Fluorinated indazoles as novel selective inhibitors of nitric oxide synthase (NOS): Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2009.07.067

In order to find new compounds with neuroprotective activity and NOS-I/NOS-II selectivity, we have designed, synthesized, and characterized 14 new NOS inhibitors with an indazole structure. The first group corresponds to 4,5,6,7-tetrahydroindazoles (4-8), the second to the N-methyl derivatives (9-12) of 7-nitro-1H-indazole (1) and 3-bromo-7-nitro-1H-indazole (2), and the latter to 4,5,6,7-tetrafluoroindazoles (13-17). Compound 13 (4,5,6,7-tetrafluoro-3-methyl-1H-indazole) inhibited NOS-I by 63% and NOS-II by 83%. Interestingly, compound 16 (4,5,6,7-tetrafluoro-3-perfluorophenyl-1H-indazole) inhibited NOS-II activity by 80%, but it did not affect to NOS-I activity. Structural comparison between these new indazoles further supports the importance of the aromatic indazole skeleton for NOS inhibition and indicate that bulky groups or N-methylation of 1 and 2 diminish their effect on NOS activity. The fluorination of the aromatic ring increased the inhibitory potency and NOS-II selectivity, suggesting that this is a promising strategy for NOS selective inhibitors.

Full text of DOI:10.1016/j.bmc.2009.07.067

Bioorganic & Medicinal Chemistry 17 (2009) 6180–6187
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorinated indazoles as novel selective inhibitors of nitric oxide synthase
(NOS): Synthesis and biological evaluation
a
a
a
b
Rosa M. Claramunt a, , Concepción López , Carlos Pérez-Medina , Marta Pérez-Torralba , José Elguero ,
*
Germaine Escames c, Darío Acuña-Castroviejo c,d,
*
a Departamento de Química Orgánica y Bio-Orgánica, Facultad de Ciencias, UNED, Senda del Rey 9, E-28040 Madrid, Spain
b Instituto de Química Médica, CSIC, Centro de Química Orgánica Manuel Lora-Tamayo, Juan de la Cierva 3, E-28006 Madrid, Spain
c Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avenida del Conocimiento s/n, E-18100 Armilla, Granada, Spain
d Laboratorio de Análisis Clínicos, Hospital Universitario San Cecilio, Avenida Dr. Olóriz s/n, 18012 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to find new compounds with neuroprotective activity and NOS-I/NOS-II selectivity, we have
designed, synthesized, and characterized 14 new NOS inhibitors with an indazole structure. The first
group corresponds to 4,5,6,7-tetrahydroindazoles (4–8), the second to the N-methyl derivatives (9–12)
of 7-nitro-1H-indazole (1) and 3-bromo-7-nitro-1H-indazole (2), and the latter to 4,5,6,7-tetrafluoroin-
dazoles (13–17). Compound 13 (4,5,6,7-tetrafluoro-3-methyl-1H-indazole) inhibited NOS-I by 63% and
NOS-II by 83%. Interestingly, compound 16 (4,5,6,7-tetrafluoro-3-perfluorophenyl-1H-indazole) inhibited
NOS-II activity by 80%, but it did not affect to NOS-I activity. Structural comparison between these new
indazoles further supports the importance of the aromatic indazole skeleton for NOS inhibition and indi-
cate that bulky groups or N-methylation of 1 and 2 diminish their effect on NOS activity. The fluorination
of the aromatic ring increased the inhibitory potency and NOS-II selectivity, suggesting that this is a
promising strategy for NOS selective inhibitors.
Received 4 May 2009
Revised 24 July 2009
Accepted 25 July 2009
Available online 6 August 2009
To our friend Dr. Sergio Erill of the Dr.
Antonio Esteve Foundation
Keywords:
Indazoles
Fluorine pharmacophore
NOS-I
NOS-II
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Nitric oxide plays a key role in the physiology and pathophysi-
ology of the cardiovascular, central nervous, and immune systems.
Nitric oxide synthases (NOS) constitute an enzyme family com-
prising three isoforms: neuronal (NOS-I or Type I), inducible (NOS-
II or Type II), and endothelial (NOS-III or Type III).1–6 NOS isoforms
are homodimers and catalyze a two-step NADPH-dependent oxida-
NO reacts with molecular oxygen, thiols, transition metal centers
and other biological targets allowing it to function as a rapidly
reversible, specific, and local signal transduction molecule as well
as a nonspecific mediator of tissue damage.17–23
tion of
L
-arginine to NO and
L
-citrulline. NOS monomers consist of
In view of the pivotal function of NO, many efforts have been
devoted to selectively inhibit any of the three NOS isoforms, and
from the several structures investigated moving away from the
amino acid arginine-like template, indazole derivatives deserve
special attention by their potency. So 7-nitro-1H-indazole (7-NI,
a reductase domain and an oxygenase domain. The first one is
homologous to cytochrome P-450 reductase and contains binding
sites for NADPH, FAD, FMN, and calmodulin (CaM).7–10 The oxygen-
ase domain binds L-arginine, the iron protoporphyrin IX (heme
prosthetic group) and tetrahydrobiopterin (H4B). The formation
of stable NOS homodimers is an H4B-substrate and heme-depen-
dent process. Dimerization of NOS is critical for the flavin-to-heme
electron transfer step, as the flow of electrons during catalysis oc-
curs in trans from the reductase domain of one monomer subunit
to the oxygenase domain of the other monomer. There is a high de-
gree of structural similarity within the critical catalytic center and
dimer interface regions between NOS isoforms.11–16
1) and 3-bromo-7-nitro-1H-indazole (NIBr, 2) at 10 lM concentra-
tion suppress NOS-I activity by 87% and 96%, respectively, the lat-
ter compound being one of the most potent inhibitors known until
now.24 In vitro inhibition of the three NOS isoforms occurs, but
in vivo the inhibition is selective for the type I NOS and the mech-
anism of such action is very well established.25–29 IC50 values for
these and other indazole derivatives are presented in Scheme 1.
The most important contribution to the understanding of the
mechanism of action of nitroindazoles is that of Getzoff and co-
workers29 that reported the X-ray structures of inducible (NOS-II)
and endothelial (NOS-III) NOS oxygenase domains cocrystallized
with 1, 2, 5- and 6-nitroindazoles (that of 2 cocrystallized with
NOS-III was previously reported by Raman et al.15). Their main
* Corresponding authors. Tel.: +34 91 3987322; fax: +34 91 3986697 (R.M.C.);
tel.: +34 958 241000x20169; fax: +34 958 819132 (D.A.-C.).
E-mail addresses: rclaramunt@ccia.uned.es (R.M. Claramunt), dacuna@ugr.es (D.
