Structure-activity and structure-property relationships of novel Nrf2 activators with a 1,2,4-oxadiazole core and their therapeutic effects on acetaminophen (APAP)-induced acute liver injury

Article abstract of DOI:10.1016/j.ejmech.2018.08.071

The antioxidant function induced by Nrf2 protects the liver from damage. We found a novel Nrf2 activator named compound 25 via structural modification of compound 1 we previously reported. In vitro, compound 25 induced Nrf2 transport into the nucleus and protected hepatocyte L02 cells from APAP-induced cytotoxicity via activating the Nrf2-ARE signaling pathway. In vivo, 25 exhibited therapeutic effects in a mouse model of acute liver injury induced by acetaminophen (APAP) by up-regulating Nrf2-dependent antioxidases and down-regulating liver injury markers in serum. Together, these results indicated that 25 is a potent Nrf2/ARE activator both in vitro and in vivo. The drug-like properties of compound 25 further revealed its potential for development as a therapeutic drug against acute liver injury.

Full text of DOI:10.1016/j.ejmech.2018.08.071

European Journal of Medicinal Chemistry 157 (2018) 1376e1394
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-activity and structure-property relationships of novel Nrf2
activators with a 1,2,4-oxadiazole core and their therapeutic effects on
acetaminophen (APAP)-induced acute liver injury
,
b
,
,
,
, ***
Li-Li Xu a c, Yu-Feng Wu c, Lei Wang a b, Cui-Cui Li c, Li Li a b, Bin Di c
,
Qi-Dong You a **, Zheng-Yu Jiang a
,
b,
, b, *
a State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
c Key Laboratory on Protein Chemistry and Structural Biology, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China
Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The antioxidant function induced by Nrf2 protects the liver from damage. We found a novel Nrf2 acti-
vator named compound 25 via structural modification of compound 1 we previously reported. In vitro,
compound 25 induced Nrf2 transport into the nucleus and protected hepatocyte L02 cells from APAP-
induced cytotoxicity via activating the Nrf2-ARE signaling pathway. In vivo, 25 exhibited therapeutic
effects in a mouse model of acute liver injury induced by acetaminophen (APAP) by up-regulating Nrf2-
dependent antioxidases and down-regulating liver injury markers in serum. Together, these results
indicated that 25 is a potent Nrf2/ARE activator both in vitro and in vivo. The drug-like properties of
compound 25 further revealed its potential for development as a therapeutic drug against acute liver
injury.
Received 22 May 2018
Received in revised form
11 August 2018
Accepted 25 August 2018
Available online 28 August 2018
Keywords:
Nrf2 activators
Oxadiazole core
Acute liver injury
Structure-activity relationship
Antioxidant function
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
the cysteine residues of Keap1 are oxidized or alkylated resulting in
structural changes in Keap1 [10,11]. Nrf2 detaches from Keap1 for
It is generally thought that the induction of nuclear factor
erythroid 2 p45-related factor 2 (Nrf2) to resist environmental
stress is a promising strategy for maintaining cellular homeostasis
[1,2]. Nrf2, as a member of the basic-leucine zipper (b-ZIP) tran-
scription factor family [3], plays a critical role in antioxidant stress
[4,5]. In a stress-free state, Nrf2 is anchored in the cytoplasm by
kelch-like ECH-associated protein 1 (Keap1) and is directed toward
ubiquitin-mediated proteasomal degradation [6,7]. This process
maintains intracytoplasmic Nrf2 at a low level [8,9]. When cells are
exposed to oxidative stress, such as reactive oxygen species (ROS),
stabilization and is no longer ubiquitinated and degraded [12,13].
Meanwhile, the newly synthesized Nrf2 accumulates and trans-
locates into the nucleus and then forms heterodimers with small
Maf proteins [8,14]. The heterodimer binds to the antioxidant
response element (ARE), participating in the regulation of antiox-
idant enzymes including NAD(P)H: quinone oxidoreductase-1
(NQO1), hemeoxygenase-1 (HO-1) and
GCS) [15,16].
g-glutamylcysteine (g-
The liver is the first organ through which oral xenobiotics pass
after being absorbed by the intestine and is the primary site of
* Corresponding author. State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China.
** Corresponding author. State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China.
*** Corresponding author. Key Laboratory on Protein Chemistry and Structural Biology, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Edu-
cation, China Pharmaceutical University, Nanjing, 210009, China.
E-mail addresses: dibin@cpu.edu.cn (B. Di), youqd@163.com (Q.-D. You), jiangzhengyucpu@163.com (Z.-Y. Jiang).
https://doi.org/10.1016/j.ejmech.2018.08.071
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1377
bioconversion and metabolism [17]. Oxidative stress is associated
2. Results
with liver diseases including fibrosis, cirrhosis and advanced he-
patocellular carcinoma (HCC) [18]. Therefore, reducing oxidative
stress plays a critical role in the treatment of liver diseases [19].
Thus, the antioxidant function of Nrf2 activation has been exten-
sively studied in the treatment of liver diseases [20e22]. Nrf2-
knockout and Keap1-knockout mouse models have been used to
study the protective function of Nrf2 activation [23,24]. Nrf2 can act
as a research target in the prevention and treatment of liver dis-
eases [25,26].
Acetaminophen (APAP) is the most common drug that causes
drug-induced liver damage clinically. After APAP is absorbed by the
body, 90% of APAP is excreted in combination with glucuronic acid or
sulfuric acid. The remaining 5%e10% of APAP is metabolized by the
cytochrome p450 (CYP450) system. APAP is metabolized to produce
n-acetyl-para-benzoquinone imine (NAPQI). The toxic metabolite
NAPQI is detoxified by glutathione (GSH) [27]. Once GSH is depleted
in hepatocytes, NAPQI reacts with the sulfhydryl groups of protein to
form an NAPQI-protein adduct. The formation of protein adducts is a
key initiation event leading to hepatotoxicity [28].
Many studies suggest that APAP-induced liver injury can trigger
the compensatory activation of Keap1-Nrf2 signaling pathway
in vivo. The possible mechanisms are as follows: 1. NAPQI depletes
GSH in hepatocytes to produce oxidative stress. 2. NAPQI directly
modifies the cysteine thiol groups of Keap1 in the cytoplasm to
alter the conformation of Keap1 and to disrupt the Keap1-Nrf2
interaction. Nrf2 is dissociated from Keap1 and translocated into
the nucleus. The above two factors work together to activate the
Nrf2 pathway. Nrf2 binds to ARE in the nucleus and promotes the
expression of detoxification-related target proteins including
NQO1, cysteine ligase catalytic subunit (GCLC) and cysteine ligase
regulatory subunit (GCLM) for detoxification. This is also a self-
protection mechanism of the body [29e31].
2.1. Structure-activity relationship study of derivatives containing a
1,2,4-oxadiazole core
2.1.1. Design, synthesis and evaluation of ring A derivatives
In our previous research, compound 1 with a p-fluorine sub-
stituent in phenyl A exhibited optimal ARE-inducing activity [32].
In this paper, compound 1 was selected as the starting compound
for further structural modifications and SAR studies. The changes in
ARE-inducing activity deserved attention when ring A was intro-
duced to multiple halogen substituents including eF, eCl and eBr
(Fig. 1). We synthesized sixteen compounds 1e16 and evaluated
their Nrf2-ARE inducing activities using the ARE-luciferase reporter
gene assay. Dimethyl fumarate (DMF) [33] and tertiary butylhy-
droquinone (tBHQ) [34] were used as positive controls (Table 1).
The synthesis route of these derivatives is summarized in Scheme 1.
The Nrf2-ARE inducing activities of all the synthetic derivatives
with halogen substituents in ring A were evaluated in stably
transfected HepG2-ARE-C8 cells using the ARE-luciferase reporter
gene assay. The position of the ꢀF substituent in the phenyl ring
was crucial to the activity of these compounds. Among them, when
ring A was substituted with a single ꢀF, the derivative with the ꢀF
substituent at the para position (1) was the most active compound,
followed by derivatives with ortho (4) or meta (5) position occu-
pied by ꢀF. When the para-position of ring A was substituted with a
single substituent of eF, eCl or eBr, the derivative with a ꢀF sub-
stituent at the para position (1) was the most active compound,
followed by those with ꢀCl (2) and ꢀBr (3) substituents at the para
position. In summary, the charge and position of the substituents
could influence the ARE-inducing activity. When ring A was
switched with a single halogen substituent, eF was preferred as the
para substituent.
We previously reported a novel Nrf2 activator known as com-
pound 1, which has a p-fluorine in the phenyl group (ring A). We
also systematically studied the structureeactivity relationship
(SAR) of ring A [32]. To obtain more potential candidate compounds
and further improve the structure-activity relationship, we per-
formed a more comprehensive structural modification of the rings
A and C. In this paper, 1 was chosen as the initial compound. To
further assess the effect of halogen substituents on ring A, we
focused on changes in ARE-inducing activity when phenyl group
was introduced into halogen substituents including eF, eCl and
eBr in various combinations. Sixteen compounds were synthesized
and evaluated using the ARE-luciferase reporter gene assay. Among
them, compound 6, bearing two eF substituents at the meta and
para positions of phenyl group, exhibited enhanced ARE-inducing
activity as compared with 1. Therefore, retaining this privileged
structure fragment, the subsequent structural modifications were
primarily focused on ring C. Twenty-five compounds were syn-
thesized. The results revealed that 25 with ring C replaced by 304-
methylpyridinyl was the most potent Nrf2 activator, presenting
enhanced ARE-inducing activity and improved physicochemical
properties (4-fold increase in intrinsic solubility compared to
compound 1). Further assessments of its biological activity indi-
cated that in normal liver cells (L02), 25 promoted the nuclear
translocation of Nrf2 and increased the expression of Nrf2-driven
downstream antioxidant proteins including NQO1 and HO-1.
Even at high concentrations, compound 25 did not show signifi-
cant cytotoxicity in L02 cells. Moreover, 25 showed in vivo thera-
peutic effects in an APAP-induced acute liver injury mouse model
by activating the Nrf2-ARE pathway. Compound 25 induced the up-
regulation of antioxidant proteins and the down-regulation of he-
patic injury markers in serum. These results demonstrated that 25
would be a potent Nrf2/ARE activator and a liver protective agent.
Next, we investigated changes in the ARE-inducing activity of
the derivatives when two halogen substituents were introduced
into ring A. When the para position of the ring A was occupied by
the eF element, the eF, eCl or eBr substituents were introduced
into the meta position. The ARE-inducing activities of those de-
rivatives showed that 6 with two eF substituents in the para and
meta positions of phenyl (3, 4-F) was the most active compound. In
addition, the para position of ring A was also occupied by the eF
substituent, and the substituents eF, eCl or eBr were introduced
into the ortho position. The ARE-inducing activity for 2-Br-4-F (13)
was stronger than that for 2-Cl-4-F (8) and 2, 4-F (10). Among these
derivatives, 6 (m, p-F) showed concentration-dependent Nrf2-ARE
inducing activity and was more potent than 1 (p-F). However,
when ring A was introduced to three substituents, the ARE-
inducing activities of the compounds decreased significantly.
2.1.2. Design, synthesis and evaluation of ring C derivatives
Among the ring A derivatives, 6 with m, p-F substituents in
phenyl A showed potent ARE-inducing activity. We subsequently
performed modifications of ring C to obtain twenty-five derivatives
while maintaining ring A as an m, p-F substituted benzene ring.
During this process, the benzo[d]imidazole group (ring C) was
replaced with multiple types of heterocyclic substituents or phenyl
(Fig. 1). The synthesis route of these derivatives is summarized in
Scheme 2.
The Nrf2-ARE inducing activities of all ring C derivatives in
HepG2-ARE-C8 cells were evaluated using the ARE-luciferase re-
porter gene assay and summarized in Table 2. Compound 25 (304-
methylpyridinyl) at 80 mM exerted an optimal induction activity
of 59.55-fold compared to the blank control, which was superior to
1 and 6 at the same concentration. The ARE-inducing activity was
decreased when ring C (benzo[d]imidazole group) was replaced

