Design, synthesis, biological evaluation and docking studies of sulfonyl isatin derivatives as monoamine oxidase and caspase-3 inhibitors

Article abstract of DOI:10.1039/c6md00228e

The aim of this study was to design novel multifunctional neuroprotective agents that would slow down or halt neurodegeneration through inhibition of MAO-A, MAO-B and caspase-3. We focused on pharmacophoric groups of known MAO-inhibitors including selegil

Full text of DOI:10.1039/c6md00228e

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DOI: 10.1039/C6MD00228E
Design, synthesis, biological evaluation and docking studies of sulfonyl isatin
derivatives as monoamine oxidase and caspase-3 inhibitors†
Mohsen Tavari, Sarel F. Malan, Jacques Joubert*
aPharmaceutical/-* Chemistry , School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville, South
Africa.
*Corresponding author at present address: School of Pharmacy, University of the Western Cape, Private Bag X17,
Bellville 7535, South Africa. Tel: +27 21959 2195; eꢀmail: jjoubert@uwc.ac.za
†The authors declare no competing interests.
Abstract:
The aim of this study was to design novel multifunctional neuroprotective agents that would slow down or halt
neurodegeneration through inhibition of MAOꢀA, MAO–B and caspaseꢀ3. We focused on pharmacophoric groups of
known MAOꢀinhibitors including selegiline (propargylamine moiety) and safinamide (fluorobenzyl moiety), and isatin
sulfonamide caspaseꢀ3 inhibitors. The synthesised compounds consisted of the isatin nucleus conjugated to a
fluorophenylsulfonyl moiety at position 5 and on selected compounds a propargylamine moiety was introduced at the Nꢀ
position of isatin. All the compounds showed promising MAOꢀA inhibitory activity (IC50: 4 – 22 µM). The compounds
without the propargylamine moiety showed better MAOꢀB inhibition (IC50: 5 – 8 µM) and caspaseꢀ3 inhibition (IC50: 25
– 29 µM). The fluorophenylsulfonyl moiety in the synthesized compounds was significant for their multifunctional
activity. In general the active compounds showed similar or improved inhibitory activities when compared to the
reference compounds isatin (MAOꢀA and –B) and ZꢀVADꢀFMK (caspaseꢀ3). Possible binding orientations of selected
isatin analogues within the active site cavities of MAOꢀA, ꢀB and caspaseꢀ3 are proposed and in silico ADMET studies
were also done. Nꢀ[(3ꢀFluorophenyl)methyl]ꢀNꢀmethylꢀ2,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide (4) and Nꢀ[(4ꢀ
fluorophenyl)methyl]ꢀNꢀmethylꢀ2,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide (5) showed the best multifunctional
neuroprotective potential and can be considered as new lead compounds for the development of alternative treatment
options for neurodegenerative disorders such as AD, PD and HD.
Keywords: Isatin, Propargylamine, Monoamine Oxidase, Caspaseꢀ3, Apoptosis, Neuroprotection
1. Introduction
Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are
neurodegenerative disorders characterised by progressive and irreversible loss of neurons from
specific regions of the brain.1,2,3 Not a single cause or process alone leads to these neurodegenerative
disorders, instead it is the result of several factors which most likely include a variety of mechanistic
pathways.3 Some of these pathways may include monoamine oxidase A and B (MAOꢀA and MAOꢀ
B) and caspases which may contribute to the apoptotic cascade and may play a role in AD, PD and
HD.
Neurotransmitter amines are catabolized by MAOꢀA and MAOꢀB and these enzymes have been
targeted for the treatment of neuropsychiatric and neurodegenerative diseases.4,5,6 MAOꢀA
metabolizes both serotonin and norepinephrine, two monoamines implicated in depressive illness
and anxiety disorders.4,7 The antidepressive effect of MAOꢀA inhibitors is associated with blocking

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the central breakdown of these monoamines thus extending their action. SinDcOeI: 1a0.1s0i3g9/nCi6fMicDa0n0t228E
proportion of patients with PD exhibit signs of depression, the inhibition of MAOꢀA in these
patients may be beneficial.8 Besides offering a potential antidepressant action in PD, MAOꢀA
inhibitors may also provide a symptomatic benefit by reducing MAOꢀA catalysed oxidation of
dopamine.4,8 The oxidative deamination reaction catalyzed by MAOꢀB, and to a lesser extent MAOꢀ
A, is one of the major catabolic pathways of dopamine in the brain. Inhibition of the MAOꢀB
enzyme leads to enhanced dopaminergic neurotransmission and it is currently a target in the
symptomatic treatment of PD.8 Furthermore, MAOꢀA and MAOꢀB inhibitors may also exert
neuroprotective effects by reducing the concentration of potentially hazardous byꢀproducts produced
by MAO catalysed neurotransmitter oxidation and other bioactive endogenous or exogenous
neurotoxins.8ꢀ11 Therefore inhibition of MAO may exert some neuroprotective effects and may be of
value in the treatment of AD, PD and HD.
Caspases are a set of intracellular enzymes strictly controlling the apoptotic pathway and have been
implicated in the development of neurodegenerative disorders.12ꢀ15 Over a dozen caspases have been
discovered so far and identified as initiating (e.g. caspases 8, 9 and 10) and executing caspases (e.g.
caspases 3, 6 and 7). Basically two biological pathways are known to initiate apoptosis, either by a
particular protein which binds to the death receptor (death receptor pathway) or by damaging DNA
and subsequently mitochondrial induction of the cell death program (mitochondrial pathway). The
apoptotic process is driven by signals from both paths cumulating in the activation of executing
caspases.12ꢀ15 Activation of caspaseꢀ3 has been observed in all kinds of apoptosis including
neurodegenerative apoptotic processes involved in disorders such as PD, AD, and HD.13ꢀ15
Diseaseꢀmodifying approaches have turned into an area of focus in research since these strategies
interrupt the initial pathologic events associated with the onset of the disease. Hindering the
neurotoxic cascade in the case of neurodegenerative diseases is an example of one of these
approaches.3,16,17 In clinical practice, multiple targeting has proceeded through the
polypharmaceutical approach by using a combination of therapeutic agents functioning on different
etiological targets independently.18,19 This strategy however has several drawbacks, such as multiple
toxicities, sideꢀeffects and drugꢀdrug interactions.20 For these reasons, one drug molecule that
possess promiscuous activity acting on more than one biological target in the neurodegenerative
cascades would be of value. The advantages associated with this strategy includes a lower likelihood
of encountering unwanted sideꢀeffects and the possibility to increase therapeutic index by simple
structural modifications.21 With this in mind, potential compounds that may provide the structural
features that can be used to design a single entity with potent, selective inhibition of caspaseꢀ3 with
MAOꢀA and/or MAOꢀB inhibitory properties were identified. The compounds selected for our
design strategy includes isatin, selegeline, rasagiline and isatin sulfonamide derivatives.

