Structural Insights of Benzenesulfonamide Analogues as NLRP3 Inflammasome Inhibitors: Design, Synthesis, and Biological Characterization

Article abstract of DOI:10.1021/acs.jmedchem.8b00733

NLRP3 inflammasome plays critical roles in a variety of human diseases and represents a promising drug target. In this study, we established the in vivo functional activities of JC124, a previously identified NLRP3 inflammasome inhibitor from our group, in mouse models of Alzheimer's disease and acute myocardial infarction. To understand the chemical space of this lead structure, a series of analogues were designed, synthesized, and biologically characterized. The results revealed the critical roles of the two substituents on the benzamide moiety of JC124. On the other hand, modifications on the sulfonamide moiety of JC124 are well tolerated. Two new lead compounds, 14 and 17, were identified with improved inhibitory potency (IC₅₀ values of 0.55 ± 0.091 and 0.42 ± 0.080 μM, respectively). Further characterization confirmed their selectivity and in vivo target engagement. Collectively, the results strongly encourage further development of more potent analogues based on this chemical scaffold.

Full text of DOI:10.1021/acs.jmedchem.8b00733

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Article
Structural insights of benzene sulfonamide analogs as NLRP3
inflammasome inhibitors: Design, synthesis, and biological characterization
Jacob W Fulp, Liu He, Stefano Toldo, Yuqi Jiang, Ashley Boice, Chunqing Guo, Xia
Li, Andrew Rolfe, Dong Sun, Antonio Abbate, Xiang-Yang Wang, and Shijun Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00733 • Publication Date (Web): 07 Jun 2018
Downloaded from http://pubs.acs.org on June 7, 2018
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Structural insights of benzene sulfonamide analogs as NLRP3 inflammasome inhibitors:
Design, synthesis, and biological characterization
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Jacob Fulpa, Liu Hea, Stefano Toldob, Yuqi Jianga, Ashley Boicea, Chunqing Guoc, Xia Lic, Andrew
Rolfd, Dong Sund, Antonio Abbateb, XiangꢀYang Wangc, Shijun Zhanga*
aDepartment of Medicinal Chemistry , bDepartment of Internal Medicine and Pauley Heart Center,
cDepartment of Human & Molecular Genetics, dDepartment of Anatomy and Neurobiology,
Virginia Commonwealth University, Richmond, Virginia, USA, 23298
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Abstract
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NLRP3 inflammasome plays critical roles in a variety of human diseases and represents a
promising drug target. In this study, we established the in vivo functional activities of JC124, a
previously identified NLRP3 inflammasome inhibitor from our group, in mouse models of
Alzheimer’s disease and acute myocardial infarction. To understand the chemical space of this lead
structure, a series of analogs were designed, synthesized, and biologically characterized. The results
revealed the critical roles of the two substituents on the benzamide moiety of JC124. On the other
hand, modifications on the sulfonamide moiety of JC124 are well tolerated. Two new lead
compounds, 14 and 17, were identified with improved inhibitory potency (IC50 of 0.55 ± 0.091 and
0.42 ± 0.080 µM, respectively). Further characterization confirmed their selectivity and in vivo
target engagement. Collectively, the results strongly encourage further development of more potent
analogs based on this chemical scaffold.
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Introduction
Inflammasomes are intracellular multiprotein complexes that tightly regulate the innate
immune response and the production of proꢀinflammatory cytokines such as interleukin (IL)ꢀ1β and
ILꢀ18.1 The inflammasomes share a similar structure and are typically formed by a sensor
component, an adaptor component (the apoptosisꢀassociated speckꢀlike protein containing a caspase
recruitment domainꢀꢀꢀASC), an effector component, typically proꢀcaspaseꢀ1, and the substrate
component (the proꢀinflammatory cytokines ILꢀ1β and ILꢀ18).2,3 The sensors recognize danger
signals such as Damage associated molecular pattern molecules (DAMPs) released during tissue
injury or stress (extracellular ATP, urate crystal, βꢀamyloid, cell debris, etc) and
Pathogen-associated molecular patterns (PAMPs), evolutionary conserved structures of the
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microbial invader. Upon assembly of the inflammasome complex, proꢀcaspaseꢀ1 will be cleaved
and activated to process proinflammatory cytokines ILꢀ1β and ILꢀ18 to their active forms, then
mediating a plethora of inflammatory responses and ultimately to one specific cell death known as
pyroptosis.3,4
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A number of inflammasome complexes have been identified and this includes the NODꢀlike
receptor (NLR) containing family such as NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4,
the absent in melanoma 2 (AIM2) inflammasome, and retinoic acidꢀinducible gene I (RIGꢀI) like
receptors (RLRs).3 Among these, the NLRP3 inflammasome, which is composed of the sensor
NLRP3, the adapter ASC, and proꢀcaspaseꢀ1, has been extensively studied and is critically involved
in the maturation of ILꢀ1β and ILꢀ18.1 Most recently, a new player, NEK7, has been added to the
NLRP3 inflammasome complex as an essential component to its activation.5ꢀ7 Emerging evidence
has suggested critical roles for the NLRP3 inflammasome and ILꢀ1β in the pathogenesis of many
human diseases, such as autoinflammatory disorders, diabetes, acute myocardial infarction (AMI),
traumatic brain injury (TBI), multiple sclerosis (MS), and Alzheimer’s disease (AD).8ꢀ20 The
pathological roles of the NLRP3 inflammasome are well illustrated by cryopyrinꢀassociated
periodic syndrome (CAPS), a group of inherited autoinflammatory diseases, caused by
gainꢀofꢀfunction mutations in the NLRP3 protein.9 Recently, studies have demonstrated that levels
of ILꢀ1β and expression of active caspaseꢀ1 were found to be elevated in AD mouse models and
AD patients.13, 17, 18, 21, 22 Notably, NLRP3ꢀ/ꢀ and Caspꢀ/ꢀ mice carrying mutations associated with
familial AD exhibited improved cognitive functions, thus clearly suggesting the essential roles of
the NLRP3 inflammasome axis in AD development.17 Most recently, ASC specks derived from
microglia has been shown to cross seed amyloidꢀβ (Aβ) in transgenic AD mice, thus support the
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role of NLRP3 inflammasome activation in driving the Aβ pathology of AD.20 NLRP3
inflammasome also plays critical roles in the inflammatory responses to myocardial injury during
AMI.23 In the early phases of AMI, the acute ischemic injury induces the expression of NLRP3
inflammasome components (priming), which concomitantly provides the stimuli leading to NLRP3
activation and formation of the macromolecular aggregate (trigger), leading to an active
inflammasome.23,24 Caspaseꢀ1 is detected in the heart starting 3 to 6 hours after ischemia and its
activity peaks between 24 and 72 hours, while low grade activation persists for weeks after the
initial insult.23,25 Reperfusion, while it effectively reduces infarct size, does not prevent activation of
the NLRP3 inflammasome and leads to further injury through caspaseꢀ1ꢀdependent inflammatory
cell death.26 To support this notion, studies by Abbate and others demonstrated that mice with
genetic deletion of NLRP3 or ASC exhibited smaller infarct size in experimental AMI model, and
reduced tendency toward adverse remodeling and heart failure, consistent with previously reported
central role of caspaseꢀ1 in AMI.25,27,28 Transgenic mice expressing constitutively active caspaseꢀ1,
on the other hand, developed adverse cardiac remodeling and heart failure.29 Collectively, these
studies strongly suggest that this pathway could be targeted for the treatment of a variety of
diseases. Indeed, several biological agents have been successfully developed and approved by FDA
by targeting this pathway as treatments for CAPS and this includes ILꢀ1 receptor antagonist
anakinra, ILꢀ1β antibody canakinumab, and decoys of ILꢀ1 receptor rilonacept.18, 30
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Although the pathogenic roles of the NLRP3 inflammasome in a variety of human disorders
are quickly emerging, the basis of NLRP3 inflammasome activation and its contribution to disease
progression remain not fully understood. It is therefore of importance to develop novel and specific
NLRP3 inflammasome inhibitors (NLRP3Is) as pharmacological tools, which will complement
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ongoing molecular and genetic studies to precisely define the role of NLRP3 inflammasome in the
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O
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H3CO
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Cl
H3CO
Glyburide
JC121
JC124
Cl
Figure 1. Chemical structures of small molecule inhibitors targeting the NLRP3
inflammasome pathway.
