Electrophilic warhead-based design of compounds preventing NLRP3 inflammasome-dependent pyroptosis

Article abstract of DOI:10.1021/jm501072b

Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death implicated in the pathogenesis of autoinflammatory diseases as well as in disorders characterized by excessive cell death and inflammation. Activation of NLRP3 inflammasome

Full text of DOI:10.1021/jm501072b

Article
pubs.acs.org/jmc
Electrophilic Warhead-Based Design of Compounds Preventing
NLRP3 Inflammasome-Dependent Pyroptosis
Mattia Cocco,† Davide Garella,† Antonella Di Stilo,† Emily Borretto,† Livio Stevanato,† Marta Giorgis,†
Elisabetta Marini,† Roberto Fantozzi,† Gianluca Miglio,*,† and Massimo Bertinaria*,†
†Dipartimento di Scienza e Tecnologia del Farmaco, Universita
̀
degli Studi di Torino, Via P. Giuria 9, 10125 Torino, Italy
S
* Supporting Information
ABSTRACT: Pyroptosis is a caspase-1-dependent pro-inflam-
matory form of programmed cell death implicated in the
pathogenesis of autoinflammatory diseases as well as in
disorders characterized by excessive cell death and inflamma-
tion. Activation of NLRP3 inflammasome is a key event in the
pyroptotic cascade. In this study, we describe the synthesis and
chemical tuning of α,β-unsaturated electrophilic warheads
toward the development of antipyroptotic compounds. Their
pharmacological evaluation and structure−activity relationships
are also described. Compound 9 was selected as a model of this
series, and it proved to be a reactive Michael acceptor,
irreversibly trapping thiol nucleophiles, which prevented both
ATP- and nigericin-triggered pyroptosis of human THP-1 cells
in a time- and concentration-dependent manner. Moreover, 9 and other structurally related compounds, inhibited caspase-1 and
NLRP3 ATPase activities. Our findings can contribute to the development of covalent, multitarget antipyroptotic compounds
targeting molecular components of the NLRP3 inflammasome regulatory pathway.
priming signal), have been shown to prime cells by inducing the
transcription and translation of NLRP3 protein itself.10,16,17
further signal (activating signal), such as ATP or nigericin, is
required to trigger the formation of the inflammasome complex
that leads to the activation of caspase-1 and eventually
pyroptosis (Figure 1).
INTRODUCTION
■
A
Pyroptosis is a pro-inflammatory form of programmed cell
death originally described in macrophages infected with Shigella
flexneri.1,2 Pores with a 1−2 nm diameter appear in the plasma
membrane of pyroptotic macrophages at early time points. Pore
formation is a caspase-1-dependent process: it dissipates
cellular ionic gradient, producing a net increased osmotic
pressure, water influx, cell swelling, and eventually osmotic lysis
and release of the pro-inflammatory intracellular contents.3
Caspase-1 activation in macrophages can also result in the
proteolytic maturation and release of pro-inflammatory
interleukins (IL)-1β and IL-18 from cells.4,5 However,
pyroptosis has been shown to occur also independently of
IL-1β and IL-186 and is also triggered in nonmyeloid cell types
either by bacterial or nonbacterial stimuli, including viruses,
microparticles, cancer chemotherapy, as well as host factors,
including misfolded proteins.7−13 In the past decade, a better,
albeit still incomplete, understanding of the biochemical
pathways leading to caspase-1 activation has been achieved.
Pro-caspase-1 undergoes proximity-induced autoproteolytic
conversion of the zymogen into active caspase-1 in large
cytosolic protein complexes termed inflammasomes.5,14,15 The
NLR family, pyrin domain-containing 3 (NLRP3) inflamma-
some is the best recognized and the most widely implicated
regulator of caspase-1 activation. Activation of NLRP3
inflammasome is often described in terms of a two-step process
requiring two signals. For example, many microbial Toll-like
receptor (TLR) ligands, such as lipopolysaccharide (LPS,
Convincing data have highlighted the pathophysiological
implications of caspase-1 activation in pyroptotic cells. Such
data suggest that pharmacological interventions aimed to
counteract this event may be a promising pharmacological
strategy to treat diseases characterized by excessive/uncon-
trolled cell death and inflammation.18−21 Systemic auto-
inflammatory diseases known as cryopyrin-associated periodic
syndromes (CAPS) are a case in point. They are rare, chronic,
disabling disorders encompassing three conditions: familial cold
autoinflammatory syndrome (FCAS, or familial cold urticaria),
Muckle−Wells syndrome (MWS), and chronic infantile
neurologic, cutaneous, articular (CINCA) syndrome (also
known as neonatal-onset multisystem inflammatory disease,
NOMID). Although the exact pathogenic mechanism under-
lying these disorders has not been fully clarified, mutations in
NLRP3/CIAS1 could result either in a gain-of-function of
NLPR3 protein or in an increase of its level, leading to a
chronic sterile state of inflammation.22−24 Moreover, the onset
of chronic inflammation, due to NLRP3 inflammasome
Received: July 17, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. A simplified view illustrating NLRP3 inflammasome activation pathways. Priming: A first signal (i.e., TLR ligands) induces the NF-κB-
mediated transcription of NLRP3 and pro-IL-1β. Activation: A second signal is then required to trigger the pro-inflammatory response. ATP,
activating the purinergic P2X7 receptor; nigericin, a K+/H+ antiport ionophore; crystalline structures (i.e., uric acid, cholesterol crystals, silica) or
misfolded protein aggregates, promote the inflammasome assembly and autoactivation. Once assembled, NLRP3 inflammasome triggers the
autoproteolytic conversion of pro-caspase-1 into active caspase-1 that promotes pyroptosis and/or IL-1β release.
activation, has recently been linked to a wide and diverse range
of disorders including type-2 diabetes mellitus, atherosclerosis,
inflammatory bowel disease, respiratory diseases, liver injury, as
well as neurodegenerative diseases, thereby expanding the
potential for therapeutic application of “NLRP3 inflammasome
blockers”.18,25−27
Some small molecules have been reported to prevent NLRP3
inflammasome-dependent pyroptosis (or caspase-1 activation as
surrogate marker) triggered by several stimuli, among them
resatorvid (1), 5Z-7-oxozeaneol (2), and bromoxone (3)
(Chart 1).28 However, knowledge of direct inhibitors of
NLRP3 inflammasome activation or assembly is limited to
(E)-3-tosylacrylonitrile (Bay 11-7082, 4),29 a compound of
synthetic origin, and parthenolide (5), a sesquiterpene lactone
extracted from feverfew (Tanacetum parthenium), which might
also target multiple stages of the NLRP3 inflammasome
signaling pathway.29 Recently, 3,4-methylenedioxy-β-nitrostyr-
ene (6), a synthetic kinase inhibitor, has been also shown to
specifically inhibit NLRP3 activation.30
Such structurally unrelated compounds share an olefin
activated by substitution with electron withdrawing group(s)
as the sole substructure similarity. Therefore, it is reasonable to
suggest that the formation of covalent adducts via Michael-type
reactions with molecular targets involved in the regulation of
the pyroptotic cascade could underlie their antipyroptotic
activity. In particular, the alkylation of the thiol group of
functional or regulatory cysteine residues in proteins involved
in the NLRP3 inflammasome regulatory network (e.g., NLRP3
and caspase-1) could be a common feature of their mechanism
of action. Nowadays, the pursuit of covalent inhibitors has
gained a renewed interest as an attractive strategy in drug
design.31 The reactivity of electrophilic functionalities can be
finely regulated, thus enabling medicinal application with
favorable toxicity, through two approaches: (1) tuning their
reactivity and (2) conferring target selectivity, thus confining
their reactivity to the target pocket.32−34
Chart 1. Structures of Compounds Inhibiting NLRP3
Activation or NLRP3-Related Signaling Pathways
A few innovative studies on small electrophilic molecules,
able to efficiently modulate different biological pathways, have
been recently reported.35
In view of the above, we designed a library of electrophilic
warheads, bearing substituted α,β-unsaturated nitrile and α,β-
unsaturated carbonyl-derived functionalities, to explore the
possibility of obtaining new compounds able to interfere with
the NLRP3 inflammasome-dependent pyroptotic cascade.
B
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Structures of designed electrophilic warheads 7−42 and reference compound 43.
The primary aim of this research is to establish the
structure−activity relationships (SARs) of the synthesized
electrophilic fragments and eventually to find a hit structure
amenable for further chemical manipulation.
tages are usually its long reaction times and variable yields that
strongly depend on the reactivity of carbonyl compound and
olefinic partner as well as on the catalyst and solvent
employed.36
Initially, the method developed by Yu and co-workers was
used.37 This method required a stoichiometric amount of 1,4-
diazabicyclo[2.2.2]octane (DABCO), as the catalyst, in a 1/1
mixture of dioxane/H2O. Under such conditions, we were only
able to efficiently react 2-pyridinecarboxaldehyde and 2-
nitrobenzaldehyde with ethyl acrylate and acrylonitrile,
obtaining the desired MBH adducts 7, 8, 22, and 23 in 44−
70% yields (Scheme 1). When the same method was applied to
We synthesized a focused library of compounds of general
structure I (Figure 2, compounds 7−42), inspired by 5, where
an α,β-unsaturated lactone substructure with an exocyclic
double bond is found. The modulation of R1 and R2 should
enable fine-tuning of reactivity of the unsaturated double bond,
while W and X variations could generate compounds with
modulated physicochemical properties, allowing enough
chemical space for further manipulation toward more selective
agents. 43 (Figure 2), a close analogue deprived of Michael
acceptor fragment, was also synthesized as reference compound
for pharmacological studies.
a
Scheme 1. Synthesis of Compounds 7−10, 22, and 23
In this paper, we present the pyroptosis inhibition rates of
five series (Figure 2, series A−E) of electrophilic fragments
together with their cytotoxicity values. We show that selected
compounds act at several stages of pyroptotic cascade because
they can inhibit directly caspase-1 and NLRP3 ATPase activity.
We also demonstrate that these compounds act as Michael
1
acceptors in physiologically similar conditions via H NMR
experiments and a thiol trapping kinetic assay.
a
Reagents and conditions: (a) W-CHO (1 mmol), CH2CHR1 (3
RESULTS AND DISCUSSION
■
mmol), DABCO (1 mmol), dioxane/H2O (1/1), rt.
Chemistry. For the synthesis of the designed compounds,
we relied on the use of the Morita−Baylis−Hillman (MBH)
reaction which involves a C−C bond formation between an
electron-deficient alkene and a carbonyl compound catalyzed
by a tertiary amine or a phosphine. This atom-economic
reaction offers several advantages making use of commercially
available starting materials and mild reaction conditions
generating flexible and multifunctional adducts; main disadvan-
less reactive aldehydes (2-halobenzaldehydes), either no
appreciable amount of product was obtained (9; Scheme 1)
or longer reaction times were required (10; Scheme 1).
We then switched to the use of a mixture of THF/MeOH/
H2O (1/1/2) as reaction solvent (Scheme 2). The use of this
medium proved successful with a range of aldehydes only when
C
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
they were reacted with acrylonitrile, affording 24, and 26−30 in
variable yields.
imidazole (Im), tetramethylethylenediamine (TMEDA), and
triphenylphosphine (PPh3) were employed as the catalysts;
chloroform, THF/MeOH/H2O (1/1/2), 1,4-dioxane/H2O (1/
1), and CH3CN/H2O (9/1) were employed as solvents. The
use of DABCO or DMAP in CH3CN/H2O (9/1) proved to be
the most efficient combination; in particular, satisfactory yield
of 9 (67%) was obtained with DABCO catalysis (Table 1, entry
4). Applying the aforementioned conditions, we successfully
scaled up MBH reaction up to 40 mmol scale without any
significant loss in yield (Table 1, entry 17). Heating at 80 °C
showed a negligible influence of temperature on MBH kinetics
(Table 1, entry 18).
a
Scheme 2. Synthesis of Compounds 9, 24, and 26−30
This procedure was then used for the synthesis of 10−19,
which were isolated in 7−96% yields after flash chromatog-
raphy (FC) (Scheme 3). To investigate whether this procedure
a
Reagents and conditions: (a) W-CHO (1 mmol), CH2CHR1 (3
a
Scheme 3. Synthesis of Compounds 10−21, 25, and 34
mmol), DABCO (1 mmol), THF/MeOH/H2O (1/1/2), rt.
Either no reaction or traces (<1%) of products were obtained
when less reactive ethyl acrylate was used (9; Scheme 2).
To obtain MBH adducts with ethyl acrylate, we studied the
reaction between 2-chlorobenzaldehyde, chosen as model
aldehyde, and ethyl acrylate under different conditions (Table
1). 2-Chlorobenzaldehyde (1 mmol) and ethyl acrylate (3
mmol) were reacted at room temperature for 7 days in four
different solvents with stoichiometric amounts of different
catalysts. DABCO, 4-(dimethylamino)pyridine (DMAP), 1,5-
diazabiciclo[5.4.0]undec-5-ene (DBU), triethylamine (Et3N),
Table 1. Optimization of Reaction Conditions for the
Synthesis of 9
a
Reagents and conditions: (a) W-CHO (1 mmol), CH2CH-R1 (3
mmol), DABCO (1 mmol), CH3CN/H2O (9/1), rt, 7 d; (b) W-CHO
(1 mmol), CH2CH-R1 (3 mmol), DABCO (1 mmol), neat, rt, 7 d;
(c) W-CHO (1 mmol), CH2CH-R1 (3 mmol), DABCO (1 mmol),
CH3CN/H2O (9/1), rt, 16 h; (d) W-CHO (1 mmol), CH2CH-R1
(1.2 mmol), DABCO (1 mmol), formamide, rt, N2 atm, 69 h; (e) W-
CHO (1 mmol), CH2CH-R1 (3 mmol), rac-proline (0.1 mmol),
NaHCO3 (0.25 mmol), DMF/H2O (9/1), rt, 5 d.
a
entry
solvent
catalyst
isolated yield (%)
1
1,4-dioxane/H2O (1/1)
THF/MeOH/H2O (1/1/2)
CHCl3
DABCO
DABCO
DABCO
DABCO
DMAP
DBU
<1
<1
2
3
4
CH3CN/H2O (9/1)
CHCl3
67
31
25
5
was not exclusive to ethyl acrylate-derived adducts, we applied
the same protocol for the reaction of 2-fluorobenzaldehyde
with acrylonitrile, obtaining 85% yield of 25. Such a result
suggests that this method can effectively be used to synthesize
both α-substituted acrylonitriles and acrylates. Ketone deriva-
tive 34 was synthesized using rac-proline/NaHCO3 in place of
DABCO as the catalyst.
