Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

Article abstract of DOI:10.1016/j.ejmech.2015.12.046

Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and anti

Full text of DOI:10.1016/j.ejmech.2015.12.046

European Journal of Medicinal Chemistry 109 (2016) 146e156
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and
-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole
derivatives as dengue virus inhibitors targeting NS5 polymerase
3
a
,
b
,
1
,
2
a
,
b
,
1
c
c
Fatiha Benmansour
, C eꢀ cilia Eydoux
, Gilles Querat , Xavier de Lamballerie ,
Bruno Canard , Karine Alvarez , Jean-Claude Guillemot a , Karine Barral a
Aix-Marseille Universit ꢀe , AFMB, AD2P, UMR 7257, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
UMR190, Emergence des Pathologies Virales, Aix-Marseille University, IRD French Institute of Research for Development, EHESP French School of Public
Health, 27 Boulevard Jean Moulin, Marseille 13005, France
a,
b
a,
b
,
b,
1
, b, *, 1, 2
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2015
Received in revised form
Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a
novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR
around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-
22 December 2015
2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues
Accepted 23 December 2015
Available online 29 December 2015
derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by
this method were shown to possess in vitro activity against the polymerase of dengue virus. The most
potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit
submicromolar activity on the four dengue virus serotypes.
Keywords:
2
1
3
1
-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-
,3,4-oxadiazole
-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-
,2,4-oxadiazole
©
2016 Elsevier Masson SAS. All rights reserved.
Dengue virus
NS5 RdRp inhibitors
Antiviral activity
1
. Introduction
by mosquito-borne Dengue, West Nile, Kunjin, Japanese encepha-
litis and Yellow fever viruses, can induce life-threatening diseases
of epidemic proportions with a devastating economic impact [2].
The four serotypes of dengue virus (DENV1-4) [3] cause the most
prevalent mosquito-borne viral disease with up to 50 to 100 million
human infections annually, leading to about 30,000 deaths; Dengue
disease has increased dramatically in urban and semi-urban areas
and has become a major international public health concern [4].
Over 2.5 billion people (over 40% of the world's population) are
now at risk from DENV. Despite recent studies in vaccine devel-
opment [5], there are currently neither a truly cross-protective
vaccine nor approved antiviral compounds to treat DENV in-
fections. In such a context, the deployment of an innovative drug
discovery approach towards the development of potent antiviral
compounds to prevent or treat DENV infections represents an ur-
gent complement to any therapeutic approach.
Flaviviruses are among the most frequent emerging and re-
emerging viruses [1]. Flavivirus infections, such as those caused
Abbreviations: DENV, dengue virus; NS, non-structural protein; RdRp, RNA-
dependent RNA polymerase; HTS, high-throughput screening; IC50, inhibitory
concentration that causes 50% reduction in enzyme activity; EC50, effective con-
centration that induces 50% of the maximum effect on infected cells; CC50, cytotoxic
concentration that reduces the infected cell number by 50%; TI, therapeutic index;
LC/MS, liquid chromatography/mass spectroscopy; SD, standard deviation; SAR,
Structure-activity relationship; SPA, scintillation proximity assay; T3P, Propyl-
phosphonic anhydride.
*
Corresponding author. Present address: Centre de Recherche en canc eꢀ rologie de
Marseille, CNRS UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille
Universit eꢀ , 13009 Marseille, France.
E-mail address: karine.barral@inserm.fr (K. Barral).
These authors contributed equally.
Present address: Centre de Recherche en canc eꢀ rologie de Marseille, CNRS
1
A validated target is the flaviviral replication machinery. Many
replication steps have been characterized, leading to the identifi-
cation of enzymes whose inhibition could block viral replication
2
UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille Universit eꢀ , 13009
Marseille, France.
http://dx.doi.org/10.1016/j.ejmech.2015.12.046
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
147
[
6]. Flaviviruses are single-stranded positive RNA viruses carrying a
importance in the antiviral activity of bromo or phenyl substituent
at position 5 of the thiophene ring and small substituents prefer-
entially halogen at positions 3 and/or 4 of the aryl ring.
On the basis of the structure of compounds 2 and 3, our series of
derivatives was meant to evaluate the impact on the antiviral ac-
tivity of amide replacement by 1,2,4- and 1,3,4-oxadiazole and aryl
ring substitution while keeping a 5-bromo or a 5-phenyl substitu-
ent at the thiophene ring.
7Me
0
cap-1 structure (
GpppA OMe-RNA) at their 5 -end [7]. The
2
0
replication of the genome is ensured by the viral replication/tran-
scription complex, composed of nonstructural proteins carrying the
polymerase, RNA unwinding and capping activities. The flaviviral
protein NS5 is carrying on its C-terminal domain (c.a. 70 kDa) an
essential RNA-dependent RNA polymerase (RdRp) that is strictly
required for the replication of flaviviruses [8], and therefore con-
stitutes a promising target for the development of anti-flaviviral
compounds [9]. Only two nucleoside analogues known as DENV
RdRp inhibitors, NITD-008 and Balapiravir, entered preclinical an-
imal safety study and clinic trial, but both were terminated due to
toxicity and lack of potency, respectively [10].
2.1. Chemistry
The synthesis of 1,2,4- or 1,3,4-oxadiazoles are commonly syn-
thesized in two steps including a first condensation of carboxylic
acid derivatives with amidoximes or acylhydrazides, respectively,
in the presence of a coupling reagent. Then, heterocycles are sub-
sequently formed by intramolecular cyclodehydration [14].
The identification of suitable starting-point compounds for the
development of new anti-DENV remains a major challenge. In such
a perspective, we set up a HTS (high-throughput screening)
approach using an enzymatic activity assay to identify novel DENV-
®
Propylphosphonic anhydride (T3P ) is a highly reactive n-propyl
2
RdRp inhibitors. From the screening campaign of the French
phosphonic acid cyclic anhydride, generally used as a coupling
agent and recently described as an efficient reagent for the one-pot
synthesis of 1,2,4-oxadiazoles and 1,3,4-oxadiazoles [15]. T3P is a
safe handling reagent with low toxicity which offers a broad
functional group tolerance under mild reaction conditions and an
easy to workup due to water soluble by-products. All these ad-
vantages over traditional reagents led us to use T3P for the one-pot
synthesis of the desired oxadiazoles. T3P acts both as a coupling
and cyclodehydration reagent and allows to the direct conversion
of carboxylic acids to 1,3,4-oxadiazoles in the presence of acylhy-
drazides and 1,2,4-oxadiazoles in the presence of amidoximes. The
trans-3-(5-bromo-2-thienyl)acrylic acid 4 and trans-3-(5-phenyl-2-
thienyl)acrylic acid 5 were prepared by reaction of malonic acid in
hot pyridine/piperidine with appropriate aldehydes via Knoeve-
nagel condensation [16] in 72% and 94%, respectively (Scheme 1).
Initially, our studies started by heating thienylacrylic acid de-
rivatives 4 and 5 with different substituted benzohydrazides in the
presence of T3P under standard conditions described by Augustine
et al. [15]. Benzohydrazides are often either commercially available
or easily accessible by reaction of substituted alkyl benzoate with
hydrazine. However, under conventional heating conditions at
National Chemical Library (http://chimiotheque-nationale.enscm.
fr/), we identified a potent hit scaffold, represented by (2E)-N-(2-
hydroxy-4-nitrophenyl)-3-(5-nitrothiophen-2-yl)prop-2-enamide
1, with an IC50 of 1.3 mM on DENV-2 RdRp assay (Fig. 1).
On the basis of the structural novelty and the encouraging
antiviral activity, a synthetic program was initiated to explore the
structure-activity relationship (SAR) around the initial HTS hit 1.
We performed a series of medicinal-chemistry-based structural
modifications. More than one hundred analogues were synthesized
and led to the identification of two candidates 2 and 3 (Fig. 1)
displaying potent inhibition activities, with EC50 from 0.04
mM to
1.38 M (compound 2) and 0.4 Me3.2 M (compound 3), on the
m
m
m
replication of DENV1-4 clinical isolates [11].
Bioisosteric replacement is an important tool in medicinal
chemistry that can be used to improve the potency, physico-
chemical properties and bioavailability. Because aniline de-
rivatives are known as functional groups that are involved in re-
actions with DNA, we turned our attention to 1,3,4-oxadiazoles and
1,2,4-oxadiazoles that have often been described as bioisosteres for
amides as a result of the increased hydrolytic and metabolic sta-
bility ring [12,13]. With the aim of developing novel analogues with
higher antiviral profile, we performed the replacement of the
amide bond in candidates 2 and 3 with 1,2,4- and 1,3,4-oxadiazoles.
