Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors (III)

Article abstract of DOI:10.1016/j.ejmech.2008.01.014

We have previously described a series of 4,5-dihydro-1H-pyrazole as moderately potent nNOS inhibitors. As a follow up of these studies, we report here the preparation and the preliminary evaluation of a series of 1-alkyl-3-benzoyl-4,5-dihydro-1H-pyrazole

Full text of DOI:10.1016/j.ejmech.2008.01.014

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2579e2591
http://www.elsevier.com/locate/ejmech
Short communication
Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase
(nNOS and iNOS) potential inhibitors (III)*
a
M. Dora Carrion , Luisa C. Lopez Cara , M. Encarnacion Camacho ,
a
a
´
´
´
Vıctor Tapias , Germaine Escames , Darıo Acuna-Castroviejo ,
b
b
b
´
´
~
Antonio Espinosa a, Miguel A. Gallo a, Antonio Entrena a,
*
a Departamento de Qu´ımica Farmace´utica y Orga´nica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n, 18071 Granada, Spain
b Departamento de Fisiolo´gica, Instituto de Biotecnolo´gica, Universidad de Granada, Spain
Received 3 December 2007; received in revised form 30 December 2007; accepted 7 January 2008
Available online 26 January 2008
Abstract
We have previously described a series of 4,5-dihydro-1H-pyrazole as moderately potent nNOS inhibitors. As a follow up of these studies, we
report here the preparation and the preliminary evaluation of a series of 1-alkyl-3-benzoyl-4,5-dihydro-1H-pyrazole and 1-alkyl-3-benzoyl-1H-
pyrazole as potential inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS). None of the reported compounds
exhibited significant iNOS or nNOS inhibition, although the 1-benzyl-3-(2-amino-5-chlorobenzoyl)-1H-pyrazole-5-carboxylic acid ethyl ester
derivative (10l), which shows an inhibition of 50% versus iNOS at a 1 mM final concentration and no activity against nNOS, is potentially
amenable of further optimization. The reasons for the inactivity of the reported series are discussed on the basis of docking studies.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Neural nitric oxide synthase; Inducible nitric oxide synthase; Inhibition; Benzoyl-pyrazole; Benzoyl-pyrazoline
1. Introduction
tone [4e6]. Finally, iNOS expression is induced in macro-
phages and other cell types by numerous inflammatory stimuli,
including endotoxin (LPS) and cytokines (e.g. IL-1), and par-
ticipates in host defense, and possibly in chronic inflammatory
processes [7].
The NO action can be physiological or pathological. An ex-
cessive NO production by nNOS is involved in different neu-
rological disorders such as Alzheimer’s disease [8], the
amyotrophic lateral sclerosis [9] and Huntington’s disease
[10]. The induction of iNOS produces a continuous and ele-
vated NO production, and is involved in a variety of diseases,
including septic shock [11], inflammatory arthritis [12], and
inflammatory bowel disease [13]. Development of NOS inhib-
itors constitutes a current strategy in the research of new com-
pounds showing interesting properties.
Nitric oxide (NO) is an important bioregulator and ubiqui-
tous messenger that mediates several normal and physiopath-
ological processes [1,2]. NO biosynthesis is mediated by
nitric oxide synthase (NOS), which catalyses the L-arginine
oxidation. Classically, three isoforms of NOS have been iden-
tified, two of them are constitutively expressed and are known
as neural NOS (nNOS) and endothelial cell NOS (eNOS),
while the third one is induced by several factors, and hence
is called inducible NOS (iNOS). nNOS is responsible of the
NO production in the central nervous system where it acts
as a neurotransmitter [3]. eNOS is found primarily in vascular
endothelium and regulates the blood pressure and the vascular
Recently, we have synthesized and evaluated a series of
NOS inhibitors with general structures 1e4 [14e17]. Com-
pounds 1 [14] and 2 [15] have a kynurenine and kynurenamine
structures, respectively, and show good nNOS inhibition.
*
For parts I and II, see Refs. [16,17].
* Corresponding author. Tel.: þ34 958 243849; fax: þ34 958 243845.
E-mail address: aentrena@ugr.es (A. Entrena).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.014

2580
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
Pyrazoline derivatives 3 [16] constitute a new class of potent
nNOS inhibitors, and 1-benzoyl-D2-pyrazoline derivatives 4
have shown a moderate selectivity in the inhibition of the
iNOS isoform [17].
derivatives 13e15 were synthesized from the corresponding
(2-nitro-5-substituted)phenyl vinyl ketones 7e9 by reaction
with ethyl diazoacetate, as previously described [17]. In this
reaction, a 1,3-dipolar cycloaddition takes place to yield the
O
O
CO2H
R2
O
O
R1
R1
N
H3CO
R2
N
O
N
H
R
N
H
NH2
NH2
NH2
1
2
3
R2
R2
N
N
CO2Et
CO2Et
R2
N
CO2Et
O
N
N
N
R1
R1
O
O
R1
O
NH2
NH2
NH2
4
5
6
R1 = H, Cl, OMe
R2 = Me, Et, Bu, Bn
R1 = H, Cl, OMe
R2 = Me, Et, Bu, Bn
In this paper the synthesis and biological evaluation as NOS
inhibitors of a series of benzoyl-pyrazoline 5 and benzoyl-pyr-
azole 6 derivatives are described. Both families of compounds
are characterized by the presence of an N1-alkyl group, and some
of them show moderate inhibition and good iNOS selectivity.
D1-pyrazoline intermediates 10e12 which suffer the appropri-
ated H-3 rearrangement to give the final product. The possibil-
ity of an H-5 rearrangement and the formation of the
regioisomeic D2-pyrazolines 16e18 has been previously dis-
cussed and rejected [18].
Compounds 13e15 were treated with the corresponding
(R2O)2SO2 or R2Br in order to obtain the N-alkyl derivatives
19ael. During the N-alkylation reaction of compounds 13e
15, an unexpected aromatization produces the pyrazole deriv-
atives 20bem. This aromatization can be explained by the
participation of the 20-NO2 group [19], which acts as an oxi-
dant, yielding smaller quantities of side products bearing
2. Results and discussion
2.1. Chemistry
Scheme 1 shows the general synthetic pathway followed in
the preparation of compounds 5 and 6. Benzoyl-D2-pyrazoline
H-3
CO2Et
H
CO2Et
R2
R2
CO2Et
CO2Et
rearrangement
HN
N
N
N
N
N
O
b)
R1
a)
N
N
H
O
+
R1
R1
R1
R1
O
O
O
NO2
13 R1 = H
NO2
NO2
NO2
20 b-m
14 R1 = OMe
15 R1 = Cl
NO2
19 a-l
7 R1 = H
8 R1 = OMe
9 R1 = Cl
H- 5
rearrangement
10-12
CO2Et
N
c)
c)
HN
R1
O
R2
R2
CO2Et
CO2Et
CO2Et
O
CO2Et
c)
CO2Et
NO2
N
N
HN
N
HN
N
HN
N
N
N
c)
d)
16-18
R1
R1
15
Cl
Cl
Cl
O
O
O
O
NH2
5 a-l
NH2
6 b-l
NH2
NO2
NH2
5 m
20 m
6 m
Scheme 1. General synthetic pathway followed in the preparation of compounds 5 and 6. (a): N2CHCO2Et; (b) (R2O)2SO2 or R2Br, K2CO3, THF or 1,4-dioxane;
(c) Fe/FeSO4, H2O; (d) c-C6H11Br, K2CO3, THF.

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2581
Table 2
nNOs and iNOS inhibition (%) observed in the presence of 1 mM concentra-
tion of compounds 5aem and 6bem
reduced nitrogen functions, which were not isolated in the
preparation of compounds 19 and 20.
Table 1 shows the reaction conditions used and the final
yield obtained for each compounds 19ael and 20bem. It
can be observed that both pyrazole and pyrazoline derivatives
are obtained with variable yields depending on both the alky-
lating agent and the reaction conditions.
The more reactive alkyl sulphates allow the use of milder
conditions in the N-alkylation reaction (THF, 70 ꢀC, 15e
22 h). The total amount of pyrazoline 19 depends on the reac-
tivity of the alkylating agent, and higher yields are obtained
with (MeO)2SO2 than with (EtO)2SO2 and (BuO)2SO2. No
significant influence of the substrate R1 substituent is observed
since compounds 13e15 give the corresponding pyrazoline
derivatives with similar yields.
The less reactive alkyl halides require the use of 1,4-diox-
ane as solvent and higher temperature (120 ꢀC, 3 h). In this
case, only the more reactive PhCH2Br gives place to small
yields of pyrazoline 19d, 19h, or 19l, while c-C6H11Br does
not react.
The aromatization seems to proceed better in an opposing
manner: the worse the alkylating agent is the higher the yield
of pyrazole derivative 20. In fact, no N-alkylation is observed
in the reaction of 15 with c-C6H11Br and only the nitropyra-
zole 20m (R2 ¼ H) is isolated. This behaviour is probably
due to a kinetic control in both processes: when alkylation is
slow, the aromatization can take place more easily.
Reduction (Fe/FeSO4) of nitro derivatives 19ael and 20be
m yields the corresponding final products 5ael and 6bem, re-
spectively. Finally, compound 5m was obtained by reduction
of compound 15.
Compounds R1
R2
% nNOS inhibitiona % iNOS inhibitiona
5a
5b
5c
5d
5e
5f
H
H
H
H
Me
ꢁ9.8 ꢂ 3.0
22.1 ꢂ 4.4
29.6 ꢂ 2.1
28.4 ꢂ 5.8
ꢁ7.8 ꢂ 4.0
1.7 ꢂ 2.7
12.7 ꢂ 5.2
ꢁ1.5 ꢂ 1.7
12.2 ꢂ 1.4
9.4 ꢂ 1.4
Et
Bu
PhCH2
OCH3 Me
OCH3 Et
0.6 ꢂ 4.0
17.1 ꢂ 0.7
18.6 ꢂ 1.2
10.5 ꢂ 2.6
22.9 ꢂ 0.9
14.7 ꢂ 3.2
10.5 ꢂ 3.6
17.3 ꢂ 0.6
19.1 ꢂ 0.7
12.5 ꢂ 2.1
5.4 ꢂ 0.9
5g
5h
5i
OCH3 Bu
OCH3 PhCH2
ꢁ10.0 ꢂ 2.3
9.3 ꢂ 1.2
Cl
Cl
Cl
Cl
Cl
H
Me
1.3 ꢂ 2.3
5j
Et
Bu
41.5 ꢂ 2.2
38.5 ꢂ 3.5
18.3 ꢂ 0.4
32.1 ꢂ 2.2
39.5 ꢂ 1.6
21.9 ꢂ 3.1
21.9 ꢂ 0.4
20.3 ꢂ 0.5
26.2 ꢂ 0.9
19.4 ꢂ 2.9
14.5 ꢂ 6.2
35.5 ꢂ 1.4
7.8 ꢂ 0.5
5k
5l
PhCH2
H
5m
6b
6c
6d
6f
Et
Bu
H
H
OCH3 Et
PhCH2
13.5 ꢂ 4.4
24.6 ꢂ 1.1
20.5 ꢂ 0.4
30.5 ꢂ 0.8
7.3 ꢂ 3.9
6g
6h
6i
OCH3 Bu
OCH3 PhCH2
Cl
Cl
Cl
Cl
Cl
Me
Et
6j
27.5 ꢂ 2.1
18.7 ꢂ 1.0
50.4 ꢂ 3.2
12.8 ꢂ 4.6
6k
6l
Bu
PhCH2
H
2.7 ꢂ 1.4
6m
a
40.2 ꢂ 2.2
Data represent the mean ꢂ SEM of the percentage of nNOS inhibition pro-
duced by 1 mM concentration of each compound. Each value is the mean of
three experiments performed by triplicate.
inhibition of both isoenzymes in the presence of 1 mM con-
centration of each final compound. In general, compounds 5
and 6 behave as weak inhibitors against both isoenzyme,
and for this reason additional biological assays are not recom-
mended. Nevertheless some conclusions can be drawn from
the experimental data.
2.2. nNOS and iNOS inhibition
Compound 5a is the simplest one in the pyrazoline series
and does not inhibit nNOS: even a small activation of the en-
zyme is observed. Changing the N-methyl group by a bigger
one, the nNOS inhibition percentage grows slightly, but not
enough to consider these compounds as good nNOS inhibitors.
Compounds 5eeh bear a 50-OMe group and in all of them the
nNOS inhibition decreases in relation to compounds 5aed, in-
dicating that the substitution in this position of the benzene
ring by a polar electron-donating group is detrimental for
the inhibition activity.
Compounds 5 and 6 have been tested for its inhibition of
both nNOS and iNOS isoforms. Table 2 illustrates the
Table 1
Reaction conditions and yields (in parenthesis) for each isolated product in the
N-alkylation reaction of compounds 13e15
Substrate
Reagenta
Compound
11 (yield)
Compound
12 (yield)
d
R1
R2
b
b
b
13
13
13
13
14
14
14
14
15
15
15
15
15
a
(MeO)2SO2
19a (84)
19b (46)
19c (8)
H
Me
b
(EtO)2SO2
20b (26)
20c (35)
20d (27)
H
Et
Bu
b
(BuO)2SO2
PhCH2Brc
(MeO)2SO2
H
Compounds 5iem bear a 50-chloro substituent and all of
them show better nNOS inhibition, indicating that an elec-
tron-withdrawing group in this position increases the nNOS
inhibition activity. The influence the N-substituent is not so
clear. Compounds 5m (R2 ¼ H), 5i (R2 ¼ Me), 5j (R2 ¼ Et)
and 5k (R2 ¼ Bu) show inhibition percentage of 32%, 1.3%,
41% and 38%, respectively. An additional increment in the
R2 volume decreases inhibition activity, and compound 5l
(R2 ¼ CH2Ph) inhibits the nNOS activity by 18%.
19d (26)
19e (84)
19f (38)
19g (33)
19h (11)
19i (82)
19j (37)
19k (29)
H
PhCH2
Me
d
OMe
OMe
OMe
OMe
Cl
b
(EtO)2SO2
20f (17)
20g (23)
20h (38)
20i (9)
Et
Bu
b
(BuO)2SO2
PhCH2Brc
(MeO)2SO2
PhCH2
Me
b
(EtO)2SO2
20j (27)
20k (28)
20l (11)
20m (55)d,e
Cl
Cl
Et
Bu
b
(BuO)2SO2
PhCH2Brc
c-C6H11Brb
19l (15)
d
Cl
Cl
PhCH2
H
Anhydrous K2CO3 was used as a base in all reactions.
THF, 70 ꢀC.
Pyrazole derivatives 6bem show higher nNOS inhibition in
relation to the pyrazoline analogs 5aem. The benzene ring is
not substituted in compounds 6bed and the N-ethyl group
gives the better inhibition percentage. Compounds 6feh bear
b
c
d
e
Dioxane, 120 ꢀC.
Not detected.
No alkylation observed.

