Development of Novel Tetrahydroquinoline Inhibitors of NLRP3 Inflammasome for Potential Treatment of DSS-Induced Mouse Colitis

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Development of Novel Tetrahydroquinoline Inhibitors of NLRP3

Inﬂammasome for Potential Treatment of DSS-Induced Mouse

Colitis

Zhen Dai, Xiao-yi Chen, Lu-yan An, Cui-cui Li, Ni Zhao, Fan Yang, Song-tao You, Chen-zhi Hou,

Kan Li, Cheng Jiang,* Qi-dong You,* Bin Di,* and Li-li Xu *

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ABSTRACT: The NLRP3 inﬂammasome is a critical component

of innate immunity, which defends internal and external threats.

However, inappropriate activation of the NLRP3 inﬂammasome

induces various human diseases. In this study, we discovered and

synthesized a series of tetrahydroquinoline inhibitors of NLRP3

inﬂammasome. Among these analogues, compound 6 exhibited

optimal NLRP3 inhibitory activity. In vitro studies indicated that

compound 6 directly bound to the NACHT domain of NLRP3 but

not to protein pyrin domain (PYD) or LRR domain, inhibited

NLRP3 ATPase activity, and blocked ASC oligomerization,

thereby inhibiting NLRP3 inﬂammasome assembly and activation.

Compound 6 speciﬁcally inhibited the NLRP3 inﬂammasome

activation, but had no eﬀect on the activation of NLRC4 or AIM2 inﬂammasomes. Furthermore, in the dextran sulfate sodium

(DSS)-induced colitis mouse model, compound 6 exhibited signiﬁcant anti-inﬂammatory activity through inhibiting NLRP3

inﬂammasome in vivo. Therefore, our study provides a potent NLRP3 inﬂammasome inhibitor, which deserves further structural

optimization as a novel therapeutic candidate for NLRP3-driven diseases.

and pathogen-associated molecular patterns (PAMPs) can

activate the NLRP3 inﬂammasome, but the molecular

mechanism of NLRP3 inﬂammasome activation remains

poorly understood.⁶⁻⁸NLRP3 inﬂammasome is involved in

the pathogenesis of multiple diseases, including IBD,

hereditary cryopyrin-associated periodic syndromes (CAPSs),

Muckle−Wells syndrome (MWS), type 2 diabetes, athero-

sclerosis, obesity, gout, and others.¹³⁻¹⁶

The activation process of NLRP3 inﬂammasome includes

two steps: priming and activation.¹⁷In the priming step,

inﬂammatory stimuli activate the nuclear factor-κB (NF-κB)

signaling pathway through targeting PRRs such as toll-like

receptors (TLRs) and others.^18,19NF-κB activation increases

the protein expressions of NLRP3, pro-IL-1β, and pro-caspase-

1 and induces the post-translational modiﬁcation of NLRP3.¹²

The next activation step is triggered by extracellular ATP or

bacterial toxins, thereby promoting caspase-1 activation and

INTRODUCTION

■

Inﬂammatory bowel diseases (IBD), including ulcerative colitis

and Crohn’s disease, are chronic immune system-mediated

diseases with features of high morbidity and recurrence rate in

the gastrointestinal tract.^1,2IBD severely aﬀects the quality of

life of patients and signiﬁcantly increases the risk of colon

cancer. However, the exact etiology of IBD is very complicated

and remains elusive. It is now widely accepted that the

development of IBD is implicated in the excessive activation of

immune system and the production of chronic proinﬂamma-

tory cytokines, including IL-1β, IL-6, IL-18, and TNF-α.^3,4

A

previous study has revealed that the production and secretion

of cytokines were regulated by some NOD-like receptor

(NLR) family members.⁵Among them, the NOD-, LRR-, and

pyrin domain-containing protein 3 (NLRP3) inﬂammasome is

one of the most studied and composed of NLRP3, apoptosis-

associated speck-like protein containing a CARD (ASC) and

caspase-1.⁶⁻⁹The assembly of this multiprotein complex

promotes caspase-1-mediated cleavage of pro-IL-1β and pro-

IL-18, resulting in the mature and functional IL-1β and IL-18

production.^10,11NLRP3 is the unique pattern recognition

receptor (PRR), which can be activated by a wide variety of

stimuli such as bacteria, virus and fungal pathogens, uric acid

crystal, ATP, and others.¹²Damage-associated molecular

patterns (DAMPs) released during tissue damage or stress

Received: November 6, 2020

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A

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Figure 1. Four hit compounds with tetrahydroquinoline core were discovered through pharmacophore-based virtual screening. (A). The best

pharmacophore hypothesis for NLRP3 inhibitors screening. The distance between the centroids and the 3D spatial arrangements are represented in

solid blue lines. The pharmacophore features are color-coded: green, hydrogen-bond donor; cyan, hydrophobic group; and orange, aromatic ring.

(B). Binding modes of the hit compounds with pharmacophore model. (C). Chemical structures of the tetrahydroquinoline analogues. (D).

Inhibitory activities of the four hits against IL-1β at the concentrations of 20 and 50 μM tested by enzyme-linked immunosorbent assay (ELISA).

Data are expressed as the inhibition rate (%) of IL-1β release versus dimethyl sulfoxide (DMSO) group mean standard error of the mean (s.e.m.)

(n = 3) from three independent experiments. The p-values referred to the comparison between negative control and indicated compounds

treatment and were analyzed by unpaired, two-tailed Student’s t-test, NS: not signiﬁcant, *p < 0.05, **p < 0.01.

the subsequent maturation and secretion of IL-1β and IL-18.

To study the IBD, DSS, a classic mouse colitis trigger is used to

establish acute colitis mouse models. The DSS-induced colitis

is characterized by some clinical symptoms that mimic IBD

including the general inﬂammatory processes associated with

histopathology.^20,21Previous studies showed that DSS can

cause lysosomal damage and activate NLRP3 inﬂammasome,

which plays an important role in IBD.^22,23Therefore, the

NLRP3 inﬂammasome can be considered as a novel

therapeutic target for IBD.

The NLRP3 protein consists of the N-terminal protein pyrin

domain (PYD) domain, the central NACHT domain, and the

C-terminal LRR domain. Among these domains, the main

function of the PYD domain is to maintain NLRP3 in the

inactive conformation, recruit ASC protein through PYD-PYD

interaction, and promote the formation of NLRP3 inﬂamma-

some complex.²⁴⁻²⁶LRR domain in several innate immune

receptors is mainly responsible for the recognition of

pathogenic microorganisms and dangerous molecules. How-

ever, it has been reported that the LRR domain is not required

for NLRP3 self-regulation and inﬂammasome activation.²⁶The

truncated NLRP3 protein lacking LRR domain is fully

activated by some canonical NLRP3 inﬂammasome triggers

such as nigericin, ATP, SiO₂, and others.²⁶More recently, the

LRR domain of NLRP3 is involved in the partial response to

some speciﬁc stimuli (H₂O₂and Listeria monocytogenes).²⁷In

particular, the NACHT nucleotide binding domain of NLRP3

is responsible for the ATP-dependent oligomerization

process.²⁸Therefore, it has been found that a decrease in

ATPase activity blocks NLRP3 auto-oligomerization and its

intermolecular binding to ASC, which is one of the key steps in

activating inﬂammasome.^28,29It is a focus of future research to

elucidate the molecular mechanisms of NLRP3-driven diseases

to develop potent NLRP3 inhibitors and assess their

therapeutic potential.

To date, some NLRP3 inhibitors have been reported,

including those that directly block NLRP3 protein and ones

indirectly inhibit inﬂammasome components as well as related

signaling events. One of the main drawbacks of indirect

inhibitors is that their exact targets have not been fully

elucidated; thus, there are potential risks caused by oﬀ-target

eﬀects.¹²Compared with blocking cytokines, directly inhibiting

NLRP3 inﬂammasome is a speciﬁc, low-cost, and eﬀective anti-

inﬂammatory strategy. Several direct inhibitors of NLRP3

inﬂammasome have been discovered to date. Among them, the

diarylsulfonylurea compound MCC950 (originally reported as

CRID3/CP-456773), with a half-maximum inhibitory concen-

tration (IC₅₀) of 7.5 nM in bone marrow-derived macrophages

(BMDMs) and 8.1 nM in human monocyte-derived macro-

phages (HMDMs), respectively, is the most potent NLRP3

inhibitor.^30,31MCC950 speciﬁcally blocks NLRP3 inﬂamma-

some activation through inhibiting ASC oligomerization and

the ATPase activity of NLRP3 NACHT domain,^30,31which

demonstrates prominent therapeutic potential in multiple

NLRP3-associated disease models, including Alzheimer’s

disease,³²atherosclerosis,³³diabetes,³⁴colitis,³⁵CAPS, and

experimental autoimmune encephalomyelitis.³¹

Here, we report the development of a series of NLRP3

small-molecule inhibitors with tetrahydroquinoline cores.

G514-0208 was selected as the starting point for further

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a

Scheme 1. Synthetic Route of Tetrahydroquinoline Derivatives

a

Reagents and conditions: (a) MsCl, Et₃N, CH₂Cl₂, r.t.; (b) 10% Pd/C, H₂, CH₂Cl₂, r.t.; (c) RSO₂Cl, Et₃N, CH₂Cl₂, r.t.

Table 1. Inhibitory Activity of All of the Tetrahydroquinoline Derivatives Measured by ELISA

a

inhibitory rates of IL-1β (%)

b

compd.

R

20 μM

50 μM

IC₅₀(μM) (cytotoxity)

1

2

3

4

5

6

7

8

p-nitrile phenyl

28.71 1.56

54.50 4.59

0.35 3.06

42.77 0.37

61.43 1.51

73.19 1.90

44.96 6.27

48.21 5.61

50.19 1.54

15.35 3.45

7.76 7.13

30.71 7.81

39.10 6.52

2.51 0.21

63.18 3.62

29.24 2.88

34.21 1.69

10.84 4.10

29.54 2.31

6.70 0.90

52.42 6.32

28.98 1.56

24.41 1.94

41.50 6.03

51.23 5.26

75.94 10.31

6.35 2.26

64.98 2.86

84.38 3.57

55.07 8.71

68.82 5.70

24.86 3.86

75.97 7.30

91.75 2.44

89.00 7.51

92.23 4.18

79.66 8.83

97.19 7.92

95.45 1.29

88.71 3.01

93.83 4.27

63.16 9.15

86.56 3.30

82.18 6.12

88.04 1.63

74.89 2.07

90.97 3.68

109.78 8.08

37.74 4.67

95.13 4.37

89.87 1.00

72.00 1.41

101.70 1.25

101.84 1.25

101.47 1.54

84.91 8.55

85.48 1.09

104.24 0.60

35.91

8.32

p-triﬂuoromethyl phenyl

3,5-bis(triﬂuoromethyl) phenyl

p-nitrophenyl

o-nitrophenyl

p-acetylphenyl

1-naphthyl

34.68

65.26

>100

97.17

>100

66.64

>100

71.84

>100

94.56

90.90

78.37

>100

71.14

>100

98.80

95.55

>100

98.83

2-naphthyl

p-biphenyl

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

p-chlorophenyl

p-bromophenyl

p-ﬂuorophenyl

m-ﬂuorophenyl

o-ﬂuorophenyl

p-t-butylphenyl

p-methoxyphenyl

p-methylphenyl

p-acetylaminophenyl

p-(methoxycarbonyl)phenyl

2-thienyl

3-pyridyl

phenyl

methyl

ethyl

n-propyl

n-butyl

benzyl

2-(1,3-dioxoisoindolin-2-yl) ethyl

MCC950

c

29

a

ELISA analysis of IL-1β in serum from THP-M cells pretreated with various compounds for 24 h at doses of 20 and 50 μM and then stimulated

b

with LPS for 3 h and ATP for 1 h. After treatment of the tested compounds for 72 h, cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazoliumbromide (MTT) assay and expressed as IC₅₀, median inhibitory concentration. MCC950 served as positive control, while

c

DMSO served as vehicle control. All data were expressed as mean s.e.m. (n = 3) from three independent experiments. The concentrations of

MCC950 were 500 nM and 2 μM.

optimization. Among these analogues, compound 6 exhibited

the most potent inhibitory activity against ATP-induced IL-1β

release. In vitro experiments demonstrated that compound 6

mainly inhibited the activation phase of NLRP3 inﬂamma-

some. Further research showed that compound 6 directly

bound to the NLRP3 NACHT domain but not to PYD or LRR

domain, inhibited NLRP3 ATPase activity, and blocked ATP-

induced ASC oligomerization. We also demonstrated that

compound 6 had a signiﬁcant therapeutic eﬀect on the DSS-

induced colitis mouse model.

RESULTS AND DISCUSSION

■

Virtual Screening for NLRP3 Inhibitors. Virtual screen-

ing of a database of small compounds resulted in the discovery

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Figure 2. (A) Structures of tetrahydroquinoline derivatives used for further screening. (B) ELISA analysis of cleaved IL-1β from THP-M cells

treated with diﬀerent compounds at concentrations of 1, 5, 10, 15, 30, 45, 60, and 100 μM, and then stimulated with LPS and ATP. All data were

expressed as mean s.e.m. (n = 3) from three independent experiments.

of several hit compounds for inhibiting NLRP3 inﬂammasome.

