The inhibition of catechol O-methyltransferase and monoamine oxidase by tetralone and indanone derivatives substituted with the nitrocatechol moiety

Article abstract of DOI:10.1016/j.bioorg.2021.105130

The enzymes, catechol O-methyltransferase (COMT) and monoamine oxidase (MAO) are important drug targets, and inhibitors of these enzymes are established therapy for symptomatic Parkinson's disease (PD). COMT inhibitors enhance the bioavailability of levodopa to the brain, and therefore are combined with levodopa for the treatment of motor fluctuations in PD. Inhibitors of the MAO-B isoform, in turn, are used as monotherapy or in conjunction with levodopa in PD, and function by reducing the central degradation of dopamine. It has been reported that 1-tetralone and 1-indanone derivatives are potent and specific inhibitors of MAO-B, while compounds containing the nitrocatechol moiety (e.g. tolcapone and entacapone) are often potent COMT inhibitors. The present study attempted to discover compounds that exhibit dual COMT and MAO-B inhibition by synthesizing series of 1-tetralone, 1-indanone and related derivatives substituted with the nitrocatechol moiety. These compounds are structurally related to series of nitrocatechol derivatives of chalcone that have recently been investigated as potential dual COMT/MAO inhibitors. The results show that 4-chromanone derivative (7) is the most promising dual inhibitor with IC₅₀ values of 0.57 and 7.26 μM for COMT and MAO-B, respectively, followed by 1-tetralone derivative (4d) with IC₅₀ values of 0.42 and 7.83 μM for COMT and MAO-B, respectively. Based on their potent inhibition of COMT, it may be concluded that nitrocatechol compounds investigated in this study are appropriate for peripheral COMT inhibition, which represents an important strategy in the treatment of PD.

Full text of DOI:10.1016/j.bioorg.2021.105130

Bioorganic Chemistry 114 (2021) 105130
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The inhibition of catechol O-methyltransferase and monoamine oxidase by
tetralone and indanone derivatives substituted with the
nitrocatechol moiety
,
*
a
,
,b
Andries D. de Beera, Lesetja J. Legoabea , Anel Petzer b, Jacobus P. Petzera
´
a Centre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2520, South Africa
b Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom 2520, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
The enzymes, catechol O-methyltransferase (COMT) and monoamine oxidase (MAO) are important drug targets,
and inhibitors of these enzymes are established therapy for symptomatic Parkinson’s disease (PD). COMT in-
hibitors enhance the bioavailability of levodopa to the brain, and therefore are combined with levodopa for the
treatment of motor fluctuations in PD. Inhibitors of the MAO-B isoform, in turn, are used as monotherapy or in
conjunction with levodopa in PD, and function by reducing the central degradation of dopamine. It has been
reported that 1-tetralone and 1-indanone derivatives are potent and specific inhibitors of MAO-B, while com-
pounds containing the nitrocatechol moiety (e.g. tolcapone and entacapone) are often potent COMT inhibitors.
The present study attempted to discover compounds that exhibit dual COMT and MAO-B inhibition by synthe-
sizing series of 1-tetralone, 1-indanone and related derivatives substituted with the nitrocatechol moiety. These
compounds are structurally related to series of nitrocatechol derivatives of chalcone that have recently been
investigated as potential dual COMT/MAO inhibitors. The results show that 4-chromanone derivative (7) is the
Catechol O-methyltransferase
COMT
Monoamine oxidase
MAO
Parkinson’s disease
Chalcone
Tetralone
Indanone
Chromanone
most promising dual inhibitor with IC50 values of 0.57 and 7.26
μ
M for COMT and MAO-B, respectively, followed
by 1-tetralone derivative (4d) with IC50 values of 0.42 and 7.83
μM for COMT and MAO-B, respectively. Based on
their potent inhibition of COMT, it may be concluded that nitrocatechol compounds investigated in this study are
appropriate for peripheral COMT inhibition, which represents an important strategy in the treatment of PD.
1. Introduction
treatment with these compounds, COMT inhibition also reduces the
conversion (via O-methylation) of levodopa to 3-O-methyldopa, a
Catechol O-methyltransferase (COMT) is a magnesium ion (Mg2+
)
metabolite which competes with levodopa for uptake at the blood–brain
barrier [3,4]. Clinical observations suggest that poor response to levo-
dopa therapy could be linked to high plasma levels of 3-O-methyldopa
[8]. While the peripheral inhibition of COMT is an important strategy in
PD, inhibitors that target centrally located COMT may represent a future
treatment for schizophrenia. COMT plays a unique role in regulating
dopamine levels and function in the prefrontal cortex, and centrally
acting inhibitors could therefore represent a potential treatment of
cognitive disorder in schizophrenia and in other conditions involving
dopaminergic dysfunction in the prefrontal region [9]. Among the
COMT inhibitors in clinical use, tolcapone is the only drug that acts in
the central nervous system, while entacapone and opicapone are
peripherally-acting inhibitors (Fig. 1) [10].
dependent enzyme that catalyzes the transfer of a methyl group from its
cofactor, S-adenosyl-L-methionine (SAM), to a hydroxyl group of a
catechol substrate. COMT substrates are of both endogenous and exog-
enous origin and notably include neurotransmitters such as dopamine
and noradrenaline, and the metabolic precursor of dopamine, levodopa
[1,2]. The metabolism of neurotransmitters by COMT results in the
termination of their physiological activities, and this enzyme is therefore
of therapeutic relevance. Inhibitors of COMT are generally used for the
treatment of Parkinson’s disease (PD) and act by reducing the peripheral
metabolism of levodopa [3–6]. This increases the bioavailability of
levodopa to the brain and COMT inhibitors are frequently combined
with levodopa as first-line add-on therapy of motor fluctuations in PD
[7]. In addition to enhancing peripheral levodopa levels following
Another enzyme that plays a key role in the metabolism of dopamine
* Corresponding author.
E-mail addresses: lesetja.legoabe@nwu.ac.za (L.J. Legoabe), 12264954@nwu.ac.za (A. Petzer), jacques.petzer@nwu.ac.za (J.P. Petzer).
https://doi.org/10.1016/j.bioorg.2021.105130
Received 21 September 2020; Received in revised form 23 June 2021; Accepted 24 June 2021
Available online 28 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
in neurotransmitter metabolism and thus relevant drug targets for
neurological disorders, most notably PD, previous studies have
attempted to discover compounds that inhibit both COMT and MAO. In
this respect nitrocatechol derivatives of chalcone were investigated as
dual inhibitors [18,19]. The nitrocatechol moiety is considered privi-
leged for COMT inhibition and is present in all clinically used COMT
inhibitors (e.g. tolcapone, entacapone, opicapone), while chalcones
have been reported to act as high potency and specific inhibitors of
MAO-B [20]. Examples of nitrocatechol derivatives of chalcone with
dual COMT/MAO inhibition are given in Fig. 2. While these derivatives
are high potency inhibitors of COMT, only moderate MAO-B inhibition
was observed. For example, chalcone 1 is a potent COMT inhibitor (IC50
= 0.29 µM) while acting as a moderately potent MAO-B inhibitor (IC50
= 13.9 µM) [18]. Chalcone 2 is representative of a particularly potent
COMT inhibitor that has been reported in these literature studies (IC50
= 0.14 µM) [19]. Interestingly, conversion of the chalcones to the cor-
responding pyrazoline derivatives resulted in enhanced COMT inhibi-
tion compared to the chalcones. The pyrazoline derivative 3 was thus
the most potent COMT inhibitor reported in these literature studies with
O
O
HO
HO
HO
N
CN
HO
NO2
NO2
Tolcapone
Entacapone
Cl
N
O
HO
HO
N
N
Cl
O
NO2
Opicapone
Fig. 1. The structures of nitrocatechol inhibitors of COMT.
an IC50 value of 0.048
reference COMT inhibitor, entacapone, with a reported IC50 value of
0.23 M. The possibility that dual COMT/MAO-B inhibition may be
μM [19]. This is significantly more potent than the
and other neurotransmitters is monoamine oxidase (MAO). The two
MAO isoforms, MAO-A and MAO-B, catalyze the oxidation of amine
substrates, and similar to COMT, also are important drug targets for the
treatment of neuropsychiatric and neurodegenerative disorders [11,12].
