Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950

Article abstract of DOI:10.1021/acsmedchemlett.6b00198

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

Full text of DOI:10.1021/acsmedchemlett.6b00198

Letter
pubs.acs.org/acsmedchemlett
Identification, Synthesis, and Biological Evaluation of the Major
Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950
Manohar Salla,† Mark S. Butler,† Ruby Pelingon,† Geraldine Kaeslin,† Daniel E. Croker,† Janet C. Reid,†
Jong Min Baek,‡ Paul V. Bernhardt,‡ Elizabeth M. J. Gillam,‡ Matthew A. Cooper,*,†
and Avril A. B. Robertson*,†
†Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
‡School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia
S
* Supporting Information
ABSTRACT: MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing
protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of
MCC950 with human liver microsomes, and subsequent analysis by HPLC−MS/MS, revealed a major metabolite, where
hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were
synthesized, and coelution using HPLC−MS/MS confirmed the structure of the metabolite. Further synthesis of individual
enantiomers and coelution studies using a chiral column in HPLC−MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-
1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950
with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the
major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested
for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived
macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar
inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.
KEYWORDS: NLRP3, inflammasome, MCC950, microsome, metabolite, cytochrome P450
issues. Elucidating the metabolic fate of drug candidates is a key
requirement of regulatory bodies.8−10 Sometimes these
metabolites guide the development of new and improved
drugs.11 In this Letter, we report the identification, synthesis,
and NLRP3 inhibitory activity of the major human microsomal
metabolite of the NLRP3 inflammasome inhibitor MCC950
and identify the P450 enzymes, which catalyze its formation.
For the metabolite identification, MCC950 was incubated at
37 °C with human liver microsomes and the cofactor NADPH
for 120 min. The reaction was quenched with ice cold CH3CN,
vortexed, and centrifuged. The supernatant was analyzed using
(+)- and (−)-QTOF-ESI-MS/MS, and one major metabolite
was identified (Figure 1). In the (−)-ESI-MS the major
metabolite showed a strong [M−H]− mass ion peak at m/z
419.1, while the (+)-ESI-MS showed a reduced intensity for the
[M + H]+ mass ion peak and two facile losses of water (SI).
he NLRP3 inflammasome acts as a key mediator of
Tinflammatory responses through the activation of caspase-
1 leading to processing and release of the pro-inflammatory
cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18).1,2
NLRP3 is associated with several diseases such as cryopyrin-
associated periodic syndrome (CAPS), type 2 diabetes,
atherosclerosis, asthma, gouty arthritis, and inflammatory
central nervous system (CNS) diseases.3−5 Recently, a potent
and selective small molecule inhibitor of the NLRP3
inflammasome, MCC950 (Figure 1), was reported (IC50 7.5
nM) with early promise for treatment of inflammatory
diseases.6
The detection and identification of in vivo metabolites is an
important part of the drug discovery and development process.7
Metabolically labile sites can be modified to improve the lead
compounds ADME properties. The majority of drugs are
eliminated from the body by metabolism as a detoxification
process. However, metabolites may have pharmacological
activity that influence the efficacy of the therapeutic or can
be responsible for potential toxicity and drug−drug interaction
Received: May 16, 2016
Accepted: September 27, 2016
© XXXX American Chemical Society
A
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Figure 1. (A) (+)-QTOF-ESI-MS TIC. (B) XIC chromatogram of major human liver microsomal metabolite reaction showing the structure of
MCC950 and the major metabolite.
The (−)-ESI-MS/MS spectra of MCC950 and its metabolite
showed common fragments for the furan sulfonamide (m/z
204) (SI), which indicated that the oxygen was in the
1,2,3,5,6,7-hexahydro-s-indacene unit. The facile double dehy-
dration of the metabolite ruled out phenolic oxidation, and
three possible metabolite structures 1−3 were proposed and
synthesized (Figure 2).
Scheme 1. Synthesis of Key Intermediates 8 and 9
a
Reagents and conditions: (a) 3-chloropropionyl chloride, AlCl3,
DCM, 2 h, rt, 85%; (b) H2SO4, 48 h, 55−60 °C; (c) H2SO4 and
HNO3 (1:1), 1 h, 0−5 °C, 42%, 11%, 5% of 8, 9, and 10, respectively.
