The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues

Article abstract of DOI:10.1016/j.bioorg.2015.02.005

Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogue

Full text of DOI:10.1016/j.bioorg.2015.02.005

Bioorganic Chemistry 59 (2015) 117–123
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The adenosine receptor affinities and monoamine oxidase B inhibitory
properties of sulfanylphthalimide analogues
a
b
a
Mietha M. Van der Walt a, , Gisella Terre’Blanche , Anél Petzer , Jacobus P. Petzer
⇑
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Centre of Excellence for Pharmaceutical Sciences, 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:
Received 17 November 2014
Available online 18 February 2015
Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective
inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties
of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes)
and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as
Parkinson’s disease and Alzheimer’s disease, dual-target-directed drugs that antagonize adenosine recep-
tors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylph-
thalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a
number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated,
5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adeno-
Keywords:
Adenosine A1 receptors
Adenosine A2A receptors
Monoamine oxidase B
Sulfanylphthalimides
Parkinson’s disease
Alzheimer’s disease
sine A1 receptors with a Ki value of 0.369
lM. This compound is reported to also inhibit MAO-B with
an IC50 value of 0.020 M. Such dual-target-directed compounds may act synergistic in the treatment
l
of Parkinson’s disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhi-
bition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find
therapeutic value in Alzheimer’s disease: antagonism of the A1 receptor may be beneficial in the treat-
ment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In
neurological diseases, such as Parkinson’s disease and Alzheimer’s disease, dual-target-directed drugs
are expected to be advantageous over single-target treatments.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
[2]. The inadequacies of dopamine replacement therapy emphasise
the need for more effective drugs for the treatment of PD. One non-
Parkinson’s disease (PD) is a progressive neurodegenerative dis-
order, characterized pathologically by the loss of dopaminergic
nigrostriatal neurons and clinically by disabling movement disor-
ders [1]. The current therapy of PD relies mainly on dopamine
replacement strategies with L-DOPA, the metabolic precursor of
dopamine, and dopamine agonist drugs as the most useful agents
[2]. Although these strategies are highly effective in controlling
the early stages of the disease, long-term treatment is associated
with drug-related complications such as loss of drug efficacy, the
onset of dyskinesia and the occurrence of psychosis and depression
dopaminergic approach that has been explored, is the blockade of
adenosine receptors [3]. Four subtypes of adenosine receptors have
been identified in the central nervous system (CNS), the A1, A2A, A2B
and A3 subtypes, of which adenosine A1 and A2A receptors repre-
sent targets for the development of PD therapies [4]. Adenosine
A2A receptors are mainly localized in the striatum where A2A recep-
tor activation opposes dopamine D2 receptor signalling [4,5].
Adenosine A2A antagonists, in turn, have been shown to facilitate
dopamine receptor signalling and thereby restore motor function
in animal models of Parkinson’s disease [3]. In this respect, adeno-
sine A2A antagonists potentiate the motor benefit of L-DOPA in
Parkinson’s disease patients, without the potentiation of L-DOPA-
associated dyskinesia [6]. In contrast to adenosine A2A receptors,
adenosine A1 receptors are widely expressed in the CNS. In the
striatum, adenosine A1 receptor stimulation inhibits dopamine
release [7]. Conversely, blockade of A1 receptors facilitates dopa-
mine release in the striatum and potentiates dopamine mediated
responses [7]. Additionally, it has been reported that adenosine
Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; CNS, central
nervous system; MAO, monoamine oxidase; [3H]DPCPX, [3H]-dipropyl-
8-cyclopentylxanthine; [3H]NECA, N-[3H]-ethyladenosin-50-uronamide; CPA,
N6-cyclopentyladenosine;
DMPX,
3,7-dimethyl-1-propargylxanthine;
SAR,
Structure–activity relationship; SEM, standard error of the mean; SD, standard
deviation.
⇑
Corresponding author. Fax: +27 18 2994243.
E-mail address: 13035134@nwu.ac.za (M.M. Van der Walt).
http://dx.doi.org/10.1016/j.bioorg.2015.02.005
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

118
M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
A1 receptor antagonists may improve learning and memory in ani-
mal models [7]. Adenosine A1 receptor antagonists may thus also
find therapeutic application by enhancing cognitive function in PD.
Furthermore, adenosine A1 and A2A receptors may also find
therapeutic value in Alzheimer’s disease (AD), another well-known
neurological disorder. Pathologically, AD may be characterized by a
marked loss of hippocampal and cortical neurons that clinically
produces an impairment of memory and cognitive ability [8].
Selective adenosine A1 receptor antagonists have been indicated
to possess therapeutic value in the treatment of AD’s cognitive dys-
function [9]. This may be attributed to the CNS stimulatory effects
associated with adenosine A1 receptor antagonists [9]. Adenosine
A2A receptor antagonists show promise in the treatment of AD,
due to their neuroprotective effects observed during preclinical
studies [9,10].
