Discovery and optimization of 4-oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127021

Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of NLRP3 with IC₅₀ of 2.4 μM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.

Full text of DOI:10.1016/j.bmcl.2020.127021

Journal Pre-proofs
Discovery and Optimization of 4-Oxo-2-thioxo-thiazolidinones as NOD-like
receptor (NLR) family, pyrin domain–containing protein 3 (NLRP3) Inhibitors
Yun Chen, Hongbin He, Hua Jiang, Li Li, Zhiyu Hu, Huiying Huang, Qingyan
Xu, Rongbin Zhou, Xianming Deng
PII:
S0960-894X(20)30091-3
DOI:
https://doi.org/10.1016/j.bmcl.2020.127021
BMCL 127021
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
28 November 2019
17 January 2020
5 February 2020
Please cite this article as: Chen, Y., He, H., Jiang, H., Li, L., Hu, Z., Huang, H., Xu, Q., Zhou, R., Deng, X., Discovery
and Optimization of 4-Oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family, pyrin domain–containing
protein 3 (NLRP3) Inhibitors, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/
j.bmcl.2020.127021
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Discovery and Optimization of 4-Oxo-2-thioxo-thiazolidinones as NOD-like
receptor (NLR) family, pyrin domain–containing protein 3 (NLRP3) Inhibitors
Yun Chena,1, Hongbin Heb,1, Hua Jiangb,1, Li Lia, Zhiyu Hua, Huiying Huanga, Qingyan Xua, Rongbin
Zhoub,*zrb1980@ustc.edu.cn, Xianming Denga,*xmdeng@xmu.edu.cn
aState-Province Joint Engineering Laboratory of Targeted Drugs from Natural Products, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102,
China
bInstitute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, School of
Life Sciences and Medical Center
⁎Corresponding authors.
1These authors contributed equally to this work
Graphical abstract
Abstract
Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been
recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-
guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of
NLRP3 with IC50 of 2.4 μM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the
development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.
Keywords:NLRP3, thiazolidinones, Structure-activity relationship (SAR)
Introduction
Inflammasomes are large, cytosolic, multimeric protein complexes and respond to a diverse set of pathogen-associated molecular
patterns (PAMPs) and danger-associated molecular patterns (DAMPs).1-3 Once activated, assembly of the NLRP3 inflammasome
promote the maturation and secretion of important pro-inflammatory cytokines such as IL-1β and IL-18.4-5 Whereas numerous
inflammasomes have been identified,6 NLRP3 is the best characterized and the most elusive one.7 Aberrant NLRP3 activation has
previously been implicated in several auto-inflammatory and auto-immune diseases such as Alzheimer’s disease, type 2 diabetes
mellitus, atherogenesis and gout.8-11 Among the NLRP3 inhibitors identified so far, including benzenesulfonamide-based compounds,12-
13 novel boron compound series,14 acrylate and acrylamide derivatives,15-16 OLT1177,17 BAY 11-708218 and MNS,19 there is no evidence
showing that these inhibitors can specifically and directly inhibit NLRP3 itself.20 Therefore, the development of novel and specific
NLRP3 inhibitors for the treatment of NLRP3-driven diseases is urgently needed. In this work, we reported the identification,
characterization, and structure-activity relationship (SAR) for a novel chemotype of inhibitors of NLRP3. Iterative rounds of
optimization efforts led to the discovery of 4-oxo-2-thioxo-thiazolidinone derivative, 9, as a potent and specific NLRP3 inhibitor.

