Antioxidant and acetylcholinesterase inhibition properties of novel bromophenol derivatives

Article abstract of DOI:10.1016/j.bioorg.2015.04.006

In this study, series of novel bromophenol derivatives were synthesized and investigated for their antioxidant and AChE inhibition properties. Novel brominated diarylmethanones were obtained from the acylation reactions of benzoic acids with substituted b

Full text of DOI:10.1016/j.bioorg.2015.04.006

Accepted Manuscript
Antioxidant and Acetylcholinesterase Inhibition Properties of Novel Bromo-
phenol Derivatives
Necla Öztaşkın, Yasin Çetinkaya, Parham Taslimi, Süleyman Göksu, İlhami
Gülçin
PII:
S0045-2068(15)00034-6
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.04.006
YBIOO 1806
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
12 March 2015
Please cite this article as: N. Öztaşkın, Y. Çetinkaya, P. Taslimi, S. Göksu, İ. Gülçin, Antioxidant and
Acetylcholinesterase Inhibition Properties of Novel Bromophenol Derivatives, Bioorganic Chemistry (2015), doi:
http://dx.doi.org/10.1016/j.bioorg.2015.04.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antioxidant and Acetylcholinesterase Inhibition Properties of
Novel Bromophenol Derivatives
Necla Öztaşkına, Yasin Çetinkayab, Parham Taslimia, Süleyman Göksua,*, İlhami Gülçina,c,*
aDepartment of Chemistry, Faculty of Science, Atatürk University, 25240-Erzurum, Turkey
bOltu Vocational School, Department of Food Technology, Atatürk University, 25400-Oltu-
Erzurum, Turkey
cDepartment of Zoology, College of Science, King Saud University, Riyadh-Saudi Arabia
*Corresponding authors:
Prof. Dr. Süleyman Göksu, phone: +90 442 2314389; fax: +904422360948;
e-mail: sgoksu@atauni.edu.tr
Prof. Dr. İlhami Gülçin, phone: +904422314375, fax: +904422314109;
e-mail: igulcin@atauni.edu.tr
Running title: Biological activities of novel bromophenol derivatives
1

ABSTRACT
In this study, series of novel bromophenol derivatives were synthesized and investigated for
their antioxidant and AChE inhibition properties. Novel brominated diarylmethanones were
obtained from the acylation reactions of benzoic acids with substituted benzenes. One of the
bromodiarylmethanone was synthesized from the bromination of diarylmethanone with
molecular bromine. All diarylmethanones were converted into their bromophenol derivatives
with BBr3. The antioxidant activities of all synthesized compounds were elucidated by using
various bioanalytical assays. Radical scavenging activities of compounds 10-24 were
•+
·
evaluated by means of DPPH and ABTS scavenging activities. In addition, reducing ability
of 10-24 were determined by Fe3+, Cu2+, and [Fe3+-(TPTZ)2]3 reducing activities. α-
Tocopherol, trolox, BHA, and BHT were used as positive antioxidant and radical scavenger
·, ABTS•+
molecules. On the other hand, IC50 values were calculated for DPPH
scavenging,
and AChE inhibition effects of novel compounds. The results obtained from the current
studies clearly show that novel bromophenol derivatives 20-24 have considerable antioxidant,
antiradical, and AChE inhibition effects.
Keywords: Acetylcholinesterase; Antioxidant activity; Bromophenols; Diarylmethanones;
Demethylation; Enzyme inhibition
2

1. INTRODUCTION
Naturally occurring bromophenols are abundant in marine life [1]. They are mostly isolated
from the marine algae as a very large and diverse group of eukaryotic organisms [2].
Recently, the total syntheses of these compounds have also attracted the synthetic
community’s attention [3], because it has been demonstrated that natural and synthetic
bromophenols have diverse biological activities [4]. For instance, Vidalol A (1) and Vidalol B
(2), which are isolated from the Caribbean marine red alga Vidalia, were shown to have anti-
inflammatory properties [5]. On the other hand, bromophenols 3-5 isolated from the brown
alga Leathesia nana showed cytotoxicity [6] and anticancer [7] activities. In another study,
Wang et al. has reported the aldose reductase inhibitory activity of the natural product 6 [8].
Furthermore, radical-scavenging properties of 6 [9], antioxidant activities of 1 [10] and 6 [11]
have also been investigated. Recently, the total synthesis and the biological activities of some
natural bromophenols have been performed by our group [12-15]. In these studies, the human
carbonic anhydrase inhibitory actions of 2 [12], 4-6 [13a,b], their synthetic derivatives [14]
and paroxonase inhibitory properties [15] of some related compounds were evaluated (Figure
1).
Figure 1 here
The oxidative stress has been a major research interest due to its implicated role in
human diseases such as cancer, atherosclerosis, and brain dysfunction [16]. Free radicals,
•-
especially reactive oxygen species (ROS), such as superoxide anion radical (O2 ), hydroxyl
radical (HO·), and hydrogen peroxide (H2O2) generated during metabolism can damage
macromolecules including fatty acids, proteins, DNA, and other macromolecules, which
cause various diseases including neurodegenerative diseases, cardiovascular diseases, cancers
and aging-related disorders [17]. Many degenerative disorders are initiated by the reaction of
ROS with human body cell constituents. All aerobic organisms have antioxidant defenses,
including both antioxidant enzymes and antioxidant food constituents to remove or repair the
damaged biomolecules [18a-c]. Antioxidants are bioactive compounds commonly used to
preserve the quality of food products by protecting them against oxidative rancidity [19]. In
the case of oxidative stress, the most susceptible molecules for ROS attack are lipids, proteins,
and nucleic acids [20]. The most injurious effects on the viability of the cells and human
health are oxidative changes in nucleic acids, which cause mutagenic processes and cancer
[21]. Antioxidant compounds protect the human body from free radicals and many chronic
cardiovascular diseases, cancers, and ageing by capturing free radicals [22]. Also, these
3

bioactive compounds can scavenge free radicals or ROS and increase shelf life by retarding
the process of lipid peroxidation, which is one of the major reasons for deterioration of food
and pharmaceutical products during processing and storage [23a,b]. They retard the progress
of many chronic diseases as well as lipid peroxidation. Recently, there is a growing interest in
safer antioxidants for food and pharmaceutical applications, and a growing trend in consumer
preferences toward safer antioxidants [24a-d].
Alzheimer’s disease (AD) is a fatal and chronic neurodegenerative disease that usually
starts slowly and gets worse over time. It was reported that the disease process is associated
with tangles and plaques in the brain. There were 35 million people worldwide with AD, and
there has been no effective treatment developed for this disease up to now. The use of
acetylcholinesterase inhibitors (AChEIs) to block the cholinergic degradation of acetylcholine
(Ach) is therefore considered to be a promising approach for the treatment of AD [25a-c]. At
the present, the main clinical treatment strategy for AD is the use of AChEIs such as
Donepezil, Rivastigmine, and Galantamine. These drugs modestly improve the memory and
cognitive function but do not prevent progressive neurodegenerative effects because of the
complexity of AD pathogenesis. Based on these discoveries, many different approaches to
treat AD have been developed. In particular, the use of AChEIs for this multifaceted disease
has received much attraction [25a-c].
As it can be seen from the brief description given above, bromophenols show useful
biological activities. In our previous studies, we synthesized bromophenols containing
polybromides in their structures. The synthesis and biological evaluation of novel
bromophenols having only a single bromine atom will be important for synthetic and
biological features. In this context, here we report the synthesis and determination of
antioxidant, antiradical and AChE inhibition effects of five novel bromophenols.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The reactions of acids or alcohols with benzene related compounds in the presence of
polyphosphoric acid (PPA) afford diarylmethanones [26a,b]. It is obvious from the structure
of 9 that an electrophilic aromatic substitution carried out on this compound can lead to two
different products. However, methoxy group has more steric hindrance than a bromine atom.
4

