Structure-Based Drug Design of Potent Pyrazole Derivatives against Rhinovirus Replication

Article abstract of DOI:10.1021/acs.jmedchem.8b00931

Rhinoviruses (RVs) have been linked to exacerbations of many pulmonary diseases, thus increasing morbidity and/or mortality in subjects at risk. Unfortunately, the wide variety of RV genotypes constitutes a major hindrance for the development of Rhinovirus replication inhibitors. In the current investigation, we have developed a novel series of pyrazole derivatives that potently inhibit the Rhinovirus replication. Compounds 10e and 10h behave as early stage inhibitors of Rhinovirus infection with a broad-spectrum activity against RV-A and RV-B species (EC₅₀ < 0.1 μM). We also evaluate the dynamics of the emerging resistance of these promising compounds and their in vitro genotoxicity. Molecular docking experiments shed light on the pharmacophoric elements interacting with residues of the drug-binding pocket.

Full text of DOI:10.1021/acs.jmedchem.8b00931

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Article
Structure-based Drug Design of Potent Pyrazole
Derivatives against Rhinovirus Replication
Laurene Da Costa, Els Scheers, Antonio Coluccia, Adriano Casulli, Manon
Roche, Carole Di Giorgio, Johan Neyts, Thierry Terme, Roberto Cirilli,
Giuseppe La Regina, Romano Silvestri, Carmen Mirabelli, and Patrice Vanelle
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Aug 2018
Downloaded from http://pubs.acs.org on August 29, 2018
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Structure-based Drug Design of Potent Pyrazole
Derivatives against Rhinovirus Replication
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Laurène Da Costa,†,# Els Scheers,‡,# Antonio Coluccia,§ Adriano Casulli,∞ Manon Roche,†
Carole Di Giorgio,ʟ Johan Neyts,‡ Thierry Terme,† Roberto Cirilli,ǁ Giuseppe La Regina,§
Romano Silvestri,§ Carmen Mirabelli,‡ Patrice Vanelle†,*
†
Aix-Marseille Univ, Institut de Chimie Radicalaire, Laboratoire de Pharmacochimie
Radicalaire, UMR 7273 CNRS, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
‡
KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega
Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven,
Belgium
§ Department of Drug Chemistry and Technologies, Sapienza University of Rome, Laboratory
affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, I-00185
Roma, Italy
∞
WHO Collaborating Centre for the Epidemiology, Detection and Control of Cystic and
Alveolar Echinococcosis. European Reference Laboratory for Parasites. Department of
Infectious Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161, Rome, Italy
ǁ Centro nazionale per il controllo e la valutazione dei farmaci, Istituto Superiore di Sanità, Viale
Regina Elena 299, I-00161 Roma, Italy
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Aix-Marseille Univ, CNRS, IRD, Avignon Université, IMBE UMR 7263, Laboratoire de
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Mutagénèse Environnementale, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
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KEYWORDS: RV, VP1 protein, capsid binder, chiral inhibitors, heterocycles.
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ABSTRACT: Rhinoviruses (RVs) have been linked to exacerbations of many pulmonary
diseases, thus increasing morbidity and/or mortality in subjects at risk. Unfortunately, the wide
variety of RV genotypes constitutes a major hindrance for the development of Rhinovirus
replication inhibitors. In the current investigation, we have developed a novel series of pyrazole
derivatives that potently inhibits the Rhinovirus replication. Compounds 10e and 10h behave as
early-stage inhibitors of Rhinovirus infection with a broad-spectrum activity against RV-A and
RV-B species (EC50 < 0.1 µM). We also evaluate the dynamics of the emerging resistance of
these promising compounds and their in vitro genotoxicity. Molecular docking experiments shed
light on the pharmacophoric elements interacting with residues of the drug-binding pocket.
INTRODUCTION
Rhinoviruses (RVs) are among the most common viruses worldwide. With a respiratory tract
tropism, these infectious agents of the Picornaviridae family (genus Enterovirus) are the major
causative agents of upper respiratory tract infections.1,2 Besides being the etiological agent of the
“common cold”, they are also associated with acute otitis media (AOM) development in
pediatric populations,3,4,5 and sinusitis.6,7 For the past decade, RVs have been identified as the
cause of exacerbations of chronic pulmonary diseases in infants, the elderly, and
immunocompromised patients.8,9 RVs have a strong impact on asthma-related morbidity and
mortality in these populations.10,11,12,13 RV infections have also been reported to lead to
worsening of chronic obstructive pulmonary disease (COPD),14,15 and cystic fibrosis.16,17
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Currently, more than three hundred million people suffer from chronic pulmonary illnesses.
Industrial and academic laboratories are therefore reviving research on RVs, seeking to provide
new therapeutic options.
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As commonly observed in the Picornavirales order, RVs are composed of a positive-sense
single-stranded RNA surrounded by a protein shell called capsid. To date, 167 types of RVs have
been identified by molecular-based phylogenetics and classified in three species, namely RV-A,
RV-B, and RV-C.18,19 Following entry, viral RNA is translated into a single polyprotein, which is
further processed into four structural and seven non-structural proteins required for the
replication. The structural proteins (VP1-VP4) form the characteristic icosahedral capsid of all
members of the genus Enterovirus.20 The assembly of these four viral proteins has specific
structural features enabling it to serve as a cellular binding site: a star-shaped dome on the
icosahedral 5-fold axis from five VP1 units and a depression surrounding the dome that forms a
“canyon”.21,22,23 The majority of RV-A and RV-B binds to ICAM-1, one of the members of the
immunoglobulin superfamily, through the “canyon”24 but the pentameric dome of the minor
group (10% of all RV-A and RV-B) has an affinity for the low-density lipoprotein receptor
(LDL-R).25 Furthermore, it was recently reported that RV-C15 uses cadherin-related family
member 3 (CDHR3) as its cellular receptor.26,27 Viruses of the Enterovirus genus, and in
particular RV-A and RV-B, are characterized by the presence of a hydrophobic cavity under the
floor of the canyon. In many RVs, this cavity contains a small lipid molecule of cellular origin,
called “pocket factor”.28 This molecule stabilizes the viral capsid and takes part in the capsid
uncoating process during viral entry.
The high genetic and antigenic variability across RVs hampers the development of vaccines,
prompting the development of antiviral compounds instead. To do so, conserved proteins like the
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viral protease 3C (rupintrivir),29,30 and a conserved process like viral entry (capsid binders) were
identified as antiviral targets. More specific, capsid binders target the hydrophobic pocket and
block virus uncoating and/or attachment.31,32,33,34,35 Despite forty years of extensive antiviral
research, no antiviral drug has been approved yet for the treatment of rhinovirus infections. The
best-studied and now reference capsid binder is pleconaril (WIN 63843, 1),36 developed by
Schering-Plough. However, its clinical development for the treatment of RV infections was
halted due to poor in vivo activity and rapid selection of drug-resistant variants. Two capsid
binders are now in the advanced phase of development: vapendavir (BTA-798, 2) (Aviragen
Therapeutics®, clinical trials phase IIb, NCT01175226) (Figure 1A) and AZN001 (AstraZeneca,
pre-clinical trials),37 proving that research on this class of inhibitors is actively pursued.