Through high-throughput screening (HTS) campaign on an in-house bioactive compound library, CFTRinh-172 (1) was identified
as a potent ‘hit’ for blocking NLRP3 inflammasome activation (Figure 1A), which is a reported inhibitor of the cystic fibrosis
transmembrane conductance regulator (CFTR) channel.21 CFTRinh-172 exhibited a dose-dependent inhibitory activity on monosodium
urate crystals (MSU)-induced caspase-1 activation and IL-1β secretion in LPS-primed bone marrow–derived macrophages (BMDMs)
(Figure 1B and 1C). Having identified 1 as a promising ‘hit’, a library (2−21) bearing thiazolidinone core was designed and synthesized
using a concise three-step synthetic route (Schemes 1−2). Briefly, reaction of commercially available amines with an excess of
thiophosgene under basic conditions followed by cyclization in the presence of methyl 2-sulfanylacetate afforded 24a−k in moderate
yield. Connection between the thiazolidinones and aromatic aldehydes under microwave-assisted conditions afforded the desired 4-oxo-
2-thioxo-3-substituted-thiazolidin-5-ylidene-aromatic derivatives 1−15, and 18−21 (Scheme 1). 2,4-dioxo-3-substituted-thiazolidin-5-
ylidene-aromatic derivatives 16−17 were prepared as described in Scheme 2. The geometry of the exocyclic double bond is assumed to
be
(Z)
configuration
due
to
steric
effects.22
Using 1 as a lead, the SAR for 2-thioxothiazolidin-4-one was explored, utilizing inhibition of IL-1β secretion in BMDMs and
intracellular chloride efflux in HT29 cells as readout to measure the inhibitory activity against NLRP3 inflammasome and CFTR,
respectively. We first investigated the effects of modification to the aryl ring (R2) by introducing a hydrophobic group or a hydrophilic
group resulted in compounds 2−8. As shown in Table 1, introduction of 4-methylpiperazin-1-yl, morpholino and 4-hydroxyl-piperidin-
1-yl groups to 1 led to 2−4, respectively, which all showed slightly improved NLRP3 inhibitory activity. When the trifluoromethyl
group was replaced with trifluoromethoxy and nitro groups (2 vs 5, 3 vs 6, 2 vs 7), respectively, the anti-inflammatory activity was
maintained. However, compound 8 containing substituent with anilino group dramatically abolished the inhibitory activity (32%)
against NLRP3 at 10 μM. These modifications all yielded compounds possessing less potent activities against CFTR, which provided
the improved NLRP3 selectivity.
We next explored the effects of introducing additional carbon linker between phenyl ring and thiazolidinone core, resulting
compounds 9−11 with improved NLRP3 inhibitory activities, having IC50s of 2.40, 5.91 and 4.32 μM, respectively (Table 2).
Surprisingly, compounds 9−11 didn’t show CFTR inhibitory activities at 20 μM. These results indicated that proper length of the linker
appears to be critical for achieving both activity and selectivity against NLRP3. Replacement the carboxyl group with tetrazolyl
bioisostere yielded compounds 12−14, which exhibited a slight decrease of inhibitory potency against NLRP3 compared with their
counterparts (9−11). Compound 15 containing a substituent with 4-methylpiperazin-1-yl group showed a sharp loss of inhibitory
activity against NLRP3, suggesting a limited tolerance for 4-amide derivatization under the condition of one carbon linker (n = 1). The
distinct SAR profiles were observed with different carbon linker (1 vs 9, and 2 vs 15).
Subsequently, 2,4-dioxo-thiazolidinedione derivatives (16−17) were synthesized and evaluated, which exhibited decrease activities
against NLRP3. This result suggested the positive impact of the thioxo group (X = S) in the thiazolidinone core. Finally, the
substitutents of R4 ranging from 3-trifluoromethoxylphenyl (18), 3-fluorophenyl (19), 3-pyridyl (20) to biphenyl (21), were investigated
resulting in decreased NLRP3 inhibitory activities.
The SAR exploration of 2-thioxothiazolidin-4-one scaffold revealed that the one carbon spacer between phenyl ring and
thiazolidinone core (n = 1), the thioxo group (X = S), 4-carboxyl group (R3) and 3-trifluoromethylphenyl group (R4) were key structural
features for achieving both potent inhibitory activity and selectivity against NLRP3. The most active compound 9 inhibited IL-1β
secretion in LPS-primed BMDMs with micromolar cellular IC50 value of 2.40 μM, but not blocked CFTR activity at a concentration of
20 μM.

