Discovery of chalcone analogues as novel NLRP3 inflammasome inhibitors with potent anti-inflammation activities

Article abstract of DOI:10.1016/j.ejmech.2021.113417

NLRP3 inflammasome activation plays a critical role in inflammation and its related disorders. Herein we report a hit-to-lead effort resulting in the discovery of a novel and potent class of NLRP3 inflammasome inhibitors. Among these, the most potent lead

Full text of DOI:10.1016/j.ejmech.2021.113417

European Journal of Medicinal Chemistry 219 (2021) 113417
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of chalcone analogues as novel NLRP3 inflammasome
inhibitors with potent anti-inflammation activities
Cheng Zhang 1, Hu Yue 1, Ping Sun 1, Lei Hua, Shuli Liang, Yitao Ou, Dan Wu, Xinyi Wu,
Hao Chen, Ying Hao, 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
Article history:
NLRP3 inflammasome activation plays a critical role in inflammation and its related disorders. Herein we
report a hit-to-lead effort resulting in the discovery of a novel and potent class of NLRP3 inflammasome
inhibitors. Among these, the most potent lead 40 exhibited improved inhibitory potency and almost no
toxicity. Further mechanistic study indicated that compound 40 inhibited the NLRP3 inflammasome
activation via suppressing ROS production. More importantly, treatment with 40 showed remarkable
therapeutic effects on LPS-induced sepsis and DSS-induced colitis. This study encourages further
development of more potent inhibitors based on this chemical scaffold and provides a chemical tool to
identify its cellular binding target.
Received 1 March 2021
Received in revised form
22 March 2021
Accepted 23 March 2021
Available online 1 April 2021
Keywords:
NLRP3 inflammasome
Inhibitor
© 2021 Elsevier Masson SAS. All rights reserved.
Michael acceptor
1. Introduction
Increasing evidence shows that NLRP3 inflammasome is closely
related to various human diseases, such as peritonitis, colonic
NLRP3 inflammasome is a prospective drug target against
inflammation and its related diseases [1]. It is a protein complex in
the cytoplasm composed of the inflammasome sensor molecule
(NLRP3 protein), the adaptor protein ASC and the effector compo-
nent (pro-caspase-1) [2]. The activation of NLRP3 inflammasome
requires a two-stage process: a priming stage and an activation
stage. The priming step is referred to the upregulation of tran-
inflammation, nonalcoholic steatohepatitis (NASH), and Alzheimer’s
disease (AD) [8e15]. NLRP3 inflammasome activation involves
several mechanisms: intracellular ion flux (such as Kþ efflux) [16],
release of lysosomal resident cathepsin B [17], production of reactive
oxygen species (ROS) [18], and ubiquitin/deubiquitination post-
translation modification of NLRP3 [19]. Yet there is no unified state-
ment about its main activation mechanism. As shown in Fig. 1, many
small molecules showed different levels of inhibitory activities on
NLRP3 inflammasome [20e28]. Among these, most inhibitors
exhibited a micromolar inhibitory activity. The most potent MCC950
scription of NLRP3 and prointerleukin-1b (pro-IL-1b) through the
nuclear factor kappa B (NF- B) pathway after pathogen-associated
k
molecular patterns (PAMPs) or damage associated molecular pat-
terns (DAMPs) stimulation of a Toll-like receptor (TLR). In the sec-
ond activation step, the primed cell is stimulated by a second
stimulus (typically a DAMP in sterile inflammation), which causes
activation of the NLRP3 inflammasome [3e5]. The activated NLRP3
inflammasome induces pro-caspase-1 self-cleavage into active
inhibits IL-1b release with high potency (IC50 in BMDMs: 7.5 nM) and
specificity [20]. However, it was not developed as it was found to
induce liver toxicity in clinical trials, which may be caused by its
metabolically reactive furan moiety or high drug dose of 1200 mg per
day [1]. Notably, Somalix (Inflazome’s), IFM-2427 (IFM Tre/Novar-
tis’s), and NT-0167 (Nodthera’s) are currently undergoing phase II or I
clinical trials [29]. In addition, another NLRP3 inhibitor OLT1177 (non-
caspase-1, which further cleaves pro-IL-1b into mature IL-1b and
releases it into extracellular [6,7].
dose dependence, IC50: ~0.1 mM in J744A.1) [21], chemically different
from MCC950, is currently undergoing phase II clinical trials for
treatment of osteoarthritis, but it is also facing the same test of high-
dose drug safety (1000 mg doses of OLT1177 daily) [1]. Therefore,
there is still great enthusiasm in seeking novel types of small-
molecule inhibitors against NLRP3 inflammasome activation.
* Corresponding author.
** Corresponding author.
E-mail addresses: huwenhui@gzhmu.edu.cn (W. Hu), yzj23@gzhmu.edu.cn
(Z. Yang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113417
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Fig. 1. Compound structures and potency of identified NLRP3 inhibitors.
To address this issue, we screened compounds that are either in
clinical use or have reached late-stage clinical trials and identified
(summarized in Fig. 2: Box A and B). This further systematic SAR
exploration led to the final lead compound. The design of com-
pounds 2e15 was mainly to understand the tolerability of bilateral
regions to various functional group substitutions. Analogues 16e29
were designed to examine the importance of substituents of phenyl
elafibranor (GFT505) as an inhibitor against IL-1
by NLRP3 inflammasome. GFT505, developed by the French com-
pany, was a peroxisome proliferation-activated receptor- (PPAR-
) dual agonist as a drug candidate to treat NASH [30,31]. Un-
b release mediated
a/d
a
/d
in b position to their biological activities. Then, a further design of
fortunately, in May 2020, it was disclosed to fail to hit the pre-
defined primary endpoint of NASH with an improvement of
fibrosis, and terminated in a Phase III study. Despite this, the
chalcone scaffold was at least proved to possess good safety. In our
screening model, GFT505 showed low inhibitory potency toward
compounds 30e40 was to evaluate whether the replacement of
methylthiol moiety with various groups could improve biological
activity.
The preparation of the designed compounds was achieved by
the conditions as described in Scheme 1. Briefly, compounds 2e4,
16e32 and 35e40 were obtained by the ClaisenꢀSchmidt
condensation of substituted acetophenones 1aꢀi and benzalde-
hyde 2aꢀm. Treatment of 3 with various isocyanates in toluene at
70 ꢁC gave compounds 5e8 (Scheme 1). Reaction of 17 with the
corresponding bromides or propyl isocyanate to afford 10, 15 or 14.
Compounds 11e13 were synthesized by the substitution reaction of
17 with various bromocarboxylates in the presence of K2CO3 fol-
lowed by hydrolysis. Using different equivalent ratios of m-CPBA in
CH2Cl2, thioether 17 was oxidized to compounds 33 and 34,
respectively.
NLRP3 inflammasome mediated IL-1b release with IC50 of ~40 mM.
In addition, its calculated lipophilicity (cLogP) is high, 5.1. Generally,
lower cLogP as well as lower molecular weight (MW) are beneficial
to achieve good druggable profiles [32,33]. Another undeniable fact
is that lead-to-drug optimization generally tends to raise cLogP and
MW. All in all, GFT505 is not yet qualified as a lead compound and
requires far more effort to be optimized. Therefore, it is clear to
increase its potency while reducing cLogP and MW to make it a
suitable lead compound.
Herein, we describe work that leads to the identification of a
potent NLRP3 inhibitor for the treatment of inflammation-related
diseases. Furthermore, the mechanism of NLRP3 inflammasome
2.2. Development of structure-activity relationship (SAR)
inhibition is primarily studied due to its ability to inhibit IL-1
b
release from macrophages and to affect the NLRP3 activation.
Finally, in vivo pharmacological studies in animal models of sepsis
and colitis show relevant therapeutic effects on inflammation.
A cellular model has been established in our previous study
using murine macrophages cell line J774A.1, in which the release of
IL-1b is medicated via the NLRP3 inflammasome upon stimulation
with lipopolysaccharide (LPS) and Nigericin [26]. The enzyme-
linked immunosorbent assay (ELISA) was used to detect the level
2. Results and discussion
of IL-1
tively weak inhibitory potency with an IC50 of 39.6
replacement of the 2-carboxyisopropanyl with methyl group (2)
M). In
the beginning, considering its facile synthesis, we used
compound 2 as a template to study the effect of methylthiol sub-
stituent at 40 position on the activity. As shown in Table 1, amino
group replacing methylthiol at 4’ position (3) significantly
b
secretion. As shown in Table 1, GFT505 exhibited a rela-
2.1. Design and synthesis of GFT505 analogues
mM. The
GFT505 (1) includes
a
pharmacologically active chalcone
exhibited slightly diminished inhibitory activity (IC50 ¼ 49.5
m
structure bearing a hydroxyisobutyric acid, methylthiol group, and
two methyl groups in positions 4, 4’ and 3 (or 5), respectively. Thus,
we started a tentative structure-activity relationship (SAR) study
and designed an initial set of 14 analogues that were synthesized
and tested against NLRP3 inflammasome mediated IL-1
b
release
improved the inhibitory activity with an IC50 of 5.7 mM. Due to the
2

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Fig. 2. Chemical structures of the designed analogues of GFT505.
Scheme 1. Chemical synthesis of the designed analogues. Reagents and conditions: (a) 4 M HCl in MeOH, rt, 24 h; (b) NaOH, MeOH, rt, 24 h; (c) various isocyanates, toluene, 70 ꢁC,
overnight; (d) various bromides or bromocarboxylates, K2CO3, DMF, 90 ꢁC, 6 h; (e) LiOH, THF, H2O, 50 ꢁC, overnight; (f) propyl isocyanate, TEA, CH2Cl2, rt, 5 h; (f) m-CPBA (33:1.0 eq,
34: 2.6 eq), CH2Cl2, 0 ꢁC or rt, 5 h.
3

