Synthesis of new indazole derivatives as potential antioxidant agents

Article abstract of DOI:10.1007/s00044-013-0835-6

New indazole derivatives were synthesized by reacting hydrazine hydrate and its derivatives with cyclohexenone derivatives, which in turn prepared from respective chalcones. The chemical structure of newly synthesized compounds was well characterized by using ¹H NMR, ¹³C NMR, IR and mass spectral data. All the synthesized products were screened for their antioxidant properties. All indazole derivatives exhibited noticeable DPPH radical scavenging activity, reducing power capacity and total antioxidant capacity in comparison with the respective standards. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0835-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2368–2376
DOI 10.1007/s00044-013-0835-6
ORIGINAL RESEARCH
Synthesis of new indazole derivatives as potential antioxidant
agents
•
Majal Sapnakumari Badiadka Narayana
•
•
Balladka Kunhanna Sarojini Leelavathi Narayana Madhu
Received: 28 June 2013 / Accepted: 7 October 2013 / Published online: 17 October 2013
Ó Springer Science+Business Media New York 2013
Abstract New indazole derivatives were synthesized by
reacting hydrazine hydrate and its derivatives with cyclo-
hexenone derivatives, which in turn prepared from
respective chalcones. The chemical structure of newly
synthesized compounds was well characterized by using 1H
NMR, 13C NMR, IR and mass spectral data. All the syn-
thesized products were screened for their antioxidant
properties. All indazole derivatives exhibited noticeable
DPPH radical scavenging activity, reducing power capacity
and total antioxidant capacity in comparison with the
respective standards.
of research, as they can protect the human body from free
radicals and reduce the risk of many chronic diseases, such
as atherosclerosis, stroke, diabetes, Alzheimer’s disease,
some forms of cancer and oxidative stress responsible for
DNA, protein and membrane damage. Antioxidants are
also believed to play a very important role in the body
defence system against reactive oxygen species (ROS)
which are the dangerous by products generated during
normal cellular respiration. Supplementation with antioxi-
dants may help to maintain an adequate antioxidant status
and therefore, the normal physiological function of a living
system (Van Acker et al., 1996). Hence, there is consid-
erable interest in the discovery and development of effi-
cient synthetic or natural antioxidants.
Keywords Chalcone ꢀ Cyclohexenone ꢀ Indazole ꢀ
Antioxidant activity
Many synthetic antioxidants which are characterized by
a better antioxidant activity than natural antioxidants and
are more easily available, which are being used in a wide
variety of food products. Butylated hydroxytoluene (BHT)
and butylated hydroxyanisole (BHA) were originally
developed as preservatives to protect petroleum from oxi-
dative gumming and in personal care products. However,
these compounds have been used as antioxidants in human
foods since 1954 and are perhaps the most common anti-
oxidants used in foods today (Sherwin, 1976). Edaravone
(3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free
radical scavenger, is used for treatment of patients with
acute brain infarction (Kokura et al., 2005). The use of
synthetic antioxidants in consumer products has attracted a
great deal of research interest in synthetic antioxidants.
Fluorination of any molecule increases the lipophilicity
and thereby causing the higher partitioning of the antioxi-
dant agents into the biomembranes of cells (Ortial et al.,
2006). Also, the heterocyclic systems with chlorine or
bromine substituent show better antioxidant activity (Inami
et al., 2012; Hossain et al., 2009). Indazole nucleus is an
Introduction
An antioxidant is a substance that when present at small
concentrations compared to those of an oxidizable sub-
strate, significantly delays or prevents oxidation of that
substrate (Kumar, 2011). The search for new molecules
with antioxidant properties is an exceptionally active area
M. Sapnakumari ꢀ B. Narayana (&)
Department of Studies in Chemistry, Mangalore University,
Mangalagangotri, Mangalore 574199, Karnataka, India
e-mail: nbadiadka@yahoo.co.uk
B. K. Sarojini
Research Department of Chemistry, P.A. College of
Engineering, Nadupadavu, Mangalore 574153, Karnataka, India
L. N. Madhu
Post Graduation Department of Biochemistry, St. Aloysius
College, Mangalore 575003, India
123

Med Chem Res (2014) 23:2368–2376
2369
important class of nitrogen containing heterocyclic moiety
that was extensively used as key building block for the
synthesis of various pharmaceutically important agents.
Indazole exhibits a variety of pharmacological properties
like anticancer, antiasthametic, antipyretic, antiviral, anti-
microbial, cytostatic properties, antiinflammatory, anti
HIV, antitumor, hypoglycemic, antiprotozoal, antihyper-
tensive and tyrosine kinase inhibitor activities (Pawar
et al., 2012; Thangadurai et al., 2012; Liu et al., 2007;
Thomas et al., 2008). Survey of the literature in the recent
past revealed that indazole derivatives possessed signifi-
cant antioxidant activities (Samshuddin et al., 2013; Shakil
et al., 2013). Indazole is also a core part of various bio-
active molecules like adjudin, a phase three molecule for
male human contraceptive pill, which is a derivative of 1H-
indazole-3-carboxylic acid. It has shown to possess potent
anti-spermatogenic activity. In view of above observations
and also the reported antioxidant activity of indazoles
(Samshuddin et al. 2013), it was thought worthwhile to
synthesize new indazole derivatives 3a–c, 4a–c, 5a–c and
6a–c from cyclohexenone derivatives. All the synthesized
derivatives were characterized by spectral data and
screened for their in vitro antioxidant properties.
ophenyl/4-bromophenyl)-6-(4-fluorophenyl)-2-(4-nitro-
phenyl)-1,2,4,5-tetrahydro-3H-indazol-3-one 5a–c and
4-(phenyl/4-chlorophenyl/4-bromophenyl)-2-(2,4-dinitro-
phenyl)-6-(4-fluorophenyl)-1,2,4,5-tetrahydro-3H-indazol-
3-one 6a–c, respectively (Scheme 1).
