Synthesis and in vitro evaluation of N-substituted maleimide derivatives as selective monoglyceride lipase inhibitors

Article abstract of DOI:10.1021/jm900461w

The endocannabinoid 2-arachidonoylglycerol (2-AG) plays a major role in many physiological processes, and its action is quickly terminated via enzymatic hydrolysis catalyzed by monoglyceride lipase (MGL). Regulating its endogenous level could offer therapeutic opportunities; however, few selective MGL inhibitors have been described so far. Here, we describe the synthesis of N-substituted maleimides and their pharmacological evaluation on the recombinant human fatty acid amide hydrolase (FAAH) and on the purified human MGL. A few N-arylmaleimides were previously described (Saario, S. M.; Salo, O. M.; Nevalainen, T.; Poso, A.; Laitinen, J. T.; Jarvinen, T.; Niemi, R. Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2-Arachidonoylglycerol in Rat Cerebellar Membranes. Chem. Biol. 2005, 12, 649-656) as MGL inhibitors, and along these lines, we present a new set of maleimide derivatives that showed low micromolar IC50 and high selectivity toward MGL vs FAAH. Then, structure-activity relationships have been investigated and, for instance, 1-biphenyl-4-ylmethylmaleimide inhibits MGL with an IC50 value of 790 nM. Furthermore, rapid dilution experiments reveal that these compounds act as irreversible inhibitors. In conclusion, N-substituted maleimides constitute a promising class of potent and selective MGL inhibitors.

Full text of DOI:10.1021/jm900461w

7410 J. Med. Chem. 2009, 52, 7410–7420
DOI: 10.1021/jm900461w
Synthesis and in Vitro Evaluation of N-Substituted Maleimide Derivatives as
Selective Monoglyceride Lipase Inhibitors
Nicolas Matuszak, Giulio G. Muccioli,† Geoffray Labar, and Didier M. Lambert*
ꢀ
Endocannabinoid and Cannabinoid Research Group, Drug Design and Discovery Centre, Unite de Chimie Pharmaceutique et de Radiopharmacie,
Louvain Drug Research Institute, Ecole de Pharmacie, Faculte de Medecine, Universite Catholique de Louvain, 73 Avenue E. Mounier UCL-
ꢀ
ꢀ
ꢀ
CMFA (7340), B-1200 Bruxelles, Belgium. †Present address: Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, Louvain Drug
ꢀ
Research Institute, Universite Catholique de Louvain, 72 Avenue E. Mounier UCL-CHAM (7230), B-1200 Bruxelles, Belgium.
Received April 10, 2009
The endocannabinoid 2-arachidonoylglycerol (2-AG) plays a major role in many physiological
processes, and its action is quickly terminated via enzymatic hydrolysis catalyzed by monoglyceride
lipase (MGL). Regulating its endogenous level could offer therapeutic opportunities; however, few
selective MGL inhibitors have been described so far. Here, we describe the synthesis of N-substituted
maleimides and their pharmacological evaluation on the recombinant human fatty acid amide
hydrolase (FAAH) and on the purified human MGL. A few N-arylmaleimides were previously
described (Saario, S. M.; Salo, O. M.; Nevalainen, T.; Poso, A.; Laitinen, J. T.; Jarvinen, T.; Niemi,
R. Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of
2-Arachidonoylglycerol in Rat Cerebellar Membranes. Chem. Biol. 2005, 12, 649-656) as MGL
inhibitors, and along these lines, we present a new set of maleimide derivatives that showed low
micromolar IC50 and high selectivity toward MGL vs FAAH. Then, structure-activity relationships
have been investigated and, for instance, 1-biphenyl-4-ylmethylmaleimide inhibits MGL with an
IC50 value of 790 nM. Furthermore, rapid dilution experiments reveal that these compounds act as
irreversible inhibitors. In conclusion, N-substituted maleimides constitute a promising class of potent
and selective MGL inhibitors.
Introduction
receptors,10,11 which is known to act as a retrograde messen-
ger.12 MGL has been purified, and its cDNA was isolated and
cloned from adipose tissue.4,5 This protein, containing two
lipase motives (active serine motif GXSXG and the HG
dipeptide) with a Ser122/His269/Asp239 catalytic triad
(evidenced by mutagenesis studies4), is predicted to belong
to the R/β hydrolase fold family and is found abundant in
brain tissue, particularly at presynaptic terminals13 and in
areas of the brain where CB1 cannabinoid receptors are also
localized. The relevance of MGL to 2-AG metabolism has
been shown by Dinh et al. who demonstrated that over-
expression of MGL in rat neurons reduced the accumulation
of 2-AG14 and that RNA interference-mediated silencing of
MGL expression enhanced 2-AG accumulation in HeLa
cells.15 However, they showed that half of the 2-AG hydro-
lyzing activity remains detectable after MGL was immunode-
pleted from cytosolic brain fractions, suggesting that other
metabolic pathways are involved in 2-AG degradation.15
More recently, Muccioli and co-workers discovered a 2-AG
hydrolase activity in microglial cells16 distinct from that of
MGL which could be assigned to ABDH6.17 This underlines
the need of new efficient pharmacological tools such as
purified enzymes or selective and efficient MGL inhibitors.
Furthermore, as the sequence homology between murine and
human proteins is only 83%,18 the use of a human enzyme
can provide relevant information about the enzyme and
will minimize the differences in drug-enzyme interactions
between in vitro and in vivo effects in human.
Modulating the level of the endocannabinoids anandamide
and 2-arachidonoylglycerol (2-AGa) through their biosynth-
esis or degradation pathways can constitute a therapeutic
approach for numerous pathological states, including multi-
ple sclerosis, pain and inflammation, and different types of
cancer.2 To date, four enzymes responsible for the inactiva-
tion of endocannabinoids via their hydrolysis have been
characterized at the molecular level: fatty acid amide hydro-
lase (FAAH),3 monoglyceride lipase (MGL),4,5 a recently
discovered type-2 FAAH,6 N-acylethanolamine hydrolyzing
acid amidase (NAAA).7 Moreover, two serine hydrolases,
namely, ABHD6 and ABHD12, have been proposed by
Blankman et al.8 as new 2-AG hydrolyzing enzymes in mouse
brain.
MGL is a 33 kDa serine hydrolase hydrolyzing the 2- or 1
(3)-ester bond of monoglycerides to fatty acids and glycerol.9
MGL hydrolyzes one of the major endocannabinoids, 2-AG,
an endogenous full agonist at CB1 and CB2 cannabinoid
*To whom correspondence should be addressed. Phone: þ32-2-764-
7347. Fax: þ32-2-764-7363. E-mail: didier.lambert@uclouvain.be.
a Abbreviations: 2-AG, 2-arachidonoylglycerol; ATFMK, arachido-
nyltrifluoromethylketone; BSA, bovine serum albumin; CB, cannabi-
noid; DEAD, diethylazodicarboxylate; FAAH, fatty acid amide
hydrolase; MAFP, methylarachidonyl fluorophosphonate; MGL,
monoglyceride lipase; NAAA, N-acylethanolamine-hydrolyzing acid
amidase; NAM, N-arachidonylmaleimide; 2-OG, 2-oleoylglycerol; Ph,
phenyl; PPh3, triphenylphosphine; THF, tetrahydrofuran.
r
pubs.acs.org/jmc
Published on Web 07/07/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7411
Table 1. Structures of Previously Reported MGL Inhibitorsb
a Values were from the original publication of Long21 from mouse recombinant enzymes. No SEM was provided in the reference. b Determination of
their selectivity profile of known MGL inhibitors on hMGL versus hFAAH. Values are expressed as mean ( SEM from three independent experiments
performed in duplicate.
