Fluorescent polycyclic ligands for nitric oxide synthase (NOS) inhibition

Article abstract of DOI:10.1016/j.bmc.2008.08.049

In recent years polycyclic compounds have been shown to exhibit pharmacological profiles of importance in the symptomatic and proposed curative treatment of neurodegenerative diseases (e.g., Parkinson's and Alzheimer's disease). These structures also show modification and improvement of the pharmacokinetic and pharmacodynamic properties of drugs in current use. Nitric oxide (NO) is a molecular messenger involved in a number of physiological processes in mammals. It is synthesised by nitric oxide synthase (NOS) from l-arginine and its overproduction could lead to a number of neurological disorders. The aim of this study was to synthesise a series of novel indazole, indole and other fluorescent derivatives conjugated to polycyclic structures for evaluation in NOS assays. NOS is a target system where fluorescent techniques and fluorescently labelled NOS inhibitors can be used for detecting the biophysical properties of enzyme-ligand interactions and thus facilitate development of novel inhibitors of neurodegeneration. This could lead to a greater insight into the neuroprotective mechanism and a possible cure/treatment for neurodegenerative diseases. A series of compounds incorporating polycyclic structures such as 3-hydroxy-4-aza-8-oxoheptacyclo[9.4.1.0.^2,100.^3,140.^4,90.^9,130^12,15]tetradecane and amantadine as well as suitable fluorescent moieties were selected for synthesis. In the biological evaluation the oxyhaemoglobin (oxyHb) assay was employed to determine the activity of the novel compounds at an enzymatic level of NOS. IC₅₀ values of the novel fluorescent compounds were compared to that of aminoguanidine (AG) and 7-nitroindazole (7-NI), two known NOS inhibitors, and showed moderate to high affinity (IC₅₀ values ranging from 7.73 μM to 0.291 μM) for the NOS enzyme.

Full text of DOI:10.1016/j.bmc.2008.08.049

Bioorganic & Medicinal Chemistry 16 (2008) 8952–8958
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent polycyclic ligands for nitric oxide synthase (NOS) inhibition
*
Jacques Joubert, Sandra van Dyk, Sarel F. Malan
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag 6001, 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:
In recent years polycyclic compounds have been shown to exhibit pharmacological profiles of importance
in the symptomatic and proposed curative treatment of neurodegenerative diseases (e.g., Parkinson’s and
Alzheimer’s disease). These structures also show modification and improvement of the pharmacokinetic
and pharmacodynamic properties of drugs in current use. Nitric oxide (NO) is a molecular messenger
involved in a number of physiological processes in mammals. It is synthesised by nitric oxide synthase
Received 20 May 2008
Revised 19 August 2008
Accepted 22 August 2008
Available online 27 August 2008
(NOS) from L-arginine and its overproduction could lead to a number of neurological disorders. The
Keywords:
aim of this study was to synthesise a series of novel indazole, indole and other fluorescent derivatives
conjugated to polycyclic structures for evaluation in NOS assays. NOS is a target system where fluores-
cent techniques and fluorescently labelled NOS inhibitors can be used for detecting the biophysical prop-
erties of enzyme–ligand interactions and thus facilitate development of novel inhibitors of
neurodegeneration. This could lead to a greater insight into the neuroprotective mechanism and a pos-
sible cure/treatment for neurodegenerative diseases. A series of compounds incorporating polycyclic
structures such as 3-hydroxy-4-aza-8-oxoheptacyclo[9.4.1.0.2,100.3,140.4,90.9,13012,15]tetradecane and
amantadine as well as suitable fluorescent moieties were selected for synthesis. In the biological evalu-
ation the oxyhaemoglobin (oxyHb) assay was employed to determine the activity of the novel com-
pounds at an enzymatic level of NOS. IC50 values of the novel fluorescent compounds were compared
to that of aminoguanidine (AG) and 7-nitroindazole (7-NI), two known NOS inhibitors, and showed mod-
Fluorescent inhibitors
Nitric oxide synthase
Polycyclic cage
Neuroprotection
erate to high affinity (IC50 values ranging from 7.73
l
M to 0.291
lM) for the NOS enzyme.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the intramolecular photocyclisation of the Diels Alder adduct of p-
benzoquinone and cyclopentadiene.5 (Scheme 1)
Investigations into the synthesis and chemistry of novel satu-
rated polycyclic hydrocarbon ‘cage’ compounds have been the
aim of several research groups. The medicinal potential of these
compounds was realised with the discovery that amantadine (3)
exhibits antiviral activity. Subsequent to this discovery, it was
found that amantadine could be benificial to patients with
Parkinson’s disease. It expresses its anti-Parkinsonian activity by
increasing extracellular dopamine (DA) levels via DA re-uptake
inhibition1 or DA release and NMDA receptor antagonism.2 Interest
in the pharmacology of polycyclic cage amines was further stimu-
lated when the dimethyl derivative of amantadine, memantine (4),
was found to be a clinically well tolerated NMDA receptor
antagonist.3
Nitric oxide synthases are a family of enzymes in the body that
contributes to neurotransmission, the immune system and vaso-
dilitation. It does so by synthesis of nitric oxide and
from the terminal nitrogen atom of -arginine in the presence of
NADPH and oxygen7 (O2) (Scheme 2) via the intermediate NG-hy-
droxy-
-arginine.8
L-citrulline
L
L
Three distinct NOS enzymes have been identified and character-
ised as products of different genes, with different subcellular local-
isation, regulation, catalytic properties, and inhibitor sensitivity;
neuronal NOS (nNOS) and endothelial NOS (eNOS), which are con-
stitutively expressed, and inducible NOS (iNOS).7,8 nNOS and eNOS
are physiologically activated by steroid hormones or neurotrans-
mitters such as NO, dopamine, glutamate and glycine that increase
A structural similarity exists between the polycyclic cage struc-
ture of adamantane amines and that of the pentacycloundecyl
amines.4 Pentacycloundecylamines derivatives (2) are derived
from Cookson’s diketone (Pentacyclo[5.4.02,6.03,10.05,9]undecane-
8,11-dione) (1), the so called ‘bird cage’ compound, obtained from
CH3
NH2
NH2
NHR
O
O
3
4
2
O
1
H3C
* Corresponding author. Tel.: +27 18 2992266; fax: +27 18 2994243.
