Novel chromone and xanthone derivatives: Synthesis and ROS/RNS scavenging activities

Article abstract of DOI:10.1016/j.ejmech.2016.03.043

Chromones and xanthones are oxygen-containing heterocyclic compounds acknowledged by their antioxidant properties. In an effort to develop novel agents with improved activity, a series of compounds belonging to these chemical classes were prepared. Their syntheses involve the condensation of appropriate 2-methyl-4H-chromen-4-ones, obtained via Baker-Venkataraman rearrangement, with (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde to provide the corresponding 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-dien-1-yl]-4H-chromen-4-ones. Subsequent electrocyclization and oxidation of these compounds led to the synthesis of 1-aryl-9H-xanthen-9-ones. After cleavage of the protecting groups, hydroxylated chromones and xanthones were assessed as scavenging agents against both reactive oxygen species (ROS) [superoxide radical (O₂^?-), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), singlet oxygen (¹O₂), and peroxyl radical (ROO^?)] and reactive nitrogen species (RNS) [nitric oxide (^?NO) and peroxynitrite anion (ONOO^-)]. Generally, all the tested new hydroxylated chromones and xanthones exhibited scavenger effects dependent on the concentration, with IC₅₀ values found in the micromolar range. Some of them were shown to have improved scavenging activity when compared with previously reported analogues, allowing the inference of preliminary conclusions on the structure-activity relationship.

Full text of DOI:10.1016/j.ejmech.2016.03.043

European Journal of Medicinal Chemistry 115 (2016) 381e392
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel chromone and xanthone derivatives: Synthesis and ROS/RNS
scavenging activities
,
1
b,
Carina Proença a , Helio M.T. Albuquerque 1, Daniela Ribeiro a, Marisa Freitas a,
ꢀ
Clementina M.M. Santos b *, Artur M.S. Silva b, Eduarda Fernandes a
,
c
,
, **
a UCIBIO-REQUIMTE, Applied Chemistry Laboratory, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo
Ferreira, 228, 4050-313 Porto, Portugal
b Department of Chemistry & QOPNA, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
c
ꢀ
School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolonia, 5300-253 Bragança, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Chromones and xanthones are oxygen-containing heterocyclic compounds acknowledged by their
antioxidant properties. In an effort to develop novel agents with improved activity, a series of com-
pounds belonging to these chemical classes were prepared. Their syntheses involve the condensation of
appropriate 2-methyl-4H-chromen-4-ones, obtained via Baker-Venkataraman rearrangement, with (E)-
Received 13 November 2015
Received in revised form
16 March 2016
Accepted 17 March 2016
Available online 21 March 2016
3-(3,4-dimethoxyphenyl)acrylaldehyde
to
provide
the
corresponding
2-[(1E,3E)-4-(3,4-
dimethoxyphenyl)buta-1,3-dien-1-yl]-4H-chromen-4-ones. Subsequent electrocyclization and oxida-
tion of these compounds led to the synthesis of 1-aryl-9H-xanthen-9-ones. After cleavage of the pro-
tecting groups, hydroxylated chromones and xanthones were assessed as scavenging agents against both
reactive oxygen species (ROS) [superoxide radical (Oꢀ2-), hydrogen peroxide (H2O2), hypochlorous acid
(HOCl), singlet oxygen (1O2), and peroxyl radical (ROOꢀ)] and reactive nitrogen species (RNS) [nitric oxide
(ꢀNO) and peroxynitrite anion (ONOOꢁ)]. Generally, all the tested new hydroxylated chromones and
xanthones exhibited scavenger effects dependent on the concentration, with IC50 values found in the
micromolar range. Some of them were shown to have improved scavenging activity when compared
with previously reported analogues, allowing the inference of preliminary conclusions on the structure
eactivity relationship.
Keywords:
Chromones
Xanthones
Reactive oxygen species
Reactive nitrogen species
Antioxidant activity
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
They are characterized by the presence of a huge diversity of
functional groups associated to a range of pharmacological prop-
Chromones (4H-chromen-4-ones) constitute an important class
of oxygenated heterocyclic compounds that commonly occur in
nature [1]. Their large variety of biological and pharmacological
effects, dependent on structural features such as the type, number
and position of substituents attached to the chromone nucleus, is
well-documented and several derivatives are even currently used
as therapeutic agents [2e4].
erties that includes anti-inflammatory, antimicrobial, antioxidant,
cardiovascular protective, hepatoprotective and cytotoxic activities
[6e10].
Despite the successful application of certain natural compounds
as drugs, their structurally limited accessibility imposed by
biosynthetic pathways, the complex and time consuming process
of extraction and purification or even their economically unfeasible
synthesis on a gram-scale, often make them not candidate drugs for
scientific and pharmaceutical industry in the quest for new thera-
peutic hits [11]. Being aware of this knowledge, the development of
new synthetic procedures applied for the synthesis of more effi-
cient bioactive compounds constitutes an interesting and hot
challenge for organic chemists.
9H-Xanthen-9-ones or simply xanthones are another class of
naturally occurring oxygen-containing heterocyclic compounds [5].
* Corresponding author. School of Agriculture, Polytechnic Institute of Bragança,
ꢀ
Campus de Santa Apolonia, 5300-253 Bragança, Portugal.
Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) occurred in biological systems through a variety of sources
and under normal and controlled conditions mediate and regulate a
** Corresponding author.
E-mail addresses: clems@ipb.pt (C.M.M. Santos), egracas@ff.up.pt (E. Fernandes).
Equal contribution to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.03.043
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

382
C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
variety of physiological functions. However, ROS and RNS over-
accumulation, known as oxidative stress, caused by the imbal-
ance between their generation and elimination, results in severe
deleterious effects on various cell constituents as DNA, RNA, lipids,
and proteins [12,13] and it is consequently implicated in numerous
physiopathologies such metabolic disorders, cellular aging,
inflammation, cardiovascular dysfunction, neurodegenerative dis-
eases and carcinogenesis [12e15].
these 1,3-diketones were treated with a catalytic amount of p-tol-
uenesulfonic acid (p-TSA) in DMSO to afford the desired 2-methyl-
4H-chromen-4-ones 5a-5c. The most important structural features
in the 1H NMR spectra of chromones 5a-5c are in each case a singlet
at
tons and a singlet at
Furthermore, the 13C NMR spectra of chromones 5a-5c showed
signals at 19.7e20.6, 110.3e111.8 and 166.2e178.0 ppm assigned
d
2.30e2.40 ppm corresponding to the resonance of 2-CH3 pro-
d
6.07e6.18 ppm corresponding to H-3.
d
The maintenance of ROS/RNS balance is carried out by endog-
enous enzymatic antioxidant defenses such as superoxide dis-
mutase, glutathione peroxidase, thioredoxin reductase and catalase
and by non-enzymatic compounds such as glutathione, uric acid
and coenzyme Q. If the internal production of antioxidants is not
enough to neutralize all the ROS/RNS produced, a series of exoge-
nous non-enzymatic antioxidants can be provided from human diet
that includes carotenoids, phenolic compounds, and flavonoids,
among others [16]. Thus, in the demand for new antioxidant agents,
the design of novel and more effective scavengers of ROS and RNS is
considered a key topic for several research groups. Continuing our
interest in this area herein we report the synthesis of novel chro-
mone and xanthone derivatives, as well as the scavenging activity
of their hydroxyl constituents against ROS [superoxide radical (Oꢀ2-),
hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxy-
gen (1O2), and peroxyl radical (ROOꢀ)] and RNS [nitric oxide (ꢀNO)
and peroxynitrite anion (ONOOꢁ)].
to resonances of 2-CH3, C-3 and C-4, respectively.
The required (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde 9 was
synthesized through a two-step method starting with an acid-
catalysed iodination of 1,2-dimethoxybenzene 7 with N-iodo-
succinimide (NIS), followed by
a palladium-catalysed cross-
coupling reaction with acrolein diethyl acetal (Scheme 1) and its
structure was confirmed by NMR spectroscopy. The 1H NMR
spectrum of this aldehyde showed: i) two singlets at
3.94 ppm due to the resonances of 3- and 4-OCH3; two doublets at
6.62 and 7.42 ppm attributed to the resonances of the H- and H-
vinyl protons, respectively, in a trans configuration (JH b 15.8 Hz);
d 3.93 and
d
a
b
a
,H
and a doublet at high frequency value (d 9.66 ppm) assigned to the
CHO resonance. The structure of this aldehyde was also confirmed
by the 13C NMR signals of CHO (193.5 ppm), 3,4-(OCH3)2 (55.8 and
55.9 ppm), and of C-a (126.6 ppm) and C-b (152.8 ppm).
The reactivity of 2-methyl-4H-chromen-4-ones has been the
main topic of several works [18]. Among them, there are a number
of condensation reactions with carbonyl compounds to afford new
heterocyclic systems. Following our previous work involving
condensation, electrocyclization and oxidation reactions [19], we
have synthesized chromones 10a-10c and xanthones 11a-11c,
respectively. Thus, the 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-
1,3-dien-1-yl]-4H-chromen-4-ones 10a-10c were prepared in good
yields by the condensation of 2-methyl-4H-chromen-4-ones 5a-5c
with (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde 9 in the presence
of 4 equiv of sodium ethoxide (generated in situ), at room tem-
perature (Scheme 2). The most important features in the 1H NMR
spectra of 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-dien-1-yl]-
4H-chromen-4-ones 10a-10c are in each case: i) the absence of the
singlet due to the CH3 proton resonance of the precursor 2-
2. Results and discussion
2.1. Chemistry
The required starting 2-methyl-4H-chromen-4-ones 5a-5c were
synthesized by the Baker-Venkataraman method in moderate
overall yields [17] (Scheme 1). It started by the monoprotection of
2’-hydroxyacetophenones 1b-1c with dimethyl sulfate in refluxing
acetone. The next steps involved the acetylation of the protected 2’-
hydroxyacetophenones 2a-2c followed by the base-catalysed
Baker-Venkataraman rearrangement of the formed ester 3a-3c,
leading to the 1,3-diketone derivatives 4a-4c in good yields. Finally,
Scheme 1.

C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
383
Scheme 2.
methylchromones 5a-5c; ii) the change in the multiplicity of pro-
tons H- and H- from doublets to multiplets, indicating that these
protons are now included in a 1,3-diene chain. The resonances of C-
(Scheme 1, vide experimental). The 1H NMR spectrum of chromone
5d showed particular signals assigned as two singlets at 3.38 and
5.36 ppm and two multiplets at 3.55e3.58 and 3.83e3.86 ppm
corresponding to the resonances of the protons of MEM moiety. On
a
b
d
d
a
d
, C-
122.1e122.5, 136.9e137.4, 124.8e125.2 and 138.5e138.8 ppm,
b, C-g and C-d
carbons appear in 13C NMR spectra at
the other hand, its carbon resonances can be observed at d 59.0,
respectively, while that of the carbonyl carbon appear at
177.7e178.3 ppm. The electrocyclization and oxidation reactions of
2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-dien-1-yl]-4H-chro-
men-4-ones 10a-10c were performed under refluxing 1,2,4-
trichlorobenzene (TCB) with a catalytic amount of iodine to afford
the desired 1-(3,4-dimethoxyphenyl)-9H-xanthen-9-ones 11a-11c
in moderate to good yields (Scheme 2). During this step the 5-OMe-
protecting group of chromone 10b was cleaved giving the corre-
sponding 8-hydroxyxanthone 11b. This was confirmed from the 1H
68.0, 71.4 and 93.2 ppm in the 13C NMR spectrum of chromone 5d.