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L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
Fig. 1. The structures of the derivatives containing a 1,2,4-oxadiazole core.
Table 1
Nrf2-inducing activity of sixteen derivatives containing a 1,2,4-oxadiazole core and halogen substituents measured using the luciferase reporter gene assay.
Cpd.
R
ARE-inducing activity in luciferase reporter assay (m
M)a,b
0.01
0.1
1
20
40
80
1
2
3
4
5
6
7
8
4-F
4-Cl
4-Br
2-F
3-F
3,4-F
2,3-F
2-Cl-4-F
3-Cl-4-F
2,4-F
3-Cl
2,5-F
2-Br-4-F
3-Br-4-F
2,4,5-F
5-Cl-2,4-F
1.57 0.29
0.33 0.05
0.22 0.02
2.01 0.24
1.21 0.44
1.46 0.18
1.77 0.18
0.68 0.15
1.04 0.28
1.62 0.18
1.74 0.08
2.31 0.01
1.01 0.13
1.68 0.21
1.55 0.59
1.94 0.38
0.82 0.04
0.02
2.49 0.67
0.72 0.06
0.25 0.05
4.51 0.30
2.69 0.29
6.21 0.68
2.22 0.18
2.98 0.12
3.88 0.34
2.17 0.47
3.23 0.37
4.15 1.35
2.65 0.02
1.36 0.44
1.86 0.51
1.18 0.32
1.00 0.07
0.2
10.23 0.66
1.64 0.18
0.87 0.12
12.04 1.50
4.49 2.79
12.35 0.51
3.70 0.17
4.22 1.28
3.22 0.24
2.48 0.48
4.78 0.34
5.24 0.02
4.18 1.48
1.89 0.09
2.15 1.29
1.26 0.16
1.10 0.08
2
27.28 1.26
15.49 1.03
13.41 0.72
12.70 2.90
8.83 1.33
24.03 0.80
4.55 0.12
11.8 0.36
7.53 0.05
4.58 0.46
12.79 1.01
16.85 1.21
12.10 0.06
3.46 0.22
3.95 0.09
3.35 1.23
1.65 0.35
20
40.81 0.22
18.52 0.98
3.44 1.51
15.04 1.60
12.38 2.01
42.61 2.43
10.69 1.80
14.71 0.42
9.59 0.25
7.34 0.61
13.19 0.21
20.20 1.46
21.59 0.61
3.92 0.64
4.36 0.04
5.14 0.64
2.97 0.45
40
44.40 0.89
14.14 0.49
2.75 0.15
19.09 1.01
14.10 1.12
49.86 0.30
16.51 4.11
18.31 2.05
12.49 0.21
8.36 0.58
19.15 0.70
21.87 2.70
25.15 0.41
4.84 0.98
7.92 1.56
7.61 0.63
ND
9
10
11
12
13
14
15
16
DMF
80
tBHQ
1.18 0.08
1.25 0.01
1.24 0.04
1.41 0.07
2.14 0.10
5.27 0.41
a
The inducing activity of the compound was calculated compared to the blank control, and the data are presented as the mean SEM of three separate experiments.
All compounds showed no significant anti-proliferative effects at the concentration of 80 mM.
b
with pyridine (17, 18, 19), pyrazine (22) and pyrimidine (23)
compared to that of 1. In addition, the ARE-inducing activity
decreased when the eF substituent was imported into pyridine (20,
21) compared with that of 1. However, the replacement of the
benzo[d]imidazole group with (304-methylpyridinyl) (25)
enhanced the ARE-inducing activity compared to that of 1. In
contrast, the ARE-inducing activity was significantly reduced when
the benzo[d]imidazole group was substituted with 203-
methylpyridinyl (24) and 205-methylpyridinyl (26). This result
showed that the position of the methyl substituent on pyridine
played an important role in maintaining its activity. Substitution of
the benzo[d]imidazole group with an indole ring (27), naphthalene
ring (28) and biphenyl (29) also resulted in a decrease in ARE-
inducing activity compared with that of 1. Next, we explored the
change in ARE-inducing activity when the benzo[d]imidazole
group was replaced with phenyl. The para position of the benzene
ring was replaced with electron-donating groups, including eCH3
(38), eOCH3 (39), and etertiary butyl (40) or weak electron-
withdrawing substituent of eBr (35), which were superior to the
strong electron-withdrawing substituents, including eF (30, 31,
32), eCl (33) and eacetyl (37). Unlike ring A, the introduction of
electron-donating groups to ring
C promoted the Nrf2-ARE

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1379
Scheme 1. Synthetic route of 1,2,4-oxadiazole derivatives with halogen substituents.
Reagents and conditions: (a) 5 M HCl, HCOOH, reflux, 4 h, 94%; (b) K2CO3, EtOH, H2NOH$HCl, reflux, 12 h, 50%; (c) CDI, DMF, 110 ꢁC, 12e18 h.
Scheme 2. Synthetic route of 1,2,4-oxadiazole derivatives of ring C.
Reagents and conditions: (a) K2CO3, EtOH, H2NOH$HCl, reflux, 12 h; (b) CDI, DMF, 110 ꢁC, 12e18 h.
inducing activity, while electron-withdrawing groups decreased
this activity. Meanwhile, no substitution or multiple substitutions
in ring C impaired the Nrf2-ARE inducing activity.
benzyl (38) and benzene ring with methoxyl groups (39) rendered
poor solubility. Taken together, compound 25, which presented
potent ARE-inducing activity and appropriate physicochemical
properties, was selected for further pharmacological studies.
In summary, the SAR studies could be described as follows: (1)
For phenyl ring A replaced with a single halogen substituent, the
introduction of para-F up-regulated the Nrf2-ARE inducing activity.
(2) When two halogen substituents were introduced into phenyl
ring A and the para position was replaced with eF, the meta posi-
tion was preferential to eF (3eF) and the ortho position was pref-
erential to eBr (2eBr). (3) Introduction of three substituents into
ring A significantly decreased the ARE-inducing activity. (4) When
ring C was replaced with phenyl and nitrogen-containing hetero-
cyclic rings, the introduction of electron-donating substituents into
ring C promoted the Nrf2-ARE inducing activity, while electron-
withdrawing substituents impaired this activity. (5) The ARE-
inducing activity decreased when ring C was replaced with
phenyl having zero or multiple substituents.
2.3. Pharmacological results
2.3.1. Cytotoxicities of the selected derivatives
For compounds having excellent Nrf2-ARE inducing activity in
the luciferase reporter gene assay, the normal liver cell line L02 was
selected to test for cytotoxicity by the MTT method. L02 cells were
treated with derivatives (Fig. 2) having potent ARE-inducing ac-
tivities for 24 h at concentrations of 0.2, 2, 20, 40, 80 and 160 mM.
tBHQ was used as the positive control. Compound 25 and tBHQ
exhibited no more than 7% cytotoxicity even at concentrations up to
160 mM. Compounds 1 and 6 showed no more than 20% cytotoxicity
in L02 cells at the high concentration of 160 mM.
2.2. Physicochemical properties of the active compounds
2.3.2. Effects of 25 on the Nrf2-ARE pathway
Nrf2, which escapes ubiquitin degradation, can translocate into
the nucleus and bind to ARE, resulting in the up-regulation of Nrf2-
dependent downstream markers. We investigated the modulation
of 25 on Nrf2 and Nrf2-dependent proteins including NQO1 and
HO-1 in L02 cells. Compound 25 increased the protein expression of
NQO1, HO-1 and Nrf2 in L02 cells in time-dependent and
concentration-dependent manners (Fig. 3A and B). To confirm that
the up-regulation of downstream antioxidant proteins was caused
by Nrf2 activation, siRNA was designed to purposefully down-
regulate the Nrf2 mRNA transcription. The qRT-PCR results
showed that when the Nrf2 gene transcription level was purpose-
fully down-regulated to 30% by Nrf2 siRNA, the mRNA transcrip-
tions of NQO1, GCLM and HO-1 were also down-regulated. The
To identify potential drug-like compounds, we determined the
physicochemical properties of several compounds that exhibited
potent ARE-inducing activity using the luciferase reporter gene
assay. LogD (pH 7.4) and intrinsic aqueous solubility were deter-
mined on the Gemini Profiler instrument (pION) by the “gold-
standard” Avdeef-Bucher potentiometric titration method. As
shown in Table 3, although the logD7.4 values remained the same,
the solubilities of all the selected compounds changed significantly.
Among them, the intrinsic solubility of 25 (250
which was 4 times that of compound 1 (intrinsic solubility was
66.90 M) and 2.5 times that of compound 6 (intrinsic solubility
was 99
mM) was optimal,
m
m
M). The introduction of hydrophilic groups such as 304-
methylpyridinyl (25), 203-methylpyridinyl (24) and 205-
methylpyridinyl (26) significantly improved the solubility. The
introduction of hydrophobic substituents including biphenyl (29),
addition of 25 (20 mM) obviously up-regulated the mRNA tran-
scription levels of Nrf2 and its downstream genes including NQO1,
GCLM and HO-1 (Fig. 3C and 3E). The above results indicated that

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L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
Table 2
Nrf2-inducing activity of ring C derivatives containing a 1,2,4-oxadiazole core as determined by the luciferase reporter gene assay.
Cpd.
R
AREeinducing activity in luciferase reporter assay (m
M)a,b
0.01
0.1
1
20
40
80
1
1.57 0.29
2.49 0.67
10.23 0.66
27.28 1.26
40.81 0.22
44.40 0.89
6
1.46 0.18
0.65 0.35
6.21 0.68
0.83 0.02
12.35 0.51
1.05 1.35
24.03 0.81
1.31 0.14
42.61 2.43
1.32 0.42
49.86 0.31
2.55 0.07
17
18
19
1.08 0.26
1.45 0.91
1.62 0.67
1.71 0.70
1.07 0.31
2.02 0.70
1.29 0.47
3.15 1.20
1.72 0.07
3.15 0.49
2.88 0.86
4.15 0.35
20
21
0.96 0.30
1.15 0.14
1.97 0.31
1.25 0.07
2.17 0.07
1.56 0.14
2.95 0.84
1.75 0.07
5.79 0.90
2.52 0.56
6.35 0.98
2.95 0.21
22
1.13 0.33
1.43 0.40
1.95 0.07
1.81 0.01
2.83 0.84
3.35 0.38
23
24
0.92 0.31
1.16 0.38
1.06 0.26
1.08 0.01
1.07 0.53
2.65 0.81
2.05 0.35
3.96 1.14
2.95 0.49
4.46 0.68
5.82 0.74
5.68 0.14
25
26
2.75 0.21
0.63 0.11
3.46 0.28
1.06 0.02
16.1 0.98
2.04 0.51
24.66 0.14
8.96 0.44
48.73 0.28
9.76 0.28
59.55 0.49
10.58 0.14

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1381
Table 2 (continued )
Cpd.
R
AREeinducing activity in luciferase reporter assay (m
M)a,b
0.01
0.1
1
20
40
80
27
0.83 0.31
1.88 0.47
2.01 0.01
2.36 0.17
3.77 0.38
4.02 0.35
28
29
1.16 0.12
1.15 0.07
1.27 0.21
1.25 0.35
1.52 0.35
6.75 0.38
1.96 0.30
8.54 1.18
1.96 0.35
3.57 0.49
10.76 1.07
14.73 0.84
30
3.85 0.63
3.96 1.13
4.25 0.49
4.85 0.07
5.91 0.28
6.95 1.34
31
32
0.92 0.11
1.76 0.37
1.81 0.05
2.12 0.03
2.05 0.42
2.05 0.42
2.76 0.12
3.75 0.21
3.45 0.21
5.68 0.14
5.46 0.47
6.93 0.76
33
34
2.85 0.21
0.58 0.08
3.06 0.14
1.16 0.05
3.85 0.35
1.76 0.53
7.65 0.35
1.98 0.01
8.29 0.28
2.63 0.15
9.95 0.35
3.63 0.60
35
36
2.68 0.28
1.42 0.14
3.78 0.42
1.36 0.08
4.49 0.14
1.73 0.17
8.65 0.56
1.73 0.17
12.15 0.07
2.68 0.03
18.25 1.06
2.56 0.01
37
1.25 0.07
2.03 0.54
2.04 0.12
2.61 0.58
3.78 0.10
5.01 1.11
38
39
0.67 0.17
1.63 0.22
1.22 0.03
1.87 0.67
1.85 0.28
5.35 0.07
2.51 0.65
6.62 0.49
10.17 1.06
16.35 0.03
14.17 0.14
15.18 1.50
(continued on next page)

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L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
Table 2 (continued )
Cpd.
R
AREeinducing activity in luciferase reporter assay (m
M)a,b
0.01
0.1
1
20
40
80
40
1.35 0.67
2.88 0.14
2.91 0.44
8.35 0.60
13.23 0.83
24.02 1.30
41
1.87 0.28
1.91 0.02
2.15 0.91
3.45 0.49
3.55 0.07
4.45 0.07
DMF
tBHQ
0.82 0.04
1.18 0.08
1.00 0.07
1.25 0.01
1.10 0.08
1.24 0.04
1.65 0.35
1.41 0.07
2.97 0.45
2.14 0.10
ND
5.27 0.41
a
The inductivity of the compound was calculated compared to the blank control, and the data are presented as the mean SEM of three separate experiments.
All compounds showed no significant anti-proliferative effects at the concentration of 80 mM.
b
Table 3
25 could induce the accumulation of Nrf2 in the nucleus. An
immunofluorescence assay confirmed that 25 induced Nrf2 accu-
mulation in the nucleus of L02 cells. The green staining represents
Nrf2, blue staining represents nucleus and the merge represents
the nuclear import of Nrf2. The strong fluorescence in the nucleus
Physicochemical properties of the selected compounds.
Compound
LogD, pH 7.4
Intrinsic solubility, (mM)
1
6
3.37
3.30
2.86
3.07
2.65
4.75
3.91
4.16
3.91
3.92
66.90
99
200
250
220
16
77
35
86
91
24
25
26
29
35
38
39
40
indicated that Nrf2 translocated into the nucleus in
a
concentration-dependent manner compared to that in the control
group (Fig. 4). The data further supported that 25 could induce Nrf2
translocation into the nucleus.
2.3.3. Protection of L02 cells from APAP-induced cytotoxicity by 25
As a normal hepatic cell line, L02 cells are commonly used in
hepatotoxicity studies induced by various hepatotoxic agents. It is
well known that APAP overdose can cause liver damage. We
selected three concentrations including 5,10 and 20 mM, to evaluate
the protective effects of 25 on APAP-induced hepatocyte injury.
APAP (8 mM) was used to establish a cell damage model of L02 cells.
L02 cells were plated in 96-well plates for 12 h and pretreated with
25 at different concentrations ranging from 5 mM to 20 mM for 24 h.
The MTT cell proliferation assay showed that the L02 cells were
damaged after APAP treatment for 24 h. We observed that 25
increased the proliferative capacity of (8 mM) APAP-challenged
L02 cells in a concentration-dependent manner. Compound 25 at
20 mM increased the cell viability to approximately 80%, similar to
tBHQ and DMF (Fig. 5). Additionally, to explore whether the liver
protection of 25 was dependent on Nrf2 activation, we performed
siRNA Nrf2 interference assays. The cell viability of L02 cells
decreased to 50% when co-treated with siRNA Nrf2 and 25 (20 mM)
compared to that in cells treated with 25 alone at the same con-
centration. All the results showed that 25 increased the cell viability
in the hepatocyte injury model by Nrf2 activation.
Fig. 2. Cytotoxicity evaluation of selected derivatives of ring A (6) and ring C (25)
containing a 1,2,4-oxadiazole core in L02 cells. L02 cells were plated in a 96-well plate
for 12 h and subsequently treated with different concentrations (0, 0.2, 2, 20, 40, 80,
160 mM) of the selected derivatives for another 24 h. Compound 1 and tBHQ were used
as positive controls. The cell viability was measured by the MTT assay. All data
represent the mean SD of three independent experiments.
2.3.4. Therapeutic effects of 25 on APAP-induced acute liver injury
in a mouse model
To further investigate the potential protective effects of 25 on an
APAP-induced acute liver injury mouse model, we measured the
serum content of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST), which are regarded as common markers of
liver damage. The contents of the model group increased remark-
ably compared to the normal group (Fig. 6A and B). Following
treatment with different concentrations of 25 (10 mg/kg, 50 mg/kg
and 100 mg/kg, respectively), the levels of ALT and AST were
decreased in a concentration-dependent manner compared to
those in the APAP model group. These results indicated that 25
exhibited potential therapeutic effects on APAP-induced acute liver
the up-regulation of downstream antioxidant enzymes caused by
25 was based on Nrf2 activation. A subsequent qRT-PCR assay also
confirmed the results that 25 could up-regulate the mRNA tran-
scriptions of Nrf2, HO-1, NQO1, GCLC and GCLM in a concentration
dependent manner (Fig. 3F). All these data indicated that 25 pro-
tected L02 cells through activating Nrf2-ARE antioxidant signaling
pathway.
Induction of the transcription of antioxidant genes by activating
the Nrf2-ARE cytoprotective pathway requires the transfer of Nrf2
from the cytoplasm to the nucleus. Therefore, we explored whether

