Discovery of N-phenyl-1-(phenylsulfonamido)cyclopropane-1-carboxamide analogs as NLRP3 inflammasome inhibitors

Article abstract of DOI:10.1007/s00044-021-02740-7

Two series of novel NLRP3 inflammasome inhibitors are designed, synthesized, and evaluated in an effort to develop diversified analogs based on the N-(phenylcarbamoyl)benzenesulfonamide scaffold. SAR studies reveal that the sulfonylurea linker can tolerate chemical modifications with either simply changing over the position of carbonyl and sulfonyl group or structurally flexibly inserting a cyclopropyl group, leading to identification of several more potent and diversified NLRP3 antagonists (e.g., 9) with low nanomolar inhibitory activities. Further studies indicate that these two series of compounds with low cytotoxicity exhibited weak effects on the generation of NO and TNF-a. The findings may serve as good starting points for the development of more potent NLRP3 inflammasome inhibitors as valuable pharmacological probes or potential drug candidates.

Full text of DOI:10.1007/s00044-021-02740-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1294–1308
https://doi.org/10.1007/s00044-021-02740-7
ORIGINAL RESEARCH
Discovery of N-phenyl-1-(phenylsulfonamido)cyclopropane-1-
carboxamide analogs as NLRP3 inflammasome inhibitors
Wanwan Li1 Zhongqiang Cao1 Junjie Cheng1 Feiyu Chen1 Shuai Li1 Yiwei Huang1 Long Tai Zheng1
Na Ye
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Received: 26 March 2021 / Accepted: 3 May 2021 / Published online: 16 May 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Two series of novel NLRP3 inflammasome inhibitors are designed, synthesized, and evaluated in an effort to develop
diversified analogs based on the N-(phenylcarbamoyl)benzenesulfonamide scaffold. SAR studies reveal that the sulfonylurea
linker can tolerate chemical modifications with either simply changing over the position of carbonyl and sulfonyl group or
structurally flexibly inserting a cyclopropyl group, leading to identification of several more potent and diversified NLRP3
antagonists (e.g., 9) with low nanomolar inhibitory activities. Further studies indicate that these two series of compounds
with low cytotoxicity exhibited weak effects on the generation of NO and TNF-a. The findings may serve as good starting
points for the development of more potent NLRP3 inflammasome inhibitors as valuable pharmacological probes or potential
drug candidates.
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Keywords Microglia cells Lipopolysaccharide (LPS) NLRP3 inflammasome Nigericin IL-1β Inhibitors
Introduction
reactive oxygen species (ROS) [2]. As a proinflammatory
cytokine, IL-1β is produced by activated microglia cells to
trigger inflammatory responses in brain [3]. Unlike tumor
necrosis factor α (TNF-α) and IL-6, secretion of IL-1β needs
activation of NLR family pyrin domain containing 3
(NLRP3) mediated inflammasome besides activation of
nuclear factor-kappa B (NF-κB) signaling pathway [4]. Once
secreted, IL-1β can stimulate the surrounding microglia or
astrocytes to produce another proinflammatory factors such
as TNF-α, iNOS, COX-2, and IL-6, which contributed to
neuronal cell death and injuries [3]. Therefore, inhibition of
NLRP3 inflammasome activation would be an effective
therapeutic strategy for neuroinflamamtory diseases.
Microglia-mediated neuroinflammation actively implicated
in the progression of neurodegenerative diseases such as
Parkinson’s disease, Alzheimer’s disease, Multiple sclerosis,
and HIV-associated dementia [1]. As the resident macro-
phage cells in brain, microglia continually monitor sur-
rounding tissue to maintain homeostasis of brain in
physiological conditions, but in neurodegenerative or
inflammatory conditions, it is often activated and can pro-
duce number of proinflammatory factors including IL-1β as
well as neurotoxic mediators such as nitric oxide (NO) and
NLRP3 inflammasome as the most well-known inflam-
masome is a cytosolic multiprotein signaling complex that
mediates the secretion of potent inflammatory mediators [5–
7], and is associated with the pathogenesis of many com-
mon neurodegenerative diseases [8–22] including Parkin-
son’s disease, Alzheimer disease, and ischemic stroke.
Activation of the NLRP3 inflammasome is known to be
controlled by activation of two step signal, priming step
(signal 1) and activating step (signal 2). The signal 1 is
induced by activation of transcriptional factor NF-κB,
resulting in upregulation of NLPR3 and pro-IL-1β. The
signal 2 is triggered by various intra- or extra-stimuli such
as extracellular ATP, nigericin, viral RNA, lysosomal
These authors contributed equally: Wanwan Li, Zhongqiang Cao
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02740-7.
* Long Tai Zheng
zhenglongtai@suda.edu.cn
* Na Ye
yena@suda.edu.cn
1
Jiangsu Key Laboratory of Neuropsychiatric Diseases and College
of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu
215123, China

Medicinal Chemistry Research (2021) 30:1294–1308
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Fig. 1 Small molecule inhibitors
targeting the NLRP3
inflammasome pathway
Fig. 2 Drug design strategy for the current work
damage, aggregated amyloid beta, ROS production, or
mitochondrial metabolism following signal 1 activation.
Upon response to those signaling events, NLRP3 initiates
recruitment of the adapter apoptosis-associated speck-like
protein containing a C-terminal caspase recruitment domain
(ASC) and caspase-1, resulting in production of mature IL-
1β and IL-18 [6, 7].
Many small molecules have been developed as direct or
indirect NLRP3 inflammasome inhibitors (e.g., Fig. 1), but
there are still no NLRP3 antagonists approved for ther-
apeutic use [6, 7, 23–25]. One of the most well-
characterized NLRP3 inflammasome inhibitors is the sul-
fonylurea analog—MCC950 (also called CRID3 and
CP‑446,773), which was initially identified by Perregaux
macrophages [35, 36]. Later, three different research groups
reported that MCC950 can tolerate its furan moiety to be
modified with phenyl rings [37–39]. Currently, its sulfo-
nylurea linker was reported to be replaced with N‑cyano-
sulfoximineurea [38] and ester substituted linker [40]
resulting in nanomolar activity in THP1 cells and human
peripheral blood mononuclear cells, respectively.
As depicted in Fig. 2, we designed and synthesized two
newly diversified series as NLPR3 inflammasome inhibitors
by chemical modifications of the sulfonylurea linker on the
N-(phenylcarbamoyl)benzenesulfonamide
scaffold
of
MCC950 and replacement of its furan ring B with phenyl
rings. Series I was designed by changing over the position
of carbonyl and sulfonyl group in the sulfonylurea linker,
while series II was designed to make the compact linker
flexible by inserting a cyclopropyl group in the middle in
view of versatile cyclopropyl fragments frequently appeared
in preclinical/clinical drug molecules [41]. Herein, we
report that such structural modifications on the N-(phe-
nylcarbamoyl)benzenesulfonamide scaffold have resulted in
the discovery of several structural diverse NLRP3 inflam-
masome inhibitors such as 9 with low nanomolar inhibitory
activities.
et al. as the most potent inhibitor of IL‑1β release (IC50
=
8.1 nM in bone-marrow-derived macrophage (BMDM)
cells) [26]. MCC950 as a useful tool compound displays
efficacy in a wide range of animal disease models [7]
including Alzheimer’s disease [27], Parkinson’s disease
[19], multiple sclerosis [28], myocardial infarction [29],
atherosclerosis [30], stroke [31], asthma and allergic airway
inflammation [32], and cryopyrin-associated periodic syn-
dromes [16]. Although MCC950 was tested in phase II
clinical trials for rheumatoid arthritis, it was not progressed
further due to its liver toxicity [7]. The combination of its
metabolically reactive furan moiety and sulfonylurea linker,
which may generate the cis-enedione and isocyanate in vivo
respectively, and subsequent conjugate with biological
macromolecules, might underlie the observed toxicity
[33, 34]. In 2016, Robertson group provided first insight
into the SAR of the hexahydroindacene moiety of MCC950,
and found that this tricyclic moiety is largely indispensable
for good bioactivity in human monocyte-derived
Results and discussion
Chemistry
The synthesis of new derivatives based on N-(phe-
nylcarbamoyl)benzenesulfonamide scaffold with chemical
optimizations on the linker and phenyl ring B is outlined in
Schemes 1 and 2. 1,2,3,5,6,7-hexahydro-s-indacen-4-amine 1,

