Synthesis and biological evaluation of indazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.01.027

The inhibition of neuronal and inducible nitric oxide synthases (nNOS and iNOS) by a series of 36 indazoles has been evaluated, showing that most of the assayed derivatives are better iNOS than nNOS inhibitors. A parabolic model relating the iNOS inhibiti

Full text of DOI:10.1016/j.ejmech.2011.01.027

European Journal of Medicinal Chemistry 46 (2011) 1439e1447
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and biological evaluation of indazole derivatives
,
a
c,
Rosa M. Claramunt a , Concepción López , Ana López d, Carlos Pérez-Medina a, Marta Pérez-Torralba a,
*
Ibon Alkorta b, José Elguero b, Germaine Escames c d, 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 Departamento de Fisiología, Facultad de Medicina, Avenida de Madrid 11, 18012 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of neuronal and inducible nitric oxide synthases (nNOS and iNOS) by a series of 36
indazoles has been evaluated, showing that most of the assayed derivatives are better iNOS than nNOS
inhibitors. A parabolic model relating the iNOS inhibition percentage with the difference, Erel, between
stacking and apical interaction energies of indazoles with the active site of the NOS enzyme has been
established.
Received 8 October 2010
Received in revised form
11 January 2011
Accepted 14 January 2011
Available online 28 January 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
In memoriam of our friend Professor
Concepción Foces-Foces.
Keywords:
Indazoles
iNOS
nNOS
Modeling
1. Introduction
Pharmacia & Upjohn group with U-19451A [5] where they explored
the iNOS/nNOS selectivity. Save the first three 7-nitro derivatives,
A family of enzymes known as nitric oxide synthases (NOS)
catalyzes the oxidation of -arginine to -citrulline and nitric oxide
1e3, 3-methylindazole (5), the three 3-bromo-7-nitro derivatives,
15e17, 3,7-dinitroindazole (18), 3-hydroxyindazole (19) and 3-
hydroxy-7-nitroindazole (23), the remaining 26 indazoles contain
fluorine substituents. To date, only our group has explored the
inhibitory activity of fluoroindazoles against iNOS.
This is our third paper on the inhibitory properties of indazoles
against NOS. In the first one [6], we reported the percentage of
nNOS and iNOS inhibition of eleven indazoles at 1 mM concentra-
tion: 1, 2, 3, 8, 12, 13, 14, 15, 16, 17 and 22, whose values will be used
in this paper. In the second one we proposed a theoretical model
based on the stacking of indazoles above the heme [7].
L
L
(NO), a molecule that plays an important role in the regulation of
blood pressure, neurotransmission, and the immune response.
Three isoforms of nitric oxide synthase have been identified in
different tissues. Neuronal (nNOS) and endothelial (eNOS) nitric
oxide synthases are expressed constitutively and are calcium/
calmodulin dependent; inducible nitric oxide synthase (iNOS) is
expressed in response to inflammatory or immunologic stimuli and
its activity is independent of calmodulin. All NOS isoforms are
protoporphyrin IX heme enzymes and require NADPH, FAD, FMN
and tetrahydrobiopterin (H4B) as cofactors [1e4].
We present here the results of our investigation on the selec-
tivity of inducible iNOS compared to constitutive nNOS of a series of
indazoles depicted in Fig. 1, in a similar approach to that of the
2. Results and discussion
2.1. In vitro NOS inhibition
The 36 indazoles studied in this article have been tested for their
inhibitory properties employing the method reported by Bredt et al.
for the determination of NOS activity (Section 4.1). 25 of these
indazoles are studied for the first time; each value reported inTable 1
is the average of three measurements.
* Corresponding author.
** Corresponding author. Departamento de Fisiología, Facultad de Medicina,
Avenida de Madrid 11, 18012 Granada, Spain.
E-mail addresses: rclaramunt@ccia.uned.es (R.M. Claramunt), dacuna@ugr.es
(D. Acuña-Castroviejo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.027

1440
R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
F
F
Me
N
Me
F
N
N Me
N
N
N
N
N
N
N
N
N
F
F
Me
H
H
H
H
NO2
NO2
3
NO2
F
1
4
5
2
6
F
F
F
F
Me
N
Me
Me
N
CF3
N
CF3
N
Me
N
F
F
O2N
F
F
N
N
N
N
N
N
N
F
F
F
H
H
H
H
H
H
F
8
F
F
F
NO2
11
7
10
12
9
F
F
NO2
N
N
H
C6F5
Br
Br
N Me
Br
N
N
H
C6H5
N
N
H
F
F
F
F
N
N
N
H
N
N
Me
NO2
18
F
F
NO2
NO2
17
NO2
14
16
15
13
F
OH
N
N
H
F
OH
N
N
H
OH
N
N
H
OH
OH
N
N
H
OH
N
F
F
N
N
F
F
N
F
H
H
F
NO2
23
NO2
F
F
19
21
20
24
22
F
CO2H
EtO2C
F
MeO2C
F
OH
EtO2
N
OH
N
OH
N
N
H
OH
OH
N
OH
MeO2C
F
F
F
HO2C
F
C
F
N
N
N
H
N
N
N
N
H
F
F
F
H
H
H
F
F
F
F
F
F
F
F
F
29
25
27
28
30
26
F
F
F
OH
OH
N
OH
F
OH
N
OH
N
N
H
OH
N
N
H
F
F
F
F
F
F
F
N
N
N
N
N
N
H
MeO2C
HO2C
EtO2C
F
H
H
H
F
F
F
MeO2C
CO2H
EtO2C
32
31
33
35
34
36
Fig. 1. The indazoles series.
Some values of iNOS and nNOS percentages of inhibition
(marked w) for compounds 37, 38 and 39 (Fig. 2) have been esti-
mated. Bland-Ward and Moore described that the iNOS inhibitory
properties of indazole itself (37) should be weak [8], and conse-
quently we have used a 45% value. The IC50 mM values of 7-
methoxyindazole (38) were established by Boucher et al.:
nNOS ¼ 1.5 and iNOS ¼ 5.5 [11], and since the percentage of inhi-
bition against nNOS was known (80.0), [9] we have estimated the
effect on iNOS to be similar but slightly lower, 70%. Vichard et al.
