Discovery of N -(3-(1-Methyl-1,2,3,6-tetrahydropyridin-4-yl)-1 H -indol-6-yl) thiophene-2-carboximidamide as a selective inhibitor of human neuronal nitric oxide synthase (nNOS) for the treatment of pain

Article abstract of DOI:10.1021/jm201063u

3,6-Disubstituted indole derivatives were designed, synthesized, and evaluated as inhibitors of human nitric oxide synthase (NOS). Bulky amine containing substitution on the 3-position of the indole ring such as an azabicyclic system showed better selectivity over 5- and 6-membered cyclic amine substitutions. Compound (-)-19 showed the best selectivity for neuronal NOS over endothelial NOS (90-fold) and inducible NOS (309-fold) among the current series. Compounds 16 and (-)-19 were shown to be either inactive or very weak inhibitors of human cytochrome P450 enzymes, indicating a low potential for drug-drug interactions. Compound 16 was shown to reverse thermal hyperalgesia in vivo in the Chung model of neuropathic pain. Compound 16 was also devoid of any significant vasoconstrictive effect in human coronary arteries, associated with the inhibition of human eNOS. These results suggest that 16 may be a useful tool for evaluating the potential role of selective nNOS inhibitors in the treatment of pain such as migraine and CTTH.

Full text of DOI:10.1021/jm201063u

ARTICLE
pubs.acs.org/jmc
Discovery of N-(3-(1-Methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indol-6-yl)
thiophene-2-carboximidamide as a Selective Inhibitor of Human
Neuronal Nitric Oxide Synthase (nNOS) for the Treatment of Pain
Subhash C. Annedi,*,† Shawn P. Maddaford,† Gabriela Mladenova,† Jailall Ramnauth,† Suman Rakhit,†
John S. Andrews,† David K. H. Lee,‡ Dongqin Zhang,§ Frank Porreca,§ David Bunton,|| and Lee Christie||
†NeurAxon Inc., 2395 Speakman Drive, Suite 1001, Mississauga, Ontario, L5K 1B3, Canada
‡NoAb BioDiscoveries Inc., 2820 Argentia Road, Mississauga, Ontario, L5N 8G4, Canada
§Department of Pharmacology, University of Arizona, 1501 North Campbell Avenue, Tucson, Arizona 85724, United States
Biopta Ltd., Weipers Centre, Garscube Estate, Bearsden Road, Glasgow G61 1QH, U.K.
ABSTRACT: 3,6-Disubstituted indole derivatives were designed, synthesized, and
evaluated as inhibitors of human nitric oxide synthase (NOS). Bulky amine containing
substitution on the 3-position of the indole ring such as an azabicyclic system showed
better selectivity over 5- and 6-membered cyclic amine substitutions. Compound (À)-
19 showed the best selectivity for neuronal NOS over endothelial NOS (90-fold) and
inducible NOS (309-fold) among the current series. Compounds 16 and (À)-19 were
shown to be either inactive or very weak inhibitors of human cytochrome P450
enzymes, indicating a low potential for drugÀdrug interactions. Compound 16 was
shown to reverse thermal hyperalgesia in vivo in the Chung model of neuropathic pain.
Compound 16 was also devoid of any significant vasoconstrictive effect in human
coronary arteries, associated with the inhibition of human eNOS. These results suggest that 16 may be a useful tool for evaluating the
potential role of selective nNOS inhibitors in the treatment of pain such as migraine and CTTH.
’ INTRODUCTION
the headache pain intensity on the visual analogue scale (2 h after
the treatment) when compared to placebo, however the effect was
less pronounced when compared to the migraine study.13 Com-
pounds 2 and 3 are the most extensively studied nonpeptidic NOS
inhibitors and provided the foundation for elucidating the phar-
macological role of selective nNOS inhibitors in several disorders
including nociception, opioid-induced side effects, antidepressant,
and anxiolytic properties (Figure 1).14À16 Compound 4 is also a
selective nNOS inhibitor has been shown to provide greater
neuroprotection (44%) than 2 (22%) or 3 (8%) in a gerbil model
of global cerebral ischemia.17 The selective inhibition of nNOS or
iNOS over the eNOS isoform is necessary in order to avoid the
cardiovascular liabilities associated with eNOS.18,19 Numerous
attempts have been made toward the design and synthesis of
selective NOS inhibitors targeting both the L-arginine and BH4
binding sites.20À22 The early NOS inhibitor design, which is
based on L-arginine (substrate), produced only nonselective
NOS inhibitors. However, subsequent designed inhibitors based
on the available crystal structures of NOS enzymes23 that targeted
the arginine binding site or BH4 cofactor site as well as dimeriza-
tion inhibitors have been shown to be more potent and selective
among NOS isoforms.20À22
Nitric oxide (NO) is one of the most studied cell-signaling
molecules, believed to be involved in a number of physio-
logical processes such as inflammation, regulation of blood
pressure, opioid tolerance, immunity, and neurotransmission.1,2
Nitric oxide synthase (NOS) converts L-arginine to L-citrulline
and produces NO, utilizing NADPH and O2 as substrates and
flavin adenine dinucleotide (FAD), flavin mononucleotide
(FMN), (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-
tetrahydropteridin-4(1H)-one (BH4), and heme as cofactors.3,4
NOS consist of three isoforms: neuronal NOS (nNOS or NOS1),
a constitutive form thought to play a role in neurotransmission;
endothelial NOS (eNOS or NOS3), which is also constitutive
and plays a role in regulating vascular tone and platelet aggrega-
tion; inducible NOS (iNOS or NOS2), which is generally
produced during bacterial infection, tumor cell cytolysis, and
inflammation.
