New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus capsid-binders

Article abstract of DOI:10.1016/j.bmc.2010.06.103

Substituted (E)-3-styryl-4H-chromen-4-ones 1a-d, 3-[(1E,3E)-4-phenylbuta-1, 3-dienyl]-4H-chromen-4-ones 2a-d, (E)-3-styryl-2H-chromenes 3a-d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a-d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC₅₀ values of 0.20 μM and 1.38 μM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.

Full text of DOI:10.1016/j.bmc.2010.06.103

Bioorganic & Medicinal Chemistry 18 (2010) 6480–6488
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus
capsid-binders
Cinzia Conti a, Nicoletta Desideri b,
*
a Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Scienze di Sanità Pubblica, Sezione di Microbiologia, Università‘La Sapienza’ di Roma, P.le A. Moro, 5, 00185 Roma, Italy
b Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università ‘La Sapienza’, P.le A. Moro, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-
ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes
4a–d were designed and synthesized to improve the anti-picornavirus activity of previously tested ana-
logues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14
and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although
considerable differences were observed in the sensitivity of viruses to each compound. Generally, both
HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length
as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as
Received 19 April 2010
Revised 24 June 2010
Accepted 29 June 2010
Available online 30 July 2010
Keywords:
2H-Chromenes
4H-Chromen-4-ones
Antivirals
the most effective inhibitor of both HRVs showing IC50 values of 0.20 lM and 1.38 lM towards serotype
1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high ther-
apeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structur-
ally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus
infection, probably at the adsorption and/or uncoating level.
Human rhinoviruses
Enterovirus 71
Capsid binder
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the eradication of poliovirus from developed countries, EV71 has
emerged as major cause of neurologic threat in the world.
Picornaviruses are a group of small, non-enveloped, positive
stranded RNA viruses that constitute one of the largest virus fam-
ilies and comprise several pathogens implicated in an extensive
range of clinical manifestations which affect humans as well as
animals. Human rhinoviruses (HRVs) and enteroviruses (EVs) are
two related genera within this virus family.
HRVs represent the most important aetiological agents of
common cold.1 The over 100 viral serotypes can be divided into
two distinct groups, A and B, on the basis of their susceptibility to
capsid-binding compounds.2,3 Although HRV infections are often
mild and self-limiting in healthy adults, the socioeconomic impact
caused by missed school or work days is enormous. Moreover,
increasing evidences describe the link between HRV infections and
several serious upper and lower tract complications such as asthma,
chronic bronchitis and otitis media.4
Although EV71 usually causes hand, foot and mouth disease, a mild
exanthematous infection occurring mainly in young children, it is
also associated with acute neurological diseases including aseptic
meningitis, acute flaccid paralysis and even fatal neurogenic pul-
monary edema.6 In the last decades a significant increase in epi-
demic activity of EV71 has been observed and severe outbreaks
have been reported in various parts of the world, particularly in
the Asia-Pacific region.7–10
The difficulty of vaccine development for the majority of
picornaviruses makes the search of efficacious drugs a necessity.
Although extensive efforts have been devoted to the search for effec-
tive agents, to date no drug has been approved by the FDA for clinical
use, and the patient care remains symptomatic. Each step during the
life cycle of picornaviruses has been considered a potential target for
antiviral compounds, however only inhibitors of capsid functions
and of 3C protease demonstrated in vivo activity. Since it is well
recognized that the structures of viral capsid and 3C protease are
relatively conserved among different serotypes, molecules acting
on each of these targets could be promising anti-picornavirus agents
with a broad-spectrum of activity.11
EVs comprise poliovirus, echoviruses, coxsackieviruses, and
numbered enteroviruses. The clinical manifestations associated
with EVs range from mild illnesses such as fever and rash to poten-
tially severe and life-threatening infections, such as meningitis,
encephalitis, myocarditis, poliomyelitis, and neonatal sepsis.5 After
Recently, we prepared and tested against HRV serotype 1B and 14
a series of (Z)-3-benzylidenechromans, 3-benzyl-2H-chromenes
and 3-benzylchromans12 related to the most active synthetic
* Corresponding author. Tel.: +39 06 49913892; fax: +39 06 49693268.
E-mail address: nicoletta.desideri@uniroma1.it (N. Desideri).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.103

C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
6481
3(2H)-isoflavenes13–15 and homoisoflavones16–18 previously stud-
ied by us. Several members within these families of inhibitors
showed submicromolar potency against HRV 1B coupled with a high
therapeutic index. On the contrary, HRV 14, a representative sero-
type A, was only weakly inhibited.12 Mechanism of action studies
indicated that, similarly to related flavanoids,19,20 the new com-
pounds behave as capsid-binders and interfere with very early
events of virus multiplication.12 The different activity against HRV
1B and 14 has been attributed to the size of the compound-binding
site into the capsid protein VP1 varying with the specific serotype.
Previous research on capsid-binding compounds indicated that the
viral group B binding site accommodates molecules with shorter
chains, while long-chained compounds are routinely more active
against viral group A rhinoviruses.21 Subtle differences in the size
and shape of the hydrophobic pocket have been described also
among EVs. In the case EV71, the binding pocket was shown to
accommodate long molecules such as pyridyl imidazolidinones.22,23
Therefore, we planned to increase the length of the linker chain be-
tween the heterocycle and the phenyl moiety in order to occupy
more efficiently the capsid pocket of picornaviruses.
In this paper, we describe the synthesis of (E)-3-styryl-4H-
chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-
chromen-4-ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and
3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a–d designed
to improve the anti-picornavirus spectrum of activity of the previ-
ously tested analogues. The antiviral potency of the new derivatives
was evaluated against HRV 1B and 14, representative serotypes for
group B and A of HRVs, and against EV-71 selected among EVs for
its clinical significance.
ꢁ16 Hz) that prove the trans configuration for both double bonds
of the dienic system.
(E)-3-Styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-
1,3-dienyl]-2H-chromenes 4a and 4d were synthesized according
to the two step procedure shown in Scheme 2. For this purpose,
2H-chromene-3-carbaldehydes 5a and 5d were prepared with
some modifications of the literature procedures,25,26 by refluxing
the appropriate 2-hydroxybenzaldehyde and acrolein in the pres-
ence of potassium carbonate. The subsequent Horner–Wads-
worth–Emmons reaction of 2H-chromene-3-carbaldehydes 5a
and 5d with commercially available diethyl benzylphosphonates
or diethyl trans-cinnamylphosphonate, carried out using sodium
hydride as base, provided only the (E) isomers 3a–d, 4a and 4d.
The stereochemistry of compounds was established on the basis
of the coupling constant values of the protons in the chain
(J ꢀb = 16.5 Hz and ꢁ15.6 Hz for 3a–d and 4a,d, respectively).
a
As diethyl trans-4-chlorocinnamylphosphonate is not commer-
cially available, an alternative method was adopted to prepare
3-[(1E,3E)-4-(4-chlorophenyl)buta-1,3-dienyl]-2H-chromenes 4b
and 4c (Scheme 3). The target compounds (4b and 4c) were obtained
by the Horner-Wadsworth-Emmons reaction of diethyl 4-chloro-
benzylphosphonate with the appropriate (E)-3-(2H-chromen-
3-yl)acrylaldehyde (6a and 6d), obtained by the Wittig reaction of
the corresponding 2H-chromene-3-carbaldehyde (5a and 5d) with
[(1,3-dioxolan-2-yl)methyl]triphenylphosphonium bromide, fol-
lowed by removal of the protecting group accomplished with oxalic
acid. Also this method provided only the isomers with trans config-
uration of both double bonds (J ꢀb and J ꢀd ꢁ15.6 Hz).
a
c
2.2. Antiviral tests
2. Results and discussion
2.1. Chemistry
Initially, all the synthesized compounds (1a–d, 2a–d, 3a–d and
4a–d) were evaluated for effects on morphology, viability and
growth of HeLa and Hep-2 cells, human cell lines suitable for the rep-
lication of HRVs and EVs, respectively. Morphological alterations
were scored microscopically, and the action of compounds on loga-
rithmic cell growth was determined by the XTT colorimetric meth-
od.27 The maximum non-cytotoxic concentration (MNTC) and the
50% cytotoxic concentration (TC50) of compounds are reported in
Table 1. The MNTC was expressed as the highest dose tested that
did not produce any toxic effect or reduction of cell growth after
3 days incubation at 37 °C. The TC50 was indicated as the concentra-
tion of compound reducing the cell viability by 50% as compared
with the control. Generally, compounds presented low cytotoxicity
against HeLa cells, with the exception of 6-chloro-3-[(1E,3E)-
4-(4-chlorophenyl)buta-1,3-dienyl]-4H-chromen-4-one 2c and
3-[(1E,3E)-4-(4-chlorophenyl)buta-1,3-dienyl]-2H-chromene 4b.
