1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.07.022

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole r

Full text of DOI:10.1016/j.bmcl.2011.07.022

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5301–5304
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,5-Disubstituted indole derivatives as selective human neuronal nitric
oxide synthase inhibitors
⇑
Paul Renton, Joanne Speed, Shawn Maddaford, Subhash C. Annedi , Jailall Ramnauth, Suman Rakhit,
John Andrews
NeurAxon Inc., 2395 Speakman Drive, Suite #1001, Mississauga, ON, Canada L5K 1B3
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors
of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitu-
tions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at
the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and
13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (À)-15 and (+)-15) showed increased inhib-
itory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most
Received 31 May 2011
Revised 4 July 2011
Accepted 6 July 2011
Available online 14 July 2011
Keywords:
1,5-Disubstitued indole derivatives
Nitric oxide
Nitric oxide synthase
Nitric oxide synthase inhibitors
Selective neuronal nitric oxide synthase
inhibitors
potent compound of the series for human nNOS (IC50 = 0.02
over the eNOS (eNOS/nNOS = 96-fold) and iNOS (iNOS/nNOS = 850-fold) isoforms.
Ó 2011 Elsevier Ltd. All rights reserved.
lM) (S)-12 showed very good selectivity
Nitric oxide (NO), which is produced from
L
-arginine by the ni-
binding site produced only nonselective NOS inhibitors. However,
later generations of peptidic and nonpeptidic NOS inhibitors tar-
geting the arginine binding site or Biopterin co-factor site as well
as dimerization inhibitors have been shown to be more potent
and selective among the NOS isoforms.7–9
tric oxide synthase (NOS) enzyme, is one of the most studied neu-
romodulators.1,2 NOS consists of three isoforms: neuronal NOS
(nNOS), is a constitutive form expressed in neuronal cells and
thought to play a role in neurotransmission and the endothelial
isoform (eNOS), is also a constitutive form plays a role in regulating
vascular tone and platelet aggregation. The third isoform is an
inducible form (iNOS) found in macrophage cells, which produce
NO during bacterial infection or tumor cell cytolysis. Overproduc-
tion of NO by the individual NOS isoforms, such as nNOS and/or
iNOS plays an important role in the treatment of several disorders
including septic shock, stroke, neurodegenerative disorders
(Parkinson’s, ALS, MS) and pain (migraine, CTTH, visceral and neu-
ropathic).3 The selective inhibition of nNOS and/or iNOS over the
eNOS isoform is required in order to avoid the cardiovascular lia-
bilities associated with eNOS inhibition.4
The pharmacophore model 1 (Fig. 1) that describes the arginine
binding site of the NOS enzyme contains an amidine group (guani-
dine isostere) and a basic amine group, attached to the indole core
(central aryl scaffold). The amidine group makes an important
bidentate interaction with the conserved glutamic acid residue;
whereas the basic amine is known to improve potency and selectiv-
ity among the NOS isoforms for compounds with a thiophene ami-
dine group (2 and 3).10–12 An increased potency and selectivity
was observed for nNOS isoform over eNOS and iNOS isoforms with
an amino substitution on thiophene amidine compounds containing
either a benzothiazole or dihydroquinoline core.10,11 Our design
strategy is based on attaching a basic amine side chain and thio-
phene amidine group onto an indole core, which is an important
structural component in many pharmaceutical agents and also re-
ferred to as ‘privileged structure’ (capable of binding to many recep-
tors).13 In our continued search for new small molecule drug-like
selective nNOS inhibitors,10 herein we report the design and synthe-
sisof a seriesof1,5-disubstitutedindole derivativesand theirbiolog-
ical activity evaluations against all human NOS isoforms.
