Novel inhibitors of nitric oxide synthase with antioxidant properties

Article abstract of DOI:10.1016/j.ejmech.2012.01.002

We previously described a series of imidazole-based inhibitors substituted at N-1 with an arylethanone chain as interesting inhibitors of neuronal nitric oxide synthase (nNOS), endowed with good selectivity vs endothelial nitric oxide synthase (eNOS). As

Full text of DOI:10.1016/j.ejmech.2012.01.002

European Journal of Medicinal Chemistry 49 (2012) 118e126
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel inhibitors of nitric oxide synthase with antioxidant properties
,
a
a
a
a
Loredana Salerno a , Maria N. Modica , Giuseppe Romeo , Valeria Pittalà , Maria A. Siracusa ,
*
Maria E. Amato b, Rosaria Acquaviva a, Claudia Di Giacomo a, Valeria Sorrenti a
a Dipartimento di Scienze del Farmaco, Università degli Studi di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
b Dipartimento di Chimica, Università di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously described a series of imidazole-based inhibitors substituted at N-1 with an arylethanone
chain as interesting inhibitors of neuronal nitric oxide synthase (nNOS), endowed with good selectivity
vs endothelial nitric oxide synthase (eNOS). As a follow up of these studies, several analogs characterized
by the presence of substituted imidazoles or other mono or bicyclic nitrogen-containing heterocycles
instead of simple imidazole were synthesized, and their biological evaluation as in vitro inhibitors of both
nNOS and eNOS is described herein. Most of these compounds showed improved nNOS and eNOS
inhibitory activity with respect to reference inhibitors. Selected compounds were also tested to analyze
their antioxidant properties. Some of them displayed good capacity to scavenge free radicals and ability
to reduce lipid peroxidation.
Received 24 June 2011
Received in revised form
16 December 2011
Accepted 4 January 2012
Available online 11 January 2012
Keywords:
NOS inhibitors
nNOS
Ó 2012 Elsevier Masson SAS. All rights reserved.
eNOS
Antioxidants
Imidazoles
Ethanones
1. Introduction
of systemic blood pressure and for the inhibition of platelet
aggregation; in addition, NO constitutively generated, can serve as
The free radical gas nitric oxide (NO) acts as signaling molecule
in various tissues and is responsible for both physiological and
pathological effects; it is synthesized by nitric oxide synthase (NOS)
neuromodulator in non-adrenergic, non-cholinergic nerve endings
of the gastrointestinal, and genitourinary systems; NO derived from
iNOS has mainly the biological significance of cytotoxic agent in the
normal immune or inflammatory response [10e14].
from L-arginine (L-Arg) and molecular oxygen. Three isoforms of
this enzyme have been identified so far: neuronal (nNOS), inducible
(iNOS), and endothelial (eNOS) NOS; all three isoforms are consti-
tuted by a N-terminal catalytic oxygenase domain which binds Fe-
Nevertheless, overproduction of NO, especially by nNOS and
iNOS, is associated with various diseases states such as neuro-
degeneration, stroke, migraine and chronic headache, Parkinson,
Alzheimer, and Huntington diseases (nNOS), hypotensive crises
during septic shock, arthritis, colitis, tissue damage, cancer, and
various kinds of inflammatory states (iNOS) [15e20]. Besides iNOS
overexpression [21e23], recently it has been reported that eNOS is
overexpressed in various type of cancer and may be involved in
tumor angiogenesis [24e26].
Therefore, the use of substances with inhibitory properties on all
the NOS isoforms has great therapeutic potential for the treatment
of the above-mentioned diseases. Consequently, over the last
twenty years the design and synthesis of NOS inhibitors have
received much attention. As a result of both classical and compu-
tational studies, some very potent and selective inhibitors of nNOS
and iNOS have been nowadays identified [27e32].
protoporphyrin IX (heme), the substrate L-Arg, and (6R)-5,6,7,8-
tetrahydrobiopterin (BH4), by a central linker region that binds
calmodulin (CaM), and by a C-terminal reductase domain with
binding sites for flavines (FAD, FMN), and NADPH cofactors; the
heme domain provides the site for L-Arg oxidation, while FAD and
FMN shuttle electrons from NADPH to the Fe-heme; the cofactor
BH4 is fundamental for the catalytic process, stabilizing the active
homodimeric form of the enzyme [1e5].
NO, produced by all three isoforms, is involved in a number of
physiological processes in mammalians [6e9]. In particular, NO
generated by nNOS is an important neurotransmitter for brain
development, memory, learning, brain perception, and long-term
potentiation; NO produced by eNOS is responsible for the control
At the beginning of our studies on the synthesis of NOS inhibi-
tors, we selected imidazole nucleus as template because imidazole
itself and simple imidazole derivatives were reported in the early
literature as inhibitors of various isoforms of NOS [33,34]. On these
* Corresponding author. Tel.: þ39 095 7384024; fax: þ39 095 222239.
E-mail address: l.salerno@unict.it (L. Salerno).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.002

L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
119
basis, our research group has conducted extensive investigation of
a number of imidazole derivatives variously substituted at the N-1
[35e37]. Some of them showed interesting inhibitory activity on
nNOS against eNOS and iNOS, in particular compounds carrying an
arylethanone chain; then, we selected the neuronal isoform as
main target. Compounds which showed the best biological profile
in terms of activity on nNOS and selectivity vs eNOS, i.e. 2-(1H-
imidazol-1-yl)-1-(4-nitrophenyl)-ethanone 1 and 2-(2-methyl-1H-
imidazol-1-yl)-1-(4-nitrophenyl)-ethanone 2 (Fig. 1), were also
investigated in order to elucidate their mechanism of action and
R
O
NO2
N
N
1: R = H
2: R = CH3
3: R = CH(CH3)2
Fig. 1. Chemical structures of compounds 1e3.
resulted to inhibit nNOS “non-competitively” vs the substrate L-Arg
and “competitively” vs the cofactor BH4, without interaction with
heme iron [38].
R1
O
R2
Br
N
R
R
(a)
N
N
R3
R1
+
O
N
H
R2
18-21
R3
4-13
18: R = SO2CF3
22-34
19: R = H
20: R = Br
21: R = NO2
O
R2
R1
R1
NO2
N
(b)
N
+
N
21
R3
N
H
R2
R3
14-16
36-38
N
O
(c)
NO2
N
N
21
+
N
H
N
N
39
17
22: R = SO2CF3, R1 = H, R2 = H, R3 = H
23: R = H, R1 = CH3, R2 = H, R3 = H
24: R = Br, R1 = CH3, R2 = H, R3 = H
25: R = NO2, R1 = H, R2 = CH3, R3 = H
26: R = NO2, R1 = H, R2 = H, R3 = CH3
27: R = H, R1 = iC3H7, R2 = H, R3 = H
28: R = Br, R1 = iC3H7, R2 = H, R3 = H
29: R = NO2, R1 = C3H7, R2 = H, R3 = H
30: R = NO2, R1 = C6H5, R2 = H, R3 =H
31: R = NO2, R1 = H, R2 = C6H5, R3 = H
32: R = NO2, R1 = H, R2 = NO2, R3 = H
33: R = NO2, R1 = C2H5, R2 = CH3, R3 = H
34: R = NO2, R1 = H, R2 = C6H5, R3 = C6H5
36: R1 = H, R2 = H, R3 = H
4: R1 = H, R2 = H, R3 = H
5: R1 = CH3, R2 = H, R3 = H
6: R1 = H, R2 = CH3, R3 = H
7: R1 = iC3H7, R2 = H, R3 = H
8: R1 = C3H7, R2 = H, R3 = H
9: R1 = C6H5, R2 = H, R3 = H
10: R1 = H, R2 = C6H5, R3 = H
11: R1 = H, R2 = NO2, R3 = H
12: R1 = C2H5, R2 = CH3, R3 = H
13: R1 = H, R2 = C6H5, R3 = C6H5
14: R1 = H, R2 = H, R3 = H
15: R1 = CH3, R2 = H, R3 = H
16: R1 = CH3, R2 = H, R3 = CH3
37: R1 = H, R2 = H, R3 = CH3
38: R1 = CH3, R2 = H, R3 = CH3
Scheme 1. Reagents and conditions: (a) K2CO3, dry DMF, r.t., 1.5 h; (b) NaNH2, dry DMF, 60 ꢀC, 1.5 h; (c) TEA, CHCl3, 0 ꢀC, 4 h.

