Design, synthesis and in vitro evaluation of novel chroman-4-one, chroman, and 2H-chromene derivatives as human rhinovirus capsid-binding inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.10.060

As part of an effort to generate broad-spectrum inhibitors of rhinovirus replication, novel series of (E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a-e, (E)-3-(3-phenylprop-2-yn-1-ylidene)chroman-4-ones 2a and 2b, (Z)-3-[(E)-3-phenylallylidene]chromans 3a

Full text of DOI:10.1016/j.bmc.2011.10.060

Bioorganic & Medicinal Chemistry 19 (2011) 7357–7364
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and in vitro evaluation of novel chroman-4-one, chroman,
and 2H-chromene derivatives as human rhinovirus capsid-binding inhibitors
Cinzia Conti a, Luca Proietti Monaco b, 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 Rome, 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:
As part of an effort to generate broad-spectrum inhibitors of rhinovirus replication, novel series of (E)-3-
[(E)-3-phenylallylidene]chroman-4-ones 1a–e, (E)-3-(3-phenylprop-2-yn-1-ylidene)chroman-4-ones 2a
and 2b, (Z)-3-[(E)-3-phenylallylidene]chromans 3a–e, and (E)-3-(3-phenylprop-1-en-1-yl)-2H-chrom-
enes 4a–d were designed and synthesized. All the compounds were tested in vitro for their efficacy
against infection by human rhinovirus (HRV) 1B and 14, two representative serotypes for rhinovirus
group B and A, respectively. Most of the analogues were found to be potent and selective inhibitors of
both HRVs, although HRV 1B was generally more susceptible than HRV 14. Mechanism of action studies
of (E)-6-chloro-3-(3-phenylprop-1-en-1-yl)-2H-chromene 4b, the most potent compound on HRV 1B
infection, suggested that 4b behaves as a capsid-binder probably acting at the uncoating level.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 12 July 2011
Revised 18 October 2011
Accepted 19 October 2011
Available online 26 October 2011
Keywords:
Chroman-4-ones
Chromans
2H-Chromenes
Rhinoviruses
Capsid-binders
1. Introduction
respectively. Several of these inhibitors presented submicromolar
potency against HRV 1B coupled with high therapeutic index. On
Human rhinoviruses (HRVs), members of the Picornaviridae
family, represent the most frequent etiological agents of the com-
mon cold and are associated with upper respiratory tract complica-
tions such as otitis media and sinusitis.1 Although HRVs often
induce a mild and self-limited respiratory illness in humans, the
prevalence and the recurrent nature of HRV infections explain their
high medical and socio-economic impact in term of healthcare and
lost productivity. In addition, HRV infections can also affect the
lower respiratory airways and can exacerbate chronic respiratory
disorders such as asthma and chronic obstructive pulmonary dis-
ease.2,3 Since the multiplicity of serotypes makes the development
of an effective vaccine impracticable, constant efforts have been
devoted to the discovery and development of efficacious antiviral
agents. However, at present, no antiviral therapy has been ap-
proved for the treatment of rhinovirus infections.
Pursuing our researches on small molecules with anti-picornavirus
activity, we recently devoted our attention to a series of (Z)-3-ben-
zylidenechromans, 3-benzyl-2H-chromenes and 3-benzylchro-
mans4 related to the most active synthetic 3(2H)-isoflavenes5–8
and homoisoflavones9–11 previously studied by us. The antiviral po-
tency of these compounds was evaluated in vitro against HRV 1B and
14, selected as representative serotypes for group B and A of HRVs,
the contrary, HRV 14 infection was only weakly inhibited.4 Similarly
to related flavanoids,8,12,13 these analogues behaved as capsid-bind-
ers and interfered with very early events of HRV multiplication.4 The
explanation for the different sensibility of HRV 1B and 14 to these
chromans could reside in the size of the compound-binding site
which differs depending upon the specific serotype. Previous re-
search on capsid-binding compounds demonstrated that viral group
B binding site accommodates molecules with shorter chains, while
long-chained compounds are routinely more active against group
A.14
To develop compounds with broad-spectrum activity, we mod-
ified the linker chain length between the heterocycle and the phe-
nyl moiety. A linear, unsaturated chain containing two or four
carbon atoms was introduced as a linker. Optimum activity against
both HRV serotypes was achieved with the unsaturated 2-carbon
chain analogues, (E)-3-styryl-2H-chromenes. Surprisingly, despite
the larger size of the HRV 14 capsid binding site, elongation of
the linker chain from two to four carbon atoms resulted in a loss
of activity against this serotype.15 Mechanism of action studies
indicated that also these compounds behaved as capsid-binders
interfering with the early stage(s) of rhinovirus infection.15
These observations prompted us to further explore the effect of
some simple structural modification of this scaffold. In the present
paper, we describe the synthesis, antiviral activity and mechanism
of action of new analogues containing an unsaturated 3-carbon
chain between the two rings. The antiviral potency of the new
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.060

7358
C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
O
R'
R'
H
R
1 2a
,
R = R' = H
O
O
1 2b
,
R = Cl, R' = H
R = R' = Cl
O
O
1a-e
(i)
(i)
1c
1d
R = H, R' = Cl
R
1e R = F, R' = H
O
O
H
R
O
2a, b
Scheme 1. Reagents and conditions: (i) 85% H3PO4, 80 °C, 4 h.
(E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a–e, (E)-3-(3-phe-
nylprop-2-yn-1-ylidene)chroman-4-ones 2a and 2b, (Z)-3-[(E)-
3-phenylallylidene]chromans 3a–e, and (E)-3-(3-phenylprop-1-
en-1-yl)-2H-chromenes 4a–d was evaluated against infection of
HeLa cells by HRV 1B and 14, representative serotypes for group B
and A of HRVs, respectively.
O
R'
R
O
1a-e
2. Results and discussion
2.1. Chemistry
(i)
O
R'
(E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a–e and (E)-3-
(3-phenylprop-2-yn-1-ylidene)chroman-4-ones 2a and 2b were
prepared by acid-catalyzed condensation of chroman-4-ones with
trans-cinnamaldehydes and 3-phenylpropiolaldehyde, respec-
tively. The reaction was carried out by heating the mixture in
85% phosphoric acid for 4 h (Scheme 1). The 1H NMR spectra of
the new chromanones 1a–e suggest the trans-configuration of
R
R
3a-e
+
O
R'
R'
the exocyclic double bond. In fact, the signals of Ha and Hb appear
around 7.5 ppm and 7.00 ppm, respectively. In the 1H NMR spectra
of the cis-isomers, these signals would be shifted at higher and
lower field, respectively, due to the effect of carbonyl group. The
4a-e
+
coupling constant values of Hb and Hc (Jb– from 15.3 to 15.5 Hz)
c
indicate also the trans-configuration of this double bond. The ste-
reochemistry of these compounds was confirmed by 2D NOESY
experiments. The strong NOE cross peaks between H2 and Hb
O
R
and between Ha and Hc demonstrate the trans-configuration for
5a-e
both double bonds.
In a similar manner, the chemical shift of the proton in the chain
(around 7.00 ppm) indicates the trans-configuration of the exocy-
clic double bond of (E)-3-(3-phenylprop-2-yn-1-ylidene)chro-
man-4-ones 2a and 2b.