E-mail address: sarel.malan@nwu.ac.za (S.F. Malan).
Scheme 1. Structural similarities between the polycyclic compounds.4
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.049

J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
8953
Table 1
Fluorescent
NH+2
H2N
O
H2N
H2N
NOH
derivatives
of
3-hydroxy-4-aza-8-oxohepta-
cyclo[9.4.1.0.2,10.03,14.04,9.09,13.012,15]tetradecane
NH
NH
NH
NADPH
O2
NADPH
O2
N
O
O
N
COO-
COO-
H3N+
H3N+
COO-
H3N+
R
R =
Scheme 2. Reaction catalysed by NOS.6
H3C
the intracellular calcium concentrations. iNOS, in contrast, is Ca2+
independent and is expressed in a broad range of cell types. This
form of NOS is induced after stimulation with cytokines and expo-
sure to microbial products. After permanent activation, it continu-
ously produces high concentrations of NO.9
NOS is the only known enzyme that has several cofactors
including NADPH, flavin adenine dinucleotide (FAD), flavin mono-
nucleotide (FMN), heme, tetrahydrobiopterin (BH4) and calmodu-
lin.6 Although NO mediates several physiological functions, a
number of disease states are associated with either the overpro-
duction or underproduction of NO, making the NOS pathway an
attractive target for the development of therapeutics.7 Overpro-
duction by NOS has been implicated in a number of clinical disor-
ders, including acute (stroke) and chronic (Alzheimer’s, Parkinson’s
and Huntington’s diseases) neurodegenerative diseases, convul-
sions and pain.10
O ---
NH
O
O
O ---
N
N
H
5
6
Table 2
Fluorescent derivatives of amantadine
R
R =
The majority of known NOS inhibitors are nonselective or iNOS
selective and only a few compounds are able to selectively inhibit
nNOS. These include 7-NI,11 1-(2-trifluoromethyl-phenyl)-imida-
zole (TRIM),12 some aromatic amidines13 and amino acid derivatives
(for example, some aminoguanidines).14,15 TRIM has been reported
to be relatively selective for nNOS, but with low potency while the
nitroindazole family were found to be potent nNOS inhibitors.11,12
Molecular tools capable of providing mechanistic insights into the
production of NO and/or the inhibition of the NOS enzymes thus
remain of interest. The development of neuroprotective agents is
therefore orientated towards the synthesis of novel structures that
interfere with a specific step of the complex chemical signalling
system involving NOS and the inhibition of the enzyme itself.
In recent years the use of fluorescent detection methods, that is,
confocal laser scanning microscopy, flow cytometry and image
analysis, in nonradioactive assays have found widespread applica-
bility in receptor and enzyme pharmacology. Fluorescent ligands
are used to determine receptor and enzyme properties like recep-
tor internalisation and sub cellular localisation, the thermodynam-
ics and kinetics of ligand binding and to assess the nature of the
microenvironment of the ligand binding site.16–18
The main objective of this study was to synthesise fluorescent
NOS inhibitors and to explore their neuroprotective ability/poten-
tial. The fluorescent compounds for this study were selected on the
basis of their spectroscopic properties, ease of synthesis and struc-
tural similarities to 7-NI to exhibit NOS inhibition. It is hypothe-
sised that the novel fluorescent compounds may have the ability
to inhibit NOS, as the compounds selected as fluorescent ligands
have structural similarities to 7-NI, which is reported to be a selec-
tive nNOS inhibitor.11,19 The fluorescent compounds synthesised
include N-methylanthranilic acid, indazole-3-carboxylic acid, 1-
fluoro-2,4-dinitrobenzene, 1-cyanoisoindole, 1-thiocyanoisoindole
and 1-nitroisoindole conjugated to 3-hydroxy-4-aza-8-oxohepta-
cyclo[9.4.1.02,10.03,14.04,9.09,13.012,15]tetradecane (Table 1) and
amantadine (Table 2).
H3C
NH ---
NH
O
NO2
O
N
NH ---
N
N
H
O2N
7
8
9
NC
NCS
O2N
--- N
--- N
--- N
10
11
12
primary alcohol and amine, respectively. The dinitrobenzene
complex was obtained through amination (9) with the primary
amine of amantadine and the fluorescent isoindoles were obtained
with the reaction of o-phthaldialdehyde with amantadine in the
presence of sodium cyanide, sodium thiocyanate or sodium nitrate
to form the fluorescent isoindoles (10, 11, 12).
