Synthesis and antioxidant properties of (3,4-dihydroxyphenyl)(2,3,4- trihydroxyphenyl)methanone and its derivatives

Article abstract of DOI:10.1002/ardp.201100272

(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone (5) and its two derivatives with bromine were synthesized from reactions such as bromination and demethylation of (3,4-dimethoxyphenyl)(2,3,4-trimethoxyphenyl)methanone (6). The Wolf-Kishner reduction product (9) of 6 and its three derivatives with bromine were obtained. 4-(3,4-Dihydroxybenzyl)benzene-1,2,3-triol and its dibromide derivative (16) were also synthesized from 9 and the corresponding dibromide derivative. The in vitro antioxidant activities of nine new compounds synthesized in these reactions were determined by analyzing the radical scavenging activities of bromophenols for 2,2′-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), N,N-dimethyl-p-phenylenediamine (DMPD), and the superoxide anion radical (O₂^-?) and examining the total reducing power through Fe³⁺-Fe²⁺ transformation, FRAP and CUPRAC assays and the ferrous ions (Fe²⁺) chelating activities. Moreover, the results of these activities were compared to those of standard antioxidant compounds such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and trolox. The results showed that the synthesized bromophenols had effective antioxidant power. The phenol 5 with two phenolic rings and five phenolic hydroxyl groups was the most potent antioxidant and radical scavenger. In conclusion, the new compounds are promising molecules to be used owing to their potential antioxidant properties. (3,4-Dihydroxyphenyl) (2,3,4-trihydroxyphenyl)methanone (5) and its two derivatives with bromine were synthesized. 4-(3,4-Dihydroxybenzyl)benzene-1,2,3-triol and its dibromide derivative (16) were also synthesized. The in vitro antioxidant activities of nine new compounds were determined, showing that the synthesized bromophenols had effective antioxidant power. The phenol 5 with two phenolic rings and five phenolic hydroxyl groups was the most potent antioxidant and radical scavenger.

Full text of DOI:10.1002/ardp.201100272

Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
323
Full Paper
Synthesis and Antioxidant Properties of (3,4-Dihydroxyphenyl)-
(2,3,4-trihydroxyphenyl)methanone and Its Derivatives
1
1
1,3
Yasin C¸ etinkaya1,2, Hu¨lya Goc¸er , Abdullah Menzek , and Ilhami Gulc¸in
¨
˙
¨
1 Department of Chemistry, Faculty of Science, Atatu¨rk University, Erzurum, Turkey
2 Atatu¨rk University, Oltu Vocational School, Department of Food Technology, Oltu-Erzurum, Turkey
3 Faculty of Sciences and Letters, Agri Ibrahim Cecen University, Agri, Turkey
(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone (5) and its two derivatives with bromine were
synthesized from reactions such as bromination and demethylation of (3,4-dimethoxyphenyl)(2,3,4-
trimethoxyphenyl)methanone (6). The Wolf-Kishner reduction product (9) of 6 and its three derivatives
with bromine were obtained. 4-(3,4-Dihydroxybenzyl)benzene-1,2,3-triol and its dibromide derivative (16)
were also synthesized from 9 and the corresponding dibromide derivative. The in vitro antioxidant activities
of nine new compounds synthesized in these reactions were determined by analyzing the radical
scavenging activities of bromophenols for 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),
1,1-diphenyl-2-picryl-hydrazyl (DPPH), N,N-dimethyl-p-phenylenediamine (DMPD), and the superoxide
anion radical (Oꢀ2ꢁ) and examining the total reducing power through Fe3þ-Fe2þ transformation, FRAP
and CUPRAC assays and the ferrous ions (Fe2þ) chelating activities. Moreover, the results of these activities
were compared to those of standard antioxidant compounds such as butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), a-tocopherol, and trolox. The results showed that the synthesized
bromophenols had effective antioxidant power. The phenol 5 with two phenolic rings and five
phenolic hydroxyl groups was the most potent antioxidant and radical scavenger. In conclusion, the
new compounds are promising molecules to be used owing to their potential antioxidant properties.
Keywords: Antioxidant activity / Bromination / Bromophenols / Phenols / Radical scavenging
Received: July 29, 2011; Revised: October 3, 2011; Accepted: October 11, 2011
DOI 10.1002/ardp.201100272
O
Br
O
Br
OH
Introduction
OH
OH
Br
OH
OH
OH
OH
HO
HO
HO
Bromophenols, abundantly found in marine life, have
some important biological activities [1, 2]. Bromophenols
1–3 are natural products [3–5] and some of them exhibit
enzyme inhibition [6–7], cytotoxicity [8], feeding deterrent
[3], antioxidant [9, 10] and microbial [11, 12] activities. Most
derivatives of compound 4 with Br also exhibit enzyme
inhibition [7] and antioxidant [10] activities. Compound 5,
similar to 4, has five OH groups. The compound 5 and its
derivatives, especially with Br, may exhibit important bio-
logical activities such as antioxidant activity.
R'
R
Br
OH
OH
OH
=
=
H
Br R'
1
2
3
R
R
R
R'
4
5
=
=
=
H
,
=
R' Br
Oxidative stress is characterized by an increased concen-
tration of intracellular oxidizing species, such as reactive
oxygen species (ROS), and is often accompanied by the loss
of antioxidant defense capacity. ROS are continuously pro-
duced by the body’s normal use of oxygen such as respiration
and some cell mediated immune functions. ROS include free
radicals such as superoxide anion radicals (Oꢀ2ꢁ), hydroxyl
radicals (OHꢀ), and nonfree radical species such as hydrogen
˙
Correspondence: Ilhami Gu¨lc¸in, Department of Chemistry, Faculty of
Sciences and Letters, Atatu¨rk University, 25240-Erzurum, Turkey.
E-mail: igulcin@atauni.edu.tr
Fax: þ90 4422360948
Additional correspondence: Abdullah Menzek
E-mail: amenzek@atauni.edu.tr
Fax: þ90 4422360948
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

324
Y. C¸ etinkaya et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
peroxide (H2O2) and singlet oxygen (1O2) [13, 14]. It is well
known that excessive ROS attack many organs and induce
oxidative damage directly to such critical biological mol-
ecules as lipoproteins, membrane lipids, polyunsaturated
fatty acids, carbohydrates and nucleotides causing lipid per-
oxidation and protein oxidation [15, 16]. Metabolic oxidative
stress has been implicated, directly or indirectly, in the
development of diseases and degenerative processes, includ-
ing inflammation, cancer, dementia and physiological aging
[17]. Antioxidants have recently emerged as a way of mini-
mizing the biomolecular damage caused by the attack of ROS
to vital constituents of living organisms [18, 19]. Antioxidants
protect the quality of foods by retarding oxidative breakdown
of the lipid components [20]. Commercial antioxidants are
generally synthetic compounds [21]. It is well known that
bromophenols display antioxidant and radical scavenging
activities [10]. Therefore, there is a great interest towards
new synthetic [10, 22–25] and natural [26–34] antioxidants
which can represent a good pharmacological alternative to
counteract oxidative stress [35].
To synthesize compounds without CO from 6, the Wolff-
Kishner reduction of ketone 6 was performed and diaryl-
methane derivative
9 was synthesized in high yield
(Scheme 2). Bromination of aromatic compounds with ceric
ammonium nitrate (CAN)/LiBr is selective bromination [39].
Brominations of 9 with different equivalents of LiBr/CAN
were also performed at RT by applying the same procedure.
Its reaction with 1.1 equivalents of LiBr/CAN gave a mixture
of three products while its reaction with 2.2 equivalents of
LiBr/CAN gave a sole product. The products were isolated and
their structures were determined. The product produced
in the reaction as sole product was dibromide 12, and the
products found in the mixture were bromides 10, 11 and 12.
