Arylnitroalkenes as scavengers of macrophage-generated oxidants

Article abstract of DOI:10.1016/j.ejmech.2013.12.029

Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization intermediates, whose concentrations are tightly regula

Full text of DOI:10.1016/j.ejmech.2013.12.029

European Journal of Medicinal Chemistry 74 (2014) 31e40
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Arylnitroalkenes as scavengers of macrophage-generated oxidants
,
,
Laura Celano a b, Claudio Carabio a, Renata Frache c, Nicolás Cataldo a c, Hugo Cerecetto c,
**
Mercedes González c , Leonor Thomson
,
a,b,
*
a Enzimología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
b Center for Free Radicals and Biomedical Research, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, Montevideo 11800, Uruguay
c Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several
pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization in-
termediates, whose concentrations are tightly regulated by specialized enzyme repertoires and should
remain undisturbed by the addition of exogenous antioxidant molecules, as already demonstrated by
intervention studies with antioxidant vitamins. Our goal was to develop specific antioxidants able to
scavenge peroxynitrite anion, as well the radicals derived from the homolytic decomposition of its
conjugated acid, nitrogen dioxide and hydroxyl radical. Fourteen substituted nitroalkenes, seven 4-
substituted 1-(2-nitro-1Z-ethenyl)benzene, and seven 4-substituted (2-nitro-1Z-propenyl)benzene,
with different stereochemical and electronic characteristics were synthesized and tested. Compounds
with the electron donor group N,N-dimethylamino showed the highest reaction rates against perox-
ynitrite, and also reacted with its homolytic decomposition products, ꢀOH and ꢀNO2. While 1,1-
dimethylamino-4-(2-nitro-1Z-ethenyl)benzene came up as a lead for future developments without the
risk of interfering with signalization pathways, since it was highly specific for peroxynitrite and per-
oxynitrite derived radicals, its methylated analogous 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)ben-
Received 21 October 2013
Received in revised form
19 December 2013
Accepted 21 December 2013
Available online 3 January 2014
Keywords:
Nitroalkenes
Peroxynitrite
Nitric oxide
Nitrogen dioxide
Scavengers
Antioxidants
zene was less specific and also reacted with NO and O2 ꢁ, the biological precursor of H2O2.
Ó 2014 Elsevier Masson SAS. All rights reserved.
ꢀ
ꢀ
1. Introduction
defences. However, an imbalance in the normal relationship be-
tween oxidants and physiological antioxidants is in the basis of
Reactive species (RS, it will be used in general to account for
both oxygen and nitrogen species) are continuously produced by
normal metabolism, and its deleterious effects on biological mol-
ecules are kept under control by an organized array of antioxidant
several pathologies.
Although macrophage respiratory burst with the consequent
generation of RS is a key weapon against pathogens, if excessive or
unresolved could cause organ dysfunction. In particular, perox-
ynitrite the product of the diffusion-controlled reaction between
two radicals, nitric oxide and superoxide, is a relevant biological
oxidant and a nitrating agent [1].
Several human diseases including rheumatoid arthritis [2],
types 1 and 2 diabetes [3,4], cardiovascular disease [5e8], and
metabolic syndrome [9], involve activation of the immune system
and the associated oxidative stress as contributors to their etiology
and progression. However, therapeutically targeting oxidative
stress in various diseases has proven more problematic than first
anticipated. Many natural and synthetic antioxidants have been
tested as potential therapeutic tools with variable results [10].
Among them, nitro-fatty acids (NO2-FA), that are electrophilic
Michael acceptors, react with nucleophiles such as cysteine thio-
Abbreviations: RS, reactive species; NO2-FA, nitro-fatty acids; LPS, lipopolysac-
charide; Fe2þ-Cyt c, Fe2þ-cytochrome c; Fe3þ-Cyt c, Fe3þ-cytochrome c; DHR,
dihydrorhodamine; RH, rhodamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; DAF-FM, 4-amino-5-methylamino-20,70 difluoro-
fluorescein diacetate; IFNg, interferon-gamma; NOS, nitric oxide synthase; 1400W,
N-(3-(aminomethyl)benzyl) acetamidine; MetHb, methemoglobin; Hb, hemoglo-
bin; PMA, phorbol myristate acetate; DMSO, dimethyl sulfoxide; HbO2, oxyhemo-
globin; DMEM, Dulbecco’s modified Eagle medium; dPBS, Dulbecco’s phosphate
buffer saline; SOD, superoxide dismutase.
* Corresponding author. Enzimología, Instituto de Química Biológica, Facultad de
Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay.
Tel.: þ598 25258618x7214.
** Corresponding author. Grupo de Química Medicinal, Laboratorio de Química
Orgánica, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo
11400, Uruguay. Tel.: þ598 25258618xx7216.
late, imidazole ring of histidine and -amino group of lysine resi-
dues [11]. In vitro studies showed that NO2eFAs inhibit neutrophil
and platelet function, vascular smooth muscle cell proliferation,
3
E-mail addresses: megonzal@fq.edu.uy (M. González), leonorthomson@gmail.
com, lthomson@fcien.edu.uy (L. Thomson).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.029

32
L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
expression of endothelial adhesion molecule, lipopolysaccharide
(LPS)-induced macrophage activation and transmigration [12e14].
Additionally, we found that nitroalkene (I) (Fig. 1) was able to
2.2. ꢀNO release
ꢀ
The ability of the synthesized compounds to release NO was
addressed first measuring hemoglobin oxidation by 100 M of the
release NO in the absence and the presence of cysteine at 37 ꢂC
m
ꢀ
[15].
compounds. Exclusively nitroalkenes 1b, 2b, 1f and 2f released
Knowing that the biological activity of this nitroalkene was due
to the nitroethenyl moiety, we developed a molecular simplification
programme analyzing herein simpler nitroalkene derivatives.