2582
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
a 50-methoxy group and they are as potent as compounds 6be
d, indicating that this type of substitution does not signifi-
cantly affect the inhibition. Finally, in compounds 6iem, the
benzene ring bears a 50-chloro substituent and these com-
pounds show better inhibition percentages in this series, like
in pyrazoline derivatives 5iem. Similarly to compounds 5ie
m, the influence of the N-alkyl (R2) group is not so clear.
Compounds 6m (R2 ¼ H) and 6j (R2 ¼ Et) are the more potent
inhibitors (40% and 35%, respectively), and an additional in-
crease in the R2 volume decreases the inhibition.
From the previous data, it can be concluded that the 50-Cl
substituent is the best one for the nNOS inhibition in both fam-
ilies of compounds 5 and 6, and R2 ¼ Et or R2 ¼ Bu is the
more appropriated substituents in the N-1 position of both pyr-
azole and pyrazoline moieties.
Some of these compounds also inhibit iNOS (Table 2).
Among pyrazoline derivatives, the inhibition percentages are
low; in fact only compound 5i (R1 ¼ Cl, R2 ¼ Me) shows
a 23% of inhibition, and changing the N-Me group by any
other one diminishes the iNOS inhibition. On the other
hand, compounds belonging to the pyrazole family 6 show
higher inhibition percentages than the corresponding pyrazo-
line compounds 5. Among them, compounds 6d, 6h and 6l
show 13%, 30% and 50% inhibition percentages, respectively,
indicating that the N-CH2Ph group is the best one for the
activity. In both families of compounds, the 50-Cl substitution
is better for the inhibition than the 50-OMe group, and this is
better than a non-substituted benzene ring. 50-Cl substituent
is also preferred in the N-acyl benzoyl-pyrazolines 4 [17].
2.3. Docking studies
Several iNOS and nNOS crystal structures can be found in
the Brookhaven protein databank. A recent paper [20] de-
scribes the crystal structure of Nu-propyl-L-arginine (NPA),
a nNOS selective inhibitor, co-crystallized with the heme oxy-
genase domain of both nNOS (PDB id: 1QW6) and iNOS
(PDB id: 1QW4). NPA binds to nNOS (or iNOS) in the en-
zyme active site, similarly to the natural substrate L-arginine.
The stronger interaction between the inhibitor and the enzyme
is the reinforced hydrogen bond formed between the guanidi-
nium moiety of the inhibitor and Glu592 (Glu371 in iNOS)
carboxylate (Fig. 1). On the other hand, the NPA amine group
also forms an hydrogen bond with one of the Glu592 carbox-
ylate O atoms, while the NPA carboxylate interacts by means
of hydrogen bonds with both the Tyr562 (Tyr341 in iNOS) hy-
droxyl and Gln478 N32-H atoms.
In the same paper, authors justify the nNOS selectivity of
Nu-propyl-L-arginine as a consequence of the mutation of
Asn498 in nNOS by a Thr277 in iNOS. These residues are
A
B
Glu592
Glu371
Gln478
Gln257
Arg481
Tyr562
Arg260
Tyr341
Asn498
Thr277
Tyr482
Tyr261
Fig. 1. Detailed views of Nu-propyl-L-arginine inside the binding site of the heme oxygenase domain in both nNOS (A) and iNOS (B) complexes’ crystal struc-
tures. Dotted blue lines represent hydrogen bonds between the inhibitor and the enzymes. A reinforced hydrogen bond between the positively charged ligand gua-
nidinium moiety and Glu592 (nNOS) or Glu371 (iNOS) is the main interaction stabilizing the complex. Additional hydrogen bonds between the ligand amine
group and Glu592 (or Glu371), and the ligand carboxylate and Tyr562 (or Tyr341) also contribute to the complex stability. In iNOS Thr277 (Asn498 in
nNOS) modifies the Arg260 side-chain (blue arrow), which in turn alters the orientation of Gln257 side-chain (red arrow) and prevents the formation of the hy-
drogen bond between this residue and the ligand, being this the reason of the higher nNOS selectivity of this inhibitor (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.).

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2583
situated in the substrate access channel of both enzymes, rel-
atively far away from the binding site (Fig. 1). In nNOS, the
Asn498 side-chain interacts with Arg481 side-chain, which
in turn stabilises the Gln478 side-chain, allowing the forma-
tion of the hydrogen bond between Gln478 N32-H and one
of the NPA carboxylate O atoms. Instead of that, in iNOS
the corresponding Thr277 side-chain forms a hydrogen bond
with Tyr261, allowing the rotation of Arg260 side-chain
(Fig. 1, blue arrow). This rotation modifies the Gln257 side-
chain orientation (Fig. 1, red arrow) preventing the formation
of the hydrogen bond with the NPA carboxylate. As a conse-
quence, the iNOS/NPA complex will be less stable and this
could be the reason for the observed selectivity of this inhib-
itor for nNOS.
Glide program [21] has been used for the docking studies.
Three dimensional structures of the heme oxygenase domain
of both nNOS and iNOS isoenzymes have been obtained
from 1QW6 and 1QW4 crystal structures, respectively. Prepa-
ration of enzyme, the heme group and ligands has been per-
formed following standard procedures included in the Glide
program suite.
In general, Glide’s Gscore values obtained for complexes of
compounds 9bem and 10bem in both nNOS and iNOS bind-
ing sites are small (about ꢁ3.50 kcal/mol) indicating that these
complexes are weak, and consequently both families of com-
pounds are weak inhibitors. No linear relationships have been
found between Gscore values and the inhibition percentage.
One pose has been obtained for compounds 6bem inside
nNOS binding site. Fig. 2 shows such a pose for compound
6g. It can be observed that the ligand CO2Et group is situated
almost parallel to the heme group, in a similar position to the
NPA guanidinium moiety, probably due to a favourable inter-
action between the Fe atom and the electron-rich p-system of
the ester group. The aminophenyl moiety of the ligand points
towards Glu592, and only one hydrogen bond is formed
between one of the Glu592 carboxylate O atom and the
20-NH2 group of the ligand. Finally, the ligand N-alkyl sub-
stituent is situated into a binding pocket near both heme
side-chains (red arc). This binding pocket is big enough to ac-
commodate even an N-CH2Ph substituent, and for this reason,
all compounds 6bem show the same preferred pose.
In compounds 5aem, the pyrazoline C-5 atom is chiral and
two enantiomers must be considered. Nevertheless, the bind-
ing zone below the Fe atom is relatively big and the CO2Et
substituent of both R and S enantiomers can be accommodated
easily in this zone. For this reason, the preferred pose of com-
pounds 5aem inside nNOS is equivalent to that of their ana-
logs 6bem, being very small the differences in Gscore values
between both families of compounds and between R and S en-
antiomers of compounds 5aem. Compounds 5aem are
slightly worse nNOS inhibitors than compounds 6bem, and
the reason could be the existence of the two enantiomers
which form two complexes, one of them slightly less stable.
Docking experiments performed with compounds 5aem
and 6bem on iNOS indicate that both families behave in
a similar way. Gscore values are small in all compounds,
about of the same order than in nNOS experiments. Never-
theless, the main pose obtained for these compounds in
iNOS is slightly different to that described for nNOS.
Fig. 2 shows the pose obtained for compound 6g in the
iNOS binding site. It can be observed that the CO2Et group
of the ligand is situated near the Fe atom and almost parallel
to the heme group, similarly to the nNOS/6g complex. On
the other hand, in iNOS the Gln257 side-chain is rotated in
relation to its homologous residue in nNOS (Gln478) (vide
supra), and this side-chain prevents the ligand aminophenyl
moiety to be situated near to Glu371. Consequently, the li-
gand N-butyl substituent is accommodated between Gln257
and Glu371 (red arrow), while the aminophenyl moiety is sit-
uated near the heme side-chains.
A
B
C
Glu371
Glu592
Gln257
Gln478
nNOS/6g
iNOS/6g
iNOS/6l
Fig. 2. The preferred poses obtained for compound 6g in nNOS (A) and iNOS (B), and for compound 6l in iNOS (C). Rotation of iNOS Gln257 side-chain in
relation to the same nNOS residue (Gln478) changes the relative situation of 6g aminophenyl and N-butyl groups inside both enzymes. The bigger N-CH2Ph sub-
stituent of compound 6l cannot be accommodated between Gln257 and Glu371 (right) and yields a different pose that could explain the higher activity and se-
lectivity of this molecule (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

