Synthesis and antirhinovirus activity of new 3-benzyl chromene and chroman derivatives

Article abstract of DOI:10.1016/j.bmc.2009.03.051

A series of 3-benzyl chromenes and chromans were synthesized and tested in vitro against human rhinovirus (HRV) 1B and 14, two representative serotypes for rhinovirus group B and A, respectively. All the new compounds, with the exception of 3-benzyl-2H-chromene (3a), showed a potent activity against HRV serotype 1B within micro or submicromolar range (IC₅₀s from 0.11 to 6.62 μM). The low cytotoxicity of all the derivatives resulted in compounds with high therapeutic index (TI). On the contrary, HRV 14 infection was only weakly inhibited by the majority of these compounds. The 3-benzylidenechromans 2b and 2c showed the highest anti-HRV 1B activity (IC₅₀ 0.12 and 0.11 μM, respectively) coupled with remarkable TI (625.00 and 340.91, respectively). Mechanism of action studies on (Z)-3-(4-chlorobenzylidene)chroman (2b) suggest that the new compounds behave as capsid binders and interfere with very early stages of HRV 1B replication, similarly to related flavanoids.

Full text of DOI:10.1016/j.bmc.2009.03.051

Bioorganic & Medicinal Chemistry 17 (2009) 3720–3727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antirhinovirus activity of new 3-benzyl chromene
and chroman derivatives
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:
A series of 3-benzyl chromenes and chromans were synthesized and tested in vitro against human rhi-
novirus (HRV) 1B and 14, two representative serotypes for rhinovirus group B and A, respectively. All
the new compounds, with the exception of 3-benzyl-2H-chromene (3a), showed a potent activity against
HRV serotype 1B within micro or submicromolar range (IC50s from 0.11 to 6.62 lM). The low cytotoxicity
of all the derivatives resulted in compounds with high therapeutic index (TI). On the contrary, HRV 14
Received 12 November 2008
Revised 24 March 2009
Accepted 25 March 2009
Available online 1 April 2009
infection was only weakly inhibited by the majority of these compounds. The 3-benzylidenechromans
2b and 2c showed the highest anti-HRV 1B activity (IC50 0.12 and 0.11 lM, respectively) coupled with
remarkable TI (625.00 and 340.91, respectively). Mechanism of action studies on (Z)-3-(4-chlorobenzyl-
idene)chroman (2b) suggest that the new compounds behave as capsid binders and interfere with very
early stages of HRV 1B replication, similarly to related flavanoids.
Keywords:
Chromenes
Chromans
Rhinovirus
Antiviral activity
Mechanism of action
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
gested that they specifically interfere with some early events of
viral replication. In the case of HRV, 40,6-dicyanoflavan delayed
The human rhinoviruses (HRV) are important pathogens caus-
ing most of the upper respiratory tract infections in humans.1
Although these infections are often mild and self-limiting, the im-
pact on human productivity and on medical costs is enormous.
HRV infections are also associated with several serious medical
complications such as otitis media, chronic bronchitis and asthma.2
Since the more than one hundred serotypes of HRVs make the
development of a vaccine impractical, extensive efforts have been
focused on the development of effective antiviral agents for the
treatment of HRV infections. However, despite the in vitro activity
of several compounds, to date only few drugs have shown efficacy
in humans and none has been approved for clinical use.3
Among the structurally different classes of molecules inhibiting
picornavirus replication in vitro, we focused our attention on nat-
ural and synthetic flavanoids and flavonoids. Several flavans, isofl-
avans, 3(2H)-isoflavenes and homo-isoflavonoids exhibited a broad
spectrum of antipicornavirus activity.4–9 Studies on the mecha-
nism of action of selected flavanoids on HRV and EV infections sug-
the uncoating kinetic of neutral-red encapsidated virus and pre-
vented mild acid or thermal inactivation of virus infectivity, sug-
gesting an action at the uncoating level due to a stabilizing effect
on virion capsid conformation.10 In the case of poliovirus, 3(2H)-
isoflavene was found to exert its action during the uncoating step.
Analysis of mutations conferring resistance provides strong evi-
dence that 3(2H)-isoflavene inserts into a hydrophobic pocket in-
side capsid protein VP1, increasing the stability of the viral
particle and making the virus resistant to uncoating.11 The capsid
protein VP1 is considered a promising therapeutic target since sev-
eral compounds which have reached clinical evaluation were
shown to bind to this viral protein.12 The presence of so-called cap-
sid-binding molecules appears to stabilize the protein coat, pre-
venting adsorption of the virus to the cell and/or uncoating.12
In continuation of the search for more potent and highly selec-
tive analogues, we designed (Z)-3-benzylidenechromans (2a–d), 3-
benzyl-2H-chromenes (3a–d) and 3-benzylchromans (4a–d, 6a–d,
7a, 7d) related to the most active synthetic 3(2H)-isoflavenes4–6
and homoisoflavones7–9 previously studied by us.
Abbreviations: ATCC, American Type Culture Collection; EV, enterovirus; FCS,
foetal calf serum; HRV, human rhinovirus; IC50, 50% inhibitory concentration;
MEM, minimum essential medium; MNTC, maximum non-cytotoxic concentration;
PBS, phosphate buffered saline; PFU, plaque forming unit; TC50, 50% cytotoxic
concentration; TI, therapeutic index; VP, viral protein; XTT, 2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt.
2. Results and discussion
2.1. Chemistry
The synthesis of the designed (Z)-3-benzylidenechromans 2a–d
was achieved starting from the corresponding (E)-3-benzylidench-
* Corresponding author. Tel.: +39 06 49913892; fax: +39 06 491491.
E-mail address: nicoletta.desideri@uniroma1.it (N. Desideri).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.051

C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
3721
roman-4-ones 1a–d obtained by condensation of the appropriate
O
O
R'
chroman-4-one with the suitable benzaldehyde. The reaction was
conducted in the presence of 85% phosphoric acid or dry hydro-
chloric acid in ethyl alcohol following previously described general
methods.7 Subsequent reaction of (E)-3-benzylidenchroman-4-
ones 1a–d with lithium aluminium hydride in the presence of alu-
minium chloride, selectively reduced the carbonyl group to a
methylene group with some isomerization of the double bond
(Scheme 1). The obtained mixture of (Z)-3-benzylidenechromans
2a–d and 3-benzyl-2H-chromenes 3a–d (about in 3:1 ratio) was
separated by column chromatography. The stereochemistry of
the double bond of (Z)-3-benzylidenechromans 2a–d was estab-
lished by 2D NOESY experiments. The strong NOE cross peaks be-
tween H4 and vinylic proton, and also between H2 and H20, H60
unequivocally indicated the cis configuration of this double bond.