Herein, we report on the SAR around 3-phenyl-5-[(E)-2-(thiophen-
ꢀ
80 C [15],corresponding 1,2-diacylhydrazides were formed exclu-
sively and could be isolated in good yields (see example for 1,2-
diacylhydrazides 6 and 7 in Scheme 1). Further heating of the re-
action mixture for another 24 h did not afford the desired 1,3,4-
2
-yl)ethenyl]-1,2,4-oxadiazole
and
5-phenyl-2-[2-(2-thienyl)
oxadiazoles.
The
cyclodehydration
reaction
of
1,2-
ethenyl]-1,3,4-oxadiazole scaffolds and describe in depth the
in vitro antiviral properties of analogues derived from an efficient
two-step process.
diacylhydrazides is often reported in the literature by employing
toxic cyclodehydrating reagents such as SOCl2 or POCl3 [17e20].
Upon treatment with POCl3, 1,2-diacylhydrazides 6 and 7 under-
went cyclization to the expected 1,3,4-oxadiazoles 8 and 9 with 49%
and 87% yields, respectively (Scheme 1 and Table 1 - Method A).
Because our aim was to quickly develop a convenient and robust
reaction protocol for the synthesis of 1,3,4-oxadiazole libraries from
readily available building blocks, we optimized this protocol by
using microwave heating conditions known to short dramatically
reaction times and increase product purities and yields [21e24].
After few reaction optimization such as performing the reaction
2
. Results and discussion
Recently, we explored SAR around the N-phenyl-3-
arylacrylamide core from compound 1 (Fig. 1). Various modifica-
tions were studied with among others additional functionalities on
the aromatic and the thiophene rings [11]. This study revealed the
ꢀ
under microwaves at higher temperature (100e160 C) using T3P
(2.5e5 eq) and Et3N (3e5 eq) in various solvents such AcOEt,
CH3CN and DMF, we were able to conclude that use of T3P (3 eq,
ꢀ
5
2
0% solution in AcOEt) and Et3N (5 eq) in AcOEt at 160 C for
0e30 min achieved optimum yield (these reactions were conve-
niently monitored by LC/MS analysis). Thus, under microwave
heating conditions, a higher conversion of 1,3,4-oxadiazole was
obtained in a much shorter time (Table 1 - Method B). Throughout
our study, thienylacrylic acids were kept constant while the
substituted benzohydrazides were varied, whereby 1,3,4-
oxadiazoles 8e28 were isolated in moderate to good yields.
Fig. 1. Chemical structures of HTS hit 1 and potent analogues 2 and 3.

148
F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
Scheme 1. Synthesis of thienyl acrylic acids 4e5 and 1,3,4-oxadiazole analogues 8e9 (Method A).
Table 1
®
Synthesis of 1,3,4-oxadiazoles from thienyl acrylic acids and substituted benzohydrazides with Method A or Method B. (a) T3P, Et
reflux 1 h (Method A); or (c) T3P, Et
3
3
N, AcOEt, reflux, 1e24 h then (b) POCl ,
®
ꢀ
3
N, AcOEt, microwave, 160 C, 20e30 min (Method B).
H
O
N
N N
O
S
NH2
S
R1
Method A (a and b)
or Method B (c)
R1
+
R2
O
OH
R3
4
5
R = Br
R = Ph
1
1
R2 R3
Entry
Product
Method A yield (%)
29
Method B yield (%)
32
Entry
Product
Method B yield (%)
49
8
18
19
20
9
47
30
24
63
41
43
1
1
0
1
38
55
21
45
1
1
1
1
1
1
2
3
4
5
6
7
10
53
25
23
14
60
28
15
24
66
48
32
22
23
24
25
26
27
15
11
38
27
33
55
28
18
As shown in Table 1, the reaction conditions were compatible
Method B, purification issues due to low solubility have been
encountered, but it remains the most convenient and fast reaction
protocol for the synthesis of 1,3,4-oxadiazole libraries which would
also be amenable for automation. For this reason, compounds
18e28 were rapidly and exclusively synthesized following the
optimized procedure (Method B) in moderate yields.
with diverse halogens, methoxy and basic nitrogen tertiary amine
substituents. For entries 8e17, both methods A and B were carried
out and final yields were compared. In almost all cases, higher
yields were obtained under our optimized conditions (Method B).
Only three cases reacted more efficiently with Method A (entries 9,
1
3 and 17), which did not require a chromatographic purification to
In the second part of our study, we focused on the synthesis of
some 1,2,4-oxadiazole bioisoster derivatives and started by heating
isolate final products with good purity. Indeed, in a few cases with

F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
149
thienylacrylic acid derivative 4 with different commercial ami-
doximes in the presence of T3P under standard conditions
described by Augustine et al. (Scheme 2 - Method C) [15]. Once
position 5 of the thiophene ring were inactive (IC50 ꢁ 100
m
M, data
not shown). These results also validated bioisosteric replacements
for the amide moiety with 1,3,4-oxadiazoles and 1,2,4-oxadiazoles,
since 14 of the synthesized derivatives showed an efficient inhibi-
ꢀ
again, under conventional heating conditions at 80 C, corre-
sponding O-acylamidoxime intermediates 29e32 were formed
exclusively with low yields (4e9%). Further heating of the reaction
mixture under reflux for another 48 h did not afford the desired
tion of DENV-2 RdRp activity with IC50 values below 15 mM.
As shown in Table 2, the effect of the substituents on the phenyl
ring adjacent to the oxadiazole was explored and small substituents
preferentially halogen in meta- and/or para-position were privi-
leged. Replacement of the bromine on the thiophene ring with the
phenyl resulted in lower potency compounds (Table 2 see 9, 11, 13,
15, 17, 19, 21 and 23). Based on these preliminary findings, 1,3,4 and
1,2,4-oxadiazole derivatives subsequently synthesized contain a
brominated thiophene. Regarding the substituents at the aryl ring
adjacent to the oxadiazole, halogen (Cl, Br and F), trifluoromethyl
and methoxy were well tolerated, while substitution at position 4
with a dimethylamine substituent (compounds 20 and 21) led to a
slightly lower inhibition. Compounds 8, 10, 33 and 34 with chlorine
in meta- and/or para-position produced the best inhibitory effect
1,2,4-oxadiazoles and did not consumed more starting materiel.
The cyclodehydration reaction of O-acylamidoximes is generally
ꢀ
performed at high temperature (above 80 C) in solvents such as
DMF [25,26] or pyridine [27,28] or at room temperature in the
presence of a strongly basic reagent such as TBAF [29,30]. Both
cyclodehydration reaction conditions of O-acylamidoximes in-
termediates were performed and the use of TBAF (0.1 eq) in THF at
room temperature showed a higher efficiency (80e90% yields
determined by LC/MS analysis, Scheme 2 - Method C).
In the same perspective of improving procedure for the syn-
thesis of some 1,2,4-oxadiazole derivatives from readily available
building blocks, we performed the reaction described by Augustine
et al. [15]. by using microwave heating conditions. As previously,
after few reaction optimization, we were able to conclude that use
with IC50 values below 10
mM as well as compounds 16 and 18 with
a CF at position 4 and bromine at position 3, respectively.
3
of T3P (3 eq, 50% solution in AcOEt) and Et3N (5 eq) in AcOEt at
2.3. Antiviral evaluation on DENV-infected cells
ꢀ
160 C for 30 min achieved optimum yield. For this study, the
thienylacrylic acid 4 was kept constant while the substituted ami-
doximes were varied, whereby 1,2,4-oxadiazoles 33e36 were iso-
lated with rather low yields (Scheme 2 - Method D). The increase in
heating time and/or temperature did not improve the reaction ef-
ficiency and resulted in starting materials degradation. Neverthe-
less, under microwave heating conditions, a higher conversion of
The most efficient inhibitors 8e10, 14, 16e18, 24, 25, 27, 28, 33
and 34 identified on DENV-2 RdRp (IC50 ꢂ 15
mM) were evaluated
for their potential inhibitory effect on the in vitro replication of
DENV in Vero cells. The strains used are not cultured-adapted and
none exhibited cytopathic effects in Vero E6 cells up to 7 days after
infection. We therefore relied on measuring the viral RNA copy
numbers in the supernatant as a proxy of viral production. Condi-
tions were set up so that the viral replication was still in the
exponential growth curve and had not attained a plateau. Viral RNA
in the cell supernatants were extracted and measured by real-time
RT-PCR taking the amount of viral RNA in the non-treated cells as
reference. Mean inhibitions of viral production upon addition of
compounds in each triplicate were plotted and the best four pa-
rameters sigmoidal curve was used to describe and calculate EC50
values (Table 3).
1,2,4-oxadiazoles were obtained in a much shorter time.
2.2. Fluorescent assay for DENV-2 RdRp activity
The oxadiazole derivatives 8e28 and 33e36 were firstly evalu-
ated for inhibitory activity in a DENV-2 RdRp assay (Table 2). IC50
®
values were determined using a PicoGreen fluorescent assay (see
experimental section). Hinokiflavone was used as positive control
®
for this PicoGreen fluorescent assay and displayed an IC50 value of
0
.45 ± 0.04
m
M [31].