Excessive NO or production by individual isoforms such as
nNOS and iNOS plays an important role in the development of
several disorders including septic shock, stroke, neurodegenera-
tive disorders (Parkinson’s, ALS and MS), and pain (migraine,
CTTH, visceral, and neuropathic).5À11 For example, 1, a non-
selective NOS inhibitor, was able to show 67% antimigraine
effect compared to placebo in a randomized double-blind clinical
study with 15 migraine patients.12 A similar randomized double-
blind, crossover clinical study was conducted on 16 patients with
CTTH using 1 (6 mg/kg). Compound 1 significantly reduced
The simplified pharmacophore model adapted by our group
for competitively inhibiting NOS at the substrate binding site
Received: August 8, 2011
Published: September 16, 2011
r
2011 American Chemical Society
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Scheme 2a
a (a) N-Methyl-4-piperidone, pyrrolidine, EtOH, reflux or quinuclidin-
3-one HCl, KOH, MeOH:H2O, reflux; (b) (i) H2N-NH2 H2O, Raney-
3
3
Ni, MeOH, reflux or Pd-C/H2, EtOH, rt; (ii) 11, EtOH, rt; (c) SFC
chiral chromatographic separation.
As part of our ongoing efforts to find small molecule selective
nNOS inhibitors,24,26À28 herein we report the synthesis
and biological activity evaluations of 3,6-disubstituted indole
derivatives that led to the identification of 16 as a lead candidate
among the series. The results disclosed in this report identify 16
as a candidate to investigate the role of selective nNOS inhibitors
for the treatment of neuropathic pain and potentially primary
headaches such as migraine and CTTH.
Figure 1. Most studied literature examples (1À3) and thiophene
amidine compounds (4À6) with basic amine side chain.
Scheme 1a
’ RESULTS AND DISCUSSION
Chemistry. 3,6-Disubstituted indole derivatives were pre-
pared as shown in Schemes 1 and 2. The substitution on
3-position of the indole ring was introduced by a key carbonÀ
carbon bond formation reaction between the protected 6-amino
indole 8 and N-methylmaleimide under acidic conditions
(Scheme 1).30 Reduction of both the 1-methyl-2,5-dioxopyrro-
lidine ring and N-benzoyl group was carried out with LiAlH4 at
room temperature to obtain compound 10. Deprotection of the
benzyl group under hydrogenation conditions followed by
coupling with the thiophene-2-carbimidothioate 1131 provided
the final compound 12.
a (a) BzCl, Et3N, THF, rt; (b) N-methylmaleimide, AcOH, reflux; (c)
LiAlH4, THF, rt; (d) (i) Pd(OH)2, EtOH, rt, (ii) 11, EtOH, rt.
Condensation of 6-nitro-1H-indole (13) with either N-methyl-
4-piperidone or quinuclidin-3-one hydrochloride was carried out
under basic conditions to obtain the 3-substituted indole deri-
vatives 14 and 15, respectively (Scheme 2).32,33 Reduction of the
nitro group in 14 was performed under hydrogenation condi-
tions with hydrazine hydrate. A mixture of two intermediate
amines (5:2) was observed due to the partial reduction of the
1-methyl-1,2,3,6-tetrahydropyridine ring attributable to the pro-
longed reaction conditions. The mixture of amines was coupled
to the thiophene-2-carbimidothioate 11 to obtain the final
compounds 16 and 17 in 50% and 20%, respectively, which
were separated by column chromatography on silica gel.
The reduction of nitro group in compound 15 was performed
under hydrogenation conditions with hydrazine hydrate to
obtain the amine intermediate, which was then coupled to the
thiophene-2-carbimidothioate 11 to obtain the final compound
18 (Scheme 2). To avoid the reduction of the 1-azabicyclo-
[2.2.2]oct-2-ene ring system, the reaction was heated to reflux for
a maximum of 5 min in a preheated oil bath. The consumption of
the starting nitro compound 15 was evident by the disappearance
of the yellow color of the solution. Reduction of both the nitro
contains a guanidine isosteric group and a basic amine group
both attached to a central aryl scaffold.24 The amidine group
makes an important bidentate interaction with the conserved
glutamic acid residue to achieve the necessary potency, whereas
the basic amine (4À6) is shown to contribute to nNOS isoform
selectivity.24À28 However, this model is somewhat simplified
because several other factors are likely important for achieving
isoform selectivity.22,23 Our design strategy is based on an indole
core, which has been an important structural component in many
pharmaceutical agents and also referred to as “privileged struc-
ture” (capable of binding to many receptors).29 In our previous
reports with 2-aminobenzothiazoles (5)24 and disubstituted
indole derivatives (6),26,27 we were able to show that the
selectivity for nNOS isoform over eNOS and iNOS isoforms
can be increased by attaching a bulky or cyclic amine side chain to
the central aryl scaffold. On the basis of these results, in our
present strategy, selected 5- and 6-membered and bicyclic amine
side chains were introduced onto the 3-position of the indole ring
while keeping the thiophene amidine group fixed at 6-position.
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Table 1. Inhibition of Human NOS Enzymes by 3,6-Disubstituted Indole Derivatives
a Values reported in parentheses are 95% confidence intervals. In a radiometric method, inhibitory activities were measured by the conversion of
[3H]-L-arginine into [3H]-L-citrulline. b Compound 1 and L-NAME are known nonselective NOS inhibitors; tested for comparison. C Compound 6 is
previously reported selective nNOS inhibitor, included for comparison.
group as well as the 1-azabicyclo[2.2.2]oct-2-ene ring system was
carried out under standard hydrogenation conditions with
palladium on carbon to provide the amine intermediate, which
was reacted with thiophene-2-carbimidothioate 11 to obtain the
final compound 19 as a mixture of enantiomers (Scheme 2). The
racemic mixture 19 was separated using semipreparative chiral
HPLC techniques to obtain the pure enantiomers (À)-19 and
(+)-19. The absolute stereochemistry of the separated enantio-
mers was not determined at this time.
