(E)-2-Styrylchromones as potential anti-norovirus agents

Article abstract of DOI:10.1016/j.bmc.2010.05.006

Human noroviruses (NoV) are now recognized as the most frequent cause of outbreaks and sporadic cases of acute gastroenteritis. Despite the significant economic impact and considerable morbidity of norovirus disease, no drug or vaccine is currently available to treat or prevent this disease, therefore the discovery of anti-norovirus drugs is urgent. In the present work, a total of 12 structure related chromone and (E)-2-styrylchromones were evaluated for their potential anti-norovirus activity using the murine norovirus (MNV) as a surrogate model for human NoV. From the 12 compounds studied, six (E)-2-styrylchromones were found to have with interesting anti-norovirus activity. The best compounds of the series were (E)-5-hydroxy-2-styrylchromone and (E)-4′-methoxy-2-styrylchromone with an IC₅₀ ≈ 7 μM. A first insight into the mechanism of action of these compounds was possible. An interesting relationship between the anti-norovirus activity and the chemical structure was observed. The present study points out that the (E)-2-styrylchromones skeleton is an important one which deserves to be developed and further explored as new antiviral drugs against NoV.

Full text of DOI:10.1016/j.bmc.2010.05.006

Bioorganic & Medicinal Chemistry 18 (2010) 4195–4201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(E)-2-Styrylchromones as potential anti-norovirus agents
Joana Rocha-Pereira a,b, Ricardo Cunha a, Diana C. G. A. Pinto c, Artur M. S. Silva c,
Maria São José Nascimento a,b,
*
a Serviço de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal
b CEQUIMED-UP, Faculdade de Farmácia, Universidade do Porto, Portugal
c Departamento de Química & QOPNA, Universidade de Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Human noroviruses (NoV) are now recognized as the most frequent cause of outbreaks and sporadic
cases of acute gastroenteritis. Despite the significant economic impact and considerable morbidity of
norovirus disease, no drug or vaccine is currently available to treat or prevent this disease, therefore
the discovery of anti-norovirus drugs is urgent.
In the present work, a total of 12 structure related chromone and (E)-2-styrylchromones were evaluated
for their potential anti-norovirus activity using the murine norovirus (MNV) as a surrogate model for
human NoV.
Received 8 January 2010
Revised 19 April 2010
Accepted 4 May 2010
Available online 7 May 2010
Keywords:
Norovirus
MNV
Antiviral activity
(E)-2-Styrylchromones
From the 12 compounds studied, six (E)-2-styrylchromones were found to have with interesting anti-
norovirus activity. The best compounds of the series were (E)-5-hydroxy-2-styrylchromone and (E)-40-
methoxy-2-styrylchromone with an IC50 ꢀ 7
lM. A first insight into the mechanism of action of these
compounds was possible. An interesting relationship between the anti-norovirus activity and the chem-
ical structure was observed. The present study points out that the (E)-2-styrylchromones skeleton is an
important one which deserves to be developed and further explored as new antiviral drugs against NoV.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
sic infectivity assay (plaque reduction assay) constitutes a good
strategy to discover active compounds against NoV and has been
Human noroviruses (NoV) are now recognized as the most fre-
quent cause of outbreaks and sporadic cases of acute gastroenteri-
tis.1,2 They affect people of all age groups and often occur in
crowded locations, usually causing a short-term, self-limiting dis-
ease. However, complications derived from severe dehydration
may occur in infants and the elderly because they are more sensitive
to volume depletion. Moreover norovirus infection has put healthy
people in intensive care and has been associated with chronic diar-
rhea among transplant patients.3 Despite the significant economic
impact and considerable morbidity of norovirus disease, no drug
or vaccine is currently available to treat or prevent this disease,
therefore the discovery of anti-norovirus drugs is urgent.
Little is known about the biology of human NoV, which is
mainly due to the absence of a cell culture system or small animal
model.4 Murine norovirus (MNV) is able to replicate in the murine
macrophage cell line RAW 264.7 and this system is considered to-
day the best surrogate model for human NoV.5,6 MNV is genetically
related to non-cultivable human NoV and it is likely that many fun-
damental mechanisms of replication and biochemical features are
conserved between these viruses.6 Therefore, screening chemical
compounds against the MNV/RAW 264.7 surrogate system by clas-
frequently used in disinfection and inactivation studies of NoV.7–9
The replication strategy of NoV is not well understood but its
family, Caliciviridae, shares many characteristics with the family
Picornaviridae.4,10,11 Both include ssRNA(+) virus that encode a
large polyprotein matured by proteolytic cleavage into a viral pro-
tease and a RNA-dependent RNA polymerase, among others. Thus,
it is possible that some inhibitors of picornavirus replication may
also have an effect on norovirus replication. Many flavonoids of
natural or synthetic origin have been shown to inhibit the replica-
tion of picornavirus, including poliovirus, coxsackievirus A/B and
rhinovirus.11–15 Recently, 2-styrylchromones have emerged as a
new class of flavonoid-type compounds with activity against rhi-
novirus, a picornavirus.16–18 Therefore, the aim of the present work
was to study a series of structure related chromones and (E)-2-sty-
rylchromones (Fig. 1) for their potential anti-norovirus activity,
establish a structure–activity relationship and get some insights
into the mechanism of action of the most active compounds.
2. Results
2.1. Synthesis
Chromone (2) were obtained by a Claisen condensation of the
* Corresponding author.
