4-Substituted indazoles as new inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2007.03.024

A series of halo-1-H-indazoles has been synthesized and evaluated for its inhibitory activity on neuronal nitric oxide synthase. Introduction of bromine at the C4 position of the indazole ring system provided a compound almost as potent as the reference compound, that is, 7-nitroindazole (7-NI). The importance of position 4 is further demonstrated by the synthesis and pharmacological evaluation of the 4-nitroindazole which was also a potent inhibitor of NOS activity. These compounds also exhibited in vivo NOS inhibitory activity, as attested by potent antinociceptive effects following systemic administration.

Full text of DOI:10.1016/j.bmcl.2007.03.024

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3177–3180
4-Substituted indazoles as new inhibitors of neuronal
nitric oxide synthase
Michel Boulouard,a Pascale Schumann-Bard,a,b Sabrina Butt-Gueulle,a Elodie Lohou,a
a
a,
a
*
´
Silvia Stiebing, Valerie Collot and Sylvain Rault
aUPRES-EA 3915—Centre d’Etudes et de Recherche sur le Me´dicament de Normandie (CERMN)—UFR des Sciences
Pharmaceutiques, rue Vaube´nard, Caen, France
bCNRS UMR 6185—Universite´ de Caen Basse-Normandie, Bd H Becquerel, 14032 Caen, France
Received 22 December 2006; revised 7 March 2007; accepted 9 March 2007
Available online 14 March 2007
Abstract—A series of halo-1-H-indazoles has been synthesized and evaluated for its inhibitory activity on neuronal nitric oxide syn-
thase. Introduction of bromine at the C4 position of the indazole ring system provided a compound almost as potent as the reference
compound, that is, 7-nitroindazole (7-NI). The importance of position 4 is further demonstrated by the synthesis and pharmaco-
logical evaluation of the 4-nitroindazole which was also a potent inhibitor of NOS activity. These compounds also exhibited
in vivo NOS inhibitory activity, as attested by potent antinociceptive effects following systemic administration.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The importance of nitric oxide (NO) as a biological mes-
senger in numerous physiological processes has been
demonstrated in a growing fashion over the last decades.
This molecule is indeed involved in various fundamental
functions such as neurotransmission,1 blood pressure
and blood flow regulation,2 platelet aggregation and
inflammation.3 On the other hand, overproduction of
NO plays a role in a variety of disorders, such as septic
shock, pain,4 ischaemia5 and several neurodegenerative
diseases.6 NO is synthesized in several cell types from
L-arginine by different isoforms of nitric oxide synthase
(NOS). To date, three isoforms have been cloned: neuro-
nal (nNOS) and endothelial (eNOS) types, which are
both constitutive and calcium dependent, and an induc-
ible, calcium independent one (iNOS).7 Since these iso-
forms possess a distinct cellular localisation and are
differentially regulated, they represent specific targets
for potential therapeutical approaches. Development
of selective inhibitors of one of these isoforms is there-
fore of considerable interest, both for a therapeutical
purpose and for their use as specific pharmacological
tools. For example, NO of neuronal origin is involved
in pain transmission8,9 and constitutes thus a potential
target for antinociceptive drugs. Likewise, nNOS and/
or iNOS10 should be selectively targeted for neuropro-
tection against ischaemia, while the eNOS isoform,
which has beneficial cerebrovascular effects, should be
leaved unaffected.11 Numerous nNOS inhibitors have
therefore been developed over the last decade but only
few present both potency and a clear selectivity towards
this isoform.5 The first developed inhibitors belong to
the L-arginine analogues family12 and are mostly not
selective for the neuronal isoform. Another series of
inhibitors is constituted by heterocycles such as substi-
tuted indazoles or imidazoles. The nitroindazole family
(with 7-nitroindazole, 7-NI, as the lead compound) are
potent nNOS inhibitors but their selectivity over the
other isoforms remains low, at least in vitro.8,13 We al-
ready partially performed the pharmacomodulation of
the indazole nucleus, especially on the 7 position, in or-
der to develop novel potent and specific inhibitors of
nNOS and we showed that the nitro-substitution was
not absolutely necessary for the biological activity of
the indazole ring.14 We rather demonstrated the inhibi-
tory properties against NOS activity of the 7-methoxy-
substituted indazole, which presents strong similarities
with the mechanism of inhibition of 7-NI.15 In order
to improve our knowledge of the structure–activity rela-
tionships of the indazole derivatives, we aimed here to
pharmacomodulate more extensively the indazole ring
Keywords: Nitric oxide; Indazole; Neuronal nitric oxide synthase;
Inhibitor; Antinociceptive.