Developing small molecule selective NOS inhibitors is an ongo-
ing challenge, due to the high similarity of the active sites of the
three isoforms, where 16 out of 18 residues within 6 Å of the sub-
strate-binding pocket are identical.5 Structure-based inhibitor de-
sign became
a useful tool with the availability of crystal
structures of NOS enzymes with the only exception of human
nNOS isoform.6 Early NOS inhibitor design that focused mainly
on L-arginine (substrate) type compounds targeting the substrate
Synthesis of 1,5-disubstituted indole derivatives was carried out
according to the general procedures described in Schemes 1–6. The
3-carbon side chain was introduced at the 1-position of 5-nitro-1
H-indole using a two-stage alkylation procedure (Scheme 1). The
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.022

5302
P. Renton et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5301–5304
Aryl Scaffold
O2N
O2N
X
i
H
N
R
N
R
N
Cl
Indole
core
H
50-62%
N
19
NH
27-30
Amidine Anchor
attached at 5-position
X = S or O
Various Basic amines
Attached at 1-position
H
N
ii
1
X
NH.HI/HBr
SR
X
NH
N
H
N
S
N
H
N
S
26
X = S, R = Me
NH
NH
Cl
31 X = O, R = Bn
R
NH
9-12: X = S
50-98%
13: X = O
N
N
S
H
N
N
;
; 28 and 10: R =
27 and 9: R =
3
2 (ARL17477)
F
29 and 11: R =
; 30, 12 and 13: R =
N
N
Figure 1. Pharmacophore model (1) for selective nNOS inhibitor design. Literature
thiophene amidine compounds with basic amine side chain (2 and 3).
Scheme 2. (i) K2CO3, DMF, 80 °C; (ii) (a) Pd-C/H2, EtOH, RT; (b) 26 or 31, EtOH, RT.
alkylation of 5-nitro-1H-indole (19) with 1-chloro-3-iodopropane
under basic conditions gave the chloro intermediate 20. The chloro
group in compound 20 was substituted with various acyclic or cyc-
lic amines to obtain the intermediates 21–25 in excellent yield. The
nitro group was then reduced to the corresponding amino group
under hydrogenation conditions and coupled with the thiophene-
2-carbimidothioate 26 to provide the final compounds 4–8.14
The alkylation of 5-nitro-1H-indole (19) with various
2-chloroethyl amino derivatives in presence of potassium carbon-
ate gave intermediates 27–30 (Scheme 2). The nitro group was
reduced to amino group and coupled with either thiophene-
2-carbimidothioate 26 or furan-2-carbimidothioate 31 to give the
final compounds 9–13.15 During the alkylation reaction with
1-(2-chloroethyl)-2-methylpyrrolidine, rearrangement (ring open-
ing/quarternization) was observed to obtain a 7-membered prod-
uct 32 (15%) along with the desired product 30 (Scheme 3).16
Product 30 and rearranged product 32 were separated by using sil-
ica gel column chromatography. The nitro group in compound 32
was reduced to the corresponding amino group as described above
O2N
O2N
O2N
i
N
N
+
N
H
N
Cl
19
N
N
32
(Minor, 15%)
30 (Major, 49%)
H
N
ii
50%
32
X
NH
N
N
14: X = S
15:
X = O
Scheme 3. (i) K2CO3, DMF, 80 °C; (ii) (a) Pd-C/H2, EtOH, RT; (b) 26 or 31, EtOH, RT.
O2N
O2N
ii
i
N
74%
87-100%
N
H
O2N
O2N
i
O2N
+
N
N
N
19
N
N
H
(S)
Cl
Cl
20
19
77%
H
N
O2N
N
iii
32
S
(S)-30
NH
N
R
N
NH.HI
SMe
S
H
N
S
ii
26
50-70%
(S)-30
R
HN
78%
N
4-8
21-25
(S)
N
22
5:
;
;
21
23
4:
;
and
R =
N
H
and
and
R =
R =
25
N
(S)-12
N
H
N
24
7:
;
and
R =
6:
Scheme 4. (i) K2CO3, DMF, 80 °C; (ii) (a) Pd-C/H2, EtOH, RT; (b) 26 or 31, EtOH, RT.
N
O
8:
NH
and
R =
and coupled to thiophene-2-carbimidothioate 26 and furan-2-car-
bimidothioate 31 to obtain the final compounds 14 and 15,
respectively.
