Antioxidant capacity of 2-(3,5-diaryl-4,5-dihydro-1h-pyrazol-1-yl)-4- phenylthiazoles

Article abstract of DOI:10.2174/157018010792929513

The antioxidant capacity of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazoles was evaluated. The values of antioxidant capacities of compounds 2d and 2e were found to be, respectively, 2,700 ± 150 and 3,135 ± 230 TE by the ORAC method, correspo

Full text of DOI:10.2174/157018010792929513

Letters in Drug Design & Discovery, 2010, 7, 657-660
657
Antioxidant Capacity of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazoles
F.A.N. Silvaa,b, L. Pizzutic,d,f, F.H. Quinac,d, S.P. Souzad, P.F. Rosalese, G.M. Siqueirae,
C.M.P. Pereira*,b,d,e, S.B.M. Barros*,b and D.P. Rivelli*,b
aLaboratório de Patologia, Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas,
Universidade de São Paulo, 05508-000, São Paulo, SP, Brazil
bLaboratório de Análises Toxicológicas, Departamento de Farmácia, Faculdade de Ciências Farmacêuticas,
Universidade de São Paulo, 05508-000, São Paulo, SP, Brazil
cInstituto de Química, Universidade de São Paulo, 05513-970, São Paulo, SP, Brazil
dCentro de Capacitação e Pesquisa em Meio Ambiente, Universidade de São Paulo, 11573-000, Cubatão, SP, Brazil
eDepartamento de Química e Geociências, Universidade Federal de Pelotas, 96010-900, Pelotas, RS, Brazil
fFaculdade de Ciências Exatas e Tecnologia, Universidade Federal da Grande Dourados, 79804-970, Dourados, MS,
Brazil
Received April 04, 2010: Revised June 02, 2010: Accepted July 11, 2010
Abstract: The antioxidant capacity of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazoles was evaluated. The
values of antioxidant capacities of compounds 2d and 2e were found to be, respectively, 2,700 ± 150 and 3,135 ± 230 TE
by the ORAC method, corresponding to a significant antioxidant capacity.
Keywords: Antioxidant, Antioxidant synthetic, Green chemistry, Thiazole, Trolox, ORAC.
INTRODUCTION
recent publication, we reported the antioxidant capacity of
phenylpyrazole using the ORAC method [14]. In continua-
tion of our research program, we report here the antioxidant
evaluation of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazoles, synthetic derivatives compounds of facile
preparation (Scheme 1).
Thiazole derivatives possess a broad spectrum of biologi-
cal activities, which have attracted much recent attention
from medicinal chemists [1-5]. Several drugs containing the
thiazole nucleus, such as blenoxane, bleomycine and tiazo-
furin, are known antineoplastic agents [6].
The antioxidant capacity of several thiazolyl compounds
has also been reported [7-9]. Recently, antioxidants have
become a topic of increasing interest [10-13]. Characteriza-
tion of the antioxidant capacity of natural and synthetic
compounds with potential pharmacological activity is of
great interest to medical and nutritional experts, to health and
food science researchers and to the public in general [10-12].
Although there is no total antioxidant capacity assay based
on a single, easily executable chemical reaction, there are
numerous published methods that claim to measure total
antioxidant capacity in vitro [10-12]. The ORAC method for
determining antioxidant capacity uses: (a) AAPH (an azo
radical initiator) which degrades the fluorescence of the
probe; (b) the molecular probe fluorescein (FL) for monitor-
ing the reaction progress and (c) the antioxidant of interest.
In the presence of an antioxidant, the decrease in fluores-
cence is inhibited. The advantage of the ORAC approach is
that it provides kinetics parameters (area under the curve)
equally well for antioxidants that exhibit distinct lag phases
and for those samples that have no lag phases [10-12]. In
RESULTS AND DISCUSSION
A preliminary ORAC assay screening was carried out to
determine which compounds exhibited the most significant
antioxidant capacity. This led to the selection of 2-(5-(3-
nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-phen-
ylthiazole (2d) and 2-(5-(4-methoxyphenyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (2e) for more
careful quantification Table 1, because the other compounds
did not show measurable antioxidant potential by the ORAC
method.
O
N
Br
Ar
N
N
Ph
Ar
N
KOH
N
S
H2N
S
Ph
*Address correspondence to these authors at the Laboratório de Análises
Toxicológicas, Departamento de Farmácia, Faculdade de Ciências Farma-
cêuticas, Universidade de São Paulo, 05508-000, São Paulo, SP, Brazil;
Tel: 55 11 30913631, +55 1138132197; Fax: 55 11 38132197, +55
1138132197; E-mails: smbarros@usp.br, diogopineda@gmail.com,
claudio.martin@pq.cnpq.br
(1)
(2)
Scheme 1. Preparation of 4-phenylthiazole 2a-2e.
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