The structures of compounds 3a–c, 4a–c, 5a–c and 6a–
c were confirmed by their spectral data. The IR spectra of
the compounds 3a–c showed stretching bands at
3,041–3,307 cm-1 due to NH group of indazole moiety,
while strong stretching band in the region
1
1,598–1,685 cm-1 attributed to carbonyl group. In the H
NMR spectra, two singlets appeared at d 9.73–9.84 and
11.62–11.73 ppm were due to the NH proton of indazole
ring. Two doublets of doublets for each compound were
due to the two protons attached to C-5 carbon, attached to
the chiral carbon C-4. One more doublet of doublet, in the
region d 4.21–4.26 ppm was due to a proton attached to
chiral carbon C-4. Similarly, a singlet observed at d
6.71–6.76 ppm could be due to methylene proton of C-7.
13C NMR spectrum showed signals in the region d
40.03–40.07 and 42.56–42.83 ppm was due to the aliphatic
CH2 and CH carbons, respectively. The signals in the
region d 138.66–139.64, 117.72–119.67, 109.36–109.83
and 153.13–157.21 ppm were observed for carbons C-6,
C-7, C-8 and C-9, respectively. The amide carbonyl carbon
appeared at d 158.64–159.86 ppm. LCMS spectra sup-
ported the formation of indazole derivatives. Elemental
analysis also gave satisfactory results for all the com-
pounds. Similarly, the structures of N-substituted indazole
derivatives 4a–c, 5a–c and 6a–c were confirmed by IR, 1H
NMR, 13C NMR and mass spectral data. The detailed
spectral data are given in the experimental section.
Results and discussion
Chemistry
Synthesis of cyclohexenone derivatives 2a–c were carried
out by reacting chalcones 1a–c with ethyl acetoacetate in
the presence of base as described in our previous study
(Kant et al., 2012). The chalcones 1a–c, in turn prepared by
the Claisen–Schmidt condensation of 4-fluoroacetophe-
none with different aldehydes. The cyclocondensation of
acetoacetic ester with chalcones led to the generation of
two chiral centes at C-1 and C-6 in cyclohexenones. As the
explored reaction was not stereoselective, both the con-
figurations of the chiral carbon atoms were expected to be
noticed in the synthesized cyclohexenones, which would
result in a mixture of diastereomers. No effort was under-
taken to separate the diastereomeric cyclohexenones and
was used as such for further reaction. The cyclohexenone
derivatives, ethyl 6-(phenyl/4-chlorophenyl/4-bromophe-
nyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-carbox-
ylate 2a–c, which contain 1,3-dicarbonyl system, reacted
with hydrazine hydrate/phenyl hydrazine/4-nitrophenyl
hydrazine/2,4-dinitrophenyl hydrazine in acidic medium
resulted in the formation of indazole derivatives, 4-(phenyl/
4-chlorophenyl/4-bromophenyl)-6-(4-fluorophenyl)-1,2,4,
5-tetrahydro-3H-indazol-3-one 3a–c, 4-(phenyl/4-chlor-
ophenyl/4-bromophenyl)-6-(4-fluorophenyl)-2-phenyl-1,2,
4,5-tetrahydro-3H-indazol-3-one 4a–c, 4-(phenyl/4-chlor-
Antioxidant activity
DPPH radical scavenging assay
DPPH is one of the stable and commercially available
organic nitrogen radical and has a UV–visible absorption
maximum at 517 nm (Huang et al., 2005). A rapid, simple,
accurate and inexpensive method to measure antioxidant
capacity of substances involves the use of this free radical.
It is widely used to test the ability of compounds to act as
free radical scavengers or hydrogen donors. Antioxidants
tested on DPPH were also found extremely effective in cell
systems (Tiwari, 2004). This simple test further provides
information on the ability of a compound to contribute
electrons during antioxidant action (Tiwari, 2004). The odd
electron in the DPPH free radical gives strong absorption
maximum at 517 nm and is purple in colour. The colour
changes from purple to yellow as the molar absorptivity of
the DPPH radical at 517 nm reduce upon the transfer of
123

2370
Med Chem Res (2014) 23:2368–2376
acidic H-atom from the compound to DPPH radical to form
DPPH-H. The resulting decolourization is stoichiometric
with respect to number of electrons captured. The results
are summarized in Table 1.
Table 1 Antioxidant activity of synthesized compounds
Compounds
% DPPH
scavenging
assay
Reducing power
assay (FRAP)
Total
antioxidant
capacity
The free radical scavenging power of indazoles can be
attributed to OH or NH groups in the indazole. Among the
tested compounds, compounds 4a–c and 5a–c showed very
high radical scavenging capacity with concentration of
1 mg/mL in comparison with the standard Glutathione. The
high radical scavenging capacity of the compounds 4a–
c was due to the presence of phenyl ring in the indazole.
The scavenging capacity of compounds 5a–c is decreased
slightly because of the electron withdrawing nitro group.
The presence of two nitro groups is the reason for the lower
scavenging capacity of the compound 6a–c. However, in
case of compound 3a–c, more tautomerism is possible due
to the presence of free NH group. Therefore, their scav-
enging capacities are less (Fig. 1).