From a medicinal chemistry point of view, few MGL
inhibitors are available and they often lack selectivity or
have not been evaluated on both human MGL and human
FAAH.19,20 A recent breakthrough has been done with
the report of 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl-
(hydroxy)methyl)piperidine-1-carboxylate (JZL184)21 as a
potent and selective MGL inhibitor. The administration of
this MGL inhibitor clearly demonstrated that a selective
blockade of 2-arachidonoylglycerol hydrolysis, without
modifying anandamide levels in the brain, produces canna-
binoid behavioral effects. Apart from this compound, MGL
can be inhibited by various nonspecific serine hydrolase
inhibitors, such as methylarachidonyl fluorophosphonate
(MAFP), hexadecylsulfonyl fluoride, and phenylmethylsul-
fonyl fluoride.9,22 On the basis of MGL sensitivity to free
sulfhydryl group-modifying agents such as p-chloromercur-
ibenzoate and mercury chloride, Saario and co-workers1
investigated the sensitivity of MGL to maleimide deri-
vatives, well-known Michael acceptors. Among them,
N-arachidonylmaleimide (NAM, 1) with a reported IC50
value of 140 nM was the most active in the series. Although
1 was not the most potent MGL inhibitor, it exhibits a
promising selectivity profile. As a preliminary experiment,
the inhibitory potential of human-MGL vs human-FAAH
has been measured for five different MGL inhibitors
(Table 1) 1, MAFP, arachidonyltrifluoromethylketone

7412 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Matuszak et al.
Scheme 1. Synthesis of the N-Phenylmaleimides (2-28), N-Alkylphenylmaleimides (29-36), N-Phenylsuccinimide (37), N-Phenylph-
talimide (38), 3-Methylphenylmaleimide (39), 3,4-Dimethylphenylmaleimide (40), and 1,3-Diphenylmaleimide (41)a
a Reagents and conditions: (a) stirred in diethyl ether for 1 h and filtered off; (b) then stirred in acetic anhydride with sodium acetate (0.5 equiv) for
30 min on a steam bath.
Scheme 2. Synthesis of N-Alkylmaleimides (44-46)a
neopentyl alcohol, triphenylphosphine (PPh3), and diethy-
lazodicarboxylate (DEAD) in anhydrous tetrahydrofuran
(THF), as outlined in Scheme 2.
N-Phenylsuccinimide (37) and N-phenylphtalimide (38)
were obtained following method A, starting from succinic
anhydride and phthalic anhydride, respectively. Using the
same method but starting from citraconic anhydride, 2,3-
dimethylmaleic anhydride, and 2-phenylmaleic anhydride
gave 3-methyl-1-phenylmaleimide (39), 3,4-dimethyl-1-phe-
nylmaleimide (40), and 1,3-diphenylmaleimide (41), respec-
tively. The bismaleimide derivative (49) was synthesized
using 1,4-benzenedimethanamine and maleic anhydride
(method A).
Structure-Activity Relationships. As a first step, we
wanted to test the hypothesis stating that 2 inhibits MGL
through a nucleophilic attack on its olefinic double bond.
Indeed, Saario et al. showed that the succinimide analogues
of the active maleimide were devoid of any activity against
MGL.1 However, those data were obtained on a tissue
preparation rather than using a pure MGL preparation,
and therefore, the substrate hydrolysis seen by Saario and
colleagues could have been at least partially the result of a
2-oleoylglycerol hydrolysis through a non-MGL activity.
Thus, we decided either to remove the double bond of the
maleimide moiety such in 37 or to render it unreactive
with respect to a Michael addition such as for phenylphtha-
limide 38. Both compounds were unable to inhibit MGL,
even when tested at millimolar concentrations (Table 2).
We also tested if increasing the steric hindrance around
the double bond (39-41) would be as detrimental to the
activity as removing it. Indeed the double bond of com-
pounds 39-41 is less accessible to the enzyme nucleophilic
attack, resulting in a reduction of about half of MGL activity
when tested at 1 mM.
a Reagents and conditions: (a) Pd(PPh3)4 (1 equiv), neopentyl alcohol
(0.5 equiv), DEAD (1 equiv) stirred overnight in THF.
(ATFMK), biphenyl-3-ylcarbamic acid cyclohexyl ester
(URB602), 5-biphenyl-4-ylmethyltetrazole-1-carboxylic acid
dimethylamide (LY2183240). On the basis of the obser-
vation that 1 and N-phenylmaleimide (2) both appeared to
be good MGL inhibitors1 with a high selectivity profile, we
report here the synthesis of a series of N-phenylmaleimides,
N-alkylmaleimides, and bismaleimides and their pharmaco-
logical characterization on both human recombinant FAAH
and MGL. We describe the structure-activity relationships
among these three subfamilies of maleimides and their selec-
tivity profile toward MGL, with respect to some of the few
known MGL inhibitors. Their mechanism of inhibition has
also been investigated.
Results
Chemistry. The N-substituted maleimides described here-
in were synthesized following two main routes. The first one
(Scheme 1, method A23) involves a substituted aniline or an
aliphatic amine and maleic anhydride as building blocks,
whereas the second one requires a primary alcohol and
maleimide (Scheme 2, method B24). The N-phenylmalei-
mides (2-35) were readily obtained using method A by
reacting the desired substituted aniline with maleic anhy-
dride in diethyl ether, leading to the corresponding
N-phenylmaleanic acid, which precipitates in diethyl ether.
Without any further purification, this open intermediate was
subsequently cyclized in acetic anhydride in the presence of
sodium acetate to the N-phenylmaleimide of interest (2-35).
1-Biphenyl-4-ylmethylmaleimide (36) and the N-alkylmalei-
mide derivatives (44-46) were obtained using the Mitsuno-
bu procedure (method B). Therefore, the relevant alkyl
alcohol was stirred with maleimide in the presence of
Taken together, these data confirm that the presence of a
freely accessible double bond is a key parameter to MGL
inhibition by maleimide derivatives. Since the double bond is
essential to the inhibition, the maleimide motif was kept
unchanged and the effect of the N-substitution on the
inhibitory potency was explored (Table 3). Three series of
compounds, N-phenylmaleimides (2-35), N-alkylmalei-
mides (42-46), and bismaleimides (47-53), were evaluated
on purified human MGL activity.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7413
being obtained with N-(3-iodophenyl)maleimide (27, IC50=
2.24 μM). Upon analysis of the data reported in Table 3, it is
apparent that the size of the halogen has little influence on
the inhibition; indeed, regardless of the position, there was
no significant difference between the fluorine-substituted
compounds (17-19) and the iodine-substituted compounds
(26-28). For instance, N-(4-fluorophenyl)maleimide (19,
Table 2. Structures and Inhibition Potential of N-Phenylmaleimide,
N-Phenylsuccinimide, N-Phenylphtalimide, and Hindered Double-
Bond Maleimides toward hMGL and hFAAH Activitya
IC50 = 5.18 μM), N-(4-chlorophenyl)maleimide (22, IC50
=
7.24 μM), N-(4-bromophenyl)maleimide (25, IC50=4.37 μM),
and N-(4-iodophenyl)maleimide (28,IC50=4.34 μM) all showed
a similar inhibitory potency toward MGL. However, the posi-
tion of the substituent had some influence on the inhibition
potential; as for all the halogens, position 3 was preferred to
positions 2 and 4. A possible explanation could be that the
attractive inductive effect of the halogens in position 3 lowers the
electronic density of the double bond which in turn makes it
more sensitive to a nucleophilic attack. Moreover, this series still
showed good selectivity toward MGL, with most IC50 values for
FAAH inhibition being above 200 μM.