In this work, we constructed a three-dimensional (3D)

pharmacophore model based on common features of ligands,

including two hydrogen-bond acceptors, two hydrophobe, and

one ring aromatic pharmacophore features (Figure 1A). A total

of 1596 candidate compounds were obtained in the Chemdiv

library through pharmacophore-based virtual screening, which

were further ﬁltered and veriﬁed by the cross-docking

procedure. From these candidates, 1507 compounds success-

fully docked into the active sites of the NLRP3 protein (PDB:

6NPY) by Libdock and CDOCKER (DS 3.0), in which 20

compounds with preferable docking scores and binding models

were obtained (Table S3 in the Supporting Information). In

vitro experiment showed that four compounds with tetrahy-

droquinoline skeleton structure exhibited desired IL-1β

inhibitory activity in a concentration-dependent manner

(Figure 1C,D).

As compound G514-0208 exhibited the most potent

inhibition on NLRP3 inﬂammasome, we then designed and

synthesized a series of compounds based on tetrahydroquino-

line core with multiple sulfonamide groups. These compounds

inhibited the production of NLRP3 inﬂammasome-dependent

IL-1β to varying degrees. The 7-disubstituted sulfonamide

group on the tetrahydroquinoline core of G514-0208 was

replaced by substituents with diﬀerent electronic and steric

eﬀects, including aryl, aromatic heterocyclic groups and alkyl

groups, to study the eﬀect on NLRP3 inﬂammasome-

dependent IL-1β release.

Tetrahydroquinoline derivatives were synthesized according

to the synthetic route shown in Scheme 1. Commercially

available 7-nitro-tetrahydroquinolin was reacted with meth-

anesulfonyl chloride at room temperature to obtain 1-

(methylsulfonyl)-7-nitro-1,2,3,4-tetrahydroquinoline in 91.2%

yield. The nitro group of compound I-1 was reduced by 10%

palladium carbon and converted into 1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-amine (I-2) in dichloromethane

with a yield of 83.3%. Target compounds (1−28) were

synthesized through compound I-2 reacting with sulfonyl

chlorides containing diﬀerent substituents, using triethylamine

as a base in dichloromethane.

inﬂammasome, leading to the release of IL-1β and IL-18.^36,37

In this study, ATP and nigericin were used as NLRP3

inﬂammasome activators to explore the inhibitory activity of

compound 6. To stimulate NLRP3 inﬂammasome, we adopt

the model of phorbol myristate acetate (PMA)-stimulated

THP-1 human leukemic monocyte cells to be diﬀerentiated

into macrophages (THP-Ms), as previously described.³⁸THP-

Ms were exposed to LPS (100 ng/mL) for 3 h and then

stimulated with ATP (5 mM) or nigericin (10 μM) for 1 h.

Many reports have conﬁrmed that IL-1β is the most potent

proinﬂammatory cytokine activated by caspase-1 and involves

in a variety of NLRP3-driven diseases.³⁹Therefore, the

inhibitory potencies of the analogues on NLRP3 inﬂamma-

some were evaluated by testing the level of IL-1β using ELISA.

As shown in Table 1, most synthetic tetrahydroquinoline

derivatives exhibited excellent inhibitory activity against

mature IL-1β release at a high concentration of 50 μM,

while the compounds with diﬀerent R-substituents exhibited

signiﬁcantly diﬀerent inhibitory eﬀects at a lower dose of 20

μM. In general, the benzene ring of aryl sulfonamides (4, 5, 6)

bearing electron-deﬁcient groups showed better IL-1β

inhibitory activity than those compounds (16, 17, 18) with

electron-rich groups. Among them, the activity of compound 3

with multiple triﬂuoromethyl groups was remarkably de-

creased, which may be attributed to its poor solubility.

However, the data revealed that all tetrahydroquinoline

derivatives of which the R-position was substituted with

halogen-substituted benzene rings showed poor anti-inﬂam-

matory activity. In comparison to the results of 4-methyl

benzene sulfonamides (17), the activity of compound 15 (p-t-

butylbenzene sulfonamide) was increased. It is speculated that

the steric eﬀect of R-substituted phenyl plays an important

role. We also examined the eﬀects of heteroaromatic

substituents at the R-position on the NLRP3 inﬂammasome-

driven IL-1β secretion. The activity of compound 21

containing pyridine at R-position was better than that of

compound 20 with thiophene, which may be ascribed to the

slightly stronger polarity of the pyridine ring than the

thiophene ring.

The next step was to investigate the inhibitory activity of the

tetrahydroquinoline derivatives bearing alkyl substituents at

the R-positions. When the R-position of tetrahydroquinoline

was replaced with alkyl groups, the activity of -n-butyl > -n-

propyl > -n- ethyl ≈ -n- methyl suggested that increasing the

Preliminary SAR of the Tetrahydroquinoline Deriva-

tives. Exogenous microbial stimuli such as nigericin and

endogenous danger signals such as ATP are classical NLRP3

inﬂammasome triggers, both of which can activate NLRP3

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Figure 3. Compound 6 inhibited the NLRP3 inﬂammasome activation induced by ATP or nigericin. (A) Western blots of cell lysates from THP-M

cells treated with compound 6 at doses of 15−45 μM and then stimulated with LPS and ATP. (B, C) ELISA of cleaved IL-1β (B) and cleaved

caspase-1 (C) in culture supernatants from THP-M cells treated with various doses of compound 6 and then induced by LPS and ATP. (D, E)

ELISA of IL-1β in supernatant from THP-M cells pretreated with (D) CY-09 or (E) compound 6 and then stimulated with LPS, followed by ATP.

(F, G) ELISA of IL-1β in supernatant from LPS-primed THP-M cells treated with compound 6 and then stimulated with (F) NLRP3 activator

ATP or (G) nigericin. (H) ELISA of IL-1β in supernatant from BMDM cells pretreated with compound 6 or CY-09 and then stimulated with LPS

and ATP. (I) ELISA of IL-1β in supernatant from LPS-primed BMDM cells treated with compound 6 or CY-09, following stimulation with ATP.

The data were expressed as mean s.e.m. (n = 3) from three independent experiments. The p-values referred to the comparison between negative

control and indicated compounds treatment and were analyzed by unpaired, two-tailed Student’s t-test, NS: not signiﬁcant, *p < 0.05, **p < 0.01,

***p < 0.001, ****p < 0.0001.

chain length of alkyl substituents may improve the activity of

the synthesized compounds. In addition, compared with

tetrahydroquinoline derivatives containing phenyl groups

(22), compounds 7, 8, and 9 exhibited stronger NLRP3

inﬂammasome inhibitory activity, proving that polyaromatic

substituents were more preferred.

In summary, for the aryl substituents on the amino sulfonyl

group, the steric property of R-substituted groups on the

tetrahydroquinoline derivatives has a signiﬁcant eﬀect on

inhibiting NLRP3 inﬂammasome-dependent IL-1β production.

It is worth noting that long-chain and polyaromatic

substituents at the R-position are beneﬁcial for increasing the

anti-inﬂammatory activity of the synthetic compounds with

tetrahydroquinoline cores.

Given ELISA assays revealed that these analogues exhibited

desirable inhibitory activity against NLRP3 inﬂammasome,

their cytotoxicity was also measured by MTT assay. The data

showed that the IC₅₀values of most synthesized compounds

were above 100 μM, indicating that these inhibitors did not

exhibit cytotoxicity on THP-M cells (Table 1). We next

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Figure 4. Compound 6 had slight inhibitory eﬀects on the priming of NLRP3 inﬂammasome through inhibiting NF-κB. (A) Western blots of cell

lysates from THP-M cells treated with compound 6 and then stimulated with LPS for 3 h (compound 6 before LPS). (B) Western blots of cell

lysates from THP-M cells stimulated with LPS for 3 h and treated with compound 6 (compound 6 after LPS). (C) THP-M cells were pretreated

with compound 6 for 24 h followed by LPS stimulation for 4 h. Protein levels of p65, phosphorylated p65 (p-p65), IκBα, and phosphorylated IκBα

were analyzed by western blot. (D) The mRNA levels of NLRP3, caspase-1, and IL-1β were determined with real-time PCR. Glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) was used as an internal reference. (E). ELISA of TNF-α in culture supernatants of THP-Ms pretreated with

compound 6 and then stimulated with LPS (100 ng/mL), followed by ATP (5 mM). The data were expressed as mean s.e.m. (n = 3) from three

independent experiments. The p-values referred to the comparison between negative control and indicated compounds treatment and were

analyzed by unpaired, two-tailed Student’s t-test, NS: not signiﬁcant, *p < 0.05, **p < 0.01.

selected some compounds with better activity for the second

round of screening to further determine the most potent

NLRP3 inﬂammasome inhibitor. As shown in Figure 2, these

compounds remarkably inhibited the production of IL-1β in a

concentration-dependent manner. Among these analogues,

compound 6 showed the most potent inhibitory activity

against the NLRP3 inﬂammasome, with an IC₅₀of

approximately 7.8 μM. As the result, compound 6 was chosen

for further pharmacological studies in vitro and in vivo.

Compound 6 Inhibits NLRP3 Inﬂammasome Activa-

tion. To further conﬁrm the inhibitory eﬀect of compound 6

on NLRP3 inﬂammasome, we next investigated the eﬀects of

compound 6 on the priming and activation stages of NLRP3

inﬂammasome. THP-M cells were pretreated with various

doses of compound 6 and then stimulated with LPS and

extracellular ATP or nigericin. The data revealed that

compound 6 dose-dependently inhibited the secretion of

cleaved IL-1β and p20 subunit of active caspase-1 in

supernatants from THP-M cells (Figure 3A−C). CY-09 served

as the positive control (Figure 3D), which was reported to be

eﬀective in inhibiting NLRP3 inﬂammasome activation.⁴⁰As

shown in Figure 3E,F, the secretion of mature IL-1β was

signiﬁcantly inhibited in THP-M cells treated with compound

6 before or after LPS priming, suggesting that compound 6 had

a signiﬁcant inhibitory eﬀect on NLRP3 activation. Nigericin, a

K⁺/H⁺ionophore, activates NLRP3 inﬂammasome through

forming a nuclear pore and triggering K⁺eﬄux.^36,41

Compound 6 also blocked the nigericin-induced IL-1β release

in a concentration-dependent manner (Figure 3G). In

addition, BMDMs, the primary murine cells, were used to

test and verify the eﬀects of compound 6. As shown in Figure

3H,I, before or after LPS priming, compound 6 exhibited

signiﬁcant inhibitory eﬀects on NLRP3 inﬂammasome-related

IL-1β production in BMDMs. These results indicated that

compound 6 potently inhibited the activation stage of NLPR3

inﬂammasome.

We next tested whether compound 6 could suppress the

upstream signaling of NLRP3 inﬂammasome. As shown in

Figure 4A,B, compound 6 slightly inhibited the protein

expressions of NLRP3 and pro-IL-1β in THP-M cells treated

with 6 prior to LPS stimulation, compared to compound 6

treatment after LPS priming. The priming stage of NLRP3

inﬂammasome formation is mediated by NF-κB pathway. In

normal cells, NF-κB is inhibited through the formation of

heterotrimer (p65, p50, and inhibitors of NF-κB subunits

IκBα) in the cytoplasm.⁴²LPS triggering TLRs activates IκB

kinase (IKK) complex, which subsequently leads to IκBα

phosphorylation, ubiquitination, and proteasome-mediated

degradation. The activation of IKK complex licenses p50−

p65 heterodimer, the most common form of NF-κB, to

translocate to the nucleus, bind to speciﬁc DNA sequences,

and enhance target gene transcription.⁴³The eﬀect of

compound 6 on the NF-κB signaling was investigated. As

shown in Figure 4C, compound 6 had weak inhibitory ability

on the phosphorylation of NF-κB. Because IκBα transcription

is mediated by NF-κB pathway and its negative feedback

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Figure 5. Compound 6 directly bound to the NLRP3 NACHT domain and inhibited NLRP3 ATPase activity. (A) NLRP3 ATPase activity assay in

the presence of various concentrations of compound 6 or equal-volume phosphate-buﬀered saline (PBS). (B). Schematic representation of human

NLRP3 domains. (C). Kinetics of the interactions between NLRP3 domains and compound 6 determined by BLI assay. (D−F) BLI dose-response

association and dissociation curves of compound 6 and PYD domain (D), NACHT domain (E), and LRR domain (F) of NLRP3, respectively. The

results were expressed as mean s.e.m. (n = 3) carried out in triplicate from three independent experiments. Statistics were analyzed by unpaired,

two-tailed Student’s t-test, **p < 0.01,***p < 0.001.

function for NF-κB, compound 6 had dose-dependent

inhibitory eﬀect on the IκBα expression after LPS stimulation

for 4 h. Moreover, the result of real-time polymerase chain

reaction (RT-PCR) showed that compound 6 treatment

caused a slight decrease in the IL-1β mRNA level, but there

was no signiﬁcant diﬀerence in NLRP3 or caspase-1 mRNA

levels (Figure 4D). In addition, we investigated the eﬀect of

compound 6 on the LPS-induced TNF-α production.

