1.88 0.18 0.40 0.03
41%*200
2.66 0.29
1.72 0.21
l
M
108 18
0.42 0.05 0.26 0.05
1.22 0.21 0.37 0.09
0.62 0.10 0.22 0.03
0.39 0.05 0.17 0.01
Positive controls
Rutin
Ebselen
2.53 0.37
—
—
—
0.50 0.03
2.01 0.22
HOCl-induced oxidation at a concentration of 100
lM. For lower
*
Scavenging effect (mean%) at the highest tested concentration (in superscript).
concentrations, the scavenging activity of the tested xanthones

C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
6779
100
80
60
40
20
0
1c
2b
2c
3a
3b
3c
12.5
50.0
100.0
200.0
Concentration (µM)
ꢀꢁ
Figure 2. O2 scavenging activity of 2,3-diarylxanthones 1c, 2b–3c. Each column represents the values obtained from five experiments, performed in triplicate
(mean SEM).
100
1c
2b
2c
80
60
40
20
0
3a
3b
3c
3.125
6.250
25.000
100.000
Concentration (µM)
Figure 3. HOCl scavenging activity of 2,3-diarylxanthones 1c, 2b–3c. Each column represents the values obtained from five experiments, performed in triplicate
(mean SEM).
was visibly more affected by the presence of the catechol group
rather than the number of hydroxyl substituents. This is clear by
the analysis of the results, which shows that derivatives 3a–c in
addition of compounds 1c and 2c were all more effective scaveng-
Other interesting feature could be suggested by the analysis of
Figure 4, which presents the results of the 1O2 scavenging activity
of 2,3-diarylxanthones possessing at least a catechol unit. We can
observe that, for concentrations of 10–20 lM, the presence of
ers than compound 2b, at 6.25 and 3.125
l
M concentration (Fig. 3).
two catechol groups in compound 3c seems to be essential for
the high scavenging potential of this derivative. In contrast, for
the lower concentrations, the number of catechol units does not
seem to bring any advantage to the scavenging activity, all the
derivatives presenting similar effects.
The data presented in Table 1 show that all the hydroxyxant-
hones tested provided higher ORAC values than the endogenous
antioxidant ascorbic acid. The ROOÅ scavenging activity of these
compounds seems to mostly depend on the hydroxylation pattern
In agreement with the results from other ROS, the 1O2 scaveng-
ing activity of 2,3-diarylxanthones depends particularly on the OH-
substitution pattern. Compounds with a catechol unit presented a
considerably higher effect than those lacking this feature as it can
be confirmed by the IC50 values. 2,3-Diarylxanthones 1c, 2c, 3a–c
presented IC50 values in a range of 2.5–6.8
1b, 2a,b, having a phenol instead of a catechol ring, were notice-
ably less potent, with IC50s rounding 58.4–80 M (Table 1).
lM and compounds
l

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C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
100
80
60
40
20
0
1c
2c
3a
3b
3c
0.156
0.625
2.500
10.000
20.000
Concentration (µM)
Figure 4. 1O2 scavenging activity of 2,3-diarylxanthones 1c, 2c, 3a–c. Each column represents the values obtained from five experiments, performed in triplicate
(mean SEM).
in the aryl groups. However, in this assay, the presence of phenol
groups seems to be extremely important for the scavenging effect.
The highest ORAC value was obtained for compound 2b, a xan-
thone bearing two phenol groups as substituents. The introduction
of an additional OH substituent drastically decreases the scaveng-
ing activity as it is evident by the results of compounds 3a–c.
2,3-Diarylxanthone 3c, a derivative with two ortho-dihydroxyl
groups, provided the lowest ORAC value from all the tested
hydroxyxanthone. These observations are not in accordance to
the knowledge about structure–activity relationship of flavonoids
and phenolic compounds. The ortho-dihydroxyl substitution is
particularly important to the ROOÅ absorbing activity of those
classes of compounds.34–36 The mechanism involves the H-atom
donating ability of the antioxidant to the ROOÅ, thus acting as chain
radical terminator.
Å
The NO scavenging activity indicates that the OH-substitution
pattern in the aryl groups is the main factor responsible for the
scavenging effect of this RNS. The compounds with a catechol
Å
group play an important role as protectors of the NO-dependent
oxidation of DAF-2 (Fig. 5). Yet, this group of compounds is also
much more effective than rutin, a flavonoid, which is a well-known
ÅNO scavenger. The other 2,3-diarylxanthones provided a much
weaker effect, as can be confirmed by the analysis of the results
presented in Table 2.