Based on the above considerations, the discovery of new adeno-
sine receptor antagonists is pursued by several research groups. Of
particular interest are reports that certain adenosine receptor
antagonists, such as xanthine and benzothiazinone derivatives,
also act as inhibitors of the enzyme monoamine oxidase (MAO) B
[11,12]. For example, (E)-8-(3-chlorostyryl)caffeine (CSC, 1;
Ki = 26–54 nM) [13–15], a frequently used reference A2A antago-
nist, also is a highly potent MAO-B inhibitor with a Ki value of
80.6 nM (Fig. 1) [11,16]. In the CNS, MAO-B represents a major
catabolic enzyme of dopamine, and inhibitors of MAO-B have been
used in PD to conserve depleted dopamine reserves [17]. MAO-B
inhibitors are thus established therapy in PD and are often used
in combination with L-DOPA, or as mono-therapy in early PD
[17]. There is also evidence that MAO-B inhibition protect against
neurodegeneration in PD, presumably by reducing the formation
of neurotoxic aldehyde species and hydrogen peroxide, which are
by-products of the MAO catalytic cycle [18]. Additionally, the
neuroprotective properties associated with MAO-B may also
be beneficial for AD treatment [9].
In the current study we have attempted to discover additional
dual-target-directed compounds that antagonize adenosine recep-
tors and inhibit MAO-B. A recent report has shown that a series of
5-sulfanylphthalimide analogues (2) are highly potent inhibitors of
MAO-B [19]. Certain 5-sulfanylphthalimides were also potent inhi-
bitors of the MAO-A isoform. For example, 5-[(4-bromobenzyl)sul-
fanyl]phthalimide (2h) inhibits human MAO-A and MAO-B with
IC50 values of 0.273 lM and 0.0074 lM, respectively. MAO-A
inhibitors are clinically used to treat depressive illness, and since
a significant proportion of PD patients also exhibit signs of depres-
sion, the MAO-A inhibitory activities of these homologues may be
beneficial in PD therapy [18]. Based on the favourable MAO inhibi-
tion profile of the reported 5-sulfanylphthalimides, the current
study examined the possibility that these compounds also may
possess affinities for adenosine receptors. For this purpose, the
affinities of 9 of the reported 5-sulfanylphthalimides for rat adeno-
sine A1 and A2A receptors were evaluated. To sample chemical
space, phthalimides containing the phenylsulfanyl (2a), benzylsul-
fanyl (2b) and phenylethylsulfanyl (2c) substituents on C5 were
selected (Table 1). 5-(Phenylsulfanyl)- and 5-(benzylsulfanyl)ph-
thalimide were further substituted on the phenyl and benzyl rings
with halogens (F, Cl and Br) and an alkyl (OCH3) group to yield 2d–
i. We have further extended the series of sulfanylphthalimides by
synthesizing a homologous series of 4-sulfanylphthalimides, com-
pounds 3a–i. The affinities of these homologues for rat adenosine
A1 and A2A receptors, as well as their inhibition potencies towards
human MAO-A and MAO-B were subsequently measured and
compared to those of the 5-sulfanylphthalimides.
2. Results and discussion
2.1. Chemistry
The 5-sulfanylphthalimide analogues were synthesized as
previously reported [19]. The 4-sulfanylphthalimide analogues
(3a–i) were synthesized according to a similar protocol by reacting
an appropriate thiol with 3-nitrophthalimide in the presence of
K2CO3 (Fig. 2). Acetone served as solvent for these reactions. The
target compounds were obtained with moderate to high yields
(30–70%), and were purified by crystallization as cited in the
Dual-target-directed compounds that antagonize adenosine
receptors and inhibit MAO-B may thus act synergistic in the treat-
ment of PD: antagonism of the A1 and A2A receptors may facilitate
dopamine release and signalling, while MAO-B inhibition may
reduce the central catabolism of dopamine. Therefore, such com-
pounds may have enhanced therapeutic value in PD. Additionally,
it may be reasoned that dual-target directed compounds may find
therapeutic value in the treatment of AD: antagonism of the A1
receptor may be beneficial in the treatment of cognitive dysfunc-
tion, while antagonism of the A2A receptor and MAO-B inhibition
may exhibit neuroprotective effects. In neurological diseases, such
as PD and AD, dual-target-directed therapeutics is expected to be
advantageous over single-target treatments [9,10].
Table 1
The Ki values for the binding of 5-sulfanylphthalimide analogues 2 to rat adenosine A1
and A2A receptors, and the reported IC50 values for the inhibition of human MAO-A
and MAO-B.
O
3
R
5
N
H
1
O
O
Ki (
l
M)a
IC50 (l
M)b
Cl
N
N
N
R
A1
A2A
MAO-A
MAO-B
No affinityc
No affinityc
No affinityc
No affinityc
No affinityc
No affinityc
No affinityc
No affinityc
No affinityc
8.03
O
N
2a
2b
2c
2d
2e
2f
2g
2h
2i
C6H5S–
C6H5CH2S–
0.648 0.11
0.595 0.04
1.88 0.32
No affinityc
No affinityc
36.7 1.19
No affinityc
0.676 0.19
0.369 0.02
0.986
0.0045
0.030
1
1.92
2.27
1.68
1.01
0.958
0.506
0.273
1.63
C6H5(CH2)2S–
(4-ClC6H4)S–
(4-BrC6H4)S–
(4-FC6H4)CH2S–
(4-ClC6H4)CH2S–
(4-BrC6H4)CH2S–
(4-OCH3C6H4)CH2S–
0.457
0.364
0.0068
0.0056
0.0074
0.020
R
O
S
O
5
S
4
R
N
H
N
H
a
Values are expressed as the mean standard error of the mean (SEM) of
O
2
duplicate determinations.