2. Results and discussion
The synthesised compounds were all obtained as oils or amor-
phous solids from chromatography or were crystallised from or-
ganic solvents and the structures were confirmed using 1H and
13C NMR, MS and IR. The oxyhaemoglobin (oxyHb) assay20 was em-
ployed to determine the activity of the novel compounds at an
enzymatic level of NOS. This assay is principally based on the reac-
tion of NO with oxyHb and the formation of methaemoglobin
(metHb). In order to determine the amount of NO formed, the
change in absorbance difference between 401 and 421 nm is
measured during the initial linear phase of the reaction (Fig. 1). If
The anthranilic and indazole complexes were obtained through
the intermediate complexes with CDI and DCC and yielded the
fluorescent esters (5, 6) and amides (7, 8) on reaction with the

8954
J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
Aminoguanidine
Compound 6
Compound 8
Compound 10
Compound 11
Compound 12
7-NI
110
100
90
80
70
60
50
40
30
20
10
0
-8
-7
-6
-5
-4
-3
-2
-1
log[Compounds] (M)
Figure 1. A typical spectrophotometric recording of compound 10 at a specific
concentration. Continuous scans between 390 nm and 430 nm were performed as
the oxyHb was converted to metHb.
Figure 3. Inhibition curves of compounds with meaningful activities are superim-
posed to compare their IC50 values, 7-NI and aminoguanidine were used as
reference compounds.
cyanoisoindole adamantane compound showed a 10-fold increase
in activity when compared to the 1-tiocyanoisoindole adamantane
and 1-nitroisoindole adamantane compounds (11 and 12) (Fig. 3
and Table 3).
All compounds showed an acceptable difference of excitation
and emission wavelengths and Stoke shifts varied from 29 to
80 nm. Compound 6, the second most potent compound showed
the highest Stoke shift of 80 nm (Table 3).
the change in absorbance at 401 nm is plotted against time and the
change in absorbance over time at 421 nm is subtracted, the slope
of this resulting curve is an indication of the increase in molar
amount of metHb, and is identical to the molar amount of NO gen-
erated (Fig. 2). From this inhibition data the IC50 values were calcu-
lated and compared.
The inhibition curves of the selected compounds were superim-
posed on a single graph and the IC50 values were calculated. From
the calculated IC50 values; compounds 6, 8, 10, 11 and 12 revealed
promising results as possible NOS inhibitors. 100% inhibition of
NOS could not be obtained for the novel synthesised fluorescent
3. Conclusion
structures as solubility becomes
concentrations.
When the compounds are compared to 7-NI (Fig. 3), one can
clearly see that none of the structures showed as high activity as
a limiting factor at higher
We have identified a series of fluorescent structures with mod-
erate to high affinity for the NOS enzyme, which may be utilised for
further in vitro and in vivo studies using modern imaging tech-
niques (e.g., confocal laser scanning microscopy, flow cytometry
or multiphoton microscopy).
The potential of these novel fluorescent polycyclic structures as
NOS inhibitors with structural similarity to 7-NI, a selective nNOS
inhibitor,11,17 the documented attenuation of excessive influx of
Ca2+ into neuronal cells by NMDA receptor and L-type Ca2+ channel
modulation22 and effects on dopamine re-uptake and release,23
indicate that these novel compounds may find application as mul-
tipotent drugs in neuroprotection.
7-NI (IC50 = 0.111
lM). All the compounds however showed more
potent inhibitory activity than aminoguanidine (IC50 = 19.41
l
M)
(Fig. 3). Aminoguanidine is reported to be a selective iNOS inhibi-
tor21 and the lower activity observed could, to a certain degree, be
attributed to this fact. Compounds 5, 7 and 9 showed low or no
inhibition (Table 3) of the NOS enzyme when compared to 7-NI
and aminoguanidine.
The indazole structures, compounds 6 (IC50 = 0.35
(IC50 = 2.53 M) showed significant NOS activity. Both of these
compounds exhibit better inhibition of the enzyme than the free
indazole-3-carboxylic compound, IC50 = 9.65 M; (Fig. 4). This con-
lM) and 8
l
These compounds thus have potential as useful pharmacolog-
ical tools to investigate enzyme–ligand or receptor–ligand inter-
actions in the quest for effective neuroprotective strategies and
l
firms that both the polycyclic tetradecane and the adamantane
moieties increase the activity of the indazole structures. The in-
creased activity of the compounds conjugated to the polycyclic
structures could be because of the higher lipophilicity and mem-
brane permeability of these compounds.
Compound 10 proved to be the best inhibitor of the novel fluo-
rescent compounds with a potent IC50 value of 0.291 lM. This 1-
could lead to
mechanism.
a greater insight into the neuroprotective
In order to more accurately determine the selectivity of the no-
vel inhibitors, inhibition studies with individual NOS isoforms uti-
lising a larger series of derivatives will be conducted in further
investigations. Additional assays on the NMDA receptor, voltage
gated Ca2+ channel, MAO-B enzyme and blood–brain barrier per-
meability will furthermore elaborate on potential value of these
compounds.