A. Reference compounds in anti-RV therapeutic research
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B. Previous pharmacomodulation work
TOE-END
H-bond with
Y128
π-stacking with
Y128/Y152
π-stacking
with Y197
PORE
Figure 1. A. Structure of known capsid binders 1 and 2 of RVs; B. “Pore” side
pharmacomodulations: antiviral activity against RV-B14 and key ligand/protein interactions.
Since 2014, we have been developing a new chiral scaffold with a promising antirhinoviral
profile against RV-B. The hit compound 3 (LPCRW_0005) revealed the same antiviral profile
and the same binding pocket as pleconaril 1.38,39 Extensive pharmacomodulations were required
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to extend the scaffold to fill the “pore” side within this hydrophobic pocket.40 Thus, we
discovered new hits, compounds 4, substituted with pyrazole or triazole moieties, with a potent
antiviral activity against RV-B14 (Figure 1B).41
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Here, we carried out a new heterocyclic pharmacomodulations aimed to lengthen our scaffold.
At the “pore” side, we evaluated the 4-pyridinyl, 1H-pyrazol-1-yl, or 1H-triazol-1-yl analogues.
Whereas at the “toe-end” side, we studied the introduction of heteroaromatic 5-member rings and
halogen atoms. The latter were placed at position 4 of ring A as suggested by docking studies
and, in addition, they kept the same interatomic distances as 1. This led us to identify two
analogues with an efficacy in the nanomolar range. Both their broad-spectrum activity (RV-A
and RV-B) and their antiviral mechanism of action were explored. Moreover, due to the presence
of a nitro group on cycle A, we evaluated their in vitro genotoxicity using a micronucleus test. In
addition, in vitro emerging resistance against RV-B14 was assessed and compared with 1.
Evaluations of the new structure-activity relationships (SARs) were also performed based on in
silico molecular docking.
RESULTS
Synthesis of the heterocyclic antirhinoviral library.
The synthetic routes for all derivatives are described in Scheme 1. The 2-nitrobenzyl chlorides
6a-6d were prepared by reduction of carboxylic acids 5a-5d with a stabilized BH3.THF solution,
and subsequent chlorination with SOCl2.
The hetero-functionalization on the “pore” side was carried out using an organic electron
donor, tetrakis(dimethylamino)ethylene (TDAE). This agent generates a carbanion from benzyl
chloride by two sequential single-electron transfer (SET).42 A nucleophilic addition on the C=O
bond of aldehyde derivatives then yields to form the chiral secondary alcohol linker (Scheme 1,
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route c). Thus, pyrazole and triazole derivatives 9c-9f were obtained from commercial reagents
(4-(1H-pyrazol-1-yl)benzaldehyde and 4-(1H-1,2,4-triazol-1-yl)benzaldehyde) and 6a-6c. For
pyridinic analogues, we prepared upstream the aldehyde 8 from a Suzuki-Miyaura cross-
coupling reaction (Scheme 1, route d) and a TDAE strategy led us to compounds 9a and 9b.
Finally, the brominated compounds 9b, 9c and 9f underwent in a final pallado-catalyzed
coupling to functionalize the "toe-end" position (Scheme 1, route e). Pd(PPh3)2Cl2 was used as
catalyst to prepare the pyridinic analogues 10a-10c with yields between 66 and 78%. The last
compounds bearing a pyrazole or a triazole nucleus (10d-10j) were prepared with the highly
reactive catalyst Pd(dppf)Cl2, providing up to 96% yields.
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Scheme 1. General procedure for the synthesis of the heterocyclic antirhinoviral librarya
a Reagents and conditions: (a) BH3-THF, THF, rt, 3 days; (b) SOCl2, Et3N, CH2Cl2, rt, 12 h,
79-92% (2 steps); (c) 8, 4-(1H-pyrazol-1-yl)benzaldehyde or 4-(1H-1,2,4-triazol-1-
yl)benzaldehyde, TDAE, DMF, 1) -20 °C, 1 h, 2) rt, 12 h, 13-35%; (d) pyridin-4-ylboronic acid,
Pd(PPh3)4, K2CO3, DMF/Water, MW (130 °C), 30 min, 85%; (e) boronic acid derivative,
Pd(PPh3)2Cl2, K2CO3, dioxane, 80 °C, 12 h, 66-78% for 10a-10c or boronic acid derivative,
Pd(dppf)Cl2, K2CO3, dioxane/water, 80 °C, 12 h, 54-96% for 10d-10j.
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Identification of antirhinoviral compounds via CPE reduction assays
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The newly-synthesized compounds were evaluated for in vitro antiviral activity against RV-
B14 in a cell-based CPE reduction assay on HeLa Rh cells. Antiviral activity is expressed as the
concentration at which 50% of virus-induced CPE was inhibited (EC50). Compound cytotoxicity
(CC50) was assessed in parallel in compound-treated, uninfected cells. Pleconaril 1 was used as a
reference capsid binder. Compounds were classified according to the moieties inserted on the
“pore” side: 4-pyridinyl, 1H-pyrazol-1-yl, or 1H-triazol-1-yl. From there, the antirhinoviral
properties of (aza)-heterocycles as well as halogens (chlorine, bromine, and fluorine) were
studied.
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We first assessed the antiviral activity of the pyridinic analogues 9a, 9b, and 10a-10c but we
could not detect an improvement in EC50 compared to the hits 4 (Table 1). In contrast, 6 out of
11 compounds bearing a pyrazole or a triazole nucleus (9c-9f and 10d-10j) on the “pore” side
were more potent against RV-B14 than 4, especially those substituted with a 1-methyl-1H-
pyrazol-4-yl group towards the “toe-end” side (10e and 10h), with EC50 values of 0.2 µM and SI
> 1235. Substitution of a trifluoromethyl group on the pyrazole moiety (10d and 10g) slightly
increased the EC50 values of the compounds and significantly affected the cytotoxicity of the
respective compounds (CC50 = 14-21 µM), thereby decreasing their selectivity index. The
dimethylisoxazol moieties (10f and 10i) induced a complete loss of antiviral activity against RV-
B14. These observations underline the significance of steric hindrance on the "toe-end" side.
Brominated analogues 9c and 9f showed a similar antiviral activity but a less desirable SI due
to a considerable anti-metabolic activity (CC50 < 39 µM). To overcome this toxicity, the efficacy
of chlorinated (9d) and fluorinated (9e) compounds was evaluated. By providing fluorine or
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chlorine atom, bioisostere of bromine, the cytotoxic effects on HeLa cells were significantly
improved (CC50 > 100).
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Overall, these findings indicate that the introduction of a methylpyrazole or chlorine can
improve antiviral activity with negligible effect on cell toxicity, thus improving SI. The two most
promising hits 10e and 10h displayed characteristics warranting further investigation.
