Antitrypanosomal and antioxidant properties of 4-hydroxycoumarins derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.07.013

In the present communication we prepared a series of six 4-hydroxycoumarin derivatives, isosters of quercetin, recognized as an antioxidant natural compound, with the aim of evaluating the antitrypanosomal activity against Trypanosoma cruzi, the parasite

Full text of DOI:10.1016/j.bmcl.2012.07.013

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5569–5573
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antitrypanosomal and antioxidant properties of 4-hydroxycoumarins
derivatives
Fernanda Pérez-Cruz a, Silvia Serra b,c, , Giovanna Delogu c, Michel Lapier a, Juan Diego Maya d,
⇑
Claudio Olea-Azar a, Lourdes Santana b, Eugenio Uriarte b
a Departamento de Química Inorgánica y Analitica, Laboratorio de radicales libres y antioxidantes, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile,
Casilla 233, Santiago 1, Chile
b Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
c Dipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
d Departamento de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present communication we prepared a series of six 4-hydroxycoumarin derivatives, isosters of
quercetin, recognized as an antioxidant natural compound, with the aim of evaluating the antitrypanos-
omal activity against Trypanosoma cruzi, the parasite responsible for Chagas disease, and the antioxidant
properties. We have used the 4-hydroxycoumarin moiety (compound 1) as the molecular template for
the synthesis of compounds 2–7. These derivates have shown moderate trypanocidal activity. However
they have been proved to be good antioxidants. In particular compound 7 is the most active antioxidant
and it is, therefore, a potential candidate for a successful employment in conditions characterized by free
radicals overproduction.
Received 26 May 2012
Revised 30 June 2012
Accepted 4 July 2012
Available online 13 July 2012
Keywords:
4-Hydroxycoumarins
Trypanosoma cruzi
Chagas disease
Ó 2012 Elsevier Ltd. All rights reserved.
Antioxidant properties
Phenolic compounds are one of the major families of secondary
metabolites in plants and they include a broad group of molecules.
They have shown important properties such as antioxidant and can
act protecting from degenerative diseases in which reactive oxygen
species (ROS) are involved.1–4 These compounds contain one or
more aromatic benzene rings with one or more hydroxyl groups
and their properties are related to their chemical structure. Cou-
marins are one of the most abundant molecules of naturally occur-
ring poliphenolic compounds. A lot of them have been identified
from natural sources, especially from green plants. Due to their
structural features they are important building blocks in the natu-
ral product and synthetic chemistry areas. Coumarins are recog-
nized to possess anti-inflammatory, antioxidant, antiallergic,
hepatoprotective, antithrombotic, antiviral, enzymatic inhibition
and anticarcinogenic properties.5–13 The hydroxycoumarins are
typical phenolic compounds and can act as potent metal chelators
and free radical scavengers.14,15 Flavonoids are another important
group of natural phenolic compounds that are known for their bio-
logical activities, including the important antioxidant properties.16
The quercetin is one of the most powerful and effective antioxidant
flavonoid present in nature.17
Antioxidants are a class of substances of many diverse chemical
structures that are capable of decrease or prevent oxidation of
other sensitive molecules through different mechanisms like che-
lation of active metal ions, free radicals scavenging or inhibition
of pro-oxidant enzymes.18 Free radicals are very unstable mole-
cules responsible of aging, cellular membrane and DNA damage,
and possibly serious diseases like cancer and heart condition. It
has been found that many hydroxycoumarins affect the formation
and scavenging of ROS, exhibiting antioxidant tissue-protective
properties.19,20
Furthermore ROS produce oxidative stress that contributes to
Chagas disease progression.21 This illness, known also as American
Trypanosomiasis, is caused by the protozoan parasite, Trypanosoma
cruzi (T. cruzi).22 It is a serious threat to health in Central and South
America, and it is one of the most important emerging parasitic
disease in developed countries.23,24 The parasite has a complex life
cycle, which involves obligatory passage through four stages.25,26
The infection is transmitted to humans predominantly by insect
vector, blood transfusion or by transmission from mother to fetus.