[12] have reported the IC50 mM values for 7-cyanoindazole (39):
nNOS ¼ 25 and iNOS ¼ 110 (these much larger values determined
by a different technique should not be compared with those of
other authors) and also for 7-nitroindazole (1), nNOS ¼ 25 and
iNOS ¼ 50. Therefore, we have used the same value for nNOS (88%,
as for 1) and reduced the value for iNOS to 80%.
A perusal of Table 1 shows that the most potent iNOS inhibitors
are, in decreasing order, 4 (98.7) > 15 (97.0) > 10 (95.6) > 9 (92.0) >
24 (91.0) > 18 (89.3) > 21 (89.1) > 1 (84.0) > 8 (83.0) (Fig. 3).
Meanwhile the nNOS inhibitory potency decreases as follows: 15
(95.0) > 9 (90.0) > 1 (88.0) > 10 (85.4) (Fig. 4).
Regarding selectivity we can say that the majority of the assayed
indazoles are better iNOS than nNOS inhibitors. At a concentration of
1 mM, compounds 18 (40-fold selective),14 (10-fold), 24 (6-fold) and
4 (2-fold) showgood promise as possible iNOS selective inhibitors. At
the same concentration, the other potent iNOS inhibitors
(compounds 1, 9 and 15) also show high potency vs. nNOS and thus
poor selectivity between the isoforms. However, these compounds
deserve to be studied more in depth before ruling out selectivity.
All indazoles bearing a carboxylic or an ester substituent, 25e36,
proved to be moderate iNOS inhibitors and very weak nNOS ones,

R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
1441
Table 1
iNOS and nNOS % inhibition in the presence of indazoles. Values from Ref. [6] are
underlined. Except when indicated, the concentration of these compounds used in
the NOS assays was 1 mM.
N
N
N
N
N
N
H
H
H
CN
39
OMe
38
Compound
iNOS
84.0
nNOS
37
1 (7-Nitro-1H-indazole)
88.0
27.3
29.0
43.3
33.7a
52.1
58.4
63.0
90.0
85.4
69.9
41.0
11.0
8.0
2 (1-Methyl-7-nitro-1H-indazole)
3 (2-Methyl-7-nitro-2H-indazole)
4 (4,5,6,7-Tetrafluoro-1H-indazole)
5 (3-Methyl-1H-indazole)
25.0
36.0
98.7
77.4
74.3
69.2
83.0
92.0
95.6
80.1
21.0
37.0
80.0
97.0
10.0
7.0
Fig. 2. Three literature indazoles.
and 24. 7-Nitroindazole (1) is commercially available and was used
without further purification. Compounds 5, 8, 11 and 12 can be
found in Ref. [18] whereas indazoles 2, 3, 13, 14, 16, 17 and 22 in Ref.
[6]. Difluoro-3-methylindazoles 6, 7, 9 and 10 are reported in Ref.
[19] and 3-hydroxyindazole (19) and 3-hydroxy-7-nitroindazole
(23) in Ref. [20]. Finally, trifluoro-3-hydroxy-indazolecarboxylic
acids and esters, 25e36, are described in Ref. [21].
In order to prioritize comparability over correct nomenclature,
we have named all the compounds as substituted indazoles even in
the case of the 3-hydroxy groups that in accordance with IUPAC
rules are indazol-3-ols. Also, for the sake of clarity we avoided the
use of the prefix 1H for unsubstituted indazoles in the text. See
Table 1 for the full name of all derivatives.
6 (3-Methyl-4,6-difluoro-1H-indazole)
7 (3-Methyl-6,7-difluoro-1H-indazole)
8 (3-Methyl-4,5,6,7-tetrafluoro-1H-indazole)
9 (3-Methyl-4,6-difluoro-7-nitro-1H-indazole)
10 (3-Methyl-6,7-difluoro-5-nitro-1H-indazole)
11 (3-Trifluoromethyl-1H-indazole)
12 (3-Trifluoromethyl-4,5,6,7-tetrafluoro-1H-indazole)
13 (3-Phenyl-4,5,6,7-tetrafluoro-1H-indazole)
14 (3-Pentafluorophenyl-4,5,6,7-tetrafluoro-1H-indazole)
15 (3-Bromo-7-nitro-1H-indazole)
95.0
22.0
29.0
2.1
16 (1-Methyl-3-bromo-7-nitro-1H-indazole)
17 (2-Methyl-3-bromo-7-nitro-2H-indazole)
18 (3,7-Dinitro-1H-indazole)
89.3
55.1
48.1
89.1
6.0
19 (3-Hydroxy-1H-indazole)
31.4b
11.1
57.1
23.0
18.9
14.9
1.8
20 (3-Hydroxy-4,6-difluoro-1H-indazole)
21 (3-Hydroxy-6,7-difluoro-1H-indazole)
22 (3-Hydroxy-4,5,6,7-tetrafluoro-1H-indazole)
23 (3-Hydroxy-7-nitro-1H-indazole)
24 (3-Hydroxy-4,6-difluoro-7-nitro-1H-indazole)
25 (3-Hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic acid)
26 (3-Hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic acid
methyl ester)
4,5,6,7-Tetrafluoroindazole (4) was prepared, not without diffi-
culty, according to Scheme 1.
8.2
When pentafluorobenzaldehyde (40) reacted with hydrazine in
the standard conditions to prepare indazoles, only azine 42 was
isolated [22]. To obtain hydrazone 41 the reaction was carried out in
ethanol/water at low temperature and high-dilution. When 41 was
heated in toluene or N,N0-dimethylformamide the only product
isolated was 42. These failed attempts are in agreement with the
results reported by Lukin et al. [23] about the difficulty to cyclize
hydrazones derived from benzaldehydes when compared to those
derived from acetophenones, which could be due to an unfavorable
conformation of the former [24].
However, according to Lukin, the formation of indazoles from
hydrazones occurs through a hydrazineehydrazone intermediate
like 43 (in the original article without fluorine substituents) that
cyclizes into 3-hydrazineindazoline 44 to afford, after elimination
of a molecule of hydrazine, the desired indazole 4. Therefore, we
carried out the reaction of 40 with an excess of hydrazine (1:5) and,
although we obtained the desired product 4 in 13% yield, we also
isolated a large amount of the isomeric hydrazineehydrazone 45.