Brominations of 9 with 5.1 equivalents of LiBr/CAN were
performed at RT so as to obtain products with more Br than
two. Dibromide 12 was also obtained from the reaction as
sole product (Scheme 2).
Reactions of 6–9 and 12 with BBr3 in CH2Cl2 were per-
formed at 0–258C in order to synthesize the phenol and
bromophenol derivatives and examine their antioxidant
properties. Phenol 5 and bromophenol derivatives 13–16
were synthesized from these demethylation reactions
(Scheme 2).
The aim of this study was to synthesize the compound 5
derivatives and investigate the effects of their biological
activities including ferric ions (Fe3þ) and cupric ions (Cu2þ
)
þ
reducing power, DPPHꢀ, ABTSꢀþ, DMPDꢀ , H2O2 and Oꢀ2ꢁ scav-
enging and ferrous ions (Fe2þ) chelating activity methods.
Antioxidant activity
Antioxidant compounds play important roles in the scaveng-
ing and inhibition of free radicals. Therefore, investigating
and finding new sources of antioxidants have gained
importance. The antioxidant potential of bromophenols
Results and discussion
Synthesis
þ
was analysed by DPPHꢀ scavenging, ABTSꢀ scavenging,
(3,4-Dimethoxyphenyl)(2,3,4-trimethoxyphenyl)methanone
(6) [36] was obtained from the reaction of 3,4-dimethoxyben-
zoic acid and 1,2,3-trimethoxybenzene in polyphosphoric
acid (PPA) by the known method (Scheme 1) [7, 10, 37, 38].
According to literature method [36, 39], bromination reactions
of 6 with 1.1 and 6.0 equivalents (equiv.) of ceric ammonium
nitrate (CAN)/LiBr at room temperature (RT) were performed.
A monobromide 7 and a dibromide 8 were obtained from
these reactions as the sole products (Scheme 1).
þ
DMPDꢀ scavenging, O2ꢀꢁ scavenging, Fe3þ-Fe2þ transform-
ation, cupric ion (Cu2þ) reducing and ferrous ion (Fe2þ
)
chelating assays.
Radical scavenging activity
Many diseases such as Parkinson’s disease, Alzheimer’s dis-
ease, atherosclerosis, inflammation and reperfusion injury
have been linked to ROS-mediated damage of biological
3 4-dimethoxybenzoic acid
,
a
e
M
O
e
r
M
O
B
O
O
e
M
O
O
7
e
e
e
e
O
M
O
M
M
e
e
M
M
O
b
c
e
M
O
e
O
M
O
M
O
e
O
M
O
e
M
O
e
r
M
O
B
e
OM
r
B
8
6
Scheme 1. Reagents and conditions: (a) 1,2,3-trimethoxybenzene-PPA, 808C, 1.5 h, 94%; (b) LiBr (1.1 equiv.)/CAN (1.1 equiv.),
CH3CN, RT, 3 d, 97%; (c) LiBr (6.0 equiv.)/CAN (6.0 equiv.), CH3CN, RT, 6 d, 95%.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
Synthesis and Antioxidant Properties of Phenols
325
Br
OMe
Br
OMe
Br
OMe
OMe
OMe
OMe
OMe
OMe
+
+
MeO
MeO
MeO
OMe
OMe
OMe
OMe
Br
10
11
12
5%
38%
52%
b
R1
O
OH
OMe
OH
OH
OMe
OMe
a
6
HO
MeO
OH
R2
OMe
e
13 R1 = H, R2 = Br
14 R1 = R2 = Br
9
c
or d
O
OH
R
OH
OH
OH
OH
OH
HO
12
HO
OH
OH
R
5
15 R = H
16 R = Br
Scheme 2. Reagents and conditions: (a) KOH-NH2NH2/(OHCH2)2, 110–1908C, 3 d, 83%; (b) LiBr (1.1 equiv.)/CAN (1.1 equiv.), CH3CN,
RT, 3 d; (c) LiBr (2.1 equiv.)/CAN (2.1 equiv.), CH3CN, RT, 3 d, 95%; (d) LiBr (5.1 equiv.)/CAN (5.1 equiv.), CH3CN, RT, 3 d, 98%; (e) BBr3,
CH2Cl2, 96%.
macromolecules which arises from an imbalance between
radical-generating and radical-scavenging systems [40].
ticated instrumentation is required. These chromogens are
easy to use, have a high sensitivity, and allow for rapid
analysis of the antioxidant activity of a large number of
samples. These assays have been applied to determine the
antioxidant activity of food components [40, 44].
þ
DPPHꢀ, ABTSꢀþ, DMPDꢀ and Oꢀ2ꢁ radical scavenging assays
are widely used to determine the radical scavenging abilities
of various samples [41–43]. The radical scavenging activity of
bromophenols was determined by DPPHꢀ, ABTSꢀþ, DMPDꢀþ
and Oꢀ2ꢁ scavenging assays in the present study. The efficient
concentration EC50 or concentration necessary to decrease
The antiradical activity of bromophenols or standard com-
pounds can be determined by assessing the scavenging
activity on DPPH radicals. In this assay, the purple chrom-
ogen radical DPPHꢀ is reduced by antioxidant/reducing com-
pounds to the corresponding pale yellow hydrazine [45, 46]. A
freshly prepared DPPH solution exhibits a deep purple colour
with an absorption maximum at 517 nm. This purple colour
generally disappears when an antioxidant is present in the
medium. Thus, antioxidant molecules can quench DPPH free
radicals by providing hydrogen atoms or by electron
donation, conceivably via a free-radical attack on the DPPH
molecule, and convert them to a colourless product. In this
assay, we measure the DPPH initial absorbance, and the
absorbance once the potential antioxidant has been added.
The reduction of absorbance is a measure of the free DPPH
due to the action of the antioxidant. The scavenging capacity
is generally evaluated in organic media by monitoring the
absorbance decrease at 517 nm until the absorbance remains
constant [47]. This method has been widely used to evaluate
þ
the initial DPPHꢀ, ABTSꢀþ, DMPDꢀ and Oꢀ2ꢁ concentration by
50% (in mM) was then obtained for each compound. The
results are summarized in Table 1. When an antioxidant is
added to the radicals, there is a degree of decolorization
owing to the presence of the antioxidants,þwhich reverse
the formation of the DPPHꢀ radical, ABTSꢀ and DMPDꢀþ
cation:
DPPHꢀ þ AH ! DPPHꢁH þ Aꢀ
ABTSꢀþ þ AH ! ABTSþ þ Aꢀ
DMPDꢀþ þ AH ! DMPDþ þ Aꢀ
These methods are rapid; a sample analysis takes 15 min in
total and little manpower, no expensive reagents or sophis-
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Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
þ
Table 1. EC50 values (mM) for DPPHꢀ, ABTSꢀþ, DMPDꢀ and O2ꢀꢁ radical scavenging assays of bromophenols and standard antioxidants
(ABTS: 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), DPPH: 1,1-diphenyl-2-picryl-hydrazyl, DMPD: N,N-dimethyl-p-
phenylenediamine, and Oꢀ2ꢁ: superoxide anion radicals, BHA: butylated hydroxyanisole, BHT: butylated hydroxytoluene; EC50
the antioxidant concentration (mM) necessary to decrease the initial radical concentration by 50%).