For this reason, an electrophilic aromatic substitution of these kinds of compounds react at the
ortho- position of −Br substituent and at the para- position of an −OMe group [26b].
Therefore, benzoic acids 7 and 8 were reacted with commercially available 3-bromoanisole
(9) in the presence of PPA to give diarylmethanons 10 and 11 in moderate yields. On the
other hand, 2-substituted 1,4-dimethoxy benzenes (especially Br or Me substituted ones)
undergo electrophilic aromatic substitution at the ortho- position of the OMe group and the
para- position of the substituent [27]. Similarly, the reactions of acids 12 and 13 with a
known 2-bromo-1,4-dimethoxy benzene (14) [28] afforded diarylmethanones 15 and 16 [29]
in 76% and 50% yield, as expected (Scheme 1).
Scheme 1 here
Again the reaction of carboxylic acid 12 with 1,3-dimethoxy benzene (17) in the presence of
PPA produced the compound 18 [30]. Selective bromination of aromatic compounds is an
important route for the synthesis of novel target compounds. There are several reagents used
for this purpose. Molecular bromine (Br2) is one of these reagents mostly used for the
bromination of activated aromatic compounds [31]. To extend our research on the synthesis of
another mono brominated compound, the bromination of 18 with 1.01 equiv. of Br2 at room
temperature (rt) for 12 hour (h) afforded mono brominated diarylmethanone 19 with a yield of
62% (Scheme 2).
Scheme 2 here
Demethylation of aryl methyl ethers is one of the most convenient methods for the synthesis
of biologically active phenolic compounds [32a,b]. BBr3 is one of the most widely-used
reagents for the O-demethylation of aryl methyl ethers [33]. Hence, demethylation reactions
o
of diarylmethanones 10, 11, 15, 16, and 19 with BBr3 at 0-25 C under N2 for 24 h furnished
novel bromophenols 20-24 in 91, 80, 92, 93, and 83% yields, respectively (Scheme 3).
5

The structures of the synthesized compounds were elucidated with 1H-, 13C-NMR, IR
and elemental analysis.
Scheme 3 here
2.1. Biochemistry-Antioxidant Properties
Lipid peroxidation in foods has become a major concern with the increased use of
polyunsaturated vegetable, fish, or microbial oils for health benefits. It not only produces
undesirable off-flavours, but also decreases the nutritional quality and safety of food products
[34a,b], which are unacceptable to consumers. Among the methods employed to retard or
inhibit oxidation of lipids, addition of antioxidants is the most effective way [35a,b]. On the
other hand, phenolic compounds have attracted wide range of interest because of their health
benefits, biological features and impact on multiple sensory attributes of food including
colour, flavour and astringency of foods [36a,b]. They are thought to be an integral part of
both human and animal diets and frequently used for pharmacological applications [37a-c].
Phenolic compounds are secondary plant metabolites, structure of which includes one or more
aromatic rings substituted by one or more hydroxyl groups [36a]. They are secondary plant
metabolites and naturally present in almost all plant materials, including food and
pharmaceutical products of plant origin [38a-d]. The antioxidant capacities of novel
bromophenols are determined by using several antioxidant assays. For this purpose,
[Fe(CN)6]-3-[Fe(CN)6]-4 reduction method is the first used antioxidant evaluation of novel
bromophenols [39a,b]. Table 1 shows that novel bromophenols have effective [Fe(CN)6]-3
reducing ability and these differences were statistically very significant (p<0.01). Increasing
[Fe (CN)6]-4 absorbance of the reaction mixture indicates increased reducing capacity, due to
an increase in the complex formation. Reducing ability of novel bromophenols and positive
controls showed the following order: 23 (2.563 0.003, r2: 0.9895) > BHA (2.354 0.003, r2:
0.9575) > 21 (2.123 0.011, r2: 0.9999) ≈ 20 (2.094 0.004, r2: 0.9711) > 22 (1.947 0.003, r2:
0.9757) > 24 (1.163 0.002, r2: 0.9700) > BHT (1.405 0.003, r2: 0.9630) > α-tocopherol
(0.927 0.008, r2: 0.9819) > trolox (0.893 0.002, r2: 0.9930). On the other hand, compounds
10-19 as precursors of novel bromophenols 20-24 not having any phenolic groups in the
aromatic rings are located in the para- position. These compounds demonstrated to have weak
6

antioxidant activities. The results clearly showed that novel bromophenol 23 had the most
powerful Fe3+ ability and changed yellow colour of the test solution to green or blue,
depending on their reducing properties. In this molecule, both hydroxyl groups (-OH) are
located in aromatic ring at the para- position (Table 1). It was reported that phenolic
compounds are not active antioxidants, unless they contain substituents at either ortho- or
para- positions. Both positions have increased the electron density at the -OH groups and
lowered the oxygen-hydrogen bond energy, in effect increasing the reactivity towards the
lipid free radicals. Substituents of phenolic compounds at the meta- positions have rather
limited effects. Electronic and steric effects are responsible for the antioxidant activities and
stoichiometric factors of the chain-breaking phenolic antioxidant compounds [38c,40]. For
elucidation of the hydrogen abstraction mechanism and reducing ability of phenolic
antioxidant compounds in the chain process of autoxidation, molecular orbital theory has been
applied [41].
Cupric ions (Cu2+) reducing assays are based on reduction the reduction of Cu2+ to Cu+. Cu+-
neocuproine complexes occur in the presence of neocuproine. This complex had maximum
absorption at 450 nm [42]. Also, this assay is cost-effective, selective, stable, rapid and
suitable for antioxidants regardless of hydrophilicity or chemical type [40a]. The Cu2+
reducing capacities of novel bromophenols and standard compounds were shown in Table 1.
Cu2+ reducing ability of novel bromophenols and standard compounds at the same
concentration (20 µg/mL) exhibited the following order: 23 (2.018 0.005, r2: 0.9997) ≈ 21
(2.014 0.004, r2: 0.9984) > 22 (1.966 0.002, r2: 0.9707) ≈ BHA (1.962 0.002, r2: 0.9999) >
20 (1.602 0.001, r2: 0.9430) > BHT (1.407 0,004, r2: 0.9972) > trolox (1.005 0.002, r2:
0.9992) > α-tocopherol (0.854 0.003, r2: 0.9998) > 24 (0.655 0.003, r2: 0.9692). As in
Fe[(CN)6]3 reducing ability, the most powerful cupric ions (Cu2+) reducing ability was
observed in novel bromophenol 23. Additionally, buried thiols groups (-SH) of proteins
release in urea buffer at near of pH 7.0 and react with reagent. So, this method can be used for
measurement of reducing activity of antioxidants, which include thiol groups such as
glutathione [43].
Table 1 here
The FRAP assay measures the ability of phenolic antioxidant compounds to reduce the
ferric 2,4,6-tripyridyl-s-triazine complex [Fe3+-(TPTZ)2]3+ to the intensely blue coloured
ferrous complex [Fe2+-(TPTZ)2]2+ in acidic medium [44a,b]. FRAP assay is one of the widely
used, simple and convenient method for determining the antioxidant activity [45].
7