120
L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
Some neurological disorders, such as cerebral ischemia and
mouse recombinant iNOS. Some of them were also tested for
analyzing their antioxidant properties. To this purpose, their abili-
ties to quench a stable radical, to scavenge superoxide anion, and to
inhibit in vitro lipid peroxidation, were evaluated.
stroke, are related to enhanced formation of free radical species
both of nitrogen (NOꢁ) and oxygen (O2 ꢂ) in brain tissue [39,40];
ꢁ
in fact inhibition of nNOS as well as scavenging reactive oxygen
species has been shown to result in an enhanced neuronal
survival after cerebral ischemia [41]. Consequently, combination
therapy with substances having different mechanisms of action
could be a good strategy for the treatment of the above-
mentioned diseases, but often it suffers of the possibility of
drugedrug interactions. An alternative approach is to combine
multiple activities in the same compound, for example inhibition
of nNOS and antioxidant activities [42e44]. Since compounds
possessing imidazole ring have been found to exhibit antioxi-
dant activities [45e47], we have recently tested the antioxidant
properties of a selected series of imidazole derivatives previously
synthesized in our laboratory as nNOS inhibitors, in particular
compounds 1, 2 and 2-[2-(1-methylethyl)-1H-imidazol-1-yl]-1-
2. Chemistry
ThepreparationoftitlecompoundsisoutlinedinSchemes1and2.
Synthetic procedures described in literature were taken into account
[49e52] and slightly modified. In brief, imidazole derivatives 22e34,
benzimidazole derivatives 46e48, pyridoimidazole derivatives 49,
51, and benzotriazole derivatives 50 and 52 were obtained by alkyl-
ation of heterocycles 4e13, 40e43, respectively, with appropriate 1-
aryl-2-bromo-ethanone 18e21, using dry dimethylformamide as
solvent, potassiumcarbonateasbase, atroomtemperatureandunder
nitrogen atmosphere. Pyrazole derivatives 36e38 were prepared by
alkylation of corresponding pyrazole 14e16 with 2-bromo-40-nitro-
acetophenone 21 in dry dimethylformamide, sodium amide, at 60 ꢀC,
and under nitrogen atmosphere. Triazole derivative 39 was obtained
by reaction of 1,2,4 triazole 17 with 21 in chloroform and triethyl-
amine, at 0 ꢀC, under nitrogen atmosphere. Finally, indazole deriva-
tives 53 and 54 were synthesized by reaction of opportune indazole
44 and 45 with an excess of alkylating agent 21 at 120 ꢀC for 1 h in the
absence of solvent. Yields were in the range 29e75%. Purification of
most compounds was usually performed by flash chromatography.
All the synthesized compounds were characterized by analytical, IR
and 1H NMR spectral data. The alkylation of some starting azoles
potentially can lead to more than one regioisomers; in particular, in
somecasesoneprevalentregioisomerwasobtained(31, 32, 33, 37, 53,
54); in other cases, the formation of a mixture of two regioisomers
(25, 26, 49, 50, 51, 52) occurred; these last isomers were separated by
flash chromatography. In order to assign the correct structures for the
above-mentioned regioisomers, correlation spectroscopy (nuclear
overhauser effect spectroscopy, NOESY) was also used.
(4-nitrophenyl)-ethanone
3 [48] (Fig. 1). Obtained results
showed that compounds 1 and 2 had good capacity to scavenge
free radicals and to reduce lipid peroxidation; so, possessing
multiple activities in the same molecule, they could be consid-
ered useful for therapy of brain diseases caused by an over-
production of radical species, overcoming difficulties derived
from a therapy with drugs possessing different mechanisms of
action.
With the aim to improve the observed inhibitory activity on
nNOS, in this paper we describe the synthesis of a new series of
compounds containing an arylethanone chain in which two main
structural modifications were carried out with respect to the
reference compounds 1e3: (i) Introduction of various substituents
at the C-2, C-4, and C-5 of the imidazole nucleus, (ii) Substitution of
imidazole nucleus with mono and bicyclic N-containing heterocy-
cles. All synthesized compounds were tested to evaluate the
inhibitory properties toward rat recombinant nNOS; selected
compounds were also tested on endothelial cell (EC) line eNOS and
Scheme 2. Reagents and conditions: (a) K2CO3, dry DMF, r.t., 1.5 h; (b) 120 ꢀC, 1 h.

L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
121
3. Results and discussion
compound 22 carrying a 4-trifluoromethylsulfonyl substituent,
residue able to combine electronic and steric properties of both
nitro and phenyl in the same substituent, according to the principal
properties approach [53]. Compound 22 resulted less potent than
reference compounds, so the nitro group was maintained in the
most of the next synthesized compounds. Further modifications on
the structure of new potential inhibitors were then realized on the
imidazole moiety. Investigation of the mode of action of
compounds 1 and 2, has revealed that these compounds inhibited
3.1. NOS inhibition
New synthesized compounds were tested in in vitro enzymatic
assays to evaluate their ability to inhibit various NOS isoforms.
Results, expressed as Ki or percentage of inhibition, are summarized
in Table 1, and are in the micromolar range. All compounds were
tested on nNOS; compounds were tested on eNOS when Ki nNOS
was ꢃ120
m
M; compounds that resulted active against nNOS and
nNOS “non-competitively” vs the substrate L-Arg and “competi-
less active against eNOS were tested also on iNOS. Compounds 1e3
(Fig. 1), previously synthesized in our laboratory, were used as
reference substances since they have been proven to be good
inhibitors of nNOS endowed with good selectivity vs eNOS [35]. The
first target of this work was to improve this inhibitory activity.
Reference compounds 1e3 can be considered as constituted by two
main portions linked by an ethanone chain, i.e. the imidazole
nucleus, and the 4-nitrophenyl moiety. In our previous study we
extensively investigated the effect of the substituents on the phenyl
ring on nNOS inhibition, concluding that the most potent
compounds carried a nitro or a phenyl group at the 4-position [35].
In order to complete this study, in this work firstly we synthesized
tively” vs the cofactor BH4, without interaction with heme iron [38].
These results were in contrast with those reported in literature
regarding the mechanism of inhibition on various isoforms of NOS
caused by simple imidazoles, such as 1, 2, and 4-phenylimidazole
[33,34]. These last compounds inhibited nNOS by acting as sixth
coordination ligand of the heme iron in the place of oxygen and the
order of potency was 1-phenylimidazole > 2-phenylimidazole > 4-
phenylimidazole. In terms of structureeactivity relationships these
results were justified by the reduction of electron density in
imidazole nucleus caused by the steric hindrance induced by
phenyl ring [33]. On the other hand, we reported that compounds
carrying a methyl or 1-methylethyl substituent at the C-2 position
of the imidazole nucleus, such as 2 and 3, were more effective in
inhibiting nNOS and less effective in inhibiting eNOS than the
unsubstituted analog 1, affording more selective compounds
[35,38]. On these basis and in order to improve the observed
inhibitory activity and better understand structureeactivity rela-
tionships within this class of compounds, in this work we designed
and synthesized compounds 23e34 (Table 1) characterized by the
presence of an imidazole nucleus substituted with methyl, propyl,
1-methylethyl, phenyl or nitro groups at C-2, C-4, and C-5 positions.
As a general trend, compounds 23e34, in particular those in
Table 1
Inhibitory properties of compounds 22e39, 46e54.
Compound
Ki nNOS (
m
M)a
Ki eNOS (
m
M)a
% of inhibition
of iNOS (500 mM)
22
23
24
25
26
27
28
29
30
31
32
33
34
35c
36
37
38
39
46
47
48
49
50
51
52
53
54
1d
450
>500
620
85
NTb
NTb
NTb
>2000
45
NTb
NTb
NTb
4
90
NTb
NTb
NTb
NTb
NTb
4.6
750
450
40
40
45
NTb
NTb
45
which the substituted imidazoles are coupled with
a 4-
nitrophenylethanone chain, maintained or improved the inhibi-
tory activity on nNOS with respect to reference compounds 1e3.
