3-Phenylalkyl-2H-chromenes and -chromans as novel rhinovirus infection inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.02.012

Following our studies on structure-activity relationships of anti-rhinovirus chromene and chroman derivatives, we designed and synthesized new series of 3-phenylalkyl-2H-chromenes and -chromans bearing differently sized, aliphatic linker chains between the two cycles. The cytotoxicity and the antiviral activity of the new compounds on human rhinovirus (HRV) serotype 1B and 14 infection were evaluated in HeLa cell cultures. Most of the tested compounds interfered with HRV1B multiplication in the micromolar or submicromolar concentrations while HRV14 was less susceptible. 3-[3-(4-Chlorophenyl)propyl]chroman (9c) was selected for preliminary mechanism of action studies due to its potent activity against both serotypes (IC₅₀ of 0.48?μM and 1.36?μM towards HRV1B and 14, respectively) coupled with high selectivity (SI?=?206.18 and 73.26, respectively). Results of time of addition/removal studies suggest that 9c, similarly to related derivatives, behaves as a capsid binder interfering with some early events of the HRV1B infectious cycle.

Full text of DOI:10.1016/j.bmc.2017.02.012

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-Phenylalkyl-2H-chromenes and -chromans as novel rhinovirus
infection inhibitors
Cinzia Conti a, Luca Proietti Monaco b, Nicoletta Desideri b,
⇑
a Dipartimento di Scienze di Sanità Pubblica, Sezione di Microbiologia, Università ‘‘La Sapienza” di Roma, P.le A. Moro, 5, 00185 Roma, Italy
b 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:
Following our studies on structure-activity relationships of anti-rhinovirus chromene and chroman
derivatives, we designed and synthesized new series of 3-phenylalkyl-2H-chromenes and -chromans
bearing differently sized, aliphatic linker chains between the two cycles. The cytotoxicity and the antivi-
ral activity of the new compounds on human rhinovirus (HRV) serotype 1B and 14 infection were eval-
uated in HeLa cell cultures. Most of the tested compounds interfered with HRV1B multiplication in the
micromolar or submicromolar concentrations while HRV14 was less susceptible. 3-[3-(4-Chlorophenyl)
propyl]chroman (9c) was selected for preliminary mechanism of action studies due to its potent activity
Received 5 October 2016
Revised 2 February 2017
Accepted 7 February 2017
Available online xxxx
Keywords:
2H-Chromenes
Chromans
Rhinovirus
Antiviral activity
Capsid binders
against both serotypes (IC50 of 0.48 lM and 1.36 lM towards HRV1B and 14, respectively) coupled with
high selectivity (SI = 206.18 and 73.26, respectively). Results of time of addition/removal studies suggest
that 9c, similarly to related derivatives, behaves as a capsid binder interfering with some early events of
the HRV1B infectious cycle.
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
into clinical trials. For example, clinical studies demonstrated that
intranasal prophylaxis with Pirodavir lowered HRV replication and
Human Rhinoviruses (HRVs) belong to the genus Enterovirus of
the Picornaviridae family. The over 160 serotypes of HRVs are
responsible for common cold, a mild illness of the upper respira-
tory tract. Although not important in terms of clinical morbidity,
the high incidence of the disease in adults as well as in children
produces significant clinical and economic burden worldwide.1
Beside cold, HRV infection has been clinically associated with dif-
ferent respiratory tract complications such as bronchiolitis, pneu-
monia, sinusitis and acute otitis media, and has been implicated
in exacerbations of chronic respiratory disorders, such as asthma,
cystic fibrosis and chronic obstructive pulmonary disease (COPD).2
Recently, experimental HRV infection in COPD patients established
a causative relationship between virus infection and exacerba-
tions,3 moreover COPD exacerbations were predicted by the World
Health Organization to become one of the major cause of death in
the next decades worldwide.4
shedding rates,5 and double-blind placebo-controlled natural cold
trials with oral administration of Pleconaril documented a reduc-
tion in illness duration and severity.6,7 However, because of safety
concerns due to adverse effects,8 further investigations have been
discontinued.9 Vapendavir, a new 1,2-benzisoxazole analog of Piro-
davir, significantly reduced viral load in experimental HRV infec-
tion studies with healthy volunteers.10 It is still under clinical
evaluation for treatment of HRV-induced exacerbations of
asthma.11,12 Therefore, to date, the search for antiviral compounds
is still open.
Viral attachment to the host cell membrane and uncoating of
the RNA genome represent suitable targets of the HRV life cycle
for antiviral intervention and prevention of infection. Based on
receptor usage, HRVs can be distinguished into a major and a
minor group. Major group HRVs bind and gain entry into the cell
cytoplasm through a complex interaction with intercellular adhe-
sion molecule-1 (ICAM-1).13–15 Minor group HRVs bind to low den-
sity lipoprotein receptor (LDLR) family16 and enter the cells by an
endocytotic pH-dependent pathway.17
Due to the large number of serotypes, vaccination against HRVs
is not feasible at present. Despite intensive efforts in antiviral
search and development, so far specific drugs have not been
approved for clinical use, although several compounds progressed
Several small molecules, belonging to different chemical
classes, were found to effectively interfere with the early stages
of HRV infection.18 Among these, the so-called capsid-binders are
able to directly interact with virion shell proteins thus hindering
⇑
Corresponding author.
E-mail address: nicoletta.desideri@uniroma1.it (N. Desideri).
http://dx.doi.org/10.1016/j.bmc.2017.02.012
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

2
C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
virus adsorption and/or hampering genome uncoating, probably
through an alteration of capsid flexibility. Interference by capsid-
binding molecules with RNA uncoating was observed for all stud-
ied serotypes independent of receptor grouping. On the contrary,
inhibition of virus binding to the cell surface was found to be
dependent on individual serotype or compound tested.19
(1a-f) with oxalyl chloride in dry dichloromethane. The obtained
acyl chlorides were reacted with a solution of trimethylsilyldia-
zomethane (TMSCHN2), a stable and safe substituted for hazardous
diazomethane, in dry acetonitrile. The intermediate diazoketones
were treated in situ with 48% aqueous HBr to afford the desired bro-
momethylketones (2a-f) in excellent yields. Finally, the reaction of
2a-f with (2-hydroxybenzyl)triphenylphosphonium bromide in
the presence of tert-BuOK allowed the alkylation of the phenolic
hydroxyl group and the cyclization by an intramolecular Wittig
reaction to give the desired 2H-chromenes (3a-f).
During our long-lasting research program, we explored the anti-
enterovirus properties of synthetic analogues of natural fla-
vanoids20–23 and flavonoids.24–30 Several derivatives showed a
potent and broad antipicornavirus spectrum of efficacy and
behaved as capsid-binders preventing virus adsorption to the host
cell and/or uncoating of the viral genome.23,31,32 Starting from the
promising activity of isoflavanoids20–23 and homoisoflavonoids,25,30
more recently, we designed and synthesized structurally related 3-
benzyl-2H-chromenes, 3-benzylchromans and (Z)-3-benzyli-
denechromans.33 These capsid-binder derivatives showed activity
within micro or submicromolar range against HRV serotype 1B,
while only weakly affected the replication of serotype 14.33 The dif-
ferent sensibility of the two serotypes could be explained according
to the different size of compound-binding site within VP1 capsid
protein. X-ray crystallographic studies on a variety of HRV sero-
types confirmed the diverse size of the hydrophobic pocket into
the capsid protein VP1 and revealed that larger molecules occupy
more efficiently the HRV14 binding site.34–41 Therefore, in order
to obtain analogues with a broader anti-HRV spectrum of activity,
we increased the length of the spacer between the heterocycle
(chromone, chroman-4-one, chroman or 2H-chromene) and the
phenyl or pyridine moiety.42–44 A linear, unsaturated chain contain-
ing from two to four carbon atoms was introduced as a linker
between the two rings.42–44 Optimum activity against both HRV
serotypes was achieved by introducing an unsaturated two carbon
chain joining the 2H-chromene to the benzene ring.42 Mechanism
of action studies on (E)-3-styryl-2H-chromene confirmed an inter-
ference with the early stages of HRV1B replication.42
In this paper, we describe the synthesis and the anti–HRV activ-
ity of new series of 3-phenylalkyl-2H-chromenes and -chromans
designed to further explore the structure-activity relationships of
2H-chromene and chroman HRV inhibitors. Starting from the
hypothesis that more flexible molecules could be capable to insert
more easily into the canyon structure and then to occupy more
efficiently the binding site of different HRV serotypes, linear, satu-
rated polymethylene chains ranging from two to five carbon atoms
were introduced between heterocyclic and phenyl rings (3a,d-f
and 4a,d-f). In addition, maintaining the 2-carbon bridge between
2H-chromene and benzene ring, a small series of branched-chain
linker compounds (3c, 3b and 13) was synthesized and tested for
anti-HRV activity in cell cultures.
Subsequent catalytic hydrogenation of 3-phenylalkyl-2H-
chromenes 3a,d-f afforded the corresponding 3-(3-phenylalkyl)
chromans 4a,d-f with high yields (Scheme 2).