Traditionally, the assay is conducted at acidic pH (3.6) to maintain iron solubility. The results,
in terms of the reducing abilities of the novel bromophenols and standard antioxidant
compounds, were as follows: 23 (2.093 0.004, r2: 0.9709) ≈ 20 (2.012 0.001, r2: 0.9591) ≥
21 (1.986 0.004, r2: 0.9916) ≥ 22 (1.974 0.005, r2: 0.9546) ≥ BHA (1.869 0.001, r2: 0.9806)
> trolox (1.742 0.002, r2: 0.9997) > BHT (1.328 0.004, r2: 0.9986) > α-tocopherol
(1.123 0.002, r2: 0.9986) > 24 (0.401 0.002, r2: 0.9404) (Table 1). These results emphasized
that novel bromophenols had notable Fe3+ reducing ability and electron donor properties for
neutralizing free radicals.
The novel bromophenol 23 has two phenolic groups at para- positions. Thereby, this
novel bromophenol is expected to have a strong reducing activity. The presence of different
substituents in the phenol backbone structure modulates their antioxidant property, in
particular their hydrogen-donating capacity. In addition, the presence of a carbonyl group,
such as aromatic acid, ester, or lactone, enhanced its antioxidant activity [40a]. Moreover, it is
well known that steric and electronic effects are responsible for the antioxidant activities and
stoichiometric factors of the chain-breaking phenolic antioxidants [38c]. The activity of an
antioxidant molecule also increases when its carbonyl group is separated from the aromatic
ring. For example, cinnamic acid is more effective antioxidant than benzoic acid. Steric
hindrance of the phenolic hydroxyls by a neighbouring inert group, such as methoxy groups,
enhanced its antioxidant activity [40a,38c].
The radical scavenging properties of novel bromophenols were investigated by DPPH·
and ABTS•+ scavenging assays [46a-c]. DPPH· is a stable nitrogen-centred free radial and has
been well used to employ the ability of free-radical scavenging properties or hydrogen
donation of compounds in food, medicinal, and pharmaceutical materials [47a-c]. This assay
is a fast and simple radical scavenging method. DPPH radical scavenging was broadly
accepted for the in vitro evaluation of natural substance antioxidant potential due to its
simplicity and low cost [48a-c]. In this study, DPPH· scavenging assay was used for the
primary screening of free radical scavenging activity of novel bromophenols. Consequently,
novel bromophenols exhibit a remarkable DPPH free radical scavenging activity. As it can be
seen by Table 2, IC50 values of DPPH· scavenging effects of novel bromophenols and
standard radical scavengers increased in the order: 23 (14.15 µg/mL) ≈ 22 (14.74 µg/mL) ≥ 21
(15.40 µg/mL) ≈ 20 (15.75 µg/mL) > 24 (28.92 µg/mL). A lower IC50 value indicates
powerful DPPH· scavenging activity of novel bromophenols. On the other hand, BHA, BHT,
α-tocopherol and trolox demonstrated IC50 values of 22.35, 34.65, 21.65 and 16.11 µg/mL,
respectively. Also, IC50 values of precursors (10-19) used for the synthesis of (20-24) are
8

ranging of 221.37-346.50 µg/mL. These results clearly revealed that bromophenols (20-24)
had notable DPPH· scavenging ability and electron donor properties for neutralizing free
radicals.
Table 2 here
In ABTS•+ scavenging assay, ABTS is oxidized by oxidants like K2S2O8 and MnO2 to
its radical cation (ABTS•+), which is bright green-blue in colour and has an absorption
maximum of 734 nm [49a-c]. The absorbance of the ABTS•+ solution decreases when an
electron is donated by an antioxidant to quench the free radical. This assay has been used for
both hydrophilic and lipophilic antioxidants such as tocopherols and carotenoids [50]. ABTS•+
radicals are more reactive than DPPH radicals and applicable for both hydrophilic and
lipophilic compounds [51a-c]. As it can be seen by Table 2, novel bromophenols (20-24)
show important ABTS•+ scavenging properties. IC50 values of ABTS•+ scavenging of novel
bromophenols (20-24) increased in the following order: 23 (2.23 µg/mL) ≈ 20 (2.55 µg/mL) ≈
21 (2.58 µg/mL) ≈ 22 (2.61 µg/mL) > 24 (22.35 µg/mL). A lower IC50 value indicates
powerful ABTS•+ scavenging activity. Also, IC50 values of precursors (10-19) of novel
bromophenols (20-24) are ranging of 138.60-138.60 µg/mL. In addition, BHA (2.72 µg/mL),
BHT (2.88 µg/mL), α-tocopherol (4.81 µg/mL) and trolox (2.86 µg/mL) were used as positive
controls for ABTS•+ scavenging activity.
AChE is found in the brain and in erythrocytes with high concentrations and it is a
crucial enzyme for the nervous system. AChE inhibitors are used in the treatment of several
neuromuscular diseases, and in the treatment of AD [25a]. The activity (%)–[Bromophenols]
graphs were drawn and the half maximal inhibitory concentration (IC50) values of novel
bromophenols demonstrated 50% inhibition of AChE isoenzymes were calculated after
suitable dilutions. Ki values for novel bromophenols were determined for AChE. Ki is defined
as the binding affinity constant of the novel bromophenols to AChE. To determine the Ki
values, each novel bromophenols was tested at three different concentrations [52a-d].
Lineweaver–Burk curves were drawn in detail as described previously [53a-c]. For
descriptions of inhibitory effects, researchers often used an IC50 value; however, Ki constant
is a more suitable parameter. Ki values were calculated from Lineweaver–Burk graphs
(Figures 2). In this study, both the IC50 and Ki parameters of novel bromophenols were
calculated (Table 2). The most powerful AChE inhibition ability observed for novel
bromophenol 22. Figure 2 shows IC50 and Ki graphs of novel bromophenol 22.
Figure 2 here
9

We are very experienced by our previous studies that the Ki values of phenolic
dopamine-related compounds were generally found in the range of 33.04-131.68 nM [54a]. In
contrast to the phenolic compounds, arylmethyl ethers such as sulphonamides incorporating
the tetralin scaffold demonstrated Ki values in the range of 56.04-537.3 µM [54b].
Furthermore, it was reported that Galantamine, which is used for the treatment of AD and
various other memory impairments, has been shown the lower AChE inhibition activity
(EC50: 3.70 mM) [54b]. In the last part of the study, AChE was also highly inhibited by novel
bromophenol derivatives (10-24) at the low nanomolar inhibition with Ki values in the range
of 2.78-5.88 nM (Table 2). These results clearly showed that all novel synthesized compounds
(10-24) demonstrated powerful AChE inhibition properties. The most powerful inhibition was
observed for novel bromophenol 23 with a Ki value of 2.78 nM. All the remaining newly
synthesized compounds reported here were also showed highly efficient inhibition constants
against AChE. On the other hand, tacrine, which first centrally acting cholinesterase inhibitor
approved for the treatment of AD demonstrated Ki value of 7.64 nM.
3. CONCLUSION
In conclusion, five diarylmethanones incorporating bromine and methoxy functional groups
were synthesized by starting from benzoic acids. Five novel bromophenols (20-24) were
synthesized from these compounds via O-demethylation with BBr3. The antioxidant activities
and AChE inhibition properties of all synthesized compounds were evaluated. Novel
bromophenols were found to be effective AChE inhibitors. Different assays including
reducing power, DPPH· and ABTS•+ radical scavenging activities showed that the synthesized
bromophenols were also effective antioxidants. When compared with the standard
antioxidants such as popular synthetic antioxidants BHA, BHT, α-tocopherol and trolox, the
synthesized compounds reported in this paper are most potent antioxidants. Based on the
information given above, the synthesized novel bromophenols can be used for minimizing or
preventing lipid peroxidation in food, medical and pharmaceutical products, retarding the
formation of toxic oxidation products, maintaining nutritional quality and prolonging the shelf
life of pharmaceuticals.
10

