Structural insights of sulfonamide-based NLRP3 inflammasome inhibitors: design, synthesis, and biological characterization

Article abstract of DOI:10.1007/s00044-020-02692-4

NLRP3 inflammasome has recently attracted much attention as a potentially druggable target to develop potential therapeutics for inflammatory and neurodegenerative disorders. In our continuing studies, structure–activity relationship studies were conducted based on a newly identified NLRP3 inhibitor, YQ-II-128, from our laboratory to understand the structural features and improve aqueous solubility. The results revealed that steric interactions at the propoxyl and amide domain of YQ-II-128 are important for the observed inhibitory potency on the NLRP3 inflammasome. The results also identified the amide domain to incorporate polar moieties to improve solubility and potentially pharmacokinetic properties. As a result, analog 10 was identified as a selective NLRP3 inhibitor with comparable potency while significantly improved aqueous solubility. Collectively, these findings strongly encourage further optmization of 10 to develop analogs with improved pharmacokinetic properties as potential NLRP3-targted therapeutics.

Full text of DOI:10.1007/s00044-020-02692-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:473–482
https://doi.org/10.1007/s00044-020-02692-4
ORIGINAL RESEARCH
Structural insights of sulfonamide-based NLRP3 inflammasome
inhibitors: design, synthesis, and biological characterization
1
Yiming Xu1 Matteo Scipioni1 Hallie Blevins1 Shijun Zhang
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●
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Received: 16 November 2020 / Accepted: 8 December 2020 / Published online: 13 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
NLRP3 inflammasome has recently attracted much attention as a potentially druggable target to develop potential
therapeutics for inflammatory and neurodegenerative disorders. In our continuing studies, structure–activity relationship
studies were conducted based on a newly identified NLRP3 inhibitor, YQ-II-128, from our laboratory to understand the
structural features and improve aqueous solubility. The results revealed that steric interactions at the propoxyl and amide
domain of YQ-II-128 are important for the observed inhibitory potency on the NLRP3 inflammasome. The results also
identified the amide domain to incorporate polar moieties to improve solubility and potentially pharmacokinetic properties.
As a result, analog 10 was identified as a selective NLRP3 inhibitor with comparable potency while significantly improved
aqueous solubility. Collectively, these findings strongly encourage further optmization of 10 to develop analogs with
improved pharmacokinetic properties as potential NLRP3-targted therapeutics.
●
●
Keywords NLRP3 inflammasome SAR Medicinal chemistry
Introduction
other proteins into their active forms, including the pro-
inflammatory cytokines of interleukin (IL)-1β and IL-18
[2–6]. Among the inflammasomes that have been identified
and studied, the NLRP3 inflammasome is activated by
plethora of stimuli and plays a significant role in the
maturation and production of IL-1β and IL-18 [6]. The
pathological roles of NLRP3 inflammasome dysregulation
is evidenced by the gain-of-function mutations of NLRP3 in
cryopyrin-associated periodic syndrome [7]. Recently,
emerging evidence have linked the dysregulation of the
NLRP3 inflammasome to a variety of human diseases,
particularly those that are accompanied by the upregulation
of inflammatory cytokines including inflammatory and
neurodegenerative disorders [7–15]. Therefore, inhibiting
the production and release of these inflammatory mediators
would be of therapeutic value, highlighting the translational
promise of this protein complex to develop effective
NLRP3-targeted therapies for human diseases. This notion
is further supported by the successful development of
canakinumab, an IL-1β-neutralizing antibody; anakinra, a
recombinant IL-1 receptor antagonist; and rilonacept, a
decoy receptor that binds IL-1β and IL-1α [16–18].
The innate immune system is at the forefront of immune
responses in the detection of antigens and foreign invaders
in the body [1]. These include pathogen associated mole-
cular patterns (PAMPs) and damage associated molecular
patterns (DAMPs), which are recognized through pattern
recognition receptors (PRRs) [1, 2]. Amongst the char-
acterized PRRs, the NOD-like receptors (NLRs), absent in
melanoma 2 (AIM2)-like receptors, and retinoic acid-
inducible gene I-like receptors recognize PAMPS and
DAMPs in the cytosolic environment [2]. Architecturally,
these cytosolic PRRs form a multiprotein complex called
the inflammasome and consist of a sensor component, e.g.,
NLR, an adapter component (the apoptosis-associated
speck-like protein containing
a
caspase-recruitment
domain, ASC), and an effector component (pro-caspase-1)
which is then converted into its active form, caspase-1, by
proximity-induced cleavage [3]. Caspase-1 can then cleave
* Shijun Zhang
Recently, small molecule inhibitors that directly inhibit
the NLRP3 protein or indirectly interrupt the NLRP3
inflammasome signaling pathway have been reported [19].
