Inhibition of monoamine oxidase by selected C5- and C6-substituted isatin analogues

Article abstract of DOI:10.1016/j.bmc.2010.11.028

Previous studies have shown that (E)-5-styrylisatin and (E)-6-styrylisatin are reversible inhibitors of human monoamine oxidase (MAO) A and B. Both homologues are reported to exhibit selective binding to the MAO-B isoform with (E)-5-styrylisatin being the most potent inhibitor. To further investigate these structure-activity relationships (SAR), in the present study, additional C5- and C6-substituted isatin analogues were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. With the exception of 5-phenylisatin, all of the analogues examined were selective MAO-B inhibitors. The C5-substituted isatins exhibited higher binding affinities to MAO-B than the corresponding C6-substituted homologues. The most potent MAO-B inhibitor, 5-(4-phenylbutyl) isatin, exhibited an IC₅₀ value of 0.66 nM, approximately 13-fold more potent than (E)-5-styrylisatin and 18,500-fold more potent than isatin. The most potent MAO-A inhibitor was found to be 5-phenylisatin with an IC ₅₀ value of 562 nM. The results document that substitution at C5 with a variety of substituents is a general strategy for enhancing the MAO-B inhibition potency of isatin. Possible binding orientations of selected isatin analogues within the active site cavities of MAO-A and MAO-B are proposed.

Full text of DOI:10.1016/j.bmc.2010.11.028

Bioorganic & Medicinal Chemistry 19 (2011) 261–274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of monoamine oxidase by selected C5- and C6-substituted
isatin analogues
⇑
Clarina I. Manley-King, Jacobus J. Bergh, Jacobus P. Petzer
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies have shown that (E)-5-styrylisatin and (E)-6-styrylisatin are reversible inhibitors of
human monoamine oxidase (MAO) A and B. Both homologues are reported to exhibit selective binding
to the MAO-B isoform with (E)-5-styrylisatin being the most potent inhibitor. To further investigate these
structure–activity relationships (SAR), in the present study, additional C5- and C6-substituted isatin ana-
logues were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. With the
exception of 5-phenylisatin, all of the analogues examined were selective MAO-B inhibitors. The C5-
substituted isatins exhibited higher binding affinities to MAO-B than the corresponding C6-substituted
homologues. The most potent MAO-B inhibitor, 5-(4-phenylbutyl)isatin, exhibited an IC50 value of
0.66 nM, approximately 13-fold more potent than (E)-5-styrylisatin and 18,500-fold more potent than
isatin. The most potent MAO-A inhibitor was found to be 5-phenylisatin with an IC50 value of 562 nM.
The results document that substitution at C5 with a variety of substituents is a general strategy for
enhancing the MAO-B inhibition potency of isatin. Possible binding orientations of selected isatin ana-
logues within the active site cavities of MAO-A and MAO-B are proposed.
Received 21 September 2010
Revised 3 November 2010
Accepted 9 November 2010
Available online 13 November 2010
Keywords:
Monoamine oxidase
Reversible inhibition
Selectivity
Competitive inhibition
Isatin
Molecular docking
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
are dependent on the inhibition of the catabolism of serotonin,
norepinephrine and dopamine in the brain which leads to in-
Monoamine oxidase (MAO) A and B are flavin adenine dinucle-
otide (FAD) containing enzymes which are tightly anchored to the
mitochondrial outer membrane.1 Although MAO-A and MAO-B are
encoded by separate genes, they share approximately 70% amino
acid sequence identity.2 The X-ray crystal structures of recombi-
nant human MAO-A3 and MAO-B1 have shown that the active site
amino acid residues and their relative geometries are also highly
conserved between the 2 enzymes and only 6 of the 16 active site
residues differ between the 2 isozymes.3 Despite these similarities,
the enzymes have unique substrate and inhibitor specificities. For
example, MAO-A catalyses the oxidation of serotonin and norepi-
nephrine and is irreversibly inhibited by clorgyline while MAO-B
preferentially utilises benzylamine as substrate and is irreversibly
inhibited by (R)-deprenyl. Both isoforms utilise dopamine as sub-
strate.4 In addition, literature reports a variety of small molecule
inhibitors with selectivities towards the two enzymes ranging
from negligible to several orders of a magnitude.5,6
creased levels of these neurotransmitters.4 MAO-A inhibitors are
particularly effective in the treatment of depression in elderly pa-
tients.4,7 Inhibitors of MAO-B are employed in the treatment of
neurodegenerative disorders such as Parkinson’s disease (PD).
MAO-B appears to be the major dopamine metabolizing enzyme
in the basal ganglia, and inhibitors of this enzyme may conserve
the depleted dopamine stores in the PD brain. This may lead to
enhanced dopaminergic neurotransmission and consequently
symptomatic relief of the symptoms of PD.8–10 MAO-B inhibitors
may also increase the elevation of dopamine levels in the basal
ganglia following levodopa treatment11 and are therefore used as
adjuvant to levodopa therapy in PD.12 Besides providing symptom-
atic relief, MAO-B inhibitors may also protect against further neu-
rodegeneration in PD by reducing the levels of potentially toxic
byproducts such as H2O2 and dopaldehyde which form as a result
of the oxidative metabolism of dopamine.13 MAO-B inhibitors may
be particularly relevant in the therapy in age-related neurodegen-
erative disorders such as PD since MAO-B activity and density
increase in most brain regions with age.14,15
Because MAO-A and MAO-B catalyses the catabolism of neuro-
transmitter amines, they are considered attractive drug targets in
the therapy of neurological disorders. Both reversible and irrevers-
ible inhibitors of MAO-A are used to treat depressive illness and
anxiety disorder. The antidepressant effect of MAO-A inhibitors
The endogenous small molecule inhibitor isatin (1) (Fig. 1) is
reported to be a reversible inhibitor of both human MAO-A and
MAO-B with enzyme–inhibitor dissociation constants (Ki values)
of 15 lM and 3 l
M for the two isozymes, respectively.16 The
three-dimensional structure of recombinant human MAO-B with
isatin bound to the active site shows that isatin is located in the
⇑
Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.028

262
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
O
O
N
N
N
O
O
N
Cl
O
N
H
Isatin (1)
8-(3-Chlorobenzyloxy)caffeine (6)
O
5
H
CH3
O
O
NH2
N
N
F
H
H
(E)-5-Styrylisatin (2)
O
O
Safinamide (7)
CHO
O
6
N
H
Cl
(E)-6-Styrylisatin (3)
O
O
O
Figure 1. The structures of isatin (1), (E)-5-styrylisatin (2) and (E)-6-styrylisatin (3).
7-(3-Chlorobenzyloxy)-4-formylcoumarin (8)
Figure 3. The structures of 8-(3-chlorobenzyloxy)caffeine (6), safinamide (7) and 7-
(3-chlorobenzyloxy)-4-formylcoumarin (8).
substrate cavity in close proximity to the FAD co-factor where it is
involved in hydrogen bonding with conserved water molecules.17
Since isatin binds within the substrate cavity, the entrance cavity
of the enzyme is unoccupied. Based on this observation we have
recently synthesized (E)-5-styrylisatin (2) and (E)-6-styrylisatin
(3) in an attempt to enhance the binding affinity of isatin to
MAO-B.18 The results documented that both (E)-styrylisatin ana-
logues exhibited significantly higher binding affinities than isatin
with the C5-substituted isomer being the more potent inhibitor
of the two isomers. Modelling studies suggested that the (E)-sty-
rylisatin analogues binds to the MAO-B active site with the isatin
dioxoindolyl ring bound to the substrate cavity while the styryl
side chain extends into the entrance cavity. The interaction of
the styryl side chain with the entrance cavity amino acid residues
may allow for more productive binding with the enzyme compared
to isatin and hence more potent inhibition.18 In accordance with
this analysis the small molecule caffeine (4) (Fig. 2), which is ex-
pected to bind to either the substrate or entrance cavity, is a weak
MAO-B inhibitor with a Ki value of 3.6 mM.18 The C8 chlorostyryl
substituted analogue, (E)-8-(3-chlorostyryl)caffeine [CSC, (5)],
however was found to be a potent reversible inhibitor with a Ki
human MAO-B with a Ki value of 0.036 l
M, approximately 105-fold
more potently than caffeine. Modelling studies have shown that
the caffeine ring is located within the substrate cavity of the en-
zyme while the benzyloxy side chain binds within the entrance
cavity. Again, the improved inhibition of the 8-benzyloxycaffeiene
analogues compared to caffeine may be explained by binding inter-
actions between the benzyloxy side chain and the entrance cavity
of MAO-B. The view that the benzyloxy side chain binds within the
entrance cavity is supported by the three-dimensional structure of
a complex between safinamide (7) and recombinant human MAO-
B which shows that the 3-fluorobenzyloxy side chain of safinamide
occupies the entrance cavity while the propanamidyl moiety is
located within the substrate cavity.22 Similarly, the structure of a
complex between 7-(3-chlorobenzyloxy)-4-formylcoumarin (8)
and human MAO-B shows that the 3-chlorobenzyloxy side chain
binds in the entrance cavity of the enzyme with the coumarin ring
occupying the substrate cavity.22
While the three-dimensional complex between isatin and MAO-
A has not yet been determined, modelling studies have been per-
formed with (E)-5-styrylisatin (2) and (E)-6-styrylisatin (3).18
These suggest that, similar to its binding mode within MAO-B,
the dioxoindolyl rings of both isomers occupy the space in close
proximity to the FAD co-factor with their respective styryl side
chains extending towards the entrance of the active site. Notably,
(E)-5-styrylisatin exhibited a 19-fold higher binding affinity to
MAO-A than isatin while the C6-substituted isomer (3) had a sim-
ilar binding affinity to that of isatin.18 The lack of enhancement of
the MAO-A binding affinity by C6 styryl substitution is not well
understood.
value of 0.086 l
M.19,20 The higher affinity of CSC for the MAO-B
active site may be explained by the additional productive interac-
tions of the chlorostyryl side chain within the entrance cavity.
We have recently shown that the MAO-B binding affinity of
caffeine may also be enhanced by substitution with a variety of
benzyloxy side chains at C8 of the caffeine ring.21 For example,
8-(3-chlorobenzyloxy)caffeine (6) (Fig. 3) inhibits recombinant
O
N
N
To further investigate these structure–activity relationships
(SAR), in the present study, we have synthesized additional C5-
and C6-substituted isatin analogues and evaluated them as inhib-
itors of recombinant human MAO-A and MAO-B. One of the goals
of this study was to determine if C5-substituted isatin analogues
are in general better MAO-B inhibitors than the corresponding C6
isomers as observed with the (E)-styrylisatin analogues. Further-
more, this study also aimed to determine the effect of C5- and
C6-substitution of isatin on MAO-A inhibition activity. As dis-
cussed above, compared to isatin, (E)-5-styrylisatin (2) was found
to be a better MAO-A inhibitor while the C6-substituted isomer
(3) had a similar inhibition potency to that of isatin.18 Among the
N
O
N
Caffeine (4)
O
N
N
N
Cl
O
N
(E)-8-(3-Chlorostyryl)caffeine (CSC) (5)
Figure 2. The structures of caffeine (4) and (E)-8-(3-chlorostyryl)caffeine [CSC, (5)].