3,7-Dinitroindazole (18) has been described in the literature by
two different groups both using the same synthetic approach
(Scheme 2) involving the nitration of 7-nitroindazole (1) to afford
2,7-dinitroindazole (46) that on heating in anisole [25] or amyl
alcohol [26] solution was converted into 18. We have followed the
method described in Ref. [25], with similar results to those
reported.
3-Hydroxy-4,6-difluoroindazole (20) and 3-hydroxy-6,7-
difluoroindazole (21) were prepared in a similar way to other 3-
hydroxyindazoles described in a previous paper [21] (Scheme 3).
Secondary products resulting from the nucleophilic replacement of
the fluorine atoms in para-position to the ester group were also
detected.
Indazoles 20 and 21 have CAS Registry numbers (887567-77-3
and 1000343-93-0, respectively) but only the first one appears to
have been described in a patent [27]. Finally, nitration of compound
20 affords 3-hydroxy-4,6-difluoro-7-nitroindazole (24) in good
yield (Scheme 3).
91.0
62.6
22.6
6.3
27 (3-Hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic acid
ethyl ester)
38.7
1.2
28 (3-Hydroxy-4,6,7-trifluoro-1H-indazole-5-carboxylic acid)
29 (3-Hydroxy-4,6,7-trifluoro-1H-indazole-5-carboxylic acid
methyl ester)
16.3
48.9
12.1
13.3
30 (3-Hydroxy-4,6,7-trifluoro-1H-indazole-5-carboxylic acid
ethyl ester)
51.8
2.5
31 (3-Hydroxy-4,5,7-trifluoro-1H-indazole-6-carboxylic acid)
32 (3-Hydroxy-4,5,7-trifluoro-1H-indazole-6-carboxylic acid
methyl ester)
42.0
46.2
5.4
19.7
33 (3-Hydroxy-4,5,7-trifluoro-1H-indazole-6-carboxylic acid
ethyl ester)
53.5
10.2
34 (3-Hydroxy-4,5,6-trifluoro-1H-indazole-7-carboxylic acid)
35 (3-Hydroxy-4,5,6-trifluoro-1H-indazole-7-carboxylic acid
methyl ester)
40.7
49.6
11.1
5.1
36 (3-Hydroxy-4,5,6-trifluoro-1H-indazole-7-carboxylic acid
ethyl ester)
43.7
5.6
37 (1H-indazole) [8]
38 (7-Methoxy-1H-indazole) [9]
39 (7-Cyano-1H-indazole) [12]
w45
w70
w80
11.2
80.0
w88
a
The related 3-ethyl-1H-indazole has a % of nNOS inhibition of 18.8 [8].
3-Hydroxy-1H-indazole (19) is inactive towards nNOS (IC50 (mM) > 1000) [6,10].
b
being 3-hydroxy-5,6,7-trifluoroindazole-4-carboxylic acid (25)
(iNOS ¼ 62.6%, nNOS ¼ 1.8% e 35-fold selective) the most promising
candidate.
A FreeeWilson analysis using presenceeabsence
matrices [13,14] of these 12 indazoles affords the values gathered in
Table 2.
Coefficients in Table 2 indicate small sensitivity to the nature of
R (H, CH3, C2H5) and the position (4, 5, 6, 7) of the substituent, but
are always larger for iNOS than for nNOS.
If one wants to exclude nitroaromatic compounds to avoid
toxicity problems [15e17], then the most interesting indazole is 4
distantly followed by 21 and 8. In fact 7-cyanoindazole (39) and its
3-bromo derivative were prepared [12] to by-pass the aforemen-
tioned problems shown by the nitroindazoles 1 and 15.
2.2. Chemistry
2.3. Modeling studies
We have already reported the preparation and physicochemical
In our previous work we advocated the use of the difference Erel
properties of the 36 assayed compounds save those of 4, 18, 20, 21
of stacking and apical interaction energies between indazoles and

1442
R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
Fig. 3. Percentage of residual activity of iNOS in the presence of the tested compounds.
porphyrines to estimate the inhibitory potency of these compounds
[7]. The model was based on crystallographic studies [28e30],
replacing the Fe by Zn for computational ease. The NOS inhibitory
activity (in percentage) was related, in an exponential relationship,
with the difference, Erel, between the stacking and the apical
(lone-pair) interaction energies as follows: % inhibition ¼ 104:0ꢀ
comparative analyses making use of the values we found in the
NIST database [33]: pyridine basicity 930, imidazole basicity 942.8
and acidity 1466, pyrazole basicity 894.1 and acidity 1480, indazole
basicity 900.8 and acidity 1457 kJ molꢀ1. Using these values, the
following trendlines can be calculated:
PA ¼ ð75 ꢂ 38Þ þ ð0:92 ꢂ 0:04Þ basicity; n ¼ 4; R2
2:21 ꢁ eðE =8:54Þ. A similar result was obtained using stacking
rel
ꢀ
ꢁ
energies instead of Erel. Compound 14, C13HF9N2, due to its exces-
sive size was excluded from the calculations.
The interaction of indazoles with the active site of the enzyme
involves not only stacking but hydrogen bonding (HB) of the NeH
(indazole)/O]C and NeH/N(indazole) type. Then, we explored
a model including NeH HB acidity and N: HB basicity using the
¼ 0:996 differences ꢂ 2:7 kJ molꢀ1
(1)
(2)
D
H ¼ ð506 ꢂ 81Þ þ ð0:64 ꢂ 0:05Þ acidity; n ¼ 3; R2
ꢀ
ꢁ
¼ 0:993 differences ꢀ 26 kJ molꢀ1
thermodynamic acidity (
DH) and basicity (PA or proton affinity),
Although the experimental gas-phase acidities are systemati-
since it is known that they are proportional for structurally related
compounds [7,31,32]. Table 3 contains all the information we have
gathered on this problem, including data for different heterocycles
(pyridine, imidazole and pyrazole) of known experimental basicity
(PA) and acidity (DH).
