Benzyloxynitrostyrene analogues – A novel class of selective and highly potent inhibitors of monoamine oxidase B

Article abstract of DOI:10.1016/j.ejmech.2016.11.016

This study examines a series of novel 3-benzyloxy-β-nitrostyrene analogues as a novel class of inhibitors of the monoamine oxidase (MAO) enzymes. MAO inhibitors are considered useful for the treatment of depression and Parkinson's disease, and have recently attracted attention as potential therapeutic agents for a range of disorders including Alzheimer's disease, prostate cancer and certain cardiomyopathies. This study shows that the 3-benzyloxy-β-nitrostyrene analogues are potent inhibitors of the MAO-B isoform with IC₅₀values in the nanomolar range (39–565 nM). Significantly, effectiveness towards MAO-B inhibition seems to be governed by the introduction of a 4″-fluoro-substituent on the benzyloxy ring, with compound 2b exhibiting the highest degree of MAO-B inhibition potency (IC₅₀= 0.039 μM) and selectivity (SI = 166) among the compounds investigated. Since some of the 3-benzyloxy-β-nitrostyrene analogues possess potencies that are comparable to that of the reversible inhibitor, safinamide (IC₅₀= 0.080 μM), it may be concluded that this class may be promising leads for the development of reversible and selective MAO-B inhibitors, that may be useful for the management of Parkinson's disease.

Full text of DOI:10.1016/j.ejmech.2016.11.016

European Journal of Medicinal Chemistry 125 (2017) 1193e1199
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Benzyloxynitrostyrene analogues e A novel class of selective and
highly potent inhibitors of monoamine oxidase B
,
,
,
b
a, b
Mietha M. Van der Walt a *, Gisella Terre’Blanche a b, Jacobus P. Petzer a , Anel Petzer
ꢀ
a Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa
b 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:
This study examines a series of novel 3-benzyloxy-b-nitrostyrene analogues as a novel class of inhibitors
Received 5 September 2016
Received in revised form
14 October 2016
Accepted 7 November 2016
Available online 9 November 2016
of the monoamine oxidase (MAO) enzymes. MAO inhibitors are considered useful for the treatment of
depression and Parkinson's disease, and have recently attracted attention as potential therapeutic agents
for a range of disorders including Alzheimer's disease, prostate cancer and certain cardiomyopathies. This
study shows that the 3-benzyloxy-b-nitrostyrene analogues are potent inhibitors of the MAO-B isoform
with IC50 values in the nanomolar range (39e565 nM). Significantly, effectiveness towards MAO-B in-
hibition seems to be governed by the introduction of a 400-fluoro-substituent on the benzyloxy ring, with
Keywords:
Benzyloxynitrostyrene
3-benzyloxy-b-nitrostyrene analogues
Slow reversible
Monoamine oxidase
MAO-B
Inhibition
Parkinson's disease
compound 2b exhibiting the highest degree of MAO-B inhibition potency (IC50 ¼ 0.039
tivity (SI ¼ 166) among the compounds investigated. Since some of the 3-benzyloxy-
analogues possess potencies that are comparable to that of the reversible inhibitor, safinamide
M), it may be concluded that this class may be promising leads for the development of
m
M) and selec-
b
-nitrostyrene
(IC50 ¼ 0.080
m
reversible and selective MAO-B inhibitors, that may be useful for the management of Parkinson's disease.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
of the 2-phenylethylamine, a trace amine which may further in-
crease neurotransmission via release of dopamine and norepi-
The monoamine oxidases (MAOs) are flavine adenine dinucle-
otide (FAD) dependent enzymes bound to the outer membrane of
mitochondria [1,2]. These enzymes consist of two isoforms, MAO-A
and MAO-B, and are responsible for the oxidative deamination of
neurotransmitters and dietary amines [3]. Due to their roles in the
metabolism of neurotransmitters, the MAOs are important targets
for the development of drugs for the treatment of neuropsychiatric
and neurodegenerative disorders. Inhibitors of MAO-A and MAO-B
have thus been used for the treatment of depression and Parkin-
son's disease, respectively [2,4]. In depression, MAO-A inhibitors
act by blocking the central metabolism of serotonin and norepi-
nephrine, while in Parkinson's disease MAO-B inhibitors act by
preventing the central breakdown of dopamine [5]. MAO-B in-
nephrine [7,8]. The beneficial effects of MAO-B inhibitors in
Parkinson's disease, however, extend beyond restoration of dopa-
minergic neurotransmission and MAO-B inhibitors have been re-
ported to possess neuroprotective properties [9]. This protective
effect is, in part, due to the reduction of potentially harmful by-
products of the MAO catalytic cycle [10]. These are hydrogen
peroxide and intermediate aldehydes, which may damage bio-
molecules if not rapidly cleared [5]. Hydrogen peroxide, in partic-
ular, is a substrate for the Fenton reaction and forms the highly
reactive and injurious hydroxyl radical [5]. Since MAO-B activity in
the brain increases with age [11], this isoform seems to be more
relevant (than MAO-A) for dopamine metabolism and hydrogen
peroxide formation in Parkinson's disease. To further underscore
the clinical importance of the MAOs, MAO-B inhibitors are also
under investigation for the treatment of Alzheimer's disease [12]
and as an aid to smoking cessation [13], while MAO-A inhibitors
may find future application in the treatment of certain cardiomy-
opathies [14] and prostate cancer [15].
hibitors are frequently used in combination with L-dopa, the
metabolic precursor of dopamine, in Parkinson's disease and pro-
vide symptomatic relief by conserving the depleted central dopa-
mine supply [6]. Inhibition of MAO-B also elevates the central levels
Two important considerations for MAO inhibitor design are
reversibility and specificity of inhibition. While irreversible MAO-A
inhibitors have been used for the treatment of depression, these
drugs have fallen out of favour due to a potentially fatal increase in
* Corresponding author.