The next step in the structure-activity relationships
studies reported here consisted of adding a phenyl ring to
the N-phenylmaleimide moiety, resulting in three N-biphe-
nylmaleimide derivatives (29-31, Table 4). When com-
pared to 2 (IC50=15.90 μM), there was a 10-fold increase
in MGL inhibition with IC50 values around 2 μM for
29-31. Note that, as previously observed, the position of
the phenyl substituent had no influence on MGL inhibition.
As the electronic effect of the phenyl substituent cannot
explain here the evolution of the inhibitory potential,
it seems that the steric effect of the biphenyl moiety or a
π-π interaction with an aromatic residue in the active site
of MGL could play a role in the enhancement of inhibition.
Contrary to what is observed for MGL, the phenyl sub-
stituent position has a great influence on the inhibition of
a Values are expressed as mean ( SEM from three independent
experiments performed in duplicate for the IC50 values. In the absence
of IC50 values, percentages of inhibition at 1 mM are given.
In order to study the importance of the alkyl chain length
and position on the phenyl ring, a series of alkyl-substituted
N-phenylmaleimides (i.e., N-(alkylphenyl)maleimides, 2-12)
was synthesized. The N-(alkylphenyl)maleimides showed
MGL inhibition properties with IC50 values ranging from
2.00 to 14.10 μM. The best inhibition was observed with
compounds bearing a methyl or an ethyl group in position
2. In order to investigate the influence of a longer alkyl chain
linked to the phenyl ring, we also tested N-(4-heptylphenyl)-
maleimide 12. Only small differences in the MGL inhibitory
activity were observed when the side chain in position 4 of the
phenyl ring was lengthened from two (8, IC50 = 2.82 μM)
to seven carbons (12, IC50=2.12 μM). On the contrary, if the
side chain in position 4 is shortened to one carbon (5, IC50=
14.10 μM) or is replaced by H (2, IC50 = 15.9 μM), the
inhibitory activity is significantly reduced. Note that these
N-(alkylphenyl)maleimides showed a good selectivity profile
for MGL toward FAAH, with IC50 values on FAAH ranging
from 65 μM to values above 500 μM (Table 3).
We next synthesized compounds with more hydrophilic
substituents, such as hydroxy or alkoxy substituents (13-
16), on the phenyl ring. N-(4-Hydroxyphenyl)maleimide (13,
IC50 = 12.9 μM) had a similar potency compared with 2,
whereas the alkoxy-substituted compounds (14-16) showed
inhibition values ranging between 5.75 μM (16) and 6.92 μM
(14). These two series of compounds retained the same
selectivity factor of 100-300 for MGL compared to FAAH.
The following step in this series was to investigate the
effect of halogen substitution on the phenyl ring. Twelve
N-(halogenophenyl)maleimide derivatives varying by the
nature and position of the halogen atom were synthesized
(17-28). These compounds showed inhibition activity, with
IC50 values ranging from 2.24 to 7.24 μM, the best inhibition
FAAH activity, with 1-biphenyl-4-ylmaleimide (31, IC50=
5.89 μM) being 30 times more active than 1-biphenyl-
2-ylmaleimide (29, IC50=180 μM).
Interestingly, introducing a methylene spacer between the
maleimide and the 4-biphenyl motif led to a further increase
of MGL inhibition, as observed with 36 (IC50=0.79 μM).
Because the presence of a methylene spacer increased
MGL inhibition, we investigated the influence of the spacer
arm between the maleimide moiety and the phenyl ring.
Thus, we synthesized three derivatives characterized by a
spacer of increasing length between the maleimide and the
phenyl moieties (32-34, Table 4). All three compounds,
1-benzylmaleimide (32), 1-phenethylmaleimide (33), and
1-(4-phenylbutyl)maleimide (34), proved to be more potent
compared to 2.
The compounds gained in activity when the phenyl or
biphenyl moiety is spaced out from the maleimide ring; the
IC50 value is 3-7 times lower when the phenyl ring is spaced
out from the maleimide backbone (compound 2 vs 32-34)
and is also 2 times better when there is a methylene between
the maleimide ring and the biphenyl moiety (compound 31 vs
36). Finally, as seen with the other series, all these com-
pounds retained selectivity for MGL vs FAAH.
Our second major series of N-substituted maleimides was
the N-alkylmaleimides (Table 5). Indeed, N-ethylmaleimide
(43) was shown to inhibit the hydrolysis of 2-AG in rat
cerebellar membranes with an IC50 value of 53 μM.1 In our
hands, by the use of a purified human recombinant MGL, 43
inhibited 2-OG hydrolysis with an IC50 value of 16.6 μM.
The shorter analogue, N-methylmaleimide (42), was slightly

7414 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Matuszak et al.
Table 3. hMGL and hFAAH Inhibition by N-Phenylmaleimide Derivatives 3-28a
IC50 (μM)
compd
R
R0
R00
hMGL
hFAAH
selectivity
3
4
CH3
H
H
CH3
H
H
2.69 ( 0.33
2.75 ( 0.48
14.10 ( 1.70
2.00 ( 143
4.68 ( 0.57
2.82 ( 0.27
5.89 ( 0.57
7.86 ( 0.95
2.88 ( 0.43
2.12 ( 0.35
12.90 ( 2.2
6.92 ( 0.84
6.60 ( 0.47
5.75 ( 0.56
4.47 ( 0.55
2.39 ( 0.35
5.18 ( 0.45
4.17 ( 0.51
2.82 ( 0.20
7.24 ( 1.10
2.92 ( 0.52
4.27 ( 0.31
4.37 ( 0.53
5.01 ( 0.36
2.24 ( 0.22
4.34 ( 0.80
>500
>500
>200
>200
>150
>150
22
5
H
CH2CH3
H
CH3
H
>500
>500
6
H
CH2CH3
7
H
H
CH2CH3
(CH2)2CH3
103 ( 7
163 ( 12
186 ( 65
65 ( 10
120 ( 18
87 ( 18
288 ( 50
>500
8
H
H
H
H
H
H
H
H
OCH3
H
H
F
58
32
9
H
CH(CH3)2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
CH(CH3)2
(CH2)6CH3
9
40
H
H
41
30
H
OH
H
OCH3
H
78
H
>500
>500
>200
>200
63
H
OCH3
H
F
295 ( 50
89 ( 16
120 ( 21
214 ( 88
102 ( 26
112 ( 11
>500
H
H
37
23
H
H
H
Cl
H
H
Br
H
H
I
F
Cl
H
H
77
39
H
H
Cl
H
7
75
Br
H
H
151 ( 58
51 ( 12
200 ( 124
62 ( 11
60 ( 4
27
50
H
Br
H
I
37
45
H
H
H
H
I
14
a Values are expressed as the mean ( SEM from three independent experiments performed in duplicate.
Table 4. Inhibition Values of N-Biphenylmaleimides (29-31), N-Al-
kylphenylmaleimides (32-34), 1-(4-Ethylbenzyl)maleimide (35), and 1-
Biphenyl-4-ylmethylmaleimide (36) on hMGL and hFAAH Activitya
Table 5. Inhibition Values of N-Methylmaleimide (42), N-Ethylmalei-
mide (43), and N-Alkylmaleimides (44-46) on hMGL and hFAAHa
IC50 (μM)
IC50 (μM)
compd
R
hMGL
hFAAH
selectivity
1.95
compd
n
R
R0
R00
hMGL
hFAAH
selectivity
1
42
43
44
45
46
arachidonyl
methyl
ethyl
1.12 ( 0.14
24.5 ( 3.0
16.6 ( 1.6
3.55 ( 0.43
2.69 ( 1.38
0.93 ( 0.16
2.18 ( 0.62
29
30
31
32
33
34
35
36
0
0
0
1
2
4
1
1
Ph
H
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
1.55 ( 0.23
2.34 ( 0.29
180 ( 57
49 ( 6.0
116
21
4
40% at 1 mM
35% at 1 mM
40.7 ( 8.2
0% at 1 mM
26% at 1 mM
H
Ph
H
H
H
1.62 ( 0.16 5.89 ( 2.0
nonyl
hexadecyl
oleyl
12
5.14 ( 0.76
7.61 ( 1.13
2.14 ( 0.49
186 ( 38
249 ( 79
85 ( 13
36
33
39
35
73
a values are expressed as mean ( SEM from 3 independent experi-
ments performed in duplicate for the IC50 values, in absence of IC50
values, percentages of inhibition at 1 mM are given).
ethyl 3.55 ( 0.53
Ph
123 ( 12
0.79 ( 0.23 16.6 ( 3.8
a Values are expressed as the mean ( SEM from three independent
experiments performed in duplicate.
more active, with IC50 values of 0.93 and 1.12 μM, respectively,
even if we can postulate that the introduction of a double bond
in the alkyl chain allows a better interaction with MGL.