4. EXPERIMENTAL
4.1. General
All chemicals and solvents are commercially provided and were used after distillation or
treatment with drying agents. IR spectra were obtained from solutions in 0.1 mm cells with a
Perkin-Elmer spectrophotometer. Melting points were determined on a capillary melting
apparatus (BUCHI 530) and are uncorrected. The 1H- and 13C-NMR spectra were recorded on
a 400 (100) MHz Varian spectrometer; δ in ppm, Me4Si as the internal standard. Elemental
analyses were performed on a Leco CHNS-932 apparatus. All column chromatography was
performed on with silica gel (60-mesh, Merck). TLC is preparative thin layer chromatography
prepared from 1 mm of silica gel 60 PF (Merck) on glass plates.
4.2. Chemistry
The synthesis of 2-bromo-1,4-dimethoxy benzene (14) was carried out according to the
literature procedure [28].
4.2.1. General procedure for the synthesis of diarylmethanones [Synthesis of (2-bromo-4-
methoxyphenyl)(3,4-dimethoxyphenyl)methanone (10)]
In order to prepare PPA (polyphosphoric acid), a mixture of H3PO4 (6.4 g) and P2O5 (11.5 g)
o
in 150 mL beaker was stirred at 80 C for 15 min. 1-Bromo-3-methoxybenzene (9) (3.08 g,
16.47 mmol) and 3,4-dimethoxybenzoic acid (7) (2 g, 10.98 mmol) were added to prepared
o
PPA solution and the mixture was stirred with a glass stick at 80 C for 1.5 h. After the
completion of the reaction, cold water was added to the reaction mixture and then the organic
layer was extracted with EtOAc (3x150 mL). The combined organic phases were dried over
Na2SO4 and the solvent was evaporated under reduced pressure. The residue was purified by
column chromatography with silica gel (30 g) by eluting with hexane:ethyl acetate (9:1).
Recrystallization of the solid with hexane-ethyl acetate gave 2-bromo-4-methoxyphenyl)(3,4-
o
dimethoxyphenyl)methanone (10) (2.32 g) as a white solid with 60% yield. Mp: 140-142 C.
1H NMR (400 MHz, CDCl3): δ 7.53 (d, 1H, J = 1.8 Hz), 7.28 (d, 1H, A part of AB system, J =
8.5 Hz,), 7.23 (dd, 1H, A part of AB system, J = 8.4, 1.8 Hz), 7.17 (d, 1H, J = 2.3 Hz), 6.91
(dd, 1H, B part of AB system, J = 8.5, 2.3 Hz), 6.83 (d, 1H, B part of AB system, J = 8.4 Hz),
11

3.93 (s, 6H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 194.5, 161.2, 153.9, 149.40, 133.2,
130.7, 130.0, 126.4, 121.1, 118.8, 113.2, 111.4, 110.1, 77.6, 77.3, 76.9, 56.3, 56.2, 55.9. IR
(cm-1, CH2Cl2): 3082, 3006, 2938, 2839, 1657, 1597, 1559, 1512, 1491, 1463, 1417, 1341,
1298, 1267, 1232, 1183, 1130, 1031, 984, 914, 864. Anal. Calcd. for C16H15BrO4: C 54.72; H
4.31, Found: C 54.76; H 4.28.
4.2.2. 2-Bromo-4-methoxyphenyl)(2,3-dimethoxyphenyl)methanone (11)
The reaction of 1-bromo-3-methoxybenzene (9) (3.08 g, 16.47 mmol) with 2,3-dimethoxy
benzoic acid (8) (2.0 g, 10.98 mmol) according to the general procedure described at 4.2.1
afforded (2-bromo-4-methoxyphenyl)(2,3-dimethoxyphenyl)methanone (11) (2.13 g, 55%
o
yield) in 1.5 h. Yellow solid. Mp: 88-90 C. 1H NMR (400 MHz, CDCl3): δ 7.39 (d, 1H, A
part of AB system, J = 8.6 Hz), 7.15 (d, 1H, J = 2.4 Hz), 7.13 – 7.03 (m, 3H), 6.83 (dd, 1H, B
13
part of AB system, J = 8.6, 2.4 Hz), 3.87 (s, 3H), 3.83 (s, 3H), 3.64 (s, 3H). C NMR (100
MHz, CDCl3): δ 195.0, 161.9, 153.1, 148.4, 134.2, 133.6, 132.7, 124.2, 121.9, 119.4, 115.8,
112.9, 61.7, 56.2, 55.9. IR (cm-1, CH2Cl2): 3082, 3004, 2940, 2838, 1668, 1594, 1559, 1478,
1440, 1426, 1390, 1314, 1266, 1231, 1199, 1184, 1077, 1033, 1003, 964,863. Anal. Calcld.
for C16H15BrO4: C 54.72; H 4.31, Found: C 54.59; H 4.28.
4.2.3. (4-Bromo-2,5-dimethoxyphenyl)(4-methoxyphenyl)methanone (15)
The general procedure described above at 4.2.1 was applied to 2-bromo-1,4-
dimethoxybenzene (14) (1.07 g, 4,93 mmol) and 4-methoxybenzoic acid (12) (0.5 g, 3,29
mmol) to give (4-bromo-2,5-dimethoxyphenyl)(4-methoxyphenyl)methanone (15) (0.88 g,
o
76%) in 1.5 h. White solid. Mp: 130-132 C. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, 2H, J =
13
8.9 Hz), 7.20 (s, 1H), 6.92-6.90 (m, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.68 (s, 3H). C NMR
(100 MHz, CDCl3): δ 194.1, 164.0, 151.4, 150.40, 132.5, 130.5, 129.1, 117.6, 114.27, 113.8,
113.0, 57.1, 56.8, 55.7. IR (cm-1, CH2Cl2): 3074, 3006, 2940, 2842, 1659, 1599, 1574, 1509,
1490, 1464, 1441, 1380, 1316, 1216, 1170, 1128, 1049, 1030, 982, 912. Anal. Calcld. for
C16H15BrO4: C, 54.72; H, 4.31; Found: C, 54.68; H, 4.36.
4.2.4. (4-Bromo-2,5-dimethoxyphenyl)(phenyl)methanone (16)
12

