Synthesis, evaluation and application of polycyclic fluorescent analogues as N-methyl-d-aspartate receptor and voltage gated calcium channel ligands

Article abstract of DOI:10.1016/j.ejmech.2011.08.008

A series of polycyclic fluorescent ligands were synthesised and evaluated in murine striatal synaptoneurosomes for N-methyl-d-aspartate receptor (NMDAR) mediated calcium flux inhibition and inhibition of calcium influx through voltage gated calcium channe

Full text of DOI:10.1016/j.ejmech.2011.08.008

European Journal of Medicinal Chemistry 46 (2011) 5010e5020
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, evaluation and application of polycyclic fluorescent analogues as
N-methyl- -aspartate receptor and voltage gated calcium channel ligands
D
,
,b,
Jacques Joubert a b, Sandra van Dyk a, Ivan R. Green c, Sarel F. Malan a
*
a Pharmaceutical Chemistry, North-West University, Private Bag 6001, Potchefstroom 2520, South Africa
b School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
c Department of Chemistry, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of polycyclic fluorescent ligands were synthesised and evaluated in murine striatal synapto-
neurosomes for N-methyl- -aspartate receptor (NMDAR) mediated calcium flux inhibition and inhibition
Received 18 July 2011
Received in revised form
4 August 2011
Accepted 5 August 2011
Available online 11 August 2011
D
of calcium influx through voltage gated calcium channels (VGCC). Amantadine (a) and N-(1-adamantyl)-
1,3-propanediamine (c) substituted with 1-cyanoisoindole (3), indazole (5), dinitrobenzene (7, 8), dansyl
(9, 10) and coumarin (11) moieties showed moderate to high inhibition of the NMDAR. A high degree of
VGCC inhibition was observed for the cyanoisoindole compounds (3, 4) the dansyl compounds (9, 10) and
the coumarin compound (12). Fluorophores conjugated to hydroxy-4-aza-8-oxoheptacyclotetradecane
(13, 14) did not exhibit any significant VGCC inhibition, but the indazole conjugate (14) showed prom-
ising NMDAR activity. Dose response curves were calculated for selected NMDAR inhibitors (8e11) and
N-[3-(1-adamantylamino)propyl]-5-dimethylaminonaphthalene-1-sulfonamide (10) exhibited the
highest activity of the novel compounds. Compound 10 was further used as a fluorescent NMDAR ligand
in a fluorescent competition assay utilizing MK-801, NGP1-01 and amantadine as known NMDAR
inhibitors to demonstrate the possible applications of the novel fluorescent compounds. These small
molecule fluorescent ligands can be considered as possible pharmacological tools in assay development
and/or other investigations in the study of neurodegeneration.
Keywords:
Polycyclic
Amantadine
NMDAR
Calcium channels
Fluorescent ligands
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
disorders such as PD and AD [4]. Overstimulation of the NMDAR byan
excess of the endogenous neurotransmitter glutamate during path-
Neurodegeneration and the development of neuroprotective
agents have in recent years become an increasingly important focus
of research. Much time and effort have gone into establishing the
cause and markers of disorders like Parkinson’s disease (PD) and
Alzheimer’s disease (AD) as they negatively influence the quality of
life of millions of people around the world [1]. Neurodegeneration
can be described as the process by which certain neurons in the
central nervous system (CNS), and especially the brain, are damaged
by a variety of mechanisms [2]. These disorders are characterized by
progressive and irreversible loss of neurons from specific regions of
the brain [3]. Amongst the mechanisms implicated to be responsible
for neuronalcelldeath, we havefocused our interestonexcitotoxicity.
ological conditions leads to excessive influx of extracellular calcium
into neuronal cells resulting in cell death, a process known as exci-
totoxicity [5]. Calcium entry through voltage gated calcium channels
(VGCC) also contributes to calcium overload and mitochondrial
disruption that leads to the recruitment or release of mediators
responsible for the activation of an apoptotic cascade and ultimately,
in cell death [4]. Several authors have described the link between
glutamate toxicity and calcium influx through the NMDAR and VGCC
[4e7]. The subsequent calcium overload and observed excitotoxicity
lead to the idea that glutamate toxicity participated in neuronal cell
death [6]. This link also established a specific rationale for targeting
theNMDARand/orVGCCforselectiveblockadeof extraneous calcium
fluxes, thus allowing subsequent reduction in glutamate-induced
calcium influx [7].
The N-methyl-D-aspartate receptor (NMDAR) has been suggested
as a drug target through its involvement in neurodegenerative
Several polycyclic compounds (Fig. 1) have been identified as
NMDAR antagonists. This includes the high-affinity noncompetitive
NMDAR antagonists (MK-801 and PCP) and low affinity uncom-
petitive NMDAR antagonists (amantadine, memantine and NGP1-
01) [8]. MK-801 and PCP bind to the PCP binding site located
* Corresponding author. School of Pharmacy, University of the Western Cape,
Private Bag X17, Bellville 7535, South Africa. Tel.: þ27 21959 3190; fax: þ27 21959
1588.
E-mail address: sfmalan@uwc.ac.za (S.F. Malan).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.008

J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
5011
CH3
site [21]. The greatest challenge in the design of fluorometric assays
lies in the development of a fluorescent ligand while maintaining
potency and accommodating steric demands of the fluorophore,
especially when the fluorescent moiety is conjugated to a small
molecule. Nonetheless, there have been several reports of successful
fluorophore-conjugation with small molecule ligands [22,23], and
recent advances in fluorometric assays utilising fluorescent ligands
[22] has encouraged pursuit of this approach.
H
N
NH2
NH2
H3C
O
NGP-101
Amantadine
Memantine
In our efforts to design a fluorescent polycyclic NMDAR and/or
calcium ligand we utilized the amantadine and 3-hydroxy-4-aza-8-
oxoheptacyclotetradecane polycyclic moieties as the NMDAR and/or
calcium channel inhibitors conjugated to various fluorescent ligands.
The adamantine and polycyclic moieties were selected because of
their known affinity for VGCC and uncompetitive NMDAR channel
antagonism which modulates rather than blocks the NMDAR
channel completely and allows normal neuronal functioning [6,9]. It
is hypothesized that these polycyclic moieties will be effective
scaffolds to conjugate to small molecule fluorescent compounds and
to use these molecules to gain a better understanding of the
mechanism of action involved in modulation of NMDAR and/or
calcium channel activity utilizing fluorescent high-throughput
assays.
HN
N
O
O
CH3
MK-801 (Dizocilpine)
PCP (Phencyclidine)
Pentacycloundecane
Fig. 1. Representative polycyclic structures as NMDAR and/or calcium channel
modulators [8,9].
within the ion-channel pore of the NMDAR [9]. This antagonism is
use dependent in that the PCP site is only accessible when the ion
channel pore is in an open, or activated state [9]. The block is
enhanced by increases in open channel probability. This phenom-
enon implies that compounds with pronounced use-dependency
will have greater affinity for the brain region where over-
stimulation occurs [10]. Once bound, the blocker can be trapped by
channel closure. Amantadine, memantine and NGP1-01 modulate
the NMDAR channel, rather than blocking it completelyas is the case
for the high-affinity noncompetitive NMDAR antagonists [6,9].
Thus, the low-affinity uncompetitive NMDAR channel antagonists
only modulate the NMDAR channel during excessive calcium flux
and remain in the channel long enough to reduce excessive calcium
influx, operating in a use dependent manner that allows for normal
neuronal functioning [9]. Because of this low affinity, amantadine
and NGP1-01 are speculated to leave the receptor site before the
channel closes [8e10]. NGP1-01 is described as having a dual
mechanism of action by blocking both the NMDAR and l-type
calcium channels, with its neuroprotective activity confirmed by
in vivo studies [11e16].
Radioligand binding techniques have been widely used to study
receptor pharmacology and physiology [17]. Geldenhuys et al.
described the characterisation of polycyclic compounds derived
from the pentacycloundecane structure and their interaction with
NMDAR by means of radioligand binding studies [8]. Despite the
usefulness and sensitivity of radioligand binding techniques, the
use of alternative methods (for instance fluorescent techniques) to
study receptor-ligand binding interactions may provide informa-
tion not readily accessible by conventional radioreceptor tech-
niques and circumvent some of the drawbacks, such as high cost,
disposal, health hazard and potential technical implications, asso-
ciated with this methodology [18]. Techniques to visualize physi-
ological or pathophysiological changes in living cells have become
increasingly important in biomedical sciences. Fluorescent probes
are excellent tools to analyze and clarify the roles of biomolecules
in living cells, affording high spatial and temporal resolution via
microscopic imaging. The development of tools for probing bio-
logical events has thus become an area of intense interest [19,20].
