Phenylpyrrole derivatives as neural and inducible nitric oxide synthase (nNOS and iNOS) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.11.013

We have previously described a series of 3-phenyl-4,5-dihydro-1H-pyrazole derivatives as moderately potent nNOS inhibitors. As a follow up of these studies, several new 5-phenyl-1H-pyrrole-2-carboxamide derivatives have been synthesized, and their biologi

Full text of DOI:10.1016/j.ejmech.2008.11.013

European Journal of Medicinal Chemistry 44 (2009) 2655–2666
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Phenylpyrrole derivatives as neural and inducible nitric oxide synthase
(nNOS and iNOS) inhibitors
a
a
a
b
a
´
´
´
´
Luisa C. Lopez Cara , M. Encarnacion Camacho , M. Dora Carrion , Vıctor Tapias , Miguel A. Gallo ,
b
b
a
a,
*
´
˜
Germaine Escames , Darıo Acuna-Castroviejo , Antonio Espinosa , Antonio Entrena
a
´
Departamento de Qu´ımica Farmace´utica y Organica, Facultad de Farmacia, Universidad de Granada, Granada, Spain
b Departamento de Fisiolo´gica, Instituto de Biotecnolo´gica, Universidad de Granada, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously described a series of 3-phenyl-4,5-dihydro-1H-pyrazole derivatives as moderately
potent nNOS inhibitors. As a follow up of these studies, several new 5-phenyl-1H-pyrrole-2-carboxamide
derivatives have been synthesized, and their biological evaluation as in vitro inhibitors of both neural and
inducible Nitric Oxide Synthase (nNOS and iNOS) is described. Some of these compounds show good
iNOS/nNOS selectivity and the more potent compounds 5-(2-aminophenyl)-1H-pyrrole-2-carboxilic acid
methylamide (QFF205) and cyclopentylamide (QFF212) have been tested as regulators of the in vivo
nNOS and iNOS activity. Both compounds prevented the increment of the inducible NOS activity in both
cytosol (iNOS) and mitochondria (i-mtNOS) observed in the MPTP model of Parkinson’s disease.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Received 5 November 2008
Received in revised form
26 November 2008
Accepted 27 November 2008
Available online 6 December 2008
Keywords:
Neural NOS
Inducible NOS
Mitochondrial NOS
Inhibitors
Phenylpyrrole derivatives
1. Introduction
More recently, a mitochondrial-localized NOS isoform situated
in the internal membrane of the mitochondria (mtNOS) was
Nitric Oxide (NO) is an important bioregulator and an ubiqui-
tous biomessenger involved in several physiological and patho-
logical processes such as vasodilatation [1], non-specific host
defense [2], ischemia reperfusion injury [3] chronic or acute
inflammation [4], and neurological disorders like Alzheimer’s
disease [5] amyotrophic lateral sclerosis [6] and Huntington’s
disease [7].
discovered [15]. Even there was a controversy among the type(s)
of mtNOS in terms of their classification as constitutive or induc-
ible [16], in a recent paper the existence of both constitutive and
inducible mitochondrial NOS has been proven (c-mtNOS and
i-mtNOS, respectively) [17].
eNOS is involved in the regulation of smooth muscle relaxation
and blood pressure, and in the inhibition of platelet aggregation
[18] while nNOS has been shown to regulate neuronal transmission
and cerebral blood flow [19]. The major function of iNOS is thought
to serve in host defense mechanism [20]. Both mitochondrial
c-mtNOS and i-mtNOS are involved in the NO production in the
mitochondria that in turn controls the bioenergetic processes
inside this organelle [21].
It has also been reported that an uncontrolled NO production by
iNOS causes diseases such shock condition [22], inflammatory
arthritis [23] chronic ileitis and colitis [24]. NO overproduction by
nNOS produces neurotoxicity, and this fact has been associated
with several neurological disorders such as Alzheimer’s disease [5],
amyotrophic lateral sclerosis [6] and Huntington’s disease [7].
Recent reports showed iNOS activation and inflammatory reaction
in neurodegenerative processes such as Parkinson’s disease and
Alzheimer’s disease [25].
In mammals, NO is synthesized from L-arginine in various cell
types (neurons [8], endothelial cells [9] and macrophages [10]) by
a family of nitric oxide synthase (NOS) [11] isoenzymes, with
consumption of molecular oxygen, NADPH and other cofactors [12].
Based on its endogenous regulation, NOSs have been structurally
classified as constitutive NOS (cNOS), that requires Ca2þ/Calmod-
ulin (CaCAM) for its activation [13] and inducible NOS (iNOS), which
is CaCAM independent [14]. cNOS has been subdivided into endo-
thelial (eNOS) and neuronal (nNOS) attending to its localization in
the vascular endothelium and in the brain, respectively. The
inducible isoform (iNOS) is present in macrophages activated by
inflammatory cytokines or by lipopolysaccharide (LPS).
* 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.2008.11.013

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Thus nNOS and iNOS represent a therapeutic target since inhi-
molecules, docking studies of compounds 8a–v inside both nNOS
and iNOS binding sites have been tackled using Schro¨dinger soft-
ware [41]. Phenylpyrazoles 4 have been also studied for comparison
with the new molecules.
bition of these enzymes can help in the treatment of several
disorders, and a selective inhibition of one of these isoforms would
be desired. On the other hand, selective inhibitors may also
constitute useful pharmacological tools in the research of NO
biological functions.
Melatonin 1 is a hormone synthesized by many organs and
tissues of the organisms including the pineal gland [26] that shows
inhibitory effects in rat [27] and human [28] central nervous system
(CNS), being this the reason for its anticonvulsant and neuro-
protective properties [29]. Diverse experiments have suggested
that melatonin attenuates glutamate-mediated responses in the rat
striatum [30] and this inhibitory effect takes place through the
inhibition of nNOS [31–33]. nNOS inhibition by melatonin has
demonstrated to be dose-dependent and calmodulin-dependent
[34].
Our search group has reported several nNOS inhibitors with
a kynurenine structure 2, showing a significant nNOS inhibition
activity [34,35]. A second type of nNOS inhibitors described by our
research group show a kinurenamine structure 3 [36] among them
the main melatonin brain metabolite (AMK: 3, R1 ¼ MeO, R2 ¼ Me).
All these compounds inhibit nNOS in a dose-dependent manner,
and it has been found that AMK rather than melatonin is the active
metabolite against nNOS in rat striatum [37]. We have also pub-
lished a new relatively potent and less flexible nNOS inhibitors of
general formula 4, bearing a 4,5-dihydro-1H-pyrazole moiety [38].
Finally, 3-benzoyl-4,5-dihydro-1H-pyrazole derivatives 5 [39], 6
[40], and 3-benzoyl-1H-pyrazoles 7 [40], show moderated both
nNOS and iNOS inhibition, but in some cases a iNOS selectivity is
observed.
Potential maps needed for docking experiments were generated
using Glide program, from the heme oxygenase domain of both
isoenzymes, obtained from the crystal structures of Nu-propil-
L-
arginine co-crystallized with both nNOS (PDB id: 1QW6) and iNOS
(PDB id: 1QW4) [42]. 3D structures of compounds 8a–v were
generated from fragment libraries, and optimized using the Mac-
romodel module. Glide program was used for docking the ligands
using the XP option.
Two different poses have been found for phenylpyrazolines 4
inside nNOS binding site, depending on the presence or the absence
of 50-substituent in these molecules. In all cases, the benzene ring
lies almost parallel to the heme group, and the pyrazoline ring is
situated between Gln478 and Glu592. This last residue is crucial for
the interaction between NOS and L-arginine, since it forms an
electrostatic reinforced hydrogen bond with the substrated, and
also interacts with almost all NOS inhibitors.
When compound 4 has a non-substituted benzene ring (R1 ¼ H),
the 20-NH2 group points toward Glu592, forming a hydrogen bond
with this residue. Fig. 1A shows the best pose for compound 4m
(R1 ¼ H, R2 ¼ c-C3H5). Nevertheless, when compound 4 bears a 50-
substituent (R1 ¼ OMe or Cl), this group will interact with Phe584
in a pose similar to that of Fig. 1A. For this reason, the benzene ring
rotates, and the best pose for 50-substituted pyrazoline derivatives
shows the 20-NH2 group pointing to the heme side chains. Fig. 1B
shows as an example the best pose found for compound 3-(2-
amino-5-methoxyphenyl)-2,3-dihydro-1H-pyrazole 4f (R1 ¼ OMe,
In this paper we describe a new type of NOS inhibitors with
general structure 8, bearing a pyrrole moiety. These new molecules
show moderate in vitro nNOS and iNOS inhibition and in some cases
iNOS selectivity. A preliminary in vivo study of the more active
compounds is also presented. Two compounds reduce the in vivo
NOS activity in cytosol and mitochondria in the MPTP model
Parkinson’s disease.
R2 ¼ c-C4H7). In this case, no hydrogen bond is observed between
the nNOS binding site and the inhibitor.
Pyrrole derivatives 8a–v behave similarly to pyrazolines 4, and
two different poses have been found inside nNOS binding site for
these derivatives (Fig. 1C, D). Compounds 8a–k bear a 50-substitued
benzene ring, and the main pose obtained for these molecules is
similar to that of 50-substituted pyrazolines 4. Fig. 1C shows the
pose for compound 8d inside nNOS. Nevertheless, it can be
observed that in this case two hydrogen bonds are formed between
both the pyrrole NH and the amide NH bonds, and one Glu592
carboxylate O atom.
