Synthesis and antioxidant properties of pulvinic acids analogues

Article abstract of DOI:10.1016/j.bmc.2010.09.037

The synthesis of three types of pulvinic acid analogues, using a diversity-oriented strategy starting from a single compound, dimethyl l-tartrate, is described. Lacey-Dieckmann condensation, alcohol dehydration and Suzuki-Miyaura cross-couplings were empl

Full text of DOI:10.1016/j.bmc.2010.09.037

Bioorganic & Medicinal Chemistry 18 (2010) 7931–7939
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antioxidant properties of pulvinic acids analogues
Brice Nadal a, Sophie A.-L. Thetiot-Laurent a, Serge Pin b, Jean-Philippe Renault b, Damien Cressier c,
Ghassoub Rima c, Antoine Le Roux d, Stéphane Meunier d, Alain Wagner d, Claude Lion e, Thierry Le Gall a,
⇑
a CEA Saclay, iBiTecS, Service de Chimie Bioorganique et de Marquage, Bât. 547, 91191 Gif-sur-Yvette, France
b CEA Saclay, IRaMiS, Service Interdisciplinaire sur les Systèmes Moléculaires et les Matériaux/UMR 3299 CNRS/CEA, Laboratoire de Radiolyse, 91191 Gif-sur-Yvette, France
c Laboratoire Hétérochimie Fondamentale et Appliquée, UMR 5069-CNRS/UPS, Université Paul Sabatier, 118, route de Narbonne, 31062 Toulouse Cedex 9, France
d Laboratoire des Systèmes Chimiques Fonctionnels, UMR/CNRS 7199, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France
e ITODYS, Université Paris 7, CNRS UMR 7086, Bât. Lavoisier, 15 rue Jean Antoine de Baïf, 75205 Paris Cedex 13, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 April 2010
Revised 9 September 2010
Accepted 16 September 2010
Available online 25 September 2010
The synthesis of three types of pulvinic acid analogues, using a diversity-oriented strategy starting from a
single compound, dimethyl -tartrate, is described. Lacey–Dieckmann condensation, alcohol dehydration
L
and Suzuki–Miyaura cross-couplings were employed in the course of the analogues syntheses. The eval-
uation of the antioxidant properties of the 28 synthesized analogues was carried out using antioxidant
capacity assays (protection of thymidine and b-carotene) and free radical scavenging assays (DPPH
radical and ABTS radical cation). This allowed to assess the relative influence of the groups bonded to
the tetronic ring and to the exocyclic double bond on the activity, as well as the importance of this
exocyclic double bond. It was shown that the presence of an electron-donating group on the 3-position
of the tetronic ring had a beneficial effect. It was shown in several assays that the presence of the
exocyclic bond was not crucial to the activity.
Keywords:
Pulvinic acids
Dieckmann condensation
Antioxidant
Free radicals
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
recently reported.10 This test measured the protection of the DNA
base thymidine submitted to three types of oxidative stresses. It
While oxygen is essential to life, its metabolism leads to the
production of reactive oxygen species (ROS) which can be respon-
sible for oxidative damages.1 Under normal conditions, the concen-
trations of ROS are maintained at low levels by the action of the
organism’s defences, which may be enzymes such as superoxide
dismutase or glutathione peroxidase, or small organic molecules
such as ascorbic acid (vitamin C), tocopherols (vitamin E), and
glutathione. These molecules, called antioxidants, are either
produced by the organism or acquired through alimentation. The
balance between the production and the elimination of ROS is thus
normally controlled; however, when it is perturbed, oxidative
stress is generated, leading to oxidative damages to biomolecules.
Such damages have been associated with various pathologies,
including Alzheimer’s disease2 and atherosclerosis,3 and also, more
generally, with ageing.4 A treatment by antioxidants is potentially
a way to overcome the oxidative stress, and then to improve the
patient’s condition.5 Several studies have shown beneficial effects
by antioxidant compounds such as ascorbic acid,6 resveratrol,7
and flavonoids.8 Efforts have also been made to study synthetic
antioxidants, which may be derived from naturally ones.9
allowed identifying norbadione A (1)11,12 (Fig. 1), a pigment of
the mushrooms Xerocomus badius (bay boletus) and Pisolithus
tinctorius, as a potent antioxidant. It efficiently protected thymi-
dine towards
c-irradiation, thus showing a potential activity as
radioprotective agent, and also towards UV-exposure in the pres-
ence of H2O2. However, this product was found to be inefficient
towards another oxidative stress, the Fenton-type oxidation, which
was instead rather typical of a pro-oxidant activity.13 Further stud-
ies showed that a structurally related pigment, di-O-methylatrom-
entic acid (2), efficiently protected thymidine towards the three
stress conditions, and did not show any pro-oxidant behavior.
Compound 2 belongs to a larger pigment family typical of boletes,
and also found in lichens, pulvinic acids, the simpler of which is
pulvinic acid (3).14 On the basis of these results, it was of interest
to study the structure–activity relationship of various pulvinic acid
analogues.
The pulvinic acid structure may be seen as a template to which
many modifications can be made. Pulvinic acids are characterized
by a tetronic acid moiety which is substituted by an aryl group
and by a hydroxycarbonylalkylidene moiety. The natural pigments
differ by the nature of the aryl groups, which are often
hydroxylated.
In the course of a study aiming at evaluating the antioxidant
activity of hundreds of compounds, a high-throughput screening
test that made use of a competitive immunoassay technique was
We recently reported the antioxidant properties of analogues in
which one of the aryl groups has been replaced by an alkyl group,15
and now wish to disclose the synthesis and antioxidant activity
evaluation of three types of pulvinic acid analogues (Fig. 2). The
⇑
Corresponding author. Tel.: +33 16908 7991; fax: +33 16908 7105.
E-mail address: thierry.legall@cea.fr (T. Le Gall).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.037

7932
B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
R1
CO2H
O
O
O
OH
R1
8
LiHMDS
O
HO
O
CO2Me
MeO2C
DCC, DMAP
THF, -78 ºC
O
CO2Me
OH
CH2Cl2
MeO2C
or nBu4NF
OH
7
THF
O
OH
R1
9
OH
HO
OH
R1
O
O
OH
HO2C
CO2H
CO2Me
OH
(CF3CO)2O
NEt3, CH2Cl2
O
CO2Me
O
O
norbadione A
5
4
(1)
HO
Scheme 1. Synthesis of tetronic acids 5 and alkenes 4.
MeO
OH
OH
CO2H
CO2H
diol with acids 8 in the presence of dicyclohexylcarbodiimide
(DCC) and catalytic 4-(dimethylamino)pyridine (DMAP) afforded
the corresponding esters 9a–i (Table 1).