In order to check the validity of the calculated values before
using them for modelization purposes, we have carried out two
cally underestimated by the calculations, they are proportional (Eq.
(2)) and therefore can be used in the models of activity.
In a previous report, we related iNOS activity with the Erel for
a total of eleven indazoles, suggesting the existence of an asymp-
totic model [6]. Now, with a larger number of compounds available,
we consider that a parabolic model is a better fit than the former.
Parabolic relationships (corresponding to polynomial models of
Fig. 4. Percentage of residual activity of nNOS in the presence of the tested compounds.

R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
1443
Table 2
NO2
N
Contribution of the different substituents and positions to the activity of the 12
indazoles bearing a CO2R substituent.
Δ
N
N
NO2
N
N
N
Substituent
iNOS
nNOS
H
H
NO2
NO2
NO2
CO2H
40 ꢂ 7
42 ꢂ 7
47 ꢂ 7
0.93
8 ꢂ 3
11 ꢂ 3
5 ꢂ 3
0.76
46
CO2CH3
CO2C2H5
R2
1
18
Scheme 2. Synthesis of indazole 18.
4-CO2R
5-CO2R
6-CO2R
7-CO2R
R2
41 ꢂ 8
39 ꢂ 8
47 ꢂ 8
45 ꢂ 8
0.93
3 ꢂ 3
9 ꢂ 3
12 ꢂ 3
7 ꢂ 3
0.80
ꢀ
ꢁ
% nNOS ¼ ꢀð325 ꢂ 284Þ ꢀ ð0:62 ꢂ 0:26Þ Er2el =10
þ ð0:50 ꢂ 0:32ÞPA; n
¼ 13; R2 ¼ 0:35
(5)
(6)
Selectivity ð
D
¼ % iNOS ꢀ % nNOSÞ
order 2) were introduced by Hansch and soon became classical in
medicinal chemistry [14,34,35].
Fig. 5 represents the variation of iNOS inhibition percentage
with Erel after excluding compounds 12 (3-trifluoromethyl-4,5,6,7-
tetrafluoroindazole) and 21 (3-hydroxy-6,7-difluoroindazole). The
trendline corresponds to:
¼ ð365 ꢂ 205Þ ꢀ ð0:72 ꢂ 0:42ÞErel
ꢀ ð0:55 ꢂ 0:28ÞPA; n
¼ 13; R2 ¼ 0:48
3. Conclusion
iNOS % inhibition ¼ ð95 ꢂ 3Þ ꢀ ð0:24 ꢂ 0:12ÞErel
ꢀ ð0:036 ꢂ 0:005ÞEr2el; n
For the first time the inhibitory properties of fluorinated inda-
zoles against iNOS have been evaluated. From a library of 34
indazole derivatives we have identified a very potent iNOS inhib-
itor, 4,5,6,7-tetrafluoroindazole (4), a non-nitro derivative that
constitutes a promising candidate for future developments.
We advocate the use of computational modeling of the inter-
action between a large series of indazole candidates with Zn-
porphyrins and proceed to synthesize and test only those that the
model predicts to be active and selective. Even though these
predictions are not entirely precise they suffice to know where to
address the research.
¼ 13; R2 ¼ 0:81
(3)
The curve reaches its maximum (100%) for values of Erel close to
0 (no experimental data correspond to it).
The only other property that appears to have an effect on some
inhibition data is the thermodynamic basicity, PA. The acidity does
not appear to have an influence of the inhibitory properties. Eqs.
(4)e(6) can be calculated from the data reported in Tables 1 and 3,
and although only the first one is significant, the other two suggest
a necessary criterion for selectivity iNOS vs. nNOS: the indazole
must have low basicity.
4. Experimental
%iNOS ¼ ð189ꢂ67Þꢀð0:22ꢂ0:11ÞErel
4.1. Assay of iNOS/nNOS activities
ꢀ
ꢁ
ꢀð0:30ꢂ0:07Þ Er2el =10ꢀð0:11ꢂ0:08ÞPA; n
¼ 13;R2 ¼ 0:85
L
-Arginine, L
-citruline, N-(2-hydroxymethyl)piperazine-N0-(2-
(4)
ethanesulfonic acid) (HEPES), DL-dithiothreitol (DTT), leupeptin,
F
F
F
F
F
NH2
H
F
F
N
F
N2H4
F
F
CHO
F
F
F
toluene or DMF
EtOH/H2O
N
N
H
low temp.
H
high-dilution
F
F
F
F
F
F
40
41
42
5 N2H4
F
NH2
H
NH2
F
N
F
F
N
F
F
F
NHNH2
H
F
F
F
F
F
F
F
H
+
N H
N
N
H
N
H
H2NHN
NHNH2
F
F
F
43
44
4
45
Scheme 1. Synthesis of indazole 4.

1444
R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
R1
R2
R1
F
R1
OH
N
OH
N
CO2H
F
CO2CH3
F
SOCl2
MeOH
N2H4
KNO3
H2SO4
N
F
N
F
F
F
R2
NO2
H
R2
H
47, R1 = F, R2 = H
20, R1 = F, R2 = H
49, R1 = F, R2 = H
50, R1 = H, R2 = F
24 (from 20)
48, R1 = H, R2 = F
21, R1 = H, R2 = F
Scheme 3. Synthesis of indazoles 20, 21 and 24.
aprotinin, pepstatin, phenylmethylsulfonyl-fluoride (PMSF), hypo-
(Naþ form) and eluted with 1.2 mL of water. -3H-citrulline was
L
xantine-9-
b
-
D
-ribofuranosid (inosine), ethylene glycol-bis-(2-ami-
quantified by liquid scintillation counting in a Beckman LS-6000
noethylether)-N,N,N0,N0-tetraacetic acid (EGTA), bovine serum
albumin (BSA), Dowex-50W (50 ꢁ 8e200), FAD, NADPH and
system (Beckman Coulter, Fullerton, CA, USA). The retention of
L
-3H-arginine in this process was greater then 98%.
5,6,7,8-tetrahydro-
obtained from SigmaeAldrich Química (Spain).
(58 Ci/mmol) was obtained from Amersham (Amersham Biosci-
ences, Spain). Tris(hydroxymethyl)-aminometane (TriseHCl) and
calcium chloride were obtained from Merck (Spain).