2. Results and discussion
2.1. Drug design
In compounds 8l–v (R1 ¼ H) the benzene ring is not substituted,
and the main pose found for these molecules is similar to that of
unsubstituted pyrazolines. Fig. 1D shows, the 8s/nNOS complex,
and it can be observed that the 20-NH2 group is pointing toward
Glu592. In this complex, one hydrogen bond is formed between one
Phenylpyrazolines 4 have been proven to be good nNOS inhib-
itors [38], and compounds 8 have been designed from phenyl-
pyrazolines 4, substituting the pyrazole moiety by a pyrrole ring. In
order to test the potentiality as NOS inhibitors of these new

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2657
Fig. 1. (A, B) The preferred poses found for 3-(2-aminophenyl)-2,3-dihydro-1H-pyrazole (4m, R1 ¼ H, R2 ¼ c-C3H5), and 3-(2-amino-5-methoxyphenyl)-2,3-dihydro-1H-pyrazole (4f,
R1 ¼ OMe, R2 ¼ c-C4H7) inside the nNOS binding site. The preferred pose found for compounds 8d (C) and 8s (D) in nNOS. These molecules interact with Glu592 by means of two
hydrogen bonds (red dotted lines). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). (E) The preferred pose
obtained for compound 8s inside iNOS, in this case three hydrogen bonds are formed with Glu371.
O atom of Glu592 carboxylate, and the 20-NH2 group. An additional
hydrogen bond is formed between the same O atom of Glu592
carboxylate and the ligand amide NH bond.
9b is also commercially available, and 5-methoxy-2-nitro-
benzaldehyde 9a was prepared by O-methylation of 5-hidroxy-2-
nitrobenzaldehyde (CH3I/K2CO3 in THF) [39].
Similar poses have been obtained inside the iNOS binding site
for compounds 8a–v. Nevertheless, in this isoenzyme, Gln257 is
rotated in relation to its equivalent residue in nNOS (Gln478). For
this reason, the c-C5H9 substituent in compound 8s (Fig. 1E) is
shifted in relation to Fig. 1D, and this slight modification of the
ligand geometry allows the formation of a third hydrogen bond
between the 20NH2 and the Glu371 carboxylate.
Docking studies indicate that compounds 8a–v can fit inside
nNOS binding site with reasonable geometries and that they can
interact with the enzyme by means of several hydrogen bonds that
are not present in the complexes formed by pyrazolines 4. For this
reason, it can be expected that these new molecules should be
better nNOS inhibitors than pyrazolines 4. Docking studies also
indicate that compounds 8a–v can bind correctly inside the iNOS
binding site. These reasons prompted us to the synthesis and
biological evaluation of these molecules.
Azides derivatives 11a–c cyclizise and yield the corresponding
nitrophenylpyrrole derivatives 12a–c when heated in p-xylene.
Compound 12c was treated with CH3I under basic conditions and in
the presence of 18-crown-6 ether to yield compound 12d.
Two alternative procedures were employed in the synthesis of
nitrocarboxamide derivatives 14a–v from compounds 12a–d.
Compounds 14a, 14g, 14l and 14v were directly obtained from 12a–
d, respectively, by treatment with NH4Cl/NH4OH [46]. In all other
cases, the ester moiety of compounds 12a–c was previously
hydrolyzed (NaOH, then AcOH) [47] to yield the carboxylic acid
derivatives 13a–c, which in turn were transformed into the acyl
chloride (SOCl2) and treated with the appropriated amine (R3NH2/
TEA) to yield the corresponding N-substituted carboxamide 14 [48].
Finally, compounds 8a–v were obtained by reduction of the
nitro group in the corresponding derivative 14a–v, performed by
treatment with Fe/FeSO4 [49].
2.2. Chemistry
2.3. In vitro NOS inhibition
Scheme 1 represents the general synthetic pathway followed in
the preparation of the final 5-phenyl-1H-pyrrole-2-carboxamide
derivatives described in this paper. Two main structural modifica-
tions were performed in these molecules: i) modification of the
amide chain; and, ii) substitution of the benzene H-50 atom. The
pyrrolic ring has been constructed by means of the Hemetsberger
reaction [43]. The synthetic pathway begins with the reaction of 2-
nitro-cinnamaldehyde derivatives 10a–c with ethyl azidoacetate
to yield the corresponding 2-azido-5-(2-nitro-5-substituted-
phenyl)-penta-2,4-dienoic acid ethyl ester 11a–c [44]. While 2-
nitrocinnamaldehyde 10c is commercially available, compounds 10a
and 10b have been prepared from the corresponding 2-nitro-5-
substituted-benzaldehyde 9a–b, by reaction with Ph3P]CHCHO
according to the Wittig reaction [45]. 5-Chloro-2-nitrobenzaldehyde
Table 1 shows the in vitro inhibition percentage of nNOS and
iNOS isoforms produced by
a 1 mM concentration of each
compound 8a–v, compared with the control assays. In general,
compounds 8a–v behave as weak inhibitors against both isoen-
zyme, and for this reason additional biological assays are not
recommended. Nevertheless some conclusions can be drawn from
the experimental data.
Compounds 8a–f bear a 50-methoxy substituent (R1 ¼ OMe) in
the benzene ring. Among them, 8a and 8b (R2 ¼ H, Me) do not
inhibit nNOS. An increment in the volume of N-caboxamide
substituent increases the inhibition percentage, compounds 8c
(R2 ¼ Pr, 43%) and 8d (R2 ¼ c-C3H5, 32%) being the best inhibitors in
this series of compounds. In compounds 8e and 8f (R1 ¼ OMe,

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L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
Scheme 1. General synthetic pathway followed in the preparation of compounds 8a–v. a): Ph3P]CHCHO; b): N3CH2CO2Et, OHꢀ; c): thermolysis, p-xylene; d): MeI/OHꢀ, 18-crown-
6; e): i) NaOH 1 N; ii) AcOH; f): i) SOCl2; ii) R3NH2/TEA, CH2Cl2; g) NH4Cl/NH4OH; h): Fe/FeSO4.
R2 ¼ c-C5H9, CH2Ph), a decreasing of the nNOS inhibition can be
observed again.
(R2 ¼ Pr, 33%) and 8j (R2 ¼ Bu, 36%) also show good inhibition
percentage, indicating that a R3 must be a group with a moderate
volume.
Finally, compounds 8l–v show a low inhibition percentage and
some of them produce an activation of the enzyme. This fact indi-
cates that a non-substituted benzene moiety is detrimental for the
nNOS inhibition activity.
Table 1 also shows the iNOS inhibition observed in the presence
of 1 mM concentration of compounds 8. Unfortunately, no clear
relationship between the structure and the activity can be
observed. Since compounds 8g–k do not inhibit iNOS, it seems that
Compounds 8g–k bear a 50-chloro substituent in the benzene
ring (R1 ¼ Cl) and show higher nNOS inhibition percentage, indi-
cating that this substituent is the better one for the inhibition of
this enzyme. The importance of the 50-chloro substituent for the
nNOS inhibition has been previously described in compounds 4
[38], 5 [39], and 6–7 [40], since the better nNOS inhibitors
belonging to these families of compounds have such substituent in
its benzene moiety. Compound 8k with an N-cyclopropyl substit-
uent is the best inhibitor in this series (48%). Compounds 8i
Table 1
In vitro nNOs and iNOS inhibition (%) observed in the presence of 1 mM concentration of compounds 8a–v.
Compound
Codea
R1
R2
R3
% nNOS inhibitionb
% iNOS inhibitionb
8a
8b
8c
8d
8e
8f
8g
8h
8i
QFF193
QFF194
QFF195
QFF196
QFF197
QFF198
QFF199
QFF200
QFF201
QFF202
QFF203
QFF204
QFF205
QFF206
QFF207
QFF208
QFF210
QFF211
QFF212
QFF213
QFF209
QFF214
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Me
Pr
c-C3H5
c-C5H9
CH2Ph
H
Me
Et
Bu
c-C3H5
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
ꢀ18.14 ꢁ 2.01
ꢀ47.97 ꢁ 2.6
43.45 ꢁ 3.38
32.54 ꢁ 2.63
15.33 ꢁ 2.42
8.89 ꢁ 1.13
8.95 ꢁ 0.50
26.17 ꢁ 6.85
6.78 ꢁ 3.93
21.36 ꢁ 4.68
26.25 ꢁ 3.22
22.41 ꢁ 1.94
5.34 ꢁ 2.34
1.27 ꢁ 3.39
2.8 ꢁ 1.75
34.48 ꢁ 0.99
17.11 ꢁ 0.74
33.40 ꢁ 2.46
36.46 ꢁ 4.13
48.07 ꢁ 1.30
15.01 ꢁ 2.66
ꢀ12.79 ꢁ 0.21
4.15 ꢁ 0.97
8j
8k
8l
3.33 ꢁ 1.27
7.20 ꢁ 0.31
3.07 ꢁ 1.79
32.68 ꢁ 2.78
20.49 ꢁ 5.19
13.17 ꢁ 5.2
7.53 ꢁ 2.76
ꢀ1.1 ꢁ 1.75
20.1 ꢁ 4.78
52.79 ꢁ 1.7
17.20 ꢁ 7.54
28.11 ꢁ 2.39
15.97 ꢁ 1.62
8m
8n
8o
8p
8q
8r
8s
8t
Pr
Bu
ꢀ12.93 ꢁ 2.17
ꢀ0.01 ꢁ 1.80
2.92 ꢁ 0.87
8.44 ꢁ 1.87
5.36 ꢁ 3.19
c-C3H5
c-C4H7
c-C5H9
c-C6H11
CH2Ph
H
H
H
H
13.40 ꢁ 0.46
7.52 ꢁ 2.50
9.56 ꢁ 1.19
8u
8v
a
Internal code used in the identification of each compound.
Data represent the mean ꢁ SEM of the percentage of nNOS and iNOS inhibition produced by 1 mM concentration of each compound. Each value is the mean of three
b
experiments performed by triplicate in homogenates of four rat striata in each one.

L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
2659
a 50-Cl substituent is detrimental for the activity and consequently
a 50-MeO substituent or an unsubstituted benzene ring are
preferable.