Å
Some of us had already studied the NO scavenging activity
of some flavones and 2-styrylchromones.37,38 In general, the
100
1c
2c
3a
3b
3c
80
60
40
20
0
0.156
0.625
2.500
10.000
Concentration (µM)
Figure 5. ÅNO scavenging activity of 2,3-diarylxanthones 1c, 2c, 3a–c. Each column represents the values obtained from five experiments, performed in triplicate
(mean SEM).

C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
6781
compounds presenting a catechol moiety, have shown to be more
cases, IC50 values under 1
lM. The results obtained are largely
potent than those lacking this unit. The same behaviour was ob-
served in the present study and the tested 2,3-diarylxanthones pre-
sented similar effects on the ÅNO scavenging activity to those
presented by the structural similar compounds. Other authors also
affected by the number and position of hydroxyl groups in the
xanthone core. The ROOÅ scavenging activity seems to depend
mostly on the presence of phenolic groups while for the other
tested ROS and RNS, xanthones with a catechol ring were the most
potent scavengers. These findings allowed the establishment of
structure–activity relationships and that 2,3-diarylxanthones can
have a promising future as antioxidant pharmacophores.
Å
reported that some flavonoids were good NO scavengers.39 Thus,
considering the structural similarity between the above-referred
compounds, it is very likely that the studied hydroxyxanthones
might present some therapeutic value.
Concerning ONOOꢁ, all the studied hydroxy-2,3-diarylxanthon-
es were able to scavenge this RNS in a very efficient way. Appar-
ently, the scavenging effect is related to the number and position
of the hydroxyl substituents on the D and E-rings. However, these
features have a different contribution when the assays are per-
formed in the absence or in the presence of NaHCO3. Figure 6 de-
scribes the ONOOꢁ scavenging activity of the catecholic-2,3-
diarylxanthones, in the absence of bicarbonate. We can observe
that by increasing the number of hydroxyl substituents, higher is
the scavenging effect of these compounds. Derivatives 2c, 3b and
3c were visibly more active than the compounds 1c and 3a, which
possesses only two hydroxyl groups. Heijnen et al.40 studied the
ONOOꢁ scavenging of substituted phenols and several flavonoids
and concluded that the activity is positively influenced by the
number of OH groups and by the presence of catechol units in
the above-referred molecules. When we performed this assay in
the presence of bicarbonate the results seemed to be dependent
on the position of the ortho-dihydroxyl moiety, which were some-
times favourable and others unfavourable to the scavenging activ-
ity, depending on the concentration of the antioxidant species
(Fig. 7). The reaction between ONOOꢁ and CO2 is very fast
4. Materials and methods
4.1. Chemicals
All the chemicals and reagents were of analytical grade. Ascor-
bic acid, DAF-2, dihydrolipoic acid, DHR, ebselen, 30% hydrogen
peroxide, lucigenin, b-nicotinamide adenine dinucleotide (NADH),
nitroblue tetrazolium chloride (NBT), 3-(aminopropyl)-1-hydro-
xy-3-isopropyl-2-oxo-1-triazene (NOC-5), penicillamine, phena-
zine methosulfate (PMS), sodium hypochlorite solution, with 4%
available chlorine, rutin and tiron were obtained from Sigma–Al-
drich (St. Louis, USA).
a,
a0-Azodiisobutyramidine dihydrochloride
(AAPH), histidine, and trolox were obtained from Fluka Chemie
GmbH (Steinheim, Germany). Fluorescein sodium salt and querce-
tin were obtained from Aldrich (Milwaukee, USA). All the other re-
agents were obtained from Merck (Darmstadt, Germany).
The polyhydroxy-2,3-diarylxanthones 1a–3c were synthesized
according to procedures previously described in the literature10
and showed to possess identical spectroscopic and analytical
data.8–10 Briefly, 2,3-diarylxanthones 1–3 have been obtained by
a five steps chemical sequence starting from simple starting mate-
rials (Scheme 1).10 The first step of this synthesis involves the
esterification of 20-hydroxyacetophenone 4 with cinnamoyl chlo-
ride derivatives 5a–c (5a is commercial and 5b,c are prepared
in situ from the corresponding cinnamic acids and phosphorous
oxychloride in dry pyridine). Baker–Venkataraman rearrangement
of esters 6a–c with potassium hydroxide in DMSO afforded b-dike-
tones 7a–c; these were brominated and cyclodehydrated by treat-
ment with PTT (phenyltrimethylammonium tribromide), leading
to the synthesis of 2-bromo-2-styrylchromones 8a–c. The Heck
reaction of bromochromones 8a–c with styrene derivatives 9a–c
(K = 3–5.8 ꢂ 104 Mꢁ1 sꢁ1 41,42
)
and results in the formation of the
nitrosoperoxycarbonate anion (ONOOCO2ꢁ), whose decomposition
leads to the formation of different species including the highly
reactive NO2 and CO3 radicals.43 Thus, the observed variation in
the scavenging activities is probably due to the different ability
to scavenge the resulting radicals.
