Design, synthesis and evaluation of an NLRP3 inhibitor diazirine photoaffinity probe

Article abstract of DOI:10.1016/j.tetlet.2020.151849

The NLRP3 inhibitor MCC950/CRID3 ameliorates a remarkable number of inflammatory disorders in animal models. Herein we describe a trifluoromethyl phenyl diazirine (TPD) photoaffinity probe, called TPD-950-Br, to probe the molecular interactions of MCC950. We show that TPD-950-Br covalently captures proximal species upon photo-activation and inhibits IL-1β production in an NLRP3 inhibitor assay.

Full text of DOI:10.1016/j.tetlet.2020.151849

Journal Pre-proofs
Design, Synthesis and Evaluation of an NLRP3 Inhibitor Diazirine Photoaf‐
finity Probe
James R. Hill, Rebecca C. Coll, Kate Schroder, Avril A.B. Robertson
PII:
S0040-4039(20)30289-6
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.151849
TETL 151849
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 February 2020
13 March 2020
13 March 2020
Please cite this article as: Hill, J.R., Coll, R.C., Schroder, K., Robertson, A.A.B., Design, Synthesis and
Evaluation of an NLRP3 Inhibitor Diazirine Photoaffinity Probe, Tetrahedron Letters (2020), doi: https://doi.org/
10.1016/j.tetlet.2020.151849
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Design, Synthesis and Evaluation of an NLRP3 Inhibitor Diazirine
Photoaffinity Probe
James R. Hill,*a Rebecca C. Coll,a,b Kate Schrodera and Avril A. B. Robertson*a,c
a. Institute for Molecular Bioscience, The University of Queensland, Australia.
b. Current address: The Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom.
c. School of Chemistry and Molecular Biosciences, The University of Queensland, Australia.
The NLRP3 inhibitor MCC950/CRID3 ameliorates a remarkable number of inflammatory disorders in animal
models. Herein we describe a trifluoromethyl phenyl diazirine (TPD) photoaffinity probe, called TPD-950-Br, to
probe the molecular interactions of MCC950. We show that TPD-950-Br covalently captures proximal species
upon photo-activation and inhibits IL-1β production in an NLRP3 inhibitor assay.
A fundamental aspect of drug discovery is the use of chemical probes to interrogate biological systems.1
Photoaffinity probes are masked reactive compounds that can preferentially interact with a molecular target.
Ultraviolet (UV) photo-activation unveils a reactive intermediate that irreversibly binds proximal species.2 This is
useful for probing transient molecular interactions, for example, target identification studies.3 This requires
incorporation of a photo-cross-linking unit, such as a benzophenone, diazirine or aryl azide.
The molecular target and mechanism of action of MCC950 (also called CP-456,773 and CRID3) has been in
question since the compound was reported in 2001, as a potent inhibitor of interleukin (IL)-1β activity.4 The
molecular target of MCC950 was originally thought to be glutathione transferase omega 1; although, this was
subsequently disproven.5,6 In 2015, MCC950 was demonstrated to selectively inhibit IL-1β production derived from
NLRP3 (NOD [nucleotide oligomerisation domain], LRR [leucine-rich repeat] and pyrin domain-containing protein
3), with a half-maximal inhibitory concentration (IC50) of 8 nM.7 MCC950 was suggested to target NLRP3 or a closely
associated protein.7 MCC950 then became a widely used tool compound for determining the role of NLRP3 in
various inflammatory diseases and fuelled a flurry of interest in NLRP3 inhibitor development.8,9 However, at this
stage the precise mechanism of action was unknown and there was no evidence of a physical interaction between
MCC950 and NLRP3. In 2019, the understanding of MCC950’s molecular target and mechanism of action was further
elucidated. Using photoaffinity labelling, a drug affinity responsive target stability assay and surface plasmon
resonance, we demonstrated that MCC950 directly and reversibly interacts with the ATP-hydrolysis motif of
NLRP3.10 Additionally, Tapia-Abellán and co-workers. reported that MCC950 binding closes the ‘open’ conformation
of NLRP3.11 Later work by Vande Walle and co-workers. similarly used photoaffinity labelling to conclude that
MCC950 interferes with NLRP3-ATP binding.12
The two MCC950 photoaffinity probes reported to date, BP-950 and PAL-CRID3, both utilise the benzophenone
photo-cross-linking unit (Fig. 1).10,12 Benzophenones are the largest of the popular photo-cross-linkers. This size
affects the NLRP3-inhibitor potency of benzophenone-MCC950 analogues, which are of approximately micromolar
potency, compared to the 8 nM potency of the parent compound.10,12 Diazirines on the other hand, have a small
steric footprint, allowing them to be nested within a compounds’ biologically active motif with less perturbation.13,14
Moreover, using various photoaffinity probes reduces the chance of target misidentification, improves the
likelihood that all potential interactions are probed and provides insights into the overall structure of the inhibitor
binding pocket.2,15 Herein we describe the design, synthesis and evaluation of an NLRP3-inhibitory photoaffinity
probe, whereby the trifluoromethyl phenyl diazirine (TPD) photo-cross-linker is nested within the biologically active
motif.
BP-950
IC50 = 1360 nM
O
O
O
H
N
S
N
N
H
H
O
O
alkyne
benzophenone
purification tag
photo-cross-linker
O
O
O
O
H
N
O
S
O
N
N
H
H
O
PAL-CRID3
IC50 = 731 nM
Figure 1. Benzophenone-MCC950 photoaffinity probes.