3a
17.89 2.5
19.78 1.19
16.23 1.23
94 2.11
1.2 0.25
0.63 0.2
8.4 0.5
3b
4.75
1
3c
1.143 0.25
1.492 0.33
1.945 0.33
0.802 0.25
0.95 0.1
1.17 0.37
2.59 0.44
3.95 0.35
1.97 0.34
6.22 1.99
0.69 0.15
0.58 0.15
7.83 1.11
16.17 1.34
4.69 0.85
–
4a
4b
90.15 2.53
96.77 2.55
84 .43 1.53
89.56 1.02
85.33 1.95
19.56 3.85
4.21 1.21
2.34 1.95
92.09 1.09
4c
5a
5b
0.47 0.09
0.64 0.1
5c
6a
0.75 0.1
6b
3.26 0.2
6c
0.76 0.1
Standard
0.105 0.02
2
NH
Scheme 1 Synthesis of
indazole derivatives 3a–c, 4a–c,
5a–c and 6a–c
1
HN
9
7
3
O
17
18
8
NH NH
H O
2
2
2
15
12
16
6
4
14
13
EtOH/H2SO4
10
11
5
reflux, 12 hr
19
21
F
R
20
3a-c
O
24
25
23
26
2
1
22
N
NH
HN
27
1a-c
F
R
NH
2
9
3
R= Cl, H, Br
7
O
8
15
17
CH COCH COOC
H
5
3
2
2
EtOH/H2SO4
16
6
14
4
18
19
10
11
5
reflux, 12 hr
C H
2
5
21
O
O
13
F
R
12
20
4a-c
24
O
NO
2
23
25
26
2
1
22
N
HN
27
NH
2a-c
NH
2
F
R
9
3
7
O
8
O N
2
15
12
17
16
6
4
14
13
18
EtOH/H2SO4
10
11
5
reflux, 12 hr
19
21
F
R
20
5a-c
24
27
23
O N
2
NO
2
25
26
NO
2
2
N
1
22
HN
NH
NH
9
3
2
7
O
8
O N
15
17
2
16
6
4
14
13
18
19
EtOH/H2SO4
10
11
5
reflux, 12 hr
21
F
R
12
20
6a-c
123

Med Chem Res (2014) 23:2368–2376
Reducing power assay
2371
6c showed moderate antioxidant capacity. Among them,
the compounds 6b and 3a reduced Mo(VI) to Mo(V) in a
better way (Fig. 3).
The reducing capacity of a compound may serve as a
significant indicator of its potential antioxidant activity
(Oktay et al., 2003). Compounds with reducing power
indicate that they give up electrons and can reduce the
oxidized intermediates of lipid peroxidation processes, so
that they can act as primary and secondary antioxidants
(Chanda and Dave, 2009). Substance, which has reducing
ability, react with potassium ferricyanide (Fe3?) to form
potassium ferrocyanide (Fe2?), which then react with ferric
chloride to form ferric ferrous complex that has an
absorption maximum at 700 nm. Increased absorbance of
the reaction mixture indicates the increased reducing
power. The results are summarized in Table 1.
Experimental section
Melting points were taken in open capillary tubes and were
uncorrected. The purity of the compounds was confirmed
by thin layer chromatography using Merck silica gel 60
F
254-coated aluminium plates using ethyl acetate : n-hex-
ane (1:3, v/v) as solvent system. IR spectra were recorded
on Shimadzu-FTIR Infrared spectrometer in KBr (kmax in
cm-1). H NMR (400 MHz) spectra were recorded on a
1
Bruker AMX 400 spectrometer, with 5 mm PABBO BB-
1H TUBES with TMS as internal standard. LCMS was
obtained using Agilent 1200 series LC and Micromass zQ
spectrometer. Electrospray (ESI) method was used for
ionization. Elemental analysis was carried out using VA-
RIO EL-III (Elementar Analysensysteme GmBH). A
UV–Visible spectrophotometer (SHIMADZU, Model No.:
Reducing power assay is expressed in effective con-
centration (mg/mL) equivalent of 0.5 absorbance Ellagic
acid. Among the tested compounds, many compounds
showed significant reducing power capacity in comparison
with the standard Ellagic acid. Compounds 3b, 4c, 5a, 5b,
5c, 6a and 6c showed good reducing power capacity while
compounds 3a, 3c, 4a and 4b exhibited moderate reducing
power capacity. Although, the presence of free NH group
contributes to the reducing power assay, the net activity
depends on the different substituents present in the mole-
cule (Fig. 2).
Reducing Power Assay
Total antioxidant capacity
The total antioxidant capacity of synthesized compounds
was determined by the phosphomolybdenum assay method
(Prieto and Pineda, 1999) which is based on the reduction
of Mo(VI) to Mo(V) by the compound and subsequent
formation of a green phosphate-Mo(V) complex in acidic
condition.
The antioxidant activity was expressed as the number of
gram equivalents of ascorbic acid. Among the newly pre-
pared indazole derivatives, compounds 3a, 5a, 6a, 6b and
Fig. 2 Comparison of reducing power assay of newly synthesized
indazole derivatives
Total Antioxidant Capacity Assay
DPPH Radical Scavenging Assay
Fig. 3 Comparison of total antioxidant capacity of newly synthesized
indazole derivatives
Fig. 1 Comparison of DPPH scavenging assay of newly synthesized
indazole derivatives
123

2372
Med Chem Res (2014) 23:2368–2376
UV-2550) with 1 cm matched quartz cells was used for the
absorbance measurements.
JXA = 3.8 Hz, JXB = 8.4 Hz), 6.76(s, 1H, CH),
7.21–7.29(m, 9H, Ar–H), 9.84(s, 1H, NH)11.73(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.07 (ali-
phatic CH2, C-5), 42.79 (aliphatic CH, C-4), 159.86 (amide
C=O, C-3), 138.66(C-6), 118.64(C-7), 109.83(C-8),
157.21(C-9), 115.43(C-12, C-14), 126.43(C-19), 127.55(C-
17, C-21), 128.36(C-11, C-15), 128.58(C-18, C-20),
135.85(C-10), 140.59(C-16), 161.19(C-13). LCMS: m/z
Synthesis of cyclohexenone derivatives 2a–c was car-
ried out by refluxing chalcones 1a–c with ethyl acetoace-
tate in ethanol in the presence of sodium hydroxide as
described in our previous study (Kant et al., 2012).
Synthesis of indazole derivatives
307.16 (M ? 1).
C H N analysis; calculated for
4-(4-Chlorophenyl)-6-(4-fluorophenyl)-1,2,4,5-tetrahydro-
C19H15FN2O: C, 74.50; H, 4.94; N, 9.14. Found: C, 74.46;
H, 4.99; N, 9.11 %.