As shown in Scheme 3, the chloro substituted 3-(3-phenyl-
propyl)chromans (9a-c) were prepared by catalytic hydrogenation
of the appropriate mixture of (Z)-3-[(E)-3-phenylallylidene]-chro-
man, (E)-3-(3-phenyl) prop-1-en-1-yl)-2H-chromene, and 3-cin-
namyl-2H-chromene (6, 7, 8a; 6, 7, 8b or 6, 7, 8c). As previously
reported, these mixtures were obtained by reduction of the corre-
sponding (E)-3-[(E)-3-phenylallylidene]chroman-4-one (5a-c)
with lithium aluminium hydride.43
The synthesis of (E)-3-(1-phenylpropen-2-yl)-2H-chromene (13)
started from the preparation of 3-acetyl-2H-chromene (10) by reac-
tionof2-hydroxybenzaldehydeand3-buten-2-oneinthepresenceof
1,4-diazabicyclo-[2.2.2]-octane (DABCO) in chloroform and water
(1:1), at room temperature. The reduction of the obtained 3-acetyl-
2H-chromene (10) with sodium borohydride in methanol provided
the corresponding alcohol (11). This intermediate was treated with
triphenylphosphine hydrobromide in dry acetonitrile to yield [1-
(2H-chromen-3-yl)ethyl]triphenylphosphonium bromide (12) with
goodyield. Finally, thetarget compound13was obtainedbytheWit-
tig reaction with benzaldehyde (Scheme 4).
(E)-3-(2H-Chromen-3-yl)-1-phenylprop-2-en-1-one 15 was
synthesized according to the two step procedure shown in
Scheme 5. 2H-Chromene-3-carbaldehyde 14 was prepared as pre-
viously reported by refluxing 2-hydroxybenzaldehyde and acrolein
in the presence of potassium carbonate.42 The subsequent Horner–
Wadsworth–Emmons reaction of 14 with commercially available
diethyl (2-oxo-2-phenylethyl)phosphonate, carried out using
sodium hydride as base in dry THF, provided only the (E) isomer
15. The stereochemistry of compounds was established on the
basis of the coupling constant value of the protons in the chain
(J -b = 16.0 Hz).
a
2.2. Antiviral activity
The results on cytotoxicity and anti-rhinovirus evaluation of the
new synthesized compounds (3a-f, 4a, 4d-f, 9a-c, 13 and 15) are
reported in Table 1.
2. Results and discussion
2.1. Chemistry
Cytotoxicity is indicated as maximum non-cytotoxic concentra-
tion (MNCC) and 50% cytotoxic concentration (CC50). Starting from
MNCC, the dose-dependent effect of compounds on HRV serotype
1B and HRV serotype 14 infection was evaluated in HeLa cell cul-
tures by a plaque reduction test. HRV 1B and 14 were taken as rep-
The designed 3-phenylalkyl-2H-chromenes (3a-f) were obtained
modifyingthe synthetic strategydeveloped to prepare 3-benzyl-2H-
chromene derivatives45 (Scheme 1). For this purpose, the required
bromomethyl-phenylalkylketones (2a-f) were prepared in good
overall yields starting from the treatment of the appropriate acids
resentative serotypes for antiviral group
B and A of HRVs,
respectively. Antiviral group A and B were defined on the basis of
Scheme 1. Reagents and conditions: (i) CO2C12, dry CH2C12, 0 °C, rt, 3 h; (ii) TMSCHN2, dry CH3CN, 0 °C, 2 h; (iii) HBr 48%, 0 °C, rt, 18 h; (iv) tert-BuOK, dry THF, rt, 23 h.
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 2. Reagents and conditions: (i) Pd/C 10%, dry dioxane, rt, 30 min.
Scheme 3. Reagents and conditions: (i) LiAlH4, AlCl3 dry Et2O, rt 1 h and reflux 2.5 h; (i) Pd/C 10%, dry dioxane, rt, 15 min.
Scheme 4. Reagents and conditions: (i) DABCO, CHC13/H2O (1:1), rt, 7 days; (ii) NaBU, MeOH, rt, 6 h; (iii) triphenylphosphine hydrobromide, dry CH3CN, reflux, 5 h; (iv) tert-
BuOK, dry THF, rt, 23 h.
Scheme 5. Reagents and conditions: (i) K2CO3, dry dioxane, reflux, 4 h; (ii) NaH, dry THF, r.t. 20 h.
contrasting susceptibilities of HRVs to a selected panel of capsid-
binding compounds of different length.46,47
percent reduction of HRV14 plaque number was found lower than
50% at the MNCC while they exhibited an anti-HRV1B effect within
the micromolar or submicromolar range. When compared to
BW683C, all the synthesized compounds were less potent against
HRV1B; instead some of them were active against serotype 14, dif-
ferently from BW683C which was shown ineffective against this
serotype.48
Within chromene derivatives, compounds containing a spacer
of two carbon atoms between heterocyclic and phenyl moieties,
3-phenylethyl-2H-chromene 3a showed high potency (IC50 = 0.48
The antiviral activity is expressed as the compound concentra-
tion required to produce a 50% reduction of plaque number with
respect to mock-treated virus-infected cultures (IC50) (Table 1).
When a 50% reduction of plaque count is not achieved, the percent-
age inhibition obtained at the MNCC is reported in parentheses.
The selectivity index (SI), calculated by the CC50/IC50 ratio, is also
reported in Table 1. 40,6-Dichloroflavan (BW683C), an inhibitor of
group B serotypes, has been utilized as a control for activity.
Generally, the new compounds were able to dose-dependently
inhibit the replication of both serotypes, although HRV14 was
always less susceptible than HRV1B. For several compounds, the
l
M) and good selectivity against HRV1B (SI = 103.09) but low effi-
cacy against serotype 14. The introduction of a methyl group in the
-position of the linker chain between the two cycles (3b) resulted
a
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

4
C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Cytotoxicity and anti-rhinovirus activity of 3-phenylalkyl-2H-chromenes (3a-f), 3-(3-phenylalkyl)chromans (4a,d-f, 9a-c), (E)-3-(1-phenylpropen-2-yl)-2H-chromene (13) and
(E)-3-(2H-chromen-3-yl)-1-phenylprop-2-en-1-one (15).
O
O
R
R
R1
R1
n
n
3a,d-f
4a,d-f; 9a-c
Comp.
n
R
R1
MNCC (
l
M)a
CC50
(l
M)b
IC50
(l
M)c HRV 1B
SId
IC50
(l
M)c HRV 14
SId
3a
4a
3d
4d
9a
9b
9c
3e
4e
3f
2
2
3
3
3
3
3
4
4
5
5
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
25.00
3.12
50.00
6.25
0.48 0.03
0.49 0.05
2.37 0.18
9.59 0.43
1.79 0.14
0.56 0.03
0.48 0.02
0.54 0.04
0.71 0.08
0.56 0.07
4.74 0.37
IN
104.17
12.75
84.39
>25.00 (26%)
>3.12 (16%)
40.47 0.66
>100 (37%)
25.00 1.21
30.00 1.08
1.36 0.09
12.70 0.15
>50.00 (25%)
>50.00 (37%)
33.03 1.11
>12.50 (31%)
–
–
4.94
–
8.00
6.67
73.53
7.87
–
–
6.05
–
100.00
100.00
100.00
100.00
50.00
50.00
50.00
50.00
100.00
12.50
200.00
200.00
200.00
200.00
100.00
100.00
100.00
100.00
200.00
25.00
20.85
111.73
357.14
208.33
185.18
140.84
178.57
42.19
4f
3b
O
O
O
O
3c
6.25
12.50
25.00
50.00
1.17 0.51
10.68
>6.25 (5%)
–
13
12.50
25.00
25.00
IN
IN
15
IN
NT
NAe
O
BW683C
>25.00e
0.025 0.001
>1000
–
Cl
O
Cl
Values shown in this table are the means the standard deviations of two independent assays done in triplicate.
a
The maximum non-cytotoxic concentration (MNCC) 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 CC50 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 MNCC was reported in parentheses.
d
The selectivity index (SI) value was equal to CC50/IC50
Not active up to the MNCC.
.
e
in a loss of activity against HRV1B. In contrast, the shift of the
methyl group to the b-position led to the isomer 3c endowed with
improved potency and selectivity when compared to 4d. In partic-
ular, 3-[3-(4-chlorophenyl)propyl]chroman (9c) displayed the
good activity against HRV1B (IC50 = 1.17
(SI = 10.68). Both analogues exhibited a modest inhibitory effect
on HRV14 replication. In addition, the presence of -methyl sub-
l
M) but low selectivity
highest level of activity against both serotypes (IC50 of 0.48
lM
and 1.36 M towards HRV 1B and 14, respectively) coupled with
l
a
high selectivity (SI = 206.18 and 73.26, respectively). Therefore,
stituent coupled with a double bound in the chain (13) abolished
the efficacy towards both serotypes. For these reasons, the impact
on activity of branched chains was not further explored. Instead,
the reduction of the chromene ring to give the 3-phenylethylchro-
mans (4a) maintained the anti-HRV1B efficacy within the submi-
this chroman emerged as a good candidate for further studies.
