Synthesis of 4-hydroxycoumarin and 2,4-quinolinediol derivatives and evaluation of their effects on the viability of HepG2 cells and human hepatocytes culture

Article abstract of DOI:10.1016/j.ejmech.2004.07.006

We report here the synthesis of aromatic coumarins and aromatic α-quinolones which were evaluated in vitro for their protective potentialities against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage on human liver cell death, i.e., human hepatom

Full text of DOI:10.1016/j.ejmech.2004.07.006

European Journal of Medicinal Chemistry 39 (2004) 931–937
www.elsevier.com/locate/ejmech
Original article
Synthesis of 4-hydroxycoumarin and 2,4-quinolinediol derivatives
and evaluation of their effects on the viability of HepG2 cells
and human hepatocytes culture
Bernard Refouvelet a,*, Catherine Guyon a,Yves Jacquot a,1, Corinne Girard b, Hervé Fein b,c
Françoise Bévalot b, Jean-François Robert a, Bruno Heyd d, Georges Mantion d,
Lysiane Richert c, Alain Xicluna a
,
a Laboratoire de chimie organique et thérapeutique, faculté de médecine et de pharmacie, université de Franche-Comté, place Saint-Jacques,
25030 Besançon cedex, France
b Laboratoire de pharmacognosie, EA 482, faculté de médecine et de pharmacie, université de Franche-Comté, place Saint-Jacques,
25030 Besançon cedex, France
c Laboratoire de biologie cellulaire, EA 479, faculté de médecine et de pharmacie, université de Franche-Comté, place Saint-Jacques,
25030 Besançon cedex, France
d Service de chirurgie digestive et vasculaire, hôpital Jean Minjoz, 25000 Besançon, France
Received 24 February 2003; received in revised form 8 July 2004; accepted 12 July 2004
Available online 15 September 2004
Abstract
We report here the synthesis of aromatic coumarins and aromatic a-quinolones which were evaluated in vitro for their protective
potentialities against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage on human liver cell death, i.e., human hepatoma HepG2 cell
line and human hepatocytes in primary culture. We found that the presence of a benzylidene at the 3-position or a heterocycle with N and S
heteroatoms on the benzopyrone or quinolone system was essential for the protective effect of these compounds against t-BHP-induced
decrease in viability of cells. We found also that a methoxy group on the aromatic ring systems decreased this potential. t-BHP-induced
cytotoxicity in primary cultures of human hepatocytes could be therefore prevented by these compounds suggesting that they could display
hepatoprotective effects in humans.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Coumarins; Quinolones; Human liver cells; Cell viability
1. Introduction
Tert-butyl hydroperoxide (t-BHP) is a well known inducer
of oxidative stress in biological systems and it is believed that
reactive free radical intermediates including peroxy, alkoxy
and carbon centred radicals are mediators of peroxide-
dependant injury [10,11]. The human hepatoma cell line
HepG2 is classically used [12–18] to investigate the mecha-
nisms of the cytotoxic or cytoprotective effects of xenobio-
tics. In particular, t-BHP-induced oxidative stress, its conse-
quent cytotoxicity, and the protective effects of various
compounds against this toxicity have been recently described
using this specific cell line [19–21].
During these last 30 years, a great number of heterocyclic
coumarins or quinolones have been synthesized and their
biological activities extensively evaluated. Indeed, heterocy-
clic coumarins and quinolones display some interesting phar-
macological activities, particularly as anticoagulants, photo-
sensibilizing drugs, estrogen mimics or antiproliferative
compounds [1–9].
* Corresponding author.
E-mail address: bernard.refouvelet@univ-fcomte.fr (B. Refouvelet).
1 Present address. UMR 7613, case 45, université Pierre et Marie Curie, 4,
place Jussieu, 75252, Paris Cedex 05, France. Tel.: +33-1-44-27-26-78;
fax: +33-1-44-27-38-43; jacquot@ccr.jussieu.fr (Y. Jacquot).
