Design, synthesis, and screening of sulfonylurea-derived NLRP3 inflammasome inhibitors

Article abstract of DOI:10.1007/s00044-019-02466-7

Inflammasomes are multiprotein assemblies that produce robust inflammatory responses upon stimulation with pathogen- and/or danger-associated molecular patterns. Uncontrolled inflammasome activation has been linked to the pathophysiology of a wide array of disorders including life-threatening pathogenic infections, e.g., Francisella tularensis. There has been a great deal of interest in the development of small molecule inflammasome inhibitors. Using computational modeling based on chalcone derivatives, we have developed novel tertiary sulfonylurea compounds as inhibitors of the NLRP3 inflammasome. The polar enone functional alert of chalcone was replaced with a sulfonylurea scaffold while maintaining the relative positions of the two aromatic rings. These compounds were evaluated for their ability to inhibit NLRP3 and AIM2 inflammasome activation triggered by Francisella tularensis infection.

Full text of DOI:10.1007/s00044-019-02466-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02466-7
ORIGINAL RESEARCH
Design, synthesis, and screening of sulfonylurea-derived NLRP3
inflammasome inhibitors
Amol A. Kulkarni1 Ayyiliath M. Sajith1 Trevor T. Duarte2 Anahis Tena2 Charles T. Spencer2 J. Phillip Bowen3
●
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Received: 19 July 2019 / Accepted: 29 October 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Inflammasomes are multiprotein assemblies that produce robust inflammatory responses upon stimulation with pathogen-
and/or danger-associated molecular patterns. Uncontrolled inflammasome activation has been linked to the pathophysiology
of a wide array of disorders including life-threatening pathogenic infections, e.g., Francisella tularensis. There has been a
great deal of interest in the development of small molecule inflammasome inhibitors. Using computational modeling based
on chalcone derivatives, we have developed novel tertiary sulfonylurea compounds as inhibitors of the NLRP3
inflammasome. The polar enone functional alert of chalcone was replaced with a sulfonylurea scaffold while maintaining the
relative positions of the two aromatic rings. These compounds were evaluated for their ability to inhibit NLRP3 and AIM2
inflammasome activation triggered by Francisella tularensis infection.
Introduction
associated molecular patterns (PAMPs) (Petrilli et al.
2007). Of particular interest is sensing of DAMPs and
PAMPs through the nucleotide binding domain leucine-rich
repeat, pyrin-containing 3 (NLRP3) inflammasome. Aber-
rant activation of the NLRP3 inflammasome is shown to
play a crucial role in the pathogenesis of neurodegenerative
diseases (e.g., Alzheimer’s disease, type-2 diabetes, obesity,
multiple sclerosis) and life-threatening pathogenic infec-
tions, including the Gram-negative bacterium Francisella
tularensis, causative agent of human tularemia (Atianand
et al. 2011).
Sensing of DAMPs or PAMPs results in the formation of
the multimeric inflammasome complex and autoproteolysis
of procaspase 1. Active caspase 1 cleaves interleukin 1β
(IL-1β) and interleukin-18 (IL-18) from their precursor
forms to the active form (Thornberry et al. ; Martinon et al.
2002; Agostini et al. 2004). The release of active IL-1β and
IL-18 subsequently trigger the expression of other inflam-
matory cytokines, such as IL-6, TNF-α, IL-12, IFN-γ, etc
(Henry and Monack 2007; Mariathasan et al. 2005; Weiss
et al. 2007). This response, however, must be tightly con-
trolled as its dysregulation can result in the pathological
cytokine storm (Tisoncik et al. 2012). In the case of
F. tularensis infection, excessive pro-inflammatory cyto-
kine production has been shown to progress into edema,
hemorrhage, hypovolemic shock, acute respiratory distress
syndrome, and, if left untreated, death (Mares et al. 2008;
Sharma et al. 2011). Mice deficient in inflammasome
Inflammasomes are oligomeric multiprotein complexes that
function as pattern recognition receptors to trigger the
initiation of the inflammasome cascade. The core subunit is
typically comprised of procaspase 1 (the ultimate effector
protein that initiates inflammation) and apoptosis-associated
speck-like protein containing a caspase recruitment domain
(ASC), which functions as an adaptor protein between
procaspase 1 and a variety of sensor proteins that recognize
molecular pattern motifs (Rathinam et al. 2016). Activation
of the inflammasome plays a crucial role in a variety of
pathophysiological responses when exposed to danger-
associated molecular patterns (DAMPs) and pathogen-
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02466-7) contains supplementary
material, which is available to authorized users.