4. Experimental
4.1. Chemistry: general procedures
Unless otherwise specified, materials were obtained from com-
mercial suppliers and used without further purifications. All reac-
tions were monitored by thin-layer chromatography on 0.20 mm
thick aluminum silica gel sheets (AlugramÒ SIL G/UV254, Kieselgel
60, Macherey-Nagel, Düren, Germany). Visualisation was achieved
using UV light (254 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 (0.063–0.2 mm, Merck) except when otherwise stated.
Figure 2. The change in absorbance at 401 nm and 421 nm versus time was
calculated and the difference of the respective slope values [(mdA(401 nm)) ꢀ (m-
dA(421 nm))] gives an indication of enzyme activity.

J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
8955
Table 3
NOS enzyme inhibition data and fluorescent properties of compounds
O
N
R
NH
O
R
Compounds 7-12
Log IC50
Compounds 5-6
a
a
Compound
R (fluorophore)
Mp (°C)
% Yield
(lM)
kex (nm)
kem (nm)
5
6
7
8
2-Methylaminobenzoate
1H-indazole-3-carboxylate
2-Methylaminobenzamide
1H-indazole-3-carboxamide
2,4-Dinitrophenyl-1-amine
1-Cyanoisoindole
213
190
209
212
300
160
213
210
—
36
16
25
44
50
37
21
25
—
ꢀ2.93
ꢀ4.22
ꢀ2.53
ꢀ3.52
—
ꢀ4.52
ꢀ3.88
ꢀ3.74
ꢀ4.96
ꢀ2.72
ꢀ3.28
368
330
366
340
396
358
352
332
—
415
410
415
400
449
395
409
393
—
9
10
11
12
7-NIb
AGc
I-3-Cd
1-Thiocyanoisoindole
1-Nitroisoindole
164
266
—
—
—
360
—
420
kex = excitation k; kem = emission k.
a
At 10ꢀ5 M in absolute ethanol at 25 °C.
7-Nitroindazole.
Aminoguanidine.
Indazole-3-carboxylic acid.
b
c
d
4.2.1. 3-Hydroxy-4-aza-8-oxoheptacyclo[9.4.1.02,10.03,14.04,9.-
09,13.012,15]tetradecane
110
100
90
80
70
60
50
40
30
20
10
0
Indazole-3-carboxylic acid
Compound 6
Compound 8
Pentacyclo[5.4.02,6.03,10.05,9]-undecane-8,11-dione (3 g, 17.24
mmol) was dissolved in 30 ml tetrahydrofuran and cooled down
to 5 °C while stirring in an ice bath. 3-amino-1-propanol
(1.105 ml, 17.22 mmol) dissolved in 6 ml THF, was added slowly
with continued stirring of the reaction mixture at 5 °C. The carbi-
nolamine started precipitating after approximately 15 min, but
the reaction was allowed to reach completion for an additional
30 min. Water was removed from the precipitate azeotropically
by refluxing it in 60 ml dry benzene using a Dean-Stark apparatus
for 1 h or until no more water was collected in the trap. The excess
benzene was removed under reduced pressure and the rearranged
cage structure, a yellow oil, was crystallised from THF to render the
-8
-7
-6
-5
-4
-3
-2
-1
log[Compounds] (M)
Figure 4. Inhibitory curves of the indazole test compounds, showing the increased
activities of the compounds conjugated with the tetradecane (6) and adamantane
(8).
final product as
12.987 mmol, 75.33%).
14H17NO2; Mp: 170–172 °C; 1 H NMR (300 MHz, CDCl3) dH:
a colourless crystalline solid (Yield: 3 g,
C
4.97–3.94 (br s, 1H), 3.85–3.74 (m, 2H), 3.73–3.67 (m, 2H), 3.02–
2.53 (3 ꢁ m, 8H), 1.80:1.52 (AB-q, 2H, J = 10.58 Hz), 1.75–1.55 (m,
2H). 13 C NMR (75 MHz, CDCl3) dC: 101.45 (2 ꢁ s), 62.69 (t), 54.97
(d), 53.14 (d), 45.73 (d), 44.63 (d), 44.00 (d), 42.92 (d), 42.41 (t),
41.67 (t), 41.48 (d), 41.01 (d), 24.33 (t); MS (EI, 70 eV) m/z: 231
The MS spectra were recorded on an analytical VG 70-70E mass
spectrometer using electron ionisation (EI) at 70 eV. Melting points
were determined using a Stuart SMP-300 melting point apparatus
and capillary tubes. The melting points are uncorrected. IR spectra
were recorded on a Nicolet Magna—IR 550 spectrometer. Samples
were applied either as film or incorporated in KBr pellets. 1H and
13C spectra were obtained using a Varian Gemini 300 spectrometer
at a frequency of 300.075 MHz and 75.462 MHz, respectively. All
chemical shifts are reported in parts per million (ppm) relative to
the signal from TMS (d = 0) added to an appropriate deuterated sol-
vent. The following abbreviations are used to describe the multi-
plicity of the respective signals: s—singlet, br s—broad singlet, d—
doublet, dd—doublet of doublets, t—triplet, q—quartet and m—
multiplet.
(M+), 174, 151, 139, 91, 41, 28; IR (KBr)
1320, 1166 cmꢀ1
mmax: 3446, 1484, 1346,
.
4.2.2. 3-{4-Aza-8-oxo-heptacyclo[0.4.1.02,10.03,14.04,9 09,13
012,15]tetradecyl}-2-(methylamino)benzoate (5)
.
