Synthesis and evaluation of 2-substituted 4(3H)-quinazolinone thioether derivatives as monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.09.032

In the present study, a series of fourteen 2-mercapto-4(3H)-quinazolinone derivatives was synthesised and evaluated as potential inhibitors of the human monoamine oxidase (MAO) enzymes. Quinazolinone is the oxidised form of quinazoline, and although this class has not yet been extensively explored as MAO inhibitors, it has been shown to possess a wide variety of biological activities. Among the quinazolinone derivatives investigated, seven compounds (IC₅₀ < 1 μM) proved to be potent and specific MAO-B inhibitors, with the most potent inhibitor, 2-[(3-iodobenzyl)thio]quinazolin-4(3H)-one, exhibiting an IC₅₀ value of 0.142 μM. Further investigation showed that this inhibitor is a reversible and competitive inhibitor of MAO-B with a K_i value of 0.068 μM. None of the test compounds were MAO-A inhibitors. Analysis of the structure-activity relationships (SARs) for MAO-B inhibition shows that substitution on the C2 position of quinazolinone with a benzylthio moiety bearing a Cl, Br or I on the meta position yields the most potent inhibitors of the series. In contrast, substitution with the unsubstituted benzylthio moiety (IC₅₀ = 3.03 μM) resulted in significantly weaker inhibition activity towards MAO-B. This study suggests that quinazolinones are promising leads for the development of selective MAO-B inhibitors which may be used for the treatment of neurodegenerative disorders such as Parkinson's disease.

Full text of DOI:10.1016/j.bmc.2018.09.032

Accepted Manuscript
Synthesis and evaluation of 2-substituted 4(3H)-quinazolinone thioether deriv-
atives as monoamine oxidase inhibitors
Malikotsi A. Qhobosheane, Anél Petzer, Jacobus P. Petzer, Lesetja J. Legoabe
PII:
S0968-0896(18)31321-X
https://doi.org/10.1016/j.bmc.2018.09.032
BMC 14557
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
21 July 2018
12 September 2018
24 September 2018
Please cite this article as: Qhobosheane, M.A., Petzer, A., Petzer, J.P., Legoabe, L.J., Synthesis and evaluation of
2-substituted 4(3H)-quinazolinone thioether derivatives as monoamine oxidase inhibitors, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.032
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Synthesis and evaluation of 2-substituted 4(3H)-quinazolinone thioether
derivatives as monoamine oxidase inhibitors
Malikotsi A. Qhobosheane a, Anél Petzer a,b, Jacobus P. Petzer a,b, Lesetja J. Legoabe a, *
a
Centre of Excellence for Pharmaceutical Sciences, 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
*Corresponding author at: Centre of Excellence for Pharmaceutical Sciences, North-West University,
Private Bag X6001, Potchefstroom 2520, South Africa (L.J. Legoabe).
E-mail address: lesetja.legoabe@nwu.ac.za (L.J. Legoabe).
E-mail addresses:
Malikotsi A. Qhobosheane: qhobosheane.ma@gmail.com
Anél Petzer: 12264954@nwu.ac.za
Jacobus P. Petzer: jacques.petzer@nwu.ac.za
Lesetja J. Legoabe: lesetja.legoabe@nwu.ac.za
1

Abstract
In the present study, a series of fourteen 2-mercapto-4(3H)-quinazolinone derivatives was synthesised
and evaluated as potential inhibitors of the human monoamine oxidase (MAO) enzymes. Quinazolinone
is the oxidised form of quinazoline, and although this class has not yet been extensively explored as
MAO inhibitors, it has been shown to possess a wide variety of biological activities. Among the
quinazolinone derivatives investigated, seven compounds (IC50 < 1 µM) proved to be potent and
specific MAO-B inhibitors, with the most potent inhibitor, 2-[(3-iodobenzyl)thio]quinazolin-4(3H)-
one, exhibiting an IC50 value of 0.142 μM. Further investigation showed that this inhibitor is a reversible
and competitive inhibitor of MAO-B with a Ki value of 0.068 µM. None of the test compounds were
MAO-A inhibitors. Analysis of the structure-activity relationships (SARs) for MAO-B inhibition shows
that substitution on the C2 position of quinazolinone with a benzylthio moiety bearing a Cl, Br or I on
the meta position yields the most potent inhibitors of the series. In contrast, substitution with the
unsubstituted benzylthio moiety (IC50 = 3.03 µM) resulted in significantly weaker inhibition activity
towards MAO-B. This study suggests that quinazolinones are promising leads for the development of
selective MAO-B inhibitors which may be used for the treatment of neurodegenerative disorders such
as Parkinson’s disease.
Keywords: monoamine oxidase, MAO, inhibition, reversible, quinazolinone, Parkinson’s disease
2