The majority of the compounds exhibited a similar toxicity
against Hep-2 cells, while 1a, 3a and 3c were more toxic against this
cell line.
The method described by Silva et al.24 was used for the synthe-
sis of (E)-3-styryl-4H-chromen-4-ones 1a–d. This procedure
involves the condensation of 4-oxo-4H-chromene-3-carbaldehy-
des with 5 M equiv of phenylacetic acids in the presence of potas-
sium tert-butoxide and refluxing dry pyridine. Under these
conditions, a Knoevenagel-type reaction, followed by decarboxyl-
ation, took place and the (E)-3-styryl-4H-chromen-4-ones 1a–d
were diastereoselectively obtained (Scheme 1). The coupling con-
stant values of the vinylic protons (J
configuration of this double bond.
ꢁ16 Hz) indicate the trans
a
ꢀb
The same experimental conditions were also successfully ap-
plied to the condensation of 4-oxo-4H-chromene-3-carbaldehydes
with (E)-4-phenylbut-3-enoic acids in order to prepare 3-[(1E,3E)-
4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d (Scheme 1).
The stereoselectivity of the methods was confirmed by the
coupling constant values of the vinylic protons (J
and J
cꢀd
a
ꢀb
Scheme 1. Reagents: (i) tert-BuOK, dry Py, reflux.

6482
C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
Scheme 2. Reagents and conditions: (i) K2CO3, dry dioxane, reflux, 4 h; (ii) NaH, dry THF, rt 20 h.
The inhibitory activity of compounds on the replication of HRV
1B, HRV 14 and EV71 was evaluated by a plaque reduction assay,
starting from the MNTC. HRV 1B and 14 were utilized as
representative serotypes for group B and A of HRVs, while EV71
was chosen as a clinically relevant member within EVs. The com-
pound concentration required to produce a 50% reduction of plaque
number with respect to mock-treated virus-infected cultures (IC50
)
is reported in Table 1. When the IC50 value was higher than the
MNTC, the percentage inhibitionat this dose is reported in parenthe-
ses. The therapeutic index (TI), expressed as TC50 versus IC50 ratio,
was calculated (Table 1). 40,6-Dichloroflavan (BW683C), an inhibitor
of group B serotypes, was included as a control.28
The new compounds interfered with the replication of both
HRV serotypes and EV71 dose-dependently, although considerable
differences were observed in sensitivity of each virus. EV71 was
generally less susceptible than both HRVs and the percent reduc-
tion of plaque number was found always lower than 50% up to
the MNTC. Despite the low potency of all compounds against
EV71, derivatives 2a–d and 4a–d, with a longer linker chain
between the heterocycle and the phenyl moiety, produced a
higher percent of inhibition with respect to the corresponding
shorter analogues 1a–d and 3a–d. Unfortunately, the most active
Scheme 3. Reagents and conditions: (i) tert-BuOK, dry THF, rt 24 h and reflux 6 h;
(ii) (COOH)2, H2O, rt, overnight; (iii) NaH, dry THF, rt 22 h.
Table 1
Cytotoxicity and anti-picornavirus activity of (E)-3-styryl-4H-chromen-4-ones (1a–d) and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones (2a–d), (E)-3-styryl-2H-
chromenes (3a–d) and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes (4a–d)
MNTCa
(lM)
TC50
(l
M)
IC50
(
lM) HRV 1B
TId
IC50
(l
M) HRV 14
TId
MNTCe
(l
M)
IC50 (lM)E71
b
c
c
c
Compd
R
R0
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
H
H
Cl
Cl
H
H
>200.00f
25.00
25.00
25.00
25.00
25.00
3.12
50.00
25.00
50.00
25.00
50.00
50.00
3.12
>200.00f
50.00
50.00
50.00
50.00
50.00
6.25
100.00
50.00
100.00
50.00
100.00
100.00
6.25
3.12
4.54
2.19
3.22
5.27
4.09
0.71
1.6
0.20
0.40
7.13
11.20
30.83
3.10
0.54
6.27
>64.16
11.01
22.19
15.53
9.49
12.22
8.80
62.5
250.00
250.00
7.01
8.93
3.24
4.36
5.52
25 (24.1%)
25 (33.1%)
15.00
>45.87
9.06
—
50.00
25.00
25.00
25.00
25.00
25.00
3.12
50.00
12.50
50.00
12.50
50.00
50.00
3.12
50.00 (34.6%)
25.00 (21.6%)
25.00 (35.7%)
25.00 (37.0%)
25.00 (39.8%)
25.00 (49.1%)
3.12 (46.6%)
50.00 (46.7%)
12.50 (36.1%)
50.00 (49.7%)
12.50 (46.6%)
50.00 (30.8%)
50.00 (29.5%)
3.12 (32.9%)
6.25 (39.1%)
12.50 (25.4%)
10.00 (14%)
Cl
Cl
H
—
H
3.33
12.28
—
H
Cl
Cl
H
4.07
Cl
Cl
H
3.12 (36.2%)
50.00 (48.3%)
1.38
18.39
8.28
11.17
50.00
3.12 (30.4%)
6.25 (12.8%)
12.50 (37.2%)
NAg
—
H
36.23
5.44
6.04
8.95
2.00
—
—
—
—
H
Cl
Cl
H
Cl
Cl
H
H
Cl
Cl
H
Cl
Cl
H
2.00
23.15
3.99
6.25
12.50
25.00
12.50
25.00
>25.00
6.25
12.50
10.00
4d
BW683C
0.026
a
The maximum non-cytotoxic concentration (MNTC) was the highest dose tested that did not produce any cytotoxic effect and reduction in viability of HeLa cells, or on cell
growth after 3 days of incubation at 37 °C.
b
The TC50 value was the concentration of compound which reduced the HeLa cell viability by 50%, as compared with the control.
The IC50 value was the dose of compound reducing the plaque number by 50% and was calculated by plotting the log of drug concentration versus the percentage of
c
plaque reduction. When a 50% reduction was not achieved, the percent of inhibition obtained at the MNTC was reported in parentheses.
d
The therapeutic index (TI) value was equal to TC50/IC50
The maximum non-cytotoxic concentration (MNTC) was the highest dose tested that did not produce any cytotoxic effect and reduction in viability of Hep cells, or on cell
.
e
growth after 3 days of incubation at 37 °C.
f
The saturation concentration in cell culture medium was found to be lower than MNTC and TC50
Not active up to the highest concentration tested (MNTC).
.
g

C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
6483
compounds, 2c and 4b, were also the most toxic for Hep-2 cells
(MNTC = 3.12 V). Therefore, it was impossible to test both ana-
logues at concentrations higher than 3.12 M.