Thus, compared to the nonyl derivative, the increase
of the alkyl chain length results in less soluble derivatives
without significant gain in activity. These compounds retained
selectivity toward MGL inhibition, with FAAH inhibition
IC50 values in the millimolar range. Note that the somewhat
lower activity for 1 compared to what was first reported by
less active (IC50=24.5 μM). As might be expected, a longer
substituent such as the one of N-nonylmaleimide (44) resulted
in an increased inhibition of MGL (46, IC50=3.55 μM). Note
that the activity was only slightly modified by a further increase
of the alkyl chain length as observed with N-hexadecylmalei-
mide (45, IC50=2.69 μM). Interestingly, the closest substrate
analogues (i.e., N-oleylmaleimide (46) and 1) were only slightly

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7415
Saario et al.1 is in line with what we previously reported25,26
and to what Blankman et al. found using rat brain
membranes.8
As these compounds are assumed to act via a Michael
addition on the cysteine residues of the enzyme, preincuba-
tion of the enzyme with maleimide inhibitors would facilitate
and enhance the covalent addition, thus increasing the
inhibitory potential of such compounds. As a first approach,
we investigated the influence of different preincubation times
on the inhibitory potency of N-phenylmaleimide 2 and1,
4-bis-(malimido)xylene 49 (Figure 1). These compounds
showed higher inhibition values following 15 and 30 min of
preincubation with the enzyme. The most relevant effect of
preincubation was noticed after 30 min, demonstrating that
the inhibitory effect of such compounds could be enhanced
by preincubating them with the enzyme.
Then, we investigated whether these compounds act as
reversible or irreversible MGL inhibitors. MAFP and
ATFMK were used as controls for an irreversible and
reversible MGL inhibition, respectively.26-28 We tested
1 and two of our best MGL inhibitors so far: 36 and 49
(Figure 2).
Saario et al.1 suggested the presence of two cysteine
residues (Cys 208 and Cys 242) in the vicinity of MGL active
site that would explain the activity of the maleimide deriva-
tives. Thus, we hypothesized that a compound bearing two
reactive moieties (i.e., two maleimides) could be more prone
to potently inhibit MGL. This hypothesis was investigated
by testing a series of bismaleimides derivatives varying by the
nature of their linker (Scheme 3 and Table 6).
Two close analogues of 2, phenyl-1,3-bismaleimide (47)
and phenyl-1,4-bismaleimide (48), were first assayed against
MGL. As illustrated in Table 5, 47 demonstrated low
micromolar inhibition value toward MGL. However, no
significant difference was observed between the 1,3- and
the 1,4- analogues. These compounds are 8 times more
potent on MGL than 2. We next increased the flexibility of
the linker by synthesizing compounds 49 and 50, which
inhibited MGL with IC50 values of 1.70 and 2.24 μM,
respectively. Finally, compounds with an even more flexible
linker (i.e., butyl (51), hexyl (52), and bis-1,2-diethoxyethane
(53)) were assayed without noting any further increase of the
activity (IC50 values of 28.8, 1.70, and 1.95 μM, respectively).
When 50 (IC50=1.70 μM) was compared with its analogue
lacking the second maleimide moiety (35, IC50=3.55 μM), it
appears that a second maleimide moiety does not result in an
increased inhibitory potency. Furthermore, 51 and 52
showed IC50 values of the same magnitude as their corre-
sponding N-alkylmaleimides analogues. This suggests that
either reacting with one residue is sufficient to fully inhibit
the enzyme or that the two potentially reactive cysteine
residues are not close enough to allow a single molecule to
react with both of them.
After rapid dilution, MAFP, known as irreversible inhi-
bitor, still inhibited 83% of MGL activity while ATFMK,
reported as reversible one, only inhibited 26% of MGL
Table 6. Structures and Inhibitory Activities of the Bismaleimides
Assayed in This Studya Regarding hMGL and hFAAH
IC50 (μM)
compd
hMGL
hFAAH
selectivity
47
48
49
50
51
52
53
2.03 ( 0.30
2.22 ( 0.45
1.70 ( 0.21
2.24 ( 0.30
28.8 ( 6.60
1.70 ( 0.16
1.95 ( 0.34
37.2 ( 11.8
13.49 ( 2.90
16.6 ( 2.90
10.7 ( 1.03
8.32 ( 2.65
18.2 ( 2.69
80% at 1 mM
18
6
Figure 1. Influence of the preincubation of 2 and 49 on the inhibi-
tion of the purified human MGL activity.
10
5
N-Phenylmaleimide (2) and 1,4-bis(malimido)xylene (49) were pre-
incubated at their IC50 value for 0, 15, and 30 min with human
purified MGL. Data shown are the mean of three experiments
performed in duplicate. Statistical significance was assessed by one-
way ANOVA followed by a Dunett post-test (/, P < 0.05; //, P <
0.01) relative to no-preincubated samples.
0.3
11
a IC50 values or percent of inhibition at 1 mM. In the absence of IC50
values, percentages of inhibition at 1 mM are given.
Scheme 3. Structure of the Bismaleimides Used in This Study

7416 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Matuszak et al.
compared to their FAAH activity. It seems determinant for
the enzyme inhibition that the maleimide moiety is substituted
by a large lipophilic group (biphenyl) and spaced out from
the maleimide ring: indeed, the best inhibitor of our series is
36, with an IC50 value of 790 nM on MGL and a selectivity
index of 73 toward FAAH. Previously described MGL
inhibitors have been assessed for their potency in stress
induced analgesia32 or their antinociceptive effects,31,33,34
and compounds like 36 or 46 could produce the same bene-
ficial effects. Furthermore, the maleimide derivatives’ me-
chanism of inhibition is comparable to that of 1, which was
recently shown to enhance 2-AG effects in vivo.29 In compar-
ison to the high lipophilicity of 1 (clogP value of 6.12) and of
46 (clogP value of 6.12), compound 36 exhibited a higher
inhibitory potential as well as a reduced lipophilicity (clogP
value of 2.89). This could represent a new horizon in the
treatment of diseases where the imbalance of endocannabi-
noids has been proved to play a major role, like in CNS
diseases and in the treatment of pain and psychiatric disorders
(depression, anxiety, etc.).
Figure 2. Irreversible inhibition assay of several MGL inhibitors
(MAFP, ATFMK, 1, 36, 49) with purified human MGL.
All compounds were assayed at 40 times their IC50 value except
ATFMK at 20 times its IC50 value on human purified MGL.
Dimethyl sulfoxide was taken as a control. Data shown are the
mean of three experiments performed in duplicate. Statistical
significance was assessed by one-way ANOVA followed by a
Dunett post-test (/, P < 0.05; //, P < 0.01), relative to DMSO.