Although compound 6 had a little inhibitory eﬀect on the

TNF-α secretion at a high concentration (45 μM), it almost

exhibited no inhibition on the TNF-α level at the

concentrations of 5, 10, 15, and 30 μM, at which it eﬀectively

acted on NLRP3 inﬂammasome activation (Figures 4E and

3E−I). Collectively, these results suggested that compound 6

had a slight eﬀect on the priming of NLRP3 inﬂammasome

through inhibiting NF-κB, whereas the inhibitory eﬀect on

NLRP3 inﬂammasome activation was predominant.

Compound 6 Binds to NLRP3 NACHT Domain and

Inhibits NLRP3 ATPase Activity. We explored the

mechanism of how compound 6 inhibited the activation of

NLRP3 inﬂammasome. A previous study showed that the

NLRP3 ATPase activity is required for NLRP3 inﬂammasome

assembly and activation.²⁸The eﬀect of compound 6 on the

NLRP3 ATPase activity was then tested. Puriﬁed human

NLRP3 protein was incubated with compound 6 at indicated

concentrations for 30 min. ATP was then added into the

reaction mixtures and incubated at 37 °C for 1 h. The amount

of ATP hydrolysis was determined by luminescent kinase assay

of the residual amount of ATP. According to the experimental

result of ATPase activity, compound 6 exhibited a dose-

dependent inhibition on NLRP3 ATPase activity (Figure 5A).

The binding aﬃnity of compound 6 with three functional

NLRP3 domains (PYD, NACHT, and LRR) was next tested

using biolayer interferometry (BLI) assay. The tested proteins

used as the receptor protein were biotinylated with N-

succinimidyl-^D-biotin at a 1:5 stoichiometric ratio and

immobilized onto Super Streptavidin (SSA) biosensors. The

equilibrium dissociation constants (K_D) value is calculated by

K_off/K_on, which represents the binding aﬃnity between ligands

and receptor proteins.⁴⁴The binding curves and the K_Dvalues

were used to evaluate the binding aﬃnity between the tested

proteins and compound 6. The results of BLI assay showed

that the K_Dof compound 6 with NLRP3 PYD domain and

NACHT domain were 8.77 M and 17.2 μM, respectively,

whereas compound 6 did not bind to the LRR domain (Figure

5C−F), indicating that compound 6 bound to the NLRP3

NACHT domain but not to other domains. Based on these

results, compound 6 directly bound to the NLRP3 NACHT

domain and blocked NLRP3 ATPase activity, thus inhibiting

NLRP3 inﬂammasome activation.

Compound 6 Inhibits ASC Oligomerization and

NLRP3 Inﬂammasome Assembly. Previous studies have

demonstrated that the oligomerization of ASC is a critical

event for subsequent NLRP3 inﬂammasome assembly and

activation.^45,46We tested whether compound 6 has any eﬀect

on the ATP-induced ASC specks formation and used MCC950

as a positive control. THP-M cells were treated with

compound 6 and then stimulated with LPS and ATP. Once

ATP triggered NLRP3 activation, the formation of ASC

oligomerization was observed by immunoﬂuorescence and

western blotting (Figure 6A,B). The data showed that

compound 6 had concentration-dependent suppression on

the ASC oligomerization, but had no eﬀect on the ASC level in

cell lysates. We also evaluated the eﬀect of compound 6 on the

NLRP3-ASC interaction, which is critical for NLRP3

inﬂammasome assembly. Experimental results demonstrated

compound 6 slightly inhibited ATP-induced interaction

between ASC and NLRP3 in THP-M cells (Figure 6C). The

above results indicated that compound 6 inhibited the

assembly and activation of NLRP3 inﬂammasome.

Compound 6 Speciﬁcally Inhibits NLRP3 Inﬂamma-

some without Blocking K⁺Eﬄux or ROS Production. To

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Figure 6. Compound 6 blocks NLRP3 inﬂammasome-related ASC oligomerization dose-dependently. (A) THP-M cells were treated with

compound 6 or MCC950 (30 μM) and then stimulated with LPS (3 h) and ATP for 1 h. The cells were lysed in PBS containing Triton X-100. The

Triton X-100-insoluble pellets and Triton X-100-soluble fraction were collected and used to analyze ASC oligomerization by western blotting. (B)

THP-M cells treated as (A) were ﬁxed with 4% paraformaldehyde followed by ASC and 4′,6-diamidino-2-phenylindole (DAPI) staining. The ASC

speck images were collected with a confocal scanning microscope (Zeiss LSM 700). Scale bars: 10 μm. (C) Co-IP and western blots of interaction

of ASC and NLRP3 in THP-M cells pretreated with various concentrations of compound 6 and then stimulated with LPS and ATP. The results

were obtained from three independent experiments.

conﬁrm the speciﬁc inhibitory eﬀect of compound 6 on the

NLRP3 inﬂammasome, we also investigated whether 6 could

inhibit the activation of other inﬂammasomes such as NOD-

like receptor family CARD-containing 4 (NLRC4) and absent

in melanoma 2 (AIM2) inﬂammasome. The results showed

that compound 6 had no eﬀect on NLRC4 inﬂammasome

triggered by ﬂagellin of Salmonella typhimurium (Figure 7A).

Besides, transfected poly-(dA:dT) (poly-deoxyadenylic−deox-

ythymidylic acid sodium salt)-induced AIM2 inﬂammasome-

related IL-1β secretion was not obviously aﬀected by

compound 6 (Figure 7B). However, under the same

conditions, compound 6 showed signiﬁcant inhibitory activity

against ATP-induced NLRP3 inﬂammasome in LPS-primed

THP-M cells (Figure 7C).

Previous studies showed that a decrease in intracellular

potassium concentration is a common upstream event during

the NLRP3 inﬂammasome activation and required for caspase-

1 activation via the P2X7 (purinergic receptor P2X, ligand-

gated ion channel, 7) receptor.^47,48Thus, we further explored

whether compound 6 could inﬂuence K⁺eﬄux. ATP treatment

induced a signiﬁcant decrease of intracellular potassium in

LPS-primed THP-M cells, but it could not be blocked by

compound 6 even at a high concentration of 45 μM (Figure

7D). Mitochondrial damage and the generation of reactive

oxygen species (ROS) are involved in the NLRP3 inﬂamma-

some activation.⁴⁹Our results showed that compound 6 had

no eﬀect on the mitochondrial membrane potential decrease

(Figure 7E) or ROS production (Figure 7F) induced by

extracellular ATP.

Taken together, these data indicated that compound 6 was a

speciﬁc inhibitor for NLRP3 inﬂammasome activation, and its

inhibitory eﬀect was not attributed to preventing potassium

eﬄux or inhibiting ROS production.

Compound 6 Inhibits NLRP3 Activation In Vivo and

Attenuates Colitis Severity in Mice Model. DSS is the

most common inducer of colitis and increases colonic

permeability due to its toxicity to epithelial cells.⁵⁰A previous

study proved that DSS-induced colitis is an experimental

NLRP3 inﬂammasome-mediated inﬂammation.²²To explore

the anti-inﬂammatory activity of compound 6 in vivo, we

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Figure 7. Compound 6 speciﬁcally inhibited NLRP3 inﬂammasome, but not NLRC4 or AIM2 inﬂammasome, and did not block K⁺eﬄux or ROS

production. (A−C) Production of IL-1β in supernatant from LPS-primed THP-M cells treated with compound 6 at doses of 5−15 μM and ﬂagellin

of S. typhimurium (A), transfected poly(dA:dT) (B) or ATP (C). (D) Concentrations of intracellular K⁺were determined by inductively coupled

plasma mass spectrometry (ICP-MS) in THP-M cells induced by LPS and ATP. (E, F) Confocal microscopy analyses were performed in THP-M

cells treated with compound 6 (30 μM) and then stimulated with LPS and ATP, followed by being stained with MitoTracker Red (E) or MitoSOX

(F). Nuclei were stained with DAPI. The results were expressed as mean s.e.m. (n = 3) from three independent experiments. Statistics were

analyzed by unpaired, two-tailed Student’s t-test, NS: not signiﬁcant, *p < 0.05.

adopted a mouse model of DSS-induced acute ulcerative

colitis, as previously reported.^51,52Acute colitis was induced by

administration of DSS in drinking water for 7 days. Compound

6 was given by gavage from day 1 to day 10. The body weights

of mice and symptoms of colitis were recorded every day

during the study. After 2.5% DSS treatment, C57BL/6 mice

exhibited acute clinical symptoms including bodyweight loss,

diarrhea, and grossly bloody stools. Compared with the DSS

group, oral administration of compound 6 at 40 and 80 mg/kg

alleviated bodyweight loss in mice during the onset of colitis

(Figure 8A). As shown in Figure 8B, compound 6 dose-

dependently reduced the disease activity index (DAI), which

was used to evaluate the severity of DSS-induced colitis. In

addition, the administration of compound 6 signiﬁcantly

attenuated the shortening of the colon length caused by DSS

treatment (Figure 8C,D).

ameliorated these symptoms and showed strong protective

eﬀects against DSS-induced inﬂammasome activation in the

acute colitis model (Figure 9A,D). To investigate the eﬀects of

compound 6 on NLRP3 inﬂammasome in vivo, we evaluated

the expression of NLRP3 inﬂammasome-related proteins in

DSS-induced colitis. The in vivo results suggested that the

administration of compound 6 signiﬁcantly downregulated the

protein levels of NLRP3 and IL-1β in the colon tissues of DSS-

induced NLRP3 inﬂammasome-related colitis mouse model

(Figure 9B,C,E). The NF-κB signaling pathway regulates many

inﬂammation-related gene transcription in IBD.⁵³Our results

showed that compound 6 reduced the RELA (p65) subunit of

NF-κB in the colons of DSS-stimulated mice. In addition, the

results of ELISA showed that compound 6 decreased the

secretion of IL-1β in a dose-dependent manner in colon tissues

(Figure 9F), indicating that compound 6 inhibited NLRP3

inﬂammasome activation in vivo. Taken together, these results

indicated that compound 6 eﬀectively alleviated the severity of

DSS-induced ulcerative colitis in mouse.

The histopathological analysis showed severe pathological

changes mainly including mucosal damage, crypt necrosis, and

inﬁltration of inﬂammatory cells in the lamina propria in the

DSS-induced colitis mouse model. Compound 6 signiﬁcantly

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Figure 8. Compound 6 treatment alleviates the symptoms of DSS-induced colitis, including weight loss, diarrhea, bloody stools, and shortening of

colon. C57BL/6 mice were administrated with 2.5% DSS for 7 days and fed with drinking water for another 3 days. Compound 6 (40, 80 mg/kg)

was given by gavage from day 1 to day 10. (A) Changes in bodyweight of mice were measured and presented as a percent of bodyweight change (n

= 8 per group). (B) DAI was calculated (n = 8 per group). (C) The length of colon was measured (n = 8 per group). (D) The colon was

photographed. The results were expressed as mean s.e.m. Statistics were analyzed by unpaired, two-tailed Student’s t-test, ****p < 0.0001.

inhibited the production of NLRP3 inﬂammasome-dependent

IL-1β. Next, the compound G514-0208 was selected as the

starting point and 28 tetrahydroquinoline derivatives were

synthesized for the preliminary SAR research of R-substituents.

The results suggested that compounds with sterically hindered

substituents or long-chain groups were preferred for increasing

the inhibitory activity against IL-1β. Among the synthetic

derivatives, compound 6 showed the most potent inhibitory

activity against IL-1β release and was selected for further

pharmacological evaluation. In vitro studies proved that

compound 6 mainly inhibited the activation of NLRP3

inﬂammasome. In addition, compound 6 directly bound to

the NLRP3 NACHT domain but not to PYD or LRR domain,

blocked NLRP3 ATPase activity, and suppressed NLRP3

inﬂammasome-related ASC oligomerization. Moreover, com-

pound 6 treatment had no eﬀect on AIM2 or NLRC4

inﬂammasome and did not block potassium eﬄux. In vivo

experiments revealed that compound 6 treatment remarkably

inhibited the expressions of related proteins such as NLRP3

and IL-1β, and attenuated the severity of NLRP3 inﬂamma-

some-driven colitis.

