Inhibition of monoamine oxidase B by N-methyl-2-phenylmaleimides

Article abstract of DOI:10.1016/j.bmc.2009.03.005

Based on a recent report that 1-methyl-3-phenylpyrrolyl analogues are moderately potent reversible inhibitors of the enzyme monoamine oxidase B (MAO-B), a series of structurally related N-methyl-2-phenylmaleimidyl analogues has been prepared and evaluated

Full text of DOI:10.1016/j.bmc.2009.03.005

Bioorganic & Medicinal Chemistry 17 (2009) 3104–3110
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of monoamine oxidase B by N-methyl-2-phenylmaleimides
Clarina I. Manley-King a, Gisella Terre’Blanche a, Neal Castagnoli Jr. b, Jacobus J. Bergh a, Jacobus P. Petzer a,
*
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Department of Chemistry, Virginia Tech and Edward Via College of Osteopathic Medicine, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a recent report that 1-methyl-3-phenylpyrrolyl analogues are moderately potent reversible
inhibitors of the enzyme monoamine oxidase B (MAO-B), a series of structurally related N-methyl-2-
phenylmaleimidyl analogues has been prepared and evaluated as inhibitors of MAO-B. In general, the
maleimides were more potent competitive inhibitors than the corresponding pyrrolyl analogues.
N-Methyl-2-phenylmaleimide was found to be the most potent inhibitor with an enzyme–inhibitor dis-
Received 15 January 2009
Revised 2 March 2009
Accepted 3 March 2009
Available online 9 March 2009
sociation constant (Ki value) of 3.49
lM, approximately 30-fold more potent than 1-methyl-3-phenylpyr-
Keywords:
role (Ki = 118 M). This difference in activities may be dependent upon the ability of the maleimidyl
l
Monoamine oxidase B
Reversible inhibitors
Competitive inhibition
Maleimide
heterocyclic system to act as a hydrogen bond acceptor. This is in correspondence with literature reports
which suggest that hydrogen bond formation is involved in stabilizing inhibitor–MAO-B complexes. Also
reported here is a brief kinetic study of the hydrolysis of the N-methyl-2-phenylmaleimidyl analogues in
aqueous solution. The findings of the inhibition studies are discussed with reference to the rate and
extent of hydrolysis.
Hydrolysis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
gen and pyrrole NH are hydrogen bonded to ordered water mole-
cules present in the active site.4
It was recently demonstrated that a series of 1-methyl-3-phe-
nylpyrrolyl analogues (1a–g) act as moderately potent competitive
inhibitors of the enzyme, monoamine oxidase B (MAO-B) (Fig. 1).1
The most potent analogue was 1-methyl-(3-trifluoromethyl-
phenyl)pyrrole (1e) with an enzyme–inhibitor dissociation
Therefore, the inclusion of hydrophilic substituents that could
displace active site water molecules and establish hydrogen bond
interactions with active site residues and residual water molecules
should result in increased affinity of a reversible inhibitor for MAO-
B.4 N-Methyl-2-phenylmaleimide (5a) (Fig. 1) is an example of a
modified 1-methyl-3-phenylpyrrolyl system that could lead to
hydrogen bonding in the active site of MAO-B. In contrast to the
pyrrolyl analogues N-methyl-2-phenylmaleimides may interact
with MAO-B via both hydrogen bonding and hydrophobic burial
and hence may be more potent inhibitors of the enzyme. In the
constant (Ki value) of 6.55
1-methyl-3-phenylpyrrole (1a) with a Ki value of 118
l
M. The least potent inhibitor was
M. Since
l
1-methyl-3-phenylpyrroles probably bind to the active site of
MAO-B principally via hydrophobic interactions, we speculate that
modification of the structure to include hydrogen bond acceptors
may enhance binding affinity. Literature supports the idea that
reversible MAO-B inhibitors may be stabilized via hydrogen bond-
ing in the active site of the enzyme.2 For example, the three-
dimensional structure of recombinant human MAO-B co-crystal-
lized with safinamide (2) (Fig. 2) has shown that the amidyl
functional group acts as both hydrogen bond donor and acceptor
with Gln206 and an ordered water molecule in the substrate cavity
of the enzyme.3 Likewise, 7-(3-chlorobenzyloxy)-4-formylcouma-
rin (3) binds with its coumarin moiety in the substrate cavity
space, where the aldehyde oxygen acts as a hydrogen bond accep-
tor for the hydroxyl group of Tyr435 and a conserved water mole-
cule.3 Structural analysis of the small molecule inhibitor, isatin (4),
in complex with human MAO-B, shows that the C-2 carbonyl oxy-
C6H4X
C6H4X
O
O
N
N
CH3
CH3
1a-g
5a-g
a: X = H
e: X = 3-CF3
f: X = 3-CH3
g: X = 3-OCH3
b: X = 3-Cl
c: X = 3-Br
d: X = 3-F
* Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
Figure 1. The structures of compounds discussed in the text: 1-methyl-3-phenyl-
pyrrolyl (1a–g) and N-methyl-2-phenylmaleimidyl (5a–g) analogues.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.005

C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
3105
H
CH3
O
X
NH2
i, ii
N
+
O
O
N
F
H
O
NH2
6
7
Safinamide (2)
CHO
X
X
Cl
Cl
O
3
O
O
iii
O
O
O
O
O
N
N
5a-g
8
O
N
Scheme 1. Synthetic pathway to N-methyl-2-phenylmaleimides (5a–g). Key: (i)
NaNO2, HCl, 0 °C; (ii) sodium acetate, CuCl2, 0 °C; (iii) 2,6-lutidine, isopropanol.