E-mail
addresses:
dalenevanderwalt@gmail.com,
13035134@nwu.ac.za
(M.M. Van der Walt).
http://dx.doi.org/10.1016/j.ejmech.2016.11.016
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

1194
M.M. Van der Walt et al. / European Journal of Medicinal Chemistry 125 (2017) 1193e1199
blood-pressure that may occur when combined with tyramine-
containing foods [5,7]. The first generation MAO-B inhibitors are
known as irreversible inhibitors. Irreversible or “suicide” inhibitors
of MAO-B bind to the enzyme reversibly at first, but after oxidation
they form a covalent interaction with the enzyme, rendering it
permanently unavailable for amine metabolism [16]. In addition,
high or repeated concentrations of irreversible MAO-B inhibitors
lead to a loss of MAO-B selectivity [17] and thus contribute to the
potential adverse effect of tyramine-induced hypertension
(“cheese-reaction”) that is often associated with irreversible MAO-
A inhibition. On the other hand, reversible, competitive inhibitors
bind non-covalently to the active site of the enzyme [16], thus
allowing for competition with the substrate. Two irreversible MAO-
(Fig. 1). This was observed when compound 1b gained MAO-B in-
hibition activity and selectivity, while the compound lacking a
benzyloxy ring exhibited no MAO-B inhibition activity [21].
Halogen substitution on the benzyloxy ring was also explored in
the alkanamide derivatives and it was found that substitution on
the 300 position (meta) was favourable for MAO-B inhibition [22]. In
addition, acetophenone derivatives (1a) showed favourable MAO-B
inhibition with halogen substitution on the 400 position (para) of the
benzyloxy ring [20]. Based on the latter observations, it is plausible
to consider benzyloxy substitution to improve the inhibition po-
tency of a scaffold intended for MAO-B inhibition. It is noteworthy
that benzyloxy substitution is not the only structural modification
that may improve MAO-B inhibition potency. In the case of com-
pound 1b, the isopropylamine substituent was well tolerated for
MAO-B inhibition [21], the propionamide moiety (1c) was shown to
be favourable for MAO-B inhibition [1] and furthermore the small
acetyl group (1a) of the acetophenone derivatives also displayed
MAO-B inhibition [20]. However, introducing a thioanisole to the
amine (1d) resulted in a loss for both MAO-A and -B inhibition
(Fig. 1).
B
inhibitors, rasagiline (IC50
(IC50 ¼ 0.020 M), are currently in use for the treatment of Par-
kinson's disease [18,19], while a reversible inhibitor, safinamide
(IC50 ¼ 0.080 M), has entered clinical trials [1,19]. Therefore, the
¼
0.070 mM) and selegiline
m
m
MAO-B isoform is of therapeutic importance in PD and the need
exists to develop potent, selective and reversible MAO-B inhibitors.
A wide range of MAO-B inhibitors possess a benzyloxy side
chain to enhance inhibition potency. Recently a series of aceto-
phenone derivatives were evaluated for MAO activity. The most
potent MAO-B inhibitor 30-(400-chlorobenzyloxy)acetophenone (1a)
possessed a 30-benzyloxy on the acetophenone scaffold [20]. Other
benzyloxy containing examples include the amphetamine deriva-
tive [(4-benzyloxy)phenyl]-2-aminopropane (1b) possessing an
unsubstituted benzyloxy side chain [21], whereas the alkanamide
derivative, safinamide (1c) contains a meta substituted (300 position)
fluorine on the benzyloxy ring [1]. In both cases (1b and 1c), the
addition of a benzyloxy substituent seemed to enhance MAO-B
inhibition potency and selectivity over the MAO-A isoform
In analogy to the above observations the present study in-
vestigates the MAO inhibition properties of a series of novel 3-
benzyloxy-b-nitrostyrene analogues (2aek). These 3-benzyloxy-
b-nitrostyrenes will structurally possess a benzyloxy side chain on
the 30-position of the nitrostyrene scaffold similar to the substitu-
tion pattern of compound 1a (Fig. 1). In addition, by analogy with
compounds 1a and 1b various substituents (F, Cl, Br, CF3, CH3) were
explored at the 300 and 400 positions of the benzyloxy ring. This is the
first report of the MAO inhibition properties of the benzyloxyni-
trostyrene class of compounds, and as will be shown, good potency
MAO-B inhibitors exist among these compounds.
isopropylamine
NH2
benzyloxy
CH3
O
1b
MAO-B Ki: 0.71μM (r)
alkanamide
CH3
benzyloxy
benzyloxy
acetyl
Cl
4''
4''
3''
nitroethene
NO2
O
NH2
benzyloxy
3''
N
H
O
O
3'
CH
F
O
3
O
2a-k
nitrostyrene scaffold
1a
MAO-B IC50: 0.011μM (h)
1c
MAO-B IC50: 0.08 μM (h)
methylthioamphetamine
SCH3
CH3
N
H
benzyloxy
O
1d
MAO-B Ki: > 100 μM (r)
Fig. 1. Structures of 30-(400-chlorobenzyloxy)acetophenone (1a), [(4-benzyloxy)phenyl]-2-aminopropane (1b), 2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (1c), N-4-
benzyloxybenzyl-4-methylthioamphetamine (1d) as well as the general structure of the novel 3-benzyloxy- -nitrostyrene derivatives (2a-k).