* Amol A. Kulkarni
amol.kulkarni@howard.edu
1
Department of Pharmaceutical Sciences, College of Pharmacy,
Howard University, Washington, DC 20059, USA
2
Department of Biological Sciences, University of Texas at El Paso,
El Paso, TX 79968, USA
3
Department of Pharmaceutical Sciences, College of Pharmacy,
Mercer University, Atlanta, GA 30341, USA

Medicinal Chemistry Research
activation are more resistant to F. tularensis infection
(Mariathasan et al. 2005; Periasamy et al. 2016). Moreover,
mice deficient in both IL-1β and IL-18, products of
inflammasome activation, are more susceptible to F. tular-
ensis infection compared with the wild type mice. Con-
versely, mice deficient in only one of these products are
protected (Collazo et al. 2006). Therefore, suppression but
not ablation of inflammasome function seems to be a pro-
mising approach for preventing the overactivation of the
inflammatory cytokine storm during F. tularensis infection.
Given the central role of the NLRP3 inflammasome in
this and other pathological and pathophysiological pro-
cesses, there is a profound interest in the development of
small molecules with NLRP3 inhibitory activity. Glyburide,
a drug used in the treatment of type-2 diabetes was shown to
display NLRP3 inhibitory activity at high millimolar con-
centrations (Lamkanfi et al. 2009). In addition, MCC950, a
sulfonylurea-based compound, showed promising activity
in animal models of multiple sclerosis (Coll et al. 2015).
Polyphenolic compounds such as curcumin (Yin et al.
2018), resveratrol (Chang et al. 2015), isoliquiritigenin
(Honda et al. 2014), have been identified as NLRP3
inflammasome inhibitors. Small molecules that form cova-
lent interactions with NLRP3 inflammasome are also
reported. However, the structure of neither the NLRP3
inflammasome nor its individual component proteins has
been determined. This lack of structural details has proven
to be a major impediment in the rational design of small
molecule inhibitors capable of specifically inhibiting the
uncontrolled inflammasome activation. In this article, we
report the synthesis of rationally-designed small molecule
NLRP3 inflammasome inhibitors using computational
chemistry and pharmacophore modeling. The cyclic, ter-
tiary sulfonylurea compounds were geometry optimized at
the Hartree–Fock level of theory using the 6–31 G(d) basis
set using Spartan ’16 molecular modeling software (Pro
2018). The pharmacophoric regions were aligned using
curcumin derivatives as the lead molecule. These novel
rationally-designed inflammasome inhibitors were screened
in vitro against Francisella tularensis-induced NLRP3
inflammasome activation.
but this does not preclude this natural product as a scaffold
for drug development (Capuzzi et al. 2017). Most recently,
curcumin was shown to bind to dual-specificity tyrosine-
regulated kinase 2 (Banerjee et al. 2018). Also, curcumin
and the chalcone derivative isoliquiritigenin are known
inhibitors of the NLRP3 inflammasome. Curcumin, how-
ever, has a low plasma half-life because of its facile
degradation via a retro-aldol pathway (Anand et al. 2007). It
also metabolized into pharmacologically inactive glucur-
onide metabolite. Thus, chemical and enzymatic instability
of this natural product, present some serious challenges
from the drug discovery perspective.
Previous studies have shown that removal of the
β-diketone moiety significantly improves the chemical sta-
bility, but there still remain serious pharmacokinetic con-
cerns (Robinson et al. 2003, 2005). The curcuminoids and
chalcones are two promising structural scaffolds that can be
used for the development of potential inflammasome inhi-
bitors (Fig. 1). Nevertheless, aromatic hydroxyl derivatives
of the curcuminoids and chalcones are still vulnerable to
facile phenolic glucuronidation. Moreover, the α,
β-unsaturated carbonyl moiety can undergo potential and
problematic Michael additions with glutathione. We envi-
sioned using chalcone and curcuminoid scaffolds as our
starting point for the development of new series of potential
inflammasome inhibitors using the following stratagem.