3
O
b
Values obtained from reference [19].
No affinity observed at
c
a maximum concentration of 100 lM of the test
Fig. 1. The structure of CSC (1) and the general structures of 5-sulfanylphthalimide
analogues (2) and 4-sulfanylphthalimide analogues (3) are also shown.
compound.

M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
119
R
NO2
S
100
O
O
a,b
R-SH
75
50
25
N
H
+
N
H
O
O
Fig. 2. Synthetic pathway to 4-sulfanylphthalimide analogues (3a–i). Reagents and
conditions: (a) K2CO3, acetone, reflux, 24 h; (b) HCl (6 N).
supplementary. In each instance, the structures of the target com-
pounds were verified by 1H NMR, 13C NMR and mass spectrometry
(MS). The purities of the test compounds were evaluated by high
performance liquid chromatography (HPLC) as cited in the supple-
mentary. The molecular structure of 3i was elucidated by X-ray
crystallography and confirms substitution on the C4 position of
the phthalimide moiety (Fig. 3).
-2
0
2
4
6
Log [2b]
Fig. 4. The replicate sigmoidal dose–response curves for the binding of 2b
(expressed in nM) to rat A1 receptors. The bound cpm values were adjusted for
nonspecific binding and are expressed as percentage of the cpm values recorded in
the absence of 2b.
(DMPX) which exhibit Ki values for binding to A1 and A2A receptors
of 12 lM and 8.6 lM, respectively [14]. While no clear structure–
2.2. In vitro binding to A1 and A2A receptors
activity relationships (SARs) are apparent, it may be concluded that
the sulfanylphthalimide analogues possess higher affinities for
adenosine A1 receptors than A2A receptors since, for those homo-
logues which exhibit affinities for A1 receptors, the binding affini-
ties at A1 receptors are superior to those recorded at A2A receptors.
The affinities of the sulfanylphthalimide analogues for rat ade-
nosine A1 and A2A receptors were evaluated by the radioligand
binding protocol described in literature [15,20]. The binding of
the test compounds to adenosine A1 receptors were evaluated by
measuring the displacement of 1,3-[3H]-dipropyl-8-cyclopentylx-
anthine ([3H]DPCPX) from rat whole brain membranes (Fig. 4).
Binding of the test compounds to adenosine A2A receptor, in turn,
was evaluated by measuring the displacement of N-[3H]ethy-
ladenosin-50-uronamide ([3H]NECA) from rat striatal membranes.
The adenosine A2A receptor in vitro binding studies were carried
out in the presence of N6-cyclopentyladenosine (CPA) to minimize
the binding of [3H]NECA to A1 receptors. The results of these bind-
ing studies are given in Tables 1 and 2. The results show that
among the sulfanylphthalimide analogues (2 and 3) evaluated, 11
compounds possess affinity for adenosine A1 receptors while 3
homologues exhibit affinity for A2A receptors. It is noteworthy that
none of the 5-sulfanylphthalimide analogues (2) possess affinity
2.3. In vitro inhibition of MAO-B
In order to evaluate the possibility that the sulfanylphthalimide
analogues may act as dual-target-directed compounds, the
4-sulfanylphthalimide analogues (3a–i) were examined as
inhibitors of recombinant human MAO-A and MAO-B. The human
MAO inhibitory properties of the 5-sulfanylphthalimide analogues
(2a–i) have been previously reported [19]. The enzymatic activities
were determined fluorometrically by measuring the extent to
which the mixed MAO-A/B substrate, kynuramine, is oxidized
by the MAOs [21,22]. Kynuramine is oxidized by MAO-A and
MAO-B to yield 4-hydroxyquinoline, a fluorescent metabolite
(kex = 310 nm; kem = 400 nm). Under the experimental conditions,
neither kynuramine nor the test inhibitors exhibit fluorescence.
The MAO inhibition potencies of the sulfanylphthalimide
analogues are expressed as the corresponding IC50 values, and
are given in Tables 1 and 2.
