Synthesis and evaluation of 1,2,4-oxadiazole derivatives as potential anti-inflammatory agents by inhibiting NF-κB signaling pathway in LPS-stimulated RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2020.127373

In this study, a series of compounds with 1,2,4-oxadiazole core was designed and synthesized for the optimization of JC01, an anti-inflammatory hit identified from our in-house compound library using NF-κB pathway luciferase assay and NO production assay.

Full text of DOI:10.1016/j.bmcl.2020.127373

Bioorganic&MedicinalChemistryLetters30(2020)127373
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and evaluation of 1,2,4-oxadiazole derivatives as potential anti-
inflammatory agents by inhibiting NF-κB signaling pathway in LPS-
stimulated RAW 264.7 cells
⁎
Yu-Ying Zhanga, Qian-Qian Zhanga, Juan Zhanga, , Jia-Li Songa, Jia-Cheng Lia, Ke Hanb,
⁎
⁎
Jin-Tian Huangc, Cheng-Shi Jianga, , Hua Zhanga,
a School of Biological Science and Technology, University of Jinan, Jinan 250022, China
b Department of Traumatic Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China
c Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250021, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, a series of compounds with 1,2,4-oxadiazole core was designed and synthesized for the optimi-
zation of JC01, an anti-inflammatory hit identified from our in-house compound library using NF-κB pathway
luciferase assay and NO production assay. All the synthetic compounds 1–29 have been screened for their anti-
inflammatory effects by evaluating their inhibition against LPS-induced NO release, and compound 17 exhibited
the highest activity. Western blotting and immunofluorescence analysis revealed that 17 prominently inhibited
LPS-induced activation of NF-κB in RAW264.7 cells and blocked the phosphorylation of p65. Consistent with
these results, it was found that 17 prevented the nuclear translocation of NF-κB induced by LPS. These data
highlighted 17 as a promising anti-inflammatory agent by inhibiting NF-κB activity.
1,2,4-oxadiazole
Structural optimization
Anti-inflammatory activity
NO production
NF-κB
Inflammation is a defensive response on exposure to harmful stimuli
such as pathogens and irritants, with quintessential features like red-
ness, swelling, pain, heat and dysfunction.1 The pathology of in-
flammation is a complicated procedure by the organism to remove the
injurious stimuli and to initiate the healing process of the tissues.2
However, in certain circumstances, inflammation is also deemed to be a
major risk factor for the pathogenesis of chronic diseases, including
arthritis, diabetes, asthma, atherosclerosis and even cancer,3–7 and its
process involves in the activated macrophages such as RAW 264.7 cells.
Macrophages play a crucial role in the innate immune response, and
they protect cells from injury induced by both exogenous and en-
dogenous factors.8,9 The LPS-stimulated RAW264.7 cell is usually em-
ployed as a classical inflammatory cell model,10 and the activated
macrophages secrete pro-inflammatory mediators including tumor ne-
crosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β),
metalloproteinases, nitric oxide (NO) and inducible nitric oxide syn-
thase (iNO).11–13 Meanwhile, the activation of nuclear factor-κB (NF-
κB) signaling pathways, which have been found to play important roles
in inflammatory reactions and diseases, mediates the release of pro-
inflammatory factors in monocytes/macrophages. Therefore, treat-
ments aimed at inhibiting NF-κB may have potential therapeutic ad-
vantages for inflammatory diseases.14,15. In past decades, non-steroidal
anti-inflammatory drugs (NSAIDs) and immune drugs have been ap-
plied in some inflammatory diseases.16,17
To discover potential anti-inflammatory agents targeting the in-
hibition of NF-κB activation, an initial NF-κB inhibitor, 1,2,4-oxadiazole
derivative JC01 (Fig. 1), was screened out from our in-house compound
library of 5387 compounds by using NF-κB pathway luciferase
assay.18,19 This compound exhibited an IC50 value of 13.83
2.71 μM
against TNF-α-induced NF-κB activation, which was further confirmed
by western blotting and immunofluorescence analysis (Fig. S1 in
Supporting Information). Then the anti-inflammatory activity of JC01
was evaluated by testing its inhibition on the production of NO in LPS-
stimulated RAW264.7 cells. NO is an important inflammatory mediator
which can be produced by activated macrophages. The result from
Table 1 showed that JC01 had a suppressive effect on the production of
NO in LPS-stimulated RAW264.7 cells, with an IC50 value of
26.96
1.31 μM (data shown in Table 2). These initial results in-
dicated that JC01 could serve as a good starting molecule for further
optimization to develop novel anti-inflammatory agents through in-
hibiting NF-κB activation. Herein, we report the design, synthesis,
biological evaluation and mechanistic of a series of JC01-derived 1,2,4-
oxadiazoles.
Literature survey demonstrates that 1,2,4-oxadiazole is a common
⁎ Corresponding authors.
E-mail addresses: bio_zhangj@ujn.edu.cn (J. Zhang), bio_jiangcs@ujn.edu.cn (C.-S. Jiang), bio_zhangh@ujn.edu.cn (H. Zhang).
https://doi.org/10.1016/j.bmcl.2020.127373
Received 18 May 2020; Accepted 27 June 2020
Availableonline11July2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Y.-Y. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127373
Fig. 1. The workflow of discovery of 1,2,4-oxadiazoles as anti-inflammatory agents.
heterocycle in various anti-inflammatory compounds,20 and some in-
dole derivatives have also been identified as NF-κB inhibitors21,22 and
anti-inflammatory agents.23,24 Thus, both 1,2,4-oxadiazole and indole
fragments were preserved in the first step modification of JC01, and the
effect of substituting position of 1,2,4-oxadiazole on indole ring on the