E-mail address: saojose@ff.up.pt (M. S. J. Nascimento).
appropriate 20-hydroxyacetophenones and methyl formate19 lead-
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.006

4196
J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
ing to the 2-hydroxy-4-chromanone (1), which was then dehy-
drated by treatment with a catalytic amount of iodine in refluxing
DMSO (Scheme 1). This dehydration method is currently used in
the synthesis of 2-substituted chromones20,21 and is now applied
for the first time to 2-unsubstituted chromone.
eneacetophenones with hydrogen peroxide (Scheme 4). (E)-3-
Methoxy-2-styrylchromones (17) was obtained by methylation of
(E)-3-hydroxy-2-styrylchromone (15) with methyl sulfate in alka-
line medium.18
(E)-2-Styrylchromones (4–9) have been prepared by aldol con-
densation/oxidative cyclization procedure22 (Scheme 2). The base-
catalysed aldol condensation of 20-hydroxyacetophenones with
cinnamaldehydes gave the corresponding 20-hydroxycinnamylid-
eneacetophenones (3) in good yields (85–93%). The oxidative cycli-
zation of (3) with a catalytic amount of iodine in refluxing DMSO
for half an hour yielded 2-styrylchromones (4–6) in good yields
(85–93%).23 However, the cyclodehydrogenation of cinnamylidene-
acetophenones bearing a 60-benzyloxy group (R1 = OBn, R2 = R3 = H)
lead to 5-hydroxy-2-styrylchromone (7) with a longer reflux time
(2 h). This means that it is possible to obtain the starting material
oxidative cyclization and further debenzylation of the formed chro-
mone (6) in one step, leading to 5-hydroxy-2-styrylchromone (7).24
In the oxidative cyclization of 20-hydroxycinnamylideneacetophe-
nones unsubstituted in the vinylic moiety, only the isomer (E)-2-sty-
rylchromones (4–7) were obtained, whereas in the case of 20-
2.2. Cytotoxicity of chromones and (E)-2-styrylchromones
Prior to evaluating the anti-norovirus activity of compounds it
was first necessary to establish which was their highest concentra-
tion which did not cause significant toxicity to RAW cells (MNTC).
Their potential cytotoxicity was determined by measuring the MTT
reduction capacity of RAW cells. Concentrations of compounds
associated with a toxicity <10% (MNTC90) were considered ideal
but those concentrations associated with a MTT reduction capacity
P70% (MNTC70) were still considered tolerable.27,28 Hence, the
MNTC90 and MNTC70 values of all the compounds were determined
together with their CC50 (concentration of compounds that causes
50% cytotoxicity) and are summarized in Table 1.
Results showed that compounds 8 and 12 had no significant
toxicity for RAW cells even at the highest concentration tested
(MNTC90 >150
ited a tolerable toxicity (MNTC70 >150
pounds presented relatively large margin of security that
enabled further testing for anti-norovirus activity at concentra-
tions of 150 M. On the contrary, RAW cells treated with 150
l
M), while compounds 2, 4, 5, 6, 7, 13 and 14 exhib-
hydroxycinnamylideneacetophenone bearing a
c
-methyl group
lM). Hence, all these com-
(R2 = CH3, R1 = R3 = H) both isomers (E)-2-
a-methylstyrylchromone
a
(8) and (Z)-2-a-methylstyrylchromone (9) were formed, the (E)
being obtained in higher proportion.23
l
lM
(E)-40-Methyl-2-styrylchromone (12) and (E)-40-methoxy-2-
styrylchromone (13) have been prepared by the Baker–Venkatar-
aman approach starting from 20-hydroxyacetophenone and
cinnamic acid derivatives (Scheme 3). 20-Cinnamoyloxyaceto-
phenones (10) were obtained from the reaction of the 20-hydroxy-
acetophenone with the appropriate cinnamoyl chlorides, prepared
in situ from cinnamic acids and phosphorous oxychloride. The Ba-
ker–Venkataraman rearrangement of (10) into the corresponding
1,3-diketones, which exists in solution as their enolic form (11),
was performed upon treatment with sodium hydroxide in DMSO
at room temperature. The treatment (11) with a mixture of DMSO
and p-toluenesulfonic acid led to their cyclodehydration yielding
the expected (E)-2-styrylchromone (12, 13).25 (E)-40-Hydroxy-2-
styrylchromone (14) was obtained by demethylation of (E)-40-
methoxy-2-styrylchromone (13) with boron tribromide (see Sec-
tion 5).
of compounds 15, 16 and 17 showed a different toxicity profile.
For these compounds, an acceptable percentage of viable cells
(>70%) was only observed with concentrations 675
pound 17 and 618.75 M for compounds 15 and 16.
Taken together, these results led us to consider 50
l
M for com-
l
lM as the
maximum concentration of all compounds to be tested for the
screening of antiviral activity, except for compounds 15 and 16
for which only concentrations 610 lM were considered.
2.3. Anti-norovirus activity of chromones and (E)-2-styryl-
chromones
The anti-norovirus activity of chromone (2) and (E)-2-styrylchr-
omones (4–8, 12–17) was evaluated by plaque reduction assay.
The IC50 of each compound studied together with their selectivity
index (SI) are summarized in Table 1.