*
Corresponding author. Tel.: +33 231936458; fax: +33 231931188;
e-mail: valerie.collot@unicaen.fr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.024

3178
M. Boulouard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3177–3180
by exploring systematically all positions from 4 to 7. To
this end, we synthesized and evaluated the NOS inhibi-
tory properties of 4- to 7-halogeno-substituted indaz-
oles. Due to its difficult chemical access, substitution
at the position 4 had indeed been until now poorly inves-
tigated. Our present results reveal that substitution of
the indazole nucleus on the latter position led to active
compounds, we investigated this position in more detail
with a reference substituent, that is, a nitro-group.
the corresponding diazonium tetrafluoroborate salt 2a–
m in excellent yields. Cyclization of these salts, pro-
moted by potassium acetate and 18-crown-6 in dry chlo-
roform, gave indazoles 3a–m in good yields (Scheme 1
and Table 1).22
6-Iodoindazole 3n was obtained according to Scheme
3.23 Unfortunately, the same pathway applied to
7-aminoindazole did not allow access to the correspond-
ing 7-iodoindazole.
Most of the syntheses of the indazoles substituted on the
six-membered ring reported in the literature proceed
from benzene precursors in which the pyrazole moiety
was generated by ring closure starting from isatines,
phenylhydrazones or o-toluidines.16–19 Amongst these
reactions, we found that the most efficient proceeded
by a phase transfer catalyzed reaction from o-meth-
ylbenzenediazonium tetrafluoroborates20 as presented
in Scheme 1. Indeed, in our laboratory, the successful
method for the preparation of indazoles substituted by
electrondonating or electronwithdrawing14,21 groups
prompted us to apply it to the synthesis of indazoles
bearing halogen atoms in 4, 5, 6 or 7 position (Scheme
1).
4-Nitroindazole 3o was obtained starting from the cor-
responding o-toluidine according to Scheme 1 in 63%
overall yield.
Effects of the derivatives on nNOS activity were evalu-
ated in vitro on rat cerebellum homogenates. IC50 were
determined from the NOS inhibition curves constructed
with four concentrations (0.1, 1, 10 and 100 lM). Enzy-
matic activities were assayed by monitoring the conver-
sion of L-[3H]arginine to L-[3H] citrulline according to a
previously described method12 (1 mM CaCl2, 200 lM
b-NADPH, 0.88 lM L-arginine, 0.12 lM L-[3H]argi-
nine, 15 min at 37 ꢁC). Basal activity represented
120 15 pmol citrulline formed/mg protein/h and the al-
ready described lead compound, 7-NI (dissolved in
DMSO 0.4%), exhibited an IC50 value of 0.6 0.2 lM
(mean SEM; n = 5 experiments).
Our first concern was to prepare commercially unavail-
able aniline precursors 1d and 1l. Following classical
Sandmeyer procedure and reduction we were able to
prepare according to Scheme 2 1c and 1k, respectively,
in 85% and 83% overall yields.
As shown in Table 2, the 7-substituted tested indazoles
displayed a consistent inhibitory effect against the
NOS activity (albeit lower than the reference compound
7-nitroindazole, 7-NI), confirming the already known
importance of this position on the indazole nucleus,13,14
on condition that the substitution is not a bulky one.24,25
Interestingly, our results revealed the importance of the
Indazoles 3a–m were obtained by diazotization of
2-methylanilines 1a–m with aqueous sodium nitrite solu-
tion in fluoroboric acid (50% solution in water) to give
CH3
NH2
CH3
+
i
ii
X
N
X
X
-
Table 1. Formation of halo-1-H-indazoles 3a–m
N2BF4
N
H
X
Diazonium
2a–m (%)
X
Indazole 3a–m
(%)
1a-m
2a-m
3a-m
Scheme 1. Synthesis of halo-1-H-indazoles 3a–m. Reagents and
conditions: (i) 1—HBF4 aq 50%; 2—NaNO2 aq, 0 ꢁC; (ii) AcOK, 18-
crown-6, CHCl3, rt.
