Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic derivatives. Potential use as anti-inflammatory and anti-atherogenic agents

Article abstract of DOI:10.1016/j.ejmech.2018.09.062

We have previously shown the antioxidant and anti-inflammatory properties of several para-substituted arylnitroalkenes. Since oxidative stress and inflammation are key processes that drive the initiation and progression of atherosclerosis, in the present work the antioxidant, anti-inflammatory and anti-atherogenic properties of an extended library of aryl-nitroaliphatic derivatives, including several newly designed nitroalkanes, was explored. The antioxidant capacity of the nitroaliphatic compounds, measured using the oxygen radical absorbance capacity assay (ORAC) showed that the p-methylthiophenyl-derivatives were about three times more effective than Trolox to prevent fluorescein oxidation, independently of the presence or the absence of the double bond next to the nitro group. The peroxyl radical scavenger capacity of the p-dimethylaminophenyl-derivatives was even higher, being the reduced form of these compounds even more active. In fact, while the antioxidant capacity of 1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene and 1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was 4.2 ± 0.1 and 5.4 ± 0.1 Trolox Eq/mol, respectively; ORAC values obtained with the ethyl and the propyl derivatives were 10 ± 1 and 13 ± 2 Trolox Eq/mol, respectively. The p-dimethylamino-derivatives, especially the nitroalkanes, were also able to prevent LDL oxidation mediated by peroxyl radicals. Oxygen consumption due to the oxidation of fatty acids was delayed in the presence of the dimethylamino substituted compounds, only the alkanes interrupted the chain of lipid oxidations decreasing the rate of oxygen consumption. Although the formation of foam cells in the presence of oxidized-LDL (oxLDL) remained unaffected, the molecules containing the dimethylamino moiety were able to decrease the expression of IL-1β in LPS/INF-γ challenged macrophages.

Full text of DOI:10.1016/j.ejmech.2018.09.062

Accepted Manuscript
Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic derivatives.
Potential use as anti-inflammatory and anti-atherogenic agents
Nicolás Cataldo, Bruno Musetti, Laura Celano, Claudio Carabio, Adriana Cassina,
Hugo Cerecetto, Mercedes González, Leonor Thomson
PII:
S0223-5234(18)30840-7
10.1016/j.ejmech.2018.09.062
EJMECH 10773
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 July 2018
Revised Date: 4 September 2018
Accepted Date: 25 September 2018
Please cite this article as: Nicolá. Cataldo, B. Musetti, L. Celano, C. Carabio, A. Cassina, H. Cerecetto,
M. González, L. Thomson, Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic
derivatives. Potential use as anti-inflammatory and anti-atherogenic agents, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.062.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic
derivatives. Potential use as anti-inflammatory and anti-atherogenic agents
Nicolás Cataldoa,b,*, Bruno Musettia,b,*, Laura Celanoa,1, Claudio Carabioa, Adriana
Cassinac,d, Hugo Cerecettob, Mercedes Gonzálezb†, Leonor Thomsona,c**
aLaboratorio de Enzimología, Instituto de Química Biológica, Facultad de Ciencias,
Universidad de la República. Iguá 4225, Montevideo, 11400, Uruguay.
bGrupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de
Ciencias, Universidad de la República, Iguá 4225, Montevideo, 11400, Uruguay
cCenter for Free Radical and Biomedical Research, Facultad de Medicina,
Universidad de la República, General Flores 2125, Montevideo, 11800, Uruguay.
dDepartamento de Bioquímica, Facultad de Medicina, Universidad de la República.
General Flores 2125, Montevideo, 11800, Uruguay.
†Professor Mercedes Gonzalez passed away on May 21,2018.
*Both authors contributed equally to this work.
**Corresponding author: L. Thomson Facultad de Ciencias, Universidad de la
República, Iguá 4225, 11400, Montevideo, Uruguay. Phone: 598-25258618 ext.
7214. E-mails: lthomson@fcien.edu.uy.
Abbreviations: oxLDL, oxidized low-density lipoprotein; LDL, low density
lipoprotein; ORAC: oxygen radical absorbance capacity assay; AAPH, 2,2'-azo-bis
(2-amidinopropane)
dihydrochloride;
DMSO,
dimethyl
sulfoxide;
LPS,
lipopolysaccharide (endotoxin) of the outer membrane of Gram-negative bacteria,
INF-γ, interferon gamma.
1
Present Address: Bioquímica y Microbiología General, Universidad Tecnológica (UTEC), Cno.
Alejandro Malcom s/n, La Paz, Colonia, 70200, Uruguay.
1

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ABSTRACT
We have previously shown the antioxidant and anti-inflammatory properties of
several para-substituted arylnitroalkenes. Since oxidative stress and inflammation
are key processes that drive the initiation and progression of atherosclerosis, in the
present work the antioxidant, anti-inflammatory and anti-atherogenic properties of an
extended library of aryl-nitroaliphatic derivatives, including several newly designed
nitroalkanes, was explored. The antioxidant capacity of the nitroaliphatic compounds,
measured using the oxygen radical absorbance capacity assay (ORAC) showed that
the p-methylthiophenyl-derivatives were about three times more effective than Trolox
to prevent fluorescein oxidation, independently of the presence or the absence of the
double bond next to the nitro group. The peroxyl radical scavenger capacity of the p-
dimethylaminophenyl-derivatives was even higher, being the reduced form of these
compounds even more active. In fact, while the antioxidant capacity of 1-
dimethylamino-4-(2-nitro-1Z-ethenyl)benzene and 1-dimethylamino-4-(2-nitro-1Z-
propenyl)benzene was 4.2 ± 0.1 and 5.4 ± 0.1 Trolox Eq/mol, respectively; ORAC
values obtained with the ethyl and the propyl derivatives were 10 ± 1 and 13 ± 2
Trolox Eq/mol, respectively. The p-dimethylamino-derivatives, especially the
nitroalkanes, were also able to prevent LDL oxidation mediated by peroxyl radicals.
Oxygen consumption due to the oxidation of fatty acids was delayed in the presence
of the dimethylamino substituted compounds, only the alkanes interrupted the chain
of lipid oxidations decreasing the rate of oxygen consumption. Although the
formation of foam cells in the presence of oxidized-LDL (oxLDL) remained
unaffected, the molecules containing the dimethylamino moiety were able to
decrease the expression of IL-1β in LPS/INF-γ challenged macrophages.
Key words: arylnitroalkanes, atherosclerosis, antioxidants, anti-inflammatory
2