Figure 2. Proposed structures of the major human microsomal
metabolite of MCC950.
by reacting with concentrated H2SO4/HNO3 (1:1) at 0 °C.
Three nitrated products were separated by column chromatog-
raphy and identified as major isomer 8, and minor isomers 9
and 10. During the analysis of compounds 8−10, surprisingly
we noticed some discrepancies between our 1H NMR data and
that previously reported by Urban et al.12 We confirmed the
structures of the three isomers 8−10 by 2D NMR analysis and
single crystal X-ray crystallography for each compound (SI) and
showed that 9 was incorrectly assigned by Urban et al.12
For the synthesis of compound 1 (Scheme 2), 8-nitro-
3,5,6,7-tetrahydro-s-indacen-1(2H)-one (8) was reduced to the
corresponding amine 11 by hydrogenation using 10% Pd/C as
catalyst under hydrogen atmosphere (1 atm) at room
temperature.14 Amine 11 was converted to the isocyanate
intermediate 12 using Boc2O and DMAP in CH3CN at room
temperature, then reacted in situ with 4-(2-hydroxypropan-2-
yl)furan-2-sulfonamide 13 using NaH as base in anhydrous
THF at room temperature to give sulfonylurea intermediate
14.15 Reduction of the ketone intermediate using NaBH4 in
The synthesis of compounds 1−3 (Figure 2) required key
intermediates 8 and 9 (Scheme 1); these intermediates were
synthesized by procedures reported by Urban et al.12 Initially,
3-chloropropionyl chloride was prepared from 3-chloropro-
pionic acid using oxalyl chloride with a catalytic amount of
DMF in anhydrous DCM at room temperature.13 The resulting
3-chloropropionyl chloride was subsequently reacted with
indane in the presence of aluminum trichloride in anhydrous
DCM at room temperature to give chloroketone 5 in 85% yield.
Chloroketone 5 was cyclized by heating in the presence of
concentrated sulfuric acid at 55−60 °C for 48 h and was found
to be a mixture of ketone 6 with small amounts of regioisomer
1
7, as confirmed by H NMR spectroscopy. These regioisomers
were difficult to separate at this stage, and therefore, the
mixture was used for the next step. Nitration of the
regioisomeric ketones was achieved under standard conditions
B
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Scheme 2. Synthesis of Compound 1
Scheme 3. Synthesis of Compound 3
a
Reagents and conditions: (a) 10% Pd/C, H2 (1 atm), MeOH, rt, 2 h,
93%; (b) Boc2O, DMAP, CH3CN, rt 30 min; (c) NaH, THF, rt, 16 h;
67% (2-steps); (d) NaBH4, MeOH, rt, 3 h, 86%.
a
Reagents and conditions: (a) NaBH4, MeOH, rt, 2 h, 97%; (b) p-
TSA, toluene, reflux, 2 h, 80%; (c) m-CPBA, DCM, rt, 16 h, 82%; (d)
ZnI2/NaCNBH4, DCE, reflux, 4 h, 86%; (e) 10% Pd/C, H2 (1 atm),
MeOH, 16 h, 69%; (f) Boc2O, DMAP, CH3CN, rt, 30 min; (g) NaH,
THF, rt, 16 h, 39% (2-steps).
MeOH at room temperature for 3 h gave desired product 1 as a
racemic mixture.
Compound 2 racemate (Figure 2) was synthesized via the
same procedures used for compound 1 (Scheme 2) but using 4-
nitro-3,5,6,7-tetrahydro-s-indacen-1(2H)-one (9) as the starting
material.
Scheme 4. Synthesis of Compound 2a and 2b
Our initial route to synthesis of compound 3 is shown in
Scheme 3. The ketone group of 8-nitro-3,5,6,7-tetrahydro-s-
indacen-1(2H)-one (8) was reduced and dehydrated as before
to give the corresponding olefin 16.16 However, conversion to
alcohol intermediate 18 directly from olefin 16 was challenging.
Several methods were investigated, without success, including
TiCl4/NaBH4 and hydroboration using BH3·THF followed by
oxidation with H2O2/NaOH.17,18 An alternative method
proceeded via the epoxide 17, which was successfully produced
by oxidation of olefin 16 using m-CPBA in DCM at room
temperature.19 To open the epoxide ring regioselectively,
toward desired alcohol 18, we followed a procedure reported by
Finkielsztein et al. using ZnI2/NaCNBH4 in DCE under reflux
conditions for 4 h.20 Intermediate 18 was then reduced to the
aniline 19 and converted to the isocyanate 20 as before.