The design of a MCC950 photoaffinity probe was guided by prior studies of NLRP3 sulfonylurea inhibitors (Fig.
2).4,7,16–19 These studies indicated that: i) the sulfonylurea and (tricyclic) s-hexahydroindacene moiety are largely
indispensable for nanomolar NLRP3 inhibitory–activity; ii) halogens are tolerated at the 4-position of the s-
hexahydroindacene; iii) the furan can be replaced with numerous aromatic groups; and iv) this aromatic group
tolerates a broad range of substituents.
MCC950
TPD-950-Br
NLRP3 inhibitor
NLRP3 inhibitor photoaffinity probe
Br
H
O
O O
N
N
O
HO
O
O
S
S
F3C
N
N
N
N
H
H
H
H
O
TPD
Br
increasing modification tolerated
photo-cross-linker
MS tag
Figure 2. Design of TPD950-Br, an NLRP3-inhibitory photoaffinity probe.
Considering these structure activity relationships (SAR), TPD-950-Br was designed by incorporating two new
features (Fig. 2). The furan of MCC950 was replaced with a trifluoromethyl phenyl diazirine (TPD) moiety and an
aromatic proton was replaced by bromine. These changes were envisaged to cause minimal perturbation to NLRP3-
inhibitory activity. A TPD was selected over other photo-cross-linking units due to its small topological footprint,
activation at non-protein-damaging wavelengths (~355 nm), and reduced tendency for non-specific labelling.20 The
bromine was incorporated to provide a mass spectrometry (MS) tag, due to the naturally occurring 1:1 79Br - 81Br
ratio.21 In its photo-inactive form, TPD-950-Br was anticipated to behave in the same manner as other sulfonylurea
NLRP3 inhibitors — as a reversible inhibitor. However, when photo-activated, the resulting reactive carbene was
intended to cross-link irreversibly to spatially proximal species.
TPD-950-Br, like many TPD-containing photoaffinity probes, required a lengthy synthesis. This synthesis has four
phases (Scheme 1): i) substituting the trifluoromethyl acetophenone starting material with a synthetic handle, ii)
installing the TPD moiety, and iii) functionalising the TPD with a sulfonamide, and iv) incorporating the TPD into the
final bioactive compound. The instability of diazirine and sulfonylurea functional groups limited the feasible reaction
conditions.
Desired substitution
TPD synthesis
TPD functionalisation
Incorporation into bioactive molecule
O
N
N
N
N
O O
Br
O
O O
N
N
NO2
S
NO2
F3C
F3C
NH2
F3C
S
F3C
N
N
H
H
2
6
9
TPD-950-Br
Scheme 1. Design of TPD950-Br, an NLRP3-inhibitory photoaffinity probe.
To provide a synthetic handle for subsequent modifications and to create the desired substitution pattern,
commercially available trifluoromethyl acetophenone (1) was nitrated, using nitric and sulfuric acid, to afford meta-
nitro trifluoromethyl acetophenone 2 (Scheme 2, A). The nitro group served as a protecting group, for what would
eventually be reduced to the aniline.
13,22
Compound 2 then underwent a four step reaction sequence, common in TPD synthesis (Scheme 2, B-E)
Treating 2 with hydroxylamine hydrochloride and pyridine, converted the carbonyl to the E- and Z-oxime isomers
(3). We initially performed column chromatography after this reaction; however, later replicates found that the
crude oxime could be used without purification. Oxime 3 was then treated with tosyl chloride, 4-
dimethylaminopyridine (DMAP) and triethylamine, affording N-tosylate 4. Reaction with liquid ammonia caused
nucleophilic attack and intramolecular cyclisation to form diaziridine 5. Subsequent oxidation using iodine and
trimethylamine in dichloromethane afforded diazirine 6. Each reaction in this TPD sequence afforded good to
excellent yields (77-99%).