This includes MCC950 [20], CY-09 [21], Oridonin [22],
szhang2@vcu.edu
1
Department of Medicinal Chemistry, Virginia Commonwealth
University, Richmond, VA 23298, USA

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Fig. 1 Small molecule inhibitors
of the NLRP3 inflammasome
Tranilast [23], MNS [24], OLT1177 [25], INF39 [26], and
JC124 [27] (Fig. 1), among others. The demonstrated
in vivo efficacy from various disease models by some of
these inhibitors further supported the translational potential
of targeting this protein complex [28–32]. Our laboratory
has recently developed sulfonamide-based NLRP3 inhibi-
tors and medicinal chemistry campaign led to a lead com-
pound, YQ-II-128, that shows promising potency and
selectivity to NLRP3 over other inflammasomes [27, 33]
(Fig. 2). Furthermore, our ongoing studies support the direct
interaction of YQ-II-128 with the NLRP3 protein (unpub-
lished data). Although preliminary pharmacokinetic studies
demonstrated YQ-II-128 is a blood brain barrier penetrant,
its permeability is limited and this compound also exhibits
poor oral bioavailability, likely due to poor GI solubility
and high first-pass effects. Herein, we report the structural
modifications of YQ-II-128 to understand how structural
modifications on this chemical scaffold impact biological
activity and to improve the aqueous solubility of these
analogs by structure activity relationship (SAR) studies.
Fig. 2 Development of YQ-II-128 as a NLRP3 inhibitor
changed to a piperazine as our previous SAR studies sug-
gested the tolerance of a tertiary sulfonamide at this position,
and tertiary sulfonamide may increase the BBB penetration
[27, 34]. Analogs 5–13 were designed to replace the
thiophen-3-yl acetyl moiety with amino acids or acid with an
amine moiety to improve aqueous solubility.
The chemical syntheses were achieved by the conditions
shown in Scheme 1. Briefly, intermediate 14 was synthesized
as we described previously [27]. Upon coupling with pro-
pargylamine or piperazine, reactions with hydrazine yielded 16
and 18, respectively. Aldehydes 21, 24, and 26 were obtained
by alkylation of chlorosalicylaldehyde 19, which was followed
by reductive amination with 16 and 18 to give 22, 25, 27, or
28, respectively. Finally, coupling reactions of 22, 25, 27, or
28 with corresponding acids in the presence of EDCI and
HOBt to yield target compounds 2–13, respectively.
Molecular design and chemistry
To improve the aqueous solubility and explore whether fur-
ther modifications of YQ-II-128 will improve its inhibitory
potency, a series of analogs were designed and synthesized.
As shown in Fig. 3, structural modifications were mainly
focused on three positions of YQ-II-128 to incorporate amine
or hydroxyl containing moieties. Previous SAR study sug-
gested the important roles of the propoxyl substituent of the
lead compound. Therefore, analogs 2 and 3 are designed to
include a morpholine or OH at the terminal position of the
propoxyl substituent. In analog 4, the propargyl moiety was
Results and discussion
After chemical synthesis, the target compounds were tested
for their inhibitory potencies on the production of IL-1β in

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475
Fig. 3 Chemical structures of the
designed analogs of YQ-II-128
mouse macrophage J774A.1 cells upon activation with
LPS/ATP, a well-established assay for the NLRP3 inflam-
masome activity. As showed in Table 1, incorporation of a
morpholine or an OH at the terminal position of the pro-
poxyl substituent, as evidenced by 2 and 3, led to a
decreased inhibitory potency (nine- and sixfold, respec-
tively) compared to that of YQ-II-128. This may hint that
steric and hydrophobic interactions at this position are
important for the biological activity. Replacement of the
propargyl moiety in YQ-II-128 with a piperazine moiety
also led to a fourfold loss of potency under the current
experimental settings. Our previous SAR studies of JC124
demonstrated a comparable potency by the same structural
modification at this position. This may suggest that analogs
based on the scaffolds of JC124 and YQ-II-128 interact
with the NLRP3 protein differently. But comparison of a
full spectra of SAR studies are warranted to draw any
conclusion. Surprisingly, the analogs with an amino acid
moiety on the amide position (compounds 5–9) all showed
reduced inhibitory potency compared to that of YQ-II-128
with analog 9 being the most potent in this series. The
results may hint that an aromatic moiety is needed at this
position to optimally interact with the NLRP3 protein.
Adding an NH2 moiety to the alpha-carbon of the thiophen-
3-yl acetamide resulted an analog 10 with approximately
twofold loss of potency. Interestingly, the S-isomer (10) is
2× more potent than the R-isomer (11), thus suggesting a
role of stereochemistry on biological activity in this domain.
Extension of one carbon on the amide domain as reflected
by analogs 12 and 13 retained the inhibitory potency as that
of 10. The results of 12 and 13 also indicate that the sub-
stitution position on the thiophene ring is not critical for
biological activity. The results of 10–13 also support the
notion that the presence of an aromatic moiety is important
for biological activity in the amide domain.
