Antioxidant activity of benzoxazolinonic and benzothiazolinonic derivatives in the LDL oxidation model

Article abstract of DOI:10.1016/j.bmc.2009.09.016

A series of benzazolone compounds were synthesized utilizing benzoxazolinonic and benzothiazolinonic heterocycles as the building unit. The antioxidant activity of these compounds was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO₄ or 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective action of these compounds against the cytotoxicity was evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The best antioxidant activities were observed for phenolic compounds 13 and 14b with ratio R = 2.5, 3.2 (5 μM). Both of these test substances increased the cell viability significantly as indicated by MTT assay and LDH release assay.

Full text of DOI:10.1016/j.bmc.2009.09.016

Bioorganic & Medicinal Chemistry 17 (2009) 7823–7830
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antioxidant activity of benzoxazolinonic and benzothiazolinonic derivatives
in the LDL oxidation model
Ismaël Yekini a, Fatma Hammoudi a, Françoise Martin-Nizard b, Saïd Yous a, Nicolas Lebegue a,
Pascal Berthelot a, Pascal Carato a,
*
a Faculté de Pharmacie, Université Lille Nord de France, Laboratoire de Chimie Thérapeutique (EA 1043)3, Rue du Professeur Laguesse, BP 83, 59006 LILLE Cedex, France
b Institut Pasteur de Lille, Département d’Athérosclérose, INSERM U545, Lille F-59019, Université Lille Nord de France, Faculté de Pharmacie et de Médecine, Lille F-59006, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2009
Accepted 10 September 2009
Available online 15 September 2009
A series of benzazolone compounds were synthesized utilizing benzoxazolinonic and benzothiazolinonic
heterocycles as the building unit. The antioxidant activity of these compounds was determined by
inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO4 or 2,20-azobis
(2-amidinopropane) dihydrochloride (AAPH). The protective action of these compounds against the cyto-
toxicity was evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells
(BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The best antioxidant activities were observed for
Keywords:
Melatonin
Pinoline
Benzazolones
Lipid oxidation
Antioxidant
phenolic compounds 13 and 14b with ratio R = 2.5, 3.2 (5
the cell viability significantly as indicated by MTT assay and LDH release assay.
Ó 2009 Elsevier Ltd. All rights reserved.
lM). Both of these test substances increased
1. Introduction
plasma infiltrate the intimal space of the arteries and are oxidized
by free radicals. There is considerable experimental evidence to
show that several different antioxidant compounds given at high
pharmacological doses are effective in decreasing both LDL oxida-
tion and atherogenesis in animals.8 In humans, supplementation
with antioxidants combined at physiological doses is incapable of
inhibiting coronary heart disease in primary prevention.9 Antioxi-
dants would have to be given at high pharmacological doses in hu-
mans to inhibit ex vivo Cu2+-induced LDL oxidation.10
The pineal gland secretes a number of pineal indoles including
melatonin. Melatonin (N-acetyl-5-methoxytryptamine), a highly
conserved naturally occurring molecule, is a pineal hormone that
regulates circadian rhythms and also protects against oxidative
stress-induced tissue damage, lipid peroxidation, and apoptosis.1,2
Antioxidant actions of melatonin are observed at different levels
including attenuation of radical formation, which is also referred
to as radical avoidance. Although melatonin efficiently interacts
with various reactive oxygen species (ROS) and reactive nitrogen
species as well as with organic radicals, it also upregulates antiox-
idant enzymes and downregulates pro-oxidant enzymes.3–6
Cardiovascular diseases (coronary heart disease, stroke, etc.),
depending on atherosclerosis development, remain the major
cause of death in most developed countries. Atherosclerosis is a
chronic vascular disease in which inflammation and oxidative
stress are commonly implicated as major causative factors. Early
stages of plaque development involve endothelial activation in-
duced by inflammatory cytokines, oxidized low-density lipopro-
tein (ox-LDL), and/or changes in endothelial shear stress. It has
been clearly demonstrated that hypercholesterolemia secondary
to high LDL plasma levels and subsequent oxidation of these lipo-
proteins are the major causes of atherosclerosis in humans and dif-
ferent animal models.7 LDL particles which accumulate in the
Duell 11 tested the capacity of melatonin in inhibiting the oxida-
tion of LDL in a standardized in vitro system. They showed that
melatonin had no antioxidant activity at physiologic concentra-
tions and only moderate antioxidant activity at concentrations that
were 4–6 orders of magnitude greater than peak physiologic con-
centrations. Therefore it seems interesting to study the free radical
scavenging and antioxidant activity of melatonin derivatives that
act as strong antioxidants and that could not act on circadian
rhythm.
The potent endogenous antioxidant melatonin possesses sev-
eral analogs or metabolites, and several effects attributable to mel-
atonin have been suggested to be mediated by these metabolites.12
Pinoline (6-methoxy-1,2,3,4-tetrahydro-b-carboline), a minor cyc-
lic metabolite of melatonin13, has been shown to inhibit lipid per-
oxidation in different model systems.14,15 Starting from pinoline,
previous research were realized in our laboratory to lead a b-carb-
oline compound GWC22.16 This compound has been shown to
display better antioxidant properties than melatonin toward per-
oxyl-induced peroxidation of a linoleate model system.17–19
* Corresponding author. Tel.: +33 3 20964966; fax: +33 3 20 964913.
E-mail address: pascal.carato@univ-lille2.fr (P. Carato).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.016

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I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
benzazolones (1a and 1b). From 3-methyl-2(3H)-benzazolone (1a
X = O, S
GWC22
and 1b), 6-bromoethyl-3-methyl-2(3H)-benzazolone (3a and 3b)
were obtained by a Friedel–Craft acid-catalyzed acylation22,23 fol-
lowed by a reduction of the ketone carbonyl group.23,24 While com-
pound 5b was obtained via a Delepine condensation25 of the
bromoethyl derivative (3b) with hexamethylenetetramine in good
yield (72%), application of this procedure to access compound 5a
was unsuccessful. Derivative 5a was obtained in two steps with
good yield (74%), starting from compound 3a in a Gabriel reaction,
the phthalimidoethyl derivative (4) was obtained in dimethylform-
amide with phthalimide potassium salt, then a reaction of hydrazi-
nolysis in ethanol afforded compound 5a.26 This methodology
could not be applied to benzoxazolone derivative (3b), the hydra-
zinolysis also opens the oxazole ring.
