Monoamine oxidase inhibition by C4-substituted phthalonitriles

Article abstract of DOI:10.1016/j.bioorg.2011.10.003

It was recently reported that a series of C5-substituted phthalimides are remarkably potent reversible inhibitors of recombinant human monoamine oxidase (MAO) B. Modeling studies suggested that the phthalimide ring forms numerous polar interactions with the polar region of the MAO-B substrate cavity while the C5 side chain extends to, and interacts via Van der Waals interactions with the hydrophobic regions of the enzyme entrance cavity. Interactions with both cavities appear to be requirements for high affinity binding. In the present study we have examined an analogs series of C4-substituted phthalonitriles as potential human MAO inhibitors. The phthalonitriles were found to be highly potent reversible MAO-B inhibitors with most analogs exhibiting IC₅₀ values in the low nM range. The phthalonitriles also interacted with human MAO-A, although with lower binding affinities compared to MAO-B. Modeling studies suggest that the high binding affinities of the phthalonitriles to MAO-B may depend, at least in part, on the formation of polar interactions between the nitrile functional groups and the enzyme substrate cavity. Examination of a homologs series of benzonitriles established that the phthalonitrile moiety is more optimal for MAO-B inhibition than the corresponding benzonitrile moiety, and that C3-substituted benzonitriles are better MAO-B inhibitors than C4-substituted benzonitriles. Since elimination of the nitrile functional group yielded compounds with only moderate MAO-B inhibition potencies, it may be concluded that this functional group is privileged for MAO-B inhibition.

Full text of DOI:10.1016/j.bioorg.2011.10.003

Bioorganic Chemistry 40 (2012) 114–124
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Monoamine oxidase inhibition by C4-substituted phthalonitriles
⇑
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:
Received 13 September 2011
Available online 29 October 2011
It was recently reported that a series of C5-substituted phthalimides are remarkably potent reversible
inhibitors of recombinant human monoamine oxidase (MAO) B. Modeling studies suggested that the
phthalimide ring forms numerous polar interactions with the polar region of the MAO-B substrate cavity
while the C5 side chain extends to, and interacts via Van der Waals interactions with the hydrophobic
regions of the enzyme entrance cavity. Interactions with both cavities appear to be requirements for high
affinity binding. In the present study we have examined an analogs series of C4-substituted phthalonitr-
iles as potential human MAO inhibitors. The phthalonitriles were found to be highly potent reversible
MAO-B inhibitors with most analogs exhibiting IC50 values in the low nM range. The phthalonitriles also
interacted with human MAO-A, although with lower binding affinities compared to MAO-B. Modeling
studies suggest that the high binding affinities of the phthalonitriles to MAO-B may depend, at least in
part, on the formation of polar interactions between the nitrile functional groups and the enzyme sub-
strate cavity. Examination of a homologs series of benzonitriles established that the phthalonitrile moiety
is more optimal for MAO-B inhibition than the corresponding benzonitrile moiety, and that C3-substi-
tuted benzonitriles are better MAO-B inhibitors than C4-substituted benzonitriles. Since elimination of
the nitrile functional group yielded compounds with only moderate MAO-B inhibition potencies, it
may be concluded that this functional group is privileged for MAO-B inhibition.
Keywords:
Monoamine oxidase
Reversible inhibition
Phthalonitrile
Phthalimide
Benzonitrile
Molecular docking
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
action of dopamine in the brain, but also enhance dopamine levels
after treatment with levodopa, the metabolic precursor of dopa-
Monoamine oxidase A and B (MAO-A and -B) play essential
roles in the oxidation of neurotransmitter amines in the brain
and peripheral tissues [1]. MAO-A selectively catalyzes the oxida-
tion of serotonin while dopamine, epinephrine and norepinephrine
are substrates for both MAO isoforms [2]. The false neurotransmit-
ters, benzylamine and b-phenylethylamine, are metabolized by
MAO-B [2,3].
Based on their roles in the catabolism of monoamine neuro-
transmitters, the MAO enzymes are considered to be useful drug
targets [2,4,5]. Since both MAO isoforms are found in the human
brain, inhibitors of these enzymes are employed in neuropsychiat-
ric and neurodegenerative diseases [6,7]. MAO-A inhibitors are
used in the treatment of anxiety disorder and depressive illness
[2,8]. MAO-B inhibitors, in turn, reduce the catabolism of dopa-
mine in the basal ganglia and are employed in the treatment of
Parkinson’s disease [9,10]. MAO-B inhibitors not only prolong the
mine [11,12]. For these reasons, MAO-B inhibitors are frequently
combined with levodopa in Parkinson’s disease therapy [5,10].
Interestingly, MAO-B inhibitors are also thought to protect against
the neurodegenerative processes associated with Parkinson’s dis-
ease [13]. This effect may, in part, be attributed to the reduction
of harmful metabolic by-products such as dopanal and H2O2 that
arise from the MAO-B catalyzed oxidation of dopamine [14–18].
Of significance is the observation that MAO-A activity remains con-
stant with age while MAO-B levels and activity increase up to 4–5
fold in most brain regions, including the basal ganglia [19–21]. In
the aged parkinsonian brain, the inhibition of MAO-B catalyzed
dopamine turnover may therefore be particularly relevant since
it would lead to a reduction of harmful dopamine-derived oxida-
tion products and possibly protection against further neuronal
damage.
The availability of the three-dimensional structures of both
MAO-A and -B greatly assists in the design of new inhibitors of
these enzymes [22,23]. The active site of MAO-A consists of a single
cavity while the active site of MAO-B is comprised of two separate
spaces, an entrance cavity and substrate cavity [22]. The MAO-B
active site cavities are normally separated by the side chain of
Ile-199, but upon binding of larger cavity-filling ligands, the
Abbreviations: CCDC, Cambridge crystallographic data centre; DMSO, dimethyl
sulfoxide; FAD, flavin adenine dinucleotide; MAO, monoamine oxidase; PDB,
protein data bank; RMSD, root mean square deviation.
⇑
Corresponding author. Fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.10.003

C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
115
Ile-199 may adopt an alternate conformation which allows for the
CN
CN
R
4
4
fusion of the two cavities [24]. X-ray crystallography has shown
that relatively large MAO-B inhibitors, such as safinamide (1)
(Fig. 1), adopt an orientation in the active site that places the polar
end of the molecule, the propanamidyl moiety, in the polar region
of the substrate cavity, near the FAD, while the apolar fluoroben-
zyloxy side chain extends into the hydrophobic entrance cavity
[25]. This dual interaction mode may, to a large degree, explain
the high binding affinity of safinamide for the MAO-B active site.
Several other examples exist of relatively large cavity-filling
MAO-B inhibitors which undergo polar and apolar interactions
with the substrate and entrance cavities, respectively. For example,
the chlorobenzyloxy side chain of 7-(3-chlorobenzyloxy)-4-form-
ylcoumarin (2) binds in the entrance cavity of the enzyme with
the polar coumarin ring interacting with the polar region of the
substrate cavity [25]. A recent report documents that a series of
C5-substituted phthalimides (3) are exceptionally potent revers-
ible inhibitors of recombinant human MAO-B [26]. Modeling stud-
ies suggested that the polar functional groups of the phthalimide
ring forms numerous interactions with the polar region of the
MAO-B substrate cavity while the apolar C5 side chain extends
to, and interacts via Van der Waals interactions with the hydropho-
bic entrance cavity of the enzyme. Similar to safinamide, interac-
tions with both the substrate and entrance cavities may explain
the high binding affinities of the C5-substituted phthalimides to
MAO-B. Based on the MAO inhibition properties of the phthali-
mides, in the present study a homologs series of C4-substituted
phthalonitriles (4) were synthesized and examined as potential hu-
man MAO-A and -B inhibitors (Fig. 2). With the selection of C4 side
chains of relatively low polarity suited for binding within the en-
trance cavity, the C4-substituted phthalonitriles may exhibit high
binding affinities to MAO-B since the nitrile functional group is
highly polar and may interact with the polar regions in the sub-
strate cavity. These compounds may therefore also exhibit a dual
mode of binding to the MAO-B active site, a property which is
thought to be favorable for high affinity binding. The notion that
nitriles may undergo polar interactions with the active site of
MAO-B is supported by the observation that the nitrile functional
group is considered to be a bioisostere of water and has been used
in drug design to displace water molecules from the binding sites
of proteins [27]. For example, the introduction of nitrile groups
at the appropriate sites of quinazoline and benzotriazine inhibitors
of scytalone dehydratase improved the inhibition potencies by sev-
eral orders of magnitude [28]. To examine the potential role that
R
3
CN
5
4
O
R
6
Fig. 2. The structures of C4-substituted phthalonitrile (4), C3- and C4-substituted
benzonitriles (5) benzyl phenyl ether (6) analogues.
nitrile groups may play in the binding of the C4-substituted pht-
halonitriles to MAO-A and -B, a series of C3- and C4-substituted
benzonitriles (5) were synthesized and examined as inhibitors.