The (E)-3-hydroxy-2-styrylchromones (15, 16) were obtained
by the Algar–Flynn–Oyamada type-reaction,26 which consists in
the oxidative cyclisation of the appropriate 20-hydroxycinnamylid-
Chromone 2 and (E)-2-styrylchromones 5, 6, and 14 showed no
anti-norovirus activity even when tested at the highest concentra-
tion of 50
lM. Compounds 15 and 16 also showed no antiviral
R2
O
O
O
R3
Chromone (2)
R1
O
E
R1 = R2 = R3 = H, ( )-2-Styrylchromone ( )
4
12
R
1 = R3 = H, R2 = CH , ( )-4'-Methyl-2-styrylchromone (
)
E
E
E
3
R1 = R3 = H, R2 = OCH , ( )-4'-Methoxy-2-styrylchromone (
)
13
3
14
R
R
1 = R3 = H, R2 = OH, ( )-4'-Hydroxy-2-styrylchromone (
)
O
O
1 = R3 = H, R2 = CH , ( )-4'-Chloro-2-styrylchromone ( )
5
E
3
R2
R
1 = C6H5CH2, R2 = R3 = H, ( )-5-Benzyloxy-2-styrylchromone ( )
6
E
R1
R
R
1 = OH, R2 = R3 = H, ( )-5-Hydroxy-2-styrylchromone ( )
7
8
E
1 = R2 = H, R3 = CH , ( )-α-Methyl-2-styrylchromone ( )
E
3
R1 = OH, R2 = H, (E)-3-Hydroxy-2-styrylchromone (
)
16
R
1 = OCH3, R2 = H, (E)-3-Methoxy-2-styrylchromone (
18
)
17
R
1 = OH, R2 = CH3, (E)-3-Hydroxy-α-methyl-2-styrylchromone (
)
Figure 1. Chemical structures of chromones (2) and (E)-2-styrylchromones (4–8, 12–17).

J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
4197
HCO2CH3
O
OH
O
I2, DMSO
Na (s)
(82%)
90ºC
(75%)
OH
O
O
O
(1)
Scheme 1. Synthesis of chromone (2).
(2)
CHO
R2
R1
O
R1
R3
R2
NaOH, H2O
+
MeOH
(85-93%)
OH
OH
O
(3)
R3
When R1 =OBn, R2=R3=H
I2 (catalytic), DMSO,
reflux, 2 h
I2 (catalytic), DMSO,
reflux, 30 min.
R3
O
R2
O
O
O
CH3
R1
O
(4) R1=R2=R3=H (85%)
OH
O
(5) R1=R2=H, R3=Cl (93%)
(6) R1=OBn, R2=R3=H (93%)
(8) R1=R3=H, R2=CH3 (85%)
(9) (11%)
(7) (81%)
Scheme 2. Synthesis of (E)-2-styrylchromones (4–9).
R
CO2H
O
R
POCl3, dry py
KOH
O
O
+
60ºC
DMSO
OH
O
OH
O
HO
(11)
p-Toluenesulfonic
(80-83%)
(85-94%)
R
(10)
acid, DMSO, 100ºC
R
OH
When R = OCH3
CH2Cl2 BBr3
-78ºC to room temp
(95%)
O
O
O
,
O
(12) R = CH3 (90%)
(13) R = OCH3 (88%)
(14)
Scheme 3. Synthesis of (E)-2-styrylchromones (12–14).
effect, but the maximum concentration tested was only 10
to their toxicity to RAW cells.
lM due
rylchromone (13) the most active compounds with IC50 values of
7.0 M and 7.4 M, respectively. These two compounds (7 and
l
l
However, an anti-norovirus activity was observed for (E)-2-sty-
rylchromones 4, 7, 8, 12, 13 and 17. Their antiviral effect ranged
13) exerted their anti-norovirus activity in a dose-dependent
way, presenting no significant cytotoxicity for effective concentra-
tions (Fig. 2). Consequently, they presented high selectivity index
(SI) values (>21.6 and >13.5, respectively).
from moderate (IC50 = 20–50
lM) to potent (IC50 <10 lM), being
(E)-5-hydroxy-2-styrylchromone (7) and (E)-40-methoxy-2-sty-

4198
J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
R
O
O
B
O
O
A
R
OH
O
3
( )
When R=H
OH
OCH3
(15) R = H (76%)
(16) R = CH3 (72%)
(17) (95%)
A. H2O2 (35%), NaOH/H2O, EtOH
B. (CH3)2SO4 K2CO3 acetone, reflux
,
,
Scheme 4. Synthesis of (E)-3-substituted-2-styrylchromones (15–17).
Table 1
Cytotoxicity and anti-norovirus activity of chromones (2) and (E)-2-styrylchromones (4–8, 12–17)
SId
a
b
b
c
Compounds
CC50
(lM)
MNTC70
(lM)
MNTC90
(lM)
IC50 (lM)
Chromone (2)
(E)-2-Styrylchromone (4)
(E)-40-Chloro-2-styrylchromone (5)
(E)-5-Benzyloxy-2-styrylchromone (6)
(E)-5-Hydroxy-2-styrylchromone (7)
>150*
>150
>150
>150
>150
>150
>150
>100
>150*
>150*
>150
>150
>150
>150
>150
>150
>100
>150*
18.75
18.75
75
37.5
75
75
>50* (29.9%)
17.7 6.5
>50 (7.6%)
>50 (39.4%)
7.0 0.7
—
>8.5
—
75
—
4.69
>150
>150
50
>21.6
>8.3
>6.3
>13.5
—
—
—
>4.0
(E)-
a
-Methyl-2-styrylchromone (8)
18.0 5.2
23.7 7.5
7.4 1.3
(E)-40-Methyl-2-styrylchromone (12)
(E)-40-Methoxy-2-styrylchromone (13)
(E)-40-Hydroxy-2-styrylchromone (14)
(E)-3-Hydroxy-2-styrylchromone (15)
<2.34* (86%)
2.34
>50* (21.1%)
>10 (6.8%)
>10 (40.8%)
37.9 6.3
52.1 10.6
46.7 8.7
>150
(E)-3-Hydroxy-
(E)-3-Methoxy-2-styrylchromone (17)
a-methyl-2-styrylchromone (16)
<2.34 (81%)
37.5
a
Results of CC50 (concentrations of compounds that cause 50% cytotoxicity when compared to control cells) show means SEM of 3–4 independent experiments
performed in duplicate.
