Synthesis of deuterium-labelled analogues of NLRP3 inflammasome inhibitor MCC950

Article abstract of DOI:10.1016/j.bmcl.2017.12.054

This study describes the syntheses of di, tetra and hexa deuterated analogues of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor MCC950. In di and tetra deuterated analogues, deuteriums were incorporated into the 1,2,3,5,6,7-hexahydro-s-indacene moiety, whereas in the hexa deuterated MCC950 deuteriums were incorporated into the 2-(furan-3-yl)propan-2-ol moiety. The di deuterated MCC950 analogue was synthesised from 4-amino-3,5,6,7-tetrahydro-s-indacen-1(2H)-one 5. Tetra deuterated analogues were synthesised in 10 chemical steps starting with 5-bromo-2,3-dihydro-1H-inden-1-one 9, whereas the hexa deuterated analogue was synthesised in four chemical steps starting with ethyl-3-furoate 24. All of the compounds exhibited similar activity to MCC950 (IC₅₀ = 8 nM). These deuterated analogues are useful as internal standards in LC-MS analyses of biological samples from in vivo studies.

Full text of DOI:10.1016/j.bmcl.2017.12.054

Accepted Manuscript
Synthesis of deuterium-labelled analogues of NLRP3 inflammasome inhibitor
MCC950
Manohar Salla, Mark S. Butler, Nicholas L. Massey, Janet C. Reid, Matthew A.
Cooper, Avril A.B. Robertson
PII:
S0960-894X(17)31228-3
https://doi.org/10.1016/j.bmcl.2017.12.054
BMCL 25510
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 November 2017
22 December 2017
23 December 2017
Please cite this article as: Salla, M., Butler, M.S., Massey, N.L., Reid, J.C., Cooper, M.A., Robertson, A.A.B.,
Synthesis of deuterium-labelled analogues of NLRP3 inflammasome inhibitor MCC950, Bioorganic & Medicinal
Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.054
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of deuterium-labelled analogues of NLRP3 inflammasome
inhibitor MCC950
Manohar Salla a, Mark S. Butler a, Nicholas L. Masseya, Janet C. Reid a, Matthew A. Cooper a, b*
and Avril A. B. Robertson a*
a Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072,
Australia
b Inflazome Ltd., The Tower, Trinity TEC, Pearse Street, Dublin, Ireland
ABSTRACT
This study describes the syntheses of di, tetra and hexa deuterated analogues of the NOD-like
receptor pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor MCC950. In di
and tetra deuterated analogues, deuteriums were incorporated into the 1,2,3,5,6,7-hexahydro-s-
indacene moiety, whereas in the hexa deuterated MCC950 deuteriums were incorporated into the
2-(furan-3-yl)propan-2-ol moiety. The di deuterated MCC950 analogue was synthesised from 4-
amino-3,5,6,7-tetrahydro-s-indacen-1(2H)-one 5. Tetra deuterated analogues were synthesised in
10 chemical steps starting with 5-bromo-2,3-dihydro-1H-inden-1-one 9, whereas the hexa
deuterated analogue was synthesised in four chemical steps starting with ethyl-3-furoate 24. All
of the compounds exhibited similar activity to MCC950 (IC50 = 8 nM). These deuterated
analogues are useful as internal standards in LC-MS analyses of biological samples from in vivo
studies.
1

The NLRP3 inflammasome is a key mediator of inflammatory processes. Its abnormal
activation is involved in the pathogenesis of inherited disorders such as cryopyrin-associated
periodic syndrome (CAPS) and complex inflammatory diseases, including metabolic disorders
such as type 2 diabetes, atherosclerosis, and gouty arthritis.1 Increasing evidence indicates that
NLRP3 is also implicated in several central nervous system (CNS) diseases including multiple
sclerosis, Alzheimer’s and Parkinson’s diseases.2,3 A small molecule inhibitor of NLRP3
inflammasome, MCC950 (CRID3),4,5 has been reported with IC50 8 nM in human cell based
assays, and impressive in vivo activity in multiple proof of concept models of inflammatory
disease.6-8 This molecule is also widely used as a tool to understand the role of NLRP3 in
biological processes.9-11
Figure 1. Structure of MCC950
Isotopically labelled compounds can be used as internal standards in quantification of drugs and
associated metabolites in biological samples by using liquid chromatography‐mass spectrometry
(LC‐MS).12,13 Indeed, we recently identified and reported the major metabolite of MCC950.14
In this study, deuterium-labelled analogues of MCC950 have been synthesised for use as
isotopic standards in pharmacokinetic analysis. Deuterium has been included in multiple
positions of MCC950 such that the synthetic precursors can be used towards additional standards
in the future.
2