Current treatment is based on old and quite unspecific drugs: nif-
urtimox (Nfx) and benznidazole (Bzn). In fact this therapy is highly
toxic and usually ineffective in the chronic stages.27–30 During the
course of T. Cruzi infection and disease development, ROS can be
produced as a consequence of tissue destruction caused by toxic
secretions of parasite, immune-mediated cytotoxic reactions, and
⇑
Corresponding author.
E-mail address: silvserra@tiscali.it (S. Serra).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.013

5570
F. Pérez-Cruz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5569–5573
secondary damage in the myocardium. Therefore, interventions
with antioxidant compounds, that reduce the generation or the ef-
fects of ROS, may exert beneficial effects in preventing or arresting
oxidative damage.31
Given the known antioxidant properties attributed to the cou-
marin skeleton and to the flavonoid compounds like quercetin
(Fig. 1), which presents a hydroxyl group in the pyron ring, in
the present work a series of six 4-hydroxycoumarin derivatives
were synthesized (compounds 2–7).
In fact these compounds can be considered isosters of the men-
tioned flavonoid. We have used the 4-hydroxycoumarin (com-
pound 1) as the molecular template in the design and the
synthesis of these derivatives that present also an aryl group at
C3 (Fig. 1).
OH
O
O
R1
I
R1
(a)
O
O
O
I: R1 = H,
: R1 = Cl
II
R2
R3
(b)
B(OH)2
R2
R3
Hydroxy, methoxy, methyl and/or chloro substituents were
introduced in the 3-phenyl ring, whereas the six position in the
coumarin moiety was not substituted/or substituted with a chloro
atom.
OH
O
2: R1 = H, R2 = H, R3 = H
R1
3: R1 = H, R2 = OMe, R3 = H
: R1 = H, R2 = Cl, R3 = H
: R1 = H, R2 = Me, R3 = H
: R1 = H, R2 = H, R3 = OH
: R1 = Cl, R2 = H, R3 = OH
4
5
6
7
O
Then the antitrypanosomal activity against Trypanosoma cruzi
and the antioxidant properties of compounds 1–7 were assessed.
The key step for the synthesis of the 3-arylcoumarin skeleton
was achieved by a palladium-catalyzed Suzuki coupling reaction
between phenyliodonium zwitterion salts and the conveniently
substituted phenyl boronic acids.32–36 Initially we have synthe-
sized the different phenyliodonium coumarinate species (I–II) that
are electrophilic molecules with a positive charge at iodine atom
delocalized to the neighboring oxygen.35 Then we have carried
out the palladium-catalyzed coupling reaction using Pd(OAc)2 as
catalyst and P(t-Bu)3 as ligand to afford the compounds 2–7 in
good yields.32,33,36
Scheme 1. Reagents and conditions: (a) PhI(OAc)2, Na2CO3, H2O, rt, 14 h; (b)
Pd(OAc)2, P(t-Bu)3, LiOH, DME/H20, rt, 24–48 h.
Table 1
% Trypanocidal activity results for the compounds 1–7 and nifurtimox (Nfx)
Compounds
% Trypanocidal activity at (10 l )
mol Lꢀ1
a
1
2
3
4
5
6
7
Nfx
21.1 0.4
1.6 0.2
30.7 2.2
16.7 6.9
The reaction conditions are delineated in the Scheme 1.
The antitrypanosomal activity for all synthesized compounds
was evaluated using MTT assays.37,38
3.9 3.3
The results reported in Table 1 have shown that derivatives 2, 4,
and 5 respectively with any substituent, a chlorine atom or a
methyl group at p-position in the 3-phenyl ring present antitry-
panosomal activity. However, these compounds have less activity
compared with positive control nifurtimox. On the other hand
the simple 4-hydroxycoumarin (compound 1) and compounds 3,
6 and 7 with methoxy or hydroxyl substituents in the 3-phenyl
ring do not show any activity.
In the present series is clearly observed how the absence of the
3-phenyl ring or the introduction of methoxy or hydroxyl groups in
this causes a considerably decrease of antitrypanosomal activity.