In a first step, the 14 compounds were screened on DENV-1
Indonesia and DENV-2 Trinidad using four two fold dilutions in
duplicates. Crude effective dose (EC50) and cytotoxicity (CC50) were
determined. NITD-008, an adenosine analog that potently inhibits
DENV was used as positive control [32]. Compounds for which EC50
According to the results obtained for the hundred of analogues
synthesized previously and leading to the identification of candi-
dates 2 and 3 (Fig. 1) [11], this study revealed the importance in the
antiviral activity of bromo or phenyl substituent at position 5 of the
thiophene ring. Indeed, compounds 22 and 23 having bromo or
phenyl substituent at position 5 of the thiophene ring, respectively,
was above 20 mM or for which the Selectivity Index (CC50/EC50) was
below 10 (eg compounds 8 and 25) were discontinued. The most
efficient inhibitors 18, 24, 27, 33 and 34 were then fully evaluated
using clinical strains in the four serotypes. Compounds 18 and 27
were active against DENV-2 RdRp with IC50 values of 16.1
mM and
18.8 M, whereas the equivalent compounds unsubstituted at
m
Scheme 2. Synthesis of 1,2,4-oxadiazoles from 3-(5-bromo-2-thienyl)acrylic acid 4 and substituted amidoximes with Method C or Method D.

150
F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
Table 2
IC50 determination of 1,3,4-oxadiazole 8e28 and 1,2,4-oxadiazole 33e36 derivatives on DENV-2 RdRp activity.
Entry
Structure
IC50
(
m
M) a
Entry
Structure
IC50 (m
M) a
8
6.0 ± 1.0
21
41.9 ± 10.8
9
14.1 ± 2.4
3.5 ± 0.5
22
23
24
25
26
27
16.1 ± 2.2
18.8 ± 3.3
10.4 ± 1.8
10.9 ± 0.6
16.3 ± 4.2
11.0 ± 1.8
1
1
1
1
1
0
1
2
3
4
24.3 ± 3.0
16.3 ± 1.4
17.4 ± 1.8
13.7 ± 3.3
1
1
1
5
6
7
22.0 ± 3.4
4.8 ± 0.4
11.8 ± 1.6
28
33
11.1 ± 1.0
O
N
N
2.2 ± 0.1
S
Br
N
Cl
Cl
1
1
8
9
4.0 ± 0.5
34
35
O
9.1 ± 1.3
S
Br
N
53.9 ± 15.5
O
N
32.6 ± 4.8
S
Br
N
OCF
3
20
20.0 ± 3.7
36
O
N
21 ± 2.1
S
Br
N
CF
3
a
DENV-2 RdRp: IC50
(
mM) are mean values ± SD (n ¼ 3).
Table 3
EC50 and CC50 determination for inhibitors 8e10, 14, 16e18, 24, 25, 27, 28, 33 and 34 on DENV-1 to -4 clinical isolates.
Compounds
Vero E6
CC50 M)
DENV-1
EC50 M)
DENV-2
EC50 M)
DENV-3
EC50 M)
DENV-4
EC50 M)
(
m
(
m
(
m
(
m
(m
Indonesia
Trinidad
Martinique
Bolivia
Dakar
Martinique
8
9
30
5.9
4.7
e
e
e
e
>100
>100
>100
>100
>100
>100
>100
70
>100
55
>100
>100
>100
>20
>20
15
>20
>20
9.1
11.0
7.6
6.9
>20
>20
>20
>20
>20
5.8
10.0
7.9
12
e
e
e
e
1
1
1
1
1
2
2
2
2
3
3
0
4
6
7
8
4
5
7
8
3
4
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4.9
7.0
e
e
5.7
7.0
e
12.7
e
14.0
e
5
e
9.8
e
10
e
e
>20
2
2.5
>20
2.7
2.6
e
8.5
e
3.7
3.0
0.12
5
3.0
0.08
3
3.0
0.03
NITD008
0.86
e
0.82
e: not determined.

F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
151
exhibited specific antiviral activity against the four serotypes with a
mean EC50 below 10 M (range 4.9 Me12 M). 1,2,4-Oxadiazole
derivatives 33 and 34 were the most potent against the four sero-
types with a mean EC50 of 4.1 M (range 2 Me8.5 M) and 2.8
range 2.5 Me3.0 M), respectively, and a selectivity index up to
more 50 folds. Once again, compounds with bromine on the thio-
phene ring and chlorine (or bromine) substituent in meta- or para-
position at the aryl ring adjacent to the oxadiazole displayed the
most potent inhibition activities on DENV-1 to -4 clinical isolates.
water, 200 mL ice and 200 mL concentrated hydrochloric acid to
yield a solid. The precipitating solid was filtered off, washed with
water and dried under pressure to give the corresponding 3-(2-
thienyl)acrylic acid derivative.
m
m
m
m
m
m
mM
(
m
m
4.1.1.1. 3-(5-bromo-2-thienyl)acrylic acid 4. 1H NMR (DMSO-d6,
250 MHz):
J ¼ 3.9 Hz, 1H), 7.28 (d, J ¼ 3.9 Hz, 1H), 6.16 (d, J ¼ 15.8 Hz, 1H);
NMR (DMSO-d6, 62 MHz):
¼ 167.53,141.25,136.28,132.85,132.28,
d
¼ 12.46 (bs, 1H), 7.67 (d, J ¼ 15.7 Hz, 1H), 7.36 (d,
13
C
d
-
1
18.74, 115.42; LC/MS (ESI): 230.87 [MꢄH] and isotopic peak
þ
3
. Conclusion
232.88; HRMS (FAB) cald for C
H
7 4
O
2
SBr (M þ H) 230.9115, found
2
30.9131; purity > 99%; beige powder. Yield ¼ 72%.
A rapid and easily accessible chemical pathway was developed
to synthesize new 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-
,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-
,2,4-oxadiazole derivatives in moderate to good yield. These
4.1.1.2. 3-(5-phenyl-2-thienyl)acrylic acid 5. 1H NMR (DMSO-d6,
250 MHz):
¼ 12.16 (bs, 1H), 7.50e7.63 (m, 3H), 7.36e7.43 (m, 2H),
7.17e7.35 (m, 3H), 6.03 (d, J ¼ 15.7 Hz, 1H); C NMR (DMSO-d6,
62 MHz):
¼ 167.24, 146.11, 138.10, 136.66, 133.45, 132.90, 129.22,
1
1
d
13
compounds were evaluated for anti-DENV-2 RdRp activity, and 14
of them showed an efficient inhibition of RdRp activity with IC50
d
-
128.50, 125.57, 124.89, 117.29; LC/MS (ESI): 228.96 [MꢄH] and
þ
þ
values below 15
mM. The most potent inhibitors were tested in
231.10 [MþH] ; HRMS (FAB) cald for C13
11
H O
2
S (M þ H) 231.0480,
infected cells model to evaluate their efficacy. Among them, com-
pounds 18, 27, 33 and 34 displayed inhibition activities on the
replication of DENV1-4 clinical isolates with EC50 in low micro-
molar range.
Since several crystal structures of DENV RdRp have been
determined [33,34], structural efforts are ongoing in order to obtain
a crystallographic complex with these inhibitors to further define
their mechanism of inhibition and potentially highlight a novel
allosteric binding site. These advances should be greatly helpful in
drug design and inhibitor development against dengue virus.
found 231.0486; purity > 99%; slight yellow powder. Yield ¼ 94%.
4.1.2. General procedure for two steps synthesis of 1,3,4-oxadiazole
derivatives 8e17 (Method A) and general procedure for one-step
synthesis of 1,3,4-oxadiazole derivatives 8e28 (Method B)
Method A
To a stirred solution of acrylic acid (200 mg, 1 eq) and
substituted hydrazide (1 eq) in dry ethyl acetate (5e10 mL) were
®
added dropwise T3P (2.5 eq, 50% solution in EtOAc) and triethyl-
4
. Experimental section
amine (3 eq), and the mixture was refluxed under argon atmo-
sphere for 1 he24 h. The completion of reaction was confirmed by
TLC. The mixture was cooled to room temperature, and poured into
ice/water. The precipitating solid was filtered off, washed with
water, cyclohexan and dried under pressure to give the pure diac-
ylhydrazine derivative. An example of diacylhydrazine derivatives
6e7 synthesis is given below.