H
Isatin (4)
Figure 2. The structures of compounds discussed in the text: safinamide (2),
7-(3-chlorobenzyloxy)-4-formylcoumarin (3) and isatin (4).
N-methyl-2-phenylmaleimides were recrystallized twice from eth-
anol. Even though this resulted in relatively low yields, it was
found to be effective in removing residual amounts of the Diels–Al-
der adduct as judged by 1H NMR. The presence of these contami-
nants could also be conveniently detected via neutral aluminum
oxide TLC (see Section 4). The structures and purity of the target
compounds were verified by mass spectrometry, 1H NMR and 13C
NMR. For those compounds previously reported, the physical data
and melting points obtained were compared to the corresponding
literature values as cited in Section 4.
present study we have prepared and evaluated a series of N-
methyl-2-phenylmaleimides (5a–g) as potential MAO-B inhibitors
and compared the potencies of inhibition to that of the correspond-
ing pyrrolyl analogues (1a–g). As with the series of pyrrolyl ana-
logues different substituents at C-3 of the phenyl ring were
examined.
The double bond of the maleimide ring has been shown to un-
dergo conjugate addition reactions with thiol groups at physiolog-
ical pH to form a stable thio ether bond. This chemistry has been
exploited in the modification, quantitation and analysis of cysteine
containing proteins.5 Since MAO-B contains an active site cysteinyl
residue (Cys172),6 the possibility exists that the maleimides inves-
tigated here may interact irreversibly with the enzyme and hence
the time-dependency of the interaction between N-methyl-2-
phenylmaleimide (5a) and MAO-B has been studied. In accordance
with this idea both recombinant human MAO-A and MAO-B are
inactivated by N-ethylmaleimide.7 Also, since N-alkylmaleimides
are reported to undergo hydroxide ion-mediated hydrolysis,8 the
rate of hydrolysis of N-methyl-2-phenylmaleimide in aqueous
solution also was examined. The findings of the inhibition studies
are discussed with reference to the reversibility of inhibition and
rate and extent of hydrolysis.
2.2. Enzymology
In the present study we have examined the MAO-B inhibition
potential of N-methyl-2-phenylmaleimides (5a–g) using the mito-
chondrial fraction obtained from baboon liver tissue as enzyme
source. Baboon liver tissue exhibits a high degree of MAO-B cata-
lytic activity while being devoid of MAO-A activity.14 Also, the
interaction of reversible inhibitors with MAO-B obtained from ba-
boon liver tissue appears to be similar to the interaction with the
human form of the enzyme since inhibitors are approximately
equipotent with both enzyme sources.15 As substrate for the
MAO-B activity measurements, 1-methyl-4-(1-methylpyrrol-2-
yl)-1,2,3,6-tetrahydropyridine (MMTP) was used. MMTP
undergoes MAO-B catalyzed ring
a-carbon oxidation to yield the
corresponding dihydropyridinium species MMDP+.14 Since MMDP+
absorbs light maximally at a wavelength of 420 nm, the concentra-
tion of this species can be conveniently estimated via spectropho-
tometry. None of the N-methyl-2-phenylmaleimides (5a–g)
evaluated here absorbs light at this wavelength (see Section 4).
The MAO-B inhibitory properties of 5a–g first were investigated
in order to determine whether the test inhibitors act as reversible
inhibitors or time-dependent inactivators of the enzyme. As stated
in the introduction, enzyme inactivation could be mediated if the
maleimide double bond were to undergo a conjugate addition
reaction with the active site cysteinyl residue (Cys172) of MAO-
B. For this study 5a was selected as a representative test com-
pound. Baboon liver mitochondria were preincubated with 5a
2. Results
2.1. Chemistry
The N-methyl-2-phenylmaleimidyl analogues (5a–g) examined
in this study were conveniently prepared according to a previously
reported procedure (Scheme 1).9 In the first step the appropriately
substituted aniline (6) is diazotized and the resulting diazo inter-
mediate treated at –10 °C with N-methylmaleimide (7), a modified
Meerwein reaction.10,11 The target N-methyl-2-phenylmaleimides
(5a–g) were obtained following thermal dehydrohalogenation of
the intermediate chlorosuccinimides (8) in the presence of 2,6-lu-
tidine. The formation of Diels–Alder adducts has been reported as
side products in the preparation of maleimides according to this
synthetic route.12 This side reaction could be partially suppressed
by dilution of the lutidine with isopropanol and by reduction of
the heating time.9 Following purification by column chromatogra-
phy13 to remove substantial amounts of ‘diazo resins’, the final
(14
lM) for periods of 0, 15, 30, and 60 min and the rates of
MAO-B catalyzed oxidation of MMTP (90
l
M) to MMDP+ were
measured.16 As shown in Figure 3 the MAO-B oxidation rate
slightly increases with increased preincubation time of 5a with
the enzyme. The increase of catalytic activity may be due to the
hydrolysis of the maleimidyl inhibitor in aqueous solution to yield

3106
C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
20
15
6
5
4
3
2
1
0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
10
5
0
-10 -5
0
5
10 15 20
[I], µM
0
15
30
60
Preincubation time (min)
Figure 3. The rates of the baboon liver mitochondrial MAO-B catalyzed oxidation of
MMTP (90 lM). The enzyme preparation was preincubated for various periods of
time (0–60 min) with 5a (14
-0.01
0.00
0.01
0.02
0.03
0.04
l
M). The rates are expressed as nmol MMDP+ formed/
1/[S], µM-1
mg protein/min.
