The monoamine oxidase inhibition properties of C6- and N1-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives

Article abstract of DOI:10.1007/s11030-019-09960-5

Abstract: Quinazolinone compounds are of interest in medicinal chemistry since they display a wide range of biological properties. In the present study, a series of C6- and N1-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives were synthesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO). Some of these quinazolinones are structurally related to a series of 3,4-dihydro-2(1H)-quinolinone derivatives, which have previously been reported to act as specific inhibitors of MAO-B. The results document that, among 37 compounds synthesised, seven displayed IC₅₀ values < 1?μM for the inhibition of MAO-B. The most potent MAO-A inhibitor exhibits an IC₅₀ value of 7.43?μM while the most potent MAO-B inhibitor possesses an IC₅₀ value of 0.269?μM. Good-potency MAO inhibition was only observed among C6-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives with N1-substitution yielding comparatively low-potency inhibition. MAO-B-specific inhibitors such as some of the quinazolinone compounds investigated here may act as leads for the design of therapies for neurodegenerative disorders such as Parkinson’s disease. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-019-09960-5

Molecular Diversity
https://doi.org/10.1007/s11030-019-09960-5
ORIGINAL ARTICLE
The monoamine oxidase inhibition properties of C6‑
and N1‑substituted 3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
derivatives
Lereze Marais1 · Anél Petzer1,2 · Jacobus P. Petzer1,2 · Lesetja J. Legoabe1
Received: 22 March 2019 / Accepted: 15 May 2019
© Springer Nature Switzerland AG 2019
Abstract
Quinazolinone compounds are of interest in medicinal chemistry since they display a wide range of biological properties.
In the present study, a series of C6- and N1-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives were syn-
thesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO). Some of these quinazolinones are
structurally related to a series of 3,4-dihydro-2(1H)-quinolinone derivatives, which have previously been reported to act as
specifc inhibitors of MAO-B. The results document that, among 37 compounds synthesised, seven displayed IC50 values
<1 µM for the inhibition of MAO-B. The most potent MAO-A inhibitor exhibits an IC50 value of 7.43 µM while the most
potent MAO-B inhibitor possesses an IC50 value of 0.269 µM. Good-potency MAO inhibition was only observed among
C6-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives with N1-substitution yielding comparatively low-
potency inhibition. MAO-B-specifc inhibitors such as some of the quinazolinone compounds investigated here may act as
leads for the design of therapies for neurodegenerative disorders such as Parkinson’s disease.
Graphical abstract
Keywords Monoamine oxidase · MAO · Inhibition · Reversible · Quinazolinone · Neurodegenerative disorders
Introduction
The monoamine oxidase (MAO) enzymes are favin ade-
nine dinucleotide (FAD)-containing enzymes that catalyse
Electronic supplementary material The online version of this
the oxidation of neurotransmitter and dietary amines in
both the peripheral and central tissues [1]. Two isoforms of
MAO are expressed, MAO-A and MAO-B, which are prod-
ucts of distinct genes [2]. Although MAO-A and MAO-B
share a high degree of sequence and structural similarity,
article (https://doi.org/10.1007/s11030-019-09960-5) contains
supplementary material, which is available to authorized users.
* Lesetja J. Legoabe
lesetja.legoabe@nwu.ac.za
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
Fig. 1 Structures of selected MAO substrates and inhibitors
they exhibit diferent substrate and inhibitor specifcities
[1, 3, 4]. MAO-A metabolises serotonin and is specifcally
inhibited by the propargylamine compound clorgyline, while
MAO-B metabolises the arylalkylamines, benzylamine
and 2-phenethylamine and is specifcally inhibited by (R)-
deprenyl (Fig. 1). Certain substrates such as dopamine, epi-
nephrine, tyramine and norepinephrine are oxidised by both
MAO isoforms, while a number of inhibitors (e.g. tranylcy-
promine) also do not display isoform specifcity [1].
damage mediated by hydrogen peroxide, a by-product of
MAO catalysis [5]. Currently, two irreversible acting pro-
pargylamine compounds, (R)-deprenyl and rasagiline, are
clinically used for the management of Parkinson’s disease,
while a reversible inhibitor, safnamide, has recently been
approved [1, 9]. MAO-A inhibitors that are used in the clinic
for the treatment of depression include tranylcypromine,
phenelzine, moclobemide, toloxatone and brofaromine [1]. It
should be cautioned that reversibility of MAO-A inhibition
is an important consideration since irreversible inhibitors
such as tranylcypromine and phenelzine may cause a severe
hypertensive crisis when combined with food that contains
the sympathomimetic amine, tyramine [10, 11]. With the
irreversible inhibition of MAO-A in the gastrointestinal tract
and vascular endothelium, dietary tyramine gains access to
the circulatory system which leads to the release of norepi-
nephrine from the sympathetic neurons and subsequently a
hypertensive reaction [5, 12]. In some instances, this hyper-
tensive reaction can be fatal. This led to the development
of reversible MAO-A inhibitors such as moclobemide and
brofaromine, which possess a lower liability for increases in
blood pressure [13, 14]. MAO-B-specifc inhibitors also do
not cause tyramine-induced hypertension since MAO-B is
absent from the gut tissues [5].
Because of their involvement in the catabolism of neu-
rotransmitter amines, MAO-A and MAO-B are considered
targets for the treatment of neurological disorders [1, 5]. In
this respect, MAO-A inhibitors are used in the treatment of
depression and anxiety, while MAO-B inhibitors are often
combined with l-dopa in the treatment of Parkinson’s dis-
ease [1, 6]. In Parkinson’s disease, MAO-B inhibitors block
the MAO-B-catalysed metabolism of dopamine in the brain
and thus enhance dopaminergic neurotransmission. In
combination with l-dopa, MAO-B inhibitors may enhance
dopamine levels derived from the conversion of l-dopa to
dopamine and may thus allow for a reduction in the dose
of l-dopa required for a therapeutic efect [7, 8]. MAO-B
inhibitors may also protect against neuronal injury and neu-
rodegeneration in Parkinson’s disease by reducing oxidative
1 3

Molecular Diversity
3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives. In
total, 26 derivatives with C6-substitution (5a–m; 6a–m) and
11 derivatives with N1-substitution (7a–e; 8a–f) were syn-
thesised. The present study is the frst investigation of the
MAO inhibition properties of 3-methyl-3,4-dihydroquina-
zolin-2(1H)-one derivatives.
Results and discussion
Chemistry
In the current study, two series of 3-methyl-3,4-dihydro-
quinazolin-2(1H)-ones were synthesised. The frst series
consists of C6-substituted 3-methyl-3,4-dihydroquinazo-
lin-2(1H)-one derivatives 5a–m and 6a–m for which ben-
zoyl/acyl and cinnamoyl C6-substitutions were considered,
respectively. The C6-substituted benzoyl/acyl derivatives (5)
were synthesised by Friedel–Crafts acylation (Fig. 3). To a
solution of aluminium trichloride and 3-methyl-3,4-dihydro-
quinazolin-2(1H)-one in carbon disulphide, the appropriate
acyl chloride or bromide was added. The reaction mixture
was heated under refux (50 °C) for 24 h, and on completion,
the reaction was terminated by the addition of a mixture
of ice and water. The product was collected by fltration,
dried in a convection oven and recrystallised from ethanol.
Attempts to synthesise the C6-substituted cinnamoyl deriva-
tives by direct acylation failed, and with the exception of
6b, the target cinnamoyl derivatives 6 were thus synthesised
from 6-acetyl-3-methyl-3,4-dihydroquinazolin-2(1H)-one
(5l). Derivative 6b was synthesised by employing 5m as
starting material. To a solution of 5l or 5m in a mixture
of hydrochloric acid (32%) and methanol, an appropri-
ate benzaldehyde (or cinnamaldehyde for 6c) was added.
The reaction was heated under refux for 24–48 h, and on
completion, the product was isolated and recrystallised as
described above.
Fig. 2 Structures of 4(3H)-quinazolinone (1–3) and 3,4-dihydro-
2(1H)-quinolinone (4) derivatives
Because of their biochemical and clinical signifcance,
the discovery and development of MAO inhibitors are
being actively pursued [15]. Quinazolinone is a heterocyclic
nitrogen containing system composed of fused phenyl and
pyrimidinone (pyrimidone) rings. Over the years, natural
and synthetic quinazolinones have attracted attention since
they display a variety of biological activities [16–19] includ-
ing MAO inhibition [20–24]. Other biological activities of
quinazolinones include antimicrobial, anticonvulsant, anti-
cancer, antimalarial, antihypertensive, anti-infammatory,
antidiabetic, antitumor, anticholinesterase, dihydrofolate
reductase inhibition, as well as the inhibition of cellular
phosphorylation and kinases [25]. As mentioned, the MAO
inhibitory properties of quinazolinone derivatives have also
been reported [20–22]. For example, Khattab and co-work-
ers synthesised a series of 4(3H)-quinazolinone derivatives
and evaluated them as potential inhibitors of MAO-A and
MAO-B. Compounds 1 (IC50 =3.6 nM), 2 (IC50 =2.8 nM)
and 3 (IC50 =2.1 nM) were found to be most active inhibi-
tors and displayed MAO-A inhibition that was comparable
to the reference inhibitor, clorgyline (Fig. 2) [22]. Quina-
zolinones that have been studied as MAO inhibitors are
from the 4(3H)-quinazolinone class of compounds, while
2(1H)-quinazolinones have not yet been investigated. Based
on promising properties of 4(3H)-quinazolinones, we envis-
age that isomeric 2(1H)-quinazolinone derivatives may
also act as MAO inhibitors. In support of this, structurally
related compounds such as 3,4-dihydro-2(1H)-quinolinone
derivatives are known to potently and specifcally inhibit
MAO-B. For example, among a series of 3,4-dihydro-
2(1H)-quinolinone derivatives substituted on the C6 and C7
positions, the most active compound (4) exhibits an IC50
value of 0.086 µM [26, 27]. Based on these fndings, the
present study synthesises series of C6- and N1-substituted
The target 3-methyl-3,4-dihydroquinazolin-2(1H)-one
derivatives 5 and 6 were obtained in yields of 13–100%.
The structures were determined by 1H NMR, 13C NMR and
mass spectrometry, while purities were estimated by HPLC
as cited in Experimental section. The successful synthesis of
the cinnamoyl derivatives 6 was verifed by the presence of
the 1H NMR signals of the vinylic protons of the substituent.
The second series, the N1-substituted 3-methyl-
3,4-dihydroquinazolin-2(1H)-one derivatives 7a–e and
8a–f, was synthesised according to the pathway shown in
Fig. 4. 3,4-Dihydro-3-methyl-2(1H)-quinazolinone was
dissolved in N,N-dimethylformamide (DMF) and treated
with sodium hydride at 0 °C. The reaction was stirred for
20 min, and an appropriate alkyl bromide or alkyl chlo-
ride was subsequently added. The reaction was stirred for
a further 1 h at 0 °C, and after completion was partitioned
1 3