pathogenesis of related human diseases, and as potential therapeutics. To this end, several small
molecules have recently been reported to inhibit the NLRP3 inflammasome signaling pathway with
different or unknown mechanisms of action (MOA) (Figure 1).31 Among these inhibitors, glyburide
is an antiꢀdiabetic drug promoting insulin release and has shown inhibitory activity on NLRP3
inflammasome in myeloid cells in vitro.32 In contrast, glipizide, another sulfonylurea antiꢀdiabetic
agent, lacks this inhibitory effect on the NLRP3 inflammasome.11 This suggests that the observed
inhibitory effects on NLRP3 inflammasome by glyburide is independent from its actions on the
K
ATP channels which are involved in insulin release. Further studies suggested that the sulfonyl and
benzamide moieties within this structure are necessary for the observed inhibitory activity.32
Although the observed antiꢀinflammatory properties of glyburide suggest beneficial effects, further
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development of this compound is limited by the need of high doses that potentially induce lethal
hypoglycemia.
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Our laboratories have recently designed and developed a sulfonamide analog JC121 based on
the structure of glyburide (Figure 1).33ꢀ35 Biological characterization established that JC121 is a
selective NLRP3I and exhibits promising in vivo protective activities in mouse AMI models.
Furthermore, as summarized in Figure 2, our studies suggested that JC121 may directly interfere
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Figure 2. Proposed mechanism of action for the benzene sulfonamide analogs as NLRP3Is.
with the formation of the NLRP3 inflammasome complex,34 based on the facts that 1) it blocks
ASC aggregation; 2) it does not directly inhibit caspaseꢀ1 activation as NLRC4 and AIM2 pathways
are not affected by JC121; and 3) it blocks the release of ILꢀ1β and the activation of caspaseꢀ1 in
macrophages expressing constitutively active NLRP3 from mutant mice. Our ongoing studies also
demonstrated the direct binding interactions of our inhibitor with the NLRP3 protein (unpublished
data). Based on the structure of JC121, we further developed a methylated analog, JC124, to
balance the hydrophilicity and hydrophobicity (Figure 1). Our initial studies established that JC124
is an active and selective NLRP3I.36 Our studies also demonstrated the in vivo efficacy of this
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compound in reducing AD pathology in a transgenic mouse AD model.36 Herein, we report further
characterization of JC124 in a transgenic AD mouse model and a mouse AMI mode to confirm its
therapeutic effects. Furthermore, structure activity relationship (SAR) studies were conducted to
help understand and explore the chemical space of JC124, providing guidance for further structural
optimization/refinement of this chemical scaffold.
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Results and Discussion
Therapeutic effects of JC124 on cognitive functions in APP/PS1 mice. Our previous studies in
TgCRND8 mice, a widely used mouse AD model,37,38 demonstrated that treatment with JC124 (50
mg/kg) modified multiple AD pathologies, including betaꢀamyloid (Aβ) deposit, oxidative stress,
inflammatory responses, and synaptic degeneration.36 With the promising activity on AD
pathologies, we decided to examine whether such modifications by JC124 can lead
Figure 3. Effects of JC124 treatment on cognitive functions in APP/PS1 mice. At the age of 7
months old, animals were randomly divided into the following experimental groups: WT mice:
vehicle group (n=4), JC124 treatment group (n=6). Tg mice: vehicle group (n=5), JC124
treatment group (n=7). Starting at the age of 7 months old, animals were treated for 8 weeks (5
times/week). After completing treatment, animals were tested on cognitive functions using
NOR (A) and MWM (B and C) methods.
to improved cognitive functions. We then employed APP/PS1 mice, another widely used mouse
AD model,39 to test JC124 treatment with a battery of behavioral tests including Novel Object
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Recognition (NOR) and Morris Water Maze (MWM). In our previous studies, moderate
improvement on AD pathology was observed for JC124 at a dose of 50 mg/kg,36 we therefore
decided to use a higher dose, 100 mg/kg, in APP/PS1 mice for the behavioral studies. Treatment
started at the age of 7 months old and continued for 8 weeks by oral administration (5 times/week).
This protocol mimics the therapeutic settings since significant AD pathologies have developed by 7
months old in APP/PS1 mice.39 As shown in Figure 3A, compared to the wild type (WT) mice,
transgenic APP/PS1 (Tg) mice in both vehicle or JC124 treated groups showed a discrimination
index (DI) lower than 50%, which suggested cognitive deficits of Tg mice. JC124 treatment
showed a trend to improve cognitive function with higher DI in JC124ꢀtreated group compared to
the vehicleꢀtreated group. However, the difference did not reach a statistical significance. In MWM
fixedꢀplatform test, mice in all groups showed improved learning with time during the 4 days
latency trials, however, at the last day of trial, WT vehicle group had shorter latency to find goal
platform than the Tg groups, indicating better performance in WT vehicle group in this test (Figure
3B). Notably, in the MWM fixed platform probe trial test, Tg animals treated with JC124 spent
significantly longer time in the goal quadran than the vehicleꢀtreated Tg group, suggesting
improved memory function by JC124 treatment (Figure 3C). A noted, in the MWM probe trial test
of WT animals, JC124 treated mice spent shorter time in the goal quadran compared to the vehicle
treated group. Although the difference did not reach stastistical significance, the observed
difference may indicate some degree of immune supressive effects by JC124 treatemnt in normal
aniamls that could affect cognitive function. This speculation needs to be confirmed in future study
by examining the systemic immune responses. Collectively, under therapeutic trial settings, we
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found that JC124 exhibits beneficial effects on cognition as evidenced by the improved memory
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function (probe trial).
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Figure 4. Effects of JC124 in an experimental model of AMI. Mice (n=6 for each group)
were subjected to I/R for 30 min or 75 min. Then infarct size (A) was measured by TTC
staining and serum troponin I level (B) was determined by ELISA.
Therapeutic effects of JC124 in an experimental model of AMI. To further confirm the in vivo
efficacy of JC124, we examined its effects to protect cardiac functions in an experimental AMI
model induced by transient myocardial ischemia followed by reperfusion (I/R model), in which the
NLRP3 inflammasome has been indicated critical roles on postꢀischemia inflammatory responses
and heart failure.25,40 To explore the effective doses of JC124 and based on the preliminary
pharmacokinetic data of JC124 (unpublished data), we selected a lower dose of 30 mg/kg in this
experiment. Two different I/R models were used to evaluate the protective effects of JC124: 1)
coronary ligation was continued for 30 min; and 2) a more severe model in which coronary ligation
was continued for 75 min. As shown in Figure 4, treatment with JC124 significantly limited the
infarct size as measured with triphenyltetrazolium chloride (TTC) staining or troponin I levels in
both models when compared to the vehicle treatment group, thus further confirming the in vivo
efficacy of JC124 at this dose.