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Table 1
Inhibitory potency of analogues 2e15 on IL-1b release mediated by NLRP3 inflammasome activation in J774A.1 Cells.
b
d
Compd
R1
R2
IC50
(
m
M) a
cLogP
5.11
LE c
LLE
GFT505 (1)
SMe
39.6 7.5
0.22
ꢀ0.71
2
3
4
5
SMe
NH2
NMe2
Me
Me
Me
Me
49.5 4.2
5.7 0.2
25.9 1.0
33.2 0.3
5.20
3.82
5.02
4.89
0.27
0.34
0.27
0.23
ꢀ0.90
1.42
ꢀ0.43
ꢀ0.41
6
7
Me
Me
9.6 2.0
5.11
6.21
0.25
0.20
ꢀ0.09
ꢀ1.58
23.5 11.7
8
9
-CH3
38.7 4.2
6.46
4.49
0.19
n.d.e
ꢀ2.05
SMe
>40.0
n.d.e
10
11
SMe
SMe
12.7 1.0
31.7 6.0
4.33
5.33
0.28
0.23
0.57
ꢀ0.83
12
SMe
>40.0
5.04
n.d.e
n.d.e
13
14
SMe
SMe
28.9 0.1
22.3 4.1
5.37
4.91
0.22
0.25
ꢀ0.83
ꢀ0.26
15
SMe
21.9 2.8
5.63
0.21
ꢀ0.97
a
IC50 values are the means of three experiments.
Calculated logarithm of the octanol/water distribution coefficient using ChemBioDraw Ultra 14.0.
b
c
LE ¼ ¡1.37✕log (IC50)/N(heavy atom)
LLE ¼ ¡log (IC50) ¡ cLogP.
n. d.: not determined.
.
d
e
potential genotoxicity of aromatic amines, the amino group
in 3 was derivatized into dimethylamino (4) and different ureido
groups (5, 6, 7, and 8), which resulted in a significant drop in po-
tency, but most of them are more potent than 2. These data suggest
that amine derivatives have potential benefits for improving ac-
tivity. On the right-hand side, structure modifications of carbox-
yisopropanyl moiety in GFT505 were also relatively tolerated. For
example, the prolonged-carbon-chain acid 11 and 13, amide
analog 14 and installation of water-soluble morpholine 15 showed
slightly increased activities on inhibiting NLRP3-mediated IL-
ligand lipophilicity efficiency (LLE) values: LE is an assessment of
the efficiency of potency per heavy atom in a ligand or measuring
the potency regarding ligand size, while LLE refers to an approxi-
mate measure of the balance between potency and lipophilicity
[34e37]. Therefore, the LE and LLE values were valuable guides for
our hit optimization which aimed to increase potency while
reducing MW and lipophilicity (cLogP). In the evolution of GFT505
to 10, the LE and LLE values were improved from 0.22 to 0.28 and
from ꢀ0.71 to 0.57, respectively.
Next, we chose to maintain the C-1 vinyl ketone group of
1
b
release. Removal of two branched methyl groups or insertion of
GFT505 while studying the SAR of aromatic substituent at b posi-
vinylidene group did not improve the potency. Notably, when car-
boxyisopropanyl was replaced with a hydroxyethoxyl moiety as
in 10, the inhibitory potency was improved by 3-fold as compared
to GFT505. These data seem to indicate that a suitable hydrogen
bonding at C-4 position is potentially beneficial to inhibitory
activity..
tion on the inhibitor core (Table 2). A ring-fused ester of 10 formed
compound 16, which hindered the hydrogen bonding of the orig-
inal alcohol as a hydrogen bond donor, resulting in a 2-fold potency
loss (IC50 ¼ 24.2
removing carboxyisopropanyl moiety to expose the OH group of
phenol (17) significantly improved the inhibitory potency to 2.1 M,
mM) as compared to 10 (Table 2). As intended,
m
During optimization of GFT505, our synthetic compound quality
was monitored by analyzing two metrics, ligand efficiency (LE) and
a >18-fold increase as compared to that of GFT505. The quality
metrics, LE and LLE, was increased to 0.37 and 1.16. An addition of
4

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Table 2
Table 3
Inhibitory potency of analogues 16e29 on IL-1
inflammasome activation in J774A.1 Cells.
b
release mediated by NLRP3
Inhibitory potency of analogues 30e40 on IL-1
inflammasome activation in J774A.1 Cells.
b release mediated by NLRP3
Compd
R3
IC50
(
m
M)a
cLogPb
4.19
LEc
LLEd
0.43
Compd
R4
IC50
(
m
M)a
cLogPb
LEc
LLEd
16
24.2 2.3
0.30
30
31
32
33
F
Cl
I
12.3 0.4
3.9 0.3
28.6 6.0
10.6 0.3
4.07
4.64
5.05
2.69
0.34
0.37
0.31
0.31
0.84
0.77
ꢀ0.51
2.28
17
2.1 0.6
4.52
0.37
1.16
18
19
20
21
22
23
3.5 0.2
4.03
3.62
4.07
3.47
3.86
4.06
0.36
0.35
0.35
0.33
0.34
0.34
1.43
1.22
0.99
1.53
1.11
0.84
34
12.9 0.2
2.63
0.29
2.26
14.6 0.4
8.8 1.0
35
36
37
38
29.4 2.9
>40.0
5.74
5.63
6.12
4.90
0.25
n.d.e
n.d.e
0.24
ꢀ1.21
n.d.e
n.d.e
0.40
10.0 1.4
10.7 0.8
12.7 0.23
>40.0
5.0 0.1
39
40
3.4 0.1
1.3 0.1
3.88
3.60
0.28
0.34
1.59
2.29
24
25
9.7 0.1
5.3 1.8
4.30
4.57
0.34
0.34
0.72
0.71
a
IC50 values are the means of three experiments.
Calculated logarithm of the octanol/water distribution coefficient using Chem-
b
26
27
>40.0
>40.0
4.62
3.25
n.d.e
n.d.e
n.d.e
n.d.e
BioDraw Ultra 14.0.
c
LE ¼ ¡1.37✕log (IC50)/N(heavy atom)
.
d
LLE ¼ ¡log (IC50) ¡ cLogP.
e
n. d.: not determined.
28
11.7 1.1
1.6 0.3
5.57
3.02
0.29
0.40
ꢀ0.64
halogen atoms (F, Cl, I), only chloro substitution (31) could maintain
comparable potency against IL-1 release. Then, methylthio group
b
29
2.77
was oxidized into sulfoxides (33) and sulfones (34) to decrease
cLogP values, but the inhibitory potency decreased by 5e7 fold. An
obvious potency loss was also observed when we introduced the
lipophilic groups, such as phenyl (35) and 2-thienyl (36, 37), with
the increasing the cLogP values of above 5. In contrast, substitution
of hydrophilic fragments (38, 39) at C-40 contributed to the recov-
ery of inhibitory potency. The acrylamide group exists in many
marketed drugs, and its incorporation can improve the drug-like
properties of drug candidates [38e41]. Thus, we installed the
acrylamide group at C-4’, and the resulting compound 40 is the
a
IC50 values are the means of three experiments.
Calculated logarithm of the octanol/water distribution coefficient using Chem-
b
BioDraw Ultra 14.0.
c
LE ¼ ¡1.37✕log (IC50)/N(heavy atom)
.
d
LLE ¼ ¡log (IC50) ¡ cLogP.
e
n. d.: not determined.
another phenolic hydroxyl (29: IC50 ¼ 1.6
improvement in IL-1 inhibition. The importance of hydrogen bond
donor is further supported by the activity results of 18
M) as a 4-CO2H substitution with comparable inhibi-
mM) further led to a slight
most potent one in all series of analogues with an IC50 of 1.3 mM. In
b
addition, the cLogP value of 40, compared with 17, was further
reduced from 4.5 to 3.6 while improving LLE value from 1.16 to 2.29,
and the LE value was maintained.
(IC50 ¼ 3.4
m
tory potency. Relative to other inhibitors, removal (19, 20) or sub-
stitution (21e24, 27, 28) or a positional change (25, 26) of two
methyl groups did not improve the inhibitory potency on IL-
2.3. Drug-likeness evaluation
1b release in J774A.1 cells. Among the compounds, 29 is the most
potent inhibitor against NLRP3 inflammasome activation, but it
suffers from poor solubility. Taken together, compound 17 was
selected for further modification based on the increase of potency,
LE, and LLE.
To the class of compounds having a Michael acceptor, weak
electrophilicity is a necessary requirement for the development of
safe covalent drugs [22]. Therefore, GFT505, 2, 17, and 40, con-
taining a, b-unsaturated carbonyl structure, were tested for reac-
Finally, we decided to modify the substituent at 40 position in 17
to further improve the drug-like properties. In this position, an
appropriate lipophilic group was critical for inhibitory activity
(Table 3). When we replaced the methylthio group with different
tivity as Michael acceptor. Compounds were reacted with an excess
amount of glutathione (GSH, 20 eq.) in phosphate buffer solution
(pH ¼ 7.4) at 37 ꢁC using DMSO as the cosolvent. Interestingly,
GFT505 showed much higher reactivity than 17 and 40 (Fig. 3). We
5