.-
N-Methylanthranilic acid (0.390 g, 2.583 mmol) was added to a
stirred solution of N,N0-carbonyldiimidazole (0.421 g, 2.583 mmol)
in anhydrous tetrahydrofuran (25 ml). After 24 h 3-Hydroxy-4-
aza-8-oxoheptacyclo[9.4.1.02,10.03,14.04,9.09,13.012,15]tetradecane
(0.6 g, 2.583 mmol) in tetrahydrofuran (10 ml) was added and the
mixture was allowed to react for 72 h at room temperature. The
precipitate was filtered and washed with cold THF (2ꢁ 15 ml)
yielding the product as a light yellow powder (Yield: 336 mg,
0.82 mmol, 36%).
4.2. Synthesis
The well-described Cookson’s diketone, pentacyclo[5.4.0.02,6
.
03,10.05,9]undecane-8,11-dione (1), was synthesised according to
C
22H21N3O3; Mp: 213 °C; 1 H NMR (300 MHz, CDCl3) dH: 8.14–
8.11 (dd, 2H, J = 8.19, 0.89 Hz), 7.70–7.67 (dd, 2H, J = 8.38,
the published method (Cookson et al., 1964, 1958).5,24

8956
J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
0.94 Hz), 7.42–7.36 (m, 3H), 7.26–7.21 (m, 3H) 3.89–3.09 (2 ꢁ m,
4H), 2.95–2.62 (3 ꢁ m, 8H), 2.89–2.87 (d, 2H), 1.89–1.12 (2 ꢁ m,
4H). 13C NMR (75 MHz, CDCl3) dC: 166.60 (s), 152.23 (s), 134,75
(d), 131.93 (d), 114.29 (d), 110.68 (d), 109.74 (s), 63.05 (t), 55.52
(2 ꢁ d), 43.93 (d), 43.91 (t), 43.88 (t), 29.46 (q), 24.00 (t); MS (EI,
product as a white amorphous solid. (Yield: 288 mg, 0.768 mmol,
41.5%).
C
18H21N3O; Mp: 212 °C; 1 H NMR (300 MHz, CDCl3) dH: 8.14–
8.11 (dd, 2H, J = 8.19, 0.89 Hz), 7.70–7.67 (dd, 2H, J = 8.38,
0.94 Hz), 7.42–7.36 (m, 3H), 7.26–7.21 (m, 3H), 2.21 (s, 3H),
2.12–2.11 (d, 6H, J = 2.96 Hz), 1.76–1.75 (m, 6H). 13C NMR
(75 MHz, CDCl3) dC: 163.47 (s), 140.97 (s), 139.37 (m), 127.06
(m), 123.93 (s), 122.64 (m), 121.66 (d), 51.28 (s), 40.49 (3 ꢁ t),
35.65 (3 ꢁ t), 29.63 (s), 28.99 (3 ꢁ d); MS (EI, 70 eV) m/z: 295
70 eV) m/z: 364 (M+), 231, 230, 214, 134, 69, 43; IR (KBr) mmax
:
3377, 2957, 2360, 1687, 1520, 1343, 1226, 1180 cmꢀ1
.
4.2.3. 3-{4-Aza-8-oxo-heptacyclo[0.4.1.02,10.03,14.04,9 09,13
.
.-
012,15]tetradecyl}-1H-indazole-3-carboxylate (6)
(M+), 238, 135, 91, 43, 28; IR (KBr)
mmax: 3459, 2911, 2854, 1672,
1H-Indazole-3-carboxylic acid (0.702 g, 4.323 mmol), 3-hy-
droxy-4-aza-8-oxoheptacyclo-[9.4.1.02,10.03,14.04,9.09,13.012,15]-
tetradecane (0.702 g, 4.323 mmol) and dimethylaminopyridine
(0.09 g, 0.737 mmol) was dissolved in dried dichloromethane
(40 ml). The mixture was cooled to 5 °C using an external ice bath.
N,N0-dicyclohexylcarbodiimide (DCC; 1.5 g, 7.27 mmol) was added
in molar excess and the mixture was stirred for an additional 5 min
at 5 °C. Thereafter the mixture was stirred for 48 h at room temper-
ature. After 48 h the solvents were removed in vacuo and the res-
idue suspended in 50 ml water, extracted with DCM (3ꢁ 25 ml)
and dried over MgSO4. The solvent was removed and yielded a col-
ourless oil. Resolution of the product mixture was accomplished by
column chromatography with Ethyl acetate/DCM/PE, 1:1:1, and
with EtOH/THF, 1:1 (Rf = 0.69), yielding the product as a white
powder (Yield: 255 mg, 0.68 mmol, 16%).
1493, 1197, 856 cmꢀ1
.
4.2.6. N-(2,4-Dinitrophenyl)adamantan-1-amine (9)
1-Fluoro-2,4-dinitrobenzene (0.707 ml, 2.5 mmol), amantadine
hydrochloride (0.378 g, 2.5 mmol) and K2CO3 (0.691 g, 5 mmol)
was dissolved in 50 ml absolute acetonitrile. The pH was adjusted
to 8–9 with triethylamine. The reaction mixture was stirred in the
dark for 48 h, where after the mixture was filtered, the precipitate
extracted with DCM (3 ꢁ 25 ml) and dried over MgSO4. The solvent
was removed in vacuo rendering the product as a bright yellow
amorphous solid (Yield: 403 mg, 1.268 mmol, 50%).
