VP1 crystal structure-guided exploration and optimization of 4,5-dimethoxybenzene-based inhibitors of rhinovirus 14 infection

Article abstract of DOI:10.1016/j.ejmech.2016.03.049

Human rhinoviruses (HRV) are the predominant cause of common colds and flu-like illnesses, but are also responsible for virus-induced exacerbations of asthma and chronic obstructive pulmonary disease. However, to date, no drug has been approved yet for clinical use. In this study, we present the results of the structure-based lead optimization of a class of new small-molecule inhibitors that we previously reported to bind into the pocket beneath the canyon of the VP1 protein. A small series of analogues that we designed based on the available structure and interaction data were synthesized and evaluated for their potency to inhibit the replication of HRV serotype 14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (3v) was found to be a potent inhibitor exhibiting micromolar activity (EC₅₀ Combining double low line 3.4 ± 1.0 μM) with a toxicity for HeLa cells that was significantly lower than that of our previous hit (LPCRW-0005, CC₅₀ Combining double low line 104.0 ± 22.2 μM; 3v, CC₅₀ > 263 μM).

Full text of DOI:10.1016/j.ejmech.2016.03.049

Accepted Manuscript
VP1 crystal structure-guided exploration and optimization of 4,5-dimethoxybenzene-
based inhibitors of rhinovirus 14 infection
Laurène Da Costa, Manon Roche, Els Scheers, Antonio Coluccia, Johan Neyts,
Thierry Terme, Pieter Leyssen, Romano Silvestri, Patrice Vanelle
PII:
S0223-5234(16)30231-8
10.1016/j.ejmech.2016.03.049
EJMECH 8474
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 February 2016
Revised Date: 17 March 2016
Accepted Date: 18 March 2016
Please cite this article as: L. Da Costa, M. Roche, E. Scheers, A. Coluccia, J. Neyts, T. Terme, P.
Leyssen, R. Silvestri, P. Vanelle, VP1 crystal structure-guided exploration and optimization of 4,5-
dimethoxybenzene-based inhibitors of rhinovirus 14 infection, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.03.049.
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VP1 crystal structure-guided exploration and optimization
Leave this area blank for abstract info.
of 4,5-dimethoxybenzene-based inhibitors of rhinovirus
14 infection
Laurène Da Costa,1 Manon Roche,1 Els Scheers,2 Antonio Coluccia,3 Johan Neyts,2 Thierry Terme,1 Pieter Leyssen,2 Romano Silvestri,3 and Patrice
Vanelle.1
1 Aix-Marseille Université, Institut de Chimie Radicalaire, UMR 7273 CNRS, 27 Boulevard Jean Moulin, Marseille, France.
2 KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
3 Institut Pasteur Italy, Department of Drug Chemistry and Technologies, Sapienza University, I-00185 Rome, Italy.

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VP1 crystal structure-guided exploration and optimization of 4,5-
dimethoxybenzene-based inhibitors of rhinovirus 14 infection
Laurène Da Costa,1 Manon Roche,1 Els Scheers,2 Antonio Coluccia,3 Johan Neyts,2** Thierry
Terme,1 Pieter Leyssen,2 Romano Silvestri,3*** and Patrice Vanelle.1*
1 Aix-Marseille Université, Institut de Chimie Radicalaire, UMR 7273 CNRS, 27 Boulevard Jean Moulin,
Marseille, France.
2 KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical
Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
3 Institut Pasteur Italy, Department of Drug Chemistry and Technologies, Sapienza University, I-00185 Rome,
Italy.
*
Corresponding author for chemistry. Tel.: +33-4-91835580; fax: 33-4-91-79-46-77; e-mail:
patrice.vanelle@univ-amu.fr
** Corresponding author for biology. Tel.: +32-16-332883; fax: 32-16-337340; e-mail:
johan.neyts@rega.kuleuven.be
*** Corresponding author for in silico design. Tel.: +39-06-4991-3800; fax: 39-6-4991-3993; e-mail:
romano.silvestri@uniroma1.it
Abstract
Human rhinoviruses (HRV) are the predominant cause of common colds and flu-like
illnesses, but are also responsible for virus-induced exacerbations of asthma and chronic
obstructive pulmonary disease. However, to date, no drug has been approved yet for clinical
use. In this study, we present the results of the structure-based lead optimization of a class of
new small-molecule inhibitors that we previously reported to bind into the pocket beneath the
canyon of the VP1 protein. A small series of analogues that we designed based on the
available structure and interaction data were synthesized and evaluated for their potency to
inhibit the replication of HRV serotype 14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-
yl)phenyl)ethanol (3v) was found to be a potent inhibitor exhibiting micromolar activity (EC50
= 3.4 ± 1.0 µM) with a toxicity for HeLa cells that was significantly lower than that of our
previous hit (LPCRW_0005, CC50 = 104.0 ± 22.2 µM; 3v, CC50 > 263 µM).
Keywords
VP1 protein, HRV14, capsid inhibitors, in silico design, TDAE.

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1. Introduction
Human rhinoviruses (HRV) are single-stranded, positive-sense RNA viruses that
belong to the family of Picornaviridae. Based on whole-genome sequencing and taxonomic
analysis, these human pathogens can be divided into three genetically distinct HRV groups,
designated groups A, B, and C. Each group contains multiple serotypes [1-2]. HRVs are
pathogens of the respiratory tract and are a major cause of acute, predominantly mild and self-
limiting infections, the so-called “common cold”. The incidence of the disease usually
dramatically increases during fall, and HRVs are responsible for up to 80% of colds [3]. The
clinical symptoms are a runny nose and coughing, and less frequently, wheezing, otitis media,
sinusitis, or even pneumonia. Several studies demonstrated that HRVs-induced disease can be
more acute in children or immunodeficient patients [4]. Overall, HRV infections have a major
economic impact through loss of productivity and strain on the healthcare system. Currently,
treatment is limited to symptomatic therapy with over-the-counter medicines. The
development of vaccines for prevention is not (yet) an option because of the large number of
serotypes [5]. Over recent decades, several small-molecule inhibitors have been developed to
treat the common cold caused by rhinovirus infection [6]. However, none of these candidates
reached the market, mainly because the side-effects of the treatment did not outweigh the
burden of the disease or due to lack of efficacy in clinical studies. Recently, the causative link
between rhinovirus infection, exacerbation of asthma and chronic obstructive pulmonary
disease (COPD) [7] has triggered, both in academic circles and in pharmaceutical industry, a
new interest in the development of an anti-rhinoviral drug [8].
Different stages of the virus replication cycle can be targeted by small-molecule
inhibitors. In particular, the capsid protein VP1 has shown promise for the development of
antiviral molecules, as it contains a large pocket beneath the receptor-binding canyon [9].
Strikingly, this has led to the discovery and development of a variety of structurally different
inhibitors [10]. Our research teams recently reported a novel chemical scaffold of VP1
inhibitors characterized by a 4,5-dimethoxybenzene skeleton [11]. The base structure of this
compound class resulted from a synthesis method using Tetrakis(DimethylAmino)Ethylene
(TDAE). This is an organic reducing agent [12] which reacts with haloalkyl derivatives to
generate an anion under mild conditions via two sequential transfers of one electron. This
carbanion is able to react with various electrophiles, such as aromatic aldehydes, α-ketoester,
α-ketomalonate, α-ketolactam, and sulfonimine derivatives [13-14]. This methodology was
used to explore the structure-activity relationships of 4,5-dimethoxybenzene derivatives [11].