b
MNTC70 and MNTC90 are maximum non-toxic concentrations (the highest concentration tested in which the MTT reduction capacity of RAW cells was at least 70% or 90%
when compared to control cells). When a >90% reduction was not achieved, results at the minimum concentration tested were reported in parentheses.
c
Results of IC50 (concentrations of compounds that reduced the plaque number by 50% when compared to control cells) show means SEM of 3–7 independent
experiments performed in duplicate. When a 50% reduction was not achieved, the results obtained at the maximum concentration tested (10 or 50 lM) were reported in
parentheses.
d
The selectivity index (SI) was the CC50/IC50
.
*
Results from two independent experiments performed in duplicate.
2.4. Effect of time of addition of compounds in the anti-
norovirus activity
after the viral infection and for a continuous period of 48 h. These re-
sults indicate that the studied compounds interfere more with steps
of viral replication that occur during the 48 h, rather than those
occurring during the 1 h of infection.
In an attempt to understand if compounds 7 and 13 interfere with
an early or a late step of norovirus replication cycle, two time-points
of compoundadditionweretestedand comparedwith standardcon-
ditions (Table 2). The anti-norovirus activity of compounds signifi-
cantly decreased (P <0.0001) when they were present only during
the 1 h of viral infection, according to our protocol. However, the po-
tent effect of these compounds remained when they were added
2.5. Anti-norovirus activity versus anti-rhinovirus activity of
(E)-2-styrylchromones
A review of published studies concerning the antiviral activity
of (E)-2-styrylchromones17,18 common to the present study was
(E)-4'-methoxy-2-styrylchromone
(E)-5-hydroxy-2-styrylchromone
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
1
10
100
0.1
1
10
100
Concentration (μM)
Concentration (μM)
Figure 2. Cytotoxicity (
) and anti-norovirus activity (
) of (E)-5-hydroxy-2-styrylchromone (7) and (E)-40-methoxy-2-styrylchromone (13).

J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
4199
Table 2
tionship between the anti-norovirus activity and the chemical
structure was observed. Hence, when comparing the IC50 of chro-
mone 2 (>50 lM) with (E)-2-styrylchromone 4 (17.7 lM), it is
Effect of time of addition of (E)-5-hydroxy-2-styrylchromone (7) and (E)-40-methoxy-
2-styrylchromone (13)
Compounds
IC50
(
l
M)
IC50
(
l
M)
IC50
(
l
M)
remarkable to note that the introduction of the (E)-2-styryl group
in the chromone moiety was responsible for the appearance of the
anti-norovirus effect. Moreover, the nature of the 40-substituent in
the 2-styrylchromone moiety influenced the potency of the anti-
norovirus effect as demonstrated when the activities of 2-styrylchr-
1 h + 48 ha
1 h onlyb
48 hc
(E)-5-Hydroxy-2-
6.9 0.4
39.0 3.8
8.2 1.3
8.4*
stryrylchromone (7)
(E)-40-Methoxy-2-
stryrylchromone (13)
6.9*
47.7*,
omones 12 and 13 were compared (IC50 23.7 lM vs 7.4 lM, respec-
tively). The 40-methoxy group showed to be the best substituent,
conferring to compound 13 an activity three times superior to the
40-methyl substituted compound 12. On the contrary, the 40-chloro
and 40-hydroxy substituents of compounds 5 and 14 were responsi-
ble for a loss of antiviral activity. It was also observed that the nature
of the 5-substituent of (E)-2-styrylchromones 6 and 7 was relevant
for the anti-norovirus activity. When comparing their IC50 with that
Results show means SEM of three independent experiments performed in
duplicate.
Effect in MNV replication of different times of exposure to compounds 7 and 13:
during the hour of viral infection plus 48 h in the overlay (standard conditions).
Only during the hour of viral infection.
Only for 48 h in the overlay.
Results from two independent experiments performed in duplicate.
A significant difference (P <0.0001) was observed relatively to the standard
a
b
c
*
conditions.
of (E)-2-styrylchromone 4 (17.7
droxy substituent of compound 7 was responsible for the appear-
ance of one of the most potent anti-norovirus effects (7.0 M)
while the 5-benzyloxy group in compound 6 was responsible for a
loss of antiviral activity (>50 M). Additionally, it was curious to
lM) we can observe that the 5-hy-
conducted and a comparison of the antiviral activities was at-
tempted (Table 3). As observed, some of these compounds showed
to be active against both norovirus and rhinovirus, while others
were only effective against one of these viruses. It was noticeable
that the most potent compound for rhinovirus (17) presented only
a modest anti-norovirus activity whereas one of the most active
compounds against NoV, 40-methoxy-2-stryrylchromone (13),
was considered by the authors to be inactive for HRV 14, although
it presented a slight antiviral effect against HRV 1B.17 These differ-
ent profiles of antiviral effect of 2-styrylchromones against these
two viruses probably reflect a different interaction of the com-
pounds with norovirus and rhinovirus.
l
l
note that the 3-hydroxy group of (E)-2-styrylchromones appears
to confer cytotoxicity to compounds 15 and 16, the only ones that
present this substituent and exhibit high toxicity (CC50 ꢀ 50
lM).