Figure 4. Lineweaver–Burk plots of the oxidation of MMTP by baboon liver MAO-B
in the absence (filled circles) and presence of various concentrations of 5b (open
C6H5
circles, 4 lM; filled triangles, 8 lM; open triangles, 16 lM). The rates are expressed
as nmol MMDP+ formed/mg protein/min and the inset is the replot of the slopes
versus the inhibitor concentration.
CO2H
H
O
N
mitochondria (0.15 mg protein/mL) and MMTP at a final concen-
C6H5
tration of 90 lM were added and the rates of the MAO-B catalyzed
CH3
oxidation of MMTP to MMDP+ were measured (see Section 4).
When 5b was preincubated for a period of 0 min, the rate of
MMDP+ formation was 47% ( 0.3%) of the rate measured in the ab-
sence of 5b. When 5b was preincubated for 30, 60, 120, 180 and
240 min the rates of MMDP+ formation were 52% ( 1.4%), 66%
( 0.4%), 68% ( 0.4%), 80% ( 0.4%) and 84% ( 0.9%), respectively, of
the rate measured in the absence of 5b. These data show that an
increase in preincubation time results in a reduction of the inhibi-
tion potency of 5b. Since an increase in preincubation time is asso-
ciated with a larger extent of hydrolysis, these results are in
accordance with the notion that the hydrolysis products are not
MAO-B inhibitors or at least weak inhibitors compared to the par-
ent maleimide.
9a
OH-
O
O
N
C6H5
H
N
CH3
5a
CH3
CO2H
O
9b
Scheme 2. The proposed hydrolysis of N-methyl-2-phenylmaleimides (5a) in
aqueous solution to yield the isomeric maleamic acids 9a and/or 9b.
one or a mixture of the corresponding carboxy acids (Scheme 2).
Should such hydrolysis render the maleimide inhibitor inactive,
the result would be an increase of MAO-B catalytic activity with in-
creased preincubation time. This analysis is consistent with time
dependency of the putative hydrolysis (see below). Since there is
no decrease of MAO-B activity with increased incubation time, it
can also be concluded that 5a interacts reversibly with the active
site of MAO-B. The reversibility of enzyme inhibition by 5a–g
was well supported by the linear Lineweaver–Burk plots con-
structed from the kinetic data. As shown by example with
N-methyl-2-(3-chlorophenyl)maleimide (5b), the Lineweaver–
Burk plots were indicative of competitive inhibition for the
inhibitors tested (Fig. 4). Although this result suggests that N-
methyl-2-phenylmaleimides are not alkylated by the thiol group
of the active site cysteinyl residue (Cys172), the possibility that
MAO-B is inactivated at a very slow rate can not be excluded. For
example, N-ethylmaleimide at a concentration of 0.8 mM is re-
ported to inactivate recombinant human MAO-B with a half-time
of 8 h.7
The enzyme–inhibitor dissociation constants (Ki values) for the
inhibition of MAO-B by the N-methyl-2-phenylmaleimides (5a–g)
evaluated here as well as those previously reported1 for the corre-
sponding 1-methyl-3-phenylpyrrolyl analogues (1a–g) are listed in
Table 1. The same assay conditions, enzyme source and substrate
that were used for the evaluation of the pyrrolyl inhibitors1 were
also employed here for the evaluation of the maleimidyl inhibitors.