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Medicinal Chemistry Research (2021) 30:1294–1308
Scheme 1 The synthesis of series I compounds. Reagents and con-
ditions: (a) (i) ClSO2NCO, Et3N, t-BuOH, CH2Cl2, 0 °C, rt, 2 h; (ii)
conc. HCl:EtOAc = 1:2, rt, 1 h; 50% for two steps. (b) RCOOH, CDI,
DBU, THF, 70 °C, 24 h, 20–97%. (c) NaBH4, EtOH, 80 °C, 12 h,
70%. (d) conc. HCl:EtOAc = 1:2, rt, 2 h, 97%. (e) Acryloyl chloride,
DIPEA, DMF, rt, 3 h, 38%
Scheme 2 The synthesis of
series II compounds. Reagents
and conditions: (a) (i) EDCI,
HOBt, Et3N, DCM, rt, 24 h; (ii)
conc. HCl: EtOAc = 1: 2, rt, 1 h;
59% for two steps. (b) RCOOH,
EDCI, HOBt, Et3N, DCM, rt,
24 h, 27–91%. (c) RSO2Cl,
pydine, DCM, rt, 16 h, 45–56%.
(d) NaBH4, EtOH, 80 °C, 12 h,
42%. (e) conc. HCl: EtOAc = 1:
2, rt, 2 h, 91%. (f) Acryloyl
chloride, DIPEA, DMF, rt, 3 h,
52%. (g) LiOH, THF, H2O, rt,
2 h, 48%. (h) Cu, MeNH2,
100 °C, 22 h, 43%
prepared from commercially available 2,3-dihydro-1H-
indene by following a literature procedure [42], was used as
the key intermediate. Condensation of 1 with chlorosulfonyl
isocyanate and 1-((tert-butoxycarbonyl)amino)cyclopro-
pane-1-carboxylic acid, and then deprotection, provide key
intermediates 2 and 13 in 50% and 59% overall yield,
respectively. As outlined in Scheme 1, series I compounds
3–7 and 9–10 were prepared by condensation of 2 with the
commercially available acids under the treatment of CDI
and DBU in 20–97% yields. Similarly, amidation or sul-
fonamidation of amide 13 with the commercially available
corresponding acids or sulfonyl chloride yielded series II
compounds 14–18, 20, 23–25, 27, and 29–30 in 33–91%
yields (Scheme 2). Subsequently, reduction of alcohols 7
and 18 with NaBH4 at reflux yield 8 and 19 in 70% and
42%, respectively. Hydrolysis of esters 25 with LiOH
yielded corresponding acids 26 in 48% yield. Deprotection
of Boc group in 10 and 20 obtained amines 11 and 21 in
97% and 91% yield, followed by acylation with acryloyl
chloride led to 12 and 22 in 38% and 52% yield, respec-
tively. The synthesized final compounds were analyzed by
1H NMR, 13C NMR, and high-resolution mass spectra
(HRMS).
Cytotoxicity of the target compounds 3–12 and 14–
30
Since cytotoxicity may affect the inhibitory effects of
compounds on cytokines production in microglia cells, we
firstly evaluated the effect of compounds on cell viability by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide (MTT) assay in BV-2 microglia cells. As shown in
Fig. 3, most compounds at concentrations of 20 μM showed
no obvious cytotoxicity in BV-2 microglia cells, with the
fact that the relative cell viability is higher than 95% except
for that of compound 10 (88.34 1.68%). Thus, 20-μM
concentration was chosen for subsequent screening
experiments.

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Table 2 The inhibitory effect of series II compounds on the production
of IL-1β in LPS/nigericin-stimulated primary microglia cells
No.
X
Ar
Inhibition rate of IL-1β (%)a
10 µM 20 µM
Fig. 3 The effect of compounds on the viability of BV-2
microglia cells
14
CO 4-F-Ph
38.50 1.51 50.80 3.02
47.42 4.37 67.73 2.92
49.56 1.26 64.10 2.53
51.55 4.37 67.74 2.92
56.68 3.02 67.95 1.51
57.75 4.54 65.39 1.51
51.55 4.37 63.56 2.92
59.88 4.54 66.67 1.51
15
CO 4-Me-Ph
16
CO 4-OMe-Ph
CO 4-CF3-Ph
Table 1 The inhibitory effect of series I compounds on the production
of IL-1β in LPS/nigericin-stimulated primary microglia cells
17
18
CO 4-COMe-Ph
CO 4-CHOHMe-Ph
CO 4-NHBoc-Ph
CO 4-NH2-Ph
19
20
21
22
CO 4-Acrylamide-Ph
CO 2-C(Me)2OH-Furan
CO 4-C(Me)2OH-Ph
CO 3-COOMe-Ph
CO 3-COOH-Ph
CO 3-NHMe
–
–
–
45.76
45.76 4.36
48.30
0
No.
R
Inhibition rate of IL-1β (%)a
23
24
0
10 µM
20 µM
25
68.31 2.53 75.64 0.63
50.45 2.53 64.10 1.26
38.50 4.54 50.84 3.02
70.10 1.26 70.51 1.26
65.63 2.53 70.51 1.26
73.20 2.92 87.63 2.92
3
F
50.90 0.63
56.70 2.92
59.88 4.54
–
55.64 0.63
65.32 1.46
66.67 1.51
26
4
Me
28
5
OMe
CF3
29
SO2 4-OMe-Ph
SO2 4-CF3-Ph
6
48.38 0
30
bMCC950
7
Ac
41.71 3.02
57.75 1.51
60.95 3.02
–
55.13 1.51
64.10 3.02
69.23 3.02
8
CHOHMe
i-Pr
aThe primary microglia cells were primed with LPS (100 ng/ml) for 3 h
and then the cells were pretreated with compounds (10 and 20 μM) for
30 min before stimulation of nigericin (25 μM). After treatment of
nigericin for 30 min, the amounts of IL-1β in cell culture supernatants
were measured by ELISA. MCC950 served as positive control, while
DMSO served as vehicle control. All data were expressed as mean S.
D (n = 3) and are representative of three independent experiments
9
10
NHBoc
NH2
48.38 0
11
–
45.76 7.19
62.82 1.89
87.63 2.92
12
Acrylamide
54.03 2.53
73.20 2.92
bMCC950
bThe concentrations of MCC950 were 100 nM and 1 μM. “−”
indicates that the activity is not tested
aThe primary microglia cells were primed with LPS (100 ng/ml) for 3 h
(10 and 20 μM) 30 min before stimulation of nigericin (25 μM). After
treatment of nigericin for 30 min, the amounts of IL-1β in cell culture
supernatants were measured by ELISA. MCC950 served as positive
control, while DMSO served as vehicle control. All data were
expressed as mean S.D (n = 3) and representative of three indepen-
dent experiments
(ELISA) (Tables 1 and 2). Compounds with inhibition at
20 μM higher than 50% were further evaluated for the
second round of screening at 10 μM. And the inhibition of
reference compound MCC950 is 87.6% and 73.2% at 1 μM
and 100 nM, respectively.
bThe concentrations of MCC950 were 100 nM and 1 μM. “−”
indicates that the activity is not tested
As shown in Table 1, we initially investigated the electronic
effect of substitutions on the phenyl ring B in series I. Intro-
duction with electro-withdrawing groups as in compounds 3,
6, and 7 generally led to a slight loss of activity compared to
that of compounds 4, 5, and 8–9 with electro-donating groups.
The most potent compound 9 with a bulky isopropyl group
displays an inhibitory activity of 61% at 10 µM. Unexpectedly,
compounds with bulky group, hydrogen bond donor or
Michael receptor (as in compound 10–12), all decrease
potency, suggesting that the substitutions on the phenyl ring B
play an important role in NLRP3 inhibition.
In vitro evaluation on IL-1β production inhibition
The NLRP3 inhibition of each compound was first tested at
a concentration of 20 μM by measuring IL-1β secretion
from primary microglia cells, the innate immune cells in the
central nervous system (CNS). Lipopolysaccharide (LPS)-
primed primary microglia cells were pretreated with the test
compounds, and then stimulated with inflammasome acti-
vator nigericin. The levels of IL-1β in cell culture media
were measured by enzyme-linked immunosorbent assay