O
O
O
O
Several conditions were tested in order to minimize the forma-
tion of diesters. The formation of diesters was limited to less than
20% by using 1 equiv of acid and 1 equiv of DCC; the reagents were
added at 0 °C, and then the reaction mixture was stirred at room
temperature. The diesters formed were readily removed by silica
gel chromatography, and were not usually isolated. Esters
9a–i were obtained in 38–67% yield.
OMe
di-O-methylatromentic acid
pulvinic acid
(2)
(3)
Figure 1. Norbadione A and pulvinic acids.
The acetoacetyl derivative of dimethyl
prepared using another method (Scheme 2). Thus, treatment of
dimethyl
-tartrate with 2,2,6-trimethyl-1,3-dioxin-4-one 1017 in
L-tartrate 9j was
OH
OH
R1
O
R1
O
CO2Me
L
p-xylene at reflux afforded compound 9j in 55% yield.
O
O
CO2Me
OH
The preparation of tetronic acids via a Dieckmann condensation
was first described by Lacey18 and has since been frequently em-
ployed, especially in the course of syntheses of natural products
containing a tetronic acid moiety.19 Brandänge et al. have reported
the Dieckmann condensation of dimethyl O-acetyltartrate using
lithium hexamethyldisilazide as base.20 Two conditions were em-
ployed for the cyclization of esters 9 to the corresponding tetronic
acids 5, depending on the nature of the substituent R1 (Table 1).
Compounds 5a–g were obtained, in 42–79% yield, by treatment
of the esters with 3 equiv of lithium hexamethyldisilazide
4
5
MeO
OH
CO2Me
R2
O
O
6
Figure 2. Structures of targeted pulvinic acids analogues 4, 5 and 6.
aim was in the first place to assess the relative effect on the activity
of the group R1, which is bound to the lactone ring, and the group
R2, which is placed on the exocyclic double bond; variously substi-
tuted compounds 4 and 6 would inform us on that matter. We also
wished to evaluate the importance of the double bond by compar-
ing the activities of compounds 4 versus 5. Compounds 5 also pres-
ent some analogies with ascorbic acid, which is a tetronic acid
substituted by a 1,2-dihydroxyethyl group.
Table 1
Synthesis of esters 9 and tetronic acids 5
Entry
R1
Acid
9 (% yield)
Basea
5 (% yield)
1
2
3
4
5
6
7
8
9
4-(MeO)C6H4
Ph
4-MeC6H4
4-BrC6H4
4-FC6H4
4-(BnO)C6H4
2-Thienyl
4-(NO2)C6H4
BnOC(O)
CH3C(O)
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a (65)
9b (67)
9c (57)
9d (59)
9e (52)
9f (62)
9g (55)
9h (42)
9i (40)
9jb
LiHMDS
LiHMDS
LiHMDS
LiHMDS
LiHMDS
LiHMDS
LiHMDS
Bu4NF
5a (64)
5b (59)
5c (70)
5d (79)
5e (55)
5f (42)
5g (54)
5h (60)
5i (58)
5j (50)
In this paper, we give a full account on the synthesis of nine
alkenes 4, ten tetronic acid derivatives 5, and nine methyl pulvi-
nates 6, which were all obtained from a single precursor, dimethyl
Bu4NF
Bu4NF
L
-tartrate, using a strategy that we previously reported in a
10
—
preliminary communication.16 The antioxidant properties of these
compounds were also evaluated, using two kinds of systems: the
thymidine protection immuno-enzymatic assay and the b-carotene
bleaching assay may be viewed as antioxidant capacity assays,
while the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and the
a
Base employed for the conversion of compound 9 to tetronic acid 5.
Compound 9j was prepared as described in Scheme 2.
b
O
O
2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic
method are free radical scavenging assays.
acid)
(ABTS)
O
O
10
OH
O
2. Results and discussion
2.1. Synthesis of analogues
O
CO2Me
MeO2C
CO2Me
MeO2C
9j
p-xylene
reflux
OH
OH
7
55%
Compounds 4 and 5 were prepared from dimethyl
L
-tartrate 7
as summarized in Scheme 1. In the first step, esterification of the
Scheme 2. Synthesis of the acetoacetic ester 9j.

B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
7933
(LiHMDS) in THF at ꢀ78 °C, according to the method described by
Brandänge.
In the case of alkene 4a, the two isomers were fully
characterized.16 In infrared, a strong band at 2594 cmꢀ1, corre-
sponding to a chelated hydroxyl, was observed for the E-alkene,
while a strong band at 3524 cmꢀ1, due to a nonchelated hydroxyl,
was observed for the Z-alkene.23 It is then not surprising that the
E-isomers, in which the hydroxyl group is chelated by the ester func-
tion, are distinctly less polar than the Z-isomers. Hence the synthetic
sequence described allowed to prepare various compounds 4, in
which the tetronic acid moiety is substituted by a methoxycarbon-
ylmethylene in position 5 and by various aryl and heteroaryl groups
in position 3. The synthesis of the alkenes 4 was easily performed on
a multi-gram scale.
Several methyl pulvinates 6a–h, in which a 4-methoxyphenyl
group is attached to the tetronic acid moiety, and which contain
various R2 aryl or heteroaryl substituents were prepared from
alkene 4a as reported previously (Scheme 3).16 The main features
of their preparation were the iodination of alkene 4a leading to
iodide 11, which was accompanied by an inversion of the double
bond configuration, and the Suzuki–Miyaura cross-couplings
carried out from iodide 11 and various boronates 12, using the
conditions reported by Occhiato et al.24 It is worthy of note that
these cross-couplings were carried out using iodide 11, in which
the enol function was left unprotected. The physical and spectro-
scopic characteristics of the known compound 6a were in
agreement with the reported values.25
However, these conditions were not satisfactory for the prepa-
ration of tetronic acid 5h, leading to degradation products. We
then found that a mere treatment of esters 9h–j with tetrabutyl-
ammonium fluoride21 in THF at room temperature allowed the
clean formation of the corresponding tetronic acids 5h–j, obtained
in 50–60% yield. In compounds 9h–j, R1 is an electron-withdrawing
group (4-nitrophenyl, benzyl ester and acetyl); hence the adjacent
methylene protons are more acidic and the use of a weaker base
was possible. It is noteworthy that all the tetronic acids prepared
were isolated as stereochemically pure isomers, indicating that
no epimerization had occurred under the cyclization conditions.
Hydrogenolysis of benzyl ether 5f under usual conditions
(H2, 10% Pd/C, MeOH–DMF) afforded quantitatively the corre-
sponding phenol 5k.