Therefore, the reduction of (E)-3-benzylidenchroman-4-ones 1a–
d to (Z)-3-benzylidenechromans 2a–d occurred with the retention
of configuration, but the E/Z descriptors changed because of the
change in priority at C3.
R
1a-d
(i)
R
O
R'
+
O
R'
+
R
R
R
2 a-d
3a-d
O
R'
+
O
R'
+
R
R
O
5a-d
4 a-d
O
O
R'
R'
+
OH
OH
6 a-d
7a, d
On the contrary, (E)-3-benzylidenchroman-4-ones 1a–d were
reduced to a complex mixture of partially and fully reduced prod-
ucts when treated with sodium cyanoborohydride in the presence
of zinc iodide (Scheme 2). Complete separation of all the reaction
products was achieved by column chromatography. The saturated
cis-chromanols 6a–d were the predominant products isolated
(about in 40% yield) from this reaction. The stereochemistry of
the diastereoisomeric chromanols 6a–d and 7a,d was assigned on
the basis of NMR data which was compared with literature values
reported for 6a and 7a.13–15
Since 3-benzyl-2H-chromenes 3a–d were obtained in poor yield
by reduction of (E)-3-benzylidenchroman-4-ones 1a–d, the three-
step procedure previously described by us was followed to synthe-
size these compounds.16 The synthesis began with the O-alkylation
of the appropriate 2-(hydroxymethyl)phenol with 1-bromo-3-phe-
nylpropan-2-one. The intermediate ethers were then converted to
the corresponding triphenylphosphonium bromides and finally cy-
clized by an intramolecular Wittig reaction.16
1, 2, 3, 4, 5, 6, 7 a R = R' = H
1, 2, 3, 4, 5, 6 b
R = H, R' = Cl
R = R' = Cl
1, 2, 3, 4, 5, 6 c
1, 2, 3, 4, 5, 6, 7 d R = Cl, R' = H
Compound (Yield %)
2a (6)
3a (0.5)
4a (8)
2b (7)
3b (2)
4b (7)
2c (11) 2d (12)
3c (3)
4c (4)
3d (0.5)
4d (6)
5a (10) 5b (12) 5c (11) 5d (12)
6a (37) 6b (39) 6c (39) 6d (44)
7a (4)
7b (0)
7c (0)
7d (6)
Scheme 2. Reagents and conditions: (i) NaCNBH4, ZnI2, ClCH2CH2Cl, reflux, 20 h.
O
O
R'
R'
As shown in Scheme 3, 3-benzylchromans 4a–d were synthe-
sized in good yield by reduction 3-benzylchroman-4-ones 5a–d7
with lithium aluminium hydride in the presence of aluminium
chloride.
R
R
4, 5 a R = R' = H
4, 5 b R = H, R' = Cl
4, 5 c R = R' = Cl
4, 5 d R = Cl, R' = H
5a-d
(i)
O
4 a-d
O
O
R'
+
R
Scheme 3. Reagents and conditions: (i) LiAlH4, AlCl3, Et2O, reflux, 30 min.
O
(ii)
(i)
1a, b
2.2. Antiviral tests
1c, d
In preliminary studies, the cytotoxicity of all the new synthe-
sized compounds (1a–d, 2a–d, 3a–d, 4a–d, 5a–d, 6a–d, 7a,d) was
determined by evaluating the effects on morphology, viability
and growth of HeLa (Ohio) 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.17
The inhibitory activity of all the compounds on HRV replication
was evaluated in a plaque reduction assay, starting from the max-
imum non-cytotoxic concentration (MNTC), as already described.5
A previous systematic evaluation of a panel of capsid-binder com-
pounds against all serotyped HRVs established the existence of two
virus groups, called groups A and B, with contrasting susceptibili-
ties for these antivirals. Group B contains twice as many serotypes
as group A, and accounts for five times as many colds as group A
O
O
R'
R
1a-d
(iii)
O
O
R'
R'
+
R
R
2 a-d
3a-d
1, 2, 3 a R = R' = H
1, 2, 3 b R = H, R' = Cl
1, 2, 3 c
R = R' = Cl
1, 2, 3 d R = Cl, R' = H
Scheme 1. Reagents and conditions: (i) 85% H3PO4, 80 °C, 6 h; (ii) HCl gas, EtOH, rt,
24 h; (iii) LiAlH4, AlCl3, Et2O, reflux, 30 min.

3722
C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
serotypes.18,19 On the basis of these observations, we utilized HRV
1B and 14 as representative serotypes for group B and A, respec-
tively. The results of cytotoxicity and antiviral activity are reported
in Table 1. 40,6-Dichloroflavan (BW683C), an inhibitor of group B
serotypes, was included as a control.20
2.3. Mechanism of action study
On the basis of both high anti-HRV 1B activity and therapeutic
index (IC50 = 0.12
lM
and TI = 625), (Z)-3-(4-chlorobenzyli-
dene)chroman (2b) was chosen to clarify the mechanism of antivi-
ral action by evaluating the effects produced either on virus
particles or multiplication.