Curcumin can be divided into three critical regions
(Fig. 2). Both regions A and C are comprised of substituted
aromatic rings, which we hypothesized, played a critical
role in binding. The tolerance in the distance between the
centroids of these two rings and their orientation have not
been explored fully. Region B (red) is comprised of an
electrophilic α,β-unsaturated carbonyl system, shown below
in an enol form. Our goal was to modify this electrophilic
linker, which connects the two aromatic regions, to elim-
inate the α,β-unsaturated carbonyl system.
The electrophilic enone linker region can undergo
Michael (1,4-addition) reactions with various nucleophiles
to form covalent bonds. While there are drugs with the
enone functional group, it is considered a structural alert
because of the potential for depleting glutathione con-
centrations. Previously we (Kumari et al. 2015) and others
(Deck et al. 2018) have shown that the linker region can be
modified. Figure 3 shows that a similar division of the
enone and dienone into three regions.
Results and discussion
Molecular modeling and chemistry
We used the two aromatic pharmacophoric centers and
the hydrogen-bond acceptor pharmacophoric center of the
carbonyl of chalcone as our starting point. Figure 4 shows
the alignment of curcumin and chalcone. We hypothesized
that the linker (region B) could be replaced with a sulfo-
nylurea. One scaffold that connects the two aromatic ring
systems with a six-membered sulfonylurea is shown in
Fig. 5. The sulfonyl group both in terms of electrostatics
Curcumin, a bis-phenolic natural product isolated from the
rhizomes of Curcuma longa, has been reported to have
diverse and promising pharmacological activity that could
be used to treat a wide array of disease states. The fact that
curcumin has a number of biological effects has labeled
curcumin as a Pan-assay interference compound (PAINS),

Medicinal Chemistry Research
Fig. 1 The structure of curcumin
in the keto-enol form is shown.
This natural product has served
as the lead structure for the
development of two different
classes of derivatives: the
chalcones and the mono-ketone
curcuminoids
O
OH
H3CO
HO
OCH3
OH
Curcumin
(enol form)
O
O
R1
R1
HO
R2
R2
Chalcones
Curcuminoids
aromatic ring systems because we were interested in
exploring what tolerance of the putative binding site and to
develop novel lead compounds.
Chalcone and a series of diaromatic sulfonylurea struc-
tures were energy minimized at the Hartree–Fock level of
theory using the 6–31 G(d) basis set. Frequency calculations
were carried out to verify that minimum energy structures
were achieved. While we do not necessarily expect the
bioactive conformations to be identical with energy minima,
we decided to use the latter as a first approximation. Using
the pharmacophoric pattern of chalcone as our model, we
were able to align the six-membered and five-membered
sulfonylurea molecular structures with an excellent fit
(Fig. 6). Note the proximity of the pharmacophoric carbonyl
oxygen electron density with the sulfonyl oxygen electron
density. The pharmacophore model has the two aromatic
rings of the sulfonylureas occupying essentially the same
3D space as chalcone. Presumably there is enough con-
formational flexibility of the aromatic rings to provide an
even better structural fit, but energy minimized structures
are shown in Fig. 6. The sulfonyl oxygen atoms occupy
similar conformational space as the carbonyl oxygen of
chalcone.
H
O
O
H3CO
HO
OCH3
OH
A
C
B
Fig. 2 The lead structure of curcumin is divided into three regions.