NO production was firstly taken into mind, which led to the preparation
of analogues 1–3. The cytotoxicity of 1–3 towards RAW264.7 cells at
20 μM were initially evaluated using MTT assay with doxorubicin as
control drug. The results from Table 1 showed that only compound 3
with 1,2,4-oxadiazole on C-5 position of indole ring was non-toxic to-
ward RAW 264.7 cells with the cell viability of 79.38%, compared with
1 and 2 (cell viability < 50%). Next, the anti-inflammatory activity of
1 was determined in the model of NO production in RAW264.7 cells.
The result showed that inhibition ratio of 3 (46.76%) against NO pro-
duction was close to that of hit 1. Taking these results into considera-
tion, the C-5 substitution of 1,2,4-oxadiazole on indole was thought to
be optimal.
towards RAW 264.7cells, compared with the vehicle control (Fig. 2).
Thus, 17 at specific concentration was used in the subsequent in-
vestigation.
As NF-κB is an important transcription factor that regulates the
expression of most pro-inflammatory cytokines such as iNOS, IL-6 and
TNF-α, the NF-κB signaling pathway was then investigated as a likely
target underpinning the anti-inflammatory action of 17. Indeed, in the
immunofluorescence analysis, overlaid fluorescence microscopic
images of LPS-stimulated RAW 264.7 cells revealed that treatment of
17 at 20 μM impeded LPS-stimulated nuclear translocation of p65
(Fig. 3A). Then, we investigated the critical proteins of this signaling
pathway by western blotting to determine the effect of 17 on the NF-κB
activity, as shown in Fig. 3B. Compound 17 strongly suppressed the
phosphorylation of p65 which is indicative of activation of p65 for NF-
κB transcriptional activity, as well as the phosphorylation and de-
gradation of IκB-α, in a dose-dependent manner. Collectively, the anti-
inflammatory effect of compound 17 was at least in part mediated by
inhibition of the NF-κB signaling pathway.
Next, the 5-(1,2,4-oxadiazol-3-yl)-1H-indole core was reasonably
kept, and different substituents were introduced to C-3 position of ox-
adiazol ring to produce 16 analogues (4–19). Most compounds of
analogues 4–14 were toxic toward RAW264.7, and only 4, and 13 were
non-toxic and had inhibition ratio of 47.22%, and 53.94% against NO
production, respectively. In order to decrease the toxicity of this series
of compounds, the benzene ring was replaced by other ring systems,
such as tetrahydropyran (15), cyclopentene (16), furan (17), cyclo-
hexane (18) and cyclobutane (19). It was to our delight that compound
17 had little effect on the growth of RAW 264.7 at 20 μM with cell
viability of 98.96%, suggesting that 17 was non-toxic towards
RAW264.7 cells. Then, compound 17 showed a reduction on NO pro-
duction with inhibitory ratio of 86.60% in comparison with LPS-treated
group. Further, the IC50 value of 17 against NO production was further
To determine the protective effect of 17 on RAW264.7 macrophage
inflammatory responses induced by LPS, we analyzed the mRNA ex-
pression levels of IL-6, IL-1β and TNF-α by real-time PCR. As shown in
Fig. 4A–C, the expressions of IL-6, IL-1β and TNF-α induced by LPS
were significantly up-regulated at the indicated time points, whereas
dose-dependent reductions in LPS stimulated IL-6, IL-1β and TNF-α
mRNA expression levels were observed in macrophages after co-in-
cubation with 17 (p < 0.05). Thus, 17 showed significant inhibition
on IL-6, IL-1β and TNF-α mRNA expressions. At the same time, the
effect of 17 pre-treatment on iNOS synthesis was also investigated.
iNOS was detected on western blot of the cell lysate after exposure to
LPS. As shown in Fig. 4D, the production of iNOS was up-regulated after
the LPS stimulus. However, pre-treatment of the cells with 17 sig-
nificantly reduced the increase in iNOS production.
determined to be 12.84
0.21 μM. In addition, compound 17 showed
NF-κB inhibitory activity with an IC50 value of 1.35
κB pathway luciferase assay (Table 2).
0.39 μM in NF-
In summary, twenty-nine 1,2,4-oxadiazole derivatives were de-
signed and prepared in order to optimize the anti-inflammatory profile
of hit JC01. The bioassay results highlighted compound 17 as the most
promising anti-inflammatory agent, which could significantly inhibit
NO production and NF-κB activation and had significant effects on the
inhibition of pro-inflammatory mediators. The results demonstrated
that 17 decreased NO and iNOS productions in RAW 264.7 macrophage
cells after LPS stimulation. The anti-inflammatory activity of 17 may be
mediated by the NF-κB activation mechanism in the RAW 264.7 cells.
These results indicated that 17 could function as a lead compound
deserving further studies for developing anti-inflammatory drugs.
Alternatively, the 5-phenyl-1,2,4-oxadiazole fragment of hit JC01
was reserved while the indole ring was replaced by various other
groups, which led to the synthesis of analogues 20–29. Except com-
pounds 20, 21 and 26, the others were non-toxic towards RAW 264.7
cells and showed inhibitory activity against NO production with in-
hibitory ratios ranging from 34.28% to 63.58%.
The synthesis of 1,2,4-oxadiazoles JC01 and 1–29 was achieved in
one-step route summarized in Schemes 1 and 2. All of these compounds
were smoothly prepared from the cyclization of substituted benzoic
acids with different hydroxylcarboximidamides under microwave con-
dition. The detailed synthetic procedure and data of 1H, 13C NMR and
HRMS for target compounds were included in Supplementary Material.
Since the potential NF-κB inhibitor 17 showed improved activity
compared with JC01 in both LPS-induced NO production and NF-κB
pathway luciferase assays, it was taken as a model compound for the
follow-up tests. The further results of the MTT assay demonstrated that
17 in the concentration range from 0 to 100 μM had no cytotoxicity
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
2