1. Introduction
The human monoamine oxidases (MAOs) are mitochondrial bound flavin-dependent enzymes that
catalyse the oxidative deamination of neurotransmitters such as serotonin, dopamine, epinephrine and
norepinephrine.1 The MAO enzymes exist as two isoforms, MAO-A and MAO-B, which are
approximately 70% identical on the amino acid sequence level.2,3 In spite of this, the MAOs differ in
substrate and inhibitor specificities, and tissue distribution. For example, MAO-B is specifically and
irreversibly inhibited by selegiline and rasagiline, and preferentially metabolises benzylamine and
phenylethylamine.1 On the other hand, the MAO-A isoform has specificity for bulkier endogenous
amines such as serotonin, and is irreversibly inhibited by clorgyline.1 Dopamine, epinephrine,
norepinephrine, tyramine and tryptamine are considered substrates for both forms of the enzyme.1 The
MAO enzymes are found in most tissues including the brain, gut, liver, placenta, platelets and
lymphocytes.4 MAO-B is exclusively expressed in platelets and is the main form in human liver tissue,5
while MAO-A is the main form in human placental and gut tissues.6,7 In the brain, the two MAOs are
not evenly distributed with MAO-B being the main form in the basal ganglia.8 The main function of
MAO is to metabolise and terminate the actions of neurotransmitter amines. In the peripheral tissues,
the MAOs also function as metabolic barriers, with MAO-A regulating the entry of biogenic amines
such as tyramine into the systemic circulation,9,10 while MAO-B in the microvessels of the blood-brain
barrier limits the entry of certain trace amines such as phenylethylamine into the central nervous
system.11,12
An imbalance of the concentrations of monoamine neurotransmitters in the brain is responsible for
many of the clinical symptoms of various neurological disorders including depression and Parkinson’s
disease. In this respect, MAO-A metabolises serotonin in the brain, and specific MAO-A inhibitors are
thus used clinically for the treatment of depression and anxiety disorder.13 MAO-B on the other hand is
a key dopamine metabolising enzyme in the brain and MAO-B specific inhibitors are used for the
treatment of Parkinson’s disease.14 In Parkinson’s disease, MAO-B inhibitors are often combined with
L-dopa in an attempt to enhance the therapeutic efficacy of L-dopa.8 MAO-B inhibitors may also protect
against neurodegeneration in Parkinson’s disease by reducing levels of hydrogen peroxide and aldehyde
3

compounds, potentially harmful by-products of MAO-B catalysis.8,15 Since brain MAO-B activity
increases with age, the involvement of hydrogen peroxide and aldehyde derivatives in
neurodegeneration in Parkinson’s disease may be particularly relevant.16
Although MAO-A also metabolises dopamine in the brain,8 MAO-A inhibitors are not used in
Parkinson’s disease due to concerns over potentially serious elevations in blood-pressure when
combined with antiparkinsonian drugs such as L-dopa.17 In fact drugs that inhibit MAO-A, particularly
irreversible inhibitors, are used with caution in the clinic because of their ability to cause a potentially
lethal hypertensive crisis when combined with tyramine-containing food.9,10,18 This is known as the
“cheese reaction” and is the result of the inhibition of intestinal MAO-A, which normally inactivates
tyramine, an indirectly acting sympathomimetic amine, present in certain food such as cheese. When
MAO-A is inhibited, tyramine reaches the systemic circulation leading to the release of norepinephrine
from the peripheral adrenergic neurons and a rapid blood-pressure increase. MAO-B specific inhibitors
and reversible MAO-A inhibitors are not associated with the cheese reaction.18
Numerous scaffolds have been explored as leads for the design of potent MAO inhibitors. Among these
is the quinazolinone moiety. It has been demonstrated that quinazolinone derivatives containing the
hydrazine and pyrazoline moieties (e.g. 1 and 2) are highly potent MAO inhibitors (Fig. 1).19 Other
4(3H)-quinazolinones that have been reported as MAO inhibitors include structures 3–5 as
representative examples.20–24 Based on these reports, substitution on C2 of the 4(3H)-quinazolinone was
considered in the present study. The benzyl, pyridinylmethyl and substituted benzyl moieties were
linked, via a thiol ether, to 4(3H)-quinazolinone to yield 6a–k (Fig. 2). For the purpose of this study,
benzylthio substitution was selected since it is similar to the benzyloxy substituent which is present in
numerous experimental MAO-B inhibitors as well as in safinamide. For cavity-spanning MAO-B
inhibitors, the benzyloxy moiety interacts with the entrance cavity via Van der Waals interactions, thus
enhancing binding affinity. Since halogen substitution was shown to increase MAO inhibition potency
in previous studies, the 4(3H)-quinazolinone thioether derivatives were substituted with halogens on
the meta and para positions of the benzyl ring. For comparison with benzyloxy substitution, the 2-oxo-
4

2-phenylethyl (6l), 3-phenylpropyl (6m) and 2-phenoxyethyl (6n) substituents were also explored in
this study. Based on the previous studies, these 4(3H)-quinazolinone derivatives may act as potential
MAO inhibitors. Although the scaffold has been described, substitution via the thioether linkage at C2
has only been described by one previous report. The present study is the first comprehensive
investigation of the MAO inhibition properties of 4(3H)-quinazolinone thioether derivatives, and the
first report of high potency inhibition by members of this class.
Fig. 1. The structures of quinazolinone derivatives 1–5.
Fig. 2. The general structure of the 4(3H)-quinazolinone thioether derivatives 6a–n.
5

