Benzisothiazolinone as a useful template for the design of new monoacylglycerol lipase inhibitors: Investigation of the target residues and comparison with octhilinone

Article abstract of DOI:10.1016/j.bmcl.2011.10.026

The regulation of 2-arachidonoylglycerol (2-AG) levels is a major issue as 2-AG has been proven to participate in numerous physiopathological phenomena such as neuroprotection or analgesia. Octhilinone, a cysteine-reagent compound, has recently been shown to inhibit in the nanomolar range monoacylglycerol lipase (MAGL), the major enzyme responsible for the degradation of 2-AG. Here, we further investigate the mechanism by which octhilinone and its benzisothiazolinone analog inhibit human MAGL. We also provide new information on the structural requirements for MAGL inhibition by these compounds. Finally, we describe for N-octylbenzisothiazolinone a mode of inhibition which is partially different from that described for octhilinone, especially with regard to the targeted cysteine residues in the vicinity of the catalytic site.

Full text of DOI:10.1016/j.bmcl.2011.10.026

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7321–7324
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzisothiazolinone as a useful template for the design of new
monoacylglycerol lipase inhibitors: Investigation of the target residues
and comparison with octhilinone
Nicolas Matuszak a, Bouazza Es Saadi a, Geoffray Labar a, Jacqueline Marchand-Brynaert b,
Didier M. Lambert a,
⇑
a Université catholique de Louvain, Louvain Drug Research Institute, Medicinal Chemistry Department (CMFA), 73 Avenue E. Mounier, bte B1.73.10, B-1200 Bruxelles, Belgium
b Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Laboratoire de Chimie Organique et Médicinale, Bâtiment Lavoisier, Place Louis Pasteur
L4.01.02, B-1348 Louvain-La-Neuve, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The regulation of 2-arachidonoylglycerol (2-AG) levels is a major issue as 2-AG has been proven to par-
ticipate in numerous physiopathological phenomena such as neuroprotection or analgesia. Octhilinone, a
cysteine-reagent compound, has recently been shown to inhibit in the nanomolar range monoacylglyc-
erol lipase (MAGL), the major enzyme responsible for the degradation of 2-AG. Here, we further investi-
gate the mechanism by which octhilinone and its benzisothiazolinone analog inhibit human MAGL. We
also provide new information on the structural requirements for MAGL inhibition by these compounds.
Finally, we describe for N-octylbenzisothiazolinone a mode of inhibition which is partially different from
that described for octhilinone, especially with regard to the targeted cysteine residues in the vicinity of
the catalytic site.
Received 12 September 2011
Revised 5 October 2011
Accepted 7 October 2011
Available online 18 October 2011
Keywords:
Monoacylglycerol lipase
2-Arachidonoylglycerol
Benzisothiazolinone
Octhilinone
Ó 2011 Elsevier Ltd. All rights reserved.