We report here the synthesis of benzothiazinic derivatives
of coumarin (2a–b) and quinolones (2c–d) (Fig. 1) as well as
3-benzylidene derivatives of coumarins (5a–b) and quinolo-
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.07.006

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B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
Fig. 1. Synthesis of quadricyclic coumarins and quinolones.
nes (5c–d) (Fig. 2) [22–26]. We were investigated their effect
on t-BHP-induced cytotoxicity in HepG2 cells and primary
cultures of human hepatocytes, widely used for the evalua-
tion of hepatotoxic and hepatoprotective compounds in hu-
mans [27–29]. We were also tested the potential hepatopro-
tective activity of the reported derivatives and their effects on
t-BHP-induced cytotoxicity in primary cultures of human
hepatocytes.
and conducted to a non-isolable enaminone intermediate
[26]. Subject to a nucleophilic attack at the 3-position of the
coumarin or the quinolone, the bis(o-aminophenyl)disulfde
(DAPDS) motif was cleaved and the enaminone led to the
desired products 2a–d by an intramolecular cyclisation. The
expected compounds were crystallized from DMSO or DMF
on cooling at room temperature and were isolated by filtra-
tion under vacuum. Their structures were assigned from
1
FTIR and H-NMR spectroscopy, EI-MS spectrometry and
elemental analysis.
The 3-benzylidenecoumarins 5a–b and 3-benzylidene-
quinolones 5c–d were prepared as shown in Fig. 2. The
Knovenagel condensation of 4-hydroxycoumarins 1a–b or
2,4-quinolinediols 1c–d on the substituted benzaldehydes (3,
4) carried out in pyridine gave compounds 5a–d in only one
diastereoisomeric form (Z). These compounds were crystal-
lized from alcohol and were isolated by filtration under
vacuum.
2. Chemistry
The 6,12-dihydrobenzopyrano[3,4-b][1,4]benzothiazin-
6-ones 2a–b and 6,12-dihydroquinolo [3,4-b][1,4]benzo-
thiazin-6-ones 2c–d were prepared as shown in Fig. 1. The
quadricyclic system was synthesized according to the
method of Tabakovic [3,22,25]. The addition of 2-amino-
thiophenol on the substituted 4-hydroxycoumarins 1a–b or
on the substituted 2,4-quinolinediols 1c–d was made in polar
aprotic solvents such as DMSO or DMF. This addition was
accompanied by the elimination of one molecule of water
The structural determination of compounds 5a–d was
realised on the basis of NMR studies. The stereochemistry
(Z) of compounds (5a–d) was easily determined by simple
Fig. 2. Synthesis of benzylidene coumarins or quinolones.

B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
933
1
examination of H-NMR spectra and comparison with the
data reported in the literature [30,31]. For compounds 5a–d
the (Z) configuration has been characterised by the presence
of an intramolecular hydrogen bond.
3. Results and discussion
Among the synthesized products, the coumarin 2a was
particularly interesting for its protective activity against
t-BHP-induced cytotoxicity in the cultured human hepatoma
HepG2 cell line. Actually, it was shown that a concentration-
dependent protective effect on 3 mM t-BMP-induced cyto-
toxicity in HepG2 cells (Fig. 3b). Of note, 2a displayed a
complete protection at 10 µM comparable to that of quercetin
(tested as a reference antioxidant compound) at the same
concentration (Fig. 3c) [32–35]. A significant effect on
HepG2 cell line viability was also observed with t-BHP from
1 mM and about 50% cell mortality was observed at 3 mM
t-BHP (Fig. 3a).
Table 1 shows the percentage of HepG2 cells viability
co-incubated for 3 h with 3 mM t-BHP in the absence or in
the presence of the tested compounds at various concentra-
tions.
As shown in this table, the presence of a benzylidene
group at the 3-position (compounds 5a–d) or a heterocycle
with two heteroatoms on the coumarin or the quinolone
(2a–d) system appeared essential for the protective effect of
coumarin and quinolone derivatives. Note that compounds
with a methoxy group on the aromatic ring (2b, 5b) are
totally devoid of cytoprotective activity. Our data confirm
also recent studies from other groups [19–21], which were
reported that the human hepatoma cell line HepG2 provides a
useful model to study compounds with antagonist effects on
t-BHP-induced oxidative stress and cell damage.
In order to assess how the data obtained with the human
hepatoma cell line HepG2 correlate with those obtained with
primary cultures of human hepatocytes, the protective effects
of compound 2a were also assessed on primary cultures of
human hepatocytes isolated from three different donors.