MAO-A specifically metabolizes serotonin and noradrenaline in the
brain, and inhibitors of this enzyme are established antidepressant
agents [13,14]. MAO-B inhibitors, in turn, are used in the treatment of
PD, and act by reducing the MAO-B catalyzed metabolism of dopamine
in the central nervous system [14,15]. Similar to COMT, inhibitors of
MAO-B may be combined with levodopa in PD therapy where they
enhance central dopamine levels following levodopa treatment [16,17].
It is hypothesized that, since MAO-B and COMT metabolize dopamine
and levodopa, dual inhibitors of these enzymes may act synergistically
to enhance dopaminergic neurotransmission. This may lead to superior
symptomatic treatment in PD. MAO-B inhibitors may also possess neu-
roprotective properties in PD. This action is based on the reduction of
MAO-catalyzed hydrogen peroxide formation in the brain, while reac-
tive oxygen species derived from hydrogen peroxide have been impli-
cated in neurodegeneration in PD [11].
μ
beneficial in PD has been investigated before. In a clinical study, the
effect of dual inhibition of these enzymes was investigated in levodopa-
treated patients with PD. The findings showed that entacapone
improved the clinical response to levodopa, and this improvement was
more marked when combined with selegiline, a MAO-B specific inhibi-
tor [21].
To expand on the structure–activity relationships (SARs) for dual
COMT/MAO inhibition, this study investigates additional chalcone de-
rivatives that contain the nitrocatechol moiety. This study focused on
structures that may be viewed as ring-closed analogues of chalcone, and
included 1-tetralone (4a–f) and 1-indanone (5a–e) derivatives, as well
as 3-coumaranone (6), 4-chromanone (7) and 4-thiochromanone (8)
derivatives (Table 1). With this approach it is anticipated that the
chalcone structure will confer MAO-B inhibition while the nitrocatechol
will facilitate interaction with COMT. This is the first time that these
compounds will be investigated as potential dual MAO/COMT in-
hibitors, and in particular, the possibility that 1-tetralone, 1-indanone
and related derivatives may inhibit COMT has not been investigated
Based on the observation that both COMT and MAO are key enzymes
O
N
N
O
O
HO
HO
HO
HO
HO
HO
CN
Br
NO2
COMT: IC50 0.29
NO2
COMT: IC50 0.14
OH
µM
µM
µM
NO2
COMT: IC50 0.048
1
2
3
=
=
=
µM
µM
µM
µM
=
=
=
=
MAO-A: IC50 76.1
MAO-A: IC50 43.9
MAO-A: IC50 76.6
µM
=
=
MAO-B: IC50 13.9
MAO-B: IC50 55.8
MAO-B: IC50 83.2
µM
F
O
O
O
O
O
O
O
O
O
O
9
10
11
12
>
=
=
=
=
=
MAO-A: IC50 100
=
MAO-A: IC50 0.792
MAO-A: IC50 0.555
MAO-A: IC50 26.7
=
MAO-B: IC50 0.062
µM
µM
µM
µM
µM
µM
µM
MAO-B: IC50 0.0086
MAO-B: IC50 0.063
MAO-B: IC50 0.015
µM
Fig. 2. The structures of compounds that have been investigated as COMT and MAO inhibitors [18,19,22,24,25,26].
2

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
Table 1
IC50 values for the inhibition of COMT and MAO by the tetralone, indanone and related derivatives. The IC50 values of reference inhibitors are also
given.
IC50 (µM)a
SIb
R5
R6
R7
MAO-A
MAO-B
COMT
4a
-H
-H
-H
42.3 ± 2.71
37.9 ± 1.09
36.6 ± 3.38
37.4 ± 0.98
30.3 ± 1.93
53.1 ± 3.19
34.3 ± 3.19
37.4 ± 0.247
47.0 ± 6.43
29.4 ± 2.42
32.7 ± 1.26
41.8 ± 5.72
46.0 ± 5.26
32.5 ± 3.37
5.02 ± 0.45
–
15.4 ± 0.64
45.9 ± 1.93
29.6 ± 2.31
7.83 ± 0.63
11.0 ± 0.31
27.6 ± 1.45
20.7 ± 0.96
60.5 ± 4.74
24.2 ± 3.56
14.2 ± 2.73
15.0 ± 0.39
17.3 ± 0.98
7.26 ± 0.65
11.2 ± 1.10
2.56 ± 0.21
–
0.46 ± 0.06
0.37 ± 0.07
0.79 ± 0.07
0.42 ± 0.18
0.71 ± 0.09
0.24 ± 0.05
0.21 ± 0.06
0.21 ± 0.03
0.16 ± 0.007
0.17 ± 0.04
0.38 ± 0.072
0.19 ± 0.05
0.57 ± 0.05
1.69 ± 0.29
–
2.75
0.83
1.24
4.78
2.75
1.92
1.66
0.62
1.94
2.07
2.18
2.42
6.34
2.90
1.96
–
4b
-H
–OCH3
–OCH3
-H
–OCH3
4c
-H
-H
4d
–OCH3
-H
4e
-H
-H
–OCH3
4f
-H
–NH2
-H
-H
–
5a
-H
5b
–OCH3
–OCH3
–
5c
–OCH2O-
–
5d
–OCH3
-H
-H
–OH
–
–
5e
–OH
–
6
–
–
–
–
7
–
8
–
–
Curcumin
Entacapone
0.23c
a
All values expressed as mean ± SD of triplicate determinations.
Selectivity index: SI = IC50(MAO-A)/IC50(MAO-B).
Value taken from reference [19].
b
c
before. It is interesting to note that 1-tetralone, 1-indanone, 3-coumara-
none and 4-chromanone derivatives have previously been reported to
act as potent and specific inhibitors of MAO-B [22–26]. For example, 1-
tetralone and 1-indanone compounds 9 and 10 are reported to inhibit
MAO-B with IC50 values of 0.063 and 0.015 µM, respectively, while 3-
coumaranone and 4-chromanone derivatives 11 and 12 exhibit IC50
values of 0.062 and 0.0086 µM for MAO-B.
2.2. MAO inhibition
The IC50 values for the inhibition of MAO-A and MAO-B were
measured using the commercially available recombinant human en-
zymes [30,31]. Kynuramine served as substrate for the measurement of
the enzyme catalytic rates of both isoforms of the MAO [32]. Kynur-
amine is oxidized by the MAOs to yield 4-hydroxyquinoline, which can
conveniently be measured by fluorescence spectrophotometry. By
measuring MAO activities in the presence of a concentration range
(0.003–100 µM) of the test inhibitors, sigmoidal plots of enzyme cata-
lytic rate versus inhibitor concentration (Log[I]) were constructed from
which the IC50 values were obtained. Examples of such sigmoidal plots
are given in Fig. 4.
