Novel sulfanylphthalimide analogues as highly potent inhibitors of monoamine oxidase B

Article abstract of DOI:10.1016/j.bmcl.2012.08.113

Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression and neurodegenerative diseases, respectively. Base

Full text of DOI:10.1016/j.bmcl.2012.08.113

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6632–6635
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel sulfanylphthalimide analogues as highly potent inhibitors of
monoamine oxidase B
Mietha M. Van der Walt a, Gisella Terre’Blanche a, Anél Petzer b, Jacobus P. Petzer a,
⇑
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Unit for Drug Research and Development, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two
isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression
and neurodegenerative diseases, respectively. Based on a recent report that the phthalimide moiety may
be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of
5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results
document that 5-sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of
the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor,
Received 12 July 2012
Revised 16 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
Isatin
Phthalimide
Sulfanylphthalimide
Monoamine oxidase
MAO
5-(benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 lM for the inhibition of MAO-B with a
427-fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent
an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkin-
sonian therapy.
Inhibition
Ó 2012 Elsevier Ltd. All rights reserved.
Monoamine oxidase A and B (MAO-A and -B) are flavine ade-
nine dinucleotide (FAD) containing enzymes, bound to the outer
membrane of mitochondria.1,2 These enzymes catalyze the oxida-
tive deamination of neurotransmitter and dietary amines thereby
terminating their physiological actions.3 Although MAO-A and -B
share 70% sequence identity, they exhibit different substrate and
inhibitor specificities.4 MAO-A metabolizes serotonin, adrenaline
and noradrenaline and inhibitors of this enzyme are in use for
the treatment of clinical depression and anxiety.5 MAO-B preferen-
tially metabolizes the dietary amine, 2-phenylethylamine, and may
therefore act as a metabolic brain barrier, limiting the entry of this
false neurotransmitter into the central nervous system.6,7 Since
MAO-B also catabolyzes dopamine in the brain, inhibitors of this
enzyme are used in the treatment of Parkinson’s disease.5,7,8 In Par-
kinson’s disease, MAO-B inhibitors conserve the depleted supply of
central dopamine and enhance dopamine levels following adminis-
tration of levodopa, the metabolic precursor of dopamine.9 For
these reasons, MAO-B inhibitors are frequently combined with
levodopa in Parkinson’s disease therapy. It should be noted that
MAO-A also metabolizes dopamine in the primate brain, and
MAO-A inhibitors may consequently also elevate dopamine levels
in the central nervous system.9 In addition, MAO-A inhibitors
may be employed to treat non-motor symptoms of Parkinson’s
disease such as depression and anxiety.3,10 MAO-A inhibitors in
conjunction with levodopa should, however, be used with caution
since this may lead to a severe hypertensive response.11
A wide variety of heterocyclic moieties have been employed in
the design of MAO inhibitors. Among these are isatin (1), an endog-
enous small molecule inhibitor of MAO-A and -B, and caffeine (3)
(Fig. 1).12–14 Phthalimide (2), an isomer of isatin, has also recently
been reported to be a potentially useful scaffold for the design of
MAO-B selective inhibitors.15 Although phthalimide is a weak
MAO inhibitor, substitution on C5 yields structures endowed with
highly potent and selective MAO-B inhibitory activities. In contrast,
N-substitution yields structures that are essentially devoid of MAO
inhibitory properties.15 The MAO-B inhibitory properties of isatin
O
3
5
O
2
N
O
N
H
N
N
N
Isatin (1)
8
O
O
3
1
5
Caffeine (3)
N
H
O
Phthalimide (2)
⇑
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 isatin (1), phthalimide (2) and caffeine (3).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.113

M. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6632–6635
6633
Table 1
The 1H NMR and 13C NMR chemical shifts for the NH proton and carbonyl C1 and C3
O
O
O
O
of phthalimide analogues 8a–k
5
5
O
N
H
O
N
H
3
R
5
4
6
O
O
N
N H
O
N
N
N
1
N
N
8
N
O
O
8
S
O
N
O
R
NH
C1/C3
5
7
8a
8b
8c
8d
8e
8f
8g
8h
8i
–S-(CH2)-C6H5
11.28a
11.29a
11.29a
11.29a
11.28a
8.20b
168.8a
168.8a
168.7a
168.8a
168.8a
167.7b
168.5a
168.5a
168.8a
167.6b
167.9b
168.8a
168.8a
168.8a
168.8a
168.8a
167.8b
168.6a
168.6a
168.8a
167.8b
168.0b
–S-(CH2)-(4-Cl-C6H4)
–S-(CH2)-(4-Br-C6H4)
–S-(CH2)-(4-F-C6H4)
–S-(CH2)-(4-OCH3-C6H4)
–S-C6H5
Figure 2. The structures of 5-benzyloxyisatin (4), 8-benzyloxycaffeine (5), 5-
benzyloxyphthalimide (6) and 8-(benzylsulfanyl)caffeine (7).
a
O
O
–S-(4-Cl-C6H4)
–S-(4-Br-C6H4)
–S-(CH2)2-C6H5
–S-C6H11
11.35
11.36
a
11.28a
a,b
N
H
N H
R
SH
a
R
8j
8k
11.30
8.12b
O2N
S
–S-(CH2)2-CH(CH3)2
O
O
a
NMR experiments conducted in DMSO-d6.
b
NMR experiments conducted in CDCl3.
Scheme 1. Synthetic pathway to 5-sulfanylphthalimide analogues. Reagents and
conditions: (a) K2CO3, acetone, reflux, 24 h; (b) HCl (6 N).
Table 2
and caffeine may similarly be enhanced by substitution on the C5
and C6 positions of isatin and the C8 position of caffeine. In this re-
gard, the benzyloxy substituent has been shown to be particularly
favorable, and benzyloxy substitution of isatin, caffeine and
phthalimide yields compounds 4–6 which are several orders of
magnitude more potent MAO-B inhibitors than the parent com-
pounds (Fig. 2).13–15 In all instances, halogen substitution on the
benzyloxy ring further improves inhibition potency. Modeling
studies have shown that productive interactions of the benzyoxy
side chain with the MAO-B entrance cavity may be responsible
for this behavior. Interestingly, the benzylsulfanyl side chain ap-
pears to exhibit similar properties to that of the benzyloxy moiety,
since a series of 8-(benzylsulfanyl)caffeine (7) analogues has re-
cently been shown to exhibit similar MAO-B inhibition potencies
to those of the 8-benzyloxycaffeine (5) analogues.16 Based on these
analyses, the present study examines the possibility that ben-
zylsulfanyl substitution on C5 of phthalimide (to yield 8a) would
also lead to highly potent MAO-B inhibition. For this purpose, the
effect that substitution (Cl, Br, F and OCH3) on the benzylsulfanyl
ring has on MAO inhibition will be explored. In addition, this study
also determines the effect on MAO inhibition by the phenylsulfa-
nyl, (2-phenylethyl)sulfanyl, cyclohexylsulfanyl and (3-methylbu-
tyl)sulfanyl substituents. This study therefore aims to discover
new highly potent MAO-B inhibitors and to contribute to the struc-
ture–activity relationships (SARs) of MAO inhibition by phthali-
mide derived compounds.
The IC50 values for the inhibition of recombinant human MAO-A and -B by 5-