Y.-Y. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127373
Table 1
Table 1 (continued)
Effects of compounds on the cell viability of RAW 264.7 for 24 h and their
inhibition ratio against NO production at 20 μM.
Compound
Structure
Cell viabilitya
95.68%
Inhibition ratio
of NO
Compound
Structure
Cell viabilitya
Inhibition ratio
production
of NO
production
15
2.75%
JC01
1
64.29%
45.71%
16
17
18
19
24.28%
98.96%
68.81%
85.38%
–
b
11.61%
-
86.60%
–
2
3
31.02%
79.38%
–
46.76%
57.58%
4
85.27%
47.22%
20
21
22
23
24
25
26
42.13%
46.20%
88.97%
85.39%
90.42%
86.24%
28.16%
–
5
6
7
8
9
51.17%
23.59%
16.75%
17.24%
26.44%
–
–
–
–
–
–
43.13%
34.28%
59.89%
63.58%
–
10
11
12
36.55%
41.31%
44.95%
–
–
–
27
28
29
89.59%
85.17%
92.81%
39.33%
48.24%
41.64%
a
Cell viability was expressed as percent cell viability when compared to that
of DMSO vehicle control cells (100%), and cell viability more than 75% at
20 μM was considered to be non-toxic.
13
14
88.08%
22.79%
53.94%
–
b
Not tested.
3

Y.-Y. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127373
Table 2
Results of inhibition on NF-κB activation and NO production.
Compound
IC50 (NF-κB, μM)a
IC50 (NO production, μM)a
JC01
13.83
1.35
0.03
–
2.71
0.39
0.007
26.96
12.84
1.31
0.21
17
b
Bortezomib
Dexamethasone
-
4.16
0.23
a
b
IC50 value was taken as a mean from three independent experiments.
Not tested.
Scheme 1. Synthesis of JC01 and 1–19. Reagents
and conditions: (a) EDCI, HOBT, DIEA, DMF, mi-
crowave heating at 180 °C, 20 min.
Scheme 2. Synthesis of 20–29. Reagents and conditions: (a) EDCI, HOBT, DIEA, DMF, microwave heating at 180 °C, 20 min.
Fig. 2. Effect of 17 on viability of RAW264.7 cells analyzed by MTT method.
4

Y.-Y. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127373
Fig. 3. Inhibition of LPS-stimulated inflammatory
response by 17 through suppression of NF-kB
pathway. (A) Immunofluorescence staining of p65 in
RAW264.7 cells after treatment with 17 for 1 h,
followed by 24 h of stimulation of LPS (1 μg/mL) or
vehicle. Scale bar, 50 μm; representative images
from three individual experiments with similar re-
sults shown. (B) RAW264.7 cells were treated with
various concentrations of 17 for 45 min; phospho-
p65, p65, IκBα and β-Actin were detected by
Western blot. Similar results were obtained in three
independent experiments. The results were ex-
pressed as mean
SD (n = 3). *p < 0.05 vs.
control group; #p < 0.05 vs LPS-induced group.
5

Y.-Y. Zhang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127373
Fig. 4. (A, B and C) Effects of 17 on IL-6(A), IL-1β (B) and TNFα (C) mRNA expressions in LPS-stimulated RAW264.7 macrophages measured by qRT-PCR. RAW264.7
cells were co-treated with different concentrations of 17 and LPS (1 μg/mL) for 24 h. Graphs represent relative expression values, the ratio of different gene to
reference gene. Data are expressed as mean
SD (n = 3); compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001. (D) Western blotting analysis of
iNOS in RAW264.7 cells after LPS stimulation. Data are expressed as mean
##p < 0.01.
SD (n = 3); compared with control, ***p < 0.001; compared with group LPS,
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