b

M.M. Van der Walt et al. / European Journal of Medicinal Chemistry 125 (2017) 1193e1199
1195
O
O
X
HO
O
Br
H
i
+
X
H
a:X = H
3
b:X = 4-F
c:X = 4-Cl
d:X = 4-Br
e:X = 4-CF3
f:X = 4-CH3
ii, iii, iv
X
O
NO2
g:X = 3-F
h:X = 3-Cl
i: X = 3-Br
j: X = 3-CF3
2
k: X= 3-CH3
Scheme 1. Synthetic pathway to 3-benzyloxy-b
-nitrostyrene derivatives (2aek). Reagents and conditions: (i) K2CO3, DMF, r.t., 48 h; (ii) nitromethane, 0 ꢀC; (iii) sodium methoxide,
10 min, r.t.; (iv) 5 N HCl (aq), 15 min, 0 ꢀC.
2. Results and discussion
2b, the meta (300) substituted compound 2g (IC50 ¼ 0.103
mM) was
approximately 2.6-fold less potent. Furthermore, compound 2a d
possessing an unsubstituted benzyloxy ring d resulted in the
second-highest degree of MAO-B inhibition activity with an IC50
2.1. Chemistry
The desired 3-benzyloxy-
b
-nitrostyrene derivatives (2aek)
value of 0.085
compounds 2a and 2b presented inhibition activities in the same
range as selegiline (IC50 0.020 M) and rasagiline
(IC50 ¼ 0.070 M) that are currently on the market [16] and safi-
namide (IC50 ¼ 0.080 M) that has been evaluated in clinical trials
mM and 2.2-fold less potent than 2b. Noteworthy,
were successfully synthesized by a two-step protocol as outlined in
Scheme 1. First, the appropriate benzyloxybenzaldehyde (3aek)
was obtained after 3-hydroxybenzaldehyde was reacted with a
commercially available and suitable substituted benzyl bromide.
Thereafter, the crude benzyloxybenzaldehyde (3aek) was reacted
with nitromethane to afford the proposed test compounds (2aek).
The desired compounds were obtained in fair yields (22e73%) after
purification via recrystallization. The reaction conditions and
compound characterization are described in the experimental
Section (4.1). In each instance, the structures of the novel com-
pounds (2aek) were verified by 1H NMR, 13C NMR and mass
spectrometry and the purities were evaluated by HPLC analysis, as
cited in the supplementary material.
¼
m
m
m
[18]. MAO-B inhibition potency may be influenced by substitution
with bulky groups (Br, CF3) to such an extent that it may even lead
to a loss of MAO activity. This was demonstrated by the finding that
2i (300-Br), 2j (300-CF3) and 2e (400-CF3) resulted in IC50 values
ranging between 0.106 and 0.273
mM for MAO-B inhibition and that
2d (400-Br) was inactive for both MAO isoforms at the maximum
tested concentration (100 mM). The introduction of a methyl group
at the meta (300) position (2k) of the benzyloxy ring resulted in the
lowest inhibition potency reported for either of the MAO isoforms.
2.2. In vitro evaluation of MAO-A and MAO-B inhibition properties
Table 1
The IC50 values for the inhibition of recombinant human MAO-A and MAO-B by 3-
benzyloxy-
b
-nitrostyrene derivatives (2aek).
The 3-benzyloxy-b-nitrostyrene analogues (2aek) were evalu-
ated as inhibitors of MAO-A and MAO-B using the recombinant
human enzymes. MAO activity was measured fluorometrically [23]
by recording the rate of MAO-catalysed oxidation of kynuramine, a
mixed (MAO-A/B) substrate [24]. Kynuramine is oxidised by the
4''
X
3''
O
NO2
MAOs to ultimately yield 4-hydroxyquinoline,
a fluorescent
metabolite. The inhibition potencies of 2aek are expressed as the
mean IC50 values of triplicate determinations. The IC50 values and
MAO-B selectivity index (SI) d [IC50 (MAO-A)]/[IC50 (MAO-B)] d
for inhibitory effect were calculated for each test compound and are
documented in Table 1.
2a-k
X
IC50 SD (
m
M)a
SIb
hMAO-A
hMAO-B
To explore structure-activity relationships (SARs) of MAO inhi-
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
3.65 0.15
6.46 1.14
7.59 1.14
>100c
6.60 0.41
9.36 0.89
4.20 0.58
3.52 0.77
6.63 0.74
8.78 0.45
11.8 0.98
0.085 0.016
0.039 0.002
0.144 0.021
>100c
0.106 0.001
0.192 0.006
0.103 0.004
0.105 0.032
0.262 0.058
0.273 0.040
0.565 0.024
43
400-F
166
53
bition by the 3-benzyloxy-
ring was substituted on the meta (300) and para (400) positions with
halogens (F, Cl, Br) and alkyl groups (CF3, CH3). The 3-benzyloxy-
nitrostyrenes were generally found to be specific and potent MAO-
B inhibitors, with IC50 values ranging from 0.039 to 0.565 M.
b-nitrostyrene analogues, the benzyloxy
400-Cl
400-Br
400-CF3
400-CH3
300-F
300-Cl
300-Br
300-CF3
300-CH3
n.d.d
62
b
-
49
41
34
25
32
21
m
However, compound 2d displayed no inhibitory activity for either
MAO isoforms (Table 1).