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Design and synthesis of JC124 analogs. With the confirmation of in vivo efficacy, we decided to
conduct SAR studies of JC124 to understand the chemical space around this lead structure. In
addition, the results of SAR studies may help shed light on whether this chemical scaffold functions
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SO2NHCH3
SO2NHCH3
O
NH
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NH
H3CO
H3CO
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Cl
Cl
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CF3
OH
NH
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Cl
H3CO
N
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NH
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Cl
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OCH3
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Figure 5. Chemical structures of the designed analogs of JC124.
by a single mechanism of action, thus providing valuable information on target interaction. The
design of JC124 congeners was mainly focused on the phenyl domain of the benzamide moiety and
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the sulfonamide domain to understand the influence of substituents on their biological activities
(Figure 5). Compounds 1ꢀ4 were designed to examine the importance of the 2ꢀCH3O and 5ꢀCl to
their biological activities using the deꢀconstruction strategy. Analogs 5ꢀ9 were designed to
investigate how the positional change of the CH3Oꢀ and Clꢀ substituents will influence their
inhibitory activities. The results of JC124 suggested that the sulfonamide moiety could be
optimized to improve biological activity. Therefore, analogs 10ꢀ21 were designed to understand the
space at this domain for further optimization process. To evaluate whether a cyclized version of
sulfonamide analogs will provide improved inhibitory activity, compounds 22ꢀ26 were designed.
We also designed analogs 27 and 28 to investigate how the linkage length between the two phenyl
rings will impact the biological activity.
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The
chemical syntheses of the designed analogs were successfully achieved by employing the
conditions detailed in Schemes 1 and 2. Briefly, condensation of 2ꢀphenylethylamine 29 with
phthalic anhydride gave 30, which was on sulfonation with chlorosufonic acid to give intermediate
1. Reaction of 31 with methylamine afforded intermediate 32. Refluxing of 32 with NH2NH2 in
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ethanol followed by coupling reactions with corresponding substituted benzoic acids under standard
peptide coupling conditions yielded analogs 1ꢀ9 (Scheme 1). Compounds 10ꢀ26 were synthesized
following the conditions listed in Scheme 2. Coupling of 29 with 5ꢀchloroꢀ2ꢀmethoxybenzoic acid
34 followed by sulfonation afforded sulfonyl chloride intermediate 36. Reaction of intermediate 36
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with various
amines afforded compounds 10ꢀ26. Similarly, compounds 27 and 28 were obtained by following
the same strategies with 3ꢀphenylpropylamine 37 or 4ꢀphenylbutylamine 38 as starting material,
respectively.
Evaluation of the inhibitory potency for the designed analogs on ILꢀ1β release in J774A.1
cells. Our previous studies have established a cellular model using mouse macrophage J774A.1
cells in which the release of ILꢀ1β is mediated through the activation of NLRP3 inflammasome
upon stimulation with lipopolysaccharide (LPS) and adenosine triphosphate (ATP).33 The level of
secreted ILꢀ1β was monitored by an enzymeꢀlinked immunosorbent assay (ELISA). As shown in
Table 1, lead compound JC124 showed an inhibitory potency of 3.25 ꢁM under the current
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experimental conditions. Removal of 2ꢀOCH3 or 5ꢀCl resulted in significant decrease of inhibitory
potency (> 4 fold) as demonstrated by analogs 1 and 2. The importance of the 2ꢀOCH3 and 5ꢀCl
was further demonstrated by analog 3 in which both substituents were removed with a complete
loss of inhibitory activity on ILꢀ1β release. When the 2ꢀOCH3 was deꢀmethylated to a 2ꢀOH moiety,
a reduced inhibitory potency (~3 fold) was observed in analog 4. The results of JC124 and 4
suggest that modification of the 4ꢀOH moiety to other alkoxyl groups at this position could lead to
improved inhibitory potency. Move of the 5ꢀCl to different positions on the phenyl ring all led to
significant decrease of inhibitory potency on ILꢀ1β release in J774A.1 cells as evidenced by analogs
5ꢀ7. Interestingly, positional change of the 2ꢀOCH3 to the metaꢀposition led to an analog 8 with
comparable potency to that of JC124, while change to the paraꢀposition, as demonstrated by 9, led
to significant loss of the inhibitory potency. This may indicate that the electronic effects by the
CH3O substituent is not an essential factor for the observed biological activity. Taken together, the
results of 5ꢀ9 strongly suggest the important roles of the 2,5ꢀdisubstitution on the observed
inhibition of ILꢀ1β release under the experimental conditions.
10
11
12
13
14
15
16
17
18
19
20
21
22
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26
27
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Table 1. Inhibitory potency of the designed analogs on the production of ILꢀ1β by J774A.1 cells
upon stimulation with LPS/ATP.
Compound
R1
R2
n=
1
IC50 (µM)
3.25 ± 1.34
13.36 ± 2.26
17.10 ± 4.32
JC124
1
1.
2.
1
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146.56 ± 26.31
10.48 ± 0.091
31.78 ± 3.19
3.
4.
5.
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17
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1
18.031 ± 0.27
6.
1
1
44.42 ± 6.32
5.83 ± 1.36
7.
8.
1
32.75 ± 3.78
9.
1
1
2.01 ± 0.43
1.64 ± 0.20
10.
11.
1
1
1
1.40 ± 0.62
0.97 ± 0.053
0.55 ± 0.091
12.
13.
14.
1
1
1
1
1
1
1
2.08 ± 0.59
1.31 ± 0.16
0.42 ± 0.080
0.63 ± 0.22
1.50 ± 0.78
1.90 ± 0.20
0.81 ± 0.12
15.
16.
17.
18.
19.
20.
21.
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1.66 ± 1.21
3.53 ± 1.68
5.01 ± 1.33
22.
23.
24.
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1
1
3.40 ± 0.62
3.44 ± 0.97
25.
26.
2
3
2.03 ± 0.61
1.74 ± 0.61
27.
28.
Structural modifications at the sulfonamide moiety, on the other hand, were well tolerated. As
shown in Table 1, with the increase of the size of the substituents on the sulfonamide N, the
inhibitory potency improves on ILꢀ1β release as reflected by analogs 10ꢀ14. Compound 14 with a
diꢀbutyl substitution on the sulfonamide N is the most potent one within this series of analogs with
an IC50 of 0.55 µM. With this observation, we then turned our attention to more bulky substituents
at this position. As shown by 15, aniline substitution on the sulfonamide moiety resulted in a
comparable inhibitory potency to that of JC124. Notably, when the benzene ring was replaced with
a benzyl moiety as in 16, the inhibitory potency was increased by 2.5 fold compared to that of
JC124. Substitution on the benzyl moiety with a 4ꢀOCH3 as illustrated by 17 further improved the
inhibitory potency to 0.42 µM, > 3 fold increase compared to that of 16. Analog 18 with a 4ꢀOH at
this position exhibited comparable potency as 17. This may suggest that an electronꢀdonating group
at this specific position favors the inhibitory activity on ILꢀ1β release under the LPS/ATP challenge
conditions in J774A.1 cells. This notion is further supported by the results of 19 and 20 as a 4ꢀCl or
4ꢀCF3 substitution, both electronꢀwithdrawing, led to a comparable or decreased inhibitory potency
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to that of 16. Further structural extension at this position by a phenylethyl moiety, as shown in 21,
resulted in slight improvement when compared to 16. Analogs with a heterocyclic moiety on the
sulfonamide moiety, as demonstrated by compounds 22ꢀ26, exhibited comparable inhibitory
potency as that of JC124. The results of the monoꢀ or disubstituted analogs suggested that bulky
substituents with rotational flexibility are preferred to providing analogs with improved inhibitory
potency on ILꢀ1β release. Structural extension slightly improved the inhibitory potency under the
current experimental conditions as evidenced by 27 and 28 with an IC50 of 2.03 and 1.74 µM,
respectively. This is in agreement with the results of compounds 17 and 21, thus suggesting that
hydrophobic interactions at this domain may be important for the observed inhibition on ILꢀ1β
release.
