3,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase (nNOS) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.031

A series of 3,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). Various guanidine isosteric groups were explored at the 5-position of the indole ring, while keeping the basic ami

Full text of DOI:10.1016/j.bmcl.2012.01.031

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1980–1984
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3,5-Disubstituted indole derivatives as selective human neuronal nitric
oxide synthase (nNOS) inhibitors
Subhash C. Annedi a, , Shawn P. Maddaford a, Jailall Ramnauth a, Paul Renton a, Joanne Speed a,
⇑
Suman Rakhit a, John S. Andrews a, Frank Porreca b
a NeurAxon Inc., 2395 Speakman Drive, Suite #1001, Mississauga, ON, Canada L5K 1B3
b Department of Pharmacology, University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of
human nitric oxide synthase (NOS). Various guanidine isosteric groups were explored at the 5-position of
the indole ring, while keeping the basic amine side chain such as N-methylpiperidine ring, fixed at the
3-position of the indole ring. Compounds having 2-thiophene amidine and 2-furanyl amidine groups
(7, 8, 10 and 12) showed increased activity for human neuronal NOS and good selectivity over endothelial
and inducible NOS isoforms. Compound 8 was shown to reverse (10 mg/kg, ip) thermal hyperalgesia in
the L5/L6 spinal nerve ligation (neuropathic pain) model and was devoid of any significant drug–drug
interaction potential due to cytochrome P450 inhibition or cardiovascular liabilities associated with
the inhibition of endothelial NOS.
Received 19 December 2011
Revised 10 January 2012
Accepted 11 January 2012
Available online 21 January 2012
Keywords:
3,5-Disubstituted indole derivatives
Nitric oxide
Selective neuronal nitric oxide synthase
inhibitors
Ó 2012 Elsevier Ltd. All rights reserved.
Migraine
Neuropathic pain
Nitric oxide (NO), synthesized by three distinct but closely re-
lated isoforms of nitric oxide synthase (NOS), is a reactive free rad-
ical gas involved in a wide range of physiological functions and
pathophysiological states.1 Neuronal NOS (nNOS) and endothelial
NOS (eNOS) are constitutively expressed, whereas inducible NOS
(iNOS) is expressed during bacterial infection and inflammation.2
Overproduction or excess NO produced by NOS, particularly nNOS,
can lead to the development of several disorders such as septic
shock, stroke, pain (migraine, CTTH, visceral, and neuropathic),
and neurodegenerative disorders (Parkinson’s disease, MS, and
Alzheimer’s disease).3,4 To gain the therapeutic benefits of nNOS
inhibition, it is necessary to achieve selective nNOS inhibition to
preserve the important roles of eNOS and iNOS in controlling blood
pressure and in immune response, respectively.5,6
NOS, up to 100-fold selectivity for nNOS over the eNOS isoform
was achieved.9 The important contributing factor of a guanidine
group for NOS inhibition has became one of the most promising
strategies for early selective NOS inhibition design.10 Over the
course of more than two decades, various structural classes of
selective NOS inhibitors such as guanidines, isothioureas, ami-
dines, aminopyridines, thioureas, imidazoles, indazoles, indoles,
quinolones and several others were identified.11 In particular, ami-
dine and aminopyridine derivatives attracted more attention
among the selective nNOS inhibitors due to their druglike nature
with better PK/PD properties.8
The pharmacophore model adapted by our group that targets
the arginine binding site of the NOS enzyme contains a guanidine
isosteric group and a basic amine group, attached to a central aryl
scaffold as described earlier.12 The amidine group makes an impor-
tant bidentate interaction with the conserved glutamic acid resi-
due; whereas the basic amine is shown to improve potency and
selectivity among the NOS isoforms (1 and 2, Fig. 1).12–14 On the
basis of our previous results with 3,6-disubstituted indole deriva-
tives (2, Fig. 1),15 we selected the N-methylpiperidine ring as the
basic amine side chain at 3-position of the indole ring and ex-
tended our study to explore various guanidine isosteric groups at
the 5-position of the indole ring for the optimization process. In
our continued search for new small molecule druglike selective
nNOS inhibitors,12–16 herein we report the design, synthesis, and
biological activity evaluations of a series of 3,5-disubstituted
First generation NOS inhibitors, which were modified analogs of
either L-arginine (substrate) or L-citrulline (product), were non-
selective among the NOS isoforms.7 However, later generations of
peptidic as well as non-peptidic NOS inhibitors designed based
on the combination of X-ray crystallography and enzyme mutation
studies were proven to be selective among the NOS isoforms.8 By
exploring the one amino acid difference (Asp597 in nNOS vs
Asn368 in eNOS) in the active site of both neuronal and endothelial
⇑
Corresponding author. Tel.: +1 416 673 8457; fax: +1 905 823 5836.