R1 = H, CH3, Bz
R2 = H, C3H7, C9H19
N
CH3
N
X
N
O
H
O
N
R2
O
CH3
O
CH3
O
N
H
6-MBAO
OR1
Figure 1. Design of benzoxazolinonic and benzothiazolinonic derivatives.
6-Methoxy-2-benzoxazolone (6-MBAO) was described in the
literature as exhibiting an antioxidative potency similar to that of
melatonin.20 Both the structures, GWC22 and 6-methoxy-2-benz-
oxazolinone, allowed to design a new family of benzoxazolinonic
and benzothiazolinonic derivatives with potent antioxidant activi-
ties (Fig. 1). Benzoxazolone and the sulfur bioisoster, that is, ben-
zothiazolone, were frequently used for medicinal chemistry.21 In
continuation with previous studies on the biologically active b-
carboline derivatives of melatonin, this work aimed to study the
possible protective effect of novel benzazolone derivatives.
In this family, we have modulated the nature of the heterocycle
with benzoxazolone (X = O) and benzothiazolone (X = S), the
length of the linear chain on the nitrogen atom of compounds with
propyl and nonyl groups. Various substituents (R1) were intro-
duced on the 3-position of the phenyl ring with R1 as hydrogen
atom, methyl, or benzyl groups. Then, the oxazolinonic ring of
the benzoxazolone heterocycle was open in basic media to give
the corresponding aminophenol derivatives.
Another way was attempted in four steps to synthesize com-
pound 5b. 3-Methyl-2(3H)-benzoxazolone was formylated using
hexamethylenetetramine in polyphosphoric acid (PPA),27 to afford
compound 6 . The formyl derivative 6 was condensed with nitro-
methane in the presence of a catalytic amount of ammonium ace-
tate to afford the expected nitrovinyl product 7 by a Henry
reaction.28,29 Reduction of the
a,b-unsaturated nitroalkene 7 to
the desired saturated compound 5b was performed with sodium
borohydride in a mixture of chloroform and isopropanol in the
presence of silica gel, followed by hydrogenation over Pd/C. This
way gave low yield of 29% while compound 5b was obtained using
the first way in three steps with 47% yield.
Scheme 2 illustrates the procedures used for the synthesis of
benzazolinonic analogs of GWC22. Under Schotten–Baumann reac-
tion conditions, aminoethyl compounds 5a–b were reacted with
phenyl acyl chloride derivatives to furnish the expected amide
derivatives 9a–c. Classical Bischler–Napieralski cyclization affor-
ded, in the presence of phosphorous oxychloride, compounds
10a–c, which were reduced in methanol by sodium borohydride
to give derivatives 11a–c. Attempts using Pictet–Spengler reac-
tion30 in CH2Cl2 with TFA, compound 5a or 5b, and the desired
benzaldehyde, to furnish compounds 11a–c were unsuccessful.
Alkylation of compounds 11a–c by n-propyl or n-nonyl iodide, in
acetonitrile with potassium carbonate, provided the corresponding
2. Results and discussion
2.1. Chemistry
Scheme 1 illustrates the procedures used for the synthesis of
the required primary amines 5a–b starting from 3-methyl-2(3H)-
CH3
N
O
BrCOCH2Br
X
(CH2)6N4
CH3
CH3
N
1a : X= S
1b : X= O
AlCl3
DMF
PPA
N
O
O
O
O
X
O
CHO
Br
2a : X= S
2b : X= O
6
O
CH3CO2NH4
CH3NO2
Et3SiH
TFA
CH3CO2H
CH3
N
3a : X= S CH3
3b : X= O
O
N
NK
DMF
O
CH3
O
O
X
NO2
Br
X= S
N
7
O
O
NaBH4
Silice
iPrOH
CHCl3
1) (CH2)6N4
CHCl3
2) HCl, EtOH
X= O
S
N
4
O
CH3
N
CH3
N
H2N-NH2
EtOH
H2 - Pd/C
MeOH
O
O
X
NO2
NH2
8
5a : X= S
5b : X= O
Scheme 1. Synthesis of 6-ethylamino-3-methyl-2(3H)-benzazolones (5a and 5b).

I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
7825
R1O
OR1
CH3
N
Cl
CH3
CH3
N
N
O
POCl3
O
N
O
O
O
K2CO3
H2O - CHCl3
X
X
X
NH2
N
H
10a : X= S R1= CH3
10b : X= O R1= CH3
10c : X= O R1= Bz
5a : X= S
5b : X= O
9a : X= S R1= CH3
9b : X= O R1= CH3
9c : X= O R1= Bz
OR1
R1O
HO
R1O
CH3
CH3
N
CH3
N
IR2
R2
N
H2 - Pd/C
MeOH
NaBH4
MeOH
K2CO3
N
N
NH
O
O
O
CH3CN
X
O
X
1) H2O - MeOH
NaOH
13
12a : X= S R1= CH3 R2= C3H7
12b : X= O R1= CH3 R2= C3H7
12c : X= O R1= Bz R2= C3H7
12d : X= O R1= CH3 R2= C9H19
11a : X= S R1= CH3
11b : X= O R1= CH3
11c : X= O R1= Bz
2) HCl
R1O
CH3
HN
N
14b : R1= CH3
14c : R1= H
HO
Scheme 2. Synthesis of final compounds 12a–d, 13, and 14b–c.
tertiary amines (12a–d). Deprotection reaction of the benzyl group
of compound 12c, in methanol with Pd/C under atmospheric pres-
sure of hydrogen, gave compound 13 with phenolic group. Com-
pounds 12b and 13, in a solution of water/methanol with sodium
hydroxide, afforded after acidification the aminophenolic com-
pounds 14b and 14c.
fore, the free radical scavenging mechanism contributes toward
the inhibitory action of these compounds.