1 3 4 5a
R = R' = H
,
,
,
1 3 4 5b
,
,
,
R = Cl, R' = H
R = R' = Cl
1 3 4 5c
,
, ,
1 3 4 5d
,
,
,
R = H, R' = Cl
Subsequent reaction of (E)-3-[(E)-3-phenylallylidene]chroman-
4-ones 1a–e with lithium aluminum hydride in the presence of alu-
minum chloride, reduced the carbonyl group to a methylene group
with some isomerization ofthe exocyclicdouble bond (5a–e) or both
the double bonds (4a–e) (Scheme 2). The presence of small amounts
of (E)-3-(3-phenylprop-1-en-1-yl)-2H-chromenes 4a–e and 3-cin-
namyl-2H-chromenes 5a–e together with (Z)-3-[(E)-3-(3-chloro-
phenyl)allylidene]chromans 3a–e was evident in the 1H NMR
spectra of the crude reaction products. The main product was
isolated from the mixture by column chromatography and further
purified by crystallization. The stereochemistry of the exocyclic
double bond of (Z)-3-[(E)-3-(3-chlorophenyl)allylidene]chromans
3a–e was established by 2D NOESY experiments. The strong NOE
1, 3, 4, 5e R = F, R' = H
Scheme 2. Reagents and conditions: (i) LIAIH4, AlCl3, dry Et2O, rt 1 h and reflux
2.5 h.
retention of the exocyclic double bond configuration, but the E/Z
descriptors change because of the change in priority at C3. The cou-
pling constant values of Hb and H
NMR spectra suggest the trans-configuration of the second double
bond. The strong NOE cross peaks between H
configuration.
As shown in Scheme 3, (E)-3-(3-phenylprop-1-en-1-yl)-2H-
chromenes 4a–d were conveniently obtained by the Wittig reaction
of the appropriate 2H-chromene-3-carbaldehyde and phenethyltri-
phenylphosphonium bromide, using sodium ethoxide as base. The
coupling constant values of the vinylic protons (J –b = 15.9 Hz) indi-
cate the trans-configuration of the double bond in the chain.
c
(Jb– = 15.3 or 15.4 Hz) in the 1H
c
a and Hc confirm this
cross peaks between H4 and Ha unequivocally indicate the
cis-configuration of this double bond. Therefore, the reduction of
(E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a–e to (Z)-3-[(E)-
3-(3-chlorophenyl)allylidene]chromans 3a–e occurs with the
a

C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
7359
exhibited low cytotoxicity showing TC50 ranging from 25 to
100 M.
R'
O
l
+
O
The dose-dependent inhibitory activity of compounds on HRV
1B and HRV 14 replication was evaluated in vitro by a plaque
reduction assay, starting from the MNTC. A previous systematic
evaluation of a panel of capsid-binding compounds against all
HRVs established the existence of two virus groups, called groups
A and B, with contrasting susceptibilities for these anti-rhinovirus-
es. Group B contains twice as many serotypes as group A, and ac-
counts for five times as many colds as group A serotypes.17,18 In
our research, we utilized HRV 1B and 14 as representative sero-
types for group B and A, respectively. The results of antiviral activ-
ity are expressed as compound concentration required to produce
a 50% reduction of plaque number with respect to mock-treated
virus-infected cultures (IC50) (Table 1). When the IC50 value is
not achieved up to the MNTC, the percentage inhibition at this dose
is reported in parentheses. The therapeutic index (TI), expressed as
TC50 versus IC50 ratio, was calculated and reported in Table 1. 40,6-
Dichloroflavan (BW683C), an inhibitor of group B serotypes, was
included as a control.19
Br-
Ph3P
R
H
(i)
O
R'
R
4a-d
4a
4b
4c
4d
R = R' = H
R = Cl, R' = H
R = R' = Cl
R = H, R' = Cl
Scheme 3. Reagents and conditions: (i) EtONa, absolute EtOH, reflux, 2.5 h.
Although BW683C is at present one of the most active com-
pounds against HRV 1B and group B serotypes, it was generally
inactive against group A of HRVs.17–19 None of the new compounds
was more potent than the reference compound BW683C against
HRV 1B. Differently, they were found to be effective also against
serotype 14 infection indicating a wider anti-HRV spectrum.
Although HRV 1B was generally found more susceptible than
HRV 14 to the action of the new compounds, several derivatives
showed activity in the micromolar range against both HRV sero-
types. Only compounds 1c, 3c and 4a exhibited a higher potency
against serotype 14.
2.2. Antiviral tests
The results of the biological evaluation of the new compounds
(1a–e, 2a, 2b, 3a–e and 4a–d) are presented in Table 1. Each com-
pound was first tested for its effects on morphology, viability and
growth of HeLa cells, a human cell line suitable for the replication
of HRVs. Morphological alterations were scored microscopically,
and the action of the compounds on logarithmic cell growth was
determined by the XTT colorimetric method.16 The cytotoxicity
of compounds is referred as maximum non-cytotoxic concentra-
tion (MNTC) and 50% cytotoxic concentration (TC50). The MNTC
is the highest dose that did not produce any toxic effect or reduc-
tion of cell growth after 3 day incubation at 37 °C. The TC50 is the
concentration of compound reducing the cell viability by 50% as
compared with the control. In general, all the new compounds
In the (E)-3-[(E)-3-phenylallylidene]chroman-4-one series 1a–e,
all the analogues showed potent anti-HRV 1B activity (IC50s ranging
from 0.85 to 4.98 lM) and selectivity (TIs from 7.46 to 50.50). On the
contrary, only 40,6-dichloro and 6-fluoro derivatives (1c and 1e)
exhibited IC50s lower than MNTCs against HRV 14. Notably, com-
pound 1c was the most potent and selective compound among all
Table 1
Cytotoxicity and anti-picornavirus activity of (E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a–e, (E)-3-(3-phenylprop-2-yn-1-ylidene)chroman-4-ones 2a and 2b, (Z)-3-[(E)-3-
phenylallylidene]chromans 3a–e, and (E)-3-(3-phenylprop-1-en-1-yl)-2H-chromenes 4a–d
Comp
R
R0
MNTC (
l
M)a
TC50 (
l
M)b
IC50 (
l
M)c HRV 1B
TId
IC50 (
l
M)c HRV 14
TId
1a
1b
1c
1d
1e
2a
2b
3a
3b
3c
3d
3e
4a
4b
4c
4d
BW683C
H
Cl
Cl
H
F
H
Cl
H
Cl
Cl
H
F
H
Cl
Cl
H
—
H
H
Cl
Cl
H
H
H
H
H
Cl
Cl
H
H
H
Cl
Cl
—
12.50
25.00
12.50
25.00
25.00
25.00
50.00
50.00
25.00
12.50
25.00
50.00
50.00
12.50
25.00
50.00
25.00
25.00
50.00
25.00
50.00
50.00
50.00
75.00
100.00
50.00
25.00
50.00
75.00
75.00
25.00
100.00
100.00
>25.00e
0.85
0.99
3.35
2.54
4.98
9.30
7.89
0.99
2.79
11.49
1.39
5.57
13.59
0.42
9.73
3.61
29.41
50.50
7.46
19.68
10.04
5.38
12.50 (7.2%)
25.00 (48.6%)
1.42
25.00 (17.1%)
17.02
20.00
30.41
47.59
25.00 (30.3%)
8.27
—
—
17.61
—
2.94
2.50
2.47
2.10
—
3.02
8.31
2.05
6.76
—
9.51
101.01
17.92
2.18
35.97
13.46
5.52
59.52
10.28
27.70
>961
6.02
36.51
11.09
12.50 (23.0%)
25.00 (27.0%)
45.10
—
2.22
—
0.026
NAf
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. At this concentration, residual ethanol in culture medium was verified not to be cytotoxic.
b
The TC50 value was the concentration of compound which reduced the HeLa cell viability by 50%, as compared with the control. At this concentration, residual ethanol in
culture medium was verified not to be cytotoxic.
c
The IC50 value was the dose of compound reducing the plaque number by 50% and was calculated by plotting the drug concentration versus the percentage of 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 saturation concentration in cell culture medium was found to be lower than TC50
Not active up to the highest concentration tested (MNTC).