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From this series, LPCRW_0005 (Figure 1, EC50 = 2.2 ± 0.5 µM; CC50 = 104.0 ± 22.2 µM)
was selected as prototype compound and was, in parallel with pleconaril (Figure 1, EC50 = 0.2
± 0.1 µM; CC50 = 50.2 ± 5.0 µM), the subject of a detailed study. We examined its molecular
mechanism of action and solved the crystal structure of the compound bound to the VP1
protein (PDB ID: 4PDW) [15].
Figure 1. Rhinovirus replication inhibitors.
Several key interactions were identified by superimposition of the binding modes of
LPCRW_0005 and pleconaril: (i) the open end of the pocket is left empty by LPCRW_0005;
(ii) an extra density was observed in the crystal structure data that could be modelled as a
glycerol. Given that pleconaril is a more voluminous compound than LPCRW_0005, it should
be possible to manipulate the 4,5-dimethoxybenzene scaffold to optimize the interactions
within the drug-binding pocket. Our previous study [11] encouraged this strategy, with
LPCRM_0108, which bears a biphenyl group as extra hydrophobic ring (C-ring) (Figure 1,
EC50 = 1.97 ± 0.10 ꢀM).
Instead of pursuing a solely bio-assay-guided strategy for the further optimization of
this compound class, it was decided to make use of the available crystal structure data and to
use docking experiments (in silico design) to focus the chemistry effort. During this study, the
results of which are reported here, five series of potential inhibitors of rhinovirus replication
were designed, synthesized, and evaluated for antiviral activity on the replication of
rhinovirus in a virus-cell-based assay. The base scaffold and its nomenclature are summarized
in Figure 2.
Figure 2. Structure and nomenclature of the 4,5-dimethoxybenzene derivatives investigated in the present study.

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2. Results and discussion
2.1. Design strategy
To test the performance of the available in silico design platform, we first carried out a
cross-docking analysis using PLANTS [16], Glide [17], and Autodock [18] docking
algorithms to explore four different HRV14 capsid PDB structures (see Experimental
section). The best scoring pose for each docking algorithm was compared with the
corresponding crystallographic conformation in terms of the root-mean-square deviation
(rmsd) of atomic positions. Given the consistent rmsd values (see Supporting Information),
docking experiments were performed by means of PLANTS. The PLANTS LPCRW_0005
binding mode was superimposed on the co-crystallized complex, revealing the same
interactions previously reported [15]. Moreover, the docking study led to the identification of
other hydrophobic interactions between the A-ring and residues V188 and M224 (Figure 3).
Figure 3. PLANTS binding mode of LPCRW_0005 (magenta) versus co-crystallized binding pose of LPCRW_0005 (cyan).
Closest residues are reported as green lines, the rest of the structure of VP1 is shown as ribbons. H-bonds are depicted as
dotted black lines.
To complete the model before embarking on to a drug design effort, the binding mode
of the active LPCRM_0108 was also studied (Figure 4). This molecule engaged in the same
interactions that were observed for LPCRW_0005, but it was also observed that its C-ring
engages in additional positive interactions with the hydrophobic pocket. These hydrophobic
contacts with L106 and F126, together with a π-π interaction with Y197, are in agreement
with the slightly better antiviral activity of LPCRM_0108 as compared to LPCRW_0005.

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Figure 4. Binding mode of the proposed scaffold (magenta) versus co-crystallized binding pose of LPCRW_0005 (cyan).
Closest residues are reported as green lines, the VP1 backbone is shown as a ribbon. H-bonds are depicted as dotted black
lines.
To extend the scaffold with an extra hydrophobic residue in an attempt to further
improve the antiviral activity, a total of five options were explored: introduction of (i) a para-
substituted C-ring, (ii) a meta-substituted C-ring, (iii) an ortho-substituted C-ring, (iv) a meta-
substituted B-ring by aryl group and, (v) a para-substituted B-ring by heterocycle (Figure 5).
Figure 5. Overview of the modifications proposed during the in silico-assisted optimization of the structure-activity
relationships.
From the docking experiments, the para (3a-3j), meta (3k-3o), and ortho (3p-3s) -
substituted C-ring derivatives showed a binding mode consistent with that proposed for the
parent compounds (Figure 6A, B and, C). Among the three substitution patterns, the

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substituents in para position pointed toward the open end of the VP1 pocket (Figure 6A). The
substituents in meta position filled a hydrophobic pocket formed by L116 and V122, engaging
in additional interactions (Figure 6B). Furthermore, the nitro (3n) and hydroxymethyl (3o)
derivatives established a hydrogen bond with L106. The ortho substituents appear to lead to
non-coplanarity of B- and C-rings, and this might affect the hydrophobic stabilization of the
C-ring. It is worth noting that the nitro (3r) and hydroxymethyl (3s) derivatives established an
H-bond with the L106 backbone (Figure 6C).
In contrast, the meta-substitution of the B-ring by an aryl group (compounds 3t and
3u) induced a completely different binding mode. None of the binding interactions described
for LPCRW_0005 could be observed (data not shown). When the C-ring was replaced with a
heterocycle (3v-3y), the PLANTS proposed binding mode was consistent with that of
LPCRW_0005 and all the interactions previously described were maintained. Nevertheless,
all heterocycles appear to lead to hydrophobic interactions with Y197 and F126 (Figure 6D).
A
B
C
D
Figure 6. PLANTS binding mode of Panel A: para-substituted C-ring derivatives 3a (pink), 3b (white), 3e (magenta), and
3h (orange) versus co-crystallized conformation of LPCRW_0005 (cyan); Panel B: meta-substituted C-ring derivatives 3k
(green), 3l (magenta), 3m (red), 3n (yellow), and 3o (brown); Panel C: ortho-substituted C-ring derivatives 3r (pink), 3s
(brown); Panel D: heterocycle C-ring derivatives 3v (white), 3w (magenta), 3x (green), and 3y (brown). Closest residues are
reported as green lines, the VP1 skeleton is shown as ribbon structure. H-bonds are depicted as dotted black lines.

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The results of the in silico design study were used iteratively with chemistry and
biological evaluation rounds to rationalize and focus the chemistry effort in the next step
towards the further optimization of the 4,5-dimethoxybenzene derivatives.
2.2. Chemistry
To synthesize the 4,5-dimethoxybenzene derivatives of interest, we followed a
synthetic pathway involving two steps: a Suzuki cross-coupling reaction and a nucleophilic
addition using TDAE. This synthesis strategy for compounds 3a-3y is illustrated in Scheme 1.
Scheme 1. General pathway for the synthesis of 4,5-dimethoxybenzene derivatives. Reagents and conditions: (a) NaBH4,
MeOH/CH2Cl2, rt, 12 h, 99%; (b) SOCl2 , Et3N, CH2Cl2, rt, 12 h, 98%; (c) Pd(PPh3)4, K2CO3, DMF/H2O, MW (130 °C), 0.5-
1 h, 42-99%; (d) TDAE (1.1 equiv.), DMF, 1. -20 °C, 1 h, 2. rt, 12 h, 15-92%.
The starting material, 1-(chloromethyl)-4,5-dimethoxy-2-nitrobenzene 1, was prepared
via a first reductive reaction followed by chlorination. While, most of the required aromatic
aldehydes 2 were commercially, sixteen of them were prepared using a simple cross-coupling
aryl-aryl or aryl-heterocycle reaction between a phenylboronic acid and the 3- or 4-
bromobenzaldehyde. The use of microwave irradiation allowed us to obtain these compounds
2a-2p in 42-99% yields.
All compounds (3a-3y) of this series were generated by a TDAE-initiated reaction on
intermediate 1 with benzaldehyde derivatives under classical TDAE conditions already
described [11]. This synthesis strategy involved 3 equiv. of aldehyde in the presence of 1.1
equiv. of TDAE, and allowed us to synthesize the compounds with average yields of 55%.
2.3. In vitro evaluation of biological activity
The antiviral activity of all compounds and of the reference compound pleconaril was
evaluated in an in vitro cytopathic effect reduction (CPE) assay against HRV serotypes 14
(group B rhinoviruses), and 2 (group A rhinoviruses). No activity was observed against HRV