When the reaction protocol was used for the synthesis of
compounds bearing an aliphatic moiety in W position,
contrasting results were obtained. The reaction between
acetaldehyde and ethyl acrylate afforded modest yield (28%)
of 20. However, when more lipophilic cyclohexane-carbox-
aldehyde was used, no reaction occurred. To obtain 21, we
adapted a reported procedure38 employing DABCO in
formamide under a nitrogen atmosphere. In such conditions,
we were able to obtain the desired compound in 33% isolated
yield.
6
CHCl3
7
CHCl3
Et3N
8
CHCl3
Im
9
CHCl3
TMEDA
PPh3
10
11
12
13
14
15
16
17
18
CHCl3
complex mixture
b
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
CH3CN/H2O (9/1)
DMAP
DBU
42
<1
Et3N
Im
2
<1
TMEDA
PPh3
complex mixture
c
DABCO
DABCO
63
d
68
a
All reactions were run on 2-chlorobenzaldheyde (1 mmol) with ethyl
acrylate (3 mmol), catalyst (1 mmol) in 3 mL of the reported solvent.
About 4% of the Michael type byproduct 2-((2-chlorophenyl)(3-
ethoxy-3-oxopropoxy)methyl)acrylate (31) was obtained. Reaction
run on 40 mmol of 2-chlorobenzaldheyde with ethyl acrylate (120
mmol) and DABCO (40 mmol). Reaction performed on 20 mmol
scale at 80 °C.
b
c
Interestingly, the reaction performed using DMAP catalysis
to obtain 9 (Table 1, entry 11) generated a second minor
product (31) in addition to the desired compound. Compound
31 was generated by oxa-Michael addition of 9 on ethyl acrylate
d
D
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of Compounds 9, 15, 16, and 31−33
a
Reagents and conditions: (a) W-CHO (1 mmol), ethyl acrylate (3 mmol), DMAP (1 mmol), CH3CN/H2O (9/1), rt, 7d.
a
double bond. Because we were interested in studying the effect
of O-alkylation on a small series of chloro-substituted
compounds, we employed DMAP-catalyzed MBH reaction to
synthesize compounds 32 and 33, the O-alkylated analogues of
15 and 16, without optimizing the reaction conditions (Scheme
4).
Scheme 6. Synthesis of Compounds 37−42
To obtain compounds with R1 substituent variation, 9 was
converted into the corresponding acid 35 by treatment with
methanolic KOH and subsequently coupled to propanamine to
afford the amide 36. Reference compound 43 was obtained via
double-bond reduction of 9 using H2 and 5% Pd/C in ethyl
acetate (Scheme 5).
a
Reagents and conditions: (a) ethyl propiolate, MgI2, CH2Cl2, 0 °C,
18 h; (b) PhB(OH)2, Cs2CO3, Pd(OAc)2, DME/H2O (1/1), rt, 24 h;
(c) MeMgBr, LiCuBr2, −40 °C, 40 min.
cultures exposed to ATP (Figure 3A). Moreover, as shown in
Figure 3B, a significant increase in lactate dehydrogenase
a
Scheme 5. Synthesis of Compounds 35, 36, and 43
a
Reagents and conditions: (a) KOH 0.9 M, MeOH, rt, 18 h; (b)
Figure 3. Effects of ATP and nigericin on THP-1 cells. (A) PMA-
differentiated and LPS-primed THP-1 cells were exposed to ATP (5
mM, 1 h). Representative images showing morphological changes
related to the ATP-triggered cell death. The arrow indicates a cell
undergoing pyroptosis, as indicated by a measurable size increase,
followed by cytoplasmic swelling and cell lysis within a few minutes of
ATP exposure. (B) PMA-differentiated and LPS-primed THP-1 cells
were exposed to vehicle alone, ATP (5 mM, 1 h), or nigericin (10 μM,
1.5 h) in serum-free medium. LDH activity was measured in the
collected supernatants by a colorimetric assay. Data are expressed as
absorbance at λ= 490 nm and are the mean SEM of at least six
experiments run in triplicate. ** p < 0.01 vs vehicle alone.
HOBt, HBTU, DIPEA, DMF/CH2Cl2 (5/2), rt, 18 h; (c) H2, Pd/C
(5%), EtOAc, rt, 10 min.
To obtain R2-modulated compounds, functionalization of the
double bond in the β-position was accomplished starting from
(E)- and (Z)-iodoacrylates 37 and 38, synthesized via one-step
MBH reaction between 2-chlorobenzaldehyde and ethyl
propiolate, followed by iodination with MgI2. Obtained iodo-
derivatives were reacted in a Pd-catalyzed Suzuki reaction with
phenylboronic acid to furnish (E)-β-phenyl acrylate 39 (48%)
and its (Z)-isomer 40 (44%) or in a LiCuBr2 catalyzed Gilman
reaction with methyl magnesium bromide to give (E)- and (Z)-
crotonates 41 and 42 (Scheme 6). Isomer identification was
determined by comparison of 1H NMR spectra of similar
compounds, the olefinic proton appearing at higher ppm in the
(E)-isomer (i.e., 8.10 ppm in 37) than in the (Z)-isomer (i.e.,
7.16 ppm in 38).39
Antipyroptotic Effect, Cytotoxicity, and Structure−
Activity Relationships of Synthesized Compounds.
Pyroptosis was studied in phorbol myristate acetate (PMA)-
differentiated and LPS-primed THP-1 cells exposed to ATP (5
mM; 1 h) or nigericin (10 μM, 1.5 h) as previously
described.28,40 Dying cells with increased size which eventually
undergo to cytoplasmic swelling and cell lysis through the
rupture of the plasma membrane were observed in the cell
(LDH) activity, used as a marker of cell death, was measured in
the supernatant of ATP− (41.9 3.6%; p < 0.01, ANOVA and
Bonferroni’s post-test) or nigericin-treated cells (43.3 4.3%; p
< 0.01) compared to vehicle alone. These findings are
consistent with previous studies and indicate that this
experimental model allow the study of pyroptosis.
To explore the pharmacological properties of the synthesized
compounds, their ability to prevent the ATP-triggered cell
death was initially studied. The synthesized compounds were
assumed to act by forming covalent adducts with yet undefined
molecular targets, and thus, to evaluate the relative efficacy,
they were initially added at 10 μM, 1 h before the ATP
exposure. In addition, given that adduct formation may result in
E
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Inhibitory Effect of Synthesized Compounds on Pyroptotic Cell Death of THP-1 Cells and Cytotoxic Properties in HK-
2 Cells
a
b
c
a
b
c
compd
pyroptosis decrease (%)
cytotoxicity TC50 (μM)
cLogP
compd
pyroptosis decrease (%)
cytotoxicity TC50 (μM)
cLogP
5
100.3 6.3
54.3 6.3
63.7 13.3
75.1 2.6
82.3 7.8
53.7 4.2
45.8 1.6
44.8 4.2
50.5 4.1
76.6 5.8
72.4 1.4
78.4 11.5
26.8 0.3
37.8 1.3
59.6 5.4
64.3 3.6
52.7 2.7
97.9 5.7
83.0 3.7
20.1 5.5
53.4 6.5
7.8 1.1
67.0 3.4
25.4 2.5
55.9 5.5
64.5 7.8
28.9 2.2
34,9 8,4
45.2 6.4
70.5 2.6
18.8 5.1
45.8 7.2
56.7 5.2
22.5 5.5
>100
3.03
1.36
1.90
2.79
1.97
2.77
2.30
2.76
2.75
3.36
3.33
3.28
2.31
2.42
1.02
2.44
1.03
1.78
2.19
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
30.5 5.6
81.3 4.5
54.1 3.6
53.5 4.4
51.5 7.9
14.1 5.8
58.3 7.6
68.8 7.2
43.9 7.8
12.1 1.2
42.0 6.9
31.2 6.8
5.1 7.4
12.9 1.5
21.8 4.4
22.3 2.4
10.1 2.5
16.3 1.9
36.4 3.4
>100
1.56
2.44
1.77
2.62
2.72
2.70
3.94
4.30
4.36
2.23
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
>100
>100
1.9 8.2
>100
d
1.85
83.5 2.2
6.4 2.2
5.9 5.2
>100
2.42
3.09
3.09
4.15
4.15
3.37
3.37
2.66
17.6 4.0
55.5 4.5
52.5 2.0
31.8 4.2
36.2 4.2
1.6 2.5
>100
20.5 7.1
3.6 1.4
11.6 2.5
73.8 7.2
80.1 3.0
75.5 4.2
a
PMA-differentiated and LPS-primed THP-1 cells were pretreated with either vehicle alone or each compound (10 μM; 1 h). Pyroptosis was
triggered with ATP (5 mM), and LDH activity was measured in the collected supernatant 1 h after ATP exposure by a colorimetric assay. Data are
b
expressed as percent of pyroptosis decrease vs vehicle alone. HK-2 cells were exposed to increasing concentrations (0.1−100 μM) of each
compounds, and cell viability was measured at 72 h by the MTT assay. Pharmacological and toxicological data are expressed as mean SEM of at
c
d
least four experiments run in triplicate. Calculated with ALOGPS 2.1 (http://www.vcclab.org/lab/alogps2.1). cLogP calculated for neutral form.
cytotoxicity, their effects on the viability of human renal
epithelial cells (HK-2) were also evaluated by MTT assay.
None of the assessed compounds (10 μM, 1 h) exerted direct
inhibition of LDH activity (data not shown), thus indicating
that measurement of LDH activity can be used as a marker to
study the antipyroptotic effect of these compounds. 5 was
adopted as a positive reference compound and abolished cell
death (Table 2). Pyroptosis of THP-1 cells was significantly (p
< 0.05 vs vehicle alone) prevented by most of the synthesized
(14.1 5.8% to 97.9 5.5%) respectively; p = 0.844; Wilcoxon
signed rank test). However, the median of the TC50 for the
effects on the viability of HK-2 cells was significantly lower for
the nitrile compared that for the ester series (18.4 μM (3.6
1.4 to 36.4 3.4 μM) vs 54.7 μM (7.8 1.1 to 70.5 2.6
μM); p = 0.014). This suggests that nitrile series development
can be discontinued due to its higher cytotoxicity. These
findings indicate that the nature of the R1 and W groups
significantly influences the pharmacological and toxicological
activity of these compounds.
compounds (Table 2). The effects ranged from 1.6
2.5%
(43) to 97.9 5.7% (23), thus indicating that the entire
To better understand the role of the R1 group, the effects of
ethylketone 34, carboxylic acid 35, and propylamide 36 were
studied (D-series) (Figure 2). Cell death was significantly
prevented by 35 (42.0 6.9%; p < 0.01) and 36 (31.2 6.8%;
p < 0.05) but not by 34 (12.1 1.2%). Moreover, 35 and 36
were both significantly (p < 0.01) less effective and less toxic
than 9 (Table 2). These findings not only confirm the role of
R1 in determining the pharmacological activity of these α,β-
unsaturated compounds but also indicate that, compared to the
cyano, carboxylic, and carboxyamido groups, the ester group is
associated with an overall more favorable pharmacotoxico-
logical profile. As a result, further experiments were focused on
the ester series (Figure 2, A-series).
spectrum of potential efficacy was observed. Half-maximal
toxicity concentrations (TC50) values obtained in cytotoxicity
test, namely the molar concentration of compounds that
decreases cell viability of HK-2 cells by 50%, ranged within >3-
Log units, from 1.9 8.2 μM (34) to >100 μM (21, 31−33,
35, 39, and 40), thereby indicating a large variety in
toxicological activities (Table 2).
9 was significantly more effective in preventing cell death
compared to its saturated analogue 43 (75.1 2.6% vs 1.6
2.5%, p < 0.01). Contrarily, the two compounds exerted
comparable effects on the viability of HK-2 cells because the
TC50 were 67.0
3.4 and 75.5
4.2 μM, respectively.
Therefore, the α,β-unsaturated carbonyl moiety is a structural
feature strictly related to the antipyroptotic activity of 9, thus
consolidating our hypothesis that this substructure is the
putative pharmacophore of known inflammasome inhibitors.
Results show that both compounds belonging to the ester
series (A-series) and to the nitrile series (B-series) were able to
significantly prevent pyroptotic cell death of THP-1 cells.
Inspection of data obtained for esters 7−12, 15, and 16 and
corresponding nitriles 22−27, 29, and 30 revealed no
significant difference in the overall antipyroptotic efficacy
(median effect: 63.7% (45.8 1.6% to 82.3 7.8%) vs 53.4%
To explore the role of chlorine substitutions of the W-phenyl
group, the effects of different monochloro-substituted isomers
were first studied. Compared to the ortho-chloro-substituted 9,
both the meta-(13) and para-chloro-substituted (14) com-
pounds were significantly (p < 0.01) less effective and more
toxic (Table 2). Subsequently, the effects of 8−11, 18, and 19,
bearing different substituents in the ortho-position of the W-
benzene ring, were considered. Cell death was significantly
prevented by all these compounds (p < 0.05). In addition, the
amplitude of the effects decreased linearly as follows F ≥ Cl ≥
NO2 ≥ Br > H > Me > OMe (Supporting Information, Figure
F
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Summary of SAR for studied electrophilic compounds.
S1), but differences were significant (p < 0.05) only when
electron-withdrawing substituents were compared to electron-
donating ones. Cell viability of HK-2 cells was significantly
decreased by all these compounds. A comparative analysis of
the TC50 revealed that 8 (ortho-NO2) and 10 (ortho-F) were
markedly more toxic than other compounds (Supporting
Information, Figure S1). Data taken together indicate that the
antipyroptotic efficacy and cytotoxicity of these α,β-unsaturated
esters are significantly influenced by the electronic properties of
the substituent at the ortho-position of benzene ring in W. In
addition, the more favorable pharmacological profile was
achieved by the chlorine-substituted 9.
The effects of dichloro-substituted compounds 15 (2,6-
dichloro−), 16 (2,4-dichloro−), and 17 (3,5-dichloro−) were
studied. Cell death was significantly prevented by all these
compounds (72.4 1.4% to 78.4 11.5%), and their effects
were not significantly different from those exerted by 9.
However, 15 and 17 proved to be significantly (p < 0.05) more
38. In addition, these compounds markedly decreased cell
viability of HK-2 cells, with TC50 values significantly lower (p <
0.01) than 9. Pyroptosis was instead significantly (p < 0.01)
decreased by compounds 39−42. Compared to 9, both β-
phenyl-substituted (39, 40) and β-methyl-substituted (41, 42)
derivatives were significantly less effective (p < 0.05), and no
differences were detected within both isomeric pair of
compounds. Consistently, compared to 9, β-substitution led
to increased TC50 values from 73.8 7.2 μM (41) to >100 μM
(39 and 40). These findings indicate that R2 substituent also
influences the pharmacological and toxicological activity of
these compounds. Moreover, the geometry of the double bond
plays a minor role in determining their activity (Figure 4).
