Discovery of Second-Generation NLRP3 Inflammasome Inhibitors: Design, Synthesis, and Biological Characterization

Article abstract of DOI:10.1021/acs.jmedchem.9b01155

NLRP3 inflammasomes have recently emerged as an attractive drug target for neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as selective inhibitors of NLRP3 inflammasomes. Initial characterization of the lead HL

Full text of DOI:10.1021/acs.jmedchem.9b01155

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Article
Discovery of second-generation NLRP3 inflammasome
inhibitors: Design, synthesis, and biological characterization
Yuqi Jiang, Liu He, Jakob C. Green, Hallie Blevins, Chunqing Guo, Sulay Patel, Matthew
Sean Halquist, MaryPeace McRae, Jurgen Venitz, Xiang-Yang Wang, and Shijun Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01155 • Publication Date (Web): 18 Oct 2019
Downloaded from pubs.acs.org on October 19, 2019
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Discovery of second-generation NLRP3 inflammasome inhibitors: Design, synthesis, and
biological characterization
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Yuqi Jianga#, Liu Hea, Jakob Greena, Hallie Blevinsa, Chunqing Guob, Sulay Harsiddhbhai Patelc,
Matthew S. Halquistd, MaryPeace McRaec, Jürgen Venitzd, Xiang-Yang Wangb, Shijun Zhanga*
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aDepartment of Medicinal Chemistry, bDepartment of Human & Molecular Genetics, cDepartment
of Pharmacotherapy and Outcomes, dDepartment of Pharmaceutics, Virginia Commonwealth
University, Richmond, Virginia, USA, 23298
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Abstract
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NLRP3 inflammasome has recently emerged as an attractive drug target for
neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as
selective inhibitors of NLRP3 inflammasome. Initial characterization of the lead HL16
demonstrated improved however non-selective inhibition on the NLRP3 inflammasome. Structure-
activity relationship studies of HL16 identified a new lead, 17 (YQ128), with an IC50 of 0.30 ± 0.01
µM. Further studies from in vitro and in vivo models confirmed its selective inhibition on the
NLRP3 inflammaome and its brain penetration. Furthermore, pharmacokinetic studies in rats at 20
mg/kg indicated extensive systemic clearance and tissue distribution, leading to a half-life of 6.6
hours. However, the oral bioavailability is estimated to be only 10%, which may reflect limited GI
permeability and possibly high first-pass effects. Collectively, these findings strongly encourage
development of more potent analogs with improved pharmacokinetic properties from this new
chemical scaffold.
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Introduction
Inflammasomes are important components of the innate immunity to initiate immune
responses through pattern recognition receptors (PRRs).1 Three major types of PRRs have been
identified and characterized to regulate the functions of inflammasomes and this includes the NOD-
like receptor (NLR) containing family, the absent in melanoma 2 (AIM2), and retinoic acid-
inducible gene I (RIG-I) like receptors (RLRs).2 Upon recognition of the damage associated
molecular pattern molecules (DAMPs) released during tissue injury or stress and/or pathogen-
associated molecular patterns (PAMPs), inflammasome complexes are assembled to include a
sensor component, an adaptor component (the apoptosis-associated speck-like protein containing a
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caspase recruitment domain---ASC), and an effector component, typically pro-caspase-1, and the
substrate component.2, 3 Subsequently, pro-caspase-1 will be cleaved and activated to produce pro-
inflammatory cytokines interleukin (IL)-1β and IL-18.2, 4 Among the members of the NLR family,
the NLRP3 inflammasome plays essential roles in the maturation and production of IL-1β and IL-
18.1 Notably, emerging studies have shown dysregulation of this inflammasome and IL-1β in the
pathogenesis of many human diseases, such as autoinflammatory disorders, diabetes, and
neurodegenerative disorders.5-12 Therefore, NLRP3 inflammasome has attracted extensive interests
as a promising target of drug development for pathological conditions in which the dysregulation or
overactivation of this inflammasome is evident.
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Several small molecule inhibitors have recently been reported to block the NLRP3
inflammasome pathways. This includes MCC950,13-15, Bay 11-7082,16 CY-09,17 Oridonin,18
Tranilast,19 INF39,20 Glyburide,21, 22 JC124.23, 24 among which MCC950 has been used in many
studies as a pharmacological tool to demonstrate NLRP3 inflammasome as a viable drug target to
development therapeutics for human diseases (Figure 1).13, 15 Our research group has recently
designed and developed sulfonamide analogs as active NLRP3 inflammasome inhibitors and
potential therapeutics for Alzheimer’s disease (AD), multiple sclerosis (MS) and traumatic brain
injury (TBI).23-26 Furthermore, our studies suggested that the sulfonamide analogs directly interfere
with the formation of the NLRP3 inflammasome complex.24, 25 Studies of one of our lead
compounds JC124 have shown both in vitro and in vivo activities in animal models of AD23 and
TBI,26 thus strongly suggesting further development of analogs based on this chemical scaffold with
improved potency and drug like properties. Herein, we report the design and discovery of a series of
compounds from a new chemical scaffold. Structure-activity relationship (SAR) studies were
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conducted to understand the contributions of different structural features of the lead structure and to
provide guidance for further structural optimization/refinement of this chemical scaffold.
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CH3
OH
O
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OH
H
O
F3C
HO
S
H3C
O
O
O
S
O
N
OH
O
N
CN
N
H
S
H3C
H3C
H
H
O
OH
O
O
O
H3C CH3
CH3
Bay 11-7082
CY-09
Oridonin
MCC950
NH
CH3
HN
HN
O
O S O
O S O
O
Cl
O
O
COOEt
N
H
O
OH
O
NH
Cl
O
NH
Cl
Tranilast
INF39
H3CO
H3CO
JC124
Glyburide
Figure 1. Structures of small molecule inhibitors targeting the NLRP3 inflammasome
pathway.
Results and Discussion
Design of a new chemical scaffold as NLRP3 inhibitors. Our SAR studies of JC124 (1, Figure
2) suggested that only limited modifications can be tolerated on the phenyl ring of the benzamide
moiety.24 To expand the scope for structural variations and optimization of this lead structure, we
designed a new chemical scaffold exemplified by HL16 (2, Figure 2). In this structure, we changed
the amide functional group to an appendix position of the structure and this should allow
introduction of a variety of substituents to explore the SAR and to optimize the biological activity.
Specifically, we incorporated a propargyl substituent on the sulfonamide moiety and an acrylamide
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CH3
HN
HN
O S O
O S O
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O
NH
N
H3CO
H3CO
O
Cl
Cl
HL16 (2)
JC124 (1)
Figure 2. Designed new chemical scaffold from JC124.
moiety based on the results of our chemical probe studies (unpublished data). Biological
characterization from murine macrophage J774A.1 cells that release IL-1β upon the activation of
NLRP3 inflammasome by lipopolysaccharide (LPS) and adenosine triphosphate (ATP)27
established an IC50 of 1.30 ± 0.23 µM for HL16 (Figure 3A), 2.5-fold increase compared that of
JC124. The inhibitory activity of HL16 was also confirmed in mouse peritoneal macrophages
(Figure 3B). However, when the selectivity was examined (J774A.1 cells were stimulated with
LPS/poly(dA:dT) or LPS/flagellin to activate the NLRC4 and AIM2 inflammasome, respectively),
HL16 also significantly inhibited NLRC4 and AIM2 inflammasomes at 10 µM concentration
(Figure 3C).