Furthermore, a series of homologs (6) devoid of the nitrile func-
tional group was also investigated as potential MAO inhibitors.
Similar to the C5-substituted phthalimides, alkyl- and aryloxy side
chains were selected for the purpose of this study since these have
been shown to enhance the MAO-A and -B binding affinities of a
variety of scaffolds including caffeine, isatin and phthalimide
[26,29,30]. Previous studies have suggested that alkyl- and aryloxy
side chains, with a relatively larger degree of conformational free-
dom as a result of rotation about the carbon–oxygen ether bond,
may be better suited for binding to MAO-A than relatively rigid
structures [29,31]. Although intended for binding to MAO-B, the
C4-substituted phthalonitriles examined here may therefore also
act as MAO-A inhibitors.
2. Results
2.1. Chemistry
In the present study a series of C4-substituted phthalonitrile
(4a–i), C3- and C4-substituted benzonitrile (5a–h) and benzyl phe-
nyl ether analogs (6a–d) were synthesized with the aim of exam-
ining their MAO inhibitory properties. The target phthalonitrile
analogs (4a–i) were synthesized by reacting 4-nitrophthalonitrile
with the appropriate alcohol in the presence of potassium carbon-
ate in dimethyl sulfoxide (DMSO) (Scheme 1) [32]. The phthalo-
nitriles were in most cases obtained in relatively good yields (up
to 84%). The substituted benzonitrile analogs (5a–h) investigated
in this study were synthesized in a similar manner by reacting
the 3- or 4-nitrobenzonitrile with an appropriate alcohol [32].
H
CH3
NH2
N
CN
CN
O2N
CN
CN
O
F
H
O
4
R
O
a
OH
R
+
1
2
CHO
4a-i
4
Cl
a
O2N
OH
Br
+
R
R
O
O
O
O
3
R
R
CN
CN
5a-h
6b-d
O
HO
O
b
+
R
5
NH
3
O
Scheme 1. Synthetic pathway to C4-substituted phthalonitrile analogues (4a–i),
C3- and C4-substituted benzonitrile analogues (5a–h) and benzyl phenyl ether
analogues (6b–d). Reagents and conditions: (a) K2CO3, DMSO, argon, (b) K2CO3,
acetone.
Fig. 1. The structures of safinamide (1), 7-(3-chlorobenzyloxy)-4-formylcoumarin
(2) and C5-substituted phthalimide (3).

116
C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
Table 1
For this reaction, yields of 22–61% were obtained. With the excep-
tion of 6a which is commercially available, the benzyl phenyl ether
analogs (6b–d) were synthesized by reacting phenol with an alkyl
bromide in the presence of potassium carbonate in acetone. Yields
of 54–67% were obtained. In each instance, the structures and puri-
ties of the new compounds were verified by 1H NMR, 13C NMR,
mass spectrometry and HPLC analysis as cited in Section 4.
The IC50 values for the inhibition of recombinant human MAO-A and -B by
phthalonitrile analogues 4a–i.a
CN
CN
R
IC50
(
lM)
SIb
R
MAO-A
MAO-B
6.02 0.33
2.2. MAO inhibition studies
4a
4b
4c
4d
4e
4f
4g
4h
4i
C6H5O
C6H5CH2O
25.2 0.042
1.79 0.016
1.31 0.059
0.652 0.067
0.399 0.026
5.05 0.113
19.2 0.771
0.642 0.217
2.45 0.231
4
0.0079 0.001
0.018 0.0003
0.136 0.016
0.0066 0.0003
0.244 0.060
0.568 0.055
0.0048 0.0001
0.226 0.019
227
73
5
To examine the MAO inhibition potencies of the phthalonitrile
(4a–i), benzonitrile (5a–h) and benzyl phenyl ether analogs (6a–
d), recombinant human MAO-A and -B were employed as enzyme
sources. As enzyme substrate the mixed MAO-A/B substrate,
kynuramine, was used. Kynuramine, which exhibits similar Km val-
C6H5CH2CH2O
C6H5(CH2)3O
(E)-C6H5CH@CHCH2O
2-Naphthalenyl-O
4-BrC6H4O
60
21
34
134
11
4-BrC6H4CH2O
4-BrC6H4CH2CH2O
ues towards the two enzymes (16.1 lM and 22.7 lM for MAO-A
and -B, respectively) is oxidized to yield 4-hydroxyquinoline, a
fluorescent compound which is readily measurable in the presence
of the non-fluorescent substrate [29]. At the excitation and emis-
sion wavelengths (kex = 310 nm, kem = 400 nm) and inhibitor con-
centrations used, none of the test inhibitors fluoresced, or
quenched the fluorescence of 4-hydroxyquinoline. The inhibition
potencies of the test inhibitors are expressed as the IC50 values
which were determined from sigmoidal dose–response curves as
shown by example in Fig. 3.
a
All values are expressed as the mean SD of duplicate determinations.
b
The selectivity index is the selectivity for the MAO-B isoform and is given as the
ratio of Ki(MAO-A)/Ki(MAO-B).
C4 substituent is also a strategy to enhance the MAO-B inhibition
potencies of C4-substituted phthalonitrile analogs. The finding that
increasing size or length of the C4 substituent leads to enhanced
MAO-B inhibition will be explored with the aid of molecular mod-
eling below. Interestingly, the introduction of an ethenyl double
bond into the C4 side chain, as observed with the phenylpropenyl-
oxy derivative 4e, also resulted in potent MAO-B inhibition
2.2.1. MAO-B inhibition studies
The MAO-B inhibition potencies of the C4-substituted phthalo-
nitrile analogs (4a–i) are presented in Table 1. With the exception
(IC50 = 0.0066 lM). In an attempt to further enhance the MAO-B
inhibition potencies of the phthalonitrile analogs, the phenyl rings
of the C4 substituent of selected homologs (4a–c) were substituted
with bromine. It has previously been documented that bromine
substitution on the phenyl rings of alkyloxy- and aryloxy substi-
tuted phthalimide analogs leads to further enhancement of binding
to MAO-B [26]. The results show that bromine substitution of pht-
halonitrile 4a (to yield 4g) results in an IC50 value for the inhibition
of 4a (IC50 = 6.02
be exceptionally potent MAO-B inhibitors with IC50 values in the
nM range (0.005–0.57 M). It is significant that 4a contains the
lM), all the phthalonitrile analogs were found to
l
shortest C4 substituent (–OC6H5) among the phthalonitrile analogs
examined. Increasing the length of the C4 side chain by only one
methylene unit (–OCH2C6H5) yields compound 4b with an IC50 va-
lue of 0.0079 lM. Compound 4b is approximately 750 fold more
of MAO-B of 0.568
(IC50 = 6.02 M). The bromine substituted homologs of 4b and 4c
were also found to be potent MAO-B inhibitors with recorded
IC50 values of 0.0048 M for 4h and 0.226 M for 4i. The bromine
substituted compound 4h is therefore only slightly more potent
than its unsubstituted homolog 4b (IC50 = 0.0079 M) as an
MAO-B inhibitor while 4i is a weaker inhibitor than its unsubsti-
tuted homolog 4c (IC50 = 0.018 M). These results indicate that
lM, 10 fold more potent than 4a
potent as an MAO-B inhibitor than is 4a. In fact, 4b is the third
most potent MAO-B inhibitor among the phthalonitrile analogs.
Further increasing the length of the C4 side chain yields the pheny-
l
l
l
lethoxy- and phenylpropoxy substituted homologs 5c (IC50
=
0.018 M) and 5d (IC50 = 0.136 M), which were also found to be
l
l
l
potent MAO-B inhibitors. Also of significance is the observation
that replacement of the phenoxy phenyl ring of 4a with a napht-
halenyl ring to yield compound 4f, results in enhanced MAO-B
inhibition potency. Compound 4f (IC50 = 0.244 lM) is approxi-
mately 24 fold more potent than 4a as an MAO-B inhibitor. This
l
bromine substitution on the phenyl ring of the C4 substituent en-
hances the MAO-B inhibition potencies of weaker phthalonitrile
inhibitors such as 4a to a large degree while its effects on the
MAO-B inhibition potencies of the more potent analogs (4b, 4c)
are less significant and may even lead to a loss of inhibition
activity.