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1383
Fig. 3. (A) Time-dependent expression of Nrf2, HO-1 and NQO1 affected by 25. After treatment with 20 mM 25 for 0, 2, 4, 8, 12, 24 h, cell lysates were prepared from L02 cells and
subjected to western blot analysis. (B) Concentration-dependent expression of Nrf2, HO-1 and NQO1 affected by 25. After treatment with 25 at different concentrations for 24 h, cell
lysates were prepared from L02 cells and subjected to western blot analysis. Densitometric analysis was performed for each protein. All results were normalized to actin expression
and are presented as the mean SD of three individual experiments. The data were analyzed using ImageJ 1.44p. (C) The mRNA transcription of Nrf2 after treatment with Nrf2
siRNA and 25. L02 cells were treated with Nrf2 siRNA (50 nM), 25 (20
siRNA and 25 (20 M). The L02 cells were treated with 25 (20 M), Nrf2 siRNA (50 nM) or 25 (20
as the blank control. (E) The mRNA transcription of Nrf2 and its downstream markers including NQO1, GCLM and HO-1 after treatment with Nrf2 siRNA and 25. L02 cells were
treated with Nrf2 siRNA (50 nM), 25 (20 M) or Nrf2 siRNA (50 nM) and 25 (20 M) separately. All genes transcription was determined by qRT-PCR. (F) Quantitative real-time PCR
analysis of Nrf2, HO-1, NQO1, GCLC and GCLM in L02 cells. All genes transcription levels were determined after treatment with different concentrations (0, 2.5, 5, 10, 20, 40 M) of 25
mM) or Nrf2 siRNA (50 nM) and 25 (20
mM). (D) Western blot analysis of Nrf2 protein after exposure to Nrf2
m
m
m
M) plus Nrf2 siRNA (50 nM). Additional L02 cells were treated with DMSO for use
m
m
m
for 12 h. GAPDH was used as the control for normal expression of these genes. The values shown are the means SDs (n ¼ 3). *p < 0.05, **p < 0.01, ***p < 0.001, statistically
significant difference from the non-treated blank control group.

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L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
Fig. 4. Immunofluorescence staining of Nrf2 in L02 cells. Nrf2 and nuclei were labeled with DyLight 488 and DAPI, respectively. Green, Nrf2; blue, nuclei. Compound 25 (5 and
20 M, respectively) was added and compared to the untreated control group. Cells treated with tBHQ (80 M) served as the positive control (scale bar 20 m). (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article.)
m
m
m
Fig. 5. Cytoprotection by 25 against damage induced by 8 mM APAP. (A) The mRNA transcription of Nrf2 after treatment of Nrf2 siRNA and 25. The L02 cells were treated with Nrf2
siRNA (50 nM), 25 (20
were treated with 25 (20
were plated in a 96-well plate for 12 h and subsequently treated with 25 at 5, 10 and 20
(50 nM) and evaluated after the administration of compound 25 at 20
m
M) or Nrf2 siRNA (50 nM) and 25 (20
M), Nrf2 siRNA (50 nM) or 25 (20
m
m
M), respectively. (B) Western blot analysis of Nrf2 protein after exposure to Nrf2 siRNA and 25 (20
M) plus Nrf2 siRNA (50 nM). Additional L02 cells were treated with DMSO for use as the blank control. (C) L02 cells
M separately for another 24 h. An additional group was pre-treated with siRNA Nrf2
M. After the growth medium was replaced with medium containing APAP (8 mM) and incubation was
mM). The L02 cells
m
m
m
continued for 24 h, the cell viability was measured by the MTT assay. tBHQ and DMF were used as positive controls. All data represent the mean SD of three independent ex-
periments. *p < 0.05, **p < 0.01, statistically significant difference from the non-treated blank control group.
injury in mice and prevented APAP hepatotoxicity by reducing the
expression of liver damage markers including ALT and AST in
mouse serum.
Next, western blot and qRT-PCR analyses were used to confirm
whether the liver protection by 25 was based on Nrf2-ARE pathway
activation. The protein expression levels of Nrf2, HO-1 and NQO1 at

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1385
Fig. 6. (A&B) Protective effects of 25 on APAP-intoxicated mice. Mice received an intraperitoneal injection of 25 at 10 or 50 or 100 mg/kg (n ¼ 10) once a day for seven days. The
control group received the same volume of saline. After the seventh gavage of 25 or saline, the animals received a single injection of APAP (300 mg/kg). All mice were sacrificed at
24 h after APAP injection to obtain blood and liver samples. ALT (A) and AST (B) levels in mice serum. (C&D) Effects of 25 on the expression of Nrf2, HO-1 and NQO1 in APAP-
intoxicated mice. The protein levels of Nrf2, HO-1 and NQO1 (C) were measured by western blot analysis as described in the materials and methods section. The protein
expression levels were normalized to those of b-actin. The gene transcription levels of Nrf2, HO-1, NQO1 and GCLM (D) were measured by qRT-PCR analysis as described in the
materials and methods section. The gene transcription levels were normalized to those of GAPDH. (E&F&G) Effects of 25 on the activities and protein expression of hepatic
antioxidant enzymes in APAP-intoxicated mice. The contents of GSH (E), GST (F) and SOD (G) were measured as described in the materials and methods section. (H) Representative
histological images of liver sections stained with hematoxylin and eosin (H&E) to evaluate the protective effects of 25. The mice were intraperitoneally injected with 25 at 10, 50 or
100 mg/kg once a day for seven days. The control group received the same volume of saline. After the seventh gavage of 25 or saline, the animals received a single injection of APAP
(300 mg/kg). All mice were sacrificed at 24 h after APAP injection to perform H&E staining. Typical images were chosen from each experimental group. The original magnification
was 10 ꢂ 20. All data represent the means SD. Significant differences were presented as ***p< 0.001, **p < 0.01 or *p < 0.05 compared with the APAP group.
different concentrations of 25 were significantly higher than those
in the model group (Fig. 6C). Subsequently, qRT-PCR was performed
to examine the relative levels of Nrf2, HO-1, NQO1 and GCLM mRNA
transcripts (Fig. 6D). Treatment with 25 (50 mg/kg and 100 mg/kg)
elevated the relative levels of the Nrf2, HO-1, NQO1 and GCLM
mRNA transcripts in a concentration-dependent manner. Thus, 25

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L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
could activate the Nrf2-ARE pathway at both the protein and mRNA
transcription levels, resulting in ideal therapeutic effects on the
APAP-induced liver injury mouse model.
caused by differences in their spatial conformations.
Next, we performed further biological activity assessments and
found that 25 could increase the transcription of Nrf2 downstream
target genes including GCLM, NQO1 and HO-1. Compound 25
showed almost no cytotoxicity in normal liver cells (L02). Addi-
tionally, 25 could protect the liver against APAP-induced acute liver
damage in mice via activating the Nrf2-ARE pathway. Compound
25 up-regulated antioxidant enzymes including GSH, GST and SOD
and down-regulated the liver injury markers of AST and ALT in
serum.
However, as a potent Nrf2 activator, 25 exhibits some indis-
tinctive issues that need to be further validated. According to our
experiment and research experience, we know that the absence of
a, b-unsaturated carbonyl groups excludes the possibility of a
Michael addition reaction activating the Nrf2-ARE pathway
[39e41].
Additionally, the regulation of compound 25 on antioxidant
enzymes (GSH, GST and SOD), which eliminate ROS and alleviate
oxidative stress, was determined (Fig. 6E and F & G). The induction
fold of antioxidases in the APAP-challenged model group was
significantly decreased. Pretreatment with different concentrations
of 25 (100 mg/kg, 50 mg/kg, 10 mg/kg) prior to APAP treatment
significantly enhanced the release of GSH, GST and SOD in a
concentration-dependent manner. As shown in Fig. 6H, histological
analysis of liver tissue was performed using hematoxylin and eosin
(H&E) staining. In the control group, the hepatocytes presented
central rounded vesicular nucleus, while APAP triggered severe
hepatocyte damage including massive cell necrosis (shown in APAP
model group). Pretreatment of 25 reduced the APAP-induced liver
damage in a concentration-dependent manner. Treatment with 25
significantly reduced the number of necrotic cells. The hepatic ar-
chitecture following treatment with 25 at 100 mg/kg was approx-
imately normal to that in the control group. Together, these results
indicated that the therapeutic effects of 25 on APAP-induced acute
liver injury in a mouse model were achieved by activating the Nrf2-
ARE pathway. Compound 25 provides a promising therapeutic
strategy to prevent APAP-induced liver injury.
Above all, SAR studies on the 1,2,4-oxadiazole core provided
guidance for subsequent structural modifications. The in vivo re-
sults regarding resistance to liver injury indicate that 25 is a
promising novel hepato-protective agent that activates the Nrf2-
ARE pathway. Compound 25 could be regarded as a potent Nrf2
activator for the treatment of APAP-induced liver injury.
4. Experimental section
3. Conclusion and discussion
4.1. General information
Activating Nrf2 to resist environmental stress is generally
believed to be a promising strategy [35]. Nrf2 up-regulates the
transcriptions of several antioxidant and electrophile detoxifying
genes via AREs present in their transcriptional promoters [36].
Activators of the Keap1-Nrf2-ARE pathway can induce the expres-
sion of phase II antioxidant enzymes, which further exert chemo-
preventive effects against a series of inflammation-related diseases
including uncontrolled inflammation, cancer, neurodegenerative
diseases, cardiovascular diseases and aging [37,38]. Therefore,
discovering efficient activators of the Nrf2-ARE pathway is a
promising approach and is urgently required.
We reported a series of novel Nrf2 activators with a 1,2,4-
oxadiazole core and investigated the primary SAR of ring A. All
analogues had three parts, including rings A, B and C. Among the
derivatives, compound 1 (wherein p-fluorine was substituted in
phenyl ring A) showed potent ARE-inducing activity [32]. Next, we
investigated the changes in ARE-inducing activity when halogen
substituents were introduced into phenyl ring A in various com-
binations. We synthesized sixteen compounds and evaluated their
Nrf2-ARE inducing activities via the luciferase reporter gene assay.
Among these compounds, 6 (m, p-F in ring A) showed more potent
ARE-inducing activity than 1. Based on compound 6 with the
optimal ring A structure (m, p-F), we subsequently modified ring C
and obtained twenty-five derivatives. Compound 25 (304-
All reagents were purchased from commercial organizations.
Organic solvent was concentrated in a rotary evaporator (Büchi
Rotavapor) under reduced pressure below 55 ꢁC degrees. Reactions
were monitored by thin-layer chromatography (TLC) on 0.25 mm
silica gel plates (GF254) and visualized under UV light. Melting
points were measured with a Melt-Temp II apparatus. The 1H NMR
and 13C NMR spectra were recorded on a Bruker AV-300 instrument
using deuterated solvents with tetramethylsilane (TMS) as the in-
ternal standard. The purity (ꢃ95%) of the compounds was verified
by HPLC performed on an Agilent C18 (4.6 mm ꢂ 150 mm, 3.5
mm)
column using a mixture of solvent methanol/water at a flow rate of
0.5 mL/min and peak detection at 254 nm under UV. ESI-mass
spectra and high-resolution mass spectra (HRMS) were recorded
on a Water Q-Tof micro mass spectrometer. IR spectra were deter-
mined on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film.
4.2. Synthesis
4.2.1. Compounds 1e16 were synthesized using a similar method
4.2.1.1. 1H-benzo[d]imidazole-5-carbonitrile (43).
3,4-diaminobenzonitrile (42, 2.5 g; 18.75 mmol; 1.0 equiv) was
dissolved in 5 M aqueous HCl (110 mL). The mixture was heated at
105 ꢁC to reflux for 12 h. After stopping the reaction, the mixture
was cooled to room temperature, basified with aqueous ammonia
and stored overnight. The generated solid product was collected by
filtration and washed with ice water. The products could be used
without further purification. Yield: 2.5 g (94.0%). 1H NMR
methylpyridineyl) at 80 mM exerted an optimal inducing activity
that was 59.55-fold higher than that of 1 and 6 at the same con-
centration. The positions of the substituents on the phenyl or pyr-
idine group did affect the Nrf2-ARE inducing activity. In contrast to
ring A, electron-donating substituents in ring C promoted the Nrf2-
ARE inducing activity, while electron-withdrawing substituents
impaired this activity. Meanwhile, either no substituents or mul-
tiple substituents in ring C resulted in a decrease in ARE-inducing
activity.
The protective effect of compound 25 on hepatocyte L02 cells
and mouse liver tissues was studied in this paper. In addition, 25
exhibited a four-fold higher solubility compared to 1. Despite the
similar solubility of 24, 25 and 26, there were significant differences
in ARE-inducing activities of them. This result may have been
(300 MHz, DMSO)
J ¼ 4.17 Hz, 1H), 7.45 (d, J ¼ 8.34 Hz, 1H). MS m/z: 144.2 [M þ H]
d
12.47 (s, 1H), 8.34 (s, 1H), 8.03 (s, 1H), 7.62 (þd,
.
4.2.1.2. (Z)-N0-Hydroxy-1H-benzo[d]imidazole-5-carboximidamide
(44). Compound 43 (2.5 g; 17.5 mmol; 1.0 equiv) was dissolved in
EtOH (60 mL) treated with K2CO3 (4.32 g; 31.5 mmol; 1.8 equiv) and
H2NOH$HCl (2.25 g; 32.4 mmol; 1.8 equiv) and heated to reflux for
12 h. The mixture was diluted with diethyl ether when cooled to
room temperature. The product was collected by filtration, washed
with water, and dried under an infrared lamp. The compound was
used without further purification. Yield: 1.54 g (50%). 1H NMR

