Discovery of a series of ester-substituted NLRP3 inflammasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127560

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Aberrant activation by a wide range of exogenous and endogenous signals can lead to chronic, low-grade inflammation. It has attracted a great deal of interest as a drug target due to the association with diseases of large unmet medical need such as Alzheimer's disease, Parkinson's disease, arthritis, and cancer. To date, no drugs specifically targeting inhibition of the NLRP3 inflammasome have been approved. In this work, we used the known NLRP3 inflammasome inhibitor CP-456,773 (aka CRID3 or MCC 950) as our starting point and undertook a Structure-Activity Relationship (SAR) analysis and subsequent scaffold-hopping exercise. This resulted in the rational design of a series of novel ester-substituted urea compounds that are highly potent and selective NLRP3 inflammasome inhibitors, as exemplified by compounds 44 and 45. It is hypothesized that the ester moiety acts as a highly permeable delivery vehicle and is subsequently hydrolyzed to the carboxylic acid active species by carboxylesterase enzymes. These molecules are greatly differentiated from the state-of-the-art and offer potential in the treatment of NLRP3-driven diseases, particularly where tissue penetration is required.

Full text of DOI:10.1016/j.bmcl.2020.127560

Bioorganic&MedicinalChemistryLetters30(2020)127560
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a series of ester-substituted NLRP3 inflammasome inhibitors
⁎
David Harrisona, , Nicolas Boutardc, Krzysztof Brzozkac, Marta Bugajc, Stefan Chmielewskic,
Anna Cierpichc, John R. Doedensb, Charles-Henry R.Y. Fabritiusc, Christopher A. Gabelb,
Michal Galezowskic, Piotr Kowalczykc, Oleksandr Levenetsc, Magdalena Mroczkowskac,
Katarzyna Palicac, Roderick A. Porterd, David Schultzc, Marta Sowinskac, Grzegorz Topolnickic,
Piotr Urbanskic, Jakub Woyciechowskic, Alan P. Watta
a NodThera Ltd., Suite 8, The Mansion, Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, United Kingdom
b NodThera Inc., 454 N 34th Street, Seattle, WA 98103, USA
c Selvita S.A. (now Ryvu Therapeutics), Park Life Science, ul. Bobrzynskiego 14, 30-348, Kraków, Poland
d Rod Porter Consultancy, 89 Back Street, Ashwell, Baldock, Hertfordshire SG7 5PG, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
The NLRP3 inflammasome is a component of the innate immune system involved in the production of proin-
flammatory cytokines. Aberrant activation by a wide range of exogenous and endogenous signals can lead to
chronic, low-grade inflammation. It has attracted a great deal of interest as a drug target due to the association
with diseases of large unmet medical need such as Alzheimer’s disease, Parkinson’s disease, arthritis, and cancer.
To date, no drugs specifically targeting inhibition of the NLRP3 inflammasome have been approved. In this work,
we used the known NLRP3 inflammasome inhibitor CP-456,773 (aka CRID3 or MCC 950) as our starting point
and undertook a Structure-Activity Relationship (SAR) analysis and subsequent scaffold-hopping exercise. This
resulted in the rational design of a series of novel ester-substituted urea compounds that are highly potent and
selective NLRP3 inflammasome inhibitors, as exemplified by compounds 44 and 45. It is hypothesized that the
ester moiety acts as a highly permeable delivery vehicle and is subsequently hydrolyzed to the carboxylic acid
active species by carboxylesterase enzymes. These molecules are greatly differentiated from the state-of-the-art
and offer potential in the treatment of NLRP3-driven diseases, particularly where tissue penetration is required.
NLRP3
Inflammasome
Interleukin-1β
Ester
Carboxylesterase
Prodrug
Letter
(BMDMs), Kupffer cells, peritoneal macrophages and alveolar macro-
phages, can form inflammasomes.