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
263
C5- and C6-substituents chosen for this study was the benzyloxy
side chain which has been shown to enhance the binding affinity
of caffeine to the active site of both MAO-A and MAO-B.21 Other
substituents considered in this study include the phenoxy, 2-phen-
ylethyl, 4-phenylbutyl, phenyl and 4-chlorophenoxy groups. We
have also examined the importance of the isatin moiety for binding
to MAO-A and MAO-B by comparing the inhibition potencies of the
C5- and C6-substituted isatins with those of the corresponding ani-
line analogues. With this comparison the importance of the car-
bonyl functional groups of the dioxoindolyl ring for binding to
the MAO isozymes may be determined. Literature reports that
the NH and the C2 carbonyl oxygen of isatin are hydrogen bonded
to water molecules in the substrate cavity of MAO-B.17 Similar
interactions may also be possible between the aniline NH2 and
the active sites of the MAO enzymes.
Figure 4. The X-ray crystallographic structure of 9d shown with displacement
ellipsoids.
the pH of the filtrates to 3 and then <1 and column chromatogra-
phy (see Section 4). While the Sandmeyer methodology is more
frequently used for the synthesis of isatin analogues, the low solu-
bility of the starting anilines in the aqueous reaction medium made
this procedure unsuitable for the synthesis of the target isatin ana-
logues.24 The successful formation of the isatin ring system of the
target compounds were verified by the presence of a 13C NMR sig-
nal at 181–185 ppm which corresponds to carbonyl C3 and a signal
at 159–161 ppm which corresponds to carbonyl C2 (Table 1).25
With the exception of 4-(2-phenylethyl)aniline (10c), 3-(2-
phenylethyl)aniline (10d) and 4-(4-phenylbutyl)aniline (10i) all
of the anilines required for the synthesis of the isatin analogues
were commercially available. Anilines 10c and 10d were synthe-
sized by reacting diethyl 4- or diethyl 3-nitrobenzylphosphonate
(11a, b)26 with benzaldehyde (12) to yield the 4- or 3-nitrostilb-
enes (13a, b), respectively (Scheme 2).27 Catalytic hydrogenation
of the nitrostilbenes in the presence of Pd/C yielded the corre-
sponding anilines (10c, d). Aniline 10i was similarly synthesized
by reacting diethyl 4-nitrobenzylphosphonate (11a) with cinna-
maldehyde (14) to obtain 1-nitro-4-[(1E,3E)-4-phenylbuta-1,3-
dien-1-yl]benzene (15). Again, hydrogenation of 15 afforded the
corresponding aniline 10i.
2. Results
2.1. Chemistry
In the present study a series of 10 C5- and C6-substituted isatin
analogues (9a–j) were synthesized with the aim of examining their
MAO inhibitory properties. The C5-substituted isatin analogues
(9a, c, e, g, i, j) were synthesized by treating the appropriately
C4-substituted aniline (10a, c, e, g, i, j) with diethyl ketomalonate
in the presence of acetic acid according to the literature description
(Scheme 1).23 The C6-substituted isatin analogues (9b, d, f, h) were
similarly synthesized, from the C3-substituted aniline derivatives
(10b, d, f, h) and diethyl ketomalonate. While the latter reaction
may also give the corresponding C4-substituted isatin analogues,
only a single product was isolated from the reaction mixtures. 1H
NMR indicated that in each instance these were the C6-substituted
analogues as evidenced by the singlet corresponding to the C4 pro-
ton (9b, 6.46 ppm; 9d, 6.61 ppm; 6f, 6.28 ppm; 9h, 7.09 ppm). This
is in accordance to the literature report that the reaction between
C3-substituted aniline derivatives and diethyl ketomalonate yields
the corresponding C6-substituted isatins.23 Furthermore, the
molecular structure of 9d was elucidated by X-ray crystallography
and confirms substitution at the C6 position (Fig. 4). Although the
target compounds were obtained in low yields (1.2–9.3%) the crys-
talline products were of a high degree of purity as judged by HPLC
(see Section 4). These low yields may be due to resinification and
the formation of side products. Fortunately, the desired isatins
could be obtained via a combination of filtration steps, adjusting
2.2. MAO inhibition studies—isatin analogues
To determine the MAO-A and MAO-B inhibition potencies of the
test inhibitors, the extent by which different concentrations of a
test inhibitor reduces the rate of the MAO-catalysed oxidation of
kynuramine, a mixed MAO-A/B substrate, was measured. For this
purpose the recombinant human MAO-A and MAO-B enzymes
were employed.28 Kynuramine is non-fluorescent until undergoing
Table 1
13C NMR chemical shifts for carbonyl C2 and C3 of isatin derivatives 9a–ja
O
R1
R2
R1
R2
4
5
6
a, b
O
O
R1
3
3
5
N
NH2
O
H
10a-j
9a-j
2
R2
6
N
R1
R2
H
9
a
b
c
d
e
f
C6H5CH2O
H
H
C6H5CH2O
H
R1
R2
H
C2
C3
C6H5CH2CH2
9a
9b
9c
9d
9e
9f
9g
9h
9i
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
159.5
160.5
159.5
159.9
159.6
160.1
159.6
159.8
159.5
159.5
184.6
181.5
184.5
183.7
184.1
181.8
184.4
183.7
184.6
184.0
H
C6H5CH2CH2
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
H
C6H5O
H
H
C6H5O
H
g
h
i
C6H5
H
C6H5
H
C6H5(CH2)4
4-ClC6H4O
H
j
H
C6H5(CH2)4
4-ClC6H4O
9j
H
Scheme 1. Synthetic route to C5- and C6-substituted isatin analogues (9a–j).
Reagents and conditions: (a) diethyl ketomalonate, CH3CO2H, 120 °C; (b) air, 120 °C.
a
The NMR experiments were conducted in DMSO-d6 (see Section 4).

264
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
b
R1
a
CHO
+
NO2
(EtO)2
P
NO2
NH2
O
R2
12
13a, b
11a, b
R1 = C6H5CH2CH2
R2 = H
10c:
R1 = H
R2 = C6H5CH2CH2
10d:
NO2
CHO
R1
NH2
NO2
b
+
a
(EtO)2
P
R2
O
14
15
11a
R1 = C6H5(CH2)4
R2 = H
10i:
Scheme 2. Synthetic route to aniline derivatives 10c, 10d and 10i. Reagents and conditions: (a) NaOEt; (b) H2 (atm), Pd/C (10%), rt.
MAO-catalysed oxidative deamination and subsequent ring closure
to yield 4-hydroxyquinoline, a fluorescent metabolite. The concen-
trations of the MAO-generated 4-hydroxyquinoline in the incuba-
tion mixtures was determined by comparing the fluorescence
emitted by the samples to that of known amounts of authentic
4-hydroxyquinoline.28 At the excitation (310 nm) and emission
(400 nm) wavelengths and inhibitor concentrations used in this
study, none of the test inhibitors fluoresced or quenched the fluo-
rescence of 4-hydroxyquinoline. The inhibition potencies of the
test inhibitors were expressed as the IC50 values (Fig. 5). To allow
for the calculation of the selectivity index [SI = Ki(MAO-A)/Ki(-
MAO-B)], the experimentally determined IC50 values were con-
verted to the corresponding Ki values for the inhibition of MAO-A
and MAO-B according to the Cheng–Prusoff equation.20,29
30
25
20
15
10
5
A
-3
-2
-1
0
1
2
3
Log[I]
The IC50 values for the inhibition of MAO-A and -B by isatin ana-
logues 9a–j are presented in Table 2. For comparison, the inhibition
potencies of isatin (1), (E)-5-styrylisatin (2) and (E)-6-styrylisatin
(3) were also measured and are given in Table 2. The MAO-A and
-B inhibition potencies of compounds 1–3 have been previously re-
ported using the purified recombinant human enzymes which
were expressed in Pichia pastoris.16,18 The present account reports
the inhibition data using membrane bound recombinant human
MAO-A and -B from insect cells. In accordance with the literature,
isatin was found to be a moderately potent inhibitor of MAO-A and
4
3
2
1
-B with IC50 values of 31.8 lM and 12.4 lM, respectively. Based on
the selectivity index (Table 2) isatin is approximately 1.57-fold
more selective for MAO-B than for the A isoform. Also in agreement
with the literature18 was the finding that (E)-5-styrylisatin (2) is a
potent inhibitor of both MAO-A and -B with IC50 values of
B
-3
-2
-1
0
1
2
3
Log[I]
0.233 lM and 0.009 lM, respectively. In fact, (E)-5-styrylisatin
Figure 5. The sigmoidal dose–response curve of the initial rates of oxidation of
kynuramine by recombinant human MAO-A (Panel A) and recombinant human
MAO-B (Panel B) versus the logarithm of concentration of inhibitor 9g (expressed in
nM). The determinations were carried out in duplicate and the values are expressed
as the mean SD. The concentrations of kynuramine used were 45 lM and 30 lM
for the studies with MAO-A and MAO-B, respectively, and the rate data are
was the second most potent MAO-B inhibitor examined in this
study and approximately 1300-fold more potent than was isatin.
Also in agreement with literature,18 (E)-6-styrylisatin was a rela-
tively potent MAO-B inhibitor, while exhibiting moderately potent
MAO-A inhibitory activity. Compared to the C5-substituted isomer
2, (E)-6-styrylisatin was approximately 68-fold less potent as an
MAO-B inhibitor.
expressed as nmol 4-hydroxyquinoline formed/min/mg protein.
The most potent MAO-B inhibitor among the examined com-
pounds was 5-(4-phenylbutyl)isatin (9i) with an IC50 value of
0.66 nM, approximately 13-fold more potent than (E)-5-styrylisat-
in and 18,500-fold more potent than isatin. The observation that
the 4-phenylbutyl group is the longest side chain considered in this
study indicates that longer side chains enhance the MAO-B inhibi-
tion potency of isatin to a larger extent compared to relatively
shorter side chains. In contrast to its effect on the MAO-B inhibition
potency, the 4-phenylbutyl side chain did not enhance the MAO-A
inhibition potency of isatin to a great extent. Compound 9i only
moderately inhibited MAO-A with an IC50 value of 2.19 lM,
approximately 14-fold more potent than the MAO-A inhibition
potency of isatin. The only isatin analogues examined in this study
which potently inhibited MAO-A were (E)-5-styrylisatin (2) and