Acuña-Castroviejo).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.067

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
6181
Br
N
N
NO2
N
1
2
N
N
N
H
H
NOS-I: 0.71
NOS-II: 5.8
ref. 24
NO2
NOS-I: 0.62
NOS-II: 3.2
ref. 24
NOS-I: 0.17
NOS-II: 0.29
ref. 24
NO2
NO2
NO2
Cl
O2N
N
N
N
N
N
N
N
N
O2N
O2N
NOS-I: 32
NOS-I: 3.1
NOS-I: 47
H
H
H
NOS-I: 158
H
ref. 30,31
ref. 30,31
ref. 30,31
ref. 30,31
N
N
N
N
N
N
N
N
H NOS-I: > 100
H NOS-I: 100
H NOS-I: > 100
NOS-I: no effect
H
OH
OMe
OEt
OPr
OBu
OMe
Cl
ref. 32,33
ref. 32,33
ref. 32,33
ref. 32,33
MeO
N
N
N
N
3
N
N
N
N
MeO
Cl
H
H
H
H NOS-I: 60
NOS-I: 200
ref. 32,33
NOS-I: 700
NOS-I: 500
ref. 32,33
ref. 32,33
ref. 32,33
Cl
Br
I
Cl
Br
I
N
N
N
N
N
N
N
N
H
NOS-I: 44
NOS-I: 59
H
H
H
NOS-I: 22
NOS-I: 4.2
ref. 31
ref. 31
ref. 31
ref. 31
N
N
N
N
N
N
N
N
H
Br
NOS-I: 14
NOS-I: 2.4
NOS-I: 72
H
H
H
NOS-I: 4.3
Br
ref. 31
ref. 31
ref. 31
ref. 31
N
N
N
N
NOS-I: 17
H
H
NOS-I: 37
ref. 31
ref. 31
Et
Cl
OH
N
N
N
N
N
N
N
N
H NOS-I: 178
H NOS-I: 310
H NOS-I: 100
H
NOS-I: >1000
ref. 30,34
ref. 30,34
ref. 30,34
ref. 30,34
Br
H2N
O2N
N
N
N
N
N
N
H2N
N
N
H
NOS-I: >1000
H
NOS-I: 115
ref. 30,34
H NOS-I: >1000
NO
2 NOS-I: 275
NH2
ref. 30,34
ref. 30,34
ref. 30,34
Scheme 1. IC50 (lM) of literature indazoles.
conclusion is that there are two modes of binding named (accord-
ing to their hydrogen bonds) O–N and N–N (Scheme 2). Compound
2 adopts the O–N conformation while 6- and 7-nitroindazoles pre-
fer the N–N one. Compound 1 crystallizes in both conformations.
Porubsky et al. have reported the structure of indazole coordi-
nated through its lone pair (LP model) to the heme iron (Scheme 2)
of cytochrome P-450 2E1 like 4-methylpyrazole as well as histidine
residues.30
Other indazoles (nitro, chloro, bromo, and iodo) with different
substitution patterns were also found to be active but with higher
IC50 than 1 and 2. Amino and alkoxy indazoles afforded poor inhi-
bition results, save 7-methoxy-1H-indazole (7-MI, 3) that showed
unexpected activity.31–36 We have summarized in Scheme 1 all
the available information concerning indazoles and NOS previous
to the present work.
From all these data, it appears that: (i) the aromatic indazole
skeleton with both hydrogen acceptor and donor nitrogen atoms,
seems to be crucial for the inhibitory action, (ii) hydrogen bonding
acceptor groups (electron withdrawing and electron donating) in
the benzo ring increase the activity in the order 7 > 4 > 6 > 5 posi-
tions, (iii) substitution at position 3 by an electronegative group
such as bromine increases the potency too, but its effect is weaker
than substitution at position 7, and (iv) bulky groups diminish the
activity due to steric constraints in the enzyme active center.
In this report we describe our studies on three groups of inda-
zoles, a total of 14 derivatives depicted in Scheme 3. The first cor-
N
H
N
H
C
C
O
O
O
O
N
H
N
N
H
N
N
H
N
N
N
N
N
N
O
O
N
N
N
N
N
N
N
N
N
LP mode
O-N mode
N-N mode
Scheme 2. The different binding modes of indazoles to heme.

6182
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
O
O
O
O
Me
N
Me
N
N
N
Me Me
Me
N H
Me
Me
Me
Me
N
N
N
N
N
4
6
8
H
5
H
H
7
H
NOS-I: 0%
NOS-II: 4%
NOS-I: 2%
NOS-I: 9%
NOS-I: 19%
NOS-II: 23%
NOS-I: 6%
NOS-II: 0%
NOS-II: 22%
NOS-II: 20%
N
N
N
N
N Me
N
H
Me
NO2
NO2
NO2
1
9
10
NOS-I: 29%
NOS-II: 36%
NOS-I: 27%
NOS-I: 88%
NOS-II: 25%
NOS-II: 84%
Br
Br
Br
N
N
H
N
N
Me
N Me
N
NO2
NO2
NO2
2
11
12
NOS-I: 29%
NOS-II: 7%
NOS-I: 95%
NOS-I: 22%
F
NOS-II: 97%
NOS-II: 10%
F
F
F
F
F
F
F
F
OH
Me
CF3
F
F
F
F
F
F
F
F
F
F
F
N
N
H
N
N
H
N
N
H
N
N
N
N
H
H
14
F
F
F
F
F
13
17
15
16
NOS-I: 33%
NOS-II: 6%
NOS-I: 41%
NOS-II: 21%
NOS-I: 11%
NOS-II: 37%
NOS-I: 63%
NOS-II: 83%
NOS-I: 8%
NOS-II: 80%
Scheme 3. Structures of the studied compounds with% of inhibition (1 mM).
responds to 4,5,6,7-tetrahydroindazoles (4–8), the second to the N-
methyl derivatives (9–12) of 7-nitro-1H-indazole (1) and 3-bromo-
7-nitro-1H-indazole (2), and the latter to 4,5,6,7-tetrafluoroindaz-
oles (13–17).
These different structural types provide insights on the influ-
ence of steric effects, suppression of hydrogen bond donors (NH),
and charge distribution inversion37 of the benzo ring in the fluori-
nated indazoles resulting in the discovery of a new class of potent
and selective inhibitors of NOS-II activity.
of hydrazines with cyclohexanones or mono or bifunctional are-
nes.38–42
1(2),5,6,7-Tetrahydro-4H-indazol-4-ones (4–7) were obtained
from 1,3-cyclohexanediones instead of cyclohexanone and fully de-
scribed by us in a structural paper.43 ( )-4,5,6,7-Tetrahydro-7,8,8-
trimethyl-4,7-menthane-2H-indazole (8) was prepared by the same
method starting from ( )-camphor (18) via ( )-(Z)-3-(hydroxy-
methylene)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (19).
In what concerns fluorinated indazoles, 3-methyl-4,5,6,7-tetra-
fluoro-1H-indazole (13) and 3-trifluoromethyl-4,5,6,7-tetrafluoro-
1H-indazole (14) were prepared by ring-closure reaction of
octafluoro- and pentafluoro-acetophenone with hydrazine and
published already.37,44 The same method, as shown in Scheme 4,
has been applied in this study to the synthesis of indazoles
(15–17) but using pentafluorobenzophenone (20), decafluorobenzo-
phenone (21), and the methyl ester of 2,3,4,5,6-pentafluorobenzoic
2. Results and discussion
2.1. Chemistry
Indazoles are synthesized by formation of one or two bonds of
the pyrazole ring and the most common route uses the reaction
F
F
NNH2
F
R
4
3a
7a
F
F
F
3
Δ
20 R = C6H5
21 R = C6F5
R
F
5
N
1
2
N
H
F
F
F
O
F
6
7
HF
F
F
F
H2O
HF
R
H2N-NH2
Toluene
15 R = C6H5, 90% yield
16 R = C6F5, 90% yield
F
F
O
F
R
22 R = OCH3
F
F
F
Δ
H2O
R
N
N
H
NHNH2
F
F
17 R = OH, 28% yield
Scheme 4. General synthetic pathway followed in the preparation of fluoroindazoles 15–17.