2584
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
This pose is common to all compounds except 6l and 6h,
which show a different pose. Fig. 2 also shows the preferred
pose obtained for compound 6l. The N-CH2Ph substituent is
too big to be accommodated between Gln257 and Glu371,
and this part of the binding site is fulfilled by the CO2Et group
(blue arrow), while the N-CH2Ph occupies the zone below the
heme group. Finally, the aminophenyl moiety is situated near
the heme side-chain, and a hydrogen bond between the ligand
20-NH2 group and one of the heme carboxylates is formed.
This hydrogen bond can be formed in this complex due to
the big volume of the N-CH2Ph group, which shifts the mole-
cule far away from the heme group and allows the rotation of
the aminophenyl moiety in order to form the hydrogen bond.
In general, all complexes have been demonstrated to be
weak, and this can be due to two different reasons. On the
one hand, the reinforced hydrogen bond described for L-argi-
nine and Nu-propyl-L-arginine (which is also present in other
known and potent NOS inhibitors) is not formed by our com-
pounds, and this can be one reason for the small Gscore value
and consequently the lack of potency of these compounds.
On the other hand, Fig. 2 shows that in all complexes the
ligand phenyl ring and CO bond are not coplanar, and the ex-
pected conjugation among both moieties is broken. The addi-
tional energy necessary for the ligand to adopt such
conformations in the complexes is detrimental for the stability
of the complexes and hence this could be another reason for
the small activity of these molecules.
Glide’s Gscore values are the sum of several terms of en-
ergy, among them a term defining the internal torsional energy
of the ligand conformer. In our ligands, this term partially re-
flects the lack of conjugation between the phenyl ring and the
CO bond among other rotational components, and is always
positive giving place to the observed small negative Gscore
values.
Nevertheless, a real quantification of internal torsional en-
ergy inside the protein force field is difficult to assess and ad-
ditional calculations are needed. For this reason, ab initio
calculations have been performed using model compounds
in order to estimate the rotational barrier around the PheCO
bond as well as the possible influence of the lack of conjuga-
tion between both the CO bond and the phenyl ring on the sta-
bility of the complex.
Fig. 3. (A) The preferred calculated conformation (Gaussian 98) of both 3-(2-
aminobenzoyl)-2,3-dihydro-1H-pyrazole 21 and 3-(2-aminobenzoyl)-1H-pyra-
zole 22, considered as model compounds of compounds 5 and 6. (B) Variation
of the relative energy (kcal/mol) in compounds 21 and 22 during the rotation
around the PheCO bond.
the object of minimizing the electrostatic interactions between
both heteroatoms.
This conformation has been used as starting point, and a re-
laxed scan around the PheCO bond has been performed by
means of Gaussian program. Fig. 3 also shows the energy var-
iation during the rotation. It can be observed that in both mol-
ecules energy grows continuously when rotation takes places
from 20ꢀ to 100ꢀ, being the difference between them very
small. A higher energy increment in compounds 6 would be
expected since in these molecules conjugation is extended
over the benzene ring, the CO, the pyrazole moiety and the
CO2Et group. Nevertheless, ab initio calculation on model
compounds indicates a similar energy increment in both
molecules.
In our complexes, the ligand PheCO torsional angle value
has a value between 70ꢀ and 95ꢀ, and hence ligands need 4e
6 kcal/mol to adopt such conformation. Such energy comes
from the enzymeeligand interaction and consequently the
complexes are weaker.
In a first stage, a conformational search of both 3-(2-amino-
benzoyl)-4,5-dihydro-1H-pyrazole 21 and 3-(2-aminoben-
zoyl)-1H-pyrazole 22 (considered as model compounds of 5
and 6, respectively) using MacroModel [22] program has
been tackled, in order to identify the more stable conformer
of these molecules. Further, the preferred conformer has
been optimized by means of Gaussian 98 program [23] using
the HartreeeFock Hamiltonian at a 6-31G* level. Fig. 3 shows
such conformer for both compounds, and it can be observed
that the CO bond and the benzene ring lie almost in the
same plane, defining a dihedral angle of about 20ꢀ. An intra-
molecular hydrogen bond between the 20-NH2 and the CO
groups also stabilized such conformation. Finally, the CO
group and the pyrazole (or pyrazoline) C]N double bond
are situated in an antiperiplanar disposition, probably with
3. Conclusions
In general, compounds 5aem and 6bem behave as weak
inhibitors of both iNOS and nNOS. Docking studies indicate
that the putative complexes formed by these molecules inside
the nNOS and iNOS binding sites are not too stable, and this is

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2585
the reason for the small inhibition percentage observed. The
small stability of these complexes seems to be mainly due to
two factors: (i) the lack of the reinforced hydrogen bond
formed between the ligand and Glu592 (nNOS) or Glu371
(iNOS) and observed in nNOS/Nu-propyl-L-arginine complex;
and (ii) an unfavorable ligand conformation that weakens the
PheCO conjugation.
1.4 Hz, 1H), 7.50 (dd, J ¼ 7.5, 1.4 Hz, 1H), 4.25 (m, 3H), 3.46
(dd, J ¼ 17.3, 13.1 Hz, 1H), 3.32 (dd, J ¼ 17.3, 10.4 Hz, 1H),
3.10 (s, 3H), 1.32 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 186.28, 169.44, 148.26, 144.97, 135.44, 133.62, 130.42,
129.46, 123.92, 68.29, 62.01, 39.79, 34.69, 14.22 ppm; MS
(LSIMS) m/z 328.0909 (M þ Na)þ, calcd mass for
C14H15N3O5Na: 328.0909.
Compound 6l shows the better iNOS inhibition percentage
and the better iNOS/nNOS selectivity of all studied com-
pound. These two facts could be due to the different binding
mode of such compound observed in the iNOS complex. Fur-
ther modifications of this molecule could serve to obtain more
potent and selective iNOS inhibitors.
4.1.2. 1-Ethyl-3-(2-nitrobenzoyl)-4,5-dihydro-1H-pyrazole-
5-carboxylic acid ethyl ester, 19b
Yield 46%, 1H NMR (CDCl3): d ¼ 8.02 (dd, J ¼ 8.1,
1.1 Hz, 1H), 7.67 (dt, J ¼ 7.7, 7.5, 1.1 Hz, 1H), 7.56 (dt,
J ¼ 8.1, 7.7, 1.6 Hz, 1H), 7.50 (dd, J ¼ 7.5, 1.6 Hz, 1H),
4.37 (dd, J ¼ 13.0, 10.4 Hz, 1H), 4.23 (q, J ¼ 7.1 Hz, 2H),
3.37 (m, 4H), 1.29 (t, J ¼ 7.1 Hz, 3H), 1.12 ppm (t,
J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.08, 169.81,
148.23, 144.58, 135.39, 133.48, 130.56, 129.55, 123.75,
66.05, 61.94, 46.95, 34.44, 14.18, 12.46; MS (LSIMS) m/z
342.106724 (M þ Na)þ, calcd mass for C15H17N3O5Na:
342.106591.
4. Experimental section
Melting points were determined using an Electrothermal-
1
1A-6301 apparatus and are uncorrected. H NMR and 13C
NMR spectra were recorded on a Bruker AMX 300 spectrom-
eter operating at 75.479 MHz for 13C and 300.160 MHz for 1H
and on a Bruker ARX 400 spectrometer operating at
400.132 MHz for 1H and 100.623 MHz for 13C, in CDCl3
(at concentration of ca. 27 mg/mL in all cases). The center
of each peak of CDCl3 [7.26 ppm (1H) and 77.0 ppm (13C)]
was used as internal reference in a 5 mm 13C/1H dual probe
(Wilmad, No. 528-PP). The temperature of the sample was
maintained at 297 K. The peaks are reported in parts per mil-
lion (d). High-resolution mass spectroscopy (HRMS) was car-
ried out on a VG AutoSpec Q high-resolution mass
spectrometer (Fisons Instruments). Elemental analyses were
performed on a Perkin Elmer 240 C and agreed with theoret-
ical values within ꢂ0.4%. Flash chromatography was carried
out using silica gel 60, 230e240 mesh (Merck), and the sol-
vent mixture reported within parentheses was used as eluent.
4.1.3. 1-Butyl-3-(2-nitrobenzoyl)-4,5-dihydro-1H-pyrazole-
5-carboxylic acid ethyl ester, 19c
Yield 8%, 1H NMR (CDCl3): d ¼ 8.02 (dd, J ¼ 8.1, 1.2 Hz,
1H), 7.67 (dt, J ¼ 7.7, 7.5, 1.2 Hz, 1H), 7.56 (dt, J ¼ 8.1, 7.7,
1.6 Hz, 1H), 7.50 (dd, J ¼ 7.5, 1.6 Hz, 1H), 4.37 (dd, J ¼ 12.4,
10.2 Hz, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H), 3.33 (m, 4H), 1.50 (m,
2H), 1.28 (m, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H), 0.86 ppm (t,
J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.10, 169.86,
148.15, 143.99, 135.47, 133.56, 130.36, 129.57, 123.82,
66.39, 62.01, 51.92, 34.38, 29.52, 19.97, 14.23, 13.75 ppm;
MS (LSIMS) m/z 370.1355 (M þ Na)þ, calcd mass for
C17H21N3O5Na: 370.1378.
4.1.4. 1-Benzyl-3-(2-nitrobenzoyl)-4,5-
4.1. Preparation of 1-alkyl-3-(2-nitro-5-substituted-
benzoyl)-4,5-dihydro-1H-pyrazole-5-carboxylic acid
ethyl esters (19ael) and 1-alkyl-3-(2-nitro-5-
substituted-benzoyl)-1H-pyrazole-5-carboxylic acid
ethyl esters (20bem)
dihydro-1H-pyrazole-5-carboxylic acid ethyl ester, 19d
Yield 26%, 1H NMR (CDCl3): d ¼ 8.09 (dd, J ¼ 8.1,
1.1 Hz, 1H), 7.70 (dt, J ¼ 7.7, 7.5, 1.1 Hz, 1H), 7.59 (dt,
J ¼ 8.1, 7.7, 1.5 Hz, 1H), 7.50 (dd, J ¼ 7.5, 1.5 Hz, 1H),
7.30 (m, 3H), 7.13 (m, 2H), 4.68 (d, J ¼ 14.8 Hz, 1H), 4.46
(d, J ¼ 14.8 Hz, 1H), 4.16 (m, 3H), 3.37 (dd, J ¼ 17.4,
12.9 Hz, 1H), 3.29 (dd, J ¼ 17.4, 11.4 Hz, 1H), 1.26 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.51, 169.60,
147.82, 145.10, 135.57, 134.87, 133.69, 130.42, 129.41,
128.81, 128.77, 128.18, 123.96, 64.71, 61.93, 56.33, 34.40,
14.16 ppm; MS (LSIMS) m/z 404.1220 (M þ Na)þ, calcd
mass for C20H19N3O5Na: 404.1222.
To a solution of the corresponding 3-(2-nitro-5-substituted-
benzoyl)-4,5-dihydro-1H-pyrazole-5-carboxylic acid ethyl es-
ters 13e15 (1 mmol) in THF (4 mL) or 1,4-dioxane (7 mL),
2.58 mmol of K2CO3 and 2.12 mmol of the alkylating agent
were added. The mixture was refluxed and stirred, and after
cooling the K2CO3 precipitated was filtered off and the filtrate
was concentrated in vacuo. The residue was solved in water
(100 mL) and extracted with ethyl acetate (3 ꢃ 30 mL), and
the combined layers were dried (Na2SO4), filtered and concen-
trated to yield a residue that was purified by flash chromatog-
raphy (ethyl acetate/hexane 1:9).
4.1.5. 3-(5-Methoxy-2-nitrobenzoyl)-1-methyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19e
Yield 84%, 1H NMR (CDCl3): d ¼ 8.01 (d, J ¼ 9.1 Hz, 1H),
6.93 (dd, J ¼ 9.1, 2.8 Hz, 1H), 6.81 (d, J ¼ 2.8 Hz, 1H), 4.20
(q, J ¼ 7.1 Hz, 2H), 3.83 (s, 3H), 3.39 (dd, J ¼ 17.3,
13.0 Hz, 1H), 3.25 (dd, J ¼ 17.3, 10.7 Hz, 1H), 3.03 (s, 3H),
1.25 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 186.17, 169.32, 163.57, 144.76, 140.24, 137.98, 126.38,
114.97, 113.74, 68.08, 61.08, 56.05, 39.65, 34.50,
4.1.1. 1-Methyl-3-(2-nitrobenzoyl)-4,5-
dihydro-1H-pyrazole-5-carboxylic acid ethyl ester, 19a
Yield 84%, 1H NMR (CDCl3): d ¼ 8.05 (dd, J ¼ 8.1, 1.1 Hz,
1H), 7.69 (dt, J ¼ 7.8, 7.5, 1.1 Hz, 1H), 7.58 (dt, J ¼ 8.1, 7.8,