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Isatin (Figure 1) is a reversible inhibitor of both human MAOꢀA and MAOꢀB with Ki values of 15
µM and 3 M, respectively.22 Previous studies have shown that isatin is located in the substrate
cavity of MAOꢀB in close proximity to the flavin adenine dinucleotide (FAD) coꢀfactor.23 Since
isatin binds within the substrate cavity, the entrance cavity of the enzyme is unoccupied. Therefore
the 5 or 6 position of isatin can be substituted with a functional moiety that can occupy the entrance
cavity and allow for more productive binding with the enzyme compared to isatin alone and
potentially promote more potent inhibition. Studies have also identified the 5 position as optimal for
substitution for improved MAO activity.24 Safinamide is also a MAO inhibitor and the complex
between safinamide and MAOꢀB shows that the 3ꢀfluorobenzyloxy side chain of safinamide
occupies the entrance cavity.25 Therefore it is proposed that substituting the 5 position of isatin with
a fluorobenzyl moiety may improve the MAO activity of isatin.
Entrance cavity
N
F
Proposed MAO interactions
NH
Rasagaline
Substrate binding site
Selegiline
O
O
N
S
O
O
N
O
R
N
O
S
O
R
O
O
N
H
O
FAD-cofactor
N
H
S3 Pocket
F
Proposed caspase 3 interactions
Isatin
Isatin-5-sulfonamide
derivatives
Cysteine163
binding site
H
O
O
N
NH2
S
NH
O
O
N
F
O
S2 Pocket
O
Safinamide
Figure 1: Design strategy and proposed MAO and caspaseꢀ3 interactions of the target compounds
Rasagiline and selegiline (Figure 1) are propargylamine derivatives that irreversibly inhibit MAOꢀB
by binding to the FAD coꢀfactor of the MAOꢀB enzyme. They also have promising neuroprotective
activities in vivo.26 It has been established that the neuroprotective effects of these compounds can
be attributed to their propargyl moiety.11,27,28 This observation was made on the grounds that
propargylamine itself exerts the same neuroprotective effects as offered by rasagiline. It has also
been established that the MAO inhibiting activity is not a prerequisite for the neuroprotection
provided by rasagiline, selegiline and propargylamine as these compounds have been shown to
inhibit apoptosis through other antiꢀapoptotic mechanisms.29ꢀ32 Substituting the isatin moiety at the
Nꢀposition with a propargylamine moiety might thus increase the MAO inhibitoryꢀ and antiꢀ
apoptotic activity of isatin. Figure 1 (top right) shows the interactions of the proposed compounds.
Isatin sulfonamides have been identified as potent, nonꢀpeptide, selective inhibitors of caspasesꢀ
3.12,33ꢀ36 These compounds are said to be superior to peptideꢀbased inhibitors because they possess
higher metabolic stability and cell permeability. They are also able to differentiate between

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apoptosis and necrosis since caspasesꢀ3 is only involved in apoptosis.36 The active site of capaseꢀ3
consist out of four pockets S1ꢀS4 with the S2 and S3ꢀpockets being the most important for caspaseꢀ3
inhibitory activity and selectivity.35,36,45 Recent studies revealed the configuration and binding
interactions of isatin sulfonamides within the active site of caspaseꢀ3.36 The 3ꢀcarbonyl group of
isatin forms an interaction with cystein163 within the active site of caspaseꢀ3.35,36 This position is
generally not modified due to loss of activity or change in mode of action. The hydrophobic S2ꢀ
pocket and S3ꢀpocket accommodates the sulfonamide and a number of structurally distinct
conjugates such as a pyrrolidine moiety in the S2ꢀpocket and aliphatic or aromatic moieties
connected to the pyrrolidine reaching into the S3ꢀpocket.35,36 With the above binding orientations
observed in previous studies we propose in figure 1 (bottom right) the general structure of our
designed compounds. We propose that the 3ꢀcarbonyl will interact with cystein163 with the
sulphonamide moiety sitting in the S2ꢀpocket and the Nꢀfluorophenyl moiety reaching into the S3ꢀ
pocket conferring potential caspaseꢀ3 inhibitory activity.36
With the focus being on the development of multifunctional neuroprotective drugs for AD, PD
and/or HD, it was thus a rational decision to incorporate isatin sulfonamideꢀ, fluorophenyl and
propargylamine functional groups into the structures of the compounds to be synthesised (Figure 1).
This strategy could provide compounds that are able to slow down or stop the apoptotic process
through caspaseꢀ3 inhibition and provide some degree of neuroprotection through MAO inhibition.
2. Results and Discussions
2.1 Chemistry
The 5ꢀfluorophenylsulfonyl isatin derivatives (3 ꢀ 8) were synthesized utilizing a three step process
(Scheme 1). The synthesis of 5ꢀchlorosulfonyl isatin (2) was accomplished starting from
commercially available isatinꢀ5ꢀsulfonic acid sodium salt dihydrate (1) and reacting it with
phosphorous oxychloride using tetramethylene sulfone as solvent to form the sulfonyl chloride
derivative (2). In the following step, compound 2 was conjugated to the fluorophenylamine
derivative with the fluoroꢀfunction at either the ortho, meta or para position through a nucleophilic
substitution reaction on the chlorosulfonyl moiety. The resultant compounds (3 ꢀ 5) were conjugated
on the N position of the isatin moiety with propargyl bromide using microwave assisted synthesis
(130 °C, 150 W, 40 min) through a nucleophilic substitution reaction to give the final compounds (6
– 8). Compound 9 was synthesised using a similar microwave synthesis procedure as described for
compounds 6 – 8 (Scheme 2). The structures and purity were confirmed by nuclear magnetic
resonance spectroscopy and mass spectrometry.

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O
S
O
H
Na
O
O
O
Cl
S
F
H
POCl3
O
O
.H2O
+
O
O
N
H
TMS, 60 oC, 6 h
N
H
1
2
HN
CHCl3/ THF
H
DIPEA
rt, 1 h
F
H
N
Propargyl bromide
O
O
O
60% NaH, DMF
MW, 130 oC,
S
O
O
O
N
N
H
150 W, 40 min
1
2
3
1
2
3
(3) R = F, R = H, R = H
(6) R = F, R = H, R = H
1
2
3
1
2
3
(4) R = H, R = F, R = H
(7) R = H, R = F, R = H
1
2
3
1
2
3
(8) R = H, R = H, R = F
(5) R = H, R = H, R = F
Scheme 1: The general synthesis procedure of the novel isatin compounds (3 – 8).
O
O
Propargyl bromide
O
O
60% NaH, DMF
MW, 95 oC,
N
N
H
150 W, 40 min
9
Scheme 2: Microwave assisted synthesis of compound 9.
2.2. MAO-A and MAO-B studies
2.2.1. MAO inhibition studies
To determine the MAOꢀA and MAOꢀB inhibition potencies of the test compounds, the extent by
which different concentrations of a test inhibitor reduces the MAOꢀcatalysed oxidation of
kynuramine, a mixed MAOꢀA/B substrate, was measured. Recombinant human MAOꢀA and MAOꢀ
B enzymes were used.37 Kynuramine is nonꢀfluorescent until undergoing MAOꢀcatalysed oxidative
deamination and subsequent ring closure to yield 4ꢀhydroxyquinoline, a fluorescent metabolite. The
concentrations of the MAOꢀgenerated 4ꢀhydroxyquinoline in the incubation mixtures was
determined by comparing the fluorescence emitted by the inhibitor samples to that of control
samples containing no inhibitor.37 The inhibition potencies of the test inhibitors were expressed as
IC50 values. IC50 values were converted to the corresponding Ki values for the inhibition of MAOꢀA
and MAOꢀB according to the Cheng–Prusoff equation.38 The IC50 and Ki values for the inhibition of
MAOꢀA and ꢀB by the isatin analogues 3 – 9 are presented in Table 1. The inhibition potencies of
the reference compounds; isatin, selegiline and clorgyline, were also measured and are given in
Table 1. Isatin was found to be a moderately potent MAOꢀA and ꢀB inhibitor with IC50 values of
22.54 µM and 12.07 µM respectively, which corresponds well with that reported in the literature.22
All the novel test compounds (3 – 9) showed significant MAOꢀA inhibition (IC50: 4.31 – 22.75
µM). The introduction of the fluorophenylsulfonyl moiety on the 5 position of isatin resulted in
compounds that retained MAOꢀA inhibitory activity (3 and 4, IC50 = 21.73 and 22.75 µM) and