StructureÀActivity Relationship (SAR) Studies. All com-
pounds were converted into their corresponding dihydrochlor-
ide salts to improve their water solubility. Table 1 displays the in
vitro activity of 3,6-disubstituted indole derivatives, determined
as IC50 values against all three human NOS isoforms. Human
NOS inhibitory activities were determined by measuring the
conversion of [3H]-L-arginine to [3H]-L-citrulline using a radio-
metric assay. The current design strategy of attaching a mono-
and bicyclic basic amine at 3-position of the indole ring is adapted
from our previous results with 2-aminobenzothiazole and sub-
stituted indole derivatives, where bulky and cyclic amine sub-
stitutions provided better potency and selectivity for the nNOS
isoform.24,26,27 It was observed that a bulky substitution such as
an azabicyclic system in 18, (()-19, (À)-19, and (+)-19 showed
better selectivity over 5- and 6-membered cyclic amine substitu-
tion in 12, 16, and 17 and is consistent with our previous
observation with 2-aminobenzothiazole and substituted indole
derivatives.24,26,27 At the same time, the 6-membered cyclic
amine in 17 improved the potency and selectivity relative to
5-membered cyclic amine substitution in 12. Compound (À)-19
with an azabicyclic amine showed the best selectivity for human
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Table 2. Inhibition of Human Cytochrome P450 Enzymes by
16 and (À)-19
IC50 (μM)
CYP subtype
substrate
16
(À)-19
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
CYP3A4
CEC
MFC
CEC
AMMC
BFC
>100
∼72.7
40.1
NAa
NAa
∼49.6
>100
∼100
>100
∼63.2
74.8
BQ
>100
a NA: no inhibition observed over the concentration range tested.
Figure 2. Compound 16 attenuates thermal hyperalgesia in the L5/L6
SNL model of neuropathic pain in a dose-dependent manner.
nNOS over eNOS (90-fold) and iNOS (309-fold) among the
current series. On the basis of their potency and selectivity,
compounds 16 and (À)-19 were selected for further evaluation
in various in vitro and in vivo assays.
relaxation is due to the inhibition of eNOS, the addition of L-
arginine (substrate for eNOS) would recover the relaxation due
to ACh. ACh-mediated vasorelaxation effects of L-NAME
(positive control) and 16 were tested (1 μM, 10 μM, 100
μM) in the absence and presence of L-arginine to determine
whether any effects were due to the inhibition of human eNOS.
L-NAME at all concentrations inhibited responses to ACh in a
dose-dependent manner (Figure 3A). The inhibitory effect of
L-NAME (100 μM) was reversed with the addition of L-arginine
(1 mM), which is a substrate for eNOS (Figure 3B). No
significant inhibition with 16 on the ability of the blood vessels
to respond to ACh was observed at any tested concentration
(Figure 4A). Furthermore, the addition of L-arginine did not
significantly affect the responses to ACh in the presence of 16,
providing further evidence that 16 did not have an effect on
agonist-mediated activation of human eNOS (Figure 4B). The
data suggests that 16 would be devoid of any vasoconstrictive
effect at concentrations that are physiologically relevant based
on its human eNOS potency. The lack of vasoconstrictive effect
of 16 is consistent with its weak inhibitory potency in the human
eNOS enzyme (IC50 = 26.5 μM).
Cytochrome P450 Enzyme Inhibition Studies. Cytochrome
P450 enzyme inhibition studies were performed to assess
the potential for metabolism-based drugÀdrug interactions
(Table 2), which is an inevitable hurdle during the drug devel-
opment process.34 This will also rule out the inhibitory activity
with cytochrome P450 enzymes, where compounds that bind to
heme iron (for example, imidazole-containing compounds)35
have shown to be potent inhibitors of cytochrome P450 enzymes
that are closely related to NOS. Hence, compounds 16 and (À)-
19 were tested in a range (0.0457À100 μM) of concentrations
against the five major human cytochrome P450 enzymes and the
IC50 values were reported in Table 2. Both compounds showed
very little drugÀdrug interaction potential, as the compounds are
not active or very weak inhibitors of the five major human
cytochrome P450 enzymes.
The Chung or Spinal Nerve Ligation (SNL) Model Studies.
On the basis of NOS inhibitory and selectivity data and in vitro
CYP inhibition data, 16 was selected for further profiling in an
in vivo model of neuropathic pain (Figure 2).36 The Chung or
spinal nerve ligation (SNL) model involves ligation of both the
L5 and L6 spinal nerves of one side of the rat, which will produce
thermal and mechanical allodynia of the affected foot. With-
drawal latency to application of radiant heat to the paw was
tested. Intraperitoneal administration of 16 at 10 mg/kg and
60 mg/kg resulted in reversal of thermal hyperalgesia in a dose-
dependent manner. A reversal of thermal hyperalgesia was
observed between 60 and 120 min, with a maximum effect of
67% reversal at 90 min following a single dose of 60 mg/kg
(intraperitoneal).
eNOS Mediated Vasoconstriction Effect on Isolated
Human Resistance Arteries. Compound 16 was assessed for
the contractile response (inhibition of acetylcholine-mediated
vasorelaxation) on isolated human resistance arteries to assess
the undesirable cardiovascular effect associated with the inhibi-
tion of eNOS in comparison to a known nonselective NOS
inhibitor, L-nitro arginine methyl ester (L-NAME) (Figures 3
and 4) and a negative (vehicle) control.18,19 The arteries were
preconstricted with U46619, a thromboxane A2 (TxA2) mi-
metic agent, and then exposed to acetylcholine (ACh), an
endothelium, and nitric oxide dependent vasodilator. The
response to ACh provides the information on the activity of
eNOS in an active human biological tissue and thereby any
inhibitory effect of 16 on eNOS. Furthermore, if inhibition of
’ CONCLUSIONS
In conclusion, a series of selective 3,6-disubstitued indole
derivatives were synthesized and shown to be selective inhibitors
of human nNOS over eNOS and iNOS isoforms. A highly
bulky amino-substituent in the 3-position of the indole ring such
as an azabicyclic amine provided better selectivity over the
smaller 5- or 6-membered cyclic amine substitution. Compounds
16 and (À)-19 with better selectivity for human nNOS over
eNOS were shown to be inactive or very weak inhibitors of
human cytochrome P450 enzymes, indicating an unlikely poten-
tial for CYP-450 dependent drugÀdrug interactions. Intraper-
itoneal administration of 16 was also shown to reverse the
thermal hyperalgesia in the SNL model of neuropathic pain.
The lack of inhibition of ACh-mediated vasorelaxation in isolated
human resistance arteries by 16 mitigates a cardiovascular effect
associated with the inhibition of human eNOS. The significant
results with 16 presented in this communication provide an
opportunity to investigate the role that the nNOS enzyme plays
in neuropathic pain as well as in other clinical indications of pain
such as migraine and CTTH.