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Compounds 14 and 17 are selective NLRP3Is. After the establishment of inhibitory
potency on ILꢀ1β release in J774A.1 cells for the designed analogs, we selected 14 and 17, the two
most potent analogs, for further characterization in mouse bone marrow derived macrophages
(BMDMs) and in mice. As shown in Figures 6A, both 14 and 17 dose dependently suppressed the
release of ILꢀ1β from BMDMs upon LPS/ATP challenge with an IC50 of 0.12 ± 0.067 and 0.36 ±
0.043 µM, respectively. Analog 14 is ~4 times more potent in BMDMs than in J774A.1 cells to
inhibit the release
of ILꢀ1β, while the potency of 17 is comparable in these two cell populations. This may suggest that
BMDMs are more sensitive to 14 under the current experimental conditions, consistent with our
previous studies.41 We next examined the selectivity of 14 and 17 to NLRP3 inflammasome. To this
end, J774A.1 cells were stimulated with LPS/poly(dA:dT) or LPS/flagellin to activate the NLRC4
and AIM2 inflammasome, respectively. As shown in Figures 6B and 6C, treatment of J774A.1 cells
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with 14 or 17 under these experimental conditions did not significantly interfere with the production
of ILꢀ1β (by ANOVA oneꢀway analysis), thus confirming the relatively specific inhibition of
NLRP3 inflammasome by 14 and 17. Consistent with the reported results,36,42 no inhibitory activity
was observed for JC124 and MCC950, a recently reported and potent inhibitor that targets the
NLRP3 inflammasome pathway,42 under these conditions as well. The results from the selectivity
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Figure 6. Compounds 14 and 17 blocks NLRP3 inflammasome activation and ILꢀ1β production in
primary macrophages and LPSꢀchallenged mice. (A) BMDMs were primed with LPS (1 ꢁg/mL) for
4.5 h and then treated with indicated doses of 14 or 17 when adding ATP (5 mM) stimulation for 30
min. ILꢀβ in the culture media was assayed by ELISA. (B and C) J774A.1 cells were treated with
LPS (1 µg/mL) and test compounds (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
ILꢀ1β were measured by ELISA. (D) Serum levels of ILꢀ1β from C57BL/6 mice pretreated with 14
and 17 (0.5 mg/kg) or vehicle control were measured by ELISA 2.5 h after i.p. injection of LPS (20
mg/kg)
studies also serve as an indirect 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. This is consistent with our previously reported results.34 Finally, we tested
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the in vivo engagement of the NLRP3 inflammasome by 14 and 17. Mice were pretreated with 14 or
17 before intraperitoneal injection of LPS, which has been shown to trigger ILꢀ1β production in a
NLRP3ꢀdependent manner.43 MCC950 was tested as a positive control. As shown in Figure 6D,
serum levels of ILꢀ1β was sharply reduced by the treatment of all three compounds at the tested
dose, thus strongly suggesting the engagement of the NLRP3 inflammasome pathway in the
observed effects by 14 and 17. Although compounds 14 and 17 exhibit significantly improved
potency when compared to JC124, they are still less potent than MCC950. However, compounds
14 and 17 exhibited comparable activity to MCC950 in this in vivo LPSꢀchallenge experiment. This
could be caused by different pharmacokinetic properties of these analogs as well as the unique
MOA of our inhibitors. Further studies are warranted to investigate and confirm these speculations.
Conclusion
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In our efforts to develop small molecule inhibitors by targeting the NLRP3 inflammasome, a
sulfonamide based chemical scaffold was identied to provide analogs as active NLRP3Is. In this
study, we further confirmed the in vivo activity of JC124, a recently identified lead inhibitor from
our group, in a transgenic AD mouse model and a mouse AMI model. The results clearly provided
confidence for further develop of new analogs based on this chemcial scaffold. Further SAR studies
revealed the positions on the structure of JC124 for further optimization. Our results demonstrated
that the 2ꢀOCH3 and 5ꢀCl are essential to the inhibitory activity on the NLRP3 inflammasome as
positional change or removal of them led to significant decrease of inhibitory activity. On the other
hand, structural modifications on the Nꢀsubstituents of the sulfonamide moiety are tolerated and
bulky groups tend to provide analogs with improved potency. As a result of this SAR study, two
new lead compounds, 14 and 17, were identified with improved inhibitory potency. Further
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biological characterization in BMDMs and J774A.1 cells confirmed the inhibitory potency and
selectivity of these two lead compounds to the NLRP3 inflammasome. More importantly, studies in
LPSꢀchallenged mice, a mouse model in which the release of ILꢀ1β is NLRP3 inflammasome
dependent, confirmed that both 14 and 17 signifiantly suppressed the production of ILꢀ1β, thus
confirming the in vivo engagement of this inflammasome complex by these two compounds.
Collectively, the results strongly encourage further studies and development of new analogs based
on this chemical scaffold as NLRP3Is and explore their potential therapeutic applications.
Experimental Section
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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 thinꢀlayer 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 (200ꢀ300 mesh,
1
Fisher Scientific) using solvents as indicated. HNMR and 13CNMR spectra were routinely
recorded on Bruker ARX 400 spectrometer. The NMR solvent used was CDCl3 or DMSOꢀd6 as
indicated. Tetramethylsilane (TMS) was used as internal standard. The purity of target NLRPIs was
determined by HPLC using Varian 100ꢀ5 C18 250 x 4.6 mm column with UV detection (288 nm)
(50% acetonitrile/50% H2O/0.1 % trifluoroacetic acid (TFA) and 80% methanol/19.9% H2O/0.1%
TFA two solvent systems) to be ≥ 95%.
3ꢀchloroꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (1). Compound 1 was prepared
1
from 3ꢀChlorobenzoic acid (0.47 mmol) following Method C in 91% yield. H NMR (400 MHz,
DMSOꢀd6) δ 8.69 (t, J= 5.44 Hz, 1H), 7.83 (t, J = 1.76 Hz, 1H), 7.76 (dt, J = 7.80, 1.35 Hz, 1H),
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7.70 (d, J = 8.28 Hz, 2H), 7.61ꢀ 7.59 (m, 1H), 7.52 ꢀ 7.46 (m, 4H), 3.53 (q, J = 6.64 Hz, 2H), 2.95
5
6
7
13
(t, J = 7.16 Hz, 2H), 2.39 (d, J = 5.04 Hz, 3H); C NMR (100 MHz, DMSOꢀd6) δ 164.8, 144.3,
8
9
137.2, 136.5, 133.1, 130.9, 130.8, 129.4, 126.9, 126.7, 125.8, 40.4, 34.6, 28.6.
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2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (2) Compound 2 was prepared
from 2ꢀmethoxybenzoic acid (0.47 mmol) following Method C in 65% yield. 1H NMR (400 MHz,
DMSOꢀd6) δ 8.18 (t, J= 5.58 Hz, 1H), 7.70 ꢀ 7.74 (m, 3H), 7.50 (d, J = 8.38 Hz, 2H), 7.45 (dt, J =
2.01, 7.91 Hz 1H), 7.38 (q, J = 5.03 Hz, 1H), 7.11 (d, J = 8.20 Hz, 1H), 7.02 (dt, J = 7.57, 0.78 Hz,
1H), 3.81 (s, 3H), 3.57 (q, J = 6.60 Hz, 2H), 2.94 (t, J = 7.02 Hz, 2H), 2.41 (d, J = 5.04 Hz, 3H);
13C NMR (100 MHz, DMSOꢀd6) δ 164.9, 156.9 144.4, 137.2, 132.1, 130.3, 129.5, 126.7, 123.0,
120.0, 111.9, 55.8, 40.1, 34.7, 28.6.
Nꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (3) Compound 3 was prepared starting
from benzoic acid (0.47 mmol) following Method C in 56% yield. 1H NMR (400 MHz,
DMSOꢀd6) δ 8.61 (t, J = 5.46 Hz, 1H), 7.85 (d, J = 8.00 Hz, 2H), 7.75 (d, J = 8.00 Hz, 2H), 7.59 ꢀ
7.48 (m, 5H), 7.41 (q, J = 5.01 Hz, 1H), 3.59 (q, J = 6.68 Hz, 2H), 3.00 (t, J = 7.20 Hz, 2H), 2.44
13
(d, J = 5.04 Hz, 3H); C NMR (100 MHz, DMSOꢀd6) δ 164.2, 144.5, 137.1, 134.5, 131.0, 129.4,
128.2, 127.0, 126.7, 40.3, 34.8, 28.6.
5ꢀchloroꢀ2ꢀhydroxyꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (4). Compound 4
was prepared from 5ꢀchlorosalicylic acid (0.47 mmol) following Method C in 60% yield. 1H
NMR (400 MHz, DMSOꢀd6) δ 8.94 (t, J= 5.46 Hz, 1H), 7.71 (d, J = 8.28 Hz, 2H), 7.48 (d, J = 8.28
Hz, 2H), 7.42 (dd, J = 9.03, 2.69 Hz, 1H), 7.37 (q, J = 5.03 Hz, 1H), 6.93 (d, J = 8.84 Hz, 1H), 3.58
13
(q, J = 6.64 Hz, 2H), 2.96 (t, J = 7.14 Hz, 2H), 2.39 (d, J = 5.04 Hz, 3H); C NMR (100 MHz,
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DMSOꢀd6) δ 167.4 , 158.4 144.1, 137.2, 133.2, 129.4, 127.3, 126.8, 122.9, 119.3, 116.9, 40.1, 34.5,
28.6.
7
8
9
3ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (5). Compound 5
was prepared from 3ꢀchloroꢀ2ꢀmethoxybenzoic acid (0.27 mmol) following Method C in 40% yield.
1H NMR (400 MHz, DMSOꢀd6) δ 8.46 (t, J = 5.48 Hz, 1H), 7.77 (d, J = 8.24 Hz, 2H), 7.62 (dd, J =
8.16, 1.53 Hz, 1H), 7.56 (d, J = 8.24 Hz, 2H), 7.44 ꢀ 7.40 (m, 2H), 7.24 (t, J = 7.84 Hz, 1H), 3.73 (s,
3H), 3.61 (q, J = 6.59 Hz, 2H), 3.01 (t, J = 7.04 Hz, 2H), 2.46 (d, J = 5.04 Hz, 3H); 13C NMR (100
MHz, DMSOꢀd6) δ 165.1, 152.8, 144.3, 137.2, 132.1, 131.7, 129.4, 128.2, 127.1, 126.7, 125.0,
61.5, 34.5, 28.6.
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4ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ((methylamino)sulfinyl)phenethyl)benzamide (6). Compound 6
was prepared from 4ꢀchloroꢀ2ꢀmethoxybenzoic acid (0.54 mmol) following Method C in 60% yield.
1H NMR (400 MHz, DMSOꢀd6) δ 8.22 (t, J = 5.58 Hz, 1H), 7.77 (d, J = 8.33 Hz, 2H), 7.74 (d, J =
8.32 Hz, 1H), 7.53 (d, J = 8.32 Hz, 2H), 7.42 (q, J = 4.84 Hz, 1H), 7.25 (d, J = 1.88 Hz, 1H), 7.13
(dd, J = 8.34, 1.95 Hz, 1H), 3.88 (s, 3H), 3.60 (q, J = 6.95 Hz, 2H), 2.98 (t, J = 6.97 Hz, 2H), 2.45
13
(d, J = 4.88 Hz, 3H); C NMR (100 MHz, DMSOꢀd6) δ 164.0, 157.6, 144.4, 137.2, 136.3, 131.7,
129.4, 126.7, 122.1, 120.5, 112.5, 56.3, 40.1, 34.7 28.6.
2ꢀchloroꢀ6ꢀmethoxyꢀNꢀ(4ꢀ((methylamino)sulfinyl)phenethyl)benzamide (7). Compound 7
was prepared from 2ꢀChloroꢀ6ꢀmethoxybenzoic acid (0.54 mmol) following Method C in 62%
yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.49 (t, J = 5.05 Hz, 1H), 7.70 (d, J = 8.32 Hz, 2H), 7.50
(d, J = 8.32 Hz, 2H), 7.39 ꢀ 7.31 (m, 2H), 7.04 ꢀ 7.01 (m, 2H), 3.75 (s, 3H), 3.49 (q, J = 7.01 Hz,
2H), 2.91 (t, J = 7.01 Hz, 2H) 2.41 (d, J = 5.04 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.9,
156.9, 144.3, 137.1, 130.4, 129.5, 127.2, 126.6, 120.9, 110.3, 56.0, 34.5, 28.6.
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3ꢀchloroꢀ5ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (8).
Compound
5
6
7
8
9
8 was prepared from 3ꢀchloroꢀ5ꢀmethoxybenzoic acid (0.36 mmol) following Method C in 70%
yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.66 (t, J = 5.48 Hz, 1H), 7.70 (d, J = 8.33 Hz, 2H), 7.46
(d, J = 8.33 Hz, 2H), 7.42 ꢀ 7.41 (m, 1H), 7.36 (q, J = 5.03 Hz, 1H), 7.34 ꢀ 7.32 (m, 1H), 7.19 ꢀ 7.18
(m, 1H), 3.82 (s, 3H), 3.53 (q, J = 6.64 Hz, 2H), 2.94 (t, J = 7.14 Hz, 2H), 2.40 (d, J = 5.04 Hz,
3H); 13C NMR (100 MHz, DMSOꢀd6) δ 164.6, 160.1, 144.3, 137.3, 137.2, 133.8, 129.4, 126.7,
119.1, 111.9, 55.81, 40.4, 34.6, 28.6.
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3ꢀchloroꢀ4ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenethyl)benzamide (9).
Compound 9
was prepared from 3ꢀchloroꢀ4ꢀmethoxybenzoic acid (0.36 mmol) following Method C in 70% yield.
1H NMR (400 MHz, DMSOꢀd6) δ 8.59 (t, J = 5.46 Hz, 1H), 7.94 (d, J = 2.24 Hz, 1H), 7.85 (dd, J =
2.24, 8.71 Hz, 1H), 7.75 (d, J = 8.29 Hz, 2H), 7.51 (d, J = 8.28 Hz, 2H), 7.40 (q, J = 5.06 Hz, 1H),
7.27 (d, J = 8.72 Hz, 1H), 3.96 (s, 3H), 3.56 (q, J = 7.06 Hz, 2H), 2.98 (t, J = 7.06 Hz, 2H), 2.44 (d,
J = 5.06 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ 164.5, 156.7, 144.4, 137.2, 129.4, 128.7,
127.7, 127.5, 126.7, 120.8, 112.3, 56.4, 40.3, 34.8, 28.6.