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Fig. 3. Reactivity of GFT505, 2, 17 and 40 with glutathione as Michael acceptors.
proposed that a stronger electron-donating conjugation of pheno-
(BMDMs) with 40 before nigericin stimulation. 40 produced a
late anion in 17 and 40, formed by deprotonation of PBS solution,
concentration-dependent inhibition of IL-1
b secretion by ELISA in
resulted in significantly reduced electrophilic reactivity of
a, b-
both cell models (Fig. 4A and B). Western blot experiments showed
unsaturated ketone with GSH. The importance of para-phenol is
supported by the higher reactivity results of 2 (a methyl ether of
17). These findings indicate 17 and 40 can be regarded as a “safe”
covalent and irreversible inhibitor with weak electrophilicity.
These also suggest the possible presence of specific covalent
binding to biological nucleophiles and a reduced possibility of
triggering hypersensitivity reactions [22].
As shown in Table 4, our lead compound 40 possessed excellent
physicochemical profiles to obey Lipinski’s rule of five or Veber’s
parameters. The safety of GFT505, 17 and 40 was evaluated with
cytotoxic activities against normal human embryonic kidney
that 40 blocked caspase-1 p20 maturation and IL-1
dose-dependent manner, but did not affect pro-IL-1
b
b
secretion in a
, pro-caspase-
1, NLRP3, or ASC in cell lysates (Fig. 4C). This suggests 40 acts in the
NLRP3 activation step rather than the priming step. The lactate
dehydrogenase (LDH) activity, used as a marker of cell pyroptosis,
was measured in the supernatant of nigericin-treated J774A.1 cells.
Pyroptosis induced by LPS/nigericin was significantly prevented by
compound 40 (Fig. 4D). Furthermore, 40 had not inhibitory effect
on AIM2 or NLRC4 inflammasome activation (Fig. 4E and F). The
results demonstrate that 40 specifically inhibits NLRP3
inflammasome-dependent caspase-1 activation and IL-1b secretion
293T cell line (HEK293T). Compound 40 (TC50 ¼ 155.6
similar cytotoxicity to GFT505 (TC50 ¼ 141.5 M), but the cytotox-
icity was lower than 17 (TC50 ¼ 86.2 M) toward HEK293T cells.
m
M) showed
via acting in the NLRP3 activation step.
m
m
2.5. Biological mechanism study
This also suggests that the potency and cytotoxicity of 40 do not
entirely depend on its Michael-acceptor reactivity toward thiols. As
a result, the selectivity index [ratio of TC50 (HEK-293T) versus IC50
(J774A.1)] for 40 was high up to 119.7. These findings suggest that
40 as an NLRP3 inhibitor may be safe for normal human cells when
administrated. Therefore, compound 40, which showed potent in-
Chalcones were thought to have various pharmacological ac-
tivities via the regulation of ROS apoptotic pathway [42e44]. ROS
has also shed light on NLRP3 inflammasome activation step. On the
basis of the effects of chalcones on ROS, we hypothesized that
compound 40 might inhibit this NLRP3 inflammasome activation
through suppressing ROS production. As expected, treatment with
40 remarkably decreased the intracellular ROS generation accessed
by Muse Cell Analyzer (Fig. 5A and B) and a fluorescence microplate
reader (Fig. 5C). Next, we detected mitochondrial ROS (mtROS) by
utilizing a more specific fluorescent probe MitoSOX Red (mito-
chondrial superoxide indicator). As shown in Fig. 5D and E, LPS/
nigericin stimulation induced mitochondrial ROS production and
hibition of IL-1b release and possessed good drug-like properties,
was selected as a promising lead for further investigation.
2.4. Study on the selectivity of compound 40 to inhibit
inflammasome
To confirm the effect of 40 on NLRP3 inflammasome activation,
its cytotoxic activity was firstly evaluated. Compound 40 did not
show any toxicity against J774A.1 cells in 2 h of incubation, and only
high-concentration drug treatment showed slight cytotoxicity in
IL-1b release in J774A.1 cells. A mitochondria-targeted antioxidant
Mitoquinone (MitoQ) was adopted as a positive reference drug.
Obviously, MitoQ decreased mtROS generation, which could
contribute to the inhibition of IL-1b secretion. For compound 40,
24
h of incubation (Fig. S1). Then, we treated LPS-primed
our results reveal that its effect on the inhibition of NLRP3
inflammasome activation may also depend on mtROS reduction.
mtROS production is closely linked to mitochondrial dysfunction,
which was thought to be an important signal for NLRP3 inflam-
masome activation. Taken together, a direct correlation between
J774A.1 cells and mouse bone marrow-derived macrophages
Table 4
Properties of compounds 16 and 40.
Compd MW
GFT505 384.5
HBD HBA cLogPa tPSA IC50 M)b TC50 M)c SId
(m (m
the extent of the ROS inhibition and the activity against IL-1
b
1
1
2
5
3
3
5.1
4.5
3.6
63.6 39.6
37.3 2.1
66.4 1.3
141.5 7.9 3.6
86.2 12.8 41.0
155.6 9.1 119.7
release suggests that 40 inhibits NLRP3 inflammasome activation
via suppressing ROS production.
17
40
298.4
321.4
a
Calculated logarithm of the octanol/water distribution coefficient using Chem-
2.6. In vivo efficacy of compound 40
BioDraw Ultra 14.0.
b
50% inhibition concentration of IL-1b in J774A.1 cells.
50% cytotoxic concentration against HEK-293T.
c
To verify the potential of 40 to suppress inflammatory re-
sponses, we first investigated its effect on inhibiting the NLRP3-
d
The selectivity index (SI) is the ratio of TC50 (HEK-293T) versus IC50 (J774A.1).
6

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Fig. 4. Compound 40 specifically suppresses NLRP3 inflammasome activation and prevents pyroptosis in vitro. (A-D) LPS primed-macrophages were treated with various doses of
compound 40 for 1h and then stimulated with nigericin in J774A.1 cells (A) and BMDMs (B) for another 1 h, then cells culture supernatants were analyzed by ELISA for IL-1 (A-B).
N ¼ 3. The value of LDH activity was normalized to that of Control. N ¼ 5. J774A.1 cells stimulated as usual, Western blot analysis of cleaved IL-1 , activated caspase-1 (P20) in
culture supernatants (SN) and NLRP3, pro-IL-1 , pro-caspase-1, ASC in lysates of J774A.1 cells (C). Supernatants were also analyzed by the LDH release assay for pyroptosis (D). (E, F)
ELISA of IL-1
b
b
b
b
in SN from LPS primed-J774A.1 cells treated with compound 40 for 1h and then stimulated with FLA-ST Ultrapure (E) or poly (dA:dT) (F). N ¼ 5. Data are represented
as mean SEM. One-Way ANOVA Statistics were used for statistical analysis and analyzed using an: Compared with LPS/nigericin treated group, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5. Compound 40 inhibits ROS production in vitro. (A-C) LPS-primed J774A.1 cells were treated with various dose of compound 40 for 1 h and then stimulated with nigericin for
another 1 h. Then, the cells were loaded with DHE or DCFH-DA for intracellular ROS measurement assessed by Muse Cell Analyzer (n ¼ 3) (A, B) and a fluorescence microplate
reader (n ¼ 5) (C). (DeE) LPS primed-J774A.1 cells were treated with MitoQ or 40 (D, E) for 1h and stimulated with nigericin for another 1 h, and then medium supernatants were
analyzed by ELISA for IL-1
with LPS/nigericin treated group, *P < 0.05, **P < 0.01, ***P < 0.001.
b
(D) and the cells were stained with MitoSOX Red (5
mM) for mtROS measurement analyzed by a fluorescence microplate reader (n ¼ 5) (E). Compared
driven inflammation in mice models of LPS-induced sepsis [20]. As
showed significantly increased IL-1b level in lavage fluid. Our re-
shown in Fig. 6, mice were injected intraperitoneally with LPS and
sults showed that oral administration of 40 obviously reduced the
7

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
Fig. 6. Compound 40 alleviates peritoneal inflammation in sepsis. (A and B) ELISA of IL-1
b
in the serum (A) or peritoneal cavity (B) of C57BL/6 mice intraperitoneally injected with
LPS (20 mg/kg) with or without compound 40 (25 mg/kg or 100 mg/kg). Data are representative of n ¼ 4e6 and are means SEM. Compared with LPS treated group, *P < 0.05,
**P < 0.01, ***P < 0.001.
level of IL-1
b
in both serum and peritoneal fluid in a dose-
(Fig. 7E) as compared to the control group. Intragastrical adminis-
tration of inhibitor 40 (25 and 100 mg/kg) significantly dose-
dependently attenuated the decrease in colon length and fecal
occult blood. In particular, IL-1b levels in colonic tissue, associated
with the induction of colitis, were assessed by ELISA assays. Our
results showed that treatments with 40 significantly prevented the
increments of colonic IL-1b levels as compared to the control group.
Taken together, our findings show that 40 can exert beneficial
effects on inflammatory diseases including sepsis and colitis by
dependent manner. Thus, these results suggest that the inhibitor
40 effectively prevents acute inflammation in vivo in sepsis.
Next, we tested the therapeutic potential of 40 in vivo using a
model of dextran sodium sulfate (DSS)-induced ulcerative colitis
[45,46]. Seven days after being fed with 2% DSS-containing drinking
water, the inflamed mice were characterized by a shortened colonic
length (Fig. 7A and B) caused by fecal occlumency and increasing
severity of fecal occult blood (Fig. 7C and D) and inflammation
Fig. 7. Compound 40 ameliorates DSS-induced ulcerative colitis in mice. (A) The colon was photographed. (B) The length of the colon was measured when the mice were sacrificed.
(C and D) Feces were collected from mice per day, and blood content was tested in fecal occult blood. (E) Protein levels of IL-1b in colon homogenates were examined by ELISA.
N ¼ 5e7. Data are represented as mean and SEM. Compared with Control group, ##P < 0.01, ###P < 0.001; Compared with DSS treated group, **P < 0.01, ***P < 0.001.
8