2j
2k
The 400-fluoro-substituted compound 2b was observed to exhibit
the highest degree of MAO-B inhibition potency (IC50 ¼ 0.039
mM)
a
All IC50 values are expressed as the mean SD of triplicate determinations.
The selectivity index refers to the MAO-B isoform and is given as the ratio of
and selectivity (SI ¼ 166) among the compounds investigated.
Improved MAO-B inhibition potency may be governed by fluorine
substitution on the benzyloxy ring system, with para (400) substi-
tution (2b) preferred over meta (300) substitution (2g). Compared to
b
[IC50 (MAO-A)]/[IC50 (MAO-B)].
c
No MAO inhibition was observed at a maximum tested concentration of 100 mM.
The value was not determined.
d

1196
M.M. Van der Walt et al. / European Journal of Medicinal Chemistry 125 (2017) 1193e1199
Similarly, 2f showed the weakest MAO-A and -B inhibitions among
the para (400) substituted test compounds (2bef). In general, methyl
substitution did not seem to improve MAO inhibition of the
3-benzyloxy-b-nitrostyrenes. The MAO-B inhibition potency of the
test compounds (2aek) varied in the following order:
2b > 2a > 2g > 2h > 2e > 2c > 2f > 2i > 2j > 2k > 2d.
In contrast to MAO-B, the MAO inhibition results revealed that
the test compounds (2aek) act as weak inhibitors of the MAO-A
enzyme. With the exception of 2d, which did not inhibit MAO-A,
compounds 2a and 2h exhibited the greatest MAO-A inhibition
150
100
50
potencies, with the IC50 values recorded as 3.64 and 3.52
respectively. These values are comparable to toloxatone (3.26
m
M,
0
mM)
(R)-deprenyl
__2_b___
2b
No inhibitor
__________________
[25], a MAO-A inhibitor used for the treatment of depression. The
MAO-A inhibition properties may be therapeutically beneficial in
PD and should not be discarded. Inhibitors of MAO-A are indicated
for treating depressive illness [6], a non-motor symptom of PD that
is often neglected during conventional treatment [26].
not dialyzed
dialyzed
Fig. 2. Reversibility of inhibition of MAO-B by 2b. The MAO-B enzyme was pre-
incubated with 2b at 4 ꢁ IC50 for 15 min, dialyzed for 24 h and the residual MAO-B
enzyme activity was subsequently measured (2b-dialyzed). MAO-B was similarly
preincubated in the absence (no inhibitor-dialyzed) and presence of the irreversible
MAO-B inhibitor, (R)-deprenyl ((R)-deprenyl-dialyzed), and dialyzed. For comparison,
the residual MAO-B activity of undialyzed mixtures of MAO-B with 2b is also shown
(2b-not dialyzed).
2.3. In vitro evaluation of the reversibility of MAO-B inhibition
The 3-benzyloxy-
selective for MAO-B over the MAO-A isoform. As mentioned above,
compound 2b (IC50 ¼ 0.039 M) possessed the highest MAO-B
b-nitrostyrene analogues were found to be
3. Conclusion
m
The aim of the study reported here was to identify potent, se-
lective and reversible inhibitors of the MAO-B enzyme. This was
achieved by the successful synthesis of the novel 3-benzyloxy-b-
inhibitory potency among the test compounds (2aek). For this
reason, 2b was selected as representative compound to determine
if the benzyloxynitrostyrene class of MAO-B inhibitors act as
reversible or irreversible enzyme inhibitors. The reversibility of
MAO-B inhibition by 2b was investigated by dialysis. For this pur-
pose, the recovery of the MAO-B enzymatic activity after dialysis of
mixtures of MAO-B and 2b was measured [24]. A negative control
was obtained by performing dialyses of MAO-B in the absence of
the inhibitor and a positive control by employing a similar dialysis
in the presence of the known irreversible inhibitor, (R)-deprenyl.
The MAO-B enzyme and the inhibitors (at a concentration equal to
4 ꢁ IC50) were firstly pre-incubated for 15 min, and subsequently
dialyzed for 24 h. The reactions were diluted 2-fold with the
addition of kynuramine and the residual MAO-B catalytic rates
were measured. For comparison, the undialyzed mixtures of 2b and
MAO-B were also recorded.
nitrostyrenes (2aek) and these compounds were recorded as
highly potent MAO-B inhibitors with IC50 values in the low nano-
molar range (39e565 nM). Among the compounds evaluated 2b
(IC50 ¼ 0.039
was the most potent inhibitor with the greatest selectivity
(SI ¼ 166). The good potency MAO-B inhibition by 3-benzyloxy-
m b-nitrostyrene,
M), the 400-F substituted 3-benzyloxy-
b
-
nitrostyrene may be explained by their structural similarity to 30-
(400-chlorobenzyloxy)acetophenone (1a) and safinamide (1c), and it
may be suggested that these compounds exhibit similar binding
modes to human MAO-B as safinamide. On examination it was
found that substitution with bulky groups (Br, CF3, CH3) in general
led to lower MAO-B inhibition. Furthermore, the reversible dialysis
of 2b with MAO-B showed possible tight-binding interaction.