With these new NLRP3 inhibitors in hand that demonstrated excellent inhibitory potency, the most potent compounds (2−7 and 9),
as well as 1, were tested against human liver microsomes (HLM) and mouse liver microsomes (MLM) in the presence of an NADPH
regenerating system (Table 3). Of these, the stability of 1 was limited in mouse liver microsomes (t1/2 = 12.6 min) but was better in
human microsomes (t1/2 = 277.2 min). The morpholino-amide derivatives 3 and 6 exhibited much better metabolic stability (t1/2 >145
and 187.3 min, respectively), than 4-methylpiperazin-1-yl-amide derivatives (2, 5 and 7) and 4-hydroxypiperidin-1-yl-amide derivative
(4) in human microsomes. Compound 9 exhibited the best in vitro microsomal stability in MLM with lowest clearance values (CLhep
20.7 mL/min/kg) and longest half-life (t1/2 > 145.0 min) among these analogs.
<
The effects of compound 9 on NLRP3 inflammasome activation were also further characterized in BMDMs. Compound 9
exhibited time-dependent inhibitory activities on IL-1β secretion at the dose of 5 µM in LPS-primed BMDMs when stimulated with
different NLRP3 agonists, including nigericin, ATP and MSU (Figure 2A–C). Similarly, when LPS-primed BMDMs were treat ed with
an increasing concentration of NLRP3 agonists along with 9 (5 µM), secreted IL-1β was also inhibited in a dose-dependent manner
(Figure 2D–F). In contrast, 9 had no effects on tumor necrosis factor-α (TNF-α) production (Figure 2G) and could not inhibit NLRC4 or
AIM2 inflammasome activation induced by Salmonella infection or dsDNA analog Poly(dA:dT) transfection, respectively (Figure 2H
and 2I), which demonstrates that 9 is a specific inhibitor for NLRP3 inflammasome. Compound 9 also blocked nigericin or ATP-
induced BMDMs death (Fig. S1).
Then, the pharmacokinetic properties of compound 9 were further evaluated in Sprague-Dawley rats administered a single
intravenous or oral dose. Compound 9 exhibited moderate pharmacokinetics with a half-life of 1.8 hours, an area under the curve of
1928 (h·ng)/mL, and a bioavailability of 27% (Table S1).

Compound 9 has been proven to be a potent, selective, and
direct inhibitor of NLRP3.23 The in vivo experimental results
showed that inhibition of NLRP3 by compound 9 can
significantly prevent NLRP3-dependent acute inflammation in
mice model of cryopyrin-associated autoinflammatory syndrome
(CAPS) and reverse NLRP3-dependent metabolic disorders in
diabetic mice. Furthermore, compound 9 is active ex vivo for
monocytes from healthy individuals or synovial fluid cells from
patients with gout (for a more comprehensive in vivo efficacy
study, please see ref 23).
10. Duewell, P.; Kono, H.; Rayner, K. J.; Sirois, C. M.;
Vladimer, G.; Bauernfeind, F. G.; Abela, G. S.; Franchi, L.;
Nunez, G.; Schnurr, M.; Espevik, T.; Lien, E.; Fitzgerald,
K. A.; Rock, K. L.; Moore, K. J.; Wright, S. D.; Hornung,
V.; Latz, E., NLRP3 inflammasomes are required for
atherogenesis and activated by cholesterol crystals. Nature
2010, 464 (7293), 1357-1361.
11. Martinon, F.; Petrilli, V.; Mayor, A.; Tardivel, A.; Tschopp,
J., Gout-associated uric acid crystals activate the NALP3
inflammasome. Nature 2006, 440 (7081), 237-241.
