Inhibition of monoamine oxidase by C5-substituted phthalimide analogues

Article abstract of DOI:10.1016/j.bmc.2011.06.070

Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors of human monoamine oxidase (MAO) A and B. In general, C5- and C6-substitution of isatin leads to enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selective binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the substrate cavities of MAO-A and -B. Based on these observations and the close structural resemblances between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and evaluated as MAO inhibitors. While phthalimide and N-aryl-substituted phthalimides were found to be weak MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of recombinant human MAO-B with IC₅₀ values ranging from 0.007 to 2.5 μM and moderately potent reversible inhibitors of recombinant human MAO-A with IC₅₀ values ranging from 0.22 to 9.0 μM. By employing molecular docking the importance of hydrogen bonding between the active sites of MAO-A and -B and the phthalimide inhibitors are highlighted.

Full text of DOI:10.1016/j.bmc.2011.06.070

Bioorganic & Medicinal Chemistry 19 (2011) 4829–4840
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of monoamine oxidase by C5-substituted phthalimide analogues
⇑
Clarina I. Manley-King, Jacobus J. Bergh, Jacobus P. Petzer
Pharmaceutical Chemistry, 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:
Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors
of human monoamine oxidase (MAO) A and B. In general, C5- and C6-substitution of isatin leads to
enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selec-
tive binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring
binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between
the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the sub-
strate cavities of MAO-A and -B. Based on these observations and the close structural resemblances
between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and eval-
uated as MAO inhibitors. While phthalimide and N-aryl-substituted phthalimides were found to be weak
MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of
Received 20 May 2011
Revised 20 June 2011
Accepted 26 June 2011
Available online 29 June 2011
Keywords:
Monoamine oxidase
Reversible inhibition
Phthalimide
Isatin
recombinant human MAO-B with IC50 values ranging from 0.007 to 2.5
ible inhibitors of recombinant human MAO-A with IC50 values ranging from 0.22 to 9.0
l
M and moderately potent revers-
M. By employing
Molecular docking
l
molecular docking the importance of hydrogen bonding between the active sites of MAO-A and -B and
the phthalimide inhibitors are highlighted.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Of particular interest is the observation that MAO-B is the most
abundant form in the human basal ganglia.11,12
Monoamine oxidase A and B (MAO-A and -B) are flavin adenine
dinucleotide (FAD)-containing enzymes which are attached to the
outer membrane of mitochondria.1 The FAD cofactors are cova-
lently bound to the MAO enzymes via a thio ether linkage between
The principal biochemical function of the MAO’s is thought to be
the catalysis of the -carbon oxidation of a variety of monoamine
a
neurotransmitters and dietary amines. In addition the MAO’s also
oxidize amine containing xenobiotic drugs and toxins. For example,
serotonin acts as the preferential substrate for MAO-A while the
false neurotransmitters, benzylamine and b-phenylethylamine,
are selectively oxidized by MAO-B.13,14 Dopamine, epinephrine
and norepinephrine are substrates for both isoforms.14,15 These
observations suggest that the MAO’s do not only function to termi-
nate the central action of neurotransmitter amines, but may also
protect neurons form stimulation by extraneous amines by degrad-
ing false neurotransmitters.3 The MAO’s have also been shown to
activate neurotoxins. For example, the central oxidation and subse-
quent activation of the parkinsonian inducing pro-neurotoxin, 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is dependent
upon the action of MAO-B.16 In vitro experiments have shown that
MPTP is a substrate for both isoforms. The MAO-A isoform, how-
ever, is rapidly inactivated by reactive intermediates which form
when MPTP is oxidized by MAO-A and therefore does not signifi-
cantly contribute to the oxidation of MPTP in vivo.16
the side chain of a cysteinyl residue and the C8a-position of the
FAD.2 In both MAO isoforms this cysteine residue is part of the con-
served pentapeptide Ser-Gly-Gly-Cys-Tyr. Even though MAO-A and
-B are encoded by different genes, they share approximately 70%
amino acid sequence identity.3 Both MAO genes are located on
the X chromosome and consist of 527 and 520 amino acids, respec-
tively. These findings, together with observation that the genes ex-
hibit identical exon-intron organizations, suggest that the two
isoforms are derived from a common ancestral gene.3,4
The MAO genes are expressed in a variety of central and periph-
eral tissues including the liver, brain, blood platelets, placenta and
gastrointestinal tract. Within these tissues the concentrations and
activities of the MAO’s differ. MAO-B activity is found almost
exclusively in human blood platelets5 while MAO-A activity is
exclusively present in the human placental6 and gut tissues.7 With-
in the human liver, MAO-B is the most abundant isoform.8 Both
MAO isoforms are found in the human brain, although they are
differently distributed9 with MAO-B present in higher levels.10,11
The location of MAO-A and -B in the brain and its role in the
degradation of monoamine neurotransmitters have made these
enzymes attractive targets for the development of drugs for the
treatment of neuropsychiatric and neurodegenerative diseases.
For example, reversible and irreversible inhibitors of MAO-A are
used to treat anxiety disorder and depression. Based on the
⇑
Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.070

4830
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
monoamine hypothesis of depression, MAO-A inhibitors reduce
the degradation of serotonin, norepinephrine and dopamine in
the brain which leads to increased levels of these neurotransmit-
ters in the brain and a resulting antidepressant effect.14 MAO-A
inhibitors are particularly useful in the management of depression
in elderly patients.14,17 MAO-B inhibitors are currently clinically
used in the treatment of Parkinson’s disease. Since MAO-B appears
to be primarily responsible for dopamine catabolism in the basal
ganglia,12,18 inhibitors may conserve the depleted dopamine sup-
ply in this brain region, enhance the concentration of dopamine
that is derived from administered levodopa, the metabolic precur-
sor of dopamine and prolong the action of dopamine.19,20 Based on
these observations, MAO-B inhibitors are frequently combined
with levodopa in the therapy of Parkinson’s disease.21 Also of
importance is the observation that MAO-B levels and activity in-
crease in most brain regions, including the basal ganglia, with
age.11,22,23 In contrast, MAO-A activity remains constant with
age.10 Considering that Parkinson’s disease occurs primarily in el-
derly patients, the age-associated increase of MAO-B and the con-
sequent increase of MAO-B catalyzed dopamine turnover make
MAO-B inhibitors particularly relevant in Parkinson’s disease ther-
apy. Of importance is the observation that in the primate brain,
MAO-A also contribute to the catabolism of dopamine. For exam-
ple, the selective irreversible inhibitor of MAO-A, clorgyline, is re-
ported to enhance the concentration of dopamine in the striatum
of primates treated with levodopa and the degree of this elevation
is comparable to that obtained with (R)-deprenyl, a selective irre-
versible inhibitor of MAO-B.19 Nonselective MAO-A/B inhibitors
may therefore be more efficacious in prolonging the effect of dopa-
mine in the basal ganglia.15
O
O
N
1
H
O
O
O
5
O
O
N
2
3
H
H
6
N
O
Figure 1. The structures of isatin (1), 5-benzyloxyisatin (2) and 6-benzyloxyisatin
(3).
O
N
H
4
O
Figure 2. The structure of phthalimide (4).
A significant disadvantage of MAO-A inhibition is the occur-
rence cardiovascular effects when combined with indirectly acting
sympathomimetic amines such as tyramine which is present is cer-
tain foods.15,24 This interaction occurs because tyramine is nor-
mally metabolized by MAO-A in the gut wall which reduces the
amounts of tyramine that enters the systemic circulation. Irrevers-
ible inhibition of MAO-A prevents the normal catabolism of
tyramine in the gut and hence leads to increased systemic concen-
trations of tyramine. In contrast, this interaction does not occur
with reversible inhibitors since the inhibitor is readily displaced
by tyramine which is subsequently normally metabolized by
MAO-A. These observations suggest that when designing MAO-A
selective inhibitors or MAO-A/B mixed inhibitors, the inhibitors
should interact reversibly with MAO-A. Another advantage of
reversible inhibition is that, following withdrawal of the drug, en-
zyme activity is recovered quickly upon elimination of the drug
from the tissues. In contrast, after terminating treatment with irre-
versible MAO inhibitors, recovery of enzyme activity may require
several weeks since the turnover rate for the biosynthesis of
MAO in the human brain may be as much as 40 days.25
enzyme is unoccupied. Based on this observation and modeling
studies C5- and C6-substituted isatin analogues were previously
examined as potential inhibitors of recombinant human MAO-A
and -B.28,29 In general, the isatin analogues proved to be potent
and selective MAO-B inhibitors with selected members also exhib-
iting high binding affinities towards MAO-A. Isatin analogues bear-
ing C5- and C6-benzyloxy substituents (2 and 3) were particularly
potent reversible MAO-B inhibitors with IC50 values of 0.103 and
0.138 l
M, respectively.29 The high binding affinities of the substi-
tuted isatin analogues to MAO-B may be explained by modeling
studies which suggest that the isatin dioxoindolyl ring is bound
to the substrate cavity while the C5 and C6 substituents extends
into the entrance cavity. The additional productive interactions of
the C5 and C6 substituents with the entrance cavity amino acid
residues may allow for higher binding affinities to the enzyme
compared to isatin and hence more potent inhibition.28,29 Isatin
may therefore be considered as a promising scaffold for the design
of MAO inhibitors. Based on these observations the present study
examines a series of phthalimide analogues as potential inhibitors
of MAO-A and -B. Phthalimide (4) is an isomer of isatin and appro-
priately substituted phthalimide analogues may therefore also
possess affinity for the MAO isozymes (Fig. 2). To explore this pos-
sibility, 5-benzyloxyphthalimide (5a) and other selected 5-alkyl-
oxy- and 5-aryloxyphthalimides (5b–i) were synthesized and
evaluated as inhibitors of MAO-A and -B (Scheme 1). As discussed
above, the benzyloxy side chain was selected as initial substituent
in this study since it has been shown to enhance the binding affin-
ity of isatin to particularly MAO-B.
For these reasons several studies are underway to discover new
MAO inhibitors which bind reversibly to the enzymes. An example
of a reversible MAO-A/B mixed inhibitor is isatin (1) (Fig. 1). Isatin
is an endogenous small molecule which inhibits both human MAO-
A and -B with enzyme-inhibitor dissociation constants (Ki values)
of 15 lM and 3 l
M for the two isozymes, respectively.26 While
the X-ray crystal structure of isatin bound to MAO-A has not yet
been determined, the three-dimensional structure of isatin com-
plexed within the active site of recombinant human MAO-B is
known.27 The structure model shows that isatin is bound within
the substrate cavity of MAO-B with the C2 carbonyl oxygen of
the dioxoindolyl moiety directed towards the FAD cofactor. Here
the NH and the C2 carbonyl oxygen of isatin are stabilized by
hydrogen bonding to water molecules present in the substrate cav-
ity of MAO-B.27 Of interest is the observation that with isatin lo-
cated in the MAO-B substrate cavity, the entrance cavity of the
2. Results and discussion
2.1. Chemistry
In the present study selected 5-alkyloxy- and 5-aryloxyphthal-
imides (5a–i) were synthesized with the aim of examining their