2. Results and discussion
2.1. Chemistry
The 1-tetralone (4a–f), 1-indanone (5a–e), 3-coumaranone (6), 4-
chromanone (7) and 4-thiochromanone (8) derivatives given in table
1 were synthesized as shown in Fig. 3. In the first step 5-nitrovanillin
(13) was treated with aluminum trichloride (AlCl3) in the presence of
pyridine [22,27]. This yielded the catechol intermediate, 3,4-dihydroxy-
5-nitrobenzaldehyde (14), which was reacted in acidic conditions (HCl/
methanol) with the appropriate 1-tetralone (15), 1-indanone (16), 3-
coumaranone (17), 4-chromanone (18) or 4-thiochromanone (19) re-
agent to yield the desired 1-tetralone (4a–f), 1-indanone (5a–e), 3-cou-
maranone (6), 4-chromanone (7) and 4-thiochromanone (8) derivatives
[28]. The chalcones (20a–c) given in table 2 were similarly synthesized
via the Claisen-Schmidt condensation reaction in acidic conditions
(Fig. 3). For this purpose, the appropriate acetophenone (21) was
reacted with 3,4-dihydroxy-5-nitrobenzaldehyde (14). The yields that
were obtained were good, ranging between 77 and 91% for the ring-
closed chalcones. The final products were purified by crystallization,
characterised by NMR and MS, and purities were estimated by HPLC.
Since the NMR spectra show the presence of only one isomer, it is
postulated that for these derivatives, the carbonyl and nitrocatechol
moieties are trans with respect to each other [29].
The IC50 values of the study compounds are given in table 1. None of
the compounds were high potency MAO inhibitors with IC50 values
>29.4 µM for MAO-A, and >7.26 µM for MAO-B. The most potent MAO
inhibitor was 7 with an IC50 value of 7.26 µM for the inhibition of MAO-
B. Compound 7 also possesses the highest degree of specificity for MAO-
B with a selectivity index (SI) value of 6.3. Compound 7 is a 4-chroma-
none derivative, which suggests that the ether oxygen in in the ring
system may be involved in productive interactions with the MAO-B
active site. This is supported by the observation that replacement of
the oxygen by sulphur (compound 8) leads to a slight decrease in MAO-B
inhibition activity (IC50 = 11.2 µM). When these results are compared to
the corresponding 1-tetralone compound 4a (IC50 = 15.4 µM), it may be
concluded that the addition of an oxygen in the 1-tetralone ring in-
creases MAO-B inhibition activity, although to a small degree.
The second most potent MAO-B inhibitor of this study is 1-tetralone
4d with an IC50 of 7.83 µM. This compound is substituted with a
methoxy on C5 of the 1-tetralone moiety. Substitution with a methoxy
on C7 (compound 4e) yielded slightly lower inhibition potency (IC50
=
11.0 µM), while substitution with a methoxy on C6 (compound 4c)
yielded a further reduction in potency (IC50 = 29.6 µM). Disubstitution
3

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
O
R6
R5
O
O
R7
R6
NO2
NO2
OH
5a-e
HO
OH
O
R6
R5
OH
NO2
4a-f
O
R6
6
O
b
O
HO
OH
b
b
R5
R7
R6
16
O
R6
17
R5
15
O
O
NO2
OH
NO2
OH
H
H
a
OH
OCH3
13
14
O
O
R1
O
S
R3
21
O
18
R2
b
O
b
b
NO2
OH
O
19
NO2
OH
O
O
R1
20a-c
OH
NO2
OH
7
R2
OH
S
OH
8
Fig. 3. The synthesis of 1-tetralone (4a–f), 1-indanone (5a–e), 3-coumaranone (6), 4-chromanone (7) and 4-thiochromanone (8) derivatives, as well as chalcones
(20a–c). Key: (a) AlCl3, pyridine, CHCl3, reflux, 24 h; (b) HCl, methanol, reflux, 24–26 h.
Table 2
IC50 values for the inhibition of COMT and MAO by chalcone
100
75
50
25
0
derivatives.
IC50 (µM)a
SIb
R1
R2
MAO-A
MAO-B
COMT
20a
20b
20c
-H
-H
42.5 ± 3.35
48.6 ± 0.66
42.0 ± 2.58
39.8 ± 4.93
18.0 ± 4.01
28.5 ± 1.85
0.89 ± 0.25
0.91 ± 0.14
0.41 ± 0.03
1.07
2.70
1.47
-Br
-Br
-H
–CN
a
All values expressed as mean ± SD of triplicate determinations.
Selectivity index: SI = IC50(MAO-A)/IC50(MAO-B).
b
-3 -2 -1
0
1
2
3
with the methoxy on C6 and C7 yielded compound 4b, which exhibited a
further decrease in MAO-B inhibition potency (IC50 = 45.9 µM)
compared to the corresponding monosubstituted analogues. Substitu-
tion of the methoxy group for an amine group (compound 4f) slightly
enhanced MAO-B inhibition (compare with 4c).
Log [I]
Fig. 4. Sigmoidal plots for the inhibition of MAO-B by compounds 4d (tri-
angles) and 7 (circles).
Other structure–activity relationships (SARs) may be derived from
the MAO-B inhibition data. 1-Indanone derivative 5b (IC50 = 60.5 µM) is
a relatively weak MAO-B inhibitor, and when compared to compound
5d (IC50 = 14.2 µM) showed that monomethoxy substitution leads to
improved potency compared to disubstitution. The methylenedioxy
substituted compound (5c; IC50 = 24.2 µM) is a more potent inhibitor
compared to the dimethoxy substituted compound 5b, which may be
due to reduced steric size of 5c. Interestingly, when the methoxy group
of compound 5d (IC50 = 14.2 µM) is substituted with a hydroxyl to yield
5e (IC50 = 15.0 µM), MAO-B inhibition potency remains unchanged. 3-
Coumaranone (6; IC50 = 17.3 µM) is approximately equal in potency to
the corresponding 1-indanone 5e (IC50 = 15.0 µM). Considering the
MAO-B inhibition potency of 5a (IC50 = 20.7 µM) it may be concluded
that, in general, monosubstitution with either hydroxyl or methoxy
groups increases MAO-B inhibition potency (compare 5a vs. 5d and 5e).
While the goal of this study was to discover dual COMT/MAO-B in-
hibitors, the study compounds were also evaluated as MAO-A inhibitors.
The results show that only compounds 4b and 5b are selective for MAO-
A. The most potent inhibition of MAO-A was achieved with compound
4

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
5d with an IC50 value of 29.4 µM. Overall the compounds showed lower
potency MAO-A inhibition compared to MAO-B, which may be expected
since chalcones are known to be MAO-B specific inhibitors. The mod-
erate inhibition of MAO-A observed with some compounds could,
however, be useful in alleviating non-motor symptoms of PD such as
depression. Compounds such as 5d that inhibit both MAO-A and MAO-B
(although weakly) may possess multiple mechanisms relevant to PD.