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Table 1. In vitro biological activity of racemic compounds against RV-B14.a
« Toe-end »
side
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9
« Pore »
side
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EC50 RV-B14
CC50 HeLa
(µM)c
SId
Entry
(rac)-Compd
R1
4-pyridinyl
R2
(µM)b
1
2
COOCH3
Br
6.4 ± 1.1
> 264
> 250
> 41
9a
9b
4-pyridinyl
4-pyridinyl
0.59 ± 0.01
1.3 ± 0.5
> 424
3
4
5
> 248
> 86
> 191
> 72
10a
10b
10c
4-pyridinyl
4-pyridinyl
1.2 ± 0.2
1.0 ± 0.3
> 100
> 100
6
7
8
1H-pyrazol-1-yl
1H-pyrazol-1-yl
1H-pyrazol-1-yl
Br
Cl
F
0.25 ± 0.06
0.128 ± 0.004
8.7 ± 0.1
22.5 ± 0.7
> 100
90
9c
9d
9e
> 781
> 12
> 100
9
1H-pyrazol-1-yl
1H-pyrazol-1-yl
1H-pyrazol-1-yl
0.5 ± 0.3
0.2 ± 0.1
> 247
14 ± 12
> 247
29
> 1235
-
10d
10e
10f
10
11
14 ± 3
12
13
1H-1,2,4-triazol-1-yl Br
1H-1,2,4-triazol-1-yl
0.25 ± 0.07
0.4 ± 0.2
39 ± 1
21 ± 4
156
52
9f
10g
14
15
16
1H-1,2,4-triazol-1-yl
1H-1,2,4-triazol-1-yl
1H-1,2,4-triazol-1-yl
pleconaril
0.18 ± 0.05
> 247
> 256
> 256
> 1422
-
10h
10i
10j
1
0.4 ± 0.2
44 ± 14
> 131
110
17
0.03 ± 0.02
> 4039
a
b
All values are expressed as median ± Med. Abs. Dev. EC50 = 50% effective concentration
c
(concentration at which 50% inhibition of virus-induced cell death is observed). CC50 = 50%
cytotoxic concentration (concentration at which 50% adverse effect is observed on host cell
viability). d SI = selectivity index for RV-B14, SI = CC50/EC50. (rac) = racemic.
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Absolute configuration affects 10e and 10h pharmacological activities.
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Having previously observed that chirality significantly influences the activity of this class of
compounds,41 we separated and characterized the enantiomers for compounds 10e and 10h. The
antiviral activity of the separated enantiomers was then tested against RV-B14 in a cell-based
CPE reduction assay.
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The enantioseparation was successfully carried out by HPLC on the polysaccharide-based
Chiralpak IG-3 chiral stationary phase. The enantiomers collected on a semipreparative scale
were submitted to stereochemical characterization according to the multi-step methodology
previously described.41 To determine the absolute configuration of the enantiomers 10h was used
a Mosher’s protocol whereas the stereochemistry of 10e was assigned by a correlative method
based on circular dichroism (CD) measurements. This detail methodology is described in the
Supporting Information.
Similar to the previous series, we found an increased antiviral activity and selectivity index for
the (S)-enantiomers (Table 2). The fold increase of EC50 of these enantiopure compounds
compared to the racemic mixture was more pronounced than that observed in the previous
series.41 Despite (R)-10e enantiomer retained the antiviral activity, compound (S)-10e gained 5.7-
fold antiviral potency, whereas (S)-10h was 3 times more active than the racemic counterpart.
For the enantiopure compounds, an antiviral activity similar to or higher than that of 1 was
reached. In summary, we identified two new hit compounds as active as pleconaril 1, in the
context of in vitro RV-B14 infection. Furthermore, we were able to demonstrate that the HPLC
enantioseparation is a mandatory step to significantly improve the antiviral activity and
selectivity index of this class of compounds.
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Table 2. In vitro biological activity of enantiopure compounds against RV-B14.a
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CC50 HeLa
(µM)c
EC50 RV-B14
(µM)b
SId
Entry
Compd
9
1
2
3
4
5
6
7
(rac)-10e
(S)-10e
(R)-10e
(rac)-10h
(S)-10h
(R)-10h
1
0.2 ± 0.1
0.035 ± 0.006
2.5 ± 0.9
> 1000
> 342
> 5000
> 9771
> 136
3883
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> 342
0.18 ± 0.05
0.06 ± 0.03
> 128
699 ± 63
> 342
> 5700
-
> 342
0.03 ± 0.02
> 131
> 4039
a
b
All values are expressed as median ± Med. Abs. Dev. EC50 = 50% effective concentration
c
(concentration at which 50% inhibition of virus-induced cell death is observed). CC50 = 50%
cytotoxic concentration (concentration at which 50% adverse effect is observed on host cell
viability). d SI = selectivity index for RV-B14, SI = CC50/EC50. (rac) = racemic.
Pan-RV activities of 10e and 10h
The antiviral activity of the most promising compounds of the series, compounds 10e and 10h,
and their enantioseparated counterparts was evaluated against a representative panel of
rhinoviruses belonging to type A (RV-A02, RV-A08, RV-A28, RV-A85, and RV-A89), and type
B (RV-B14, RV-B42, and RV-B70) (Table 3).
All the compounds were found to be active against RV-group B in a similar range of
concentration, with the exception of RV-B42 type, which is known to be naturally resistant to
pleconaril-like capsid binders.46 Interestingly, for the first time, this class of compounds showed
antiviral activity against A-group RVs, in particular where RV-A89 infection was involved.
Remarkably, in the case of the only type A-virus sensitive to compounds 10e and 10h, the (R)-
enantiomers were more active than the (S)-counterparts. This switch in enantiomer activity could
reflect specific properties of the pocket. An in-depth study of the primary VP1 sequence
highlighted an additional H-bond with the (R)-enantiomers (Figure 5).
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Table 3. Evaluation of pan-RV activities of the enantiomers of 10e and 10h.a
5
6
7
8
9
EC50
1
EC50 (R)-10h
EC50 (S)-10e
EC50 (R)-10e EC50 (S)-10h
Types
(µM)b
(µM)b
(µM)b
> 128
(µM)b
> 128
(µM)b
> 128
RV-A02
RV-A08
RV-A28
RV-A85
RV-A89
RV-B14
RV-B42
RV-B70
0.015 ± 0.002
0.49 ± 0.09
0.015 ± 0.008
0.011 ± 0.002
0.0040 ± 0.0001
0.03 ± 0.02
> 128
> 128
> 128
> 128
1.5 ± 0.4
> 128
> 128
> 20
RV-A
RV-B
> 128
> 128
> 128
> 128
> 128
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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31
32
33
34
35
36
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38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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59
60
> 128
> 128
> 128
> 128
2.4 ± 0.5
0.035 ± 0.006
> 128
0.190 ± 0.001
2.5 ± 0.9
> 128
> 128
0.06 ± 0.03
> 128
> 128
0.167 ± 0.007
0.017 ± 0.006
> 128
0.05 ± 0.03
a
b
All values are expressed as median ± Med. Abs. Dev. EC50 = 50% effective concentration
(concentration at which 50% inhibition of virus-induced cell death is observed).