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1387
(300 MHz, DMSO)
d
13.10 (s, 1H), 10.86 (s, 1H), 8.50 (s, 1H), 8.32 (s,
4.2.1.8. 3-(1H-benzo[d]imidazol-6-yl)-5-(3,4-2fluorophenyl)-1,2,4-
oxadiazol (6). Yield 32%. White solid; m. p. 220e221 ꢁC. 1H NMR
2H), 8.03 (s, 1H), 7.74 (d, J ¼ 4.23 Hz, 1H), 7.58 (d, J ¼ 4.23 Hz, 1H).
þ
MS: 177.3 [M þH]
.
(300 MHz, DMSO)
d 12.79 (s, 1H), 8.39 (s, 1H), 8.30e8.19 (m, 2H),
8.19e8.04 (m, 1H), 8.02e7.90 (m, 1H), 7.82e7.66 (m, 2H). IR (cmꢀ1
,
KBr film): 3441 (ꢀNH), 1623, 1400, 1101, 754. HRMS (ESI): calcd for
4.2.1.3. 3-(1H-Benzo[d]imidazol-6-yl)-5-(4-fluorophenyl)-1,2,4-
oxadiazole (1). 4-fluoro benzoic acid (0.16 g, 1.14 mmol, 1.0 equiv)
was dissolved in DMF (3.0 mL) treated with carbonyldiimidazole
(0.184 g, 1.14 mmol, 1.0 equiv) and stirred at room temperature for
1 h. Compound 44 (0.2 g, 1.14 mmol, 1.0 equiv) was added in the
mixed solution and an additional 3 mL of DMF was added to the
system. Next, the temperature was increased to 110 ꢁC, and stirring
was continued for 12e18 h. After cooling, the mixture was diluted
using water and saturated aqueous NaHCO3 solution. The generated
solid product was collected by filtration and washed with saturated
aqueous NaHCO3 solution. Yield 26.2%. White solid; m. p.
C
15H9F2N4O [M þ H]þ 299.0739, found 299.0743. HPLC (80%
methanol in water): tR ¼ 8.67 min, 97.53%.
4.2.1.9. 3-(1H-benzo[d]imidazol-6-yl)-5-(2,3-2fluorophenyl)-1,2,4-
oxadiazol (7). Yield 33%. White solid; m. p. 219e220 ꢁC. 1H NMR
(300 MHz, DMSO)
(m, 1H), 7.95 (dd, J ¼ 8.44, 1.34 Hz,1H), 7.90e7.74 (m, 2H), 7.57e7.43
(m, 1H). 13C NMR (75 MHz, DMSO)
169.22, 159.98, 159.96, 158.41,
d 12.47 (s, 1H), 8.40 (s, 1H), 8.32 (s, 1H), 8.11e8.01
d
158.38, 156.78, 155.03, 155.01, 122.84, 122.72, 122.52, 122.40, 119.98,
119.87, 119.66, 119.55, 117.36, 117.34, 117.01, 116.99. IR (cmꢀ1, KBr
film): 3448 (ꢀNH), 1629, 1400, 1125, 989. HRMS (ESI): calcd for
186e187 ꢁC. 1H NMR (300 MHz, DMSO)
8.30e8.27 (m, 3H), 7.95 (d, J ¼ 8.22 Hz, 1H), 7.78 (s, 1H), 7.55e7.49
(m, 2H). 13C NMR (125 MHz, DMSO)
174.128, 168.867, 164.780 (d,
d 12.80 (s, 1H), 8.40 (s, 1H),
C
15H9F2N4O [M þ H]þ 299.0739, found 299.074. HPLC (80% meth-
anol in water): tR ¼ 9.29 min, 97.86%.
d
J ¼ 251.13 Hz), 143.950, 130.581 (d, J ¼ 9.25 Hz), 120.731, 120.150,
119.626, 116.609 (d, J ¼ 22.34 Hz), 115.861, 114.816. IR (cmꢀ1, KBr
film): 3419 (ꢀNH), 1620, 1095, 1023, 805. HRMS (ESI): calcd for
4.2.1.10. 3-(1H-benzo[d]imidazol-6-yl)-5-(2-chloro-4-fluorophenyl)-
1,2,4-oxadiazol (8). Yield 24.9%. White solid; m. p. 277e278 ꢁC. 1H
NMR (300 MHz, DMSO)
d 12.80 (s, 1H), 8.55e8.22 (m, 3H),
C
15H10FN4O [M þ H]þ 281.0833, found 281.0843. HPLC (80%
8.01e7.80 (m, 3H), 7.56e7.50 (m, 1H), 13C NMR (75 MHz, DMSO)
methanol in water): tR ¼ 7.43 min, 98.88%.
d
172.92, 168.64, 165.65, 162.27, 144.13, 134.25, 134.14, 134.12,
134.00, 119.65, 119.60, 119.02, 118.68, 115.74, 115.45. IR (cmꢀ1, KBr
4.2.1.4. 3-(1H-benzo[d]imidazol-6-yl)-5-(4-chlorophenyl)-1,2,4-
film): 3448 (ꢀNH), 1637, 1400, 1089, 989. HRMS (ESI): calcd for
oxadiazol (2). Yield 23.8%. White solid; m. p. 263e264 ꢁC. 1H NMR
C
15H9ClFN4O [M þ H]þ 315.0443, found 315.0449. HPLC (80%
(300 MHz, DMSO)
d 12.79 (s, 1H), 8.51e8.48 (m, 2H), 8.22 (d,
methanol in water): tR ¼ 8.62 min, 96.11%.
J ¼ 8.61 Hz, 2H), 8.11e8.09 (m, 1H), 7.94e7.76 (m, 3H). 13C NMR
(75 MHz, DMSO)
d 175.075, 168.899, 145.221, 144.105, 133.199,
4.2.1.11. 3-(1H-benzo[d]imidazol-6-yl)-5-(3-chloro-4-fluorophenyl)-
1,2,4-oxadiazol (9). Yield 13%. White solid; m. p. 253e254 ꢁC. 1H
129.489, 129.489, 127.845, 127.845, 123.461, 120.853, 119.960,
118.335, 114.496, 112.632, 111.092. IR (cmꢀ1, KBr film): 3435 (ꢀNH),
1611, 1106, 1021, 805. HRMS (ESI): calcd for C15H10ClN4O [M þ H]þ
297.0538, found 297.0535. HPLC (80% methanol in water):
tR ¼ 15.44 min, 97.40%.
NMR (300 MHz, DMSO)
d
7.93e7.76 (m, 5H), 7.57 (dd, J ¼ 5.6, 2.0 Hz,
1H), 7.31 (dd, J ¼ 8.9, 7.5 Hz, 1H). 13C NMR (75 MHz, DMSO)
d 173.68,
162.11, 158.75, 144.62, 144.37, 138.68, 138.11, 130.69, 130.13, 129.73,
129.61,129.47, 128.25,121.80,121.17, 120.25, 118.91,118.62. IR (cmꢀ1
,
KBr film): 3448 (ꢀNH), 1637, 1400, 1091, 988. HRMS (ESI): calcd for
C
15H9ClFN4O [M þ H]þ 315.0443, found 315.0449. HPLC (80%
4.2.1.5. 3-(1H-benzo[d]imidazol-6-yl)-5-(4-bromophenyl)-1,2,4-
oxadiazol (3). Yield 0.266 g (34.4%). White solid; m. p. 244e245 ꢁC.
methanol in water): tR ¼ 11.53 min, 97.11%.
1H NMR (300 MHz, DMSO)
d 12.79 (s, 1H), 8.40 (s, 1H), 8.33 (s, 1H),
4.2.1.12. 3-(1H-benzo[d]imidazol-6-yl)-5-(2,4-2fluorophenyl)-1,2,4-
oxadiazol (10). Yield 13%. White solid; m. p. 258e259 ꢁC. 1H NMR
8.16 (d, J ¼ 8.52 Hz, 2H), 7.95 (d, J ¼ 8.82 Hz, 1H), 7.90 (d, J ¼ 8.49 Hz,
2H), 7.78 (d, J ¼ 8.04 Hz, 1H). IR (cmꢀ1, KBr film): 3417 (ꢀNH), 1611,
1098, 1029, 799. HRMS (ESI): calcd for C15H10BrN4O [M þ H]þ
341.0033, found 341.0023. HPLC (80% methanol in water):
tR ¼ 17.20 min, 98.30%.
(300 MHz, DMSO) d 12.78 (s, 1H), 8.39e8.26 (m, 3H), 7.93e7.41 (m,
3H), 7.39e7.36 (m,1H). IR (cmꢀ1, KBr film): 3448 (ꢀNH),1623, 1400,
1090, 959. HRMS (ESI): calcd for C15H9F2N4O [M þ H]þ 299.0739,
found 299.0743. HPLC (80% methanol in water): tR ¼ 6.68 min,
98.22%.
4.2.1.6. 3-(1H-benzo[d]imidazol-6-yl)-5-(2-fluorophenyl)-1,2,4-
oxadiazol (4). Yield 38.9%. Brown solid; m. p. 233e236 ꢁC. 1H NMR
4.2.1.13. 3-(1H-benzo[d]imidazol-6-yl)-5-(3-chlorophenyl)-1,2,4-
(300 MHz, DMSO) d 8.38 (s,1H), 8.33 (s,1H), 8.30e8.20 (m,1H), 7.94
oxadiazol (11). Yield 13.3%. White solid; m. p. 258e259 ꢁC. 1H NMR
(d, J ¼ 7.27 Hz, 1H), 7.86e7.73 (m, 2H), 7.62e7.44 (m, 2H). 13C NMR
(300 MHz, DMSO)
d
8.51e8.32 (m, 2H), 8.18 (s, 2H), 8.04e7.79 (m,
174.33, 169.46, 144.57, 134.59,
(75 MHz, DMSO)
d 169.20, 162.11, 158.70, 144.86, 141.08, 139.09,
4H). 13C NMR (75 MHz, DMSO)
d
136.01, 135.90, 131.30, 125.91, 125.86, 121.16, 119.85, 117.84, 117.56,
116.56, 115.35. IR (cmꢀ1, KBr film): 3446 (ꢀNH), 1622, 1400, 1091,
750. HRMS (ESI): calcd for C15H10FN4O [M þ H]þ 281.0833, found
281.0835. HPLC (80% methanol in water): tR ¼ 6.01 min, 95.11%.
133.43, 131.96, 127.79, 127.00, 125.81, 121.28, 119.95, 116.45, 116.37,
115.40, 115.36. IR (cmꢀ1, KBr film): 3447 (ꢀNH), 1629, 1400, 1092,
987. HRMS (ESI): calcd for C15 H10ClN4O [M þ H]þ 297.0538, found
297.0546. HPLC (80% methanol in water): tR ¼ 10.57 min, 98.23%.
4.2.1.7. 3-(1H-benzo[d]imidazol-6-yl)-5-(3-fluorophenyl)-1,2,4-
oxadiazol (5). Yield 54%. White solid; m. p. 232e233 ꢁC. 1H NMR
4.2.1.14. 3-(1H-benzo[d]imidazol-6-yl)-5-(2,5-2fluorophenyl)-1,2,4-
oxadiazol (12). Yield 47.6%. White solid; m. p. 261e262 ꢁC. 1H NMR
(300 MHz, DMSO)
3H), 7.82e7.66 (m, 2H), 7.66e7.55 (m, 1H). 13C NMR (75 MHz,
DMSO) 174.52, 174.48, 169.49, 164.27, 161.01, 144.56, 132.41,
132.30,125.95, 125.84,124.66,124.62,120.80,120.52,115.20,114.88.
IR (cmꢀ1, KBr film): 3447 (ꢀNH), 1629, 1400, 1102, 754. HRMS (ESI):
calcd for C15H10FN4O [M þ H]þ 281.0833, found 281.0839. HPLC
(80% methanol in water): tR ¼ 7.57 min, 95.73%.
d
12.79 (s, 1H), 8.40e8.33 (m, 2H), 8.10e7.90 (m,
(300 MHz, DMSO)
d
12.79 (s, 1H), 8.39e8.32 (m, 2H), 8.06e7.78 (m,
169.17, 159.94,
2H), 7.72e7.58 (m, 3H). 13C NMR (75 MHz, DMSO)
d
d
159.91, 158.36, 158.33, 156.73, 156.69, 154.99, 154.96, 144.55, 122.77,
122.65, 122.45, 122.33, 121.27, 119.91, 119.80, 119.60, 119.48, 117.26,
117.25, 116.91, 116.89, 113.58, 113.46, 113.39, 113.27. IR (cmꢀ1, KBr
film): 3448 (ꢀNH), 1629, 1400, 1088, 990. HRMS (ESI): calcd for
C
15H9F2N4O [M þ H]þ 299.0739, found 299.0743. HPLC (80%