.
e
f
.

7360
C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
the new derivatives against serotype 14 (IC50 = 1.42
while it was about twofold less potent and selective against serotype
1B (IC50 = 3.35 M, TI = 7.46).
When the chroman-4-one ring in 1a-e series was replaced by a
chroman ring to give analogues 3a–e, the inhibitory activity
against serotype 1B was generally retained (IC50s ranging from
l
M, TI = 17.61)
(Fig. 1A) or 56 °C (Fig. 1B) (3.8 and 2.9 log PFU/mL, respectively). In
the presence of 4b, the drop in virus infectivity was significantly
reduced (2.8 and 1.8 log PFU/mL, respectively) and the protective
effect towards low pH and thermal inactivation was 1.0 and 1.1
log, respectively. Exposure of HRVs to mild acid or heat induces
conformational changes of the virion capsid structure similar to
those produced during the uncoating of the viral particles inside
the host cells.20,21 The presence of capsid-binders within the
hydrophobic pocket of VP1 protein results in resistance to acid
and thermal inactivation due to a reduction of capsid flexibil-
ity.22,23 Taken together, our data suggest that 4b could act as a cap-
sid-binder. However, binding of 4b was reversible by dilution as
indicated by results on virus infectivity. A similar behaviour has al-
ready been described for (Z)-3-(4-chlorobenzylidene)chroman and
(E)-3-styryl-2H-chromene, two related compounds recently stud-
ies by us.4,15
l
0.99 to 11.49 lM), and only the 6-chlorochroman 3b exhibited
an IC50 higher than MNTC against serotype 14.
In both (E)-3-[(E)-3-phenylallylidene]chroman-4-one and (Z)-3-
[(E)-3-(3-chlorophenyl)allylidene]chroman series, the replacement
of the chlorine at the 6 position in compounds 1b and 3b with fluo-
rine to give the compounds 1e and 3e resulted in a marked reduc-
tion in activity against HRV 1B. On the contrary, the potency
against serotype 14 was enhanced by this substitution, although
the 6-fluoro analogues 1e and 3e displayed a higher activity
against serotype 1B. A similar behaviour was observed against both
serotypes when the double bond in the chain of (E)-3-[(E)-3-phe-
nylallylidene]chroman-4-ones 1a and 1b was replaced by a triple
bond to give the corresponding (E)-3-(3-phenylprop-2-yn-1-yli-
dene)chroman-4-ones 2a and 2b.
The isomerisation of both double bonds in (Z)-3-[(E)-3-(3-chlo-
rophenyl)allylidene]chromans 3a–d to provide inhibitors 4a–d,
generally led to a significant loss in potency or a modest improve-
ment in activity. The only exception to this generalization was the
6-chloro analogue 4b which was >sixfold more potent than corre-
sponding 3b against HRV 1B and showed the highest inhibitory
The antiviral action of 4b (42 lM) towards different stages of
HRV 1B multiplication in HeLa cells was investigated under one-
step growth conditions. 4b was present: (i) during the entire time
of virus replication, (ii) during virus binding to the cell membrane
only, (iii) added or removed at different time intervals after virus
attachment to the cells in the cold.
The highest level of inhibition (88.5%) was reached when 4b
was added to cells together with the virus inoculum and main-
tained until the end of virus multiplication. A similar effect was ob-
served when 4b was added immediately after virus adsorption
period (1 h at 4 °C, time 0). Also the addition of 4b 15, 30, 45 or
60 min after virus binding still produced a high reduction (above
80%) in virus yield. Instead, when 4b was added at later times (2,
4 or 6 h after virus binding), inhibition significantly dropped
(31%, 12% and 1%, respectively, Fig. 2).
activity (IC50 = 0.42
(TI = 59.52).
lM) coupled with remarkable selectivity
2.3. Mechanism of action studies
(E)-6-Chloro-3-(3-phenylprop-1-en-1-yl)-2H-chromene 4b, the
most potent compound against HRV 1B (IC50 = 0.42 lM), was se-
lected to clarify the mechanism of antiviral action by evaluating
the effects produced on both virus particles and multiplication.
The virus-neutralizing effect of 4b on HRV 1B infectivity was
investigated by incubating a virus suspension at high titre with
Pretreatment of HeLa cells with 4b before HRV infection or the
presence of 4b during the time of virus adsorption only (1 h, 4 °C)
caused only a minimal reduction in virus yield (6% and 16%, respec-
tively) (data not shown). Both results indicate that 4b does not hin-
der cellular receptors for virus nor interfere with HRV 1B
attachment to the host cell membrane.
the compound at a concentration of 42
l
M (100 times the IC50).
In experiments where 4b was added at the end of virus adsorp-
tion (0 time) and removed after 30 min or 1 h treatment of infected
cells (33 °C), it produced a reduction in virus yield of approxi-
mately 35%. A time-dependent increase of inhibition was observed
when the compound was removed after longer incubation times
and the highest inhibition (93%) was achieved when 4b was re-
moved after 6 h of incubation with the infected cells (Fig. 3).
Taken together, our results demonstrate that also (E)-6-chloro-
3-(3-phenylprop-1-en-1-yl)-2H-chromene (4b) exerts an early
activity during HRV multiplication, although it does not interfere
After serial 10-fold dilutions to achieve non inhibitory concentra-
tions of free compound, the infectivity titers of mock- and 4b-trea-
ted virus suspensions were found to be similar (5.33 ꢀ 106 PFU/mL
and 5.11 ꢀ 106 PFU/mL, respectively). These results indicate that
4b does not damage virus particles.
In stabilization studies, 4b (42 lM) significantly protected HRV
1B infectivity against inactivation by both mild acid and heat treat-
ments. As shown in Figure 1, in the absence of 4b, the infectivity of
control virus decreased significantly when exposed to either pH 5
8
8
A
B
7
6
5
4
3
2
1
0
7
HRVC
HRVT
4b
HRVC
HRVT
4b
6
5
4
3
2
1
0
HRVC
HRVT
4b
HRVC
HRVT
4b
HRVC: untreated virus,
HRVC: untreated virus,
HRVT: virus exposed to pH 5.0,
HRVT: virus heated at 56 °C,
4b: virus heated at 56 °C in the presence of 4b.
4b: virus exposed to pH 5.0 in the presence of 4b.
Figure 1. Protective effect of (E)-6-chloro-3-(3-phenylprop-1-en-1-yl)-2H-chromene 4b on acid (A) and thermal inactivation (B) of HRV 1B infectivity.