With the exception of 3-benzyl-2H-chromene 3a, all chromenes
and chromans tested showed high antiviral activity against HRV 1B
within micro or submicromolar range (IC50s ranging from 0.11
l
M
Initially, the direct effect of 2b on HRV 1B infectivity and stabil-
ity was investigated. The virus-neutralizing action was studied
incubating HRV 1B at high titre with the compound at a concentra-
to 6.62 M). Generally, the potent inhibitory activity on HRV 1B
l
coupled with the low cytotoxicity resulted in compounds with
high therapeutic index (TI). In contrast, only a modest inhibition
of HRV 14 replication was observed up to MNTCs.
tion of 12 lM, corresponding to 100 times the IC50. After 10-fold
dilutions to achieve non-inhibitory concentrations of free com-
pound, the infectivity titres of mock- and 2b-treated virus suspen-
sions were found to be similar (3.9 ꢀ 107 PFU/mL and
4.1 ꢀ 107 PFU/mL, respectively). The same results were observed
Among the (Z)-3-benzylidenechromans 2a–d, 3-benzyl-2H-
chromenes 3a–d and 3-benzylchroman-4-ols 6a–d, the anti-HRV
1B effect was more pronounced for the 40-chloro and 40,6-dichloro
analogues (compounds b and c, respectively) than for the corre-
sponding unsubstituted or 6-chloro substituted derivatives (com-
pounds a and d, respectively). Therefore, a chlorine at the 40-
position appears a key element for optimum activity. In fact, the
(Z)-3-benzylidenechromans 2b and 2c were the most potent com-
even at a higher concentration (36
the contrary, 2b (12
l
M) (4.0 ꢀ 107 PFU/mL). On
l
M) significantly protected HRV 1B against
inactivation by mild acid or heat treatments. Results reported in
Figure 1 show that, 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). In the presence of 2b, the drop
in HRV infectivity was significantly reduced and the protective ef-
fect towards low pH and thermal inactivation was 3.2 and 1.4 log,
respectively. Taken together, these results indicate that 2b stabi-
lized the virion capsid conformation without modifying the virus
infectivity. Therefore, binding of compound is important to the
inhibitory action, although it was reversible by dilution.
pounds in this novel family of inhibitors, with IC50s of 0.12
0.11 M, and TIs of 625.00 and 340.91, respectively.
In contrast, among the 3-benzylchromans 4a–d, the unsubsti-
tuted compound 4a exhibited the highest activity (IC50 = 0.23 M),
the corresponding 40,6-dichloro derivative 4c showed comparable
potency (IC50 = 0.29 M), while 3-(4-chlorobenzyl)chroman 4b
was threefold less effective (IC50 = 0.69 M) than the parent com-
lM and
l
l
l
l
pound 4a. In addition, these three 3-benzylchromans combined a
very low cytotoxicity with a submicromolar effect on the HRV 1B
infection.
Although all the 3-benzylchroman-4-ols (6a–d, 7a and 7d)
exhibited an anti-HRV 1B effect within micromolar range, they
showed a marked reduction in potency in comparison with the
corresponding chromans unsubstituted at the 4-position (4a–d).
The antiviral activity of 2b (12 lM or 36 lM) towards different
stages of 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. At both concentrations, maximal
inhibition (more than 99%) was achieved when compound 2b was
Table 1
Cytotoxicity and anti-rhinovirus activity of (Z)-3-benzylidenechromans (2a–d), 3-benzyl-2H-chromenes (3a–d), 3-benzylchromans (4a–d), (cis)-3-benzylchroman-4-ols (6a–d)
and (trans)-3-benzylchroman-4-ols (7a, 7d)
MNTCa
(
lM)
TC50
(l
M)
IC50 (lM)
TId
b
c
Compound
R
R0
HRV 1B
HRV 14
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
6a
6b
6c
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
H
25.00
12.50
1.56
25.00
25.00
6.25
1.56
3.12
25.00
6.25
12.50
25.00
25.00
12.50
25.00
12.50
25.00
6.25
150
75
6.62
21.59
22.66
0.12e
0.11
12.50 (21.3%)
1.56 (14.2%)
25.00 (43.3%)
25.00 (23.9%)e
6.25 (12.8%)
1.56 (11.2%)
3.12 (19.3%)
25.00 (41.4%)
6.25 (12.4%)
12.50 (33.0%)
25.00 (35.3%)
25.00 (15.9%)
NAg
625.00
340.91
119.94
—
441.18
250.00
31.25
326.09
>289.86
258.62
>172.41
42.98
40.32
101.35
30.61
>55.71
24.00
>961
37.5
150
150
150
37.5
75
1.34
25.00 (36.8%)e
0.34
H
Cl
Cl
H
0.15
2.40
75
0.23e
0.69
H
>200f
75
Cl
Cl
H
0.29e
1.16
>200f
150
75
3.49e
1.86e
1.48
H
Cl
Cl
H
150
75
25.00 (7.0%)
12.50 (47.7%)
NAg
6d
7a
7d
BW683C
2.45
>200f
150
3.59e
6.25
Cl
6.25 (27.3%)
25.00
>25.00f
0.026
NAg
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 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 against HRV 1B.
Compound producing a reduction (from 70% to 50%) in the mean viral plaque size, beside an effect on plaque number.
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
.
g

C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
3723
8
7
6
5
4
3
2
1
0
8
A
B
7
6
HRVC
HRVC
HRVT
2b
5
HRVT
4
2b
3
2
1
0
HRVC
HRVT
2b
HRVC
HRVT
2b
HRVC:
HRVT:
HRVC:
HRVT:
Untreated virus,
Virus heated at 56 ºC,
Untreated virus,
Virus exposed to pH 5.0,
2b:
Virus heated at 56 ºC in the presence of 2b.
2b:
2b
.
Virus exposed to pH 5.0 in the presence of
Figure 1. Protective effect of 2b on acid (A) and thermal inactivation (B) of HRV 1B infectivity.
added to cells together with the virus and maintained until the end
of HRV multiplication. When the compound was added to infected
HeLa cells immediately after virus binding, the reduction in virus
yield was still 94%. Instead, when 2b was added 30 min after virus
3
2.5
2
12 uM
36 uM
binding, inhibition dropped to 44% at 36 lM and 6% at 12 lM
1.5
1
(Fig. 2). At the same time, removal of 2b from infected cells after
only 30 min of contact still produced more than 93% suppression
in virus production. Similar levels of inhibition were achieved
when the compound was removed after longer incubation times
(Fig. 3).
0.5
0
0.5 h
1 h
2 h
4h
Remarkably, maximal reduction of virus yield was also ob-
served when 2b was present during the time of virus adsorption
only (1 h, 4 °C) (Fig. 4). Instead, treatment of HeLa cells (1 h, 4 °C)
with 2b before HRV infection, produced only a moderate level of
virus yield reduction (Fig. 4). Together, these results suggest that
the compound interferes with virus attachment to the cell surface
and that the inhibitory effect on HRV 1B binding is not mediated by
an interaction of compound with cellular receptors.