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serotype 2 (data not shown). The data for activity against HRV14 are summarized in the
tables below.
2.3.1. Para-substituted C-ring
The introduction of a group with either a hydrogen bond acceptor, hydrophobic or
hydrophilic properties on the C-ring produced compounds with high cytotoxic properties for
HeLa cells. Only compound 3b, bearing a NO2 group, proved to only slightly inhibit
rhinovirus-induced cell death (3b: EC50 = 4.7 ± 0.6 µM; CC50 = 21.0 ± 6.0 µM). To sum up,
the presence of a C-ring substituted in para-position induced high toxicity for HeLa cells and
did not give rise to compounds with a better antiviral profile than LPCRW_0005 (Table 1).
Table 1. In vitro evaluation of the antiviral activity of para-substituted C-ring derivatives against HRV14.
a, b
a, b
Compound
EC50
CC50
SI
> 4.7
4.7 ± 0.1
ND
3a
21.0 ± 6.0
> 245
4.5
3b
3c
4.7 ± 0.6
>
245
ND
> 244
> 244
ND
ND
ND
ND
ND
ND
ND
3d
3e
3f
>
>
>
6.0
4.7
4.1 ± 1.3
4.7 ± 0.1
> 244
244
3g
3h
3i
> 4.5
4.1
> 4.3
4.5 ± 0.3
4.1 ± 0.2
4.3 ± 0.1
>
3j
EC50 = 50% Effective Concentration (concentration at which 50% inhibition of virus-induced cell death is observed).
CC50 = 50% Cytostatic/Cytotoxic Concentration (concentration at which 50% adverse effect is observed on host cell viability).
SI = Selectivity Index for HRV14, SI = CC50/EC50
ND = not determined.
.
a All values are in ꢀM, expressed as median ± Med. Abs. Dev.
b In assay conditions: pleconaril EC50 = 0.2 ± 0.1 µM; CC50 = 50.2 ± 5.0 µM.
2.3.2. Meta-substituted C-ring
The effect of the five substitutions, i.e. CN, NO2, Cl, F, and a hydroxymethyl group,
were also evaluated in the meta-position. The presence of a C-3’ substitution increased
activity (Table 2). In fact, for compounds 3k, 3l, and 3o, EC50 values less than 2 µM were
observed. However, the extension of the scaffold by introduction of a C-ring substituted in
meta-position did not reduce the toxicity of these compounds (SI on average of 3.5).

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Table 2. In vitro evaluation of the antiviral activity of meta-substituted C-ring derivatives against HRV14.
a, b
a, b
Compound
EC50
CC50
SI
5.2 ± 1.0
5.9 ± 1.1
7.4 ± 2.3
5.5 ± 0.7
6.0 ± 1.7
3.7
1.4 ± 0.2
3k
3l
3.5
3.4
2.5
4.3
1.7 ± 0.1
2.2 ± 0.3
3m
3n
2.2 ± 0.9
1.4 ± 0.4
3o
Legend to the table: see Table 1.
2.3.3. Ortho-substituted C-ring
A smaller series of ortho-substituted C-ring derivatives was also explored (Table 3). In
contrast to what would be expected from the in silico model prediction, compounds 3r and 3s
revealed an anti-rhinovirus potency similar to that of the meta-substituted C-ring (3r: EC50 =
1.5 ± 0.1 µM; 3s: EC50 = 3.0 ± 0.1 µM) with slightly lower toxicity for HeLa cells.
Table 3. In vitro evaluation of the antiviral activity of ortho-substituted C-ring derivatives against HRV14.
a, b
a, b
Compound
EC50
CC50
SI
> 9.3
> 5.0
9.3 ± 3.6
5.0 ± 2.0
9.5 ± 1.9
24.0 ± 2.0
ND
3p
ND
6.3
8.0
3q
3r
1.5 ± 0.1
3.0 ± 0.1
3s
Legend to the table: see Table 1.
2.3.4. Meta-substituted B-ring by aryl group
In an attempt to further stabilize the interaction of the compounds with the
hydrophobic pocket, the influence of a phenyl group in meta-position of the B-ring was
explored (Table 4). This strategy was quickly abandoned, as no antiviral effect could be
observed for any of the compounds for an improvement of the cytotoxicity.
Table 4. In vitro evaluation of the antiviral activity of meta-substituted B-ring derivatives against HRV14.
a, b
a, b
Compound
EC50
CC50
SI
> 9.3
> 5.0
9.3 ± 3.6
5.0 ± 2.0
ND
3t
ND
3u
Legend to the table: see Table 1.

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2.3.5. Heterocyclic C-ring
In a fifth series of compounds, we assessed the influence of heterocyclic substituents
(i.e. pyridine, furan and thiophene) in para-position on B-ring (Table 5). Introduction of a 3-
or 4-substituted pyridine (compounds 3v, and 3w) yielded compounds with potent antiviral
activity. Although the EC50 values were similar to those obtained for the compounds reported
above (3v: EC50 = 3.4 ± 1.0 µM; 3w: EC50 = 2.0 ± 0.8 µM), these modifications resulted in a
significantly reduced toxic effect on HeLa cells (3v and 3w: CC50 > 263 µM). Furthermore,
the introduction of thiophene and furan heterocycles improved the antiviral activity to EC50
values lower than 2 µM (3x: EC50 = 1.6 ± 0.2 µM; 3y: EC50 = 1.6 ± 0.2 µM), although the
CC50 also shifted to lower concentrations, yielding SI values lower than those of 3v and 3w.
According to the hit selection criteria (i.e. the cell and monolayer morphology of treated,
infected cells should, following microscopic inspection, look like uninfected, and untreated
control cells), compound 3v presented, in vitro, a promising hit profile.
Table 5. In vitro evaluation of the antiviral activity of heterocycle para-substituted B-ring derivatives against HRV14.
a, b
a, b
Compound
EC50
CC50
SI
3.4 ± 1.0
2.0 ± 0.8
1.6 ± 0.2
1.6 ± 0.2
> 263
> 263
77.3
131.5
6.9
3v
3w
11.0 ± 6.0
5.3 ± 0.1
3x
3.3
3y
Legend: see table 1.
3. Conclusion
In summary, novel 4,5-dimethoxybenzene derivatives were designed to better fills the
drug-binding pocket of capsid protein VP1 of HRV serotype 14. Structure-based guided
design, efficient synthesis and subsequent evaluation in a virus-cell-based assay led to the
discovery of a new and potent inhibitor of rhinovirus replication (3v) exhibiting micromolar
activity (EC50 = 3.4 ± 1.0 µM) and a CC50 that is significantly higher than that of our previous
hit (LPCRW_0005, CC50 = 104.0 ± 22.2 µM; 3v, CC50 > 263 µM).
Follow-up studies will be conducted to explore structure-based strategies with the
purpose to broaden the antiviral activity of this compound series to other rhinovirus types.

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4. Experimental section
4.1. Molecular Modeling
All molecular modeling studies were performed on a MacPro dual 2.66 GHz Xeon
running Ubuntu 12. The HRV capsid structures were downloaded from PDB [20]: 4PDW
[15]; 1NCQ [21]; 2HWB [22]; 2HWC [22]; 1R09 [23]. Hydrogen atoms were added to the
protein, using Molecular Operating Environment (MOE) 2007.09 [24] and minimized,
keeping all the heavy atoms fixed until a rmsd gradient of 0.05 kcal.mol-1.Å-1 was reached.
Ligand structures were built with MOE and minimized using the MMFF94x force field until a
rmsd gradient of 0.05 kcal.mol-1.Å-1 was reached. The images depicted in this report were
generated by Pymol [25].
4.2. Chemistry
4.2.1. General
Commercially available reagents and solvents were used without further purification.
Microwave-assisted reactions were performed in 10-20 mL sealed vials using a Biotage®
Initiator Classic Microwave oven; temperatures were measured with an IR-sensor and
reaction times are given as hold times. Reactions were monitored by thin-layer
chromatography (plates coated with silica gel 60 F254 from Merck) and by LC-MS analyses
with a Thermo Scientific Accela High Speed LC System® coupled with a single quadrupole
mass spectrometer Thermo MSQ Plus®. The RP-HPLC column used is a Thermo Hypersil
Gold® 50 x 2.1 mm (C18 bounded), with particles of 1.9 µm diameter. Analysis was 8 min
running with a MeOH/H2O eluent gradient from 50:50 to 95:05. Silica gel 60 (70-230 mesh
from Macherey-Nagel) was used for flash chromatography. Melting points were measured in
open capillary tubes with Büchi apparatus and are uncorrected. 1H and 13C NMR spectra were
recorded at room temperature in deuterated solvents on a Brüker Avance-250 instrument (250
MHz), a Brüker Avance III nanobay-300 MHz or a Brüker Avance III nanobay-400 MHz.
Chemical shifts (δ) are reported in parts per million (ppm) relative to TMS as internal
13
standard or relative to the solvent [1H: δ(DMSO) = 2.50 ppm, δ(CDCl3) = 7.26 ppm; C
δ(DMSO) = 39.52 ppm, δ(CDCl3) = 77.16 ppm]. Data are reported as follows: chemical shift,
multiplicity (s = singulet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, and m
= multiplet), coupling constant in Hertz, and integration. Two-dimensional spectroscopy
(HSQC and HMBC) was used to assist in the assignment and the data are not reported.