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
reducing the level of pro-inflammatory cytokine IL-1
b
mediated by
methylthioacetophenone 1a (200 mg, 1.20 mmol) and 3,5-
Dimethyl-4-methoxybenzaldehyde 2a (197 mg, 1.20 mmol) ac-
cording to the general synthetic procedure A as a white solid
NLPR3 activation. Thus, these suggest that the blockade of NLPR3
activation could represent a promising drug target for inflamma-
tion and its related disease.
(290 mg, yield 77%). 1H NMR (400 MHz, CDCl3)
d 7.97e7.93 (m, 2H),
7.72 (d, J ¼ 15.6 Hz, 1H), 7.42 (d, J ¼ 15.6 Hz, 1H), 7.33e7.27 (m, 2H),
3. Conclusions
3.75 (s, 3H), 2.53 (s, 3H), 2.32 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
189.4, 159.4, 145.6, 144.6, 134.8, 131.7, 130.6, 129.4, 129.1, 125.2,
A clinical compound library screen was used to identify a novel
NLRP3-inflammasome inhibitor elafibranor (GFT505), which was
clinically proved to possess good safety. Starting from the low po-
tency and low LLE hit, we optimized the chalcone pharmacophore
by utilizing both simplification and replacement strategies guided
by SAR and structure information. A tentative SAR study demon-
strated that a suitable hydrogen bond donor at C-4 position is
conducive to inhibitory activity. A breakthrough came from the
removal of carboxyisopropanyl moiety with releasing the hydrogen
bonding of phenol, which effectively propelled the potency into the
low micromolar level. Further SAR study revealed the 3, 5-
dimethyl in phenol moiety are essential to the potency on inhib-
iting NLRP3 inflammasome activation as positional change or
removal of them led to a decrease of inhibitory activity. Then,
structural modifications on the methylthio moiety led to a new lead
compound 40, which is approximately 30 times more potent
against NLRP-3 inflammasome activation and lower toxicity than
that of GFT505 at the cellular level. Indeed, 40 is a preferable lead of
good drug-like properties, such as lower MW and lipophilicity
(cLogP), higher LE and LLE. Further mechanistical characterization
indicate the inhibitory potency of 40 against the NLRP3 inflam-
masome activation via suppressing ROS production. The in vivo
ability of 40 to alleviate sepsis and colitis after oral administration
was confirmed. Moreover, 40, used as a chemical probe, is under-
going chemoproteomic studies to identify its cellular binding
target, which will direct further pharmacochemical investigation.
Collectively, the results strongly support further chemical devel-
opment of 40 as a promising lead for NLRP3 inhibitors and explore
their therapeutic potential for inflammation and related diseases.
120.7, 59.9, 16.4, 15.0; HRMS (ESI) calcd for C19H20O2S (M þ H)þ
313.1257, found 313.1278.
(E)-1-(4-aminophenyl)-3-(4-methoxy-3,5-dimethylphenyl)
prop-2-en-1-one (3). Compound
3 was prepared from 4-
aminoacetophenone 1b (1.00 g, 7.40 mmol) and 2a (1.22 g,
7.40 mmol) according to the general synthetic procedure A as a
yellow solid (1.30 g, yield 62%). 1H NMR (400 MHz, DMSO‑d6)
d
7.97e7.87 (m, 2H), 7.75 (d, J ¼ 15.4 Hz, 1H), 7.57e7.42 (m, 3H),
6.69e6.56 (m, 2H), 6.15 (s, 2H), 3.68 (s, 3H), 2.25 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6) 185.8, 158.3, 153.8, 141.3, 131.1, 130.9, 130.6,
d
129.2, 125.5, 121.1, 112.7, 59.4, 15.9; HRMS (ESI) calcd for C18H19NO2
(M þ H)þ 282.1489, 282.1489.
(E)-1-(4-(dimethylamino)phenyl)-3-(4-methoxy-3,5-
dimethylphenyl)prop-2-en-1-one (4) Compound 4 was prepared
from 4-dimethylaminoacetophenone 1c (126 mg, 0.77 mmol) and
2a (126 mg, 0.77 mmol) according to the general synthetic pro-
cedure A as a yellow solid (177 mg, yield 75%). 1H NMR (400 MHz,
DMSO‑d6)
(m, 3H), 6.80e6.70 (m, 2H), 3.68 (s, 3H), 3.04 (s, 6H), 2.26 (s, 6H);
13C NMR (100 MHz, DMSO‑d6)
186.1, 158.4, 153.3, 141.5, 130.9,
d
8.14e8.00 (m, 2H), 7.78 (d, J ¼ 15.5 Hz, 1H), 7.59e7.51
d
130.7, 130.6, 129.2, 125.3, 121.1, 110.8, 59.4, 39.6, 15.8; HRMS (ESI)
calcd for C20H23NO2 (M þ H)þ 310.1802, found 310.1804.4.1.
(E)-3-(2,3-dihydrobenzofuran-5-yl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (16). Compound 16 was prepared from
1a (90 mg, 0.54 mmol) and 2,3-dihydrobenzofuran-5-carbaldehyde
2c (80 mg, 0.54 mmol) according to the general synthetic procedure
A as a yellow solid (118 mg, yield 74%). 1H NMR (400 MHz,
DMSO‑d6)
d
8.12e8.05 (m, 2H), 7.85 (d, J ¼ 1.7 Hz, 1H), 7.77 (d,
J ¼ 15.4 Hz,1H), 7.69 (d, J ¼ 15.4 Hz,1H), 7.61 (dd, J ¼ 8.3, 1.9 Hz,1H),
7.42e7.36 (m, 2H), 6.84 (d, J ¼ 8.3 Hz, 1H), 4.61 (t, J ¼ 8.7 Hz, 2H),
3.23 (t, J ¼ 8.7 Hz, 2H), 2.55 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
4. Experimental
d
187.7, 162.2, 145.2, 144.2, 134.1, 130.7, 128.9, 128.6, 127.5, 125.4,
4.1. Chemistry
124.9, 118.6, 109.3, 71.8, 28.6, 14.0; HRMS (ESI) calcd for C18H16O2S
(M þ H)þ 297.0944, found 297.0967.
4.1.1. General
Unless otherwise mentioned, all reactions were carried out
under a nitrogen atmosphere with anhydrous solvents from com-
mercial sources (Alfa, Innochem, and Shanghai Chemical Reagent
Company) without further purification. Yields refer to chromato-
graphically homogeneous materials, unless otherwise stated. Re-
actions were monitored by thin-layer chromatography carried out
on 0.25 mm Yantai silica gel plates (60F-254). Visualization was
achieved using UV light, or phosphomolybdic acid in ethanol fol-
lowed by heating. Tsingdao silica gel (200e300 mesh) was used for
flash column chromatography. NMR spectra were recorded with a
400 MHz (1H NMR, 400 MHz; 13C NMR, 100 MHz) spectrometer.
Data are reported as follows: chemical shift, multiplicity
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, br ¼ broad,
m ¼ multiplet), coupling constants, and integration.
4.1.3. General produce B for the preparation of 5ꢀ8
To a solution of 3 (1.0 equiv.) in toluene (1.5 mL) was added
corresponding isocyanate (2.0 equiv.). The mixture was stirred at
70 ꢁC overnight. The forming precipitate was filtered, washed with
toluene and dried to obtain target compounds.
(E)-1-isopropyl-3-(4-(3-(4-methoxy-3,5-dimethylphenyl)
acryloyl)phenyl)urea (5). Compound 5 was prepared from 3
(50 mg, 0.18 mmol) and isopropyl isocyanate (30 mg, 0.36 mmol)
according to the general synthetic procedure B as a yellow solid
(48 mg, yield 74%). 1H NMR (400 MHz, DMSO‑d6)
d 8.78 (s, 1H),
8.10e8.03 (m, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H), 7.63e7.52 (m, 5H), 6.19
(d, J ¼ 7.5 Hz, 1H), 3.84e3.74 (m, 1H), 3.69 (s, 3H), 2.26 (s, 6H), 1.11
(d, J ¼ 6.5 Hz, 6H); 13C NMR (100 MHz, DMSO‑d6)
d 187.1, 158.7,
154.0, 145.3, 142.7, 131.0, 130.4, 130.3, 130.1, 129.5, 120.8, 116.6, 59.4,
41.1, 22.9, 15.9; HRMS (ESI) calcd for C22H26N2O3 (M þ H)þ
367.2016, found 367.2013.
4.1.2. General produce a for the preparation of 2ꢀ4, 16
To a solution of 1aꢀc (1.0 equiv.) and 2a or 2c (1.0 equiv.) in
methanol was added sodium hydroxide (10.0 equiv.). The solution
was stirred at room temperature for 24 h. The forming precipitate
was filtered and recrystallized in methanol to give title compounds
2e4.
(E)-1-(4-(3-(4-methoxy-3,5-dimethylphenyl)acryloyl)
phenyl)-3-propylurea (6). Compound 6 was prepared from 3
(50 mg, 0.18 mmol) and propyl isocyanate (30 mg, 0.36 mmol)
according to the general synthetic procedure B as a yellow solid
(49 mg, yield 74%). 1H NMR (400 MHz, DMSO‑d6)
d 8.91 (s, 1H),
(E)-3-(4-methoxy-3,5-dimethylphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (2). Compound 2 was prepared from 4-
8.11e8.00 (m, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H), 7.69e7.49 (m, 5H), 6.33
(t, J ¼ 5.7 Hz, 1H), 3.69 (s, 3H), 3.11e3.02 (m, 2H), 2.26 (s, 6H),
9