Experimental Section
Chemistry. All reagents were purchased from commercial
sources (Sigma-Aldrich or Acros) and were used without further
purification. Solvents were of analytical grade. Bismaleimidobu-
tane (1-(4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)butyl)-1H-maleimide)
(51), bismaleimidohexane (1-(6-(2,5-dioxo-2H-pyrrol-1(5H)-yl)-
hexyl)-1H-maleimide) (52), and 1,8-bis-maleimidodiethyleneglycol
(53) were bought from Thermo Fisher Scientific, and 1,10-(methy-
lenedi-4,1-phenylene)bismaleimide (50) was purchased from
Acros Organics. N,N0-(1,3-phenylene)dimaleimide (47), N,N0-
(1,4-phenylene)dimaleimide (48), N-methylmaleimide (42), and
N-ethylmaleimide (43) were from Sigma-Aldrich, and N-arachi-
donylmaleimide (1) was bought from Cayman Chemicals. Nuclear
magnetic resonance (1H NMR, 13C NMR) spectra were recorded
at room temperature on a Bruker Avance 400 operating at
activity. All three maleimides tested showed similar beha-
vior compared with the one observed with MAFP, thus
still inhibiting from 69% to 86% of MGL activity after a
400-fold dilution into assay buffer. As expected, these results
taken together validated the hypothesis that these com-
pounds act as covalent inhibitors of MGL and that the
inhibitory potential of such inhibitors is enhanced by a
preincubation with the enzyme.
Conclusion
Here, we report the synthesis and structure-activity rela-
tionship studies of maleimide-type MGL inhibitors. The
maleimide family could present many advantages. First, used
as probes, they could give new interesting data about the
enzyme’s environment, as they react with particular amino
acids. Indeed, recently, using mass spectrometry and muta-
tional analysis, Zvonok and co-workers28,29 gave new infor-
mation about the recombinant enzyme catalytic site using 1,
confirming Saario’s first hypothesis and pushing us forward
to use our inhibitors as “informers” about the local environ-
ment of the enzyme. Second, as Michael acceptors, they are
good irreversible inhibitors, and thus, the role of MGL in
2-AG degradation could be efficiently evaluated. Indeed,
Burston et al. demonstrated that 1 could reveal the in vitro
activity of 2-AG and enhance the effects of 2-AG through
inhibition of MGL.30 However, this class of agents also
presents some limitations. Indeed, their high reactivity with
free thiols may cause them to cross-react with other proteins
that depend on cysteine residues for their function.
From a therapeutic point of view, the design of endocan-
nabinoid hydrolases inhibitors may be a chance for the
treatment of several pathological states such as inflammatory
pain31 or anxiety. Indeed, the elevation of the endocannabi-
noidlevels directlywhere endocannabinoidsare producedand
needed would allow for higher selectivity compared to the
systemic use of cannabinoid agonists.
The inhibitors presented here demonstrated good inhibi-
tion toward MGL with IC50 values in the low micromolar
range for most of them. Besides their relative potency, it is
important to note thatthese inhibitors are quite selective when
1
400 MHz for H and 100 MHz for 13C. Chemical shifts (δ) are
reported relative to the tetramethylsilane peak set at 0.00 ppm. In
the case of multiplets, the signals are reported as intervals. Signals
were abbreviated as s, singlet; d, doublet; t, triplet; q, quadruplet;
m, multiplet. Melting points (mp) were determined in open
capillaries using an Electrothermal 9100 apparatus and are re-
ported uncorrected. Mass spectra were recorded on a Finningan
MAT 44S, with an ionization voltage of 70 eV.
All tested compounds were at least 95% pure on the basis of
HPLC-MS using a LTQ Orbitrap mass spectrometer (Thermo-
Fisher Scientifc) coupled to an Accela HPLC system (Thermo-
Fisher Scientific). Separation was achieved using a Luna LC-18
column (5 μM, 4.60 mm ꢀ 250 mm) (from Phenomenex).
Elemental analyses were performed for original compounds
on a Carlo Erba EA 1108 analyzer (Carlo Erba, Milano, Italy)
and are within (0.4% of the theoretical values; values are
presented in the Supporting Information. Liquid film infrared
(IR) spectra were recorded using a Perkin-Elmer FT-IR 286
spectrometer, and values are reported as λ in cm-1; relevant
peaks are presented in the Supporting Information.
General Procedures. 3-Phenylaniline (30a). 30a was obtained
through a Suzuki coupling. Briefly, to a stirred solution of
3-bromoaniline (2.2 mL, 20 mmol) in toluene (100 mL) were
added Pd(PPh3)4 (0.924 g, 0.80 mmol), a solution of Na2CO3
(16.59 g, 20 mmol) in H2O (50 mL), and a solution of phenyl-
boronic acid (4.98 g, 40 mmol) in EtOH (50 mL) under N2
atmosphere. The mixture was vigorously stirred under reflux
overnight. Once the mixture cooled, water was added, and
the mixture was extracted with CH2Cl2. The combined
organic layers were dried (Na2SO4) and concentrated under
reduced pressure. Purification of the residue by silica column

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7417
chromatography (hexane/EtOAc 1:4) gave 3-phenylaniline
as a yellow solid (3.26 g, 96%). GC-MS m/z 170 (MHþ).
1H NMR (CDCl3) δ 6.67 (d, 1H), 6.90 (s, 1H), 6.99 (d, 1H),
7.22 (t, 1H), 7.32 (t, 1H), 7.55 (d, 2H), 7.40 (t, 2H). 13C NMR
(CDCl3) δ 114.0, 114.2, 117.8, 127.1, 127.2, 128.6, 129.7, 141.4,
142.5, 146.6.
1-(4-Heptylphenyl)maleimide (12). White-off crystalline solid.
Yield: 74%. Mp 55-56 °C. GC-MS m/z 272 (MHþ). 1H NMR
(DMSO-d6) δ 0.86 (t, 3H), 1.28 (d, 8H), 1.58 (t, 2H), 2.60 (t, 2H),
7.16 (d, 2H), 7.21 (d, 2H), 7.29 (d, 2H). 13C NMR (CDCl3)
δ 13.8, 22.0, 28.4, 28.5, 30.8, 31.2, 34.6, 126.6, 128.6, 129.0,
134.6, 142.0, 170.0.
General Procedure for the Preparation of N-Arylmaleimides
(Method A) (Compounds 2-36). In a 100 mL three-necked flask
provided with a stirrer, a reflux condenser, and a dropping
funnel were placed 1.96 g (20 mmol) of maleic anhydride (or
succinic anhydride) and 25 mL of diethyl ether. The maleic
anhydride dissolved upon stirring, at which point a solution of
1 equiv (20 mmol) of the appropriate aniline in 5 mL of diethyl
ether was run through the dropping funnel. The resulting thick
suspension was stirred at room temperature for 1 h and was then
cooled in an ice bath. The N-substituted maleanic acid was
recovered by filtration and dried and subsequently added to a
flask containing a solution of anhydrous sodium acetate (0.65 g,
8 mmol) in acetic anhydride (6.7 mL) and stirred over a steam
bath for 30 min. The reaction mixture was then cooled to
room temperature in a cold water bath and was then poured
into 100 mL of an ice-water mixture. The precipitated product
was recovered by filtration, washed three times with 30 mL
portions of ice-cold water, and dried. The crude N-substituted
maleimide was recrystallized from ethanol/water to afford the
desired product.
1-(4-Hydroxyphenyl)maleimide (13). Golden crystalline solid.
Yield: 87%. Mp 188-190 °C. GC-MS m/z 190 (MHþ). 1H
NMR (DMSO-d6) δ 2.29 (s, 2H), 7.19 (s, 2H), 7.25 (m, 2H), 7.38
(m, 2H). 13C NMR (DMSO-d6) δ 122.3, 127.9, 134.6, 149.5,
169.8.