2586
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
14.04 ppm; MS (LSIMS) m/z 358.1013 (M þ Na)þ, calcd
(dd, J ¼ 13.3, 10.0 Hz, 1H), 4.25 (q, J ¼ 7.1 Hz, 2H), 3.38
(m, 4H), 1.32 (t, J ¼ 7.1 Hz, 3H), 1.13 ppm (J ¼ 7.2 Hz,
3H); 13C NMR (CDCl3): d ¼ 184.29, 169.60, 146.35, 143.96,
140.18, 136.96, 130.23, 129.63, 125.25, 66.06, 62.04, 46.89,
34.22, 14.18, 12.53 ppm; MS (LSIMS) m/z 376.0673
(M þ Na)þ, calcd mass for C15H16N3O5ClNa: 376.0676.
mass for C15H17N3O6Na: 358.1015.
4.1.6. 1-Ethyl-3-(5-methoxy-2-nitrobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19f
Yield 38%, 1H NMR (CDCl3): d ¼ 8.03 (d, J ¼ 9.1 Hz, 1H),
6.96 (dd, J ¼ 9.1, 2.8 Hz, 1H), 6.86 (d, J ¼ 2.8 Hz, 1H), 4.35
(dd, J ¼ 13.1, 10.5 Hz, 1H), 4.21 (q, J ¼ 7.1 Hz, 2H), 3.87
(s, 3H), 3.34 (m, 4H), 1.27 (t, J ¼ 7.1 Hz, 3H), 1.08 ppm (t,
J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.19, 169.87,
163.63, 144.59, 140.88, 138.09, 126.35, 115.22, 113.90,
65.95, 61.92, 56.16, 46.30, 34.47, 14.17, 12.43 ppm; MS
(LSIMS) m/z 350.1355 (M þ H)þ, calcd mass for
C16H20N3O6: 350.1352.
4.1.11.
1-Butyl-3-(5-chloro-2-nitrobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19k
Yield 29%, 1H NMR (CDCl3): d ¼ 7.98 (d, J ¼ 8.7 Hz, 1H),
7.50 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.45 (d, J ¼ 2.3 Hz, 1H), 4.46
(dd, J ¼ 13.3, 10.3 Hz, 1H), 4.23 (q, J ¼ 7.1 Hz, 2H), 3.33
(m, 4H), 1.50 (m, 2H), 1.29 (t, J ¼ 7.1 Hz, 3H), 1.26 (m,
2H), 0.86 ppm (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3):
d ¼ 184.20, 169.56, 146.28, 143.71, 140.14, 137.04, 130.19,
129.62, 125.27, 66.36, 62.03, 51.81, 34.13, 29.54, 19.92,
14.17, 13.66 ppm; MS (LSIMS) m/z 404.0990 (M þ Na)þ,
calcd mass for C17H20N3O5ClNa: 404.0989.
4.1.7. 1-Butyl-3-(5-methoxy-2-nitrobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19g
Yield 33%, 1H NMR (CDCl3): d ¼ 8.05 (d, J ¼ 9.1 Hz, 1H),
6.97 (dd, 1H, J ¼ 9.1, 2.8 Hz, 1H), 6.87 (d, 1H, J ¼ 2.8 Hz,
1H), 4.36 (dd, J ¼ 13.2, 10.3 Hz, 1H), 4.22 (q, J ¼ 7.1 Hz,
2H), 3.88 (s, 3H), 3.32 (m, 4H), 1.48 (m, 2H), 1.29 (t,
J ¼ 7.1 Hz, 3H), 1.23 (m, 2H), 0.84 ppm (t, J ¼ 7.2 Hz, 3H);
13C NMR (CDCl3): d ¼ 186.14, 169.87, 163.61, 143.98,
140.85, 138.20, 126.36, 115.24, 113.83, 66.31, 61.93, 56.87,
51.87, 34.41, 29.49, 19.93, 14.19, 13.68 ppm; MS (LSIMS)
m/z 400.1488 (M þ Na)þ, calcd mass for C18H23N3O6Na:
400.1484.
4.1.12. 1-Benzyl-3-(5-chloro-2-nitrobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19l
Yield 12%, 1H NMR (CDCl3): d ¼ 8.04 (d, J ¼ 8.7 Hz, 1H),
7.54 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.46 (d, J ¼ 2.3 Hz, 1H), 7.31
(m, 3H), 7.13 (m, 2H), 4.70 (d, J ¼ 15.1 Hz, 1H), 4.48 (d,
J ¼ 15.1 Hz, 1H), 4.17 (m, 3H), 3.37 (dd, J ¼ 17.3, 12.5 Hz,
1H), 3.27 (dd, J ¼ 17.3, 11.5 Hz, 1H), 1.27 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 184.70, 169.41,
145.98, 144.56, 140.37, 137.09, 134.71, 130.32, 129.52,
128.83, 128.78, 128.27, 125.47, 64.77, 62.02, 56.28, 34.17,
14.15 ppm; MS (LSIMS) m/z 438.0835 (M þ Na)þ, calcd
mass for C20H18N3O5ClNa: 438.0834.
4.1.8. 1-Benzyl-3-(5-methoxy-2-nitrobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19h
Yield 15%, 1H NMR (CDCl3): d ¼ 8.12 (d, J ¼ 9.1 Hz, 1H),
7.30 (m, 3H), 7.12 (m, 2H), 7.00 (dd, J ¼ 9.1, 2.8 Hz, 1H), 6.88
(d, J ¼ 2.8 Hz, 1H), 4.69 (d, J ¼ 14.8 Hz, 2H), 4.45 (d,
J ¼ 14.8 Hz, 1H), 4.17 (m, 3H), 3.91 (s, 3H), 3.36 (dd,
J ¼ 17.3 Hz, 12.8 Hz, 1H), 3.29 (dd, J ¼ 17.3, 11.7 Hz, 1H),
1.26 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.59,
169.67, 163.79, 145.21, 140.52, 138.27, 134.86, 128.82,
128.75, 128.17, 126.56, 115.32, 113.75, 64.67, 61.92, 56.32,
56.20, 34.42, 14.16 ppm; MS (LSIMS) m/z 434.1322
(M þ Na)þ, calcd mass for C21H21N3O6Na: 434.1328.
4.1.13. 1-Ethyl-3-(2-nitrobenzoyl)-1H-pyrazole-5-
carboxylic acid ethyl ester, 20b
Yield 26%, mp 91e93 ꢀC; 1H NMR (CDCl3): d ¼ 8.12 (dd,
J ¼ 8.1, 1.1 Hz, 1H), 7.75 (dt, J ¼ 7.7, 7.5, 1.1 Hz, 1H), 7.65
(dt, J ¼ 8.1, 7.7, 1.6 Hz, 1H), 7.59 (dd, J ¼ 7.5, 1.6 Hz, 1H),
7.45 (s, 1H), 4.51 (q, J ¼ 7.2 Hz, 2H), 4.35 (q, J ¼ 7.1 Hz,
2H), 1.37 (t, J ¼ 7.1 Hz, 3H), 1.34 ppm (t, J ¼ 7.2 Hz, 3H);
13C NMR (CDCl3): d ¼ 186.74, 159.14, 148.38, 135.27,
133.84, 130.93, 129.61, 123.91, 112.73, 61.54, 48.10, 15.26,
14.22 ppm; MS (LSIMS) m/z 340.0909 (M þ Na)þ, calcd
mass for C15H15N3O5Na: 340.0909.
4.1.9. 3-(5-Chloro-2-nitrobenzoyl)-1-methyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19i
1
Yield 82%, mp 83e85 ꢀC; H NMR (CDCl3): d ¼ 8.01 (d,
J ¼ 8.7 Hz, 1H), 7.52 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.44 (d,
J ¼ 2.3 Hz, 1H), 4.27 (m, 3H), 3.44 (dd, J ¼ 17.4, 13.3 Hz,
1H), 3.30 (dd, J ¼ 17.4, 10.3 Hz, 1H), 3.12 (s, 3H),
1.31 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 184.47, 169.24, 146.33, 144.28, 140.31, 136.99, 130.27,
129.49, 125.41, 68.18, 62.07, 39.64, 34.44, 14.20 ppm; MS
(LSIMS) m/z 362.0515 (M þ Na)þ, calcd mass for
C14H14ClN3O5Na: 362.0519.
4.1.14. 1-Butyl-3-(2-nitrobenzoyl)-1H-pyrazole-5-
carboxylic acid ethyl ester, 20c
Yield 35%, 1H NMR (CDCl3): d ¼ 8.12 (dd, J ¼ 8.1,
1.2 Hz, 1H), 7.75 (dt, J ¼ 7.7, 7.4, 1.2 Hz, 1H), 7.65 (dt,
J ¼ 8.1, 7.7, 1.5 Hz, 1H), 7.58 (dd, J ¼ 7.4, 1.5 Hz, 1H),
7.46 (s, 1H), 4.46 (t, J ¼ 7.2 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz,
2H), 1.71 (m, 2H), 1.37 (t, J ¼ 7.1 Hz, 3H), 1.23 (m, 2H),
0.87 ppm (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3):
d ¼ 186.82, 159.19, 148.29, 148.17, 135.35, 134.14, 133.88,
130.90, 129.57, 123.94, 112.69, 61.51, 52.54, 32.20, 19.61,
14.22, 13.56 ppm; MS (LSIMS) m/z 346.1406 (M þ H)þ,
calcd mass for C17H20N3O5: 346.1402.
4.1.10.
3-(5-Chloro-2-nitrobenzoyl)-1-ethyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 19j
Yield 40%, 1H NMR (CDCl3): d ¼ 7.98 (d, J ¼ 8.7 Hz, 1H),
7.53 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.49 (d, J ¼ 2.2 Hz, 1H), 4.38