10e and 10h act as early-stage inhibitors of Rhinovirus replication.
To confirm the mode of action of the newly-synthetized analogues, a time-of-addition
experiment was performed (Figure 2). Compounds 10e and 10h and the most active
enantioseparated counterparts were added at selected time points before, during, and after
infection. Viral replication was quantified 8 hours after infection by the amount of intracellular
viral RNA. We observed that both the racemic mixtures of 10e and 10h and the purified
enantiomers (S)-10e and (S)-10h interfered with RV-B14 replication at an early stage. In
particular, the compounds were able to prevent virus replication only when added 2 hours before
or at the time of infection, which resulted in a decreased intracellular viral RNA load 8 hours
after infection. The inhibitory effect of the molecules was completely lost when added 2 or more
hours after infection (no reduction in viral RNA load compared to the virus control (VC))
indicating that the compounds act at an early stage of RV replication, similar to 1 and the initial
hit 3 (LPCRW_0005).39 These observations indicate that the pharmacomodulations did not alter
the mechanism of action of this series of compounds.
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Figure 2. Time of drug-addition experiment. Cells were treated with the compounds 10e, 10h
and the respective enantiomers at selected time points pre- and post-infection (RV-B14).
Pleconaril (1) was used as a positive control for early stage inhibition of viral replication. Cells
were harvested 8 hours post infection and viral RNA was quantified by RT-qPCR. Similar to 1,
compounds 10e and 10h and their active enantiomers interfere with RV-B14 replication at an
early stage.
Drug-resistance develops later with compounds 10e and 10h than with pleconaril 1.
The rapid development of resistance against capsid binders such as pleconaril is a major
obstacle both in vitro and in vivo, as demonstrated by several clinical trials. To evaluate the
dynamics of emerging resistance, RV-B14 was passaged in the presence of 10e, 10h and 1 at
their in vitro EC50, 2xEC50 and 4xEC50 (Table 4). In the sub-optimal concentration (in vitro
EC50), the three compounds developed resistance by passage 3, with 10e-treated cultures less
likely to develop resistance by passage 4 (only 50% of infected-treated cultures showed viral-
induced CPE). However, at the higher concentration of 2xEC50 and 4xEC50, required for
efficacious antiviral treatment, resistance developed more rapidly in 1-treated than in 10e- and
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10h-treated cultures. Although it is difficult to ultimately conclude on in vitro evolutionary
experiments, these data may suggest that RV-B14 is less prone to develop resistance in presence
of compound 10e or 10h than in presence of 1.
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Table 4. RV-B14 induced CPE at passage 1-4 (P1-4) in presence of compounds 10e, 10h, or
pleconaril 1.
Compd
10e
xEC50
P1a
P2a
P3a
P4a
4
2
1
4
2
1
4
2
1
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
50%
0%
0%
100%
100%
0%
0%
50%
0%
25%
75%
25%
100%
100%
100%
0%
50%
50%
0%
50%
100%
25%
100%
100%
100%
10h
1
100%
VC
aPercentage of wells showing full CPE (n=4). VC: virus control.
Next, in order to correlate the resistant phenotype with eventual drug-resistant virus variants,
the VP1 genomic region from the cultures treated with compounds 10e and 10h at passage 4 was
analyzed by sequencing. Virus harvested from cultures that proved to be equally sensitive to the
compound after four passages and without any sign of viral-induced CPE, showed no mutations,
except for four (out of 14) cultures that carried either the amino acid substitution A150V or
V191E. Both mutations were previously associated with in vitro resistance to 1.43 On the other
hand, the virus harvested from drug-resistant cultures carried one of the mutations A150V,
C199W, or N105S. As mentioned above, both A150V but also position C199 (C199Y variant)
were previously described for in vitro resistance to 1.44 The N105S substitution is a new variant
associated with 10h resistance.
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Table 5. Mutations detected in the VP1 gene of RV-B14 cultures 1-4 (C1-C4) after 4 passages in
presence of compounds 10e or 10h.
7
8
9
Compd
10e
xEC50
C1
C2
C3
C4
10
11
12
13
14
15
16
17
18
19
20
21
22
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45
46
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A150V
A150V
C199W
A150V
N105S
C199W
A150V
A150V
C199W
-
N105S
C199W
-
-
-
-
-
4
2
1
4
2
1
V191E
-
-
-
-
10h
C199W
C199W
Green: no CPE. Red: full CPE.
In vitro genotoxicity study
A SAR study of the reference compound 3 highlighted the significance of the nitro group: the
38
absence of NO2 or replacement with an amine or chlorine (unpublished data) abolished
antiviral activity against RV-B14 (EC50 RV-B14 > 306 µM). Indeed, the presence of a nitro
group led us to evaluate the potential clastogenic and/or aneugenic activities of the most active
enantiomers (S)-10e and (S)-10h. The nitroaromatics can be reduced in vivo to form several
reactive intermediates (nitro radical, nitroso, nitroxyl radical, or aromatic N-oxide). In this
research, their genotoxic effects were evaluated using an in vitro micronucleus clastogenicity
study.45 This assay detects compounds that damage chromosomes or interfere with the cell
division apparatus, producing abnormal DNA fragments. It is currently a prerequisite for future
drug development, especially in the framework of research on nitro compounds.
The test was conducted by incubating CHO cells in culture with the test compounds at
increasing concentrations (1 µM to 100 µM), with and without metabolic activation. After
exposure, the cells were cultured for a specific period of time allowing micronuclei to form in
interphase daughter cells in the presence of a blocking agent of cytokinesis (cytochalasin B). The
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harvested cells in interphase were subjected to specific staining and analysis for the presence of
micronuclei.
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8
9
All results are presented in Tables A and B (Supporting Information). No significant increase
in micronucleated cell rates was observed for any of the non-cytotoxic concentrations, with or
without the metabolizing mixture. These findings indicate that compounds (S)-10e and (S)-10h
did not exert cytogenetic effects on CHO cells in culture, and did not produce metabolites with
cytogenetic effects. The negative results for these capsid binders in the in vitro micronucleus
assay, indicate that they are devoided of clastogenic and/or aneugenic activities under these
experimental conditions.
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Molecular modelling
The reported derivatives were studied via docking experiments to gain insight into the
compounds binding mode. The binding pocket was fixed at the capsid site and the computations
were carried out according to a previously reported procedure.40
First, we evaluated the most profitable ring A position for the introduction of the substituents
(R2) listed in Table 1. The proposed docking poses showed that the compounds bearing the R2
substituents at position 4 had the better docking score and a binding mode superimposable with
that of the reference compounds 1, 3 and 4a.