Subsequent reaction with sulfonamide intermediate 13 gave the
desired product 3.
To confirm the structure of the major metabolite of
MCC950, initially we coeluted the major metabolite with
each of the synthesized compounds 1−3 (Figure 2) using
HPLC-(−)-ESI-SRM-MS/MS. MCC950 was incubated with
human liver microsomes and NADPH, then sampled at t = 0;
three further samples were taken at t = 120 min, and each were
spiked with one of the synthesized compounds. Only
compound 2 coeluted with the major metabolite of
MCC950, thereby confirming the structure of the major
metabolite formed in human liver microsomes.
a
Reagents and conditions: (a) (S)-(−)-CBS, BH3·Me2S, THF, 0 °C,
30 min, 84%; (b) (R)-(+)-CBS, BH3·Me2S, THF, 0 °C, 30 min, 30%.
Further details in SI.
To confirm the stereochemistry of the metabolite, we
synthesized individual enantiomers (2a and 2b, Scheme 4) to
use in coelution studies. Enantioselective reduction of
intermediate 9 (Scheme 4) with (−) or (+) CBS catalyst and
BH3·Me2S reducing agent gave the corresponding alcohols 21a
and 21b.21,22 The (S)-(−)-CBS reagent produced (R)-alcohol
C
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Letter
(21a, yield 84%) in enantiomeric ratio 96:4 and (R)-(+)-CBS
reagent produced (S)-alcohol (21b) in enantiomeric ratio
80:20 as determined by chiral HPLC. The (S)-alcohol (21b,
yield 30%) was further purified by chiral semipreparative HPLC
to 99:1 enantiomeric ratio. Compounds 21a and 21b were
reduced to their corresponding amines by hydrogenation using
10% Pd/C catalyst under hydrogen atmosphere (1 atm) at
room temperature. Subsequent conversion to the isocyanate
and in situ reaction with 4-(2-hydroxypropan-2-yl)furan-2-
sulfonamide 13, as before, give desired products 2a and 2b in
22% and 7% yield, respectively (see SI).
To confirm the structure of the major metabolite of
MCC950, we coeluted with each of the synthesized compounds
2a and 2b (Scheme 4) using a chiral column in HPLC-
(−)-ESI-SRM-MS/MS. Only compound 2a (R-isomer)
coeluted thereby confirming stereochemistry of the major
metabolite.
The major P450 isoforms responsible for the metabolism of
MCC950 were then investigated. Metabolism of MCC950 by
recombinant P450 enzymes was analyzed by incubation at 37
°C using enzymes coexpressed with NADPH-cytochrome P450
reductase (hCPR) in Escherichia coli membrane fractions. The
following forms were assessed: P450 1A2, 2A6, 2A13, 2B6,
2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2S1, 2U1, 2W1, 3A4,
3A5, and 4A11. Membranes containing hCPR but no P450
were used as a negative control. The results detailed in Table 1
show eight of the 17 tested isoforms can form the metabolite,
particularly CYP2A6 and 2C9.
metabolite 2a (IC50 1238 nM) was 170-fold less active than
MCC950 but more active than the S-isomer 2b (IC50 6352
nM). Comparing the regioisomers, compound 3 (IC50 1828
nM) had almost similar activity to compound 2 (IC50 1971
nM), while interestingly compound 1 had IC50 in the
nanomolar range (232 nM). The cytotoxicity of MCC950
and the hydroxylated isomers 1−3 was tested using human
kidney and liver cell lines HEK293 or HepG2, and no evidence
of toxicity was found. These results indicate, in the
hexahydroindacene moiety, hydroxylation is generally detri-
mental to NLRP3 inhibitory activity but is better tolerated in
compound 1 and could be further explored in future studies.
This work gives the first indication as to the likely metabolic
fate of MCC950 while simultaneously revealing SAR of the
hexahydroindacene moiety. Future work will focus on
characterizing MCC950 metabolism in vivo and examining
the effects of MCC950 and the metabolite 2a on CYP
inhibition and CYP induction processes.