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
265
Table 2
The IC50 values and calculated Ki values for the inhibition of recombinant human MAO-A and -B by isatin derivatives 9a–ja
O
R1
O
R2
N
H
9
IC50
(
lM)
Kib
(lM)
SIc
R1
R2
MAO-A
MAO-B
0.103 0.011
0.138 0.005
1.40 0.124
9.93 4.54
1.54 0.575
9.91 0.752
1.19 0.173
8.52 0.260
0.00066 0.00001
0.066 0.008
12.4 0.693
0.009 0.001
0.617 0.011
MAO-A
MAO-B
9a
9b
9c
9d
9e
9f
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
H
C6H5(CH2)4
4-ClC6H4O
H
(E)-C6H5CH@CH
H
H
4.62 0.148
72.4 26.3
4.88 2.03
6.93 0.842
9.44 0.356
62.2 8.25
0.562 0.026
4.64 0.556
2.19 0.218
12.2 1.22
31.8 4.50
0.233 0.006
2.72 0.082
1.22
19.1
1.29
1.83
2.49
16.4
0.148
1.22
0.577
3.21
8.38
0.044
0.059
0.603
4.28
0.663
4.27
0.513
3.67
0.00028
0.028
5.34
27.4
321
2.13
0.427
3.75
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
H
3.84
9g
9h
9i
0.289
0.333
2030
113
9j
H
H
H
Isatin
2
3
1.57
15.8
2.70
0.061
0.717
0.004
0.266
(E)-C6H5CH@CH
a
All values are expressed as the mean SD of duplicate determinations.
b
The Ki values were calculated from the experimentally measured IC50 values according to the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 45
M for human MAO-A and [S] = 30 M and Km (kynuramine) = 22.7
M for human MAO-B.21,29
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of Ki(MAO-A)/Ki(MAO-B).
lM and
Km (kynuramine) = 16.1
l
l
l
c
(E)-5-phenylisatin (9g) with IC50 values of 0.233
respectively. The other homologues examined all exhibited IC50
values towards MAO-A in the M range. Interestingly, compounds
2 and 9g are the only C5-substituted isatin analogues with a side
chain phenyl ring, that is, conjugated to the isatin ring system. Also
noteworthy is the observation that the majority (seven) of the isat-
in analogues examined displayed selectivity for the MAO-B isoform
(Table 2).
l
M and 0.562
l
M,
and (E)-2-styrylbenzimidazolyl analogues.30 For example, 8-(3-
chlorobenzyloxy)caffeine is reported to inhibit human MAO-B with
l
an IC50 value of 0.107
than the unsubstituted analogue, 8-benzyloxycaffeine, with an
IC50 value of 1.77
M.21
lM, approximately 16-fold more potently
l
2.3. MAO inhibition studies—aniline analogues
The C5- and C6-benzyloxy substituted isatin analogues (9a, b)
were also found to be potent MAO-B inhibitors with IC50 values
of 0.103 lM and 0.138 lM, respectively. As stated in the Introduc-
tion, this finding is in agreement with literature reports that the
benzyloxy side chain enhances the binding affinity of small mole-
cules such as caffeine to the active site of MAO-B.21 Interestingly,
the C6-benzyloxy substituted analogue 9b was the weakest
MAO-A inhibitor among the isatin analogues.
As mentioned in Section 1, the importance of the isatin moiety
for binding to MAO-A and MAO-B was also examined by comparing
the inhibition potencies of the C5- and C6-substituted isatins with
those of the corresponding para- and meta-substituted anilines.
Inspection of the X-ray crystal structure of isatin in complex with
human MAO-B suggests that the dioxoindolyl NH and the C2 car-
bonyl oxygen are involved in stabilizing hydrogen bond interac-
tions with water molecules in the substrate cavity of MAO-B.17
Since similar interactions may also be possible between the aniline
NH2 and the active sites of the MAO enzymes, the anilines may also
possess MAO inhibitory properties. By comparing the MAO inhibi-
tion potencies of the isatins with those of the anilines, the impor-
tance of the lactam and C2 carbonyl functional groups of the
dioxoindolyl ring for binding to the MAO isozymes may be
evaluated.
Compared to the benzyloxy substituted isatin analogues (9a, b),
5-(2-phenylethyl)isatin (9c) and 6-(2-phenylethyl)isatin (9d) were
relatively weaker MAO-B inhibitors with IC50 values of 1.40
lM
and 9.93 M, respectively. Compounds 9c was approximately 13-
l
fold less potent that the corresponding C5-benzyloxy substituted
isatin analogues (9a) while 9d was approximately 71-fold less
potent than the corresponding C6-benzyloxy substituted isatin
analogue 9b. Similarly, 5-phenoxyisatin (9e), 6-phenoxyisatin
(9f), 5-phenylisatin (9g) and 6-phenylisatin (9h) were also found
to be relatively weaker MAO-B inhibitors than the benzyloxy
substituted analogues (9a, b). It can therefore be concluded that
the 2-phenylethyl, phenoxy and phenyl side chains do not increase
the MAO-B binding affinity of isatin to the same extent observed
for the (E)-styryl and benzyloxy side chains.
The IC50 values for the inhibition of recombinant human MAO-A
and -B by the aniline analogues (10a–l) are presented in Table 3.
All of the anilines evaluated were found to be relatively weak
MAO-A inhibitors with the most potent compound (10k) exhibit-
ing an IC50 value of 25.3
MAO-B inhibitors with the most potent inhibitor, the 4-phenylbu-
tyl substituted aniline (10i), exhibiting an IC50 value of 5.55 M.
lM. The anilines were also relatively weak
l
While 5-phenoxyisatin (9e) was found to be moderately potent
With the exception of 10c, 10e and 10h, all of the anilines exam-
ined displayed selectivity for the MAO-B isoform (Table 3). This
isoform selectivity is similar to that observed for the isatin
analogues.
MAO-B inhibitor (IC50 = 1.54
phenoxy analogue 9j proved to be a potent MAO-B inhibitor with
an IC50 value of 0.066 M. This result demonstrates the ability of
lM), the C5-substituted 4-chloro-
l
halogen substitution at a side chain phenyl ring to enhance binding
affinity of reversible inhibitors to MAO-B. This effect is similar to
that observed for 8-benzyloxycaffeinyl,21 (E)-8-styrylcaffeinyl19
Interestingly, the 4-phenylbutyl substituted isatin analogue
was also the most potent MAO-B inhibitor among the isatin ana-
logues. Inspection of the inhibition data in Tables 2 and 3 shows