Figure 3. HL16 inhibits NLRP3 inflammasome. J774A.1 cells (A) or mouse peritoneal
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macrophages (B) were primed with LPS (1 μg/mL) for 4.5 h and then treated with indicated
concentrations of HL16 when adding ATP (5 mM) stimulation for 30 min. IL-β in the culture media
was assayed by ELISA. (C) J774A.1 cells were treated with LPS (1 µg/mL) and HL16 (10 µM) for
1 h. Flagellin (1 µg/mL) was added and allowed to incubate for 6 hr or (Poly(dA:dT)) (4 µg/ml) for
8 hr. The supernatants were collected and levels of IL-1β were measured by ELISA. Data are
expressed as mean ± SEM from at least 3 independent experiments with at least triplicates for each
experiment. Statisitical analysis by student t-test.
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Further testing in mice challenged with LPS also demonstrated that treatment with HL16 (10
mg/kg) led to significant suppression of both IL-1β (Figure 4A) and TNF-α (Figure 4B), while
MCC950 (10 mg/kg), the known NLRP3 inhibitor13-15 as a positive control, only inhibited IL-1β,
suggesting the nonspecific action of HL16.
Figure 4. HL16 nonselectively suppressed the production of cytokines in vivo. Serum levels of IL-
1β (A) and TNF-α (B) from C57BL/6 mice (n=4 per group) pretreated with HL16 (10 mg/kg) or
MCC950 (10 mg/kg) by intraperitoneal injection (i.p.) were measured by ELISA 2.5 h after i.p.
injection of LPS (50 mg/kg). Data are expressed as mean ± SD. Statistical analysis by student t-test.
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HN
O S O
6
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26:
27:
CH3
CH3
10
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CH3
N
CH3
28:
O
O
S
R
Cl
26-28
HN
HN
HN
O S O
O S O
O S O
2 (HL16)
N
N
R
N
O
R
H3CO
O
H3CO
H3C
Cl
Cl
Cl
3-8
9-25
N
9:
16:
3:
CH3
O
O
O
O
NH
O
O
17:
18:
CH3
10:
11:
12:
4:
5:
6:
S
HN
O S O
O
O
O
OCH3
O
O
S
S
19:
20:
O
S
O
O
O
N
N
O
7:
8:
13:
14:
N
O
S
O
N
H3C
S
CH3
21:
22:
N
R
24: Br
25: F
N
O
O
N
NH
N
15:
NH
O
N
23:
N
O
Figure 5. Chemical structures of the designed analogues of HL16.
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Structural exploration of HL16 by SAR studies. To explore whether structural modifications of
HL16 will improve inhibitory potency and selectivity to NLRP3 inflammasome, a series of analogs
were designed and synthesized. As shown in Figure 5, structural modifications were mainly
focused on two positions of HL16: the appendix amide and the methoxy group on the benzyl
moiety. To evaluate the role of the chlorine substitution, we also designed analogs with a fluorine or
bromine at this position. In total, twenty-six analogs were designed and synthesized.
The chemical syntheses were achieved by the conditions outlined in Schemes 1 and 2.
Briefly, reaction of sulfonyl chloride 29 with propargylamine gave 30, followed by de-protection
using methylhydrazine in benzene to provide the amine 31. Aldehyde 32 was reacted with
iodomethane or 1-bromopropane to obtain compound 33 or 35, respectively. Reductive amination
of 33 with 31 in the presence of NaBH3CN yielded 34, which on coupling reaction with various
carboxylic acids in the presence of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole (HOBt) gave the target compounds 2-6. Alkylation of 34 with
allyl bromide or 1-bromopropane afforded 7 and 8, respectively. Similarly, compound 9-25 were
synthetized following the conditions outlined in schemes 1 and 2 with 35, 37, or 40 as the starting
material.
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Scheme 1a
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O
O
O
O
S Cl
O
O
5
6
7
8
S NH
O
N
S NH
O
b
H3CO
O
N
a
H2N
O
O
31
29
30
1
9
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HN
O S O
HN
O S O
CHO
CHO
H3CO
d
c
e
HO
31
Cl
Cl
2-8
32
33
N
NH
R
34
H3CO
Cl
Cl
f
HN
HN
O S O
O S O
CHO
h
g
O
H3C
31
Cl
9-23
35
N
NH
R
O
H3C
36
H3C
Cl
Cl
HN
HN
O S O
O S O
CHO
CHO
HO
f
g
O
i
H3C
31
R
R
24-25
37
38a-38b
N
37a: Br
37b: F
NH
O
O
R
S
39a-39b
H3C
O
H3C
R
aReagents and conditions: (a) Propargylamine, TEA, DCM; (b) Methylhydrazine, benzene; (c)
Iodomethane, K2CO3, DMF; (d)TEA, Acetic acid, NaCNBH3; (e) various acids, EDCI, HOBt,
TEA, DCM; For 7 and 8: Allylbromide or 1-Bromopropane, K2CO3, DMF; (f) 1-Bromopropane,
K2CO3, DMF; (g) TEA, Acetic acid, NaCNBH3; (h) various acids, EDCI, HOBt, TEA, DCM. (i) 3-
Thiopheneacetic acid, EDCI, HOBt, TEA, DCM.
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Scheme 2a
5
6
7
HN
HN
O S O
O S O
8
9
CHO
HO
CHO
b
c
O
a
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R
31
Cl
Cl
NH
N
O
40a-40c
32
O
O
S
R
40a:
40b:
R
CH3
CH3
Cl
Cl
26-28
41a-41c
CH3
40c:
CH3
aReagents and conditions: (a) Bromobutane or 1-Bromopentane or 2-Bromobutane, K2CO3, DMF
(b) TEA, Acetic acid, NaCNBH3; (c) 3-Thiopheneacetic acid, EDCI, HOBt, TEA, DCM.
After synthesis, the analogs were tested using the J774A.1 cell model upon challenge with
LPS/ATP to measure their inhibitory potencies on the release of IL-1β by an enzyme-linked
immunosorbent assay (ELISA). As shown in Table 1, saturation of the acrylamide to a propanamide
as represented by compound 3 retained the inhibitory potency.. Chain extension to a hexanamide as
in 4 did not lead to improved inhibitory potency. Notably, replacement with a 2-phenylacetamide
lead to ~2-fold increase of inhibitory potency as evidenced by compound 5. Bioisoteric replacement
of the phenyl ring of 5 with a thiophene (6) retained its inhibitory potency. To evaluate whether the
appendix amide is essential to the biological activity, compounds 7 and 8 were designed and tested.
The results of these two analogs suggest that a tertiary amine can still maintain the inhibitory
potency. Replacement of the methoxy substituent in the structure of HL16 with a propoxyl one as
illustrated by 9 led to a slight improvement of inhibitory potency. Therefore, in the following
analogs, we kept this propoxyl substituent and varied the appendix amide to evaluate their effects
on the inhibitory potency. Surprisingly, the combination of a 2-phenylacetamide and a propoxyl
substituent in compound 10 did not result in increased inhibitory potency compared to 5 or 9.
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Introduction of a para-methoxy on the phenyl ring of 10 led to reduction of inhibitory potency as
seen by compound 11. However, structural extension to a 3-phenylpropanamide in 12 retained the
inhibitory potency. Bioisosteric replacement of the phenyl ring of the 2-phenylacetamide with a 2-
(13) or 3-pyridine (14), or 3-indole (15) retained the inhibitory potency while replacement with an
imidazole-4-yl moiety (16) led to a ~2-fold reduction of inhibitory potency. Notably, replacement
of the phenyl ring of the 2-phenylacetamide with a 3-thiophene (17) further increased the inhibitory
potency by 2-fold. Change of the 2-phenylacetamide of 10 to a furan-3-carboxamide (18),
thiophene-3-carboxamide (19), 1,3-thiazole-4-carboxamide (20), or (2,5-diaza-1-thiazole)-3-
carboxamide (21) all slightly increased the inhibitory potency while replacement with (1,2,4-
traizole)-3-carboxamide (22) and pyrazine-2-carboxamide (23) led to slight reduction of inhibitory
potency. Based on these observations, we further replaced the Cl of compound 17 with a Br (24) or
F (25) and this led to reduction of inhibitory potency. We also evaluated how the propoxyl
substituent of 17 can be further modified in analogs 26-28. The results demonstrated that increase
of chain length or steric hindrance did not improve the inhibitory potency. Although some of the
structural modifications represented by these analogs only led to slight improvements of inhibitory
potency, overall, the SAR studies identified critical postions to optimize and improve inhibitory
potency for the design of next generation analogs.