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
1.54e1.39 (m, 2H), 0.88 (t, J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz,
phenyl)prop-2-en-1-one (21). Compound 21 was prepared from
1a (300 mg, 1.80 mmol) and 4-hydroxy-3-methoxybenzaldehyde
2g (274 mg, 1.80 mmol) according to the general synthetic pro-
cedure C as a yellow solid (275 mg, yield 51%). 1H NMR (400 MHz,
DMSO‑d6) d 187.1, 158.7, 154.8, 145.4, 142.7, 131.0, 130.4, 130.4, 129.5,
120.8, 116.6, 59.4, 40.9, 22.9, 15.9, 11.4; HRMS (ESI) calcd for
C
22H26N2O3 (M þ H)þ 367.2016, found 367.2019.
(E)-1-(4-fluorophenyl)-3-(4-(3-(4-methoxy-3,5-
DMSO‑d6)
d
9.68 (s, 1H), 8.11e8.02 (m, 2H), 7.75 (d, J ¼ 15.4 Hz, 1H),
dimethylphenyl)acryloyl)phenyl)urea (7). Compound 7 was pre-
pared from 3 (50 mg, 0.18 mmol) and 4-fluorophenyl isocyanate
(48 mg, 0.36 mmol) according to the general synthetic procedure B
7.67 (d, J ¼ 15.4 Hz, 1H), 7.51 (d, J ¼ 2.0 Hz, 1H), 7.43e7.35 (m, 2H),
7.28 (dd, J ¼ 8.3, 1.9 Hz, 1H), 6.84 (d, J ¼ 8.1 Hz, 1H), 3.87 (s, 3H), 2.55
(s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 187.8, 149.7, 148.0, 145.1,
as a yellow solid (70 mg, 94%). 1H NMR (400 MHz, DMSO‑d6)
d
9.14
144.6, 134.2, 128.9, 126.3, 124.9, 124.1, 118.5, 115.6, 111.7, 55.8, 14.0;
HRMS (ESI) calcd for C17H16O3S (M þ H)þ 301.0893, found 301.0911.
(E)-3-(3-fluoro-4-hydroxyphenyl)-1-(4-(methylthio)phenyl)
prop-2-en-1-one (22). Compound 22 was prepared from 1a
(300 mg, 1.80 mmol) and 3-fluoro-4-hydroxybenzaldehyde 2h
(252 mg,1.80 mmol) according to the general synthetic procedure C
as a yellow solid (155 mg, yield 30%). 1H NMR (400 MHz, DMSO‑d6)
(s, 1H), 8.85 (s, 1H), 8.18e8.04 (m, 2H), 7.82 (d, J ¼ 15.5 Hz, 1H),
7.67e7.55 (m, 5H), 7.53e7.44 (m, 2H), 7.21e7.08 (m, 2H), 3.69 (s,
3H), 2.26 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
d 187.2, 158.7, 157.6
(d, J ¼ 237.0 Hz),152.3,144.4,142.9,135.6 (d, J ¼ 2.6 Hz),131.2,130.9,
130.3, 130.0, 129.5, 120.3 (d, J ¼ 7.9 Hz), 120.3, 117.3, 115.4 (d,
J ¼ 22.3 Hz) 59.4, 15.8; HRMS (ESI) calcd for C25H23FN2O3 (M þ H)þ
419.1765, found 419.1777.
d
10.51 (s, 1H), 8.12e8.03 (m, 2H), 7.85 (dd, J ¼ 12.6, 2.1 Hz, 1H), 7.79
(E)-1-(4-(3-(4-methoxy-3,5-dimethylphenyl)acryloyl)
phenyl)-3-(p-tolyl)urea (8). Compound 8 was prepared from 3
(50 mg, 0.18 mmol) and 4-tolyl isocyanate (47 mg, 0.36 mmol)
according to the general synthetic procedure B as a yellow solid
(d, J ¼ 15.5 Hz, 1H), 7.64 (d, J ¼ 15.5 Hz, 1H), 7.52e7.47 (m, 1H),
7.41e7.36 (m, 2H), 7.06e6.88 (m, 1H), 2.55 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6)
d
187.7, 151.2 (d, J ¼ 241.6 Hz), 147.5 (d,
J ¼ 12.5 Hz), 145.3, 143.0 (d, J ¼ 2.5 Hz), 133.9, 129.0, 126.9 (d,
J ¼ 2.6 Hz), 126.7 (d, J ¼ 6.6 Hz), 124.9, 120.0, 117.8 (d, J ¼ 3.3 Hz),
115.8 (d, J ¼ 18.6 Hz), 14.0; HRMS (ESI) calcd for C16H13FO2S
(M þ H)þ 289.0693, found 289.0712.
(66 mg, yield 89%). 1H NMR (400 MHz, DMSO‑d6)
d 9.10 (s, 1H), 8.70
(s, 1H), 8.18e8.01 (m, 2H), 7.82 (d, J ¼ 15.4 Hz, 1H), 7.67e7.50 (m,
5H), 7.41e7.28 (m, 2H), 7.16e7.02 (m, 2H), 3.69 (s, 3H), 2.27 (s, 6H),
2.25 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
187.2, 158.7, 152.2,
(E)-3-(2-fluoro-4-hydroxyphenyl)-1-(4-(methylthio)phenyl)
prop-2-en-1-one (23). Compound 23 was prepared from 1a
(300 mg, 1.80 mmol) and 2-fluoro-4-hydroxybenzaldehyde 2i
(252 mg,1.80 mmol) according to the general synthetic procedure C
as a yellow solid (96 mg, yield 18%). 1H NMR (400 MHz, DMSO‑d6)
144.5, 142.9, 136.7, 131.1, 131.1, 130.9,130.3,130.0,129.5, 129.2, 120.7,
118.6, 117.2, 59.4, 20.4, 15.8; HRMS (ESI) calcd for C26H26N2O3
(M þ H)þ 415.2016, found 415.2021.
4.1.4. General produce C for the preparation of 17, 19e32, 35ꢀ40
To a solution of 4 M HCl in methanol were added 1a or 1dꢀl (1.0
equiv.) and 2b or 2eꢀo (1.0 equiv.). The solution was stirred at room
temperature for 24 h. The forming precipitate was filtered and
dried to obtain target compounds.
d
10.58 (s, 1H), 8.09e8.01 (m, 2H), 7.94 (t, J ¼ 8.8 Hz, 1H), 7.81e7.70
(m, 2H), 7.43e7.35 (m, 2H), 6.72 (dd, J ¼ 8.6, 2.3 Hz, 1H), 6.66 (dd,
J ¼ 12.6, 2.3 Hz, 1H), 2.55 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
187.7, 162.2(d, J ¼ 250.0 Hz), 161.7 (d, J ¼ 12.6 Hz), 145.4, 135.5 (d,
J ¼ 3.7 Hz), 133.9, 130.3 (d, J ¼ 4.5 Hz), 128.9, 125.0, 120.1 (d,
J ¼ 4.3 Hz), 113.4 (d, J ¼ 11.5 Hz), 112.64 (d, J ¼ 2.3 Hz), 102.9 (d,
J ¼ 24.1 Hz), 14.0; HRMS (ESI) calcd for C16H13FO2S (M þ H)þ
289.0693, found 289.0708.
(E)-3-(3-chloro-4-hydroxyphenyl)-1-(4-(methylthio)phenyl)
prop-2-en-1-one (24). Compound 24 was prepared from 1a
(300 mg, 1.80 mmol) and 3-chloro-4-hydroxybenzaldehyde 2j
(282 mg,1.80 mmol) according to the general synthetic procedure C
as a yellow solid (124 mg, yield 23%). 1H NMR (400 MHz, DMSO‑d6)
(E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (17). Compound 17 was prepared from 4-
methylthioacetophenone 1a (2.33 g, 14.00 mmol) and 3,5-
dimethyl-4-hydroxybenzaldehyde 2b (2.10 g, 14.00 mmol) ac-
cording to the general synthetic procedure C as a yellow solid
(3.20 g, yield 77%). 1H NMR (400 MHz, DMSO‑d6)
d 8.94 (s, 1H),
8.14e8.02 (m, 2H), 7.71 (d, J ¼ 15.4 Hz, 1H), 7.60 (d, J ¼ 15.4 Hz, 1H),
7.49 (s, 2H), 7.43e7.34 (m, 2H), 2.55 (s, 3H), 2.20 (s, 6H); 13C NMR
(100 MHz, DMSO‑d6)
d
187.8, 156.3, 145.2, 144.6, 134.2, 129.7, 129.0,
d
10.81 (s, 1H), 8.14e8.06 (m, 2H), 8.02 (d, J ¼ 2.1 Hz, 1H), 7.81 (d,
125.8, 125.0, 124.7, 118.2, 16.7, 14.0; HRMS (ESI) calcd for C18H18O2S
J ¼ 15.5 Hz, 1H), 7.67e7.60 (m, 2H), 7.41e7.36 (m, 2H), 7.02 (d,
(M þ H)þ 299.1100, found 299.1106.
J ¼ 8.4 Hz, 1H), 2.55 (s, 1H); 13C NMR (100 MHz, DMSO‑d6)
d 187.7,
(E)-3-(4-hydroxyphenyl)-1-(4-(methylthio)phenyl)prop-2-
en-1-one (19). Compound 19 was prepared from 1a (680 mg,
4.09 mmol) and 4-hydroxybenzaldehyde 2e (500 mg, 4.09 mmol)
according to the general synthetic procedure C as a yellow solid
155.3, 145.3, 142.7, 133.9, 130.1, 129.7, 129.0, 127.2, 124.9, 120.5,
119.8, 116.7, 14.0; HRMS (ESI) calcd for C16H13ClO2S (M þ H)þ
305.0398, found 305.0411.
(E)-3-(4-hydroxy-2,5-dimethylphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (25). Compound 25 was prepared from
1a (300 mg, 1.80 mmol) and 4-hydroxy-2,5-dimethylbenzaldehyde
2k (271 mg, 1.80 mmol) according to the general synthetic pro-
cedure C as a yellow solid (382 mg, yield 71%). 1H NMR (400 MHz,
(667 mg, yield 60%). 1H NMR (400 MHz, DMSO‑d6)
d
10.11 (s, 1H),
8.10e8.03 (m, 2H), 7.76e7.64 (m, 4H), 7.43e7.32 (m, 2H), 6.88e6.80
(m, 2H), 2.55 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
187.8, 160.1,
d
145.1, 144.2, 134.1, 131.0, 128.9, 125.9, 124.9, 118.3, 115.9, 14.0; HRMS
(ESI) calcd for C16H14O2S (M þ H)þ 271.0787, found 271.0812.
(E)-3-(4-hydroxy-3-methylphenyl)-1-(4-(methylthio)phenyl)
prop-2-en-1-one (20). Compound 20 was prepared from 1a
(300 mg, 1.80 mmol) and 4-hydroxy-3-methylbenzaldehyde 2f
(245 mg, 1.80 mmol) according to the general synthetic procedure C
as a yellow solid (325 mg, yield 63%). 1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6)
d
9.87 (s, 1H), 8.17e8.01 (m, 2H), 7.92 (d, J ¼ 15.3 Hz, 1H),
7.81 (s, 1H), 7.64 (d, J ¼ 15.3 Hz, 1H), 7.45e7.28 (m, 2H), 6.67 (s, 1H),
2.55 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
187.7, 158.1, 145.0, 141.0, 137.8, 134.2, 129.5, 128.9, 124.9, 123.9,
122.4, 118.4, 116.6, 19.0, 15.5, 14.0; HRMS (ESI) calcd for C18H18O2S
(M þ H)þ 299.1100, found 299.1114.
d
10.00 (s, 1H), 8.12e7.99 (m, 2H), 7.73e7.66 (m, 2H), 7.63 (d,
(E)-3-(4-hydroxy-2,6-dimethylphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (26). Compound 26 was prepared from
1a (300 mg, 1.80 mmol) and 4-hydroxy-2,6-dimethylbenzaldehyde
2l (271 mg, 1.80 mmol) according to the general synthetic pro-
cedure C as a yellow solid (256 mg, yield 48%). 1H NMR (400 MHz,
J ¼ 15.4 Hz, 1H), 7.51 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.41e7.35 (m, 2H), 6.85
(d, J ¼ 8.3 Hz, 1H), 2.55 (s, 3H), 2.17 (s, 3H); 13C NMR (100 MHz,
DMSO‑d6) d 187.7,158.4,145.0,144.4,134.2,131.3,128.9,125.7,124.9,
124.7, 118.0, 114.9, 15.9, 14.0; HRMS (ESI) calcd for C17H16O2S
(M þ H)þ 285.0944, found 285.0968.
DMSO‑d6) 1H NMR (400 MHz, DMSO‑d6)
d
9.73 (s, 1H),
d 8.02e7.95
(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(methylthio)
(m, 2H), 7.85 (d, J ¼ 15.8 Hz, 1H), 7.40e7.35 (m, 2H), 7.28 (d,
10