Table 2
Total NOS activities (pmol
in both the cytosol and mitochondria cell fractions isolated from the substantia nigra
(SN) in mice treated with MPTP, MPTP/melatonin (aMT), MPTP/QFF-205 (8m) and
MPTP/QFF-212 (8s).
L
-[3H]-citrulline ꢂ (mg of protein)ꢀ1 ꢂ minꢀ1) measured
Regarding the influence of the N-substituent over the activity,
the available information is also confused: compounds 8b and 8m
(R2 ¼ Me) or 8n (R2 ¼ Et) show moderate inhibition percentages;
compound 8d (R1 ¼ MeO, R2 ¼ c-C3H5) also show a moderate
inhibition, but compound 8q (R1 ¼ Cl, R2 ¼ c-C3H5) does not inhibit
iNOS. On the other hand, a further increase in the R2 volume gives
place to an increment in the inhibition activity. Compounds 8e
(26%, R1 ¼ MeO, R2 ¼ c–C5H9), 8f (22%, R1 ¼ MeO, R2 ¼ CH2Ph), and
8u (28%, R1 ¼ H, R2 ¼ CH2Ph) show moderate inhibition, and
compound 8s (52%, R1 ¼ H, R2 ¼ c-C5H9) is the best inhibitor in all
the series.
In contrast with what can be expected from the docking studies,
compounds 8a–v behave as weak NOS inhibitors, and this can be
due to two different reasons. The first one is related with the fact
that the scoring function used in Glide could not properly evaluate
the interaction between the iron atom of the heme group and the
benzene ring of the ligand, giving poor predictions for the score of
each complex. The second one is that these molecules could act as
non-competitive inhibitors.
Regarding the second possibility, we have found that melatonin
1 [34], kynurerine 2 (R ¼ Me) [34], and AMK (3, R1 ¼ OMe, R2 ¼ Me)
[37] behave as non-competitive nNOS inhibitors. We have also
described that the incorporation of increasing amounts of calmod-
ulin (CaM) in the incubation medium resulted in a progressive loss
of the efficiency to inhibit nNOS. On the other hand, these molecules
bound Ca–CaM complex, indicating that this interaction is respon-
sible of nNOS inhibition.
Compound
Cytosol
nNOS
Mitochondria
c-mtNOS
iNOS
i-mtNOS
Control
MPTP
aMT
8m
95.44 ꢁ 0.86
83.65 ꢁ 0.37
19.91 ꢁ 1.93
65.53 ꢁ 1.84
74.35 ꢁ 1.93
2.82 ꢁ 0.09
29.37 ꢁ 0.68
19.02 ꢁ 1.73
5.78 ꢁ 0.84
6.53 ꢁ 0.29
28.64 ꢁ 0.62
25.86 ꢁ 0.54
13.75 ꢁ 0.32
9.95 ꢁ 0.98
14.1 ꢁ 1.83
22.08 ꢁ 0.12
43.75 ꢁ 0.97
23.60 ꢁ 2.18
7.46 ꢁ 0.98
9.45 ꢁ 0.81
8s
also inhibits the mitochondrial i-mtNOS activity and expression
[21,51] and the nNOS activity [52] It has also found that melatonin
shows neuroprotection properties in different models, including
MPTP-induced Parkinsonism [28a,53].
For these reasons, the more active iNOS inhibitors 8m and 8s
have been selected to test their ability to reduce the in vivo NOS
activity in both the cytosol and the mitochondria in the substantia
nigra (SN) of the MPTP Parkinson’s disease. Table 2 shows the total
experimental NOS activities found in each cell fraction, and Fig. 2
shows the NOS relative activities in both cell fractions considering
the NOS activities in control animals as 100%.
MPTP administration (see Section
4 for details) slightly
decreases the nNOS (83.6 ꢁ 0.4 vs. 95.4 ꢁ 0.9 pmol/min/mg prot)
and c-mtNOS (25.9 ꢁ 0.5 vs. 28.6 ꢁ 0.6 pmol/min/mg prot) activi-
ties in cytosol and mitochondria, respectively. Melatonin adminis-
tration before the MPTP treatment significantly reduced nNOS
(19.9 ꢁ 1.9 vs. 83.6 ꢁ 0.4 pmol/min/mg prot) and c-mtNOS
(13.7 ꢁ 0.3 vs. 25.9 ꢁ 0.5 pmol/min/mg prot) activities in MPTP-
treated mice (Table 2). Administration of compounds 8m and 8s
reduced the nNOS activity in a lesser extent than melatonin, but
they provoke a stronger reduction in c-mtNOS activity than that
produced by melatonin (Table 2).
The iNOS activity present in the cytosol of control animals is
almost undetectable, while MPTP administration increased it up to
10 times (29.4 ꢁ 0.7 vs. 2.8 ꢁ 0.1 pmol/min/mg prot). Melatonin
administration partially counteracted the effect of MPTP on iNOS
activity (19.0 ꢁ 1.7 pmol/min/mg prot), while administration of
compounds 8m and 8s significantly reduces the MPTP-induced
iNOS activity to control values (5.8 ꢁ 0.8 and 6.5 ꢁ 0.3 pmol/min/
mg, respectively). On the other hand, MPTP increased 2 times the i-
mtNOS activity (43.7 ꢁ 1.0 vs. 22.1 ꢁ0.1 pmol/min/mg prot),
whereas melatonin absolutely prevented this effect of MPTP
Compounds 8a–v do not bind calmodulin (data not shown) and
this mechanism seem to be not applicable to the inhibition of nNOS
by these molecules.
iNOS is not regulated by CaM, since CaM is bound to it with high
affinity and functions as a permanent enzyme subunit. For this
reason, interaction of compounds 8a–v with CaM is not a suitable
mechanism for iNOS inhibition.
Nevertheless, the contrast between docking studies and in vitro
experimental values seems to suggest another type of mechanism,
probably due to the interaction with other part of the enzyme.
2.4. In vivo assays
It has been described that melatonin 1 reduces the iNOS activity
and expression in several inflammatory models [50]. Melatonin
Fig. 2. Relative NOS activities (%) measured in both the cytosol (up) and mitochondria (down) cell fractions isolated from the substantia nigra (SN) in mice treated with MPTP,
MPTP/melatonin (aMT), MPTP/QFF-205 (8m) and MPTP/QFF-212 (8s). C represents control animals treated with the vehicle (ethanol/saline). Data represents means ꢁ SEM of seven
experiments performed by triplicate in homogenates of four SN in each one. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control.

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(23.6 ꢁ 2.2 pmol/min/mg prot). Interestingly, compounds 8m and
8s were much more efficient than melatonin in reducing i-mtNOS
activity in MPTP-treated animals (7.5 ꢁ1.0 and 9.4 ꢁ 0.8 pmol/min/
mg prot, respectively) (Table 2).
4.1.1. 5-Methoxy-2-nitrocinnamaldehyde, 10a
(87%); mp. 123–125 ꢃC; MS (LSIMS) m/z 230.0427 (M þ Na)þ,
Calcd. Mass for C10H9NO4Na 230.0429.
Since compounds 8m and 8s are not potent iNOS in vitro
inhibitors, it seems that a direct interaction with this enzyme is not
the molecular mechanism for the observed in vivo activities. More in
deep studies are needed in order to clarify the biological mechanism
by which these molecules diminish the NOS activities in vivo. At
present we are testing two possibilities: i) Compounds 8m and 8s
can be metabolized so that their in vivo activity can be due to
a common metabolite that interacts with iNOS, and ii) These
molecules, instead of a direct blockade of the NOS activity could
modify the genomic expression of iNOS (or i-mtNOS), diminishing
by this indirect route the activity of these enzymes. The last possi-
bility seems to be more probable. In fact, melatonin exerts some of
its functions through its interaction with ROR/RZR nuclear receptors
[28a,54] and compounds 8a–v could behave in a similar way.
Even if the molecular mechanism is still unknown, compounds
8m and 8s selectively decrease the NOS activity due to the induc-
ible isoforms of this enzyme in both cytosol and mitochondria.
Since i-NOS and i-mtNOS are those that suffer higher alteration in
several physiological disorders, the potentiality of these molecules
in the development of compounds with interesting pharmacolog-
ical properties is clear.
4.1.2. 5-Chloro-2-nitrocinnamaldehyde, 10b
(84%); mp. 180–181 ꢃC; MS (LSIMS) m/z 233.9942 (M þ Na)þ,
Calcd. Mass for C9H6NO3ClNa 233.9933.
4.2. Preparation of a-azido-5-(2-nitro-5-substituted-phenyl)-2,4-
pentadienoic acid ethyl esther 11a–c. General method
To stirred solution of ethyl azidoacetate (69.12 mmol) and the
appropriate 2-nitro-5-substituted-cinnamadehyde 10a–c (13.
43 mmol) in dry ethanol, a solution of sodium ethanolate (70 mmol
of Na in 60 mL of dry ethanol) was added dropwise. The reaction
mixture was stirred under argon atmosphere at ꢀ20 ꢃC for 4.5 h,
and then poured into water. The aqueous mixture was extracted
with ethyl acetate (3 ꢂ 50 mL), and the combined organic layers
were dried (Na2SO4), filtered, and concentrated to yield a crude
material which was recrystallized from methanol/diethyl ether or
ethyl acetate/hexane. Compounds 11a–c are unstable and sponta-
neously tend to cyclize to the 2-arylpyrroles derivatives, and these
compounds were not isolated for this reason.
4.3. Preparation of 5-(2-nitro-5-substituted-phenyl)-1H-pyrrole-2-
carboxylic acid ethyl ester 12a–c. General method
3. Conclusions
The appropriated azides 11a–c were suspended in p-xilene and
In summary, the more interesting findings in this paper are two:
i) compounds 8m and 8s show selectivity in the in vitro inhibition
of the iNOS isoform; and ii) both compounds produce a strong
reduction of both the iNOS (cytosol) and i-mtNOS (mitochondria) in
vivo activities induced by the toxin administration in the MPTP
Parkinson model.
heated (60–125 ꢃC) for 7–24 h. Evaporation of the solvent allows to
obtain
a solid that was recrystallized or purified by flash
chromatography.