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2587
4.1.15. 1-Benzyl-3-(2-nitrobenzoyl)-1H-pyrazole-5-
MS (LSIMS) m/z 360.0361 (M þ Na)þ, calcd mass for
carboxylic acid ethyl ester, 20d
C14H12N3O5ClNa: 360.0363.
1
Yield 27%, mp 106e108 ꢀC; H NMR (CDCl3): d ¼ 8.15
(dd, J ¼ 8.1, 1.0 Hz, 1H), 7.76 (dt, J ¼ 7.7, 7.5, 1.0 Hz, 1H),
7.66 (dt, J ¼ 8.1, 7.7, 1.5 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.5 Hz,
1H), 7.51 (s, 1H), 7.26 (m, 3H), 7.12 (m, 2H), 5.66 (s, 2H),
4.30 (q, J ¼ 7.1 Hz, 2H), 1.32 ppm (t, J ¼ 7.1 Hz, 3H); 13C
NMR (CDCl3): d ¼ 186.84, 159.02, 148.76, 148.08, 135.90,
135.33, 134.29, 133.96, 130.96, 129.58, 128.64, 128.05,
127.59, 124.02, 113.05, 61.61, 55.95, 14.17 ppm; MS
(LSIMS) m/z 402.1064 (M þ Na)þ, calcd mass for
C20H17N3O5Na: 402.1065.
4.1.20. 3-(5-Chloro-2-nitrobenzoyl)-1-ethyl-1H-pyrazole-5-
carboxylic acid ethyl ester, 20j
1
Yield 27%, mp 100e102 ꢀC; H NMR (CDCl3): d ¼ 8.07 (d,
J ¼ 8.7 Hz, 1H), 7.59 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.53 (d,
J ¼ 2.3 Hz, 1H), 7.45 (s, 1H), 4.50 (q, J ¼ 7.2 Hz, 2H), 4.34 (q,
J ¼ 7.1 Hz, 2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.31 ppm (t, J ¼ 7.1 Hz,
3H); 13C NMR (CDCl3): d ¼ 185.17, 159.04, 147.97, 146.43,
140.64, 136.74, 134.05, 130.83, 129.64, 125.40, 112.75, 61.62,
48.19, 15.26, 14.22 ppm; MS (LSIMS) m/z 374.0522 (M þ Na)þ,
calcd mass for C15H14N3O5ClNa: 374.051968.
4.1.16. 1-Ethyl-3-(5-methoxy-2-nitrobenzoyl)-1H-pyrazole-
5-carboxylic acid ethyl ester, 20f
4.1.21. 1-Butyl-3-(5-chloro-2-nitrobenzoyl)-1H-pyrazole-5-
Yield 17%, mp 104e106 ꢀC; 1H NMR (CDCl3): d ¼ 8.13 (d,
J ¼ 9.1 Hz, 1H), 7.43 (s, 1H), 7.05 (dd, J ¼ 9.1, 2.8 Hz, 1H),
6.99 (d, J ¼ 2.8 Hz, 1H), 4.51 (q, J ¼ 7.2 Hz, 2H), 4.34 (q,
J ¼ 7.1 Hz, 2H), 3.91 (s, 3H), 1.31 ppm (t, J ¼ 7.2 Hz, 6H);
13C NMR (CDCl3): d ¼ 186.72, 163.92, 159.14, 148.34,
140.94, 137.95, 133.81, 126.50, 115.57, 114.07, 112.72,
61.52, 56.24, 48.11, 15.53, 14.22 ppm; MS (LSIMS) m/z
340.0909 (M þ H)þ, calcd mass for C16H18N3O6: 348.1195.
carboxylic acid ethyl ester, 20k
1
Yield 28%, mp 58e60 ꢀC; H NMR (CDCl3): d ¼ 8.08 (d,
J ¼ 8.7 Hz, 1H), 7.60 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.53 (d,
J ¼ 2.2 Hz, 1H), 7.46 (s, 1H), 4.46 (t, J ¼ 7.3 Hz, 2H), 4.35
(q, J ¼ 7.1 Hz, 2H), 1.69 (m, 2H), 1.37 (t, J ¼ 7.1 Hz, 3H),
1.23 (m, 2H), 0.87 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR
(CDCl3): d ¼ 185.22, 159.07, 147.88, 146.32, 140.64, 136.82,
134.32, 130.79, 129.61, 125.42, 112.69, 61.59, 52.59, 32.18,
19.59, 14.21, 13.53 ppm; MS (LSIMS) m/z 402.0827
(M þ Na)þ, calcd mass for C17H18N3O5ClNa: 402.0832.
4.1.17. 1-Butyl-3-(5-methoxy-2-nitrobenzoyl)-1H-pyrazole-
5-carboxylic acid ethyl ester, 20g
4.1.22. 1-Benzyl-3-(5-chloro-2-nitrobenzoyl)-1H-pyrazole-
Yield 23%, 1H NMR (CDCl3): d ¼ 8.15 (d, J ¼ 9.1 Hz, 1H),
7.45 (s, 1H), 7.06 (dd, J ¼ 9.1, 2.8 Hz, 1H), 6.97 (d, J ¼ 2.8 Hz,
1H), 4.47 (t, J ¼ 7.3 Hz, 2H), 4.35 (q, J ¼ 7.1 Hz, 2H), 3.92 (s,
3H), 1.72 (m, 2H), 1.37 (t, J ¼ 7.1 Hz, 3H), 1.23 (m, 2H),
0.88 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.79,
163.98, 159.14, 148.26, 140.90, 138.03, 134.07, 126.51,
115.60, 113.99, 112.69, 61.49, 56.23, 52.54, 32.26, 19.91,
14.26, 13.57 ppm; MS (LSIMS) m/z 398.1323 (M þ Na)þ,
calcd mass for C18H21N3O6Na: 398.1328.
5-carboxylic acid ethyl ester, 20l
1
Yield 11%, mp 135e136 ꢀC; H NMR (CDCl3): d ¼ 8.16
(d, J ¼ 8.7 Hz, 1H), 7.61 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.56 (d,
J ¼ 2.3 Hz, 1H), 7.51 (s, 1H), 7.26 (m, 3H), 7.12 (m, 2H),
5.66 (s, 2H), 4.31 (q, J ¼ 7.1 Hz, 1H), 1.32 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 185.30, 158.95,
148.33, 146.25, 140.80, 136.76, 135.76, 134.45, 130.92,
129.67, 128.70, 128.14, 127.61, 125.35, 113.10, 61.73,
56.01, 14.20 ppm; MS (LSIMS) m/z 436.0670 (M þ Na)þ,
calcd mass for C20H16N3O5ClNa: 436.0676.
4.1.18. 1-Benzyl-3-(5-methoxy-2-nitrobenzoyl)-1H-
pyrazole-5-carboxylic acid ethyl ester, 20h
4.1.23. 3-(5-Chloro-2-nitrobenzoyl)-1H-pyrazole-5-
carboxylic acid ethyl ester, 20m
Yield 38%, 1H NMR (CDCl3): d ¼ 8.16 (d, J ¼ 9.1 Hz, 1H),
7.49 (s, 1H), 7.26 (m, 3H), 7.12 (m, 2H), 7.06 (dd, J ¼ 9.1,
2.7 Hz, 1H), 6.98 (d, J ¼ 2.7 Hz, 1H), 5.66 (s, 2H), 4.29 (q,
J ¼ 7.1 Hz, 2H), 3.91 (s, 3H), 1.31 ppm (t, J ¼ 7.1 Hz, 3H);
13C NMR (CDCl3): d ¼ 186.78, 163.99, 159.01, 148.72,
140.82, 137.96, 135.94, 134.23, 128.64, 128.02, 127.55,
126.59, 115.64, 114.06, 113.02, 61.59, 56.25, 55.91,
14.16 ppm; MS (LSIMS) m/z 432.1174 (M þ Na)þ, calcd
mass for C21H19N3O6Na: 432.1171.
Yield 55%, mp 199e201 ꢀC; 1H NMR ((CD3)2CO):
d ¼ 13.71 (br s, 1H), 8.24 (d, J ¼ 8.8 Hz, 1H), 7.81 (dd,
J ¼ 8.8, 2.3 Hz, 1H), 7.75 (br s, 1H), 7.38 (s, 1H), 4.36 (q,
J ¼ 7.1 Hz, 2H), 1.33 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR
((CD3)2CO): d ¼ 185.22, 158.37, 151.14, 146.39, 140.05,
137.05, 136.30, 131.17, 129.37, 126.02, 109.10, 61.49,
13.62 ppm; MS (LSIMS) m/z 324.0390 (M þ H)þ, calcd
mass for C13H11N3O5Cl: 324.0387.
4.1.19. 3-(5-Chloro-2-nitrobenzoyl)-1-methyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 20i
4.2. General procedure for the preparation of
compounds 5aem and 6bem
Yield 9%, mp 128e130 ꢀC; 1H NMR (CDCl3): d ¼ 8.11 (d,
J ¼ 8.7 Hz, 1H), 7.60 (dd, J ¼ 8.7, 2.3 Hz, 1H), 7.52 (d,
J ¼ 2.3 Hz, 1H), 7.48 (s, 1H), 4.36 (q, J ¼ 7.1 Hz, 2H), 4.10
(s, 3H), 1.38 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 185.21, 159.21, 147.94, 145.99, 140.79, 136.92, 134.85,
130.78, 129.47, 125.51, 112.57, 61.68, 40.58, 14.24 ppm;
To a suspension of the corresponding nitroarenes 15, 19ae
l, 20bem (0.524 mmol) in water, a mixture of Fe (5.24 mmol)
and FeSO4 (0.524 mmol) was added. The reaction mixture was
refluxing from 3 h, filtered through Celite and extracted with
dichloromethane (3 ꢃ 15 mL) and ethyl acetate (3 ꢃ 15 mL).