Figure 2. Structures of deuterium-labelled MCC950 analogues
The synthesis of compound 1 (Figure 2) required key intermediate 5 (Scheme 1), which was
synthesised by procedures reported in our previous publication.14 Intermediate 5 was converted
into deuterium labelled amine intermediate 6 by using LiAlD4/AlCl3 in anhydrous Et2O at room
temperature.15 The structure of the amine 6 was confirmed by LC-MS and NMR analyses.
Detection of the desired mass, m/z 176 [M+H]+ on (+)-ESI-LC-MS, disappearance of the
carbonyl carbon signal, appearance of a new carbon signal with reduced intensity in the aliphatic
region (13C NMR), and no additional signals in 1H NMR spectra confirmed the structure of
amine 6. Amine 6 was converted into the corresponding isocyanate intermediate 7 using Boc2O
and DMAP in acetonitrile at room temperature.16 Subsequent reaction with 4-(2-hydroxypropan-
2-yl)furan-2-sulfonamide 8 in presence of NaH gave the desired product 1 in 98% isotopic purity
(LC-MS).17 In the (–)-ESI-LC-MS analysis, the desired m/z 405 [M–H]– (406 Da) was detected,
which was 2 Dalton (Da) higher mass than MCC950 (404 Da, m/z 403 [M–H]–). The 1H NMR
spectra showed two protons less than MCC950 in the hexahydroindacene moiety at the site of
deuterium incorporation confirming the structure of compound 1.
3

Scheme 1. Reagents and conditions: (a) LiAlD4, AlCl3, Et2O, rt, 4 h, 32% (b) Boc2O, DMAP,
CH3CN, 30 min (c) NaH, THF, rt, 16 h, 63% (2 steps).
The synthesis of compounds 2 and 3 (Figure 2) required two key intermediates 16 and 17
(Scheme 2); these intermediates were synthesised using commercially available 5-bromo-2,3-
dihydro-1H-inden-1-one (Scheme 2) as starting material. Reaction with LiAlD4/AlCl3 gave
deuterated intermediate 10 in excellent yield. Heck coupling with t-butyl acrylate gave alkene
intermediate 11,18 which was reduced to intermediate 12 by hydrogenation using Pd/C as catalyst
over 1 h. The quantity of catalyst (10% wt/wt) was key to the success of this conversion. In
initial efforts to convert intermediate 11 to 12, with excess loading of catalyst (20% wt/wt) and
prolonged reaction duration (typically overnight), >50% of deuterium to hydrogen exchange was
observed. The optimal reaction conditions were found to be 10% catalyst loading with 1 hour
reaction time under a hydrogen atmosphere. Deprotection of the t-butyl group was achieved
using trifluoroacetic acid to obtain 13 in quantitative yield. The acid intermediate 13 was first
chlorinated using oxalyl chloride and catalytic DMF at room temperature for 1 h, then subjected
to cyclization via Friedel–Crafts acylation using AlCl3 catalyst in 1,2 dichloroethane (DCE).
During this cyclisation, we observed ca. 15% (by LC-MS) of regioisomer 15; this was separated
by column chromatography. 1H NMR spectra of intermediate 14 showed two singlets as
expected in the aromatic region, and two doublets for regioisomer 15, confirming the structure of
the regioisomers. Nitration of the intermediate 14 using concentrated H2SO4/HNO3 (1:1) at 0 °C
gave a mixture of regiosiomers 16 and 17. The regioisomers were separated by column
chromatography and the minor isomer was identified as 16 and the major as 17.14 The structures
of intermediates 16 and 17 were confirmed by comparing the NMR spectra with their hydrogen
4