Tetronic acids 5a–k were notably characterized by the presence,
in the 1H NMR spectra, of a doublet at 5.04–5.42 ppm, correspond-
ing to the proton on the lactone ring, and
a
doublet at
4.78–5.00 ppm corresponding to the
a-hydroxyl proton. Interest-
ingly, in 1H NMR, compound 5j appears as a mixture of two stereo-
isomers, due to an enolization of the acetyl carbonyl function
(Fig. 3). For example the protons of the two methyl groups appear
as singlets at d = 2.57 and 2.59 ppm.
Alcohols 5a–e, 5g–k were then converted to the corresponding
alkenes in dehydrating conditions, using trifluoroacetic anhydride
(3 equiv) and triethylamine (6 equiv) in the presence of a catalytic
amount of 4-dimethylaminopyridine (DMAP), according to a previ-
ously reported method (Table 2).22 However, the dehydration of
ketone 5j could not be obtained under these conditions and other
methods tested also failed. For compound 5k, which contains a
phenol function, higher amounts of reagents have been employed.
While alkene 4i was obtained as a pure Z-isomer, the other al-
kenes 4a–h were obtained as mixtures of stereoisomers, in which
the E-isomer accounted usually for 10–20%. The isomers were dis-
tinguished on the basis of the chemical shift of the ethylenic pro-
ton, which appears at upper field in the E-isomer (for compounds
4a, dE = 6.15 ppm, dZ = 5.91 ppm). In each case, the two isomers
were easily separated, and only the major, more polar Z-isomer
was usually recovered. The yields of the reactions from compounds
3, in which the aryl group is substituted either by an electron-
withdrawing or electron-donating function, were generally good.
However, lower yields were obtained from 2-thienyl derivative
5g (37%) and from phenol 5k (37%).
Methyl pulvinate 6i, which contains a 2-furyl group, was pre-
pared according to a different procedure, using 2-furylboronic acid
instead of the corresponding boronate.26 The expected compound
was isolated in 35% yield (Scheme 4).
Methyl pulvinates 6a–i were thus conveniently prepared from
readily available dimethyl
L-tartrate, using a four-step flexible
MeO
1) 0.5 N NaOH (1 equiv)
acetone, H2O
OH
2) N-chlorosuccinimide
AcOH, THF, 50 ºC
O
O
50%
CO2Me
4a
O
MeO
R2
B
12
O
OH
CO2Me
I
PdCl2(PPh3)2
2 M Na2CO3
O
O
THF, reflux
50-80%
OH
O
O
11
CO2Me
OH
HO
CO2Me
OH
6, 12
R2
MeO
6a, 12a C6H5
O
O
O
OH
O
6b, 12b 3-(HO)C6H4
6c, 12c 4-ClC6H4
CO2Me
R2
Figure 3. Structures of compound 5j.
6d, 12d 3-(F3C)C6H4
6e, 12e 2-(F3C)C6H4
O
O
6f, 12f
4-AcC6H4
6
Table 2
Synthesis of alkenes 4a–h
6g, 12g 3-thienyl
6h, 12h 3-furyl
Entry
R1
Tetronic acid
Alkene
Yielda (%)
Scheme 3. Synthesis of methyl pulvinates 6.
1
2
3
4
5
6
7
8
9
4-(MeO)C6H4
Ph
4-MeC6H4
4-BrC6H4
4-FC6H4
4-(HO)C6H4
2-Thienyl
4-(NO2)C6H4
BnOC(O)
5a
5b
5c
5d
5e
5k
5g
5h
5i
4a
4b
4c
4d
4e
4f
4g
4h
4i
83
62
83
63
74
44b
37
73
83
MeO
OH
Pd(PPh3)4
K3PO4
O
CO2Me
O
+
11
B(OH)2
THF, reflux
O
O
13
6i
a
Yield of isolated Z-isomer.
9 Equiv of Et3N and 4.5 equiv of (CF3)2CO were employed.
b
Scheme 4. Synthesis of methyl pulvinate 6i.

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B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
Table 3
Thymidine protection
approach, characterized by the fact that no functional group
protection was needed. The antioxidant properties of methyl pulv-
inates 6a–i, tetronic acids 5a–e and 5g–j and alkenes 4a–h were
then evaluated as described below.
Compound Thymidine
protection (%)
Thymidine
Thymidine
protection (%)
Fentonc
protection (%) UV/
radiolysisa
H2O2
b
Reference compound
2.2. Antioxidant assays
Trolox
5.2 0.2
4.4 1.0
11.7 7.5
Hydroxy esters
A series of assays were then performed in order to evaluate the
antioxidant properties of the synthesized compounds.27 Firstly,
the protection of thymidine towards three types of oxidative
5a
5b
5c
5d
5e
5f
5g
5h
5i
87.4 1.5
62.0 0.6
42.7 0.4
60.2 1.2
39.0 0.5
37.4 3.5
40.3 2.2
60.9 1.8
47.9 0.8
5.6 1.8
24.9 0.2
33.2 2.0
43.8 14.7
37.3 1.3
55.3 1.0
51.0 1.4
16.1 3.6
33.4 0.7
16.8 1.9
7.4 2.3
25.8 8.6
31.0 5.0
7.0 4.5
37.6 3.7
55.2 2.7
21.1 0.2
0.0 3.6
70.7 1.2
2.6 1.6
0.0 0.1
conditions (c-rays, UV/H2O2, and Fenton oxidation) was determined
using the previously described immuno-enzymatic assay.10,13 Then,
the radical scavenging properties of the compounds were studied
using assays based on two oxidants, the radical 2,2-diphenyl-1-pic-
rylhydrazyl (DPPHÅ) and the radical cation of (2,20-azinobis-(3-eth-
ylbenzothiazoline-6-sulfonic acid) (ABTSÅ+). Finally the evaluation
of b-carotene protection in a lipidic emulsion under oxidative
conditions by the compounds was realized. In each case, reference
compounds, that is, known antioxidant compounds were also
tested.