HO
TsO
F3C
O
O
N
N
NO2
NO2
NO2
A)
B)
C)
F3C
F3C
F3C
1
2
3
4
N N
HN NH
N N
F3C
D)
E)
F)
G)
NH2
NO2
NO2
F3C
F3C
5
6
7
Br
N N
O O
S
N N
F3C
O O
S
O
N N
F3C
O O
S
K)
H)
F3C
NH2
Cl
N
N
H
H
8
9
TPD-950-Br
Br
Br
I)
J)
H2N
H2N
OCN
10
11
12
Scheme 2. TPD-950-Br Synthesis. Reagents and conditions: A) H2SO4, HNO3, -5 °C to r.t., 2 h, 95%. B) NH2OH.HCl, pyridine, reflux, 4 h, 99%. C) TsCl, DMAP,
Et3N, CH2Cl2 r.t., 16 h, 77%. D) NH3, -78 °C to r.t., 24 h, 99%. E) I2, Et3N, r.t., 2 h, 98%. F) Na2S2O4, THF/water, r.t., 16 h, 56%. G) i. NaNO2, HCl, water, <5 °C,
15 min; ii. SO2, CuCl, <5 °C, 2 h, 68%. H) NH3, THF, -78 °C to r.t., 3 h, 92%. I) NBS, NH4OAc, CH3CN, r.t., 10 min, 98%. J) Triphosgene, Et3N, THF, r.t., 4 h, 96%.
K) NaH, THF, r.t., 16 h, 55%.
With the diazirine ring now formed, we turned our attention to functionalising the TPD moiety with a
sulfonamide (Scheme 2, F-H); being mindful of TPD sensitivity towards high temperatures, harsh reducing agents
and strong acids.13 Nitro TPD 6 was reduced to TPD aniline 7 using sodium dithionite. Wixe and co-workers have
demonstrated TPD aniline 7 can also be prepared via an N-Boc protection/deprotection strategy.23 Next, we utilised
a Sandmeyer-style, copper(I)-catalysed nucleophilic aromatic substitution reaction to convert TPD aniline 7 to TPD
sulfonyl chloride 8. This involved treating 7 with aqueous sodium nitrite and hydrochloric acid to form the diazonium
salt in situ.24 To this, an aqueous solution of sulfur dioxide and cuprous chloride was added, yielding TPD sulfonyl
chloride 8 (68%) and trace quantities of the sulfonic acid. An attempt to perform the same reaction in acetonitrile-
water solutions resulted primarily in the sulfonic acid. The aqueous method is suggested to favour sulfonyl chloride
formation, over the sulfonic acid, as the former precipitates before hydrolysis can occur.25 Without purification, the
crude TPD sulfonyl chloride (8) was treated with liquid ammonia, affording sulfonamide 9.
Next, we set about incorporating sulfonamide 9 into the biologically active compound (Scheme 2, I-K). Aniline
10 was brominated with N-bromosuccinimide (NBS) to afford bromo aniline 11. Then, using triphosgene and
triethylamine, converted to isocyanate 12. Sulfonamide 9 was prepared as the sodium salt using sodium hydride
and reacted with isocyanate 12 to allow the sulfonylurea condensation and the formation of TPD-950-Br. The
structure was confirmed via nuclear magnetic resonance (NMR) and high resolution (HR) MS.
To confirm that TPD-950-Br could undergo photo-cross-linking, we irradiated it in deuterated methanol and
observed the formation of the methanol adduct 13 by 19F NMR and tandem MS. TPD photolysis proceeds via the
formation of a carbene, directly or via the linear diazo. Carbenes then undergo carbon-hydrogen or heteroatom-
hydrogen insertion with proximal species.26 This is exemplified in Scheme 3, whereby TPD-950-Br exposed to UV
forms carbene 14, directly or via linear diazo 15. Carbene 14 then undergoes deuterium-oxygen insertion to form
methanol adduct 13. Both the 79Br and 81Br methanol-adducts (13) were observed by tandem MS. However, the
fragmentation of 13 in MS/MS may weaken the isotopic signature when analysing tryptic peptides.
The trifluoromethyl group associated with the TPD, diazo and methanol adduct each exhibit characteristic 19F
NMR shifts of 67, 59 and 78 ppm, respectively.27 During UV exposure of TPD-950-Br, 19F NMR spectra were recorded
at various intervals over 14 minutes. Plotting the relative 19F NMR integrals, the photolysis of TPD-950-Br can be
observed, simultaneously with the formation of diazo 15 and methanol adduct 13 (Fig. 3). This photolysis provides
guidance for future cell-based photo-cross-linking experiments.