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
6183
Table 2
acid (22), respectively. The low yield in the latter example is due to
the fact that substitution of the fluorine by hydrazine occurs in
ortho and para position with respect to the ester group,45 so be-
sides 4,5,6,7-tetrafluoro-1H-indazole-3-ol (17), the methyl
2,3,5,6-tetrafluoro-4-hydrazinylbenzoate is formed.
Finally, derivatives 9–12 have been synthesized from the lead
indazole scaffolds, 7-nitro-1H-indazole (1) and 3-bromo-7-nitro-
1H-indazole (2), by methylation in dry methanol and sodium
methoxide with methyl iodide as alkylating agent.46
NOS-I and NOS-II inhibition in the presence of the nitroindazoles assayed (com-
pounds 9–12) and 7-nitro-1H-indazole and 3-bromo-7-nitro-1H-indazole (com-
pounds 1 and 2, respectively)
Compound
% Inhibition NOS-I
88
27.3 0.3
% Inhibition NOS-II
1
9
10
2
11
12
4
16
25
36
97
10
7
4
3
1
1
2
1
29
95
22
29
5
1
1
1
2.2. In vitro NOS inhibition
Data represent the mean SEM of the percentage of NOS-I and NOS-II inhibition
produced by 1 mM of each compound. Each value is the mean of three experiments
performed by triplicate in homogenates of rat lung samples.
Table 1 and Figure 1 illustrate the NOS-I and NOS-II inhibition in
the presence of 1 mM of the four tetrahydroindazolones [1(2),
5,6,7-tetrahydro-4H-indazole-4-one (4), 6,6-dimethyl-1(2),5,6,7-
tetrahydro-4H-indazole-4-one (5), 3-methyl-1(2),5,6,7-tetrahydro-
4H-indazole-4-one (6), 3,6,6-trimethyl-1(2),5,6,7-tetrahydro-4H-
indazole-4-one (7)], and ( )-4,5,6,7-tetrahydro-7,8,8-trimethyl-
4,7-methane-2H-indazole (8). It can be seen that none of these
compounds show an important inhibitory activity, with 1(2),5,
6,7-tetrahydro-4H-indazole-4-one (4) the most active of them,
showing only a 20% of NOS inhibition. These results further sup-
port the importance of the aromatic indazole (benzo[c]pyrazole)
skeleton for NOS inhibition, and suggest that bulky groups dimin-
ish their activity due to steric constraints in the enzyme active
center.
Table 2 and Figure 2 represent the NOS-I and NOS-II inhibition
in the presence of 1 mM of the known inhibitors 7-nitroindazole
(1) and 3-bromo-7-nitroindazole (2), and their N-methylated
derivatives 1-methyl-7-nitro-1H-indazole (9), 2-methyl-7-nitro-
2H-indazole (10), 3-bromo-1-methyl-7-nitro-1H-indazole (11),
and 3-bromo-2-methyl-7-nitro-2H-indazole (12). Compared with
Figure 2. Percent of NOS-I and NOS-II activities in the presence of 1 mM
concentration of the nitroindazoles assayed (compounds 9–12) and 7-nitro-1H-
indazole (compound 1) and 3-bromo-7-nitro-1H-indazole (compound 2), as com-
pared with those of untreated samples (control). Each value is the mean of three
experiments performed by triplicate in homogenates of rat lung samples. **P <0.01
and ***P <0.001 versus control.
Table 1
NOS-I and NOS-II inhibition in the presence of the assayed 4,5,6,7-tetrahydro-
indazoles (compounds 4–8)
1 and 2, their methylated derivatives show relatively weak activity.
Compounds 9 and 10 inhibit both NOS-I and NOS-II by about 25%,
whereas compounds 11 and 12 display a slight selective inhibition
of NOS-I. Anyway, the low inhibitory ability of these compounds
suggests that N-methylation of 1 and 2 prevents their effect on
NOS activity.
Compound
% Inhibition NOS-I
% Inhibition NOS-II
4
5
6
7
8
19
6
1
3
2
2
1
23
2
1
1
1
7
2
2
4
2
22
20
9
Table 3 and Figure 3 show the NOS-I and NOS-II inhibition in
the presence of 1 mM of the tetrafluoroindazoles 4,5,6,7-tetra-
fluoro-3-methyl-1H-indazole (13), 4,5,6,7-tetrafluoro-3-trifluoro-
methyl-1H-indazole (14), 3-phenyl-4,5,6,7-tetrafluoro-1H-inda-
zole (15), 4,5,6,7-tetrafluoro-3-perfluorophenyl-1H-indazole (16),
and 4,5,6,7-tetrafluoro-1H-indazole-3-ol (17). This group of com-
pounds was the most active against NOS activity. Specifically, com-
pound 13 inhibited NOS-I by 63% and NOS-II by 83%, respectively,
suggesting a slight selectivity for NOS-II. Interestingly, compound
16 inhibited NOS-II activity by 80%, but it did not affect to NOS-I
Data represent the mean SEM of the percentage of NOS-I and NOS-II inhibition
produced by 1 mM of each compound. Each value is the mean of three experiments
performed by triplicate in homogenates of rat lung samples.
Table 3
NOS-I and NOS-II inhibition in the presence of the 4,5,6,7-tetrafluoro-1H-indazoles
assayed (Compounds 13-17).
Compound
% Inhibition NOS-I
% Inhibition NOS-II
13
14
15
16
17
63
41
11
8
7
2
2
3
3
83
21
37
80
6
1
1
2
1
7
Figure 1. Percent of NOS-I and NOS-II activities in the presence of 1 mM
concentration of the 4,5,6,7-tetrahydroindazoles assayed (compounds 4–8), as
compared with those of untreated samples (control). Each value is the mean of
three experiments performed by triplicate in homogenates of rat lung samples. *P
<0.05 and **P <0.01 versus control.
23
Data represent the mean SEM of the percentage of NOS-I and NOS-II inhibition
produced by 1 mM of each compound. Each value is the mean of three experiments
performed by triplicate in homogenates of rat lung samples.

6184
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
Of the 14 indazole derivatives studied, 4,5,6,7-tetrafluoro-1H-
indazoles 13 and 16 show good NOS inhibition although with dif-
ferent selectivity, the latter being a selective inhibitor of the NOS-II
isoform. Selective inhibition of NOS-II is of great interest from a
therapeutic point of view being potentially useful for treating sep-
sis, neurodegenerative disorders, diabetes, and arthritis. In this re-
gard, the comparative study of the 14 compounds assayed strongly
supports that fluorination of the benzene moiety of the indazoles is
a promising strategy in searching for potent and selective NOS
inhibitors. Their binding mode to heme (O–N vs N–N, Scheme 2)
is under study. The LP mode is probably reserved to basic inda-
zoles, like alkoxy-substituted ones (Scheme 1).
4. Experimental
4.1. Chemistry
Figure 3. Percent of NOS-I and NOS-II activities in the presence of 1 mM
concentration of the 4,5,6,7-tetrafluoro-1H-indazoles assayed (compounds 13–
17), as compared with those of untreated samples (control). Each value is the mean
of three experiments performed by triplicate in homogenates of rat lung samples.
