Synthesis and antioxidant activity evaluation of a syringic hydrazones family

Article abstract of DOI:10.1016/j.ejmech.2010.03.031

A novel series of hydrazones derived from syringaldehyde and their antioxidant properties have been explored. Several employed methods such as scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS? ⁺) radical cation expressed as Trolox equivalent antioxidant capacity (TEAC), inhibition of superoxide anion (O₂?^-) generation and of human cell-mediated low-density lipoprotein oxidation (monitored by the formation of TBARS) exhibited their potent antioxidant properties. The carbonyl scavenger efficacy was also evaluated by measuring the ability to decrease the protein carbonyl content in cells challenged with oxidized LDL. Inthis report, we discuss about the synthesis of hydrazones and their dual biological role, antioxidant and carbonyl scavenger for further application in atherosclerosis.

Full text of DOI:10.1016/j.ejmech.2010.03.031

European Journal of Medicinal Chemistry 45 (2010) 3019e3026
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antioxidant activity evaluation of a syringic hydrazones family
,
,
b
,
,
,b
Nadji Belkheiri a b, Benaissa Bouguerne a c, Florence Bedos-Belval a b, Hubert Duran a
,
Corinne Bernis c, Robert Salvayre c, Anne Nègre-Salvayre c , Michel Baltas
,
a,b,
**
*
a Université de Toulouse, UPS, LSPCMIB (Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique), 118 Route de Narbonne, F-31062 Toulouse Cedex 9, France
b CNRS, LSPCMIB (Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique), 118 Route de Narbonne, F-31062 Toulouse Cedex 9, France
c INSERM I2MR 858, IFR 31 CHU Rangueil, L3 Building, Avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of hydrazones derived from syringaldehyde and their antioxidant properties have been
explored. Several employed methods such as scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical and 2,20-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTSꢀþ) radical cation expressed as
Trolox equivalent antioxidant capacity (TEAC), inhibition of superoxide anion (Oꢀ2ꢁ) generation and of
human cell-mediated low-density lipoprotein oxidation (monitored by the formation of TBARS)
exhibited their potent antioxidant properties. The carbonyl scavenger efficacy was also evaluated by
measuring the ability to decrease the protein carbonyl content in cells challenged with oxidized LDL. In
this report, we discuss about the synthesis of hydrazones and their dual biological role, antioxidant and
carbonyl scavenger for further application in atherosclerosis.
Received 1 December 2009
Received in revised form
17 March 2010
Accepted 20 March 2010
Available online 25 March 2010
Keywords:
Syringic hydrazones
Antioxidants
Oxidative stress
Carbonyl stress
LDL oxidation
Atherosclerosis
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
cellular membranes resulting in the generation of reactive carbonyl
compounds (RCC) that react rapidly with free amino groups and
Oxidative stress is a well-known mechanism that is responsible
for the development of vascular damage. Different pathogenic
stimuli involved in cardiovascular diseases, such as activated
macrophages, hyperglycaemia, oxidized low-density lipoprotein
(LDL), exert their harmful effects, at least partially, through an
increased local generation of reactive oxygen species. Reactive
Oxygen Species (ROS) are normally produced throughout oxygen
metabolism and play a major role in physiological and pathological
cell redox signalling [1]. Oxidative stress results from an imbalance
between i) an overproduction of ROS by the different cellular
sources such as the mitochondrial respiratory chain, nicotinamide
adenine dinucleotide phosphate hydride oxidases (NADPHOXs or
NOXs), xanthine oxidase, lipoxygenases, cytochromes P450, and
other oxidases and ii) decreased cellular and plasma antioxidant
defenses [1]. Besides the direct free radical attack of cellular
components, oxidative stress induces a lipid peroxidation of
thiol residues on proteins thereby forming adducts involved in the
“carbonyl stress” [2,3]. Both oxidative and carbonyl stress alter
protein function and trigger the accumulation of modified proteins,
which results in inflammation and apoptosis [3,4]. ROS and RCC are
implicated in the pathophysiology of ageing and oxidative stress-
associated pathologies such as diabetes, neurodegenerative
diseases, atherosclerosis and cardiovascular complications [5,6].
Low-density lipoproteins (LDL) become atherogenic after under-
going oxidation within the vascular wall, in the presence of tran-
sition metals and ROS generated by vascular cells [7]. Oxidized LDL
are involved in the formation of early atherosclerotic lesions (foam
cell deposition as fatty streaks), and in their evolution toward more
advanced states characterized by endothelial dysfunction, chronic
inflammatory responses and toxic events leading to plaque rupture
and thrombosis [7,8].
Antioxidants inhibit the generation of ROS and the subsequent
formation of lipid peroxidation products, thereby preventing both
oxidative and carbonyl stress. Most antioxidants prevent LDL
oxidation in cell-dependent and cell-free systems, and delay the
formation of atherosclerotic lesions in animal models for athero-
sclerosis such as apoE-/-mice [9]. Discrepancies exist about their
efficiency against atherosclerosis in humans; this resulting at least
in part, from their bioavailability within the plaque, their chemical
* Corresponding author.
** Corresponding author. Université de Toulouse, UPS, LSPCMIB (Laboratoire de
Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique), 118 Route de
Narbonne, F-31062 Toulouse Cedex 9, France. Tel.: þ33 (0) 5 61 55 62 89; fax: þ33
(0) 5 61 55 60 11.
E-mail address: baltas@chimie.ups-tlse.fr (M. Baltas).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.031

3020
N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
H3CO
HO
H3CO
N NHR
EtOH,
6-12h
+
RNHNH2,
(Y = HCl
Y
CHO
HO
H3CO
H3CO
for 1, 3, 4 and 6 only)
Cpd
ID
1
2
3
4
5
6
7
8
9
10
F
H3C
N
CF3
N N
N
N
HN
NH
R
F
S
N
H2N
O
Cl
Cl
O
O
O
O
Yield
75%
97%
82%
47%
48%
86% 89% 97%
64%
90%
Scheme 1. Synthesis of syringic hydrazones.
nature and their inability to scavenge RCCs, once adducts are
formed on proteins [10]. However antioxidants remain very effi-
cient in preventing the early atherosclerotic lesions and inflam-
matory events implicated in the evolution of the lesions toward
more advanced states [9,10].
and commercially available organic nitrogen radicals. DPPH radicals
are considered as a representative method for the preliminary
screening of compounds able to scavenge activated oxygen species,
since they are more stable and easier to handle than oxygen free
radicals and DPPH assay has been largely used to measure the
antioxidant activity of different phenolic compounds.
We have recently reported that hydrazine derivatives such as
hydralazine, exhibit efficient antiatherogenic activity mediated by
their carbonyl scavenger activity [11]. However their antioxidant
capacity is moderate, limiting their use as antiatherogenic agents.