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Figure 3. (A) Effect of L-NAME on the responses to ACh. (B) Effect of L-arginine (1 mM) on response to ACh in the presence of L-NAME.
Figure 4. (A) Effect of 16 on the responses to ACh. (B) Effect of L-arginine (1 mM) on response to ACh in the presence of 16.
’ EXPERIMENTAL SECTION
to obtain the title compound (3.55 g, 99%) as a solid. 1H NMR (CDCl3)
δ 6.53 (t, 1H, J = 2.4 Hz), 6.95 (dd, 1H, J = 2.1, 8.4 Hz), 7.21 (t, 1H, J =
2.7 Hz), 7.48À7.60 (m, 4H), 7.88À7.92 (m, 3H), 8.27 (brs, 2H). ESI-
MS (m/z, %): 259 (M + Na, 80), 237 (MH+, 100).
All the reactions were performed under an atmosphere of argon and
stirred magnetically unless otherwise noted. Commercial reagents and
anhydrous solvents were used as received without further purification.
Reactions were monitored by analytical TLC using precoated silica gel
aluminum plates (Sigma-Aldrich, 0.2 mm, 60 Å) and were visualized
with UV light or stained appropriately. Flash column chromatography
was performed using Silicycle Siliaflash F60 (40À63 μm) silica gel. The
1H NMR spectra were obtained on a Bruker 300 MHz spectrometer.
Low and high resolution mass spectra were obtained on an applied
Biosystems/MDS Sciex QstarXL hybrid quadrupole/TOF instrument
using electrospray ionization. Chemical purity was determined by
Agilent 1100 HPLC system using Zorbax, SB-C18 reverse phase
column, and the purity was determined to be g95% until unless
specified. The chiral purity was determined by CHIRALPAK AD-H
column and determined to be g99%. No attempts were made to
optimize the yields.
N-(3-(1-Methyl-2,5-dioxopyrrolidin-3-yl)-1H-indol-6-yl)-
benzamide (9). A solution of N-(1H-indol-6-yl)benzamide (8) (3.5 g,
14.81 mmol) and N-methylmaleimide (4.07 g, 37.03 mmol) in glacial
acetic acid (100 mL) was refluxed for 56 h. The reaction was brought to
room temperature, and acetic acid was evaporated. The crude was taken
into ethyl acetate, washed with satd NaHCO3 solution and brine and
dried (Na2SO4). Solvent was evaporated, and crude was purified by
column chromatography (EtOAc:hexanes, 1:3 to 1:1) on silica gel to
obtain the title compound (2.32 g, 45%) as a foam. 1H NMR (DMSO-
d6) δ 2.80 (dd, 1H, J = 5.1, 18.0 Hz), 2.92 (s, 3H), 3.23 (dd, 1H, J = 9.3,
18.0 Hz), 4.34 (dd, 1H, J = 5.1, 9.3 Hz), 7.26À7.36 (m, 2H), 7.45À7.60
(m, 4H), 7.95À7.98 (m, 2H), 8.07 (s, 1H), 10.17 (s, 1H), 11.02 (s, 1H).
ESI-MS (m/z, %): 370 (M + Na, 100), 348 (MH+, 58).
N-(1H-Indol-6-yl)benzamide (8). A solution of 1H-indol-6-
amine (7) (2.0 g, 15.13 mmol) in dry THF (30 mL) was treated with
Et3N (6.32 mL, 45.39 mmol), followed by benzoyl chloride (1.84 mL,
15.88 mmol) at 0 °C. The reaction was brought to room temperature
and stirred for 1 h. The reaction was diluted with water and product was
extracted into ethyl acetate. The combined ethyl acetate layer was
washed with brine, dried (Na2SO4), and solvent was evaporated to
obtain the crude product. The crude was diluted with ethyl acetate
(25 mL) followed by hexanes (150 mL), and the precipitate was filtered
N-Benzyl-3-(1-methylpyrrolidin-3-yl)-1H-indol-6-amine
(10). A solution of N-(3-(1-methyl-2,5-dioxopyrrolidin-3-yl)-1H-indol-
6-yl)benzamide (9) (2.28 g, 6.56 mmol) in dry THF (30 mL) was
treated with LiAlH4 (2.49 g, 65.63 mmol) portionwise over a period of
45 min at 0 °C. The reaction was brought to room temperature and
stirred for 48 h. The reaction was quenched with sodium sulfate
decahydrate (8.0 g), followed by careful addition of water (9 mL) at
0 °C, and stirred for 30 min at room temperature. The reaction was
diluted with ethyl acetate, filtered, and washed with ethyl acetate.
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The combined ethyl acetate layer was evaporated, and the crude was
purified by column chromatography (2 M NH3 in MeOH:CH2Cl2, 5:95
to 1:9) on silica gel to obtain the title compound (0.44 g, 22%) as a foam.
1H NMR (DMSO-d6) δ 1.76À1.87 (m, 1H), 2.11À2.23 (m, 1H), 2.27
(s, 3H), 2.36 (t, 1H, J = 8.4 Hz), 2.42À2.48 (m, 1H), 2.64À2.71 (m,
1H), 2.90 (t, 1H, J = 8.1 Hz), 3.37À3.45 (m, 1H), 4.26 (d, 2H, J = 5.7
Hz), 5.88 (t, 1H, J = 6.0 Hz), 6.34 (d, 1H, J = 0.9 Hz), 6.45 (dd, 1H, J =
1.8, 8.4 Hz), 6.72 (d, 1H, J = 1.2 Hz), 7.17À7.38 (m, 6H), 10.11 (s, 1H).
ESI-MS (m/z, %): 306 (MH+, 100). ESI-HRMS calculated for
C20H24N3 (MH+), 306.1964; observed, 306.1967.