For comparison with the ring-closed derivatives, this study also
synthesized a small series of open-chain chalcones that contain the
nitrocatechol moiety (Table 2). The chalcones (20a–c) also are not
potent MAO-B inhibitors. Compound 20b was the most potent MAO-B
inhibitor with an IC50 value of 18.0 µM. Compound 20a shows that
unsubstituted chalcones has lower potency, and by adding electron
withdrawing groups to the unsaturated (enone) system (compounds
20b–c) an increase in MAO-B inhibition potency was observed, with
both compounds substituted with bromine on the enone system. All
chalcones are specific inhibitors of MAO-B compared to the MAO-A
isoform.
nitrocatechol derivatives of chalcone are potent COMT inhibitors, much
modification of the chalcone structure is tolerated without a loss (or
significant gain) of inhibition activity. A possible explanation for this
may be proposed when considering the three-dimensional structure of
the COMT enzyme. The active site of COMT is located in a shallow
groove at the surface of the enzyme. The nitrocatechol moiety forms an
extensive interaction network that involves coordination to the mag-
nesium ion and hydrogen bonding to polar amino acid residues [36,37].
This network is crucial for the binding of nitrocatechol compounds to
COMT, and is responsible for the high binding affinities of nitrocatechol
compounds. Most nitrocatechol inhibitors (e.g. tolcapone) extend to-
wards the exterior of the enzyme, which limits the possibility of further
significant interactions by side chain attached to the nitrocatechol [38].
To show this, 5a was computationally docked into an active site model
of rat COMT complexed with 3,5-dinitrocatechol (PDB code: 1VID) [36]
according to the literature protocol [19]. As per literature description,
the preparation of the protein model and ligand was carried out with
Discovery Studio, while docking was done with AutoDock Vina [39].
Only solutions where the nitrocatechol of 5a exhibits a similar binding
mode to those of 3,5-dinitrocatechol and tolcapone were considered as
probable, and are presented here. The result is given in Fig. 6, and show
that the nitrocatechol moiety undergoes a similar interaction network
compared to 3,5-dinitrocatechol and tolcapone [38]. In this respect, the
catechol OH groups coordinate with the magnesium ion (Mg2+), while
hydrogen bonding occurs with Lys144, Asn170 and Glu199. The
substituted indanone moiety of 5a projects out of the active site and has
the potential for very limited interactions (Fig. 6B). Underscoring its
minor role in inhibitor binding, is the finding that at least two different
binding orientations are predicted for the 1-indanone moiety.
2.3. COMT inhibition
For the COMT inhibition studies, the soluble fractions obtained from
homogenates of rat liver tissue served as enzyme source [18,33,34].
Ethical approval for the collection of the tissue and the use thereof for
this research was obtained from the Research Ethics Committee of the
North-West University (ethics approval number: NWU-00564-19-A5).
To measure COMT activity, esculetin (6,7-dihydroxycoumarin) was
used as substrate. The methylation of esculetin (to yield scopoletin) by
COMT was monitored by fluorescence spectrophotometry [35]. By
measuring COMT catalytic activity in the presence of a concentration
series of the test inhibitors (0.003–80 µM), sigmoidal plots of enzyme
catalytic rate versus inhibitor concentration (Log[I]) were constructed
from which the IC50 values were obtained. Examples of such sigmoidal
plots are given in Fig. 5.
Although the COMT inhibition activities of the study compounds are
similar, some SARs may be derived. Among the 1-indanone derivatives,
the hydroxyl substituted compound 5e (IC50 = 0.38 µM) is the weakest
inhibitor compared to the unstubtituted (5a), methoxy substituted (5b,
5d) and the methylenedioxy compound (5c). Among the 1-tetralone
derivatives, it is interesting to note that the amine substituted com-
pound 4f (IC50 = 0.24 µM) is the most potent COMT inhibitor. It is also
noteworthy that 3-coumaranone 6 (IC50 = 0.19 µM) is a more potent
inhibitor than 4-chromanone 7 (IC50 = 0.57 µM), which in turn is more
potent compared to 4-thiochromanone 8 (IC50 = 1.69 µM). A molecular
basis for the difference in activity between 7 and 8 is not apparent. The
docking study shows that the substituent attached to the nitrocatechol
projects out of the active site and undergoes limited interactions. Among
these, the 5-membered ring derivative is thus more advantageous for
COMT inhibition compared to the 6-membered-ring derivatives.
Accordingly, unsubstituted 1-indanone 5a (IC50 = 0.21 µM) is a more
potent COMT inhibitor that the corresponding unsubstituted 1-tetralone
4a (IC50 = 0.46 µM). The unsubstituted open-chain chalcone 20a (IC50
= 0.89 µM) in turn is a weaker COMT inhibitor compared to the cor-
responding unsubstituted 1-tetralone (4a) and 1-indanone (5a) de-
rivatives, which suggests that the strategy of ring closure of the
chalcones (to yield 1-tetralone and 1-indanone) may be appropriate for
enhancing COMT inhibition, although to a relatively small degree.
The IC50 values for the inhibition of COMT are given in table 1. The
inhibition data show that the nitrocatechol compounds of this study are
all inhibitors of COMT. With the exception of the 4-thiochromanone
derivative 8 (IC50 = 1.69 µM), all compounds are submicromolar
(IC50 < 1 µM) COMT inhibitors. These include both the ring-closed (4–7)
and open-chain chalcones (20a–c) with IC50 values ranging from to 0.16
(5c) to 0.91 µM (20b). The inhibition potencies are similar to that
recorded for entacapone under identical experimental conditions [19].
This result suggests that a wide diversity of nitrocatechol compounds
may act as potent COMT inhibitors with the principal structural
requirement being the presence of the nitrocatechol moiety. Although
100
75
50
25
0
3. Conclusion
The present study attempted to discover compounds that exhibit dual
COMT and MAO-B inhibition. For this purpose 1-tetralone, 1-indanone
and related derivatives were substituted with the nitrocatechol moiety.
Nitrocatechol compounds are well-known to potently inhibit COMT,
while the study compounds are related to the structure of chalcone, a
class known to act as specific inhibitors of MAO-B. The results show that,
with the exception of the 4-thiochromanone derivative (8), all com-
pounds are potent COMT inhibitors with IC50 < 1 µM. All the nitro-
catechol compounds were relatively weak MAO inhibitors while being
very potent COMT inhibitors. This demonstrates that the nitrocatechol
moiety is excellent for COMT inhibition while being poor for MAO
-3 -2 -1
0
1
2
3
Log [I]
Fig. 5. Sigmoidal plots for the inhibition of COMT by compounds 5c (triangles)
and 5d (circles).
5

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
Fig. 6. Two docked orientations of 5a (yellow and blue) in the active site of rat COMT. Panel A shows the molecular interactions with the active site, while Panel B
illustrates that the 1-indanone moiety extends from the active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
inhibition. The substituent attached to the nitrocatechol moiety thus
plays a relatively smaller role in determining binding to these enzymes.
The MAO-B inhibition potencies of the study compounds, however, were
moderate to weak. In spite of the weak MAO inhibition recorded in this
study, compound 7 has improved MAO-B inhibition compared to liter-
ature nitrocatechol compounds (e.g. 1–3). 4-Chromanone derivative 7
was the most promising dual inhibitor with an IC50 value for the inhi-
bition of COMT of 0.57 µM, and an IC50 value for the inhibition of MAO-
B of 7.26 µM. The 1-tetralone derivative 4d, was also noteworthy as a
potentially dual acting inhibitor with IC50 values for the inhibition of
COMT and MAO-B of 0.42 and 7.83 µM, respectively.
their potent inhibition of the COMT enzyme, it may be concluded that
the 1-tetralone, 1-indanone and related derivatives of this study are
more appropriate for peripheral COMT inhibition, which represents an
important strategy in the treatment of PD. The moderate MAO-B inhi-
bition of some compounds in conjunction with the low likelihood of
brain penetration, in turn, suggest that these compounds would not be
effective as MAO-B inhibitors in PD.
