Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group

Article abstract of DOI:10.1016/j.bmc.2013.05.016

In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide synthase (iNOS) of these compounds. In gener

Full text of DOI:10.1016/j.bmc.2013.05.016

Bioorganic & Medicinal Chemistry 21 (2013) 4132–4142
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole
derivatives as potential nNOS/iNOS selective inhibitors.
Part 2: Influence of diverse substituents in both the phenyl moiety
and the acyl group
M. Dora Carrión a, Mariem Chayah a, Antonio Entrena a, Ana López b, Miguel A. Gallo a,
Darío Acuña-Castroviejo b, M. Encarnación Camacho a,
⇑
a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Spain
b Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de La Salud, Universidad de Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric
oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide
synthase (iNOS) of these compounds. In general, we can confirm that these pyrazoles are nNOS selective
inhibitors. In addition, taking these compounds as a reference, we have designed and synthesized a series
of new derivatives by modification of the heterocycle in 1-position, and by introduction of electron-
donating or electron-withdrawing substituents in the aromatic ring. These derivatives have been evalu-
ated as nNOS and iNOS inhibitors in order to identify new compounds with improved activity and selec-
tivity. Compound 3r, with three methoxy electron-donating groups in the phenyl moiety, is the most
potent nNOS inhibitor, showing good selectivity nNOS/iNOS.
Received 12 March 2013
Revised 30 April 2013
Accepted 8 May 2013
Available online 17 May 2013
Keywords:
4,5-Dihydro-1H-pyrazoles
Inhibition
Neuronal nitric oxide synthase (nNOS)
Inducible nitric oxide synthase (iNOS)
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
macrophages and microglia in response to inflammatory stimuli
and it is not Ca2+ dependent;5 finally, a mitochondrial-localized
Nitric oxide (NO) is an inorganic free radical that has diverse
physiological roles, including regulation of blood pressure, neuro-
transmission and macrophage defense systems.1 NO is synthesized
NOS isoform situated in the internal membrane of mitochondria
(mtNOS) has been discovered,6 and the existence of constitutive
(c-mtNOS) and inducible (i-mtNOS) has been proved.7,8
from the enzyme catalysis of
L
-arginine to
L
-citrulline in several cell
While eNOS-generated NO plays a role in vascular regulation,
the potential therapeutic utility of NOS inhibitors is restricted to
the selective inhibition of the neuronal or inducible isoforms.9 In
particular, an overproduction of NO by nNOS has been associated
with strokes,10 migraine headaches,11 or neurodegenerative disor-
ders such as Parkinson12 and Alzheimer’s diseases.13 On the other
hand, an overproduction of NO by iNOS causes inflammatory bo-
wel disease, arthritis, and neuropathic pain.14,15 Also, inflamma-
tory reaction in Parkinson’s disease is associated with iNOS16 and
i-mtNOS induction.17 Thus nNOS and iNOS represent a therapeutic
target since inhibition of these enzymes can help in the treatment
of several disorders, and a selective inhibition of one of these iso-
forms would be desired.
Previously, we have described a series of nNOS inhibitors with a
kynurenine 1,18 kynurenamine 2,19 or 4,5-dihydro-1H-pyrazole
structure 3.20 Furthermore, we have published 3-benzoyl-1-acyl-
4,5-dihydro-1H-pyrazole 4,21 3-benzoyl-1-alkyl-1H-pyrazole 522
and 5-phenyl-1H-pyrrole-2-carboxamide derivatives 6.23 These
compounds show moderate inhibition values of nNOS and iNOS,
types by a family of nitric oxide synthase (NOS) isoenzymes, with
consumption of molecular oxygen, nicotinamide adenine dinucleo-
tide phosphate (NADPH) and other cofactors such as flavin adenine
dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiop-
terin (BH4) and iron protoporphyrin (heme). The NOS enzymes are
homodimeric proteins consisting of a C-terminal reductase domain
and a N-terminal catalitic oxigenase domain connected by a
calmodulin-binding linker. The C-terminal reductase domain
transfers electrons from NADPH through FAD and FMN, to the
N-terminal oxygenase domain, which binds L-arginine, BH4 and
heme.2 Several distinct isoforms of NOS have been discovered.
Two of them, neuronal NOS (nNOS) and endothelial NOS (eNOS)
are constitutively expressed in different tissues and are activated
by Ca2+, and they play a role in neurotransmission3 and blood-ves-
sel dilation,4 respectively; inducible NOS (iNOS) is expressed by
⇑
Corresponding author. Tel.: +34 958 243844; fax: +34 958 243845.
E-mail address: ecamacho@ugr.es (M.E. Camacho).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.016

M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4133
Figure 1. nNOS and iNOS inhibitors synthesized by our research group.
with hydrazine in ethanol produced the 4,5-dihydro-1H-pyrazoles
28–33 which, in situ, were transformed with the acyl chloride into
derivatives 34a–r. Finally, reduction of the nitro group with SnCl2
in ethanol yielded the final compounds 3a–r (see Scheme 1).28
2.2. Biological data
The biological activity of the new compounds 3a–r as inhibitors
of both iNOS and nNOS has been evaluated by means of in vitro as-
says using recombinant isoenzymes. We have made the assays
using a 1 mM concentration of each compound in order to identify
the more potent and selective derivatives.
Figure 3 shows the percentages of residual nNOS and iNOS
activity in the presence of each compound in relation to the con-
trol, and Table 1 shows the inhibition percentages versus both
isoenzymes.
Figure 2. Structure of new 4,5-dihydro-1H-pyrazole derivatives 3a–r.
although in some cases iNOS selectivity is observed. Finally, we
have reported a group of selective iNOS/nNOS inhibitors wearing
a 4,5-dihydro-1,3,4-thiadiazole ring 7 (see Fig. 1).24
In this paper we describe a new group of 3-phenyl-4,5-dihydro-
1H-pryrazole compounds, and we evaluate their in vitro inhibition
by both neuronal and inducible NOS. In these compounds, we have
increased the activation and deactivation of the aromatic ring
keeping the cyclopropyl and phenyl groups, as well as the size
and flexibility of the phenyl moiety, with the aim of finding new
and more potent and selective nNOS inhibitors. Thus, the benzene
ring has been substituted with one or several electron-withdraw-
ing (5-Cl and 4,5-Cl2) or electron-donating [5-OMe, 4,5-(OMe)2
and 2,3,4-(OMe)3] substituents in order to analyse the influence
of the aromatic ring charge distribution on the inhibitory activity.
In addition, the size of the R2 substituent has also been increased
(Ph, CH2Ph and CH2CH2Ph) to test the importance of the steric ef-
fects in the NOS inhibition and selectivity.
In general, compounds 3a–r behave as weak inhibitors against
iNOS, since only two compounds (3g and 3h) show an inhibition
of around 50% at the concentration of 1 mM. Nevertheless, some
conclusions can be inferred from the experimental data. From a
qualitative point of view, the influence of R1 and R2 over the iNOS
activity is variable. When R2 = c-C3H5, compounds with the best
inhibitory activity have electron-donating substituents in R1, and
the activity increases with the number of these substituents, 3o
(30.1%) being one of the best iNOS inhibitors with three methoxy
groups in the aromatic ring. On the other hand, when R2 = Ph,
CH2Ph or CH2CH2Ph, the most potent compounds wear two elec-
tron-withdrawing groups in the aromatic ring, and therefore 3g
(49.2%, R2 = CH2Ph) and 3h (50.2%, R2 = CH2CH2Ph) are the best
iNOS inhibitors.
Table 1 also shows the nNOS inhibition values in presence of
1 mM concentration of compounds 3a–r. These compounds behave
as better inhibitors against nNOS, since five molecules show an
inhibition percentage higher than 48%. In general, molecules with
electron-donating groups in R1 behave as good nNOS inhibitors,
with the exception of compounds 3i and 3o, which are inactive.
In this series, 3n (R1 = 4,5-(OMe)2, R2 = CH2CH2Ph) and 3q
(R1 = 2,3,4-(OMe)3, R2 = CH2Ph) show moderate inhibition values
(55.3% and 48.4%, respectively), 3p (R1 = 2,3,4-(OMe)3, R2 = Ph)
shows good percentage of inhibition (62.9%), and compound 3r
(R1 = 2,3,4-(OMe)3, R2 = CH2CH2Ph) is the best nNOS inhibitor
(82.5%). In the series with electron-withdrawing substituents, the
most potent molecule is 3e (R1 = 4,5-Cl2, R2 = c-C3H5) with a mod-
erate inhibition (50.5%). As shown, compounds with electron-
donating groups in R1 are better nNOS inhibitors than those with
electron-withdrawing ones, and a non-substituted benzene moiety
2. Results and discussion
2.1. Chemistry
3,4-Dichlorobenzaldehyde 8 was transformed into the 4,5-di-
chloro-2-nitrobenzaldehyde 12 by treatment with a mixture of
fuming nitric acid (100%) and concentrated H2SO4.25 2,3,4-Tri-
methoxy-6-nitrobenzaldehyde 15 was obtained by reaction of
2,3,4-trimethoxybenzaldehyde 9 with a mixture of HNO3 and
AcOH.26 These nitroderivatives and the commercially available 2-
nitrobenzaldehyde 10, 5-chloro-2-nitrobenzaldehyde 11, 5-meth-
oxy-2-nitrobenzaldehyde 13 and 4,5-dimethoxy-2-nitrobenzalde-
hyde 14 were transformed into the final compounds 3a–r.20
Therefore, these derivatives were treated with vinyl-magnesium
bromide to yield the corresponding allylic alcohols 16–2120,27
quantitatively, which were further oxidized (Jones reagent) to ob-
tain the enone derivatives 22–27. Reaction of the enones 22–27

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M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
Scheme 1. General synthetic pathway followed in the preparation of compounds 3a–r.
Figure 3. Percentages of the nNOS and iNOS activities in the presence of 1 mM of the 4,5-dihydro-1H-pyrazole derivatives assayed (compounds 3a–al) compared to control.
Each value is the mean of three experiments performed in triplicate in the presence of recombinant iNOS or nNOS.
is not beneficial for the nNOS inhibitory activity. Moreover, with
regard to the acyl group, the increase of size and flexibility im-
proves the nNOS inhibition in most cases, being the phenylpropa-
noyl groups one of the most interesting.