N-(3-(1-Methylpyrrolidin-3-yl)-1H-indol-6-yl) thiophene-2-
carboximidamide (12). A solution of N-benzyl-3-(1-methylpyrroli-
din-3-yl)-1H-indol-6-amine (10) (0.42 g, 1.37 mmol) in absolute
ethanol (5 mL) was treated with 20% Pd(OH)2 on carbon (0.5 g),
purged with hydrogen gas, and stirred under hydrogen (balloon
pressure) for 48 h. The reaction was filtered through a pad of Celite
and washed with ethanol. The combined ethanol layer was treated with
methyl thiophene-2-carbimidothioate hydroiodide (11) (0.78 g, 2.75
mmol) at room temperature and stirred for 48 h. The reaction was
basified with satd NaHCO3 solution, and product was extracted into
CH2Cl2. The combined CH2Cl2 layer was washed with brine and dried
(Na2SO4). Solvent was evaporated, and crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2, 1:9) on silica gel to
obtain the title compound (0.285 g, 64%) as a solid. 1H NMR (DMSO-
d6) δ 1.86À1.95 (m, 1H), 2.18À2.27 (m, 1H), 2.45À2.59 (m, 2H),
2.68À2.76 (m, 1H), 2.96 (t, 1H, J = 8.1 Hz), 3.45À3.56 (m, 1H), 6.29
(brs, 2H), 6.54 (dd, 1H, J = 1.2, 8.1 Hz), 6.78 (s, 1H), 6.99 (d, 1H, J = 1.8
Hz), 7.09 (t, 1H, J = 4.2 Hz), 7.49 (d, 1H, J = 8.1 Hz), 7.58 (d, 1H, J = 4.8
Hz), 7.71 (d, 1H, J = 3.6 Hz), 10.51 (s, 1H). ESI-MS (m/z, %): 325
(MH+, 38), 282 (31), 163 (100). ESI-HRMS calculated for C18H21N4S
(MH+), 325.1481; observed, 325.1495.
The combined organic layer was evaporated, and crude was purified by
column chromatography (2 M NH3 in MeOH:CH2Cl2, 1:9) on silica gel
to obtain the intermediate mixture of amines in 5:2 ratio.
A solution of the above intermediate mixture of amines in dry EtOH
(10 mL) was treated with methyl thiophene-2-carbimidothioate hydro-
iodide (11) (0.33 g, 1.165 mmol) at room temperature and stirred
for 24 h. The reaction was worked-up and purified as described for 12 to
obtain the title compounds 16 (0.085 g, 50%) and 17 (0.04 g, 20%).
Compound 16, foam; 1H NMR (DMSO-d6) δ 2.28 (s, 3H), 2.50À2.57
(m, 4H), 3.00À3.04 (m, 2H), 6.09 (s, 1H), 6.31 (brs, 1H), 6.59 (dd, 1H,
J = 1.2, 8.4 Hz), 6.82 (s, 1H), 7.09 (dd, 1H, J = 3.6, 4.9 Hz), 7.24 (d, 1H,
J = 2.1 Hz), 7.59 (d, 1H, J = 5.1 Hz), 7.70À7.73 (m, 2H), 10.85 (s, 1H).
1
ESI-MS (m/z, %), 337 (M+, 100). Compound 17, foam; H NMR
(DMSO-d6) δ 1.62À1.75 (m, 2H), 1.90À1.94 (m, 2H), 2.02À2.09
(m, 2H), 2.22 (s, 3H), 2.64À2.72 (m, 1H), 2.85À2.89 (m, 2H), 6.31
(brs, 1H), 6.53 (dd, 1H, J = 1.2, 8.2 Hz), 6.79 (s, 1H), 6.94 (d, 1H, J = 1.8
Hz), 7.09 (dd, 1H, J = 3.6, 4.9 Hz), 7.45 (d, 1H, J = 8.4 Hz), 7.59 (d, 1H,
J = 4.2 Hz), 7.72 (d, 1H, J = 3.6 Hz), 10.53 (brs, 1H). ESI-MS (m/z, %),
339 (M+, 100).
N-(3-(1-Azabicyclo[2.2.2]oct-2-en-3-yl)-1H-indol-6-yl)thi-
ophene-2-carboximidamide (18). 3-(6-Nitro-1H-indol-3-yl)-1-
azabicyclo[2.2.2]oct-2-ene (15) (0.4 g, 1.48 mmol) in dry methanol
(10 mL) was treated with Raney nickel (∼0.05 g) and hydrazine hydrate
(0.46 mL, 1.02 mmol). The resulting mixture was placed in a preheated
oil bath and refluxed for 2 min or until yellow color disappears. The
reaction was worked-up and purified as described for 16 and 17 to obtain
the amine intermediate (3-(1-azabicyclo[2.2.2]oct-2-en-3-yl)-1H-indol-
1
6-amine) (0.35 g, quantitative) as a foam. H NMR (DMSO-d6) δ
1.42À1.54 (m, 2H), 1.62À1.74 (m, 2H), 2.52À2.56 (m, 2H),
2.86À2.94 (m, 2H), 2.98À3.02 (m, 1H), 4.75 (s, 2H), 6.41 (dd, 1H,
J = 2.1, 8.4 Hz), 6.53 (d, 1H, J = 1.8 Hz), 6.69 (s, 1H), 7.16 (d, 1H, J = 2.4
Hz), 7.35 (d, 1H, J = 8.4 Hz), 10.57 (s, 1H). ESI-MS (m/z, %): 240
(MH+, 100).
Prepared from the above amine intermediate (3-(1-azabicyclo[2.2.2]-
oct-2-en-3-yl)-1H-indol-6-amine) (0.33 g, 1.37 mmol) and methyl
thiophene-2-carbimidothioate hydroiodide (11) (0.78 g, 2.75 mmol)
as described for 12 to obtain the title compound (0.42 g, 81%) as a solid.
1H NMR (DMSO-d6) δ 1.44À1.56 (m, 2H), 1.66À1.78 (m, 2H),
2.52À2.58 (m, 2H), 2.89À2.98 (m, 2H), 3.04À3.10 (m, 1H), 6.29
(s, 2H), 6.61 (dd, 1H, J = 1.8, 8.7 Hz), 6.78 (s, 1H), 6.83 (s, 1H), 7.09
(t, 1H, J = 4.2 Hz), 7.39 (d, 1H, J = 2.4 Hz), 7.59 (d, 1H, J = 4.8 Hz), 7.63
(d, 1H, J = 8.4 Hz), 7.72 (d, 1H, J = 3.3 Hz), 10.97 (s, 1H). ESI-MS (m/z
%): 349 (MH+, 95), 161 (100). ESI-HRMS calculated for C20H21N4S
(MH+), 349.1481; observed, 349.1494.