266
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
Table 3
The IC50 values and calculated Ki values for the inhibition of recombinant human MAO-A and -B by aniline derivatives 10a–la
R1
R2
NH2
10
IC50
(l
M)
Kib
(
lM)
SIc
R1
R2
MAO-A
MAO-B
MAO-A
MAO-B
10a
10b
10c
10d
10e
10f
10g
10h
10i
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
H
C6H5(CH2)4
4-ClC6H4O
(E)-C6H5CH@CH
H
H
191 53.5
466 75.2
131 6.22
12.8 4.47
23.4 5.65
135 0.011
115 7.85
303 8.77
140 42.1
50.3
123
34.5
93.5
24.1
—
—
18.1
—
5.51
10.1
58.1
49.5
131
60.3
—
9.13
12.2
0.594
1.89
0.185
C6H5CH2O
H
C6H5CH2CH2
H
C6H5O
H
C6H5
H
H
H
355 38.8
91.4 21.1
No inhibitiond
No inhibitiond
68.6 1.32
No inhibitiond
29.3 1.33
25.3 2.01
30.4 4.70
—
No inhibitiond
No inhibitiond
5.55 0.492
16.8 1.74
6.34 3.41
5.74 1.05
—
—
—
1.07
2.44
3.24
—
2.39
7.24
2.73
2.47
10j
10k
10l
7.72
6.67
8.01
(E)-C6H5CH@CH
a
All values are expressed as the mean SD of duplicate determinations.
b
The Ki values were calculated from the experimentally measured IC50 values according to the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 45
M for human MAO-A and [S] = 30 M and Km (kynuramine) = 22.7
M for human MAO-B.21,29
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of Ki(MAO-A)/Ki(MAO-B).
lM and
Km (kynuramine) = 16.1
l
l
l
c
d
No inhibition observed at a maximum tested concentration of 100 lM.
that the order of the MAO-B inhibition potencies of the aniline ana-
logues is similar to that of the isatin analogues. For example, the
aniline analogues substituted with styryl, benzyloxy and 4-chloro-
phenoxy side chains at the para- and meta-positions were more po-
tent MAO-B inhibitors than the corresponding 2-phenylethyl,
phenoxy and phenyl substituted anilines. Similarly, the C5- and
C6-substituted styryl, benzyloxy and 4-chlorophenoxy isatin ana-
logues were more potent MAO-B inhibitors than the corresponding
2-phenylethyl, phenoxy and phenyl substituted isatins. These data
suggests that the isatin and aniline analogues exhibit similar bind-
ing modes to the active site of MAO-B. Previous modelling stud-
ies18 have suggested that (E)-5-styrylisatin (2) and (E)-6-
styrylisatin (3) bind to the MAO-B active site with the dioxoindolyl
rings of both isomers occupying the substrate cavity space in close
proximity to the FAD co-factor while their respective styryl side
chains extends towards the entrance cavity of the enzyme. Should
the aniline analogues exhibit as similar binding mode, the aniline
moiety is expected to also occupy the relatively polar substrate
cavity31 where the NH2 may be involved in hydrogen bond interac-
tions. The para- and meta-substituted side chains are expected to
extend towards the entrance cavity of the enzyme. By employing
molecular docking studies, possible binding modes and interac-
tions of selected aniline and isatin analogues within an MAO-B ac-
tive site model will be proposed below.
able to propose that the carbonyl oxygen of isatin and C5- and
C6-substituted isatin analogues also may act as hydrogen bond
acceptors in the MAO-A active site. Since the aniline analogues
examined here do not possess carbonyl functional groups they lack
the additional stabilizing interactions with the MAO active sites
that these functional groups provide and are hence weaker inhib-
itors than the isatins. Another factor that may contribute to stabi-
lizing isatins, and not anilines, within the active sites of MAO-A
and -B are possible
p–p stacking interactions between the dioxo-
indolyl ring and the amide of an active site Gln residue. In MAO-
A, Gln-215 is reported to undergo stacking interactions with har-
mine3 while in MAO-B Gln-206 may similarly interact with bound
ligands.
Since both aniline and isatin are expected to be uncharged in
the buffer used for the inhibition studies (pH 7.4), differing ionisa-
tion states of the aniline NH2 and isatin lactam NH do not explain
the difference in binding affinities to the MAO enzymes. Also, since
aminyl substrates of MAO are reported to bind as the deprotonated
amines to the active sites of these enzymes, it may be expected
that the uncharged aniline and isatin species are the active
inhibitors.11,32
2.4. Reversibility studies
The finding that the aniline analogues are weaker MAO-A and -
B inhibitors than the corresponding isatin analogues indicates that
the lactam and C2 carbonyl functional groups of the dioxoindolyl
ring are important structural features for binding to the MAO ac-
tive sites. A possible explanation may be that the dioxoindolyl car-
bonyl oxygens act as hydrogen bond acceptors within the substrate
cavities of MAO-A and -B thereby providing additional stabilization
of the inhibitor–enzyme complex. In accordance with this pro-
posal, the three-dimensional structure of isatin bound to human
MAO-B has shown the C2 carbonyl oxygen to be hydrogen bonded
to ordered water molecules in the substrate cavity.17 While the
structure of isatin bound to MAO-A has not yet been determined
the architectures of the MAO-A and -B active sites are similar,3
especially in the vicinity of the FAD co-factor where hydrogen
bonding between isatin and MAO-B occur. It is therefore reason-
With the finding that a several C5- and C6-substituted isatin
analogues are potent MAO-A and -B inhibitors, this study further
aimed to investigate whether the observed enzyme inhibition is
reversible or irreversible. For this purpose the time dependence
of MAO-A and -B inhibition by one representative inhibitor, com-
pound 9c, was evaluated. The MAO-A and -B inhibition potencies
of compound 9c are relatively lower compared to other isatin ana-
logues evaluated in this study. Since these studies are conducted at
concentrations equal to the IC50 values of the test compound, this
would allow for the use of relatively higher inhibitor concentra-
tions compared to the concentrations that would be needed for
more potent compounds. A possible hydrolysis event (see below)
is expected to have a smaller effect (over the 60 min experimental
time) on higher concentrations of an inhibitor and would yield re-
sults that are better interpretable. Recombinant human MAO-B

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
267
was preincubated with 9c for periods of 0, 15, 30 and 60 min and
the residual rates of the MAO-A and -B catalysed oxidation of
kynuramine were measured. For this purpose, the concentrations
relatively small. Time-dependent inhibition studies with other
isatin analogues yielded similar results (data not shown).
To further examine the modes of MAO-A and -B inhibition, sets
of Lineweaver–Burk plots were constructed for the inhibition of
both enzymes by 9c, the selected representative inhibitor (Fig. 7A
and B). Inspection of the Lineweaver–Burk plots suggests that 9c
inhibits both MAO-A and -B competitively since the plots are linear
and intersect at the y-axis. These findings lend further support for
the finding that 9c interacts reversibly with the active sites of
human MAO-A and -B and is in accordance with literature which
reports that both isatin16 and (E)-styrylisatin analogues18 are com-
petitive inhibitors of recombinant human MAO-A and -B.
of 9c chosen were 9.76
lM for the incubations with MAO-A and
2.80 M for the incubations with MAO-B. These concentrations
l
are approximately twofold the measured IC50 values for the inhibi-
tion of the respective enzymes by 9c.
As shown in Figure 6A and B, there is no time-dependent reduc-
tion in the rates of MAO-A and -B catalysed oxidation of kynur-
amine when compound 9c is preincubated with the enzyme for
various periods of time. From this result it may be concluded that
the inhibition of MAO-A and -B is reversible, at least for the time
period (60 min) and at the inhibitor concentrations (2 Â IC50) eval-
uated. Interestingly, marked increases of both the MAO-A and -B
catalytic rate with increased preincubation time of 9c with the en-
zymes are observed. One possible explanation for this observation
is that 9c, and probably the other isatin analogues, undergo slow
hydrolysis in the aqueous buffer (pH 7.4) used for the inhibition
studies. Isatins are known to undergo C–N bond fission to yield
the ring-opened amino acid. This process is reversible and acidifi-
cation reforms the isatin.33 Considering that an incubation time of
20 min was chosen for determining the inhibition potencies of the
isatin analogues, the recorded IC50 values (Table 2) may be an
underestimation of the MAO-A and -B inhibition potencies. Since
relatively little loss of inhibition potency of the isatin analogues
is observed between the 15 min and 30 min time points, the effect
of hydrolysis on the measured IC50 values is expected to be
2.5. Molecular modelling studies
The findings of this study show that while the isatin analogues
are in general good MAO-A and -B inhibitors, the corresponding
aniline analogues act as weak inhibitors. As discussed above, one
possible reason for this observation is that, in addition to the po-
tential hydrogen bonding interactions provided by the lactam
nitrogen, the isatin carbonyl oxygens may interact via hydrogen
bonding with MAO-A and -B active site residues and water mole-
cules in the vicinity of the FAD co-factor. This would in turn lead
to additional stabilization of the inhibitor–enzyme complex. Since
the aniline analogues do not possess carbonyl functional groups
similar stabilizing interactions between the anilines and the
MAO-A and -B active sites are absent. For the anilines, the anilinic
0.60
A
A
8
0.45
0.30
0.15
0.00
6
4
2
0
0
15
30
60
-0.02
0.00
0.02
1/[S]
0.04
0.06
Incubation time (min)
3
2
1
0
B
B
1.6
1.2
0.8
0.4
0.0
0
15
30
60
-0.02
0.00
0.02
1/[S]
0.04
0.06
Incubation time (min)
Figure 6. Time-dependant inhibition of the recombinant human MAO-A (Panel A)
and recombinant human MAO-B (Panel B) catalysed oxidation of kynuramine by
compound 9c. The enzymes were preincubated for various periods of time (0–
Figure 7. Lineweaver–Burk plots of the recombinant human MAO-A (Panel A) and
recombinant human MAO-B (Panel B) catalysed oxidation of kynuramine in the
absence (filled squares) and presence of various concentrations of 9c. For the
60 min) with 9c at concentrations of 9.76
respectively. The concentrations of kynuramine used were 45
l
M and 2.80
l
M for MAO-A and MAO-B,
M and 30 M for the
l
l
studies with MAO-A (Panel A) the concentrations of 9c were: 2.5
squares), 5 M (filled circles), 10 M (open circles). For the studies with MAO-B
(Panel B) the concentrations of 9c were: 0.125 M (open squares), 0.25 M (filled
circles), 0.5 M (open circles). The rates (V) are expressed as nmol product formed/
min/mg protein.
lM (open
studies with MAO-A and MAO-B, respectively, and the rate data are expressed as
nmol 4-hydroxyquinoline formed/min/mg protein. The catalytic rates recorded in
the absence of the inhibitors are 12.6 1.05 and 6.94 0.02 nmol/min/mg for MAO-
A and -B, respectively.
l
l
l
l
l