10
11
12
13
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15
16
17
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Table 1. Inhibitory potency of the designed analogs on the production of IL-1β by J774A.1
cells upon stimulation with LPS/ATPa.
O
R2
R1
N
S NH
O
OR3
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Cpd
2
(HL16)
R1
R2
R3
IC50 (μM)a
1.30±0.23
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
CH3
3
4
5
6
7
8
1.47±0.20
1.93±0.11
0.69±0.15
0.90±0.06
1.78±0.21
1.15±0.12
O
CH3
10
11
12
13
14
15
16
17
18
19
20
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22
23
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25
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O
O
O
S
CH3
CH3
CH3
CH3
9
Cl
Cl
Cl
0.97±0.12
0.87±0.02
1.49±0.07
O
10
11
O
O
O
OCH3
CH3
12
Cl
0.62±0.02
CH3
CH3
13
14
Cl
Cl
0.73±0.07
0.89±0.08
O
O
N
N
O
NH
CH3
15
Cl
0.79±0.07
N
CH3
CH3
16
17
Cl
Cl
1.66±0.10
0.30±0.01
O
O
NH
S
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O
S
S
CH3
CH3
CH3
18
Cl
Cl
Cl
0.47±0.04
0.31±0.04
0.58±0.09
O
O
O
19
20
10
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N
N
S
CH3
21
Cl
0.46±0.03
N
O
O
N
N
NH
CH3
CH3
CH3
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23
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Cl
Cl
1.06±0.04
0.88±0.09
0.91±0.09
0.67±0.07
0.55±0.05
0.47±0.14
0.78±0.19
N
N
O
O
Br
F
S
S
S
S
S
CH3
O
O
O
O
Cl
Cl
Cl
CH3
CH3
CH3
CH3
aThe inhibitory potency was derived from results of at least 3 independent experiments
with at least triplicates for each experiment; data are shown as mean ± SEM.
Compound 17 is a selective inhibitor of NLRP3 inflammasome. After the establishment of
inhibitory potency on IL-1β release in J774A.1 cells for the designed analogs, we selected 17
(YQ128), the most potent analog among this series, for further characterization. Before moving to
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test its selectivity, we confirmed its inhibitory activity in primary mouse peritoneal macrophages.
As shown in Figures 6A, both 17 and MCC950, the known NLRP3 inhibitor as a positive control,
dose dependently suppressed the release of IL-1β from peritoneal macrophages upon LPS/ATP
challenge with an IC50 of 1.59 ± 0.60 and 0.04 ± 0.0008 µM, respectively. Compound 17 is ~5
times less potent in peritoneal macrophagess than in J774A.1 cells to inhibit the release of IL-1β.
This may suggest that J774A.1 are more sensitive to 17 under the current experimental conditions.
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We next examined the effects of 17 on NLRC4 and AIM2 inflammasomes. As shown in Figure 6B,
treatment of J774A.1 cells with 17 under these experimental conditions did not significantly
interfere with the production of IL-1β by NLRC4 or AIM2 inflammasome (by student t-test
analysis), thus suggesting the specific inhibition of NLRP3 inflammasome by 17. We then tested
the in vivo engagement of and selectivity to the NLRP3 inflammasome by 17. C57BL/6 mice (n=4
per group) were pretreated with 17 or MCC950 (positive control) at 10 mg/kg before
intraperitoneal injection of LPS, which has been shown to trigger IL-1β production in a NLRP3-
dependent manner.28 As shown in Figure 6C, serum level of IL-1β was significantly reduced while
no significant inhibition on the TNF-α level (Figure 6D) was observed by the treatment of both
compounds at the tested dose, thus strongly suggesting the selective in vivo engagement of NLRP3
inflammasome in the observed effects by 17 and MCC950. Lastly, we confirmed the selective
inhibition on NLRP3 inflammasome by 17 in nlrp3-/- mice (n=3 per group). As expected, upon
stimulation with LPS, nlrp3 deficiency abolished the production of IL-1β while the production of
TNF-α is normal (Figure 6E). Treatment of nlrp3-/- mice with 17 (10 mg/kg) did not produce
inhibition on the level of TNF-α and this again confirmed the selective inhibition on the NLRP3
inflammasome by our compound. The results from the selectivity studies serve as an indirect
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evidence to support the MOA that analogs derived from this chemical scaffold interfere with the
NLRP3 inflammasome complex, instead of the upstream priming step by the LPS, which is
consistent with our previously reported results.25
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Figure 6. Compound 17 selectively inhibits the NLRP3 inflammasome. (A) Mouse peritoneal
macrophages were primed with LPS (1 μg/mL) for 4.5 h and then treated with indicated compounds
at indicated concentrations when adding ATP (5 mM) stimulation for 30 min. IL-β in the culture
media was assayed by ELISA. (B) J774A.1 cells were treated with LPS (1 µg/mL) and 17 (10 µM)
for 1 h. Flagellin (1 µg/mL) was added and allowed to incubate for 6 hr or (Poly(dA:dT)) (4 µg/ml)
for 8 hr. The supernatants were collected and levels of IL-1β were measured by ELISA. Serum
levels of IL-1β.(C) and TNF-α (D) from C57BL/6 (n=4 per group) mice pretreated 17 (10 mg/kg)
or MCC950 (10 mg/kg) were measured by ELISA 2.5 h after i.p. injection of LPS (50 mg/kg). (E)
Serum levels of IL-1β and TNF-α under indicated treatment conditions (both 17 and MCC950 were
tested at 10 mg/kg) of nlrp3-/- mice (n=3 per group) were measured by ELISA 2.5 h after i.p.
injection of LPS (25 mg/kg). For in vitro assasys, data are expressed as mean ± SEM from at least 3
independent experiments with at least triplicates for each experiment. For in vivo assays, data are
expressed as mean ± SD. Statistical analysis by student t-test.
Compound 17 is a BBB penetrant but shows poor oral pharmacokinetic properties. Since our
ultimate goal is to develop small molecule inhibitors of the NLRP3 inflammasome as potential
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therapeutics for neurodegenerative disorders, especially for AD, effective drug candidates need to
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cross the blood-brian barrier (BBB) and reach the brain tissue and be suitable for chronic once-daily
oral administration. To test whether 17 is a brain penetrant, we first determined its permeability and
transport directionality using immortalized human cerebral microvascular endothelial cells
hCMEC/D3 as the human BBB model.29 This model expresses functional efflux transporters such
as P-glycoprotein which are also expressed at the BBB; it has been widely used as a surrogate for
human BBB.29, 30 Apparent permeability (Papp) was calculated using Papp = (dX/dT • Vr )/(A • Co),
where dX/dT is the mass of transported compound (X) over time (T), Vr is the volume in the
receiver compartment, A is the surface area of the membrane insert, and Co is the initial
concentration in the donor compartment.30 We first examined the potential cytotoxicity of 17 on
hCMEC/D3 cells to rule out any potential interference with results interpretation, and the results
demonstrated that 17 at 20 µM did not show significant toxic effects on these cells (Figure 7A).