4. Experimental section
4.1. Chemical and instrumentation
As discussed in the introduction, the possibility that the study com-
pounds may act in the central nervous system is an important consid-
eration. For these compounds, central COMT inhibition would be
relevant to the treatment of cognitive disorder in schizophrenia and in
other conditions involving dopaminergic dysfunction in the prefrontal
region [9]. Centrally active COMT inhibitors may also reduce central
dopamine metabolism and this enhances dopaminergic neurotransmis-
sion in PD. COMT inhibitors are usually administered in conjunction
with levodopa, and may further improve the therapeutic efficacy of
levodopa [10]. Similarly, for MAO-B inhibitors to be relevant to PD,
brain penetration and central inhibition of MAO-B is a requirement. To
estimate if the compounds of this study are likely to penetrate the
blood–brain barrier, applicable physicochemical properties were
calculated and interpreted with the SwissADME tool, provided by the
Swiss Institute of Informatics [40]. To predict brain penetration and oral
bioavailability, the SwissADME tool uses chemical structure and certain
physiochemical attributes to produce a bioavailability radar. Among the
properties that are considered are lipophilicity, size, polarity, solubility,
flexibility and saturation to produce a plot which shows whether a
compound is considered drug-like. These attributes as well as molecular
weight, molecular refractivity and polar surface area (tPSA) are used to
predict biological barrier permeation, specifically gastrointestinal ab-
sorption and brain permeation. The compounds of this study show
negative in silico brain penetration (Table S1, supplementary material),
primarily because of the high tPSA (>80 Å2). The compounds, however,
show good absorption from the gastrointestinal tract, and no rule ex-
ceptions with respect to the rules of Lipinski [41]. It should be noted that
for tolcapone, negative in silico brain penetration is also predicted,
which correlates with limited brain penetration reported for this com-
pound (brain to plasma ratio up to 0.01) [42,43]. In spite of this, there is
evidence that tolcapone is a central nervous system active COMT in-
hibitor. The prediction, however, highlights the polarity of the nitro-
catechol moiety, and that this represents a limitation for brain
penetration by this class of compounds. Based on these calculations and
All starting materials were obtained from Sigma-Aldrich, and were as
used without further purification. Carbon (13C) and proton (1H) NMR
spectra were recorded on a Bruker Advance III 600 spectrometer at
frequencies of 600 MHz and 151 MHz, respectively. DMSO d6 served as
solvent for all compounds. Multiplicities are given as s (singlet),
d (doublet), dd (doublet of doublets), ddd (doublet of doublet of dou-
blets), t (triplet) and m (multiplet). Silica gel 60 (Merck) with UV254
fluorescent indicator was used for the thin layer chromatography (TLC).
Melting points were measured by means of differential scanning calo-
rimetry (DSC) with a Mettler DSC 3 Star System (Mettler Toledo, Grei-
fensee, Switzerland). High resolution mass spectra (HRMS) were
recorded with a Bruker micrOTOF-Q II mass spectrometer in atmo-
spheric pressure chemical ionization (APCI) mode.
For the MAO inhibition studies, the substrate (kynuramine dihy-
drobromide) and microsomes from insect cells containing recombinant
human MAO-A and MAO-B (5 mg protein/mL) were obtained from
Sigma-Aldrich, while a Varian Cary Eclipse fluorescence spectropho-
tometer was used to measure fluorescence intensities at end-point. For
the COMT inhibition studies, MgCl2, S-adenosyl-L-methionine, esculetin
and L-cysteine were obtained from Sigma-Aldrich, while a SpectraMax
iD3 (Molecular Devices) multi-mode microplate reader was used for the
continuous measurement of fluorescence intensities.
4.2. Purity determination
The purities of the synthesized compounds were determined by high
performance liquid chromatography (HPLC). These analyses were car-
ried out with an Agilent 1200 series HPLC system equipped with a
quaternary pump and an Agilent 1200 series diode array detector. HPLC
grade acetonitrile (Merck) and Milli-Q water (Millipore) were used as
mobile phase in a ratio of 30:70, with a flow rate of 1 mL/min. A Venusil
XBP C18 column (4.60 × 150 mm, 5 µm) was used for the separation.
The concentration of the acetonitrile in the mobile phase was increased
6

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
linearly to 85% over a period of 5 min. Each run lasted 15 min and a
period of 5 min was allowed for equilibrium between runs. A volume of
10 µL of the test compound solution was injected to the system, and the
eluent was monitored at wavelengths of 210, 230, 254 and 280 nm. The
test compounds were dissolved in a mixture of methanol and acetonitrile
(50:50) at a concentration of 1 mM. The purities of compounds 5c and
20c could not be determined since these compounds are too poorly
soluble in methanol and acetonitrile.
3.05 (t, J = 6.0 Hz, 2H), 3.84 (s, 3H), 6.90 (d, J = 2.1 Hz, 1H), 6.94 (dd,
J = 8.7, 2.4 Hz, 1H), 7.23 (d, J = 1.7 Hz, 1H), 7.53 – 7.47 (m, 2H), 7.91
(d, J = 8.7 Hz, 1H), 10.53 (s, 2H). 13C NMR (151 MHz, DMSO d6) δ
26.62, 28.14, 55.56, 112.31, 113.76, 116.57, 119.00, 120.22, 126.24,
129.88, 133.34, 135.24, 137.43, 145.90, 147.54, 163.29, 168.56,
185.14. APCI-HRMS m/z: calc. for C18H16NO6 (MH+), 342.0972,
found 342.0973. Purity (HPLC – 210 nm): 99.5%. MP:223.94 ◦C.
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-5-methoxy-3,4-
dihydronaphthalen-1(2H)-one (4d)
The title compound was prepared to a yield of 82.8% as a dusty
yellow powder. 1H NMR (600 MHz, DMSO d6) δ2.86 (t, J = 6.5 Hz, 2H),
3.05 (t, J = 5.9 Hz, 2H), 3.84 (s, 3H), 7.26 – 7.24 (m, 2H), 7.37 (t, J =
8.0 Hz, 1H), 7.50 (d, J = 1.7 Hz, 1H), 7.53 (s, 1H), 7.57 (d, J = 7.8 Hz,
1H), 10.55 (s, 2H). 13C NMR (151 MHz, DMSO d6) δ 20.88, 25.91,
55.84, 115.08, 116.80, 119.02, 120.30, 125.70, 127.44, 131.66, 133.69,
133.97, 134.93, 137.51, 142.12, 147.56, 156.10, 186.61. APCI-
HRMS m/z: calc. for C18H16NO6 (MH+), 342.0972, found 342.0977.
Purity (HPLC – 210 nm): 98.8%. MP: 238.83 ◦C.
4.3. Synthesis of the 3,4-dihydroxy-5-nitrobenzaldehyde (14)
To a dry round bottom flask, 5-nitrovanillin (13, 25.4 mmol) and
aluminum trichloride (27.9 mmol) were placed, and 50 mL chloroform
was added. The mixture was cooled to 0˚C in an ice bath, and pyridine
(111 mmol) was added dropwise. The ice bath was removed and the
reaction mixture was heated under reflux in an atmosphere of nitrogen
for 24 h. The chloroform was removed under reduced pressure, and the
crude product was quenched with the addition of 100 mL aqueous HCl
(20%). This mixture was stirred for 1 h at room temperature. The water
phase was extracted with three portions of ethyl acetate (50 mL), and the
combined organic phases were dried over anhydrous MgSO4. The ethyl
acetate was removed under reduced pressure and the crude was hot
filtered with toluene (50 mL) to produce yellow crystals [27].