CONCLUSIONS

■

Inappropriate activation of NLRP3 inﬂammasomes is

associated with the occurrence and development of a wide

variety of acute and chronic inﬂammatory diseases including

IBD,²³gout,⁵⁴atherosclerosis,⁵⁵type 2 diabetes,⁵⁶and

Alzheimer’s disease.⁵⁷Therefore, inhibiting the NLRP3

inﬂammasome activation has been considered as a potent

therapeutic strategy for inﬂammatory diseases. There have

been a number of NLRP3 inﬂammasome inhibitors reported,

including CY-09,⁴⁰sulforaphane,⁵⁸isoliquiritigenin,⁵⁹β-

hydroxybutyrate (BHB),⁶⁰ﬂufenamic acid,⁶¹mefenamic

acid,⁶¹3,4-methylenedioxy-β-nitrostyrene (MNS),⁶²partheno-

lide,⁶³BAY 11-7082,⁶³INF39,⁶⁴Oridonin,⁶⁵Tranilast,⁶⁶and

MCC950.³¹These compounds have been developed and

demonstrated to be eﬀective in cell levels or animal models of

related diseases. However, most of them are indirect and

nonspeciﬁc inhibitors with multiple bioactivities, which may

cause adverse reactions. It should be noted that a few speciﬁc

inhibitors have been described. Apart from IL-1β blockers such

as canakinumab, anakinra, and rilonacept, there is still no

approved NLRP3 inﬂammasome-dependent inhibitor listed.¹²

Given that IL-1β is not the only product of NLRP3

inﬂammasome activation but also produced by other

inﬂammasomes, direct NLRP3 inhibitors have potentially

improved safety and eﬀectiveness compared to cytokine IL-1

receptor antagonists. Thus, there is an urgent desire for

discovering and developing speciﬁc and direct targeting

inhibitors of the NLRP3 inﬂammasome in the clinical

treatment for NLRP3-driven diseases.

In summary, compound 6 is a speciﬁc inhibitor of NLRP3

inﬂammasome, which is characterized by ease of chemical

synthesis and modiﬁcation as well as low toxicity. Both in the

in vitro and in vivo studies, compound 6 exhibits good

inhibitory activity against NLRP3 inﬂammasome. It provides a

potential promising lead compound for further structural

optimization to developing a new therapeutic approach for

NLRP3 inﬂammasome-driven diseases.

In this study, we demonstrated that compound 6 was a

potent and speciﬁc inhibitor of NLRP3. It directly bound to

NLRP3 NACHT domain and showed inhibitory activity

against NLRP3 inﬂammasome both in cells and mice. We

initially obtained four tetrahydroquinoline compounds that

EXPERIMENTAL SECTION

■

1

Chemistry. H NMR and ¹³C NMR spectra were recorded on

Bruker 300 and 75 MHz spectrometers, respectively, in CDCl₃or

DMSO-d₆with tetramethyl silane as internal reference standard. The

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Figure 9. Compound 6 inhibits NLRP3 inﬂammasome in vivo and shows a protective eﬀect on C57BL/6 mice with DSS-induced ulcerative colitis.

(A) Serial sections of paraﬃn-embedded colon tissues were stained with hematoxylin and eosin (H&E). The result shown was from one

representative paraﬃnized specimen out of six studied (n = 6). Magniﬁcation: ×200. (B, C) Immunohistochemistry (IHC) of NLRP3 and IL-1β in

colonic tissue section. Positive staining is in brown. The result shown was from one representative paraﬃnized specimen out of six studied (n = 6).

Magniﬁcation: ×200. (D) Histopathological scores of each group were determined. (E) The protein levels of NLRP3, IL-1β, p65, and

phosphorylated p65 in colon were examined by western blotting. (F) The protein level of the cleaved IL-1β in colon homogenates was determined

by ELISA. Data were presented as mean s.e.m. (n = 8). Statistics were analyzed by unpaired, two-tailed Student’s t-test, *p < 0.05, **p < 0.01,

***p < 0.001.

data are presented as follows: chemical shift (δ), multiplicity (s =

singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br =

broad), coupling constants (J) in hertz (Hz). Reactions were

monitored by thin-layer chromatography (TLC) on 0.25 mm silica

gel plates (GF254) and visualized with UV light. The purity (>95%)

of the compounds was determined by HPLC performed on an Agilent

C18 (4.6 mm × 250 mm, 5 μm) column and peak detection at 254

nm with UV. High-resolution mass spectra (HRMS) were recorded in

positive-ion mode by electrospray ionization (ESI) using a time-of-

ﬂight mass spectrometer. The melting points of compounds were

obtained on a Mettler Toledo MP50 instrument. IR spectra (KBr

disks) were recorded with a Thermo Fisher NICOLET 6700 FT-IR

spectrometer. Column chromatography was performed using a 300−

400 mesh silica gel and technical-grade solvents. Compounds G505-

0320, G505-0416, G505-0411, and G514-0208 were purchased from

Topscience (http://www.tsbiochem.com). Unless otherwise noted,

all of the reagents and solvents used in this work were purchased from

commercial suppliers and used as received without further

puriﬁcation.

Chemical Synthesis. 1-(Methylsulfonyl)-7-nitro-1,2,3,4-tetrahy-

droquinoline (I-1). 7-Nitro-1,2,3,4-tetrahydroquinoline (48.0 g, 0.27

mol) was mixed with triethylamine (74.8 mL,0.54 mol) in 100 mL of

dichloromethane. The mixture was stirred in an ice bath for 15 min.

Methanesulfonyl chloride (21 mL, 0.27 mol) was dropwise added into

the reaction mixture. After that, the ice bath was removed and the

reaction was stirred at room temperature until TLC showed complete

consumption of 7-nitro-1,2,3,4-tetrahydroquinoline. The mixture was

washed with dilute hydrochloric acid and saturated salt solution, dried

with anhydrous sodium sulfate, and the solvent was evaporated under

reduced pressure. The crude product was recrystallized in 60 mL of

methanol for 1 h. After ﬁltering, the desired product was collected as a

white solid (62.9 g, 91.2% yield). ¹H NMR (300 MHz, CDCl₃) δ 8.56

(d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.4, 2.2 Hz, 1H), 7.31 (d, J = 8.2 Hz,

1H), 3.88−3.84 (m, 2H), 3.03 (s, 3H), 2.97 (t, J = 6.6 Hz, 2H),

2.13−2.04 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 149.34, 140.33,

138.77, 133.03, 121.23, 119.81, 48.84, 41.79, 30.12, 24.34. HRMS

(ESI) calcd for C₁₀H₁₂N₂NaO₄S [M + Na]⁺279.0410, found

279.0306. HPLC (10−100% methanol in water), t_R= 18.00 min,

>99.99%.

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1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-amine (I-2). 1-

(Methylsulfonyl)-7-nitro-1,2,3,4-tetrahydroquinoline (I-1) (19.0 g,

74.2 mmol) was dissolved in 60 mL of dichloromethane. After adding

10% Pd on carbon (1.9 g), the mixture was stirred under hydrogen

atmosphere at room temperature until TLC monitored the

disappearance of raw materials. Then, the reaction mixture was

ﬁltered and the ﬁltrate was concentrated under reduced pressure. The

crude product was puriﬁed by recrystallizing in methanol, to give the

7.24 (d, J = 2.1 Hz, 1H), 6.89 (dd, J = 8.1, 2.2 Hz, 1H), 3.73−3.69

(m, 2H), 2.99 (s, 3H), 2.87 (t, J = 6.4 Hz, 2H), 1.95−1.91 (m, 2H).

¹³C NMR (75 MHz, DMSO-d₆) δ 154.03, 146.20, 140.65, 135.71,

134.13, 133.50, 132.85, 129.42, 128.13, 127.35, 48.81, 41.16, 29.57,

24.03. IR (cm⁻¹, KBr ﬁlm): 1609.79, 1573.63, 1530.24, 1492.63,

1389.46, 1347.03, 1317.14, 1167.20, 854.79. HRMS (ESI) calcd for

C₂₂H₂₄N₅O₁₀S₃[M + NH₄]⁺614.0680, found 614.0691. HPLC (60%

acetonitrile in water), t_R= 12.01 min, >99.99%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-2-nitro-N-

((2-nitrophenyl)sulfonyl)benzenesulfonamide (5). White solid, yield

56.9%, m.p. 178.9−179.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 8.23

(d, J = 7.7 Hz, 2H), 8.08−7.95 (m, 6H), 7.49 (d, J = 1.6 Hz, 1H),

7.29 (d, J = 8.2 Hz, 1H), 7.02 (dd, J = 8.1, 1.7 Hz, 1H), 3.70−3.67

(m, 2H), 2.85−2.82 (m, 5H), 1.92−1.88 (m, 2H). ¹³C NMR (75

MHz, DMSO-d₆) δ 148.14, 137.83, 137.60, 133.30, 133.19, 132.16,

131.01, 129.56, 129.54, 127.51, 125.55, 125.21, 46.30, 38.04, 27.04,

21.42. IR (cm⁻¹, KBr ﬁlm): 1548.56, 1495.04, 1440.56, 1387.05,

1369.69, 1344.14, 1176.85. HRMS (ESI) calcd for C₂₂H₂₄N₅O₁₀S₃

[M + NH₄]⁺614.0680, found 614.0710. HPLC (60% acetonitrile in

water), t_R= 6.72 min, 99.55%.

4-Acetyl-N-((4-acetylphenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (6). White

solid, yield 57.5%, m.p. 189.1−190.6 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.21 (d, J = 8.5 Hz, 4H), 7.99 (d, J = 8.5 Hz, 4H), 7.31−

7.26 (m, 2H), 6.78 (dd, J = 8.1, 2.0 Hz, 1H), 3.72−3.69 (m, 2H), 2.96

(s, 3H), 2.85 (t, J = 6.4 Hz, 2H), 2.68 (s, 6H), 1.94−1.90 (m, 2H).

¹³C NMR (75 MHz, DMSO-d₆) δ 197.69, 142.22, 141.56, 137.98,

1

target compound (14.0 g, 83.3% yield) as a white solid. H NMR

(300 MHz, CDCl₃) δ 7.11 (d, J = 2.0 Hz, 1H), 6.90 (d, J = 8.1 Hz,

1H), 6.45 (dd, J = 8.1, 2.1 Hz, 1H), 3.81−3.77 (m, 2H), 3.63 (s, 2H),

2.89 (s, 3H), 2.74 (t, J = 6.6 Hz, 2H), 1.98−1.90 (m, 2H). ¹³C NMR

(75 MHz, CDCl₃) δ 145.29, 137.48, 130.39, 118.65, 111.98, 108.82,

46.64, 38.35, 26.34, 22.29. HRMS (ESI) calcd for C₁₀H₁₅N₂O₂S [M +

H]⁺227.0849, found 227.0878. HPLC (10−100% methanol in

water), t_R= 9.43 min, >97.60%.

General Procedure for the Preparation of Compounds (1−

28). 4-Cyano-N-((4-cyanophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (1). 1-(Meth-

ylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-amine (I-2) (200 mg, 0.885

mmol) was dissolved in 5 mL of dichloromethane. After adding

triethylamine (368 μL, 2.66 mmol), 4-cyanobenzenesulfonyl chloride

(356 mg, 1.77 mmol) diluted in 5 mL of dichloromethane solution

was added dropwise to the mixture. The reaction mixture was stirred

at room temperature overnight. After stopping the reaction, the

mixture was diluted with dichloromethane and washed with dilute

hydrochloric acid, saturated sodium bicarbonate solution, and

saturated saltwater. The organic layer was separated and dried over

anhydrous sodium sulfate. The solvent was removed by evaporating

under reduced pressure. The crude product was puriﬁed by

recrystallizing in methanol to give the target compound 1 (394 mg,

132.56, 131.43, 131.26, 129.71, 128.83, 126.70, 124.55, 46.20, 38.45,

27.58, 26.98, 21.42. IR (cm⁻¹, KBr ﬁlm): 1694.16, 1602.56, 1572.66,

1492.15, 1384.16, 1340.77, 1172.99, 854.31. HRMS (ESI) calcd for

C₂₆H₃₀N₃O₈S₃[M + NH₄]⁺608.1190, found 608.1229. HPLC (60%

acetonitrile in water), t_R= 8.08 min, 99.09%.