C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
7361
100
90
80
70
60
50
40
30
20
10
0
chromans 3a–e, and (E)-3-(3-phenylprop-1-en-1-yl)-2H-chrom-
enes 4a–d. As expected several compounds exhibited a wide spec-
trum of anti-HRV activity coupled with a high therapeutic index.
The data obtained from stabilization and time of addition/removal
studies are in agreement with the capsid-binder hypothesis. Time
of addition/removal experiments suggest an interference with the
uncoating process of viral genome without an action on HRV adsorp-
tion to the host cell membrane.
4. Experimental
4.1. Chemistry
Chemicals were purchased from Sigma–Aldrich or Alfa Aesar
and used without further purification. Melting points were deter-
mined on a Stenford Research Systems OptiMelt (MPA-100) appa-
ratus and are uncorrected. 1H NMR and 13C NMR spectra were
detected with a Bruker AM-400 spectrometer, using TMS as inter-
nal standard. IR spectra were recorded on a FT-IR PerkinElmer
Spectrum 1000. All compounds were routinely checked by thin-
layer chromatography (TLC) and 1H NMR. TLC was performed on
silica gel or aluminium oxide fluorescent coated plates. Compo-
nents were visualised by UV light. Elemental analyses (C, H, Cl)
of all new compounds were within 0.4% of theoretical values.
2H-Chromene-3-carbaldehyde,15 6-chloro-2H-chromene-3-carbal-
dehyde,15 phenethyltriphenylphosphonium bromide24 and 4-chlo-
rophenethyltriphenylphosphonium bromide24 were synthesized
following the procedure previously described.
EC
0'
15'
30'
45'
1 h
2 h
4 h
6 h
Figure 2. Effect of varying the time of addition of 4b (42
l
M) on the inhibition of
HRV 1B multiplication under one-step growth conditions. Virus yield was deter-
mined by plaque assay. Virus control titre was 2.4 ꢀ 104 PFU/mL. EC: 4b was
present during the entire infection cycle (1 h at 4 °C and 10 h at 33 °C). 00, 150, 300,
450, 1, 2, 4 and 6 h: compound was added at different times (00, 150, 300, 450, 1, 2, 4,
and 6 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).
100
90
80
70
60
50
40
30
20
10
0
4.1.1. General procedure for the synthesis of the (E)-3-[(E)-3-
phenylallylidene]chroman-4-ones (1a–e) and (E)-3-(3-phenyl-
prop-2-yn-1-ylidene)chroman-4-ones (2a,b)
A mixture of the appropriate chroman-4-one (10 mmol) and
trans-cinnamaldehyde (10 mmol) or 3-phenylpropiolaldehyde
(10 mmol), in 85% phosphoric acid (63 mL) was heated at 80 °C
for 4 h while stirring. After cooling the mixture was diluted with
ice and water. The precipitate was filtered off, washed with water
and crystallized.
15'
30'
45'
1 h
2 h
4 h
6 h
Figure 3. Effect of varying the time of removal of 4b (42
l
M) on the inhibition of
HRV 1B replication under one-step growth conditions. Virus yield was determined
by plaque assay. Virus control titre was 2.4 ꢀ 104 PFU/mL. 150, 300, 450, 1, 2, 4 and
6 h: compound 4b was added after virus adsorption (1 h at 4 °C, time 0) and
removed after different lengths of incubation (150, 300, 450, 1, 2, 4 and 6 h) at 33 °C.
4.1.1.1. (E)-3-[(E)-3-Phenylallylidene]chroman-4-one
(1a). Yield: 80%, mp = 115–119 °C from ethyl alcohol. IR (KBr):
1664 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.01 (dd, 1H, H5,
.
J5–6 = 7.8 Hz, J5–7 = 1.8 Hz), 7.54–7.45 (m, 4H, H20, H60, H7, H
7.40–7.34 (3H, H30–H50), 7.09 (d, 1H, H
, Jb– = 15.5 Hz), 7.08–
6.97 (m, 3H, H6, H8, Hb), 5.26 (d, 2H, H2, J2- = 1.6 Hz). 13C NMR
a),
with cell receptor recognition by the virus. Maximal reduction in
virus yield is achieved when 4b is added to infected cells within
the first hour of infection or removed from infected cells after 6 h
of treatment. These data suggest an interference by 4b during
the uncoating process.
The results obtained with the chromene 4b significantly differ
from those previously described for related compounds such as
(Z)-3-(4-chlorobenzylidene)chroman and (E)-3-styryl-2H-chro-
mene.4,15 Maximal inhibition of virus yield was observed when
both these compounds were present during the time of virus
adsorption only. A similar reduction was noticed when compounds
were removed 30 min after virus binding.4,15 On the contrary, both
analogues did not modify virus yield when added only 1 h after
virus infection. Therefore, modification in shape and length of the
backbone as in the new (E)-6-chloro-3-(3-phenylprop-1-en-1-yl)-
2H-chromene 4b does not allow interaction of this molecule with
virus capsid structures involved in receptor recognition.
c
c
a
(CDCl3, 100 MHz):
d (ppm) 181.92, 161.44, 143.30, 136.06,
135.97, 135.56, 129.52, 128.92, 127.92, 127.67, 127.45, 122.45,
121.88, 121.64, 117.86, 66.99. Anal. calcd for C18H14O2: C, 82.42;
H, 5.38. Found: C, 82.58; H, 5.46.
4.1.1.2. (E)-6-Chloro-3-[(E)-3-phenylallylidene]chroman-4-one
(1b). Yield: 85%, mp = 159–160 °C from ethyl alcohol. IR (KBr):
1669 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 7.96 (d, 1H, H5, J
.
5–7 = 2.7 Hz), 7.54–7.49 (3H, H20, H60, H
H30–H50), 7.11 (d, 1H, H
, Jb– = 15.4 Hz), 6.99 (dd, 1H, Hb J
b = 11.6 Hz, Jb– = 15.4 Hz), 6.95 (d, 1H, H8, J7–8 = 8.8 Hz), 5.26 (d,
a), 7.43–7.35 (m, 4H, H7,
c
c
a–
c
2H, H2, J2- = 1.6 Hz). 13C NMR (CDCl3, 100 MHz):
d (ppm)
a
180.81, 159.85, 143.98, 136.79, 135.93, 135.35, 129.71, 128.96,
128.82, 127.54, 127.38, 127.31, 123.27, 121.46, 119.58, 67.14. Anal.
calcd for C18H13ClO2: C, 72.85; H, 4.42; Cl, 11.95. Found: C, 72.97;
H, 4.45; Cl, 11.80.
3. Conclusion
4.1.1.3. (E)-6-Chloro-3-[(E)-3-(4-chlorophenyl)allylidene]chro-
man-4-one (1c). Yield: 81%, mp = 168–176 °C from ethyl ace-
In the present study we focused our attention onto the design,
synthesis and anti-HRV activity of new series of (E)-3-[(E)-3-phenyl-
allylidene]chroman-4-ones 1a–e, (E)-3-(3-phenylprop-2-yn-1-yli-
dene)chroman-4-ones 2a and 2b, (Z)-3-[(E)-3-phenylallylidene]
tate. IR (KBr): 1654 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
7.95 (d, 1H, H5, J5–7 = 2.7 Hz), 7.50–7.44 (3H, H20, H60, H
a), 7.41

7362
C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
(dd, 1H, H7, J7–8 = 8.8 Hz, J5–7 = 2.7 Hz), 7.36 (d, 2H, H30, H50, J2 –
at 0 °C, and the mixture was poured into 2 N hydrochloric acid.
