Discovery of dronedarone and its analogues as NLRP3 inflammasome inhibitors with potent anti-inflammation activity

Article abstract of DOI:10.1016/j.bmcl.2021.128160

Inhibiting NLRP3 inflammasome activation is a prospective therapeutic strategy for uncontrolled inflammatory diseases. It is the first time that dronedarone, a multiply ion channel blocker, was identified as a NLRP3-inflammasome inhibitor with an IC₅₀ value of 6.84 μM against IL-1β release. A series of novel 5-amide benzofuran derivatives were designed and synthesized as NLRP3-inflammasome inhibitors. Compound 8c showed slightly increased activity (IC₅₀ = 3.85 μM) against IL-1β release. Notably, treatment with 8c could significantly inhibit NLRP3-mediated IL-1β release and ameliorate peritoneal inflammation in a mouse model of sepsis. Collectively, 8c is a promising lead compound for further chemical development as a NLRP3 inhibitor with anti-inflammation effects.

Full text of DOI:10.1016/j.bmcl.2021.128160

Bioorg. Med. Chem. Lett. 46 (2021) 128160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of dronedarone and its analogues as NLRP3 inflammasome
inhibitors with potent anti-inflammation activity
Hao Chena, Xiuhui Chena, Ping Suna, Dan Wu, Hu Yue, Jintao Pan, Xinxuan Li, Cheng Zhang,
Xinyi Wu, Lei Hua, Wenhui Hu*, Zhongjin Yang*
Key Laboratory of Molecular Target & Clinical Pharmacology and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & the Fifth Affiliated
Hospital, Guangzhou Medical University, Guangzhou, Guangdong 511436, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inhibiting NLRP3 inflammasome activation is a prospective therapeutic strategy for uncontrolled inflammatory
diseases. It is the first time that dronedarone, a multiply ion channel blocker, was identified as a NLRP3-
NLRP3 inflammasome
Inhibitor
inflammasome inhibitor with an IC50 value of 6.84 μM against IL-1β release. A series of novel 5-amide benzo-
IL-1β
furan derivatives were designed and synthesized as NLRP3-inflammasome inhibitors. Compound 8c showed
Dronedarone
slightly increased activity (IC50 = 3.85 μM) against IL-1β release. Notably, treatment with 8c could significantly
inhibit NLRP3-mediated IL-1β release and ameliorate peritoneal inflammation in a mouse model of sepsis.
Collectively, 8c is a promising lead compound for further chemical development as a NLRP3 inhibitor with anti-
inflammation effects.
Targeting NLRP3 inflammasome is a new and hot research field with
strong potential to treat inflammatory diseases.1,2 Over-activation of
NLRP3 inflammasome is closely related to various diseases, such as
peritonitis, hyperinflammation following virus infection, inflammatory
bowel disease, Alzheimer’s disease (AD), stroke, gout and nonalcoholic
steatohepatitis (NASH).3–8 Notably, NLRP3ꢀ /ꢀ mice exhibited improved
pathology in a mice model for these diseases.3 NLRP3 inflammasome is a
multiprotein complex that has been extensively studied.9 It is an oligo-
meric protein formed by activation of the sensor protein NLRP3 and then
recruitment of ASC and procaspase-1.10 NLRP3 activation relies on
intracellular secondary signals triggered pathogen-associated molecular
patterns (PAMPs) or damage-associated molecular patterns
(DAMPs),11,12 unlike other inflammasome sensors (AIM2, NLRP1, or
NLRC4) as the direct recognition of external stimuli.10 To date, there are
various intracellular secondary signals to activate NLRP3, such as
intracellular ion fluxes, lysosomal resident cathepsin B, reactive oxygen
species (ROS), and ubiquitin/deubiquitination post-translation modifi-
cation of NLRP3.13 Among these, intracellular ion fluxes are common
and critical triggers signal of NLRP3 inflammasome activation.14
Ion fluxes, including K+ efflux,10,13 Ca2+ mobilization,12 and Clꢀ
efflux,15 have been early proposed to be related to the release of
proinflammatory cytokine IL-1β mediated by NLRP3 inflammasome
activation. Indeed, recent studies found that many ion channel blockers
could inhibit NLRP3 inflammasome activation (Figure 1). For example,
glyburide, a selective inhibitor of ATP-sensitive K+ channel, inhibits IL-
1β release in LPS-activated human monocytes with an IC50 of 12