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INTRODUCTION
Since cardiovascular and neurological complications of atherosclerosis are the
main causes of morbidity and mortality worldwide [1], and because the attempt to
improve cardiovascular outcomes have been unsuccessful, there is an urgent need
of new therapies to increase life expectancy and improve the quality of life of
atherosclerotic patients. Lowering cholesterol excess, restoring normal levels of
blood pressure and commitment to a heart healthy lifestyle are key strategies to
prevent major cardiac events, however intervention in additional mechanisms like
vascular inflammation and oxidative stress are also important therapeutic targets in
atherosclerosis [2]. For example, the activation of inflammasomes, in particular the
nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin
domain 3 (NLRP3) inflammasome, is now emerging as a critical molecular
mechanism for many degenerative diseases, including atherosclerosis [3,4]. A
plethora of danger signals are able to trigger the assembly of the NLRP3
inflammasome, including molecular patterns derived from pathogens (PAMPs),
including lipopolysaccharides of the outer membrane of bacteria (like LPS), but also
modified endogenous molecules with the so called danger associated molecular
patterns (DAMPs) [5].
Oxidation and nitration of proteins and lipids generate neo-epitopes which are
recognized as DAMPs by specific cell receptors [6–8]. In fact, oxidized LDL (oxLDL)
is recognized by TLR4/6 and scavenger receptors triggering inflammatory cascades.
Phagocytosis of the modified lipoprotein will ultimately transform subendothelial
macrophages in lipid-laden foam cells [9,10]. Uncontrolled accumulation of
cholesterol crystals, from LDL, damages the lysosomal membrane causing the
leakage of peptidases into the cytosol and the hydrolytic activation of caspase 1 [11–
14]. Simultaneously, NFκB dependent pathways cause the expression and assembly
of the NLRP3 inflammasome with the subsequent activation of caspase 1. Caspase
1 is the hydrolytic enzyme responsible for activating IL-1β, an important mediator of
the inflammatory response [15–17]. Although several intents were done to decrease
the inflammatory response by the use of non-steroidal anti-inflammatory and
biological agents, disappointing results were found during phase III trials [18,19].
In a previous report we developed a series of simple arylnitroalkene derivatives
3

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ꢀ
with different stereoelectronic properties, able to release NO and to scavenge the
•
highly reactive products of peroxynitrite decomposition, •OH and NO2 [20]. Our lead
compounds, especially 1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene (1) and 1-
dimethylamino-4-(2-nitro-1Z-propenyl)benzene (2), and to a lesser extent 5-(2-nitro-
1Z-propenyl)benzo[d][1,3]dioxol (4) (Figure 1A), also shown significant analgesic,
anti-inflammatory, and anti-arthritic properties in animal models [21]. The
pharmacologic effects were as least in part mediated by inhibition of prostaglandin H
synthase, but the compounds were also able to prevent the activation of the matrix
metallopeptidase 9 (MMP-9) [21]. Since IL-1β has been shown to induce MMP-9
production through activation of p42/p44 MAPK, p38 MAPK, JNK, and NF-κB
pathways [22], the anti-inflammatory properties of the nitroalkene derivatives could
also be due to interference in one or more steps related to or triggered by the
assembly of the inflammasome, and in consequence could be suitable for the
protection of the cardiovascular system as a multitarget therapy, by preventing LDL
oxidation and also ameliorating inflammation. To test this hypothesis and with the
interest of knowing more about the relationship between the structure and bioactivity,
the capacity of twenty aryl-nitroaliphatic compounds to react with peroxyl radicals
preventing LDL oxidation and ameliorate the production of IL-1β by LPS/INF-γ
challenged macrophages was analyzed.
Since in our previous studies [20,21] we found that the presence of electron donor
moieties at the phenyl group play relevant roles for the bioactivity, we designed new
p-substituted-phenyl derivatives with similar electronic properties (Figure 1B). On the
other hand, to explore the relevance of the double bond next to the nitro group in the
nitroalkene moiety and since little is known about the bioactivity of nitroalkanes, we
designed and tested several reduced derivatives (Figure 1B).
4