methanol
adduct
diazirine
CD3OD
UV
Br
Br
O
O
O O
N
N
O
O
D3C
O
D
S
S
F3C
N
N
- N2
F3C
N
N
H
H
H
H
TPD-950-Br
13
UV
UV
CD3OD
O
- N2
diazo
N
N
Br
Br
carbene
F3C
O
UV
O
S
O
O
S
O
- N2
N
N
H
F3C
N
N
H
H
H
15
14
Figure 3. Photolysis of TPD-950-Br measured by 19F NMR.
Scheme 3. Photo-cross-linking of TPD-950-Br to deuterated methanol.
To confirm biological activity, TPD-950-Br was screened in a cell-based NLRP3 inhibitor assay. IL-1β release from
murine bone-marrow derived macrophages was measured, as per previously reported methods.7,16 The IL-1β IC50
of TPD-950-Br was 164 nM (pIC50 = 6.8 ± 0.1, n = 4). In comparison, the IL-1β IC50 of MCC950 = 8 nM (pIC50 = 8.1).7
From this we infer that TPD-950-Br has retained its NLRP3 inhibitor properties, consistent with the literature NLRP3
sulfonylurea SAR. Specifically, replacing the furan with a TPD group and introducing a bromine on the s-
hexahydroindacene only resulted in a 20-fold reduction in potency.
In summary, we have synthesised TPD-950-Br, a diazirine-containing analogue of the widely studied NLRP3
inhibitor MCC950. In doing so, we have expanded the scope of TPD-compatible nucleophilic aromatic substitutions.
The photo-cross-linking properties of TPD-950-Br were confirmed using 19F NMR and tandem MS. Our NLRP3
inhibitor assay demonstrated that TPD-950-Br is a potent inhibitor of IL-1β production (IC50 = 164 nM), in line with
the literature SAR. TPD-950-Br may be useful for studying MCC950-protein interactions.
Experimental
Please see supplementary for experimental details.
Conflicts of interest
RC, AR and KS are co-inventors on granted patents (US 10,538,487, EP 3259253) and patent applications
(WO2018215818, WO2017140778, WO2016131098) for NLRP3 inhibitors, which are licensed to Inflazome Ltd, a
company headquartered in Dublin, Ireland. Inflazome is developing drugs that target the NLRP3 inflammasome to
address unmet clinical needs in inflammatory disease.
Acknowledgements
We thank Alun Jones and David Edwards for conducting MS experiments, and Nicholas Massey, Joshua Dickinson
and Beth Rayner for contributions to diazirine synthesis. This work was supported by the National Health and
Medical Research Council of Australia (Fellowship no. 1141131 to KS; Project Grant no. 1086786 to AR and KS), the
Australian Research Council (Fellowship no. FT130100361 to KS), the Institute for Molecular Bioscience (Research
Advancement Award to JH) and The University of Queensland (Postdoctoral Fellowships to RC and Research
Scholarship to JH).
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
RC, AR and KS are co-inventors on granted patents (US 10,538,487, EP 3259253) and patent applications
(WO2018215818, WO2017140778, WO2016131098) for NLRP3 inhibitors, which are licensed to Inflazome Ltd, a
company headquartered in Dublin, Ireland. Inflazome is developing drugs that target the NLRP3 inflammasome to
address unmet clinical needs in inflammatory disease.

Highlights




Synthesis of TPD-950-Br, a trifluoromethyl phenyl diazirine-analogue of MCC950.
TPD-950-Br is a potent NLRP3 inhibitor, IC50 = 164 nM.
TPD-950-Br cross-links with methanol when UV irradiated.
TPD-950-Br is synthesised via a Sandmeyer-style sulfochlorination.