μ
M.16,17
Another K+ channel blocker β-hydroxybutyrate (BHB) inhibits IL-1β
release in LPS-primed mouse bone marrow-derived macrophages
(BMDMs) with an IC50 of 2 ~ 10 mM.18 As a Ca2+ channel blocker,
nimodipine inhibits Aβ-stimulated IL-1β release from microglia cells
with ~ 48% inhibition at 36 nM.19 The Clꢀ channel blocker ethacrynic
acid inhibits ATP-induced release of IL-1β in human monocytes with the
IC50 of 3
μ
M.20 These findings suggest that blocking ion channel blockers
such as K+, Ca2+, or Clꢀ is a potential strategy against NLRP3 inflam-
masome activation.
Inspiringly, multiply blocking of ion fluxes may have beneficial ef-
fects on inhibition of NLRP3 inflammasome activation. Dronedarone, a
marketed drug, is a multiply ion channel blocker, which can inhibit K+,
Ca2+ and Na+ fluxes.21,22 As expected, in the model of LPS and nigericin-
stimulated NLRP3 inflammasome activation in J774A.1 cells,23 drone-
darone showed good inhibitory potency toward NLRP3 inflammasome
mediated IL-1β release with IC50 of 6.84 μM. The potential of drone-
darone to inhibit NLRP3 inflammasome drove us to identify NLRP3 in-
hibitors with increased potency.
* Corresponding authors.
E-mail addresses: huwenhui@gzhmu.edu.cn (W. Hu), yzj23@gzhmu.edu.cn (Z. Yang).
a
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128160
Received 22 March 2021; Received in revised form 25 May 2021; Accepted 25 May 2021
Available online 29 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128160
Fig. 1. Reported inhibitors of IL-1β as ion channel blockers.
Fig. 2. Structures of dronedarone, 6 and 13.
In the beginning, we found that the intermediate 6 and 13 (Figure 2),
Scheme 2, similar to the previous synthetic route from 2 to 5, interme-
diate 13 was obtained from 10 in three steps. Next, sulfonylation reac-
tion27 of 13 and chlorosulfonic acid afforded intermediate 14.
Treatment of 14 with oxalyl chloride,28 and then condensation with the
corresponding amines29 provided compound 15a ~ 15g in the yield of
25%~62%.
removal of methanesulfonyl or methanesulfonamide, led to the loss of
potency on inhibiting IL-1β release mediated by NLRP3 inflammasome
(IC50 > 100 μM). As the sulfonamide moiety seems to significantly affect
NLRP3 inflammasome inhibiting activity, we focused our interest on
modifying the C-5 aminosulfonyl group of dronedarone.
The preparation of various derivatives of dronedarone is illustrated
in Scheme 1 and Scheme 2 according to similar reported procedures.24,25
Commercially available 2-(bromomethyl)-4-nitrophenol underwent
Wittig reaction25 to provide benzofuran derivative 2. Friedel-Craft
acylation reaction26 of 2 with 4-methoxybenzoyl chloride in the pres-
ence of AlCl3 to afford the intermediate 3 which, upon demethylation
using AlCl3 in chlorobenzene, provided intermediate 4. O-alkylation of 4
with N-butyl-N-(3-chloropropyl)butan-1-amine in the presence of po-
tassium carbonate offered 5, which was further reduced with iron power
to provide the amine 6.25 Compound 7a ~ 7j, 8a ~ 8g, 9a ~ 9k were
obtained from intermediate 6 with various substrate (sulfonyl chlorides,
acyl chlorides, isocyanic acids or aminosulfonyl chlorides) by N-acyla-
tion24 or nucleophilic addition27 in the yield of 12%~85%. As shown in
To evaluate the ability of the compounds to inhibit NLRP3 inflam-
masome activation, all the synthesized compounds were tested for their
ability to inhibit the release of IL-1β mediated via the NLRP3 inflam-
masome activation using LPS and nigericin-stimulated J774A.1 cells. As
shown in Table 1, the prototype dronedarone showed good inhibitory
activity with an IC50 value of 6.84 μM and was used as a benchmark to
compare the potency of other analogs. As mentioned earlier, the sulfonyl
at the C5-position amino group significantly affects the activities against
NLRP3 inflammasome, so we first investigated the effect of different
sizes of R substituents on sulfonamide. In general, small R group on the
C-5 aminosulfonyl moiety resulted in favorable inhibitory activity,
which increased in an order of cyclopropyl < vinyl < Et < Me with IC50
values of 45.5, 25.3, 19.0, and 6.84 μM, respectively. The six-member
2