5ꢀchloroꢀNꢀ(4ꢀ(N,Nꢀdiethylsulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide (10). Compound 10
was prepared from 36 (0.39 mmol) and diethylamine (1.95 mmol) following Method B in 87%
yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.26 (t, J = 5.77 Hz, 1H), 7.73 (d, J = 8.38 Hz, 1H), 7.60
(d, J = 2.70 Hz, 1H), 7.51 ꢀ 7.46 (m, 3H), 7.16 (d, J = 9.54 Hz, 1H), 3.81 (s, 3H), 3.57 (q, J = 6.93
Hz, 2H), 3.96 (q, J = 7.60 Hz, 4H), 2.95 (t, J = 7.60 Hz, 2H), 1.04 (t, J = 7.25 Hz, 6H); 13C NMR
(100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.5, 137.7, 137.4, 129.6, 129.5, 129.7, 124.8, 124.3, 114.2,
56.2, 41.7, 40.1, 34.6, 14.0.
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀpropylsulfamoyl)phenethyl)benzamide (11). Compound 11
was prepared from 36 (0.39 mmol) and propylamine (1.95 mmol) following method B in 61%
yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.24 (t, J = 3.50 Hz, 1H), 7.72 (d, J = 8.20 Hz, 2H), 7.61
(d, J = 2.08 Hz, 1H), 7.51 ꢀ 7.46 (m, 3H), 7.15 (d, J = 8.88 Hz, 1H), 3.81 (s, 3H), 3.55 (q, J = 6.60
Hz, 2H), 2.92 (t, J = 7.02 Hz, 2H), 2.67 (q, J = 6.68 Hz, 2H), 1.36 (sex, J = 7.16 Hz, 2H), 0.77 (t, J
= 7.38 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.1, 138.6, 131.5, 129.6, 129.4,
126.5, 124.9, 124.3, 114.2, 56.2, 44.3, 40.1, 34.6, 22.3, 11.1.
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5ꢀchloroꢀNꢀ(4ꢀ(N,Nꢀdipropylsulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide (12). Compound
12 was prepared from 36 (0.39 mmol) and dipropylamine (1.95 mmol) following following Method
B in 75% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.24 (t, J = 5.50 Hz, 1H), 7.72 ( d, J = 8.28 Hz,
2H), 7.61 (d, J = 2.80 Hz, 1H), 7.51 ꢀ 7.47 (m, 3H), 7.15 (d, J = 8.88 Hz, 1H), 3.83 (s, 3H), 3.56
(q, J = 6.58 Hz, 2H), 3.00 (t, J = 7.54 Hz, 4H), 2.94 (t, J = 6.96 Hz, 2H), 1.46 (sex, J = 7.47 Hz,
4H), 0.80 (t, J = 7.38 Hz, 6H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.5, 137.4, 131.4,
129.6, 129.5, 126.8, 124.8, 124.3, 114.2, 56.3, 49.7, 40.1, 34.6, 21.6, 10.9.
Nꢀ(4ꢀ(Nꢀbutylsulfamoyl)phenethyl)ꢀ5ꢀchloroꢀ2ꢀmethoxybenzamide (13). Compound 13 was
prepared from 36 (0.39 mmol) and butylamine (1.95 mmol) following following method B in 56%
yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.25 (t, J = 5.58 Hz, 1H), 7.73 (d, J = 8.32 Hz, 2H), 7.62
(d, J = 2.80 Hz, 1H), 7.51 ꢀ 7.47 (m, 3H), 7.15 (d, J = 8.92 Hz, 1H), 3.82 (s, 3H), 3.55 (q, J = 6.60
Hz, 2H), 2.93 (t, J = 7.00 Hz, 2H), 2.71 (q, J = 6.60 Hz, 2H), 1.37 ꢀ 1.17 (m, 4H), 0.78 (t, J = 7.28
Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.1, 138.5, 131.4, 129.5, 129.4, 126.5,
124.9, 124.3, 114.2, 56.2, 42.2, 40.2, 34.6, 31.0, 19.4, 13.4.
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5ꢀchloroꢀNꢀ(4ꢀ(N,Nꢀdibutylsulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide (14). Compound
14 was prepared from compound 36 (0.39 mmol) and dibutylamine (1.95 mmol) following method
B in 86% yield. (400 MHz, DMSOꢀd6) δ 8.26 (t, J = 5.42 Hz, 1H), 7.71 (d, J = 8.32 Hz, 2H), 7.63
(d, J = 2.80 Hz, 1H), 7.51 ꢀ 7.47 (m, 3H), 7.15 (d, J = 8.92 Hz, 1H), 3.82 (s, 3H), 3.54 (q, J = 6.53
Hz, 2H), 3.02 (t, J = 7.52 Hz, 4H), 2.93 (t, J = 7.00 Hz, 2H), 1.41 (qun, J = 7.52 Hz, 4H), 1.22 (sex,
J = 7.40 Hz, 4H), .837 (t, J = 7.34 Hz, 6H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7,
144.1, 138.5, 131.4, 129.5, 129.4, 126.5, 124.9, 124.3, 114.2, 56.2, 42.2, 40.2, 34.6, 31.0, 19.2,
13.5.
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀphenylsulfamoyl)phenethyl)benzamide (15). Compound 15
was prepared from compound 36 (0.26 mmol) and aniline (1.30 mmol) following method B in 80%
1
yield. Purity was confirmed by HPLC: H NMR (400 MHz, DMSOꢀd6) δ 8.25 (t, J = 5.56 Hz,
1H), 7.70 (d, J = 8.36 Hz, 2H), 7.61 (d, J = 2.80 Hz, 1H), 7.49 (dd, J = 8.88, 2.89 Hz, 1H), 7.42 (d,
J = 8.36 Hz, 2H), 7.19 (d, J = 2.78 Hz, 2H), 7.13 (d, J = 8.92 Hz, 1H), 7.09 (d, J = 4.34 Hz, 2H),
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7.00 (t, J = 7.64 Hz, 1H), 3.73 (s, 3H), 3.50 (q, J = 6.56 Hz, 2H), 2.87 (t, J = 7.02 Hz, 3H); C
NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.6, 144.8, 137.7, 137.5, 131.4, 129.5, 129.4, 129.1, 126.7,
124.8, 124.3, 123.9, 119.9, 114.1, 56.1, 34.5, 30.6.
Nꢀ(4ꢀ(Nꢀbenzylsulfamoyl)phenethyl)ꢀ5ꢀchloroꢀ2ꢀmethoxybenzamide (16). Compound 16
was prepared from compound 36 (1.28 mmol) and benzylamine (6.40 mmol) following method B in
72% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (t, J = 5.56 Hz, 1H), 8.09 (t, J = 5.90 Hz, 1H),
7.76 (d, J = 8.32 Hz, 2H), 7.65 (d, J = 2.08 Hz, 1H) 7.50 (dd, J = 9.05, 3.15 Hz, 1H), 7.47 (d, J =
8.32 Hz, 2H), 7.29 ꢀ 7.19 (m, 3H), 7.16 (d, J = 8.92 Hz, 1H), 3.97 (d, J = 5.60 Hz, 2H), 3.83 (s,
3H), 3.56 (q, J = 6.62 Hz, 2H), 2.93 (t, J = 7.06 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6,
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155.7, 144.2, 138.7, 137.6, 131.5, 129.5, 129.4, 128.2, 127.5, 127.1, 126.6, 124.9, 124.4, 114.2,
56.3, 46.1, 40.2, 34.6.
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀ(4ꢀmethoxybenzyl)sulfamoyl)phenethyl)benzamide
(17).