3H-indazol-3-one (3a)
This compound was prepared by refluxing ethyl 6-(4-
chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with hydrazine hydrate (0.01 mol)
in 20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a yellow solid with
73 % yield. M. p. 147–149 °C. IR (KBr, kmax in cm-1):
3115 (NH), 3047 (Ar–H), 1598(C=O), 1230 (C–F). 1H
NMR (400 MHz, DMSO-d6, d ppm): 2.80 (dd, 1H, HA,
JAB = 16.8 Hz, JAX = 3.8 Hz), 3.10 (dd, 1H, HB,
JBA = 16.6 Hz, JBX = 8.4 Hz),4.21 (dd, 1H, HX,
JXA = 3.6 Hz, JXB = 8.4 Hz), 6.71(s, 1H, CH),
7.13–7.27(m, 8H, Ar–H), 9.73(s, 1H, NH)11.62(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.05 (ali-
phatic CH2, C-5), 42.83 (aliphatic CH, C-4), 158.74 (amide
C=O, C-3), 139.64 (C-6), 117.72(C-7), 109.83(C-8),
155.52(C-9), 115.45(C-12, C-14), 128.38(C-11, C-15),
128.62(C-18, C-20), 129.67(C-17, C-21), 131.54(C-19),
135.81(C-10), 138.94(C-16), 162.19(C-13). LCMS: m/z
4-(4-Bromophenyl)-6-(4-fluorophenyl)-1,2,4,5-tetrahydro-
3H-indazol-3-one (3c)
This compound was prepared by refluxing ethyl 6-(4-
bromophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with hydrazine hydrate (0.01 mol)
in 20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a yellow solid with
76 % yield. m. p. 144–146 °C. IR (KBr, kmax in cm-1):
1
3094 (NH), 3037 (Ar–H), 1637 (C=O), 1241 (C–F). H
NMR (400 MHz, DMSO-d6, d ppm): 2.81 (dd, 1H, HA,
JAB = 16.6 Hz, JAX = 3.6 Hz), 3.11 (dd, 1H, HB,
JBA = 16.6 Hz, JBX = 8.8 Hz),4.22 (dd, 1H, HX,
JXA = 3.8 Hz, JXB = 8.6 Hz), 6.73(s, 1H, CH),
7.18–7.37(m, 8H, Ar–H), 9.78(s, 1H, NH), 11.67(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.03 (ali-
phatic CH2, C-5), 42.56 (aliphatic CH, C-4), 158.64 (amide
C=O, C-3), 139.63(C-6), 119.67(C-7), 109.36(C-8),
153.13(C-9), 115.33(C-12, C-14), 120.32(C-19), 128.52(C-
11, C-15), 130.53(C-17, C-21), 131.65(C-18, C-20),
135.56(C-10), 139.74(C-16), 162.14(C-13). LCMS: m/z
341.2 (M ? 1).
C
H
N
analysis; calculated for
C19H14ClFN2O: C, 66.97; H, 4.14; N, 8.22. Found: C,
66.91; H, 4.23; N, 8.18 %.
386.04 (M ? 1).
C H N analysis; calculated for
6-(4-Fluorophenyl)-4-phenyl-1,2,4,5-tetrahydro-3H-
C19H14BrFN2O: C, 59.24; H, 3.66; N, 7.27. Found: C,
59.20; H, 3.69; N, 7.23 %.
indazol-3-one (3b)
This compound was prepared by refluxing ethyl 4-(4-
fluorophenyl)-2-oxo-6-phenylcyclohex-3-ene-1-carbox-
ylate (0.01 mol) with hydrazine hydrate (0.01 mol) in
20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a yellow solid with
74 % yield. M. p. 150–152 °C. IR (KBr, kmax in cm-1):
3098 (NH), 3032 (Ar–H), 1631(C=O), 1233 (C–F). 1H
NMR (400 MHz, DMSO-d6, d ppm): 2.84 (dd, 1H, HA,
JAB = 16.6 Hz, JAX = 3.6 Hz), 3.13 (dd, 1H, HB,
JBA = 16.4 Hz, JBX = 8.2 Hz),4.26 (dd, 1H, HX,
4-(4-Chlorophenyl)-6-(4-fluorophenyl)-2-phenyl-1,2,4,5-
tetrahydro-3H-indazol-3-one (4a)
This compound was prepared by refluxing ethyl 6-(4-
chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with phenyl hydrazine (0.01 mol)
in 20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a yellow solid with
62 % yield. m. p. 170–172 °C. IR (KBr, kmax in cm-1):
3059 (NH), 3039 (Ar–H), 1647(C=O), 1238 (C–F). 1H
123

Med Chem Res (2014) 23:2368–2376
2373
NMR (400 MHz, DMSO-d6, d ppm): 2.91 (dd, 1H, HA,
JAB = 16.4 Hz, JAX = 3.7 Hz), 3,21(dd, 1H, HB,
JBA = 17.1 Hz, JBX = 8.12 Hz),4.21 (broad s, 1H, HX),
6.91(s, 1H, CH), 7.13–7.79(m, 14H, Ar–H), 11.23(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.09 (ali-
phatic CH2, C-5), 43.34 (aliphatic CH, C-4), 166.92 (amide
C=O, C-3), 139.61(C-6), 117.74(C-7), 109.79(C-8),
155.52(C-9), 113.28(C-23, C-27), 115.43(C-12, C-14),
119.27(C-25), 128.19(C-11, C-15), 128.53(C-18, C-20),
129.23(C-17, C-21), 129.38(C-24, C-26), 131.5(C-
19),135.02(C-10), 136.32(C-22), 138.84(C-16), 162.25(C-
13). LCMS: m/z 416.8 (M? ? 1). C H N analysis; calcu-
lated for C25H18ClFN2O: C, 72.03; H, 4.35; N, 6.72.