2.3. Mechanism of action studies
The mechanism of action of the most powerful synthesized ana-
log (9c) within the new series has been studied under one cycle of
HRV1B growth in HeLa cells. The concentration of compound uti-
cromolar range (IC50 = 0.49 lM), although 4a showed only a
modest selectivity index (SI = 12.75). The activity towards serotype
14 remained modest for both phenylethyl derivatives 3a and 4a.
The elongation up to 5 carbon atoms of the linear, poly-
methylene spacer connecting 2H-chromene with phenyl ring
resulted in analogues 3d-f showing low cytotoxicity and potent
inhibitory activity against HRV1B. The corresponding chroman
analogues (4d-f), although exhibiting a low cytotoxicity, demon-
strated a lower potency than chromenes. Both series of compounds
were endowed with a weak activity against HRV14.
lized was 48.5 lM, a hundred times the IC50 value towards HRV1B.
Maximal inhibition of virus production (94%) was achieved
when 9c was present during the entire time of virus infection
(Fig. 1, bar T). The same level of inhibition was observed when
9c was present only during the time of virus adsorption (1 h,
4 °C) (Fig. 1, bar B) indicating a successful interference with virus
binding to the host cells. Also treatment of cells with 9c for 1 h
(4 °C) prior to infection reduced by 90% the virus yield (Fig. 1,
bar P) suggesting an ability of compound to adsorb to the cell sur-
face in a suitable position to prevent infection. This result could be
The introduction of chlorine at the 40 and/or 6 position of the 3-
(3-phenylpropyl)chroman 4d led to derivatives 9a-c showing
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 1. Effect of addition of 9c at different times during HRV1B one-step growth cycle in HeLa cells. Virus yield was determined by plaque assay after 10 h of incubation at
33 °C. Virus yield of mock-treated HRV1B infected cultures was 4.1 ꢀ 104 PFU/mL. P: Cells were treated with 9c for 1 h at 4 °C before virus binding (1 h at 4 °C). B: Cells were
treated with 9c during virus binding (1 h at 4 °C). T: Cells were treated with 9c during virus binding and virus multiplication (10 h at 33 °C). 0, 300, 1 h, 2 h, 3 h, 4 h: 9c was
added to infected cells at different times after virus binding (time 0) and maintained for the remaining infection time (up to 10 h at 33 °C).
100
due to the ability of membrane-bound compound to interact with
90
80
70
60
50
40
30
20
10
0
HRV particles at the cell membrane level thus inhibiting viral can-
yon function(s). Alternatively, binding of 9c to the cell surface
could hinder cell receptor recognition by the virus either through
a specific interaction with receptor molecules or by a steric hin-
drance effect. Similarly to our results, Tisdale and Selway49
reported that pretreatment of cultures with BW683C produced
an inhibition of HRV1B yield equal to that obtained when the com-
pound was added with virus.
Addition of compound immediately after virus adsorption (time
0) until the end of virus growth still resulted in 90% reduction of
HRV1B progeny yield (Fig. 1, bar 0). This result indicates that 9c,
beside an action on virus attachment to the cell membrane, is also
able to interfere with some event(s) of virus multiplication. Addi-
tion of 9c to the infected culture medium after only 30 min-1 h
post binding resulted less effective (50% and 40% reduction, respec-
tively) and the inhibition was lost after a 2 h delay or more (Fig. 1,
bar 2h, 3h, 4h). All these results indicate that 9c acts on the early
stages of rhinovirus multiplication.
In a parallel set of experiments 9c was added to the cells imme-
diately after virus adsorption (time 0) and removed after different
times of incubation at 33 °C. Removal of compound from 15 min up
to 1 h post infection (p.i.) caused a reduction of virus yield ranging
from 47% to 69% indicating that the antiviral effect of 9c was par-
tially reversible (Fig. 2). Removal of compound at later times p.i.
(2 h, 4 h, and 6 h) did not significantly affect virus yield confirming
an action of 9c on the early stages of HRV infection (Fig. 2).
A virucidal test was set up to verify an effect on virus particles.
After a 1 h exposure (33 °C) of HRV1B to 9c, the infectivity titers of
treated and mock-treated virus suspensions were found similar
(mock-treated virus 1.63 ꢀ 107 PFU/mL, 9c-treated virus
1.38 ꢀ 107 PFU/mL). On the contrary, in stabilization studies, 9c
protected HRV1B infectivity against inactivation by both mild acid
and heat treatments. Exposure to mild acid produced a drop in
infectivity of 3.1 log. In the presence of 9c, the reduction in infec-
tivity was only 0.7 log (Fig. 3). In a similar manner, heat treatment
significantly lowered virus infectivity (by 5.4 log) while in the pres-
ence of 9c the reduction was 4.2 log (Fig. 4). All together these
results suggest that 9c behaves as a capsid binder and that the
inhibitory effect is reversible by dilution.
15'
30'
1h
2h
4h
6h
Fig. 2. Effect of removal of 9c at different times during HRV1B one-step growth
cycle in HeLa cells. Virus yield was determined by plaque assay after 10 h of
incubation at 33 °C. Virus yield of mock-treated HRV1B infected cultures was
4.1 ꢀ 104 PFU/mL. 150, 300, 1 h, 2 h, 4 h, 6 h: 9c was added to cells after virus binding
(1 h at 4 °C, time 0) and removed after different incubation times at 33 °C. The
cultures were further incubated in maintenance medium without drug up to 10 h
(33 °C).
for cytotoxicity and antiviral activity on HRV serotype 1B and 14
infection in HeLa cell cultures. 3-[3-(4-Chlorophenyl)propyl]chro-
man (9c) showed potent activity against both serotypes and high
selectivity. Preliminary mechanism of action studies, carried out
on HRV serotype 1B as a model, suggest that 9c affect functions
associated with the viral capsid, including virus attachment to cell
membrane and the uncoating of viral genome.
3. Experimental
3.1. Chemistry
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. 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
In conclusion, new series of 3-phenylalkyl-2H-chromenes and -
chromans bearing aliphatic spacers ranging from two to five C
atoms between the two cycles have been synthesized and tested
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

6
C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
8
7
6
5
4
3
2
1
0
V
pH 5
9c
Fig. 3. Protective effect of 9c on acid inactivation of HRV1B infectivity. V: Untreated virus. pH 5: Virus exposed to pH 5.0 for 30 min. 9c: Virus exposed to pH 5.0 in the
presence of 9c.
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied by column chromatography on silica gel eluting with a mix-
ture of ethyl acetate/light petroleum (1:2).
8
7
6
5
4
3
2
1
0
3.1.1.1. 1-Bromo-4-phenylbutan-2-one (2a). Yield: 72%, mp = 43–
44 °C. IR (KBr): 1718 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
7.29 (t, 2H, H30, H50 J2 -3 = J3 -4 = 7.6 Hz), 7.22–7.17 (m, 3H, H20,
H40, H60), 3.84 (s, 2H, 2H1), 2.98–2.94 (m, 4H, 2H3, 2H4). 13C
NMR (CDCl3, 100 MHz): d (ppm): 200.63, 136.23, 130.29, 128.96,
128.60, 40.82, 33.85, 27.10.
0
0
0
0
3.1.1.2. 1-Bromo-4-phenylpentan-2-one (2b). Yield: 76%, mp = 46–
47 °C. IR (KBr): 1718 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
7.26 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.19–7.14 (m, 3H, H20,
H40, H60), 3.70 (s, 2H, 2H1), 3.32–3.27 (m, 1H, H4), 2.89 (dd, 1H,
1H3, Jgem = 16.4 Hz, J3-4 = 6.8 Hz), 2.82 (dd, 1H, 1H3, Jgem = 16.4 Hz,
J3-4 = 7.6 Hz), 1.25 (d, 3H, CH3, JCH3–CH = 6.8 Hz). 13C NMR (CDCl3,
100 MHz): d (ppm) 200.50, 145.44, 128.55, 126.66, 126.45, 47.93,
35.46, 34.95, 21.79.
0
0
0
0
V
56°C
9c
Fig. 4. Protective effect of 9c on thermal inactivation of HRV1B infectivity. V:
Untreated virus. 56 °C: Virus incubated at 56 °C for 20 min. 9c: Virus incubated at
56 °C for 20 min in the presence of 9c.
1H NMR. TLC was performed on silica gel or aluminium oxide flu-
orescent coated plates (Merck, Kieselgel or Aluminium oxide 60
F254). Components were visualised by UV light. Elemental analy-
ses (C, H, N, Cl) of new compounds were within 0.4% of theoretical
values. 2H-Chromene-3-carbaldehyde was synthesized following
the procedure previously described.42
3.1.1.3. 1-Bromo-3-methyl-4-phenylbutan-2-one (2c). Yield: 75%,
mp = 48–49 °C. IR (KBr): 1713 cmꢁ1
.