:
þ
þ
DPPHꢀ
scavenging
ABTSꢀ
scavenging
DMPDꢀ
scavenging
Oꢀ2ꢁ
scavenging
Metal chelating
Ferrozine
Bipyridyl
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
BHA
BHT
a-Tocopherol
Trolox
5
400
171
109
233
87
162
77
99
198
71
85
36
84
423
144
191
47
265
161
195
193
20
160
87
50
232
80
68
55
78
56
62
54
52
46
90
129
73
97
67
26
85
68
53
50
51
31
70
38
98
73
84
29
25
24
29
18
27
25
9
36
18
67
23
57
227
103
70
91
116
38
42
47
3
9
10
11
12
13
14
15
16
108
19
93
66
the free radical scavenging activity of various antioxidant
substances [48, 49]. This method is based on the reduction of
DPPH in alcoholic solutions in the presence of a hydrogen-
donating antioxidant due to the formation of the non-radical
form DPPH-H in the reaction [30]. The dark colour of the DPPH
radical becomes lighter due to the antioxidant compound,
causing a decrease in the absorbance at 517 nm. The DPPH
free radical scavenging activities of bromophenols and
standard antioxidants (BHA, BHT, a-tocopherol and trolox)
were investigated.
As can be seen in Table 1, the DPPHꢀ, ABTSꢀþ, DMPDꢀþ and
Oꢀ2ꢁ radical scavenging activities of the new synthesized
compounds (5 and 9–16) were evaluated. Lower IC50 values
indicate higher DPPHꢀ radical scavenging ability. In this
respect, all of the newly synthesized compounds (5 and 9–
16) exhibited radical scavenging abilities on DPPHꢀ
when compared to standard antioxidants such as BAH,
BHT, a-tocopherol and trolox. As can seen in Table 1, the
most effective IC50 value was found in phenol compound 15
as 36 mM (r2: 0.961). On the other hand, IC50 values of
four standard compounds were changed from 109 to 400 mM.
Another effective method to measure the radical scavenging
activity is the ABTS radical cation decolourisation assay, which
showed similar results to those obtained in the DPPH reaction.
Similar to the DPPH radical scavenging activity, the new com-
pounds (5 and 9–16) had effective ABTS radical scavenging
activity. The ABTS radical scavenging activities of the new
compounds (5 and 9–16) and standard antioxidants
decreased in the following order: 15 (19 mM) ꢂ 13 (20 mM) <
5 (47 mM) < BHA (84 mM) < 16 (93 mM) < 14 (108 mM) < a-
12 (193 mM) ꢃ 11 (195 mM) < 9 (265 mM) < BHT (423 mM),
respectively. These results show that the newly synthesized
compounds have effective ABTS radical scavenging activity
(Table 1).
Another assay used to measure the radical scavenging
þ
activity involves the decolourisation of the DMPDꢀ cation
radical, similar to the DPPH scavenging and ABTS cation
þ
radical decolourisation assay. DMPDꢀ has a maximum
absorbance at 505 nm. Antioxidant compounds or radicþal
scavengers, which can act as hydrogen donors for DMPDꢀ
,
þ
decrease the absorbance at 505 nm of DMPDꢀ [50]. As it is
seen in Table 1, the new compounds (5 and 9–16) have
þ
effectivþe DMPDꢀ radical scavenging activity and their
DMPDꢀ scavenging capacity decreased in the following
order: 16 (46 mM) < a-tocopherol (50 mM)
ꢃ
15 (52 mM)
ꢃ 14 (54 mM) ꢃ 10 (55 mM) ꢃ 12 (56 mM) < 13 (62 mM) < 9
(68 mM) < 11 (78 mM) < 5 (80 mM) < BHT (87 mM) < BHA
(160 mM) < trolox (191 mM). These results show that thþe
newly synthesised compounds have marked DMPDꢀ
scavenging activity.
Superoxide radical anion (Oꢀ2ꢁ) is produced as a result of
the donation of one electron to oxygen. This radical arises
either from several metabolic processes or following oxy-
gen activation by irradiation. This process can generate
other more harmful ROS, such as H2O2, OHꢀ, HOCl and
1O2 [40, 51]. Two different in vitro superoxide-generating
systems are commonly used. The first one is a xanthine
oxidase–hypoxanthine system and the other one is a
riboflavin–methionine–illuminate system. In this study,
we preferred the riboflavin–methionine–illuminate system
to generate superoxide anion radicals.
tocopherol (144 mM) < 10 (161 mM) < trolox (191 mM)
ꢃ
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Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
Synthesis and Antioxidant Properties of Phenols
327
The greatest inhibition of superoxide radical formation
was observed in bromophenol compound 16 (Table 1). IC50
value of this compound (3 mM) was lower than that of the
used standard and bromophenol compounds.
the new compounds interfered with the formation of the
ferrous-ferrozine complex. It suggests that bromophenol
compounds have chelating activity and are able to capture
ferrous ion before ferrozine [14]. The present study demon-
strated that the new compounds bound ferrous ions (Fe2þ).
Metal-binding capacity was investigated by assessing the abil-
ity of the antioxidants to compete with the indicator ferro-
zine to complex with ferrous ions (Fe2þ) in solution [13].
Bromophenol compounds had strong chelating effect on
ferrous ions (Fe2þ). All new compounds exhibited effective
chelation of ferrous ion (Fe2þ). As can be seen in Table 1,
the IC50 values of the ferrous ion (Fe2þ) chelating effects of the
new compounds ranged between 30–100 mM.
The riboflavin–methionine–illuminate system was used to
generate superoxide anion radicals (Oꢀ2ꢁ) in this study. Then,
superoxide anion radicals reduced NBT2þ to produce forma-
zan which was blue in colour. Antioxidants inhibit the for-
mation of blue NBT [52]. Superoxide anion radicals appear
indirectly when the assay is performed under aerobic con-
ditions. In the presence of an antioxidant molecule that can
donate an electron to NBT, the typical purple colour of for-
mazan decays and this change can be monitored spectropho-
tometrically at 560 nm. Antioxidants have the ability to
inhibit the formation of NBT and scavenge superoxide anion
radicals. The decrease observed in absorbance at 560 nm in
the presence of antioxidants indicates the scavenging of
superoxide anions in the reaction mixture [53].
A significant drawback of this complexation reaction in
measuring the presence of antioxidant chelator is that the
reaction is affected by both the antioxidant-Fe2þ and ferro-
zine-Fe2þ complex formation constants, and the competition
between the two chelators for binding to iron. Thus, a weak
antioxidant iron chelator would be seriously underestimated
in quantitative determination. From a nutritional point of
view, it is not yet possible to assess the role of a weak anti-
oxidant iron chelator in preventing the Fenton reaction
in vivo. Nonetheless, this reaction serves as a convenient assay
to assess the iron chelating activity of antioxidants.
Antioxidants inhibit the formation of blue NBT [52]. The
two principal reactions are involved in this assay are [54]:
2NBTHꢀ ! NBT þ NBTH2 ðFormazanÞ ðaÞ
NBTHꢀ þ O2 $ NBT þ Oꢀ2ꢁ ðA quasi equilibriumÞ ðbÞ
2þ
3 Ferrozine þ FeðH2OÞ ! Fe-ðFerrozineÞ43ꢁ þ 6H2O
When riboflavin is photochemically activated, it reacts
with NBT to generate NBTHꢀ [55], which leads to formazan
according to reaction (a). In the presence of oxygen, radical
species are controlled by quasi-equilibrium (b). Thus, super-
oxide anion radicals appear indirectly when the assay is
performed under aerobic conditions. In the presence of an
antioxidant molecule that can donate an electron to NBT, the
typical purple colour of formazan decays, which can be
followed spectrophotometrically at 560 nm. Antioxidants
are able to inhibit the formation of NBT and scavenge super-
oxide anion radicals. The decrease in absorbance at 560 nm
in the presence of antioxidants indicates the scavenging of
superoxide anions in the reaction mixture [43].
6
The metal chelating capacity was significant since it
reduced the concentration of the catalyzing transition metal
in lipid peroxidation. It was reported that chelating agents
are effective as secondary antioxidants because they reduce
the redox potential, thereby stabilizing the oxidized form of
the metal ions [56].