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
J ¼ 15.8 Hz, 1H), 6.56 (s, 2H), 2.53 (s, 3H), 2.34 (s, 6H); 13C NMR
DMSO‑d6)
d
188.3, 156.5, 145.4,137.7, 137.3, 130.2,129.8,125.7, 124.7,
(100 MHz, DMSO‑d6)
d
188.1, 158.0, 145.4, 141.6, 139.8, 134.1, 129.0,
117.9,101.4,16.6; HRMS (ESI) calcd for C17H15IO2 (M þ H)þ 379.0189,
125.1, 124.6, 124.4, 115.8, 21.6, 14.0; HRMS (ESI) calcd for C18H18O2S
found 379.0197.
(M þ H)þ 299.1100, found 299.1113.
(E)-1-([1,1′-biphenyl]-4-yl)-3-(4-hydroxy-3,5-
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (27). Compound 27 was prepared from
dimethylphenyl)prop-2-en-1-one (35). Compound 35 was pre-
pared from 1-([1,10-biphenyl]-4-yl)ethan-1-one 1g (131 mg,
0.67 mmol) and 2b (100 mg, 0.67 mmol) according to the general
synthetic procedure C as a yellow solid (102 mg, yield 47%). 1H NMR
1a
(300
mg,
1.80
mmol)
and
4-hydroxy-3,5-
dimethoxybenzaldehyde 2m (328 mg, 1.80 mmol) according to
the general synthetic procedure C as a yellow solid (265 mg, yield
(400 MHz, DMSO‑d6) d 8.27e8.18 (m, 2H), 7.87e7.81 (m, 2H),
45%). 1H NMR (400 MHz, DMSO‑d6)
d
9.04 (s, 1H), 8.13e8.06 (m,
7.80e7.71 (m, 3H), 7.65 (d, J ¼ 15.5 Hz, 1H), 7.55e7.47 (m, 4H),
2H), 7.78 (d, J ¼ 15.4 Hz, 1H), 7.67 (d, J ¼ 15.4 Hz, 1H), 7.43e7.36 (m,
7.46e7.39 (m, 1H), 2.21 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
2H), 7.20 (s, 2H), 3.85 (s, 6H), 2.56 (s, 3H); 13C NMR (100 MHz,
d 188.5, 156.5, 145.0, 144.3, 139.1, 136.9, 129.9, 129.3, 128.5, 127.1,
DMSO‑d6)
d
187.8, 148.1, 145.1, 145.0, 138.7, 134.1, 129.0, 125.1, 124.9,
127.0, 125.8, 124.8, 118.4, 16.7; HRMS (ESI) calcd for C23H20O2
118.9, 106.9, 56.2, 14.0; HRMS (ESI) calcd for C18H18O4S (M þ H)þ
(M þ H)þ 329.1536, found 329.1537.
331.0999, found 331.1000.
(E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-(thiophen-2-yl)
phenyl)prop-2-en-1-one (36). Compound 36 was prepared from
1-(4-(thiophen-2-yl) phenyl)ethan-1-one 1h (100 mg, 0.49 mmol)
and 2b (74 mg, 0.49 mmol) according to the general synthetic
procedure C as a yellow solid (63 mg, yield 38%). 1H NMR (400 MHz,
(E)-3-(3,5-diethyl-4-hydroxyphenyl)-1-(4-(methylthio)
phenyl)prop-2-en-1-one (28). Compound 28 was prepared from
1a (100 mg, 0.60 mmol) and 3,5-diethyl-4-hydroxybenzaldehyde
2n (107 mg, 0.60 mmol) according to the general synthetic pro-
cedure C as a yellow solid (136 mg, yield 69%). 1H NMR (400 MHz,
DMSO‑d6)
7.78e7.70 (m, 2H), 7.69e7.60 (m, 2H), 7.51 (s, 2H), 7.20 (dd, J ¼ 5.1,
3.7 Hz, 1H), 2.22 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
187.9,
d 8.96 (s, 1H), 8.24e8.12 (m, 2H), 7.88e7.79 (m, 2H),
DMSO‑d6)
d
8.81 (s, 1H), 8.19e7.99 (m, 2H), 7.72 (d, J ¼ 15.4 Hz, 1H),
7.64 (d, J ¼ 15.4 Hz, 1H), 7.48 (s, 2H), 7.42e7.34 (m, 2H), 2.63 (q,
d
J ¼ 7.5 Hz, 4H), 2.55 (s, 3H), 1.17 (t, J ¼ 7.5 Hz, 6H); 13C NMR
156.4, 144.9, 142.2, 137.7, 136.7, 129.8, 129.4, 128.9, 127.5, 125.7,
125.5, 125.4, 124.7, 118.2, 16.6; HRMS (ESI) calcd for C21H18O2S
(M þ H)þ 335.1100, found 35.1103.
(100 MHz, DMSO‑d6)
d 187.7, 155.2, 145.0, 144.8, 134.2, 130.9, 128.9,
128.1, 126.1, 124.9, 118.1, 23.0, 14.4, 14.0; HRMS (ESI) calcd for
C
20H22O2S (M þ H)þ 327.1413, found 327.1429.
(E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-(5-
(E)-3-(3,4-dihydroxyphenyl)-1-(4-(methylthio)phenyl)prop-
methylthiophen-2-yl)phenyl)prop-2-en-1-one (37). Compound
37 was prepared from 1-(4-(5-methylthiophen-2-yl)phenyl)ethan-
1-one 1i (100 mg, 0.46 mmol) and 2b (69 mg, 0.46 mmol) according
to the general synthetic procedure C as a yellow solid (55 mg, yield
2-en-1-one (29). Compound 29 was prepared from 1a (1.20 g,
7.24 mmol) and 3,4-dihydroxybenzaldehyde 2o (1.00 g, 7.24 mmol)
according to the general synthetic procedure C as a yellow solid
(960 mg, yield 46%). 1H NMR (400 MHz, DMSO‑d6)
d
9.61 (s, 1H),
34%). 1H NMR (400 MHz, DMSO‑d6)
d 8.95 (s, 1H), 8.18e8.08 (m,
9.25 (s, 1H), 8.14e7.93 (m, 2H), 7.66e7.53 (m, 2H), 7.43e7.34 (m,
2H), 7.26 (d, J ¼ 2.1 Hz, 1H), 7.18 (dd, J ¼ 8.2, 2.1 Hz, 1H), 6.81 (d,
2H), 7.80e7.68 (m, 3H), 7.62 (d, J ¼ 15.4 Hz, 1H), 7.55e7.46 (m, 3H),
6.94e6.81 (m, 1H), 2.50e2.46 (m, 3H), 2.21 (s, 6H); 13C NMR
J ¼ 8.2 Hz, 1H), 2.55 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
187.8,
(100 MHz, DMSO‑d6) d 187.8, 156.3, 144.7, 141.2, 139.7, 138.0, 136.3,
148.7, 145.6, 145.1, 144.7, 134.2, 128.9, 126.4, 125.0, 122.2, 118.3,
115.8, 115.6, 14.0; HRMS (ESI) calcd for C16H14O3S (M þ H)þ
287.0736, found 287.0740.
129.7, 129.4, 127.4, 125.8, 125.5, 124.8, 124.7, 118.2, 16.6, 15.2; HRMS
(ESI) calcd for C22H20O2S (M þ H)þ 349.1257, found 349.1261.
(E)-4-fluoro-N-(4-(3-(4-hydroxy-3,5-dimethylphenyl)
(E)-1-(4-fluorophenyl)-3-(4-hydroxy-3,5-dimethylphenyl)
prop-2-en-1-one (30). Compound 30 was prepared from 1-(4-
fluorophenyl)ethan-1-one 1d (500 mg, 3.62 mmol) and 2b
(543 mg, 3.62 mmol) according to the general synthetic procedure
C as a yellow solid (618 mg, yield 63%). 1H NMR (400 MHz,
acryloyl)phenyl)benzenesulfon-amide (38). Compound 38 was
prepared from N-(4-acetylphenyl)-4-fluorobenzenesulfonamide 1j
(100 mg, 0.42 mmol) and 2b (63 mg, 0.42 mmol) according to the
general synthetic procedure C as a yellow solid (88 mg, yield 49%).
1H NMR (400 MHz, DMSO‑d6)
d 8.93 (s, 1H), 8.07e7.99 (m, 2H),
DMSO‑d6)
d
8.94 (s, 1H), 8.29e8.09 (m, 2H), 7.71 (d, J ¼ 15.4 Hz, 1H),
7.95e7.86 (m, 2H), 7.62 (d, J ¼ 15.4 Hz, 1H), 7.55 (d, J ¼ 15.4 Hz, 1H),
7.62 (d, J ¼ 15.4 Hz, 1H), 7.48 (s, 2H), 7.41e7.31 (m, 2H); 13C NMR
7.49e7.37 (m, 4H), 7.32e7.20 (m, 2H), 2.19 (s, 6H); 13C NMR
(100 MHz, DMSO‑d6)
d
187.4, 164.9 (d, J ¼ 251.3 Hz), 156.4, 145.1,
(100 MHz, DMSO‑d6)
d
187.4, 164.6 (d, J ¼ 252.3 Hz), 156.3, 144.5,
134.7 (d, J ¼ 2.9 Hz), 131.3 (d, J ¼ 9.2 Hz), 129.7, 125.7, 124.7, 118.0,
115.7 (d, J ¼ 21.7 Hz), 16.6; HRMS (ESI) calcd for C17H15FO2 (M þ H)þ
271.1129, found 271.1131.
(E)-1-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethylphenyl)
prop-2-en-1-one (31). Compound 31 was prepared from 1-(4-
chlorophenyl)ethan-1-one 1e (103 mg, 0.67 mmol) and 2b
(100 mg, 0.67 mmol) according to the general synthetic procedure
C as a yellow solid (128 mg, yield 67%). 1H NMR (400 MHz,
141.8, 135.7 (d, J ¼ 2.9 Hz), 133.4, 130.0, 129.9 (d, J ¼ 9.7 Hz), 129.6,
125.8, 124.7, 118.4, 118.1, 116.8 (d, J ¼ 23.0 Hz), 16.6; HRMS (ESI)
calcd for C23H20FNO4S (M þ H)þ 426.1170, found 426.1175.
(E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-{[(propan-2-yl)
sulfamoyl]amino}phenyl)prop-2-en-1-one (39). Compound 39
was prepared from N-(4-acetylphenyl) isopropyl amino-
sulfonamide 1k (50 mg, 0.20 mmol) and 2b (29 mg, 0.20 mmol)
according to the general synthetic procedure C as a yellow solid
DMSO‑d6)
d
8.18e8.10 (m, 2H), 7.70 (d, J ¼ 15.4 Hz, 1H), 7.65e7.58
(28 mg, yield 37%). 1H NMR (400 MHz, DMSO‑d6)
d 10.22 (s, 1H),
(m, 3H), 7.49 (s, 2H), 2.21 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
8.90 (s, 1H), 8.13e8.01 (m, 2H), 7.77e7.64 (m, 2H), 7.57 (d,
J ¼ 15.4 Hz, 1H), 7.47 (s, 2H), 7.29e7.17 (m, 2H), 3.34e3.29 (m, 1H),
2.20 (s, 6H), 0.99 (d, J ¼ 6.5 Hz, 6H); 13C NMR (100 MHz, DMSO‑d6)
d
187.7, 156.5, 145.4, 137.7, 136.7, 130.3, 129.8, 128.8, 125.6, 124.7,
117.9, 16.5; HRMS (ESI) calcd for C17H15ClO2 (M þ H)þ 287.0833,
found 287.0832.
d
187.4, 156.2, 144.1, 143.6, 131.5, 129.9, 129.6, 125.9, 124.7, 118.3,
(E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-iodophenyl)
prop-2-en-1-one (32). Compound 32 was prepared from 1-(4-
iodophenyl)ethan-1-one 1f (1.64 g, 6.66 mmol) and 2b (1.00 g,
6.66 mmol) according to the general synthetic procedure C as a
yellow solid (1.10 g, yield 44%). 1H NMR (400 MHz, DMSO‑d6)
116.5, 45.2, 23.1, 16.6; HRMS (ESI) calcd for C20H24N2O4S (M þ H)þ
389.1560, found 389.1532.
(E)-N-(4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)phenyl)
acrylamide(40). Compound 40 was prepared from N-(4-
acetylphenyl)acrylamide 1l (100 mg, 0.53 mmol) and 2b (79 mg,
0.53 mmol) according to the general synthetic procedure C as a
yellow solid (112 mg, yield 66%). 1H NMR (400 MHz, DMSO‑d6)
d
7.96e7.92 (m, 2H), 7.91e7.86 (m, 2H), 7.67 (d, J ¼ 15.5 Hz, 1H), 7.61
(d, J ¼ 15.5 Hz, 3H), 7.49 (s, 2H), 2.20 (s, 6H); 13C NMR (100 MHz,
11