Regarding the nNOS/iNOS selectivity, it can be observed that the
most active compounds which have electron-donating groups in
R1, show better selectivity versus nNOS than compounds with
electron-withdrawing or neutral substituents, and 3p (R1 = 2,3,4-
(OMe)3, R2 = Ph) is the most selective one of all the tested com-
pounds. Furthermore, compound 3r, with three electron-donating
groups in the phenyl moiety is the most potent nNOS inhibitor,
showing good selectivity versus nNOS. On the other hand, com-
pound 3h (R1 = 4,5-Cl2, R2 = CH2CH2Ph) behaves as a selective
iNOS/nNOS inhibitor, with a moderate iNOS inhibition (50.2%).

M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4135
Table 1
the increase of size and flexibility of the R2 radical led us to the
preparation of compound 3r, which is more potent than 3ab, pre-
viously described.
In vitro nNOS and iNOS inhibition (%) observed in the presence of 1 mM concentration
of compounds 3a–r
Compound
R1
R2
% Inhib. iNOSa
% Inhib. nNOSa
As regards iNOS inhibition of all tested compounds, the best re-
sults have been obtained with the deactivation of the aromatic ring
(R1 = 4,5-Cl2) and the increase of size and flexibility of the phenyl
ring (R2 = CH2Ph and CH2CH2Ph) (derivatives 3g and 3h). However,
derivatives 3a–al are, in general, inactive versus iNOS and, as a re-
sult, they can be considered inhibitors with good nNOS selectivity.
Table 3 shows the IC50 values measured for the nNOS inhibition
of derivatives 3n, 3p and 3r observed in the initial in vitro assay,
which ranges between 0.40 and 0.88 mM, being 3r the most potent
inhibitor of all compounds of the series.
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
H
5-Cl
5-Cl
4,5-Cl2
4,5-Cl2
4,5-Cl2
4,5-Cl2
CH2Ph
CH2CH2Ph
CH2Ph
CH2CH2Ph
c-C3H5
Ph
CH2Ph
CH2CH2Ph
CH2Ph
CH2CH2Ph
c-C3H5
Ph
2.31 0.31
5.64 0.74
34.62 1.73
31.37 2.43
15.53 0.23
36.10 8.50
49.22 0.38
50.17 0.24
15.0 1.58
21.19 1.33
25.21 1.38
28.9 1.97
21.0 2.38
23.72 1.04
30.07 3.21
8.14 1.56
23.39 3.23
18.29 4.74
2.68 0.27
7.36 0.96
18.24 2.35
32.36 3.23
50.48 3.12
0.62 1.13
21.59 1.67
2.97 0.32
0.03 2.03
25.93 2.04
19.65 1.23
25.14 2.17
35.07 2.28
55.33 0.21
0.05 1.25
62.87 3.25
48.36 2.87
82.45 3.26
5-OMe
5-OMe
4,5-(OMe)2
4,5-(OMe)2
4,5-(OMe)2
4,5-(OMe)2
2,3,4-(OMe)3
2,3,4-(OMe)3
2,3,4-(OMe)3
2,3,4-(OMe)3
CH2Ph
2.3. Docking studies
CH2CH2Ph
c-C3H5
Ph
CH2Ph
CH2CH2Ph
nNOS and iNOS crystal structures have been obtained from the
Brookhaven protein databank (PDB ID: 1QW6 and 1QW4,29 respec-
tively). Schrödinger software30 has been used for the docking stud-
ies. The preparation of the enzyme and the ligands has been done
following standard procedures included in the Schrödinger suite.
The protein preparation is performed using the Protein Preparation
Wizard module,31 and 3D structures of compounds 3a–r were gen-
erated from fragment libraries, and optimized using the Macro-
model module. The LigPrep32 program suite was used for the
ligands preparation, and Glide program was utilized for docking
the ligands using the SP option.
Glide’s Gscore values obtained for compounds 3a–r complexes
in both nNOS and iNOS binding sites are small (between ꢀ3.00
and ꢀ5.50 kcal/mol) indicating that these complexes are weak,
and consequently the compounds are moderated inhibitors.
Three different poses have been obtained for compounds 3a–r
inside the nNOS binding site. Figure 433 illustrates, as an example,
the preferred pose observed for compounds 3r, 3p and 3d, respec-
tively. In compound 3r (pose a), the phenyl moiety is situated be-
a
Data represent the percentage of nNOS and iNOS inhibition produced by 1 mM
concentration of each compound. Each value is the mean of three experiments
performed by triplicate using recombinant iNOS and nNOS enzymes.
Table 2
In vitro nNOS and iNOS inhibition (%) observed in the presence of 1 mM concentration
of compounds 3s–al
Compound
R1
R2
% Inhib. iNOSa
% Inhib. nNOSb
3s
3t
3u
3v
3x
3y
3z
3aa
3ab
3ac
3ad
3ae
3af
3ag
3ah
3ai
3aj
3ak
3al
H
H
H
H
H
5-Cl
5-Cl
5-Cl
Me
Et
Pr
c-C3H5
Ph
Me
Et
Pr
c-C3H5
Ph
Me
Et
Pr
Bu
c-C3H5
c-C4H7
c-C5H9
c-C6H11
Ph
7.73 1.16
6.88 0.95
0.35 1.75
14.80 1.64
8.7 2.18
1.03 2.31
13.72 2.88
0.29 3.26
18.89 2.16
17.04 2.55
20.39 0.42
20.39 1.53
13.09 1.2
0.18 1.4
33.69 3.62
36.47 4.52
52.39 2.24
38.79 3.18
57.05 3.13
47.58 4.01
46.15 5.66
34.43 3.70
70.24 5.60
61.12 3.11
38.04 1.53
53.27 2.83
34.70 1.32
49.76 1.53
62.24 4.68
38.30 3.33
49.87 4.13
62.20 1.91
58.92 3.55
5-Cl
5-Cl
low the heme group establishing
a p–p interaction. The
trimethoxyphenyl ring adopts a position that allows the interac-
tion of 40-OMe and 50-OMe groups with Arg481 through two
hydrogen bonds. The 20-NH2 group forms a hydrogen bond with
one of the carboxylate moieties of the heme group. The coplanar
disposition of the pyrazoline and the amide carbonyl group rein-
forces the conjugation between both moieties contributing to the
stability of the conformation. Moreover, this conformation in-
creases the length of the molecule, which allows the methoxy
groups to establish van der Waals (vdW) interactions with Ala497.
Compounds that are not able to interact with Arg481 remain in
an intermediate position between the heme group and this amino-
acid. This is the case of 3p. In pose b, the ligand is orientated in a
similar manner as pose a showed for the compound 3r. The phenyl
ring is situated below the heme group but interact, by means of
one hydrogen bond, with Asn569. Another hydrogen bond is estab-
lished between 20-NH2 and one of the carboxylate moieties of the
heme group. The conformation of the ligand 3p seems to be also
stable since C = O and the pyrazoline are coplanar which favors
the conjugation between them.
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
19.90 2.11
1.91 2.1
1.85 1.2
13.25 1.32
12.18 0.55
a
Data represent the percentage of nNOS and iNOS inhibition produced by 1 mM
concentration of each compound. Each value is the mean of three experiments
performed by triplicate using recombinant iNOS and nNOS enzymes.
b
See Ref. 20.
In addition, Figure 3 also shows the data of the iNOS activity of
compounds 3s–al, and Table 2 includes the data of the iNOS inhi-
bition for these compounds (not published before), as well as their
nNOS values previously described,20 in order to compare them.
These compounds have weak values of iNOS inhibition and they
can be considered as selective inhibitors of nNOS versus iNOS.
In general, the best nNOS inhibition values of derivatives 3s–al
were obtained with the c-C3H5 and Ph groups in R2, 3ab (R1 = 5-Cl,
R2 = c-C3H5) being the most active compound of this group, show-
ing good selectivity versus nNOS.
When we carried out different modifications in both, the aro-
matic moiety and the acyl group, we observed that the deactiva-
tion of the aromatic ring in the dichloro derivatives in relation to
the monochloro ones produced a considerable loss of activity.
Moreover, although the greatest activation of the aromatic ring
in compounds with R2 = c-C3H5 produced a drastic loss of activity,
Table 3
IC50 values (mM) for the inhibition of nNOS activity by the pyrazoline derivatives 3n,
3p and 3r
Compound
R1
R2
IC50 (mM)
3n
3p
3r
4,5-(OCH3)2
3,2,4-(OCH3)3
3,2,4-(OCH3)3
CH2CH2Ph
Ph
CH2CH2Ph
0.88
0.53
0.40

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M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
Figure 4. Detailed view of the main poses obtained for compounds 3r (pose a), 3p (pose b) and 3d (pose c) in the nNOS binding site. Dotted lines indicate hydrogen bond
interactions between the ligand and the residues of the enzyme.
Finally, Figure 4 also shows the preferred pose for compound 3d
(pose c). In this pose, there are two hydrogen bonds established by
the 20-NH2 group. One of them implies Glu592 and the other one
implies one of the carboxylate moieties of the heme group. The
phenyl ring is also situated, as in poses a and b, below the heme
group.
arginine residues does not allow the accommodation of molecules
longer than compound 3f. These aminoacids represent a steric
impediment.
The third pose in iNOS for compound 3q is represented in Fig-
ure 5c. This pose is similar to the pose of compound 3r inside nNOS
binding side (Fig. 4a). However, the conformation of Arg260 in
iNOS reduces the available volume under the carboxylate heme
moieties forcing the ligand 3q to remain in an intermediate plane
without any interaction with the enzyme.
It is clear that the volume of R1 and the volume and length of R2
influence the orientation and the disposition of the ligands inside
the binding site. Additionally, the conformation of Arg260 in iNOS
and Arg481 in nNOS, and consequently, the conformation of other
residues in both binding sites, seem to be decisive on the interac-
tion with the protein as well as on the orientation of the ligands in-
side the binding cavity (Fig. 6).
Regarding iNOS, docking studies indicate the existence of three
different poses for compounds 3a–r. As an example, Figure 5 shows
the preferred poses for compounds 3h, 3f and 3q, respectively.
The rotation of Arg260 in iNOS in relation to Arg481 in nNOS
diminished the volume available bellow this residue, and forces
compound 3h to adopt a totally different orientation inside iNOS
in relation to nNOS. Pose a shows a hydrogen bond between 20-
NH2 and Ser256 whose conformation changes respect to the con-
formation of Ser477 in nNOS because of the Arg260 rotation men-
tioned above. The phenyl and the pyrazoline rings are in a coplanar
disposition which reinforces the conjugation between C@N double
bond and the phenyl ring, and allows the formation of an intramo-
lecular hydrogen bond between the 20-NH2 group and the pyrazo-
line N-2 atom. This situation contributes to the stability of this
conformation. Furthermore, 40- and 50-Cl atoms interact by means
of vdW interactions with Tyr485 and Asn115 favoring the stability
of the conformation.