After the establishment of inhibitory potency on IL-1β
release in J774A.1 cells, we selected 10 and 12, the most
potent analogs among this series, to evaluate their selec-
tivity on the NLRP3 inflammasome. To this end,
J774A.1 cells were stimulated with LPS/poly(dA:dT) or
LPS/flagellin to activate the NLRC4 and AIM2 inflam-
masome, respectively. As shown in Fig. 4, compound 12
also significantly inhibited the NLRC4 and AIM2
inflammasomes at 10 µM while compound 10 did not
show significant inhibition on these inflammasomes at this
concentration. The results suggest that although no dif-
ference was observed on the inhibition of IL-1β produc-
tion upon activation of the NLRP3 inflammasome by both
compounds, structurally extended analog 12 lost the
selective inhibition on NLRP3 inflammasome. This also
suggests that steric interaction at this domain is limited
with the NLRP3 protein. We also measured the solubility
of 10 and YQ-II-128 in water at pH of 7.4. As expected,
analog 10 with a primary NH2 moiety shows improved
solubility of 19.5 4.7 μg/ml, compared to YQ-II-128 at
6.3 1.1 μg/ml.

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Scheme 1 Reactants and
conditions: a propargylamine,
TEA, DCM; b d NH2NH2,
EtOH, 60 °C; c 1-(tert-
Butoxycarbonyl) piperazine,
TEA, DCM; e 1, 2-
Dibromoethane, K2CO3,
CH3CN; f morphine, K2CO3,
CH3CN; g acetic acid,
NaCNBH3; h various acids,
EDCI, HOBt, TEA, DCM; i
TBSCl, imidazole, DCM; j 1M
TBAF, THF; k 1-
bromopropane, K2CO3, DMF; l
TFA, DCM
Summary
Experimental section
In our efforts to develop small molecule inhibitors by tar-
geting the NLRP3 inflammasome, we conducted SAR stu-
dies of a lead NLRP3 inhibitor, YQ-II-128, that was
recently identified from our laboratory from a sulfonamide
based chemical scaffold. The design of the new analogs was
focused on incoporation of polar moieties, such as amine
and OH, to improve aquesous solubility. The results
revealed that steric interactions play important roles at the
propoxyl and the amide domains of YQ-II-128. Further-
more, the presence of an aromatic moiety is necessary for
optimla inhibitory potency at the amide domain. Among the
series of analogs designed and charatcerized herein, analog
10 showed slightly reduced inhibitory potency while
maintained selective inhibition on the NLRP3 inflamma-
some, and exhibited improved aqeous solubility. Collec-
tively, the results strongly encourage further charactrization
and development of analogs of 10 as NLRP3 inhibitors and
exploring their therapeutic potentials for human diseases.
Materials and instrumentation
All chemicals and reagents used in the experiments were of
analytical grade and obtained from Sigma-Aldrich. Opera-
tion process was monitored by thin layer chromatography
(precoated silica gel 60 F254 plates, EMD Chemicals) and
visualized with UV light or by treatment with phospho-
molybdic acid. Flash chromatography was performed on
silica gel (200–300 mesh, Fisher Scientific) using solvents
as indicated. 1H-NMR and 13C-NMR spectra were routinely
recorded on a Bruker ARX 400 spectrometer. The NMR
solvent used was CDCl3 or DMSO-d6 as indicated. Tetra-
methylsilane was used as internal standard.
Chemistry
4-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-N-(prop-2-yn-1-yl)-
benzenesulfonamide (15).

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477
Table 1 Inhibitory potency of the designed analogs on the production of IL-1β by J774A.1 cells upon stimulation with LPS/ATP
Compound R1
R2
R3
IC50 (μM)
2.9 0.9
1.8 0.1
1.2 0.2
2
3
4
4.5 0.9
3.4 0.2
3.9 1.5
2.1 0.5
1.2 0.1
0.7 0.1
1.4 0.4
0.7 0.2
5
6
7
8
9
10
11
12
0.9 0.1
13
0.3 0.01
YQ-II-128

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Fig. 4 A, B J774A.1 cells were primed with LPS (1 µg/ml) and acti-
vated with ATP (5 mM), flagellin (1 µg/ml), or poly(dA:dT) (4 µg/ml).
Compounds were added when ATP, flagellin or poly(dA:dT) was
added. Supernatants were then collected for IL-1β analysis by ELISA.
Experiments were conducted in triplicate and repeated at least three
time and Error bars represent SEM. Statistical analysis by Student t test
To the solution of 14 (3.0 g, 8.5 mmol) in acetonitrile
(50 ml) were added propargylamine (1.1 ml, 17.0 mmol)
and trimethylamine (2.4 ml, 17.0 mmol). The solution was
stirred at room temperature (RT) for 4 h. The solvent was
removed by vacuum. Dichloromethane (DCM) was added
and the organic layer was washed with H2O, dried over
Na2SO4, concentrated, and purified by column chromato-
graphy (DCM/MeOH = 100/1) to give 15 (2.3 g, 75%) as a
white solid. 1H NMR (400 MHz CDCl3): δ 3.04 (t, J = 7.44
Hz, 2H), 3.74–3.76 (m, 2H), 3.91 (t, J = 7.32 Hz, 2H), 4.67
(s, 1H), 7.31–7.39 (m, 2H), 7.63–7.77 (m, 6H).
washed with CH3CN and evaporated to dry. The desired
product was purified by column with hexane/acetate = 15:1
as eluent in 85% yield. 1H NMR (400 MHz, Chloroform-d)
δ 10.39 (d, J = 1.2 Hz, 1H), 7.92–7.63 (m, 1H), 7.42 (dt,
J = 9.1, 1.9 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 4.56–4.19
(m, 2H), 3.86–3.50 (m, 2H).