268
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
nitrogen is the only functional group that could undergo hydrogen
bonding in the vicinity of the FAD co-factor. To provide additional
insight, the binding modes of 5-benzyloxyisatin (9a) and its corre-
sponding aniline, 4-benzyloxyaniline (10a), in MAO-A and -B were
examined using molecular docking.
The structures of human MAO-A co-crystallized with harmine
(PDB entry: 2Z5X)3 and human MAO-B co-crystallized with safina-
mide (PDB entry: 2V5Z)22 were selected and molecular docking
was carried out according to a modification of a previously re-
ported protocol with the LigandFit application of the Discovery
Studio 1.7 modelling software (Accelrys).21 These models were se-
lected based on the high resolution of the crystallographic struc-
tures. Furthermore, in the complex between MAO-B and
safinamide, the side chain of Ile-199 is rotated out of the normal
conformation. This allows for the fusion of the entrance and sub-
strate cavities which is a necessity for the binding of relatively
large inhibitors which span both the entrance and substrate cavi-
ties.16 The active site of MAO-A on the other hand consists of a sin-
gle cavity. The valences of the FAD co-factor and the co-crystallised
ligands were corrected, hydrogen atoms were added to the MAO-A
and -B models and the models were subjected to a three-step
energy minimisation procedure with the protein backbone con-
strained (see Section 4). After the energy minimisation procedure,
the backbone constraint was removed and the co-crystallised
ligands were deleted from the models. The structures of 9a and
10a were constructed and geometry optimised within Discovery
Studio and subsequently docked into the protein models with
the LigandFit application of Discovery Studio. The docked inhibitor
orientations and conformations were further refined with the
Smart Minimizer algorithm in Discovery Studio and 10 possible
binding solutions were computed for each inhibitor. The accuracy
of this procedure was evaluated by redocking the co-crystallized li-
gands, harmine and safinamide, into the active sites of MAO-A and
-B, respectively. After each inhibitor was docked three times the
best-ranked orientations of harmine and safinamide exhibited
RMSD values of 0.64 Å and 1.54 Å, respectively, from the position
of the co-crystallized ligand. This protocol was therefore deemed
to be suitable for the docking of inhibitors into the active site of
MAO-B.
The best-ranked docking solution of isatin derivative 9a within
the active site of MAO-B shows that the dioxoindolyl ring binds
within the substrate cavity in close proximity of the FAD co-factor
(Fig. 8A). This binding orientation of the dioxoindolyl ring is similar
to that observed for isatin co-crystallized within the active site of
recombinant human MAO-B17 and for (E)-5-styrylisatin previously
docked into an MAO-B model.18 The C5 benzyloxy side chain of 9a
extends beyond the boundary defined by the side chain of Ile-199
into the entrance cavity of the enzyme. This binding orientation is
similar to that observed for the co-crystallized inhibitor, safina-
mide, which also spans both active site cavities.22 A variety of
relatively large inhibitors such as trans,trans-farnesol16 and 1,4-di-
phenyl-2-butene17 are also reported to traverse both MAO-B active
site cavities. Within the hydrophobic environment of the entrance
cavity, the benzyloxy side chain is most likely stabilized by Van der
Waals interactions.31 These interactions may, in part, explain the
enhanced MAO-B inhibition potencies of C5- and C6-substituted
isatin analogues compared to isatin (Table 2). In contrast to the
substituted isatin analogues, isatin binds only within the substrate
cavity and does not interact with the entrance cavity residues. Of
importance is the observation that the 2-oxo and NH functional
groups of 9a interact via hydrogen bonding with water molecules
present in the active site. In the three-dimensional structure of
isatin bound to human MAO-B, the C2 carbonyl oxygen of isatin
is also hydrogen bonded to an ordered water molecule in the
substrate cavity.17 The NH functional group may also undergo
hydrogen bonding with the phenolic OH of Tyr-435. Another
Figure 8. Representation of the best-ranked docking solution for the binding of
isatin analogue 9a (orange coloured, Panel A) and aniline analogue 10 (cyan
coloured, Panel B) in the active site of MAO-B (2V5Z.pdb).22 The illustrations were
generated with PyMOL.41
significant interaction between 9a and the MAO-B active site is a
possible
p–p interaction between the isatin ring and the amide
functional group of the Gln-206 side chain with an interplane dis-
tance of approximately 3.5 Å3. As discussed above, this interaction
may also, in part, explain the enhanced binding affinity of the isatin
analogues to MAO-B compared to the aniline analogues.
In the best-ranked docking solution of aniline derivative 10a
within the active site of MAO-B, the NH2 functional group binds
in the polar region of the substrate cavity in the vicinity of the
FAD co-factor and the ‘aromatic sandwich’ defined by Tyr-398
and Tyr-435 (Fig. 8B). This is consistent with the proposal that
the NH2 moiety of aminyl substrates of MAO-B is recognised by
the same aromatic residues.34 Within the substrate cavity, the
NH2 functional group possibly forms hydrogen bonds with a water
molecule and the C4 carbonyl oxygen of the flavin. Similar to what
has been observed for the MAO-B–9a complex, the benzyloxy side
chain of 10a also extends towards the entrance cavity of the en-
zyme where it is possibly stabilized via Van der Waals interactions.

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
269
One of the most notable differences between the binding modes of
9a and 10a within the MAO-B active site is the presence of the
additional hydrogen bond interaction between the 2-oxo group
of 9a and a water molecule. In accordance with the analysis above,
this interaction may, in part, explain the enhanced MAO-B inhibi-
tion potencies of the isatin analogues compared to the correspond-
ing aniline analogues.
Examination of the binding modes of 9a (Fig. 9A) and 10a
(Fig. 9B) within the MAO-A active site, reveals that these inhibitors
adopt similar orientations to those observed in the MAO-B active
site. The isatin ring of 9a is located in close proximity to the FAD
co-factor and both the C2 and C3 carbonyl oxygens are hydrogen
bonded to active site water molecules. Interestingly, in contrast
to the binding orientations of isatin17 and 9a in the human MAO-
B active site, in the MAO-A active site, the dioxoindolyl ring of 9a
is rotated through ꢀ180°. The dioxoindolyl ring of 9a possibly
forms a
p–p interaction with the amide functional group of the
Gln-215 side chain with an interplane distance of approximately
3.6 Å3. Inhibitor 10a, on the other hand, is only hydrogen bonded
via its anilinic NH2 to a water molecule and possibly the C4 car-
bonyl oxygen of the flavin. The observation that the MAO-A–9a
complex is stabilized by hydrogen bonding to both the 2- and 3-
oxo groups while the MAO-A–10a complex is by hydrogen bonding
to only the NH2 may, in part, explain the enhanced MAO-A inhibi-
tion potencies of the isatin analogues compared to the correspond-
ing aniline analogues.
3. Discussion
In the present study a series of 10 C5- and C6-substituted isatin
analogues (9a–j) were synthesized and evaluated as MAO inhibi-
tors. The results document that the isatin analogues are reversible
competitive inhibitors of both MAO isoforms and in most instances
exhibit selectivity for MAO-B (Table 2). Of the 10 analogues, only 3
compounds (9d, g and h) were selective for MAO-A. It can there-
fore be concluded that, similar to isatin (1), C5- and C6-substituted
isatin analogues act in general as MAO-B selective inhibitors. Also
noteworthy is the finding that C5- and C6-substitution of isatin in
general leads to a considerable enhancement of the MAO-B inhibi-
tion potencies of the test compounds while having, with the excep-
tion of 9g, a smaller effect on the MAO-A inhibition potencies. The
results also document that, without exception, the C5-substituted
isatin analogues are more potent inhibitors of MAO-A and -B than
the corresponding C6-substituted isatin homologues. This finding
is similar to the observation that (E)-5-styrylisatin (2) is a better
MAO-A and -B inhibitor than the C6-substituted analogue (E)-6-
styrylisatin (3). It can therefore be concluded that C5-substitution
of isatin is more optimal than C6-substition for improving the MAO
inhibition potencies of isatin. The finding that C5- and C6-substitu-
tion considerably enhances the MAO-B inhibition potency of isatin
is in agreement with the view that the C5- and C6-side chains
interact with the entrance cavity amino acid residues in order to
allow for more productive interactions with the enzyme compared
to isatin and hence more potent inhibition.
A possible explanation for the higher binding affinities of the
C5-substituted isatin analogues towards MAO-B compared to
MAO-A may be found by examining the docked binding modes of
9a in the active sites of human MAO-A and -B. While the dioxoind-
olyl ring of 9a adopts a similar binding orientation in MAO-B to
that observed for isatin in an MAO-B–isatin X-ray crystal structure
model,17 in the MAO-A active site the dioxoindolyl ring of 9a is
rotated through approximately 180° (Fig. 10). This alternative
binding orientation in the MAO-A active site may be less optimal
for the formation of stabilizing interactions with the active site
residues and water molecules. These results is in agreement with
previous docking studies which showed that the isatin ring of
Figure 9. Representation of the best-ranked docking solution for the binding of
isatin analogue 9a (orange coloured, Panel A) and aniline analogue 10 (cyan
coloured, Panel B) in the active site of MAO-A (2Z5X.pdb).3 The illustrations were
generated with PyMOL.41
(E)-5-styrylisatin also occupies the alternative binding orientation
in the MAO-A active site18 and suggests that other C6-substituted
isatin analogues also adopts the alternative orientation in MAO-A.
A similar analysis may explain the higher binding affinities of
the C5-substituted isatin analogues towards MAO-B compared to
the binding affinities of the corresponding C6-substituted isatin
analogues. In the docked binding mode of 9b in the active site of
human MAO-B, the dioxoindolyl ring of 9b is also rotated through
ꢀ180° compared to the orientation of 9a (Fig. 11). Compound 9b
therefore occupies a less optimal binding orientation in MAO-B
than 9a, and hence exhibits a lower MAO-B inhibition potency than
inhibitor 9a. This alternative binding orientation of the dioxoindol-
yl ring of 9b in the MAO-B active site is similar to that observed for
(E)-6-styrylisatin previously docked into an MAO-B model18 and is
most probably shared by other C6-substituted isatin analogues.
In this study we have also examined the importance of the isat-
in moiety for binding to MAO-A and MAO-B by comparing the inhi-
bition potencies of the C5- and C6-substituted isatins with those of