Another method that is widely used in measuring the
metal chelating activity is 2,20-bipyridine-ferrous ion (Fe2þ
)
chelating activity. Metal ions can cause lipid peroxidation
which, in turn, can produce free radicals and lipid peroxides
[45]. Therefore, metal chelating activity indicates the antiox-
idant property. The decreased absorbance in the reaction
mixture indicates higher metal chelating ability. 2,20-
Bipyridine forms a complex with free Fe2þ but not with
Fe2þ that is bound to other chelators. Thus, a decrease in
the amount of 2,20-bipyridine-Fe2þ complex formed after
treatment indicates the presence of antioxidant chelators.
The 2,20-bipyridine-Fe2þ complex produced a chromophore
with absorbance that could be measured at 522 nm.
According to the results, bromophenols had effective ferrous
ion (Fe2þ) binding afinity. The IC50 values of metal chelating
by 2,20-bipyridine ranged between 9–84 mM. The results
obtained from both metal chelating methods clearly showed
that compound 12 had the most powerful ferrous ions metal
chelating effect (Table 1). The compound 12 had five methoxy
Metal ion chelating activity
One measurement of the metal-chelating activity of an
antioxidant is based on the absorbance measurement of
the Fe2þ-ferrozine complex after the prior treatment of a
ferrous ion solution with test material. The metal chelating
capacity was significant since it reduced the concentration of
the catalyzing transition metal in lipid peroxidation. It was
reported that chelating agents are effective as secondary
antioxidants because they reduce the redox potential [43].
The data obtained from Table 1 reveal that bromophenol
compounds possess marked capacity for iron binding,
suggesting that their main action as a peroxidation inhibitor
may be related to their iron binding capacity. In this assay,
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Y. C¸ etinkaya et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
groups (–OCH3). This compound may chelate the ferrous ion
(Fe2þ) with its five methoxy groups (–OCH3). The compounds
with structures containing two or more of these functional
groups in a favorable structure–function configuration can
show ferrous ion (Fe2þ) chelation activity [32].
bromophenols and standard compounds. The presence of
different substituents in the phenol backbone structure
modulates their antioxidant property, in particular their
hydrogen-donating capacity. In general, unsubstituted
phenol is inactive as hydrogen donor and monophenol is a
less efficient antioxidant than polyphenol. The introduction
of an electron-donating group such as a hydroxyl group in
the ortho or para position increases the antioxidant activity
of phenol. In addition, the reducing power of a phenolic
molecule increases with the presence of an extra hydroxyl
group on the phenolic ring. When the structure of compound
5 was compared to compound 4, it is clearly seen that com-
pound 5 had an extra phenolic hydroxyl group (–OH) than
compound 4. Because of this phenolic hydroxyl group (–OH),
Reducing power determination
The reduction capacity of a compound may serve as a sig-
nificant indicator of its potential antioxidant activity.
Antioxidant compounds are able to donate electrons to reac-
tive radicals converting them into more stable and unreac-
tive species [57]. Reducing powers of the newly synthesized
compounds (5 and 9–16) were investigated by FRAP and
CUPRAC assays.
Antioxidant compounds reduce Fe3þ-ferricyanide com-
plexes to the ferrous (Fe2þ) form. The Prussian blue coloured
complex is formed by adding FeCl3 to the ferrous (Fe2þ) form.
Therefore, the amount of reduction can be determined by
measuring the formation of Perl’s Prussian blue at 700 nm
[58]. In this assay, the yellow colour of the test solution turned
to green or blue depending on the reducing power of the
antioxidant. A higher absorbance means a higher ferric
reducing power.
compound 5 (2.029) demonstrated higher ferrous (Fe2þ
)
reducing activity than compound 4 (1.737). Ferric ion
(Fe3þ) reduction is often used as an indicator of electron-
donating activity which is an important mechanism of phe-
nolic antioxidants [59].
The CUPRAC method is also used to determine the
reducing powers of antioxidant compounds [60]. This
method is based on the reduction of Cu2þ to Cuþ by anti-
oxidants in the presence of neocuproine [61]. In this assay, a
higher absorbance indicates higher cupric ion (Cu2þ) reduc-
ing ability. Of all compounds, bromophenol compound 5
(0.794 ꢅ 0.061) has the highest cupric ion (Cu2þ) reducing
activity (Table 2). Also, the cupric ion (Cu2þ) reducing activity
absorbance values of the synthesized new compounds (5 and
9–16) ranged from 0.129 to 0.794 at the concentration of
10 mg/mL. These values were found to be 0.759 for resveratrol
[14], 1.085 for cepharanthine, 1.336 for fangchinoline [45],
0.350 for eugenol [56], 0.568 for propolis [16], 0.190–1.009
for some 5,10-dihydroindeno[1,2-b]indole derivatives [62].
Reducing power of bioactive compounds or food compone-
nets reflects the electron donating capacity and is associated
with antioxidant activity. Bioactive compounds with antiox-
idant effects can be reductants and inactivate oxidants [63].
The reducing capacity of a bioactive compound can be
measured by the direct reduction of Fe[(CN)6]3 to Fe[(CN)6]2.
Addition of free Fe3þ to the reduced product leads to the
formation of the intense Perl’s Prussian blue complex,
Fe4[Fe(CN-)6]3, which has a strong absorbance at 700 nm [43].
Reductant
FeðCNÞ6 ꢁꢁꢁꢁꢁꢁꢁꢁ! FeðCNÞ6
3ꢁ
4ꢁ
FeðCNÞ46ꢁ þ FeCl3 ꢁꢁꢁꢁꢁ! Fe4½FeðCNÞ ꢄ
6 3
As shown in Table 2, the synthesized new compounds
(5 and 9–16) demonstrated effective Fe3þ-Fe2þ reducing
power. Especially, phenol
5
had the best activity
(2.029 ꢅ 0.199) when compared to the other synthesized
Table 2. Reducing power of synthesised bromophenols and
standard compounds by Fe3þ-Fe2þ transformation ability, Cu2þ-Cuþ
reducing ability and FRAP assay (BHA: butylated hydroxyanisole,
BHT: butylated hydroxytoluene).
Fe3þ-Fe2þ
reducing
power
Cu2þ-Cuþ
reducing
power
FRAP
assay
BHA
BHT
Trolox
1.900 ꢅ 0.178
0.789 ꢅ 0.265
0.283 ꢅ 0.072
0.728 ꢅ 0.091
0.519 ꢅ 0.233
0.260 ꢅ 0.039
0.345 ꢅ 0.073
0.794 ꢅ 0.061
0.129 ꢅ 0.007
0.202 ꢅ 0.032
0.139 ꢅ 0.025
0.162 ꢅ 0.041
0.667 ꢅ 0.026
0.306 ꢅ 0.057
0.743 ꢅ 0.066
0.638 ꢅ 0.015
0.508 ꢅ 0.367
2.402 ꢅ 0.259
1.510 ꢅ 0.165
1.159 ꢅ 0.346
0.538 ꢅ 0.381
0.807 ꢅ 0.320
2.108 ꢅ 0.027
0.574 ꢅ 0.348
0.588 ꢅ 0.239
0.952 ꢅ 0.286
2.398 ꢅ 0.262
1.080 ꢅ 0.189
2.590 ꢅ 0.237
Reductant
FeðCNÞ6 ꢁꢁꢁꢁꢁꢁꢁꢁ! FeðCNÞ6
3ꢁ
4ꢁ
a-Tocopherol 0.532 ꢅ 0.021
5
9
2.029 ꢅ 0.199
0.300 ꢅ 0.283
0.118 ꢅ 0.013
0.126 ꢅ 0.039
0.122 ꢅ 0.019
1.971 ꢅ 0.163
0.490 ꢅ 0.099
1.768 ꢅ 0.214
1.556 ꢅ 0.345
4ꢁ
FeðCNÞ6 þ Fe3þꢁꢁꢁꢁꢁ! Fe4½FeðCNÞ6ꢄ3
10
11
12
13
14
15
16
An increase in absorbance of the reaction mixture would
indicate an increase in the reducing capacity due to an
increase in the formation of the complex. There are a number
of assays designed to measure overall antioxidant activity, or
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Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
Synthesis and Antioxidant Properties of Phenols
329
reducing potential, as an indication of a host’s total capacity
to withstand free radical stress. The ferric ions (Fe3þ) reduc-
ing antioxidant power assay takes advantage of a single
electron transfer in which a ferric salt is used as an oxidant
[33]. In this assay, the yellow colour of the test solution
changes to various shades of green and blue depending on
the reducing power of antioxidant samples. The reducing
capacity of a compound may serve as a significant indicator
of its potential antioxidant activity [26]. This method is not
only cost-effective and rapid, it is also robust, selective and
suitable for a variety of antioxidants regardless of chemical
type or hydrophilicity [19].