On the other hand the activity improves enormously in presence
of a p-chloro atom in the 3-phenyl ring of the 4-hydroxycoumarin.
In fact the compound 4 that presents this substituent is the best of
a
52.5 2.2
a
Inactive at concentration tested.
the evaluated series with around 30% of trypanocidal activity at
10
mol Lꢀ1
These preliminary findings and especially the antitrypanosomal
capacity found for compound 4, encourage us to the future struc-
tural optimization of these compounds.
The capacity of scavenging peroxyl radicals was studied
through the oxygen radical absorbance capacity method using
fluorescence-based technology of detection measurements
(ORAC-FL).39,40 This assay gives an relative index refering to the
hydrosoluble standard molecule (trolox), a vitamine E derivative.
Results are expressed as ORAC values and are tabulated in Table
2.41 They take account the induction time, initial rate and the range
of total inhibition obtained for each value.42
l
.
OH
O
O
OH
OH
The oxidation occurs due to exposition of the fluorophore, in
this case fluorescein (FL), to the peroxyl radical leading to decay
of fluorescence emission through time.40 In ORAC assays, the loss
OH
OH
HO
O
O
Table 2
1
compound
quercetin
ORAC values and (%) scavenging of hydroxyl radical calculated for compounds 1–7
R'
OH
Compounds
ORAC values
% scavenging
R
1
2
3
4
5
6
7
4.2 0.26
4.4 0.16
5.7 0.30
3.9 0.15
6.5 0.23
4.9 0.35
7.7 0.54
61.9 3.4
45.1 2.6
46.3 2.5
31.3 4.7
34.6 4.5
40.2 3.4
100 4.5
O
O
4-hydroxycoumarin derivatives
Figure 1. Chemical structure of compound 1, quercetin and 4-hydroxycoumarin
derivatives.

F. Pérez-Cruz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5569–5573
5571
of fluorescence of fluorescein generally shows an induction time
and is reliant on the oxidation potential of antioxidant molecules.
It refers to the time in which the FL is protected against peroxyl
radicals in presence of antioxidant molecules. This behavior is
associated to competitive reaction between radical both probe
and antioxidant.43 We obtained profile of fluorescence measure
at 528 nm versus the incubation time at different concentration
for all 4-hydroxycoumarin derivatives. Then the area under the
curve (AUC) was calculated for all studied compounds.
The highest ORAC values are found for compounds 5 and 7 (6.5
and 7.7, respectively). The results are comparable with quercetin
(7.28) and catechin (6.76), used as reference compounds.39
Figure 2a shows the kinetic profiles obtained in presence of
increasing concentration of compound 7. For all coumarin deriva-
tives the area under the curve (AUCNET) of the kinetic profiles de-
pend on the concentration of the additive as it is presented in
Figure 2b.
The different ORAC values are related to the substituents pres-
ent in the coumarin skeleton. We have used the commercially 4-
hydroxycoumarin, compound 1, as patron molecule. When it was
added a phenyl group at 3 position (compound 2), the delocaliza-
tion of semiquinone radical improved, influencing ORAC value. In
the compound 3, in which the phenyl ring presents a p-methoxy
group, and in the compound 5 with a p-methyl group, an incre-
ment in the antioxidant capacity was observed. This fact can be
explained taking into account the strong electron donating effect
that increases the electronic density around the hydroxyl group,
favoring the hydrogen atom transfer mechanism (HAT). On the
contrary in the compound 4, that presents as substituent on its
phenyl ring an electron withdrawing group like a chloro atom,
the antioxidant capacity decreases. The positive effect, although
smaller, is observed for the compound 6 with a m-hydroxyl group
on the 3-phenyl ring. This substituent, together with one chlorine
atom on the 6 position of the coumarin skeleton leads to the com-
pound 7, which resulted to be the best of the evaluated series.
So it has been found that the presence, the number and the po-
sition of electron donating groups (hydroxyl, methoxy or methyl)
on the 3-phenyl ring and the presence of an electron withdrawing
group (chlorine atom) at 6 position on the coumarin skeleton are
important structural factors that contribute to the increment of
the antioxidant capacity of these 4-hydroxycoumarins.