result reveals that increasing the size of the aromatic ring of the
3
2
1
To examine the requirement of the presence of both nitrile
groups of the phthalonitrile analogs for MAO-B inhibition, a series
of benzonitriles (5a–h) was examined as MAO-B inhibitors. The
importance of the position of the nitrile group on the phenyl ring
with respect of the alkyloxy side chain was also investigated by
synthesizing both the C3- and C4-substituted benzonitriles. The
MAO inhibition potencies of the C3- and C4-substituted benzonitr-
iles are presented in Table 2. Compounds 5a and 5b, the benzoni-
trile analogs of phthalonitrile 4b, were both found to be weaker
MAO-B inhibitors than 4b (IC50 = 0.0079
0.785 M and 0.249 M, respectively. Similarly, compounds 5c
and 5d, the benzonitrile analogs of phthalonitrile 4e, were also
weaker MAO-B inhibitors than 4e (IC50 = 0.0066 M) with IC50
values of 0.376 M and 0.174 M, respectively. The same trend is
observed for compounds 5e (IC50 = 0.266 M) and 5f (IC50
0.041 M) which were weaker MAO-B inhibitors than their
lM) with IC50 values of
-4
-2
0
2
l
l
Log [4d]
l
Fig. 3. The sigmoidal dose-response curve of the initial oxidation rate of human
MAO-B vs. the logarithm of the concentration of 4d (expressed in M). The
determinations were carried out in duplicate and the catalytic rates are expressed
l
l
l
l
=
as nmoles 4-hydroxyquinoline formed/min/mg protein.
l

C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
117
Table 2
2.2.2. MAO-A inhibition studies
The IC50 values for the inhibition of recombinant human MAO-A and -B by
As shown in Table 1 the C4-substituted phthalonitrile analogs
(4a–i) are also inhibitors of human MAO-A. As evident from the
selectivity index (SI) values all the phthalonitriles examined here
are however selective inhibitors for the MAO-B isoform. The most
selective inhibitor, compound 4b, was 227 fold more selective for
MAO-B than for MAO-A. In view of the high inhibition potency of
4b, this compound may represent a promising MAO-B selective
inhibitor. Although the phthalonitriles were weaker MAO-A inhib-
itors, several compounds displayed IC50 values in the nM range. For
example, the most potent MAO-A inhibitor, compound 4e, exhib-
benzonitrile analogues 5a–h.a
R
CN
IC50
(
l
M)
SIb
R
MAO-A
MAO-B
5a
5b
5c
5d
5e
5f
4-C6H5CH2O
3-C6H5CH2O
32.2 2.93
13.0 2.95
0.785 0.049
0.249 0.054
0.376 0.039
0.174 0.028
0.266 0.004
0.041 0.019
1.35 0.074
1.39 0.322
41
52
42
21
9
64
9
50
4-(E)-C6H5CH@CHCH2O
3-(E)-C6H5CH@CHCH2O
4-(4-BrC6H4)CH2O
3-(4-BrC6H4)CH2O
4-(4-BrC6H4)CH2CH2O
4-C6H5CH2O(CH2)2O
15.9 0.636
3.60 0.565
2.44 0.115
2.610 0.173
11.85 8.21
69.5 3.58
ited an IC50 value of 0.399 lM. Interestingly, similar to the results
obtained with MAO-B, increasing the length of the C4 side chain
was associated with an increase in MAO-A inhibition potency.
For example, among the phthalonitriles, the phenoxy substituted
homolog (4a) was the weakest MAO-A inhibitor with an IC50 value
5g
5h
a
All values are expressed as the mean SD of duplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the
of 25.2
benzyloxy (4b) and phenylethoxy (4c) substituted homologs with
IC50 values of 1.79 M and 1.31 M, respectively. Further increas-
ing the C4 side chain, as demonstrated by the phenylpropoxy
(4d; IC50 = 0.652 M) and phenylpropenyloxy (4e; IC50
0.399 M) substituted homologs, yielded even more potent
lM. Increasing the length of the C4 substituent yielded the
b
ratio of Ki(MAO-A)/Ki(MAO-B).
l
l
l
=
corresponding phthalonitrile analog 4h (IC50 = 0.0048 lM). These
l
results demonstrate that the phthalonitrile moiety is more optimal
for MAO-B inhibition than the corresponding benzonitrile moiety.
Also, the results show C3-substituted benzonitriles are better
MAO-B inhibitors than C4-substituted benzonitriles. For example,
C3-substituted benzonitrile 5b is 3 fold more potent than its corre-
sponding C4-substituted isomer 5a, while 5d is 2 fold more potent
than its corresponding C4-substituted benzonitrile analog 5c. Sim-
ilarly, 5f, is 6 fold more potent as a MAO-B inhibitor than its corre-
sponding C4-substituted isomer 5e. It may therefore be concluded
that meta placement of the nitrile and alkyloxy groups results in
more productive interactions with the MAO-B active site than para
placement of these groups.
The significance of the nitrile functional groups for binding of
the C4-substituted phthalonitriles to MAO-B was further investi-
gated by examining the MAO-B inhibition potencies of a series of
benzyl phenyl ether analogs (6a–d) which are devoid of the nitrile
group. As shown in Table 3 the benzyl phenyl ether analogs proved
to be moderately potent inhibitors of MAO-B with IC50 values in
MAO-A inhibition. Bromine substitution on the phenyl ring of the
C4 side chain did not enhance MAO-A inhibition activity to a large
extent. For example, the bromine substituted analog 4h exhibited
an IC50 value for the inhibition of MAO-A of 0.642
more potent than its unsubstituted homolog, 4b (IC50 = 1.79
Bromine substitution of 4a and 4c, to yield compounds 4g and 4i,
was however associated with reduced MAO-A inhibition potency.
The C3- and C4-substituted benzonitrile analogs (5a–h) were
also found to be MAO-A inhibitors (Table 2). Similar to the phthalo-
nitriles, the benzonitriles were however selective inhibitors of the
MAO-B isoform. In general, the series of benzonitriles were weaker
MAO-A inhibitors than the corresponding phthalonitrile analogs.
For example, compounds 5a and 5b, the benzonitrile analogs of
l
M, only 2.7 fold
l
M).
phthalonitrile 4b (IC50 = 1.79
inhibitors with IC50 values of 32.2
Benzonitriles 5c (IC50 = 15.9 M) and 5d (IC50 = 3.60
weaker MAO-A inhibitors than their corresponding phthalonitrile
analog 4e (IC50 = 0.399 M). The same trend was observed for the
l
M) were relatively weak MAO-A
M and 13.0 M, respectively.
M) were also
l
l
l
l
l
the lM range (1.3–11.8 lM). These inhibitors are therefore weaker
benzonitrile analogs 5e and 5f which were approximately 4 fold
weaker MAO-A inhibitors that the corresponding phthalonitrile
homolog 4h. These results demonstrate that, as for MAO-B, the
phthalonitrile moiety results in enhanced MAO-A inhibition com-
pared to the corresponding benzonitrile moiety. In general, the
C3-substituted benzonitriles were found to be more potent MAO-
A inhibitors than the C4-substituted benzonitriles. For example,
C3-substituted benzonitrile 5b was found to be 2.5 fold more po-
tent than the corresponding C4-substituted isomer 5a, while 5d
was found to be 4 fold more potent than its corresponding C4-
substituted isomer 5c. The C4-substituted phthalonitrile 5e and
its C3-substituted isomer 5f exhibited similar inhibition potencies
towards MAO-A. Analogous to the inhibition results obtained with
MAO-B, it may therefore be concluded that meta placement of the
nitrile and alkyloxy groups, in general, may yield improved MAO-A
inhibition activity compared to para placement of these groups.
The importance of the nitrile functional groups for interaction of
the phthalonitrile and benzonitrile analogs with MAO-A was fur-
ther investigated by determining the MAO-A inhibitory properties
of the series of benzyl phenyl ether analogs (6a–d) which is
devoid of the nitrile group (Table 3). Compared to the inhibition
potencies of the phthalonitriles and benzonitriles, the benzyl phe-
nyl ether analogs were found to be weak inhibitors of MAO-A with
MAO-B inhibitors when compared to the corresponding phthalo-
nitriles and the benzonitriles which contain the same alkyloxy sub-
stituents. For example, the benzyloxy substituted phthalonitrile
(4b) and benzonitriles (5a and 5b) are 8–839 fold more potent as
MAO-B inhibitors than benzyl phenyl ether (6a). As shown in Ta-
ble 4, compounds 6b–d were similarly weaker inhibitors than their
nitrile containing homologs. From this result it may be concluded
that the nitrile functional group is a requirement for high affinity
binding of the phthalonitrile and benzonitrile analogs to the
MAO-B active site.
Table 3
The IC50 values for the inhibition of recombinant human MAO-A and -B by analogues
6a–d.a
IC50
(lM)
SIb
MAO-A
MAO-B
6a
6b
6c
6d
C6H5CH2O C6H5
No Inh.c
145 18.5
65.5 25.5
102 12.0
6.63 0.59
1.32 0.003
3.05 0.487
11.8 2.10
–
(E)-C6H5CH = CHCH2O C6H5
(4-BrC6H4)CH2O C6H5
(4-BrC6H4) CH2CH2O C6H5
110
21
9
a
All values are expressed as the mean SD of duplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the
b
IC50 values ranging from 65.5 lM to 145 lM. Benzyl phenyl ether
(6a) was found to be devoid of MAO-A inhibition properties. As
ratio of Ki(MAO-A)/Ki(MAO-B).
c
No inhibition observed at a maximal concentration of 100 lM of the test
inhibitor.
shown in Table 4 the phthalonitrile (4) and benzonitrile (5) analogs

118
C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
Table 4
A comparison of the IC50 values for the inhibition of MAO-B and -A by compounds 6 to the IC50 values of the corresponding compounds 4 and 5.