Molecular Diversity
Fig. 3 Synthesis of C6-substi-
tuted 3-methyl-3,4-dihydro-
quinazolin-2(1H)-one deriva-
tives 5 and 6. Key a AlCl3,
CS2, refux, 24 h; b HCl (32%)/
methanol, refux, 24–48 h
Fig. 4 Synthesis of N1-sub-
stituted 3-methyl-3,4-dihydro-
quinazolin-2(1H)-one deriva-
tives 7 and 8. Key a NaH, DMF,
0 °C
between water and ethyl acetate. The organic phase was
dried over MgSO4 and then concentrated in vacuo, and
the resulting residue was recrystallised from ethanol,
ethyl acetate or a mixture of petroleum ether and ethyl
acetate/ethanol. Compounds 7b, 7c, 7e, 8a and 8c were
purified by column chromatography. The 3-methyl-3,4-di-
hydroquinazolin-2(1H)-one derivatives were obtained in
yields of 10–67%. The structures were verified, as cited
IC50 values for the inhibition of MAO
The IC50 values for the inhibition of MAO-A and MAO-B
were determined by employing the human recombinant
enzymes [28, 29]. Kynuramine, a nonspecifc MAO sub-
strate, was used to measure the catalytic rates of both
enzyme isoforms. Kynuramine is non-fuorescent, and after
MAO-catalysed oxidative deamination yields 4-hydroxyqui-
noline, a metabolite which fuoresces in alkaline media. The
rate of kynuramine oxidation was thus determined by meas-
uring the production of 4-hydroxyquinoline by fuorescence
spectrophotometry (λex =310, λem =400) at the endpoint of
the enzyme reaction. The enzyme reactions contained sub-
strate (50 µM) and test inhibitor (0.003–100 µM) in potas-
sium phosphate bufer (pH 7.4, 100 µM). After the reactions
were initiated with the addition of the enzyme, the reactions
were incubated for 20 min at 37 °C and terminated with the
addition of sodium hydroxide (2 N). Control reactions were
conducted in the absence of inhibitor. IC50 values were esti-
mated by constructing sigmoidal plots of enzyme catalytic
rate versus the logarithm of inhibitor concentration. Exam-
ples of such plots are shown in Fig. 5.
1
in Experimental section, by H NMR, 13C NMR and
mass spectrometry, and purity was estimated by HPLC.
Interestingly, this procedure yielded both the 3-methyl-
3,4-dihydroquinazolin-2(1H)-one derivatives 7a–e and
the 3-methyl-3,4-dihydroquinazolin-2(1H,3H)-diones,
1
compounds 8a–f. For 7a–e, H NMR spectra exhibit a
singlet (2H) at approximately 4.5 ppm for the methylene
protons at position 4, which was absent for compound
8a–f. Furthermore, in the 13C NMR spectra of 8a–f,
an additional carbonyl carbon signal at approximately
162 ppm was observed.
1 3

Molecular Diversity
The IC50 values for the inhibition of the human MAOs by
3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives are
given in Tables 1, 2, 3 and 4. From the results, it is evident
that the 3-methyl-3,4-dihydroquinazolin-2(1H)-one deriva-
tives 5–8 are weak MAO-A inhibitors with IC50 >7.43 µM.
Only three compounds (e.g. 6b, 6g and 6h) possess
IC50 < 15 µM for the inhibition of MAO-A. Furthermore,
with the exception of 5c, 5j and 7a–d, none of the com-
pounds evaluated are specifc for the MAO-A isoform. These
compounds are, however, very weak inhibitors of MAO-A.
While no clear trend exists for the inhibition of MAO-A, it
is noteworthy that none of the 3-methyl-3,4-dihydroquina-
zolin-2(1H,3H)-diones (8a–f) exhibited inhibition, even at
a maximal tested concentration of 100 µM. Furthermore,
the three most potent MAO-A inhibitors, 6b, 6g and 6h, are
all cinnamoyl-substituted 3-methyl-3,4-dihydroquinazolin-
2(1H)-one derivatives.
100
75
50
25
0
-3
-2
-1
0
1
2
3
Log[I]
Fig. 5 Sigmoidal plots for the inhibition of MAO-A and MAO-B by
6b (open circles) and 6c (flled circles), respectively
Table 1 IC50 values for the
inhibition of recombinant
human MAO-A and
MAO-B by 3-methyl-3,4-
dihydroquinazolin-2(1H)-one
derivatives 5
aAll values are expressed as the mean standard deviation (SD) of triplicate determinations
bSelectivity index (SI)=IC50(MAO-A)/IC50(MAO-B)
cNo inhibition observed at a maximal tested concentration of 100 µM
dValue obtained from literature [34]
1 3

Molecular Diversity
Table 2 IC50 values for the inhibition of recombinant human MAO-A and MAO-B by 3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives 6
X
IC50 (µM)a
MAO A
SIb
MAO B
6a
6b
6c
6d
6e
6f
H
–
–
No inhc
7.43 0.178
24.7 2.36
No inhc
24.6 3.80
78.1 9.16
8.86 0.532
11.1 1.63
44.2 7.10
52.6 2.93
No inhc
1.06 0.066
2.54 0.103
0.269 0.071
3.72 0.044
3.16 0.460
0.607 0.034
1.23 0.289
0.406 0.024
0.350 0.030
0.416 0.029
0.446 0.082
1.01 0.101
0.513 0.031
–
2.9
91.8
–
4-OCH3
4-OH
4-Cl
4-CN
4-CF3
3-Cl
2-Cl
4-F
7.8
128.7
7.2
27.3
126.3
126.4
–
6g
6h
6i
6j
6k
6l
4-Br
3-F
21.8 0.566
21.6
–
6m
No inhc
See Table 1 for footnotes
In general, the 3-methyl-3,4-dihydroquinazolin-2(1H)-
one derivatives are more potent MAO-B inhibitors. Among
the 37 compounds synthesised, seven displayed IC50 <1 µM
for the inhibition of MAO-B. The most potent MAO-B inhib-
itor (6c) possesses an IC50 value of 0.269 µM. Good-potency
MAO inhibition was only observed among C6-substituted
3-methyl-3,4-dihydroquinazolin-2(1H)-one derivatives,
specifcally among the cinnamoyl-substituted compounds
6, which all display IC50 values<3.72 µM. Among the ben-
zoyl/acyl-substituted derivatives 5, only two compounds (5d
and 5g) possess IC50 <10 µM of the inhibition of MAO-B.
With N1-substitution, no MAO-B inhibition was observed,
even at a maximal tested concentration of 100 µM.
Table 3 IC50 values for the inhibition of recombinant human MAO-A
and MAO-B by 3-methyl-3,4-dihydroquinazolin-2(1H)-one deriva-
tives 7
The following structure–activity relationships (SARs)
for MAO inhibition were observed. Substitution with chlo-
rine in the para and fuorine in meta positions of the ben-
zoyl moiety led to more potent MAO-B inhibitors (5d and
5g) among the 3-methyl-3,4-dihydroquinazolin-2(1H)-one
derivatives 5. The position of substituent is important since
the para (5e) and ortho (5f) fuorine-substituted derivatives
are lower potency MAO-B inhibitors. Replacement of the
benzoyl phenyl ring (5a) with the cyclohexyl (5c) led to
the loss of MAO-B inhibition activity, suggesting that the
aromatic ring is essential for activity. This is supported by
X
IC50 (µM)a
MAO A
SIb
MAO B
7a
7b
7c
7d
7e
4-Cl
3-Br
4-Br
4-I
86.7 4.97
50.2 7.22
47.9 3.17
55.8 5.12
No inhc
No inhc
No inhc
No inhc
No inhc
No inhc
–
–
–
–
–
4-CF3
See Table 1 for footnotes
1 3