2. Results and discussion
2.1. Chemistry
The 4(3H)-quinazolinone thioether derivatives (6a–n) were synthesised in poor to excellent yields (5-
95%) by employing the nucleophilic substitution reaction shown in Fig. 3. Commercially available 2-
mercapto-4(3H)-quinazolinone (7) and an appropriate arylalkyl bromide or chloride were suspended in
ethanol (10 mL) in the presence of K2CO3 or NaOH, and stirred for 2 h. With the exception of 6n, the
target products were precipitated with the addition of ice-cold water, collected by filtration and dried.
Compounds 6b and 6n were isolated by extraction to ethyl acetate. The crude products obtained were
purified by recrystallisation from ethyl acetate, and the structures and purities of the target compounds
1
were verified by H NMR, 13C NMR, mass spectrometry and HPLC analyses as cited in the
experimental section. Thin-layer chromatography shows that the low yields obtained for certain
reactions are a result of incomplete reactions. In addition, for more polar compounds, solvent-solvent
extraction resulted in loss of the product, thus reducing the yield.
Fig. 3. Route for the synthesis of 4(3H)-quinazolinone thioether derivatives 6a–n.
2.2. IC50 values for the inhibition of MAO
Recombinant human MAO-A and MAO-B were employed to evaluate the MAO inhibitory properties
of the newly synthesised compounds.25 Kynuramine served as enzyme substrate for both MAO-A and
MAO-B. Kynuramine is oxidised to yield 4-hydroxyquinoline as ultimate product, a metabolite that
fluoresces (λex = 310 nm; λem = 400 nm) in alkaline media and can thus be readily measured by
fluorescence spectrophotometry.26 By measuring the fluorescence of 4-hydroxyquinoline in the
presence and absence of the test inhibitors (10 and 100 µM), it was established that the 4(3H)-
quinazolinone thioether derivatives do not fluoresce under the specific assay conditions, and thus do
6

not affect with the fluorescence measurements. A typical enzyme reaction contained the substrate (50
µM) and the test inhibitors (at 0.003–100 µM), and were initiated with addition of enzymes. As negative
control, enzyme reactions were also carried out in absence of inhibitor. The reactions were incubated at
37 °C for 20 min and terminated by the addition of sodium hydroxide (2 N). The rate of formation of
4-hydroxyquinoline in each incubation was calculated from the fluorescence measurements and these
data were fitted to a sigmoidal curve of rate versus the logarithm of inhibitor concentration (Fig. 4). The
IC50 values for the inhibition of MAO were estimated from these plots. For comparison, the known
reversible MAO-A inhibitor, toloxatone (IC50 = 3.92 μM), and the MAO-B inhibitor, lazabemide (IC50
= 0.091 μM), were included as positive controls and were evaluated under identical conditions.27
100
75
50
25
0
-3
-2
-1
0
1
2
3
Log[I]
Fig. 4. Sigmoidal curve of rate versus the logarithm of inhibitor concentration for the inhibition of
MAO-B by 6c (IC50 = 0.566 µM; filled circles) and 6k (IC50 = 0.142 µM; open circles).
The IC50 values for the inhibition of MAO by the 2-substituted 4(3H)-quinazolinone thioether
derivatives are reported in table 1. The results show that 2-substituted 4(3H)-quinazolinones are indeed
MAO inhibitors, with IC50 ranging from 0.142 to 31.8 μM for the inhibition of the MAO-B isoform.
The results further demonstrate that the majority of the test inhibitors (12 of 14) are selective for MAO-
B, with no compounds displaying inhibition towards the MAO-A isoform. With the exception of 6c and
6h, those derivatives bearing a halogen on the meta-position of the benzyl ring (6i, 6j and 6k) are more
7

potent than the corresponding para-substituted derivatives. Compound 6k, the meta-iodobenzyl
derivative, is the most potent inhibitor among the evaluated compounds (IC50 = 0.142 μM). However,
its para-iodo substituted isomer, 6f, is devoid of inhibition activity for MAO-B. Since the meta and the
para isomers 6f and 6k can be considered as having similar lipophilicities and very similar electronic
properties, the loss of activity of the para-iodo compound could likely be attributed to steric effects. In
this respect, steric bulk at the meta position seems to be beneficial while being detrimental at the para
position. This is supported by the observation that for both the chlorine and bromine substituted
compounds, meta substitution yields more potent MAO-B inhibition than para substitution, presumably
due to the steric bulk of the halogens. The weaker inhibition potency observed for 6h (meta-F) compared
to 6c (para-F) further supports the notion that low steric bulk on the meta position reduces inhibition
potency while low steric bulk para is beneficial for inhibition.
It is interesting to note that substitution on the benzyl ring with both halogens (F, Cl, Br, I) and alkyl
(CF3) substituents enhances MAO-B inhibitory potency relative to the derivative bearing an
unsubstituted benzyl ring (6a). In fact, with the exception of 6f, all halogen-substituted derivatives act
as more potent MAO-B inhibitors than 6a. Also of note is that the 3-phenylpropyl (6m) and 2-
phenoxyethyl (6n) substituted compounds exhibited similar MAO-B inhibition potencies compared to
6a, which may be attributed to the absence of substituents (e.g. halogens) on the side chain phenyl rings.
The pyridine substituted compound, 6b, did not inhibit either MAO isoforms, which suggests that non-
polar, lipophilic phenyl-containing substituents are more appropriate for MAO-B inhibition compared
to the pyridine ring. The 2-oxo-2-phenylethyl substituted derivative 6l also displayed relatively poor
MAO-B inhibition, which shows that introduction of an oxo group to the side chain linker reduces
MAO-B inhibition. These results demonstrate the appropriate substitution and substitution pattern of
4(3H)-quinazolinones via thioether linkage for the design of MAO-B inhibitors.
8