In conclusion, this study shows, for the first time, that the
benzyloxynitrostyrene class of compounds act as inhibitors of
MAO-B, with good potencies comparing well with the reversible
inhibitor safinamide and the irreversible inhibitors selegiline and
rasagiline. Considering the above, the novel synthetic 3-benzyloxy-
It was found that the MAO-B enzyme's catalytic activities were
recovered to 38% of the control levels. For comparison, the undia-
lyzed mixtures of 2b with MAO-B were also recorded and found to
be recovered to 37%. As expected, the enzymatic catalytic activity of
the irreversible inhibitor, (R)-deprenyl, was not recovered (5% of
control). Taking the latter into consideration, the results indicate
that inhibition of MAO-B by 2b cannot be readily reversed by
dialysis. This observation may be explained by a non-covalent
tight-binding behaviour of 2b to the MAO-B enzyme [27]. Various
examples of this type of MAO-B inhibition in the literature exists.
For example, chromone derivatives have been reported to possess
non-covalent tight-binding interactions [28,29], while a series of
reversible and tight-binding MAO-B selective inhibitors has also
been described [30]. Furthermore, slowly-reversible MAO-B inhi-
bition was demonstrated by fluoxetine and norfluoxetine after
dialysis [31]. According to Harfenist and co-workers [32] values of
percentage of reversibility between 0 and 20% indicates that
binding of a compound to the enzyme is essentially not reversible
by dialysis, between 20 and 80% indicate slow or partial dialysis in
the time allowed, while values higher than 80% shows that binding
to the enzyme is readily reversible. Since MAO-B inhibition was
only partially reversed by dialysis, it is therefore plausible, that
compound 2b may act as a potent MAO-B inhibitor (in vitro)
involving a tight-binding interaction (Fig. 2). However, further
investigation is needed.
b-nitrostyrene analogues demonstrated to be potent, selective in-
hibitors of MAO-B rather than the MAO-A isoform and for this
reason are promising drug candidates worth testing for PD treat-
ment in future.
4. Experimental section
4.1. Chemistry
All starting materials, reagents and solvents were purchased
from Sigma-Aldrich and were used without further purification.
The structures of the novel synthesized 3-benzyloxy-b-nitro-
styrene analogues were elucidated by proton (1H) and carbon (13C)-
NMR spectra and recorded on a BrukerAvance III 600 spectrometer
in deuterochloroform (CDCl3). The chemical shifts are expressed in
parts per million (d) relative to the signal of tetramethylsilane and
coupling constants J in hertz (Hz). Spin multiplicities are abbrevi-
ated as follow: s (singlet), d (doublet), t (triplet), q (quartet) or m
(multiplet). Furthermore, high resolution mass spectra (HRMS)
were recorded with a BrukermicrOTOF-Q II mass spectrometer in

M.M. Van der Walt et al. / European Journal of Medicinal Chemistry 125 (2017) 1193e1199
1197
atmospheric-pressure chemical ionization (APCI) mode. Melting
4.2.2. 1-[(4-fluorophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2b)
The title compound (light yellow crystals) was prepared from 3-
hydroxybenzaldehyde, 4-fluorobenzyl bromide and nitromethane
in a yield of 45%: mp 95.2e96.0 ꢀC (ethyl acetate); 1H NMR (Bru-
points (mp) were measured with a Buchi M-545 melting point
apparatus and are uncorrected. The degree of purity for each of the
test compounds (2aek) was estimated by HPLC analysis, which was
carried out with an Agilent 1100 HPLC system equipped with a
quaternary pump and an Agilent 1100 series diode array detector.
Each HPLC run lasted 15 min and a time period of 5 min was
kerAvance III 600, CDCl3)
d 5.04 (s, 2H), 7.06e7.09 (m, 4H), 7.14 (d,
1H, J ¼ 7.9 Hz), 7.35 (t,1H, J ¼ 7.9 Hz), 7.39 (q, 2H, J ¼ 8.7 Hz), 7.53 (d,
allowed for equilibration between runs. A volume of 20 mL of so-
1H, J ¼ 13.6 Hz), 7.94 (d, 1H, J ¼ 13.6 Hz); 13C NMR (BrukerAvance III
lutions of the test compounds in acetonitrile (1 mM) was injected
into the HPLC system and the eluent was monitored at wavelengths
of 254 nm.
600, CDCl3) d 69.5, 115.0, 115.6, 115.7, 118.6, 112.1, 129.3, 129.4, 130.5,
131.4, 132.0, 132.0, 137.4, 138.9, 159.1, 161.8, 163.4; APCI-HRMS m/z:
calcd for C15H13FNO3 (MHþ), 274.0879, found 274.0856; Purity
(HPLC): 100%.
4.2. Synthetic preparation
4.2.3. 1-[(4-chlorophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2c)
General synthetic procedure to prepare the 3-benzyloxy-
b-
nitrostyrene analogues.