does not react rapidly with some antioxidants such as gluta-
thione. However, we are of the opinion that the FRAP assay is
still suitable for assessment of antioxidant activities of new
compounds because only a limited amount of plant gluta-
thione is absorbed by humans [69]. As can be seen in Table 2,
all compounds showed marked [Fe3þ-(TPTZ)2]3þ-[Fe2þ-(TPTZ)2]2þ
reducing abilities. However, the most powerful
[Fe3þ-(TPTZ)2]3þ reducing power was observed in bromophe-
nol compound 16 (2.590 ꢅ 0.237). This activity was greater
than that of BHA (0.508 ꢅ 0.367), BHT (2.402 ꢅ 0.259),
a-tocopherol (1.159 ꢅ 0.346) and trolox (1.510 ꢅ 0.165).
The FRAP assay measures the ability of antioxidants Conclusion
to reduce the ferric 2,4,6-tripyridyl-s-triazine complex
[Fe3þ-(TPTZ)2]3þ to the intensely blue coloured ferrous com-
plex [Fe2þ-(TPTZ)2]2þ in acidic medium. As can be seen in
Scheme 3, the reaction measures reduction of ferric 2,4,6-
tripyridyl-s-triazine (TPTZ) to a colored product [64].
Syntheses of phenols (5 and 15) and bromophenols (13, 14 and
16) were performed in two steps. The first step was the
synthesis of their derivatives with methoxides 6–12 because
phenolic OH groups in reactions were usually protected as
arylmethylethers [5, 10, 15, 17, 70–73]. The second step was
the demethylation of the corresponding compounds.
FRAP values are calculated by measuring the absorbance
increase at 593 nm and relating it to a ferrous ions standard
solution or to an antioxidant standard solution such as
ascorbic acid. This method has also better reproducibility
and higher sample throughput [65]. As the FRAP assay
measures the reducing capacity based upon reduction of
ferric ion, antioxidants acting by radical quenching such
as thiols and carotenoids in particular will not be determined
[66, 67]. In this respect, this method is very suitable in order
to determine the reducing capacity of the new compounds.
We selected the FRAP assay to evaluate the antioxidant activi-
ties of the new phenolic compounds for the following
reasons. Firstly, the FRAP assay treats the antioxidants in
the sample as reductants in a redox-linked colorimetric reac-
tion. Secondly, the procedure of FRAP assay is relatively
simple and easy to be standardized. This method has been
frequently used to conduct rapid evaluations on the total
antioxidant capacities of various food and beverages as well
as pure compounds [68]. Furthermore, it has been applied to
measure the antioxidant activity of polyphenols [66]. One
probable disadvantage of this assay is the fact that this assay
(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl) methanone
(5), its monobromide 13 and dibromide 14 derivatives were
synthesized from corresponding reactions such as bromina-
tion and demethylation of (3,4-dimethoxyphenyl)(2,3,4-tri-
methoxyphenyl) methanone (6). The Wolf-Kishner reduction
product (9) of 6 reacted with LiBr/CAN to produce three
bromides derivatives 10–12. 4-(3,4-Dihydroxybenzyl)-
benzene-1,2,3-triol (15) and its dibromide derivative 16 were
also synthesized by demethylation of 9 and dibromide 12,
respectively. Nine new compounds were synthesized in these
reactions and their structures were determined.
In the context of our study, we have determined antiox-
idant, antiradical and metal chelating effects of the newly
synthesized compounds (5 and 9–16) and four standard anti-
oxidant molecules (BHA, BHT, a-tocopherol and trolox). The
resultsþclearly indicate that the most effective DPPHꢀ, ABTSꢀþ
,
DMPDꢀ and Oꢀ2ꢁ radical scavenging activities were found in
phenol 15. This synthesized phenol was found as the most
potent radical scavenger. On the other hand, phenol 5
N
N
N
N
N
N
N
N
N
N
N
N
N
Fe2+
N
N
Fe3+
N
N
N
N
N
N
N
N
N
Fe3+-TPTZ
Fe2+-TPTZ
Scheme 3. [Fe3þ-(TPTZ)2]3þ-[Fe3þ-(TPTZ)2]2þ reduction reaction for the FRAP assay.
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330
Y. C¸ etinkaya et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
water (2 ꢆ 20 mL), and brine (20 mL). Then it was dried over
Na2SO4 and the solvents were evaporated. The residue was sub-
jected to column chromatography on silica gel (SiO2, 110 g) and
eluted using EtOAc/hexane (1:9) to give dibromide 12 (47 mg,
5%), monobromide 10 (299 mg, 38%) and monobromide 11
(409 mg, 52%), respectively.
obtained maximum reducing power in three different
assays.
Experimental section
General information
1-Bromo-5-(3,4-dimethoxybenzyl)-2,3,4-
All chemicals and solvents are commercially available and were
used after distillation or treatment with drying agents. Melting
points were determined on a capillary melting apparatus (BUCHI
530) and are uncorrected. IR spectra were obtained from
solutions in 0.1 mm cells with a Perkin-Elmer spectropho-
tometer. The 1H- and 13C-NMR spectra were recorded on a 200
(50) and 400 (100)-MHz Varian spectrometer; d in ppm, Me4Si as
the internal standard. Elemental analyses were performed on a
Leco CHNS-932 apparatus. All column chromatography was per-
formed on silica gel (60-mesh, Merck). PLC is preparative thick-
layer chromatography: 1 mm of silica gel 60 PF (Merck) on glass
plates.
trimethoxybenzene (10)
Liquid as pale yellow. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.00
(bs, aromatic, 1H), 6.79 (d, A part of AB-system, J ¼ 8.1 Hz, aromatic,
1H), 6.73 (s, aromatic, 1H), 6.71 (dd, B part of AB-system, J ¼ 8.2,
1.83 Hz, aromatic, 1H), 3.91 (s, OCH3, 3H), 3.87 (s, OCH3, 3H), 3.85
(s, OCH3, 6H), 3.83 (s, CH2, 2H), 3.76 (s, OCH3, 3H). 13C-NMR
(100 MHz, CDCl3) d 151.55 (C), 149.91 (C), 149.14 (C), 147.73
(C), 133.04 (C), 132.20 (C), 127.93 (CH), 127.59 (C), 121.06 (CH),
112.52 (CH), 111.51 (CH), 111.47 (C), 61.19 (OCH3), 61.15 (OCH3),
61.04 (OCH3), 56.12 (OCH3), 56.10 (OCH3), 35.44 (-CH2–).