30.83 lM to 0.54 l
M) and only the 40,6-dichloro analogue 4c was
active within a submicromolar range.
l
l
On the contrary, several compounds of these series showed
significant antiviral activity against both HRV serotypes, although
they generally exhibited a higher potency against serotype 1B.
Within the chromen-4-one series (1a–d and 2a–d), 6-chloro-3-
[(1E,3E)-4-(4-chlorophenyl)buta-1,3-dienyl]-4H-chromen-4-one
2c was the most potent compound against HRV 1B, with an IC50 of
2.3. Mechanism of action study
Due to its potent in vitro activity against both HRV serotypes
(IC50 = 0.20 lM and 1.38 lM, respectively) and high therapeutic
indexes (TI = 250.00 and 36.23, respectively), (E)-3-styryl-2H-chro-
mene 3a was chosen to investigate the mechanism of the antiviral
action. The effects produced either on HRV 1B virus particles or
HRV 1B multiplication have been evaluated.
Initially, the action of 3a on virus infectivity and stability was
investigated. The virus-neutralizing potential was studied by incu-
bating HRV 1B at high titre with the compound at the concentration
0.71 lM. Potency was reduced when one (2b and 2d) or both the
chlorine atoms (2a) were removed or when the linker chain was
shortened (1c). Despite the high in vitro anti-HRV 1B potency, 2c
showed a modest therapeutic index (TI = 8.80) coupled with a
weak activity against HRV 14. On the contrary, although both
unsubstituted chromones (1a and 2a) and 40-chloro substituted
chromones (1b and 2b) exhibited a lower activity than 2c against
serotype 1B, they showed comparable potency against both HRV
serotypes. In addition to the broader spectrum of anti-HRV activity,
(E)-3-styryl-4H-chromen-4-one 1a exhibited low cytotoxicity
resulting in high therapeutic indexes (TI >64.1 and >45.87 against
HRV 1B and 14, respectively).
Compared to compound 2c, the replacement of the chromen-4-
one moiety with the chromene ring (4c) produced a slight
improvement in anti-HRV 1B potency (IC50 = 0.54 lM) and an
higher selectivity (TI = 23.15), while the anti-HRV 14 inhibitory
activity became even lower.
of 20 lM, corresponding to 100 times the IC50. After serial 10-fold
dilutions to achieve non-inhibitory concentrations of free com-
pound, the infectivity titres of mock- and 3a-treated virus suspen-
sions were found to be similar (2.86 ꢂ 107 PFU/mL and 2.66 ꢂ
107 PFU/mL, respectively), indicating that 3a does not damage virus
particles. In stabilization studies, 3a (20 lM) was found to protect
HRV 1B against inactivation by mild acid or heat treatment. As
shown in Figure 1, in the absence of compound, the infectivity of
control virus decreased significantly when exposed to either pH 5
(Fig. 1A) or 56 °C (Fig. 1B) (4.5 and 3.1 log PFU/mL, respectively).
In the presence of 3a, the drop in virus infectivity was reduced
(3.5 and 2.1 log PFU/mL, respectively) and the protective effect to-
wards inactivation was 1.0 log for both treatments. It is known that
exposure to mild acid or heat would cause conformational changes
of the virion capsid structure similar to those produced during the
uncoating of the viral genome.29,30 Binding of so-called capsid-
binders within the hydrophobic pocket of VP1 protein results in
resistance to acid and thermal inactivation due to a reduction of
capsid flexibility.31,32 Taken together, our results on stabilization
of HRV infectivity suggest that 3a could act as a capsid-binder.
However, binding of 3a was reversible by dilution as indicated by
results on virus infectivity. A similar behaviour has also been ob-
served for 40,6-dicyanoflavan and (Z)-3-(4-chlorobenzylidene)chro-
man, related compounds previously studies by us.10,12
Remarkably, all the 3- styryl-2H-chromenes tested (3a–d) dis-
played a broad-spectrum of anti-HRV activity. 3-Benzyl-2H-chro-
mene 3a showed the highest level of activity against both
serotypes (IC50 of 0.20 lM and 1.38 lM towards HRV 1B and 14,
respectively) and significant selectivity (TI = 250 and 36.23, respec-
tively). All modifications in this series resulted in diminished activ-
ity compared with 3a. In particular, the introduction of a chlorine
atom at the 40 position (3b) caused a slight decrease in the inhibi-
tory effect against HRV 1B and a dramatic loss of activity against
HRV 14 (2- and 13-fold, respectively). When a chlorine atom was
introduced at the 6 position (3d) or at both 40 and 6 positions
(3c), the reduction in potency and selectivity was substantial for
both serotypes.
The antiviral activity of 3a (20 lM) towards different stages of
Surprisingly, despite the larger size of the HRV 14 capsid bind-
ing site, increasing the linker chain length from two (3a–d) to four
(4a–d) carbon atoms resulted in a loss of activity against this sero-
type. Moreover, the chromen-4-one derivatives 4a–d displayed a
variable degree of potency against HRV 1B (IC50 ranging from
HRV 1B multiplication was investigated under one-step growth
conditions. Compound was continuously present during the entire
time of virus replication, during virus binding to the cell membrane
only or added or removed at different time intervals after virus
adsorption in the cold.
B
A
8
8
7
6
5
4
3
2
1
0
7
HRVC
HRVC
HRVT
3a
6
HRVT
5
3a
4
3
2
1
0
HRVC
HRVT
3a
HRVC
HRVT
3a
HRVC : untreated virus,
HRVT: virus exposed to pH 5.0,
3a: virus exposed to pH 5.0 in the presence of 3a.
HRVC: untreated virus,
HRVT: virus heated at 56 °C,
3a: virus heated at 56 °C in the presence of 3a.
Figure 1. Protective effect of 3a on acid (A) and thermal inactivation (B) of HRV 1B infectivity.

6484
C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
suppression in virus yield of 91.8%, confirming an early activity
1.2
1
of this compound. Similar levels of inhibition were achieved when
3a was removed after 1 h or longer incubation times (Fig. 3).
The analysis of data from time of addition/removal studies are
in agreement with the capsid-binder hypothesis, as suggested by
the interfering activity of 3a during virus binding and the very
early events of virus life cycle. In fact, the finding that a comparable
efficacy is achieved when 3a is present during the entire replica-
tion time or during virus binding only, strongly suggests a direct
interaction with virus particle. A similar behaviour has already
been described for the previously studied (Z)-3-(4-chlorobenzyli-
dene)chroman.12 However, differently from (Z)-3-(4-chlorobenzyl-
idene)chroman, the activity of 3a is significantly exerted also
within 30 min following virus attachment (91.8% inhibition). At
this incubation time, (Z)-3-(4-chlorobenzylidene)chroman pro-
duced only a 44% reduction of virus yield even at a higher concen-
0.8
0.6
0.4
0.2
0
EC
0 h
0.5 h
1 h
2 h
4 h
Figure 2. Effect of varying the time of addition of 3a (20
lM) on the inhibition of
HRV 1B replication under one-step growth conditions. Virus yield was determined
by plaque assay. Virus control titre was 3.82 ꢂ 106 PFU/mL. EC: 3a was present
during the entire infection cycle (1 h at 4 °C and 10 h at 33 °C). 0, 0.5, 1, 2 and 4 h:
compound was added at different times (0, 0.5, 1, 2 and 4 h) after the virus
adsorption period (1 h at 4 °C, time 0) and maintained until the end of virus
multiplication (up to 10 h at 33 °C).
tration (36 lM vs 20 lM). This finding could reflect a better ability
of 3a to enter the cells and reach the intracellular virus target.