Cysteine
Even if the recreational use of Cannabis sativa has been known
for centuries, the pharmacology of the endocannabinoid system
has only been explored for less than 50 years, characterizing year
after year the receptors, the endogenous ligands and the degrada-
tion enzymes. The concept of targeting these later components of
the endocannabinoid machinery as a strategy to elevate endocan-
nabinoid levels is recent, and the design of inhibitors of the metab-
olism of the endocannabinoids has allowed to access new
information about these enzymes. Among the endocannabinoids,
2-arachidonoylglycerol (2-AG) binds both CB1 and CB2 cannabinoid
receptors as an agonist, and is responsible for numerous physiolog-
ical effects. For instance, 2-AG-induced CB1 cannabinoid receptor
activation leads to neuroprotection,1 anxiolysis2 and antinocicep-
tion.2,3 However, these effects are quickly terminated by several
2-AG hydrolyzing enzymes, the guardians of 2-AG pools. The main
actor of this process is monoacylglycerol lipase (MAGL), a serine
hydrolase that cleaves monoacylglycerols. Nevertheless, two
additional enzymes, ABHD12 and ABHD6, have been demonstrated
to participate in 2-AG degradation in the brain for, respectively, 9%
and 4% of total 2-AG hydrolysis.4 Even if ABHD12 is supposed to
guard intracellular pools of 2-AG,5 the role of ABHD6, highly ex-
pressed in BV-2 cells, is yet to be defined. FAAH is also implicated
in 2-AG metabolism but at a lesser extent (2%).4 Two major strat-
egies are developed to inhibit MAGL, and by doing so, elevating
2-AG levels: a first class of inhibitors targets the serine residue
which belongs to the catalytic triad such as the carbamates
URB602 or JZL184. This strategy has proven to be efficient, and
has allowed the characterization of some pharmacological conse-
quences of the inhibition of MAGL.6–8 Recently, Labar and co-work-
ers reported the structure of MAGL which crystallized as a dimer
while at the same time, Bertrand et al., published the crystal struc-
ture of human MAGL in complex with the inhibitor SAR269. These
reports as well as studies on the murine enzyme confirmed the
identity of the catalytic triad, composed of the serine122, the his-
tidine269 and the aspartate239 (rat and human MAGL share
83.8% of amino acid sequence identity9–11) and confirmed the
importance of the cysteine residues (Cys201, Cys208, Cys242)
located at the vicinity of the catalytic site.12 Therefore, another
strategy consists in targeting these cysteine residues. We and oth-
ers previously reported a series of maleimides that bound cysteine
residues and among which some compounds selectively inhibited
human MAGL in the nanomolar range.13,14 Among the cysteine-tar-
geting inhibitors, isothiazolinones and benzisothiazolinones have
also been shown to inhibit rat MAGL with nanomolar potencies.15
King and colleagues reported that the inhibition of such com-
⇑
Corresponding author. Tel.: +32 2 764 7347; fax: +32 2 764 7363.
E-mail address: didier.lambert@uclouvain.be (D.M. Lambert).
pounds occurred through
a partially reversible mechanism,
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.026

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N. Matuszak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7321–7324
As a starting point, octhilinone (1) and N-octylbenzisothiazoli-
none (2a) (Fig. 1) were evaluated on the inhibition of human MAGL,
following the same conditions as King15 (preincubation of 10 min at
37 °C, and incubation of 10 min at 37 °C with the rat enzyme). In
these conditions, compounds 1 and 2a inhibited human recombi-
1 (octhilinone)
2a
nant MAGL with IC50 values of 0.14 0.08 and 0.66 0.19 lM,
respectively. These affinities are 1.5–11 times inferior to those pub-
lished on the rat purified enzyme by King and co-workers (IC50 value
88 12 nM for octhilinone and 59 7 nM for 2a). We also evaluated
both compounds on humanrecombinantFAAH, whichwas inhibited
by octhilinone and 2a with IC50 values of 18.9 5.3
lM and
35.2 6.0 M, respectively. These compounds retained selectivity
l
for MAGL, while being 53–135 times more potent on MAGL than
on FAAH.
We next wanted to discriminate the determinant structural
features for MAGL inhibition by 2a. We modified the structure of
the benzisothiazolinone while conserving the octyl side chain by
changing the sulfur atom of the benzisothiazolinone scaffold,
either by a carbonyl group (generating a phtalimide, 6), a methy-
lene group (generating an isoindolinone, 7), or an oxygen (for the
benzisoxazolone, 5) (Fig. 2).
3
4a
Figure 1. Structures of octhilinone (1), 2-octylbenzo[d]isothiazol-3(2H)-one (2a),
1-biphenyl-4-ylmethyl-maleimide
[d]isothiazol-3(2H)-one (4a).