E-mail address: sannedi@neuraxon.com (S.C. Annedi).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.031

S. C. Annedi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1980–1984
1981
hydroiodide, benzyl furan-2-carbimidothioate hydrobromide, and
methyl benzimidate hydrochloride to obtain 11–13, respectively.
The reduction of the nitro group in 5 was carried out as described
above to obtain the free amine, which was protected with benzoy-
lisothiocyanate to obtain 14 (Scheme 2). The benzoyl group was re-
moved under strong basic conditions to obtain the free amine 15,
which was alkylated either with iodomethane or iodoethane to ob-
tain the S-alkyl thiourea derivatives 16 and 17, respectively.
H
N
N
S
NH
N
NH
S
N
H
N
N
H
1
2
All compounds were converted into their corresponding dihy-
drochloride salts,20 and their inhibitory activities were measured
against all three human NOS isoforms (Table 1). In a radiometric
method, inhibitory activities were measured by the conversion of
Figure 1. Recently reported selective human nNOS inhibitors.
[3H]- -arginine into [3H]- -citrulline.21 Compound 7 with 2-thio-
L L
phene amidine attached directly to the 5-position of the indole ring
was the most potent inhibitor for nNOS (0.19 M) and showed the
indole derivatives, which led to the identification of 7 and 8 as lead
candidates.
l
best selectivity (118- and 105-fold) over eNOS and iNOS isoforms,
respectively, among the current series. 2-Furanyl amidine com-
pounds (8 and 12) were less potent (twofold) for nNOS isoform
when compared to the corresponding 2-thiophene amidine com-
pounds (7 and 11). In general, compounds with 2-thiophene ami-
dine and 2-furanyl amidine groups (7, 8, 10, 11, and 12) showed
very good potency for human nNOS isoform, when compared to
bulky groups such as 5-methyl-2-thiophene amidine, phenyl ami-
dine and carbamothioyl benzamide in 9, 13, and 14, respectively.
Reduction of the 1-methyl-1,2,3,6-tetrahydropyridine ring (7 and
8) into 1-methylpiperidine ring (11 and 12) resulted in 4–5 times
weaker potency for nNOS isoform with reduced selectivity over
the eNOS and iNOS isoforms. Extension of the 2-thiophene amidine
group by one carbon (10) resulted in weaker potency (ꢀ7-fold) for
nNOS isoform with reduced selectivity over the eNOS and iNOS iso-
forms, when compared to 7. Substitution of the 2-thiophene ami-
dine in 7 either with 5-methyl-2-thiophene amidine or phenyl
amidine in 9 and 13, resulted in loss of potency (up to 50-fold)
for nNOS isoform and selectivity over the eNOS isoform (eNOS/
nNOS = 118 for 7 vs 6 and 14 for 9 and 13, respectively). S-Alkyl
thiourea derivatives (16 and 17) also showed weaker potency (up
to 25-fold) for nNOS isoform with reduced selectivity over eNOS
Synthesis of 3,5-disubstituted indole derivatives was carried
out according to the general procedures described in Schemes 1
and 2. The substitution on 3-position of the indole ring in 3 and
4 was introduced by a key carbon–carbon bond formation reaction
with N-methyl-4-piperidone under basic conditions to obtain 5
and 6, respectively, as described earlier (Scheme 1).17 Reduction
of the nitro group in 5 was performed under hydrogenation condi-
tions with hydrazine hydrate to obtain the corresponding amine,
which was coupled to methyl thiophene-2-carbimidothioate
hydroiodide, benzyl furan-2-carbimidothioate hydrobromide, and
5-methylthiophene-2-carboximidothioic acid methyl ester hyd-
roiodide to obtain the target compounds 7, 8, and 9, respectively.18,19
To avoid the reduction of the 1-methyl-1,2,3,6-tetrahydropyridine
ring, the reduction reaction was carried out in a pre-heated oil bath
for a maximum of 5–10 min. or until the yellow color disappears.
