Polycyclic propargylamine and acetylene derivatives as multifunctional neuroprotective agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.039

The aim of this study was to design drug-like molecules with multiple neuroprotective mechanisms which would ultimately inhibit N-methyl-d-aspartate (NMDA) receptors, block L-type voltage gated calcium channels (VGCC) and inhibit apoptotic processes as we

Full text of DOI:10.1016/j.ejmech.2014.04.039

European Journal of Medicinal Chemistry 80 (2014) 122e134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Polycyclic propargylamine and acetylene derivatives as
multifunctional neuroprotective agents
Frank T. Zindo a, Quinton R. Barber b, Jacques Joubert a, Jacobus J. Bergh b,
,b,
Jacobus P. Petzer b, Sarel F. Malan a
*
a Pharmaceutical Chemistry, School of Pharmacy, University of The Western Cape, Private Bag X17, Bellville 7535, South Africa
b Pharmaceutical Chemistry, 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:
The aim of this study was to design drug-like molecules with multiple neuroprotective mechanisms
which would ultimately inhibit N-methyl-D-aspartate (NMDA) receptors, block L-type voltage gated
Received 10 December 2013
Received in revised form
2 April 2014
Accepted 11 April 2014
Available online 13 April 2014
calcium channels (VGCC) and inhibit apoptotic processes as well as the monoamine oxidase-B (MAO-B)
enzyme in the central nervous system. These types of compounds may act as neuroprotective and
symptomatic drugs for disorders such as Alzheimer’s and Parkinson’s disease. In designing the com-
pounds we focused on the structures of rasagiline and selegiline, two well known MAO-B inhibitors and
proposed neuroprotective agents. Based on this consideration, the compounds synthesised all contain
the propargylamine functional group of rasagiline and selegiline or a derivative thereof, conjugated to
various polycyclic cage moieties. Being non-polar, these polycyclic moieties have been shown to aid in
the transport of conjugated compounds across the bloodebrain barrier, as well as cell membranes and
have secondary positive neuroprotective effects. All novel synthesised polycyclic derivatives proved to
have significant anti-apoptotic activity (p < 0.05) which was comparable to the positive control, sele-
giline. Four compounds (12, 15 and 16) showed promising VGCC and NMDA receptor channel inhibitory
activity ranging from 18% to 59% in micromolar concentrations and compared favourably to the reference
compounds. In the MAO-B assay, 8-phenyl-ethynyl-8-hydroxypentacycloundecane (10), exhibited MAO-
Keywords:
Polycyclic
Propargylamine
Neuroprotection
Apoptosis
B inhibition of 73.32% at 300
much as 40% when compared to the control experiments.
Ó 2014 Elsevier Masson SAS. All rights reserved.
mM. This compound also reduced the percentage of apoptotic cells by as
1. Introduction
by an intrinsic cell suicide program known as apoptosis [4e7]. This
process is triggered by several stimuli, and consists of numerous
pathways and cascades, each one having an influence on the other,
ultimately leading to cell death [4e7]. One such pathway is the
excitotoxic process which leads to apoptosis. Excitotoxicity is a
result of activation of postsynaptic receptors; including NMDA re-
ceptors, 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) proprio-
nate (AMPA), and kainate receptors. Upon their activation, these
receptors open their associated ion channel to allow the influx of
Ca2þ and Naþ ions. The excessive influx of calcium together with
any calcium release from intracellular compartments can over-
whelm Ca2þ-regulatory mechanisms and lead to cell death [8,9].
This mechanism of cell death suggests that the receptors and their
associated calcium channels serve as drug target sites for curbing
neurodegeneration. Several compounds, including polycyclic
amines such as amantadine (2), NGP1-01 (3), MK-801 (4), and
phencyclidine (PCP, 5) have been reported to show inhibitory ac-
tivities on NMDA receptors and calcium channels (Fig. 1) [10].
The pathology of neurodegenerative disorders, such as Parkin-
son’s disease (PD) and Alzheimer’s disease (AD), is caused by the
abnormal loss of neuronal cells in certain areas of the brain [1]. It
consequently causes an imbalance of certain neurotransmitter
levels in the brain, giving rise to the characteristic signs and
symptoms of these disorders [2,3]. Ultimately, it compromises the
normal functionality and well-being of the individual suffering
from the disease [3], thus making it an absolute necessity to create
drugs which would halt this neuronal breakdown process and aid
in treating the signs and symptoms of neurodegenerative disorders.
The abnormal death of neurons in the central nervous system of
individuals suffering from neurodegenerative diseases takes place
* Corresponding author. Present address: School of Pharmacy, University of the
Western Cape, Private Bag X17, Bellville 7535, South Africa.
E-mail address: sfmalan@uwc.ac.za (S.F. Malan).
http://dx.doi.org/10.1016/j.ejmech.2014.04.039
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
123
Fig. 1. Representative polycyclic cage compounds (1e3), NMDA receptor and calcium channel modulators - NGP1-01 (3), MK-801 (4) and phencyclidine (PCP, 5), the neuroprotective
MAO-B inhibitors - rasagiline (6) and selegiline (7), and propargylamine (8).
The oxidative deamination reaction catalysed by monoamine
oxidase-B (MAO-B) is one of the major catabolic pathways of
dopamine in the brain. Inhibition of this enzyme lead to enhanced
dopaminergic neurotransmission and is currently used in the
symptomatic treatment of PD [11e14]. Furthermore, MAO-B in-
hibitors may also exert a neuroprotective effect by reducing the
concentrations of potentially hazardous by-products produced by
MAO-B-catalysed dopamine oxidation [15]. In PD and AD it has
been shown that there are age-related elevated levels of MAO-B,
which not only act indirectly as a trigger to the apoptotic process,
but also give rise to some of the signs and symptoms associated
with these disorders [16e18].
In the current study, the approach was to develop multifunc-
tional drugs which would halt the apoptotic neuronal breakdown
process and eliminate some of the signs and symptoms of diseases
such as AD and PD by: (a) Inhibiting NMDA receptors and blocking
L-type voltage gated calcium channels (VGCC) thus regulating the
Ca2þ influx mediated excitotoxic process; (b) Inhibiting the MAO-B
enzyme thus allowing increase in dopamine levels in the CNS and
reducing the levels of the highly oxidative products produced by
the activity of this enzyme; (c) Possess anti-apoptotic activity to
halt the natural neuronal cell death process. With this in mind, we
focused on the structures of pentacycloundecane (1), amantadine
(2), rasagiline (6) and selegiline (7, Fig. 1).
cellular membranes, including the selectively permeable bloode
brain barrier, and increases its affinity for lipophilic regions in
target proteins [23,24]. In addition, these polycyclic moieties afford
metabolic stability, thereby prolonging the pharmacological effect
of a drug, leading to a reduction of dosing frequency and improving
patient compliance thereof [25]. The known NMDA receptor
channel antagonism of these cage compounds, combined with their
L-type calcium channel blocking activity, suggest that these poly-
cyclic cage moieties may potentially serve as therapeutic agents for
neurodegenerative disorders [10,23,26e28].
With the focus being on the development of multifunctional
drugs, it was thus a rational decision to incorporate polycyclic cage
moieties and propargylamine functional groups or derivatives
thereof into the structures of the novel compounds to be syn-
thesised, thus giving rise to a series of compounds with the
inherent therapeutic profiles of the contributing moieties (i.e.
NMDA receptor channel antagonism, L-type calcium channel
blocking activity, MAO-B inhibitory activity and anti-apoptotic ac-
tivity). A single compound exhibiting such an array of multifunc-
tional neuroprotective activities may curb the neurodegenerative
process more effectively than a compound which functions on only
one of the many drug target sites available.
Rasagiline and selegiline are second generation propargylamine
derivatives that irreversibly inhibit brain MAO-B, and have prom-
ising neuroprotective activities [19]. After several years of study and
research, it has been established that the neuroprotective effects of
rasagiline and selegiline can be attributed to its propargyl moiety
[16e18]. This observation was made on the grounds that prop-
argylamine (8), itself exerts the same neuroprotective effects as
offered by rasagiline. It has also been established that the MAO-B
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
anti-apoptotic mechanisms that may contribute to their possible
disease-modifying activities [20,21]. Since MAO-B activity is
increased in both AD and PD, MAO-B inhibitors may be of further
therapeutic benefit [22].
2. Results and discussion
2.1. Synthesis
In synthesising the novel polycyclic compounds (9e16, Fig. 2),
propargylamine, propargylbromide or ethynyl magnesium bromide
was reacted with the PCU (1, Cookson’s diketone) or amantadine (2)
scaffold to give the final compounds (Fig. 2). Each compound was
synthesised to evaluate the activity and benefit of the presence of a
certain group of atoms in the molecule. These groups included the
following: a terminal acetylene group (9), an acetylene group be-
tween two non-polar groups (10), a secondary propargylamine
connected to an oxa-PCU structure (11, 12), a tertiary propargyl-
amine conjugated to an aza-PCU structure (13, 14) and an ada-
mantane structure (15, 16).
Polycyclic cage compounds, such as pentacyloundecane (PCU,1),
amantadine (2) and NGP1-01 (3) have various biological applica-
tions, with special interest in the symptomatic and proposed
curative treatment of neurodegenerative diseases [23]. These
compounds can be used to modify and improve the pharmacoki-
netic and pharmacodynamic properties of drugs and it is apparent
from literature that the polycyclic cage is useful as both a scaffold
for side-chain attachment as well as for improving a drug’s lip-
ophilicity [23]. This lipophilicity enhances a drug’s transport across
Starting from the PCU diketone (1) or the methyl-diketone (a,
Scheme 1), depending on the compound to be synthesised, the
reaction proceeded by conjugation of propargylamine through
reductive amination with sodium borohydride, following steps i-iv
to give the oxa-derivatives,11 and 12. The aza derivatives,13 and 14,
were synthesised utilising sodium cyanoborohydride as reducing
agent. Compound 9 was synthesised via the Grignard reaction
through conjugation of ethynyl magnesium bromide with the
methyl-diketone producing the title compound 9 (Scheme 1).

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F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
Fig. 2. Synthesised acetylene (9 and 10) and propagylamine (11e16) polycyclic compounds evaluated for anti-apoptotic activity, calcium modulating effects and MAO-B inhibition.
Scheme 1. Reagents and conditions: (i) THF, ꢀ10 ꢁC, 45 min; ii) benzene, DeaneStark, reflux, 1 h; (iii) THF/MeOH, NaBH4, rt, 24 h; 11, 10%; 12, 27% (iv) THF/MeOH, NaCNBH4, rt, 24 h;
13, 16%; 14, 4%; (v) THF, ethynyl magnesium bromide, rt, 21 h, 9, 16%.