Found: C, 71.98; H, 4.37; N, 6.69 %.
in ethanol. The product was obtained as a yellow solid with
51 % yield. M. p. 188–190 °C. IR (KBr, kmax in cm-1):
3041 (NH), 2939 (Ar–H), 1654 (C=O), 1236 (C–F). H
1
NMR (400 MHz, DMSO-d6, d ppm): 2.89 (dd, 1H, HA,
JAB = 16 Hz, JAX = 3.8 Hz), 3.18 (dd, 1H, HB,
JBA = 16.4 Hz, JBX = 7.8 Hz), 4.38 (broad s, 1H, HX),
6.89(s, 1H, CH), 7.11–7.88 (m, 14H, Ar–H), 11.24(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.07 (ali-
phatic CH2, C-5), 43.36 (aliphatic CH, C-4), 167.02 (amide
C=O, C-3), 139.66(C-6), 117.69(C-7), 109.83(C-8),
155.58(C-9), 113.58(C-23, C-27), 115.43(C-12, C-14),
119.37(C-25), 120.3(C-19), 128.19(C-11, C-15), 129.47(C-
24, C-26), 130.09(C-17, C-21), 131.61(C-18, C-20),
135.26(C-10), 136.36(C-22), 139.72(C-16), 162.51(C-13).
LCMS: m/z 461.05 (M? ? 1). C H N analysis; calculated
for C25H18FN2O: C, 65.09; H, 3.93; N, 6.07. Found: C,
65.04; H, 3.94; N, 6.04 %.
6-(4-Fluorophenyl)-2,4-diphenyl-1,2,4,5-tetrahydro-3H-
indazol-3-one (4b)
This compound was prepared by refluxing ethyl 4-(4-
fluorophenyl)-2-oxo-6-phenylcyclohex-3-ene-1-carbox-
ylate (0.01 mol) with phenyl hydrazine (0.01 mol) in
20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a yellow solid with
43 % yield. M. p. 192–194 °C. IR (KBr, kmax in cm-1):
3062 (NH), 3022 (Ar–H), 1685(C=O), 1240 (C–F). 1H
NMR (400 MHz, DMSO-d6, d ppm): 2.89 (dd, 1H, HA,
JAB = 16.8 Hz, JAX = 3.62 Hz), 3.19 (dd, 1H, HB,
JBA = 17.2 Hz, JBX = 8.04 Hz), 4.38 (broad s, 1H, HX),
6.88 (s, 1H, CH), 7.10–7.77 (m, 15H, Ar–H), 11.21(s, 1H,
NH). 13C NMR (100 MHz, CDCl3, d ppm): 40.04 (ali-
phatic CH2, C-5), 43.48 (aliphatic CH, C-4), 166.29 (amide
C=O, C-3), 139.64(C-6), 117.74(C-7), 109.67(C-8),
155.49(C-9), 113.36(C-23, C-27), 115.45(C-12, C-14),
119.27(C-25), 126.6(C-19), 127.37(C-17, C-21), 128.38(C-
11, C-15), 128.71(C-18, C-20), 129.38(C-24, C-26),
135.22(C-10), 136.54(C-22), 140.67(C-16), 162.25(C-13).
LCMS: m/z 382.12 (M? ? 1). C H N analysis; calculated
for C25H19FN2O: C, 78.52; H, 5.01; N, 7.33. Found: C,
78.48; H, 5.04; N, 7.30 %.
4-(4-Chlorophenyl)-6-(4-fluorophenyl)-2-(4-nitrophenyl)-
1,2,4,5-tetrahydro-3H-indazol-3-one (5a)
This compound was prepared by refluxing ethyl 6-(4-
chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with 4-nitrophenyl hydrazine
(0.01 mol) in 20 mL ethanol in the presence of concen-
trated sulphuric acid (0.5 mL). The reaction mixture was
cooled and poured into ice-cold water (50 mL). The pre-
cipitate obtained after neutralization was collected by fil-
tration and recrystallized in ethanol. The product was
obtained as
a orange solid with 49 % yield. M.
p. 119–121 °C. IR (KBr, kmax in cm-1): 3124 (NH), 3042
(Ar–H), 1678 (C=O), 1234 (C–F) 1534 (NO2). H NMR
1
(400 MHz, DMSO-d6, d ppm): 2.92 (broad d, 1H, CH2-HA,
JAB = 16.8 Hz), 3.20 (dd, 1H, CH2-HB, JBA = 16.4 Hz),
4.40 (broad s, 1H, CH-HX), 6.92 (s, 1H, CH), 7.03–8.40
(m, 12H, Ar–H), 11.98 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3, d ppm): 40.81(aliphatic CH2, C-5), 42.64 (aliphatic
CH, C-4), 166.98 (amide C=O, C-3), 139.69(C-6),
117.74(C-7), 109.84(C-8), 155.61(C-9), 114.18(C-23,
C-27), 115.42(C-12, C-14), 121.61(C-24, C-26), 128.04(C-
11, C-15), 128.82(C-18, C-20), 129.24(C-17, C-21),
131.57(C-19), 135.12(C-10), 138.84(C-16), 138.85(C-25),
142.44(C-22), 162.19(C-13). LCMS: m/z 507.2 (M ? 1).
C H N analysis; calculated for C25H17ClFN3O3: C, 65.01;
H, 3.71; N, 9.10 Found: C, 64. 98; H, 3.75; N, 9.06 %.