In order to study the antioxidant reactivity of all 3-phenyl-4-
hydroxycoumarin derivatives synthesized against hydroxyl radi-
cals, we have adapted a non-catalytic and competitive Fenton sys-
tem in which the spin trap DMPO and the antioxidant molecules
compete for hydroxyl radicals.44,45
The ESR spectrum shows four hyperfine lines, due to the DMPO-
OH adduct formation. The intensity of the spectrum decreases
(a)
4000
(b)
3500
3000
2500
2000
1500
1000
500
0
-500
0,0
5,0x10-7
1,0x10-6
1,5x10-6
2,0x10-6
Compound 7 / M
Figure 3. (a) ESR spectrum for adduct DMPO-OH without antioxidant molecule
(black) and in presence of compound 1 (red); (b) ESR spectrum for adduct DMPO-
OH without antioxidant molecule (black) and in presence of compound 7 (red).
Figure 2. (a) ORAC profile for compound 7; (b) graphic AUCNET versus concentra-
tion compound 7.

5572
F. Pérez-Cruz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5569–5573
19. Symeonidis, T.; Chamilos, M.; Hadjipavlou-Litina, D. J.; Kallitsakis, M.; Litinas,
K. E. Bioorg. Med. Chem. Lett. 2009, 19, 1139.
20. Nishiyama, T.; Ohnishi, J.; Hashiguchi, Y. Biosci. Biotechnol. Biochem. 2001, 65,
1127.
when a 3-aryl-4-hydroxycoumarin derivative is added into the sys-
tem. Figure 3a shows the experiment carried out for the compound
1 in which a 61.9% of scavenging hydroxyl radicals was obtained. In
the spectrum of Figure 3b we can observe for the compound 7 a to-
tal scavenging. This response was observed for all derivatives and
the percentage of the hydroxyl radical scavenging activity is illus-
trated in Table 2.41 The order of best reactivity against hydroxyl
radical was 7 > 1 > 3 > 2 > 6 > 5 > 4.
In conclusion, the values obtained in the study for the antitry-
panosomal activity have not shown experacted results. Only com-
pounds 2, 4, and 5 have an appreciable activity but still lower than
nifurtimox, used in the treatment of Chagas disease. We can use
these results for a structural optimization of these series of com-
pounds. On the other hand, we have confirmed the good antioxi-
dant activity of 4-hydroxycoumarin derivatives. Their antioxidant
activity is significantly affected by the introduction of a phenyl
moiety at the C3 position. Also when a substituent with electron
donating effect is present in the phenyl group, the antioxidant
capacity increases. The results of the antioxidant assay using ESR
showed higher reactivity against hydroxyl radical for compound
1 and 7. A very interesting finding is that derivative 7 is very reac-
tive and present good antioxidant capacity against hydroxyl and
peroxyl radicals. Based of these results, we can conclude that the
compound 7 is a potential candidate for a successful employment
in conditions characterized by an overproduction of free radicals.
21. De Oliveira, T. B.; Pedrosa, R. C.; Filho, D. W. Int. J. Cardiol. 2007, 116, 357.
22. El Sayed, N. M.; Myler, P. J.; Bartholomeu, D. C., et al Science 2005, 309, 409.
23. Schofield, C. J.; Jannin, J.; Salvatella, R. Trends Parasitol. 2006, 22, 583.
24. Lepesheva, G. I.; Zaitseva, N. G.; Zhou, W.; Liu, J.; Hill, G. C.; Warterman, M. R. J.
Biol. Chem. 2006, 281, 3577.
25. Bern, C.; Montgomery, S. P.; Herwaldt, B. L.; Rassi, A., Jr.; Marin-Neto, J. A.;
Dantas, R. O.; Maguire, J. H.; Acquatella, H.; Morillo, C.; Kirchhoff, L. V.; Gilman,
R. H.; Reyes, P. A.; Salvatella, R.; Moore, A. C. J. Am. Med. Assoc. 2007, 298, 2171.
26. Castro, J. A.; de Mecca, M. M.; Bartel, L. C. Hum. Exp. Toxicol. 2006, 25, 471.
27. Jakson, Y.; Alirol, E.; Jetaz, L.; Wolff, H.; Combescure, C.; Chappuis, F. Clin. Infect.
Dis. 2010, 51, e69.