**P <0.01 and ***P <0.001 versus control.
Melting points for compounds 8–12 and 17 were determined by
DSC on a Seiko DSC 220C connected to a Model SSC5200H Disk Sta-
tion and for compounds 15 and 16, a ThermoGalen hot stage
microscope was used. Thermograms (sample size 0.003–0.0010 g)
were recorded at the scanning rate of 2.0 °C minꢀ1. Thin-layer
chromatography (TLC) was performed with Merck Silica Gel (60
F254). Compounds were detected with a 254-nm UV lamp. Silica
gel (60–320 mesh) was employed for routine column chromatog-
raphy separations with the indicated eluent. Elemental analyses
for carbon, hydrogen, and nitrogen were carried out by the Micro-
analytical Service of the Universidad Complutense of Madrid on a
Perkin–Elmer 240 analyzer.
Solution spectra were recorded, at 300 K save specified, on a
Bruker DRX 400 (9.4 T, 400.13 MHz for 1H, 376.50 for 19F,
100.62 MHz for 13C and 40.56 MHz for 15N) spectrometer with a
5-mm inverse detection H–X probe equipped with a z-gradient coil
for 1H, 13C, and 15N. For 19F NMR experiments, a 5-mm inverse
detection QNP probe equipped with a z-gradient coil was used.
Chemical shifts (d in ppm) are given from internal solvents, CDCl3
(7.26), DMSO-d6 (2.49), for 1H and CDCl3 (77.0), DMSO-d6 (39.5) for
13C. External references, CFCl3 (0.00) for 19F and CH3NO2 (0.00) for
15N NMR were used. 2D (1H–1H) gs-COSY and inverse proton de-
tected heteronuclear shift correlation spectra, (1H–13C) gs-HMQC,
(1H–13C) gs-HMBC, (1H–15N) gs-HMQC, and (1H–15N) gs-HMBC,
were acquired and processed using standard Bruker NMR software
and in non-phase-sensitive mode.47 Gradient selection was
achieved through a 5% sine truncated shaped pulse gradient of
1 ms. Variable temperature experiments were recorded on the
same spectrometer. A Bruker BVT3000 temperature unit was used
to control the temperature of the cooling gas stream and an ex-
changer to achieve low temperatures.
activity. So, this compound 16 show an important selectivity for
NOS-II inhibition. Compounds 14 and 15 behave as partial selective
inhibitors of NOS-I and NOS-II, respectively, whereas compound 17
was the least active of all tetrafluoroindazoles assayed. Because of
the selective activity compound 16, the inhibition of NOS-II activity
was studied in the presence of different concentrations of this
compound. Table 4 shows the results of these experiments. Com-
pound 16 inhibits the activity of NOS-II in a dose-dependent man-
ner with an IC50 of 0.4 mM. The fact that fluorination of the
benzene ring could improve the aromatic stacking, together with
the possibility that fluorine atoms may form weak hydrogen
bounds in the active center, may account for the activity of these
fluorinated molecules.
3. Conclusions
A major challenge of modern medicine is to design compounds
that modulate specific enzymes while leaving related isozymes
unaffected. Very few studies report both the NOS-I and NOS-II
inhibitory properties of indazoles (Scheme 1).24 The most interest-
ing data reported in Schemes 1 and 3 can be summarized as
follows:
NOS-I:
Less than 1 lM (in red): 1, 2 and 2,7-dinitroindazole.
Between 1 and 10
lM (in blue): 4-nitro, 7-chloro, 4-bromo, and
6-bromoindazole.
Percentages of inhibition higher than 80% (in red): 1 and 2.
Percentages of inhibition higher than 60% (in blue): 13.
NOS-II:
7-Nitro-1H-indazole (1) is commercially available and used
without further purification. The following compounds were pre-
pared according to published procedures: 3-bromo-7-nitro-1H-
indazole (2),46 1(2),5,6,7-tetrahydro-4H-indazol-4-one (4),43 6,6-
dimethyl-1(2),5,6,7-tetrahydro-4H-indazol-4-one (5),43 3-methyl-
1(2),5,6,7-tetrahydro-4H-indazol-4-one (6),43 3,6,6-trimethyl-1(2),
5,6,7-tetrahydro-4H-indazol-4-one (7),43 3-methyl-4,5,6,7-tetra-
fluoro-1H-indazole (13),44 and 3-trifluoromethyl-4,5,6,7-tetra-
fluoro-1H-indazole (14).37
Less than 1
lM: 2.
Between 1 and 10
lM: 1 and 2,7-dinitroindazole.
Percentages of inhibition higher than 80% (in red): 1, 2, and 13.
Percentages of inhibition higher than 60% (in blue): 16.
Table 4
Dose–response curve for NOS-II activity in the presence of compound 16
4.2. ( )-4,5,6,7-Tetrahydro-7,8,8-trimehyl-4,7-menthane-2H-
Concentration (mM)
NOS-II activity
indazole (8)
1
0.5
0.1
20
58
90
1
3
3
In a round-bottomed flask equipped with reflux condenser and
magnetic stirrer a slurry of NaH (3.26 g, 0.14 mol) in THF (60 mL)
was prepared, ( )-camphor (18) (4.0 g, 26 mmol) was added and
gas evolution commenced immediately. The mixture was refluxed
Data represent the mean SEM of the percentage of NOS-I and NOS-II inhibition
produced by 1 mM of each compound. Each value is the mean of three experiments
performed by triplicate in homogenates of rat lung samples.

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
6185
for 25 min until gas evolution ceased and was then cooled to room
temperature. Ethyl formate (8.4 mL, 0.1 mol) was added dropwise
over 1 h, and the mixture was then stirred for an additional 16 h.
After quenching (2 mL of 2-propanol and 60 mL of water), the mix-
ture was treated with Et2O (3 ꢁ 25 mL) and then acidified to pH 1
with concentrated HCl. The oil was extracted with Et2O
(3 ꢁ 25 mL), the extracts were dried with anhydrous sodium sul-
fate and solvent was removed under reduced pressure to afford
( )-(Z)-3-(hydroxymethylene)-1,7,7-trimethylbicyclo[2.2.1]hep-
tan-2-one (19) as a brown solid (2.5 g, 55%). 1H NMR (DMSO-d6) d
0,71 (s, 3H, CH3-b), 0,79 (s, 3H, CH3-a), 0.86 (s, 3H, CH3 (C1)), 1.22
(m, 2H, H5), 1.60 (m, 1H, H6ax), 1.89 (m, 1H, H6ec), 2.74 (d,
3J = 3.8 Hz, 1H, H4), 7.18 (d, 3J = 4.9 Hz, 1H, @CH(OH)), 10.17 (d,
3J = 6.1 Hz, 1H, OH).
A solution of 19 (2.5 g, 14 mmol) and hydrazine hydrate 55%
(0.89 g, 15 mmol) in MeOH (150 mL) was refluxed for 41 h. Solvent
was removed under reduced pressure to afford 8 as a yellow solid
(2.4 g, 95%) The product was purified by sublimation and crystalli-
zation in water; mp. 139.1 °C (DSC),48 enantiomer (+) 144–145 °C].