The synthesis of compound 10 commenced with the Cookson’s
diketone (Scheme 2), which was mono-protected as the corre-
sponding ethylene acetal. The remaining ketone was reduced with
lithium aluminium hydride and subsequent hydrolysis furnished
the hydroxyl-ketone. Wolff-Kisher reduction of the hydroxyl-
ketone under Huang-Minlon conditions gave the mono-alcohol,
followed by oxidation with chromium trioxide to yield the mono-
ketone (b). The mono-ketone (b) was conjugated to phenyl-
acetylene in the presence of a potassium fluoride and alumina so-
lution to yield the title compound 10 (Scheme 2).
with subsequent reduction, utilising LiAlH to give N-benzyl-ada-
mantan-1-amine (c, Scheme 3). Propargylbromide was conjugated
to (c), via a nucleophilic SN2 substitution reaction, to produce
compound 15. Compound 16 was synthesised by means of micro-
wave irradiation in the presence of an excess amount of prop-
argylbromide (Scheme 3).
The PCU structures, compounds (9e14), were obtained and
evaluated as racemic mixtures and the structure and geometry of
representative compounds were recently confirmed using single X-
ray crystallography and standard spectroscopy techniques [29].
The synthesis of compound 15 commenced through the conju-
gation of the primary amine of amantadine (2) and benzaldehyde
2.2. Anti-apoptotic activity
The compounds’ anti-apoptotic activity were evaluated in vitro
using the DePsipherÔ kit, which marks changes in the mitochon-
drial membrane potential that takes place during apoptosis [30].
The quantitative and qualitative detection of apoptosis were done
by means of flow cytometry, which made it possible to determine
the percentage of cells that were still viable in the samples after
treatment with the synthesised compounds. For this purpose SK-
NBE(2) neuroblastoma cells were used, and apoptosis was
induced using a serum-deprivation model. Control experiment 1
was included to evaluate the viability status of the cells, in the
absence of test compound, with 1.25% dimethyl sulphoxide (DMSO,
Fig. 3). The viability data was used to compare the data generated
with the test compounds in order to determine if the test com-
pounds attenuated the progression of the apoptotic process. With
this control experiment the effect of 1.25% DMSO was also evalu-
ated. Control experiment 2 was used to evaluate the viability of the
cells and the progression of the apoptotic process in the absence of
DMSO. When comparing the values of control experiments 1 and 2,
Scheme 2. Reagents and conditions: (i) p-TsOH (cat), benzene, DeaneStark, reflux,
3.5 h, 95% (ii) LiAlH4, Et2O, reflux, 2 h then 6% aq HCl, rt, 2 h, 61%; (iii) NH2.NH2.H2O,
diethylene glycol, 120 ꢁC, 1.5 h, then KOH, 190 ꢁC, 3 h, 33%; (iv) CrO3, H2O, 94% aq
CH3COOH, 90 ꢁC, 4 h, b, 84%; (v) KF/alumina, 60 ꢁC, 12 h, 10, 73%.

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
125
Scheme 3. Reagents and conditions: (i) EtOH, rt, 4 days, 59%; (ii) eH2O; (iii) NaBH4, reflux, 12 h, c, 59%; (iv) DMF, rt, 24 h; 15, 25%; (v) Excess propargylbromide, aq NaOH, mi-
crowave (25 min, 80 ꢁCe100 ꢁC, 250 W); 16, 91%.
Fig. 3. Anti-apoptotic activity of the test compounds (*p < 0.05; **p < 0.01; ***p < 0.001).
it is clear that even though the final DMSO concentration in the
samples was only 1.25%, it had a negative effect on the viability of
the cells. In control experiment 2, which did not contain any DMSO,
cell viability increased with 7.19%. It can thus be argued that the
serum deprivation was not the only factor inducing apoptosis, but
also the DMSO, which served as co-solvent for the test compounds.
Control experiment 3 was used to evaluate the influence of re-
introduction of serum rich medium, which significantly attenu-
ated the apoptotic process. This control experiment was used to
estimate the potencies of the test compounds as anti-apoptotic
agents. Control experiment 4 was included to analyse and deter-
mine the viability status of the cells, when no apoptosis had been
induced, and conditions had been favourable to the end of the
experiment. It was also included to ensure that the cells analysed
were indeed healthy before inducing apoptosis (see Section 4.3.1.
for a complete explanation of the experimental procedure).
control experiment 1, compounds 9e16 and NGP1-01 (3) all had
significant anti-apoptotic activity improving cell health between 9%
and 41%. The anti-apoptotic activity of 9e15 were the most sig-
nificant (p < 0.001; Fig. 3). The compound with the highest anti-
apoptotic activity was 9, where all cultures treated exhibited only
0.5% apoptotic cells in the analysed samples. Compound 10 was
slightly less active than compound 9, with only 2.8% of the cells
showing apoptosis (Fig. 3). Studying the results of these experi-
ments, it is clear that the anti-apoptotic activity of propargylamine
can most probably be attributed to the acetylene group, as com-
pounds 9 and 10 have equivalent or even higher activity than
compounds 11e16. For activity, this acetylene group can either be
terminal (9) or between two non-polar groups (10), with the ter-
minal acetylene group having slightly higher activity. It was also
confirmed that when linked to a polycyclic cage structure, prop-
argylamine and acetylene derivatives displayed increased anti-
apoptotic activity. Further investigation must be conducted to
ascertain whether this reported activity is solely due to the acety-
lene group, the cage moiety, or the acetylene group in conjunction
with a nearby electronegative atom/group.
The novel compounds were all tested in triplicate at three
different concentrations (1 mM, 100
control, selegiline, was evaluated in triplicate at two concentrations
(100 M and 10 M, Fig. 3). Included in the assay was the prototype
mM and 10 mM). The positive
m
m
polycyclic cage moiety, 8-benzylamino-8,11-oxapentacyclo
[5.4.02,6.03,10.05,9]undecane (NGP1-01) [31], which was also evalu-
ated in triplicate at the highest concentration (1 mM). NGP1-01 is a
well-known NMDA receptor channel inhibitor and calcium channel
antagonist, and was evaluated for anti-apoptotic activity, as an
abnormal increase in calcium levels can act as a trigger for
apoptosis [10]. The positive control, selegiline, had 12e19% more
cells that were healthy and wherein apoptosis had either not been
induced or reached the level of mitochondrial depolarisation,
compared to control experiment 1 (Fig. 3). When compared to
Comparing the activity of compounds 11 and 13, it seems that
the tertiary propargylamine had slightly higher activity than the
secondary propargylamine, but the oxa- and aza-compounds in
general had comparable activity activities. In the case of the oxa-
compounds (11, 12), the methylated cage showed increased activ-
ity. With the aza compounds (13, 14), this was not observed. The
adamantane compounds (15, 16) had significantly lower activities
than that of the pentacycloundecane compounds 9e13. Solubility
problems were also experienced with compounds 15 and 16 at
higher concentrations, which may contribute to the observed lower

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F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
anti-apoptotic action. It could also be attributed to the possibility
that the pentacycloundecanes may have an increased ability to
cross cell membranes due to their high degree of lipophilicity [23],
which would result in increased intracellular concentrations of the
compounds. Comparing the percentage healthy cells of the samples
treated with test compounds 9, 10, 12 and 13, with that of the
control experiment 4, which is representative of healthy cells in a
favourable environment, it is interesting to note that these samples
had more healthy cells than that of control experiment 4. This may
be attributed to the compounds inhibiting even baseline apoptotic
processes.
Although NGP1-01 exhibited significant anti-apoptotic activity,
the activities of the synthesised compounds (9e16) were shown to
be higher in comparison. This might be due to NGP1-01 only
inhibiting one of the triggers of apoptosis, namely excitotoxicity
due to calcium flux into the neuronal cells, whereas the newly
synthesised compounds possibly inhibit the apoptotic cascade,
directly or indirectly, at more than one point. However, further
evaluations of these compounds are necessary to ascertain this.
MK-801, a commercially available potent high affinity NMDAR
channel blocker and NGP1-01.
The results obtained showed variable activities from the com-
pound series (Table 1). Compound 16, an adamantane derivative,
showed the best VGCC inhibitory activity within this series (45%).
This adamantine derived propargylamine compound showed ac-
tivity higher compared to that of NGP1-01 (7, 27%). Although
structurally similar to 16, the substitution of the aromatic moiety of
16 with a propargyl moiety to give the di-substituted propargyl-
amine adamantane compound (15), lead to a significant decrease in
VGCC activity (18%). This observation indicates the importance of
including the benzylamine moiety within these structures for
optimal VGCC activity. The exact mechanism in which these com-
pounds interact with the VGCC for activity is thus far unexplored.
The presence of the polycyclic cage moieties in the series of com-
pounds and similarities to NGP1-01 and other polycyclic cage
benzylamines, supports a mechanism earlier described for NGP1-
01 as being frequency- and voltage- dependent open calcium
channel blockers [33].
These compounds also exhibited of the highest NMDAR channel
inhibitory activities within this test series (52% and 59% for 15 and
16 respectively). This high activity might be attributed to the in-
clusion of the adamantane moiety, which has known NMDAR
inhibitory activity. Within the pentacycloundecane (PCU) test se-
ries, compounds 10 (19%), 12 (26%) and 13 (18%) showed moderate
VGCC blocking effects when compared to that of NGP1-01 (27%).
The remaining PCU derivatives showed low (9 and 14) to no VGCC
activity (11). It is interesting that the inclusion of a methyl group to
the structure of the inactive oxa-PCU compound 11 to give com-
pound 12 improved the VGCC activity by 26%. However, this trend
was not observed for the aza-PCU compounds 13 (11%) and 14
(18%). NMDAR channel inhibition was observed for PCU compounds
10 (23%) and 12 (38%), which showed improved inhibitory activity
when compared to NGP1-01 (18%). Compound 12 showed dual
action on both VGCC (26%) and NMDAR (38%). This finding suggests
that the inclusion of the methyl substituent contributes towards
binding and activity on both these drug targets as the compound
exhibited activity comparable to, and better than NGP1-01 on both
the VGCC and NMDAR. Comparing the activities obtained with
compound 12 to that of compound 14 (VGCC e 11%, NMDAR e 11%),
suggests that the oxa compounds have better VGCC and NMDAR
inhibitory activity compared to aza compounds. Removal of the
methyl substituent from 14 to give compound 13 increased VGCC
inhibitory activity to 18% but decreased NMDAR inhibitory action to
only 4%. Compound 10, which resembles NGP1-01 in the aromatic
moiety, also showed dual action on the VGCC (19%) and NMDAR
2.3. VGCC and NMDA receptor channel activity
All polycyclic propargylamine and acetylene derivatives (9e16)
were screened at 100 mM for their potential inhibitory activity on
VGCC and/or NMDA receptor (NMDAR) channel. They were
assessed using the fluorescent ratiometric indicator, Mag-Fura-2/
AM, and a fluorescent plate reader. KCl mediated calcium influx
in murine synaptoneurosomes was used to evaluate the influence
of the test compounds on the calcium influx via VGCC. NMDA/
Glycine mediated calcium influx was used to evaluate the influence
of the test compounds on calcium influx via the NMDA receptor
channel. Both assays were carried out with reference to standard
controls. Results are shown in Table 1.