1-(2-Methoxyphenyl)maleimide (14). Brown crystalline solid.
Yield: 88%. Mp 109-111 °C. GC-MS m/z 204 (MHþ). 1H
NMR (CDCl3) δ 6.82 (s, 2H), 7.02 (q, 2H), 7.16 (d, 1H), 7.39
(t, 1H), 3.78 (s, 3H). 13C NMR (CDCl3) δ 55.8, 112.1, 119.8,
130.0, 134.5, 155.4, 169.7.
1-(3-Methoxyphenyl)maleimide (15). Yellow crystalline solid.
Yield: 76%. Mp 68-70 °C. GC-MS m/z 204 (MHþ). 1H NMR
(DMSO-d6) δ 3.77 (s, 3H), 6.91 (m, 2H), 6.98 (d, 1H), 7.17
(s, 2H), 7.38 (t, 1H). 13C NMR (DMSO-d6) δ 55.9, 112.7, 113.2,
118.9, 129.5, 132.5, 134.5, 159.3, 170.0.
1-(4-Methoxyphenyl)maleimide (16). Green crystalline solid.
Yield: 84%. Mp 136-138 °C. GC-MS m/z 204 (MHþ). 1H
NMR (CDCl3) δ 3.81 (s, 3H), 6.81 (s, 2H), 6.97 (s, 2H), 7.22
(d, 2H). 13C NMR (CDCl3) δ 55.5, 114.5, 123.9, 127.6, 134.1,
159.2, 169.8.
1-(2-Fluorophenyl)maleimide (17). Pale-yellow crystalline so-
1-(2-Methylphenyl)maleimide (3). Beige crystalline solid. Yield:
1
lid. Yield: 62%. Mp 74-75 °C. GC-MS m/z 192 (MHþ). H
84%. Mp 47-48 °C. GC-MS m/z 188 (MHþ). 1H NMR (CDCl3)
δ 2.12 (s, 3H), 6.73 (s, 2H), 7.08 (s, 1H), 7.23-7.30 (m, 3H). 13
C
NMR (DMSO-d6) δ 7.50-7.56 (m, 1H), 7.40-7.47 (m, 2H),
7.35 (t, 1H), 7.27 (s, 2H). 13C NMR (CDCl3) δ 116.7, 116.9,
124.7, 129.8, 130.7, 130.8, 134.7, 168.7.
NMR (CDCl3) δ 17.8, 126.8, 128.7, 129.3, 130.1, 131.0, 131.0,
134.2, 136.9, 169.6.
1-(3-Fluorophenyl)maleimide (18). Yellow crystalline solid.
Yield: 48%. Mp 61-62 °C. GC-MS m/z 192 (MHþ). 1H NMR
(DMSO-d6) δ 7.20 (s, 2H), 7.32 (d, 2H), 7.70 (d, 2H). 13C NMR
(CDCl3) δ 125.9, 128.0, 128.4, 133.9, 141.1, 169.3.
1-(3-Methylphenyl)maleimide (4). Yellow crystalline solid.
Yield: 78%. Mp 35-38 °C. GC-MS m/z 188 (MHþ). 1H
NMR (DMSO-d6) δ 3.36 (t, 1H), 7.21 (s, 1H), 7.17 (s, 2H), 7.12
(d, 2H). 13C NMR (CDCl3) δ 21.0, 123.0, 126.5, 128.5, 128.6,
130.7, 133.8, 138.9, 169.3.
1-(4-Methylphenyl)maleimide (5). Yellow powder. Yield: 91%.
Mp 158-160 °C. GC-MS m/z 188 (MHþ). 1H NMR (DMSO-d6)
δ 2.38 (s, 3H), 6.82 (s, 2H), 7.20 (d, 2H), 7.26 (d, 2H). 13C NMR
(CDCl3) δ 21.1, 126.0, 128.5, 129.8, 134.2, 138.1, 169.7.
1-(2-Ethylphenyl)maleimide (6). White crystalline solid. Yield:
73%. Mp 60-61 °C. GC-MS m/z 202 (MHþ). 1H NMR
(CDCl3) δ 1.13 (t, 3H), 2.44 (q, 2H), 7.06 (d, 1H), 7.26 (t, 1H),
7.34-7.39 (m, 2H). 13C NMR (CDCl3) δ 14.2, 24.3, 126.8, 129.0,
129.3, 129.5,129.7, 134.3, 142.4, 170.0.
1-(4-Fluorophenyl)maleimide (19). Yellow crystalline solid.
Yield: 72%. Mp 146-148 °C. GC-MS m/z 192 (MHþ). 1H
NMR (CDCl3) δ 6.83 (s, 2H), 7.14 (t, 2H), 7.32(q, 2H). 13C
NMR (CDCl3) δ 116.0, 127.2, 127.9, 134.2, 160.6, 169.4.
1-(2-Chlorophenyl)maleimide (20). Orange crystalline solid.
Yield: 86%. Mp 74-75 °C. GC-MS m/z 208 (MHþ). 1H NMR
(CDCl3) δ 6.88 (s, 2H), 7.26 (d, 1H), 7.37 (m, 2H), 7.53 (d, 2H). 13
NMR (CDCl3) δ 127.6, 129.0, 130.3, 130.6, 133.1, 134.4, 168.7.
C
1-(3-Chlorophenyl)maleimide (21). Yellow crystalline solid.
Yield: 77%. Mp 66-69 °C. GC-MS m/z 208 (MHþ). 1H NMR
(CDCl3) δ 6.84 (s, 2H), 7.28 (d, 1H), 7.34 (d, 2H), 7.38 (d, 1H), 7.40
(t, 1H). 13C NMR (CDCl3) δ 124.0, 126.1, 128.1, 130.1, 132.4,
134.3, 135.7, 169.0.
1-(3-Ethylphenyl)maleimide (7). Yellow crystalline solid. Yield:
68%. Mp 80-82 °C. GC-MS m/z202 (MHþ). 1H NMR (DMSO-
d6) δ 1.19 (t, 3H), 3.64 (q, 2H), 7.14 (t, 2H), 7.17 (s, 2H), 7.25 (d,
1H), 7.39 (t, 1H). 13C NMR (DMSO-d6) δ 15.0, 27.5, 123.8, 125.8,
126.9, 128.4, 131.2, 134.3, 144.2, 169.6.
1-(4-Chlorophenyl)maleimide (22). Yellow crystalline solid.
Yield: 94%. Mp 114-115 °C. GC-MS m/z 208 (MHþ). 1H
NMR (CDCl3) δ 6.84 (s, 2H), 7.25 (d, 2H), 7.58 (d, 2H). 13C
NMR (CDCl3) δ 119.4, 125.2, 128.2, 130.2, 132.2, 170.0.
1-(2-Bromophenyl)maleimide (23). Pale-white crystalline so-
1-(4-Ethylphenyl)maleimide (8). Golden solid. Yield: 89%.
Mp 65-68 °C. GC-MS m/z 202 (MHþ). 1H NMR (CDCl3) δ
1.24 (t, 3H), 2.65 (q, 2H), 6.81 (d, 2H), 7.21-7.28 (m, 2H). 13
C
1
lid. Yield: 76%. Mp 78-80 °C. GC-MS m/z 253 (MHþ). H
NMR (CDCl3) δ 15.5, 28.6, 100.1, 126.2, 128.7, 128.8, 128.9,
134.3, 144.4, 169.8.