The apical-to-basolateral (A to B) and basolateral-to-apical (B to A) Papp of 17 was 5.21 ± 0.56 x
10-6 and 1.11 ± 0.12 x 10-6 cm/sec, respectively (Figure 7B). Thus, 17 exhibits an efflux ratio of
0.22, suggesting that 17 is not likely subject to active efflux. We next confirmed the in vivo BBB
penetration of 17 in C57BL/6 mice (n=3 per time point) by oral (PO) administration (20 mg/kg,
single dose). To accurately quantify the amount of 17 delivered to the brain, we perfused the mice
brain to wash out the vascular blood completely prior to collecting and homogenizing brain tissues.
Plasma and brain homogenate samples were analyzed by Liquid Chromatography Tandem Mass
Spectrometry (LC-MS/MS). As shown in Table 2, compound 17 appeared in plasma with the
highest concentration at as early as 30 min after PO administration. Brain concentrations of
compound 17 after 0.5, 1, and 4 h were 21.3, 20.5, and 6.6 ng/g, respectively, indicating that 17
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penetrated into the CNS after oral administration at the tested dose. Furthermore, the brain-to-
plasma concertation ratio increases with time. However, after proximate conversion (based on the
assumption that 1 g of brain tissue is around 1 mL in volume), the brain concentration of 17 was
around 12 nM after 4 h of oral ingestion, 25 fold less than its in vitro IC50 value. Lastly, we
evaluated the basic pharmacokinetic (PK) properties of 17 in Sprague-Dawley rats (n=3 and 11
serial plasma samples over a period of 24 h; plasma concentrations were quantified by LC-MS/MS)
after a dose of 20 mg/kg via intravenous (IV) and PO administration. The results showed that after
IV administration, 17 exhibits extensive extravascular distribution with a large steady-state volume
of distribution (Vdss) of 8.5 L/kg and rapid total clearance (CLtot) of 41 mL/min/kg, approaching
hepatic (and renal) blood flow with the possibility of extrahepatic clearance mechanisms. This
resulted in an intermediate terminal plasma half-life (t1/2) of 6.6 h after IV administration. After oral
administration of an ad-hoc suspension using 10% Cremophor EL in PBS, this compound shows
delayed gastrointestinal absorption with a tmax and cmax of 12 h and 73 ng/mL, respectively (Fig.
7C); oral bioavailability (Foral) was estimated as 10%. This suggests the possibility of poor GI
solubility/permeability and/or high first-pass effects at the tested dose of 20 mg/kg PO.
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Table 2. BBB penetration of 17 (PO, 20 mg/kg) in mice (n=3, mean ± SD) at various time
points
0.5 h
1 h
4 h
Brain (ng/g)
21.25 ± 16.90
277.33 ± 182.15
20.48 ± 4.19
57.60 ± 55.65
0.36
6.58 ± 11.39
8.00 ± 10.51
0.82
Plasma (ng/mL)
Brain-to-Plasma ratio 0.077
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Figure 7. Permeability of 17 in hCMEC/D3 cells and PK properties in rats. (A) hCMEC/D3 cells
were treated with 17 (20 µM) for 2 h, then cell viability was measured using the Live/DeadTM kit.
(B) hCMEC/D3 cells were plated on transwell filters. Compound 17 (20 µM) was added to either
the apical or basolateral side, then samples were analyzed by HPLC to determine flux (A-B: apical-
to-basolateral; B-A: basolateral-to-apical) at indicated time points. (C) Spraque-Dawley rats were
treated with 17 (20 mg/kg) via IV and PO (n=3 per rout). Plasma samples were collected at
indicated time points and analyzed by LC-MS/MS. Data are expressed as mean ± SEM for studies
in hCMEC/D3 cells from 3 independent experiments with at least triplicates for each experiement,
as mean ± SD for studies in rats.
Conclusions
In our continuing efforts to develop small molecule inhibitors of the NLRP3 inflammasome,
a new chemical scaffold represented by HL16 was designed to allow more scope for structural
modifications. Although HL16 exhibited improved inhibitory potency on IL-1β production from
J774A.1 cells compared to our previously reported inhibitor, it suffered the loss of selectivity to the
NLRP3 inflammasome. Further SAR studies of HL16 established that the 2-OCH3 can be modified
to improve inhibitory potency. The acrylamide domain of HL16 can tolerate strucutral
modifications and a heteroaromatic acetamide tend to provide analogs with improved potency. As a
result of this SAR study, one new lead compound, 17, was identified with a more than 4-fold
increased inhibitory potency compared to HL16. Biological characterization in murine peritoneal
macrophages and J774A.1 cells confirmed its inhibitory potency and selectivity to the NLRP3
inflammasome. More importantly, studies in LPS-challenged mice, a mouse model in which the
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release of IL-1β is NLRP3 inflammasome dependent, demonstrated that 17 after a single dose of 10
mg/kg signifiantly and selectively suppressed the production of IL-1β, but not TNF-α, thus
supporting its in vivo engagement of NLRP3 inflammasome. In addition, results from studies in
nlrp3-/- mice echoed its selective engagement of NLRP3 inflammsome since no inhibitory activity
on the production of TNF-α was observed. Studies from both in vitro and in vivo models supported
that 17 can cross the BBB to reach the CNS and is not likely subject to efflux transport. However,
while PK studies in rats suggested adequate systemic characteristics with a plasma t1/2 of 6.6 hours,
oral bioavailability was quite low (10%), likely due to poor GI solubility and possibly high first-
pass effects. Collectively, these findings strongly encourage further development of new analogs
based on this chemical scaffold with improved PK properties as inhibitors of the NLRP3
inflammasome and explore their potential therapeutic applications.
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Experimental Section
Chemistry. Reagents and solvents were obtained from commercial suppliers and used as
received unless otherwise indicated. All reactions were carried out under inert atmosphere (N2)
unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC) (precoated
silica gel 60 F254 plates, EMD Chemicals) and visualized with UV light or by treatment with
Phosphomolybdic acid (PMA). Flash chromatography was performed on silica gel (200-300 mesh,
Fisher Scientific) using solvents as indicated. 1HNMR and 13CNMR spectra were routinely
recorded on Bruker ARX 400 spectrometer. The NMR solvent used was CDCl3 or DMSO-d6 as
indicated. Tetramethylsilane (TMS) was used as internal standard. The purity of target compounds
was determined by HPLC using Varian 100-5 C18 250 x 4.6 mm column with UV detection (230
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nm) (60% acetonitrile/40% H2O/0.1 % trifluoroacetic acid (TFA) and 80% methanol/19.9%
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H2O/0.1% TFA, two solvent systems) to be ≥ 95%.
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Method C. N-(5-chloro-2-methoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)acryl-
amide (2). Acrylic acid (0.03 g, 0.4 mmol) and HOBt (0.068 g, 0.5 mmol) were dissolved in
anhydrous DCM (20 mL) under ice bath followed by the addition of EDCI (0.096 g, 0.5 mmol).