These results confirmed that the presence of 4-nitrophenyl linked
to the ethanone chain was critical for nNOS inhibition and that the
introduction of a substituent on the imidazole nucleus, with the
only exception of 4-NO2 (32), gave a good contribution to the
inhibitory properties. Among these substituted imidazoles 23e34,
37.5
70
120
75
36.25
230
40
120
75
NTb
NTb
NTb
NTb
30.6
NTb
24
25
NTb
60
63
65
30
90
40
2-(4-methyl-1H-imidazol-1-yl)-1-(4-nitrophenyl)-ethanone
25
NTb
NTb
NTb
3.5
was the most interesting since maintained the activity on nNOS and
selectivity vs eNOS observed for the 2-methyl analog 2.
Owing to the close structural class of cytochrome P450 with
NOS enzymes, cytochrome P450 inhibition assay was performed for
compound 25. Our results showed that even at concentrations high
47.5
480
46.6
13.75
31.66
12.5
35.83
22.5
32.5
66.25
105
74
NTb
2.75
37.5
70.25
32.5
45
15
4
NTb
NTb
12
as 500
at 500
chrome P450.
mM, compound 25 had no substantial effect (% of inhibition
30
60
75
>5000
>2000
500
mM ¼ 13%) on the catalytic activity of microsomal cyto-
NTb
31.6
32
Since results described so far clearly indicated that the presence of
unsubstituted imidazole nucleus was not crucial for nNOS inhibition,
we designed, synthesized and tested compounds 35e39 containing
pyrrole, pyrazole or triazole, and compounds 46e54 containing
benzimidazole, pyridoimidazole, benzotriazole or indazole instead of
imidazole. Among monocyclic compounds 35e39, only pyrrole
derivative 35 (Table 1), prepared according to reference [54], gave
a drastic reduction of activity whereas pyrazole andtriazole derivatives
36e39 showed inhibitory activity on nNOS comparable or better than
reference compounds 1e3, although showing less selectivity vs eNOS.
Also bicyclic derivatives 46e54 were generally more potent toward
nNOS than reference compounds 1e3; however, this increase of
potency was observed also against eNOS, consequently they resulted
less selective than reference compounds. In particular the benzimid-
azole derivative 47, being about 17-fold more potent on eNOS than
nNOS, could be useful as lead compound to modify in order to improve
2e
3f
22.5
25
a
Inhibition constants were obtained by measuring percentage of inhibition with
at least three concentrations of inhibitor as described in Ref. [66].
b
Not tested.
Structure of compound 35 [54]:
c
NO2
N
O
d
According to Ref. [35].
According to Ref. [38].
According to Ref. [48].
e
f

122
L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
Table 2
Table 4
DPPH radical scavenging activities of compounds 25, 29, 48, 50.
Effect of compounds 25, 29, 50 on lipid hydroperoxide levels in human plasma
incubated at 37 ꢀC for 2 h.
Compound
500
m
Ma
100
m
Ma
50
m
Ma
IC50 (mM)
Compound
100
m
Ma
50
m
Ma
25
m
Ma
IC50
(
m
M)
LogP
25
29
48
50
1b
2b
3b
41%
56%
45%
31%
17%
35%
19%
21%
12%
25%
11%
14%
>500
262
>500
>500
415
25
29
50
1b
2b
3b
69%
44%
57%
65%
31%
48%
53%
30%
37%
10
>100
17
18
1.75
>100
1.48
2.17
2.54
1.024
1.11
1.98
165
230
a
a
Values had a standard deviation of ꢃ10% (n ꢅ 3).
Values had a standard deviation of ꢃ10% (n ꢅ 3).
b
b
According to Ref. [48].
According to Ref. [48].
this selectivity [24e26], whereas 5,6-dimethylbenzimidazole deriva-
tive 48 and benzotriazole derivative 50 resulted the most interesting
compounds in terms of potency against nNOS and selectivity vs eNOS.
Results regarding iNOS activity for selected compounds 25, 31,
36, 38, 48, 49, 50, and 53 evidenced that, even if most of the
compounds disclosed showed an increased activity for both nNOS
and eNOS isoforms, they were less active toward iNOS isoform with
xanthine/xanthine oxidase system. Compounds 25, 29, 48, and 50
inhibited superoxide anion formation in a dose-dependent manner
and this activity was generally comparable or better than reference
compounds 1e3 (Table 3). As a general trend, in this test all
compounds resulted more effective than in the previous one.
Antioxidant activity in an “ex vivo cell-free system” was evaluated by
measuring antilipoperoxidative capacity in human plasma incu-
bated with or without some of the synthesized compounds for 2 h
at 37 ꢀC. We selected compounds 25, 29, and 50 since they were the
most active as scavengers of free radicals in the previous assays. All
these compounds were able to inhibit lipid peroxidation and their
potency was similar or only slightly less than reference compounds
1e3 (Table 4). Since lipophilicity can play a role in the capacity of
compounds to inhibit lipid peroxidation, usually increasing distri-
bution of the compounds in the membrane lipid bilayer [45], we
calculated LogP (cLogP) of compounds 25, 29, and 50. Results show
that there is any correlation between cLogP and IC50 values because
compounds with the highest activity, 25 and 29 (Table 4), showed
the lowest and the highest value of cLogP, respectively.
% of inhibition at 500 mM ranging between 4% and 30%.
3.2. Antioxidant properties
The second target of this work was the evaluation of the anti-
oxidant properties of selected compounds endowed with good
nNOS inhibition. The aim of combining in the same molecule
inhibition of nNOS and antioxidant activities [55] is to reduce
neurological damage caused by overproduction of nitrogen and
oxygen radical species. Selected compounds were 25 and 29 among
monocyclic derivatives, 48 and 50 among bicyclic derivatives.
Antioxidant properties were firstly evaluated by two in vitro tests:
(i) Quenching of 1-diphenyl-2-picrylhydrazyl (DPPH) radical; (ii)
Scavenger effect on superoxide anion. Then, compounds 25, 29, and
50 were screened in an “ex vivo cell-free system” to test lipid per-
oxidation inhibitory activity. Results are listed in Tables 2e4 and are
expressed as percentage of inhibition at different concentrations.
IC50 values were calculated for the most interesting compounds.
Free radical scavenging activity of the selected compounds was
tested by their ability to bleach the stable DPPH radical. This assay
provides information on the reactivity of test compounds with
a stable free radical. Because of its odd electron, DPPH gives a strong
4. Conclusions
Compounds 22e39, 46e59, containing an arylethanone chain
linked to mono or bicyclic nitrogen-containing heterocycles, were
synthesized and their inhibitory activity against the three isoforms
of NOS was evaluated. Most of them resulted more potent against
nNOS and eNOS than reference compounds 1e3 and showed very
weak activity against iNOS. The most potent inhibitors of nNOS
resulted 48 and 50, whereas the most interesting compound both
for potency and selectivity was 25.
Some compounds (25, 29, 48, and 50) were selected to test their
ability to quench a stable radical, to scavenge superoxide anion and
to inhibit in vitro lipid peroxidation. Tested compounds resulted
more effective in scavenging superoxide anion than in quenching
DPPH radical. Generally, their potency was similar or slightly less
with respect to reference compounds 1e3. Compound 25, being an
interesting inhibitor of nNOS endowed with good selectivity vs
eNOS, iNOS and cytochrome P450, and having good antioxidant
properties, emerged from these studies. In consideration of these
results, it is reasonable to affirm that the combination of nNOS
inhibition and antioxidant activities in the same molecule is
possible and is a promising target aimed to provide new thera-
peutic strategies. Further studies on this class of molecules are in
progress to increase selectivity and antioxidant properties.
absorption band at
l
¼ 517 nm in visible spectroscopy (deep violet
color). As this electron becomes paired off in the presence of a free
radical scavenger, the absorption vanishes, and the resulting
decolorization is stoichiometric with respect to the number of
electrons taken up. In this assay, most of tested compounds showed
a DPPH quenching dose-dependent capacity, usually less than
reference compounds 1e3 (Table 2). The superoxide anion scav-
enging capacity of selected compounds was investigated by means
of a method which excludes the Fenton-type reaction and the
Table 3
Superoxide ion scavenging activities of compounds 25, 29, 48, 50.