Figure 3. Effect of varying the time of removal of 2b (12
l
M or 36
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 3.8 ꢀ 105 PFU/mL. Compound
was added after virus adsorption period (1 h at 4 °C, time 0) and removed at
different lengths of incubation (0.5, 1, 2 h).
6
5
4
3
2
1
0
The overall analysis of data from inactivation and stabilization
studies of HRV infectivity are consistent with 2b acting as a cap-
sid-binder, although binding was reversible by dilution. This mech-
anism of action is shared with several structurally related anti-
picornavirus agents such as 3(2H)-isoflavene.11 Previous research
on capsid-binding compounds demonstrate that optimum activity
is associated with a high degree of occupancy of the pocket. There-
fore, the difference in activity by 2b observed against HRV 1B and
3
2.5
2
12 uM
36 uM
CV
A
B
C
D
E
F
Figure 4. Effect of 2b on HRV 1B attachment to the cell surface and on membrane
receptors for virus. Virus yield was determined by plaque assay after one cycle of
1.5
1
virus growth (1 h at 4 °C and 10 h at 33 °C). CV: Virus control. A (12
l
M) and D
M): Cells exposed to 2b for 1 h at 4 °C before virus attachment (1 h at 4 °C). B
M) and E (36 M): Cells exposed to 2b during the time of virus attachment
M) and F (36 M): Cells exposed to 2b during virus attachment
(36
(12
l
l
0.5
0
l
(1 h at 4 °C). C (12
l
l
(1 h at 4 °C) and replication (10 h at 33 °C).
EC
1 h
2 h
0
0.5 h
Figure 2. Effect of varying the time of addition of 2b (12
lM or 36 lM) on the
14 could be attributed to the relative size of the binding site within
the capsid protein VP1. In fact, the viral group B binding site
accommodates shorter molecules, while longer compounds were
routinely more active against serotype 14.21 In agreement with
the capsid-binder hypothesis, data from time of addition/removal
inhibition of HRV 1B replication under one-step growth conditions. Virus yield was
determined by plaque assay. Virus control titre was 3.8 ꢀ 105 PFU/mL. EC: 2b was
present during the entire infection cycle (1 h at 4 °C and 10 h at 33 °C). 0, 0.5 h, 1 h,
2 h: compound was added at different times (0, 0.5, 1, 2 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).

3724
C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
studies indicate that 2b interferes with very early event(s) of virus
infection process, as already reported for several related com-
pounds.6,10,11,22 Moreover, the finding that an equal efficacy is
achieved when 2b is present during the entire replication time or
during virus binding only, strongly suggests that this compound
exerts its antiviral effect by a direct interaction with virus particles
during HRV attachment to the cell surface. This observation is fur-
ther supported by the absence of a direct action on cell membrane
receptors for virus in pre-treatment studies.
Mechanism of action studies of previously synthesized com-
pounds, such as 40,6-dicyanoflavan and isoflavans, suggested an
interference with the uncoating of viral genome without an action
on HRV adsorption.10,22 Remarkably, (Z)-3-(4-chlorobenzyli-
dene)chroman (2b), besides an effect at the uncoating level, as sug-
gested by protection of virus infectivity against low pH and
thermal inactivation, demonstrated a significant activity also dur-
ing the HRV attachment step to the host cell membrane.
4.1.1.1. (Z)-3-Benzylidenechroman (2a). Yield: 37%, colourless
1
oil. H NMR (CDCl3, 400 MHz): d (ppm) 7.37–7.18 (m, 5H, H20–
H60), 7.13–7.08 (m, 2H, H5, H7), 6.90 (dt, 1H, H6, J5–6
=
J6–7 = 7.4 Hz, J6–8 = 1.1 Hz), 6.84 (dd, 1H, H8, J7–8 = 8.0 Hz,
J6–8 = 1.1 Hz), 6.69 (s, 1H, H9), 4,83 (s, 2H, H2), 3.66 (s, 2H, H4).
Anal. Calcd for C16H14O: C, 86.45; H, 6.35. Found: C, 86.73; H, 6.30.
4.1.1.2. (Z)-3-(4-Chlorobenzylidene)chroman (2b). Yield: 42%,
mp = 77–79 °C from n-hexane. 1H NMR (CDCl3, 400 MHz):
d
(ppm) 7.32 (d, 2H, H30, H50, J2 –3 = 8.6 Hz), 7.13–7.08 (m, 4H, H20,
H60, H5, H7), 6.91 (dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.1 Hz),
6.84 (dd, 1H, H8, J7–8 = 8.0 Hz, J6–8 = 1.1 Hz), 6.63 (s, 1H, H9), 4,79
(s, 2H, H2), 3.65 (s, 2H, H4). Anal. Calcd for C16H13ClO: C, 74.85;
H, 5.10; Cl, 13.81. Found: C, 74.98; H, 5.25; Cl, 14.00.
0
0
4.1.1.3. (Z)-6-Chloro-3-(4-chlorobenzylidene)chroman (2c). Yield:
28%, mp = 71–72 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d
(ppm) 7.33 (d, 2H, H30, H50, J2 –3 = 8.3 Hz), 7.11 (d, 2H, H20, H60, J2 –
0
0
0
30
= 8.4 Hz), 7.07–7.05 (m, 2H, H5, H7), 6.76 (d, 1H, H8, J7–
3. Conclusion
8 = 8.6 Hz), 6.64 (s, 1H, H9), 4,77 (s, 2H, H2), 3.61 (s, 2H, H4). Anal.
Calcd for C16H12Cl2O: C, 66.00; H, 4.15; Cl, 24.35. Found: C, 66.12;
H, 4.22; Cl, 24.15.
A series of 3-benzyl chromene and chroman derivatives was
synthesized and tested for its anti HRV activity and cytotoxicity
in vitro. Several members of this new family of inhibitors showed
submicromolar potency against HRV 1B coupled with a high ther-
apeutic index.
Similarly to related flavanoids, the new compounds behaved as
capsid-binders and interfered with very early events of virus
multiplication.