5-chloro-2-(2-morpholinoethoxy)benzaldehyde (21)
Compound 20 (2.5 g, 9.5 mmol) and morphine (0.83 ml,
9.5 mmol) were dissolved in dry CH3CN (30 ml), then
K2CO3 (1.96 g, 14.22 mmol) was added and refluxed for
overnight. The reaction mixture was cooled to RT and fil-
tered, the organic layer was collected, evaporated and pur-
ified by column using acetate/methanol = 40:1 as eluent
(yield: 78%).
4-(2-Aminoethyl)-N-(prop-2-yn-1-yl)benzenesulfona-
mide (16).
Hydrazine (0.8 ml, 25.0 mmol) was added dropwise to a
solution of 15 (1.8 g, 5.0 mmol) in EtOH (30 ml). After
addition, the solution was heated to 60 °C for 1 h. Then H2O
(100 ml) was added and extracted with DCM (3 × 20 ml).
The organic layer was collected, dried over Na2SO4, and
concentrated to give compound 15 as a yellow liquid
without purification (0.94 g, 79%). 1H NMR (400 MHz
MeOD): δ 2.47 (d, J = 2.56 Hz, 1H), 2.84–2.95 (m, 4H),
3.76 (d, J = 2.52 Hz, 2H), 7.44 (d, J = 8.32 Hz, 2H), 7.83
(d, J = 8.36 Hz, 2H).
1H NMR (400 MHz, Chloroform-d) δ 10.33 (s, 1H), 7.72
(d, J = 2.7 Hz, 1H), 7.42 (dd, J = 8.9, 2.7 Hz, 1H), 6.89 (d,
J = 8.9 Hz, 1H), 4.20 (s, 2H), 3.69 (s, 4H), 2.85 (s, 2H),
2.58 (s, 4H).
5-chloro-2-(2-hydroxyethoxy)benzaldehyde (23)
Compound 19 (1.56 g, 10 mmol) was added drop wise
over 15 min to a vigorously stirred solution of sodium
hydroxide (0.4 g, 10 mmol) in water (15 ml), and then 2-
Bromoethanol (709 μL, 10 mmol) was added dropwise and
the resulting solution was heated to reflux for 16 h. The
solution was cooled and maintained at about 10 °C while
sodium hydroxide was added until the solution was strongly
alkaline (pH = 10). The reaction mixture was extracted with
DCM (×4). The combined organic layers were dried and
purified by column (hexane: acetate = 1:1) (yield: 71%).
1H NMR (400 MHz, Chloroform-d) δ 10.33 (d, J = 13.5
Hz, 1H), 7.70 (dd, J = 6.0, 2.8 Hz, 1H), 7.41 (dd, J = 8.9,
2.8 Hz, 1H), 6.99–6.82 (m, 1H), 4.12 (q, J = 8.6, 6.4 Hz,
2H), 3.96 (p, J = 5.2, 4.7 Hz, 2H).
tert-butyl4-((4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)phe-
nyl)sulfonyl)piperazine-1-carboxylate (17)
Compound 17 was synthetized following the procedure
of compound 15.
1H NMR (400 MHz, Chloroform-d) δ 7.91–7.82 (m, 1H),
7.74 (m, 2H), 7.65 (h, J = 3.7, 3.1 Hz, 2H), 7.59–7.49 (m,
1H), 7.40 (m, 1H), 7.34–7.29 (m, 1H), 3.93 (m, 2H), 3.43
(m, 4H), 3.36–2.97 (m, 4H), 2.84 (d, J = 5.9 Hz, 2H),
1.40–1.32 (m, 9H).
2-(2-bromoethoxy)-5-chlorobenzaldehyde (20)
Compound 19 (3 g, 19 mmol) and 1, 2-Dibromoethane
(16.5 ml, 190 mmol) were dissolved in dry CH3CN (50 ml),
then K2CO3 (3.93 g, 28.5 mmol) was added and refluxed for
18 h. The reaction mixture was cooled to RT and filtered,
2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5-chlor-
obenzaldehyde (24)
TBSCl (0.68 g, 4.56 mmol) was added to the solution of
compound 23 (0.45 g, 2.28 mmol) and imidazole (0.23 g,

Medicinal Chemistry Research (2021) 30:473–482
479
3.42 mmol) in DCM (10 ml). The mixture was stirred at RT
overnight. After reaction completion, the DCM was washed
with DI water and brine, evaporated and purified by column
(hexane/acetate = 20:1) to yield the product as a white solid
in 89% yield.
followed by the addition of TEA (292 μL, 2.1 mmol). The
mixture was stirred at RT for 30 min. Compound 22 (0.49 g,
1.0 mmol) was added and the reaction was stirred at RT for
3 h. The reactant was then washed with saturated NaHCO3,
and brine. DCM layer was collected and concentrated and
purified by column chromatography (DCM/MeOH = 30/1)
to give desired compound as a white solid in 69% yield.