Scheme 1. (i) NaH, 1-chloro-3-iodopropane, DMF; (ii) RH, K2CO3, KI, AcCN, reflux;
(iii) (a) Pd-C/H2, EtOH, RT; (b) 26, EtOH, RT.

P. Renton et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5301–5304
5303
In contrast to the synthesis of 2- or 3-carbon flexible side chain
analogs (4–13), the synthesis of rigid side chain analogs 16 and 17
Br
Br
N
i
ii
was achieved by an indolization reaction using
a modified
50%
NO2
Leimgruber–Batcho indole synthesis.21 Condensation of substi-
tuted o-nitro toluene 33 with dimethylformamide dimethyl acetal
in presence of pyrrolidine gave intermediate 34 (Scheme 5). The
enamine 34 was converted into acetal 35 using chlorotrimethylsi-
lane in methanol under refluxing conditions. The nitro group
in compound 35 was converted into an amino group using sodium
dithionite, followed by reductive amination with either
8-methyl-8-azabicyclo[3.2.1]octan-3-one or quinuclidin-3-one
gave intermediates 36 and 37.22,23 The indolization reaction of
intermediates 36 and 37 was carried under strong acidic condi-
tions to provide the substituted 5-bromo-1H-indole derivatives
38 and 39 in quantitative yield. The bromo group in intermediates
38 and 39 was converted into the corresponding animo group
using standard Buchwald-Hartwig amination reaction condi-
tions,24 followed by the coupling of the amine to the thiophene-
2-carbimidothioate 26 provided the final compounds 16 and 17,
respectively.
NO2
33
34
Br
OMe
Br
iii. a
OMe
iv
100%
OMe
b R=O
36-60%
NH
R
36 and 37
OMe
NO2
35
H
N
Br
v
S
35-80%
N
R
NH
N
R
38 and 39
16
17
and
37 39
17:
36 38
16:
R =
;
,
and
R =
,
and
N
N
A standard reductive amination reaction between 4-bromoani-
line 40 and 1-methylpiperidin-4-one in presence of triacetoxy-
borohydride and acetic acid gave the intermediate 41 (Scheme
6). Intermediate 41 was chloroacetylated using chloroacetonitrile
in presence of BCl3 to obtain compound 42.25 The reductive cycli-
zation (indolization) of compound 42 with sodium borohydride
in presence of sodium hydroxide was carried out to provide the
substituted 5-bromo-indole 43. The indole derivative 43 was con-
verted into final compound 18 using standard Buchwald–Hartwig
amination, followed by coupling to the thiophene-2-carbimido-
thioate 26 as described above.
All compounds were converted into their corresponding
dihydrochloride salts (except 7, which is a trihydrochloride salt)
to improve their water solubility,26 and their inhibitory activities
measured against all three human NOS isoforms (Table 1).27 All
compounds displayed sub-micromolar potencies for human nNOS
isoform and good selectivity over the human eNOS and iNOS
isoforms. Compound (S)-12 was identified as the most potent
Scheme 5. (i) DMFDMA, pyrrolidine, DMF, 110 °C; (ii) chlorotrimethylsilane,
MeOH, reflux; (iii) (a) sodium dithionite, NaHCO3, EtOH, reflux; (b) Na2SO4,
NaBH(OAc)3, AcOH, RT, (iv) 1 N HCl, MeOH, reflux; (v) (a) Pd2(dba)3, LiHMDS, PtBu3,
THF, reflux; (b) 26, EtOH, RT.