Fig. 4a shows that t-BHP-induces a significant effect on the
cell viability of human hepatocytes with 50% cell viability at
0.3 mM t-BHP. It thus appeared that primary cultures of
human hepatocytes were much more sensitive to t-BHP-
induced oxidative stress than the cell line HepG2. Actually,
we have previously reported that HepG2 cells express only in
part some fundamental metabolic pathways [13] and mimic
in part the pleiotropic responses to xenobiotics [14] of pri-
mary cultures of human hepatocytes. It could be possible that
the metabolic pathways potentially involved in the produc-
tion of reactive free radical intermediates [10–11] are only
partly expressed in HepG2 cells.Another possibility could be
that the HepG2 cell line of human liver origin is more
resistant to oxidative stress-induced damages than their pri-
mary human hepatocyte counterparts, due to a higher expres-
sion of defence systems against oxidative stress. As we re-
Fig. 3
.
Cytotoxic effect of tert-butylhydroperoxide in HepG2 cells. Results
are reported as mean SEM of three independent experiments. * Values
significantly different from control, P < 0.05 (Tuckey’s test). (a) Of 3 mM
t-BPH-induced cytotoxicity in HepG2 cells (b) Protective effect of com-
pound 2a (c) of quercetin. Results are reported as mean SEM of three
independent experiments. * Values significantly different from control,
P < 0.05 (Tuckey’s test).
a Values significantly different from (t-BHP 3 + 10 mM 2a or quercetin).
cently described that the antioxidant status in primary
cultures of rat hepatocytes was depressed compared to livers
[36], a similar pattern in primary cultures of human hepato-
cytes could explain their greater sensitivity to oxidative
stress. Further studies are required to explore this hypothesis.
The well-known antioxidant compound quercetin [32–35]
displayed
a concentration-dependent protective effect
against t-BHP (0.3 mM) induced cytotoxicity in primary
cultures of human hepatocytes with a complete protection at
10 µM (Fig. 4c). The benzopyrone derivative 2a, by achie-

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B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
Table 1
Protective effect of synthesized compounds at different concentrations on 3 mM t-BHP-induced cytotoxicity in HepG2 cells
Compounds
X
R1
R2
Concentrations of tested compounds (µM)
0
0.5
1
5
10
2a
2b
5a
5b
2c
5c
2d
5d
O
H
–
54
53
57
58
61
63
54
41
4
6
1
2
4
7
2
2
71 4 a
76 4 a
89 3 a
104 4 a
O
OCH3
H
–
42
63
51
76
93 3 a
58
63 2 a
6
2
2
2
44
72
43 4 a
88 7 a
2
4
48
80
40 2 a
9
3
45
73
45 2 a
82 4 a
8
6
O
H
O
H
OCH3
NH
NH
NCH3
NCH3
H
–
78
83
83 4 a
62 4 a
1
1
H
H
–
81
4
80
3
H
5
74 3 a
77 3 a
79 5 a
65 6 a
H
H
HepG2 cells were exposed to 3 mM t-BHP together with various concentrations of a given tested compound. Cell viability was expressed as percentage of
HepG2 cells viability in the absence of t-BHP (0 mM). Results are reported as mean SEM of two or three independent experiments.
a Significantly different from 3 mM t-BPH alone, P < 0.05 (Tuckey’s test).
ving considerable protection from concentrations as low as
0.5 µm (Fig. 4b), appeared to display even a higher protecting
potential.
Melting points were determined on a Büchi No510 appa-
ratus and were uncorrected. Thin layer chromatography
(TLC) was carried out on anAlugram Sil G/UV254 plate with
appropriate solvents. Microanalyses were carried out by the
Service Central d’Analyses, Centre National de la Recherche
Scientifique, Vernaison (France).
4. Conclusion
In conclusion, the present work confirms that the human
hepatoma cell line HepG2 constitutes a reliable model to
study compounds with protective potentialities against
t-BHP-induced cytotoxicity. Our results suggest that the pre-
sence of a benzylidene group at the 3-position or a heterocy-
cle with two heteroatoms on the coumarin or on the quino-
lone system is essential for a protective potential and that a
methoxy group on the aromatic ring system decreases drama-
tically this protection. The observation that those compounds
also antagonised t-BHP-induced cytotoxicity in primary cul-
tures of human hepatocytes suggests that they could display
interesting hepatoprotective effects in humans. The disco-
very of 6,12-dihydro-1-benzopyrano[3,4-b][1,4]benzo-
thiazin-6-one 2a as a cytoprotective compound against hepa-
tic oxidative stress at 0.5 µM may conduct to a promising
pharmacomodulation for the discovery of new potential
drugs.