Compound
100
m
Ma
50
m
Ma
25
m
Ma
IC50 (mM)
25
29
48
50
1c
56%
62%
45%
51%
23%
34%
37%
35%
20%
22%
0%
66
38
>100b
85
26%
5. Experimental procedures
165
70
185
2c
5.1. Chemistry: general methods
3c
a
Values had a standard deviation of ꢃ10% (n ꢅ 3).
Melting points were determined in a Gallemkamp apparatus
with an MFB-59 digital thermometer in glass capillary tubes and
are uncorrected. Elemental analyses for C, H, N were within ꢄ0.4%
b
IC50 was not calculated because compound was not soluble at concentration
ꢅ100 M.
According to Ref. [48].
m
c

L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
123
of theoretical values and were performed on a Carlo Erba Elemental
Analyzer Mod.1108 apparatus. The IR spectra were recorded in KBr
disks on a PerkineElmer 1600 series FTeIR spectrometer. 1D-1H
NMR spectra were determined with a Varian Inova Unity 200
(200 MHz), instrument in DMSO-d6 solution; 13C NMR spectra and
2D g-COSY and NOESY experiments were determined with a Varian
Unity Inova 500 (500 MHz) instrument in CDCl3. Chemical shifts are
J ¼ 8.4 Hz, 2H, aromatic); 13C NMR (500 MHz, CDCl3)
d
9.0, 50.9,
124.3, 127.3, 127.6, 129.12,137.5, 138.5, 151.1, 190.5. Anal. C12H11N3O3
(C, H, N).
5.2.4. 2-[2-(1-Methylethyl)-1H-imidazol-1-yl]-1-phenyl-ethanone
(27)
The title compound was isolated as white powder (75%); mp
in
d
values (ppm) using tetramethylsilane as the internal standard;
99e100 ꢀC; IR (KBr) cmꢂ1 2933, 1687; 1H NMR (DMSO-d6)
d 1.11 (s,
coupling constants (J) are given in Hz. Signal multiplicities are
characterized as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad signal). All the synthesized compounds were
tested for purity on TLC on Merck plates (Kieselgel 60 F254) and
3H, CH3), 1.52 (s, 3H, CH3), 2.79e2.94 (m, 1H, CH), 5.70 (s, 2H, CH2),
6.78 (s, 1H, imidazole), 6.94 (s, 1H, imidazole), 7.56e7.76 (m, 3H,
aromatic), 8.05e8.09 (m, 2H, aromatic). Anal. C14H16N2O (C, H, N).
spots were visualized under the UV light (
l
¼ 254 and 366 nm).
5.2.5. 1-(4-Bromophenyl)-2-[2-(1-methylethyl)-1H-imidazol-1-yl]-
ethanone (28)
Preparative column chromatography was performed using Merck
silicaegel 60 (230e400 mesh). All chemicals and solvents were
reagent grade and were purchased from commercial sources. 2-
Bromo-1-[(4-trifluromethylsulfonyl)phenyl]-ethanone 18 was
prepared according to literature [56]. Analytical and spectral data of
compounds 23, 24, 46, and 50, are in agreement with literature
[57e60].
The title compound was isolated as white powder (70%); mp
154e155 ꢀC; IR (KBr) cmꢂ1 2930, 1694; 1H NMR (DMSO-d6)
d 1.11 (s,
3H, CH3), 1.45 (s, 3H, CH3), 2.79e2.91 (m, 1H, CH), 5.77 (s, 2H, CH2),
6.79 (d, J ¼ 1.3 Hz, 1H, imidazole), 6.93 (d, J ¼ 1.3 Hz 1H, imidazole),
7.80e7.86 (m, 2H, aromatic), 7.96e8.03 (m, 2H, aromatic). Anal
C14H15BrN2O (C, H, N).
5.2. General procedure for the synthesis of imidazole,
benzimidazole, pyridoimidazole, and benzotriazole derivatives
22e34, 46e52
5.2.6. 1-(4-Nitrophenyl)-2-(2-propyl-1H-imidazol-1-yl)-ethanone
(29)
The title compound was isolated as orange powder (62%); mp
158e160 ꢀC; IR (KBr) cmꢂ1 1693, 1523, 1345; 1H NMR (DMSO-d6)
A solution of imidazole 4e13, benzimidazole 40, 41, pyr-
idoimidazole 42 or benzotriazole 43 (23 mmol) in dry DMF (15 mL)
and K2CO3 (11.5 mmol) was stirred for 10 min at 0e5 ꢀC, under
nitrogen atmosphere. After this time, appropriate 1-aryl-2-bromo-
ethanone 18e21 (23 mmol) was added and the mixture was stirred
for 1.5 h then poured into ice water. The resulting crude material
was extracted with dichloromethane (3 ꢆ 50 mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure; the obtained residue
were purified by means of flash chromatography performed using
silica gel 60 (230e400 mesh) and a mixture of ethyl acetate/
methanol 8:2 v/v or dichloromethane/methanol 9.5/0.5 v/v (only
for purification of 25 and 26, 49 and 51, 50 and 52, respectively) as
eluent. By use of this procedure, the subsequent compounds were
obtained:
d
0.88 (t, J ¼ 7.2 Hz, 3H, CH3), 1.50e1.71 (m, 2H CH2CH2CH3), 2.47 (t,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 5.76 (s, 2H, CH2), 6.78e6.79 (m, 1H,
imidazole), 6.98e6.99 (m, 1H, imidazole), 8.26e8.30 (m, 2H,
aromatic), 8.40e8.44 (m, 2H, aromatic). Anal.C14H15N3O3 (C, H, N).
5.2.7. 1-(4-Nitrophenyl)-2-(2-phenyl-1H-imidazol-1-yl)-ethanone
(30)
The title compound was isolated as orange powder (50%); mp
149e150 ꢀC; IR (KBr) cmꢂ1 1714, 1526, 1354; 1H NMR (DMSO-d6)
d
5.45 (s, 2H, CH2), 7.02 (d, J ¼ 1.4 Hz, 1H, imidazole), 7.25 (d,
J ¼ 1.4 Hz, 1H, imidazole), 7.38e7.48 (m, 5H, aromatic), 8.02e8.07
(m, 2H, aromatic), 8.30e8.36 (m, 2H, aromatic). Anal. C17H13N3O3
(C, H, N).
5.2.8. 1-(4-Nitrophenyl)-2-(4-phenyl-1H-imidazol-1-yl)-ethanone
(31)
5.2.1. 2-(1H-Imidazol-1-yl)-1-(4-trifluoromethylsulfonylphenyl)-
ethanone (22)
The title compound was isolated as orange powder (45%); mp
188e190 ꢀC; IR (KBr) cmꢂ1 1718, 1526, 1354; 1H NMR (500 MHz,
The title compound was isolated as yellow powder (38%); mp
CDCl3)
d
5.47 (s, 2H, CH2), 7.25e7.28 (m, 2H, aromatic þ imidazole),
98e100 ꢀC; IR (KBr) cmꢂ1 1690, 1373, 1215; 1H NMR (DMSO-d6)
d
7.39 (dd, J ¼ 7.2 and 7.8 Hz, 2H, aromatic), 7.59 (s, 1H, imidazole),
5.84 (s, 2H, CH2), 6.96 (br s, 1H, imidazole), 7.14 (br s, 1H, imid-
azole), 7.63 (s, 1H, imidazole), 8.35e8.41 (m, 4H, aromatic). Anal.
C12H9F3N2O3S (C, H, N, S).