1H NMR (CDCl3, 400 MHz): d
(ppm) 7.24 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.16 (t, 1H, H40,
0
0
0
0
J4 -5 = 7.6 Hz), 7.10 (d, 2H, H20, H60, J2 -3 = 7.6 Hz), 3.76 (s, 2H,
2H1), 3.14–3.07 (m, 1H, H3), 2.92 (dd, 1H, 1H4, Jgem = 13.6 Hz,
J3-4 = 7.2 Hz), 2.82 (dd, 1H, 1H4, Jgem = 13.6 Hz, J3-4 = 7.2 Hz), 1.09
(d, 3H, CH3, JCH3-CH = 6.8 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm)
204.95, 139.01, 128.89, 128.63, 126.60, 45.68, 39.73, 34.31, 16.94.
0
0
0
0
3.1.1. General procedure for the synthesis of the 1-
bromomethylphenylalkyl-2-ones (2a-f)
Oxalyl chloride (23 mmol) was added dropwise to a solution of
the appropriate phenylalkyl acid (1a-f) (10 mmol) in dry CH2Cl2
(18.5 mL) cooled at 0 °C. After complete addition, the mixture
was stirred at room temperature for 3 h. After this period, the mix-
ture was evaporated to dryness and the obtained phenylalkyl acyl
chloride was used for the next reaction without further purifica-
tion. 2N trimethylsilyldiazomethane in dry Et2O (27.3 mL) was
added dropwise to a solution of the appropriate phenylalkyl acyl
chloride (10 mmol) in dry CH3CN (13.5 mL) cooled at 0 °C. After
stirring at 0 °C for 2 h, 48% aqueous HBr (15.3 mL) was added drop-
wise to the mixture cooled at 0 °C. After complete addition, the
mixture was stirred at room temperature overnight, then diluted
with water and extracted with ethyl acetate. The organic layer
was washed with brine and NaHCO3 solution, then dried over
3.1.1.4. 1-Bromo-5-phenylpentan-2-one (2d). Yield: 30%, mp = 51–
50 °C. IR (KBr): 1715 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
7.20 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.18–7.15 (m, 3H, H20,
H60, H40), 3.83 (s 2H, 2H1), 2.66–2.61 (m, 4H, 2H3, 2H5), 1.94 (quin,
2H, 2H4 J3-4 = J4-5 = 7.6 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm)
201.74, 141.19, 128.45, 128.08, 126.08, 38.91, 34.83, 33.37, 25.23.
0
0
0
0
3.1.1.5. 1-Bromo-6-phenylhexan-2-one (2e). Yield: 58%, mp = 48–
49 °C. IR (KBr): 1715 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
7.19 (m, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.17–7.08 (m, 3H, H20,
H60, H40), 3.73 (s, 2H, 2H0), 2.52 (t, 2H, 2H3, J3-4 = 7.2 Hz), 2.46
(t, 2H, 2H6, J5-6 = 7.2 Hz), 1.57–1.48 (m, 4H, 2H4, 2H5). 13C NMR
0
0
0
0
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
(CDCl3, 100 MHz): d (ppm) 201.25, 141.93, 128.27, 128.23, 125.69,
39.28, 35.47, 34.71, 30.55, 23.27.
123.09, 121.29, 118.18, 115.27, 67.21, 41.37, 39.41, 18.04. Anal.
Calcd for C18H18O: C, 86.36; H, 7.25. Found: C, 86.57; H, 7.29.
3.1.2.4. 3-(3-Phenylpropyl)-2H-chromene (3d). Yield: 43%, mp = 43–
3.1.1.6. 1-Bromo-7-phenylheptan-2-one (2f). Yield: 47%, mp = 47–
45 °C from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR (CDCl3,
.
48 °C. IR (KBr): 1717 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm)
.
400 MHz): 7.24 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.17–7.12
(m, 3H, H20, H60, H40), 7.05 (dt, 1H, H7, J7-8 = J6-7 = 8.0 Hz,
J5-7 = 1.6 Hz), 6.87 (dd, 1H, H5, J5-6 = 8.0 Hz, J5-7 = 1.6 Hz), 6.79 (dt,
1H, H6, J6-7 = J5-6 = 8.0 Hz, J6-8 = 1.2 Hz), 6.74 (dd, 1H, H8,
J7-8 = 8.0 Hz, J6-8 = 1.2 Hz), 6.10 (s, 1H, H4), 4.60 (s, 1H, H2), 2.59
(t, 2H, 2H9, J9-10 = 7.6 Hz), 2.02 (t, 1H, 2H11, J10-11 = 7.6 Hz), 1.77
(quin, 2H, 2H10, J9-10 = J10-11 = 7.6 Hz). 13C NMR (CDCl3,
7.25 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.15–7.13 (m, 3H, H20,
H60, H40), 3.82 (s, 2H, 2H1), 2.61–2.57 (m, 4H, 2H3, 2H7), 1.66–
1.57 (m, 4H, 2H4, 2H6), 1.36–1.28 (m, 2H, 2H5). 13C NMR (CDCl3,
100 MHz): d (ppm) 201.95, 142.35, 128.34, 128.24, 125.66, 39.61,
35.64, 34.35, 31.09, 28.54, 23.61.
0
0
0
0
0
0
0
0
3.1.2. General procedure for the synthesis of the phenylalkyl-2H-
chromenes (3a-f)
100 MHz):
d (ppm) 153.16, 142.02, 134.97, 128.62, 128.58,
128.35, 126.14, 126.10, 123.12, 121.47, 119.04, 115.48, 68.51,
35.59, 32.81, 28.60. Anal. Calcd for C18H18O: C, 86.36; H, 7.25.
Found: C, 86.67; H, 7.23.
Tert-BuOK (10 mmol) was added to a solution of (2-hydroxy-
benzyl)triphenylphosphonium bromide (10 mmol) in dry THF
(43 mL) at room temperature. After 10 min, a solution of the appro-
priate bromomethylphenylketone (2a-f) (10 mmol) in dry THF
(44 mL) was added dropwise. After 2 h, a new portion of tert-BuOK
(10 mmol) was added and the mixture was stirred for 24 h at room
temperature under nitrogen. After this period, ice and water were
added and the mixture was neutralized and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered and evaporated to dryness. The solid product
was chromatographed on silica gel column eluting with a mixture
of CHCl3/light petroleum (1:1) and further purified by crystalliza-
tion by n-hexane.
3.1.2.5. 3-(4-Phenylbutyl)-2H-chromene (3e). Yield: 42%, mp = 45–
46 °C from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR (CDCl3,
.
0
0
0
0
400 MHz): 7.27 (t, 2H,, J2 -3 = J3 -4 = 7.8 Hz), 7.19–7.15 (m, 3H,
H20, H60, H40), 7.02 (dt, 1H, H7, J6-7 = J7-8 = 7.6 Hz, J7-5 = 2.0 Hz),
6.90 (dd, 1H, H5, J5-6 = 7.6 Hz, J7-5 = 2.0 Hz), 6.82 (dt, 1H, H6,
J5-6 = J6-7 = 7.6 Hz, J6-8 = 1.2 Hz), 6.74 (dd, 1H, H8, J7-8 = 7.6 Hz,
J6-8 = 1.2 Hz), 6.10 (s, 1H, H4), 4.60 (s, 2H, H2), 2,63 (t, 2H, 2H9,
J9-10 = 7.8 Hz), 2.08 (t, 2H, 2H12, J11-12 = 7.8 Hz), 1.68–1.62 (m,
2H, 2H11), 1.55–1.49 (m, 2H, 2H10). 13C NMR (CDCl3, 100 MHz):
d (ppm) 152.88, 142.26, 135.01, 128.38, 128.31, 128.07, 125.87,
125.75, 122.94, 121.25, 118.69, 115.22, 68.32, 35.69, 33.07, 30.99,
26.36. Anal. Calcd for C19H20O: C, 86.32; H, 7.63. Found: C, 86.01;
H, 7.66.
3.1.2.1. 3-Phenethyl-2H-chromene (3a). Yield: 22%, mp = 39–40 °C
from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
(ppm) 7.22 (t, 2H, H30, H50, J2 -3 = 7.6 = J3 -4 = 7.6 Hz), 7.21–7.17
(m, 3H, H20, H60, H40), 7.06 (dt, 1H, H7, J6-7 = J7-8 = 7.4 Hz,
J5-7 = 1.6 Hz), 7.03 (dd, 1H, H5, J5-6 = 7.4 Hz, J5-7 = 1.6 Hz), 6.90
(dt, 1H, H6, J5-6 = J6-7 = 7.4 Hz, J6-8 = 1.2 Hz), 6.83 (dd, 1H, H8,
J7-8 = 7.4 Hz, J6-8 = 1.2 Hz), 6.15 (s, 1H, H4), 4.66 (s, 2H, H2), 2.80
(t, 2H, H10, J9-10 = 8.0 Hz), 2.36 (t, 2H, H9, J9-10 = 8.0 Hz). 13C NMR
0
0
0
0
3.1.2.6. 3-(5-Phenylpentyl)-2H-chromene (3f). Yield: 55%, mp = 45–
46 °C from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR (CDCl3,
.