5.1.1. 6,12-Dihydrobenzopyrano[3,4b][1,4]benzothiazin-6-
one (2a)
The 4-hydroxycoumarin (1a) (50 mmol) and
2-aminothiophenol (60 mmol) were added to 20 ml of
DMSO and heated to 140 °C for 12 h. On cooling at room
temperature, the product crystallized and was filtered under
vacuum. Recrystallization from ethanol gave 2a in 60%
yield; mp > 300 °C. IR (KBr) (cm–1): 3335, 3010, 1668,
1
1618, 1015, 737; H-NMR (DMSO-d6) d (ppm): 6.90–7.10
(4H, m, Ar–H), 7.50 (2H, m Ar), 7.65 (1H, dd, 4J = 6.5 Hz,
3J = 7.5 Hz, H-2), 8.20 (1H, m, 3J = 7.5 Hz, H-1), 9.00 (1H,
s, NH). MS (m/z): 267 [M+]; C15H9NO2S.
5.1.2.
6,12-Dihydro-3-methoxybenzopyrano[3,4-b][1,4]
benzothiazin-6-one (2b)
This compound was obtained in 65% yield from
7-methoxy-4-hydroxycoumarin (1b) according to the gene-
ral procedure used for the synthesis of 2a; mp > 300°C. IR
(KBr) (cm–1): 3335, 3010, 2934, 1670, 1616, 1028, 750;
1H-NMR (DMSO-d6) d (ppm): 3.85 (3H, s, OCH3), 6.85
(2H, m, Ar-H), 6.98 (1H, d, 3J = 2.5 Hz, H-4), 6.95 (2H, m,
Ar-H), 7.02 (1H, dd, 4J = 2.5 Hz, 3J = 9.1 Hz, H-2), 8.10 (1H,
d, H-1), 9.01 (1H, s, NH). MS (m/z) = 297 [M+].
C16H11NO3S.
5. Experimental protocols
5.1. Chemistry
All compounds were characterized using the methods of
elemental analyses, IR spectroscopy, NMR spectroscopy and
MS spectrometry. Infrared spectra were recorded on a Shi-
madzu FTIR-8201 PC spectrometer in potassium bromide
pellets for solids and as liquid films for oils (m in cm–1).
Proton NMR spectra were recorded on a Bruker AC
200 spectrometer. The samples were dissolved in DMSO-d6
or in CDCI3. All measurements were performed at 293 K and
the chemical shift values were referenced to tetramethylsi-
lane (CEA Saclay, France) as internal standard. Chemical
shift values and IR data for all compounds are summarized in
the experimental part and are in agreement with the proposed
structures.
5.1.3. 6,12-Dihydroquinolo[3,4-b][1,4]benzothiazin-6-one
(2c)
Quinolin-2,4-diol (1c) (50 mmol) and 2-aminothiophenol
(60 mmol) were added to 15 ml of DMF and heated at 120 °C
for 12 h. On cooling at room temperature, the product crys-
tallized and was filtered under vacuum. Recrystallization
from ethanol gave (2c) in 65% yield: mp > 300 °C. IR (KBr)
(cm–1): 3335, 2820, 1668, 1654, 1606, 748; 1H-NMR
(DMSO-d6) d (ppm): 7.05–7.70 (7H, m, Ar–H), 8.10 (1H, m,
H–1), 9.10 (1H, s, NH), 12.00 (1H, s, NH). MS (m/z): 266
[M+]. C15H10N2OS.

B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
935
m, Ar–H), 8.05 (1H, d, H–1), 9.00 (1H, s, NH), 12.00 (1H, s,
NH); MS (m/z) = 280 [M+]. Anal. calculated for
C16H12N2OS: C, 68.57, H, 4.29, N, 10.00, S, 11.43. Found:
C, 68.66, H, 4.22, N, 9.95, S, 11.37.
5.1.5. (Z)-2,4-Dihydro-3-benzylidenebenzopyran-2,4-dione
(5a)
The 4-hydroxycoumarin (1a) (0.01 mol) and 0.012 mole
of benzaldehyde (3) were added to 10 ml of pyridine and
refluxed for 3 h. Once cooling at room temperature, the
mixture was rapidly added to 100 ml of cold alcohol and
vigorously stirred. The product crystallized and was filtered
under vacuum. Recrystallization from ethanol gave 5a in
75% yield; mp > 230 °C. IR (KBr) (cm–1): 3030, 1654, 1605,
1164, 748; 1H-NMR (DMSO-d6) d (pm): 7.10–7.30 (10H, m,
Ar–H), 6.90 (1H, s), 9.10 (1H, s); MS (m/z): 218 [M+].