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
4831
O
O
O
R
CN
CN
R
a, b
a
N
H
O
R
NH2
+
N
R
5
8
7
O
O
6a-b
O
R
R
R
a
b
c
C6H5O
f
2-Naphthalenyl-O
4-BrC6H4CH2O
4-BrC6H4CH2CH2O
4-BrC6H4O
a
C6H5
C6H5CH2O
g
h
i
b
C6H5CH2
C6H5CH2CH2O
C6H5(CH2)3O
Scheme 2. The synthetic route to the N-substituted phthalimide analogues 6a and
6b. Reagents: (a) glacial acetic acid.
d
e
(E)-C6H5CH=CHCH2O
Scheme 1. The synthetic route to the 5-alkyloxy- and 5-aryloxyphthalimides (5a–i)
that were examined in this study. Reagents: (a) NaOC2H5; (b) HNO3 (10%).
were employed.29,33,34 The MAO-A/B mixed substrate, kynuramine,
was used to measure the catalytic rates of the enzymes in the ab-
sence and presence of various concentrations of the test inhibi-
tors.33 Kynuramine is oxidized by the MAO enzymes to yield the
fluorescent metabolite, 4-hydroxyquinoline. Concentration mea-
surements of 4-hydroxyquinoline were conveniently carried out
by fluorescence spectrophotometry (excitation wavelength of
310; emission wavelength of 400 nm) since none of the inhibitors
investigated in this study fluoresced at these excitation/emission
wavelengths or quenched the fluorescence of 4-hydroxyquinoline
at the inhibitor concentrations used.35 The inhibition potencies of
the test inhibitors were calculated from the experimentally ob-
tained sigmoidal dose–response curves and expressed as the IC50
values (Fig. 3). Using the Cheng-Prusoff equation,36,37 the isoform
selectivity index [SI = Ki(MAO-A)/Ki(MAO-B)] of each inhibitor
was calculated. For this purpose the experimentally determined
IC50 values were converted to the corresponding Ki values for the
inhibition of MAO-A and MAO-B.
Table 1
1H NMR and 13C NMR chemical shifts for the NH proton and carbonyl C1 and C3 of
phthalimide analogues 5a–ia
O
R
3
5
N
H
1
5
O
R
NH
C1/C3
5a
5b
5c
5d
5e
5f
5g
5h
5i
C6H5O
11.26
11.18
11.16
11.63
11.18
11.32
11.19
11.18
11.33
168.4
168.8
168.8
168.8
168.8
168.4
168.8
168.8
168.4
168.6
168.9
168.9
168.9
168.9
168.6
168.9
168.9
168.6
C6H5CH2O
C6H5CH2CH2O
C6H5(CH2)3O
(E)-C6H5CH@CHCH2O
2-Naphthalenyl-O
4-BrC6H4CH2O
The recombinant human MAO inhibition data for the 5-alkyl-
oxy- and 5-aryloxyphthalimide analogues (5a–i) are presented in
Table 2. For comparison, the reported MAO-A and -B inhibition
potencies of isatin (1), 5-benzyloxyisatin (2) and 6-benzyloxyisatin
(3) are also given.29 Compared to isatin (1), 5-benzyloxyisatin (2)
and 6-benzyloxyisatin (3), the phthalimides were in general supe-
rior MAO-B inhibitors with IC50 values ranging from 0.007–
4-BrC6H4CH2CH2O
4-BrC6H4O
a
The NMR experiments were conducted in DMSO-d6 (see Section 4).
MAO inhibitory properties. The target phthalimides were prepared
from the corresponding C4-substituted phthalonitriles (7a–i) as
shown in Scheme 1. The phthalonitriles were treated with a solu-
tion of sodium ethoxide followed by dilute nitric acid to yield the
phthalimide analogues 5a–i in poor to good yields (10–81%).30
The successful formation of the phthalimide ring system of the tar-
get compounds were verified by the presence of two 13C NMR sig-
nals at 168.4–168.9 ppm which corresponds to the carbonyl
carbons at C1 and C3 and the presence of a 1H NMR signal at
approximately 11 ppm which corresponds to the phthalimide NH
proton (Table 1). The C4-substituted phthalonitriles (7a–i) re-
quired for these synthesis were obtained by reacting 4-nitropht-
halonitrile with the appropriate alcohol in the presence of
potassium carbonate in dimethyl sulfoxide (DMSO) as described
previously.31 The N-substituted phthalimide analogues 6a–c were,
in turn, prepared by reacting phthalic anhydride (8) with the
appropriate amine in glacial acetic acid (Scheme 2).32 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 experimental section.
2.49
phthalimide (IC50 = 2.49
tor than 5-benzyloxyisatin (IC50 = 0.103
(IC50 = 0.138 M) but still approximately fivefold more potent than
isatin (IC50 = 12.4 M). Increasing the length of the C5 substituent
enhances the MAO-B inhibition potencies of the phthalimide ana-
logues by several orders of a magnitude. For example, substitution
at C5 with a benzyloxy moiety yields compound 5b with an IC50
l
M. The only exception was 5a, the phenoxy substituted
M), which were a weaker MAO-B inhibi-
M) and 6-benzyloxyistin
l
l
l
l
value of 0.043 lM, approximately 58-fold more potent than the
phenoxy substituted phthalimide 5a. Similarly, substitution with
the phenylethoxy (5c) and phenylpropoxy (5d) side chains also
yielded exceptionally potent MAO-B inhibitors with IC50 values
of 0.017 lM and 0.050 lM, respectively. Even introduction of an
ethenyl double bond into the phthalimide C5 side chain, as ob-
served with the phenylpropenyloxy derivative 5e, yielded a potent
MAO-B inhibitor (IC50 = 0.046 lM). Interestingly, replacing the
phenyl ring of the C5 substituent of 5a with a naphthalenyl ring
yielded the exceptionally potent MAO-B inhibitor 5f with an IC50
value 0.033 lM. Compound 5f is 75-fold more potent than 5a as
an MAO-B inhibitor. This result further demonstrates that increas-
ing the length or size of the C5 substituent enhances the MAO-B
inhibition potencies of the phthalimide analogues. This result also
shows that the length or size of the substituent may be increased
by increasing the length of the oxy linker between the phthalimide
and phenyl rings or by modifying the phenyl ring to enhance its
size, by for example replacement with a naphthalenyl ring. To
2.2. MAO inhibition studies
To examine the 5-alkyloxy- and 5-aryloxyphthalimide ana-
logues 5a–i as potential inhibitors of MAO-A and -B, microsomes
from insect cells containing the recombinant human enzymes