The potent activity presented by (E)-5-hydroxy-2-styrylchro-
mone (7) and (E)-40-methoxy-2-styrylchromone (13) justifies fur-
ther investigation of the mechanism of action underlying their
anti-norovirus effect. A first insight into the mechanism of action
of these compounds was reached by studying the effect of their
addition at different time-points, which revealed that compounds
7 and 13 interfere more with steps of virus life cycle that follow
the entrance of virus in cells. This may include either the blocking
of viral enzymes involved in RNA replication (e.g., RNA-dependent
RNA polymerase) or in proteolytic maturation, or an interference
with virus release. Inhibitors of viral polymerases have shown to
be related to antiproliferative drugs targeting cellular polymerase
in highly replicating tumor cells.29 In a previous study, our team
demonstrated that (E)-40-methoxy-2-styrylchromone was a potent
inhibitor of the growth of a variety of human tumor cell lines,
being much less efficient in inhibiting non-tumor human cells.30
This ability of preferentially suppressing highly replicating tumor
cells and the capacity of (E)-40-methoxy-2-styrylchromone (13)
to inhibit a late step of norovirus life cycle raise the possibility of
this compound targeting the polymerase of norovirus. However,
the present experimental data allows only this initial hypothesis
for the mechanism of action of these compounds. Additional stud-
ies are needed to unveil the specific viral target of these (E)-2-sty-
rychromones, which will include the generation of drug-resistant
virus and their molecular characterization.
3. Discussion
In the present work, a total of twelve structure related chro-
mone (2) and (E)-2-styrylchromones (4–8, 12–17) were evaluated
for their potential anti-norovirus activity using the human NoV
surrogate model MNV/RAW 264.7 cells. The choice of this class
of flavonoid-type compounds was based on the previously de-
scribed anti-rhinovirus activity of 2-styrylchromones16–18 and the
similarity of physical properties and replication strategy between
picornavirus and calicivirus families.
From the 12 compounds studied, six (E)-2-styrylchromones
exhibited interesting anti-norovirus activity, being (E)-5-hydro-
xy-2-styrylchromone (7) and (E)-40-methoxy-2-styrylchromone
(13) the best compounds of the series, with an IC50 ꢀ 7
lM. More-
over, the high SI values presented by these compounds (>21.6 and
>13.5, respectively) confers them an apparently good toxicological
profile to be developed as antiviral drugs.
Although it may not be accurate to draw a structure–activity rela-
tionship from this small group of compounds, an interesting rela-
4. Conclusion
Table 3
Anti-norovirus versus anti-rhinovirus activity of (E)-2-styrylchromones (4, 5, 12–15,
17)
To our knowledge, this is the first study reporting anti-noro-
virus activity of 2-styrylchromones. The interesting activity of
(E)-5-hydroxy-2-styrylchromone and (E)-40-methoxy-2-styrylchr-
omone against norovirus led us to conclude that the (E)-2-sty-
rylchromones skeleton is an important one, which deserves to
be developed and explored as novel antiviral drugs against this
important human pathogen.
Compounds
MNV
IC50
HRV 1B
IC50
HRV 14
IC50
a
b
b
(l
M)
(l
M)
(lM)
(E)-2-Styrylchromone (4)
17.7 6.5
>50
23.7 7.5
7.4 1.3
>50
6.19
> 12.5
>6.25
>3.12
9.29
>25
(E)-40-Chloro-2-stryrylchromone (5)
(E)-40-Methyl-2-stryrylchromone (12)
(E)-40-Methoxy-2-stryrylchromone (13)
(E)-40-Hydroxy-2-stryrylchromone (14)
(E)-3-Hydroxy-2-stryrylchromone (15)
(E)-3-Methoxy-2-stryrylchromone (17)
6.19
>6.25
Inactive
6.41
9.76
1.6
5. Experimental
>10
37.9 6.3
5.13
1.1
5.1. General methods
MNV = murine norovirus; HRV = human rhinovirus (strains 1B and 14).
a
Results of anti-norovirus activity obtained in the present study.
b
Melting points were determined on a BUCHI Melting point appa-
ratus and are uncorrected. NMR spectra were recorded on Bruker
Results of anti-rhinovirus activity published by other authors (Desideri et al.,
2000; Desideri et al., 2003).

4200
J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
Avance 300 (300.13 MHz for 1H and 75.47 MHz for 13C) and Bruker
Avance 500 (500.13 MHz for 1H and 125.76 MHz for 13C) spectrom-
eters. Chemical shifts (d) are reported in ppm values (d) and cou-
pling constants (J) in hertz. The internal standard was TMS. 1H
assignments were made using two-dimensional gradient selected
correlation spectroscopy (gCOSY) and nuclear Overhauser effect
spectroscopy (NOESY; 800 ms mixing time), experiments, while
13C assignments were made using two-dimensional gradient se-
lected heteronuclear single quantum coherence (gHSQC) and
two-dimensional gradient selected heteronuclear multiple quan-
tum coherence (gHMBC) (delays for one bond and long-range J C/
H couplings were optimized for 145 and 7 Hz, respectively) exper-
iments. Mass spectra (EI, 70 eV) were measured on a VG Autospec
Q and M mass spectrometers.