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Medicinal Chemistry Research (2021) 30:1294–1308
Table 3 The IC50 values of potent compounds in inhibiting the
production of IL-1β in primary microglia cells
In series II as shown in Table 2, a flexible linker by
inserting a cyclopropyl group, results in a dramatically dif-
ferent SAR result from series I. Their activities are indepen-
dent on the electronic and steric effect of substitutions on the
4-position of phenyl ring B (as in compounds 14–18). The
compounds with electro-withdrawing groups such as F (e.g.,
14 vs. 3) reduce potency, while compounds with CF3, and Ac
group enhance potency (e.g., 17 vs. 6; 18 vs. 7). In addition,
the compounds with electro-donating groups such as Me and
OMe (e.g., 15 vs. 4; 16 vs. 5) reduce potency, while com-
pounds with NHBoc and NH2 group enhance potency (e.g., 20
vs. 10; 21 vs. 11). These results suggest that the 4-position of
the phenyl ring B of series II can tolerate chemical mod-
ifications with either electron-withdrawing or -donating
groups. Compound 22 with a covalent warhead, which may
interact with NLRP3 proteins, is also inactive. Moreover,
introduction of the same furan B ring as MCC950 (as in
compound 23) lost potency. Alternatively, introduction of the
phenyl ring B with the same substitution, 2-hydroxypropan-2-
yl group, still lost activity. Surprisingly, switching the sub-
stitution to 3-position (as in compounds 25–38) leads to the
discovery of ester 25 as the most potent compound in series II,
with an inhibitory activity of 68.3% at 10 µM. To our great
delight, the potency also boosts by replacement an amide with
a sulfamide linker in compounds 16–17, leading to com-
pounds 29–30 with an inhibitory activity of 65–70% at 10 µM
(Fig. 3).
No
cLogPa
TPSAb
IL-1β
IC50 (µM)c
4
3.07
3.09
2.73
4.03
4.25
3.26
3.07
3.91
2.75
3.29
2.60
3.54
2.54
75.27
84.50
95.50
75.27
58.20
75.27
78.42
96.53
84.22
84.50
84.50
75.27
108.64
6.36 0.80
6.64 0.82
8.00 0.90
4.48 0.65
8.60 0.93
6.07 0.78
7.93 0.90
7.53 0.88
7.74 0.89
2.05 0.31
2.12 0.33
2.78 0.44
–
5
8
9
17
18
19
20
21
25
29
30
MCC950
ahttp://146.107.217.178/lab/alogps/start.html
bhttps://www.molinspiration.com/cgi-bin/properties#
cThe primary microglia cells were primed with LPS (100 ng/ml) for 3 h
and then the cells were pretreated with compounds for 30 min before
stimulation of nigericin (25 μM). After treatment of nigericin for
30 min, the amounts of IL-1β in cell culture supernatants were
measured by ELISA. All data were expressed as mean S.D (n = 3)
and are representative of three independent experiments
All these above findings suggest that the sulfonylurea
linker on the N-(phenylcarbamoyl)benzenesulfonamide
scaffold by replacing furan ring B of MCC950 with phenyl
rings can tolerate chemical modifications with either simply
changing over the position of carbonyl and sulfonyl group
or inserting a cyclopropyl group to be flexible.
concentration dependently inhibited the production of IL-
1β with an IC50 value of 30.62 1.49 nM (Fig. 5A, B).
Compound 9 is slightly more lipophilic than compounds
25, 29, and 30, and its TPSA value is slightly lower than
the other three compounds (Table 3), indicating that
compound 9 may have better membrane permeability
[43]. Distinct IC50 value of primary microglia cells and
BMDMs may be attributed to distinct expression of drug-
metabolism-related genes or different cell membrane
permeability between these two cell types.
Compounds 9, 25, 29–30 inhibit the production of
IL-1β in a concentration-dependent manner
We selected compounds with inhibition of the production
of IL-1β at 10 μM higher than 50% to further determine
their half-maximal inhibitory concentration (IC50, Table
3). Calculated partition coefficient (clogP) and topologi-
cal polar surface area (TPSA) are the fundamental phy-
sicochemical properties to evaluate their drug-like
properties. All these selected compounds have the
acceptable clogP and TPSA values for CNS drug candi-
dates (Table 3). Among these analogs, compound 9 in
series I and compounds 25 and 29–30 in series II
remarkably inhibited the production of IL-1β in a
concentration-dependent manner in primary microglia
cells, with IC50 values of 2.05–4.48 μM (Fig. 4). Com-
pounds 9, 25, and 29–30 were also evaluated their inhi-
bition on the production of IL-1β in the BMDMs
stimulated by LPS/ATP. Surprisingly, only compound 9
We also evaluated the effect of compound 9 on TNF-a
production in LPS/ATP-treated BMDM. As shown in Fig.
5C, D, compound 9 did not impaired TNF-a secretion,
suggesting that the inhibition of IL-1β production was
NLRP3-specific.
In vitro evaluation of NO inhibition
LPS induces expression of pro-IL-1β and NLRP3 in
microglia cells via activation of NF-κB signaling pathway
[44, 45]. NO is an important neuroinflammatory factor that
is secreted by LPS-activated microglia cells and contributes
to neuronal damage and apoptosis, and its production is
independent of NLRP3 inflammasome activation, but
dependent on activation of NF-κB signaling pathway [46–
49]. Therefore, NO production may partly reflect the levels

Medicinal Chemistry Research (2021) 30:1294–1308
1299
Fig. 4 Compounds 9, 25, 29,
and 30 block NLRP3
inflammasome activation in
primary microglia cells. A–D
The primary microglia cells
were primed with LPS (100 ng/
ml) for 3 h and then the cells
were pretreated with indicated
concentration of compounds for
30 min before stimulation of
nigericin (25 μM). After
treatment of nigericin for
30 min, the amounts of IL-1β in
cell culture supernatants were
measured by ELISA. All data
were expressed as mean S.D
(n = 3) and are representative of
three independent experiments.
**P < 0.01 and ***P < 0.001
compared to LPS/nigericin-
treated alone group
Fig. 6 The effects of compounds on NO production in LPS-activated
BV-2 microglia cells. BV-2 microglia cells were pretreated with
compounds (20 μM) for 30 min and then incubated with LPS for 24 h.
The production of NO in cell culture supernatant was measured by
Griess agents. The formula to calculate the percent of inhibition is
following: % inhibition = [1 − (NO amounts of compound treatment
in the presence of LPS − NO amounts of compound alone treatment)/
(NO amounts of LPS alone treatment − NO amounts of control)] ×
100. Data shown as mean S.D. (n = 3)
Fig. 5 Compounds 9 and MCC950 suppress ATP-induced NLRP3
inflammasome activation in BMDMs. The BMDMs were primed with
LPS (100 ng/ml) for 3 h and then the cells were pretreated with indi-
cated concentration of compounds for 30 min before stimulation of
ATP (3 mM). After treatment of nigericin for 30 min, the amounts of
IL-1β (A, B) or TNF-α (C, D) in cell culture supernatants were
measured by a specific ELISA kit, respectively. All data were
expressed as mean S.D (n = 3) and are representative of three
independent experiments. **P < 0.01 and ***P < 0.001 compared to
LPS/ATP alone treated group
of pro-IL-1β and NLRP3 in LPS-activated microglia cells.
We thus further confirmed whether our compounds inhib-
ited the activation of NF-κB signaling pathway by evalu-
ating their inhibitory effects on LPS-induced NO
generation. As shown in Fig. 6, all these NLRP3 inhibitors
exhibited weak inhibitory activity on NO production by
<40% inhibition at 20 µM, indicating that inhibition of IL-
1β production was independent of signal 1.