5j
5k
63.7 2.4
29.4 1.9
93.4 4.4
Alkenes
4a
4b
4c
4d
4e
4f
4g
4h
4i
73.8 0.8
69.4 2.9
30.0 0.1
51.7 0.5
52.9 1.0
57.2 3.4
51.4 3.0
58.5 1.7
23.1 2.6
30.7 4.3
37.7 1.0
52.7 0.8
41.2 0.5
69.3 4.0
32.6 4.2
23.6 4.9
31.5 0.4
13.9 4.4
32.0 5.0
45.8 6.1
14.1 3.2
27.1 0.5
28.3 0.7
82.7 2.1
0.0 0.6
2.2.1. Protection of thymidine
The degradation of the DNA base thymidine under oxidative
treatment is well known to lead to numerous compounds.28 In this
method, thymidine was oxidized under a variety of conditions, and
the amount of remaining intact thymidine was determined owing
to a competitive immuno-enzymatic assay, which makes use of a
specific anti-thymidine monoclonal antibody.10 The protection of
thymidine by the synthesized compounds under three types of oxi-
dative conditions are summarized in Table 3. The first oxidative
condition involved an irradiation at 340 Gy, using a cesium-137
source; the second one consisted in a UV irradiation of a hydrogen
peroxide solution k = 254 nm, 1.75 J cmꢀ2); finally, Fenton condi-
tions, a treatment with Fe2+/EDTA and hydrogen peroxide, were
also employed.
As previously stated, differences in the protection effect are
expected to be observed, depending on the oxidation conditions.13
Both the radiolysis of water and the homolytic cleavage of the
oxygen-oxygen bond of hydrogen peroxide under UV irradiation
lead to the formation of hydroxyl radicals, which would then react
with thymidine. In these cases, compounds would efficiently pro-
tect thymidine by trapping first the hydroxyl radical or by breaking
peroxidation chains. Regarding the Fenton system, it is supposed to
generate, along with hydroxyl radicals, iron-containing oxidants.29
In previous work, it was shown that under the Fenton conditions,
the protective effects observed were significantly different from
those obtained using the two other conditions.13
40.7 0.4
0.3 0.4
Methyl pulvinates
6a
6b
6c
6d
6e
6f
6g
6h
6i
46.4 3.2
42.9 1.7
50.0 0.1
45.0 1.0
35.8 2.1
46.1 3.4
41.1 1.2
34.8 0.5
36.0 6.2
28.7 1.6
58.0 3.8
91.6 2.8
41.7 4.1
33.9 3.7
14.8 3.5
41.1 3.8
45.2 9.5
13.8 1.8
0.0 2.0
34.2 0.8
22.6 0.9
46.3 1.7
17.6 0.4
34.3 1.2
76.0 2.4
77.0 1.2
62.0 0.3
a
Thymidine protection under
[compound] = 50
M in 12.5 mM phosphate buffer, 340 Gy, source: 137Cs.
Thymidine protection under UV/H2O2 exposure: [thymidine] = 70
c-radiation exposure: [thymidine] = 15 lM,
l
b
l
M, [com-
pound] = 100
lM, [H2O2] = 5 mM in 12.5 mM phosphate buffer, k = 254 nm,
1.75 J cmꢀ2
.
c
Thymidine protection under UV/H2O2 exposure: [thymidine] = 70
lM, [com-
pound] = 100
l
M, Fe2+/EDTA/H2O2 (1:1:100) 640 mM in 11.4 mM phosphate buffer,
30 min. All experiments were performed in duplicate and averaged.
The results obtained for the thymidine protection when using
the UV irradiation of a hydrogen peroxide solution reflects the
same tendency. For example, compounds 5i and 4i were poorly
efficient in both types of conditions. However the amplitude of
the protection observed for the whole series of products was lesser
than in the former tests. The best result (50.0%) was obtained for
methyl pulvinate 6b. It should be noted that the protection given
by the methyl pulvinates 6 lies in a short range (about 30–50%).
In the Fenton-type oxidation, hydroxy esters 5c, 5g, 5i, 5j,
bearing a R1 group 4-tolyl, 2-thienyl, benzyl ester and acetyl,
respectively, were found to be completely inactive, which may
reveal a pro-oxidant activity for these compounds. On the contrary,
4-nitrophenyl derivative 5h was very efficient, and phenol 5k led
to more than 90% protection. Alkenes 4g, containing a 2-thienyl
group, and 4i, bearing a benzyl ester were inactive, as the corre-
sponding hydroxy esters, while phenol 4f had the best activity.
Among the methyl pulvinates, compound 6b in which R1 is a
3-hydroxyphenyl group was the most efficient protector and
phenyl-substituted 6a was also quite efficient. Among these com-
pounds, furan derivatives 6h and 6i, and 2-(trifluoromethyl)phenyl
derivative 6e were found to be poorly active (less than 15%
protection).
Most of the hydroxy esters 5 led to a good protection of
thymidine in the test using c
-rays. Compound 5b in which R1 is
a phenyl group gave a protection of 62%. Compounds 5d, 5h, and
5k in which the aromatic ring is 4-substituted by a bromide, a nitro
group and a hydroxyl function, respectively, led to similar results.
In this series, compound 5j, which is substituted by an acetyl
group, was inefficient, while 5a, which contains a 4-methoxy-
phenyl group, gave the best result. Alkenes 4 were also generally
good protectors (over 50%, except for 4-tolyl derivative 4c and
benzyl ester 4i). Among these alkenes, 4a, which is substituted
by a 4-methoxyphenyl group, was the most efficient. In general,
there were not great differences in activity between an alkene 4
and the corresponding hydroxy ester 5 having the same R1 group.
Methyl pulvinates 6 are all substituted by the 4-methoxyphenyl
group at the 3-position; however, they were usually found to be
less efficient than alkene 4a. Compounds 6g–i, substituted by a
heterocyclic ring on the exocyclic double bond (R2), gave the best
protection.

B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
7935
Table 4
In summary, the thymidine assays showed that the tested com-
pounds which did not bear a R1 aryl group displayed little activity.
Usually, an electron-donating R1 group was preferable, although 4-
nitrophenyl-substituted 5h protected efficiently thymidine against
Fenton-type oxidation. Both 4-methoxyphenyl-substituted hydro-
xy ester 5a and alkene 4a were very active in the test involving
Scavenging activity of compounds for DPPH radical and ABTS radical cation
a,b,c
Compound
DPPH assay 1/EC50
ABTS assay TEACd (mM)
Reference compounds
Ascorbic acid
Gallic acid
4.56
11.72
4.88
0.64 0.01
1.36 0.02
1
Trolox
c
-rays; it is noteworthy that compound 5a protected thymidine
as efficiently as norbadione A (84%).10
Hydroxy esters
5a
5b
5c
5d
5e
5f
5g
5h
5i
3.44
3.57
3.27
2.81
4.20
3.12
2.25
—
1.55 0.09
1.94 0.20
1.54 0.15
4.91 1.42
2.94 0.48
2.16 0.11
2.2.2. Scavenging activity of compounds for DPPH radical and
ABTS radical cation
Both radicals DPPHÅ and ABTSÅ+ (Fig. 4) are frequently used to
evaluate antioxidant activities of natural as well as synthetic
compounds.