10.05 (s, 1 H, 40-OH). 13C NMR (125.77 MHz, DMSO-d6): d 109.0
(C-3), 116.0 (C-30,50), 116.9 (C-
), 118.2 (C-8), 123.6 (C-10), 124.8
a
(C-5), 125.2 (C-6), 126.1 (C-10), 129.8 (C-20,60), 134.2 (C-7), 136.9
(C-b), 155.5 (C-9), 159.4 (C-40), 162.5 (C-2), 176.9 (C-4). ESI+-MS:
m/z 287 (20) (M+Na)+, 265 (100) (M+H)+. HRMS-EI: Calcd for
C17H13O3 264.0786; found: 264.0796.
5.3. Samples
Stock solutions (60 mM) of each compound 2, 4–8 and 12–17
were prepared in DMSO (drug vehicle) and kept at ꢂ20 °C. Appro-
priate dilutions were freshly prepared just prior to every assay.
5.4. Cell culture
5.2. Chemical compounds
The murine macrophage cell line RAW 264.7 was used to prop-
agate MNV. Cells were grown in DMEM supplemented with 2 mM
Chromone (2) was obtained by condensation of the 20-hydroxy-
acetophenone and methyl formate and their structural character-
isation is as follow.
L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U penicil-
lin/mL, 100 g/mL streptomycin and 10% (growth medium) or 2%
l
(maintenance medium) of inactivated fetal bovine serum (FBS) at
37 °C in a humidified atmosphere of 5% CO2. Confluent RAW cells
were used in all assays and were obtained by plating 5.0 ꢃ 105 via-
ble cells/mL in growth media followed by a 24 h incubation period.
Culture media was then removed and replaced by maintenance
medium.
Chromone (2): Mp 58–60 °C; 1H NMR (300.13 Hz, CDCl3): d
(ppm) 6.18 (d, 1 H, J = 6.0 Hz, H-3), 7.22 (ddd, 1 H, J = 0.9, 7.7 and
7.8 Hz, H-6), 7.26 (dd, 1 H, J = 0.9 and 8.2 Hz, H-8), 7.50 (ddd, 1
H, J = 1.7, 7.7 and 8.2 Hz H-7), 7.79 (d, 1 H, J = 6.0 Hz, H-2), 7.98
(dd, 1 H, J = 1.7 and 8.2 Hz H-7). 13C NMR (75.47 Hz, CDCl3): d
(ppm) 112.0 (C-3), 117.4 (C-8), 123.9 (C-10), 124.4 (C-6), 124.6
(C-5), 133.0 (C-7), 154.9 (C-2), 155.6 (C-9), 176.6 (C-4). EI-MS: m/
z 146 (100) M+ꢁ, 120 (17), 118 (45), 92 (34), 90 (20), 74 (12), 63
(24), 53 (10). Anal. Calcd for C9H6O2 (146.04): C, 73.97; H, 4.14.
Found: C, 74.21; H, 4.10.
The first overlay media for viral plaque assay was prepared in
E-MEM 2 ꢃ without phenol red and supplemented with 4 mM
L-
glutamine, 0.2 mM non essential amino acids, 30 mM HEPES,
0.23% sodium bicarbonate, 2 mM sodium pyruvate, 200 U penicil-
lin/mL, 200 lg/mL streptomycin and 10% FBS. The second overlay
(E)-2-Styrylchromones (4–8, 12, 13) have been prepared by the
Baker–Venkataraman method or the aldol condensation/oxidative
cyclization procedure22 and have shown spectroscopic and
analytical data identical to those previously reported: (E)-2-sty-
rylchromone (4), (E)-40-chloro-2-styrylchromone (5), a-methyl-
2-styrylchromone (8);23 (E)-40-methyl-2-styrylchromone (12)
and (E)-40-methoxy-2-styrylchromone (13);25 (E)-5-benzyloxy-2-
styrylchromone (6) and (E)-5-hydroxy-2-styrylchromone (7).24
The (E)-3-alkoxy-2-styrylchromones (15–17) were obtained by
the Algar–Flynn–Oyamada type-reaction.26 Compounds (15, 17)
have shown spectroscopic and analytical data identical to those
media contained in addition neutral red at a final concentration of
0.6%.
5.5. Virus
MNV (virus strain MNV-1.CW1) was provided by Herbert W.
Virgin (Washington University, St. Louis, USA). MNV was propa-
gated by infecting confluent RAW cells cultured in maintenance
medium and harvested when cytopathic effect stopped progress-
ing. MNV stocks were obtained by submitting infected cells to
three consecutive freeze–thaw cycles followed by centrifugation
to partially purify virus from cell lysates being the supernatants
stored at ꢂ80 °C.