1388
L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
methanol in water): tR ¼ 6.64 min, 96.08%.
8.85 (s, 1H), 8.55 (d, J ¼ 3.45 Hz, 1H), 8.01 (d, J ¼ 7.41 Hz, 1H),
7.42e7.37 (m, 1H), 6.00 (s, 2H). EI-MS m/z: 136.1 [M]þ. HPLC (80%
methanol in water): tR ¼ 4.87 min, 95.38%.
4.2.1.15. 3-(1H-benzo[d]imidazol-6-yl)-5-(2-bromo-4-fluorophenyl)-
1,2,4-oxadiazol (13). Yield 32.9%. White solid; m. p. 233e234 ꢁC. 1H
NMR (300 MHz, DMSO)
8.10e7.96 (m, 2H), 7.94e7.76 (m, 2H), 7.58e7.18 (m, 1H). 13C NMR
(75 MHz, DMSO) 169.14, 165.87, 162.47, 144.66, 134.89, 134.76,
d
12.80 (s, 1H), 8.55e8.19 (m, 3H),
4.2.2.4. (Z)-N0-Hydroxy-5-fluoropyridine-2-carboxamidine
Yield: 1.2 g (37.7%). m. p. 186e187 ꢁC. 1H NMR (300 MHz, DMSO)
(48).
d
d
9.96 (s, 1H), 8.55 (d, J ¼ 2.88 Hz, 1H), 7.93e7.88 (m, 1H), 7.78e7.71
134.04, 131.03, 125.45, 122.53, 122.20, 119.66, 118.26, 116.50, 116.21.
IR (cmꢀ1, KBr film): 3446 (ꢀNH), 1628, 1400, 1091, 988. HRMS (ESI):
calcd for C15H9BrFN4O [M þ H]þ 358.9938, found 358.9946. HPLC
(80% methanol in water): tR ¼ 8.73 min, 95.34%.
(m, 1H), 5.85 (s, 2H). EI-MS m/z: 154.1 [M]þ. HPLC (80% methanol in
water): tR ¼ 7.57 min, 98.58%.
4.2.2.5. (Z)-N0-Hydroxy-6-methylpyridine-2-carboxamidine
Yield: 1.7 g (53%). m. p. 179e180 ꢁC. 1H NMR (300 MHz, DMSO)
(49).
4.2.1.16. 3-(1H-benzo[d]imidazol-6-yl)-5-(3-bromo-4-fluorophenyl)-
1,2,4-oxadiazol (14). Yield 5.4%. White solid; m. p. 268e269 ꢁC. 1H
d
9.74 (s, 1H), 8.71 (d, J ¼ 1.65 Hz, 1H), 7.89 (dd, J ¼ 2.10, 8.07 Hz, 1H),
7.24 (d, J ¼ 8.10 Hz, 1H), 5.93 (s, 2H), 2.46 (s, 3H). EI-MS m/z: 150.1
NMR (300 MHz, DMSO)
1H), 8.30e8.29 (m, 1H), 8.02 (s, 1H), 7.97e7.78 (m, 1H), 7.56 (s, 1H),
7.16 (s, 1H). 13C NMR (75 MHz, DMSO)
169.15, 165.86, 162.48,
d 12.81 (s, 1H), 8.58 (s, 1H), 8.41e8.32 (m,
[M]þ. HPLC (80% methanol in water): tR ¼ 5.87 min, 97.61%.
d
4.2.2.6. (Z)-N0-Hydroxy-pyrazine-2-carboxamidine (50). Yield: 1.4 g
144.61, 134.89, 134.76, 134.04, 131.04, 125.45, 122.53, 122.20, 119.65,
118.25, 116.50, 116.21. IR (cmꢀ1, KBr film): 3448 (ꢀNH), 1637, 1400,
1074, 988. HRMS (ESI): calcd for C15H9BrFN4O [M þ H]þ 358.9938,
found 358.9942. HPLC (80% methanol in water): tR ¼ 5.82 min,
98.86%.
(42.6%). m. p. 219e220 ꢁC. 1H NMR (300 MHz, DMSO)
d 10.19 (s, 1H),
8.83 (d, J ¼ 4.86 Hz, 2H), 7.49 (t, J ¼ 4.86 Hz, 1H), 5.84 (s, 2H). EI-MS
m/z: 137.1[M]þ. HPLC (80% methanol in water): tR ¼ 7.15 min,
98.53%.
4.2.2.7. (Z)-N0-Hydroxy-pyrimidine-2-carboxamidine
Yield: 1.5 g (45.7%). m. p. 211e212 ꢁC. 1H NMR (300 MHz, DMSO)
(51).
4.2.1.17. 3-(1H-benzo[d]imidazol-6-yl)-5-(2,4,5-3fluorophenyl)-
1,2,4-oxadiazol (15). Yield 7%. White solid; m. p. 216e217 ꢁC. 1H
d
10.19 (s, 1H), 8.83 (d, J ¼ 4.86 Hz, 2H), 7.51 (t, J ¼ 4.86 Hz, 1H), 5.84
NMR (300 MHz, DMSO)
7.94e7.77 (m, 1H), 7.75 (s, 1H), 7.20 (s, 1H). 13C NMR (75 MHz,
DMSO) 169.68, 158.28, 158.23, 154.95, 154.89, 144.65, 138.71,
d
8.36e8.26 (m, 2H), 8.13e8.07 (m, 1H),
(s, 2H). EI-MS m/z: 137.1[M]þ. HPLC (80% methanol in water):
tR ¼ 8.34 min, 99.25%.
d
138.37, 137.63, 130.02, 129.91, 129.68, 121.36, 121.18, 120.31, 113.03,
112.79, 112.70, 112.68. IR (cmꢀ1, KBr film): 3448 (ꢀNH), 1638, 1400,
1092, 988. HRMS (ESI): calcd for C15H8F3N4O [M þ H]þ 317.0645,
found 317.0644. HPLC (80% methanol in water): tR ¼ 4.91 min,
95.46%.
4.2.2.8. (Z)-N0-Hydroxy-1H-indole-4-carboxamidine
Yield: 1.1 g (35.7%). m. p. 189e190 ꢁC. 1H NMR (300 MHz, DMSO)
(52).
d
11.19 (s,1H), 9.57 (s,1H), 7.42e7.23 (m, 3H), 7.11e7.05 (m,1H), 6.82
(d, J ¼ 2.49 Hz, 1H), 5.68 (s, 2H). EI-MS m/z: 174.1[M]þ. HPLC (80%
methanol in water): tR ¼ 7.51 min, 96.65%.
4.2.1.18. 3-(1H-benzo[d]imidazol-6-yl)-5-(2,4-2fluoro-5-
chlorophenyl)-1,2,4-oxadiazol (16). Yield 10.3%. White solid; m. p.
4.2.2.9. (Z)-N0-Hydroxy-naphthalene-2-carboxamidine
Yield: 1.3 g (42.7%). m. p. 206e207 ꢁC. 1H NMR (300 MHz, DMSO)
8.01e7.85 (m, 3H), 7.80 (s, 1H), 7.77e7.68 (m, 2H), 7.69e7.48 (m,
5H), 4.59 (s, 2H), 3.87 (d, J ¼ 13.80 Hz, 1H), 3.38 (d, J ¼ 13.80 Hz, 1H).
EI-MS m/z: 185.1[M]þ. HPLC (80% methanol in water):
tR ¼ 5.34 min, 95.64%.
(53).
217e218 ꢁC. 1H NMR (300 MHz, DMSO)
d
12.80 (s, 1H), 8.57 (s, 1H),
d
8.38 (s,1H), 8.16 (s,1H), 8.01e7.69 (m,1H), 7.50 (s,1H). IR (cmꢀ1, KBr
film): 3449 (ꢀNH), 1637, 1400, 1090, 987. 13C NMR (75 MHz, DMSO)
d
169.26, 158.26, 154.88, 151.78, 137.83, 137.75, 130.32, 120.12,
120.07, 119.11, 118.77, 118.75, 114.16, 113.80. HRMS (ESI): calcd for
C
15H8ClF2N4O [M þ H]þ 332.0645, found 332.0644. HPLC (80%
4.2.2.10. (Z)-N0-hydroxy-[1,10-biphenyl]-4-carboximidamide
Yield: 1.0 g (33.7%). m. p. 146e147 ꢁC. 1H NMR (300 MHz, DMSO)
(54).
methanol in water): tR ¼ 4.92 min, 95.53%.
d
9.69 (s, 1H), 7.78e7.65 (m, 6H), 7.49e7.34 (m, 3H), 5.86 (s, 2H). EI-
4.2.2. Intermediates 45~76 were synthesized using a similar
method
MS m/z: 211.1[M]þ. HPLC (80% methanol in water): tR ¼ 9.53 min,
96.53%.
4.2.2.1. (Z)-N0-Hydroxy-pyridine-2-carboxamidine
(45).
2-cyanopyridine (2.5 g, 24.01 mmol) was dissolved in EtOH (60 mL)
treated with K2CO3 (5.97 g; 43.22 mmol; 1.8 equiv) and H2NOH$HCl
(3.0 g; 43.22 mmol; 1.8 equiv) and heated to reflux for 12 h. The
mixture was diluted with diethyl ether when it was cooled to room
temperature. The product was collected by filtration, washed with
water, and dried under an infrared lamp. The compound was used
without further purification. Yield: 1.5 g (45.5%). m. p. 184e185 ꢁC.
4.2.2.11. (Z)-N0-hydroxy-4-methylbenzimidamide (55). Yield: 1.4 g
(43.7%). m. p. 193e194 ꢁC. 1H NMR (300 MHz, DMSO)
d 9.55 (s, 1H),
7.55 (d, J ¼ 8.16 Hz, 2H), 7.16 (d, J ¼ 7.98 Hz, 2H), 5.75 (s, 2H), 2.30 (s,
3H). EI-MS m/z: 149.1[M]þ. HPLC (80% methanol in water):
tR ¼ 7.54 min, 97.86%.
4.2.2.12. (Z)-4-Acetyl-N0-hydroxybenzimidamide (56). Yield: 1.5 g
1H NMR (300 MHz, DMSO)
d
9.93 (s, 1H), 8.56e8.54 (m, 1H),
(48%). m. p. 194e195 ꢁC. 1H NMR (300 MHz, DMSO)
d 7.85 (d,
7.87e7.77 (m, 2H), 7.42e7.37 (m, 1H), 5.84 (s, 2H). EI-MS m/z: 136.1
J ¼ 7.51 Hz, 2H), 7.38 (d, J ¼ 7.51 Hz, 2H), 4.59 (s, 2H), 3.83 (d,
J ¼ 13.80 Hz, 1H), 3.37 (d, J ¼ 13.80 Hz, 1H), 2.51 (s, 3H). EI-MS m/z:
177.1[M]þ. HPLC (80% methanol in water): tR ¼ 6.75 min, 96.45%.
[M]þ. HPLC (80% methanol in water): tR ¼ 6.57 min, 97.68%.
4.2.2.2. (Z)-N0-Hydroxy-pyridine-4-carboxamidine (46). Yield: 1.3 g
(39.5%). m. p. 185e186 ꢁC. 1H NMR (300 MHz, DMSO)
d
10.06 (s, 1H),
4.2.2.13. (Z)-2-Fluoro-N0-hydroxybenzimidamide (57). Yield: 1.6 g
8.58e8.55 (m, 2H), 7.65e7.62 (m, 2H), 6.03 (s, 2H). EI-MS m/z: 136.1
(50.3%). m. p. 146e147 ꢁC. 1H NMR (300 MHz, DMSO)
d 9.68 (s, 1H),
[M]þ. HPLC (80% methanol in water): tR ¼ 5.57 min, 96.78%.
7.52e7.38 (m, 2H), 7.29e7.17 (m, 2H), 5.81 (s, 2H). EI-MS m/z: 153.1
[M]þ. HPLC (80% methanol in water): tR ¼ 5.65 min, 98.85%.
4.2.2.3. (Z)-N0-Hydroxy-pyridine-3-carboxamidine (47). Yield: 1.6 g
(48.6%). m. p. 187e188 ꢁC. 1H NMR (300 MHz, DMSO)
d
9.85 (s, 1H),
4.2.2.14. (Z)-3-Fluoro-N0-hydroxybenzimidamide (58). Yield: 1.4 g