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Elemental analyses were carried out at the Spectropole, Faculté des Sciences de Saint Jérôme,
Marseille (France) and accurate mass measurements (HRMS) were recorded on a TOF
spectrometer, realized by Spectropole of Faculté des Sciences de Saint Jérôme, Marseille
(France).
4.2.2. 1-(Chloromethyl)-4,5-dimethoxy-2-nitrobenzene (1)
Step (a): To a solution of a 6-nitroveratraldehyde (20.0 g, 95 mmol) in a mixture of
MeOH/CH2Cl2 (800 mL; 3:1), NaBH4 (7.6 g, 199 mmol) was slowly added at 0 °C. After
stirring overnight at room temperature, the reaction was quenched by addition of water. The
organic layer was washed with water and brine, dried over Na2SO4, filtered off and
concentrated under reduced pressure. After purification by column chromatography on silica
gel (CH2Cl2), the pure product (4,5-dimethoxy-2-nitrophenyl)methanol (19.7 g, 92 mmol) was
obtained as a yellow solid. Yield, 99%.
Step (b): To a solution of (4,5-dimethoxy-2-nitrophenyl)methanol (19.5 g, 91 mmol)
in CH2Cl2 (55 mL) was added a few drops of triethylamine, and SOCl2 (1.3 mL, 18 mmol) at
0 °C. After stirring overnight at room temperature, the reaction was quenched by addition of
EtOH. The organic layer was washed with water and brine, dried over Na2SO4, filtered off
and concentrated under reduced pressure. After purification by column chromatography on
silica gel (Cyclohexane/EtOAc 7:3), the pure product 1 (20.7 g, 90 mmol) was obtained as a
1
yellow solid. Yield, 98%. m.p: 59.9 °C. H NMR (250 MHz, CDCl3) δ 7.68 (s, 1H), 7.09 (s,
13
1H), 5.00 (s, 2H), 4.00 (s, 3H), 3.96 (s, 3H). C NMR (100 MHz, CDCl3) δ 153.5, 148.8,
140.4, 127.3, 112.9, 108.5, 56.6, 56.5, 43.7.
4.2.3. General procedure A for Suzuki cross-coupling reactions
To a solution of a 3- or 4-bromobenzaldehyde in a mixture DMF/water (3:1; 14.8
mL/mmol) were added boronic acid derivative (1.5 equiv.), Pd(PPh3)4 (5 mol%), and K2CO3
(2 equiv.). The reaction mixture was heated under microwave irradiation (130 °C) until TLC
showed complete conversion of the starting material (0.5-1 h). After cooling, the mixture was
extracted with dichloromethane. The combined organic layers were washed with water and
brine, dried over Na2SO4, filtered off, and concentrated under reduced pressure to afford the
corresponding crude product 2.
Compounds 2a-2p were synthesized following the procedure described above from the
corresponding boronic acids.

ACCEPTED MANUSCRIPT
4.2.4. General procedure B for TDAE reaction with various electrophiles
To a glass vessel capable of being sealed with Teflon cap (for microwave vials) were
added 1 and benzaldehyde derivative (3 equiv.). The vessel was capped and then, evacuated
and backfilled with N2 (process repeated 3X). Anhydrous DMF (3.5 mL/mmol) was
introduced and the solution was vigorously stirred for 20 min at -20 °C. TDAE was added
slowly and the mixture was stirred for one hour. Then, the reaction was stirred at room
temperature overnight. After LC-MS analysis clearly showed that the chloride had been
totally consumed, the reaction was hydrolysed with distilled water. The mixture was then
extracted with dichloromethane. The combined organic layers were washed with water and
brine, dried over Na2SO4, filtered off and concentrated under reduced pressure to afford the
corresponding crude product 3.
4.2.4.1. 4'-(2-(4,5-Dimethoxy-2-nitrophenyl)-1-hydroxyethyl)-[1,1'-biphenyl]-4-carbonitrile
(3a). The crude product was prepared according to procedure B starting from 1 (112 mg, 0.49
mmol) and 4’-formyl-[1,1’-biphenyl]-4-carbonitrile (301 mg, 1.45 mmol). After purification
by column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (93 mg, 0.23
mmol) was obtained as a yellow solid. Yield, 48%. m.p: 185 °C; 1H NMR (400 MHz, DMSO-
d6) δ 7.91 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.55 (s, 1H),
7.46 (d, J = 8.3 Hz, 2H), 6.87 (s, 1H), 5.47 (d, J = 5.0 Hz, 1H), 4.87-4.82 (m, 1H), 3.83 (s,
3H), 3.76 (s, 3H), 3.26 (dd, J = 13.2, 4.6 Hz, 1H), 3.18 (dd, J = 13.2, 8.4 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6) δ 152.1, 147.0, 146.1, 144.5, 141.7, 136.9, 132.9 (2C), 128.3, 127.4
(2C), 126.8 (2C), 126.6 (2C), 118.9, 115.4, 109.9, 107.9, 72.3, 56.0 (2C), 42.3. Anal. Calcd.
for C23H20N2O5 (%): C, 68.31; H, 4.98; N, 6.93. Found (%): C, 67.79; H, 4.89; N, 6.76.
4.2.4.2. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-nitro-[1,1'-biphenyl]-4-yl)ethanol (3b). The
crude product was prepared according to procedure B starting from 1 (78 mg, 0.34 mmol) and
4’-nitro-[1,1’-biphenyl]-4-carbaldehyde 2a (200 mg, 0.88 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (73 mg, 0.17
mmol) was obtained as a yellow solid. Yield, 51%. m.p: 202 °C; 1H NMR (250 MHz, DMSO-
d6) δ 8.30 (d, J = 8.9 Hz, 2H), 7.96 (d, J = 8.9 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.55 (s, 1H),
7.48 (d, J = 8.3 Hz, 2H), 6.88 (s, 1H), 5.51 (d, J = 4.9 Hz, 1H), 4.88-4.81 (m, 1H), 3.83 (s,
13
3H), 3.76 (s, 3H), 3.30-3.14 (m, 2H). C NMR (75 MHz, DMSO-d6) δ 152.5, 147.4, 147.0,
146.9, 146.9, 142.1, 136.9, 128.7, 128.1 (2C), 127.4 (2C), 127.1 (2C), 124.6 (2C), 115.8,