equivalents already confirmed by X-ray crystallography.14 The 1H NMR of the deuterated
compounds 16 and 17 (Scheme 2) showed a triplet instead of multiplet for protons on C-6 carbon
and no signal was observed for deuteriums on C-7 carbon.
Scheme 2. Reagents and conditions: (a) LiAlD4, AlCl3, Et2O, rt, 4 h, 94% (b) t-Butyl acrylate,
Pd(OAc)2, Tri(p-tolyl)phosphine, Et3N, DMF, 100 °C, 47% (c) 10% Pd/C, H2 (1 atm), MeOH, rt,
1 h, 97% (d) CF3COOH, CH2Cl2, rt, 98% (e) i) (CO)2Cl2, DMF (cat.) ii) AlCl3, DCE, rt, 1 h, 72%
(f) con. HNO3/H2SO4, 0 °C, 1 h, 16%, 62% of 16 and 17 respectively.
After confirming the structures (using unlabelled 16) for the synthesis of compound 2 (Scheme
3), the nitro intermediate 16 was reduced to the corresponding amine 18 by hydrogenation using
Pd/C as catalyst, followed by reaction with LiAlD4/AlCl3 to give the tetra deuterium-labelled
amine 19. This key amine intermediate 19 was converted into the corresponding isocyanate 20 as
before, followed by reaction with sulfonamide 8 in presence of NaH to give the desired product 2
in 98% isotopic purity (LC-MS).
5

Scheme 3. Reagents and conditions: (a) 10% Pd/C, H2 (1 atm), MeOH, rt, 1 h, 94% (b) LiAlD4,
AlCl3, Et2O, rt, 4 h, 31% (c) Boc2O, DMAP, CH3CN, 30 min (d) NaH, THF, rt, 16 h, 68% (2
steps).
Compound 3 (Figure 2) was synthesised from 8-nitro-3,5,6,7-tetrahydro-s-indacen-1(2H)-
one-7,7-d2 17 (Scheme 4) via the same procedures used for compound 2 (Scheme 3) in 97.3%
isotopic purity (LC-MS). In the (–)-ESI-LC-MS analysis for compounds 2 and 3 (408 Da), the
desired m/z 407 [M–H]– was detected, 4 Da higher than MCC950 (404 Da, m/z 403 [M–H]–). 1H
NMR spectra showed four protons less than MCC950 in the hexahydroindacene moiety where
deuteriums were incorporated, confirming the structures of compounds 2 and 3.
6

Scheme 4. Reagents and conditions: (a) 10% Pd/C, H2 (1 atm), MeOH, rt, 1 h, 89% (b) LiAlD4,
AlCl3, Et2O, rt, 4 h, 40% (c) Boc2O, DMAP, CH3CN, 30 min (d) NaH, THF, rt, 16 h, 56% (2
steps).
Compound 4 (Scheme 5) was synthesised from ethyl-3-furoate as starting material. This was
reacted with chlorosulfonic acid in anhydrous CH2Cl2 for 72 hours at room temperature. Pyridine
was then added at –10 °C followed by phosphorous pentachloride (PCl5) and the resulting
solution stirred for 16 h at room temperature to give sulfonyl chloride 25. This sulfonylchloride
was treated with liquid NH3 at –78 °C to obtain sulfonamide 26 in excellent yield without any
purification. Intermediate 26 was reacted with in situ generated CD3MgI in Et2O at room
temperature to give the desired deuterium-labelled sulfonamide intermediate 27 in high yield.
The sulfonamide 27 was coupled with previously synthesised isocyanate 28 as before to give the
desired sulfonylurea 4 in 100% isotopic purity (LC-MS). In the (–)-ESI-LC-MS analysis for
compound 4 (410 Da), the desired m/z 409 [M–H]– was detected, 6 Da higher than MCC950
(404 Da, m/z 403 [M–H]–). In the 1H NMR spectrum, no signal was detected for deuterium-
labelled methyl groups, confirming the structure of compound 4.
Scheme 5. Reagents and conditions: (a) i) ClSO3H, CH2Cl2, rt, 72 h ii) Pyridine, PCl5, rt, 12 h,
79% (b) NH3, rt, 3 h, 94% (c) CD3MgI, Et2O, rt, 16 h, 89% (d) NaH, THF, rt, 16 h, 47%.
Compounds 1-4 were tested for their NLRP3 inflammasome inhibitory activity in a human
cell based assay. Assays were conducted in two or three biological repeats performed in triplicate
and the results are listed in Table 1. NLRP3-induced production and release of the pro-
inflammatory cytokine interleukine-1β (IL-1β) from lipopolysaccharide (LPS) primed human
7