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1389
(44%). m. p. 180e181 ꢁC. 1H NMR (300 MHz, DMSO)
d
9.79 (s, 1H),
4.2.3. Compound 17e41 were synthesized using a similar method
7.65e7.37 (m, 3H), 7.26e7.16 (m, 1H), 5.90 (s, 2H). EI-MS m/z: 153.1
4.2.3.1. 5-(3,4-difluorophenyl)-3-(pyridin-2-yl)-1,2,4-oxadiazole
(17). 3,4-difluorobenzoic acid (0.46 g, 2.92 mmol, 1.0 equiv) was
dissolved in DMF (3.0 mL) treated with carbonyldiimidazole (0.48 g,
2.92 mmol, 1.0 equiv) and stirred at room temperature for 1 h. 45
(0.4 g, 2.92 mmol, 1.0 equiv) was added in the mixed solution and
another 3 mL DMF was added in the system. Then the temperature
was increased to 110 ꢁC and stirring was continued for 12e18 h.
After cooling, the mixture was diluted by means of water and
saturated aqueous NaHCO3 solution. The generated solid product
was collected by filtration and washed with saturated aqueous
NaHCO3 solution. Yield: 0.345 g (45.6%). White solid; m. p.
[M]þ. HPLC (80% methanol in water): tR ¼ 6.34 min, 97.85%.
4.2.2.15. (Z)-4-Fluoro-N0-hydroxybenzimidamide (59). Yield: 1.43 g
(45%). m. p. 222e223 ꢁC. 1H NMR (300 MHz, DMSO)
d 9.65 (s, 1H),
7.73e7.68 (m, 2H), 7.25e7.16 (m, 2H), 5.85 (s, 2H). EI-MS m/z: 153.1
[M]þ. HPLC (80% methanol in water): tR ¼ 7.27 min, 98.73%.
4.2.2.16. (Z)-3-Methoxy-N0-hydroxybenzimidamide (60). Yield: 1.6 g
(51%). m. p. 205e206 ꢁC. 1H NMR (300 MHz, DMSO)
d 9.63 (s, 1H),
7.30e7.21 (m, 3H), 6.95e6.91 (m, 1H), 5.81 (s, 2H), 3.76 (s, 3H). EI-
MS m/z: 165.1[M]þ. HPLC (80% methanol in water): tR ¼ 6.64 min,
99.10%.
144e145 ꢁC. 1H NMR (300 MHz, DMSO)
2H), 7.98e7.96 (m, 2H), 7.90e7.64 (m, 2H). 13C NMR (75 MHz,
DMSO) 173.688, 168.412, 164.187, 161.069, 150.291, 145.234,
d 8.80 (s, 1H), 8.18e8.04 (m,
d
4.2.2.17. (Z)-3,4-Difluoro-N0-hydroxybenzimidamide
Yield: 1.0 g (32%). m. p. 189e190 ꢁC. 1H NMR (300 MHz, DMSO)
9.77 (s, 1H), 7.87e7.39 (m, 3H), 5.94 (s, 2H). EI-MS m/z: 171.1[M]þ.
(61).
137.666, 126.260, 126.050, 123.403, 111.596, 111.221, 108.896. HRMS
(ESI): calcd for C13H7F2N3ONa [M þ Na]þ 282.04494, found
282.04609. HPLC (80% methanol in water): tR ¼ 9.82 min, 96.93%.
d
HPLC (80% methanol in water): tR ¼ 8.56 min, 95.74%.
4.2.3.2. 5-(3,4-difluorophenyl)-3-(pyridin-4-yl)-1,2,4-oxadiazole
(18). Yield: 0.304 g (40.2%). White solid; m. p. 168e169 ꢁC. 1H NMR
4.2.2.18. (Z)-4-Chloro-N0-hydroxybenzimidamide (62). Yield: 1.1 g
(35.4%). m. p. 184e185 ꢁC. 1H NMR (300 MHz, DMSO)
d
7.37 (d,
(300 MHz, DMSO)
d
8.87 (s, 2H), 8.03e7.92 (m, 4H), 7.81e7.67 (m,
175.59, 168.55, 165.81, 165.64,
J ¼ 7.53 Hz, 2H), 7.22 (d, J ¼ 7.53 Hz, 2H), 4.59 (s, 2H), 3.79 (d,
J ¼ 13.80 Hz, 1H), 3.34 (d, J ¼ 13.80 Hz, 1H). EI-MS m/z: 169.1[M]þ.
HPLC (80% methanol in water): tR ¼ 6.89 min, 97.63%.
1H). 13C NMR (75 MHz, DMSO)
d
162.51, 162.35, 152.34, 134.44, 127.30, 122.38, 113.18, 113.06, 112.93,
112.81, 110.89, 110.55, 110.20. HRMS (ESI): calcd for C13H8F2N3O
[M þ H]þ 260.06299, found 260.06408. HPLC (80% methanol in
water): tR ¼ 8.34 min, 99.25%.
4.2.2.19. (Z)-2-Bromo-N0-hydroxybenzimidamide (63). Yield: 1.2 g
(41%). m. p. 191e192 ꢁC. 1H NMR (300 MHz, DMSO)
d 7.87 (s, 1H),
7.71e7.56 (m, 2H), 7.39e7.17 (m, 3H). EI-MS m/z: 214.1[M]þ. HPLC
(80% methanol in water): tR ¼ 7.63 min, 97.71%.
4.2.3.3. 5-(3,4-difluorophenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole
(19). Yield: 0.295 g (39.2%). White solid; m. p. 123e124 ꢁC. 1H NMR
(300 MHz, DMSO)
8.55e8.07 (m, 3H), 7.75e7.41 (m, 2H). 13C NMR (75 MHz, DMSO)
167.37, 167.18, 163.83, 151.27, 151.14, 133.77, 131.41, 131.28, 130.25,
d 9.24e9.21 (m, 1H), 8.82e8.77 (m, 1H),
4.2.2.20. (Z)-4-(tert-butyl)-N0-hydroxybenzimidamide
Yield: 1.1 g (36.4%). m. p. 177e178 ꢁC. 1H NMR (300 MHz, DMSO)
(64).
d
d
9.56 (s, 1H), 7.59 (d, J ¼ 8.40 Hz, 2H), 7.37 (d, J ¼ 8.43 Hz, 2H), 5.75
129.80, 121.41, 121.02, 117.47, 117.17, 112.00. HRMS (ESI): calcd for
C
methanol in water): tR ¼ 9.63min, 97.43%.
(s, 2H), 1.28 (s, 9H). EI-MS m/z: 191.1[M]þ. HPLC (80% methanol in
water): tR ¼ 7.85 min, 96.43%.
13H8F2N3O [M þ H]þ 260.06299, found 260.06581. HPLC (80%
4.2.2.21. (Z)-N0-Hydroxy-6-methylpyridine-3-carboxamidine
Yield: 1.4 g (40%). m. p. 172e173 ꢁC. 1H NMR (300 MHz, DMSO)
(65).
4.2.3.4. 5-(3,4-difluorophenyl)-3-(5-fluoropyridin-2-yl)-1,2,4-
oxadiazole (20). Yield: 0.315 g (44.3%). White solid; m. p.
d
9.74 (s, 1H), 8.71 (d, J ¼ 1.65 Hz, 1H), 7.89 (dd, J ¼ 2.10, 8.07 Hz, 1H),
162e163 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.84 (s, 1H), 8.29e7.25 (m,
1H), 8.06e7.91 (m, 3H), 7.79e7.72 (m, 1H). 13C NMR (75 MHz,
DMSO) 169.02, 165.79, 165.62, 163.26, 162.50, 162.33, 159.83,
7.24 (d, J ¼ 8.10 Hz, 1H), 5.93 (s, 2H), 2.46 (s, 3H). EI-MS m/z: 150.1
[M]þ. HPLC (80% methanol in water): tR ¼ 8.67 min, 98.63%.
d
143.17, 140.50, 140.17, 126.75, 126.68, 126.06, 125.81, 113.05, 112.93,
112.81, 112.68, 110.71, 110.37, 110.03. HRMS (ESI): calcd for
4.2.2.22. (Z)-N0-Hydroxy-4-methylpyridine-2-carboxamidine (66).
Yield: 1.0 g (31%). m. p. 167e168 ꢁC. 1H NMR (300 MHz, DMSO)
C
13H7F3N3O [M þ H]þ 278.05357, found 278.05565. HPLC (80%
d
9.86 (s, 1H), 8.40 (d, J ¼ 4.95 Hz, 1H), 7.67 (s, 1H), 7.22 (d,
methanol in water): tR ¼ 10.51 min, 97.51%.
J ¼ 4.95 Hz, 1H), 5.81 (s, 2H), 2.33 (s, 3H). EI-MS m/z: 150.1[M]þ.
HPLC (80% methanol in water): tR ¼ 8.14 min, 99.75%.
4.2.3.5. 5-(3,4-difluorophenyl)-3-(3-fluoropyridin-2-yl)-1,2,4-
oxadiazole (21). Yield: 0.345 g (48.5%). White solid; m. p.
4.2.2.23. (Z)-N0-Hydroxy-3-fluoropyridine-2-carboxamidine
Yield: 1.3 g (41%). m. p. 179e180 ꢁC. 1H NMR (300 MHz, DMSO)
(67).
146e147 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.67 (d, J ¼ 2.85 Hz, 1H),
8.06e7.99 (m, 1H), 7.89e7.87 (m, 2H), 7.80e7.67 (m, 2H). 13C NMR
(75 MHz, DMSO) 165.79, 165.62, 162.49, 162.32, 161.08, 147.73,
d
10.13 (s, 1H), 8.46e8.44 (m, 1H), 7.53e7.47 (m, 1H), 5.85 (s, 2H). EI-
d
MS m/z: 154.1[M]þ. HPLC (80% methanol in water): tR ¼ 8.62 min,
147.66, 129.66, 129.60, 127.01, 126.77, 113.05, 112.92, 112.81, 112.68,
110.72, 110.37, 110.03. HRMS (ESI): calcd for C13H6F3N3ONa [M þ
Na]þ 300.03552, found 300.03671. HPLC (80% methanol in water):
tR ¼ 10.71 min, 98.81%.
99.43%.
4.2.2.24. (Z)-3,5-Dimethoxy-N0-hydroxybenzimidamide
Yield: 1.4 g (46.7%). m. p. 209e210 ꢁC. 1H NMR (300 MHz, DMSO)
(68).
d
9.63 (s, 1H), 6.84 (d, J ¼ 2.04 Hz, 1H), 6.49 (t, J ¼ 1.98 Hz, 1H), 5.80
4.2.3.6. 5-(3,4-difluorophenyl)-3-(pyrazin-2-yl)-1,2,4-oxadiazole
(22). Yield: 0.345 g (46.0%). White solid; m. p. 217e218 ꢁC. 1H NMR
(s, 2H), 3.74 (s, 6H). EI-MS m/z: 195.1[M]þ. HPLC (80% methanol in
water): tR ¼ 9.61 min, 98.71%.
(300 MHz, DMSO)
d
9.38 (s, 1H), 8.92 (s, 2H), 8.22e8.18 (m, 1H), 7.74
156.00, 147.74,
(s, 1H), 7.20 (s, 1H). 13C NMR (75 MHz, DMSO)
d
4.2.2.25. (Z)-4-Bromo-N0-hydroxybenzimidamide (69). Yield: 1.2 g
145.72, 144.42, 141.86, 137.77, 130.20, 126.73, 125.89, 125.85, 120.28,
117.52, 117.21. HRMS (ESI): calcd for C12H7F2N4O [M þ H]þ
261.05824, found 261.05841. HPLC (80% methanol in water):
tR ¼ 9.63 min, 97.68%.
(41%). m. p. 174e175 ꢁC. 1H NMR (300 MHz, DMSO)
d 9.76 (s, 1H),
7.64e7.54 (m, 4H), 5.88 (s, 2H). EI-MS m/z: 214.0[M]þ. HPLC (80%
methanol in water): tR ¼ 7.92 min, 99.17%.