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CHO
R
R
A)
MeNO2 or EtNO2
NaBH4
Ar
Ar
Ar
NO2
NO2
n-butNH2 / EtOH / reflux
THF-MeOH / 0 oC to rt
1
2
3
4
5
6
7
8
9
, -R= -H, -Ar= -Ph-4-(NMe2)
, -R= -Me, -Ar= -Ph-4-(NMe2)
, -R= -H, -Ar= -Ph-3,4-(-OCH2O-)
, -R= -Me, -Ar= -Ph-3,4-(-OCH2O-)
, -R= -H, -Ar= -Ph-4-OMe
, -R= -Me, -Ar= -Ph-4-OMe
, -R= -H, -Ar= -Ph-4-SMe
, -R= -H, -Ar= -Ph-4-(NHAc)
, -R= -H, -Ar= -2-thienyl
17, -R= -H, -Ar= -Ph-4-(NMe2)
18, -R= -Me, -Ar= -Ph-4-(NMe2)
19, -R= -H, -Ar= -Ph-4-SMe
20, -R= -H, -Ar= =Ph-4-(NHAc)
10, -R= -Me, -Ar= -2-thienyl
11, -R= -H, -Ar= -Ph
12, -R= -Me, -Ar= -Ph
13, -R= -H, -Ar= -Ph-4-Cl
14, -R= -Me, -Ar= -Ph-4-Cl
15, -R= -H, -Ar= -Ph-4-Br
16, -R= -Me, -Ar= -Ph4-Br
B)
R
NO2
X
structural modifications
proposed herein
Figure 1. Studied derivatives. A. Synthetic procedures for nitroalkenes (obtained
as Z-isomers) and nitroalkanes. B. Design of new compounds.
RESULTS
1.
Synthesis
The synthesis of the already reported library of arylnitroalkenes was performed via
Henry reaction as described [20,21]. In addition two new unsaturated derivatives 1-
methylthio-4-(2-nitro-1Z-ethenyl)benzene (7) and the 1-acetylamino-4-(2-nitro-1Z-
ethenyl)benzene (8) were generated (Figure 1A). The synthesis of the reduced
compounds, 1-dimethylamino-4-(2-nitroethyl)benzene (17), 1-dimethylamino-4-(2-
nitropropenyl)benzene (18), 1-methylthio-4-(2-nitroethyl)benzene (19) and 1-
acetylamino-4-(2-nitroethyl)benzene (20), was performed by mild reduction of the
double bond from the appropriate nitroalkene (1, 2, 7 and 8, respectively) using
sodium borohydride. Methanol was used as co-solvent, since the methoxylation of
borohydride favors the selective alkene-reduction leaving the nitro group unchanged
[23,24]. Derivatives 17-20 (Figure 1A) were obtained in good to very good yields (61-
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86 %) after chromatographic purifications. The new products were structurally
characterized by 1H NMR and 13C spectroscopy, and their purity established by TLC
1
and elemental microanalysis (C, H, N, S) (Table S1). In the H NMR spectra, the
presence of the nitroalkanes of interest, 17-20 (Figure 1A), was evidenced by the
appearance of two triplets or triplets of doublets at high field (δ ~4.8 and ~3.2 ppm)
due to the loss of the unsaturation, provoking that the methylene protons became
flanked by two neighboring protons each and the consequent disappearance of the
characteristic nitroalkene-olefinic protons at low field (δ ~7.7 and ~7.5 ppm), (see
examples in Figure S1). For nitroalkane 19 the diastereotopic properties of the
methylene protons belonging to the nitroethyl-moiety (triplets of doublets) were
1
clearly observed in the H NMR spectrum (Figure S1)). The study of the proton-
carbon correlations was carried out through HSQC and HMBC experiments. The Z-
isomeric form of the new nitroalkenes, 7 and 8, was confirmed using the coupling
constants H-H for the alkene-systems and by NOE-diff experiments.
2. Reaction with peroxyl radicals
In view of the already reported results on the reaction of aryl nitroalkenes with
biologically generated oxidants [20,21] we decided to explore the reactivity of these
kind of derivatives with peroxyl radicals using an standard technique, the oxygen
radical absorbance capacity (ORAC). This technique measures the loss of auto-
fluorescence of fluorescein after oxidation by the products of the thermal
decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The main
degradation pathways of AAPH in aqueous solutions, hydrolysis (Eq. 1) and thermal
decomposition (Eq. 2), generate unstable carbon centered radicals (R●) [25].
ꢅꢆ ꢅꢈ
ꢇ
AAPH +HOꢀ ꢉꢁ.ꢊꢂꢊꢃꢊꢁꢊꢄꢊꢊꢊꢋ R•ꢁ + R•ꢌ
(1)
ꢅꢎ ꢅꢈ
ꢏ
AAPH ꢉꢌ.ꢊꢁꢊꢍꢊꢁꢊꢄꢊꢊꢊꢋ Nꢌ + 2R•ꢁ
(2)
The radicals react at almost diffusion-controlled rate with molecular oxygen to yield
peroxyl radical (R–OO●) (Eq. 3), which decomposes mainly to alkoxyl radicals (Eq.
4).
2R• + 2Oꢌ → 2ROO•
2ROO• → 2RO• + Oꢌ
(3)
(4)
Both, peroxyl and alkoxyl radicals react fast with reduced targets (XH) (Eq. 5).
•
•ꢐ •ꢑ
ROO RO + XH → ROOH ROH + X
ꢐ
ꢑ
(5)
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Figure 2. Oxygen radical antioxidant capacity. Representative time courses of
fluorescein (7.5 nM) oxidation by AAPH (30 mM) at 37 °C in 75 mM phosphate buffer
pH 7.4, 100 µM DTPA, in the presence of Trolox (3-24 µM) (A) and 1 (1.5-7.5 µM)
(B) at λexc 485 nm and λem 512 nm. Fluorescein oxidation in the absence of
antioxidants was represented by dashed lines. Numbers above lines denote
antioxidant concentration (µM). Insets show linear fit of the areas under the curves
(AUC) as a function of antioxidant concentration. AUC values are expressed as
media ± SD of three independent determinations.
The capacity of the selected compounds to trap both radicals preventing fluorescein
oxidation was compared against the same property of 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), a hydrosoluble analogous of vitamin
E used as standard (Figure 2). The dimethylamino derivatives, 1, 2, 17 and 18, were
the most potent antioxidants (Table 1), confirming our previous results for the
nitroalkenes (1 and 2) and showing a potential higher antioxidant capacity for both
saturated analogous (nitroalkanes 17 and 18) [20,21]. In fact, the capacity to trap
peroxyl radicals by compounds 1 and 2 was more than 4 times higher than the
capacity of Trolox, but surprisingly, the reduction of the double bond on the side
chain of both dimethylamino derivatives to generate compounds 17 and 18,
increased significantly the antioxidant capacity of the products. Actually, 18 was 10
times more effective than Trolox to prevent the loss of fluorescence while the
antioxidant capacity of 17 was even higher (Table 1).
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Table.1. ORAC values of the analyzed compounds.
EFa
Derivative
TEqb,c
Derivative
TEqb,c
13 ± 2
10 ± 1
1
2
17
18
4.2 ± 0.1
5.4 ± 0.1
3
0.72 ± 0.01
1.6 ± 0.2
4
5
0.46 ± 0.03
0.46 ± 0.02
3.5 ± 0.3
medd
6
7
19
20
2.8 ± 0.2
0.26 ± 0.06
0.63 ± 0.07
0.43 ± 0.07
0.54 ± 0.01
0.29 ± 0.01
0.37 ± 0.06
0.44 ± 0.01
0.39 ± 0.02
0.44 ± 0.04
0.45 ± 0.06
8
9e
10e
11
12
13
14
15
16
wef
med-iewg
a EF: electronic effect of the aryl substituent.
b Trolox equivalents (TEq) were determined by ORAC as in Fig. 2.
c Results are the average of three independent experiments ± SD.
d med: electron-donor group by mesomeric effect.
e bioisosters of derivatives 11 and 12, respectively.
f we: without electronic effect.
g
med-iew: electron-donor group by mesomeric effect and electron-withdrawing
group by inductive effect.
The antioxidant capacity of the derivatives bearing the 1,3-dioxol system was higher
than the one observed with Trolox in the molecule bearing the nitropropenyl
framework (4) and was slightly lower than 1 in the molecule with the nitroethenyl
framework (3), the differences between both compounds was in concordance with
our previous reports [20,21]. While the compounds carrying other substituents,
including the methoxy (5, 6), hydrogen (11, 12) and halide (13, 14, 15, and 16)
groups were 50% less effective than Trolox, without any consistent difference
attributable to the presence of the additional methyl group in R (Table 1).
Additionally, we studied the antioxidant ability of derivatives 9 and 10 (Table 1), that
hold a thiophene heterocycle instead of the phenyl group, potential bioisosters of
derivatives 11 and 12, respectively. Derivatives 9 and 10 displayed higher activity
than 11 and 12, probably as a result of the mesomeric-electron donor ability of the
sulfur in the heterocycle.
8