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128160
Scheme 1. Chemical synthesis of 7a ~ 7j, 8a ~ 8g, 9a ~ 9k. Reagents and conditions: (a) Ph3P, DCM, reflux, 1 h; (b) CH3(CH2)3COCl, Et3N, PhMe, reflux, 1 h; (c) 4-
methoxybenzoyl chloride, AlCl3, DCM, rt, 24 h; (d) AlCl3, PhCl, 80 ◦C, 2 h; (e) N-butyl-N-(3-chloropropyl)butan-1-amine, K2CO3, KI, DMF, 65 ◦C, 5 h; (f) Fe, NH4Cl,
H2O, EtOH, reflux, 1 h; (g) various sulfonyl chlorides, PhMe, Et3N, rt, 2 h; (h) various acyl chlorides, DCM, Et3N, rt, 2 h; (i) various isocyanic acids, DCM, Et3N, 0 ◦C,
2 h.
Scheme 2. Chemical synthesis of 15a ~ 15g. Reagents and conditions: (a) 4-methoxybenzoyl chloride, AlCl3, DCM, rt, 24 h; (b) AlCl3, PhCl, 80 ◦C, 2 h; (c) N-butyl-
N-(3-chloropropyl)butan-1-amine, K2CO3, KI, DMF, 65 ◦C, 5 h; (d) ClSO3H, rt, 12 h; (e) Oxalyl chloride, DMF, DCM, 0 ◦C, 2 h; (f) various amine, Et3N, rt, 12 h.
ring substituents (cyclohexyl, 3-pyridyl, and phenyl) resulted in a 1.5 to
5.0-fold decrease of activities, as compared to that of dronedarone. The
para-substituted phenyl derivatives 7g ~ 7j (Table 1), had no
improvement in potency. The reversal of the C-5 aminosulfonyl group of
dronedarone led to compound 15b which is 3.2 times less potent as an
inhibitor of NLRP3 inflammasome. The attempt to change different
types of amino groups (15a ~ 15g) failed to improve the inhibitory
activities. Bioisosteric replacement is usually a tool of the utmost
importance widely used in analogs design. Therefore, the replacement of
methylsulfonamide function by an acetamido group showed similar
inhibitory activity. Furthermore, various substitutions of R group in this
series exhibited comparable activity to that of the parent compound, and
bioisosteres, had relatively negative effects on inhibition of IL-1β
releasing via NLRP3 inflammasome. For example, the subseries of urea
9a ~ 9h and aminosulfonamide 9i ~ 9k exhibited low inhibitory with
IC50 values of 13.3 ~ 74.8 μM and 7.65 ~ 13.4 μM, respectively. The
potent activity of compound 8c indicated that the compound has the
potential to treat NLRP3 inflammasome-associated inflammatory
diseases.
As the compound 8c showed the most potent inhibition of IL-1β
release, 8c was further selected to investigate its preliminary mecha-
nism. Compound 8c exhibited a concentration-dependent inhibition of
IL-1β release in J774A.1 cells (Figure 3A). The result of immunoblot
analysis (Figure 3B) showed that 8c dose-dependently decreased the
amount of caspase-1 p20 and IL-1β in supernatants from the 8c-treated
cells. Compound 8c did not affect the expression levels of pro-IL-1β, pro-
8c showed more potent activity with an IC50 value of 3.85
μM. The other
sulfonamide bioisosteres, urea and aminosulfonamide function
3