Consequently, we synthesized and tested different nitroethenyl
derivatives containing substituents in the 4-position of the phenyl
ring, with different stereoelectronic properties. In this kind of sys-
tems, the nitro group is distant from the aromatic ring and conju-
gated via the ethylene system, allowing long-distance transmission
of electronic effects [16]. In addition, the bioisoster of the phenyl
moiety, 2-thienyl group, was also studied (Fig. 1). The reactivity of
these nitroalkenes as scavengers of different RS, peroxynitrite and
its derivatives radicals, was assayed chemically. In vitro analysis in
murine macrophages was performed to determine the capacity to
scavenge peroxynitrite derived or precursor radicals.
measurable amounts of ꢀNO under our experimental conditions
ꢀ
(Table 1). The rate constants of NO release were similar to previ-
ously reported values for well known ꢀNO donors [19], ranging from
0.06 to 0.25 ꢄ 10ꢁ3 minꢁ1 for the N,N-dimethylamino derivatives
(1b and 2b), and from 0.48 to 1.32 ꢄ 10ꢁ3 minꢁ1 for the [1,3]dioxole
derivatives (1f and 2f). ꢀNO release was also demonstrated for
nitroalkenes 1b, 2b, 1f and 2f with an electrochemical sensor, with
similar results (Fig. 2).
2.3. Reactivity with peroxynitrite
2.3.1. Second order rate constant estimation with peroxynitrite
The reactivity of the studied nitroalkenes as antioxidants was
analyzed by their reaction with peroxynitrite, an important bio-
logical oxidant that reacts by one or two electron mechanisms.
Since the rate constant of Fe2þ-cytochrome c (Fe2þ-Cyt c) oxidation
by peroxynitrite is already known (k ¼ 2.4 ꢄ 104 Mꢁ1 sꢁ1 at pH 7.4,
and 37 ꢂC) [20] the magnitude of this oxidation can be easily
determined at 550 nm, through competition experiments per-
formed to determine the second order rate constant for the reaction
between peroxynitrite and the panel of nitroalkenes. Given that
pseudo-first order conditions were unattainable due to solubility
limitations, nitroalkenes were used at the same concentration as
Fe2þ-Cyt c (360 mM). Under those conditions it can be assumed that
w90% of peroxynitrite reacts directly with the targets instead of
2. Results
2.1. Chemistry
2.1.1. Synthesis
Fourteen nitroalkenes were obtained via Henry reaction. The
process took place in one pot using the corresponding aromatic
aldehyde and nitromethane or nitroethane in the presence of cat-
alytic amounts of n-butylamine (Scheme 1) [15,17,18]. All nitro-
alkenes were characterized by 1H NMR, COSY experiments, MS, and
UV spectroscopy (Table 1). They were obtained as a unique isomer
around the double bound. In all cases the Z isomer was obtained,
this was determined by NOE experiments (see Supplementary
Material). The purity of the synthesized compounds was estab-
lished by TLC and microanalysis. Only compounds with analytical
results for C, H and N, within ꢃ0.4 of the theoretical values were
considered pure enough.
undergoing homolysis, which occurs with a rate constant of 0.9 sꢁ1
.
Since the concentration ratio between the Fe3þ-Cyt c generated in
the absence and in the presence of the nitroalkene competitor is
proportional to the relationship between the kinetic constants, the
desired rate constant can be estimated using Eq. (1) [21,22].
Fe2þꢁCyt c
½
ꢅ
0
ln
½Fe2þꢁCyt cꢅ0ꢁ½Fe3þꢁCyt cꢅN
½Nitroalkeneꢅ0
k1
k2
¼
(1)
ln
½Nitroalkeneꢅ0ꢁ½PnitroalkeneꢅN
where the concentration of Pnitroalkene was calculated as the dif-
ference between Fe3þ-Cyt c concentration obtained after perox-
ynitrite exposure in the absence and in the presence of nitroalkene.
The values obtained were w103 Mꢁ1 sꢁ1 (Table 2) one order of
magnitude lower than the reported rate constant for the reaction
between peroxynitrite and Fe2þ-Cyt c. The substituents at the 4-
position of the aromatic ring increased in a different extent the
reactivity of the compounds with peroxynitrite. In particular, the
N,N-dimethylamino substituted derivatives, 1b and 2b, reacted
with peroxynitrite more than 2 times faster than the unsubstituted
ones, 1a and 2a. Regarding the relationship between the rate con-
stants of series 1 and 2, except for 2f, were equal or slightly lower in
series 1. The kinetic constants obtained for the direct reaction of
peroxynitrite with nitroalkenes were in the same order than with
glutathione [23] but lower than the reaction with oxyhemoglobin
[24] and the amino acid selenocysteine [25]. Higher reaction rates
were described for other synthetic scavengers, like manganese
porphyrins (105 to 107 Mꢁ1 sꢁ1
(<107 Mꢁ1 ꢁ1) [27].
) [26] and iron porphyrins
s
2.3.2. Reactivity with peroxynitrite derived radicals
Kinetic results showed that the fourteen studied nitroalkenes
reacted directly with peroxynitrite, but in addition they might
interact with the radicals derived from it, ꢀNO2 and ꢀOH.
Fig. 1. Design of nitroalkenes based on benzofuroxan containing nitroethenyl moiety
releasing ꢀNO.

L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
33
Scheme 1. Synthesis of the studied nitroalkenes.
Dihydrorhodamine (DHR), is oxidized by peroxynitrite indirectly,
2.4. Biology
via its radical decomposition products leading to rhodamine (RH)
production [28]. The results of DHR protection are shown in Fig. 3.
Most nitroalkenes, except for 2e, were able to protect DHR
2.4.1. Reaction with peroxynitrite generated by J774 cells
Since peroxynitrite is generated under inflammatory conditions
as a continuous flux, we tested the reactivity of nitroalkenes 1b, 2b,
1f, and 2f with peroxynitrite derived radicals generated by acti-
vated murine macrophages J774 cells. Previously, the toxicity of
nitroalkenes was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) cell viability assay. After 2 h
(23.5 mM) from peroxynitrite (50 mM) with varying efficiency. Most
propenyl derivatives (series 2) showed a lower protection on DHR
oxidation than the corresponding ethenyl homologous (series 1).