Inflammasomes are multiprotein complexes first identified and
named by Jürg Tschopp as the molecular machinery by which caspases
are activated.1 Upon activation these complexes are formed in the cy-
tosol and consist of an upstream sensor protein of the NOD-like (nu-
cleotide-binding oligomerization domain-containing protein) receptor
(NLR) family, the adaptor protein ASC (apoptosis-associated speck-like
protein containing a CARD) and the effector procaspase. Active cas-
pases in turn process proinflammatory cytokines such as interleukin-1β
(IL-1β) and interleukin-18 (IL-18) from their inactive to active forms.
Most immune cells of the monocyte-macrophage lineage, such as per-
ipheral blood monocytes, bone marrow-derived macrophages
The NLRP3 (NOD-like receptor, Leucine-rich Repeat and Pyrin do-
main-containing protein 3) inflammasome is of particular therapeutic
interest because of its links to human disease. NLRP3 activation is a
two-step signalling process. The first step, known as priming, occurs
when endogenous cytokines or microbial-derived molecules (e.g. lipo-
polysaccharide (LPS)) bind to extracellular receptors such as toll-like
receptors, IL-1 receptors and tumor necrosis factor (TNF) receptors.2
This upregulates the transcription and production of intracellular
NLRP3, proIL-1β and proIL-18 through activation of transcription
factor NF-κB. The second step, termed activation, requires a second
stimulus. This leads to NLRP3 inflammasome assembly, the cleavage of
Abbreviations: NLRP3, NOD-like receptor, Leucine-rich Repeat and Pyrin domain-containing protein 3; NOD-like, nucleotide-binding oligomerization domain-
containing protein-like; ASC, apoptosis-associated speck-like protein containing a CARD; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor;
NSAIDs, non-steroidal anti-inflammatory drugs; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; BMDMs, bone marrow-derived macrophages;
PBMC, peripheral blood mononuclear cells; ATP, adenosine triphosphate; PAMPA, parallel artificial membrane permeability assay; CES, carboxylesterase
⁎ Corresponding author.
E-mail address: david.harrison@nodthera.com (D. Harrison).
https://doi.org/10.1016/j.bmcl.2020.127560
Received 14 July 2020; Received in revised form 25 August 2020; Accepted 14 September 2020
Availableonline18September2020
0960-894X/CrownCopyright©2020PublishedbyElsevierLtd.Allrightsreserved.

D. Harrison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127560
procaspase-1 to the active form caspase-1, and the release of mature IL-
1β and IL-18. Caspase-1 also cleaves and activates Gasdermin-D
(GSDMD),3 a protein that forms pores in the cell membrane, leading to
pyroptosis (programmed inflammatory cell death) which facilitates
release of inflammatory cytokines into extracellular space.
Table 1
Truncation of CP-456773.
Compound
Structure
PBMC IL-1β IC50 (µM)a
CP-456,773
0.030
The NLRP3 inflammasome is formed upon activation by a wide
range of second stimuli, categorized as either pathogen-associated
molecular patterns (PAMPs) or danger-associated molecular patterns
(DAMPs). PAMPs can be peptidoglycan, and both viral or bacterial RNA
and DNA. DAMPs include adenosine triphosphate (ATP), beta-amyloid,
ceramides, monosodium urate crystals, cholesterol crystals, calcium
pyrophosphate dihydrate crystals, fatty acids, silica, alum and asbestos.
This broad range of activating ‘sterile’ danger signals is unique to the
NLRP3 inflammasome and it therefore has great potential as a drug
target in a wide range of human diseases. Therapeutic intervention to
inhibit NLRP3 inflammasome activation could have clinical benefits for
conditions where chronic, low-grade inflammation caused by in-
appropriate activation of the NLRP3 inflammasome is implicated. These
include Alzheimer’s disease,4 Parkinson’s disease,5 gout,6 type II dia-
betes7,8 and atherosclerosis.9
1
2
> 20
> 20
3
4
5
0.45
1.8
Various small molecules have been reported to inhibit NLRP3 in
both in vitro and in vivo studies, the structures of some of these are
shown in Fig. 1.