In pose b, 3f is orientated towards the opposite side of the bind-
ing cavity. The –NH2 group forms a hydrogen bond with Glu371.
The phenyl Cl atoms interact with the guanidinium groups of the
Arg375, Arg382 and Arg260. However, the ligand conformation
seems to be not too stable since the conjugation between the phe-
nyl ring and the C@N bond is very weak due to the fact that the
pyrazoline and the phenyl rings are not coplanar. Compound 3h
cannot adopt this pose because the pocket formed by the three
2.4. Discussion
The substrate binding sites of the NOS isoforms oxygenase do-
mains present a high level of aminoacids conservation and struc-
tural similarity. However, some differences outside the active site
and a conformational flexibility inside allow the adoption of differ-
ent conformations and the establishment of different interactions
which explains the preference of the compound for one or another
isoenzyme.
The main difference between both nNOS and iNOS binding site
is the orientation of the side-chain of Arg481 (or Arg260 in iNOS)
which controls the orientation of Gln478 (or Gln257 in iNOS),
and which in turn depends on the mutation of Asn498 in nNOS
by Thr277 in iNOS. Therefore, the difference in the side-chain
Figure 5. Detailed view of the main poses obtained for compounds 3h (pose a), 3f (pose b) and 3q (pose c) in the iNOS binding site. Dotted lines indicate hydrogen bond
interactions between the ligand and the residues of the enzyme and the intramolecular hydrogen bond.

M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4137
Figure 6. View of the surface of nNOS and iNOS binding site and the orientation of the compounds inside.
orientation of Arg481 in nNOS with regard to Arg260 in iNOS could
explain the selectivity and potency observed in some 4,5-dihydro-
1H-pyrazole derivatives described in this paper.
have reported the results on iNOS and nNOS inhibition of com-
pounds 3a–r, by means of an activation and deactivation of the
aromatic ring, and an increase of the size and flexibility of the R2
group in this type of structures. In this report, the increase of size
of R2 substituent improves the iNOS, as well as the nNOS inhibitory
activity. Nevertheless, regarding the R1 substituent in the aromatic
ring, electron-withdrawing groups are good for iNOS inhibition,
whereas electron-donating substituents improve the values of
nNOS inhibition. This fact is confirmed by docking studies which
show the better orientation of 3h in iNOS and 3r in nNOS. In addi-
tion, structures 3s–al exhibit a nNOS/iNOS selectivity. Among all
compounds, 3g and 3h are the best iNOS inhibitors (about 50% of
inhibition), and derivative 3r is the most potent nNOS inhibitor
of all the tested compounds, with a good nNOS/iNOS selectivity.
Thus, such a compound as 3r can be an interesting starting point
for possible new alternatives in neurodegenerative diseases such
as Alzheimer or Parkinson.
In poses b and c (Fig. 4) obtained for these molecules inside the
nNOS binding site, ligands adopt a conformation that allows the
formation of two hydrogen bonds. The first one, usually, formed
by the –NH2 group with one of the carboxylate moieties of the
heme group. The second bond, in all –OMe derivatives, is formed
between –OMe group and the nearest residue. In the case of com-
pounds with R1 = H, Cl, this second H-bond cannot be formed or is
established by the –NH2 group such is the case in compound 3d
(pose c, Fig. 4). In pose a, two –OMe groups interact by mean of
two hydrogen bonds with Arg482, the main difference in the bind-
ing cavity between nNOS and iNOS. This fact, together with vdW
interactions with Ala497, could explain the highest selectivity
and potency observed in compound 3r. Besides, the coplanar dis-
position of R2 phenyl ring below the heme group enables a p–cat-
ion interaction with Fe3+ ion. Also, the conjugation between the
pyrazoline and C@O double bond as a consequence of the coplan-
arity provides more stability to the conformation of the ligands as
shown in pose a and b (Fig. 2).
4. Experimental section
4.1. Chemistry
In iNOS, the available volume for binding the ligand is smaller
compared with nNOS,29 as it is illustrated in Fig. 6. The rotation of
iNOS Arg260 side-chain in relation to the nNOS Arg481 side-chain
is involved in this fact. In pose a and b (Fig. 5), the ligands have an
opposite disposition in the binding site. In pose b, one hydrogen
4.1.1. Material and methods
Melting points were determined using an Electrothermal-1A-
6301 apparatus and are uncorrected. 1H and 13C NMR spectra were
bond is formed between the –NH2 group and Glu371.
p–cation
recorded on
a Bruker AMX 300 spectrometer operating at
interaction between the R2 phenyl ring and heme group Fe3+ is weak
due to the lack of coplanarity of both rings. The disubstituted phenyl
ring is aimed towards the pocket formed by Arg375, Arg382 and
Arg260. This pocket represents a steric impediment, for compounds
with bulky R2, to adopt pose b. Therefore, compounds like 3h is ori-
entated to the opposite site of the binding cavity (Fig. 6). In pose a
(Fig. 5), the ligand establishes a hydrogen bond with Ser256 across
the –NH2 group. Another intramolecular hydrogen bond, between –
NH2 and the pyrazoline N-2, stabilizes the conformation. This orien-
tation allows the Cl atoms to interact by means of vdW interaction
with Tyr485 and Asn115 which is mutated by Leu337 in nNOS. The
conformational stability and the particular orientation of this com-
pound could explain its higher iNOS selectivity and potency com-
pared with the rest of the compounds.
75.49 MHz for 13C and 300.20 for 1H and on a Bruker ARX 400 spec-
trometer operating at 400.17 MHz for 1H and 125.69 MHz for 13C,
in CDCl3 (at concentration of ca. 27 mg mLꢀ1 in all cases) The cen-
ter 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 ppm (d). High-resolution mass
spectroscopy (HRMS) was carried out on a VG AutoSpec Q high-
resolution mass spectrometer (Fision Instruments). Flash chroma-
tography was carried out using silica gel 60, 230–240 mesh (Merk),
and the solvent mixture reported within parentheses was used as
eluent.
4.1.2. Preparation of 1-(2-nitrophenyl-substituted)prop-2-en-1-
ol (18, 21) general method
On the other hand, iNOS poses a and c (Fig. 5) are the preferred
ones for most of the pyrazolines with methoxy groups. In both
cases, these compounds have weak interactions with the enzyme.
This fact could explain the lack of iNOS activity in these derivatives.
To a ꢀ70 °C solution of 4.75 mmol of the corresponding 2-nitro-
benzaldehyde (12, 15) in 20 mL of THF was added 6.65 mmol of
vinylmagnesium bromide. After being stirred for 3.5 h, the mixture
was quenched with 20 mL of 0.010 N HCl and diluted with 100 mL
of ethyl acetate and extracted. The separated organic phase was
washed with brine, dried, then concentrated in vacuo to obtain a
residue that was purified by column chromatography (EtOAc/hex-
ane 1:10).
3. Conclusions
In summary, as an extension of the 4,5-dihydro-1H-pyrazole
derivatives 3s–al previously synthesized, in the present study we

4138
M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4.1.2.1.
(18).
1-(4,5-Dichloro-2-nitrophenyl)prop-2-en-1-ol
isolation, the pyrazoline was dissolved in dry CH2Cl2, and triethyl-
amine (1.5 mmol) and the acyl chloride (1.4 mmol) were added at
room temperature under nitrogen atmosphere. The reaction mix-
ture was stirred for 3 h, filtered and washed with 10% HCl, 2 M
NaOH, H2O, and brine. The organic phase was dried and filtered.
Evaporation of the solvent rendered a residue that was purified
by column chromatography (EtOAc/hexane 1:2).
Mp: 40–41 °C. Yield: 65%; 1H NMR (400.17 MHz, CDCl3)
d: 8.07 (s, 1H, H-30), 7.91 (s, 1H, H-60), 6.05–5.97 (m, 1H, H-2), 5.84
(d, 1H, J1–2 = 5.5 Hz, H-1), 5.42 (dd, 1H, J3a–3b = 1.2 Hz, J3a–
2 = 17.2 Hz, H-3a), 5.28 (dd, 1H, J3b–3a = 1.2 Hz, J3b–2 = 11.8 Hz, H-
13
3b). C NMR (125.69 MHz, CDCl3) d: 141.35 (C-20), 140.37 (C-50),
139.83 (C-2), 135.18 (C-40), 134.15 (C-10), 133.11 (C-60), 129.11
(C-30), 119.81 (C-3), 71.90 (C-1). MS (ESI): [M+1]+ = 2475.9832.
4.1.4.1. 3-(2-Nitrophenyl)-1-phenylacetyl-4,5-dihydro-1H-pyra-
4.1.2.2.
(21).
1-(6-Nitro-2,3,4-trimethoxyphenyl)prop-2-en-1-ol
zole (34a).
Mp: 116–117 °C. Yield: 75%; 1H NMR
Mp: 90–93 °C. Yield: 100%; 1H NMR (400.17 MHz,
(400.17 MHz, CDCl3) d: 7.93–7.17 (3 m, 9H, –CH–arom), 4.09 (t,
2H, J5–4 = 10.2 Hz, H-5), 4.01 (s, 2H, CO-CH2), 3.20 (t, 2H, J4–
5 = 10.2 Hz, H-4). 13C NMR (75.49 MHz, CDCl3) d: 170.18 (CO),
152.81 (C-3), 148.59 (C-20), 132.58 (C-100), 130.73, 130.41, 130.23
(C-40, C-50, C-60), 129.62, 128.48, 126.80 (C-200–C-600), 124.54 (C-
10), 124.26 (C-30), 44.65 (C-5), 40.91 (CO-CH2), 33.63 (C-4). MS
(ESI): [M+H]+ = 310.1102.
CDCl3) d: 7.24 (s, 1H, H-50), 6–19–6.15 (m, 1H, H-2), 5.63 (d, 1H,
J1–2 = 4.1 Hz, H-1), 5.33–5.02 (m, 2H, H-3a, H-3b), 3.99, 3.97, 3.96
(3s, 9H, 20-OCH3, 30-OCH3, 40-OCH3).13C NMR (75.49 MHz, CDCl3)
d: 155.94 (C-20), 153.46 (C-40); 152.77 (C-30), 146.84 (C-60),
139.52 (C-2), 121.25 (C-10), 114.75 (C-3), 104.49 (C-50), 69.03 (C-
1), 63.03 (30-OCH3), 61.26 (20-OCH3), 56.93 (40-OCH3). MS (LSIMS):
[M+Na]+ = 292.0792.