The mixture was stirred at r.t. for 30 min. Compound 34 (0.20 g, 0.5 mmol) and TEA (0.07 mL) in
DCM (30 mL) were added directly. The reaction was stirred at r.t. for 3 h. Then DCM solution was
washed with 1N HCl, saturated NaHCO3 and brine for 3 times, dried over anhydrous Na2SO4 and
evaporated under vacuum. The crude product was purified by chromatography using EtOAc–
hexane (1:1) as the mobile phase, to obtain 2 as a white solid (0.088 g, yield: 47%).1H-NMR (400
MHz CDCl3): δ 2.03-2.04 (m, 1H), 2.84-2.91 (m, 2H), 3.51-3.58 (m, 2H), 3.74-3.76 (m, 5H), 4.37-
4.55 (m, 2H), 4.89-4.91 (m, 1H), 5.60-5.64 (m, 1H), 6.31-6.45 (m, 2H), 6.74 (d, J=8.68 Hz, 1H),
6.92 (d, J=2.52 Hz, 1H), 7.09-7.22 (m, 2H), 7.27 (d, J=8.24 Hz, 1H), 7.72-7.77 (m, 2H). 13C
NMR (100 MHz, CDCl3): δ 167.1, 156.0, 144.8, 138.3, 129.5, 129.2, 128.7, 128.3, 127.8, 127.6,
127.3, 126.6, 125.9, 111.5, 77.9, 73.1, 55.8, 48.7, 33.8, 32.9, 29.7. HRMS (AP-ESI) m/z calcd for
C22H23ClN2O4S [M + Na]+ 469.0965, found 469.1003.
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N-(5-chloro-2-methoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)propion-amide
(3). Compound 3 was prepared from propanoic acid and 34 following the procedure of Method C in
55% yield. 1H-NMR (400 MHz DMSO-d6): δ 0.93-0.98 (m, 3H), 2.24-2.36 (m, 2H), 2.82 (t, J=7.44
Hz, 1H), 2.95 (t, J=7.52 Hz, 1H), 3.03-3.05 (m, 1H), 3.49 (t, J=7.36 Hz, 1H), 3.54 (t, J=7.48 Hz,
1H), 3.64-3.66 (m, 2H), 3.82 (d, J=5.12 Hz, 3H), 4.44 (d, J=8.32 Hz, 2H), 6.98-7.07 (m, 2H), 7.27-
7.36 (m, 1H), 7.39 (d, J=8.16 Hz, 1H), 7.47 (d, J=8.20 Hz, 1H), 7.70-7.74 (m, 2H), 8.05-8.10 (m,
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1H). 13C NMR (100 MHz, DMSO-d6): δ 173.1, 155.7, 144.3, 138.3, 129.5, 129.3, 128.1, 127.5,
127.0, 126.8, 126.5, 124.2, 112.6, 79.3, 74.6, 55.7, 48.5, 46.9, 34.1, 31.9, 25.4, 9.4. HRMS (AP-
ESI) m/z calcd for C22H25ClN2O4S [M + Na]+ 471.1121, found 471.1156.
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N-(5-chloro-2-methoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)hexan-amide (4).
Compound 4 was prepared from hexanoic acid acid and 34 following the procedure of Method C in
62% yield. 1H-NMR (400 MHz DMSO-d6): δ 0.82-0.88 (m, 3H), 1.18-1.29 (m, 4H), 1.43-1.48 (m,
2H), 2.26 (t, J=7.32 Hz, 2H), 2.83 (t, J=7.68 Hz, 1H), 2.94 (t, J=7.32 Hz, 1H), 3.03-3.05 (m, 1H),
3.50 (t, J=7.40 Hz, 1H), 3.56 (t, J=7.28 Hz, 1H), 3.64-3.66 (m, 2H), 3.82 (d, J=3.92 Hz, 3H), 4.44
(d, J=7.40 Hz, 2H), 6.99 (dd, J1=2.68 Hz, J2=9.48 Hz, 1H), 7.01-7.07 (m, 1H), 7.27-7.37 (m, 1H),
7.40 (d, J=8.32 Hz, 1H), 7.46 (d, J=8.36 Hz, 1H), 7.70-7.75 (m, 2H), 8.05-8.10 (m, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 172.4, 155.7, 144.3, 138.4, 129.6, 129.3, 128.1, 127.6, 126.9, 126.8,
124.2, 112.6, 79.3, 74.6, 55.7, 48.5, 46.9, 34.1, 31.9, 31.6, 30.9, 24.5, 22.0, 13.9. HRMS (AP-ESI)
m/z calcd for C25H31ClN2O4S [M + Na]+ 513.1591, found 513.1599.
N-(5-chloro-2-methoxybenzyl)-2-phenyl-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)pheneth-
yl)aceamide (5). Compound 5 was prepared from 2-phenyl acetic acid and 34 following the
procedure of Method C in 69% yield. 1H-NMR (400 MHz DMSO-d6): δ 2.83 (t, J=7.20 Hz, 1H),
2.89 (t, J=7.56 Hz, 1H), 3.02-3.03 (m, 1H), 3.49 (t, J=7.00 Hz, 1H), 3.58 (t, J=7.00 Hz, 1H), 3.64-
3.69 (m, 4H), 3.82 (d, J=2.72 Hz, 3H), 4.47 (d, J=8.88 Hz, 2H), 6.99 (dd, J1=2.64 Hz, J2=16.64 Hz,
1H), 7.07 (dd, J1=8.80 Hz, J2=17.36 Hz, 1H), 7.13-7.36 (m, 7H), 7.45 (d, J=8.40 Hz, 1H), 7.69 (d,
J=8.28 Hz, 1H), 7.76 (d, J=8.83 Hz, 1H), 8.05-8.11 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ
170.6, 155.8, 144.1, 138.5, 135.7, 129.6, 129.3, 129.0, 128.9, 128.3, 127.8, 127.6, 127.2, 126.9,
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126.4, 124.3, 112.7, 79.3, 74.6, 55.8, 48.9, 46.7, 42.8, 34.1, 31.9. HRMS (AP-ESI) m/z calcd for
C27H27ClN2O4S [M + Na]+ 533.1278, found 533.1285.
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N-(5-chloro-2-methoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(thiophen-3-
yl)acetamide (6). Compound 6 was prepared from thiophene-3-yl-acetic acid and 34 following the
procedure of Method C in 57% yield. 1H-NMR (400 MHz DMSO-d6): δ 2.79-2.89 (m, 2H), 3.02-
3.04 (m, 1H), 3.47 (t, J=7.56 Hz, 1H), 3.59 (t, J=7.40 Hz, 1H), 3.64-3.69 (m, 4H), 3.81 (s, 3H),
4.46 (d, J=4.68 Hz, 2H), 6.93-7.07 (m, 3H), 7.25-7.29 (m, 3H), 7.44-7.50 (m, 2H), 7.70 (d, J=8.32
Hz, 1H), 7.75 (d, J=8.32 Hz, 1H), 8.05-8.10 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 170.3,
155.7, 144.1, 138.5, 135.5, 129.6, 129.2, 128.6, 128.2, 127.7, 127.2, 127.0, 126.8, 125.9, 124.3,
122.4, 112.7, 79.3, 74.6, 55.8, 48.9, 46.7, 34.6, 34.3, 31.9. HRMS (AP-ESI) m/z calcd for
C25H25ClN2O4S2 [M + Na]+ 539.0842, found 539.0866.
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Method D. 4-(2-(allyl(5-chloro-2-methoxybenzyl)amino)ethyl)-N-(prop-2-yn-1-yl)benzenesul-
fonamide (7). A mixture of 34 (0.2 g, 0.5 mmol) and K2CO3 (0.14 g, 1.0 mmol) in DMF was stirred
at room temperature. Then allyl bromide (0.06 g, 0.5 mmol) was added and the mixture was stirred
at room temperature for 6 h. After removing DMF under reduced pressure, the residue was taken up
to DCM. The DCM solution was washed with water for 3 times, dried over anhydrous Na2SO4,
filtered and evaporated. The crude product was purified by chromatography using EtOAc–Hexane
(1:1) as the mobile phase, to obtain 7 as a white solid (0.058 g, yield: 27%). 1H-NMR (400 MHz
DMSO-d6): δ 2.78-2.84 (m, 4H), 3.06 (t, J=2.48 Hz, 1H), 3.69 (s, 2H), 3.76-3.80 (m, 5H), 4.03 (d,
J=2.52 Hz, 2H), 5.19-5.28 (m, 2H), 5.66-5.76 (m, 1H), 6.98 (d, J=8.76 Hz, 1H), 7.26 (dd, J1=2.72
Hz, J2=8.68 Hz, 1H), 7.31 (d, J=2.76 Hz, 1H), 7.46 (d, J=8.32 Hz, 2H), 7.76 (d, J=8.36 Hz, 2H).