Molecular Diversity
Table 4 IC50 values for the
inhibition of recombinant
human MAO-A and
MAO-B by 3-methyl-3,4-
dihydroquinazolin-2,4(1H,3H)-
diones derivatives 8
See Table 1 for footnotes
the observation that the compounds bearing non-aromatic
alkyl or alkyl halide substituents, 5i–m, are also devoid of
MAO-B inhibition activity. Compounds substituted with
heteroaromatic groups, such as furanyl, (5h), also do not
inhibit MAO-B which indicates that heteroaromatic groups
are less appropriate than the phenyl. Interestingly, phenyl-
and benzyl-containing compounds (5a) (IC50 = 51.7 µM)
and 5b (IC50 =50.9 µM) possess similar MAO-A inhibition
potencies, while a reduction and loss of MAO-A inhibition
were observed when the phenyl group was replaced with the
cyclohexyl (5c) and furanyl (5h) groups, respectively.
In contrast to the benzoyl-substituted derivative 5a
(IC50 = 30.8 µM), cinnamoyl substitution to yield 6a
(IC50 = 1.06 µM) resulted in more potent MAO-B inhibi-
tion. Compound 6c (IC50 =0.269 µM), in turn, is approxi-
mately fourfold more potent than compound 6a, which
shows that extension of conjugation (by replacing the styryl
group with a phenylbutadienyl) further enhances MAO-B
inhibition activity. Recently, it has been reported that com-
pounds containing α, β, γ, δ-diunsaturated ketones, such
as cinnamylidene acetophenones, display antioxidant and
anti-infammatory activities [30]. Compound 6c may thus
be a candidate compound that displays multiple biological
activities relevant to the treatment of Parkinson’s disease
including MAO-B inhibition, antioxidant and anti-infam-
matory activities. MAO inhibitors with antioxidant and anti-
infammatory properties would be benefcial in the treatment
of Parkinson’s disease, since infammation and oxidative
stress have been linked to the pathogenesis of Parkinson’s
disease [31]. Substitution with halogens and alkyl groups
(e.g. 6f–m) on the cinnamoyl phenyl ring leads to improved
MAO-B inhibition activity compared to substitution with
a polar hydroxyl group (e.g. 6e). Compound 6a is devoid
MAO-A inhibition activity; however, extension of conjuga-
tion (e.g. 6c) results in a compound with MAO-A inhibition,
although weak. Substitution on the cinnamoyl phenyl ring of
6a with the para nitrile (6g) and trifuoromethyl (6h) groups
yields the second and third most potent MAO-A inhibitors
of the study. Substitution with a smaller halogen (fuorine)
on the cinnamoyl phenyl ring produced compounds which
lacked MAO-A inhibition activity (6k and 6m), although
substitution with bromine and chlorine produced active
MAO-A inhibitors (6f, 6i, 6j and 6l). Interestingly, com-
pound 6e, the hydroxy-substituted compound, was active as
a MAO-A inhibitor (IC50 =24.6 µM), although the methoxy-
substituted homologue (6d) was found to be inactive. For
compounds 6, in general, the electron-withdrawing halogen
substituents yielded more potent MAO-B inhibition com-
pared to electron-releasing substituents (OCH3 and OH).
Although the N1-substituted 3-methyl-3,4-dihydroquina-
zolin-2(1H)-one derivatives (7a–d) are weak MAO-A inhibi-
tors, they exhibited specifcity for the MAO-A isoform. In
contrast, the 3-methyl-3,4-dihydroquinazolin-2,4(1H,3H)-
diones (8a–f) were found to be inactive against both
MAO isoforms. For the 3-methyl-3,4-dihydroquinazolin-
2,4(1H,3H)-diones, a number of structural modifcations
were explored; however, no MAO inhibition was observed.
It may thus be concluded that 3-methyl-3,4-dihydroquina-
zolin-2,4(1H,3H)-diones are not suitable for the design
of MAO inhibitors. Among the N-substituted 3-methyl-
3,4-dihydroquinazolin-2(1H)-one derivatives, substitution
with halogens at the appropriate position on the benzyl
1 3

Molecular Diversity
Fig. 6 Reversibility of inhibi-
tion of MAO-A and MAO-B
by compounds 6b and 6c,
(a)
(b)
100
75
50
25
0
100
75
50
25
0
respectively. MAO-A was
pre-incubated in the absence of
inhibitor and presence of 6b and
pargyline (a), and MAO-B was
pre-incubated in the absence of
inhibitor and presence of 6c and
(R)-deprenyl (b). After dialysis,
the residual enzyme activities
were measured. For compari-
son, the activities of undialysed
mixtures of the MAOs and
the test inhibitors were also
measured
NI
6b parg
dialysed
6b
NI
6c
depr
6c
undialysed
dialysed
undialysed
moiety produced compounds with MAO-A inhibition activ-
ity (7a–d), with compound 7c displaying the highest inhi-
bition potency (IC50 =47.9 µM). Interestingly, the MAO-A
inhibition potencies of the bromine-substituted compounds
(7b and 7c) were within experimental error identical, which
shows that the position (para versus meta) is not an impor-
tant consideration.
inhibitors, pargyline and (R)-deprenyl, served as positive
controls, while dialysis of the enzymes in the absence of
inhibitor served as negative control. For comparison, the
MAO activities of undialysed mixtures of the MAOs and
the test inhibitors were also measured.
For the negative control (absence of the inhibitor), the
residual activity was set to 100%. As shown by the results,
6b and 6c are reversible MAO inhibitors since dialysis
restores MAO-A and MAO-B activity to 98% and 86%,
respectively. Inhibition, however, persists in undialysed
mixtures of MAO-A and MAO-B with these test inhibi-
tors, with the residual activity at 59% and 41%, respec-
tively. Dialysis of the mixtures containing the MAOs and
the irreversible inhibitors, pargyline and (R)-deprenyl, does
not restore activity with the residual activities at 1.6% and
3.1%, respectively.
Reversibility of MAO inhibition
As mentioned, reversibility of MAO inhibition, especially
of the MAO-A isoform, is an important consideration since
irreversible MAO-A inhibitors may lead to a potentially fatal
hypertensive crisis when combined with certain food [11].
Furthermore, compared to reversible inhibition, irreversible
inhibitors also may have the disadvantage of slow and vari-
able rates of enzyme recovery after drug withdrawal [32,
33]. For these reasons, we have set out to investigate the
reversibility of MAO inhibition by selecting the 3-methyl-
3,4-dihydroquinazolin-2(1H)-one derivatives. Compounds
6b and 6c were selected to investigate the reversibility of
MAO-A and MAO-B inhibition, respectively. Since these
compounds do not possess functional groups [e.g. propar-
gylamine, hydrazine, cyclopropylamine, haloallylamine or
N-(2-aminoethyl)carboxamide] that are associated with irre-
versible MAO inhibition, it is expected that 6b and 6c would
act as reversible inhibitors. Dialysis was used to investigate
the reversibility of inhibition. Compounds 6b and 6c, at
concentrations of 4 × IC50, were combined with the MAO
enzyme and pre-incubated for 20 min followed by dialy-
sis for 24 h. Following dialysis, the reactions were diluted
twofold with the addition of kynuramine to yield an inhibi-
tor concentration of 2×IC50. The residual enzyme activities
were measured and are presented graphically (Fig. 6). Simi-
lar dialysis experiments in the presence of the irreversible
Mode of inhibition
Lineweaver–Burk plots were constructed to investigate the
modes of MAO-A and MAO-B inhibition by 6b and 6c,
respectively. MAO activity were measured at eight diferent
kynuramine concentrations (15–250 µM), in the absence and
presence of fve diferent concentrations of the inhibitors. As
shown in Fig. 7, the Lineweaver–Burk plots for the inhibi-
tion of both MAO-A and MAO-B are linear and have com-
mon y-intercepts. This is typical for competitive inhibition,
and from these data, it may be concluded that 6b and 6c are
competitive inhibitors of the MAO isozymes, respectively.
This further supports the fnding of the dialysis studies that
the inhibition is reversible. From replots of the slopes of the
Lineweaver–Burk plots versus inhibitor concentration, Ki
values of 6.9 and 0.16 µM for the inhibition of MAO-A and
MAO-B by 6b and 6c, respectively, are estimated.
1 3