Fresh synaptoneurosomes were prepared [32] from rat brain
homogenate and incubated with the ratiometric fluorescent cal-
cium indicator, Fura-2/AM. The synthesized compounds were
incubated for 30 min and a 140 mM KCl or a 100 mM NMDA/Glycine
solution, in the VGCC and NMDA assay respectively, was added to
depolarize the cell membranes or to stimulate calcium flux through
the respective channels. Calcium influx was then monitored based
on the fluorescence intensity relative to that of a negative control
(without inhibiting compound) over a 5 min period. Two positive
controls were included in the VGCC assay; nimodipine,
a
commercially available dihydropyridine L-type calcium channel
blocker and the prototype pentacycloundecane compound NGP1-
01. Two positive controls were also included in the NMDAR assay;
Table 1
The biological activities of test compounds as indication of potential neuroprotective activity.
Compound
Molecular
formula
Molecular weight (g/mol)
% Viable cells e
apoptosis induced [10
% VGCC inhibition [100
m
M]
% NMDA receptor
% MAO-B inhibition
[300 M]
m
M]
inhibition [100
m
M]
m
Propagylamine
Selegiline
NGP1-01
9
10
11
12
13
C3H5N
55.079
187.281
265.350
214.260
262.346
212.275
226.302
213.275
227.302
227.345
279.419
418.440
221.297
79***
77***
67**
n.d.
n.d.
27**
10
19
Inactive
26
18*
11
18*
n.d.
n.d.
18*
Inactive
23
Inactive
38***
4
11
52***
59*
19.93*
C
C
C
C
C
C
C
C
C
C
C
C
13H17
18H19NO
14H14O2
N
93.33***
14.57*
Inactive
73.32***
Inactive
11.67*
5.83
10.83*
14.00*
20.13*
n.d.
100***
98***
88***
97***
93***
67***
68***
70***
Inactive
n.d.
19H18
O
14H15NO
15H17NO
14H15NO
15H17NO
16H21
20H25
14
15
16
N
N
45*
100***
Inactive
Nimodipine
MK-801
21H26N2O7
16H15
n.d.
100***
N
n.d.
Statistical analysis was performed on raw data, with asterisks indicating significant inhibitory effect [(*) p < 0.05, (**) p < 0.001, (***) p < 0.0001] when compared to the
control. n.d. ¼ not determined.

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
127
Fig. 4. Comparison between the putative binding modes of safinamide and compound 10 to the human MAO-B enzyme active site. The compounds and FAD cofactor in bold lines
(online indicated in green and red respectively) with their respective binding interactions with the amino acid residues (sticks) shown on the right of each enzyme cavity rep-
resentation. Safinamide and compound 10 show productive interactions with amino acid residues Ile-199 and Gln 205, and Ile-199, respectively.
(23%) as seen with NGP1-01, a known VGCC and NMDAR antago-
nist. It is suggested that these compounds (9e16) exhibit a
blockade of the NMDA channel which is consistent with uncom-
petitive antagonism, similar to that reported for memantine.
Memantine shares the phencyclidine (PCP)/tenocyclidine (TCP)/
MK-801/ketamine binding site inside the NMDA channel pore.
Radio-ligand binding studies with [3H]MK-801 and [3H]TCP,
however, showed little or no displacement of PCU compounds,
including NGP1-01, from this binding site. The functional block of
calcium observed for the PCU structures needs to be further
explored to elucidate the exact mechanism of action. We hypoth-
esize that the series described in this paper will exhibit a similar
mechanism of action and act as direct NMDA channel blockers [10].
consistently better inhibitors than the rest of the series and it can
be speculated that these compounds were able to block the
entrance cavity of the MAO-B enzyme active site (see Fig. 4). This
may possibly be attributed to the planar character of the phenyl
ring and possibly to the extended planar character of compound 10.
It has also been reported in literature that planar compounds
frequently act as potent inhibitors of MAO-B [34,35]. Compound 10
contains both a hydroxyl and an extended planar phenyl moiety,
which might further explain the significantly higher activity of 10
compared to that of the other synthesised compounds.
2.4.1. MAO-B molecular modelling
Computer-assisted simulated docking of compounds 9e16, us-
ing Molecular Operating Environment (MOE), was carried out in an
attempt to clarify the MAO-B inhibition activity e or lack thereof e
for these structures [36]. The best-ranked docking solutions of the
test compounds examined, showed that the proposed inhibitors
occupy only the entrance cavity and barely access the substrate
cavity of the MAO-B enzyme (PDB ID: 2V5Z, Fig. 4) [37]. In general,
the amino acid residues between 120 and 220 are important in
conferring substrate selectivity of MAO-B [38], most importantly
residues Ile-199 (situated in the entrance cavity) and Gln-205
(situated in the substrate cavity). Interaction with these amino
acid residues may confer a compound MAO-B inhibitory activity as
shown by safinamide (Fig. 4), a known MAO-B inhibitor.
2.4. MAO-B inhibitory activity
The synthesised compounds were also evaluated in vitro at
various concentrations as inhibitors of MAO-B, using a spectro-
photometric assay that utilised MMTP, an analogue of the neuro-
toxin MPTP as substrate, with baboon liver mitochondria serving as
enzyme source. The potency of MAO-B inhibition was expressed as
percentage inhibition of the MAO-B enzyme (Table 1). Only com-
pound 10 showed significant MAO-B inhibition activity when
compared to that of selegiline (93.22%), inhibiting the MAO-B
enzyme by 73.32% at 300 mM. The rest of the compounds in the
series either had no activity (9, 11) or exhibited very limited inhi-
bition, ranging from 5.83 to 20.13% (12e16). Comparing the MAO-B
inhibition potencies of 11 with 12 and 13 with 14 reveals that in
both cases the methyl group on the polycyclic cage, appeared to
slightly increase MAO-B inhibition activity of the compounds. This
may be ascribed to the enhanced interaction obtained with the
non-polar entrance regions of the MAO-B enzyme.
For comparison, NGP1-01 was also evaluated as a MAO-B in-
hibitor, but no significant activity was observed. The compounds
containing a phenyl side chain (10, 15 and selegiline), were
For compound 10 it was shown that the polycyclic moiety is
stabilised within the entrance cavity with the extended planar
phenyl moiety able to traverse deeper into the enzyme cavity and
form the necessary interactions with Ile-199 (Fig. 4). This produc-
tive interaction between the phenyl function and the active site
residue Ile-199 of the substrate cavity and/or the stabilisation of the
polycyclic moiety to actively block the entrance cavity, might
explain the MAO-B inhibitory activity observed for 10. All the
polycyclic moieties are stabilised within the hydrophobic entrance
cavity and were unable to traverse deeper into the substrate cavity,

128
F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
to from the necessary binding interactions with the flavin adenine
dinucleotide (FAD) cofactor, which would have resulted in binding
interactions and an increase the activity of these compounds
(Fig. 4) [39,40]. Based on the molecular modelling results we are
currently in process of developing a series of optimized PCU-
propargylamine hybrid molecules with binding interactions and
molecular orientations which might lead to enhanced MAO-B ac-
tivity for these compounds. This series will enable the propargyl
moiety to penetrate deeper into the cavity within close proximity of
the FAD co-factor, similar to selegiline and rasagiline (data not
shown) [19]. We also envision that the multifunctional activities
shown for the current propargylamine derivatives will be retained
in the new series.
spectrometer at 70 eV and 100 ꢁC. All HREI samples were intro-
duced by a heated probe and perfluorokerosene was used as
reference standard. 1H and 13C NMR spectra were acquired on a
Bruker Avance III 600 MHz spectrometer, with the 1H spectra
recorded at a frequency of 600.170 MHz and the 13C spectra at
150.913 MHz. Tetramethylsilane (TMS) was used as internal stan-
dard, with CDCl3 as solvent. 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 respec-
tive signals: s e singlet; br s e broad singlet; d e doublet; dd e
doublet of doublets; t e triplet; m e multiplet; and AB-q e AB
quartet. The multiplicity of the identified carbons was confirmed
with DEPT-spectra. Microwave synthesis was performed using a
CEM DiscoverÔ microwave synthesis system.
3. Conclusion
4.2. Synthesis of compounds
This study aimed to develop compounds that would ultimately
inhibit NMDA receptors, block VGCC and inhibit apoptotic pro-
cesses as well as the MAO-B enzyme. Based on the results it can be
concluded that compounds 10, 12, 15 and 16 showed the best range
of activities. These compounds however showed little to no activity
on the MAO-B enzyme except for 8-phenyl-ethynyl-8-hydroxy-
pentacycloundecane (10) which inhibited the MAO-B enzyme by
4.2.1. Pentacyclo[5.4.1.02,6.03,10.05,9]undecane-8-11-dione (1)
As described by Cookson et al. (1958, 1964) [41].
4.2.2. 1-Methyl-pentacyclo[5.4.1.02,6.03,10.05,9]undecane-8-11-dione
(a)
As described by Marchand et al. (1984) [42].
73.32% at 300 mM. The compounds in the series all had significant
anti-apoptotic activity comparable (14e16) to and better (9e13)
than selegiline. Although the findings suggest compounds 9 and 11
to have little or no activity on the VGCC, NMDA receptors and MAO-
B enzyme, the ability of these compounds to significantly inhibit
apoptosis suggests that the compounds exhibit their neuro-
protective action through some other mechanism(s) unexplored in
this study. Having significant anti-apoptotic activity, this series of
compounds might present interesting lead compounds in the
design and development of more potent inhibitors. Further in vitro
and in vivo studies into the anti-apoptotic cascade, dopamine
transmission and bloodebrain barrier permeability of these novel
compounds are in process. Findings from these studies will elabo-
rate on their potential as neuroprotective agents and might identify
the point(s) at which these compounds inhibit the apoptotic pro-
cess. The polycyclic propargylamines and acetylene derivatives
thus have potential as novel multifunctional neuroprotective
agents and further investigation is necessary to determine their
maximum benefit in the treatment of neurodegenerative disorders.
4.2.3. Pentacyclo[5.4.1.02,6.03,10.05,9]undecane-8-one (b)
As described by Dekker and Oliver (1979) [43].
4.2.4. 8-Benzylamino-8,11-oxapentacyclo[5.4.02,6.03,10.05,9
undecane (NGP1-01, 3)
]
As described by Van der Schyf et al. (1986) [31].
4.2.5. 1-Methyl-8-ethynyl-11-hydroxy-8,11-oxapentacyclo
[5.4.02,6.03,10.05,9]undecane (9)
A solution of 1-methyl-pentacyclo[5.4.1.02,6.03,10.05,9]undecane-
8-11-dione (a) (1.88 g; 10 mmol) in dry tetrahydrofuran (10 mL)
was added to an excess of ethynyl magnesium bromide (1.0 M so-
lution in tetrahydrofuran) (2.838 mL; 22 mmol) under argon, whilst
stirring. The resulting mixture was stirred at ambient temperature
for 21 h. The reaction mixture was poured over saturated aqueous
ammonium chloride (NH4Cl) solution (100 mL), and the resulting
suspension was extracted with diethyl ether (3 ꢂ 25 mL). The
combined organic extracts were washed sequentially with water
(25 mL) and brine (25 mL). The solvent was dried (Na2SO4) and
filtered, and the filtrate was concentrated under reduced pressure.