ACCEPTED MANUSCRIPT
108.3, 72.7, 56.4 (2C), 42.7. Anal. Calcd. for C22H20N2O7 (%): C, 62.26; H, 4.75; N, 6.60.
Found (%): C, 62.28; H, 4.70; N, 6.51.
4.2.4.3. 4'-(2-(4,5-Dimethoxy-2-nitrophenyl)-1-hydroxyethyl)-[1,1'-biphenyl]-4-carbaldehyde
(3c). The crude product was prepared according to procedure B starting from 1 (74 mg, 0.32
mmol) and [1,1’-biphenyl]-4,4'-dicarbaldehyde 2b (200 mg, 0.95 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (80 mg, 0.20
mmol) was obtained as a yellow solid. Yield, 62%. m.p: 163 °C; 1H NMR (250 MHz, DMSO-
d6) δ 10.05 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.3 Hz,
2H), 7.55 (s, 1H), 7.46 (d, J = 8.3 Hz, 2H), 6.87 (s, 1H), 5.49 (d, J = 4.9 Hz, 1H), 4.87-4.80
13
(m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.30-3.14 (m, 2H). C NMR (75 MHz, DMSO-d6) δ
192.7, 152.1, 147.0, 145.9, 145.7, 141.7, 137.4, 135.0, 130.1 (2C), 128.3, 127.2 (2C), 126.8
(2C), 126.5 (2C), 115.3, 107.9, 72.3, 55.9 (2C), 42.3. Anal. Calcd. for C23H21NO6 (%): C,
67.80; H, 5.20; N, 3.44. Found (%): C, 67.34; H, 5.14; N, 3.39.
4.2.4.4. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)ethanol
(3d). The crude product was prepared according to procedure B starting from 1 (152 mg, 0.65
mmol) and 4'-(hydroxymethyl)-[1,1'-biphenyl]-4-carbaldehyde 2c (418 mg, 1.97 mmol). After
purification by column chromatography on silica gel (CH2Cl2/EtOAc 8:2), the pure product
(39 mg, 0.09 mmol) was obtained as a light yellow solid. Yield, 15%. m.p: 180 °C; 1H NMR
(250 MHz, DMSO-d6) δ 7.61 (d, J = 8.1 Hz, 4H), 7.55 (s, 1H), 7.39 (d, J = 8.1, 4H), 6.84 (s,
1H), 5.41 (d, J = 4.9 Hz, 1H), 5.21 (t, J = 5.7 Hz, 1H), 4.84-4.77 (m, 1H), 4.53 (d, J = 5.7 Hz,
13
2H), 3.83 (s, 3H), 3.75 (s, 3H), 3.29-3.14 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 152.0,
146.9, 144.5, 141.6 (2C), 138.7, 138.4, 128.4, 127.0 (2C), 126.4 (2C), 126.2 (2C), 126.1 (2C),
115.3, 107.8, 72.4, 62.6, 55.9 (2C), 42.4. Anal. Calcd. for C23H23NO6 (%): C, 67.47; H, 5.66;
N, 3.42. Found (%): C, 66.94; H, 5.68; N, 3.23.
4.2.4.5. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)ethanol
(3e). The crude product was prepared according to procedure B starting from 1 (62 mg, 0.27
mmol) and 4'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbaldehyde 2d (200 mg, 0.80 mmol).
After purification by column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure
1
product (109 mg, 0.24 mmol) was obtained as a white solid. Yield, 92%. m.p: 134 °C; H
NMR (250 MHz, DMSO-d6) δ 7.89 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.71 (d, J =
8.1 Hz, 2H), 7.55 (s, 1H), 7.45 (d, J = 8.1 Hz, 2H), 6.86 (s, 1H), 5.48 (d, J = 4.9 Hz, 1H),
4.87-4.80 (m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.30-3.14 (m, 2H). 13C NMR (100 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ 152.0, 146.9, 145.8, 144.0, 141.6, 137.2, 128.3, 127.7 (q, J = 32 Hz, 1C), 127.3
(2C), 126.7 (2C), 126.5 (2C), 125.7 (q, J = 4 Hz, 2C), 124.4 (q, J = 270 Hz, 1C), 115.3, 107.8,
72.3, 55.9 (2C), 42.4. Anal. Calcd. for C23H20F3NO5 (%): C, 61.74; H, 4.51; N, 3.13. Found
(%): C, 61.31; H, 4.43; N, 3.07.
4.2.4.6. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-methyl-[1,1'-biphenyl]-4-yl)ethanol (3f). The
crude product was prepared according to procedure B starting from 1 (77 mg, 0.33 mmol) and
4'-methyl-[1,1'-biphenyl]-4-carbaldehyde (196 mg, 0.99 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (70 mg, 0.18 mmol) was
1
obtained as a yellow solid. Yield, 54%. m.p: 109 °C; H NMR (250 MHz, DMSO-d6) δ 7.59
(d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.54 (s, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.25 (d, J
= 8.0 Hz, 2H), 6.83 (s, 1H), 5.41 (d, J = 4.8 Hz, 1H), 4.83-4.76 (m, 1H), 3.82 (s, 3H), 3.74 (s,
13
3H), 3.27-3.12 (m, 2H), 2.33 (s, 3H). C NMR (75 MHz, DMSO-d6) δ 152.0, 146.9, 144.3,
141.7, 138.7, 137.2, 136.6, 129.5 (2C), 128.4, 126.4 (2C), 126.3 (2C), 126.0 (2C), 115.3,
107.8, 72.4, 55.9, 55.9, 42.4, 20.6. Anal. Calcd. for C23H23NO5 (%): C, 70.21; H, 5.89; N,
3.56. Found (%): C, 70.16; H, 6.16; N, 3.39.
4.2.4.7. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-methoxy-[1,1'-biphenyl]-4-yl)ethanol (3g).
The crude product was prepared according to procedure B starting from 1 (55 mg, 0.24 mmol)
and 4’-methoxy-[1,1’-biphenyl]-4-carbaldehyde (152 mg, 0.71 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (62 mg, 0.15
1
mmol) was obtained as an off-white solid. Yield, 64%. m.p: 137 °C; H NMR (250 MHz,
DMSO-d6) δ 7.59 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.37 (d, J = 8.2
Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 6.84 (s, 1H), 5.41 (d, J = 4.7 Hz, 1H), 4.83-4.76 (m, 1H),
3.82 (s, 3H), 3.79 (s, 3H), 3.75 (s, 3H), 3.28-3.13 (m, 2H). 13C NMR (62.5 MHz, DMSO-d6) δ
158.7, 152.0, 146.9, 143.6, 141.6, 138.3, 132.3, 128.2, 127.3 (2C), 126.1 (2C), 125.5 (2C),
115.3, 114.2 (2C), 108.0, 72.2, 55.9, 55.8, 55.0, 42.1. Anal. Calcd. for C23H23NO6 (%): C,
67.47; H, 5.66; N, 3.42. Found (%): C, 67.57; H, 5.73; N, 3.37.
4.2.4.8. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4'-fluoro-[1,1'-biphenyl]-4-yl)ethanol (3h). The
crude product was prepared according to procedure B starting from 1 (104 mg, 0.45 mmol)
and 4'-fluoro-[1,1'-biphenyl]-4-carbaldehyde (270 mg, 1.35 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (100 mg, 0.25
mmol) was obtained as a yellow solid. Yield, 56%. m.p: 131 °C; 1H NMR (250 MHz, DMSO-
d6) δ 7.68 (dd, JHH = 8.9 Hz, JHF = 5.5 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.54 (s, 1H), 7.39 (d,