The nitroalkenes containing N,N-dimethylamino-, 1b and 2b, the
[1,3]dioxole substituent, 1f, and thienyl moiety, 1g, showed the
highest protection, preventing DHR oxidation in 59, 55, 45 and 44%,
respectively. Although no significant differences due to the nature
of the substituent were observed on the direct reaction, determined
by the competition assay, these electron-donor groups, N,N-dime-
thylaminophenyl, 4-(benzo[d] [1,3]dioxol-5-yl) and 2-thienyl,
of incubation in the presence of 10 and 20
mM of nitroalkenes,
survivals higher than 85% were obtained, demonstrating that
within the time of the assay macrophages are not affected in their
integrity by the four studied compounds. A cellular flux of perox-
ynitrite was established through macrophage simultaneous stim-
showed greater ability to trap peroxynitrite derived radicals, OH
ulation for ꢀNO and Oꢁ2 ꢀ production and DHR (15
mM) oxidation was
ꢀ
and ꢀNO2. These results obtained by bolus addition of peroxynitrite
are only an approximation to what is expected to occur in biological
conditions. The more effective peroxynitrite scavenger and ꢀNO
releasing agents, the N,N-dimethylamino derivatives, 1b and 2b,
and the [1,3]dioxole derivatives, 1f and 2f, were selected to explore
their capacity to trap peroxynitrite generated by activated
macrophages.
followed for 120 min in the absence and the presence of nitro-
alkenes (10
mM). A 100% of DHR oxidation corresponded to
0.07 ꢃ 0.01 RFU minꢁ1. The N,N-dimethylamino derivatives, 1b and
2b (65.4 and 73% respectively), and the nitropropenylbenzodioxole
2f (63%) were the most active molecules against peroxynitrite
derived radicals, while the nitroethenyl derivative 1f showed a
lower protection (37.4%) (Fig. 4).
Table 1
UV spectral characteristics and ꢀNO releasing properties of the studied compounds.
b
Cpd.
lmax
3
(mMꢁ1 cmꢁ1
)
ꢀNO release (ꢄ10ꢁ3 minꢁ1
)
1a
1b
NM
362
461
NM
NM
359
375
291
356
316
365a
446
320
324
351
366
363
NA
3.9
4.9
NA
NA
3.9
3.2
3.4
1.1
27.7
2.0
3.1
6.0
21.4
8.9
9.5
10.5
0
0.25 ꢃ 0.03
1c
1d
1e
1f
0
0
0
0.06 ꢃ 0.02
1g
2a
2b
0
0
0.48 ꢃ 0.06
2c
2d
2e
2f
0
0
0
1.32 ꢃ 0.14
2g
0
NM e no maximum inside the explored wavelengths (280e700 nm).
NA e not applicable.
Fig. 2. ꢀNO release. Representative profile obtained with the ꢀNO-sensor by addition of
a
Shoulder.
increasing concentrations of nitroalkene 2b (40e80
mM, as indicated by the arrows), to
b
Determined as MetHb formation at 401 nm.
the chamber containing 75 mM phosphate, pH 7.4, at 37 ꢂC.

34
L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
Table 2
Reactivity with peroxynitrite.
a,b,c
Cpd.
k (Mꢁ1 sꢁ1
)
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2.4 ꢃ 0.6 ꢄ 103
6.2 ꢃ 0.3 ꢄ 103
4.5 ꢃ 0.9 ꢄ 103
5.9 ꢃ 1.0 ꢄ 103
5.6 ꢃ 0.7 ꢄ 103
2.4 ꢃ 0.6 ꢄ 103
4.9 ꢃ 0.3 ꢄ 103
2.4 ꢃ 0.8 ꢄ 103
5.2 ꢃ 0.9 ꢄ 103
2.5 ꢃ 0.3 ꢄ 103
5.4 ꢃ 0.5 ꢄ 103
4.5 ꢃ 0.9 ꢄ 103
5.4 ꢃ 0.9 ꢄ 103
2.6 ꢃ 0.5 ꢄ 103
2g
a
Data are the mean ꢃ SD of three independent
experiments.
b
c
Nitroalkenes were assayed at 1.5e13
The second order rate constants for the reaction
mM.
Figꢁ. 4. DHR oxidation by J774-cells generated peroxynitrite. Simultaneous ꢀNO and
between nitroalkenes and peroxynitrite were
determined by a competition assay.
(400 U mLꢁ1)/LPS (8
m
g mLꢁ1) addition, fol-
ꢀ
O2 production were induced by INF
g
lowed after 5 h by PMA (4
m
g mLꢁ1). DHR (15
M) were added together with DHR. Data are
mM) oxidation was measured at 520 nm
(
lexc ¼ 485 nm) for 1 h. Nitroalkenes (10
m
means ꢃ SD of triplicates from a representative experiment. Significant differences
with and without nitroalkenes (*p < 0.05), and between 1b and 1f (**p < 0.05) were
found.
2.4.2. Trapping of peroxynitrite radical precursors
Since peroxynitrite production by activated macrophages arises
from the simultaneous production of NO and O2 ꢁ, we decided to
explore the reactivity of the selected compounds, 1b, 2b, 1f, and 2f,
with the individual radical precursors. The capacity of these com-
ꢀ
ꢀ
interferon-gamma (INF-
g
)/LPS challenged macrophages was tested
M) increased near
M). Hemoglobin
ꢀ
(Fig. 5B). Basal NO production (2.21 ꢃ 0.01
m
threefold after macrophage activation (6.1 ꢃ 1.1
m
pounds (10
evaluated
m
M) to prevent peroxynitrite generation from ꢀNO, was
measuring
4-amino-5-methylamino-20,70-difluoro-
oxidation was mediated by ꢀNO, since the nitric oxide synthase
(NOS) inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W,
100 mM) prevented methemoglobin (MetHb) increase. The com-
pounds 2b, 1f and 2f also decreased hemoglobin (Hb) oxidation
(w70%), but it remained unchanged in the presence of 1b. Results
on Hb oxidation as well as the already mentioned with DAF-FM
fluorescein diacetate (DAF-FM) oxidation. All nitroalkenes were
able to protect DAF-FM oxidation by N2O3, a ꢀNO derived molecule,
in a significant extent (Fig. 5A). The pair 1b and 2b showed
important differences in the degree of protection between them
(28% and 74%, respectively), but the [1,3]dioxole derivatives, 1f and
2f, had similar effects (51.9% and 51.6% of protection, respectively).