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Compound 17 was prepared from compound 36 (0.26 mmol) and 4ꢀmethoxybenzylamine (1.30
mmol) following method B in 69% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.28 (t, J = 5.46 Hz,
1H), 8.00 (t, J = 6.22 Hz, 1H), 7.75 (d, J = 8.12 Hz, 2H), 7.64 (d, J = 2.68 Hz, 1H), 7.51 (dd, J =
8.99, 2.39 Hz, 1H), 7.46 (d, J = 8.20 Hz, 2H), 7.17 (d, J = 8.92 Hz, 1H), 7.13 (d, J = 8.56 Hz, 2H),
6.83 (d, J = 8.60 Hz, 2H), 3.90 (d, J = 6.20 Hz, 2H), 3.83 (s, 3H), 3.72 (s, 3H), 3.56 (q, J = 6.55 Hz,
2H), 2.94 (t, J = 7.04 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 158.4, 155.7, 144.2, 138.7,
131.5, 129.5, 129.4, 128.9, 126.6, 126.7, 124.9, 124.3, 114.2, 113.6, 56.3, 55.03, 45.7, 40.2, 34.7.
5ꢀchloroꢀNꢀ(4ꢀ(Nꢀ(4ꢀhydroxybenzyl)sulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide
(18).
Compound 18 was prepared from compound 36 (0.39 mmol) and 4ꢀ(aminomethyl)phenol (1.95
mmol) following method B in 51% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 9.30 (br. s., 1H), 8.28
(t, J = 5.46 Hz, 1H), 7.92 (t, J = 6.20 Hz, 1H), 7.74 (d, J = 8.20 Hz, 2H), 7.64 (d, J = 2.80 Hz, 1H),
7.50 (dd, J = 8.96, 2.89 Hz, 1H), 7.46 (d, J = 8.24 Hz, 2H), 7.16 (d, J = 8.92 Hz, 1H), 7.00 (d, J =
8.04 Hz, 2H), 6.65 (d, J = 8.44 Hz, 2H), 3.83 ꢀ 3.82 (m, 5H), 3.55 (q, J = 6.60 Hz, 2H), 2.93 (t, J =
13
7.02 Hz, 2H); C NMR (100 MHz, DMSOꢀd6) δ 163.6, 156.5, 155.7, 144.2, 138.7, 131.5, 129.5,
129.4, 128.4, 127.6, 124.9, 124.3, 114.9 114.2, 56.4, 56.3, 45.8 40.3, 34.7.
5ꢀchloroꢀNꢀ(4ꢀ(Nꢀ(4ꢀchlorobenzyl)sulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide
(19).
Compound 19 was prepared from compound 36 (0.39 mmol) and 4ꢀchlorobenzylamine (1.95 mmol)
following method B in 45% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.28 (t, J = 5.58 Hz, 1H),
8.14 (t, J = 6.26 Hz, 1H), 7.74 (d, J = 8.32 Hz, 2H), 7.64 (d, J = 2.80 Hz, 1H), 7.50 (dd, J = 8.79,
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2.77 Hz, 1H), 7.46 (d, J = 8.28 Hz, 2H), 7.32 (d, J = 8.56 Hz, 2H), 7.24 (d, J = 8.56 Hz, 2H), 7.16
(d, J = 8.92 Hz, 1H), 3.97 (d, J = 6.12 Hz, 2H), 3.82 (s, 3H), 3.55 (q, J = 6.63 Hz, 2H), 2.93 (t, J =
7.08 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.3, 138.6, 136.8, 131.7, 131.5,
129.6, 129.5, 129.4, 128.1, 126.5, 124.9, 124.3, 114.2, 56.2, 45.3 40.2, 34.7.
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5ꢀchloroꢀNꢀ(4ꢀ(Nꢀ(4ꢀ(trifluoromethyl)benzyl)sulfamoyl)phenethyl)ꢀ2ꢀmethoxybenzamide
(20). Compound 20 was prepared from compound 36 (0.39 mmol) and 4ꢀ(trifluoromethyl)benzyl-
amine (1.95 mmol) following method B in 48% yield. 1H NMR (400 MHz, DMSOꢀd6) δ
8.23ꢀ8.27 (m, 2H), 7.74 (d, J = 7.88 Hz, 2H), 7.62ꢀ7.64 (m, 3H), 7.51 (d, J = 8.88 Hz, 1H),
7.44ꢀ7.46 (m, 4H), 7.17 (d, J = 8.84 Hz, 1H), 4.09 (d, J = 5.92 Hz, 2H), 3.82 (s, 3H), 3.56 (q, J =
6.24 Hz, 2H), 2.93 (t, J = 6.80 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.3,
142.6, 138.5, 131.4, 129.4, 128.1, 126.5, 125.0, 124.9, 124.8, 124.3, 114.1, 56.2, 45.5, 40.2, 34.6.
5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(Nꢀphenethylsulfamoyl)phenethyl)benzamide (21). Compound
21 was prepared from compound 36 (0.32 mmol) and phenylethylamine (1.60 mmol) following
method B in 69% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.25 (t, J = 5.58 Hz, 1H), 7.74 (d, J =
8.26 Hz, 2H), 7.66 (t, J = 5.78 Hz, 1H), 7.63 (d, J = 2.76, Hz, 1H), 7.51 ꢀ 7.46 (m, 3H), 7.25 (d, J =
7.52 Hz, 2H), 7.20 (d, J = 7.24 Hz, 1H), 7.14 (d, J = 8.44 Hz, 2H), 3.80 (s, 3H), 3.55 (q, J = 6.58
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Hz 2H), 2.98 ꢀ 2.91 (m, 4H), 2.68 (t, J = 7.50 Hz, 2H); C NMR (100 MHz, DMSOꢀd6) δ 163.6,
155.7, 144.3, 138.7, 138.3, 131.4, 129.5, 128.6, 128.3, 126.6, 126.2, 124.9, 124.3, 114.1, 56.2, 40.2,
44.0, 35.3, 34.6, 18.6.
Nꢀ(4ꢀ(azetidinꢀ1ꢀylsulfonyl)phenethyl)ꢀ5ꢀchloroꢀ2ꢀmethoxybenzamide (22) Compound 22
was prepared from compound 36 (0.39 mmol) azetidine hydrochloride (1.95 mmol) following
method B in 64% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.29 (t, J = 5.52 Hz, 1H), 7.74 (d, J =
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8.24 Hz, 2H), 7.60 ꢀ 7.56 (m, 3H), 7.50 (dd, J = 8.92, 2.80 Hz, 1H), 7.15 (d, J = 8.88 Hz, 1H), 3.83
(s, 3H), 3.64 (t, J = 7.66 Hz, 4H), 3.58 (q, J = 6.63 Hz, 2H), 2.98 (t, J = 7.00 Hz, 2H) 1.99 ꢀ 1.92
(m, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 145.5, 131.6, 131.4, 129.8, 129.4,
128.2, 124.9, 124.3, 114.2, 56.2, 50.7, 40.1, 34.8, 14.7
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(pyrrolidinꢀ1ꢀylsulfonyl)phenethyl)benzamide (23). Compound
23 was prepared from compound 36 (0.26 mmol) and pyrrolidine (1.30 mmol) following method B
in 73% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.25 (t, J = 5.68 Hz, 1H), 7.73 (d, J = 8.28 Hz,
2H), 7.59 (q, J = 2.80 Hz, 1H), 7.52 ꢀ 7.48 (m, 3H), 7.15 (d, J = 8.88 Hz, 1H), 3.82 (s, 3H), 3.56 (q,
J = 6.60 Hz, 2H), 3.12 (t, J = 6.74 Hz, 4H), 2.94 (t, J = 6.98 Hz, 2H), 1.62 ꢀ 1.54 (m, 4H); 13C NMR
(100 MHz, DMSOꢀd6) δ 163.6, 155.7, 144.9, 134.2, 131.4, 129.6, 129.4, 127.3, 124.9, 124.3, 114.2,
56.2, 47.7, 40.1, 34.7, 24.6.