L
-biopterin dihydrocloride (H4-biopterin) were
A total of 25 compounds were assayed for nNOS/iNOS activities:
L
-[3H]-arginine
4,5,6,7-tetrafluoro-1H-indazole (4); 3-methyl-1H-indazole (5);
3-methyl-4,6-difluoro-1H-indazole (6); 3-methyl-6,7-difluoro-1H-
indazole (7); 3-methyl-4,6-difluoro-7-nitro-1H-indazole (9);
3-methyl-6,7-difluoro-5-nitro-1H-indazole (10); 3-trifluoromethyl-
1H-indazole (11); 3,7-dinitro-1H-indazole (18); 3-hydroxy-
1H-indazole (19); 3-hydroxy-4,6-difluoro-1H-indazole (20);
3-hydroxy-6,7-difluoro-1H-indazole (21); 3-hydroxy-7-nitro-1H-
indazole (23); 3-hydroxy-4,6-difluoro-7-nitro-1H-indazole (24);
For iNOS activity determination, male Wistar rats (3-months
old, 220e250 g) were breed and kept in the University animal
facility on a 12 h light/12 h dark cycle at 22 ꢂ 2 ꢃC, 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 [36]. Briefly, three days before
the experiment the jugular vein was cannulated under i.p. equi-
thesin anesthesia (1 mL/kg) for LPS administration. Rats were i.v.
injected with 10 mg/kg b.w. LPS (E. coli 0127:B8, SigmaeAldrich,
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 ꢃC in liquid nitrogen. Pieces of
lungs were homogenized (0.1 g/mL) in ice-cold buffer (25 mM Tris,
3-hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic
acid
(25);
3-hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic acid methyl
ester (26); 3-hydroxy-5,6,7-trifluoro-1H-indazole-4-carboxylic acid
ethyl ester (27); 3-hydroxy-4,6,7-trifluoro-1H-indazole-5-carbox-
ylic acid (28); 3-hydroxy-4,6,7-trifluoro-1H-indazole-5-carboxylic
acid methyl ester (29); 3-hydroxy-4,6,7-trifluoro-1H-indazole-
5-carboxylic acid ethyl ester (30); 3-hydroxy-4,5,7-trifluoro-
1H-indazole-6-carboxylic acid (31); 3-hydroxy-4,5,7-trifluoro-
1H-indazole-6-carboxylic acid methyl ester (32); 3-hydroxy-4,5,7-
trifluoro-1H-indazole-6-carboxylic acid ethyl ester (33); 3-hydroxy-
4,5,6-trifluoro-1H-indazole-7-carboxylic acid (34); 3-hydroxy-
4,5,6-trifluoro-1H-indazole-7-carboxylic acid methyl ester (35);
3-hydroxy-4,5,6-trifluoro-1H-indazole-7-carboxylic acid ethyl ester
(36). Except when indicated, the concentration of these compounds
used in the NOS assays was 1 mM.
0.5 mM DTT, 10 mg/mL pepstatin, 10 mg/mL leupeptin, 10 mg/mL
aprotinin, 1 mM PMSF, pH 7.6) at 0e4 ꢃC [37]. The crude homoge-
nate was centrifuged at 2500g at 4 ꢃC for 5 min, and aliquots of the
supernatant were either stored at ꢀ80 ꢃC for total protein deter-
mination [38] or used immediately for NOS activity measurement.
For nNOS activity determination, untreated male Wistar rats
(200e250 g) were killed by cervical dislocation and striata were
quickly collected and immediately used to measure nNOS activity.
Upon removal, tissues were cooled in an ice-cold buffer (25 mM
4.2. Chemistry
Melting points for compounds 4, 20, 21 and 24 were determined
by DSC on a Seiko DSC 220C connected to a Model SSC5200H Disk
Tris, 0.5 mM DTT, 10 mg/mL leupeptin, 10 mg/mL pepstatin, 10 mg/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 homogenates
were centrifuged 5 min at 1000g, and an aliquot of the supernatant
was frozen at ꢀ80 ꢃC for total protein determination [38].
Table 3
PA (basicity),
D
H (acidity), Ei stacking energies, Ei apical energies, Erel (Ei stacking ꢀ Ei
apical). M05-2x/6-311þG(d) calculations including BSSE. All values in kJ molꢀ1
.
NOS activity was measured by the method of Bredt et al. [39],
Ligand
PA
D
H
Stacking
Apical
Erel
monitoring the conversion of
Enzyme activity was referred as pmol
The final incubation volume was 100
sample added to prewarmed (37 ꢃC) buffer to give a final concen-
tration of 25 mM Tris, 1 mM DTT, 30 M H4-biopterin, 10 M FAD,
0.5 mM inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 -arginine
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 L
-3H-arginine to -3H-citrulline.
Pyridine
Imidazole
Pyrazole
1
4
5
8
9
10
12
15
18
19
21
24
37
38
39
927.8
945.5
891.4
860.2
839.2
918.3
862.7
858.8
844.4
803.9
843.3
793.7
901.4
872.0
849.4
898.7
911.2
859.2
e
e
e
e
e
e
L
-3H-citrulline/min/mg prot.