The title compound (light yellow needles) was prepared from 3-
hydroxybenzaldehyde, 4-chlorobenzyl bromide and nitromethane
in a yield of 64%: mp 104.9e106.7 ꢀC (ethyl acetate); 1H NMR
The 3-benzyloxy-b-nitrostyrene analogues (2aek) were pre-
pared via the synthesis of benzyloxybenzaldehydes followed by
appropriately substituting the aromatic aldehydes with nitro-
methane (Scheme 1). The benzyloxybenzaldehyde derivatives
(3aek) were prepared according to a protocol described in the
literature [33]. To a stirred solution of 3-hydroxybenzaldehyde
(16.4 mmol) in N,N-dimethylformamide (60 mL), potassium car-
bonate (24.59 mmol) was added in successive portions at room
temperature. The appropriately substituted benzyl bromide
(16.4 mmol) was subsequently added and stirring continued for
48 h. The reaction was quenched by pouring the reaction mixture
into cold water (50 mL). Compounds 3aee and 3g resulted in a
precipitate that was collected via filtration and washed with
100 mL cold water. However, compounds 3f and 3hek yielded an
oily residue that was extracted with ethyl acetate, dried with
magnesium sulphate and then the combined organic phase was
evaporated to dryness. All the intermediates (3aek) obtained were
used without further purification in the next step.
(BrukerAvance III 600, CDCl3) d 5.05 (s, 2H), 7.06e7.08 (m, 2H), 7.14
(d, 1H, J ¼ 7.9 Hz), 7.34e7.36 (m, 5H), 7.53 (d, 1H, J ¼ 13.6 Hz), 7.93
(d, 1H, J ¼ 13.6 Hz); 13C NMR (BrukerAvance III 600, CDCl3)
d 69.4,
115.0, 118.6, 122.1, 128.7, 128.9, 130.5, 131.4, 134.0, 134.7, 137.4, 138.8,
159.0; APCI-HRMS m/z: calcd for C15H13ClNO3 (MHþ), 290.0584,
found 290.0602; Purity (HPLC): 100%.
4.2.4. 1-[(4-bromophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2d)
The title compound (light yellow needles) was prepared from 3-
hydroxybenzaldehyde, 4-bromobenzyl bromide and nitromethane
in a yield of 64%: mp 95.8e97.0 ꢀC (ethyl acetate); 1H NMR (Bru-
kerAvance III 600, CDCl3)
d 5.03 (s, 2H), 7.05e7.08 (m, 2H), 7.14 (d,
1H, J ¼ 7.9 Hz), 7.29 (d, 2H, J ¼ 8.3 Hz), 7.35 (t, 1H, J ¼ 7.9 Hz),
7.50e7.54 (m, 3H), 7.93 (d, 1H, J ¼ 13.6 Hz); 13C NMR (BrukerAvance
III 600, CDCl3)
d 69.4, 115.0, 118.6, 122.1, 122.2, 129.0, 130.5, 131.4,
The 3-benzyloxy-b-nitrostyrene analogues (2aek) examined in
131.8, 135.2, 137.4, 138.8, 159.0; APCI-HRMS m/z: calcd for
this study was prepared by the protocol described by Tanaka and
co-workers [34]. Nitromethane (12.2 mmol) and sodium meth-
oxide (28% methanol solution, 9.8 mmol) was added together and
cooled to 0 ꢀC. In a separate round-bottom flask 9.4 mmol of the
appropriate benzyloxybenzaldehyde intermediate (3aek) was
mixed with methanol (20 mL) and sodium methoxide (28% meth-
anol solution, 1.48 mmol). The latter mixture was added slowly to
the first and stirring continued for 10 min at room temperature.
After the reaction mixture was cooled to 0 ꢀC, 5 N aqueous hy-
drochloric acid solution (40 mL) was added and the reaction
mixture was stirred for 15 min at room temperature. The resulting
precipitate was collected via filtration to yield the crude 3-
C
15H13BrNO3 (MHþ), 334.0079, found 334.0077; Purity (HPLC):
100%.
4.2.5. 1-[(4-(trifluoromethyl)phenyl)methoxy]-3-[(E)-2-
nitroethenyl]benzene (2e)
The title compound (light yellow crystals) was prepared from 3-
hydroxybenzaldehyde, 4-trifluoromethylbenzyl bromide and
nitromethane in a yield of 43%: mp 95.9e96.5 ꢀC (methanol); 1H
NMR (BrukerAvance III 600, CDCl3) d 5.15 (s, 2H), 7.07e7.10 (m, 2H),
7.16 (d, 1H, J ¼ 7.5 Hz), 7.37 (t,1H, J ¼ 7.9 Hz), 7.54 (dd, 3H, J ¼ 3.4 Hz,
J ¼ 11.3 Hz), 7.65 (d, 2H, J ¼ 8.3 Hz), 7.94 (d, 1H, J ¼ 13.6 Hz); 13C
benzyloxy-
b
-nitrostyrene analogues. The desired 3-benzyloxy-
b
-
NMR (BrukerAvance III 600, CDCl3) d 69.3, 115.0, 118.5, 122.2, 123.1,
nitrostyrene analogues (2aek) were obtained after recrystalliza-
tion from an appropriate solvent. The NMR results, of all the test
compounds, indicated the exclusive formation of an Eeisomer, as
corroborated by a significant coupling constant (J) of approximately
13 Hz.
124.9, 125.7, 127.4, 130.3, 130.5, 130.6, 131.5, 137.5, 138.8, 140.3,
159.0; APCI-HRMS m/z: calcd for C16H12F3NO3 (M), 323.0848, found
323.0758; Purity (HPLC): 100%.