5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(piperidinꢀ1ꢀylsulfonyl)phenethyl)benzamide (24). Compound
24 was prepared from compound 36 (0.26 mmol) and piperidine (1.30 mmol) following method B
in 70% yield. 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 2.76 Hz, 1H), 7.78 (br. s., 1H), 7.71 (d, J
= 8.28 Hz, 2H), 7.40 (d, J = 8.30 Hz, 1H), 7.38 (dd, J = 2.80, 8.80 Hz, 2H), 6.88 (d, J = 8.78 Hz,
1H), 3.79 (s, 3H), 3.76 (q, J = 6.78 Hz, 2H), 2.99 (t, J = 5.50 Hz, 2H), 3.01 (t, J = 6.80 Hz, 4H),
1.64 (quin, J = 5.71 Hz, 4H), 1.42 (quin, J = 5.96 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ
163.6, 155.6, 145.0, 133.3, 131.4, 129.6, 129.4, 127.4, 124.9, 124.3, 114.1, 56.2, 46.5, 40.1, 34.7,
24.6, 22.8.
5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(piperazinꢀ1ꢀylsulfonyl)phenethyl)benzamide (25). Compound 25
was prepared from compound 36 (0.39 mmol) and piperazine (1.95 mmol) following method B in
60% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.29 (t, J = 5.52 Hz, 1H), 7.66 (d, J = 8.24 Hz, 2H),
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7.62 (d, J = 2.76 Hz, 1H), 7.54 ꢀ 7.49 (m, 3H), 7.16 (d, J = 8.92 Hz, 1H), 3.83 (s, 3H), 3.55 (q, J =
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7.06 Hz, 2H), 2.95 (t, J = 7.06 Hz, 2H), 2.77 ꢀ 2.70 (m, 8H); C NMR (100 MHz, DMSOꢀd6) δ
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34.7.
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5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(morpholinosulfonyl)phenethyl)benzamide (26).
Compound
26 was prepared from compound 36 (0.39 mmol) and morpholine (1.95 mmol) following method B
in 91% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (t, J = 5.56 Hz, 1H), 7.68 (d, J = 8.24 Hz,
2H), 7.60 (d, J = 2.80 Hz, 1H), 7.55 (d, J = 8.24 Hz, 2H), 7.49 (dd, J = 9.01, 2.83 Hz, 1H), 7.15 (d,
J = 8.92 Hz, 1H), 3.82 (s, 3H), 3.62 (t, J = 4.66 Hz, 4H), 3.57 (q, J = 6.56 Hz, 2H), 2.96 (t, J =
13
6.98 Hz, 2H), 2.84 (t, J = 4.62 Hz, 4H); C NMR (100 MHz, DMSOꢀd6) δ 163.6, 155.7, 145.5,
132.3, 131.4, 129.7, 129.4, 127.7, 124.9, 124.3, 114.1, 65.2, 56.2, 45.9, 40.1, 34.7.
5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(3ꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenyl)propyl)benzamide
(27).
Compound 27 was prepared from 41 (0.37 mmol) and methylamine HCl (0.19 mmol) following
Method B in 72% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.29 (t, J = 5.30 Hz, 1H), 7.70 (d, J =
8.28 Hz, 2H), 7.64 (d, J = 2.76 Hz, 1H), 7.51 (dd, J = 9.91, 2.91 Hz, 1H), 7.47 (d, J = 8.24 Hz, 2H),
7.37 (q, J = 5.01 Hz, 1H), 7.17 (d, J = 8.88 Hz, 1H), 3.88 (s, 3H), 3.28 (q, J = 6.18 Hz, 2H), 2.73 (t,
13
J = 7.66 Hz, 2H), 2.40 (t, J = 5.04 Hz, 3H), 1.85 (quin, J = 7.32 Hz, 2H); C NMR (100 MHz,
DMSOꢀd6) δ 163.8, 155.6, 146.7, 136.8, 131.2, 129.3, 129.0 125.7, 125.5, 124.2, 114.1, 56.3, 38.7,
32.3, 30.3 28.6.
5ꢀchloroꢀ2ꢀmethoxyꢀNꢀ(4ꢀ(4ꢀ(Nꢀmethylsulfamoyl)phenyl)butyl)benzamide (28). Compound
28 was prepared from 42 (0.37 mmol) and methylamine HCl (0.19 mmol) following Method B in
84% yield. 1H NMR (400 MHz, DMSOꢀd6) δ 8.24 (t, J = 5.54 Hz, 1H), 7.70 (d, J = 8.24 Hz, 2H),
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5.05 Hz, 1H), 7.16 (d, J = 8.88 Hz, 1H), 3.85 (s, 3H), 3.29 (q, J = 6.67 Hz, 2H), 2.71 (t, J = 7.50
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Hz, 2H), 2.40 (d, J = 5.04 Hz, 3H), 1.65 (quin, J =8.15 Hz, 2H), 1.54 (quin, J =7.06 Hz, 2H); C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (100 MHz, DMSOꢀd6) δ 163.7, 155.6, 147.2, 136.7, 131.2, 129.3, 129.0, 126.7, 125.5, 124.3,
114.1, 56.3, 38.8, 34.5, 28.7, 28.6, 27.9.
2ꢀphenethylisoindolineꢀ1,3ꢀdione (30). Phthalic anhydride (103.20 mmol) and
2ꢀphenylethylamine 29 (82.50 mmol) were added to acetic acid (30 mL), and refluxed for 3 h. The
solution was cooled to room temperature and was poured into iceꢀwater. The precipitate was
1
filtered and purified by recrystallization in ethanol to give 30 as a white solid, (17.25 g, 33%). H
NMR (400 MHz, DMSOꢀd6) δ 7.84 ꢀ 7.81 (m, 4H), 7.27 ꢀ 7.16 (m, 5H), 3.82 (t, J = 7.36 Hz, 2H),
2.93 (t, J = 7.34 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 167.6, 138.2, 134.4, 131.5, 128.6,
128.4 126.4, 122.3, 38.8, 33.6
Method A. 4ꢀ(2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)ethyl)benzenesulfonyl chloride (31). To the
solution of 30 (59.74 mmol) in dichloromethane (DCM) was added chlorosulfonic acid (15 mL) at
ꢀ20 ºC. Then the solution was heated to 60 ºC for 1 h. The solution was cooled to room temperature
and poured onto crushed ice. The product was extracted into DCM, concentrated, and purified by
column chromatography to give 31 as a white solid (17.05 g, 82%). 1H NMR (400 MHz, DMSOꢀd6)
δ 7.83 ꢀ 7.82 (m, 4H), 7.49 (d, J = 7.55 Hz, 2H), 7.16 (d, J = 7.56 Hz, 2H), 3.81 (t, J = 7.16 Hz,
2H), 2.92 (t, J = 7.16 Hz, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 167.6, 146.1, 138.7, 134.4, 131.4,
127.9, 125.6, 123.0, 38.6, 33.4.
Method B. 4ꢀ(2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)ethyl)ꢀNꢀmethylbenzenesulfonamide (32). To the
solution of compound 31 (10.40 mmol) in DCM (200 mL) was added methylamine (52.00 mmol)
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