The condensation reaction of chromone 5d with (E)-3-(3,4-
dimethoxyphenyl)acrylaldehyde
9
and subsequent electro-
cyclization and oxidation reactions occurred as already mentioned
for the methoxylated chromones 5a-5c. Finally, the MEM group
was easily removed in a couple hours by reaction with BBr3. Thus,
the most important characteristics in the 1H NMR spectra of com-
pounds 12a-12c and 13a-13c are: i) the singlets corresponding to
the resonances of OH protons of the catechol moiety at
NMR spectrum by the presence of a singlet at
d
12.60 ppm assigned
d 9.08e9.42 ppm for derivatives 12a-12c and at d 8.86e8.94 ppm
to the resonance of the 8-OH proton. The most relevant structural
for derivatives 13a-13c; ii) the singlets assigned to the resonances
of OH protons at position 5 of chromone 12b and of position 8 of
features present in the 1H NMR spectra of compounds 11a-11c are
three double doublets at
d
7.17e7.19, 7.65e7.73 and 7.47e7.51 ppm,
xanthone 13b, at
singlets attributed to OH protons at position 7 of chromone 12c and
of position 6 of xanthone 13c at 10.81e10.89 ppm, respectively.
d 12.59e12.77 ppm, respectively; and iii) the
due to the resonances of H-2, H-3 and H-4, respectively, which
means that the cyclization to xanthones occurred. The HMBC and
HSQC spectra of xanthones 11a-11c allowed to assign the carbon
d
resonances of C-2 (
4 ( 117.2e117.5 ppm) and the newly formed quaternary carbons C-
9a ( 117.9e119.4 ppm) and C-1 ( 143.7e144.0 ppm. Finally and in
d 127.3e127.8 ppm), C-3 (d 132.7e134.2 ppm), C-
2.2. ROS and RNS scavenging activity studies
d
d
d
In this study, novel chromone 12a-12c and xanthone 13a-13c
derivatives were subjected to preliminary screening of their anti-
oxidant potential in non-cellular systems, through their ability to
scavenge ROS and RNS involved in oxidative stress processes. All
compounds possess a 3,4-dihydroxyphenyl moiety, known as
catechol unit, in the B-ring of chromone and in the D-ring of
xanthone, an important feature for the improved scavenging ac-
tivity of ROS/RNS presented by several phenolic compounds
[20e22].
order to obtain the desired hydroxylated derivatives 12a-12c and
13a-13c, the deprotection reaction of respectively 10a-10c and 11a-
11c, occurred in the presence of boron tribromide (BBr3) in dry
CH2Cl2 (Scheme 2). Unfortunately, the deprotection step was not
straightforward for 7-OMe-chromone 10c and 6-OMe-xanthone
11c, which was not achieved even after several days of reaction
with BBr3. This drawback was overcome by changing the protecting
4’-hydroxy group of 20,4’-dihydroxyacetophenone 1c with
Xanthones 13a-13c were able to prevent the Oꢀ2ꢁ-dependent
methoxyethoxymethyl
chloride
(MEMCl).
The
Baker-
Venkataraman procedure for the synthesis of chromone 5d was
similar to the described for the methoxylated chromones 5a-5c,
however, it required one more OHeprotection step due to the
release of the MEM group during the cyclodehydration reaction
reduction of nitroblue tetrazolium chloride (NBT) in
concentration-dependent manner (Table 1). Derivatives 13a and
13c were the most active tested compounds presenting IC50 values
a
of 55 4 and 61
2 mM, respectively, and similar to the positive

384
C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
Table 1
Scavenging activity of the tested compounds against superoxide radical (Oꢀ2-), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and singlet oxygen (1O2) (IC50
mean SEM).
,
Compound
R1
R2
IC50
Oꢀ2-
(mM)
H2O2
143
125 13
121
HOCl
32
94 15
17
1O2
Chromones
12a
12b
H
H
OH
H
OH
H
e
e
8
3
1.3 0.1
1.1 0.2
1.6 0.4
mMa
12c
36%200
9
3
Xanthones
13a
mMa
H
H
55
4
12%1000
158 18
4.4 0.7
13b
13c
H
OH
OH
H
80 11
658 23
518 26
96
31
8
3
10
7.3 0.6
2
61
60
2
Positive control
Quercetin
7
1338 42
2.4 0.3
1.5 0.2
a
Scavenging effect (mean %) at the highest tested concentration (in superscript).
control quercetin (IC50 60
7
m
M). Derivative 13b were less active
presented by trihydroxyxanthones when compared with dihy-
droxyxanthones [26,27].
with an IC50 of 80 11 M. These results seem to indicate that the
m
presence of the OH group in position 8 of the xanthone core is
disadvantageous for the Oꢀ2ꢁ scavenging activity since this deriva-
tive is less effective than their parent compound 13a and their 6-OH
derivative, 13c. This comes in agreement with previous studies on
flavonoids that the strong intramolecular hydrogen bond between
the OH group peri to the carbonyl group is generally disadvanta-
geous for the scavenging activity of these compounds [23]. In
addition, the new 1-arylxanthones 13a-13c were less potent scav-
engers than other xanthones possessing a catechol group at posi-
tion 2 or 3 of their skeleton [24]. It was not possible to determine
the scavenging effect of chromones 12a and 12b due to the pre-
cipitation that occurred in the tested system. Chromone 12c caused
36% inhibition of absorbance at its maximum concentration
The HOCl-induced oxidation of dihydrorhodamine 123 (DHR)
was efficiently prevented by all the tested compounds, in a
concentration-dependent manner. Compounds 12a, 12c and 13c
proved to be the most active ones, providing IC50 values of
32
3
mM, 17
3
mM and 31
3 mM, respectively (Table 1). The
results showed that the presence of a 7-OH group in chromone 12c
led to a higher scavenging effect while the introduction of a 5-OH
group, chromone 12b, led to a weaker scavenger then their
parent compound, chromone 12a. The importance of these OH
groups on the A-ring were previously reported for the HOCl scav-
enging activity of flavones (analogues of chromones 12a-12c
without the 1,3-diene system) and 2-styrylchromones (analogues
of chromones 12a-12c lacking one double bond), related to the
ability to promote the chlorination at C-6 and C-8 positions, via an
electrophilic aromatic substitution [28]. On the other hand, the
additional double bond found in the new chromones seems to be
unfavourable for the HOCl scavenging activity since these com-
pounds were less potent scavengers than the previously tested 2-
styrylchromones [29]. Similarly to the above described for the
scavenging effect against H2O2, the 6-OH substitution (compound
(200 m
M). The lack of reactivity of these chromones towards Oꢀ2ꢁ is
not in accordance to the knowledge about structureeactivity rela-
tionship of several flavonoids and phenolic compounds, bearing a
catechol or styryl moiety, to scavenge this ROS [20,21,25].
All the tested compounds proved to be effective scavengers of
H2O2 in a concentration-dependent manner, except for compound
13a, which only reached 12% effect at the highest tested concen-
tration (1000
m
M) (Table 1). Chromones 12a-12c (IC50 values of
13c, IC50 31
enging of HOCl in comparison to the 8-OH derivative (compound
13b, IC50 96 M) or even their absence in the main core
(compound 13a, IC50 158 18 M). The 2,3-diarylxanthones
3 mM) in the tested xanthones favoured the scav-
143 M, 125
8
m
13 M and 121 M, respectively) were
m
9 m
noticeably more active than xanthones 13a-13c. All the tested
chromones were even more active than the positive control quer-
8 m
m
cetin (IC50 1338 42
m
M). These results indicate that the presence
already reported were found to be more active than the tested 1-
arylxanthones, suggesting the importance of the catechol group
at C-2 or C-3 rather than in C-1 to their reaction with this ROS [24].
All of the tested compounds were able to scavenge 1O2 in a
concentration-dependent manner. Chromones 12a-12c (IC50 values
of 7-OH and 5-OH groups is favourable to the reaction of chro-
mones with H2O2 but no influence was observed for the position of
the OH groups in the chromone core since no meaningful differ-
ences were found for derivatives 12b and 12c. Similar to chro-
mones, the presence of OH groups in B-ring of the xanthone moiety
is an important structural feature. However, their presence at C-6
(compound 13c, IC50 518 26
for the H2O2 scavenging effect in comparison to its presence at C-8
(compound 13b, IC50 658 23 M). The study developed by Santos
of 1.3 0.1
considerably more effective than xanthones 13a-13c (IC50 values of
4.4 0.7 M, 10 M and 7.3 0.6 M, respectively) (Table 1).
Moreover, chromones 12a and 12b showed a slightly higher effect
than the positive control, quercetin (IC50 1.5 0.2 M). Analysing
mM, 1.1 0.2 mM and 1.6 0.4 mM, respectively) were
mM) seems to have a positive effect
m
2
m
m
m
m
et al. [24] showed that xanthones possessing catechol groups at
positions 2 or 3 of the xanthone backbone have no effect in pre-
venting the oxidation of lucigenin promoted by H2O2. Taking into
consideration that these xanthones have no OH groups in B-ring,
we can infer that their inclusion appears to be important to
enhance the scavenging properties against this ROS. This finding is
also corroborated with the high inhibitory potential on H2O2
the results we can conclude that chromones 12a-12c were shown
to be more effective scavengers of 1O2 than the 2-styrylchromones
and flavones with comparable structures [29], indicating that the
additional double bonds at C-2 of the chromone moiety clearly
influence the scavenger effect, increasing its activity. Another
important feature is the presence of the 5-OH group to improve the
1O2 scavenging potential in contrast to their substitution at C-7 that

C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
385
brings no advantage [29]. In what concerns to xanthones, the
absence of OH groups in B-ring increases 1O2 scavenging activity
when compared to its presence at C-6 or C-8 of the xanthone
backbone, the 6-OH derivative (13c) being more active than the 8-
OH derivative (13b). Curiously, a different behaviour is observed to
chromones increases the scavenging activity against ꢀNO when
compared to its presence in position 5 or 7, being more effective in
the former case. Chromones 12a-12c were much weaker scaven-
gers than the structurally related 2-styrylchromones tested by
Gomes et al., indicating that the introduction of a double bond at C-
2 decreases the scavenging capacity [29]. With respect to xanthone
derivatives, the more active was compound 13c followed by com-
a
-mangostin (a trihydroxyxanthone) that presented a stronger
scavenger activity than -mangostin (a dihydroxyxanthone) [26].
g
The results from the Oxygen Radical Absorbance Capacity
(ORAC) assay are listed in Table 2. All the tested compounds were
able to delay the loss of fluorescence, preventing the ROOꢀ-
dependent fluorescein oxidation, in a concentration-dependent
manner. In fact, it has been previously referred that the presence
of a catechol unit plays an essential role in what concerns the ROOꢀ
scavenging activity of phenolic compounds, delaying or preventing
lipid peroxidation [21,30]. All of compounds achieved similar ORAC
values which were apparently superior to the value presented by
the water-soluble vitamin E analogue trolox, used as standard. In
fact, if we consider that compounds 12a-12c and 13a-13c are ex-
pected to trap 4 peroxyl radicals similar to quercetin while trolox
only traps 2 [31], the tested compounds are slightly less effective
than trolox and clearly less effective than quercetin (Table 2). The
results indicate that chromone 12c (1.63 0.09) has higher scav-
enging effect, followed by chromones 12a (1.44 0.01) and 12b
pound 13b (IC50 1.5
0.1 mM) and compound 13a (IC50
1.99 0.09 M). These results demonstrate that the presence of an
m
OH group in position 6 of tꢀhe A-ring increases the scavenging ac-
tivity of xanthones against NO when compared to its presence in
position 8 and even more in their absence in that ring.