We report here the results of our efforts to design a polycyclic
fluorescent ligand for the NMDAR and possibly VGCC with the aim of
enabling development of fluorometric assays to assist in the quest
for effective neuroprotective strategies. Fluorometric assays offer
several advantages over their radiometric counterparts viz; to
determine properties like receptor internalization and sub cellular
localization, the thermodynamics and kinetics of ligand binding and
to assess the nature of the microenvironment of the ligand binding
2. Chemistry
The fluorescent ligands for this study were selected on the basis of
their spectroscopic properties and structural requirements for VGCC
and NMDARinhibition. These ligands were successfullyconjugated to
amantadine (a) and 3-hydroxy-4-aza-8-oxoheptacyclotetradecane
(e) by means of direct conjugation employing methods previously
described [24,25]. 3-Hydroxy-4-aza-8-oxoheptacyclotetradecane (e)
was synthesized by reacting 3-aminopropanol with the well-
described Cooksen diketone (d) [26,27] with subsequent intra-
molecular cyclisation and dehydration to form e [28]. To reduce
structural hindrances and achieve potentially greater affinity for the
VGCC and the NMDAR, we also synthesised an adamantine molecule
with
a 3-carbon spacer [N-(1-adamantyl)-1,3-propanediamine]
separating the conjugated fluorophore and the amantadine moiety.
N-(1-Adamantyl)-1,3-propanediamine (c) was synthesized by
Michael additionutilizing acrylonitrile to give 3-(1-adamantylamino)
propionitrile (b) and subsequent reduction of the nitrile with LiAlH to
form the desired primary amine (c, Fig. 2).
The fluorescent molecules utilized for evaluation of VGCC and
NMDAR activity included: N-methylanthranilic acid, indazole-3-
carboxylic acid, 1-fluoro-2,4-dinitobenzene, 1-cyanoisoindole, dan-
syl chloride and coumarin-3-carboxylic acid. These compounds were
conjugated to amantadine, N-(1-adamantyl)-1,3-propanediamine
and 3-hydroxy-4-aza-8-oxoheptacyclotetradecane by various
synthetic methods including activation chemistry, amination, ami-
dation and esterfication. The synthesised compounds (Table 1) were
all obtained as oils or amorphous solids from chromatography or
were crystallized from organic solvents. Structures were confirmed
using 1H and 13C NMR, IR and MS.
3. Pharmacology
NMDAR and VGCC activity of the test compounds were evaluated
on murine synaptoneurosomes utilizing the fluorescent ratiometric
calcium indicator, Mag-Fura-2/AM, and a Cary EclipseÒ fluorescence
spectrometer system [29]. All novel fluorescent compounds and
controls were tested at 100
mM, to identify active VGCC and/or
NMDAR calcium influxinhibitors. NMDAR dose response curves were
calculated for selected compounds which showed a high degree of
NMDAR inhibition. From these studies N-[3-(1-adamantylamino)
propyl]-5-dimethylaminonaphthalene-1-sulfonamide (10) was

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J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
LiAlH
1) NaHCO3(aq)
Diethyl ether
NH HCl 2) Acrylonitrile
2.
N
H
N
N
H
NH2
c
a
b
Benzene
-H2O
OH
THF
OH
+
H2N
OH
N
H
O
O
3-aminopropanol
O
d
O
N
O
N
OH
HO
e
Fig. 2. General reaction schemes for the synthesis of N-(1-adamantyl)-1,3-propanediamine (c) and 3-hydroxy-4-aza-8-oxoheptacyclotetradecane (e).
identified as a potent NMDAR inhibitor. Compound 10 was further
utilized as a fluorescent NMDAR ligand in a competition assay where
we utilized different polycyclic NMDAR inhibitors to compete with
compound 10 for binding of the NMDAR and demonstrate the
possible application(s) of these novel fluorescent ligands.
inhibitor NGP1-01. In the ratiometric imaging experiments using
Mag-fura-2AM as fluorescent calcium indicator, application of high
concentration potassium (140 mM) resulted in an increase in the
fluorescent ratio in the presence of extracellular calcium. This
increase in fluorescence indicates an influx of calcium through VGCC.
The reference compounds nimodipine, a commercially available
dihydropyridine l-type calcium channel blocker, NGP1-01 and
amantadine exhibited statistically significant average decreases in
fluorescence, and subsequent decrease in calcium influx, 87.71%,
29.88% and 13.87% respectively, when compared to the KCl control
treated synaptoneurosomes (Fig. 3). Synaptoneurosomes incubated
with test compounds 3, 4, 9, 10 and 12 showed the most significant
inhibition of calcium influx with decreases in fluorescence of 45.76%,
4. Results and discussion
4.1. VGCC calcium influx inhibition
The amantadine and 3-hydroxy-4-aza-8-oxoheptacyclotetradec-
ane fluorescent compounds were evaluated for VGCC activity
because of their structural similarities towards the calcium channel
Table 1
Fluorescent derivatives conjugated to amantadine (a), N-(1-adamantyl)-1,3-propanediamine (c) and 3-hydroxy-4-aza-8-oxoheptacyclotetradecane (e).
O
a
c
e
R
N
H
R1
N
R2
R2 = 13, 14
R = 1, 3, 5, 7, 9, 11
R1 = 2, 4, 6, 8, 10, 12
H
N
O
NC
N
O
HN
N
H
N
N
H
5, 6
1, 2
3, 4
NO2
O
O
H
N
O
H
N
S
N
N
H
O
NO2
O
7, 8
9, 10
11, 12
O
O
HN
O
O
N
N
H
13
14

J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
5013
fluorescence of 24.65%,15.21% and 16.38%. Test derivatives 1, 2, 5, 6, 7
and 8 had weak VGCC activity.
We found that the increase in chain length between the
amantadine moiety and the fluorophores generally lead to an
increase in calcium channel blocking activity, whereas direct
attachment of the fluorophores to amantadine noticeably reduced
the activity for calcium channel blockage. For the cyanoisoindole
compounds (3 and 4) and the dansyl compounds (9 and 10) an
increase in chain length led to a significant (p < 0.05) increase in
activity (Fig. 3 and Table 2). The same tendency (though not
significant; p > 0.05) was observed for the anthranilic (1, 2), inda-
zole (5, 6), and the coumarin (11, 12) derivatives. This correlated
with the study by Liebenberg et al. which found that an increase in
chain length between the polycyclic and aromatic moieties led to
an increase in calcium channel antagonistic activity [30]. It can be
argued that an increase in chain length might indicate that a deeper
immersion into the calcium channel may be obtained for stronger
interaction with the putative binding site. The tetradecane fluo-
rescent moieties (13, 14) have low calcium channel activity, only
slightly better than amantadine. The compounds with significant
VGCC activity (Table 2) all contain bicyclic aromatic functionalities
(3, 4, 9, 10 and 11) in their structures, showing a significant
increased degree of VGCC inhibition when compared to their
mono-aromatic counterparts (1, 2, 7, 8 and 13). The isondole (3, 4),
dansyl (9, 10) and coumarin (11) functional moieties contained in
these structures are described throughout literature as privileged
structures. Further development of these structures however needs
to be conducted to elaborate on their true potential as VGCC
Fig. 3. Screening of test compounds (100
m
M, n ¼ 9) for antagonism of KCl-mediated
calcium influx via VGCC into murine striatal synaptoneurosomes. Each bar represents
mean percentage of control values ꢀ SEM. Abbreviations are: control (C), nimodipine
(N), NGP1-01 (P) and amantadine (A). Statistical analysis (ANOVA) was performed on
raw data, with asterisks signifying significant inhibitory effect [(*) p < 0.05, (**)
p < 0.01 and (***) p < 0.001)] when compared to the control (100%).
54.08%, 55.23%, 63.26%and 32.01%, respectively. All these compounds
demonstrated significantly better inhibition (p < 0.05) than the
reference polycyclic compounds NGP1-01 and amantadine (Fig. 3 and
Table 2), with compound 10 exhibiting the highest activity (63.26%).
Compounds 11, 13 and 14 also had notable decreases (p < 0.05) in
Table 2
Summary of effects the polycyclic fluorescent derivatives and related compounds on KCl induced calcium influx inhibition (at 100 mM), inhibition of NMDA/glycine (N/G)
induced calcium influx (at 100
mM) and fluorescent properties of the compounds.
O
R
N
H
R
N
R
Compounds 1, 3, 5, 7, 9, 11
Compounds 2, 4, 6, 8, 10, 12
Compounds 13, 14
Compound
R (Fluorophore)
% Ca2þ-flux after KCl stimulationa,c
% Ca2þ -flux after N/G stimulationa,c
cLog P
l
ex (nm)b
l
em (nm)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
N-Methylanthranilamido
N-Methylanthranilamido
1-Cyanoisoindole
1-Cyanoisoindole
1H-Indazole-3 carboxamido
1H-Indazole-3 carboxamido
2,4-Dinitrophenyl-1-amine
2,4-Dinitrophenyl-1-amine
Dansylamido
Dansylamido
Coumarin-3-carboxamido
Coumarin-3-carboxamido
2-Methylaminobenzoate
1H-Indazole-3-carboxylate
e
e
e
e
e
e
e
93.20 ꢀ 2.69*
87.91 ꢀ 1.12**
54.24 ꢀ 5.45***
45.92 ꢀ 2.03***
93.43 ꢀ 2.34*
86.91 ꢀ 0.11*
89.14 ꢀ 4.10*
88.50 ꢀ 2.09*
44.77 ꢀ 4.70***
36.74 ꢀ 6.47***
75.35 ꢀ 11.0*
67.99 ꢀ 9.37**
84.79 ꢀ 5.76*
83.62 ꢀ 2.54**
12.29 ꢀ 1.66***
86.13 ꢀ 3.63*
n.d.