Molecular Diversity
the C6-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-
one derivatives is the increase in potency when the conju-
gation is extended by replacing the cinnamyl group with
a cinnamylidene group. In this respect, the cinnamylidene
derivative 6c is the most potent MAO-B inhibitor of the
series. Some derivatives also exhibited MAO-A inhibition,
with 6b in particular displaying an IC50 value of 7.43 µM.
This potency is comparable to reference inhibitors such as
toloxatone, which is reported to inhibit MAO-A with an IC50
value of 3.92 µM [34]. Since dopamine is metabolised by
both MAO isoforms, nonspecifc MAO inhibitors such as
6b may be considered useful for the therapy of Parkinson’s
disease. The present study also shows that the 3-methyl-
3,4-dihydroquinazolin-2(1H)-one class of compounds are
reversible inhibitors of both MAO-A and MAO-B. This is
signifcant since reversible MAO inhibitors are less likely to
cause tyramine-induced hypertension than irreversible MAO
inhibitors. Based on these fndings, it may be concluded that
C6-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one
derivatives are promising MAO-B inhibitors for the treat-
ment of Parkinson’s disease, while nonspecifc MAO inhibi-
tors may also fnd application in the treatment of depression,
which often is a comorbid condition of Parkinson’s disease.
(a)
(b)
Experimental section
Chemicals and instrumentation
Unless otherwise specifed, all chemicals were obtained
from Sigma-Aldrich and were used without further puri-
fcation. Proton (1H) and carbon (13C) NMR spectra were
recorded in deuterated dimethyl sulfoxide (DMSO-d6) or
deuterochloroform (CDCl3) with a Bruker Avance III 600
spectrometer at frequencies of 600 MHz and 151 MHz,
respectively. MestReNova was used to process and analyse
Fig. 7 Lineweaver–Burk plots of human MAO-A and MAO-B cata-
lytic activities in the absence (flled squares) and presence of various
concentrations of 6b (a) and 6c (b), respectively. For MAO-A, the
concentrations of 6b were 1.86 µM (open squares), 3.72 µM (flled
circles), 5.58 µM (open circles), 7.43 µM (triangles) and 9.29 µM
(diamonds). For the studies with MAO-B, the concentrations of 6c
were 0.067 µM (open squares), 0.134 µM (fled circles), 0.202 µM
(open circles), 0.269 µM (triangles) and 0.336 µM (diamonds)
1
NMR data. H NMR data are reported by providing the
chemical shift (δ), the integration and multiplicity of the
signals. The chemical shifts (δ) are given in parts per million
(ppm) and the coupling constants (J) in Hz. Spin multiplici-
ties are given as singlet (s), doublet (d), doublet of doublets
(dd), doublet of doublet of doublets (ddd), triplet (t), tri-
plet of doublet (td) and multiplet (m). High-resolution mass
spectra (HRMS) were recorded on a Bruker micrOTOF-QII
mass spectrometer in atmospheric-pressure chemical ioni-
sation (APCI) mode. Melting points (mp) were determined
with a Buchi B-545 melting point apparatus and are uncor-
rected. Thin-layer chromatography (TLC) was carried out to
monitor the progress of reactions. Silica gel 60 (Merck) F254
sheets were used for TLC, and the developed sheets were
visualised under a UV254 light or by staining with iodine
vapour. A Varian Cary Eclipse instrument was employed
for fuorescence spectrophotometry. For the enzymology,
Conclusions
The present study shows that a number of compounds among
a series of C6-substituted 3-methyl-3,4-dihydroquinazolin-
2(1H)-one derivatives act as reversible and specifc MAO-B
inhibitors. Such compounds are thus suitable leads for the
development of selective MAO-B inhibitors that may fnd
application in neurodegenerative disorders such as Parkin-
son’s disease. The N1-substituted 3-methyl-3,4-dihydro-
quinazolin-2(1H)-one derivatives (7) proved to be MAO-
A-specific inhibitors, although the inhibition potencies
were weak. An interesting SAR for MAO-B inhibition by
1 3

Molecular Diversity
microsomes from insect cells containing recombinant human
MAO-A and MAO-B (5 mg protein/mL) and kynuramine
dihydrobromide were obtained from Sigma-Aldrich.
APCI-HRMS m/z calcd C16H21N2O2 (MH+), 273.1597,
found 273.1583. Purity (HPLC): 98.8%.
6‑(4‑Chlorobenzoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5d) The title compound was prepared in a
General procedure for synthesis of 5a–m
1
yield of 69%: mp 236.3–242.1 °C. H NMR (600 MHz,
Friedel–Crafts acylation was employed to synthesise the
C6-substituted 3-methyl-3,4-dihydroquinazolin-2(1H)-one
derivatives. Aluminium trichloride (4.62 mmol), 3-methyl-
3,4-dihydroquinazolin-2(1H)-one (3.08 mmol) and the
appropriate acyl chloride or bromide (3.08 mmol) were dis-
solved in carbon disulphide (6 mL). The reaction was heated
under refux (50 °C) for 24 h. The progress of the reaction
was monitored by silica gel TLC with ethyl acetate as mobile
phase. On completion of the reaction, 30 mL of a mixture
of ice and water was added. The precipitate that formed was
collected by fltration and dried in a convection oven over-
night. The crude product was purifed by recrystallisation
from a suitable solvent (ethanol).
DMSO) δ 9.71 (s, 1H), 7.71–7.64 (m, 2H), 7.62–7.52 (m,
4H), 6.87 (d, J = 8.3 Hz, 1H), 4.46 (s, 2H), 2.86 (s, 3H).
13C NMR (151 MHz, DMSO) δ 193.04, 153.04, 142.39,
136.87, 136.47, 131.14, 130.73, 129.13, 128.56, 127.97,
117.78, 112.99, 49.39, 33.88; APCI-HRMS m/z calcd for
C16H14ClN2O2 (MH+), 301.0738, found 301.0756. Purity
(HPLC): 99.4%.
6‑(4‑Fluorobenzoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5e) The title compound was prepared in a
1
yield of 64%: mp 233.6–236.2 °C. H NMR (600 MHz,
DMSO) δ 9.69 (s, 1H), 7.81–7.68 (m, 2H), 7.58–7.51 (m,
2H), 7.40–7.28 (m, 2H), 6.87 (d, J = 8.3 Hz, 1H), 4.46
(s, 2H), 2.85 (d, J = 12.4 Hz, 3H). 13C NMR (151 MHz,
DMSO) δ 192.85, 165.12, 163.46, 153.08, 142.24, 134.30,
132.18, 132.11, 130.68, 129.38, 127.91, 117.76, 115.57,
115.42, 112.96, 49.41, 33.89; APCI-HRMS m/z calcd for
C16H14FN2O2 (MH+), 285.1033, found 285.1059. Purity
(HPLC): 98.9%.
6‑Benzoyl‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(5a) The title compound was prepared in a yield of 39%: mp
235.6–258.4 °C. 1H NMR (600 MHz, DMSO) δ 9.69 (s, 1H),
7.66 (d, J = 7.6 Hz, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.57 (d,
J=8.3 Hz, 1H), 7.54–7.51 (m, 3H), 6.87 (d, J=8.3 Hz, 1H),
4.46 (s, 2H), 2.85 (d, J=11.5 Hz, 3H). 13C NMR (151 MHz,
DMSO) δ 194.18, 153.08, 142.20, 137.78, 132.02, 130.72,
129.47, 129.25, 128.44, 127.93, 117.72, 112.94, 49.41,
33.88; APCI-HRMS m/z calcd for C16H14N2O2 (MH+),
267.1128, found 267.1124. Purity (HPLC): 96%.
6‑(2‑Fluorobenzoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5f) The title compound was prepared in a
1
yield of 52%: mp 268.3–268.5 °C. H NMR (600 MHz,
DMSO) δ 9.74 (s, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.58–7.51
(m, 2H), 7.49 (dd, J=7.4, 1.7 Hz, 1H), 7.38–7.31 (m, 2H),
6.86 (d, J = 8.4 Hz, 1H), 4.45 (s, 2H), 2.84 (s, 3H). 13C
NMR (151 MHz, DMSO) δ 190.81, 159.72, 158.08, 152.93,
143.11, 132.94, 132.89, 130.55, 130.06, 130.04, 129.50,
127.57, 127.05, 126.95, 124.74, 124.72, 117.95, 116.23,
116.09, 113.19, 49.30, 33.86; APCI-HRMS m/z calcd for
C16H14FN2O2 (MH+), 285.1033, found 285.1021. Purity
(HPLC): 99.2%.
3‑Methyl‑6‑(2‑phenylacetyl)‑3,4‑dihydroquinazolin‑2(1H)‑one
(5b) The title compound was prepared in a yield of 53%:
1
mp 259.4–259.5 °C. H NMR (600 MHz, DMSO) δ 9.66
(s, 1H), 7.88 (dd, J=8.4, 1.6 Hz, 1H), 7.82 (s, 1H), 7.30 (t,
J=7.5 Hz, 2H), 7.26 (d, J=7.1 Hz, 2H), 7.23 (d, J=7.2 Hz,
1H), 6.85 (d, J = 8.4 Hz, 1H), 4.47 (s, 2H), 4.27 (s, 2H),
2.87 (s, 3H). 13C NMR (151 MHz, DMSO) δ 196.25,
153.49, 142.81, 135.94, 130.07, 129.83, 129.75, 128.77,
126.99, 126.87, 118.18, 113.48, 49.96, 44.71, 34.37; APCI-
HRMS m/z calcd for C17H17N2O2 (MH+), 281.1285, found
281.1285. Purity (HPLC): 92.8%.
6‑(3‑Fluorobenzoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5g) The title compound was prepared in a
1
yield of 81%: mp 243.9–354.9 °C. H NMR (600 MHz,
DMSO) δ 9.72 (s, 1H), 7.58 (ddd, J=13.7, 8.0, 4.6 Hz, 3H),
7.52–7.42 (m, 3H), 6.88 (d, J=8.3 Hz, 1H), 4.47 (s, 2H),
2.86 (s, 3H). 13C NMR (151 MHz, DMSO) δ 192.78, 162.62,
160.99, 153.01, 142.52, 140.12, 140.08, 130.83, 130.67,
130.62, 128.92, 128.03, 125.41, 118.91, 118.77, 117.82,
115.74, 115.59, 113.03, 49.37, 33.87; APCI-HRMS m/z
calcd for C16H14FN2O2 (MH+), 285.1033, found 285.1017.
Purity (HPLC): 99.0%.
6‑(Cyclohexanecarbonyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5c) The title compound was prepared in a
1
yield of 39%: mp 207.3–242.2 °C. H NMR (600 MHz,
DMSO) δ 9.62 (s, 1H), 7.78 (dd, J=8.4, 1.6 Hz, 1H), 7.73
(s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.47 (s, 2H), 3.29 (dd,
J=12.6, 9.7 Hz, 1H), 2.87 (s, 3H), 1.80–1.70 (m, 4H), 1.67
(d, J = 12.8 Hz, 1H), 1.36 (ddd, J = 23.4, 17.3, 10.8 Hz,
4H), 1.18 (d, J = 12.6 Hz, 1H). 13C NMR (151 MHz,
DMSO) δ 201.74, 153.56, 142.56, 129.28, 129.22, 126.69,
118.23, 113.54, 49.99, 44.41, 34.37, 29.70, 26.10, 25.69;
6‑(Furan‑2‑carbonyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5h) The title compound was prepared in
1 3