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-7-methoxy-3,4-
dihydronaphthalen-1(2H)-one (4e)
The title compound was prepared to a yield of 87.1% as a yellow
powder. 1H NMR (600 MHz, DMSO d6) δ 2.08 (s, 2H), 2.88 (t, J = 6.4
Hz, 2H), 3.05 (t, J = 5.8 Hz, 2H), 3.80 (s. 3H), 7.17 (dd, J = 8.4, 208 Hz,
1H), 7.25 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 8.4, 1H), 7.42 (d, J = 2.8 Hz,
1H), 7.51 (d, J = 1.8 Hz, 1H), 7.56 (s, 1H), 10.55 (s, 2H). 13C NMR (151
MHz, DMSO d6) δ 26.96, 30.72, 55.33, 110.16, 116.81, 120.33, 120.91,
125.70, 129.94, 133.67, 134.23, 135.04, 135.81, 137.49, 142.13,
4.4. Synthesis of 1-tetralone (4a–f), 1-indanone (5a–e), 3-coumaranone
(6), 4-chromanone (7) and 4-thiochromanone (8) derivatives, as well as
chalcones 20a–c
147.55,
158.22,
186.33.
APCI-HRMS m/z:
calc.
for -
C
18H16NO6 (MH+), 342.0972, found 342.0973. Purity (HPLC – 210
3,4-Dihydroxy-5-nitrobenzaldehyde (14, 1.092 mmol) and the
appropriate 1-tetralone (15), 1-indanone (16), 3-coumaranone (17) or
acetophenone (21) reagents, or 4-chromanone (18) or 4-thiochroma-
none (19) (1.092 mmol) were dissolved in a mixture of 6 mL HCl
(32%) and 4 mL methanol, and heated under reflux for 24–26 h. The
progress of the reactions was monitored with silica gel TLC with a
mixture of petroleum ether and ethyl acetate (1:1) as mobile phase.
Upon completion, the reaction was quenched with addition ice water
(50 mL), upon which a precipitate formed. The precipitated material
was collected by filtration and dried at 60˚C for 48 h before further
analysis. Compound 5b and 20c were purified by recrystallisation from
ethanol and acetonitrile, respectively.
nm): 100%. MP: 229.88 ◦C.
(2E)-6-Amino-2-(3,4-dihydroxy-5-nitrobenzylidene)-3,4-dihy-
dronaphthalen-1(2H)-one (4f)
The title compound was prepared to a yield of 78.6% as a reddish
orange powder. 1H NMR (600 MHz, DMSO d6) δ 2.77 (t, J, = 6.4 Hz,
6H), 2.98 (t, J = 5.9 Hz, 6H). 6.46 (s, 3H), 6.60 (dd, J = 8.5, 1.8 Hz, 3H),
7.23 (d, J = 1.9 Hz, 3H), 7.46 – 7.39 (m, 6H), 7.50 (d, J = 1.9 Hz, 1H),
7.73 (d, J = 8.5 Hz, 3H), 7.97 (d, J = 1.9 Hz, 1H), 9.80 (s, 1H). 13C NMR
(151 MHz, DMSO d6) δ 26.75, 28.31, 115.83, 116.28, 120.39, 126.23,
127.01, 130.10, 123.04, 136.12, 137.51, 141.67, 145.50, 147.52,
148.36,
184.13,
190.65.
APCI-HRMS m/z:
calc.
for -
C
17H15N2O5 (MH+), 327.0975, found 327.0978. Purity (HPLC – 210
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-3,4-dihydronaph-
thalen-1(2H)-one (4a)
nm): 98.3%. MP: 263.90 ◦C.
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-2,3-dihydro-1H-
The title compound was prepared to a yield of 89.54% as an orange
powder. 1H NMR (600 MHz, DMSO d6) δ 2.90 (t, J = 6.2 Hz, 2H), 3.03
(t, J = 6.1 Hz, 2H), 7.22 (s, 1H), 7.39–7.32 (m, 2H), 7.47 (s, 1H), 7.54
(dd, J = 14.0, 6.3 Hz, 2H), 7.90 (d, J = 7.8 Hz, 1H). 13C NMR (151 MHz,
DMSO d6) δ 27.07, 28.18, 117.15, 120.88, 126.14, 127.58, 127.87,
129.05, 133.14, 134.21, 134.65, 135.65, 137.68, 142.76, 143.85,
inden-1-one (5a)
The title compound was prepared in a yield of 79.6% as a bright
yellow powder. 1H NMR (600 MHz, DMSO d6) δ 4.07 (s, 2H), 7.42 (s,
1H), 7.51 – 7.46 (m, 2H), 7.70 (ddd, J = 14.7, 10.6, 4.2 Hz, 2H), 7.76
(dd, J = 10.1, 4.8 Hz, 2H), 10.69 (s, 2H). 13C NMR (151 MHz, DMSO d6)
δ 31.75, 118.07, 120.23, 123.63, 125.51, 126.74, 127.79, 131.47,
134.26, 134.94, 137.23, 137.83, 142.99, 147.88, 149.83, 193.15. APCI-
HRMS m/z: calc. for C16H12NO5 (MH+), 298.0710, found 298.0734.
Purity (HPLC – 210 nm): 98.9%. MP: 255.00 ◦C
148.00,
187.28.
APCI-HRMS m/z:
calc.
for -
C
17H14NO5 (MH+), 312.0866, found 312.0884. Purity (HPLC – 210
nm): 100%. MP: 205.49 ◦C.
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-6,7-dimethoxy-3,4-
dihydronaphthalen-1(2H)-one (4b)
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-5,6-dimethoxy-2,3-
dihydro-1H-inden-1-one (5b)
The title compound was prepared to a yield of 85.88% as a bright
yellow powder. 1H NMR (600 MHz, DMSO d6) δ 2.86 (t, J = 6.0 Hz, 2H),
3.01 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 3.83 (s, 3H), 6.90 (s, 1H), 7.21 (s,
1H), 7.39 (s, 1H), 7.46 (d, J = 2.2 Hz, 2H). 13C NMR (151 MHz,
DMSO d6) δ 27.20, 27.79, 55.80, 56.16, 109.22, 110.97, 116.79,
120.67, 125.86, 126.21, 133.54, 135.60, 137.64, 138.79, 142.35,
147.84, 148.19, 153.86, 185.64. APCI-HRMS m/z: calc. for -
The crude was recrystallized from ethanol to produce the title
compound in a yield of 87.6% as a yellow powder. 1H NMR (600 MHz,
DMSO d6) δ 3.82 (s, 3H), 3.90 (s, 3H), 3.93 (s, 2H), 7.20 (d, J = 12.0 Hz,
2H), 7.29 (s, 1H), 7.44 (s, 1H), 7.71 (s, 1H), 10.62 (s, 2H). 13C NMR (151
MHz, DMSO d6) δ 31.83, 56.10, 56.47, 104.95, 108.55, 117.79, 120.91,
126.16, 130.02, 130.36, 135.59, 138.14, 143.19, 145.31, 148.29,
19H18NO7 (MH+), 372.1078, found 372.1073. Purity (HPLC – 210
149.81,
155.75,
192.07.
APCI-HRMS m/z:
calc.
for -
C
C
18H16NO7 (MH+), 358.0921, found 358.0924. Purity (HPLC – 210
nm): 95.4%. MP: 241.00 ◦C.
nm): 95%. MP: 264.06 ◦C.