Moreover, we have also reported that phosphonocinnamic deriva-
tives obtained by reaction between syringaldehyde and activated
phosphonates possess interesting dual biological activities as
antioxidant and cytoprotective properties [12]. More recently, we
have designed biaryl compounds based on 5,50-bisvanillin scaffold
possessing identical or different substitutions: the known phos-
phonodiester and/or fluorophosphonoester moiety and a hydra-
zone function [13]. The best compounds in term of antioxidant and
cytoprotective effects was found to be the symmetrical compound
bearing two hydralazine scaffolds while all other ones were much
less potent. In continuation of this work and in order to get insight
on the efficiency of the hydrazone functionality, we report here the
synthesis of simpler monomeric hydrazono-derivatives obtained
through syringaldehyde coupling with a series of hydrazines and
their ability to block oxidation parameters. Their potency have been
evaluated through their impact on the 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) assay [14], Trolox equivalent antioxidant capacity
(TEAC) [15], the carbonyl and Oꢀ2ꢁ scavenging activities.
This method is based on measuring the continual absorbance
decrease of the ethanol solution of the stable free nitrogen radical
DPPH at 517 nm, in the presence of the derivatives tested. All tested
hydrazones present UV absorbance between 230 and 350 nm, and
do not interfere with the kinetic measurement of DPPH absorbance
decrease at 517 nm. The radical scavenging activity was expressed
as the molar amount of antioxidant necessary to decrease the initial
DPPH concentration by 50% (efficient concentration) EC50 (Fig. 1).
The DPPHꢀ absorbance decrease in presence of antioxidants is
due to hydrogen transfer from the antioxidant, thus forming the
DPPH-H stable compound. Ethanolic DPPHꢀ solutions used at
different concentrations were stable (checked over a 60 min
period). A significant decrease in the concentration of DPPHꢀ radical
due to the scavenging ability of some of the antioxidants was
observed. The DPPHꢀ absorbance values were respectively 0.16,
0.34, 0.38, 0.38, 0.48, 0.50, 0.64, 0.65, 0.75, 0.92 and 1.00 with
DPPHꢀ initial optical absorbance of 1.01 at 517 nm. The IC50 values
were determined for all compounds and reported in Fig. 1.
Compounds possessing a pyridine ring adjacent to the carbonyl
group have the lowest IC50 values among the synthesized hydra-
zones (17 and 18
As shown in Scheme 2 for compound 8, the hydrogen atom of
the isoniazid moiety in position from the hydrazone function is
mM for compounds 8 and 7 respectively).
a
2. Chemistry
highly activated. Compound 8 may easily undergo abstraction of
Syringic hydrazones described were readily synthesized
according to the procedures outlined in Scheme 1. Commercially
available syringaldehyde was refluxed in absolute ethanol with the
different hydrazines during 6e12 h. The reaction was followed by
TLC until completion. The solution was then cooled to room
temperature and the resulting precipitate was collected by filtra-
tion to provide the corresponding hydrazones as hydrochloride salt
or not, depending on the nature of commercially available hydra-
zines in good to excellent yield (Scheme 1).
120
>103
100
80
60
40
20
0
3. Pharmacology
3.1. Scavenging radical activity on 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical
1
2
3
4
5
6
7
8
9
10
Trolox
Fig. 1. IC50: concentration needed for reducing DPPHꢀ absorption by 50% at 517 nm.
Values are average of 3 independent measurements for each compound. Errors are
ꢂlog SD (standard deviation).
Radical scavenging potency of the compounds tested was
assessed in vitro by the DPPH assay. DPPH is one of the few stable

N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
3021
N
N
N
N
N
NO2
NO2
O
O
N
N
O
N
N
O
O
N
N
NH
N
N
N
O2N
NO2
O2N
NO2
NH
N
N
N
+
+
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OCH3
H3CO
OH
OH
OH
OH
OH
DPPH-H
DPPH
(E)-N'-(4-hydroxy-3,5-dimethoxybenzylidene)nicotinohydrazide intermediate radicals
Scheme 2. Purposed reaction of DPPHꢀ with nicotinohydrazide derivative 8.
one hydrogen atom from this position. Hydrogen atom abstraction
from phenolic ring under the examined conditions is quite difficult
due to its strong intramolecular bond with the adjacent methoxy
groups. As a consequence the reaction of DPPH radicals may create
nitrogen radical on the antioxidant considered. This is in agreement
with the recent work of Dolenc et al. [17] concerning the autoxi-
dation process of hydrazones. After studying structurally different
hydrazones, the authors proposed for those possessing at least one
hydrogen atom on nitrogen, the formation of Nꢀ centered radical
(hydrazonyl radical) which is stabilized through conjugation
forming delocalisated Cꢀ centered radicals. The different DPPHꢀ
scavenging properties (IC50) of the synthesized hydrazones may
reflect the relative ability for the NeH hydrogen atom abstraction
from the considered compound.
persulphate to give the relatively stable (over 24 h) ABTS radical.
The concentration of ABTSꢀþ is measured at 734 nm [16]. Thus the
antioxidant-induced reduction of the ABTSꢀþ concentration is
directly related to the antioxidant capacity of the compound being
tested.
ABTS radicals are involved in an electron transfer process. All
screening compounds exhibited at various levels radical cation
scavenging activity. There were no differences between the TEAC
values for the syringic hydrazones at 1 min, 4 min and 6 min (data
not shown).
As seen in Fig. 2, compounds 7 and 8 were the most effective
ABTSꢀþ radical scavengers depending on concentration range
(0e15
hydrazone concentrations, the effect being less pronounced for
m
M). ABTSꢀþ absorbance decreases significantly with all
hydrazone 8 at concentrations above 12 mM. The scavenging effects
on ABTSꢀþ decreased in the order 7 > 8 > 9 > 10 > 4 ¼ Trolox ¼
3.2. Scavenging radical activity on 2,20-azinobis(3-ethylbenz-
thiazoline-6-sulfonic acid) (ABTSꢀþ) radical cation
5 > 2 > 3 > 1 >6.
Table 1 summarised the results obtained in terms of Trolox
equivalents capacity. Three hydrazones, among the tested ones, 7, 8
and 9 possess TEAC values more than 15% of the Trolox value while
the other compounds possess equivalent or inferior values. This
might be due to a better transfer of radical and better resonance
stabilisation of the radical species for theses compounds (Table 1).
The antioxidative activity of syringic hydrazones was also
evaluated by measuring the Trolox equivalent antioxidant capacity
values (TEAC).This method measures the relative ability of antiox-
idant substances to scavenge the radical cation 2,20-azinobis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTSꢀþ
) compared with
a standard amount of the synthetic antioxidant Trolox (6-hydroxy-
2.5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analogue.
The TEAC value is defined as the concentration of standard Trolox
with the same antioxidant capacity as 1 mM concentration of the
antioxidant compound under investigation. This assay is based on
the generation and detection of a coloured long-lived specific
radical cation; ABTS is chemically oxidized by potassium
3.3. Effect of syringic hydrazones on LDL oxidation and superoxide
anion generation
The protective effect of syringic hydrazones was studied on LDL
oxidation induced either by cell contact with human microvascular
endothelial cells (HMEC-1) (this system allows to mimic the
pathophysiological events occurring in the vascular wall) [18], or by
two “cell-free systems” e.g. i) oxidation by copper ions at 37 ꢃC,
which initiates radical chain reaction with lipoperoxides pre-
formed in LDL, thereby generating peroxy radicals, hydroxides,
hydroperoxides and aldehydes [19], and ii) UV-C irradiation at
0,7
Trolox
0,6
8
7
0,5
9
Table 1
0,4
0,3
0,2
0,1
0
Determination of antioxidant activity as TEAC of syringic hydrazones by ABTS.