4.1. Chemistry
All reagents and solvents were purchased from Sigma Aldrich,
Acros or Alfa aesar companies. Analytical thin layer chromatogra-
phies (TLC) were performed on silica gel 60F 254 aluminium plates
(
Merck) of 0.2 mm thickness with appropriate solvents. The spots
0
were examined with UV light (
Preparative flash column chromatographies were performed using
silica gel (Merck) G60 230-240 under compressed air. Microwave-
assisted syntheses were performed by using a Biotage Initiator
Classic. The H NMR and C NMR spectra were determined with a
BRUCKER AMX 250 MHz or with a JEOL JNM-ECS 400 MHz.
Chemical shifts are expressed in ppm and coupling constants (J) are
in hertz (s ¼ singlet, bs ¼ broad singlet, d ¼ doublet, bd ¼ broad
doublet, dd ¼ double doublet, ddd ¼ double double doublet,
t ¼ triplet, m ¼ multiplet, dm ¼ double multiplet). ESI Mass Spectra
were obtained on a Thermo Scientific MSQ™ Plus mass spec-
trometer. HPLC was performed on a Thermo Scientific Accela (600)
LC systems equipped with a PDA detector. Samples were eluted at
l
¼ 254 nm) and Cericdip Spray.
4.1.2.1. N -[(2E)-3-(5-bromothiophene-2-yl)prop-2-enoyl]-3-
1
chlorobenzohydrazide 6. H NMR (DMSO-d6, 250 MHz):
d
¼ 10.74
(s, 1H), 10.33 (s, 1H), 8.02 (bs, 1H), 7.95 (d, J ¼ 7.7 Hz, 1H), 7.75 (d,
®
J ¼ 15.6 Hz, 1H), 7.76e7.61 (m, 2H), 7.41 (d, J ¼ 3.9 Hz, 1H), 7.37 (d,
1
13
-
J ¼ 3.8 Hz, 1H), 6.52 (d, J ¼ 15.6 Hz, 1H); LC/MS (ESI): 382.84 [MꢄH]
and isotopic peak: 384.81; purity ¼ 95%. Yield ¼ 59%.
0
4.1.2.2. 3-chloro-N -[(2E)-3-(5-phenylthiophene-2-yl)prop-2-enoyl]
benzohydrazide 7. H RMN (DMSO-d , 250 MHz):
1
6
d
¼ 10.78 (s, 1H),
10.51 (s, 1H), 8.14 (s, 1H), 8.07 (d, J ¼ 7.3 Hz, 1H), 7.98e7.83 (m, 4H),
7.80e7.72 (m, 2H), 7.70e7.54 (m, 4H), 6.70 (d, J ¼ 15.5 Hz, 1H); LC/
ꢄ
MS (ESI): 380.99 [MꢄH] ; purity
Yield ¼ 54%.
¼
95%; yellow powder.
flow rate of 400
mL/min using a linear gradient 5e100% B in 6 min or
The corresponding diacylhydrazine derivative was placed under
1
2
HCO
0 min. Reverse phase (RP) chromatography was carried out on a
3
argon atmosphere and POCl was added dropwise (10 mL). The
.1 ꢃ 100 mm Hypersil GOLD PFP column. Eluant A: water þ0.1%
H. High Resolution
mixture was refluxed for 1h30. The mixture was cooled to room
temperature, and poured into ice/water. The precipitating solid was
filtered off, washed with water, cyclohexane and dried under
pressure to give the desired 1,3,4-oxadiazole derivative.
2
H and eluant B: acetonitrile þ 0.1% HCO
2
Mass Spectra (HRMS) were obtained on a SYNAPT G2S WATERS
mass spectrometer.
4.1.1. General procedure for the synthesis of the 3-(5-bromo-2-
Method B
thienyl)acrylic acid 4 and 3-(5-phenyl-2-thienyl)acrylic 5 via
Knoevenagel condensation
To a stirred solution of acrylic acid (200 mg, 1 eq) and
2
-Thiophenecarboxyaldehyde derivative (1 eq, 30 mmol) was
substituted hydrazide (1 eq) in dry ethyl acetate (5e10 mL) were
added dropwise T3P (3 eq, 50% solution in EtOAc) and triethyl-
amine (5 eq). The reaction vessel was sealed under argon atmo-
sphere and the mixture was heated under microwave conditions at
®
dissolved in pyridine (60 mL) and piperidine (3 mL). After addition
of malonic acid (1.2 eq, 36 mmol), the mixture was refluxed for 2 h.
After cooling, the mixture was poured into a mixture of 200 mL

152
F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
ꢀ
1
60 C for 20 min to 30 min. The completion of reaction was
confirmed by TLC. The mixture was cooled to room temperature.
The organic layer was washed twice with 1N HCl, once with H
and once with brine. The organic layer was dried under Na SO and
powder. Method A e purity ¼ 83%; Two-step yield ¼ 10%. Method B
e purity ¼ 95%; Yield ¼ 28%.
2
O
2
4
4.1.2.8. 2-(4-fluorophenyl)-5-[(E)-2-(5-phenylthiophen-2-yl)
1
concentrated under reduced pressure. The crude product was pu-
rified by silica gel flash chromatography (cyclohexan/EtOAc or
DCM/Methanol) to afford the desired 1,3,4-oxadiazole derivative.
ethenyl]-1,3,4-oxadiazole 13.
H
NMR (DMSO-d6, 400 MHz):
d
¼ 8.12 (dd, J ¼ 8.7 Hz and J ¼ 5.3 Hz, 2H), 7.88 (d, J ¼ 16.1 Hz, 1H),
7.68 (d, J ¼ 7.4 Hz, 2H), 7.55 (bs, 2H), 7.47e7.41 (m, 4H), 7.34 (d,
13
6
J ¼ 7.3 Hz, 1H), 6.97 (d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d ,
101 MHz):
4
.1.2.3. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(3-
d
¼ 164.35, 163.06, 146.55, 139.33, 133.58, 132.65, 132.39,
1
6
chlorophenyl)-1,3,4-oxadiazole 8. H NMR (DMSO-d , 400 MHz):
¼ 8.16 (t, J ¼ 1.9 Hz,1H), 8.11 (dt, J ¼ 7.7 Hz and J ¼ 1.3 Hz,1H), 7.99
d, J ¼ 16.1 Hz,1H), 7.79 (ddd, J ¼ 8.1 Hz and J ¼ 2.1 Hz and J ¼ 1.2 Hz,
129.92, 129.83, 129.79, 129.01, 126.13, 125.60, 120.60, 117.32, 117.10,
þ
d
(
1
1
d
1
[
C
108.63; LC/MS (ESI): 349.05 [MþH] ; HRMS (TOF, ESIþ) cald for
þ
C
20
H
14
N
2
OFS (M þ H) 349.0811, found 349.0808; yellow powder.
H), 7.72 (t, J ¼ 7.9 Hz,1H), 7.49 (d, J ¼ 4.0 Hz,1H), 7.38 (d, J ¼ 3.9 Hz,
Method A e purity ¼ 95%; Two-step yield ¼ 53%. Method B e
13
6
H), 7.12 (d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d , 101 MHz):
¼ 164.40, 162.62, 141.82, 134.58, 132.41, 132.29, 131.97, 131.93,
purity ¼ 98%; Yield ¼ 15%.
31.83, 126.63, 125.75, 125.71, 115.38, 109.27; LC/MS (ESI): 368.95
4.1.2.9. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(4-
þ
1
MþH] and isotopic peak: 370.94; HRMS (TOF, ESIþ) cald for
methoxyphenyl)-1,3,4-oxadiazole
250 MHz):
(d, J ¼ 3.5 Hz,1H), 7.32 (d, J ¼ 3.6 Hz,1H), 7.18 (d, J ¼ 8.5 Hz, 2H), 7.02
14.
H
NMR
(DMSO-d6,
þ
14
H
9
N
2
OSClBr (M þ H) 366.9307, found 366.9306; beige powder.
d
¼ 8.03 (d, J ¼ 8.4 Hz, 2H), 7.84 (d, J ¼ 16.2 Hz, 1H), 7.41
Method A e purity ¼ 85%; Two-step yield ¼ 29%. Method B e
1
3
6
purity ¼ 96%; Yield ¼ 32%.
(d, J ¼ 16.2 Hz, 1H), 3.88 (s, 3H); C NMR (DMSO-d , 101 MHz):
d
¼ 163.78, 163.67, 162.68, 141.99, 132.41, 131.66, 131.08, 129.05,
þ
4
.1.2.4. 2-(3-chlorophenyl)-5-[(E)-2-(5-phenylthiophen-2-yl)
ethenyl]-1,3,4-oxadiazole 9. H NMR (DMSO-d6, 250 MHz):
116.13, 115.44, 115.08, 109.67, 56.10; LC/MS (ESI): 362.86 [MþH]
1
d
¼ 7.96
and isotopic peak: 364.89; HRMS (TOF, ESIþ) cald for
þ
(
(
bs, 1H), 7.91 (d, J ¼ 7.4 Hz, 1H), 7.82 (d, J ¼ 16.1 Hz, 1H), 7.56e7.44
C
15
H
12
N
2
O
2
SBr (M þ H) 362.9803, found 362.9804; beige powder.
m, 6H), 7.31 (t, J ¼ 7.3 Hz, 2H), 7.23 (d, J ¼ 7.1 Hz, 1H), 6.87 (d,
Method A e purity ¼ 99%; Two-step yield ¼ 25%. Method B e
þ
J ¼ 16.2 Hz, 1H); LC/MS (ESI): 364.99 [MþH] ; HRMS (TOF, ESIþ)
purity ¼ 97%; Yield ¼ 24%.