13C NMR (100 MHz, DMSO-d6): δ 155.7, 146.3, 136.0, 132.2, 130.6, 129.4, 128.1, 127.3, 127.1,
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123.9, 119.4, 112.1, 76.9, 76.2, 55.6, 49.7, 48.9, 46.6, 36.0, 35.4. HRMS (AP-ESI) m/z calcd for
C22H25ClN2O3S [M + H]+ 433.1347, found 433.1338.
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4-(2-((5-chloro-2-methoxybenzyl)(propyl)amino)ethyl)-N-(prop-2-yn-1-yl)benzenesul-
fonamide (8). Compound 8 was prepared from 1-bromopropane and 34 following the procedure of
Method D in 21% yield. 21% yield. 1H-NMR (400 MHz DMSO-d6): δ 0.86 (t, J=7.36 Hz, 3H),
1.48-1.57 (m, 2H), 2.78-2.83 (m, 4H), 3.04-3.09 (m, 3H), 3.68 (s, 2H), 3.76 (s, 3H), 4.09 (d, J=2.48
Hz, 2H), 6.98 (d, J=8.72 Hz, 1H), 7.26 (dd, J1=2.76 Hz, J2=8.68 Hz, 1H), 7.31 (d, J=2.76 Hz, 1H),
7.45 (d, J=8.36 Hz, 2H), 7.73 (d, J=8.32 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 155.7, 146.1,
136.1, 130.6, 129.3, 128.1, 127.2, 127.1, 123.9, 112.1, 77.5, 75.9, 55.6, 49.7, 48.2, 46.6, 36.2, 35.4,
20.4, 10.9. HRMS (AP-ESI) m/z calcd for C22H27ClN2O3S [M + H]+ 435.1503, found 435.1457.
N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)acryl-amide (9).
compound 9 was synthetized from acrylic acid and compound 36 following the procedure of
Method C in 58% yield. 1H-NMR (400 MHz DMSO-d6): δ 0.95-1.00 (m, 3H), 1.70-1.76 (m, 2H),
2.86 (t, J=7.64 Hz, 1H), 2.93 (t, J=7.68 Hz, 1H), 3.02-3.04 (m, 1H), 3.54-3.69 (m, 4H), 3.98 (t,
J=6.44 Hz, 2H), 4.44-4.53 (m, 2H), 5.63-5.69 (m, 1H), 6.09-6.20 (m, 1H), 6.65-6.76 (m, 1H), 6.99-
7.10 (m, 2H), 7.26-7.46 (m, 3H), 7.73 (d, J=8.16 Hz, 2H), 8.04-8.06 (m, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 165.6, 155.2, 144.1, 138.4, 129.6, 129.2, 128.3, 127.9, 127.8, 127.3, 126.8, 123.9,
113.4, 79.3, 74.6, 69.6, 48.4, 47.2, 34.7, 31.9, 21.9, 10.5. HRMS (AP-ESI) m/z calcd for
C24H27ClN2O4S [M + Na]+ 497.1277, found 497.1248.
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N-(5-chloro-2-propoxybenzyl)-2-phenyl-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)pheneth-
yl)acetamide (10). Compound 10 was prepared from 2-phenylacetic acid and 36 following the
procedure of Method C in 49% yield as a white solid. 1H-NMR. (400 MHz DMSO-d6): δ 0.93-0.99
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(m, 3H), 1.70-1.73 (m, 2H), 2.80 (t, J=7.52 Hz, 1H), 2.87 (t, J=7.44 Hz, 1H), 3.01-3.02 (m, 1H),
3.45-3.49 (m, 1H), 3.60 (t, J=7.56 Hz, 1H), 3.64-3.68 (m, 4H), 3.92-3.98 (m, 2H), 4.46 (t, J=14.96
Hz, 2H), 6.98-7.05 (m, 2H), 7.13-7.33 (m, 7H), 7.42 (d, J=8.16 Hz, 1H), 7.68 (d, J=8.32 Hz, 1H),
7.74 (d, J=8.32 Hz, 1H), 8.05-8.08 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 170.5, 155.2,
144.1, 138.5, 135.7, 129.5, 129.2, 129.1, 128.9, 128.3, 127.9, 127.6, 127.3, 126.8, 126.4, 124.1,
113.4, 79.3, 74.6, 69.5, 48.8, 46.6, 42.9, 34.1, 31.9, 21.9, 10.5. HRMS (AP-ESI) m/z calcd for
C29H31ClN2O4S [M + Na]+ 561.1591, found 561.1595.
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N-(5-chloro-2-propoxybenzyl)-2-(4-methoxyphenyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamo-
yl)phenethyl)acetamide (11). Compound 11 was prepared from 2-(4-methoxy)-phenylacetic acid
and 36 following the procedure of Method C in 51% yield as a white solid. 1H-NMR (400 MHz
DMSO-d6): δ 0.95-1.00 (m, 3H), 1.71-1.76 (m, 2H), 2.85 (t, J=7.52 Hz, 1H), 2.97 (t, J=7.40 Hz,
1H), 3.02-3.03 (m, 1H), 3.51 (t, J=6.96 Hz, 1H), 3.57-3.67 (m, 5H), 3.72-3.77 (m, 3H), 3.94-3.99
(m, 2H), 4.45 (d, J=4.00 Hz, 2H), 6.87-7.10 (m, 5H), 7.20-7.31 (m, 2H), 7.37 (d, J=8.20 Hz, 1H),
7.45 (d, J=8.24 Hz, 1H), 7.71 (d, J=8.32 Hz, 1H), 7.77 (d, J=8.28 Hz, 1H), 8.03-8.09 (m, 1H). 13C
NMR (100 MHz, DMSO-d6): δ 170.7, 156.7, 155.0, 144.2, 138.5, 130.6, 130.4, 129.5, 129.2, 128.1,
127.9, 127.5, 127.0, 126.8, 124.3, 123.9, 120.2, 113.3, 110.6, 79.3, 74.6, 69.6, 55.4, 49.0, 47.1,
43.2, 34.3, 31.9, 21.9, 10.5. HRMS (AP-ESI) m/z calcd for C30H33ClN2O5S [M + Na]+ 591.1696,
found 591.1656.
N-(5-chloro-2-propoxybenzyl)-3-phenyl-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)pheneth-
yl)propanamide (12). Compound 12 was prepared from 3-phenylpropanoic acid and 36 following
the procedure of Method C in 55% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.90-
0.99 (m, 3H), 1.64-1.75 (m, 2H), 2.57-2.64 (m, 2H), 2.76-2.88 (m, 4H), 3.01-3.05 (m, 1H), 3.49
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(t, J=7.40 Hz, 1H), 3.55 (t, J=7.32 Hz, 1H), 3.63-3.66 (m, 2H), 3.91-3.96 (m, 2H), 4.38-4.44 (m,
2H), 6.99-7.04 (m, 2H), 7.16-7.32 (m, 6H), 7.35 (d, J=8.36 Hz, 1H), 7.40 (d, J=8.32 Hz, 1H),
7.69-7.73 (m, 2H), 8.05-8.10 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 171.6, 155.1, 144.2,
141.3, 138.4, 129.5, 129.2, 128.3, 128.2, 128.1, 127.7, 127.4, 127.3, 126.8, 125.8, 124.0, 113.4,
79.3, 74.6, 69.6, 48.3, 46.9, 34.1, 33.4, 31.9, 30.7, 21.9, 10.5. HRMS (AP-ESI) m/z calcd for
C30H33ClN2O4S [M + Na]+ 575.1747, found 575.1750.