28. Perez-Molina, J. A.; Perez-Ayala, A.; Moreno, S.; Fernandez-Gonzalez, M. C.;
Zamora, J.; Lopez-Velez, R. J. Antimicrob. Chemother. 2009, 64, 1139.
29. Bern, C. N. Eng. J. Med. 2011, 364, 2527.
30. Cerecetto, E.; Gonzalez, M. Curr. Top. Med. Chem. 2006, 22, 583.
31. Gupta, S.; Wen, J. J.; Garg, N. J. Oxidative Stress in Chagas Disease. Interdiscip.
Perspect. Infect. Dis. 2009, 190354.
32. Zhu, Q.; Wu, J.; Fathi, R.; Yang, Z. Org. Lett. 2002, 4, 3333.
33. Goldoni, L.; Cravotto, G.; Penoni, A.; Tollari, S.; Palmisano, G. Synlett 2005, 6,
927.
34. Serra, S.; Ferino, G.; Matos, M. J.; Vázquez-Rodríguez, S.; Delogu, G.; Viña, D.;
Cadoni, E.; Santana, L.; Uriarte, E. Bioorg. Med. Chem. Lett. 2012, 22, 258.
35. General procedure for the preparation of 3-phenyliodonium coumarinates (I–II):
iodobenzene diacetate (10 mmol) was suspended in a solution of Na2CO3
(10 mmol) in water (100 mL) and was stirred for 30 min at room temperature.
To this solution was added a mixture of the corresponding 4-hydroxycoumarin
(10 mmol) and Na2CO3 (10 mmol) in water (100 mL). After the mixture was
stirred at room temperature for 14 h, the precipitate was collected by filtration,
washed with water (5 ꢁ 20 mL) and dried under vacuum. The resulting white
solid was used without further purification.
36. General procedure for the preparation of 3-aryl-4-hydroxycoumarins (2-7): a
degassed solution of appropriated phenyl boronic acid (1.21 mmol) and P(t-
But)3 (0.109 mmol) in DME and H2O (4:1, 12.5 mL) was added to a mixture of
iodonium ylide (0.55 mmol), LiOH/H2O (1.65 mmol) and Pd(OAc)2
(0.027 mmol) under argon at room temperature. After being stirred at the
same temperature for 24–48 h. The resulting mixture was purified by FC
(hexane/ethyl acetate, 7:3) to give the desired compound.
Acknowledgements
Thanks to Italian Ministry (PRIN 2008, F21J10000010001),
Spanish Ministry (PS09/00501) and Xunta de Galicia
(09CSA030203PR). S. Serra thanks Regione Sardegna for the grant
(PR-MAB-A2009-613), Fernanda Pérez-Cruz gratefully acknowl-
edges CONICYT-Chile for Doctoral fellowship and Scholarship sup-
port for doctoral theses CONICYT-Chile N° 24110059. Claudio Olea-
Azar acknowledges to FONDECYT (Chile) for project 1110029. Juan
Diego Maya acknowledges to FONDECYT 1090078 and Anillo ACT
112.
4-Hydroxy-3-(30-hydroxyphenyl)coumarin (6). It was obtained with yield 58%.
Mp: 265–267 °C. 1H NMR (DMSO-d6) d (ppm): 6.76 (m, 2H, H40, H60), 7.41 (m,
5H, H6, H7, H8, H20, H50), 7.94 (s, 1H, H5). 13C NMR (DMSO-d6) d (ppm): 80.3,
93.0, 101.2, 106.7, 115.1, 116.7, 118.3, 122.1, 124.2, 124.5, 129.5, 132.8, 152.7,
157.5, 160.1. MS m/z (%): 254 (M+, 48), 134 (26), 121 (100), 65 (28). Anal. Calcd
for C15H10O4: C, 70.86; H, 3.96. Found: C, 70.88; H, 3.98.