24.63 4.37
43.34 8.33
82.69 34.80
0.60 0.02
DPPH is a stable, commercially available radical, characterized
by an absorption band at 515 nm. Its reduction by an antioxidant
compound leads to a decolorisation which can be monitored by a
spectrophotometer as described by Brand-Williams et al.30 The as-
say was adapted in order to be performed in 96-well plates. For
each compound, ten concentrations were employed and the per-
centage of remaining DPPH was determined at a steady state.
The efficient concentration EC50 or concentration necessary to
decrease the initial DPPHÅ concentration by 50% (in mol/L of
antioxidant/mol/L of DPPHÅ), was then obtained for each com-
pound. The results, summarized in the Table 4, are expressed in
terms of antiradical power (ARP = 1/EC50); the higher is this value,
the more active is the tested compound.
Most compounds were able to scavenge the DPPH radical.
Methyl pulvinates 6 were found to behave similarly, with ARP val-
ues in the range 2.97–4.08. Alkenes 4 were slightly less active, the
more efficient compound being 4-methoxyphenyl derivative 4a.
2-Thienyl derivative 4g and 4-nitrophenyl derivative 4h were
inactive, as were hydroxy esters 5h, 5i, 5j. Among the other hydro-
xy esters, phenyl-substituted 5b and 4-fluorophenyl-substituted
5e had the best activities.
The ABTS assay allows to measure the ability of an antioxidant
compound to scavenge the radical cation ABTSÅ+, which is gener-
ated by oxidation of the ABTS salt with a strong oxidant such as
potassium persulfate.31 The reduction of the blue-green radical
cation was monitored at 740 nm with a spectrophotometer. Sev-
eral concentrations of compound were employed and for each con-
centration, the absorbance was measured until a steady state was
reached. This allowed determining the EC50 value (concentration
necessary to decrease the initial absorbance at 740 nm by 50%)
for each compound. The results were expressed in terms of TEAC
(Trolox Equivalent Antioxidant Capacity: concentration of antioxi-
dant that gives the same percentage change of absorbance of
ABTSÅ+ as that of 1 mM Trolox).
Hydroxy esters led to results that varied largely, thus showing a
strong effect of the nature of the R1 group on the scavenging
activity of these compounds. Derivatives containing 2-thienyl,
4-nitrophenyl and benzyl ester groups displayed only weak activ-
ities. Among the other compounds, those having a phenyl or an
electron-donating R1 group (compounds 5a, 5b, 5c, and 5f) led to
TEAC between 1.54 and 2.16 mM. Phenol 5k was found to be more
active than Trolox. Alkenes 4 were usually less potent than the cor-
responding hydroxy esters 5 bearing the same substituent R1.
—
—
3.00
5j
5k
Alkenes
4a
4b
4c
4d
4e
4f
4g
4h
4i
3.60
3.22
2.56
1.17
1.97
2.85
—
1.31 0.06
46.19 5.48
6.51 1.93
17.71 5.44
15.40 3.11
0.48 0.01
—
0.93
18.70 3.09
18.51 0.49
Methyl pulvinates
6a
6b
6c
6d
6e
6f
6g
6h
6i
3.20
3.68
3.37
4.08
—
3.27
2.97
3.35
3.42
1.67 0.30
1.56 0.10
3.97 0.25
2.75 0.30
12.11 6.68
6.18 0.68
2.69 0.24
1.06 0.15
1.16 0.23
a
DPPH radical scavenging capacity assay: [DPPH] = 100 lM in ethanol, [com-
pound] = 0–1 10ꢀ3 M in ethanol (10 concentrations), k = 515 nm; EC50 = antioxidant
concentration necessary to decrease the initial [DPPHÅ] by 50%; experiments were
performed in duplicate.
b
[DPPH]/EC50 = antiradical power; the higher is this value, the more efficient is
the antioxidant compound.
c
No value is indicated in the cases where no steady state has been reached after
1 h.
ABTS radical cation decolorisation assay: an ABTSÅ+ solution (absor-
d
bance = 0.70 0.02 at k = 740 nm) and compound solutions of varying concentra-
tions were mixed, then the absorbance was measured until a steady state is
observed; the EC50 (concentration necessary to decrease the initial absorbance at
740 nm by 50%) was determined; experiments were performed in triplicate; results
are reported using TEAC.
However, 4-methoxyphenyl derivative 4a had a TEAC of 1.31 mM
and compound 4f, which bears a phenol function, was more active
than Trolox.
Among methyl pulvinates 6, which all contain a 4-methoxy-
phenyl R1 group, the furan derivatives 6h and 6i displayed an
activity similar to that of Trolox. The other compounds were less
good ABTSÅ+ scavengers; it should be noted that the products 6c,
6d, 6e, and 6f, having an electron-withdrawing R2 group, were
significantly less efficient.
In these assays, compounds bearing in the 3-position of the
tetronic ring a phenyl nucleus substituted by an electron-donating
substituent were then the best scavengers, and phenols 5k and 4f
were especially efficient in the ABTS assay.
O2N
SO3H
S
Ph
HO3S
N
S
N
N
NO2
N
N
2.2.3. b-Carotene bleaching assay
Ph
N
Et
ABTS·+
Figure 4. Structures of DPPHÅ and ABTSÅ+
It was of interest to evaluate the efficiency of the synthesized
compounds as scavengers of oxygen-centered radicals such as
lipoperoxyl radicals, which play an important role in the ROS-med-
iated degradation of membranes, hence in atherosclerosis.
O2N
Et
DPPH·
.

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B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
In this assay, the reaction of b-carotene with linoleic acid
allowed to evaluate the respective influences of the modified sub-
stituents R1 and R2, and also to assess the importance of the pres-
ence of an exocyclic double bond.
oxidation products in an emulsified, aqueous system, induced a
color change which was monitored spectrophotometrically, at
k = 470 nm, in the presence of an antioxidant (at two concentra-
The observed results are in agreement with the assumption
previously made15 that the antioxidant capacity of the pulvinic
acids comes from the possibility, for the radical initially formed
on the enol function oxygen atom, which is deprotonated at
neutral pH, to be delocalize on the aromatic nucleus present at
the 3-position of the lactone (Scheme 5). The stabilization of the
radical does not appear to be sufficient when this aromatic group
is replaced by a carbonyl function (acetyl or benzyl ester).
The difference of activity, in tests such as the thymidine protec-
tions: 116 lM and 24 l
M).32–34 The percentage of protection of
b-carotene was determined after 4 h for each compound. It should
be noted that when the oxidation of b-carotene is higher in the
presence of an antioxidant than in a control experiment in the
absence of compound, then the protection is negative. Selected
results obtained for well-known antioxidants and for the most
active compounds are summarized in Figure 5.