4.1.1.4. (Z)-3-Benzylidene-6-chlorochroman (2d). Yield: 32%,
mp = 68–70 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d (ppm)
7.40–7.25 (m, 3H, H30–H50), 7.18 (d, 2H, H20, H60, J2 –3 = 7.2 Hz),
7.07–7.04 (m, 2H, H5, H7), 6.76 (d, 1H, H8, J7–8 = 8.6 Hz), 6.70 (s,
1H, H9), 4,81 (s, 2H, H2), 3.61 (s, 2H, H4). Anal. Calcd for C16H13ClO:
C, 74.85; H, 5.10; Cl, 13.81. Found: C, 74.99; H, 5.08; Cl, 13.96.
0
0
4. Experimental
4.1. Chemistry
4.1.1.5. 3-Benzyl-2H-chromene (3a). Yield: 12%, colourless oil
(lit.23 31 °C). 1H NMR (CDCl3, 400 MHz): d (ppm) 7.29–7.16 (m,
5H, H20–H60), 7.02 (ddd, 1H, H7, J6–7 = 7.4 Hz, J7–8 = 8.0 Hz,
J5–7 = 1.7 Hz), 6.88 (dd, 1H, H5, J5–6 = 7.4 Hz, J5–7 = 1.7 Hz), 6.80
(dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.2 Hz), 6.74 (dd, 1H, H8,
J7–8 = 8.0 Hz, J6–8 = 1.2 Hz), 6.11 (s, 1H, H4), 4,59 (s, 2H, H2), 3.34
(s, 2H, H9). Anal. Calcd for C16H14O: C, 86.45; H, 6.35. Found: C,
86.67; H, 6.23.
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 spectra were detected with a
Bruker AM-400 spectrometer, using TMS as internal 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 (Merck, Kieselgel or Aluminium
oxide 60 F254). Components were visualized by UV light. Elemen-
tal analyses (C, H, Cl) of all new compounds were within 0.4% of
theoretical values. (E)-3-Benzylidenechroman-4-ones (1a–d) were
synthesized following the procedure previously described.7 3-Ben-
zyl-2H-chromenes (3a–d)16 and 3-benzylchroman-4-ones (5a–d)7
were prepared with high yields according to the methods previ-
ously reported.
4.1.1.6. 3-(4-Chlorobenzyl)-2H-chromene
(3b). Yield:
17%,
mp = 74–75 °C from n-hexane. 1H NMR (CDCl3, 400 MHz):
d
(ppm) 7.29 (d, 2H, H30, H50, J2 –3 = 8.6 Hz), 7.16 (d, 2H, H20, H60, J
= 8.6 Hz), 7.07 (ddd, 1H, H7, J6–7 = 7.4 Hz, J7–8 = 8.0 Hz, J5–
0
0
20–30
7 = 1.6 Hz), 6.92 (dd, 1H, H5, J5–6 = 7.4 Hz, J5–7 = 1.6 Hz), 6.84 (dt,
1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.1 Hz), 6.76 (dd, 1H, H8, J7–
8 = 8.0 Hz, J6–8 = 1.1 Hz), 6.14 (s, 1H, H4), 4,62 (s, 2H, H2), 3.38 (s,
2H, H9). Anal. Calcd for C16H13ClO: C, 74.85; H, 5.10; Cl, 13.81.
Found: C, 74.65; H, 5.24; Cl, 13.99.
4.1.1.7. 6-Chloro-3-(4-chlorobenzyl)-2H-chromene (3c). Yield:
8%, mp = 105–106 °C from n-hexane. 1H NMR (CDCl3, 400 MHz):
d (ppm) 7.30 (d, 2H, H30, H50, J2 –3 = 8.6 Hz), 7.15 (d, 2H, H20, H60,
4.1.1. General procedure for the reduction of 1a–d with lithium
aluminium hydride and aluminium chloride
0
0
0
0
J2 –3 = 8.6 Hz), 7.00 (dd, 1H, H7, J7–8 = 8.6 Hz, J5–7 = 2.5 Hz), 6.89
(d, 1H, H5, J5–7 = 2.5 Hz), 6.68 (d, 1H, H8, J7–8 = 8.6 Hz), 6.06 (s,
1H, H4), 4,62 (s, 2H, H2), 3.38 (s, 2H, H9). Anal. Calcd for
C16H12Cl2O: C, 66.00; H, 4.15; Cl, 24.35. Found: C, 66.25; H, 4.13;
Cl, 24.46.
A solution of the appropriate (E)-3-benzylidenechroman-4-one
(1a–d) (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 refluxed for 30 min. Excess
of reducing reagent was destroyed by adding ethyl acetate at
0 °C, and the mixture was poured into 2 N hydrochloric acid. The
ether layer was washed with brine, dried over anhydrous sodium
sulfate, filtered and evaporated to dryness. The obtained mixture
of the corresponding (Z)-3-benzylidenechromans (2a–d) and 3-
benzyl-2H-chromenes (3a–d) (about 3:1 ratio) were separated by
column chromatography on silica gel eluting with light petroleum.
4.1.1.8. 3-Benzyl-6-chloro-2H-chromene
(3d). Yield:
10%,
mp = 99–100 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d
(ppm) 7.34–7.19 (m, 5H, H20–H60), 6.99 (dd, 1H, H7, J7–8 = 8.5 Hz,
J5–7 = 2.5 Hz), 6.88 (d, 1H, H5, J5–7 = 2.5 Hz), 6.66 (d, 1H, H8,
J7–8 = 8.5 Hz), 6.07 (s, 1H, H4), 4,63 (s, 2H, H2), 3.40 (s, 2H, H9).
Anal. Calcd for C16H13ClO: C, 74.85; H, 5.10; Cl, 13.81. Found: C,
74.57; H, 5.06; Cl, 14.08.

C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
3725
4.1.2. General procedure for the synthesis of the 3-
benzylchromans (4a–d)
3-benzylidenechroman-4-one (1a–d) (10 mmol) in 1,2-dichloro-
ethane (50 mL). The reaction mixture was refluxed for 20 h. After
cooling to room temperature, the mixture was poured into an
ice-cold mixture of saturated ammonium chloride solution con-
taining 10% of 6 N hydrochloric acid (200 mL). After stirring until
the gas evolution ceased, the mixture was extracted with ethyl
acetate. The combined organic phases were washed with brine,
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness. The residue was chromatographed on silica gel eluting
with light petroleum to separate (Z)-3-benzylidenechroman (2a–
d), 3-benzyl-2H-chromene (3a–d) and 3-benzylchroman (4a–d).