1390
L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
4.2.3.7. 5-(3,4-difluorophenyl)-3-(pyrimidin-2-yl)-1,2,4-oxadiazole
(23). Yield: 0.295 g (39.3%). White solid; m. p. 202e203 ꢁC. 1H NMR
(ESI): calcd for C20H13F2N2O [M þ H]þ 335.09905, found 335.09987.
HPLC (80% methanol in water): tR ¼ 12.54 min, 96.29%.
(300 MHz, DMSO)
d
9.09 (d, J ¼ 4.89 Hz, 2H), 7.93 (d, J ¼ 5.55 Hz,
2H), 7.82e7.67 (m, 2H). 13C NMR (75 MHz, DMSO)
d
175.53, 168.53,
4.2.3.14. 5-(3,4-difluorophenyl)-3-(2-fluorophenyl)-1,2,4-oxadiazole
(30). Yield: 0.365 g (50.6%). White solid; m. p. 135e136 ꢁC. 1H NMR
165.80, 165.63, 162.50, 162.33, 159.64, 156.37, 127.76, 124.42, 113.06,
112.94, 112.82, 112.69, 110.73, 110.39, 110.05. HRMS (ESI): calcd for
(300 MHz, DMSO)
d
8.66 (d, J ¼ 4.14 Hz, 1H), 8.04e7.98 (m, 1H),
C
12H7F2N4O [M þ H]þ 261.05824, found 261.05841. HPLC (80%
7.88e7.73 (m, 3H), 7.08 (t, J ¼ 8.99 Hz, 2H). 13C NMR (75 MHz,
methanol in water): tR ¼ 9.89 min, 98.74%.
DMSO) d 165.65, 165.58, 162.00, 158.61, 154.81, 154.65, 151.96,
151.79, 151.44, 151.28, 148.67, 148.49, 134.23, 134.11, 131.04, 131.02,
126.40, 126.35, 126.29, 126.24, 125.54, 125.50, 119.69, 119.45, 118.03,
117.77, 117.40, 117.13. HRMS (ESI): calcd for C14H8F3N2O [M þ H]þ
277.05832, found 277.05884. HPLC (80% methanol in water):
tR ¼ 9.53 min, 97.97%.
4.2.3.8. 5-(3,4-difluorophenyl)-3-(6-methylpyridin-2-yl)-1,2,4-
oxadiazole (24). Yield: 0.285 g (39.6%). White solid; m. p.
107e108 ꢁC. 1H NMR (300 MHz, DMSO)
d
9.07 (d, J ¼ 5.40 Hz, 1H),
8.28e8.23 (m, 1H), 7.83e7.73 (m, 2H), 7.49e7.43 (m, 1H), 7.03 (t,
J ¼ 9.00 Hz, 1H), 2.56 (s, 3H). 13C NMR (75 MHz, DMSO)
d 166.80,
161.69, 147.64, 147.58, 135.17, 135.11, 125.68, 125.66, 124.03, 123.94,
120.23, 119.69, 119.45, 118.07, 117.80, 117.74, 116.07, 115.75, 42.22,
42.14. HRMS (ESI): calcd for C14H10F2N3O [M þ H]þ 274.07864,
found 274.08060. HPLC (80% methanol in water): tR ¼ 11.26 min,
96.62%.
4.2.3.15. 5-(3,4-difluorophenyl)-3-(3-fluorophenyl)-1,2,4-oxadiazole
(31). Yield: 0.324 g (45.0%). White solid; m. p. 162e163 ꢁC. 1H NMR
(300 MHz, DMSO)
7.88e7.76 (m,1H), 7.72e7.63 (m,1H), 7.53e7.47 (m,1H), 7.20 (s, 1H).
13C NMR (75 MHz, DMSO)
164.36, 161.11, 155.92, 152.60, 137.79,
d 8.28e8.13 (m, 2H), 8.00e7.93 (m, 1H),
d
137.72, 132.18, 132.07, 130.19, 126.60, 125.75, 125.70, 123.81, 123.77,
120.23, 120.19, 119.35, 119.07, 117.37, 117.06, 114.41, 114.10. HRMS
(ESI): calcd for C14H8F3N2O [M þ H]þ 277.05832, found 277.05884.
HPLC (80% methanol in water): tR ¼ 10.26 min, 98.19%.
4.2.3.9. 5-(3,4-difluorophenyl)-3-(6-methylpyridin-3-yl)-1,2,4-
oxadiazole (25). Yield: 0.325 g (45.1%). White solid; m. p.
244e245 ꢁC. 1H NMR (300 MHz, DMSO)
d
9.09 (s, 1H), 8.28 (d,
J ¼ 7.92 Hz,1H), 7.88e7.85 (m, 2H), 7.73e7.67 (m, 1H), 7.49e7.46 (m,
1H), 2.56 (s, 3H). 13C NMR (75 MHz, DMSO)
168.24, 165.82, 165.65,
d
4.2.3.16. 5-(3,4-difluorophenyl)-3-(4-fluorophenyl)-1,2,4-oxadiazole
(32). Yield: 0.353 g (49.0%). White solid; m. p. 140e141 ꢁC. 1H NMR
163.11, 162.53, 162.35, 148.68, 136.23, 127.45, 125.07, 120.74, 113.17,
113.04, 112.92, 112.79, 110.77, 110.43, 110.09, 25.59. HRMS (ESI):
calcd for C14H10F2N3O [M þ H]þ 274.07864, found 274.07934. HPLC
(80% methanol in water): tR ¼ 10.47 min, 97.53%.
(300 MHz, DMSO)
7.85e7.70 (m, 1H), 7.51e7.38 (m, 2H). 13C NMR (75 MHz, DMSO)
161.99,158.60,154.81,154.64,151.96,151.78,151.44,151.28,134.24,
d 8.26e8.20 (m, 1H), 8.19e8.06 (m, 3H),
d
134.13, 131.04, 131.02, 126.41, 126.36, 126.30, 126.25, 125.56, 125.51,
119.70, 119.46, 118.04, 117.78, 117.41, 117.14. HRMS (ESI): calcd for
4.2.3.10. 5-(3,4-difluorophenyl)-3-(4-methylpyridin-2-yl)-1,2,4-
oxadiazole (26). Yield: 0.34 g (47.2%). White solid; m. p.
C
14H8F3N2O [M þ H]þ 277.05832, found 277.05884. HPLC (80%
148e149 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.63 (d, J ¼ 4.47 Hz, 1H),
methanol in water): tR ¼ 10.47 min, 98.54%.
8.00 (s, 1H), 7.89 (d, J ¼ 4.83 Hz, 2H), 7.74e7.68 (m, 2H), 7.46 (d,
J ¼ 3.66 Hz, 2H), 2.45 (s, 3H). 13C NMR (75 MHz, DMSO)
d
165.79,
4.2.3.17. 5-(3,4-difluorophenyl)-3-(4-chlorophenyl)-1,2,4-oxadiazole
(33). Yield: 0.334 g (48.6%). White solid; m. p. 243e244 ꢁC. 1H NMR
165.62, 162.49, 162.32, 161.08, 147.73, 147.66, 129.66, 129.60, 127.01,
126.77, 113.05, 112.92, 112.81, 112.68, 110.72, 110.37, 110.03, 15.69.
HRMS (ESI): calcd for C14H9F2N3ONa [M þ Na]þ 300.03552, found
300.03671. HPLC (80% methanol in water): tR ¼ 10.93 min, 99.13%.
(300 MHz, DMSO)
d 8.26e8.20 (m, 1H), 8.19e8.06 (m, 3H),
7.85e7.70 (m, 1H), 7.51e7.38 (m, 2H). HRMS (ESI): calcd for
C
14H7ClF2N2O [M þ H]þ 293.67832, found 293.67884. HPLC (80%
methanol in water): tR ¼ 13.47 min, 98.84%.
4.2.3.11. 5-(3,4-difluorophenyl)-3-(1H-indol-4-yl)-1,2,4-oxadiazole
(27). Yield: 0.34 g (53.1%). White solid; m. p. 229e230 ꢁC. 1H NMR
4.2.3.18. 5-(3,4-difluorophenyl)-3-(2-bromophenyl)-1,2,4-oxadiazole
(34). Yield: 0.302 g (47.9%). White solid; m. p. 187e188 ꢁC. 1H NMR
(300 MHz, DMSO)
(m, 1H), 7.93e7.56 (m, 4H), 7.32e7.26 (m, 1H), 7.08 (s, 1H). 13C NMR
(75 MHz, DMSO) 170.55, 165.81, 165.63, 162.51, 162.34, 137.91,
d
11.52 (s, 1H), 8.26 (t, J ¼ 8.46 Hz, 1H), 8.10e8.08
(300 MHz, DMSO)
d 8.30e8.14 (m, 2H), 8.02e7.87 (m, 2H), 7.73 (s,
d
1H), 7.65e7.53 (m, 2H). HRMS (ESI): calcd for C14H6BrF2N2O [M -
H]e 334.96371, found 334.96375. HPLC (80% methanol in water):
tR ¼ 10.19 min, 98.43%.
128.78, 125.88, 122.31, 122.15, 121.63, 116.68, 112.99, 112.86, 112.75,
112.62, 110.39, 110.05, 109.71, 103.61, 100.75. HRMS (ESI): calcd for
C
16H10F2N3O [M þ H]þ 298.07864, found 298.07909. HPLC (80%
methanol in water): tR ¼ 10.72 min, 98.91%.
4.2.3.19. 5-(3,4-difluorophenyl)-3-(4-bromophenyl)-1,2,4-oxadiazole
(35). Yield: 0.343 g (54.4%). White solid; m. p. 131e132 ꢁC. 1H NMR
4.2.3.12. 5-(3,4-difluorophenyl)-3-(naphthalen-2-yl)-1,2,4-
oxadiazole (28). Yield: 0.293 g (44.2%). White solid; m. p.
(300 MHz, DMSO)
d
8.25e28.21 (m, 1H), 8.15e28.99 (m, 3H),
168.66, 165.28,
7.82e28.72 (m, 3H), 13C NMR (75 MHz, DMSO)
d
207e208 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.56e8.49 (m, 1H),
165.63, 162.51, 162.33, 136.00, 132.95, 130.91, 129.29, 127.45, 127.21,
123.75, 113.11, 112.98, 112.86, 112.74, 110.76, 110.42, 110.08. HRMS
(ESI): calcd for C14H6BrF2N2O [M - H]e 334.96371, found 334.96375.
HPLC (80% methanol in water): tR ¼ 9.28 min, 97.91%.
8.22e7.96 (m, 4H), 7.70e7.55 (m, 2H), 7.36 (m, 1H), 7.27 (m, 1H),
7.07 (m, 1H). HRMS (ESI): calcd for C18H10F2N2O [M þ H]þ
309.29905, found 309.29987. HPLC (80% methanol in water):
tR ¼ 11.54 min, 98.71%.
4.2.3.20. 3,5-bis(3,4-difluorophenyl)-1,2,4-oxadiazole
Yield: 0.298 g (43.8%). White solid; m. p. 207e208 ꢁC. 1H NMR
(300 MHz, DMSO)
7.92 (d, J ¼ 5.52 Hz, 2H), 7.77e7.74 (m, 3H),
7.72e7.54 (m, 1H). 13C NMR (75 MHz, DMSO)
154.90, 151.32,
130.73, 130.69, 124.47, 124.42, 124.38, 124.33, 120.25, 119.97, 118.58,
118.58, 118.23, 118.00, 117.45, 117.43, 116.70, 116.44. HRMS (ESI):
calcd for C14H6F4N2O [M þ H] þ 295.21371, found 295.21375. HPLC
(80% methanol in water): tR ¼ 9.34 min, 96.91%.
(36).
4.2.3.13. 5-(3,4-difluorophenyl)-3-([1,10-biphenyl]-4-yl)-1,2,4-
oxadiazole (29). Yield: 0.313 g (43.2%). White solid; m. p.
d
151e152 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.19e8.13 (m, 2H),
8.02e7.87 (m, 4H), 7.76e7.67 (m, 3H), 7.53e7.17 (m, 3H). 13C NMR
(75 MHz, DMSO) 168.08, 164.27, 164.10, 160.98, 160.81, 143.16,
d
d
138.78, 128.87, 128.04, 127.56, 127.26, 126.63, 126.22, 126.07, 124.53,
117.92, 111.47, 111.35, 111.23, 111.10, 109.02, 108.68, 108.34. HRMS

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1391
4.2.3.21. 5-(3,4-difluorophenyl)-3-(4-acetyl phenyl)-1,2,4-oxadiazole
(37). Yield: 0.302 g (44.8%). White solid; m. p. 230e231 ꢁC. 1H
NMR (300 MHz, DMSO)
8.07 (d, J ¼ 8.34 Hz, 2H), 7.87e7.84 (m,
4H), 7.72e7.66 (m, 1H), 2.19 (s, 3H). 13C NMR (75 MHz, DMSO)
174.81,169.47,165.76,165.59,162.47,162.30,153.60,141.36,128.91,
4.3.2. Compound dilutions
All compounds were dissolved in DMSO at 10 mmol per liter as
stock solutions and stored at ꢀ20 ꢁC. Fresh cell-culture medium
was used to dilute the stock solutions.
d
d
128.48, 127.58, 126.95, 126.63, 112.95, 112.83, 112.71, 112.58, 110.56,
110.22, 109.88, 12.65. HRMS (ESI): calcd for C16H9F2N2O2 [M - H]e
299.06376, found 299.06550. HPLC (80% methanol in water):
tR ¼ 10.34 min, 97.65%.
4.3.3. MTT analysis
Cytotoxicity was determined using the MTT assay. MTT was
purchased from Sigma (St. Louis, MO). It was dissolved in
phosphate-buffered saline (PBS) at a concentration of 5 mg/mL as
the stock solution and stored at ꢀ20 ꢁC. After the cells were treated
with a density gradient of test compounds or DMSO for 24 h,
4.2.3.22. 5-(3,4-difluorophenyl)-3-(p-tolyl)-1,2,4-oxadiazole
Yield: 0.356 g (49.4%). White solid; m. p. 150e151 ꢁC. 1H NMR
(300 MHz, DMSO) 8.09e7.86 (m, 4H), 7.84e7.81 (m, 1H),
7.76e7.66 (m, 2H), 2.59 (s, 3H). 13C NMR (75 MHz, DMSO)
169.85,
(38).
20.0
mL of MTT solution (5 mg/mL) was added to each well of a 96-
well plate for 4 h. Next, the solution was removed, and 150.0
mL of
d
DMSO was added to each well to dissolve the water-soluble MTT-
formazan crystals. The absorbance values (OD value) were recorded
at 570 nm by an Elx800 absorbance microplate reader (BioTek,
Vermont, USA).
d
165.82, 165.64, 162.36, 143.24, 137.37, 131.81, 131.21, 128.47, 124.41,
119.48, 116.65, 113.00, 112.88, 112.76, 112.63, 110.52, 110.18, 109.83,
22.47. HRMS (ESI): calcd for C15H11F2N2O [M þ H]þ 273.0834, found
273.08384. HPLC (80% methanol in water): tR ¼ 9.34 min, 98.78%.
IC50 ¼ [1 - (ODtest- ODblank) / (ODcontrol-ODblank)]*100%
4.2.3.23. 5-(3,4-difluorophenyl)-3-(3-methoxyphenyl)-1,2,4-
oxadiazole (39). Yield: 0.401 g (57.8%). White solid; m. p.
122e123 ꢁC. 1H NMR (300 MHz, DMSO)
d
7.88 (s, 2H), 7.82e7.45 (m,
4H), 7.21e7.19 (m, 1H), 3.86 (s, 3H). 13C NMR (75 MHz, DMSO)
169.74, 165.81, 165.64, 162.51, 162.34, 161.08, 131.88, 128.39,
4.3.4. ARE-luciferase activity assay
The experimental procedures were performed as reported pre-
viously [35]. HepG2-ARE-C8 cells stably expressing ARE-luciferase
were seeded onto 48-well plates at a density of 5 ꢂ 104 cells/well
and incubated overnight before incubation with test compounds.
The cells were treated with different concentrations of the test
compounds, with tBHQ serving as the positive control, DMSO
serving as the negative control, and the luciferase cell culture lysis
reagent serving as the blank. After 24 h of treatment, the medium
d
120.80, 119.04, 113.53, 112.99, 112.86, 112.74, 112.62, 110.54, 110.20,
109.85, 56.76. HRMS (ESI): calcd for C15H11F2N2O2 [M þ H]þ
289.07831, found 289.0786. HPLC (80% methanol in water):
tR ¼ 8.79 min, 98.31%.
4.2.3.24. 3-(4-(tert-butyl)phenyl)-5-(3,4-difluorophenyl)-1,2,4-
oxadiazole (40). Yield: 0.386 g (59.1%). White solid; m. p.
was removed and 400.0
Next, the cells were lysed in the luciferase cell culture lysis reagent.
After centrifugation, 20.0 L of the supernatant was used for
mL of cold PBS was added to each well.
110e111 ꢁC. 1H NMR (300 MHz, DMSO)
d
7.99 (d, J ¼ 8.31 Hz, 2H),
7.85 (d, J ¼ 5.55 Hz, 2H), 7.69e7.59 (m, 3H), 1.32 (s, 9H). 13C NMR
m
(75 MHz, DMSO)
d 169.74, 165.80, 165.62, 162.50, 162.33, 156.11,
determining the luciferase activity according to the protocol pro-
vided by the manufacturer (Promega, Madison, WI). The luciferase
activity was measured by a Luminoskan Ascent luminometer
(Thermo Scientific, USA). The data were obtained in triplicates and
expressed as fold induction over control.
128.69, 128.35, 127.63, 127.46, 126.27, 124.39, 113.00, 112.88, 112.76,
112.63,110.56,110.22,109.88, 36.15, 32.32, 32.24, 32.13. HRMS (ESI):
calcd for C18H17F2N2O [M þ H]þ 315.13035, found 315.1312. HPLC
(80% methanol in water): tR ¼ 8.98 min, 97.10%.
4.2.3.25. 5-(3,4-difluorophenyl)-3-(3,5-dimethoxyphenyl)-1,2,4-
oxadiazole (41). Yield: 0.341 g (52.4%). White solid; m. p.
Inductivity ¼ (RLUtest ꢀ RLUblank)/(RLUcontrol ꢀ RLUblank)
RLU ¼ relative light unit
123e124 ꢁC. 1H NMR (300 MHz, DMSO)
d
8.22e7.94 (m, 2H),
7.76e7.70 (m, 1H), 7.19e7.15 (m, 2H), 6.72 (s, 3H), 3.83 (s, 6H). 13
NMR (75 MHz, DMSO) 168.60, 161.34, 154.74, 154.58, 151.92,
C
d
151.74, 151.38, 151.21, 148.63, 148.45, 127.99, 126.32, 126.28, 126.22,
126.17, 119.61, 119.37, 117.97, 117.71, 116.00, 115.67, 105.30, 103.96,
42.20, 42.12. HRMS (ESI): calcd for C16H13F2N2O3 [M þ H]þ
319.08888, found 319.08992. HPLC (80% methanol in water):
tR ¼ 9.34 min, 95.91%.
4.3.5. Western blotting
Anti-NQO1 (sc-271116) antibodies were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b-actin (AP0060)
and anti-Nrf2 (BS1258) were purchased from Bioworlde (Bio-
worlde, USA). Anti-HO-1 (no. 5853 S) was purchased from Cell
Signaling Technology (USA). The cells were washed once with ice-
cold PBS and dissociated with 1 mL of 1 ꢂ trypsin. The cells were
4.3. Pharmacology
4.3.1. Cell culture conditions
centrifuged at 2000 rpm and lysed in 80.0 mL of lysis buffer, which
HepG2 cells stably transfected with an ARE luciferase reporter
(HepG2-ARE-C8) were kindly provided by Professor A. N. Tony
Kong (Rutgers University, Piscataway, NJ) and Prof. Rong Hu (China
Pharmaceutical University). Cells were cultured in DMEM (Gibco,
USA) with 10% (v/v) fetal bovine serum (FBS, Gibco, USA) in a hu-
midified atmosphere of 5% CO2 and 95% air at 37 ꢁC. L02 cells (Cell
Bank of Shanghai Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences) were cultured in DMEM (Gibco, USA) supplemented with
10% (v/v) FBS in a humidified atmosphere of 5% CO2 and 95% air at
37 ꢁC.
contained 150.0 mM NaCl, 1% NP-40, 50.0 mM Tris-HCl, pH 7.5,
1 mM NaF, EDTA, DTT, Leu, and PMSF for 1 h. The cells were
centrifuged again at 12000 rpm for 20 min at 4 ꢁC. The supernatants
were collected, and the protein concentrations of the whole cell
lysates were determined by the BCA assay with Varioskan Flash
(Thermo, Waltham, MA) at 562 nm. The samples were stored
at ꢀ80 ꢁC until use. The Nuclear-Cytosol Extraction Kit (KeyGEN, NJ,
China) was used to isolate the nuclear and cytosolic protein ac-
cording to the protocol. The collected proteins were stored
at ꢀ80 ꢁC until use.
Equal amounts of total protein extracts were separated by SDS-