1H NMR (400 MHz, DMSO-d6) δ 8.00 (dt, J = 13.8, 5.9
Hz, 1H), 7.63 (ddd, J = 24.4, 9.6, 3.1 Hz, 2H), 7.44–7.32
(m, 2H), 7.28–7.08 (m, 3H), 7.05–6.78 (m, 3H), 4.38 (d,
J = 6.6 Hz, 2H), 4.05 (dd, J = 10.4, 5.2 Hz, 2H), 3.70–3.27
(m, 9H), 3.00–2.48 (m, 5H), 2.37 (d, J = 9.3 Hz, 4H). 13C
NMR (101 MHz, DMSO) δ 170.72, 144.60, 139.01,
136.00, 130.04, 129.69, 129.15, 128.97, 128.53, 128.17,
127.32, 127.25, 126.38, 122.72, 114.07, 79.82, 75.05,
66.61, 53.93, 47.00, 43.59, 35.36, 34.80, 34.61, 33.32,
32.37. HRMS (AP-ESI) m/z: calcd for C30H34ClN3O5S2 [M
+H]+ 616.1707; found, 616.1711.
1H NMR (400 MHz, Chloroform-d) δ 10.51–10.12 (m,
1H), 7.70 (dd, J = 2.9, 1.4 Hz, 1H), 7.40 (ddd, J = 11.4,
9.2, 2.7 Hz, 1H), 6.90 (dd, J = 9.0, 4.4 Hz, 1H), 4.11 (dt,
J = 17.5, 4.8 Hz, 2H), 3.94 (dt, J = 14.1, 4.8 Hz, 2H),
0.91–0.72 (m, 9H), 0.04–0.01 (m, 6H).
5-chloro-2-propoxybenzaldehyde (26)
A mixture of compound 19 (1.6 g, 10 mmol), 1-
bromopropane (1.3 g, 11.0 mmol) and and K2CO3 (2.75
g, 20 mmol) in DMF (30 ml) was stirred at RT for 48 h.
DMF was evaporated under reduced pressure, and the
residue was taken up to DCM (30 ml). The DCM solution
was washed with water thrice, dried over anhydrous
Na2SO4, filtered, and evaporated to obtain product as a
yellow solid and was used in the next step without any
further purification (yield: 85%).
Compounds 3–-13 were synthesized following the pro-
cedure of compound 2.
1H NMR (400 MHz DMSO-d6): δ 1.03 (t, J = 7.40 Hz,
3H), 1.75–1.84 (m, 2H), 4.12 (t, J = 6.40 Hz, 2H), 7.29 (d,
J = 8.92 Hz, 1H), 7.62 (d, J = 2.80 Hz, 1H), 7.69 (dd, J1 =
2.84 Hz, J2 = 8.96 Hz, 1H), 10.33 (s, 1H).
4-(2-((5-chloro-2-propoxybenzyl)amino)ethyl)-N-(prop-
2-yn-1-yl)benzenesulfonamide (28)
Compound 26 (2.0 g, 10 mmol) and compound 16 (2.4 g,
10 mmol) were dissolved in 1, 2-dichloroethane (30 ml) and
stirred at RT for 1 h. Then, acetic acid (343 μL, 6 mmol)
was added, and the mixture was stirred at RT for another 1
h. NaCNBH3 (0.67 g, 10 mmol) in methanol was added
portion wise. Then, the reaction was stirred at RT for 4 h.
The reactant was washed with water, brine, dried over
anhydrous Na2SO4, and evaporated under vacuum. The
crude product was purified by chromatography using hex-
ane/acetate = 1/1 to get target compound as oil in
57% yield.
N-(5-chloro-2-(2-hydroxyethoxy)benzyl)-N-(4-(N-(prop-2-
yn-1-yl)sulfamoyl)phenethyl)-2-(thiophen-3-yl)acetamide (3)
1H NMR (400 MHz, DMSO-d6) δ 8.06 (dt, J = 12.2, 5.9
Hz, 1H), 7.76-7.64 (m, 2H), 7.51–7.41 (m, 2H), 7.38–7.12
(m, 3H), 7.08–6.91 (m, 3H), 4.88 (q, J = 6.0 Hz, 1H), 4.53
(d, J = 3.2 Hz, 2H), 4.05 (dt, J = 13.4, 4.9 Hz, 2H),
3.78–3.41 (m, 7H), 3.02 (dt, J = 3.6, 2.4 Hz, 1H), 2.83 (dt,
J = 33.0, 7.6 Hz, 2H). 13C NMR (101 MHz, DMSO) δ
170.80, 155.73, 144.01, 138.97, 136.00, 130.06, 129.69,
129.14, 128.67, 128.04, 127.31, 126.39, 124.57, 122.83,
114.05, 79.82, 75.05, 70.83, 60.04, 49.31, 46.83, 35.19,
33.28, 32.37. HRMS (AP-ESI) m/z: calcd for
C26H27ClN2O5S2 [M+Na]+ 569.0947; found, 569.0948.