Molecular Diversity
1
a yield of 100%: mp 244.8–248.2 H NMR (600 MHz,
DMSO) δ 9.72 (s, 1H), 8.09 (dd, J=1.7, 0.7 Hz, 1H), 7.79
(dd, J=8.4, 1.7 Hz, 1H), 7.74 (s, 1H), 7.39 (d, J=3.5 Hz,
1H), 6.92 (d, J=8.4 Hz, 1H), 6.78 (dd, J=3.6, 1.7 Hz, 1H),
4.51 (s, 2H), 2.88 (s, 3H). 13C NMR (151 MHz, DMSO)
δ 180.24, 153.54, 151.98, 148.40, 142.68, 130.29, 129.84,
127.68, 120.74, 118.25, 113.57, 112.99, 49.95, 34.39; APCI-
HRMS m/z calcd for C14H13N2O3 (MH+), 257.0920, found
257.0902. Purity (HPLC): 99.3%.
3‑Methyl‑6‑propionyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(5m) The title compound was prepared in a yield of
80%: mp 301.0–312.6 °C. 1H NMR (600 MHz, DMSO) δ
9.63 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 6.83
(d, J=8.4 Hz, 1H), 4.47 (s, 2H), 2.94 (q, J=7.2 Hz, 2H),
2.87 (s, 3H), 1.06 (t, J=7.2 Hz, 3H). 13C NMR (151 MHz,
DMSO) δ 199.03, 153.57, 142.54, 130.16, 128.99, 126.37,
118.05, 113.42, 49.98, 34.37, 31.17, 8.83; APCI-HRMS m/z
calcd for C12H15N2O2 (MH+), 219.1128, found 219.1137.
Purity (HPLC): 99.1%.
6‑(2‑Chloroacetyl)‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(5i) The title compound was prepared in a yield of 81%:
General procedure for preparation of compounds
6a–m
1
mp 249.8–252.1 °C. H NMR (600 MHz, DMSO) δ 9.72
(s, 1H), 7.80 (dd, J=8.4, 1.9 Hz, 1H), 7.76 (s, 1H), 6.85 (d,
J=8.4 Hz, 1H), 5.07 (s, 2H), 4.47 (s, 2H), 2.88 (s, 3H). 13
C
6-Acetyl-3-methyl-3,4-dihydroquinazolin-2(1H)-one (5l)
for the synthesis of 6a and 6c–m or 3-methyl-6-propionyl-
3,4-dihydroquinazolin-2(1H)-one (5m) (1.375 mmol) for the
synthesis of 6b and the appropriate benzaldehyde (or cinna-
maldehyde for 6c) (1.375 mmol) were dissolved in a mixture
of hydrochloric acid (32%; 5.5 mL) and methanol (3.67 mL).
The reaction was heated under refux for 24 to 48 h, and the
progress was monitored by using silica gel TLC with ethyl
acetate as the mobile phase. Upon completion, 30 mL of
a mixture of ice and water was added to the reaction. The
residue that formed was collected by fltration and dried in a
convection oven overnight. The crude product was purifed
by recrystallisation from ethanol.
NMR (151 MHz, DMSO) δ 190.11, 153.39, 143.35, 129.70,
127.66, 126.97, 118.27, 113.53, 49.87, 47.60, 34.37; APCI-
HRMS m/z calcd for C11H12ClN2O2 (MH+), 239.0581, found
239.0584. Purity (HPLC): 99.4%.
6‑(3‑Chloropropanoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5j) The title compound was prepared in a
1
yield of 79%: mp 171.7–171.8 °C. H NMR (600 MHz,
DMSO) δ 9.68 (s, 1H), 7.80 (dd, J = 8.4, 1.9 Hz, 1H),
7.76 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.47 (s, 2H), 3.91
(t, J = 6.3 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H), 2.88 (s, 3H).
13C NMR (151 MHz, DMSO) δ 195.36, 153.47, 142.99,
129.84, 129.26, 126.65, 118.15, 113.50, 49.93, 34.38; APCI-
HRMS m/z calcd for C12H14ClN2O2 (MH+), 253.0738, found
253.0736. Purity (HPLC): 96.3%.
6‑Cinnamoyl‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(6a) The title compound was prepared in a yield of 40%: mp
249.9–250.0 °C. 1H NMR (600 MHz, DMSO) δ 9.70 (s, 1H),
8.04–7.97 (m, 2H), 7.95–7.85 (m, 3H), 7.71 (d, J=15.6 Hz,
1H), 7.57–7.44 (m, 3H), 6.89 (d, J =8.4 Hz, 1H), 4.52 (s,
2H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.45,
153.49, 143.50, 142.85, 135.32, 131.06, 130.91, 129.80,
129.38, 129.22, 127.28, 122.42, 118.28, 113.61, 50.03,
34.42; APCI-HRMS m/z calcd for C18H17N2O2 (MH+),
293.1284, found 293.1281. Purity (HPLC): 91.9%.
6‑(4‑Chlorobutanoyl)‑3‑methyl‑3,4‑dihydroquinazo-
lin‑2(1H)‑one (5k) The title compound was prepared in a
1
yield of 57%: mp 180.8–238.3 °C. H NMR (600 MHz,
DMSO) δ 9.65 (s, 1H), 7.79 (dd, J = 8.4, 1.8 Hz, 1H),
7.74 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.47 (s, 2H), 3.70
(t, J = 6.7 Hz, 2H), 3.08 (t, J= 7.1 Hz, 2H), 2.87 (s, 3H),
2.08–2.02 (m, 2H). 13C NMR (151 MHz, DMSO) δ 197.46,
153.52, 142.76, 130.03, 129.06, 126.47, 118.11, 113.47,
49.95, 45.44, 35.13, 34.37, 27.47; APCI-HRMS m/z calcd
for C13H16ClN2O2 (MH+), 267.0894, found 267.0896. Purity
(HPLC): 94.5%.
(Z)‑3‑Methyl‑6‑(2‑methyl‑3‑phenylacryloyl)‑3,4‑dihydroquina-
zolin‑2(1H)‑one (6b) The title compound was prepared in
1
a yield of 17%: mp 122.8–122.9 °C. H NMR (600 MHz,
DMSO) δ 9.64 (s, 1H), 7.61 (dd, J=8.3, 1.8 Hz, 1H), 7.56
(s, 1H), 7.51 (d, J= 7.4 Hz, 2H), 7.45 (t, J= 7.7 Hz, 2H),
7.38 (d, J=7.3 Hz, 1H), 7.05 (s, 1H), 6.87 (d, J=8.3 Hz,
1H), 4.49 (s, 2H), 2.87 (s, 3H), 2.15 (d, J = 1.3 Hz, 3H).
13C NMR (151 MHz, DMSO) δ 197.38, 153.64, 142.16,
139.32, 136.65, 135.95, 130.74, 130.55, 130.12, 128.97,
128.92, 128.04, 118.09, 113.32, 49.96, 34.36, 15.34; APCI-
HRMS m/z calcd for C19H19N2O2 (MH+), 307.1441, found
307.1436. Purity (HPLC): 91.8%.
6‑Acetyl‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one (5l) The
title compound was prepared in a yield of 76%: mp 213.3–
224.8 °C. 1H NMR (600 MHz, DMSO) δ 9.65 (s, 1H), 7.77
(dd, J=8.3, 1.9 Hz, 1H), 7.72 (s, 1H), 6.83 (d, J=8.3 Hz,
1H), 4.47 (s, 2H), 2.87 (s, 3H), 2.48 (s, 3H). 13C NMR
(151 MHz, DMSO) δ 196.47, 153.53, 142.69, 130.48,
129.39, 126.74, 118.03, 113.40, 49.95, 34.37, 26.77; APCI-
HRMS m/z calcd for C11H13N2O2 (MH+), 205.0971, found
205.0973. Purity (HPLC): 98.9%.
1 3