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Medicinal Chemistry Research (2021) 30:1294–1308
Conclusions
water. The organic layer was dried over Na2SO4, filtered,
and concentrated in vacuum to give the crude product. The
residue was purified by column chromatography on silica
gel [Petroleum ether (PE)/EtOAc = 3/1].
Two series of novel NLRP3 inflammasome inhibitors based
on the N-(phenylcarbamoyl)benzenesulfonamide scaffold
by replacing furan ring B of MCC950 with phenyl rings
have been designed and synthesized. The SAR results
indicate that their sulfonylurea linker can tolerate chemical
modifications with either simply changing over the position
of carbonyl and sulfonyl group or structurally flexibly
inserting a cyclopropyl group. All these modification efforts
allow us to further tune MCC950 to achieve more potent
and structurally diverse NLRP3 inflammasome inhibitors,
e.g., compound 9 with an IC50 value of 30.62 nM in
BMDMs stimulated by LPS/ATP. Compound 9 may hold
promise as a potential drug candidate toward novel ther-
apeutics against human diseases, and serve as a valuable
pharmacological probe to elucidate the physiological func-
tions of NLRP3 inflammasome.
To remove the Boc protective group, the product from
above was dissolved in 6 mL 1:2 (v/v) mix of conc. HCl:EA
and let stay at rt for 1 h. After concentrated and neutralized
with ammonia, the reaction mixture was extracted with
DCM (30 mL × 2). The combined organic layer was washed
with brine (30 mL). dried over anhydrous Na2SO4, filtered,
and concentrated in vacuum to give the crude product. The
residue was purified by column chromatography on silica
gel (PE/EtOAc = 2/1), giving product 2 as white solid
(192 mg, 44%).1H NMR (400 MHz, DMSO-d6) δ 8.24 (s,
1H), 6.96 (s, 1H), 6.72 (s, 2H), 2.93 (t, J = 7.1 Hz, 4H),
2.79 (t, J = 7.1 Hz, 4H), 1.98–1.89 (m, 4H).
General procedures 1 (GP1)
A mixture of benzoic acids (1 eq) and CDI (1.1 eq) in THF
(2 mL/mmol) was heated at 70 °C for 45 min under a
nitrogen atmosphere, cooled down and added 2 (1.1 eq) and
DBU (3 eq). The mixture was stirred for 20–24 h. The
reaction mixture was diluted with DCM (30 mL), and
washed with 1-N HCl and brine (30 mL). The organic layer
was dried over anhydrous Na2SO4, filtered, and con-
centrated in vacuum to give the crude product. The residue
was purified by column chromatography on silica gel to
afford corresponding products.
Experimental
Chemistry
All commercially available starting materials and solvents
were reagent grade, and used without further purification.
Reactions were performed under a nitrogen atmosphere in
dry glassware with magnetic stirring. Column chromato-
graphy was carried out on silica gel (200–300 mesh). All
reactions were monitored using thin layer chromatography
on silica gel plates. Visualization of the developed chroma-
tograms was performed with detection by UV (254 and
365 nm). 1H NMR spectra were recorded on 400 or 600 MHz
(100 or 150 MHz for 13C NMR) agilent NMR spectrometer.
1H and 13C NMR spectra were recorded with TMS as an
internal reference. Chemical shifts were expressed in ppm,
and J values were given in Hz. HRMS were recorded on a
GCT PremierTM (CI) Mass Spectrometer or an Agilent 6540
UHD Accurate-Mass Q-TOF LC/MS.
3-Fluoro-N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoyl)benzamide (3)
According to GP1, 4-fluorobenzoic acid (21 mg, 0.3 mmol)
and 2 (88 mg, 0.35 mmol) were converted to the desired
product 3 (19 mg, 17%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 11.95 (s, 1H), 9.71 (s, 1H), 7.97
(dd, J = 8.2, 5.6 Hz, 2H), 7.34 (t, J = 8.7 Hz, 2H), 7.00 (s,
1H), 2.78 (m, 8H), 1.92–1.81 (m, 4H). 13C NMR (101-
MHz, DMSO-d6) δ 165.2 (d, JC-F = 251.96), 165.1, 144.0,
141.3, 131.5 (d, JC-F = 9.44), 129.3 (d, JC-F = 2.83), 128.4,
120.0, 116.1 (d, JC-F = 322.11), 33.0, 30.9, 25.8. HRMS
(ESI) calcd for C19H20FN2O3S [M + H]+: 375.1179, found
375.1176.
N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoylcarbamate (2)
To a stirred solution of chlorosulfonyl isocyanate (0.20 ml,
1.75 mmol) in anhydrous dichloromethane (3 mL) at 0 °C
was added t-butanol (0.16 mL, 1.75 mmol) in the same
solvent. After a period of 30 min, the resulting solution and
triethylamine (0.25 mL, 1.75 mmol) were slowly added into
a solution of 1 (300 mg, 1.75 mmol) in 2 mL of dichlor-
omethane. The reaction did not rise above 5 °C. The
resulting reaction solution was allowed to warm up to room
temperature for over 2 h. The reaction mixture diluted with
30 mL of dichloromethane, washed with 1-N HCl and
N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)sulfamoyl)-3-
methylbenzamide (4)
According to GP1, 4-methylbenzoic acid (20 mg,
0.3 mmol) and 2 (88 mg, 0.35 mmol) were converted to the
1
desired product 4 (46 mg, 41%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 11.82 (s, 1H), 9.62 (s, 1H), 7.80 (d,
J = 7.8 Hz, 2H), 7.30 (d, J = 7.7 Hz, 2H), 6.99 (s, 1H),

Medicinal Chemistry Research (2021) 30:1294–1308
1301
2.83–2.72 (m, 8H), 2.36 (s, 3H), 1.91–1.81 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ 166.1, 143.9, 143.5, 141.2,
130.1, 129.6, 128.7, 128.6, 119.9, 33.0, 30.9, 25.8, 21.5.
HRMS (ESI) calcd for C20H23N2O3S [M + H]+: 371.1429,
found 371.1427.
converted to the desired product 9 (58 mg, 60%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 9.62
(s, 1H), 7.82 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 7.4 Hz, 2H),
7.00 (s, 1H), 3.00–2.92 (m, 1H), 2.83–2.73 (m, 8H),
1.91–1.80 (m, 4H), 1.22 (s, 3H), 1.21 (s, 3H).13C NMR
(151 MHz, DMSO-D6) δ 166.0, 153.9, 143.9, 141.2, 130.5,
128.8, 128.5, 127.0, 119.9, 33.9, 33.0, 30.9, 25.8, 23.9.
HRMS (ESI) calcd for C22H27N2O3S [M + H]+: 399.1742,
found 399.1738.
N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)sulfamoyl)-4-
methoxybenzamide (5)
According to GP1, 4-methoxybenzoic acid (44 mg,
0.29 mmol) and 2 (80 mg, 0.32 mmol) were converted to the
desired product 5 (22 mg, 20%) as a white solid. H NMR
tert-Butyl(4-((N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoyl)carbamoyl)phenyl)carbamate (10)
1
(400 MHz, DMSO-d6) δ 11.71 (s, 1H), 9.57 (s, 1H), 7.89 (d,
J = 8.5 Hz, 2H), 7.03 (d, J = 8.6 Hz, 2H), 6.99 (s, 1H), 3.83
(s, 3H), 2.77 (m, 8H), 1.90–1.81 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ 165.5, 163.2, 143.9, 141.3, 130.7,
128.6, 124.8, 119.9, 114.3, 56.0, 33.0, 30.9, 25.8. HRMS
(ESI) calcd for C20H23N2O4S [M + H]+: 387.1379, found
387.1374.
According to GP1, 4-((tert-butoxycarbonyl)amino) benzoic
acid (60 mg, 0.25 mmol) and 2 (71 mg, 0.28 mmol) were
converted to the desired product 10 (60 mg, 51%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.76
(s, 1H), 9.57 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.54 (d, J =
8.2 Hz, 2H), 6.99 (s, 1H), 2.88–2.67 (m, 8H), 1.93–1.81 (m,
4H), 1.48 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 165.5,
153.0, 144.2, 143.9, 141.3, 129.7, 128.5, 125.8, 119.9,
117.6, 80.2, 33.0, 30.8, 28.5, 25.8. HRMS (ESI) calcd for
C24H30N3O5S [M + H]+: 472.1906, found 472.1902.
N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)sulfamoyl)-3-
(trifluoromethyl)benzamide (6)
According to GP1, 4-(trifluoromethyl)benzoic acid (34 mg,
0.18 mmol) and 2 (50 mg, 0.20 mmol) were converted to the
desired product 6 (42 mg, 55%) as a white solid. H NMR
(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)sulfamoyl)-4-(1-
hydroxyethyl)benzamide (8)
1
(400 MHz, DMSO-d6) δ 12.20 (s, 1H), 9.82 (s, 1H), 8.06 (d,
J = 7.7 Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.01 (s, 1H), 2.77
(m, 8H), 1.93–1.81 (m, 4H). 13C NMR (151 MHz, DMSO-
d6) δ 165.2, 144.0, 141.2, 136.6, 132.7 (q, JC-F = 32.1),
126.1 (d, JC-F = 3.38), 126.0 (q, JC-F = 272.99), 120.0,
33.0, 30.9, 25.8. HRMS (ESI) calcd for C20H20F3N2O3S [M
+ H]+: 425.1147, found 425.1141.
Compound 7 (100 mg, 0.25 mmol) was dissolved in 2-ml
ethanol, and sodium borohydride (12 mg, 0.30 mmol) was
added to the mixture. The reaction was refluxed at 80 °C for
12 h. The reaction mixture was extracted with DCM
(30 mL × 2), and washed with brine. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated in
vacuum to give the crude product. The residue was purified
by column chromatography on silica gel (PE/EtOAc = 1/1)
Acetyl-N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoyl)benzamide (7)
1
to the desired product 8 (70 mg, 70%) as a white solid. H
NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 9.64 (s, 1H),
7.85 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 7.00 (s,
1H), 5.33 (s, 1H), 4.78 (d, J = 6.1 Hz, 1H), 2.84–2.72 (m,
8H), 1.92–1.83 (m, 4H), 1.33 (d, J = 6.4 Hz, 3H).13C NMR
(101 MHz, DMSO-d6) δ 166.0, 152.8, 143.9, 141.3, 131.1,
128.5, 128.5, 125.9, 120.0, 68.2, 33.0, 30.9, 26.1, 25.9.
HRMS (ESI) calcd for C21H25N2O4S [M + H]+: 401.1535,
found 401.1530.
According to GP1, 4-acetylbenzoic acid (30 mg,
0.18 mmol) and 2 (50 mg, 0.20 mmol) were converted to the
desired product 7 (26 mg, 36%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 12.09 (s, 1H), 9.76 (s, 1H), 8.03 (d,
J = 8.0 Hz, 2H), 7.97 (d, J = 8.1 Hz, 2H), 6.98 (s, 1H),
2.80–2.69 (m, 8H), 2.60 (s, 3H), 1.90–1.79 (m, 4H). 13C
NMR (151 MHz, DMSO-d6) δ 198.1, 165.5, 144.0, 141.2,
140.1, 136.4, 128.9, 128.7, 128.3, 120.0, 33.0, 30.9, 27.5,
25.8. HRMS (ESI) calcd for C21H23N2O4S [M + H]+:
399.1379, found 399.1374.
1
Amino-N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoyl)benzamide (11)
N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)sulfamoyl)-4-
isopropylbenzamidee (9)
To remove the Boc protective group, compound 10 (60 mg,
0.13 mmol) from above was dissolved in 1:2 (v/v) mix of
conc. HCl: EtOAc (6 mL) and let stay at rt for 1 h. After
concentrated and neutralized with ammonia, the reaction
mixture was extracted with DCM (30 mL × 2). The
According to GP1, 4-isopropylbenzoic acid (40 mg,
0.24 mmol) and compound 2 (67 mg, 0.26 mmol) were