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improved MAOꢀA inhibitory activity (5, IC50 = 8.26 µM) compared to isatin. Further substitution of
compounds 3 – 5 with a propargylamine moeity (6 – 8) on the Nꢀposition of the isatin moiety in
general led to a twoꢀfold increase in MAOꢀA inhibitory activity. The most potent MAOꢀA inhibitor
among the examined compounds was the paraꢀsubstituted fluoro compound (8), with an IC50 value
of 4.31 µM, approximately 5.2ꢀfold more potent than isatin. In general the introduction of the
fluorophenylsulfonylꢀ and propargylamine moiety to the structure of isatin led to improved MAOꢀA
inhibitory activity. None of the test compounds were as potent as clorgyline (IC50 = 0.001 µM).
The test compounds were also evaluated for MAOꢀB inhibitory activity. The inhibitors without the
propargylamine moiety showed around a two fold increase in MAOꢀB activity (4 and 5, IC50 = 8.32
and 5.96 µM) compared to isatin with the exception of the ortho fluoro substituted compound 3
(IC50 > 100 µM). This suggests that the meta and para fluorophenylsulfonyl substituents were better
accommodated in the MAOꢀB cavity. Introduction of the propargylamine moiety at the N position of
isatin lead to a significant decrease in MAOꢀB activity. Compounds 7 and 8 were 8.4 and 16.8 fold
less active than their nonꢀpropargylamine containing counterparts 4 and 5. This could indicate that
the interaction between the propargyl moiety and the FAD coꢀfactor of MAOꢀB is not present in
these structures as is the case in selegiline.39 Another reason may be that the substrate cavity where
the isatin moiety binds cannot accommodate the additional propargylamine group or the orientation
of the isatin moiety has changed and is no longer conducive for productive binding interactions at
this site. None of the test compounds were as potent as selegiline (IC50 = 0.020 µM). Compounds 6,
7 and 8 containing the propargylamine functional group shows better activity towards the MAOꢀA
enzyme (relative selectivity, SI: 10 – 24). Compounds 4 and 5 are dually active inhibitors of MAOꢀ
A and MAOꢀB with compound 5 being the best dual active inhibitor of MAOꢀA (IC50 = 8.26 µM)
and MAOꢀB (IC50 = 5.96 µM) in this series.
Table 1: In vitro IC50 values of the test compounds (3 – 9) for MAOꢀA, MAOꢀB and caspaseꢀ3.
R2
R3
R1
O
O
O
O
N
O
N
S
O
N
R4
9
( )
3
8
)
(
-
Compound
R1 R2 R3 R4
MAOꢀA
IC50 uM
MAOꢀA
MAOꢀB
IC50 uM
MAOꢀB
SI
Caspaseꢀ3
Ki uMa
Ki uMa
MAOꢀAb IC50 uM
F
H
F
H
H
F
H
H
F
H
H
F
H
H
H
21.63
22.75
8.26
5.70
6.00
2.18
2.48
2.81
1.14
> 100*
8.32
5.96
128.2
70.58
> 100*
n.d.
>7.55
1.37
1.18
22.25
10.81
>23.91
60.96
25.08
29.26
44.74
51.49
> 100*
3
4
5
6
7
8
H
H
F
H
H
3.58
2.57
55.18
30.38
n.d.
Propargyl 9.43
Propargyl 10.67
Propargyl 4.31
H

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ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
30.91
8.14
5.94
24.06
0.0003
n.d.
35.19
12.07
0.020
n.d.
15.15
5.20
0.009
n.d.
2.12
> 100*
> 100*
> 100*
> 100*
40.00
9
Isatin
22.54
67.25
0.001
n.d.
0.54
0.0002
n.d.
Selegiline
Clorgyline
ZꢀVADꢀFMK
n.d.
n.d.
n.d.
aThe experimentally determined IC50 values were used to calculate the Ki values according to the equation by Cheng and
Prusoff: Ki = IC50/(1 + [S]/Km) with [S] = 45 ꢁM and Km (Kynuramine) = 16.1 ꢁM for MAOꢀA; and [S] = 30 ꢁM and Km
(Kynuramine) = 22.67 ꢁM for MAOꢀB respectively.38 bRelative selectivity (SI) for MAOꢀA is defined by the ratio of
IC50(MAOꢀB)/IC50(MAOꢀA) for each compound. *Maximum concentration tested before precipitation of the test
compound occurred.
2.2.2. MAO reversibility studies
As several of the novel test compounds showed promising MAOꢀA and/or ꢀB inhibition and
observing that inclusion of the propargylamine either decreased or didn’t significantly influence
activity, this study further aimed to investigate whether the observed enzyme inhibition is reversible
or irreversible. For this purpose the time dependence of MAOꢀA and ꢀB inhibition by the active
compounds (IC50 < 100 µM, Table 1) were evaluated. Recombinant human MAOꢀA and MAOꢀB
was preincubated with the test compounds for periods of 0, 15, 30 and 60 min and the residual rates
of the MAOꢀA and ꢀB catalysed oxidation of kynuramine were measured. For this purpose, the
concentrations of the test compounds chosen were approximately twofold the measured IC50 values
for the inhibition of the respective enzymes. Clorgyline, with known irreversible inhibition for
MAOꢀA,40 and selegiline, with known irreversible inhibition for the MAOꢀB,39 were used as a
reference compounds.
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
0
15
30
60
0
15
30
60
0
15
30
60
Clorgyline
Compound 5
Compound 8
Figure 2: Timeꢀdependant inhibition of the recombinant human MAOꢀA catalysed oxidation of kynuramine
by clorgyline, 5 and 8. The enzyme was preincubated for various periods of time (0–60 min, xꢀaxis) with
clorgyline, 5 and 8 at concentrations of 0.002 ꢁM, 16.52 ꢁM and 8.62 ꢁM. The concentration of kynuramine
used was 45 ꢁM and the rate data are expressed as 4ꢀhydroxyquinoline formed (measured in RFU)/min/mg
protein.