4-(4-Bromophenyl)-6-(4-fluorophenyl)-2-phenyl-1,2,4,5-
tetrahydro-3H-indazol-3-one (4c)
This compound was prepared by refluxing ethyl 6-(4-
bromophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with phenyl hydrazine (0.01 mol)
in 20 mL ethanol in the presence of concentrated sulphuric
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
6-(4-Fluorophenyl)-2-(4-nitrophenyl)-4-phenyl-1,2,4,5-
tetrahydro-3H-indazol-3-one (5b)
This compound was prepared by refluxing ethyl 4-(4-
fluorophenyl)-2-oxo-6-phenylcyclohex-3-ene-1-carbox-
ylate (0.01 mol) with 4-nitrophenyl hydrazine (0.01 mol)
in 20 mL ethanol in the presence of concentrated sulphuric
123

2374
Med Chem Res (2014) 23:2368–2376
acid (0.5 mL). The reaction mixture was cooled and poured
into ice-cold water (50 mL). The precipitate obtained after
neutralization was collected by filtration and recrystallized
in ethanol. The product was obtained as a orange solid with
66 % yield. M. p. 121–123 °C. IR (KBr, kmax in cm-1):
3307 (NH), 3113 (Ar–H), 1618 (C=O), 1278 (C–F), 1519
(NO2). 1H NMR (400 MHz, DMSO-d6, d ppm): 2.95
(broad d, 1H, CH2-HA, JAB = 16.4 Hz), 3.18 (dd, 1H,
CH2-HB, JBA = 16.6 Hz), 4.37 (broad s, 1H, CH-HX), 6.88
(s, 1H, CH), 7.07–8.43 (m, 13H, Ar–H), 11.93 (s, 1H, NH).
13C NMR (100 MHz, CDCl3, d ppm): 40.77 (aliphatic
CH2, C-5), 43.49 (aliphatic CH, C-4), 166.86 (amide C=O,
C-3), 139.48(C-6), 117.69(C-7), 109.79(C-8), 155.58(C-9),
114.14(C-23, C-27), 115.41(C-12, C-14), 121.64(C-24,
C-26), 126.08(C-19), 127.84(C-17, C-21), 128.02(C-11,
C-15), 128.72(C-18, C-20), 135.10(C-10), 138.79(C-16),
138.85(C-25), 142.41(C-22), 162.20(C-13). LCMS: m/z
carboxylate (0.01 mol) with 2,4-dinitrophenyl hydrazine
(0.01 mol) in 20 mL ethanol in the presence of concen-
trated sulphuric acid (0.5 mL). The reaction mixture was
cooled and poured into ice-cold water (50 mL). The pre-
cipitate obtained after neutralization was collected by fil-
tration and recrystallized in ethanol. The product was
obtained as
a yellow solid with 81 % yield. M.
p. 155–157 °C. IR (KBr, kmax in cm-1): 3129 (NH), 3129
(Ar–H), 1647 (C=O), 1234 (C–F), 1547 (NO2). H NMR
1
(400 MHz, DMSO-d6, d ppm): 3.02 (broad d, 1H, CH2-HA,
JAB = 16.4 Hz), 3.26 (dd, 1H, CH2-HB, JBA = 16.4 Hz),
4.46 (broad s, 1H, CH-HX), 6.99 (s, 1H, CH), 7.14–8.49
(m, 12H, Ar–H), 11.98 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3, d ppm): 40.83 (aliphatic CH2, C-5), 43.62 (ali-
phatic CH, C-4), 166.94 (amide C=O, C-3), 139.67 (C-6),
117.73 (C-7), 109.82 (C-8),155.60 (C-9), 115.04(C-24),
115.44(C-12,
C-14),
119.24(C-26),
127.70(C-22),
473.4 (M ? 1).
C
H
N
analysis; calculated for
128.05(C-11, C-15), 128.79(C-18, C-20), 129.21(C-17,
C-21), 131.56(C-19), 132.86(C-27), 135.11(C-10),
138.85(C-16), 139.81(C-23), 143.42(C-25), 162.17(C-13).
LCMS: m/z 462.4 (M ? 1). C H N analysis; calculated for
C25H16ClFN4O5: C, 59.24; H, 3.18; N, 11.05. Found: C,
59.21; H, 3.21; N, 11.01 %.
C25H18FN3O3: C, 70.25; H, 4.24; N, 9.83. Found: C, 70.22;
H, 4.27; N, 9.80 %.
4-(4-Bromophenyl)-6-(4-fluorophenyl)-2-(4-nitrophenyl)-
1,2,4,5-tetrahydro-3H-indazol-3-one (5c)
This compound was prepared by refluxing ethyl 6-(4-
bromophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with 4-nitrophenyl hydrazine
(0.01 mol) in 20 mL ethanol in the presence of concentrated
sulphuric acid (0.5 mL). The reaction mixture was cooled and
poured into ice-cold water (50 mL). The precipitate obtained
after neutralization was collected by filtration and recrystal-
lized in ethanol. The product was obtained as a orange solid
with 54 % yield. M. p. 121–123 °C. IR (KBr, kmax in cm-1):
3105 (NH), 3113 (Ar–H), 1622 (C=O), 1276 (C–F) 1523
(NO2). 1H NMR (400 MHz, DMSO-d6, d ppm): 2.93 (broad
d, 1H, CH2-HA, JAB = 16.2 Hz), 3.23 (dd, 1H, CH2-HB,
JBA = 16.6 Hz), 4.42 (broad s, 1H, CH-HX), 6.94 (s, 1H,
CH), 7.06–8.43 (m, 12H, Ar–H), 11.99 (s, 1H, NH). 13C
NMR (100 MHz, CDCl3, d ppm): 40.79 (aliphatic CH2, C-5),
43.47 (aliphatic CH, C-4), 166.93 (amide C=O, C-3),
139.94(C-6), 117.71(C-7), 109.81(C-8), 155.63(C-9),
114.19(C-23, C-27), 115.39(C-12, C-14), 120.35(C-19),
121.67(C-24, C-26), 127.84(C-11, C-15), 130.04(C-17,
C-21), 131.63(C-18, C-20), 135.13(C-10), 138.86(C-25),
139.72(C-16), 142.47(C-22), 162.17(C-13). LCMS: m/z 552.2
(M ? 1). C H N analysis; calculated for C25H17BrFN3O3: C,
59.30; H, 3.38; N, 8.30. Found: C, 59.27; H, 3.41; N, 8.26 %.