for A2A receptors at a maximal tested concentration of 100 lM,
while compounds among both the 5-sulfanylphthalimide (2) and
4-sulfanylphthalimide (3) analogues were found to bind to A1
receptors. Among the compounds evaluated, compound 2i was
found to possess the highest binding affinity to adenosine A1
receptors with a Ki value of 0.369
pound is 3b, which exhibits the second highest binding affinity
to adenosine A1 receptors (Ki = 0.548 M). This compound also
possesses affinity for adenosine A2A receptors with a Ki value of
0.980 M. Among the series of sulfanylphthalimides, 3b thus pos-
lM. Another noteworthy com-
The results show that, although none of the 4-sulfanylphthalim-
ide analogues (3a–i) were MAO-A inhibitors (up to a maximal con-
l
centration of 100
inhibitors. The most potent MAO-B inhibitor, compound 3f exhib-
ited an IC50 value of 0.290 M. Other notable MAO-B inhibitors
with submicromolar IC50 values were 3g (IC50 = 0.460 M) and 3e
(IC50 = 0.443 M). These MAO-B inhibition potencies are, however,
modest compared to the dual-target-directed compound, CSC,
which inhibits MAO-B with an IC50 value of 0.146 M [23]. Com-
lM), most homologues (7 of 9) were MAO-B
l
l
sesses the highest binding affinity to adenosine A2A receptors and
represents a compound with dual affinities for A1 and A2A recep-
tors. It should be noted that although the affinities for adenosine
receptors recorded here are relatively low, it is comparable to first
generation antagonists such as 3,7-dimethyl-1-propargylxanthine
l
l
l
pared to the MAO inhibitory properties of the previously reported
5-sulfanylphthalimide analogues (2a–i), the 4-sulfanylphthalimide
analogues are also modest inhibitors. For example, the 5-sul-
fanylphthalimide analogues (2a–i) are reported to all inhibit
human MAO-A with 3 homologues exhibiting IC50 values in the
submicromolar range. The 5-sulfanylphthalimide analogues are
also in each instance more potent MAO-B inhibitors than the 4-
substituted homologues with all IC50 values in the submicromolar
range. In this respect, compound 2i is particularly noteworthy
since it not only represents a highly potent MAO-B inhibitor
(IC50 = 0.020
adenosine A1 receptor (Ki = 0.369
evaluated. This compound may thus be considered as the most
l
M), but also exhibits the highest affinity for the
l
M) among the compounds
Fig. 3. The X-ray crystallographic structure of 3i shown with displacement
ellipsoids.

120
M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
Table 2
The Ki values for the binding of 4-sulfanylphthalimide analogues 3 to rat adenosine A1 and A2A receptors, and the experimental IC50 values for the inhibition of human MAO-A and
MAO-B.
R
O
4
3
N
H
1
O
Ki (l
M)a
IC50 (l
M)b
R
A1
A2A
MAO-A
MAO-B
3a
3b
3c
3d
3e
3f
3g
3h
3i
C6H5S–
C6H5CH2S–
7.72 1.95
0.548 0.03
0.649 0.02
No affinityc
No affinityc
6.30 0.28
No affinityc
No affinityc
1.73 0.45
No affinityc
0.980 0.15
17.0 0.30
No affinityc
No affinityc
12.7 0.09
No affinityc
No affinityc
No affinityc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
No inhibitionc
9.17 0.58
1.88 1.52
3.90 1.77
1.39 0.50
0.443 0.14
0.290 0.07
0.460 0.24
No inhibitionc
No inhibitionc
C6H5(CH2)2S–
(4-ClC6H4)S–
(4-BrC6H4)S–
(4-FC6H4)CH2S–
(4-ClC6H4)CH2S–
(4-BrC6H4)CH2S–
(4-OCH3C6H4)CH2S–
a
b
c
Values are expressed as the mean SEM of duplicate determinations.
Values are expressed as the mean standard deviation (SD) of triplicate determinations.
No affinity/inhibition observed at a maximum concentration of 100 lM of the test compound.
weaker MAO-B inhibitor (IC50 = 1.88
able as a compound acting at both adenosine receptors and MAO-B.
l
M), which makes it less suit-
O
5
O
N
H
3. Conclusion
O
4
The aim of the present study was to examine the possibility of
developing dual-target-directed drugs that act as antagonists of
adenosine receptors and inhibitors of MAO-B. Such compounds
may act synergistic and offer enhanced benefit in the treatment
of neurological diseases such as PD and AD. PD treatment: antag-
onism of the adenosine A2A and A1 receptors facilitate dopamine
receptor signalling and dopamine release, respectively, while
MAO-B inhibition may reduce the central catabolism of dopamine.
AD treatment: antagonism of the adenosine A1 receptor may be
beneficial for the cognitive dysfuntion, while antagonism of the
adenosine A2A receptor and MAO-B inhibition display neuroprotec-
tive effects. Compound 2i represents the most promising dual-tar-
get-directed compound identified in this study. Although 2i
possesses no affinity for adenosine A2A receptors, it exhibits the
highest affinity for the A1 receptor among the compounds evaluat-
ed, and also is a highly potent MAO-B inhibitor. This compound
may thus be considered as a promising candidate for dual action
at adenosine A1 receptors and MAO-B. Compound 2h also is a note-
worthy dual-target-directed compound since it is a potent inhibi-
tor of both MAO-A and MAO-B, while also possessing good
affinity for adenosine A1 receptors. Although a limited number of
derivatives were examined, and the adenosine receptor affinities
may be considered as modest, this study provides ‘‘proof of con-
cept’’ for the proposal that sulfanylphthalimide analogues may
serve as promising leads for the design of dual acting adenosine
antagonists and MAO inhibitors. This study also suggests that com-
pounds that are structurally similar to the sulfanylphthalimides
may represent leads for the design of dual-target-directed
compounds. In this respect, a preliminary survey reveals that
5-benzyloxyphthalimide (4) [24] and 5-benzyloxyisatin (5) [25]
(Fig. 5), compounds known to inhibit MAO-B, also possess modest
affinities for adenosine A1 receptors (Table 3). Compounds 4 and 5
O
5
O
O
N
H
5
Fig. 5. The structures of 5-benzyloxyphthalimide (4) and 5-benzyloxyisatin (5).