It would be interesting to evaluate if 3a can produce similar ef-
fects against HRV 14 infection. The results could shed more lights
to the capsid-binding hypothesis.
3. Conclusion
Maximal inhibition (92%) was achieved when 3a was added to
cells together with the virus and maintained until the end of
HRV multiplication. Also when the compound was added to in-
fected HeLa cells immediately after virus binding or 30 min after
virus binding, a strong reduction in virus yield (91.4% and 90.0%,
respectively) was still observed. However, when 3a was added
1 h after virus binding, inhibition dropped to 7.7%. Virus yield
was unaffected when the compound was added 2 or 4 h post infec-
tion (Fig. 2). These data indicate that 3a interferes with early
event(s) of virus replication.
New series of (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,
3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d, (E)-3-sty-
ryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-
2H-chromenes 4a–d have been synthesized and tested for their
cytotoxicity and anti-HRV or EV71 activity in vitro. Some derivatives
displayed a broad-spectrum of anti-HRV activity whereas only a
modest efficacy was observed towards EV71. (E)-3-Styryl-2H-chro-
mene 3a was the most active anti-HRV agent.
Mechanism of action studies suggest that the new compounds
affect functions associated with the viral capsid, including virus
attachment to cell membrane and probably the uncoating of viral
genome.
Remarkably, when 3a was present during the time of virus
adsorption only (1 h, 4 °C) a 91.7% reduction of virus yield was still
observed (Fig. 3). At the same time, the incubation (1 h, 4 °C) of
HeLa cells with 3a before virus infection did not modify the virus
yield (data not shown), thus ruling out an effect at the cellular
receptor level. Therefore, interaction of 3a with HRV during the
adsorption step probably hinders capsid structure(s) responsible
for successful virus binding to the host cell membrane.
4. Experimental
4.1. Chemistry
When 3a was added immediately after virus adsorption and
removed after only 30 min of contact at 33 °C, it still produced a
Chemicals were purchased from Sigma–Aldrich and used with-
out further purification. Melting points were determined on a Stan-
ford Research Systems OptiMelt (MPA-100) apparatus and are
uncorrected. 1H NMR spectra were detected with a Bruker AM-
400 spectrometer, using TMS as internal standard. IR spectra were
recorded on a FT-IR Perkin–Elmer Spectrum 1000. All compounds
were routinely checked by thin-layer chromatography (TLC) and
1H NMR. TLC was performed on silica gel or aluminium oxide fluo-
rescent coated plates (Merck, Kieselgel or Aluminium oxide 60
F254). Components were visualised by UV light. Elemental analy-
ses (C, H, Cl) of all new compounds were within 0.4% of theoret-
ical values. (E)-4-(4-Chlorophenyl)but-3-enoic acid was prepared
as previously described.33
1.2
1
0.8
0.6
0.4
0.2
0
4.1.1. General procedure for the synthesis of the (E)-3-styryl-4H-
chromen-4-ones (1a–d) and 3-[(1E,3E)-4-phenylbuta-1,3-
dienyl]-4H-chromen-4-ones (2a–d)
0
0.5 h
1 h
2 h
4 h
To a solution of the appropriate 4-oxo-4H-chromene-3-carbal-
dehyde (10 mmol) and phenylacetic acid or (E)-4-phenylbut-3-
enoic acid (50 mmol) in dry pyridine (110 mL), tert-BuOK
(15 mmol) was added. The mixture was refluxed until complete
disappearance of 4-oxo-4H-chromene-3-carbaldehyde. After cool-
ing the mixture was diluted with ice and water, and acidified to
pH 2 with 2 N HCl. The precipitate was filtered, washed with water
Figure 3. Effect of varying the time of removal of 3a (20
l
M) on the inhibition of
HRV 1B replication under one-step growth conditions. Virus yield was determined
by plaque assay after 10 h of incubation at 33 °C. Virus control titre was
3.82 ꢂ 106 PFU/mL. 0: 3a was present during the time of virus adsorption only
(1 h, 4 °C) and removed at the time indicated as 0. 0.5, 1, 2 and 4 h: compound was
added after virus adsorption period (1 h at 4 °C, time 0) and removed after different
lengths of incubation (0.5, 1, 2 and 4 h) at 33 °C.

C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
6485
and purified by column chromatography on silica gel eluting with a
1:2 mixture of ethyl acetate and light petroleum.
15.5 Hz), 7.43 (d, 1H, H8, J7–8 = 8.9 Hz), 7.37 (d, 2H, H20, H60,
J2 –3 = 8.5 Hz), 7.30 (d, 2H, H30, H50, J2 –3 = 8.5 Hz), 6.87 (dd, 1H,
0
0
0
0
Hb, J ꢀb = 15.7 Hz, Jbꢀ = 10.5 Hz), 6.66 (d, 1H, Ha, J ꢀb = 15.7 Hz),
a
a
c
4.1.1.1. (E)-3-Styryl-4H-chromen-4-one (1a). Yield: 57%, mp =
167–168 °C (lit. 168–169 °C)34 from ethyl alcohol. The spectroscopic
data of the compound thus obtained were identical to those previ-
ously reported.34
6.50 (d, 1H, Hd, J ꢀd = 15.5 Hz). 13C NMR (CDCl3, 100 MHz): d
c
(ppm) 175.2, 154.1, 153.1, 135.7, 133.8, 133.4, 132.8, 132.5,
131.3, 130.0, 128.9, 127.6, 125.7, 125.0, 122.9, 121.8, 119.8. Anal.
Calcd for C19H12Cl2O2: C, 66.49; H, 3.52; Cl, 20.66. Found: C,
66.73; H, 3.53; Cl, 20.58.
4.1.1.2. (E)-3-(4-Chlorostyryl)-4H-chromen-4-one (1b). Yield:
72%, mp = 154–156 °C (lit. 159–160 °C)34 from ethyl alcohol. The
compound exhibited spectroscopic data identical to those previ-
ously reported.34
4.1.1.8. 6-Chloro-3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chro-
men-4-one (2d). Yield: 69%, mp = 172–173 °C from ethyl alcohol.
IR (KBr): 1644 cmꢀ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.24 (d,
.
1H, H5, J5–7 = 2.6 Hz), 8.04 (s, 1H, H2), 7.60 (dd, 1H, H7, J7–8
=
4.1.1.3. (E)-6-Chloro-3-(4-chlorostyryl)-4H-chromen-4-one (1c).
Yield: 47%, mp = 204–206 °C from ethyl acetate. IR (KBr): 1642
8.9 Hz, J5–7 = 2.6 Hz), 7.52–7.41 (m, 4H, H
c
, H8, H20, H60), 7.34 (t,
2H, H30, H50, J2 –3 = J3 –4 = 7.5 Hz), 7.24 (t, 1H, H40, J3 –4 = 7.5 Hz),
6.91 (dd, 1H, Hb, J ꢀb = 15.6 Hz, Jbꢀ = 10.6 Hz), 6.72 (d, 1H, Ha,
0
0
0
0
0
0
cmꢀ1
.
1H NMR (CDCl3, 400 MHz): d (ppm) 8.25 (d, 1H, H5,
a
c
J5–7 = 2.5 Hz), 8.09 (s, 1H, H2), 7.63–7.58 (m, 2H, H7, Hb), 7.46–
J
ꢀb = 15.6 Hz), 6.51 (d, 1H, Hd, J ꢀd = 15.6 Hz). 13C NMR (CDCl3,
a
c
7.44 (m, 3H, H20, H60, H8), 7.32 (d, 2H, H30, H50, J2 –3
=
100 MHz): d (ppm) 175.2, 154.1, 152.9, 137.2, 134.0, 133.7,133.0,
131.2, 129.4, 128.7, 127.8, 126.5, 125.7, 125.0, 122.2, 122.0,
119.8. Anal. Calcd for C19H13ClO2: C, 73.91; H, 4.24; Cl, 11.48.
Found: C, 73.77; H, 4.44; Cl, 11.62.