1H NMR (CDCl3) d 10.10 (br s, 1H, NH), 7.08 (s, 1H, H3), 2.77 (d,
3J = 4.0, 1H, H4); 1.15 and 2.07 (ddd, 2H, H5ax and H5eq), 1.29
and 1.84 (ddd, 2H, H6ax and H6eq), 1.15 (s, 3H, CH3-7), 0.95 (s,
3H, CH3-a), 0.65 (s, 3H, CH3-b); 13C NMR (CDCl3) d 119.8 (d,
1J = 184.5, C3), 125.7 (s, C3a), 47.1 (d, 1J = 145.0, C4), 27.8 (t,
1J = 132.2, C5), 33.7 (t, 1J = 132.4, C6), 49.9 (s, C7), 165.8 (s, C7a),
61.0 (s, C8) 20.4 (q, 1J = 124.7, CH3-b), 19.3 (q, 1J = 124.9, CH3-a),
10.8 (q, 1J = 125.2, CH3-7).
in dry methanol (10 mL). Then, sodium methoxyde (0.07 g,
1.3 mmol) and 0.25 g of methyl iodide (0.11 mL, 1.8 mmol) were
added. The mixture was heated to reflux for 2 days and then the
solvent was removed under reduced pressure. Water (10 mL)
was added and the residue was extracted with chloroform
(3 ꢁ 15 mL). The organic layers were combined, dried (Na2SO4),
and concentrated to afford a crude formed mainly by the two iso-
mers. After silica gel chromatography with (hexane/ethyl acetate
30:1), 11 was obtained first (0.24, 37%) and increasing to 1:1 to af-
ford 12 (0.29, 44%). Mp (11): 161.1 °C (DSC) (160–162 °C).46 Mp
(12): 200.5 °C (DSC) (194–196 °C).46 Anal. Calcd for C8H6BrN3O2
(11 and 12): C, 37.25; H, 2.51; N, 16.32. Found: C, 37.37; H, 2.47;
N, 16.26. (11) 1H NMR (DMSO-d6) d 8.03 (dd, 3J = 7.9, 4J = 0.7, 1H,
3
H4), 7.43 (dd, 3J = 3J = 7.9, 1H, H5), 8.28 (dd, J = 7.8, 4J = 0.7, 1H,
H6), 4.13 (s, 3H, CH3); 13C NMR (DMSO-d6) d 121.1 (d, 3J = 4.0,
C3), 127.3 (d, 2J = 9.4, C3a), 127.2 (dd, 1J = 167.0, 3J = 8.5, C4),
121.2 (d, 1J = 168.8, C5), 126.0 (ddd, 1J = 167.2, 2J = 2.9, 3J = 7.7,
C6), 135.0 (d, 3J = 9.2, C7), 131.9 (dd, 3J = 3J = 6.9, C7a), 40.9 (q,
1J = 142.5, CH3); 15N NMR (DMSO-d6) d ꢀ198.9 (N1), ꢀ53.6 (N2).
(12) 1H NMR (DMSO-d6) d 8.02 (dd, 3J = 7.9, 4J = 0.8, 1H, H4), 7.31
(dd, 3J = 3J = 7.9, 1H, H5), 8.35 (dd, 3J = 7.9, 4J = 0.8, 1H, H6), 4.24
(s, 3H, CH3); 13C NMR (DMSO-d6) d 111.0 (m, C3), 124.9 (d,
2J = 9.2, C3a), 128.3 (dd, 1J = 166.5, 3J = 8.1, C4), 120.8 (d,
1J = 167.4, C5), 125.9 (ddd, 1J = 167.4, 2J = 2.9, 3J = 9.5, C6), 136.4
(d, 3J = 7.7, C7), 139.3 (dd, 3J = 3J = 6.9, C7a), 39.5 (q, 1J = 142.0,
CH3); 15N NMR (DMSO-d6) d ꢀ87.1 (N1), ꢀ155.2 (N2).
4.5. 3-Phenyl-4,5,6,7-tetrafluoro-1H-indazole (15)
4.3. 1-Methyl-7-nitro-1H-indazole (9) and 2-methyl-7-nitro-
2H-indazole (10)
In a round-bottomed flask equipped with reflux condenser, pen-
tafluorobenzophenone (20) (0.70 g, 2.5 mmol) was dissolved in
toluene (30 mL), hydrazine monohydrate (0.40 g, 8.0 mmol) was
added and the mixture refluxed during 6 h. After this period, the
mixture was cooled and the solvent was removed under reduced
pressure. The product was purified by silica gel chromatography
(hexane/ethyl ether 15:1) to afford 15 (0.67, 90%) as a white solid.
Mp 164–165 °C; Anal. Calcd for C13H6F4N2: C, 58.66; H, 2.27; N,
10.52. Found: C, 58.58; H, 2.45; N, 10.40. 1H NMR (CDCl3) d 11.48
(br s, 1H, NH), 7.57 (m, 2H, Hm), 7.45 (m, 3H, Ho, Hp); 19F NMR
In a round-bottomed flask equipped with reflux condenser, 7-
nitro-1H-indazole (1) (1.0 g, 6.1 mmol) was dissolved in dry meth-
anol (40 mL). Then, sodium methoxyde (0.42 g, 7.4 mmol) and
1.75 g of methyl iodide (0.77 mL, 12.3 mmol) were added. The mix-
ture was heated to reflux for 2 days and then the solvent was re-
moved under reduced pressure. Water (10 mL) was added and
the residue was extracted with chloroform (3 ꢁ 15 mL). The organ-
ic layers were combined, dried (Na2SO4), and concentrated to af-
3
5
ford
a
crude formed by the two isomers and the starting
(CDCl3) d ꢀ140.9 (dd, JF5 = 19.3, JF7 = 19.3, F4), ꢀ165.2 (ddd,
3
4
3
material. After silica gel chromatography with hexane/ethyl ace-
tate 10:1, 9 was obtained first (0.21, 19%) and reaching 1:1 to final-
ly afford 10 (0.32, 29%). Mp (9): 100.5 °C (DSC) (98–100 °C).45,49 Mp
(10): 144.9 °C (DSC) (145 °C).46 Anal. Calcd for C8H7N3O2 (9 and
10): C, 54.11; H, 4.20; N, 23.17. Found: C, 54.06; H, 4.10; N, 22.45.
Compound 9 1H NMR (DMSO-d6) d 8.37 (s, 1H, H3), 8.23 (dd,
3J = 7.8, 4J = 1.0, 1H, H4), 7.32 (dd, 3J = 3J = 7.8, 1H, H5), 8.17 (dd,
3J = 7.8, 4J = 1.0, 1H, H6), 4.13 (s, 3H, CH3); 13C NMR (DMSO-d6) d
134.3 (dd, 1J = 193.2, 3J = 2.4, C3), 128.5 (ddd, 2J = 2J = 9.9, 3J = 2.5,
C3a), 128.6 (dd, 1J = 165.8, 3J = 8.4, C4), 120.1 (d, 1J = 167.2, C5),
124.5 (ddd, 1J = 166.5, 2J = 2.8, 3J = 8.1, C6), 134.8 (m, C7), 130.5
(m, C7a), 40.9 (q, 1J = 141.3, CH3); 15N NMR (DMSO-d6) d ꢀ200.3
(N1), ꢀ46.3 (N2). (10) 1H NMR (DMSO-d6) d 8.73 (s, 1H, H3), 8.25
(dd, 3J = 7.9, 4J = 0.9, 1H, H4), 7.23 (dd, 3J = 3J = 7.9,1H, H5), 8.28
(dd, 3J = 7.9, 4J = 0.9, 1H, H6), 4.27 (s, 3H, CH3); 13C NMR (DMSO-
d6) d 127.7 (d, 1J = 195.3, 3J = 2.4, C3), 125.4 (d, 2J = 9.2, C3a),
129.9 (dd, 1J = 165.4, 3J = 8.1, C4), 119.6 (d, 1J = 166.8, C5), 124.6
(ddd, 1J = 165.4, 2J = 2.7, 3J = 9.1, C6), 136.4 (m, C7), 139.6 (ddd,
2J = 3J = 3J = 6.7, C7a), 40.6 (q, 1J = 156.6, CH3); 15N NMR (DMSO-
d6) d ꢀ92.3 (N1), ꢀ152.5 (N2).