Å
ꢀꢁ
In conclusion, the results obtained in the present study revealed
new promising 2,3-diarylxanthones with outstanding ROS and RNS
scavenging properties. In fact, some of the studied derivatives
proved to be extremely efficient scavengers, showing, in some
100
1c
2c
3a
3b
3c
80
60
40
20
0
0.125
0.250
0.500
1.000
Concentration (µM)
Figure 6. ONOOꢁ scavenging activity of 2,3-diarylxanthones 1c, 2c, 3a–c. Each column represents the values obtained from five experiments, performed in triplicate
(mean SEM).

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C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
120
100
80
60
40
20
0
1c
2c
3a
3b
3c
0.156
0.313
1.250
5.000
Concentration (µM)
Figure 7. ONOOꢁ scavenging activity of 2,3-diarylxanthones 1c, 2c, 3a–c, in the presence of NaHCO3 25 mM. Each column represents the values obtained from five
experiments, performed in triplicate (mean SEM).
yielded 2,3-diarylxanthones 10a–c, 11a–c and 12a–c. The final
step of our synthetic route consisted on the demethylation of these
methoxy-2,3-diarylxanthones 10a–c, 11a–c and 12a–c by treat-
ment them with boron tribromide (2.5 equiv per methyl group)
in dichloromethane.
formed at 37 °C and the tested compounds were dissolved in
ethanol, as previously described.38 HOCl was daily prepared by
adjusting the pH of a 1% 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 percentage inhibition of HOCl-
induced oxidation of DHR. Each assay corresponds to five experi-
ments, conducted in triplicate.
4.2. Equipment
A microplate reader (Synergy HT, BIO-TEK), with spectrophoto-
metric, fluorimetric, and chemiluminometric detection, plus tem-
perature control capacity, was used for all ROS and RNS
scavenging assays.
4.3.4. Singlet oxygen scavenging assay
The 1O2 was generated by the thermal decomposition of a
previously synthesized water-soluble endoperoxide [disodium
3,30-(1,4-naphthalene)bispropionate (NDPO2)].44 The 1O2 scaveng-
ing activity was measured by monitoring the oxidation of non-
fluorescent DHR to fluorescent rhodamine 123, at 37 °C, after a
30 min incubation period, as described in the literature.44 Ascorbic
acid was used as positive control. The results were expressed as the
percentage inhibition of 1O2-induced oxidation of DHR. Each assay
corresponds to five experiments, conducted in triplicate.
4.3. ROS and RNS scavenging assays
4.3.1. Superoxide radical scavenging assay
ꢀꢁ
ꢀꢁ
The O2 was generated by the NADH/PMS system and the O2
scavenging activity was determined by monitoring the O2ꢀꢁ-in-
duced reduction of NBT, according to a previously developed pro-
cedure.38 The effect of the tested compounds dissolved in DMSO
was determined spectrophotometrically at 560 nm during 2 min.
The antioxidant tiron was used as positive control. The results
were expressed as the percentage inhibition of the NBT reduction
to diformazan. Each assay corresponds to five experiments, con-
ducted in triplicate.
4.3.5. Peroxyl radical scavenging assay
The ROOÅ scavenging activity was measured by monitoring the
fluorescence decay resulting from ROOÅ-induced oxidation of fluo-
rescein and expressed as the ‘oxygen radical absorbance capacity’
(ORAC), according to a described procedure.38 ROOÅ was generated
by thermal decomposition of AAPH and the tested compounds dis-
solved in acetone and subsequently diluted in phosphate buffer
(mixture 1:9). Trolox was used as the standard control in each
study. Ascorbic acid was used as positive control. The results were
expressed as ORAC values. Each assay corresponds to four experi-
ments, conducted in triplicate.