In research conducted prior to the discovery of the NLRP3 in-
flammasome, Perregaux, et. al. observed that the anti-diabetic sulfo-
nylurea glyburide (also known as glibenclamide) inhibited IL-1β post-
translational processing.10 Further optimization of glyburide led to the
discovery of the clinical candidate CP-456,773, which advanced into a
Phase II trial.11,12 A subsequent study by Coll, et. al. established that CP-
456,773 inhibited IL-1β release via selective inhibition of NLRP3 in-
flammasome activation.13 A number of other scaffolds have been as-
sociated with inhibition of NLRP3 activation. For example, Baldwin, et.
al. reported that BC23, amongst a series of oxazaborine compounds,
was an effective NLRP3 inflammasome inhibitor both in vitro and in
vivo.14 Some fenamate NSAIDs, exemplified by mefenamic acid, were
shown by Daniels, et. al., to inhibit the NLRP3 inflammasome and
showed in vivo effects in a rodent Alzheimer’s disease model.15 Other
small molecules such as 2-(4-nitrophenyl)-3H-quinazolin-4-one,16 JC-
171,17 the natural product isoliquiritigenin,18 CY-09,19 Bay 11-708220
and Fc11a-221 have also been reported to show NLRP3-inhibitory ef-
fects in various models. Various extensive reviews of small molecule
NLRP3 inflammasome inhibitors have been published.22–25
2.8
a
Values represent the numerical average of at least two experiments.
selective NLRP3 inhibitor CP-456,773 as a chemical starting point. This
represented an attractive lead as the molecule had been through pre-
clinical safety studies and advanced into the clinic. We took a de novo
approach to systematically truncate the molecule and assess the SAR of
the resultant fragment molecules. In vitro potency was assessed in a
human peripheral blood mononuclear cell (PBMC) assay. This assay
measures the ability of a compound to inhibit IL-1β release caused by
LPS and ATP stimulation. As a compound is required to permeate the
cell in order to disrupt the NLRP3 inflammasome, the potency is af-
fected by both functional activity and cell permeability.
The initial results of the truncation approach are shown in Table 1.
Removal of one or both fused cyclopentyl rings on the
Our efforts to discover novel NLRP3 inhibitors began with the
Fig. 1. Small molecule NLRP3 inhibitors.
2

D. Harrison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127560
Fig. 2. Design rationale for acid-containing compounds.
hexahydroindacene, resulting in indane (compound 1) and phenyl (2),
respectively, led to a dramatic loss of activity in the PBMC assay. From
this we concluded that the hexahydroindacene ring made an important
contribution to the potency of CP-456,773. The furan portion of the
molecule proved to be far more amenable to simplification, with the
unsubstituted furan (3), phenyl (4) and even methyl sulfone (5) all
retaining some activity in the cellular assay.
Table 3
Exploring the ester substituent.
Compound
Structure
PBMC IL-1β IC50 (µM)a
11
6.8
Encouraged by the result for 5 we sought to assess the importance of
the acidic center of the sulfonylurea and to explore alternatives for it, as
shown in Fig. 2. We designed a small set of compounds that maintained
the features of hexahydroindacene ring connected to an amide moiety
and featured an acidic center. The compounds synthesized and their
corresponding cellular PBMC assay IC50 values are shown in Table 2.
The carboxylate compound 6 and corresponding methyl ester inter-
mediate 7 were both inactive. The sulfonamide compounds 8 and 9,
chosen as they both maintained the sulfone moiety along with a weak
acidic center, were also devoid of activity. The carboxylic acid sub-
stituted urea (10) also did not show any activity. Surprisingly the
corresponding methyl ester (11), synthesized as a precursor of the acid,
12
13
1.6
1.8
14
> 20
15
16
17
> 20
> 20
> 20
Table 2
Results for acid replacement compounds.
Compound
Structure
Calculated pKa
PBMC IL-
1β IC50
(µM)a
(most acidic)
5
3.4
4.3
2.8
6
> 20
18
> 20
7
8
n/a
7.8
> 20
> 20
a
Values represent the numerical average of at least two experiments
showed a modest level of inhibition.