þ
cald for C20
14
H N
2
OSCl (M þ H) 365.0515, found 365.0513; yellow
powder. Method A e purity ¼ 90%; Two-step yield ¼ 47%. Method B
4.1.2.10. 2-(4-methoxyphenyl)-5-[(E)-2-(5-phenylthiophen-2-yl)
1
e purity ¼ 96%; Yield ¼ 24%.
ethenyl]-1,3,4-oxadiazole 15.
H NMR (DMSO-d6, 250 MHz):
d
¼ 7.96 (d, J ¼ 8.7 Hz, 2H), 7.81 (d, J ¼ 16.1 Hz, 1H), 7.65 (d,
4
.1.2.5. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(3,4-
dichlorophenyl)-1,3,4-oxadiazole 10. H NMR (DMSO-d6, 250 MHz):
J ¼ 7.2 Hz, 2H), 7.52 (bs, 2H), 7.39 (t, J ¼ 7.2 Hz, 2H), 7.31 (d,
1
J ¼ 7.1 Hz, 1H), 7.10 (d, J ¼ 8.8 Hz, 2H), 6.93 (d, J ¼ 16.1 Hz, 1H), 3.80
13
6
d
7
1
(
1
¼ 8.19 (d, J ¼ 1.9 Hz, 1H), 7.95 (dd, J ¼ 8.5 Hz and J ¼ 2.0 Hz, 1H),
(s, 3H); C NMR (DMSO-d , 101 MHz):
d
¼ 163.84, 162.76, 146.40,
.84 (d, J ¼ 16.2 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.32 (d, J ¼ 3.9 Hz,
139.48, 133.68, 132.35, 131.82, 129.74, 129.05, 128.92, 126.14, 125.55,
13
þ
H), 7.22 (d, J ¼ 3.9 Hz, 1H), 6.96 (d, J ¼ 16.2 Hz, 1H); C NMR
116.35, 115.53, 115.44, 108.86, 56.13; LC/MS (ESI): 361.04 [MþH] ;
þ
DMSO-d6, 101 MHz):
d
¼ 164.59, 162.18, 141.88, 135.31, 132.95,
HRMS (TOF, ESIþ) cald for C21
H
17
N
2
O
2
S (M þ H) 361.1011, found
32.46, 132.33, 132.12, 131.75, 128.80, 127.19, 124.39, 115.33, 109.33;
361.1009; yellow powder. Method A e purity ¼ 90%; Two-step
þ
LC/MS (ESI): 400.74 [MþH] and isotopic peaks: 402.73, 404.74;
yield ¼ 23%. Method B e purity ¼ 98%; Yield ¼ 66%.
þ
HRMS (TOF, ESIþ) cald for C14
H
8
N
2
OSCl
2
Br (M þ H) 400.8918,
found 400.8912; beige powder. Method A e purity ¼ 95%; Two-step
yield ¼ 30%. Method B e purity ¼ 98%; Yield ¼ 63%.
4.1.2.11. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-[4-(tri-
1
fluoromethyl)phenyl]-1,3,4-oxadiazole 16.
50 MHz):
(d, J ¼ 16.2 Hz, 1H), 7.45 (d, J ¼ 4.0 Hz, 1H), 7.33 (d, J ¼ 3.9 Hz, 1H),
H
NMR (DMSO-d6,
2
d
¼ 8.30 (d, J ¼ 8.1 Hz, 2H), 8.02 (d, J ¼ 8.3 Hz, 2H), 7.93
4
.1.2.6. 2-(3,4-dichlorophenyl)-5-[(E)-2-(5-phenylthiophen-2-yl)
ethenyl] -1,3,4-oxadiazole 11.
1
6
13
6
H
NMR (DMSO-d , 400 MHz):
7.09 (d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.71,
d
¼ 8.25 (d, J ¼ 1.8 Hz, 1H), 8.03 (dd, J ¼ 8.4 Hz and J ¼ 1.8 Hz, 1H),
162.82, 141.81, 132.48, 132.25, 132.12, 132.03, 127.98, 127.62, 126.95,
126.91, 125.65, 122.93, 115.53, 109.34; LC/MS (APCI): 400.86
7
2
2
(
1
.94 (d, J ¼ 16.1 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.68 (d, J ¼ 7.8 Hz,
þ
H), 7.55 (d, J ¼ 3.7 Hz,1H), 7.53 (d, J ¼ 3.8 Hz,1H), 7.34 (t, J ¼ 7.6 Hz,
[MþH] and isotopic peak: 402.72; HRMS (TOF, ESIþ) cald for
13
þ
H), 7.35 (d, J ¼ 7.6 Hz, 1H), 6.97 (d, J ¼ 16.1 Hz, 1H); C NMR
C
15
H
9
N
2
OF
3
SBr (M þ H) 400.9571, found 400.9574; yellow pow-
6
DMSO-d , 101 MHz):
d
¼ 164.61, 162.19, 147.51, 139.11, 136.25,
der. Method A e purity ¼ 85%; Two-step yield ¼ 14%. Method B e
33.82, 133.58, 132.33, 131.60, 131.36, 129.15, 128.65, 128.57, 126.06,
purity ¼ 98%; Yield ¼ 48%.
þ
1
25.97, 124.18, 123.79, 107.95; LC/MS (ESI): 398.96 [MþH] and
isotopic peak: 400.95; HRMS (TOF, ESIþ) cald for C20
13
H N
2
OSCl
2
4.1.2.12. 2-[(E)-2-(5-phenylthiophen-2-yl)ethenyl]-5-(4-
þ
1
(
M þ H) 399.0126, found 399.0122; yellow powder. Method A e
trifluoromethylphenyl)-1,3,4-oxadiazole 17.
250 MHz):
J ¼ 7.1 Hz, 2H), 7.52 (bs, 2H), 7.38 (t, J ¼ 6.9 Hz, 2H), 7.30 (d,
H NMR (DMSO-d6,
purity ¼ 96%; Two-step yield ¼ 38%. Method B e purity ¼ 96%;
Yield ¼ 55%.
d
¼ 8.21 (d, J ¼ 7.9 Hz, 2H), 7.97e7.83 (m, 3H), 7.64 (d,
13
J ¼ 7.0 Hz, 1H), 6.97 (d, J ¼ 16.0 Hz, 1H); C NMR (DMSO-d6,
101 MHz):
4
.1.2.7. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(4-
d
¼ 164.84, 162.71, 146.70, 139.19, 133.47, 132.93, 129.80,
1
6
fluorophenyl)-1,3,4-oxadiazole 12. H NMR (DMSO-d , 400 MHz):
¼ 8.15 (dd, J ¼ 8.8 Hz, 1H), 8.13 (d, J ¼ 8.8 Hz, 1H), 7.87 (d,
J ¼ 16.2 Hz, 1H), 7.48 (t, J ¼ 8.8 Hz, 2H), 7.41 (d, J ¼ 3.9 Hz, 1H), 7.31
129.77, 129.74, 129.03, 128.90, 127.93, 126.98, 126.95, 126.91, 126.88,
þ
d
126.13, 126.08, 125.62, 108.38, 105.32; LC/MS (ESI): 398.98 [MþH] ;
þ
HRMS (TOF, ESIþ) cald for C21
H
14
N
2
OF
3
S (M þ H) 399.0779, found
13
6
(
1
1
3
d, J ¼ 3.9 Hz, 1H), 7.04 (d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d ,
399.0775; yellow powder. Method A e purity ¼ 95%; Two-step
yield ¼ 60%. Method B e purity ¼ 98%; Yield ¼ 32%.