4.1.4.2. 3-(2-Nitrophenyl)-1-phenylpropanoyl-4,5-dihydro-1H-
4.1.3. Preparation of 1-(2-nitrophenyl-substituted)prop-2-en-1-
one (24, 26, 27) general method
pyrazole (34b).
Mp: 120–121 °C. Yield: 70%; 1H NMR
(300.20 MHz, CDCl3) d: 7.74 -7.09 (3 m, 9H, –CH–arom), 3.99 (t,
2H, J4–5 = 10.2 Hz, H-5), 3.08 (t, 2H, J4–5 = 10.2 Hz, H-4), 2.91 (m,
4H, CO-CH2–CH2). 13C NMR (75.49 MHz, CDCl3): d 171.64 (CO),
152.72 (C-3), 148.84 (C-20), 141.46 (C-100), 132.63 (C-40), 130.66
(C-50), 130.25 (C-60), 128.78, 128.59, 126.35 (C-200 to C-600), 126.23
(C-10), 124.44 (C-30), 44.58 (C-5), 36.02 (CO–CH2–CH2), 33.57 (C-
4), 31.34 (CO–CH2–CH2). MS (ESI): [M+Na]+ = 346.1166.
Freshly prepared Jones reagent (1.1 mL, 2.67 M) was added
dropwise to 2 mmol of the corresponding alcohol in 4 mL of ace-
tone at room temperature. After 10 min, ice-water (5 mL) was
added followed by 1 mL of saturated NaHSO3. The resulting mix-
ture was extracted with ethyl acetate. The extracts were dried
(Na2SO4) filtered, and concentrated to afford a residue that was
purified by column chromatography (EtOAc/hexane 1:10).
4.1.4.3. 3-(5-Chloro-2-nitrophenyl)-1-phenylacetyl-4,5-dihydro-
4.1.3.1.
(24).
1-(4,5-Dichloro-2-nitrophenyl)prop-2-en-1-one
1H-pyrazole (34c).
Mp: 154–156 °C. Yield: 50%; 1H NMR
Mp: 68–70 °C. Yield: 78%; 1H NMR (400.17 MHz, CDCl3)
(400.17 MHz, CDCl3) d: 7.81 (d, 1H, J3 ꢀ4 = 7.8 Hz, H-30), 7.54–
7.22 (m, 7H, H-40, H-60, H-200 to H-600), 4.07 (t, 2H, J4–5 = 10.2 Hz,
H-5), 3.98 (s, 2H, CO–CH2) 3.15 (t, 2H, J4–5 = 10.2 Hz, H-4). 13C
NMR (75.49 MHz, CDCl3) d: 170.21 (CO), 151.77 (C-3), 145.56 (C-
20), 139.13 (C-50), 135.18 (C-100), 130.58 (C-40), 130.48 (C-60),
129.77, 128.70 (C-200, C-300, C-500, C-600), 128.25 (C-10), 127.05 (C-
400), 125.95 (C-30), 44.87 (C-5), 40.99 (CO–CH2), 33.52 (C-4). MS
(LSIMS): [M+H]+ = 344.0798.
0
0
d: 8.26 (s, 1H, H-30), 7.53 (s, 1H, H-60), 6.62 (m, 1H, H-2), 6.09 (dd,
1H, J3a–3b = 1.6 Hz, J3a–2 = 10.6 Hz, H-3a), 5.91 (dd, 1H, J3b–
3a = 1.6 Hz, J3b–2 = 17.6 Hz, H-3b,). 13C NMR (75.49 MHz, CDCl3) d:
190.87 (C-1), 142.50 (C-20), 139.74 (C-50), 136.17 (C-60), 135.46
(C-40), 134.80 (C-10), 132.17 (C-3), 130.61 (C-2), 126.59 (C-30). MS
(ESI): [M+H]+ = 245.9722.
4.1.3.2.
(26).
1-(4,5-Dimethoxy-2-nitrophenyl)prop-2-en-1-one
Mp: 138–140 °C. Yield: 80%; 1H NMR (400.17 MHz,
4.1.4.4.
dihydro-1H-pyrazole (34d).
3-(5-Chloro-2-nitrophenyl)-1-phenylpropanoyl-4,5-
Mp: 160–162 °C. Yield: 60%; 1H
CDCl3) d: 7.64 (s, 1H, H-30), 6.79 (s, 1H, H-60), 6.58 (m, 2H, H-2),
5.94 (d, 1H, J3a–2 = 10.6 Hz, H-3a), 5.82 (d, 1H, J3b–2 = 17.6 Hz, H-
3b,), 3.99 (50-OCH3), 3.96 (40-OCH3). 13C NMR (125.69 MHz, CDCl3)
d: 193.56 (CO), 154.09 (C-40), 150.05 (C-50), 139.91 (C-20), 137.00
(C-2), 130.31 (C-3), 130.03 (C-10), 110.25 (C-60), 107.11 (C-30),
56.96 (40-OCH3, 50-OCH3). MS (LSIMS): [M+Na]+ = 260.0540.
NMR (400.17 MHz, CDCl3) d: 7.75 (d, 1H, H-30, J3 ꢀ4 = 8.4 Hz),
7.47–7.08 (m, 7H, H-40, H-60, H-200 to H-600), 4.01 (t, 2H, J4–
5 = 10.2 Hz, H-5), 3.05 (t, 2H, J4–5 = 10.2 Hz, H-4), 2.84–2.60 (m,
4H, COCH2–CH2). 13C NMR (75.49 MHz, CDCl3) d: 171.75 (CO),
151.83 (C-3), 146.85 (C-20), 141.35 (C-100), 139.22 (C-50), 130.55
(C-40), 130.37 (C-60), 128.79, 128.55 (C-200, C-300, C-500, C-600),
128.99 (C-100), 126.36 (C-400), 125.97 (C-30), 44.76 (C-5), 35.96 (C-
4), 33.51 (CO–CH2–CH2), 31.51 (CO–CH2–CH2). MS (LSIMS):
[M+H]+ = 358.0957.
0
0
4.1.3.3.
(27).
1-(6-Nitro-2,3,4-trimethoxyphenyl)prop-2-en-1-one
Mp: 143–145 °C. Yield: 70%; 1H NMR (400.17 MHz,
CDCl3) d: 7.55 (s, 1H, H-50), 6.64 (dd, 1H, J2–3a = 10.6 Hz, J2–
3b = 17.6 Hz, H-2), 6.08–5.67 (m, 2H, H-3a, H-3b), 4.09–3.91 (3s,
9H, 20-OCH3, 30-OCH3, 40-OCH3). 13C NMR (125.69 MHz, CDCl3) d:
187.07 (CO), 155.88 (C-40), 153.82 (C-20), 150.91 (C-60), 148.17
(C-30), 137.83 (C-2), 130.39, (C-3), 124.48 (C-10), 104.05 (C-50),
63.05, 62.45 (20-OCH3, 30-OCH3), 56.78 (40-OCH3). MS (LSIMS):
[M+Na]+ = 290.0646.
4.1.4.5. 1-Cyclopropylcarbonyl-3-(4,5-dichloro-2-nitrophenyl)-
4,5-dihydro-1H-pyrazole (34e).
Mp: 164–166 °C. Yield:
1
60%; H NMR (400.17 MHz, CDCl3) d: 7.94 (s, 1H, H-30), 7.72 (s,
1H, H-60), 4.09 (t, 2H, J5–4 = 10.6 Hz, H-5), 3.13 (t, 2H, J4–
5 = 10.6 Hz, H-4), 2.49–2.44 (m, 1H, H-100), 1.13–0.86 (m, 4H, H-
200, H-300). 13C NMR (125.69 MHz, CDCl3) d: 173.01 (CO), 150.36
(C-3), 146.82 (C-20), 137.54 (C-50), 134.68 (C-40), 131.62 (C-60),
126.33 (C-30), 126.18 (C-10), 45.25 (C-5), 33.06 (C-4), 11.81 (C-100),
8.82 (C-200, C-300). MS (LSIMS): [M+Na]+ = 350.0077.
4.1.4. Preparation of 1-acyl-3-(2-nitrophenyl-substituted)-4,5-
dihydro-1H-pyrazoles (34a–n) and 1-acy-3-(6-nitro-2,3,4-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (34o–r) general
method
The corresponding substituted-phenyl vinyl ketone (1.4 mmol)
was added to a solution of hydrazine monohydrate in ethanol
(15 mL), and the mixture was refluxed during 3 h. After this period,
the mixture was cooled, extracted with diethyl ether (4 ꢁ 10 mL),
dried Na2SO4, filtered and concentrated to dryness. Without
4.1.4.6. 1-Benzoyl-3-(4,5-dichloro-2-nitrophenyl)-4,5-dihydro-
1H-pyrazole (34f).
Mp: 130–132 °C. Yield: 50%; 1H NMR
(400.17 MHz, CDCl3) d: 7.90 (s, 1H, H-30), 7.80 (d, 2H, H-200, H-600,
0000
0000
J
= J
= 7.2 Hz), 7.63 (s, 1H, H-60), 7.45–7.38 (m, 3H, H-300, H-
2—3
6—5
400, H-500), 4.29 (t, 2H, H-5, J5–4 = 10.1 Hz), 3.18 (t, 2H, H-4,

M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4139
J4–5 = 10.1 Hz). 13C NMR (75.49 MHz, CDCl3) d: 167.52 (CO), 151.22
(C-3), 137.38 (C-100), 134.70 (C-50), 133.41 (C-40), 131.43 (C-60),
131.36 (C-400), 129.74–127.88 (C-200-C-600), 126.25 (C-30), 125.91
(C-10), 45.81 (C-5), 32.54 (C-4). MS (ESI): [M+H]+ = 364.0256.
(s, 1H, H-30), 7.48–7.42 (m, 3H, H-300 to H-500), 7.29 (s, 1H, H-60),
4.33 (t, 2H, J5–4 = 9.9 Hz, H-5), 4.00, 3.99 (2s, 6H, 40-OCH3, 50-
OCH3), 3.21 (t, 2H, J4–5 = 9.9 Hz, H-4). 13C NMR (75.49 MHz, CDCl3)
d: 165.71 (CO), 151.28 (C-3), 148.24 (C-50), 144.08 (C-40), 139.72
(C-20), 132.49 (C-100), 129.43 (C-200, C-600), 128.24 (C-400), 126.16
(C-300, C-500), 120.25 (C-10), 110.23 (C-60), 106.29 (C-30), 55.02 (40-
OCH3, 50-OCH3), 44.18 (C-5), 32.29 (C-4). MS (LSIMS):
[M+Na]+ = 378.1064.