400 MHz): d (ppm) 7.25 (t, 2H, H30, H50 J2 -3 = J3 -4 = 7.6 Hz),
7.16–7.14 (m, 3H, H20, H60, H40), 7.02 (dt, 1H, H7, J7-6 = J
7-8 = 8.0 Hz, J5-7 = 1.6 Hz), 6.88 (dd, 1H, H5, J5-6 = 8.0 Hz,
J5-7 = 1.6 Hz), 6.81 (dt, 1H, H6, J5-6 = J7-6 = 8.0 Hz, J6-8 = 1.2 Hz),
6.75 (dd, 1H, H8, J7-8 = 8.0 Hz, J6-8 = 1.2 Hz), 6.10 (s, 1H, H4), 4.63
(s, 2H, H2), 2.59 (t, 2H, 2H9, J9-10 = 7.6 Hz), 2.03 (t, 2H, 2H13, J12-
13 = 7.6 Hz), 1.63 (quin, 2H, 2H10, J9-10 = J10-11 = 7.6 Hz), 1.49 (quin,
0
0
0
0
(CDCl3, 100 MHz):
d (ppm) 152.98, 141.24, 134.44, 128.48,
128.31, 128.25, 126.14, 126.03, 122.87, 121.31, 119.22, 115.29,
68.31, 33.52, 29.71. Anal. Calcd for C17H16O: C, 86.40; H, 6.82.
Found: C, 86.73; H, 6.79.
2H, 2H12, J11-12 = J12-13 = 7.6 Hz), 1.36 (quin, 2H, 2H11, J10-11
=
J11-12 = 7.6 Hz). 13C NMR (CDCl3, 100 MHz):
d
(ppm) 152.87,
3.1.2.2. 3-(2-Phenylpropyl)-2H-chromene (3b). Yield: 58%, mp = 63–
64 °C from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR (CDCl3,
.
142.52, 135.19, 128.35, 128.25, 128.03, 125.86, 125.45, 122.96,
121.23, 118.56, 115.21, 68.32, 35.82, 33.11, 31.23, 28.88, 26.68.
Anal. Calcd for C20H22O: C, 86.29; H, 7.97. Found: C, 86.58; H, 7.96.
400 MHz): d (ppm) 7.24 (t, 2H, H30, H50, J2 -3 = 7.6 = J3 -4 = 7.6 Hz),
7.16–7.11 (m, 3H, H20, H60, H40), 6.98 (ddd, 1H, H7, J6-7 = 7.6 Hz,
J7-8 = 8.0 Hz, J5-7 = 1.6 Hz), 6.84 (dd, 1H, H5, J5-6 = 7.6 Hz, J5-
7 = 1.6 Hz), 6.77 (t, 1H, H6, J5-6 = J6-7 = 7.6 Hz), 6.73 (d, 1H, H8, J7-
8 = 8.0 Hz), 6.05 (s, 1H, H4), 4.56 (s, 2H, H2), 2.90–2.87 (m, 1H,
H10), 2.34 (dd, 1H, 1H9, Jgem = 14.4 Hz, J9-10 = 7.0 Hz), 2.26 (dd,
1H, 1H9, Jgem = 14.4 Hz, J9-10 = 8.0 Hz), 1.24 (d, 3H, CH3, JCH3-
CH = 6.8 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm) 152.89, 146.32,
133.35, 128.40, 128.13, 126.70, 126.21, 125.91, 122.84, 121.17,
120.61, 115.20, 68.22, 42.58, 37.99, 21.73. Anal. Calcd for
0
0
0
0
3.1.3. General procedure for the synthesis of 3-phenylalkylchromans
(4a,d-f, 9a-c)
A solution of the appropriate phenylalkyl-2H-chromene (3a,d-f)
or the appropriate mixture of (Z)-3-[(E)-3-phenylallylidene]chro-
man, (E)-3-(3-phenyl)prop-1-en-1-yl)-2H-chromene, and 3-cin-
namyl-2H-chromene (6, 7, 8a; 6, 7, 8b or 6, 7, 8c)43 (10 mmol) in
dry dioxane (80 mL) was hydrogenated over 10% Pd/C (15.2 mmol)
at room temperature for 30 min, under 40 psi pressure. After this
period, the catalyst was filtered off and the solvent was removed
under reduced pressure. The residue was purified by column chro-
matography on silica gel eluting with a mixture of chloroform/light
petroleum (1:1). The product was further purified by crystalliza-
tion from n-hexane.
C18H18O: C, 86.36; H, 7.25. Found: C, 86.02; H, 7.28.
3.1.2.3. 3-(1-Phenylpropan-2-yl)-2H-chromene (3c). Yield: 38%,
mp = 65–68 °C from n-hexane. IR (KBr): 1604 cmꢁ1 1H NMR
(CDCl3, 400 MHz):
(ppm) 7.23 (t, 2H, H30, H50, J2 -3
.
0
0
d
= J3 -4 = 7.6 Hz), 7.16–7.10 (m, 3H, H20, H60, H40), 7.01 (dt, 1H,
H7, J6-7 = J7-8 = 7.6 Hz, J5-7 = 1.6 Hz), 6.89 (dd, 1H, H5, J5-6 = 7.6 Hz,
J5-7 = 1.6 Hz), 6.81 (dt, 1H, H6, J5-6 = J6-7 = 7.6 Hz, J6-8 = 1.2 Hz),
6.76 (d, 1H, H8, J7-8 = 7.6 Hz), 6.11 (s, 1H, H4), 4.64 (s, 2H, H2),
2.81 (dd, 1H, H10, Jgem = 13.2 Hz, J9-10 = 6.0 Hz), 2.45 (dd, 1H,
H10, Jgem = 13.2 Hz, J9-10 = 8.0 Hz), 2.47–2.38 (m, 1H, H9), 1.05 (d,
3H, CH3, JCH3-CH = 7.2 Hz). 13C NMR (CDCl3, 100 MHz): d (ppm)
153.18, 140.01, 138.97, 129.00, 128.24, 128.18, 126.15, 126.08,
0
0
3.1.3.1. 3-Phenethylchroman (4a). Yield: 69%, mp = 42–43 °C from
n-hexane. IR (KBr): 1607 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
(ppm) 7.27 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.19–7.15 (m,
0
0
0
0
3H, H20, H60, H40), 7.06 (ddd, 1H, H7, J6-7 = 7.4 Hz, J7-8 = 8.0 Hz,
J5-7 = 1.6 Hz), 7.02 (dd, 1H, H5, J5-6 = 7.4 Hz, J5-7 = 1.6 Hz), 6.82
(dt, 1H, H6, J5-6 = J6-7 = 7.4 Hz, J6-8 = 1.2 Hz), 6.78 (dd, 1H, H8, J7-
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

8
C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
8 = 8.0 Hz, J6-8 = 1.2 Hz), 4.20 (m, 1H, 1H2, Jgem = 10.8 Hz,
J2-3 = 3.2 Hz), 3.76 (dd, 1H, 1H2, Jgem = 10.8 Hz, J2-3 = 9.2 Hz), 2.88
(dd, 1H, 1H4, Jgem = 16.2 Hz, J3-4 = 5.2 Hz), 2.78–2.66 (m, 2H,
H10), 2.49 (dd, 1H, H4, Jgem = 16.2 Hz, J3-4 = 9.6 Hz), 2.07–1.97
(m, 1H, H3), 1.75–1.56 (m, 2H, H9). 13C NMR (CDCl3, 100 MHz): d
(ppm) 154.67, 141.84, 129.88, 128.45, 128.32, 127.23, 125.96,
121.57, 120.25, 116.44, 70.38, 33.50, 33.03, 31.67, 31.49. Anal.
Calcd for C17H18O: C, 85.67; H, 7.61. Found: C, 85.41; H, 6.63.
123.29, 117.70, 70.57, 35.94, 31.75, 31.34, 31.19, 28.55. Anal. Calcd
for C18H19ClO: C, 75.38; H, 6.68; Cl, 12.36. Found: C, 75.65; H, 6.70;
Cl, 12.42.