In general, the maleimides were more potent competitive inhibi-
tors of MAO-B than the corresponding pyrrolyl analogues with Ki
values ranging from 3.49 to 11.0
6.55–118 M for the pyrrolyl analogues. N-Methyl-2-phenylmale-
imide (5a) was found to be the most potent inhibitor with a Ki va-
lue of 3.49 M, approximately 30-fold more potent than 1-methyl-
3-phenylpyrrole (1a) (Ki = 118 M). The differences in inhibition
lM for the maleimides and
l
l
l
potencies between the C-3 phenyl substituted maleimidyl (5b–g)
and the pyrrolyl (1b–g) inhibitors were more modest. For example,
N-methyl-2-(3-bromophenyl)maleimide (5c) (Ki = 6.99
only approximately twofold more potent than 1-methyl-3-(3-
bromophenyl)pyrrole (1c) (Ki = 14.3 M). The only exception was
observed for the 3-CF3 phenyl substituted analogues with pyrrolyl
1e (Ki = 6.55 M) inhibiting MAO-B more potently than the corre-
lM) was
l
To evaluate the notion that the products generated upon hydro-
lysis of the maleimidyl analogues are not MAO-B inhibitors, 5b at a
l
concentration of 13
in the aqueous incubation buffer (100 mM sodium phosphate buf-
l
M (approximately 2 Â Ki) was preincubated
sponding maleimidyl 5e (Ki = 11.0 lM). Interestingly, in contrast
to the pyrrolyl analogues considered here,1 substitution on the
fer, pH 7.4) for various time periods (0–240 min). Baboon liver
phenyl ring of the N-methyl-2-phenylmaleimidyl analogues leads

C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
3107
Table 1
80
The Ki values for the inhibition of MAO-B by the N-methyl-2-phenylmaleimides (5a–
pH 8.0
g) and the corresponding 1-methyl-3-phenylpyrrolyl analogues (1a–g)a
60
40
20
0
Ar
Ar
O
O
N
N
pH 7.4
CH3
CH3
5
1
pH 7.0
pH 6.4
400
Ar
Ki value for 5b
Ki value for 1c
a
b
c
d
e
f
C6H5
3.49
6.66
6.99
5.73
11.0
8.60
8.56
118
3-ClC6H4
3-BrC6H4
3-FC6H4
3-CF3C6H4
3-CH3C6H4
3-OCH3C6H4
20.9
14.3
38.9
6.55
56.0
41.7
0
100
200
300
Time (min)
Figure 5. The rates of hydrolysis of 5a (100 lM) in 100 mM sodium phosphate
g
buffer at pH 6.4, 7.0, 7.4 and 8.0. The temperature of the incubations was 37 °C and
the extent of hydrolytic cleavage was estimated spectrophotometrically from the
decrease in absorbance at 349 nm of the aqueous solutions of 5a. Because of the
limited solubility of 5a in water, all incubations contained 4% DMSO as cosolvent.
a
The Ki values are expressed in
The Ki values of 5b–g may be an underestimation of the potencies due to high
l
M.
b
rates of aqueous hydrolysis (see text for details).
c
Values obtained from Ref. 1.
1.4
to inhibitors with reduced inhibition potencies since unsubstituted
5a was found to be the best inhibitor. As discussed in the next sec-
tion, the relatively modest inhibition potencies of 5b–g may be a
result of a higher rate of hydrolysis in the aqueous incubation med-
ium used for the inhibition studies compared to the hydrolysis rate
of 5a.
0 min
1.2
180 min
360 min
1.0
0.8
0.6
0.4
0.2
0.0
NaOH (1 N)
2.3. The rate of hydrolysis of N-methyl-2-phenylmaleimide
As discussed in the previous section, the rates of the MAO-B cat-
alyzed oxidation of MMTP in the presence of 5a increase slightly
with increased preincubation time of 5a with the enzyme
(Fig. 3). Since N-alkylmaleimides are reported to undergo hydrox-
ide ion dependent hydrolysis,8 the possibility exists that the hydro-
lysis of 5a in the aqueous incubation medium to yield products
that are not MAO-B inhibitors could affect its apparent inhibition
potency. This process would reduce the concentration of inhibitor
5a and therefore result in an increase of the rates of MAO-B cata-
lyzed oxidation of MMTP. The observation that MAO-B catalytic
activity increases with increased preincubation time is in agree-
ment with the time dependency of the hydrolysis process. To
determine if hydrolysis of 5a, the most potent inhibitor of the ser-
ies, may have significantly affected the measured Ki value (Table 1),
the extent and rate of hydrolysis of 5a were examined in the aque-
ous incubation medium used in the enzymology. Solutions of 5a
200
250
300
350
400
450
500
Wavelength (nm)
Figure 6. The absorption spectra of a solution of 5a (100 lM, starting concentra-
tion) following incubations for different time periods (0, 180 and 360 min) in
100 mM sodium phosphate buffer at pH 7.4. The absorption spectrum of a solution
of 5a (100 lM, starting concentration) incubated for 45 min in 1 N NaOH is also
shown. The temperature of the incubations was 37 °C and DMSO (4%) was added as
cosolvent.
the rate of hydrolysis increases with increasing pH, the process is
hydroxide ion dependent. This is similar to what has been reported
for the hydrolysis of N-alkylmaleimides.8 At 360 min after initia-
tion of the experiment, the extent of hydrolysis at pH 7.0, 7.4
and 8.0 were 13%, 31.4% and 73.8%, respectively. After 30 min at
a pH of 7.4 the extent of hydrolysis was only 2.8% for a 100 lM
solution of 5a. Similarly, slow hydrolysis was also observed at low-
er concentrations of 5a. After 30 min at a pH of 7.4 the extent of
hydrolysis was approximately 1% for a 10 lM solution (results
not shown). Since the time period used for the measurement of
the Ki values was only 10 min, it may be concluded that no signif-
icant reduction of 5a concentration due to hydrolysis in the aque-
ous incubation medium occurred during this time and that
hydrolytic cleavage of 5a therefore did not significantly affect the
measured Ki value. However, a similar analysis of the aqueous sta-
bility of 5b–g revealed that, in contrast to 5a, these congeners
underwent rapid hydrolysis in 100 mM sodium phosphate buffer
(pH 7.4) at 37 °C (Fig. 7). After a 30 min incubation period, the ex-
(100 lM) in 100 mM sodium phosphate buffer (pH 6.4, 7.0, 7.4
and 8.0) were incubated at 37 °C and the remaining concentrations
of 5a in the incubations were measured spectrophotometrically
(Fig. 5). Compound 5a absorbs maximally at 349 nm. After incuba-
tions in 1 N NaOH this chromophore is replaced by a new chromo-
phore with kmax at 269 nm (Fig. 6). The reduction of absorbance at
349 nm is associated with the hydrolysis of the maleimide ring
since hydrolysis of N-methylmaleimide results in a similar reduc-
tion in absorbance at its longest wavelength of maximal absor-
bance (300 nm).8 Therefore the residual concentration of 5a in
the incubations could be calculated from the molar absorptivity
(2820 MÀ1) of 5a at 349 nm (Fig. 6).