3-(1-Methyl-1,2,3,6-tetrahydro-pyridin-4-yl)-6-nitro-1H-
indole (14). A solution of 6-nitro-1H-indole (13) (0.5 g, 3.08 mmol)
in dry EtOH (5 mL) was treated with pyrrolidine (0.77 mL,
9.25 mmol) and N-methyl-4-piperidone (0.75 mL, 6.16 mmol) at
room temperature, and the resulting solution was refluxed for 48 h.
The reaction was brought to room temperature, further cooled to 0 °C,
and the solid was filtered off. The solid was washed with ethanol and
dried to obtain the title compound (0.567 g, 72%). 1H NMR (DMSO-
d6) δ 2.28 (s, 3H), 2.50À2.58 (m, 4H), 3.00À3.05 (m, 2H), 6.18 (s,
1H), 7.83 (s, 1H), 7.89 (dd, 1H, J = 2.1, 9.0 Hz), 7.97 (d, 1H, J = 9.0
Hz), 8.31 (d, 1H, J = 2.1 Hz), 11.88 (brs, 1H). ESI-MS (m/z, %): 258
(M+, 100).
3-(6-Nitro-1H-indol-3-yl)-1-azabicyclo[2.2.2]oct-2-ene (15).
A solution of 6-nitro-1H-indole (13) (1.0 g, 6.16 mmol) in MeOH:H2O
(20 mL, 1:1) was treated with KOH (1.73 g, 30.83 mmol), followed by
3-quinoclidine hydrochloride (1.99 g, 12.33 mmol) at room temperature,
and the resulting dark-brown mixture was refluxed for 36 h. The reaction
was brought to room temperature, filtered, and washed with MeOH:H2O
(3 Â 5 mL, 1:1), followedbymethanol(5mL). Theyellowsolid wasdried
N-(3-(Quinuclidin-3-yl)-1H-indol-6-yl)thiophene-2-carboxi-
midamide (19). A solution of 3-(6-nitro-1H-indol-3-yl)-1-azabicyclo-
[2.2.2]oct-2-ene (15) (0.4 g, 1.48 mmol) in dry ethanol (10 mL) was
treated with PdÀC (∼0.05 g), purged with hydrogen gas, and stirred for
36 h at room temperature under hydrogen (balloon pressure). The
reaction was filtered through a pad of Celite and was washed with
ethanol. The combined ethanol layer was treated with methyl thio-
phene-2-carbimidothioate hydroiodide (11) (0.84 g, 2.97 mmol) at
room temperature and stirred overnight (14 h). The reaction was
worked-up and purified as described for 12 to obtain the title compound
1
under vacuum to obtain the title compound (1.4 g, 84%). H NMR
(DMSO-d6) δ 1.46À1.58 (m, 2H), 1.68À1.76 (m, 2H), 2.52À2.58 (m,
2H), 2.89À2.97 (m, 2H), 3.08À3.12 (m, 1H), 6.89 (d, 1H, J = 1.2 Hz),
7.91À7.97 (m, 3H), 8.32 (d, 1H, J = 1.2 Hz), 11.98 (s, 1H). ESI-MS
(m/z, %): 270 (MH+, 100).
N-[3-(1-Methyl-1,2,3,6-tetrahydro-pyridin-4-yl)-1H-indol-
6-yl]-thiophene-2-carboxamidine (16) and N-[3-(1-Methyl-
piperidin-4-yl)-1H-indol-6-yl]-thiophene-2-carboxamidine
(17). A solution of 3-(1-methyl-1,2,3,6-tetrahydro-pyridin-4-yl)-6-ni-
tro-1H-indole (14) (0.15 g, 0.58 mmol) in dry MeOH (5 mL) was
treated with Raney nickel (∼0.05 g) and hydrazine hydrate (0.18 mL,
5.82 mmol) at room temperature, and the resulting mixture was refluxed
for 3 h. The reaction was brought to room temperature, filtered though
a pad of Celite, and washed with MeOH: H2Cl2 (1:1, 2 Â 10 mL).
1
(0.4 g, 77%) as a solid. H NMR (DMSO-d6) δ 1.20À1.28 (m, 1H),
1.56À1.74 (m, 3H), 2.62À2.72 (m, 1H), 2.79À2.92 (m, 3H),
3.09À3.34 (m, 4H), 6.30 (s, 2H), 6.53 (d, 1H, J = 8.4 Hz), 6.79 (s,
1H), 7.07À7.10 (m, 2H), 7.38 (d, 1H, J = 8.4 Hz), 7.58 (d, 1H, J = 4.5
Hz), 7.72 (d, 1H, J = 3.0 Hz), 10.61 (s, 1H). ESI-MS (m/z, %): 351
(MH+, 38), 176 (100). ESI-HRMS calculated for C20H23N4S (MH+),
351.1637; observed, 351.1637.
N-(3-(Quinuclidin-3-yl)-1H-indol-6-yl)thiophene-2-carboxi-
midamide ((-)-19 and (+)-19). N-(3-(Quinuclidin-3-yl)-1H-indol-
6-yl)thiophene-2-carboximidamide (19) was separated using chiral
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Journal of Medicinal Chemistry
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Table 3. Gradient System Used for Determining the Chemi-
cal Purity of All Final Compounds
Table 4. The Chemical and Chiral Purity of All Final
Compounds
compound
retention
chemical
retention
chiral
time (min)
0.1% TFA in acetonitrile (%)
0.1% TFA in water (%)
time (min)
purity (%)
time (min)
purity (%)
0
0
30
50
30
0
100
70
5À7
7.5À10
12
12
7.46
7.59
7.63
7.69
7.86
9.14
9.15
93.2
96.5
96.2
92.7
97.4
95.2
96.8
NAa
NAa
NAa
NAa
NAa
6.92
33.93
NAa
NAa
NAa
NAa
NAa
100
50
16
70
17
14À15
100
18
(()-19
(À)-19
(+)-19
HPLC (30% 2-propanol (0.1% DEA)/CO2, 100 bar). Column: Chir-
alcel OD-H (2 cm  20 cm) SN 06À6079. Flow rate 65 mL/min.