CN
R
R
4
R
3
CN
CN
5
4
6
R
IC50 value ratios for the inhibition of MAO-B
IC50 value ratios for the inhibition of MAO-A
6/4a
6/5b (C4-subst.)
6/5c (C3-subst.)
6/4a
6/5b (C4-subst.)
6/5c (C3-subst.)
C6H5CH2O
(E)-C6H5CH@CHCH2O
4-BrC6H4CH2O
839
200
635
52
8
4
11
9
27
8
74
–
–
363
102
42
–
9
27
9
–
40
25
–
4-BrC6H4CH2CH2O
a
b
c
The ratio of the IC50 values of compounds 6 to the IC50 values of compounds 4.
The ratio of the IC50 values of compounds 6 to the IC50 values of compounds 5 containing C4 substituents.
The ratio of the IC50 values of compounds 6 to the IC50 values of compounds 5 containing C3 substituents.
are 9–363 fold more potent as MAO-A inhibitors than the corre-
sponding benzyl phenyl ether analogs (6). This analysis shows that
the nitrile functional group significantly enhances the binding
affinities of the substituted phthalonitrile and benzonitrile analogs
to the MAO-A.
Panel A
4
3
2
1
0
2.3. Reversibility of inhibition
Based on the highly potent MAO inhibition potencies of the pht-
halonitrile analogs, a selected phthalonitrile analog, compound 4d,
was evaluated for its ability to interact reversibly with human
MAO-A and -B. For this purpose the time dependency of the inhi-
bition was evaluated. While irreversible inhibitors would display
a time-dependent reduction of enzyme activity, in the presence
of reversible inhibitors enzyme activity would remain unchanged
regardless of the time period for which the inhibitor is incubated
with the enzyme. Compound 4d was preincubated with recombi-
nant human MAO-A or -B for various periods of time (0–60 min)
and the residual MAO catalytic activities were measured after the
addition of kynuramine to the incubations. The concentrations of
0
15
30
60
Incubation time (min)
0.8
0.6
0.4
0.2
0.0
Panel B
4d that were selected for the preincubations were 1.31
lM and
0.27 M for the incubations with MAO-A and -B, respectively.
l
These concentrations are approximately 2 fold the measured IC50
values for the inhibition of the respective enzymes by 4d. The re-
sults of these reversibility studies are presented in Fig. 4. As shown
by the bar graphs, preincubation of 4d with both MAO-A and -B
does not result in a time-dependent loss of MAO-A and -B catalytic
activities. Even after incubating 4d for a period of 60 min with the
MAO enzymes no reduction of the catalytic rates are observed.
From these results it may therefore be concluded that 4d is not a
time-dependent inhibitor of MAO-A and -B and the interactions
of this phthalonitrile analog with the MAO enzymes are reversible,
at least for the time period (0–60 min) and at the inhibitor concen-
trations (2 Â IC50) evaluated. Interestingly, a small but significant
time-dependent increase of the MAO-A catalytic rate is observed
when the enzyme is preincubated with 4d. A plausible explanation
for this behavior is not readily apparent.
0
15
30
60
Incubation time (min)
Fig. 4. The time-dependent inhibition of human MAO-A (Panel A) and MAO-B
(Panel B) by 4d. The enzymes were preincubated for various periods of time (0–
60 min) with 4d at concentrations of 1.31 lM and 0.27 lM for MAO-A and MAO-B,
respectively, and the residual catalytic rated were recorded. The catalytic rates are
expressed as nmoles 4-hydroxyquinoline formed/min/mg protein.
Lineweaver–Burk plots obtained in this manner are shown in
Fig. 5. The results show that the sets of Lineweaver–Burk plots con-
structed for the inhibition of MAO-A and -B are linear and intersect
at the y-axis. This indicates that the inhibition of the MAO enzymes
by 4d are competitive and thus provide further support that 4d is a
reversible MAO inhibitor.
To further examine the binding modes of 4d to MAO-A and -B,
the possibility that 4d acts as a competitive inhibitor of these en-
zymes was explored. For this purpose, Lineweaver–Burk plots were
constructed for the inhibition of MAO-A and –B by 4d. The initial
catalytic rates of MAO-A or -B were measured at four different con-
2.4. Molecular modeling
centrations (15–90 lM) of the substrate, kynuramine. These mea-
surements were carried out in the absence and presence of three
different concentrations of 4d. For the studies with MAO-A the
To provide additional insight into the relationships between the
MAO-A and -B inhibitory potencies and the structures of the inhib-
itors examined in this study, the binding modes of selected analogs
(4a, 4b, 5a and 5b) in the active site cavity of MAO-B and the
concentrations of 4d were 0.1625–0.65
lM while for the studies
with MAO-B the concentrations of 4d were 0.035–0.14
lM. The

C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
119
Panel A
0.4
0.3
0.2
0.1
0.0
-0.02
0.00
0.02
0.04
0.06
1/[S]
1.6
1.2
0.8
0.4
0.0
Panel B
Fig. 6. An illustration of the predicted binding orientations of 4a (green) and 4b
(orange) in the active site of MAO-B. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
-0.02
0.00
0.02
0.04
0.06
1/[S]
Fig. 5. Lineweaver-Burk plots of the catalytic rates of human MAO-A (Panel A) and
MAO-B (Panel B) in the absence (filled squares) and presence of various concen-
trations of 4d. For the studies with MAO-A (Panel A) the concentrations of 4d were:
group of Tyr-435 (Fig. 6). Interestingly, the C2 nitrile group is direc-
ted towards Tyr-60 and Phe-343 in a region of the substrate cavity
that is considered to be relatively apolar [34]. No hydrogen bond-
ing of the C2 nitrile is observed. Since the entrance cavity is a
highly hydrophobic environment the alkyloxy side chain is most
likely stabilized here via Van der Waals interactions [34]. Another
0.1625
studies with MAO-B (Panel B) the concentrations of 4d were: 0.035
squares), 0.07 M (filled circles), 0.14 M (open circles). The catalytic rates (V) are
expressed as nmol product formed/min/mg protein.
l
M (open squares), 0.325
l
M (filled circles), 0.65
lM (open circles). For the
l
M (open
l
l
potentially significant interaction is a
p–sigma interaction be-
tween the alkyloxy side chain phenyl ring and Ile-199. The docked
orientation of 4a (Fig. 6) is similar to that of 4b with the exception
binding orientation of 4b in the active site cavity of MAO-A were
examined using molecular docking.
The Discovery Studio modeling software (Accelrys) [33] was
used to carry out the molecular docking experiments according
to a modification of a previously reported protocol (see Section 4)
[29,30]. The three-dimensional structures of human MAO-A
cocrystallized with harmine (PDB entry: 2Z5X) [22] and human
MAO-B cocrystallized with safinamide (PDB entry: 2V5Z) [25] were
selected for the modeling studies. The protonation states of the
ionizable residues of the protein models were calculated and
hydrogen atoms were added accordingly. The backbone atoms of
the models were constrained and the models were subjected to
an energy minimization cascade. For the purpose of the docking
procedure the crystal waters were removed from the models with
the exception of those in the MAO-A and -B active sites that are re-
ported to be conserved and non-displaceable (see Section 4). The
structures of ligands were constructed and prepared within Dis-
covery Studio and were docked into the active sites of the MAO-
A and -B models using the CDOCKER application. When redocking
the structures of harmine and safinamide into the active sites of
the enzymes, the docked binding orientations exhibited relatively
small RMSD values of 0.77 Å and 1.66 Å, respectively, from the po-
sition of the cocrystallized ligands. This protocol may therefore be
considered suitable for investigating the potential binding modes
of inhibitors within MAO-A and -B.