(3),
and
2-(4-methylbiphenyl)benzo
We also modified the architecture of the lead, by interchanging
the reactive groups, leading to the synthesis of two compounds, a
benzoxazol-2-one 8 and a benzothiazol-2-one 9 (Fig. 2). Except for
7, compounds were prepared via a classic nucleophilic substitu-
tion, using K2CO3 as a base in acetonitrile.16 Compound 7 was
obtained by the reduction of N-octylphtalimide 6. The alkylation
of benzisothiazolone (X = S) and benzisoxazolone (X = O) by
octylbromide led to the isolation of four products (2a, 2b and 5a,
5b) since, as expected in our experimental conditions, both O-
and N-alkylation could occur. We thus tested both O-alkylated
and N-alkylated isomers on MAGL, and while the two N-substi-
tuted isomers 2a and 2-octylbenzisoxazol-3-one 5a displayed
low micromolar inhibition values on MAGL, the 3-octyloxybenz-
isothiazole (2b) weakly inhibited MAGL, and the 3-octyloxybenzis-
a
2b, 4b (X=S)
5b (X=O)
2a, 4a (X=S)
5a (X=O)
a
b
6
7
a
oxazole (5b) inhibited MAGL with an IC50 value of 67.9 15.6 lM
(Table 1, Figure 2).
Previously, we proposed 1-biphenyl-4-ylmethyl-maleimide (3,
Fig. 1) as a lead structure for MAGL inhibitors.14 Based on the data
published by King and co-workers,15 we thought that substituting a
benzisothiazolinone with a 4-methylbiphenyl group could lead to a
more potent compound than octhilinone (Fig 1). 2-[(4-Phenylphe-
nyl)methyl]isothiazol-3-one (4a) (Fig. 1) was synthesized from
benzisothiazolinone and 4-phenylbenzyl bromide, and evaluated
8 (X=O), 9 (X=S)
Figure 2. Preparation of compounds 2 and 4–9. Reagents and conditions: (a) RBr
(R = C8H17 for
CH3COOH.
2 and 5–9, R=CH2biPh for 4), K2CO3, acetonitrile; (b) Zn, HCl,
involving the cysteine C208. Here, we propose to refine the struc-
tural features which are required for MAGL inhibition by benziso-
thiazolinone-based compounds. Moreover, we further investigate
the inhibition mechanism, either for isothiazolinone or for benziso-
thiazolinone compounds on human MAGL. We show that the dif-
ferences between the rat and human enzyme can lead to
different conclusions and that a reference test on the human en-
zyme could be useful to evaluate and compare the potencies of var-
ious MAGL inhibitors.
on MAGL and FAAH. We measured an IC50 value of 0.41 0.01 lM
on MAGL, with a selectivity index of 73 for MAGL over FAAH.
Indeed, the methylbiphenyl motif allowed an increase of the inhib-
itory potential towards MAGL, compared to 2a, and offered a higher
selectivity for MAGL versus FAAH. Furthermore, the isomer 4b did
not inhibit MAGL at 1 mM, and barely inhibited 50% of FAAH activ-
ity at the same concentration.
Table 1
Inhibition values on human recombinant MAGL and FAAH
Compound
IC50 (MAGL,
l
M) or % inhibition (10À3 M)
IC50 (FAAH,
l
M) or % inhibition (10À3 M)
Selectivity (MAGL/FAAH)
1
1.09 0.19
1.66 0.38
18.9 5.3
35.2 6.0
43% 0.5
30.0 4.4
50% 1.0
44.7 7.8
31.6 4.4
17.3
21.2
—
73.0
—
0.16
0.47
—
2a
2b
4a
4b
5a
5b
6
55%
5
0.41 0.01
No inhibition at 10À3
275 76
M
67.9 15.6
No inhibition at 10À3
No inhibition at 10À3
26% 15
M
M
No inhibition at 10À3
No inhibition at 10À3
129 41
M
M
7
8
—
—
9
72% 19
309 89
—

N. Matuszak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7321–7324
7323
a
b
0min
10min
30min
100
100
75
50
25
0
75
50
25
0
-9
-8
-7
-6
-5
-4
-3
-2
-9
-8
-7
-6
-5
-4
-3
-2
Log [octhilinone]
Log [octhilinone]
c
d
100
75
50
25
0
100
75
50
25
0
-9
-8
-7
-6
-5
-4
-3
-2
-9
-8
-7
-6
-5
-4
-3
-2
Log [2a]
Log [2a]
Figure 3. MAGL inhibition by octhilinone or compound 2a after 0, 10 or 30 min of preincubation at room temperature (a, c) or 37 °C (b, d).