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Figure 3. LDL oxidation by AAPH. Oxygen consumption by LDL (0.2 mg mL-1)
alone (dotted line), AAPH (30 mM) alone (dash line) and LDL (0.2 mg mL-1) plus
AAPH (30 mM) (continuous line) was analyzed at 37o C in 75 mM phosphate buffer,
pH 7.4, using a high-resolution oxygen electrode as described in Materials and
Methods. Traces are representative of three independent determinations performed
in the same condition using a different purification batch of LDL. Numbers below
lines represent mean oxygen consumption (pmol O2 ml−1 s−1 ± SD) (n = 3)
In addition, the capacity of the molecules to prevent fluorescein oxidation was
preserved after substitution of the tertiary amine present in the nitroalkene 1 by a
thioether moiety in the nitroalkene 7, but it was significantly decreased in the
presence of an acetamido group in the phenyl moiety in nitroalkene 8, while the
reduction of the double bond in both compounds to generate nitroalkanes 19 and 20,
respectively, failed to improve the antioxidant properties of the products. Clearly,
electronic effects played relevant roles in the antioxidant profiles, thus derivatives
with negative or near to zero Hammett σp and near to zero inductive σI constants, for
p-substituents of the phenyl moiety, displayed the best profiles, i.e. σp = -0.83 and σI
= 0.06 for –N(Me)2; σp = 0.00 and σI = 0.23 for –SMe; σp = -0.15 and σI = 0.26 for –
NAc; σp = -0.27 and σI = 0.27 for –OMe; and σp = 0.23 and σI = 0.47 for –Cl [26]
9

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A
B
C
D
.
Figure 4. Effect of NA on the oxidation of LDL by AAPH. Representative traces of
oxygen consumption by 0.2 mg mL-1 LDL plus 30 mM AAPH assayed in the absence
(LDL+AAPH) and in the presence of 20 µM 1 (A), 2 (B), 17 (C) and 18 (D) as
specified at the right of each trace, the arrows indicate the addition of the molecules
during the course of the reaction. Numbers next to the lines depict the mean rate of
oxygen consumption (pmol O2 ml−1 s−1) from three independent experiments ± SD.
Statistically significant differences (****p<0.0001) were found between the rate of
oxygen consumption by the system in the absence and the presence of the analyzed
compounds.
3. Protection of LDL from peroxyl radical mediated oxidations.
Since several of the synthetic compounds, were able to effectively trap peroxyl and
alkoxyl radicals derived from AAPH decomposition, and since these radicals can
trigger lipid oxidation, the more effective compounds to prevent fluorescein oxidation
were selected to explore the capacity to prevent LDL oxidation by AAPH measuring
oxygen consumption. The rate of oxygen consumption by 30 mM AAPH at 37o C
(26.0 ± 0.7 pmol O2 ml−1 s−1) (Figure 3) was similar to the already reported
decomposition rate [25]. After reaction with oxygen, AAPH-derived peroxyl and
alkoxyl radicals (Eq. 3 and 4) can react with different biological molecules leading
mainly to the production
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Figure 5. Effect on IL-1β expression and ox-LDL internalization by J774 cells.
A. IL-1β expression by J774 cells incubated for 1 h in the absence (None) and the
presence of 1, 2, 17 and 18 (5 µM), and challenged for 4 h with INF-γ (400 U mL-1)
and LPS (8 µg mL-1) was analyzed. A representative western blot is shown on the
top, while the densitometric analysis of IL-1β bands is shown on the bottom. Bars
represent mean values of the protein bands from non-challenged cells (black bars)
and the cells treated with INF-γ plus LPS (grey bars) from three independent
experiments. (****, p<0.0001). B. A representative merged image showing J774 cells
loaded with ox-LDL DiI (red) and the nucleus stained with Hoechst 3342 (blue) is
shown on the top (Bar = 25 ꢀm). The bottom graph represents the ratio between the
fluorescence due to ox-LDL DiI (λexc 549, λem 565) and Hoechst (λexc 352, λem
461) against the concentration of 1 (●), 2 (○), 17 (■) and 18 (□). J774 cells were
incubated in 96 well plates, in the presence of increasing concentrations (0.3-5 µM)
of the compounds for 1 h and settled for 4 h with ox-LDL DiI and the fluorescence at
both wavelengths was read using a plate reader. C. Confluent cell monolayers were
incubated for 2 h in the presence of increasing concentrations (0.3-5 µM) of 1 (●), 2
(○), 17 (■) and 18 (□) and the effect on cell viability was measured by the MTT
reduction assay. D. As in C but cell viability was measured after 24 h of incubation
with the compounds.
of carbon centered radicals (X●) (Eq. 5). The reaction of X● with molecular oxygen
give rise to peroxyl radicals (XOO●) (Eq. 6). The reaction of peroxyl radicals with
another reduced molecule starts a chain of radical mediated reactions, known as
propagation phase (Eq. 7).
11