12. Lamkanfi, M.; Mueller, J. L.; Vitari, A. C.; Misaghi, S.;
Fedorova, A.; Deshayes, K.; Lee, W. P.; Hoffman, H. M.;
Dixit, V. M., Glyburide inhibits the Cryopyrin/Nalp3
inflammasome. J. Cell Biol. 2009, 187 (1), 61-70.
13. Fulp, J.; He, L.; Toldo, S.; Jiang, Y.; Boice, A.; Guo, C.; Li,
X.; Rolfe, A.; Sun, D.; Abbate, A.; Wang, X. Y.; Zhang, S.,
Structural Insights of Benzenesulfonamide Analogues as
NLRP3 Inflammasome Inhibitors: Design, Synthesis, and
Biological Characterization. J. Med. Chem. 2018.
61(12):5412-5423.
14. Baldwin, A. G.; Rivers-Auty, J.; Daniels, M. J. D.; White,
C. S.; Schwalbe, C. H.; Schilling, T.; Hammadi, H.;
Jaiyong, P.; Spencer, N. G.; England, H.; Luheshi, N. M.;
Kadirvel, M.; Lawrence, C. B.; Rothwell, N. J.; Harte, M.
K.; Bryce, R. A.; Allan, S. M.; Eder, C.; Freeman, S.;
Brough, D., Boron-Based Inhibitors of the NLRP3
Inflammasome. Cell Chem. Biol. 2017, 24 (11), 1321-1335
e5.
15. Cocco, M.; Pellegrini, C.; Martinez-Banaclocha, H.;
Giorgis, M.; Marini, E.; Costale, A.; Miglio, G.; Fornai, M.;
Antonioli, L.; Lopez-Castejon, G.; Tapia-Abellan, A.;
Angosto, D.; Hafner-Bratkovic, I.; Regazzoni, L.;
Blandizzi, C.; Pelegrin, P.; Bertinaria, M., Development of
an Acrylate Derivative Targeting the NLRP3
Inflammasome for the Treatment of Inflammatory Bowel
Disease. J. Med. Chem. 2017, 60 (9), 3656-3671.
16. Cocco, M.; Garella, D.; Di Stilo, A.; Borretto, E.;
Stevanato, L.; Giorgis, M.; Marini, E.; Fantozzi, R.; Miglio,
G.; Bertinaria, M., Electrophilic warhead-based design of
compounds preventing NLRP3 inflammasome-dependent
pyroptosis. J. Med. Chem. 2014, 57 (24), 10366-82.
17. Marchetti C; Swartzwelter B; Gamboni F; Neff CP; Richter
K; Azam T; Carta S; Tengesdal I; Nemkov T; D'Alessandro
A; Henry C; Jones GS; Goodrich SA; St Laurent JP; Jones
TM; Scribner CL; Barrow RB; Altman RD; Skouras DB;
Gattorno M; Grau V; Janciauskiene S; Rubartelli A; Joosten
LAB; CA., D., OLT1177, a β-sulfonyl nitrile compound,
safe in humans, inhibits the NLRP3 inflammasome and
reverses the metabolic cost of inflammation. Proc. Natl.
Acad. Sci. U. S. A. 2018, 115 (7), E1530-E1539.
18. Juliana, C.; Fernandes-Alnemri, T.; Wu, J.; Datta, P.;
Solorzano, L.; Yu, J. W.; Meng, R.; Quong, A. A.; Latz, E.;
Scott, C. P.; Alnemri, E. S., Anti-inflammatory compounds
parthenolide and Bay 11-7082 are direct inhibitors of the
inflammasome. J. Biol. Chem. 2010, 285 (13), 9792-9802.
19. He, Y.; Varadarajan, S.; Munoz-Planillo, R.; Burberry, A.;
Nakamura, Y.; Nunez, G., 3,4-methylenedioxy-beta-
nitrostyrene inhibits NLRP3 inflammasome activation by
blocking assembly of the inflammasome. J. Biol. Chem.
2014, 289 (2), 1142-50.