Further elution with ethyl acetate and light petroleum (1:10) al-
lowed the isolation of 3-benzylchroman-4-one (5a–d), (cis)-3-ben-
zylchroman-4-ol (6a–d) and, eventually, (trans)-3-benzylchroman-
4-ol (7a, 7d). The yields of all the compounds are reported in
Scheme 2.
A solution of the appropriate 3-benzylchroman-4-one (5a–d)
(10 mmol) in dry ethyl ether (120 mL) was added dropwise to a
suspension of lithium aluminium hydride (17.5 mmol) and alu-
minium chloride (35.0 mmol) in dry ethyl ether (20 mL). After
complete addition, the mixture was refluxed for 30 min. Excess
of reducing reagent was destroyed by adding ethyl acetate at
0 °C, and the mixture was poured into 2 N hydrochloric acid. The
ether layer was washed with brine, dried over anhydrous sodium
sulfate, filtered and evaporated to dryness. The residue was puri-
fied by column chromatography on silica gel eluting with light
petroleum, to give the corresponding 3-benzylchromans (4a–d).
4.1.2.1. 3-Benzylchroman (4a). Yield: 64%, mp = 46–48 °C from
n-hexane (lit.24 52 °C from ethyl alcohol). 1H NMR (CDCl3,
400 MHz): d (ppm) 7.23–7.08 (m, 5H, H20–H60), 6.98 (ddd, 1H,
H7, J6–7 = 7.4 Hz, J7–8 = 8.0 Hz, J5–7 = 1.6 Hz), 6.89 (dd, 1H, H5,
J5–6 = 7.4 Hz, J5–7 = 1.6 Hz), 6.75–6.70 (m, 2H, H6, H8), 4.08 (dd,
1H, H2, Jgem = 10.6 Hz, J2–3 = 3.1 Hz), 3.71 (dd, 1H, H2,
Jgem = 10.6 Hz, J2–3 = 8.4 Hz), 2.69 (dd, 1H, H4, Jgem = 16.2 Hz,
J3–4 = 4.9 Hz), 2.60 (dd, 1H, H9, Jgem = 13.6 Hz, J3–9 = 7.4 Hz), 2.51
(dd, 1H, H9, Jgem = 13.6 Hz, J3–9 = 7.6 Hz), 2.42 (dd, 1H, H4,
Jgem = 16.2 Hz, J3–4 = 8.6 Hz), 2.22 (m, 1H, H3). Anal. Calcd for
C16H16O: C, 85.68; H, 7.19. Found: C, 85.79; H, 7.33.
4.1.3.1. (cis)-3-Benzylchroman-4-ol (6a). Yield: 37%, mp = 113–
114 °C from n-hexane (lit.13 114 °C from benzene–hexane). IR
(KBr): 3440 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 7.37–7.17
.
(m, 7H, H20–H50, H5, H7), 6.89 (dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz,
J6–8 = 1.0 Hz), 6.84 (dd, 1H, H8, J7–8 = 8.2 Hz, J6–8 = 1.0 Hz), 4.52 (t,
1H, H4, J3–4 = J4-OH = 4.6 Hz), 4.14–4.07 (m, 2H, H2), 2.89 (dd, 1H,
H9, Jgem = 13.6 Hz, J3–9 = 8.4 Hz), 2.68 (dd, 1H, H9, Jgem = 13.6 Hz,
J3–9 = 7.3 Hz), 2.33 (m, 1H, H3), 1.65 (d, 1H, OH, J4-OH = 4.6 Hz).
Anal. Calcd for C16H16O2: C, 79.97; H, 6.71. Found: C, 80.07; H, 6.83.
4.1.2.2. 3-(4-Chlorobenzyl)chroman (4b). Yield: 72%, mp = 71–
72 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d (ppm) 7.28 (d,
4.1.3.2. (cis)-3-(4-Chlorobenzyl)chroman-4-ol (6b). Yield: 39%,
2H, H30, H50, J2 –3 = 8.3 Hz), 7.13 (d, 2H, H20, H60, J2 –3 = 8.3 Hz), 7.09
(ddd, 1H, H7, J6–7 = 7.4 Hz, J7–8 = 8.0 Hz, J5–7 = 1.6 Hz), 6.99 (dd, 1H,
H5, J5–6 = 7.4 Hz, J5–7 = 1.6 Hz), 6.83 (dt, 1H, H6, J5–6 =J6–7 = 7.4 Hz,
J6–8 = 1.1 Hz), 6.80 (dd, 1H, H8, J7–8 = 8.0 Hz, J6–8 = 1.1 Hz), 4.16 (dd,
1H, H2, Jgem = 10.7 Hz, J2–3 = 3.0 Hz), 3.82 (dd, 1H, H2, Jgem = 10.7 Hz,
J2–3 = 8.0 Hz), 2.80 (dd, 1H, H4, Jgem = 16.2 Hz, J3–4 = 5.3 Hz), 2.67 (dd,
1H, H9, Jgem = 13.7 Hz, J3–9 = 7.5 Hz), 2.59 (dd, 1H, H9, Jgem = 13.7 Hz,
J3–9 = 7.7 Hz), 2.50 (dd, 1H, H4, Jgem = 16.2 Hz, J3–4 = 8.3 Hz), 2.27 (m,
1H, H3). Anal. Calcd for C16H15ClO: C, 74.27; H, 5.84; Cl, 13.70. Found:
C, 74.31; H, 5.76; Cl, 13.88.
mp = 130–132 °C from n-hexane. IR (KBr): 3528 cmꢁ1
.