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X• + ꢒꢌ → XOO•
XOO• + XH → XOOH + X•
(6)
(7)
Oxygen consumption by LDL in the presence of AAPH showed the characteristic
biphasic profile of lipid oxidation, with an initial slow phase, due to the presence of
endogenous antioxidants; followed by a propagation phase of fast oxygen
consumption (Figure 3). In the first phase, the rate of oxygen consumption in the
presence of LDL plus AAPH was similar to the rate with AAPH alone, consistent with
the silent (regarding oxygen consumption) initial phase of lipid oxidation.
Approximately 7 min later the entrance in the propagation phase triggered a burst of
oxygen consumption with a rate of 138 ± 2 pmol O2 ml−1 s−1 (Figure 3).
The addition of 20 µM of the selected compounds (1, 2, 17, and 18) before AAPH
delayed the transition from the initiation to the propagation phase (Figure 4). While
the propagation phase in the presence of nitroalkenes 1 and 2 started 20 min after
the addition of AAPH, nitroalkanes 17 and 18 were even more effective protecting
LDL form peroxyl radicals mediated oxidation. In fact, in the presence of 18 the
initiation phase last 30 min, while 17 delayed the beginning of the propagation
phase in more than 40 min (Figure 4). The addition of the selected molecules during
the reaction also delayed the entrance of the system into the propagation phase. The
addition of successive boluses of 1 and 2 before the beginning of the propagation
phase delayed its appearance, however both compounds were ineffective when
added after the beginning of this phase (Figure S3). Conversely, 17 and 18 were
also able to stop the oxidation process even if added after the beginning of the
propagation phase, reversing the rate of oxygen consumption to a level close to that
of AAPH alone (Figure 4C and D).
4. In vitro analysis of the anti-inflammatory and anti-atherogenic
properties.
To explore the mechanisms involved in the anti-inflammatory properties of the
dimethylamino substituted nitroalkenes already reported [20], 1 and 2, the ability of
these compounds, together with the reduced analogues described herein, 17 and 18,
to interfere with IL-1β activation was studied. Murine macrophages (J774 cells) were
incubated for 4 hours in the presence of INF-γ plus LPS and the expression of IL-1β
was explored by western blot. As expected, the expression of IL-1β increased
significantly in cell cultures exposed to the INF-γ and LPS. The addition of the
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dimethylamino derivatives decreased significantly the expression of the cytokine,
being the saturated compounds more effective than the corresponding unsaturated
ones (Figure 5A). Since neither LDL nor oxLDL were able to stimulate the synthesis
of pro-IL-1β under our experimental conditions (Figure S3), and because the
deposition of foam cells into the artery wall is one of the earliest steps in the
progression of the atherosclerotic plaque [10], the capacity of the selected
compounds to prevent the internalization of ox-LDL by murine macrophages was
explored. The complex between oxLDL and the lipophilic fluorescent dye DiI was
effectively taken up by J774 cells (Figure 5B), as described before [27]. The
fluorescent signal appeared into the cells both in the absence and the presence of
the compounds. Moreover, in the presence of the higher concentrations used (2.5-5
µM) of 1 and 2 a significant increase in the amount of fluorescence due to the
labeled lipoprotein was observed, discarding any interference of the compounds with
the phagocytic process. To rule out the possibility that the effect of the analyzed
molecules on the expression of IL-1β were due to cellular dead, cell viability and
function were analyzed by investigating the capacity of the cellular mitochondria to
reduce the tetrazolium compound MTT. The cell survival remained unchanged after
2 and 24 h of incubation in the presence of the analyzed concentrations (0.3-5 µM)
of both nitroalkenes and nitroalkanes, being the amount of formazan formed
equivalent to the amount generated in the presence of vehicle alone (Figure 5C and
D).
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CONCLUSIONS
The main cardiovascular risk factors, including hypertension, smoking, physical
inactivity, obesity, hyperlipemia, and diabetes mellitus are associated with
inflammation and oxidative stress [28]. In animal models, vascular cell dysfunction,
intimal hypertrophy, and the formation and destabilization of atherosclerotic plaques
were associated with an increased oxidant status [29,30]. Therefore, oxidative stress
was considered as a major contributor to the development of cardiovascular
diseases and antioxidant treatments were proposed as promising therapeutic
strategies. However, the attempts to improve cardiovascular outcomes by mean of
antioxidant vitamin supplementation were unsuccessful, remaining the incidence of
major cardiovascular events unchanged [31,32]. Moreover, the increase of the risk of
lung cancer in smoker males after supplementation with β-carotene [22] discouraged
the use of liposoluble vitamins and other unspecific antioxidants in primary care.
Those findings have prompted the development of more specific antioxidants, able to
•
leave the vascular redox balance intact, preserving the normal levels of NO and
H2O2, since both have been shown as essential molecules in redox signaling. We
have already reported the capacity of several p-phenylsubstituted nitroalkenes to
scavenge oxygen and nitrogen derived oxidants [20]. In particular the dimethylamino
derivatives were able to specifically react with the radicals derived from ONOO-
•
•
•
homolysis, OH and NO2, without significant reaction with NO or H2O2 [20]. The
same compounds also showed remarkable anti-inflammatory and analgesic
properties due to inhibition of prostaglandin H synthase [21]. In consequence these
compounds can be proposed as candidates for multitarget therapy to ameliorate the
inflammatory process and the consequent oxidative stress.
To reinforce our previous findings [20,21], in the present work the potential anti-
atherogenic properties of the molecules analyzed so far and several new derivatives
was investigated. As observed before [20], the p-dimethylamino-substituted
nitroalkenes (1 and 2) and the newly synthesized nitroalkanes (17 and 18) were
highly effective antioxidants. In fact, the four compounds acted as peroxyl radical
scavengers preventing the oxidation of fluorescein and LDL. In particular,
nitroalkanes 17 and 18 acted as chain breaking antioxidants, decreasing the
consumption of oxygen due to the propagation phase of LDL-lipid oxidation. Both the
survival and the capacity of J774 cells to recognize and phagocyte oxLDL remined
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unaffected in the presence of the tested compounds (1, 2, 17 and 18). However, the
same compounds were able to interfere with one of the main products of
inflammasome activation, the expression of IL-1β. In fact, the molecules decreased
the expression of IL-1β induced by LPS and INF-γ, reinforcing the anti-inflammatory
properties of the dimethylamino-derivatives, having the nitroalkanes even better
antioxidant and anti-inflammatory properties than the original nitroalkenes.
Several biological agents with anti-inflammatory properties are being tested for the
treatment of atherosclerosis [33]. Recently, the results of the CANTOS trial
(Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) showed that
canakinumab, a monoclonal IL-1β–neutralizing antibody, reduced the risk of
recurrent cardiovascular events in patients with prior heart attack [34,35]. However,
treatments with biological agents are expensive and the risk of the long-term therapy
is still unknown or has been proven to be largely disappointing due to an increase in
the risk of opportunistic infections or cardiovascular complications [18,19]. Because
of that, the search for new compounds able to decrease the inflammatory response
associated with chronic diseases, as atherosclerosis, is a priority. In this context our
compounds can become an effective and economic alternative to delay the
progression of atherosclerosis and prevent the appearance of major cardiac events.
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MATERIALS AND METHODS
Experimental
Materials were purchased from Sigma-Aldrich Co. (USA) and Across Organic
(Janssen Pharmaceutical, Geel, Belgium). All solvents were dried and distillated with
conventional methods prior to use, and the synthesis carried under nitrogen
atmosphere. Compounds 1-6, 9-16, and 18 were prepared as previously described
[20,24]. The synthesized compounds were chemically characterized by thin layer
chromatography (TLC), nuclear magnetic resonance (NMR) and elemental
microanalyses (CHN). Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel
GmbH & Co. KG., Düren, Germany) was used for TLC. The purity of compounds
was determined by elemental microanalyses performed on a Carlo Erba Model
EA1108 elemental analyzer from 48 h-vacuum-dried samples. The analytical results
1
13
for C, H, N and S were within ± 0.4 of the theoretical values. The H and C NMR
spectra were recorded on a Bruker DPX 400 (400 MHz), using TMS as the internal
standard and with the indicated deuterated solvent; the chemical shifts are reported
in ppm (δ) and coupling constants (J) values are given in Hertz (Hz). Signal
multiplicities are represented by: s (singlet), d (doublet) and td (triplet of doublet).
NOE diff experiments were performed to establish the stereochemistry around the
double bond.
General procedure for synthesis of nitroalkenes
In a rounded flask, a mixture of the corresponding aldehyde (0.8 mmol), anhydrous
o
nitromethane (3 mL) and ammonium acetate (0.8 mmol) was stirred at 100 C for 2
h. The solvent was evaporated in vacuo and the residue was treated with water (30
mL) and extracted with ethyl acetate (3 × 20 mL). The organic layer was
successively washed with water (2 × 50 mL), HCl (1M, 2 × 25 mL) and brine (2 × 20
mL), dried with anhydrous sodium sulfate and evaporated in vacuo. The obtained
solid was washed with n-hexane to yield the corresponding product.
1
1-Methylthio-4-(2-nitro-1Z-ethenyl)benzene (7): Yellow solid. (0.11 g, 61%) H-
NMR (CDCl3, 400 MHz) δ (ppm): 7.60 (d, 1H, J=12.00), 7.47 (d, 1H, J=12.00), 7.29
13
(d, 2H, J=8.00), 7.27 (m, 2H), 2.55 (s, 3H). C-NMR (CDCl3, 100 MHz) δ (ppm):
(Found: C, 55.1; H, 4.8; N, 6.9; S, 16.3%.
145.0, 138.6, 136.1, 129.0, 125.3, 14.0.
C9H9NO2S requires C, 55.4; H, 4.7; N, 7.2; S, 16.4%).
16