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128160
Table 1
Inhibitory potency against the IL-1β production of designed compounds.
Compound
R =
X =
IC50
6.84
(μМ)
Compound
R =
X =
IC50
6.77
(μМ)
Dronedarone
Me
8a
Me
7a
Et
19.0
45.5
25.3
25.0
10.7
33.4
25.0
12.7
81.6
27.9
26.7
22.2
42.5
83.8
8b
8c
8d
8e
8f
26.7
3.85
5.82
9.03
13.3
7b
7c
7d
7e
7f
8g
9a
9b
9c
9d
9e
9f
>100
13.3
19.2
25.0
74.8
71.2
24.5
18.5
16.1
7g
7h
7i
7j
15a
15b
15d
15e
H
Me
9g
9h
N
O
O
O
O
Cl
S
N
N
N
H
H
H
15f
>100
61.1
9i
9j
11.4
13.4
7.65
O
O
O O
HN
N
S
S
S
S
N
H
N
H
15c*
15g*
O
S
O
O
O
N
N
N
H
>100
9k
O
S
O
O
O
HN
N
N
H
“*” represents structure R and X is combined in one structure.
caspase-1, NLRP3, or ASC in cell lysates. These findings suggest com-
pound 8c indeed suppresses NLRP3 Inflammasome-mediated caspase-1
activation and IL-1β secretion via acting on the inflammasome activa-
tion step.
IL-6 level in vivo (2% in peritoneal fluid and 0% in serum), as well as
TNF-
α in peritoneal fluid. TNF-α in serum was an exception which
showed an 18% reduction as compared to LPS-induced model group.
These results suggest that the inhibitor 8c may markedly suppress
NLRP3 inflammasome-related acute inflammation in sepsis.
Furthermore, we investigated whether 8c could ameliorate the sepsis
in animal models (Figure 4). Mice were injected intraperitoneally with
LPS and showed significantly increased secretion of proinflammatory
In conclusion, dronedarone, a multiply ion channel blocker, was
identified as a novel NLRP3-inflammasome inhibitor. It exhibited a good
inhibitory effect on NLRP3-inflammasome mediated IL-1β release with
cytokines IL-1β, IL-6 and TNF-α in both the serum and peritoneal lavage
fluid compared to the control group.23,30 Treatment with 8c (100 mg/
kg/d) significantly reduced the level of NLRP3-inflammasome mediated
IL-1β in both serum (42%) and peritoneal lavage fluid (22%). We
noticed that compound 8c had no or only slightly inhibitory effects on
an IC50 value of 6.84 μM. A series of 5-amide benzofuran derivatives
were designed, synthesized, and evaluated for IL-1β inhibitory activity.
Of the compounds tested, 8c showed slightly increased biological profile
exhibiting an IL-1β inhibitory activity (IC50) of 3.85
μM. Further
4

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128160
Fig. 3. Compound 8c reduces NLRP3 inflammasome-dependent IL-1β secretion. (A) LPS-primed J774A.1 cells were treated with indicated dose of 8c for 1 h and then
stimulated with nigericin for another 1 h. The level of IL-1β release in cell supernatant detected by ELISA. (B-C) Western blot analyzed the level of NLRP3-related
proteins in cell supernatant and lysate of J774A.1 cells.
Fig. 4. Compound 8c ameliorates LPS-induced sepsis in vivo. (A and B) IL-1β level from C57BL/6 mice that were treated with 100 mg/kg compound 8c before LPS
injection in the serum (A) or peritoneal lavage fluid (B) from mice intraperitoneally injected with LPS in the presence or absence of compound 8c. (C and D) ELISA of
IL-6 in the serum (C) or peritoneal lavage fluid (D) from mice intraperitoneally injected with LPS in the presence or absence of compound 8c. (E and F) ELISA of TNF-
α
in the serum (E) or peritoneal lavage fluid (F) from mice intraperitoneally injected with LPS in the presence or absence of compound 8c. Results are representative
of n = 5 and are means ± SEM. Compared with LPS treated group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Western blotting study found compound 8c suppresses NLRP3 inflam-
masome via acting on the inflammasome activation step. Importantly, in
a sepsis model in mice, the treatment with 8c indeed significantly
inhibited NLRP3-mediated IL-1β release and ameliorate peritoneal
inflammation. Collectively, the results strongly support further chemical
development of 8c as a promising lead for NLRP3 inhibitors and
exploration of their therapeutic potential for inflammatory diseases.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128160.
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