All the tested compounds were shown to efficiently prevent
ONOOꢁ-induced oxidation of DHR, in a concentration-dependent
manner. These assays were also performed in the presence of
25 mM NaHCO3 in order to mimics the physiological CO2 concen-
tration in vivo. The fast reaction between ONOOꢁ and CO2
(k2 ¼ 3e5.8 ꢂ 104
M
ꢁ1 sꢁ1) leads to the formation of other highly
reactive species that affects the reactivity of ONOOꢁ scavengers in
the reaction medium, either increasing or decreasing their effects
[32]. Generally, the compounds were shown to be equally potent
scavengers in the absence and presence of NaHCO3, with the
exception of xanthones 13a (more active in the presence of
NaHCO3) and 13c (more active in the absence of NaHCO3). A similar
conclusion can be drawn from the scavenging effects presented by
several phenolic compounds and flavonoids [29,33,34]. Chromone
12c proved to be the most active derivative, with IC50 of 0.29 0.02
(1.40
0.05), with similar ORAC values. Considering that chro-
mones 12a-12c are less active than the 2-styrylchromones studied
by Gomes et al. we can conclude that the additional double bond at
C-2 decreases the scavenging effect against this ROS [29]. Analysing
the results of xanthones, compounds 13a (1.5
0.1) and 13c
and 0.30 0.04 mM, in the absence and in the presence of NaHCO3
(1.58 0.09) are the most active ones, followed by compound 13b
(1.1 0.1). An interesting feature common to all the ROS described
is the fact that 6-OH xanthone 13c was shown to have improved
scavenging activity when compared to the 8-OH xanthone 13b.
Similar to the H2O2 scavenging assays, the ROOꢀ scavenging effects
of the tested 1-arylxanthones showed to be more efficient than the
2,3-diarylxanthones studied by Santos et al. [24]. In fact, they
concluded that, unlike to the observed for the other ROS, the
introduction of phenolic groups at C-2 or C-3 of the xanthone core
is more favourable for the scavenging of ROOꢀ that the addition of
catechol groups in these positions.
25 mM, respectively (Table 3).
Concerning the results in the absence of NaHCO3, chromones
12a-12c (IC50 range from 0.29 to 0.34
cient than xanthones 13a-13c (IC50 range from 0.39 to 0.57
(Table 3) and even than the positive control, quercetin (IC50
0.45 0.07 M) (Table 2). No meaningful differences were observed
m
M) were visibly more effi-
mM)
m
in the scavenging effect of all chromone derivatives 12a-12c, which
indicates that the catecholic B-ring, a common feature in these
compounds, is the main factor responsible for the ONOOꢁ scav-
enging capacity [29]. The lower IC50 value of xanthone 13c indicates
that the 6-OH substitution improved the ONOOꢁ scavenging effect
when compared to 8-OH substitution (xanthone 13b, IC50
The ꢀNO-induced oxidation of 4,5-diaminofluorescein (DAF-2)
was efficiently inhibited by all of the tested compounds, in a
concentration-dependent manner. Compounds 12a, 12b, 12c and
13c revealed to be the most potent scavengers with IC50 values of
0.57
0.08
mM) or their parent compound (xanthone 13a, IC50
0.51 0.08
mM). All 1-arylxanthones tested presented lower effi-
ciency than 2,3-diarylxanthones reported by Santos et al. [24].
In the presence of NaHCO3, the most active scavengers were
0.82 0.07 mM, 1.23 0.08 mM, 1.42 0.09 mM and 1.08 0.05 mM,
respectively (Table 3). The absence of OH groups in the A-ring of
compounds 12a, 12c and 13a with IC50 values of 0.34 0.05 mM,
Table 2
Scavenging activity of the tested compounds against peroxyl radical (ROOꢀ) expressed as ORAC values (mean SEM).
Compound
R1
R2
ORACROO
ꢀ
SEM (mM trolox equiv/mM compound)
Chromones
12a
12b
H
H
OH
H
OH
H
1.44 0.01
1.40 0.05
1.63 0.09
12c
Xanthones
13a
13b
H
H
OH
H
OH
H
1.5 0.1
1.1 0.1
1.58 0.09
13c
Positive controls
Quercetin
Trolox
3.4 0.3
1

386
C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
Table 3
Scavenging activity of the tested compounds against nitric oxide (ꢀNO) and peroxynitrite anion (ONOOꢁ) (with and without 25 mM NaHCO3) (IC50, mean SEM).
Compound
R1
R2
IC50
ꢀNO
(mM)
ONOOꢁ without NaHCO3
ONOOꢁ with NaHCO3
Chromones
12a
12b
H
H
OH
H
OH
H
0.82 0.07
1.23 0.08
1.42 0.09
0.33 0.02
0.34 0.03
0.29 0.02
0.34 0.05
0.4 0.1
0.30 0.04
12c
Xanthones
13a
13b
H
H
OH
H
OH
H
1.99 0.09
1.5 0.1
1.08 0.05
0.51 0.08
0.57 0.08
0.39 0.08
0.35 0.05
0.56 0.07
0.66 0.07
13c
Positive controls
Quercetin
0.77 0.07
0.45 0.07
0.68 0.09
0.30 0.04
m
M and 0.35 0.05
m
M, respectively (Table 3). Analysing
Bruker Avance 300 spectrometer (300.13 for 1H and 75.47 MHz for
13C), with CDCl3 as solvent, unless otherwise stated. Chemical shifts
(d) are reported in ppm, and coupling constants (J) in Hz. The in-
ternal standard was TMS. Positive-ion ESI mass spectra were ac-
quired with a QTOF 2 instrument. Elemental analyses were
obtained with a CHNS 932 LECO analyser (University of Aveiro).
the results in the absence and in the presence of NaHCO3, we can
state that chromones 12a-12c were shown to possess a similar
behaviour regarding the presence of OH substituents but a higher
scavenging activity than the 2-styrylchromones [29]. For xanthone
derivatives 13a-13c, the absence of OH groups in the B-ring
enhanced the ONOOꢁ scavenging activity in comparison to their
inclusion at position 8 and even more at position 6 of the xanthone
skeleton. The range of the IC50 values found for the tested 1-
arylxanthones were similar to those obtained from 2,3-
diarylxanthones [24].
4.2. Reagents and chemicals
All chemicals and solvents used were of analytical grade, ob-
tained from commercial sources and used as received or dried by
standard procedures. The endoperoxide disodium 3.3’-(1,4-
naphthalene)bispropionate (NDPO2) was synthesized according to
procedure previously described by some of the authors [35].
3. Conclusion
Novel 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-dien-1-yl]-
4H-chromen-4-ones and corresponding 1-aryl-9H-xanthen-9-ones
were prepared from simple starting materials. Generally, all the
tested new hydroxylated chromones and xanthones exhibited
scavenger effects dependent on the concentration, with IC50 values
found in the micromolar range. We can also state that no homo-
geneous behaviour was observed in the scavenging activity of the
tested compounds against the most physiologically relevant ROS
and RNS, varying in accordance with the reactive species under
study. The 7-hydroxychromone 12c appeared as the lead com-
pound being the most active for scavenging H2O2, HOCl, ROOꢀ and
ONOOꢁ.
Some of the tested compounds were shown to have improved
scavenging activity when compared with previously reported an-
alogues, possibly suggesting novel structureeactivity relationships
that need to be confirmed in future studies: the additional double
bond of the new synthesized chromones increased the 1O2 and
ONOOꢁ scavenging ability [29]; the catechol group in position C-1
of the xanthone core has a stronger effect in the H2O2 and ROOꢀ
scavenging activity rather than in positions C-2 or C-3 to their re-
action with these ROS [24]. In conclusion, the novel synthesized
chromone and xanthone derivatives seem to be promising phar-
macophores with potential therapeutic applications associated to
oxidative stress disorders.
4.3. Synthesis of methoxy-2’-hydroxyacetophenones (2b, 2c)
Dimethyl sulfate (3.1 mL, 32.7 mmol) was added to a solution of
the appropriate 2’-hydroxyacetophenone (1b, 1c) (29.7 mmol) in
acetone (60 mL) with potassium carbonate (12.3 g, 89.1 mmol). The
mixture was stirred at reflux for 20 min. The inorganic salts were
removed by filtration and washed with acetone (50 mL). The sol-
vent was evaporated to dryness and the residue was purified by
silica gel column chromatography using dichloromethane as
eluent.
60-Methoxy-2’-hydroxyacetophenone (2b) (4.69 g, 95%) and 4’-
methoxy-2’-hydroxyacetophenone (2c) (4.59 g, 93%), which showed
spectroscopic and analytical data identical to one previously re-
ported [36].
4.4. Synthesis of 1-{2-hydroxy-4-[(2-methoxyethoxy)methoxy]
pheny})ethan-1-one (2d)
Methoxyethoxylmethyl chloride (MEMCl) (4.14 mL, 36.3 mmol)
was added to a solution of 20,4’-dihydroxyacetophenone (1c)
(33 mmol) in acetone (60 mL) with potassium carbonate (9.12 g,
66 mmol). The mixture was stirred at reflux for 1 h. The inorganic
salts were removed by filtration and washed with acetone (50 mL).
The solvent was evaporated to dryness and the residue was purified
by silica gel column chromatography using dichloromethane as
4. Experimental section
4.1. Chemistry, general information
Melting points were measured in a Büchi Melting Point B-540
apparatus and are uncorrected. NMR spectra were recorded on a
eluent,
giving
1-{2-hydroxy-4-[(2-methoxyethoxy)methoxy]
phenyl}ethan-1-one (2d) in good yield (5.70 g, 72%).

C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
387
4.4.1. 1-{2-hydroxy-4-[(2-methoxyethoxy)methoxy]pheny})ethan-
H-30, J 2.5 Hz), 6.98 (dd, 1H, H-50, J 8.8, 2.5 Hz), 7.82 (d, 1H, H-60, J
8.8 Hz) ppm; 13C NMR (75 MHz, CDCl3): 21.2 (20-OCOCH3), 29.0 (C-
1-one (2d)
d
Colourless oil. 1H NMR (300 MHz, CDCl3):
d
2.57 (s, 3H, H-2),
2), 59.0 (CH3OCH2CH2OCH2O), 68.0 (CH3OCH2CH2OCH2O), 71.4
(CH3OCH2CH2OCH2O), 93.2 (CH3OCH2CH2OCH2O), 111.4 (C-30),
113.4 (C-50), 123.9 (C-10), 132.3 (C-60), 151.1 (C-20) 161.2 (C-40þ), 169.4
(20-OCOCH3), 195.8 (C-1) ppm. MS m/z (ESI, %): 305 ([MþNa] , 100).
HRMS (ESI) m/z calcd. for C14H19O6 [MþH]þ 283.1176; found
283.1175.
3.38 (s, 3H, OCH3), 3.54e3.57 (m, 2H, CH3OCH2CH2OCH2O),
3.80e3.83 (m, 2H, CH3OCH2CH2OCH2O), 5.30 (s, 1H, CH3OCH2
-
CH2OCH2O), 6.57 (dd, 1H, H-50, J 8.8, 2.4 Hz), 6.60 (d, 1H, H-30, J
2.4 Hz),7.65 (d,1H, H-60, J 8.8 Hz),12.61 (s,1H, 20-OH) ppm; 13C NMR
(75 MHz, CDCl3):
d 26.3 (C-2), 59.0 (CH3OCH2CH2OCH2O), 68.1
(CH3OCH2CH2OCH2O), 71.5 (CH3OCH2CH2OCH2O), 93.0 (CH3OCH2
-
CH2OCH2O),103.8 (C-30), 108.1 (C-50), 114.7 (C-10), 132.4 (C-60),163.5
(C-2), 164.8 (C-20), 202.8 (C-1) ppm. MS m/z (ESI, %): 241 ([MþH]þ,
100), 263 ([MþNa]þ, 64). HRMS (ESI) m/z calcd. for C12H17O5
[MþH]þ 241.1071; found 241.1073.