IR (CH2Cl2, cmꢁ1) 2928, 2850, 1590, 1513, 1460, 1418, 1403,
1292, 1236, 1139, 1082, 1037, 1006, 962. Anal. Calcd. for
(C18H21BrO5): C 54.42; H 5.33 Found C 54.47; H 5.34.
The compounds 6–8 were synthesized according to [36].
Synthesis of 1-(3,4-dimethoxybenzyl)-2,3,4-
trimethoxybenzene (9)
1-(2-Bromo-4,5-dimethoxybenzyl)-2,3,4-
trimethoxybenzene (11)
To
a
solution of the ketone
6
(2.1 g, 6.3 mmol) in
Amorphous as pale yellow. Mp 75–778C. 1H-NMR (400 MHz,
CDCl3) d (ppm): 7.03 (s, aromatic, 1H), 6.68 (d, A part of
AB-system, J ¼ 8.6 Hz, aromatic, 1H), 6.63 (s, aromatic, 1H),
6.58 (d, B part of AB-system, J ¼ 8.6 Hz, aromatic, 1H), 3.97
(s, CH2, 2H), 3.87 (s, OCH3, 3H), 3.85 (s, OCH3, 3H), 3.832
(s, OCH3, 3H), 3.828 (s, OCH3, 3H), 3.75 (s, OCH3, 3H).
13C-NMR (100 MHz, CDCl3) d 152.62 (C), 152.04 (C), 148.58 (C),
148.15 (C), 132.56 (C), 126.19 (C), 124.31 (CH), 115.67 (CH), 114.71
(C), 113.86 (CH), 113.77 (C), 107.34 (CH), 60.97 (OCH3), 60.95
(OCH3), 56.36, (OCH3), 56.15 (2 OCH3), 35.44 (CH2). IR (CH2Cl2,
cmꢁ1) 3072, 3008, 2937, 2834, 2602, 2021, 1723, 1601, 1510,
1491, 1467, 1443, 1428, 1415, 1385, 1343, 1329, 1257, 1228,
1200, 1193, 1170, 1095, 1036, 972, 937, 905. Anal. Calcd. for
(C18H21BrO5): C 54.42; H 5.33 Found C 54.50; H 5.36.
OHCH2CH2OH (4.0 mL) were added KOH (227 mg, 5.9 mmol)
and hydrazine hydrate (0.6 mL, 619 mg, 12 mmol), consecu-
tively, at RT and under N2(g). After the mixture was heated to
1108C and stirred at this temperature for 1 h, it was stirred at
1908C for 3 d. The mixture was cooled to RT, acidified with HCl
(10%), and extracted with ethyl acetate (EtOAc) (3 ꢆ 40 mL). The
combined organic phases were dried over Na2SO4 and the solvent
was evaporated. The residue with EtOAc/hexane (3:7) was
filtrated from silica gel column (SiO2, 80 g) and 9 (83%, 1.67 g)
was obtained and crystallized from methanol as white crystals
in the refrigerator at 48C. Mp 58–608C. 1H-NMR (400 MHz, CDCl3)
d (ppm): 6.79 (d, A part of AB-system, J ¼ 8.1 Hz, aromatic, 1H),
6.78 (d, A part of AB-system, J ¼ 8.5 Hz, aromatic, 1H), 6.75
(d, J ¼ 1.7 Hz, aromatic, 1H), 6.72 (dd, B part of AB-system,
J ¼ 8.1, 1.8 Hz aromatic, 1H), 6.60 (d, B part of AB-system,
J ¼ 8.5 Hz, aromatic, 1H), 3.88 (s, OCH3, 3H), 3.87 (s, OCH3,
3H), 3.85 (s, CH2, 2H), 3.84 (s, OCH3, 6H), 3.77 (s, OCH3, 3H).
13C-NMR (100 MHz, CDCl3) d 152.5 (C), 152.0 (C), 149.1 (C),
147.5 (C), 142.6 (C), 134.3 (C), 127.7 (C), 124.5 (CH), 120.9 (CH),
112.5 (CH), 111.4 (CH), 107.4 (CH), 61.0 (OCH3), 60.9 (OCH3), 56.2
1-Bromo-5-(2-bromo-4,5-dimethoxybenzyl)-2,3,4-
trimethoxybenzene (12)
Pale yellow crystals from CH2Cl2/hexane. Mp 97–1008C. 1H-NMR
(400 MHz, CDCl3) d (ppm): 7.03 (s, aromatic, 1H), 6.87 (s, aromatic,
1H), 6.64 (s, aromatic, 1H), 3.95 (s, CH2, 2H), 3.91 (s, OCH3, 3H),
3.88 (s, OCH3, 3H), 3.86 (s, OCH3, 3H), 3.84 (s, OCH3, 3H), 3.78
(s, OCH3, 3H).
(OCH3), 56.1 (OCH3), 56.0 (OCH3), 35.5 (CH2). IR (CH2Cl2, cmꢁ1
)
2991, 2940, 2836, 1601, 1513, 1494, 1465, 1442, 1417, 1311,
1275, 1260, 1204, 1164, 1136, 1100, 1094, 1027, 917. Anal.
Calcd. for (C18H22O5): C 67.91; H 6.97. Found C 67.97; H 6.99.
(C18H22O5): C 67.91 MS m/z (CI, methane) 318.1 (Mþ).
13C-NMR (100 MHz, CDCl3) d 151.56 (C), 150.00 (C), 148.67 (C),
148.44 (C), 147.64 (C), 131.31 (C), 130.75 (CH), 127.57 (CH), 115.78
(CH), 114.85 (CH), 113.94 (CH), 111.55 (C), 61.21, 61.00, 56.37,
56.26 (OCH3), 35.35 (CH2). IR (CH2Cl2, cmꢁ1) 3074, 2957, 2052,
2010, 1935, 1715, 1610, 1592, 1522. Anal. Calcd. for (C18H21BrO5):
C 45.40; H 4.23 Found C 45.58; H 4.22.
Standard procedure for bromination with LiBr/CAN
Bromination of 1-(3,4-dimethoxybenzyl)-2,3,4-
trimethoxybenzene (9) with LiBr/CAN (1 equiv.)
1-(3,4-Dimethoxybenzyl)-2,3,4-trimethoxybenzene (9)
with different equivalents (2.1 and 6.0.) of LiBr/CAN
To a solution of 6 (630 mg, 1.98 mmol) and LiBr (172 mg,
1,98 mmol) in CH3CN (15 mL) was added a solution of CAN
(1.086 g, 1.98 mmol) in CH3CN (15 mL) dropwise at RT and
under N2 over 10 min. After the solution was stirred at RT
and under N2 for 3 d, water (15 mL) was added and the mixture
was extracted with EtOAc (3 ꢆ 40 mL). The combined organic
phases were washed with solutions (5%, 2 ꢆ 20 mL) of NaHCO3,
Only dibromide 12 (287 mg, 95%) was obtained when the
reaction of
6 (200 mg, 0,6 mmol, 1.0 equiv.) with LiBr
(114 mg, 1.32 mmol, 2.1 equiv.) and CAN (724 mg, 1.32 mmol,
2.1 equiv.) was performed according to the standard procedure
described above. The reaction of 6 (200 mg, 0.6 mmol, 1.0 equiv.)
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Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
Synthesis and Antioxidant Properties of Phenols
331
with LiBr (273 mg, 3.14 mmol, 5.1 equiv.) and CAN (1.72 g,
3.14 mmol, 5.1 equiv.) at the same condition also gave dibromide
12 (293 mg, 98%) as sole product.
(CO), 151.60 (C), 149.95 (C), 148.05 (C), 144.82 (C), 133.37 (C),
130.76 (C), 128.14 (CH), 119.62 (CH), 115.96 (CH), 114.10 (C),
108.25 (CH), 99.91 (C). IR (acetone, cmꢁ1): 3467, 1970, 1739,
1630, 1514, 1474, 1434, 1373, 1190, 1099, 1019, 960. Anal.