O
Br
Br
i
Br
Cl
ii
N
NH 55%
O
NH2
.HCl
NH
N
40
64%
N
41
42
H
Br
iii
60%
iv
50%
N
S
N
N
nNOS inhibitor (IC50 = 0.02 lM) in the series with very good selec-
NH
N
N
tivity over eNOS (eNOS/nNOS = 96-fold) and iNOS (iNOS/
nNOS = 850-fold) isoforms. Compounds with N-(1-(2-(1-methyl-
pyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-
(1-methylazepan-4-yl)- side chains (14, 15, (À)-15 and (+)-15)
showed excellent selectivity for nNOS over eNOS and iNOS iso-
forms. Compound 9 with a 2-carbon linker was less selective
(threefold for nNOS and eNOS, sixfold for iNOS) compared to the
corresponding compound 4 with a 3-carbon linker. 2-Furanyl
amidines 13 and 15 showed weaker activities (ꢀ2-fold for nNOS
and eNOS isoforms), and (ꢀ2- to 6-fold for iNOS isoform) when
compared to their corresponding 2-thiophene amidine compounds
12 and 14, respectively. The separated enantiomers (R)-12 and
(S)-12 did not show any significant difference in potency or selec-
tivity among the NOS isoforms, when compared to the racemic
mixture 12. Similarly the enantiomers (À)-15 and (+)-15 also did
not show any significant difference in potency or selectivity among
the NOS isoforms in comparison to the racemate 15.
In summary, we have designed and synthesized a series of
1,5-disubstituted indole derivatives, which showed sub-micromo-
lar potencies for human nNOS with good selectivities over eNOS
and iNOS isoforms. In general compounds with N-(1-(2-(1-methyl-
pyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) or cyclic (14, 15,
(À)-15, (+)-15, 16, 17 and 18) side chains were shown to provide
better selectivities for human nNOS over eNOS and iNOS isoforms.
We have also shown that by varying the basic amine side chain, as
much as 96- and 850-fold selectivity for human nNOS over eNOS
and iNOS, respectively, can be achieved without reducing the po-
18
43
Scheme 6. (i) NaBH(OAC)3, AcOH, DCE, 55 °C; (ii) chloroacetonitrile, BCl3, DCE,
reflux; (iii) NaBH4, 1 M NaOH, EtOH, 0 °C; (iv) (a) Pd2(dba)3, LiHMDS, PtBu3, THF,
reflux; (b) 26, EtOH, RT.
Compound 12 was separated into its enantiomers (R)-12 and
(S)-12 using chiral HPLC techniques.17 The stereochemistry of the
separated enantiomers was confirmed by an independent
chiral synthesis using (S)-2-(2-chloroethyl)-1-methylpyrrolidine
(Scheme 4).18 Accordingly, 5-nitro-1H-indole (19) was treated with
(S)-2-(2-chloroethyl)-1-methylpyrrolidine in presence of potas-
sium carbonate to provide the chiral intermediate (S)-30 along
with the rearranged product 32. The nitro group in compound
(S)-30 was converted into the corresponding amino group and cou-
pled with the thiophene-2-carbimidothioate 26 to give the chiral
(S)-12. Racemic compound 12 and its separated enantiomers (R)-
12 and (S)-12 were co-injected with the synthesized chiral com-
pound (S)-12 to determine the stereochemistry of the separated
enantiomers.19 Compound 15 was also separated into its enantio-
mers (À)-15 and (+)-15 using chiral HPLC techniques,20 but no
further attempts were made to determine the stereochemistry of
compounds (À)-15 and (+)-15 at this time.