96.66 ꢀ 4.89
88.02 ꢀ 4.25
84.01 ꢀ 5.53*
86.41 ꢀ 4.12
83.70 ꢀ 0.99*
95.19 ꢀ 6.67
80.75 ꢀ 6.27*
76.59 ꢀ 9.55*
78.05 ꢀ 5.41*
62.97 ꢀ 8.21*
79.71 ꢀ 4.42*
86.77 ꢀ 5.71
86.41 ꢀ 4.11
77.89 ꢀ 8.23*
n.d.
83.10 ꢀ 2.98*
64.04 ꢀ 2.62**
86.63 ꢀ 1.03**
80.71 ꢀ 1.44**
90.99 ꢀ 6.20
77.29 ꢀ 2.11*
4.24 ꢀ 0.40
4.12 ꢀ 0.42
4.88 ꢀ 0.35
3.32 ꢀ 0.42
3.02 ꢀ 0.43
3.14 ꢀ 0.40
5.54 ꢀ 0.32
5.13 ꢀ 0.35
5.54 ꢀ 0.38
5.42 ꢀ 0.40
3.53 ꢀ 0.75
3.41 ꢀ 0.75
2.63 ꢀ 0.72
1.53 ꢀ 0.72
3.85 ꢀ 0.59
2.22 ꢀ 0.24
3.68 ꢀ 0.39
1.68 ꢀ 0.36
2.10 ꢀ 0.28
2.10 ꢀ 0.26
0.33 ꢀ 0.60
366
360
358
360
340
340
396
390
334
324
364
360
366
330
e
415
420
395
400
400
405
449
430
517
455
407
410
415
410
e
Nimodipine
Amantadine
MK-801
NGP1-01
c
d
e
e
e
e
e
70.12 ꢀ 2.69***
63.28 ꢀ 4.26***
89.33 ꢀ 3.54*
87.24 ꢀ 2.14*
e
e
e
e
e
e
e
e
Asterisks signifying statistical significance, (*) p < 0.05, (**) p < 0.001 and (***) p < 0.0001.
a
% Control ꢀ SEM (n ¼ 3).
b
At 10ꢁ5 M in DMSO at 25 ꢂC;
l
ex ¼ excitation
l;
l
em ¼ emission ; n.d. ¼ not determined.
l
c
Contol was taken as 100% calcium influx.

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J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
inhibitors and to develop structure activity relationships for this
series of fluorescent compounds (Fig. 3 and Table 2).
fluorescent molecules showed enhanced inhibition compared to
that of the references, NGP1-01 and amantadine (Fig. 4 and
Table 2), with compound 10 (37.03%) exhibiting the highest inhi-
bition of NMDAR calcium influx of the novel inhibitors, comparable
to MK-801 (35.96%). Compounds 2, 4, 12 and 13 exhibited slight,
though not statistically significant NMDAR inhibition of between
11.98% and 13.59%. Compounds 1 and 6 showed no discernable
NMDAR inhibitory activity. Dose-response curves were constructed
(Fig. 5) for selected active NMDAR inhibitors (8e11). The solubility
of these novel compounds however became a limiting factor at
higher concentrations and compounds were only tested to
Compounds 9 and 10, with high affinity for the voltage gated
calcium channels, have long wavelength fluorescence (334/517 nm
and 324/455 nm, respectively), high Stokes shifts (183 and 131,
respectively) and strong affinity for calcium channels (Table 2).
Compounds 3 and 4 also show promise as possible fluorescent
ligands for the calcium channel but have short fluorescent wave-
lengths (358/395 nm and 360/400 nm, respectively) and low Stokes
shifts (37 and 40, respectively), which could be unfavorable in some
fluorescent imaging techniques. Compounds 9 and 10 could thus be
utilized as potential fluorescent ligands for imaging of calcium
channels using modern fluorescent imaging techniques.
Intermediates b, c and e were also screened for calcium channel
activity and interesting findings were observed. N-(1-Adamantyl)-
1,3-propanediamine (c) and 3-hydroxy-4-aza-8-oxoheptacyclote-
tradecane (d) showed modest calcium channel inhibitory activity,
weaker than all reference test compounds (Table 2). Importantly,
intermediate 3-(1-adamantylamino)propionitrile (b) exhibited
a high degree of calcium influx inhibition (36.72% decrease in fluo-
rescence) superior to NGP1-01 and amantadine. This could indicate
that the nitrile group was involved in favorable binding interactions
with calcium channels and reduction thereof to the secondary amine
(compound c) drastically decreased activity. Both compounds 3 and 4
contain nitrile groups and exhibited high calcium channel blocking
activity. These important findings mayleadto furtherdevelopmentof
compounds derived from nitrile containing leads to develop potent
calcium channel inhibitors.
a maximum concentration of 100
From this data it was clear that compound 10 had the highest
activity at all concentrations (Fig. 5) with an IC50 value of 0.27
and maximum inhibition of 37.41% at a concentration of 100
mM before precipitation occurred.
mM
mM.
According to previous studies [8,31], an increase in affinity for the
NMDAR channel may be associated with an increase in hydropho-
bicity. Log P values were calculated (ACD Labs/ChemsketchÒ) for the
test compounds and were found to be higher than that of the
reference compounds (Table 2). The highest Log P values were pre-
dicted for compounds 7, 8, 9, and 10 (Log P > 5), some of the most
potent inhibitors amongst the novel inhibitors. The inhibition
observed for these derivatives correlates to the high lipophylicity,
with subsequent high permeability across biological membranes,
and favorable steric interactions of the fluorophores with the
NMDAR. In our study the compounds with a greater molecular size,
to a certain degree, lead to an increase in activity. This could however
also be attributed to the high lipophylicity of the novel compounds
and interactions of the fluorophores with certain residues in the
receptor binding pocket (Table 2 and Fig. 4). The increase in the chain
length of the adamantyl fluorophores in general gave more favour-
able steric and electronic interactions between the fluorophores and
the residues in the receptor binding pocket, than the more sterically
hindered directly conjugated moieties.
4.2. NMDAR calcium influx inhibition
The effects of the novel polycyclic fluorescent moieties of
representative experiments in the presence and absence of test
compounds and reference compounds (100
mM) on NMDA/glycine-
The compounds with biaromatic functionalities in their struc-
tures in general showed better NMDAR antagonistic activity, with
the dinitrobenzene derivatives also having significant NMDAR
activity (Table 2). The activity of the dinitrobenzene compounds
could probably be ascribed to additional interactions or inclusion of
other mechanisms of action for these derivatives in the NMDAR
channel. One hypothesis is that the nitro functionalities could
contribute to the S-nitrosylation of cysteine residues in the NMDAR
channels, thus increasing NMDAR inhibitory activity [32,33].
Further development of the high-affinity NMDAR inhibitors iden-
tified in this study will elaborate on structure activity relationships
of these compounds.
mediated calcium influx into synaptoneurosomes are shown in
Table 2 and Fig. 4. Incubation with the reference compounds MK-
801, NGP1-01 and amantadine showed statistically significant
average decreases in fluorescence of 35.96%, 13.37% and 16.9%
respectively, when compared to the NMDA/glycine control syn-
aptoneurosomes. A statistically significant (p < 0.05) decrease in
fluorescence was also observed for compounds 3, 5, 7, 8, 9, 10, 11
and 14 (16%e39%) when compared to the control. All of these
105
Compound 8
100
Compound 9
95
Compound 10
90
Compound 11
85
80
75
70
65
60
Fig. 4. Screening of test compounds (100
m
M, n ¼ 9) for antagonism NMDAR-mediated
-10
-9
-8
-7
-6
-5
-4
-3
calcium influx into murine striatal synaptoneurosomes. Each bar represents mean
percentage of control values ꢀ SEM. Abbreviations are: Control (C), MK-801 (M), NGP1-
01 (P) and amantadine (A). Statistical analysis was performed on raw data, with
asterisks signifying significant inhibitory effect [(*) p < 0.05, (**) p < 0.001] when
compared to the control (100%).
log[Compounds] (M)
Fig. 5. Inhibition curves of compounds 8e11 with meaningful NMDAR activity were
superimposed to compare their potencies at different concentrations.