1302
Medicinal Chemistry Research (2021) 30:1294–1308
combined organic phases were then washed with brine
(30 mL), dried over anhydrous Na2SO4, filtered, and con-
centrated in vacuum to give the residue. The residue was
purified by column chromatography on silica gel (DCM/
MeOH = 40/1) to the desired product 11 (46 mg, 97%) as a
gel (PE/EtOAc = 1/2), giving pure product (134 mg, 67%)
as white solid.
To a stirred suspension of the above compound (134 mg,
0.66 mmol) in DCM (2 mL), EDCI (255 mg, 1.33 mmol),
HOBt (180 mg, 1.33 mmol), and triethylamine (0.71 mL,
2 mmol) were added, followed by the compound 1 (114 mg,
0.66 mmol). The clear solution was stirred at room tempera-
ture 22 h. The reaction mixture diluted with DCM (30 mL),
and washed with 1-N HCl. The aqueous solution was
extracted with EtOAc (20 mL × 2), and the combined organic
phases were washed with brine (30 mL), dried over anhydrous
Na2SO4, filtered, and concentrated in vacuum to give the crude
product. To remove its Boc protective group, the crude pro-
duct from above was dissolved in 1:2 (v/v) mix of conc. HCl:
EtOAc (6 mL), and let stay at rt for 1 h. After concentrated and
neutralized with ammonia, the reaction mixture was extracted
with DCM (30 mL × 2). The combined organic phases were
then washed with brine (30 mL), dried over anhydrous
Na2SO4, filtered, and concentrated in vacuum to give the crude
product. The residue was purified by column chromatography
on silica gel (PE/EtOAc = 1/2) to the desired product 13
(100 mg, 59%) as a white solid. 1H NMR (400 MHz, DMSO-
d6) δ 9.52 (s, 1H), 6.92 (s, 1H), 2.79 (t, J = 7.0 Hz, 4H), 2.67
(t, J = 7.0 Hz, 4H), 2.49 (s, 2H), 1.96 (m, 4H), 1.11 (d, J =
2.8 Hz, 2H), 0.83 (d, J = 2.8 Hz, 2H).
1
white solid. H NMR (400 MHz, DMSO-d6) δ 11.29 (s,
1H), 9.35 (s, 1H), 7.63 (d, J = 8.1 Hz, 2H), 6.98 (s, 1H),
6.53 (d, J = 8.1 Hz, 2H), 5.94 (s, 2H), 2.87–2.71 (m, 8H),
1.93–1.79 (m, 4H). 13C NMR (151 MHz, DMSO-d6) δ
165.6, 153.6, 143.8, 141.2, 130.6, 128.8, 119.7, 118.8,
112.9, 33.0, 30.8, 25.8. HRMS (ESI) calcd for
C19H22N3O3S [M + H]+: 372.1382, found 372.1378.
Acrylamido-N-(N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
sulfamoyl)benzamide (12)
Compound 11 (70 mg, 0.19 mmol) and DIPEA (0.10 mL,
0.57 mmol) were dissolved in dry DMF (3 mL) and the
temperature of the solution was lowered to 0 °C. A solution
of acryloyl chloride (0.02 mL, 0.23 mmol) was added
dropwise. The reaction temperature was increased gradually
from 0 °C to room temperature and stirring was continued
3 h at room temperature. Solvent was suspended in 10%
HCl (10 mL) and then extracted with DCM (30 mL × 2).
The combined organic extracts were washed with sat.
NaHCO3 (20 mL), dried over anhydrous Na2SO4, and
evaporated to give the crude product. The residue was
purified by column chromatography on silica gel (PE/
EtOAc = 1/1) to the desired product 12 (30 mg, 38%) as a
General procedures (GP2)
To a stirred aromatic compounds (1.5 eq) in DCM (2 mL/
mmol), EDCI (1 eq), HOBt (1 eq) and triethylamine (1.5 eq)
were added, followed by compound 13 (1 eq).The clear
solution was stirred at room temperature 20–24 h. The
reaction mixture was diluted with DCM (30 mL), and
washed with 1-N HCl and water. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated in
vacuum to give the crude product. The residue was purified
by column chromatography on silica gel to afford the cor-
responding products.
1
white solid. H NMR (400 MHz, DMSO-d6) δ 11.77 (s,
1H), 10.46 (s, 1H), 9.58 (s, 1H), 7.88 (d, J = 8.3 Hz, 2H),
7.76 (d, J = 8.5 Hz, 2H), 6.99 (s, 1H), 6.46 (dd, J = 16.8,
10.1 Hz, 1H), 6.30 (d, J = 16.8 Hz, 1H), 5.81 (d, J =
10.1 Hz, 1H), 2.82–2.72 (m, 8H), 1.91–1.82 (m, 4H). 13C
NMR (151 MHz, DMSO-d6) δ 165.6, 164.0, 143.9, 143.3,
141.2, 132.0, 130.0, 128.6, 128.2, 127.5, 119.8, 119.1,
33.0, 30.9, 25.8. HRMS (ESI) calcd for C22H24N3O4S [M
+ H]+: 426.1488, found 426.1487.
1-Amino-N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
4-Fluoro-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
cyclopropanecarboxamide (13)
carbamoyl)cyclopropyl)benzamide (14)
1-Amino cyclopropanecarboxylic acid (100 mg, 0.99 mmol)
was dissolved in a mixture of dioxane (3.0 mL) and 0.5-N
NaOH (3.0 mL). To this, solution was added di-tert-butyl
dicarbonate (0.28 mL, 1.5 mmol) and the resulting mixture
stirred at rt for 15 h. The reaction mixture was concentrated
under reduced pressure, and diluted with EtOAc (30 mL)
and 1 N HCl (10 mL). The aqueous solution was extracted
with EtOAc (20 mL × 2), and the combined organic phases
were washed with brine (30 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica
According to GP2, 4-fluorobenzoic acid (51 mg,
0.36 mmol) and compound 13 (55 mg, 0.24 mmol) were
converted to the desired product 14 (65 mg, 72%) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 9.09
(s, 1H), 8.00 (m, 2H), 7.29 (t, J = 8.5 Hz, 2H), 6.94 (s, 1H),
2.78 (m, 4H), 2.64 (m, 4H), 2.00–1.86 (m, 4H), 1.39 (s, 2H),
1.04 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.9,
167.0, 164.4 (d, JC-F = 248.70), 143.0, 138.7, 131.3, 130.7
(d, JC-F = 8.92), 130.5, 118.3, 115.3 (d, JC-F = 21.65), 35.4,
32.9, 30.6, 25.6, 16.7. HRMS (ESI) calcd for C23H24FN2O2
[M + H]+: 379.1822, found 379.1819.