Applying of the procedure given at 4.2.1 was applied to 2-bromo-1,4-dimethoxybenzene (14)
(3.02 g,13.92 mmol) and benzoic acid (13) (1.7 g, 13,92 mmol) to afford (4-bromo-2,5-
dimethoxyphenyl) (phenyl)methanone (16) [28] (2.25 g 50% yield) in 1.5 h. White solid. Mp:
137-139 oC. 1H NMR (400 MHz, CDCl3): δ 7.79 (d, 2H, J = 7.2 Hz), 7.55 (t, 1H, J = 7.4 Hz),
13
7.43 (t, 2H, J = 7.7 Hz,), 7.2 (s, 1H), 6.95 (s, 1H), 3.84 (s, 3H), 3.64 (s, 3H). C NMR (100
MHz, CDCl3): δ 195.3, 151.5, 150.2, 137.5, 133.2, 129.7, 128.4, 128.3, 117.4, 114.8, 112.9,
56.9, 56.5. IR (cm-1, CH2Cl2): 3102, 3071, 3002, 2930, 2897, 2843, 1644, 1593, 1570, 1488,
1454, 1439, 1375, 1312, 1248, 1212, 1174, 1151, 1050, 1017, 982. Anal. Calcld. for
C15H13BrO3: C 56.10; H 4.08, Found: C 56.05; H 4.12.
4.2.5. Synthesis of (2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (18)
The synthesis of compound 18 was achieved from the reaction of acid 12 with 1,3-dimethoxy
benzene (17) according to the procedure described above at 4.2.1.
1H- and 13C-NMR data of (2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (18) is in
agreement with data given in the literature [30].
4.2.6. Synthesis of (5-bromo-2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (19)
To a solution of (2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (18) (0.5 g, 1.84 mmol)
in CHCl3 (50 mL) was added a solution of Br2 (0.29 g, 1.85 mmol) in CHCl3 (40 mL) at rt.
The reaction mixture was stirred at rt for 12 h. After completion of the reaction, the solvent
was evaporated under reduced pressure. The crystallization of the residue with ethyl acetate-
hexane furnished (5-bromo-2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (19) (0.4 g,
o
1
62% yield) as a white solid. Mp: 135-137 C. H NMR (400 MHz, CDCl3): δ 7.76 (d, 2H, A
part of AB system, J = 8.9 Hz), 7.53 (s, 1H), 6.90 (d, 2H, B part of AB system, J = 8.9 Hz),
13
6.52 (s, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.74 (s, 3H). C NMR (100 MHz, CDCl3): δ 193.0,
163.7, 158.7, 158.6, 134.3, 132.4, 131.1, 123.0, 113.7, 102.3, 96.9, 56.6, 56.3, 55.7. IR (cm-1,
CH2Cl2): 3003, 2966, 2933, 2837, 1632, 1587, 1507, 1458, 1433, 1387, 1310, 1282, 1253,
1210, 1169, 1018, 969, 902. Anal. Calcld. for C16H15BrO4: C 54.72; H 4.31; Found: C 54.67;
H 4.38.
13

4.2.7. General procedure for the synthesis of bromophenols [Synthesis of (2-bromo-4-
hydroxyphenyl)(3,4-dihydroxyphenyl)methanone (20)]
To a solution of 2-bromo-4-methoxyphenyl)(3,4-dimethoxyphenyl)methanone (10) (0.5 g,
1.42 mmol) in CH2Cl2, a solution of BBr3 (2.14 g, 8.54 mmol) in CH2Cl2 (8 mL) was added
drop wise within 5 min. under N2 atmosphere at 0 oC. After the mixture was allowed to warm
to room temperature, it was stirred at rt for 24 h. Methanol (30 mL) was added to the reaction
mixture drop wise at 0 oC within 10 min. The solvent was evaporated under reduced pressure.
To the residue were added EtOAc (50 mL) and H2O (50 mL). The organic layer was separated
and the H2O layer was extracted with EtOAc (2x30 mL). The combined organic phases were
dried over Na2SO4 and the solvent was evaporated under reduced pressure. (2-Bromo-4-
hydroxyphenyl)(3,4-dihydroxyphenyl)methanone (20) (0.40 g, 91% yield) was obtained as a
black viscous liquid. 1H NMR (400 MHz, CD3COCD3): δ 9.19 (s, 1H, OH), 8.77 (s, 1H, OH),
8.42 (s, 1H, OH), 7.33 (d, 1H, J = 1.9 Hz), 7.24 (d, 1H, A part of AB system, J = 8.3 Hz), 7.17
- 7.13 (m, 2H), 6.95 (dd, 1H, J = 8.2, 2.2 Hz), 6.91 (d, 1H, B part of AB system, J = 8.3 Hz).
13C NMR (100 MHz, CD3COCD3): δ 193.5, 159.3, 150.9, 145.2, 132.8, 130.7, 129.7, 124.3,
120.1, 119.8, 116.6, 115.1, 114.5. IR (cm-1, CH2Cl2): 3208, 2923, 2845, 1641, 1589, 1513,
1468, 1419, 1350, 1273, 1209, 1104, 1030, 985, 952. Anal. Calcld. for C13H9BrO4: C 50.51;
H 2.93, Found: C 50.48; H 2.30.
4.2.8. (2-Bromo-4-hydroxyphenyl)(2,3-dihydroxyphenyl)methanone (21)
General demethylation procedure described above at 4.2.8 with BBr3 (2.14 g, 8.54 mmol) was
applied to 11 (0.5 g, 1.42 mmol) to give (2-bromo-4-hydroxyphenyl)(2,3-dihydroxyphenyl)
o
1
methanone (21) (0.35 g, 80% yield) as a brown solid. Mp: 254-256 C. H NMR (400 MHz,
CD3COCD3): δ 12.05 (s, 1H, OH), 9.33 (s, 1H, OH), 8.13 (s, 1H, OH), 7.35 (d, 1H, A part of
AB system, J = 8.4 Hz), 7.21 (d, 1H, J = 2.2 Hz), 7.14 (dd, 1H), 7.01 (dd, 1H, B part of AB
system, J = 8.4, 2.2 Hz), 6.84-6.76 (m, 2H). 13C NMR (100 MHz, CD3COCD3): δ 201.8,
159.9, 151.4, 146.6, 131.1, 130.7, 124.2, 121.9, 120.1, 120.0, 119.9, 119.1, 114.7. IR (cm-1,
CH2Cl2): 3231, 3072, 1618, 1581, 1481, 1445, 1423, 1368, 1327, 1267, 1208, 1046, 1010,
890. Anal. Calcld. for C13H9BrO4: C 50.51; H 2.93 Found: C 50.45; H 3.04.
4.2.9. (4-Bromo-2,5-dihydroxyphenyl)(4-hydroxyphenyl)methanone (22)
14