previously reported,18 while (E)-3-hydroxy-
omone (16) is
new compound: Mp 123–124 °C. 1H NMR
-CH3),
a-methyl-2-styrylchr-
a
(500.13 MHz, DMSO-d6): d (ppm) 2.32 (d, 3 H, J = 1.3 Hz,
a
7.34–7.37 (m, 1 H, H-40), 7.44–7.49 (m, 5 H, H-6, H-20,60, H-30,50),
7.59 (s broad, 1 H, H-b), 7.70 (d, 1 H, J = 7.7 Hz, H-8), 7.93 (ddd, 1
H, J = 1.6, 7.0 and 7.7 Hz, H-7), 8.10 (dd, 1 H, J = 1.6 and 8.0 Hz,
5.6. Cytotoxicity by the MTT assay
The toxicity of compounds was evaluated by the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
RAW cells in 96-well plates were exposed to serial concentrations
H-5). (125.77 MHz, DMSO-d6): d (ppm) 15.6 (
a-CH3), 118.3 (C-8),
121.2 (C-10), 124.7 (C-5), 124.5 (C-6), 127.7 (C-40), 128.5 (C-20,60),
128.9 (C-
a
), 129.4 (C-30,50), 133.6 (C-7), 134.2 (C-b), 136.3 (C-10),
of each compound (in duplicate) for 48 h, being 150
lM (or
138.9 (C-3), 148.3 (C-2), 154.3 (C-9), 172.7 (C-4). ESI+-MS: 301
(35) (M+Na)+, 279 (100) (M+H)+. Anal. Calcd for C18H14O3
(278.30): C, 77.68; H, 5.07. Found: C, 77.68; H, 5.04.
100 M for compound 13) the highest concentration tested. Con-
l
trol cells were exposed to the maximum concentration of DMSO
(0.25%) and the same incubation time. After treatment, MTT solu-
tion (0.5 mg/mL) was added and plates were incubated for further
4 h to allow the reduction of MTT. Then, medium was removed and
DMSO was added to solubilize the formazan crystals. Absorbance
was measured at 550 nm. The percentage of MTT reduction was
calculated comparing the absorbance of compound treated
cells to the control cells and the CC50 value of each compound
(concentration that reduced the absorbance of treated cells by
50% when compared to control cells) was determined. Maximum
non-toxic concentration of compounds (MNTC) was defined as
those in which the MTT reduction capacity of treated cells was at
least 90% (MNTC90) or 70% (MNTC70) when compared to control
cells. Only non-toxic concentrations of compounds were further
tested for antiviral activity.
(E)-40-Hydroxy-2-styrylchromone (14): A solution of (E)-40-
methoxy-2-styrylchromone (13) (0.6 mmol) in dry CH2Cl2 was
cooled in a propan-2-ol bath at ꢂ78 °C and then a solution of
BBr3 in CH2Cl2 (1 M, 1 mL) was added drop wise. After the addition,
the bath was removed and the reaction mixture was stirred under
nitrogen, at room temperature, for 24 h. After that period, the solu-
tion was poured into ice and water. The solid obtained was filtered
off and washed with water and light petroleum. Mp 284–285 °C;
1H NMR (500.13 MHz, DMSO-d6): d (ppm) 6.40 (s, 1 H, H-3), 6.85
(d, 2 H, J = 8.6 Hz, H-30,50), 6.99 (d, 1 H, J = 16.1 Hz, H-
a), 7.47 (t,
1 H, J = 7.5 Hz, H-6), 7.59 (d, 2 H, J = 8.6 Hz, H-20,60), 7.63 (d, 1 H,
J = 16.1 Hz, H-b), 7.71 (d, 1 H, J = 7.7 Hz, H-8), 7.81 (ddd, 1 H,
J = 1.6, 7.5 and 7.7 Hz, H-7), 8.01 (dd, 1 H, J = 1.6 and 7.5 Hz, H-5),

J. Rocha-Pereira et al. / Bioorg. Med. Chem. 18 (2010) 4195–4201
4201
5.7. Anti-norovirus activity by plaque reduction assay
Supplementary data
The anti-norovirus activity of compounds was evaluated by pla-
que reduction assay. RAW cells in 6-well plates were infected with
MNV for 1 h at 37 °C and simultaneously exposed to serial concen-
trations of each compound (in duplicate) for a continuous period
of 48 h. These conditions will be from here on referred as standard.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.006.
References and notes
1. Glass, R. I.; Parashar, U. D.; Estes, M. K. N. Eng. J. Med. 2009, 361, 1776.
2. Patel, M. M.; Hall, A. J.; Vinje, J.; Parashar, U. D. J. Clin. Virol. 2009, 44, 1.
3. Widdowson, M. A.; Monroe, S. S.; Glass, R. I. Emerg. Infect. Dis. 2005, 11, 735.
4. Green, K. Y. In Fields Virology, Vol. 2; Knipe, D. M., Howley, P. M., Eds.; Lippincott
Williams & Wilkins: Philadelphia, 2007; pp 949–979.
5. Wobus, C. E.; Karst, S. M.; Thackray, L. B.; Chang, K. O. Sosnovtsev S.V.; Belliot, G.;
Krug, A.; Mackenzie, J.M.; Green, K.Y.; Virgin, H.W. PLoS Biol 2004, 2, 432.
6. Wobus, C. E.; Thackray, L. B.; Virgin, H. W. J. Virol. 2006, 80, 5104.
7. Cannon, J. L.; Papafragkou, E.; Park, G. W.; Osborne, J.; Jaykus, L. A.; Vinje, J. J.
Food Prot. 2006, 69, 2761.
8. Baert, L.; Uyttendaele, M.; Vermeersch, M.; Van Coillie, E.; Debevere, J. J. Food
Prot. 2008, 71, 1590.