Table 1. The IC50 values for the inhibition of MAO-A and MAO-B by 4(3H)-quinazolinone thioether
derivatives.
IC50 (μM)a
MAO-A
NId
SIb
>33
–
cLogPc
MAO-B
3.03 ± 0.131
3.52 ± 0.64
2.03 ± 0.64
3.57 ± 0.66
6a
6b
6c
NId
NId
NId
0.566 ± 0.024
>177
NId
NId
NId
NId
0.482 ± 0.036
0.779 ± 0.188
NId
>207
>128
–
4.12 ± 0.64
4.29 ± 0.66
4.55 ± 0.66
4.09 ± 0.65
6d
6e
6f
0.700 ± 0.005
>143
6g
NId
NId
NId
NId
NId
2.50 ± 0.112
0.230 ± 0.020
0.208 ± 0.015
0.142 ± 0.011
31.8 ± 0.707
>40
3.57 ± 0.66
4.12 ± 0.64
4.29 ± 0.66
4.55 ± 0.66
3.22 ± 0.65
6h
6i
>435
>481
>704
>3.1
6j
6k
6l
NId
NId
5.99 ± 1.60
>17
>81
4.37 ± 0.63
3.66 ± 0.64
6m
6n
1.24 ± 0.053
a All values are expressed as the mean ± standard deviation (SD) of triplicate determinations.
9

b Selectivity index (SI) = IC50(MAO-A)/IC50(MAO-B).
c Calculated with ACD/ChemSketch 1.2 (Advanced Chemistry Development).
d No inhibition observed at a maximal tested concentration of 100 µM.
2.3. Reversibility and mode of MAO-B inhibition
To examine the reversibility of MAO inhibition by the 4(3H)-quinazolinone thioether derivatives, the
recovery of enzymatic activity after dialysis of enzyme-inhibitor mixtures was measured.25 MAO-B and
a selected test inhibitor, compound 6k, at a concentration equal to 4 × IC50 were pre-incubated for 15
min and subsequently dialysed for 24 h. After dialysis, the incubations were diluted twofold with the
addition of kynuramine and the residual enzyme activity was measured. As negative control, MAO-B
was similarly dialysed in the absence of the inhibitor, and as positive control in the presence of the
irreversible inhibitor selegiline. For reversible inhibition, enzymatic activity is expected to recover to
the level of the negative control value (100%) following dialysis. In contrast, for irreversible inhibition,
enzyme activity is not expected to recover after dialysis. In this experiment, MAO-B activity was
recovered to 108% of the control value after dialysis of incubations containing MAO-B and 6k. Enzyme
activity of undialysed mixtures of MAO-B and 6k are 46% of the negative control value, while only
3% activity was recovered after dialysis of incubations containing MAO-B and selegiline. These results
are given in figure 5, and it may thus be concluded that 6k is a reversible inhibitor of MAO-B.
10

100
75
50
25
0
NI
6k sel
dialysed
6k
undialysed
Fig. 5. Reversibility of MAO-B inhibition by 6k. MAO-B was pre-incubated in the presence of 6k (at
4 × IC50) for 15 min, dialysed for 24 h and the residual enzyme activity was measured (6k – dialysed).
Similarly, MAO-B was pre-incubated and dialysed in the absence of the inhibitor (NI – dialysed) and
in the presence of the irreversible MAO-B inhibitor, selegiline (sel – dialysed). The residual activity of
undialysed mixtures of MAO-B and 6k (6k – undialysed) was also measured for comparison.
To investigate the mode of MAO-B inhibition by 6k, a set of Lineweaver-Burke plots (Fig. 6) was
constructed. The set comprised of 6 plots which were constructed by measuring enzyme activities in
the absence and presence of different inhibitor concentrations (0 µM, 1/4 × IC50, ½ × IC50, ¾ × IC50, 1 ×
IC50, 11/4 × IC50). Eight different substrate (kynuramine) concentrations ranging from 15 to 250 µM
were used for each plot. The results show that the plots are linear and intersect on the y-axis. This
indicates that 6k inhibits MAO-B competitively. For MAO-B inhibition, the Ki value was estimated by
plotting the slopes of the Lineweaver-Burke plots versus the inhibitor concentration, with the Ki equal
to 0.068 µM.
11












[I], M




1/[S]