Molecular Diversity
3‑Methyl‑6‑((2E,4Z)‑5‑phenylpenta‑2,4‑dienoyl)‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6c) The title compound was prepared
in a yield of 68%: mp 243.7–247.2 °C. 1H NMR (600 MHz,
DMSO) δ 9.70 (s, 1H), 7.87 (dd, J = 8.4, 1.7 Hz, 1H),
7.83 (s, 1H), 7.60 (d, J=7.3 Hz, 2H), 7.55–7.44 (m, 1H),
7.44–7.30 (m, 4H), 7.27–7.18 (m, 2H), 6.88 (d, J=8.4 Hz,
1H), 4.51 (s, 2H), 2.89 (s, 3H). 13C NMR (151 MHz,
DMSO) δ 187.48, 153.49, 143.92, 142.72, 141.64, 136.56,
131.14, 129.62, 129.43, 127.79, 127.68, 126.88, 125.86,
118.33, 113.62, 50.00, 34.40; APCI-HRMS m/z calcd for
C20H19N2O2 (MH+), 319.1441, found 319.1449. Purity
(HPLC): 99.6%.
title compound was prepared in a yield of 13%: mp 220.9–
224.8 °C. 1H NMR (600 MHz, DMSO) δ 9.72 (s, 1H), 8.10–
8.06 (m, 2H), 8.05–8.03 (m, 1H), 8.03–8.01 (m, 1H), 8.00–
7.92 (m, 3H), 7.73 (d, J=15.6 Hz, 1H), 6.89 (d, J=8.4 Hz,
1H), 4.52 (s, 2H), 2.90 (s, 3H). 13C NMR (151 MHz,
DMSO) δ 187.25, 153.42, 143.11, 141.28, 139.92, 133.19,
130.76, 130.02, 129.95, 129.78, 127.44, 125.74, 118.34,
113.64, 112.60, 50.01, 34.42; APCI-HRMS m/z calcd for
C19H16N3O2 (MH+), 318.1237, found 318.1250. Purity
(HPLC): 76.5%.
(E)‑3‑Methyl‑6‑(3‑(4‑(trifuoromethyl)phenyl)acryloyl)‑3,4‑di-
hydroquinazolin‑2(1H)‑one (6h) The title compound was
prepared in a yield of 26%: mp 216.9–252.0 °C. 1H NMR
(600 MHz, DMSO) δ 9.72 (s, 1H), 8.09 (d, J=8.2 Hz, 2H),
8.07–8.02 (m, 2H), 8.00 (s, 1H), 7.81 (d, J =8.3 Hz, 2H),
7.75 (d, J=15.6 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 4.52 (s,
2H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.30,
153.43, 143.06, 141.49, 139.37, 130.80, 130.45, 130.24,
129.98, 129.76, 127.39, 126.16, 126.14, 125.46, 125.12,
123.66, 118.33, 113.63, 50.01, 34.41; APCI-HRMS m/z
calcd for C19H16F3N2O2 (MH+), 361.1158, found 361.1158.
Purity (HPLC): 78.7%.
(E)‑6‑(3‑(4‑Methoxyphenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6d) The title compound was prepared
in a yield of 47%: mp 220.3–331.9 °C. 1H NMR (600 MHz,
DMSO) δ 9.70 (s, 1H), 8.03 (dd, J=8.4, 1.7 Hz, 1H), 7.98
(s, 1H), 7.91 (d, J = 15.6 Hz, 1H), 7.68 (d, J = 15.5 Hz,
1H), 7.48–7.40 (m, 2H), 7.37 (t, J=7.9 Hz, 1H), 7.02 (dd,
J=8.1, 2.4 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 4.52 (s, 2H),
3.83 (s, 3H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO)
δ 187.46, 160.12, 153.48, 143.50, 142.86, 136.72, 131.05,
130.39, 129.86, 127.26, 122.67, 121.94, 118.28, 116.84,
113.93, 113.59, 55.78, 50.04, 34.41; APCI-HRMS m/z calcd
for C19H19N2O3 (MH+), 323.1390, found 323.1389. Purity
(HPLC): 96.4%.
(E)‑6‑(3‑(3‑Chlorophenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6i) The title compound was pre-
1
pared in a yield of 49%: mp 257.7–257.9 °C. H NMR
(E)‑6‑(3‑(4‑Hydroxyphenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6e) The title compound was prepared
in a yield of 16%: mp 290.9–294.8 °C. 1H NMR (600 MHz,
DMSO) δ 10.07 (s, 1H), 9.67 (s, 1H), 8.00–7.92 (m, 2H),
7.71 (dd, J=12.0, 10.4 Hz, 3H), 7.64 (d, J=15.4 Hz, 1H),
6.88 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.6 Hz, 2H), 4.51 (s,
2H), 2.89 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.33,
160.41, 153.53, 143.95, 142.53, 131.44, 131.28, 129.54,
127.04, 126.42, 118.83, 118.19, 116.26, 113.54, 56.51,
50.06, 34.41, 19.06; APCI-HRMS m/z calcd for C18H17N2O3
(MH+), 309.1233, found 308.1145. Purity (HPLC): 98.9%.
(600 MHz, DMSO) δ 9.70 (s, 1H), 8.08–8.02 (m, 2H),
7.99 (d, J=15.4 Hz, 2H), 7.80 (d, J=6.9 Hz, 1H), 7.68 (d,
J=15.6 Hz, 1H), 7.49 (t, J=4.8 Hz, 2H), 6.89 (d, J=8.4 Hz,
1H), 4.52 (s, 2H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO)
δ 187.30, 153.45, 142.98, 141.78, 137.60, 134.27, 131.15,
130.90, 130.43, 129.95, 128.27, 128.24, 127.37, 123.96,
118.29, 113.61, 50.04, 34.42; APCI-HRMS m/z calcd for
C18H16ClN2O2 (MH+), 327.0894, found 327.0884. Purity
(HPLC): 93.2%.
(E)‑6‑(3‑(2‑Chlorophenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6j) The title compound was prepared
in a yield of 56%: mp 257.6–265.1 °C. 1H NMR (600 MHz,
DMSO) δ 9.72 (s, 1H), 8.19 (d, J=6.6 Hz, 1H), 7.9–8.0 (m),
7.58 (d, J=6.9 Hz, 1H), 7.47 (s, 2H), 6.90 (d, J=8.2 Hz,
1H), 4.52 (s, 2H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO)
δ 187.20, 153.44, 143.08, 138.06, 134.72, 132.94, 132.31,
130.77, 130.52, 129.98, 128.95, 128.14, 127.41, 125.22,
118.35, 113.65, 50.01, 34.42; APCI-HRMS m/z calcd for
C18H16ClN2O2 (MH+), 327.0894, found 327.0900. Purity
(HPLC): 92.3%.
(E)‑6‑(3‑(4‑Chlorophenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6f) The title compound was prepared
in a yield of 42%: mp 244.3–256.6 °C. 1H NMR (600 MHz,
DMSO) δ 9.70 (s, 1H), 8.02 (dd, J=8.4, 1.8 Hz, 1H), 7.98
(s, 1H), 7.92 (t, J=12.1 Hz, 3H), 7.69 (d, J=15.6 Hz, 1H),
7.53 (d, J=8.5 Hz, 2H), 6.89 (d, J=8.4 Hz, 1H), 4.52 (s,
2H), 2.88 (d, J=13.3 Hz, 3H). 13C NMR (151 MHz, DMSO)
δ 187.32, 153.46, 142.91, 142.02, 135.35, 134.31, 130.96,
130.91, 129.86, 129.41, 127.30, 123.16, 118.29, 113.60,
50.02, 34.41; APCI-HRMS m/z calcd for C18H16ClN2O2
(MH+), 327.0894, found 327.0864. Purity (HPLC): 75.4%.
(E)‑6‑(3‑(4‑Fluorophenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6k) The title compound was pre-
1
( E ) ‑ 4 ‑ ( 3 ‑ ( 3 ‑ M e t h y l ‑ 2 ‑ o x o ‑ 1 , 2 , 3 , 4 ‑ t e t r a h y d r o -
quinazolin‑6‑yl)‑3‑oxoprop‑1‑en‑1‑yl)benzonitrile (6g) The
pared in a yield of 27%: mp 241.5–253.3 °C. H NMR
(600 MHz, DMSO) δ 9.69 (s, 1H), 8.01 (dd, J=8.4, 1.9 Hz,
1 3