All compounds, designed starting from derivatives GWC22 and
6-MBAO, showed antioxidant activities. Indeed, for compounds
10b and 11b an antioxidant effect is observed at 5 lM (R = 1.8),
the antioxidant activity is lower than that of the other compounds
synthesized but higher than that of 6-methoxy-2-benzoxazolone
2.2. Antioxidant activity
and melatonine21 (R = 1.5 for 100
l
M). Benzothiazolone com-
pounds (10a, 11a, and 12a, respectively, with R = 2.5, 2.1, and 2.1
for 5 M) have more antioxidant activity than derivative GWC22
(R = 1.6 for 5 M) and their benzoxazolone analogs (10b, 11b,
12b, respectively, with R = 1, 1, 1.5 for 5 M) which could be ex-
The oxidation of LDL was determined by measuring conjugated
diene formation at 234 nm for 480 min. To eliminate the possible
role of Cu-chelation, oxidation was induced by water-soluble
2,20-azobis(2-amidinopropane) dihydrochloride (AAPH), which
generates free radical, by its spontaneous thermal decomposition.
In the presence of CuSO4 or AAPH, the OD234 curve versus time
corresponds to the typical one described by Esterbauer,31 including
the lag, propagation, and decomposition phases.
l
l
l
plained by an improved lipophilicity with the sulfur derivatives
(10a, 11a, 12a). Substitution with benzyl group induced the best
results when we compared compounds 10b, 11b (R = 1 for 5
with 10c, 11c (R = 1.5 for 5 M) and compound 12b (R = 1.5 for
M) with 12c (R = 2.2 for 5 M), this compound 12c showed
lM)
l
5
l
l
Without Cu2+ or AAPH, the kinetics of diene conjugate forma-
tion was slower, and the final concentration was dramatically low-
er after 24 h of oxidation. The lag phase for the initiation of LDL
oxidation in the presence of copper or AAPH was about 100 min.
The capacity of derivatives to reduce or inhibit copper or AAPH-ini-
tiated LDL oxidation was tested and the kinetics of LDL oxidation
led us to calculate the ratio R (Table 1).
the best antioxidant activities in this benzoxazolone series supe-
rior to GWC22. In previous paper, the authors demonstrated the
relationship between the chain length (which influences the Log P)
and the antioxidative activity.32,33 In the benzoxazolone series we
have modulated the chain length at the N-2 position with deriva-
tives 12b and 12d, but we observed similar antioxidant activities
(R = 1.5 and 1.7 for 5 lM) for these compounds. Phenolic deriva-
R represents the ratio between the length of the lag phase of the
oxidative procedure of LDL with different compounds and the
length of the one without compound (control).
tives were well described to enhance antioxidant activities, so we
synthesized phenolic compound 13, by a debenzylation reaction
of compound 12c, which displays good antioxidant activities with
Addition of 1–100
lM of tested compounds to the LDL solution
a ratio R = 2.5 for 5 lM. Starting from derivatives 12b and 13, the
induced a dose-dependent increase in the lag phase of conjugated
diene formation (data not shown). Results at 5 M are represented
in Table 1.
ring opening of the oxazole led to aminophenolic derivatives 14b
and 14c. Compound 14c due to the color at 234 nm could not be
tested for the antioxidant activities, while 14b exhibited the best
Excepted for compounds 10b and 11b, (R = 1 for 5 M Cu2+) the
study confirms that the derivatives reduce copper-induced LDL
oxidation (for 5 M Cu2+). The most antioxidant products in a copper
system were tested in AAPH oxidation system. In the presence of
the tested molecules there was a strong correlation in the lag phase
duration between these two oxidative procedures of LDL. There-
antioxidant activity with a ratio R = 3.2 for 5 lM of the series.
2.3. Cytotoxicity assays
In vivo low-density protein (LDL) oxidation is a progressive
phenomenon leading to the presence of minimally and highly

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I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
Table 1
Antioxidant activities and cytotoxicity assays of compounds 10a–c, 11a–c, 12a–d, 13, and 14b,c
R1O
R1O
R1O
CH3
HN
CH3
N
CH3
N
R2
N
N
N
O
O
X
X
HO
11a-c, 12a-d, 13
10a-c
14b-c
Compd
R1
R2
Activity (R)
M (Cu2+
LDH release in %
100
l
M (Cu2+
)
10
l
)
5
l
M (Cu2+
)
5
lM (AAPH)
Melatonin
6-MBOA
GWC 22
10a
10b
10c
11a
11b
11c
12a
12b
12c
12d
13
14b
14c
—
—
—
—
—
—
—
—
—
—
—
1.5
1.5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
—
—
—
—
—
1.6
2.5
1
1.5
2.1
1
1.5
2.1
1.5
2.2
1.7
2.5
3.2
—
—
—
1.5
2.5
—
1.4
2
—
1.6
1.9
—
2.2
—
2.4
3.3
—
—
—
>5
>5
1.8
3.2
>5
1.8
3.3
>5
3.3
>5
3.8
>5
>5
—
15.32 1.56
14.12 1.58
CH3
CH3
Bz
CH3
CH3
Bz
CH3
CH3
Bz
CH3
H
17.2 1.85
14.50 1.62
—
16.50 1.75
14.60 1.65
C3H7
C3H7
C3H7
C9H19
C3H7
C3H7
C3H7
13.85 1.48
13.50 1.45
13.20 1.42
14.10 1.51
CH3
H
R: Ratio between the length of the lag phase of the oxidative procedure of LDL with different compounds and the length of the one without compound (control).
LDH activity was expressed as the percentage of total LDH cellular release following the addition of Triton X-100 (final concentration of 0.1% v/v in ethanol). The cells were
pretreated for 24 h with 10
22.76 1.53%.
lM drugs, then for 16 h with the same concentration of drugs and 100 lg/mL of mo-LDL. The LDH release in the presence of mo-LDL alone is
oxidized LDLs in the subendothelial arterial space. Oxidized LDLs
have been reported to be cytotoxic against endothelial cells.
Furman34 in 1999 showed the differential toxicities of air (mO-LDL)
or Copper-Oxidized LDLs (Cu) toward endothelial cells. Both the
morphological aspect of the cells themselves and LDH test revealed
that mO-LDLs had cytotoxicity with up to 8 h of oxidation.