6-Chloro-4-hydroxy-3-(30-hydroxyphenyl)coumarin (7). It was obtained with
yield 69%. Mp: 283–285 °C. 1H NMR (DMSO-d6) d (ppm): 6.76 (t, J = 7.1 Hz, 2H,
H40, H60), 7.14–7.25 (m, 1H, H20), 7.37–7.48 (m, 1H, H8), 7.64–7.80 (m, 2H, H7,
H50), 7.96 (s, 1H, H5). 13C NMR (DMSO-d6) d (ppm): 92.1, 107.3, 115.2, 118.7,
121.9, 123.3, 128.4, 129.4, 132.3, 151.3, 152.5, 157.4, 159.4, 161.8, 164.9. MS
m/z (%): 288 (M+, 65), 155 (63), 154 (26), 134 (100). Anal. Calcd for C15H9ClO4:
C, 62.41; H, 3.14. Found: C, 62.39; H, 3.12.
References and notes
1. Tyagi, Y. K.; Kumar, A.; Raj, H. G.; Vohra, P.; Gupta, G.; Kumari, R.; Kumar, P.;
Gupta, R. K. Eur. J. Med. Chem. 2005, 40, 413.
2. Hamdi, N.; Puerta, C.; Valerga, P. Eur. J. Med. Chem. 2008, 43, 2541.
3. Panteleon, V.; Kostakis, I. K.; Marakos, P.; Pouli, N.; Andreado, I. Bioorg. Med.
Chem. Lett. 2008, 18, 5781.
4. Guardado-Yordi, E.; Pérez-Molina, E.; Matos, M. J.; Uriarte-Villares, E. In
Nutrition, Well-Being and Health; Bouayed, J. and Bohn, T. Eds.; InTech, 2012,
Chapter 2.
5. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.
2005, 12, 887.
6. Borges, F.; Roleira, F.; Milhazes, N.; Uriarte, E.; Santana, L. Front Med. Chem.
2009, 4, 23.
7. Matos, M. J.; Terán, C.; Pérez-Castillo, Y.; Uriarte, E.; Santana, L.; Viña, D. J. Med.
Chem. 2011, 54, 7127.
8. Matos, M. J.; Santana, L.; Uriarte, E.; Delogu, G.; Corda, M.; Fadda, M. B.; Era, B.;
Fais, A. Bioorg. Med. Chem. Lett. 2011, 21, 3342.
9. Viña, D.; Matos, M. J.; Yáñez, M.; Santana, L.; Uriarte, E. Med. Chem. Commun.
2012, 3, 213.
10. Secci, D.; Carradori, S.; Bolasco, A.; Chimenti, P.; Yáñez, M.; Ortuso, F.; Alcaro, S.
Eur. J. Med. Chem. 2011, 46, 4846.
11. Ostrov, D. A.; Hernández Prada, J. A.; Corsino, P. E.; Finton, K. A.; Le, N.; Rowe, T.
C. Antimicrob. Agents Chemother. 2007, 51, 3688.
12. Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Detsi, A.; Koufaki, M.;
Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Eur. J. Med. Chem. 2009, 44, 3020.
13. Viña, D.; Matos, M. J.; Ferino, G.; Cadoni, E.; Laguna, R.; Borges, F.; Uriarte, E.;
Santana, L. ChemMedChem 2012, 7, 464.
14. Fylaktakidou, K. C.; Hadjipavlou-Litina, D. J.; Litinas, K.; Nicolaides, D. N. Curr.
Pharm. Des. 2004, 10, 3813.
37. Muelas, S.; Suárez, M.; Pérez, R.; Rodríguez, H.; Ochoa, C.; Escario, J. A.; Gómez-
Barrio, A. Mem. Inst. Oswaldo Cruz 2002, 97, 269.