270
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
Figure 11. Representation of the best-ranked docking solution for the binding of
isatin analogue 9b (orange coloured) in the active site of MAO-B (2V5Z.pdb).22 The
illustration was generated with PyMOL.41
tetramethylsilane added to the deuterated solvent. Spin multiplic-
ities are given as s (singlet), br s (broad singlet), d (doublet), dd
(doublet of doublets), t (triplet) or m (multiplet). Direct insertion
electron impact ionisation (EIMS) and high resolution mass spectra
(HRMS) were obtained on a DFS high resolution magnetic sector
mass spectrometer (Thermo Electron Corporation). Melting points
(mp) were determined on a Stuart SMP10 melting point apparatus
and are uncorrected. Column chromatography was carried out
with Silica gel 60 (Fluka; 0.063–0.2 mm) while thin layer chroma-
tography (TLC) was carried out using silica gel 60 (Merck) with
UV254 fluorescent indicator. To determine the purity of the synthe-
sized compounds, HPLC analyses were conducted with an Agilent
1100 HPLC system equipped with a quaternary pump and an Agi-
lent 1100 series diode array detector (see Supplementary data).
HPLC grade acetonitrile (Merck) and Milli-Q water (Millipore)
was used for the chromatography. For fluorescence spectropho-
tometry, a Varian Cary Eclipse fluorescence spectrophotometer
was employed. Microsomes from insect cells containing recombi-
nant human MAO-A and -B (5 mg/mL), kynuramineÁ2HBr and
isatin were obtained from Sigma–Aldrich. (E)-5-Styrylisatin (2),
(E)-6-styrylisatin (3), aniline 10k and aniline 10l were synthesized
as described previously.18 Single-crystal X-ray diffraction analysis
was carried out with a Bruker Smart X2S diffractometer.
Figure 10. Representation of the binding orientation of isatin (Panel A) in an MAO-
B–isatin X-ray crystal structure model,17 the docked orientations of 9a in the active
sites of MAO-B (Panel B) and MAO-A (Panel C) and the docked orientation of 9b in
the MAO-B active site (Panel D).
the corresponding aniline analogues. The results showed that the
aniline analogues are weaker MAO-A and -B inhibitors than the
corresponding isatin analogues and suggests that the lactam and
C2 carbonyl functional groups of the dioxoindolyl ring are impor-
tant structural features for binding to the MAO active sites. Molec-
ular docking studies suggest that in both MAO-A and -B, the isatin
ring forms one additional hydrogen bond interaction with the ac-
tive site residues and waters of MAO-A and -B than the corre-
sponding aniline analogues. This may, in part, explain the
enhanced MAO inhibition potencies of the isatin analogues com-
pared to the corresponding aniline analogues. Another factor that
may contribute to stabilizing the complexes between the isatin
analogues and the MAO isozymes are possible
actions between the isatin ring and the amide
p
p
–
p
stacking inter-
-face of an active
4.2. Synthesis of C5- and C6-substituted isatin analogues (9a–j)
site Gln residue. In MAO-A, Gln-215 undergo stacking interactions
with isatin 9a while in MAO-B Gln-206 form stacking interactions
with 9a.
The C5- and C6-substituted isatin analogues (9a–j) investigated
in this study were synthesized according to a modification of the
literature description.23 A mixture of the appropriate C4- or C5-
substituted aniline (10, 20 mmol) and acetic acid (35 mL) was
heated at 110–120 °C to yield a clear solution. For this purpose
either the free base or hydrochloride salt of the aniline may be
used. Diethyl ketomalonate (20 mmol) was added to the reaction
and the resulting solution was heated under reflux at 110–120 °C
for 8 h. The residual acetic acid was removed by steam distillation
and the pH of the reaction mixture was adjusted to 11 with potas-
sium hydroxide (10%). The reaction was again heated under reflux
at 120 °C for a period of 18 h while a stream of air was passed con-
tinuously through the reaction mixture. The reaction was cooled to
room temperature and gravity filtered to yield a clear filtrate. The
pH of the filtrate was adjusted to 3 with hydrochloric acid (20%)
4. Experimental section
4.1. Chemicals and instrumentation
Unless otherwise noted, all starting materials were obtained
from Sigma–Aldrich and were used without purification. Proton
(1H) and carbon (13C) NMR spectra were recorded on a Varian
Gemini 300 spectrometer at frequencies of 300 MHz and 75 MHz,
respectively, and on a Bruker Avance III 600 spectrometer at fre-
quencies of 600 MHz and 150 MHz, respectively. All NMR mea-
surements were conducted in DMSO-d6 and the chemical shifts
are reported in parts per million (d) downfield from the signal of