Octhilinone was previously shown to act via a partially revers-
ible mechanism.15 We thus wanted to confirm this suggested
mechanism, and investigated whether an isothiazolinone and a
benzisothiazolinone compounds bearing the same side chain be-
haved similarly. Among enzymatic assays on MAGL, preincubation
time and incubation time are a source of difference in the apparent
IC50 values reported. In the present study, we evaluated the impact
of preincubation on the IC50 values of octhilinone and 2a at either
room temperature or 37 °C (Fig. 3), and three preincubation times
(after 0, 10, and 30 min of preincubation) (Fig. 3).17 First, an in-
crease of the apparent affinity was observed for both compounds
when comparing the inhibition obtained after 0 and 10 min of pre-
incubation, while there was little difference between 10 and 30 min
of preincubation, whatever the temperature. Second, a decrease of
the IC50 value could be also noticed for octhilinone while no appar-
ent change was observed for compound 2a when comparing the
inhibition curves after a preincubation of 10 min at either room
temperature or 37 °C. This seems to indicate that both time and
temperature parameters influence MAGL inhibition by isothiazoli-
100
75
50
25
0
wt
C201A
C208A
C242A
201T
100
75
50
25
0
-2
-6
-5
-4
-3
-2
[DTT] (M)
0
10
10
10
10
10
10
+ [octhilinone] 10-5M
-9
-8
-7
-6
-5
-4
-3
-2
Log [octhilinone]
100
100
75
50
25
0
75
50
25
0
-2
-6
-5
-4
0
-3
0
-2
0
0
[DTT] (M)
10
10
10
1
1
1
-9
-8
-7
-6
-5
-4
-3
-2
+ [2a] 10-5M
Log [2a]
Figure 4. Influence of the addition of DTT to MAGL inhibition by octhilinone and
compound 2a.
Figure 5. Impact of cysteine mutations to alanine on MAGL inhibition by
octhilinone and compound 2a.

7324
N. Matuszak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7321–7324
none compounds while the inhibition by benzisothiazolinone com-
pounds seems less sensitive to temperature.
References and notes
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11. Wall, E. M.; Cao, J.; Chen, N.; Buller, R. M.; Upton, C. Virus Res. 1997, 52, 157.
12. Labar, G.; Bauvois, C.; Borel, F.; Ferrer, J. L.; Wouters, J.; Lambert, D. M.
Chembiochem 2010, 11, 218.
13. Saario, S. M.; Salo, O. M.; Nevalainen, T.; Poso, A.; Laitinen, J. T.; Jarvinen, T.;
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14. Matuszak, N.; Muccioli, G. G.; Labar, G.; Lambert, D. M. J. Med. Chem. 2009, 52,
7410.
We also wanted to confirm the mechanism proposed by King
and coworkers, implicating MAGL cysteine residues in the inhibi-
tion process by isothiazolinone-based compounds. We first incu-
bated octhilinone or compound 2a with MAGL and then
increasing amounts of dithiothreitol (DTT) that reduces disulfide
bonds to free sulfhydryl groups were added (Fig. 4). DTT was thus
supposed to cleave the adduct formed between a cysteine of MAGL
and the benzisothiazolinone. In both cases, the activity of MAGL
was restored with increasing DTT concentrations, confirming a cys-
teine-based mechanism of inhibition.