The protective effect of melatonin derivatives against cytotoxics
mO-LDL was studied in cultured bovine aortic endothelial cells
(BAECs). This model of primary culture cells is commonly used in
our laboratory.
and 11c (R = 1.5 for 5
respectively. We observed the best antioxidant activities (R = 2.5
and 3.2, respectively for 5 M) and the lower LDH release 13.5%
and 13.2%, respectively, for compounds 13 and 14b. Cellular vital-
ity was determined by measuring mitochondrial activity in the
presence of MTT:(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide). After 16 h of incubation with mO-LDL alone
MTT reactivity is about 60%. With the different derivatives synthe-
sized, all the activities reach nearly 90 1% without differences be-
tween them.
lM), LDH release reaches 17.2% and 16.5%,
l
Normal LDL (100
minor cellular shape changes and after 16 h of incubation most
cells remained attached to the bottom of culture wells. Incubation
l
g protein/ml) in cellular medium induced
These biological properties look important in term of preven-
tion of cardiovascular diseases33,35 but the capacity of molecules
to inhibit atherogenic lipoprotein oxidation in vitro offers no pre-
diction of their capacity to inhibit in vivo atherosclerosis develop-
ment. Tailleux18,36 rated that the feeding of hypercholesterolemic
mice on an atherogenic diet supplemented with melatonin highly
increases the surface of atherosclerotic lesions in aorta and the
sensitivity of atherogenic lipoprotein to ex vivo oxidation even
though high melatonin doses inhibit lipoprotein oxidation
in vitro. A melatonin-related compound (DTBHB: N-[2-(5-meth-
oxy-1H-indol-3-yl)ethyl]-3,5-di-tert-butyl-4-hydroxybenzamide)
has been reported to strongly inhibit lipid peroxidation in vitro and
considerably increase the sensitivity of atherogenic lipoproteins to
ex vivo oxidation but did not modify atherosclerotic lesion devel-
opment in mice.
with 100 lg protein/ml mO-LDL for 16 h resulted in major mor-
phological cellular changes: the initial visual change consisted in
cell contraction, with the appearance of cytoplasmic ramification,
then some cells rounded up and detached from the bottom of the
wells. After treatment with GWC22 or melatonin derivatives the
number of detached cells decreased and fixed cells were less
shrunk.
In this study, incubation of cultured endothelial cells with mO-
LDL (100 lg protein/ml) for 16 h induced toxicity, characterized by
cellular morphological changes, an increase in cellular LDH release,
and a decrease in MTT activity. When we add benzazolinones
derivatives to the medium there is a decrease of the cellular LDH
release and an increase of MTT reactivity. Cytotoxicity was quanti-
fied using LDH release. LDH release increased when cells were trea-
ted with mO-LDL in comparison with normal LDL (22.76 1.53% vs
11.07 2.8, respectively). In the presence of normal LDL the drugs
had no significant effect on cellular LDH release, while in the pres-
ence of mO-LDL, GWC 22 and other product derivatives signifi-
cantly reduced LDH activity.
The studies in vivo are necessary to confirm the cardiovascular
protective effects of the benzazolinone derivatives.
3. Conclusion
The synthesized compounds displayed interesting antioxidant
activity superior to those of melatonin and 6-methoxy-2-benzox-
azolinone. Compounds in the benzothiazolone series gave higher
In summary our results show that the most antioxidant prod-
ucts are also the less cytotoxics. For example, for compounds 10c

I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
7827
antioxidant activities than the benzoxazolone series. The modula-
tion with a benzyloxy group on the phenyl ring was important for
antioxidant activity (10c, 11c, and 12c), identically for compounds
13 and 14b with the presence of phenolic group.
gen atmosphere and stirred for 1 day. Palladium/C was filtered and
the solvent was removed in vacuo. The oily product was dissolved
in EtOAc, and ether saturated with gaseous HCl was added. The
corresponding hydrochloride product was filtered and recrystal-
lized in methanol. Yield 78%; mp >240 °C; 1H NMR (300 MHz,
DMSO-d6), d = 3.00 (m, 4H, CH2), 3.30 (s, 3H, NCH3), 7.11 (dd, 1H,
H5, J = 7.9 Hz, J5–7 = 1.2 Hz), 7.21 (d, 1H, H4, J = 7.9 Hz), 7.30 (d,
1H, H7, J = 1.2 Hz), 8.10 (br s, 3H, NH3þ); Mass m/z 193.3 (M++1).
The oxidative modification of LDL has been alleged to play an
important role in the development of human atherosclerosis. Thus,
protecting LDL from oxidation by compounds such as melatonin
derivatives may be an effective strategy to delay or prevent the pro-
gression of the disease. The toxicity of mO-LDL exposed to melato-
nin derivatives is weaker in terms of LDH release. Because oxidation
of LDL is implicated in the pathogenesis of atherosclerosis, the
enhancement of LDL resistance to oxidation is one of the models
used by many researchers to investigate the efficacy of new drugs
as antioxidants against radicals generated in the lipophilic phase.
The remarkable capacity of these derivatives to function in vitro
as antioxidants has been demonstrated in this study. They exhibit
high antioxidant protective activity against peroxidations induced
by metal ions or peroxyl radical. Furthermore, they do not chelate
copper, suggesting that they may exert their antioxidant effect on
lipid peroxidation primarily by scavenging free radicals. The capac-
ity of molecules to inhibit atherogenic lipoprotein oxidation in vitro
offers no prediction of their capacity to inhibit in vivo atherosclero-
sis development. To demonstrate beneficial effects on atherosclero-
sis we need to make further experiments in vivo.
4.4. General procedure for the synthesis of benzamide
derivatives (9a–c)
To a solution of 10% K2CO3 aqueous (20 mL) and chloroform
(30 mL), 6-(2-ethylamino)-3-methyl-2(3H)-benzothiazolone or
benzoxazolone (5a or 5b) (10 mmol) and the desired phenyl acid
chloride (15 mmol) were added. The reaction mixture was stirred
for 2 h at room temperature. A 5% NaOH aqueous solution was
added (10 mL) and the reaction mixture was stirred for 30 min.