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120000
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
0
15
30
60
0
15
30
60
0
15
30
60
Selegiline
Compound 4
Compound 7
Figure 3: Timeꢀdependant inhibition of the recombinant human MAOꢀB (Panel B) catalysed oxidation of
kynuramine by selegiline, 4 and 7. The enzymes were preincubated for various periods of time (0–60 min, xꢀ
axis) with selegiline, 4 and 7 at concentrations of 0.020 ꢁM, 16.64 ꢁM and 141.16 ꢁM. The concentration of
kynuramine used was 30 ꢁM and the rate data are expressed as 4ꢀhydroxyquinoline formed (measured in
RFU)/min/mg protein.
As shown in Figure 2 and 3, both clorgyline and selegiline showed a timeꢀdependent reduction in
the rates of MAOꢀA and ꢀB catalysed oxidation of kynuramine, confirming irreversible inhibition.
Compounds 4, 5, 7 and 8 however showed no timeꢀdependent reduction in the rates of MAOꢀA and
ꢀB catalysed oxidation of kynuramine. From these results it can be concluded that the inhibition of
MAOꢀA and –B by the isatin derivatives is reversible, at least for the time period (60 min) and at the
inhibitor concentrations (2 × IC50) evaluated. Timeꢀdependent inhibition studies with the other isatin
derivatives (3, 6 and 9) yielded similar results (data not shown). The compounds containing the
propargylamine moiety (6 – 9) also showed reversible MAO inhibition thus indicating that the
interaction between the propargyl moiety and the FAD coꢀfactor of MAOꢀA and MAOꢀB is not
present in these structures as is the case in the irreversible inhibitors clorgyline and selegiline.39,40
2.2.3. MAO molecular modelling studies
The MAO inhibition studies show that compounds 4 and 5 are in general good MAOꢀA and ꢀB
inhibitors with their propargylamine counterparts 6 – 8 showing potent MAOꢀA inhibitory activity
and weaker MAOꢀB inhibition. To provide additional insight, the binding modes of compound 5 and
its corresponding propargylamine counterpart 8 in MAOꢀA and ꢀB were examined using molecular
docking. The structures of human MAOꢀA coꢀcrystallized with harmine (PDB entry: 2Z5X)41 and
human MAOꢀB coꢀcrystallized with safinamide (PDB entry: 2V5Z)25 were retrieved from the
Brookhaven Protein Data Bank (www.rcsb.org/pdb). Molecular docking was carried out with the Dock
application of the Molecular Operating Environment software.42 The bestꢀranked docking solution
of compound 5 within the active site of MAOꢀB shows that the isatin moiety binds in the polar
region of the substrate cavity in the vicinity of the FAD coꢀfactor and the ‘aromatic sandwich’
defined by Tyr398 and Tyr435 (Figure 4). This binding orientation of the dioxoindolyl ring is
similar to that observed for isatin coꢀcrystallized within the active site of recombinant human MAOꢀ
B.23 The C5 fluorophenylsulfonyl side chain of 5 extends past Ile199 which is situated in the

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entrance cavity of the enzyme. This binding orientation is similar to that observed for the coꢀ
crystallized inhibitor, safinamide, which also spans both active site cavities.25 Within the
hydrophobic environment of the entrance cavity, the fluorophenylsulfonyl side chain is most likely
stabilized by Van der Waals interactions.25 These interactions may, in part, explain the enhanced
MAOꢀB inhibition potencies of compounds 4 and 5 compared to isatin (Table 1). The paraꢀ
fluorophenylsulfonyl moiety exhibited a better orientation and lower binding energy when modeled
into the MAOꢀB enzyme compared to its meta and ortho counterparts (data not shown). This may
explain the improved inhibitory activity of the paraꢀfluoro substituted compounds. In contrast to 5,
the bestꢀranked docking solution of the propargylamine substituted compound 8 shows that the
isatin moiety is now situated in the entrance cavity with the fluorophenylsulfonyl side chain
spanning the substrate cavity (Figure 4). The propargylamine substitution thus induced a drastic
change in orientation of 8 and this may explain the significant reduction in MAOꢀB activity
observed for compounds 6 ꢀ 8 (Table 1).
Examination of the binding mode of 5 (Figure 4) within the MAOꢀA active site, reveals that the
isatin moiety is orientated towards the FAD coꢀfactor similar to that observed in the MAOꢀB active
site. The bestꢀranked docking solution of 8 shows that, in contrast with its MAOꢀB binding
orientation, the isatin ring is situated in the vicinity of the FAD cofactor, similar to that observed for
compound 5. The propargylamine moiety is also accommodated in the active site cavity of MAOꢀA,
however its orientation does not enable interaction with the FAD cofactor. The binding of the isatin
moiety, in both 5 and 8, in the polar region of the substrate cavity and potential interactions with
surrounding amino acid residues may be the reason why both these compounds showed good MAOꢀ
A inhibitory activity. As observed in MAOꢀB modeling experiment the paraꢀfluorophenylsulfonyl
moiety also exhibited a better orientation and lower binding energy when compared to its meta and
ortho counterparts (data not shown).

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Figure 4: Comparison between the putative binding modes of compound 5 and 8 to the human MAOꢀB (left)
and human MAOꢀA (right) enzyme active sites. FAD is shown in magenta, compound 5 in green and
compound 8 in red.
2.3 Caspase-3 studies
2.3.1 Caspase-3 inhibition studies
Inhibition of recombinant human caspaseꢀ3 by the test compounds was assessed using a
commercially available assay kit purchased from Abcam.43 This experiment is a fluorometric assay
and utilizes a synthetic peptide caspaseꢀ3 substrate DEVDꢀAFC by measuring the release of its
fluorogenic product, 7ꢀaminoꢀ4ꢀtrifluoromethyl coumarin (AFC), upon binding of the substrate with
caspaseꢀ3. The IC50 values from the caspaseꢀ3 assays are summarized in Table 1.
Compounds 3 – 7 showed the best caspaseꢀ3 inhibitory activity (IC50: 25 ꢀ 61 µM) when compared
to the known caspaseꢀ3 inhibitor ZꢀVADꢀFMK44 (IC50 = 40 µM) in this assay. Compounds 8, 9,
isatin and the selective MAO inhibitors selegiline39 and clorgyline40 were found to be weak caspaseꢀ
3 inhibitors (IC50 > 100 µM). The introduction of the propargylamine moiety into the structure of 3
to produce 6 increased the activity slightly, but not significantly, by 1.4 fold. The converse was true
for compounds 4 and 7 where the propargylamine conjugate decreased in activity more than twoꢀ
fold. Compound 9 also showed a drastic reduction in activity when compared to its counterpart 5.
Therefore, based on what was observed, the introduction of the propargylamine moeity in general
decreased the caspaseꢀ3 inhibitory ability of these compounds. This could indicate that the
propargylamine moiety does not fit optimally into the active site of caspaseꢀ3 resulting in decreased
inhibitory activity. Compounds 4 (IC50 = 25.08 µM) and 5 (IC50 = 29.26 µM) containing only the
isatin and fluorophenylsulfonyl moieties showed the best caspaseꢀ3 inhibitory activity in this series
and are more potent than isatin and ZꢀVADꢀFMK in this assay.
2.3.2 Caspase-3 molecular modelling studies
To gain some insight into the binding modes of compounds 5 and 8 they were docked into the active
site of caspaseꢀ3 (Figure 5, Panel A ꢀ D) using MOE software42. The crystal structure of the caspaseꢀ
3 protein with a coꢀcrystallized isatin sulfonamide derivative was acquired from the Protein Data
Bank (PDB ID: 1GFW).36 Figure 5 (Panel A) shows the active site and the S2 and S3ꢀpockets of
caspaseꢀ3.