2588
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
The combined organic layers’ phase was washed with brine,
dried (Na2SO4), and evaporated. The residue was recrystal-
lized from dichloromethane/hexane and/or purified by flash
chromatography using the appropriate solvents in each case.
4.2.5. 3-(2-Amino-5-methoxybenzoyl)-1-methyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5e
1
Yield 90%, mp 86e88 ꢀC; H NMR (CDCl3): d ¼ 7.98 (d,
J ¼ 3.0 Hz, 1H), 6.92 (dd, J ¼ 8.9, 3.0 Hz, 1H), 6.59 (d,
J ¼ 8.9 Hz, 1H), 5.69 (br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.03
(t, J ¼ 12.4, 11.8 Hz, 1H), 4.07 (s, 3H), 3.50 (dd, J ¼ 17.3,
12.4 Hz, 1H), 3.29 (dd, J ¼ 17.3, 11.8 Hz, 1H), 3.17 (s, 3H),
1.30 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 187.28,
170.17, 150.17, 147.55, 145.26, 122.86, 118.14, 118.12,
116.19, 67.85, 61.74, 55.86, 40.80, 37.18, 14.23 ppm; MS
(LSIMS) m/z 328.1271 (M þ Na)þ, calcd mass for
C15H19N3O4Na: 328.1273. Anal. C15H19N3O4 (C, H, N).
4.2.1. 3-(2-Aminobenzoyl)-1-methyl-4,5-dihydro-1H-
pyrazole-5-carboxylic acid ethyl ester, 5a
Yield 90%, mp 80e82 ꢀC; 1H NMR (CDCl3): d ¼ 8.33 (dd,
J ¼ 8.5, 1.5 Hz, 1H), 7.24 (ddd, J ¼ 8.4, 7.2, 1.5 Hz, 1H), 6.65
(m, 1H), 6.64 (d, J ¼ 8.4 Hz, 1H), 5.96 (br s, 2H), 4.27 (q,
J ¼ 7.1 Hz, 2H), 4.04 (t, J ¼ 12.5, 11.7 Hz, 1H), 3.51 (dd,
J ¼ 17.3, 12.5 Hz, 1H), 3.31 (dd, J ¼ 17.3, 11.7 Hz, 1H), 3.19
(s, 3H), 1.32 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 188.10, 170.24, 150.52, 147.42, 133.93, 133.83, 118.23,
116.85, 115.93, 67.95, 61.77, 40.84, 37.08, 14.26 ppm; MS
(LSIMS) m/z 298.1170 (M þ Na)þ, calcd mass for
C14H17N3O3Na: 298.1167. Anal. C14H17N3O3 (C, H, N).
4.2.6. 3-(2-Amino-5-methoxybenzoyl)-1-ethyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5f
Yield 71%, 1H NMR (CDCl3): d ¼ 8.06 (d, J ¼ 3.0 Hz, 1H),
6.93 (dd, J ¼ 8.9, 3.0 Hz, 1H), 6.61 (d, J ¼ 8.9 Hz, 1H), 5.72
(br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.17 (t, J ¼ 12.2,
12.0 Hz, 1H), 3.75 (s, 3H), 3.40 (m, 4H), 1.31 (t, J ¼ 7.1 Hz,
3H), 1.30 ppm (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3):
d ¼ 187.17, 170.65, 150.15, 147.22, 145.34, 123.11, 118.22,
118.12, 115.81, 65.59, 61.74, 56.80, 46.73, 36.90, 14.24,
12.82 ppm; MS (LSIMS) m/z 320.1603 (M þ H)þ, calcd
mass for C16H22N3O4: 320.1610. Anal. C16H21N3O4 (C, H, N).
4.2.2. 3-(2-Aminobenzoyl)-1-ethyl-4,5-dihydro-1H-
pyrazole-5-carboxylic acid ethyl ester, 5b
Yield 84%, 1H NMR (CDCl3): d ¼ 8.35 (dd, J ¼ 8.2,
1.6 Hz, 1H), 7.23 (ddd, J ¼ 8.4, 7.2, 1.6 Hz, H-40), 6.64 (m,
2H), 5.95 (br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.18 (dd,
J ¼ 12.5, 11.5 Hz, 1H), 3.41 (m, 4H), 1.31 (t, J ¼ 7.1 Hz,
3H), 1.27 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3):
d ¼ 188.13, 170.78, 150.51, 146.93, 133.90, 133.84, 118.35,
116.87, 115.93, 65.39, 61.78, 47.72, 36.91, 14.28,
12.77 ppm; MS (LSIMS) m/z 312.1317 (M þ Na)þ, calcd
mass for C15H19N3O3Na: 312.1324. Anal. C15H19N3O3 (C,
H, N).
4.2.7. 3-(2-Amino-5-methoxybenzoyl)-1-butyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5g
1
Yield 91%, mp 64e66 ꢀC; H NMR (CDCl3): d ¼ 8.05 (d,
J ¼ 2.9 Hz, 1H), 6.92 (dd, J ¼ 8.9, 2.9 Hz, 1H), 6.60 (d,
J ¼ 8.9 Hz, 1H), 5.60 (br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.20
(t, J ¼ 12.3, 11.6 Hz, 1H), 3.74 (s, 3H), 3.38 (m, 4H), 1.70 (m,
2H), 1.40 (m, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H), 0.92 ppm (t,
J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3): d ¼ 187.14, 170.62,
150.17, 146.64, 145.22, 123.08, 118.26, 115.75, 65.96, 61.73,
55.76, 52.83, 36.79, 29.92, 20.19, 14.25, 13.87 ppm; MS
(LSIMS) m/z 370.1743 (M þ Na)þ, calcd mass for
C18H25N3O4Na: 370.1742. Anal. C18H25N3O4 (C, H, N).
4.2.3. 3-(2-Aminobenzoyl)-1-butyl-4,5-dihydro-1H-
pyrazole-5-carboxylic acid ethyl ester, 5c
Yield 53%, 1H NMR (CDCl3): d ¼ 8.34 (dd, J ¼ 8.4,
1.6 Hz, 1H), 7.22 (dt, J ¼ 8.4, 7.3, 1.6 Hz, 1H), 6.64 (m,
2H), 5.94 (br s, 2H), 4.21 (m, 3H), 3.37 (m, 4H), 1.66 (m,
2H), 1.40 (m, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H), 0.93 ppm (t,
J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3): d ¼ 188.06, 170.69,
150.44, 146.26, 133.85, 133.68, 118.48, 116.83, 115.88,
65.79, 61.70, 52.78, 36.80, 29.81, 20.19, 14.24, 13.86 ppm;
MS (LSIMS) m/z 318.1814 (M þ H)þ, calcd mass for
C17H24N3O3: 318.1817. Anal. C17H23N3O3 (C, H, N).
4.2.8. 3-(2-Amino-5-methoxybenzoyl)-1-benzyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5h
Yield 75%, 1H NMR (CDCl3): d ¼ 8.06 (d, J ¼ 3.0 Hz, 1H),
7.31 (m, 5H), 6.93 (dd, J ¼ 8.9, 3.0 Hz, 1H), 6.60 (d, J ¼
8.9 Hz, 1H), 4.78 (d, J ¼ 14.4 Hz, 1H), 4.50 (d, J ¼ 14.4 Hz,
1H), 4.19 (q, J ¼ 7.2 Hz, 2H), 4.08 (t, J ¼ 12.3, 11.5 Hz,
1H), 3.64 (s, 3H), 3.43 (dd, J ¼ 17.4, 12.3 Hz, 1H), 3.31 (dd,
J ¼ 17.4, 11.5 Hz, 1H), 1.28 ppm (t, J ¼ 7.2 Hz, 3H); 13C
NMR (CDCl3): d ¼ 187.15, 170.37, 150.23, 147.73, 145.37,
135.85, 129.11, 128.11, 127.95, 123.23, 118.43, 118.07,
115.87, 64.49, 61.74, 57.07, 55.79, 36.87, 14.21 ppm; MS
(LSIMS) m/z 404.1590 (M þ Na)þ, calcd mass for
C21H23N3O4Na: 404.1586. Anal. C21H23N3O4 (C, H, N).
4.2.4. 3-(2-Aminobenzoyl)-1-benzyl-4,5-dihydro-1H-
pyrazole-5-carboxylic acid ethyl ester, 5d
Yield 63%, 1H NMR (CDCl3): d ¼ 8.40 (dd, J ¼ 8.1,
1.5 Hz, 1H), 7.30 (m, 6H), 6.67 (m, 2H), 4.82 (d,
J ¼ 14.4 Hz, 1H), 4.57 (d, J ¼ 14.4 Hz, 1H), 4.17 (q, J ¼
7.1 Hz, 2H), 4.05 (t, J ¼ 12.3, 12.2 Hz, 1H), 3.38 (dd,
J ¼ 17.3, 12.3 Hz, 1H), 3.29 (dd, J ¼ 17.3, 12.2 Hz, 1H),
1.27 ppm (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3):
d ¼ 188.19, 170.42, 150.58, 147.25, 135.53, 133.93, 129.31,
128.68, 128.04, 118.29, 116.90, 115.90, 64.11, 61.71, 56.97,
36.87, 14.21 ppm; MS (LSIMS) m/z 374.1485 (M þ Na)þ,
calcd mass for C20H21N3O3Na: 374.1480. Anal. C20H21N3O3
(C, H, N).
4.2.9. 3-(2-Amino-5-chlorobenzoyl)-1-methyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5i
1
Yield 92%, mp 82e84 ꢀC; H NMR (CDCl3): d ¼ 8.35 (d,
J ¼ 2.4 Hz, 1H), 7.15 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.57 (d,