L and consisted of 10
1489.0
1513.0
1446.8
1409.0
1482.2
1416.2
1424.5
1382.5
1358.5
1408.1
1358.4
1477.0
1444.8
1417.0
1477.9
1487.8
1428.4
e
e
m
mL
e
e
ꢀ41.1
ꢀ63.6
ꢀ82.7
ꢀ45.4
ꢀ75.3
ꢀ78.8
ꢀ45.0
ꢀ44.9
ꢀ54.4
ꢀ92.7
ꢀ61.4
ꢀ88.4
ꢀ36.7
ꢀ62.8
ꢀ57.5
ꢀ60.0
ꢀ73.1
ꢀ56.7
ꢀ66.6
ꢀ74.4
ꢀ78.4
ꢀ49.7
ꢀ55.7
ꢀ43.5
ꢀ55.1
ꢀ91.2
ꢀ75.1
ꢀ68.5
ꢀ76.2
ꢀ72.8
18.9
9.5
m
m
ꢀ26.0
mM L
21.2
L
ꢀ0.9
ꢀ0.4
4.7
m
continued for 30 min at 37 ꢃC. Control incubations were performed
in absence of NADPH. The activity of iNOS was measured in the
presence or absence of 10 mM EDTA. EDTA removes calcium from
the medium preventing the activation of nNOS; thus, in the pres-
ence of EDTA only iNOS activity is detected. The reaction was
stopped adding 400
EGTA, 175 mM
-3H-citrulline, pH 5.5. The mixture was decanted
onto a 2 mL column packed with Dowex-50W ion exchanger resin
10.8
ꢀ10.9
ꢀ37.6
29.8
ꢀ13.3
31.8
13.4
15.3
mL of cold 0.1 M HEPES containing 10 mM
L

R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
1445
110
100
90
100/0
4
10
15
24
9
18
1
8
80
39
5
70
38
60
19
50
37
40
30
-40
-30
-20
-10
0
10
20
30
40
Erel (kJ/mol)
Fig. 5. Variation of the percentage of iNOS inhibition with Erel
.
Station while for compounds 18, 41, 42 and 45 a ThermoGalen hot
stage microscope was used. Thermograms (sample size 0.003e
(1He15N) gs-HMBC, were acquired and processed using standard
Bruker NMR software and in non-phase-sensitive mode [40].
Gradient selection was achieved through a 5% sine truncated sha-
ped pulse gradient of 1 ms. Variable temperature experiments were
recorded on the same spectrometer. A Bruker BVT3000 tempera-
ture unit was used to control the temperature of the cooling gas
stream and an exchanger to achieve low temperatures.
0.0010 g) were recorded at a scanning rate of 2.0e5.0 ꢃC minꢀ1
.
Thin-layer chromatography (TLC) was performed with Merck silica
gel (60 F254). Compounds were detected with a 254-nm UV lamp.
Silica gel (60e320 mesh) was employed for routine column chro-
matography separations with the indicated eluent. Elemental
analyses for carbon, hydrogen, and nitrogen were carried out on a
PerkineElmer 240 analyzer. Exact mass was determined on a Q-TOF
LC/MS 6520 (Agilent Technologies) equipment using ESIþ.
4.2.1. 4,5,6,7-Tetrafluoro-1H-indazole (4)
In a three-neck round-bottom flask equipped with a reflux
condenser, 98% hydrazine monohydrate (0.70 g, 14 mmol) was
dissolved in THF (200 mL). The flask was placed in a bath of ice/
water and 2,3,4,5,6-pentafluorobenzaldehyde (40) (0.55 g,
2.8 mmol) dissolved in THF (150 mL) was added dropwise. The
mixture was then stirred at 70 ꢃC for 48 h. The solution was dec-
anted and solvent removed under reduced pressure to obtain
a yellowish solid which was suspended in hexane. The solid was
filtered, washed with chloroform and discarded (0.29 g). In the
mother liquor the product together with 2,3,5,6-tetrafluoro-
4-(hydrazonomethyl)phenylhydrazine (45) was present. The
separation of these two components was carried out by column
chromatography on silica gel with hexane/diethyl ether 20:1 to
afford 4 (70 mg,13%) as a white solid. Mp 191.5 ꢃC (DSC). Anal. Calcd
Solution spectra were recorded, at 300 K save specified, on
a Bruker DRX 400 (9.4 Tesla, 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 HeX 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), THF-d8 (3.58) and CD3CN (1.93) for 1H and
CDCl3 (77.0), DMSO-d6 (39.5), THF-d8 (67.4) and CD3CN (1.4
and 118.7) for 13C. And external references, CFCl3 (0.00) for 19F and
CH3NO2 (0.00) for 15N NMR were used. 2D (1He1H) gs-COSY and
inverse proton detected heteronuclear shift correlation spectra,
(1He13C) gs-HMQC, (1He13C) gs-HMBC, (1He15N) gs-HMQC, and

1446
R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
for C7H2F4N2: C, 44.23; H, 1.06; N, 14,74. Found: C, 44.34; H, 1.17; N,
4.2.4. 3-Hydroxy-4,6-difluoro-1H-indazole (20)
14.62. 1H NMR (CDCl3)
d
10.79 (br s, 1H, NH), 8.24 (d, 5JF7 ¼ 3.1, 1H,
In a three-neck round-bottom flask equipped with a reflux
condenser, methyl 2,4,6-trifluorobenzoate (49) (0.20 g, 1.1 mmol)
was dissolved in tetrahydrofuran (20 mL); then, a solution of 98%
hydrazine monohydrate (0.15 g, 3.0 mmol) in ethanol (10 mL) was
added dropwise. The mixture was heated at 70 ꢃC for 24 h. After
cooling at room temperature, the solution was decanted and the
organic solvent evaporated under vacuum. The solid residue was
washed with dichloromethane and dried to afford 20 (0.13 g, 76%)
as a white solid. Mp 293.5 ꢃC. Anal. Calcd for C7H4F2N2O: C, 49.42;
H, 2.37; N, 16.47. Found: C, 49.44; H, 2.49; N, 16.27. 1H NMR
3
5
H3); 19F NMR (CDCl3)
d
ꢀ145.5 (dd, JF5 ¼18.9, JF7 ¼ 18.9, F4),
3
3 4
ꢀ165.5 (ddd, JF4 ¼18.9, JF6 ¼ 18.9, JF7 ¼ 2.2, F5), ꢀ156.9 (dd,
3JF5 ¼18.9, JF7 ¼ 18.9, F6), ꢀ159.0 (dd, JF6 ¼ 18.9, JF4 ¼18.9, F7);
3
3
5
13C NMR (THF-d8)
d
132.2 (d, 1J ¼ 194.9, C3), 111.4 (dd, 2J ¼ 20.8,
3J ¼ 3.2, C3a), 139.6 (dddd, 1J ¼ 250.1, 2J ¼ 11.4, 3J ¼ 4J ¼ 3.8, C4),
135.8 (ddd, 1J ¼ 242.7, 2J ¼ 2J ¼ 15.5, C5), 139.8 (dddd, 1J ¼ 247.6,
2J ¼ 2J ¼ 15.1, 3J ¼ 2.5, C6), 132.7 (ddd, 1J ¼ 250.9, 2J ¼ 14.7, 3J ¼ 4.1,
C7), 126.3 (ddd, 2J ¼ 15.4, 3J ¼ 6.6, 3J ¼ 3.5, C7a); 15N NMR (THF-d8,
T ¼ 207 K)
d
ꢀ201.1 (N1), ꢀ58.6 (N2).