No apparent relationship between the antipyroptotic activity
and the lipophilicity, calculated by cLogP, was evidenced within
this class of compounds (Table 2).
Finally, the amplitude of the antipyroptotic effects exerted by
9, 10, 15−17, 23, 24, and 26 was statistically comparable to
that of 5. However, the TC50 values were significantly higher for
9, 15, and 16, suggesting a more promising toxicological profile
with respect to 5.
toxic (TC50 45.2
6.4 μM and TC50 18.8
5.1 μM,
respectively) than the monosubstituted 9, while 16 exhibited
comparable cytotoxic activity. Together, these results indicate
that chlorine substitution on the W-phenyl ring modulates both
the antipyroptotic efficacy and the cytotoxicity of these
compounds.
To further ascertain the role of the W group, the effects of
12, 20, and 21, bearing, respectively phenyl, methyl, and
cyclohexyl moieties, were examined. Comparable antipyroptotic
effects were exerted by these compounds (from 45.8 1.6% to
Time-Course and Concentration−Response Curves of
the Antipyroptotic Effect. Compounds 8−10, 32, 33, 41,
and 42, selected on the basis of their modulated efficacy, were
tested to investigate the dynamics of the antipyroptotic effect in
comparison to 5. First, time-dependency was studied. Differ-
entiated and primed THP-1 cells were pretreated (15−90 min
before exposure to ATP) with the synthesized compounds (10
μM), and cell death was measured 1 h after exposure to ATP (5
mM), as previously described. The antipyroptotic effect raised
over the pretreatment time without achieving the steady-state
(Figure 5A). Compared to the less effective compounds (41
and 42), more effective ones (8−10) were characterized by a
faster onset of antipyroptotic effects. Then, the concentration−
response relationships were studied, exposing cells to increasing
concentrations (0.001−10 μM) of each compound and
measuring LDH activity 1 h after exposure to ATP (5 mM).
As shown in Figure 5B, antipyroptotic effect increased in a
concentration-dependent manner, and the EC50 values, namely
the molar concentration of compound that produces 50% of its
maximal possible effect, were in the submicromolar range for all
tested compounds (Table 3).
64.3
3.6%). By contrast, cell viability of HK-2 cells was
differently affected and the TC50 values were from 22.5 5.5
μM (20) to >100 μM (21).
To explore the role of the hydroxyl group in determining
antipyroptotic activity and cytotoxicity of these compounds, the
effects of 9, 15, and 16 were compared to those of O-alkylated
analogues 31−33 (Figure 2, C-series). 9, 15, and 16 exerted
significantly (p < 0.05) higher effects than 31−33 (Table 2).
However, 31−33 were markedly less toxic (TC50 > 100 μM)
with respect to their nonalkylated counterparts 9, 15, and 16
(45.2 6.4 to 70.5 2.6 μM). Such results indicate that the
hydroxyl group could play a relevant role in determining the
cytotoxic activity of these compounds, while O-alkylation did
not significantly affect antipyroptotic activity of this class of
compounds.
Such results could indicate that these compounds prevent
pyroptosis of THP-1 cells by the same mechanism of action.
The affinities for the binding of these compounds to their
Lastly, the role of the R2 substituent was studied. Cell death
of THP-1 cells was not decreased by β-iodo derivatives 37 and
G
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Time-course and concentration−response curves for the
effects of representative compounds on the ATP-triggered cell death of
THP-1 cells. PMA-differentiated and LPS-primed THP-1 cells were
pretreated with vehicle alone or indicated compounds. (A) For the
time-course experiments, each compound was added at 10 μM, 15−90
min before exposure to ATP (5 mM; 1 h). (B) For the concentration−
response experiments, each compound was added at increasing
concentrations (0.001−10 μM) 1 h before exposure to ATP (5
mM; 1 h). LDH activity was measured in the collected supernatant by
Figure 6. Concentration−response curves of the effects of
representative compounds on the nigericin-triggered cell death of
THP-1 cells. PMA-differentiated and LPS-primed THP-1 cells were
pretreated with vehicle alone or indicated compounds. Each
compound was added at increasing concentrations (0.001−10 μM)
1 h before exposure to nigericin (10 μM; 1.5 h). LDH activity was
measured in the collected supernatant by a colorimetric assay. The
◆
■
□
●
effects of representative compounds (5 ( ), 8 ( ), 9 ( ), 10 ( ), 32
○
▲
△
▼
( ), 33 ( ), 41 ( ), and 42 ( )) are shown. Data are expressed as
decrease of cell death vs vehicle alone, and are the mean SEM of at
least four experiments run in triplicate.
◆
a colorimetric assay. The effects of representative compounds (5 ( ),
■
□
●
○
▲
△
▼
8 ( ), 9 ( ), 10 ( ), 32 ( ), 33 ( ), 41 ( ), and 42 ( )) are
shown. Data are expressed as decrease of cell death vs vehicle alone
and are the mean SEM of at least four experiments run in triplicate.
was significantly (p < 0.01) lower when cells were exposed to
nigericin.
Data indicate that inhibition of the ATP-triggered events
upstream of the NLRP3 inflammasome and caspase-1
activation significantly contributes to the antipyroptotic activity
of the synthesized compounds.
Chemical Reactivity of 9 as Michael Acceptor. The
biological action of covalent drugs could be correlated to their
ability to react with nucleophilic thiols of functional/regulatory
cysteine residues in protein targets. As a result, the in vitro
reactivity of 9, chosen as a model of this class of compounds,
with glutathione (GSH), was investigated. The mechanism of
the reaction between thiols and MBH adducts can follow
several pathways: (i) −OH group direct displacement, (ii) acyl
substitution, (iii) Michael conjugate addition, and (iv) Michael
addition followed by −OH displacement with allylic rearrange-
ment (Figure 7A). Products distribution depends on the
reaction conditions employed.41
Table 3. pEC50 for the Effects on the ATP- or Nigericin-
Triggered Cell Death of THP-1 Cells of Representative
Compounds 8−10, 32, 33, 41, 42, and reference 5
pEC50
compd
ATP
Nigericin
5
6.9 0.1
6.5 0.3
6.9 0.1
6.8 0.1
6.9 0.1
7.1 0.1
6.9 0.3
7.0 0.3
6.3 0.2
6.7 0.2
6.8 0.1
6.6 0.2
6.9 0.3
6.7 0.7
6.8 1.5
6.3 1.3
8
9
10
32
33
41
42
a
PMA-differentiated and LPS-primed THP-1 cells were pretreated
with vehicle alone or indicated compounds (0.001−10 μM) 1 h before
exposure to either ATP (5 mM; 1 h) or nigericin (10 μM, 1.5 h). Cell
death was determined by measuring LDH activity in the collected
supernatant by a colorimetric assay. Data are the mean SEM of at
least four experiments run in triplicate.
To mimic the physiological conditions, a solution of 9 in
D2O/DMSO-d6 was treated with a solution containing 3 mol
equiv of GSH in pD 7.4-buffered D2O. The reaction mixture
was incubated at 37 °C, and the disappearing of double bond
1
1
molecular targets did not significantly differ, as indicated by the
comparable EC50 values. The relative efficacy was related to the
rate of action underlying the antipyroptotic effect: the faster the
action, the higher the efficacy.
signals was monitored by H NMR (Figure 7B). H NMR
spectra were recorded after 5 min, 30 min, 60 min, 120 min,
360 min, and 24 h to follow the progress of the reaction. The
time-dependent decrease in the intensity of signals referred to
H1, H2, and H3 of 9 (Figure 7A), and the simultaneous
appearance of new signals: H1′ at 5.48−5.44 ppm (9c) and H1″
7.92 ppm (9d) confirmed the ability of 9 to react with
biologically relevant −SH groups in Michael-type addition.
Under these conditions, we identified the diastereomeric
mixture of derivatives 9c as the major product, together with a
little amount of the rearranged compound 9d (Figure 7A,B).
Indeed, a similar behavior was previously observed by studying
the diterpenoid jolkinolide D.42 The difference in reaction
kinetics is strongly dependent on the medium used. In running
the experiment in D2O/DMSO-d6 (9/1), about 6 h were
required to reach half of the initial concentration of 9, while,
when the experiment was carried out in a D2O/DMSO-d6 (1/
3) mixture, t1/2 for 9 was estimated to be around 4 h (Figure
Effects of Synthesized Compounds on Nigericin-
Triggered Cell Death of THP-1 Cells. To better understand
the antipyroptotic activity of this class of compounds, the
effects of selected compounds 8−10, 32, 33, 41, and 42 were
studied on the nigericin-triggered cell death40 of PMA-
differentiated and LPS-primed THP-1 cells. As shown in
Figure 3B, LDH release was significantly (p < 0.01 vs vehicle
alone) increased by nigericin (10 μM, 1.5 h), and the amplitude
of the effect was comparable to that caused by ATP. Nigericin-
triggered cell death was prevented by the synthesized
compounds (0.001−10 μM) in a concentration-dependent
manner (Figure 6), and comparable pEC50 were calculated
(Table 3). However, compared to the ATP-triggered cell death
(Figure 5B), the amplitude of the maximal antipyroptotic effect
H
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. (A) Possible pathways for the reaction of 9 with glutathione (GSH). (B) Reaction of 9 with GSH (3 equiv) in pD 7.4 D2O/DMSO-d6 (9/
1
1) at 37 °C monitored by H NMR at different time intervals. (C) Reaction of 9 with GSH (3 equiv) in pD 7.4 D2O/DMSO-d6 (1/3) at 37 °C
monitored at different time intervals. (D) 9 (a) was reacted with GSH (1 equiv). After 72 h of equilibration, the obtained adduct (b) was treated
with NEM (10 equiv) in pD 7.4 D2O/DMSO-d6 (1/3) at 37 °C. Reaction was monitored after 2 h (c) and 24 h (d) of NEM addition.
Table 4. Second-Order Reaction Rate Constants for the Reaction of Representative Compounds 5, 9, 12, 18, 24, 34−36, 40, and
42 with Cysteamine in the Kinetic Chemoassay and Antipyroptotic Activity Measured in THP-1 Cells
a
b
compd
k2 (M−1 s−1
)
pyroptosis decrease (%)
W
R1
R2
−H
−H
−H
X
5
47.9 2.9
100.3 6.3
12.1 1.2
75.1 2.6
83.0 3.7
52.5 2.0
45.8 1.6
26.8 0.3
36.2 4.2
31.2 6.8
42.0 6.9
34
9
7.16 0.92
0.866 0.006
0.777 0.005
0.588 0.043
0.524 0.031
0.423 0.025
0.126 0.003
0.126 0.005
0.117 0.006
2-Cl-C6H4−
2-Cl-C6H4−
2-Cl-C6H4−
2-Cl-C6H4−
C6H5−
2-MeO-C6H4−
2-Cl-C6H4−
2-Cl-C6H4−
2-Cl-C6H4−
−COEt
−COOEt
−CN
−COOEt
−COOEt
−COOEt
−COOEt
−CONHPr
−COOH
−H
−H
−H
−H
−H
−H
−H
−H
−H
24
40
12
18
42
36
35
(Z)-Ph
−H
−H
(Z)-Me
−H
−H
a
Compounds (0.250 mM) were reacted with equimolar amount of cysteamine in PBS pH 7.4 containing 2.5% DMSO at 37 °C. The decrease in free
(nonreacted) cysteamine was monitored using DTNB reagent at different time points during 90 min. Second-order rate constants, calculated
according to the eq 1/cSH = 1/c0SH + kt, were determined from 5−10 data points per rate plot and are expressed as mean SEM of at least three
b
separate experiments. PMA-differentiated and LPS-primed THP-1 cells were pretreated with either vehicle alone or each compound (10 μM; 1 h).
Pyroptosis was triggered with ATP (5 mM) and LDH activity was measured in the collected supernatant 1 h after ATP exposure by a colorimetric
assay. Data are expressed as percent of pyroptosis decrease vs vehicle alone.
I
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7C). This finding suggests that the properties of the
microenvironment, in the surroundings of a molecular target,
might influence the reactivity and consequently the biological
properties of these electrophilic derivatives.
behavior, showed a poor ability to prevent pyroptotic cell death.
Subgroup analysis allowed further reactivity−activity correla-
tion. Comparison of R1-modulated compounds 9, 24, 35, and
36 evidenced that strong electron withdrawing ester and cyano
groups conferred higher electrophilicity and pharmacological
activity to compounds 9 and 24 with respect to carboxy- and
carboxyamido-substituted compounds 35 and 36 (Table 4).
When we compare ester derivatives 9, 12, and 18, modulated in
the W position, again their activity increase with increasing
reactivity, ranking the order as 18 < 12 < 9 (Table 4). Finally,
comparison of R2-substituted compounds confirmed this trend,
the order of antipyroptotic efficacy being in keeping with the
ranked reactivity 9 > 40 > 42 (Table 4). Finally, reference 5
proved the most reactive agent of the series as well as the best
antipyroptotic compound.
To verify the reversibility of the reaction with thiols, a
dynamic-equilibrium experiment was performed. A mixture of 9
and GSH (1 mol equiv) was equilibrated for 72 h in a pD 7.4
mixture of D2O/DMSO-d61 (1/3), and the progress of the
reaction was monitored by H NMR. After this time, 10 mol
equiv of N-ethyl maleimide (NEM), a highly reactive thiol trap,
1
were added and H NMR spectra were recorded at different
intervals of time during 24 h. In these conditions, no evidence
of increasing of signals relative to the conjugated double bond
was observed (Figure 7D). The same results were obtained
upon 1:10 dilution of the reaction mixture (Supporting
Information, Figure S2). According to our data, these
compounds can be assumed to act as covalent, irreversible
thiol-trapping agents. The kinetics of their reactivity, together
with their physicochemical properties, might discriminate in
their biological action.
Assessment of the Michael Acceptor Activity. To
determine the chemical reactivity of the electrophilic fragments
in thia-Michael additions, we used a 96-well-format spectro-
photometric kinetic assay. We adopted a kinetic assay able to
estimate the reactivity of compounds toward cysteamine, used
as a model of a reactive thiol contained in proteins. In our assay,
the time-dependent decrease in free cysteamine concentration
was measured through the use of 5,5′-dithio-bis(2-nitrobenzoic
acid) (DTNB), which is able to react with cysteamine SH
group.43 Compounds 5, 9, 12, 18, 24, 34−36, 40, and 42,
showing widely modulated antipyroptotic activity, were tested
to verify whether different pharmacological activity was related
to a different behavior as Michael acceptors. Selected
compounds were mixed with an equimolar amount of
cysteamine in pH 7.4 phosphate-buffered solution at 37 °C.