Also, all the newly synthesized compounds were evaluated by docking. The PLANTS
proposed binding poses showed that the compounds shared a consistent binding mode, in
accordance with the one of the reference compounds 3 and 4a (Figure 3).
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Figure 3. PLANTS proposed binding mode for compounds (left panel) 9b (white), 9c (orange),
and 9f (cyan); and (right panel) 10c (cyan), 10d (white), and 10g (orange). Reference
compounds, 3 magenta, and 4a grey, also depicted as lines. Residues involved in contacts
depicted as green lines. VP1 protein depicted as yellow cartoon, H-bonds as yellow dot lines.
The binding mode analyses highlighted some key interactions: (a) the nitro-phenyl moiety had
π-π staking with Y152, (b) the linker oxygen atom formed an H-bond with Y128 side chain, and
(c) the unsubstituted phenyl ring had hydrophobic interactions with L106, V191 and M221 side
chains. The “pore”-side substituents (pyridine, pyrazole, and triazole) were involved in π-π
staking contact with Y197. The “toe-end” substituents constituted two distinct groups: the
aromatic groups formed staking contact with F186 (Figure 3, right panel); whereas the halogen
atoms established steric rather than electrostatic interactions at the hydrophobic pocket mainly
formed by Y152, F186, and M224 (Figure 3, left panel). When compounds 10d, 10e, and 10f,
were compared more closely, we observed that the dimethylisoxazole moiety led to an unrelated
binding mode, while the CF3 (10d) and the CH3 (10e) groups were superimposable and had
hydrophobic contacts with I130, A150, and V176 (Figure D, Supporting Information).
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We also studied the binding modes of the 10e and 10h enantiomers. The docking poses in
particular differed between (R) and (S) pairs. The (S)-enantiomers were in the right geometry to
form an H-bond between the chiral oxygen alcohol atom and the Y128 side chain; contrastingly,
the alcohol group of the (R)-enantiomers pointed toward the hydrophobic residue V191
destabilizing the binding (Figure 4). These findings were in accordance with the biological data
and consistent with previously reported docking poses.41
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Figure 4. PLANTS proposed binding mode for derivatives (R)-10e (orange) and (S)-10e (cyan).
Residues involved in contacts depicted as green lines. VP1 protein depicted as yellow cartoon.
H-bond is shown as yellow dot line.
The capsid binders integrate into a highly conserved pocket mainly formed by VP1.46 The
serotypes’VP1 primary sequence identities were explored to clarify the broad-range
susceptibility of the 10e and 10h compounds. Analysis focused on the residues directly involved
in interactions with the studied compounds.
For the RV-B studied isotypes (Table 4), comparing RV-B14 and RV-B70 isotypes revealed
that only M224 changed to isoleucine. This residue played a role in the stabilization of the
compounds making contact with the “toe-end” substituents. Nevertheless, this residue change did
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not affect the other contacts and the binding mode (data not shown). Actually, the more
hydrophobic nature of the isoleucine might strengthen the interaction with the pyrazole ring.
Comparison between RV-B14 and RV-B42 highlighted two residue changes: Y152 to Phe and
V191 to Leu. The F152/L191 pattern was previously described as “diagnostic” of resistance to
capsid binders.46 In particular, the bulkier leucine instead of valine might have a detrimental
steric effect on compound binding. This hypothesis was confirmed by docking studies with the
homology model for RV-B42 VP1. Indeed, the docking experiments for 10e and 10h led to an
unrelated binding mode (data not shown).
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Table 4. VP1 alignment of the amino acids of RV-B types in the binding pocket.
Amino acid position
102 104 106 116 124 126 128 150 151 152 174 175 176 186 188 191 197 198 219 221 224 245
RV-B
14
42
70
W
-
-
I
-
-
L
-
-
L
-
-
F
-
-
S
-
-
Y
-
-
A
-
-
M
-
-
Y
F
-
P
-
-
S
-
-
V
-
-
F
-
-
V
-
-
V
L
-
Y
-
-
N
-
-
N
-
-
M
-
-
M
-
I
H
-
-
We also analyzed the VP1 sequence for the RV-A isotypes (Table 5), aiming to elucidate the
great inhibitory activity of 10e and 10h on the A89 isotype. It is noteworthy that the valine 98
was peculiar to A89 isotype, and this residue was in the central part of the canyon. The docking
experiments with the RV-A89 VP1 homology model confirmed the role of V98. The smaller
valine instead of the isoleucine, of the other studied isotypes, created a small space filled by the
nitro group. This binding mode also featured in: (a) an aromatic contact between the “pore”-side
pyrazole and Y190; (b) a hydrophobic interaction between the “toe-end” pyrazole and F179 and
I217; and (c) a contact formed by the nitro-phenyl group with Y144 and I181. It should be noted
that the reported binding mode showed an H-bond with the S120 side chain for the (R)-
enantiomers alone (Figure 5).
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Table 5. VP1 alignment of the amino acids of RV-A types in the binding pocket.
5
6
7
8
9
Amino acid position
77 96
98 100 118 120 122 124 142 143 144 166 167 168 179 181 184 190 212 214 217 238
RV-A
89
2
8
28
85
I
-
-
-
-
W
-
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V
I
I
I
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L
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I
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I
L
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F
Y
I
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M
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A
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S
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V
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F
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N
-
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M
-
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I
L
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L
L
H
-
L
-
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-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Figure 5. Proposed binding mode for the compounds (R)-10e (cyan) and (S)-10e (orange) at RV-
A89 isotype. Residues of RV-A2 depicted as green lines; residues of RV-A89 depicted as grey
lines. The cartoon of A89 isotype is also shown. H-bond shown as yellow dot line.
The docking studies provided us with a general binding mode consistent with the biological
activity. Nevertheless, several other factors might be involved in the compound binding, such as:
the effect of other changed amino acids, the residues outside the binding pocket and the
compounds’ access to the pocket.
DISCUSSION
Capsid binders are still considered attractive molecules for the prophylaxis and treatment of
rhino/enterovirus infections, as witnessed by the wealth of studies/clinical trials over the recent
years. These molecules target the hydrophobic pocket beneath the floor of the canyon formed by
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approximately twenty amino acids of VP1. This viral protein is highly variable across
Rhinoviruses and Enteroviruses as well as the composition of the pocket. It is hence a challenge
to design molecules that are strong capsid binders and pan-genotype active. Here, we describe
the chemical modulation of a pleconaril-like capsid binder with greater in vitro antiviral activity
than that of 3 and 4 and a broader spectrum of activity against Rhinoviruses.
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Following enantioseparation, (S)-10h showed just a high potency against RV-B14 infection
and an increased potency compared to 1 against RV-B70 infection. In addition, both the 10e and
10h exhibited a delayed in vitro resistance development compared to 1. Although the
interpretation of in vitro evolutionary experiments is challenging, our data suggest that RV-B14
is likely less prone to develop resistance in presence of compounds 10e and 10h than in presence
of 1. As resistance development was a major problem during the clinical development phase of
1, our findings on this novel class of pleconaril-like compounds are highly encouraging.