The organic layer was washed with saturated aqueous sodium
bicarbonate and brine, than it was dried over anhydrous sodium
sulphate, filtered and evaporated to dryness. The residue was chro-
matographed on silica gel column eluting with ethyl acetate/light
petroleum (1:5 for 3a and 3e, 1:8 for 3b, 1:10 for 3c, 1:20 for 3d)
and purified by crystallization.
0
30
c
= 8.5 Hz), 7.05 (d, 1H, Hc, Jb– = 15.3 Hz), 7.03–6.92 (m, 2H, H8,
Hb), 5.25 (d, 2H, H2, J2- = 1.6 Hz). 13C NMR (CDCl3, 100 MHz): d
a
(ppm) 180.80, 159.85, 142.34, 136.35, 135.60, 135.47, 134.42,
129.23, 128.72, 128.64, 127.46, 127.23, 123.20, 121.97, 119.61,
67.10. Anal. calcd for C18H12Cl2O2: C, 65.28; H, 3.65; Cl, 21.41.
Found: C, 65.59; H, 3.73; Cl, 21.64.
4.1.1.4. (E)-3-[(E)-3-(4-Chlorophenyl)allylidene]chroman-4-one
(1d). Yield: 83%, mp = 160–163 °C from ethyl acetate. IR (KBr):
4.1.2.1. (Z)-3-[(E)-3-Phenylallylidene]chroman (3a). Yield:
54%, mp = 102–104 °C from n-hexane. 1H NMR (CDCl3, 400 MHz):
1665 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.01 (dd, 1H, H5,
.
d (ppm) 7.43 (dd, 2H, H20, H60, J2 –3 = 8.2 Hz, J2 –4 = 1.3 Hz), 7.34–
7.23 (m, 3H, H30–H50), 7.11–7.05 (m, 2H, H5, H7), 7.03 (dd, 1H,
Hb, J –b = 11.1 Hz, Jb– = 15.4 Hz), 6.91–6.84 (m, 2H, H6, H8), 6.58
0
0
0
0
J5–6 = 7.8 Hz, J5–7 = 1.8 Hz), 7.51–7.43 (4H, H7, H20, H60, H
a
), 7.34
(d, 2H, H30, H50, J2 –3 = 8.6 Hz), 7.07 (ddd, 1H, H6, J5–6 = 7.8 Hz, J6–
7 = 7.2 Hz, J6–8 = 1.1 Hz), 7.02–6.93 (m, 3H, H8, Hb, H ), 5.26 (d,
2H, H2, J2– = 1.6 Hz). 13C NMR (CDCl3, 100 MHz):
(ppm)
0
0
a
c
c
(d, 1H, H
c
, Jb– = 15.4 Hz), 6.32 (dd, 1H, H
a
, J –b = 11.1 Hz, J2–
c
a
d
= 0.9 Hz), 4.86 (d, 2H, H2, J2– = 0.9 Hz), 3.59 (s, 2H, H4). 13C
a
a
a
181.88, 161.45, 141.66, 135.66, 135.51, 135.31, 134.57, 129.68,
129.19, 128.56, 127.94, 122.41, 122.17, 121.96, 117.90, 66.97. Anal.
calcd for C18H13ClO2: C, 72.85; H, 4.42; Cl, 11.95. Found: C, 73.09;
H, 4.37; Cl, 11.78.
NMR (CDCl3, 100 MHz): d (ppm) 154.86, 137.25, 133.20, 131.87,
128.93, 128.67, 128.73, 127.31, 126.44, 126.31, 123.15, 122.79,
121.02, 116.78, 65.33, 34.23. Anal. calcd for C18H16O: C, 87.06; H,
6.49. Found: C, 87.36; H, 6.63.
4.1.1.5. (E)-6-Fluoro-3-[(E)-3-phenylallylidene]chroman-4-one
(1e). Yield: 90%, mp = 182–185 °C from ethyl acetate. IR (KBr):
4.1.2.2.
(Z)-6-Chloro-3-[(E)-3-phenylallylidene]chroman
(3b). Yield: 49%, mp = 121–124 °C from n-hexane. 1H NMR
1669 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 7.65 (dd, 1H, H5,
.
(CDCl3, 400 MHz): d (ppm) 7.42 (d, 2H, H20, H60, J2 –3 = 7.7 Hz),
7.35–7.22 (m, 3H, H30–H50), 7.07–7.04 (m, 2H, H5, H7), 7.00 (dd,
1H, Hb, J –b = 11.2 Hz, Jb– = 15.3 Hz), 6.78 (d, 1H, H8, J7–8 = 8.4),
0
0
J5–F = 8.3 Hz, J5–7 = 2.9 Hz), 7.54–7.49 (m, 3H, H20, H60, H
a), 7.42–
7.33 (3H, H30–H50), 7.19 (ddd, 1H, H7, J7–8 = 7.8 Hz, J7–F = 9.0 Hz, J
a
c
5–7 = 2.9 Hz), 7.11 (d, 1H, H
c
, Jb– = 15.5 Hz), 7.04–6.95 (m, 2H,
6.60 (d, 1H, H
c
, Jb– = 15.3 Hz), 6.33 (d, 1H, H
a
, J –b = 11.2 Hz),
c
c
a
H8, Hb), 5.24 (d, 2H, H2, J2– = 1.3 Hz). 13C NMR (CDCl3,
4.84 (s, 2H, H2), 3.56 (s, 2H, H4). 13C NMR (CDCl3, 100 MHz): d
(ppm) 153.50, 137.10, 133.68, 130.56, 128.69, 128.48, 127.85,
127.28, 126.85, 126.48, 125.67, 124.31, 122.88, 118.07, 65.39,
34.03. Anal. calcd for C18H15ClO: C, 76.46; H, 5.35; Cl, 12.54. Found:
C, 76.68; H, 5.38; Cl, 12.30.
a
100 MHz): d (ppm) 181.20, 157.64, 157.62 (d, J = 244 Hz), 143.86,
136.55, 135.96, 129.68, 128.96, 128.47, 127.53, 123.07 (d,
J = 7 Hz), 122.99 (d, J = 23 Hz), 121.54, 119.49 (d, J = 7 Hz), 112.87
(d, J = 23 Hz), 67.12. Anal. calcd for C18H13FO2: C, 77.13; H, 4.67;
F, 6.78. Found: C, 77.44; H, 4.59.
4.1.2.3. (Z)-6-Chloro-3-[(E)-3-(4-chlorophenyl)allylidene]chro-
man (3c). Yield: 36%, mp = 142–146 °C from ethyl acetate/light
4.1.1.6.
(E)-3-(3-Phenylprop-2-yn-1-ylidene)chroman-4-one
1
(2a). Yield: 50%, mp = 140 °C from n-hexane. IR (KBr): 2183,
petroleum. H NMR (CDCl3, 400 MHz): d (ppm) 7.34 (d, 2H, H20,
1662 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 8.00 (dd, 1H, H5,
.