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2589
J ¼ 8.8 Hz, 1H), 5.97 (br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.08
(t, J ¼ 12.7, 11.4 Hz, 1H), 3.47 (dd, J ¼ 17.3, 12.7 Hz, 1H),
3.28 (dd, J ¼ 17.3, 11.4 Hz, 1H), 3.21 (s, 3H), 1.31 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.60, 169.99,
148.90, 146.50, 133.61, 132.72, 120.23, 118.64, 118.12,
67.63, 61.80, 40.55, 36.71, 14.19 ppm; MS (LSIMS) m/z
332.0777 (M þ Na)þ, calcd mass for C14H16ClN3O3Na:
332.0777. Anal. C14H16ClN3O3 (C, H, N).
318.0618 (M þ Na)þ, calcd mass for C13H14ClN3O3Na:
318.0621. Anal. C13H14Cl N3O3 (C, H, N).
4.2.14. 3-(2-Aminobenzoyl)-1-ethyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6b
Yield 70%, mp 60e62 ꢀC; 1H NMR (CDCl3): d ¼ 8.27 (dd,
J ¼ 1.6, 8.6 Hz, 1H), 7.35 (s, 1H), 7.28 (dd, J ¼ 7.6, 1.6 Hz,
1H), 6.68 (m, 2H), 6.17 (br s, 2H), 4.68 (q, J ¼ 7.2 Hz, 2H),
4.36 (q, J ¼ 7.1 Hz, 2H), 1.49 (t, J ¼ 7.2 Hz, 3H), 1.38 ppm
(t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 188.89, 159.46,
151.36, 150.09, 134.45, 134.38, 132.61, 118.10, 116.97,
115.86, 114.42, 61.35, 47.90, 15.79, 14.26 ppm; MS (LSIMS)
m/z 310.1165 (M þ Na)þ, calcd mass for C15H17N3O3Na:
310.1167. Anal. C15H17N3O3 (C, H, N).
4.2.10. 3-(2-Amino-5-chlorobenzoyl)-1-ethyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5j
Yield 50%, 1H NMR (CDCl3): d ¼ 8.41 (d, J ¼ 2.5 Hz, 1H),
7.16 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.58 (d, J ¼ 8.8 Hz, 1H), 5.96 (br
s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.22 (t, J ¼ 12.8, 11.2 Hz, 1H),
3.75 (m, 4H), 1.31 (t, J ¼ 7.1 Hz, 3H), 1.29 ppm (t, J ¼ 7.2 Hz,
3H); 13C NMR (CDCl3): d ¼ 186.69, 170.49, 148.91, 146.11,
133.53, 133.00, 120.36, 118.86, 118.13, 65.39, 61.82, 47.57,
36.55, 14.24, 12.79 ppm; MS (LSIMS) m/z 346.0932
(M þ Na)þ, calcd mass for C15H18ClN3O3Na: 346.0934.
Anal. C15H18ClN3O3 (C, H, N).
4.2.15. 3-(2-Aminobenzoyl)-1-butyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6c
Yield 57%, mp 63e64 ꢀC; 1H NMR (CDCl3): d ¼ 8.27 (dd,
J ¼ 8.6, 1.4 Hz, 1H), 7.35 (s, 1H), 7.29 (dd, J ¼ 7.3, 1.4 Hz,
1H), 6.68 (m, 2H), 6.18 (br s, 2H), 4.64 (t, J ¼ 7.3 Hz, 2H),
4.36 (q, J ¼ 7.1 Hz, 2H), 1.87 (m, 2H), 1.37 (m, 2H), 1.37
(t, J ¼ 7.1 Hz, 3H), 0.95 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR
(CDCl3): d ¼ 188.89, 159.51, 151.36, 149.99, 134.43,
132.89, 118.00, 116.97, 115.84, 114.36, 61.34, 52.40, 32.67,
19.83, 14.27, 13.69 ppm; MS (LSIMS) m/z 338.1479
(M þ Na)þ, calcd mass for C17H21N3O3Na: 338.1480. Anal.
C17H21N3O3 (C, H, N).
4.2.11. 3-(2-Amino-5-chlorobenzoyl)-1-butyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5k
1
Yield 54%, mp 90e92 ꢀC; H NMR (CDCl3): d ¼ 8.40 (d,
J ¼ 2.6 Hz, 1H), 7.16 (dd, J ¼ 8.8, 2.6 Hz, 1H), 6.58 (d,
J ¼ 8.8 Hz, 1H), 5.93 (br s, 2H), 4.25 (q, J ¼ 7.1 Hz, 2H),
4.23 (t, J ¼ 12.8, 11.0 Hz, 1H), 3.39 (m, 4H), 1.70 (m, 2H),
1.42 (m, 2H), 1.31 (t, J ¼ 7.1 Hz, 3H), 0.96 ppm (t,
J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3): d ¼ 186.63, 170.48,
148.82, 145.44, 133.42, 133.01, 120.37, 118.98, 118.11,
65.76, 61.81, 52.50, 36.42, 29.86, 20.19, 14.24, 13.85 ppm;
MS (LSIMS) m/z 374.1249 (M þ Na)þ, calcd mass for
C17H22ClN3O3Na: 374.12473. Anal. C17H22ClN3O3 (C, H, N).
4.2.16. 3-(2-Aminobenzoyl)-1-benzyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6d
Yield 45%, mp 116e118 ꢀC; 1H NMR (CDCl3): d ¼ 8.32 (dd,
J ¼ 8.1, 1.5 Hz, 1H), 7.40 (s, 1H), 7.29 (m, 6H), 6.69 (m, 2H),
5.84 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 1.33 ppm (t, J ¼ 7.1 Hz,
3H); 13C NMR (CDCl3): d ¼ 188.79, 159.40, 151.42, 150.53,
136.56, 134.57, 134.53, 128.69, 128.02, 127.82, 118.02,
117.00, 115.89, 114.74, 61.46, 55.72, 14.23 ppm; MS (LSIMS)
m/z 372.1315 (M þ Na)þ, calcd mass for C20H19N3O3Na:
372.1324. Anal. C20H19N3O3 (C, H, N).
4.2.12. 3-(2-Amino-5-chlorobenzoyl)-1-benzyl-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5l
Yield 68%, 1H NMR (CDCl3): d ¼ 8.39 (d, J ¼ 2.5 Hz, 1H),
7.35 (m, 5H), 7.19 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.59 (d,
J ¼ 8.8 Hz, 1H), 5.95 (br s, 2H), 4.85 (d, J ¼ 14.5 Hz, 1H), 4.53
(d, J ¼ 14.5 Hz, 1H), 4.21 (q, J ¼ 7.1 Hz, 2H), 4.08 (t,
J ¼ 12.5 Hz, 1H), 3.30 (d, J ¼ 12.5 Hz, 2H), 1.29 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 187.03, 170.15,
148.93, 146.81, 135.19, 133.70, 133.02, 129.47, 128.81,
128.14, 120.42, 118.74, 118.15, 64.11, 61.82, 56.87, 36.39,
14.21 ppm; MS (LSIMS) m/z 408.1087 (M þ Na)þ, calcd mass
for C20H20ClN3O3Na: 408.1090. Anal. C20H20ClN3O3 (C, H, N).
4.2.17. 3-(2-Amino-5-methoxybenzoyl)-1-ethyl-
1H-pyrazole-5-carboxylic acid ethyl ester, 6f
1
Yield 75%, mp 78e80 ꢀC; H NMR (CDCl3): d ¼ 7.97 (d,
J ¼ 2.9 Hz, 1H), 7.40 (s, 1H), 6.99 (dd, J ¼ 8.9, 2.9 Hz, 1H),
6.66 (d, J ¼ 8.9 Hz, 1H), 5.90 (br s, 2H), 4.69 (q,
J ¼ 7.2 Hz, 2H), 4.36 (q, J ¼ 7.1 Hz, 2H), 3.76 (s, 3H), 1.50
(t, J ¼ 7.2 Hz, 3H), 1.38 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(CDCl3): d ¼ 188.10, 159.48, 150.26, 150.07, 146.20,
132.77, 123.70, 118.33, 117.84, 116.52, 114.53, 61.39,
55.88, 48.87, 15.70, 14.24 ppm; MS (LSIMS) m/z 340.1271
(M þ Na)þ, calcd mass for C16H19N3O4Na: 340.1273. Anal.
C16H19N3O4 (C, H, N).
4.2.13. 3-(2-Amino-5-chlorobenzoyl)-4,5-dihydro-
1H-pyrazole-5-carboxylic acid ethyl ester, 5m
1
Yield 76%, mp 122e124 ꢀC; H NMR (CDCl3): d ¼ 8.41
(d, J ¼ 2.3 Hz, 1H), 7.17 (dd, J ¼ 8.8, 2.3 Hz, 1H), 6.90 (br
s, 1H), 6.58 (d, J ¼ 8.8 Hz, 1H), 6.05 (br s, 2H), 4.42 (dd,
J ¼ 12.4, 5.4 Hz, 1H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.44 (dd,
J ¼ 17.6, 5.4 Hz, 1H), 3.29 (dd, J ¼ 17.6, 12.4 Hz, 1H),
1.27 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 187.25, 171.86, 150.70, 149.30, 134.16, 132.90, 120.42,
118.21, 62.15, 60.52, 35.44, 14.19 ppm; MS (LSIMS) m/z
4.2.18. 3-(2-Amino-5-methoxybenzoyl)-1-butyl-
1H-pyrazole-5-carboxylic acid ethyl ester, 6g
1
Yield 82%, mp 82e84 ꢀC; H NMR (CDCl3): d ¼ 7.97 (d,
J ¼ 3.0 Hz, 1H), 7.40 (s, 1H), 6.99 (dd, J ¼ 8.9, 3.0 Hz, 1H),
6.66 (d, J ¼ 8.9 Hz, 1H), 5.91 (br s, 2H), 4.64 (t, J ¼ 7.3 Hz,