4.1.4.7.
dihydro-1H-pyrazole (34g).
3-(4,5-Dichloro-2-nitrophenyl)-1-phenylacetyl-4,5-
Mp: 128–130 °C. Yield: 40%; 1H
NMR (300.20 MHz, CDCl3) d: 7.93 (s, 1H, H-30), 7.63 (s, 1H, H-60),
7.42–7.26 (m, 5H, H-200 to H-600), 4.08 (t, 2H, J5–4 = 10.4 Hz, H-5),
3,97 (s, 2H, COCH2), 3.13 (t, 2H, J4–5 = 10.5 Hz, H-4). 13C NMR
(75.49 MHz, CDCl3) d: 170.23 (CO), 150.62 (C-3), 148.05 (C-20),
137.53 (C-100), 135.07 (C-50), 134.91 (C-40), 131.64 (C-60), 129.73,
128.71 (C-200, C-300, C-500, C-600), 127.08 (C-400), 126.39 (C-30), 125.8
(C-10), 44.93 (C-5), 40.99 (COCH2), 33.25 (C-4). MS (ESI):
[M+H]+ = 378.0412.
4.1.4.13. 3-(4,5-Dimethoxy-2-nitrophenyl)-1-phenylacetyl-4,5-
dihydro-1H-pyrazole (34m).
Mp: 60–62 °C. Yield: 52%; 1H
NMR (300.20 MHz, CDCl3) d: 7.58 (s, 1H, H-30), 7.37–7.35 (m, 2H,
H-200, H-600), 7.30–7.27 (m, 2H, H-300, H-500), 7.22 (m, 1H, H-400),
6.88 (s, 1H, H-60), 4.07 (t, 2H, J5–4 = 10.2 Hz, H-5), 4.01–3.93 (m,
8H, 40-OCH3, 50-OCH3, CO–CH2), 3.10 (t, 1H, 2H, J4–5 = 10.2 Hz, H-
4). 13C NMR (75.49 MHz, CDCl3) d: 165.17 (CO), 150.64 (C-3),
148.37 (C-50), 145.33 (C-40), 136.70 (C-20), 130.86 (C-100), 124.98
4.1.4.8. 3-(4,5-Dichloro-2-nitrophenyl)-1-phenylpropanoyl-4,5-
Mp: 126–128 °C. Yield: 41%; 1H
(C-3 , C5 ), 123.87 (C-2 , C-6 ); 122.17 (C-4 ), 117.21 (C-1 ),
107.39 (C-60), 103.32 (C-30), 52.10 (40-OCH3, 50-OCH3), 40.14 (C-
5), 36.42 (CO-CH2), 29.71 (C-4). MS (LSIMS): [M+H]+ = 370.1402.
00
00
00
00
00
0
´
dihydro-1H-pyrazole (34h).
NMR (300.20 MHz, CDCl3) d: 7.86 (s, 1H, H-30), 7.54 (s, 1H, H-60),
7.34–7.20 (m, 5H, H-200 to H-600), 4.00 (t, 2H, J5–4 = 10.2 Hz, H-5),
3.04 (t, 2H, J4–5 = 10.2, H-4), 3.02–2.91 (m, 4H, COCH2CH2). 13C
NMR (75.49 MHz, CDCl3) d: 171.72 (CO), 150.57 (C-3), 148.04 (C-
20), 141.29 (C-100), 137.60 (C-50), 134.85 (C-40), 131.63 (C-60),
128.77 (C-200-C-600), 126.38 (C-30),126.33 (C-10), 44.84 (C-5), 35.92
(COCH2CH2), 33.20 (C-4), 31.34 (COCH2CH2). MS (ESI):
[M+H]+ = 392.0569.
4.1.4.14. 3-(4,5-Dimethoxy-2-nitrophenyl)-1-phenylpropanoyl)-
4,5-dihydro-1H-pyrazole (34n).
Mp: 165–167 °C. Yield 30%;
1H NMR (400.17 MHz, CDCl3) d: 7.58 (s, 1H, H-30), 7.27–7.25 (m,
4H, H-200, H-300, H-500, H-600), 7.18 (m, 1H, H-400), 6.89 (s, 1H, H-60),
4.08 (t, 2H, J5–4 = 10.2 Hz, H-5), 3.99, 3.98 (2s, 40-OCH3, 50-OCH3),
3.09 (t, 2H, J4–5 = 10.2 Hz, H-4), 3.01–2.98 (m, 4H, CO–CH2–CH2).
13C NMR (125.69 MHz, CDCl3) d: 173. 76 (CO), 157. 64 (C-3),
155.59 (C-50), 152.42 (C-40), 144.01 (C-20), 143.77 (C-100), 131.15
(C-200, C-600), 130.98 (C-300, C-500), 128.62 (C-400), 124.57 (C-10),
114.41 (C-60), 110.48 (C-30), 59.30, 59.22 (40-OCH3, 50-OCH3),
47.16 (C-5), 38.37 (C-4), 36.98 (CO–CH2–CH2), 33.62 (CO–CH2–
CH2). MS (LSIMS): [M+H]+ = 384.1559.
4.1.4.9. 3-(5-Methoxy-2-nitrophenyl)-1-phenylacetyl-4,5-dihy-
dro-1H-pyrazole (34i).
Mp: 116–117 °C. Yield: 75%; 1H
NMR (400.17 MHz, CDCl3) d: 8.05 (d, 1H, H-30 J3 –4 = 9.0 Hz),
0
0
7.40–7.19 (m, 5H, H-200 to H-600), 7.02 (dd, 1H, J4 –3 = 9.0 Hz, J4 –
0
0
0
= 2.8 Hz, H-40), 6.95 (d, 1H, J6 –4 = 2.8 Hz, H-60), 4.09 (t, 2H, H-5,
60
0
0
J5–4 = 10.6 Hz), 4.00 (s, 2H, COCH2), 3.93 (s, 3H, OCH3), 3.13 (d,
2H, J4–5 = 10.6 Hz, H-4,). 13C NMR (125.69 MHz, CDCl3) d: 172.41
(CO), 165.76 (C-50), 157.33 (C-3), 141.40 (C-20), 137.91 (C-100),
132.68, 132.16, 131.06 (C-200 to C-600), 129.94 (C-10), 129.32 (C-30),
118.40 (C-60), 117.54 (C-40), 58.78 (OCH3), 47.24 (COCH2), 43.43
(C-5), 36.99 (C-4). MS (LSIMS): [M+H]+ = 340.1297.
4.1.4.15.
1-Cyclopropylcarbonyl-3-(6-nitro-2,3,4-trimethoxy-
Mp: 100–103 °C.
phenyl)-4,5-dihydro-1H-pyrazole (34o).
1
Yield: 30%; H NMR (400.17 MHz, CDCl3) d: 7.42 (s, 1H-50), 4.07
(t, 2H, H-5, J5–4 = 10.2 Hz), 3.96, 3.95, 3.90 (3s, 20-OCH3, 30-OCH3,
40-OCH3), 3.18 (t, 2H, H-4, J4–5 = 10.2 Hz), 2.41–2.38 (m, 1H, H-
100), 1.04–0.83 (m, 4H, H-200, H-300). 13C NMR (75.49 MHz, CDCl3)
d: 172.66 (CO), 154.14 (C-3), 153.12 (C-40), 152.58 (C-30), 147.13
(C-60), 143.69 (C-20), 116.82 (C-10), 104.58 (C-50), 62.45, 61.46 (20-
OCH3, 30-OCH3), 56.81 (40-OCH3), 44.83 (C-5), 35.93 (C-4), 11.90
(C-100), 8.41 (C-200, C-300). MS (LSIMS): [M+H]+ = 350.1306.
4.1.4.10. 3-(5-Methoxy-2-nitrophenyl)-1-phenylpropanoyl-4,5-
dihydro-1H-pyrazole (34j).
Mp: 117–118 °C. Yield: 81%; 1H
NMR (400.17 MHz, CDCl3) d: 8.05 (d, 1H, J3 –4 = 9.0 Hz, H-30),
0
0
7.32–7.13 (m, 5H, H-200 to H-600), 7.02 (dd, 1H, J4 –3 = 9.0 Hz, J4 –
0
0
0
= 2.8 Hz, H-40), 6.93 (d, 1H, J6 –4 = 2.8 Hz, H-60), 4.08 (t, 2H, J5–
60
0
0
4 = 10.6 Hz, H-5), 3.92 (s, 3H, OCH3), 3.12 (d, 2H, J4–5 = 10.6 Hz, H-
4), 3.01–2.99 (m, 4H, COCH2CH2). 13C NMR (125.69 MHz, CDCl3)
d: 173.87 (CO), 165.81 (C-50), 157.24 (C-3), 143.95 (C-20), 137.91
(C-100), 132.80, 131.15, (C-200 to C-600), 130.97 (C-10), 128.62 (C-30),
118.45 (C-60), 117.40 (C-40), 58.80 (OCH3), 47.14 (C-5), 38.35
(COCH2CH2), 36.99 (COCH2CH2), 33.64 (C-4). MS (LSIMS):
[M+H]+ = 354.1453.
4.1.4.16.
dihydro-1H-pyrazole (34p).
1-Benzoyl-3-(6-nitro-2,3,4-trimethoxyphenyl)-4,5-
Mp: 102–106 °C. Yield: 20%; 1H
NMR (400.17 MHz, CDCl3) d: 7.70–7.67 (m, 2H, H-200, H-600), 7.33–
7.25 (m, 3H, H-300 to H-500), 7.18 (s, 1H, H-50), 4.23 (t, 2H, J4–
5 = 9.9 Hz, H-4), 3.87, 3.86, 3.79 (3s, 20-OCH3, 30-OCH3, 40-OCH3),
3.18 (t, 2H, J4–5 = 9.9 Hz, H-4). 13C NMR (75.49 MHz, CDCl3) d:
168.91 (CO), 155.35 (C-3), 154.91 (C-40), 153.80 (C-30), 148.12 (C-
60), 145.12 (C-20), 135.71 (C-400), 132.19 (C-300, C-500), 130.19 (C-
100), 129.08 (C-200, C-600), 117.19 (C-10), 105.71 (C-50), 63.47, 62.59
(20-OCH3, 30-OCH3), 57.06 (40-OCH3), 46.87 (C-5), 36.71 (C-4). MS
(LSIMS): [M+H]+ = 386.1312.