4832
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
Halogen substitution on the ring system of the C5 side chain may
Panel A
therefore be viewed as a general strategy to enhance the MAO-B
inhibition potency of moderate and weak phthalimide inhibitors.
The importance of the C5 substituent for MAO-B inhibition was
demonstrated by the finding that phthalimide (4) is a weak MAO-B
20
15
10
5
inhibitor with an IC50 value of 134
mately 11-fold weaker than the MAO-B inhibition potency re-
corded for isatin (IC50 = 12.4 M) and approximately 54-fold
lM (Table 3). This is approxi-
l
weaker than the inhibition potency recorded for 5a, the weakest
C5 substituted phthalimide analogue of the studied series
(IC50 = 2.49 lM). Also, the significance of the positioning of the
substituent at C5 of the phthalimide ring was demonstrated by
the finding that the N-substituted isomers 6a and 6b only inhibits
MAO-B at very high concentrations (Table 3). These results show
that a C5 substituent is critical for the MAO-B inhibition activity
of phthalimide and that the position of the substituent on the
phthalimide ring system is also an important consideration. This
behavior will be further explored below with the aid of molecular
docking studies.
-4
-3
-2
-1
0
1
Log[I]
The 5-alkyloxy- and 5-aryloxyphthalimide analogues 5a–i were
also evaluated as potential inhibitors of recombinant human MAO-
A (Table 2). With the exception of 5a, the phthalimide analogues
displayed weaker MAO-A inhibition potencies compared to their
corresponding MAO-B inhibition potencies. Based on the isoform
SI values, these analogues (5b–i) were 10- to 129-fold more potent
as MAO-B inhibitors. Compound 5a was essentially a non-selective
MAO-A/B inhibitor. The most potent MAO-A inhibitor among the
test compounds was 5g, the bromobenzyloxy substituted ana-
Panel B
6
4
2
logue, with an IC50 value of 0.22 lM. Similar to the trend observed
for the inhibition of MAO-B, bromine substitution on the phenyl
ring of the C5 substituent enhances the MAO-A inhibition poten-
cies. For example, 5g is approximately 19-fold more potent than
its corresponding unsubstituted homologue 5b (IC50 = 4.17
Similarly, bromine substituted compounds 5h (IC50 = 1.08
l
l
M).
M)
-4
-3
-2
-1
0
1
2
Log[I]
and 5i (IC50 = 1.08
sponding unsubstituted homologues 5c (IC50 = 3.58
(IC50 = 5.85 M), respectively, although to lesser extents (3–5-fold)
than observed for 5b. Also similar to MAO-B, substitution of the
phenyl ring of the C5 substituent of 5a with a naphthalenyl ring
significantly improved the MAO-A inhibition potency with inhibi-
l
M) were also more potent than their corre-
lM) and 5a
Figure 3. The sigmoidal dose–response curves of the initial rates of oxidation of
kynuramine by recombinant human MAO-A (Panel A) and recombinant human
MAO-B (Panel B) vs. the logarithm of concentration of inhibitor 5g (expressed in
M). The determinations were carried out in duplicate and the values are expressed
as the mean SD. The concentrations of kynuramine used were 45 and 30 M for
l
l
l
the studies with MAO-A and MAO-B, respectively, and the rate data are expressed
as nmoles 4-hydroxyquinoline formed/min/mg protein.
tor 5f displaying an IC50 value of 0.92 lM, approximately sixfold
more potent than 5a. In fact, 5f was the second most potent
MAO-A inhibitor among the phthalimides evaluated. Increasing
the C5 side chain length also enhanced the MAO-A inhibition
potencies of the phthalimide analogues, although by a relatively
small extent. For example the phenoxy (5a), benzyloxy (5b),
phenylethoxy (5c) and phenylpropoxy (5d) substituted analogues
examine the possibility of further enhancing the MAO-B inhibition
potency of the C5 substituted phthalimide analogues discussed
above, the phenyl rings of the C5 substituent of selected homo-
logues (5a–c) were substituted with bromine. Bromine was se-
lected since previous studies have shown that halogen
substitution, and in particular bromine substitution, on the phenyl
ring of the C8 substituent of benzyloxycaffeine34 and styrylcaf-
feine38 derivatives enhances the MAO-B inhibition potencies of
these compounds by several orders of a magnitude. In accordance
with this view, phthalimide analogue 5i, the bromine substituted
homologue of 5a exhibited an IC50 value for the inhibition of
exhibited IC50 values of 5.85, 4.17, 3.58 and 1.73
Interestingly, the phenylpropenyloxy substituted analogue 5e
(IC50 = 8.99 M) was found to be the weakest MAO-A inhibitor
lM, respectively.
l
among the phthalimides. In a previous study it was found that a
C8 substituent with a relatively large degree of flexibility is a
requirement for caffeine analogues to act as MAO-A inhibitors.
For example, 8-benzyloxycaffeine (9)34 and some of its analogues
are inhibitors of both MAO-A and -B, while (E)-8-styrylcaffeine
(10)38,39 is a MAO-B selective inhibitor with no observed affinity
for MAO-A (Fig. 4). Modeling studies have suggested that the abil-
ity of the 8-benzyloxycaffeinyl analogues to bind to the MAO-A ac-
tive site may, to a large degree, depend on the relatively large
degree of rotational freedom of the benzyloxy side chain at the car-
bon-oxygen ether bond.34 In contrast, the more conformationally
rigid (E)-styryl side chain is less well accommodated by the
MAO-A active site. A possible reason for the relatively weaker
MAO-A inhibition potency of 5e may therefore be that, due to
the ethenyl double bond, its phenylpropenyloxy side chain is less
flexible than that of the other phthalimides examined here, and
MAO-B of 0.055 lM, 45-fold more potent than 5a (IC50 = 2.49 lM).
Phthalimide analogue 5g, the bromine substituted homologue of
5b was found to be the most potent MAO-B inhibitor of the present
series with an IC50 value of 0.0069
(IC50 = 0.043 M). Noteworthy is the observation that compound
5c (IC50 = 0.017 M) is approximately fourfold more potent as an
MAO-B inhibitor than its bromine substituted homologue, 5h
(IC50 = 0.067 M). These results indicate that while halogen substi-
lM, sixfold more potent than 5b
l
l
l
tution on the phenyl ring of the C5 side chain enhances the MAO-B
inhibition potencies of weaker phthalimide inhibitors (5a) to a
large degree, its effects on the MAO-B inhibition potencies of the
more potent phthalimide analogues (5b, 5c) are less remarkable.