The progress of the reaction was monitored adding DTNB
reagent at different time points over a period of 90 min.
Second-order rate constant (k2) were obtained from the
second-order eq 1:
These data indicate that the use of kinetic cysteamine
chemoassay might help to predict the antipyroptotic activity of
acrylate-derived electrophiles.
Effects of Compounds 5, 9, 24, 35, and 42 on
Caspase-1, NLRP3 ATPase Activity, and Keap1-Nrf2
Activation. Pyroptotic cascade involves a number of
interacting proteins and NLRP3 and caspase-1 play crucial
roles in the execution of this cascade.3,4 Interestingly, NLRP3
and caspase-1 possess 45 and 12 Cys residues, respectively.
Alkylation of the Cys285 of caspase-1 has been demonstrated
to mediate the inhibitory effects of 5 on this protease.29 More
recently, 6 has been demonstrated to bind to NLRP3.30 On
these bases, caspase-1 and NLRP3 have been considered as
potential targets relating to the antipyroptotic activity of the
synthesized compounds. To investigate the mode of action of
the synthesized warheads, we checked the effects of
representative compounds 5, 9, 24, 35, and 42, selected on
the basis of their distinct chemical structures and antipyroptotic
efficacy, on caspase-1 and NLRP3 ATPase activity. Compared
to vehicle alone, caspase-1 activity, measured as the rate of
liberation of the para-nitroanilide from the caspase-1 labeled
substrate N-acetyl-Tyr-Val-Ala-Asp-para-nitroanilide (Ac-
YVAD-pNA), was decreased by these compounds at 10 μM
concentration (Supporting Information, Figure S3). The
magnitude of the inhibitory effects was in the range 25.4
3.2% (42) to 36.2 4.1% (24), with no significant difference
among them (Figure 8). Significant differences were
determined when the inhibition of the NLRP3 ATPase activity
was measured (Figure 8). In particular, compared to 5 (25.9
9.1%), a significantly more pronounced inhibition was exerted
by 9 (51.8 10.6%; p < 0.05), 24 (80.5 9.6%; p < 0.01), and
1
cSH
1
=
+ k2t
cS0H
(1)
where cSH is the concentration of nonreacted cysteamine at
time t, and c0SH is the initial concentration of cysteamine
employed. Obtained second-order rate constants are reported
in Table 4.
35 (80.2
exerted by 42 (23.3
1.4%; p < 0.01), while comparable effects were
Data analysis shows that Michael acceptor reactivity of tested
compounds paralleled well their antipyroptotic efficacy, with
the notable exception of compound 34, whose activity is
overwhelmed by an extremely high degree of cytotoxicity. In
particular, compounds 9 and 24, endowed with relatively high
k2 values (0.866 0.006 to 0.777 0.005 M−1 s−1), showed
high antipyroptotic activity. This activity was significantly
higher than that showed by slow reacting electrophiles 36, 42,
and 35 (k2 values 0.126 0.005 to 0.117 0.006 M−1 s−1).
The faster reaction rate exhibited by ester 9, compared to
corresponding nitrile 24, might be explained by the presence of
the β-OH group contributing, via an H-bonding effect, to a
stronger activation of the COOEt unit compared to the CN
unit. Compounds 40, 12, and 18, endowed with intermediate k2
23.3%). It is noteworthy that the
inhibition of caspase-1 and NLRP3 ATPase activity does not
strictly correlate with the antipyroptotic effects exerted by these
compounds (see Table 2). These findings, together with our
results on the pyroptosis triggered by nigericin and ATP, and
the published data on the complexity of the NLRP3
inflammasome regulatory circuits,15,27 suggest that the
combination of multiple actions on different targets should
be considered to explain the pharmacological efficacy of these
compounds.
Activation of the Keap1-Nrf2 pathway plays a major role in
cellular defense against oxidants and electrophiles. Under
normal conditions, Keap1 (Kelch-like ECH-associated protein
1) binds to Nrf2 (NF-E2-related factor 2) in the cytoplasm and
promotes the ubiquitination of Nrf2. Subsequently, Nrf2
protein is degraded by the proteasome. Keap1 possesses 27
values (0.588
0.043 to 0.423
0.025 M−1 s−1), behave
accordingly as mild antipyroptotic agents, with the partial
exception of compound 18, which, in spite of a fair electrophilic
J
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
60 min) with increasing concentration (0.01−10 μM) of either
5 or 9, then caspase-1 activity was measured. The kinetic
constants for the irreversible first-order reaction, kinact, and the
dissociation constants for compound binding to free enzyme, Ki
= (k−1/k1), were obtained from the observed rate constant for
inhibition, kobs, at each concentration (Figure 9). Both
Figure 8. Effects of synthesized compounds on caspase-1 (white bar)
and NLRP3 ATPase activity (black bar). Reactions were carried out in
96-well plates in 100 μL final volume at 37 °C. For the caspase-1
activity, enzyme from THP-1 cell cultures (50 U) was preincubated
(15 min) with the representative compounds (10 μM) in the reaction
buffer. Reaction rate was determined by monitoring continuously
liberation of the chromophore pNA from the caspase-1 labeled
substrate Ac-YVAD-pNA (200 μM; 1 h) at 405 nm. For the NLRP3
ATPase assay, recombinant NLRP3 (0.01 μg) was preincubated with
the same compounds (10 μM) for 15 min. ATP (250 μM) was added,
and the reaction mixtures were further incubated for 1 h at 37 °C. The
hydrolysis of ATP by NLRP3 was determined by measuring Pi content
in the reaction mixtures with Pi ColorLock Gold phosphate detection
system. Data are expressed as percentage on inhibition of enzymatic
activity vs vehicle alone and are the mean SEM of at least three
experiments run in duplicate. *p < 0.05, **p < 0.01 vs 5.
Figure 9. Time-dependency of the inhibition of caspase-1 activity by 5
and 9. Enzyme from THP-1 cell cultures (50 U) was preincubated
(15−60 min) with increasing concentration (0.01−10 μM) of either 5
( ) or 9 ( ), then caspase-1 activity was measured by monitoring
continuously liberation of the chromophore pNA from the caspase-1
labeled substrate Ac-YVAD-pNA (200 μM; 1 h) at λ= 405 nm. The
kinetic constants for the irreversible first-order reaction, kinact, and the
dissociation constants for compound binding to free enzyme, Ki =
(k−1/k1), were obtained from the observed rate constant for inhibition,
kobs, at each concentration.
○
●
compounds inhibited caspase-1 activity in a concentration-
and time-dependent manner with kinact of 3.2 1.1 s−1 (9), 4.0
0.6 s−1 (5), Ki of 9.6 3.3 μM (9), and 10.6 1.6 μM (5).
Cys residues, a subset of which (Cys151, Cys273, and Cys288)
is known to be reactive toward electrophiles.
When Nrf2 inducers inactivate Keap1 via the modification of
Cys residues, Nrf2 is stabilized, and de novo synthesized Nrf2
translocates into the nucleus. Nrf2 heterodimerizes with small
Maf proteins (musculoaponeurotic fibrosarcoma protein) and
activates target genes through antioxidant response elements,
exerting cytoprotective effects against various toxic insults.44
To evaluate whether the synthesized compounds activate the
Keap1-Nrf2 pathway, THP-1 cells were exposed (1−4 h) to
compound 9 (10 μM; here adopted as model compound) and
the expression level of marker genes encoding for Nrf2
(NFE2L2) and heme oxygenase-1 (HMOX1) was evaluated by
RT-PCR analysis. As shown in Supporting Information, Figure
S4, the expression of these genes was significantly (p < 0.01)
increased by compound 9, thus indicating that Keap1-Nrf2
pathway is indeed activated by this electrophilic compound.
However, an exposure time longer than 2 h (time-interval used
to evaluate the antipyroptotic effect) was required to measure
significant responses. Therefore, these results indicate that
activation of Keap1-Nrf2 does not contribute to the
antipyroptotic effects of this class of compounds.
CONCLUSIONS
■
Using an electrophilic fragment-based strategy, a focused library
of 36 α,β-unsaturated carbonyl- or cyano-derivatives (7−42)
was designed and prepared. Our studies showed that the
biological activity of this series of compounds is dependent on
their reactivity as Michael acceptors. Moreover, the pharmaco-
logical evaluation and the devised SAR showed that their
antipyroptotic activity and cytotoxicity can be finely modulated
through suitable choice of the substitution pattern.
Compounds 9, 15, and 16 proved interesting antipyroptotic
agents endowed with an overall more favorable pharmaco-
logical profile with respect to 5, with 9 emerging as a promising
lead amenable of further chemical development. Mechanisti-
cally we demonstrated the ability of 9, and of other selected
compounds of the series (24, 35), to inhibit NLRP3 ATPase
and caspase-1 activities; the lack of a direct correlation between
the extent of the inhibition and the antipyroptotic activity
suggest that additional proteins related to the NLRP3
inflammasome activation might be targeted by these com-
pounds. A network-based model involving not only multiple
targets but also synergistic interactions among these targets
should be considered to explain the antipyroptotic activity of
these compounds.
Taken together, these results suggest that a complex
mechanism should be considered to explain their mechanism
of action. It is reasonable that additional proteins related to the
NLRP3 inflammasome activation might be targeted by these
compounds.
Time-Dependency of the Effects of 5 and 9 on
Caspase-1 Activity. The above findings suggest that the
assessed compounds could act as irreversible inhibitors through
a time-dependent covalent addition to the thiol group of
catalytic/regulatory cysteine residues in molecular targets.
Therefore, the time-dependency of the inhibition of caspase-1
activity by 5 and 9 was studied. Enzyme was preincubated (15−
Chemoproteomic studies with 9, used as a probe, are in
progress to identify its cellular targets; the results will allow
further pharmacochemical modulation.
EXPERIMENTAL SECTION
■
General Methods. All the reactions were monitored by TLC on
Merck 60 F254 (0.25 mm) plates, which were visualized by UV
K
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
inspection and/or by spraying KMnO4 (0.5 g in 100 mL 0.1 N
NaOH). Flash chromatography (FC) purifications were performed
using Fluka silica gel with 60 mesh particles. 1H and 13C NMR spectra
were registered on Bruker Avance 300 spectrometer, at 300 and 75
MHz, respectively. Chemical shifts (δ) are given in ppm, calibrated to
tetramethylsilane (TMS) as internal standard. Low-resolution mass
spectra were recorded on a Finnigan-MAT TSQ700 in chemical
ionization mode (CI) using isobutane. GC-MS analyses were carried
out on a Agilent Technologies 6850 Network GC System gas-
chromatograph with 5973 network mass selective detector. ESI-mass
spectra were recorded on a Waters Micromass ZQ equipped with an
ESI source. Melting points were measured with a capillary apparatus
2-(Hydroxy(phenyl)methyl)acrylonitrile (27). The product was
obtained using benzaldehyde in 7 days. The crude product was
purified by FC eluting with PE/EtOAc from 9.5/0.5 to 8/2 to afford
27 as a colorless oil (0.690 mmol, 109.8 mg, 69% yield).
2-(Hydroxy(naphthalen-1-yl)methyl) acrylonitrile (28). The prod-
uct was obtained using 1-naphthaldehyde in 7 days. The crude product
was purified by FC eluting with PE/EtOAc from 9/1 to 8/2 to afford
28 as a yellowish oil (0.720 mmol, 150.7 mg, 72% yield).
2-((2,6-Dichlorophenyl)(hydroxy)methyl)acrylonitrile (29). The
product was obtained using 2,6-dichlorobenzaldehyde in 66 h. The
crude product was purified by FC eluting with PE/EtOAc from 9.5/
0.5 to 8/2 to afford 29 as a colorless oil (0.210 mmol, 47.9 mg, 21%
1
yield). MS/CI (isobutane): [M − H]+ 228. H NMR (CDCl3): δ
(Buchi 540). Purity of compounds was checked by UHPLC
̈
7.38−7.23 (m, 3H, ArH), 6.16 (s, 1H, CCH), 6.12 (s, 1H, C
CH), 6.06 (s, 1H, CH), 3.30 (br,1H, OH). 13C NMR (CDCl3): δ,
135.5, 133.8, 131.2, 131.1, 131.0, 124.1, 116.9, 70.7. Anal.
(C10H7Cl2NO) C, H, N.
(PerkinElmer) Flexar 15, equipped with UV−vis diode array detector
using a Acquity UHPLC CSH Phenyl-Hexyl 1.7 μm 2.1 mm × 50 mm
column (Waters) and H2O/CH3CN and H2O/CH3OH solvent
systems. Detection was performed at λ = 200, 215, and 254 nm.
The analytical data confirmed that the purity of the products was
≥95%. Reference compound 5 was from Enzo Biochem, Farmingdale,
NY, USA.
2-((2,4-Dichlorophenyl)(hydroxy)methyl)acrylonitrile (30). The
product was obtained using 2,4-dichlorobenzaldehyde in 68 h. The
crude product was purified by FC eluting with PE/EtOAc from 9.9/
0.1 to 7/3 to afford 30 as a yellowish oil (0.370 mmol, 84.4 mg, 37%
yield).
General Procedure for the Preparation of Compounds 9−19
and 25. To a stirred solution of appropriate aldehyde (1.00 mmol) in
CH3CN (2.7 mL), activated olefin (3.00 mmol) and water (0.3 mL)
were added. DABCO (112 mg, 1.00 mmol) was then added to the
mixture, and the reaction was allowed to stir at room temperature for 7
days. The mixture was diluted with CH2Cl2 (25 mL) and washed with
1 N HCl (3 × 25 mL) and brine (30 mL), dried (Na2SO4), and the
solvent evaporated under reduced pressure. The residue was purified
by FC as indicated. Characterization data for literature compounds 9,
16,48 11,49 14,46 18,40 10, 12, and 19,51 are reported in Supporting
Information.
Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)acrylate (9). The
product was obtained using 2-chlorobenzaldehyde and ethyl acrylate.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 9 as a colorless oil (0.670 mmol, 161.3 mg, 67% yield).
Ethyl 2-((2-Fluorophenyl)(hydroxy)methyl)acrylate (10). The
product was obtained using 2-fluorobenzaldehyde and ethyl acrylate.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 10 as a yellowish oil (0.709 mmol, 159.2 mg, 71% yield).