The residue, pale yellow oil, was successfully purified by precipi-
tation from a mixture of ethyl acetate/petroleum ether (1:3), over a
period of 24 h at ambient temperature. This yielded the pure
product as a light brown powder (yield: 0.762 g; 3.170 mmol;
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 thin-layer chromatography on
15.87%). C14H14O2; MW, 214.3 g/mol; mp. 126 ꢁC; IR (KBr) vmax
:
3260, 2125, 1211, 1031 cm-1; MS (EI, 70 eV) m/z: 214 (Mþ), 186, 169,
158, 116, 91, 77, 39; HR-ESI [MDH]D: calc. 215.1067, exp. 215.1069.
1H NMR (600 MHz, CDCl3) dH: 3.72 (s, OH), 3.12e2.37 (m, 7H, H-
2,3,5,6,7,9,10), 2.31 (s, 1H, H-13), 1.88:1.54 (AB-q, 2H, J ¼ 10.4 Hz, H-
4a,4b), 1.10 (s, 3H, CH3); 13C NMR (150 MHz, CDCl3) dC: 116.87 (1C,
C-11), 81.18 (1C, C-8), 75.30 (1C, C-12), 61.84 (1C, C-13), 57.24 (1C, C-
9), 56.91 (1C, C-7), 50.75 (1C), 47.46 (1C), 45.22 (1C), 43.49 (1C, C-4),
41.56 (1C), 39.19 (1C, C-1), 15.29 (1C, C-14).
0.20 mm thick aluminium silica gel sheets (AlugramÒ SIL G/UV254
,
Kieselgel 60, MachereyeNagel, 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
volume-to-volume basis. Chromatographic purifications were
performed on silica gel (0.063e0.2 mm, Merck) except when
otherwise stated. The mass spectra (MS) were recorded on an
analytical VG 70-70E mass spectrometer using electron ionisation
(EI) at 70 eV. Infra-red (IR) spectra were recorded on a Shimadzo IR
prestige e 21 Fourier transform infrared spectrophotometer using
KBr. Melting points were determined using a Gallenkamp and
Stuart SMP-300 melting point apparatus and capillary tubes. All the
melting points determined were recorded uncorrected. High res-
olution electron spray ionisation (HREI) mass spectra for all com-
pounds were recorded on a Waters API Q-Tof Ultima mass
4.2.6. 8-Phenylethynyl-8-hydroxy-pentacyclo[5.4.02,6.03,10.05,9
undecane (10)
Neutral alumina (60e80 mesh, 30 g) in water (150 mL) was
added to a stirred solution of potassium fluoride (20 g) in water
(150 mL). After 30 min the water was evaporated in a rotary
evaporator at 80 ꢁC. When most of the water had been removed,
the remaining mixture was heated to, and maintained at 140e
]

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
129
150 ꢁC under vacuum (5 mm Hg) overnight to afford 50 g of KF/
4.2.8. 1-Methyl-8-(N)-propargylamino-8,11-oxapentacyclo
alumina reagent. Phenylacetylene (255.350 mg; 2.500 mmol),
pentacyclo[5.4.02,6.03,10.05,9]undecane-8-one (b, 480.650 mg;
3 mmol), and KF/alumina (2.500 g) were mixed in a 25 mL flask at
60 ꢁC. The progress of the reaction was monitored by TLC. After
12 h the reaction mixture was washed with petroleum ether,
filtered, and the solvent was evaporated under vacuum, affording
a clear yellow oil. The residue was purified by means of column
chromatography, by using a versa flash silica-gel column with
petroleum ether/ethyl acetate (10:2) as eluent, to afford the
product as a light yellow wax (yield: 480 mg; 1.830 mmol; 73.18%).
[5.4.02,6.03,10.05,9]undecane (12)
1-Methyl-penta-cyclo[5.4.02,6.03,10.05,9]undecane-8-11-dione
(a, 5.460 g; 0.029 mol) was dissolved in tetrahydrofuran (50 mL)
and cooled to ꢃ10 ꢁC, while stirring on an external bath containing
an acetone/NaCl/ice mixture. Propargylamine (1.600 g; 0.029 mol)
was slowly added with continued stirring of the reaction mixture
at lowered temperature. The reaction mixture was stirred for an
additional 1½ hours to reach completion. The THF was removed
under reduced pressure, affording the carbinolamine as a red/
brown oil. Water was removed azeotropically by refluxing this
material in dry benzene (60 mL), under DeaneStark dehydrating
conditions for 1 h, or until no more water was collected in the
trap. The benzene was removed under reduced pressure, which
yielded the Schiff base as a brown oil. The Schiff base (imine) was
dissolved in a mixture of anhydrous methanol (30 mL) and
anhydrous tetrahydrofuran (150 mL). Reduction was carried out by
adding sodium borohydride (1.500 g; 0.040 mol) in excess and
stirring the mixture for 24 h at room temperature. The solvents
were removed under reduced pressure, the residue suspended in
water (100 mL) and extracted with methylene chloride
(4 ꢂ 50 mL). The combined organic fractions were washed with
water (2 ꢂ 100 mL), dried over anhydrous MgSO4 and evaporated
under reduced pressure to yield a milky dark brown oil. Purifica-
tion of the product mixture was accomplished using column
chromatography, with ethyl acetate/methylene chloride/petro-
leum ether (1:1:1) as eluent, yielding the desired amine as a dark
brown oil (Yield: 1.753 g; 7.746 mmol; 26.71%). C15H17O1N1; MW,
227.3 g/mol; IR (KBr) vmax: 3310, 3250, 2100, 1452, 1153, 1007 cm-
1; MS (EI, 70 eV) m/z: 227 (Mþ), 212, 198, 184, 158, 145, 131, 91, 77,
39; HR-ESI [MDH]D: calc. 228.1383, exp. 228.1395. 1H NMR
(600 MHz, CDCl3) dH: 4.08 (d, 1H, J ¼ 4.4 Hz, H-11), 3.57 (m, 2H, H-
12a,12b), 2.73e2.20 (m, 7H, H- 2,3,5,6,7,9,10), 2.14 (br s, NH), 2.06
(d, 1H, J ¼ 5.28 Hz, H-14), 1.86:1.51 (AB-q, 2H, J ¼ 10.4 Hz, H-
4a,4b), 1.15 (s, 3H, CH3); 13C NMR (150 MHz, CDCl3) dC: 109.73 (1C,
C-8), 87.15 (1C, C-11), 82.65 (1C, C-13), 70.95 (1C, C-14), 54.97 (1C,
C-9), 54.66 (1C, C-7), 50.74 (1C), 46.99 (1C), 43.58 (1C), 43.18 (1C),
38.91 (1C, C-1), 33.07 (1C, C-12), 19.85 (1C, C-15).
C
19H18O; MW, 262.4 g/mol; mp. 90 ꢁC; IR (KBr) vmax: 3069, 2225,
1599, 1491, 1125, 752 cmꢀ1; MS (EI, 70 eV) m/z: 262 (Mþ), 196, 183,
165, 129, 115, 91, 77; HR-ESI [MDH]D: calc. 263.1430 exp.
263.1439; 1H NMR (600 MHz, CDCl3) dH 7.36e7.13 (m, 5H, H
15,16,17,18,19), 2.77e2.28 (m, 8H, H-1,2,3,5,6,7,9,10), 1.93:1.60 (AB-
q, 2H, H-11a,11b), 1.72:1.18 (AB-q, 2H, J ¼ 10.4 Hz, H-4a,4b); 13C
NMR (150 MHz, CDCl3) dC: 131.51 (1C, C-14), 128.23 (2C, C-15,19),
128.10 (2C, C-16,18), 123.02 (1C, C-17), 94.15 (1C, C-8), 83.21 (1C, C-
13), 75.91 (1C, C-12), 51.50 (1C), 47.27 (1C), 45.20 (1C), 44.95 (1C),
42.73 (1C, C-4), 41.50 (1C), 40.61 (1C), 36.62 (1C), 34.73 (1C), 28.98
(1C, C-11).
4.2.7. 8-(N)-propargylamino-8,11-oxapentacyclo[5.4.02,6.03,10.05,9
]
undecane (11)
Pentacyclo-[5.4.02,6.03,10.05,9]undecane-8-11-dione (1,
5
g;
0.029 mol) was dissolved in tetrahydrofuran (50 mL) and cooled
to ꢃ ꢀ10 ꢁC, while stirring on an external bath, containing an
acetone/NaCl/ice mixture. Propargylamine (1.600 g; 0.029 mol)
was added drop-wise, with continued stirring of the reaction
mixture at lowered temperature. The carbinolamine started
precipitating after approximately 15 min, but the reaction was
allowed to stir for an additional 30 min to reach completion. The
carbinolamine was isolated by filtration and washed with ice cold
THF. Water was removed azeotropically by refluxing the material
in dry benzene (60 mL), under DeaneStark dehydrating conditions
for 1 h, or until no more water was collected in the trap. The
benzene was removed under reduced pressure, which yielded the
Schiff base as yellow oil. The Schiff base (imine) was then dis-
solved in a mixture of anhydrous methanol (30 mL) and anhy-
drous tetrahydrofuran (THF) (150 mL). Reduction was carried out
by adding sodium borohydride (NaBH4) (1.500 g; 0.040 mol) in
excess, and stirring the mixture for 24 h at room temperature. The
solvents were removed under reduced pressure, the residue sus-
pended in water (100 mL) and extracted with methylene chloride
(4 ꢂ 50 mL). The combined organic fractions were washed with
water (2 ꢂ 100 mL), dried over anhydrous MgSO4 and evaporated
under reduced pressure to yield a milky yellowish oil. Purification
of the product mixture was accomplished using column chroma-
tography on silica gel, with ethyl acetate/methylene chloride/pe-
troleum ether (1:1:1) as eluent. This yielded the desired amine as
a light yellow precipitate. Recrystallisation from absolute ethanol
rendered the final product as a colourless microcrystalline solid
(yield: 600 mg; 2.827 mmol; 9.75%). C14H15O1N1; MW, 213.28 g/
mol; mp. 113 ꢁC; IR (KBr) vmax: 3306, 3238, 2124, 1485, 1153,
1000 cmꢀ1; MS (EI, 70 eV) m/z: 213 (Mþ), 184, 134, 118, 91, 77, 39;
HR-ESI [MDH]D: calc. 214.1226, exp. 214.1225. 1H NMR (600 MHz,
CDCl3) dH: 4.63 (t, 1H, J ¼ 5 Hz, H-11), 3.57:3.56 (dd, 2H, J ¼ 2.5 Hz,
H-12a,12b), 2.80e2.39 (m, 8H, H-1,2,3,5,6,7,9,10), 2.26 (br s, NH),
2.21 (s, 1H, H-14), 1.88:1.52 (AB-q, 2H, J ¼ 10.4 Hz, H-4a,4b); 13C
NMR (150 MHz, CDCl3) dC: 108.98 (1C, C-8), 82.81 (1C, C-11), 82.76
(1C, C-13), 70.92 (1C, C-14), 55.46 (1C, C-7/9), 54.76 (1C, C-7/9),
44.84 (1C), 44.65 (1C), 44.54 (1C), 43.3 (1C), 43.07 (1C, C-12), 42.04
(1C), 41.54 (1C), 33.07 (1C).