Reduction of the cyano group in 6 was carried out using LiAlH4,
followed by coupling of the free amine to methyl thiophene-2-car-
bimidothioate hydroiodide provided the target compound 10. The
reduction of the nitro group as well as the 1-methyl-1,2,3,6-tetrahy-
dropyridine ring was carried out under standard hydrogenation con-
ditions using palladium on carbon to obtain the corresponding
amine, which was coupled to methyl thiophene-2-carbimidothioate
N
R
b
N
21-67%
N
H
R
R
7-10
a
N
75%
N
H
N
H
3: R = NO2
4: R = CN
5: R = NO2
6: R = CN
c
R
50-62%
N
H
11-13
H
H
N
H
7, 11: R =
, 8, 12: R =
N
,
, 9: R =
N
S
S
O
NH
NH
10: R =
NH
H
H
N
Ph
N
, 13: R =
S
NH
NH
Scheme 1. Reagents and conditions: (a) N-Methyl-4-piperidone, pyrrolidine, EtOH, reflux; (b) (i) hydrazine hydrate, Raney-Ni, MeOH, LiAlH4, THF, reflux; (ii) methyl
thiophene-2-carbimidothioate hydroiodide or benzyl furan-2-carbimidothioate hydrobromide or 5-methylthiophene-2-carboximidothioic acid methyl ester hydroiodide,
EtOH, rt; (c) (i) Pd-C/H2, EtOH, rt; (ii) methyl thiophene-2-carbimidothioate hydroiodide or benzyl furan-2-carbimidothioate hydrobromide or methyl benzimidate
hydrochloride, EtOH, rt.

1982
S. C. Annedi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1980–1984
N
N
H
N
H
N
H
N
H
N
a
Ph
b
5
R
O
S
S
N
H
N
H
14
15:
R = H
16: R = Me
17:
R = Et
Scheme 2. Reagents and conditions: (a) (i) Hydrazine hydrate, Raney-Ni, MeOH, reflux; (ii) benzoylisothiocyanate, acetone, rt, 92%. (b) (i) 2 N NaOH, THF, reflux, 79%, (ii)
lodomethane, acetone, reflux 19% or iodoethane , acetone, reflux, 25%.
Table 1
Inhibition of human NOS enzymes by 3,5-disubstituted indole derivatives
R2
R1
N
H
Compound
R1
R2
nNOS IC50
(
l
M)a
eNOS IC50
(
l
M)a
iNOS IC50
(
l
M)a
eNOS/nNOS
118
iNOS/nNOS
105
H
N
N
N
N
N
N
N
N
7
0.19 (0.11–0.31)
0.55 (0.29–1.05)
10.4 (5.7–18.6)
1.29 (0.97–1.71)
1.02 (0.68–1.52)
2.24 (0.66–7.54)
22.4 (7.51–66.4)
36.1 (26.3–49.4)
65.3 (38.3–111)
64.7 (50.4–82.8)
24.2 (16.7–34.9)
97.4 (68.1–139)
20.0 (8.63–46.7)
58.5 (28.3–120.9)
NTb
S
NH
H
N
8
66
6
106
NCc
66
O
NH
H
N
9
S
NH
H
N
10
11
12
85.0 (44.8–161)
58.2 (38.3–88.5)
107.9 (55.8–208.4)
50
24
43
S
S
NH
H
N
57
NH
H
N
48
O
NH
H
Ph
N
13
14
16
18.0 (5.92–54.5)
14.0 (9.8–19.8)
2.25 (1.34–3.78)
249 (40.9–1514)
43.0 (32.0–57.6)
36.1 (27.3–47.6)
NTb
14
3
NCc
NCc
8
NH
H
H
N
Ph
N
N
N
NTb
S
O
H
S
N
17.1 (8.5–34.1)
16
NH
S
H
N
N
17
2d
4.83 (2.46–9.4)
0.80 (0.5–1.4)
105.2 (54.4–203)
26.5 (19.5–35.9)
NTb
22
33
NCc
15
NH
12.3 (7.3–20.8)
Values reported in parentheses are 95% confidence intervals.
a
In a radiometric method, inhibitory activities were measured by the conversion of [3H]- -arginine into [3H]-
L L-citrulline.
NT = Not tested.
NC = Not calculable.
b
c
d
Compound 2 is a recently reported selective human nNOS inhibitor, included for comparison.

S. C. Annedi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1980–1984
1983
isoform, when compared to 7 and is consistent with our previous
0
20
40
60
80
Vehicle
observation with 2-aminobenzothiazole compounds.12 Overall,
2-thiophene amidine and 2-furanyl amidine groups were deter-
mined to be ideal guanidine isosteric groups for designing selective
nNOS inhibitors using this particular 3,5-disubstituted indole ser-
ies of compounds. At the same time moving the 2-thiophene ami-
dine group from 6-position of the indole ring (2) to 5-position of
the indole ring (7) was shown to increase the potency for nNOS
(fourfold) isoform and selectivity over the eNOS (3.5-fold) and
iNOS (sevenfold) isoforms and is consistent with our previous
observation with 2-aminobenzothiazole compounds.12 Com-
pounds 7 and 8 were tested for their inhibitory activity in rat nNOS
isoform and showed 12- and 5-fold weaker inhibitory activity (7:
8 (10 µM)
-9.0
-8.0
-7.0
-6.0
Acetylcholine (log M)
Figure 3. Compound
(inhibition of ACh-mediated vasorelaxation) associated with the inhibition of
human eNOS at a concentration of 10 M.