38. Trypanocidal activity was evaluated against the T. cruzi trypomastigote stage
(clone Dm28c). It was measured through the MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5 diphenyltetrazolium bromide) assay, using 0.22 mg mLꢀ1 phenazine
metosulfate (as electron carrier). In this colorimetric assay for testing the
antitrypanosomal activity, the 3-aryl-4-hydroxycoumarin derivatives,
dissolved in DMSO were added to 107 parasites mLꢀ1 at 10 mol Lꢀ1 final
l
concentration for 24 h at 37 °C. DMSO final concentration was less than 0.1% v/
v. Likewise, nifurtimox was added as positive control. After incubation time,
we determined the number of viable parasites by absorbance measures at
570 nm in a microplate spectrophotometer.
39. Ou, B.; Hampsch-Woodill, M.; Prior, R. L. J. Agric. Food Chem. 2001, 49, 4619.
40. The ORAC analyses were carried out on
a Synergy HT multi detection
microplate reader, from Bio-Tek Instruments, Inc. (Winooski, USA), using
white polystyrene 96-well plates, purchased from Nunc (Denmark).
Fluorescence was read from the top, with an excitation wavelength of 485/
20 nm and an emission filter of 528/20 nm. The plate reader was controlled by
Gen 5 software. The reaction was carried out in 75 mM sodium phosphate
buffer (pH 7.4), and 200
aryl-4-hydroxycoumarin solutions in methanol with a range of concentration
between 0.3 M and 2 M were placed in each well of 96-well plate. The
lL final volume. FL (40 nM, final concentration) and 3-
l
l
mixture was preincubated for 15 min at 37 °C, before rapidly adding the AAPH
solution (18 mM, final concentration). The microplate was immediately placed
in the reader and automatically shaken prior to each reading. The fluorescence
was recorded every 1 min for 120 min. A blank with FL and AAPH using
methanol instead of the antioxidant solution and five calibration solutions
15. Kostova, I. Mini-Rev. Med. Chem. 2006, 6, 365.
using Trolox (0.5 lM to 2.5 lM) as antioxidant were also used in each assay.
16. Procházková, D.; Boušová, I.; Wilhelmová, N. Fitoterapia 2011, 82, 513.
17. Coßskun, Ö.; Kanter, M.; Armutçu, F.; Çetin, K.; Kaybolmaz, B.; Yazgan, Ö. Eur. J.
Gen. Med. 2004, 1, 37.
The inhibition capacity was expressed as Equivalents Trolox (Tequiv), and is
quantified by integration of the area under the curve (AUCNET). All reaction
mixtures were prepared in triplicate and at least three independent assays
18. Nordberg, J.; Arnér, E. S. Free Radic. Biol. Med. 2001, 31, 1287.

F. Pérez-Cruz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5569–5573
5573
were performed for each sample. The area under the fluorescence decay curve
42. Niki, E. Free Radical Biol. Med. 2010, 49, 503.
(AUC) was calculated integrating the decay of the fluorescence where F0 is the
initial fluorescence read at 0 min and F is the fluorescence read at time. The net
AUC corresponding to the sample was calculated by subtracting the AUC
corresponding to the blank. Data processing was performed using Origin Pro 8
SR2 (Origin Lab Corporation, USA).
43. Bisby, R. H.; Brooke, R.; Navaratnam, S. Food Chem. 2008, 108, 1002.
44. Yoshimura, Y.; Inomata, T.; Nakazawa, H.; Kubo, H.; Yamaguchi, F.; Ariga, T. J.
Agric. Food Chem. 1999, 47, 4653.
45. We mixed 150
concentration 3 mM) follow by addition of 50
final concentration) and finally 50 L Hydrogen peroxide 30%. The mixture was
lL
of N,N-dimethylformamide with 50
lL NaOH (final
lL DMPO spin trap (30 mM
41. Statistical analyses were conducted using GraphPad Prism
5
software
l
(GraphPad Software, Inc. San Diego, CA). The data are expressed as
means SD. The experimental data were analyzed by one-way analysis of
variance (ANOVA), and differences between groups were assessed using
put in EPR Cell and we recorded spectrum after five minutes of reaction. All
derivatives were studied to 1 mM final concentration.
Tukey’s post-test. The level of significance was set at
experiments were replicated three times.
p <0.05, and all