0
0
J3 –4 = 8.4 Hz), 6.90 (d, 1H, Ha
, J ꢀb = 16.3 Hz). 13C NMR (CDCl3,
a
0
0
100 MHz): d (ppm) 175.3, 154.1, 153.2, 135.7, 133.8, 133.7,
131.4, 131.0, 128.9, 127.8, 125.6, 125.0, 121.6, 119.8, 119.2. Anal.
Calcd for C17H10Cl2O2: C, 64.38; H, 3.18; Cl, 22.36. Found: C,
64.50; H, 3.15; Cl, 22.60.
4.1.2. General procedure for the synthesis of 2H-chromene-3-
carbaldehydes (5a,d)
4.1.1.4. (E)-6-Chloro-3-styryl-4H-chromen-4-one (1d). Yield:
Acrolein (75 mmol) was added dropwise to a suspension of
the appropriate 2-hydroxybenzaldehyde (50 mmol) and K2CO3
(79 mmol) in dry dioxane (115 mL). The mixture was refluxed for
4 h. After cooling, the solvent was removed under reduced pressure
and the residue was distributed between dichloromethane and 2 N
NaOH. The organic phase was washed with brine, dried over anhy-
drous Na2SO4, filtered and evaporated to dryness. The residue was
purified by column chromatography on silica gel eluting with a
1:1 mixture of dichloromethane and light petroleum (1:1).
52%, mp = 198–202 °C from ethyl alcohol. IR (KBr): 1640 cmꢀ1 1H
.
NMR (CDCl3, 400 MHz): d (ppm) 8.26 (d, 1H, H5, J5–7 = 2.5 Hz),
8.11 (s, 1H, H2), 7.63–7.58 (m, 2H, H7, Hb), 7.52 (d, 2H, H20, H60,
J2 –3 = 7.9 Hz), 7.44 (d, 1H, H8, J7–8 = 8.9 Hz), 7.36 (t, 2H, H30, H50,
0
0
J2 –3 = J3 –4 = 7.9 Hz), 7.27 (t, 1H, H40, J3 –4 = 7.9 Hz), 6.96 (d, 1H,
0
0
0
0
0
0
Ha
, J –b = 16.4 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm) 175.4,
a
154.2, 153.0, 137.2, 133.7, 132.2, 131.3, 128.7, 128.0, 126.7,
125.6, 125.0, 122.0, 119.8, 118.5. Anal. Calcd for C17H11ClO2: C,
72.22; H, 3.92; Cl, 12.54. Found: C, 72.43; H, 3.82; Cl, 12.42.
4.1.2.1. 2H -Chromene-3-carbaldehyde (5a). Yield: 70%, mp =
41–42 °C (lit. 42–43 °C).26 The compound exhibited spectroscopic
data identical to those previously reported.26
4.1.1.5. 3-[(1E,3E)-4-Phenylbuta-1,3-dienyl]-4H-chromen-4-one
(2a). Yield: 46%, mp = 139–140 °C from ethyl alcohol. IR (KBr):
1644 cmꢀ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.29 (dd, 1H, H5,
.
J5–6 = 8.0 Hz, J5–7 = 1.7 Hz), 8.05 (s, 1H, H2), 7.67 (ddd, 1H, H7,
J6–7 = 7.1 Hz, J7–8 = 8.6 Hz, J5–7 = 1.7 Hz), 7.53–7.40 (m, 5H, H , H6,
H8, H20, H60), 7.33 (t, 2H, H30, H50, J2 –3 = J3 –4 = 7.4 Hz), 7.24 (t,
4.1.2.2. 6-Chloro-2H-chromene-3-carbaldehyde (5d). Yield:
81%, mp = 92–93 °C (lit. 93–95 °C).25 The compound exhibited
spectroscopic data identical to those previously reported.25
c
0
0
0
0
1H, H40, J3 –4 = 7.4 Hz), 6.92 (dd, 1H, Hb, J –b = 15.4 Hz, Jb–
=
0
0
a
c
10.6 Hz), 6.71 (d, 1H,
H
a
,
J
–b = 15.4 Hz), 6.54 (d, 1H, Hd,
4.1.3. General procedure for the synthesis of the (E)-3-styryl-2H-
chromenes (3a–d) and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-
chromenes (4a,d)
a
J
–d = 15.6 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm) 176.4, 155.8,
c
152.9, 137.3, 133.6, 133.4, 132.5, 129.6 128.6, 127.7, 126.5, 126.3,
125.2, 124.1, 122.8, 121.9, 118.0. Anal. Calcd for C19H14O2: C,
83.19; H, 5.14. Found: C, 83.32; H, 5.21.
A
solution of the appropriate diethyl benzylphosphonate
(11 mmol) or diethyl trans-cinnamylphosphonate (11 mmol) in
dry THF (30 mL) was added dropwise to a stirred suspension of
sodium hydride (11 mmol) in dry THF (15 mL) under nitrogen.
The mixture was stirred for 10 min at room temperature, then a
solution of the appropriate 2H-chromene-3-carbaldehyde (5a,d)
(10 mmol) in dry THF (15 mL) was added dropwise. After stirring
for 20 h at room temperature, water was added to the mixture
and THF was removed under reduced pressure. The solid collected
by filtration was washed with water, and purified by column chro-
matography on silica gel eluting with a mixture of dichlorometh-
ane and light petroleum (1:1 for 3a–d, and 1:3 for 4a,d).
4.1.1.6. 3-[(1E,3E)-4-(4-Chlorophenyl)buta-1,3-dienyl]-4H-chro-
men-4-one (2b). Yield: 48%, mp = 170–171 °C from ethyl alcohol.
IR (KBr): 1643 cmꢀ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.29 (dd,
.
1H, H5, J5–6 = 7.9 Hz, J5–7 = 1.7 Hz), 8.04 (s, 1H, H2), 7.67 (ddd, 1H,
H7, J6–7 = 7.2 Hz, J7–8 = 8.4 Hz, J5–7 = 1.7 Hz), 7.54–7.40 (m, 3H, H
c,
H6, H8), 7.36 (d, 2H, H20, H60, J2 –3 = 8.2 Hz), 7.29 (d, 2H, H30, H50,
0
0
0
0
J2 –3 = 8.2 Hz), 6.87 (dd, 1H, Hb, J –b = 15.4 Hz, Jb– = 10.6 Hz), 6.65
a
c
(d, 1H, Ha
, J –b = 15.4 Hz), 6.53 (d, 1H, Hd, J –d = 15.9 Hz). 13C
a
c
NMR (CDCl3, 100 MHz): d (ppm) 176.5, 155.8, 153.1, 135.8, 133.5,
133.2, 132.3, 132.1, 130.2, 128.8, 127.6, 126.3, 125.3, 124.1,
123.5, 121.8, 118.0. Anal. Calcd for C19H13ClO2: C, 73.91; H, 4.24;
Cl, 11.48. Found: C, 74.10; H, 4.32; Cl, 11.56.
4.1.3.1. (E)-3-Styryl-2H-chromene (3a). Yield: 47%, mp = 122-
124 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d (ppm) 7.44
(d, 2H, H20, H60, J2 –3 = 7.6 Hz), 7.34 (t, 2H, H30, H50, J2 –3 = J3 –4
=
=
=
0
0
0
0
0
0
7.6 Hz), 7.25 (t, 1H, H40, J3 –4 = 7.6 Hz), 7.11 (dt, 1H, H7, J6–7
J7–8 = 7.5 Hz, J5–7 = 1.5 Hz), 7.03 (dd, 1H, H5, J5–6 = 7.5 Hz, J5–7
0
0
4.1.1.7. 6-Chloro-3-[(1E,3E)-4-(4-chlorophenyl)buta-1,3-dienyl]-
4H-chromen-4-one (2c). Yield: 50%, mp = 176–178 °C from ethyl
acetate. IR (KBr): 1639 cmꢀ1
.