658 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 9
Silva et al.
Table 1. Selected Experimental Compounds (2)
Compound 2a
Compound 2b
Compound 2c
Compound 2d*
Compound 2e*
O2N
N
S
N
N
N
N
N
S
N
N
N
N
MeO
Ph
N
N
S
N
S
N
N
S
*Compound with antioxidant capacity.
The values of antioxidant capacities of 2-(5-(3-nitro-
phenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthi-
azole (2d) and 2-(5-(4-methoxyphenyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (2e) were found
to be, respectively, 2,700 ± 150 and 3,135 ± 230 μmol eq.
Trolox/g (TE) by the ORAC method (Table 2), correspond-
ing to quite good antioxidant capacity (Trolox itself is 4000
μmol/g). Of course, it should be kept in mind that in vitro
antioxidant assays only represent the observed response in
the given reaction medium, meaning that the values of the
apparent antioxidant activities might also be influenced by
differences in the chemical reaction between the free radical
used in the assay and the substance being tested.
(25 ꢁL of solvent + 150 ꢁL of 40 nM fluorescein + 25 ꢁL
AAPH) to obtain the net area under the curve (net AUC) for
each sample.
Table 2. ORAC Antioxidant Capacity as the Concentration
of Compound 2d and 2e (Values were Expressed as
Mean ± Standard Deviation)
Compound
Antioxidant Capacity
Correlation
Coefficient (r2)
2d
2e
2,700 ꢀꢁ150 μmol eq. trolox/g
3,135 ꢀꢁ230μmol eq. trolox/g
0,9722
0,9849
Using the Trolox® calibration curve, expressed as net
area under the curve versus concentration (ꢁM), the antioxi-
dant capacity of each sample can be expressed in terms of its
ꢁmol equivalents of Trolox/g ± standard deviationor
Trolox® equivalent (TE) (Table 2). This procedure was car-
ried out in triplicate for each sample at each concentration.
Thus, higher values of TE mean higher antioxidant capacity
of the sample.
ORAC Methodology, Antioxidant Evaluation
This method was employed because it uses a physiologi-
cal relevant free radical as well as its reaction medium. The
automatic ORAC assay was described by Ou et al. [10]. In
this method, an azo initiator (AAPH) decomposing at 37°C
abstracts hydrogen from sodium fluorescein, reducing its
fluorescence (485/20 nm excitation filter and 528/20 nm
emission filter). When a test compound with antioxidant
capacity is added, the reaction with fluorescein and the resul-
tant decrease in its fluorescence are delayed until the anti-
oxidant capacity of the test compound is completely ex-
hausted. The putative antioxidants were solubilized in ace-
tone and diluted in 7% randomly methylated ꢀ-cyclodextrin
solution. After appropriate dilution, 25 ꢁL of these solutions
were transferred to microplates in triplicate and 150 ꢁL of 40
nM fluorescein diluted in 75mM phosphate buffer pH 7.0,
were added. On the same microplate a control (25 ꢁL of sol-
vent + 150 ꢁL of 40 nM fluorescein) and a Trolox (standard)
curve (150 ꢁL fluorescein 40 nM + 25 ꢁL aliquot from a
Trolox® solution of known concentration) were made. To
maintain the plate temperature, 300 ꢁL of water were added
around the wells. After the incubation (37°C/30 min), 25 ꢁL
of AAPH (153 mM in 75 mM phosphate buffer, pH 7.0)
were added and the plate shaken during 10 seconds at maxi-
mum intensity. The plate reader (Synergy – BIOTEK Multi-
detection microplate reader, Winooski, VT) was pro-
grammed to record the fluorescence at each cycle from 1
minute after addition of AAPH up to 60 minutes and the area
under the fluorescence curve integrated over time by using
Gen5 software. All area values were corrected for the control
Fig. (1). Absorbance (nm) of compounds 2d and 2e in acetone.
Finally, care was taken to demonstrate that compounds
2a - 2e do not interfere with the fluorimetric analysis itself.
All five compounds fluoresce at about 450nm when exited