H60, J2 –3 = 8.5 Hz), 7.29 (d, 2H, H30, H50, J2 –3 = 8.5 Hz), 7.07–7.04
(m, 2H, H5, H7), 6.97 (dd, 1H, Hb, J –b = 11.1 Hz, Jb– = 15.4 Hz),
0
0
0
0
J5–6 = 7.8 Hz, J5–7 = 2.6 Hz), 7.55–7.48 (m, 3H, H20, H60, H7), 7.42–
7.35 (m, 3H, H30–H50), 7.07 (ddd, 1H, H6, J5–6 = 7.8 Hz, J6–
a
c
6.76 (d, 1H, H8, J7–8 = 8.0), 6.54 (d, 1H, H
c
, Jb– = 15.4 Hz), 6.31 (d,
c
7 = 8.0 Hz, J6–8 = 1.1 Hz), 7.03–7.05 (m, 2H, H8, H
a), 5.34 (d, 2H,
1H, Ha
, J –b = 11.1 Hz), 4.83 (s, 2H, H2), 3.56 (s, 2H, H4). 13C NMR
a
H2, J2– = 1.9 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm) 180.43,
(CDCl3, 100 MHz):
d (ppm) 153.42, 135.57, 133.44, 132.26,
a
161.78, 139.54, 136.13, 132.00, 129.66, 128.60, 127.89, 122.12,
122.07, 121.68, 118.14, 116.92, 104.71, 85.28, 68.60. Anal. calcd
for C18H12O2: C, 83.06; H, 4.65. Found: C, 83.37; H, 4.50.
131.24, 128.87, 128.48, 127.60, 127.32, 126.58, 125.71, 124.17,
123.42, 118.07, 65.31, 34.05. Anal. calcd for C18H14Cl2O: C, 68.15;
H, 4.45; Cl, 22.35. Found: C, 67.91; H, 4.62; Cl, 22.46.
4.1.1.7.
(E)-6-Chloro-3-(3-phenylprop-2-yn-1-ylidene)chro-
4.1.2.4. (Z)-3-[(E)-3-(4-Chlorophenyl)allylidene]chroman
(3d). Yield: 42%, mp = 135–136 °C from ethyl acetate/light
man-4-one (2b). Yield: 60%, mp = 127–131 °C from n-hexane.
IR (KBr): 2185, 1661 cmꢁ1
.
1H NMR (CDCl3, 400 MHz): d (ppm)
petroleum. H NMR (CDCl3, 400 MHz): d (ppm) 7.34 (d, 2H, H20,
1
7.95 (d, 1H, H5, J5–7 = 2.6 Hz), 7.53 (dd, 2H, H20, H60, J2 –
H60, J2 –3 = 8.5 Hz), 7.29 (d, 2H, H3’, H5’, J2 –3 = 8.5 Hz), 7.14–7.05
0
0
0
0
0
0
0
30
a c
= 7.9 Hz, J2 –5 = 1.6 Hz), 7.44 (dd, 1H, H7, J7–8 = 9.0 Hz, J5–
(m, 2H, H5, H7), 6.98 (dd, 1H, Hb, J –b = 11.1 Hz, Jb– = 15.4 Hz),
7 = 2.6 Hz), 7.41–7.36 (m, 3H, H30–H50), 7.03 (t, 1H, H
a,
J2–
6.92–6.84 (m, 2H, H6, H8, J7–8 = 8.0), 6.53 (d, 1H,
H
c
,
Jb–
= 15.4 Hz), 6.31 (d, 1H, Ha, J –b = 11.1 Hz), 4.86 (s, 2H, H2), 3.61
a
= 1.9 Hz), 6.97 (d, 1H, H8, J7–8 = 9.0 Hz), 5.33 (d, 2H, H2, J2–
a
a
c
= 1.9 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm) 179.39, 160.18,
(s, 2H, H4). 13C NMR (CDCl3, 100 MHz): d (ppm) 154.74, 135.70,
133.11, 131.78, 131.55, 130.95, 129.47, 128.84, 127.57, 127.35,
126.08, 123.66, 121.09, 116.77, 65.21, 34.23. Anal. calcd for
138.60, 135.93, 132.04, 129.81, 128.63, 127.59, 127.12, 122.46,
121.96, 119.86, 117.78, 105.45, 85.16, 68.73. Anal. calcd for
C18H11ClO2: C, 73.35; H, 3.76; Cl, 12.03. Found: C, 73.08; H, 3.91;
C18H15ClO: C, 76.46; H, 5.35; Cl, 12.54. Found: C, 76.71; H, 5.23;
Cl, 11.95.
Cl, 12.74.
4.1.2. General procedure for the synthesis of the (Z)-3-[(E)-3-
4.1.2.5.
(Z)-6-Fluoro-3-[(E)-3-phenylallylidene]chro-
phenylallylidene]chromans (3a–e)
man (3e). Yield: 40%, mp = 121–123 °C from ethyl acetate/light
1
A
solution of the appropriate (E)-3-[(E)-3-phenylallylid-
petroleum. H NMR (CDCl3, 400 MHz): d (ppm) 7.42 (d, 2H, H20,
H60, J2 –3 = 7.9 Hz), 7.35–7.22 (m, 3H, H30–H50), 7.00 (dd, 1H, Hb,
0
0
ene]chroman-4-one (1a-e) (10 mmol) in dry ethyl ether (120 mL)
was added dropwise to a suspension of lithium aluminium hydride
(17.5 mmol) and aluminium chloride (35.0 mmol) in dry ethyl
ether (20 mL). After complete addition, the mixture was stirred
at room temperature for 1 h and refluxed for 2.5 h. After cooling,
excess of reducing reagent was destroyed by adding ethyl acetate
J
a
–b = 11.2 Hz, Jb– = 15.4 Hz), 6.81–6.76 (m, 3H, H5, H7, H8), 6.59
c
(d, 1H, Hc, Jb– = 15.4 Hz), 6.32 (d, 1H, Ha, J –b = 11.2 Hz), 4.83 (s,
c a
2H, H2), 3.57 (s, 2H, H4). 13C NMR (CDCl3, 100 MHz): d (ppm)
157.30 (d, J = 236 Hz), 150.97, 137.17, 133.53, 131.13, 128.69,
127.82, 126.56, 126.47, 124.12 (d, J = 8 Hz), 122.98, 117.66 (d,

C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
7363
J = 8 Hz), 114.79 (d, J = 23 Hz), 113.95 (d, J = 23 Hz), 65.48, 34.22.
Anal. calcd for C18H15FO: C, 81.18; H, 5.68; F, 7.13. Found: C,
81.23; H, 5.72; Cl, 7.00.
6.78 (dd, 1H, H8, J7–8 = 8.2 Hz, J6–8 = 1.1 Hz), 6.32 (s, 1H, H4), 6.17
(d, 1H, –b = 15.9 Hz), 5.68 (dt, 1H, Hb, –b = 15.9 Hz,
Jb– = 6.9 Hz), 4.92 (s, 2H, H2), 3.46 (d, 2H, H
Ha
,
J
J
a
a
c
, Jb– = 6.8 Hz). 13C
c
c
NMR (CDCl3, 100 MHz): d (ppm) 153.77, 138.25, 131.43, 130.96,
129.06, 128.66, 128.21, 128.17, 127.49, 126.76, 125.56, 123.29,
121.56, 115.49, 67.00, 42.52.Anal. calcd for C18H15ClO: C, 76.46; H,
5.35; Cl, 12.54. Found: C, 76.63; H, 5.21; Cl, 12.30.