Both regions A and C, with the molecular skeleton shown in black, are
aromatic rings which are linked via an electrophilic unsaturated car-
bonyl system, region B. A line drawing of curcumin is displayed at the
bottom of the figure. The natural product was energy minimized in
Spartan at the Hartree–Fock level of theory using the 6–31 G(d) basis
set, and it is enveloped in its electrostatic potential energy surface. The
electrostatic potential energy surface is color coded, where red depicts
a more electronegative region and blue depicts a more electropositive
region
Chemical synthesis
Chemical synthesis of the tertiary sulfonylurea structures
is outlined in Scheme 1. It involves a reaction of 4-
substituted aniline derivative with the appropriate chlor-
oalkyl isocyanate resulting in the formation of acyclic
urea 2 and 3. Base-mediated cyclization of the latter fol-
lowed by reaction with the appropriate arylsulfonyl
chloride resulted in the isolated of the desired
3°-sulfonylurea products 4–7.
and length was a reasonable mimic for the region B of
chalcone and the enol form of curcumin. Our computational
studies described below indicate that diaromatic sulfony-
lurea structures can adopt conformations that place the
aromatic rings in similar positions in 3D space. Our goal
was not to necessarily have identical placement of the

Medicinal Chemistry Research
Fig. 3 The chalcone and dienone
derivatives of curcumin are
shown in the same simple two-
dimensional outline. From a
drug design perspective, the goal
is to find a linker that connects
the two aromatic rings
O
O
H3CO
HO
H3CO
HO
OCH3
OH
OCH3
OH
A
C
A
C
B
B
Fig. 6 The scaffolds of chalcone (green), the five-membered sulfo-
nylurea (blue), and the six-membered sulfonylurea (purple) are shown.
In this overlay, all of the structures have been aligned in Spartan based
on the chalcone pharmacophore model using energy minimized
structures at the Hartree–Fock level of theory with the 6–31 G(d)
basis set
Fig. 4 Chalcone (green) and the enol form of curcumin (black) were
energy minimized at the Hartree–Fock level of theory using the
6–31 G(d) basis set. Curcumin and chalcone were aligned in Spartan
using the pharmacophoric groups of the latter
O
O O
S
(Fig. 7a). Infection of macrophages by F. tularensis is
known to induce some cell death (d.n.s); however, addition
of 100 μM of the inhibitor compounds did not induce
additional cell death as measured by PI staining (Fig. 7b).
To prevent misinterpretation of data, all subsequent
experiments were performed at inflammasome inhibitor
compound concentrations at or below 100 µM.
N
N
A
C
B
Macrophage sensing of F. tularensis infection differently
relies on the NLRP3- or AIM2-dependent inflammasome in
human and mouse cells, respectively. These rationally-
designed 3°-sulfonylurea compounds were designed to
prevent inflammation by disrupting formation of the
NLRP3 inflammasome. Initiation of inflammation involves
an orderly stepwise process resulting in proteolytic pro-
cessing and release of IL-1β and subsequent signaling to
induce secretion of inflammatory cytokines, e.g., IL-6.
Functional activation of the inflammasome results in accu-
mulation of these cytokines in the supernatant providing a
rapid measure of the effects of the inhibitors. Pre-treatment
of macrophages with inflammasome inhibitor compounds
prior to infection did not alter production of either IL-1β or
IL-6 in response to infection with F. tularensis with the
inhibitor being removed prior to infection (Supplementary
Fig. 1).
Fig. 5 The sulfyonylureas system can be used to link the two aromatic
regions A and C
2.2 Biological screening
In order to prevent direct killing of the mammalian host
cells, cytotoxicity of the compounds was determined against
uninfected and infected mammalian macrophage cell lines.