4.3.1. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
ethyl ester 12a
(80 ꢃC, 12 h, ethyl acetate:hexane 1:5, 95%); mp 146–148 ꢃC; 1H-
4. Experimental section
NMR ((CD3)2CO):
1H), 6.89 (dd, 1H), 6.34 (dd, 1H), 3.96 (s, 1H); 13C-NMR (CD3)2CO):
162.70, 161.22, 141.78, 132.31, 129.55, 127.04, 124.48, 116.56,
115.78, 114.10, 110.59, and 55.79; MS (LSIMS) m/z 313.0803
(M þ Na)þ, Calcd. Mass for C14H14N2O5Na 313.0800.
d 11.15 (bs, 1H), 7.99 (d,1H), 7.17 (d, 1H), 7.08 (dd,
Melting points were determined using an Electrothermal-1A-
6301 apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra
were recorded on a Bruker AMX 300 spectrometer operating at
75.479 MHz for 13C and 300.160 for 1H and on a Bruker ARX 400
spectrometer operating at 400.132 MHz for 1H and 100.623 MHz
for 13C, in CDCl3, CD3OD, (CD3)2 CO or DMSO-d6 (at concentration of
ca. 27 mg mlꢀ1 in all cases). The center of each peak of CDCl3
[7.26 ppm (1H) and 77.0 ppm (13C)] was used as an internal refer-
ence 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
d
4.3.2. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
ethyl ester 12b
(60 ꢃC, 8 h, recrystallized from methanol/diethyl ether, 96%); mp
121–123 ꢃC; 1H-NMR ((CD3)2CO):
d
11.30 (bs, 1H), 7.99 (d, 1H), 7.82
(d, 1H), 7.65 (dd, 1H), 6.92 (dd, 1H, dd), 6.41 (dd, 1H); 13C-NMR
(CD3OD): 161.67, 147.97, 138.52, 131.98, 130.83, 130.72, 129.50,
d
reported in ppm (
d
). High-resolution mass spectroscopy (HRMS)
128.89, 126.83, 116.70, and 111.82; MS (LSIMS) m/z 317.0298
(M þ Na)þ, Calcd. Mass for C13H11N2O4ClNa 317.0305.
was carried out on a VG AutoSpec Q high-resolution mass spec-
trometer (Fisons Instruments). Elemental analyses were performed
on a Perkin Elmer 240 ꢃC and agreed with theoretical values
within ꢁ 0.4%. Flash chromatography was carried out using silica
gel 60, 230–240 mesh (Merck), and the solvent mixture reported
within parentheses was used as an eluent.
4.3.3. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid ethyl
ester 12c
(125 ꢃC, 24 h recrystallized from dichloromethane/hexane,
91%); mp 150–151 ꢃC; 1H-NMR ((CD3)2CO):
d
11.35 (bs, 1H), 7.87 (d,
1H), 7.66 (m, 2H), 7.53 (pt, 1H), 6.90 (dd, 1H), 6.25 (dd, 1H); 13C-
NMR ((CD3)2CO): 164.14, 150.22, 133.37, 132.95, 132.50, 129.84,
4.1. Preparation of 2-nitro-5-substituted-cinnamadehyde 10a–b.
d
General method
127.74, 125.78, 125.09, 117.39, and 111.31; MS (LSIMS) m/z 261.0879
(M þ H)þ, Calcd. Mass for C13H13N2O4 261.0875.
2-Nitro-5-substituted-benzaldehyde 9a,b (8.29 mmol) was
added to
a
solution of 8.29 mmol of (triphenylphosphor-
4.4. Preparation of 1-methyl-5-(2-nitrophenyl)-1H-pyrrole-2-
carboxylic acid ethyl ester 12d
anylidene)acetaldehyde in dichloromethane (25 mL). The mixture
was stirred at room temperature between 8 and 24 h, under argon
atmosphere. After this period, the mixture was concentrated to
dryness, and the obtained solid was purified by flash chromatog-
raphy (ethyl acetate:hexane 1:50).
Potassium tert-butoxide (11.6 mmol) was added to a solution of
18-crown-6 (1 mmol) in 20 mL of dry ether, and the mixture was
stirred 15 min. After that, 0.5 mmol of 12c was added, and reaction

L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
2661
mixture was stirred for another 15 min. Then, a solution of CH3I
(0.139 mmol) in 5 mL of ethyl ether was added dropwise at 0 ꢃC,
and the reaction mixture was stirred for 18 h at room temperature.
Finally, the reaction mixture was washed with water (3 ꢂ 10 mL)
and the aqueous layers extracted with ethyl acetate (3 ꢂ 10 mL).
The combined organic layer was dried (Na2SO4), filtered and
concentrated to obtain brown oil that was identified as compound
12d.
The reaction mixture was stirred for 3 h at room temperature,
washed with H2O several times and the combined aqueous layers
extracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic layers
were dried (Na2SO4), filtered, concentrated and the residue
recrystallized or purified by flash chromatography.
4.7.1. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
methylamide 14b
(91%); mp 137–139 ꢃC; 1H-NMR (CDCl3):
d
10.60 (bs, 1H), 7.90 (d,
1H), 6.99 (d, 1H), 6.87 (dd, 1H), 6.54 (pt, 1H), 6.30 (pt, 1H), 6.02 (m,
1H), 3.86 (s, 3H), 2.85 (d, 3H). 13C-NMR (CDCl3):
162.50, 161.72,
4.5. 1-Methyl-5-(2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
ethyl ester 12d
d
141.68, 130.37, 129.55, 127.47, 127.26, 116.39, 113.55, 111.12, 109.71,
56.05, and 26.21; MS (LSIMS) m/z 298.0801 (M þ Na)þ, Calcd. Mass
for C13H13N3O4Na 298.0803.
(92%); 1H-NMR (CDCl3):
1H), 7.46 (dd, 1H), 7.04 (d, 1H), 6.13 (d, 1H), 4.33 (q, 2H), 3.72 (s, 1H),
1.39 (t, 3H); 13C-NMR (CDCl3):
161.42, 149.85, 135.72, 133.41,
d 8.06 (dd, 1H), 7.70 (pt, 1H,), 7.62 (pt,
d
132.77, 129.88, 127.20, 124.45, 124.03, 117.39, 109.19, 59.98, 33.96,
and 14.51; MS (LSIMS) m/z 297.0853 (M þ Na)þ, Calcd. Mass for
C14H14N2O4Na 297.0851.
4.7.2. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
propylamide 14c
(82%); mp 141–143 ꢃC; 1H-NMR (CDCl3):
d 10.45 (bs, 1H), 7.90 (d,
1H), 6.99 (d,1H), 6.88 (dd,1H), 6.55 (d,1H), 6.32 (d,1H), 5.94 (bs,1H),
4.6. Preparation of 5-(2-nitro-5-substituted-phenyl)-1H-pyrrole-2-
carboxylic acid. 13a–c. General method
3.87 (s, 3H), 3.27 (m, 2H),1.54 (m, 2H), 0.91 (t, 3H); 13C-NMR (CDCl3):
d
162.51, 160.99, 141.73, 130.29, 129.49, 127.57, 127.27, 116.23, 113.63,
111.16, 109.49, 56.05, 41.23, 23.08, and 11.42; MS (LSIMS) m/z
326.1114 (M þ Na)þ, Calcd. Mass for C15H17N3O4Na 326.1116.
The appropriated ethyl 5-(2-nitro-5-substituted-phenyl)-1H-
pyrrole-2-carboxylate 12a–c (2.04 mmol) was stirred and dissolved
in 1 N NaOH solution (4.08 mmol) at 100 ꢃC, glacial AcOH
(4.08 mmol) was then added, and the solution was stirred at room
temperature for 1 h. The solution was extracted with ethyl acetate
(3 ꢂ 50 mL), and the combined organic layers were washed with
water, dried (Na2SO4), filtered, and concentrated to yield a crude
material that was recrystallized from ethyl acetate/hexane.
4.7.3. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclopropylamide 14d
(71%); mp 158–159 ꢃC; 1H-NMR (CDCl3):
d 10.40 (bs,1H), 7.90 (d,
1H), 6.98 (d, 1H), 6.87 (dd, 1H), 6.52 (bs, 1H), 6.30 (pt, 1H), 6.13 (bs,
1H), 3.87 (s, 3H), 2.75 (m, 1H); 0.75 (m, 2H), 0.55 (m. 2H); 13C-NMR
(CDCl3):
d 162.54, 141.68, 130.49, 129.41, 127.32, 116.29, 113.63,
111.26, 109.98, 56.07, 22.72, and 6.86; MS (LSIMS) m/z 324.0959
4.6.1. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic
acid 13a
(M þ Na)þ, Calcd. Mass for C15H15N3O4Na 324.0960.
(82%); mp 201–203 ꢃC; 1H-NMR ((CD3)2CO):
d
11.15 (bs, 1H);
7.99 (d, 1H); 7.17 (d, 1H); 7.08 (dd, 1H) 6.89 (dd, 1H); 6.34 (dd, 1H);
3.96 (bs, 1H); 13C-NMR ((CD3)2CO):
162.70, 161.22, 141.78, 132.31,
4.7.4. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclopentylamide 14e
d
(90%); mp 181–183 ꢃC; 1H-NMR (CDCl3):
d 10.70 (bs,1H), 7.89 (d,
129.55, 127.04, 124.48, 116.56, 115.78, 114.10, 110.59, and 55.79; MS
(LSIMS) m/z 285.0486 (M þ Na)þ, Calcd. Mass for C12H10N2O5Na
285.0487.