In conclusion, the metabolism of MCC950, a potent and
specific inhibitor of NLRP3 inflammasome, was investigated in
human liver microsomes. MCC950 formed a major metabolite,
the structure of which was proposed based on the analysis of
(+)- and (−)-UPLC-QTOF-ESI-MS. Three possible re-
gioisomers were synthesized, and coelution with the microsome
product confirmed the metabolite structure as N-((1-hydroxy-
1,2,3,5,6,7-hexahydro-s-indacen-4-yl) carbamoyl)-4-(2-hydroxy-
propan-2-yl)furan-2-sulfonamide (2). Synthesis of individual
enantiomers 2a and 2b followed by coelution with microsomal
metabolite using a chiral column in HPLC-(−)-ESI-SRM-MS/
MS confirmed the stereochemistry of the metabolite as R
enantiomer 2a. The R enantiomer 2a was 170-fold less potent
as an NLRP3 inhibitor than MCC950 in human cell-based
assay. MCC950 incubated with a panel of P450 enzymes
showed isoforms 1A2, 2A6, 2B6, 2C9, 2C18, 2C19, 2J2, and
3A4 are likely to be responsible for formation of the major
metabolite in human tissues, with the relative importance of
individual forms likely to depend on their expression level in
different tissues and subjects.
Table 1. P450 Isoforms That Catalyze Formation of Major
Metabolite
P450 isoform
major metabolite 2a (%), t = 2 h
1A2
2A6
2B6
2C9
2C18
2C19
2J2
0.6
2.5
0.1
4.9
1.3
1.1
1.1
1.3
ASSOCIATED CONTENT
■
3A4
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00198.
The ability of the synthesized compounds to inhibit NLRP3
inflammasome activity was investigated (Table 2). NLRP3-
induced production of the pro-inflammatory cytokine IL-1β
from LPS primed human monocyte derived macrophages,
stimulated with ATP, was tested in the presence and absence of
increasing concentrations of compounds 1−3, 2a, and 2b. The
determined IC50 values were then compared to that for
MCC950 used in this assay as positive control. The major
Experimental procedures, characterization data for all
intermediates and final compounds, descriptions of
biological assays, X-ray crystal structures of intermediates
8, 9 and 10, and LC−MS and LC−MS/MS spectra of
MCC950 and its major metabolite 2a (PDF)
AUTHOR INFORMATION
Table 2. NLRP3 Inflammasome Inhibition Activity (IL-1β
IC50) and Cytotoxicity of MCC950 and Compounds 1−3
■
Corresponding Authors
*E-mail: uqarob15@uq.edu.au. Fax: +61-7-3346-2090. Phone:
+61-7-3346-2204.
cytotoxicity
compd
IL-1β IC50 (nM) CC50 Hek293 (μM) CC50 HepG2 (μM)
*E-mail: m.cooper@uq.edu.au. Fax: +61-7-3346-2090. Phone:
+61-7-3346-2044.
MCC950
7.5
232
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
1
Funding
2
1971
1238
6352
1828
The authors are thankful to National Health and Medical
Research Council (NHMRC) for the financial support in terms
of funding (NHRMRC grant APP1086786). M.S. is supported
by an UQ International Scholarship (UQI) Ph.D. scholarship
2a
2b
3
>62.5
>62.5
D
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The authors declare no competing financial interest.
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We would like to thank David Edwards (IMB, UQ) for advice
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ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
NADPH, nicotinamide adenine dinucleotide phosphate;
HepG2, human liver hepatocellular carcinoma G2 cells;
HEK293, human embryonic kidney 293 cells; IL-1β, inter-
leukin-1β; XIC, extracted-ion chromatogram; SI, Supporting
Information; Pd/C, palladium on carbon; MeOH, methanol;
m-CPBA, meta-chloroperoxybenzoic acid; TiCl4, titanium
tetrachloride; NaH, sodium hydride; NaBH4, sodium borohy-
dride; NaCNBH4, sodium cyanoborohydride; ZnI2, zinc iodide;
BH3·THF, borane tetrahydrofuran complex; CBS, Corey−
Bakshi−Shibata; BH3·Me2S, borane dimethyl sulfide complex
(22) Teitelbaum, A. M.; Meissner, A.; Harding, R. A.; Wong, C. A.;
Aldrich, C. C.; Remmel, R. P. Synthesis, pH-dependent, and plasma
stability of Meropenem prodrugs for potential use against drug-
resistant tuberculosis. Bioorg. Med. Chem. 2013, 21, 5605−5617.
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