1H NMR (CDCl3, 400 MHz): d (ppm)
1.5 Hz), 6.91–6.81 (m, 3H, H6, H8, Hb), 6.52 (s, 1H, H4), 6.44 (d,
1H,
–b = 16.5 Hz), 4.83 (d, 2H, H2). 13C NMR (CDCl3,
100 MHz): d (ppm) 153.8, 137.0, 130.7, 129.1, 128.8, 128.0,
8.25 (d, 1H, H5, J5–7 = 2.6 Hz), 8.04 (s, 1H, H2), 7.61 (dd, 1H, H7,
J7–8 = 8.9 Hz, J5–7 = 2.6 Hz), 7.49 (m, 1H, H , Jb– = 10.6 Hz, J
Ha, J
a
c
=
c–d
c

6486
C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
127.9, 127.0, 126.6, 126.4, 124.0, 122.8, 121.5, 115.5, 65.6. Anal.
Calcd for C17H14O: C, 87.15; H, 6.02. Found: C, 86.01; H, 6.22.
122.3, 116.7, 65.6. Anal. Calcd for C19H14Cl2O: C, 69.32; H, 4.29;
Cl, 21.54. Found: C, 69.10; H, 4.42; Cl, 21.65.
4.1.3.2. (E)-3-(4-Chlorostyryl)-2H-chromene (3b). Yield: 42%,
mp = 167–170 °C from ethyl acetate. 1H NMR (CDCl3, 400 MHz): d
4.1.4. General procedure for the synthesis of (E)-3-(2H-chromen-
3-yl)acrylaldehydes (6a,d)
(ppm) 7.36 (d, 2H, H20, H60, J2 –3 = 8.5 Hz), 7.30 (d, 2H, H30, H50,
0
0
tert-BuOK (13 mmol) was added to a cooled (0 °C) and magnet-
ically stirred suspension of [(1,3-dioxolan-2-yl)methyl]triphenyl-
phosphonium bromide (13 mmol) in dry THF (100 mL). After
30 min, a solution of the appropriate 2H-chromene-3-carbalde-
hyde (5a,d) (10 mmol) in dry THF (25 mL) was added dropwise.
The mixture was stirred at room temperature for 2 h and refluxed
for 6 h. After cooling, a solution of oxalic acid (10 g) in water
(100 mL) was added and the mixture stirred overnight at room
temperature. THF was removed under reduced pressure and the
mixture extracted with Et2O. The combined organic phases were
washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, filtered and evaporated to dryness. The residue
was purified by column chromatography on silica gel eluting with
dichloromethane/light petroleum (1:1) (6b) or dichloromethane
(6c).
0
0
J2 –3 = 8.5 Hz), 7.11 (ddd, 1H, H7, J6–7 = 7.5 Hz, J7–8 = 8.0 Hz, J5–7
=
1.7 Hz), 7.03 (dd, 1H, H5, J5–6 = 7.5 Hz, J5–7 = 1.7 Hz), 6.88 (dt, 1H,
H6, J5–6 = J6–7 = 7.5 Hz, J6–8 = 1.1 Hz), 6.84–6.79 (m, 2H, H8, Hb),
6.52 (s, 1H, H4), 6.37 (d, 1H, Ha, J –b = 16.5 Hz), 5.07 (d, 2H, H2).
a
13C NMR (CDCl3, 100 MHz): d (ppm) 153.8, 135.5, 133.4, 130.4,
129.3, 128.9, 127.6, 127.2, 127.0, 126.6, 124.6, 122.6, 121.6,
115.5, 65.5. Anal. Calcd for C17H13ClO: C, 75.98; H, 4.88; Cl,
13.19. Found: C, 76.37; H, 4.99; Cl, 13.02.
4.1.3.3.
(E)-6-Chloro-3-(4-chlorostyryl)-2H-chromene
(3c).
Yield: 64%, mp = 186–190 °C from ethyl acetate. 1H NMR (CDCl3,
400 MHz): d (ppm) 7.37 (d, 2H, H20, H60, J2 –3 = 8.4 Hz), 7.30 (d,
0
0
2H, H30, H50, J2 –3 = 8.5 Hz), 7.05 (dd, 1H, H7, J7–8 = 8.5 Hz, J5–7
2.3 Hz), 7.00 (d, 1H, H5, J5–7 = 2.3 Hz), 6.81 (d, 1H, Hb, J
=
=
0
0
a
–b
16.5 Hz), 6.75 (d, 1H, H8, J7–8 = 8.5 Hz), 6.45 (s, 1H, H4), 6.40 (d,
–b = 16.5 Hz), 5.06 (d, 2H, H2). 13C NMR (CDCl3,
4.1.4.1. (E)-3-(2H-Chromen-3-yl)acrylaldehyde (6a). Yield: 46%,
mp = 173–176 °C. IR (KBr): 2840, 2720, 1700 cmꢀ1. 1H NMR (CDCl3,
400 MHz): d (ppm) 9.63 (d, 1H, CHO, Jb–CHO = 7.6 Hz), 7.25–7.15 (m,
1H,
Ha, J
a
100 MHz): d (ppm) 152.2, 135.2, 133.8, 131.6, 129.0, 128.7,
127.7, 127.6, 126.7, 126.3, 124.2, 123.2, 116.8, 115.1, 65.7. Anal.
Calcd for C17H12Cl2O: C, 67.35; H, 3.99; Cl, 23.39. Found: C,
67.52; H, 4.08; Cl, 23.47.
2H, H7, Ha), 7.14 (d, 1H, H5, J5–6 = 7.8 Hz), 6.95–6.84 (m, 3H, H4,
H6, H8), 6.05 (dd, 1H, Hb, J –b = 16.2 Hz, Jb–CHO = 7.6 Hz), 5.00 (s,
a
2H, H2). 13C NMR (CDCl3, 100 MHz): d (ppm) 193.1, 155.0, 147.6,
131.2, 131.0, 129.8, 128.5, 127.9, 126.8, 121.9, 117.5, 65.0. Anal.
Calcd for C12H10O2: C, 77.40; H, 5.41. Found: C, 77.63; H, 5.24.
4.1.3.4. (E)-6-Chloro-3-styryl-2H-chromene (3d). Yield: 90%,
mp = 154–157 °C from ethyl acetate. 1H NMR (CDCl3, 400 MHz): d
(ppm) 7.44 (d, 2H, H20, H60, J2 –3 = 7.6 Hz), 7.35 (t, 2H, H30, H50,
0
0
4.1.4.2. (E)-3-(6-Chloro-2H-chromen-3-yl)acrylaldehyde (6d).
J2 –3 = J3 –4 = 7.6 Hz), 7.26 (t, 1H, H40, J3 –4 = 7.6 Hz), 7.04 (dd, 1H,
H7, J7–8 = 8.5 Hz, J5–7 = 2.5 Hz), 6.99 (d, 1H, H5, J5–7 = 2.5 Hz), 6.84
Yield: 49%, mp = 155–156 °C. IR (KBr): 2820, 2730, 1685 cmꢀ1 1H
.