01 MHz):
d
¼ 164.11, 163.02, 141.82, 132.38, 131.74, 131.52, 129.85,
29.76, 120.48, 120.46, 117.29, 117.07, 115.23, 109.42; LC/MS (ESI):
þ
50.99 [MþH] and isotopic peak: 352.98; HRMS (TOF, ESIþ) cald
4.1.2.13. 2-(3-bromophenyl)-5-[(E)-2-(5-bromothiophen-2-yl)
OFSBr (M þ H)þ 350.9303, found 350.9599; yellow
ethenyl]- 1,3,4-oxadiazole 18. H NMR (DMSO-d , 300 MHz):
1
6
for C14
H
9
N
2

F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
153
d
¼ 8.22 (t, J ¼ 1.6 Hz, 1H), 8.08 (dm, J ¼ 8.1 Hz, 1H), 7.91 (d,
J ¼ 16.2 Hz,1H), 7.85 (dm, J ¼ 8.0 Hz,1H), 7.59 (t, J ¼ 8.0 Hz,1H), 7.42
d, J ¼ 3.9 Hz, 1H), 7.31 (d, J ¼ 3.9 Hz, 1H), 7.03 (d, J ¼ 16.2 Hz, 1H);
4.1.2.19. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(4-
chlorophenyl)-1,3,4-oxadiazole 24. H NMR (DMSO-d6, 250 MHz):
1
(
d
¼ 8.04 (d, J ¼ 8.6 Hz, 2H), 7.83 (d, J ¼ 16.2 Hz, 1H), 7.66 (d,
1
3
6
C NMR (DMSO-d , 101 MHz):
d
¼ 164.40, 162.50, 141.82, 135.18,
J ¼ 8.6 Hz, 2H), 7.37 (d, J ¼ 3.9 Hz, 1H), 7.27 (d, J ¼ 3.8 Hz, 1H), 7.00
13
6
1
32.40, 132.10, 131.95, 131.83, 129.43, 126.10, 125.90, 122.93, 115.37,
(d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.31,
þ
1
4
(
09.27; LC/MS (ESI): 410.67 [MþH] and isotopic peaks: 412.73,
14.78; purity ¼ 95%; HRMS (TOF, ESIþ) cald for C14 OSBr
M þ H) 410.8802, found 410.8798; yellow powder. Yield ¼ 49%.
163.09, 141.87, 137.32, 132.45, 131.86, 131.76, 130.14, 128.94, 122.72,
þ
H
9
N
2
2
115.35, 109.43; LC/MS (ESI): 366.74 [MþH] and isotopic peak:
þ
368.72; purity ¼ 95%; HRMS (TOF, ESIþ) cald for C14
9 2
H N OSClBr
(
M þ H)þ 366.9307, found 366.9306; yellow powder. Yield ¼ 38%.
4
.1.2.14. 2-(3-bromophenyl)-5-[(E)-2-(5-phenylthiophen-2-yl)
1
4.1.2.20. 2-(4-bromophenyl)-5-[(E)-2-(5-bromothiophen-2-yl)
ethenyl]-1,3,4-oxadiazole 19.
¼ 8.20 (d, J ¼ 1.6 Hz, 1H), 8.06 (dd, J ¼ 7.8 Hz and J ¼ 1.6 Hz, 1H),
.94 (d, J ¼ 16.1 Hz, 1H), 7.81 (m, 1H), 7.68 (d, J ¼ 8.3 Hz, 2H),
.60e7.54 (m, 3H), 7.45e7.40 (m, 2H), 7.34 (d, J ¼ 8.5 Hz, 1H), 6.98
H NMR (DMSO-d6, 400 MHz):
1
6
ethenyl]-1,3,4-oxadiazole 25.
¼ 8.02 (d, J ¼ 8.6 Hz, 2H), 7.91 (d, J ¼ 16.2 Hz, 1H), 7.86 (d,
J ¼ 8.7 Hz, 2H), 7.43 (d, J ¼ 3.9 Hz, 1H), 7.33 (d, J ¼ 3.9 Hz, 1H), 7.06
H RMN (DMSO-d , 400 MHz):
d
7
7
d
1
3
6
13
6
(d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.26,
(
1
d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.65,
1
63.13, 141.81, 133.00, 132.39, 131.84, 131.72, 128.99, 126.16, 122.98,
62.53, 146.66, 139.33,136.88, 135.20, 133.56, 132.80, 132.15,129.80,
þ
1
15.33, 109.35; LC/MS (ESI): 410.68 [MþH] and isotopic peaks:
129.50, 129.04, 126.13, 125.63, 124.63, 122.98, 108.46, 105.42; LC/MS
þ
412.69, 414.69; purity
¼ 97%; HRMS (TOF, ESIþ) cald for
(
ESI): 408.79 [MþH] and isotopic peak: 410.73; purity ¼ 96%;
C
14
H
9
N
2
OSBr
2
(M þ H)þ 410.8802, found 410.8805; yellow powder.
OSBr (M þ H)þ 409.0010, found
HRMS (TOF, ESIþ) cald for C20
14 2
H N
Yield ¼ 27%.
4
09.0006; yellow powder. Yield ¼ 41%.
4
.1.2.21. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(3-
4
.1.2.15. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-[(4-N,N-
1
6
fluorophenyl)-1,3,4-oxadiazole 26. H RMN (DMSO-d , 250 MHz):
¼ 7.99 (d, J ¼ 16.1 Hz, 1H), 7.99e7.91 (m, 2H), 7.75 (dd, J ¼ 7.9 Hz
and J ¼ 2.1 Hz, 1H), 7.58 (td, J ¼ 8.7 Hz and J ¼ 1.9 Hz, 1H), 7.49 (d,
1
6
dimethyl)phenyl]-1,3,4-oxadiazole
4
20.
H
RMN
(DMSO-d ,
d
00 MHz):
d
¼ 7.81 (d, J ¼ 8.9 Hz, 2H), 7.72 (d, J ¼ 16.1 Hz, 1H), 7.34
(
6
d
d, J ¼ 3.8 Hz, 1H), 7.25 (d, J ¼ 3.8 Hz, 1H), 6.94 (d, J ¼ 16.2 Hz, 1H),
13
J ¼ 3.9 Hz, 1H), 7.39 (d, J ¼ 3.8 Hz, 1H), 7.13 (d, J ¼ 16.2 Hz, 1H);
C
1
3
6
.80 (d, J ¼ 9.0 Hz, 2H), 2.98 (s, 6H); C NMR (DMSO-d , 101 MHz):
6
NMR (DMSO-d ,101 MHz):
d
¼ 164.41,162.83,141.85,132.46,132.39
¼ 164.52, 162.95, 152.89, 142.14, 132.35, 131.40, 130.30, 128.46,
(CeF), 132.31, 131.96, 131.87, 125.80, 123.41, 119.62, 119.41, 115.43,
þ
1
14.75, 112.28, 110.07, 109.86, 40.18; LC/MS (ESI): 375.77 [MþH]
þ
1
14.08, 113.84, 109.34; LC/MS (ESI): 350.78 [MþH] and isotopic
and isotopic peak: 377.77; purity ¼ 96%; HRMS (TOF, ESIþ) cald for
peak: 352.87; purity
¼ 97%; HRMS (TOF, ESIþ) cald for
C
H
16 15
N
3
OSBr (M þ H)þ 376.0119, found 376.0119; orange powder.
OSBrF (M þ H)þ 350.9603, found 350.9605; yellow powder.
14 9 2
C H N
Yield ¼ 33%.
Yield ¼ 43%.
4
.1.2.22. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(3,5-
4
.1.2.16. 2-(4-N,N-dimethylphenyl)-5-[(E)-2-(5-phenylthiophen-2-
1
6
1
dichlorophenyl)-1,3,4-oxadiazole 27. H RMN (DMSO-d , 400 MHz):
d
1
yl)ethenyl]-1,3,4-oxadiazole 21. H NMR (DMSO-d6, 400 MHz):
d
¼ 8.02 (d, J ¼ 1.9 Hz, 2H), 7.90 (d, J ¼ 16.1 Hz,1H), 7.87 (t, J ¼ 1.9 Hz,
¼ 7.84 (d, J ¼ 8.6 Hz, 2H), 7.79 (d, J ¼ 16.1 Hz, 1H), 7.68 (d,
H), 7.36 (d, J ¼ 3.9 Hz, 1H), 7.26 (d, J ¼ 3.9 Hz, 1H), 6.99 (d,
J ¼ 7.8 Hz, 2H), 7.55e7.51 (m, 2H), 7.42 (t, J ¼ 7.4 Hz, 2H), 7.34 (d,
13
6
J ¼ 16.1 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.72, 161.69,
J ¼ 7.2 Hz, 1H), 6.92 (d, J ¼ 16.2 Hz, 1H), 6.82 (d, J ¼ 8.6 Hz, 2H), 2.97
s, 6H); 1 C NMR (DMSO-d , 101 MHz):
3
6
141.81, 135.75, 132.48, 132.43, 131.94, 131.81, 126.98, 125.56, 115.62,
(
d
¼ 168.79, 163.13, 153.04,
þ
1
09.10; LC/MS (ESI): 400.71 [MþH] and isotopic peaks: 402.70,
146.12, 139.64, 133.72, 132.09, 131.05, 129.74, 128.92, 128.48, 126.11,
þ
404.68; purity ¼ 98%; HRMS (TOF, ESIþ) cald for C H N OSCl Br
1
25.48, 112.39, 110.42, 109.14, 39.91; LC/MS (ESI): 373.95 [MþH] ;
14
8
2
2
(M þ H)þ 400.8918, found 400.8921; yellow powder. Yield ¼ 55%.