2,3,5,6-Tetrafluoro-4-(hydrazonomethyl)phenylhydrazine (45)
was obtained as a yellow solid. Mp > 300 ꢃC. Exact mass (ESIþ) calc
(DMSO-d6) d 11.8 (br s, 1H, NH), 10.9 (br s, 1H, OH), 6.71 (ddd,
4
3
4
3JF4 ¼ 3JF6 ¼ 10.3, JH7 ¼ 1.9, 1H, H5), 7.09 (dd, JF6 ¼ 9.2, JH5 ¼1.9,
for C7H6F4N4 222.0529, found 222.0527. 1H NMR (DMSO-d6)
d
7.62
1H, H7); 19F NMR (DMSO-d6)
d
ꢀ115.9 (dd, JH5 ¼ 9.6, JF6 ¼ 8.4,
3
4
(s, 1H, eCH]Ne), 7.11 (s, 2H, H2NeNHeC1), 6.94 (s, 1H, eNHe),
F4), ꢀ111.6 (br s, F6); 13C NMR (DMSO-d6)
d 153.6 (s, C3), 98.9
4.46 (br s, 2H, ]NeNH2); 19F NMR (DMSO-d6)
d
ꢀ147.9 (m, F3 and
(d, 2J ¼ 20.2, C3a), 155.7 (dd, 1J ¼ 253.3, 3J ¼ 16.7, C4), 94.4 (dd,
2J ¼ 30.5, 2J ¼ 23.3, C5), 161.8 (dd, 1J ¼ 242.7, 3J ¼ 11.7, C6), 92.0
(dd, 2J ¼ 26.1, 4J ¼ 4.7, C7), 143.6 (dd, 3J ¼ 15.1, 3J ¼ 10.3, C7a); 15N
F5), ꢀ159.3 (m, F2 and F6); 13C NMR (DMSO-d6)
d
143.9 (ddd,
1J ¼ 246.6, 2J ¼ 12.7, 3J ¼ 3.4, C3 and C5), 136.8 (ddd, 1J ¼ 239.2,
2J ¼ 15.3, 3J ¼ 3.8, C2 and C6), 129.8 (dd, 2J ¼ 2J ¼ 10.0, C1), 126.1
(eCH]), 103.5 (dd, 2J ¼ 2J ¼ 13.2, C4).
NMR (THF-d8, T ¼ 207 K)
d
ꢀ224.1 (N1, 1J ¼ 107.1), ꢀ117.9 (N2).
Reaction of 2,3,4,5,6-pentafluorobenzaldehyde (40) with hydra-
zine monohydrate in toluene leads to 2-bis(perfluorobenzylidene)
hydrazine (42), obtained after purification by column chromatog-
raphy on silica gel with hexane/ethyl acetate 40:1 and crystallization
in chloroform as yellow needles (62%). Mp 124e130 ꢃC (lit. [22]
4.2.5. 3-Hydroxy-6,7-difluoro-1H-indazole (21)
In a three-neck round-bottom flask equipped with a reflux
condenser, methyl 2,3,4-trifluorobenzoate (50) (0.21 g, 1.1 mmol)
was dissolved in toluene (10 mL), then, a solution of 98% hydrazine
monohydrate (0.24 g, 4.7 mmol) in ethanol (10 mL) was added
dropwise. The mixture was heated at 80 ꢃC for 24 h. After cooling at
room temperature 5 mL of toluene were added, the solution was
decanted and the organic solvent evaporated under vacuum. The
solid residue was washed with pentane and 0.16 g of a crude solid
was obtained. After silica gel chromatography with (hexane/ethyl
acetate 3:1), 21 was obtained (0.11 g, 60%) as a white solid. Mp
237.3 ꢃC (DSC). Anal. Calcd for C7H4F2N2O: C, 49.42; H, 2.37; N,
131 ꢃC). 1H NMR (CDCl3)
d
8.76 (s, 1H, eCH]Ne); 13C NMR (CDCl3)
d
108.9 (m, C1), 137.9 (dm, C3 and C5), 142.9 (dm, C2 and C6), 146.0
(dm, C4), 152.3 (d, eCH]Ne); 19F NMR (CDCl3)
d
ꢀ161.5 (m, F3, F30,
F5 and F50), ꢀ148,9 (m, F4 and F40), ꢀ139,0 (m, F2, F20, F6 and F60). If
the reaction of 2,3,4,5,6-pentafluorobenzaldehyde (40) was carried
out with hydrazine monohydrate in ethanol/water at low tempera-
ture and high-dilution, perfluorobenzylidenehydrazine (41) was
obtained as awhite solid (89%). Mp 68e69 ꢃC (lit. [22] 68 ꢃC).1H NMR
16.47. Found: C, 49.43; H, 2.61; N, 16.01. 1H NMR (DMSO-d6)
d 12.1
3
4
(CDCl3)
d
6.00 (s, 2H, NH2), 7.70 (s, 1H, eCH]Ne); 13C NMR (CDCl3)
(br s, 1H, NH), 11.0 (br s, 1H, OH), 7,43 (ddd, JH5 ¼ 8.8, JF6 ¼ 4.4,
3 3 4
d
110.4 (m, C1), 128.7 (d, eCH]Ne), 137.8 (dm, C3 and C5), 140.5
5JF7 ¼ 0.7, 1H, H4), 6.99 (ddd, JF6 ¼ 11.0, JH4 ¼ 8.8, JF7 ¼6.6, 1H,
(dm, C2 and C6),144.5 (dm, C4); 19F NMR (CDCl3)
d
ꢀ163.2 (m, F3 and
H5); 19F NMR (DMSO-d6)
d
ꢀ145.2 (ddd, JF7 ¼ 20.3, JH5 ¼10.9,
3 4
3
3
F5), ꢀ155.7 (m, 1F, F4), ꢀ144.2 (m, F2 and F6).