J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
5015
to its relatively potent antagonism of NMDAR-stimulated calcium
influx into murine synaptoneurosomes (Table 2), long fluorescent
wavelength (324Ex/455Em) and high Stokes shift (131 nm). Both
amantadine and MK-801 binds to the PCP binding site deep within
the NMDAR channel [9,34]. The synthesised adamantine fluo-
rophores share the same polycyclic amine template and we
postulated that these compounds should also interact with the PCP
binding site within the ion channel of the NMDAR.
Results of the novel competition binding assay are depicted in
Figs. 6 and 7. MK-801 decreased the fluorescent intensity of the
experimental sample by 46.03%, indicating the equipotence of the
two molecules (Figs. 6 and 7). The displacement of 10 by amanta-
dine (36.30%) further confirms the binding of this fluorophore at
the PCP site. NGP1-01 has been reported to have no binding affinity
for the PCP binding site but to interact with a unique binding site,
different to that reported for MK-801 or PCP, in the NMDA receptor/
ion channel complex [8,12]. In our study NGP1-01 also competed
for binding with compound 10 (17%), but to a lesser extent to that of
amantadine and MK-801. This suggests that compound 10 probably
interacts with a further binding site, other than the PCP binding
site. One hypothesis for the decrease in fluorescent intensity of
compound 10 by NGP1-01 would be to suggest that the dansyl
moiety undergoes aromatic interaction with (an) aromatic amino
acid(s) located at the entrance of the NMDA receptor channel- the
site where NGP1-01 interacts [12]. Such an interaction would allow
the molecule to be ‘anchored’ in such a way that the adamantine
moiety can descend into the channel lumen to a depth allowed by
the linker between amantadine and the dansyl moiety. In support
of this hypothesis, the ability of MK-801 and amantadine to
compete with compound 10 for the PCP binding site may be a result
of its longer linkage, allowing the amantadine moiety to explore
deeper into the channel to reach the proximity of the PCP binding
site. This is probably the reason why the compounds incorporating
the linker in general (7, 8, 9 and 10, Table 2) showed better inhi-
bition of the NMDAR.
Fig. 6. Fluorescent intensities of NMDA/glycine-mediated competition assay utilizing
compound 10 (100 M) and known NMDAR inhibitors. Each bar represents mean
percentage values ꢀ SEM (n ¼ 9). Abbreviations are: MK-801 (M), NGP1-01 (P),
Amantadine (A) and (K) a combination of all three inhibitors (M, P and A). Statistical
analysis was performed on raw data, with asterisks indicating significant inhibitory
effect [(*) p < 0.05, (**) p < 0.001] when compared to the control (10).
m
Intermediates b, c and e were also screened for NMDAR activity
with 3-(1-adamantylamino)propionitrile (b) and 3-hydroxy-4-aza-
8-oxoheptacyclotetradecane (e) showing significant (19.29% and
22.71%, respectively) NMDAR inhibition (Table 2). 3-(1-
Adamantylamino)propionitrile (b) exhibited better activity than its
primary amine counterpart N-(1-adamantyl)-1,3-propanediamine
(c, 9.01%). This could indicate that the nitrile group is involved in
favorable binding interactions with the NMDAR. 3-Hydroxy-4-aza-
8-oxoheptacyclotetradecane exhibited good inhibitory activity
(22.71%) compared to the reference compounds and substitution
thereof with the 1H-indazole-3-carboxylate moiety (13) retained
activity (22.11%), whereas the 2-methylaminobenzoate (14) substi-
tution decreased activity (13.59%).
The combination of MK-801, amantadine and NGP1-01 resulted
in slight increase in displacement of compound 10 (55%) when
compared to the separate NMDAR inhibitors, confirming our
hypothesis that compound 10 binds to the PCP binding site as well
as to other sites on the NMDAR (Figs. 6 and 7). Compound 10 was
not displaced quantitively (100%) and this was ascribed to the
binding kinetics and equipotence of the compounds. Binding to
other cell structures also needs to be excluded in future studies.
4.3. NMDAR competition assay utilizing N-[3-(1-adamantylamino)
propyl]-5-dimethylamino-naphthalene-1-sulfonamide (10)
A competition assay was developed as proof of concept for the
potential use of these novel fluorescent polycyclic compounds in
biological applications. It could also confirm the binding site of
compound 10 when compared to known NMDAR antagonists.
Compound 10 was used as a fluorescent ligand for these assays, due
5. Conclusion
a
We described the synthesis, VGCC- and NMDAR-mediated
calcium influx inhibition studies, and demonstrated a possible
application for this novel group of amantadine and tetradecane
polycyclic fluorescent ligands. We have identified fluorescent
compounds with long wavelength fluorescence, high Stokes shifts
and affinity for the NMDAR and/or VGCC. Further investigations
need to be conducted on the displacement of compound 10 as
a novel promising ligand for channel research and utilizing the other
novel fluorescent NMDAR inhibitors discovered in these studies.
Additional fluorescent studies utilizing a fluorescent microscope or
confocal laser scanning microscopy could elaborate on the mecha-
nism of action. These compounds could also be used as direct
binding calcium channel probes and this will also be investigated for
future developments. Ultimately, the use of these ligands might
provide an attractive alternative to radioligand binding studies and
offer methods to directly study the kinetics of ligand receptor
interactions and facilitate in the development of new fluorescent
high-throughput assays. Ion channels have become important
targets in academic and pharmaceutical research alike.
200
b
150
c
100
d
e
50
400
450
500
550
600
Wavelength (nm)
Fig. 7. Fluorescent emission spectra of compound 10 (100
alone (a) (control) and in the presence of (100 M) NGP1-01 (b), amantadine (c), MK-
801 (d) and (e) consisting of all three NMDAR inhibitors (b, c, and d).
mM) with compound 10
m

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J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
Understanding how ion channels function is critical in determining
underlying disease mechanisms and drug interactions. These novel
fluorescent polycyclic compounds may also be utilised as multi-
functional neuroactive drugs showing NMDAR inhibition, VGCC
inhibition, nitric oxide synthase inhibition, antioxidant activity and
improved dopamine transmission [25]. Further studies utilizing
these structures are underway to establish their usefulness as neu-
roprotective agents and/or fluorescent ligands to be used in neuro-
degenerative disorders.
6.1.2. N-(1-Adamantyl)-1,3-propanediamine (c)
To a well-cooled suspension of 3.8 g of LiAlH in 200 ml of dry
Et2O, a solution of 12.3 g in 10 ml Et2O of 3-(1-adamantylamino)
propionitrile (b) was added dropwise. After this addition, the
reaction mixture was stirred at room temp for 3 h. With cooling,
4 ml of H2O was added, followed by 3 ml 5 N NaOH solution, and
a further 14 ml of H2O. The Et2O layer was decanted, and the solid
cake was washed with several portions (5 ꢃ 50 ml) of Et2O. The
Et2O layers were combined, filtered, dried with MgSO4 and evap-
orated in vacuo, to yield N-(1-adamantyl)-1,3-propanediamine as
a white wax (10.2 g, 48.97 mmol, 82%). Physical data: C13H24N2; mp:
wax; 1H NMR (200 MHz, MeOD) dH: 3.22e3.16 (t, J ¼ 6.2 Hz, 2H),
2.97e2.91 (t, J ¼ 6.1 Hz, 2H), 2.71e2.60 (m, 2H), 2.17e2.10 (m, 3H),
6. Experimental
6.1. Chemistry: general procedures
1.91 (bs, 3H) 1.81e1.64 (m, 12H); 13C NMR (50 MHz, MeOD) dC
:
Unless otherwise specified, materials were obtained from
commercial suppliers and used without further purifications. All
reactions were monitored by thin-layer chromatography on
42.63, 41.25, 40.38, 39.92, 38.56, 38.09, 37.78, 37.46, 32.59, 31.60,
31.55, 31.29, 30.29; IR (ATR; cmL1): 3360, 2906, 2848, 1550, 1475,
1313, 813, 792 cmꢁ1; HR-ESI [M D H]D: calcd. 209.2012, exp.
209.2018.
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. 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 SMP-300 melting point apparatus and capillary tubes.
The melting points are uncorrected. High resolution electro spray
ionisation (HR-ESI) mass spectra were recorded on a Waters API Q-
Tof Ultima mass spectrometer at 70 eV and 100 ꢂC. All HR-ESI
6.1.3. 3-Hydroxy-4-aza-8-oxoheptacyclo
[9.4.1.02,10.03,14.04,9.09,13.012,15]tetradecane (e) [24,28]
Physical data: C14H17NO2; mp: 170e172 ꢂC; 1H NMR (300 MHz,
CDCl3) dH: 4.97e3.94 (bs, 1H), 3.85e3.74 (m, 2H), 3.73e3.67 (m,
2H), 3.02e2.53 (m, 8H),1.80:1.52 (AB-q, 2H, J ¼ 10.58 Hz),1.75e1.55
(m, 2H). 13C NMR (75 MHz, CDCl3) dC: 101.45, 62.69, 54.97, 53.14,
45.73, 44.63, 44.00, 42.92, 42.41, 41.67, 41.48, 41.01, 24.33; IR (KBr;
cmL1): 3446, 1484, 1346, 1320, 1166 cmꢁ1; MS (EI, 70 eV) m/z: 231
(Mþ).