NMR (CDCl3) δ 6.86 (s, 2H), 7.25 (d, 1H), 7.31 (t, 1H), 7.42 (t,
1H), 7.70 (d, 1H). 13C NMR (CDCl3) δ 122.3, 128.4, 130.8,
133.6, 134.5, 168.8.
1-(4-Propylphenyl)maleimide (9). Golden solid. Yield: 44%.
Mp <30 °C. GC-MS m/z 216 (MHþ). 1H NMR (CDCl3) δ 1.50
(t, 3H), 2.57 (m, 2H), 3.42 (t, 2H), 7.00 (s, 2H), 7.17 (d, 2H), 7.26
(t, 2H). 13C NMR (DMSO-d6) δ 27.5, 28.1, 34.5, 36.8, 125.6,
128.2, 134.4, 141.8, 171.0.
1-(2-Isopropylphenyl)maleimide (10). Yellow crystalline solid.
Yield: 79%. Mp 114-116 °C. GC-MS m/z 216 (MHþ). 1H
NMR (CDCl3) 1.57 (s, 6H), 4.04 (s, 2H), 6.86 (s, 2H), 6.88-6.91
(m, 2H), 7.27 (d, 1H), 7.60 (d, 1H). 13C NMR (CDCl3) δ 30.9,
121.5, 127.4, 130.3, 132.3, 134.3, 169.1.
1-(4-Isopropylphenyl)maleimide (11). Yellow crystalline solid.
Yield: 55%. Mp 70-72 °C. GC-MS m/z 216 (MHþ). 1H NMR
(DMSO-d6) δ 1.22 (d, 6H), 2.93 (m, 1H), 7.16 (s, 2H), 7.23 (d,
2H), 7.35 (d, 2H). 13C NMR (DMSO-d6) δ 23.7, 33.1, 126.6,
126.7, 129.1, 134.5, 148.0, 170.0.
1-(3-Bromophenyl)maleimide (24). Golden needles. Yield:
78%. Mp 114-116 °C. GC-MS m/z 253 (MHþ). 1H NMR
(CDCl3) δ 7.26 (s, 2H), 7.34 (d, 2H), 7.51 (m, 1H), 7.56 (s, 1H).
13C NMR (CDCl3) δ 124.5, 128.9, 130.3, 130.9, 132.5, 134.3, 135.2,
169.0.
1-(4-Bromophenyl)maleimide (25). Yellow crystalline solid.
Yield: 87%. Mp 128-130 °C. GC-MS m/z 253 (MHþ). 1H
NMR (CDCl3) δ 1.32- 1.41 (m, 4H), 1.59-1.62 (m, 2H), 1.68-
1.71 (m, 2H), 2.58 (t, 2H), 3.04 (t, 2H), 7.09-7.14 (m, 3H), 7.19-
7.22 (m, 2H), 7.62 (s, 1H). 13C NMR (CDCl3) δ 121.5, 127.4, 130.3,
132.2, 134.3, 169.1.
1-(2-Iodophenyl)maleimide (26). Brown needles. Yield: 88%.
Mp 102-104 °C. GC-MS m/z 300 (MHþ). 1H NMR (CDCl3)

7418 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Matuszak et al.
δ 7.25 (s, 2H), 7.42 (d, 1H), 7.53 (t, 1H), 7.99 (d, 1H). 13C NMR
(CDCl3) δ 98.9, 129.4, 130.3, 131.0, 134.5, 139.8, 168.7.
1-(3-Iodophenyl)maleimide (27). Yellow crystalline solid. Yield:
72%. Mp 154-155 °C. GC-MS m/z 300 (MHþ). 1H NMR
(DMSO-d6) δ 7.19 (s, 2H), 7.29 (t, 1H), 7.39 (t, 1H), 7.77 (d,
2H). 13C NMR (CDCl3) δ 93.9, 126.1, 130.7, 132.8, 134.7, 134.9,
136.2, 169.5.
1,3-Diphenylmaleimide (41). 41 was obtained as 1 using
phenylmaleic anhydride instead of maleic anhydride. White
crystalline solid recrystallized from ethanol. Yield: 51%. Mp
1
116-117 °C. GC-MS m/z 250 (MHþ). H NMR (DMSO-d6)
δ 7.27 (s, 1H), 7.47 (m, 4H), 7.55 (m, 2H), 7.67 (m, 4H). 13C
NMR (DMSO-d6) δ 126.5, 126.6, 129.8, 129.9, 130.0, 130.1,
130.2, 168.4.
1-(4-Iodophenyl)maleimide (28). Yellow crystalline solid. Yield:
90%. Mp 145-147 °C. GC-MS m/z 300 (MHþ). 1H NMR
(CDCl3) δ 6.85 (s, 2H), 7.13 (d, 2H), 7.79 (d, 2H). 13C NMR
(CDCl3) δ 127.6, 131.1, 134.3, 138.3, 169.0.
1-Biphenyl-2-ylmaleimide (29). White crystalline solid. Yield:
84%. Mp 138-140 °C. GC-MS m/z 250 (MHþ). 1H NMR
(DMSO-d6) δ 7.05 (s, 2H), 7.14 (d, 2H), 7.31-7.41 (m, 4H),
7.47-7.57 (m, 4H). 13C NMR (DMSO-d6) δ 126.4, 126.5, 127.3,
127.8, 128.4, 128.9, 129.2, 133.6, 137.2, 139.8, 168.8.
1-Biphenyl-3-ylmaleimide (30). Yellow crystalline solid. Yield:
61%. Mp 76-78 °C. GC-MS m/z 250 (MHþ). 1H NMR
(CDCl3) δ 6.87 (s, 2H), 7.31-7.36 (m, 2H), 7.44 (t, 3H), 7.51-
7.60 (m, 5H). 13C NMR (CDCl3) δ 122.7, 122.9, 125.2, 124.7,
125.7, 126.8, 127.4, 129.6, 132.2, 138.1, 140.4, 167.4.
1-Biphenyl-4-ylmaleimide (31). Yellow crystalline solid. Yield:
97%. Mp 176-178 °C. GC-MS m/z 250 (MHþ). 1H NMR
(DMSO-d6) δ 6.88 (s, 2H), 7.37 (t, 1H), 7.42-7.47 (m, 4H), 7.59
(d, 2H), 7.68 (d, 2H). 13C NMR (DMSO-d6) δ.13.8, 22.0, 26.0,
27.8, 31.2, 37.0, 134.4, 171.0.
1-Benzylmaleimide (32). Beige crystalline solid. Yield: 67%.
Mp 68-71 °C. GC-MS m/z 188 (MHþ). 1H NMR (DMSO-d6)
δ 4.60 (s, 2H), 7.08 (s, 2H), 7.24-7.33 (m, 6H). 13C NMR
(CDCl3) δ 41.5, 120.1, 127.9, 127.4, 128.7, 134.3, 136.2, 170.4.
1-Phenethylmaleimide (33). Beige crystalline solid. Yield: 76%.
Mp 109-112 °C. GC-MS m/z 202 (MHþ). 1H NMR (DMSO-d6)
δ 1.06 (t, 2H), 2.82 (t, 2H), 6.97 (s, 2H), 7.15 (d, 2H), 7.20 (d, 1H),
7.27 (t, 2H). 13CNMR(DMSO-d6)δ33.5, 38.3, 126.3, 128.2, 128.5,
134.3, 138.0, 170.6.