OS (M þ H)þ
purity ¼ 99%; HRMS (TOF, ESIþ) cald for C22
20 3
H N
374.1327, found 374.1327; orange powder. Yield ¼ 45%.
4
.1.2.23. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-(3-
1
6
methoxyphenyl)-1,3,4-oxadiazole 28. H RMN (DMSO-d , 250 MHz):
d
4
.1.2.17. 2-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-5-phenyl-1,3,4-
¼ 7.96 (d, J ¼ 16.2 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.64 (d,
1
6
oxadiazole 22. H RMN (DMSO-d , 400 MHz):
J ¼ 7.6 Hz and J ¼ 1.9 Hz, 2H), 7.83 (d, J ¼ 16.2 Hz, 1H), 7.62e7.56 (m,
H), 7.38 (d, J ¼ 3.9 Hz, 1H), 7.27 (d, J ¼ 3.9 Hz, 1H), 7.01 (d,
d
¼ 8.04 (dd,
J ¼ 1.8 Hz,1H), 7.59 (d, J ¼ 7.9 Hz,1H), 7.49 (d, J ¼ 4.0 Hz,1H), 7.38 (d,
J ¼ 3.9 Hz, 1H), 7.28 (dd, J ¼ 8.2 Hz and J ¼ 2.6 Hz, 1H), 7.12 (d,
3
13
6
J ¼ 16.2 Hz, 1H), 3.93 (s, 3H); C NMR (DMSO-d , 101 MHz):
13
6
J ¼ 16.1 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 164.15, 163.81,
d
¼ 164.21, 163.72, 160.25, 141.94, 132.46, 131.86, 131.65, 131.29,
141.91, 132.60, 132.43, 131.85, 131.55, 129.96, 127.16, 123.80, 115.29,
124.98, 119.48, 118.52, 115.34, 112.08, 109.51, 56.04; LC/MS (ESI):
þ
1
09.53; LC/MS (ESI): 332.80 [MþH] and isotopic peak: 334.76;
þ
3
62.80 [MþH] and isotopic peak: 364.80; purity ¼ 98%; HRMS
purity ¼ 96%; HRMS (TOF, ESIþ) cald for C14
H
10
N
2
OSBr (M þ H)þ
SBr (M þ H)þ 362.9803, found
(
3
TOF, ESIþ) cald for C15
12 2 2
H N O
3
32.9697, found 332.9696; yellow powder. Yield ¼ 15%.
62.9807; brown solid. Yield ¼ 18%.
4
.1.2.18. 2-[(E)-2-(5-phenylthiophen-2-yl)ethenyl]-5-phenyl-1,3,4-
4.1.3. General procedure for two steps synthesis (Method C) and
one-step synthesis (Method D) of 1,2,4-oxadiazole derivatives
33e36
1
oxadiazole 23. H NMR (DMSO-d6, 400 MHz):
d
¼ 8.06 (dd,
J ¼ 7.6 Hz and J ¼ 1.9 Hz, 2H), 7.88 (d, J ¼ 16.1 Hz, 1H), 7.68 (d,
J ¼ 7.4 Hz, 2H), 7.62e7.57 (m, 3H), 7.55 (bs, 2H), 7.43 (t, J ¼ 7.6 Hz,
13
2
H), 7.34 (d, J ¼ 7.4 Hz, 1H), 6.98 (d, J ¼ 16.1 Hz, 1H); C NMR
Method C
6
(
DMSO-d , 101 MHz):
d
¼ 164.32, 163.79, 146.51, 139.35, 133.58,
132.71, 132.53, 132.35, 129.95, 129.80, 129.01, 127.17, 126.12, 125.60,
To a stirred solution of acrylic acid (200 mg, 1 eq) and
substituted amidoxime (1 eq) in dry ethyl acetate (5e10 mL) were
added dropwise ®T3P (2.5 eq, 50% solution in EtOAc) and triethyl-
amine (3 eqe5 eq), and the mixture was refluxed under argon
þ
1
23.90, 108.68; LC/MS (ESI): 330.89 [MþH] ; purity ¼ 97%; HRMS
(
TOF, ESIþ) cald for C20
H
15
N
2
OS (M þ H)þ 331.0905, found
3
31.0903; yellow powder. Yield ¼ 11%.

154
F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
atmosphere for 24 h. The completion of reaction was confirmed by
TLC. The mixture was cooled to room temperature and poured into
ice/water. The aqueous layer was extracted twice with EtOAc and
4.1.3.4. 5-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-3-[4-
1
6
trifluoro(methoxy)phenyl]-1,2,4-oxadiazole 35. H NMR (DMSO-d ,
400 MHz):
¼ 8.07 (d, J ¼ 8.9 Hz, 2H), 7.97 (d, J ¼ 16.1 Hz, 1H), 7.50
(d, J ¼ 8.8 Hz, 2H), 7.44 (d, J ¼ 4.0 Hz, 1H), 7.26 (d, J ¼ 3.9 Hz, 1H),
d
then the organic layer was washed twice with saturated NaHCO
3
,
1
3
6
and once with brine. The organic layer was dried under Na SO and
2
4
7.01 (d, J ¼ 16.1 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 175.73,
concentrated under reduced pressure. The crude product was pu-
rified by silica gel flash chromatography (cyclohexan/EtOAc) to
afford the desired acylamidoxime. An example of the acylamidox-
ime derivative 29 synthesis is given below.
167.50, 150.91, 150.89, 141.31, 135.35, 133.53, 132.53, 129.73, 125.87,
-
122.11, 116.76, 109.35; LC/MS (ESI): 415.26 [MꢄH] and isotopic
þ
peak: 417.22; HRMS (TOF, ESIþ) cald for C15
H
9
N
2
O
2
F
3
SBr (M þ H)
416.9520, found 416.9520; yellow powder. Method C e Two-step
conversion yield determined by LC/MS ¼ 5%. Method D e
purity ¼ 95%; Yield ¼ 11%.
4
.1.3.1. (E)-[amino(4-chlorophenyl)methylidene]amino-(2E)-3-(5-
1
bromothiophen-2-yl)prop-2-enoate 29.
H
NMR (acetone-d6,
2
50 MHz):
d
¼ 7.72 (d, J ¼ 8.8 Hz, 2H), 7.70 (d, J ¼ 15.7 Hz, 1H), 7.36
(
6
d, J ¼ 8.8 Hz, 2H), 7.18 (d, J ¼ 3.9 Hz, 1H), 7.09 (d, J ¼ 3.9 Hz, 1H),
4.1.3.5. 5-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-3-[(4-
-
1
6
.38 (bs, 2H), 6.33 (d, J ¼ 15.8 Hz, 1H); LC/MS (ESI): 382.74 [MꢄH]
trifluoromethyl)phenyl]-1,2,4-oxadiazole 36.
400 MHz):
¼ 8.24 (d, J ¼ 8.1 Hz, 2H), 8.08 (d, J ¼ 16.1 Hz, 1H), 7.96
(d, J ¼ 8.3 Hz, 2H), 7.53 (d, J ¼ 4.0 Hz,1H), 7.35 (d, J ¼ 3.9 Hz,1H), 7.11
H NMR (DMSO-d ,
þ
and isotopic peak: 384.81, 384.80 [MþH] and isotopic peak:
d
3
86.85; purity ¼ 98%; beige powder. Yield ¼ 9%.
13
6
The corresponding acylamidoxime (1 eq) was placed under
(d, J ¼ 16.1 Hz, 1H); C NMR (DMSO-d , 101 MHz):
d
¼ 175.95,
argon atmosphere, anhydrous THF (3 mL) and TBAF (0.1 eq, 1 M in
THF) was added. The mixture was stirred at room temperature for
167.52, 141.28, 135.51, 133.62, 132.54, 132.01, 128.33, 126.78, 126.75,
126.71, 126.67, 125.66, 122.96, 116.85, 109.25; LC/MS (ESI): 401.14
ꢄ
þ
16e18 h. The completion of reaction was confirmed by TLC. The
[MꢄH] ; HRMS (TOF, ESIþ) cald for C15
H
9
N
2
OF3SBr (M þ H)
solvent was evaporated and EtOAc was added. The organic layer
was washed twice with water and twice with brine. The organic
400.9571, found 400.9570; yellow powder. Method C e Two-step
conversion yield determined by LC/MS ¼ 3%. Method D e
purity ¼ 96%; Yield ¼ 26%.
2 4
layer was dried under Na SO and concentrated under reduced
pressure.