The best ranked docking solution for the binding of 4b to MAO-
B illustrates that the phthalonitrile moiety of the structure binds
within the substrate cavity while the alkyloxy side chain extends
towards the entrance cavity. In the substrate cavity, the C1 nitrile
group interacts via hydrogen bonding with the phenolic hydroxyl
Fig. 7. An illustration of the predicted binding orientations of 5a (green) and 5b
(orange) in the active site of MAO-B. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)

120
C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
than the aryloxy side chain of 4a does not extend far into the en-
trance cavity and does not interact with Ile-199. The apparent re-
duced degree of interaction between the entrance cavity and the
aryloxy side chain of 4a may explain the relatively low binding
affinity of this compound to MAO-B. The benzonitriles 5a and 5b
adopt almost identical orientations to 4b (Fig. 7). Similar to 4b,
the para nitrile group of 5a undergoes a hydrogen bond interaction
with the phenolic hydroxyl group of Tyr-435 while the meta nitrile
group of 5b is directed towards Tyr-60 and Phe-343, the hydropho-
bic patch in the substrate cavity. Even though no hydrogen bond-
ing is predicted for the nitrile functional group of 5b, the results
of the enzyme inhibition studies showed that C3 substituted ben-
zonitriles, such as 5b, are more potent MAO-B inhibitors than the
corresponding C4 substituted benzonitriles for which hydrogen
bonding is predicted (e.g. 5a). It may therefore be concluded that
differing polar interactions of the C4 and C3 substituted benzonitr-
iles with the substrate cavity do not account for the observed dif-
ference in MAO-B inhibition activities. It is noteworthy that similar
binding orientations were generated using the LigandFit (Accelrys)
[33] and GOLD (CCDC) [35] docking protocols (results not shown).
The predicted binding orientation of 4b within the MAO-A ac-
tive site shows that the phthalonitrile ring is bound to the sub-
strate cavity where the C2 nitrile group is involved in hydrogen
bonding with a crystal water molecule as well as with the phenolic
hydroxyl of Tyr-444 (Fig. 8). In the MAO-A active site the phthali-
mide ring is rotated through approximately 180° compared to the
orientation adopted in the MAO-B active site. This dissimilarity of
the MAO-A and -B binding orientations of a specific inhibitor is fre-
quently observed in docking studies. For example, the heterocyclic
rings of caffeine [29] as well as isatin [30] derivatives are also ro-
tated through ꢀ180° when comparing the bound orientations in
the MAO-B active site to those in the MAO-A active site. Although
not involved in polar interactions the C1 nitrile group is located in
close proximity to the FAD cofactor, approximately 3.0 Å from the
carbonyl C4 of the FAD. The polar interactions of the C2 nitrile
group appears to play a relatively large role in stabilizing the
phthalimide inhibitors within the MAO-A active site and the loss
of polar interactions between the nitrile groups and the substrate
cavity may explain the reduction of the MAO-A inhibition poten-
cies upon removal of the nitrile groups from the inhibitors. In the
MAO-A active site, residue Phe-208 is located at the position occu-
pied by Ile-199 in the MAO-B active site. As a result a
p–sigma
interaction, similar to that formed by the alkyloxy side chains with
Ile-199 in the MAO-B active site, is not present between the inhib-
itors and the MAO-A active site. The loss of this interaction may ex-
plain, at least in part, the weaker MAO-A binding affinities of the
phthalonitriles examined here compared to their corresponding
MAO-B binding affinities.
3. Discussion
In the present study it was shown that relatively simple small
molecules may be endowed with potent MAO-B inhibition prop-
erties by appropriate substitution with the nitrile functional
group. Phthalonitriles have been shown to be particularly potent
reversible MAO-B inhibitors with all of the examined structures,
except compound 4a, possessing IC50 values in the nM range.
Modeling studies suggest that the nitrile functional group inter-
acts with the polar substrate cavity of the MAO-B enzyme while
the alkyloxy side chain extends into the entrance cavity. This pro-
posed dual binding has also been observed in MAO-B crystal
structures containing other reversible inhibitors in the active site
and may be a requirement for high affinity reversible interaction
between the inhibitor and enzyme [24,25]. Structural modifica-
tions that lead to enhanced MAO-B inhibition potency include
extending the length of the alkyloxy side chain and bromine sub-
stitution on the side chain phenyl ring. Both these modifications
most probably lead to more productive interactions with the en-
trance cavity of MAO-B and hence more potent inhibition. Pht-
halonitrile analog 4a possesses a relatively short alkyloxy side
chain which does not project far into the entrance cavity and as
a result may form only weak interactions with the enzyme en-
trance cavity. This may explain the relatively weak MAO-B inhibi-
tion potency of 4a compared to the homologs containing longer
alkyloxy side chains. Bromine substitution may have the same ef-
fect as lengthening the side chain or alternatively may promote
dipole interactions within the entrance cavity thus leading to im-
proved MAO-B inhibition.
The importance of the nitrile functional group for high affinity
binding to MAO-B is demonstrated by the finding that removal of
the nitrile groups is associated with a loss of MAO-B activity. As
mentioned in the Introduction, the nitrile functional group is
highly polar and probably interacts with the polar regions in the
substrate cavity of the enzyme. Considering the high degree of loss
of MAO-B activity (up to 839 fold) when the phthalonitrile nitrile
groups are eliminated, the interactions formed by these groups
are essential for stabilizing the phthalonitrile inhibitors in the
MAO-B active site. These results show that substitution with a ni-
trile functional group may enhance the potency of reversible MAO-
B inhibitors by several orders of magnitude, a strategy that may be
applied in the design of MAO inhibitors. While the benzonitriles
were weaker MAO-B inhibitors than the corresponding phthalo-
nitriles they still exhibited high binding affinities to MAO-B, a
property that may to a large degree be attributed to the presence
of the remaining nitrile group.
Interestingly, the phthalonitriles were also found to be revers-
ible MAO-A inhibitors although with weaker potencies compared
to the inhibition of MAO-B. Phthalonitrile analogs 4d, 4e and 4h
exhibited IC50 values towards MAO-A in the nM range and since
these compounds are also potent MAO-B inhibitors they represent
potential leads for the design of mixed MAO-A/B inhibitors. The ni-
trile functional groups were also found to be a requirement for
MAO-A inhibition since elimination of these groups results in loss
Fig. 8. An illustration of the predicted binding orientation of 4b (yellow) in the
active site of MAO-A. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
121
of activity. The active site cavities of MAO-A and -B are highly con-
served with only 6 of the 16 active site residues differing between
the two isoforms [22]. The polar regions of the MAO-A and -B ac-
tive sites, in close proximity to the FAD, are in particular well con-
served. It may therefore be expected that similar polar interactions
between the MAO-A active site and the nitrile functional groups
may form compared to those formed with the MAO-B active site.
The proposed formation of these interactions may also explain
the higher binding affinities of the phthalonitrile analogs compared
to the corresponding analogs devoid of the nitrile functional group.
Similar to the results obtained with MAO-B, the benzonitriles were
weaker MAO-A inhibitors than the corresponding phthalonitriles.
This result demonstrates that, as for MAO-B, the multiple nitrile
groups cooperatively enhance binding affinity to MAO-A. The
placement of the nitrile functional group on the phenyl ring of
the benzonitrile inhibitors also affects MAO-A inhibition potency
with meta placement of the nitrile and alkyloxy groups resulting
in enhanced MAO-B inhibition compared to para placement of
these groups.
4.2.1. 4-Phenoxyphthalonitrile (4a)
The title compound was prepared from 4-nitrophthalonitrile
and phenol in a yield of 32%: mp 99–101 °C (ethanol), lit 100–
101 °C [36]. 1H NMR (DMSO-d6) d 7.19 (d, 2H, J = 7.9 Hz), 7.32 (t,
1H, J = 7.5 Hz), 7.35 (dd, 1H, J = 2.6, 9.0 Hz), 7.50 (t, 2H,
J = 8.3 Hz), 7.77 (d, 1H, J = 2.3 Hz), 8.08 (d, 2H, J = 8.7 Hz); 13C
NMR (DMSO-d6) d 108.1, 115.4, 115.9, 116.7, 120.4, 121.89,
122.7, 125.9, 130.7, 136.3, 153.8, 161.1; APCI-MS 220; APCI-HRMS
m/z: Calcd 220.0637. Found 220.0644; Purity (HPLC): 98.9%.
4.2.2. 4-Benzyloxyphthalonitrile (4b)
The title compound was prepared from 4-nitrophthalonitrile
and benzyl alcohol in a yield of 84%: mp 104–106 °C (ethanol).
1H NMR (DMSO-d6) d 5.26 (s, 2H), 7.36 (t, 1H, J = 7.5 Hz), 7.41 (t,
2H, J = 7.9 Hz), 7.46 (d, 2H, J = 7.5 Hz), 7.52 (dd, 1H, J = 2.6,
8.7 Hz), 7.85 (d, 1H, J = 2.6 Hz), 8.05 (d, 1H, J = 9.0 Hz); 13C NMR
(DMSO-d6) d 70.5, 106.1, 115.7, 116.2, 116.3, 120.5, 128.1, 128.4,
128.6, 135.4, 135.8, 161.7; EIMS 234; EI-HRMS m/z: Calcd
234.0793. Found 234.0794; Purity (HPLC): 98.4%.
4.2.3. 4-(2-Phenylethoxy)phthalonitrile (4c)
4. Experimental section
The title compound was prepared from 4-nitrophthalonitrile
and 2-phenylethanol in a yield of 24%: mp 113–114 °C (ethanol).