4.1.4.11.
1-Cyclopropylcarbonyl-3-(4,5-dimethoxy-2-nitro-
Mp: 144–146 °C.
phenyl)-4,5-dihydro-1H-pyrazole (34k).
1
Yield: 80%; H NMR (400.17 MHz, CDCl3) d: 7.60 (H-30), 6.98 (H-
60), 4.09 (t, 2H, J5–4 = 10.2 Hz, H-5), 4.00 (50-OCH3), 3.98 (40-
OCH3), 3.11 (t, 2H, J4–5 = 10.2 Hz, H-4), 2.55–2.51 (m, 1H, H-100),
1.08–0.83 (m, 4H, H-200, H-300). 13C NMR (125.69 MHz, CDCl3) d:
172.66 (CO), 155.15 (C-3), 153.22 (C-50), 149.96 (C-40), 141.90 (C-
20), 122.39 (C-10), 112.13 (C-60), 108.07 (C-30), 56.89, 56.78 (40-
OCH3, 50-OCH3), 45.14 (C-5), 34.40 (C-4), 11.89 (C-100), 8.56 (C-200,
C-300). MS (LSIMS): [M+H]+ = 320.1246.
4.1.4.17.
1-Phenylacetyl-3-(2,3,4-trimethoxy-6-nitrophenyl)-
Mp: 100–102 °C. Yield:
4,5-dihydro-1H-pyrazole (34q).
1
21%; H NMR (400.17 MHz, CDCl3) d: 7.45–7.10 (m, 6H, H-200 to
H-600, H-50), 4.08 (t, 2H, H-5, J5–4 = 10.0 Hz), 3.98, 3.97 (2s, 20-
OCH3, 30-OCH3), 3.94 (s, 40-OCH3), 3.94–3.86 (m, 2H, CO–CH2),
3.19 (t, 2H, H-4, J4–5 = 10.0 Hz).13C NMR (75.49 MHz, CDCl3) d:
169.81 (CO), 154.17 (C-3), 153.30 (C-40), 152.57 (C-30), 147.15 (C-
60), 143.70 (C-20), 135.64 (C-100), 129.64 (C-300, C-500), 128.57 (C-200,
C-600), 126.81 (C-400), 116.73 (C-10), 104.63 (C-50), 62.36, 61.48
4.1.4.12. 1-Benzoyl-3-(4,5-dimethoxy-2-nitrophenyl)-4,5-dihy-
dro-1H-pyrazole (34l).
Mp: 186–188 °C. Yield: 60%; 1H
NMR (300.20 MHz, CDCl3) d: 7.92–7.89 (m, 2H, H-200, H-600), 7.59

4140
M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
(20-OCH3, 30-OCH3), 46.83 (40-OCH3), 44.53 (C-5), 40.96 (CO–CH2),
3.97 (t, 2H, J5–4 = 10.1 Hz, H-5), 3.26 (t, 2H, J4–5 = 10.1 Hz, H-4),
3.05–2.98 (m, 4H, CO–CH2–CH2). 13C NMR (125.69 MHz, CDCl3) d:
170.33 (CO), 156.41 (C-3), 145.44 (C-20), 141.48 (C-100), 130.93 (C-
40), 128.88 (C-60), 128.62 (C-200, C-300, C-500, C-600), 126.36 (C-400),
121.39 (C-50), 117.42 (C-30), 114.60 (C-10), 42.90 (C-5), 36.27
(CO–CH2–CH2), 33.11 (C-4), 31.14 (CO–CH2–CH2). MS (LSIMS):
[M+Na]+ = 350.1036.
36.71 (C-4). MS (LSIMS): [M+H]+ = 400.4031.
4.1.4.18.
3-(6-Nitro-2,3,4-trimethoxyphenyl)-1-phenylpropa-
Mp: 104–106 °C. Yield:
noyl-4,5-dihydro-1H-pyrazole (34r).
22%; 1H NMR (400.17 MHz, CDCl3) d: 7.30–6.97 (m, 6H, H-50, H-200
to H-600), 4.10 (t, 2H, J5–4 = 10.0 Hz, H-5), 3.97, 3.95 (2s, 20-OCH3, 30-
OCH3), 3.87 (s, 40-OCH3), 3.19 (t, 2H, J4–5 = 10.0 Hz, H-4), 3.00–2.94
(m, 4H, –CO–CH2–CH2). 13C NMR (125.69 MHz, CDCl3) d: 180.21
(CO), 156.59 (C-3), 155.79 (C-40), 154.94 (C-30), 149.52 (C-60),
143.92 (C-20), 142.97 (C-100), 131.18 (C-200, C-600), 131.97 (C-300, C-
500), 128.96 (C-400), 119.05 (C-10), 107.03 (C-50), 62.23, 61.29 (20-
OCH3, 30-OCH3), 46.86 (40-OCH3), 44.27 (C-5), 38.45 (C-4), 35.85
4.1.5.5. 3-(2-Amino-4,5-dichlorophenyl)-1-cyclopropylcarbon-
yl-4,5-dihydro-1H-pyrazole (3e).
Mp: 148–149 °C. Yield:
1
80%; H NMR (400.17 MHz, CDCl3) d: 7.27 (s, 1H, H-30), 6.85 (s,
1H, H-60), 3.99 (t, 2H, J5–4 = 10.2, H-5), 3.27 (t, 2H, J4–5 = 10.2, H-
4), 2.30–2.36 (m, 1H, H-100), 1.11–0.80 (m, H-200, H-300). 13C NMR
(125.69 MHz, CDCl3) d: 178.07 (CO), 158.29 (C-3), 141.23 (C-20),
130.50 (C-40), 125.78 (C-60), 125.26 (C-50), 115.32 (C-10), 112.41
(C-30), 35.89 (C-5), 28.30 (C-4), 7.43 (C-100), 3.73 (C-200, C-300). MS
(LSIMS): [M+Na]+ = 321.0414.
(CO–CH2–CH2),
32.21
(CO–CH2–CH2).
MS
(LSIMS):
[M+H]+ = 414.1604.
4.1.5. Preparation of 1-acyl-3-(2-aminophenyl-substituted)-4,5-
dihydro-1H-pyrazoles (3a–n) and 1-acy-3-(6-aminophenyl-
2,3,4-trimethoxy-)-4,5-dihydro-1H-pyrazole (3o–r) general
method
A mixture of nitroarene (0.309 mmol) and SnCl2 (350 mg) was
dissolved on ethanol and was stirred under reflux for 2 h. The solu-
tion was quenched to pH = 7 with saturated NaHCO3 water solu-
tion, extracted with ethyl acetate (2 ꢁ 15 mL), and dried
(Na2SO4). Evaporation of the solvent gave a residue of the corre-
sponding amino-derivative that was purified by column chroma-
tography (EtOAc/hexane 1:3).
4.1.5.6.
dro-1H-pyrazole (3f).
(400.17 MHz, DMSO) d: 7.68 (d, 2H, J2
3-(2-Amino-4,5-dichlorophenyl)-1-benzoyl-4,5-dihy-
Mp: 220–223 °C. Yield: 92%; 1H NMR
00ꢀ300
¼ J6
= 8.0 Hz, H-200,
00ꢀ500
H-600), 7.52–7.42 (m, 3H, H-300, H-400, H-500), 7.40 (s, 1H, H-60), 6.89
(s, 1H, H-30), 3.99 (t, 2H, J5–4 = 9.9 Hz, H-5), 3.36 (t, 2H, J4–
5 = 9.9 Hz, H-4). 13C NMR (75.49 MHz, CDCl3) d: 168.07 (CO),
157.69 (C-3), 147.69 (C-20), 135.81 (C-100), 133.36 (C-40), 131.49
(C-60), 129.08–128.60 (C-200, C-600, C-300, C-500), 131.29 (C-400),
116.57 (C-30), 113.40 (C-10), 116.76 (C-50), 44.15 (C-5), 31.38 (C-
4). MS (ESI): [M+H]+ = 334.0514.
4.1.5.1. 3-(2-Aminophenyl)-1-phenylacetyl-4,5-dihydro-1H-pyr-
azole (3a).
Mp: 196–198 °C. Yield: 70%; 1H NMR
4.1.5.7.
dihydro-1H-pyrazole (3g).
3-(2-Amino-4,5-dichlorophenyl)-1-phenylacetyl-4,5-
Mp: 155–160 °C. Yield: 90%; 1H
(300.20 MHz, CDCl3) d: 7.39–7.20 (m, 7H, H-200 to H-600, H-40, H-
60), 6.77–6.72 (m, 2H, H-30, H-50), 5.71 (sa, 2H, NH2), 4.08 (s, 2H,
CO–CH2), 4.02 (t, 2H, J5–4 = 10 Hz, H-5), 3.34 (t, 2H, H-4, J4–
5 = 10 Hz). 13C NMR (75.49 MHz, CDCl3) d: 168.89 (CO), 157.87
(C-3), 146.99 (C-20), 135.58 (C-100), 131.30 (C-40), 129.78 (C-60),
129.28, 128.82 (C-200, C-300, C-500, C-600), 126.99 (C-400), 117.05 (C-
50), 116.24 (C-30), 112.95 (C-10), 42.93 (C-5), 41.64 (CO–CH2),
33.41 (C-4). MS (LSIMS): [M+Na]+ = 302.1267.
NMR (400.17 MHz, CDCl3) d: 7.30 (m, 6H, H-60, H-200 to H-600),
6.88 (s, 1H, H-30), 4.04–3.98 (m, 4H, COCH2, H-5); 3.25 (t, 2H, J4–
5 = 10.5 Hz, H-4).13C NMR (75.49 MHz, CDCl3) d: 168.88 (CO),
155.38 (C-3), 145.32 (C-20), 142.68 (C-100), 134.66 (C-40), 130.43
(C-60), 129.00–128.70 (C-200, C-600, C-300, C-500), 126.93 (C-400),
122.23 (C-50), 117.31 (C-30), 113.05 (C-10), 43.07 (C-5), 41.48
(CO–CH2), 33.06 (C-4). MS (ESI): [M+Na]+ = 370.0490.
4.1.5.2.
1H-pyrazole (3b).
3-(2-Aminophenyl)-1-phenylpropanoyl-4,5-dihydro-
Mp: 145–147 °C. Yield: 74%; 1H NMR
4.1.5.8.