The results (Fig. 5) show that while 5a is stable at pH 6.4, at pH
7–8 a time– and pH–dependent reduction of 5a concentration is
observed. As discussed above, the reduction of 5a concentration
may be attributed to the hydrolysis of the maleimidyl ring. Since

3108
C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
2.1 kcal/mole. This difference in energy is within the range ex-
5c
pected for hydrogen bond formation. We conclude that the forma-
tion of hydrogen bonds is an important factor in stabilizing MAO-
B–ligand complexes and should be taken into consideration when
designing MAO-B inhibitors.
80
60
40
20
0
5b,d-e
5g
5f
The relatively modest differences between the inhibition poten-
cies of 5b–g and their corresponding pyrrolyl analogues (1b–g) is
most likely the result of a high rate of hydrolysis in the aqueous
medium used for the inhibition studies. The results document that
5b–g undergo rapid hydrolysis in aqueous media and, assuming
that the respective degradation products are not MAO-B inhibitors,
the measured Ki values are likely to be an underestimation of the
true inhibition potencies of these inhibitors. In contrast, 5a exhib-
ited a relatively slow rate of hydrolysis and the measured Ki value
for the inhibition if MAO-B may be a more accurate estimation of
its inhibition potency.
5a
0
10
20
30
40
50
60
70
Time (min)
Figure 7. The rates of hydrolysis of 5a–g (100 lM) in 100 mM sodium phosphate
4. Experimental
buffer at pH 7.4. The temperature of the incubations was 37 °C and the extent of
hydrolytic cleavage was estimated spectrophotometrically from the decrease in
absorbance at 337–354 nm (see Section 4) of the aqueous solutions of 5a–g.
Because of the limited solubility of 5a–g in aqueous solvent, all incubations
contained 4% DMSO as cosolvent.
Caution: MMTP is a structural analogue of the nigrostriatal neu-
rotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and
should be handled using disposable gloves and protective eyewear.
Procedures for the safe handling of MPTP have been described
previously.18
tents of hydrolysis were 40–69% for 100 lM solutions of 5b–g. It
can therefore be concluded that significant reductions of the con-
centrations of 5b–g in the aqueous incubation medium occurred
during the MAO-B inhibition studies and that, assuming that the
respective hydrolysis products are not MAO-B inhibitors, the
measured Ki values (Table 1) are an underestimation of the inhi-
bition potencies of these inhibitors. An interesting observation is
that the set of absorption spectra generated for the hydrolysis of
5a exhibits a stable isosbestic point at 308 nm (Fig. 6). This sug-
gests that the hydrolysis reaction of 5a proceeds without forming
intermediates or multiple products.17 The absorption spectra
generated from the aqueous incubations of 5b–g, however, do
not exhibit isosbestic points which suggest that mechanisms of
degradation of 5b–g are different from that of 5a. This may
underlie the difference in aqueous stability between 5a and
5b–g.
4.1. Chemicals and instrumentation
All starting materials, unless otherwise stated, were obtained
from Sigma-Aldrich and were used without purification. The oxa-
late salt of MMTP was prepared as described previously.19 Proton
(1H) and carbon (13C) NMR spectra were recorded on a Varian
Gemini 300 spectrometer at frequencies of 300 MHz and
75 MHz, respectively. Chemical shifts are reported in parts per
million (d) downfield from the signal of tetramethylsilane added
to deuterated chloroform (CDCl3). Spin multiplicities are given as
s (singlet), d (doublet), q (quartet) or m (multiplet) and the cou-
pling constants (J) are given in hertz (Hz). Direct insertion elec-
tron impact ionization (EIMS) and high resolution (HRMS) mass
spectra were obtained with an AutoSpec ETOF (Micromass) mass
spectrometer. Melting points (mp) were determined on a Stuart
SMP10 melting point apparatus and are uncorrected. UV–vis
spectra were recorded on a Shimadzu UV-2100 double-beam
and a Shimadzu MultiSpec-1501 photodiode array spectropho-
tometer. Thin layer chromatography (TLC) was carried out with
neutral aluminum oxide 60 (Merck) containing UV254 fluorescent
indicator and benzene as mobile phase. Column chromatography
was carried out with Fluka aluminum oxide (Brockmann Activity
I).
3. Discussion
In the present study a series of N-methyl-2-phenylmaleimides
(5a–g) was evaluated as inhibitors of MAO-B and the inhibition
potencies were compared to those of the corresponding
1-methyl-3-phenylpyrrolyl analogues (1a–g). N-Methyl-2-phenyl-
maleimide (5a) was found to be the most potent inhibitor, approx-
imately 30-fold more potent than 1-methyl-3-phenylpyrrole (1a).