N-(5-chloro-2-propoxybenzyl)-N-(4-(piperazin-1-ylsul-
fonyl)phenethyl)-2-(thiophen-3-yl)acetamide (4)
1H NMR (400 MHz, DMSO-d6) δ 7.80–7.49 (m, 4H),
7.50–7.18 (m, 3H), 7.13–6.92 (m, 3H), 4.50 (d, J = 9.3 Hz,
2H), 4.24–3.50 (m, 8H), 3.03–2.70 (m, 9H), 1.77 (dt, J =
13.2, 6.7 Hz, 2H), 1.01 (q, J = 7.6 Hz, 3H). 13C NMR (101
MHz, DMSO) δ 170.74, 155.60, 144.80, 135.98, 133.41,
130.31, 129.11, 128.98, 128.23, 128.14, 127.92, 126.42,
124.42, 122.83, 113.69, 70.04, 49.35, 46.77, 44.88, 43.55,
35.28, 34.81, 22.47, 10.94. HRMS (AP-ESI) m/z: calcd for
C28H34ClN3O4S2 [M+H]+ 576.1758; found, 576.1755.
(S)-2-amino-N-(5-chloro-2-propoxybenzyl)-3-methyl-N-
(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)butanamide (5)
1H NMR (400 MHz, DMSO-d6) δ 8.28-8.06 (m, 3H),
7.80–7.66 (m, 2H), 7.51 (d, J = 8.2 Hz, 1H), 7.25 (m, 1H),
7.14–6.99 (m, 1H), 4.72–3.81 (m, 5H), 3.82-3.45 (m, 2H),
3.31–2.53 (m, 3H), 2.24–1.87 (m, 1H), 1.73 (m, 2H),
1.04–0.67 (m, 9H). 13C NMR (101 MHz, DMSO) δ 169.19,
155.65, 144.10, 138.92, 131.26, 129.95, 128.54, 127.51,
1H NMR (400 MHz DMSO-d6): δ 0.97 (t, J = 7.68 Hz,
3H), 1.64–1.73 (m, 2H), 2.81-2.84 (m, 4H), 3.05 (t, J =
2.52 Hz, 1H), 3.65–3.66 (m, 2H), 3.72 (s, 2H), 3.92 (t, J =
6.36 Hz, 2H), 6.97 (d, J = 8.72 Hz, 1H), 7.25 (dd, J1 =
2.76 Hz, J2 = 8.68 Hz, 1H), 7.33 (d, J = 2.72 Hz, 1H), 7.43
(d, J = 8.36 Hz, 2H), 7.72 (d, J = 8.36 Hz, 2H), 8.05
(s, 1H).
Compounds 22, 25 and 27 were synthesized following
the procedure of compound 28.
N-(5-chloro-2-(2-morpholinoethoxy)benzyl)-N-(4-(N-
(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(thiophen-3-yl)
acetamide (2)
3-Thiopheneacetic acid (0.3 g, 2.1 mmol), EDCI (0.4 g,
2.1 mmol) and HOBt (0.28 g, 2.1 mmol) were dissolved in
anhydrous dichloromethane (10 ml) under an ice bath

480
Medicinal Chemistry Research (2021) 30:473–482
127.32, 124.75, 114.17, 79.82, 75.07, 70.27, 54.85, 49.15,
34.86, 32.36, 30.29, 22.33, 19.21, 16.84, 10.86. HRMS
(AP-ESI) m/z: calcd for C26H34ClN3O4S [M+H]+
520.2037; found, 520.2040.
(S)-2-amino-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-
(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(thiophen-3-yl)
acetamide (10)
1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H),
7.70–7.39 (m, 3H), 7.38-7.08 (m, 3H), 7.08-6.67 (m, 3H),
5.08 (d, J = 72.0 Hz, 1H), 4.57–4.03 (m, 2H), 3.85 (m, 2H),
3.52 (d, J = 43.1 Hz, 3H), 2.95 (s, 1H), 2.86–2.56 (m, 1H),
1.61 (m, 2H), 0.87 (m, 3H). 13C NMR (101 MHz, DMSO) δ
171.79, 155.48, 143.63, 139.00, 129.90, 129.73, 128.73,
128.16, 127.77, 127.48, 127.30, 127.25, 127.12, 124.40,
113.67, 79.80, 75.03, 70.06, 50.93, 48.77, 44.26, 34.53,
32.37, 22.43, 10.92. HRMS (AP-ESI) m/z: calcd for
C27H30ClN3O4S2 [M+H]+ 560.1425; found, 560.1427.
(R)-2-amino-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-
(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(thiophen-3-yl)
acetamide (11)
(S)-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-
1-yl)sulfamoyl)phenethyl)pyrrolidine-2-carboxamide (6)
1H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.64 (M,
2H), 7.40 (d, J = 7.8 Hz, 1H), 7.34–6.85 (m, 4H),
4.55–4.15 (m, 2H), 3.88 (m, 2H), 3.56 (d, J = 18.7 Hz, 2H),
3.10–2.67 (m, 6H), 1.83–1.46 (m, 4H), 1.05 (t, J = 7.2 Hz,
4H), 0.89 (dt, J = 10.6, 7.3 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 172.38, 155.82, 143.64, 139.31, 129.97, 129.80,
128.50, 128.43, 127.32, 124.40, 114.04, 79.81, 75.08,
70.12, 58.27, 58.04, 46.57, 46.08, 32.35, 29.92, 29.76,
22.31, 10.82, 9.83. HRMS (AP-ESI) m/z: calcd for
C26H32ClN3O4S [M+H]+ 518.1880; found, 518.1875.