4.1.3. General procedure for the synthesis of the (E)-3-(3-phenyl-
prop-1-en-1-yl)-2H-chromenes (4a–d)
A 0.5 M solution of sodium ethoxide (25 mL) was added drop-
wise to a stirred suspension of the appropriate phenethyltriphenyl-
phosphonium bromide (10 mmol) in ethyl alcohol (50 mL). After
stirring for 45 min at room temperature, a solution of the appropri-
ate 2H-chromene-3-carbaldehyde (10 mmol) in ethyl alcohol
(85 mL) was added dropwise and the mixture was refluxed for
2.5 h. After cooling, water was added, ethyl alcohol was removed
under reduced pressure and the residue was extracted with ethyl
acetate. The organic layer was washed with brine, dried over anhy-
drous sodium sulphate, filtered and evaporated to dryness. The res-
idue was purified by column chromatography on silica gel eluting
with ethyl acetate/light petroleum (1:4). The product was further
purified by crystallization from n-hexane.
4.2. Virology
4.2.1. Cells
HeLa (Ohio) cells were grown at 37 °C using Eagle’s Minimum
Essential Medium (MEM) supplemented with 100 lg/mL of strep-
tomycin and 100 U/mL of penicillin G and 8% heat-inactivated foe-
tal calf serum (FCS) (growth medium). The concentration was
reduced to 2% for cell maintenance (maintenance medium).
4.2.2. Compounds
Stock solutions were made up in ethanol (1, 0.5 or 0.1 mg/mL)
and further diluted in cell culture medium shortly before use.
4.1.3.1. (E)-3-(3-Phenylprop-1-en-1-yl)-2H-chromene
(4a). Yield: 45%, mp = 89–90 °C from n-hexane. 1H NMR (CDCl3,
4.2.3. Virus
400 MHz): d (ppm) 7.31 (t, 2H, H30, H50, J2 –3 = J3 –5, = 7.5 Hz), 7.25–
7.19 (m, 3H, H20, H40, H60), 7.07 (ddd, 1H, H7, J7–8 = 8.0 Hz, J6–
7 = 7.4 Hz, J5–7 = 1.6 Hz), 6.97 (dd, 1H, H5, J5–6 = 7.4 Hz, J5–
7 = 1.6 Hz), 6.85 (dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.1 Hz), 6.78
(dd, 1H, H8, J7–8 = 8.0 Hz, J6–8 = 1.1 Hz), 6.32 (s, 1H, H4), 6.19 (d,
Reference strains of HRV type 1B and 14 were purchased from
American Type Culture Collection (ATCC). Virus stocks were pre-
pared infecting hela (Ohio) cell monolayers at a multiplicity of
infection of 0.1 PFU/cell. Infected cells were incubated at 33 °C.
When the viral-induced cytopathic effect involved most of the
cells, the cultures were freeze-thawed three times and the clarified
supernatants titrated by plaque assay, essentially as described by
Fiala and Kenny.25 The virus was stored at ꢁ80 °C until used
0
0
0
1H,
Ha, J –b = 15.9 Hz), 5.74 (dt, 1H, Hb, J –b = 15.9 Hz, Jb–
a
a
= 6.9 Hz), 4.93 (s, 2H, H2), 3.50 (d, 2H, H
c
, Jb– = 6.9 Hz). 13C
c
c
NMR (CDCl3, 100 MHz): d (ppm) 159.86, 142.39, 136.26, 135.54,
135.02, 134.42, 129.23, 128.73, 128.64, 127.56, 126.59, 123.20,
121.73, 119.52, 67.09, 42.91. Anal. calcd for C18H16O: C, 87.06; H,
6.49. Found: C, 86.95; H, 6.63.
4.2.4. XTT assay for cellular cytotoxicity
A tetrazolium-based (XTT) colorimetric assay was used to mea-
sure the cytotoxicity of compounds, as previously described.16
Briefly, HeLa cells were seeded in 96-well tissue culture plates
4.1.3.2. (E)-6-Chloro-3-(3-phenylprop-1-en-1-yl)-2H-chromene
(4b). Yield: 43%, mp = 80–81 °C from n-hexane. 1H NMR (CDCl3,
(2 ꢀ 103 cells/well) in 100
lL of growth medium with or without
400 MHz): d (ppm) 7.34 (t, 2H, H30, H50, J2 –3 = J3 –5, = 7.9 Hz), 7.26–
7.19 (m, 3H, H20, H40, H60), 7.02 (dd, 1H, H7, J7–8 = 8.5 Hz, J5–
7 = 2.5 Hz), 6.95 (d, 1H, H5, J5–7 = 2.5 Hz), 6.72 (d, 1H, H8, J7–
compounds in twofold dilutions, starting from the maximum solu-
ble concentration in cell culture medium. Triplicate wells were used
for each drug concentration to be tested. In parallel, media contain-
ing the same concentrations of ethanol were used as control in or-
der to evaluate the residual toxicity of ethanol. The plates were
incubated at 37 °C in 5% CO2-air until the untreated monolayers
0
0
0
8 = 8.5 Hz), 6.25 (s, 1H, H4), 6.19 (d, 1H, H
a, J –b = 15.9 Hz), 5.78
a
(dt, 1H, Hb, J –b = 15.9 Hz, Jb– = 6.8 Hz), 4.94 (s, 2H, H2), 3.52 (d,
a
c
2H,
Hc, d (ppm)
Jb– = 6.8 Hz). 13C NMR (CDCl3, 100 MHz):
c
152.00, 139.52, 137.16, 131.73, 130.25, 128.92, 128.66, 128.61,
128.22, 126.41, 126.06, 124.10, 120.95, 116.59, 65.79, 39.53. Anal.
calcd for C18H15ClO: C, 76.46; H, 5.35; Cl, 12.54. Found: C, 76.71; H,
5.22; Cl, 12.31.
were confluent (3 days). Then, 50 lL of XTT labelling mixture was
added to each well (final XTT concentration 0.15 mg/mL) and the
cells incubated for 4 h at 37 °C. The spectrophotometric 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 alterations on the third day of
incubation in the presence of compounds. The highest concentra-
tion of compound that did not produce any modification of mor-
phology and viability on 100% of cells was the maximum non-
4.1.3.3. (E)-6-Chloro-3-[3-(4-chlorophenyl)prop-1-en-1-yl]-2H-
chromene (4c). Yield: 41%, mp = 135–143 °C from n-hexane. 1H
NMR (CDCl3, 400 MHz):
30
d
(ppm) 7.28 (d, 2H, H30, H50, J2 –
0
= 8.1 Hz), 7.12 (d, 2H, H20, H60, J2 –3 = 8.1 Hz), 7.01 (dd, 1H, H7,
0
0
J7–8 = 8.5 Hz, J5–7 = 2.5 Hz), 6.95 (d, 1H, H5, J5–7 = 2.5 Hz), 6.71 (d,
1H, H8, J7–8 = 8.5 Hz), 6.25 (s, 1H, H4), 6.16 (d, 1H, H , J
cytotoxic concentration. The 50% cytotoxic concentration (TC50)
was indicated as the concentration of compound reducing the cell
viability by 50%, as compared with mock-treated cells.