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N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(pyri-din-4-
yl)acetamide (13). Compound 13 was prepared from 2-(pyridine-4-yl)acetic acid and 36 following
the procedure of Method C in 41% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.99-
1.05 (m, 3H), 1.75-1.81 (m, 2H), 2.88 (t, J=7.52 Hz, 1H), 3.02 (t, J=7.40 Hz, 1H), 3.08 (t, J=2.48
Hz, 1H), 3.52-3.56 (m, 1H), 3.66-3.71 (m, 3H), 3.78-3.83 (m, 2H), 3.99-4.05 (m, 2H), 4.53 (s, 2H),
7.05-7.15 (m, 2H), 7.20-7.23 (m, 2H), 7.29-7.33 (m, 1H), 7.40 (d, J=8.16 Hz, 1H), 7.53 (d, J=8.32
Hz, 1H), 7.76 (d, J=8.24 Hz, 1H), 7.82 (d, J=8.28 Hz, 1H), 8.11-8.14 (m, 1H), 8.51-8.54 (m, 2H).
13C NMR (100 MHz, DMSO-d6): δ 169.4, 156.6, 155.2, 149.3, 144.8, 144.0, 138.6, 129.6, 129.2,
128.4, 127.7, 127.0, 126.8, 124.9, 124.6, 124.1, 113.4, 79.3, 74.6, 69.6, 48.7, 47.5, 46.7, 34.3, 31.9,
21.9, 10.5. HRMS (AP-ESI) m/z calcd for C28H30ClN3O4S [M + Na]+ 562.1543, found 562.1579.
N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)-2-(pyri-din-2-
yl)acetamide (14). Compound 14 was prepared from 2-(pyridine-2-yl)acetic acid and 36 following
the procedure of Method C in 43% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.94-
1.00 (m, 3H), 1.69-1.77 (m, 2H), 2.81 (t, J=7.48 Hz, 1H), 2.96 (t, J=7.40 Hz, 1H), 3.08 (t, J=2.52
Hz, 1H), 3.49 (t, J=7.40 Hz, 1H), 3.63-3.67 (m, 3H), 3.87 (d, J=9.08 Hz, 2H), 3.93-3.97 (m, 2H),
4.46-4.52 (m, 2H), 6.97-7.34 (m, 6H), 7.46 (d, J=8.40 Hz, 1H), 7.66-7.78 (m, 3H), 8.04-8.09 (m,
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1H), 8.54-8.56 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 169.9, 156.2, 155.1, 148.9, 144.1,
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138.5, 136.6, 129.5, 129.2, 128.2, 127.8, 127.4, 127.3, 126.9, 124.1, 123.9, 121.9, 113.4, 79.3, 74.6,
69.6, 49.2, 46.7, 42.3, 34.3, 31.9, 21.9, 10.5. HRMS (AP-ESI) m/z calcd for C28H30ClN3O4S [M +
Na]+ 562.1543, found 562.1566.
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N-(5-chloro-2-propoxybenzyl)-2-(1H-indol-3-yl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)-
phenethyl)acetamide (15). Compound 15 was prepared from 2-(indole-3-yl)acetic acid and 36
following the procedure of Method C in 25% yield as a white solid. 1H-NMR (400 MHz DMSO-
d6): δ 0.92-0.99 (m, 3H), 1.69-1.73 (m, 2H), 2.74-2.80 (m, 2H), 2.99-3.02 (m, 1H), 3.48 (t, J=7.56
Hz, 1H), 3.61 (t, J=7.51 Hz, 1H), 3.63-3.66 (m, 2H), 3.71-3.76 (m, 2H), 3.92-3.97 (m, 2H), 4.46-
4.48 (m, 2H), 6.96-7.32 (m, 8H), 7.37 (d, J=8.12 Hz, 1H), 7.52 (t, J=7.60 Hz, 1H), 7.65 (d, J=8.28
Hz, 1H), 7.72 (d, J=8.28 Hz, 1H), 7.96-8.08 (m, 1H), 10.90-10.95 (m, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 171.1, 155.2, 144.2, 138.4, 136.1, 129.4, 129.2, 128.1, 127.5, 127.4, 127.1, 127.0,
126.8, 124.1, 123.5, 121.1, 118.5, 113.4, 111.4, 108.1, 79.3, 74.5, 69.6, 49.1, 46.9, 34.2, 33.0, 31.9,
21.9, 10.4. HRMS (AP-ESI) m/z calcd for C31H32ClN3O4S [M + Na]+ 600.1699, found 600.1695.
N-(5-chloro-2-propoxybenzyl)-2-(1H-imidazol-4-yl)-N-(4-(N-(prop-2-yn-1-yl)sulfamo-
yl)phenethyl)acetamide (16). Compound 16 was prepared from 2-(imidazole-4-yl)acetic acid and
36 following the procedure of Method C in 35% yield as a white solid. 1H-NMR (400 MHz DMSO-
d6): δ 0.94-1.00 (m, 3H), 1.70-1.78 (m, 2H), 2.75-2.79 (m, 2H), 3.02-3.04 (m, 1H), 3.38-3.45 (m,
1H), 3.56-3.72 (m, 5H), 3.93-3.98 (m, 2H), 4.43-4.53 (m, 2H), 6.83-6.87 (m, 1H), 7.03-7.11 (m,
2H), 7.24 (dd, J1=2.68 Hz, J2=8.72 Hz 1H), 7.30-7.32 (m, 1H), 7.45 (d, J=8.36 Hz, 1H), 7.54-7.57
(m, 1H), 7.68 (d, J=8.36 Hz, 1H), 7.74 (d, J=8.36 Hz, 1H), 8.05-8.08 (m, 1H), 11.09 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): δ 170.2, 155.0, 144.2, 138.4, 134.8, 129.5, 129.2, 128.2, 127.9, 127.4,
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127.3, 126.8, 124.1, 113.4, 79.3, 74.6, 69.5, 49.2, 46.8, 34.2, 32.9, 31.9, 21.9, 10.5. HRMS (AP-
ESI) m/z calcd for C26H29ClN4O4S [M + Na]+ 551.1495, found 551.1471.
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yl)acetamide (17). Compound 17 was prepared from 2-(thiophene-3-yl)acetic acid and 36
following the procedure of Method C in 61% yield as a white solid. 1H-NMR (400 MHz DMSO-
d6): δ 0.93-0.99 (m, 3H), 1.69-1.74 (m, 2H), 2.80 (t, J=7.58 Hz, 1H), 2.88 (t, J=7.48 Hz, 1H),
3.01-3.03 (m, 1H), 3.46 (t, J=7.48 Hz, 1H), 3.60 (t, J=7.68 Hz, 1H), 3.63-3.71 (m, 4H), 3.92-3.97
(m, 2H), 4.48 (d, J=8.28 Hz, 2H), 6.93-7.04 (m, 3H), 7.18-7.33 (m, 3H), 7.43 (d, J=7.88 Hz, 1H),
7.46-7.49 (m, 1H), 7.68 (d, J=7.88 Hz, 1H), 7.74 (d, J=7.88 Hz, 1H), 8.04-8.09 (m, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 170.2, 155.2, 144.1, 138.5, 135.5, 129.5, 129.2, 128.6, 128.3, 127.9,
127.6, 127.4, 126.8, 125.9, 124.1, 122.4, 113.4, 79.3, 74.6, 69.5, 48.9, 46.6, 34.8, 34.3, 31.9, 21.9,