4.2.9. 8-Hydroxy-(N)-propargyl-8,11-azapentacyclo
[5.4.02,6.03,10.05,9]undecane (13)
Pentacyclo-[5.4.02,6.03,10.05,9]undecane-8-11-dione (1,
5
g;
0.029 mol) was dissolved in tetrahydrofuran (50 mL) and cooled
to ꢃ ꢀ10 ꢁC, while stirring on an external bath, containing an
acetone/NaCl/ice mixture. Propargylamine (1.600 g; 0.029 mol) was
added slowly with continued stirring of the reaction mixture at
lowered temperature. The carbinolamine started precipitating after
approximately 60 min, but the reaction was stirred for an additional
45 min to reach completion. This carbinolamine was isolated by
filtration. Water was removed azeotropically by refluxing this
material in dry benzene (60 mL), under DeaneStark dehydrating
conditions for 1 h, or until no more water was collected in the trap.
The benzene was removed under reduced pressure and the Schiff
base was used without further purification. It was dissolved in a
solution of acetic acid (15 mL) and dry methanol (250 mL). To the
resulting solution was added sodium cyanoborohydride (NaBH3CN)
(2.510 g; 40 mmol) portion wise, with stirring at room temperature
over a period of 5 min. The resulting mixture was stirred at room
temperature for 2 h. The reaction mixture was then concentrated
under reduced pressure, and water (100 mL) was added to the
residue. The resulting suspension was stirred and solid sodium
bicarbonate was added portion wise until evolution of carbon di-
oxide ceased. Excess solid sodium bicarbonate (2.000 g) was added,
and the aqueous suspension was extracted with methylene chlo-
ride (4 ꢂ 50 mL). The combined extracts were washed with water

130
F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
(2 ꢂ 100 mL), dried with anhydrous magnesium sulphate and
filtered. The filtrate was concentrated under reduced pressure. A
yellow solid was thereby obtained. Purification of the product
mixture was accomplished using column chromatography on silica,
with ethyl acetate/methylene chloride/ethanol (10:5:1) as eluent.
The desired amine was obtained as an off-white powder. Recrys-
tallisation from cyclohexane rendered the final product as an off
white microcrystalline solid (Yield: 960 mg; 4.501 mmol; 15.52%).
4.2.11. N,N-Dipropargyl-adamantan-1-amine (15)
Excess propargylbromide (0.595 mL; 6.677 mmol), amantadine
(2, 302.49 mg; 2 mmol) and aqueous sodium hydroxide (NaOH)
solution (180 mg in 6 mL water; 4.5 mmol) were placed in a round-
bottom glass flask equipped with a condenser and a magnetic
stirrer. The flask was placed in a CEM discover focused microwave
synthesis system, and subjected to microwave irradiation at 80e
100 ꢁC (power 250 Watt) for 25 min. After completion of the re-
action, the product was extracted into ethyl acetate. The solvent
was then removed under reduced pressure. The unreacted aman-
tadine was removed from the residue by adding acetone to the
residue and collecting the amantadine by filtration. The acetone
was then removed under reduced pressure from the filtrate,
affording orange crystals which were recrystallised out of ethyl
acetate to afford the pure product as light yellow crystals (412 mg;
1.812 mmol; 90.61%). C16H21N1; MW, 227.35 g/mol; mp. 76 ꢁC; IR
(KBr) vmax: 3237, 2099, 1119 cmꢀ1; MS (EI, 70 eV) m/z: 227 (Mþ),
184, 144, 132, 91, 79, 53, 39; HR-ESI [MDH]D: calc. 228.1752, exp.
C
14H15O1N1; MW, 213.3 g/mol; mp. 140 ꢁC; IR (KBr) vmax: 3225,
3100, 2114, 1120, 1070, cmꢀ1; MS (EI, 70 eV) m/z: 213 (Mþ), 196, 174,
147, 134, 118, 91, 77, 39; HR-ESI [MDH]D: calc. 214.1226, exp.
214.1222; 1H NMR (600 MHz, CDCl3) dH: 3.71 (s, 1H, H-12), 3.35 (d,
1H, J ¼ 16.5 Hz, H-11), 3.00e2.43 (m, 8H, H-1,2,3,5,6,7,9,10), 2.25 (s,
1H, H-14), 1.82:1.49 (AB-q, 2H, J ¼ 10.4 Hz, H-4a,4b); 13C NMR
(150 MHz, CDCl3) dC: 125.53 (1C, C-8), 81.23 (1C, C-13), 71.37 (1C, C-
14), 65.52 (1C, C-11), 45.62 (1C, C-7/9), 43.12 (1C, C-7/9), 41.74 (1C,
C-4), 41.60 (1C), 36.79 (1C), 30.33 (1C, C-12).
228.1755; 1H NMR (600 MHz, CDCl3) dH
: 3.65 (s, 4H, H
4.2.10. 1-Methyl-8-Hydroxy-(N)-propargyl-8,11-azapentacyclo
[5.4.02,6.03,10.05,9]undecane (14)
11a,11b,14a,14b), 2.17 (s, 2H, H-13,16), 2.06 (s, 3H, H-3,5,8), 1.80 (s,
6H, H-2a,2b,6a,6b,7a,7b), 1.63e1.46 (m, 6H, H-4a,4b,9a,9b,10a,10b);
13C NMR (150 MHz, CDCl3) dC: 82.12 (2C, C-12,15), 72.08 (2C, C-
13,16), 55.45 (1C, C-1), 39.96 (3C, C-3,5,8), 36.54 (3C, C-2,6,7), 35.03
(2C, C- 11,14), 29.70 (3C, C-4,9,10).
1-Methyl-pentacyclo[5.4.02,6.03,10.05,9]undecane-8-11-dione
(b, 10.658 g; 56.620 mmol) was dissolved in tetrahydrofuran
(50 mL) and cooled to ꢃ ꢀ10 ꢁC, while stirring on an external
bath containing an acetone/NaCl/ice mixture. Propargylamine
(3.119 g; 56.620 mmol) was added drop wise with continued
stirring of the reaction mixture at lowered temperature. The
reaction mixture was stirred for 5½ hours to reach completion.
The THF was removed under reduced pressure, affording a red/
brown oil, the carbinolamine. Water was removed azeotropically
by refluxing this material in dry benzene (60 mL), under Deane
Stark dehydrating conditions for 1 h, or until no more water was
collected in the trap. The benzene was removed under reduced
pressure, which yielded the Schiff base as a dark brown oil. The
Schiff base was used without further purification. It was dis-
solved in a solution of acetic acid (30 mL) in dry methanol
(500 mL). To the resulting solution, sodium cyanoborohydride
(3.970 g; 63 mmol) was added portion wise, with stirring at room
temperature over a period of 5 min. The resulting mixture was
stirred at room temperature for 14 h. The reaction mixture was
then concentrated under reduced pressure, and water (150 mL)
was added to the residue. The resulting suspension was stirred,
and solid sodium bicarbonate was added portion wise until
evolution of carbon dioxide ceased. Excess solid sodium bicar-
bonate (3.000 g) was added, and the aqueous suspension was
extracted with methylene chloride (4 ꢂ 50 mL). The combined
extracts were washed with water (2 ꢂ 100 mL), dried with
anhydrous magnesium sulphate, and filtered. The filtrate was
concentrated under reduced pressure, leaving a deep orange oil
as residue. Purification of the product mixture was accomplished
using column chromatography on silica, with ethyl acetate/
tetrahydrofuran (5:1) as eluent. This yielded the desired amine as
a light yellow oil, which precipitated when ethanol was added.
Recrystallisation from ethanol rendered the final product as a
light yellow microcrystalline solid (Yield: 555 mg; 2.442 mmol;
4.2.12. N-Propargyl-N-benzyl-adamantan-1-amine (16)
A solution of amantadine (2, 5 g; 33.056 mmol) and benzalde-
hyde (3.51 g; 33.073 mmol) in ethanol (60 mL) was stirred for 4
days at ambient temperature under nitrogen atmosphere. The
solvents were removed in vacuo. The resulting oil was dissolved in
80 mL benzene and refluxed under DeaneStark conditions for 6 h,
where after the benzene was removed in vacou. The resulting re-
action mixture was dissolved in 60 mL ethanol and solid sodium
borohydride (NaBH4) (2.5 g; 66.085 mmol) was then added slowly
in small portions over 30 min, and stirring of the resulting sus-
pension was continued at room temperature for 30 min under ni-
trogen atmosphere. The reaction mixture was refluxed for 12 h.
After cooling to ambient temperature, the mixture was diluted with
ethanol (60 mL), and the excess sodium borohydride was destroyed
by adding aqueous hydrochloric acid (HCl) (10 mL, 5 M) drop wise.
The reaction mixture was then made alkaline, to pH 12, by adding
aqueous sodium hydroxide (NaOH) solution. Finally, the desired
product was extracted to methylene chloride (4 ꢂ 10 mL) and dried
over anhydrous magnesium sulphate (MgSO4). The solvent was
evaporated under reduced pressure to afford the intermediate
product, N-benzyl-adamantan-1-amine (c), as white crystals (yield:
4.757 g; 19.6 mmol; 59.29%). This intermediate product was sub-
sequently used in the following steps without any further purifi-
cation. A solution was made of N-benzyl-adamantan-1-amine (c,
4.757 g; 19.6 mmol) and excess propargylbromide (2.3 mL;
25.81 mmol) in dry dimethylformamide (30 mL), and left for 24 h to
react at ambient temperature. After this period of time the mixture
was diluted with water (50 mL), and an extraction was done using
methylene chloride (DCM) (3 ꢂ 50 mL) and dried over anhydrous
sodium sulphate (Na2SO4). The solvent was removed under
reduced pressure to afford a thick dark orange residue. The desired
product was precipitated out of the residue by the addition of
chloroform (30 mL), which allowed the collection of the final
product by filtration as an off-white powder (yield: 1.352 g;
4.82 mmol; 24.59%). C20H25N1; MW, 279.3 g/mol; mp. 81 ꢁC; IR
(KBr) vmax: 3210, 3062, 2091, 1495, 1130, 748 cmꢀ1; MS (EI, 70 eV)
m/z: 279 (Mþ), 236, 222, 185, 135, 91; HR-ESI [MDH]D: calc.
4.31%). C15H17O1N1; MW, 227.3 g/mol; mp. 125 ꢁC; IR (KBr) vmax
:
3225, 3078, 2124, 1119, 1070 cmꢀ1; MS (EI, 70 eV) m/z: 227 (Mþ),
188, 134, 118, 91, 77, 39; HR-ESI [MDH]D: calc. 228.1388, exp.
228.1392; 1H NMR (600 MHz, CDCl3) dH: 3.46e3.34 (m, 1H, H-11),
3.22:3.09 (d, 2H, J ¼ 17.3 Hz, H-12a,12b), 2.57e2.09 (m, 8H, H-
2,3,5,6,7,9,10,14), 1.60:1.06 (AB-q, 2H, J ¼ 10.4 Hz, H-4a,4b), 1.19
(s, 3H, CH3); 13C NMR (150 MHz, CDCl3) dC: 125.53 (1C, C-8),
80.31 (1C, C-13), 72.68 (1C, C-14), 57.40 (1C, C-11), 46.62 (1C, C-
9), 46.10 (1C, C-7), 45.09 (1C), 41.54 (1C, C-4), 37.63 (1C), 36.33
(1C), 34.52 (1C), 30.32 (1C, C-12), 21.98 (1C, C-15).