(R)-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-
1-yl)sulfamoyl)phenethyl)pyrrolidine-2-carboxamide (7)
1H NMR (400 MHz, DMSO-d6) δ 8.10 (q, J = 5.9 Hz,
1H), 7.79–7.60 (m, 2H), 7.56–7.42 (m, 1H), 7.42–6.93 (m,
4H), 4.69–4.39 (m, 2H), 4.03-3.93 (m, 2H), 3.72-3.58 (m,
2H), 3.28-2.57 (m, 6H), 2.08-1.51 (m, 4H), 1.46-1.16 (m,
4H), 0.96 (M, 3H). 13C NMR (101 MHz, DMSO) δ 169.01,
158.62, 144.07, 139.14, 129.97, 128.76, 127.32, 127.27,
126.27, 119.50, 113.91, 78.89, 75.07, 70.14, 58.34, 58.18,
47.25, 46.06, 32.36, 29.57, 24.00, 22.29, 10.93, 9.02.
HRMS (AP-ESI) m/z: calcd for C26H32ClN3O4S [M+H]+
518.1880; found, 518.1875.
(R)-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-
1-yl)sulfamoyl)phenethyl)thiazolidine-2-carboxamide (8)
1H NMR (400 MHz, DMSO-d6) δ 8.01 (m, 1H), 7.76-
7.54 (m, 2H), 7.49-7.06 (m, 3H), 7.06–6.77 (m, 2H),
4.60–4.20 (m, 2H), 4.03–3.77 (m, 2H), 3.64-3.55 (m, 2H),
3.15–2.50 (m, 6H), 1.65 (m, 2H), 1.30-0.78 (m, 6H). 13C
NMR (101 MHz, DMSO) δ 177.13, 155.82, 138.98,
138.69, 130.06, 130.03, 129.77, 128.33, 127.28, 124.55,
113.80, 79.82, 75.07, 70.09, 61.20, 56.79, 48.73, 32.37,
29.49, 29.15, 22.48, 10.95, 10.92. HRMS (AP-ESI) m/z:
calcd for C25H30ClN3O4S2 [M+Na]+ 558.1264; found,
558.1260.
1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H),
7.79–7.46 (m, 3H), 7.46–7.18 (m, 3H), 7.17–6.93 (m, 2H),
6.86 (d, J = 10.6 Hz, 1H), 5.33–4.87 (m, 1H), 4.67-4.08 (m,
2H), 3.94 (m, 2H), 3.60 (d, J = 43.7 Hz, 3H), 3.03 (s, 1H),
2.94–2.66 (m, 1H), 1.69 (m, 2H), 0.94 (m, 3H). 13C NMR
(101 MHz, DMSO) δ 171.77, 155.50, 143.65, 139.02,
129.90, 129.74, 128.74, 128.18, 127.93, 127.50, 127.32,
127.26, 127.13, 124.42, 113.70, 79.82, 75.04, 70.07, 51.10,
48.78, 44.25, 34.53, 32.37, 22.44, 10.92. HRMS (AP-ESI)
m/z: calcd for C27H30ClN3O4S2 [M+H]+ 560.1445; found,
560.1446.
(S)-2-amino-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-
(prop-2-yn-1-yl)sulfamoyl)phenethyl)-3-(thiophen-2-yl)
propanamide (12)
1H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 7.76 (d,
J = 7.8 Hz, 2H), 7.45–7.22 (m, 4H), 7.09–6.93 (m, 3H),
6.86 (d, J = 21.7 Hz, 1H), 4.45 (d, J = 35.0 Hz, 2H), 3.94
(m, 3H), 3.82–3.50 (m, 4H), 3.21–3.02 (m, 2H), 3.01–2.65
(m, 3H), 1.77 (m, 2H), 1.01 (m, 3H). 13C NMR (101 MHz,
DMSO) δ 174.92, 155.52, 143.93, 138.96, 129.82, 129.75,
128.27, 128.07, 127.96, 127.32, 127.11, 126.48, 124.80,
124.47, 113.60, 79.83, 75.00, 70.03, 55.36, 53.29, 48.83,
43.84, 36.51, 32.36, 22.47, 10.94. HRMS (AP-ESI) m/z:
calcd for C28H32ClN3O4S2 [M+H]+ 596.1420; found,
596.1417.
(R)-2-amino-N-(5-chloro-2-propoxybenzyl)-3-phenyl-N-
(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)propanamide
(9)
(S)-2-amino-N-(5-chloro-2-propoxybenzyl)-N-(4-(N-
(prop-2-yn-1-yl)sulfamoyl)phenethyl)-3-(thiophen-3-yl)
propanamide (13)
1H NMR (400 MHz, DMSO-d6) δ 7.76 (dd, J = 8.3, 3.1
Hz, 2H), 7.29 (m, 8H), 7.10–6.93 (m, 2H), 4.42–4.27 (m,
2H), 3.98 (q, J = 6.0 Hz, 2H), 3.77–3.45 (m, 3H), 3.08 (m,
1H), 2.94–2.62 (m, 4H), 2.09–1.62 (m, 4H), 1.01 (m, 3H).