3-(2-Amino-4,5-dichlorophenyl)-1-phenylpropanoyl-
Mp: 175–178 °C. Yield: 93%;
4,5-dihydro-1H-pyrazole (3h).
(300.20 MHz, CDCl3) d: 7.33–7.11 (m, 7H, H-200 to H-600, H-40, H-
60), 6.78–6.73 (m, 2H, H-30, H-50), 5.76 (sa, 2H, NH2), 4.00 (t, 2H,
J5–4 = 10 Hz, H-5), 3.34 (t, 2H, J4–5 = 10 Hz, H-4), 3.10–3.03 (m,
4H, CO–CH2–CH2). 13C NMR (75.49 MHz, CDCl3) d: 170.29 (CO),
157.67 (C-3), 146.98 (C-20), 141.64 (C-100), 131.24 (C-40), 129.75
(C-60), 128.72, 128.67 (C-200, C-300, C-500, C-600), 126.34 (C-400),
117.04 (C-50), 116.24 (C-30), 113.53 (C-10), 42.79 (C-5), 36.37
(CO–CH2–CH2), 33.26 (C-4), 31.23 (CO–CH2–CH2). MS (LSIMS):
[M+Na]+ = 316.1416.
1H NMR (300.20 MHz, CDCl3) d: 7.26 (m, 6H, H-60, H-200 to H-600),
6.92 (s, 1H, H-30), 5.05 (sa, 2H, NH2), 4.02 (t, 2H, J5–4 = 10.3 Hz, H-
5), 3.28 (t, 2H, J4–5 = 10.3 Hz, H-4), 3.05–2.98 (m, 4H, CO–CH2–
CH2).13C NMR (75.49 MHz, CDCl3) d: 170.47 (CO), 155.54 (C-3),
144.60 (C-20), 141.39 (C-100), 134.81 (C-40), 130.61 (C-60), 128.75,
128.67 (C-200, C-600, C-300, C-500, C-50), 126.43 (C-400), 120.51 (C-10),
118.05 (C-30), 43.03 (C-5), 36.22 (CO–CH2–CH2), 33.09 (C-4),
31.14 (CO–CH2–CH2). MS (ESI): [M+H]+ = 362.0812.
4.1.5.9.
dihydro-1H-pyrazole (3i).
NMR (400.17 MHz, CDCl3) d: 7.36–7.21 (m, 5H, H-200 to H-600),
3-(2-Amino-5-methoxyphenyl)-1-phenylacetyl-4,5-
4.1.5.3. 3-(2-Amino-5-chlorophenyl)-1-phenylacetyl-4,5-dihy-
Mp: 177–180 °C. Yield: 71%; 1H
dro-1H-pyrazole (3c).
Mp: 152–154 °C. Yield: 68%; 1H
NMR: (400.17 MHz, CDCl3) d: 7.33–7.26 (m, 5H, H-200 to H-600),
6.85 (dd, 1H, J4 –3 = 8.8 Hz, J4 –6 = 2.9 Hz, H-40), 6.75–6.71 (m, 2H,
H-30, H-60), 4.04 (s, 2H, CO–CH2), 3.99 (t, 2H, J5–4 = 8.8 Hz, H-5),
3.76 (s, 3H, OCH3), 3.28 (t, 2H, J4–5 = 8.8 Hz, H-4). 13C NMR
(125.69 MHz, CDCl3) d: 171.40 (CO), 159.83 (C-50), 154.02 (C-3),
143.18 (C-20), 137.91 (C-100), 131.70, 131.21 (C-200, C-300, C-500, C-
600), 129.39 (C-400), 120.64 (C-40), 120.19 (C-30), 116.82 (C-60),
113.72 (C-10), 58.70 (OCH3), 45.44 (C-5), 43.98 (CO–CH2), 35.83
(C-4). MS (ESI): [M+Na]+ = 332.1374.
0
0
0
0
7.15–7.11 (m, 2H, H-40, H-60), 6.65 (d, 1H, J3 ꢀ4 = 8.6 Hz, H-30),
4.02–3.96 (m, 4H, H-5, CO–CH2), 3.27 (t, 2H, J4–5 = 10 Hz, H-4).
13C NMR (75.49 MHz, CDCl3) d: 168.94 (–CO–), 156.65 (C-3),
145.45 (C-20), 135.35 (C-100), 130.99 (C-40), 129.20 (C-60), 128.91
(C-200 to C-600), 121.39 (C-50), 117.43 (C-30), 114.60 (C-10), 43.05
0
0
(C-5),
41.61
(CO–CH2),
33.25
(C-4).
MS
(LSIMS):
[M+Na]+ = 336.0884.
4.1.5.4.
dihydro-1H-pyrazole (3d).
NMR: (400.17 MHz, CDCl3) d: 7.30–7.19 (m, 5H, H-200 to H-600),
3-(2-Amino-5-chlorophenyl)-1-phenylpropanoyl-4,5-
4.1.5.10.
3-(2-Amino-5-methoxyphenyl)-1-phenylpropanoyl-
Mp: 165–167 °C. Yield: 69%; 1H
4,5-dihydro-1H-pyrazole (3j).
Mp: 155–157 °C. Yield: 75%;
1H NMR (400.17 MHz, CDCl3) d: 7.31–7.24 (m, 4H, H-200, H-300, H-
7.16–7.10 (m, 2H, H-40, H-60), 6.66 (d, 1H, J3 ꢀ4 = 8.6 Hz, H-30),
500, H-600), 7.20–7.18 (m, 1H, H-400), 6.86 (dd, 1H, J
= 8.8 Hz,
0 0
0
0
4ꢀ3

M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
4141
0 0
J
= 3.1 Hz, H-40), 6.76–6.72 (m, 2H, H-30, H-60), 3.97 (t, 2H, J5–
(C-50), 63.79, 63.64 (20-OCH3, 30-OCH3), 58.36 (40-OCH3), 45.78
(C-5), 38.51 (C-4), 14.49 (C-100), 10.51 (C-200, C-300). MS (LSIMS):
[M+H]+ = 320.1504.
4ꢀ6
4 = 10.3 Hz, H-5), 3.77 (s, 3H, OCH3), 3.29 (t, 2H, J4–5 = 10.3 Hz, H-
4), 3.08–2.97 (m, 4H, COCH2–CH2) 13C NMR (125.69 MHz, CDCl3)
d: 172.80 (CO), 159.61 (C-50), 154.07 (C-3), 143.99 (C-20), 143.04
(C-100), 131.07 (C-200, C-300, C-500, C-600), 128.72 (C-400), 120.52 (C-
40), 120.23 (C-30), 116.83 (C-60), 113.50 (C-10), 58.69 (OCH3),
45.30 (C-5), 38.70 (CO–CH2–CH2), 35.69 (C-4), 33.60 (CO–CH2–
CH2). MS (ESI): [M+Na]+ = 346.1531.
4.1.5.16.
dihydro-1H-pyrazole (3p).
3-(6-Amino-2,3,4-trimethoxyphenyl)-1-benzoyl-4,5-
Mp: 155–157 °C. Yield: 60%; 1H
NMR (400.17 MHz, CDCl3) d: 7.81–7.79 (m, 2H, H-200, H-600), 7.39–
7.37 (m, 3H, H-300-H-500), 5.29 (s, 1H, H-60), 4.13 (t, 2H, J5–
4 = 11.7 Hz, H-5), 3.92 (1s, 3H, 30-OCH3), 3.83, 381 (2s, 6H, 20-
4.1.5.11. 3-(2-Amino-4,5-dimethoxyphenyl)-1-cyclopropylcar-
OCH3, 40-OCH3), 3.51
(t,
2H, J4–5 = 11.7 Hz, H-4).13
C
bonyl-4,5-dihydro-1H-pyrazole (3k).
Mp: 128–130 °C. Yield:
NMR(75.49 MHz, CDCl3) d: 167.12 (CO), 156.14 (C-3), 155.54 (C-
40), 154.43 (C-20), 144.65 (C-60), 134.93 (C-30), 133.34 (C-100),
130.71 (C-300, C-500), 128.12 (C-200, C-600), 126.87 (C-400), 110.31 (C-
10), 77.18 (C-50), 61.48, 61.01 (20-OCH3, 30-OCH3), 56.26 (40-OCH3),
43.90 (C-5), 35.64 (C-4). MS (LSIMS): [M+H]+ = 356.1561.
90%; 1H NMR (400.17 MHz, CDCl3) d: 6.71 (s, 1H, H-60), 6.32 (s, 1H,
H-30), 3.97 (t, 2H, J5–4 = 9.6, H-5), 3.88 (s, 3H, 50-OCH3), 3.82 (s, 3H,
40-OCH3), 3.29 (t, 2H, J4–5 = 9.6, H-4), 2.43 (m, 1H, H-100), 1.10–0.84
(m, 2H, H-200, H-300). 13C NMR (75.49 MHz, CDCl3) d: 171.43 (CO),
157.14 (C-3), 152.41 (C-40), 142.58 (C-50), 141.35 (C-20), 113.21
(C-60), 105.87 (C-10), 100.10 (C-30), 57.32 (40-OCH3), 56.06 (50-
OCH3), 43.07 (C-5), 33.40 (C-4), 12.17 (C-100), 8.25 (C-200, C-300).
MS (LSIMS): [M+Na]+ = 312.1324.
4.1.5.17. 3-(6-Amino-2,3,4-trimethoxyphenyl)-1-phenylacetyl-
4,5-dihydro-1H-pyrazole (3q).
Mp: 158–160 °C. Yield: 60%;
1H NMR (400.17 MHz, CDCl3) d: 7.33–7.21 (m, 5H, H-200 to H-600),
5.97 (s, 1H, H-50), 4.00–3.85 (m, 4H, H-5, CO–CH2), 3.86, 3.82 (2s,
6H, 20-OCH3, 30-OCH3), 3.74 (s, 3H, 40-OCH3), 3.40 (t, 2H, J4–
5 = 10 Hz, H-4). 13C NMR (75.49 MHz, CDCl3) d: 168.66 (CO),
157.20 (C-3), 155.89 (C-40), 154.62 (C-20), 144.69 (C-60), 135.79
(C-30), 133.97 (C-100), 129.20 (C-300, C-500), 128.80 (C-200, C-600),
126.90 (C-400), 104.99 (C-10), 94.97 (C-50), 61.26 (20-OCH3, 30-
OCH3), 55.95 (40-OCH3), 43.09 (C-5), 41.70 (CO–CH2), 36.33 (C-4).