1H), 6.98 (d, 1H), 6.87 (dd, 1H), 6.52 (bs, 1H), 6.30 (bs, 1H), 5.87 (d,
1H), 4.17 (m, 1H), 3.86 (s, 3H), 1.95–1.36 (m, 8H); 13C-NMR (CDCl3):
d
162.48, 160.69, 141.66, 130.40, 129.55, 127.59, 127.20, 116.18,
113.63, 111.03, 109.62, 56.04, 51.27, 33.25, and 23.81; MS (LSIMS) m/
z 352.1273 (M þ Na)þ, Calcd. Mass for C17H19N3O4Na 352.1273.
4.6.2. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
13b
(93%); mp 197–199 ꢃC; 1H-NMR ((CD3)2CO):
d
11.30 (bs,1H), 7.99
4.7.5. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
benzylamide 14f
(d, 1H), 7.82 (d, 1H), 7.65 (dd, 1H), 6.92 (dd, 1H), 6.41 (dd, 1H); 13C-
NMR (CD3OD):
d
161.67, 147.97, 138.52, 131.98, 130.83, 130.72,
(90%); mp 181–182 ꢃC; 1H-NMR ((CD3)2CO):
d 11.27 (bs,1H), 7.96
129.50, and 128.89; MS (LSIMS) m/z 288.998 (M þ Na)þ, Calcd. Mass
(bs, 1H); 7.92 (d, 1H), 7.25 (m, 5H), 7.18 (d, 1H), 7.00 (dd, 1H), 6.88
for C11H7N2O4ClNa 288.9992.
(dd, 1H), 6.28 (dd, 1H), 4.49 (d, 2H), 3.95 (s, 3H); 13C-NMR
((CD3)2CO):
d 162.49, 160.72, 141.82, 139.90, 130.49, 129.56, 128.34,
4.6.3. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid 13c
128.23, 127.57, 126.86, 116.30, 113.60, 110.42, 110.15, 55.71, and
42.72; MS (LSIMS) m/z 374.1117 (M þ Na)þ, Calcd. Mass for
C19H17N3O4Na 374.1116.
(93%); mp 221–222 ꢃC; 1H-NMR ((CD3)2CO):
d
11.35 (bs, 1H),
7.87 (d, 1H), 7.66 (m, 2H), 7.53 (pt, 1H), 6.90 (dd, 1H), 6.25 (dd, 1H);
13C-NMR ((CD3)2CO):
164.14, 150.22, 133.37, 132.95, 132.50,
d
129.84, 127.74, 125.78, 125.09, 117.39, and 111.31; MS (LSIMS) m/z
255.0379 (M þ Na)þ, Calcd. Mass for C11H8N2O4Na 255.0381.
4.7.6. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
methylamide 14h
(79%); mp 191–193 ꢃC; 1H-NMR ((CD3)2CO):
d
11.45 (bs,1H), 7.92
(d, 1H), 7.79 (d, 1H), 7.57 (dd, 1H), 7.52 (bs, 1H), 6.78 (dd, 1H), 6.32
(dd, 1H), 2.78 (d, 3H); 13C-NMR ((CD3)2CO):
162.47, 148.51, 138.77,
4.7. Preparation of 5-(2-nitro-5-substituted-phenyl)-1H-pyrrole-2-
carboxylic acid alkylamide 14b–f, h–k, m–u. General method
d
132.17, 130.70, 129.69, 129.37, 129.28, 127.12, 111.96, 111.60, and
26.55; MS (LSIMS) m/z 280.0485 (M þ H)þ, Calcd. Mass for
C12H11N3O3Cl 280.0489.
SOCl2 (11 mmol) was added to a solution of 5-(2-nitro-5-
substituted-phenyl)-1H-pyrrole-2-carboxylic acid 13a–c (1 mmol)
in dry CH3CN (30 mL), and the reaction mixture was stirred at 65–
80 ꢃC for 5 h. After this period, the mixture was concentrated to
dryness, yielding a brown solid (the acyl chloride) that was solved
in CH2Cl2 (10 mL), and a solution of the appropriated amine (R3NH2,
2 mmol) and TEA (3 mmol) in CH2Cl2 (3 mL) was added dropwise.
4.7.7. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
ethylamide 14i
(64%); mp 143–145 ꢃC; 1H-NMR (CDCl3):
d
10.78 (bs,1H), 7.75 (d,
1H), 7.56 (d, 1H), 7.36 (dd, 1H), 6.55 (dd, 1H), 6.35 (dd, 1H), 3.91 (bs,

2662
L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
1H), 3.35 (m, 2H), 1.16 (t, 3H); 13C-NMR (CDCl3):
d
160.82, 146.74,
1H), 2.86 (m,1H), 0.65–0.52 (m, 4H); 13C-NMR ((CD3)2CO):
d 162.49,
138.47, 131.04, 128.46, 128.36, 128.30, 128.10, 125.83, 111.67, 109.92,
34.51, and 15.04; MS (LSIMS) m/z 316.0472 (M þ Na)þ, Calcd. Mass
for C13H12N3O3ClNa 316.04648.
149.65, 132.92, 132.03, 130.16, 129.49, 129.12, 127.17, 124.63, 111.24,
110.65, 23.21, and 6.40; MS (LSIMS) m/z 294.0855 (M þ Na)þ, Calcd.
Mass for C14H13N3O3Na 294.0854.
4.7.8. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
butylamide 14j
4.7.15. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclobutylamide 14r
(74%); mp 105–107 ꢃC; 1H-NMR (CDCl3):
d
10.40 (bs, 1H), 7.78 (d,
(61%); mp 170–173 ꢃC; 1H-NMR (CDCl3):
d 10.53 (bs, 1H), 7.77 (d,
1H), 7.60 (d, 1H), 7.41 (dd, 1H), 6.58 (d, 1H), 6.40 (d, 1H), 5.93 (bs,
1H), 7.58 (m, 2H), 7.44 (pt, 1H), 6.56 (pt, 1H), 6.32 (pt, 1H); 6.06 (d,
1H), 3.39 (m, 2H), 1.57 (m, 2H), 1.34 (m, 2H), 0.96 (t, 3H); 13C-NMR
1H), 4.40 (m, 1H), 2.27 (m, 2H), 1.89 (m, 2H), 1.67 (m, 2H); 13C-NMR
(CDCl3):
d
160.72, 146.69, 138.57, 130.88, 130.48, 128.36, 128.22,
(CDCl3): d 159.90, 148.73, 132.18, 131.08, 129.73, 128.27, 127.64,
128.17, 125.90, 111.83, 109.73, 39.36, 31.36, 20.17, and 13.84; MS
(LSIMS) m/z 344.0777 (M þ Na)þ, Calcd. Mass for C15H16N3O3ClNa
344.0778.
126.44, 124.21, 110.98, 109.91, 44.69, 31.45, and 15.18; MS (LSIMS)
m/z 286.1192 (M þ H)þ, Calcd. Mass for C15H16N3O3 286.1191.
4.7.16. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclopentylamide 14s
4.7.9. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclopropylamide 14k
(68%), mp 174–175 ꢃC; 1H-NMR (CDCl3):
d
10.52 (bs, 1H), 7.75 (d,
1H), 7.57 (m, 2H), 7.41 (pt, 1H), 6.53 (d, 1H), 6.31 (pt, 1H), 5.86 (d,
1H), 4.20 (m, 1H), 1.99–1.35 (m, 8H); 13C-NMR (CDCl3):
160.56,
(80%); mp 164–166 ꢃC; 1H-NMR (CDCl3):
d
10.50 (bs,1H), 7.74 (d,
1H), 7.58 (ps, 1H), 7.37 (dd, 1H), 6.52 (bs, 1H), 6.34 (bs, 1H), 6.09 (bs,
1H), 2.78 (m, 1H), 0.78–0.57 (m, 4H); 13C-NMR (CDCl3):
163.53,
d
d
148.73, 132.16, 131.08, 129.56, 128.20, 127.94, 126.42, 124.18, 110.91,
109.74, 51.24, 33.27, and 23.80; MS (LSIMS) m/z 322.1168 (M þ Na)þ,
Calcd. Mass for C16H17N3O3Na 322.1167.
148.07, 139.86, 132.25, 129.82, 129.51, 129.42, 127.18, 113.17, 111.53,
24.07, and 8.24; MS (LSIMS) m/z 328.0465 (M þ Na)þ, Calcd. Mass
for C14H12N3O3ClNa 328.0465.
4.7.17. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclohexylamide 14t
4.7.10. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
methylamide 14m
(80%); mp 171–172 ꢃC; 1H-NMR (CDCl3):
d 10.55 (bs, 1H), 7.75 (d,
(76%); mp 163–164 ꢃC; 1H-NMR (DMSO-d6):
d
11.86 (bs, 1H),
8.07 (q, 1H), 7.90 (d, 1H), 7.83 (m, 2H), 7.51 (ddd, 1H), 6.77 (dd, 1H),
6.16 (dd, 1H), 2.74 (d, 3H); 13C-NMR (DMSO-d6):
160.72, 147.83,
1H), 7.56 (m, 2H), 7.41 (pt,1H), 6.54 (d,1H), 6.32 (d,1H), 5.78 (d,1H),
3.78 (m, 1H), 1.92–1.58 (m, 5H), 1.38–1.09 (m, 5H); 13C-NMR
d
(CDCl3): d 160.04, 148.70, 132.17, 131.01, 129.53, 128.19, 128.03,
132.29, 131.22, 128.83, 128.48, 128.15, 126.00, 123.84, 110.66, 109.15,
and 25.46; MS (LSIMS) m/z 268.0699 (M þ Na)þ, Calcd. Mass for
C12H11N3O3Na 268.0698.
126.46, 124.21, 110.90, 109.66, 48.22, 33.35, 25.57, and 24.96; MS
(LSIMS) m/z 336.3434 (M þ Na)þ, Calcd. Mass for C17H19N3O3Na
336.3441.