4.6. Synthesis of 1-(2-hydroxyphenyl)butane-1,3-diones/(Z)-3-
hydroxy-1-(2-hydroxyphenyl)but-2-en-1-one (4a-4d)
Sodium hydride (536 mg, 22.3 mmol) was added to a solution of
the appropriate 2-acetylphenyl acetate (3a-3d) (14.9 mmol) in dry
THF (30 mL). The mixture was stirred under reflux for 2 h, under
nitrogen atmosphere. After that period, the solution was poured
into ice (50 g) and water (50 mL), and the pH was adjusted to 5e6
with diluted hydrochloric acid. The mixture was vigorously stirred
for 15 min. The aqueous layer was extracted with dichloromethane
(3 ꢂ 50 mL) and the solvent was evaporated to dryness. The residue
was purified by silica gel column chromatography using dichloro-
methane as eluent giving 1-(2-hydroxyphenyl)butane-1,3-diones/
(Z)-3-hydroxy-1-(2-hydroxyphenyl)but-2-en-1-ones (4a-4d) in
good yields.
4.5. Synthesis of 2-acetylphenyl acetates (3a-3d)
Acetyl chloride (5.2 mL, 73.4 mmol) was added to a solution of
the appropriate acetophenone (2a-d) (36.7 mmol) in dry pyridine
(50 mL). The solution was stirred under nitrogen atmosphere at
room temperature for 2 h. After that period, the solution was
poured into ice (100 g) and water (100 mL), and the pH was
adjusted to 4 with diluted hydrochloric acid. The mixture was
vigorously stirred for 15 min and the aqueous layer extracted with
dichloromethane (3 ꢂ 100 mL). The organic residue was dried over
anhydrous sodium sulfate and the solvent was evaporated to dry-
ness. The residue was purified by silica gel column chromatography
using dichloromethane as eluent giving the 2-acetylphenyl acetates
(3a-3d) in excellent yields.
4.6.1. 1-(2-Hydroxyphenyl)buta-1,3-dione (25%)/(Z)-3-Hydroxy-1-
(2-hydroxyphenyl)but-2-en-1-one (75%) (4a)
(1.43 g, 54%), m. p. 94e96 ꢃC. 1H NMR (300 MHz, CDCl3):
Diketone form
d 2.32 (s, 3H, CH3), 4.11 (s, 2H, H-2), 7.90 (dd, 1H, H-
4.5.1. 2-Acetylphenyl acetate (3a)
60, J 7.8, 1.5 Hz), 11.94 (s, 1H, 20-OH); Enolic form
d 2.15 (s, 3H, CH3),
(6.15 g, 94%), m.p. 83e85 ꢃC. 1H NMR (300 MHz, CDCl3):
d
2.35 (s,
6.18 (s, 1H, H-2), 6.88 (ddd, 1H, H-50, J 7.8, 7.7, 1.1 Hz), 6.97 (dd, 1H,
H-30, J 8.1, 1.1 Hz); 7.44 (ddd, 1H, H-40, J 8.1, 7.7, 1.6 Hz); 7.64 (dd, 1H,
H-60, J 7.8, 1.6 Hz); 12.08 (s, 1H, 20-OH); 14.98 (s, 1H, 3-OH) ppm; 13C
3H, 20-OCOCH3), 2.56 (s, 3H, H-2), 7.12 (dd, 1H, H-30, J 7.9, 1.1 Hz),
7.33 (ddd, 1H, H-50, J 7.7, 7.6, 1.1 Hz), 7.54 (ddd, 1H, H-40, J 7.9, 7.6,
1.7 Hz), 7.82 (dd, 1H, H-60, J 7.7, 1.7 Hz) ppm; 13C NMR (75 MHz,
NMR (125 MHz, CDCl3): Diketone form
d
30.6 (CH3), 54.5 (C-2),
CDCl3):
d
21.2 (20-OCOCH3), 29.3 (C-2), 123.8 (C-30), 126.0 (C-50),
101.2 (C-50), 119.3 (C-10), 130.7 (C-60), 137.3 (C-40), 162.8 (C-20), 199.7
130.3 (C-60), 130.6 (C-10), 133.4 (C-40), 149.0 (C-20), 169.5 (20-
OCOCH3), 197.6 (C-1) ppm. MS m/z (EI, %): 178 ([M]þ$, 4), 136 (65),
121 (100), 107 (4), 93 (8), 92 (9), 77 (7), 65 (11), 63 (8), 51 (4). Anal.
calcd for C10H10O3: C 67.41, H 5.66; found: C 67.36, H 5.90%
(C-3), 201.4 (C-1); Enolic form d
22.8 (CH3), 95.4 (C-2), 118.7 (C-30),
119.0 (C-50), 119.4 (C-10), 128.5 (C-60), 135.7 (C-40), 162.5 (C-20), 183.0
(C-3), 195.5 (C-1) ppm; MS m/z (EI, %): 178 ([M]þ$, 8), 163 (20), 160
(15), 121 (100), 120 (9), 92 (8); Anal. calcd for C10H10O3: C 67.41, H
5.66; found: C 67.38, H 5.73%.
4.5.2. 2-Acetyl-3-methoxyphenyl acetate (3b)
(6.65 g, 87%), m.p. 76e77 ꢃC. 1H NMR (300 MHz, CDCl3):
d
2.23
4.6.2. 1-(2-Hydroxy-6-methoxyphenyl)buta-1,3-dione (50%)/(Z)-3-
Hydroxy-1-(2-hydroxy-6-methoxyphenyl)but-2-en-1-one (50%)
(4b)
(s, 3H, 20-OCOCH3), 2.49 (s, 3H, H-2), 3.85 (s, 3H, OCH3), 6.71 (dd,1H,
H-30, J 8.2, 0.6 Hz), 6.82 (dd, 1H, H-50, J 8.2, 0.6 Hz), 7.34 (t, 1H, H-40, J
8.2 Hz) ppm; 13C NMR (75 MHz, CDCl3):
d
20.7 (20-OCOCH3), 31.5 (C-
(1.74 g, 56%), m. p. 91e92 ꢃC. 1H NMR (300 MHz, CDCl3): Dike-
2), 55.8 (OCH3), 108.6 (C-50), 115.0 (C-30), 123.9 (C-10), 131.0 (C-40),
147.6 (C-20),157.4 (C-60),169.2 (20-OCOCH3), 200.4 (C-1) ppm. MS m/
z (ESI, %): 231 ([MþNa]þ, 100); Anal. calcd for C11H12O4: C, 63.45; H,
5.81; found: C, 63.50; H, 5.94%
tone form
d 2.25 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.07 (s, 2H, H-2),
6.37 (dd, 1H, H-50, J 8.4, 0.8 Hz), 6.60 (dd, 1H, H-30, J 8.1, 0.9 Hz), 7.37
(t, 1H, H-40, J 8.4 Hz), 12.95 (s, 1H, 20-OH); Enolic form
d 2.12 (s, 3H,
CH3), 3.89 (s, 3H, OCH3), 6.40 (dd, 1H, H-50, J 8.1, 0.9 Hz), 6.57 (dd,
1H, H-30, J 8.4, 0.8 Hz), 6.62 (s, 1H, H-2), 7.30 (t, 1H, H-40, J 8.1 Hz),
12.49 (s, 1H, 20-OH), 15.30 (s, 1H, 3-OH) ppm; 13C NMR (75 MHz,
4.5.3. 2-Acetyl-5-methoxyphenyl acetate (3c)
Colourless oil. (7.33 g, 96%). 1H NMR (300 MHz, CDCl3):
d
2.33 (s,
CDCl3): Diketone form
d 30.1 (CH3), 55.6 (OCH3), 59.9 (C-2), 101.2
3H, 20-OCOCH3), 2.48 (s, 3H, H-2), 3.81 (s, 3H, OCH3), 6.60 (d, 1H, H-
(C-50),110.9 (C-10),111.1 (C-30),136.9 (C-40),160.7 (C-60),165.0 (C-20),
30, J 2.5 Hz), 6.79 (dd, 1H, H-50, J 8.4, 2.5 Hz), 7.81 (d, 1H, H-60, J
200.3 (C-3), 202.0 (C-1); Enolic form d 22.9 (CH3), 55.7 (OCH3), 101.6
8.4 Hz) ppm; 13C NMR (75 MHz, CDCl3):
d
20.7 (20-OCOCH3), 28.5
(C-50), 101.7 (C-2), 109.8 (C-10), 111.0 (C-30), 134.9 (C-40), 160.2 (C-60),
163.8 (C-20), 183.3 (C-3), 194.2 (C-1) ppm; MS m/z (ESI, %): 209
([MþH]þ, 10), 231 ([MþNa]þ, 100); Anal. calcd for C11H12O4: C
63.45, H 5.81; found: C 63.37, H 6.14%.
(C-2), 55.2 (OCH3),109.0 (C-30),111.1 (C-50),122.4 (C-10),132.2 (C-60),
151.0 (C-20), 163.3 (C-40), 169.0 (20-OCOCH3), 195.2 (C-1) ppm. MS m/
z (ESI, %): 231 ([MþNa]þ, 100); Anal. calcd for C11H12O4: C, 63.45; H,
5.81; found: C, 63.23; H, 5.79%.
4.6.3. 1-(2-Hydroxy-4-methoxyphenyl)buta-1,3-dione (31%)/(Z)-3-
Hydroxy-1-(2-hydroxy-4-methoxyphenyl)but-2-en-1-one (69%)
(4c)
4.5.4. 2-Acetyl-5-[(2-methoxyethoxy)methoxy]phenyl acetate (3d)
Colourless oil. (9.22 g, 89%). 1H NMR (300 MHz, CDCl3):
d 2.35 (s,
3H, 20-OCOCH3), 2.52 (s, 3H, H-2), 3.37 (s, 3H, CH3OCH2CH2OCH2O),
3.53e3.56 (m, 2H, CH3OCH2CH2OCH2O), 3.80e3.83 (m, 2H,
CH3OCH2CH2OCH2O), 5.31 (s, 2H, CH3OCH2CH2OCH2O), 6.79 (d, 1H,
(1.98 g, 64%), m. p. 62e63 ꢃC. 1H NMR (300 MHz, CDCl3):
Diketone form
d 2.24 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 3.96 (s, 2H, H-
2), 6.36 (dd, 1H, H-50, J 8.4, 0.8 Hz), 6.60 (d, 1H, H-30, J 0.9 Hz), 7.44

388
C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
(d, 1H, H-60, J 8.4 Hz), 12.40 (s, 1H, 20-OH); Enolic form
d
2.05 (s, 3H,
7.51 (t, 1H, H-7, J 8.3 Hz) ppm; 13C NMR (75 MHz, CDCl3):
d 19.7
CH3), 3.75 (s, 3H, OCH3), 5.97 (s, 1H, H-2), 6.40 (dd, 1H, H-50, J 8.1,
0.9 Hz), 6.57 (d, 1H, H-30, J 0.9 Hz),7.75 (d, 1H, H-60, J 8.4 Hz), 12.50
(s, 1H, 20-OH), 14.75 (s, 1H, 3-OH) ppm; 13C NMR (75 MHz, CDCl3):
(CH3), 56.2 (OCH3), 106.0 (C-6), 109.7 (C-8), 111.8 (C-3), 113.9 (C-4a),
133.3 (C-7), 158.3 (C-8a), 159.5 (C-5), 163.5 (C-2), 178.0 (C-4) ppm.