Antioxidant Capacity of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 9 659
below 425nm, where they absorb (Fig. 1). Thus competitive
absorption and emission can be ruled out under the condi-
tions of excitation of fluorescein 485nm. Quenching of the
fluorescence of fluorescein by compounds 2a - 2e was also
ruled out by demonstrating that the effect of addition of up to
a two-fold excess of these compounds in acetone to an aque-
ous solution of fluorescein had the made effect (due merely
to dilution of the solution) as addition of acetone alone.
127.1, 127.3, 127.5, 128.4, 128.7, 128.7, 129.0, 130.0, 140.7,
140.7, 147.5, 154.3, 165.0;
2-[5-(3-Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-4-phenylthiazole (2d): Yield (69%), white solid; mp
172–174°C; IR (KBr): ꢀ (cmꢀ1) 3410, 3246–3060, 1961–
1760, 1596, 1530, 1462, 1442, 1346, 686; 1H NMR (CDCl3,
500 MHz): ꢁ (ppm) 3.36 (dd, 1H, J=7.5 Hz, J=17.5 Hz), 4.00
(dd, 1H, J=12.1 Hz, J=17.5 Hz), 5.75 (dd, 1H, J=7.5 Hz,
J=12.1 Hz), 6.86 (s, 1H), 7.21–8.16 (m, 14H, ArH); 13C
NMR (CDCl3, 126 MHz): ꢁ (ppm) 43.2, 64.1, 103.9, 122.3,
122.8, 125.7, 126.4, 127.6, 128.5, 128.8, 129.7, 130.1, 131.0,
132.8, 134.6, 148.2, 151.4, 151.5, 164.9;
A it empts were made to quantify the antioxidant capac-
ity by the DPPH method but, unfortunately, due to the lack
of solubility of these compounds in the reaction media tested
(methanol, ethanol and isopropanol), this was not possible.
2-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl]-4-phenylthiazole (2e): Yield (81%), yellow
solid; mp 182–184°C; IR (KBr): ꢀ (cmꢀ1) 3123–2833, 1959–
Synthesis and Structure Confirmation
The 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyra-
zoles (1) were prepared according literature [15]. The 2-(3,5-
diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazoles (2)
were prepared from the 1-thiocarbamoyl-4,5-dihydro-1H-
pyrazole (1) by reaction with phenacyl bromine in ethanol in
the presence of potassium hydroxide (Scheme 1) [16].
1
1647, 1544, 1515, 1443, 1249, 789, 672; H NMR (CDCl3,
500 MHz): ꢁ (ppm) 3.29 (dd, 1H, J=6.6 Hz, J=17.4 Hz), 3.75
(s, 3H), 3.83 (dd, 1H, J=12.0 Hz, J=17.4 Hz), 5.61 (dd, 1H,
J=6.6 Hz, J=12.0 Hz), 6,78 (s, 1H), 6.84–7.76 (m, 14H,
ArH); 13C NMR (CDCl3, 126 MHz): ꢁ (ppm) 43.3, 55.2,
64.1, 103.3, 114.0, 125.8, 126.3, 127.4, 127.9, 128.3, 128.6,
129.6, 131.5, 133.8, 135.0, 151.4, 151.5, 159.0, 164.9.
The structure of compounds (2) were confirmed by NMR
and mass spectra. NMR spectra were recorded on a Bruker
1
DPX 500 spectrometer (500 MHz for H and 125 MHz for
13C) at 300 K. Low resolution mass spectra were obtained on
a Varian Saturn 2200 GC/MS spectrometer operating at 70
eV.
CONCLUSIONS
In conclusion, 2-(5-(3-nitrophenyl)-3-phenyl-4,5-dihy-
dro-1H-pyrazol-1-yl)-4-phenylthiazole (2d) and 2-(5-(4-
methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (2e) showed good antioxidant capacity com-
pared comparable to that of Trolox®. More investigations are
currently in progress to explore the reaction and relationship
structure-activity of 4-phenylthiazoles.
Selected Experimental Data for Compounds 2a–e
2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (2a): Yield (70%); yellow solid; mp 213–
215°C; IR (KBr): ꢀ (cmꢀ1) 3115–3028, 1953–1756, 1541,
1
1520, 1492, 1443, 707, 694; H NMR (CDCl3, 500 MHz): ꢁ
(ppm) 3.33 (dd, 1H, J=6.7 Hz, J=17.4 Hz), 3.90 (dd, 1H,
J=12.0 Hz, J=17.4 Hz), 5.68 (dd, 1H, J= 6.7Hz, J=12.0 Hz),
6.80 (s, 1H), 7.20–7.77 (m, 15H, ArH); 13C NMR (CDCl3,
126 MHz): ꢁ (ppm) 43.4, 64.7, 103.3, 125.8, 126.3, 126.6,
127.4, 127.7, 128.4, 128.6, 128.7, 129.7, 131.5, 135.0, 141.8,
151.5, 151.5, 164.9.
ACKNOWLEDGEMENTS
The authors are grateful to CEPEMA-USP (Centro de
Capacitação
e Pesquisa em Meio Ambiente), CNPq
(310472/2007-5, 475575/2008-3), INCT Estudos do Meio
Ambiente/CNPq (573.667/2008-0), CAPES and Fapesp for
financial support.
2-[5-(2-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-4-phenylthiazole (2b): Yield (74%); yellow solid; mp
172–174°C; IR (KBr): ꢀ (cmꢀ1) 3117–2915, 1943–1875,
1
REFERENCES
1542, 1489, 1440, 713, 683; H NMR (CDCl3, 500 MHz): ꢁ
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(dd, 1H, J=12.2 Hz, J=17.3 Hz), 5.82 (dd, 1H, J=6.9 Hz,
J=12.2 Hz), 6.78 (s, 1H), 7.10–7.74 (m, 14H, ArH); 13C
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