We further investigated this mechanism by using various MAGL
mutants, where different cystein residues were mutated to alanine
residues (Fig. 5). We confirmed the previous observations15 as the
inhibitory potency of octhilinone was reduced when Cys208 was
mutated to alanine, compared to wild-type MAGL. But, addition-
ally, its potency was also reduced in the same fashion when
Cys242 was mutated. Furthermore, the mutation of Cys201 also
reduced, however to a lesser extent, the potency of octhilinone to-
wards MAGL inhibition.
We also evaluated compound 2a on MAGL mutants, assuming it
behaved as octhilinone (Fig. 5). Indeed, both Cys201 and Cys242
mutations reduced the potency of compound 2a, as they did for oct-
hilinone. However, in this case, the mutation of Cys208 did not seem
to affect its potency, as the inhibition curve of the C208A mutant
superimposed with that of wild-type MAGL. For both octhilinone
and 2a, the mutations of the three cysteines (Cys201, Cys208,
Cys242, referred to as 201T) to alanines (C201A/C208A/C242A triple
mutant) dramatically reduced their inhibitory potency on human
MAGL.
Our results first confirm the importance of the benzisothiazoli-
none structure, the replacement of the sulfur atom by an oxygen
(compounds 5a and 5b) being the only pharmacomodulation that
allowed MAGL inhibition, although with a lower affinity than that
obtained with the benzisothiazolinone analog 2a. We improved the
original template, confirming that the use of the motif methylbi-
phenyl (compound 4a) helps gaining in selectivity for MAGL using
this scaffold. We also confirmed the cystein-based mechanism of
inhibition and showed that octhilinone 1 and its benzisothiazoli-
none analog 2a interact with different cystein residues of MAGL.
Finally, we highlighted the interest of this chemical family and
confirmed that benzisothiazolinone-based compounds are good
MAGL inhibitors candidates.
15. King, A. R.; Lodola, A.; Carmi, C.; Fu, J.; Mor, M.; Piomelli, D. Br. J. Pharmacol.
2009, 157, 974.
16. Procedure for compounds 2(a, b) and 5(a, b): 2-octylbenzo[d]isoxazol-3(2H)-
one (2a) and 3-(octyloxy)benzo[d]isoxazole (2b) were prepared by adding
benzo[d]isothiazol-3-one (Sigma–Aldrich, 3.3 mmol) and anhydrous
potassium carbonate (5 mmol) to a solution of octyl bromide (Sigma–Aldrich,
3.3 mmol) in acetonitrile, and the mixture was refluxed for 24 h. After cooling,
the solvent was evaporated and the residue was taken up in ethyl acetate and
washed with water. Solvent evaporation afforded a crude oil, which was
purified by flash chromatography (SiO2, hexane/ethyl acetate 3:1) to give both
compounds.
2-octylbenzo[d]isothiazol-3(2H)-one (2a): Yellow oil. Yield: 17%. MS (EI): m/z
264 [M+H]+. 1H NMR (CDCl3) d 0.87 (t, 3H, J = 6.3), 1.26–1.38 (m, 10H), 1.74
(quint, 2H, J = 7.3 Hz), 3.89 (t, 2H, J = 7.3 Hz), 7.39 (t, 1H, J = 7.3 Hz), 7.57 (m,
2H), 8.0 (d, 1H, J = 7.8 Hz). 13C NMR (CDCl3) d 14.0, 22.7, 26.7, 29.2, 29.3, 29.7,
31.9, 44.1, 120.4, 125.0, 125.5, 126.8, 131.7, 140.2, 165.4. IR (CHCl3, cmÀ1):
1659.