Cpd ID
TEAC
1
2
3
4
5
6
7
8
9
10
0.75 ꢂ 0.1
0.89 ꢂ 0.01
0.78 ꢂ 0.016
1.02 ꢂ 0.01
0.98 ꢂ 0.03
0.69 ꢂ 0.02
1.25 ꢂ 0.024
1.25 ꢂ 0.02
1.15 ꢂ 0.02
1.11 ꢂ 0.024
0
5
R adical scavenger concentration (µM)
10
15
Trolox
1
Fig. 2. ABTS Radical scavenging activity of syringic hydrazones compared with Trolox.
Average values of 3 independent measurements ꢂ log SD for each compound.
Average values ꢂ log SD of 3 independent measurements for each compound.

3022
N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
Table 2
Since oxidized LDL are highly cytotoxic for vascular cells, we
measured the cell viability of HMEC-1 at the end of the cell-induced
LDL oxidation process by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay. As shown in Table 2, cell-
oxidized LDL exhibited a strong cytotoxicity for HMEC-1, which was
protected byall the newly synthesized syringic hydrazones, and was
similar to the protective effect of Trolox but less to the one of 5,50-
bisvanillin bearing two hydralazine scaffolds (55e79% vs 82%) [13].
We then compared the antioxidant ability and cytoprotective
effect to the constitutive frame (Fig. 3), of three agents possessing
different hydrazone links: compound 1 with hydralazine frame
(only hydrazone link), compound 6 with aminoguanidine moiety
(free NH) and compound 8 with isoniazid fragment (hydrazide
link). Likewise, syringaldehyde, aminoguanidine or isoniazid did
not block cell-mediated LDL oxidation, while syringic isoniazid
hydrazone 8 and syringic aminoguanidine hydrazone 6 were very
efficient (Fig. 3A). The ability of these agents to inhibit LDL oxida-
tion and Oꢀ2ꢁ generation mediated by HMEC-1 was tested, and
compared to the results obtained in the presence of native hydra-
zines. Interestingly, neither syringaldehyde, nor aminoguanidine
and isoniazid were protective against oxidized LDL-induced cyto-
toxicity, while their respective monomeric derivatives 6 and 8 were
strongly efficient for inhibiting LDL oxidation and cytotoxicity as
well (Fig. 3). It is to note that hydralazine and its related monomer
exhibited comparable inhibitory properties against LDL oxidation
and cytotoxicity (Fig. 3B), in agreement with previous results
showing the strong antioxidant and cytoprotective properties of
hydralazine [11].
Determination of protective activity of syringic hydrazones on LDL oxidation.
Cpd ID
LDL oxidation
In vitro: Cu
MTT
Oꢀ2ꢁ
In vitro: UV
Cell-mediated
1
2
3
4
5
6
7
8
9
5 ꢂ 1.5
2 ꢂ 0.3
4 ꢂ 0.1
6 ꢂ 0.3
3 ꢂ 0.1
6 ꢂ 0.2
77 ꢂ 6
85 ꢂ 7
4 ꢂ 0.3
6 ꢂ 0.5
18 ꢂ 2.5
17 ꢂ 0.3
16 ꢂ 3.5
18 ꢂ 2.5
15 ꢂ 3.5
24 ꢂ 1.5
20 ꢂ 1.5
18 ꢂ 1.5
17 ꢂ 1.5
18 ꢂ 3.5
4 ꢂ 0.2
3 ꢂ 0.2
13 ꢂ 9
5 ꢂ 0.3
4 ꢂ 0.3
10 ꢂ 6
5 ꢂ 0.1
7 ꢂ 0.2
3 ꢂ 0.1
3 ꢂ 0.2
66 ꢂ 5
55 ꢂ 1
79 ꢂ 1
58 ꢂ 6
74 ꢂ 4
70 ꢂ 1
60 ꢂ 10
65 ꢂ 7
69 ꢂ 0.2
69 ꢂ 9
30 ꢂ 3
56 ꢂ 4
33 ꢂ 2
57 ꢂ 6
42 ꢂ 3
36 ꢂ 3
44 ꢂ 3
22 ꢂ 2
50 ꢂ 3
50 ꢂ 4
10
Trolox
6 ꢂ 0.2
18 ꢂ 4.5
3 ꢂ 0.1
57 ꢂ 0.1
5 ꢂ 2
254 nm, which generates polyunsaturated fatty acid (linoleate)
hydroperoxides, as well as oxysterols and aldehydes [20]. Prelimi-
nary self-toxicity experiments indicated that some syringic
hydrazone monomers exhibit a slight cytotoxicity for HMEC-1, at
concentrations higher than 100 mM. As a consequence, the
protective effect of the different agents on LDL oxidation was
studied and compared to that of Trolox [21] at a fixed concentration
of 10 mM (Table 2).
The data presented in Table 2, indicate that all the monomers
except hydrazones 7 and 8 exhibited a comparable inhibitory effect
on cell-induced and UV-induced LDL oxidation, which was similar
to that of Trolox. All the agents blocked effectively copper-induced
LDL oxidation, except compounds 7 and 8 which were inefficient
(note that isoniazid and syringaldehyde alone did not block copper-
nor UV-induced LDL oxidation, data not shown).
3.4. Carbonyl scavenger properties
All the evaluated compounds blocked efficiently cells-induced
LDL oxidation at the same level observed for 5,50-bisvanillin
bearing two hydralazine scaffolds [13]. Syringic hydrazones
inhibited by 30e50% the generation of Oꢀ2ꢁ by HMEC-1, in agree-
ment with the implication of O2ꢀꢁ in LDL oxidation, as reported [18].
However, the inhibitory effect of syringic hydrazone on O2ꢀꢁ
generation was lower than that observed with Trolox (Table 2),
suggesting that other ROS are implicated in cell-mediated LDL
oxidation.
Carbonyl stress is characterized by the formation and the
accumulation of adducts formed on proteins by aldehydes
generated during the oxidation of polyunsaturated fatty acids
[3,5]. The formation of adducts on proteins lead to a progressive
protein dysfunction finally resulting in apoptosis [11]. As an
exemple, 4-hydroxynonenal (4-HNE) is a major aldehyde gener-
ated during the peroxidation process of polyunsaturated fatty
acid in LDL [11]. 4-HNE is involved in the carbonyl stress, as it
forms adducts on proteins and induces cytotoxicity. The
Fig. 3. Cell-mediated LDL oxidation and subsequent toxicity on HMEC-1 cells of syringic hydralazine 1, syringic aminoguanidine 6 and syringic isoniazid 8. Comparison with their
constitutive frames. *Significant between the indicated values. ns: not significant between the indicated values. Syr, syringaldehyde; Is, isoniazid; AG, aminoguanidine; Hdz,
hydralazine.