9
10.2
4.0
> 20
> 20
Encouraged that a relatively low molecular weight and neutral
molecule had shown an inhibitory effect in the PBMC IL-1β assay, we
next focused on compound 11 as a new lead. An exploration of the
methyl ester commenced with a view to identifying its role. The results
of the methyl ester SAR exploration are shown in Table 3.
10
Along with the methyl ester (11), the ethyl (12) and isopropyl (13)
esters showed single digit micromolar activity in the primary assay. The
tert-butyl ester (14), however, was devoid of activity, despite being
structurally similar to the isopropyl ester. Both the secondary (15) and
tertiary amide (16) compounds were inactive (defined as having an IC50
greater than the top concentration of the assay). Two oxadiazole-con-
taining compounds designed as nonclassical methyl ester bioisosteres
(17 and 18) also failed to exhibit any activity. The similar level of
11
n/a
6.8
a
Values represent the numerical average of at least two experiments.
3

D. Harrison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127560
Table 4
Table 5
Aryl ring modification.
Alpha-substituents.
Compound
12
R1
R2
PBMC IL-1β IC50 (µM)a
Compound
R1
R2
R3
PBMC IL-1β IC50 (µM)a
Et
1.6
12
27
28
29
30
31
H
H
Et
1.6
Me
Me
H
Me
Me
Me
Me
Me
> 20
14
rac-benzyl
(S)-benzyl
(R)-benzyl
CH2-CH2-phenyl
H
> 20
6.1
19
20
Et
Et
> 20
3.1
H
H
> 20
a
Values represent the numerical average of at least two experiments.
complete loss of potency. The hexahydroindacene was therefore se-
lected as the optimal substituent for the urea.
We next turned our attention toward the position alpha- to the
carboxylate. A small number of derivatives were tested (Table 5). Gem-
dimethyl substitution (27) resulted in a complete loss of activity. The
racemic benzyl substituent (28), although weakly potent, encouraged
the synthesis of both enantiomers (29 and 30) to probe for a stereo-
chemical preference. Indeed the (R)-benzyl example, compound 30,
was found to be the eutomer and the (S)-benzyl example (29) was de-
void of activity. The phenethyl substituted compound (31) was in-
active. These findings warranted further investigation of the alpha
substituent, with a focus on aryl and heteroaryl groups linked via a
methylene spacer. Where possible, the (R)- enantiomer was profiled
based on the previously observed stereochemical preference. These
results are shown in Table 6.
21
22
Et
Et
> 20
> 20
23
24
Me
Et
> 20
> 20
A scan of ortho, meta and para hydroxy around the phenyl ring (32,
33 and 34) showed that all these substituents slightly improved po-
tency. Similarly, meta and para nitrile substitution (35 and 36) brought
about slight improvements to the potency compared with the un-
substituted phenyl. There was more of a contrast when comparing the
three pyridyl isomers; whilst the ortho pyridyl (37) offered no potency
advantage, the meta pyridyl (38) was more potent than the corre-
sponding phenyl ring and the para pyridyl (39) was highly disfavoured.
Other azines were tolerated, with the 4- and 5-pyrimidine (40 and 41)
and 3- and 4-pyridazine (42 and 43) boasting similar to or slightly
improved potency than the phenyl analogue. Large potency gains were
seen with both the 2-pyrimidine (44) and the pyrazine group (45).
The two most potent compounds, 44 and 45, were tested in the
human whole blood (WB) assay along with the reference compound CP-
456,773. This assay quantifies NLRP3 inflammasome disruption by
measuring the inhibition of IL-1β release stimulated by LPS and ATP in
human whole blood. The binding of a compound to proteins within the
blood, principally human serum albumin and α-1 glycoprotein, causes a
reduction in available concentration of compound and consequently
reduces the measured half-maximal inhibitory concentration compared
with the PBMC assay. It is well known that acidic compounds are
generally more highly protein bound than neutral compounds and that
protein binding also increases with lipophilicity.