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
4833
Table 2
The IC50 values and calculated Ki values for the inhibition of recombinant human MAO-A and -B by phthalimide analogues 5a–ia
O
R
NH
O
R
IC50
(
lM)
Kib
(
lM)
SIc
MAO-A
MAO-B
MAO-A
MAO-B
5a
5b
5c
5d
5e
5f
5g
5h
5i
1
C6H5O
C6H5CH2O
C6H5CH2CH2O
C6H5(CH2)3O
5.85 0.203
4.17 2.082
3.58 0.461
1.73 0.364
8.99 0.130
0.92 0.024
0.22 0.031
1.08 0.160
1.08 0.316
31.8
2.49 0.0057
0.043 0.0081
0.017 0.0026
0.050 0.0093
0.046 0.0053
0.033 0.0040
0.0069 0.0003
0.067 0.015
0.055 0.017
12.4
1.54
1.1
0.94
0.46
2.37
0.2
0.058
0.285
0.285
8.38
1.22
19.1
1.073
0.019
0.007
0.02
0.020
0.01
0.003
0.029
0.024
5.34
0.044
0.059
1
59
129
21
120
17
20
10
12
1.6
(E)-C6H5CH@CHCH2O
2-Naphthalenyl-O
4-BrC6H4CH2O
4-BrC6H4CH2CH2O
4-BrC6H4O
Isatind
2
3
5-Benzyloxyisatind
6-Benzyloxyisatind
4.62
72.4
0.103
0.138
27
321
a
All values are expressed as the mean SD of duplicate determinations.
The Ki values were calculated from the experimentally measured IC50 values according to the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 45
b
lM and
Km (kynuramine) = 16.1
l
M for human MAO-A and [S] = 30 M and Km (kynuramine) = 22.7
l
l
M for human MAO-B.34,36
c
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of Ki(MAO-A)/Ki(MAO-B).
Inhibition values for these compounds obtained from Ref. 29.
d
Table 3
O
The inhibition of recombinant human MAO-A and -B by phthalimide (4) and N-
substituted phthalimide analogues (6a and 6b)
N
N
N
O
8
N
O
O
O
N
O
9
N
R
N
O
R
IC50
MAO-A
165 12.5a
10.5 3.64%c
No inhibitiond
(l
M) or %inhibitionb,c
a,b
8
N
N
MAO-B
10
4
H
134 1.63a
6a
6b
C6H5
C6H5CH2
50.3 0.38%c
20.2 5.09%c
Figure 4. The structures of 8-benzyloxycaffeine (9) and (E)-8-styrylcaffeine (10).
a
b
c
The IC50 value is reported for this compound.
All values are expressed as the mean SD of duplicate determinations.
The %inhibition at a maximum tested concentration of 100 M.
No inhibition observed at a maximum tested concentration of 100
2.3. Reversibility studies
l
d
lM.
As discussed in the Introduction, from a therapeutic point of
view, reversible inhibition of the MAO isoforms may have signif-
icant advantages over the inactivation of these enzymes. Isatin
and C5- and C6-substituted isatin analogues have previously
been shown to interact reversibly with human MAO-A and -
B.28,29 The phthalimide analogues that were investigated in this
study were similarly evaluated for their ability to interact revers-
ibly with MAO-A and -B. For this purpose the time dependence
of the inhibition of human MAO-A and -B by one representative
inhibitor, phthalimide 5a, was evaluated. The concentrations of
5a that were selected for these studies were 13.42 and
therefore the least suited for binding to MAO-A. Possible explana-
tions for this behavior will be explored in more detail below using
molecular docking.
As observed for the MAO-B inhibition potencies, the C5 substit-
uents of the phthalimides are important structural features for
their observed MAO-A inhibition activities. This is illustrated by
the finding that phthalimide (4) is a weak MAO-A inhibitor with
an IC50 value of 165 lM (Table 3). This is approximately 18-fold
weaker than the MAO-A inhibition potency of the weakest C5
substituted phthalimide analogue of the studied series, compound
5.00 lM for the incubations with MAO-A and -B, respectively.
5e (IC50 = 8.99
l
M). The significance of the positioning of the sub-
These concentrations are approximately twofold the measured
IC50 values for the inhibition of the respective enzymes by 5a.
Compound 5a, at these concentrations, and recombinant human
MAO-A or -B were preincubated for periods of 0, 15, 30 and
60 min. Following the addition of the MAO-A/B mixed substrate,
kynuramine, the residual catalytic activities of MAO-A and -B
were measured.
stituent at C5 of the phthalimide ring was also demonstrated for
the inhibition of MAO-A by the finding that the N-substituted iso-
mers were not MAO-A inhibitors (6b) or very weak inhibitors (6a),
even at high concentrations (Table 3). Therefore, as for MAO-B
inhibition, a substituent at C5 is a critical feature for the MAO-A
inhibition activity of phthalimide.

4834
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
The results of the time dependent studies are presented in Fig-
time points and that the effect of hydrolysis on the measured
IC50 values should be small.
ure 5. As shown by the graphs, there are no time-dependent reduc-
tions in the catalytic rates of MAO-A and -B when phthalimide 5a is
preincubated with the enzyme preparations, even after a period of
60 min. These results lead to the conclusion that the inhibition of
MAO-A and -B by 5a is not time dependent and therefore revers-
ible, at least for the time period (0–60 min) and at the inhibitor
concentrations (2 Â IC50) evaluated. Interestingly, small but signif-
icant increases of the rates of the MAO-A and -B catalyzed oxida-
tions of kynuramine with increased preincubation time of 5a
with the enzyme preparations are observed. A possible explanation
for this behavior may be that phthalimide 5a may slowly hydrolyze
in the aqueous buffer (potassium phosphate 100 mM, pH 7.4) used
in this study for the enzyme incubations. As a result, the concentra-
tion of the active phthalimide inhibitor may slowly decrease which
results in a lesser degree of inhibition and therefore an increase in
enzyme catalytic rate. This possibility is supported by literature re-
ports that phthalimides undergo hydrolytic C–N bond fission to
yield the ring-opened phthalamic acids.40 In this study an incuba-
tion time of 20 min was selected for the determination of the
inhibition potencies of the phthalimide analogues (Table 2).
Considering that for 5a only a small loss of inhibition potency is
observed between the 15 min and 30 min time points, it may be
concluded that relatively little hydrolysis has occurred at these
To provide further support for the finding that 5a is a reversible
inhibitor of both MAO-A and -B, the mode of enzyme inhibition
was examined. For this purpose, sets of Lineweaver–Burk plots
were constructed for the inhibition of MAO-A as well as MAO-B
by inhibitor 5a. The initial rates of the MAO-A or -B catalyzed oxi-
dation of kynuramine at four different substrate concentrations
(15–90
concentrations of inhibitor 5a. For the studies with MAO-A the
concentrations of inhibitor 5a were 1.7–6.8 M while for the stud-
ies with MAO-B the concentrations of inhibitor 5a were 0.625–
2.5 M. The Lineweaver–Burk plots so obtained are presented in
lM) were recorded in the absence and presence of various
l
l
Figure 6. Inspection of these results reveals that for the inhibition
of both MAO-A and -B by 5a, the plots are linear and intersect at
the y-axis. This suggests that the mode of inhibition is competitive
and provide further support that 5a interacts reversibly with the
active sites of human MAO-A and -B.
2.4. Molecular modeling studies
The results of this study show that C5 substituted phthalimide
analogues 5a–i are inhibitors of recombinant human MAO-A and -
B. An important observation was the critical importance of the C5
substituent for the MAO-A as well as MAO-B inhibition activities of
the phthalimides. This was best demonstrated by the finding that
phthalimide (4) and N-substituted phthalimides (6a and 6b) are
weak MAO-A and -B inhibitors and in some instances were found
not to interact with the enzymes, even at high concentrations. Also
of importance was the observation that the phenylpropenyloxy
substituted analogue 5e was the weakest MAO-A inhibitor among
the phthalimides evaluated while exhibiting potent MAO-B inhibi-
tion activity. A possible explanation for this behavior may be that
due to the presence of the ethenyl double bond, the phenylprope-
nyloxy side chain of 5e is less flexible than that of the other C5
phthalimide side chains investigated, and therefore the least suited
for binding to MAO-A. Literature suggests that at least for C8-
substituted caffeines, a large degree of rotational freedom of the
C8 side chain is a requirement for MAO-A inhibition.34 To provide
additional insight, the binding modes of selected phthalimides (5e
and 6a) in the active site cavities of MAO-A and -B were examined
using molecular docking.
A
20
16
12
8
4
0
0
15
30
60
Incubation time (min)
The molecular docking experiments were carried out with the
Discovery Studio 1.7 modeling software according to a previously
reported protocol.29,34 As enzymatic models, the crystallographic
structures of human MAO-A in complex with harmine (PDB entry:
2Z5X)41 and human MAO-B in complex with safinamide (PDB en-
try: 2V5Z)42 were selected. These models were selected based on
the high resolution of the crystallographic structures and are fre-
quently used in modeling studies.29,34 The enzymatic models were
prepared as described previously.29,34 The valences of the FAD
cofactor and the co-crystallized ligands (harmine and safinamide)
were corrected, hydrogen atoms were added to the enzymatic
models and the models were subjected to a three-step energy min-
imization cascade while the backbones of the enzymes were con-
strained. This was followed by the deletion of the co-crystallized
ligands from the models and the detection of the binding cavities
by the flood-filling algorithm. All crystal water molecules were re-
moved from the enzymatic models except for those in the MAO-A
and -B active sites that are considered conserved and non-displace-
able (see Experimental). The structures of the phthalimide inhibi-
tors (5e and 6a) were constructed and their geometries
optimized within Discovery Studio and were subsequently docked
into the active sites of the MAO-A and -B models using the Ligand-
Fit application within Discovery Studio. This protocol has been
shown to be suitable for examining the binding of inhibitors to
B
3
2
1
0
0
15
30
60
Incubation time (min)
Figure 5. Time-dependant inhibition of recombinant human MAO-A (Panel A) and
recombinant human MAO-B (Panel B) by phthalimide analogue 5a. The enzymes
were preincubated for various periods of time (0–60 min) with 5a at concentrations
of 13.42
lM and 5.00
l
M for MAO-A and MAO-B, respectively. The concentrations
of the enzyme substrate, kynuramine, were 45 and 30
MAO-A and MAO-B, respectively. The catalytic rates are expressed as nmoles 4-
hydroxyquinoline formed/min/mg protein.
l
M for the studies with