MS m/z (ESI, %): 191 ([MþH]þ, 100), 213 ([MþNa]þ, 90). HRMS-EI m/
z for C11H10O3 Mþ$ calcd 190.0630, found 190.0641
Diketone form
d
30.0 (CH3), 55.6 (OCH3), 59.9 (C-2), 101.2 (C-50),
101.3 (C-30), 105.4 (C-10), 136.9 (C-60), 160.7 (C-20), 164.9 (C-40), 197.4
(C-1), 201.8 (C-3); Enolic form
d
22.1 (CH3), 55.1 (OCH3), 94.6 (C-2),
4.7.3. 7-Methoxy-2-methyl-4H-chromen-4-one (5c)
101.0 (C-30), 107.3 (C-50), 111.6 (C-10), 129.9 (C-60), 164.8 (C-20), 165þ.4
(C-40), 181.0 (C-3), 193.9 (C-1) ppm; MS m/z (ESI, %): 209 ([MþH] ,
39), 231 ([MþNa]þ, 100). HRMS-EI m/z for C11H12O4 Mþ$ calcd
208.0736, found 208.0744.
(1.38 g, 57%), m.p. 113e114 ꢃC (Lit. 106e108 ꢃC [34]). 1H NMR
(300 MHz, CDCl3):
H-3), 6.81 (d, 1H, H-8, J 2.4 Hz), 6.94 (dd, 1H, H-6, J 8.8, 2.4 Hz), 8.08
(d, 1H, H-5, J 8.8 Hz) ppm; 13C NMR (75 MHz, CDCl3):
20.4 (CH3),
d 2.35 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 6.10 (s, 1H,
d
55.7 (OCH3), 100.1 (C-8), 110.3 (C-3), 113.9 (C-6), 117.3 (C-4a), 126.9
(C-5), 158.1 (C-8a), 163.8 (C-7), 165.5 (C-2), 177.7 (C-4) ppm. MS m/z
(ESI, %): 191 ([MþH]þ, 100). Anal. calcd for C11H10O3: C, 69.46; H,
5.30; found: C, 69.43; H, 5.63%
4.6.4. 1-(2-Hydroxy-4-methoxyethoxymethoxyphenyl)buta-1,3-
dione (46%)/(Z)-3-hydroxy-1-(2-hydroxy-4-
methoxyethoxymethoxyphenyl)but-2-en-1-one (54%) (4d)
Colourless oil. (1.51 g, 36%). 1H NMR (300 MHz, CDCl3): Diketone
form
d
2.30 (s, 3H, CH3), 3.38 (s, 3H, OCH3), 3.54e3.57 (m, 2H,
4.7.4. 7-Methoxyethoxymethoxy-2-methyl-4H-chromen-4-one
CH3OCH2CH2OCH2O), 3.79e3.83 (m, 2H, CH3OCH2CH2OCH2O), 4.02
(s, 2H, H-2), 5.30 (s, 2H, CH3CH2CH2OCH2O), 6.53e6.62 (m, 2H, H-
30, H-50), 7.54e7.58 (m, 1H, H-60), 12.30 (s, 1H, 20-OH); Enolic form
(5d)
Colourless oil. (1.21 g, 81%). 1H NMR (300 MHz, (CDCl3):
d 2.36 (s,
3H, CH3), 3.38 (s, 3H, CH3OCH2CH2OCH2O), 3.55e3.58 (m, 2H,
CH3OCH2CH2OCH2O), 3.83e3.86 (m, 2H, CH3OCH2CH2OCH2O), 5.36
(s, 2H, CH3OCH2CH2OCH2O), 6.11 (s, 1H, H-3), 7.04 (dd, 1H, H-6, J 8.7,
d
2.12 (s, 3H, CH3), 3.38 (s, 3H, OCH3), 3.54e3.57 (m, 2H,
CH3OCH2CH2OCH2O), 3.79e3.83 (m, 2H, CH3OCH2CH2OCH2O), 5.30
(s, 2H, CH3CH2CH2OCH2O), 6.05 (s,1H, H-2), 6.53e6.62 (m, 2H, H-30,
H-50), 7.54e7.58 (m, 1H, H-60), 12.42 (s, 1H, 20-OH), 14.83 (s, 1H, 3-
2.4 Hz), 7.07 (d, 1H, H-8, J 2.4 Hz), 8.08 (d, 1H, H-5, J 8.7 Hz) ppm. 13
C
NMR (75 MHz, CDCl3):
d 20.4 (CH3), 59.0 (CH3OCH2CH2OCH2O),
OH) ppm; 13C NMR (75 MHz, CDCl3): Diketone form
d
30.5 (CH3),
68.0 (CH3OCH2CH2OCH2O), 71.4 (CH3OCH2CH2OCH2O), 93.2
(CH3OCH2CH2OCH2O), 103.0 (C-8), 110.2 (C-3), 115.0 (C-6), 118.1 (C-
4a), 126.9 (C-5), 157.8 (C-8a), 161.2 (C-7), 165.8 (C-2), 177.7 (C-4)
ppm. MS m/z (ESI, %): 265 ([MþH]þ, 82), 287 ([MþNa]þ, 83), 551
([2M þ Na]þ, 100). HRMS (ESI) m/z calcd. for C14H17O5 [MþH]þ
265.1076; found 265.1068.
54.5 (C-2), 59.1 (OCH3), 68.2 (CH3OCH2CH2OCH2O), 71.5
(CH3OCH2CH2OCH2O), 93.0 (CH3OCH2CH2OCH2O), 103.9 (C-50),
108.7 (C-30), 112.9 (C-10), 132.5 (C-60), 164.2 (C-20), 165.4 (C-40),
200.0 (C-1); Enolic form
(CH3CH2CH2OCH2O), 71.5
d
22.6 (CH3), 59.1 (OCH3), 68.2
(CH3CH2CH2OCH2O), 93.0
(CH3CH2CH2OCH2O). 95.0 (C-2), 104.2 (C-50), 108.2 (C-30), 112.9 (C-
10), 130.2 (C-60), 163.1 (C-20), 164.9 (C-40), 181.5 (C-3), 194.4 (C-1)
ppm. MS m/z (ESI, %): 283 ([MþH]þ, 17), 305 ([MþNa]þ, 53), 587
([2M þ Na]þ, 16). HRMS (ESI) m/z calcd. for C14H19O6 [MþH]þ
283.1176; found 283.1176.
4.7.5. 7-Hydroxy-2-methyl-4H-chromen-4-one (6)
(1.99 g, 89%), m.p. 246e247 ꢃC. 1H NMR (300 MHz, CDCl3):
d 2.33
(s, 3H, CH3), 6.09 (s, 1H, H-3), 6.80 (d, 1H, H-8, J 2.2 Hz), 6.88 (dd, 1H,
H-6, J 8.7, 2.2 Hz), 7.83 (d,1H, H-5, J 8.7 Hz),10.72 (s, 1H, 7-OH) ppm;
13C NMR (75 MHz, CDCl3):
d 19.9 (CH3), 102.1 (C-8), 109.5 (C-3),
4.7. Synthesis of 2-methyl-4H-chromen-4-ones (5a-5c, 6)
114.7 (C-6), 115.8 (C-4a), 126.5 (C-5), 157.7 (C-8a), 162.4 (C-7), 165.9
(C-2), 176.2 (C-4) ppm. MS m/z (ESI, %): 177 ([MþH]þ, 100), 199
([MþNa]þ, 15). HRMS (ESI) m/z calcd. for C10H9O3 [MþH]þ
177.0546; found 177.0548
A catalytic amount of p-toluenesulfonic acid monohydrate (p-
TSA, 1.21 g, 6.35 mmol) was added to a solution of the appropriate
1-(2-hydroxyphenyl)buta-1,3-dione/(Z)-3-hydroxy-1-(2-
hydroxyphenyl)but-2-en-1-one (4a-4d) (12.7 mmol) in DMSO
(20 mL). The mixture was stirred at 100 ꢃC under nitrogen atmo-
sphere for 2 h. After that period the mixture was poured into ice
(100 g) and water (100 mL) and the resulting mixture extracted
with ethyl ether (3 ꢂ 100 mL), being organic layer dried over
anhydrous sodium sulfate. The solvent was evaporated to dryness
and the residue purified by silica gel column chromatography using
dichloromethane as eluent giving 2-methyl-4H-chromen-4-ones
(5a-5c, 6) in very good yields.
4.8. Synthesis of 4-iodo-1,2-dimethoxybenzene (8)
To
a stirred solution of 1,2-dimethoxybenzene 7 (1 mL,
7.85 mmol) in acetonitrile (5 mL) was added N-iodosuccinimide
(NIS) (1.94 g, 8.63 mmol) and trifluoroacetic acid (TFA) (0.18 mL,
2.35 mmol). The mixture was stirred for 2 h at 80 ꢃC. After that
period the resulting mixture was poured into ice (20 g) and water
(100 mL) and a saturated solution of sodium thiosulfate was added.
The aqueous layer was extracted with dichloromethane
(3 ꢂ 100 mL), dried over anhydrous sodium sulfate and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography using dichloromethane as eluent to
give the desired 4-iodo-1,2-dimethoxybenzene 8 in excellent yield
(1.97 g, 95%). This compound showed spectroscopic and analytical
data identical to one previously reported [37].
4.7.1. 2-Methyl-4H-chromen-4-one (5a)
(1.73 g, 85%), m.p. 69e71 ꢃC (Lit. 55e56 ꢃC [36]). 1H NMR
(300 MHz, CDCl3):
(m, 2H, H-6, H-8), 7.65 (ddd, 1H, H-7, J 8.6, 7.1, 1.6 Hz), 8. 19 (dd, 1H,
H-5, J 8.0,1.6 Hz) ppm; 13C NMR (75 MHz, CDCl3):
20.6 (CH3), 110.5
d 2.40 (s, 3H, CH3), 6.18 (s, 1H, H-3), 7.36e7.44
d
(C-3), 117.8 (C-8), 123.5 (C-4a), 124.9 (C-6), 125.6 (C-5), 133.5 (C-7),
150.1 (C-8a), 166.2 (C-2), 207.0 (C-4) ppm. MS m/z (ESI, %): 161
([MþH]þ, 100), 183 ([MþNa]þ, 23). HRMS (ESI) m/z calcd. for
4.9. Synthesis of (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde (9)
C
10H9O2 [MþH]þ 161.0597; found 161.0599
To a stirred solution of 1-iodo-3,4-dimethoxybenzene 8 (0.132 g,
0.5 mmol) in DMF (2.0 mL) were added acrolein diethyl acetal
(0.229 mL,1.5 mmol), nBu4NOAc (0.302 g,1.0 mmol), K2CO3 (0.104 g,
0.75 mmol), and Pd(OAc)2 (0.003 g, 0.015 mmol) and the mixture
was stand for 4 h at 90 ꢃC. After that period, the mixture was cooled,
hydrochloric acid 2 N was slowly added and the reaction was stirred
4.7.2. 5-Methoxy-2-methyl-4H-chromen-4-one (5b)
(1.69 g, 70%), m.p. 97e98 ꢃC (Lit. 97e98 ꢃC [36]). 1H NMR
(300 MHz, CDCl3):
d 2.30 (s, 3H, CH3), 3.96 (s, 3H, OCH3), 6.07 (s, 1H,
H-3), 6.78 (dd, 1H, H-6, J 8.3, 0.6 Hz), 6.97 (dd, 1H, H-8, J 8.3, 0.6 Hz),

C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
389
at room temperature for 10 min. Then, the mixture was diluted
4.10.3. 2-[(1E,3E)-4-(3,4-Dimethoxyphenyl)buta-1,3-dien-1-yl]-7-
with ethyl ether (50 mL) and washed with water (3 ꢂ 50 mL). The
organic layer was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography with dichloromethane as eluent
to give the desired (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde 9 in
good yield (77 mg, 80%).