In the cellular environment, the compounds could be reacting with
showed that the lack of the Ca-methylation that differentiates 1b
from 2b makes the former a poorer ꢀNO scavenger. In order to study
the reactivity with O2 ꢁ, J774 cells were activated with phorbol
ꢀ
either ꢀNO or N2O3. In order to clarify this point, the effect of 50
nitroalkenes on oxyHb (50
M) oxidation by ꢀNO generated by
mM
myristate acetate (PMA, 4
m
g mLꢁ1), and the nitroalkenes werꢁe
m
ꢀ
added after 30 min. The ability of the compounds to react with O2
was studied following the superoxide dismutase inhibitable Fe3þ
-
Cyt c reduction at 550 nm (Fig. 6). Propenyl derivatives showed the
highest protective effect with significant differences (*p < 0.05)
against the condition in the absence of the compounds, being 2f the
most reactive one (45.1 ꢃ 2.8%). No protection was provided by the
ethenyl derivatives, 1b and 1f. In summary, while both [1,3]dioxole
ꢀ
derivatives (1f and 2f) as 2b reacted with NO and also with su-
peroxide anion, 1b was more specific against the radicals derived
from peroxynitrite decomposition.
3. Discussion and conclusions
Fourteen 4-substituted-benzenes, with different electro-
chemical and structural properties bearing a nitroethenyl (series 1)
or a nitropropenyl (series 2) group, were synthesized and their
antioxidant properties assessed in vitro. All reacted directly with
peroxynitrite anion. The kinetic constants determined by compe-
tition with cytochrome c, at pH 7.4 and 37 ꢂC, were in the range of
2.4e6.2 ꢄ 103 Mꢁ1 sꢁ1 (Table 2). The derivatives belonging to series
1 (with the exceptions of 1a and 1f) showed higher rate constants
than the members of series 2, suggesting that the absence of the
methyl group, in the ethenyl-lateral chain, confers enhanced
reactivity independently of the substituent group at the 4-position
of the phenyl group.
Fig. 3. Reaction with peroxynitrite-derived radicals. DHR 123 (50
a bolus additions of peroxynitrite (50
M) at 37 ꢂC in 0.1 M phosphate buffer (pH 7.4,
0.1 mM DTPA) in the absence and in the presence of nitroalkenes (400 M), and RH
production was determined at 500 nm. RH formation in the absence of nitroalkenes
mM) was exposed to
m
m
(ONOOꢁ) was 23.5
mM. Significant differences between the assayed nitroalkenes and
ONOOꢁ alone (*p < 0.05), and between nitroalkenes series 1 vs series 2 (**p < 0.05)
were found.
While most studied nitroalkenes protected the fluorescent
probe DHR from peroxynitrite-derived radicals ꢀOH and ꢀNO2, again

L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
35
Fig. 5. Nitric oxide scavenging activity. NOS2 was induced in macrophage monolayer by IFN
g
(400 U mLꢁ1) and LPS (8
m
g mLꢁ1) for 5 h. A. Oxidation of DAF-FM diacetate (5
m
M) by
N2O3 was tested in the absence (þINF
g/LPS) and the presence of nitroalkenes (10 mM) by fluorescence (lexc 485 nm/lem 520 nm). Significant differences (*p < 0.05) between the
conditions without and with nitroalkene treatment, and between nitroalkenes 1b and 2b (**p < 0.05, n ¼ 4) were obtained. B. Oxidation of intracellular hemoglobin by ꢀNO was
explored by exposing intact erythrocytes (50
Significant differences (*p < 0.05) between the conditions without and with nitroalkenes 2b, 1f and 2f were obtained. Data are the means ꢃ SD from triplicates.
m
M hemoglobin) to pre-activated near confluence J774 cells (þIFNg/LPS) in the absence and the presence of 50 mM nitroalkenes.
the 2-nitroethenyl derivatives, 1aeg, were the most efficient. In
particular, the compounds possessing the electron donor group
N,N-dimethylamino, in 1b and 2b, as well as the phenyl bioisostere,
2-thienyl-derivative in 1g, showed the highest reaction rates with
peroxynitrite (6.2, 5.2 and 4.9 ꢄ 103 Mꢁ1 sꢁ1, respectively, Table 2)
and also the greatest ability to trap the radicals generated by the
homolytic decomposition of peroxynitrous acid, ꢀOH and ꢀNO2 (58,
59 and 52%, respectively, Fig. 3). When the reactions between the
nitroalkenes 1b or 2b and the radicals ꢀOH and ꢀNO2 were analyzed
theoretically, at ab initio level, the same tendencies that the
experimental results were observed (Fig. 7A), finding that the spin
surface of the 1b-OH adduct radical was more significant than the
corresponding surface for the methyl-analog, 2b-OH adduct radical
(Fig. 7B). Additionally, both propenyl derivatives, 2b and 2f, further
explained by the highest electron-donor characteristics of both
substituents, N,N-dimethylamino and [1,3]dioxole. The same effect,
electron-donor property, could be used to explain the different
behavior of 1a and 2a respect to 1g and 2g, where the thienyl
substituent is usually a phenyl bioisoster. Although cytotoxicity has
been linked to the electrophilicity, this was not observed in our case
where all the compounds at the concentrations and times tested
showed similar survival of macrophages with values close to 100%.