Moreover, we correlated the resistance phenotype with mutations in VP1. Two of the altered
positions (A150 and C199) were previously associated with resistance to 1, whereas N105S is a
newly-identified variant specifically associated with compound 10h and/or 10e. These residues
lie in close proximity to the residues defining the hydrophobic pocket, suggesting that the
environment around the pocket is also crucial for compound-binding.
This study also shed light on the dynamics of interaction of the newly-synthesized x-analogues
with the binding pocket by means of molecular docking simulations. We were able to identify
the residue V98 in RV-A as a key residue associated with in vitro antiviral activity. In addition,
the differing enantiomer preferences in the context of RV-B or RV-A89 infection are explained.
Only the (R)-enantiomer was able to engage in an H-bond with the pocket residue S120,
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suggesting that this residue is also crucial for antiviral activity. Taken together, these data will
guide the synthesis of more potent and broad-spectrum compounds.
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CONCLUSION
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In summary, we identified a novel RV inhibitors series with a promising pharmacological
profile. Several compounds displayed sub-micromolar EC50 values against a representative panel
of RV species. In line with our previous work, this SAR study was undertaken to lengthen our
scaffold on the closed side of the hydrophobic pocket, called the "toe-end". This work confirms
the value of hydrophobic interactions with the residues I130, A150, and V176 from the “toe-end”
side. Several structures were identified, among them methyl pyrazole compounds showed to
form numerous hydrophobic interactions within the pocket. The enantiopure compounds 10e and
10h inhibited replication of RV-A and RV-B species with an EC50 between 0.017 and 0.19 µM
against RV-B14, RV-B70 and RV-A89, and were comparable to 1. Determination of the absolute
configuration via Mosher’s protocol confirms the major role played by the (S)-alcohol linker
against RV-B14 and RV-B70. Conversely, the (R)-stereocenter of 10e induced activity against
RV-A89. The docking studies showed that an H-bond with S120 plays a key role for this
activity.
The results of time-of-addition experiments, in which (S)-10e and (S)-10h show an inhibition
profile similar to that of 1 against RV-B14, and of a resistance-development study, indicate that
compounds 10 act at an early stage of the viral infection. In addition, a new point mutation
N105S on the viral capsid protein is identified. Our in vitro genotoxicity study also reveals that
derivatives 10 have no clastogenic and/or aneugenic activities in CHO cells, despite the nitro
group. In conclusion, this full-ranging investigation identifies two potent antivirals with broad-
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spectrum activity against RV-A and RV-B species. In view of their promising pharmacological
properties, we therefore intend to explore the pan-EV potential of these new pyrazole inhibitors.
7
8
9
EXPERIMENTAL SECTION
Chemistry
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General methods. Commercially available reagents and solvents were used without further
purification. 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.
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1H and C 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 standard or relative to the solvent [1H: δ(DMSO) = 2.50 ppm, δ(CDCl3) = 7.26
ppm; 13C: 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, m = multiplet, and bs = broad singlet), 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. Accurate mass measurements (HRMS) were recorded on a TOF
spectrometer, realized by Spectropôle of Faculté des Sciences de Saint Jérôme, Marseille
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(France). Purity of tested compounds was found to be > 95% by high pressure liquid
chromatography (HPLC).
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4-Bromo-1-(chloromethyl)-2-nitrobenzene (6a). Step 1: BH3 (1.0 M solution in THF, 7 mL, 7
mmol) was slowly added to a solution of 4-bromo-2-nitrobenzoic acid (2 g, 8.1 mmol) in
anhydrous THF (10 mL) at 0 °C. The mixture was stirred at room temperature for 3 days. The
mixture was quenched by dropwise addition of a saturated aqueous solution of NaHCO3. Then,
the mixture was extracted with ethyl acetate. The combined organic layers were washed with
water and brine, dried over anhydrous Na2SO4, filtered off and concentrated under reduced
pressure to afford the pure (4-bromo-2-nitrophenyl)methanol (1.3 g, 5.7 mmol, yield 71 %) as a
beige solid. Step 2: To a solution of (4-bromo-2-nitrophenyl)methanol (1.3 g, 5.7 mmol) in
CH2Cl2 (10 mL) was added a few drops of triethylamine, and SOCl2 (0.5 mL, 6.7 mmol) at 0 °C.
After stirring overnight at room temperature, the reaction was quenched by addition of an
aqueous solution NaOH 2N. The mixture was washed with water and brine, dried over
anhydrous Na2SO4, filtered off and concentrated under reduced pressure. After purification by
column chromatography on silica gel (Petroleum ether/EtOAc 8:2), the pure product 6a (1,3 g,
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5,2 mmol, yield 92%) was obtained as a beige solid. H NMR (250 MHz, CDCl3) δ 8.21 (d, J =
1.7 Hz, 1H), 7.77 (dd, J = 8.3, 1.7 Hz, 1H). 7.59 (d, J = 8.3 Hz, 1H), 4.93 (s, 2H, CH2). 13C NMR
(62.5 MHz, CDCl3) δ 148.4 (CIV,Ar), 136.9 (CHAr), 133.0 (CHAr), 131.6 (CIV,Ar), 128.4 (CHAr),
122.8 (CIV,Ar), 42.3 (CH2).
4-Chloro-1-(chloromethyl)-2-nitrobenzene (6b). Synthesized following the same procedure
used to prepare 6a, the crude product was prepared with 4-chloro-2-nitrobenzoic acid (2.3 g, 11.4
mmol). After purification by column chromatography on silica gel (CH2Cl2), the pure product 6b
(2.14 g, 10.4 mmol, yield 92%) was obtained as a brown oil. 1H NMR (250 MHz, CDCl3) δ 8.00
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(d, J = 1.6 Hz, 1H), 7,65-7,57 (m, 2H), 4,91 (s, 2H, CH2). 13C NMR (62.5 MHz, CDCl3) δ 148,2
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(CIV,Ar), 135,3 (CIV,Ar), 133,8 (CHAr), 132,8 (CHAr), 131,0 (CIV,Ar), 125,4 (CHAr), 42,2 (CH2).
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4-Fluoro-1-(chloromethyl)-2-nitrobenzene (6c). Synthesized following the same procedure
used to prepare 6a, the crude product was prepared with 4-fluoro-2-nitrobenzoic acid (1.3 g, 7.0
mmol). After purification by column chromatography on silica gel (CH2Cl2), the pure product 6c
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(1.16 g, 6.1 mmol, yield 89%) was obtained as a green oil. H NMR (250 MHz, CDCl3) δ 7,75
(dd, JHF = 8.2 Hz, JHH = 2.7 Hz, 1H), 7,68 (dd, JHH = 8.6 Hz, JHF = 5.4 Hz, 1H), 7,40-7,33 (m,
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1H), 4,91 (s, 2H, CH2). C NMR (62.5 MHz, CDCl3) δ 161.9 (d, JCF = 251 Hz, CIV,Ar), 148.4
(d, 3JCF =8 Hz, CIV,Ar), 133.5 (d, 3JCF = 8 Hz, CHAr), 128.6 (d, 4JCF = 4 Hz, CIV,Ar), 121.1 (d, 2JCF
= 21 Hz, CHAr), 113.0 (d, 2JCF = 27 Hz, CHAr), 42.2 (CH2).