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1
1-Acetylamino-4-(2-nitro-1Z-ethenyl)benzene (8): Yellow solid. (0.10g, 78%) H-
NMR (CDCl3, 400 MHz) δ (ppm): 7.69 (d, 1H, J=12.00), 7.62 (d, 1H, J=12.00), 7.36
(d, 2H, J=8.00), 7.25 (d, 2H, J=8.00), 2.06 ppm (s, 3H). 13C-NMR (CDCl3, 100 MHz)
δ (ppm): 168.9, 142.8, 138.8, 130.9, 124.4, 118.5, 39.5. (Found: C, 58.5; H, 4.5; N,
13.4%. C10H10N2O3 requires C, 58.3; H, 4.9; N, 13.6%).
General procedure for synthesis of nitroalkanes
In a 25 mL round-bottom flask the corresponding nitroalkene (0.5 mmol) was
o
dissolved in THF (1.6 mL) and MeOH (0.2 mL). After cooling at 0 C, NaBH4 (2.5
mmol) was added in four fractions and allowed to react for 40 min at room
temperature under stirring. Then the solvent was distilled in vacuo, and the residue
was treated with brine (10 mL) and extracted with ethyl ether (3 × 5 mL). The organic
layer was dried with anhydrous sodium sulfate and the solvent was evaporated in
vacuo. The compounds were purified by column chromatography (SiO2, gradient of
n-hexane:ethyl acetate).
1
1-Dimethylamino-4-(2-nitroethyl)benzene (17): Yellow solid (0.03 g, 86%). H-
NMR (CDCl3, 400 MHz) δ(ppm): 7.08 (d, 2H, J=8.00), 6.70 (d, 2H, J=8.00), 4.56 (t,
2H, J=8.00), 3.24 (t, 2H, J=8.00), 2.94 (s, 6H). 13C-NMR (CDCl3, 100 MHz) δ (ppm):
149.8, 129.0, 122.5, 112.2, 76.5, 40.3, 32.3. (Found: C, 61.6; H, 7.4; N, 14.0%.
C10H14N2O2 requires C, 61.8; H, 7.3; N, 14.4%).
1
1-Methylthio-4-(2-nitroethyl)benzene (19): Yellow oil (0.03 g, 86%). H-NMR
(CDCl3, 400 MHz) δ(ppm): 7.23 (d, 2H, J=8.00), 7.08 (d, 2H, J=8.00), 4.95 (td, 1H,
J=8.00, J=12.00), 4.80 (td, 1H, J=8.00, J=12.00), 3.19 (td, 1H, J=8.00, J=12.00),
13
3.08 (td, 1H, J=8.00, J=12.00), 2.50 (s, 3H). C-NMR (CDCl3, 100 MHz) δ (ppm):
140.0, 130.2, 128.8, 126.8, 77.0, 32.0, 14.1. (Found: C, 54.5; H, 5.6; N, 6.9; S,
16.0%. C9H11NO2S requires C, 54.8; H, 5.6; N, 7.1; S, 16.3%).
1
1-Acetylamino-4-(2-nitroethyl)benzene (20): Yellow solid (0.03 g, 78%). H-NMR
(DMSO-d6, 400 MHz) δ(ppm): 7.48 (d, 2H, J=8.00), 7.18 (d, 2H, J=8.00), 4.60 (t, 2H,
13
J=8.00), 3.30 (t, 2H, J=8.00), 2.18 (s, 3H). C-NMR (DMSO-d6, 100 MHz) δ (ppm):
168.3, 137.5, 131.0, 129.4, 120.0, 76.0, 32.3, 24.6. (Found: C, 57.5; H, 5.6; N,
13.8%. C10H12N2O3 requires C, 57.7; H, 5.8; N, 13.5%).
Preparation of stock solutions. Stock solutions were prepared fresh in DMSO.
Dilutions were made in phosphate buffer at pH and ionic strength according to each
17