ACCEPTED MANUSCRIPT
J = 8.2 Hz, 2H), 7.27 (dd, JHH = 8.9 Hz, JHF = 8.9 Hz, 2H), 6.84 (s, 1H), 5.42 (d, J = 4.9 Hz,
13
1H), 4.83-4.76 (m, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 3.27-3.12 (m, 2H). C NMR (75 MHz,
DMSO-d6) δ 162.2 (d, J = 242 Hz, 1C), 152.3, 147.2, 144.7, 141.9, 138.1, 136.7 (d, J = 3 Hz,
1C), 128.8 (d, J = 8 Hz, 2C), 128.6, 126.7 (2C), 126.5 (2C), 115.9 (d, J = 21 Hz, 2C), 115.6,
108.1, 72.6, 56.2 (2C), 42.5. Anal. Calcd. for C22H20FNO5 (%): C, 66.49; H, 5.07; N, 3.52.
Found (%): C, 67.10; H, 4.89; N, 3.42.
4.2.4.9. 1-(4'-Chloro-[1,1'-biphenyl]-4-yl)-2-(4,5-dimethoxy-2-nitrophenyl)ethanol (3i). The
crude product was prepared according to procedure B starting from 1 (107 mg, 0.46 mmol)
and 4'-chloro-[1,1'-biphenyl]-4-carbaldehyde (300 mg, 1.38 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (75 mg, 0.18
mmol) was obtained as a yellow solid. Yield, 40%. m.p: 145 °C; 1H NMR (250 MHz, DMSO-
d6) δ 7.69 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.51 (d, J = 8.5 Hz, 2H),
7.41 (d, J = 8.2 Hz, 2H), 6.86 (s, 1H), 5.45 (d, J = 4.9 Hz, 1H), 4.85-4.78 (m, 1H), 3.83 (s,
13
3H), 3.76 (s, 3H), 3.29-3.13 (m, 2H). C NMR (75 MHz, DMSO-d6) δ 152.0, 146.9, 145.1,
141.7, 138.8, 137.4, 132.1, 128.8 (2C), 128.3, 128.3 (2C), 126.4 (2C), 126.2 (2C), 115.3,
107.9, 72.3, 55.9 (2C), 42.3. Anal. Calcd. for C22H20ClNO5 (%): C, 63.85; H, 4.87; N, 3.38.
Found (%): C, 63.97; H, 4.72; N, 3.40. HRMS (ESI+) m/z Calc. for C22H20ClNO5 [M + Na]+
436.0922, found 436.0923.
4.2.4.10. 1-(4'-Bromo-[1,1'-biphenyl]-4-yl)-2-(4,5-dimethoxy-2-nitrophenyl)ethanol (3j). The
crude product was prepared according to procedure B starting from 1 (95 mg, 0.41 mmol) and
4'-bromo-[1,1'-biphenyl]-4-carbaldehyde 2e (320 mg, 1.22 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (115 mg, 0.25
mmol) was obtained as a yellow solid. Yield, 62%. m.p: 119 °C; 1H NMR (250 MHz, DMSO-
d6) δ 7.68-7.60 (m, 6H), 7.56 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 6.86 (s, 1H), 5.45 (d, J = 4.9
13
Hz, 1H), 4.86-4.79 (m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.29-3.14 (m, 2H). C NMR (100
MHz, DMSO-d6) δ 152.0, 146.9, 145.2, 141.6, 139.2, 137.5, 131.8 (2C), 128.7 (2C), 128.4,
126.5 (2C), 126.2 (2C), 120.7, 115.3, 107.8, 72.3, 55.9 (2C), 42.4. HRMS (ESI+) m/z Calc.
for C22H20BrNO5 [M + Na]+ 480.0417, found 480.0418.
4.2.4.11. 4'-(2-(4,5-Dimethoxy-2-nitrophenyl)-1-hydroxyethyl)-[1,1'-biphenyl]-3-carbonitrile
(3k). The crude product was prepared according to procedure B starting from 1 (101 mg, 0.44
mmol) and 4'-formyl-[1,1'-biphenyl]-3-carbonitrile (272 mg, 1.31 mmol). After purification
by column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (102 mg, 0.25

ACCEPTED MANUSCRIPT
mmol) was obtained as a yellow solid. Yield, 58%. m.p: 144 °C; 1H NMR (250 MHz, DMSO-
d6) δ 8.15 (s, 1H), 8.04-8.01 (m, 1H), 7.84-7.80 (m, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.70-7.63
(m, 1H), 7.55 (s, 1H), 7.44 (d, J = 8.2 Hz, 2H), 6.88 (s, 1H), 5.46 (d, J = 4.9 Hz, 1H), 4.87-
4.80 (m, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 3.33-3.13 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 152.0, 146.9, 145.8, 141.6, 141.1, 136.6, 131.4, 130.9, 130.2, 130.1, 128.4, 126.6 (2C),
126.5 (2C), 118.8, 115.3, 112.1, 107.8, 72.3, 56.0, 55.9, 42.4. Anal. Calcd. for C23H20N2O5
(%): C, 68.31; H, 4.98; N, 6.93. Found (%): C, 68.30; H, 4.93; N, 6.76.
4.2.4.12. 1-(3'-Chloro-[1,1'-biphenyl]-4-yl)-2-(4,5-dimethoxy-2-nitrophenyl)ethanol (3l). The
crude product was prepared according to procedure B starting from 1 (89 mg, 0.38 mmol) and
3'-chloro-[1,1'-biphenyl]-4-carbaldehyde (250 mg, 1.15 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (79 mg, 0.19 mmol) was
obtained as a light yellow solid. Yield, 50%. m.p: 105 °C; 1H NMR (250 MHz, DMSO-d6) δ
7.71-7.62 (m, 4H), 7.55 (s, 1H), 7.52-7.40 (m, 4H), 6.87 (s, 1H), 5.44 (d, J = 4.9 Hz, 1H),
4.86-4.79 (m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.26-3.16 (m, 2H). 13C NMR (75 MHz, DMSO-
d6) δ 152.0, 146.9, 145.5, 142.2, 141.7, 137.2, 133.8, 130.8, 128.4, 127.2, 126.5 (2C), 126.5
(2C), 126.3, 125.3, 115.3, 107.8, 72.3, 55.9 (2C), 42.4. Anal. Calcd. for C22H20ClNO5 (%): C,
63.85; H, 4.87; N, 3.38. Found (%): C, 64.09; H, 4.87; N, 3.84.
4.2.4.13. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(3'-fluoro-[1,1'-biphenyl]-4-yl)ethanol (3m).
The crude product was prepared according to procedure B starting from 1 (66 mg, 0.28 mmol)
and 3'-fluoro-[1,1'-biphenyl]-4-carbaldehyde (170 mg, 0.85 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (79 mg, 0.20
1
mmol) was obtained as a white solid. Yield, 70%. m.p: 89 °C; H NMR (300 MHz, DMSO-
d6) δ 7.67 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.52-7.48 (m, 3H), 7.42 (d, J = 8.2 Hz, 2H), 7.20-
7.15 (m, 1H), 6.86 (s, 1H), 5.40 (d, J = 4.8 Hz, 1H), 4.86-4.80 (m, 1H), 3.83 (s, 3H), 3.77 (s,
13
3H), 3.25-3.15 (m, 2H). C NMR (75 MHz, DMSO-d6) δ 162.7 (d, J = 241 Hz, 1C), 152.0,
146.9, 145.3, 142.4 (d, J = 8 Hz, 1C), 141.6, 137.3, 130.8 (d, J = 8 Hz, 1C), 128.3, 126.4
(4C), 122.5 (d, J = 3Hz, 1C), 115.3, 114.9 (d, J = 20 Hz, 1C), 113.2 (d, J = 22 Hz, 1C), 107.8,
72.2, 55.9 (2C), 42.3. HRMS (ESI+) m/z Calc. for C22H20FNO5 [M + Na]+ 420.1218, found
420.1219.
4.2.4.14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(3'-nitro-[1,1'-biphenyl]-4-yl)ethanol (3n). The
crude product was prepared according to procedure B starting from 1 (101 mg, 0.44 mmol)
and 3'-nitro-[1,1'-biphenyl]-4-carbaldehyde 2f (300 mg, 1.32 mmol). After purification by