4.2.6. 1-[(4-methylphenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2f)
4.2.1. 1-(benzyloxy)-3-[(E)-2-nitroethenyl]benzene (2a)
The title compound (light yellow needles) was prepared from 3-
hydroxybenzaldehyde, 4-methylbenzyl bromide and nitromethane
in a yield of 32%: mp 85.5e86.0 ꢀC (methanol); 1H NMR (Bruker-
The title compound (light yellow crystals) was prepared from 3-
hydroxybenzaldehyde, benzyl bromide and nitromethane in a yield
of 23%: mp 92.5e93.6 ꢀC (methanol); 1H NMR (BrukerAvance III
Avance III 600, CDCl3) d2.36 (s, 3H), 5.04 (s, 2H), 7.08e7.09 (m, 2H),
600, CDCl3)
d
5.09 (s, 2H), 7.09e7.10 (m, 2H), 7.13 (d, 1H, J ¼ 7.5 Hz),
7.12 (d, 1H, J ¼ 7.5 Hz), 7.20 (d, 2H, J ¼ 7.5 Hz), 7.32e7.36 (m, 3H),
7.33e7.36 (m, 2H), 7.38e7.43 (m, 4H), 7.53 (d, 1H, J ¼ 13.6 Hz), 7.94
7.53 (d, 1H, J ¼ 13.9 Hz), 7.94 (d, 1H, J ¼ 13.9 Hz); 13C NMR (Bru-
(d, 1H, J ¼ 13.6 Hz); 13C NMR (BrukerAvance III 600, CDCl3)
d
70.2,
kerAvance III 600, CDCl3) d 21.2, 70.1, 115.0, 118.8, 121.9, 127.6, 129.4,
115.0, 118.7, 122.0,127.4, 128.2, 128.7, 130.4, 131.3, 136.2, 137.3, 139.0,
159.2; APCI-HRMS m/z: calcd for C15H14NO3 (MHþ), 256.0974,
found 256.0979; Purity (HPLC): 100%.
130.4, 131.3, 133.1, 137.2, 138.1, 139.0, 159.3; APCI-HRMS m/z: calcd
for C16H16NO3 (MHþ), 270.1130, found 270.1135; Purity (HPLC):
100%.

1198
M.M. Van der Walt et al. / European Journal of Medicinal Chemistry 125 (2017) 1193e1199
4.2.7. 1-[(3-fluorophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2g)
270.1134; Purity (HPLC): 100%.
The title compound (yellow crystals) was prepared from 3-
hydroxybenzaldehyde, 3-fluorobenzyl bromide and nitromethane
in a yield of 22%: mp 83.9e84.6 ꢀC (methanol); 1H NMR (Bruker-
4.3. In vitro evaluation of MAO inhibition properties
4.3.1. Determination of MAO-A and MAO-B inhibition IC50 values
The determination of IC50 values for the inhibition of the MAOs
has been reported [24]. The recombinant human enzymes (Sigma-
Aldrich) were used as enzyme sources and kynuramine served as
substrate. Kynuramine is metabolised by the MAO enzymes to yield
4-hydroxyquinoline, a fluorescent compound when dissolved in
alkaline media. The formation of 4-hydroxyquinoline was thus
measured by fluorescence spectrophotometry (lex ¼ 310 nm;
lem ¼ 400 nm) [23]. The rates of MAO catalysis were recorded in the
presence of the test compounds (2aek), and sigmoidal graphs of
rate versus the logarithm of inhibitor concentration were prepared.
The IC50 values of the test compounds (2aek) were estimated from
these graphs and are reported as the mean standard deviation
(SD) of triplicate determinations (Table 1).
Avance III 600, CDCl3)
d 5.08 (s, 2H), 7.00e7.04 (m, 1H), 7.07e7.09
(m, 2H), 7.13e7.15 (m, 2H), 7.18 (d,1H, J ¼ 7.5 Hz), 7.34e7.37 (m, 2H),
7.53 (d, 1H, J ¼ 13.6 Hz), 7.94 (d, 1H, J ¼ 13.6 Hz); 13C NMR (Bru-
kerAvance III 600, CDCl3) d 69.3, 69.3, 114.1, 114.3, 115.0, 115.0, 115.2,
118.6, 122.1, 122.7, 122.7,130.3,130.3,130.5,131.4,137.4,138.8,138.8,
159.0, 162.2, 163.8; APCI-HRMS m/z: calcd for C15H13FNO3 (MHþ),
274.0879, found 274.0879; Purity (HPLC): 100%.
4.2.8. 1-[(3-chlorophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2h)
The title compound (bright yellow crystals) was prepared from
3-hydroxybenzaldehyde, 3-chlorobenzyl bromide and nitro-
methane in a yield of 73%: mp 91.6e93.8 ꢀC (methanol); 1H NMR
(BrukerAvance III 600, CDCl3) d 5.06 (s, 2H), 7.06e7.09 (m, 2H), 7.15
(d, 1H, J ¼ 7.5 Hz), 7.28e7.32 (m, 3H), 7.36 (t, 1H, J ¼ 7.9 Hz), 7.42 (br
4.3.2. Recovery of enzyme activity after dialysis
s, 1H), 7.53 (d, 1H, J ¼ 13.6 Hz), 7.94 (d, 1H, J ¼ 13.9 Hz); 13C NMR
The protocol for examining the reversibility of MAO inhibition
by dialysis has been reported [24]. The MAO-B enzyme was first
pre-incubated with compound 2b for 15 min at 37 ꢀC, and subse-
quently dialyzed at 4 ꢀC for 24 h. The inhibitor concentration in
these pre-incubations is 4 ꢁ IC50 value of 2b. After dialysis, the pre-
incubations were diluted two-fold with the addition of kynuramine
(BrukerAvance III 600, CDCl3) d 69.3, 115.0, 118.6, 122.2, 125.3, 127.4,
128.4, 130.0, 130.5, 131.4, 134.6, 137.4, 138.3, 138.8, 159.0; APCI-
HRMS m/z: calcd for
C
15H13ClNO3 (MHþ), 290.0585, found
290.0582; Purity (HPLC): 100%.