Wavelength 220 nm. Injection volume 0.3À0.5 mL, 7.3 mg/mL
methanol. First eluting enantiomer (À)-19: [α]D = À11.88 (0.706 g/
100 mL, MeOH). 1H NMR (MeOH-d4) δ 1.45À1.59 (m, 1H),
1.82À2.06 (m, 3H), 2.12 (brs, 1H), 2.92À2.99 (m, 1H), 3.06À3.16
(m, 4H), 3.44À3.49 (m, 2H), 6.74 (dd, 1H, J = 1.8, 8.4 Hz), 7.01 (d, 1H,
J = 1.5 Hz), 7.13 (dd, 1H, J = 3.6, 5.1 Hz), 7.17 (s, 1H), 7.53 (d, 1H, J =
8.1 Hz), 7.58 (dd, 1H, J = 1.2, 5.1 Hz), 7.65 (dd, 1H, J = 0.9, 3.6 Hz). ESI-
MS (m/z, %), 351 (MH+, 60) 176 (100). ESI-HRMS calculated for
C20H22N4S (MH+), 351.1637; observed, 351.1645. Second eluting
99.2
a NA: not applicable.
The reactions are carried out at 37 °C for 45 min. The reaction was
stopped by adding 20 μL of ice-cold buffer containing 100 mM HEPES,
3 mM EGTA, 3 mM EDTA, pH = 5.5. [3H]-L-citrulline was separated by
DOWEX (ion-exchange resin DOWEX 50 W X 8À400, SIGMA) and
the DOWEX was removed by spinning at 12000g for 10 min in the
centrifuge. Then 70 μL Aliquot of the supernatant was added to 100 μL
of scintillation fluid and the radio activity was counted in a liquid
scintillation counter (1450 Microbeta Jet, Wallac). Specific NOS activity
was reported as the difference between the activity recovered from the
test solution and that observed in a control sample containing 240 mM
of the inhibitor (1). All assays were performed in duplicate.
Cytochrome P450 Enzyme Inhibition Studies. The assay
used microsomes (Supersomes, GENTEST Corp.), prepared from
insect cells, expressing the various subtypes of the cytochrome P450
isozymes (CYP). The assay monitored the formation of a fluorescent
metabolite following incubation of the microsomes with a specific
fluorogenic CYP substrate. Reactions (0.2 mL) were performed in
96-well microtiter plates at 37 °C in the presence of an NADPH
regenerating system (NADP+, glucose-6-phosphate, glucose-6-phospate
dehydrogenase, and MgCl2). Inhibition of metabolic product formation
by the test compound for each enzyme was tested in the absence or
presence of varying concentrations of test compounds. An enzyme-
selective inhibitor was also tested in the assay as a positive control. All
determinations are performed in duplicate.
Efficacy in the Chung Model of Neuropathic Pain. Nerve
ligation injury was performed according to the literature procedure.36
Rats were anesthetized with halothane, and the L5 and L6 spinal nerves
were exposed, carefully isolated, and tightly ligated with 4À0 silk suture
distal to the DRG. After ensuring homeostatic stability, the wounds were
sutured and the animals allowed to recover in individual cages. This
technique produced signs of neuropathic dysesthesias, including tactile
allodynia, thermal hyperalgesia, and guarding of the affected paw, which
began on day 1 of the surgery and peaked on day 16. After a period of
recovery following the surgical intervention, rats showed enhanced
sensitivity to painful and normally nonpainful stimuli.
Contractile Effects on Human Resistance Arteries. Fresh
specimens of human resistance arteries were obtained from surgical
explant tissue with full informed consent and ethical permission from the
donor. All test tissues, having been cut into ring segments of approxi-
mately 2 mm length, were attached by 40 μm diameter wire running
through the lumen of the vessel to stainless steel heads in 10 mL
myograph baths containing Krebs-bicarbonate physiological saline
solution (PSS), aerated with 95% O2 and 5% CO2 and maintained at
a temperature of 37 °C. Changes in tension were recorded using a
Danish Myotech isometric transducer. The segments were allowed
to equilibrate for at least 30 min and were washed with PSS every
15 min during the equilibration period. Segments were processed
through a standardization procedure to reduce signal variability prior
1
enantiomer (+)-19: [α]D = +13.93 (0.933 g/100 mL, MeOH). H
NMR (MeOH-d4) δ 1.29À1.48 (m, 1H), 1.78À1.96 (m, 3H), 2.07 (brs,
1H), 2.84À2.92 (m, 1H), 3.01À3.16 (m, 4H), 3.42À3.39 (m, 2H), 6.72
(dd, 1H, J = 1.8, 8.4 Hz), 6.99 (d, 1H, J = 1.5 Hz), 7.12 (dd, 1H, J = 3.6,
5.1 Hz), 7.14 (s, 1H), 7.51 (d, 1H, J = 8.1 Hz), 7.56 (dd, 1H, J = 1.2, 5.1
Hz), 7.63 (dd, 1H, J = 0.9, 3.6 Hz). ESI-MS (m/z, %), 351 (MH+, 60)
176 (100). ESI-HRMS calculated for C20H22N4S (MH+), 351.1637;
observed, 351.1642.
General Procedure for Conversion of the Free Base to the
Corresponding Dihydrochloride Salt. A solution of the free base
(1.0 equiv) in methanol was treated with 1 M HCl solution in diethyl
ether (3.0 equiv) dropwise at room temperature. The resulting mixture
was stirred for 10 min and concentrated to dryness. The product was
dried under reduced pressure to obtain the dihydrochloride salt as a
solid. The chemical purity of the dihydrochloride salts was similar to
their corresponding free bases.
Chemical Purity. The chemical purity (Table 4) was determined
by using Agilent Zorbax, SB-C18 (3.5 μM, 2.1 mm  100 mm) column
using gradient method (0.1% TFA in acetonitrile: 0.1% TFA in water,
Table 3) with 0.5 mL/min flow rate at 254 nm. Injection volume: 5 μL
(1 mg/mL in MeOH).
Chiral Purity. The chiral purity (Table 4) was determined by
CHIRALPAK AD-H (column no. ADH0CE-LG122) column using
isocratic method (ethanol:hexanes (contains 0.2% DEA), 3:7) with
1.5 mL/min flow rate at 254 nm. Injection volume: 10 μL (1 mg/mL in
MeOH).