6.1.4. N-Adamantan-1-yl-2-(methylamino)-benzamide (1) [24]
Physical data: C18H24N2O; mp: 213 ꢂC; 1H NMR (200 MHz, CDCl3)
dH: 8.01e7.97 (dd, J ¼ 6.5 and 2.2 Hz,1H), 7.28 (m, 2H), 6.56e6.62
(m, 3H), 2.86 (s, 3H), 2.00 (s, 3H),1.86 (d, 6H),1.62e1.44 (m, 6H); 13C
NMR (50 MHz, CDCl3) dC: 175.01, 151.75, 132.74, 132.49, 117.54,
113.96, 110.05, 51.09, 40.98, 35.80, 29.74, 29.12; IR (ATR, cmL1):
samples were introduced by
a
heated probe and per-
fluorokerosene was used as reference standard. 1H and 13C NMR
spectra for all novel compounds were obtained using a Varian
Gemini 200 spectrometer at a frequency of 200 MHz and 50 MHz,
respectively. All chemical shifts are reported in parts per million
(ppm) relative to the signal from TMS (
d
¼ 0) added to an appro-
3341, 2911, 2853, 1609, 1503, 1365, 754 cmꢁ1; HR-ESI [M D H]D
:
calcd. 284.1888, exp. 284.1886.
priate deuterated solvent. The following abbreviations are used to
describe the multiplicity of the respective signals: s e singlet,
bs e broad singlet, d e doublet, dd e doublet of doublets, t e triplet,
q e quartet and m e multiplet. Compounds 1, 3, 5, 7, 9, 11, 13 and 14
were synthesised and characterised as published in previous
publications [24,25] and only the structural data of these derivatives
are presented in this paper. The well-described Cookson’s diketone,
pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8,11-dione (d), was syn-
thesised according to the published methods [26,27]. A Cary
EclipseÒ fluorescence spectrometer was used for fluorescence
measurements. The fluorescent compounds were measured at
a concentration of 10ꢁ5 M in DMSO at room temperature. Emission
spectra were recorded at the excitation maximal wavelength.
6.1.5. N-[3-(1-Adamantylamino)propyl]-2-methylamino-
benzamide (2)
To a solution of N-methylanthranilic acid (0.28 g, 1.85 mmol) in
DMF (10 ml) was added N,N-carbonyldiimidazole (0.33 g,
2.035 mmol). The resulting solution was protected from light and
warmed at 60 ꢂC for 1 h and then cooled to room temperature
before adding N-(1-adamantyl)-1,3-propanediamine (0.383 g,
1.85 mmol). The resulting solution was protected from light and
reacted for 48 h at room temperature and for 6 h at 60 ꢂC to ensure
complete reaction. The reaction mixture was added to DCM and
extracted with water acidified to pH 3 (3 ꢃ 25 ml). The water phase
was collected, basified with 5 N NaOH (pH 12) and extracted with
DCM (3 ꢃ 25 mL). The combined organic phases were collected,
dried over MgSO4 and the solvent removed in vacuo rendering the
crude product as a light yellow oil. The oil was dissolved in ethyl
acetate and N-hexane was added dropwise until the solution
became murky. The mixture was then cooled down to 5 ꢂC for
crystallization to proceed. The crystals were washed with cold ethyl
acetate (2 ꢃ 15 ml), yielding the pure product as a light yellow
crystalline solid (0.231 g, 0.68 mmol, 37%). Physical data:
C21H31N3O; mp: 99 ꢂC; 1H NMR (200 MHz, CDCl3) dH: 8.58 (bs, NH),
7.76 (bs, NH), 7.46e7.42 (dd, 1H, J ¼ 7.6 and 1.4 Hz), 7.30e7.26 (dd,
1H, J ¼ 7.6 and 1.4 Hz), 6.67e6.60 (dd, 1H, J ¼ 8.4 and 1.4 Hz),
6.59e6.52 (m, 1H), 3.51e3.48 (m, 1H), 2.86e2.79 (m, 5H), 2.06 (m,
3H), 1.75e1.60 (m, 16H); 13C NMR (50 MHz, CDCl3) dC: 169.90,
150.60, 132.55, 127.81, 115.30, 114.21, 111.01, 51.16, 42.62, 40.41,
39.88, 36.75, 29.71, 29.60, 28.95; IR (ATR; cmL1): 3302, 3275, 3215,
6.1.1. 3-(1-Adamantylamino)propionitrile (b)
Adamantane.HCl was converted into the free base by washing it
with a water solution saturated with NaHCO3 to afford the aman-
tadine free base. To a solution of 10 g of amantadine free base in
100 ml of acrylonitrile, 1 ml of H2O was added and the mixture was
heated overnight under reflux. The excess acrylonitrile was
removed in vacuo and the mixture was added to 100 ml DCM and
extracted with brine (3 ꢃ 100 ml), dried over MgSO4, filtered and
concentrated in vacuo to give 12.3 g of 3-(1-adamantylamino)pro-
pionitrile as a glassy solid (12.3 g, 60 mmol, 91%). Physical data:
C13H20N2; mp: 48 ꢂC; 1H NMR (200 MHz, CDCl3) dH: 3.00e2.87 (t,
J ¼ 6.6 Hz, 2H), 2.51e2.45 (t, J ¼ 6.6 Hz, 2H), 2.075 (s, 3H), 1.65 1.61
(m,12H); 13C NMR (50 MHz, CDCl3) dC: 118.96, 51.03, 42.83, 36.81,
36.59, 29.61, 20.21; IR (ATR; cmL1): 3307, 2899, 2846, 2246, 1153,
771 cmꢁ1; HR-ESID: calcd. 205.1700, exp. 205.1705.

J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
5017
2908, 2846, 1671, 1627, 1579, 1517, 742 cmꢁ1; HR-ESI [M D H]D
calcd. 342.2540, exp. 342.2545.
:
The oil was dissolved in ethyl acetate (25 ml) and cooled down
to ꢁ15 ꢂC while stirring on an external acetone bath. Forced
evaporation of the ethyl acetate with a mild stream of N2 gas caused
oversaturation and consequent precipitation of a white solid. The
precipitate was quickly filtered, suspended in water saturated with
NaHCO3, extracted with DCM (3 ꢃ 25 ml) and dried over MgSO4.
The solvents were removed in vacuo yielding the product as a pale
waxy solid. (Yield: 0.288 g, 0.77 mmol, 42%). Physical data:
C21H28N4O; mp: wax; 1H NMR (200 MHz, MeOD) dH: 8.41e8.37 (dd,
1H, J ¼ 8.0 and 1.2 Hz), 8.08 (bs, NH), 7.56e7.52 (dd, 1H, J ¼ 8.8 and
1.4 Hz), 7.44e7.36 (m, 1H), 7.30e7.23 (m, 1H), 3.60e3.57 (m, 1H),
2.86e2.73 (t, 1H, J ¼ 6.6 Hz), 2.067 (m, 3H), 1.92e1.86 (m, 2H),
6.1.6. N-(1-Cyano-2H-isoindol-2-yl)adamantan-1-amine (3) [24]
Physical data: C19H20N2; mp: 160 ꢂC; 1H NMR (200 MHz, CDCl3)
dH: 7.69e7.64 (m, 2H), 7.51 (s, 1H), 7.26e7.19 (m, 1H), 7.12e7.04 (m,
1H), 2.45 (m, 6H), 2.32 (s, 3H), 1.83 (m, 6H); 13C NMR (50 MHz,
CDCl3) dC: 150.64, 133.86, 125.34, 122.74, 122.39, 120.88, 117.83,
166.59, 115.88, 60.43, 43.00, 35.97, 30.03; IR (ATR, cmL1): 3140,
2910, 2850, 2189, 1182.37, 783, 752 cmꢁ1; HR-ESI [M D H]D: calcd.
276.16366, exp. 276.16265.