C16H10O3.
5.1.6. (Z)-2,4-Dihydro-3-(p-methoxybenzylidene)benzopy-
ran-2,4-dione (5b)
This compound was obtained in 77% yield from benzal-
dehyde (4) according to the general procedure used for the
synthesis of 5a; mp = 212 °C. IR (KBr) (cm–1): 3030, 1652,
1
1607, 1160, 752. H-NMR (DMSO-d6) d (ppm): 7.10–7.30
(9H, m, Ar–H), 6.90 (1H, s), 3.85 (1H, s) MS (m/z) = 280
[M+]. C17H12O4.
5.1.7. (Z)-2,4-Dihydro-3-benzylidenquinolin -2,4-dione (5c
)
This compound was obtained in 75% yield from
p-methoxybenzaldehyde (3) according to the general proce-
dure used for the synthesis of 5a; mp = 195 °C. IR (KBr)
(cm–1): 3390, 3030, 2820, 1654, 1606, 1164, 748. 1H-NMR
(DMSO-d6) d (ppm): 7.00–7.30 (10H, m, Ar–H), 6.85 (1H,
s). MS (m/z) = 249 [M+]. C16H11NO2.
5.1.8. (Z)-2,4-Dihydro-1-methyl-3-benzylidenquinolin-2,4-
dione (5d)
This compound was obtained in 78% yield from benzal-
dehyde (3) according to the general procedure used for the
synthesis of 5a; mp = 192 °C. IR (KBr) (cm–1): 3390, 3020,
1
2830, 1627, 1608, 1087, 754. H-NMR (CDCl3) d (ppm):
7.00–7.30 (10H, m, Ar-H), 6.85 (1H, s), 3.50 (3H, s); MS
Fig. 4. Cytotoxic effect of tert-butylhydroperoxide in human hepatocytes in
(m/z) = 263 [M+]. C17H13NO2.
primary culture. Results are reported as mean SEM of three independent
experiments. *Values significantly different from control, P < 0.05 (Tuc-
key’s test). (a) Hepatoprotective effect of 0.3 mM t-BPH-induced cytotoxi-
city in human hepatocytes in primary culture (b) of compound 2a (c) of
quercetin. Results are reported as mean SEM of three independent expe-
5.2. Pharmacology
Collagenase and all products for cell culture were pur-
chased from Life Technologies (Cergy Pontoise, France) and
foetal calf serum (FCS) from Eurobio (Les Ulis, France). All
other chemicals were purchased from Sigma Chemicals (St
Quentin Fallavier, France) and were of analytical grade.
Culture plastic plates were obtained from Costar (Dutscher,
France).
a
riments. Values significantly different from (t-BHP 0.3 + 10 mM 2a or
quercetin). b Values significantly different from (t-BHP 0.3 + 5 mM 2a or
quercetin).
5.1.4. 5-Methyl-6,12-dihydroquinolo[3,4-b][1,4]benzothia-
zin-6-one (2d)
This compound was obtained in 65% yield from
N-methylquinolin-2,4-diol (1d) according to the general pro-
cedure used for the synthesis of 2a: mp > 300 °C. IR (KBr)
(cm–1): 3325, 3006, 2860, 1655, 1604, 1093, 758; 1H-NMR
(DMSO-d6). d (ppm): 3.55 (3H, s, N-CH3), 7.00–7.70 (7H,
5.2.1. HepG2 cell culture
The HepG2 cell line was derived from a human hepato-
blastoma and was obtained from Dr. J.P. Beck (Strasbourg).

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B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
Cells were cultured in William’s medium containing 10%
foetal calf serum (FCS) (Gibco, France), 2 mM glutamine
and 50 µg/ml gentamycin. Cultures were maintained in 5%
CO2 in air at 37 °C. HepG2 cells were routinely maintained
in 25 cm2 flasks, the medium was renewed every 48 h, and
cells subcultured every 7 days. To study the effects of the
various tested compounds, HepG2 cells were plated in 96-
multiwell culture plates at a concentration of 0.6 104
cells/100 µl medium.
ments was calculated using the one-way analysis of variance
followed by Thuckey’s test for multiple comparisons versus
pairwise. Values were considered statistically significant at
P < 0.05.