Uninfected cells were cultured with titrating amounts of
inflammasome inhibitor for 48 h and then, stained with
propidium iodide to label dead and dying cells. These cul-
tures were imaged on a BD Pathway Confocal imager and
quantified by confocal fluorescence counting. Inflamma-
some inhibitor compounds 4, 5, and 6 were mildly cytotoxic
at the highest concentration tested (1 mM) while compound
7 was highly cytotoxic at the highest concentration

Medicinal Chemistry Research
Scheme
1 Synthesis
of
tertiary
sulfonylureas
a
2-
sulfonyl chloride, 4-fluorobenzene sulfonyl chloride or 4-
trifluromethylbenzene sulfonyl chloride (80–88% overall yield over
two steps)
chloroethylisocyanate (n = 0) or 3-chloropropylisocyanate (n = 1),
CH2Cl2, rt, (87–89%); b 1. NaH (1 equivalent), 2. 4-methoxybenzene
A
B
100
125
95
100
Untreated
Compound 4
90
85
80
75
50
25
0
Compound 5
Compound 6
Compound 7
60
50
40
10-5 10-4 10-3 10-2 10-1 100
[Inhibitor], uM
101 102 103 104
Untreated
Compound 4
Compound 6
Compound 5
Compound 7
Fig. 7 Toxicity of compounds 4–7. a Propidium iodide staining of
uninfected cells analyzed on a BD Pathway Bioimager. Untreated cell
viability is represented by the black circle. No differences in the total
cell number were observed. b Propidium iodide staining of cells
infected with F. tularensis treated with 100 μM compounds 4–7 ana-
lyzed on a BD Pathway Bioimager compared with untreated infected
cells. Data is representative of three independent experiments
Addition of 100 µM inflammasome inhibitors during
infection of mouse or human macrophages with F. tular-
ensis, however, did result in inhibition of inflammation as
measured by the accumulation of IL-1β and IL-6 in the
supernatant after 48 h. Compounds 4, 5, and 7 effectively
inhibited IL-1β production, >80% suppression, from infec-
ted mouse macrophages (Fig. 8a) while only compound 4
efficiently suppressed downstream IL-6 secretion (Fig. 8b).
Compound 6 was completely ineffective at suppressing
cytokine production in mouse cells. In infected human cells,
compounds 4, 6, and 7 mildly inhibited IL-1β production,
15‒20% suppression (Fig. 8c), while compound 7 was most
effective at inhibiting downstream IL-6 production
(Fig. 8d). These data suggested that while compound 4
strongly inhibits inflammasome activation, it is likely spe-
cific to the AIM2-mediated inflammasome rather than
NLRP3. In contrast, compound 7 has a strong specificity for
the NLRP3-mediated inflammasome.
were imaged by confocal microscopy to determine the
extent of association between ASC (red) and the NLRP3 or
AIM2 subunits (green). Resting cells segregate the fluor-
escent signals and appear either red or green (Fig. 9a,
Uninfected) while F. tularensis infection induces formation
of the inflammasome observed as the overlap of the fluor-
escent signal resulting in yellow cells (Fig. 9a, Infected)
which was unaltered by vehicle treatment (Fig. 9a, Vehicle).
Addition of Compound 4 to murine cells strongly prevented
overlap of the AIM2-ASC signal. This effect titrated out at
1 μM. In contrast, compounds 5 and 7 were less effective at
preventing overlap of the fluorescent signal. Compound 6
did not prevent overlap of the inflammasome subunit at any
concentration tested. The previously reported inhibitor,
glyburide, was also ineffective at preventing overlap of the
fluorescent signal at the concentrations tested here while
MCC950 treatment did segregate the inflammasome sub-
units. Similarly, infection of human macrophages triggered
global inflammasome formation (Fig. 9b, Infected). This
was effectively blocked by compounds 4, 5 and 7 but was
unaffected by compound 6 (Fig. 9b). Of these, compound 7
was most effective at preventing inflammasome formation.
To verify that these compounds disrupt formation of the
inflammasome, treated macrophages were stained with
antibodies against the ASC and either AIM2 (Fig. 9a) or
NLRP3 (Fig. 9b) subunits of the inflammasome. These cells

Medicinal Chemistry Research
A
C
B
D
100
80
60
40
20
0
80
60
40
20
0
Compound 4
Compound 5
Compound 6
Compound 7
80
60
40
20
0
30
Compound 4
Compound 5
Compound 6
Compound 7
20
10
0
Fig. 8 Inhibition of inflammation elicited by F. tularensis infection of
mouse (a, b) and human (c, d) macrophages. Infected macrophages
were treated with 100 μM inhibitor for 48 h and production of IL-1β
(a, c) and IL-6 (b, d) was quantified by ELISA. Representative data is
presented from three replicate experiments
Glyburide was more effective at blocking inflammasome
formation in human cells than mouse cells while MCC950
continued to be effective.
potential inhibitors. Since the production of IL-6 results
from an amplification of the inflammatory signal, these
differences are augmented and much more easily observed.