NOS Enzyme Assays. Recombinant human nNOS, eNOS, and
iNOS were produced in Baculovirus-infected Sf9 cells. In a radiometric
method, NOS activity is determined by measuring the conversion of
[3H]-L-arginine to [3H]-L-citrulline. To measure eNOS and nNOS,
10 μL of enzyme was added to 100 μL of 40 mM HEPES, pH = 7.4,
containing 2.4 mM CaCl2, 1 mM MgCl2, 1 mg/mL BSA, 1 mM EDTA,
1 mM dithiothreitol, 1 μM FMN, 1 μM FAD, 10 μM tetrahydrobiop-
terin, 1 mM NADPH, and 1.2 μM CaM. To measure iNOS, 10 μL of
enzyme was added to 100 μL of 100 mM HEPES, pH = 7.4, containing
1 mM CaCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 μM FMN, 1 μM
FAD, 10 μM tetrahydrobiopterin, 120 μM NADPH, and 100 nM CaM.
To measure enzyme inhibition, a 15 μL solution of a test substance is
added to the enzyme assay solution, followed by a preincubation time of
15 min at RT. The reaction is initiated by addition of 20 μL of L-arginine
containing 0.25 μCi of [3H] arginine/mL and 24 μM L-arginine.
The total volume of the reaction mixture is 150 μL in every well.
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Journal of Medicinal Chemistry
ARTICLE
to pharmacological intervention. All segments were then exposed to
KPSS (62.5 mM) three times to provide a reference means of
contractility.
The pharmacology was conducted in the following order:
1 Test tissue was first challenged to provide a measure of maximum
contractility.
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2 Test tissue was washed with PSS and allowed to return to baseline.
3 Test tissue was then tested for endothelial integrity by precon-
tracting the tissue with thromboxane mimetic U46619 (1 Â 10À7
M) and then adding cumulative concentrations of a known
endothelium-dependent dilator agonist (ACh; 1 Â 10À10 M to
1 Â 10À5 M). If the endothelium was intact, ACh produced
relaxations.
4 Test tissue was rinsed and allowed to return to baseline.
5 The test article was tested in the presence and absence of L-arginine
(10À3 M). Compound 16 ((L-arginine) was added at the selected
concentration for a period of 50 min. In the presence of 16
((L-arginine), all vessels were then submaximally vasoconstricted
with U46619 prior to CCRCs to ACh (1 Â 10À10 M to 1 Â 10À5
M). Following wash-out with PSS to baseline, 16 ((L-arginine)
was readded for 50 min at the next highest concentration. CCRCs
to ACh following U46619 preconstriction were then repeated. In
total, three concentrations of 16 and equivalent concentrations of
L-NAME were studied in each artery ring (10À6 M, 10À5 M and
10À4 M) in the presence and absence of 10À3 M L-arginine.
Responses were expressed as a % of the maximal contractile response
to U46619 (as a negative % change for a vasodilatory response). The %
relaxation (reversal) of U46619-mediated contractions in response to
ACh was plotted as concentration versus response. Direct contractile
effects were expressed as a % of the maximum contractile response to
KPSS (62.5 mM). Best-fit curves were constructed using nonlinear
regression.
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’ AUTHOR INFORMATION
(15) Volke, V.; Wegener, G.; Bourin, M.; Vasar, E. Antidepressant-
and anxiolytic-like effects of selective neuronal NOS inhibitor 1-(2-
trifluoromethylphenyl)-imidazole in mice. Behav. Brain Res. 2003, 140,
141–147.
Corresponding Author
*Phone: 1-416-673-8457. Fax: 1-905-823-5836. E-mail: sannedi@
neuraxon.com.
(16) Ulak, G.; Mutlu, O.; Akar, F. Y.; Komsuoglu, F. I.; Tanyeri, P.;
Erden, B. F. Neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)-
imidazole augment the effects of antidepressants acting via serotonergic
system in the forced swimming test in rats. Pharmacol. Biochem. Behav.
2008, 90, 563–568.
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Synergistic neuroprotective effects by combining an NMDA or AMPA
receptor antagonist with nitric oxide synthase inhibitors in global
cerebral ischemia. Eur. J. Pharmacol. 1999, 381, 113–119.
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M. A.; Bevan, J. A.; Fishman, M. C. Hypertension in mice lacking the gene
for endothelial nitric oxide synthase. Nature 1995, 377, 239–242.
(19) Lassen, H. L.; Iverson, H. K.; Olesen, J. A dose-response study
of nitric oxide synthase inhibition in different vascular beds in man. Eur.
J. Clin. Pharmacol. 2003, 59, 499–505.
(20) Erdal, E. P.; Litzinger, E. A.; Seo, J.; Zhu, Y.; Ji, H.; Silverman,
R. B. Selective neuronal nitric oxide synthase inhibitors. Curr. Top. Med.
Chem. 2005, 5, 603–624.
(21) Tinker, A. C.; Wallace, A. V. Selective inhibitors of inducible
nitric oxide synthase: potential agents for the treatment of inflammatory
diseases? Curr. Top. Med. Chem. 2006, 6, 77–92.
(22) Silverman, R. B. Design of selective neuronal nitric oxide
synthase inhibitors for the prevention and treatment of neurodegenera-
tive diseases. Acc. Chem. Res. 2009, 42, 439–451.
’ ACKNOWLEDGMENT
We are grateful to Asinex Ltd (Moscow, Russia) for perform-
ing the human iNOS inhibition assays. We thank the Natural
Sciences and Engineering Research Council (NSERC) of Canada
for providing an Industrial Research Fellowship to G.M.
’ ABBREVIATIONS USED
NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal
nitric oxide synthase; eNOS, endothelial nitric oxide synthase;
iNOS, inducible nitric oxide synthase; CTTH, chronic tension-
type headache; L-NAME, L-nitro arginine methyl ester; FAD,
flavin adenine dinucleotide; FMN, flavin mononucleotide; BH4,
(6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahy-
dropteridin-4(1H)-one; SNL, spinal nerve ligation; ACh,
acetylcholine
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