3.1.3.6. 6-Chloro-3-[3-(4-chlorophenyl)propyl]chroman (9b). Yield:
85%, mp = 41–42 °C from n-hexane. IR (KBr): 1484 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz): d (ppm) 7.20 (d, 2H, H30, H50, J2 -3 = 8.0 Hz),
0
0
7.05 (d, 2H, H20, H60, J2 -3 = 8.0 Hz),6.96 (dd, 1H, H7, J7-8 = 8.8 Hz,
J5-7 = 2.4 Hz), 6.92 (d, 1H, H5, J7-5 = 2.4 Hz), 6.67 (d, 1H, H8,
J7-8 = 8.8 Hz), 4.11 (dd, 1H, H2, Jgem = 10.8 Hz, J2-3 = 3.4 Hz), 3.63
(dd, 1H, H2, Jgem = 10.8 Hz, J2-3 = 9.4 Hz), 2.71 (dd, 1H, 1H4,
Jgem = 16.4 Hz, J3-4 = 4.8 Hz), 2.54 (t, 2H, 2H11, J10-11 = 7.6 Hz),
2,31 (dd, 1H, 1H4, Jgem = 16.4 Hz, J3-4 = 9.6 Hz), 1.91–1.87 (m, 1H,
H3), 1.70–1.59 (m, 2H, 2H10), 1.37–1.28 (m, 1H, 1H9), 1.28–1.19
0
0
3.1.3.2. 3-(3-Phenylpropyl)chroman (4d). Yield: 34%, mp = 41–42 °C
from n-hexane. IR (KBr): 1606 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
(ppm) 7.27 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.8 Hz), 7.18–7.15 (m,
3H, H20, H60, H40), 7.05 (t, 1H, H7, J6-7 = J7-8 = 7.8 Hz), 6.99 (d, 1H,
H5, J5-6 = 7.2 Hz), 6.83–6.76 (m, 2H, H6, H8), 4.17 (dd, 1H, 1H2,
Jgem = 10.4 Hz, J2-3 = 3.2 Hz), 3.69 (dd, 1H, 1H2, Jgem = 10.4 Hz,
J2-3 = 9.2 Hz), 2.82 (dd, 1H, 1H4, Jgem = 16.0 Hz, J3-4 = 4.0 Hz), 2.62
(t, 2H, 2H11, J10-11 = 7.6 Hz), 2.41 (dd, 1H, 1H4, Jgem = 16.0 Hz,
J3-4 = 9.6 Hz), 2.03–1.97 (m, 1H, H3), 1.77–1.66 (m, 2H, 2H10),
1.45–1.36 (m, 2H, 1H9), 1.36–1.27 (m, 2H, 1H9). 13C NMR (CDCl3,
0
0
0
0
(m, 1H, 1H9). 13C NMR (CDCl3, 100 MHz):
140.41, 131.44, 129.64, 128.28, 128.36, 127.05, 124.69, 123.15,
117.64, 70.34, 35.16, 31.58, 31.18, 30.98, 28.34. Anal. Calcd for C18
18Cl2O: C, 67.30; H, 5.65; Cl, 22.07. Found: C, 67.12; H, 5.66; Cl,
21.99.
d (ppm) 153.19,
-
H
100 MHz):
d (ppm) 154.65, 142.14, 129.83, 128.34, 128.32,
127.16, 125.79, 121.67, 120.18, 116.39, 70.49, 35.95, 32.03, 31.48,
31.36, 28.58. Anal. Calcd for C18H20O: C, 85.67; H, 7.99. Found: C,
86.85; H, 8.02.
3.1.3.7. 3-[3-(4-Chlorophenyl)propyl]chroman (9c). Yield: 16%,
mp = 42–43 °C from n-hexane. IR (KBr): 1489 cmꢁ1
.
1H NMR
(CDCl3, 400 MHz): d (ppm) 7.23 (d, 2H, H30, H50, J2 -3 = 8.4 Hz),
7.10–7.04 (m, 3H, H20, H60, H7), 7.00 (d, 1H, H5, J5-6 = 7.2 Hz),
6.84–6.77 (m, 2H, H6, H8), 4.18 (dd, 1H, 1H2, Jgem = 10.8 Hz,
J2-3 = 3.2 Hz), 3.71 (dd, 1H, 1H2, Jgem = 10.8 Hz, J2-3 = 9.2 Hz), 2.83
(dd, 1H, 1H4, Jgem = 16.2 Hz, J3-4 = 4.4 Hz), 2.59 (t, 2H, 2H11,
J10-11 = 8.0 Hz), 2,43 (dd, 1H, 1H4, Jgem = 16.2 Hz, J3-4 = 9.4 Hz),
2,04–1.95 (m, 1H, H3), 1.76–1.63 (m, 2H, 2H10), 1.45–1.36
(m, 2H, 1H9), 1.36–1.28 (m, 2H, 1H9). 13C NMR (CDCl3,
0
0
3.1.3.3. 3-(4-Phenylbutyl)chroman (4e). Yield: 86%, mp = 43–44 °C
from n-hexane. IR (KBr): 1607 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
(ppm) 7.24 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.16–7.13 (m,
3H, H20, H60, H40), 7.04 (t, 1H, H7, J7-6 = J7-8 = 7.6 Hz), 6.97 (d, 1H,
H5, J5-6 = 7.2 Hz), 6.81–6.77 (m, 2H, H6, H8), 4.14 (dd, 1H, 1H2,
Jgem = 10.4 Hz, J2-3 = 3.2 Hz), 3.67 (dd, 1H, 1H2, Jgem = 10.4 Hz, J2-
3 = 9.6 Hz), 2.78 (dd, 1H, 1H4, Jgem = 16.0 Hz, J3-4 = 4.8 Hz), 2.59 (t,
2H, 2H12, J11-12 = 7.6 Hz) 2.38 (dd, 1H, 1H4, Jgem = 16.0 Hz, J3-
4 = 9.6 Hz), 1.99–1.88 (m, 1H, H3), 1.62 (quin, 2H, 2H11, J10-
11 = J11-12 = 7.6 Hz), 1.46–1.22 (m, 4H, 2H9, 2H10). 13C NMR (CDCl3,
100 MHz): d (ppm) 154.61, 142.38, 129.81, 128.32, 128.24, 127.10,
125.65, 121.68, 120.11, 116.34, 70.48, 35.77, 31.98, 31.55, 31.48,
31.46, 26.31. Anal. Calcd for C19H22O: C, 85.67; H, 8.32. Found: C,
85.98; H, 8.29.
0
0
0
0
100 MHz):
d (ppm) 154.62, 140.56, 131.54, 129.86, 129.71,
128.44, 127.21, 121.58, 120.24, 116.41, 70.43, 35.28, 32.03, 31.47,
31.23, 28.51. Anal. Calcd for C18H19ClO: C, 75.38; H, 6.68; Cl,
12.36. Found: C, 75.63; H, 6.66; Cl, 12.32.
3.1.3.8.
1-(2H-Chromen-3-yl)ethanone
(10). 3-Buten-2-one
(40 mmol) was added dropwise to a suspension of 2-hydroxyben-
zaldehyde (20 mmol) and 1,4-diazabicyclo-[2.2.2]-octane (DABCO)
(20 mmol) in CHCl3 (14 mL) and H2O (14 mL) at room temperature.
The mixture was stirred, under nitrogen atmosphere, at room tem-
perature and for a week. After this period, the separated solid was
filtered and washed with water. The organic layer was washed
with 2 N NaOH, 2 N HCl and brine, dried over anhydrous Na2SO4,
filtered and evaporated to dryness. The obtained solid was purified
by column chromatography on silica gel eluting with a mixture of
chloroform/light petroleum (1:1). Yield: 46%. mp = 44–45 °C IR
3.1.3.4. 3-(5-Phenylpentyl)chroman (4f). Yield: 81%, mp = 42–43 °C
from n-hexane. IR (KBr): 1607 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
(ppm) 7.24 (t, 2H, H30, H50, J2 -3 = J3 -4 = 7.6 Hz), 7.15–7.13 (m,
3H, H20, H60, H40), 7.04 (t, 1H, H7, J7-6 = J7-8 = 7.8 Hz), 6.98 (d, 1H,
H5, J5-6 = 7.4 Hz), 6.82–6.77 (m, 2H, H6, H8), 4.15 (dd, 1H, 1H2,
Jgem = 10.4 Hz, J2-3 = 3.2 Hz), 3.67 (dd, 1H, 1H2, Jgem = 10.4 Hz,
J2-3 = 9.6 Hz), 2.78 (dd, 1H, 1H4, Jgem = 16.4 Hz, J3-4 = 4.8 Hz), 2.58
(t, 2H, H13, J12-13 = 7.6 Hz), 2.38 (dd, 1H, 1H4, Jgem = 16.4 Hz,
J3-4 = 9.6 Hz), 1.96–1.91 (m, 1H, H3), 1.61 (quin, 2H, 2H12, J11-
12 = J12-13 = 7.6 Hz), 1.46–1.18 (m, 6H, 2H9, 2H10, 2H11). 13C NMR
0
0
0
0
(KBr): 1637 cmꢁ1 1H NMR (CDCl3, 400 MHz): d (ppm) 7.30 (d,
.
1H, H4, J2-4 = 1.6 Hz), 7.27–7.23 (m, 1H, H7, J6-7 = 7.6 Hz,
J7-8 = 8.0 Hz, J5-7 = 1.6 Hz), 7.16 (dd, 1H, H5, J5-6 = 7.6 Hz,
J5-7 = 1.6 Hz), 6.93 (dt, 1H, H6, J5-6 = J6-7 = 7.6 Hz, J6-8 = 1.6 Hz),
6.85 (d, 1H, H8, J7-8 = 8.0 Hz), 5.00 (d, 2H, H2, J2-4 = 1.6 Hz), 2.40
(s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d (ppm) 194.65, 154.43,
132.72, 131.29, 129.56, 128.10, 120.65, 119.63, 115.13, 63.10,
23.80.