1H NMR
0
0
0
0
(CDCl3, 400 MHz): d (ppm) 7.15 (d, 2H, H30, H50, J2 –3 = 8.5 Hz),
0
0
7.06–7.03 (m, 4H, H20, H60, H5, H7), 6.74 (dt, 1H, H6, J5–6
=
J6–7 = 7.5 Hz, J6–8 = 1.2 Hz), 6.70 (dd, 1H, H8, J7–8 = 8.1 Hz,
J6–8 = 1.2 Hz), 4.31 (d, 1H, H4, J3–4 = 2.9 Hz), 3.93–3.88 (m, 2H,
H2), 2.68 (dd, 1H, H9, Jgem = 13.7 Hz, J3–9 = 8.6 Hz), 2.45 (dd, 1H,
H9, Jgem = 13.7 Hz, J3–9 = 7.2 Hz), 2.19 (m, 1H, H3), 1.90 (br s, 1H,
OH). Anal. Calcd for C16H15ClO2: C, 69.95; H, 5.50; Cl, 12.90. Found:
C, 70.03; H, 5.63; Cl, 12.98.
4.1.3.3. (cis)-6-Chloro-3-(4-chlorobenzyl)chroman-4-ol (6c). Yield:
4.1.2.3. 6-Chloro-3-(4-chlorobenzyl)chroman (4c). Yield: 20%,
mp = 75–78 °C from n-hexane. 1H NMR (CDCl3, 400 MHz): d (ppm)
39%, mp = 149–150 °C from n-hexane. IR (KBr): 3530 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz): d (ppm) 7.30 (d, 2H, H30, H50, J2 –3 = 8.3 Hz), 7.21–
7.19 (m, 3H, H20, H60, H5), 7.15 (dd, 1H, H7, J7–8 = 8.7 Hz, J5–
7 = 2.5 Hz), 6.78 (d, 1H, H8, J7–8 = 8.7 Hz), 4.46 (m, 1H, H4), 4.09–4.05
(m, 2H, H2), 2.85 (dd, 1H, H9, Jgem = 13.7 Hz, J3–9 = 8.5 Hz), 2.63 (dd,
1H, H9, Jgem = 13.7 Hz, J3–9 = 7.4 Hz), 2.25 (m, 1H, H3), 1.70 (d, 1H,
OH, J4-OH = 4.5 Hz). Anal. Calcd for C16H14Cl2O2: C, 62.15; H, 4.56; Cl,
22.93. Found: C, 62.03; H, 4.77; Cl, 23.02.
0
0
7.26 (d, 2H, H30, H50, J2 –3 = 8.5 Hz), 7.08 (d, 2H, H20, H60,
0
0
0
0
J2 –3 = 8.5 Hz), 7.01 (dd, 1H, H7, J7–8 = 8.7 Hz, J5–7 = 2.5 Hz), 6.95 (d,
1H, H5, J5–7 = 2.5 Hz), 6.71 (d, 1H, H8, J7–8 = 8.7 Hz), 4.11 (dd, 1H,
H2, Jgem = 10.6 Hz, J2–3 = 3.0 Hz), 3.77 (dd, 1H, H2, Jgem = 10.6 Hz,
J2–3 = 8.1 Hz), 2.73 (dd, 1H, H4, Jgem = 16.4 Hz, J3–4 = 5.2 Hz), 2.64
(dd, 1H, H9, Jgem = 13.7 Hz, J3–9 = 7.6 Hz), 2.54 (dd, 1H, H9,
Jgem = 13.7 Hz, J3–9 = 7.6 Hz), 2.43 (dd, 1H, H4, Jgem = 16.4 Hz,
J3–4 = 8.2 Hz), 2.23 (m, 1H, H3). Anal. Calcd for C16H14Cl2O: C,
65.55; H, 4.81; Cl, 24.18. Found: C, 66.80; H, 4.97; Cl, 24.03.
4.1.3.4. (cis)-6-Chloro-3-benzylchroman-4-ol (6d). Yield: 44%,
mp = 113–115 °C from n-hexane. IR (KBr): 3380 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz): d (ppm) 7.38–7.24 (m, 5H, H20–H60), 7.19 (d,
1H, H5, J5–7 = 2.6 Hz), 7.14 (dd, 1H, H7, J7–8 = 8.7 Hz, J5–7 = 2.6 Hz),
6.78 (d, 1H, H8, J7–8 = 8.7 Hz), 4.48 (d, 1H, H4, J3–4 = 3.2 Hz), 4.12–
4.04 (m, 2H, 2H2), 2.86 (dd, 1H, H9, Jgem = 13.6 Hz, J3–9 = 8.4 Hz),
2.68 (dd, 1H, H9, Jgem = 13.6 Hz, J3–9 = 7.3 Hz), 2.30 (m, 1H, H3),
1.66 (br s, 1H, OH). Anal. Calcd for C16H15ClO2: C, 69.95; H, 5.50;
Cl, 12.90. Found: C, 70.11; H, 5.68; Cl, 12.73.
4.1.2.4. 3-Benzyl-6-chlorochroman (4d). Yield: 56%, mp = 54–55 °C
from n-hexane. 1H NMR (CDCl3, 400 MHz): d (ppm) 7.38–7.17 (m, 5H,
H20–H60), 7.03 (dd, 1H, H7, J7–8 = 8.6 Hz, J5–7 = 2.4 Hz), 7.00 (d, 1H, H5,
J5–7 = 2.4 Hz), 6.73 (d, 1H, H8, J7–8 = 8.6 Hz), 4.17 (dd, 1H, H2,
Jgem = 10.7 Hz, J2–3 = 3.1 Hz), 3.81 (dd, 1H, H2, Jgem = 10.7 Hz,
J2–3 = 8.4 Hz), 2.79 (dd, 1H, H4, Jgem = 16.4 Hz, J3–4 = 5.1 Hz), 2.70 (dd,
1H, H9, Jgem = 13.7 Hz, J3–9 = 7.5 Hz), 2.61 (dd, 1H, H9, Jgem = 13.7 Hz,
J3–9 = 7.7 Hz), 2.49 (dd, 1H, H4, Jgem = 16.4 Hz, J3–4 = 8.6 Hz), 2.30 (m,
1H, H3). Anal. Calcd for C16H15ClO: C, 74.27; H, 5.84; Cl, 13.70. Found:
C, 74.03; H, 5.98; Cl, 13.63.
4.1.3.5. (trans)-3-Benzylchroman-4-ol (7a). Yield: 5%, mp = 143–
144 °C from n-hexane (lit.13 144 °C from benzene–hexane). IR (KBr):
3380 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 7.34–7.17 (m, 7H,
.