Among the tested compounds, methyl pulvinate 6f, which
contains a 4-acetylphenyl R2 group, was the most efficient, compa-
tion against c-radiations and the DPPH assay, between hydroxy es-
rable to vitamin E at 116
l
M, but more active at 24
l
M. Interest-
ters 5 and the corresponding alkenes 4 bearing the same R1 group
was usually not very important. This may show that the presence
of an exocyclic double bond is not essential to the compounds
activity, at least in these tests. Hence the presence of a tetronic acid
to which an aryl group is bonded in the 3-position would be the
essential feature for the antioxidant property. However, more
discrepancies were observed in the results obtained from the
corresponding 4 and 5 products in the test involving the Fenton
oxidation or the ABTS radical scavenging.
With regard to methyl pulvinates 6, a comparison with alkene
4a, which lacks a R2 substituent, shows that the R2 group does
not necessary lead to an increase of activity in most assays.
However, it is noteworthy that the more efficient compounds in
the b-carotene bleaching assay were two methyl pulvinates 6e
and 6f; the increased lipophilicity of these compounds as
compared to hydroxy esters 5 or alkenes 4 may be accounted for
this outcome.
ingly, the theoretical value of log P35 for 6f (0.54) was much
lower than that of vitamin E (9.98). The partitioning of the tested
compound between the lipophilic and the hydrophilic phases is
an important factor to be considered in this assay, since a lipophilic
antioxidant compound should be more able to scavenge the lipop-
eroxyl radicals. For example, hydrophilic ascorbic acid does not
lead to any protection, unlike vitamin E. Hence, methyl pulvinates
6, which are generally more lipophilic than hydroxy esters 5 and
alkenes 4, could be expected to protect b-carotene with more effi-
ciency. Methyl pulvinate 6e, having a 3-(trifluoromethyl)phenyl
group (log P = 2.15), also gave a good protection at 116 lM. The
other compounds that led to a protection better than 44% at this
concentration are hydroxy ester 5b, in which R1 is a phenyl group,
and alkene 4a.
2.2.4. Discussion on antioxidant assays results
The utilization of several antioxidant tests having different
characteristics was expected to afford indications on the relative
properties of the three classes of compounds that were prepared,
which can be viewed as analogues of pulvinic acids. The test that
used thymidine as the target then informs on the capacity to
protect a DNA base from oxidation, and specific informations can
also be obtained from the different stresses employed in this assay.
The DPPH and ABTS assays are frequently employed for the assess-
ment of antioxidant properties of products and were thus relevant
in this study. Finally, the b-carotene bleaching assay is a good way
to mimic a protection effect of biological membrane. This study
Among hydroxy esters 5, which are simpler analogues of pulvi-
nic acids, the most active compound in the thymidine protection
assay under
c-radiation exposure, 5a, contained the electron-
donating 4-methoxyphenyl R1 substituent. The corresponding
alkene 4a was also very active in this test, as well as in the DPPH
assay. Finally, hydroxy ester 5k, which is substituted at the 3-posi-
tion by a 4-phenol group, performed well in most assays, including
the thymidine protection under Fenton oxidation.
3. Conclusions
We have reported the flexible synthesis of three types of pulvinic
acids analogues, all derived from a single starting material, dimethyl
100
116 µM
L-tartrate. Various tetronic acids, containing a hydroxy ester moiety,
24 µM
80
were prepared by Lacey–Dieckmann condensation of the corre-
sponding esters. Dehydration of these hydroxy esters then readily
afforded the corresponding Z configurated alkenes. Differently
substituted methyl pulvinates were obtained via Suzuki–Miyaura
cross-couplings involving a vinyl iodide and several boronates.
Evaluation of the antioxidant properties of the synthesized
compounds was then carried out using a series of tests. Most
compounds have displayed a good activity. A beneficial effect of
an electron-donating group at the 3-position of the tetronic acid
moiety was observed. Methyl pulvinates 6 were found to be more
efficient in the b-carotene bleaching assay, in accordance with
their more lipophilic characteristics, as compared to the other
derivatives. Compounds 4 and 5 bearing a 4-methoxyphenyl or a
4-hydroxyphenyl group were found to be very efficient in most as-
says. These derivatives appear very interesting for further studies
owing to their good antioxidant activity. In particular, evaluation
of the anti-inflammatory and radioproprotective properties of
these compounds will be investigated. Lastly, it should be pointed
out that structural modulations can easily be performed, as the
synthesis of similar compounds can be readily achieved using the
strategy presented here.
60
40
20
0
-20
Figure 5. b-Carotene bleaching assay: aliquots (250
from b-carotene, linoleic acid and Tween 40 emulsifier in oxygen-saturated water
were added to 50 of pure ethanol antioxidant compound solution at two
concentrations (116 M and 24 M) and placed at 50 °C for 4 h. The absorbance
was measured at 470 nm, and the percentage of protection of b-carotene was
determined after 4 h; experiments were performed in triplicate and averaged.
lL) of an emulsion prepared
l
l
L
l

B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
7937
R
R
O
O
electron
transfer
R2
3
R2
O
CO2Me
O
CO2Me
O
O
R
R
O
O
R2
CO2Me
Scheme 5. Mechanism of pulvinic analogues oxidation.
R2
CO2Me
O
O
O
O
aqueous layer was extracted with AcOEt (2 ꢁ 60 mL). The com-
bined organic layers were dried (MgSO4) and filtered and concen-
trated under vacuum. Silica gel chromatography (89:10:1–70:30
4. Experimental section
4.1. General methods
CH2Cl2/MeOH/AcOH) afforded
as a white solid.
a-hydroxy ester 5b (858 mg, 59%)
THF was freshly distilled from sodium benzophenone ketyl.
Reactions were performed under an argon atmosphere. TLC: Silica
Gel 60F254 plates with detection by UV light and by an acidic
ethanolic solution of phosphomolybdic acid. Column chromatogra-
4.5. Methyl (R)-2-hydroxy-2-((R)-3-hydroxy-5-oxo-4-phenyl-
2,5-dihydrofuran-2-yl)acetate (5b)
phy: 40–63 lm silica gel. Melting points were uncorrected. NMR:
White solid; mp 188–190 °C; ½a D20
ꢂ
+150.4 (c 1.0, MeOH); IR (KBr
pellet) mmax: 3294, 3058, 3023, 2956, 2761, 1760, 1736, 1667, 1599,
400.133 and 100.624 MHz for 1H and 13C, respectively. Chemical
shifts (d) are in ppm (s = singlet, d = doublet, t = triplet, m =
multiplet, b = broad), coupling constants (J) are in Hz.