(CDCl3, 100 MHz):
d (ppm) 154.62, 142.56, 129.81, 128.32,
128.21, 127.09, 125.60, 121.72, 120.10, 116.34, 70.52, 35.88,
32.00, 31.66, 31.49, 31.35, 29.33, 26.57. Anal. Calcd for C20H24O:
C, 85.67; H, 8.63. Found: C, 86.98; H, 6.60.
3.1.3.5. 6-Chloro-3-(3-phenylpropyl)chroman (9a). Yield: 79%,
mp = 42–43 °C from n-hexane. IR (KBr): 1484 cmꢁ1
.
1H NMR
3.1.3.9. 1-(2H-Chromen-3-yl)ethanol (11). A solution of NaBH4
(10 mmol) in 2 N NaOH (5 mL) was added, in a single portion, to
a stirred solution of 10 in dry EtOH (50 mL). After stirring at room
temperature for 2 h, the solvent was evaporated to dryness under
vacuum and the residue was taken up with H2O and extracted with
Et2O. The organic phase was washed with brine, dried over anhy-
drous Na2SO4, filtered and evaporated to dryness. The white oil
was used for the next reaction without further purification. Yield:
89%, oil. 1H NMR (CDCl3, 400 MHz): d (ppm) 7.08 (dt, 1H, H7, J7-
8 = J6-7 = 8.0 Hz, J5-7 = 1.6 Hz), 6.96 (dd, 1H, H5, J5-6 = 7.6 Hz, J5-
7 = 1.6 Hz), 6.85 (m, 1H, H6, J5-6 = 7.6 Hz, J6-7 = 8.0 Hz, J6-
(CDCl3, 400 MHz):
d
(ppm) 7.28 (t, 2H, H30, H50, J2 -3
0
0
= J3 -4 = 7.6 Hz), 7.20–7.16 (m, 3H, H20, H60, H40), 7.00 (dd, 1H, H7,
J6-7 = 8.4 Hz, J5-7 = 2.4 Hz), 6.97 (d, 1H, H5, J5-7 = 2.4 Hz), 6.70 (d,
1H, H8, J7-8 = 8.4 Hz), 4.17 (dd, 1H, 1H2, Jgem = 10.8 Hz, J2-
3 = 3.2 Hz), 3.70 (dd, 1H, 1H2, Jgem = 10.8 Hz, J2-3 = 9.2 Hz), 2.80
(dd, 1H, 1H4, Jgem = 16.4 Hz, J3-4 = 4.0 Hz), 2,61 (t, 2H, 2H11, J10-
11 = 7.6 Hz), 2.39 (dd, 1H, 1H4, Jgem = 16.4 Hz, J3-4 = 9.2 Hz), 2.01–
1.95 (m, 1H, H3), 1.76–1.68 (m, 2H, 2H10), 1.45–1.36 (m, 1H,
1H9), 1.36–1.28 (m, 1H, 1H9). 13C NMR (CDCl3, 100 MHz): d
(ppm) 153.29, 142.05, 129.36, 128.37, 127.15, 125.87, 124.83,
0
0
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
8 = 1.2 Hz), 6.78 (dd, 1H, H8, J7-8 = 8.0 Hz, J6-8 = 1.2 Hz), 6.32 (s, 1H,
3.2. Virology
H4), 4.77 (s, 2H, H2), 4.36 (q, 1H, CH, JCH–CH3 = 6.4 Hz), 2.12 (bs, 1H,
OH), 1.34 (d, 3H, CH3, JCH-CH3 = 6.4 Hz). 13C NMR (CDCl3, 100 MHz):
d (ppm) 153.55, 137.48, 128.98, 126.75, 122.49, 121.49, 118.46,
115.54, 68.62, 65.51, 21.30.
3.2.1. Cells and virus
HeLa cells (Ohio strain) were grown in Eagle’s Minimum Essen-
tial Medium (MEM) with 1% non essential aminoacids, 100 g/mL
l
streptomycin, 100 U/mL penicillin G and 8% heat-inactivated foetal
calf serum (growth medium). The serum concentration was
reduced to 2% for cell maintenance (maintenance medium). Cell
monolayers were kept at 37 °C in a humidified atmosphere with
5% CO2.
HRV type 1B and type 14 were purchased from the American
Type Culture Collection (ATCC). Virus stocks were prepared infect-
ing HeLa cells at a multiplicity of infection of 0.1 PFU/cell. Infected
cells were incubated in maintenance medium at 33 °C. When the
viral-induced cytopathic effect involved most of the cells, working
stocks were prepared as cellular lysates and the clarified super-
natants titrated by plaque assay as previously described.44
3.1.4. [1-(2H-Chromen-3-yl)ethyl]triphenylphosphonium bromide
(12)
Triphenylphosphine hydrobromide (10 mmol) was added at
room temperature to a solution of the 1-(2H-chromen-3-yl)ethanol
(11) (10 mmol) in dry CH3CN (24 mL). The reaction mixture was
refluxed for 5 h. After cooling to room temperature, Et2O was
added. The white precipitate was filtered and used for the next
reaction without further purification. Yield: 45%. Mp > 200 °C 1H
NMR (CD3OD, 400 MHz): d (ppm) 7.96–7.88 (m, 15H, 3H20, 3H60,
3H30, 3H50, 3H40), 7.17–6.76 (m, 3H, H5, H6, H7), 6.24 (d, 1H, H4,
J = 4.8 Hz), 4.91–4.83 (m, 1H, H9), 4.41 (dd, 1H, 1H2, J = 14.4 Hz,
J = 3.6 Hz), 4.27 41 (dd, 1H, 1H2, J = 14.4 Hz, J = 3.6 Hz), 1.72 (dd,
3H, CH3, J = 7.2 Hz, J = 18.4 Hz).
3.2.2. Compounds
Stock solutions of compounds were made up in ethanol (1, 0.5
or 0.1 mg/mL) and further diluted in cell culture medium shortly
before use.
3.1.4.1. (E)-3-(1-Phenylprop-1-en-2-yl)-2Hchromene (13). Tert-
BuOK (10 mmol) was added to a suspension of [1-(2H-chromen-
3-yl)ethyl]triphenylphosphonium bromide (12) (10 mmol) in dry
THF (43 mL) at room temperature. After 30 min, a solution of ben-
zaldehyde (10 mmol) in dry THF (45 mL) was added dropwise. The
mixture was stirred for 24 h at room temperature under nitrogen.
After this period, ice and water was added and the mixture was
neutralized with 2 N HCl. The obtained suspension was extracted
with CHCl3 and the organic layer was washed with brine, dried
over anhydrous Na2SO4, filtered and evaporated to dryness. The
obtained solid was chromatographed on silica gel column eluting
with a mixture of CHCl3/light petroleum (1:1), and further purified
by crystallization from n-hexane. Yield: 24%, mp = 56–57 °C from
3.2.3. Cytotoxicity studies
The cytotoxicity of compounds was evaluated with a tetra-
zolium-based (XTT) colorimetric assay and by microscopical eval-
uation, as previously described.44 The highest concentration of
compound that did not affect morphology and viability in 100%
of cells was recorded as the maximum non-cytotoxic concentration
(MNCC). The 50% cytotoxic concentration (CC50) was the dose of
compound reducing the cell viability by 50% of the control
samples.
3.2.4. Determination of the 50% inhibitory concentration (IC50
)
1
n-hexane. H NMR (CDCl3, 400 MHz): d (ppm) 7.34 (t, 2H, H20,
The IC50 values were determined as described previously.44
Briefly, HeLa cells in 6-well plates were infected with a virus sus-
pension producing approximately 100 plaques per well. After incu-
bation for 1 h at 33 °C, the cells were overlaid with medium for
plaques in the presence or absence of fourfold dilutions of drugs,
starting from the MNCC. After incubation at 33 °C for 72 h, the cells
were stained and the plaques were counted. The compound con-
H6, J2 -3 = 7.6 Hz), 7.29–7.21 (m, 3H, H30, H50, H40), 7.11 (dt, 1H,
H7, J7-6 = J7-8 = 8.0 Hz, J5-7 = 1.6 Hz), 7.04 (dd, 1H, H5, J5-6 = 8.0 Hz,
J5-7 = 1.6 Hz), 6.88 (t, 1H, H6, J7-6 = J5-6 = 8.0 Hz), 6.83 (d, 1H, H8,
J7-8 = 8.0 Hz) 6.61 (s, 1H, H4), 6.45 (s, 1H, Hb), 5.10 (s, 2H, H2),
2.12 (s, 3H, CH3). 13C NMR (CDCl3, 100 MHz): d (ppm): 153.54,
137.71, 133.44, 133.24, 129.40, 128.96, 128.17, 127.13, 126.79,
126.47, 122.91, 121.46, 120.41, 115.35, 66.48, 14.88. Anal. Calcd
for C18H16O: C, 87.06; H, 6.49. Found: C, 87.32; H, 6.51.
0
0
centration reducing virus plaque formation by 50% was the IC50
.
It was calculated by plotting the percentage of plaque reduction,
with respect to the control plaque count, versus the logarithm of
compound dose.