2-(2,4-Dinitrophenyl)- 6-(4-fluorophenyl)-4-phenyl-
1,2,4,5-tetrahydro-3H-indazol-3-one (6b)
This compound was prepared by refluxing ethyl 4-(4-
fluorophenyl)-2-oxo-6-phenylcyclohex-3-ene-1-carbox-
ylate (0.01 mol) with 2,4-dinitrophenyl hydrazine
(0.01 mol) in 20 mL ethanol in the presence of concen-
trated sulphuric acid (0.5 mL). The reaction mixture was
cooled and poured into ice-cold water (50 mL). The pre-
cipitate obtained after neutralization was collected by fil-
tration and recrystallized in ethanol. The product was
obtained as
a yellow solid with 67 % yield. M.
p. 148–150 °C. IR (KBr, kmax in cm-1): 3259 (NH), 3122
(Ar–H), 1664 (C=O), 1273 (C–F) 1552 (NO2). H NMR
1
(400 MHz, DMSO-d6, d ppm): 3.05 (broad d, 1H, CH2-HA,
JAB = 16.6 Hz), 3.24 (dd, 1H, CH2-HB, JBA = 16.4 Hz),
4.49 (broad s, 1H, CH-HX), 7.01 (s, 1H, CH), 7.17–8.53
(m, 13H, Ar–H), 11.97 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3, d ppm): 40.74 (aliphatic CH2, C-5), 43.47 (ali-
phatic CH, C-4), 166.87 (amide C=O, C-3), 139.46(C-6),
117.65(C-7), 109.74(C-8), 155.57(C-9), 115.06(C-24),
115.44(C-12,
127.71(C-22), 127.81(C-17, C-21), 128.03(C-11, C-15),
128.73(C-18, C-20), 132.84(C-27), 135.14(C-10),
C-14),
119.23(C-26),
126.09(C-19),
4-(4-Chlorophenyl)-2-(2,4-dinitrophenyl)-6-(4-
139.79(C-23), 140.77(C-16), 143.43(C-25), 162.21(C-13).
LCMS: m/z 428.2 (M ? 1). C H N Analysis; Calculated for
C25H17FN4O5: C, 63.56; H, 3.63; N, 11.86. Found: C,
63.53; H, 3.67; N, 11.85 %.
fluorophenyl)-1,2,4,5-tetrahydro-3H-indazol-3-one(6a)
This compound was prepared by refluxing ethyl 6-(4-
chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
123

Med Chem Res (2014) 23:2368–2376
2375
4-(4-Bromophenyl)-2-(2,4-dinitrophenyl)-6-(4-
Reducing power assay
fluorophenyl)-1,2,4,5-tetrahydro-3H-indazol-3-one (6c)
The reducing power of the synthesized indazoles was
determined according to the method of (Oyaizu, 1986).
Different concentrations of the samples (100–1,000 lg/
mL) in DMSO (1 mL) were mixed with phosphate buffer
(2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide
(2.5 mL, 1 % solution). The mixture was incubated at
50 °C for 20 min. After which 10 % trichloroacetic acid
(2.5 mL) was added to the mixture which was then cen-
trifuged for 10 min. The upper layer of solution (2.5 mL)
was mixed with distilled water (2.5 mL) and FeCl3
(0.5 mL, 0.1 %), and then the absorbance at 700 nm was
measured using a spectrophotometer. Higher absorbance
of the reaction mixture indicated greater reducing power.
All experiments were carried out in triplicate, and the
reducing power assay was represented by effective con-
centration (mg/mL) equivalent of 0.5 absorbance Ellagic
acid.
This compound was prepared by refluxing ethyl 6-(4-
bromophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-
carboxylate (0.01 mol) with 2,4-dinitrophenyl hydrazine
(0.01 mol) in 20 mL ethanol in the presence of concen-
trated sulphuric acid (0.5 mL). The reaction mixture was
cooled and poured into ice-cold water (50 mL). The pre-
cipitate obtained after neutralization was collected by fil-
tration and recrystallized in ethanol. The product was
obtained as
a yellow solid with 62 % yield. M.
p. 153–155 °C. IR (KBr, kmax in cm-1): 3189 (NH), 3131
(Ar–H), 1652 (C=O), 1225 (C–F), 1543 (NO2). H NMR
1
(400 MHz, DMSO-d6, d ppm): 3.03 (broad d, 1H, CH2-HA,
JAB = 16.0 Hz), 3.22 (dd, 1H, CH2-HB, JBA = 16.4 Hz),
4.43 (broad s, 1H, CH-HX), 6.99 (s, 1H, CH), 7.11–8.51
(m, 12H, Ar–H), 11.978 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3, d ppm): 40.77 (aliphatic CH2, C-5), 43.48 (ali-
phatic CH, C-4), 166.95 (amide C=O, C-3), 139.51 (C-6),
117.74 (C-7), 109.84 (C-8), 155.62 (C-9), 115.04(C-24),
Total antioxidant capacity
115.36(C-12,
127.69(C-22), 128.07(C-11, C-15), 130.03(C-17, C-21),
131.61(C-18, C-20), 132.85(C-27), 135.15(C-10),
C-14),
119.24(C-26),
120.34(C-19),
The total antioxidant capacity of synthesized compounds
was determined by the phosphomolybdenum method (Pri-
eto and Pineda, 1999). About 1 mL of compound solutions
in DMSO (20 lg) was combined with 1 mL of reagent
solution (0.6 M sulphuric acid, 28 mM sodium phosphate
and 4 mM ammonium molybdate). The tubes were capped
and incubated in a boiling water bath at 95 °C for 90 min.
After that, the samples were cooled to room temperature.
The absorbance of each solution was read at 695 nm
against reagent blank using a spectrophotometer. The
results were expressed in lM of ascorbic acid equivalent
per mg of the sample.
139.69(C-16), 139.80(C-23), 143.41(C-25), 162.16(C-13).
LCMS: m/z 507.7 (M ? 1). C H N analysis; calculated for
C25H16BrFN4O5: C, 54.46; H, 2.93; N, 10.16. Found: C,
54.42; H, 2.97; N, 10.14 %.
Antioxidant activity
DPPH radical scavenging assay
Free radical scavenging activity of compounds was mea-
sured by DPPH using the reported method (Blois, 1958).