The organic layer was dried with magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue was recrystal-
lized in the appropriate solvent.
4.4.1. 3-Methoxy-N-[2-(3-methyl-2-oxo-2,3-dihydro-
benzothiazol-6-yl)ethyl]benzamide (9a)
Yield 78% (absolute ethanol); mp 130–131 °C; 1H NMR
(300 MHz, CDCl3), d = 2.95 (t, 2H, CH2, J = 7.18 Hz), 3.40 (s, 3H,
NCH3), 3.70 (t, 2H, CH2, J = 7.28 Hz), 3.85 (s, 3H, OCH3), 6.35 (s,
1H, NH), 6.97 (d, 1H, H4, J = 8.2 Hz), 7.03 (dd, 1H, H5, J5–4
= 8.02 Hz, J5–7 = 1.17 Hz), 7.18 (d, 1H, HAr, J = 7.24 Hz), 7.23 (d,
1H, H7, J = 1.17 Hz), 7.30 (m, 3H, HAr).; Mass m/z 343.4 (M++1).
4. Experimental
Compounds were purified on a glass column using Merck Silica
Gel 60 (230–400 mesh). Melting points were determined by a
Büchi 510 capillary apparatus and are uncorrected. 1H NMR spectra
were recorded on a Bruker AVANCE 300 spectrometer and chemi-
cal shifts are in ppm with TMS as internal standard. Mass spectra
were recorded on a quadripolar Finnigan Mat SSQ 710 instrument.
4.4.2. 3-Methoxy-N-[2-(3-methyl-2-oxo-2,3-dihydro-
benzoxazol-6-yl)ethyl]benzamide (9b)
Yield 69% (absolute ethanol); mp 137–138 °C; 1H NMR
(300 MHz, CDCl3), d = 2.95 (t, 2H, CH2, J = 6.5 Hz), 3.40 (s, 3H,
NCH3), 3.70 (t, 2H, CH2, J = 6.5 Hz), 3.85 (s, 3H, OCH3), 6.35 (s, 1H,
NH), 6.97 (d, 1H, H4, J = 8.2 Hz), 7.03 (dd, 1H, H5, J = 8.2 Hz
,J = 1.2 Hz), 7.18 (d, 1H, HAr, J = 7.3 Hz), 7.23 (d, 1H, H7, J = 1.2 Hz),
7.30 (m, 3H, HAr); Mass m/z 327.5 (M++1).
4.1. 3-Methyl-6-(2-nitrovinyl)-2(3H)-benzoxazolone (7)
To 35 mL of acetic acid, 6-formyl-3-methyl-2(3H)-benzoxazo-
lone (4 g, 22 mmol), ammonium acetate (3.83 g, 49 mmol) and
nitromethane (18 mL, 330 mmol) were added. The reaction mixture
was refluxed for 2 days. Water (100 mL) was introduced and the
resulting precipitate was filtered and recrystallized in methanol.
Yield 72%; mp 195–197 °C; 1H NMR (300 MHz, CDCl3), d = 3.50 (s,
3H, NCH3), 7.08 (d, 1H, H4, J = 8.2 Hz), 7.43 (d, 1H, H7, J = 1.5 Hz),
7.46 (dd, 1H, H5, J = 8.2 Hz, J5–7 = 1.5 Hz), 7.60 (d, 1H, CH,
J = 13.5 Hz), 8.06 (d, 1H, CH, J = 13.5 Hz); Mass m/z 222.2 (M++1).
4.4.3. 3-Benzyloxy-N-[2-(3-methyl-2-oxo-2,3-dihydro-benzoxazol-
6-yl)ethyl]benzamide (9c)
Yield 94% (acetonitrile); mp 178–179 °C; 1H NMR (300 MHz,
CDCl3), d = 2.96 (t, 2H, CH2, J = 6.4 Hz), 3.42 (s, 3H, NCH3), 3.70 (t,
2H, CH2, J = 6.4 Hz), 5.10 (s, 2H, OCH2), 6.20 (s, 1H, NH), 6.90 (d,
1H, H4, J = 7.9 Hz), 7.03 (dd, 1H, H5, J = 7.9 Hz, J = 1.7 Hz), 7.10 (m,
4H, HAr), 7.37 (m, 1H, H7), 7.40 (m, 5H, HAr); Mass m/z 403.3
(M++1).
4.2. 3-Methyl-6-(2-nitroethyl)-2(3H)-benzoxazolone (8)
To a mixture of chloroform/propan-2-ol (30/5, v/v), 3-methyl-6-
(2-nitrovinyl)-2(3H)-benzoxazolone (1.1 g, 4.8 mmol), silica (1.5 g)
and sodium borohydride (0.43 g, 12.1 mmol) were added. The reac-
tion mixture was stirred for 1 day at room temperature, and the
reaction was stopped by dropwise addition of acetic acid (5 mL).
The reaction mixture was stirred for an additional 15 min. The insol-
uble material was filtered and washed with dichloromethane, and
then the solvent was removed in vacuo. The resulting precipitate
was recrystallized in absolute ethanol. Yield 68%; mp 134–136 °C;
1H NMR (300 MHz, CDCl3), d = 3.25 (t, 2H, CH2, J = 7.1 Hz), 3.30 (s,
3H, NCH3), 4.85 (t, 2H, NCH2, J = 7.1 Hz), 7.12 (d, 1H, H4, J = 8.2 Hz),
7.18 (d, 1H, H5, J = 8.2 Hz), 7.31 (s, 1H, H7); Mass m/z 224.1 (M++1).
4.5. General procedure for the synthesis of derivatives 10a–c
To a solution of acetonitrile (50 mL), benzamide derivatives
(9a–c) (32 mmol) and POCl3 (3.1 mL, 320 mmol) were added. The
reaction mixture was refluxed for 1 day and then evaporated under
reduced pressure. The crude residue was washed with ethyl ace-
tate and filtered. The precipitate was recrystallized in the appropri-
ate solvent.