1392
L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
PAGE and then electroblotted onto PVDF membranes (PerkinElmer,
Northwalk, CT, USA). After blocking with 1% BSA for 1 h, the
membranes were incubated at 37 ꢁC for 1 h and then at 4 ꢁC over-
night with a primary antibody. They were treated with a DyLight
800 labeled secondary antibody at 37 ꢁC for 1 h. The membranes
were screened with the Odyssey infrared imaging system (LI-COR,
Lincoln, Nebraska, USA).
by the Institutional Animal Care and Use Committee of China
Pharmaceutical University. All animals were appropriately used in a
scientifically valid and ethical manner. The animal use approval
number of China Pharmaceutical University is SYXK(苏)2016-0011.
The animal room was environmentally controlled at 25 ꢁC, 55%e
60% humidity and 12 h light/dark cycle. 50 female C57BL/6 mice
(20e25 g) were randomly divided into five groups (n ¼ 10): Group I
(control group) received normal saline as a vehicle for 7 days. Group
II mice (APAP group) were injected with APAP (Sigma-Aldrich, St.
Louis no. 103-90-2) on the 7th day with a single dose of 300 mg/kg.
Groups III-V mice (25 þ APAP group) were injected with compound
25 at a low-dose (10 mg/kg/day) group, middle-dose (50 mg/kg/
day) group, and a high-dose (100 mg/kg/day) for 7 consecutive
days. One hour after the last administration, a single dose of APAP
(300 mg/kg) was injected to the mice of groups III-V. Both com-
pound 25 and APAP were dissolved in physiological saline. The
injection volume was calculated from the body weight of the mice
4.3.6. Immunofluorescence of L02 cells
L02 cells were grown on coverslips for 24 h then treated with
compounds at different concentrations for 12 h. The cells were
fixed and probed with an Nrf2 antibody and an DyLight 488-labeled
anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz,
CA), before being stained with the fluorochrome dye DAPI (Santa
Cruz Biotechnology, Santa Cruz, CA) to visualize the nuclei observed
under a laser scanning confocal microscope (Olympus Fluoview
FV1000, Japan) with peak excitation wavelengths of 570 and
340 nm.
and should not exceed 200 mL. After 24 h of the APAP challenge, all
mice were sacrificed to obtain blood and liver samples for further
4.3.7. qRT-PCR
biochemical analysis.
Total RNA of the L02 cells was isolated using TRIzol (Invi-
trogen). The quantification and purity of the RNA samples were
assessed by A260/A280 absorption, and RNA samples with ratios
above 1.8 were stored at ꢀ80 ꢁC for further analysis. The RNA was
reverse transcribed using the PrimeScript RT reagent kit following
the manufacturer's instructions. The following primer sequences
were used, Nrf2 sense primer 50-AACCACCCTGAAAGCACGC-30 and
antisense primer 50-TGAAATGCCGGAGTCAGAATC-3’; HO-1 sense
primer 50- ATGGCCTCCCTGTACCACATC-30 and antisense primer
50- TGTTGCGCTCAATCTCCTCCT-3’; NQO-1 sense primer 50-
CGCAGACCTTGTGATATTCCAG-30 and antisense primer 50-
4.3.11. Measurement of plasma ALT and AST levels
Blood samples from mice were centrifuged at 3000ꢂg, for
10 min at 4 ꢁC to obtain the serum supernatants. The contents of
ALT and AST in serum were measured using commercially available
kits (Nanjing Jiancheng Institute of Biotechnology, China).
4.3.12. Assay for antioxidant enzymes activity
Liver tissues were collected and homogenized in phosphate
buffer (pH 7.0). Tissue homogenates were then centrifuged at
10,000ꢂg, for 10 min at 4 ꢁC to separately obtain the supernatant
and pellet for further experiments. The protein concentration was
determined using a BCA protein assay kit (Pierce, Rockford, IL, USA).
CGTTTCTTCCATCCTTCCAGG-3’;
TTGGAGTTGCACAGCTGGATTC-30 and antisense primer 50-
TGGTTTTACCTGTGCCCACTG-3’; GCLC sense primer:
50-
GCLM
sense
primer
50-
GTCCTCAGGTGACATTCCAAGC-3’; and antisense primer: 50-
TGTTCTTCAGGGGCTCCAGTC-3’. GAPDH was used as the internal
controls (sense primer 50-AGGTCGGTGTGAACGGATTTG-30 and
antisense primer 50-TGTAGACCATGTAGTTGAGGTCA-30). Quanti-
tative real-time RT-PCR analysis of Nrf2, NQO1, HO-1, and GCLM
were performed using a STEPONE SYSTERM Fast Real Time PCR
system (Applied Biosystems). GAPDH was used for normalization.
The values are expressed as the fold increase compared with the
control. Each cycle consisted of denaturation at 95 ꢁC for 5 s
combined with annealing and extension at 60 ꢁC for 30 s. A total of
40 cycles were performed.
4.3.13. Measurement of hepatic GSH levels
The GSH level was measured using a modified protocol with a
GSH kit provided by Jiancheng Bioengineering Institute (Nanjing,
China). The observation absorbance of the reaction was read at
420 nm and the enzyme activity was calculated as mmol/g protein.
4.3.14. Measurement of hepatic superoxide dismutase (SOD)
activity
SOD activity was determined by measuring the ability of SOD to
inhibit the photochemical reduction of nitro blue tetrazolium (NBT)
according to the absorbance at 550 nm. Using the commercial kit
protocol provided by Jiancheng Bioengineering Institute (Nanjing,
China), the data were expressed as SOD U/mg protein compared
with the standard.
4.3.8. Transfection of small interfering RNA (siRNA)
A predesigned siRNA against human Nrf2 (catalog no. 115762)
and a control scrambled siRNA (catalog no. 4611) were purchased
from Biomics (Biomics, China). L02 cells were plated at a density of
7 ꢂ 105 cells per 60 mm dish. The cells were transfected with 50 nM
siRNA against Nrf2 using Lipofectamine 2000 (Invitrogen). After
24 h incubation, fresh medium was added, and the cells were
cultured for another 48 h. The cells were then treated with 20.0
compound 25 for an additional 12 h and lysed for qRT-PCR.
4.3.15. GST activity assay
The GST level was determined by a commercial kit provided by
Jiancheng Bioengineering Institute (Nanjing, China). The observa-
tion absorbance of the reaction was read at 412 nm, and the activity
was calculated as U/mg protein. GSH consumption was used as a
marker to evaluate whether APAP was a substrate for GST.
4.3.9. Statistical analysis
Statistical analyses were calculated using both one-way ANOVA
and Kruskal-Wallis tests for the multiple comparision. For all tests
only P values < 0.05 were considered statistically significant. All
descriptive data are reported as the mean SD. GraphPad Prism
was used for statistical analysis.
Ancillary information
Author contributions
4.3.10. Animals and experimental design
The manuscript was written with contributions from all authors.
Animal studies were conducted according to protocols approved
All authors have given approved the final version of the manuscript.

L.-L. Xu et al. / European Journal of Medicinal Chemistry 157 (2018) 1376e1394
1393
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Notes
The authors declare no competing financial interest.
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Acknowledgement
This study was supported by projects, [81602948], [81773581]
and [81773639] of the National Natural Science Foundation of
China, the Natural Science Foundation of Jiangsu Province of China
[no. BK20160746]; [2015ZX09101032], [2017ZX09302003] and
[2018ZX09711002-003-006] of the National Major Science and
Technology Project of China (Innovation and Development of New
Drugs); the Priority Academic Program Development of Jiangsu
Higher Education Institutions; the College Students Innovation
Project for the R&D of Novel Drugs [J1310032]; the China Post-
doctoral Science Foundation-funded project [2017M620232]; The
Program for Outstanding Scientific and Technological Innovation
Team of Jiangsu Higher Education [YY20180315004]; and Funda-
mental Research Funds for the Central Universities [2016ZPT005].
Abbreviations
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Keap1
Nrf2
ARE
b-ZIP
ROS
NQO1
HO-1
SAR
Kelch-like ECH-associated protein-1
nuclear factor erythroid 2-related factor 2
antioxidant response element
basic-leucine zipper
reactive oxygen species
NAD(P)H/quinone oxidoreductase
heme oxygenase-1
structure-activity relationship
over-the-counter
acetaminophen
glutathione
hepatocellular carcinoma
dimethyl fumarate
tertiary butylhydroquinone
mitogen-activated protein kinases
phosphoinositide 3-kinase
p38 MAP kinase
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against CdSe quantum dot-induced cytotoxicity, PLoS One 10 (2015),
e0138771.
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defence pathway: role in protection against drug-induced toxicity, Toxicology
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C.E. Goldring, H. Powell, C. Sanderson, S. Williams, L. Higgins, M. Yamamoto,
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OTC
APAP
GSH
HCC
DMF
tBHQ
MAPK
PI3K
P38
JNK
c-Jun N-terminal kinase
UPLC-MS/MS ultra-performance liquid chromatography-tandem
mass spectrometry
NBT
CYP450
NAPQI
GCLC
nitro blue tetrazolium
cytochrome p450
n-acetyl-para-benzoquinone imine
cysteine ligase catalytic subunit
cysteine ligase regulatory subunit
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injury produced by various hepatotoxicants, Oxid. Med. Cell. Longev. 2013
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GCLM
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P. Renard, J. Verrax, P.B. Calderon, AICAR induces Nrf2 activation by an AMPK-
independent mechanism in hepatocarcinoma cells, Biochem. Pharmacol. 91
(2014) 168e180.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.08.071.
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