25
26
Et
Et
> 20
> 20
a
Values represent the numerical average of at least two experiments.
potency of the methyl, ethyl and isopropyl esters and the inactivity of
the chemically and enzymatically more stable tert-butyl ester led to a
working hypothesis that the ester acts as a delivery vehicle for the
carboxylic acid. Given that the primary assay was a cellular rather than
isolated protein assay, cell permeability was required in order to
measure activity. It is likely that the ester moiety greatly improves
cellular permeation relative to the carboxylic acid, then subsequent
intracellular enzymatic cleavage delivers the active species.
The next round of modification focused on the aryl substituent ad-
jacent to the urea, the results of which are shown in Table 4. Expansion
of the 5-membered to a 6-membered alkyl ring to give compound 19
resulted in ablation of activity. Interestingly, activity could be mostly
restored by translocating the 5-membered ring to give the 5-substituted
heptahydro-cyclopenta[a]naphthalene (20). Variation of the para po-
sition of the aryl ring included chloro substitution (21) and conversion
to the 4-aza ring (22) yielded only inactive compounds. 2,6-Bis-alkyl
substituted phenyls (23 and 24), which were reasoned to impart an
orthogonal twist of the aryl group relative to the urea carbonyl and thus
mimic the hexahydroindacene, did not give promising results. Finally,
replacement of the hexahydroindacene group with other large lipo-
philic groups such as 1-adamantyl (25) or benzhydryl (26) resulted in
As shown in Table 7, in the PBMC assay the two leading compounds
from this series were equipotent to the NLRP3 inflammasome inhibitor
CP-456,773. By contrast, in the whole blood assay there was a sig-
nificant improvement for compounds 44 and 45 compared with CP-
456,773, with IC50 values approximately 20-fold and 8-fold more po-
tent, respectively. The whole blood attenuation (Δ = WB IC50 ÷ PBMC
IC50) for CP-456,773 is 97-fold, whereas for compound 44 it is only 4-
fold. CP-456,773 is an acidic sulfonylurea (estimated pKa ≈ 5) and as
such is negatively charged at physiological pH. This acidic center is
likely to contribute to the binding to plasma proteins as implied by the
high whole blood attenuation. By contrast, compounds 44 and 45 are
uncharged at physiological pH and this may explain why the
4

D. Harrison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127560
Table 6
Table 7
Methylene-spaced alpha-aryl substituents.
Comparison and further profiling of CP-456,773, 44 and 45.
Compound
CP-456,773
44
45
PBMC IL-1β IC50 (µM)a
0.030
2.9
0.036
0.16
4
0.030
0.37
12
WB IL-1β IC50 (µM)a
Δ WB/PBMC
97
Compound
R1
R2
R3
PBMC IL-1β IC50 (µM)a
Speck formation inh IC50 (µM)
TNFα inh IC50 (µM)
0.028
> 40
> 40
< 0.003
NT
0.033
2.7
0.20
7.6
30
32
(R)-benzyl
H
H
Me
Me
6.1
2.5
IL-6 inh IC50 (µM)
4.3
6.1
PAMPA Pe (nm/s)b
82
139
79.2
0
Hu blood stability (% at 1 h)c
92.4
0
Mu blood stability (% at 1 h)c
NT
33
34
H
H
Me
Me
2.0
2.7
a
Values represent the numerical average of at least two experiments.
bParallel Artificial Membrane Permeability Assay. cDefined as percentage re-
maining after incubation in whole blood at 37° C for one hour. NT = not tested.
attenuation to whole blood for both compounds is significantly reduced
compared to CP-456,773.