General Procedure for the Preparation of N-Alkylmaleimide
(Method B) (Mitsunobu Coupling) (36, 44-46). A 250 mL round-
bottom flask was charged with PPh3 (2.70 g, 10.3 mmol)
to which was added 70 mL of anhydrous THF. The resulting
clear solution was cooled to -78 °C. Diethylazodicarboxylate
(1.63 mL, 10.3 mmol) was added over a period of 2-3 min. The
yellow reaction mixture was stirred during 5 min, after which the
desired alcohol (11.3 mmol) was added over 1 min and stirred
for 5 min. Neopentyl alcohol (0.50 g, 5.7 mmol) and maleimide
(1.00 g, 10.3 mmol) were added sequentially to the reaction
mixture as solids. The resulting suspension was allowed to
remain at -78 °C for 5 min during which time most of the
maleimide dissolved. The cooling bath was then removed, and
the mixture was stirred overnight at room temperature. The
clear solution was evaporated under reduced pressure and the
resulting solid recrystallized from ethanol or purified by column
chromatography.
1-Biphenyl-4-ylmethylmaleimide (36). 36 was synthesized
starting from 4-biphenylmethanol. Yellow crystalline solid re-
crystallized from ethanol. Yield: 96%. Mp 155-159 °C. GC-
MS m/z 264 (MHþ). 1H NMR (CDCl3) δ 7.54 (d, 4H), 7.42 (t,
4H), 7.34 (t, 1H), 6.72 (s, 2H), 4.72 (s, 2H). 13C NMR (CDCl3) δ
41.2, 127.1, 127.4, 127.5, 128.8, 128.9, 134.3, 135.2, 140.7, 170.4.
N-Nonylmaleimide (44). 44 was synthesized starting from
1-nonanol. White solid purified by column chromatography
(silica) using dichloromethane as eluent. Yield: 65%. Mp 46-
49 °C. GC-MS m/z 224 (MHþ). 1H NMR (DMSO-d6) δ 0.85 (t,
3H), 1.23 (m, 12H), 1.45 (m, 2H), 3.38 (t, 2H), 7.00 (s, 2H). 13
C
1-(4-Phenylbutyl)maleimide (34). White crystalline solid. Yield:
68%. Mp 60-62 °C. GC-MS m/z 230 (MHþ). 1H NMR
(CDCl3) δ 1.67-1.57 (m, 4H), 2.64 (d, 2H), 3.54 (t, 2H), 6.67 (s,
2H), 7.16 (d, 3H), 7.30-7.24 (m, 3H). 13C NMR (CDCl3) δ 28.1,
28.5, 35.3, 37.3, 125.9, 128.4, 134.1, 142.0, 170.9.
1-(4-Ethylbenzyl)maleimide (35). White-yellow powder. Yield:
96%. Mp 47-48 °C. GC-MS m/z 216 (MHþ). 1H NMR
(DMSO-d6) δ 1.14 (m, 3H), 2.56 (m, 2H), 4.55 (s, 2H), 7.06 (s,
2H), 7.15 (m, 4H). 13C NMR (DMSO-d6) δ 15.5, 27.7, 40.2, 127.2,
127.8, 133.9, 134.6, 142.9, 170.7.
N-Phenylsuccinimide (37). 37 was synthesized similarly to 1
using succinic anhydride instead of maleic anhydride. White
crystalline solid. Yield: 89%. Mp 154-157 °C. GC-MS m/z 176
(MHþ). 1H NMR (DMSO-d6) δ 2.54 (q, 4H), 7.01 (t, 1H), 7.28
(t, 2H), 7.58 (d, 2H). 13C NMR (DMSO-d6) δ 28.7, 30.9, 118.8,
122.8, 128.6, 139.2, 170.0, 173.7.
NMR (DMSO-d6) δ 13.9, 22.0, 27.8, 31.5, 54.8, 134.3, 134.7,
171.0.
N-Palmitylmaleimide (45). 45 was synthesized starting from
1-hexadecanol. White solid recrystallized from ethanol. Yield:
93%. Mp 67-68 °C. GC-MS m/z 322 (MHþ). 1H NMR
(CDCl3) δ 0.88 (t, 3H), 1.25 (m, 26H), 1.57 (t, 2H), 3.50 (t,
2H), 6.98 (s, 2H). 13C NMR (CDCl3) δ 14.1, 22.7, 26.8, 28.6,
29.2, 29.4, 29.5, 29.6, 29.7, 32.0, 38.0, 134.1, 171.0.
N-Oleylmaleimide (46). 46 was synthesized starting from oleyl
alcohol. White solid. Purified by column chromatography
(silica) using dichloromethane as eluent. Yield: 48%. Mp
<20 °C. GC-MS m/z 348 (MHþ). 1H NMR (DMSO-d6) δ
0.85 (t, 3H), 1.23 (m, 18H), 1.47 (t, 2H), 1.97 (m, 4H), 3.37 (t,
2H), 5.32 (t, 2H), 7.00 (s, 2H). 13C NMR (DMSO-d6) δ 13.8,
22.0, 26.0, 27.8, 28.4, 28.5, 28.6, 28.7, 29.0, 31.2, 36.9, 129.5,
134.3, 171.0.
N-Phenylphtalimide (38). 38 was synthesized similarly to 1
using phthalic anhydride instead of maleic anhydride. White
crystalline solid. Yield: 84%. Mp 201-203 °C. GC-MS m/z
224 (MHþ). 1H NMR (CDCl3) δ 7.42 (m, 3H), 7.51 (t, 2H), 7.79
(d, 2H), 7.96 (d, 2H). 13C NMR (CDCl3) δ 123.8, 126.6, 128.1,
129.1, 131.7, 131.8, 134.4, 167.3.
3-Methyl-1-phenylmaleimide (39). 39 was obtained as 1 using
methylmaleic anhydride instead of maleic anhydride. White
solid recrystallized from ethanol. Yield: 37%. Mp 98-100 °C.
GC-MS m/z 188 (MHþ). 1H NMR (DMSO-d6) δ 2.06 (s, 3H),
6.05 (s, 1H), 7.04 (d, 1H), 7.28 (d, 2H), 7.60 (q, 2H). 13C NMR
(DMSO-d6) δ 21.6, 119.3, 121.1, 123.0, 123.2, 128.6, 139.3,
146.9, 168.2.
3,4-Dimethyl-1-phenylmaleimide (40). 40 was obtained as 1
using dimethylmaleic anhydride instead of maleic anhydride.
White crystalline solid recrystallized from ethanol. Yield: 40%.
Mp 78-79 °C. GC-MS m/z 202 (MHþ). 1H NMR (DMSO-d6)
2.03 (s, 3H), 7.02 (t, 1H), 7.28 (t, 2H), 7.56 (d, 2H). 13C NMR
(DMSO-d6) δ 9.1, 118.8, 122.8, 128.5, 139.2, 140.3, 166.4.
Procedure for the Preparation of the 1,4-Bis(malimido)xylene
(49). This procedure is similar to the procedure for 2. However,
2 equiv of the appropriate amine are used (40 mmol) instead of 1.
1,4-Bis(malimido)xylene (49). Beige solid. Recrystallized from
ethanol. Yield: 46%. Mp >250 °C. GC-MS m/z 297 (MHþ).
1H NMR (CDCl3) δ 4.56 (s, 4H), 7.06 (s, 4H), 7.18 (s, 4H). 13
NMR (DMSO-d6) δ 40.0, 127.4, 134.6, 135.8, 170.7.
C
Pharmacological Assays. MGL Activity Assay. Measure-
ment of radiolabeled 2-oleoylglycerol (2-OG) hydrolysis was
performed as previously described.35 Briefly, 2-OG (10 μM,
[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 in the presence of purified recombinant human
MGL26 (5 ng in 50 mM Tris buffer, pH 8.0, 0.1% BSA, 200 μL of
total volume assay) and either dimethyl sulfoxide (controls)
or tested compounds. The incubation was stopped by adding
400 μL of an ice-cold 1:1 methanol-chloroform mixture to each
tube and thorough mixing. After centrifugation at 700g for
5 min, radioactivity in the upper aqueous layer was measured

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7419
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