4.1. Chemicals and instrumentation
1H NMR (DMSO-d6)
d 3.06 (t, 2H, J = 6.8 Hz), 4.37 (t, 2H,
J = 6.8 Hz), 7.22 (m, 1H), 7.31 (m, 4H), 7.44 (dd, 1H, J = 2.6,
9.0 Hz), 7.77 (d, 1H, J = 2.6 Hz), 8.02 (d, 1H, J = 9.0 Hz); 13C NMR
(DMSO-d6) d 34.4, 69.4, 105.9, 115.7, 116.2, 116.3, 120.1, 120.3,
126.4, 128.3, 129.0, 135.7, 137.6, 161.8; APCI-MS 248; APCI-HRMS
m/z: Calcd 248.0950. Found 248.0937; Purity (HPLC): 97.2%.
Unless otherwise noted, all reagents were obtained from Sig-
ma–Aldrich and were used without further purification. Benzyl
phenyl ether (6a) was obtained from Sigma–Aldrich. Proton (1H)
and carbon (13C) NMR spectra were recorded in DMSO-d6 with a
Bruker Avance III 600 spectrometer at frequencies of 600 MHz
and 150 MHz, respectively. The NMR chemical shifts are given as
parts per million (d) downfield from the signal of tetramethylsilane
added to DMSO-d6 and spin multiplicities are given as s (singlet), d
(doublet), dd (doublet of doublets), t (triplet) or m (multiplet). Di-
rect insertion electron impact ionization (EI), atmospheric-pres-
sure chemical ionization (APCI) and high resolution mass spectra
(HRMS) were obtained on a DFS high resolution magnetic sector
mass spectrometer (Thermo Electron Corporation) in electron ion-
ization (EI) or atmospheric pressure chemical ionization (APCI)
mode. Melting points (mp) were determined on a Stuart SMP10
melting point apparatus and are uncorrected. To estimate the de-
gree of purity of the synthesized compounds, HPLC analyses were
conducted with an Agilent 1100 HPLC system equipped with a qua-
ternary pump and an Agilent 1100 series diode array detector (see
Supplementary material). HPLC grade acetonitrile (Merck) and
Milli-Q water (Millipore) were used for the chromatography. Fluo-
rescence spectrophotometry was carried out with a Varian Cary
Eclipse fluorescence spectrophotometer. Kynuramine.2HBr, 4-
hydroxyquinoline and microsomes from insect cells containing re-
combinant human MAO-A and -B (5 mg/mL) were obtained from
Sigma–Aldrich.
4.2.4. 4-(3-Phenylpropoxy)phthalonitrile (4d)
The title compound was prepared from 4-nitrophthalonitrile
and 3-phenyl-1-propanol in a yield of 24%: mp 113–114 °C (etha-
nol). 13C NMR (DMSO-d6) d 2.03 (m, 2H), 2.72 (t, 2H, J = 7.5 Hz),
4.12 (t, 2H, J = 6.4 Hz), 7.17 (t, 1H, J = 7.5 Hz), 7.21 (d, 2H,
J = 7.2 Hz), 7.27 (t, 2H, J = 7.5 Hz), 7.43 (dd, 1H, J = 2.6, 9.0 Hz),
7.75 (d, 1H, J = 2.6 Hz), 8.02 (d, 1H, J = 9.0 Hz); 13H NMR (DMSO-
d6) d 29.9, 31.2, 68.3, 105.8, 115.8, 116.2, 116.3, 120.1, 120.3,
125.9, 128.3, 128.4, 135.8, 141.1, 162.0; APCI-MS 262; APCI-HRMS
m/z: Calcd 262.1106. Found 262.1098; Purity (HPLC): 94.5%.
4.2.5. 4-{[(2E)-3-phenylprop-2-en-1-yl]oxy}phthalonitrile (4e)
The title compound was prepared from 4-nitrophthalonitrile
and cinnamyl alcohol in a yield of 70%: mp 143–145 °C (ethanol).
1H NMR (DMSO-d6) d 4.89 (d, 2H, J = 6.0 Hz), 6.50 (m, 1H), 6.80
(d, 1H, J = 15.8 Hz), 7.27 (t, 1H, J = 7.2 Hz), 7.34 (t, 2H, J = 7.5 Hz),
7.49 (m, 3H), 7.82 (s, 1H), 8.05 (d, 1H, J = 8.7 Hz); 13C NMR
(DMSO-d6) d 69.4, 106.0, 115.8, 116.2, 116.3, 120.4, 120.5, 123.3,
126.6, 128.2, 128.7, 133.8, 135.8, 161.6; EIMS 260; EI-HRMS m/z:
Calcd 260.0950. Found 260.0946; Purity (HPLC): 92.9%.
4.2.6. 4-(Naphthalen-2-yloxy)phthalonitrile (4f)
4.2. Synthesis of C4-substituted phthalonitrile analogs (4a–i)
The title compound was prepared from 4-nitrophthalonitrile
and 2-naphthol in a yield of 76%: mp 121–123 °C (ethanol). 1H
NMR (DMSO-d6) d 7.37 (dd, 1H, J = 2.6, 9.0 Hz), 7.47 (dd, 1H,
J = 2.6, 9.0 Hz), 7.54 (m, 2H), 7.710 (d, 1H, J = 1.9 Hz), 7.88 (d, 1H,
J = 2.3 Hz), 7.91 (d, 1H, J = 7.91 Hz), 7.99 (d, 1H, J = 7.91 Hz), 8.06
(d, 1H, J = 9.0 Hz), 8.11 (d, 1H, J = 8.7 Hz); 13C NMR (DMSO-d6) d
108.4, 115.4, 115.9, 116.7, 116.8, 120.0, 122.4, 123.0, 125.9,
127.0, 127.5, 127.8, 130.8, 130.9, 133.9, 136.3, 151.6, 161.1;
APCI-MS 270; APCI-HRMS m/z: Calcd 270.0793. Found 270.0795;
Purity (HPLC): 97.3%.
The C4-substituted phthalonitrile analogs (4a–i) investigated in
this study were synthesized according to the literature description
[32]. In an atmosphere of argon, a mixture of 4-nitrophthalonitrile
(17.5 mmol), the appropriate alcohol (21 mmol) and potassium
carbonate (35 mmol) in 15 mL dimethyl sulfoxide (DMSO) was
stirred at room temperature for a period of 48 h. The reaction
was poured into 100 mL ice cold water and the precipitate was iso-
lated by filtration and washed with 100 mL water. The crude mate-
rial was dissolved in ethanol, filtered through cotton wool and left
to recrystallize at room temperature. Purification of the target pht-
halonitriles required at least two successive rounds of recrystalli-
zation from ethanol.
4.2.7. 4-(4-Bromophenoxy)phthalonitrile (4g)
The title compound was prepared from 4-nitrophthalonitrile
and 4-bromophenol in a yield of 71%: mp 193–195 °C (ethanol).

122
C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
1H NMR (DMSO-d6) d 7.17 (d, 2H, J = 9.0 Hz), 7.43 (dd, 1H, J = 2.6,
8.7 Hz), 7.66 (d, 2H, J = 8.7 Hz), 7.84 (d, 1H, J = 2.6 Hz), 8.10 (d,
1H, J = 2.6 Hz); 13C NMR (DMSO-d6) d 108.6, 115.3, 115.9, 116.8,
117.8, 122.4, 122.6, 122.9, 133.4, 136.3, 153.3, 160.6; APCI-MS
298, 300; APCI-HRMS m/z: Calcd 299.9721. Found 299.9725; Purity
(HPLC): 99.8%.
7.49 (m, 4H); 13C NMR (DMSO-d6) d 68.5, 112.2, 117.7, 118.7,
120.6, 124.2, 124.6, 126.5, 128.0, 128.7, 130.9, 133.0, 136.0,
158.4; Purity (HPLC): 90.5%.
4.3.5. 4-(4-Bromobenzyloxy)benzonitrile (5e)
The title compound was prepared from 4-nitrobenzonitrile and
4-bromobenzyl alcohol in a yield of 22%: mp 118–120 °C (ethanol),
lit 130–131 °C [39]. 1H NMR (DMSO-d6) d 5.18 (s, 2H), 7.16 (d, 2H,
J = 8.7 Hz), 7.40 (d, 2H, J = 8.7 Hz), 7.58 (d, 2H, J = 8.3 Hz), 7.76 (d,
2H, J = 8.7 Hz); 13C NMR (DMSO-d6) d 68.8, 103.1, 115.9, 119.1,
121.3, 130.0, 131.4, 134.2, 135.6, 161.6; EIMS 287; EI-HRMS m/z:
Calcd 286.9946. Found 286.9953; Purity (HPLC): 98.9%.