Compounds 44 and 45 were profiled further to ensure they retained
the profile of a selective NLRP3 inflammasome inhibitor, as displayed
by CP-456,773. The ASC speck assay measures the aggregation of green
fluorescent protein (GFP) tagged ASC in THP-1 cells using confocal
microscopy. This assay uses nigericin as signal 2 (activation), rather
than ATP as used in the PBMC assay. This provides additional me-
chanistic information that the compound does not have signal 2-specific
activity, for example P2X7 antagonism. Combined with the fact that the
ASC speck assay reads out ASC speck formation rather than down-
stream events (for example caspase activation, IL-1β release), inhibition
in both the PBMC and speck assays is a strong indication that a com-
pound inhibits a common component of NLRP3 activation or in-
flammasome assembly. Both 44 and 45 show potent inhibition of ASC
speck formation in this assay, with the former having a comparable half
maximal inhibitory concentration to CP-456,773. This demonstrates
that these compounds inhibit the NLRP3 inflammasome upstream of
inflammasome formation. The TNFα and IL-6 selectivity assays measure
the inhibition of these cytokines from human PBMCs stimulated with
LPS. NLRP3 inflammasome activation results in IL-1 and IL-18 release
and therefore in the timeframe of the assay TNFα and IL-6 release
should not be inhibited by a specific NLRP3 inflammasome inhibitor.
CP-456,773 shows no effect in the TNFα and IL-6 assays up to 40 µM
concentration. Compounds 44 and 45 show a modest inhibition in both
selectivity assays when tested at higher concentrations, but with a se-
lectivity window of two orders of magnitude compared to the PBMC
assay half maximal inhibitory concentrations.
35
36
H
H
Me
Me
1.5
1.5
37
38
H
H
Me
Me
4.2
1.5
39
40
H
H
Me
Et
> 20
6.8
41
42
H
H
Me
Me
4.5
3.3
With favourable potency and selectivity profiles determined, the
membrane permeability of 44 and 45 were assessed in a PAMPA assay.
CP-456,773 demonstrated an unmeasurably low rate of membrane
permeability (< 0.003 nm/s). By contrast, 44 and 45 both had a high
rate of membrane permeation (82 and 139 nm/s, respectively).
The stability of 44 and 45 were assessed in both human and mouse
blood. It was found that both showed reasonable stability in human
whole blood, with 92 and 79 percent of the 44 and 45 remaining, re-
spectively, after one hour of incubation. This was not the case in mouse
whole blood. Here, complete metabolism of the parent molecule had
taken place after one hour of incubation. We hypothesized that the
rapid turnover of 44 and 45 in mouse blood was due to circulating
carboxylesterases (CES), which are capable of hydrolyzing ethyl esters
to carboxylic acids.25 Due to the rapid turnover of these ester com-
pounds in mouse blood, the pharmacokinetic profiles of 44 and 45 were
not assessed.
43
H
Et
6.0
44
45
H
H
Et
Et
0.036
0.030
In conclusion, a deconstruction of CP-456,773 and subsequent ra-
tional design of acid-containing compounds led to the discovery of ester
11. The corresponding carboxylic acid, 10, did not inhibit at the top
concentration of the PBMC assay. Subsequent optimization of 11 led to
the discovery of a novel series of substituted 2‐[(phenylcarbamoyl)
amino]acetate based NLRP3 inflammasome inhibitors. Two leading
a
Values represent the numerical average of at least two experiments.
5

D. Harrison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127560
exemplars from this series, 44 and 45, were further profiled to confirm
excellent potency in PBMC, whole blood and ASC speck formation in-
hibition assays, with selectivity over TNFα and IL-6 release, high rates
of membrane permeability and good stability in human whole blood.
Our working hypothesis is that the ester moiety acts as a delivery ve-
hicle (prodrug), enabling higher rates of membrane permeability
compared to the carboxylic acid. The ester is converted to the active
species by carboxylesterase enzymes. The SAR of the ester substituent
and the large difference in blood stability between human and mouse of
compounds 44 and 45 support this prodrug hypothesis. These mole-
cules offer a differentiated profile to the widely studied inhibitor CP-
456,773 and have potential utility in the treatment of diseases where
NLRP3 inflammasome inhibition is required along with high tissue and
membrane permeability.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
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doi.org/10.1016/j.bmcl.2020.127560.
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