It is critical, however, to confirm that IL-1β production is
directly suppressed by these inhibitors since we observed
that compound 5 inhibited IL-6 production but not IL-1β
production in human cells. This suggests that compound 5
may actually inhibit TLR-mediated signaling rather than the
inflammasome signaling. Further experimentation along
these lines may identify the proteins directly suppressed by
compound 5. Surprisingly, IL-1β production was con-
sistently inhibited more strongly in murine macrophages
compared with human macrophages. Though unexplained,
this may suggest a differential reliance on IL-1β-mediated
triggering of inflammation or an as yet incomplete picture of
the NLRP3 inflammasome structure.
Cumulatively, our results demonstrate that compound 4
displayed greater inhibitory activity in murine cells,
which rely more on AIM2 to sense bacterial infection and
activate the inflammasome. Compound 7 displayed
greater inhibitor activity in human cells which rely more
on NLRP3 to sense bacterial infection and activate the
inflammasome (Atianand et al. 2011). Compound 6 was
completely ineffective at inhibiting inflammasome acti-
vation. While compound 5 inhibited the production of IL-
6 it did not inhibit IL-1β. These results were consistent in
analysis of the secretion of cytokines following infection
and the overlap of fluorescent signals in formation of the
inflammasome complex.
Conclusion
Herein, we describe a strategy for the identification of
curcumin-inspired small molecule NLRP3 inflammasome
inhibitors based on pharmacophoric and structural align-
ments using geometry optimized structures with the
Hartree–Fock level of theory using the 6–31 G(d) basis set.
Unique features of our pharmacophore modeling studies
include the substitution of the sulfonylurea moiety for the
enone linker present in curcumin and chalcone, while pre-
serving the two regions with electron rich aromatic rings.
Our synthetic protocol is efficient and compatible with a
wide-array of commonly utilized functional groups.
In addition, this study establishes a general in vitro assay
system for the screening of NLRP3 inhibitors. Despite the
production of IL-1β as the direct result of inflammasome
activation, we found that the downstream production of IL-
6 in response to concomitant inflammasome activation and
TLR-mediated signaling to be a more sensitive measure of
the inhibition of inflammation. Because IL-1β is the origi-
nating signal priming immune cells to respond by inducing
inflammation, small changes in IL-1β can result in large
changes in inflammation. As a result of these small changes
in IL-1β, it is difficult to observe differences between

Medicinal Chemistry Research
Fig. 9 Inhibition of inflammasome formation. Infected murine a or
human b macrophages were treated with indicated concentrations of
inhibitors and stained for AIM2 (A, green) or NLRP3 (b, green) and
ASC (a/b, red). Uninfected cells displayed little overlap in fluorescent
signal with red and green distinctly separated whereas infected cells
displayed overlapped fluorescent signal (yellow) indicating formation
of the inflammasome. Addition of effective inhibitors resulted in
separation of the fluorescent signal. Representative figures from two
replicate experiments
Experimental section
t = triplet, q = quartet, m = multiplet, bs = broad singlet.
Low resolution ESI mass spectra were obtained on a Waters
Acquity UPLC H-Class with PDA and SQ mass detectors
using a Waters BEH C18 1.7 lm column (2.1 50 mm). High
resolution mass spectra were obtained on VG 70–70 H or
LC/MSD trapSL spectrometer operating at 70 eV using a
direct inlet system.
General
Glassware for chemical synthesis was dried in an oven
(120 °C) and was cooled under argon before use. Unless
otherwise noted, materials obtained from the commercial
suppliers were used without any purification. Reactions
were monitored via thin-layer chromatography on Analtech
silica gel plates and visualized under UV-light. Column
chromatography was performed on silica gel (23–400 mesh)
purchased from Sigma-Aldrich. Evaporation of the volatile
components was conducted under reduced pressure using a
rotary evaporator. Fourier Transform-Infrared (FT-IR)
spectra were recorded neat on a PerkinElmer Spectrum 65
instrument. Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Avance III 400 NME spectro-
meter at 400 MHz (1H) and 100 MHz (13C) respectively in
deuterated chloroform (CDCl3) solvent. The spectra were
Synthetic procedures
Chemical synthesis of compounds 2–6 has been described
previously (Xu et al. 2018).