Calcd. for (C13H8Br2O6): C 37.18; H 1.92 Found C 37.07; H 1.91.
Standard procedure for demethylation of compounds
with OMe by ether cleavage
Synthesis of (3,4-dihydroxyphenyl)(2,3,4-
4-(3,4-Dihydroxybenzyl)benzene-1,2,3-triol (15)
It was filtered from silica gel (50 g) with EtOAc/CH2Cl2 (1:1) and
obtained as pale brown solid (94%). Mp 127–1298C. 1H-NMR
(400 MHz, CD3COCD3) d (ppm): 6.70 (d, A part of AB-system,
J ¼ 8.0 Hz, aromatic, 1H), 6.70 (d, J ¼ 2.0 Hz, aromatic, 1H),
6.57 (dd, B part of AB-system, J ¼ 8.0, 2.0 Hz, aromatic, 1H),
6.39 (d, A part of AB-system, J ¼ 8.3 Hz, aromatic, 1H), 6.32
(d, B part of AB-system, J ¼ 8.3 Hz, aromatic, 1H), 3.85 (s, methyl-
enic, 2H). 13C-NMR (100 MHz, CD3COCD3) d 144.88 (C), 144.04 (C),
143.97 (C), 143.10 (C), 133.85 (C), 132.84 (C), 120.52 (CH), 120.44
(C), 120.21 (CH), 116.06 (CH), 115.10 (CH), 106.80 (CH), 34.56 (CH2).
IR (acetone, cmꢁ1): 3335, 1804, 1693, 1620, 1500, 1432, 1339,
1279, 1222, 1184, 1143, 1050, 964. (C13H12O5): C 62.90; H 4.87
Found C 62.74; H 4.88.
trihydroxyphenyl) methanone (5)
A solution of ketone 6 (400 mg, 1.2 mmol) in CH2Cl2 (15 mL) was
cooled to 08C and then a solution of BBr3 (0.8 mL, 8.30 mmol) in
CH2Cl2 (12 mL) was added dropwise under N2(g) over 5 min. After
the cold bath was removed, the mixture was stirred at RT and
under N2 for 1 day. After the reaction mixture was cooled to 08C,
methanol (25 mL) was slowly added over 15 min and then the
solvent was evaporated. After water (40 mL) and EtOAc (50 mL)
were added, the mixture was shaken. The organic phase was
separated and the water phase was extracted with EtOAc
(2 ꢆ 40 mL). The combined organic phases were washed with
Na2S2O3 (saturated, 2 ꢆ 50 mL), NaHCO3 (5%, 2 ꢆ 50 mL) and
water (2 ꢆ 100 mL), and then it was dried over Na2SO4 and the
solvent was evaporated. The phenol 5 (302 mg, 96%) was
obtained from EtOAc/hexane as yellow solid. Mp 206–2088C.
1H-NMR (400 MHz, CD3COCD3) d (ppm): 12.65 (s, OH, 1H), 8.42
(s, OH, 4H), 7.25 (d, J ¼ 2.1 Hz, aromatic, 1H), 7.20 (d, A part of
4-Bromo-6-(2-bromo-4,5-dihydroxybenzyl)benzene-1,2,3-
triol (16)
It was filtered from silica gel (50 g) with EtOAc/CH2Cl2 (1:1) and
obtained as brown solid (95%). Mp 114–1168C. 1H-NMR (400 MHz,
CD3COCD3) d (ppm): 8.08 (OH, 1H), 8.07 (OH, 1H), 7.86 (OH, 1H),
7.79 (OH, 1H), 7.62 (OH, 1H), 7.04 (s, aromatic, 1H), 6.62
(s, aromatic, 1H), 6.61 (s, aromatic, 1H), 3.85 (s, methylenic,
2H). 13C-NMR (100 MHz, CD3COCD3) d 144.98 (C), 144.75 (C),
143.91 (C), 141.74 (C), 134.43 (C), 131.08 (C), 123.12 (CH),
120.64 (C), 119.11 (CH), 117.43 (CH), 112.73 (C), 100.06 (C),
34.29 (CH2). IR (acetone, cmꢁ1): 3335, 1809, 1693, 1620, 1500,
1432, 1339, 1279, 1222, 1185, 1143, 1050, 963. (C13H10Br2O5):
C 38.46; H 2.48 Found C 38.53; H 2.47.
AB-system, J ¼ 8.9 Hz, aromatic, 1H), 7.15 (dd,
A part of
AB-system, J ¼ 2.1, 8.2 Hz, aromatic, 1H), 6.96 (d, B part of
AB-system, J ¼ 8.2 Hz, aromatic, 1H), 6.48 (d, B part of AB-system,
J ¼ 8.9 Hz, aromatic, 1H). 13C-NMR (100 MHz, CD3COCD3)
d 194.34 (CO), 155.88 (C), 153.48 (C), 152.52 (C), 149.14 (C),
131.32 (C), 127.03 (C), 125.79 (CH), 124.66 (CH), 111.51 (CH),
111.40 (CH), 109.95 (C), 106.95 (CH). IR (acetone, cmꢁ1): 2929,
2683, 1637, 1563, 1496, 1217, 1106, 1073, 1000, 930. Anal. Calcd.
for (C13H10BrO6): C 59.55; H 3.84 Found C 59.39; H 3.85.
Synthesis of phenols 5, 13–16 from 6–9 and 12
The standard procedure described above was applied. Phenols 5,
13–16 were obtained from these reactions.
Antioxidant activity determination
DPPHꢀ scavenging activity
The DPPH free radical scavenging activity of bromophenols was
evaluated by the method of Blois [46] as previously described by
Gu¨lc¸in [72]. Briefly, different concentrations (66–400 mg/mL) of
bromophenols were prepared and diluted to 3 mL with ethanol.
Then, 1 mL of ethanolic DPPH solution (0.1 mM) was added to
the samples. These samples were vortexed and incubated in the
dark at 308C for 30 min. The absorbance was measured at
517 nm against blank samples. A decrease in absorbance
indicates DPPH free radical scavenging activity.
(5-Bromo-3,4-trihydroxyphenyl)(3,4-dihydroxyphenyl)
methanone (13)
It was obtained as yellow solid (97%). Mp 232–2358C. 1H-NMR
(400 MHz, CD3COCD3) d (ppm): 7.43 (s, aromatic, 1H), 7.26
(d, J ¼ 1.7 Hz, aromatic, 1H), 7.17 (dd, A part of AB-system,
J ¼ 8.2, 1.7 Hz, aromatic, 1H), 6.99 (d, B part of AB-system,
J ¼ 8.2 Hz, aromatic, 1H). 13C-NMR (100 MHz, CD3COCD3)
d 198.35 (CO), 151.63 (C), 149.81 (C), 149.11 (C), 145.16 (CH),
133.76 (C), 129.84 (C), 127.69 (CH), 123.00 (CH), 116.63 (CH),
115.09 (CH), 113.79 (C), 99.41 (C). IR (acetone, cmꢁ1): 2695,
1926, 1706, 1625, 1520, 1487, 1440, 1365, 1123, 1001, 964,
946. Anal. Calcd. for (C13H9BrO6): C 45.77; H 2.66 Found
C 45.61; H 2.65.
ABTSꢀþ scavenging activity
The ABTSꢀþ radical scavenging activity of bromophenols was
evaluated according to the method of Re et al. [73] with minor
modifications [14]. ABTSꢀþ is blue-greenþ in colour with a charac-
teristic absorbance at 734 nm. ABTSꢀ cation radical was pro-
duced by reacting ABTS (2 mM) in H2O and potassium
persulphate (2.45 mM) at room temperature for 12 h. The
ABTSꢀþ solution was diluted with phosphate buffer (0.1 M, pH
7.4) to achieve þan absorbance of 0.750 ꢅ 0.025 at 734 nm. Then,
1 mL of ABTSꢀ solution was added to 3 mL of bromophenols
solution in methanol at different concentrations (10–30 mg/mL).