Fig. 6. Lineweaver-Burke plots of human MAO-B catalytic activities in the absence (filled squares) and
presence of various concentrations of 6k (Ki = 0.068 µM).
3. Conclusion
The present study shows that, with the appropriate substitution, C2-substituted 4(3H)-quinazolinone
thioether derivatives are potent and specific inhibitors of human MAO-B, with compound 6k being the
most potent inhibitor (IC50 = 0.142 µM). This inhibition potency is similar in range to that recorded for
lazabemide (IC50 = 0.091 μM).27 An analysis of the SARs for MAO-B inhibition shows that substitution
with a benzyl moiety bearing a halogen (Cl, Br, I) on the meta position is particularly favourable for
MAO-B inhibition.28 In contrast, pyridine substitution (e.g. 6b) leads to loss of MAO-B inhibition
activity, which suggests that an increase of polarity of the C2 substituent is unfavourable for MAO-B
inhibition. Of note is the finding that none of the derivatives inhibited MAO-A up to a maximal tested
concentration of 100 µM, and thus would not possess the liability for the cheese reaction. Based on
their good selectivity profile for MAO-B, this series of 4(3H)-quinazolinone thioether derivatives
represents promising leads for future development of novel specific MAO-B inhibitors, which have
potential in the treatment of Parkinson’s disease. In this regard, the synthetic accessibility of this class
could facilitate their further development as clinically useful agents.
12

4. Experimental section
4.1. Chemicals and instrumentation
Unless otherwise noted, all starting materials and reagents were obtained from Sigma-Aldrich and were
used without further purification. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker
Avance III 600 spectrometer at frequencies of 600 MHz and 151 MHz, respectively, with DMSO-d6
serving as NMR solvent (Merck). Chemical shifts are reported in parts per million (δ) and spin
multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of
doublets), t (triplet) and m (multiplet). The processing and analyses of the NMR data were carried out
with MestreNova. High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II
mass spectrometer in atmospheric-pressure chemical ionisation (APCI) mode. Chemical purities were
determined by HPLC (Agilent 1200 system) with a Venusil XBP C18 column (4.60 × 150 mm, 5 μm)
and a mobile phase of 30% acetonitrile and 70% MilliQ water at a flow rate of 1 mL/min. At the start
of each injection, a solvent gradient program was initiated by linearly increasing the percentage
acetonitrile to 85% over a period of 5 min. Each run lasted 15 min, 20 µL of the test compounds in
acetonitrile (1 mM) was injected into the HPLC system and the eluent was monitored at wavelengths
of 210 and 254 nm. The melting points (mp) were determined with a Buchi B-545 melting point
apparatus and are uncorrected. Thin layer chromatography (TLC) was carried out using silica gel 60
F254 precoated aluminium sheets (0.25 mm, Merck). Microsomes from insect cells containing
recombinant human MAO-A and MAO-B (5 mg protein/mL), and kynuramine dihydrobromide were
obtained from Sigma-Aldrich. Fluorescence spectrophotometry was carried out with a Varian Cary
Eclipse fluorescence spectrophotometer.
4.2. General procedure for synthesis of 4(3H)-quinazolinone derivatives
A mixture of commercially available 2-mercapto-4(3H)-quinazolinone (7, 1.68 mmol), ethanol (10 mL)
and an appropriately substituted arylalkyl halide (1.68 mmol) was stirred in the presence of K2CO3 (3.36
mmol) or NaOH (3.36 mmol) for 2 h at room temperature. The reaction products were precipitated with
the addition of ice-cold water (15 mL), collected by filtration and dried. For derivatives 6b and 6n, the
13