Beyond the partial results obtained from this work 1b highlighted
as quit specific against peroxynitrite and peroxynitrite derived
radicals, while the lack of reactivity against the two important
ꢀꢁ
signaling molecules, ꢀNO and O2 (Figs. 5 and 6), outlines this
compound as a good candidate for further studies. This compound
was one of the arylnitroalkenes analyzed able to release measur-
able amounts of ꢀNO (Table 1, and Fig. 2). Additionally, derivative 1b
adjusted to Lipinski’s rule of five. The rule serves as a guide to
determine if compounds will be orally bioavailable [29]. In this case
the analysis of compound 1b suggested that it would be orally
administered (Table 3). Since nitroarachidonate, a linear nitro-
alkene, shown at low micromolar concentrations different biolog-
ical actions including inhibition of platelet and macrophage
activation [14,30e32], thus 1b was considered as a lead and
together with structural analogous are currently being tested in
animal models of inflammation.
reacted with NO and O2 ꢁ, the radical precursors of peroxynitrite,
generated by activated macrophages (Figs. 5 and 6). When these
reactions were studied theoretically, with nitroalkenes 1b and 2f,
clear energetic preferences in favor of the [1,3]dioxole-derivative
were confirmed (Fig. 7C) as evidenced experimentally. Better
antioxidant properties that distinguish b and f pairs could be
ꢀ
ꢀ
4. Experimental
All starting materials were purchased from SigmaeAldrich Co.
(USA) and Acros Organics (Janssen Pharmaceutical, Geel, Belgium).
All solvents were dried and distilled prior to use. All the reactions
were carried out in a nitrogen atmosphere. All of the synthesized
nitroalkenes were chemically characterized by thin layer chroma-
tography (TLC), nuclear magnetic resonance (1H NMR) and
elemental microanalyses (CHN). Alugram SIL G/UV254 (Layer:
0.2 mm) (MachereyeNagel GmbH & Co. KG., Düren, Germany) was
used for TLC. The purity of nitroalkenes was determined by
elemental microanalyses performed on a Carlo Erba Model EA1108
elemental analyzer from vacuum-dried samples. The analytical
results for C, H and N, were within ꢃ0.4 of the theoretical values.
The 1H and 13 C NMR spectra were recorded on a Bruker DPX 400
(400 MHz), using TMS as the internal standard and with the indi-
cated deuterated solvent; the chemical shifts are reported in ppm
Fig. 6. Reactivity with superoxide anion. NADPH oxidase assembly was induced in J774
cells near to confluence by the addition of PMA (4
m
g mLꢁ1). After 30 min the ability of
ꢀꢁ
nitroalkenes (10
m
M) to scavenge O2 was determined by Fe3þ-Cyt c (50
m
M) reduc-
tion at 550 nm. Superoxide production in the absence of nitroalkenes (PMA) was
(d) and coupling constants (J) values are given in Hertz (Hz). Signal
multiplicities are represented by: s (singlet), d (doublet), and m
(multiplet). NOE diff experiments were performed to establish the
9.7 ꢃ 0.2
m
M minꢁ1 and in presence of SOD was 0.3 ꢃ 0.02
m
M minꢁ1. Data are the
means ꢃ SD of triplicates from a representative experiment. Significant differences
between PMA alone and plus 2b and 2f are shown (*p < 0.05, n ¼ 6).

36
L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
Fig. 7. Theoretical studies performed on the processes to obtain the different adduct radicals (A and C). Spin surfaces (pink, isovalue: 0.002) and electronic density surfaces (gray,
isovalue: 0.007) (B down) for 1b-OH adduct radical (left) and 2b-OH adduct radical (right) (B up). (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
stereochemistry around the double bond. Mass spectrometry ex-
periments were performed on a HEWLETT PACKARD MSD 5973.
Unless specified, absorbance and fluorescence were determined
using Varioskan Flash plate reader (Thermo, Vantaa, Finland).
134.8. Calculated analysis for C8H7NO2: C: 64.4; H: 4.7; N: 9.4.
Found: C: 64.0; H: 4.8; N: 9.5.
4.3. 1,1-Dimethylamino-4-(2-nitro-1Z-ethenyl)benzene (1b)
4.1. General procedure for the synthesis of 1-(2-nitro-1Z-ethenyl)
0.17 g (20%) of a red solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
arene (1aeg)
3.10 (s, 6H), 6.60 (d, 2H, J ¼ 8.00), 7.45 (d, 2H, J ¼ 8.00), 7.53 (d, 1H,
J ¼ 12.00), 8.00 (d, 1H, J ¼ 12.00); 13C NMR (CDCl3, 100 MHz)
A mixture of the corresponding aldehyde (1 eq), nitromethane
(2.1 eq), n-butylamine (0.14 mL) and EtOH (5.6 mL) was heated at
reflux until the aldehyde disappears (confirmed by TLC). The solid
was filtered off and washed with hexane to afford the corre-
sponding product.
d (ppm): 40.0,111.9, 117.2, 131.5, 132.1, 140.3,153.0. MS (EI, 70 eV) m/
z (abundance): 192 (Mþꢀ, 100%), 162 (2.30%), 145 (100%), 130
(20.3%), 77.0 (9.40%). Calculated analysis for C10H12N2O2: C: 62.5; H:
6.3; N: 14.6. Found: C: 62.8; H: 6.8; N: 14.2.
4.4. 1-Chloro-4-(2-nitro-1Z-ethenyl)benzene (1c)
4.2. 1-(2-Nitro-1Z-ethenyl)benzene (1a)
1.22 g (40%) of a white solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
2.66 g (22%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
7.45 (d, 2H, J ¼ 8.4), 7.52 (d, 2H, J ¼ 8.4), 7.58 (d,1H, J ¼ 13.6), 7.99 (d,
d
(ppm): 7.65 (d, 1H, J ¼ 14.8), 7.90 (d, 1H, J ¼ 14.8), 8.05 (m, 5H); 13
C
1H, J ¼ 13.6); 13C NMR (CDCl3, 100 MHz)
d
(ppm): 128.0, 129.5,
130.0, 133.0, 137.0, 137.5. MS (EI, 70 eV) m/z (abundance): 183 (Mþ
,
ꢀ
NMR (CDCl3, 100 MHz)
d
(ppm): 127.3, 128.6, 129.7, 130.1, 132.5,

L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
37
Table 3
100 MHz)
d (ppm): 14.7; 126.8, 127.9, 130.6, 132.3, 132.9, 135.9.