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experiment and corresponding. Controls of interference of DMSO and auto-
fluorescence of the compounds were performed.
Evaluation of the antioxidant capacity of the compounds. The antioxidant
capacity of the molecules was evaluated using the oxygen radical absorbance
capacity (ORAC) method [36]. The decay of the fluorescence at λexc 485 nm and
λem 512 nm due to oxidation of fluorescein (7.5 nM) by peroxyl radicals was
followed during 140 min in a Varioskan Flash plate reader (Thermo Electron Corp.,
Finland) in 75 mM phosphate buffer, pH 7.4, with 100 µM DTPA at 37o C. Peroxyl
radicals were generated by thermal decomposition of 30 mM 2,2'-Azobis(2-
amidinopropane) dihydrochloride (AAPH). As standard antioxidant, 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, 1.5-24 ꢀM) was used. For the
analysis, the curves of fluorescence vs time in the absence and in the presence of
the compounds (1.3-13 µM) were normalized by dividing the values by the
fluorescence at the beginning (f0)., and the area under the curves (AUC) were then
calculated using Ec. 9.
ꢖ
ꢗ
ꢙꢚꢁꢛꢄ
ꢙꢚꢁ
∑
ꢓꢔꢕ = 1 +
(9)
ꢖ
ꢘ
Were fi represents the fluorescence reading at time i. The net value was obtained by
subtracting the AUC obtained in the absence (AAPH alone) from the area in the
presence of antioxidant. The slope of a linear regression of AUC vs concentration
was used to determine Trolox equivalents for each compound, as follows (Ec. 10):
ꢜꢝꢞꢟꢞꢠ ꢡꢢ = ꢣꢤꢥꢦꢧ ꢨꢥꢩꢦꢥꢪꢫꢬ
(10)
ꢣꢤꢥꢦꢧ ꢭꢮꢥꢤꢥꢃ
Isolation of LDL from human plasma. Human plasma was obtained from blood
voluntarily donated at the Hematology Department at Hospital de Clínicas. The
procedures were in accordance with the Helsinki’s Declaration and approved by the
Institutional Committee. Each blood donor was informed of their right to refuse, the
relevance of the investigation and the privacy (identity protection) of the information
taken, an informed consent form was signed by each donor. Total blood (450 mL)
was collected in primary bags containing 63 mL of anticoagulant solution CPD (129
mM dextrose, 105 mM citrate and 16 mM phosphate, Terumo Corporation, Tokyo,
Japan) as described [37]. Blood bags were centrifuged using a Roto Silenta 63RS
o
transfusion bag centrifuge (Hettich, Germany) at 2,200 rpm at 20 C, plasma was
o
transferred to a satellite bag and saved at -20 C until use (maximum two weeks).
The low-density lipoprotein (LDL) fraction was isolated from human plasma using a
18