C
16H19N3O4; Mp: 300 °C; 1H NMR (300 MHz, CDCl3): 9.13–9.12
(d, 2H, J = 2.77 Hz), 8.18–8.13 (dd, 3H, J = 3.40, 1.18 Hz), 7.25–7.23
(d, 2H, J = 9.80 Hz), 2.21 (s, 3H), 2.12–2.11 (d, 6H, J = 2.96 Hz), 1.76–
1.75 (m, 6H), 1.55 (br s, 1H, NH); 13 C NMR (75 MHz, CDCl3) dC:
147.76 (s), 135.39 (s), 129.17 (d), 124.86 (d), 116.20 (s), 54.28 (s),
42.09 (3 ꢁ t), 36.04 (3 ꢁ t), 29.80 (3 ꢁ d); MS (EI, 70 eV) m/z: 317
C
22H24N2O2; Mp: 190 °C; 1H NMR (300 MHz, CDCl3) dH: 7.91–
7.90 (dd, 2H, J = 9.58, 1.14 Hz), 7.38–7.24 (m, 3H), 6.64–6.61 (dd,
2H, J = 8.27, 0.79 Hz), 6.57–6.51 (m, 3H), 3.89–3.094 (2 ꢁ m, 4H),
2.95–2.62 (3 ꢁ m, 8H), 2.89–2.87 (d, 2H, J = 5.99 Hz), 1.81–1.50
(2 ꢁ m, 4H). 13C NMR (75 MHz, CDCl3) dC: 163.47 (s), 140.97 (s),
139.37 (m), 127.06 (m), 123.93 (s), 122.64 (m), 121.66 (d),
110.74 (s), 56.14 (t), 49.96 (2 ꢁ d), 49.12 (t), 49.12 (d), 33.89 (s),
30.94 (q), 24.90 (t); MS (EI, 70 eV) m/z: 375 (M+), 243, 224, 145,
(M+), 196, 135, 93, 41, 28; IR (KBr)
mmax: 3439, 2926, 2855, 2360,
1541, 1338, 1146, 831 cmꢀ1
.
4.2.7. N-(1-Cyano-2H-isoindol-2yl)adamantan-1-amine (10)
Amantadine hydrochloride (0.5 g, 3.301 mmol) and NaCN
(0.162 g, 3.301 mmol) was dissolved in 20 ml methanol/water. To
this was added o-phthaldialdehyde (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 filtered and resolution of the filtrate with
flash column chromatography (ethyl acetate/PE 1:1, Rf = 0.65)
yielded the product as a white amorphous solid (Yield: 338 mg,
1.223 mmol, 37%).
98, 56, 41, 28; IR (KBr)
mmax: 3277, 2931, 2851, 2360, 1625, 1448,
1242 cmꢀ1
.
4.2.4. N-Adamantan-1-yl-2(methylamino)benzamide (7)
N-Methylanthranilic acid (0.5 g, 3.308 mmol) was added to a
stirred solution of N,N0-carbonyldiimidazole (0.53 g 3.308 mmol)
in anhydrous tetrahydrofuran (25 ml). After 24 h amantadine
hydrochloride (0.5 g, 3.308 mmol) in tetrahydrofuran (10 ml) was
added and the pH was adjusted to 8–9 with triethylamine. The
mixture was allowed to react at room temperature. After 74 h
the reaction mixture was heated to ensure complete reaction.
The precipitate was filtered and washed with cold THF (2ꢁ
15 ml) yielding the pure product as a light yellow powder (Yield:
336 mg, 0.82 mmol, 25%).
C
19H20N2; Mp: 160 °C; 1H NMR (300 MHz, CDCl3): 7.67–7.61
(2 ꢁ m, 6H), 7.48 (s, 1H), 7.24–7.03 (2 ꢁ dd, 4H, J = 7.84, 1.01 Hz;
J = 8.64, 0.99 Hz), 2.44–2.43 (d, 6H, J = 3.19 Hz) 2.301 (s, 3H),
1.82–1.80 (m, 6H): 13 C NMR (75 MHz, CDCl3) dC: 133.72 (s),
125.20 (m), 122.61 (s), 122.25 (d), 120.76 (m), 117.67 (d) 60.29
(s), 42.90 (s), 42.85 (3 ꢁ t), 35.83 (3 ꢁ t), 29.89 (3 ꢁ d); MS (EI,
70 eV) m/z: 276 (M+), 135, 93, 41, 28; IR (KBr)
mmax: 3446, 2910,
C
18H24N2O; Mp: 209 °C; 1H NMR (300 MHz, CDCl3): 7.98–7.96
2852, 2360, 1624, 1182, 783 cmꢀ1
.