In conclusion, cell-based structure activity relationship
(SAR) studies guided the discovery of compound 9, which
represents a new chemotype exhibiting potent and highly
selective NLRP3 activities. Having excellent selectivity and
favorable pharmacokinetic profile, compound 9 may serve as a
good starting point for the development of new therapeutics
against NLRP3 inflammasome related diseases.
Notes
The authors declare no competing financial interest.
Acknowledgments
This work was supported by Ministry of Science and
Technology (2017YFA0504504 and 2016YFA0502001) and the
National Natural Science Foundation of China (81661138005,
81422045, 81603131 and 91853203), the Fundamental Research
Funds for the Central Universities of China (20720190101 and
20720170067), and the Program of Introducing Talents of
Discipline to Universities (111 Project, B06016).
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inflammasome in inflammatory diseases. Nat. Rev. Drug
N
N
5
76
75
81
32
69
68
66
27
1.10
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2.57±
3.18±
2.98±
>1
Discovery 2018, 17 (8), 588-606.
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N
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6
inhibitors with improved water solubility identified by
structure-activity analysis. Bioorg. Med. Chem. 2008, 16
N
N
7
(17), 8187-8195.
22. Nitsche, C.; Schreier, V. N.; Behnam, M. A.; Kumar, A.;
H
N
8
Bartenschlager, R.; Klein, C. D., Thiazolidinone-peptide
hybrids as dengue virus protease inhibitors with antiviral
activity in cell culture. J. Med. Chem. 2013, 56 (21), 8389-
8403.
a BMDMs were treated with tested compounds at a concentration of 10 μM. b
HT29 cells were treated with tested compounds at a concentration of 20 μM. c
SI (Selectivity index) was determined by the ratio of % NLRP3 Inhibition at
d
10 μM and % CFTR Inhibition at 20 μM. BMDMs were pretreated with
23. Jiang, H.; He, H.; Chen, Y.; Huang, W.; Cheng, J.; Ye, J.;
Wang, A.; Tao, J.; Wang, C.; Liu, Q.; Jin, T.; Jiang, W.;
Deng, X.; Zhou, R., Identification of a selective and direct
NLRP3 inhibitor to treat inflammatory disorders. J. Exp.
Med. 2017, 214 (11), 3219-3238.
varied doses of tested compounds and then primed with LPS and stimulated
with nigericin. Production of IL-1β was measured by ELISA and then the
cytokine level was normalized to that of DMSO-treated control cells. IC50
values were reported as the mean ± SD.
Table 2. SAR of Compounds 9-21 for NLRP3 and CFTR
Figure 1. CFTRinh-172 inhibits NLRP3 inflammasome activation. A)
S
X
F3C
R4
Chemical structure of CFTRinh-172 (1). B) Immunoblot analysis of IL-1β and
cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs
treated with various doses of CFTRinh-172 as indicated and then stimulated
with MSU. C) ELISA of IL-1β from LPS-primed BMDMs treated with
R3 n = 1, X = S, 9, 12 and 15
CO2H
S
S
N
N
n = 2, X = S, 10 and 13
n
n = 3, X = S, 11 and 14
n = 1, X = O, 16-17
O
18-21
O
Inhibition of NLRP3 Inhibition of CFTR Inhibition of NL
CFTRinh-172 (0–20 μM) and then stimulated with MSU. Cytokine level is
Cmpd
Structure
activity(%)a
activity(%)b
activity (IC50 μ
normalized to that of DMSO-treated control cells.