The enhanced inhibition potency of 5a may be dependent upon
the ability of the carbonyl oxygen atoms of the maleimidyl hetero-
cyclic system to act as hydrogen bond acceptors. The idea that the
maleimides may be stabilized via hydrogen bonding in the active
site is in agreement with the literature. The three-dimensional
structures of human recombinant MAO-B co-crystallized with sev-
eral reversible inhibitors have documented that enzyme–inhibitor
complexes are frequently stabilized by hydrogen bonding.3,4 Sev-
eral ordered water molecules and amino acid residues (for example
Gln206 and Tyr435), which have been shown to act as hydrogen
bond donors and acceptors, are present in the active site of
MAO-B. The suggestion that, in contrast to pyrrolyl inhibitor 1a,
maleimidyl inhibitor 5a is stabilized by hydrogen bonding within
the active site of MAO-B is further supported by the observation
4.2. Synthesis of N-methyl-2-phenylmaleimidyl analogues
(5a–g)
The N-methyl-2-phenylmaleimidyl analogues (5a–g) were syn-
thesized according to a previously reported procedure.9 The appro-
priately C-3 substituted aniline derivative (102 mmol) was
dissolved in 12 N hydrochloric acid (30 mL) and water (10 mL)
and then cooled in an ice–salt bath (–10 °C). Upon cooling, the ani-
line hydrochloric acid salt precipitates from solution. To this, a
solution of sodium nitrite (153 mmol) in 16 mL water was added
dropwise over a period of 10 min with vigorous stirring. The ani-
line derivative is completely diazotized when the reaction returns
to a complete solution of the diazonium salt. N-Methylmaleimide
(102 mmol) was dissolved in 80 mL of acetone, cooled on an ice–
salt bath (À10 °C) and the diazotized aniline was added over a per-
iod of 2 min. The pH of the reaction was adjusted to 3.0 with solid
that the difference between the Ki values of 5a (Ki = 3.49
lM) and
1a (Ki = 118 M) corresponds to DDG° of approximately
l
a

C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
3109
sodium acetate followed by the addition of solid CuCl2 (2.5 g) as a
4.3. MAO-B inhibition studies
single portion.10,11 The reaction was stirred for a period of 3 h in
the ice–salt bath while the pH was periodically adjusted to 2.9.
The reaction was allowed to return to room temperature and
was stirred for an additional 18 h. If a precipitate forms (5b–e, g),
it was collected by filtration while for an oil (5a and 5f), 100 mL
water was added and the crude product was extracted to benzene
(100 mL). The benzene phase was dried over anhydrous MgSO4 and
removed under reduced pressure, while the precipitate was air
dried. To the crude product was added 2,6-lutidine (2.5 g) and iso-
propanol (12.5 mL) and the resulting solution was heated for
15 min at 100 °C. For the synthesis of 5a and 5f a solution of
100 mL of 1 N hydrochloric acid solution was added to the reac-
tion. The resulting mixture was extracted to benzene (100 mL)
and washed with an aqueous solution of 1 N hydrochloric acid
(2 Â 50 mL). Following drying of the benzene over anhydrous
MgSO4, the solvent was removed under reduced pressure. For the
synthesis of 5b–e, g. the reaction mixture was cooled and filtered
to obtain a dark brown solid. The crude products were dissolved
in a minimum amount of benzene, applied to a short aluminum
oxide column (35 Â 50 mm) and were eluted with benzene as mo-
bile phase. The collected fractions were recrystallized twice from
boiling ethanol to afford the yellow or brown crystalline products.
For previously described 5a and 5g we found the melting points to
be 146–148 °C and 145–148 °C while the reported melting points
are 147–148 °C and 146–148 °C, respectively.9 The characteriza-
tions of compounds that are previously unreported are summa-
rized below.
N-Methyl-2-(3-chlorophenyl)maleimide (5b) was prepared from
3-chloroaniline and N-methylmaleimide (7) in a yield of 6%: mp
113–115 °C; 1H NMR (CDCl3) d 3.07 (s, 3H), 6.74 (s, 1H), 7.35–
7.44 (m, 2H), 7.78–7.81 (m, 1H), 7.89–7.91 (m, 1H); 13C NMR
(CDCl3) d 23.94, 125.02, 126.64, 128.48, 130.19, 130.35, 131.05,
135.04, 142.60, 169.92, 170.26; EIMS m/z; 221 (MÅ+); HRMS calcd
221.024356, found 221.023664.
N-Methyl-2-(3-bromophenyl)maleimide (5c) was prepared from
3-bromoaniline and N-methylmaleimide (7) in a yield of 16%:
mp 116–119 °C; 1H NMR (CDCl3) d 3.06 (s, 3H), 6.73 (s, 1H),
7.28–7.33 (m, 1H), 7.55–7.58 (m, 1H), 7.82–7.85 (m, 1H), 8.03–
8.04 (m, 1H); 13C NMR (CDCl3) d 23.91, 123.00, 125.01, 127.07,
130.39, 130.58, 131.30, 133.92, 142.42, 169.85, 170.19;
EIMS m/z; 265, 267 (MÅ+); HRMS calcd 264.973840, found
264.974289.