3JF4 = 19.3, JF6 = 19.3, JF7 = 2.6, F5), ꢀ156.8 (dd, JF5 = 19.3,
3
5
4
5JF7 = 19.3, F6), ꢀ159.6 (ddd, JF6 = 19.3, JF4 = 19.3, JF5 = 2.6, F7);
13C NMR (CDCl3) d 130.8 (C3), 107.8 (dd, 2J = 18.8, 3J = 3.8, C3a),
139.5 (dddd, 1J = 253.1, 2J = 12.0, 3J = 4J = 3.8, C4), 135.7 (ddd,
1J = 246.2, 2J = 2J = 16.3, C5), 139.6 (ddd, 1J = 252.4, 2J = 2J = 14.1,
C6), 132.3 (dddd, 1J = 249.2, 2J = 12.6, 3J = 5.0, 4J = 2.5, C7), 128.3
(overlapped, C7a), 146.3 (m, Ci), 129.2 (dt, 1J = 161.0, 2J = 7.7, Cp),
128.7 (dd, 1J = 161.0, 2J = 7.7, Co), 128.4 (dm, 1J = 160.5); 15N NMR
(CDCl3, T = 213 K) d ꢀ205.1 (N1).
4.6. 4,5,6,7-Tetrafluoro-3-perfluorophenyl-1H-indazole (16)
In a round-bottomed flask equipped with reflux condenser,
decafluorobenzophenone (21) (0.50 g, 1.4 mmol) was dissolved in
toluene (20 mL), hydrazine monohydrate (0.10 g, 2.0 mmol) was
added and the reaction was refluxed for 2 h. After cooling, the sol-
vent was removed under reduced pressure and the product was
purified by silica gel chromatography (hexane/ethyl ether, 10:1)
to afford 16 (0.45 g, 90%) as a white solid. Mp 114–116 °C; Anal.
Calcd for C13HF9N2: C, 43.84; H, 0.28; N, 7.87. Found: C, 43.84; H,
0.41; N, 8.19. 1H NMR (CDCl3) d 11.38 (br s, 1H, NH); 19F NMR
3
5
4.4. 3-Bromo-1-methyl-7-nitro-1H-indazole (11) and 3-bromo-
2-methyl-7-nitro-2H-indazole (12)
(CDCl3) d ꢀ151.8 (dd, JF5 = 19.2, JF7 = 19.2, F4), ꢀ163.2 (ddd,
3JF4 = 19.2, JF6 = 19.2, JF7 = 2.6, F5), ꢀ155.1 (dd, JF5 = 19.2,
3
4
3
3
5
3JF7 = 19.2, F6), ꢀ158.6 (dd, JF6 = 19.2, JF4 = 19.2, F7), ꢀ161.7 (m,
C6F5, Fm), ꢀ148.2 (m, C6F5, Fp), ꢀ140.7 (m, C6F5, Fo); 13C NMR
(CDCl3) d 131.5 (C3), 109.9 (dd, 2J = 18.8, 3J= 2.5, C3a), 139.0 (dddd,
In a round-bottomed flask equipped with reflux condenser, 3-
bromo-7-nitro-1H-indazole (2) (0.25 g, 1.0 mmol) was dissolved

6186
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
1J = 256.2, 2J = 12.6, 3J = 4J = 4.4, C4), 136.4 (ddd, 1J = 249.1,
2J = 2J = 15.7, C5), 140.2 (dddd, 1J = 254.9, 2J = 2J = 14.4, C6), 132.6
(dddd, 1J = 251.2, 2J = 16.3, 3J = 5.0, 4J = 2.5, C7), 127.7 (ddd,
2J = 13.9, 3J = 7.5, 3J = 3.8, C7a), 145.3 (dddd, 1J = 251.2, 2J =15.7,
3J = 6.6, 3J = 3.8, Co), 142.3 (dtt, 1J = 257.5, 2J = 13.2, 3J = 5.0, Cp),
138.0 (dtd, 1J = 250.9, 2J = 14.7, 3J = 4.5, Cm), 106.5 (td, 2J = 17.2,
3J = 3.8, Ci); 15N NMR (CDCl3, T = 213 K) d ꢀ201.6 (N1).
Tris, 0.5 mM DTT, 10 lg/mL leupeptin, 10 lg/mL pepstatin, 10 lg/
mL aprotinin, 1 mM PMSF, pH 7.6). Two striata were placed in
1.25 mL of the same buffer and sonicated (10 s ꢁ 6). The crude
homogenate was centrifuged 5 min at 1000g, and an aliquot of
the supernatant was frozen at ꢀ80 °C for total protein
determination.52
NOS activity was measured by the method of Bredt et al.,53
monitoring the conversion of
The final incubation volume was 100
sample added to prewarmed (37 °C) buffer to give a final concen-
tration of 25 mM Tris, 1 mM DTT, 30 M H4-biopterin, 10
FAD, 0.5 mM inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 -argi-
nine and 40 nM -3H-arginine, pH 7.6. The reaction was started by
the addition of 10 L NADPH (0.75 mM final concentration) and
L-3H-arginine to
L-3H-citrulline.
4.7. 4,5,6,7-Tetrafluoro-1H-indazole-3-ol (17)
lL and consisted of 10
lL
In a round-bottomed flask equipped with reflux condenser,
methyl 2,3,4,5,6-pentafluorobenzoate (22) (1.0 g, 4.4 mmol) was
dissolved in toluene (10 mL), hydrazine monohydrate (0.27 g,
5.3 mmol) was added and the reaction was heated and stirred at
70 °C for 29 h. The solvent was removed under reduced pressure
and the product was purified by silica gel chromatography (dichlo-
romethane/methanol, 30:1) and crystallized from CH2Cl2/CH3OH
to afford 17 (0.26 g, 28%) as a white solid. Mp 198.0 °C (DSC). Anal.