Ethyl 2-((2-Bromophenyl)(hydroxy)methyl)acrylate (11). The
product was obtained using 2-bromobenzaldehyde and ethyl acrylate.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 11 as a colorless oil (0.959 mmol, 273.7 mg, 96% yield).
Ethyl 2-((Phenyl)(hydroxy)methyl)acrylate (12). The product was
obtained using benzaldehyde and ethyl acrylate. The crude product
was purified by FC eluting with PE/EtOAc 9/1 to afford 12 as a
colorless oil (0.450 mmol, 92.8 mg, 45% yield).
Ethyl 2-((3-Chlorophenyl)(hydroxy)methyl) acrylate (13). The
product was obtained using 3-chlorobenzaldehyde and ethyl acrylate.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 13 as a colorless oil (0.670 mmol, 161.3 mg, 67% yield). MS/CI
(isobutane): [M − H]+ 241. 1H NMR (CDCl3): δ 7.41 (s, 2H, ArH),
7.36−7.21 (m, 2H, ArH), 6.40 (s, 1H, CCH), 5.91 (s, 1H, C
CH), 5.53 (d, 1H, J = 5.4 Hz, CH), 4.20 (q, 2H, J = 7.1 Hz), 3.66 (d,
1H, J = 5.6 Hz, OH), 1.29 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3): δ
166.4, 143.8, 141.9, 134.5, 129.9, 128.2, 127.0, 126.6, 125.1, 72.9, 61.4,
14.3. Anal. (C12H13ClO3) C, H.
General Procedure for the Preparation of Compounds 7, 8,
22, and 23. To a stirred solution of aldehyde (1.00 mmol) in 1,4-
dioxane (4.5 mL), activated olefin (3.00 mmol) and water (4.5 mL)
were added. DABCO (112 mg, 1.00 mmol) was then added to the
mixture, and the reaction was allowed to stir at room temperature until
TLC showed complete or stable consumption of the aldehyde (2 h to
3 weeks). The mixture was diluted with CH2Cl2 (3 × 25 mL) and
washed with water (25 mL) and brine (30 mL), dried (Na2SO4), and
the solvent evaporated under reduced pressure. The residue was
purified by FC as indicated. Characterization data for literature
compound 7,45 8,46 22,47 and 2348 are reported in Supporting
Information.
Ethyl 2-(Hydroxy(pyridin-2-yl)methyl)acrylate (7). The product
was obtained using 2-pyridinecarboxyaldehyde and ethyl acrylate in 2
h. The crude product was purified by FC eluting with PE/EtOAc from
9/1 to 1/1 to afford 7 as a yellowish oil (0.480 mmol, 99.5 mg, 48%
yield).
Ethyl 2-(Hydroxy(2-nitrophenyl)methyl)acrylate (8). The product
was obtained using 2-nitrobenzaldehyde and ethyl acrylate in 72 h.
The crude product was purified by FC eluting with PE/EtOAc from 9/
1 to 7/3 to afford 8 as a yellowish oil (0.600 mmol, 150.7 mg, 60%
yield).
2-(Hydroxy(pyridin-2-yl)methyl)acrylonitrile (22). The product
was obtained using 2-pyridinecarboxyaldehyde and acrylonitrile in 20
h. The crude product was purified by FC eluting with PE/EtOAc 6/4
to afford 22 as a yellowish oil (0.700 mmol, 112.1 mg, 70% yield).
2-(Hydroxy(2-nitrophenyl)methyl) acrylonitrile (23). The product
was obtained using 2-nitrobenzaldehyde and acrylonitrile after 24 h.
The crude product was purified by FC eluting with PE/EtOAc 7/3 to
afford 23 as a yellowish oil (0.440 mmol, 89.8 mg, 44% yield).
General Procedure for the Preparation of Compounds 24
and 26−30. To a stirred solution of appropriate aldehyde (1.00
mmol) in THF (2 mL), acrylonitrile (197 μL, 3.00 mmol), MeOH (2
mL), and water (4 mL) were added. DABCO (112 mg, 1.00 mmol)
was then added to the mixture, and the reaction was allowed to stir at
room temperature until TLC showed complete or stable consumption
of the aldehyde (23−166 h). The mixture was diluted with CH2Cl2 (3
× 25 mL) and washed with water (25 mL) and brine (30 mL), dried
(Na2SO4), and the solvent evaporated under reduced pressure. The
crude product was purified by FC as indicated. Characterization data
for literature compounds 24, 27, 30,48 26,49 and 2850 are reported in
Supporting Information.
2-((2-Chlorophenyl)(hydroxy)methyl)acrylonitrile (24). The prod-
uct was obtained using 2-chlorobenzaldehyde in 23 h. The crude
product was purified by FC eluting with PE/EtOAc 8/2 to afford 24 as
an off-white solid (0.970 mmol, 187.8 mg, 97% yield).
2-((2-Bromophenyl)(hydroxy)methyl)acrylonitrile (26). The prod-
uct was obtained using 2-bromobenzaldehyde in 7 days of reaction.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 26 as an off-white solid (0.960 mmol, 228.6 mg, 96% yield).
Ethyl 2-((4-Chlorophenyl)(hydroxy)methyl)acrylate (14). The
product was obtained using 4-chlorobenzaldehyde and ethyl acrylate.
The crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 14 as a colorless oil (0.380 mmol, 91.5 mg, 38% yield).
Ethyl 2-((2,6-Dichlorophenyl)(hydroxy)methyl)acrylate (15). The
product was obtained using 2,6-dichlorobenzaldehyde and ethyl
acrylate. The crude product was purified by FC eluting with PE/
EtOAc 9/1 to afford 15 as a white solid (0.360 mmol, 99.0 mg, 36%
1
yield); mp 71.6−72.1 °C. GC-MS: [M]+ 274, rt, 26.3 min. H NMR
(CDCl3): δ 7.32−7.24 (m, 2H, ArH), 7.14 (dd, 1H, J = 8.7, 7.3 Hz,
ArH), 6.41 (d, 1H, J = 1.1 Hz, CCH), 6.30 (s, 1H, CH), 5.86 (d,
L
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H, J = 1.1 Hz, CCH), 4.12 (q, 2H, J = 8.6 Hz, OCH2), 3.73 (s, 1H,
OH), 1.23 (t, 3H, J = 8.6 Hz, CH3). 13C NMR (CDCl3): δ 166.0,
139.7, 135.6, 129.4, 128.9, 126.4, 69.7, 61.3, 53.6, 13.9. Anal.
(C12H12Cl2O3) C, H.
Ethyl 2-((2,4-Dichlorophenyl)(hydroxy)methyl)acrylate (16). The
product was obtained using 2,4-dichlorobenzaldehyde and ethyl
acrylate. The crude product was purified by FC eluting with PE/
EtOAc 9/1 to afford 16 as a colorless oil (0.470 mmol, 129.3 mg, 47%
yield).
Ethyl 2-((3,5-Dichlorophenyl)(hydroxy)methyl)acrylate (17). The
product was obtained using 3,5-dichlorobenzaldehyde and ethyl
acrylate. The crude product was purified by FC eluting with PE/
EtOAC 9.5/0.5 to afford 17 as a colorless oil (0.330 mmol, 90.8 mg,
33% yield). MS/CI (isobutane): [M − H]+ 275. 1H NMR (CDCl3): δ
7.26 (s, 3H, ArH), 6.38 (s, 1H, CCH), 5.86 (s, 1H, CCH), 5.45
(s, 1H, CH), 4.19 (q, 2H, J = 7.1 Hz, OCH2), 3.46 (s, br, 1H, OH),
1.24 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (CDCl3) δ 166.0, 144.9,
141.5, 134.9, 127.8, 127.2, 125, 72.4, 61.3, 14.2. Anal. (C12H12Cl2O3)
C, H.
Ethyl 2-((Hydroxy)(2-methoxyphenyl)methyl)acrylate (18). The
product was obtained using 2-methoxybenzaldehyde and ethyl
acrylate. The crude product was purified by FC eluting with PE/
EtOAc 9/1 to afford 18 as a colorless oil (0.0698 mmol, 16.5 mg, 7%
yield).
Ethyl 2-((Hydroxy)(o-tolyl)methyl)acrylate (19). The product was
obtained using neat 2-tolualdehyde and ethyl acrylate. The crude
product was purified by FC eluting with PE/EtOAc 9/1 to afford 19 as
a colorless oil (0.280 mmol, 61.7 mg, 28% yield).
2-((2-Fluorophenyl)(hydroxy)methyl)acrylonitrile (25). The prod-
uct was obtained using 2-fluorobenzaldehyde and acrylonitrile. The
crude product was purified by FC eluting with PE/EtOAc 9/1 to
afford 25 as a colorless oil (0.850 mmol, 150.6 mg, 85% yield). MS/CI
(isobutane): [M − H]+ 178. 1H NMR (CDCl3): δ 7.51−7.39 (m, 2H,
ArH), 7.14−6.93 (m, 2H, ArH), 6.01 (s, 1H, CCH), 5.93 (s, 1H,
CCH), 5.52 (s, 1H, CCH), 3.97 (s, 1H, OH); 13C NMR
(CDCl3): δ 161.5, 158.2, 130.6, 127.7, 126.3, 124.8, 116.6, 115.6, 67.9.
Anal. (C10H8FNO) C, H, N.
Ethyl 3-Hydroxy-2-methylenebutanoate (20). In a closed vessel,
to a stirred solution of acetaldehyde (56 μL, 43.9 mg, 0.997 mmol) in
CH3CN (2.7 mL), ethyl acrylate (325 μL, 3.00 mmol) and water (0.3
mL) were added. DABCO (112 mg, 1.00 mmol) was then added to
the mixture, and the reaction was allowed to stir at room temperature
overnight. The mixture was diluted with CH2Cl2 (20 mL), washed
with 1 N HCl (3 × 20 mL) and water (20 mL), dried (Na2SO4), and
the solvent evaporated under reduced pressure to give 2052 as a
colorless oil (0.280 mmol, 40.4 mg, 28%). Characterization data are
reported in Supporting Information.
Ethyl 2-(Cyclohexyl(hydroxy)methyl)acrylate (21). To a stirred
solution of cyclohexanecarboxaldehyde (121 μL, 112.1 mg, 1.00
mmol) in formamide (0.2 mL), ethyl acrylate (109 μL, 1.01 mmol)
and DABCO (112 mg, 1.00 mmol) were added and the reaction was
allowed to stir at room temperature under nitrogen for 69 h. The
mixture was diluted with Et2O (25 mL), washed with 1 N HCl (25
mL) and water (20 mL), dried (Na2SO4), and the solvent evaporated
under reduced pressure. The residue was purified by FC eluting with
PE/EtOAc 9.5/0.5 to obtain 21 as a colorless oil (0.330 mmol, 70.1
mg, 33% yield). GC-MS: [M]+ 212, rt, 22.6 min. 1H NMR (CDCl3): δ
6.25 (s, 1H, CCH), 5.72 (s, 1H, CCH), 4.24 (q, 2H, J = 7.1 Hz,
OCH2), 4.05 (d, 1H, J = 7.2 Hz. CH), 2.64 (br, 1H, OH), 1.98 (d, 1H,
J = 13.0 Hz, cy-CH), 1.88−146 (m, 6H, H cy), 1.33 (t, 3H, J = 7.1 Hz,
CH3), 1.26−0.86 (m, 4H, H cy). 13C NMR (CDCl3): δ 166.7, 141.6,
125.7, 76.6, 60.7, 42.5, 29.9, 28.1, 26.3, 26.1, 25.9, 14.1. Anal.
(C12H20O3) C, H.
evaporated under reduced pressure. The residue was purified by FC
eluting with PE/EtOAc 9/1 to obtain the desired products.
2-((2-Chlorophenyl)(3-ethoxy-3-oxopropoxy)methyl)acrylate
(31). The product (0.0399 mmol, 13.6 mg, 4%) was obtained using 2-
chlorobenzaldehyde and ethyl acrylate as a colorless oil. GC-MS: [M]+
340, rt, 24.4 min. 1H NMR (CDCl3): δ 7.47−7.06 (m, 4H, ArH), 6.38
(s, 1H, CCH), 5.71 (s, 1H, CH), 5.68 (s, 1H, CCH), 4.31−3.97
(m, 4H, 2OCH2), 3.92−3.61 (m, 2H, CH2), 2.60 (t, J = 6.4 Hz, 2H,
CH2), 1.27−1.21 (m, 6H, 2CH3). 13C NMR (CDCl3): δ 171.4, 165.7,
140.1, 137.4, 133.9, 129.5, 129.1, 128.6, 126.9, 126.5, 75.8, 65.4, 60.8,
60.5, 35.2, 14.2, 14.1. Anal. (C17H21ClO5) C, H.
2-((2,6-Dichlorophenyl)(3-ethoxy-3-oxopropoxy)methyl)acrylate
(32). The product (0.110 mmol, 41.3 mg, 11% yield) was obtained
using 2,6-dichlorobenzaldehyde and ethyl acrylate as a colorless oil.
GC-MS: [M]+ 374, rt, 30.1 min. 1H NMR (CDCl3): δ 7.51−7.21 (m,
2H, ArH), 7.16 (dd, 1H, J = 8.7, 7.2 Hz, ArH), 6.47 (d, 1H, J = 1.2 Hz,
CCH), 6.04 (s, 1H, CH), 6.01 (d, 1H, J = 1.2 Hz, CCH), 4.32−
4.01 (m, 4H, 2 CH2), 3.85−3.67 (m, 2H, CH2), 2.60 (t, J = 6.4 Hz,
2H, CH2), 1.23 (t, 3H, J = 7.1 Hz, CH3), 1.18 (t, 3H, J = 7.1 Hz CH3).
13C NMR (CDCl3): δ 171.4, 165.5, 137.6, 136.6, 133.4, 129.5, 129.3,
127.6, 76.1, 65.0, 60.7, 60.5, 35.3, 14.2, 14.0. Anal. (C17H20Cl2O5) C,
H.
2-((2,4-Dichlorophenyl)(3-ethoxy-3-oxopropoxy)methyl)acrylate
(33). The product (0.160 mmol, 60.0 mg, 16% yield) was obtained
using 2,4-dichlorobenzaldehyde and ethyl acrylate as a colorless oil.