7.83 (d, J ¼ 7.8 Hz, 2H, aromatic), 8.17 (d, J ¼ 8.4 Hz, 2H, aromatic),
8.41 (d, J ¼ 8.4 Hz, 2H, aromatic). 13C NMR (500 MHz, CDCl3)
d 52.7,
115.7, 124.3, 124.8, 127.1, 128.6, 129.2, 133.8, 138.1, 138.5, 142.9, 151.1,
190.4. Anal. C17H13N3O3 (C, H, N).
5.2.2. 2-(4-Methyl-1H-imidazol-1-yl)-1-(4-nitrophenyl)-ethanone
(25)
5.2.9. 1-(4-Nitrophenyl)-2-(4-nitro-1H-imidazol-1-yl)-ethanone
(32)
The title compound was isolated as orange powder (45%); mp
144e145 ꢀC; IR (KBr) cmꢂ1 1715, 1527, 1356; 1H NMR (500 MHz,
The title compound was isolated as orange powder (56%); mp
CDCl3)
d 2.29 (s, 3H, CH3), 5.38 (s, 2H, CH2), 6.67 (br s, 1H, imid-
223e224 ꢀC; IR (KBr) cmꢂ1 1682, 1584, 1482, 1256, 1092; 1H NMR
azole), 7.45 (br s, 1H, imidazole), 8.13 (d, J ¼ 8.3 Hz, 2H, aromatic),
(500 MHz, CDCl3)
1H, imidazole), 8.19 (d, J ¼ 8.6 Hz, 2H, aromatic), 8.45 (d, J ¼ 8.6 Hz,
2H, aromatic). 13C NMR (500 MHz, CDCl3)
54.8, 123.2, 124.4, 129.9,
d 5.55 (s, 2H, CH2), 7.50 (s, 1H, imidazole), 7.84 (s,
8.38 (d, J ¼ 8.3 Hz, 2H, aromatic); 13C NMR (500 MHz, CDCl3)
d 13.3,
52.5, 116.4, 124.4, 129.4, 137.1, 138.6, 138.9, 151.1, 190.6. Anal.
C12H11N3O3 (C, H, N).
d
138.8, 139.1, 147.2, 150.9, 192.1. Anal. C11H8N4O5 (C, H, N).
5.2.3. 2-(5-Methyl-1H-imidazol-1-yl)-1-(4-nitrophenyl)-ethanone
(26)
5.2.10. 2-(2-Ethyl-4-methyl-1H-imidazol-1-yl)-1-(4-nitrophenyl)-
ethanone (33)
The title compound was isolated as orange powder (29%); mp
The title compound was isolated as orange powder (65%); mp
179e180 ꢀC; IR (KBr) cmꢂ1 1716, 1524, 1357; 1H NMR (500 MHz,
138e139 ꢀC; IR (KBr) cmꢂ1 1702, 1529, 1345; 1H NMR (500 MHz,
CDCl3)
d
2.13 (s, 3H, CH3), 5.35 (s, 2H, CH2), 6.91 (s, 1H, imidazole),
CDCl3)
d
1.32 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3), 2.56 (q,
7.46 (s, 1H, imidazole), 8.14 (d, J ¼ 8.4 Hz, 2H, aromatic), 8.39 (d,
J ¼ 7.6 Hz, 2H, CH2CH3), 5.28 (s, 2H, CH2), 6.53 (s, 1H, imidazole),

124
L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
8.14 (d, J ¼ 8.7 Hz, 2H, aromatic), 8.40 (d, J ¼ 8.7 Hz, 2H, aromatic).
13C NMR (500 MHz, CDCl3)
12.2, 13.6, 20.0, 51.9, 116.5, 124.4, 129.1,
136.7, 138.9, 148.9, 151.1, 191.4. Anal.C14H15N3O3 (C, H, N).
under nitrogen atmosphere. After this time, 2-bromo-40-nitro-
acetophenone 21 (23 mmol) was added and the mixture was stirred
for 1.5 h. The reaction mixture was chilled and poured into ice
water and the resulting crude material was extracted with
dichloromethane (3 ꢆ 50 mL). The combined organic layers were
dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure; the obtained residue was purified by
means of flash chromatography performed using silica gel 60
(230e400 mesh) and a mixture of ethyl acetate/methanol 8:2 v/v as
eluent. By use of this procedure, the subsequent compounds were
obtained:
d
5.2.11. 2-(4,5-Diphenyl-1H-imidazol-1-yl)-1-(4-nitrophenyl)-
ethanone (34)
The title compound was isolated as orange powder (49%); mp
193e195 ꢀC; IR (KBr) cmꢂ1 1714, 1603, 1526, 1354; 1H NMR (DMSO-
d6)
d 5.62 (s, 2H, CH2), 7.10e7.29 (m, 5H, aromatic), 7.38e7.41 (m,
5H, aromatic), 7.83 (s, 1H, imidazole), 8.08e8.19 (m, 2H, aromatic),
8.24e8.40 (m, 2H, aromatic). Anal. C23H17N3O3 (C, H, N).
5.2.12. 2-(1H-Benzimidazol-1-yl)-1-(4-nitrophenyl)-ethanone (47)
The title compound was isolated as orange powder (65%); mp
189e190 ꢀC; IR (KBr) cmꢂ1 1709, 1518, 1348; 1H NMR (DMSO-d6)
5.3.1. 1-(4-Nitrophenyl)-2-(1H-pyrazol-1-yl)-ethanone (36)
The title compound was isolated as orange powder (57%); mp
206e207 ꢀC; IR (KBr) cmꢂ1 1710, 1517, 1348; 1H NMR (DMSO-d6)
d
6.12 (s, 2H, CH2), 7.17e7.27 (m, 2H, aromatic), 7.55e7.60 (m, 1H,
d
5.92 (s, 2H, CH2), 6.29e6.35 (m, 1H, pyrazole), 7.49 (d, J ¼ 1.4 Hz,
aromatic), 7.66e7.71 (m, 1H, aromatic), 8.18 (s, 1H, aromatic),
8.31e8.36 (m, 2H, aromatic), 8.43e8.47 (m, 2H, aromatic). Anal.
C15H11N3O3 (C, H, N).
1H, pyrazole), 7.75 (d, J ¼ 1.8 Hz, 1H, pyrazole), 8.23e8.27 (m, 2H,
aromatic), 8.37e8.41 (m, 2H, aromatic). Anal. C11H9N3O3 (C, H, N).
5.3.2. 2-(4-Methyl-1H-pyrazol-1-yl)-1-(4-nitrophenyl)-ethanone
(37)
5.2.13. 2-(4,5-Dimethyl-1H-benzimidazol-1-yl)-1-(4-nitrophenyl)-
ethanone (48)
The title compound was isolated as orange powder (43%); mp
The title compound was isolated as orange powder (68%); mp
115e117 ꢀC; IR (KBr) cmꢂ1 2983, 1709, 1519, 1351; 1H NMR
203e205 ꢀC; IR (KBr) cmꢂ1 2931, 1710, 1520, 1351; 1H NMR (DMSO-
(500 MHz, CDCl3)
d 2.31 (s, 3H, CH3), 5.54 (s, 2H, CH2), 6.17 (s, 1H,
d6)
d
2.28 (s, 3H, CH3), 2.31 (s, 3H, CH3), 6.04 (s, 2H, CH2), 7.33 (br s,
pyrazole), 7.40 (s, 1H, pyrazole), 8.14 (d, J ¼ 8.2 Hz, 2H, aromatic),
1H, aromatic), 7.44 (br s 1H, aromatic), 8.01 (s, 1H, aromatic),
8.30e8.38 (m, 2H, aromatic), 8.42e8.48 (m, 2H, aromatic). Anal.
C17H15N3O3 (C, H, N).
8.35 (d, J ¼ 8.2 Hz, 2H, aromatic). 13C NMR (500 MHz, CDCl3)
d 13.3,
57.7, 106.5, 123.8, 124.1, 129.3, 131.6, 139.0, 151.2, 191.6. Anal.
C12H11N3O3 (C, H, N).
5.2.14. 2-(3H-Imidazo[4,5-b]pyridin-3-yl)-1-(4-nitrophenyl)-
ethanone (49)
5.3.3. 2-(3,5-Dimethyl-1H-pyrazol-1-yl)-1-(4-nitrophenyl)-
ethanone (38)
The title compound was isolated as orange powder (34%); mp
The title compound was isolated as orange powder (65%); mp
197e199 ꢀC; IR (KBr) cmꢂ1 1702, 1522, 1350; 1H NMR (500 MHz,
122e124 ꢀC; IR (KBr) cmꢂ1 2993, 1710, 1517, 1348; 1H NMR (DMSO-
CDCl3)
d
5.83 (s, 2H, CH2), 7.31 (dd, J ¼ 4.0 and 7.0 Hz, 1H, aromatic),
d6) d 2.08 (s, 3H, CH3), 2.11 (s, 3H, CH3), 5.76 (s, 2H, CH2), 5.87 (s, 1H,
pyrazole), 8.21e8.27 (m, 2H, aromatic), 8.35e8.42 (m, 2H,
aromatic). Anal. C13H13N3O3 (C, H, N).