4.2.8. 4-(4-Bromobenzyloxy)phthalonitrile (4h)
The title compound was prepared from 4-nitrophthalonitrile
and 4-bromobenzyl alcohol in a yield of 44%: mp 144–145 °C (eth-
anol). 1H NMR (DMSO-d6) d 5.24 (s, 2H), 7.42 (d, 2H, J = 7.5 Hz),
7.51 (d, 1H, J = 8.7 Hz), 7.60 (d, 2H, J = 7.5 Hz), 7.84 (s, 1H), 8.06
(d, 1H, J = 8.7 Hz); 13C NMR (DMSO-d6) d 69.6, 106.3, 115.7,
115.8, 116.2, 116.3, 120.5, 121.6, 130.2, 131.5, 134.9, 135.8,
161.4; Purity (HPLC): 96.3%.
4.3.6. 3-(4-Bromobenzyloxy)benzonitrile (5f)
The title compound was prepared from 3-nitrobenzonitrile and
4-bromobenzyl alcohol in a yield of 29%: mp 78–79 °C (ethanol).
1H NMR (DMSO-d6) d 5.15 (s, 2H), 7.35 (dd, 1H, J = 2.3, 8.3 Hz),
7.41 (d, 3H, J = 8.3 Hz), 7.48 (d, 1H, J = 8.3 Hz), 7.50 (s, 1H), 7.59
(d, 2H, J = 8.3 Hz); 13C NMR (DMSO-d6) d 68.8, 112.2, 117.7,
118.6, 120.6, 121.2, 124.8, 130.0, 130.9, 131.4, 135.8, 158.2; EIMS
287; EI-HRMS m/z: Calcd 286.9946. Found 286.9951; Purity
(HPLC): 98.1%.
4.2.9. 4-[2-(4-Bromophenyl)ethoxy]phthalonitrile (4i)
The title compound was prepared from 4-nitrophthalonitrile
and 2-(4-bromophenyl)ethanol in a yield of 7%: mp 103–105 °C
(ethanol). 1H NMR (DMSO-d6) d 3.04 (t, 2H, J = 6.7 Hz), 4.35 (t,
2H, J = 6.7 Hz), 7.28 (d, 2H, J = 7.9 Hz), 7.43 (d, 1H, J = 8.7 Hz),
7.49 (d, 2H, J = 8.3 Hz), 7.76 (s, 1H), 8.01 (d, 1H J = 8.7 Hz); 13C
NMR (DMSO-d6) d 33.7, 69.1, 106.0, 115.7, 116.2, 116.3, 119.6,
120.1, 120.3, 131.2, 131.3, 135.8, 137.2, 161.7; EIMS 326; EI-HRMS
m/z: Calcd 326.0055. Found 326.0044; Purity (HPLC): 97.5%.
4.3.7. 4-[2-(4-Bromophenyl)ethoxy]benzonitrile (5g)
The title compound was prepared from 4-nitrobenzonitrile and
2-(4-bromophenyl)ethanol in a yield of 61%: mp 80–81 °C (etha-
nol). 1H NMR (DMSO-d6) d 3.01 (t, 2H, J = 6.8 Hz), 4.25 (t, 2H,
J = 6.8 Hz), 7.07 (d, 2H, J = 8.7 Hz), 7.27 (d, 2H, J = 8.3 Hz), 7.47 (d,
2H, J = 8.3 Hz), 7.72 (d, 2H, J = 9.0 Hz); 13C NMR (DMSO-d6) d
34.0, 68.3, 102.9, 115.7, 119.3, 119.6, 131.3, 131.4, 134.3, 137.7,
161.9; Purity (HPLC): 98.4%.
4.3. Synthesis of C3- and C4-substituted benzonitrile analogs (5a–h)
The substituted benzonitrile analogs (5a–h) investigated in this
study were synthesized by reacting 3- or 4-nitrobenzonitrile with
the appropriate alcohol according to the procedure described
above for the synthesis of the phthalonitrile analogs (4a–i) [32].
4.3.1. 4-(Benzyloxy)benzonitrile (5a)
The title compound was prepared from 4-nitrobenzonitrile and
benzyl alcohol in a yield of 47%: mp 96–98 °C (ethanol), lit 96 °C
[37]. 1H NMR (DMSO-d6) d 5.19 (s, 2H), 7.17 (d, 2H, J = 9.0 Hz),
7.34 (t, 1H, J = 7.5 Hz), 7.39 (t, 2H, J = 7.9 Hz), 7.44 (d, 2H,
J = 7.5 Hz), 7.76 (d, 2H, J = 9.0 Hz); 13C NMR (DMSO-d6) d 69.7,
103.0, 115.9, 119.1, 127.9, 128.1, 128.5, 134.2, 136.1, 161.8; EIMS
209; EI-HRMS m/z: Calcd 209.0841. Found 209.0844; Purity
(HPLC): 99.2%.
4.3.8. 4-[2-(Benzyloxy)ethoxy]benzonitrile (5h)
The title compound was prepared from 4-nitrobenzonitrile and
2-benzyloxyethanol in a yield of 24% (oil). 1H NMR (DMSO-d6) d
3.77 (t, 2H, J = 4.5 Hz), 4.22 (t, 2H, J = 4.5 Hz), 4.54 (s, 2H), 7.11
(d, 2H, J = 8.7 Hz), 7.27 (m, 1H), 7.32 (m, 4H), 7.74 (d, 2H,
J = 8.7 Hz); 13C NMR (DMSO-d6) d 67.6, 67.9, 72.1, 102.8, 115.6,
119.1, 127.4, 127.5, 128.2, 134.1, 138.2, 162.0; EIMS 253; EI-HRMS
m/z: Calcd 253.1103. Found 253.1111; Purity (HPLC): 90.4%.
4.3.2. 3-(Benzyloxy)benzonitrile (5b)
4.4. Synthesis of benzyl phenyl ether analogs (6a–d)
The title compound was prepared from 3-nitrobenzonitrile and
benzyl alcohol in a yield of 43% (oil). 1H NMR (DMSO-d6) d 5.15 (s,
2H), 7.33 (m, 2H), 7.38 (t, 3H, J = 7.5 Hz), 7.45 (m, 3H), 7.49 (m, 1H);
13C NMR (DMSO-d6) d 69.6, 112.2, 117.7, 118.6, 120.5, 124.6, 127.8,
128.0, 128.5, 130.8, 136.3, 158.4; EIMS 209; EI-HRMS m/z: Calcd
209.0841. Found 209.0841; Purity (HPLC): 92.8%.
While benzyl phenyl ether (6a) is commercially available (Sig-
ma–Aldrich), analogs 6b–d were synthesized as follows: Phenol
(0.01 mmol) and an appropriate alkyl bromide (0.01 mmol; cin-
namyl bromide, 4-bromobenzyl bromide and 4-bromophenethyl
bromide for the synthesis of 6b, 6c an 6d, respectively) were dis-
solved in 40 mL dry acetone and K2CO3 (0.01 mmol) was added.
The reaction mixture was heated under reflux for 48 h, filtered
while hot and the residual K2CO3 was washed with acetone
(10 mL). The filtrate was cooled to room temperature and concen-
trated under reduced pressure. The resulting residue was recrystal-
lized twice from ethanol to yield the target ethers [40].
4.3.3. 4-{[(2E)-3-phenylprop-2-en-1-yl]oxy}benzonitrile (5c)
The title compound was prepared from 4-nitrobenzonitrile and
cinnamyl alcohol in a yield of 34%: mp 117–118 °C (ethanol), lit
121 °C [38]. 1H NMR (DMSO-d6) d 4.83 (d, 2H, J = 5.6 Hz), 6.51
(m, 1H), 6.78 (d, 1H, J = 15.8 Hz), 7.17 (d, 2H, J = 9.0 Hz), 7.27 (t,
1H, J = 7.1 Hz), 7.35 (t, 2H, J = 7.1 Hz), 7.48 (d, 2H, J = 7.5 Hz), 7.78
(d, 2H, J = 8.7 Hz); 13C NMR (DMSO-d6) d 68.5, 102.9, 115.8,
119.1, 124.0, 126.5, 128.0, 128.7, 133.1, 134.2, 135.9, 161.7; Purity
(HPLC): 91.5%.
4.4.1. {[(2E)-3-phenylprop-2-en-1-yl]oxy}benzene (6b)
The title compound was prepared from phenol and cinnamyl
bromide in a yield of 67%: mp 66–68 °C (ethanol), lit 68 °C [38].
1H NMR (DMSO-d6) d 4.71 (d, 2H, J = 5.6 Hz), 6.51 (m, 1H), 6.75
(d, 1H, J = 16.2 Hz), 6.93 (t, 1H, J = 7.5 Hz), 6.99 (d, 2H, J = 7.9 Hz),
7.29 (m, 3H), 7.34 (t, 2H, J = 7.9 Hz), 7.47 (d, 2H, J = 7.5 Hz); 13C
NMR (DMSO-d6) d 67.9, 114.7, 120.6, 125.0, 126.4, 127.9, 128.7,
129.5, 132.3, 136.2, 158.2; Purity (HPLC): 99.5%.