1

80.0% yield) as an oﬀ-white solid. Mp 261.8−263.4 °C. H NMR

(300 MHz, DMSO-d₆) δ 8.21 (d, J = 8.6 Hz, 4H), 8.04 (d, J = 8.6 Hz,

4H), 7.30 (d, J = 8.2 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 6.86 (dd, J =

8.1, 2.1 Hz, 1H), 3.73−3.69 (m, 2H), 2.99 (s, 3H), 2.86 (t, J = 6.5 Hz,

2H), 1.97−1.89 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 145.03,

140.62, 136.96, 135.51, 134.09, 133.61, 131.79, 129.35, 127.16,

120.39, 120.26, 48.81, 41.15, 29.59, 24.02. IR (cm⁻¹, KBr ﬁlm):

2234.61, 1601.11, 1574.11, 1494.08, 1389.46, 1341.25, 1164.79,

833.58. HRMS (ESI) calcd for C₂₄H₂₄N₅O₆S₃[M + NH₄]⁺574.0883,

found 574.0890. HPLC (60% acetonitrile in water), t_R= 9.42 min,

99.75%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-(naph-

thalen-1-ylsulfonyl)naphthalene-1-sulfonamide (7). White solid,

yield 55.1%, m.p. 244.9−245.7 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

8.37 (d, J = 8.2 Hz, 2H), 8.20 (d, J = 7.4 Hz, 2H), 8.12 (d, J = 8.2 Hz,

2H), 7.89 (d, J = 8.7 Hz, 2H), 7.69−7.58 (m, 4H), 7.31 (s, 1H), 7.23

(t, J = 7.7 Hz, 2H), 7.13 (d, J = 8.2 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H),

3.63−3.60 (m, 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.68 (s, 3H), 1.91−1.83

(m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 137.38, 136.49, 133.51,

132.67, 132.35, 131.72, 130.25, 130.21, 129.23, 128.33, 127.51,

127.09, 126.51, 124.48, 124.41, 123.34, 45.61, 37.72, 26.39, 21.11. IR

(cm⁻¹, KBr ﬁlm): 1604.48, 1589.54, 1496.49, 1379.82, 1342.70,

1167.69. HRMS (ESI) calcd for C₃₀H₃₀N₃O₆S₃[M + NH₄]⁺

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-4-(tri-

ﬂuoromethyl)-N-((4-(triﬂuoromethyl)phenyl)sulfonyl)-

benzenesulfonamide (2). White solid, yield 55.4%, m.p. 188.6−190.2

624.1291, found 624.1341. HPLC (60% acetonitrile in water), t_R

=

1

°C. H NMR (300 MHz, DMSO-d₆) δ 8.11 (s, 8H), 7.32 (d, J = 8.2

19.16 min, >99.99%.

Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H), 6.89 (dd, J = 8.1, 2.1 Hz, 1H),

3.73−3.69 (m, 2H), 2.96 (s, 3H), 2.87 (t, J = 6.4 Hz, 2H), 1.98−1.89

(m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 142.47, 137.98, 134.63,

132.79, 131.45, 131.17, 129.50, 127.42, 126.71, 125.45, 124.58, 46.21,

38.47, 26.97, 21.41. IR (cm⁻¹, KBr ﬁlm): 1606.41, 1576.04, 1495.53,

1396.69, 1319.55, 1169.62, 1128.64, 837.91. HRMS (ESI) calcd for

C₂₄H₂₄F₆N₃O₆S₃[M + NH₄]⁺660.0726, found 660.0770. HPLC

(70% acetonitrile in water), t_R= 13.66 min, >99.99%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-(naph-

thalen-2-ylsulfonyl)naphthalene-2-sulfonamide (8). White solid,

yield 80.9%, m.p. 174.8−176.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

8.48 (s, 2H), 8.21 (d, J = 8.8 Hz, 2H), 8.13 (t, J = 6.9 Hz, 4H), 7.88−

7.85 (m, 2H), 7.80 (t, J = 7.5 Hz, 2H), 7.70 (t, J = 7.5 Hz, 2H), 7.39

(d, J = 1.5 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 6.77 (dd, J = 8.1, 1.6 Hz,

1H), 3.68−3.65 (m, 2H), 2.82 (t, J = 6.4 Hz, 2H), 2.77 (s, 3H),

1.91−1.88 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 137.88,

135.97, 135.44, 132.14, 131.98, 131.87, 131.07, 130.41, 130.25,

130.11, 128.46, 128.41, 126.90, 124.60, 122.74, 46.19, 38.21, 26.96,

21.46. IR (cm⁻¹, KBr ﬁlm): 1604.48, 1589.54, 1496.49, 1379.82,

1342.70, 1167.69. HRMS (ESI) calcd for C₃₀H₃₀N₃O₆S₃[M + NH₄]⁺

N-((3,5-Bis(triﬂuoromethyl)phenyl)sulfonyl)-N-(1-(methylsulfon-

yl)-1,2,3,4-tetrahydroquinolin-7-yl)-3,5-bis(triﬂuoromethyl)-

benzenesulfonamide (3). White solid, yield 92.9%, m.p. 229.8−230.0

1

°C. H NMR (300 MHz, DMSO-d₆) δ 8.71 (s, 2H), 8.35 (s, 4H),

7.31 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 6.92 (d, J = 8.3 Hz, 1H), 3.70−

3.66 (m, 2H), 2.93 (s, 3H), 2.85 (t, J = 6.3 Hz, 2H), 1.94−1.91 (m,

2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 140.67, 138.29, 133.46,

132.64, 132.36, 131.49, 130.15, 129.20, 126.52, 124.17, 121,62, 46.07,

38.63, 26.92, 21.60. IR (cm⁻¹, KBr ﬁlm): 1603.52, 1575.56, 1495.04,

1402.00, 1359.09, 1282.91, 1140.69. HRMS (ESI) calcd for

C₂₆H₂₂F₁₂N₃O₆S₃[M + NH₄]⁺796.0474, found 796.0540. HPLC

(80% acetonitrile in water), t_R= 11.19 min, >99.99%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-4-nitro-N-

((4-nitrophenyl)sulfonyl)benzenesulfonamide (4). White solid, yield

65.0%, m.p. 246.4−247.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

8.53−8.50 (m, 4H), 8.15−8.12 (m, 4H), 7.32 (d, J = 8.2 Hz, 1H),

624.1291, found 624.1328. HPLC (70% acetonitrile in water), t_R

12.84 min, 99.57%.

=

N-([1,1′-Biphenyl]-4-ylsulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tet-

rahydroquinolin-7-yl)-[1,1′-biphenyl]-4-sulfonamide (9). White

solid, yield 67.6%, m.p. 233.1−234.3 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.99−7.91(m, 8H), 7.79 (d, J = 7.4 Hz, 4H), 7.56−7.45

(m, 6H), 7.39 (s, 1H), 7.28 (d, J = 8.1 Hz, 1H), 6.80 (dd, J = 8.1, 2.0

Hz, 1H), 3.72−3.68 (m, 2H), 2.94 (s, 3H), 2.85 (t, J = 6.0 Hz, 2H),

1.96−1.88 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 146.45,

138.46, 137.93, 137.73, 132.10, 132.01, 131.14, 129.65, 129.40,

129.08, 128.14, 127.72, 126.73, 124.46, 46.22, 38.38, 27.01, 21.46. IR

(cm⁻¹, KBr ﬁlm): 1592.91, 1563.50, 1495.04, 1449.24, 1375.96,

L

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Article

1346.07, 1168.17. HRMS (ESI) calcd for C₃₄H₃₄N₃O₆S₃[M + NH₄]⁺

130.93, 129.46, 129.29, 127.10, 48.83, 41.10, 38.19, 33.75, 29.60,

24.10. IR (cm⁻¹, KBr ﬁlm): 2966.46, 2871.01, 1592.43, 1573.63,

1493.60, 1378.37, 1356.19, 1169.62, 839.37. HRMS (ESI) calcd for

C₃₀H₄₂N₃O₆S₃[M + NH₄]⁺636.2230, found 636.2283. HPLC (75%

acetonitrile in water), t_R= 18.79 min, 99.87%.

4-Methoxy-N-((4-methoxyphenyl)sulfonyl)-N-(1-(methylsulfon-

yl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (16).

White solid, yield 51.9%, m.p. 219.2−220.9 °C. ¹H NMR (300

MHz, DMSO-d₆) δ 7.75 (d, J = 8.8 Hz, 4H), 7.31 (s, 1H), 7.24−7.16

(m, 5H), 6.66 (d, J = 8.0 Hz, 1H), 3.89 (s, 6H), 3.75−3.69 (m, 2H),

2.95 (s, 3H), 2.83 (t, J = 6.3 Hz, 2H), 1.94−1.90 (m, 2H). ¹³C NMR

(75 MHz, DMSO-d₆) δ 163.71, 137.32, 131.84, 131.27, 130.40,

130.29, 130.07, 126.18, 123.99, 114.63, 55.91, 45.72, 37.86, 26.48,

20.97. IR (cm⁻¹, KBr ﬁlm): 1593.40, 1577.97, 1496.97, 1442.49,

1375.96, 1355.23, 1263.15, 1161.42, 832.13. HRMS (ESI) calcd for

C₂₄H₃₀N₃O₈S₃[M + NH₄]⁺584.1190, found 584.1218. HPLC (60%

acetonitrile in water), t_R= 8.23 min, 99.37%.

676.1604, found 676.1657. HPLC (75% acetonitrile in water), t_R

=

14.85 min, >99.99%.

4-Chloro-N-((4-chlorophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (10). White

solid, yield 62.7%, m.p. 261.4−262.6 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.88−7.85 (m, 4H), 7.80−7.77 (m, 4H), 7.29 −7.26 (m,

2H), 6.79 (dd, J = 8.1, 2.1 Hz, 1H), 3.73−3.69 (m, 2H), 2.97 (s, 3H),

2.85 (t, J = 6.5 Hz, 2H), 1.94−1.91 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 142.98, 140.55, 140.28, 135.07, 134.14, 133.89, 132.96,

132.93, 129.30, 127.11, 48.83, 41.02, 29.60, 24.04. IR (cm⁻¹, KBr

ﬁlm): 1574.59, 1493.12, 1478.17, 1380.78, 1341.25, 1165.76, 848.53,

753.55. HRMS (ESI) calcd for C₂₂H₂₄Cl₂N₃O₆S₃[M + NH₄]⁺

592.0199, found 592.0247. HPLC (70% acetonitrile in water), t_R

=

11.57 min, 99.80%.

4-Bromo-N-((4-bromophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (11). White

solid, yield 64.7%, m.p. 269.3−270.7 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.92 (d, J = 8.6 Hz, 4H), 7.76 (d, J = 8.7 Hz, 4H), 7.29−

7.25 (m, 2H), 6.78 (dd, J = 8.1, 2.0 Hz, 1H), 3.72−3.68 (m, 2H), 2.95

(s, 3H), 2.84 (t, J = 6.4 Hz, 2H), 1.96−1.88 (m, 2H). ¹³C NMR (75

MHz, DMSO-d₆) δ 138.10, 137.93, 133.29, 132.44, 131.51, 131.27,

130.28, 129.53, 126.67, 124.47, 46.21, 38.40, 26.98, 21.42. IR (cm⁻¹,

KBr ﬁlm): 1603.52, 1571.70, 1491.67, 1379.82, 1340.28, 1163.83,

823.46, 628.20. HRMS (ESI) calcd for C₂₂H₂₄Br₂N₃O₆S₃[M +

NH₄]⁺681.9168, found 681.9188. HPLC (75% acetonitrile in water),

t_R= 8.94 min, 97.41%.

4-Fluoro-N-((4-ﬂuorophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (12). White

solid, yield 57.4%, m.p. 175.2−176.0 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.21 (d, J = 8.6 Hz, 4H), 8.04 (d, J = 8.6 Hz, 4H), 7.30

(d, J = 8.2 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 6.86 (dd, J = 8.1, 2.1 Hz,

1H), 3.73−3.69 (m, 2H), 2.99 (s, 3H), 2.86 (t, J = 6.5 Hz, 2H),

1.95−1.91 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 168.49,

140.54, 137.83, 137.79, 134.96, 134.36, 133.83, 129.31, 127.13,

120.08, 48.83, 41.05, 29.59, 24.04. IR (cm⁻¹, KBr ﬁlm): 1590.50,

1492.15, 1382.23, 1338.26, 1175.88, 1159.01, 839.37. HRMS (ESI)

calcd for C₂₂H₂₄F₂N₃O₆S₃[M + NH₄]⁺560.0790, found 560.0831.

HPLC (60% acetonitrile in water), t_R= 11.72 min, 99.91%.

3-Fluoro-N-((3-ﬂuorophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (13). White

solid, yield 70.8%, m.p. 197.7−199.2 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.81−7.70 (m, 6H), 7.63 (d, J = 8.1 Hz, 2H), 7.29−7.26

(m, 2H), 6.82 (d, J = 8.0 Hz, 1H), 3.72−3.68 (m, 2H), 2.95 (s, 3H),

2.84 (t, J = 6.4 Hz, 2H), 1.93−1.89 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 162.03, 140.53, 140.43, 137.95, 132.62, 131.38, 131.29,

126.68, 124.83, 124.44, 122.66, 115.44, 46.20, 38.44, 26.98, 21.41. IR

(cm⁻¹, KBr ﬁlm): 1591.95, 1498.42, 1435.74, 1381.75, 1336.91,

1224.58, 1150.81. HRMS (ESI) calcd for C₂₂H₂₄F₂N₃O₆S₃[M +

NH₄]⁺560.0790, found 560.0825. HPLC (60% acetonitrile in water),

t_R= 13.81 min, 99.08%.

2-Fluoro-N-((2-ﬂuorophenyl)sulfonyl)-N-(1-(methylsulfonyl)-

1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (14). White

solid, yield 49.4%, m.p. 72.4−74.0 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.90−7.82 (m, 4H), 7.56−7.42 (m, 5H), 7.26 (d, J =

8.2 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 3.71−3.68 (m, 2H), 2.87−2.83

(m, 5H), 1.91−1.87 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ

158.74, 138.54, 138.42, 137.82, 132.32, 131.95, 130.93, 130.61,

127.05, 125.66, 124.84, 118.23, 46.29, 38.00, 27.02 21.32. IR (cm⁻¹,

KBr ﬁlm): 1591.95, 1498.42, 1435.74, 1381.75, 1336.91, 1224.58,

1150.81. HRMS (ESI) calcd for C₂₂H₂₄F₂N₃O₆S₃[M + NH₄]⁺

4-Methyl-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-

N-tosylbenzenesulfonamide (17). White solid, yield 61.3%, m.p.