MS (LSIMS): [M+H]+ = 370.1703.
4.1.5.12.
dihydro-1H-pyrazole (3l).
3-(2-Amino-4,5-dimethoxyphenyl)-1-benzoyl-4,5-
Mp: 128–130 °C. Yield: 66%; 1H
NMR (300.20 MHz, CDCl3) d: 7.90–7.87 (m, 2H, H-200, H-600), 7.47–
7.43 (m, 3H, H-300-H-500), 6.78 (s, 1H, H-60), 6.72 (s, 1H, H-30), 4.21
(t, 2H, J5–4 = 10.1 Hz, H-5), 3.89 (s, 3H, 50-OCH3), 3.87 (s, 3H, 40-
OCH3), 3.38 (t, 2H, J4–5 = 10.1 Hz, H-4). 13C NMR (75.49 MHz, CDCl3)
d: 165.38 (CO), 155.46 (C-3), 150.60 (C-40), 145.23 (C-50), 141.06 (C-
20), 133.34 (C-100), 129.25 (C-300, C-500), 126.45 (C-200, C-600), 126.37
(C-400), 115.06 (C-10), 111.18 (C-60), 101.65 (C-30), 55.48 (40-OCH3),
54.56 (50-OCH3), 42.10 (C-5), 31.21 (C-4). MS (ESI):
[M+Na]+ = 348.1330.
4.1.5.18. 3-(6-Amino-2,3,4-trimethoxyphenyl)-1-phenypropa-
noyl-4,5-dihydro-1H-pyrazole (3r).
Mp: 158–160 °C. Yield:
1
68%; H NMR (400.17 MHz, CDCl3) d: 7.28–7.27 (m, 5H, H-200 to
H-600), 6.03 (s, 1H, H-50), 3.95 (t, 2H, J5–4 = 10 Hz, H-5), 3.87, 3.84
(2s, 6H, 20-OCH3, 30-OCH3), 3.76 (s, 3H, 40-OCH3), 3.44 (t, 2H, J4–
5 = 10 Hz, H-4), 3.04–2.96 (m, 4H, CO–CH2–CH2). 13C NMR
(75.49 MHz, CDCl3) d: 166.11 (CO), 158.19 (C-3), 152.96 (C-40),
151.89 (C-10), 144.70 (C-60), 141.29 (C-30), 137.79 (C-100), 124.75
4.1.5.13. 3-(2-Amino-4,5-dimethoxyphenyl)-1-phenylacetyl-4,5-
dihydro-1H-pyrazole (3m).
Mp: 185–186 °C. Yield: 89%; 1H
NMR (400.17 MHz, CDCl3) d: 7.35–7.26 (m, 4H, H-200, H-300, H-500,
H-600), 7.23–7.20 (m, 1H, H-400), 6.69 (s, 1H, H-60), 6.53 (s, 1H, H-
30), 4.04 (s, 2H, CO-CH2), 3.98 (t, 2H, J5–4 = 10.2 Hz, H-5), 3.85 (s,
3H, 50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.26 (t, 2H, J4–5 = 10.2 Hz, H-
4). 13C NMR (125.69 MHz, CDCl3) d: 171.21 (CO), 159.39 (C-3),
154.74 (C-40), 145.24 (C-50), 141.38 (C-20), 137.99 (C-100), 129.16
(C-300, C-500), 128.69 (C-200, C-600), 126.83 (C-400), 115.37 (C-10),
112.78 (C-60), 101.65 (C-30), 57.05 (40-OCH3), 56.02 (50-OCH3),
42.79 (C-5), 41.16 (CO–CH2), 33.31 (C-4). MS (LSIMS):
[M+H]+ = 340.1661.
´
00
00
00
00
0
0
´
(C-2 , C-3 , C5, C-6 ), 122.36 (C-4 ), 98.09 (C-1 ), 91.30 (C-5 ),
57.89, 57.32 (20-OCH3, 30-OCH3), 52.05 (40-OCH3), 39.00 (C-5),
32.38 (C-4), 32.28 (CO–CH2–CH2), 27.36 (CO–CH2–CH2). MS
(LSIMS): [M+H]+ = 384.1832.
4.2. In vitro nNOS and iNOS activities determination
L
-Arginine,
L
-citrulline, N-(2-hydroxymethyl)piperazine-N0-
acid) (HEPES), DL-dithiothreitol (DTT),
(2-ethanesulfonic
4.1.5.14.
3-(2-Amino-4,5-dimethoxyphenyl)-1-phenylpropa-
Mp: 176–179 °C. Yield:
hypoxantine-9-b-D-ribofuranosid (inosine), ethylene glycol-bis-
noyl-4,5-dihydro-1H-pyrazole (3n).
(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA), bovine ser-
um albumin (BSA), Dowex-50 W (50 ꢁ 8–200), FAD, NADPH and
1
60%; H NMR (400.17 MHz, CDCl3) d: 7.27–7.25 (m, 4H, H-200, H-
300, H-500, H-600), 7.17–7.15 (m, 1H, H-400), 6.70 (s, 1H, H-60), 6.52
(s, 1H, H-30), 3.96 (t, 2H, J5–4 = 10.0 Hz, H-5), 3.86 (s, 3H, 50-
OCH3), 3.83 (s, 3H, 40-OCH3), 3.26 (t, 2H, J4–5 = 10.0 Hz, H-4),
3.05–2.98 (m, 4H, CO–CH2–CH2). 13C NMR (75.49 MHz, CDCl3) d:
165.43 (CO), 152.08 (C-3), 147.57 (C-40), 145.24 (C-50), 138.02 (C-
20), 136.89 (C-100), 123.98, 123.95 (C-200, C-300, C-500, C-600), 121.55
(C-400), 113.93 (C-10), 108.20 (C-60), 97.01 (C-30), 52.48 (40-OCH3),
51.44 (50-OCH3), 38.04 (C-5), 31.50 (C-4), 30.70 (CO–CH2–CH2),
25.66 (CO–CH2–CH2). MS (LSIMS): [M+H]+ = 354.1817.
5,6,7,8-tetrahydro-L-biopterin dihydrocloride (H4-biopterin) were
obtained from Sigma–Aldrich Química (Spain).
L
-[3H]-arginine
(54 Ci/mmol) was obtained from Amersham (Amersham Biosci-
ences, Spain). Tris–(hydroxymethyl)-aminometane (Tris–HCl) and
calcium chloride were obtained from Merck (Spain). Calmodulin
from bovine brain, and recombinant iNOS and nNOS were obtained
from Alexis Biochemicals (Enzo Life Sciences, Grupo Taper, Seville,
Spain).
The nNOS activity was measured by the Bredt and Snyder34
method, monitoring the conversion of
citrulline. The final incubation volume was 100
of 10 L of an aliquot of recombinant nNOS (specific activity
21.05 nmol/min/mg protein) added to a buffer with a final concen-
tration of 25 mM Tris–HCl, 1 mM DTT, 4 M H4-biopterin, 10
FAD, 0.5 mM inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 -argi-
nine, and 50 nM
-[3H]-arginine, at pH 7.6. The reaction was started
by the addition of 10 L of NADPH (0.75 mM final) and 10 L of
each 4,5-dihydro-1H-pyrazole derivative in ethanol to give a final
L L
-[3H]-arginine to -[3H]-
4.1.5.15.
3-(6-Amino-2,3,4-trimethoxyphenyl)-1-cyclopropyl-
Mp: 176–179 °C.
lL and consisted
carbonyl-4,5-dihydro-1H-pyrazole (3o).
l
1
Yield: 65%; H-NMR: (400.17 MHz, CDCl3) d: 6.02 (s, H-50), 3.93
(t, 2H, J5–4 = 10.0 Hz, H-5), 3.87, 3.84 (2s, 6H, 20-OCH3, 30-OCH3),
3.76 (s, 3H, 40-OCH3), 3.42 (t, 2H, J4–5 = 10.0 Hz, H-4), 2.42–2.39
(m, 1H, H-100), 1.08–0.83 (m, 2H, H-200, H-300). 13C NMR
(75.49 MHz, CDCl3) d: 173.83 (CO), 159.15 (C-3), 158.13 (C-40),
157.01 (C-20), 146.95 (C-60), 136.53 (C-30), 104.58 (C-10), 97.56
l
lM
lM L
L
l
l

4142
M. D. Carrión et al. / Bioorg. Med. Chem. 21 (2013) 4132–4142
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concentration of 1 mM. The tubes were vortex and incubated at
37 °C for 30 min. Control incubations were performed by the omis-
sion of NADPH. The reaction was halted by the addition of 400
lL
of cold 0.1 M HEPES, 10 mM EGTA, and 0.175 mg/mL -citrulline,
L
pH 5.5. The reaction mixture was decanted into a 2 mL column
packet with Dowex-50 W ion-exchange resin (Na+ form) and
eluted with 1.2 mL of water.
quid scintillation spectroscopy. The retention of
L
-[3H]-citrulline was quantified by li-
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
(/mg of protein/min).
L
-[3H]-citrulline produced
For iNOS activity determination, the procedure was essentially
the same as for nNOS, excepting that an aliquot of 10 L recombi-
l
nant iNOS (specific activity of 0.7 nmol/min/mg protein) was used
instead of nNOS, and the mixture was incubated in the absence of
calmodulin.
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4.3. Statistical analysis
Data are expressed as the mean SEM. One-way analysis of var-
iance, followed by the Newmane–Keuls multiple range test was
used. A P <0.05 was considered statistically significant.
Acknowledgments
This word was partially supported by Grants from the Junta de
Andalucía (P06-CTS-01941 and P07-CTS-03135) and from the
Vicerrectorado de Política Científica e Investigación de la Univers-
idad de Granada (GENIL/GREIB and GREIB.PYR_2010_03).
23. López-Cara, L. C.; Camacho, M. E.; Carrión, M. D.; Tapias, V.; Gallo, M. A.;
Escames, G.; Acuña-Castroviejo, D.; Espinosa, A.; Entrena, A. Eur. J. Med. Chem.
2009, 44, 2655.
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Supplementary data
27. Ottl, J.; Gabriel, D.; Marriott, G. Bioconjugate Chem. 1998, 9, 143.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.016.
30. Schrödinger Suite 2012 Update 2.
31. Schrödinger Suite 2012 Protein Preparation Wizard; Epik version 2.3,
Schrödinger, LLC, New York, NY, 2012; Impact version 5.8, Schrödinger, LLC,
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