8.15 (d, J ¼ 7.0 Hz, 1H, aromatic), 8.18 (s, 1H, aromatic), 8.28 (d,
J ¼ 8.8 Hz, 2H, aromatic), 8.39 (d, J ¼ 4.0 Hz, 1H, aromatic), 8.42 (d,
J ¼ 8.8 Hz, 2H, aromatic). 13C NMR (500 MHz, CDCl3)
d 48.9, 118.8,
124.3,128.4,129.3,134.9,138.6,144.1,144.5,146.8,151.0,190.5. Anal.
C14H10N4O3 (C, H, N).
5.4. Preparation of 1-(4-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)-
ethanone (39) [51]
5.2.15. 2-(1H-Imidazo[4,5-b]pyridin-1-yl)-1-(4-nitrophenyl)-
ethanone (51)
A solution of triazole 17 (10 mmol) in CHCl3 (50 mL) and TEA
(10 mmol) was stirred for 10 min at 0e5 ꢀC. After this time, 2-
bromo-40-nitroacetophenone 21 (10 mmol) was added and the
mixture was stirred for 4 h, maintaining the temperature between
0 and 5 ꢀC. The solvent was removed under vacuum, the oil residue
was poured into ice water, and the resulting crude material was
extracted with dichloromethane (3 ꢆ 50 mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure; purification by means of
flash chromatography performed using silica gel 60
(230e400 mesh) and ethyl acetate as eluent gave the title
compound (62%) as orange powder; mp 162e164 ꢀC; IR (KBr) cmꢂ1
The title compound was isolated as orange powder (48%); mp
166e168 ꢀC; IR (KBr) cmꢂ1 1708, 1519, 1349; 1H NMR (500 MHz,
CDCl3)
d
5.68 (s, 2H, CH2), 7.30 (dd, J ¼ 5.1 and 6.9 Hz, 1H, aromatic),
7.61 (d, J ¼ 6.9 Hz, 1H, aromatic), 8.19 (s, 1H, aromatic), 8.24 (d,
J ¼ 8.2 Hz, 2H, aromatic), 8.45 (d, J ¼ 8.2 Hz, 2H, aromatic), 8.65 (d,
J ¼ 5.1 Hz, 1H, aromatic). 13C NMR (500 MHz, CDCl3)
d 51.2, 118.7,
124.7, 128.1, 129.2, 135.9, 138.7, 144.2, 145.2, 147.2, 151.0, 189.7. Anal.
C14H10N4O3 (C, H, N).
5.2.16. 2-(2H-Benzotriazol-2-yl)-1-(4-nitrophenyl)-ethanone (52)
The title compound was isolated as orange powder (72%); mp
166e168 ꢀC; IR (KBr) cmꢂ1 1698, 1519, 1349; 1H NMR (500 MHz,
1694, 1519, 1346; 1H NMR (DMSO-d6)
d 6.09 (s, 2H, CH2), 8.05 (s, 1H,
triazole), 8.22e8.38 (m, 2H, aromatic), 8.40e8.45 (m, 2H, aromatic),
CDCl3)
d
6.21 (s, 2H, CH2), 7.44 (dd, J ¼ 2.8 and 6.7 Hz, 2H, aromatic),
8.52 (s, 1H, triazole). Anal. C10H8N4O3 (C, H, N).
7.90 (dd, J ¼ 2.8 and 6.7 Hz, 2H, aromatic), 8.17 (d, J ¼ 8.9 Hz, 2H,
aromatic), 8.37 (d, J ¼ 8.9 Hz, 2H, aromatic). 13C NMR (500 MHz,
5.5. General procedure for the synthesis of indazole derivatives 53
and 54
CDCl3) d 62.1, 118.2, 124.1, 127.2, 129.3, 138.7, 145.1, 151.2, 189.2. Anal.
C14H10N4O3 (C, H, N).
A mixtureof theappropriateindazole44and 45(10mmol) and2-
bromo-40-nitroacetophenone 21 (20 mmol) was heated in an oil
bath for 1 h at 120e130 ꢀC. After cooling, the reaction mixture was
treated with 15 mL of ethyl acetate and the precipitate obtained was
collected and dried. The solid was suspended in water, treated with
stoichiometric amount of 1 N NaOH, and stirred for 1 h. The resulting
5.3. General procedure for the synthesis of pyrazole derivatives
36e38
A solution of appropriate pyrazole 14e16 (23 mmol) in dry DMF
(15 mL) and NaNH2 (23 mmol) was stirred for 10 min at 60 ꢀC,

L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
125
mixture was extracted with dichloromethane (3 ꢆ 50 mL), the
combined organic layers were dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. Recrystallization
of the residue from ethanol gave the desired products as solids.
5.6.3. Cytochrome P450 activity assay
Rat spleen microsomal Cyt P450 activity was measured by
following the NADPH-dependent reduction of cytochrome c as
described in our previous research [62]. Incubations were done at
25 ꢀC for 15 min in absence and presence of different concentra-
5.5.1. 2-(2H-Indazol-2-yl)-1-(4-nitrophenyl)-ethanone (53)
The title compound was isolated as orange powder (73%); mp
198e200 ꢀC; IR (KBr) cmꢂ1 1706, 1520, 1348; 1H NMR (500 MHz,
tions of compound 25 (5e500 mM). Reaction rates were determined
by reading the absorbance of reduced cytochrome c at 550 nm.
CDCl3)
d
5.91 (s, 2H, CH2), 7.12 (dd, J ¼ 8.3 and 7.1 Hz, 1H, aromatic),
7.33 (dd, J ¼ 8.3 and 7.2 Hz, 1H, aromatic), 7.70 (dd, J ¼ 8.3 and
9.6 Hz, 2H, aromatic), 8.06 (s, 1H, aromatic), 8.20 (d, J ¼ 8.8 Hz, 2H,
aromatic), 8.37 (d, J ¼ 8.8 Hz, 2H, aromatic). 13C NMR (500 MHz,
5.7. Antioxidant tests
5.7.1. Chemicals
CDCl3)
d
59.3, 117.5, 120.2, 122.4, 124.2, 124.4, 125.5, 126.8, 129.5,
Nicotinamide-adenine dinucleotide, reduced form disodium
salt, 1-diphenyl-2-picrylhydrazyl radical (DPPH), xylenol orange
were obtained from SigmaeAldrich Co. (St. Louis, MO, USA). All
other chemicals were from Merck (Frankfurt, Germany).
138.8, 149.4, 150.8, 190.4. Anal. C15H11N3O3 (C, H, N).
5.5.2. 2-(2H-7-Nitroindazol-2-yl)-1-(4-nitrophenyl)-ethanone (54)
The title compound was isolated as orange powder (66%); mp
214e216 ꢀC; IR (KBr) cmꢂ1 1712, 1520, 1531, 1348, 1333; 1H NMR
5.7.2. Methods
(500 MHz, CDCl3)
d
6.13 (s, 2H, CH2), 7.29 (dd, J ¼ 8.2 and 7.7 Hz, 1H,
All the imidazole derivatives analyzed show a major absorption
in the 220e260 nm range.
aromatic), 8.14 (d, J ¼ 8.2 Hz, 1H, aromatic), 8.23 (d, J ¼ 8.4 Hz, 2H,
aromatic), 8.37 (s, 1H, aromatic), 8.38e8.44 (m, 3H, aromatic). 13C
NMR (500 MHz, CDCl3)
136.2, 137.9, 138.3, 140.9, 143.2, 189.4. Anal. C15H10N4O5 (C, H, N).
d
60.0, 120.7, 124.5, 125.7, 127.5, 128.7, 129.7,
5.7.2.1. Scavenger effect on superoxide anion. Superoxide anion was
generated in vitro as described by Russo et al. [63]. A total volume of
1 mL of the assay mixture contained: 100 mM triethanolamine-
diethanolamine buffer, pH 7.4, 3 mM NADH, 25 mM/12.5 mM
5.6. Enzyme assays
EDTA/MnCl2, 10 mM
different concentrations of compounds 25, 29, 48, and 50
(25e50e100
M). After 20 min incubation at 25 ꢀC, the decrease in
absorbance at
percentage of inhibition of NADH oxidation with respect to control.