6.1.7. 2-[3-(1-Adamantylamino)propyl]isoindole-1-carbonitrile (4)
N-(1-Adamantyl)-1,3-propanediamine (0.688 g, 3.301 mmol)
and NaCN (0.162 g, 3.301 mmol) was dissolved in 20 ml methanol/
water. To this was added o-phtaldialdehyde (0.442 g, 3.301 mmol)
and the pH was adjusted to 8-9 with glacial acetic acid. The reaction
mixture was protected from light and stirred at room temperature
for 24 h. The mixture was concentrated in vacuo and added to an
aqueous 1N HCl solution and extracted with DCM (3 ꢃ 30 ml) and
dried over MgSO4. The organic fraction was filtered and the
solvents removed in vacuo yielding a light brown oil. The oil was
dissolved in ethyl acetate (25 ml) and cooled down to ꢁ15 ꢂC while
stirring on an external acetone bath. Forced evaporation of the ethyl
acetate with a mild stream of N2 gas caused oversaturation and
consequent precipitation of a white solid. The precipitate was
filtered and washed with cold cyclohexane yielding the crude
product as a white amorphous solid. Purification of the product was
accomplished with column chromatography (acetone:ethyl
acetate, 3:1) and recrystallization from absolute ethanol rendered
the final product as a white microcrystalline solid (0.220 g,
1.223 mmol, 37%). Physical data: C22H29N3; mp: 295 ꢂC; 1H NMR
(200 MHz, CDCl3:CD3OD; 1:1) dH: 7.47 (s, 1H), 7.44e7.34 (m, 2H),
7.06e6.90 (m, 1H), 6.91e6.83 (m, 1H), 2.73e2.65 (t, J ¼ 7.2 Hz, 2H),
2.13e2.01 (m, 2H), 1.96 (m, 3H), 1.65e1.64 (bs, 6H), 1.54e1.38 (m,
6H); 13C NMR (50 MHz, CDCl3) dC: 131.57, 125.37, 123.95, 122.38,
120.57, 120.22, 117.08, 114.08, 57.21, 47.70, 47.30, 37.80, 36.19, 34.90,
28.58, 27.61; IR (ATR; cmL1): 2923, 2852, 2757, 2697, 2488, 2419,
2200, 1689, 1579, 769, 747 cmꢁ1; HR-ESI [M D H]D: calcd.
334.2282, exp. 334.2283.
1.75e1.74 (m, 6H), 1.68e1.56 (m, 6H); 13C NMR (50 MHz, MeOD) dC
:
163.09, 141.44, 139.64, 127.13, 122.87, 122.63, 122.87, 122.63, 122.25,
109.92, 51.15, 42.61, 38.55, 38.13, 36.81,30.31, 29.66; IR (ATR;
cmL1): 3153, 3120, 3063, 2896, 2844, 1670, 1627, 1555, 747 cmꢁ1
HR-ESI [M D H]D: calcd. 353.2336, exp. 353.2341.
;
6.1.10. N-(2,4-Dinitrophenyl)adamantan-1-amine (7) [24]
Physical data: C16H19N3O4; mp: 300 ꢂC; 1H NMR (200 MHz,
CDCl3) dH: 9.18e9.12 (d, 1H, J ¼ 2.2 Hz), 8.81 (s, 1H, NH), 8.23e8.12
(dd,1H, J ¼ 5.1 and 2.2 Hz), 7.25e7.20 (d, 1H, J ¼ 5.1 Hz), 2.23 (s, 3H),
2.18e2.08 (m, 6H), 1.82e1.73 (m, 6H); 13C NMR (50 MHz, CDCl3) dC
:
147.86, 135.51, 131.02, 129.28, 129.28, 124.97, 116.305, 29.56, 36.15,
42.20, 54.34; IR (ATR, cmL1): 3321, 3107, 2927, 2857, 1621, 1587,
1335, 1088, 916, 743 cmꢁ1; HR-ESI [M D H]D: calcd. 317.13802, exp.
317.13756.
6.1.11. N-[(3S,5S,7S)-Adamantan-1-yl]-N΄-(2,4-dinitrophenyl)
propane-1,3-diamiane (8)
1-Fluoro-2,4-dinitro-benzene (0.354 ml, 1.25 mmol), N-(1-
adamantyl)-1,3-propanediamine (0.261 g, 1.25 mmol) and K2CO3
(0.346 g, 2.5 mmol) was dissolved in 15 ml absolute acetonitrile.
The reaction mixture was stirred in the dark for 48 h, where after
the mixture was filtered rendering a yellow precipitate. The
precipitate was brought to dryness in vacuo and purified by column
chromatography (acetone:ethyl acetate, 3:1) yielding the pure
product as a yellow amorphous solid (0.411 g, 1.09 mmol, 87%).
Physical data: C19H26N4O4; mp: 168 ꢂC; 1H NMR (200 MHz, MeOH)
dH: 9.04e9.03 (d, J ¼ 2.6 Hz, 1H), 8.34e8.28 (dd, J ¼ 9.4 and 2.6 Hz
1H), 7.25e7.21 (d, J ¼ 9.4 Hz, 1H), 3.67e3.59 (t, J ¼ 7.0 Hz, 2H),
3.11e3.03 (t, J ¼ 7.0 Hz, 2H), 2.20 (bs, 3H), 2.11e1.96 (m, 2H),
6.1.8. N-Adamantan-1-yl-1H-indazole-3-carboxamide (5) [24]
Physical data: C18H21N3O; mp: 212 ꢂC 1H NMR (200 MHz, MeOD)
dH: 8.26e8.22 (dd, J ¼ 6.5 and 2.2 Hz,1H), 7.54e7.50 (dd, J ¼ 6.5 and
2.2 Hz, 1H), 7.38e7.31 (m, 1H), 7.22e7.14 (m, 1H), 2.14 (s, 3H), 1.88
(d, 6H), 1.74e1.72 (m, 6H); 13C NMR (50 MHz, MeOD) dC: 170.65,
162.00, 143.29, 127.35, 123.73, 123.66, 122.58, 111.38, 52.64, 47.78,
41.60, 36.51, 30.48; IR (ATR, cmL1): 2979, 2909, 2854, 1637, 1475,
1322, 809, 733 cmꢁ1; HR-ESI [M D H]D: calcd. 295.3886, exp.
295.3669.
1.93e1.91 (bs, 6H), 1.86e1.62 (m, 6H); 13C NMR (50 MHz, CDCl3) dC
:
149.55, 137.29, 132.04, 131.07, 124.81, 115.71, 57.51, 41.47, 39.91,
38.28, 36.77, 30.65, 27.50; IR (ATR; cmL1): 3334, 3111, 2906, 2853,
1616, 1595, 1544, 1520, 1331, 715 cmꢁ1; HR-ESI [M D H]D: calcd.
375.2027, exp. 375.2032.
6.1.12. N-Adamantan-1-yl-5-dimethyl-amino-1-
naphthalenesulfonic acid (9) [25]
Physical data: C22H28N2O2S; mp: 160 ꢂC; 1H NMR (200 MHz,
CDCl3) dH: 8.64e8.59 (dd, J ¼ 6.2 and 2.2 Hz, 1H), 8.40e8.36 (dd,
J ¼ 6.3 and 2.2 Hz, 1H) 8.28e8.24 (dd, J ¼ 6.3 and 2.1 Hz, 1H), 7.78
(br s, 1H, NH), 7.54e7.40 (m, 2H), 7.13e7.09 (dd, J ¼ 6.4 and 2.1 Hz,
1H), 2.85 (s, 6H, N(CH3)2) 1.86 (s, 3H), 1.71 (d, 6H),1.50e1.28 (m,
6H); 13C NMR (50 MHz, CDCl3) dC: 181.38, 140.35, 130.46, 129.92,
127.74, 127.21, 126.34, 123.61, 121.70, 114.69, 52.68, 45.53, 40.12,
35.38, 28.99; IR (ATR, cmL1): 3290.86, 2902.11, 2849.28, 2779.45,
1614.28, 1588.52, 1577.02, 1438.47, 1308.27, 1143.95, 1086.98,
783.93 cmꢁ1; HR-ESI [M D H]D: calcd. 385.1950, exp. 385.1944.
6.1.9. N-[3-(1-Adamantylamino)propyl]-1H-indazole-3-
carboxamide (6)
To a solution of 1H-indazole-3-carboxylic acid (0.3 g, 1.85 mmol)
in DMF (7 ml) was added N,N’-carbonyldiimidazole (0.33 g,
2.035 mmol). The resulting solution was warmed at 60 ꢂC for 2 h
and then cooled to room temperature before adding a solution of N-
(1-adamantyl)-1,3-propanediamine (0.386 g, 1.85 mmol) in 3 ml
DMF. The resulting reaction mixture was stirred for 24 h at room
temperature to completion. After 24 h the reaction mixture was
added to DCM and extracted with water acidified to pH 3
(3 ꢃ 25 ml). The water phase was collected and basified with 5 N
NaOH (pH 12) and extracted with DCM (3 x 25 mL). The combined
organic phases were collected, dried over MgSO4 and the solvent
was removed in vacuo rendering the crude product as a yellow oil.