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
271
and the resulting precipitate was removed by vacuum filtration.
The pH of the filtrate was adjusted to <1 with hydrochloric acid
(20%) and the resulting orange to red precipitate was collected
by vacuum filtration. The precipitate (dissolved in ethyl acetate)
was applied to a short silica gel column (35 Â 80 mm) and eluted
with ethyl acetate as mobile phase. Elution of the target isatin ana-
logues were monitored by silica gel TLC using ethyl acetate/petro-
leum ether (50:50) as mobile phase and the plates were visualised
under UV light (254 nm). The products thus obtained were recrys-
tallized from ethyl acetate. For previously described 9g and 9h the
melting points were found to be 208–211 °C and 231–233 °C while
the reported melting points are 208–210 °C and 235 °C,
respectively.23
yield of 9.3%: mp 185–190 °C (decomp., ethyl acetate). 1H NMR
(Bruker Avance III 600, DMSO-d6) d 6.93 (d, 1H, J = 8.7 Hz), 6.98
(d, 2H, J = 7.9 Hz), 7.10 (m, 1H), 7.12 (t, 1H, J = 7.2 Hz), 7.29 (m,
1H), 7.37 (t, 2H, J = 7.9 Hz), 11.00 (s, 1H); 13C NMR (Bruker Avance
III 600, DMSO-d6) d 113.6, 115.0, 118.0, 118.6, 123.5, 129.1, 130.1,
146.7, 151.9, 157.0, 159.6, 184.1; EIMS 239; HRMS m/z: calcd
239.0582, found 239.0574; purity (HPLC): 99.7%; UV (CH3CN) kmax
249 nm (
e e e
30,100 MÀ1), 294 nm ( 2530 MÀ1), 433 nm ( 925 MÀ1).
4.2.6. 6-Phenoxyisatin (9f)
The title compound (bright yellow crystals) was prepared from
3-phenoxyaniline (free base) (10f) and diethyl ketomalonate in a
yield of 3.0%: mp 146–150 °C (decomp., ethyl acetate/n-hexane,
1:1). 1H NMR (Bruker Avance III 600, DMSO-d6) d 6.28 (s, 1H), 6.55
(d, 1H, J = 8.7 Hz), 7.20 (d, 2H, J = 8.3 Hz), 7.31 (t, 1H, J = 7.15 Hz),
7.51 (m, 3H), 10.89 (br s, 1H); 13C NMR (Bruker Avance III 600,
DMSO-d6) d 99.9, 110.9, 112.5, 120.9, 125.8, 127.5, 130.5, 153.4,
153.8, 160.1, 166.0, 181.8; EIMS 239; HRMS m/z: calcd 239.0582,
4.2.1. 5-Benzyloxyisatin (9a)
The title compound (bright red crystals) was prepared from 4-
benzyloxyaniline hydrochloride (10a, Merck) and diethyl ketomal-
onate in a yield of 2.7%: mp 184–185 °C (ethyl acetate). 1H NMR
(Varian Gemini 300, DMSO-d6) d 5.09 (s, 2H), 6.83 (d, 1H,
J = 8.5 Hz), 7.14 (d, 1H, J = 2.6 Hz), 7.23–7.44 (m, 6H), 10.8 (s,
1H); 13C NMR (Varian Gemini 300, DMSO-d6) d 69.9, 109.9,
113.2, 118.1, 125.8, 127.7, 127.9, 128.4, 136.8, 144.8, 154.2,
159.5, 184.6; EIMS 253; HRMS m/z: calcd 253.0739, found
found 239.0582; purity (HPLC): 99.4%; UV (CH3CN) kmax 256 nm (
e
22,100 MÀ1), 316 nm ( 11,300 MÀ1), 394 nm ( 1667 MÀ1).
e
e
4.2.7. 5-Phenylisatin (9g)
The title compound (bright red crystals) was prepared from 4-
aminobiphenyl (free base) (10g) and diethyl ketomalonate in a
yield of 8.5%: mp 208–211 °C (ethyl acetate), lit. mp 208–
210 °C.23 1H NMR (Bruker Avance III 600, DMSO-d6) d 6.97 (d,
1H, J = 8.3 Hz), 7.34 (t, 1H, J = 7.5 Hz),7.43 (t, 2H, J = 7.5 Hz), 7.61
(d, 2H, J = 7.5 Hz), 7.72 (d, 1H, J = 1.5 Hz), 7.86 (dd, 1H, J = 1.9,
8.3 Hz), 11.13 (br s, 1H); 13C NMR (Bruker Avance III 600, DMSO-
d6) d 112.7, 118.4, 122.5, 126.2, 127.5, 129.0, 134.9, 136.5, 138.7,
150.0, 159.6, 184.4; EIMS 223; HRMS m/z: calcd 223.0633, found
253.0736; purity (HPLC): 99.6%; UV (CH3CN) kmax 253 nm (
e
25,900 MÀ1), 298 nm ( 2400 MÀ1), 466 nm ( 957 MÀ1).
e
e
4.2.2. 6-Benzyloxyisatin (9b)
The title compound (bright orange crystals) was prepared from
3-benzyloxyaniline (free base) (10b, Merck) and diethyl ketomalo-
nate in a yield of 1.2%: mp 243–255 °C (decomp., ethyl acetate). 1H
NMR (Varian Gemini 300, DMSO-d6) d 5.24 (s, 2H), 6.46 (d, 1H,
J = 2.2 Hz), 6.64–6.67 (m, 1H), 7.32–7.49 (m, 6H), 10.96 (br s,
1H); 13C NMR (Varian Gemini 300, DMSO-d6) d 70.0, 98.5, 109.5,
111.3, 127.3, 127.8, 128.2, 128.5, 136.0, 153.5, 160.5, 166.7,
181.5; EIMS 253; HRMS m/z: calcd 253.0739, found 253.0733; pur-
223.0632; purity (HPLC): 99.8%; UV (CH3CN) kmax 260 nm (
e
44,700 MÀ1), 430 nm ( 981 MÀ1).
e
4.2.8. 6-Phenylisatin (9h)
ity (HPLC): 99.5%; UV (CH3CN) kmax 261 nm (
e
26,500 MÀ1),
The title compound (orange-red crystals) was prepared from 3-
318 nm (
e
12,200 MÀ1), 395 nm ( 1746 MÀ1).
e
aminobiphenyl (free base) (10h) and diethyl ketomalonate in a
yield of 3.7%: mp 231–233 °C (ethyl acetate), lit. mp 235 °C.23 1
H
4.2.3. 5-(2-Phenylethyl)isatin (9c)
NMR (Bruker Avance III 600, DMSO-d6) d 7.09 (s, 1H), 7.34 (d,
1H, J = 7.5 Hz), 7.46 (t, 1H, J = 7.2 Hz), 7.51 (t, 2H, J = 7.2 Hz), 7.57
(d, 1H, J = 7.5 Hz), 7.69 (d, 2H, J = 7.5 Hz), 11.14 (s, 1H); 13C NMR
(Bruker Avance III 600, DMSO-d6) d 110.1, 116.8, 121.3, 125.3,
127.1, 129.1, 129.2, 138.9, 149.9, 151.4, 159.8, 183.7; EIMS 223;
HRMS m/z: calcd 223.0633, found 223.0628; purity (HPLC):
The title compound (bright red crystals) was prepared from 4-
(2-phenylethyl)aniline hydrochloride (10c) and diethyl ketomalo-
nate in a yield of 5.3%: mp 165–167 °C (ethyl acetate). 1H NMR
(Varian Gemini 300, DMSO-d6) d 2.82 (s, 4H), 6.80, (d, 1H,
J = 8.0 Hz), 7.15–7.28 (m, 5H), 7.36–7.43 (m, 2H), 10.95 (s, 1H);
13C NMR (Varian Gemini 300, DMSO-d6) d 36.0, 36.9, 112.0,
117.8, 124.4, 125.9, 128.2, 128.4, 136.2, 138.4, 141.1, 148.9,
159.5, 184.5; EIMS 251; HRMS m/z: calcd 251.0946, found
98.3%; UV (CH3CN) kmax 266 nm
(e (e
15,360 MÀ1), 326 nm
15,860 MÀ1), 408 nm ( 1578 MÀ1).
e
251.0941; purity (HPLC): 99.5%; UV (CH3CN) kmax 247 nm (
e
4.2.9. 5-(4-Phenylbutyl)isatin (9i)
30,100 MÀ1), 298 nm ( 3700 MÀ1), 423 nm ( 935 MÀ1).
e
e
The title compound (dark red crystals) was prepared from 4-(4-
phenylbutyl)aniline hydrochloride (10i) and diethyl ketomalonate
in a yield of 5.0%: mp 116–121 °C (ethyl acetate). 1H NMR (Bruker
Avance III 600, DMSO-d6) d 1.52 (m, 4H), 3.41 (m, 4H), 6.80 (d, 1H,
J = 7.9 Hz), 7.14 (d, 3H, J = 7.5 Hz), 7.23 (t, 2H, J = 7.5 Hz), 7.28 (s,
1H), 7.37 (d, 1H, J = 7.9 Hz), 10.95 (s, 1H); 13C NMR (Bruker Avance
III 600, DMSO-d6) d 30.4, 30.5, 34.0, 34.9, 112.1, 117.8, 124.2, 125.7,
128.26, 128.28, 136.9, 138.3, 142.1, 148.8, 159.5, 184.6; EIMS 279;
HRMS m/z: calcd 279.1259, found 239.1253; purity (HPLC): 99.7%;
4.2.4. 6-(2-Phenylethyl)isatin (9d)
The title compound (bright orange crystals) was prepared from
3-(2-phenylethyl)aniline hydrochloride (10d) and diethyl ketomal-
onate in a yield of 4.6%: mp 203–205 °C (ethyl acetate). 1H NMR
(Bruker Avance III 600, DMSO-d6) d 2.85–2.92 (m, 4H), 6.75 (s,
1H), 6.91 (d, 1H, J = 7.5 Hz), 7.17 (t, 1H, J = 7.2 Hz), 7.22–7.28 (m,
4H), 7.39 (d, 1H, J = 7.5 Hz), 11.00 (br s, 1H); 13C NMR (Bruker
Avance III 600, DMSO-d6) d 36.2, 37.7, 112.2, 115.9, 123.0, 124.7,
126.0, 128.3, 128.4, 140.9, 151.1, 153.8, 159.9, 183.7; EIMS 251;
HRMS m/z: calcd 251.0946, found 251.0940; purity (HPLC):
UV (CH3CN) kmax 247 nm (
e e
27,300 MÀ1), 298 nm ( 3400 MÀ1),
423 nm (
e
955 MÀ1).
99.8%; UV (CH3CN) kmax 249 nm
(
e
29,000 MÀ1), 305 nm
(
e
4.2.10. 5-(4-Chlorophenoxy)isatin (9j)
7530 MÀ1), 412 nm ( 1031 MÀ1).
e
The title compound (bright red crystals) was prepared from 4-
(4-chlorophenoxy)aniline (free base) (10j) and diethyl ketomalo-
nate in a yield of 5.2%: mp 255–257 °C. 1H NMR (Bruker Avance
III 600, DMSO-d6) d 6.93(d, 1H, J = 8.3 Hz), 6.99 (d, 2H, J = 9.0 Hz),
7.16 (d, 1H, J = 2.6 Hz), 7.31 (dd, 1H, J = 2.6, 8.7 Hz), 7.40 (d, 2H,
4.2.5. 5-Phenoxyisatin (9e)
The title compound (bright red crystals) was prepared from 4-
phenoxyaniline (free base) (10e) and diethyl ketomalonate in a