N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
3023
medium, but rather from the inhibition of cell-mediated ROS
generation, in agreement with the inhibition of cellular NADPH
oxidase by hydralazine previously reported [23]. It is hypothesized
that the protective (antiatherogenic) effect of syringic hydrazones
should result from their carbonyl scavenger properties and the
potent antioxidant effect against LDL oxidation (and Oꢀ2ꢁ genera-
tion), resulting from the combination of syringaldehyde with
hydrazine compounds (isoniazid) or aminoguanidine, while these
agents alone exhibit poor antioxidant activity. It is to note that
Trolox, though strongly efficient antioxidant, is unable to trap and
neutralize carbonyl compounds and aldehydes issued from lipid
peroxidation, once formed, in contrast to syringic hydrazones
which share both antioxidant and carbonyl scavenger properties.
These new and original hydrazone drugs could constitute a new
class of agents sharing both valuable antioxidant and carbonyl
scavenger activities, with potential application in oxidative stress-
associated pathologies, and particularly atherosclerosis.
5. Experimental section
5.1. Chemistry
A mixture of hydrazine (1 equivalent) and syringaldehyde (1
equivalent) was dissolved in absolute EtOH (40 mL/mmol). The
solution was heated under reflux for 6e24 h depending on hydra-
zine used and then cooled to 25 ꢃC. The precipitated solid was
collected by filtration and rinsed thoroughly with EtOH to afford
the corresponding hydrazone. If no precipitate was observed, the
reaction mixture was concentrated under reduced pressure and the
crude residue was purified by silica gel chromatography affording
the corresponding hydrazone.
Fig. 4. Cytoprotective effect of syringic hydrazones against 4-HNE-induced cytotox-
icity. *Significant between the indicated values. ns: not significant between the indi-
cated values. Syr, syringaldehyde; Is, isoniazid; AG, aminoguanidine; Hdz, hydralazine;
Tro, Trolox.
protective effect of syringic hydrazones against carbonyl stress,
was evaluated on their ability to block the cytotoxicity induced
by 4-HNE (25 mM) in HMEC-1. As shown on Fig. 4, most agents
5.1.1. (E)-4-Hydroxy-3,5-dimethoxybenzaldehyde phthalazin-
almost completely prevented 4-HNE-induced cell death, and
were much more efficient than hydrazine derivatives used alone,
particularly isoniazid. Syringic acid (used for the synthesis of
syringic hydrazones) and (used as antioxidant) were inefficient in
preventing 4-HNE-induced cytotoxicity.
1-ylhydrazone hydrochloride (1)
Yellow solid; yield 75%; m.p. 154e156 ꢃC; IR (KBr)
n
cmꢁ1: 3415
(OeH), 1630 (C]N), 1619 (C]C), 1592 (C]C arom.), 1516 (C]C
arom.), 1464 (C]C arom.), 1222 (CeO), 1122 (CeO); 1H NMR (CDCl3,
300 MHz)
d ppm: 3.84 (s, 6H, OCH3), 7.38 (s, 2H, H2,6), 8.15 (td,
J ¼ 8.2 Hz, J ¼ 1.5, 1H, H11), 8.21 (t, J ¼ 8.2 Hz, 1H, H12), 8.25 (d,
4. Conclusion
J ¼ 8.0 Hz, 1H, H13), 8.98 (s, 1H, H7), 9.04 (s, 1H, H15), 9.23 (d,
J ¼ 7.6 Hz, 1H, H10); 13C NMR (CDCl3, 75 MHz)
d ppm: 56.83 (s, 2C,
In this work, we have evaluated the antioxidant and carbonyl
scavenger capacities of a series of newly synthesized syringic
hydrazones. They have been obtained easily through coupling
reaction between syringaldehyde and suitably chosen hydrazine
possessing electron withdrawing or electron attracting groups.
Their potency against the oxidative stress was addressed by
different means: via the 1,1-diphenyl-2-picrylhydrazyl (DPPH),
Trolox equivalent antioxidant capacity (TEAC), inhibition of LDL
oxidation and of superoxide anion generation. These new
compounds are of interest because all of them were found to be
effective antioxidants possessing reducing power on the DPPHꢀ and
TBARS assays, depending on their capacity of an NeH hydrogen
abstraction and on resonance stabilisation of the phenoxy radical.
In the TBARS assay the compounds seemed at most equivalent to
the Trolox scavenging capacity, which does not reflect their overall
antioxidant activity possibly due to other mechanisms involved.
Moreover, these agents exhibit a strong carbonyl scavenger activity
evidenced by their ability to block 4-HNE-mediated cell death
(which is not prevented by antioxidants, as reported here with
Trolox).
OCH3), 106.95 (s, 2C, C2,6), 123.60 (s, 1C, C1), 125.93 (s, 1C, C10),
128.30 (s, 1C, C9), 128.64 (s, 1C, C13), 134.14 (s, 1C, C11), 136.27 (s, 1C,
C12),140.00 (s,1C, C4),145.01 (s,1C, C15),147.95 (s,1C, C14),148.66 (s,
2C, C3,5), 153.94 (s, 1C, C7), 162.24 (s, 1C, C8); UV (EtOH, 50 mM,
25 ꢃC):
l
¼ 300 nm,
e
¼ 13,220 molꢁ1 L cmꢁ1
,
l
¼ 372 nm, ¼ 22,180
e
molꢁ1 L cmꢁ1; FAB MS: m/z: 325 [M þ Hþ]; ES HRMS: [M þ Hþ]
C17H17N4O3: 325.1315 (calcd 325.1301).
5.1.2. (E)-4-Hydroxy-3,5-dimethoxybenzaldehyde -
1,3-benzothiazol-2-ylhydrazone (2)
Yellow solid; yield 97%; m.p. 219e221 ꢃC; IR (KBr)
n
cmꢁ1: 3543
(OeH); 3480 (NeH); 3185 (]CeH ethyl.), 3068 (CeH arom.), 2846
(CeH, OeCH3), 1672 (C]N ethyl.), 1608 (C]C arom.), 1510 (C]C
arom.), 1249 (OeC arom), 1219 (OeC), 1110 (OeC); 1H NMR (CDCl3,
300 MHz)
d
ppm: 3.97 (s, 6H, OCH3); 6.94 (s, 2H, H2,6); 7.18 (t, 1H,
0
J ¼ 7.8 Hz, H5 ); 7.36 (t, 1H, J ¼ 8.1 Hz, H11); 7.56 (d, 1H, J ¼ 8.1 Hz,
H10); 7.68 (d, 1H, J ¼ 7.8 Hz, H13); 7.90 (s, 1H, H7). 13C NMR (CDCl3,
75 MHz)
d ppm: 56.45 (s, 2C, OCH3); 104.67 (s, 2C, C2,6); 121,97 (s,
2C, C11); 122.06 (s, 1C, C12); 125.05 (s, 1C, C1); 126.44 (s, 2C, C10,13);
129.43 (s, 2C, C14); 129.44 (s, 1C, C9); 138.05 (s, 1C, C4); 145.46 (s, 1C,
Altogether the data suggest that the inhibition of cell-mediated
LDL oxidation by syringic hydrazones is higher than that observed
with the initial hydrazine compounds, and probably does not result
from a simple ROS-scavenger or antioxidant activity in the culture
C7); 148.63 (s, 2C, C3,5); 167.24 (s, 1C, C8). UV (EtOH, 29.92 mM,
25 ꢃC):
l
¼ 342 nm,
e
¼ 34,920 molꢁ1 L cmꢁ1; ES MS: m/z 330.4
[M þ Hþ]; ES HRMS: [M þ Hþ] C16H16N3O3S: 330.0927 (calcd
330.0912).