Medicinal Chemistry Research (2021) 30:1294–1308
1303
N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)-4-methylbenzamide(15)
white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H),
9.24 (s, 1H), 8.04 (dd, J = 17.7, 8.2 Hz, 4H), 6.94 (s, 1H),
2.80 (t, J = 6.9 Hz, 4H), 2.66 (t, J = 7.1 Hz, 4H), 2.62 (s,
3H), 1.95 (m, 7.0 Hz, 4H), 1.42 (d, J = 2.3 Hz, 2H), 1.07 (d,
J = 2.4 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 198.2,
169.8, 167.2, 143.0, 139.0, 138.8, 138.6, 130.5, 128.5,
128.2, 118.3, 35.5, 32.9, 30.6, 27.4, 25.6, 16.7. HRMS
(ESI) calcd for C25H27N2O3 [M + H]+: 403.2022, found
403.2018.
According to GP2, p-toluic acid (22 mg, 0.16 mmol) and
compound 13 (40 mg, 0.16 mmol) were converted to the
desired product 15 (20 mg, 33%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.98 (s, 1H), 7.84 (d,
J = 7.8 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 6.94 (s, 1H), 2.79
(t, J = 6.9 Hz, 4H), 2.66 (t, J = 7.0 Hz, 4H), 2.35 (s, 3H),
2.00–1.85 (m, 4H), 1.40 (d, J = 2.5 Hz, 2H), 1.04 (d, J =
2.5 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 170.0,
167.9, 143.0, 141.5, 138.6, 131.9, 130.5, 128.9, 128.2,
118.2, 35.4, 32.9, 30.6, 25.6, 21.4, 16.6. HRMS (ESI) calcd
for C24H27N2O2 [M + H]+: 375.2073, found 375.2070.
tert-Butyl(4-((1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)carbamoyl)phenyl)carbamate (20)
According to GP2, 4-((tert-butoxycarbonyl)amino)benzoic
acid (138 mg, 0.58 mmol) and compound 13 (100 mg,
0.39 mmol) were converted to the desired product 20
(91 mg, 49%) as a white solid. 1H NMR (400 MHz, DMSO-
d6) δ 9.61 (s, 1H), 9.19 (s, 1H), 8.91 (s, 1H), 7.85 (d, J =
8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 6.93 (s, 1H), 2.79 (t,
J = 6.7 Hz, 4H), 2.65 (t, J = 6.8 Hz, 4H), 2.01–1.85 (m,
4H), 1.48 (s, 9H), 1.38 (s, 2H), 1.03 (s, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 170.1, 167.5, 153.0, 143.0, 142.8,
138.6, 130.5, 129.0, 128.0, 118.2, 117.2, 79.9, 35.4, 32.9,
30.6, 28.5, 25.6, 16.6. HRMS (ESI) calcd for C28H34N3O4
[M + H]+: 476.2549, found 476.2547.
N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)-4-methoxybenzami-de (16)
According to GP2, 4-methoxybenzoic acid (53 mg,
0.35 mmol) and compound 15 (60 mg, 0.24 mmol) were
converted to the desired product 19 (78 mg, 83%) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.91
(s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H),
6.93 (s, 1H), 3.80 (s, 3H), 2.79 (t, J = 6.9 Hz, 4H), 2.65 (t,
J = 6.9 Hz, 4H), 1.97–1.87 (m, 4H), 1.38 (d, J = 2.2 Hz,
2H), 1.03 (d, J = 2.2 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 174.9, 172.2, 166.8, 147.8, 143.3, 135.3,
134.8, 131.8, 123.0, 118.3, 60.5, 40.1, 37.7, 35.4, 30.3,
21.4. HRMS (ESI) calcd for C24H27N2O3 [M + H]+:
391.2022, found 391.2015.
N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)-5-(2-hydroxypropan-2-yl)furan-2-carboxamide
(23)
To a solution of i-propylamine (0.12 mL, 0.99 mmol) in
THF (1 mL) was added 2.4 n-butyllithium (0.33 mL,
0.99 mol) in hexanes at −15 to −8 °C with stirring under a
nitrogen atmosphere. The mixture was diluted with THF
(1 mL) and cooled to −74 °C. The furan-2-carboxylic acid
(50 mg, 0.45 mmol) in THF (1 mL) was added so as to keep
the temperature at −70 to −77 °C. After 30 min in the cold,
acetone (28 mg, 0.50 mmol) in THF (1 mL) was added
dropwise while keeping the temperature below −70 °C, and
then the reaction mixture was allowed to come to room
temperature. The mixture was quenched with water, and
most of the THF was removed by evaporation under
vacuum. The aqueous residue was extracted with diethy-
lether (20 mL × 2) and acidified with 5-N HCl. The product
was extracted with diethylether (30 mL × 3), dried with
anhydrous Na2SO4, and concentrated to give crude 4-(2-
hydroxypropan-2-yl)furan-2-carboxylic acid as an oil
(50 mg, 65%).
((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)-4-(trifluoromethyl)benzamide (17)
According to GP2, 4-(trifluoromethyl)benzoic acid (90 mg,
0.46 mmol) and compound 13 (80 mg, 0.31 mmol) were
converted to the desired product 17 (61 mg, 46%) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 2H), 8.14
(d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 6.94 (s, 1H),
2.80 (t, J = 7.0 Hz, 4H), 2.66 (t, J = 7.0 Hz, 4H), 2.01–1.86
(m, 4H), 1.42 (d, J = 2.6 Hz, 2H), 1.07 (d, J = 2.6 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) δ 169.7 (q, JC-F = 272.99),
166.8, 143.0, 138.8, 138.5, 131.5 (q, JC-F = 31.79), 130.5,
129.1, 125.4 (q, JC-F = 3.68), 124.4 (q, JC-F = 272.56),
118.3, 35.4, 32.9, 30.6, 25.5, 16.7. HRMS (ESI) calcd for
C24H24F3N2O2 [M + H]+: 429.1790, found 429.1788.
4-Acetyl-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)benzamide (18)
According to GP2, the above 4-(2-hydroxypropan-2-yl)
furan-2-carboxylic acid (50 mg, 0.29 mmol) and compound
13 (51.2 mg, 0.2 mmol) were converted to the desired pro-
duct 23 (22 mg, 27%) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 8.28 (s, 1H), 7.37 (s, 1H), 7.28 (s, 1H), 7.12 (s,
According to GP2, 4-acetylbenzoic acid (68 mg,
0.41 mmol) and compound 13 (80 mg, 0.31 mmol) were
converted to the desired product 18 (108 mg, 86%) as a