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
4835
A
0.15
0.12
0.09
0.06
0.03
0.00
-0.02
0.00
0.02
1/[S]
0.04
0.06
B
0.8
0.4
0.0
Figure 7. The predicted binding orientation of 5e (orange colored) in the active site
of MAO-B (2V5Z.pdb).
as with nitrogen N5 of the flavin. Also of significance is the obser-
vation that the phthalimide ring is orientated approximately paral-
lel to the phenyl rings of the Tyr-398 and Tyr-435 side chains. This
orientation is restricted by the flat shape of the substrate cavity of
MAO-B43 and places the plane of the phthalimide ring also approx-
imately parallel to the plane of the amide functional group of the
Gln-206 side chain. With an interplane distance of approximately
-0.02
0.00
0.02
1/[S]
0.04
0.06
Figure 6. Lineweaver–Burk plots of the recombinant human MAO-A (Panel A) and
recombinant human MAO-B (Panel B) catalyzed oxidation of kynuramine in the
absence (filled squares) and presence of various concentrations of 5a. For the
studies with MAO-A (Panel A) the concentrations of 5a were. 1.7
squares), 3.4 M (filled circles), 6.8 M (open circles). For the studies with MAO-B
(Panel B) the concentrations of 5a were. 0.625 M (open squares), 1.25 M (filled
circles), 2.5 M (open circles). The rates (V) are expressed as nmol product formed/
lM (open
l
l
3.6 Å, a possible
p–p interaction between these two functional
l
l
groups may be possible.41 Also of importance is the observation
that the phthalimide imidic N binds in the space where the amino
groups of MAO-B substrates are thought to bind,44 3.3 Å from the
C4a of the FAD cofactor. Modeling studies have suggested that
the amine N of the MAO-B selective substrate, benzylamine, binds
3.7 Å from the C4a of the flavin.44 As mentioned above, the C5 side
chain of 5e extends beyond the boundary of the substrate cavity,
which is defined by the side chain of Ile-199, into entrance cavity
of the MAO-B active site. Within the hydrophobic environment of
the entrance cavity, the phenyl ring of the C5 side chain is most
likely stabilized by Van der Waals interactions.45 This binding ori-
entation is similar to that observed for the co-crystallized inhibitor,
safinamide, which also traverses both active site cavities.42 Other
relatively large inhibitors such as trans,trans-farnesol26 and 1,4-di-
phenyl-2-butene27 are also reported to span both the substrate and
entrance cavities of the MAO-B active site. For these orientations to
be possible, the side chain of Ile-199 should be rotated out of the
normal conformation to allow for the fusion of the two cavities.26
The importance of the interactions between the C5 side chain
and the entrance cavity for stabilization of the phthalimide inhib-
itors may be demonstrated by the observation that phthalimide (4)
is a very weak MAO-B inhibitor. Being a relatively small molecule
compared to the C5 substituted derivatives, phthalimide is ex-
pected to bind only to the substrate cavity, leaving the entrance
cavity unoccupied. As a result, phthalimide does not undergo sta-
bilizing interactions with the entrance cavity and is therefore a
weak MAO-B inhibitor. The interaction of the C5 side chain of the
l
min/mg protein.
MAO-A and -B since redocking of harmine (RMSD = 0.64 Å) and
safinamide (RMSD = 1.54 Å) into the active sites of the two en-
zymes, respectively, yielded binding orientations that exhibited
relatively small RMSD values from the position of the co-crystal-
lized ligands.29
The predicted binding orientation of compound 5e within the
active site model of MAO-B is displayed in Figure 7. In the MAO-
B active site, the inhibitor adopts an orientation that positions
the phthalimide ring within the substrate cavity of the enzyme
while the C5 side chain extends towards the entrance cavity. This
orientation is observed for all of the C5 substituted phthalimide
analogues examined in this study (5a–i) (results shown for 5e
only). This binding mode places the phthalimide ring in close prox-
imity to the flavin ring with the phthalimide NH proton located
only 2.2 Å from N5 of the flavin. This region, the re side of the
FAD cofactor, of the substrate cavity is considered polar and at least
4 potential hydrogen bond interactions may occur between the
phthalimide ring of 5e and the FAD cofactor and active site water
molecules in this region. These include interactions between the
carbonyl oxygen at C1 of the phthalimide ring and the integral
water molecules HOH-1155 and HOH-1351. Potential hydrogen
bond interactions may also occur between the phthalimide NH
proton and the carbonyl oxygen at C4 of the FAD cofactor as well

4836
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
phthalimide derivatives (5a–i) with the entrance cavity may also
serve to position the phthalimide ring for optimal interaction with
the polar region, the space in close proximity to the flavin. This
view is supported by the observation that even though phthali-
mide (4) may interact with the polar region of the substrate cavity
to a similar extent compared to the C5 substituted derivatives, it
still possesses weak MAO-B inhibition activity. Further support
for the view that a dual mode of binding to both the substrate
and entrance cavities of MAO-B is crucial for the potent MAO-B
inhibition activity of the phthalimide derivatives may be obtained
by examining the predicted binding orientation of the N-substi-
tuted phthalimide, 6a, in the MAO-B active site model. As shown
in Figure 8, compound 6a is orientated, similar to 5e, with the
phthalimide ring directed towards the FAD cofactor while the N-
substituent extends towards the entrance cavity of the enzyme.
In contrast to the orientation observed for 5e, the phthalimide ring
of 6a is located relatively distant from the polar region of the sub-
strate cavity, 4.4 Å from from N5 of the flavin. As a result, no poten-
tial polar interactions between the phthalimide ring of 6a and the
polar functional groups of the substrate cavity of MAO-B are ob-
served. The lack of stabilizing polar interactions between 6a and
the substrate cavity may explain the weak MAO-B inhibition activ-
ity of 6a.
To examine possible reasons for the finding that the phthali-
mide derivatives are in general MAO-B selective inhibitors, poten-
tial binding orientations of compound 5e within the MAO-A model
was also calculated. As shown in Figure 9, the phthalimide ring of
5e is orientated towards the FAD cofactor of MAO-A with the C5
side chain positioned in the region leading to the entrance of the
active site. In contrast to its predicted orientation in MAO-B, 5e
binds more distant from the FAD cofactor, 3.2 Å from N5 of the fla-
vin. In spite of this, compound 5e is within hydrogen bond contact
distance to several polar functional groups of the MAO-A active
site. The carbonyl oxygen at C1 of the phthalimide ring may form
hydrogen bonds with an active site water molecule (HOH-710)
Figure 9. The predicted binding orientation of 5e (cyan colored) in the active site of
MAO-A (2Z5X.pdb).
while the phthalimide NH proton may form possible hydrogen
bonds with the phenolic hydrogen of Tyr-444 and a water mole-
cule (HOH-739). A possible
p–p interaction between the phthali-
mide ring and the amide functional group of the Gln-215 with an
interplane distance of approximately 3.4 Å may also occur. Inter-
estingly, 5e is bound in a bent conformation with the C5 side chain
bent by about a 50° angle from the plane of the caffeinyl ring
(Fig. 10). Compound 5e exists in this conformation in the MAO-A
active site in order to avoid unfavorable interactions with the side
chain of Phe-208. In the MAO-B active site, the residue correspond-
ing to Phe-208 in MAO-A, is Ile-199. The increased size of the Phe
aromatic ring compared to the side chain of Ile prevents the side
chain of 5e from occupying an extended conformation and the
binding mode of 5e observed in MAO-B would not be possible in
the MAO-A active site.26,41 As shown in Figure 11, if 5e were to
adopt the same orientation that is observed when it is bound to
MAO-B, the C5 side chain would partially overlap with Phe-208
in the MAO-A active site. The relatively lower degree of flexibility
of the phenylpropenyloxy side chain of 5e may make it less ame-
Figure 10. The docked orientations of 5e within the active site of MAO-B (cyan
colored) and MAO-A (green) with the phthalimide rings of the two respective
orientations superimposed.
Figure 8. The predicted binding orientation of 6a (orange colored) in the active site
of MAO-B (2V5Z.pdb).