3024
N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
5.1.3. (E)-4-Hydroxy-3,5-dimethoxybenzaldehyde(2-fluorophenyl)
hydrazone hydrocloride (3)
e
¼ 19,080 molꢁ1 L cmꢁ1
,
l
¼ 315 nm,
e
¼ 19,160 molꢁ1 L cmꢁ1; ES
MS: m/z 239.2 [M þ Hþ]; ES HRMS: [M þ Hþ] C10H15N3O3: 239.1115
Green solid; yield 82%; m.p. 104e106 ꢃC; IR (KBr)
n
cmꢁ1: 3422
(calcd 239.1144).
(NeH), 3332 (OeH), 3243 (]CeH ethyl.), 3009 (CeH arom.), 2938
(CeH, OeCH3), 1619 (C]N ethyl.), 1591 (C]C arom.), 1514 (C]C
arom.), 1243 (OeC arom), 1206 (OeC), 1115 (C-F); 1H NMR (CD3OD,
5.1.7. (E)-N0-(4-Hydroxy-3,5-dimethoxybenzylidene)
nicotinohydrazide (7)
300 MHz)
d
ppm: 3.80 (s, 6H, OCH3), 6.62 (dddd, J ¼ 8.1 Hz,
Yellow solid; yield 89%; m.p. 214e216 ꢃC; IR (KBr) cmꢁ1: 3480
n
0
J ¼ 7.4 Hz, J ¼ 4.8 Hz, J ¼ 1.6 Hz, 1H, H4 ), 6.88 (s, 2H, H2,6), 6.89
(OeH), 3185 (]CeH ethyl.), 2995 (CeH arom.), 2848 (CeH,
OeCH3), 1645 (C]N ethyl.), 1600 (C]C arom.), 1580 (NeH), 1510
(C]C arom.), 1252 (OeC arom), 1214 (OeC); 1H NMR (CD3OD,
0
(ddd, J ¼ 8.4 Hz, J ¼ 4.1 Hz, J ¼ 1.3 Hz, 1H, H6 ), 6.96 (dddd,
0
J ¼ 8.1 Hz, J ¼ 7.4 Hz, J ¼ 1.3 Hz, J ¼ 1.3 Hz, 1H, H5 ), 7.42 (ddd,
J ¼ 8.4Hz, J ¼ 8.3Hz, J ¼ 1.6 Hz, 1H, H3 )7.80 (s, 1H, H7); 13C NMR
300 MHz) d ppm: 3.92 (s, 6H, OCH3), 7.20 (s, 2H, H2,6), 7.60 (dd, 1H,
0
(CD3OD, 75 MHz)
d
ppm: 55.37 (s, 2C, OCH3), 103.36 (s, 2C, C2,6),
J ¼ 7.7 Hz, J ¼ 4.6 Hz, H11), 8.23 (s, 1H, H7), 8.36 (d, J ¼ 7.8 Hz, 1H,
113.74 (d, J ¼ 3.1 Hz, 1C, C13), 114.18 (d, J ¼ 180.0 Hz, 1C, C10), 118.09
(d, J ¼ 6.8 Hz, 1C, C11), 124.35 (d, 1C, C12), 126.59 (s, 1C, C1),133.90 (d,
J ¼ 9.3 Hz, 1C, C8), 136,51 (s, 1C, C4), 139.99 (s, 1C, C7), 148.07 (s, 2C,
C3,5), 149.70 (d, J ¼ 237 Hz, 1C, C9); 19F NMR (CD3OD, 282 MHz)
H12), 8.74 (dd, J ¼ 1.1 Hz, J ¼ 4.8 Hz, 1H, H10), 9.09 (d, J ¼ 1.2 Hz, 1H,
H13). 13C NMR (CD3OD, 75 MHz)
d ppm: 55.43 (s, 2C, OCH3), 105.05
(s, 2C, C2,6), 123.84 (s, 1C, C10), 124.62 (s, 1C, C1), 129.40 (s, 1C, C8),
136.00 (s, 1C, C9), 138.36 (s, 1C, C4), 147.94 (s, 2C, C12), 148.09 (s, 2C,
C3,5), 150.36 (s, 1C, C7), 151.69 (s,1C, C11), 163.12 (s, 1C, CO); UV
d ppm: ꢁ137.96 (s, 1F); UV (EtOH, 45.24
m
M, 25 ꢃC):
e
l
¼ 244 nm,
e
¼ 16,220 molꢁ1 L cmꢁ1
,
l
¼ 290 nm,
¼ 9750 molꢁ1 L cmꢁ1
,
(EtOH, 49.76
m
M, 25 ꢃC):
l
¼ 330 nm,
e
¼ 22,080 molꢁ1 L cmꢁ1; ES
l
¼ 345 nm,
e
¼ 22,700 molꢁ1 L cmꢁ1; ES MS: m/z 291.2 [M þ Hþ];
MS: m/z 302.2 [M þ Hþ]; ES HRMS: [M þ Hþ] C15H16N3O4: 302.1143
ES HRMS: [M þ Hþ] C15H16N2O3F: 291.1135 (calcd 291.1145).
(calcd 302.1141).