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density gradient performed by adding 0.28 g KBr per mL of plasma. Then NaCl 0.15
M was added on top and ultracentrifugation was performed at 300.000 g for 90
minutes at 4 ºC. The orange band, LDL fraction, was collected and dialyzed against
phosphate buffered saline (PBS) prepared with 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4, pH 7.4. Protein concentration was evaluated at 280 nm
(ε = 1 cm-1 (mg/mL)-1).
LDL oxidation by AAPH. The capacity of the compounds to inhibit LDL oxidation by
AAPH was measured as the rate of oxygen consumption using an Oxygraph 2 K
(Oroboros Instruments Corp). Oxygen consumption by 0.2 mg/mL LDL and 30 mM
AAPH, in the presence and the absence of the compounds (20 µM) was recorded at
37°C under continuous stirring in 75 mM phosphate b uffer, pH 7.4. The rate of
oxygen consumption was calculated by means of the equipment software (DataLab)
and expressed as pmol O2 s−1. mL−1.
Fluorescent labeling and oxidation of LDL. The LDL fraction was labeled with the
lipophilic fluorescent probe (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-yl-2-
yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole (DiI, AnaSpec, Fermont, CA)
and subsequently oxidized as described before [27]. Briefly, the LDL fraction isolated
as described before was bubble with nitrogen to remove oxygen and incubated in the
presence of 50 ꢀL of DiI (3 mg/mL in DMSO) per mg of LDL protein overnight in the
dark at 37 °C. Next day the solution of DiI-LDL (0. 1 mg/mL) was incubated with 5 µM
CuSO4 at 37 °C for 24 h, protected from light. Then the DiI-oxLDL complex was
centrifuged at 30,000 g for 5 h at 4 °C. The band d istributed in the middle layer
containing DiI-oxLDL was isolated and dialyzed against PBS with 0.24 mM EDTA.
The protein content was determined by the bicinchoninic acid technique (Sigma-
Aldrich). Samples were sterilized by filtration and stored at 4 °C, in the dark for a
maximum of 3 weeks.
Macrophage J774 culture. 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) (Gibco, Invitrogen) containing glutamine (4 mM),
sodium pyruvate (110 mg L-1), glucose (4.5 g L-1), penicillin (100 U mL-1),
streptomycin (100 mg L-1), and 10 % heat-inactivated fetal bovine serum (FBS). The
cells were plated and incubated at 37 ºC in 5 % CO2 and 95 % air atmosphere.
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Foam cell formation. Following already published procedures [27,38], J774 cells at
80% confluence in 24 well plates (~2×105 cel/well) were incubated in the absence
and the presence of 5 µM of the analyzed compounds in DMEM for 1 h, after that
DiI-oxLDL (10 µg/mL) was added to the wells and the cells were incubated for 4
o
hours at 37 C and 5% CO2. Subsequently, the cells were washed three times with
PBS with 2 mg/mL BSA and twice with PBS. To stain the nuclei, the cells were
incubated for 10 minutes with 10 µg/mL Hoechst 3342 (AnaSpec, Fermont, CA) and
washed three times with PBS. Cellular uptake of DiI-oxLDL was observed using a
Zoe Fluorescent Cell Imager (Bio-Rad Laboratories Inc., Hercules, CA) using a red
filter for DiI and a blue one for Hoechst. Alternatively, the cell culture was done in 24
well plates and the cells treated in the same way, and quantifications performed
using a Varioskan Flash plate reader (Thermo Electron Corp., Finland) as described
in [39]. Results were expressed as the ratio between the fluorescence emitted by DiI
(λExc = 549 nm, λEm = 565 nm) and the fluorescence emitted by Hoechst (λExc = 352
nm, λEm = 461 nm).
Cytotoxicity analysis. Cell viability was determined by measuring the
mitochondrial-dependent reduction of MTT (Sigma Aldrich, MO, USA) to formazan
[40]. Macrophages in confluence were incubated in the presence of 0.3-10 µM of the
o
compounds for 2 and 24 h in DMEM at 37 C and 5% CO2. Then, the medium was
replaced by Dulbecco’s PBS containing MTT (0.1 mg mL-1) and the cells were
incubated at 37 ºC for 3 h. Formazan crystals were dissolved with DMSO (90:10 v/v)
in glycine buffer, containing 0.1 M NaCl, 0.5 mM EDTA, 0.1 M glycine, pH 10.5. The
absorbance was measured at 560 nm using the Varioskan Flash plate reader
(Thermo, Vantaa, Finland).
Inflammasome activation. Confluent cell monolayers were incubated in 24 well
plates in the absence and the presence of 5 µM of the dimethylamine derivatives for
1 h. The cells were activated with 400 U/mL interferon γ (IFN-γ, Sigma) and 8 µg/mL
o
of Salmonella enterica LPS (Sigma L7770) for 4 h in DMEM at 37 C, 5% CO2, as
described before [21]. Next, the cells were harvested and the proteins from complete
cell lysates were resolved in 15% SDS-PAGE, transferred to PVDF membranes and
proved with a rabbit polyclonal antibody against IL-1β (Abcam Inc., Cambridge, MA),
followed by a donkey anti-rabbit IgG H&L conjugated to Alexa Fluor 680 (Abcam
Inc., Cambridge, MA). Blots were scanned using G:BOX, Chemi XT4 (Syngene,
20

ACCEPTED MANUSCRIPT
Cambridge, UK), and band intensities analyzed using ImageJ 1.47v (NIH, USA).
Paired gels stained with Colloidal blue were run each time to account for protein load
(Figure S2)
Statistical analysis. Data are representative or were expressed as mean ± standard
deviation from at least three experiments and were analyzed by ANOVA one way
considering a significance level of p < 0.05.
21

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Acknowledgements
This research was partially supported by Comisión Sectorial de Investigaciones
Científicas (CSIC), Universidad de la República and PEDECIBA. LC and BM
received doctoral scholarships, from Agencia Nacional de Investigación e Innovación
(ANII, Uruguay).
Competing interests
None.
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Highlights
•
•
Inflammation and oxidative stress have a pivotal role on atherosclerosis
Arilnitroalkenes, and in particular the dimethylamino derivatives were shown to inhibit
both oxidative stress and inflammation
•
•
Moreover, newly synthetized arylnitroalkanes were even more effective than the
unsaturated analogous to prevent LDL oxidation and inflammasome activation
The lead compounds appear as promising multitarget therapies for atherosclerosis due
to its antioxidant and anti-inflammatory properties.