4.5.1. 5-(3-Methoxyphenyl)-3-methyl-7,8-dihydro-3H-1-thia-
3,6-diaza-benz[f]inden-2-one hydrochloride (10a)
Yield 68% (ethanol 95°); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 3.20 (t, 2H, CH2, J = 6.4 Hz), 3.35 (s, 3H, NCH3), 3.90 (s, 3H,
OCH3), 4.00 (t, 2H, CH2, J = 6.4 Hz), 7.20 (s, 1H, H4), 7.35 (m, 2H,
HAr), 7.50 (d, 1H, HAr J = 1.8 Hz), 7.60 (t, 1H, HAr, J = 7.8 Hz), 8.00
(s, 1H, H9) 9.50 (br s, 1H, NH+); Mass m/z 325.2 (M++1).
4.3. 3-Methyl-6-(2-aminoethyl)-2(3H)-benzoxazolone
hydrochloride (5b)
To 20 mL of methanol, compound 8 (1.1 g, 4.8 mmol) and Palla-
dium/C were added. The reaction mixture was placed under hydro-

7828
I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
4.5.2. 5-(3-Methoxyphenyl)-3-methyl-7,8-dihydro-3H-1-oxa-
3,6-diaza-benz[f]inden-2-one hydrochloride (10b)
was filtered and washed with ethyl acetate. The residue was
recrystallized with the appropriate solvent.
Yield 66% (ethanol 95°); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 3.20 (t, 2H, CH2, J = 7.3 Hz), 3.30 (s, 3H, NCH3), 3.85 (s, 3H,
OCH3), 4.00 (t, 2H, CH2, J = 7.3 Hz), 7.30 (m, 2H, H4, HAr), 7.40 (m,
2H, HAr), 7.60 (t, 1H, HAr, J = 7.5 Hz), 7.70 (s, 1H, H9), 10.20 (s, 1H,
NH+); Mass m/z 309.5 (M++1).
4.7.1. 5-(3-Methoxyphenyl)-3-methyl-6-propyl-5,6,7,8-
tetrahydro-3H-1-thia-3,6-diaza-benz[f] inden-2-one
hydrochloride (12a)
Yield 64% (absolute ethanol); mp >240 °C; 1H NMR (300 MHz,
DMSO-d6), d = 0.80 (t, 3H, CH3, J = 7.3 Hz), 1.50 (m, 2H, CH2), 2.30
(m, 2H, CH2), 2.50 (m, 2H, CH2) 3.10 (m, 5H, CH2, NCH3), 3.80 (s,
3H, OCH3,), 4.60 (s, 1H, CH), 6.40 (s, 1H, H4), 6.80 (m, 4H, HAr),
7.15 (s, 1H, H9), 11.60 (br s, 1H, NH+); Mass m/z 369.4 (M++1).
4.5.3. 5-(3-Benzyloxyphenyl)-3-methyl-7,8-dihydro-3H-1-oxa-
3,6-diaza-benz[f]inden-2-one hydrochloride (10c)
Yield 45% (acetonitrile); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 3.20 (t, 2H, CH2, J = 7.4 Hz), 3.30 (s, 3H, NCH3), 4.00 (t, 2H,
CH2, J = 7.4 Hz), 5.20 (s, 2H, OCH2), 7.25 (s, 1H, H4), 7.40 (m, 8H,
HAr), 7.60 (s, 1H, H9), 10.20 (br s, 1H, NH+); Mass m/z 385.6 (M++1).
4.7.2. 5-(3-Methoxyphenyl)-3-methyl-6-propyl-5,6,7,8-tetrahydro-
3H-1-oxa-3,6-diaza-benz[f]inden-2-one hydrochloride (12b)
Yield 68% (absolute ethanol); mp >240 °C; 1H NMR (300 MHz,
DMSO-d6), d = 0.80 (t, 3H, CH3, J = 7.3 Hz), 1.80 (m, 2H, CH2), 3.10
(m, 7H, CH2 CH2, NCH3,), 3.60 (m, 5H, CH2, OCH3), 5.80 (s, 1H,
CH), 6.50 (s, 1H, H4), 6.90 (m, 3H, HAr), 7.20 (s, 1H, H9), 7.40 (m,
1H, HAr), 11.50 (br s, 1H, NH+); Mass m/z 353.5 (M++1).
4.6. General procedure for the synthesis of derivatives 11a–c
In 50 mL of methanol, derivatives 10a–c (24 mmol) were solu-
bilized and then sodium borohydride (60 mg, 36 mmol) was added
portionwise. The mixture was added for 1 h at room temperature
and then evaporated under reduced pressure. Water (30 mL) and
chloroform were added to the residue. The organic layer was dried
with magnesium sulfate, filtered, and concentrated under reduced
pressure. The oily product was solubilized in dry ethyl acetate and
treated with gaseous hydrochloric acid in diethyl ether (20 mL).
The precipitate was filtered and washed with ethyl acetate. The
residue was recrystallized with the appropriate solvent.
4.7.3. 5-(3-Benzyloxyphenyl)-3-methyl-6-propyl-5,6,7,8-tetra-
hydro-3H-1-oxa-3,6-diaza-benz[f]inden-2-one hydrochloride
(12c)
Yield 76% (absolute ethanol); mp 158–159 °C; 1H NMR
(300 MHz, DMSO-d6), d = 0.80 (t, 3H, CH3, J = 7.2 Hz), 1.80 (m, 2H,
CH2), 3.30 (m, 9H, CH2, CH2, CH2, NCH3), 5.10 (s, 2H, OCH2), 5.80
(s, 1H, CH), 6.30 (s, 1H, H4), 7.15 (m, 9H, HAr), 7.20 (s, 1H, H9),
11.50 (br s, 1H, NH+); Mass m/z 429.6 (M++1).