Table 3
The Ki values for the binding of 5-benzyloxyphthalimide (4) and 5-benzyloxyisatin (5)
to rat adenosine A1 and A2A receptors, and the reported IC50 values for the inhibition
of human MAO-A and MAO-B.
Ki (
l
M)a
IC50 (lM)
A1
A2A
MAO-A
MAO-B
4
5
4.67 1.20
9.79 2.07
No affinityb
No affinityb
4.17c
4.62d
0.043c
0.103d
a
Values are expressed as the mean SEM of duplicate determinations.
b
No affinity observed at
a maximum concentration of 100 lM of the test
compound.
c
Values obtained from Ref. [24].
Values obtained from Ref. [25].
d
promising candidate for dual action at adenosine A1 receptors and
MAO-B. Although exhibiting weaker affinities at adenosine A1
receptors, 2a [Ki(A1) = 0.648
[Ki(A1) = 0.676 M] are also examples of compounds with potential
dual action at adenosine A1 receptors and MAO-B. Compound 2h
also is a potent inhibitor of MAO-A (IC50 = 0.273 M), a property
lM], 2b [Ki(A1) = 0.595 lM] and 2h
l
l
exhibit Ki values of 4.67
to adenosine A1 receptors, while inhibiting MAO-B with IC50 values
of 0.043 M and 0.103 M. In future studies, further structural
lM and 9.79 lM, respectively for binding
which may be of value in the treatment of PD-associated depres-
sion. As discussed above, compound 3b exhibits relatively good
l
l
affinities for both adenosine A1 receptors (Ki = 0.548
l
M) and A2A
optimization of these lead compounds may yield dual-target-
receptors (Ki = 0.980 M). Compound 3b was, however, a relatively
l
directed compounds with improved activities.

M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
121
binding study, the incubations contained 5 mg of the original tis-
sue weight of the whole brain membranes, 0.1 nM [3H]DPCPX,
0.2 units/mL adenosine deaminase, the test compound and 1%
DMSO. The incubations were vortexed and incubated for 60 min
at 25 °C in a shaking waterbath. Half an hour after incubation
was started, the incubations were vortexed again. The incubations
were terminated via filtration through a prewetted 2.5 cm What-
man glass microfiber filter (grade GF/B) under reduced pressure
using a Hoffeler vacuum system. The tubes were washed twice
with 4 mL ice-cold Tris buffer and the filters were washed once
more with 4 mL ice-cold Tris buffer. The damp filters were place
in scintillation vials and 4 mL of scintillation fluid (Filter-Count)
was added. The vials were shaken and incubated for two hours
before being counted (Packard Tri-CARB 2100 TR). An estimate of
the nonspecific binding was obtained from binding studies in the
4. Experimental section
4.1. Chemicals and instrumentation
Unless otherwise noted, all the reagents were obtained from
Sigma–Aldrich and were used without further purification. Proton
(1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Ultrashield Plus 600 Avance III 600
spectrometer in DMSO-d6 (Merck). The chemical shifts are report-
ed in parts per million (d) relative to the residual solvent signals.
Spin multiplicities are abbreviated as follows: s (singlet), d (dou-
blet), dd (doublet of doublets), t (triplet) or m (multiplet). High
resolution mass spectra (HRMS) were recorded on a Bruker micrO-
TOF-Q II mass spectrometer in atmospheric-pressure chemical
ionization (APCI) mode. Melting points (mp) were measured with
a Buchi M-545 melting point apparatus and are uncorrected. Sin-
gle-crystal X-ray diffraction analysis was carried out with a Bruker
SMART X2S diffractometer. 5-Benzyloxyphthalimide (4) [24] and
5-benzyloxyisatin (5) [25] were synthesized as described in
literature.
Counting of radio activity was performed using a Packard Tri-
Carb 2100 TR liquid scintillation counter. [3H]NECA (specific activ-
ity 25 Ci/mmol) and [3H]DPCPX (specific activity 120 Ci/mmol)
were obtained from Amersham Biosciences and PerkinElmer,
respectively. Adenosine deaminase (type X from calf spleen; 250
units) and CPA were from Sigma–Aldrich. WhatmanÒ GF/B
25 mm diameter filters and dimethyl sulfoxide (DMSO) were
obtained from Merck. Filter-count was purchased from Perk-
inElmer. Fluorescence spectrophotometry was carried out with a
Varian Cary Eclipse fluorescence spectrophotometer. Microsomes
from insect cells containing recombinant human MAO-A and
MAO-B (5 mg/mL) were from BD Biosciences while kynu-
ramine.2HBr was obtained from Sigma–Aldrich.
presence of 100 lM CPA. The IC50 values were determined by plot-
ting the count values (adjusted for nonspecific binding) vs. the
logarithm of the inhibitor concentrations to obtain a sigmoidal
dose–response curve. These kinetic data were fitted to the one site
competition model incorporated into the Prism software package
(GraphPad Software Inc.). The Ki values for the competitive inhibi-
tion of [3H]NECA (Kd = 15.3 nM) [20] or [3H]DPCPX (Kd = 0.36 nM)
[26] binding by the test compounds were calculated from the
IC50 values according to the Cheng and Prusoff equation [27]. All
incubations were carried out in duplicate and the Ki values are
expressed as mean standard deviation (SD).