1499, 1424, 1328, 1256, 1238, 1168, 1128, 1058, 1013, 883, 833,
765, 753, 699, 682 cmꢀ1 1H NMR (400 MHz, acetone-d6) d 7.93
;
(2H, d, J = 7.4 Hz, CHPh), 7.37 (2H, t, J = 7.4 Hz, CHPh), 7.26 (2H, t,
J = 7.4 Hz, CHPh), 5.31 (1H, d, J = 1.8 Hz, CHOC(O)), 4.81 (1H, d,
J = 1.8 Hz, CHOH), 3.82 (3H, s, OCH3); 13C NMR (100 MHz, ace-
tone-d6) d 172.2 (2C), 172.0, 131.2, 128.8, 128.2, 127.8, 102.8,
78.6, 69.6, 52.9; HRMS (ESI-TOF) calcd for C13H12NaO6 [M+Na]+
287.0532, found 287.0530.
4.2. General procedure for the synthesis of esters 9
Dicyclohexylcarbodiimide (11.58 g, 56.13 mmol) and 4-
(dimethylamino)pyridine (2.74 g, 22.45 mmol) were added simul-
taneously to a solution of 4-methoxyphenylacetic acid (9.33 g,
56.13 mmol) and (+)-dimethyl L-tartrate (10 g, 56.13 mmol) in
dry dichloromethane (400 mL) cooled at ꢀ18 °C. The solution
was stirred at room temperature for 12 h. The resulting mixture
was concentrated under vacuum to approximately 150 mL. Diethyl
ether (300 mL) was then added and the precipitate obtained was
filtered. The filtrate was washed with a saturated aqueous NH4Cl
solution (3 ꢁ 100 mL), then dried (MgSO4), filtered and concen-
trated under vacuum. Silica gel chromatography (80:20–70:30
cyclohexane/ethyl acetate) afforded ester 9a (11.9 g, 65%) as a col-
orless oil.
4.6. General procedure for the synthesis of alkenes 4
Triethylamine (2.2 mL, 15.9 mmol) was added to a suspension
of
a-hydroxyester 5b (700 mg, 2.65 mmol) and DMAP (16.2 mg,
0.13 mmol) in dry dichloromethane (160 mL) cooled at ꢀ10 °C. Tri-
fluoroacetic anhydride (0.75 mL, 7.95 mmol) was added dropwise
over 15 min. The reaction mixture was then allowed to warm to
room temperature. After stirring for 16 h, 3 N HCl (3 mL) was
added. After stirring for 1 h at room temperature, the two layers
were separated, and the aqueous layer was extracted with AcOEt
(2 ꢁ 10 mL). The combined organic layers were dried (MgSO4),
filtered and concentrated under vacuum. Silica gel chromatogra-
phy (90:10, then 50:50, then 30:70 cyclohexane/acetone) afforded
alkene (Z)-4b as an orange solid (410 mg, 62%).
4.3. Dimethyl (2R,3R)-2-hydroxy-3-(2-(4-
methoxyphenyl)acetoxy)succinate (9a)
Colorless oil; ½a D20
ꢂ
+29.8 (c 1.00, MeOH); IR (film) mmax: 3500,
3008, 2957, 2841, 2361, 2341, 1747, 1613, 1586, 1514, 1436,
1360, 1247, 1135, 1070, 1030, 980, 860, 822, 792, 729, 638, 588,
569 cmꢀ1; 1H NMR (400 MHz, CDCl3) d 7.18 (2H, d, J = 8.4 Hz, CHAr),
6.86 (2H, d, J = 8.4 Hz, CHAr), 5.44 (1H, d, J = 2.2 Hz, CHOC(O)), 4.74
(1H, d, J = 2.2 Hz, CHOH), 3.80 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.65
(2H, s, CH2Ar), 3.62 (3H, s, OCH3); 13C NMR (100 MHz, CDCl3) d
170.9, 170.6, 167.0, 158.8, 130.4, 125.3, 114.1, 73.1, 70.4, 55.3,
53.1, 52.9, 39.8; HRMS (ESI-TOF) calcd for C15H18NaO8 [M+Na]+
349.0899, found 349.0913.
4.7. Methyl (Z)-2-(3-hydroxy-5-oxo-4-phenylfuran-2(5H)-
ylidene)acetate (4b)
Orange solid; mp 210–212 °C; IR (KBr pellet)
2961, 1771, 1702, 1661, 1641, 1439, 1404, 1373, 1257, 1215, 1184,
1136, 1026, 960, 900, 843, 786, 772, 695, 597 cmꢀ1 1H NMR
mmax: 3230, 3100,
;
(400 MHz, acetone-d6) d 7.91–7.88 (2H, m, CHPh), 7.47–7.42 (2H,
m, CHPh), 7.39–7.35 (1H, m, CHPh), 5.95 (1H, s, CHCO2Me), 3.76
(3H, s, OCH3); 13C NMR (100 MHz, acetone-d6) d 167.8, 164,
162.1, 153.1, 129.6, 129.2 (2C), 129.1, 105.7, 96.2, 52.0; HRMS
(ESI-TOF) calcd for C13H10NaO5 [M+Na]+ 269.0426, found 269.0427.
4.4. General procedure for the synthesis of a-hydroxy esters 5
Synthesis of compound 5b: A solution of LiHMDS (19.1 mL, 1 M
in THF) was added to THF (32 mL) under nitrogen. The solution
was cooled to ꢀ78 °C and then a solution of compound 9b
(1.62 g, 5.47 mmol) in THF (18 mL) was added dropwise. The reac-
tion mixture was stirred for 1 h at ꢀ78 °C and then allowed to
warm to room temperature over 2 h. After cooling at 0 °C, aqueous
2 N HCl (40 mL) was added. The two layers were separated, and the
4.8. General procedure for the Suzuki–Miyaura cross-coupling
All the solvents were degassed. To a solution of iodide 11
(150 mg, 0.373 mmol) in THF (19 mL) were added Pd(PPh3)2Cl2
(13 mg, 0.019 mmol, 5 mol %), a solution of 3-hydroxyphenylbo-

7938
B. Nadal et al. / Bioorg. Med. Chem. 18 (2010) 7931–7939
ronic acid pinacol ester 12b (123 mg, 0.559 mmol, 1.5 equiv) in
THF (7 mL), and 2 M aqueous Na2CO3 (8.2 mL). The reaction mix-
ture was refluxed for 2 h under argon. After cooling to room tem-
perature, water (5 mL) and aqueous 3 N HCl (3 mL) were added.