3-Br
3-Cl
3-F
2a
2b
2c
2d
2e
2f
99
92
80
97
98
68
92
84
99
99
78
92
80
4-Br
4-Cl
4-F
3a
3b
3c
3d
3e
3f
58
57
69
49
80
51
70
74
41
98
50
91
64
3-I
4-I
4-Br
4-Cl
4-F
5-Br
5-Cl
5-F
NH2
I
I
2g
2h
2i
3g
3h
3i
CH3
NO2
CH3
NO2
CH3
NH2
i
ii
4-I
5-I
5-Br
5-Cl
5-F
6-Br
6-Cl
6-F
2j
3j
2k
2l
3k
3l
4d
5d
1d
6-Br
6-Cl
7-Br
7-Cl
2m
3m
CH3
NO2
CH3
NH2
i
i
ii
N
N
N
Br
Br
N
H
N
H
N
H
O2N
H2N
I
1l
5l
6
7
3n
Scheme 2. Synthesis of 1d and 1l. Regeants and conditions: (a) (i)
1—NaNO2, H2SO4 concd, 0 ꢁC to rt, 2 h; 2—aq KI, rt, 12 h then
Na2S2O3 aq, 80 ꢁC (91%); (ii) HCl concd, SnCl2, 0 ꢁC then 50 ꢁC, 2 h
(93%); (b) (i) HCl concd, SnCl2, 0 ꢁC then 50 ꢁC, 2 h (83%).
Scheme 3. Synthesis of 6-iodoindazole 3n. Reagents and conditions:
(i) H2 Pd/C, EtOH (92%); (ii) 1—NaNO2, HCl concd, 0 ꢁC to rt, 2 h;
2—aq KI, 50 ꢁC then rt, 12 h, then NaOH, NaHSO3 aq (28%).

M. Boulouard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3177–3180
3179
Table 2. IC50 values of the 4- to 7-substituted halo-indazoles and
4-nitroindazole against rat cerebellar nNOS (NS, non-soluble)
Table 3. Antinociceptive activity of selected 4-substituted indazole and
7-chloroindazole new derivatives, compared to 7-nitroindazole and
acetylsalicylic acid in the writhing test in mice (n = 8 per group)
Compound IC50, lM
% inhibition Vehicle
Number of
experiments
mean SEM at 100 lM (%)
Compound
Dose
(mg/kg)
Number of writhes
(mean SEM)
3a
3e
3i
2.4 0.5
72
100
58
DMSO 0.4
DMSO 0.4
DMSO 0.4
DMSO 0.4
DMSO 0.4
3
3
3
3
3
2
Saline (vehicle)
3a
24.6 1.1
11 2.1**
8.8 1.9***
9.3 2.4***
18 2.2**
9.9 2.1***
9.3 1.2***
15 2.2**
12.9 2.3**
7.9 12.3***
7.6 1***
4.3 1.5
14.3 2.9
44.3 3.2
59.3 2.2
22 4.2
4.2 0.5
17 3.1
37 3.7
NS
100
68
12.5
25
3l
3b
3f
57
61
50
Ethanol 0.4 3
3o
12.5
25
3j
68
DMSO 0.4
DMSO 0.4
PEG 0.4
3
3
3
3
3m
3d
3h
3n
3c
3g
3k
3o
100
74
50
3m
12.5
25
72
DMSO 0.4
50
12.5
>100
>100
38
35
DMSO 0.4
DMSO 0.4
DMSO 0.4
DMSO 0.4
3
3
3
3
Acetylsalicylic acid
Arachis oil (vehicle)
7-NI
22.1
1
>100
3.1 1.1
45
100
50
6.8 1.5***
** p < 0.01.
*** p < 0.001 versus control (saline for 4-bromo-, 4-nitro-, 7-chloroin-
dazole and acetylsalicylic groups; arachis oil for 7-nitroindazole)
(ANOVA and PLSD of Fischer).