References
[1] R.A. Micheli, A.N. Booth, A.L. Livingston, E.M. Bickoff, J. Med.
Chem. 5 (1962) 321–329.
[2] S. Nishimura, M. Taki, S. Takaishi, Y. Lijima, T. Akiyama, Chem.
Pharm. Bull. 48 (2000) 505–508.
[3] Y. Jacquot, L. Bermont, H. Giorgi, B. Refouvelet, G.L. Adessi,
E. Daubrosse, A. Xicluna, Eur. J. Med. Chem. 36 (2001) 127–136.
[4] S.-G. Cao, X.-H. Wu, K.-Y. Sim, B.H.K. Tan, J.J. Vittal, J.T. Pereira,
S.-H. Goh, Helv. Chim. Acta 81 (1998) 1404–1407.
[5] T. Fujioka, K. Furumi, H. Fujii, H. Okabe, K. Mihashi, Y. Nakano,
H. Matsunaga, M. Katano, M. Mori, Chem. Pharm. Bull 47 (1999)
96–100.
5.2.2. Human hepatocyte isolation and culture
Adult normal liver samples were obtained from patients
undergoing partial hepatectomy for primary or secondary
tumours. All experimental procedures were done in com-
pliance with French laws and regulations and were approved
by the National Ethics Committee. Hepatocytes were isola-
ted by a two-step collagenase perfusion as previously descri-
bed by David et al. [28].
Viable hepatocytes were seeded in 96-multiwell culture
plates at a concentration of 0.3 104 cells/100 µl DMEM
medium as previously described by Goll et al. [29].
[6] L.W.L. Woo, A. Purohit, B. Malini, M.J. Reed, B.V.L. Potter, Chem.
Biol. 7 (2000) 773–791.
[7] A. Purohit, L.W.L. Woo, D. Barrox, H.A.M. Hejaz, R.I. Nicholson,
B.V.L. Potter, M.J. Reed, Mol. Cell. Endocrinol. 171 (2001) 129–135.
[8] Y. Jacquot, A. Cleeren, J. Laios, Y. Ma, A. Boulahdour, L. Bermont,
B. Refouvelet, G. Adessi, G. Leclercq, A. Xicluna, Biol. Pharm. Bull.
25 (2002) 335–341.
5.2.3. Assay for t-BHP cytotoxicity and protection
by various compounds
[9] Y. Jacquot, B. Refouvelet, L. Bermont, G.L. Adessi, G. Leclercq,
A. Xicluna, Pharmazie 57 (2002) 233–237.
[10] A. Iannone, A. Bini, G. Jin, V. Vannini, A. Tomasi, Free Radical Res.
Comm. 19 (1993) 141–147.
[11] H. Sies, K.H.H. Summer, Eur. J. Biochem. 57 (1975) 503–512.
[12] L. Nicod, C. Viollon, A. Regnier, A. Jacqueson, L. Richert, Hum. Exp.
Toxicol. 16 (1997) 28–34.
[13] E. Alexandre, P. David, C. Viollon-Abadie, A. Azimzadeh, P. Wolf,
D. Jaeck, L. Nicod, K. Boudjema, L. Richert, Toxicol. In Vitro 13
(1999) 427–435.
5.2.3.1. Assay of t-BHP cytotoxicity. HepG2 cells and pri-
mary cultures of human hepatocytes were plated in 96-
multiwell culture plates. Twenty-four hours after plating, the
medium was discarded and fresh medium containing t-BHP
0.3 mM was added to cultures. After 3 hours, cellular viabi-
lity was determined by measuring reduction of the
tetrazolium-blue compound MTT to a blue formazan product
according to Carmichael et al. [37].
[14] V. Goll, C. Viollon-Abadie, L. Nicod, L. Richert, Hum. Exp. Toxicol.
19 (2000) 193–202.
[15] M. Colombain, V. Goll, F. Muyard, C. Girard, F. Bévalot, L. Richert,
Planta Med. 67 (2001) 644–646.
[16] M.A. Gore, M.M. Morshedi, J.F. Reidhaar-Olson, Funct. Integ.
Genomics 1 (2000) 114–126.
[17] Y. Yamamoto, M. Bnakajima, H. Yamazaki, T. Yokoi, Life Sci. 70
(2001) 471–482.