1

204.8−205.5 °C. H NMR (300 MHz, DMSO-d₆) δ 7.71 (d, J = 8.2

Hz, 4H), 7.48 (d, J = 8.1 Hz, 4H), 7.32 (s, 1H), 7.23 (d, J = 8.1 Hz,

1H), 6.69−6.63 (m, 1H), 3.72−3.69 (m, 2H), 2.93 (s, 3H), 2.83 (t, J

= 6.3 Hz, 2H), 2.45 (s, 6H), 1.94−1.90 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 145.36, 137.34, 135.82, 131.63, 131.38, 130.45, 129.91,

127.89, 126.17, 123.91, 45.71, 37.81, 26.49, 21.10, 20.97. IR (cm⁻¹,

KBr ﬁlm): 2953.93, 2928.86, 1596.77, 1575.08, 1492.63, 1454.55,

1375.48, 1343.46, 1162.72, 813.81. HRMS (ESI) calcd for

C₂₄H₃₀N₃O₆S₃[M + NH₄]⁺552.1291, found 552.1335. HPLC

(60% acetonitrile in water), t_R= 11.83 min, 98.12%.

N,N′-(((Hydrosulfonyl(1-(methylsulfonyl)-1,2,3,4-tetrahydroqui-

nolin-7-yl)amino)sulfonyl)bis(4,1-phenylene))diacetamide (18).

White solid, yield 20.0%, m.p. 210.6−211.9 °C. ¹H NMR (300

MHz, DMSO-d₆) δ 10.46 (s, 2H), 7.81 (d, J = 9.0 Hz, 4H), 7.71 (d, J

= 8.9 Hz, 4H), 7.31 (d, J = 1.8 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 6.68

(dd, J = 8.1, 1.9 Hz, 1H), 3.71−3.67 (m, 2H), 2.91 (s, 3H), 2.82 (t, J

= 6.3 Hz, 2H), 2.11 (s, 6H), 1.92−1.89 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 169.71, 145.02, 137.77, 132.27, 132.25, 131.81, 130.93,

129.77, 126.78, 124.51, 119.00, 46.22, 38.34, 26.97, 24.64, 21.45. IR

(cm⁻¹, KBr ﬁlm): 3610.57, 3367.10, 1691.75, 1586.65, 1496.01,

1371.10, 1339.42, 1164.79, 858.17. HRMS (ESI) calcd for

C₂₆H₂₈N₄NaO₈S₃[M + Na]⁺643.0961, found 643.1016. HPLC

(40% acetonitrile in water), t_R= 9.81 min, 98.92%.

Dimethyl 4,4′-((Hydrosulfonyl(1-(methylsulfonyl)-1,2,3,4-tetra-

hydroquinolin-7-yl)amino) sulfonyl)dibenzoate (19). White solid,

yield 88.9%, m.p. 250.5−252.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

8.22 (d, J = 8.5 Hz, 4H), 7.99 (d, J = 8.5 Hz, 4H), 7.30−7.25 (m,

2H), 6.77 (dd, J = 8.1, 1.9 Hz, 1H), 3.93 (s, 6H), 3.72−3.68 (m, 2H),

2.95 (s, 3H), 2.84 (t, J = 6.3 Hz, 2H), 1.94−1.90 (m, 2H). ¹³C NMR

(75 MHz, DMSO-d₆) δ 165.32, 142.53, 137.95, 135.30, 132.60,

131.34, 131.31, 130.82, 128.89, 126.73, 124.52, 53.26, 46.20, 38.41,

26.98, 21.40. IR (cm⁻¹, KBr ﬁlm): 3032.51, 2952.00, 1719.71,

1599.16, 1574.59, 1490.70, 1387.53, 1338.36, 1167.20, 1106.46.

HRMS (ESI) calcd for C₂₆H₃₀N₃O₁₀S₃[M + NH₄]⁺640.1088, found

640.1127. HPLC (60% acetonitrile in water), t_R= 7.64 min, 99.12%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-(thio-

phen-2-ylsulfonyl)thiophene-2-sulfonamide (20). White solid, yield

39.3%, m.p. 167.5−168.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 8.22

(d, J = 4.4 Hz, 2H), 7.73 (d, J = 3.0 Hz, 2H), 7.40 (d, J = 1.1 Hz, 1H),

7.30−7.25 (m, 3H), 6.75 (dd, J = 8.1, 1.5 Hz, 1H), 3.73−3.69 (m,

2H), 2.95 (s, 3H), 2.85 (t, J = 6.4 Hz, 2H), 1.96−1.88 (m, 2H). ¹³C

NMR (75 MHz, DMSO-d₆) δ 137.73, 137.51, 136.92, 136.00, 131.62,

131.37, 130.66, 128.03, 125.74, 123.44, 45.75, 37.83, 26.57, 20.90. IR

(cm⁻¹, KBr ﬁlm): 1602.56, 1572.18, 1489.74, 1451.65, 1400.07,

1377.41, 1338.84, 1165.28, 1127.95. HRMS (ESI) calcd for

C₁₈H₂₂N₃O₆S₅[M + NH₄]⁺536.0107, found 536.0145. HPLC

(55% acetonitrile in water), t_R= 8.11 min, 95.94%.

560.0790, found 560.0824. HPLC (55% acetonitrile in water), t_R

=

11.07 min, 99.31%.

4-(tert-Butyl)-N-((4-(tert-butyl)phenyl)sulfonyl)-N-(1-(methylsul-

fonyl)-1,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide (15).

White solid, yield 54.0%, m.p. 208.1−209.2 °C. ¹H NMR (300

MHz, DMSO-d₆) δ 7.75−7.67 (m, 8H), 7.27−7.24 (m, 2H), 6.73

(dd, J = 8.1, 2.1 Hz, 1H), 3.72−3.68 (m, 2H), 2.92 (s, 3H), 2.85 (t, J

= 6.4 Hz, 2H), 1.94−1.90 (m, 2H), 1.34 (s, 18H). ¹³C NMR (75

MHz, DMSO-d₆) δ 160.94, 140.46, 138.88, 134.75, 134.56, 133.62,

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-(pyri-

din-3-ylsulfonyl)pyridine-3-sulfonamide (21). White solid, yield

58.5%, m.p. 194.5−195.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

9.01−9.00 (m, 4H), 8.29 (d, J = 8.2 Hz, 2H), 7.81−7.76 (m, 2H),

M

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7.32−7.30 (m, 2H), 6.88 (dd, J = 8.1, 1.6 Hz, 1H), 3.73−3.70 (m,

2H), 2.99 (s, 3H), 2.86 (t, J = 6.4 Hz, 2H), 1.97−1.89 (m, 2H). ¹³C

NMR (75 MHz, DMSO-d₆) δ 155.13, 148.08, 137.58, 136.07, 135.05,

132.26, 131.01, 130.56, 126.15, 124.71, 123.92, 45.70, 38.03, 26.50,

20.90. IR (cm⁻¹, KBr ﬁlm): 1604.48, 1573.63, 1497.94, 1466.12,

1379.82, 1341.25, 1151.78. HRMS (ESI) calcd for C₂₀H₂₁N₄O₆S₃[M

+ H]⁺509.0686, found 509.0618. HPLC (45% acetonitrile in water),

t_R= 8.42 min, 98.42%.

7.44−7.39 (m, 11H), 7.04 (d, J = 8.2 Hz, 1H), 6.29 (dd, J = 8.1, 1.9

Hz, 1H), 4.96 (s, 4H), 3.68−3.65 (m, 2H), 3.04 (s, 3H), 2.77 (t, J =

6.4 Hz, 2H), 1.91−1.87 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ

137.46, 131.87, 131.82, 131.70, 130.50, 129.56, 129.21, 127.83,

126.53, 125.03, 60.98, 45.99, 38.96, 26.67, 21.56. IR (cm⁻¹, KBr ﬁlm):

2928.86, 2913.91, 1604.00, 1571.70, 1495.53, 1455.90, 1367.28,

1339.80, 1154.67. HRMS (ESI) calcd for C₂₄H₃₀N₃O₆S₃[M + NH₄]⁺

552.1291, found 552.1345. HPLC (60% acetonitrile in water), t_R

=

11.93 min, 95.78%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-

(phenylsulfonyl)benzenesulfonamide (22). White solid, yield 59.6%,

2-(1,3-Dioxoisoindolin-2-yl)-N-((2-(1,3-dioxoisoindolin-2-yl)-

ethyl)sulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-

yl)ethane-1-sulfonamide (28). White solid, yield 64.5%, m.p. 248.9−

1

m.p. 185.1−186.3 °C. H NMR (300 MHz, DMSO-d₆) δ 7.85−7.80

(m, 6H), 7.71−7.66 (m, 4H), 7.33 (d, J = 1.5 Hz, 1H), 7.24 (d, J =

8.1 Hz, 1H), 6.71 (dd, J = 8.1, 1.6 Hz, 1H), 3.72−3.69 (m, 2H), 2.94

(s, 3H), 2.84 (t, J = 6.4 Hz, 2H), 1.96−1.88 (m, 2H). ¹³C NMR (75

MHz, DMSO-d₆) δ 138.56, 137.39, 134.64, 131.57, 131.41, 130.55,

129.56, 127.85, 126.17, 123.94, 45.71, 37.91, 26.49, 20.93. IR (cm⁻¹,

KBr ﬁlm): 1604.97, 1579.41, 1498.09, 1448.76, 1375.48, 1341.75,

1169.13. HRMS (ESI) calcd for C₂₂H₂₆N₃O₆S₃[M + NH₄]⁺

1

250.5 °C. H NMR (300 MHz, DMSO-d₆) δ 7.89−7.82 (m, 8H),

7.61 (s, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 4.03−

4.00 (m, 8H), 3.71−3.68 (m, 2H), 3.04 (s, 3H), 2.83 (t, J = 6.3 Hz,

2H), 1.93−1.90 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 167.53,

137.92, 135.00, 132.38, 131.97, 131.18, 131.04, 126.64, 124.65,

123.65, 52.70, 46.08, 38.79, 32.01, 26.84, 21.57. IR (cm⁻¹, KBr ﬁlm):

1773.23, 1704.76, 1606.90, 1496.01, 1448.28, 1381.26, 1342.21,

1155.15. HRMS (ESI) calcd for C₃₀H₂₈N₄NaO₁₀S₃[M + Na]⁺

524.0978, found 524.1023. HPLC (60% acetonitrile in water), t_R

=

8.20 min, 98.53%.

723.0860, found 723.0878. HPLC (60% acetonitrile in water), t_R

=

N-(Methylsulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroqui-

7.57 min, 96.13%.

nolin-7-yl)methanesulfonamide (23). White solid, yield 76.9%, m.p.

1

Pharmacology. Mice. Female C57BL/6 mice, 6−8 weeks old,

used in this study were obtained from Comparative Medicine Center

of Yangzhou University. All animals were housed under a 12 h light/

dark cycle at 20−25 °C with unrestricted access to food and water for

the duration of the experiment. All animal studies were conducted

according to protocols approved by the Institutional Animal Care and

Use Committee of China Pharmaceutical University.

Reagents. Phorbol 12-myristate 13-acetate (PMA) (ab120297,

Abcam), Murine Macrophage Colony Stimulating Factor (M-CSF,

P6015, Beyotime), LPS (Sigma-Aldrich), ATP (A3377, Sigma-

Aldrich), Flagellin from S. typhimurium (Invivogen), poly-

(deoxyadenylic-thymidylic) acid sodium salt (Poly dA:dT) (P0883,

Sigma-Aldrich), Lipofectamine 2000 (Invitrogen), MitoTracker Red

(Beyotime), MitoSOX (M36008, Invitrogen), full-length human

NLRP3 protein (BPS Bioscience, cat# 40741), and truncated

human NLRP3 protein (Zoonbio Biotechnology).

Cell Culture. BMDMs were collected from C57BL/6 mice and

diﬀerentiated for 7 days in complete Dulbecco’s modiﬁed Eagle’s

medium (DMEM, Gibco, Grand Island, NY) supplemented with 15%

fetal bovine serum (FBS, Gibco, Grand Island, NY), 1% penicillin/

streptomycin (P/S), and 40 ng/mL M-CSF. Human THP-1 cells

were obtained from the Cell Bank of Shanghai, Institute of

Biochemistry and Cell Biology, Chinese Academy of Sciences

(Shanghai, China). Human THP-1 cells were cultured in RPMI

1640 medium (Gibco, Grand Island, NY), 10% FBS, and 1%

penicillin/streptomycin. The cultures were maintained in a humidiﬁed

atmosphere of 5% CO₂and 95% air at 37 °C. THP-1 cells were

diﬀerentiated into macrophages with 100 ng/mL PMA for 12 h.