4.3. MAO inhibition studies
IC50 values for the inhibition of MAO-A and MAO-B were deter-
mined using the recombinant human enzymes as described previ-
ously [22,28]. The enzymatic reactions were carried out at pH 7.4
(K2HPO4/KH2PO4 100 mM, made isotonic with KCl) to a final
volume of 500
concentrations spanning at least 3 orders of magnitude and the
MAO-A/B mixed substrate kynuramine (45 M for MAO-A and
30 M for MAO-B). DMSO, as co-solvent (4%) was added to each
lL. The reactions contained the different inhibitor
4.2. Radioligand binding studies
l
l
The collection of tissue samples for the adenosine A2A and A1
receptor binding studies was approved by the Research Ethics
Committee of the North-West University (application number
NWU-0035-10-A5). The adenosine A2A and A1 receptor binding
studies were carried out according the protocol described in lit-
erature [15,20]. For studies with the A2A receptor, the dissected
striata of male Sprague–Dawley rats were used while for the stud-
ies with the A1 receptor whole rat brain (excluding cerebellum and
brain stem) were employed. After dissection, the tissues were snap
frozen with liquid nitrogen and stored at ꢀ70 °C. The tissue were
thawed on ice, weighed and disrupted for 30 s (striata) or 90 s
(whole brain) with the aid of a Polytron homogenizer (model:
Polytron PT 10-35 GT) in 10 volumes of ice-cold 50 mM Tris buffer
(pH 7.7 at 25 °C). The resulting homogenate was centrifuged at
20,000g for 10 min at 4 °C and the pellet was resuspended in 10
volumes of ice-cold Tris buffer, again with the aid of a Polytron
homogenizer as above. The resulting suspension was recentrifuged
and the pellet obtained was suspended in Tris buffer (pH 7.7 at
25 °C) to a volume of 5 mL/g original tissue weight. The striatal
and whole brain membranes were aliquoted into microcentrifuge
tubes and stored at ꢀ70 °C until needed. The incubations were
carried out in 4 mL polypropylene tubes that were precoated with
SigmacoteÒ (Sigma–Aldrich). All incubations were prepared with
Tris buffer (pH 7.7 at 25 °C) to a volume of 1 mL. For the A2A recep-
tor binding study, the incubations contained 10 mg of the original
tissue weight of the striatal membranes, 4 nM [3H]NECA, 50 nM
CPA, 10 mM MgCl2, 0.2 units/mL adenosine deaminase, the test
compound and 1% DMSO. DMSO was used to prepare all stock
solutions of the compounds to be tested. For the A1 receptor
reaction. The enzyme reactions were initiated with the addition
of MAO-A or MAO-B (0.0075 mg protein/mL) and incubated for
20 min at 37 °C in a waterbath. After termination with the addition
of 400 lL NaOH (2 N) and 1000 lL water, the concentrations of the
MAO generated 4-hydroxyquinoline were measured by fluores-
cence spectrophotometry (kex = 310; kem = 400 nM) [18]. For this
purpose, a linear calibration curve containing authentic 4-hydrox-
yquinoline (0.047–1.56 lM) was constructed. The enzyme catalyt-
ic rates were calculated and fitted to the one site competition
model incorporated into the Prism 5 software package (GraphPad).
The IC50 values were determined in triplicate and are expressed as
mean SD.
4.4. Single-crystal X-ray diffraction analysis
X-ray diffraction data were collected on a Bruker SMART X2S
diffractometer using monochromated (doubly curved silicon crys-
tal) Mo-Ka-radiation (0.71073 Å) and employing x scan mode. For
this purpose, a yellow coloured crystal of compound 3i was mount-
ed on a Mitegen Micromount with a small amount of epoxy and
was automatically centred. Bruker APEX2 software was used for
preliminary determination of the unit cell, and the determination
of integrated intensities and unit cell refinement were performed
with Bruker SAINT system. The data were corrected for absorption
effects with SADABS using the multiscan technique. The structure
was solved with XS and subsequent structure refinements were
performed with XL [29]. The structure was refined by anisotropic
full-matrix least-squares refinement on F2: C16H13NO3S, M =
299.34 g molꢀ1, crystal size: 0.16 ꢁ 0.16 ꢁ 0.64 mm3, monoclinic,

122
M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
space group P1 21/c1, a = 12.711(5) Å, b = 5.3605(18) Å,
c = 20.449(8) Å,
= 90 °, b = 100.434(13) °, V = 1373.7(9) Å3,
Z = 4, k = 0.71073 Å,
calcd = 1.447 Mg cmꢀ3 = 0.245 mmꢀ1
T = 200(2) K, hrange = 1.63–25.07 °, reflections collected: 8049, inde-
pendent: 2410 (Rint = 0.0372), 191 parameters, final R indices
4.5.5. 4-[(4-Bromophenyl)sulfanyl]phthalimide (3e)
a
=
c
The title compound (yellow crystals) was prepared from
3-nitrophthalimide and 4-bromothiophenol in a yield of 45%: mp
270.3–271.9 °C (ethanol); 1H NMR (Bruker Avance III 600, DMSO-
d6) d 6.93 (d, 1H, J = 7.9 Hz), 7.53–7.55 (m, 3H), 7.59–7.61 (m,
1H), 7.73 (d, 2H, J = 8.3 Hz), 11.40 (s, 1H); 13C NMR (Bruker Avance
III 600, DMSO-d6) d 119.7, 123.8, 126.7, 128.4, 130.6, 133.3, 133.8,
134.9, 137.1, 137.1, 168.7, 168.7; APCI-HRMS m/z: calcd for C14H9
BrNO2S [MH]+, 333.9537, found 333.9535; Purity (HPLC): 100%.
q
,
l
,
[I > 2r(I)]: R1 = 0.0361; wR2 = 0.0990, max/min residual electron
density: 0.208/ꢀ0.232 eꢀ/Å3, goodness-of-fit on F2 1.044,
F(000) = 624. CCDC 1015897 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk.