position 4 to obtain inhibitory properties against the
NOS activity (Table 2). Indeed, whereas the 4-chloro-
and 4-fluoroindazoles are almost ineffective to inhibit
NOS activity (IC50 > 50lM), the 4-iodoindazole is a
weak inhibitor and the 4-bromoindazole is the most
active compound of this series, almost as potent as the
reference compound, that is, 7-NI.
Table 4. Effect of L-arginine pretreatment on antinociceptive activity
of 4-bromo-, 4-nitro- and 7-chloroindazoles (n = 8 per group)
Compound
Dose
(mg/kg)
Number of writhes
(mean SEM)
Saline (vehicle)
L-Arginine
3a
23.9 1.4
The importance of this position 4 is further demon-
strated by the synthesis and pharmacological evaluation
of 4-nitroindazole 3o (Table 2). We found that this com-
pound bearing an electronwithdrawing group in posi-
tion 4 was also a potent inhibitor of NOS activity.
50
50
50
50
50
50
50
22.9 2.1
10.8 0.3***
24.3 1.9###
11.8 0.3***
23.8 1.4###
12.1 1.3***
23 1.1###
L-Arginine + 3a
3o
L-Arginine + 3o
3m
In order to characterise the in vivo pharmacology of the
most potent 4-substituted indazoles, that is, the 4-nitro-
and 4-bromoindazoles, as well as the 7-chloroindazole,
these drugs were tested for their antinociceptive proper-
ties in a mouse model of nociception, the writhing test.26
These compounds (12.5, 25, 50 mg/kg, dissolved in sal-
ine), as well as two reference drugs, 7-nitroindazole
(50 mg/kg, dissolved in arachis oil) and acetylsalicylic
acid (15 mg/kg, dissolved in saline), were administered
ip, 15 min before an ip injection of 0.6% acetic acid
aqueous solution. The number of writhes was then
counted during 10 min, 10 min after acetic acid
administration.
L-Arginine + 3m
*** p < 0.001 versus saline.
### p < 0.001 versus corresponding 4- and 7-substituted indazoles
(ANOVA and PLSD of Fischer).
inhibitors of neuronal NOS. Our results show that the
position 4 seems as important as the position 7 to confer
inhibitory properties against NOS activity. The impor-
tance of this position is further demonstrated by the po-
tency of the 4-nitro-substituted indazole to inhibit NOS
activity. The in vitro properties of these 4-substituted
compounds are associated with an in vivo efficiency, as
attested by potent antinociceptive effects following sys-
temic administration. These results open novel avenues
for a better understanding of the structure–activity rela-
tionships of the indazole derivatives as NOS inhibitors.
We currently aim to compare the interactions of
4-bromoindazole, 4-nitroindazole and 7-NI with the
enzyme NOS, by modelling studies and to investigate
their selectivity against the three isoforms on adapted
in vitro enzymatic assays.
In these conditions, the three tested compounds pro-
duced a strong antinociceptive effect, statistically signif-
icant from the weakest dose and with, at the higher dose,
a maximal effect close to that of acetylsalicylic acid
(15 mg/kg) and 7-NI (50 mg/kg, ip) (Table 3).
The antinociceptive effect of the three tested indazoles
(50 mg/kg, ip) was reversed by pretreatment with L-argi-
nine (50 mg/kg, ip administered 5 min before corre-
sponding indazoles) (Table 4), suggesting that the
analgesic effect was effectively mediated by the inhibition
of NOS activity.
Acknowledgment
This research was supported by the University of Caen
Basse-Normandie.
We report here the synthesis and pharmacological eval-
uation of 4- to 7-halogeno-substituted indazoles as

3180
M. Boulouard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3177–3180
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