monocyte-derived macrophages (HMDMs), stimulated with nigericin, was tested in the presence
and absence of increasing concentrations of test compounds. The determined IC50 values were
then compared to that for MCC950 used in this assay as positive control. As anticipated, all of
the compounds exhibited similar activity to MCC950 (IC50 8 nM).
Table 1. NLRP3 inhibitory activity results for compounds 1-4
IL1-β IC50 ±SDb
a
Compound
(nM) (HMDM)
MCC950
5±4c
6±2
7±3
6±1d
5±1
1
2
3
4
aIC50 data reported as the mean of three biological replicates performed in triplicate; bStandard deviation
cPreviously reported 8 nM 4; dIC50 data reported as the mean of two biological replicates performed in triplicate.
In summary, we have successfully synthesised di, tetra and hexa deuterium labelled analogues
of NLRP3 inflammasome inhibitor MCC950. These derivatives are useful as internal standards
for LC-MS analyses of biological samples from in vivo studies. Furthermore, the synthetic routes
and intermediates developed for these deuterated tricyclic systems (compounds 1-3, Figure 2)
can be used more widely for other lead molecules now progressing towards the clinic.
Acknowledgments
The authors are thankful to National Health and Medical Research Council (NHMRC) for the
financial support in terms of funding (NHRMRC grant APP1086786). Manohar Salla is
supported by a UQ International Scholarship (UQI) Ph.D. scholarship. We would like to
acknowledge The Australian Red Cross Blood service for their supply of buffy coat tested blood
for biological assays. MAC currently holds a fractional Professorial Research Fellow
appointment at the University of Queensland with his remaining time as CEO of Inflazome Ltd.
a company headquartered in Dublin, Ireland that is developing drugs to address clinical unmet
needs in inflammatory disease by targeting the inflammasome.
8

Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/xxx/j.bmcl.2017.xx. xx.
References
1. Guo, H.; Callaway, J. B.; Ting, J. P. Nat. Med. 2015, 21, 677-687.
2. Strowig, T.; Henao-Mejia, J.; Elinav, E.; Flavell, R. Nature 2012, 481, 278- 286.
3. Walsh, J. G.; Muruve, D. A.; Power, C. Nature. Rev. Neurosci. 2014, 15, 84- 97.
4. Coll, R. C.; Robertson, A. A.; Chae, J. J.; Higgins, S. C.; Munoz-Planillo, R.; Inserra, M. C.;
Vetter, I.; Dungan, L. S.; Monks, B. G.;Stutz, A.; Croker, D. E.; Butler, M. S.; Haneklaus, M.;
Sutton, C. E.;Nunez, G.; Latz, E.; Kastner, D. L.; Mills, K. H.; Masters, S. L.;Schroder, K.;
Cooper, M. A.; O’Neill, L. A. Nat. Med. 2015, 21, 248-255.
5. Primiano, M. J.; Lefker, B. A.; Bowman, M. R.; Bree, A. G.; Hubeau, C.; Bonin, P. D.;
Mangan, M.; Dower, K.; Monks, B. G.; Cushing, L.; Wang, S.; Guzova, J.; Jiao, A.; Lin,L.L.;
Latz, E.; Hepworth, D.; Hall, J. P., J. Immunol. 2016, 197, 2421-2433.
6. Mridha, A. R.; Wree, A.; Robertson, A. A.; Yeh, M. M.; Johnson, C. D.; Van Rooyen, D. M.;
Haczeyni, F.; Teoh, N. C.; Savard, C.; Ioannou, G. N.; Masters, S. L.; Schroder, K.; Cooper, .
. M. A.; Feldstein, A. E.; Farrell, G. C. J. Hepatol. 2017, 66, 1037-1046
7. Dempsey, C.; Rubio Araiz, A.; Bryson, K. J.; Finucane, O.; Larkin, C.; Mills, E. L.;
Robertson, A. A.; Cooper, M. A.; O'Neill, L. A.; Lynch, M. A. Brain Behav. Immun. 2017,
61, 306-316.
8. Van Hout, G. P.; Bosch, L.; Ellenbroek, G. H.; de Haan, J. J.; van Solinge, W. W.; Cooper,
M. A.; Arslan, F.; de Jager, S. C.; Robertson, A. A.; Pasterkamp, G.; Hoefer, I. E. Eur. Heart
J. 2016, 38, 828-836
9. Fuster, J. J.; MacLauchlan, S.; Zuriaga, M. A.; Polackal, M. N.; Ostriker, A. C.; Chakraborty,
R.; Wu, C. L.; Sano, S.; Muralidharan, S.; Rius, C.; Vuong, J.; Jacob, S.; Muralidhar, V.;
Robertson, A. A.; Cooper, M. A.; Andres, V.; Hirschi, K. K.; Martin, K. A.; Walsh, K.
Science 2017, 355, 842-847
10. Pinar, A.; Dowling, J. K.; Bitto, N. J.; Robertson, A. A.; Latz, E.; Stewart, C. R.;
Drummond, G. R.; Cooper, M. A.; McAuley, J. L.; Tate, M. D.; Mansell, A. J. Biol. Chem.
2017, 292, 826-836.
9

11. Gaidt, M. M.; Ebert, T. S.; Chauhan, D.; Schmidt, T.; Schmid-Burgk, J. L.; Rapino, F.;
Robertson, A. A.; Cooper, M. A.; Graf, T.; Hornung, V. Immunity 2016, 44, 833-46.
12. Liu, R. H.; Lin, D.-L.; Chang, W.-T.; Liu, C.; Tsay, W.-I.; Li, J.-H.; Kuo, T.-L., Anal. Chem.
2002, 74, 618–626
13. Mutlib, A. E., Chem. Res. Toxicol. 2008, 21, 1672-1689.
14. Salla, M.; Butler, M. S.; Pelingon, R.; Kaeslin, G.; Croker, D. E.; Reid, J. C.; Baek, J. M.;
Bernhardt, P. V.; Gillam, E. M.; Cooper, M. A.; Robertson, A. A. ACS Med. Chem. Lett.
2016, 7, 1034-1038.
15. Michaelides, M.; Hansen, T.; Dai, Y.; Zhu, G.; Frey, R.; Gong, J.; Penning, T.; Curtin, M.;
Mcclellan, W.; Clark, R.; Torrent, M.; Mastracchio, A.; Kesicki, E. A.; Kluge, A. F.; Patane,
M. A.; Ji, Z.; Wang, C. WO Patent 2016044770, 2015.
16. Knölker, H. J.; Braxmeier, T.; Schlechtingen, G. Angew. Chem. 1995, 34, 2497-2500.
17. Urban, F. J.; John Jasys, V.; Raggon, J. W.; Buzon, R. A.; Hill, P. D.; Eggler, J. F.; Weaver,
J. D. Synth. Commun. 2003, 33, 2029-2043.
18. Whitcombe, N. J.; Hii, K. K. M.; Gibson, S. E. Tetrahedron 2001, 57, 7449-7476.
10

Graphical abstract
11