5.1.4. (E)-4-Hydroxy-3,5-dimethoxybenzaldehyde(4-fluorophenyl)
hydrazone hydrocloride (4)
5.1.8. (E)-N0-(4-Hydroxy-3,5-dimethoxybenzylidene)
isonicotinohydrazide (8)
Brown crystals; yield 47%; m.p. 170e174 ꢃC; IR (KBr)
n
cmꢁ1
:
Yellow solid; yield 97%; m.p. 195e198 ꢃC; IR (KBr) cmꢁ1: 3397
n
3493 (OeH), 1611 (C]N), 1584 (C]C), 1505 (C]C arom.), 1212
(OeH), 3195 (]CeH ethyl.), 2846 (CeH, OeCH3), 1646 (C]N
ethyl.), 1587 (C]C arom.), 1515 (C]C arom.), 1453 (C]C arom.),
(OeC), 1117 (C-F); 1H NMR (CD3OD, 300 MHz)
d
ppm: 3.90 (s, 6H,
OCH3), 6.95 (s, 2H, H2,6), 6.96 (dd, J ¼ 9.0 Hz, 2H, H3 ,5 ), 7.07 (dd,
1219 (CeO), 1116 (CeO); 1H NMR (DMSO-d6, 300 MHz)
d ppm: 3.80
0
0
J ¼ 9.1Hz, J ¼ 4.8 Hz, 2H, H2 ,6 ), 7.70 (s, 1H, H7); 13C NMR (CD3OD,
(s, 6H, OCH3), 7.06 (s, 2H, H2,6), 7.78 (dd, J ¼ 4.5 Hz, J ¼ 1.7 Hz, 2H,
0
0
75 MHz)
d
ppm: 55.32 (s, 2C, OCH3), 102.92 (s, 2C, C2,6), 112.71 (d,
H10,13), 8.29 (s, 1H, H7), 8.74 (dd, J ¼ 4.5 Hz, J ¼ 1.7 Hz, 2H, H11,12); 13
C
J ¼ 7.5 Hz, 2C, C9,13), 114.93 (d, J ¼ 22.5 Hz, 2C, C10,12), 127.19 (s, 1C,
C1), 137.52 (s, 1C, C7), 141.13 (d, J ¼ 209.1 Hz, 1C, C11), 142.41 (s, 1C,
C4), 148.06 (s, 2C, C3,5), 158.17 (s, 1C, C8); 19F NMR (CD3OD,
NMR (DMSO-d6, 75 MHz) d ppm: 56.47 (s, 2C, OCH3), 105.32 (s, 2C,
C2,6), 122.02 (s, 2C, C10,13), 124.65 (s, 1C, C1), 138.39 (s, 1C, C4), 140.96
(s, 1C, C9), 148.44 (s, 2C, C3,5), 150.45 (s, 1C, C7), 150.67 (s, 2C, C11,12),
282 MHz)
¼ 240 nm,
L cmꢁ1
d
ppm: ꢁ128.77 (s, 1F); UV (EtOH, 39.73
m
M, 25 ꢃC):
162.24 (s, 1C, C8); UV (EtOH, 49.66
m
M, 25 ꢃC):
l
¼ 333 nm,
l
e
¼ 17,940 molꢁ1 L cmꢁ1
,
l
¼ 313 nm,
e
¼ 19,680 molꢁ1
e
¼ 20,960 molꢁ1 L cmꢁ1; FAB MS: m/z 302 [M þ Hþ]; ES HRMS:
,
l
¼ 345 nm,
e
¼ 25,370 molꢁ1 L cmꢁ1 ES MS: m/z 291.1
[M þ Hþ] C15H16N3O4: 302.1148 (calcd 302.1141).
[M þ Hþ]; ES HRMS: [M þ Hþ] C10H15N3O3: 291.1118 (calcd
291.1107).
5.1.9. (E)-N0-(4-Hydroxy-3,5-dimethoxybenzylidene)-3-methyl-1H-
pyrazole-5-carbohydrazide (9)
5.1.5. (E)-4-Hydroxy-3,5-dimethoxybenzaldehyde[2,6-dichloro-4-
Yellow solid; yield 64%; m.p. 223e225 ꢃC; IR (KBr) cmꢁ1: 3433
n
(trifluoromethyl)phenyl] hydrazone (5)
(NeH), 3293 (OeH), 3208 (]CeH ethyl.), 3117 (CeH arom.), 2964
(CeH, OeCH3), 1664 (C]O ethyl.), 1621 (C]N ethyl.), 1590 (C]C
arom.), 1516 (C]C arom.), 1327 (C-N), 1241 (OeC arom), 1216
White crystals; yield 48%. m.p. 161e163 ꢃC; IR (KBr)
n :
cmꢁ1
3430 (NeH), 3295 (OeH), 3080 (]CeH ethyl.), 3007 (CeH arom.),
2945 (CeH, OeCH3), 1607 (C]N ethyl.), 1587 (C]C arom.), 1514
(C]C arom.), 1261 (OeC arom), 1209 (OeC), 1116 (CeF), 1104
(OeC); 1H NMR (CD3OD, 300 MHz)
d
ppm: 2.36 (s, 3H, CH3), 3.92 (s,
6H, OCH3), 6.62 (s, 1H, H10), 7.21 (s, 2H, H2,6), 8.20 (s, 1H, H7); 13C
NMR (CD3OD, 75 MHz) ppm: 10.80 (s, 1C, CH3), 56.49 (s, 2C,
(CeCl); 1H NMR (CD3OD, 300 MHz)
d
ppm: 3.89 (s, 6H, OCH3), 7.01
(s, 2H, H2,6), 7.64 (s, 2H, H3 ,5 ), 7.97 (s, 1H, H7); 13C NMR (CD3OD,
75 MHz) ppm: 55.32 (s, 2C, OCH3), 103.52 (s, 2C, C2,6), 123.09 (q,
d
0
0
OCH3),104.95 (s, 2C, C2,6),105.27 (s,1C, C10),125.32 (s,1C, C1),138.22
(s, 1C, C4), 140.46 (s, 1C, C11), 146.49 (s, C, C7), 148.29 (s, 2C,
d
J ¼ 34 Hz, 1C, C11), 123.20 (q, J ¼ 270.9 Hz, 1C, CF3), 123.87 (s, 2C,
C9,13), 126.15 (s, 1C, C1), 126.17 (q, J ¼ 3.5 Hz, 2C, C10,12), 136.91 (s, 1C,
C4), 141.55 (s, 1C, C8), 143.15 (s, 1C, C7), 148.05 (s, 2C, C3,5). 19F NMR
C3,5),148.59 (s, 1C, C9), 158.70 (s, 1C, C8); UV (EtOH, 40 m
M, 25 ꢃC):
l
¼ 240 nm,
e
¼ 18,430 molꢁ1 L cmꢁ1
,
l
¼ 327 nm,
e
¼ 27,500 molꢁ1
L cmꢁ1
;
ES MS: m/z 305.2 [M þ Hþ]; ES HRMS: [M þ Hþ]
(CD3OD, 282 MHz)
d
ppm: ꢁ63.55 (s, 1F); UV (EtOH, 56.25
m
M, 25 ꢃC):
C14H17N4O4: 305.1252 (calcd 305.1250).
l
¼ 273 nm,
e
¼ 6860 molꢁ1 L cmꢁ1
,
l
¼ 340 nm,
e
¼ 19,270 molꢁ1
L cmꢁ1; ES MS: m/z 409.22 [M þ Hþ]; ES HRMS: [M þ Hþ] C16H14N2O3
5.1.10. (E)-Benzyl-2-(4-hydroxy-3,5-dimethoxybenzylidene)
Cl2F3: 409.0319 (calcd 409.0334).