Briefly, 1 mM solution of DPPH in ethanol was prepared,
and this solution (1 mL) was added to sample solutions
1 mg/ml of distiled water. The mixture was shaken vig-
orously and allowed to stand at room temperature for
20 min. The incubation was done under dark condition.
Then the absorbance was measured at 517 nm in a spec-
trophotometer. Lower absorbance of the reaction mixture
indicated higher free radical scavenging activity. The
capability to scavenge the DPPH radical was calculated
using the following equation:
Conclusions
New indazole derivatives were prepared using cyclohexe-
none derivatives as building blocks. All the derivatives
were characterized by H NMR, 13C NMR, IR and mass
1
spectral data. Newly synthesized compounds were
screened for their antioxidant properties. Most of the
indazole derivatives exhibited noticeable DPPH radical
scavenging activity, reducing power capacity and total
antioxidant capacity in comparison with the standards.
DPPH scavenging effect ð%Þ ¼ ðA0 ꢁ A1=A0Þ ꢂ 100;
Acknowledgments The authors are thankful to the Director, IISc,
Bangalore for NMR data. BN thanks the UGC for financial assistance
through BSR one time grant for the purchase of chemicals. MS thanks
DST for providing financial help for the research work through
INSPIRE Fellowship.
where A0 is the absorbance of the control reaction, and A1
is the absorbance in the presence of the samples or stan-
dards. Each sample was assayed at 1 mg/mL, and all
experiments were carried out in triplicate.
123

2376
Med Chem Res (2014) 23:2368–2376
Oyaizu M (1986) Studies on products of the browning reaction.
Antioxidative activities of browning reaction products prepared
from glucosamine. Jpn J Nutr 44:307–315
Pawar MP, Vyas K, Shah NM, Nimavat K (2012) Synthesis and
antimicrobial activity of some new indazolone derivatives from
1-(3,5-dibromo-2-hydroxy-4 methyl phenyl) ethanone. IJPRS
1(3):211–216
Prieto P, Pineda M (1999) Spectrophotometric quantitation of
antioxidant capacity through the formation of a phosphomolyb-
denum complex: specific application to the determination of
vitamin E. Anal Biochem 269:337–341
Samshuddin S, Narayana B, Sarojini BK, Madhu LN (2013) A study
on the reactions of alkyl 4,6-bis(4-fluorophenyl)-2-oxocyclohex-
3-ene-1-carboxylate and in vitro antioxidant activity of deriva-
tives. Med Chem Res 22:3002–3011
Shakil NA, Manish KS, Sathiyendiran M, Kumar J, Jasdeep CP
(2013) Microwave synthesis, characterization and bio-efficacy
evaluation of novel chalcone based 6-carbethoxy-2-cyclohexen-
1-one and 2H-indazol-3-ol derivatives. Eur J Med Chem
59:120–131
Sherwin ER (1976) Antioxidants for vegetable oils. J Am Oil Chem
Soc 53:430–436
Thangadurai A, Minu M, Wakode SS, Agrawal S, Narasimhan B
(2012) Indazole: a medicinally important heterocyclic moiety.
Med Chem Res 21:1509–1523
References
Blois MS (1958) Antioxidant determinations by the use of a stable
free radical. Nature 181:1199–1200
Chanda S, Dave R (2009) In vitro models for antioxidant activity
evaluation and some medicinal plants possessing antioxidant
properties: an overview. Afr J Microbiol Res 3:981–996
Hossain MM, Shaha SK, Aziz F (2009) Antioxidant potential study of
some synthesized N-heterocycles. Bangladesh Med Res Counc
Bull 35:49–52
Huang D, Boxin OU, Prior RL (2005) The Chemistry behind
antioxidant capacity assays. J Agric Food Chem 53:1841–1856
Inami K, Iizuka Y, Furukawa M, Nakanishi I, Ohkubo K, Fukuhara K,
Fukuzumi S, Mochizuki M (2012) Chlorine atom substitution
influences radical scavenging activity of 6-chromanol. Bioorg
Med Chem 20:4049–4055
Kant R, Gupta VK, Kapoor K, Sapnakumari M, Narayana B, Sarojini
BK (2012) Ethyl 6-(4-bromophenyl)-4-(4-fluorophenyl)-2-oxo-
cyclohex-3-ene-1-carboxylate. Acta Crystallogr E68:o2917–
o2918
Kokura S, Yoshida N, Sakamoto N, Ishikawa T, Takagi T,
Higashihara H, Nakabe N, Handa O, Naito Y, Yoshikawa T
(2005) The radical scavenger edaravone. Cancer Lett
229:223–233
Kumar S (2011) Radicals and antioxidants: human and food system.
Adv Appl Sci Res 2:129–135
Liu ZW, Zhang T, Yang Z (2007) Involvement of nitric oxide in
spatial memory deficits in status epilepticus rats. Neurochem Res
32:1875–1883
Oktay M, Gulcin I, Kufrevioglu OI (2003) Determination of in vitro
antioxidant activity of fennel (Foeniculum vulgare) seed
extracts. LWT Food Sci Tech 36:263–271
Ortial S, Durand G, Poeggeler B, Polidori A, Pappolla MA, Boker J,
Hardeland R, Pucci B (2006) Fluorinated amphiphilic amino
acid derivatives as antioxidant carriers: a new class of protective
agents. J Med Chem 49:2812–2820
Thomas B, Saravanan KS, Kochupurackal P, Mohanakumar KP
(2008) In vitro and in vivo evidences that antioxidant action
contributes to the neuroprotective effects of the neuronal nitric
oxide synthase and monoamine oxidase-B inhibitor, 7-nitroin-
dazole. Neurochem Int 52:990–1001
Tiwari AK (2004) Antioxidants: new generation therapeutic base for
treatment of polygenic disorders. Curr Sci 86:1092–1102
Van Acker SABE, Van den Vijgh WJF, Bast F (1996) Structural
aspects of antioxidant activity of flavonoids. Free Radic Biol
Med 20:331–342
123