Calcd for C7H2F4N2O: C, 40.79; H, 0.98; N, 13.59. Found: C, 40.87; H,
1.24; N, 13.62. 1H NMR (DMSO-d6) d 12.71 (br s, 1H, NH), 11.32 (br
s, 1H, OH); 19F NMR (DMSO-d6) d ꢀ148.8 (dd, 3JF5 = 19.5, 5JF7 = 19.5,
l
lM
lM L
L
l
continued for 30 min at 37 °C. Control incubations were performed
in absence of NADPH. The activity of NOS-II was measured in the
presence or absence of 10 mM EDTA. EDTA removes calcium from
the medium preventing the activation of NOS-I; thus, in the pres-
ence of EDTA only NOS-II is detected. The reaction was stopped
adding 400
175 mM -3H-citrulline, pH 5.5. The mixture was decanted onto a
2 mL column packed with Dowex-50 W ion exchanger resin (Na+
form) and eluted with 1.2 mL of water. -3H-citrulline was quanti-
fied by liquid scintillation counting in a Beckman LS-6000 system
(Beckman Coulter, Fullerton, CA, USA). The retention of -3H-argi-
nine in this process was greater then 98%. NOS activity was
measured by monitoring the conversion of -3H-arginine to
-3H-
-3H-citrulline/
lL of cold 0.1 M HEPES containing 10 mM EGTA,
L
3
3
F4), ꢀ170.7 (dd, JF4 = 19.5, JF6 = 19.5, F5), ꢀ159.1 (br s, F6),
3
5
4
ꢀ160.5 (ddd, JF6 = 19.5, JF4 = 19.5, JF5 = 4.5, F7; 13C NMR
(DMSO-d6) d 154.7 (d, 3J= 2.4, C3), 98.8 (dd, 2J = 18.5, 3J = 4.8,
C3a), 138.6 (dddd, 1J = 250.6, 2J = 11.8, 3J = 4J = 3.9, C4), 132.7
L
L
(ddd, 1J = 239.5, 2J = 2J
=
15.5, C5), 138.9 (dddd, 1J = 246.7,
2J = 2J = 15.4, 3J= 1.8, C6), 131.8 (dddd, 1J = 248.2, 2J = 13.8, 3J = 5.0,
4J = 2.2, C7), 127.6 (ddd, 2J = 13.9, 3J = 7.8, 3J = 3.9, C7a);15N NMR
(THF-d8, T = 207 K) d ꢀ233.4 (N1), ꢀ111.9 (N2).
L
L
citrulline. Enzyme activity was referred as pmol
min/mg prot.
L
A total of 16 compounds were assayed for NOS-I/NOS-II activi-
ties: 1(2),5,6,7-tetrahydro-4H-indazole-4-one (4), 6,6-dimethyl-
1(2),5,6,7-tetrahydro-4H-indazole-4-one (5), 3-methyl-1(2),5,6,7-
tetrahydro-4H-indazole-4-one (6), 3,6,6-trimethyl-1(2),5,6,7-tet-
rahydro-4H-indazole-4-one (7), ( )-4,5,6,7-tetrahydro-7,8,8-tri-
methyl-4,7-methane-2H-indazole (8), 7-nitro-1H-indazole (7NI,
1), 3-bromo-7-nitro-1H-indazole (NIBr, 2), 1-methyl-7-nitro-1H-
indazole (9), 2-methyl-7-nitro-2H-indazole (10), 3-bromo-1-
methyl-7-nitro-1H-indazole (11), 3-bromo-2-methyl-7-nitro-2H-
indazole (12), 4,5,6,7-tetrafluoro-3-methyl-1H-indazole (13),
4,5,6,7-tetrafluoro-3-trifluoromethyl-1H-indazole (14), 3-phenyl-
4,5,6,7-tetrafluoro-1H-indazole (15), 4,5,6,7-tetrafluoro-3-perflu-
orophenyl-1H-indazole (16) y 4,5,6,7-tetrafluoro-1H-indazole-3-
ol (17). Except when indicated, the concentration of these com-
pounds used in the NOS assays was 1 mM.
4.8. Assay of NOS-I/NOS-II activities
L
-Arginine, L
-citrulline, N-(2-hydroxymethyl)piperazine-N0-(2-
ethanesulfonic acid) (HEPES), DL-dithiothreitol (DTT), leupeptin,
aprotinin, pepstatin, phenylmethylsulfonylfluoride (PMSF), hypo-
xanthine-9-b-D-ribofuranosid (NOS-IIine), ethylene glycol-bis-(2-
aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA), bovine serum
albumin (BSA), Dowex-50 W (50 ꢁ 8-200), FAD, NADPH and
5,6,7,8-tetrahydro-L-biopterin dihydrochloride were obtained from
Sigma–Aldrich Química (Spain). L-3H-arginine (58 Ci/mmol) was
obtained from Amersham (Amersham Biosciences, Spain).
Tris(hydroxymethyl)aminomethane (Tris–HCl) and calcium chlo-
ride were obtained from Merck (Spain).
For NOS-II activity determination, male Wistar rats (3-months
old, 220–250 g) were bred and kept in the University’s animal facil-
ity on a 12 h light/12 h dark cycle at 22 2 °C, on regular chow and
tap water until the day of the experiment. All experiments were
performed according to the Spanish Government Guide and the
European Community Guide for animal care. The experimental
paradigm was published elsewhere.50 Briefly, three days before
the experiment the jugular vein was cannulated under ip equithe-
sin anesthesia (1 mL/kg) for LPS administration. Rats were iv in-
jected with 10 mg/kg bw LPS (E. coli 0127:B8, Sigma–Aldrich,
Madrid, Spain) dissolved in 0.3 mL of saline. Six hours after LPS
injection, animals were killed by decapitation. Lungs were quickly
collected, washed, and frozen to ꢀ80 °C in liquid nitrogen. Pieces of
lungs were homogenized (0.1 g/mL) in ice-cold buffer (25 mM Tris,
4.9. Statistical analysis
Data are expressed at the mean SEM. One-way analysis of var-
iance, followed by the Newman–Keuls multiple range test was
used. A P <0.05 value was considered statistically significant.
Acknowledgments
This work has been partially supported by Grants CTQ2007-
62113 and SAF2005-07991-C02-01 (Ministerio de Educación y
Ciencia, Spain), PI08-1664 (Instituto de Salud Carlos III, Spain),
and P07-CTS-03135 (Consejería de Innovación, Ciencia y Empresa,
Junta de Andalucía, Spain).
0.5 mM DTT, 10 lg/mL pepstatin, 10 lg/mL leupeptin, 10 lg/mL
aprotinin, 1 mM PMSF, pH 7.6) at 0–4 °C.51 The crude homogenate
were centrifuged at 2500g at 4 °C for 5 min, and aliquots of the
supernatant were either stored at ꢀ80 °C for total protein determi-
nation52 or used immediately for NOS activity measurement.
For NOS-I activity determination, untreated male Wistar rats
(200–250 g) were killed by cervical dislocation and striata were
quickly collected and immediately used to measure NOS-I activity.
Upon removal, tissues were cooled in an ice-cold buffer (25 mM
References and notes
1. Knowles, R. G.; Moncada, S. Biochem. J. 1994, 298, 249.
2. Bredt, D. S.; Hwang, P. M.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 682.
3. Fösterman, U.; Schmidt, H. H. H. W.; Pollock, J. S.; Sheng, H.; Mitchell, J. A.;
Warner, T. D.; Nakane, M.; Murad, F. Biochem. Pharmacol. 1991, 42, 1849.
4. Nathan, C.; Xie, Q. W. Cell 1994, 78, 915.
5. Moncada, S.; Higgs, A.; Furchgott, R. Pharmacol. Rev. 1997, 49, 137.

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 6180–6187
6187
6. Lincoln, J.; Hoyle, C. H. V.; Burnstock, G. Biomedical Research Topics; Cambridge
University Press, 1997.