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A
B
S2
S3
C
D
Figure 5: Caspaseꢀ3 active site cavity (Panel A) and interaction map (Panel B ꢀ D) displaying the coꢀ
crystallized inhibitor (B) and synthesized inhibitors 5 (C) and 8 (D). Panel A shows the coꢀcrystalized ligand
(green), compound 5 (light blue), compound 8 (red) and the four binding pockets (S1 – S4).
Modelling of the coꢀcrystallized inhibitor into the active site of caspaseꢀ3 (Panel B) showed that the
methyl amine on the isatin moiety interacts with the MetA61 amino acid residue in the active site
pocket. The carbonyl moieties interact through hydrogen bonding with CysA163 and GlyA122. The
pyrrolidinyl and phenoxy moieties of the coꢀcrystallized inhibitor fitted well into the S2 and S3
pockets of caspaseꢀ3 respectively and may form hydrophobic interactions with surrounding amino
acid residues. Similarly to the coꢀcrystallized inhibitor, the carbonyl of the isatin part of 5 generates
a hydrogen bond interaction with GlyA122 and is within close proximity to CysA163 (Panel C). The
sulfonamide moiety of 5 plunges into the hydrophobic S2 pocket and generates a hydrogen bond
interaction with Arg207. These interactions and orientations were similar to what was observed
previously for other isatin sulfonamideꢀbased caspaseꢀ3 inhibitors.36,45 The benzene ring of 5 was
not able to traverse into the S3 pocket of caspaseꢀ3 as observed for similar compounds in previous
studies.35,36,45 Furthermore, compared to 5, compound 8 does not fit as well in the active pocket
where no binding interactions were predicted for the carbonyl moieties of the isatin part (Panel D)

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and the propargylamine moiety of 8 moved the isatin functional group further away from CysA163
and GlyA122. This unfavourable position of 8 in the active site may explain the reduced caspaseꢀ3
inhibitory activity (IC50 > 100 µM) when compared to compound 5 (IC50 = 29 µM). Judging from
what has been observed, we have come to recognize that the activity of the compounds can be
improved by extending the distance between the nitrogen atom and the aromatic ring to better fit
into the S3 pocket. Other functional groups should also be explored for optimal orientation and
binding interactions in the S2 pocket.
2.4 In silico ADMET studies
The Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of selected
compounds (4, 7 and 8) were calculated using webꢀbased applications. Blood–Brain Barrier (BBB)
penetration, Human Intestinal Absorption (HIA), Cacoꢀ2 cell permeability and AMES test were
calculated using admetSAR.46 LC50 values (Lethal concentration 50% in µmol) were predicted using
http://lazar.inꢀsilico.de/predict. The predicted ADMET data is summarized in Table 2.
Table 2: Predicted ADMET properties of the selected compounds
Compound
BBB
penetration
HIAa
(%)
Cacoꢀ2
AMES
Acute toxicity
permeabilityb toxicityc LC50d µmol
%
CNS
activity
Isatin
4
5
7
99
69
69
78
78
95
+
+
+
+
+
+
+
100
100
100
99
99
99
+
+
+
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
298
526
526
688
688
165
125
8
Selegilline
ZꢀVADꢀFMK 63
97
aHuman Intestinal Absorption; bThe values more than 50 was considered as +; cMutagenic potential of chemicals; dEPA
fathead minnow acute toxicity.
Based on the predicted values for BBB penetration, all of the selected compounds should be able to
penetrate into the central nervous system. Moreover, the selected compounds did not show either
acute toxicity according to the calculated LC50 values nor mutagenic effect with respect to the
AMES test data. The ADMET profiles of the other synthesised compounds (3, 6 and 9) were similar
to that reported for compounds 4, 7 and 8 (data not shown). This data has significance indicating
that the new isatin based compounds may have good ADMET profiles thus encouraging further drug
design and development endeavours utilizing these compounds as lead structures.
3. Conclusion
The synthesized compounds, especially compounds 4 and 5, showed inhibition of the MAOꢀA,
MAOꢀB and caspaseꢀ3 enzymes at low micro molar concentrations. The role of the
fluorophenylamine moiety in the synthesized compounds was significant for their multifunctional
activity as shown by compounds 4 and 5 having good inhibitory activity towards MAOꢀA, MAOꢀB

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and also excellent inhibitory activity against caspaseꢀ3 making them ideal candidates for further
antiꢀapoptotic studies, lead compound development and multifunctional drug design. The
introduction of the propargylamine moiety only increased the MAOꢀA inhibitory activity, this was
shown by compounds 6, 7 and 8 which exhibit some MAOꢀA selectivity with low MAOꢀB and
caspaseꢀ3 inhibitory activities. Although these compounds did not show good caspaseꢀ3 activity,
they should be tested for general antiꢀapoptotic activity because the propargylamine functional
group in these compounds might act in a completely different manner than what was tested for in
this assay.28ꢀ32 These compounds thus have potential as novel multifunctional neuroprotective agents
against AD, PD and/or HD and further investigation is necessary to determine their maximum
benefit in the treatment of neurodegenerative disorders.
4. Experimental
4.1. Chemistry: General procedures
Unless otherwise specified, materials were obtained from commercial suppliers and used without
further purification. All reactions were monitored by thinꢀlayer chromatography on 0.20 mm thick
aluminium silica gel sheets (Alugram® SIL G/UV254, Kieselgel 60, MachereyꢀNagel, Düren,
Germany). Visualisation was achieved using UV light (254 nm and 366 nm), an ethanol solution of
ninhydrin or iodine vapours, with mobile phases prepared on a volumeꢀtoꢀvolume basis.
Chromatographic purifications were performed on silica gel (0.063–0.2 mm, Sigma Aldrich) except
when otherwise stated. The MS spectra were recorded on a Perkin Elmer Flexar SQ 300 mass
spectrometer by means of direct injection with a syringe pump. High resolution electron spray
ionisation (HREI) mass spectra for all compounds were recorded on a Waters API QꢀTof Ultima
mass spectrometer at 70 eV and 100 °C. All HREI samples were introduced by a heated probe and
perfluorokerosene was used as reference standard. The IR spectra were recorded on a Perkin Elmer
Spectrum 400 spectrometer, fitted with a diamond attenuated total reflectance (ATR) attachment.
Melting points were determined using a Stuart SMPꢀ300 melting point apparatus and capillary
13
tubes. All the melting points determined were recorded uncorrected. 1H and C NMR spectra were
determined using a Bruker Avance III HD spectrometer at a frequency of 400 MHz and 100 MHz,
respectively. Tetramethylsilane (TMS) was used as internal standard. All chemical shifts are
reported in parts per million (ppm), relative to the internal standard. The following abbreviations are
used to indicate the multiplicities of the respective signals: s ꢀ singlet; br s ꢀ broad singlet; d ꢀ
doublet; dd ꢀ doublet of doublets; t – triplet and m ꢀ multiplet. Microwave synthesis was performed
using a CEM DiscoverTM microwave synthesis system.
4.1.2. Synthesis of compounds
4.1.3. 2,3-Dioxoindoline-5-sulfonyl chloride (2)