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-6-methoxy-3,4-
dihydronaphthalen-1(2H)-one (4c)
(6E)-6-(3,4-dihydroxy-5-nitrobenzylidene)-6,7-dihydro-5H-
indeno[5,6-d][1,3]dioxol-5-one (5c)
The title compound was prepared to a yield of 77.86% as a bright
orange powder. 1H NMR (600 MHz, DMSO d6) δ 2.92 (t, J = 6.4 Hz, 2H),
The title compound was prepared to a yield of 88.1% as a dark yellow
7

A.D. de Beer et al.
Bioorganic Chemistry 114 (2021) 105130
powder. 1H NMR (600 MHz, DMSO d6) δ 3.94 (s, 2H), 6.19 (m, 2H),
7.18 (d, J = 12.5 Hz, 2H), 7.29 (s, 1H), 7.71 (d, J = 1.9 Hz, 1H), 7.44 (d,
en-1-one (20b)
The title compound was prepared to a yield of 77.6% as a dull yellow
powder. 1H NMR (600 MHz, DMSO d6) δ 7.53 (s, 1H), 7.66 (d, J = 15.5
Hz, 1H), 7.79 (t, J = 11.2 Hz, 3H), 7.96 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H),
10.59 (s, 2H). 13C NMR (151 MHz, DMSO d6) δ 116.14, 118.99, 121.13,
125.40, 127.13, 130.58, 131.85, 136.58, 137.87, 143.12, 143.70,
J
= 1.8 Hz, 1H), 10.63 (s, 2H). APCI-HRMS m/z: calc. for -
C
17H12NO7 (MH+), 342.0608, found 342.0611. MP: 293.36 ◦C.
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-5-methoxy-2,3-
dihydro-1H-inden-1-one (5d)
The title compound was prepared to a yield of 84.15% as a dark
orange powder. 1H NMR (600 MHz, DMSO d6) δ 7.74 – 7.67 (m, 2H),
7.44 (s, 1H), 7.30 (s, 1H), 7.17 (s, 1H), 7.01 (d, J = 8.5 Hz, 1H), 3.99 (s,
2H), 3.88 (s, 3H). 13C NMR (151 MHz, DMSO d6) δ 31.81, 110.24,
115.43, 117.53, 120.41, 125.46, 125.68, 130.06, 130.51, 134.90,
137.74, 142.79, 147.86, 152.71, 164.96, 191.38. APCI-HRMS m/z: calc.
for C17H14NO6 (MH+), 328.0816, found 328.0823. Purity (HPLC – 210
nm): 94.3%. MP: 250.83 ◦C.
147.85,
188.07.
APCI-HRMS m/z:
calc.
for -
C
15H11BrNO5 (MH+), 363.9815, found 363.981. Purity (HPLC – 280
nm): 97.7%. MP: 235.55 ◦C.
4-[(2Z)-2-Bromo-3-(3,4-dihydroxy-5-nitrophenyl)prop-2-
enoyl]benzonitrile (20c)
The crude was recrystallized from acetonitrile to produce a yield of
21.4% as a dull yellow powder. 1H NMR (600 MHz, DMSO d6) δ 7.17 (d,
J = 16.0 Hz, 1H), 7.40 (dd, J = 24.3, 15.8 Hz, 2H), 7.56 (s, 1H), 7.68 (d,
(2E)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-5-hydroxy-2,3-dihy-
dro-1H-inden-1-one (5e)
J = 16.0 Hz, 1H), 7.89 – 7.77 (m, 2H), 7.93 (s, 1H), 10.69 (s, 1H). 13
C
NMR (151 MHz, DMSO d6) δ 116.02, 118.98, 121.51, 125.56, 128.57,
The title compound was prepared to a yield of 84.7% as a mustard
yellow powder. 1H NMR (600 MHz, DMSO d6) δ 3.96 (s, 1H), 6.86 (dd,
J = 8.4, 2.1 Hz, 1H), 6.96 (d, J = 1.7 Hz, 1H), 7.28 (s, 1H), 7.46 (d, J =
2.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 2.0 Hz, 1H), 10.66 (s,
2H). 13C NMR (151 MHz, DMSO d6) δ 31.62, 112.00, 116.25, 117.56,
120.17, 125.82, 125.87, 129.29, 120.60, 135.20, 137.87, 142.60,
147.84, 152.86, 163.99, 191.23. APCI-HRMS m/z: calc. for -
128.82, 133.19, 137.60, 137.92, 142.61, 143.50, 147.84, 188.95. APCI-
HRMS m/z:
calc.
for C16H10
[
81Br]N2O5 (MH+), 389.9674,
found 389.0609. MP: 215 ◦C.
4.5. Measurement of IC50 values for the inhibition of MAO
MAO catalytic reactions were carried out in 96-well microtiter plates
(white) to a final volume of 200 µL. The reactions were carried out in
potassium phosphate buffer (100 mM, pH 7.4) and kynuramine (50 µM)
served as substrate for both MAO isoforms. The test inhibitors were
evaluated at concentrations of 0.003–100 µM, and were initially dis-
solved in DMSO. The final concentration of DMSO in the enzyme re-
actions were 4% (v/v). The reactions were initiated with the addition of
MAO-A (0.0075 mg protein/mL) or MAO-B (0.015 mg protein/mL), and
incubated for 20 min at 37 ˚C. At end-point, the reactions were termi-
nated with the addition of 80 µL NaOH (2 N), and 4-hydroxyquinoline
formed by MAO catalysis was quantitated by fluorescence spectropho-
tometry (λex = 310 nm; λem = 400 nm). By employing a calibration curve
prepared with authentic 4-hydroxyquinoline, the enzyme catalytic rates
were calculated and sigmoidal plots of rate versus the inhibitor con-
centration (log[I]) were constructed. These kinetic data were fitted to
the one site competition model incorporated into the Prism 5 software
package (GraphPad) and the IC50 values of the test compounds were
estimated. The IC50 values were determined in triplicate and expressed
as mean ± standard deviation (SD) [24].
C
16H12NO6 (MH+), 314.0659, found 314.0662. Purity (HPLC – 210
nm): 99.7%. MP: 170.47 ◦C.
(2Z)-2-(3,4-Dihydroxy-5-nitrobenzylidene)-6-hydroxy-1-benzo-
furan-3(2H)-one (6)
The title compound was prepared to a yield of 89.4% as a brownish
orange powder. 1H NMR (600 MHz, DMSO d6) δ 6.760–06.96 (m, 6H),
7.62 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 2.0 Hz, 2H), 7.90 (d, J = 1.9 Hz,
2H), 10.68 (s, 3H), 11.26 (s, 2H). 13C NMR (151 MHz, DMSO d6) δ
98.46, 109.40, 112.86, 113.20, 118.45, 120.57, 122.73, 126.11, 137.69,
143.02, 146.97, 147.83, 166.59, 167.73, 181.22. APCI-HRMS m/z: calc.
for C15H10NO7 (MH+), 316.0452, found 316.0450. Purity (HPLC – 210
nm): 98.7%. MP: 244.38 ◦C.