4.7.11. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
ethylamide 14n
4.7.18. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
benzylamide 14u
(48%); mp 207–208 ꢃC; 1H-NMR (DMSO-d6):
d
11.86 (bs, 1H),
8.10 (t, 1H), 7.91 (d, 1H), 7.66 (m, 2H), 7.51 (pt, 1H), 6.80 (pt,1H), 6.15
(pt, 1H), 3.24 (m, 2H), 1.10 (t, 3H); 13C-NMR (DMSO-d6):
160.01,
(70%); mp 137–138 ꢃC; 1H-NMR (CD3OD):
d
7.82 (d, 1H), 7.65 (m,
2H), 7.51 (pt, 1H), 7.32 (m, 5H), 6.88 (d, 1H), 6.26 (d, 1H), 5.78 (bs,
1H), 4.54 (s, 2H); 13C-NMR (CD3OD):
160.20,147.86,139.77,132.34,
d
d
147.86, 132.31, 131.22, 128.85, 128.57, 128.17, 126.02, 123.85, 110.77,
109.13, 33.28, and 14.98; MS (LSIMS) m/z 282.0855 (M þ Na)þ,
Calcd. Mass for C13H13N3O3Na 282.0854.
131.26, 129.20, 128.26, 128.16, 128.16, 127.10, 126.63, 125.95, 123.87,
111.21, 109.24, and 41.84; MS (LSIMS) m/z 344.1020 (M þ Na)þ,
Calcd. Mass for C18H15N3O3Na 344.1011.
4.7.12. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
propylamide 14o
4.8. Preparation of 5-(2-nitro-5-substituted-phenyl)-1H-pyrrole-2-
carboxamide 14a, g, l, v. General method
(65%); mp 187–189 ꢃC; 1H-NMR (CD3OD):
d
7.83 (d, 1H), 7.65 (m,
2H), 7.52 (ddd, 1H), 6.82 (d, 1H), 6.25 (d, 1H), 3.31 (m, 2H), 1.62 (m,
2H), 0.98 (t, 3H); 13C-NMR (CDCl3):
160.79, 148.73, 132.34, 130.84,
5-(2-Nitro-5-substituted-phenyl)-1H-pyrrole-2-carboxylic acid
ethyl ester 12a–d (1.92 mmol) and NH4Cl (27.4 mmol) were added
to a concentrated NH4OH solution (60 mL), and the mixture was
heated overnight at 100 ꢃC in a pressure reactor. After cooling, the
resulting precipitate was filtered off and the aqueous solution was
extracted with AcOEt (3 ꢂ 30 mL). The combined organic layers
were dried (MgSO4), filtered and concentrated to yield a solid that
was recrystallized (AcOEt/C6H12).
d
129.33, 128.41, 127.83, 126.27, 124.34, 111.14, 109.59, 41.27, 23.15,
and 11.49; MS (LSIMS) m/z 296.1013 (M þ Na)þ, Calcd. Mass for
C14H15N3O3Na 296.1011.
4.7.13. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
butylamide 14p
(82%); mp 169–170 ꢃC; 1H-NMR ((CD3)2CO):
d 11.29 (bs,1H), 7.86
(dd, 1H), 7.77 (dd, 1H), 7.69 (pt, 1H), 7.55 (pt, 1H), 7.49 (bs, 1H), 6.79
(dd, 1H), 6.24 (dd, 1H), 3.27 (m, 2H), 1.49 (m, 2H), 1.32 (m, 2H), 0.87
4.8.1. 5-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-2-carboxamide
14a
(t, 3H); 13C-NMR ((CD3)2CO):
d
160.55, 148.90, 132.18, 131.32,
(94%); mp 123–125 ꢃC; 1H-NMR ((CD3)2CO):
d
11.98 (bs,1H), 7.95
(d, 1H), 7.20 (d, 1H), 7.05 (dd, 1H), 6.87 (dd, 1H), 6.30 (dd, 1H), 3.99
(s, 3H); 13C-NMR ((CD3)2CO):
162.47,162.09,141.73,130.23,129.36,
129.30, 128.94, 128.34, 126.48, 123.89, 110.18, 109.88, 38.66, 31.89,
19.92, and 13.30; MS (LSIMS) m/z 310.1168 (M þ Na)þ, Calcd. Mass
for C15H17N3O3Na 310.1167.
d
128.16, 126.80, 115.98, 113.70, 111.07, 110.12, and 55.67; MS (LSIMS)
m/z 284.0645 (M þ Na)þ, Calcd. Mass for C12H11N3O4Na 284.0647.
4.7.14. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxylic acid
cyclopropylamide 14q
4.8.2. 5-(5-Chloro-2-nitrophenyl)-1H-pyrrole-2-carboxamide 14g
(80%); mp 214–215 ꢃC; 1H-NMR ((CD3)2CO):
d
11.50 (bs, 1H), 7.86
(81%); mp 220–222 ꢃC; 1H-NMR ((CD3)2CO):
d
11.22 (bs,1H), 7.93
(d, 1H), 7.77 (d, 1H), 7.69 (t, 1H), 7.54 (m, 2H), 6.76 (d, 1H), 6.24 (d,
(d, 1H), 7.83 (d, 1H), 7.56 (dd, 1H), 7.30 (bs, 1H), 6.56 (bs, 1H), 6.90

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2663
(dd,1H), 6.30 (dd,1H); 13C-NMR ((CD3)2CO):
131.56, 129.62, 128.92, 126.62, 112.23, and 111.41; MS (LSIMS) m/z
288.0154 (M þ Na)þ, Calcd. Mass for C11H8N3O3ClNa 288.0152.
d
162.69,147.87,138.28,
118.99, 114.96, 113.69, 110.41, 109.49, 55.89, 22.71, and 7.01; MS
(LSIMS) m/z 294.1219 (M þ Na)þ, Calcd. Mass for C15H17N3O2Na
294.1218; Anal. C15H17N3O2 (C, H, N).
4.8.3. 5-(2-Nitrophenyl)-1H-pyrrole-2-carboxamide 14l
4.9.5. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxylic
acid cyclopentylamide 8e
(90%); mp 137–138 ꢃC; 1H-NMR (CDCl3):
d
9.90 (bs, 1H), 7.80 (d,
1H), 7.59 (m, 2H), 7.45 (pt, 1H), 6.64 (dd, 1H), 6.38 (pt, 1H), 5.68 (bs,
2H); 13C-NMR (CD3CD):
169.51, 149.99, 133.05, 132.92, 131.32,
(55%), mp 185–187 ꢃC; 1H-NMR (DMSO-d6):
d 11.30 (bs, 1H), 7.77
d
(d, 1H), 6.89 (d, 1H), 6.83 (d, 1H), 6.70 (d, 1H), 6.62 (dd, 1H), 6.36 (d,
128.72, 127.81, 124.80, 114.23, and 110.74; MS (LSIMS) m/z 254.0546
(M þ Na)þ, Calcd. Mass for C11H9N3O3Na 254.0542.
1H), 4.56 (bs, 2H), 4.17 (m, 1H), 3.67 (s, 3H), 1.93–1.38 (m, 8H); 13C-
NMR (DMSO-d6):
d 160.47, 151.75, 139.23, 132.99, 127.26, 118.73,
117.96, 114.88, 113.84, 112.18, 108.56, 55.92, 50.89, 32.84, and 24.11;
MS (LSIMS) m/z 322.1532 (M þ Na)þ, Calcd. Mass for C17H21N3O2Na
322.1531; Anal. C17H21N3O2 (C, H, N).
4.8.4. 1-Methyl-5-(2-nitrophenyl)-1H-pyrrole-2-carboxamide 14v
(55%); mp 138–140 ꢃC; 1H-NMR (CD3COD):
d
8.05 (dd, 1H), 7.82
(pt, 1H), 7.73 (pt, 1H), 7.59 (dd, 1H), 6.88 (d, 1H), 6.05 (d, 1H), 3.71 (s,
3H); 13C-NMR (CD3COD):
164.13, 151.14, 134.39, 134.25, 133.55,
d
4.9.6. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxylic
acid benzylamide 8f
130.80, 127.73, 124.84, 113.37, 108.99, and 34.06; MS (LSIMS) m/z
268.0701 (M þ Na)þ, Calcd. Mass for C12H11N3O3Na 268.0698.
(72%); mp 178–180 ꢃC; 1H-NMR (CD3OD):
d
7.28 (m, 5H), 6.91
(m, 2H), 6.80 (d, 1H), 6.71 (dd, 1H), 6.43 (d, 1H), 4.53 (s, 2H), 3.74 (s,
3H); 13C-NMR (CD3OD):
163.58, 154.46, 140.50, 139.05, 135.03,
4.9. Preparation of 5-(2-amino-5-subtituted-phenyl)-1H-pyrrole-
2-carboxylic acid alkylamide 8a–v. General method
d
129.50, 128.47, 128.10, 127.09, 121.14, 119.60, 115.89, 114.70, 113.33,
109.92, 56.21, and 43.93; MS (LSIMS) m/z 344.1380 (M þ Na)þ,
Calcd. Mass for C19H19N3O2Na 344.1375; Anal. C19H19N3O2 (C, H, N).
Fe (5.24 mmol) and FeSO4 (0.524 mmol) were suspended in
water and the corresponding nitroarene 14a–v (0.524 mmol) was
added over the reaction mixture and refluxed from 3 to 5 h. After
cooling, the reaction mixture is filtered through Celite and washed
thoroughly with dichloromethane. The aqueous phase was extracted
with dichloromethane (3 ꢂ15 ml) and ethyl acetate (3 ꢂ 15 ml). The
combined organic layers were washed with brine, dried (Na2SO4),
filtered and concentrated to yield a residue that was purified by
recrystallization (CH2Cl2/C6H12) or by flash chromatography.