reaction was extracted to ethyl acetate (60 mL), dried over anhydrous magnesium sulfate and the solvent
was removed in vacuo. The target products were purified by recrystallisation from appropriate solvents.
To monitor the progress of the reactions, silica gel TLC was performed with a mobile phase that
consisted of petroleum ether and ethyl acetate (3:1).
4.2.1. 2-(Benzylthio)quinazolin-4(3H)-one (6a)
The title compound was prepared in a yield of 93%: mp 212.4–213.1 °C (ethanol), (lit. 214–216).29 1
H
NMR (600 MHz, DMSO-d6) δ 12.59 (s, 1H), 8.04 (dd, J = 7.9, 1.3 Hz, 1H), 7.78 (ddd, J = 8.5, 7.3, 1.6
Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.51 – 7.46 (m, 2H), 7.46 – 7.40 (m, 1H), 7.33 (dd, J = 10.3, 4.7 Hz,
2H), 7.28 – 7.22 (m, 1H), 4.50 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.83, 155.91, 148.76,
137.87, 135.10, 129.67, 128.92, 127.75, 126.53, 126.38, 126.14, 120.49, 34.01. APCI-HRMS m/z:
calcd for C15H13N2OS, 269.0743, found 269.0755. Purity (HPLC): 99.2%.
4.2.2. 2-[(Pyridin-4-ylmethyl)thio]quinazolin-4(3H)-one (6b)
The title compound was prepared in a yield of 5%: mp 336.6–336.7 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.65 (s, 1H), 8.49 (d, J = 5.9 Hz, 2H), 8.01 (dd, J = 7.9, 1.3 Hz, 1H), 7.78 – 7.72 (m, 1H), 7.57
(d, J = 8.0 Hz, 1H), 7.51 – 7.47 (m, 2H), 7.44 – 7.38 (m, 1H), 4.47 (s, 2H). 13C NMR (151 MHz, DMSO-
d6) δ 161.37, 154.84, 149.60, 148.16, 147.02, 134.61, 126.05, 125.89, 125.75, 124.19, 120.01, 32.13.
APCI-HRMS m/z: calcd for C14H12N3OS (MH+), 270.0696, found 270.0705. Purity (HPLC): 97.1%.
4.2.3. 2-[(4-Fluorobenzyl)thio]quinazolin-4(3H)-one (6c)
The title compound was prepared in a yield of 67%: mp 200.4–202.5 °C (ethanol), (lit. 217–219).29 1
H
NMR (600 MHz, DMSO-d6) δ 12.56 (s, 1H), 8.04 – 7.98 (m, 1H), 7.78 – 7.70 (m, 1H), 7.57 (d, J = 8.1
Hz, 1H), 7.51 (dd, J = 8.4, 5.7 Hz, 2H), 7.43 – 7.35 (m, 1H), 7.12 (t, J = 8.8 Hz, 2H), 4.46 (s, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 161.73, 161.35 (d, J = 243.4 Hz), 155.83, 148.41, 134.48, 133.96 (d, J
= 3.1 Hz), 131.20 (d, J = 8.2 Hz), 126.07, 125.84, 125.53, 120.08, 115.18 (d, J = 21.4 Hz), 32.69. APCI-
HRMS m/z: calcd for C15H12FN2OS (MH+), 287.0649, found 287.0660. Purity (HPLC): 99.1%.
4.2.4. 2-[(4-Chlorobenzyl)thio]quinazoline-4(3H)-one (6d)
The title compound was prepared in a yield of 90%: mp 228.7–232.3 °C.30 1H NMR (600 MHz, DMSO-
d6) δ 12.58 (s, 1H), 8.04 – 7.98 (m, 1H), 7.79 – 7.72 (m, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.4
Hz, 2H), 7.41 (dd, J = 11.1, 4.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 4.46 (s, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 161.28, 155.08, 148.28, 136.81, 134.69, 131.86, 131.09, 128.36, 126.07, 125.78, 120.00,
105.03, 32.69. APCI-HRMS m/z: calcd for C15H12ClN2OS (MH+), 303.0353, found 303.0384. Purity
(HPLC): 96.5%.
14

4.2.5. 2-[(4-Bromobenzyl)thio]quinazolin-4(3H)-one (6e)
The title compound was prepared in a yield of 95%: mp 242.6–245.0 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.60 (s, 1H), 8.04 – 7.98 (m, 1H), 7.75 (s, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H),
7.45 – 7.37 (m, 3H), 4.44 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.54, 155.51, 148.31, 137.34,
134.59, 131.44, 131.28, 126.07, 125.89, 125.66, 120.35, 120.04, 32.75. APCI-HRMS m/z: calcd for
C15H12BrN2OS (M+2H+), 346.9848, found 348.9869. Purity (HPLC): 100.0%.
4.2.6. 2-[(4-Iodobenzyl)thio]quinazolin-4(3H)-one (6f)
The title compound was prepared in a yield of 93%: mp 251.3–261.3 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.59 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.1 Hz,
13
1H), 7.41 (s, 1H), 7.29 (d, J = 8.2 Hz, 2H), 4.42 (s, 2H). C NMR (151 MHz, DMSO-d6) δ 161.18,
154.96, 148.28, 137.56, 137.15, 134.72, 131.57, 126.05, 125.80, 121.95, 120.01, 93.27, 32.91. APCI-
HRMS m/z: calcd for C15H12IN2OS (MH+), 394.9698, found 394.9710. Purity (HPLC): 96.2%.
4.2.7. 2-[(4-Trifluoromethylbenzyl)thio]quinazolin-4(3H)-one (6g)
The title compound was prepared in a yield of 82%: mp 217.3–219.7 °C.31 1H NMR (600 MHz, DMSO-
d6) δ 12.61 (s, 1H), 8.03 – 7.97 (m, 1H), 7.77 – 7.69 (m, 3H), 7.66 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1
Hz, 1H), 7.39 (s, 1H), 4.54 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.87, 155.76, 148.42, 143.08,
134.43, 130.00, 127.72 (q, J = 31.8 Hz), 126.07, 125.82, 125.52, 125.22 (q, J = 3.6 Hz), 123.36, 120.11,
32.82. APCI-HRMS m/z: calcd for C16H12F3N2SO (MH+), 337.0617, found 337.0626. Purity (HPLC):
98.1%.
4.2.8. 2-[(3-Fluorobenzyl)thio]quinazolin-4(3H)-one (6h)
The title compound was prepared in a yield of 63%: mp 193.7–266.1 °C (ethanol). 1H NMR (600 MHz,
DMSO-d6) δ 12.60 (s, 1H), 8.01 (dd, J = 7.9, 0.9 Hz, 1H), 7.76 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.44
– 7.38 (m, 1H), 7.33 (dd, J = 12.2, 6.9 Hz, 3H), 7.06 (s, 1H), 4.48 (s, 2H). 13C NMR (151 MHz, DMSO-
d6) δ 161.96 (d, J = 243.5 Hz), 161.51, 155.39, 148.28, 140.66, 134.62, 130.33 (d, J = 8.4 Hz), 126.09,
125.85, 125.68, 125.34 (d, J = 2.6 Hz), 120.05, 115.95 (d, J = 21.7 Hz), 114.09, 32.86. APCI-HRMS
m/z: calcd for C15H12FN2OS (MH+), 287.0649, found 287.0650. Purity (HPLC): 98.2%.
4.2.9. 2-[(3-Chlorobenzyl)thio]quinazolin-4(3H)-one (6i)
The title compound was prepared in a yield of 86%: mp 209.6–212.7 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.60 (s, 1H), 8.02 (dd, J = 7.9, 1.1 Hz, 1H), 7.79 – 7.73 (m, 1H), 7.61 – 7.54 (m, 2H), 7.48 –
13
7.37 (m, 2H), 7.36 – 7.25 (m, 2H), 4.47 (s, 2H). C NMR (151 MHz, DMSO-d6) δ 161.36, 155.17,
148.22, 140.39, 134.69, 132.82, 130.26, 129.11, 127.96, 127.20, 126.09, 125.84, 125.76, 120.03, 32.77.
APCI-HRMS m/z: calcd for C15H12ClN2OS (MH+), 303.0353, found 303.0367. Purity (HPLC): 97.8%.
15