(dd, 2H, J = 7.48, 0.821 Hz), 7.49 (br s, 1H, NH), 7.29–7.24 (m,
3H), 6.60–6.52 (m, 3H), 6.59–6.57 (dd, 2H, J = 8.43, 1.28 Hz), 2.85
(d, 3H), 2.00 (s, 3H), 1.87– 1.60 (d, 6H), 1.56–1.47 (m, 6H); 13C
NMR (75 MHz, CDCl3) dC: 151.64 (s), 132.60 (d), 132.42 (d),
117.37 (s) 113.90 (d), 109.984 (d), 51.28 (s), 40.49 (3 ꢁ t), 35.65
(3 ꢁ t), 29.63 (s), 28.99 (3 ꢁ d); MS (EI, 70 eV) m/z: 284 (M+),
4.2.8. N-(1-Thiocyano-2H-isoindol-2yl)adamantan-1-amine
(11)
Amantadine hydrochloride (0.5 g, 3.301 mmol) and NaSCN
(0.27 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 mix-
ture was protected from light and stirred at room temperature for
48 h. The reaction mixture was filtered and resolution of the fil-
trate with flash column chromatography (ethyl acetate/PE 1:1,
Rf = 0.56) yielded the product as an orange amorphous solid (Yield:
210 mg, 0.68 mmol, 21%).
151, 135, 94, 41, 28; IR (KBr)
848, 755 cmꢀ1
mmax: 3338, 2924, 1635, 1454, 1172,
.
4.2.5. N-Adamantan-1-yl-1H-indazole-3-carboxamide (8)
To solution of 1H-indazole-3-carboxylic acid (0.3 g,
a
1.85 mmol) in DMF (7 ml) was added N,N0-carbonyldiimidazole
(0.33 g, 2.035 mmol). The resulting solution was warmed at 60 °C
for 2 h and then cooled to room temperature before adding a solu-
tion of amantadine hydrochloride (0.280 g, 1.85 mmol) in DMF
(3 ml) and triethylamine (0.77 ml). The resulting solution was re-
acted for 24 h at room temperature. Thereafter the precipitate
was filtered and washed with cold THF (2ꢁ 15 ml), yielding the
C
19H20N2S; Mp: 213 °C; 1H NMR (300 MHz, CDCl3) dH: 7.76 (s,
1H), 7.74–7.41 (2 ꢁ dd, 4H, J = 9.24, 1.88 Hz; J = 13.43, 6.80 Hz),
7.39–7.24 (2 ꢁ m, 6H), 2.29–2.28 (d, 6H, J = 20.29, 2.89 Hz), 2.13
(s, 3H), 1.79–1.67 (m, 6H); 13 C NMR (75 MHz, CDCl3) dC: 140.86
(s), 134.69 (m), 130.71 (s), 127.72 (d), 123.11 (m), 122.24 (d),
55.47 (s), 47.43 (s), 40.06 (3 ꢁ t), 36.35 (3 ꢁ t), 32.62 (3 ꢁ d); MS

J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
8957
(EI, 70 eV) m/z: 307 (M+), 261, 163, 135, 91, 41, 28; IR (KBr) mmax
3139, 2910, 2851, 2190, 1421, 1182, 783 cmꢀ1
:
Extractions and storage of tissue samples prior to the assay were
carried out at 0–4 °C to avoid loss of enzyme activity. Fresh rat brain
was weighed in 50 ml pre-cooled Falcon tubes and placed on ice.
After rinsing with ice cold extraction buffer, a measured volume of
extraction buffer (5 ml/g tissue) was added to the tissue. The sample
was then homogenised with a mechanical homogeniser while the
temperature was maintained at 4 °C. After 10 s of homogenisation
.
4.2.9. N-(1-Nitro-2H-isoindol-2yl)adamantan-1-amine (12)
Amantadine hydrochloride (0.5 g, 3.301 mmol) and NaNO2
(0.27 g, 3.301 mmol) was dissolved in 20 ml methanol/water. To
this was added o-phthaldialdehyde (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 72 h. The reaction mixture was filtered and resolution of the fil-
trate with flash column chromatography (ethyl acetate/PE 1:1,
Rf = 0.73) yielded the product as a light yellow amorphous solid
(Yield: 245 mg, 0.827 mmol, 25%).
the PMSF (10 lM/ml of extraction buffer) was added to the mixture
and it was homogenised for a further 30 s. The homogenate was then
centrifuged at 12,000g for 10 min. Once the supernatant was col-
lected, it was divided into 2 ml aliquots which were assayed imme-
diately or snap freezed and stored at ꢀ70 °C.
The oxyhaemoglobin solution was prepared by carefully dis-
C
18H20N2O2; Mp: 210 °C; 1H NMR (300 MHz, CDCl3) dH: 7.82 (s,
solving the haemoglobin crystals (25 mg) in 1000 ll of cold HEPES
1H), 7.75–7.60 (2 ꢁ dd, 2H, J = 8.32, 3.26 Hz; J = 8.24, 2.96), 7.48–
7.24 (2 ꢁ m, 2H), 2.27–2.12 (d, 6H, J = 10.02 Hz), 2.10 (s, 3H),
1.78–1.67 (m, 6H); 13C NMR (75 MHz, CDCl3) dC: 147.76 (s),
135.39 (m), 129.17 (s), 129.17 (d), 124.95 (m), 116.14 (d) 54.23
(s), 42.07 (3 ꢁ t), 36.08 (3 ꢁ t), 32.46 (3 ꢁ d); MS (EI, 70 eV) m/z:
buffer21 and subsequent reduction with excess sodium dithionate
(0.958 mg). The solution immediately changed from brownish
red (mixture of oxyHb and metHb) to a dark red (deoxyhaemoglo-
bin) colour after the reductant was added. Oxygenation was car-
ried out by blowing 100% oxygen over the surface while the
solution was gently swirled for 15 min. The gradual colour change
from dark red to bright red was indicative of the oxygenation of
haemoglobin. Desalting and purification was performed by passing
the resulting oxyHb solution through a Sephadex G-25 column.