Demethylation of (4-bromo-2,5-dimethoxyphenyl)(4-methoxyphenyl)methanone (15) (0.70 g,
1.99 mmol) with BBr3 (3.00 g, 11.96 mmol) for 24 h afforded (4-bromo-2,5-
dihydroxyphenyl)(4-hydroxyphenyl)methanone (22) (0.57 g, 92%) as an orange solid. Mp:
o
1
201-203 C. H NMR (400 MHz, CD3COCD3): δ 11.19 (s, 1H, OH), 9.32 (s, 1H, OH), 8.61
(s, 1H, OH), 7.72 (d, 2H, A part of AB system, J = 8.6 Hz), 7.29 (s, 1H), 7.24 (s, 1H), 7.02 (d,
2H, B part of AB system, J = 8.6 Hz). 13C NMR (100 MHz, CD3COCD3): δ 199.4, 162.6,
156.1, 147.0, 133.0, 130.0, 122.6, 120.5, 119.3, 118.8, 116.1. IR (cm-1, CH2Cl2): 3242, 3177,
3046, 2855, 1710, 1677, 1605, 1576, 1507, 1485, 1439, 1396, 1317, 1266, 1233, 1163, 998,
891. Anal. Calcld. for C13H9BrO4: C 50.51; H 2.93, Found: C 50.48; H 3.01.
4.2.10. (4-Bromo-2,5-dihydroxyphenyl)(phenyl)methanone (23)
Demethylation of (4-bromo-2,5-dimethoxyphenyl)(phenyl)methanone (16) (0.3 g, 0.93 mmol)
with BBr3 (1.4 g, 5.6 mmol) for 24 h yielded (4-bromo-2,5-dihydroxyphenyl)(phenyl)
o
methanone (23) (0.25 g, 93% yield) as an orange solid. Mp: 130-132 C. 1H NMR (400 MHz,
CD3OD): δ 7.61 (d, 2H, J = 7.1 Hz), 7.53 (t, 1H, J = 7.4 Hz), 7.44 (t, 2H, J = 7.5 Hz), 7.10 (s,
1H), 6.97 (s, 1H), 4.78 (bs, OH of CD3OD and 2OH of ArOH). 13C NMR (100 MHz,
CD3OD): δ 201.4, 155.9, 147.9, 139.2, 133.4, 130.2, 129.5, 122.9, 121.2, 119.9, 119.1. IR
(cm-1, CH2Cl2): 3327, 2965, 2833, 1648, 1593, 1575, 1491, 1456, 1440, 1377, 1276, 1260,
1214,1179, 1052, 1020, 985, 863. Anal. Calcld. for C13H9BrO3: C 53.27; H 3.09, Found: C
53.20; H 3.14.
4.2.11. (5-Bromo-2,4-dihydroxyphenyl)(4-hydroxyphenyl)methanone (24)
Demethylation of (5-bromo-2,4-dimethoxyphenyl)(4-methoxyphenyl)methanone (19) (0.9 g,
2.56 mmol) with BBr3 (3.85 g, 15.38 mmol) for 24h gave (5-bromo-2,4-dihydroxyphenyl)(4-
o
1
hydroxyphenyl) methanone (24) (0.66 g, 83%) as a yellow solid. Mp: 218-220 C. H NMR
(400 MHz, CD3COCD3): δ 12.47 (s, 1H, OH), 10.29 (s, 1H, OH), 9.36 (s, 1H, OH), 7.81 (s,
1H), 7.67 (d, 2H, A part of AB system, J = 8.6 Hz), 7.04 (d, 2H, B part of AB system, J = 8.6
Hz), 6.63 (s, 1H). 13C NMR (100 MHz, CD3COCD3): δ 198.7, 165.6, 162.3, 161.4, 138.4,
132.6, 130.0, 116.2, 114.9, 104.9, 100.3. IR (cm-1, CH2Cl2): 3417, 3057, 3016, 2876, 1579,
1510, 1448, 1422, 1384, 1334, 1241, 1208, 1168, 1147, 1114, 973, 926. Anal. Calcld. for
C13H9BrO4: C 50.51; H 2.93; Found: C 50.47; H 3.02.
15

4.3. Determination of Antioxidant Activity
·, ABTS•+
, DMPD•+
For measurement of antioxidant activities of novel bromophenols, DPPH
•-
and O2 scavenging, metal chelating and AChE inhibition assays were used. The antioxidant
activities were determined by the following bioanalytical assays:
4.3.1. Fe3+-Fe2+ reducing assay
For determination of Fe3+ reducing ability of novel bromophenols Fe3+(CN-)6-Fe2+(CN-)6
reduction method was used [55a,b]. Briefly, different concentrations of novel bromophenols
(10-30 µg/mL) in 0.75 mL of deionized H2O were added with 1.25 mL of phosphate buffer
(0.2 M, pH 6.6) and 1.25 mL of potassium ferricyanide [K3Fe(CN)6] (1%). Then, the solution
was incubated at 50oC during 20 min. After incubation period, 1.25 mL of trichloroacetic acid
(TCA) was added (10%). Lastly, 0.5 mL of FeCl3 (0.1%) was transferred to this mixture and
the absorbance value was measured at 700 nm in a spectrophotometer [56].
4.3.2. CUPRAC Assay
This assay is based on utilising the Cu2+- neocuproine reagent as the chromogenic oxidising
agent. To the mixture of 1 mL of Cu2+-neocuproine (7.5x10-3 M) and CH3COONH4 (1 M)
buffer solutions were transferred to a test tube, which contains different concentrations of
novel bromophenols (10-30 µg/mL). Total volume was completed with distilled H2O to 2 mL
and shaken vigorously. Absorbance of samples was recorded at 450 nm after 30 min [42,57].
4.3.3. FRAP assay
[Fe3+-(TPTZ)2]3+ reducing values of novel bromophenols are determined by measuring the
absorbance increase at 593 nm and relating it to a ferrous ions standard solution or to an
antioxidant standard solution. The change in absorbance is proportional to the combined
[Fe3+-(TPTZ)2]3+ reducing value of novel bromophenols [40a,43b].
4.3.4. ABTS•+ scavenging assay
16

ABTS radical cation was generated by the interaction of ABTS (7 mM) and K2S2O8 (2.45
mM) [50] described previously [51a]. This solution was diluted with methanol until the
absorbance in the samples reached 0.750 0.05 at 734 nm. Then, 1 mL of ABTS•+ solution
was supplement to 3 mL of novel bromophenols and control solutions. The extent of
decolorization is calculated as percentage reduction of absorbance.
4.3.5. DMPD•+ scavenging assay
DMPD•+ scavenging ability of novel bromophenols was performed according to the methods
described previously [26a, 58]. The scavenging capability of DMPD•+ radical of the sample
was monitored in spectrophotometer at 505 nm [40a].
4.3.6. DPPH• scavenging assay
The DPPH radical scavenging activity of novel bromophenols was performed according to the
method developed by Blois et al [59a,b]. Evaluation of the scavenging effect on DPPH
radicals is described by Gülçin et al. [60a-c]. In this assay, the antioxidants were able to
reduce the stable radical DPPH to the yellow colored DPPH2. DPPH• scavenging assay uses
the stable radical and this method was described previously in detailed [61a-c].
4.3.9. Acetylcholinesterase inhibition assay
The inhibition effects of novel bromophenols on AChE activities were measured according to
the Ellman’s method [25a, 62]. AChI and 5,5’-dithio-bis(2-nitro-benzoic)acid (DTNB) were
used as substrate for this enzymatic reaction. To this end, 100 µL of Tris/HCl buffer (1 M, pH
8.0) and 10 µL of novel bromophenols solution at different concentrations and 50 µL AChE
(5.32x10-3 U) solution were mixed and incubated for 10 min at 25°C. Then 50 µL of DTNB
(0.5 mM) was transferred. Then the reaction was initiated by the addition of 50 µL of AChI.
The hydrolysis of AChI was recorded spectrophotometrically by the formation of yellow 5-
thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine at a
wavelength of 412 nm.
17