Lipinsky’s physicochemical properties for 1b.
Calculated analysis for C9H9NO2: C: 66.2; H: 5.6; N: 8.6. Found: C:
65.7; H: 6.1; N: 7.7. In complete agreement with previous data [17].
Lipinski descriptors
Cpd.
log P
MW
nON
4
nOHNH
0
4.11. 1,1-Dimethylamino-4-(2-nitro-1Z-propenyl)benzene (2b)
1b
2.21
192.2
1.23 g (63%) of a red solid. 1H NMR (CDCl3, 400 MHz)
2.52 (s, 3H), 3.07 (s, 6H), 6.74 (d, 2H, J ¼ 8.6), 7.43 (d, 2H, J ¼ 8.6),
8.11 (s, 1H); 13C NMR (CDCl3, 100 MHz)
(ppm): 14.4, 40.0, 111.8,
119.4, 132.6, 135.0, 142.7, 151.5. Calculated analysis for C11H14N2O2:
C: 64.1; H: 6.8; N: 13.6. Found: C: 65.0; H: 5.9; N: 13.0. In complete
agreement with previous data [17].
d (ppm):
33.8%), 170.2 (24%), 124.2 (16.7%), 107 (41.4%), 102.2 (11.7%). Calcu-
lated analysis for C8H6ClNO2: C: 52.3; H: 3.3; N: 7.6. Found: C: 52.8;
H: 3.9; N: 7.1.
d
4.5. 1-Bromo-4-(2-nitro-1Z-ethenyl)benzene (1d)
4.12. 1-Chloro-4-(2-nitro-1Z-propenyl)benzene (2c)
0.75 g (30%) of a white solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
7.55 (d, 1H, J ¼ 13.6), 7.62 (d, 2H, J ¼ 2.0), 7.64 (d, 1H, J ¼ 2.0), 7.97 (d,
2.39 g (70%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
1H, J ¼ 13.6); 13C NMR (CDCl3,100 MHz)
d
(ppm): 126.3,127.9,130.5,
d
(ppm): 2.46 (s, 3H), 7.39 (d, 2H, J ¼ 8.6), 7.46 (d, 2H, J ¼ 8.6), 8.05
132.0, 132.5, 145.5. MS (EI, 70 eV) m/z (abundance): 229.0 (Mþ þ 2,
37.0%), 227.0 (Mþꢀ, 37.5), 170.0 (97.2%), 169.1 (7.0%), 156.0 (26.6%),
100.0 (13.0%). Calculated analysis for C8H6BrNO2: C: 42.1; H: 2.6; N:
6.1. Found: C: 41.9; H: 3.1; N: 6.6.
ꢀ
(s, 1H); 13C NMR (CDCl3, 100 MHz)
d
(ppm): 14.5; 129.0, 129.5, 131.5,
134.5, 139.0, 139.5. Calculated analysis for C9H8ClNO2: C: 54.7; H:
4.1; N: 7.1. Found: C: 54.9; H: 4.2; N: 7.0. In complete agreement
with previous data [17].
4.6. 1-Methoxy-4-(2-nitro-1Z-ethenyl)benzene (1e)
4.13. 1-Bromo-4-(2-nitro-1Z-propenyl)benzene (2d)
0.40 g (15%) of a white solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.27 g (29%) of a white solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
3.89 (s, 3H), 7.16 (d, 2H, J ¼ 8.0), 7.52 (d, 1H, J ¼ 13.6), 7.55 (d, 2H,
2.45 (s, 3H), 7.32 (d, 2H, J ¼ 8.4), 7.62 (d, 2H, J ¼ 8.8), 8.02 (s, 1H); 13
C
J ¼ 8.0), 8.01 (d, 1H, J ¼ 13.6); 13C NMR (CDCl3, 100 MHz)
d (ppm):
NMR (CDCl3, 100 MHz)
d (ppm): 14.0, 124.0, 131.5, 132.0, 132.5,
55.7, 114.7, 126.5, 127.0, 131.5, 132.0, 153.0. MS (EI, 70 eV) m/z
(abundance): 179 (Mþꢀ, 46.8%), 115 (5.80%), 107 (35.7%). Calculated
analysis for C9H9NO3: C: 60.3; H: 5.1; N: 7.8. Found: C: 59.9; H: 5.5;
N: 7.2.
148.0. MS (EI, 70 eV) m/z (abundance): 243 (Mþ þ 2, 14.9%), 241
(Mþꢀ, 15.2), 100 (14.9%). Calculated analysis for C9H8BrNO2: C: 44.7;
H: 3.3; N: 5.8. Found: C: 45.1; H: 3.2; N: 5.4.
ꢀ
4.14. 1-Methoxy-4-(2-nitro-1Z-propenyl)benzene (2e)
4.7. 5-(2-Nitro-1Z-ethenyl)benzo[d][1,3]dioxol (1f)
0.62 g (62%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
0.75 g (30%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
d
(ppm): 2.50 (s, 3H), 3.89 (s, 3H), 7.00 (d, 2H, J ¼ 8.8), 7.45 (d, 2H,
d
(ppm): 7.55 (d, 1H, J ¼ 13.6), 7.62 (d, 2H, J ¼ 8.0), 7.64 (d, 1H,
J ¼ 8.8), 8.09 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d (ppm): 14.5, 55.5,
114.0, 126.0, 127.5, 132.0, 142.0, 155.0. Calculated analysis for
J ¼ 8.0), 7.97 (d, 1H, J ¼ 13.6); 13C NMR (CDCl3, 100 MHz)
d (ppm):
101.5, 107.0, 109.0, 124.0, 127.0, 134.0, 139.5, 149.0, 151.0. MS (EI,
70 eV) m/z (abundance): 193.0 (Mþꢀ, 37.0%), 170.0 (97.2%), 169.1
(7.0%),156.0 (26.6%),100.0 (13.0%). Calculated analysis for C9H9NO3:
C: 56.0; H: 3.6; N: 7.2. Found: C: 55.9; H: 3.5; N: 7.1.
C
10H11NO3: C: 62.2; H: 5.7; N: 7.2. Found: C: 62.0; H: 5.9; N:7.4.