4.2.10. 2-[(3-Bromobenzyl)thio]quinazoline-4(3H)-one (6j)
The title compound was prepared in a yield of 79%: mp 218.9–219.9 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.58 (s, 1H), 8.01 (dd, J = 7.9, 0.9 Hz, 1H), 7.76 (s, 1H), 7.71 (s, 1H), 7.58 (d, J = 8.1 Hz, 1H),
7.49 (d, J = 7.7 Hz, 1H), 7.42 (dd, J = 5.4, 4.1 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H), 4.46 (s, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 161.46, 155.25, 148.26, 140.72, 134.72, 132.05, 130.59, 130.10, 128.38,
126.13, 125.86, 125.79, 121.45, 120.05, 32.75. APCI-HRMS m/z: calcd for C15H12BrN2OS (M+2H+),
346.9848, found 348.9825. Purity (HPLC): 98.2%.
4.2.11. 2-[(3-Iodobenzyl)thio]quinazolin-4(3H)-one (6k)
The title compound was prepared in a yield of 87%: mp 228.5–230.9 °C. 1H NMR (600 MHz, DMSO-
d6) δ 12.59 (s, 1H), 8.02 (dd, J = 7.9, 1.0 Hz, 1H), 7.90 (s, 1H), 7.77 (s, 1H), 7.62 – 7.55 (m, 2H), 7.51
13
(d, J = 7.8 Hz, 1H), 7.45 – 7.39 (m, 1H), 7.11 (t, J = 7.8 Hz, 1H), 4.42 (s, 2H). C NMR (151 MHz,
DMSO-d6) δ 161.23, 140.47, 137.93, 135.89, 134.72, 130.56, 128.72, 127.26, 126.09, 125.81, 120.01,
118.40, 94.61, 77.42, 32.62. APCI-HRMS m/z: calcd for C15H12IN2OS (MH+), 394.9709, found
394.9704. Purity (HPLC): 98.8%.
4.2.12. 2-[(2-Oxo-2-phenylethyl)thio]quinazolin-4(3H)-one (6l)
The title compound was prepared in a yield of 6%: mp 263.7–265.9 °C (ethanol), (lit. 196–197).32 1
H
NMR (600 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.09 (d, J = 7.7 Hz, 2H), 7.99 (d, J = 7.8 Hz, 1H), 7.72 –
7.61 (m, 2H), 7.58 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 4.84 (s, 2H).
13C NMR (151 MHz, DMSO-d6) δ 193.76, 161.15, 155.02, 148.05, 136.22, 134.56, 133.50, 128.79,
128.32, 126.03, 125.66, 124.61, 119.79, 37.50. APCI-HRMS m/z: calcd for C16H13N2O2S (MH+),
297.0692, found 297.0723. Purity (HPLC): 99.2%.
4.2.13. 2-[(3-Phenylpropyl)thio]quinazolin-4(3H)-one (6m)
The title compound was prepared in a yield of 39%: mp 51.0–51.1 °C. 1H NMR (600 MHz, DMSO-d6)
δ 12.54 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.72 (dd, J = 11.2, 4.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.38
(t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.24 – 7.13 (m, 3H), 3.18 (t, J = 7.3 Hz, 2H), 2.71 (t, J =
7.5 Hz, 2H), 2.05 – 1.92 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.67, 156.29, 148.50, 141.14,
134.44, 128.40, 128.36, 126.04, 125.92, 125.78, 125.41, 120.02, 34.19, 30.50, 29.12. APCI-HRMS m/z:
calcd for C17H17N2OS (MH+), 297.1056, found 297.1080. Purity (HPLC): 98.4%.
4.2.14. 2-[(Phenoxyethyl)thio]quinazolin-4(3H)-one (6n)
The title compound was prepared in a yield of 32%: mp 176.0–178.7 °C (ethyl acetate). 1H NMR (600
MHz, DMSO-d6) δ 12.64 (s, 1H), 8.03 (dd, J = 7.9, 1.3 Hz, 1H), 7.79 – 7.73 (m, 1H), 7.52 (d, J = 8.1
Hz, 1H), 7.44 – 7.38 (m, 1H), 7.32 – 7.26 (m, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H),
4.27 (t, J = 6.6 Hz, 2H), 3.60 (t, J = 6.6 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.23, 158.03,
16