[18] F. Carubbi, M.E. Guicciardi, M. Concazri, P. Loria, M. Bertolotti,
N. Carilli, Bicochem. Biophys. Acta 1580 (2002) 31–39.
[19] K.R. Martin, M.L. Failla, J.C. Smith, J. Nutr. 126 (1996) 2098–2106.
[20] J.A. Kim, Y.S. Kang, S.H. Lee, Y.S. Lee, Free Radical Res. 33 (2000)
267–277.
[21] Q. Feg, Y. Torii, K. Uchda, Y. Nakamura, Y. Hara, T. Osawa, J. Agric.
Food Chem. 2 (2002) 213–220.
[22] A. Jayashree, V.S. Rao, M. Darbarwar, Synth. Commun. 20 (7) (1990)
919–924.
5.2.3.2. Assay of protection against t-BHP cytotoxicity. The
various compounds were first tested for their effects on the
viability of HepG2 cells and primary cultures of human
hepatocytes. HepG2 cells and primary cultures of human
hepatocytes were plated in 96-multiwell culture plates.
Twenty-four hours after plating, the medium was discarded
and fresh medium containing the compounds at 0–10 µM
were added to cell cultures. After 24 h, cellular viability was
determined by measuring the reduction for the tetrazolium-
blue compound MTT to formazan. All tested compounds did
not affect HepG2 cell viability and primary cultures of hu-
man hepatocytes up to 10 µM (data not shown). For this
reason, the protective effects of the various compounds at
concentrations 0–10 µM were evaluated.
The potential protective effect was tested by co-
incubating for 3 h a given tested compound at 0.5, 1.0, 5.0 or
10 µM with t-BHP (3 mM) for HepG2 cells and t-BHP
(0.3 mM) for primary cultures of human hepatocytes. Cellu-
lar viability was then determined by measuring reduction of
the tetrazolium-blue compound MTT to formazan blue.
[23] N.P. Buu-Hoï, M. Mangane, P. Jacquignon, J. Chem. Soc. (C) (1966)
50–52.
[24] M. Vanhaelen, R. Vanhaelen-Fastré, Pharm. Acta Helv. 51 (1976)
307–312.
[25] K. Tabakovic, I. Tabakovic, M. Trkovnik, A. Juric, N. Trinajstic, J.
Heterocyclic Chem. 17 (1980) 801–803.
[26] B.S. Reddy, M. Darbarwar, J. Indian Chem. Soc. 62 (1985) 377–379.
[27] P. Maurel, Drug Delivery Rev. 22 (1996) 105–132.
[28] P. David, C. Viollon-Abadie, E. Alexandre, L. Nicod, P. Wolf,
D. Jeack, K. Boudjema, L. Richert, Hum. Exp. Toxicol. 17 (1998)
544–553.
5.2.4. Statistical analysis
The statistical significance of the difference between the
mean results of the least two to three independent experi-

B. Refouvelet et al. / European Journal of Medicinal Chemistry 39 (2004) 931–937
937
[29] V. Goll, E.Alexandre, C.Viollon-Abadie, L. Nicod, D. Jaeck, L. Rich-
ert, Toxicol. Appl. Pharmacol. 160 (1999) 21–32.
[30] A. Da Settimo, A.-M. Marini, G. Primofiore, F. Da Settimo, S. Sal-
erno, C. La Mota, G. Pardit, P.L. Ferrarini, C. Mori, J. Heterocyclic
Chem. 37 (2000) 379–382.
[31] W. Davey, J.E. Gwilt, J. Chem. Soc. (1957) 1008–1019.
[32] A.M. Clement, J.K. Chipman, Carcinogenesis 19 (9) (1998) 1583–
1589.
[34] N. Sugihara, T. Arakawa, M. Onhnishi, K. Furuno, Free Radical Biol.
Med. 27 (11 and 12) (1999) 1313–1323.
[35] H. Nagata, S. Takekoshi, T. Takagi, T. Honma, K. Watanabe, J. Tokai,
Exp. Clin. Med. 24 (1) (1999) 1–11.
[36] L. Richert, D. Binda, G. Hamilton, C. Viollon-Abadie, E. Alexandre,
D. Bigot-Lasserre, R. Bars, P. Cassolo, E. LeCluyse, Toxicol. In Vitro
16 (2002) 89–99.
[33] H. Wang, J.A. Joseph, Free Radical Biol. Med. 27 (5 and 6) (1999)
683–694.
[37] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell,
Cancer Res. 47 (1987) 936–942.