0
0
0
0
0
0
NMR (CDCl3, 400 MHz): d (ppm) 9.63 (d, 1H, CHO, Jb–CHO = 7.5 Hz),
(d, 1H, Hb J –b = 16.5 Hz), 6.75 (d, 1H, H8, J7–8 = 8.5 Hz), 6.47 (d,
a
7.19–7.14 (m, 2H, H7, H ), 7.08 (d, 1H, H5, J = 2.3 Hz), 6.80–6.77
a
5–7
1H, Ha
, J –b = 16.5 Hz), 6.43 (s, 1H, H4), 5.08 (d, 2H, H2). 13C
a
(m, 2H, H4, H8), 6.08 (dd, 1H, Hb, J –b = 15.9 Hz, Jb–CHO = 7.5 Hz),
a
4.99 (s, 2H, H2). 13C NMR (CDCl3, 100 MHz): d (ppm) 193.0,
153.1, 147.8, 131.1, 131.0, 129.6, 127.9, 127.6, 126.7, 122.8,
117.4, 65.1. Anal. Calcd for C12H9ClO2: C, 65.32; H, 4.11; Cl,
16.07. Found: C, 65.11; H, 4.23; Cl, 16.37.
NMR (CDCl3, 100 MHz): d (ppm) 152.2, 136.7, 131.8, 128.9, 128.8,
128.5, 128.1, 126.5, 126.2, 126.1, 124.1, 122.7, 116.7, 65.7. Anal.
Calcd for C17H13ClO: C, 75.98; H, 4.88; Cl, 13.19. Found: C, 76.25;
H, 5.01; Cl, 13.29.
4.1.5. General procedure for the synthesis of 3-[(1E,3E)-4-(4-
chlorophenyl)buta-1,3-dienyl]-2H-chromenes (4b,c)
4.1.3.5. 3-[(1E,3E)-4-Phenylbuta-1,3-dienyl]-2H-chromene (4a).
Yield: 35%, mp = 162–167 °C from ethyl acetate. 1H NMR (CDCl3,
A solution of diethyl 4-chlorobenzylphosphonate (11 mmol) in
dry THF (30 mL) was added dropwise to a stirred suspension of
sodium hydride (11 mmol) in dry THF (15 mL) under nitrogen.
The mixture was stirred 10 min at room temperature, then a
solution of the appropriate (E)-3-(2H-chromen-3-yl)acrylaldehyde
(6a,d) (10 mmol) in dry THF (15 mL) was added dropwise. After
stirring for 22 h at room temperature, water was added to the
mixture and THF removed under reduced pressure. The solid ob-
tained was removed by filtration, and crystallized from ethyl ace-
tate (4b) or purified by column chromatography on silica gel
eluting with a 1:4 mixture of ethyl acetate and light petroleum
(4c).
400 MHz): d (ppm) 7.42 (d, 2H, H20, H60, J2 –3 = 7.7 Hz), 7.33 (t,
0
0
2H, H30, H50, J2 –3 = J3 –4 = 7.7 Hz), 7.23 (t, 1H, H40, J3 –4 = 7.7 Hz),
7.10 (dt, 1H, H7, J6–7 = J7–8 = 7.9 Hz, J5–7 = 1.6 Hz), 7.01 (dd, 1H,
H5, J5–6 = 7.4 Hz, J5–7 = 1.6 Hz), 6.90–6.80 (m, 3H, H6, H8, Hb),
0
0
0
0
0
0
6.63 (d, 1H, H
a, J –b = 15.5 Hz), 6.43 (s, 1H, H4), 6.42 (d, 1H, Hd,
a
Jd– = 15.7 Hz), 6.31 (dd, 1H, H
c
c
, Jd– = 15.7 Hz, Jb– = 10.2 Hz),
c c
5.02 (s, 2H, H2). 13C NMR (CDCl3, 100 MHz): d (ppm) 153.7,
137.2, 133.4, 130.9, 130.6, 129.1, 129.0, 128.8, 128.7, 127.7,
126.9, 126.4, 123.6, 122.9, 121.5, 115.5, 65.5. Anal. Calcd for
C19H16O: C, 87.66; H, 6.19. Found: C, 87.90; H, 6.22.
4.1.3.6. 6-Chloro-3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chro-
mene (4d). Yield: 43%, mp = 152–158 °C from ethyl acetate. 1H
4.1.5.1. 3-[(1E,3E)-4-(4-Chlorophenyl)buta-1,3-dienyl]-2H-chro-
mene (4b). Yield: 60%, mp = 203–204 °C from ethyl acetate. 1H
NMR (CDCl3, 400 MHz): d (ppm) 7.42 (d, 2H, H20, H60, J2 –3
=
0
0
NMR (CDCl3, 400 MHz): d (ppm) 7.33 (d, 2H, H20, H60, J2 –3
=
7.7 Hz), 7.33 (t, 2H, H30, H50, J2 –3 = J3 –4 = 7.7 Hz), 7.24 (t, 1H, H40,
0
0
0
0
0
0
8.5 Hz), 7.28 (d, 2H, H30, H50, J2 –3 = 8.5 Hz), 7.10 (dt, 1H, H7,
J6–7 = J7–8 = 7.8 Hz, J5–7 = 1.6 Hz), 7.01 (dd, 1H, H5, J5–6 = 7.4 Hz,
0
0
0
0
J3 –4 = 7.7 Hz), 7.03 (dd, 1H, H7, J7–8 = 8.5 Hz, J5–7 = 2.5 Hz), 6.97
(d, 1H, H5, J5–7 = 2.5 Hz), 6.86 (dd, 1H, Hb, J –b = 15.6 Hz, Jb–
=
c
a
J5–7 = 1.6 Hz), 6.90–6.78 (m, 3H, H6, H8, Hb), 6.57 (d, 1H, Ha,
9.5 Hz), 6.74 (d, 1H, H8, J7–8 = 8.5 Hz), 6.65 (d, 1H,
Ha,
J
–b = 15.6 Hz), 6.46 (s, 1H, H4), 6.43 (d, 1H, Hd, Jd– = 15.6 Hz),
J
–b = 15.6 Hz), 6.41 (d, 1H, Hd, Jd– = 15.8 Hz), 6.36 (s, 1H, H4),
a
c
a
c
6.34 (dd, 1H, H
c
, Jd– = 15.8 Hz, Jb– = 9.5 Hz), 5.01 (s, 2H, H2). 13C
6.31 (dd, 1H, H
c
, Jd– = 15.6 Hz, Jb– = 10.2 Hz), 5.01 (s, 2H, H2).
c
c
c
c
13C NMR (CDCl3, 100 MHz): d (ppm) 153.8, 135.7, 133.3, 131.9,
NMR (CDCl3, 100 MHz): d (ppm) 152.2, 137.0, 134.1, 132.0, 130.0,
131.1, 130.8, 129.6, 129.2, 128.9, 128.5, 127.6, 126.9, 124.0,
129.8, 128.8, 128.7, 128.5, 127.9, 126.5, 126.3, 126.2, 124.3,

C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
6487
122.8, 121.6, 115.5, 65.5. Anal. Calcd for C19H15ClO: C, 77.42; H,
5.13; Cl, 12.03. Found: C, 77.29; H, 5.24; Cl, 12.22.
alterations on the third day of incubation in the presence of com-
pounds. The highest concentration of compound that did not pro-
duce any modification of morphology and viability on 100% of cells
was the maximum non-cytotoxic concentration. The 50% cytotoxic
concentration (TC50) was indicated as the concentration of com-
pound reducing the cell viability by 50%, as compared with
mock-treated cells.