2590
M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2H), 4.36 (q, J ¼ 7.1 Hz, 2H), 3.76 (s, 3H), 1.88 (m, 2H), 1.38 (t,
J ¼ 7.1 Hz, 3H), 1.36 (m, 2H), 0.94 ppm (t, J ¼ 7.3 Hz, 3H); 13C
NMR (CDCl3): d ¼ 188.11, 159.52, 150.21, 150.09, 146.23,
133.04, 123.78, 118.33, 117.85, 116.47, 114.50, 61.36, 55.87,
52.39, 32.65, 19.87, 14.26, 13.71 ppm; MS (LSIMS) m/z
368.1587 (M þ Na)þ, calcd mass for C18H23N3O4Na:
368.1586. Anal. C18H23N3O4 (C, H, N).
4.2.23. 3-(2-Amino-5-chlorobenzoyl)-1-benzyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6l
1
Yield 54%, mp 135e136 ꢀC; H NMR (CDCl3): d ¼ 8.42
(d, J ¼ 2.4 Hz, 1H), 7.35 (m, 6H), 7.24 (dd, J ¼ 8.8, 2.4 Hz,
1H), 6.65 (d, J ¼ 8.8 Hz, 1H), 6.20 (br s, 2H), 5.85 (s, 2H),
4.34 (q, J ¼ 7.1 Hz, 2H), 1.35 ppm (t, J ¼ 7.1 Hz, 3H); 13C
NMR (CDCl3): d ¼ 187.63, 159.38, 150.07, 149.92, 136.30,
134.49, 133.75, 133.26, 128.82, 128.26, 128.23, 120.30,
118.84, 114.65, 61.56, 55.71, 14.25 ppm; MS (LSIMS) m/z
406.0933 (M þ Na)þ, calcd mass for C20H18ClN3O3Na:
406.0934. Anal. C20H18ClN3O3 (C, H, N).
4.2.19. 3-(2-Amino-5-methoxybenzoyl)-1-benzyl-
1H-pyrazole-5-carboxylic acid ethyl ester, 6h
1
Yield 75%, mp 116e118 ꢀC; H NMR (CDCl3): d ¼ 7.95
(d, J ¼ 3.0 Hz, 1H), 7.44 (1H, s), 7.28 (m, 5H), 6.98 (dd,
J ¼ 9.0, 3.0 Hz, 1H), 6.65 (d, J ¼ 9.0 Hz, 1H), 5.84 (s, 2H),
4.34 (q, J ¼ 7.1 Hz, 2H), 3.62 (s, 3H), 1.35 ppm (t,
J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3): d ¼ 187.93, 159.47,
150.85, 150.08, 146.34, 136.58, 133.28, 128.70, 128.05,
127.89, 124.13, 118.39, 117.60, 116.14, 114.75, 61.50,
55.76, 55.58, 14.22 ppm; MS (LSIMS) m/z 402.1423
(M þ Na)þ, calcd mass for C21H21N3O4Na: 402.1429. Anal.
C21H21N3O4 (C, H, N).
4.2.24. 3-(2-Amino-5-chlorobenzoyl)-1H-pyrazole-5-
carboxilic acid ethyl ester, 6m
1
Yield 78%, mp 136e138 ꢀC; H NMR (CDCl3): d ¼ 11.62
(br s, 1H), 8.11 (s, 1H), 7.33 (s, 1H), 7.26 (dd, J ¼ 8.9, 2.5 Hz,
1H), 6.67 (d, J ¼ 8.9 Hz, 1H), 6.17 (br s, 2H), 4.44 (q,
J ¼ 7.1 Hz, 2H), 1.41 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR
(CDCl3): d ¼ 185.70, 160.46, 149.80, 135.03, 132.13,
120.67, 118.66, 117.90, 111.78, 61.81, 14.34 ppm; MS
(LSIMS) m/z 316.0469 (M þ Na)þ, calcd mass for
C13H12ClN3O3Na: 316.0464. Anal. C13H12ClN3O3 (C, H, N).
4.2.20. 3-(2-Amino-5-chlorobenzoyl)-1-methyl-
1H-pyrazole-5-carboxylic acid ethyl ester, 6i
1
Yield 78%, mp 112e114 ꢀC; H NMR (CDCl3): d ¼ 8.36
4.3. Striatal nNOS activity determination
(d, J ¼ 2.5 Hz, 1H), 7.37 (s, 1H), 7.22 (dd, J ¼ 8.9, 2.5 Hz,
1H), 6.64 (d, J ¼ 8.9 Hz, 1H), 6.20 (br s, 2H), 4.36 (q,
J ¼ 7.1 Hz, 2H), 4.28 (s, 3H), 1.38 ppm (t, J ¼ 7.1 Hz, 3H);
13C NMR (CDCl3): d ¼ 187.50, 159.52, 149.85, 149.63,
134.43, 133.71, 133.32, 120.29, 118.43, 118.36, 114.28,
61.49, 40.49, 14.27 ppm; MS (LSIMS) m/z 330.0623
(M þ Na)þ, calcd mass for C14H14ClN3O3Na: 330.0621.
Anal. C14H14ClN3O3 (C, H, N).
L-Arginine, L-citruline, N-(2-hydroxymethyl)piperazine-N0-
(2-ethanesulfonic acid) (HEPES), DL-dithiothreitol (DTT),
leupeptin, aprotinin, pepstatin, phenylmethyl-sulfonylfluoride
(PMSF), hypoxantine-9-b-D-ribofuranosid (inosine), ethylene
glycol-bis-(2-aminoethylether)-N,N,N0,N0-tetraacetic
acid
(EGTA), bovine serum albumin (BSA), Dowex-50W
(50 ꢃ 8-200), FAD, NADPH and 5,6,7,8-tetrahydro-L-bio-
pterin dihydrochloride (H4-biopterin) were obtained from
SigmaeAldrich Quımica (Spain). L-[3H]-arginine (58 Ci/
´
4.2.21. 3-(2-Amino-5-chlorobenzoyl)-1-ethyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6j
mmol) was obtained from Amersham (Amersham Biosci-
ences, Spain). Tris-(hydroxymethyl)-aminomethane (Tris-
HCl) and calcium chloride were obtained from Merck
(Spain).
1
Yield 80%, mp 111e113 ꢀC; H NMR (CDCl3): d ¼ 8.38
(d, J ¼ 2.4 Hz, 1H), 7.37 (s, 1H), 7.23 (dd, J ¼ 8.9, 2.4 Hz,
1H), 6.64 (d, J ¼ 8.9 Hz, 1H), 6.19 (br s, 2H), 4.69 (q,
J ¼ 7.2 Hz, 2H), 4.37 (q, J ¼ 7.1 Hz, 2H), 1.51 (t, J ¼ 7.2 Hz,
3H), 1.38 ppm (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3):
d ¼ 187.65, 159.36, 149.87, 149.69, 134.38, 133.52, 132.91,
120.27, 118.48, 118.33, 114.46, 61.44, 47.99, 15.71,
14.26 ppm; MS (LSIMS) m/z 321.0876 (M)þ, calcd mass for
C15H16ClN3O3: 321.0880. Anal. C15H16ClN3O3 (C, H, N).
Rats male Wistar (200e250 g) were maintained in the Uni-
versity’s facility in a 12 h/12 h light/dark cycle at 22 ꢂ 2 ꢀC
and with free access to food and tap water. All experiments
were performed according to the Spanish Government Guide
and the European Community Guide for animal care.
Rats were killed by cervical dislocation, and the striata
were quickly collected and immediately used to measure
NOS activity. Upon removal, the tissues were cooled in ice-
cold homogenizing buffer (25 mM Tris-HCl, 0.5 mM DTT,
10 mg/mL leupeptin, 10 mg/mL pepstatine, 10 mg/mL aproti-
nine, 1 mM PMSF, pH 7.6). Two striata were placed in
1.25 mL of the same buffer and homogenized in a Polytron
(10 s ꢃ 6). The crude homogenate was centrifuged for 5 min
at 1000 g, and aliquots of the supernatant were either stored
at ꢁ20 ꢀC for total protein determination or used immediately
to measure NOS activity. The nNOS activity was measured by
the Bredt and Snyder [24] method, monitoring the conversion
of L-[3H]-arginine to L-[3H]-citruline. The final incubation vol-
ume was 100 mL and consisted of 10 mL crude homogenate
4.2.22. 3-(2-Amino-5-chlorobenzoyl)-1-butyl-1H-pyrazole-
5-carboxylic acid ethyl ester, 6k
1
Yield 77%, mp 87e88 ꢀC; H NMR (CDCl3): d ¼ 8.38 (d,
J ¼ 2.5 Hz, 1H), 7.38 (s, 1H), 7.23 (dd, J ¼ 8.9, 2.5 Hz, 1H),
6.64 (d, J ¼ 8.9 Hz, 1H), 6.19 (br s, 2H), 4.65 (t, J ¼ 7.3 Hz,
2H), 4.37 (q, J ¼ 7.1 Hz, 2H), 1.89 (m, 2H), 1.40 (m, 2H),
1.38 (t, J ¼ 7.1 Hz, 3H), 0.97 ppm (t, J ¼ 7.3 Hz, 3H); 13C
NMR (CDCl3): d ¼ 187.76, 159.43, 149.88, 149.55, 134.43,
133.61, 133.18, 120.28, 118.45, 118.35, 114.43, 61.47,
52.47, 32.60, 19.84, 14.30, 13.73 ppm; MS (LSIMS) m/z
372.1090 (M þ Na)þ, calcd mass for C17H20ClN3O3Na:
372.1090. Anal. C17H20ClN3O3 (C, H, N).

M.D. Carrio´n et al. / European Journal of Medicinal Chemistry 43 (2008) 2579e2591
2591
added to a buffer to give a final concentration of 25 mM Tris-
Acknowledgment
HCl, 1 mM DTT, 30 mM H4-biopterin, 10 mM FAD, 0.5 mM
inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 mM L-arginine,
and 50 nM L-[3H]-arginine, at pH 7.6. The reaction was started
by the addition of 10 mL of NADPH (0.75 mM final) and
10 mL of each pyrazole derivative in DMSO to give a final
concentration of 1 mM. The tubes were vortex and incubated
at 37 ꢀC for 30 min. Control incubations were performed by
the omission of NADPH. The reaction was halted by the addi-
tion of 400 mL of cold 0.1 M HEPES, 10 mM EGTA, and
0.175 mg/mL L-citruline, pH 5.5. The reaction mixture was
decanted into a 2 mL column packet with Dowex-50W ion-ex-
change resin (Naþ form) and eluted with 1.2 mL of water. L-
[3H]-citruline was quantified by liquid scintillation spectros-
copy. The retention of L-[3H]-arginine in this process was
greater than 98%. Specific enzyme activity was determined
by subtracting the control value, which usually amounted to
less than 1% of the radioactivity added. The nNOS activity
was expressed as picomoles of L-[3H]-citruline produced
This work was partially supported by grants from the Min-
´
isterio de Ciencia y Tecnologıa (SAF2005-07991-C02-01 and
SAF2005-07991-C02-02) and from the Junta de Andalucia
(P06-CTS-01941).
References
[1] S. Pfeiffer, B. Mayer, B. Hemmens, Angew. Chem., Int. Ed. 38 (1999)
1714e1731.
[2] J.F. Kerwin, J.R. Lancaster, P.L. Feldman, J. Med. Chem. 38 (1995)
4343e4362.
[3] D.S. Bredt, S.H. Snyder, Annu. Rev. Biochem. 63 (1994) 175e195.
[4] R.F. Furchgott, J.V. Zawadzki, Nature 288 (1980) 373e376.
[5] L.J. Ignarro, G.M. Buga, G.K.S. Wood, R.E. Byrns, G. Chadhuri, Proc.
Natl. Acad. Sci. U.S.A. 84 (1987) 9265e9269.
[6] R.M.J. Palmer, A.G. Ferrige, S. Moncada, Nature 327 (1987) 524e526.
[7] D.J. Stuehr, H.J. Cho, N.S. Know, M.F. Weise, C.F. Nathan, Proc. Natl.
Acad. Sci. U.S.A. 88 (1991) 7773e7777.
[8] D.T. Yew, H.W. Wong, W.P. Li, H.W. Lai, W.H. Yu, Neuroscience 89
(1999) 675e686.
(mg of protein)ꢁ1 minꢁ1
.
[9] N.K. Wong, M.J. Strong, Eur. J. Cell Biol. 77 (1998) 338e343.
[10] P.J. Norris, H.J. Waldvogel, R.L. Faull, D.R. Love, P.C. Emson, Neuro-
science 4 (1996) 1037e1047.
4.4. iNOS activity determination
[11] A. Petros, D. Bennett, P. Vallance, Lancet 338 (1991) 1157e1158.
[12] N. McCartney-Francis, J.B. Allen, D.E. Mizel, J.E. Albina, C.F. Nathan,
S.M. Wahl, J. Exp. Med. 178 (1993) 749e754.
iNOS induction was achieved by intravenous injection of
lipopolysaccharide (LPS) 20 mg/kg. Eight hours after the ad-
ministration, rats were killed by cervical dislocation, and lungs
were quickly collected homogenized in ice-cold homogeniz-
ing buffer (1 mg tissue/15 mL buffer, 25 mM Tris-HCl,
0.5 mM DTT, 10 mg/mL leupeptin, 10 mg/mL pepstatine,
10 mg/mL aprotinine, 1 mM PMSF, pH 7.6). The crude ho-
mogenate was incubated in the presence of EDTA 10 mM to
eliminate the nNOS activity that could exist. iNOs activity
was measured using the same procedure described above for
the determination of nNOS activity.
[13] S. Moncada, J. R. Soc. Med. 92 (1999) 164e169.
´
[14] M.E. Camacho, J. Leon, M.D. Carrion, A. Entrena, G. Escames,
´
~
H. Khaldy, D. Acuna-Castroviejo, M.A. Gallo, A. Espinosa, J. Med.
Chem. 45 (2002) 263e274.
´
[15] A. Entrena, M.E. Camacho, M.D. Carrion, L.C. Lopez-Cara, G. Velasco,
´
´
J. Leon, G. Escames, D. Acuna-Castroviejo, V. Tapias, M.A. Gallo,
~
´
A. Vivo, A. Espinosa, J. Med. Chem. 48 (2005) 8174e8181.
´
[16] M.E. Camacho, J. Leon, A. Entrena, G. Velasco, M.D. Carrion,
G. Escames, A. Vivo, D. Acuna-Castroviejo, M.A. Gallo, A. Espinosa,
´
´
~
J. Med. Chem. 47 (2004) 5641e5650.
´
[17] M.D. Carrion, M.E. Camacho, J. Leon, G. Escames, V. Tapias, D. Acuna-
Castroviejo, M.A. Gallo, A. Espinosa, Tetrahedron 60 (2004) 4051e4069.
´
~
´
[18] M.D. Carrion, L.C. Lopez-Cara, M.E. Camacho, A. Entrena, M.A. Gallo,
´
A. Espinosa, Magn. Reson. Chem. 43 (2005) 1063e1065.
´
´
[19] L.C. Lopez-Cara, M.E. Camacho, M.D. Carrion, M.A. Gallo,
A. Espinosa, A. Entrena, Tetrahedron Lett. 47 (2006) 6239e6242.
[20] R. Fedorov, E. Hartmann, D.K. Ghosh, I. Schlichting, J. Biol. Chem. 278
(2003) 45818e45825.
4.5. Statistical analysis
Data are expressed as the mean ꢂ SEM. One-way analysis
of variance, followed by the NewmaneKeuls multiple range
test was used. A P < 0.05 value was considered statistically
significant.
[21] Glide, Version 4.5, Schrodinger, LLC, New York, NY, 2007.
¨
[22] MacroModel, Version 9.5, Schrodinger, LLC, New York, NY, 2007.
¨
[23] Gaussian 98, Revision A.7, Gaussian, Inc., Pittsburgh, PA, 1998.
[24] D.S. Bredt, S.H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 682e685.