6.1.13. N-[3-(1-Adamantylamino)propyl]-5-dimethylaminonaphtha-
lene-1-sulfonamide (10)
N-(1-Adamantyl)-1,3-propanediamine (0.23 g, 1.1 mmol) was
dissolved in THF (15 ml) and triethylamine (0.30 g, 2.2 mmol) and

5018
J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
cooled on an ice bath. Dansyl chloride (0.30 g, 1.1 mmol) in THF
(5 ml) was added and the mixture was stirred for 1 h in the ice bath
and 24 h at room temperature. The solvents were removed in vacuo
rendering a bright yellow oil with a blue luminescence. The yellow
oil was added to water acidified to pH 3 (HClaq) and extracted with
DCM (3 ꢃ 20 ml), the aqueous layer was collected and made basic
with a 10 M NaOH solution to pH 12 and extracted with DCM
(3 ꢃ 20 ml). The combined organic layers were washed with 10%
NaHCO3 solution (5 ꢃ 10 ml) and water (3 ꢃ 10 ml), dried with
MgSO4 and concentrated in vacuo. The DCM was removed in vacuo
to render a light yellow oil. The oil was dissolved in acetone and
crystals formed on standing. Recrystallization from acetone
rendered the desired product as yellowish crystals (0.302 g,
0.68 mmol, 62%). Physical data: C25H35N3O2S; mp: 163 ꢂC; 1H NMR
(200 MHz, CDCl3) dH: 8.54e8.49 (d, J ¼ 8.4 Hz, 1H), 8.38e8.34 (d,
J ¼ 9.6 Hz, 1H), 8.26e8.22 (dd, J ¼ 7.4 and 1.2 Hz, 1H), 7.58e7.47 (m,
2H), 7.19e7.16 (d, J ¼ 7.4 Hz, 1H), 3.09e3.01 (t, J ¼ 5.6 Hz, 2H),
2.61e2.56 (t, J ¼ 5.6 Hz, 2H), 2.03 (bs, 3H), 1.70e1.54 (m, 6H),
110.68, 109.74, 63.05, 55.52, 43.93, 43.91, 43.88, 29.46, 24.00; IR
(KBr, cmL1): 3377, 2957, 2360, 1687, 1520, 1343, 1226, 1180 cmꢁ1
MS (EI, 70 eV) m/z: 364 (Mþ).
;
6.1.17. 3-{4-Aza-8-oxo-heptacyclo[0.4.1.02,10.03,14.04,9 09,13
.
.012,15
]
tetradecyl}-1H-indazole-3-carboxylate (14) [24]
Physical data: C22H24N2O2; mp: 190 ꢂC; 1H NMR (300 MHz,
CDCl3) dH: 7.91e7.90 (dd, 2H, J ¼ 9.58, 1.14 Hz), 7.38e7.24 (m, 3H),
6.64e6.61 (dd, 2H, J ¼ 8.27, 0.79 Hz), 6.57e6.51 (m, 3H), 3.89e3.094
(m, 4 H), 2.95e2.62 (m, 8H), 2.89e2.87 (d, 2H, J ¼ 5.99 Hz),
1.81e1.50 (m, 4H). 13C NMR (75 MHz, CDCl3) dC: 163.47, 140.97,
139.37, 127.06, 123.93, 122.64, 121.66, 110.74, 56.14, 49.96, 49.12,
49.12, 33.89, 30.94, 24.90; IR (KBr, cmL1): 3277, 2931, 2851, 2360,
1625, 1448, 1242 cmꢁ1; MS (EI, 70 eV) m/z: 375 (Mþ).
7. Biological evaluation
7.1. Imaging experiments using Mag-fura-2AM
1.53e1.51 (bs, 6H), 1.48e1.40 (m, 2H); 13C NMR (50 MHz, CDCl3) dC
:
152.00, 135.47, 130.05, 130.03, 129.84, 120.47, 128.19, 123.29, 119.53,
115.12, 50.90, 45.52, 44.36, 42.51, 40.10, 38.72, 29.59, 28.63; IR
(ATR; cmL1): 3282, 2904, 2847, 2782, 1614, 1589, 1321, 1141,
791 cmꢁ1; HR-ESI [M D H]D: calcd. 442.2523, exp. 442.2528.
7.1.1. Materials
All chemicals were of analytical grade or spectroscopy grade and
were purchased from SigmaeAldrich (UK) and Merck (St. Louis,
MO, USA).
6.1.14. N-(1-Adamantyl)-2-oxo-chromene-3-carboxamide (11) [25]
Physical data: C20H21NO3; mp: 300 ꢂC; 1H NMR (600 MHz,
CDCl3) dH: 8.90 (s,1H,), 7.71e7.62 (m, 2H), 7.41e7.33 (m, 2H), 2.05 (s,
3H), 1.75e1.64 (m, 12H); 13C NMR (150 MHz, CDCl3) dC: 161.60,
159.89, 154.30, 147.64, 133.69, 129.64, 125.13, 119.53, 118.72, 116.48,
52.28, 41.35, 36.34, 30.88, 29.39; IR (ATR, cmL1): 3341, 2911, 2853,
1609,1503,1365, 754 cmꢁ1; HR-ESI [M D H]D: calcd. 323.1515, exp.
323.1521.
7.1.2. Animals
The study protocol was approved by the Ethics Committee for
Research on Experimental Animals of the North-West University
(Potchefstroom Campus). Male SpragueeDawley rats were sacri-
ficed by decapitation and the brain tissue was removed and kept on
ice for homogenation. After homogenation, the aliquoted brain
homogenate was used immediately or stored at ꢁ4 ꢂC and used
within two days.
6.1.15. N-(1-Adamantyl)-2-oxo-chromene-3-carboxamide (12)
To a solution of coumarin carboxyxlic acid (0.352 g, 1.85 mmol)
in DMF (7 ml) was added N,N΄-carbonyldiimidazole (0.33 g,
2.035 mmol) in one portion. The resulting solution was stirred at
60 ꢂC for 2 h, where after a solution of N-(1-Adamantyl)-1,3-
propanediamine (0.386 g, 1.85 mmol), in DMF (3 ml) and was
added. The resulting solution was reacted for 72 h at room
temperature. It was quenched with a saturated solution of NaCl(aq)
(50 ml). The compound was extracted with ethyl acetate
(3 ꢃ 30 ml). The combined organic layers were successively washed
with 4% citric acid (30 ml), water (30 ml), 4% NaHCO3 solution
(30 ml), and water (30 ml). The organic layer was dried over
anhydrous MgSO4, and the solvent was removed in vacuo to provide
the crude product as a white crystalline solid. The product was
purified by recrystallization from ethyl acetate/petroleum ether
(1:1) to afford a white crystalline solid (0.332 g, 0.87 mmol, 47%).
Physical data: C28H28N2O3; mp: 211 ꢂC 1H NMR (200 MHz, MeOD)
dH: 8.96 (bs, NH), 8.90 (s, 1H), 7.71e7.62 (m, 2H), 7.42e7.34 (m, 2H),
3.56e3.50 (t, J ¼ 6.8 Hz, 2H), 2.79e2.66 (t, J ¼ 6.8 Hz, 2H), 2.06 (s,
3H), 1.82e1.74 (m, 1H), 1.74e1.64 (m, 12H); 13C NMR (50 MHz,
MeOD) dC: 161.70, 161.40, 154.53, 148.25, 134.05, 128.86, 125.33,
118.79, 118.69, 116.72, 50.93, 42.64, 38.26, 38.01, 36.82, 30.72,
29.68; IR (ATR; cmL1): 3292, 2902, 2885, 1724, 1709, 1641, 1607,
1568, 759 cmꢁ1; HR-ESI [M D H]D: calcd. 381.2173, exp. 381.2178.
7.1.3. General methods
The fluorescent ratiometric indicator, Mag-Fura-2/AM, and
a Cary-EclipseÒ fluorescence spectrometer were used to evaluate
the influence of the test compounds on calcium homeostasis via
the NMDA receptor channel utilizing murine synaptoneurosomes.
Procedures similar to those of published studies were used to
prepare the synaptoneurosomes and solutions, and to establish
the techniques for experimental measurement of fluorescence
[29]. All data analysis, calculation and graphs were done using
Prism 4.02Ò (GraphPhad, Sorrento Valley, CA). Data in bar charts
are presented as % values of initial fluorescence with each bar
representing the mean ꢀ SEM. Data analysis was carried out using
the Student Newman Keuls multiple range test. Concentration-
curve data were analyzed by non-linear least squares fit to
a four parameter logistic equation using Prism 4.02Ò (GraphPhad,
Sorrento Valley, CA). The level of significance was accepted at
p < 0.05.
7.1.4. Preparation of synaptoneurosomes
Male adult SpragueeDawley rats were sacrificed by decapi-
tation, whole brains were removed and synaptoneurosomes were
prepared by techniques of Hollingsworth et al., slightly modified
[35]. The brains from two rats were homogenized (4 strokes by
hand using a homogenizer) in 30 ml of ice-cold Krebs-bicar-
bonate buffer (NaCl, 118 mM; KCl, 4.7 mM; MgCl, 1.18 mM; CaCl2
1.2 mM; NaHCO3 24.9 mM; KH2PO4 1.2 mM; and glucose, 10 mM).