4.3.4. 3-{[(2E)-3-phenylprop-2-en-1-yl]oxy}benzonitrile (5d)
The title compound was prepared from 3-nitrobenzonitrile and
cinnamyl alcohol in a yield of 29% (yellow oil). 1H NMR (DMSO-d6)
d 4.79 (d, 2H, J = 6.0 Hz), 6.50 (m, 1H), 6.77 (d, 1H, J = 16.2 Hz), 7.27
(t, 1H, J = 7.5 Hz), 7.34 (t, 3H, J = 7.5 Hz), 7.40 (d, 1H, J = 7.5 Hz),

C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
123
4.4.2. 1-Bromo-4-(phenoxymethyl)benzene (6c)
and 0.135 lM for MAO-A and MAO-B, respectively, which are
The title compound was prepared from phenol and 4-bromob-
enzyl bromide in a yield of 58%: mp 96–98 °C (ethanol), lit 92.5–
93.5 °C [41]. 1H NMR (DMSO-d6) d 5.06 (s, 2H), 6.93 (t, 1H,
J = 7.5 Hz), 6.98 (d, 2H, J = 8.3 Hz), 7.28 (t, 2H, J = 7.5 Hz), 7.39 (d,
2H, J = 7.9 Hz), 7.56 (d, 2H, J = 8.3 Hz); 13C NMR (DMSO-d6) d
68.2, 114.8. 120.8, 120.9, 129.5, 129.7, 131.3, 136.6, 158.1; Purity
(HPLC): 99.5%.
approximately equal to the IC50 values for the inhibition of the
respective enzymes by 4d. The reactions were terminated with
the addition of 200 lL NaOH (2 N) and 1200 lL distilled water
and the rates of formation of 4-hydroxyquinoline were measured
as described above. All measurements were carried out in triplicate
and are expressed as mean SD [29,30].
4.7. Construction of Lineweaver–Burk plots
4.4.3. 1-Bromo-4-(2-phenoxyethyl)benzene (6d)
The title compound was prepared from phenol and 4-bromo-
phenethyl bromide in a yield of 54% (yellow oil). 1H NMR
(DMSO-d6) d 2.99 (t, 2H, J = 6.8 Hz), 4.15 (t, 2H, J = 2.8 Hz), 6.90
(m, 3H), 7.26 (t, 2H, J = 8.3 Hz), 7.29 (d, 2H, J = 8.3 Hz), 7.49 (d,
2H, J = 8.3 Hz); 13C NMR (DMSO-d6) d 34.2, 67.6, 114.4, 119.4,
120.6, 129.5, 131.1, 131.3, 138.0, 158.3; Purity (HPLC): 97.1%.
Sets of Lineweaver–Burk plots were constructed for the inhibi-
tion of MAO-A and -B by a selected inhibitor, compound 4d. The
initial MAO catalytic rates were measured at four different kynur-
amine concentrations (15–90
tor and then in the presence of three different concentrations of 4d.
For this purpose the concentrations of 4d were 0.1625–0.65
and 0.035–0.14 M for the inhibition studies with MAO-A and -B,
lM), firstly in the absence of inhibi-
l
M
l
4.5. IC50 determinations for the inhibition of human MAO
respectively. The concentrations of recombinant human MAO-A
and -B used for these measurements were 0.015 mg/mL. All enzy-
matic reactions and measurements were carried out as described
above. Linear regression analysis was performed using GraphPad
Prism [29,30].
Microsomes containing recombinant human MAO-A and -B
(5 mg/mL) were pre-aliquoted and stored at À70 °C. Enzymatic
reactions were prepared in potassium phosphate buffer (100 mM,
pH 7.4, made isotonic with KCl) and contained kynuramine at con-
centrations of 45
and -B, respectively. Various concentrations of the test inhibitor
(0–100 M) were added and the reactions were initiated with
the addition of the MAO enzymes (0.0075 mg/mL). All stock solu-
tions of the test inhibitors were prepared in DMSO and added to
the reactions to yield a final concentration of 4% (v/v) DMSO. The
l
M and 30
l
M for the incubations with MAO-A
4.8. Molecular modeling studies
l
Molecular docking and manipulations of protein models were
carried out in the Windows based Discovery Studio 1.7 molecular
modeling software (Accelrys) [33]. The test ligands were con-
structed within Discovery Studio, the hydrogen atoms were added
according to the appropriate protonation states at pH 7.4 and the
geometries were briefly optimized in Discovery Studio using a Dre-
iding-like forcefield (5000 iterations). The ligands were subse-
quently prepared for the docking simulations utilizing the
Prepare Ligands application of Discovery Studio and atom potential
types and partial charges were assigned with the Momany and
Rone CHARMm forcefield. The X-ray crystallographic structures
of MAO-A cocrystallized with harmine (PDB code: 2Z5X) [22] and
MAO-B cocrystallized with safinamide (PDB code: 2V5Z) [25] were
obtained from the Brookhaven Protein Data Bank (www.rcsb.org/
pdb). The protonation states of the ionizable amino acids residues
were calculated at pH 7.4 and hydrogen atoms were added accord-
ingly to the protein models. The valences of the FAD cofactors (oxi-
dized state) and cocrystallized ligands were corrected and the
models were automatically typed with the Momany and Rone
CHARMm forcefield. After a fixed constraint was applied to the
protein backbone, the models were energy minimized using the
Smart Minimizer algorithm. The Smart Minimizer algorithm per-
forms a steepest descent minimization (1000 steps) with a RMS
gradient tolerance of 3, followed by conjugate gradient minimiza-
tion. For this procedure the implicit generalized Born solvation
model with molecular volume was used with the dielectric con-
stant set to 4. The cocrystallized ligands and the backbone con-
straints were subsequently deleted from the models and the
binding sites were identified by a flood-filling algorithm. In both
the MAO-A and -B protein models, the cocrystallized water mole-
cules were deleted with the exception of three active site waters.
Since structures of MAO-B have shown that three active site water
molecules (HOH 1155, 1170 and 1351; A-chain of 2V5Z) are con-
served, all in the vicinity of the FAD cofactor they were retained
for the simulations [25]. In the MAO-A structure, the crystal waters
which occupy the analogous positions in the MAO-A active site
(HOH 710, 718 and 739; 2Z5X) to those waters in the MAO-B active
site cited above were retained. Docking was subsequently carried
out with the CDOCKER protocol allowing for the generation of 10
random ligand conformations and a heating target temperature
of 700 K in full potential mode. The docked ligands were finally
final volumes of the reactions were 500
lowed to incubate for 20 minutes at 37 °C and were terminated
with the addition of 200 L NaOH (2 N) and 1200 L distilled
lL. The reactions were al-
l
l
water. The reactions were centrifuged for 10 min at 16,000g and
the fluorescence of the MAO generated 4-hydroxyquinoline in
the supernatant fractions were measured (kex = 310 nm, kem
=
400 nm) [42]. The concentrations of 4-hydroxyquinoline were
determined with a linear calibration curve constructed from
solutions of authentic 4-hydroxyquinoline (0.047–1.50
calibration standards were prepared to a volume of 500
potassium phosphate buffer and contained 4% DMSO. To each stan-
dard was added 200 L NaOH (2 N) and 1200 L distilled water.
lM). The
lL in
l
l
IC50 values were determined by graphing the initial MAO catalytic
rates versus the logarithm of the inhibitor concentration to obtain
a sigmoidal dose–response curve. Each curve was constructed from
nine different inhibitor concentrations spanning at least three or-
ders of magnitude. These data were fitted to the one site competi-
tion model (GraphPad Prism) and the IC50 values were determined
in duplicate and are expressed as mean standard deviation (SD)
[29].
4.6. Time-dependent inhibition studies
To examine the reversibility of MAO inhibition, time-dependent
inhibition studies were carried with 4d as selected inhibitor. All
preincubations and incubations were carried out in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl). Com-
pound 4d, at concentrations of 1.31 lM and 0.27 lM for MAO-A
and -B, respectively, were preincubated with the enzymes
(0.03 mg/mL) for periods of 0, 15, 30, 60 min at 37 °C. These con-
centrations of 4d are 2 fold the measured IC50 values for the inhi-
bition of the respective MAO enzymes. Kynuramine at
concentrations of 45 lM and 30 lM for the incubations with
MAO-A and -B, respectively, were then added to the preincuba-
tions to yield a final enzyme concentration of 0.015 mg/mL. The
resulting reactions (500
lL final volume) were incubated at 37 °C
for 15 minutes. The final concentrations of 4d were 0.655
lM

124
C.I. Manley-King et al. / Bioorganic Chemistry 40 (2012) 114–124
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