The aqueous layer was extracted with ethyl acetate (3 ꢁ 50 mL).
The combined organic layers were dried (MgSO4), filtered and
concentrated under vacuum. Silica gel chromatography (70:30
cyclohexane/AcOEt) afforded 6b as a yellow solid (100 mg, 73%).
made in triplicate and then averaged. A curve representing the per-
centage of reduction of ABTSÅ+ as a function of the concentration of
tested compound was then drawn. This allowed to determine the
EC50 (concentration for which the initial absorbance at 740 nm de-
creased by 50%). For greater clarity, the results will be presented on
charts using the term TEAC (Trolox Equivalent Antioxidant Capac-
ity; the lower is this value, the more active is the compound).
4.10.4. b-Carotene bleaching assay
4.9. Methyl (E)-2-(3-hydroxy-4-(4-methoxyphenyl)-5-oxofuran-
2(5H)-ylidene)-2-(3-hydroxyphenyl)acetate (6b)
The method described by Marco32 and modified by Miller33 and
Taga et al.34 was adapted for a microplate lector. In a flask were
introduced 2 mL of a 2 g Lꢀ1 dichloromethane solution of b-caro-
tene), 90 mg of linoleic acid and 240 mg of Tween 40 emulsifier.
The solution was then mixed and evaporated under argon flow.
Oxygen-saturated water (50 mL) was added to the residue and
Yellow solid; mp 180–181 °C; IR (KBr pellet)
2839, 2408, 1746, 1671, 1600, 1475, 1439, 1374, 1310, 1280,
1254, 1182, 1070, 838 cmꢀ1 1H NMR (400 MHz, CDCl3) d 13.56
mmax: 3366, 2958,
;
(1H, s, OH), 8.12 (2H, d, J = 9.1 Hz, CHAr), 7.27 (1H, t, J = 7.9 Hz,
CHAr), 6.97 (2H, d, J = 9.1 Hz, CHAr), 6.86 (1H, ddd, J = 7.9, 2.4,
0.9 Hz, CHAr), 6.81 (1H, ddd, J = 7.9, 1.6, 0.9 Hz, CHAr), 6.74 (1H,
dd, J = 2.4, 1.6 Hz, CHAr), 3.87 (3H, s, OCH3), 3.85 (3H, s, OCH3);
13C NMR (100 MHz, CDCl3) d 171.7, 166.4, 159.7, 158.7, 155.4,
155.1, 133.5, 129.5 (3C), 122.6, 121.7, 117.2, 115.8, 115.0, 114.1
(2C), 105.5, 55.4, 54.5; HRMS (ESI-TOF) calcd for C20H16NaO7
[M+Na]+ 391.0794, found 391.0793.
then the solution was vortexed for 30 s. Aliquots (250
emulsion were transferred to microplates well containing 50
lL) of this
l
L
of pure ethanol antioxidant compound solution. A zero reading
was taken at 470 nm immediately after adding the emulsion to
the antioxidant solution. The reaction was thermostated at 50 °C
and the kinetics was plotted over 4 h. The last point of the measure
was taken after 4 h. (Multiskan FC, Thermofischer microplate
lector).
The antioxidant activity was expressed by the percentage of
inhibition of b-carotene bleaching with regard to the control by
the following equation: P = [1 ꢀ (A0 ꢀ At)/(Ac0 ꢀ Act)] ꢁ 100, where
A0 is the initial absorbance, At is the absorbance after 4 h, Ac0 is
the initial absorbance without antioxidant compound, Act is the
absorbance after 4 h without antioxidant compound. All determi-
nations were made in triplicate and then averaged. The evaluation
4.10. Antioxidant assays
4.10.1. Thymidine protection assay under
exposure, and Fenton oxidation
c-rays, UV/H2O2
These procedures have been previously described.10,13
4.10.2. DPPH radical scavenging capacity assay
was performed using two concentrations (24
tested compound.
lM, 116 lM) of
The DPPHÅ method of Brand-Williams et al.30 was modified for
this assay. The assay was performed in a 96-well plate. A 200
solution of DPPHÅ was prepared in pure ethanol. In each well were
added 100 L of a sample at varying concentrations in ethanol
(0–1 10ꢀ3 M) and 100 L of the DPPHÅ solution. Samples were
lM
Acknowledgments
l
l
Financial support by the Délégation Générale pour l’Armement
(DGA) is gratefully acknowledged. We gratefully thank Marie-
Claire Nevers and Hervé Volland (CEA/DSV/iBiTec-S/SPI) for
providing the anti-thymidine monoclonal antibody and for
experimental support and dr Vincent Favaudon (Institut Curie,
Orsay) for access to the gamma-irradiator.
prepared in duplicate, and ten different concentrations were em-
ployed. The plate was read every 5 min at 515 nm for a period of
1 h, using a spectrophotometer SpectraMax Plus384 (Molecular
Devices). For each concentration, a kinetic curve was plotted. The
percentages of remaining DPPHÅ at the steady state were deter-
mined. Hence, the measurement of antioxidant activity was
possible only for compounds for which the kinetics of reaction
made it possible to reach a steady state before 1 h. Then for each
compound, a curve showing the percentage of remaining DPPHÅ
versus the molar ratio of antioxidant to DPPHÅ was plotted, allow-
ing the determination of the EC50 values (EC50 antioxidant concen-
tration necessary to decrease the initial DPPHÅ concentration by
50%). As recommended by Brand-Williams et al., the results are
presented in the terms of antiradical power (ARP = 1/EC50).
Supplementary data
Supplementary data (physical and spectroscopic characteristics
of synthesized compounds) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2010.09.037.
References and notes
1. Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 3rd ed.;
University Press: Oxford, 1999.
2. Perry, G.; Cash, A. D.; Smith, M. A. J. Biomed. Biotechnol. 2002, 2, 120.
3. (a) Behl, C.; Moosmann, B. Free Radical Biol. Med. 2002, 33, 182; (b) Lusis, A. J.
Nature 2000, 407, 233.
4.10.3. ABTS radical cation decolorisation assay
A 7 mM solution of ABTS salt in water was added to a 140 mM
solution of potassium persulfate to obtain a final concentration of
3.5 mM. The mixture was stirred at room temperature in the dark
for one night to form ABTS radical cation. Before use, the solution
was diluted in pure ethanol so as to obtain an absorbance of 0.70
(0.02) at 740 nm, at 30 °C (Multiskan FC, Thermofischer microplate
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35. log P = logarithm of the partition coefficient (octanol/water); log P indicated
are theoretical values obtained using ChemDraw software.