methoxy-4H-chromen-4-one (10c)
(337 mg, 77%), m. p. 187e189 ꢃC. 1H NMR (300 MHz, CDCl3):
3.91 (s, 3H, 7-OCH3), 3.92 (s, 3H, 40-OCH3), 3.93 (s, 3H, 30-OCH3),
d
6.16 (s, 1H, H-3), 6.32 (d, 1H, H-
H-
), 6.86 (d, 1H, H-50, J 9.0 Hz), 6.88 (d, 1H, H-8, J 2.4 Hz), 6.93 (dd,
1H, H-6, J 8.8, 2.4 Hz), 7.01 (br s, 1H, H-20), 7.03 (d, 1H, H-60, J 9.0 Hz),
7.35 (ddd, 1H, H- , J 15.2, 7.7, 2.4 Hz), 8.07 (d,1H, H-5, J 8.8 Hz) ppm;
13C NMR (75 MHz, CDCl3):
55.7, 55.8, 55.9 (3 ꢂ OCH3), 100.2 (C-8),
108.7 (C-20), 109.8 (C-3), 111.1 (C-50), 113.8 (C-6), 117.9 (C-4a), 121.0
(C-60), 122.5 (C- ), 126.9 (C-5), 129.3 (C-10) 137.0 (C-
), 125.2 (C- ),
138.6 (C-
a, J 15.2 Hz), 6.78e6.90 (m, 2H, H-g,
d
b
4.9.1. (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde (9)
d
M.p. 80e82 ꢃC. 1H NMR (300 MHz, CDCl3):
d
3.93 (s, 3H, 3-
, J 15.8, 7.8 Hz), 6.91
(d, 1H, H-5, J 8.3 Hz), 7.08 (d, 1H, H-2, J 2.0 Hz), 7.17 (dd, 1H, H-6, J
8.3, 2.0 Hz), 7.42 (d, 1H, H- , J 15.8 Hz), 9.66 (d, 1H, CHO, J 7.8 Hz)
ppm. 13C NMR (75 MHz, CDCl3):
55.8 and 55.9 (3,4-OCH3), 109.7
(C-2), 111.0 (C-5), 123.3 (C-6), 126.6 (C- ), 126.9 (C-1), 149.3 (C-3),
OCH3), 3.94 (s, 3H, 4-OCH3), 6.62 (dd, 1H, H-
a
a
g
b
d
), 149.1 (C-30), 149.9 (C-40), 157.6 (C-8a), 161.6 (C-2), 164.0
b
(C-7), 177.7 (C-4) ppm. MS m/z (ESI, %): 365 ([MþH]þ, 100), 387
([MþNa]þ, 28). Anal. calcd for C22H20O5: C 72.51, H 5.53; found: C
72.89, H 5.70%.
d
a
151.9 (C-4), 152.8 (C-b
), 193.5 (CHO) ppm. MS m/z (ESIþ, %): 193
([MþH]þ, 95), 215 ([MþNa]þ, 100). HRMS (ESIþ) m/z calcd. for
4.10.4. 7-[(2-Methoxyethoxy)methoxy]-2-[(1E,3E)-4-(3,4-
C
11H13O3 [MþH]þ 193.0859; found 193.0855.
dimethoxyphenyl)buta-1,3-dien-1-yl]-4H-chromen-4-one (10d)
Orange oil. (395 mg, 75%). 1H NMR (300 MHz, CDCl3):
d 3.40 (s,
4.10. Synthesis of 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-
dien-1-yl]-4H-chromen-4-ones (10a-10d)
3H, CH3OCH2CH2OCH2O), 3.58e361 (m, 2H, CH3OCH2CH2OCH2O),
3.86e3.89 (m, 2H, CH3OCH2CH2OCH2O), 3.92 (s, 3H, 40-OCH3), 3.95
(s, 3H, 30-OCH3), 5.40 (s, 2H, CH3OCH2CH2OCH2O), 6.19 (s, 1H, H-3),
Sodium (0.11 g, 4.8 mmol) was gradually added to dry ethanol
(5 mL). The appropriate 2-methyl-4H-chromen-4-one 5a-5d
(1.2 mmol) and (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde
6.32 (d, 1H, H-
7.02e7.08 (m, 3H, H-20,60, H-6), 7.18 (d, 1H, H-8, J 2.3 Hz), 7.36e7.45
(m, 1H, H-
), 8.09 (d, 1H, H-5, J 8.8 Hz) ppm; 13C NMR (75 MHz,
CDCl3):
55.8 (30-OCH3), 55.9 (40-OCH3), 59.0 (CH3OCH2CH2O-
a, J 15.2 Hz), 6.85e9.93 (m, 3H, H-g, H-d
, H-50),
9
b
(0.29 g, 1.5 mmol) were added and the reaction mixture allowed to
stand at room temperature for 12 h. The solution was then poured
into ice (20 g) and water (30 mL) and the pH adjusted to 4 with
diluted hydrochloric acid. The obtained solid was removed by
filtration, taken in dichloromethane (50 mL) and purified by silica
gel column chromatography using dichloromethane as eluent. The
solvent was removed to dryness and the residue was recrystallized
from ethanol to provide the 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)
buta-1,3-dien-1-yl]-4H-chromen-4-ones 10a-10d, in moderate to
good yields.
d
CH2O), 68.1 (CH3OCH2CH2OCH2O), 71.4 (CH3OCH2CH2OCH2O), 93.3
(CH3OCH2CH2OCH2O), 102.9 (C-8), 108.8 (C-20), 109.8 (C-3), 111.1 (C-
50), 115.1 (C-6), 118.7 (C-4a), 121.1 (C-60), 122.4 (C-
a
), 125.2 (C-
g),
126.9 (C-5), 129.4 (C-10), 137.4 (C- ), 149.1 (C-30), 150.0
b), 138.9 (C-d
(C-40), 157.4 (C-8a), 161.5 (C-7), 161.8 (C-2), 177.9 (C-4) ppm. MS m/z
(ESI, %): 439 ([MþH]þ, 100), 461 ([MþNa]þ, 10). HRMS (ESI) m/z
calcd. for C25H27O7 [MþH]þ 439.1757; found 439.1736.
4.11. Synthesis of 1-(3,4-dimethoxyphenyl)-9H-xanthen-9-ones
(11a-11d)
4.10.1. 2-[(1E,3E)-4-(3,4-Dimethoxyphenyl)buta-1,3-dien-1-yl]-
4H-chromen-4-one (10a)
Iodine (18 mg, 0.07 mmol) was added to a solution of the
appropriate chromone 10a-10d (0.35 mmol) in 1,2,4-
trichlorobenzene (5 mL) and the mixture was refluxed for 48 h.
After this period, the solution was directly purified by silica gel
column chromatography using light petroleum ether as eluent to
remove the 1,2,4-trichlorobenzene and then dichloromethane to
recover the desired product. The solvent was evaporated to dryness
and the residue was recrystallized from ethanol to give the 1-(3,4-
dimethoxyphenyl)-9H-xanthen-9-ones 11a-11d, in moderate
yields.
(249 mg, 62%), m. p. 146e147 ꢃC. 1H NMR (300 MHz, CDCl3):
d
3.83 (s, 3H, 40-OCH3), 3.86 (s, 3H, 30-OCH3), 6.14 (s, 1H, H-3), 6.20
(d, 1H, H-
(m, 2H, H-20, H-60), 7.24e7.32 (m, 2H, H-
8.5, 0.9 Hz), 7.56 (ddd, 1H, H-7, J 8.5, 7.0, 1.7), 8.09 (dd, 1H, H-5, J 8.0,
1.7) ppm; 13C NMR (75 MHz, CDCl3): 55.5 (30-OCH3), 55.6 (40-
OCH3), 108.5 (C-20), 109.5 (C-3), 110.8 (C-50), 117.4 (C-8), 120.9 (C-60),
122.1 (C- ), 123.7 (C-4a), 124.5 (C-6), 124.8 (C- ), 125.2 (C-5), 129.0
(C-10), 133.2 (C-7), 137.4 (C- ), 148.8 (C-30), 149.7 (C-40),
), 138.8 (C-
a
, J 15.2 Hz), 6.68e6.81 (m, 3H, H-
g, H- d
, H-50), 6.92e6.96
b, H-6) 7.38 (dd, 1H, H-8, J
d
a
g
b
d
155.6 (C-8a), 161.7 (C-2) 177.9 (C-4) ppm. MS m/z (ESI, %): 335
([MþH]þ, 100), 357 ([MþNa]þ, 25). Anal. calcd for C21H18O4: C
75.43, H 5.43; found: C 75.54, H 5.71%.
4.11.1. 1-(3,4-Dimethoxyphenyl)-9H-xanthen-9-one (11a)
(76 mg, 65%), m. p. 164.0e164.9 ꢃC. 1H NMR (300 MHz, CDCl3):
d
3.88 (s, 1H, 30-OCH3), 3.96 (s, 1H, 40-OCH3), 6.87e6.97 (m, 3H, H-20,
4.10.2. 5-Methoxy-2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-
dien-1-yl]-4H-chromen-4-one (10b)
H-50, H-60), 7.19 (dd, 1H, H-2, J 7.3, 1.2 Hz), 7.33 (ddd, 1H, H-7, J 7.8,
7.1, 1.1 Hz), 7.48 (d, 1H, H-5, J 8.7 Hz), 7.51 (dd, 1H, H-4, J 9.1, 1.2 Hz),
(332 mg, 76%), m. p. 173e174 ꢃC. 1H NMR (300 MHz, CDCl3):
7.66e7.73 (m, 2H, H-3, H-6), 8.20 (dd,1H, H-8, J 7.8,1.6 Hz) ppm; 13
C
d
3.91 (s, 3H, 40-OCH3), 3.94 (s, 3H, 30-OCH3), 3.98 (s, 3H, 5-OCH3),
NMR (75 MHz, CDCl3):
d
55.8 (2 ꢂ OCH3), 110.4 (C-50), 112.2 (C-20),
6.16 (s, 1H, H-3), 6.27 (d, 1H, H-
a
, J 15.2 Hz), 6.77e6.89 (m, 4H, H-50,
117.4 (C-5), 117.5 (C-4), 119.4 (C-9a), 120.8 (C-60), 122.8 (C-8a), 123.8
(C-7), 126.9 (C-8), 127.4 (C-2), 133.4 (C-3), 134.4 (C-10), 134.5 (C-6),
144.0 (C-1), 148.1 (C-40), 148.1 (C-30), 155.3 (C-4b), 157.3 (C-4a),
176.9 (C-9) ppm. MS m/z (ESI, %): 333 ([MþH]þ, 75), 355 ([MþNa]þ,
100). HRMS-EI m/z for C21H16O4 Mþ$ calcd 332.1049, found
332.1041.