Molecular Diversity
1H), 7.99–7.93 (m, 3H), 7.87 (d, J=15.6 Hz, 1H), 7.71 (d,
J=15.6 Hz, 1H), 7.31 (t, J=8.8 Hz, 2H), 6.89 (d, J=8.4 Hz,
1H), 4.52 (s, 2H), 2.90 (s, 3H). 13C NMR (151 MHz,
DMSO) δ 187.37, 164.60, 162.95, 153.47, 142.84, 142.26,
132.02, 132.00, 131.56, 131.51, 131.04, 129.81, 127.25,
122.33, 118.27, 116.46, 116.31, 113.59, 50.04, 34.41; APCI-
HRMS m/z calcd for C18H16FN2O2 (MH+), 311.1190, found
310.1112. Purity (HPLC): 95.4%.
gel column chromatography with a mixture of petroleum
ether and ethyl acetate (2:1) serving as mobile phase.
1‑(4‑Chlorobenzyl)‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(7a) The title compound was prepared in a yield of 26%: mp
1
118.9–123.9 °C (ethanol). H NMR (600 MHz, CDCl3) δ
7.28–7.24 (m, 2H), 7.19 (d, J=8.3 Hz, 2H), 7.13–7.03 (m,
2H), 6.94 (t, J=7.4 Hz, 1H), 6.63 (d, J=8.2 Hz, 1H), 5.08
(s, 2H), 4.46 (s, 2H), 3.09 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 155.04, 138.40, 136.38, 132.55, 128.79, 128.18,
127.79, 125.57, 121.95, 119.56, 113.69, 50.47, 46.22, 35.83;
APCI-HRMS m/z calcd for C16H16ClN2O (MH+), 287.0945,
found 301.0731. Purity (HPLC): 97.4%.
(E)‑6‑(3‑(4‑Bromophenyl)acryloyl)‑3‑methyl‑3,4‑dihydro-
quinazolin‑2(1H)‑one (6l) The title compound was prepared
in a yield of 15%: mp 260.3–265.0 °C. 1H NMR (600 MHz,
DMSO) δ 9.70 (s, 1H), 8.01 (dd, J=8.4, 1.8 Hz, 1H), 7.98
(s, 1H), 7.94 (d, J=15.6 Hz, 1H), 7.84 (d, J=8.5 Hz, 2H),
7.66 (d, J=8.3 Hz, 3H), 6.89 (d, J=8.4 Hz, 1H), 4.52 (s,
2H), 2.90 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.33,
153.46, 142.92, 142.12, 134.63, 132.33, 131.13, 130.96,
129.87, 127.31, 124.23, 123.21, 118.29, 113.61, 50.03,
34.42; APCI-HRMS m/z calcd for C18H16BrN2O2 (MH+),
371.0389, found 372.0323. Purity (HPLC): 96.8%.
1‑(3‑Bromobenzyl)‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(7b) The title compound was prepared in a yield of 67%:
1
mp 96.8–100.1 °C (petroleum ether: ethyl acetate). H
NMR (600 MHz, CDCl3) δ 7.41 (d, J=8.4 Hz, 2H), 7.13 (d,
J=8.3 Hz, 2H), 7.09 (d, J=8.0 Hz, 1H), 7.06 (d, J=7.4 Hz,
1H), 6.94 (t, J=7.4 Hz, 1H), 6.62 (d, J=8.2 Hz, 1H), 5.06
(s, 2H), 4.46 (s, 2H), 3.09 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 155.03, 138.39, 136.93, 131.73, 128.19, 128.16,
125.58, 121.96, 120.62, 119.56, 113.68, 50.48, 46.27, 35.83;
APCI-HRMS m/z calcd for C16H16BrN2O (MH+), 331.0440,
found 331.0407. Purity (HPLC): 98.4%.
(E)‑6‑(3‑(3‑Fluorophenyl)acryloyl)‑3‑methyl‑3,4‑dihydroquina-
zolin‑2(1H)‑one (6m) The title compound was prepared in
1
a yield of 23%: mp 317.3–336.2 °C. H NMR (600 MHz,
DMSO) δ 9.70 (s, 1H), 8.03 (dd, J = 8.4, 1.9 Hz, 1H),
8.00–7.95 (m, 2H), 7.83 (d, J = 10.1 Hz, 1H), 7.72–7.65
(m, 2H), 7.50 (dd, J=7.9, 1.7 Hz, 1H), 7.28 (d, J=2.4 Hz,
1‑(4‑Bromobenzyl)‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(7c) The title compound was prepared in a yield of 57%: mp
1H), 6.89 (d, J=8.4 Hz, 1H), 4.52 (s, 2H), 2.90 (s, 3H). 13
C
1
NMR (151 MHz, DMSO) δ 187.33, 163.79, 162.18, 153.45,
142.96, 142.02, 137.95, 137.89, 131.33, 131.28, 130.90,
129.91, 127.36, 125.93, 123.88, 118.28, 117.61, 117.47,
115.04, 114.89, 113.62, 50.03, 34.41; APCI-HRMS m/z
calcd for C18H16FN2O2 (MH+), 311.1190, found 311.1200.
Purity (HPLC): 84.6%.
095.8–098.6 °C (petroleum ether: ethyl acetate). H NMR
(600 MHz, CDCl3) δ 7.44–7.39 (m, 2H), 7.16–7.03 (m, 4H),
6.94 (td, J=7.4, 0.6 Hz, 1H), 6.62 (d, J=8.2 Hz, 1H), 5.06
(s, 2H), 4.46 (s, 2H), 3.09 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 155.02, 138.39, 136.93, 131.73, 128.19, 128.16,
125.58, 121.96, 120.62, 119.56, 113.68, 50.47, 46.27, 35.83;
APCI-HRMS m/z calcd for C16H16BrN2O (MH+), 331.0440,
found 331.0440. Purity (HPLC): 99.2%.
General synthesis of 7a–e and 8a–f
Commercially available 3-methyl-3,4-dihydroquinazolin-
2(1H)-one (1.85 mmol) and sodium hydride (3.7 mmol)
were dissolved in anhydrous DMF. The reaction was
stirred for 20 min at 0 °C. The appropriate alkyl bromide
or alkyl chloride (1.85 mmol) was added to the mixture,
and stirring was continued for 1 h at 0 °C. The reaction
progress was monitored by TLC with petroleum ether
and ethyl acetate (2:1) as the mobile phase. Upon com-
pletion, 30 mL of a mixture of ice and water was added
and the mixture was extracted to 40 mL ethyl acetate. The
organic phase was dried over MgSO4, fltered and evapo-
rated in vacuo. In most instances, the residue was purifed
by recrystallisation from ethanol, ethyl acetate or from a
mixture of petroleum ether and ethyl acetate/ethanol (1:1).
Compounds 7b, 7c, 7e, 8a and 8c were purifed by silica
1‑(4‑Iodobenzyl)‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H)‑one
(7d) The title compound was prepared in a yield of 11%: mp
1
106.8–109.1 °C (petroleum ether: ethyl acetate). H NMR
(600 MHz, CDCl3) δ 7.61 (d, J=8.3 Hz, 2H), 7.12–7.03 (m,
2H), 7.01 (d, J=8.2 Hz, 2H), 6.95 (d, J=7.4 Hz, 1H), 6.62
(d, J=8.2 Hz, 1H), 5.06 (s, 2H), 4.46 (s, 2H), 3.09 (s, 3H),
1.65 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 155.02, 138.39,
137.68, 137.65, 128.44, 128.20, 125.58, 121.96, 119.56,
113.68, 92.07, 50.47, 46.35, 35.84; APCI-HRMS m/z calcd
for C16H16IN2O (MH+), 379.0301, found 379.0278. Purity
(HPLC): 95.4%.
3‑Methyl‑1‑(4‑(triflouromethyl)benzyl)‑3,4‑dihydroquina-
zolin‑2(1H)‑one (7e) The title compound was prepared
in a yield of 25%: mp 086.4–088.9 °C (petroleum ether:
ethyl acetate). 1H NMR (600 MHz, CDCl3) δ 7.53 (s, 1H),
1 3