153.27, 134.67, 129.56, 126.07, 125.87, 125.72, 120.85, 120.02, 114.52, 65.87, 30.69, 28.56. APCI-
HRMS m/z: calcd for C16H15N2O2S (MH+), 299.0849, found, 299.0884. Purity (HPLC): 96.3%.
4.3. Protocol for the measurement of IC50 values
The IC50 values for inhibition of MAO-A and MAO-B were measured according to the literature
method.25 The recombinant human MAOs served as enzyme sources in this study and the enzyme
reactions were carried out in white 96-well microtiter plates (Eppendorf) in potassium phosphate buffer
(100 mM, pH 7.4, made isotonic with KCl). The final volume of the reactions was 200 μL and contained
kynuramine (50 μM) and the test inhibitors at concentrations of 0.003–100 μM. Stock solutions of the
inhibitors were prepared in DMSO and added to the reactions to yield a final DMSO concentration of
4%. The reactions were initiated with the addition of MAO-A (0.0075 mg protein/mL) or MAO-B
(0.015 mg protein/mL), incubated for 20 min at 37 °C in a convection oven, and terminated with 80 μL
NaOH (2 N). At endpoint, 4-hydroxyquinoline generated by the oxidation of kynuramine was measured
by fluorescence spectrophotometry (λex = 310 nm; λem = 400 nm) and quantitated with a linear
calibration curve constructed with authentic 4-hydroxyquinoline (0.047–1.56 μM). The inhibition data
were fitted to the one site competition model incorporated into the Prism 5 software package (GraphPad)
and the IC50 values were determined from the resulting sigmoidal plots (rate versus the logarithm of
inhibitor concentration). IC50 values were determined in triplicate and are expressed as mean ± standard
deviation (SD).
4.4. Dialysis of enzyme-inhibitor mixtures
The reversibility of MAO-B inhibition by a selected 4(3H)-quinazolinone thioether derivative (6k) was
examined by dialysis using Slide-A-Lyzer® dialysis cassettes (Thermo Scientific) with a molecular
weight cut-off of 10 000 and sample volume capacity of 0.3–5 mL.25 A mixture of MAO-B (0.03 mg/ml)
and 6k, at a concentration equal to fourfold the IC50, was prepared in potassium phosphate buffer (100
mM, pH 7.4, containing 5% sucrose) to a final volume of 0.8 mL and placed in the dialysis cassettes.
DMSO (4%) was added to the mixtures as co-solvent. These mixtures were pre-incubated for 15 min at
37 °C and subsequently dialysed at 4 °C in 80 mL dialysis buffer (100 mM potassium phosphate, pH
17

7.4, containing 5% sucrose). The dialysis buffer was replaced at 3 h and 7 h after the start of dialysis.
As controls, MAO-B was similarly pre-incubated and dialysed in the presence of the irreversible MAO-
B inhibitor, selegiline (IC50 = 0.079 μM), as well as in the absence of the inhibitor.33 After 24 h of
dialysis, the dialysed samples (250 μL) were diluted twofold with the addition of kynuramine to yield
a final inhibitor concentration of 2 × IC50 and a final kynuramine concentration of 50 μM. These enzyme
reactions were incubated for 20 min at 37 °C, after which they were terminated with addition of 400 μL
NaOH (2 N) and 1000 μL of water. The residual MAO activities were measured by fluorescence
spectrophotometry as described for the IC50 determinations. For comparison, undialysed mixtures of
MAO-B and 6k were maintained at 4 °C for 24 h and thereafter diluted and assayed as above. All
reactions were carried out in triplicate, and the residual enzyme catalytic rates were expressed as mean
± SD.
4.5. Construction of Lineweaver-Burk plots and Ki determination
The mode of MAO-B inhibition by 6k was investigated by constructing a set of six Lineweaver-Burk
plots.25 The first plot was constructed in the absence of inhibitor, while the remaining plots were
constructed in the presence of five different inhibitor concentrations (1/4 x IC50, ½ x IC50, ¾ x IC50, 1 x
IC50, 11/4 x IC50). The substrate, kynuramine, was used at concentrations ranging from 15-250 μM, while
the final concentration of MAO-B was 0.015 mg/mL. All enzyme reactions and catalytic activity
measurements were carried out as described above for the IC50 determinations. Linear regression
analysis was performed using the Prism version 5.0 software package. The Ki value was estimated from
a plot of the slopes of the Lineweaver-Burke plots versus inhibitor concentration where the x-axis
intercept equals –Ki.
Conflict of interest
The authors declare no conflict of interest.
18

Acknowledgements
The NMR and MS spectra were recorded by André Joubert and Johan Jordaan of the SASOL Centre of
Chemistry, North-West University. This work is based on the research supported by National Research
Foundation of South Africa (Grant specific unique reference numbers (UID) 96135, 96180, 85642).
The Grant holders acknowledge that opinions, findings and conclusions or recommendations 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.
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Graphical abstract
100
75
50
25
0
-3 -2 -1
0
1
2
3
Log[I]
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