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2,3ꢀDioxoindolineꢀ5ꢀsulfonyl chloride was synthesised using a modified procedure from the
literature.47 A mixture of isatinꢀ5ꢀsulfonic acid sodium salt dihydrate (4 g, 14.04 mmol, 1 eq.) was
dissolved in tetramethylene sulfone (20 mL) and phosphorous oxychloride (7.4 mL, 79.2 mmol, 5.6
eq.) was added dropwise. The mixture was heated at 60 °C for 3 h. The reaction was allowed to cool
to room temperature and was added dropwise to a stirring slurry of crushed ice/CH2Cl2 (200 g/200
g). After the ice had melted, the mixture was separated and the organic layer retained, from which
the CH2Cl2 removed was by evaporation, diluted with ethyl acetate (200 mL), washed with H2O (10
× 80 mL) and evaporated to dryness. The crude product was purified by flash column
chromatography using hexane: ethyl acetate in a ratio of 1:1 as eluent to yield the title compound
2,3ꢀdioxoindolineꢀ5ꢀsulfonyl chloride (1) as a bright yellow solid (2.45 g, 9.986 mmol, 71%). The
physical properties of compound 1 were similar to that reported in the literature.46,47
4.1.4. N-[(2-Fluorophenyl)methyl]-N-methyl-2,3-dioxo-2,3-dihydro-1H-indole-5-sulfon-amide
(3)
A
solution of 1ꢀ(2ꢀfluorophenyl)ꢀNꢀmethylmethanamine (140 mg, 1.01 mmol) and
diisopropylethylamine (127.74 ml, 1.332 mmol) in CHCl3 (0.7 mL) was added dropwise to a stirred
solution of 2,3ꢀdioxoindolineꢀ5ꢀsulfonyl chloride (245.64 mg, 1 mmol) in CHCl3/THF (1:1, 13 mL)
at room temperature. The resulting solution was stirred at room temperature for 1 h and the solvent
was removed in vacuo. The crude product was purified by flash column chromatography using
hexane:ethyl acetate in a ratio of 1:1 as eluent to obtain Nꢀ[(2ꢀfluorophenyl)methyl]ꢀNꢀmethylꢀ2,3ꢀ
dioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide as a brown wax (219.46 mg, 0.63 mmol, 63%).
Physical data: C16H13FN2O4S; mp: wax; 1H NMR (400 MHz, CD3OD) δH: 8.06ꢀ8.03 (dd, J = 1.81;
7.92 Hz, 1H), 7.94 (s, 1H), 7.84ꢀ7.82 (br s, NH), 7.45ꢀ7.41 (m, 1H), 7.36ꢀ7.31 (m, 2H), 7.18ꢀ7.16
(m, 1H), 7.14ꢀ7.07 (m, 1H), 4.29 (s, CH2), 2.68 (s, CH3); 13C NMR (100 MHz, CD3OD) δC:;
181.17, 162.34, 159.89, 145.52, 132.22, 130.89, 130.53, 128.94, 124.22, 122.75, 118.18, 115.13,
112.54, 55.87, 33.81; MS (DIꢀESIꢀMS) [M+H]+: 349.11; HR-ESI [M+H]+: calc. 349.0653, exp.
349.0659; IR (ATR, cmꢀ1) vmax: 3352.00, 2924.27, 1747.87, 1614.86, 1333.82, 1151.89 cmꢀ1.
4.1.5. N-[(3-Fluorophenyl)methyl]-N-methyl-2,3-dioxo-2,3-dihydro-1H-indole-5-sulfon-amide
(4)
The title compound (4) was prepared from 1ꢀ(3ꢀfluorophenyl)ꢀNꢀmethylmethanamine (140 mg, 1.01
mmol) following the same procedure as compound 3 rendering Nꢀ[(3ꢀfluorophenyl)methyl]ꢀNꢀ
methylꢀ2,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide as a light yellow powder (236.88 mg, 0.68
1
mmol, 68 %). Physical data: C16H13FN2O4S; mp: 153ꢀ155 °C; H NMR (400 MHz, CD3OD) δH:
8.08ꢀ8.03 (dd, J = 1.80; 7.93 Hz, 1H), 7.94 (s, 1H), 7.83ꢀ7.82 (br s, NH), 7.37ꢀ7.32 (m, 2H), 7.14ꢀ
13
7.00 (2 x m, 3H), 4.16 (s, CH2), 2.63 (s, CH3); C NMR (100 MHz, CD3OD) δC: 182.64, 164.27,
161.83, 145.53, 138.92, 132.23, 130.56, 130.16, 129.00, 123.77, 118.21, 114.70, 112.59, 53.17,

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33.70; MS (DIꢀESIꢀMS) [M+H]+: 349.02; HR-ESI [M+H]+: calc. 349.0653, exp. 349.0645; IR
(ATR, cmꢀ1) vmax: 3280.90, 2924.97, 1750.43, 1614.86, 1336.01, 1147.72 cmꢀ1.
4.1.6. N-[(4-Fluorophenyl)methyl]-N-methyl-2,3-dioxo-2,3-dihydro-1H-indole-5-sulfon-amide
(5)
The title compound (5) was prepared from 1ꢀ(4ꢀfluorophenyl)ꢀNꢀmethylmethanamine (140 mg, 1.01
mmol) following the same procedure as compound 3 rendering Nꢀ[(4ꢀfluorophenyl)methyl]ꢀNꢀ
methylꢀ2,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonꢀamide as a strong yellow powder (254.30 mg,
1
0.73 mmol, 73 %). Physical data: C16H13FN2O4S; mp: 150ꢀ152 °C; H NMR (400 MHz, CD3OD)
δH: 8.05ꢀ8.03 (dd, J = 1.81; 7.93 Hz, 1H), 7.93 (s, 1H), 7.83ꢀ7.82 (br s, NH), 7.36ꢀ7.32 (m, 2H),
7.14ꢀ7.05 (m, 3H), 4.12 (s, CH2), 2.63 (s, CH3); 13C NMR (100 MHz, CD3OD) δC: 182.43, 165.25,
161.33, 147.00, 138.40, 133.55, 131.60, 131.52, 124.66, 116.65, 116.61, 114.08, 111.65, 54.27,
34.98; MS (DIꢀESIꢀMS) [M+H]+: 349.01; HR-ESI [M+H]+: calc. 349.0653, exp. 349.0644; IR
(ATR, cmꢀ1) vmax: 3291.61, 2926.54, 1752.12, 1614.86, 1334.42, 1146.03 cmꢀ1.
4.1.7.
N-[(2-fluorophenyl)methyl]-N-methyl-2,3-dioxo-1-(prop-2-yn-1-yl)-2,3-di-hydro-1H-
indole-5-sulfonamide (6)
In a microwave compatible glassꢀvessel, a stirred solution of Nꢀ[(2ꢀfluorophenyl)methyl]ꢀNꢀmethylꢀ
2,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide (3, 200 mg, 0.57 mmol) dissolved in DMF (3.5 ml)
was treated with NaH (60%, 0.16 ml, 0.29 mmol) at room temperature. The mixture was stirred for
10 minutes and then propargyl bromide (0.100 ml, 0.63 mmol) was slowly added while stirring.
Subsequently the vessel was sealed and the mixture was stirred at 130 °C for 40 minutes using
microwave irradiation (maximum power = 150 W). Thereafter the mixture was added to 35 ml of
water, extracted with 60 ml of ethyl acetate, washed with 35 ml of saturated NaCl and dried in
vacuo. After solvent removal, the crude product was purified by column chromatography using
hexane:ethyl acetate in a ratio of 70:30 as eluent to obtain Nꢀ[(2ꢀfluorophenyl)methyl]ꢀNꢀmethylꢀ
2,3ꢀdioxoꢀ1ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ2,3ꢀdiꢀhydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide as a yellow wax (78.05 mg,
1
0.202 mmol, 32 %). Physical data: C19H15FN2O4S; mp: wax; H NMR (400 MHz, CDCl3) δH:
8.12ꢀ8.09 (dd, J = 1.82; 7.93 Hz, 1H), 8.04 (s, 1H), 7.83ꢀ7.82 (br s, NH), 7.46ꢀ7.42 (m, 1H), 7.30ꢀ
7.25 (m, 2H), 7.18ꢀ7.16 (m, 1H), 7.14ꢀ7.02 (m, 1H), 4.60 (s, CH2), 4.29 (s, CH2), 2.72 (s, CH3), 2.37
(s, CH); 13C NMR (100 MHz, CDCl3) δC: 181.01, 162.23, 159.77, 145.22, 134.39, 130.79, 130.07,
129.99, 124.63, 122.20, 117.59, 115.61, 111.59, 74.89, 74.22, 47.23, 34.76, 29.85; MS (DIꢀESIꢀ
MS) [M+H]+: 387.20; HR-ESI [M+H]+: calc. 387.0809, exp. 378.0812; IR (ATR, cmꢀ1) vmax
:
3289.30, 2921.50, 1695.22, 1621.10, 1334.17, 1152.49 cmꢀ1.
4.1.8. N-[(3-fluorophenyl)methyl]-N-methyl-2,3-dioxo-1-(prop-2-yn-1-yl)-2,3-di-hydro-1H-
indole-5-sulfonamide (7)