10.5. HRMS (AP-ESI) m/z calcd for C27H29ClN2O4S2 [M + Na]+ 567.1155, found 567.1177.
N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)furan-3-
carboxamide (18). Compound 18 was prepared from 2-(furan-3-yl)acetic acid and 36 following the
procedure of Method C in 53% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.91-0.98
(m, 3H), 1.67-1.72 (m, 2H), 2.86-2.92 (m, 2H), 3.03 (t, J=2.52 Hz, 1H), 3.58-3.66 (m, 4H), 3.95-
4.04 (m, 2H), 4.59 (s, 2H), 6.53-6.62 (m, 1H), 7.05 (d, J=8.84 Hz, 1H), 7.12-7.18 (m, 1H), 7.32-
7.40 (m, 3H), 7.69-7.72 (m, 3H), 7.89-7.99 (m, 1H), 8.08 (t, J=5.92 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 164.2, 155.2, 150.7, 145.9, 143.4, 138.4, 129.3, 128.4, 128.2, 127.9, 127.3, 126.8,
124.0, 120.9, 113.4, 110.2, 79.3, 74.5, 69.6, 50.0, 47.4, 34.2, 31.9, 21.9, 10.4. HRMS (AP-ESI) m/z
calcd for C26H27ClN2O5S [M + Na]+ 537.1227, found 537.1219.
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carboxamide (19). Compound 19 was prepared from 3-thiophene carboxylic acid and 36 following
the procedure of Method C in 48% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.86-
0.99 (m, 3H), 1.63-1.74 (m, 2H), 2.91-3.02 (m, 3H), 3.59 (t, J=7.44 Hz, 2H), 3.65 (s, 2H), 3.91-
3.98 (m, 2H), 4.47-4.64 (m, 2H), 7.03-7.33 (m, 5H), 7.42 (s, 1H), 7.59-7.71 (m, 4H), 8.07 (s, 1H).
13C NMR (100 MHz, DMSO-d6): δ 166.5, 155.2, 144.0, 138.4, 136.4, 129.3, 128.1, 127.5, 126.9,
126.8, 126.6, 124.0, 113.4, 79.3, 74.5, 69.6, 49.8, 47.8, 34.1, 31.9, 21.9, 10.4. HRMS (AP-ESI) m/z
calcd for C26H27ClN2O4S2 [M + Na]+ 553.1000, found 553.1026.
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N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)thiazole-4-
carboxamide (20). Compound 20 was prepared from 4-thiazole carboxylic acid and 36 following
the procedure of Method C in 44% yield as a white solid. 1H-NMR (400 MHz DMSO-d6): δ 0.85-
1.02 (m, 3H), 1.58-1.79 (m, 2H), 2.93-3.04 (m, 3H), 3.58-3.67 (m, 3H), 3.81-3.90 (m, 2H), 4.01 (t,
J=6.32 Hz, 1H), 4.66-4.81 (m, 2H), 6.98-7.31 (m, 4H), 7.42 (d, J=7.92 Hz, 1H), 7.67 (d, J=7.92 Hz,
1H), 7.74 (d, J=7.92 Hz, 1H), 8.04-8.21 (m, 2H) ), 9.13-9.24 (m, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 163.6, 155.3, 153.9, 150.9, 144.1, 138.3, 129.2, 128.2, 127.9, 127.7, 127.6, 126.8,
124.9, 123.9, 113.3, 79.4, 74.6, 69.6, 49.7, 47.0, 34.6, 31.9, 22.0, 10.5. HRMS (AP-ESI) m/z calcd
for C25H26ClN3O4S2 [M + Na]+ 554.0951, found 554.0970.
N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)-1,2,5-
thiadiazole-3-carboxamide (21). Compound 21 was prepared from 3-1,2,5-thiadiazole carboxylic
acid and 36 following the procedure of Method C in 40% yield as a white solid. 1H-NMR (400
MHz DMSO-d6): δ 0.80-1.01 (m, 3H), 1.52-1.78 (m, 2H), 2.92-3.05 (m, 3H), 3.64-3.69 (m, 3H),
3.82-3.88 (m, 2H), 3.98-4.00 (m, 1H), 4.72-4.74 (m, 2H), 6.98-7.08 (m, 1H), 7.22-7.34 (m, 3H),
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7.43 (d, J=8.24 Hz, 1H), 7.66 (d, J=8.32 Hz, 1H), 7.74 (d, J=8.20 Hz, 1H), 8.06-8.09 (m, 1H) ),
8.82-9.10 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 161.6, 157.0, 155.4, 152.5, 143.8, 138.5,
129.3, 128.6, 128.2, 126.9, 126.7, 123.9, 113.5, 79.4, 74.5, 69.7, 49.3, 47.1, 34.2, 31.9, 21.9, 10.5.
HRMS (AP-ESI) m/z calcd for C24H25ClN4O4S2 [M + Na]+ 555.0904, found 555.0917.
N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)-1H-1,2,4-
triazole-3-carboxamide (22). Compound 22 was prepared from 3-1,2,4-triazole carboxylic acid
and 36 following the procedure of Method C in 36% yield as a white solid. 1H-NMR (400 MHz
DMSO-d6): δ 0.85-1.01 (m, 3H), 1.58-1.77 (m, 2H), 2.89-3.04 (m, 3H), 3.58-3.67 (m, 3H), 3.89 (t,
J=6.40 Hz, 1H), 3.97-4.17 (m, 2H), 4.65 (s, 2H), 6.98-7.06 (m, 1H), 7.21-7.41 (m, 4H), 7.69 (d,
J=7.92 Hz, 1H), 7.74 (d, J=8.28 Hz, 1H), 8.03-8.08 (m, 1H), 8.65-8.72 (m, 1H), 14.42-14.89 (m,
1H). 13C NMR (100 MHz, DMSO-d6): δ 162.3, 155.3, 152.7, 143.8, 143.4, 138.4, 129.2, 128.2,
128.1, 127.3, 127.1, 126.8, 123.9, 113.4, 79.3, 74.5, 69.7, 49.5, 46.6, 34.6, 31.9, 21.9, 10.5. HRMS
(AP-ESI) m/z calcd for C24H26ClN5O4S [M + Na]+ 538.1292, found 538.1315.
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23
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26
27
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35
36
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N-(5-chloro-2-propoxybenzyl)-N-(4-(N-(prop-2-yn-1-yl)sulfamoyl)phenethyl)pyrazine-2-
carboxamide (23). Compound 23 was prepared from 2-1,4-diazine carboxylic acid and 36
following the procedure of Method C in 38% yield as a white solid. 1H-NMR (400 MHz DMSO-
d6): δ 0.81-1.03 (m, 3H), 1.54-1.79 (m, 2H), 2.92-3.04 (m, 3H), 3.63-3.69 (m, 4H), 3.84-4.03 (m,
2H), 4.57-4.71 (m, 2H), 6.96-7.08 (m, 1H), 7.20-7.34 (m, 3H), 7.45 (d, J=8.24 Hz, 1H), 7.65 (d,
J=8.28 Hz, 1H), 7.75 (d, J=8.24 Hz, 1H), 8.08 (t, J=5.92 Hz, 1H), 8.52-8.82 (m, 3H). 13C NMR
(100 MHz, DMSO-d6): δ 166.5, 155.3, 149.4, 145.5, 144.4, 143.2, 142.8, 138.5, 129.3, 128.5,
128.1, 127.9, 127.1, 126.8, 126.7, 123.9, 113.5, 79.5, 74.6, 69.7, 49.5, 47.1, 34.1, 31.9, 22.0, 10.5.
HRMS (AP-ESI) m/z calcd for C26H27ClN4O4S [M + Na]+ 549.1339, found 549.1340.
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