4.6.1. 5-(3-Methoxyphenyl)-3-methyl-5,6,7,8-tetrahydro-3H-1-
thia-3,6-diaza-benz[f]inden-2-one hydrochloride (11a)
4.7.4. 5-(3-Methoxyphenyl)-3-methyl-6-nonyl-5,6,7,8-tetrahydro-
3H-1-oxa-3,6-diaza-benz[f]inden-2-one hydrochloride (12d)
Yield 63% (acetonitrile); mp 142–143 °C; 1H NMR (300 MHz,
DMSO-d6), d = 0.80 (t, 3H, CH3, J = 7.1 Hz), 1,10–1,40 (m, 12H,
CH2), 1.50 (m, 2H, CH2), 2.50 (m, 2H, CH2), 2.70 (m, 2H, CH2),
3.20 (m, 2H, CH2), 3.70 (s, 3H, NCH3), 3.70 (s, 3H, OCH3), 5.40 (s,
1H, CH), 6.50 (s, 1H, H4), 6.80 (d, 1H, HAr, J = 7.8 Hz), 7.00 (m, 2H,
HAr), 7.20 (s, 1H, H9), 7.35 (m, 1H, HAr), 11.10 (br s, 1H, NH+); Mass
m/z 437.6 (M++1).
Yield 83% (ethanol 95°); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 3.20–3.45 (m, 7H, CH2, CH2, NCH3), 3.75 (s, 3H, OCH3), 5.80
(s, 1H, CH), 6.70 (s, 1H, H4), 6.85 (d, 1H, HAr, J = 7.8 Hz), 7.00 (dd, 1H,
HAr, J = 8.2 Hz, J = 2.0 Hz), 7.10 (t, 1H, HAr, J = 1.8 Hz, J = 2.0 Hz), 7.30
(t, 1H, HAr, J = 8.2 Hz, J = 7.8 Hz), 7.60 (s, 1H, H9), 9.70 (br s, 1H,
NH2þ), 10.50 (br s, 1H, NH2+); Mass m/z 327.4 (M++1).
4.6.2. 5-(3-Methoxyphenyl)-3-methyl-5,6,7,8-tetrahydro-3H-1-
oxa-3,6-diaza-benz[f]inden-2-one hydrochloride (11b)
Yield 74% (ethanol 95°); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 3.10 (m, 7H, CH2, CH2, NCH3), 3.75 (s, 3H, OCH3), 5.75 (s, 1H,
4.7.5. 5-(3-Hydroxyphenyl)-3-methyl-6-propyl-5,6,7,8-tetrahydro-
3H-1-thia-3,6-diaza-benz[f]inden-2-one hydrochloride(13)
CH), 6.60 (s, 1H, H4), 6.80 (d, 1H, HAr, J = 7.6 Hz), 7.05 (dd, 1H, HAr
,
To a solution of compound 12c (10 mmol) in methanol was
added 10% Pd/C (100 mg). The resulting solution was stirred at
room temperature under atmospheric pressure of hydrogen for
1 day. The solution was concentrated to dryness. The residue was
purified by column chromatography using dichloromethane/meth-
anol (90/10, v/v) as eluent to afford compound 13. The oily product
was solubilized in dry ethyl acetate and treated with gaseous
hydrochloric acid in diethyl ether (20 mL). The precipitate was fil-
tered and washed with ethyl acetate. The residue was recrystal-
lized in acetonitrile. Yield 70%; mp 230–231 °C; 1H NMR
(300 MHz, CDCl3), d = 0.80 (t, 3H, CH3, J = 7.3 Hz), 1.50 (m, 2H,
CH2), 2.30 (m, 1H, CH2), 2.45 (m, 1H, CH2), 2.60 (m, 1H, CH2),
2.90 (m, 1H, CH2), 3.05 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.20 (m,
3H, NCH3), 4.55 (s, 1H, CH), 6.40 (s, 1H, H4), 6.70 (m, 2H, HAr),
J = 7.6 Hz, J = 1.1 Hz), 7.10 (s, 1H, HAr), 7.30 (s, 1H, H9), 7.40 (t, 1H,
HAr, J = 7.6 Hz, J = 7.9 Hz), 9.70 (br s, 1H, NH2+), 10.50 (br s, 1H,
NH2þ); Mass m/z 311.5 (M++1).
4.6.3. 5-(3-Benzyloxyphenyl)-3-methyl-5,6,7,8-tetrahydro-3H-
1-oxa-3,6-diaza-benz[f]inden-2-one hydrochloride (11c)
Yield 91% (acetonitrile); mp >240 °C; 1H NMR (300 MHz, DMSO-d6),
d = 3.30(m,7H,CH2, CH2,NCH3), 5.10 (s, 2H, OCH2), 5.75 (s, 1H, CH), 6.55
(s, 1H, H4), 6.90 (d, 1H, HAr, J = 7.8 Hz), 7.05 (t, 1H, HAr, J = 1.5 Hz), 7.10
(dd, 1H, HAr, J = 7.7 Hz, J = 1.9 Hz), 7.40 (m, 7H, H9, HAr), 9.70 (br s, 1H,
NH2þ), 10.50 (br s, 1H, NH2+); Mass m/z 387.3 (M++1).
4.7. General procedure for the synthesis of derivatives 12a–d
6.80 (d, 1H, HAr, J = 7.5 Hz), 6.95 (s, 1H, H9), 7.20 (t, 1H, HAr
,
Propyl or nonyl iodide (10 mmol) was added to a solution of
compounds 8a–c (10 mmol) and potassium carbonate (4.2 g,
30 mmol) in CH3CN. The reaction mixture was refluxed for 1 day
and then evaporated. Ethyl acetate (80 mL) and water (50 mL)
were added. The organic layer was dried with magnesium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography using dichloromethane/meth-
anol (98/2, v/v) as eluent to afford compounds 12a–d. The oily
product was solubilized in dry ethyl acetate and treated with gas-
eous hydrochloric acid in diethyl ether (20 mL). The precipitate
J = 7.8 Hz), 11.00 (br s, 1H, NH+); Mass m/z 339.5 (M++1).