sulfanylphthalimides 8a–k
O
3
R
5
N H
1
O
R
IC50
(
l
M)a
SIb
MAO-A
MAO-B
8a
8b
8c
8d
8e
8f
8g
8h
8i
–S-(CH2)-C6H5
1.92 0.172
0.506 0.032
0.273 0.041
0.958 0.003
1.63 0.023
8.03 0.622
1.68 0.343
1.01 0.053
2.27 0.136
1.03 0.062
0.380 0.042
0.0045 0.0004
0.0056 0.0003
0.0074 0.0029
0.0068 0.0009
0.020 0.0042
0.986 0.026
0.457 0.093
0.364 0.050
427
90
37
141
82
8.1
3.7
2.8
76
–S-(CH2)-(4-Cl-C6H4)
–S-(CH2)-(4-Br-C6H4)
–S-(CH2)-(4-F-C6H4)
–S-(CH2)-(4-OCH3-C6H4)
–S-C6H5
–S-(4-Cl-C6H4)
–S-(4-Br-C6H4)
–S-(CH2)2-C6H5
–S-C6H11
–S-(CH2)2-CH(CH3)2
0.030 0.0094
0.179 0.0082
0.015 0.0033
8j
8k
5.8
26
a
All values are expressed as the mean SD of triplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the
b
ratio of [IC50(MAO-A)]/[IC50(MAO-B)].
The enzyme catalytic activities in the absence and presence of
the test inhibitors were determined by fluorometrically measuring
the MAO-catalyzed formation of 4-hydroxyquinoline from the
mixed MAO-A/B substrate, kynuramine.14,18 This approach was
suitable for evaluating the MAO inhibitory properties of all the 5-
sulfanylphthalimides. The inhibition potencies of the test com-
pounds were calculated from the sigmoidal dose–response curves
and are expressed as the corresponding IC50 values. These IC50 val-
ues as well as the selectivity index (SI) values [SI = IC50(MAO-A)/
(IC50(MAO-B)] are given in Table 2. The results document that all
of the 5-sulfanylphthalimides are potent inhibitors of human
The 5-sulfanylphthalimides (8a–k) were conveniently synthe-
sized according to a previously described protocol (Scheme 1).17
The appropriate thiol reagents were reacted with 5-nitrophthali-
mide in the presence of K2CO3 to yield the target compounds in
low to good yields (4–76%). The 5-sulfanylphthalimides were
purified via crystallization from an appropriate solvent. In each in-
stance, the structures and purities of the target compounds were
verified by 1H NMR, 13C NMR, mass spectrometry and HPLC analy-
sis as cited in the supplementary data. The presence of two 13C
NMR signals at 167.6–168.8 ppm, which corresponds to the
carbonyl carbons at C1 and C3, and
a
1H NMR signal at
MAO-B with IC50 values ranging from 0.0045 to 0.986 lM. In accor-
8.12–8.20 ppm (CDCl3) or 11.28–11.36 ppm (DMSO-d6), which
corresponds to the phthalimide NH proton, confirmed that the
presence of the phthalimide ring (Table 1).
dance with expectation (see introduction), benzylsulfanyl substitu-
tion of phthalimide to yield 8a, resulted in highly potent MAO-B
inhibition. An IC50 value of 0.0045 lM was recorded for 8a. In fact
8a proved to be the most potent MAO-B inhibitor among the
To examine the MAO inhibitory properties of the 5-sulfanylph-
thalimides, recombinant human MAO-A and -B were employed.18
compounds of the present series. Compared to its C5 benzyloxy