8 did not show any significant vasoconstrictive effect
IC50 = 2.3 lM, 8: IC50 = 2.6 lM), respectively.
To investigate the potential therapeutic application of these
new nNOS inhibitors, 8 was selected for further profiling in an
in vivo neuropathic pain model (Fig. 2). 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 produce thermal and mechan-
ical allodynia of the affected paw.22 The thermal hyperalgesia was
tested by the withdrawal latency to application of radiant heat to
the paw. Intraperitoneal administration of 8 at 10 mg/kg resulted
in reversal of thermal hyperalgesia between 30 and 120 min with
a maximum effect of 94% reversal at 30 min (Fig. 2). After the po-
sitive in vivo results with 8, it was tested for five major human
cytochrome P450 enzyme inhibition studies to assess the potential
for metabolism-based drug–drug interactions. This will also rule
out any inhibitory activity with cytochrome P450 enzymes that
are closely related to NOS.23 Compound 8 was tested in a range
l
inhibition of human eNOS at a concentration of 10 lM (Fig. 3). This
result suggests that 8 is devoid of any cardiovascular liabilities
associated with the inhibition of eNOS based on its eNOS potency
(36.1 lM), and is consistent with our previous observation with
the reference compound 2.15
In conclusion, a series of 3,5-disubstituted indole derivatives
was designed, synthesized and shown to be selective inhibitors
of human nNOS. In this study, we picked the most promising piper-
idine basic amine side chain at 3-position of the indole ring and ex-
plored various guanidine isosteric groups at the 5-position of the
indole ring. Two of the guanidine isosteric groups (2-thiophene
amidine and 2-furanyl amidine) were shown to be sub-micromolar
potent at human nNOS and very selective over the eNOS and iNOS
isoforms. The potential therapeutic application of these com-
pounds was demonstrated by the reversal of thermal hyperalgesia
in an in vivo model of neuropathic pain with 8. The weak inhibitory
activity at the cytochrome P450 enzymes and the lack of eNOS
mediated vasoconstrictive effect associated with eNOS inhibition
with 8 mitigates drug–drug interaction potential as well as the car-
diovascular liabilities associated with these selective nNOS inhibi-
tors, respectively. Further lead optimization (based on 7 and 8) and
various in vivo activity evaluations are underway and will be com-
municated in due course.
of concentrations (up to 100
cytochrome P450 enzymes. Compound
drug–drug interaction potential, as the compound was not active
(CYP 2C9, CYP 2C19, and CYP 3A4: IC50 > 100 M) or very weak
(CYP 2D6: IC50 = 20.5 M) or weak (CYP 1A2: IC50 = 9.52 M)
inhibitor of the five major human cytochrome P450 enzymes.
lM) against the five major human
8
showed very little
l
l
l
Compound 8 was also assessed for the contractile response
(inhibition of acetylcholine (ACh)-mediated vasorelaxation) on iso-
lated human resistance arteries to assess the undesirable cardio-
vascular liability associated with the inhibition of eNOS isoform
(Fig. 3).5,24 The arteries were preconstricted with U46619, a throm-
boxane A2 (TxA2) mimetic agent, and then exposed to ACh, an
endothelium and nitric oxide dependent vasodilator as described
earlier.15 The response to ACh provides information on the activity
of eNOS in an active human biological tissue and thereby any
inhibitory effect of 8 on the eNOS isoform. Compound 8 did not
possess any significant vasoconstrictive effect associated with the
Acknowledgments
We are grateful to NoAb BioDiscoveries Inc. (Mississauga, ON,
Canada) for performing the human n and eNOS inhibition assays
and CYP 450 inhibition studies, Asinex Ltd (Moscow, Russia) for
performing the human iNOS inhibition assays and Biopta Ltd (Glas-
gow, UK) for studying the contractile effect on human resistance
arteries.
25
8 (10 mg/kg, i.p.)
20
15
10
5
References and notes
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L L-citrulline. The enzymatic
reaction was carried out in the presence or absence of varying concentrations
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