4.3.2. Hydrogen peroxide scavenging assay
The H2O2 scavenging activity was measured using a previously
described chemiluminescence methodology based on the H2O2-in-
duced oxidation of lucigenin.38 The tested compounds were dis-
solved in DMSO and the chemiluminescence assays were
performed at 37 °C. The endogenous antioxidant ascorbic acid
was used as positive control. The results were expressed as the per-
centage inhibition of the H2O2-induced oxidation of lucigenin. Each
assay corresponds to five experiments, conducted in triplicate.
4.3.6. Nitric oxide scavenging assay
Å
The NO scavenging activity was measured by monitoring the
ÅNO-induced oxidation of non-fluorescent DAF-2 to the fluorescent
triazolofluorescein (DAF-2T), using a previously described meth-
od.38 ÅNO was generated by NOC-5, the tested compounds dis-
solved in DMSO and the fluorimetric signal was detected after a
30 min incubation period, at 37 °C. Rutin was used as positive con-
trol. The results were expressed as the percentage inhibition of
4.3.3. Hypochlorous acid scavenging assay
The HOCl was measured by monitoring the HOCl-induced oxi-
dation of DHR to rhodamine 123. The fluorimetric assays were per-

C. M. M. Santos et al. / Bioorg. Med. Chem. 18 (2010) 6776–6784
6783
O
R2
R1
R2
R1
COX
O
O
A
OH
O
5a-c
6a-c
4
R1
R2
B
R1
R2
R4
R3
C
O
O
OH
O
OH
7a-c
Br
8a-c
9a-c
a) R3 = R4 = H
b) R3 = OMe, R4 = H
c) R3 = R4 = OMe
Compounds 5, 6, 7, 8
a) R1 = R2 = H
b) R1 = OMe, R2 = H
c) R1 = R2 = OMe
Diketone
Form
D
R5
R1
O
O
R6
R8
O
O
R2
R4
E
R7
R3
1a-c, 2a-c, 3a-c
1a) R5 = R6 = R7 = R8 = H
10a-c, 11a-c, 12a-c
10a) R1 = R2 = R3 = R4 = H
10b) R1 = R2 = R4 = H; R3 = OMe
10c) R1 = R2 = H; R3 = R4 = OMe
11a) R1 = OMe; R2 = R3 = R4 = H
11b) R1 = R3 = OMe; R2 = R4 = H
11c) R1 = R3 = R4 = OMe; R2 = H
12a) R1 = R2 = OMe; R3 = R4 = H
12b) R1 = R2 = R3 = OMe; R4 = H
12c) R1 = R2 = R3 = R4 = OMe
1b) R5 = R6 = R8 = H; R7 = OH
1c) R5 = R6 = H; R7 = R8 = OH
2a) R5 = OH; R6 = R7 = R8 = H
2b) R5 = R7 = OH; R6 = R8 = H
2c) R5 = R7 = R8 = OH; R6 = H
3a) R5 = R6 = OH; R7 = R8 = H
3b) R5 = R6 = R7 = OH; R8 = H
3c) R5 = R6 = R7 = R8 = OH
A: X = Cl, Dry Py, room temp., 2h or X = OH, Dry Py, POCl3, 60ºC, 2h
B: DMSO, KOH, room temp., 2 h
C: THF, PTT, room temp., 12 h
D: NMP, Pd(PPh3)4, PPh3, Et3N, 160ºC to reflux E: CH2Cl2, BBr3, -78 ºC to room temp., N2, 1-4h
Scheme 1.
ÅNO-induced oxidation of DAF-2. Each assay corresponds to five
experiments, conducted in triplicate.
the Organic Chemistry Research Unit. M.F. and A.G. acknowledge
Fundação para a Ciência e a Tecnologia (FCT) and Fundo Social
Europeu (FSE) their Ph.D. (SFRH/BD/28502/2006) and post-doc-
toral (SFRH/BPD/63179/2009) Grant, respectively.
4.3.7. Peroxynitrite scavenging assay
The ONOOꢁ scavenging activity was measured by monitoring
the ONOOꢁ-induced oxidation of non-fluorescent DHR to fluores-
cent rhodamine 123, as previously described.38 ONOOꢁ was syn-
thesized as reported in the literature by some of the authors.45
The assays were performed at 37 °C, the tested compounds dis-
solved in DMSO and the fluorimetric signal detected after a
2 min incubation period. Ebselen was used as positive 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 in vivo. This evaluation is important because,
under physiological conditions, the reaction between ONOOꢁ and
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