GC-MS: [M]+ 374, rt, 30.1 min. 1H NMR (CDCl3): δ 7.39−7.23 (m,
3H, ArH), 6.39 (s, 1H, CCH), 5.72 (s, 1H, CH), 5.65 (s, 1H, C
CH), 4.23−4.07 (m, 4H, 2CH2), 3.97−3.47 (m, 2H, CH2), 2.59 (t,
2H, J = 6.3 Hz, CH2), 1.27−1.21 (m, 6H, 2CH3). 13C NMR (CDCl3):
δ 171.3, 165.5, 139.7, 135.8, 134.6, 134.2, 129.6, 129.3, 127.3, 126.6,
75.4, 65.4, 61.2, 60.5, 35.1, 14.2, 14.0. Anal. (C17H20Cl2O5) C, H.
2-((2-Chlorophenyl)(hydroxy)methyl)pent-1-en-3-one (34).
To a stirred solution of 2-chlorobenzaldehyde (113 μL, 141.0 mg,
1.00 mmol) in DMF (900 μL) and water (100 μL), ethyl vinyl ketone
(297 μL, 252.4 mg, 3.00 mmol), rac-proline (12.1 mg, 0.105 mmol),
and NaHCO3 (21 mg, 0.25 mmol) were added. The solution was
allowed to stir for 5 days at room temperature. The mixture was
diluted with water (20 mL) and extracted with CH2Cl2 (3 × 25 mL).
The combined organic layers were dried (Na2SO4), and the solvent
evaporated under reduced pressure. The product was purified by FC
eluting with PE/EtOAc (9/1) to afford 34 as a colorless oil (0.300
1
mmol, 67.4 mg, 30% yield). MS/CI (isobutane): [M − H]+ 225. H
NMR (CDCl3): δ 7.57−7.17 (m, 4H, ArH), 6.16 (d, 1H, J = 0.7 Hz,
CCH), 5.98 (d, 1H, J = 4.4 Hz, CH), 5.63 (d, 1H, J = 0.7 Hz, C
CH), 3.56 (d, 1H, J = 4.6 Hz, OH), 2.77 (q, 2H, J = 7.3 Hz, OCH2),
1.11 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (CDCl3): δ 203.1, 148.2,
138.8, 132.5, 129.4, 129.3, 128.0, 126.9, 125.9, 68.8, 31.4, 8.1. Anal.
(C12H13ClO2) C, H.
2-((2-Chlorophenyl)(hydroxy)methyl)acrylic Acid (35). To a
stirred solution of 9 (1.50 g, 6.23 mmol) in MeOH (3 mL), an
aqueous KOH 0.9 M solution (8.7 mL) was added. The mixture was
allowed to stir at room temperature overnight. The mixture was
diluted with water (20 mL) and extracted with CH2Cl2 (3 × 20 mL),
washed with brine (20 mL), dried (Na2SO4), and the solvent
evaporated under reduced pressure. The product was purified by FC
eluting with CH2Cl2/MeOH 9.5/0.5 to obtain 35 as a white solid
(4.61 mmol, 980.2 mg, 74% yield); mp 81.6−83.4 °C. 1H NMR
(DMSO-d6): δ 12.44 (br, 1H, COOH), 7.70−7.00 (m, 4H, ArH), 6.21
(s, 1H, CCH), 5.80 (s, 1H, CH), 5.64 (s, 1H, CCH), 3.44 (br,
1H, OH). 13C NMR (DMSO-d6): δ 167.8, 144.4, 141.2, 133.2, 130.0,
129.7, 129.2, 127.9, 125.3, 67.9. Anal. (C10H9ClO3) C, H.
2-((2-Chlorophenyl)(hydroxy)methyl)-N-propylacrylamide
(36). To a stirred solution of 35 (0.17 g, 0.800 mmol) in a 2/5
CH2Cl2/DMF mixture (7 mL), HBTU (455 mg, 1.20 mmol), HOBt
(160 mg, 0.120 mmol), and DIPEA (155 mg, 1.20 mmol) were
successively added. n-Propylamine (71 mg, 1.20 mmol) was then
added, and the reaction mixture was allowed to stir at room
temperature overnight. The mixture was diluted with 10% NaHCO3
aqueous solution, extracted with CH2Cl2 (3 × 20 mL), and washed
with brine (20 mL). The organic layers were combined, dried
General Procedure for the Preparation of Compounds 31−
33. To a stirred solution of aldehyde (1.00 mmol) in CH3CN (2.7
mL), activated olefin (3.00 mmol) and water (0.3 mL) were added.
DMAP (122 mg, 1.00 mmol) was then added to the mixture, and the
reaction was allowed to stir at room temperature for 7 days. The
mixture was diluted with CH2Cl2 (25 mL), washed with 1 N HCl (3 ×
25 mL) and brine (30 mL), dried (Na2SO4), and the solvent
M
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(Na2SO4), and the solvent evaporated under reduced pressure. The
product was purified by FC eluting with PE/EtOAc/MeOH 9/0.8/0.2
to afford 36 as a white solid (0.520 mmol, 131.9 mg, 65% yield); mp
82.1−83.5 °C. MS/CI (isobutane): [M − H]+ 254. 1H NMR (CDCl3)
δ 7.64 (d, 1H, J = 7.8 Hz, ArH), 7.40−7.07 (m, 3H, ArH), 6.24 (s, 1H,
NH), 5.82 (d, 1H, J = 4.9 Hz, CH), 5.70 (s, 1H, CCH), 5.26 (s, 1H,
CCH), 4.81 (d, 1H, J = 5.1 Hz, OH), 3.32−3.18 (m, 2H, CH2),
1.60−1.41 (m, 2H, CH2), 0.87 (t, 3H, J = 7.4 Hz, CH3). 13C NMR
(CDCl3): δ 162.3, 151.2, 139.1, 137.2, 136.1, 131.2, 130.4, 129.4,
128.5, 128.2, 41.2, 22.8, 11.5. Anal. (C13H16ClNO2) C, H, N.
Preparation of Compounds 37 and 38. To a stirred solution of
MgI2 (1.33 g, 4.80 mmol) in dry CH2Cl2 (19 mL) at 0 °C, a solution
of ethyl propiolate (490 μL, 474.3 mg, 4.84 mmol) in dry CH2Cl2 (10
mL) was added dropwise, followed by a solution of 2-chlorobenzalde-
hyde (562.3 mg, 4.00 mmol) in dry CH2Cl2 (5 mL). The reaction was
allowed to stir at room temperature overnight; after this time, the
mixture was treated with 10% Na2S2O3 soln (20 mL). The mixture was
then extracted with CH2Cl2 (25 mL), washed with H2O (3 × 20 mL),
and purified by FC eluting with CH2Cl2/PE 6/4.
deep blue (15 min). The phases were separated, and the aqueous layer
was further extracted with EtOAc (2 × 20 mL). The combined organic
extracts were dried (Na2SO4) and concentrated under reduced
pressure. The crude product was purified by FC eluting with PE/
EtOAc (9/1) to afford 41 as a colorless oil (0.660 mmol, 168.1 mg,
66% yield). MS/CI (isobutane): [M − H]+ 255. 1H NMR (CDCl3): δ
7.65 (d, 1H, J = 7.5 Hz, ArH), 7.36−7.14 (m, 3H, ArH), 6.97 (q, 1H, J
= 7.2 Hz, CCH), 5.93 (s, 1H, CH), 4.34 (s, 1H, OH), 4.09 (q, 2H, J
= 7.1 Hz, OCH2), 1.95 (d, 3H, J = 7.2 Hz, CCCH3), 1.20 (t, J = 7.1
Hz, 3H, CH3). 13C NMR (CDCl3): δ 167.9, 141.5, 139.4, 133.6, 133.1,
129.8, 128.9, 128.7, 127.1, 67.9, 61.3, 15.2, 14.5. Anal. (C13H15ClO3)
C, H.
(Z)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)but-2-enoate
(42). This compound was synthesized with the same procedure
reported for 41 from iodoacrylate 38 (0.620 mmol, 157.9 mg, 62%
1
yield); colorless oil. MS/CI (isobutane): [M − H]+ 255. H NMR
(CDCl3) δ, 7.53 (d, 1H, J = 7.6 Hz, ArH), 7.41−7.11 (m, 3H, ArH),
6.09 (q, 1H, J = 7.2 Hz, CCH), 5.87 (s, 1H, CH), 4.18 (q, 2H, J =
7.1 Hz, OCH2), 3.41 (s, 1H, OH), 1.98 (d, 3H, J = 7.2 Hz, C
CCH3), 1.22 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (CDCl3) δ 167.9,
139.7, 139.4, 133.6, 133.1, 129.8, 129.2, 128.5, 127.3, 71.5, 61.1, 16.1,
14.6. Anal. (C13H15ClO3) C, H.
(E)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)-3-iodoacrylate
(37). Yellowish oil (0.920 mmol, 337.3 mg, 23% yield). MS/CI
1
(isobutane): [M − H]+ 367. H NMR (CDCl3): δ 8.10 (s, 1H, C
Ethyl 3-(2-Chlorophenyl)-3-hydroxy-2-methylpropanoate
(43). To a stirred suspension of 5% Pd/C (212 mg) in EtOAc (5
mL), a solution of 9 (240.7 mg, 1 mmol) in EtOAc (5 mL) was added.
The mixture was hydrogenated (1 bar) for 10 min at room
temperature. The flask was evacuated and the mixture filtered through
Celite. The solvent was evaporated under reduced pressure, and the
obtained residue was purified by FC eluting with PE/EtOAc from 9.8/
0.2 to 7/3 to afford 43 as a yellowish oil (0.180 mmol, 43.7 mg, 18%
yield). MS/CI (isobutane): [M − H]+ 243. 1H NMR (CDCl3): δ 7.62
(d, 1H, J = 7.5 Hz, ArH), 7.40−7.12 (m, 3H, ArH), 5.52 (s, 1H, CH),
4.21 (q, 2H, J = 7.2 Hz, OCH2), 3.30 (br, 1H, OH), 3.01−2.91 (m,
1H, CH), 1.29 (t, 3H, J = 7.1 Hz, CH3), 1.06 (d, 3H, J = 7.3 Hz, CH3).
13C NMR (CDCl3): δ 176.1, 141.8, 138.6, 133.0, 128.6, 128.2, 126.8,
CH), 7.56 (dd, 1H, J = 8.54, 10.56 Hz, ArH), 7.41−7.18 (m, 3H,
ArH), 5.92 (s, 1H, CH), 4.18 (q, 2H, J = 7.1 Hz, OCH2), 3.53 (br, 1H,
OH), 1.24 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (CDCl3): δ 165.9,
143.8, 137.4, 132.7, 129.5, 129.4, 128.1, 127.1, 87.5, 71.9, 61.5, 13.9.
Anal. (C12H12ClIO) C, H.
(Z)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)-3-iodoacrylate
(38). Pale-orange oil (2.68 mmol, 982.4 mg, 67% yield). MS/CI
1
(isobutane): [M − ]+ 367. H NMR (CDCl3): δ 7.49 (d, 1H, J = 7.0
Hz, ArH), 7.43−7.21 (m, 3H, ArH), 7.16 (s, 1H, CCH), 4.25 (q, J =
7.2 Hz, 2H, OCH2), 2.47 (br, 1H, CH), 3.53 (s, 1H, OH), 1.26 (t, 3H,
CH3). 13C NMR (CDCl3): δ 163.9, 141.6, 138.0, 133.3, 129.6, 129.0,
128.3, 126.6, 101.7, 74.3, 61.8, 14.0. Anal. (C12H12ClIO) C, H.
Preparation of Compounds 39 and 40. To a stirred mixture of
compounds 37 and 38 (98.0 mg, 0.127 mmol) in DME/H2O 1/1 kept
under nitrogen at room temperature, Cs2CO3 (261 mg, 0.801 mmol),
phenylboronic acid (36.0 mg, 0.297 mmol), and Pd(OAc)2 (6.0 mg,
0.0267 mmol) were successively added. The reaction was allowed to
stir for 24 h at room temperature, then the mixture was diluted with
EtOAc (10 mL), washed with water (10 mL), dried (Na2SO4), and the
organic solvent evaporated under reduced pressure. The crude mixture
was purified by FC eluting with PE/EtOAc from 9.5/0.5 to 9/1 to
afford compound 39 (0.0609 mmol, 19.3 mg, 48% yield) as the first
eluting isomer and compound 40 (0.0559 mmol, 17.7 mg, 44% yield).
(E)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)-3-phenyl acrylate
76.5, 60.9, 47.3, 14.7, 14.3. Anal. (C12H15ClO3) C, H.
Reaction of Derivative 9 with Glutathione. To a solution of 9
(0.0500 mmol) in DMSO-d6 (50 μL), a pD 7.4-buffered solution of
GSH (0.150 mmol.) in D2O (450 μL) was added and the reaction
mixture was kept at 37 °C. The course of the reaction was followed by
1H NMR at 300 MHz. DMSO-d6 signal was used as the internal
1
standard for calibration. H NMR spectra were recorded after 5, 30,
60, 120, 240, 360 min, and 24 h. The same experiment was repeated
using DMSO-d6 (500 μL) and pD 7.4-buffered solution of GSH in
D2O (167 μL), and TMS was used as internal standard for calibration.
Relative amount of compounds were calculated by peak integration.
1
Compound 9c was not isolated: ESI-MS [M − H+] 548. H NMR
1
(39). MS/CI (isobutane): [M − H]+ 317. H NMR (CDCl3): δ 7.84
(D2O/DMSO-d6 9/1): δ 7.64−7.32 (m, 4H, ArH), 5.48−5.44 (m, 1H,
CHOH), 4.80−4.73 (m, 1H, CHCys), 4.25−3.95 (m, 2H, OCH2),
3.78−3.74 (m, 4H, CH2Gly, CH2SG), 3.33 (dd, 1H, J = 14.3, 4.2 Hz,
CHGlu), 2.98 (dd, 2H, J = 14.0, 10.0 Hz, CH2Cys), 2.92−2.79 (m,
1H, CHCH2SG), 2.59−2.48 (m, 2H, CH2Glu), 2.22−2.11 (m, 2H,
CH2Glu), 1.26−1.03 (m, 3H, CH3).
(s, 1H, CCH), 7.61 (d, 1H, J = 7.6 Hz, ArH), 7.38 (s, 5H, ArH),
7.33−7.13 (m, 3H, ArH), 6.00 (s, 1H, CH), 4.22 (q, 2H, J = 7.1 Hz,
OCH2), 3.10 (s, br, 1H, OH), 1.29 (t, 3H, J = 7.1 Hz, CH3). 13C NMR
(CDCl3): δ 168.0, 142.2, 139.3, 134.6, 133.1, 130.9, 129.7, 129.3,
129.1, 128.7, 128.6, 128.3, 126.4, 68.4, 61.2, 14.1. Anal. (C18H17ClO3)
C, H.