(3E)-3-(3,4-Dihydroxy-5-nitrobenzylidene)-2,3-dihydro-4H-
chromen-4-one (7)
The title compound was prepared to a yield of 78.2% as an orange
yellow powder. 1H NMR (600 MHz, DMSO d6) δ 5.36 (s, 2H), 7.03 (d, J
= 8.1 Hz, 1H), 7.20–7.06 (m, 2H), 7.41 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H),
7.91–7.76 (m, 1H) .13C NMR (151 MHz, DMSO d6) δ 67.51, 117.65,
118.22, 120.64, 121.67, 122.36, 124.54, 127.55, 130.56, 135.36,
136.63, 137.79, 143.26, 147.98, 160.85, 181.30. APCI-HRMS m/z: calc.
for C16H12NO6 (MH+), 314.0659, found 314.0656. Purity (HPLC – 210
nm): 99.5%. MP: 224.68 ◦C.
4.6. Measurement of IC50 values for the inhibition of COMT
The soluble fraction prepared from homogenates of rat liver tissue
served as source of the COMT enzyme. Sprague Dawley rats were bred,
supplied and housed at the Vivarium of the Preclinical Drug Develop-
ment Platform at the Potchefstroom campus of the North-West Univer-
sity (NWU) (SAVC reg no. FR15/13458; SANAS GLP compliance no.
G0019). Experiments were approved by the AnimCare animal research
ethics committee (NHREC reg. number AREC-130913-015) at the NWU.
All animals were maintained and procedures performed in accordance
with the code of ethics in research, training and testing of drugs in South
Africa, and complied with national legislation. Ethical approval for the
collection and use of animal tissue was obtained from the Research
Ethics Committee, NWU with the following approval number: NWU-
00564-19-A5. The liver tissue was prepared as reported in literature
[33,34]. In this respect, frozen liver tissue was washed with ice cold
saline and cut into smaller pieces. Three volumes sodium phosphate
buffer (25 mM, pH 7.8, containing 0.5 mM dithiothreitol) was added,
and the tissue was homogenized for 2 min with a polytron homogenizer.
The homogenate was centrifuged at 100,000g for 30 min at 4 ◦C, and the
supernatant was stored as soluble COMT at –86 ◦C.
(3Z)-3-(3,4-Dihydroxy-5-nitrobenzylidene)-2,3-dihydro-4H-thi-
ochromen-4-one (8)
The title compound was produced to a yield of 90.6% as an orange
powder. 1H NMR (600 MHz, DMSO d6) δ 4.22 (s, 2H), 7.20 (s, 1H), 7.31
(t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.46 – 7.51 (m, 3H),8.02 (d,
J = 7.9 Hz, 1H). 13C NMR (151 MHz, DMSO d6) δ 28.78, 117.11,
120.45, 125.20, 126.41, 128.37, 130.21, 132.15, 132.93, 133.96,
135.55, 137.92, 140.97, 142.94, 148.17, 185.43. APCI-HRMS m/z: calc.
for C16H12NO5S (MH+), 330.0431, found 330.0438. Purity (HPLC – 210
nm): 100%. MP: 216.82 ◦C.
(2E)-3-(3,4-Dihydroxy-5-nitrophenyl)-1-phenylprop-2-en-1-one
(20a)
The title compound was prepared to a yield of 72% as a bright yellow
powder. 1H NMR (600 MHz, DMSO d6) δ 7.56 (dd, J = 15.4, 7.7 Hz, 3H),
7.66 (dd, J = 14.8, 6.1 Hz, 2H), 7.82 (d, J = 15.5, 1H), 7.96 (s, 1H), 8.15
(d, J = 7.7 Hz, 2H), 10.60 (s, 2H). 13C NMR (151 MHz, DMSO d6) δ
30.73, 116.04, 118.97, 121.51, 125.56, 128.57, 128.82, 133.18, 137.61,
137.89, 142.61, 143.55, 147.86, 188.95. APCI-HRMS m/z: calc. for -
C
15H12NO5 (MH+), 286.0710, found 286.0717. Purity (HPLC – 280
To measure the catalytic activity of COMT in the presence of the test
inhibitors, the method reported in literature was used [35]. The enzyme
reactions were prepared in 96-well microtiter plates (black) to a volume
nm): 96.5%. MP: 223.98 ◦C.
(2Z)-2-Bromo-3-(3,4-dihydroxy-5-nitrophenyl)-1-phenylprop-2-
8

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Bioorganic Chemistry 114 (2021) 105130
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of 200 µL. Sodium phosphate buffer (100 mM, pH 7.4) served as reaction
buffer. The reactions contained MgCl2 (2 mM), S-adenosyl-L-methionine
(1 mM), L-cysteine (20 mM), esculetin (20 µM) and the test inhibitor
(0.003–80 µM). Stock solutions of the inhibitor were prepared in DMSO
and added to the reactions to give a final concentration of 4% DMSO.
The reactions were preincubated at 37 ◦C for 15 min, and subsequently
initiated with the addition of COMT (24 µL of the supernatant). The
fluorescence intensities of the reactions were measured continuously
(λex = 355 mn; λem = 460 nm) for 500 s. The slopes obtained from graphs
of fluorescence intensity versus time were used to construct sigmoidal
dose–response curves of slope versus inhibitor concentration (log[I]).
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Molecular docking was carried out with Discovery Studio 3.1
(Accelrys) and AutoDock Vina according to the protocol reported in
literature [19,39]. The protein model of rat COMT (PDB code: 1VID) was
prepared and the ligand was constructed in Discovery Studio, while
docking was carried out with AutoDock Vina. Illustrations were pre-
pared with the PyMOL molecular graphics system [44].
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Declaration of Competing Interest
The author declare that there is no conflict of interest.
Acknowledgements
[19] R. Hitge, S. Smit, A. Petzer, J.P. Petzer, Evaluation of nitrocatechol chalcone and
pyrazoline derivatives as inhibitors of catechol-O-methyltransferase and
monoamine oxidase, Bioorg Med Chem Lett. 30 (12) (2020), 127188, https://doi.
org/10.1016/j.bmcl.2020.127188.
[20] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yanez,
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This work was financially supported by the North-West University
and National Research Foundation of South Africa [Grant specific
unique reference numbers (UID) 85642 and 96180]. The Grant holders
acknowledge that opinions, findings and conclusions or recommenda-
tions expressed in any publication generated by the NRF supported
research are that of the authors, and that the NRF accepts no liability
whatsoever in this regard.
[21] J. Lyytinen, S. Kaakkola, S. Ahtila, P. Tuomainen, H. Teravainen, Simultaneous
MAO-B and COMT inhibition in L-Dopa-treated patients with Parkinson’s disease,
Mov Disord. 12 (4) (1997) 497–505, https://doi.org/10.1002/mds.870120404.
[22] L.J. Legoabe, A. Petzer, J.P. Petzer, alpha-Tetralone derivatives as inhibitors of
monoamine oxidase, Bioorg Med Chem Lett. 24 (12) (2014) 2758–2763, https://
doi.org/10.1016/j.bmcl.2014.04.021.
[23] L.J. Legoabe, A. Petzer, J.P. Petzer, The Synthesis and Evaluation of C7-Substituted
alpha-Tetralone Derivatives as Inhibitors of Monoamine Oxidase, Chem Biol Drug
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[24] S. Mostert, A. Petzer, J.P. Petzer, Indanones as high-potency reversible inhibitors of
monoamine oxidase, ChemMedChem. 10 (5) (2015) 862–873, https://doi.org/
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105130.
[25] A.S. Van Dyk, J.P. Petzer, A. Petzer, L.J. Legoabe, 3-Coumaranone derivatives as
inhibitors of monoamine oxidase, Drug Des Devel Ther. 9 (2015) 5479–5489,
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selective and reversible monoamine oxidase B inhibitors with nanomolar potency,
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