H-6, H-g, H-d
), 7.03e7.08 (m, 3H, H-20, H-60, H-8), 7.33 (ddd, 1H, H-
b
, J 15.2, 7.6, 2.5 Hz), 7.54 (t, 1H, H-7, J ¼ 8.4 Hz) ppm; 13C NMR
(125 MHz, CDCl3):
d
55.8 (30-OCH3), 55.9 (40-OCH3), 56.4 (5-OCH3),
106.2 (C-6), 108.8 (C-20), 109.9 (C-8), 111.1 (C-50), 111.5 (C-3), 114.6
(C-4a), 121.0 (C-60), 122.1 (C- ), 129.4 (C-10), 133.5 (C-7),
), 125.2 (C-
136.9 (C- ), 138.5 (C-
a
g
b
d
), 149.1 (C-30), 149.9 (C-40), 158.0 (C-8a), 159.7
(C-5), 159.7 (C-2), 178.3 (C-4) ppm. MS m/z (ESI, %): 365 ([MþH]þ,
4.11.2. 8-Hydroxy-1-(3,4-dimethoxyphenyl)-9H-xanthen-9-one
(11b)
100), 387 ([MþNa]þ, 38), 751 ([2M þ Na]þ, 89). Anal. calcd for
C
22H20O5: C 72.51, H 5.53; found: C 72.29, H 5.75%.
(43 mg, 35%), m. p. 167e168 ꢃC. 1H NMR (300 MHz, CDCl3):

390
C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
d
3.88 (s, 3H, 40-OCH3), 3.96 (s, 3H, 30-OCH3), 6.74 (dd, 1H, H-5, J 8.3,
4.12.1. 2-[(1E,3E)-4-(3,4-Dihydroxyphenyl)buta-1,3-dien-1-yl]-4H-
0.8 Hz), 6.88e6.97 (m, 4H, H-20, H-50, H-60, H-7), 7.18 (dd, 1H, H-2, J
7.6, 1.2 Hz), 7.47 (dd, 1H, H-4, J 8.2, 1.2 Hz), 7.56 (t, 1H, H-6, J 8.3 Hz),
7.69 (dd, 1H, H-3, J 8.2, 7.6 Hz), 12.60 (s, 1H, OH) ppm; 13C NMR
chromen-4-one (12a)
(65 mg, 79%), m. p. 254e256 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
d
6.37 (s, 1H, H-3), 6.55 (d, 1H, H-
8.1 Hz), 6.81e6.95 (m, 3H, H- , H-
, H-60), 6.99 (d,1H, H-20, J 2.0 Hz),
7.43e7.52 (m, 2H, H-6, H- ), 7.66 (d,1H, H-8, J 8.4 Hz), 7.80 (ddd, 1H,
H-7, J 8.4, 7.0, 1.6 Hz), 8.00 (dd, 1H, H-5, J 7.8, 1.6 Hz), 9.11 (s, 1H, 40-
OH), 9.42 (s, 1H, 30-OH) ppm; 13C NMR [75 MHz, (CD3)2SO]:
108.9
(C-3), 113.8 (C-20), 115.9 (C-50), 118.1 (C-8), 119.9 (C-60), 121.5 (C-
),
123.5 (C-
), 124.2 (C-5), 124.8 (C-6), 125.2 (C-10), 127.9 (C-10), 134.1
(C-7), 138.3 (C-
a
, J 15.2 Hz), 6.76 (d, 1H, H-50, J
(75 MHz, CDCl3):
d
55.7 (30-OCH3), 55.9 (40-OCH3), 106.4 (C-50),
g
d
109.4 (C-8a), 110.3 (C-5), 110.4 (C-7), 112.4 (C-20), 117.3 (C-4), 117.9
(C-9a), 120.7 (C-60), 127.8 (C-2), 133.9 (C-10) 134.2 (C-3), 136.4 (C-6),
143.7 (C-1), 148.0 (C-40), 148.4 (C-30), 155.5 (C-4b), 157.3 (C-4a),
161.9 (C-8), 182.5 (C-9) ppm. MS m/z (ESI, %): 349 ([MþH]þ, 73), 371
([MþNa]þ, 100). Anal. calcd for C21H16O5: C 72.41, H 4.63; found: C
72.23, H 4.76%.
b
d
a
g
b
), 139.9 (C-d
), 145.6 (C-30), 147.1 (C-40), 155.5 (C-9),
162.1 (C-2), 176.8 (C-4) ppm. MS m/z (ESI, %): 307 ([MþH]þ, 100),
353 ([Mþ2Na]þ, 48). HRMS (ESI) m/z calcd. for C19H15O4 [MþH]þ
307.0970; found 307.0958.
4.11.3. 6-Methoxy-1-(3,4-dimethoxyphenyl)-9H-xanthen-9-one
(11c)
(53 mg, 42%), m. p. 189e190 ꢃC. 1H NMR (300 MHz, CDCl3):
4.12.2. 5-Hydroxy-2-[(1E,3E)-4-(3,4-dihydroxyphenyl)buta-1,3-
dien-1-yl]-4H-chromen-4-one (12b)
d
3.88 (s, 3H, 40-OCH3), 3.90 (s, 3H, 6-OCH3), 3.94 (s, 3H, 30-OCH3),
6.84e6.95 (m, 5H, H-20, H-50, H-60, H-5, H-7), 7.15 (dd, 1H, H-2, J 7.7,
1.2 Hz), 7.44 (dd, 1H, H-4, J 8.2, 1.2 Hz), 7.62 (dd, 1H, H-3, J 8.2,
7.7 Hz), 8.09 (d, 1H, H-8, J 9.0 Hz) ppm; 13C NMR (75 MHz, CDCl3):
(65 mg, 75%), m. p. 269e270 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
d
6.43 (s, 1H, H-3), 6.54 (d, 1H, H-
8.0 Hz), 6.79 (d, 1H, H-6, J 8.2 Hz), 6.84e6.95 (m, 3H, H-60, H-
7.00 (d, 1H, H-20, J 1.8 Hz), 7.08 (d, 1H, H-8, J 8.2 Hz), 7.53 (dd, 1H, H-
, J 15.3, 9.9 Hz), 7.65 (t, 1H, H-7, J 8.2 Hz), 9.08 (s, 1H, 40-OH), 9.41 (s,
1H, 30-OH), 12.77 (s, 1H, 5-OH) ppm; 13C NMR [75 MHz, (CD3)2SO]:
107.1 (C-8), 107.3 (C-3), 110.1 (C-10), 110.8 (C-6), 113.9 (C-20), 115.9
(C-50), 120.1 (C-60), 120.7 (C- ), 127.7 (C-10), 135.8 (C-7),
), 124.0 (C-
139.7 (C- ), 140.9 (C-
a
, J 15.3 Hz), 6.76 (d, 1H, H-50, J
g, H-d),
d
55.69, 55.70, 55.74 (6.30,40-OCH3), 99.5 (C-5), 110.2 (C-50), 112.1 (C-
20), 112.9 (C-7), 116.6 (C-8a), 117.2 (C-4), 119.3 (C-9a), 120.7 (C-60),
127.3 (C-2), 128.4 (C-8) 132.7 (C-3), 134.5 (C-10), 143.8 (C-1), 147.9
(C-40), 148.0 (C-30),157.0 (C-4b),157.2 (C-4a),164.7 (C-6), 175.9 (C-9)
ppm. MS m/z (ESI, %): 363 ([MþH]þ, 60), 385 ([MþNa]þ, 100), 747
([2M þ Na]þ, 76). Anal. calcd for C22H18O5: C 72.92, H 5.01; found: C
72.97, H 5.24%.
b
d
a
g
b
d
), 145.6 (C-30), 147.3 (C-40), 155.7 (C-9), 159.9
(C-5), 163.7 (C-2), 182.8 (C-4) ppm. MS m/z (ESI, %): 323 ([MþH]þ,
80), 345 ([MþNa]þ, 23). HRMS (ESI) m/z calcd. for C19H15O5 [MþH]þ
323.0919; found 323.0911.
4.11.4. 6-[(2-Methoxyethoxy)methoxy]-1-(3,4-dimethoxyphenyl)-
9H-xanthen-9-one (11d)
Yellow oil. (73 mg, 48%). 1H NMR (300 MHz, CDCl3):
d 3.39 (s, 3H,
4.12.3. 7-Hydroxy-2-[(1E,3E)-4-(3,4-dihydroxyphenyl)buta-1,3-
dien-1-yl]-4H-chromen-4-one (12c)
CH3OCH2CH2OCH2O), 3.56e3.59 (m, 2H, CH3OCH2CH2OCH2O),
3.85e3.88 (m, 2H, CH3OCH2CH2OCH2O), 3.88 (s, 3H, 40-OCH3), 3.94
(s, 3H, 30-OCH3), 5.39 (s, 2H, CH3OCH2CH2OCH2O), 6.86 (d, 1H, H-20,
J 2.1 Hz), 6.88 (dd, 1H, H-60, J 8.1, 2.1 Hz), 6.94 (d, 1H, H-50, J 8.1 Hz),
6.98 (dd,1H, H-7, J 8.9, 2.3 Hz), 7.11 (d,1H, H-5, J 2.3 Hz), 7.17 (dd,1H,
H-2, J 7.7, 1.1 Hz), 7.47 (dd, 1H, H-4, J 8.4, 1.1 Hz), 7.65 (dd, 1H, H-3, J
8.4, 7.7 Hz), 8.11 (d, 1H, H-8, J 8.9 Hz) ppm; 13C NMR (75 MHz,
(71 mg, 82%), m. p. 281e282 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
d
6.22 (s, 1H, H-3), 6.49 (d, 1H, H-
8.2 Hz), 6.80e6.94 (m, 5H, H-6, H-8, H-60, H-
20, J 1.9 Hz), 7.41 (dd, 1H, H-
, J 15.3, 9.4 Hz), 7.83 (d, 1H, H-5, J
9.3 Hz), 9.12 (s, 1H, 40-OH), 9.41 (s, 1H, 30-OH), 10.81 (s, 1H, 7-OH)
ppm; 13C NMR [75 MHz, (CD3)2SO]:
102.3 (C-8), 108.8 (C-3), 113.7
(C-20), 114.6 (C-6), 115.9 (C-50), 116.3 (C-4a), 119.8 (C-60), 121.7 (C-
),
), 145.6
a
, J 15.3 Hz), 6.75 (d, 1H, H-50, J
g
, H-d), 6.97 (d, 1H, H-
b
d
a
CDCl3):
(CH3OCH2CH2OCH2O), 71.5 (CH3OCH2CH2OCH2O), 93.3 (CH3OCH2
d
55.8 (30,40-OCH3), 59.1 (CH3OCH2CH2OCH2O), 68.2
124.2 (C-g b), 139.4 (C-d
), 126.5 (C-5), 127.9 (C-10), 137.5 (C-
-
(C-30), 147.0 (C-40), 157.3 (C-8a), 161.4 (C-2), 162.7 (C-7), 176.3 (C-4)
ppm. MS m/z (ESI, %): 323 ([MþH]þ, 35), 345 ([MþNa]þ, 27). HRMS
(ESI) m/z calcd. for C19H15O5 [MþH]þ 323.0919; found 323.0908.
CH2OCH2O), 102.5 (C-5), 110.3 (C-50), 112.1 (C-20), 114.0 (C-7), 117.4
(C-4), 117.5 (C-8a), 119.4 (C-9a), 120.8 (C-60), 127.4 (C-2), 128.5 (C-8),
133.0 (C-3), 134.5 (C-10), 143.9 (C-1), 148.0 and 148.1 (C-30,40), 156.8
(C-4b), 157.4 (C-4a), 162.2 (C-6), 176.1 (C-9) ppm. MS m/z (ESI, %):
437 ([MþH]þ,100), 459 ([MþNa]þ, 73), 895 ([2M þ Na]þ, 34). HRMS
(ESI): m/z calcd. for C25H25O7 [MþH]þ 437.1600; found 437.1611.