Reversibility of the Reaction of 9 with Glutathione. A
solution of 9 (0.100 mmol) in DMSO-d6 (3.2 mL) was treated with
pD 7.4 solution of GSH (0.100 mmol) in D2O (1.80 mL). The
mixture was stirred at 37 °C, monitoring the progress of the reaction
by 1H NMR. After 72 h, a 1H NMR spectrum was recorded. An
aliquot of the mixture (500 μL) was treated with 10 mol equiv of
(Z)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)-3-phenyl acrylate
1
(40). MS/CI (isobutane): [M − H]+ 317. H NMR (CDCl3): δ 7.66
(d, 1H, J = 7.5 Hz, ArH), 7.45−7.19 (m, 8H, ArH), 6.85 (s, 1H, C
CH), 6.01 (d, 1H, J = 2.3 Hz, CH), 4.07 (q, 2H, J = 7.1 Hz, OCH2),
2.75 (s, br, 1H, OH), 1.27 (t, 3H, J = 7.2 Hz, CH3). 13C NMR
(CDCl3): δ 168.7, 138.2, 135.6, 135.3, 134.2, 132.8, 129.5, 129.1,
128.4, 128.3, 128.2, 128.0, 127.0, 71.9, 60.9, 13.5. Anal. (C18H17ClO3)
C, H.
1
NEM dissolved in DMSO-d6 (180 μL). H NMR were recorded after
2 and 24 h of NEM addition.
Kinetic Cysteamine Chemoassay. The thiol assay was
performed in 96-well plates using 100 mM phosphate buffer (pH
7.4) with 500 μM EDTA as the solvent system. DTNB reagent was
prepared with 0.014 mmol DTNB (Sigma-Aldrich, Saint Luis, MO,
USA) and 0.5 mmol sodium hydrogen carbonate dissolved in 25 mL
of 100 mM phosphate buffer (pH 7.2). All measurements were done
in a Multilabel plate reader (Victor X4, PerkinElmer, Waltham, MA,
USA) at 37 °C. To perform the assay, DMSO solutions of compounds
(10 mM) and water solution of cysteamine (Sigma-Aldrich) (10 mM)
(E)-Ethyl 2-((2-Chlorophenyl)(hydroxy)methyl)but-2-enoate
(41). To a solution of iodoacrylate 37 (366.6 mg, 1.00 mmol) in dry
THF (6 mL) kept at −40 °C, under nitrogen, a freshly prepared 0.5 M
solution of LiCuBr2 (400 μL, 0.200 mmol) was added via a gastight
syringe. To the obtained mixture, a 3 M solution of MeMgBr in Et2O
(1.0 mL, 3.00 mmol) was added dropwise over 10 min at −40 °C. The
reaction mixture was allowed to stir for 30 min and then quenched by
dropwise addition of saturated aq NH4Cl. The mixture was diluted
with water, and the solution was stirred until the aqueous phase turned
N
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were diluted in the phosphate buffer to give a concentration of 0.5
mM. An equal amount of both solutions were combined, mixed, and
the kinetic measurements started immediately. At various time points
(over a time of 90 min), 150 μL of DTNB reagent were added and
after 1 min absorption was measured at λ = 405 nm. The
concentration of the remaining nonreacted cysteamine was determined
via a cysteamine calibration curve of thiol content vs absorbance
(cysteamine concentration ranging from 0.03 to 0.35 mM). Rate
constants of reaction between cysteamine and the electrophilic
compounds were determined employing second-order kinetics; k2
(M−1 s−1) values were gained from the time-dependent decay of
cysteamine concentration, cSH, by fitting the data of individual
experiments to the second order eq 1. For every tested compound,
the reaction rate constant was determined from at least three replicates
(separate experiments), taking 5−8 data points per rate plot for high
and low reactive compounds and 8−10 data points for compounds
with intermediate reactivity. The associated r2 values were >0.99.
Cell Cultures. Human continuous myelomonocytic THP-1 cells
were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland)
supplemented with fetal bovine serum (10%; Lonza), L-glutamine (2
mM; Lonza), penicillin (100 IU/mL; Lonza), and streptomycin (100
mg/mL; Lonza). Cell culture medium was replaced every 2−3 days,
and the cultures were maintained at 37 °C, and 5% CO2 in a fully
humidified incubator.
CHAPS, pH 7.5, 10 mM DTT) and monitoring liberation of the
chromophore pNA from the caspase-1-specific labeled substrate Ac-
YVAD-pNA (200 μM, 1 h; Enzo Biochem) in the Victor X4 at a λ =
405 nm. Time-dependent inhibition of caspase-1 activity was based on
a two-step model of a second-order reaction followed by an
irreversible first-order reaction as described.54
Measurement of NLRP3-ATPase Activity. NLRP3 ATPase
activity was measured as described by He and colleagues,30 with minor
modifications. Recombinant NLRP3 (0.01 μg; Abnova, Taipei,
Taiwan) was incubated with the assessed compounds in the reaction
buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1
mM EDTA) for 15 min. ATP (250 μM) was added, and the reaction
mixtures were further incubated for 1 h at 37 °C. The hydrolysis of
ATP by NLRP3 was determined by Pi ColorLock Gold (Innova
Bioscience, Cambridge, UK) phosphate detection system according to
the instructions.
Data Analysis. Results are expressed as either mean SEM or
median (range) of n experiments. Significance was assessed with either
ANOVA and Bonferroni’s post-test or Wilcoxon signed rank test, with
p < 0.05 as the cutoff. Concentration−response data were analyzed by
sigmoidal regression model using the software OriginPro version 8.0
(Microcal Software, Northampton, MA, USA).
ASSOCIATED CONTENT
* Supporting Information
■
Immortalized proximal tubule epithelial cell line from normal adult
human kidney were cultured in Dulbecco’s Modified Eagle Medium
(DMEM; Lonza) supplemented with fetal bovine serum (10%), L-
glutamine (2 mM), penicillin (100 IU/mL), and streptomycin (100
mg/mL). Cell culture medium was replaced every 2−3 days, and the
cultures were maintained at 37 °C and 5% CO2 in a fully humidified
incubator.
S
Effect of the ortho-C6H4-substituent, 1H NMR dilution
experiment, assay for caspase-1 activity, Keap1-Nrf2 activation
studies, characterization data of reported compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
In Vitro Models of Pyroptosis. Pyroptosis was studied as
previously described.28,40 The day before each experiment, cells were
plated in 48-well culture plates (75 × 103 cells/well) and were
differentiated into monocyte-like cells by treatment with PMA (50
nM, 24 h; Sigma-Aldrich). PMA-differentiated THP-1 cells were
washed twice with phosphate-buffered saline (PBS) and primed with
LPS (5 μg/mL, 4 h; Sigma-Aldrich) in serum-free medium. Cell death
was triggered with either ATP (5 mM, 1 h; Sigma-Aldrich) or nigericin
AUTHOR INFORMATION
Corresponding Authors
*For G.M.: phone, +39-011-6707955; fax, +39-011-6707688;
E-mail, gianluca.miglio@unito.it.
*For M.B.: phone, +39-011-6707738; fax, +39-011-6707687; E-
mail, massimo.bertinaria@unito.it.
Author Contributions
All authors have given approval to the final version of the
■
(10 μM, 1.5 h; Adipogen International, Schutzenstrasse, Switzerland).
̈
Cell death was quantified by using the CytoTox 96 nonradioactive
cytotoxicity assay (Promega Corporation, Madison, MI, USA), based
on a colorimetric measurement of LDH activity in the collected
supernatants. In particular, LDH activity was measured trough a
NADH-coupled enzymatic assay:
manuscript.
Notes
The authors declare no competing financial interest.
lactate + NAD+ → pyruvate + NADH + H+
(1)
ACKNOWLEDGMENTS
■
NADH + iodonitrotetrazolium → NAD+ + formazan
(2)
This research was supported by funding of the University of
Turin, Ricerca Locale 2013 (to M.B. and G.M.) and Ricerca
Locale 2012 (to A.D.S. and to R.F.). The contribution of Dr
Annalisa Costale for UHPLC analysis and Dr Luisa Boffa for
GC/MS analysis of compounds is gratefully acknowledged.
Authors wish to thank Prof Grazia Lombardi, Department of
Drug Science, University of Piemonte Orientale, Italy and Prof
Giovanni Camussi, Dipartimento di Scienze Mediche, Uni-
versity of Turin, Italy for kind gift of THP-1 and HK-2 cell
lines, respectively.
Reactions 1 and 2 were catalyzed by LDH (from the collected
supernatants) and diaphorase, respectively. Formazan concentration
was determined by measuring absorbance in the Victor X4
(PerkinElmer) at a λ = 490 nm. Cell death was expressed according
to the manufacture’s instruction.
Cytotoxic Assay. HK-2 cells were plated in 96-wells culture plates
(5 × 103 cells/well) and were exposed to increasing concentrations
(0.1−100 μM) of each compound. The cultures were maintained at 37
°C and 5% CO2 in a fully humidified incubator. Cell viability was
measured at 72 h by the MTT assay. This is a colorimetric assay based
on the conversion of the water-soluble 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) to an
insoluble purple formazan by actively respiring cells. The formazan
was solubilized with DMSO and its concentration determined by
measuring absorbance in the Victor X4 at a λ = 570 nm.
Measurement of Caspase-1 Activity. Caspase-1 activity was
determined by the spectrophotometric assay as previously described
by Thornberry53 with minor modifications. Enzyme was prepared
from THP-1 cell cultures as described. Reactions were carried out in
96-well plates in 100 μL final volume at 37 °C by diluting enzyme (50
U) in the reaction buffer (100 mM HEPES, 10% sucrose, 0.1%
ABBREVIATIONS USED
■
Ac-YVAD-pNA, N-acetyl-Tyr-Val-Ala-Asp-para-nitroanilide;
ASC, apoptosis-associated speck-like protein containing a
CARD; CAPS, cryopirin-associate periodic syndrome; CARD,
caspase recruitment domain; CHAPS, 3-[(3-cholamidopropyl)-
dimethylammonio]-1- propanesulfonate; CIAS1, cold-induced
autoinflammatory syndrome 1; CINCA, chronic infantile
neurologic, cutaneous, articular; DMEM, Dulbecco’s Modified
Eagle Medium; DMSO-d6, hexadeuterodimethyl sulfoxide;
O
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(13) Codolo, G.; Plotegher, N.; Pozzobon, T.; Brucale, M.; Tessari,
I.; Bubacco, L.; de Bernard, M. Triggering of inflammasome by
aggregated α-synuclein, an inflammatory response in synucleinopa-
thies. PLoS One 2013, 8, e55375.
(14) Martinon, F.; Burns, K.; Tschopp, J. The inflammasome: a
molecular platform triggering activation of inflammatory caspases and
processing of proIL-beta. Mol. Cell 2002, 10, 417−426.
(15) (a) Rathinam, V. A.; Vanaja, S. K.; Fitzgerald, K. A. Regulation
of inflammasome signaling. Nature Immunol. 2012, 13, 333−342.
(b) Sutterwala, F. S.; Haasken, S.; Cassel, S. L. Mechanism of NLRP3
inflammasome activation. Ann. N. Y. Acad. Sci. 2014, 1319, 82−95.
(16) Mariathasan, S.; Weiss, D. S.; Newton, K.; McBride, J.;
O’Rourke, K.; Roose-Girma, M.; Lee, W. P.; Weinrauch, Y.; Monack,
D. M.; Dixit, V. M. Cryopyrin activates the inflammasome in response
to toxins and ATP. Nature 2006, 440, 228−232.
DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); EWG, electron
withdrawing group; FC, flash chromatography; FCAS, familial
cold autoinflammatory syndrome; GSH, glutathione; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HMOX1,
heme oxygenase-1; IL, interleukin; Im, imidazole; Keap1,
Kelch-like ECH-associated protein 1; LDH, lactate dehydro-
genase; LPS, lipopolysaccharide; LRR, leucine-rich repeat
domain; Maf, musculoaponeurotic fibrosarcoma protein;
MBH, Morita−Baylis−Hillman; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide; MWS, Muckle-Wells
syndrome; NACHT, NAIP, CIIA, HET-E and TP1 domain;
NEM, N-ethylmaleimide; NLRP3, NLR family, pyrin domain-
containing 3; NOMID, neonatal-onset multisystem inflamma-
tory disease; Nrf2, NF-E2-related factor 2; PBS, phosphate-
buffered saline; PMA, phorbol myristate acetate; PYD, pyrin
domain; SEM, standard error of the mean; TLR, Toll-like
receptor
(17) Bauernfeind, F. G.; Horvath, G.; Stutz, A.; Alnemri, E. S.;
MacDonald, K.; Speert, D.; Fernandes-Alnemri, T.; Wu, J.; Monks, B.
G.; Fitzgerald, K. A.; Hornung, V.; Latz, E. Cutting edge: NF-kappaB
activating pattern recognition and cytokine receptors license NLRP3
inflammasome activation by regulating NLRP3 expression. J. Immunol.
2009, 183, 787−791.
REFERENCES
■
(18) Dinarello, C. A.; Simon, A.; Van der Meer, J. W. Treating
inflammation by blocking interleukin-1 in a broad spectrum of
diseases. Nature Rev. Drug Discovery 2012, 11, 633−652.
(1) Chen, Y.; Smith, M. R.; Thirumalai, K.; Zychlinsky, A. A bacterial
invasin induces macrophage apoptosis by binding directly to ICE.
EMBO J. 1996, 15, 3853−3860.
́ ́
(19) Lopez-Castejon, G.; Pelegrín, P. Current status of inflamma-
(2) Hilbi, H.; Moss, J. E.; Hersh, D.; Chen, Y.; Arondel, J.; Banerjee,
S.; Flavell, R. A.; Yuan, J.; Sansonetti, P. J.; Zychlinsky, A. Shigella-
induced apoptosis is dependent on caspase-1 which binds to IpaB. J.
Biol. Chem. 1998, 273, 32895−32900.
(3) Fink, S. L.; Cookson, B. T. Caspase-1-dependent pore formation
during pyroptosis leads to osmotic lysis of infected host macrophages.
Cell. Microbiol. 2006, 8, 1812−1825.
(4) Brennan, M. A.; Cookson, B. T. Salmonella induces macrophage
death by caspase-1-dependent necrosis. Mol. Microbiol. 2000, 38, 31−
40.
some blockers as anti-inflammatory drugs. Expert Opin. Invest. Drugs
2012, 21, 995−1007.
(20) Walsh, J. G.; Muruve, D. A.; Power, C. Inflammasomes in the
CNS. Nature Rev. Neurosci. 2014, 15, 84−97.