Methyl 4-(chloromethyl)-3-nitrobenzoate (6d). Synthesized following the same procedure
used to prepare 6a, 4-(methoxycarbonyl)-2-nitrobenzoic acid (1.0 g, 4.4 mmol) was used to
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obtain the pure product 6d (903 mg, 3.9 mmol, yield 79%) as a brown oil. H NMR (250 MHz,
CDCl3) δ 8.61 (d, J = 1.7 Hz, 1H), 8.24 (dd, J = 8.0, 1.7 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 4.97
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(s, 2H, CH2), 3.95 (s, 3H, CH3). C NMR (62.5 MHz, CDCl3) δ 164.5 (C=O), 147.9 (CIV,Ar),
136.7 (CIV,Ar), 134.2 (CHAr), 131.9 (CHAr), 131.6 (CIV,Ar), 126.2 (CHAr), 52.9 (CH3), 42.4 (CH2).
General procedure A for the synthesis of 9. To a glass vessel suitable for sealing with Teflon
cap (for microwave vials) were added the benzyl chloride derivative 6a-6d (1 equiv.) and
benzaldehyde derivative (3 equiv.). The vessel was capped and then evacuated and backfilled
with N2 (thrice-repeated process). Anhydrous DMF (3.5 mL/mmol) was introduced and the
solution was vigorously stirred for 20 min at -20 °C. TDAE (1.06 equiv.) was added slowly and
the mixture was stirred for one hour. Then, the reaction was stirred at room temperature
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overnight. After LC-MS analysis clearly showed that the chloride had been totally consumed, the
reaction was hydrolyzed with water. The mixture was then extracted with ethyl acetate. The
combined organic layers were washed with water and brine, dried over anhydrous Na2SO4,
filtered off, and concentrated under reduced pressure to afford the corresponding crude product.
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Methyl 4-(2-hydroxy-2-(4-(pyridin-4-yl)phenyl)ethyl)-3-nitrobenzoate (9a). The crude
product was prepared according to procedure A starting from 6d (100 mg, 0.43 mmol) and 8
(239 mg, 1.30 mmol). After purification by column chromatography on silica gel
(CH2Cl2/Diethyl ether 9:1 to 6:4), the pure product 9a (64 mg, 0.17 mmol, yield 39%) was
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obtained as a yellow solid. m.p: 188 °C. H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 6.1 Hz,
2H), 8.37 (d, J = 1.6 Hz, 1H), 8.15 (dd, J = 8.1, 1.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.71 (d, J
= 6.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.2 Hz,2H), 5.59 (d, J = 4.7 Hz, 1H, OH),
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4.88-4.84 (m, 1H), 3.90 (s, 3H, CH3), 3.31-3.24 (m, 2H). C NMR (100 MHz, DMSO-d6) δ
164.6 (C=O), 150.2 (2xCHAr), 150.0 (CIV,Ar), 146.7 (CIV,Ar), 146.2 (CIV,Ar), 138.4 (CHAr), 135.8
(CIV,Ar), 134.2 (CIV,Ar), 132.4 (CHAr), 129.0 (CIV,Ar), 126.6 (2xCHAr), 126.4 (2xCHAr), 124.6
(CHAr), 121.0 (2xCHAr), 72.0 (CH), 52.6 (CH3), 41.6 (CH2). HRMS (ESI+) m/z Calc. for
C21H18N2O5 [M+H]+ 379.1288, found 379.1288.
2-(4-Bromo-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (9b). The crude product was
prepared according to procedure A starting from 6a (500 mg, 1.99 mmol) and 8 (1097 mg, 5.98
mmol). After purification by column chromatography on silica gel (EtOAc/Cyclohexane 6:4 to
7:3), the pure product 9b (215 mg, 0.54 mmol, yield 27%) was obtained as light a yellow solid.
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m.p: 177 °C. H NMR (300 MHz, DMSO-d6) δ 8.62 (d, J = 5.8 Hz, 2H), 8.11 (d, J = 1.8 Hz,
1H), 7.82 (dd, J = 8.3, 1.8 Hz, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 5.8 Hz, 2H), 7.46 (d, J =
8.3 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H), 5.53 (d, J = 4.7 Hz, 1H, OH), 4.84-4.78 (m, 1H), 3,23-3,10
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(m, 2H). C NMR (75 MHz, DMSO-d6) δ 150.7 (CIV,Ar), 150.2 (2xCHAr), 146.7 (CIV,Ar), 146.2
(CIV,Ar), 135.8 (CIV,Ar), 135.2 (CHAr), 135.1 (CIV,Ar), 132.3 (CIV,Ar), 126.6 (2xCHAr), 126.5
(CIV,Ar), 126.4 (2xCHAr), 121.0 (2xCHAr), 119.3 (CHAr), 71.9 (CHAr), 40.9 (CH2). HRMS (ESI+)
m/z Calc. for C19H15BrN2O3 [M+H]+ 399.0339, found 399.0339.
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1-(4-(1H-Pyrazol-1-yl)phenyl)-2-(4-bromo-2-nitrophenyl)ethanol (9c). The crude product
was prepared according to procedure A starting from 6a (500 mg, 1.99 mmol) and 4-(1H-
pyrazol-1-yl)benzaldehyde (1031 mg, 5.98 mmol). After purification by column chromatography
on silica gel (Petroleum ether/EtOAc 7:3), the pure product 9c (230 mg, 0.59 mmol, yield 30%)
was obtained as a light orange solid. m.p: 164 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J =
2.6 Hz, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 8.3, 2.0 Hz, 1H), 7.79 (d, J = 8.6 Hz, 2H),
7.73 (d, J = 1.5 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H), 6.53 (dd, J = 2.6, 1.5
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Hz, 1H), 5.54 (d, J = 4,6 Hz, 1H, OH), 4.80-4.76 (m, 1H), 3.17-3.15 (m, 2H). C NMR (100
MHz, DMSO-d6) δ 150.7 (CIV,Ar), 142.8 (CIV,Ar), 140.8 (CHAr), 138.7 (CIV,Ar), 135.2 (CIV,Ar),
135.1 (CHAr), 132.3 (CHAr), 127.6 (CHAr), 126.7 (2xCHAr), 126.5 (CIV,Ar), 119.4 (CHAr), 118.0
(2xCHAr), 107.7 (CHAr), 71.8 (CH), 40.1 (CH2). HRMS (ESI+) m/z Calc. for C17H14BrN3O3
[M+H]+ 388.0291, found 388.0294.