4.15. 5-(2-Nitro-1Z-ethenyl)benzo[d][1,3]dioxol (2f)
0.49 g (37%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
4.8. 2-(2-Nitro-1Z-ethenyl)thiophene (1g)
d
(ppm): 2.48 (s, 3H), 6.06 (s, 2H), 6.90 (d, 1H, J ¼ 8.0), 6.96 (d, 1H,
J ¼ 1.6), 7.02 (d, 1H, J ¼ 8.0), 8.04 (s, 1H); 13C NMR (CDCl3, 100 MHz)
0.45 g (20%) of a white yellow solid. 1H NMR (CDCl3, 400 MHz)
d
(ppm): 14.1, 101.8, 108.8, 109.5, 125.9, 126.2, 133.7, 146.1, 148.3,
d
(ppm): 7.58 (d, 1H, J ¼ 13.6), 7.21 (d, 1H, J ¼ 3.8), 7.45 (d, 1H,
149.3. MS (IE, 70 eV) m/z (abundancia): 207 (Mþꢀ, 63.7%), 160
(70.2%), 149 (11.3%), 121 (5.60%), 103 (100%), 92 (12.9%), 77 (48.4%).
Calculated analysis for C10H9NO4: C: 58.0; H: 4.4; N: 6.8. Found: C:
58.2; H: 4.6; N: 6.3.
J ¼ 3.6), 7.66 (d, 1H, J ¼ 5.0), 8.03 (d, 1H, J ¼ 13.6); 13C NMR (CDCl3,
100 MHz)
d (ppm): 128.7, 129.3, 132.4, 134.2, 135.0, 146.1. MS (EI,
70 eV) m/z (abundance): 171.0 (Mþꢀ, 28.4%), 156.0 (12.5%), 96.0
(25.0%). Calculated analysis for C7H9NO2S: C: 49.1; H: 5.3; N: 8.2.
Found: C: 48.9; H: 5.6; N: 8.1.
4.16. 1-(2-Nitro-1Z-propenyl)thiophene (2g)
4.9. General procedure for the synthesis of 1-(2-nitro-1Z-propenyl)
arene (2aeg)
1.01 g (35%) of a yellow solid. 1H NMR (CDCl3, 400 MHz)
d (ppm):
2.58 (s, 3H), 7.20 (d, 1H, J ¼ 3.8), 7.44 (d, 1H, J ¼ 3.60), 7.65 (d, 1H,
J ¼ 5.0), 8.30 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d (ppm): 14.6, 127.6,
A mixture of the corresponding aldehyde (1 eq), nitroethane
(2.1 eq), n-butylamine (0.14 mL) and EtOH (5.6 mL) was heated at
reflux until disappear of aldehyde (checked by TLC). The solid was
filtered off and washed with hexane to afford the corresponding
product.
128.6, 132.0, 135.0, 135.5, 145.0. Calculated analysis for C7H7NO2S:
C: 49.7; H: 4.1; N: 8.3. Found: C: 48.9; H: 4.3; N: 8.1.
4.17. Preparation of nitroalkenes in solution
4.10. 1-(2-Nitro-1Z-propenyl)benzene (2a)
All nitroalkenes stock solutions were prepared fresh in DMSO. In
the in vitro experiments, final DMSO concentration was less than
0.5% to avoid cell toxicity. Dilutions were made in phosphate buffer
at pH and ionic strength according to each experiment and
2.33 g (45%) of a yellow solid. 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 2.39 (s, 3H), 7.48 (m, 5H), 8.09 (s, 1H); 13C NMR (CDCl3,

38
L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
corresponding DMSO and compound absorbance or fluorescence
4.22. Macrophage J774 culture and activation
controls were made.
The murine macrophage-like cell line J774 (ATCC-TIB-67,
American Type Culture Collection) was maintained by passage in
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco,
Invitrogen) containing glutamine (4 mM), sodium pyruvate
(110 mg Lꢁ1), glucose (4.5 g Lꢁ1), penicillin (100 U mLꢁ1), strepto-
mycin (100 mg Lꢁ1), and 10% heat-inactivated fetal cow serum.
Macrophages were plated and incubated at 37 ꢂC in 5% CO2 and 95%
air atmosphere. To minimize DMEM absorbance interference, ex-
periments were performed using Dulbecco’s phosphate buffer sa-
line (dPBS) containing CaCl2 (0.9 mM), MgCl2 (0.5 mM), KCl
(2.7 mM), NaH2PO4 (1.5 mM), NaCl (137 mM), Na2HPO4 (8.1 mM),
ꢀ
4.18. Evaluation of NO release by the synthesized compounds
The rate of ꢀNO release was determined by measuring the
oxidation of oxyhemoglobin (HbO2) to MetHb at 401 nm. The re-
action was started by adding the tested compounds (100
mM) to the
wells of 96-well plates containing 50 M HbO2 in 75 mM phosphate
m
(0.1 mM DTPA, pH 7.4) and absorbance was measured at 401 nm for
1 h, at 37 ꢂC. The rates of ꢀNO release were calculated from the slope
of the initial straight portion of each curve using
D
3 ¼
3 401MetHb ꢁ
3
401HbO2 ¼ 57 ꢃ 2 mMꢁ1 cmꢁ1 [33], and are the
glucose (5.5 mM) and L-arginine (1 mM).
average of at least three independent determinations. To confirm
the results observed with nitroalkenes 1b, 2b, 1g and 2g, ꢀNO
release was also determined using a ꢀNO sensor (WPI, Apollo 4000),
under the same conditions.