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Medicinal Chemistry Research (2021) 30:1294–1308
1H), 6.98 (s, 1H), 6.32 (s, 1H), 2.87 (t, J = 7.0 Hz, 4H),
2.78 (t, J = 6.3 Hz, 4H), 2.13–1.99 (m, 4H), 1.70 (s, 2H),
1.61 (s, 6H), 1.18 (s, 2H). 13C NMR (151 MHz, CDCl3) δ
169.5, 169.3, 149.3, 143.9, 137.1, 134.3, 129.5, 129.0,
118.5, 116.3, 115.2, 111.3, 77.2, 77.0, 76.8, 36.2, 32.9,
30.8, 29.7, 25.6, 16.8. HRMS (ESI) calcd for C24H29N2O4
[M + H]+: 409.2127, found 409.2122.
129.7, 121.3, 118.1, 112.7, 35.4, 32.9, 30.6, 25.6, 16.6.
HRMS (ESI) calcd for C25H27N2O4 [M + H]+: 419.1971,
found 419.1969.
3-Bromo-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)benzamide (27)
According to GP2, 3-bromobenzoic acid (86 mg,
0.43 mmol) and compound 13 (74 mg, 0.29 mmol) were
converted to the desired product 25 (76 mg, 60%) as a white
(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)-4-(2-hydroxypropan-2-yl)benzamide (24)
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H), 9.18
4-Isopropylbenzoic acid (100 mg, 0.60 mmol) was dis-
solved in a solution of potassium hydroxide (82 mg,
1.46 mmol) in water (2.5 mL). To the reaction mixture was
added a solution of potassium permanganate (192 mg,
1.21 mmol) in water (2.5 mL). The combined mixture was
allowed to stir at 60 °C for 2 h. The reaction mixture was
cooled to 0 °C and treated with ethylene glycol and cooled
to 0 °C. The solid were removed by filtration and the filtrate
was acidified to pH 1 by addition of 6-N HCl solution. The
solid was removed by filtration, and the fitrate was extracted
with diethylether (30 mL × 2). The combined organic
extracts were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (PE/EtOAc = 1/1), giving 4
(2-hydroxypropan-2-yl)benzoic acid (102 mg, 42%) as a
white solid.
(s, 1H), 8.16 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.73 (d, J =
7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 6.95 (s, 1H), 2.80 (t, J =
6.9 Hz, 4H), 2.66 (t, J = 7.0 Hz, 4H), 2.01–1.88 (m, 4H),
1.40 (d, J = 2.4 Hz, 2H), 1.05 (d, J = 2.4 Hz, 2H).13C NMR
(151 MHz, DMSO-d6) δ 169.8, 166.6, 143.0, 138.8, 137.0,
134.3, 131.0, 130.7, 130.5, 127.3, 121.8, 118.3, 35.4, 33.0,
30.6, 25.6, 16.6. HRMS (ESI) calcd for C23H24BrN2O2
[M + H]+: 439.1021, found 439.1021.
Acetyl-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)benzamide (19)
Compound 18 (70 mg, 0.17 mmol) was dissolved in 2 ml of
ethanol and sodium borohydride (34 mg, 0.90 mg) was
added to the mixture. The reaction was refluxed at 80 °C for
12 h. The reaction mixture was diluted with 30 mL of
dichloromethane, and washed with brine. The organic layer
was dried over anhydrous Na2SO4, filtered, and con-
centrated in vacuum to give the crude product. The residue
was purified by column chromatography on silica gel (PE/
EtOAc = 1/2) to the desired product 19 (29 mg, 42%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H),
9.00 (s, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.0 Hz,
2H), 6.94 (s, 1H), 5.27 (d, J = 4.0 Hz, 1H), 4.84–4.70 (m,
1H), 2.79 (t, J = 6.9 Hz, 4H), 2.66 (t, J = 7.0 Hz, 4H),
1.98–1.89 (m, 4H), 1.40 (d, J = 2.2 Hz, 2H), 1.31 (d, J =
6.3 Hz, 3H), 1.04 (d, J = 2.3 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 170.0, 167.9, 151.2, 143.02, 138.6, 133.0,
130.5, 128.0, 125.2, 118.2, 68.2, 40.4, 40.2, 40.1, 40.0,
39.8, 39.7, 39.5, 35.4, 32.9, 30.6, 26.4, 25.6, 16.6. HRMS
(ESI) calcd forC25H29N2O3 [M + H]+: 405.2178, found
405.2178.
According to GP2, the above 4-(2-hydroxypropan-2-yl)
benzoic acid (102 mg, 0.56 mmol) and compound 13
(96 mg, 0.38 mmol) were converted to the desired product
1
24 (52 mg, 33%) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.20 (s, 1H), 8.99 (s, 1H), 7.88 (d, J = 8.1 Hz,
2H), 7.52 (d, J = 8.2 Hz, 2H), 6.93 (s, 1H), 5.13 (s, 1H),
2.79 (t, J = 6.9 Hz, 4H), 2.66 (t, J = 6.9 Hz, 4H), 2.01–1.86
(m, 4H), 1.42 (s, 6H),1.40 (d, J = 2.5 Hz, 2H) 1.04 (d, J =
2.2 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 170.0,
167.9, 154.3, 143.0, 138.6, 132.4, 130.5, 127.8, 124.5,
118.2, 71.2, 35.4, 32.9, 32.3, 30.6, 25.6, 16.6. HRMS (ESI)
calcd for C26H31N2O3 [M + H]+: 419.2335, found
419.2334.
Methyl 3-((1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)carbamoyl-benzoate (25)
According to GP2, 3-(methoxycarbonyl)benzoic acid
(104 mg, 0.58 mmol) and compound 13 (100 mg, 0.4 mmol)
were converted to the desired product 25 (152 mg, 89%) as
Amino-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
carbamoyl)cyclopropyl)benzamide (21)
1
a white solid. H NMR (400 MHz, DMSO-d6) δ 9.31 (s,
To remove the Boc protective group, the compound 20
(100 mg, 0.21 mmol) was dissolved in 6 mL 1:2 (v/v) mix
of conc. HCl:EtOAc and let stay at rt for 1 h. It was then
concentrated down on rotovap and then on high vacuum.
After neutralizing with ammonia, the reaction mixture was
diluted with DCM (30 mL × 2), and washed with brine. The
1H), 9.28 (s, 1H), 8.54 (s, 1H), 8.20 (d, J = 7.4 Hz, 1H),
8.10 (d, J = 7.4 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 6.94 (s,
1H), 3.89 (s, 3H), 2.79 (m, 4H), 2.66 (m, 4H), 2.00–1.86 (m,
4H), 1.40 (s, 2H), 1.06 (s, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 170.4, 168.1, 152.2, 143.0, 138.3, 130.5,