4.5.6. 4-[(4-Fluorobenzyl)sulfanyl]phthalimide (3f)
The title compound (yellow needles) was prepared from
3-nitrophthalimide and 4-fluorobenzyl mercaptan in a yield of
70%: mp 179.8–180.2 °C (ethanol/ethyl acetate); 1H NMR (Bruker
Avance III 600, DMSO-d6) d 4.36 (s, 2H), 7.15 (t, 2H, J = 8.7 Hz),
7.47–7.51 (m, 3H), 7.66–7.70 (m, 2H), 11.30 (s, 1H); 13C NMR
(Bruker Avance III 600, DMSO-d6) d 33.4, 115.5, 115.6, 118.9,
126.8, 130.3, 131.2, 131.2, 132.5, 133.9, 134.7, 137.7, 160.8,
162.4, 168.9, 169.0; APCI-HRMS m/z: calcd for C15H11FNO2S
[MH]+, 288.0489, found 288.0495; Purity (HPLC): 100%.
4.5. Synthesis of the 4-sulfanylphthalimide analogues (3a–i)
A mixture of the appropriate thiol (6 mmol), 3-nitrophthalimide
(4 mmol) and K2CO3 (10 mmol) in 20 mL acetone was heated under
reflux for 24 h. The reaction was cooled to room temperature and
diluted with 150 mL water. The mixture was subsequently acidi-
fied to pH 2 with 6 N HCl. The resulting precipitate was collected
by filtration and dried at 50 °C [19].
4.5.7. 4-[(4-Chlorobenzyl)sulfanyl]phthalimide (3g)
4.5.1. 4-(Phenylsulfanyl)phthalimide (3a)
The title compound (yellow crystals) was prepared from
3-nitrophthalimide and 4-chlorobenzyl mercaptan in a yield of
61%: mp 233.5–239.4 °C (ethanol/ethyl acetate); 1H NMR (Bruker
Avance III 600, DMSO-d6) d 4.37 (s, 2H), 7.37 (d, 2H, J = 8.3 Hz),
7.47 (d, 2H, J = 8.7 Hz), 7.49–7.51 (m, 1H), 7.66–7.67 (m, 2H),
11.30 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-d6) d 33.4,
119.0, 126.9, 128.7, 130.3, 131.0, 132.2, 133.9, 134.7, 135.6,
137.5, 168.9, 169.0; APCI-HRMS m/z: calcd for C15H11ClNO2S
[MH]+, 304.0199, found 304.0099; Purity (HPLC): 100%.
The title compound (yellow crystals) was prepared from
3-nitrophthalimide and thiophenol in a yield of 62%: mp 209.0–
210.4 °C (ethanol); 1H NMR (Bruker Avance III 600, DMSO-d6) d
6.85 (d, 1H, J = 7.9 Hz), 7.51 (d, 1H, J = 7.2 Hz), 7.54–7.58 (m, 4H),
7.60–7.62 (m, 2H), 11.40 (s, 1H); 13C NMR (Bruker Avance III
600, DMSO-d6) d 119.3, 126.3, 128.5, 130.1, 130.1, 130.4, 133.8,
134.7, 135.5, 138.2, 168.7, 168.7; APCI-HRMS m/z: calcd for
C
14H10NO2S [MH]+, 256.0432, found 256.0426; Purity (HPLC): 100%.
4.5.8. 4-[(4-Bromobenzyl)sulfanyl]phthalimide (3h)
4.5.2. 4-(Benzylsulfanyl)phthalimide (3b)
The title compound (light yellow crystals) was prepared from
3-nitrophthalimide and 4-bromobenzyl mercaptan in a yield of
65%: mp 261.3–262.1 °C (ethanol/ethyl acetate); 1H NMR (Bruker
Avance III 600, DMSO-d6) d 4.38 (s, 2H), 7.42 (d, 2H, J = 8.7 Hz),
7.51–7.53 (m, 3H), 7.67–7.70 (m, 2H), 11.31 (s, 1H); 13C NMR
(Bruker Avance III 600, DMSO-d6) d 33.2, 118.8, 120.5, 126.8,
130.2, 131.2, 131.5, 133.8, 134.5, 135.9, 137.3, 168.6, 168.8;
APCI-HRMS m/z: calcd for C15H11BrNO2S [MH]+, 347.9694, found
347.9591; Purity (HPLC): 100%.