Method D
4.2. IC50 determination based on fluorescent DENV RdRp assay
To a stirred solution of acrylic acid (200 mg, 1 eq) and
The compound's concentrations leading to 50% inhibition of
DENV-2 RdRp mediated RNA synthesis was determined in IC50
buffer (50 mM HEPES pH 8.0,10 mM KCl, 2 mM MnCl , 2 mM MgCl ,
2 2
substituted (1 eq) in dry ethyl acetate (5e10 mL) were added
®
dropwise T3P (3 eq, 50% solution in EtOAc) and triethylamine (5
eq). The reaction vessel was sealed under argon atmosphere and
the mixture was heated under microwave conditions at 160 C for
10 mM DTT) containing 100 nM of homopolymeric uridine RNA
template, 20 nM of DENV-2 RdRp and 7 various concentrations of
ꢀ
3
0 min. The completion of reaction was confirmed by TLC. The
mixture was cooled to room temperature. The organic layer was
washed twice with 1N HCl, once with H O and once with brine. The
organic layer was dried under Na SO and concentrated under
compound. Five ranges of inhibitor were available (0.01e5
mM/
0.1e50 M/0.5e50 M/1 to 100 M/5 to 400 M). According to the
m
m
m
m
2
inhibitory potency of the compound tested, a range was selected to
obtain the more accurate IC50 based on the best repartition of the
points surrounding the range. Hinokiflavone was purchased from
Extrasynthese (ref 1017) and used as positive control for this
2
4
reduced pressure. The crude product was purified by silica gel flash
chromatography (cyclohexan/EtOAc) to afford the desired 1,2,4-
oxadiazole derivative.
®
PicoGreen fluorescent assay.
Reactions were conducted in 40
plate. All experiments were robotized by using a BioMek 3000
automate (Beckman). 2 L of each diluted compounds in 100%
DMSO were added in wells to the chosen concentration (5% DMSO
final concentration). For each assay, the enzyme mix was distrib-
mL volume on a 96-well Nunc
4
.1.3.2. 5-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-3-(4-
1
chlorophenyl)-1,2,4-oxadiazole 33. H NMR (DMSO-d6, 400 MHz):
¼ 8.02 (d, J ¼ 8.5 Hz, 2H), 7.99 (d, J ¼ 16.3 Hz, 1H), 7.62 (d,
J ¼ 8.5 Hz, 2H), 7.48 (d, J ¼ 4.0 Hz, 1H), 7.30 (d, J ¼ 3.9 Hz, 1H), 7.05
m
d
13
(
1
d, J ¼ 16.2 Hz, 1H); C NMR (DMSO-d6, 101 MHz):
d
¼ 175.74,
uted in wells. Reactions were started by the addition of the
ꢀ
67.75, 141.37, 136.84, 135.39, 133.61, 132.60, 129.99, 129.33, 125.62,
nucleotide mix (100
m
M ATP) and were incubated at 30 C for
ꢄ
1
3
16.82, 109.42; LC/MS (ESI): 364.80 [MꢄH] and isotopic peak:
10 min. Reaction assays were stopped by the addition of 20
mL EDTA
þ
66.77, 366.73 [MþH] and isotopic peak: 368.77; HRMS (TOF,
100 mM. Positive and negative controls consisted respectively of a
reaction mix with 5% DMSO final concentration or EDTA 100 mM
instead of compounds.
þ
ESIþ) cald for C14
9
H N
2
OSClBr (M þ H) 366.9307, found 366.9307;
yellow powder. Method C e Two-step conversion yield determined
by LC/MS ¼ 8%. Method D e purity ¼ 95%; Yield ¼ 7%.
Reaction mixes were then transferred to Greiner plate (ref
®
6
75076) using a Biomek NX automate (Beckman). Picogreen
ꢀ
4
.1.3.3. 5-[(E)-2-(5-bromothiophen-2-yl)ethenyl]-3-(3-
fluorescent reagent from Molecular probes was diluted to 1/800 in
1
chlorophenyl)-1,2,4-oxadiazole 34. H NMR (DMSO-d6, 400 MHz):
TE buffer according data manufacturer and 60 ml of reagent were
d
¼ 8.07 (d, J ¼ 16.0 Hz, 1H), 8.02e7.97 (m, 2H), 7.66e7.59 (m, 1H),
distributed into each well of the Greiner plate. The plate was
incubated 5 min in the dark at room temperature and the fluo-
rescence signal was then read at 480 nm (excitation) and 530 nm
(emission) using a TecanSafire2.
7
.63 (t, J ¼ 7.8 Hz, 1H), 7.52 (d, J ¼ 4.0 Hz, 1H), 7.35 (d, J ¼ 3.9 Hz, 1H),
13
7.09 (d, J ¼ 16.1 Hz, 1H); C NMR (DMSO-d6, 101 MHz): ¼ 175.79,
d
1
67.43, 141.30, 135.46, 134.40, 133.56, 132.54, 131.93, 131.83, 128.70,
-
1
27.05, 126.10, 116.80, 109.27; LC/MS (APCI): 366.59 [M-H ] and
IC50 was determined using the equation: % of active
þ
2
isotopic peak 368.56, 368.70 [M þ H ] and isotopic peak: 370.10;
enzyme ¼ 100/(1þ(I) /IC50), where I is the concentration of in-
hibitor and 100% of activity is the fluorescence intensity without
inhibitor. IC50 was determined from curve-fitting using Prism
software. For each value, results were obtained using triplicate in a
single experiment.
þ
HRMS (TOF, ESIþ) cald for C14
H
9
N
2
OSClBr (M þ H) 366.9307,
found 366.9310; pale orange powder. Method C e Two-step con-
version yield determined by LC/MS ¼ 8%. Method D e purity ¼ 97%;
Yield ¼ 14%.

F. Benmansour et al. / European Journal of Medicinal Chemistry 109 (2016) 146e156
155
4.3. DENV-infected cells assay
Y ¼ 100/(m0/m1)m2). This macro allows determining the best curve
fit and the m1 and m2 parameters, where m1 corresponds to EC50
.
Cells and viruses. Vero E6 cells were purchased from ATCC and
were cultivated in Eagle MEM (Gibco), with 1% Pen Strep (Gibco),1%
NEAA (non essential amino acids, Gibco), 7.5% fetal bovine serum
4
.3.3. CC50 determination on Vero E6 cell lines
Cells were plated and cultivated in the same day and conditions
as the antiviral assay. Six two-fold serial dilutions of the com-
pounds (beginning at 100 M final concentration, down to 3.1 M),
in triplicates or duplicates, were added to the wells. At day 4, the
medium was removed and replaced with 80 L of culture medium
(
Gibco). For cytotoxicity and antiviral assays, cells were plated in 96
wells plate using 2.5% FCS instead of 7.5%. DENV strains were iso-
lated from clinical samples, except for DENV-2 Trinidad which was
passaged 55 times in suckling mouse brains. They were passaged
less than 3 times in total in mosquito C6/36 cells and then Vero E6
cells.
m
m
m
containing 10
m
l of CellTiter-Blue cell viability reagent (Promega)
ꢀ
and incubated at 37 C for 90 min. Fluorescence was read using an
DENV-1 Indonesia (strain JKT 1186 TVP 949, WRCEVA (World
Reference Center for Emerging viruses and arboviruses)); DENV-2
Martinique (strain H/IMTSSA-MART/98-703): PMID:11027645;
DENV-2 Trinidad (strain 1751 TC 544, WRCEVA); DENV-3 Bolivia
Infinite M200 Pro plate reader from TECAN.
Acknowledgments
(
strain 4025, PMID:19505253); DENV-4 Dakar (strain HD34460,
We wish to thank Justine N eꢀ rot for technical assistance. We
especially thank Florence Mahuteau-Betzer (CNRS UMR9187,
INSERM U119) for providing the Institut Curie chemical library in
the context of the French National Chemical Library. This work was
supported by the project SILVER (Health-F3-2010-260644) of the
European Union 7th Framework Program, the Region PACA and the
IHU Infectiopole.
WRCEVA); DENV-4 Martinique (strain 017, isolated in the labora-
tory, unpublished).
4
.3.1. Virus yield reduction assay
The amount of each dengue virus in the assay has been cali-
brated by trial so that the virus replication at day 4 is still in the
ascending phase of the growth curve before the beginning of the
plateau. MOI ranged from 0.01 to 0.1. One day prior to infection
4
Appendix A. Supplementary data
5
ꢃ 10 Vero E6 cells were seeded in 100
mL of medium (with 2.5%
FCS) in each wells of a 96-well titer plates. The next day, 8 two-fold
serial dilutions of the compounds (beginning at 20 M final con-
centration, down to 0.16 M), in triplicates or duplicates, were
added to the cells (25 L/well, in 2.5% FCS containing medium). Four
Virus Control (VC) wells (per virus) were supplemented with 25
medium containing 0.1% DMSO and four cells control wells were
supplemented with 50 L of medium. Fifteen minutes later, 25 L of
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.046.
m
m
m
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