Inﬂammasome Stimulation. Human THP-1 cells were seeded at 5

× 10⁵/mL in a 48-well plate and diﬀerentiated with 100 ng/mL PMA

overnight. The medium was removed the following morning and

replaced with fresh 1640 medium containing DMSO (1:200),

compound 6, or CY-09 for 24 h. The cells were then primed with

LPS (100 ng/mL) for 3 h. After that, the NLRP3 activator ATP (5

mM) was added into the culture for another 1 h.

233.7−235.0 °C. H NMR (300 MHz, DMSO-d₆) δ 7.58 (d, J = 1.2

Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.22 (dd, J = 8.1, 1.6 Hz, 1H),

3.74−3.70 (m, 2H), 3.50 (s, 6H), 3.07 (s, 3H), 2.85 (t, J = 6.5 Hz,

2H), 1.98−1.90 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 137.22,

131.54, 131.48, 130.43, 126.16, 123.99, 45.58, 42.75, 38.30, 26.28,

21.15. IR (cm⁻¹, KBr ﬁlm): 2967.91, 2937.51, 1607.86, 1577.49,

1501.31, 1451.45, 1362.46, 1339.68, 1137.92. HRMS (ESI) calcd for

C₁₂H₂₂N₃O₆S₃[M + NH₄]⁺400.0665, found 400.0716. HPLC (45%

acetonitrile in water), t_R= 7.21 min, 99.58%.

N-(Ethylsulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroquino-

lin-7-yl)ethanesulfonamide (24). White solid, yield 60.6%, m.p.

1

121.1−122.3 °C. H NMR (300 MHz, DMSO-d₆) δ 7.61 (d, J = 1.8

Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.16 (dd, J = 8.1, 2.0 Hz, 1H),

3.73−3.69 (m, 2H), 3.61 (q, J = 7.3 Hz, 4H), 3.05 (s, 3H), 2.83 (t, J =

6.4 Hz, 2H), 1.97−1.89 (m, 2H), 1.33 (t, J = 7.3 Hz, 6H). ¹³C NMR

(75 MHz, DMSO-d₆) δ 137.66, 131.98, 131.76, 130.88, 126.90,

124.72, 50.04, 46.12, 38.52, 26.85, 21.54, 8.14. IR (cm⁻¹, KBr ﬁlm):

2988.16, 2945.73, 1606.41, 1577.97, 1500.35, 1449.73, 1368.73,

1345.59, 1150.33. HRMS (ESI) calcd for C₁₄H₂₆N₃O₆S₃[M + NH₄]⁺

428.0978, found 428.0995. HPLC (50% acetonitrile in water), t_R

=

6.61 min, >99.99%.

N-(1-(Methylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)-N-

(propylsulfonyl)propane-1-sulfonamide (25). White solid, yield

64.4%, m.p. 135.9−137.2 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

7.59 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.16 (dd, J = 8.1, 1.8 Hz, 1H),

3.73−3.69 (m, 2H), 3.60−3.55 (m, 4H), 3.05 (s, 3H), 2.84 (t, J = 6.4

Hz, 2H), 1.96−1.91 (m, 2H), 1.86−1.74 (m, 4H), 0.99 (t, J = 7.4 Hz,

6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 137.63, 131.91, 131.80,

130.89, 126.92, 124.72, 56.83, 46.12, 38.52, 26.84, 21.53, 17.08, 12.89.

IR (cm⁻¹, KBr ﬁlm): 2978.04, 2940.43, 2883.06, 1607.38, 1577.49,

1499.87, 1462.72, 1368.25, 1334.98, 1150.81. HRMS (ESI) calcd for

C₁₆H₃₀N₃O₆S₃[M + NH₄]⁺456.1291, found 456.1323. HPLC (55%

acetonitrile in water), t_R= 8.60 min, >99.99%.

N-(Butylsulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroquino-

lin-7-yl)butane-1-sulfonamide (26). White solid, yield 51.0%, m.p.

BMDMs were seeded at 1 × 10⁶/mL in the 48-well plate. After 12

h, the medium was removed and treated with 100 ng/mL LPS in fresh

complete DMEM for 3 h. After that, the medium was removed and

the cells were treated with compound 6 or CY-09 for 24 h. The

NLRP3 activator ATP (5 mM) was then added into the culture for

another 1 h.

To validate the eﬀect of compound 6 on the NLRP3

inﬂammasome activation, the experiment of compound 6 treatment

after LPS was performed. Human THP-1 cells were seeded at 5 ×

10⁵/mL in the 48-well plate and diﬀerentiated with 100 ng/mL PMA

overnight. The medium was removed the following morning and

replaced with LPS (100 ng/mL) in fresh 1640 medium for 3 h. After

that, the medium containing LPS was removed and compound 6 at

1

115.6−116.4 °C. H NMR (300 MHz, DMSO-d₆) δ 7.60 (s, 1H),

7.28 (d, J = 8.2 Hz, 1H), 7.17 (dd, J = 8.1, 1.9 Hz, 1H), 3.73−3.69

(m, 2H), 3.62−3.57 (m, 4H), 3.05 (s, 3H), 2.83 (t, J = 6.3 Hz, 2H),

1.94−1.91 (m, 2H), 1.79−1.69 (m, 4H), 1.47−1.35 (m, 4H), 0.89 (t,

J = 7.3 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 137.64, 131.95,

131.76, 130.90, 126.88, 124.67, 54.95, 46.13, 38.53, 26.85, 25.15,

21.54, 21.07, 13.69. IR (cm⁻¹, KBr ﬁlm): 2971.77, 2876.79, 1604.97,

1575.08, 1497.94, 1468.05, 1370.18, 1335.46, 1150.81. HRMS (ESI)

calcd for C₁₈H₃₄N₃O₆S₃[M + NH₄]⁺484.1604, found 484.1646.

HPLC (60% acetonitrile in water), t_R= 9.76 min, 99.61%.

N-(Benzylsulfonyl)-N-(1-(methylsulfonyl)-1,2,3,4-tetrahydroqui-

nolin-7-yl)-1-phenylmethanesulfonamide (27). White solid, yield

55.1%, m.p.158.6−159.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

N

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Article

indicated concentrations was added to the culture for 24 h. NLRP3

activator ATP (5 mM) or nigericin (10 μM) was then added into the

culture for another 1 h.

For induction of AIM2/NLRC4 inﬂammasome activation, THP1-

M cells were treated with LPS (100 ng/mL). After 3 h, the medium

was removed and replaced with serum-free media containing 6 for 24

h; then, the cells were stimulated with poly(deoxyadenylic-

thymidylic) acid sodium salt (Poly dA:dT) (1 μg/mL) transfected

with lipofectamine 2000 for 4 h or ﬂagellin (100 ng/mL) from S.

typhimurium for 2 h.

ELISA. Supernatants from THP-M cell culture or serum were

analyzed for human IL-1β (Boster, Wuhan, China), cleaved caspase-1

(ab219633, Abcam), TNF-α (Boster, Wuhan, China), and mouse IL-

1β (Boster, Wuhan, China) according to manufacturer’s instruction.

MTT. Cytotoxicity was determined by MTT assay. Human THP-1

cells were plated at the concentration of 5 × 10³/well in a 96-well

plate and diﬀerentiated with 100 ng/mL PMA overnight. Super-

natants were removed and replaced with fresh medium the following

morning. The cells were then treated with synthesized compounds at

concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μM or vehicle

DMSO for 72 h. Following incubation of the compounds, MTT (5

mg/mL, 20.0 μL) was added into each well of 96-well plates for 4 h.

The medium was removed, and 150.0 μL of DMSO was added into

each well to dissolve the MTT-formazan crystals. MCC950 and

DMSO were used as the positive control and vehicle control,

respectively. The absorbance value (OD value) was recorded at 570

nm by an Absorbance Microplate Reader (Thermo Multiskan FC).

The cellular proliferation inhibition rate (CPIR) was calculated as

follows:

Quantitative real-time PCR (qRT-PCR) was performed on cDNA

using AceQ qPCR SYBR Green Master Mix (Q111-02, Vazyme) with

the LightCycler 96 Real-Time PCR System (Roche). GAPDH was

used for normalization. The values are expressed as fold of the

control. The primer sequences of gene according to the previous

report³⁸were as follows:

NLRP3-F 5′-AACATTCGGAGATTGTGGTTGGG-3′;

NLRP3-R 5′-GTGCGTGAGATTCTGATTAGTGCTG-3′;

Caspase 1-F 5′-TTACAGACAAGGGTGCTGAACAA-3′;

Caspase 1-R 5′-TGAGGAGCTGGAAAGGAAGAAAG-3′;

IL-1β-F 5′-AGGCTGCTCTGGGATTC-3′;

IL-1β-R 5′-GCCACAACAACTGACGC-3′;

GAPDH-F 5′-AAGGTCGGAGTCAACGGATTT-3′;

GAPDH-R 5′-AGATGATGACCCTTTTGGCTC-3′.

Co-immunoprecipitation. After treatment with compound 6, LPS,

and ATP, the THP-M cells (1 × 10⁷/mL) were collected and lysed

with IP lysis buﬀer containing protease inhibitor. IgG (1.0 μg) and A/

G-beads (20 μL) were added into a cell lysate supernatant and

incubated on a plate shaker at 4 °C for 1 h. The mixture was

centrifuged at 2000g and 4 °C for 5 min, and the supernatant was

obtained. The antibody of ASC (1:100, ab151700, Abcam) was added

to the lysate supernatant (the same amount of normal IgG used for

precipitation as a negative control) and incubated at 4 °C overnight.

The A/G-beads (20 μL) were added into the cell lysate supernatant

and incubated at 4 °C for 2 h. The immunoprecipitated complex was

obtained by a centrifuge at 1000g for 5 min at 4 °C and then analyzed

by western blot.

Intracellular K⁺Measurement. For measurement of intracellular

potassium, human THP-1 cells were seeded at 2 × 10⁵/mL in 12-well

plates and diﬀerentiated with 100 ng/mL PMA overnight. The next

day, the medium was removed and replaced with fresh medium

containing DMSO (1:200) and compound 6 (15−45 μM) for 24 h.

After that, the cells were stimulated with 100 ng/mL LPS for 3 h and

then with ATP (5 mM) for 1 h. The culture medium was completely

removed. The cells were washed three times with a potassium-free

buﬀer (139 mM NaCl, 1.7 mM NaH₂PO₄, and 10 mM Na₂HPO₄, pH

7.2), and 65% ultrapure HNO₃was added, as described by Zhou et

al.⁶⁵The samples were transferred to glass bottles, heated for 30 min

at 80 °C, and 10 mL of double-distilled water was added. The

detection of intracellular potassium was performed by a Thermo

Fisher ICAP-Q using scandium as the internal standard.

NLRP3 ATPase Activity. Human recombinant protein NLRP3, in a

full-length form, was obtained from BPS Bioscience (MW = 120 kD,

cat# 40741). The NLRP3 protein (0.875 μg/well) was incubated with

indicated concentrations of compound 6 in the reaction buﬀer (150

mM NaCl, 25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM EDTA)

at 37 °C for 15 min. ATP (1 μM) was then added, and the reaction

mixtures were further incubated at 37 °C for another 1 h. The

hydrolysis of ATP by NLRP3 was detected by the residual amount of

ATP with Kinase-Lumi Luminescent kinase kit (Beyotime, S0150S)

according to manufacturer’s protocol. The result of NLRP3 ATPase

activity was expressed as the percentage of remaining ATPase activity

to the vehicle-treated enzyme.

BLI Binding Studies. The NHS-biotin reagent (10 mM) was added

into the truncated NLRP3 protein (PYD domain, NACHT domain,

and LRR domain) solution (0.5 mg/mL) at a 5:1 ratio and incubated

at 4 °C for 2 h. The excessive biotin reagent was removed with

dialysis membranes (MWCO: 3.5 kD). Biotinylated human

recombinant protein was added into a 96-well plate and immobilized

on SSA biosensors (ForteBio). SSA tips were prewetted with PBS.

Compound 6 (10 mM) was diluted in PBS to 200 μL at four

concentrations (5, 20, 40, and 80 μM, respectively). The NLRP3 PYD

domain, NACHT domain, and LRR domain (Zoonbio Biotechnol-

ogy) were biotinylated with N-succinimidyl-^D-biotin at a 1:5

stoichiometric ratio. PBS (200 μL) was added to the 96-well plate

as a nonspeciﬁc binding agent for each concentration. The BLI assay

consists of ﬁve steps: (1) baseline acquisition; (2) the target protein

loading on the SSA biosensors; (3) second baseline acquisition; (4)

ligand association, measurement of K_on; and (5) ligand dissociation,

measurement of k_off. The binding data were collected at 25 °C. The

CPIR (%) = [1 − (OD_test− OD_blank)/(OD

− OD_blank)]

control

× 100%

Western Blotting. The cells were centrifuged at 2000 rpm and

lysed in 80.0 μL of lysis buﬀer (P0013, Beyotime) containing 20.0

mM Tris (pH 7.5), 150.0 mM NaCl, 1% Triton X-100, sodium

pyrophosphate, β-glycerophosphate, ethylenediamine tetraacetic acid

(EDTA), Na₃VO₄, leupeptin, and 1% PMSF in an ice bath for 1 h.