9. Baert, L.; Wobus, C. E.; Van Coillie, E.; Thackray, L. B.; Debevere, J.; Uyttendaele,
M. Appl. Environ. Microbiol. 2008, 74, 543.
10. Ng, K. K.; Pendas-Franco, N.; Rojo, J.; Boga, J. A.; Machin, A.; Alonso, J. M.; Parra,
F. J. Biol. Chem. 2004, 279, 16638.
The highest concentration tested was 50
lM for all the compounds
but 10 M for compounds 15 and 16. A MNV suspension producing
l
20–80 plaque forming units (PFU) per well was used. After viral
infection period, culture media was removed and the first agarose
overlay (containing equal concentrations of compounds) was added
and incubated for 24 h. After this incubation period the second over-
lay with neutral red was added. Plates were incubated for more 24 h
and then PFU were counted. Control cells were infected with the
same MNV suspension and exposed to the maximum concentration
of DMSO (0.084%) and the same incubation time. The IC50 value of
each compound (concentration of compound that reduced the pla-
que number by 50% when compared to control cells) was deter-
mined. The SI value of each compound was calculated as CC50/IC50
.
11. De Palma, A. M.; Vliegen, I.; De Clercq, E.; Neyts, J. Med. Res. Rev. 2008, 28,
823.
12. Bauer, D. J.; Selway, J. W.; Batchelor, J. F.; Tisdale, M.; Caldwell, I. C.; Young, D.
A. Nature 1981, 292, 369.
13. Conti, C.; Mastromarino, P.; Sgro, R.; Desideri, N. Antiviral Chem. Chemother.
1998, 9, 511.
14. Robin, V.; Irurzun, A.; Amoros, M.; Boustie, J.; Carrasco, L. Antiviral Chem.
Chemother. 2001, 12, 283.
15. Tait, S.; Salvati, A. L.; Desideri, N.; Fiore, L. Antiviral Res. 2006, 72, 252.
16. Conti, C.; Mastromarino, P.; Goldoni, P.; Portalone, G.; Desideri, N. Antiviral
Chem. Chemother. 2005, 16, 267.
17. Desideri, N.; Conti, C.; Mastromarino, P.; Mastropaolo, F. Antiviral Chem.
Chemother. 2000, 11, 373.
18. Desideri, N.; Mastromarino, P.; Conti, C. Antiviral Chem. Chemother. 2003, 14,
195.
19. G.P. Ellis, in: G.P. Ellis (Ed.), General methods of preparing chromones in
Chromenes, Chromanones and Chromones, John Wiley & Sons, New York, 1977,
pp. 495-555, Chapter IX.
For the dose–response studies of (E)-5-hydroxy-2-styrylchro-
mone (7) and (E)-40-methoxy-2-styrylchromone (13), standard
conditions were used but compounds were tested starting at the
highest concentration of 100 lM to allow a more detailed study.
The anti-norovirus effect of (E)-5-hydroxy-2-styrylchromone (7)
and (E)-40-methoxy-2-styrylchromone (13) was further evaluated
by addition of compounds at two different time-points. Briefly,
RAW cells were exposed to serial concentrations of compounds 7
and 13 only during the hour of viral infection (1 h at 37 °C) or only
for 48 h in the overlay after viral infection. An experiment with
the standard conditions was run simultaneously. The IC50 values
for the different time-points were determined as described above.
20. Makrandi, J. K.; Kumari, V. Chem. Ind. 1988, 630.
5.8. Statistics
21. Santos, C. M. M.; Silva, A. M. S.; Cavaleiro, J. A. S. Eur. J. Org. Chem. 2003, 4575.
22. Price, W. A.; Silva, A. M. S.; Cavaleiro, J. A. S. Heterocycles 1993, 36, 2601.
23. Silva, A. M. S.; Pinto, D. C. G. A.; Tavares, H. R.; Cavaleiro, J. A. S.; Jimeno, M. L.;
Elguero, J. Eur. J. Org. Chem. 1998, 2031.
24. Pinto, D. C. G. A.; Silva, A. M. S.; Cavaleiro, J. A. S. Heterocycl. Commun. 1996, 2,
145.
Results are expressed as mean values SEM (standard error of the
mean). Statistical comparisons were made using one-way analysis
of variance followed by the Bonferroni’s multiple comparison post
hoc test.
25. Pinto, D. C. G. A.; Silva, A. M. S.; Cavaleiro, J. A. S.; Levai, A.; Patonay, T. J.
Heterocycl. Chem. 1998, 35, 217.
26. Gormley, T. R.; O’Sullivan, W. I. Tetrahedron 1973, 29, 369.
27. Cerqueira, F.; Cidade, H.; van Ufford, L.; Beukelman, C.; Kijjoa, A.; Nascimento,
M. S. Int. Immunopharmacol. 2008, 8, 597.
Acknowledgements
28. Cerqueira, F.; Cordeiro-da-Silva, A.; Araujo, N.; Cidade, H.; Kijjoa, A.;
Nascimento, M. S. Life Sci. 2003, 73, 2321.
29. Müller, B. H.-G. Kraüsslich. In Handbook of Experimental Pharmacology, Vol. 189;
Krausslich, H.-G., Bartenschlager, R., Eds.; Springer-Verlag: Berlin, 2009; pp 2–
24.
Thanks are due to Fundação para a Ciência e a Tecnologia (FCT),
I&D units 4040/2007 (CEQUIMED-UP) and 62/94 (QOPNA), FEDER,
POCI for financial support. The authors thank also FCT for the Ph.D.
grant of Joana Rocha-Pereira (SFRH/BD/48156/2008). Authors are
also grateful to Herbert W. Virgin for the generous provision of
the MNV.
30. Marinho, J.; Pedro, M.; Pinto, D. C. G. A.; Silva, A. M. S.; Cavaleiro, J. A. S.; Sunkel,
C. E.; Nascimento, M. S. J. Biochem. Pharmacol. 2008, 75, 826.