4.8. General procedure for the synthesis of derivatives 14b and
14c
To a solution of compound 12b or 13 (10 mmol) in methanol/
water (20/10) was added sodium hydroxide (1.6 g, 40 mmol). The
reaction mixture was refluxed for 1 h and then evaporated. Water
(50 mL) was added. The aqueous solution was extracted with
diethyl ether, acidified (pH 3) with 6 M HCl solution, and then

I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
7829
the pH was adjusted to 8 with aqueous solution of sodium bicar-
bonate. The precipitate was extracted with ethyl acetate. The or-
ganic layer was dried with magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified
by column chromatography using dichloromethane/methanol
(90/10, v/v) as eluent to afford compounds 14b–c. The oily product
14b was solubilized in dry ethyl acetate and treated with gaseous
hydrochloric acid in diethyl ether (20 mL). The precipitate was fil-
tered and washed with ethyl acetate.
plates. 10
l
L drugs in methanol solution (1
l
M) were added to
the culture medium of cells for 24 h. Then cells were incubated
at 37 °C during a period of 16 h in the presence of LDL or minimally
oxidized LDL (mO-LDL) at 100
methanol or drugs in methanol solution (final concentration
10
M).41 After 16 h of incubation, Lactate Dehydrogenase (LDH)
lg/mL with an addition of 10 lL
l
activity in the medium was measured by the spectrometric analy-
sis of NAD reduction using the LDH kit (sigma, Saint Quentin Falla-
vier, France). LDH activity was expressed as the percentage of total
LDH cellular release following the addition of Triton X-100 (final
concentration of 0.1% v/v in ethanol). The results are the mean
( SD) of two different incubations each performed in triplicate.
Cellular vitality was determined by measuring mitochondrial
activity in the presence of MTT:(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide). MTT is reduced to purple formazan
4.8.1. 6-Hydroxy-1-(3-methoxyphenyl)-7-methylamino-2-
propyl-1,2,3,4-tetrahydro-isoquinoline hydrochloride (14b)
Yield 58% (acetonitrile); mp >240 °C; 1H NMR (300 MHz, DMSO-
d6), d = 0.80 (t, 3H, CH3, J = 7.1 Hz), 1.80 (m, 2H, CH2), 2.70 (s, 3H,
NCH3), 3.00 (m, 2H, CH2), 3.40 (m, 2H, CH2), 3.70 (m, 2H, CH2),
3.85 (s, 3H, OCH3), 5.60 (s, 1H, CH), 6.60 (s, 1H, H8), 7.00 (m, 4H,
H5, HAr), 7.30 (s, 1H, OH), 7.40 (m, 1H, HAr), 11.30 (br s, 2H, NH,
NH+); Mass m/z 327.3 (M++1).
in the mitochondria of living cells. 100 lL of a 5 mg solution of MTT
in PBS were added to each culture well and incubated for 5 h. Fol-
lowing this period, the medium was removed and the crystals were
dissolved in 4 mL of dimethylformamide. Absorbance was read at
590 nm, and the results were expressed as the percentage of the
value of control wells.42
4.8.2. 6-Hydroxy-1-(3-hydroxyphenyl)-7-methylamino-2-
propyl-1,2,3,4-tetrahydro-isoquinoline (14c)
Yield 68%; mp 140–141 °C; 1H NMR (300 MHz, DMSO-d6),
d = 0.80 (t, 3H, CH3, J = 7.2 Hz), 1.60 (m, 2H, CH2), 2.70 (m, 9H,
CH2, CH2, CH2, NCH3), 4.60 (s, 1H, CH), 6.00 (s, 1H, H8), 6.20 (s,
1H, HAr), 6.50 (s, 1H, H5), 6.70 (dd, 1H, HAr, J = 7.9 Hz, J = 1.7 Hz),
6.80 (d, 1H, HAr, J = 7.4 Hz), 7.10 (t, 1H, HAr, J = 7.9 Hz, J = 7.4 Hz);
Mass m/z 313.3 (M++1).
5.3. Data analysis
The results were expressed as mean SD when a minimal num-
ber of three independent experiments were performed in tripli-
cate. Differences between the experimental results were tested
using the Mann Whitney test.
5. Antioxidant activity
Acknowledgments
5.1. Low density lipoprotein (LDL) oxidation inhibition test
The 300 MHz NMR facilities were funded by the Région Nord-
Pas de Calais (France), the Ministère de la Jeunesse, de l’Education
Nationale et de la Recherche (MJENR), and the Fonds Européens de
Développement Régional (FEDER).
Human LDLs were isolated from freshly drawn blood from
healthy, normolipidemic, fasting volunteers. LDLs were isolated
by sequential density gradient ultracentrifugation in sodium bro-
mide density solutions in the density range of 1.019–1.063 g/mL
as previously reported.37 The protein concentration was deter-
mined by Peterson’s method38 using bovine serum albumin as
the standard. Oxidation of LDL was initiated by copper.
References and notes
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fered-saline (PBS), pH 7.4, under N2 at 4 °C. Then oxidation was in-
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duced at 30 °C by adding 20
160 L of LDL (125 g of protein/mL) in the presence or absence of
20 l derivatives from 1 M to 10 M or PBS for control. During
lL of 16.6 lM CuSO4 or 2 mM AAPH to
l
l
l
l
l
copper or AAPH-induced LDL oxidation, diene-conjugated forma-
tion was followed by the measurement of optical density at
234 nm every 10 min for 8 h at 30 °C with a thermostated Spectra
Max Plus Molecular Devices spectrophotometer (96 wells, Molecu-
lar Devices Corporation Sunnyvale, California 94089).39 Drug activ-
ities were expressed in R, the ratio between the length of the lag
phase of the oxidative procedure of LDL in the presence of different
compounds and the length of the one without compound (control).
As their antioxidant capacity can be characterized by the evolution
of the LDL oxidation curves along with their concentration, each
compound was tested at concentrations of 1–100 lM. Air-oxidized
LDLs (mO-LDL) are obtained by incubation of LDL at 30 °C without
CuSO4 or AAPH.
5.2. Cellular cytotoxicity
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scribed by Gospodarowicz40 and cultured in Dubelco’s-modified
Eagle’s medium (DMEM) supplemented with 10% newborn calf
serum, 600 ng/mL glutamine, and 100 U/mL penicillin. BAECs were
used under passage 10. BAECs were then subcultured in 6-well
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Chem. 1995, 30, 715.

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I. Yekini et al. / Bioorg. Med. Chem. 17 (2009) 7823–7830
24. Caignard, D. H.; Vaccher, M. P.; Bonte, J. P.; Lesieur, D.; Cazin, J. C. Farmaco
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34. Furman, C.; Martin-Nizard, F.; Fruchart, J. C.; Duriez, P.; Teissier, E. J. Biochem.
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