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
d
10.51 (s, 1H), 8.94 (s, 1H), 8.17e8.11 (m, 2H), 7.87e7.81 (m, 2H),
(ESI) calcd for C22H22O4S (M þ H)þ 383.1312, found 383.1315.
(E)-5-(2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-oxoprop-
1-en-1-yl)phenoxy)pentanoic acid (13). The corresponding ester
was prepared from 17 (200 mg, 0.67 mmol) and ethyl 5-
bromopentanoate (420 mg, 2.01 mmol) according to step A of
general synthetic procedure D as a yellow oil (240 mg, 84%).
Compound 13 was prepared from the above ester (180 mg,
0.42 mmol) according to step B of general synthetic procedure D as
a yellow solid (121 mg, yield 72%). 1H NMR (400 MHz, DMSO‑d6)
7.71 (d, J ¼ 15.4 Hz, 1H), 7.60 (d, J ¼ 15.4 Hz, 1H), 7.48 (s, 2H), 6.48
(dd, J ¼ 17.0, 10.0 Hz, 1H), 6.32 (dd, J ¼ 17.0, 2.0 Hz, 1H), 5.82 (dd,
J ¼ 10.0, 2.0 Hz, 1H), 2.20 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
d
187.5, 163.7, 156.3, 144.4, 143.2, 133.1, 131.6, 129.8, 129.7, 128.0,
125.9, 124.8, 118.9, 118.3, 16.6; HRMS (ESI) calcd for C20H19NO2
(M þ H)þ 322.1438, found 322.1437.
4.1.5. General produce D for the preparation of 9, 11ꢀ13
d
8.12e8.06 (m, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H), 7.62 (d, J ¼ 15.5 Hz,
Step A: Preparation of carboxylic ester. To a solution of 17 (1.0
equiv.) in anhydrous DMF were added corresponding bromo-
carboxylates (3.0 equiv.) and potassium carbonate (5.0 equiv.).
The solution was heated to 90 ꢁC for 6 h, then the solution was
diluted with water and extracted with ethyl acetate. The organic
layers were combined, concentrated and purified by column
chromatography to give the desired ester.
1H), 7.57 (s, 2H), 7.43e7.36 (m, 2H), 3.77 (t, J ¼ 5.8 Hz, 2H), 2.56 (s,
3H), 2.31 (t, J ¼ 6.9 Hz, 2H), 2.25 (s, 6H), 1.77e1.70 (m, 4H); 13C NMR
(100 MHz, DMSO‑d6)
d 187.8, 174.5, 157.9, 145.4, 143.6, 133.9, 131.1,
130.1, 129.6, 129.0, 124.9, 120.5, 71.5, 33.5, 29.4, 21.3, 16.0, 14.0;
HRMS (ESI) calcd for C23H26O4S (M þ H)þ 399.1625, found
399.1629.
Step B: Hydrolysis of ester. To a solution of the above ester (1.0
equiv.) in THF/H2O (1:1, v/v) was added LiOH (6.0 equiv.). The
solution was stirred at 50 ꢁC overnight, then the solution was
adjusted to pH ¼ 2 with hydrochloric acid (1 M) and extracted
with ethyl acetate. The organic layers were combined, concen-
trated and purified by column chromatography to obtain target
compounds.
4.1.6. (E)-3-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-1-(4-
(methylthio)phenyl)prop-2-en-1-one (10)
To a solution of 17 (100 mg, 0.34 mmol) in anhydrous DMF
(2 mL) were added 2-bromoethanol (125 mg, 1.00 mmol) and po-
tassium carbonate (232 mg, 1.68 mmol). The solution was heated to
90 ꢁC for 6 h, then the solution was diluted with water and
extracted with ethyl acetate. The organic layers were combined,
concentrated and purified by column chromatography to give
compound 10 as a yellow solid (65 mg, yield 57%). 1H NMR
(E)-2-(2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-oxoprop-
1-en-1-yl)phenoxy)acetic acid (9). The corresponding ester was
prepared from 17 (200 mg, 0.67 mmol) and ethyl bromoacetate
(336 mg, 2.01 mmol) according to step A of general synthetic
procedure D as a yellow oil (183 mg, yield 71%). Compound 9 was
prepared from the above ester (300 mg, 0.78 mmol) according to
step B of general synthetic procedure D as a yellow solid (180 mg,
(400 MHz, DMSO‑d6)
d
8.12e8.06 (m, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H),
7.62 (d, J ¼ 15.5 Hz, 1H), 7.56 (s, 2H), 7.42e7.37 (m, 2H), 4.91 (t,
J ¼ 5.5 Hz, 1H), 3.84e3.79 (m, 2H), 3.74e3.68 (m, 2H), 2.56 (s, 3H),
2.28 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
d 187.8, 157.9, 145.4,
143.6, 133.9, 131.2, 130.0, 129.7, 129.0, 125.0, 120.5, 74.0, 60.5, 16.0,
14.0; HRMS (ESI) calcd for C20H22O3S (M þ H)þ 343.1362, found
343.1364.
yield 65%). 1H NMR (400 MHz, DMSO‑d6)
d 8.14e8.03 (m, 2H), 7.80
(d, J ¼ 15.5 Hz, 1H), 7.61 (d, J ¼ 15.5 Hz, 1H), 7.56 (s, 2H), 7.43e7.29
(m, 2H), 4.43 (s, 2H), 2.55 (s, 3H), 2.27 (s, 6H); 13C NMR (100 MHz,
4.1.7. (E)-2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-
DMSO‑d6)
d
187.9, 170.2, 157.4, 145.5, 143.5, 133.9, 131.1, 130.5, 129.7,
en-1-yl)phenyl propylcarbamate (14)
129.1, 125.0, 120.8, 68.8, 16.1, 14.0; HRMS (ESI) calcd for C20H20O4S
To a solution of 17 (50 mg, 0.17 mmol) and triethylamine (4 mL,
(M þ H)þ 357.1155, found 357.1159.
0.03 mmol) in anhydrous CH2Cl2 (1.5 mL) was added propyl iso-
cyanate (21 mg, 0.25 mmol). The mixture was stirred at room
temperature for 5 h, then the solution was concentrated and pu-
rified by column chromatography to give compound 14 as a white
(E)-2-(2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-oxoprop-
1-en-1-yl)phenoxy)butanoic acid (11). The corresponding ester
was prepared from 17 (200 mg, 0.67 mmol) and methyl 2-
bromobutanoate (363 mg, 2.01 mmol) according to step A of gen-
eral synthetic procedure D as a yellow oil (210 mg, yield 79%).
Compound 11 was prepared from the above ester (200 mg,
0.50 mmol) according to step B of general synthetic procedure D as
a yellow solid (120 mg, yield 62%). 1H NMR (400 MHz, DMSO‑d6)
solid (46 mg, yield 72%). 1H NMR (400 MHz, CDCl3)
d 7.99e7.90 (m,
2H), 7.72 (d, J ¼ 15.6 Hz, 1H), 7.42 (d, J ¼ 15.6 Hz, 1H), 7.36e7.27 (m,
4H), 5.21 (t, J ¼ 6.1 Hz, 1H), 3.29e3.17 (m, 2H), 2.53 (s, 3H), 2.22 (s,
6H), 1.66e1.53 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz,
CDCl3)
d 189.3, 153.8, 150.1, 145.6, 144.3, 134.6, 132.3, 132.0, 129.1,
d
8.14e8.04 (m, 2H), 7.80 (d, J ¼ 15.6 Hz, 1H), 7.61 (d, J ¼ 15.6 Hz,
128.8, 125.2, 121.3, 43.1, 23.3, 16.4, 14.9, 11.3; HRMS (ESI) calcd for
C
1H), 7.55 (s, 2H), 7.44e7.35 (m, 2H), 4.44 (t, J ¼ 5.9 Hz, 1H), 2.55 (s,
22H25NO3S (M þ H)þ 384.1628, found 384.1631.
3H), 2.28 (s, 6H), 1.97e1.81 (m, 2H), 0.98 (t, J ¼ 7.4 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6)
d
187.8, 172.1, 157.0, 145.5, 143.5, 133.9, 130.8,
4.1.8. (E)-3-(3,5-dimethyl-4-(3-morpholinopropoxy)phenyl)-1-(4-
(methylthio)phenyl)prop-2-en-1-one (15)
129.9,129.9,129.1, 125.0, 120.6, 81.2, 25.7,16.9, 14.0, 9.0; HRMS (ESI)
calcd for C22H24O4S (M þ H)þ 385.1468, found 385.1473.
To a solution of 17 (100 mg, 0.34 mmol) in anhydrous DMF
(1 mL) were added 4-(3-chloropropyl)morpholine (110 mg,
0.67 mmol) and potassium carbonate (232 mg, 1.68 mmol). The
solution was heated to 90 ꢁC for 6 h, then the solution was diluted
with water and extracted with ethyl acetate. The organic layers
were combined, concentrated and purified by column chromatog-
raphy to give compound 15 as a white solid (101 mg, yield 71%). 1H
(E)-2-((2,6-dimethyl-4-(3-(4-(methylthio)phenyl)-3-
oxoprop-1-en-1-yl)phenoxy)methyl) acrylic acid (12). The cor-
responding ester was prepared from 17 (200 mg, 0.67 mmol) and
methyl 2-(bromomethyl)acrylate (360 mg, 2.01 mmol) according to
step A of general synthetic procedure D as a yellow oil (185 mg,
yield 70%). Compound 12 was prepared from the above ester
(185 mg, 0.47 mmol) according to step B of general synthetic pro-
cedure D as a yellow solid (55 mg, yield 31%). 1H NMR (400 MHz,
NMR (400 MHz, CDCl3)
d
8.00e7.86 (m, 2H), 7.71 (d, J ¼ 15.6 Hz,1H),
7.41 (d, J ¼ 15.6 Hz, 1H), 7.34e7.27 (m, 4H), 3.84 (t, J ¼ 6.3 Hz, 2H),
3.73 (t, J ¼ 4.7 Hz, 4H), 2.59 (t, J ¼ 7.3 Hz, 2H), 2.53 (s, 3H), 2.48 (t,
J ¼ 4.7 Hz, 4H), 2.30 (s, 6H), 2.05e1.95 (m, 2H); 13C NMR (100 MHz,
CDCl3)
d
7.98e7.94 (m, 2H), 7.73 (d, J ¼ 15.6 Hz, 1H), 7.42 (d,
J ¼ 15.6 Hz, 1H), 7.35e7.28 (m, 4H), 6.60e6.56 (m, 1H), 6.29e6.25
(m, 1H), 4.56e4.52 (m, 2H), 2.54 (s, 3H), 2.31 (s, 6H); 13C NMR
CDCl3)
d 189.3, 158.3, 145.5, 144.6, 134.8, 131.8, 130.5, 129.3, 129.0,
(100 MHz, DMSO‑d6)
131.3, 130.4, 129.7, 129.1, 126.7, 125.0, 120.8, 70.2, 16.1, 14.0; HRMS
d
187.9, 166.8, 157.5, 145.5, 143.6, 137.6, 133.9,
125.2, 120.6, 70.3, 67.1, 55.5, 53.8, 27.5, 16.5, 15.0; HRMS (ESI) calcd
for C25H31NO3S (M þ H)þ 426.2097, found 426.2101.
12