4.2.9. 1-[(3-bromophenyl)methoxy]-3-[(E)-2-nitroethenyl]benzene
(2i)
(50 mM final concentration) and the reactions were incubated for a
further 20 min at 37 ꢀC. Finally, 4-hydroxyquinoline concentrations
were measured by fluorescence spectrophotometry. All experi-
ments were carried out in triplicate and the catalytic rate data are
reported as mean SD (Fig. 2). Similar experiments carried out in
the absence of inhibitor and presence of (R)-deprenyl was also
included in this study. Furthermore, the rates of undialyzed pre-
incubations of MAO and the 2b were also measured.
The title compound (bright yellow crystals) was prepared from
3-hydroxybenzaldehyde, 3-bromobenzyl bromide and nitro-
methane in a yield of 37%: mp 96.4e99.5 ꢀC (methanol/ethyl ace-
tate); 1H NMR (BrukerAvance III 600, CDCl3)
d 5.05 (s, 2H),
7.06e7.09 (m, 2H), 7.15 (d,1H, J ¼ 7.5 Hz), 7.26 (t,1H, J ¼ 7.9 Hz), 7.35
(q, 2H, J ¼ 7.9 Hz), 7.46 (d, 1H, J ¼ 8.3 Hz), 7.54 (d, 1H, J ¼ 13.9 Hz),
7.58 (br s, 1H), 7.94 (d, 1H, J ¼ 13.5 Hz); 13C NMR (BrukerAvance III
600, CDCl3)
d 69.2, 115.0, 118.6, 122.2, 122.8, 125.8, 130.3, 130.3,
Author contributions
130.5, 131.3, 131.4, 137.4, 138.5, 138.8, 158.9; APCI-HRMS m/z: calcd
for C15H13BrNO3 (MHþ), 334.0079, found 334.0086; Purity (HPLC):
100%.
MMvdW designed the study concept. The organic synthesis was
conducted by MMvdW, while AP performed the biological evalua-
tions. The raw biological data was processed by AP and JP. The
interpretation and analysis of the NMR, MS, HPLC and biological
results were performed by MMvdW and GT. MMvdW and GT
drafted, revised and wrote the manuscript with feedback and
suggestions from JP and AP.
4.2.10. 1-[(3-(trifluoromethyl)phenyl)methoxy]-3-[(E)-2-
nitroethenyl]benzene (2j)
The title compound (bright yellow crystals) was prepared from
3-hydroxybenzaldehyde, 3-trifluoromethylbenzyl bromide and
nitromethane in a yield of 54%: mp 88.8e89.7 ꢀC (methanol); 1H
NMR (BrukerAvance III 600, CDCl3) d5.13 (s, 2H), 7.09e7.12 (m, 2H),
Acknowledgements
7.17 (d, 1H, J ¼ 7.5 Hz), 7.37 (t, 1H, J ¼ 7.9 Hz), 7.51e7.56 (m, 2H), 7.61
(t, 2H, J ¼ 7.5 Hz), 7.70 (br s, 1H),7.95 (d, 1H, J ¼ 13.6 Hz); 13C NMR
We are grateful to Dr. J. Jordaan and Mr. A. Joubert of the SASOL
Centre for Chemistry, North-West University, for recording the
NMR and MS spectra, respectively. We are also thankful to Prof. J. du
Preez for HPLC analysis and a special thanks to Prof P.J. Milne for his
insightful suggestions and comments. Financial support for this
work was provided by the North-West University, the National
Research Foundation and the Medical Research Council, South
Africa.
(BrukerAvance III 600, CDCl3) d 69.4, 115.0, 118.5, 122.3, 123.0, 124.0,
124.1, 124.1, 124.1, 124.9, 125.0,125.0,125.1, 125.1, 129.2,130.6,130.6,
131.0, 131.2, 131.5, 137.3, 137.5, 138.8, 159.0; APCI-HRMS m/z: calcd
for C16H13F3NO3 (MHþ), 324.0848, found 324.0850; Purity (HPLC):
100%.
4.2.11. 1-[(3-methylphenyl)methoxy]-3-[(E)-2-nitroethenyl]
benzene (2k)
The title compound (light yellow crystals) was prepared from 3-
hydroxybenzaldehyde, 3-methylbenzyl bromide and nitromethane
in a yield of 34%: mp 77.8e79.4 ꢀC (methanol); 1H NMR (Bruker-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.016.
Avance III 600, CDCl3) d 2.37 (s, 3H), 5.05 (s, 2H), 7.09e7.10 (m, 2H),
7.12e7.16 (m, 2H), 7.21e7.27 (m, 2H), 7.28 (t, 1H, J ¼ 7.5 Hz), 7.35 (t,
1H, J ¼ 7.9 Hz), 7.53 (d, 1H, J ¼ 13.9 Hz), 7.94 (d, 1H, J ¼ 13.6 Hz); 13
C
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