ACCEPTED MANUSCRIPT
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (120 mg, 0.28
mmol) was obtained as a yellow solid. Yield, 65%. m.p: 163 °C; 1H NMR (250 MHz, DMSO-
d6) δ 8.43 (s, 1H), 8.23-8.14 (m, 2H), 7.78-7.73 (m, 3H), 7.55 (s, 1H), 7.47 (d, J = 8.1 Hz,
2H), 6.89 (s, 1H), 5.48 (d, J = 4.9 Hz, 1H), 4.88-4.81 (m, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 3.31-
13
3.14 (m, 2H). C NMR (75 MHz, DMSO-d6) δ 152.0, 148.5, 146.9, 146.0, 141.7, 141.6,
136.4, 133.1, 130.5, 128.3, 126.7 (2C), 126.6 (2C), 122.0, 120.9, 115.3, 107.8, 72.3, 56.0,
55.9, 42.4. Anal. Calcd. for C22H20N2O7 (%): C, 62.26; H, 4.75; N, 6.60. Found (%): C, 62.25;
H, 4.64; N, 6.53.
4.2.4.15. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(3'-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)ethanol
(3o). The crude product was prepared according to procedure B starting from 1 (90 mg, 0.39
mmol) and 3'-(hydroxymethyl)-[1,1'-biphenyl]-4-carbaldehyde 2g (248 mg, 1.17 mmol). After
purification by column chromatography on silica gel (CH2Cl2/EtOAc 8:2), the pure product
(73 mg, 0.18 mmol) was obtained as a yellow solid. Yield, 46%. m.p: 173 °C; 1H NMR (250
MHz, DMSO-d6) δ 7.63-7.60 (m, 3H), 7.55 (s, 1H), 7.53-7.50 (m, 1H), 7.42-7.37 (m, 3H),
7.31-7.28 (m, 1H), 6.84 (s, 1H), 5.42 (d, J = 4.8 Hz, 1H), 5.24 (t, J = 5.8 Hz, 1H), 4.85-4.78
13
(m, 1H), 4.56 (d, J = 5.2 Hz, 2H), 3.82 (s, 3H), 3.75 (s, 3H), 3.29-3.14 (m, 2H). C NMR
(100 MHz, DMSO-d6) δ 152.0, 146.9, 144.6, 143.2, 141.7, 139.8, 139.0, 128.7, 128.4, 126.4
(2C), 126.3 (2C), 125.4, 124.9, 124.6, 115.3, 107.8, 72.4, 62.9, 55.9 (2C), 42.4. Anal. Calcd.
for C23H23NO6 (%): C, 67.47; H, 5.66; N, 3.42. Found (%): C, 67.04; H, 5.59; N, 3.62.
4.2.4.16. 4'-(2-(4,5-Dimethoxy-2-nitrophenyl)-1-hydroxyethyl)-[1,1'-biphenyl]-2-carbonitrile
(3p). The crude product was prepared according to procedure B starting from 1 (71 mg, 0.31
mmol) and 4'-formyl-[1,1'-biphenyl]-2-carbonitrile 2h (190 mg, 0.92 mmol). After
purification by column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product
1
(63 mg, 0.16 mmol) was obtained as a white solid. Yield, 51%. m.p: 120 °C; H NMR (300
MHz, DMSO-d6) δ 7.95-7.92 (m, 1H), 7.82-7.77 (m, 1H), 7.62-7.54 (m, 5H), 7.48 (d, J = 8.2
Hz, 2H), 6.85 (s, 1H), 5.47 (d, J = 4.8 Hz, 1H), 4.89-4.84 (m, 1H), 3.83 (s, 3H), 3.77 (s, 3H),
3.27-3.17 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 152.0, 146.9, 146.0, 144.4, 141.6, 136.4,
133.8, 133.4, 130.0, 128.3 (2C), 128.2, 128.0, 126.1 (2C), 118.5, 115.3, 110.1, 107.9, 72.3,
56.0, 55.9, 42.2. Anal. Calcd. for C23H20N2O5 (%): C, 68.31; H, 4.98; N, 6.93. Found (%): C,
68.07; H, 4.86; N, 6.85.
4.2.4.17. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(2'-fluoro-[1,1'-biphenyl]-4-yl)ethanol (3q). The
crude product was prepared according to procedure B starting from 1 (123 mg, 0.53 mmol)

ACCEPTED MANUSCRIPT
and 2'-fluoro-[1,1'-biphenyl]-4-carbaldehyde 2i (320 mg, 1.60 mmol). After purification by
column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (104 mg, 0.26
mmol) was obtained as a yellow solid. Yield, 50%. m.p: 106 °C; 1H NMR (300 MHz, DMSO-
d6) δ 7.55 (s, 1H), 7.53-7.48 (m, 3H), 7.43-7.37 (m, 3H), 7.32-7.26 (m, 2H), 6.82 (s, 1H), 5.42
13
(d, J = 4.8 Hz, 1H), 4.86-4.80 (m, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.27-3.16 (m, 2H). C
NMR (75 MHz, DMSO-d6) δ 159.0 (d, J = 244 Hz, 1C), 152.0, 146.9, 145.0, 141.6, 133.6,
130.6 (d, J = 3 Hz, 1C), 129.3 (d, J = 8 Hz, 1C), 128.4, 128.4, 128.3, 128.1 (d, J = 14 Hz, 1C),
126.0 (2C), 124.8 (d, J = 3 Hz, 1C), 116.0 (d, J = 22 Hz, 1C), 115.3, 107.8, 72.3, 55.9, 55.9,
42.3. HRMS (ESI+) m/z Calc. for C22H20FNO5 [M + Na]+ 420.1218, found 420.1214.
4.2.4.18. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(2'-nitro-[1,1'-biphenyl]-4-yl)ethanol (3r). The
crude product was prepared according to procedure B starting from 1 (74 mg, 0.32 mmol) and
2'-nitro-[1,1'-biphenyl]-4-carbaldehyde 2j (220 mg, 0.97 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (58 mg, 0.14 mmol) was
obtained as a light yellow solid. Yield, 44%. m.p: 164 °C; 1H NMR (300 MHz, DMSO-d6) δ
7.96 (d, J = 7.9 Hz, 1H), 7.79-7.74 (m, 1H), 7.65-7.53 (m, 3H), 7.41 (d, J = 7.9 Hz, 2H), 7.30
(d, J = 7.9 Hz, 2H), 6.84 (s, 1H), 5.45 (d, J = 4.7 Hz, 1H), 4.86-4.80 (m, 1H), 3.83 (s, 3H),
3.78 (s, 3H), 3.25-3.17 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 152.0, 148.9, 146.9, 145.5,
141.6, 135.4, 134.8, 132.7, 131.7, 128.7, 128.2, 127.4 (2C), 126.2 (2C), 123.9, 115.3, 107.9,
72.3, 55.9 (2C), 42.3. Anal. Calcd. for C22H20N2O7 (%): C, 62.26; H, 4.75; N, 6.60. Found
(%): C, 62.21; H, 4.66; N, 6.47.
4.2.4.19. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(2'-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)ethanol
(3s). The crude product was prepared according to procedure B starting from 1 (138 mg, 0.60
mmol) and 2'-(hydroxymethyl)-[1,1'-biphenyl]-4-carbaldehyde 2k (380 mg, 1.79 mmol).
After purification by column chromatography on silica gel (CH2Cl2/EtOAc 7:3), the pure
product (124 mg, 0.31 mmol) was obtained as a light yellow solid. Yield, 51%. m.p: 143 °C;
1H NMR (300 MHz, DMSO-d6) δ 7.59-7.57 (m, 1H), 7.55 (s, 1H), 7.40-7.30 (m, 6H), 7.21-
7.19 (m, 1H), 6.85 (s, 1H), 5.40 (d, J = 4.7 Hz, 1H), 5.09 (t, J = 5.3 Hz, 1H), 4.86-4.80 (m,
13
1H), 4.41 (d, J = 5.3 Hz, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.27-3.17 (m, 2H). C NMR (75
MHz, DMSO-d6) δ 152.0, 146.9, 144.1, 141.7, 139.9, 139.2, 139.0, 129.2, 128.6 (2C), 128.3,
127.9, 127.1, 126.7, 125.6 (2C), 115.3, 107.8, 72.4, 60.7, 55.9 (2C), 42.3. HRMS (ESI+) m/z
Calc. for C23H23NO6 [M + Na]+ 432.1418, found 432.1418.