SOD (40 mU/ml) was used as reference compound.
b-mercapto-ethanol; samples contained
5.6.1. NOS isoenzymes
Neuronal rat recombinant nitric oxide synthase isolated from
a Baculovirus overexpression system in SF9 cells, was purchased
from ALEXIS. Inducible recombinant nitric oxide synthase isolated
from mouse macrophages were purchased from SigmaeAldrich.
Endothelial nitric oxide synthase was prepared by a transformed
endothelial cell (EC) line from the heart of C57BL/6 mice (H5V). EC
(passage 5) were cultured in flasks (Falcon, Becton Dickinson) until
confluent in Dulbecco’s modified Eagle’s medium (Life Technolo-
gies), with 10% fetal calf serum, 1 mM glutamine and antibiotics and
incubated at 37 ꢀC in a humidified 5% CO2 atmosphere. The medium
was changed every 2 days and subcultures were performed every
4e5 days following treatment with trypsin-EDTA. ECs were scraped
andwashed inphosphate-bufferedsaline. Approximately1 ꢆ109 ECs
were suspended in 1.5 mL of 50 mM TriseHCl pH 7.4 containing
10 mM EDTA, 5 mM glucose, 1.15% w/v KCl, 0.1 mM DTT, 2 mg/l
leupeptin, 2 mg/l pepstatin, and 44 mg/l phenylmethylsulfonyl
fluoride. The cell suspensions were homogenized by sonication
twice for 5 s with a Soniprep and then centrifuged at 0 ꢀC (20,000ꢆ g
for 20 min). The supernatant was then used for the enzymatic assay.
m
l
¼ 340 nm was measured. Results are expressed as
5.7.2.2. Quenching of DPPH. The free radical-scavenging capacity of
different concentrations of 25, 29, 48, and 50, was tested by their
ability to bleach the stable 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH) [64]. The reaction mixture contained 86
different concentrations of compounds 25, 29, 32, 48, and 50
(50e100e500 M) in 1 mL of ethanol. After 10 min at room
temperature the absorbance was recorded at 517 nm. Results are
expressed as percentage decrease in absorbance at
¼ 517 nm with
respect to control. Trolox (20 M) was used as reference compound.
mM DPPH and
m
l
m
5.7.2.3. Determination of lipid hydroperoxide levels in the plasma of
a healthy donor. Plasmatic lipid hydroperoxide levels were evalu-
ated by oxidation of Feþ2 to Feþ3 in the presence of xylenol orange
(FOX assay) at
l
¼ 560 nm [65]. Heparinized venous blood was
collected after overnight fasting; plasma was separated by centri-
5.6.2. Enzymatic assay
fugation at 800 g for 20 min. Plasma aliquots (500 ml) were diluted
The assay for NO synthase activities was carried out according to
Hevel and Marletta [61], measuring the rate of conversion of
oxyhemoglobin to methemoglobin using a Hitachi UV 2000 spec-
trophotometer. A reference cuvette was charged with 5 mM
oxyhemoglobin (human A0, purchased from SIGMA) in 100 mM
1:1 with oxygenated PBS and incubated at 37 ꢀC for 2 h with or
without different concentrations of compounds 25, 29, and 50
(10e50e100
tained using hydrogen peroxide (0.2e20
m
M) in a total volume of 1 mL. Calibration was ob-
M). Results are expressed
m
as percentage of inhibition respect to control (plasma incubated in
absence of test compounds).
Hepes (pH 7.4) to a final volume of 500
tained: 50
-arginine, imM Mgþþ (required only for iNOS),
170 M Dithiothreitol (DTT), 100 M NADPH, 4 M FAD, 12 M BH4,
1 mM Caþþ (required only for eNOS and nNOS), 20 U/ml Calmod-
ulin (required only for eNOS and nNOS), 10 l of DMSO (or the same
ml. A typical sample con-
mM L
m
m
m
m
5.7.3. Partition coefficient
Calculation of cLogP of compounds 25, 29, and 50, was afforded
using an Advanced Chemistry Development (ACD/Labs) Software
Solaris (4.67).
m
volume of DMSO solution of test compounds to a final concentra-
tion of 500, 100 and 50 mM), enzymatic extract (1.2 U/ml for nNOS
and iNOS and supernatant derived for 1 ꢆ109 ECs eNOS), and 5
mM
oxyhemoglobin in 100 mM Hepes, pH 7.4. The Hepes buffer was
preheated prior to use. Under these conditions, the NO formed
reacts with oxyhemoglobin to yield methemoglobin which can be
Acknowledgments
This work was supported by grants from the Italian MIUR and
the University of Catania.
measured at
l
¼ 401 nm.

126
L. Salerno et al. / European Journal of Medicinal Chemistry 49 (2012) 118e126
[36] L. Salerno, V. Sorrenti, F. Guerrera, M.C. Sarvà, M.A. Siracusa, C. Di Giacomo,
References
A. Vanella, Pharm. Pharmacol. Commun. 5 (1999) 491e494.
[37] C. Di Giacomo, V. Sorrenti, L. Salerno, V. Cardile, F. Guerrera, M.A. Siracusa,
M. Avitabile, A. Vanella, Exp. Biol. Med. 228 (2003) 486e490.
[38] V. Sorrenti, C. Di Giacomo, L. Salerno, M.A. Siracusa, F. Guerrera, A. Vanella,
Nitric Oxide 5 (2001) 32e38.
[39] U. Dirnagll, C. Iadecola, M.A. Moskowitz, Trends Neurosci. 22 (1999) 391e397.
[40] S.A. Lipton, P.A. Rosemberg, N. Engl. J. Med. 330 (1994) 139e143.
[41] B. Spinnewyn, S. Cornet, M. Auget, P.E. Chabrier, J. Cerebr. Blood Flow. Metab.
19 (1999) 139e143.
[42] P.E. Chabrier, M. Auget, B. Spinnewyn, S. Auvin, S. Cornet, C. Demerle-Pallardy,
C. Guilmard-Favre, J.G. Marin, B. Pignol, V. Gillard-Roubert, C. Roussillot-
Charnet, J. Schultz, I. Viossat, D. Bigg, S. Moncada, Proc. Natl. Acad. Sci. U.S.A.
96 (1999) 10824e10829.
[43] S. Auvin, M. Auget, E. Navet, J.H. Harnett, I. Viossat, J. Schulz, D. Bigg,
P.E. Chabrier, Bioorg. Med. Chem. Lett. 13 (2003) 209e212.
[44] O. Vajragupta, C. Boonyarat, Y. Murakami, M. Tohda, K. Musatmoto, A.J. Olson,
H. Watanabe, Free Radical Res. 40 (2006) 685e695.
[45] K. Watanabe, Y. Morinaka, Y. Hayashi, M. Shinoda, H. Nishi, N. Fukushima,
T. Watanabe, A. Ishibashi, S. Yuki, M. Tanaka, Bioorg. Med. Chem. Lett. 18
(2008) 1478e1483.
[46] T. Hussain, H.L. Siddiqui, M. Zia-ur-Rehman, M.M. Yasinzai, M. Parvez, Eur. J.
Med. Chem. 44 (2009) 4654e4660.
[47] D. Olender, J. Zwawiak, V. Lukianchuk, R. Lesyk, A. Kropacz, A. Fojutowsk,
L. Zaprutko, Eur. J. Med. Chem. 44 (2009) 645e652.
[48] V. Sorrenti, L. Salerno, C. Di Giacomo, R. Acquaviva, M.A. Siracusa, A. Vanella,
Nitric Oxide 14 (2006) 45e60.