1
280.2065, exp. 280.2063; H NMR (600 MHz, CDCl3) dH: 7.35e7.20
(m, 5H, H- 16,17,18,19,20), 3.84 (s, 2H, H-14a,14b), 3.32 (s, 2H, H-
11a,11b), 2.15 (s, 3H, H-3,5,8), 2.09 (s, 1H, H-13), 1.90 (s, 6H, H-

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
131
2a,2b,6a,6b,7a,7b), 1.65 (s, 6H, H-4a,4b,9a,9b,10a,10b); 13C NMR
(150 MHz, CDCl3) dC: 141.11 (1C, C-15), 128.65 (2C, C-16,20), 128.18
(2C, C-17,19), 126.66 (1C, C-18), 83.22 (1C, C 12), 72.08 (1C, C-13),
55.05 (1C, C-1), 48.98 (1C, C-14), 40.53 (3C, C-3,5,8), 36.77 (3C, C-
2,6,7), 34.79 (1C, C-11), 29.86 (3C, C-4,9,10).
106 cells per 2 mL of serum deprived medium containing the test
compounds, which were dissolved in dimethyl sulphoxide. Final
concentrations of samples to be analysed contained 1.25% (v/v)
DMSO. The cells were then incubated under normal conditions for a
period of 36 h before being analysed. The test compounds (6e13)
were all tested in triplicate at three different concentrations (1 mM,
4.3. Biological evaluations
100
mM and 10
mM). The positive control, selegiline, was tested in
triplicate at two concentrations (100
m
M and 10 M). Included into
m
4.3.1. Apoptosis detection e DePsipherÔ assay
the research was NGP1-01 which was also tested in triplicate at the
highest concentration (1 mM).
4.3.1.1.4. Cells used for control experiments. In this study four
control experiments were included.
DePsipherÔ utilises a lipophilic cation (5,50,6,60-tetrachloro-
1,10,3,30-tetraethylbenzimidazolyl carbocyaniniodide), which can be
used as a mitochondrial activity marker to evaluate the viability of a
cell population, detecting early apoptosis, and evaluating the effect
of drugs on the cell population [30]. The cation aggregates upon
membrane polarisation, forming an orange-red fluorescent com-
pound. If the potential is disturbed, the dye cannot access the
transmembrane space and remains orange-red or reverts to its
green monomeric form. Thus, healthy cells present both the poly-
meric (orange-red) and monomeric (green) form of the cation, with
the monomeric form residing in the cytoplasm and the aggregated
form in the transmembrane space. It can be concluded that in
comparison with healthy cells, wherein apoptosis has not been
induced, the ratio of orange-red fluorescence to green fluorescence
of apoptotic cells will be lower. The fluorescence can be observed
and measured by flow cytometry with the red aggregates having
absorption/emission maxima of 585/590 nm, and the green
monomers absorption/emission maxima of 510/527 nm.
4.3.1.1.4.1. Control experiment 1
After the cells reached 80% confluency, the serum rich medium
was replaced with serum deprived medium. The cells were then
incubated under normal conditions for a period of 24 h. After being
incubated in the serum deprived medium, the medium was removed
by centrifugation. The cells were subsequently seeded in 24-well
plates, with each well containing a concentration of 1 x 106 cells
per 2 mL of serum deprived medium and DMSO (1.25% (v/v)). After
this, the cells were incubated under normal conditions (37 ꢁC in a
5% CO2 and 95% O2 humidified atmosphere) for a period of 36 h
before being analysed. Control experiment 1 was included to
evaluate the viability status of the cells in the absence of test
compound. The viability data will be compared to the data gener-
ated with the test compounds in order to determine if the test
compounds attenuated the progression of the apoptotic process.
With this control experiment, the effect of DMSO was also
evaluated.
4.3.1.1. Biological material
4.3.1.1.1. Cell cultivation. In the current study the SK-N-BE(2)
neuroblastoma cells were used to evaluate the anti-apoptotic ac-
tivity of the synthesised compounds. The cells were cultivated in
serum-rich growing medium, consisting of F12 nutrient (F12)
supplemented with 10% foetal bovine serum (FBS), 1% penicillin/
streptomycin (Pen/Strep) (10 000 units/100 mL stock) and 0.1%
4.3.1.1.4.2. Control experiment 2
After the cells reached 80% confluency, the serum rich medium
was replaced with serum deprived medium. The cells were then
incubated under normal conditions for a period of 24 h. After being
incubated in the serum deprived medium, the medium was removed
by centrifugation. The cells were subsequently seeded in 24-well
plates, with each well containing a concentration of 1 x 106 cells
per 2 mL of serum deprived medium with no DMSO content. The
cells were incubated under normal conditions for a period of 36 h
before being analysed. Control experiment 2 was used to evaluate
the effect of DMSO on the viability of the cells and on the pro-
gression of the apoptotic process.
fungizone (FZ) (2.5
mg/mL units stock). They were incubated at
37 ꢁC in a 5% CO2 and 95% O2 humidified atmosphere. The cells
duplicated every 30e48 h and when a confluency of 80% was
reached, the adherent cells were detached from the flask bottom by
means of trypsination. To ensure complete detachment of the cells,
they were incubated with trypsine for approximately 10 min. After
trypsination, the cells were seeded in new flasks at a density of no
less than 1/6 confluency.
4.3.1.1.4.3. Control experiment 3
After the cells reached 80% confluency, the serum rich medium
was replaced with serum deprived medium. The cells were subse-
quently incubated under normal conditions for a period of 24 h.
After being incubated in the serum deprived medium, the medium
was removed by centrifugation. The cells were then seeded in 24-
well plates, with each well containing a concentration of 1 x
106 cells per 2 mL of fresh serum rich medium with no DMSO pre-
sent. After this the cells were incubated under normal conditions
for a period of 36 h before being analysed. Since the re-introduction
of serum rich medium is expected to significantly attenuate the
apoptotic process, this control experiment will be used to estimate
the potencies of the test compounds as anti-apoptotic agents.
4.3.1.1.4.4. Control experiment 4
After the cells reached 80% confluency, the serum rich medium
was replaced with fresh serum rich medium. The cells were then
incubated under normal conditions for a period of 24 h. After being
incubated in the serum rich medium, the medium was removed by
centrifugation. The cells were subsequently seeded in 24-well
plates, with each well containing a concentration of 1 ꢂ 106 cells
per 2 mL of serum rich medium with no DMSO present. The cells
were incubated under normal conditions for a period of 36 h before
being analysed. Control experiment 4 was included to analyse and
determine the viability status of the cells, when no apoptosis had
4.3.1.1.2. Induction of apoptosis. In the current study, trophic
factor deprivation/serum starvation was used to induce apoptosis
in cells. With growth factors being essential to the normal devel-
opment and survival of neuronal cells, a shortage thereof would
cause neurons to compete with one another for neurotrophic fac-
tors, with the unsuccessful ones dying. With no serum (neuro-
trophic factors) available, all the cells will die in a given time. The
serum deprived medium consisted of F12, Pen/Strep and FZ in the
same ratio as mentioned in Section 4.3.2.2.
4.3.1.1.3. Cells used to evaluate anti-apoptotic activity of com-
pounds. After the cells reached 80% confluency, the serum rich
medium was replaced with serum deprived medium. The cells
were then incubated under normal conditions (see Section 4.3.2.2)
for a period of 24 h, which was sufficient time for apoptosis to be
induced. Examining the cells under the microscope after this period
of time, it was clear that their morphology had changed. The cells
were no longer attached to the bottom of the flask, and their shape
had also changed from the sprouting appearance to a spherical
shape, being indicative of unhealthy cells. After being incubated in
the serum deprived medium for the indicated time, the medium
was removed, using centrifugation. The cells were then seeded in
24-well plates, with each well containing a concentration of 1 x

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F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
been induced, and conditions had been favourable to the end. It was
also included to ensure that the cells analysed were indeed healthy
before inducing apoptosis.
The data generated by these flow cytometry experiments were
used to determine if the test compounds have anti-apoptotic
properties on cultured cells. The values utilised to quantify cell
viability were determined by the flow cytometer. Equation (1) was
used to calculate the percentage of cells which were still viable in
the samples analysed. In determining this value, the amount of cells
in quadrant 1 (Q1), quadrant 2 (Q2) and quadrant 3 (Q3) and the
total amount of events analysed (Qtotal) were utilised.
4.3.1.2. Assay procedure. The cells of each sample were harvested
consecutively as follows: After incubating the cells for 36 h, the
medium was removed from the cells using centrifugation. The cells
that had adhered to the bottom of the flasks were removed by
trypsination. The trypsine/cell mixture was added to the cells from
which the medium had been removed. The trypsine was removed
from the cells by centrifugation at 500 ꢂ g for 5 min at room
temperature. The cells of each sample were then resuspended in
% Viable cells ¼ ½ðQ1 þ Q2Þ=ðQtotal ꢀ Q3Þꢄ ꢂ 100
(1)
The results confirmed that a significant percentage of the cells
had indeed coloured successfully with the dye. The percentage of
the coloured cells that had fluorescence intensity in the FL2 chan-
nel, gave a quantitative indication of the number of healthy cells
present in each sample, wherein apoptosis had not progressed or
been induced. In control experiment 1, 41.62% of the cells were
apoptotic, with only 58.38% remaining healthy. This indicates that
the method of apoptosis induction had indeed been successful.
Comparing the values of control experiments 1 and 2, it is clear that
even though the final DMSO content in the samples was only 1.25%,
it had a negative effect on the health of the cells. In control
experiment 2, which did not contain any DMSO, there were 7.19%
more cells that were healthy. It can thus be said that the serum
deprivation was not the only factor inducing apoptosis, but also the
DMSO which served as solvent for the test compounds.
1 mL of diluted DePsipher solution, consisting of 1 mL DePsipher dye
and 1 mL of pre-warmed F12 medium. The cells were incubated for
20 min at 37 ꢁC and 5% CO2. The samples were washed twice in PBS,
and centrifuged at 500 ꢂ g between each wash. The cells were
subsequently resuspended in 1 mL PBS and immediately taken to
be analysed on the flow cytometer. The samples were kept shielded
from light until analysis, since the DePsipherÔ agent is light
sensitive.
4.3.1.3. Data analysis. Analysis of the samples were performed on a
BD FacsCaliburÒ flow cytometer [Becton Dickinson, San Jose (USA)],
equipped with a 15 mV 488 nm, air-cooled argon-ion laser. Cells
were gated in a forward scatter/side scatter plot to exclude debris.