hydrazinecarboxylate (10)
Yellow solid; yield 90%; m.p. 115e117 ꢃC; IR (KBr)
n
cmꢁ1: 3350
5.1.6. (E)-2-(4-Hydroxy-3,5-dimethoxybenzylidene)
(OeH), 3243 (]CeH ethyl.), 3071 (CeH arom.), 2964 (CeH,
OeCH3), 1707 (C]O), 1673 (C]N ethyl.), 1588 (C]C arom.), 1548
(NeH), 1513 (C]C arom.), 1245 (OeC arom), 1217 (OeC); 1H NMR
hydrazinecarboximidamide hydrochloride (6)
Yellow solid; yield 86%; m.p. 226e228 ꢃC; IR (KBr)
n
cmꢁ1: 3387
(OeH), 3320 (NeH), 3200 (]CeH ethyl.), 3094 (CeH arom.), 2967
(CeH, OeCH3), 1689 (C]N ethyl.), 1609 (C]C arom.), 1519 (C]C
arom.), 1469 (NeH), 1115 (OeC arom); 1H NMR (CD3OD, 300 MHz)
(CD3OD, 300 MHz)
(s, 2H, H2,6), 7.29 (m, 5H, Har), 7.74 (s, 1H, H7); 13C NMR (CD3OD,
75 MHz) ppm: 55.40 (s, 2C, OCH3), 66.66 (s, 2C, C9), 104.34 (s, 2C,
d ppm: 3.77 (s, 6H, OCH3), 5.13 (s, 2H, H9), 6.95
d
d
ppm: 3.92 (s, 6H, OCH3), 7.14 (s, 2H, H2,6), 8.00 (s, 1H, H7), 13C NMR
C2,6), 125.17 (s, 1C, C1), 127.17 (s, 1C, Car), 127.72 (s, 2C, Car), 127.86 (s,
2C, Car), 136.47 (s, 1C, C4), 137.50 (s, 1C, C10), 145.78 (s, 1C, C7), 148.36
(CD3OD, 75 MHz)
124.03 (s, 1C, C1), 137.25 (s, 1C, C4), 148.15 (s, 2C, C3,5), 148.52 (s, 2C,
C7), 208.7 (s, 1C, C8); UV (EtOH, 50
M, 25 ꢃC):
¼ 234 nm,
d ppm: 55.53 (s, 2C, OCH3), 105.00 (s, 2C, C2,6),
(s,2C, C3,5), 155.10 (s, 1C, CO); UV (EtOH, 45.24 m
M, 25 ꢃC):
m
l
l
¼ 233 nm,
e
¼ 25,040 molꢁ1 L cmꢁ1
;
l
¼ 308 nm,
e
¼ 23,080 molꢁ1

N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
3025
L cmꢁ1; ES MS: m/z 331.2 [M þ Hþ]; ES HRMS: [M þ Hþ] C17H19N2O5:
acid (EDTA), as previously indicated [11]. LDL oxidation and the
antioxidant effect of syringic hydrazones were evaluated using
three different protocols, two cell-free systems (copper- and UV-
mediated oxidation) and one cell-mediated oxidation system by
331.1281 (calcd 331.1294).
5.2. DPPH scavenging activity
HMEC-1. Trolox (10 mM) was used as reference inhibitor of LDL
The DPPHꢀ solution has a deep violet colour. Radical scavenging
activity of antioxidant compounds can be measured spectropho-
tometrically at 517 nm. The DPPH radical shows absorbance at
517 nm, that decreases by the bleaching of a violet coloured ethanol
solution upon reduction by an antioxidant or a radical through
hydrogen atom or electron transfer to the odd electron in DPPHꢀ
producing the pale yellow non-radical form (DPPH-H). This
decrease is proportional to the increase of non-radical forms of
DPPH [22]. Briefly, 6 mM solution of DPPHꢀ was prepared in ethanol
oxidation, for the 3 methods. The extent of LDL oxidation was
monitored by the determination of the thiobarbituric acid reactive
substances (TBARS) content in the culture medium after 6 h incu-
bation. The amount of TBARS formed in the absence of any agent
was: cell-mediated oxidation,10.2e15.4 nmol TBARS/mg apoB; UV-
induced oxidation, 6.4e9.7 nmol TBARS/mg apoB; copper-medi-
ated LDL oxidation, 21.6e27.8 nmol TBARS/mg apoB. The results are
expressed as % of TBARS formed in the presence of the different
agents vs TBARS formed in the absence of antioxidants.
and, 90
mL of this solution was added to 2.1 mL of hydrazones
solution in ethanol at different concentrations (5e500
m
M). The
- Copper-induced LDL oxidation: LDL solutions (0.5 mg apoB/mL
NaCl 150 mM, pH 7.3) were incubated for 3 h at 37 ꢃC, in the
absorbance was measured at t ¼ 0 min (A0) and after 60 min
incubation at room temperature (A60). Percentage inhibition of
DPPH free radical (I%) was calculated in the following way: I% ¼
100 * (A0 ꢁ A60)/A0. IC50 represents concentration of the tested
compound, providing 50% inhibition of DPPH radicals. All experi-
ments were carried out in triplicate. The 6-hydroxyl-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) was used as antioxidant
reference [14].
presence of CuSO4
different syringic hydrazones used at the fixed concentration of
10 M. At the end, the oxidation of LDL was monitored by the
5 mM, as reported [11e23] and of the
m
determination of the TBARS content, using the fluorimetric
procedure of Yagi [24].
- UV-induced LDL oxidation: LDL solutions (1 mg apoB/mL NaCl
150 mM, pH 7.3 containing or not syringic hydrazones, 10 mM)
were irradiated for 2 hours, as a thin film (5 mm) in an open
5.3. ABTSꢀþ scavenging activity
beaker placed 10 cm under the UV-C source (HNS 30W OFR
Osram UV-C tube, l
max 254 nm, 0.5 mW/cm2) [11]. At the end,
The second test is based on the ability of antiradical molecules to
quench the ABTSꢀþ, a blueegreen chromophore with characteristic
absorption at 734 nm. The addition of antioxidants to the pre-
formed radical cation reduces it to ABTS, determining a decolouri-
zation. In this method, an antioxidant is added to a pre-formed
ABTSꢀrþadical solution, and after a fixed time period, the remaining
ABTS is quantified spectrophotometrically at 734 nm [22]. The
ABTSꢀþ was produced by reacting 7 mM ABTS in H2O with 2.45 mM
potassium persulfate (K2S2O8), stored in the dark at room
temperature for 12e16 h before use (the radical was stable for more
than two days under these conditions). The ABTSꢀþ solution was
further diluted in ethanol until the initial absorbance value ꢀoþf
the TBARS content was determined [24].
- Cell-mediated LDL oxidation: HMEC-1 diluted in DMEM (Dul-
becco’s modified Eagle’s medium) cultured medium containing
10% foetal calf serum and antibiotics were seeded in 24 mul-
tiwell plates. After 24 h, the standard culture medium (on
sparse proliferative cells), was removed and replaced by
serum-free Roswell Park Memorial Institute (RPMI-1640)
medium containing native LDL (100
(1
M), and HMEC-1. This mixture was incubated at 37 ꢃC for
6 h, in the presence (or the absence) of syringic hydrazones or
Trolox (10 M each) [11e24]. The same experiment was also
mg apoB/mL) and CuSO4
m
m
conducted for syringaldehyde, hydralazine, isoniazid and
aminoguanidine. At the end of the incubation, the LDL-con-
taining medium was used for determining TBARS [24]. The
results were expressed as % ꢂ S.E. of TBARS content in LDL
oxidized in the absence of antioxidant. Statistical analyses were
performed by ANOVA (Tukey test e SigmaStat software).
0.70 ꢂ 0.02 at 734 nm at 30 ꢃC. Then, in a 3 mL solution of ABTS
,
was added an ethanolic solution of the compound under study so
that their final concentrations vary between 0 and 15 mM. In each
case, a 20e80% decrease in the initial absorbance of the reaction
solution was achieved.