3.1.4.2. (E)-3-(2H-Chromen-3-yl)-1-phenylprop-2-en-1-one (15). A
solution of diethyl (2-oxo-2-phenethyl)phosphonate (11 mmol)
in dry THF (30 mL) was added dropwise to a stirred suspension
of NaH (11 mmol) in dry THF (15 mL) under nitrogen. The mixture
was stirred for 10 min at room temperature, then a solution of 2H-
chromene-3-carbaldehyde (14) (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 was collected by filtration, washed with water,
and chromatographed on silica gel column, eluting with a mixture
of dichloromethane and light petroleum (1:1). The product was
further purified by crystallization from n-hexane. Yield: 72%,
mp = 150–151 °C (lit. 148 °C)50 from n-hexane. IR (KBr):
3.2.5. Virus inactivation and stabilization
In inactivation studies, HRV1B was exposed or not to 9c
(48.5
ten-fold dilutions. In stabilization studies, HRV1B was incubated
with or without 9c (48.5 M) for 1 h at 33 °C before mild acid or
lM) for 1 h at 33 °C and titrated by plaque assay after serial
l
thermal treatment. For mild acid treatment, the pH of the mixtures
was lowered to 5 by addition of 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 incu-
bated for 20 min at 56 °C (pH 7.2) and then refrigerated on ice.
Samples were diluted ten-fold serially and titrated by plaque assay.
1651 cmꢁ1
.
1H NMR (CD3OD, 400 MHz): d (ppm) 8.02 (d, 2H, H20,
H60, J2 -3 = 7.4 Hz), 7.64 (t, 1H, H40), 7.60–7.52 (m, 3H, H30, H50,
), 7.22–7.16 (m, 2H, H5, H7), 7.10 (d, 1H, Hb, J -b = 16.0 Hz),
0
0
3.2.6. Time-reduction studies of virus yield
Ha
HeLa cells were infected at a multiplicity of 5 in the presence or
a
7.00 (s, 1H, H4), 6.93 (t, 1H, H6, J6-7 = J5-6 = 7.2 Hz), 6.84 (d, 1H,
H8, J7-8 = 8.0 Hz), 5.13 (s, 2H, H2). 13C NMR (CDCl3, 100 MHz): d
(ppm) 190.09, 154.66, 141.69, 135.70, 132.80, 132.25, 131.03,
129.01, 128.65, 128.41, 128.13, 121.96, 121.83, 120.63, 115.88,
65.28. Anal. Calcd for C18H14O2: C, 82.42; H, 5.38. Found: C,
82.19; H, 5.40.
absence of 9c (48.5
binding was taken as 0 time. Then, MEM with or without 9c
(48.5 M) was added and the temperature raised to 33 °C. Single-
cycle conditions were achieved by incubating the cells at 33 °C
for 10 h post infection (phi.). The cultures were freeze-thawed
three times and virus yield from clarified supernatants detected
lM) for 1 h in the cold (4 °C). The end of virus
l
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012

10
C. Conti et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
22. Desideri N, Conti C, Sestili I, Tomao P, Stein ML, Orsi N. Antiviral Chem
Chemother. 1992;3:195.
23. Genovese D, Conti C, Tomao P, et al. Antiviral Res. 1995;27:123.
24. Desideri N, Conti C, Sestili I, Tomao P, Stein ML, Orsi N. Antiviral Chem
Chemother. 1995;6:298.
by plaque assay. To determine which stage of virus replication was
affected by 9c, the compound was added or removed from HRV-
infected cultures at different times p.i. (0, 150, 300, 1 h, 2 h, 4 h,
6 h) and the cultures incubated at 33 °C up to 10 h p.i. The virus
yield was determined by plaque assay.
25. Desideri N, Olivieri S, Stein ML, Sgro R, Orsi N, Conti C. Antiviral Chem
Chemother. 1997;8:545.
26. Conti C, Mastromarino P, Sgrò R, Desideri N. Antiviral Chem Chemother.
1998;9:511.
27. Desideri N, Conti C, Mastromarino P, Mastropaolo F. Antiviral Chem Chemother.
2000;11:373.
28. Desideri N, Mastromarino P, Conti C. Antiviral Chem Chemother. 2003;14:195.
29. Conti C, Mastromarino P, Goldoni P, Portalone G, Desideri N. Antiviral Chem
Chemother. 2005;16:267.
30. Tait S, Salvati AL, Desideri N, Fiore L. Antiviral Res. 2006;72:252–255.
31. Conti C, Tomao P, Genovese D, Desideri N, Stein ML, Orsi N. Antimicrob Agents
Chemother. 1992;36:95.
References
1. Fendrick AM, Monto AS, Nightengale B, Sarnes M. Arch Intern Med.
2003;163:487.
2. Bochkov YA, Gern JE. Curr Allergy Asthma Rep. 2016;16:30 (and references
therein).
3. Gunawardana N, Finney L, Johnston SL, Mallia P. Antiviral Res. 2014;102:95.
4. http://www.who.int/respiratory/copd/en/.
5. Hayden FG, Andries K, Janssen PA. Antimicrob Agents Chemother. 1992;36:727.
6. Hayden FG, Herrington DT, Coats TL, et al. Clin Infect Dis. 2003;36:1523.
7. Pevear DC, Hayden FG, Demenczuk TM, Barone LR, McKinlay MA, Collett MS.
Antimicrob Agents Chemother. 2005;49:4492.
8. Ma JD, Nafziger AN, Rhodes G, Liu S, Gartung AM, Bertino Jr JS. J Clin Pharmacol.
2006;46:103.
9. Senior K. Lancet Infect Dis. 2002;2:264.
10. Lambert JN. 51th Interscience Conference on Antimicrobial Agents Chemotherapy;
2011. Presentation V-1557.
32. Salvati AL, De Dominicis A, Tait S, Canitano A, Lahm A, Fiore L. Antimicrob Agents
Chemother. 2004;48:2233–2243.
33. Conti C, Desideri N. Bioorg Med Chem. 2009;17:3720.
34. Rossmann MG, Arnold E, Erickson JW, et al. Nature. 1985;317:145.
35. Kim SS, Smith TJ, Chapman MS, et al. J Mol Biol. 1989;210:91–111.
36. Arnold E, Rossmann MG. J Mol Biol. 1990;211:763–801.
37. Kim KH, Willingmann P, Gong ZX, et al. J Mol Biol. 1993;230:206–226.
38. Diana GD, Mallamo JP. J Mol Biol. 1993;230:227.
39. Hadfield AT, Lee W, Zhao R, et al. Structure. 1997;5:427–441.
40. Verdaguer N, Blaas D, Fita I. J Mol Biol. 2000;300:1179–1194.
41. Zhao R, Pevear DC, Kremer MJ, et al. Structure. 1996;4:1205–1220.
42. Conti C, Desideri N. Bioorg Med Chem. 2010;18:6480.
43. Conti C, Proietti Monaco L, Desideri N. Bioorg Med Chem. 2011;19:7357.
44. Conti C, Proietti Monaco L, Desideri N. Bioorg Med Chem. 2014;22:1201.
45. Desideri N. Lett Org Chem. 2006;3:546.
46. Andries K, Dewindt B, Snoeks J, et al. J Virol. 1990;64:1117.
47. Andries K, Dewindt B, Snoeks J, Willebrords R, Stokbroekx R, Lewi P. J Antiviral
Res. 1991;16:213.
11. http://www.biotapharma.com/index.php/pipeline.
12. Feil SC, Hamilton S, Krippner GY, et al. ACS Med Chem Lett. 2012;3:303.
13. Greve JM, Davis G, Meyer AM, et al. Cell. 1989;56:839.
14. Staunton DE, Merluzzi VJ, Rothlein R, Barton R, Marlin SD, Springer TA. Cell.
1989;56:849.
15. Tomassini JE, Graham D, DeWitt CM, Lineberger DW, Rodkey JA, Colonno RJ.
Proc Natl Acad Sci USA. 1989;86:4907.
16. Hofer F, Gruenberger M, Kowalski H, et al. Proc Natl Acad Sci USA. 1994;91:1839.
17. Fuchs R, Blaas D. Adv Virol. 2012;826301.
18. De Palma AM, Vliegen I, De Clercq E, Neyts J. Med Res Rev. 2008;28:823 (and
references therein).
19. Dewindt B, van Eemeren K, Andries K. Antiviral Res. 1994;25:67.
20. Burali C, Desideri N, Stein ML, Conti C, Orsi N. Eur J Med Chem. 1987;22:119.
21. Conti C, Desideri N, Orsi N, Sestili I, Stein ML. Eur J Med Chem. 1990;25:725.
48. Bauer DJ, Selway JWT, Batchelor JF, Tisdale M, Caldwell JC, Young DAB. Nature.
1981;292:369.
49. Tisdale M, Selway JWT. J Gen Virol. 1983;64:795.
50. Majumdar KC, Ansary I, Shyam PK, Roy B. Synlett. 2012;23:1225.
Please cite this article in press as: Conti C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.012