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The title compound (7) was prepared from compound 4 (200 mg, 0.57 mmol) following the same
procedure as compound 6 rendering Nꢀ[(3ꢀfluorophenyl)methyl]ꢀNꢀmethylꢀ2,3ꢀdioxoꢀ1ꢀ(propꢀ2ꢀynꢀ
1ꢀyl)ꢀ2,3ꢀdiꢀhydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide as a yellowish orange powder (48.54 mg, 0.125 mmol,
1
22 %). Physical data: C19H15FN2O4S; mp: 149ꢀ153 °C; H NMR (400 MHz, CDCl3) δH: 8.14ꢀ8.11
(dd, J = 1.81; 7.93 Hz, 1H), 8.06 (s, 1H), 7.32ꢀ7.26 (m, 2H), 7.09ꢀ6.99 (2 x m, 3H), 4.61 (s, CH2),
4.20 (s, CH2), 2.68 (s, CH3), 2.38 (s, CH); 13C NMR 100 MHz, CDCl3) δC: 181.01, 162.23, 159.77,
145.22, 134.39, 130.79, 130.07, 129.99, 124.63, 122.20, 117.59, 115.61, 111.59, 74.89, 74.22,
47.23, 34.76, 29.85; MS (DIꢀESIꢀMS) [M+H]+: 387.15; HR-ESI [M+H]+: calc. 387.0809, exp.
387.0811; IR (ATR, cmꢀ1) vmax: 3304.55, 2918.37, 1737.30, 1612.86, 1345.08, 1150.80 cmꢀ1.
4.1.9. N-[(4-fluorophenyl)methyl]-N-methyl-2,3-dioxo-1-(prop-2-yn-1-yl)-2,3-dihydro-1H-
indole-5-sulfonamide (8)
The title compound (8) was prepared from compound 5 (200 mg, 0.57 mmol) following the same
procedure as compound 6 rendering Nꢀ[(4ꢀfluorophenyl)methyl]ꢀNꢀmethylꢀ2,3ꢀdioxoꢀ1ꢀ(propꢀ2ꢀynꢀ
1ꢀyl)ꢀ2,3ꢀdihydroꢀ1Hꢀindoleꢀ5ꢀsulfonamide as a yellowish brown wax (92.50 mg, 0.239 mmol, 42
1
%). Physical data: C19H15FN2O4S; mp: wax; H NMR (400 MHz, CDCl3) δH: 8.13ꢀ8.10 (dd, J =
1.80; 7.93 Hz, 1H), 8.03 (s, 1H), 7.36ꢀ7.32 (m, 2H), 7.18ꢀ7.04 (m, 3H), 4.60 (s, CH2), 4.29 (s, CH2),
2.72 (s, CH3), 2.37 (s, CH); 13C NMR (100 MHz, CDCl3) δC: 181.01, 164.00, 161.80, 145.29,
137.73, 134.22, 130.45, 130.37, 124.44, 117.64, 115.32, 115.26, 111.65, 74.86, 74.25, 53.67, 34.59,
29.87; MS (DIꢀESIꢀMS) [M+H]+: 387.15; HR-ESI [M+H]+: calc. 387.0809, exp. 387.0812; IR
(ATR, cmꢀ1) vmax: 3275.75, 2917.70, 1744.44, 1610.19, 1323.93, 1152.49 cmꢀ1.
4.1.10. 1-(Prop-2-yn-1-yl)-1H-indole-2,3-dione (9)
In a microwave compatible glassꢀvessel, a stirred solution of isatin (147 mg, 1 mmol) dissolved in
DMF (6ml) was treated with NaH (60%, 0.55 ml, 1 mmol) at room temperature. The mixture was
stirred for 10 minutes and then propargyl bromide (0.175 ml, 1.1 mmol) was slowly added while
stirring. Subsequently the vessel was sealed and the mixture was stirred at 95 °C for 10 minutes
using microwave irradiation (maximum power = 150 W). Thereafter the mixture was added to 60 ml
of water, extracted with 100 ml of ethyl acetate, washed with 60 ml of saturated NaCl and then
washed with 60 ml water and dried in vacuo. After solvent removal, the crude product was purified
by column chromatography using hexane:ethyl acetate in a ratio of 1:1 as eluent to obtain 1ꢀ(propꢀ2ꢀ
ynꢀ1ꢀyl)ꢀ1Hꢀindoleꢀ2,3ꢀdione as an orange powder (131.48 mg, 0.71 mmol, 71 %). Physical data:
C11H7NO2; mp: 159ꢀ161 °C; 1H NMR (400 MHz, CDCl3) δH: 7.63 (m, 2H), 7.18ꢀ7.11 (m, 2H), 4.55
(s, CH2), 2.30 (s, CH). 13C NMR (100 MHz, CDCl3) δC: 181.23, 165.23, 159.77, 145.23, 137.36,
134.39, 130.13, 130.05, 124.67, 116.88, 115.62, 115.40, 111.59, 75.30, 74.22, 53.46, 34.36, 29.70;
MS (DIꢀESIꢀMS) [M+H]+: 186.05; HR-ESI [M+H]+: calc. 186.0550, exp. 186.0547; IR (ATR, cmꢀ
1) vmax: 3263.86, 2964.14, 1737.80, 1614.86 cmꢀ1.