Declaration of interest
The authors report there is no conflict of interests.
Acknowledgments
We indebted to Atatürk University for the financial supports of this work. Also, İ.G. would
like to extend his sincere appreciation to the Research Chairs Program at King Saud
University for funding this research.
REFERENCES
1. G. W. Gribble, J. Nat. Prod. 55 (1992) 1353–1395.
2. J. W. Blunt, B. R Copp, W. P. Hu, M. H. Munro, G. P. T. Northcote, M. R. Prinsep, Nat. Prod.
Rep. 25 (2008) 35-94.
3. J.C. Morris, Nat. Prod. Rep. 30 (2013) 783-805.
4. J.W. Blunt, B.R Copp, R.A. Keyzers, M.H.G. Munro, M.R. Prinsep, Nat. Prod. Rep. 29 (2012)
144-222.
5. D. F. Wiemer, D. D. Idler, W. Fenical, Experientia 47 (1991) 851-853.
6. X. Xu, F. Song, S. Wang, S. Li, F. Xiao, J. Zhao, Y. Yang, S. Shang, L. Yang, J. Shi, J. Nat. Prod.
67 (2004) 1661–1666.
7. D. Shi, J. Li, S. Guo, H. Su, X. Fan, Chin. J. Ocean Lim. 27 (2009) 277–282.
8. W. Wang, Y. Okada, H. Shi, Y. Wang, T. Okuyama, J. Nat. Prod. 68 (2005) 620–622.
9. X.J. Duan, X. M. Li, B.G. Wang, J. Nat. Prod. 70 (2007) 1210–1213.
10. H.T. Balaydın, I. Gülçin, A. Menzek, S. Göksu, E. Şahin, J. Enzyme Inhib. Med. Chem. 25 (2010)
685–695.
11. W. Zhao, X. Feng, S. Ban, W. Lin, Q. Li, Bioorg. Med. Chem. Lett. 20 (2010) 4132–4134.
−428.
12. H. T. Balaydın, M. Şentürk, S. Göksu, A. Menzek, Eur. J. Med. Chem. 54 (2012) 423
13. a) Y. Akbaba, H. T. Balaydın, A. Menzek, S. Göksu, E. Şahin, D. Ekinci, Arch. Pharm. 346
(2013) 447-454; b) H. T. Balaydın, H. Soyut, D. Ekinci, S. Göksu, Ş. Beydemir, A. Menzek, E.
Şahin, J. Enzyme Inhib. Med. Chem. 27 (2012) 43-50.
14. Y. Çetinkaya, H. Göçer, İ. Gülçin, A. Menzek, Arch. Pharm. 347 (2014) 354-359.
15. Y. Akbaba, C. Türkeş, L. Polat, H. Söyüt, E. Şahin, A. Menzek, S. Göksu, Ş. Beydemir, J.
Enzyme Inhib Med Chem. 28 (2013) 1073-1079.
16. J.B.L. Tan, Y.Y. Lim, Food Chem. 172 (2015) 814-822.
17. J. Gao, T. Zhang, Z.Y. Jin, X.M. Xu, J.H. Wang, X.Q. Zha, H.Q. Chen, Food Chem. 169 (2015)
430-438.
18

18. a) İ. Gülçin, Innov. Food Sci. Emerg. 11 (2010) 210-218; b) S. Çakmakçı, E.F. Topdaş, P. Kalın,
H. Han, P. Şekerci, L. Polat Kose, İ. Gülçin, Int. J. Food Sci. Technol. 50 (2015) 472-481; c)
M.H. Sehitoglu, H. Han, P. Kalin, İ. Gülçin, A. Ozkan, H.Y. Aboul-Enein, J. Enzyme Inhib. Med.
Chem. 30 (2015) 264-269.
19. N. Nakatani, Antioxidants from spices and herbs. Shahidi F. (Ed.), Natural antioxidants,
chemistry, health effects, and applications, AOCS press. 1997, pp. 64-75.
20. M. Materska, M. Konopacka, J. Rogoliński, K. Slosarek, Food Chem. 168 (2015) 546-553.
21. L. Castro, B.A. Freeman, Nutrition, 17 (2001) 161-165.
22. S. Samaram, H. Mirhosseini, C.P. Tan, H.M. Ghazali, S. Bordbar, A. Serjouie, Food Chem, 172
(2015) 7–17.
23. a) B. Halliwell, Ann Rev Nut. 16 (1997) 33–50. b) İ. Gülçin, R. Elias, A. Gepdiremen, A. Chea,
F. Topal, J. Enzyme Inhib. Med. Chem. 25 (2010) 44-53.
24. a) İ. Gülçin, Toxicology. 217 (2006) 213-220; b) İ. Gülçin, Life Sci. 78 (2006) 803-811; c) İ.
Gülçin, Amino Acids. 32 (2007) 431-438; d) İ. Gülçin, E. Bursal, H.M. Şehitoğlu, M. Bilsel, A.C.
Gören, Food Chem. Toxicol. 48 (2010) 2227-2238.
25. a) H. Gocer, F. Topal, M. Topal, M. Küçük, D. Teke, İ. Gülçin, S.H. Alwasel, C.T. Supuran, J.
Enzyme Inhib. Med. Chem. (2015) doi.org/10.3109/14756366.2015.1036051; b) E. A. Van der
Zee, B. Platt, G. Riedel, Behav. Brain. Res. 221 (2011) 583-586; c) A. Cavalli, M.L. Bolognesi,
A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini, C. Melchiorre, J. Med. Chem. 51 (2008) 347-
372.
26. a) Y. Çetinkaya, H. Göçer, S. Göksu, İ. Gülçin, J. Enzyme Inhib. Med. Chem. 29 (2014) 168–
174; b) H. T. Balaydın, Y. Akbaba, A. Menzek, E. Şahin, S. Göksu, Arkivoc. XIV (2009) 75-87.
27. J. M. Wagner, C. J. McElhinny, A.H. Lewin, F. I. Carroll, Tetrahedron Asymm. 14 (2003) 2119-
2125.
28. S. Etienne, M. Matt, T. Oster, M. Samadi, M. Beley, Tetrahedron. 64 (2008) 9619-9624.
29. C. J. Aquino, J. L. Collins, D. J. Cowan, Y. Wu, PCT Int. Appl. 2011, WO 2011137135 A1
20111103, Chem. Abstr. 155 (2011) 615381.
30. A. Ahlburg, A. T. Lindhardt, R. H. Taaning, A. E. Modvig, T. Skrydstrup, J. Org. Chem. 78
(2013) 10310-10318.
31. S. Göksu, M. T. Uğuz, H. Ozdemir, H. Seçen, Turk J. Chem. 29 (2005) 199-205.
32. a) S. Göksu, H. Seçen, Y. Sütbeyaz, Helv. Chim. Acta. 89 (2006) 270-273; b) H. Göçer, A.
Akıncıoğlu, N. Öztaşkın, S. Göksu, İ. Gülçin, Arch. Pharm. 346 (2013) 783-792.
33. Y. Akbaba, H. T. Balaydın, S. Göksu, E. Şahin, A. Menzek, Helv. Chim. Acta. 93 (2010) 1127-
1135.
34. a) D.J. McClements, E.A. Decker, J. Food Sci. 65 (2000) 1270-1282; b) L. Zou, C.C. Akoh, Food
Chem. 168 (2015) 504-511.
19