The cells were centrifuged at 12 000 rpm for 20 min at 4 °C, and the

supernatant was obtained. The protein concentration was determined

by the bicinchoninic acid (BCA) assay with a Pierce BCA Protein

Assay kit (23227, Thermo) at 562 nm.

The protein samples were separated by sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred

to poly(vinylidene diﬂuoride) (PVDF) membranes (Immobilon-PSQ

Membrane, Merck Millipore) using wet-transfer system. The

membranes were blocked with 1% bovine serum albumin (BSA) in

TBS-T 0.5% for 2 h at room temperature. Membranes were then

incubated with primary antibody diluted in 1% BSA in TBS-T for 2 h

at room temperature and at 4 °C overnight. Following this, the

membranes were treated with horseradish peroxidase (HRP)-

conjugated secondary antibody in TBS-T for 1.5 h. The membranes

were incubated with an ECL Western Blotting detection reagent

(P90719, Millipore) and visualized with the ChemiDoc Imaging

Systems (Chemidoc XRS+, Bio-Rad).

Primary antibodies used in the studies were IL-1β antibody

(1:1000, 3A6, Cell Signaling Technology), pro-caspase-1 antibody

(1:1000, D7F10, Cell Signaling Technology), NLRP3 antibody

(1:500, D4D8T, Cell Signaling Technology), ASC antibody

(1:1000, E1E31, Cell Signaling Technology), β-actin antibody

(1:1000, MKA005B, MyBioScience), Phospho-NF-κB (1:1000,

93H1, Cell Signaling Technology), NF-κB (1:1000, 11014, Signalway

Antibody), Phospho-IκBα (1:500, 11152, Signalway Antibody), IκBα

(1:1000, 48695, Signalway Antibody), and GAPDH antibody

(1:2000, LT05123, Litho). Secondary HRP-conjugated antibodies

used were anti-rabbit IgG (1:2000, A0208, Beyotime) and anti-mouse

IgG (1:2000, A0216, Beyotime).

qRT-PCR. Gene expression was analyzed by qRT-PCR. Total RNA

was extracted using TRIzon (155960-26) and reversely transcripted to

cDNA using PrimeScript RT Master Mix (RR036A, Takara).

O

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equilibrium dissociation constant (K_D) values were calculated using

the equation (K_D= K_off/K_on).⁶⁷The binding aﬃnity of compound 6

with human recombinant NLRP3 protein was determined with Octet

Red96 system (ForteBio) and analyzed with ForteBio Octet Data

Analysis software (10.0).

epithelium missing (4 points). No signiﬁcant lesions were scored as 0

points. The scores of the above three indicators are multiplied by the

proportion of the lesion area (participation ratio) as the ﬁnal score.

The average score (mean s.e.m.) of each animal in each group was

calculated, and t-test among diﬀerent groups was performed.

IHC. Samples were prepared from formalin-ﬁxed, paraﬃn-

embedded colon tissue. Stains against anti-IL-1β (Cell Signaling

Technology, 1:100) and anti-NLRP3 (Cell Signaling Technology,

1:100) were performed according to the kit protocol (KeyGEN, NJ,

China). Histological sections of mice colons were incubated in a 100

°C water bath in 10 mM sodium citrate buﬀer with 0.1% Tween 20

for 20 min using microwave. The sections were blocked with 5%

hydrogen peroxide and 4% peptone casein for 20 min to diminish

nonspeciﬁc staining. The slides were then incubated with primary

antibodies in PBS containing 5% BSA and 10% goat serum at 37 °C

for 2 h. Biotinylated secondary anti-rabbit antibodies were then added

and incubated at room temperature for 30 min. Streptavidin-HRP was

added, and the sections were visualized with DAB substrate after 30

min. Finally, counterstaining was carried out using hematoxylin, as

previously reported.⁶⁹

Confocal Microscopy. Human THP-1 cells were seeded at 1 ×

10⁵/mL in a Petri dish and diﬀerentiated with 100 ng/mL PMA

overnight. The following morning, the culture medium was replaced

with fresh media containing compound 6 (30 μM) for 24 h. THP-M

cells were then stimulated with LPS (100 ng/mL) for 3 h and ATP (5

mM) for 1 h. After removing the medium, the cells were stained with

MitoTracker Red (50 nM) for 30 min or MitoSOX (5 μM) for 10

min. After washing three times with PBS, the cells were ﬁxed with 4%

PFA at room temperature for 30 min and then washed three times

with PBS. The cells were stained with DAPI (2 μM) for 30 min and

washed with PBS three times. The cells were visualized under a

confocal scanning microscope (Zeiss LSM 700).

ASC Oligomerization Assay. THP-1 cells were seeded at 1 × 10⁶/

mL in T25 cell ﬂasks and stimulated with 100 ng/mL PMA for 5 h to

diﬀerentiate to THP-M cells. The cell supernatant was removed, and

fresh 1640 medium was added to the cells overnight. The next

morning, the medium was removed and the cells were treated with

compound 6 or MCC950 for 24 h. After that, the cells were

stimulated with LPS (100 ng/mL) for 3 h and ATP (5 mM) for 1 h.

The cells were lysed in PBS containing 0.5% Triton X-100 at 4 °C for

1 h. The cell lysates were centrifuged at 10 000 rpm for 15 min at 4

°C. The supernatant (Triton X-100-soluble fraction) was removed

and stored at −80 °C. The pellets (Triton X-100 insoluble fraction)

were washed twice with PBS and resuspended in 500 μL of PBS.

Disuccinimidyl suberate (DSS, 2 mM) dissolved in dry DMSO was

added to the resuspended pellets. The mixture was incubated at room

temperature for 30 min and then centrifuged at 10 000 rpm for 15

min at 4 °C. After the supernatant was removed, the cross-linked

pellets were resuspended in 50 μL of sample buﬀer and boiled for 5

min. The samples were analyzed by immunoblotting.

Immunoﬂuorescence. The THP-M cells treated as described

above were ﬁxed with 4% paraformaldehyde at room temperature for

30 min and washed with PBS three times. The cells were then

permeabilized with 0.1% Triton X-100 for 25 min and blocked with

5% bovine serum albumin (BSA) in 0.5% TBS-T for 2 h at room

temperature. The cells were washed PBS three times and incubated

with rabbit anti-ASC (1:100) at 4 °C overnight. After washing with

PBS three times, the cells were incubated with Rhodamine-labeled

anti-rabbit IgG antibody for 90 min at room temperature. Nuclei were

stained with DAPI (2 μM) for 30 min and washed with PBS three

times. Immunoﬂuorescence analyses were performed using a Zeiss

LSM 700.

DSS-Induced Colitis Model and Treatment. The mice were

randomly assigned to four groups as follows: blank control, DSS-

treated, and compound 6 (40 and 80 mg/kg)-treated groups. The

blank control was treated with drinking water for 10 days till

sacriﬁced. Acute colitis was induced in female C57BL/6 by

administration of 2.5% (wt/vol) DSS in drinking water (days 1−7).

Compound 6 (40 and 80 mg/kg) was dissolved in aqueous solution

containing 0.1% Tween 80 and 0.5% sodium carboxymethyl cellulose

(CMC-Na) and given by oral administration from day 1 to day 10.

The blank group and DSS-treated group were also given equal volume

of same solvent by gavage.

Clinical Scoring and Histological Analysis. Bodyweight, stool

consistency, and the presence of gross blood in feces and at the anus

were observed and recorded every day during the experiment. The

DAI was used to evaluate the severity of the disease and calculated by

assigning well-established and validated scores according to a previous

report.⁶⁸Brieﬂy, the mean value of the following parameters was used

for calculation: (a) diarrhea (0 point = normal, 2 points = loose

stools, 4 points = watery diarrhea); (b) hematochezia (0 point = no

bleeding, 2 points = slight bleeding, 4 points = gross bleeding). On

day 14 following induction of colitis, the mice were sacriﬁced. Colons

were collected and washed with normal saline. The lengths of colons

were then measured. The colon of the mice was ﬁxed with 10%

formalin. The samples were then dehydrated, embedded in paraﬃn.

Histological sections of the mice colons were stained by H&E and

observed by optical microscopy. The severity of colitis was evaluated

with lesion score. (1) The severity of inﬂammation of the intestinal

wall was recorded as 0.5 point, 1 point, 2 points, and 3 points,

representing extremely mild, mild, moderate, and severe inﬂamma-

tion, respectively. (2) The severity of the lesion is recorded as 1 point,

2 points, and 3 points. That is, the lesion is located in the mucosal

layer (1 point), mucosa (2 points), and submucosa or transmural

injury (3 points). (3) The degree of crypt damage was evaluated by

1/3 crypt damage (1 point), 2/3 crypt damage (2 points), crypt loss

but surface epithelium intact (3 points), and crypt and surface

Statistical analysis. The data were expressed as mean

s.e.m.

Statistical analysis preformed was an unpaired, two-tailed Student’s t-

test using GraphPad Prism software. The accepted levels of

signiﬁcance were *p < 0.05, **p < 0.01, ***p < 0.001,****p <

0.0001, NS: not signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01924.

■

sı

¹H NMR, ¹³C NMR, and HPLC spectra analysis of all

synthetic compounds; amino acid sequences of NLRP3

PYD domain, NACHT domain, and LRR domain;

pharmacophore generation; pharmacophore and dock-

ing screening; small molecules in training set and testing

set; and the activity tests of selected ligands (PDF)

Molecular formula strings (CSV)

Recommended compound characterization checklist

(PDF)

Recommended compound characterization checklist

(XLS)

AUTHOR INFORMATION

Corresponding Authors

■

Cheng Jiang − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China;

Phone: +86-025-83271414; Email: jc@cpu.edu.cn

Qi-dong You − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Phone: +86-025-83271414;

Email: youqd@163.com

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01924

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Author Contributions

Bin Di − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China;

orcid.org/0000-0002-1907-1736; Phone: +86-025-

83271414; Email: dibin@cpu.edu.cn

Li-li Xu − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China;

orcid.org/0000-0001-6899-971X; Phone: +86-025-

83271414; Email: 1620174420@cpu.edu.cn

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript. L.L.X. and Z.D. designed the research. Z.D.,

X.Y.C., L.Y.A., C.C.L., N.Z., F.Y., S.T.Y., C.Z.H., and K.L.

performed the experiments of this study. L.L.X. and Z.D. wrote

the original draft. C.J., Q.D.Y., and B.D. reviewed and edited

the paper.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This study was supported by projects [81803354] and

[81773693] of the National Natural Science Foundation of

China; [2018YFC0807402] of National Key R&D Program of

China; [BK20180564] of the Natural Science Foundation of

Jiangsu Province of China; [2017M620232] of the China

Postdoctoral Science Foundation-funded project; and

[CPU2018GY02] of Double First Class Innovation Team of

China Pharmaceutical University. The authors thank the public

platform of the State Key Laboratory of Natural Medicines for

assistance of BLI assays.

Authors

Zhen Dai − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Xiao-yi Chen − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Lu-yan An − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Cui-cui Li − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Ni Zhao − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Fan Yang − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Song-tao You − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Chen-zhi Hou − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

Kan Li − Jiangsu Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing

210009, China; Key Laboratory of Drug Quality Control

and Pharmacovigilance, Ministry of Education, China

Pharmaceutical University, Nanjing 210009, China

ABBREVIATIONS

■

IBD, inﬂammatory bowel disease; NLR, NOD-like receptor;

NLRP3, NOD-like receptor family pyrin domain-containing 3;

ASC, apoptosis-associated speck-like protein containing a

CARD; PRR, pattern recognition receptor; DAMPs, damage-

associated molecular patterns; PAMPs, pathogen-associated

molecular patterns; CAPS, cryopyrin-associated periodic

syndrome; MWS, Muckle−Wells syndrome; NF-κB, nuclear

factor-κB; TLRs, toll-like receptors; DSS, dextran sulfate

sodium; BLI, biolayer interferometry; NLRC4, NOD-like

receptor family CARD-containing 4; AIM2, absent in

melanoma 2; poly-(dA:dT), poly-deoxyadenylic−deoxythymi-

dylic acid sodium salt; ROS, reactive oxygen species; ICP-MS,

inductively coupled plasma mass spectrometry; BHB, β-

hydroxybutyrate; MNS, 3,4-methylenedioxy-β-nitrostyrene;

HRMS, high-resolution mass spectra; ESI, electrospray

ionization; ELISA, enzyme-linked immunosorbent assay;

DAI, disease activity index; IHC, immunohistochemistry

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https://dx.doi.org/10.1021/acs.jmedchem.0c01924

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Development of Novel Tetrahydroquinoline Inhibitors of NLRP3 Inflammasome for Potential Treatment of DSS-Induced Mouse Colitis

DOI: 10.1021/acs.jmedchem.0c01924

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Full text of DOI:10.1021/acs.jmedchem.0c01924

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