1-(4-(trifluoromethyl)phenyl)-3-((4-(trifluoromethyl)phe-
nyl)sulfonyl)tetrahydropyrimidin-2(1H)-one (7): A round
bottom flask (10 mL capacity) equipped with a magnetic stir
bar was charged with a solution of acyclic urea (70 mg,
0.25 mmole, 1.0 equiv) in THF (2 mL). The solution was
cooled to 0 °C for 15 min. NaH (30 mg, 0.75 mmole,
3.0 equiv, 60% suspension in oil) was slowly added. The
reaction was stirred for 15 mins at 0 °C for 30 min, slowly
brought to RT, and stirred at RT for 12 h. After
12 h, 4-trifluoromethylbenzenesulfonyl chloride (68 mg,
0.28 mmole, 1.1 equiv) was added and the reaction was
stirred at RT for additional 4 h. The reaction mixture was
1
calibrated against residual solvent peaks (7.24 ppm for H
and 77.0 ppm for 13C). Chemical shifts (δ) and coupling
constants (J) are reported in parts per million (ppm) and
Hertz (Hz) respectively. The following abbreviations were
used to explain multiplicities: s = singlet, d = doublet,

Medicinal Chemistry Research
poured into separatory funnel containing ethyl acetate and
1 M HCl (10 mL each). The layers were separated, organic
layer was washed with distilled H2O (2 × 10 mL), and dried
using anhydrous Na2SO4. Solvent was removed using a
rotary evaporator. The crude product was purified using
silica gel flash column chromatography as a white solid.
Activation and inhibition of inflammasome
Murine macrophages or PMA-differentiated THP-1 cells
were plated to a density of 1 × 105 cells per well in flat
bottomed 96 well plates. Cells were infected (MOI 40) for
2 h with F. tularensis live vaccine strain (LVS). Following
infection, cells were washed twice with PBS pH 7.2, cul-
tured in fresh culture media containing 50 µg/mL genta-
mycin and incubated for an additional 16–24 h. For
compound testing, cells were incubated with 1 mM to 1 nM
final concentration of test compound for 2 h pre-infection or
for 24 h post-infection in RPMI media containing 50 µg/mL
gentamycin. The following day conditioned media was
collected and stored at −20 °C for cytokine analysis. Pro-
inflammatory cytokines (IL-1β and IL-6) were indepen-
dently measured by sandwich ELISA following manu-
facturer’s protocol (BD Bioscience, San Jose, CA). For each
experiment, 50 µL of undiluted conditioned media was
assayed.
1
7. H NMR (400 MHz, CDCl3) δ 8.15–8.13 (d, 2 H),
7.76–7.74 (d, J = 12.6 Hz, 2 H), 7.60–7.57 (d, J = 8.3 Hz,
2 H), 7.35–7.33 (m, 2H), 4.14–4.11 (t, 2H), 3.74–3.71
(t, 2H), 2.31–2.25 (m, 2H). 13C NMR (100 MHz, CDCl3):
149.98, 144.14, 142.42, 134.41, 128.47, 125.67, 125.64,
125.60, 125.56, 125.38, 125.26, 125.22, 125.18, 125.42,
123.87, 121.72, 121.16, 48.43, 44.90, 22.19. IR cm−1
:
2917.3, 2854.6, 1673.8, 1611, 1473.8, 1426.7, 1313,
1163.9, 1061.9, 1101.1, 842.21, 716.69. HRMS (ESI) m/z
453.0706 [m + H]+.