6634
M. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6632–6635
substituted
(IC50 = 0.043
phthalimide
homologue,
compound
6
l
M), compound 8a is approximately eightfold more
100
75
50
25
0
potent as a MAO-B inhibitor.15 Compound 8a also proved to be a
more potent MAO-B inhibitor than 5-benzyloxyisatin (4)
(IC50 = 0.103
and 8-(benzylsulfanyl)caffeine (7) (IC50 = 1.86
on the benzylsulfanyl ring with Cl, Br, F and OCH3, to yield 8b–e,
also resulted in highly potent inhibition, with these homologues
l
M),13 8-benzyloxycaffeine (5) (IC50 = 1.77
l
M)14
l
M).16 Substitution
exhibiting IC50 values of 0.0056–0.020 lM. It is interesting to note
that particularly the halogen substituted homologues 8b–d are
weaker MAO-B inhibitors than compound 8a. This is in contrast
to the results obtained with 8-benzyloxycaffeine (5), 5-ben-
zyloxyphthalimide (6) and 8-(benzylsulfanyl)caffeine (7), where
particularly bromine substitution on the phenyl ring leads to an
enhancement in MAO-B inhibitory potency.14–16 From these results
it may therefore be concluded that 5-(benzylsulfanyl)phthalimides
are highly potent MAO-B inhibitors and superior to the lead struc-
tures, compounds 4–7, of this study.
l
r
y
C50
=
C50
o
t
n
e
r
i
I
I
b
i
x
x
h
p
n
I
Although still considered as a potent MAO-B inhibitor, the
1
1
.
De
0
-
o
)
]
N
=
I
phenylsulfanyl homologue 8f (IC50 = 0.986
lM) was the weakest
[
R
(
]
I
[
inhibitor of the present series. In this case, however, halogen sub-
stitution in the phenyl ring to yield compounds 8g
(IC50 = 0.457 lM) and 8h (IC50 = 0.364 lM), resulted in enhanced
MAO-B inhibition. The (2-phenylethyl)sulfanyl substituted homo-
logue 8i was also found to be a potent MAO-B inhibitor with an
Figure 3. The reversibility of the inhibition of MAO-B by 8a. The enzyme was
preincubated with 8a at 10 Â IC50 and 100 Â IC50 for 30 min and then diluted to
0.1 Â IC50 and 1 Â IC50, respectively. For comparison, (R)-deprenyl, at 10 Â IC50 was
similarly incubated with MAO-B and diluted to 0.1 Â IC50. The residual enzyme
activities were subsequently measured.
IC50 value of 0.030 lM, approximately sixfold weaker than 8a.
Since both 8f and 8i are weaker MAO-B inhibitor than 8a, it
may be concluded that the benzylsulfanyl side chain is particu-
larly suitable for enhancing the MAO-B inhibitory potency of
phthalimide, and that neither a reduction in side chain length
(to yield 8f), nor an increase in chain length (to yield 8i) would
further increase inhibitory activity. The general suitability of
5-sulfanylphthalimides for MAO-B inhibition was further
demonstrated with the finding that the cyclohexylsulfanyl (8j)
and (3-methylbutyl)sulfanyl (8k) substituted homologues are also
(10 Â IC50), the MAO-B activities were not recovered (2.1% of con-
trol). Interestingly, after dilution of the 8a–MAO-B complex to
0.1 Â IC50 and 1 Â IC50, the enzyme activities are not recovered
to 90% and 50%, respectively, as expected. This result suggests
that, for the inhibition of MAO-B, 8a may possess a quasi-revers-
ible or tight-binding component.
In conclusion, the present study shows that 5-sulfanylphthali-
mides are potent and selective inhibitors of MAO-B. In this regard,
the benzylsulfanyl side chain is particularly suitable for enhancing
the MAO-B inhibitory potency of phthalimide. It is noteworthy that
potent MAO-B inhibitors with IC50 values of 0.179
0.015 M, respectively.
lM and
l
The 5-sulfanylphthalimides were found to also act as inhibitors
of MAO-A. Four homologues, 8b–d and 8k, exhibited IC50 values in
the nanomolar range, with 8c being the most potent MAO-A inhib-
compound 8a (IC50 = 0.0045
more potent than phthalimide (2) (IC50 = 134
l
M) is approximately 30,000-fold
l
M).15 This illus-
trates the importance of the C5 side chain for MAO-B inhibitory
activity. Based on their MAO-B inhibition potencies and appropri-
ate selectivity profiles, this study concludes that 5-sulfanylphthal-
imides are suitable lead compounds for the development of
antiparkinsonian drugs. From a design point of view it is notewor-
thy that a wide variety of C5 substituents yield 5-sulfanylphthali-
mides with potent MAO-B inhibitory actions. This suggests that
structural modifications made to the C5 side chain in order to im-
prove the properties of the compound are less likely to reduce
MAO-B inhibition potency.
itor with an IC50 value of 0.273 lM. As evident from the SI values,
all of the 5-sulfanylphthalimides were, however, selective inhibi-
tors of the MAO-B isoform. The most potent MAO-B inhibitor of
the series, 8a, is also the most selective inhibitor with an SI value
of 427. This compound may therefore be considered the most suit-
able homologue of the series where MAO-B selectivity is desired.
As already noted, MAO-A inhibition may lead to adverse effects
when combined with certain antiparkinsonian therapies. In con-
trast, 8c may be considered as a potent MAO inhibitor with com-
paratively low isoform selectivity.
Since it is reported that the benzyloxyphthalimide class of
MAO inhibitors interacts reversibly with MAO, the current study
examined if this property is also shared by sulfanylphthali-
mides.15 For this purpose, the reversibility of MAO-B inhibition
by the most potent sulfanylphthalimide MAO-B inhibitor, com-
pound 8a, was investigated by measuring the degree of enzyme
recovery after dilution of the enzyme-inhibitor complex. MAO-B
was preincubated with 8a at concentrations of 10 Â IC50 and
100 Â IC50 for 30 min and then diluted to 0.1 Â IC50 and
1 Â IC50, respectively.19 The results show that after dilution of
the enzyme-inhibitor complexes to concentrations of 8a equal
to 0.1 Â IC50 and 1 Â IC50, the MAO-B catalytic activities are
recovered to levels of approximately 72% and 28%, respectively,
of the control value (Fig. 3). This behavior is consistent with a
reversible interaction of 8a with MAO-B. In contrast, incubation
of MAO-B with the irreversible inhibitor (R)-deprenyl
Acknowledgments
We are grateful to André Joubert of the SASOL Centre for Chem-
istry, North-West University for recording the NMR spectra, and
the Mass Spectrometry Service, University of the Witwatersrand
for the MS analyses. Financial support for this work was provided
by the North-West University, the National Research Foundation
and the Medical Research Council, South Africa.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.113.

M. M. Van der Walt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6632–6635
6635
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