MAO-B activity measurements were carried out in sodium
phosphate buffer (100 mM, pH 7.4) with mitochondria isolated
from baboon liver tissue.1,20,21 The MAO-A and -B mixed substrate,
MMTP14 served as substrate for the inhibition studies. The final
volume of the incubations were 500
chondrial isolate (0.15 mg protein/mL), MMTP (30–120
l
L and contained the mito-
M), and
l
various concentrations of the test inhibitors. The limited solubility
of the test compounds in aqueous solution required the use of 4%
DMSO as cosolvent. Following incubation at 37 °C for 10 min, the
enzyme reactions were terminated by the addition of 10 lL per-
chloric acid (70%) and the samples were cleared via centrifugation
at 16,000g for 10 min. The concentrations of the MAO-B generated
product, MMDP+, were measured spectrophotometrically at a
wavelength of 420 nm (e
= 25,000 MÀ1).14 Ki values were deter-
mined by reploting the slopes of the Lineweaver–Burk plots versus
the inhibitor concentration and the Ki value was determined from
the x-axis intercept (intercept = –Ki). Each Ki value reported here is
representative of a single determination where the correlation
coefficient (R2 value) of the replot of the slopes versus the inhibitor
concentrations was at least 0.99.
4.4. Time-dependent inhibition studies
To determine whether maleimide 5a acts as a reversible inhib-
itor or as a time-dependent inactivator of MAO-B, baboon liver
mitochondria (0.3 mg of protein/mL) were preincubated with 5a
for periods of 0, 15, 30 and 60 min at 37 °C.14 The preincubation
solvent was 100 mM sodium phosphate buffer (pH 7.4) and the
concentration of 5a was 14
90 M, was then incubated at 37 °C for 15 min with 0.15 mg pro-
tein/mL of the preincubated mitochondria. The final volumes of
these incubations were 500 L and the final concentration of the
5a was 7 M, approximately double the Ki value (3.49 M) of 5a
for the inhibition of MAO-B. Following termination of the reactions
lM. MMTP, at final concentration of
l
l
l
l
by the addition of 10 lL of perchloric acid (70%), the concentra-
tions of MMDP+ were measured as outlined above. These experi-
ments were carried out in triplicate and the values are expressed
as mean standard error of the mean (SEM).1
To determine whether the products generated upon hydrolysis
of maleimide 5b act as inhibitors of MAO-B, 5b was preincubated
in the aqueous incubation buffer (100 mM sodium phosphate buf-
fer, pH 7.4) for various time periods (0, 30, 60, 120, 180, 240 min)
at 37 °C. Baboon liver mitochondria (0.15 mg protein/mL) and
N-Methyl-2-(3-fluorophenyl)maleimide (5d) was prepared
from 3-fluoroaniline and N-methylmaleimide (7) in a yield of
31%: mp 155–158 °C; 1H NMR (CDCl3) d 3.05 (s, 3H), 6.73 (s,
1H), 7.11–7.17 (m, 1H), 7.36–7.44 (m, 1H), 7.63–7.69 (m,
2H); 13C NMR (CDCl3) d 23.87, 115.47 (d), 117.99 (d), 124.21,
124.93, 130.50 (d), 142.61 (d), 161.12, 164.40, 169.93,
170.26; EIMS m/z; 205 (MÅ+); HRMS calcd 205.053907, found
205.053180.
MMTP at a final concentration of 90
tion was continued at 37 °C for 15 min. The final volumes of these
incubations were 500 L and the final concentration of 5b was
13 M, approximately double the Ki value (6.66 M). Incubations
lM were added and incuba-
l
l
l
carried out in the absence of 5b were included as controls. All incu-
bations contained 4% DMSO as cosolvent. The reactions were ter-
N-Methyl-2-(3-trifluoromethylphenyl)maleimide (5e) was pre-
pared from 3-trifluoromethylaniline and N-methylmaleimide (7)
in a yield of 9%: mp 103–105 °C; 1H NMR (CDCl3) d 3.07 (s, 3H),
6.81 (s, 1H), 7.55–7.60 (m, 1H), 7.68–7.71 (m, 1H), 8.07–8.10 (m,
1H), 8.15–8.18 (m, 1H); 13C NMR (CDCl3) d 23.93, 121.82, 125.25
(d), 125.38 (d), 127.45 (q), 129.48 (d), 131.56 (q), 131.65, 142.45,
169.75, 170.19; EIMS m/z; 255 (MÅ+); HRMS calcd 255.050713,
found 255.053127.
minated by the addition of 10 lL of perchloric acid (70%) and the
concentrations of MMDP+ were measured as outlined above. These
experiments were carried out in triplicate and the values are ex-
pressed as mean standard error of the mean (SEM).