4JH4 ¼ 3.1, F6), e158.7 (dd, JF6 ¼ 20.3, JH5 ¼ 6.5, F7); 13C NMR
(DMSO-d6)
d
155.9 (s, C3), 112.7 (d, 3J ¼ 4.3, C3a), 116.5 (dd, 2J ¼ 9.6,
4.2.2. 3,7-Dinitro-1H-indazole (18)
3J ¼ 4.5, C4),109.1 (d, 2J ¼ 21.0, C5),148.2 (dd, 1J ¼ 241.2, 3J ¼ 9.0, C6),
135.0 (dd, 1J ¼ 247.3, 3J ¼ 16.3, C7), 131.7 (dd, 2J ¼ 11.6, 3J ¼ 3.6, C7a);
This compound was prepared according to Ref. [25] and
obtained as a yellow solid. Mp 220e222 ꢃC (lit. [25] 220 ꢃC). 1H
15N NMR (THF-d8, T ¼ 207 K)
d
ꢀ234.4 (N1), ꢀ115.1 (N2).
NMR (CD3CN)
d
12.83 (br s,1H, NH), 7.65 (t,1H, 3J ¼ 8.0, H5), 8.48 (d,
1H, 3J ¼ 7.9, H6), 8.62 (d, 1H, 3J ¼ 8.1, H4); 13C NMR (CD3CN)
d
151.5
4.2.6. 3-Hydroxy-4,6-difluoro-7-nitro-1H-indazole (24)
(m, C3), 120.2 (d, 2J ¼ 9.5, C3a), 130.3 (dd, 1J ¼ 171.7, 3J ¼ 8.6, C4),
126.4 (d, 1J ¼ 168.7, C5), 126.8 (ddd, 1J ¼ 168.5, 2J ¼ 2.3, 3J ¼ 8.6, C6),
134.9 (m, C7), 135.4 (dd, 3J ¼ 3J ¼ 6.7, C7a); 15N NMR (CD3CN)
A round-bottomed flask containing 3-hydroxy-4,6-difluoro-
1H-indazole (20) (0.06 g, 0.35 mmol) and potassium nitrate
(0.04 g, 0.4 mmol) was placed in an iceewater bath. Then,
concentrated sulphuric acid 95e98% conc. (0.8 mL, d ¼ 1.84 g/mL)
was added dropwise, and the mixture kept at 0e4 ꢃC for 1 h. The
mixture was stirred at room temperature for 24 h and poured into
iceewater (5 mL). The solution was extracted with ethyl acetate
(3 ꢁ 20 mL), dried with anhydrous sodium sulfate and solvent was
removed under reduced pressure to afford a yellow solid. The
product was purified by silica gel chromatography with (hexane/
ethyl ether 4:1), to afford 24 (0.045 g, 60%) as a yellow solid. Mp
217.4 ꢃC (DSC). Anal. Calcd for C8H6F2N3O2: C, 39.08; H, 1.41; N,
d
ꢀ14.2 (NO2), the other nitrogen atoms were not detected.
4.2.3. General procedure for esterifications
A solution of 2,4,6-trifluorobenzoic acid (47) or 2,3,4-tri-
fluorobenzoic acid (48) (0.3 g, 1.7 mmol) in dry methanol (10 mL)
was prepared in a three-necked, round-bottom flask fitted with
a reflux condenser and a calcium chloride tube. The flask was
placed in an ice/water bath, thionyl chloride (1.4 mL) was then
added dropwise, and the mixture was heated at 60e65 ꢃC for three
days under argon. After removal of solvent, the resulting residue
was purified by extraction with pentane in the case of 49 and using
column chromatography on silica gel (hexane/diethyl ether 6:1)
for 50.
19.53; Found: C, 39.36; H, 1.61; N, 19.00. 1H NMR (DMSO-d6)
d 12.8
(br s, 1H, NH), 11.7 (br s, 1H, OH), 7.13 (dd, 3JF6 ¼ 12.4, 3JF4 ¼ 9.9, 1H,
4
3
H5); 19F NMR (DMSO-d6)
d
ꢀ101.2 (dd, JF6 ¼ 17.7, JH5 ¼ 9.8,
F4), ꢀ110.3 (br s, F6); 13C NMR (DMSO-d6)
d 154.6 (s, C3), 101.5 (d,
Methyl 2,4,6-trifluorobenzoate (49): Colorless oil, yield 85% (lit.
2J ¼ 20.1, C3a), 159.2 (dd, 1J ¼ 265.4, 3J ¼ 16.8, C4), 96.4 (dd,
2J ¼ 28.7, 2J ¼ 25.5, C5), 158.4 (dd, 1J ¼ 265.9, 3J ¼ 14.4, C6), 118.6
(dd, 2J ¼ 8.8, 4J ¼ 5.0, C7), 136.2 (dd, 3J ¼ 11.3, 3J ¼ 2.5, C7a); 15N
[41] Bp 105e110 ꢃC/15 mmHg). 1H NMR (CDCl3)
6.72 (dd, 3J ¼ 3J ¼ 8.3, 2H, H3 and H5).
d 3.94 (s, 3H, OCH3),
Methyl 2,3,4-trifluorobenzoate (50): Colorless oil, yield 81%. 1H
NMR (THF-d8, T ¼ 207 K)
d
ꢀ216.3 (1J ¼ 112.3, N1), ꢀ112.4 (N2), the
NMR (CDCl3) d 3.95 (s, 3H, OCH3), 7.03 (m, 1H, H6), 7.74 (m, 1H, H5).
NO2 signal was not detected.

R.M. Claramunt et al. / European Journal of Medicinal Chemistry 46 (2011) 1439e1447
1447
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This work has been partially supported by Grants CTQ2007-
62113, SAF2005-07991-C02-01 and CTQ2010-16122 (Ministerio de
Ciencia e Innovación, MICINN, Spain), PI08-1664 (Instituto de Salud
Carlos III, Spain), and P07-CTS-03135 (Consejería de Innovación,
Ciencia y Empresa, Junta de Andalucía, Spain). The help of Dr. Ma
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