a
a
–
b = 15.9 Hz), 5.72 (dt, 1H, Hb, J –b = 15.9 Hz, Jb– = 6.7 Hz), 4.91 (s,
a
c
2H, H2), 3.46 (d, 2H,
100 MHz):
H
c
,
Jb– = 6.7 Hz). 13C NMR (CDCl3,
c
d
(ppm) 152.06, 137.96, 132.28, 131.52, 130.01,
4.2.5. Determination of the 50% inhibitory concentration (IC50)
129.49, 129.33, 128.74, 128.37, 126.14, 124.02, 121.31, 116.64,
65.77, 38.79. Anal. calcd for C18H14Cl2O: C, 68.15; H, 4.45; Cl,
22.35. Found: C, 68.35; H, 4.25; Cl, 22.51.
The IC50 values were determined as described previously.7
Briefly, monolayers of HeLa 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, the virus inoculum was
removed and the cells were overlaid with medium for plaques, in
the presence or absence of fourfold dilutions of drugs. After three
days of incubation at 33 °C, the cells were stained with a neutral
red solution (0.2 mg/mL) in pH 7.4 phosphate buffered saline
(PBS) and the plaques were counted. The IC50 was expressed as
the concentration of drug reducing the plaque number by 50% as
4.1.3.4. (E)-3-[3-(4-Chlorophenyl)prop-1-en-1-yl]-2H-chromene
(4d). Yield: 48%, mp = 90–91 °C from n-hexane. 1H NMR (CDCl3,
400 MHz): d (ppm) 7.28 (d, 2H, H30, H50, J2 –3 = 8.3 Hz), 7.13 (d, 2H,
0
0
H20, H60, J2 –3 = 8.3 Hz), 7.08 (dd, 1H, H7, J7–8 = 8.2 Hz, J6–7 = 7.4 Hz,
J5–7 = 1.6 Hz), 6.98 (dd, 1H, H5, J5–6 = 7.4 Hz,
J5–7 = 1.6 Hz), 6.86 (dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.1 Hz),
0
0

7364
C. Conti et al. / Bioorg. Med. Chem. 19 (2011) 7357–7364
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 log-
arithm of compound dose. Triplicate wells were utilized for each
drug concentration.
Acknowledgements
We gratefully acknowledge the financial support from ‘Istituto
Pasteur-Fondazione Cenci Bolognetti’, Università degli Studi di
Roma ‘La Sapienza’. We also thank Dr. Ivano Pindinello for techni-
cal assistance.
4.2.6. Virus inactivation and stabilization
For virus inactivation studies, HRV 1B suspensions with or with-
References and notes
out 4b (42 lM) were incubated at 33 °C for 1 h. After serial 10-fold
dilutions, virus titres were measured by plaque assay on HeLa cell
monolayers.
1. Heikkinen, T.; Jarvinen, A. Lancet 2003, 361, 51.
2. Hayden, F. G. Rev. Med. Virol. 2004, 14, 17.
3. Peltola, V.; Waris, M.; Osterback, R.; Susi, P.; Hyypia, T.; Ruuskanen, O. J. Clin.
Virol. 2008, 43, 411.
For virus stabilization studies, the virus was incubated with or
4. Conti, C.; Desideri, N. Bioorg. Med. Chem. 2009, 17, 3720.
5. Burali, C.; Desideri, N.; Stein, M. L.; Conti, C.; Orsi, N. Eur. J. Med. Chem. 1987, 22,
119.
6. Conti, C.; Desideri, N.; Orsi, N.; Sestili, I.; Stein, M. L. Eur. J. Med. Chem. 1990, 25,
725.
7. Desideri, N.; Conti, C.; Sestili, I.; Tomao, P.; Stein, M. L.; Orsi, N. Antiviral Chem.
Chemother. 1992, 3, 195.
8. Genovese, D.; Conti, C.; Tomao, P.; Desideri, N.; Stein, M. L.; Catone, S.; Fiore, L.
Antiviral Res. 1995, 27, 123.
without 4b (42 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 5 by adding 0.2 M acetate buffer (pH 5). After incubation
at 33 °C for 30 min, the mixtures were neutralized with 0.85 M Tris
base. For thermal treatment, the mixtures were incubated 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.
9. Desideri, N.; Olivieri, S.; Stein, M. L.; Sgro, R.; Orsi, N.; Conti, C. Antiviral Chem.
Chemother. 1997, 8, 545.
10. Quaglia, M. G.; Desideri, N.; Boss, E.; Sgro, R.; Conti, C. Chirality 1999, 11, 495.
11. Tait, S.; Salvati, A. L.; Desideri, N.; Fiore, L. Antiviral Res. 2006, 72, 252.
12. Conti, C.; Tomao, P.; Genovese, D.; Desideri, N.; Stein, M. L.; Orsi, N. Antimicrob.
Agents Chemother. 1992, 36, 95.
13. Salvati, A. L.; De Dominicis, A.; Tait, S.; Canitano, A.; Lahm, A.; Fiore, L.
Antimicrob. Agents Chemother. 2004, 48, 2233.
4.2.7. Virus yield reduction assays
Confluent monolayers of HeLa cells in 24-well plates were in-
fected at a multiplicity of five in the presence or absence of 4b
(42 lM). The infection was synchronized by allowing HRV 1B to
14. Diana, G. D. Curr. Med. Chem.-Anti-Infective Agents 2003, 2, 1.
15. Conti, C.; Desideri, N. Bioorg. Med. Chem. 2010, 18, 6480.
16. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger,
T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827.
17. Andries, K.; Dewindt, B.; Snoeks, J.; Wouters, L.; Moereels, H.; Lewi, P. J.;
Janssen, P. A. J. J. Virol. 1990, 64, 1117.
18. Andries, K.; Dewindt, B.; Snoeks, J.; Willebrords, R.; Stokbroekx, R.; Lewi, P. J.
Antiviral Res. 1991, 16, 213.
19. Bauer, D. J.; Selway, J. W. T.; Batchelor, J. F.; Tisdale, M.; Caldwell, J. C.; Young,
D. A. B. Nature 1981, 292, 369.
20. Lonberg-Holm, K.; Korant, B. D. J. Virol. 1972, 9, 29.
21. Lonberg-Holm, K.; Yin, F. H. J. Virol. 1973, 12, 114.
22. Rombaut, B.; Vrijsen, R.; Boeye, A. Antiviral Res. 1985, 67.
23. Rombaut, B.; Andries, K.; Boeye, A. J. Gen. Virol. 1991, 72, 2153.
24. Morgan, T. K. Jr. U.S. Patent 45993441, 1986.
bind in the cold (4 °C). After 1 h, the inoculum was removed by
washing thrice with cold PBS. The end of virus binding is indicated
as 0 time. Then, MEM with or without the compound (42 lM) was
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 4b, the drug was added or removed from
HRV-infected cells at various times p.i. (0, 150, 300, 450, 1, 2, 4 and
6 h) and the cultures incubated at 33 °C up to 10 h p.i. The virus
yield was determined as above.
25. Fiala, M.; Kenny, G. E. J. Bacteriol. 1966, 92, 1710.