3-(octyloxy)benzo[d]isothiazole (2b): Yellow oil. Yield: 23%. MS (EI): m/z 264
[M+H]+. 1H NMR (CDCl3) d 0.89 (s, 3H), 1.26–1.38 (m, 8H), 1.49 (quint, 2H,
J = 8 Hz), 1.87 (quint, 2H, J = 6.6 Hz), 4.53 (t, 2H, J = 6.6 Hz), 7.37 (t, 1H,
J = 7.5 Hz); 7.50 (t, 1H, J = 7.5 Hz), 7.76 (d, 1H, J = 8 Hz), 7.91 (d, 1H, J = 8 Hz).
13C NMR (CDCl3) d 14.3, 22.8, 26.2, 29.2, 29.4, 29.5, 32.0, 69.0, 120.3, 123.3,
124.5, 125.7, 128.7, 151.7, 163.4. HR-MS (EI): calculated 263.13384, found
263.13325.
2-octylbenzo[d]isoxazol-3(2H)-one (5a): Yellowish oil. Yield: 34%. MS (EI): m/z
248 [M+H]+. 1H NMR (CDCl3) d 0.87 (s, 3H), 1.26 (m, 10H), 1.81 (quint, 2H,
J = 7.1 Hz), 4.00 (s, 2H), 7.26 (t, 2H, J = 7.6 Hz), 7.60 (t, 1H, J = 4.0 Hz), 7.84(d,
1H, J = 4.0 Hz). 13C NMR (CDCl3) d 14.2, 21.2, 22.8, 26.8, 27.9, 29.2, 31.9, 46.1,
109.9, 116.9, 123.5, 124.5, 133.1, 160.0, 162.3. IR (CHCl3, cmÀ1): 1692. HR-MS
(EI): calculated 248.16505, found 248.16528.
3-(octyloxy)benzo[d]isoxazole (5b): Colorless oil. Yield: 30%. MS (EI): m/z 248
[M+H]+. 1H NMR (CDCl3) d 0.89 (s, 3H), 1.29–1.31 (m, 10H), 1.89 (quint, 2H,
J = 7.1 Hz), 4.44 (s, 2H), 7.65 (d, H, J = 7.2 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.42 (d,
J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H). 13C NMR (DMSO-d6) d 14.2, 22.8, 25.9,
29.1, 29.3, 29.4, 31.9, 70.6, 110.3, 114.6, 121.0, 122.9, 130.4, 164.0, 166.8. HR-
MS (EI): calculated 248.16505, found 248.16536.
Acknowledgements
The UCL (Université Catholique de Louvain) and the F.R.S.-FNRS
(Fonds de la Recherche Scientifique, Belgium) are gratefully
acknowledged for financial support (FRFC no. 2.4604.09 and FRSM
no. 3.4552.11 grants). This work is partially supported by the Inter-
university Attraction Pole program (IAP P6/P19 PROFUSA). J.M-B is
senior research associate of the Belgian F.R.S.-FNRS
17. MAGL activity assay: Measurement of radiolabeled 2-oleoylglycerol (2-OG)
hydrolysis was performed as previously described.18 2-OG (50
lL, 10 lM final
concentration; [3H]-2-OG 50,000 dpm, American Radiolabeled Chemicals in
Tris buffer, pH 8.0, 50 mM with 0.6% BSA) was incubated at 37 °C for 10 min the
presence of purified recombinant human MAGL18 (5 ng of enzyme in 50 mM
Tris buffer, pH 8.0, 0.1% BSA, 200
lL of total volume assay). The incubation was
stopped by adding 400 L of an ice-cold 1:1 methanol chloroform mixture to
l
each tube and thorough mixing. After centrifugation at 700 g for 5 min,
radioactivity in the upper aqueous layer was measured by liquid scintillation.
Blanks (i.e., tubes containing no enzyme) were made for each experiment and
the values subtracted to all the other values.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.026.
18. Labar, G.; Bauvois, C.; Muccioli, G. G.; Wouters, J.; Lambert, D. M. Chembiochem
2007, 8, 1293.