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4.2. Recombinant human MAO-A and MAO-B inhibition studies
The MAO assays were conducted using a similar procedure as previously described.37 Recombinant
human MAOꢀA and ꢀB (5 mg/mL) were obtained from Sigma–Aldrich and were preꢀaliquoted and
stored at ꢀ70 °C. All enzymatic reactions were carried out to a final volume of 250 µL in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl, 20.2 mM) and contained kynuramine
as substrate, MAOꢀA or MAOꢀB (0.0075 mg/mL) and various concentrations of the test inhibitor
(0–1000 µM). The final concentrations of kynuramine in the reactions were 45 µM and 30 µM for
MAOꢀA and ꢀB, respectively. 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
incubated for 20 min at 37 °C and were terminated with the addition of 100 µL NaOH (2 N). Finally
250 µL from each eppendorf vial was added to individual black plate wells. The fluorescence of the
MAO generated 4ꢀhydroxyquinoline in the supernatant fractions were measured using a Biotek
fluorescent microplate reader at an excitation wavelength of 310 nm and an emission wavelength of
400 nm. The percentage MAO inhibition at each concentration was calculated using the following
equation:
% Inhibition = ((Control Fluorescence – Inhibitor Fluorescence)/Control Fluorescence)) x 100.
IC50 values were determined by plotting the % inhibition versus the logarithm of the inhibitor
concentration to obtain a sigmoidal dose–response 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 triplicate and are expressed as mean ± standard deviation (SD). The IC50 values
were converted to the corresponding Ki values according to the equation by Cheng and Prusoff: Ki
= IC50/(1 + [S]/Km).38
4.3. Time-dependant inhibition studies
To investigate whether the observed enzyme inhibition is reversible or irreversible, timeꢀdependant
inhibition studies were carried out with the active inhibitors. Recombinant human MAOꢀA or
human MAOꢀB (0.03 mg/mL) were preincubated for periods of 0, 15, 30, 60 min at 37°C. The
concentrations of inhibitor in these incubations were twofold the measured IC50 values for the
inhibition of MAO. The incubations were carried out in potassium phosphate buffer (100 mM, pH
7.4, made isotonic with KCl). A final concentration of 45 ꢁM kynuramine for MAOꢀA and 30 ꢁM
kynuramine for MAOꢀB 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 250 ꢁL
and the final concentration of the inhibitors were approximately equal to the IC50 values for the
inhibition of the respective enzyme. The final enzyme concentrations of the MAO preparations were
0.015 mg/mL. The reactions were terminated with 100 ꢁL NaOH (2 N) and a volume of 600 ꢁL

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distilled water was added to each reaction. The rates of formation of 4ꢀhydroxyquinoline were
measured and quantified as described above. All measurements were carried out in triplicate and are
expressed as mean ± SD.
4.4. Caspase-3 inhibition studies
Recombinant human caspaseꢀ3 and its peptideꢀspecific substrate AcꢀDEVDꢀAMC (AcꢀAspꢀ Gluꢀ
ValꢀAspꢀAMC) were purchased as part of a caspaseꢀ3 inhibitor drug detection kit from Abcam
(ab102491).43 The assay was performed exactly as set out by the kit protocol. The reaction rates
showing inhibitory potencies of the inhibitors were assessed by measuring the accumulation of the
cleaved fluorogenic product AMC (7ꢀaminoꢀ4ꢀmethylcoumarin) with a Biotek fluorescent
microplate reader at excitation and emission wavelengths of 360 and 460 nm, respectively. All
assays were performed at a volume of 150 µL at 37 °C in reaction buffer. Buffers contained the test
compounds in DMSO at various concentrations (0–1000 µM). Recombinant caspaseꢀ3 was diluted
into the appropriate buffer to a concentration of 0.5 units per assay. After 10 min incubation time,
the peptide substrate (end concentration 50 µM) was added and incubated for a further 60 min where
after the fluorescence was measured of the caspaseꢀ3 generated AMC. The percentage caspaseꢀ3
inhibition at each concentration was calculated using the following equation:
% Inhibition = ((Control Fluorescence – Inhibitor Fluorescence)/Control Fluorescence)) x 100.
IC50 values were determined by plotting the % inhibition versus the logarithm of the inhibitor
concentration to obtain a sigmoidal dose–response 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 triplicate and are expressed as mean ± standard deviation (SD).
4.5. Molecular modelling studies
4.5.1. MAO-A and MAO-B docking studies
Computerꢀassisted docking were carried out using the CHARMm force field and the human MAOꢀA
crystal structure (PDB ID: 2Z5X)41 and MAOꢀB crystal structure (PDB ID: 2V5Z),25 which were
recovered from the Brookhaven Protein Database (www.rcsb.org/pdb). Docking simulation was
performed on the test compounds using Molecular Operating Environment (MOE)42 with the
following protocol. (1) Enzyme structures were checked for missing atoms, bonds and contacts. (2)
Hydrogen atoms were added to the enzyme structure. Bound ligands were manually deleted and the
ordered water molecules were retained (3) The ligand molecules were constructed using the builder
module and were energy minimized. (4) The active site was generated using the MOEꢀAlpha Site
Finder. (5) Ligands were docked within the MAOꢀA and MAOꢀB active sites using MOEDock with
simulated annealing utilised as the search protocol and CHARMm molecular mechanics force field.
(6) The lowest energy conformation of the docked ligand complex was selected and subjected to a

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further energy minimization using CHARMm force field with all possible bond rotations and
chiralities explored. To determine the accuracy of this docking protocol, the coꢀcrystallised ligand,
harmine (PDB ID: 2Z5X), was reꢀdocked into the MAOꢀA active site and the coꢀcrystallised ligand,
safinamide (PDB ID: 2V5Z), was reꢀdocked into the MAOꢀB active site. This procedure was
repeated three times and the best ranked solutions of harmine and safinamide exhibited RMSD values
of 0.42 Å and 1.34 Å from the position of the coꢀcrystallised ligand for MAOꢀA and MAOꢀB,
respectively. In general, RMSD values smaller than 2.0 Å indicate that the docking protocol is
capable of accurately predicting the binding orientation of the coꢀcrystallised ligand.23,48 These
protocols were thus deemed to be suitable for the docking of inhibitors into the active site models of
MAOꢀA and MAOꢀB.
4.5.2. Caspase-3 docking studies
The dock function of MOE was used to predict the interactions and binding modes of the synthesized
inhibitors in the caspaseꢀ3 active site. The crystal structure of the caspaseꢀ3 protein with the coꢀ
crystallized isatin sulfonamide derivative was acquired from the Protein Data Bank (PDB ID:
1GFW).36 The docking procedure followed the same protocol as described in 4.5.1. To determine the
accuracy of this docking protocol, the coꢀcrystallised ligand, was reꢀdocked into the caspaseꢀ3 active
site. This procedure was repeated three times and the best ranked solution exhibited an RMSD value
of 1.15 Å from the position of the coꢀcrystallised ligand for caspaseꢀ3. In general, RMSD values
smaller than 2.0 Å generally indicate that the docking protocol is capable of accurately predicting the
binding orientation of the coꢀcrystallised ligand.23,48 This protocols was thus deemed to be suitable
for the docking of inhibitors into the active site model of caspaseꢀ3.
Acknowledgements
We are grateful to the NorthꢀWest University, University of the Western Cape, Medical Research
Council of South Africa and the National Research Foundation of South Africa for financial support.
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GRAPHICAL ABSTRACT
Design, synthesis, biological evaluation and docking studies of sulfonyl isatin
derivatives as monoamine oxidase and caspase-3 inhibitors
Mohsen Tavari, Sarel F. Malan, Jacques Joubert*
aPharmaceutical Chemistry , School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville, South
Africa.
R
F
O
O
N
N
R
S
O
O
N
H
O
O
N
Selegiline
S
Isatin-5-sulfonamide
derivatives
O
O
N
H
NH2
Caspase-3,
MAO-A and -B inhibition
NH
F
O
O
Safinamide
Sulfonyl isatin derivatives as multifunctional agents showing monoamine oxidase and caspaseꢀ3 inhibitory
activities in the low micromolar range.