4.9.7. 5-(2-Amino-5-chlorophenyl)-1H-pyrrole-2-carboxamide 8g
(59%); mp 197–198 ꢃC; 1H-NMR (CDCl3):
d
9.95 (bs, 1H), 7.31 (d,
1H), 7.08 (dd, 1H), 6.70 (d, 1H), 6.69 (pt, 1H), 6.46 (pt, 1H), 5.70 (bs,
2H), 3.98 (bs, 2H); 13C-NMR ((CD3)2CO):
162.29, 144.19, 131.97,
d
128.24, 127.76, 127.07, 121.94, 119.29, 117.40, 111.56, and 108.86; MS
(LSIMS) m/z 258.9987 (M þ Na)þ, Calcd. Mass for C11H10N3OClNa
258.9992; Anal. C11H10N3OCl (C, H, N).
4.9.1. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxamide
4.9.8. 5-(2-Amino-5-chlorophenyl)-1H-pyrrole-2-carboxylic acid
methylamide 8h
8a
(72%); mp 123–125 ꢃC; 1H-NMR (CDCl3):
d
10.21 (bs, 1H), 6.70
(ps, 1H), 6.90 (m, 2H), 6.67 (pt, 1H), 6.44 (pt, 1H), 5.83 (bs, 2H), 3.73
(s, 3H); 13C-NMR (CDCl3):
162.83, 153.36, 136.99, 134.33, 124.98,
(62%); mp 175–176 ꢃC; 1H-NMR (CDCl3):
d
10.05 (bs, 1H), 7.24 (d,
1H), 7.05 (dd, 1H), 6.68 (d, 1H), 6.58 (dd, 1H), 6.40 (dd, 1H), 5.94 (bs,
1H), 4.00 (bs, 2H), 2.95 (d, 3H); 13C-NMR ((CD3)2CO):
161.29,
d
d
119.68, 118.94, 115.05, 113.72, 111.86, 109.27, and 55.87; MS (LSIMS)
m/z 254.0902 (M þ Na)þ, Calcd. Mass for C12H13N3O2Na 254.0905;
Anal. C12H13N3O2 (C, H, N).
144.20, 131.48, 128.28, 127.67, 127.51, 121.37, 119.37, 117.29, 110.15,
108.75, and 25.21; MS (LSIMS) m/z 272.0566 (M þ Na)þ, Calcd. Mass
for C12H12N3OClNa 272.0566; Anal. C12H12N3OCl (C, H, N).
4.9.2. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxylic
acid methylamide 8b
4.9.9. 5-(2-Amino-5-chlorophenyl)-1H-pyrrole-2-carboxylic acid
ethylamide 8i
(48%); mp 202–204 ꢃC; 1H-NMR (CDCl3):
d
10.05 (bs, 1H), 6.87
(d, 1H), 6.72 (m, 2H), 6.58 (dd, 1H), 6.42 (dd, 1H), 5.98 (bs, 1H), 3.75
(s, 3H), 2.95 (d, 3H); 13C-NMR (CDCl3):
161.79, 153.51, 136.77,
(71%); mp 183–185 ꢃC; 1H-NMR ((CD3)2CO):
d 10.80 (bs, 1H),
7.42 (bs, 1H), 7.32 (d, 1H), 7.02 (dd, 1H), 6.82 (m, 2H), 6.43 (pt, 1H),
d
4.85 (bs, 2H), 3.34 (m, 2H), 1.12 (t, 3H); 13C-NMR ((CD3)2CO):
133.28, 126.07, 119.98, 118.96, 114.94, 113.64, 109.77, 109.03, 55.90,
and 26.27; MS (LSIMS) m/z 268.1066 (M þ Na)þ, Calcd. Mass for
C13H15N3O2Na 268.1062; Anal. C13H15N3O2 (C, H, N).
d 160.60, 144.13, 130.57, 128.19, 127.60, 127.46, 121.28, 119.30, 117.20,
110.23, 108.66, 33.70, and 15.53; MS (LSIMS) m/z 286.0722
(M þ Na)þ, Calcd. Mass for C13H14N3OClNa 286.0723; Anal.
C13H14N3OCl (C, H, N).
4.9.3. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxylic
acid propylamide 8c
4.9.10. 5-(2-Amino-5-chlorophenyl)-1H-pyrrole-2-carboxylic acid
butylamide 8j
(62%); mp 183–184 ꢃC; 1H-NMR (CDCl3):
d 10.00 (bs, 1H), 6.87
(d, 1H), 6.73 (m, 2H), 6.58 (pt, 1H), 6.43 (pt, 1H), 5.93 (bs, 1H), 3.73
(68%); mp 161–163 ꢃC; 1H-NMR (CDCl3):
d 10.09 (bs, 1H), 7.22 (d,
(s, 3H), 3.56 (m, 2H), 1.60 (m, 2H), 0.96 (t, 3H); 13C-NMR (CDCl3):
1H), 7.03 (dd, 1H), 6.66 (d, 1H), 6.58 (pt, 1H), 6.40 (pt, 1H), 5.92 (bs,
1H), 3.38 (m, 2H), 4.00 (bs, 2H), 1.55 (m, 2H), 1.37 (m, 2H), 0.93 (t,
d
161.13, 153.64, 136.00, 133.19, 126.24, 119.13, 114.91, 113.66, 109.73,
109.04, 55.87, 41.26, 23.17, and 11.45; MS (LSIMS) m/z 296.1377
(M þ Na)þ, Calcd. Mass for C15H19N3O2Na 296.1374; Anal.
C15H19N3O2 (C, H, N).
3H); 13C-NMR (CDCl3):
d 160.98, 141.95, 131.80, 128.46, 126.63,
124.02, 119.93, 118.02, 109.66, 109.40, 39.32, 31.99, 20.19, and 13.87;
MS (LSIMS) m/z 314.1037 (M þ Na)þ, Calcd. Mass for C15H18N3OClNa
314.1036; Anal. C15H18N3OCl (C, H, N).
4.9.4. 5-(2-Amino-5-methoxyphenyl)-1H-pyrrole-2-carboxylic
acid cyclopropylamide 8d
d 10.04 (bs, 1H), 6.86
(m, 1H), 6.72 (m, 2H), 6.56 (bs, 1H), 6.40 (pt, 1H), 6.10 (bs, 1H), 3.75
4.9.11. 5-(2-Amino-5-chlorophenyl)-1H-pyrrole-2-carboxylic acid
cyclopropylamide 8k
(77%); mp 195–196 ꢃC; 1H-NMR (CDCl3):
(68%); mp 166–167 ꢃC; 1H-NMR ((CD3)2CO):
d 10.95 (bs, 1H),
13
(s, 3H), 3.75 (bs, 2H), 2.81 (m, 1H), 0.82 (m, 2H), 0.60 (m, 2H);
7.47 (bs, 1H), 7.30 (d, 1H), 7.00 (dd, 1H), 6.82 (m, 2H), 6.41 (dd,
C-NMR (CDCl3):
d 158.85, 153.50, 136.85, 133.50, 125.87, 119.90,
1H), 4.81 (bs, 2H), 2.82 (m, 1H), 0.68–0.49 (m, 4H); 13C-NMR

2664
L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
((CD3)2CO):
d
161.91, 144.18, 131.68, 128.28, 127.70, 127.37, 121.47,
1H), 6.05 (d, 1H), 4.52 (m, 1H), 3.93 (bs, 2H), 2.37 (m, 2H), 1.92 (m,
2H), 1.73 (m, 2H); 13C-NMR (CDCl3):
160.12, 143.71, 133.36, 128.89,
128.84, 125.99, 119.17, 118.41, 116.69, 109.79, 108.94, 44.76, 31.76,
and 15.20; MS (LSIMS) m/z 278.1269 (M þ Na)þ, Calcd. Mass for
C15H17N3ONa 278.1269; Anal. C15H17N3O (C, H, N).
119.42, 117.32, 110.64, 108.75, 22.46, and 5.59; MS (LSIMS) m/z
298.0723 (M þ Na)þ, Calcd. Mass for C14H14N3OClNa 298.0723;
Anal. C14H14N3OCl (C, H, N).
d
4.9.12. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxamide 8l
(64%); mp 104–105 ꢃC; 1H-NMR (CDCl3):
d
9.75 (bs,1H), 7.27 (dd,
1H), 7.11 (pt, 1H), 6.80 (pt, 1H), 6.74 (d, 1H), 6.69 (pt, 1H), 6.43 (pt,
1H), 5.80 (bs, 2H), 4.00 (bs, 2H); 13C-NMR (CDCl3):
157.35, 138.35,
4.9.19. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
cyclopentylamide 8s
d
(69%); mp 163–165 ꢃC; 1H-NMR (CDCl3):
d 9.85 (bs, 1H); 7.27 (d,
128.79, 123.65, 123.54, 119.56, 113.84, 112.81, 111.41, 106.42, and
103.84; MS (LSIMS) m/z 224.0801 (M þ Na)þ, Calcd. Mass for
C11H11N3ONa 224.0799; Anal. C11H11N3O (C, H, N).
1H), 7.11 (pt, 1H), 6.80 (pt, 1H), 6.75 (dd, 1H), 6.58 (dd, 1H), 6.40 (pt,
1H), 5.83 (d, 1H), 4.33 (m, 1H), 4.00 (bs, 2H), 2.04 (m, 2H), 1.66 (m,
2H), 1.45 (m, 2H); 13C-NMR (CDCl3):
d 160.73, 143.72, 133.12, 128.85,
128.82, 126.24, 119.17, 118.41, 116.68, 109.52, 108.87, 51.25, 33.40,
and 23.85; MS (LSIMS) m/z 270.1608 (M þ H)þ, Calcd. Mass for
C16H20N3O 270.1606; Anal. C16H19N3O (C, H, N).
4.9.13. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
methylamide 8m
(68%); mp 155–157 ꢃC; 1H-NMR (CDCl3):
d
9.83 (bs, 1H), 7.28 (d,
1H), 7.12 (pt,1H), 6.82 (t,1H), 6.76 (d,1H), 6.61 (pt,1H), 6.42 (pt,1H),
4.00 (bs, 2H), 5.97 (bs, 1H), 2.97 (d, 3H); 13C-NMR (CDCl3):
161.76,
4.9.20. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
cyclohexylamide 8t
d
143.67, 133.16, 128.86, 126.01, 119.22, 118.35, 116.72, 109.74, 108.95,
and 26.27; MS (LSIMS) m/z 238.0959 (M þ Na)þ, Calcd. Mass for
C12H13N3ONa 238.0958; Anal. C12H13N3O (C, H, N).