Medicinal Chemistry Research (2021) 30:1294–1308
1305
organic layer was dried over Na2SO4, filtered, and con-
centrated in vacuum to give the crude product. The residue
was purified by column chromatography on silica gel (PE/
EtOAc = 1/2) to the desired product 21 (72 mg, 91%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H),
8.60 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 6.92 (s, 1H), 6.53 (d,
J = 8.4 Hz, 2H), 5.63 (s, 2H), 2.79 (t, J = 6.9 Hz, 4H), 2.65
(t, J = 7.0 Hz, 4H), 1.97–1.83 (m, 4H), 1.36 (d, J = 2.5 Hz,
2H), 0.99 (d, J = 2.5 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 170.4, 168.1, 152.2, 143.0, 138.3, 130.5,
129.7, 121.3, 118.1, 112.7, 35.4, 32.9, 30.6, 25.6, 16.6.
HRMS (ESI) calcd for C23H26N3O2 [M + H]+: 376.2025,
found 376.2024.
and concentrated in vacuum to give the crude product. The
residue was purified by column chromatography on silica
gel (DCM/MeOH = 40/1) to the desired product 26 (47 mg,
1
48%) as a white solid. H NMR (400 MHz, DMSO-d6) δ
13.07 (s, 1H), δ 9.30 (s, 1H), 9.25 (s, 1H), 8.53 (s, 1H), 8.16
(d, J = 7.6 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.58 (t, J =
7.7 Hz, 1H), 6.94 (s, 1H), 2.79 (t, J = 6.9 Hz, 4H), 2.66
(t, J = 7.0 Hz, 4H), 1.99–1.89 (m, 4H), 1.40 (s, 2H), 1.06 (d,
J = 2.4 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.9,
167.5, 167.3, 143.0, 138.8, 135.2, 132.4, 132.2, 131.2,
130.5, 129.2, 128.7, 118.3, 35.4, 33.0, 30.6, 25.6, 16.7.
HRMS (ESI) calcd for C24H25N2O4 [M + H]+: 405.1814,
found 405.1815.
4-Acrylamido-N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)
N-(1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
carbamoyl)cyclopropyl)benzamide (22)
cyclopropyl)-3-(methylamino)benzamide (28)
Compound 21 (106 mg, 0.28 mmol) and DIPEA (0.10 mL,
0.57 mmol) were dissolved in dry DMF (2 mL) and the
temperature of the solution was lowered to 0 °C. A solution
of acryloyl chloride (0.03 mL, 0.31 mmol) was added
dropwise. The reaction temperature was increased gradually
from 0 °C to room temperature and stirring was continued
3 h at room temperature. Solvent was suspended in 10%
HCl (10 mL) and then extracted with DCM (30 mL × 2).
The combined organic extracts were washed with saturated
solution of Na2CO3 (20 mL), dried over anhydrous Na2SO4,
and evaporated to give the crude product. The residue was
purified by column chromatography on silica gel (DCM/
MeOH = 40/1) to the desired product 22 (62 mg, 52%) as a
A magnetic stirrer bar, compound 27 (50 mg, 0.11 mmol),
methylamine (2.2 mL, 16 mmol, a 40% aqueous solution),
and copper powder (4 mg, 0.006 mmol) were added to a
screw-capped tube (15 mL) under air. The reaction mixture
was stirred and heated at 100 °C, and the mixture was
reacted for 22 h. Spin dry under vacuum. The residue was
purified by column chromatography on silica gel (PE/
EtOAc = 1.5/1) to the desired product 28 (19 mg, 43%) as
yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.24
(t, J = 7.8 Hz, 1H), 7.09 (s, 1H), 7.05–6.96 (m, 2H), 6.77 (d,
J = 7.9 Hz, 1H), 2.88 (m, 7H), 2.80 (t, J = 7.1 Hz, 4H),
2.11–1.99 (m, 4H), 1.73 (s, 2H), 1.18 (s, 2H). 13C NMR
(151 MHz, CDCl3) δ 169.5, 169.3, 149.3, 143.9, 137.1,
134.3, 129.5, 129.0, 118.5, 116.3, 115.2, 111.3, 77.2, 77.0,
76.8, 36.2, 32.9, 30.8, 29.7, 25.6, 16.8. HRMS (ESI) calcd
for C24H28N3O2 [M + H]+: 390.2182, found 390.2176.
1
white solid. H NMR (400 MHz, DMSO-d6) δ 10.37 (s,
1H), 9.22 (s, 1H), 8.97 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H),
7.73 (d, J = 8.5 Hz, 2H), 6.94 (s, 1H), 6.45 (dd, J = 16.9,
10.1 Hz, 1H), 6.29 (d, J = 16.1 Hz, 1H), 5.79 (d, J =
11.0 Hz, 1H), 2.79 (t, J = 6.8 Hz, 4H), 2.66 (t, J = 6.9 Hz,
4H), 2.02–1.87 (m, 4H), 1.39 (d, J = 2.2 Hz, 2H), 1.04 (d,
J = 2.2 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 174.8,
172.1, 168.6, 147.8, 146.8, 143.4, 136.8, 135.3, 134.3,
133.9, 132.7, 123.4, 123.0, 45.2, 45.1, 45.0, 44.8, 44.7,
44.5, 44.4, 44.3, 40.1, 37.7, 35.4, 30.3, 21.4. HRMS (ESI)
calcd for C26H28N3O3 [M + H]+: 430.2131, found
430.2127.
(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)-1-(4-
methoxyphenylsulfonamido)cyclopropanecarboxamide (29)
Into a 50-mL round-bottom flask equipped with a magnetic
stir bar and under nitrogen was weighted 4-
methoxybenzene-1-sulfonyl chloride (62 mg, 0.3 mmol) in
dichloromethane (10 mL). The suspension was cooled to
0 °C and then compound 13 (50 mg, 0.2 mmol) was added,
followed by the addition of pyridine (0.03 mL, 0.4 mmol).
The resulting suspension was stirred at room temperature
for 16 h. The reaction mixture was poured into a 60-mL
separatory funnel and washed with 1-N aqueous HCl
solution (20 mL), water (20 mL), and brine (20 mL). The
organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. Purification by
column chromatography through silica gel, eluting with
100% DCM to 5% MeOH in DCM as a gradient afforded
3-((1-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)
cyclopropyl)carbamoyl)benzoic acid (26)
Compound 25 (100 mg, 0.24 mmol) was dissolved in THF,
and then dissolved lithium hydroxide (11.5 mg, 0.48 mmol)
in water was added dropwise into the above mixture and
stirred at room temperature for 2 h. Spin dry THF, add citric
acid to neutralize to weak acidity. The reaction mixture was
diluted with DCM (30 mL × 2), and washed with brine. The
organic layer was dried over anhydrous Na2SO4, filtered,
1
compound 29 as a white solid (48 mg, 56%). H NMR
(400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.58 (s, 1H), 7.74 (d,

1306
Medicinal Chemistry Research (2021) 30:1294–1308
J = 8.0 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 6.95 (s, 1H), 3.83
(s, 3H), 2.80 (t, J = 6.7 Hz, 4H), 2.61 (t, J = 6.8 Hz, 4H),
1.96 (dd, J = m, 4H), 1.15 (s, 2H), 0.81 (s, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 169.5, 162.8, 143.2, 138.0, 133.8,
130.1, 129.1, 118.3, 114.8, 56.1, 36.7, 32.9, 30.6, 25.6,
14.5. HRMS (ESI) calcd for C23H27N2O4S [M + H]+:
427.1692, found 427.1685.
incubated in complete medium with 50-mg/ml recombinant
mouse M-CSF for 7 days. Animal study was approved by
Institutional Review Board of Soochow University and was
performed in accordance with the guidelines published in
the National Institutes of Health.
Cell viability test
N-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-1-(4-
(trifluoromethyl)phenylsulfonamido)
cyclopropanecarboxamide (30)
BV-2 microglia cell viability was measured by MTT
reagent as described previously [50].
Enzyme-linked immunosorbent assay (ELISA)
Into a 50-mL round-bottom flask equipped with a magnetic
stir bar and under nitrogen was weighted 4-(trifluoromethyl)
benzene-1-sulfonyl chloride (75 mg, 0.3 mmol) in DCM
(10 mL). The suspension was cooled to 0 °C and then
compound 13 (50 mg, 0.2 mmol) was added, followed by
the addition of pyridine (0.03 mL, 0.4 mmol). The resulting
suspension was stirred at room temperature for 16 h. The
reaction mixture was poured into a 60-mL separatory funnel
and washed with 1-N aqueous HCl solution (3 × 20 mL),
water (20 mL), and brine (20 mL). The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. Purification by column chromato-
graphy through silica gel, eluting with 100% DCM to 2.5%
MeOH in DCM as a gradient afforded compound 30 as a
The levels of IL-1β or TNF-α in the supernatants of cells
were measured by IL-1β or TNF-α specific ELISA kit
according to the instruction of the manufacturer [50].
Nitrite quantification
The levels of NO in the supernatants of cells were detected
using Griess reagent as described previously [47, 48].
Acknowledgements This work was supported by the National Natural
Science Foundation of China (31970909, 81703330, and 81372688),
the Natural Science Foundation of Jiangsu Province (BK20170347),
the Priority Academic Program Development of the Jiangsu Higher
Education Institutes (PAPD), Suzhou Municipal Science and Tech-
nology Bureau (SYS2020092), and the Jiangsu Key Laboratory of
Neuropsychiatric Diseases (BM2013003).
1
white solid (39 mg, 45%). H NMR (400 MHz, DMSO-d6)
δ 9.04 (s, 1H), 8.99 (s, 1H), 8.01 (dd, J = 15.4, 8.0 Hz, 4H),
6.94 (s, 1H), 2.78 (m, 4H), 2.55 (m, 4H), 1.98–1.89 (m, 4H),
1.23 (s, 2H), 0.87 (s, 2H).13C NMR (151 MHz, DMSO-d6)
δ 169.1, 146.3, 143.2, 138.2, 132.7 (q, JC-F = 32.09) 130.0,
128.0, 127.0 (q, JC-F = 3.78), 124.5 (q, JC-F = 272.49),
118.4, 36.8, 32.9, 30.5, 25.5, 14.7. HRMS (ESI) calcd for
C23H24F3N2O3S [M + H]+: 465.1460, found 465.1456.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Cell culture
References
BV-2 murine microglia cells were grown in DMEM sup-
plemented with 10% heat-inactivated FBS, penicillin (100
U/ml), streptomycin (10 μg/ml), and 5% CO2 at 37 °C. Rat
primary microglia were isolated from 1- to 2-day-old neo-
natal Sprague-Dawley rats as described previously [50]. In
brief, the chopped cerebral cortex were digested with papain
(2 mg/ml) 37 °C for 30 min and then mechanistically dis-
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mm flasks and cultured at 37 °C, 5% CO2. Fourteen days
after culturing, the microglia cells were collected by shaking
and centrifugation. Murine BMDM were prepared from
femoral bone marrow cells as described previously [37]. In
brief, bone marrow cells were obtained by flushing method.
After removing contaminated red cells using erythrocytes
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