Biological evaluation
Cell culture
Microscopic analysis of inflammasome formation and
THP-1 monocytes (ATCC TIB-202, Manassas, VA) were
maintained in RPMI-1640 containing 2 mM glutamine,
2 mM glucose supplemented with 10% FBS and 10 mM 2-
mercaptoethanol. For differentiation, THP-1 cells were
plated in 96 well plates at 1 × 105 cells per well and incu-
bated for 24 h (37 °C, 5% CO2) in the presence of 100 nM
phorbol myristate acetate (PMA). Following differentiation,
cells were allowed to recover for 24 h in fresh culture media
prior to use in subsequent experiments. J virus immortalized
bone marrow derived murine macrophages (BEI Resources
NR-9456, Manassas, VA) were maintained in DMEM
containing 2 mM glutamine, 2 mM glucose supplemented
with 10% FBS, 1% non-essential amino acids, 1% sodium
pyruvate and 1% HEPES.
activation
Murine macrophages or PMA-differentiated THP-1 cells
were plated in flat bottomed 96 well plates at a cell density
of 1 × 105 cells per well. Cells were incubated with
compounds A-D (100 μM to 1 nM, in seven ten-fold
dilutions) for 24 h. Cells were infected (MOI 40) for 2 h
with F. tularensis live vaccine strain (LVS). Following
infection, cells were washed twice with PBS pH 7.2,
cultured in fresh culture media containing 50 µg/mL
gentamycin and incubated for an additional 16–24 h.
Macrophages were fixed for 15 min at room temperature
with 4% formaldehyde in PBS and then were washed
three times with PBS. Nonspecific binding in cells was
blocked with 1% bovine serum albumin and cells were
permeabilized with 0.1% Triton X-100 for 5 min. Cells
were blocked with 1% bovine serum albumin in PBST for
30 minutes and then incubated with anti-ASC (Santa Cruz
Biotechnology), anti-NLRP3 and anti-ASC antibodies
(Abcam) for 1 h (Ke et al. 2017; Granata et al. 2015;
Baroja-Mazo et al. 2014) and imaged on Zeiss LSM700
at ×20 or ×63 magnification.
Compound preparation
All compounds were dissolved in DMSO and stored at
−20 °C until use. Working stocks (10 mM) were diluted in
DMSO, placed in amber borosilicate glass tubes and stored
at 4 °C.
Toxicity assay
Computational
Murine macrophages or PMA-differentiated THP-1 cells
were plated in flat bottomed 96 well plates at a cell density
of 1 × 104 cells per well. Cells were incubated with com-
pounds A-D (1 mM to 10 nM, in six 10-fold dilutions) for
24 h. After incubation macrophages were stained with
propidium iodide and Hoechst 33342 (1 µg/ml final con-
centration) and analyzed for viability on a BD pathway 855
bioimager as described previously (Lema et al. 2011).
All the calculations were carried out in the Spartan mole-
cular modeling program in the ground state in the gas phase.
Chalcone and the diaromatic sulfonylurea structures were
energy minimized using the Equilibrium Geometry option
in Spartan at the Hartree–Fock level of theory using the
6–31 G(d) basis set and with molecular mechanics
(MMFF). For each geometry optimization, a frequency

Medicinal Chemistry Research
calculation was used to verify that an energy minimum
structure was located successfully.
Acknowledgements This work was supported in part by the District of
Columbia Center for AIDS Research (DC CFAR; AI117970) and
Research Centers in Minority Institutions (RCMI), Howard University.
Biological research reported in this article was supported by the
National Institute of General Medical Sciences of the National Insti-
tutes of Health under linked Award Numbers RL5GM118969,
TL4GM118971 and UL1GM118970. The content is solely the
responsibility of the authors and does not necessarily represent the
official views of the National Institutes of Health.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
The final stable conformers for the sulfonylurea struc-
tures were achieved manually by initially generating the
+60, −60, and 180 conformations for the flexible dihedral
angles followed by energy minimization as described
above. Additionally, rotational profiles were generated to
verify the results.
While there are multiple alignments possible, the struc-
tures were aligned using the Geometry option in Spartan is
based on the chalcone pharmacophore model described in
the paper. The energy minimized structures achieved with
Hartree–Fock level of theory with the 6–31 G(d) basis set
and MMFF were used. Alignments based on either the
molecular structures or the aromatic ring pharmacophoric
centers gave similar results.
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