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
4837
observed with increasing length or size of the C5 substituent and
halogen substitution on the aromatic ring of the C5 side chain. Both
these structural modifications are expected to lead to more pro-
ductive Van der Waals interactions between the C5 side chain
and the entrance cavity. Since the phenyl ring of the C5 side chain
is most likely stabilized to a large degree by Van der Waals inter-
actions within the entrance cavity, halogen substitution on the
phenyl ring (5g–i) is expected to enhance the productive interac-
tions of the inhibitor with the entrance cavity of MAO-B via hydro-
phobic burial and dipole interactions. The enhancement of MAO-B
inhibition potencies by halogen substitution has been previously
reported.34 For example, chlorine and bromine substitution on
the benzyloxy phenyl ring of a series of 8-benzyloxycaffeines en-
hances the MAO-B inhibition potencies 20- and 22-fold, respec-
tively.34 Modeling studies suggest that this effect of halogen
substitution is also dependent on the enhancement of productive
hydrophobic and dipole interactions between the benzyloxy side
chain and the MAO-B entrance cavity.34
The C5 substituted phthalimide analogues 5a–i were also
reversible inhibitors of MAO-A. With the exception of 5a which
is non-selective, the phthalimide analogues are however MAO-B
selective inhibitors. Interestingly, similar to the results obtained
with MAO-B, increasing the C5 side chain length and substitution
with halogens on the aromatic ring of the C5 side chain also en-
hanced the MAO-A inhibition activities of the phthalimide ana-
logues, although to a lesser extent as observed for MAO-B. These
results suggest that the C5 side chain undergoes significant pro-
ductive interactions with the MAO-A active site. The importance
of the C5 side chain for binding to MAO-A was further demon-
strated by the finding that phthalimide (4) is a weak MAO-A inhib-
itor. The findings that the N-substituted phthalimides are not
MAO-A inhibitors (6b) or very weak inhibitors (6a) demonstrate
that, as for MAO-B, the position of the side chain is an important
consideration. Based on the analysis of possible binding modes of
5e within the MAO-A active site it is proposed that relatively rigid
C5 side chains are less suitable for adopting orientations that avoid
unfavorable interactions with the MAO-A active site residues and
inhibitors containing such side chains are therefore expected to
possess lower affinities for MAO-A. Modeling studies suggests that
although several polar interactions are possible between the
phthalimide ring of the C5 substituted phthalimide analogues
and the active site of MAO-A, the phthalimide analogues, do not
bind in close proximity to the FAD cofactor. Since the phthalimide
rings of these inhibitors are positioned very close to the flavin
when binding in MAO-B, these results suggest that inhibitor bind-
ing in close proximity to the FAD cofactor is more favorable than
binding more distant. These differing binding positions adopted
by the inhibitors in MAO-A and -B may explain the observed higher
binding affinities for the MAO-B active site.
Figure 11. Illustration of the overlaid active sites of human MAO-A and -B. The
binding orientation of compound 5e (orange colored) as docked within the active
site of MAO-B is shown here in the MAO-A active site. The active site residues of
MAO-A are displayed in gray. Residue Ile-199 in MAO-B is displayed in magenta
while residue Phe-208 in MAO-A is displayed in cyan.
nable for adopting orientations that avoid unfavorable interactions
with the MAO-A active site residues. This may explain its lower
binding affinity to MAO-A. Although 5e is predicted to undergo
several polar interactions with the active site of MAO-A, the results
of the inhibition studies shows that it is approximately 120-fold
more potent as an MAO-B inhibitor than an MAO-A inhibitor. This
suggests that inhibitors that bind in close proximity to the FAD
cofactor of MAO-A and -B would exhibit improved binding affini-
ties compared to inhibitors that bind more distant.
3. Conclusion
In the present study, a series of C5 substituted phthalimide ana-
logues 5a–i were synthesized and evaluated as inhibitors of recom-
binant human MAO-A and -B. The results demonstrate that the
analogues are exceptionally potent reversible MAO-B inhibitors
with most analogues exhibiting IC50 values in the lower nM range.
Analysis of the structure-activity relationships (SAR) reveals that
increasing the length or size of the C5 substituent enhances the
MAO-B inhibition potencies of the phthalimide analogues. Halogen
substitution on the ring system of the C5 side chain also enhances
MAO-B inhibition potency, particularly of the weaker phthalimide
inhibitors. Importantly, the position of the substituent at C5 is an
important structural feature since N-substituted phthalimides
(6a and 6b) are weak MAO-B inhibitors. Further evidence for the
importance of the C5 substituent for binding to MAO-B is provided
by the finding that phthalimide (4) is a weak MAO-B inhibitor.
Modeling studies suggest that both polar interactions between
the phthalimide ring and the polar region of the MAO-B substrate
cavity and Van der Waals interactions between the C5 side chain
and the entrance cavity are required for high affinity binding.45
The view that interactions between the C5 side chain and the en-
trance cavity are important for inhibitor binding is supported by
the finding that an enhancement of MAO-B inhibition activity is
In conclusion, C5 substituted phthalimides exhibit high binding
affinities for MAO-B and are therefore promising lead compounds
for the design of novel selective reversible inhibitors of MAO-B.
In addition, certain analogues such as 5g, which also possesses po-
tent MAO-A inhibition activities, may act as lead compounds for
the design of non-selective reversible MAO-A/B inhibitors.
4. Experimental section
4.1. Chemicals and instrumentation
Unless differently stated, all reagents and starting materials
were obtained from Sigma–Aldrich and were used without further
purification. Proton (1H) and carbon (13C) NMR spectra were re-
corded in DMSO-d6 with a Bruker Avance III 600 spectrometer at
frequencies of 600 MHz and 150 MHz, respectively. The NMR