272
C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
J = 8.7 Hz), 11.05 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-
d6) d 113.7, 115.5, 118.7, 119.5, 127.1, 129.4, 129.9, 147.0, 151.4,
156.1, 159.5, 184.0; EIMS 273; HRMS m/z: calcd 273.0193, found
drich and were pre-aliquoted and stored at À70 °C. All enzymatic
reactions were carried out to a final volume of 500 L in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl,
20.2 mM) and contained kynuramine as substrate, MAO-A or
MAO-B (0.0075 mg/mL) and various concentrations of the test
l
273.0192; purity (HPLC): 99.4%; UV (CH3CN) kmax 249 nm (
e
34,400 MÀ1), 430 nm ( 980 MÀ1).
e
inhibitor (0–100
lM). The final concentrations of kynuramine in
4.3. Synthesis of aniline analogues
the reactions were 45
l
M and 30 M where MAO-A and -B, respec-
l
tively, were used as enzymes. Stock solutions of the test inhibitors
were prepared in DMSO and added to the reactions to yield a final
concentration of 4% (v/v) DMSO. The reactions were carried out for
With the exception of 10c, 10d and 10i all the anilines required
for the synthesis of the isatin analogues were commercially avail-
able. Sodium (105 mmol) was allowed to react with ethanol
(109 mL) in small portions over a period of 2 h. The resulting mix-
ture was added at room temperature to diethyl 4- or diethyl 3-
nitrobenzylphosphonate (11a, b, 100 mmol)26 and benzaldehyde
(12, 100 mmol) or cinnamaldehyde (14, 100 mmol) dissolved in
ethanol (164 mL) over a period of 1.5 h. Stirring was continued
for 20 h and the thick yellow precipitate was collected by vacuum
filtration and washed with 40 mL ethanol followed by 40 mL petro-
leum ether.27 The nitro-functionalized intermediates 13a, 13b and
15 thus obtained (yield 69–71%) were used without further purifi-
cation in the following reaction. The nitro derivatives (25 mmol)
were suspended in 400 mL methanol and the reaction flask was
purged with argon. A quantity of 3% (of the weight of the nitro
derivative) of Pd/C (10%) was prewet with 2 mL water and added
to 10 mL methanol. This mixture was carefully added to the reac-
tion. The atmosphere was replaced by hydrogen and the reaction
was stirred at room temperature for 24 h during which the reac-
tion became a clear solution. The catalyst was removed by filtra-
tion through a bed of Celite and the methanol solvent was
evaporated under reduced pressure. The resulting aniline deriva-
tive was converted to the corresponding hydrochloride salt in
CH2Cl2 (70 mL). A volume of 150 mL diethyl ether may be added
to the acidic CH2Cl2 solution to facilitate precipitation of the salt
(yield 41–95%). The melting points of the hydrochloride salts of
10c, 10d and 10i were found to be 203–210 °C (decomp.), 164–
181 °C and 174–180 °C, respectively.
20 min at 37 °C and were terminated with the addition of 200
lL
NaOH (2 N). After the addition of distiled water (1200 L) to each
l
reaction, the reactions were centrifuged for 10 minutes at
16,000g. To determine the concentrations of the MAO-generated
4-hydroxyquinoline in the reactions, the fluorescence of the super-
natant at an excitation wavelength of 310 nm and an emission
wavelength of 400 nm were measured.28 Quantitative estimations
of 4-hydroxyquinoline were made with the aid of a linear calibra-
tion curve ranging from 0.047 to 1.56
dissolved in potassium phosphate buffer (100 mM, pH 7.4). Each
calibration standard was prepared to a final volume of 500 L in
potassium phosphate buffer (100 mM, pH 7.4) and contained 4%
DMSO. To each standard was also added 200 L NaOH (2 N) and
1200 L distiled water. IC50 values were determined by plotting
lM of the reference standard
l
l
l
the initial rate of oxidation versus the logarithm of the inhibitor
concentration to obtain a sigmoidal dose–response curve. For this
purpose, nine different inhibitor concentrations spanning at least
three orders of a magnitude were used for each sigmoidal curve.
This kinetic data were fitted to the one site competition model
incorporated into the Prism software package (GraphPad) and the
IC50 values were determined in duplicate and are expressed as
mean standard deviation (SD).21 The IC50 values were converted
to the corresponding Ki values according to the equation by Cheng
and Prusoff: Ki = IC50/(1 + [S]/Km).29
4.5. Time-dependant inhibition studies
4.3.1. Hydrochloride salt of 4-(2-phenylethyl)aniline (10c)
To investigate whether the observed enzyme inhibition is revers-
ible or irreversible, time-dependant inhibition studies were carried
with a selected inhibitor, 9c. Recombinant human MAO-A or human
MAO-B (0.03 mg/mL) were preincubated for periods of 0, 15, 30,
60 min at 37 °C with compound 9c. The concentrations of inhibitor
The title compound was prepared from 4-nitrostilbene (13a) in
a yield of 94.9%: mp 203–210 °C (decomp.), lit. mp 210 °C.35,36 1
H
NMR (Varian Gemini 300, DMSO-d6) d 2.87 (m, 4H), 7.13–7.33
(m, 9H), 10.39 (br s, 3H); 13C NMR (Varian Gemini 300, DMSO-
d6) d 36.4, 36.8, 123.0, 125.9, 128.2, 128.4, 129.56, 129.62, 141.1,
141.4; EIMS 197.
9c in these incubations were 9.76 lM and 2.80 lM for the incuba-
tions with MAO-A and MAO-B, respectively. These concentrations
are twofold the measured IC50 values for the inhibition of the respec-
tive MAO preparations by 9c and the incubations were carried out in
potassium phosphate buffer (100 mM, pH 7.4, made isotonic with
4.3.2. Hydrochloride salt of 3-(2-phenylethyl)aniline (10d)
The title compound was prepared from 3-nitrostilbene (13b) in
a yield of 65.1%: mp 164–181 °C (methanol/ethyl acetate, 1:2).36
1H NMR (Bruker Avance III 600, DMSO-d6) d 2.84–2.91 (m, 4H),
7.16 (t, 1H, J = 7.2 Hz), 7.21–7.27 (m, 7H), 7.36 (t, 1H, J = 7.5 Hz),
10.50 (br s, 3H); 13C NMR (Bruker Avance III 600, DMSO-d6) d
36.6, 36.7, 120.8, 123.0, 126.0, 128.1, 128.3, 128.4, 129.5, 131.9,
141.1, 143.4; EIMS 197.
KCl). A final concentration of 45
30 M kynuramine for MAO-B were then added to the preincubated
enzyme preparations and the resulting reactions were incubated at
37 °C for 15 min. The final volumes of these incubations were 500
and the final concentrations of 9c were 4.88 M and 1.40 M for
lM kynuramine for MAO-A and
l
lL
l
l
MAO-A and MAO-B, respectively. These concentrations of the inhib-
itor are approximately equal to the IC50 values for the inhibition of
the respective enzyme preparations by 9c. The final enzyme concen-
trations of the MAO preparations were 0.015 mg/mL. The reactions
4.3.3. Hydrochloride salt of 4-(4-phenylbutyl)aniline (10i)
The title compound was prepared from 1-nitro-4-[(1E,3E)-4-phe-
nylbuta-1,3-dien-1-yl]benzene (15) in a yield of 40.9%: mp 174–
180 °C (methanol/ethyl acetate, 1:1).37 1H NMR (Varian Gemini 300,
DMSO-d6) d 1.55 (m, 4H), 2.57 (m, 4H), 7.11–7.31 (m, 9H), 10.41 (br
s, 3H); 13C NMR (Varian Gemini 300, DMSO-d6) d 30.5, 30.6, 34.3,
34.9, 123.1, 125.6, 128.3, 129.42, 129.45, 142.1, 142.2; EIMS 225.
were terminated with 200 lL NaOH (2 N) and a volume of 1200 lL
distiled water was added to each reaction. The rates of formation
of 4-hydroxyquinoline were measured and quantified as described
above. All measurements were carried out in triplicate and are
expressed as mean SD.21,38,39
4.4. Recombinant human MAO-A and -B inhibition studies
4.6. Mode of inhibition
Microsomes prepared from insect cells expressing recombinant
human MAO-A and -B (5 mg/mL) were obtained from Sigma–Al-
To examine the modes of MAO-A and -B inhibition, sets of
Lineweaver–Burk plots were constructed for the inhibition of both

C. I. Manley-King et al. / Bioorg. Med. Chem. 19 (2011) 261–274
273
enzymes by a selected representative inhibitor, compound 9c. For
this purpose the initial rates of oxidation of kynuramine at four dif-
on a Mitegen Micromount with a small amount of epoxy. Bruker
APEX2 software was used for preliminary determination of the unit
cell and the determination of integrated intensities and unit cell
refinement were performed with Bruker SAINT system. The data
were corrected for absorption effects with SADABS using the mul-
tiscan technique. The structure was solved with XS42 and subse-
quent structure refinements were performed with XL.42 The
structure was refined by anisotropic full-matrix least-squares
refinement on F2: C16H13NO2, M = 251.28 g molÀ1, crystal size:
0.05 Â 0.5 Â 0.5 mm3, orthorhombic, space group P21 21 21,
ferent substrate concentrations (15–90
presence of three different concentrations of the inhibitor were
measured. The concentrations of inhibitor 9c were 2.5–10
and 0.125–0.5 M for the inhibition studies with MAO-A and -B,
lM) in the absence and
lM
l
respectively, while the concentration of the MAO preparation in
the incubations were 0.015 mg/mL. The enzymatic reactions and
measurements were carried out as described above. Linear regres-
sion analysis was performed using the Prism software package.21
a = 4.7763(10) Å, b = 9.835(3) Å, c = 27.125(7) Å,
V = 1274.2(5) Å3, Z = 4, calcd = 1.310 g cmÀ3
a
= b =
c = 90°,
4.7. Molecular modelling studies
q
,
l
= 0.087 mmÀ1
,
k = 0.71073 Å, T = 300(2) K, hrange = 1.50–20.19°, reflections col-
lected: 5430, independent: 1234 (Rint = 0.0371), 172 parameters, fi-
The molecular docking studies were carried out in the Windows
based Discovery Studio 1.7 molecular modelling software.40 The li-
gands to be docked (9a, 9b and 10) were constructed Discovery
Studio and the hydrogen atoms were added according to the
appropriate protonation states at pH 7. The geometries were
briefly optimised in Discovery Studio using a fast Dreiding-like
forcefield (1000 iterations) and atom potential types and partial
charges were subsequently automatically assigned with the
Momany and Rone CHARMm forcefield. The X-ray crystallographic
structures of MAO-A co-crystallized with harmine (PDB code:
2Z5X)3 and MAO-B co-crystallized with safinamide (PDB code:
2V5Z)22 were retrieved from the Brookhaven Protein Data Bank
(www.rcsb.org/pdb). Hydrogen atoms were added to the receptor
models according to the appropriate protonation states of the ion-
izable amino acids at pH 7. The valences of the FAD co-factors (oxi-
dised state) and co-crystallized ligands were also corrected and
hydrogen atoms were added according to the appropriate proton-
ation states at pH 7. The resulting models were automatically
typed with the Momany and Rone CHARMm forcefield, the protein
backbone was constrained and the models were subjected to a
three step energy minimisation cascade. The first step was a steep-
est descent minimisation which was followed by conjugate gradi-
ent minimisation. For both protocols the termination criteria was
set to a maximum of 2500 steps or a minimum value of 0.1 for
the root mean square of the energy gradient. The third step was
an adopted basis Newton–Rapheson minimisation with the termi-
nation criteria set to a maximum of 5000 steps or a minimum value
for the root mean square of the energy gradient of 0.01. For this
minimisation cascade the implicit generalised Born solvation mod-
el with simple switching was used with the dielectric constant set
to 4. The models were subsequently deprived of the co-crystallized
ligands and the backbone constraints and the binding site was
identified by a flood-filling algorithm. All crystal waters were re-
tained for the modelling studies and include the water molecules
which are involved in hydrogen bonding with the C2 carbonyl oxy-
gen of isatin as shown by the crystal structure of isatin in complex
with MAO-B.17 Automated docking was subsequently carried out
with the LigandFit application of Discovery Studio and 10 docking
solutions were allowed for each ligand. The docking protocol uses
total ligand flexibility whereby the final ligand conformations are
determined by the Monte Carlo conformation search method set
to a variable number of trial runs. The docked ligands were further
refined using in situ ligand minimisation with the Smart Minimizer
algorithm. Unless otherwise specified (see above), all the applica-
tion modules within Discovery Studio were set to their default val-
ues.21 The illustrations were generated in PyMOL.41
nal
R indices [I >2r(I)]: R1 = 0.0417; wR2 = 0.1142, max/min
residual electron density: 0.168/À0.211 eÀ/Å3, goodness-of-fit on
F2 1.105, F(0 0 0) = 528. CCDC 794009 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk.
Acknowledgements
The NMR and MS spectra and X-ray diffraction data were re-
corded by André Joubert and Johan Jordaan of the SASOL Centre
for Chemistry, North-West University. This work was supported
by grants from the National Research Foundation and the Medical
Research Council, South Africa.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.028.
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