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
4.1.9. (E)-4-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)
benzoic acid (18)
activation, J774A.1 cells were stimulated for 5 h with 1
and treatment for 1 h with compounds, and then the cells were
infected for 4 h with FLA-ST Ultrapure (2.5 g/mL) (Invitrogen, USA)
or cells were transfected with poly (dA:dT) (0.25 g/mL) (Invi-
mg/mL LPS
To a solution of 1a (366 mg, 2.20 mmol) in methanol (10 mL)
was added 4-formylbenzoic acid 2d (300 mg, 2.00 mmol), followed
by 40% sodium hydroxide solution (4 mL). The mixture was stirred
at room temperature for 24 h. Subsequently, the solution was
adjusted to pH ¼ 2 and then the precipitate was filtered, washed
with methanol and dried to give compound 18 as a yellow solid
m
m
trogen, USA) for 4 h using Lipofectamine 3000 (Invitrogen, USA).
4.2.2. Enzyme-linked immunosorbent assay
Supernatants from cell culture, peritoneal fluid, serum or colon
homogenates were analyzed for mouse IL-1b (Invitrogen, USA)
(415 mg, yield 70%).1H NMR (400 MHz, DMSO‑d6)
d
8.14e8.10 (m,
2H), 8.03 (d, J ¼ 15.6 Hz, 1H), 8.00e7.97 (m, 4H), 7.76 (d, J ¼ 15.6 Hz,
according to the manufacturer’s instructions.
1H), 7.47e7.35 (m, 2H), 2.56 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
187.9, 167.2, 145.9, 142.4, 138.4, 133.6, 129.7, 129.2, 128.8, 125.0,
4.2.3. Cell viability assessment by CCK-8 assay
123.8, 14.0; HRMS (ESI) calcd for C17H14O3S (M þ H)þ 299.0736,
Cells were cultured in 96-well plates and grown to 80%e90%
found 299.0740.
confluence, and pretreated withcompoundsfor 24h at 37 ꢁC with 5%
CO2, then 10 m
L/well CCK8 reagent was added for 1 h at 37 ꢁC with 5%
4.1.10. (E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-(methylsulfinyl)
phenyl)prop-2-en-1-one (33)
CO2. Absorbance was measured using an Epoch 2 Microplate Reader
at 450 nm (BioTek Instruments, Inc., Winooski, VT, USA).
3-Chloroperbenzoic acid (m-CPBA) (289 mg, 1.68 mmol) was
slowly added to the solution of 17 (500 mg, 1.68 mmol) in anhy-
drous CH2Cl2 (15 mL) under stirring at 0 ꢁC. The mixture was stirred
at room temperature for 5 h, then the solution was filtered and the
filter cake was purified by column chromatography to obtain
compound 33 as a yellow solid (354 mg, yield 67%). 1H NMR
4.2.4. Pyroptosis detection
J774A.1 cells were stimulated as usual, the cells supernatants
were evaluated for LDH release using LDH detection kit (Beyotime,
China) according to the manufacturer’s protocol.
(400 MHz, DMSO‑d6)
(m, 2H), 7.73 (d, J ¼ 15.5 Hz, 1H), 7.65 (d, J ¼ 15.5 Hz, 1H), 7.50 (s,
2H), 2.81 (s, 3H), 2.21 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
188.4,
156.6, 151.0, 145.7, 139.8, 129.9, 129.1, 125.6, 124.7, 123.9, 118.2, 43.1,
16.6; HRMS (ESI) calcd for C18H18O3S (M þ H)þ 315.1049, found
315.1051.
d
8.99 (s, 1H), 8.31e8.25 (m, 2H), 7.87e7.82
4.2.5. Western blotting analysis
Cells samples were lysed in a RIPA Lysis Buffer (Beyotime, China)
with protease inhibitors for 30 min at 4 ꢁC. The proteins in lysate or
supernatants were separated by 12% SDS-polyacrylamide gel,
transferred to PVDF membrane, and hybridized with antibodies.
Immunoreactive bands were detected by ECL (ThermoFisher Sci-
d
entific, USA). Anti-mouse IL-1b (p17; cat# AF-401-NA) was from
4.1.11. (E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(4-
R&D Systems (USA). Anti-mouse caspase-1 (p20; cat# AG-20B-
(methylsulfonyl)phenyl)prop-2-en-1-one (34)
0042), anti-NLRP3 (cat# AG-20B-0014) and anti-ASC (cat# AG-
3-Chloroperbenzoic acid (m-CPBA) (754 mg, 4.37 mmol) was
slowly added to the solution of 17 (500 mg, 1.68 mmol) in anhy-
drous CH2Cl2 (15 mL) under stirring. The mixture was stirred at
room temperature for 5 h, then the solution was filtered and the
filter cake was purified by column chromatography to obtain
compound 34 as a yellow solid (338 mg, yield 61%). 1H NMR
25B-0006) were from AdipoGen (USA). Anti-b-actin (cat#
P30002) was bought from Abmart (China). HRP Conjugated anti-
Rabbit lgG (H þ L) (cat# W4011), HRP Conjugated anti-Mouse lgG
(H þ L) (cat# W4021), and HRP Conjugated Donkey Anti-Goat IgG
(cat# V8051) were bought from Promega (USA).
(400 MHz, DMSO‑d6)
(d, J ¼ 15.6 Hz, 1H), 7.66 (d, J ¼ 15.6 Hz, 1H), 7.52 (s, 2H), 3.31 (s, 3H),
2.20 (s, 6H); 13C NMR (100 MHz, DMSO‑d6)
188.4, 156.9, 146.4,
d
8.38e8.27 (m, 2H), 8.15e8.02 (m, 2H), 7.72
4.2.6. MitoSOX red label
After being stimulated as normal, J774A.1 cells were loaded with
d
5 mM MitoSOX Red (mitochondrial superoxide indicator) in the dark
143.9, 142.0, 130.1, 129.3, 127.5, 125.5, 124.8, 118.1, 43.4, 16.7; HRMS
for 30 min at 37 ꢁC. The fluorescence intensity was measured using
a microplate reader Ex/Em ¼ 510/580 nm. All data was obtained
from experiments with at least three replicates.
(ESI) calcd for C18H18O4S (M þ H)þ 331.0999, found 331.0996.
4.1.12. Reaction analogues with glutathione
To a solution of each test compound (0.01 mmol) in DMSO
(3.3 mL) and D-PBS (1.7 mL) was added GSH (0.20 mmol). The
mixture was stirred at 37 ꢁC. The course of reaction was monitored
by HPLC at different time intervals.
4.2.7. Intracellular ROS detection
The total ROS in cells was detected by DCFH-DA assay or Muse
Oxidative stress kit. The Cells plated in a 96-well black plate were
stimulated as usual, and then the cells were incubated with 20 mM
DCFH-DA (Sigma-Aldrich, USA) at 37 ꢁC for 30 min. The fluores-
cence intensity was measured by a fluorescence microplate reader
at 485 nm for excitation and 528 nm for emission. For muse
4.2. Biology
4.2.1. Cell culture and stimulation
detection, the cells were harvested and probed with 10 mM dihy-
The murine macrophage cell line J774A.1 was purchased from
Guangzhou Jennio Biotech Co., Ltd (China). J774A.1 cells were
cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone,
USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA)
at 37 ꢁC with 5% CO2. Mouse bone marrow-derived macrophages
(BMDMs) were collected from mice tibia and femoral bone marrow
and cultured in RPMI 1640 medium with 10% FBS, 1 mM Penicillin-
Streptomycin solution, 1 mM GlutaMax and 20 ng/mL M-CSF. For
NLRP3 inflammasome activation, macrophages primed with 1
mL LPS (Sigma-Aldrich, USA) for 5 h and treated with compounds
for 1 h, and then the cells were stimulated with 10 M nigericin
(Invitrogen, USA) for 1 h. For NLRC4 or AIM2 inflammasome
droethidium (DHE (Abmart, China)) for 30 min at 37 ꢁC in the dark.
The fluorescence intensity of the Cells was analyzed by a Muse Cell
Analyzer (Merck Millipore, Germany). All data were analyzed in at
least three experiments.
4.2.8. Animal experiments
All animal experiments were performed according to the
Guidelines for the Care and Use of Laboratory Animals and were
approved by the Ethics Committee of Guangzhou Medical Univer-
sity (NO. 2020035). Animal studies are reported in compliance with
the ARRIVE guidelines. All animals were raised under a 12 h light/
dark cycle at 24 1 ꢁC with free access to food and water.
mg/
m
13

C. Zhang, H. Yue, P. Sun et al.
European Journal of Medicinal Chemistry 219 (2021) 113417
inflammasomes are activated in Alzheimer’s disease, Mol. Neurodegener. 11
4.2.8.1. Induction of sepsis and drug treatments. Female C57BL/6
mice (6e8 weeks, n ¼ 6 per group) were injected intraperitoneally
with LPS (20 mg/kg) in the presence or absence of compound 40
(pre-treat orally 3 days, 25 mg/kg/day and 100 mg/kg/day). After
6 h, the serum samples and peritoneal fluid were collected to tested
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
L.A.B. Joosten, C.A. Dinarello, OLT1177, a b-sulfonyl nitrile compound, safe in
humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of
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compounds parthenolide and Bay 11-7082 are direct inhibitors of the
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Acknowledgments
[23] M. Cocco, C. Pellegrini, H. Martínez-Banaclocha, M. Giorgis, E. Marini,
This work was supported by the National Natural Science
Foundation of China (NSFC) (NO. 81803364 to Z. Yang and NO.
81872743 to W. Hu).
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A. Costale, G. Miglio, M. Fornai, L. Antonioli, G. Lopez-Castejon, A. Tapia-
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[25] Y. Jiang, L. He, J. Green, H. Blevins, C. Guo, S.H. Patel, M.S. Halquist, M. McRae,
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113417.
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