4838
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
chemical shifts are reported in parts per million (d) downfield from
the signal of tetramethylsilane added to the deuterated solvent and
spin multiplicities are given as s (singlet), d (doublet), dd (doublet
of doublets), t (triplet) or m (multiplet). Mass spectra (MS) and
high resolution mass spectra (HRMS) were obtained on a DFS high
resolution magnetic sector mass spectrometer (Thermo Electron
Corporation) in atmospheric pressure chemical ionization (APCI)
mode. Melting points (mp) were determined on a Stuart SMP10
melting point apparatus and are uncorrected. The degrees of purity
of the target compounds were estimated by HPLC analyses using
an Agilent 1200 HPLC system equipped with a quaternary pump
and an Agilent 1200 series diode array detector (see Supplemen-
tary data). HPLC grade acetonitrile (Merck) and Milli-Q water (Mil-
lipore) were used for the chromatography. A Varian Cary Eclipse
fluorescence spectrophotometer was used for all fluorescence mea-
surements. KynuramineÁ2HBr, 4-hydroxyquinoline and micro-
somes from insect cells containing recombinant human MAO-A
and -B (5 mg/mL) were obtained from Sigma–Aldrich.
J = 7.9 Hz), 4.58 (t, 2H, J = 6.0 Hz), 7.64 (t, 1H, J = 7.2 Hz), 7.68 (d,
2H, J = 7.5 Hz), 7.74 (m, 4H), 8.17 (d, 1H, J = 9.0 Hz), 11.63 (s, 1H);
13C NMR (600 MHz, DMSO-d6) d 30.1, 31.4, 67.9, 108.3, 120.3,
124.4, 124.8, 125.9, 128.3, 128.4, 135.3, 141.2 163.7, 168.8,
168.9; APCI-MS 281; APCI-HRMS m/z: calcd 281.1052, found
281.1041; Purity (HPLC): 97.4%.
4.2.5. 5-{[(2E)-3-Phenylprop-2-en-1-yl]oxy}phthalimide (5e)
The title compound was prepared from 4-{[(2E)-3-phenylprop-
2-en-1-yl]oxy}phthalonitrile (7e) in a yield of 45%: mp 181–183 °C
(ethanol). 1H NMR (600 MHz, DMSO-d6) d 4.88 (d, 2H, J = 5.7 Hz),
6.50 (m, 1H), 6.78 (d, 1H, J = 15.8 Hz), 7.26 (t, 1H, J = 7.2 Hz), 7.32
(m, 4H), 7.47 (d, 2H, J = 7.2 Hz), 7.72 (d, 1H, J = 7.9 Hz), 11.18 (s,
1H); 13C NMR (600 MHz, DMSO-d6) d 69.0, 108.6, 120.5, 124.0,
124.6, 124.8, 126.5, 128.1, 128.7, 133.1, 135.2, 136.0, 163.3,
168.8, 168.9; APCI-MS 279; APCI-HRMS m/z: calcd 279.0895, found
279.0883; Purity (HPLC): 97.4%.
4.2.6. 5-(Naphthalen-2-yloxy)phthalimide (5f)
4.2. Synthesis of C5-substituted phthalimide analogues (5a–i)
The title compound was prepared from 4-(naphthalen-2-
yloxy)phthalonitrile (7f) in a yield of 42%: mp 205–209 °C (etha-
nol). 1H NMR (600 MHz, DMSO-d6) d 7.28 (d, 1H, J = 1.9 Hz), 7.37
(dd, 1H, J = 2.3, 9.0 Hz), 7.41 (dd, 1H, J = 2.3, 8.3 Hz), 7.53 (m, 2H),
7.65 (d, 1H, J = 2.3 Hz), 7.83 (d, 1H, J = 7.9 Hz), 7.89 (d, 1H,
J = 7.9 Hz), 7.97 (d, 1H, J = 7.9 Hz), 8.05 (d, 1H, J = 9.0 Hz), 11.32
(s, 1H); 13C NMR (600 MHz, DMSO-d6) d 111.5, 116.2, 120.2,
122.9, 125.4, 125.7, 126.7, 127.0, 127.4, 127.8, 130.6, 130.7,
133.9, 135.3, 152.5, 162.5, 168.4, 168.6; APCI-MS 289; APCI-HRMS
m/z: calcd 289.0739, found 289.0730; Purity (HPLC): 99.6%.
The C5-substituted phthalimide analogues (5a–i) investigated
in this study were synthesized according to the literature descrip-
tion.30 The appropriate C4-substituted phthalonitrile analogue
(7a–i) (7 mmol) was added at 15 °C to a solution of sodium ethox-
ide (prepared from 0.25 g sodium and 15 mL ethanol). The reaction
mixture was stirred at 15 °C for a period of 2 h and poured into
25 mL nitric acid (10%). The mixture was stirred for 30 min and
the resulting precipitate was collected by filtration, washed with
150 mL water and dried. The material was purified by at least
two successive rounds of recrystallization from ethanol.
4.2.7. 5-(4-Bromobenzyloxy)phthalimide (5g)
The title compound was prepared from 4-(4-bromobenzyl-
oxy)phthalonitrile (2g) in a yield of 60%: mp 249–251 °C (ethanol).
1H NMR (600 MHz, DMSO-d6) d 5.26 (s, 2H), 7.35 (d, 1H, J = 8.7 Hz),
7.38 (s, 1H), 7.43 (d, 2H, J = 7.9 Hz), 7.59 (d, 2H, J = 7.9 Hz), 7.73 (d,
1H, J = 8.3 Hz), 11.19 (s, 1H); 13C NMR (600 MHz, DMSO-d6) d 69.3,
108.6, 120.7, 121.3, 124.8, 130.0, 131.5, 135.2, 135.6, 163.1, 168.8,
168.9; APCI-MS 330; APCI-HRMS m/z: calcd 330.9844, found
330.9897; Purity (HPLC): 98.6%.
4.2.1. 5-Phenoxyphthalimide (5a)
The title compound was prepared from 4-phenoxyphthalonitri-
le (7a) in a yield of 24%: mp 171–173 °C (ethanol). 1H NMR
(600 MHz, DMSO-d6) d 7.18 (m, 3H), 7.29 (t, 1H, J = 7.53 Hz), 7.33
(dd, 1H, J = 2.3, 8.3 Hz), 7.49 (t, 2H, J = 7.9 Hz), 7.81 (d, 1H,
J = 7.91 Hz), 11.26 (s, 1H); 13C NMR (600 MHz, DMSO-d6) d 111.0,
120.3, 122.6, 125.3, 126.5, 130.6, 135.3, 154.7, 162.6, 168.4,
168.6; APCI-MS 239; APCI-HRMS m/z: calcd 239.0582, found
239.0580; Purity (HPLC): 99.6%.
4.2.8. 5-[2-(4-Bromophenyl)ethoxy]phthalimide (5h)
The title compound was prepared from 4-[2-(4-bromophe-
nyl)ethoxy]phthalonitrile (7h) in a yield of 81%: mp 220 °C (etha-
nol). 1H NMR (600 MHz, DMSO-d6) d 3.05 (t, 2H, J = 6.4 Hz), 4.36
(t, 2H, J = 6.4 Hz), 7.27 (d, 1H, J = 8.3 Hz), 7.30 (m, 3H), 7.49 (d,
2H, J = 7.5 Hz), 7.71 (d, 1H, 8.3 Hz), 11.18 (s, 1H); 13C NMR
(600 MHz, DMSO-d6) d 34.0, 68.8, 108.4, 119.6, 120.3, 124.5,
124.8, 131.2, 131.3, 135.3, 137.6, 163.4, 168.8, 168.9; APCI-MS
345, 347; APCI-HRMS m/z: calcd 345.0001, found 345.0001; Purity
(HPLC): 97.2%.
4.2.2. 5-Benzyloxyphthalimide (5b)
The title compound was prepared from 4-benzyloxyphthalonit-
rile (7b) in a yield of 84%: mp 159–161 °C (ethanol). 1H NMR
(600 MHz, DMSO-d6) d 5.28 (s, 2H), 7.36 (m, 2H), 7.40 (m, 3H),
7.47 (d, 2H, J = 7.5 Hz), 7.74 (d, 1H, J = 8.3 Hz), 11.18 (s, 1H); 13C
NMR (600 MHz, DMSO-d6) d 70.1, 108.6, 120.7, 124.6, 124.8,
127.8, 128.1, 128.5, 135.2, 136.1, 163.3, 168.8, 168.9; APCI-MS
253; APCI-HRMS m/z: calcd 253.0739, found 253.0743; Purity
(HPLC): 99.8%.
4.2.9. 5-(4-Bromophenoxy)phthalimide (5i)
4.2.3. 5-(2-Phenylethoxy)phthalimide (5c)
The title compound was prepared from 4-(4-bromophen-
oxy)phthalonitrile (7i) in a yield of 48%: mp 204–206 °C (ethanol).
1H NMR (600 MHz, DMSO-d6) d 7.14 (d, 2H, J = 8.7 Hz), 7.26 (d, 1H,
J = 1.9 Hz), 7.36 (dd, 1H, J = 2.3, 8.3 Hz), 7.65 (d, 2H, J = 8.7 Hz), 7.82
(d. 1H, J = 8.3 Hz), 11.33 (s, 1H); 13C NMR (600 MHz, DMSO-d6) d
111.6, 117.1, 122.3, 123.0, 125.4, 127.0, 133.3, 135.3, 154.3,
161.9, 168.4, 168.6; APCI-MS 317, 319; APCI-HRMS m/z: calcd
316.9688, found 316.9685; Purity (HPLC): 99.8%.
The title compound was prepared from 4-(2-phenylethoxy)pht-
halonitrile (7c) in a yield of 10%: mp 167–169 °C (ethanol). 1H NMR
(600 MHz, DMSO-d6)
d 3.06 (t, 2H, J = 6.8 Hz), 4.36 (t, 2H,
J = 6.8 Hz), 7.21 (t, 1H, J = 7.2 Hz), 7.31 (m, 6H), 7.70 (d, 1H,
J = 8.3 Hz), 11.16 (s, 1H); 13C NMR (600 MHz, DMSO-d6) d 34.6,
69.1, 108.4, 120.3, 124.5, 124.8, 126.4, 128.4, 129.0, 135.3, 138.0,
163.5, 168.8, 168.9; APCI-MS 267; APCI-HRMS m/z: calcd
267.0895, found 267.0891; Purity (HPLC): 99.9%.
4.3. Synthesis of N-substituted phthalimide analogues (6a and
6b)
4.2.4. 5-(3-Phenylpropoxy)phthalimide (5d)
The title compound was prepared from 4-(3-phenylprop-
oxy)phthalonitrile (7d) in a yield of 15%: mp 147–149 °C (ethanol).
The N-substituted phthalimide analogues (6a and 6b) investi-
gated in this study were synthesized according to the published
1H NMR (600 MHz, DMSO-d6)
d
2.50 (m, 2H), 3.20 (t, 2H,