4.1.5.2. 6-Chloro-3-[(1E,3E)-4-(4-chlorophenyl)buta-1,3-dienyl]-
2H-chromene (4c). Yield: 67%, mp = 193–194 °C from ethyl ace-
1
tate. H NMR (CDCl3, 400 MHz): d (ppm) 7.34 (d, 2H, H20, H60,
J2 –3 = 8.6 Hz), 7.29 (d, 2H, H30, H50, J2 –3 = 8.6 Hz), 7.03 (dd, 1H,
H7, J7–8 = 8.5 Hz, J5–7 = 2.5 Hz), 6.98 (d, 1H, H5, J5–7 = 2.5 Hz), 6.83
0
0
0
0
(dd, 1H, Hb, J –b = 15.6 Hz, Jb– = 10.1 Hz), 6.74 (d, 1H, H8, J7–8
=
a
c
4.2.5. Determination of the 50% inhibitory concentration (IC50
)
8.5 Hz), 6.59 (d, 1H,
Ha
,
J
–b = 15.6 Hz), 6.42 (d, 1H, Hd,
a
Confluent monolayers of HeLa or Hep-2 cells in 6-well plates
were infected with a virus suspension producing approximately
100 plaques per well. After 1 h of incubation at 33 °C for HRV or
1 h at 37 °C for EV 71, the virus inoculum was removed and the
cells were overlaid with medium for plaques, in the presence or
absence of fourfold dilution of drugs. After three days of incubation
at 33 °C (HRV) or two days at 37 °C (EV 71), the cells were stained
with a neutral red solution at 0.2 mg/mL in pH 7.4 phosphate buf-
fered saline (PBS) and the plaques were counted. The IC50 value
was the concentration of drug reducing the plaque number by
50% as compared with mock-treated control. It was calculated
from a dose/response curve obtained by plotting the percentage
of plaque reduction, with respect to the control plaque count,
versus the logarithm of compound dose. Triplicate wells were
utilized for each drug concentration.
Jd– = 15.7 Hz), 6.37 (s, 1H, H4), 6.32 (dd, 1H, Hc, Jd– = 15.7 Hz,
c
(ppm) 152.2, 135.6, 133.5, 132.6, 131.9, 130.6, 129.4, 129.3,
128.9, 128.6, 127.6, 126.3, 126.2, 124.2, 122.7, 116.7, 65.6. Anal.
Calcd for C19H14Cl2O: C, 69.32; H, 4.29; Cl, 21.54. Found: C,
69.10; H, 4.42; Cl, 21.65.
c
c
Jb– = 10.1 Hz), 5.01 (s, 2H, H2). 13C NMR (CDCl3, 100 MHz): d
4.2. Virology
4.2.1. Cells
HeLa (Ohio) cells were grown at 37 °C in a humidified atmo-
sphere of 5% CO2 using Eagle’s minimum essential medium
(MEM) supplemented with 100 lg/mL of streptomycin, 100 U/mL
of penicillin G and 8% heat-inactivated foetal calf serum (FCS)
(growth medium). Hep-2 (human epithelioma) cells were cultured
as above using Dulbecco’s modified Eagle’s medium (high glucose).
The serum concentration was reduced to 2% for cell maintenance
(maintenance medium).
4.2.6. Virus inactivation and stabilization
For virus inactivation studies, HRV 1B suspensions with or with-
out 3a (20 lM) were incubated at 33 °C for 1 h. After serial 10-fold
dilutions, virus titres were measured by plaque assay on HeLa cell
4.2.2. Compounds
monolayers.
Stock solutions of compounds were prepared in ethanol (1, 0.5
or 0.1 mg/mL) and further diluted in tissue culture medium shortly
before use.
For virus stabilization studies, the virus was incubated with or
without 3a (20 lM) for 1 h at 33 °C before mild acid or thermal
treatment. For mild acid treatment, the pH of the mixtures was ad-
justed to five by adding 0.2 M acetate buffer (pH 5). After incuba-
tion at 33 °C for 30 min, the mixtures were neutralized with
0.85 M Tris base. For thermal treatment, the mixtures were incu-
bated for 20 min at 56 °C (pH 7.2) and then refrigerated on ice.
All samples were diluted 10-fold serially and titrated by plaque
assay on HeLa cell monolayers.
4.2.3. Viruses
Reference strains of HRV type 1B and 14 were purchased from
American Type Culture Collection (ATCC). Virus stocks were pre-
pared by inoculating HeLa (Ohio) cell monolayers at a low multiplic-
ity of infection (0.1 PFU/cell) for 1 h at 33 °C. Infected cells were then
incubated at 33 °C in maintenance medium. When the viral-induced
cytopathic effect involved most of the cells, the cultures were freeze-
thawed three times and the clarified supernatants stored in aliquots
at ꢀ80 °C until use. The virus was titrated by plaque assay, essen-
tially the same as described by Fiala and Kenny.35
4.2.7. Virus yield reduction assays
Confluent monolayers of HeLa cells in 24-well plates were
infected at a multiplicity of five in the presence or absence of 3a
(20 lM). The infection was synchronized by allowing HRV 1B to
attach in the cold (4 °C). After 1 h, the inoculum was removed by
EV71 reference strain (ATCC, VR-784) was propagated and titrated
by plaque assay in Hep-2 cells at 37 °C as previously reported.15
washing thrice with PBS. The end of virus binding is indicated as
0 time. Then, MEM with or without the compound (20 lM) was
4.2.4. XTT assay for cellular cytotoxicity
A tetrazolium-based (XTT) colorimetric assay was used to mea-
sure the cytotoxicity of compounds. This assay is based on the
cleavage of the yellow tetrazolium salt XTT to form an orange for-
mazan dye by viable, active cells. The formazan dye formed is sol-
uble in aqueous solution and is directly quantified using a scanning
multiwell spectrophotometer.27 Cells were seeded in 96-well tis-
added and the temperature raised to 33 °C to permit internaliza-
tion. Single-cycle conditions were achieved by incubating the cells
at 33 °C for 10 h post-infection (p.i.). The cultures were freeze-
thawed three times, cell debris removed by low-speed centrifuga-
tion in the cold and the supernatants titrated by plaque assay on
HeLa cell monolayers. To determine which stage of virus replica-
tion was affected by 3a, the drug was added or removed from
HRV-infected cells at various times p.i. (0.5, 1, 2, 4 h) and the
cultures incubated at 33 °C up to 10 h p.i. The virus yield was
determined as above.
sue culture plates at 2 ꢂ 103 cells for each well in 100
lL of growth
medium with or without compounds in twofold dilutions, starting
from the maximum soluble concentration in cell culture medium.
Quadruplicate wells were used for each drug concentration to be
tested. The plates were incubated at 37 °C in 5% CO2-air until the
untreated monolayers were confluent (3 days). Then, 50 lL of
XTT labelling mixture was added to each well (final XTT concentra-
tion 0.15 mg/mL) and the cells incubated for 4 h at 37 °C. The spec-
trophotometric absorbance of the samples was measured using an
ELISA reader at 492 nm with a reference wavelength at 690 nm.
Cytotoxicity was also scored microscopically as morphological
Acknowledgement
We gratefully acknowledge the financial support from ‘Istituto
Pasteur-Fondazione Cenci Bolognetti’, Università degli Studi di
Roma ‘La Sapienza’.

6488
C. Conti, N. Desideri / Bioorg. Med. Chem. 18 (2010) 6480–6488
15. Genovese, D.; Conti, C.; Tomao, P.; Desideri, N.; Stein, M. L.; Catone, S.; Fiore, L.
Supplementary data
Antiviral Res. 1995, 27, 123.
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Chemother. 1997, 8, 545.
17. Quaglia, M. G.; Desideri, N.; Boss, E.; Sgro, R.; Conti, C. Chirality 1999,
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19. Conti, C.; Tomao, P.; Genovese, D.; Desideri, N.; Stein, M. L.; Orsi, N. Antimicrob.
Agents Chemother. 1992, 36, 95.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.103. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
20. Salvati, A. L.; De Dominicis, A.; Tait, S.; Canitano, A.; Lahm, A.; Fiore, L.
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