1-(4-(1H-Pyrazol-1-yl)phenyl)-2-(4-chloro-2-nitrophenyl)ethanol (9d). The crude product
was prepared according to procedure A starting from 6b (200 mg, 0.97 mmol) and 4-(1H-
pyrazol-1-yl)benzaldehyde (251 mg, 1.45 mmol, 1.5 equiv.). After purification by column
chromatography on silica gel (Petroleum ether/EtOAc 8:2 to 5:5), the pure product 9d (68 mg,
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0.19 mmol, yield 20%) was obtained as light a yellow solid. m.p: 167 °C. H NMR (250 MHz,
DMSO-d6) δ 8.48 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.73 (d,
J = 8.6 Hz, 2H), 7.69 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 6.54
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(dd, J = 2.4, 2.2 Hz, 1H), 5.56 (d, J = 4.5 Hz, 1H, OH), 4.82-4.75 (m, 1H), 3.22-3.17 (m, 2H).
13C NMR (75 MHz, DMSO-d6) δ 150.5 (CIV,Ar), 142.8 (CIV,Ar), 140.7 (CHAr), 138.6 (CIV,Ar),
134.9 (CHAr), 132.3 (CIV,Ar), 131.9 (CHAr), 131.5 (CIV,Ar), 127.5 (CHAr), 126.7 (2xCHAr), 123.7
(CHAr), 118.0 (2xCHAr), 107.6 (CHAr), 71.8 (CH), 40.9 (CH2). HRMS (ESI+) m/z Calc. for
C17H14ClN3O3 [M+H]+ 344.0796, found 344.0795.
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1-(4-(1H-Pyrazol-1-yl)phenyl)-2-(4-fluoro-2-nitrophenyl)ethanol (9e). The crude product was
prepared according to procedure A starting from 6c (200 mg, 1.05 mmol) and 4-(1H-pyrazol-1-
yl)benzaldehyde (273 mg, 1.58 mmol, 1.5 equiv.). After purification by column chromatography
on silica gel (Petroleum ether/EtOAc 8:2 to 5:5), the pure product 9e (120 mg, 0.36 mmol, yield
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35%) was obtained as light a yellow solid. m.p: 145 °C. H NMR (250 MHz, DMSO-d6) δ 8.48
(d, J = 2.3 Hz, 1H), 7.85 (dd, J = 8.9, 2.4 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 1.5 Hz,
1H), 7.58-7.46 (m, 2H), 7.39 (d, J = 8.5 Hz, 2H), 6.53 (dd, J = 2.3, 1.5 Hz, 1H), 5.53 (d, J = 4.6
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Hz, 1H, OH), 4.81-4.74 (m, 1H), 3.19-3.17 (m, 2H). C NMR (62.5 MHz, DMSO-d6) δ 159.9
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(d, JCF = 245 Hz, CIV,Ar), 150.4 (d, JCF = 8 Hz, CIV,Ar), 143.0 (CIV,Ar), 140.8 (CHAr), 138.7
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(CIV,Ar), 135.2 (d, JCF = 8 Hz, CHAr), 129.3 (d, JCF = 4 Hz, CIV,Ar), 127.6 (CHAr), 126.7
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(2xCHAr), 119.8 (d, JCF = 21 Hz, CHAr), 118.0 (2xCHAr), 111.5 (d, JCF = 26 Hz, CHAr), 107.8
(CHAr), 72.0 (CH), 40.9 (CH2). HRMS (ESI+) m/z Calc. for C17H14FN3O3 [M+H]+ 328.1092,
found 328.1090.
1-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-2-(4-bromo-2-nitrophenyl)ethanol (9f). The crude
product was prepared according to procedure A starting from 6a (500 mg, 1.99 mmol) and 4-
(1H-1,2,4-triazol-1-yl)benzaldehyde (1037 mg, 5.98 mmol). After purification by column
chromatography on silica gel (EtOAc/Petroleum ether 8:2), the pure product 9f (101 mg, 0.26
mmol, yield 13%) was obtained as a yellow solid. m.p: 192 °C. 1H NMR (250 MHz, DMSO-d6)
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δ 9.29 (s, 1H), 8.23 (s, 1H), 8.11 (d, J = 2,1 Hz, 1H), 7.83 (dd, J = 8.3, 2.1 Hz, 1H), 7.82 (d, J =
8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7,42 (d, J = 8.3 Hz, 1H), 5,61 (d, J = 4.7 Hz, 1H, OH),
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4.84-4.77 (m, 1H), 3.18-3.15 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 152.3 (CHAr), 150.7
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(CIV,Ar), 144.6 (CHAr), 142.2 (CIV,Ar), 135.6 (CIV,Ar), 135.2 (CIV,Ar), 135.1 (CHAr), 132.2 (CHAr),
126.9 (2xCHAr), 126.5 (CIV,Ar), 119.4 (CHAr), 119.1 (2xCHAr), 71.7 (CH), 40.9 (CH2). HRMS
(ESI+) m/z Calc. for C16H13BrN4O3 [M+H]+ 389.0244, found 389.0242.
General procedure B for the synthesis of 10a-10c. To a sealed glass vial were added the
brominated derivative (1 equiv.), boronic acid derivative (2 equiv.), Pd(PPh3)2Cl2 (3 mol%), and
K2CO3 (2 equiv.). The vessel was capped and then, evacuated and backfilled with N2 (process
repeated 3X). The dioxane (8 mL/mmol) was introduced and the solution was heated at 80 °C
overnight. After cooling, the mixture was extracted with ethyl acetate. The combined organic
layers were washed with water and brine, dried over anhydrous Na2SO4, filtered off, and
concentrated under reduced pressure to afford the corresponding crude product 10a-10c.
2-(2-Nitro-4-(thiophen-2-yl)phenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (10a). The crude
product was prepared according to procedure B starting from 9b (80 mg, 0.20 mmol) and
thiophen-2-ylboronic acid (52 mg, 0.40 mmol). After purification by column chromatography on
silica gel (Petroleum ether/EtOAc 7:3), the pure product 10a (53 mg, 0.13 mmol, yield 66%) was
obtained as a light yellow solid. m.p: 217 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.62 (d, J = 3.4
Hz, 2H), 8.13 (s, 1H), 7.90-7.64 (m, 7H), 7.53-7.46 (m, 3H), 7.19-7.17 (m, 1H), 5.53 (d, J = 4.9
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Hz, 1H, OH), 4.89-4.79 (m, 1H), 3.26-3.13 (m, 2H). C NMR (75 MHz, DMSO-d6) δ 150.6
(CIV,Ar), 150.2 (2xCHAr), 146.7 (CIV,Ar), 146.4 (CIV,Ar), 140.6 (CHAr), 135.7 (CIV,Ar), 134.1
(CIV,Ar), 133.2 (CHAr), 131.8 (CIV,Ar), 128.9 (CIV,Ar), 128.7 (CHAr), 126.9 (CHAr), 126.5
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