The title compound (light yellow needles) was prepared from
3-nitrophthalimide and benzyl mercaptan in a yield of 30%: mp
211.5–213.5 °C (ethanol); 1H NMR (Bruker Avance III 600, DMSO-
d6) d 4.39 (s, 2H), 7.25–7.28 (m, 1H), 7.32–7.35 (m, 2H), 7.46 (d,
2H, J = 7.5 Hz), 7.51 (d, 1H, J = 6.8 Hz), 7.67–7.73 (m, 2H), 11.30
(s, 1H); 13C NMR (Bruker Avance III 600, DMSO-d6) d 34.0, 118.6,
126.7, 127.4, 128.6, 129.0, 130.1, 133.8, 134.5, 136.2, 137.8,
168.7, 168.9; APCI-HRMS m/z: calcd for
270.0589, found 270.0557; Purity (HPLC): 100%.
C
15H12NO2S [MH]+,
4.5.9. 4-[(4-Methoxybenzyl)sulfanyl]phthalimide (3i)
4.5.3. 4-[(2-Phenylethyl)sulfanyl]phthalimide (3c)
The title compound (yellow crystals) was prepared from
3-nitrophthalimide and 4-methoxybenzyl mercaptan in a yield of
44%: mp 220.7–223.7 °C (ethanol); 1H NMR (Bruker Avance III
600, DMSO-d6) d 3.71 (s, 3H), 4.30 (s, 2H), 6.88 (d, 2H, J = 8.3 Hz),
7.36 (d, 2H, J = 8.7 Hz), 7.49 (d, 1H, J = 6.8 Hz), 7.66–7.71 (m, 2H),
11.31 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-d6) d 33.7,
55.2, 114.2, 118.8, 126.7, 127.9, 130.3, 130.4, 133.9, 134.7, 138.2,
The title compound (white needles) was prepared from
3-nitrophthalimide and 2-phenylethyl mercaptan in a yield of
31%: mp 150.2–151.6 °C (ethanol); 1H NMR (Bruker Avance III
600, DMSO-d6) d 2.96 (t, 2H, J = 7.6 Hz), 3.35 (t, 2H, J = 7.6 Hz),
7.20–7.23 (m, 1H), 7.28–7.32 (m, 4H), 7.51 (dd, 1H, J = 1.5,
7.9 Hz), 7.69–7.72 (m, 2H), 11.28 (s, 1H); 13C NMR (Bruker Avance
III 600, DMSO-d6) d 30.9, 33.9, 118.4, 126.4, 126.8, 128.4, 128.6,
129.8, 133.9, 134.6, 137.8, 139.7, 168.7, 168.8; APCI-HRMS m/z:
calcd for C16H14NO2S [MH]+, 284.0745, found 284.0744; Purity
(HPLC): 100%.
158.7, 169.0, 169.0; APCI-HRMS m/z: calcd for
C16H12NO3S
[MꢀH]+, 298.0538, found 298.0538; Purity (HPLC): 100%.
4.6. HPLC traces of the 4-sulfanylphthalimide analogues (3a–i)
4.5.4. 4-[(4-Chlorophenyl)sulfanyl]phthalimide (3d)
The degree of purity for each of the synthesized 4-sulfanylph-
thalimide analogues (3a-i) were estimated with HPLC analyses,
which were carried out with an Agilent 1100 HPLC system
equipped with a quaternary pump and an Agilent 1100 series diode
array detector. Milli-Q water (Millipore) and HPLC grade acetoni-
trile (Merck) were used for the chromatography of compounds
3a, 3b, 3c and 3f, and HPLC grade tetrahydrofuran (Sigma–Aldrich)
were used for the chromatography of compounds 3d, 3e, 3g, 3h
The title compound (yellow crystals) was prepared from
3-nitrophthalimide and 4-chlorothiophenol in a yield of 53%: mp
268.9–271.0 °C (ethanol/ethyl acetate); 1H NMR (Bruker Avance
III 600, DMSO-d6)
d 6.92 (d, 1H, J = 7.9 Hz), 7.54 (d, 1H,
J = 7.2 Hz), 7.58–7.63 (m, 5H), 11.41 (s, 1H); 13C NMR (Bruker
Avance III 600, DMSO-d6) d 119.6, 126.6, 127.8, 130.4, 130.5,
133.8, 134.9, 135.1, 137.0, 137.4, 168.7, 168.7; APCI-HRMS m/z:
calcd for C14H9ClNO2S [MH]+, 290.0043, found 290.0037; Purity
(HPLC): 100%.
and 3i. A Venusil XBP C18 column (4.60 ꢁ 150 mm, 5
lm) was
used and the mobile phase consisted initially of 30% acetonitrile

M.M. Van der Walt et al. / Bioorganic Chemistry 59 (2015) 117–123
[4] K. Xu, E. Bastia, M. Schwarzschild, Pharmacol. Ther. 105 (2005) 267–310.
123
or tetrahydrofuran and 70% MilliQ water at a flow rate of 1 mL/min.
At the start of each HPLC run a solvent gradient program was
initiated by linearly increasing the composition of the acetonitrile
or tetrahydrofuran in the mobile phase to 85% acetonitrile or
tetrahydrofuran over a period of 5 min. Each HPLC run lasted
15 min and a time period of 5 min was allowed for equilibration
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The NMR and MS spectra and X-ray diffraction data were
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any publication generated by the MRC supported research are that
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