Green and red fluorescence were detected in the corresponding FL-
1 and FL-2 photomultipliers through 530 nm (FITC) or 585 nm (PE/
PI) bandpass filters respectively. In generating the data, the flow
cytometer was set to analyse 50 000 events/cells. Figs. 1e3
(Supplementary Material) contains representative pseudo-colour
graphs of every sample, which was generated using FlowJoÒ,
based on the data of the flow cytometry. On the x-axis the FL-1
(green) fluorescence intensity is plotted, and on the y-axis the FL-
2 (red) fluorescence intensity is plotted. Both intensities are
measured in MFI (mean fluorescence intensity). Each spot is
representative of a single cell analysed with the flow cytometer. The
graphs are divided into four separate quadrants, each containing a
population of cells, which exhibit the same characteristics. In the
upper left quadrant (Q1) lies all the cells which emit only red
fluorescence, whereas the cells which emit only green fluorescence
can be found in the lower right quadrant (Q4). The cells which emit
both wavelengths of fluorescence can be found in the upper right
quadrant (Q2), and the cells emitting neither of the two in the
lower left quadrant (Q3). The following can be concluded by taking
note of the explanations of the different quadrants as well as the
characteristics of cells in each of these quadrants: As healthy cells
contain both the monomeric and polymeric form of the cation dye,
and would thus have intensity in both the FL1 and FL2 channels
used to analyse the samples, the MFI values of these cells will lie in
Q2 (see control experiment 4). The cells with MFI values in Q1 have
only red fluorescence, which might be due to low concentrations of
the dye inside the cell, indicating that all the monomers were
polymerised. All these cells can thus also be said to be healthy,
together with these found in Q2. Cells wherein apoptosis have been
induced only contain the monomeric form of the dye, will not
colour red at all, and will thus have MFI values in Q4. The cells lying
in Q3 have not been dyed successfully or intensively enough, and
have thus not coloured either red or green. From the pseudo-colour
graphs it is clear that there was a difference in the number of
apoptotic/healthy cells present in the different samples. Control
experiment 1 had the least MFI values in Q1þQ2, and control
experiment 4 the most. The MFI values of the other samples were
either between the values of control experiments 1 and 4, or just
above that of control experiment 4 (All pseudo-colour graphs are
supplied in the Supplementary Material).
4.3.2. NMDA and VGCC assays
4.3.2.1. Animals. The study protocol was approved by the Ethics
Committee for Research on Experimental Animals of the University
of the Western Cape (SRIRC 2012/06/13). Adult Winstar rats were
sacrificed by decapitation and the brain tissue was removed and
kept on ice for homogenation. After homogenation, the alliquoted
brain homogenate was immediately used in the subsequent assay.
4.3.2.2. Methods. The fluorescent ratiometric indicator, Mag-Fura-
2/AM, and a Bio-TekÒ fluorescence plate reader were used to
evaluate the influence of the test compounds on calcium homeo-
stasis via the VGCC and NMDA receptor channels utilizing murine
synaptoneurosomes. Preparation of synaptoneurosomes, solutions
and experimental techniques were similar to those of published
studies [26,44a–d]. All data analysis, calculation and graphs were
done using Prism 6.0Ò (GraphPhad, La Jolla, CA). Data analysis was
carried out using the Student Newman Keuls multiple range test
and the level of significance was accepted at p < 0.05.
4.3.2.3. Preparation of synaptoneuromes. Adult Winstar rats were
used. Rats were sacrificed by decapitation, and the whole brains
were removed. Whole-brain synaptoneurosomes were prepared by
the techniques of Hollingsworth et al. [45], modified slightly. The
brain from one rat was homogenized (8 strokes by hand using a
glass homogenizer) in 30 mL of ice-cold incubation buffer (NaCl,
118 mM; KCl, 4.7 mM; MgCl, 1.18 mM; CaCl2 0.1 mM; HEPES, 20 mM
and glucose, 30.9 mM pH 7.4). From this step forward the ho-
mogenate was kept ice-cold at all times to minimize proteolysis
throughout the isolation procedure. The tissue suspension was
placed in a 50 mL polycarbonate tube and then centrifuged for
5 min at 1000 g and 0 ꢁC using a Labofuge 20Ò centrifuge. After
centrifugation, the supernatant was decanted into a 50 ml poly-
carbonate tube and placed on ice. The supernatant was then
divided into 2 mL eppendorf vials whose weight had been previ-
ously recorded. This was followed by a second centrifugation at
15000 g for 20 min at 0 ꢁC. The supernatant was discarded and the
mass of the resulting pellet was calculated. Sufficient calcium-free
buffer (NaCl, 118 mM; KCl, 4.7 mM; MgCl, 1.18 mM; HEPES, 20 mM

F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
133
and glucose, 30.9 mM pH 7.4) was added to obtain a 3 mg/ml
protein concentration (protein yield is approximately 10 mg/g of
tissue) [45]
4.3.2.8. Percentage inhibition calculations. The data obtained from
the fluorescent readings of each well were expressed in the form of
a ratio (340 nm reading/380 nm reading). This ratio indicated the
net movement of Ca2þ ions across the membrane as it represents
the ratio between unbound-Ca2þ and Ca2þ-bound to Fura-2 AM.
The average ratio over a 10 s interval after 1 min of stimulation was
then subtracted from the average ratio of over the last 10 s interval
to give the net change in Ca2þ movement (Nc) across the mem-
brane over the 5 min period. This calculation was performed for
each individual well and the averages of wells containing the same
test compound were calculated (NcAve). These averages were used
to calculate the percentage inhibition of the test compounds rela-
tive to the control by use of the following equation:
4.3.2.4. General procedure for loading FURA-2 AM and incubating
test compounds. Experiments were carried out at 37 ꢁC and fluo-
rescence was measured with a flourescent plate reader (Bio-TekÒ).
1990
allowed to reach room temperature, thereafter 10
(1 mM in DMSO) was added to produce a final concentration of
M. Synaptoneurosomes were then incubated at 37 ꢁC for 30 min
ml of synaptoneurosome suspension prepared above was
ml of Fura-2 AM
5
m
after which the suspension was centrifuged on a desktop centrifuge
at 7000 g for 5 min and the supernatant decanted to remove all
extracellular Fura-2 AM. The resulting pellet was resuspended in
2 mM CaCl2 containing buffer to obtain a final protein concentra-
tion of 0.6 mg/ml.
% inhibition ¼ ½NcAveðControlÞ ꢀ NcAveðTest compoundÞꢄ
ꢂ =NcAveðControlÞ ꢂ 100 %
(2)
4.3.2.5. Measurement of intracellular calcium. For the screening test
10 mM stock solutions of the compounds in DMSO were prepared,
4.4. Monoamine oxidase B inhibition assay
with the control containing 1% DMSO and no test compound. 2
the individual stock solutions were added to a 96 well plate in
triplicate followed by the addition of 200 l of synaptoneurosomal-
Fura-2 AM solution prepared above. This gave rise to a final con-
centration of 100 M of the compounds. The 96 well plate was
ml of
The mitochondrial fraction of baboon liver tissue, as source of
the MAO-B enzyme, was isolated as described previously by Salach
& Wyler, 1987 [46], and stored at ꢀ70 ꢁC. Following addition of an
equal volume of sodium phosphate buffer (100 mM, pH 7.4) con-
taining glycerol (50%, w/v) to the mitochondrial isolate, the protein
concentration was determined by the method of Bradford using
bovine serum albumin as reference standard [47]. In the current
m
m
shaken and incubated for 30 min at 37 ꢁC and used immediately
after incubation. The measurement was then performed at 37 ꢁC in
a 96 well plate using dual wavelength excitation at 340 nm and
380 nm. The resting fluorescence was measured at 510 nm after
which the changes in fluorescence intensity following the addition
study, MMTP (Km ¼ 68.3 ꢃ 1.60
mM for baboon liver MAO-B) served
as substrate for the inhibition studies.
of 10 ml depolarizing solution using auto-injectors was recorded
over a period of 5 min. The changes in fluorescence indicated the
effect of the test compound on calcium flux.
The enzymatic reactions were conducted in sodium phosphate
buffer (100 mM, pH 7.4) and contained MMTP (50 mM), the mito-
chondrial isolate (0.15 mg protein/mL) and various concentrations
of the test compounds, spanning at least three orders of magnitude
(0.3e300
this study were 0.3
300 M. The final volume of the incubations was 500
solutions of the inhibitors were prepared in DMSO and were added
to the incubation mixtures to yield a final DMSO concentration of
4% (v/v) as concentrations higher than 4% are reported to inhibit
MAO-B [48]. The reactions were incubated at 37 ꢁC for 10 min and
m
M). The concentration of the test compounds used in
M, 1 M, 3 M, 10 M, 30 M, 100 M and
L. The stock
4.3.2.6. KCl mediated calcium stimulation. The wavelengths
selected were 340 nm and 380 nm for excitation and 510 nm for
emission, with a runtime of 5 min with 5 ms intervals. The test
compound was incubated for 30 min at 37 ꢁC and used immediately
after incubation. The procedure was initiated and kept at 37 ꢁC. At
m
m
m
m
m
m
m
m
10 s into the recording 10 ml of KCl (140 mM) depolarization solu-
tion (5.4 mM NaCl, 140 mM KCl, 10 mM NaHCO3, 1.4 mM CaCl2,
0.9 mM MgSO4, 5.5 mM Glucose monohydrate, 0.6 mM KH2PO4,
0.6 mM Na2HPO4, and 20 mM HEPES. pH adjusted to 7.4 with
NaOH) was added to the membrane preparation to depolarize the
synaptoneurosomes and activate the calcium flux (addition was
done using auto-injectors). Experiments were repeated three times
on different tissue preparations with three determinations in each
replicate.
then terminated by the addition of 10 mL perchloric acid (70%). The
samples were centrifuged at 16 000 g for 10 min, and the con-
centrations of the MAO-B generated product, MMDPþ [49], were
measured spectrophotometrically at 420 nm (ε ¼ 25 000 Mꢀ1) in
the supernatant fractions.
The enzyme inhibition activities of the test compounds were
determined using Equation (2) to calculate the inhibition potencies
of the compounds. These potencies are expressed as percentage
inhibition (% inh) of the enzyme. In determining these values, the
concentration of MMDPþ produced in the absence of a test com-
pound (C0) and the concentration of MMDPþ produced by MAO-B
in the presence of a maximal concentration of the test compound
(C300) were utilised. All determinations were conducted in dupli-
cate, yielding the percentage inhibition as an average of the two
sets of data.
4.3.2.7. NMDA/glycine mediated calcium stimulation. The wave-
lengths selected were 340 nm and 380 nm (excitation) and 510 nm
(emission) with a runtime of 5 min with 5 ms intervals. The test
compound was incubated for 30 min at 37 ꢁC and used immediately
after incubation. The procedure was initiated and kept at 37 ꢁC. At
10 s into the recording 10 ml of NMDA/Glycine (0.1 mM) depolari-
zation solution (0.1 mM CaCl2.2H2O, 0.1 mM NMDA, 0.1 mM
Glycine, 118 mM NaCl, 4.7 mM KCl, 30.9 mM Glucose monohydrate
and 20 mM HEPES. pH adjusted to 7.4 with NaOH) was added to the
membrane preparation to depolarize the synaptoneurosomes and
activate the calcium flux (Addition was done using auto-injectors).