H20–H50, H5, H7), 6.96 (dt, 1H, H6, J5–6 = J6–7 = 7.4 Hz, J6–8 = 1.0 Hz),
6.88 (dd, 1H, H8, J7–8 = 8.2 Hz, J6–8 = 1.0 Hz), 4.55 (t, 1H, H4,
J3–4 = J4-OH = 4.6 Hz), 4.23 (dd, 1H, H2, Jgem = 11.1 Hz, J2–3 = 2.6 Hz),
3.98 (dd, 1H, H2, Jgem = 11.1 Hz, J2–3 = 4.5 Hz), 2.71 (dd, 1H, H9,
Jgem = 13.8 Hz, J3–9 = 6.5 Hz), 2.54 (dd, 1H, H9, Jgem = 13.8 Hz,
4.1.3. General procedure for the reduction of 1a–d with sodium
cyanoborohydride in the presence of zinc iodide
Solid zinc iodide (15 mmol) and sodium cyanoborohydride
(75 mmol) were added at room temperature to a solution of (E)-

3726
C. Conti, N. Desideri / Bioorg. Med. Chem. 17 (2009) 3720–3727
J3–9 = 9.2 Hz), 2.23 (m, 1H, H3), 1.82 (d, 1H, OH, J4-OH = 4.6 Hz). Anal.
Calcd for C16H16O2: C, 79.97; H, 6.71. Found: C, 79.75; H, 6.84.
4.2.5. Determination of the 50% inhibitory concentration (IC50)
The IC50 values were determined as described previously.5
Briefly, confluent monolayers of HeLa cells in 6-well plates were
infected with a virus suspension producing approximately 100 pla-
ques 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 neu-
tral red solution at 0.2 mg mLꢁ1 in pH 7.4 phosphate buffered sal-
ine (PBS) and the plaques were counted. The IC50 was expressed as
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 log-
arithm of compound dose. Triplicate wells were utilized for each
drug concentration.
4.1.3.6. (trans)-3-Benzyl-6-chlorochroman-4-ol (7d). Yield: 6%,
mp = 137–139 °C from n-hexane. IR (KBr): 3380 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz): d (ppm) 7.33–7.15 (m, 7H, H20–H60, H5, H7),
6.81 (d, 1H, H8, J7–8 = 8.7 Hz), 4.46 (d, 1H, H4, J3–4 = 4.2 Hz), 4.21
(dd, 1H, H2, Jgem = 11.2 Hz, J2–3 = 2.9 Hz), 3.97 (dd, 1H, H2,
Jgem = 11.2 Hz, J2–3 = 4.5 Hz), 2.72 (dd, 1H, H9, Jgem = 13.8 Hz,
J3–9 = 6.4 Hz), 2.52 (dd, 1H, H9, Jgem = 13.8 Hz, J3–9 = 9.0 Hz), 2.22
(m, 1H, H3), 1.68 (sa, 1H, OH). Anal. Calcd for C16H15ClO2: C,
69.95; H, 5.50; Cl, 12.90. Found: C, 70.21; H, 5.43; Cl, 12.98.
4.2. Virology
4.2.1. Cells
HeLa (Ohio) cells were routinely grown at 37 °C using Eagle0s
Minimum Essential Medium (MEM) supplemented with
4.2.6. Virus inactivation and stabilization
For virus inactivation studies, HRV 1B suspensions with or with-
out 2b at different concentrations (12 lM and 36 lM) were incu-
100 l
g mLꢁ1 of streptomycin and 100 U/mL of penicillin G and
bated at 33 °C for 1 h. After serial 10-fold dilutions, virus titres
were measured by plaque assay on HeLa cell monolayers.
For virus stabilization studies, the virus was incubated with or
8% heat-inactivated foetal calf serum (FCS) (growth medium).
The concentration was reduced to 2% for cell maintenance (main-
tenance medium).
without 2b at different concentrations (12 lM and 36 lM) for
1 h at 33 °C before mild acid or thermal treatment. For mild acid
treatment, the pH of the mixtures was adjusted 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.
4.2.2. Compounds
Stock solutions were prepared in ethanol (1, 0.5 or 0.1 mg mLꢁ1
and further diluted in tissue culture medium shortly before use.
)
4.2.3. Virus
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 multi-
plicity of infection (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 clar-
ified supernatants titrated by plaque assay, essentially as described
by Fiala and Kenny.25 The virus was stored in aliquots at ꢁ80 °C
until used.
4.2.7. Virus yield reduction assays
Confluent monolayers of HeLa cells in 24-well plates were in-
fected at a multiplicity of 5 in the presence or absence of 2b
(12 lM or 36 lM). The infection was synchronized by allowing
HRV 1B to attach in the cold (4 °C). After 1 h, the inoculum was re-
moved by washing thrice with PBS. The end of virus binding is indi-
cated as 0 time. Then, MEM with or without the compound (12
lM
or 36 M) was added and the temperature raised to 33 °C to permit
l
4.2.4. XTT assay for cellular cytotoxicity
internalization. Single-cycle conditions were achieved by incubat-
ing 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 cen-
trifugation in the cold and the supernatants titrated by plaque as-
say on HeLa cell monolayers. To determine which stage of virus
replication was affected by 2b, 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.
A tetrazolium-based (XTT) colorimetric assay was used to
measure the cytotoxicity of compounds. This assay is based on
the cleavage of the yellow tetrazolium salt XTT to form an or-
ange formazan dye by viable, active cells. The formazan dye
formed is soluble in aqueous solution and is directly quantified
using a scanning multiwell spectrophotometer.17 HeLa cells were
seeded in 96 well tissue culture plates at 2 ꢀ 103 cells for well in
100 lL of growth medium with or without compounds in two-
fold 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 conflu-
Acknowledgements
We gratefully acknowledge the financial support from ‘Istituto
Pasteur-Fondazione Cenci Bolognetti’, Università degli Studi di
Roma ‘La Sapienza’.
ent (3 days). Then, 50 lL of XTT labelling mixture was added to
each well (final XTT concentration 0.15 mg mLꢁ1) 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
concentration of compound that did not produce any modifica-
tion of morphology and viability on 100% of cells was the max-
imum 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.
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