From this step forward the homogenate was kept ice-cold at all
times to minimize proteolysis throughout the isolation proce-
dure. The tissue suspension was placed in a 50 ml polycarbon ate
tube and centrifuged for 15 min at 1100ꢃ g and 0 ꢂC. After
centrifugation, the pellet was resuspended by hand in 30 ml fresh
incubation buffer and centrifuged again as described above. The
6.1.16. 3-{4-Aza-8-oxo-heptacyclo[0.4.1.02,10.03,14.04,9 09,13
tetradecyl}-2-(methylamino)benzoate (13) [24]
.
.012,15
]
Physical data: C22H21N3O3; mp: 213 ꢂC; 1H NMR (300 MHz,
CDCl3) dH: 8.14e8.11 (dd, 2H, J ¼ 8.19, 0.89 Hz), 7.70e7.67 (dd, 2H,
J ¼ 8.38, 0.94 Hz), 7.42e7.36 (m, 3H), 7.26e7.21 (m, 3H) 3.89e3.09
(m, 4H), 2.95e2.62 (m, 8H), 2.89e2.87 (d, 2H),1.89e1.12 (m, 4H).13C
NMR (75 MHz, CDCl3) dC: 166.60, 152.23, 134.75, 131.93, 114.29,

J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
5019
pellet was gently resuspended in Krebs-bicarbonate buffer at
a protein concentration of 3 mg/ml [36]. Protein yield was 10 mg/g
tissue.
were incubated with the respective compound and because these
novel compounds showed VGCC flux inhibition it was co-
incubated with the selective voltage gated calcium channel
inhibitor, nimodipine (10
mM), to block VGCC thus preventing
7.1.5. General procedure for loading FURA-2AM and incubating test
compounds
calcium flux through this channel and allowing selective evalua-
tion of the compounds for the NMDAR. The procedure was initi-
Experiments were carried out at 37 ꢂC and fluorescence was
measured with a spectrofluorimeter equipped with a water-
jacketed cuvette holder. The synaptoneurosome suspension
prepared was allowed to reach room temperature, thereafter Fura-
2 AM (5 mM in DMSO e protect solution from light) was added to
ated and kept at 37 ꢂC, at 10 s into the recording 100
m
l of krebs-
HEPES buffer containing NMDA (100
m
M) and glycine (100 M)
m
was added to the membrane preparation to stimulate calcium
influx through the NMDAR channels. The addition of NMDA
(100
mM) and glycine (100 mM) resulted in activation of the
a final concentration of 5
m
M. Synaptoneurosomes were incubated
NMDAR- mediated calcium flux. Treatments were repeated three
times on different tissue preparations with three determinations
in each replicate.
at 37 ꢂC for 10 min, diluted with Krebs-bicarbonate buffer at room
temperature to a final concentration of 0.6 mg/ml and kept at room
temperature and protected from light until used. Immediately
before the experiment, 0.25 ml of the Fura-2 AM synaptoneur-
osomal suspension was centrifuged for 10 s in a desk Hermle Z
100 MÒ Microfuge. The supernatant was discarded and the pellet
resuspended in 0.75 ml of 37 ꢂC Krebs-HEPES buffer (20 mM HEPES
substituting for NaHCO3 and adjusted with NaOH to pH 7.4). For the
7.2. NMDAR competition assay utilizing N-[3-(1-adamantylamino)
propyl]-5-dimethylamino-naphthalene-1-sulfonamide (10)
Murine synaptoneurosomes were prepared as described in 7.1.4.
Experiments were carried out at 37 ꢂC on a spectrofluorimeter. The
synaptoneurosome suspension was allowed to reach room
temperature and diluted with Krebs-bicarbonate buffer at room
temperature to a final concentration of 0.6 mg/ml. Immediately
before the experiment, 0.5 ml of the synaptoneurosomal suspen-
sion was centrifuged for 10 s in a desk Hermle Z 100 MÒ Microfuge.
The supernatant was discarded and the pellet was resuspended in
1.5 ml of 37 ꢂC Krebs-HEPES buffer. Compound 10 as control (100%)
and compound 10 with test derivative(s) were dissolved in DMSO
and applied to the synaptoneurosomal preparation at a final
screening test 100
compounds were prepared in DMSO (final DMSO concentration of
0.1%). 0.75 l of the stock solution was diluted in 0.75 ml Krebs-
HEPES buffer solution to obtain a 100 M concentration of the
mM stock solutions of the references and test
m
m
test compounds. Control experiments received 0.1% DMSO to
compensate for possible effects caused by addition of DMSO in
experiments. For the dose response curve, the test compound
selected from the screening test was incubated at different
concentrations, in a log-scale.
concentration of 100
(final DMSO concentration was kept lower than 0.1%). The reaction
was initiated by the addition of 100 l Krebs-HEPES buffer con-
taining NMDA (100 M) and glycine (100 M) to the synapto-
mM for 10 and the individual test derivatives
7.1.6. KCl-mediated Ca2þ stimulation
The wavelengths selected for the detection of intracellular
calcium utilizing Fura-2 AM were 344 nm (excitation) and 506 nm
(emission) as described in the experimental protocols of previous
papers [29]. The optics position was set at the bottom and sensi-
tivity set at 100 with a runtime of 40 s with 150 ms intervals. The
test compounds were incubated with the synaptoneurosomes for
5 min at 37 ꢂC and used immediately after incubation. The
procedure was initiated and kept at 37 ꢂC. At 10 s into the
m
m
m
neurosomal preparation and incubated for 30 min at 37 ꢂC. After
30 min the synaptoneurosomal suspension was centrifuged for 10 s
in a desk Hermle Z 100 MÒ Microfuge and the supernatant dis-
carded. At the high test concentration of compound 10 the fluo-
rescent intensities could not be measured before washing of the
synaptoneurosomal pellet because of extremely high measure-
ments. The pellet was resuspended in incubation buffer (1.5 ml)
and measured spectrofluorometrically at the excitation and emis-
sion values of compound 10 (324/455 nm) as there was no spectral
shift observed. Compound 10 was used as the control with 100%
fluorescent intensity and the decrease in fluorescence caused by
the known NMDAR inhibitors were an indication of displacement
of compound 10. All the known NMDAR antagonists did to some
degree compete with compound 10 (Figs. 6 and 7) for binding with
the NMDAR.
recording 100 ml of KCl (140 mM) depolarization solution (43 mM
NaCl, 140 mM KCl, 10 mM NaHCO3, 1.4 mM CaCl2, 0.9 mM MgSO4,
5.5 mM Glucose, and 20 mM HEPES, pH 7.4) was added to the
membrane preparation to depolarize the synaptoneurosomes and
activate calcium influx. Taking the KCI-induced increase in fluo-
rescence as 100%, the percentage that each test compound inhibits
the calcium flux, subsequently reducing the fluorescence, can be
compared to the reference compounds. To gain a more accurate
interpretation of the results, the initial average fluorescence
values before application of high concentration potassium (base
line) were subtracted from the maximum increase in fluorescence
for representative experiments in synaptoneurosomes incubated
with test compounds, reference compounds and controls. Stan-
dard errors of mean (SEM) values were incorporated as error bars
(Fig. 3). Treatments were repeated three times on different tissue
preparations with three determinations in each replicate. All the
test compounds significantly decreased the KCl- mediated calcium
flux (p < 0.05).
8. Statistical analysis
All data analysis, calculation and graphs were done using
Prism 4.02Ò (GraphPhad, Sorrento Valley, CA). Data in bar charts
are presented as % values of initial fluorescence with each bar
representing the mean ꢀ SEM. Concentration-curve data was
analyzed by non-linear least squares fit to a four parameter
logistic equation using Prism 4.02Ò. Data analysis was carried out
using the Student Newman Keuls multiple range test of test
compounds versus controls in the screening studies. The level of
significance was accepted at p < 0.05. Bonferroni’s Multiple
Comparison, one way analysis of variance (ANOVA) was per-
formed on selected data to indicate significant differences
between test compounds (p < 0.05 was considered to be
a statistically significant difference).
7.1.7. NMDA/glycine mediated Ca2þ stimulation
The wavelengths selected for the detection of intracellular
calcium utilizing Fura-2 AM were 344 nm (excitation) and 506 nm
(emission) as described earlier [29]. The optics position was set at
the bottom and sensitivity set at 100 with a runtime of 40 s with
150 ms intervals. The test compound was incubated for 5 min at
37 ꢂC and used immediately after incubation. Synaptoneurosomes

5020
J. Joubert et al. / European Journal of Medicinal Chemistry 46 (2011) 5010e5020
[20] S.F. Malan, A. Van Marle, W.M. Menge, V. Zuliana, M. Hoffman, H. Timmerman,
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Western Cape, Medicine Research Council of South Africa and the
National Research Foundation of South Africa for financial support.
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