13C NMR (101 MHz, DMSO) δ 175.39, 155.53, 143.96,
138.92, 129.79, 129.73, 129.63, 128.57, 128.31, 128.09,
127.29, 126.63, 124.65, 124.40, 113.77, 79.82, 75.01,
70.00, 53.13, 48.62, 43.69, 42.75, 35.07, 32.36, 22.46,
10.95. HRMS (AP-ESI) m/z: calcd for C30H34ClN3O4S [M
+H]+ 568.2037; found, 568.2039.
1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 15.2 Hz,
1H), 7.76 (dd, J = 8.3, 3.2 Hz, 2H), 7.52-7.22 (m, 4H), 7.18
(dd, J = 8.9, 2.4 Hz, 1H), 7.12–6.85 (m, 3H), 4.40 (d, J =
4.2 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H), 3.86 (t, J = 6.9 Hz,
1H), 3.75–3.45 (m, 4H), 3.15–3.01 (m, 1H), 2.99–2.63 (m,
4H), 1.85–1.65 (m, 2H), 1.01 (m, 3H). 13C NMR (101
MHz, DMSO) δ 175.39, 155.52, 143.99, 139.27, 129.87,
129.74, 129.48, 128.33, 128.09, 127.29, 127.27, 126.10,
124.43, 122.50, 113.61, 79.82, 75.03, 70.01, 52.55, 52.36,

Medicinal Chemistry Research (2021) 30:473–482
481
48.69, 43.74, 36.91, 32.35, 22.47, 10.91. HRMS (AP-ESI)
m/z: calcd for C28H32ClN3O4S2 [M+H]+ 596.1420; found,
596.1421.
20 min each. Supernatants were analyzed using a Varian
ProStar HPLC equipped with a Variable Wavelength
UV–Visible Detector at 230 nm and Microsorb-MV C18
column 250 × 4.6 mm, isocratic solvent system A/B 60/40
or 75/25 in 12 min (10 and YQ-II-128, respectively), A
consisting of 0.1% TFA acetonitrile, B consisting of 0.1%
TFA water, flow rate of 1 ml/min at room temperature.
Standard curves were constructed by dissolving compounds
in methanol or acetonitrile (10 and YQ-II-128, respectively)
and making serial dilutions.
Biological assays
Cells
J774A.1 murine macrophage cells and was purchased from
American Type Cell Culture (ATCC, Manassas, VA).
J774A.1 cells were cultured in Dulbecco’s modified Eagle
medium (DMEM) with 10% fetal bovine serum (FBS), 1%
penicillin/streptomycin. J774A.1 cells were used under
passage 50 for the experiments. For cell-based assays, at
least three independent experiments with at least triplicates
for each data point of each experiment were conducted.
Data accessibility
The datasets supporting this article have been included and
could be found in the manuscript.
Acknowledgements The work was supported in part by the NIA of the
NIH under award number R01AG058673 (SZ), Alzheimer’s Drug
Discovery Foundation 20150601 (SZ).
IL-1β assays in J774A.1 cells
J774A.1 cells were plated into 96-well plates (1 × 105 cells/
well) for 16 h in growth medium. Cells were primed with
Escherichia coli 0111:B4 LPS (Sigma-Aldrich) (final con-
centration: 1 μg/ml) for 4.5 h. Next, test compounds (0.1,
0.3, 1.0, 3.0, and 10.0 μM) were added and incubated for
30 min, followed by adding ATP (5 mM). After 30 min, the
supernatants were collected, and the level of IL-1β was
measured with a mouse IL-1β ELISA kit (DuoSet ELISA,
R&D Systems) following the manufacturer’s instructions.
Author contributions YX and MS performed organic synthesis and
biological characterization. HB tested the solubility. SZ designed the
research. SZ and YX wrote the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Inhibition on the NLRC4 and AIM2 inflammasome assays
J774A.1 cells were plated into 96-well plates (1 × 105 cells/
well) for 16 h in growth medium. Cells were treated with
LPS (1 μg/ml) and test compounds for 1 h (10 μM). Fla-
gellin or poly-deoxyadenylic−deoxythymidylic acid
sodium salt (poly(dA:dT)) was used to induce the formation
of the NLRC4 and the AIM2 inflammasomes. Flagellin
(Enzo Life Sciences, Farmingdale, NY), isolated from
Salmonella typhimurium strain 14028, was added in
DMEM (Invitrogen) without FBS to the plate (1 μg/ml) and
allowed to incubate for 6 h. Flagellin cell-transfection was
accomplished utilizing the Polyplus transfection kit (PUL-
Sin, New York, NY). For AIM2 activation, cells were
incubated with poly(dA:dT) (4 μg/ml) (InvivoGen, San
Diego, CA) for 8 h. The supernatants were collected, and
levels of IL-1β were measured with a mouse IL-1β ELISA
kit following manufacturer’s instructions.
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