These samples were vortexed and incubated in the dark for
(5-Bromo-2,3,4-trihydroxyphenyl)(2-bromo-4,5-
dihydroxyphenyl) methanone (14)
It was filtered from silica gel (50 g) with EtOAc/CH2Cl2 (1:1) and
obtained as brown solid (95%). Mp 204–2078C. 1H-NMR (400 MHz,
CD3COCD3) d (ppm): 7.16 (s, aromatic, 1H), 7.07 (s, aromatic, 1H),
6.96 (s, aromatic, 1H). 13C-NMR (100 MHz, CD3COCD3) d 198.35
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332
Y. C¸ etinkaya et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
30 min. After 30 min, the absorbance at 734 nm was measured
for each concentration relative to a blank. Decreased absorbance
centrations (10–30 mg/mL) of bromophenols. The total volume
was adjusted to 2 mL with distilled water, and the reaction was
mixed well. The tubes were kept at room temperature.
After 30 min of incubation, the absorbance was measured
at 450 nm against a blank. Increased absorbance indicates
increased Cu2þ-Cuþ reduction.
þ
of the samples indicates ABTSꢀ cation radical scavenging
activity.
DMPDꢀþ scavenging activity
The DMPD radical scavenging ability of bromophenols was deter-
mined by the method of Fogliano et al. [74] with slight modifi-
cations by Gu¨lc¸in [75]. Thisþassay is based on the capacity of the
extract to inhibit DMPDꢀ cation radical formation. Briefly,
105 mg of DMPD was dissolved in 5 mL of distilled water.
Then, 1 mL of this solutþion was added to 100 mL of 0.1 M acetate
buffer (pH 5.3). DMPDꢀ was produced by adding 0.3 mL ferric
chloride (0.05 M) to this solution. Different concentrations of
standard antioxidants or bromophenols (10–30 mg/mL) were
added, and the total volume was adjusted to 0.5 mL with distilled
water. One millilitre of the DMPDꢀþ solution was directly added
to the reaction mixture. The reaction mixtures were vortexed
and incubated in the dark for 15 min. The absorbance was
measured at 505 nm.
Ferrous ion (Fe2þ) chelating activity by 2,20-bipyridine
reagent
For determination of ferrous ion (Fe2þ) chelating activity, two
distinct methods were used: ferrous ion (Fe2þ) chelating activity
by ferrozine reagent and ferrous ion (Fe2þ) chelating activity by
2,20-bipyridine reagent.
Firstly, the ferrous ion (Fe2þ) chelating activity of bromophe-
nol compounds was measured according to the method of Re
et al. [73] as previously explained [75, 76]. Briefly, different
concentrations (10–30 mg/mL) of bromophenols in 0.25 mL
methanol were added to 0.25 mL of ferrous sulphate (0.2 mM).
The reaction was initiated by the addition of 1 mL Tris-HCl buffer
(pH 7.4) and 1 mL 2,20-bipyridine (0.2% in 0.2 M HCl) dissolved in
2.5 mL methanol. The total volume was adjusted to 5 mL with
distilled water. The mixture was shaken vigorously and kept at
room temperature for 10 min. Then absorbance was measured at
522 nm.
Superoxide anion radical scavenging activity
The superoxide radical scavenging activity of bromophenols was
determined by the riboflavin–methionine–illuminate assay [62].
This assay is based on the capacity of the extract to inhibit the
photochemical reduction of nitro blue tetrazolium (NBT). The
total volume of the reaction mixture was 3 mL, and the concen-
tration of bromophenols was 30 mg/mL. The concentrations of
riboflavin, methionine and NBT were 1.33 ꢆ 10ꢁ5, 4.46 ꢆ 10ꢁ5
and 8.15 ꢆ 10ꢁ8 M, respectively. The photo-induced reactions
were performed using fluorescent lamps (20 W). The reaction
mixture was illuminated for 40 min at 258C. The photochemi-
cally reduced riboflavin generates Oꢀ2ꢁ, which reduces NBT to
form blue formazan. The absorbance was measured at 560 nm. A
decrease in absorbance indicates increased superoxide anion
scavenging activity. The un-illuminated reaction mixture was
used as a blank.
Ferrous ion (Fe2þ) chelating activity by ferrozine reagent
Ferrous ions (Fe2þ) chelating activity was measured by inhibiting
the formation of Fe2þ-ferrozine complex after treatment of test
material with Fe2þ, following the method of Dinis et al. [77]. Fe2þ
-
chelating ability of bromophenol compounds was monitored by
the absorbance of the ferrous iron-ferrozine complex at 562 nm.
Briefly, different concentrations of bromophenol compunds (10–
30 mg/mL) in 0.4 mL methanol were added to a solution of
0.6 mM FeCl2 (0.1 mL). The reaction was initiated by the addition
of 5 mM ferrozine (0.1 mL) dissolved in methanol. Then, the
mixture was shaken vigorously and left at room temperature
for 10 min. Absorbance of the solution was then measured
spectrophotometrically at 562 nm [29]. The percentage of inhi-
bition of ferrozine-Fe2þ or 2,20-bipyridine-Fe2þ complexes for-
mation was calculated by using the formula given below:
Fe3þ reducing activity
The reducing power of bromophenols was measured according
to the Fe3þ-Fe2þ transfomation with slight modifications [13].
According to this method, the reduction of Fe3þ to Fe2þ is
determined by measuring the absorbance of Perl’s Prussian blue
complex. Briefly, different concentrations (10–30 mg/mL) of bro-
mophenols in distilled water (0.75 mL) were mixed with 1 mL of
sodium phosphate buffer (0.2 M, pH 6.6) and 1 mL (1%) of pot-
assium ferricyanide [K3Fe(CN)6]. The mixture was incubated at
508C for 20 min. Afterwards, the reaction mixture was acidified
with 1 mL of trichloroacetic acid (10%). Finally, 0.25 mL of FeCl3
(0.1%) was added to this solution. The absorbance of the mixture
at 700 nm was measured. A decrease in absorbance indicates
increased ferric reducing power.
ꢀ
ꢁ
lS
lC
Bound ferrous ionsð%Þ ¼ 1 ꢁ
ꢆ 100
where lC is the absorbance of control and lS is the absorbance in
the presence of bromophenols or standards. The control contains
only Fe2þ and ferrozine or 2,20-bipyridine [13, 58].
Statistical analysis
Each experiment was performed in triplicate. The data are
reported as the mean ꢅ standard deviation and were analysed
by SPSS (version 17.0 SPSS Inc.). One-way analysis of variance
was performed by ANOVA procedures. Significant differences
between the means were determined by Duncan’s multiple
range tests. P <0.05 was considered significant.
Cupric ion (Cu2þ) reducing-CUPRAC assay
The cupric ion (Cu2þ) reducing power of bromophenols was
determined by the method proposed by Apak et al. [60] with
minor modifications [10]. Briefly, 0.25 mL of 10 mM CuCl2
solution, 0.25 mL of 7.5 mM ethanolic neocuproine solution
and 0.25 mL of ammonium acetate buffer solution (1 M) were
added to a test tube and mixed with 0.25 mL of different con-
The authors are indebted to the Department of Chemistry and
Atatu¨rk University for financial support of this work (Project No.: BAB-
2009/84).
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www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 345, 323–334
Synthesis and Antioxidant Properties of Phenols
333
˙
The authors have declared no conflict of interest.
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