(21) So, A.; Ives, A.; Joosten, L. A. B.; Busso, N. Targeting
inflammasomes in rheumatic diseases. Nature Rev. Rheumatol. 2013, 9,
391−399.
(22) Agostini, L.; Burns, K.; McDermott, M. F.; Hawkins, P. N.;
Tschopp, J. NALP3 forms an IL-1β-processing inflammasome with
increased activity in Muckle−Wells autoinflammatory disorder.
Immunity 2004, 20, 319−325.
(5) Lamkanfi, M.; Dixit, V. M. Mechanism and functions of
inflammasomes. Cell 2014, 157, 1013−1022.
(23) Goldbach-Mansky, R.; Dailey, N. J.; Canna, S. W.; Gelabert, A.;
Jones, J.; Rubin, B. I.; Kim, H. J.; Brewer, C.; Zalewski, C.; Wiggs, E.;
Hill, S.; Turner, M. L.; Karp, B. I.; Aksentijevich, I.; Pucino, F.; Penzak,
S. R.; Haverkamp, M. H.; Stein, L.; Adams, B.; Moore, T. L.;
Fuhlbrigge, R. C.; Shaham, B.; Jarvis, J. N.; O’Neil, K.; Vehe, R. K.;
Beitz, L. O.; Gardner, G.; Hannan, W. P.; Warren, R. W.; Horn, W.;
Cole, J. .L; Paul, S. M.; Hawkins, P. N.; Pham, T. H.; Snyder, C.;
Wesley, R. A.; Hoffmann, S. C.; Holland, S. M.; Butman, J. A.; Kastner,
D. L. Neonatal-onset multisystem inflammatory disease responsive to
interleukin-1beta inhibition. N. Engl. J. Med. 2006, 355, 581−592.
(24) Gattorno, M.; Tassi, S.; Carta, S.; Delfino, L.; Ferlito, F.;
Pelagatti, M. A.; D’Osualdo, A.; Buoncompagni, A.; Alpigiani, M. G.;
Alessio, M.; Martini, A.; Rubartelli, A. Pattern of interleukin-1β
secretion in response to lipopolysaccharide and ATP before and after
interleukin-1 blockade in patients with CIAS1 mutations. Arthritis
Rheum. 2007, 56, 3138−3148.
(25) (a) Di Virgilio, F. The therapeutic potential of modifying
inflammasomes and NOD-like receptors. Pharmacol. Rev. 2013, 65,
872−905. (b) Davis, B. K.; Wen, H.; Ting, J. P.-Y. The inflammasome
NLRs in immunity, inflammation and associated diseases. Annu. Rev.
Immunol. 2011, 29, 707−735.
(26) De Nardo, D.; Latz, E. NLRP3 inflammasomes link
inflammation and metabolic diseases. Trends Immunol. 2011, 32,
373−379.
(6) Monack, D. M.; Detweiler, C. S.; Falkow, S. Salmonella
pathogenicity island 2-dependent macrophage death is mediated in
part by the host cysteine protease caspase-1. Cell. Microbiol. 2001, 3,
825−837.
(7) Frantz, S.; Ducharme, A.; Sawyer, D.; Rohde, L. E.; Kobzik, L.;
Fukazawa, R.; Tracey, D.; Allen, H.; Lee, R. T.; Kelly, R. A. Targeted
deletion of caspase-1 reduces early mortality and left ventricular
dilatation following myocardial infarction. J. Mol. Cell. Cardiol. 2003,
35, 685−694.
(8) Hoegen, T.; Tremel, N.; Klein, M.; Angele, B.; Wagner, H.;
Kirschning, C.; Pfister, H. W.; Fontana, A.; Hammerschmidt, S.;
Koedel, U. The NLRP3 inflammasome contributes to brain injury in
pneumococcal meningitis and is activated through ATP-dependent
lysosomal cathepsin B release. J. Immunol. 2011, 187, 5440−5451.
(9) Gulbransen, B. D.; Bashashati, M.; Hirota, S. A.; Gui, X.; Roberts,
J. A.; MacDonald, J. A.; Muruve, D. A.; McKay, D. M.; Beck, P. L.;
Mawe, G. M.; Thompson, R. J.; Sharkey, K. A. Activation of neuronal
P2X7 receptor-pannexin-1 mediates death of enteric neurons during
colitis. Nature Med. 2012, 18, 600−604.
(10) Halle, A.; Hornung, V.; Petzold, G. C.; Stewart, C. R.; Monks, B.
G.; Reinheckel, T.; Fitzgerald, K. A.; Latz, E.; Moore, K. J.; Golenbock,
D. T. The NALP3 inflammasome is involved in the innate immune
response to amyloid-beta. Nature Immunol. 2008, 9, 857−865.
(11) Heneka, M. T.; Kummer, M. P.; Stutz, A.; Delekate, A.;
Schwartz, S.; Vieira-Saecker, A.; Griep, A.; Axt, D.; Remus, A.; Tzeng,
T. C.; Gelpi, E.; Halle, A.; Korte, M.; Latz, E.; Golenbock, D. T.
NLRP3 is activated in Alzheimer’s disease and contributes to
pathology in APP/PS1 mice. Nature 2013, 493, 674−678.
(12) Shi, F.; Yang, Y.; Kouadir, M.; Fu, Y.; Yang, L.; Zhou, X.; Yin,
X.; Zhao, D. Inhibition of phagocytosis and lysosomal acidification
suppresses neurotoxic prion peptide-induced NALP3 inflammasome
activation in BV2 microglia. J. Neuroimmunol. 2013, 260, 121−125.
(27) Haneklaus, M.; O’Neill, L. AJ; Coll, R. C. Modulatory
mechanisms controlling the NLRP3 inflammasome in inflammation:
recent developments. Curr. Opin. Immunol. 2013, 25, 40−45.
(28) Gong, Y. N.; Wang, X.; Wang, J.; Yang, Z.; Li, S.; Yang, J.; Liu,
L.; Lei, X.; Shao, F. Chemical probing reveals insights into the
signaling mechanism of inflammasome activation. Cell Res. 2010, 20,
1289−1305.
(29) Juliana, C.; Fernandes-Alnemri, T.; Wu, J.; Datta, P.; Solorzano,
L.; Yu, J. W.; Meng, R.; Quong, A. A.; Latz, E.; Scott, C. P.; Alnemri, E.
P
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
S. Anti-inflammatory compounds parthenolide and Bay 11-7082 are
direct inhibitors of the inflammasome. J. Biol. Chem. 2010, 285, 9792−
9802.
(30) He, Y.; Varandarajan, S.; Munoz-Planillo, R.; Burberry, A.;
Nakamura, Y.; Nunez, G. 3,4-Methylenedioxy-β-nitrostyrene inhibits
NLRP3 activation by blocking assembly of the inflammasome. J. Biol.
Chem. 2014, 289, 1142−1150.
(31) (a) Wilson, A. J.; Kerns, J. K.; Callahn, J. F.; Moody, C. J. Keep
calm, and carry on covalently. J. Med. Chem. 2013, 56, 7463−7476.
(b) Kalguktar, A. S.; Dalvie, D. K. Drug discovery for a new generation
of covalent drugs. Expert Opin. Drug Discovery 2012, 7, 561−581.
(c) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of
covalent drugs. Nature Rev. Drug Discovery 2011, 10, 307−317.
(32) Johansson, M. H. Reversible Michael additions: covalent
inhibitors and prodrugs. Mini-Rev. Med. Chem. 2012, 12, 1330−1344.
(33) Amslinger, S. The tunable functionality of α,β-unsaturated
carbonyl compounds enables their differential application in biological
systems. ChemMedChem 2010, 5, 351−356.
(34) Avonto, C.; Taglialatela-Scafati, O.; Pollastro, F.; Minassi, A.; Di
Marzo, V.; De Petrocellis, L.; Appendino, G. An NMR spectroscopic
method to identify and classify thiol-trapping agents: revival of Michael
acceptors for drug discovery? Angew. Chem., Int. Ed. 2011, 50, 467−
471.
(35) (a) Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I.
M.; Taunton, J. Electrophilic fragment-based design of reversible
covalent kinase inhibitors. J. Am. Chem. Soc. 2013, 135, 5298−5301.
(b) Zheng, S.; Santosh Laxmi, Y. R.; David, E.; Dinkova-Kostova, A.
T.; Shiavoni, K. H.; Ren, Y.; Trevino, I.; Bumeister, R.; Ojima, I.;
Wigley, W. C.; Bliska, J. B.; Mierke, D. F.; Honda, T. Synthesis,
chemical reactivity as Michael acceptors, and biological potency of
monocyclic cyanoenones, novel and highly potent anti-inflammatory
and cytoprotective agents. J. Med. Chem. 2012, 55, 4837−4846.
(c) Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A fragment-based method
to discover irreversible covalent inhibitors of cysteine proteases. J. Med.
(44) Ma, Q. Role of Nrf2 in Oxidative Stress and Toxicity. Annu. Rev.
Pharmacol. Toxicol. 2013, 53, 401−426.
(45) Bode, M. L.; Kaye, P. T. Indolizine studies. Part 2. Synthesis and
NMR spectroscopic analysis of 2-substituted indolizines. J. Chem. Soc.,
Perkin Trans. 1 1993, 15, 1809−1813.
(46) Yuan, K.; Song, H. L.; Hu, Y.; Wu, X. Y. Chiral
phosphinthiourea-catalyzed asymmetric Morita−Baylis−Hillman re-
actions of acrylates with aromatic aldehydes. Tetrahedron Lett. 2009,
65, 8185−8190.
(47) Barbosa, T. P.; Junior, C. G. L.; Silva, F. P. L.; Lopes, H. M.;
Figueirido, L. R. F.; Sousa, S. C. O.; Batista, G. N.; Da Silva, T. G.;
Silva, T. M. S.; De Oliveira, M. R.; Vasconcellos, M. L. L. Improved
synthesis of seven aromatic Baylis−Hillman adducts (BHA):
evaluation against Artemia saliva Leach. and Leishmania chagasi. Eur.
J. Med. Chem. 2009, 44, 1726−1730.
(48) Singh, S. A.; Shat, S. V. Antimicrobial potential of 3-hydroxy-2-
methylene-3-phenylpropionic acid derivatives. Acta Pharm. 2011, 61,
447−455.
(49) Coehlo, F.; Veronese, D.; Pavam, C. H.; de Paula, V. I.; Buffon,
R. Palladium-catalyzed carbonylative cyclization of Baylis−Hillman
adducts. An efficient approach for the stereoselective synthesis of 3-
alkenyl phthalides. Tetrahedron 2006, 62, 4563−4572.
(50) Reddy, L. R.; Rao, K. R. Rate acceleration of the Baylis−Hillman
reaction using crystalline cyclodextrin complexes. Org. Prep. Proced. Int.
2000, 32, 185−203.
(51) Singha Roy, S. J.; Mukherjee, S. Enantioselctive desymmetriza-
tion of prochiral 1,3-dinitropropanes via organocatalytic allylic
alkylation. Chem. Commun. 2014, 50, 121−123.
(52) Drewes, S. E.; Emslie, N. D. Necic acid synthesis. Part 1. Total
synthesis of integerrinic acid. J. Chem. Soc., Perkin Trans. 1 1982,
2079−2083.
(53) Thornberry, N. A. Interleukin-1β converting enzyme. Methods
Enzymol. 1994, 244, 615−631.
(54) Gierse, J. K.; Koboldt, C. M.; Walker, M. C.; Seibert, K.;
Isakson, P. C. Kinetic basis for selective inhibition of cyclo-oxygenases.
Biochem. J. 1999, 339, 607−614.
Chem. 2014, 57, 4969−4974. (d) Al-Rifai, N.; Rucker, H.; Amslinger,
̈
S. Opening or closing the lock? When reactivity is the key to biological
activity. Chem.Eur. J. 2013, 19, 15384−15395.
(36) Zhao, M. X.; Wei, Y.; Shi, M. Morita−Baylis−Hillman Reaction.
In The Chemistry of the Morita−Baylis−Hillman Reaction; RSC
Catalysis Series No. 8; Shi, M., Wang, F. J., Zhao, M. X., Wei, Y.,
Eds; RSC Publishing: Cambridge, 2011; pp 1−78.
(37) Yu, C.; Liu, B.; Hu, L. Efficient Baylis−Hillman reaction using
stoichiometric base catalyst and an aqueous medium. J. Org. Chem.
2001, 66, 5413−5418.
(38) Aggarwal, V. K.; Dean, D. K.; Mereu, A.; Williams, R. Rate
acceleration of the Baylis−Hillman reaction in polar solvents (water
and formamide). Dominant role of hydrogen bonding, not hydro-
phobic effects, is implicated. J. Org. Chem. 2002, 67, 510−514.
(39) (a) Ranatunga, S.; Kim, J. S.; Pal, U.; Del Valle, J. R. An ester
enolate−Claisen rearrangement route to substituted 4-alkylidenepro-
lines. Studies toward a definitive structural revision of lucentamycin A.
J. Org. Chem. 2011, 76, 8962−8976. (b) Wei, H.-X.; Hu, J.; Jasoni, R.
L.; Li, G.; Pare, P. W. Synthesis of substituted α-(hydroxymethyl)-β-
́
iodoacrylates via MgI2-promoted stereoselective aldol coupling. Helv.
Chim. Acta 2004, 87, 2359−2363.
(40) Hentze, H.; Lin, X. Y.; Choi, M. S.; Porter, A. G. Critical role for
cathepsin B in mediating caspase-1-dependent interleukin-18 matura-
tion and caspase-1-independent necrosis triggered by the microbial
toxin nigericin. Cell Death Differ. 2003, 10, 956−968.
(41) Deane, P. O.; Guthrie, J. J.; Kaye, P. T.; Whittaker, R. E. Regio-
and diastereoselectivity in the thiomethylation of α-(1-hydroxyalkyl)-
acrylate derivatives. Synth. Commun. 1998, 28, 2601−2611.
(42) Sakakura, A.; Takayanagi, Y.; Shimogawa, H.; Kigoshi, H.
Jolkinolide D pharmacophore: synthesis and reaction with biomole-
cules. Tetrahedron 2004, 60, 7067−7075.
(43) Maydt, D.; De Spirt, S.; Muschelknautz, C.; Sthal, W.; Muller, T.
̈
J. J. Chemical reactivity and biological activity of chalcones and other
α,β-unsaturated carbonyl compounds. Xenobiotica 2013, 43, 711−718.
Q
dx.doi.org/10.1021/jm501072b | J. Med. Chem. XXXX, XXX, XXX−XXX