The absorbance was recorded at 0, 1, 4 and 6 min. The scav-
enging capacity of test compounds was calculated using the
following equation: ABTSꢀþ scavenging effect (%) ¼ 100(A0 ꢁ Ac)/A0,
where A0 is absorbance of a control lacking any radical scavenger
and Ac is absorbance of the remaining ABTSꢀþ in the presence of
scavenger. Graphs of antioxidant concentration versus percent
absorbance reduction were then plotted. The concentration of
Trolox giving the same percentage reduction of absorbance at
734 nm as the 1 mM antioxidant solution was calculated from the
three point graphs. The results were thus expressed as Trolox
equivalent antioxidant capacity (TEAC) values. For each hydrazone
and each concentration, measurements were performed in tripli-
cate. Absorbance values were corrected for radical decay using
blank solutions.
5.4.2. Cytoprotective and carbonyl scavenger effect
The subsequent toxicity of cell-oxidized LDL (in the absence or
presence of the different agents), was evaluated on HMEC-1 after
24 h incubation, using the MTT test [11,24]. Results were
expressed as % of cell viability vs the unstimulated control
(without LDL). The residual cell viability in cell incubated with
LDL in the absence of agents was around 15e21%. The carbonyl
scavenger effect of the various compounds was indirectly
measured by evaluating their cytoprotective effect on the cyto-
toxicity induced by 4-hydroxynonenal (4-HNE), an aldehyde
formed during the peroxidation of polyunsaturated fatty acids,
and involved in the formation of adducts on proteins. The effect
of the newly synthesized agents was compared to that observed
with Trolox, a classical antioxidant unable to block 4-HNE-
induced cytotoxicity.
5.4. Biochemical experiments
5.4.1. LDL oxidation
LDL were isolated by ultracentrifugation from the pooled
plasma of healthy normolipidemic human subjects and dialyzed
against PBS containing 100 mmol/L of ethylenediaminetetraacetic
5.4.3. Superoxide anion generation
Superoxide anion (Oꢀ2ꢁ) production was measured as the
superoxide dismutase (SOD)-inhibit able reduction of cytochrome

3026
N. Belkheiri et al. / European Journal of Medicinal Chemistry 45 (2010) 3019e3026
C and expressed as % of Oꢀ2ꢁ generated in HMEC-1 incubated 1 h
with LDL in the presence of agents vs HMEC-1 incubated with
LDL alone (8.2e10.6 nmol/h/mg protein). All these data are
a mean of 6 separate experiments þ/ꢁSEM. Briefly, sub confluent
HMEC-1 were pre-incubated for 30 and 60 min, in 1 mL phenol
References
[1] H. Sies, Am. J. Med. 91 (1991) 31Se38S.
[2] H. Esterbauer, R.J. Schaur, H. Zollner, Free Radic. Biol. Med. 11 (1991) 81e128.
[3] K. Uchida, Free Radic. Biol. Med. 28 (2000) 1685e1696.
[4] G. Leonarduzzi, M.C. Arkan, H. Basaga, E. Chiarpotto, A. Sevanian, G. Poli, Lipid
Free Radic. Biol. Med. 28 (2000) 1370e1378.
[5] E.R. Stadtman, Ann. N.Y. Acad. Sci. 928 (2001) 22e38.
[6] A. Negre-Salvayre, C. Coatrieux, C. Ingueneau, R. Salvayre, Br. J. Pharmacol. 153
(2008) 6e20.
[7] D. Steinberg, J. Biol. Chem. 272 (1997) 20963e20966.
[8] A.J. Lusis, Nature 407 (2000) 233e241.
[9] R. Stocker, Curr. Opin. Lipidol. 10 (1999) 589e597.
[10] R. Brigelius-Flohe, D. Kluth, A. Banning, Mol. Nutr. Food Res. 49 (2005)
1083e1089.
red free RPMI containing cytochrome C (20
SOD (100 mU/L), LDL (100 g apoB/mL) and the different hydra-
zones or Trolox (10
M). The Oꢀ2ꢁ specific reduction of cyto-
mM) with or without
m
m
chrome C was determined spectrophotometrically at 550 nm
(difference in absorbance with or without SOD), according to
McCord and Fridovitch [25] and under the previously used
conditions [23].
[11] S. Galvani, C. Coatrieux, M. Elbaz, M.H. Grazide, J.C. Thiers, A. Parini, K. Uchida,
N. Kamar, L. Rostaing, M. Baltas, R. Salvayre, A. Nègre-Salvayre, Free Radic.
Biol. Med. 45 (2008) 1457e1467.
5.4.4. 4-HNE-induced cytotoxicity
[12] C. Lapeyre, M. Deloménède, F. Bedos-Belval, H. Duran, A. Negre-Salvayre,
M. Baltas, J. Med. Chem. 48 (2005) 8115e8124.
[13] M. Delomenède, F. Bedos-Belval, H. Duran, C. Vindis, M. Baltas, A. Nègre-Sal-
vayre, J. Med. Chem. 51 (2008) 3171e3181.
Subconfluent HMEC-1 were overnight incubated in serum-free
Hank’s medium in the presence of 4-HNE (25
mM), and the different
molecules tested at a fix concentration of 10
mM. The cytotoxic
[14] M.C. Foti, C. Daquino, C. Geraci, J. Org. Chem. 69 (2004) 2309e2314.
[15] N. Nenadis, L.-F. Wang, M. Tsimidou, H.-Y. Zhang, J. Agric. Food Chem. 52
(2004) 4669e4674.
[16] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free
Radic. Biol. Med. 26 (1999) 1231e1237.
effect of 4-HNE was evaluated by MTT. Data are expressed as % of
the untreated control. Statistical analysis was performed by ANOVA
(Tukey test e SigmaStat software).
[17] M. Harej, D. Dolenc, J. Org. Chem. 72 (2007) 7214e7221.
[18] J.W. Heinecke, M. Kawamura, L. Suzuki, A. Chait, J. Lipid Res. 34 (1993)
2051e2061.
[19] O. Ziouzenkova, A. Sevanian, P.M. Abuja, P. Ramos, H. Esterbauer, Free Radic.
Biol. Med. 24 (1998) 607e623.
Acknowledgement
[20] I. Pinchuk, D. Lichtenberg, Free Radic. Res. 24 (1996) 351e360.
[21] R. Albertini, P.M. Abuja, Free Radic. Res. 30 (1999) 181e188.
[22] P. Molyneux, Songklanakarin, J. Sci. Technol. 26 (2004) 211e219.
[23] L. Mabile, O. Meilhac, I. Escargueil-Blanc, M. Troly, M.T. Pieraggi, R. Salvayre,
A. Nègre-Salvayre, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 1575e1582.
[24] K. Yagi, Chem. Phys. Lipids 45 (1987) 337e351.
The authors acknowledge the financial support from INSERM,
CNRS, Université Paul Sabatier and COST B35. Nadji Belkheiri is
recipient of a grant from algerian government. Benaissa Bouguerne
is recipient of the Ministère de l’Enseignement Supérieur et de la
Recherche.
[25] J.M. McCord, I. Fridovich, J. Biol. Chem. 244 (1969) 6049e6055.