The addition of NMDA (0.1 mM) and Gly (0.1 mM) resulted in
activation of NMDAR mediated calcium flux. Experiments were
repeated three times on different tissue preparations with three
determinations in each replicate.
% inhibition ¼ 100 ꢀ ½ðC300 ꢂ 100Þ=C0ꢄ
(3)
4.5. Molecular modelling
Computer-assisted docking were carried out using the
CHARMm force field and the human MAO-B crystal structure (PDB
ID: 2V5Z) [37], which were recovered from the Brookhaven Protein

134
F.T. Zindo et al. / European Journal of Medicinal Chemistry 80 (2014) 122e134
[14] J.P. Finberg, J. Wang, K. Bankiewich, J. Harvey-White, I.J. Kopin, D.S. Goldstein,
Journal of Neural Transmission Supplement 52 (1998) 279.
[15] M.B.H. Youdim, Y.S. Bakhle, British Journal of Pharmacology 147 (2006) S287.
[16] A. Nicotra, F. Pierucci, H. Parvez, O. Senatori, Neurotoxicology 25 (2004) 155.
[17] J.S. Fowler, N.D. Volkow, G.J. Wang, J. Logan, N. Pappas, C. Shea, R. MacGregor,
Neurobiology of Aging 18 (1997) 431.
[18] B. Karolewicz, V. Klimek, H. Zhu, K. Szebeni, E. Nail, C.A. Stockmeier,
L. Johnson, G.A. Ordway, Brain Research 1043 (2005) 57.
[19] J.J. Chen, D.M. Swope, K. Dashtipour, Clinical Therapeutics 9 (2007) 1825.
[20] O. Bar-Am, O. Weinreb, T. Amit, M.B.H. Youdim, FASEB 19 (2005) 1899.
[21] P.H. Yu, B.A. Davis, A.A. Boulton, Journal of Medicinal Chemistry 35 (1992)
3705.
Database (www.rcsb.org/pdb). Docking simulations were per-
formed on the synthesised compounds (9e16) using Molecular
Operating Environment (MOE) [36] 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 including 58W and 47W retained (important for the
formation of a triad [LyseH2Oeflavin N(5)] and the hydrophilic
section required for recognition and directionality of the substrate
amine functionality) [50] as seen with safinamide (Fig. 4). (3) The
ligand molecules were constructed using the builder module and
were energy minimized. (4) The active site was generated using the
MOE-Alpha Site Finder. (5) Ligands were docked within the MAO-B
active site 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 further energy minimization using
CHARMm force field with all possible bond rotations and chiralities
explored. To determine the accuracy of this docking protocol, the
co-crystallised ligand, safinamide (PDB ID: 2V5Z), was re-docked
into the MAO-B active site. This procedure was repeated three
times and the best ranked solutions of safinamide exhibited an
[22] S. Mandel, O. Weinreb, T. Amit, M.B.H. Youdim, Brain. Research Reviews 48
(2005) 379.
[23] J. Joubert, W.J. Geldenhuys, C.J. Van der Schyf, D.W. Oliver, H.G. Kruger,
T. Govender, S.F. Malan, Chemmedchem 7 (2012) 375.
[24] J. Zah, G. Terre’blanche, E. Erasmus, S.F. Malan, Bioorganic
& Medicinal
Chemistry 11 (2003) 3569.
[25] K.B. Brookes, P.W. Hickmott, K.K. Jutle, C.A. Schreyer, SAJC 45 (1992) 8.
[26] J. Joubert, S. Van Dyk, I.R. Green, S.F. Malan, Bioorganic & Medicinal Chemistry
19 (2011) 3935.
[27] J. Joubert, S. Van Dyk, I.R. Green, S.F. Malan, European Journal of Medicinal
Chemistry 46 (2011) 5010.
[28] H.J.R. Lemmer, J. Joubert, S. van Dyk, F.H. van der Westhuizen, S.F. Malan,
Medicinal Chemistry. [Medicinal Chemistry (Shariqah (United Arab Emir-
ates))] 8 (2012) 361.
[29] J. Joubert, H. Samsodien, Q.R. Baber, D.L. Cruickshank, M.R. Caira, S.F. Malan,
Journal of Chemical Crystallography 44 (2014) 194.
[30] Mitochondrial Membrane Potential Disruption Assay. DePsipherÔ assay kit.
http://www.rndsystems.com/product_detail_objectname_mitochondrial_
membrane_disruption.aspx (accessed 31.03.14.).
[31] C.J. Van der Schyf, G.J. Squier, W.A. Coetzee, Pharmacological Research Com-
munications 18 (1986) 407.
[32] E.B. Hollingsworth, E.T. McNeal, J.L. Burton, R.J. Williams, J.W. Daly,
C.R. Creveling, The Journal of Neuroscience: The Official Journal of the Society
for Neuroscience 5 (1985) 2240.
[33] J.J. Van der Walt, C.J. Van der Schyf, J.M. Van Rooyen, J. De Jager, M.N. Van
Aarde, South African Journal of Science 84 (1988) 448e450.
[34] A.E. Medvedev, A.S. Ivanov, N.S. Kamyshanskaya, A.Z. Kirkel, T.A. Moskvitina,
V.Z. Gorkin, N.Y. Li, V.Y. Marshakov, Journal of. Biochemistry and Molecular
Biology 36 (1995) 113.
ꢀ
RMSD value of 1.34 A from the position of the co-crystallised ligand.
ꢀ
In general, RMSD values smaller than 2.0 A generally indicate that
the docking protocol is capable of accurately predicting the binding
orientation of the co-crystallised ligand [50,51]. This protocol was
thus deemed to be suitable for the docking of inhibitors into the
active site model of MAO-B.
Acknowledgements
We are grateful to the North-West University, University of the
Western Cape, Medical Research Council of South Africa and the
National Research Foundation of South Africa for financial support.
[35] J.P. Petzer, N.C. Castagnoli, M.A. Schwarzschild, J. Chen, C. Van der Schyf,
Journal of Neurotherapy 6 (2009) 141.
[36] Molecular Operating Environment (MOE), Version 2011.10. http//www.
chemcomp.com.
[37] C. Binda, J. Wang, L. Pisani, C. Caccia, A. Carotti, P. Salvati, D.E. Edmondson,
A. Mattevi, Journal of Medicinal Chemistry 50 (2007) 5848.
[38] Y. Tsugeno, I. Hirashiki, F. Ogata, A. Ito, Journal of Biochemistry 118 (1995)
974e980.
[39] A.L. Maycock, R.H. Abeles, J.I. Salach, T.P. Singer, Biochemistry 15 (1976) 114.
[40] D.J. Mitchell, D. Nikolic, E. Rivera, S.O. Sablin, S. Choi, R.B. van Breemen,
T.P. Singer, R.B. Silverman, Biochemistry 40 (2001) 5447.
[41] R.C. Cookson, E. Crundwell, R.R. Hill, J. Hudec, Chemistry and Industry (1958)
1003.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.039
References
[42] A.P. Marchand, S.C. SuriI, A.D. Earlywine, D.R. Powell, D. Van der Helm, The
Journal of Organic Chemistry 49 (1984) 670.
[43] T.G. Dekker, D.W. Oliver, SAJC 32 (1979) 45.
[1] H.P. Rang, M.M. Dale, J.M. Ritter, Rang and Dale’s Pharmacology, Elsevier,
Philadelphia, 2007, p. 508 (Chapter 35).
[2] T. Araki, T. Kumagai, K. Tanaka, M. Matsubara, H. Kato, Y. Itoyama, Y. Imai,
Brain: A Journal of Neurology 918 (2001) 176.
[3] (a) T. Alexi, C.V. Borlongan, R.L.M. Faull, C.E. Williams, R.G. Clark,
P.D. Gluckman, P.D. Gluckman, P.E. Huges, Progress in Neurobiology 60 (2000)
409;
(b) J.G. Greene, J.T. Greenamyre, Progress in Neurobiology 48 (1996) 613.
[4] J.U. Schweichel, H.J. Merker, Teratology 7 (1973) 253.
[5] M.P. Mattson, Nature Reviews 1 (2000) 120.
[44] (a) S.J. Benavide, Y. Claustre, B. Scatton, The Journal of Neuroscience: the
Official Journal of the Society for Neuroscience 8 (1988) 3607;
(b) W.J. Geldenhuys, S.F. Malan, J.R. Bloomquist, C.J. Van der Schyf, Bioorganic
& Medicinal Chemistry 15 (2007) 1525;
(c) K. Resch, W. Imm, E. Ferber, D.F.H. Wallach, H. Fischer, Die Natur-
wissenschaften 58 (1971) 220;
(d) A.K. Stout, I. Reynolds, The Journal of Neuroscience: the Official Journal of
the Society for Neuroscience 89 (1999) 91.
[6] N.J. Holbrook, G.R. Martin, R.A. Lockshin (Eds.), Cellular Aging and Cell Death,
WileyeLiss, New York, 1996, p. 319.
[7] A.H. Wyllie, J.F.R. Kerr, A.R. Currie, International Review of Cytology 68 (1980)
251.
[8] M. Arundine, M. Tymianski, Molecular mechanisms of calcium-dependent
neurodegeneration in excitotoxicity, Cell Calcium 34 (4e5) (2013) 325.
[9] M. Arundine, M. Tymianski, Cellular and Molecular Life Sciences: CMLS 61
(2004) 657.
[10] W.J. Geldenhuys, S.F. Malan, T. Murugensan, C.J. Van der Schyf,
J.R. Bloomquist, Bioorganic & Medicinal Chemistry 12 (2004) 1799.
[11] M.B. Youdim, G.G. Collins, M. Sandler, A.B. Bevan Jones, C.M. Pare,
W.J. Nicholson, Nature 236 (1972) 225.
[45] E.B. Hollingsworth, E.T. McNeal, J.L. Burton, R.J. Williams, J.W. Daly,
C.R. Creveling, The Journal of Neuroscience: the Official Journal of the Society
for Neuroscience 5 (1985) 2240.
[46] J.I. Salach, W. Weyler, Methods in Enzymology 142 (1987) 627.
[47] M.M. Bradford, Analytical Biochemistry 72 (1976) 248.
[48] C. Gnerre, M. Catto, F. Leonetti, P. Weber, P.A. Carrupt, C. Altomare, A. Carotti,
B.J. Testa, Journal of Medicinal Chemistry 43 (2000) 4747.
[49] H. Inoue, K. Castagnoli, C.J. Van der Schyf, S. Mabic, K. Igarashi, N. Castagnoli,
Journal of Pharmacology and Experimental Therapeutics 291 (1999) 856.
[50] C. Binda, M. Li, F. Hubalek, N. Restelli, D.E. Edmondson, A. Mattevi, PNAS 100
(2003) 9753.
[51] J. Boström, J.R. Greenwood, J. Gottfries, Journal of Molecular Graphics &
Modelling 21 (2003) 449.
[12] G.G. Collins, M. Sandler, E.D. Williams, M.B. Youdim, Nature 225 (1970) 817.
[13] D.A. Di Monte, L.E. DeLanney, I. Irwin, J.E. Royland, P. Chan, M.W. Jakowec,
J.W. Langston, Brain Research 738 (1996) 53.