ACCEPTED MANUSCRIPT
4.2.4.20. 3'-(2-(4,5-Dimethoxy-2-nitrophenyl)-1-hydroxyethyl)-[1,1'-biphenyl]-4-carbonitrile
(3t). The crude product was prepared according to procedure B starting from 1 (86 mg, 0.37
mmol) and 3'-formyl-[1,1'-biphenyl]-4-carbonitrile 2l (230 mg, 1.11 mmol). After purification
by column chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (75 mg, 0.19
1
mmol) was obtained as a light yellow solid. Yield, 50%. m.p: 152 °C; H NMR (250 MHz,
DMSO-d6) δ 7.94 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.64-7.60 (m, 2H), 7.54 (s,
1H), 7.49-7.36 (m, 2H), 6.86 (s, 1H), 5.50 (d, J = 4.8 Hz, 1H), 4.90-4.83 (m, 1H), 3.82 (s,
3H), 3.75 (s, 3H), 3.34-3.18 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 152.0, 146.9, 146.4,
144.9, 141.7, 138.0, 132.9 (2C), 128.9, 128.3, 127.5 (2C), 126.2, 125.7, 124.5, 118.9, 115.3,
110.0, 107.8, 72.5, 55.9 (2C), 42.4. Anal. Calcd. for C23H20N2O5 (%): C, 68.31; H, 4.98; N,
6.93. Found (%): C, 68.39; H, 4.93; N, 6.73.
4.2.4.21. 1-(4'-Chloro-[1,1'-biphenyl]-3-yl)-2-(4,5-dimethoxy-2-nitrophenyl)ethanol (3u). The
crude product was prepared according to procedure B starting from 1 (82 mg, 0.35 mmol) and
4'-chloro-[1,1'-biphenyl]-3-carbaldehyde (230 mg, 1.06 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (56 mg, 0.13 mmol) was
obtained as a yellow solid. Yield, 39%. m.p: 81 °C; 1H NMR (250 MHz, DMSO-d6) δ 7.65 (d,
J = 8.5 Hz, 2H), 7.57-7.51 (m, 5H), 7.45-7.39 (m, 1H), 7.33-7.30 (m, 1H), 6.86 (s, 1H), 5.47
13
(d, J = 4.9 Hz, 1H), 4.88-4.81 (m, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.29-3.17 (m, 2H). C
NMR (75 MHz, DMSO-d6) δ 152.1, 147.0, 146.2, 141.8, 139.3, 138.7, 132.4, 128.9 (2C),
128.8, 128.5 (2C), 128.4, 125.4, 125.3, 124.1, 115.4, 107.9, 72.7, 56.0 (2C), 42.4. Anal.
Calcd. for C22H20ClNO5 (%): C, 63.85; H, 4.87; N, 3.38. Found (%): C, 64.00; H, 4.79; N,
3.36.
4.2.4.22. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (3v). The crude
product was prepared according to procedure B starting from 1 (109 mg, 0.47 mmol) and 4-
(pyridin-4-yl)benzaldehyde 2m (260 mg, 1.42 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 6:4), the pure product (89 mg, 0.23 mmol) was
1
obtained as a yellow solid. Yield, 50%. m.p: 195 °C; H NMR (250 MHz, DMSO-d6) δ 8.62
(d, J = 5.8 Hz, 2H), 7.77 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 5.8 Hz, 2H), 7.55 (s, 1H), 7.47 (d, J
= 8.1 Hz, 2H), 6.87 (s, 1H), 5.50 (d, J = 4.9 Hz, 1H), 4.88-4.82 (m, 1H), 3.83 (s, 3H), 3.76 (s,
13
3H), 3.30-3.14 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 152.0, 150.2 (2C), 146.9, 146.8,
146.7, 141.6, 135.7, 128.3, 126.6 (2C), 126.5 (2C), 121.1 (2C), 115.3, 107.8, 72.3, 56.0, 55.9,

ACCEPTED MANUSCRIPT
42.4. Anal. Calcd. for C21H20N2O5 (%): C, 66.31; H, 5.30; N, 7.36. Found (%): C, 65.93; H,
5.32; N, 7.26.
4.2.4.23. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-3-yl)phenyl)ethanol (3w). The crude
product was prepared according to procedure B starting from 1 (81 mg, 0.35 mmol) and 4-
(pyridin-3-yl)benzaldehyde 2n (192 mg, 1.05 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 6:4), the pure product (74 mg, 0.19 mmol) was
obtained as a light yellow solid. Yield, 56%. m.p: 164 °C; 1H NMR (250 MHz, DMSO-d6) δ
8.89 (d, J = 1.8 Hz, 1H), 8.56 (dd, J = 4.7, 1.4 Hz, 1H), 8.09-8.04 (m, 1H), 7.69 (d, J = 8.2
Hz, 2H), 7.55 (s, 1H), 7.50-7.44 (m, 3H), 6.87 (s, 1H), 5.47 (d, J = 4.9 Hz, 1H), 4.87-4.80 (m,
13
1H), 3.83 (s, 3H), 3.77 (s, 3H), 3.30-3.14 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 152.0,
148.4, 147.6, 146.9, 145.5, 141.6, 135.7, 135.4, 134.0, 128.4, 126.6 (2C), 126.5 (2C), 123.9,
115.3, 107.8, 72.3, 56.0, 55.9, 42.4. HRMS (ESI+) m/z Calc. for C21H20N2O5 [M + H]+
381.1445, found 381.1446.
4.2.4.24. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(thiophen-2-yl)phenyl)ethanol (3x). The crude
product was prepared according to procedure B starting from 1 (101 mg, 0.44 mmol) and 4-
(thiophen-2-yl)benzaldehyde 2o (246 mg, 1.31 mmol). After purification by column
chromatography on silica gel (CH2Cl2/EtOAc 9:1), the pure product (92 mg, 0.24 mmol) was
obtained as a light yellow solid. Yield, 55%. m.p: 144 °C; 1H NMR (250 MHz, DMSO-d6) δ
7.62 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.53 (dd, J = 5.1, 0.9 Hz, 1H), 7.49 (dd, J = 3.7, 0.9,
1H), 7.35 (d, J = 8.2 Hz, 2H), 7.13 (dd, J = 5.1, 3.7 Hz, 1H), 6.85 (s, 1H), 5.44 (d, J = 4.8 Hz,
13
1H), 4.83-4.76 (m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.28-3.13 (m, 2H). C NMR (75 MHz,
DMSO-d6) δ 152.0, 146.9, 144.8, 143.3, 141.6, 132.4, 128.4, 128.2, 126.5 (2C), 125.3, 125.0
(2C), 123.4, 115.3, 107.8, 72.2, 55.9 (2C), 42.2. Anal. Calcd. for C20H19NO5S (%): C, 62.32;
H, 4.97; N, 3.63; S, 8.32. Found (%): C, 61.97; H, 4.91; N, 3.63; S, 8.11.
4.2.4.25. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(furan-3-yl)phenyl)ethanol (3y). The crude
product was prepared according to procedure B starting from 1 (64 mg, 0.28 mmol) and 4-
(furan-3-yl)benzaldehyde 2p (143 mg, 0.83 mmol). After purification by column
chromatography on silica gel (CH2Cl2), the pure product (43 mg, 0.12 mmol) was obtained as
a yellow solid. Yield, 43%. m.p: 133 °C; 1H NMR (250 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.74-
7.73 (m, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.54 (s, 1H), 7.31 (d, J = 8.1 Hz, 1H), 6.95-6.94 (m,
1H), 6.84 (s, 1H), 5.39 (d, J = 4.8 Hz, 1H), 4.80-4.73 (m, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.26-
13
3.12 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 152.0, 146.9, 144.2, 144.1, 141.6, 139.1,

ACCEPTED MANUSCRIPT
130.6, 128.4, 126.2 (2C), 125.7, 125.2 (2C), 115.3, 108.7, 107.8, 72.4, 55.9 (2C), 42.4. Anal.
Calcd. for C20H19NO6 (%): C, 65.03; H, 5.18; N, 3.79. Found (%): C, 65.03; H, 5.08; N, 3.75.
Acknowledgments
This work was supported by the Centre National de la Recherche Scientifique. We express
our thanks to Youssef Kabri, and Omar Khoumeri which were an enormous help in my
everyday work and to Vincent Remusat for 1H and 13C NMR spectra recording. The antiviral
work was performed by Els Scheers, Ph.D. in Rega Institute for Medical Resaerch.
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
Highlights
VP1 crystal structure-guided exploration and optimization of 4,5-dimethoxybenzene-
based inhibitors of rhinovirus 14 infection
1. Description of potent derivatives with HRV serotype 14 activity.
2. Use of docking experiments (in silico design) to focus the chemistry effort.
3. Presentation of Structure Activity Relationships with topological approach.
4. The synthesis is straightforward and mainly achieved by using TDAE.