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
4839
procedure.32 A mixture of the appropriate amine (12 mmol) and
phthalic anhydride (8; 10 mmol) were heated under reflux in 1 M
acetic acid (20 mL) for 5 h. The reaction was allowed to cool to
room temperature, water (100 mL) was added and the precipitated
product was collected by filtration. The target N-substituted
phthalimide analogues were recrystallized from 95% ethanol. The
recorded melting points of 6a and 6b were 207–209 °C and 215–
217 °C, respectively, while the reported melting points are 209–
211 °C32 and 215 °C,46 respectively.
the final concentrations of 5a were 6.71
l
M and 2.5
l
M for MAO-A
and MAO-B, respectively. These concentrations of 5a are approxi-
mately equal to the IC50 values for the inhibition of the respective
enzyme preparations. The reactions were incubated for a further
15 min at 37 °C and subsequently terminated with 400
lL NaOH
(2 N). A volume of 1000 L distilled water was added to each reac-
l
tion and the rates of the MAO-catalyzed production of 4-hydroxy-
quinoline were measured as described above. All measurements
were carried out in triplicate and are expressed as mean SD.29,34
4.4. The determination of IC50 values for the inhibition of
human MAO-A and -B
4.6. Construction of Lineweaver–Burk plots
To evaluate the mode of MAO-A and -B inhibition, sets of
Lineweaver–Burk plots for the inhibition of these enzymes by com-
pound 5a were constructed. By employing four different concen-
Microsomes (5 mg/mL) from baculovirus infected insect cells
expressing recombinant human MAO-A or -B were obtained from
Sigma-Aldrich and were pre-aliquoted (33
l
L) and stored at
trations of the substrate, kynuramine (15, 30, 60 and 90 lM), the
À70 °C. All enzymatic reactions were carried out in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl,
20.2 mM) in microcentrifuge tubes (2 mL). The volumes of the
initial catalytic rates of the recombinant human enzymes were
measured in the absence and presence of three different concen-
trations of 5a. For the studies with MAO-A the concentrations of
reactions were 500
lL and the concentrations of MAO-A or MAO-
5a were 1.7, 3.4 and 6.8
lM. For the studies with MAO-B the con-
B in the reactions were 0.0075 mg/mL. The stock solutions of the
test inhibitors were prepared in DMSO and were added to the reac-
tions to yield concentrations of the inhibitors of 0–100 lM and a
concentration of 4% (v/v) DMSO. The MAO-A/B mixed substrate,
kynuramine, was added to the reactions to yield final concentra-
centrations of 5a were 0.625, 1.25 and 2.5
l
M. The enzymatic reac-
tions and measurements were carried out as described above for
the determination of the IC50 values with the only exception that
the concentrations of MAO-A and -B in the reactions were
0.015 mg/mL. Linear regression analysis was performed using the
Prism 5 software package.34
tions of 45 and 30 lM where MAO-A and -B, respectively, were
used as enzymes. The reactions were incubated at 37 °C in a water
bath for a period of 20 min and subsequently terminated with the
4.7. Molecular modeling studies
addition of 400 lL NaOH (2 N). A volume of 1000 lL distilled water
was added to each reaction, and the concentrations of the MAO
generated 4-hydroxyquinoline in the reactions were measured by
fluorescence spectrophotometry (excitation wavelength 310 nm,
emission wavelength 400 nm).33 Quantification of 4-hydroxyquin-
oline was achieved with a linear calibration curve constructed
The Windows based Discovery Studio 1.7 molecular modeling
software (Accelrys) was used for the molecular docking of selected
inhibitors (5e and 6a) into the active site cavities of X-ray crystal-
lographic models of MAO-A and -B. The inhibitors (5e and 6a) were
drawn in Discovery Studio, hydrogen atoms were added according
to the appropriate protonation states at pH 7.4 and the geometries
were briefly optimizated using a fast Dreiding-like forcefield (1000
iterations) available for this purpose in Discovery Studio. The atom
potential types and partial charges were then automatically as-
signed with the Momany and Rone CHARMm forcefield.
from solutions of authentic 4-hydroxyquinoline (0.047–1.5
dissolved in 500 L potassium phosphate buffer (100 mM, pH
7.4, made isotonic with KCl, 20.2 mM). Each calibration standard
also contained 400 L NaOH (2 N) and 1000 L distilled water. Sig-
lM)
l
l
l
moidal dose–response curves were constructed by plotting the ini-
tial rates of MAO catalyzed kynuramine oxidation versus the
logarithm of the inhibitor concentration. Each curve, was con-
structed from 9 to 10 inhibitor concentrations spanning at least 3
orders of a magnitude. The inhibition data were fitted to the one
site competition model incorporated into the Prism 5 software
package (GraphPad). All experiments were carried out in duplicate
and the IC50 values are expressed as mean standard deviation
(SD).34 The IC50 values were converted to the corresponding Ki val-
The X-ray crystallographic structures of MAO-A in complex
with harmine (PDB code: 2Z5X)41 and MAO-B in complex with safi-
namide (PDB code: 2V5Z)42 were acquired from the RCSB Protein
Data Bank (www.rcsb.org/pdb). Hydrogen atoms were added to
the crystallographic structures according to the appropriate pro-
tonation states of the ionizable amino acids at pH 7.4. The valences
of the FAD cofactors (oxidized state) and co-crystallized ligands
(harmine and safinamide) were corrected, hydrogen atoms were
added according to the appropriate protonation states at pH 7.4.
The resulting models were automatically typed with the Momany
and Rone CHARMm forcefield, the protein backbone was con-
strained and the models were subjected to a three step energy
minimization cascade. The first step, a steepest descent minimiza-
tion step was followed by a conjugate gradient minimization step.
For both minimization steps the termination criteria was set to a
maximum of 2500 steps or a minimum value of 0.1 for the root
mean square of the energy gradient. The third step was an adopted
basis Newton-Rapheson minimization step with the termination
criteria set to a maximum of 5000 steps or a minimum value for
the root mean square of the energy gradient of 0.01. For all steps
of this minimization cascade the implicit generalized Born solva-
tion model with simple switching was used with the dielectric con-
stant set to 4. The co-crystallized ligands and backbone constraints
were subsequently removed from the active sites of the MAO-A
and -B crystallographic structures and the binding sites were iden-
tified by a flood-filling algorithm. The crystallized water molecules
were also removed from the crystallographic structures with the
ues according to the equation by Cheng and Prusoff: Ki = IC50
/
(1 + [S]/Km).36
4.5. Time-dependence of inhibition
For the purpose of the time dependent studies compound 5a
was selected as a representative inhibitor of the series. Compound
5a and recombinant human MAO-A or human MAO-B (0.03 mg/
mL) were preincubated for periods of 0, 15, 30, 60 min at 37 °C
in potassium phosphate buffer (100 mM, pH 7.4, made isotonic
with KCl, 20.2 mM). The concentrations of 5a employed for these
reactions were 13.42 lM and 5.00 lM for the incubations with
MAO-A and -B, respectively. These are approximately twofold the
measured IC50 values for the inhibition of MAO-A and -B, respec-
tively. To the preincubated enzyme preparations were then added
the MAO-A/B mixed substrate, kynuramine, at final concentrations
of 45
respectively. The volumes of these incubations were 500
nal concentrations of the MAO preparations were 0.015 mg/mL and
lM and 30
lM for the reactions containing MAO-A and -B,
lL, the fi-

4840
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 4829–4840
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parameters for the docking runs were set to their default values
and ten possible binding solutions were computed for each docked
ligand. The best-ranked binding conformation of each ligand was
determined according to the DockScore values. The illustrations
47
were prepared in PYMOL
.
27. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
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NMR spectra were recorded by André Joubert of the SASOL
Centre for Chemistry, North-West University while the MS spectra
were recorded by Marelize Ferreira of the Mass Spectrometry
Service, School of Chemistry, University of the Witwatersrand. Sup-
port with the HPLC analysis was provided by Jan du Preez from the
Analytical Technology Laboratory, North-West University. This
work was supported by grants from the National Research Founda-
tion and the Medical Research Council, South Africa.
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Supplementary data
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Chem. 2006, 14, 3512.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.070.
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