[49] D. Nardi, A. Tajana, A. Leonardi, R. Pennini, F. Portioli, M.J. Magistretti,
A. Subissi, J. Med. Chem. 24 (1981) 727e731.
[50] M. Nazarinia, A. Sharifian, A. Shafiee, J. Heterocycl. Chem. 32 (1995) 223e225.
[51] G. Roman, J.Z. Vlahakis, D. Vukomanovic, K. Nakatsu, W.A. Szarek, Chem-
MedChem 5 (2010) 1541e1555.
[52] J. Elguero, A. Frucher, R. Jacquier, Bull. Soc. Chim. Fr. 66 (1966) 2075e2084.
[53] B. Skagerberg, D. Bonelli, S. Clementi, G. Cruciani, G. Ebert, Quant, Struct-Act.
Relat. 8 (1989) 32e38.
[54] G. Stefanchich, M. Artico, F. Corelli, S. Massa, Synthesis (1983) 757e759.
[55] A.P. Fernandez, A. Pozo-Rodrigalvarez, J. Serrano, R. Martinez-Murrillo, Curr.
Pharm. Des 16 (2010) 2837e2850.
[56] L. M. Yagupol’skii, B.E. Gruz, Zhurnal Obshchei Khimii 31 (1961) 1315e1320.
[57] B. Egner, F. Giordanetto, T. Inghardt, WO 2006068594 (2006).
[58] S. Ganguly, B. Razdan, Indian J. Heterocycl. Chem. 14 (2005) 253e254.
[59] R. Pellicciari, M. Curini, N. Spagnoli, Archiv. der Pharmazie 317 (1984) 38e41.
[60] C.-Y. Wu, K.-Y. King, C.-J. Kuo, J.-M. Fang, Y.-T. Wu, M.-Y.- Ho, C.-L. Liao, J.-
J. Shie, P.-H. Liang, C.-H. Wong, Chem. Biol. 13 (2006) 261e268.
[61] J.M. Hevel, M.A. Marletta, in: L. Packer (Ed.), Methods in Enzymology, vol. 233,
Academic Press, San Diego, 1994, pp. 250e258.
[62] A. Campisi, C. Di Giacomo, V. sorenti, R. Castana, C. La Delfa, A. Vanella, Acta
Europea Fertilitatis 25 (1994) 295e297.
[63] A. Russo, R. Acquaviva, A. Campisi, V. Sorrenti, C. Di Giacomo, G. Virgata,
A. Vanella, Cell Biol. Toxicol. 16 (2000) 91e98.
[1] S. Daff, Nitric Oxide 23 (2010) 1e11.
[2] L. Zhou, D.Y. Zhu, Nitric Oxide 20 (2009) 223e230.
[3] H. Li, T.L. Poulos, J. Inorg. Chem. 99 (2005) 293e305.
[4] W.K. Alderton, C.E. Cooper, R.G. Knowles, Biochem. J. 357 (2001) 593e615.
[5] M.A. Marletta, J. Biol. Chem. 268 (1993) 12231e12234.
[6] R.M.J. Palmer, A.G. Ferrige, S. Moncada, Nature 327 (1987) 524e526.
[7] L.J. Ignarro, G.M. Buga, G.M. Wood, R.E. Byrns, G. Chauduri, Proc. Natl. Acad.
Sci. U.S.A. 84 (1987) 9265e9269.
[8] T.M. Dawson, V.L. Dawson, S.H. Snyder, Ann. Neurol. 32 (1992) 297e311.
[9] P.L. Feldman, O.W. Griffith, D.J. Stuehr, Chem. Eng. News 20 (1993) 26e38.
[10] S. Moncada, E.A. Higgs, Br. J. Pharmacol. 147 (2006) S193eS201.
[11] D. Regoli, Curr. Pharm. Des. 10 (2004) 1667e1676.
[12] G. Dijkstra, H. van Goor, P.L. Jansen, H. Moshage, Curr. Opin. Investig. Drugs 5
(2004) 529e536.
[13] J.F. Kerwin, J.R. Lancaster, P.L. Feldman, J. Med. Chem. 38 (1995) 4343e4362.
[14] S. Moncada, E.A. Higgs, Pharmacol. Rev. 43 (1991) 109e142.
[15] S. Ashina, L. Bendtsen, M. Ashina, Curr. Pain Headache Rep. 9 (2005) pp.
415e422.
[16] C.L. Gibson, T.C. Coughlan, S.P. Murphy, Glia 50 (2005) 417e426.
[17] F.R. Cochran, J. Selph, P. Sherman, Med. Res. Rev. 16 (1996) 547e563.
[18] R. Pannu, I. Singh, Neurochem. Int. 49 (2006) 170e182.
[19] Y.Z. Wang, Y.Q. Cao, J.N. Wu, M. Chen, X.Y. Cha, World J. Gastroenterol. 11
(2005) 46e50.
[20] B.A. Narayanan, N.K. Narayanan, B. Simi, B.S. Reddy, Cancer Res. 63 (2003)
972e979.
[21] A. Orucevic, J. Bechberger, A.M. Green, R.A. Shapiro, T.R. Billiar, P.K. Lala, Int. J.
Cancer 81 (1999) 889e896.
[22] J. Svec, P. Ergang, V. Mandys, M. Kment, J. Pácha, Int. J. Exp. Pathol. 91 (2010)
44e53.
[23] J.P. Peng, S. Zheng, Z.X. Xiao, S.Z. Zhang, J. Zhejiang, Univ. Sci. 4 (2003) 221e227.
[24] P. Lukes, H. Pàcovà, T. Kucera, D. Vesely, J. Martinek, J. Asti, Folia Biol. 54
(2008) 141e145.
[25] K.H. Lim, B.B. Ancrile, D.F. Kashatus, C.M. Counter, Nature 452 (2008)
646e649.
[26] Z.J. Shang, J.R. Li, J. Oral Pathol. Med. 34 (2005) 134e139.
[27] R.B. Silverman, Acc. Chem. Res. 42 (2009) 439e451.
[28] E.P. Erdal, E.A. Litzinger, J. Seo, Y. Zhu, H. Ji, R.B. Silverman, Curr. Top. Med.
Chem. 5 (2005) 603e624.
[29] K.T. Symons, M.E. Massari, P.M. Nguyen, T.T. Lee, J. Roppe, C. Bonnefous,
J.E. Payne, N.D. Smith, S.A. Noble, M. Sablad, N. Rozenkrants, Y. Zhang, T.S. Rao,
A.K. Shiau, C.A. Hassig, Mol. Pharmacol. 76 (2009) 153e162.
[30] A. Tafi, L. Angeli, G. Venturini, M. Travagli, F. Corelli, M. Botta, Curr. Med.
Chem. 13 (2006) 1929e1946.
[31] A.C. Tinker, A.V. Wallace, Curr. Top. Med. Chem. 6 (2006) 77e92.
[32] L. Salerno, V. Sorrenti, C. Di Giacomo, G. Romeo, M.A. Siracusa, Curr. Pharm.
Des. 8 (2002) 177e200.
[33] D.J. Wolff, G.A. Datto, R.A. Samatovicz, R.A. Tempsick, J. Biol. Chem. 268 (1993)
9425e9429.
[34] R.M. Chabin, E. Mc Cauley, J.R. Calaycay, T.M. Kelly, K.L. Mac Nau, G.C. Wolfe,
N.I. Hutchinson, S. Madhusudanaraja, J.A. Schmidt, J.W. Kozarich, K.K. Wong,
Biochemistry 35 (1996) 9567e9575.
[64] F. Bonina, A. Saija, A. Tomaino, R. Lo Cascio, P. Rapisarda, J.C. Dederen, Int. J.
Cosm. Sci. 20 (1998) 331e342.
[65] S.P. Wolff, in: L. Packer (Ed.), Methods in Enzymology, 233, Academic Press,
San Diego, 1994, pp. 182e189 1994.
[35] L. Salerno, V. Sorrenti, F. Guerrera, M.C. Sarvà, M.A. Siracusa, C. Di Giacomo,
A. Vanella, Pharmazie 54 (1999) 685e690.
[66] M. Dixon, Biochem. J. 55 (1953) 170e171.