Molecular Diversity
7.51–7.45 (m, 1H), 7.41 (d, J=6.4 Hz, 2H), 7.13–7.05 (m,
2H), 6.95 (td, J=7.4, 0.7 Hz, 1H), 6.61 (d, J=8.2 Hz, 1H),
5.17 (s, 2H), 4.48 (s, 2H), 3.10 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 154.99, 139.01, 138.36, 129.67, 129.18, 128.27,
125.67, 123.89, 123.86, 123.21, 123.19, 123.17, 122.07,
119.58, 113.56, 50.48, 46.52, 35.86; APCI-HRMS m/z calcd
for C17H16F3N2O (MH+), 321.1209, found 321.1240. Purity
(HPLC): 99.1%.
134.83, 129.55, 128.87, 123.08, 121.29, 115.53, 114.42,
114.32, 65.19, 43.45, 28.43; APCI-HRMS m/z calcd for
C17H17N2O3 (MH+), 297.1233, found 297.1229. Purity
(HPLC): 94.1%.
4‑((3‑Methyl‑2,4‑dioxo‑3,4‑dihydroquinazolin‑2(1H)‑yl)
methyl)benzonitrile (8e) The title compound was prepared
in a yield of 30%: mp 194.5–281.9 °C (petroleum ether:
ethanol). 1H NMR (600 MHz, DMSO) δ 8.09 (dd, J=7.8,
1.1 Hz, 1H), 7.81 (d, J= 8.2 Hz, 2H), 7.68–7.62 (m, 1H),
7.52 (d, J = 8.2 Hz, 2H), 7.28 (t, J = 7.5 Hz, 1H), 7.21
(d, J = 8.5 Hz, 1H), 5.47 (s, 2H), 3.37 (s, 3H). 13C NMR
(151 MHz, DMSO) δ 161.80, 151.49, 142.78, 139.82,
135.63, 133.03, 128.49, 127.98, 123.46, 119.20, 115.72,
115.12, 110.54, 46.65, 28.79; APCI-HRMS m/z calcd for
C17H14N3O2 (MH+), 292.1080, found 292.1059. Purity
(HPLC): 97.0%.
1‑(3‑Chlorobenz yl)‑3‑methyl‑3, 4‑dihydroquinazo-
lin‑2(1H,3H)‑dione (8a) The title compound was prepared in
a yield of 15%: mp 127.6–130.0 °C (petroleum ether: ethyl
1
acetate). H NMR (600 MHz, CDCl3) δ 8.26 (dd, J= 7.9,
1.4 Hz, 1H), 7.57 (ddd, J=8.6, 7.3, 1.6 Hz, 1H), 7.29–7.22
(m, 4H), 7.14 (d, J=6.8 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H),
5.36 (s, 2H), 3.56 (s, 3H). 13C NMR (151 MHz, CDCl3)
δ 161.89, 151.55, 139.61, 137.85, 135.13, 134.98, 130.30,
129.13, 128.01, 126.60, 124.63, 123.30, 115.69, 114.09,
46.93, 28.73; APCI-HRMS m/z calcd for C16H14ClN2O2
(MH+), 301.0738, found 301.0738. Purity (HPLC): 99.8%.
1 ‑ ( 3 ‑ I o d o b e n z y l ) ‑ 3 ‑ m e t h y l ‑ 3 , 4 ‑ d i h y d r o q u i n a z o -
lin‑2(1H,3H)‑dione (8f) The title compound was prepared
1
in a yield of 34%: mp 131.4–134.0 °C (ethyl acetate). H
1‑Benzyl‑3‑methyl‑3,4‑dihydroquinazolin‑2(1H,3H)‑dione
(8b) The title compound was prepared in a yield of 11%:
NMR (600 MHz, DMSO) δ 8.08 (dd, J=7.8, 1.5 Hz, 1H),
7.76 (s, 1H), 7.70–7.61 (m, 2H), 7.33–7.24 (m, 3H), 7.12
(t, J = 7.8 Hz, 1H), 5.35 (s, 2H), 3.37 (s, 3H). 13C NMR
(151 MHz, DMSO) δ 161.80, 151.49, 139.91, 139.55,
136.54, 135.60, 135.55, 131.25, 128.42, 126.34, 123.38,
115.68, 115.22, 95.67, 46.17, 28.80; APCI-HRMS m/z calcd
for C16H14IN2O2 (MH+), 393.0094, found 393.0085. Purity
(HPLC): 96.3%.
1
mp 155.2–156.1 °C (ethanol: petroleum ether). H NMR
(600 MHz, CDCl3) δ 8.25 (dd, J = 7.9, 1.6 Hz, 1H), 7.54
(ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.34 (t, J = 7.4 Hz, 2H),
7.26 (dd, J = 7.6, 3.3 Hz, 3H), 7.24–7.21 (m, 1H), 7.12
(d, J = 8.4 Hz, 1H), 5.39 (s, 2H), 3.56 (s, 3H). 13C NMR
(151 MHz, CDCl3) δ 162.01, 151.62, 139.86, 135.71,
135.00, 128.99, 127.68, 126.46, 123.08, 115.65, 114.37,
47.40, 28.70; APCI-HRMS m/z calcd for C16H15N2O2
(MH+), 267.1128, found 267.1118. Purity (HPLC): 100%.
The determination of IC50 values for MAO inhibition
The IC50 values were measured according to the literature
procedure [28]. Recombinant human MAO-A and MAO-B
were used as enzyme sources, and kynuramine served as
enzyme–substrate. The enzyme reactions were carried out to
a volume of 200 µL in white polypropylene 96-well microti-
ter plates. The reactions consisted of substrate (50 µM) and
the test inhibitors (0.003–100 µM) dissolved in potassium
phosphate bufer (pH 7.4, 100 mM). The reactions were
initiated with the addition of MAO-A (0.0075 mg/mL) or
MAO-B (0.015 mg/mL) and were incubated for 20 min at
37 °C. At endpoint, the reactions were terminated with the
addition of 80 µL NaOH (2 N) and the fuorescence was
measured (λex = 310, λem = 400). Quantitative estimations
of 4-hydroxyquinoline were made by constructing a linear
calibration curve (0.047–1.50 µM). From these data, the
enzyme catalytic rates were calculated and the IC50 values
were estimated by constructing sigmoidal plots of rate ver-
sus the logarithm of inhibitor concentration. For this pur-
pose, the data were ftted to the one-site competition model
incorporated into the Prism 5 software package. All IC50
3‑Methyl‑1‑phenethyl‑3,4‑dihydroquinazolin‑2(1H,3H)‑dione
(8c) The title compound was prepared in a yield of 25%:
1
mp 143.8–145.3 °C (petroleum ether: ethyl acetate). H
NMR (600 MHz, CDCl3) δ 8.26 (dd, J=7.9, 1.3 Hz, 1H),
7.71–7.64 (m, 1H), 7.36–7.29 (m, 4H), 7.27–7.22 (m, 3H),
4.37–4.31 (m, 2H), 3.50 (s, 3H), 3.06–2.99 (m, 2H). 13C
NMR (151 MHz, CDCl3) δ 161.96, 150.90, 139.55, 137.74,
135.08, 129.24, 128.82, 128.81, 126.91, 122.90, 115.63,
113.32, 45.18, 33.61, 28.42; APCI-HRMS m/z calcd for
C17H17N2O2 (MH+), 281.1284, found 281.1262. Purity
(HPLC): 99.6%.
3‑Methyl‑1‑(2‑phenox yethyl)‑3,4‑dihydroquinazo-
lin‑2(1H,3H)‑dione (8d) The title compound was prepared
in a yield of 10%: mp 148.8–165.3 °C (ethanol). 1H NMR
(600 MHz, CDCl3) δ 8.23 (dd, J=7.9, 1.5 Hz, 1H), 7.70–
7.65 (m, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.30–7.22 (m, 3H),
6.94 (t, J = 7.3 Hz, 1H), 6.83 (d, J = 8.2 Hz, 2H), 4.55 (t,
J=5.8 Hz, 2H), 4.34 (t, J=5.8 Hz, 2H), 3.49 (s, 3H). 13
C
NMR (151 MHz, CDCl3) δ 161.95, 158.14, 151.25, 140.32,
1 3

Molecular Diversity
values were measured in triplicate and are reported as the
mean standard deviation (SD).
The Ki value was estimated from a replot of the slopes of
the Lineweaver–Burke plots versus inhibitor concentration
(x-axis intercept equals –Ki).
Determination of reversibility of inhibition
by dialysis
Electronic supplementary material
Reversibility of inhibition was determined by dialysis. Dial-
ysis was carried out with Slide-A-Lyzer dialysis cassettes
with a molecular weight cut-of of 10,000 and a sample
volume of 0.5–3 mL. For this purpose, the literature pro-
cedure was followed [28]. MAO-A or MAO-B (0.03 mg/
mL) and the test inhibitors (at concentrations of 4 × IC50)
were dissolved in potassium phosphate bufer (100 mM, pH
7.4) containing 5% sucrose and pre-incubated for 20 min
at 37 °C. Stock solutions of the inhibitors were prepared
in DMSO and added to yield a fnal concentration of 4%
DMSO. The samples were subsequently dialysed for 24 h at
4 °C with the dialysis bufer (potassium phosphate bufer,
100 mM, pH 7.4, containing 5% sucrose) being replaced
with fresh bufer at 3 h and 7 h after dialysis was started.
As positive controls, MAO-A and MAO-B were pre-incu-
bated and dialysed in the presence of the irreversible inhibi-
tors, pargyline [IC50(MAO-A) = 13 µM] and (R)-deprenyl
[IC50(MAO-B) = 0.079 µM], respectively [35, 36]. The
concentrations of the irreversible inhibitors were 4×IC50.
Dialysis of the enzymes was also carried out in the absence
of inhibitor and served as negative control. After 24 h, the
dialysis samples were diluted twofold with the addition
kynuramine to yield an inhibitor concentration of 2 ×IC50
and a kynuramine concentration of 50 µM. These reactions
were incubated for 20 min at 37 °C, terminated with the
addition of NaOH (2 N), and the fuorescence of the sam-
ples was measured as described above. The residual enzyme
activities were calculated and expressed as mean SD of
triplicate determinations. For comparison, undialysed mix-
tures of the test inhibitors and the MAOs were maintained
at 4 °C for the same time period (24 h), and the residual
enzyme catalytic rates were measured.
1H NMR, 13C NMR, mass spectra and HPLC traces for the
synthesised compounds.
Acknowledgements André Joubert and Johan Jordaan of the SASOL
Centre for Chemistry, North-West University, recorded the NMR and
MS spectra.
Funding This work was supported by grants from the National
Research Foundation and the Medical Research Council of South
Africa (Grant specifc unique reference Numbers (UID) 85642, 96180,
916135). Opinions expressed and conclusions arrived at are those of
the authors and therefore the NRF does not accept any liability in
regard thereto.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
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Afliations
Lereze Marais1 · Anél Petzer1,2 · Jacobus P. Petzer1,2 · Lesetja J. Legoabe1
1
2
Centre of Excellence for Pharmaceutical Sciences, North-
West University, Private Bag X6001, Potchefstroom 2520,
South Africa
Pharmaceutical Chemistry, School of Pharmacy, North-West
University, Private Bag X6001, Potchefstroom 2520,
South Africa
1 3