4.12.4. 1-(3,4-Dihydroxyphenyl)-9H-xanthen-9-one (13a)
(62 mg, 76%), m. p. 259e260 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
d
6.56 (dd, 1H, H-60, J 8.0, 2.1 Hz), 6.68 (d, 1H, H-20, J 2.1 Hz), 6.74 (d,
1H, H-50, J 8.0 Hz), 7.15 (dd, 1H, H-2, J 7.4, 0.8 Hz), 7.43 (dd, 1H, H-7, J
8.5, 7.2 Hz), 7.60 (dd, 1H, H-4, J 7.8, 0.8 Hz), 7.64 (d,1H, H-5, J 8.4 Hz),
7.79 (dd, 1H, H-3, J 7.8, 7.4 Hz), 7.84 (ddd, 1H, H-6, J 8.4, 7.2, 1.5 Hz),
8.02 (dd,1H, H-8, J 8.5,1.5 Hz), 8.87 (s,1H, 30-OH), 8.94 (s,1H, 40-OH)
4.12. Synthesis of 2-[(1E,3E)-4-(3,4-dihydroxyphenyl)buta-1,3-
dien-1-yl]-4H-chromen-4-ones (12a-12c) and 1-(3,4-
dihydroxyphenyl)-9H-xanthen-9-ones (13a-13c)
ppm; 13C NMR [75 MHz, (CD3)2SO]: 114.8 (C-50), 116.5 (C-20), 117.1
d
A solution of 1 M boron tribromide in dichloromethane
(2.5 equiv per methyl or MEM group to be removed) was added to a
solution of the appropriate 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)
buta-1,3-dien-1-yl]-4H-chromen-4-ones 10a-10c or 1-(3,4-
dimethoxyphenyl)-9H-xanthen-9-ones 11a-11c (0.27 mmol) in
freshly distilled dichloromethane (10 mL), under nitrogen atmo-
sphere at ꢁ78 ꢃC. The mixture was stirred at room temperature for
3e4 h. After that period, the mixture was slowly added of ice (50 g)
and water (100 mL) and vigorously stirred for 2 h to allow the
formation of a precipitate. The obtained solid was removed by
filtration, washed with water (3 ꢂ 50 mL) and light petroleum ether
(3 ꢂ 50 mL), and finally dried under vacuum to afford the expected
hydroxylated derivatives 12a-12c and 13a-13c, in good yields.
(C-4), 117.6 (C-5), 118.6 (C-9a), 119.7 (C-60), 122.2 (C-8a), 124.1 (C-7),
126.1 (C-8), 127.4 (C-2), 132.6 (C-10), 134.0 (C-3), 135.0 (C-6), 143.8
(C-1), 144.2 (C-30), 144.6 (C-40), 154.7 (C-4b), 156.8 (C-4a), 175.8 (C-
9) ppm. MS m/z (ESI, %): 305 ([MþH]þ,100), 327 ([MþNa]þ, 81), 343
([MþK]þ, 15), 631 ([2M þ Na]þ, 41). HRMS (ESI) m/z calcd. for
C
19H13O4 [MþH]þ 305.0814; found 305.0802.
4.12.5. 8-Hydroxy-1-(3,4-dihydroxyphenyl)-9H-xanthen-9-one
(13b)
(75 mg, 87%), m. p. 266e268 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
d
6.60 (dd, 1H, H-60, J 8.0, 2.1 Hz), 6.72 (d, 1H, H-20, J 2.1 Hz), 6.75 (d,
1H, H-50, J 8.0 Hz), 6.77 (dd,1H, H-7, J 8.4, 0.8 Hz), 7.05 (dd,1H, H-5, J
8.4, 0.8 Hz), 7.17 (dd, 1H, H-4, J 7.5, 1.1 Hz), 7.60 (dd, 1H, H-2, J 8.5,

C. Proença et al. / European Journal of Medicinal Chemistry 115 (2016) 381e392
391
1.1 Hz), 7.70 (t, 1H, H-6, J 8.4 Hz), 7.83 (dd, 1H, H-3, J 8.5, 7.5 Hz),
12.59 (s, 1H, 8-OH) ppm; 13C NMR [75 MHz, (CD3)2SO]:
106.7 (C-
assays were performed for the tested compounds (final concen-
d
trations of 6.25e400
mM) at the emission wavelength 528 20 nm
5), 108.9 (C-8a), 110.1 (C-7), 114.9 (C-50), 116.4 (C-20), 117.0 (C-2),
117.3 (C-9a), 119.7 (C-60), 127.8 (C-4), 132.3 (C-10), 135.0 (C-3), 137.1
(C-6), 143.7 (C-1), 144.3 (C-30), 144.8 (C-4’), 155.0 (C-4b), 156.9 (C-
4a), 161.1 (C-8), 182.2 (C-9) ppm; MS m/z (ESI, %): 321 ([MþH]þ,
100), 343 ([MþNa]þ, 47). HRMS (ESI) m/z calcd. for C19H13O5
[MþH]þ 321.0763; found 321.0752.
with excitation at 480
immediately after the plate introduction.
20 nm and the signals were measured
4.13.5. Singlet oxygen scavenging assay
The 1O2 was generated by the thermal decomposition at 37 ꢃC of
the previously synthesized NDPO2 [35]. The 1O2 scavenging activity
was measured by monitoring the oxidation of non-fluorescent DHR
to fluorescent rhodamine 123, in the presence of the tested com-
4.12.6. 6-Hydroxy-1-(3,4-dihydroxyphenyl)-9H-xanthen-9-one
(13c)
pounds (final concentrations of 0.3125e20
mM for compounds 12a-
(67 mg, 78%), m. p. 306e307 ꢃC. 1H NMR [300 MHz, (CD3)2SO]:
12c and 0.625e80 M for compounds 13a-c), after a 30 min incu-
m
d
6.53 (dd, 1H, H-60, J 8.0, 2.0 Hz), 6.64 (d, 1H, H-20, J 2.0 Hz), 6.72 (d,
bation period, according to a described procedure [29].
1H, H-50, J 8.0 Hz), 6.84e6.87 (m, 2H, H-5, H-7), 7.10 (d, 1H, H-2, J
7.5 Hz), 7.53 (d,1H, H-4, J 8.1 Hz), 7.72 (dd,1H, H-3, J 8.1, 7.5 Hz), 7.85
(d, 1H, H-8, J 9.3 Hz), 8.86 (s, 1H, 40-OH), 8.90 (s, 1H, 30-OH), 10.89 (s,
4.13.6. Peroxyl radical scavenging assay
The ROOꢀ scavenging activity was measured by monitoring the
fluorescence decay resulting from ROOꢀ-induced oxidation of
fluorescein and expressed as the ‘Oxygen Radical Absorbance Ca-
pacity’ (ORAC), as reported in the literature [38]. ROOꢀ was gener-
ated by thermal decomposition of AAPH at 37 ꢃC and the tested
1H, 6-OH) ppm; 13C NMR [75 MHz, (CD3)2SO]:
d 101.7 (C-5), 114.0
(C-7), 114.8 (C-50), 115.2 (C-8a), 116.6 (C-20), 117.0 (C-4), 118.7 (C-9a),
119.8 (C-60), 127.4 (C-2), 128.2 (C-8), 133.0 (C-10), 133.5 (C-3), 143.8
(C-1), 144.2 (C-30), 144.6 (C-40), 156.7 (C-4a), 156.9 (C-4b), 163.6 (C-
6), 174.8 (C-9) ppm. MS m/z (ESI, %): 321 ([MþH]þ, 25), 343
([MþNa]þ, 31). HRMS (ESI): m/z calcd. for C19H13O5 [MþH]þ
321.0763; found 321.0752.
compounds used at final concentrations of 0.5e10
concentrations of 0.156e5
each study.
mM. Trolox (final
mM) was used as the standard control in
4.13. ROS and RNS scavenging assays
4.13.7. Nitric oxide scavenging assay
The ꢀNO scavenging activity was measured by monitoring the
ꢀNO-induced oxidation of non-fluorescent DAF-2 to the fluꢀorescent
triazolofluorescein (DAF-2T), as previously reported [29]. NO was
generated by NOC-5, the tested compounds used at final concen-
4.13.1. General information
To perform all ROS and RNS-scavenging assays, the compounds
under study as well as the positive control, quercetin, were dis-
solved in DMSO, except for the HOCl scavenging assay (dissolved in
ethanol). The assays were undertaken at 37 ꢃC, except for super-
oxide radical scavenging assay, that was performed at room tem-
perature. Quercetin was used as positive control. In each assay, at
least four independent experiments were performed, using 5e7
concentrations in triplicate, to obtain the IC50 values, which was
calculated from the curves of percentage of inhibition versus com-
trations of 0.25e8
mM and the fluorimetric signal was detected after
a
30 min incubation period, at the emission wavelength
528 20 nm with excitation at 485 20 nm.
4.13.8. 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 reported [29]. ONOOꢁ was
synthesized as described before [39]. The fluorimetric signal
detected after a 2 min incubation period and the tested compounds
pound concentration, using the GraphPad Prism
5 software
(GraphPad Inc. La Jolla, CA). The results were expressed as the
percentage inhibition of the reactive specie-induced oxidation of
the probe.
with final concentrations of 0.156e5 mM. In a parallel set of ex-
A microplate reader (Synergy HT, BIO-TEK) for fluorescence,
absorbance in UVeVis and luminescence measurements, plus
temperature control capacity, was used for all of the ROS and RNS
scavenging assays.
periments, the assays were performed in the presence of 25 mM
NaHCO3 in order to simulate the physiological CO2 concentration
in vivo. This monitorization is important because, under physio-
logical conditions, the reaction between ONOOꢁ and CO2 is pre-
dominant, with a very fast rate constant (k2 ¼ 3e5.8 ꢂ 104 Mꢁ1 sꢁ1
)
4.13.2. Superoxide radical scavenging assay
[32].
Oꢀ2ꢁ was generated by the NADH/PMS/O2 system. The scav-
enging activity was determined by monitoring Oꢀ2ꢁ-induced
reduction of NBT, as previously reported [29]. The effects of the
Acknowledgements
tested compounds (final concentrations of 25e200
determined spectrophotometrically at 560 nm during 2 min.
mM) were
ꢀ
Sincere thanks are expressed to Faculdade de Farmacia da Uni-
ꢀ
versidade do Porto, Universidade de Aveiro, Instituto Politecnico de
~
^
Bragança, Fundaçao para a Ciencia e a Tecnologia (FCT, Portugal),
European Union, QREN, FEDER and COMPETE funding UCIBIO-
REQUIMTE (FCT UID/Multi/04378/2013) and QOPNA (FCT UID/QUI/
00062/2013) Research Units and also to the Portuguese National
4.13.3. Hydrogen peroxide scavenging assay
The H2O2 scavenging activity was measured using a previously
reported chemiluminescence procedure based on the H2O2-
induced oxidation of lucigenin [29]. The chemiluminescence signal
was recorded immediately after the plate introduction in the
microplate reader using the tested compounds at final concentra-
ꢀ
NMR Network (RNRMN). Helio M.T. Albuquerque and Marisa Frei-
tas acknowledge FCT for the financial support to their PhD (SFRH/
BD/86277/2012) and Pos-doc (SFRH/BPD/76909/2011) grants,
respectively, in the ambit of “QREN e POPH e Tipologia 4.1 e
tions of 31.25e1000 mM.
~
Formaçao Avançada”, co-sponsored by FSE and by national funds of
MCTES.
4.13.4. Hypochlorous acid scavenging assay
The HOCl was measured by monitoring the HOCl-induced
oxidation of DHR to rhodamine 123, as previously described [29].
HOCl was daily prepared by adjusting the pH of a 1% solution of
NaOCl to 6.2 with dropwise addition of 10% H2SO4. The fluorimetric
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