4.5. Hydrolysis studies
Measurements of the extent of hydrolysis of 5a (5–100 lM)
N-Methyl-2-(3-methylphenyl)maleimide (5f) was prepared from
3-methylaniline and N-methylmaleimide (7) in a yield of 4%: mp
91–93 °C; 1H NMR (CDCl3) d 2.38 (s, 3H), 3.05 (s, 3H), 6.68 (s,
1H), 7.24-7.34 (m, 2H), 7.68–7.70 (m, 2H); 13C NMR (CDCl3) d
21.37, 23.78, 123.72, 125.71, 128.70, 128.79, 129.04, 131.88,
138.64, 144.14, 170.45, 170.76; EIMS m/z; 201 (MÅ+); HRMS calcd
201.078979, found 201.078442.
were carried out in 100 mM sodium phosphate buffer at pH 6.4,
7.0, 7.4 and 8.0. All incubations were conducted in quartz absorp-
tion cuvettes (Hellma) to a volume of 2 mL and contained a final
concentration of 4% DMSO as cosolvent. The incubation tempera-
ture was maintained at 37 °C with a Shimadzu CPS controller.
UV–vis scans were recorded at 30 min intervals over a range of
230–500 nm with a Shimadzu MultiSpec-1501 photodiode array

3110
C. I. Manley-King et al. / Bioorg. Med. Chem. 17 (2009) 3104–3110
3. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
4. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A 2003, 100, 9750.
5. Knight, P. Biochem. J. 1979, 179, 191.
6. Binda, C.; Newton-Vinson, P.; Hubálek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22.
7. Hubálek, F.; Pohl, J.; Edmondson, D. E. J. Biol. Chem. 2003, 278, 28612.
8. Matsui, S.; Aida, H. J. Chem. Soc., Perkin Trans. 2 1978, 1277.
9. McKenzie, T. C.; Epstein, J. W.; Fanshawe, W. J.; Dixon, J. S.; Osterberg, A. C.;
Wennogle, L. P.; Regan, B. A.; Abel, M. S.; Meyerson, L. R. J. Med. Chem. 1984, 27,
628.
spectrophotometer. Since hydrolysis of the maleimide ring results
in bleaching of absorbance at 349 nm, the residual concentration of
5a in the incubations were calculated from the molar absorptivity
(2820 MÀ1) of 5a at this wavelength. The same procedure was fol-
lowed for the aqueous stability measurements of 5b–g and the
reduction of absorbance at the following wavelengths were re-
corded at 10 min intervals: 5b 342 nm (3100 MÀ1), 5c 343 nm
(3240 MÀ1), 5d 343 nm (3210 MÀ1), 5e 337 nm (2860 MÀ1), 5f
352 nm (3860 MÀ1), 5 g 354 nm (3780 MÀ1). All values are ex-
pressed as mean SEM of duplicate determinations.
10. Rondestvedt, C. S. Org. React 1976, 24, 225.
11. Izzo, P. T. J. Org. Chem. 1963, 28, 1713.
12. Epstein, J. W.; McKenzie, T. C.; Lovell, F. M.; Perkinson, N. A. J. Chem. Soc., Chem.
Commun. 1980, 314.
Acknowledgments
13. Rondestvedt, C. S.; Kalm, M. J.; Vogl, O. J. Am. Chem. Soc. 1956, 78, 6115.
14. Inoue, H.; Castagnoli, K.; Van der Schyf, C. J.; Mabic, S.; Igarashi, K.; Castagnoli,
N., Jr. J. Pharmacol. Exp. Ther. 1999, 291, 856.
The NMR spectra were recorded by André Joubert while the MS
spectra were recorded by Johan Jordaan and Louis Fourie of the SA-
SOL Centre for Chemistry, North-West University. This work was
supported by grants from the National Research Foundation and
the Medical Research Council, South Africa.
15. Petzer, J. P.; Steyn, S.; Castagnoli, K. P.; Chen, J. F.; Schwarzschild, M. A.; Van der
Schyf, C. J.; Castagnoli, N., Jr. Bioorg. Med. Chem. 2003, 11, 1299.
16. Khalil, A. A.; Steyn, S.; Castagnoli, N., Jr. Chem. Res. Toxicol. 2000, 13, 31.
17. Berlett, B. S.; Levine, R. L.; Stadtman, E. R. Anal. Biochem. 2000, 287, 329.
18. Pitts, S. M.; Markey, S. P.; Murphy, D. L.; Weisz, A. In MPTP: A Neurotoxin
Producing a Parkinsonian Syndrome; Markey, S. P., Castagnoli, N., Jr., Trevor, A. J.,
Kopin, I. J., Eds.; Academic: New York, 1986; pp 703–716.
19. Bissel, P.; Bigley, M. C.; Castagnoli, K.; Castagnoli, N., Jr. Bioorg. Med. Chem.
2002, 10, 3031.
References and notes
20. Van den Berg, D.; Zoellner, K. R.; Ogunrombi, M. O.; Malan, S. F.; Terre’Blanche,
G., ; Castagnoli, N., Jr.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. 2007, 15, 3692.
21. Vlok, N.; Malan, S. F.; Castagnoli, N., Jr.; Bergh, J. J.; Petzer, J. P. Bioorg. Med.
Chem. 2006, 14, 3512.
1. Ogunrombi, M. O.; Malan, S. F.; Terre’Blanche, G.; Castagnoli, N., Jr.; Bergh, J. J.;
Petzer, J. P. Bioorg. Med. Chem. 2008, 16, 2463.
2. Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti, F.; Catto, M.;
Carrupt, P. A.; Reist, M. J. Med. Chem. 2006, 49, 6264.