9
NId
NI
NI
2.40±0.10
5.91±0.24
4.32±0.09
R3
CO2H
CO2H
CO2H
=
83
74
68
Figure 2. Compound
9
specifically inhibits NLRP3 inflammasome
10
activation. (A-C) ELISA of IL-1β in supernatants from LPS-primed BMDMs
treated with 9 (5 μM) and then stimulated with nigericin (A), ATP1(1B), MSU
(C) for indicated time. (D–F) ELISA of IL-1β in supernatants from LPS-
N
N
12
67
67
72
NI
NI
NI
5.58±0.13
6.50±0.12
4.85±0.35
primed BMDMs treated with 9 (5 μM) and then stimulated with various doses
N
N
H
of nigericin (10 µM) (D), ATP (2.5 mM) (E) or MSU (150 µg/mL) (F). (G)
N
N
Production of TNF-α (as measured by ELISA) from LPS-primed BMDMs
13
N
N
treated with 9 (5 μM) and stimulated with nigericin (10 µM), ATP (2.5 mM)
H
or MSU (150 µg/mL). (H-I) ELISA of IL-1β in supernatants from LPS-
N
N
primed BMDMs treated with 9 (5 μM) and then transfected with poly(dA:dT)
14
N
N
H
(0.5 µg/ml) for various time points by using of Lipofectamine 2000
O
(Invitrogen) (H), or infected by Salmonella typhimurium (multiplicity of
15
35
67
10
23
>10
N
N
infection) (I) with different time.
CO2H
16
5.96±0.08
N
N
Scheme 1. Synthesis of Compounds 1-15 and 18-21a
17
63
NI
5.61±0.14
N
N
H
aReagents and conditions: (a) NaHCO3, CH2Cl2, H2O, 0 °C to room= F3CO
R4
18
29
63
63
73
12
NI
NI
11
>10
temperature (RT), overnight; (b) Methyl 2-sulfanylacetate, TEA, CH2Cl2, 0
°C to RT, overnight; (c) Substituted aromatic aldehydes, NaOAc, HOAc,
F
microwave, 130 °C, 0.5 h.
19
3.52±0.03
6.27±0.16
7.63±0.36
Scheme 2. Synthesis of Compounds 16-17a
N
20
aReagents and conditions: (a) K2CO3, DMF, 0 °C to RT, 12 h; (b) Substituted
aromatic aldehydes, NaOAc, HOAc, microwave, 130 °C, 0.5 h.
21
Table 1. SAR of Compounds 1-8 for NLRP3 and CFTR
a BMDMs were treated with tested compounds at a concentration of 10 μM. b
HT29 cells were treated with tested compounds at a concentration of 20 μM. c
BMDMs were pretreated with varied doses of tested compounds and then
primed with LPS and stimulated with nigericin. Production of IL-1β was
measured by ELISA and then the cytokine level was normalized to that of
R1
S
O
R1 = CF3, 1-4, 8
R1 = OCF3, 5-6
R1 = NO2, 7
S
R2
N
d
O
DMSO-treated control cells. IC50 values where reported as the mean ± SD.
SIc
Inhibition of
Inhibition of
Inhibition of NLRP3
NI means no inhibition (% inhibition @ 20 μM < 10).
R2
NLRP3 activity(%)a CFTR activity(%)b
activity (IC50 μM)d
NLRP3/CFTR
Table 3. In vitro Metabolic Stability of Compounds 1-7 and 9
OH
71
81
86
77
0.83
6.39±0.33
in HLM and MLMa
N
N
1.05
2.69±0.08
Cmpd
2.74±0.31
HLM
CLhep
(min) (mL/min/kg) (min) (mL/min/kg)
MLM
t1/2
t1/2
CLhep
N
O
81
80
74
72
1.09
1.11
1
2
3
277.2
15.6
>145
2.8
15.3
<4.7
12.6
1.7
>145
69.8
86.6
<20.7
N
OH
2.44±0.24

4
5
6
7
9
44.1
27.1
187.3
13.8
10.3
12.8
3.9
15.8
<4.7
2.8
4
3.7
23.9
1.8
>145
12.6
1.7
82.4
83
58.1
86.5
<20.7
69.8
86.6
82.4
>145
Testosterone 277.2
Propranolol
Clozapine
15.6
44.1
15.3
10.3
4
at1/2, half-life. CLhep, hepatic clearance. Testosterone, propranolol, and
clozapine were used as control.