The oxyHb is eluted as a single bright-red band. The front and back
edges were discarded.
297 (M+), 163, 135, 91, 41, 28. IR (KBr)
mmax: 3446, 2906, 1666,
1453, 1221, 730 cmꢀ1
.
4.3. Biological evaluation
4.3.1. Materials
All chemicals were of analytical grade or spectroscopy grade
and was purchased from Sigma–Aldrich (UK) and Merck (St. Louis,
MO, USA).
The concentration of oxyHb was calculated by methods de-
scribed by Feelisch26 and Hevel.27,28 Ten microlitres of the oxyHb
stock solution was added to 2990
and the absolute absorbance was determined in triplicate at
415 nm against a blank buffer. The concentration of oxyHb (CoxyHb
ll of HEPES buffer in a cuvette
4.3.2. Animals
)
The study protocol was approved by the Ethics Committee for
Research on Experimental Animals of the North-West University
(Potchefstroom Campus). Male Sprague–Dawley 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 snap freezed with liquid N2 and stored at ꢀ70 °C.
was calculated with the following Eq. (1) using a molar extinction
coefficient E414(oxyHb) of 131.0 mMꢀ1 cmꢀ1
A415 nm ꢁ 300 ðdilution factorÞ
.
CoxyHb
¼
ð1Þ
E415ðoxyHbÞ
Using the above equation, the final calculated oxyHb concentra-
tion was found to be 0.76 mM. The stock solution was then ali-
4.3.3. Methods
quoted in 200
ll units, snap freezed with liquid N2 and stored at
Spectrophotometric scans were recorded using a Varian Cary-
50Ò UV–vis spectrophotometer. The slope values were calculated
at specific wavelengths and calculated from the inhibition data.
All data analysis, calculation and graphs were done using Prism
4.02Ò (GraphPhad, Sorrento Valley, CA). All data are presented as
means SEM. Data analysis was carried out using a one-way anal-
ysis of variance, followed by the Student–Newman–Keuls multiple
range test. The level of significance was accepted at p < 0.05.
NOS assay procedure: HEPES buffer (100 mM) was prepared by
dissolving the HEPES in double-distilled water and brought to pH
7.4 by the addition of 4 N NaOH at 37 °C. This can be stored for sev-
eral weeks at 4 °C. The extraction buffer was prepared by dissolving
sucrose (320 mM), HEPES (20 mM), and ethylenediaminetetra-acetic
acid (1 mM) in double-distilled water and adjusting its pH to 7.4 at
room temperature by addition of 10% HCl.20 The following consti-
tuents were then added to the final concentrations indicated:
ꢀ70 °C.
Calcium chloride solution (CaCl2; 12.5 mM),
L
-arginine (1 mM)
and NADPH (5 mM) were prepared in HEPES buffer.
The test compounds were dissolved in HEPES buffer, methanol,
tetrahydrofuran or DMSO, the concentration of the organic sol-
vents did not exceed 2% of the final incubation concentrations. This
gave a series of concentrations in the micromolar range. 7-Nitroin-
dazole was dissolved in methanol and aminoguanidine was dis-
solved in the HEPES buffer. These two compounds were used as
the reference compounds in final concentrations ranging from
10
l
M to 10 mM.
Oxyhaemoglobin, CaCl2,
then diluted in the HEPES buffer to give final concentrations
250 M CaCl2 and 1 mM -arginine.
L-arginine and the test compound was
l
L
The reaction mixture was prewarmed for 3 min to the required
assay temperature of 37 °C and the reaction was started by the
0.1 mM D/L dithiotherol (DTT), 0.5
l
M leupeptin, soybean-trypsin
addition of NADPH and the tissue extract (100
rat brain homogenate) with a final NADPH concentration of
100 M. After establishing the baseline, continuous scans with a
scan rate of 600 nm/min every 10 s were recorded between 390
and 430 nm. The conversion of oxyHb to metHb was monitored
over a period of 10 min.
ll) (in the form of
inhibitor (10 g/ml) and aprotin (2
l
lg/ml). The extraction buffer
was then made up to its final volume with distilled water and
distributed into aliquots (typically 50 ml per aliquot) and stored
at ꢀ20 °C until required.
l
Phenylmethylsulfonyl fluoride (PMSF; 10 mg/ml) is unstable in
aqueous solution and is not included in the buffer at this stage, but
prepared as a solution in absolute ethanol, stored at ꢀ20 °C, and
added to the extraction buffer during the extraction procedure.
The composition of the extraction buffer is designed to permit
extraction of NOS from tissues without breaking intracellular
organelles and minimising proteolysis.25
4.4. Fluorescence spectrometry
A Cary EclipseÒ fluorescence spectrometer was used for fluores-
cence measurements. The fluorescent compounds were measured
at a concentration of 10ꢀ5 M in absolute ethanol at room temper-

8958
J. Joubert et al. / Bioorg. Med. Chem. 16 (2008) 8952–8958
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Pharmacol. 1995, 116, 2349.
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ature. Emission spectra were recorded at the excitation maximal
wavelength.
Acknowledgements
We are grateful to the National Research Foundation for finan-
cial support.
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