ꢀ
4.23. Induction of NO synthesis
Near confluent macrophage monolayers were exposed to
400 U mLꢁ1 murine recombinant interferon-gamma (IFN
g
, Sigma)
g mLꢁ1 of Escherichia coli LPS (serotype 0127:B8, Sigma),
known inductors of NOS2. Nitric oxide production was evaluated
after 5 h in the presence and the absence of nitroalkenes (10 M) by
the fluorescent probe DAF-FM (5 M), which permeates into cells
4.19. Synthesis of peroxynitrite
plus 8
m
The stock solutions were prepared from acidified hydrogen
peroxide and sodium nitrite in a tandem quenched-flow mixing
apparatus [34]. The solutions were treated with manganese dioxide
to remove residual hydrogen peroxide. The concentration of per-
oxynitrite was determined from its absorbance at 302 nm
m
m
and is quickly transformed into the water-soluble DAF-FM by
ꢀ
cytosolic esterases. When DAF-FM is oxidized by N2O3, a NO de-
rivative, becomes
a
fluorescent triazol
(
lexc
¼
485 nm,
(
3
¼ 1670 Mꢁ1 cmꢁ1) [35]. The concentration of nitrite present as
lem ¼ 520 nm) [41]. The increase in fluorescence was recorded for
contaminant was measured with the Griess’s reagent [36] after
decay of peroxynitrite to nitrate in monobasic sodium phosphate
solution. Nitrite content was always less than 30% of peroxynitrite.
Peroxynitrite was diluted in 0.1 M NaOH immediately before use.
Stock solutions were stored at ꢁ80 ꢂC, used only once after thaw-
ing, and then discarded.
60 min at 37 ꢂC. The reactivity of nitroalkenes with ꢀNO was tested
by OxyHb oxidation to MetHb. Red blood cells with 50
Hb were added after NOS2 induction in the absence and together
with nitroalkenes (50 M) addition. A known NOS2 inhibitor
1400W (100
M) was used as control. After 1 h incubation at 37 ꢂC
in 5% CO2 and 95% air atmosphere, 500 L of supernatant were
mM of total
m
m
m
frozen at ꢁ80 ꢂC to facilitate red blood cells disruption and
centrifuged at 18,000 ꢄ g to pellet the membranes. Hemoglobin
spectra were recorded in a Varian Cary 50 spectrophotometer and
MetHb formation was calculated from absorbance at 577 and
630 nm [42].
4.20. Competition kinetic assay
The second order rate constant between peroxynitrite (100
and nitroalkenes (360 M) was estimated by competition assay
with Fe2þ-Cyt c (360
M) [22]. Cytochrome c initial concentration
mM)
m
m
ꢀꢁ
used was calculated to surpass peroxynitrite homolytic decompo-
sition as 10 times the ratio between the first order rate constant of
peroxynitrite homolytic decomposition (0.9 sꢁ1) [37] and the sec-
ond order rate constant of the reaction between peroxynitrite and
Fe2þ-Cyt c (k ¼ 2.4 ꢄ 104 Mꢁ1 sꢁ1 at pH 7.4, and 37 ꢂC) [20]. Fe2þ-Cyt
c was obtained from Fe3þ-Cyt c (SigmaeAldrich, MO, USA) by
addition of a known excess of sodium dithionite, followed by gel
filtration to remove the excess of reductant immediately before use
sodium dithionite stock solution was prepared in degassed NaOH
4.24. Induction of O2 production
Assembly of NADPH oxidase was triggered by the addition of
ꢀꢁ
PMA (4
m
g mLꢁ1), a non-particulate stimuli that lead to O2
production, to the cell culture. Activated macrophages start to
ꢀꢁ
release O2 immediately after NADPH oxidase activation and it
remains for 90e120 min with a rate of 0.3e0.4 nmol minꢁ1 mgꢁ1
[43]. After 30 min when the culture achieved a maximal produc-
tion of O2 ꢁ, the capability of nitroalkenes (10
mM) to scavenge
ꢀ
O2 was determined by Fe3þ-Cyt c (50
mM) reduction inhibitable
ꢀꢁ
(0.1
N)
and
quantified
by
ferricyanide
reduction
(
3
¼ 1020 Mꢁ1 cmꢁ1) assuming a 2:1 stoichiometry [38]. The
420
by superoxide dismutase (SOD, E.C.1.15.1.1., 0.5
mM, SigmaeAldrich,
Fe2þ-Cyt
c
oxidation
was
recorded
at
550
nm
MO, USA). Nitroalkenes or SOD (600 U mLꢁ1) were added in dPBS.
The Fe2þ-Cyt c formation was recorded during 2 min at 550 nm
[39,44].
(
3
¼ 21,000 Mꢁ1 cmꢁ1), as previously described [39]. The sam-
550
ples were equilibrated at 37 ꢂC for 2 min before peroxynitrite
addition as a single bolus under vigorous vortexing in phosphate
buffer (0.1 M, pH 7.4, 0.1 mM DTPA). Reverse order of addition
controls were performed.
4.25. Induction of ONOOꢁ production
ꢀ
The macrophages J774 were induced for NO production. Afteꢁr
ꢀ
4.21. Scavenge of peroxynitrite derived free radicals
5 h NADPH oxidase assembly was activated with PMA for O2
ꢀ
production. This led to the simultaneous production of NO and
DHR 123 (50
single bolus of peroxynitrite (50
presence of nitroalkenes (400 M), in phosphate buffer (0.1 M, pH
m
M, SigmaeAldrich, MO, USA) was exposed to a
Oꢁ2 and resulted in peroxynitrite close to stoichiometric yields
mM), in the absence and in the
[43]. Oxidation of DHR (15
presence and in the absence of nitroalkenes (10
lowed for 120 min by fluorescence
lem ¼ 520 nm).
m
M) by activated macrophages in the
M) was fol-
485 nm,
m
m
¼
7.4, 0.1 mM DTPA) at 37 ꢂC. RH production was determined at
(
lexc
500 nm (
3
¼ 78.8 mMꢁ1 cmꢁ1) [40].
500nm

L. Celano et al. / European Journal of Medicinal Chemistry 74 (2014) 31e40
39
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pH 10.5) (90:10 v/v) and absorbance at 560 nm was read.
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4.28. Theoretical studies
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The geometry of each compound and adduct was fully optimized by
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Competing interests
None.
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This research was partially supported by Comisión Sectorial de
Investigaciones Científicas (CSIC), Universidad de la República and
PEDECIBA. LC, received doctoral and CC, RF, and NC research initi-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.029.
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