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P. Renton et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5301–5304
Table 1
Inhibition of human NOS enzymes by 1,5-disubstituted indole derivatives
H
N
X
NH
N
R
Compound
X
R
nNOS IC50
(l
M)a
eNOS IC50
(
l
M)a
iNOS IC50
(l
M)a
eNOS/nNOS
iNOS/nNOS
4
5
6
7
8
9
10
11
S
S
S
S
S
S
S
S
S
S
S
O
S
O
O
O
S
S
S
N-(1-(3-(dimethylamino)propyl)-
N-(1-(3-(cyclopropylamino)propyl)-
N-(1-(3-morpholinopropyl)-
N-(1-(3-((1-ethylpyrrolidin-2-yl)methylamino)propyl)-
N-(1-(3-adamantanaminopropyl)-
N-(1-(2-(dimethylamino)ethyl)-
0.47
0.26
0.66
0.13
0.36
0.17
0.17
0.09
0.07
0.06
0.02
0.25
0.12
0.43
0.40
0.56
0.76
0.36
0.41
12.4
8.72
6.0
6.33
3.15
3.70
9.2
95.1
34
41.6
NTb
18
15.7
4.61
9.8
14
22
17
32.7
7.6
49
26
34
9
48
9
199
131
63
NCc
50
22
54
71
64
56
96
56
94
89
60
50
25
33
38
93
27
N-(1-(2-(piperidin-1-yl)ethyl)-
N-(1-(2-(1-methylpiperidin-2-yl)ethyl)-
N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-
N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-
N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-
N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-
N-(1-(1-methylazepan-4-yl)-
N-(1-(1-methylazepan-4-yl)-
N-(1-(1-methylazepan-4-yl)-
N-(1-(1-methylazepan-4-yl)-
N-(1-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-
N-(1-(quinuclidin-3-yl)-
6.4
109
200
367
850
131
62
113
49
128
14
12
4.49
3.37
1.92
13.9
11.5
38.5
24.5
28.5
18.9
12.1
15.8
(R)-12
(S)-12
13
14
15
(À)-15
(+)-15
16
17
18
20
73
11
29
79
36
N-(1-(1-methylpiperidin-4-yl)-
15
a
b
c
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.
16. DeVita, R. J.; Hollings, D. D.; Goulet, M. T.; Wyvratt, M. J.; Fisher, M. H.; Lo, J.-L.;
Yang, Y. T.; Cheng, K.; Smith, R. G. Bioorg. Med. Chem. Lett. 1999, 9, 2615.
17. Enantiomeric separation of (R)-12 and (S)-12 was achieved using chiracel OD–
H column (20 Â 250 mm) by isocratic elution with ethanol: hexane (contains
0.1% DEA), 8:92 (flow rate: 5.6 mL/min, wavelength: 230 nm).
18. Vernier, J.-M.; El-Abdellaoui, H.; Holsenback, H.; Cosford, N. D. P.; Bleicher, L.;
Barker, G.; Bontempi, B.; Chavez-Noriega, L.; Menzaghi, F.; Rao, T. S.; Reid, R.;
Sacaan, A. I.; Suto, C.; Washburn, M.; Lloyd, G. K.; McDonald, I. A. J. Med. Chem.
1999, 42, 1684.
tency for nNOS (compound (S)-12). The most potent and selective
human nNOS inhibitor from the series compound (S)-12 has been
selected for further evaluation in a variety of in vivo pain models
and the results will be communicated in due course.
Acknowledgments
19. Co-injections were performed using analytical AD-H column by isocratic
elution with IPA: hexanes (contains 0.1% DEA), 15:85 (flow rate: 1 mL/min,
wavelength: 254 nm).
20. Enantiomeric separation of (À)-15 and (+)-15 was achieved by chiral HPLC
separation using chiralpak AD-H column (5 Â 50 cm) by isocratic elution with
100% AcCN containing 0.1% DEA (flow rate: 300 mL/min, wavelength: 300 nm).
We are grateful to NoAb BioDiscoveries Inc. (Mississauga, ON,
Canada) and Asinex Ltd (Moscow, Russia) for performing the
human NOS inhibition assays.
References and notes
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measured by the conversion of [3H]-
enzymatic reaction was carried out in the presence or absence of varying
concentrations of the inhibitor in water. The negatively charged [3H]-
citrulline was separated from the positively charged [3H]-
-arginine using
L L-citrulline. The
-arginine into [3H]-
L-
L
resin beads. Inhibition of enzyme activity by the inhibitor is measured by
dividing the enzymatic conversion in the presence of inhibitor divided by the
enzymatic conversion in the absence of inhibitor. IC50 value is the
concentration of compound that gives rise to 50% inhibition. All assays were
performed in duplicate.
14. Bercot-Vatteroni, M. Ann. Chim. 1962, 7, 303.
15. Collins, J. L.; Shearer, B. G.; Oplinger, J. A.; Lee, S.; Garvey, E. P.; Salter, M.; Duffy,
C.; Burnette, T. C.; Furfine, E. S. J. Med. Chem. 1998, 41, 2858.