(59%); mp 152-154 ꢃC; 1H-NMR (CDCl3):
d 9.98 (bs, 1H), 7.26 (d,
1H), 7.10 (pt, 1H), 6.78 (pt, 1H), 6.76 (dd, 1H), 6.60 (dd, 1H), 6.40 (dd,
1H), 5.83 (d, 1H), 3.85 (bs, 2H), 3.88 (m, 1H), 1.99–1.14 (m, 10H); 13C-
NMR (CDCl3):
d 160.24, 143.36, 133.08, 128.92, 128.77, 126.36,
4.9.14. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
ethylamide 8n
119.32, 118.62, 116.79, 109.63, 108.91, 48.23, 33.45, and 25.00; MS
(LSIMS) m/z 306.1588 (M þ Na)þ, Calcd. Mass for C17H21N3ONa
306.1582; Anal. C17H21N3O (C, H, N).
(48%); mp 145–146 ꢃC; 1H-NMR (CDCl3):
d
10.85 (bs,1H), 7.27 (d,
1H), 7.11 (t, 1H), 6.80 (t, 1H), 6.75 (d, 1H), 6.61 (pt, 1H), 6.41 (pt, 1H),
5.95 (bs, 1H), 3.44 (m, 2H), 1.22 (t, 3H); 13C-NMR (CDCl3):
161.03,
d
4.9.21. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
benzylamide 8u
143.70, 133.19, 128.87, 128.81, 126.10, 119.16, 118.39, 116.67, 109.68,
108.90, 34.40, and 15.18; MS (LSIMS) m/z 252.1113 (M þ Na)þ, Calcd.
Mass for C13H15N3ONa 252.1113; Anal. C13H15N3O (C, H, N).
(52%); mp 178–180 ꢃC; 1H-NMR ((CD3)2CO):
d 11.40 (bs, 1H),
8.55 (t,1H), 7.26 (m, 6H), 7.27 (pt, 1H), 6.89 (d, 1H), 6.82 (d, 1H), 6.68
(pt, 1H), 6.35 (d, 1H), 5.75 (bs, 2H), 4.38 (d, 2H); 13C-NMR 100 MHz,
4.9.15. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
propylamide 8o
((CD3)2CO): d 160.36, 140.00, 132.45, 129.06, 128.31, 128.00, 127.27,
126.76, 126.56, 118.43, 116.68, 111.88, 108.32, and 41.98; MS (LSIMS)
m/z 314.1271 (M þ Na)þ, Calcd. Mass for C18H17N3ONa 314.1269;
Anal. C18H17N3O (C, H, N).
(51%); mp 115–116 ꢃC; 1H-NMR (CDCl3):
d 10.00 (bs, 1H), 7.26
(dd, 1H), 7.09 (pt, 1H), 6.78 (pt, 1H), 6.73 (dd, 1H), 6.62 (dd, 1H), 6.40
(dd, 1H), 6.03 (bs, 1H), 3.98 (bs, 2H), 3.83 (m, 2H), 1.58 (m, 2H), 0.94
(t, 3H); 13C-NMR (CDCl3):
d
161.12, 143.73, 133.19, 128.82, 128.76,
4.9.22. 5-(2-Aminophenyl)-1-methyl-1H-pyrrole-2-carboxamide
126.11, 119.06, 118.35, 116.57, 109.69, 108.87, 41.17, 23.17, and 11.46;
MS (LSIMS) m/z 266.1262 (M þ Na)þ, Calcd. Mass for C14H17N3ONa
266.1269; Anal. C14H17N3O (C, H, N).
8v
(50%); mp 122–123 ꢃC; 1H-NMR (CD3OD):
d
7.22 (pt, 1H), 7.10 (d,
1H), 7.00 (d, 1H), 6.92 (d, 1H), 6.85 (pt, 1H), 6.12 (d, 1H), 3.61 (s, 1H);
13C-NMR (CD3OD):
163.84, 145.56, 135.40, 132.07, 132.15, 126.91,
d
4.9.16. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
butylamide 8p
120.01, 118.08, 113.45, 108.82, and 33.92; MS (LSIMS) m/z 238.0961
(M þ Na)þ, Calcd. Mass for C12H13N3ONa 238.0956; Anal.
C12H13N3O (C, H, N).
(54%); mp 79–80 ꢃC; 1H-NMR (CDCl3):
d 9.79 (bs, 1H), 7.27 (dd,
1H), 7.11 (pt, 1H), 6.80 (pt, 1H), 6.75 (d, 1H), 6.59 (pt, 1H), 6.41 (pt,
1H), 5.90 (bs, 1H), 4.10 (bs, 2H), 3.40 (m, 2H), 1.56 (m, 2H), 1.39 (m,
4.10. In vitro striatal nNOS activity determination
2H), 0.95 (t, 3H); 13C-NMR (CDCl3):
d 161.09, 143.78, 133.20, 128.89,
128.85, 126.18, 119.18, 118.42, 116.71, 109.52, 108.88, 39.29, 31.97,
20.18, and 13.85; MS (LSIMS) m/z 280.1428 (M þ Na)þ, Calcd. Mass
for C15H19N3ONa 280.1425; Anal. C15H19N3O (C, H, N).
L
-Arginine,
ethanesulfonic acid) (HEPES),
aprotinin, pepstatin, phenylmethyl-sulfonylfluoride (PMSF), hypo-
L
-citrulline, N-(2-hydroxymethyl)piperazine-N0-(2-
-dithiothreitol (DTT), leupeptin,
D,L
xantine-9-b-D-ribofuranosid (inosine), ethylene glycol-bis-(2-ami-
4.9.17. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
cyclopropylamide 8q
noethylether)-N,N,N0,N0,-tetraacetic acid (EGTA), bovine serum
albumin (BSA), Dowex-50W (50 ꢂ 8–200), FAD, NADPH and 5,6,7,8-
(49%); mp 137–138 ꢃC; 1H-NMR (CDCl3):
d
9.90 (bs,1H), 7.27 (dd,
tetrahydro-L-biopterin dihydrochloride (H4-biopterin) were
1H), 7.09 (pt, 1H), 6.78 (pt, 1H), 6.73 (d, 1H), 6.60 (bs, 1H), 6.38 (pt,
1H), 6.22 (bs, 1H), 4.00 (bs), 2.81 (m, 1H), 0.81 (m, 2H), 0.59 (m, 2H);
obtained from Sigma–Aldrich Quı´mica (Spain). L
-[3H]-arginine
(58 Ci/mmol) was obtained from Amersham (Amersham Biosci-
ences, Spain). Tris-(hydroxymethyl)-aminomethane (Tris–HCl) and
calcium chloride were obtained from Merck (Spain).
Male Wistar rats (2020–250 g) were used for the in vitro NOS
determination. Animals were maintained in the University’s facility
in a 12 h:12 h light/dark cycle at 22 ꢁ 2 ꢃC and with free access to
food and tap water. All experiments were performed according to
the Spanish Government Guide and the European Community.
Rats were killed by cervical dislocation, and the striata were
quickly collected and immediately used to measure NOS activity.
13C-NMR (CDCl3):
d 162.55, 143.73, 133.46, 128.87, 125.81, 119.14,
118.31, 116.67, 110.25, 108.99, 22.67, and 6.94; MS (LSIMS) m/z
242.1287 (M þ H)þ, Calcd. Mass for C14H16N3O 242.1293; Anal.
C14H15N3O (C, H, N).
4.9.18. 5-(2-Aminophenyl)-1H-pyrrole-2-carboxylic acid
cyclobutylamide 8r
(67%); mp 163–165 ꢃC; 1H-NMR (CDCl3):
d
9.85 (bs,1H), 7.26 (dd,
1H), 7.11 (pt, 1H), 6.80 (pt, 1H), 6.75 (d, 1H), 6.61 (dd, H-3), 6.40 (pt,

L.C. Lo´pez Cara et al. / European Journal of Medicinal Chemistry 44 (2009) 2655–2666
2665
Upon removal, the tissues were cooled in ice-cold homogenizing
buffer (25 mM Tris–HCl, 0.5 mM DTT, 10 g/mL leupeptin, 10 g/mL
pepstatine, 10 g/mL aprotinine, 1 mM PMSF, pH 7.6). Two striata
4.13. Statistical analysis
m
m
m
Data are expressed as the mean ꢁ SEM. One-way analysis of
variance, followed by the Newman–Keuls multiple range test was
used. A P < 0.05 value was considered statistically significant.
were placed in 1.25 mL of the same buffer and homogenized in
a Polytron (10 s ꢂ 6). The crude homogenate was centrifuged for
5 min at 1000g, and aliquots of the supernatant were either stored
at ꢀ20 ꢃC for total protein determination [55] or used immediately
to measure NOS activity. The nNOS activity was measured by the
Acknowledgements
Bredt and Snyder method [56], monitoring the conversion of
arginine to
-[3H]-citrulline. The final incubation volume was
100 L and consisted of 10 L crude homogenate added to a buffer
to give a final concentration of 25 mM Tris–HCl, 1 mM DTT, 30
H4-biopterin, 10 M FAD, 0.5 mM inosine, 0.5 mg/mL BSA, 0.1 mM
CaCl2, 10 -arginine, 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 pyrazole derivative in DMSO to give a final
L
-[3H]-
This work was partially supported by grants from the Ministerio
de Ciencia y Tecnologı´a (SAF2005-07991-C02-01 and SAF2005-
07991-C02-02) and from the Junta de Andalucia (P06-CTS-01941).
L
m
m
mM
m
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