7. Leone, A. M.; Palmer, R. M. J.; Knowles, R. G.; Francis, P. L.; Ashton, D. S.;
Moncada, S. J. Biol. Chem. 1991, 266, 23790.
8. Schmidt, H. H. H.; Lohman, W. S. M.; Walter, U. Biochim. Biophys. Acta 1993,
1178, 153.
32. Boulouard, M.; Schumann-Bard, P.; Butt-Gueulle, S.; Lohou, E.; Stiebeng, S.;
Collot, V.; Rault, S. Bioorg. Med. Chem. Lett. 2007, 17, 3177.
33. Collot, V.; Sopkova-de Oliveira Santos, J.; Schumann-Bard, P.; Colloc’h, N.;
Mackenzie, E. T.; Rault, S. J. Enzyme Inhib. Med. Chem. 2003, 18, 195.
34. Tuyman, A.; Pérollier, C.; Frapart, Y.; Schumann-Bard, P.; Collot, V.; Rault, S.;
Boucher, J. L. Nitric Oxide 2003, 9, 86.
9. Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.; Feldman, P. L.; Wiseman,
J. J. Biol. Chem. 1991, 266, 6259.
35. Babbedge, R.; Bland-Ward, C. P. A.; Hart, S. L.; Moore, P. K. Br. J. Pharmacol.
1993, 110, 225.
10. Kerwin, F. K., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. J. Med. Chem. 1995, 38, 4343.
11. Raman, C. S.; Li, H.; Martásek, P.; Král, B. S. S.; Masters, T. L.; Poulos, T. L. Cell
1998, 95, 935.
36. Claramunt, R. M.; Sanz del Castillo, D.; Elguero, J.; Nioche, P.; Raman, C. S.;
Martasek, P.; Masters, B. S. S. Drugs of the Future 2002, 27 (Suppl. A): XVIIth
Symposium on Medicinal Chemistry, 177.
12. Klatt, P.; Schmid, M.; Leopold, E.; Schmidt, K.; Werner, E. R.; Mayer, B. J. Biol.
Chem. 1994, 269, 13861.
13. Pfeiffer, S.; Mayer, B.; Hemmens, B. Angew. Chem., Int. Ed. 1999, 38, 1714.
14. Siddhanta, U.; Presta, A.; Fan, B.; Wolan, D.; Rousseau, D. L.; Stuehr, D. J. J. Biol.
Chem. 1998, 273, 18950.
15. Raman, C. S.; Li, H.; Martásek, P.; Southan, G.; Masters, B. S. S.; Poulos, T. L.
Biochemistry 2001, 40, 13448.
16. Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 191.
17. Butler, A.; Nicholson, R. Life, Death and Nitric Oxide; Royal Society of Chemistry,
2003.
37. Teichert, J.; Oulié, P.; Jacob, K.; Vendier, L.; Etienne, M.; Claramunt, R. M.; López,
C.; Pérez Medina, C.; Alkorta, I.; Elguero, J. New J. Chem. 2007, 31, 936.
38. Elguero, J.. In Comprehensive Heterocyclic Chemistry; Katritzky, A. R., Rees, C. W.,
Eds.; Pergamon Press: Oxford, 1984; Vol. 5, pp 167–343.
39. Elguero, J.. In Comprehensive Heterocyclic Chemistry II; Katritzky, A. R., Rees, C.
W., Scriven, E. F. V., Eds.; Elsevier: Oxford, 1996; Vol. 3, pp 1–75.
40. Stadlbauer, W.. In Science of Synthesis; Georg-Thieme: New York, 2002; Vol.
2.12, pp 227–324.
41. Elguero, J.; Goya, P.; Jagerovic, N.; Silva, A. M. S.. In Targets in Heterocyclic
Systems; Italian Society of Chemistry: Roma, 2002; Vol. 6, pp 52–98.
42. Karno, M.; Milata, V.; Kada, R.; Ilavsky, D. Curr. Org. Chem. 2004, 8, 695.
43. Claramunt, R. M.; López, C.; Pérez Medina, C.; Pinilla, E.; Torres, M. R.; Elguero,
J. Tetrahedron 2006, 62, 11704.
18. Loscalzo, J.; Welch, G. Prog. Cardiovasc. Dis. 1995, 38, 87.
19. Nitric Oxide in the Nervous System; Jenner, P., Vincent, S., Eds.; Academic Press,
1995.
20. Yun, H.-Y.; Dawson, V. L.; Dawson, T. M. Mol. Psychiat. 1997, 2, 300.
21. Bogdan, C. Nat. Immunol. 2001, 2, 907.
44. Hathaway, B. A.; Day, G.; Lewis, M.; Glaser, R. J. Chem. Soc., Perkin Trans. 2 1998,
2713.
22. Wu, K. K. Encyclopedia of Life Sciences; Wiley, 2001.
23. Ladecola, C. Trends Neurosci. 1997, 20, 132.
24. Bland-Ward, P. A.; Moore, P. K. Life Sci. 1995, 57, 131.
25. Medhurst, A. D.; Greenlees, C.; Parsons, A. A.; Smith, S. J. Eur. J. Pharmacol. 1994,
256, R5.
45. Burdon, J.; Hollyhead, W. B.; Tatlow, J. C. J. Chem. Soc. 1965, 6336.
46. Benchidmi, M.; Bouchet, P.; Lazaro, R. J. Heterocycl. Chem. 1979, 16, 1599.
47. Berger, S.; Braun, S. In 200 and more NMR Experiments; Wiley-VCH: Weinheim,
2004.
48. Brunner, H.; Scheck, T. Chem. Ber. 1992, 125, 701.
26. Reiner, A.; Zagvazdin, Y. Trends Pharmacol. Sci. 1998, 19, 348.
27. Wolff, D. J.; Gribbin, B. J. Arch. Biochem. Biophys. 1994, 311, 300.
28. Chen, P. F.; Tsai, A. L.; Berka, V.; Wu, K. K. J. Biol. Chem. 1997, 272, 6114.
29. Rosenfeld, R. J.; Garcin, E. D.; Panda, K.; Andersson, G.; Åberg, A.; Wallace, A. V.;
Morris, G. M.; Olson, A. J.; Stuehr, D. J.; Tainer, J. A.; Getzoff, E. D. Biochemistry
2002, 41, 13915.
49. Bouissane, L.; El Kazzouli, S. E.; Léger, J.-M.; Jarry, C.; Rakib, E. M.; Khouili, M.;
Guillaumet, G. Tetrahedron 2005, 61, 8218.
50. Crespo, E.; Macías, M.; Pozo, D.; Escames, G.; Martin, M.; Vives, F.; Guerrero, J.
M.; Acuña-Castroviejo, D. FASEB J. 1999, 13, 1537.
51. Escames, G.; León, J.; Macías, M.; Khaldy, H.; Acuña-Castroviejo, D. FASEB J.
2003, 17, 932.
30. Porubsky, P. R.; Meneely, K. M.; Scott, E. E. J. Biol. Chem. 2008, 283, 33698.
31. Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.; Siracusa, M. A. Curr. Pharm.
Des. 2002, 8, 177.
52. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
53. Bredt, D. S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9030.