35. a) F. Shahidi, Y. Zhong, Chem. Soc. Rev. 39 (2010) 4067-4079; b) T. Waraho, D.J. McClements,
E.A. Decker, Trends Food Sci. Technol. 22 (2011) 3-13.
36. a) H. Rodrìguez, J.A. Curiel, J.M. Landete, B. de Las Rivas, F.L. de Felipe, C. Gòmez-Cordovés,
Int. J. Food Microbiol. 132 (2009) 79-90; b) P. Filannino, Y. Bai, R. Di Cagno, M. Gobbetti,
M.G. Gänzle, Food Microbiol. 46 (2015) 272-279.
37. a) M. Şentürk, İ. Gülçin, A. Daştan, Ö.İ. Küfrevioğlu, C.T. Supuran, Bioorg. Med. Chem. 17
(2009) 3207-3211; b) A. Innocenti, S.B. Öztürk Sarıkaya, İ. Gülçin, C.T. Supuran, Bioorg. Med.
Chem. 18 (2010) 2159-2164; c) A. Innocenti, İ. Gülçin, A. Scozzafava, C.T. Supuran, Bioorg.
Med. Chem. Lett. 20 (2010) 5050-5053.
38. a) S.B. Öztürk Sarıkaya, İ. Gülçin, C.T. Supuran, Chem. Biol. Drug Des. 75 (2010) 515-520; b)
S.B. Öztürk Sarıkaya, F. Topal, M. Şentürk, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. Lett.
21 (2011) 4259-4262; c) H. Göçer, İ. Gülçin, Int. J. Food Sci Nut. 62 (2011) 821-825; d) İ.
Gülçin, S. Beydemir, Mini Rev. Med. Chem. 13 (2013) 408-430.
39. a) İ. Gülçin, A. Daştan, J. Enzyme Inhib. Med. Chem. 22 (2007) 685-695; b) İ. Gülçin, M.
Elmastaş, H.Y. Aboul-Enein, Arab. J. Chem. 5 (2012) 489-499.
40. a) İ. Gülçin, Arch Toxicol. 86 (2012) 345-391; b) L.R.C. Barclay, M.R. Vinqvist, K. Mukai, S.
Itoh, H. Morimoto, J. Org. Chem. 58 (1993) 7416–7420.
41. S. Tomiyama, S. Sakai, T. Nishiyama, F. Yamada, Bull. Chem. Soc. Jpn. 66 (1993) 299-304.
42. R. Apak, K. Güçlü, M. Özyürek, S.E. Karademir, E. Erçağ, Int. J. Food Sci. Nut. 57 (2006) 292-
304.
43. a) E. Köksal, İ. Gülçin, S.B. Öztürk Sarıkaya, E. Bursal, J. Enzyme Inhib. Med. Chem. 24 (2009)
395-405; b) E. Bursal, E. Köksal, İ. Gülçin, G. Bilsel, A.C. Gören, Food Res. Int. 51 (2013) 66-
74.
44. a) I.F.F. Benzie, J.J. Strain, Method Enzymol. 299 (1999) 15–27; b) Y. Çetinkaya, H. Göçer, A.
Menzek, İ. Gülçin, Arch. Pharm. 345 (2012) 323-334.
45. W.D. Li, L.S. Ren, Z.B. Lin, Chin. J. New Drug. 11 (2002) 928-931.
46. a) Gülçin, İ., Topal, F., Oztürk Sarikaya, S.B., Bursal, E., Gören, A.C., Bilsel M., Rec. Nat. Prod.
5 (2011) 158-175; b) Köksal, E., Bursal, E., Dikici, E., Tozoğlu, F., Gülçin, İ., J. Med. Plants Res.
5 (2011) 217-222; c) Gülçin, İ., J. Med. Food, 14 (2011) 975-985.
47. a) İ. Gülçin, Z. Huyut, M. Elmastaş, H.Y. Aboul-Enein, Arab. J. Chem. 3 (2010) 43-53; b) İ.
Gülçin, E. Kirecci, E. Akkemik, F. Topal, O. Hisar, Turk. J. Biol. 34 (2010) 175-188; c) H.
Şerbetçi Tohma, İ. Gülçin, Int. J. Food Propert. 13 (2010) 657-671.
48. a) E. Niki, Free Radical Biol. Med. 49 (2010) 503-515; b) K. Valentova, F. Sersen, J. Ulrichova,
J. Agric. Food Chem. 53 (2005) 5577-5582; c) H.T. Balaydın, İ. Gülçin, A. Menzek, S. Göksu, E.
Şahin, J. Enzyme Inhib. Med. Chem. 25 (2010) 685-695.
20

49. a) J.K. Winkler-Moser, H.S. Hwang, E.L. Bakota, D.A. Palmquist, Food Chem. 169 (2015) 92-
101; b) M.E. Büyükokuroğlu, İ. Gülçin, Pharmacog Mag. 4 (2009) 189-195; c) O. Talaz, İ.
Gülçin, S. Göksu, N. Saracoglu, Bioorg. Med. Chem. 17 (2009) 6583-6589.
50. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radical Biol.
Med. 26 (1999) 1231-1237.
51. a) İ. Gülçin, Chem. Biol. Interact. 179 (2009) 71-80; b) İ. Gülçin, R. Elias, A. Gepdiremen, K.
Taoubi, E. Köksal, Wood Sci Technol. 43 (2009) 195-212; a) T. Ak, İ. Gülçin, Chem. Biol.
Inteearct. 174 (2008) 27-37.
52. a) Y. Akbaba, A. Akıncıoğlu, H. Göçer, S. Göksu, İ. Gülçin, C.T. Supuran, J. Enzyme Inhib.
Med. Chem. 29 (2014) 35-42; b) Y. Akbaba, E. Bastem, F. Topal, İ. Gülçin, A. Maraş, S. Göksu,
Arch. Pharm. 347 (2014) 950–957; c) S. Göksu, A. Naderi, Y. Akbaba, P. Kalın, A. Akıncıoğlu,
İ. Gulcin, S. Durdaği, R.E. Salmas, Bioorg. Chem. 56 (2014) 75-82; d) M. Boztaş, Y. Çetinkaya,
M. Topal, İ. Gülçin, A. Menzek, E. Şahin, M. Tanc, C.T. Supuran, J. Med. Chem. 58 (2015) 640-
650.
53. a) H. Göçer, A. Akıncıoğlu, S. Göksu, İ. Gülçin, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 30
(2015) 316-320; b) K. Aksu, M. Nar, M. Tanç, D. Vullo, İ. Gülçin, S. Göksu, F. Tümer, C.T.
Supuran, Bioorg. Med. Chem. 21 (2013) 2925-2931; c) A. Akıncıoğlu, Y. Akbaba, H. Göçer, S.
Göksu, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. 21 (2013) 1379-1385.
54. a) H. Göçer, A. Akıncıoğlu, N. Öztaşkın, S. Göksu, İ. Gülçin, Arch. Pharm. 346 (2013) 783-792;
b) A. Akıncıoğlu, M. Nar, İ. Gülçin, S. Göksu, Arch. Pharm. 347 (2014) 68-76.
55. a) İ. Gülçin, J. Enzyme Inhib. Med. Chem. 23 (2008) 149-152; b) İ. Gülçin, A.Z. Tel, E. Kirecci,
Int. J. Food Propert. 11 (2008) 450-471.
56. İ. Gülçin, M. Elmastas, H.Y. Aboul-Enein, Phytother. Res. 21 (2007) 354-361.
57. İ. Gülçin, J. Enzyme Inhib. Med. Chem. 23 (2008) 871-876.
58. V. Fogliano, V. Verde, G. Randazzo, A. Ritieni, J. Agric. Food Chem. 47 (1999) 1035–1040.
59. a) M.S. Blois, Nature. 26 (1958) 1199-1200; b) İ. Gülçin, M. Elmastas, H.Y. Aboul-Enein,
Phytother. Res. 21 (2007) 354-361.
60. a) İ. Gülçin, R. Elias, A. Gepdiremen, L. Boyer, Eur. Food Res. Technol. 223 (2006) 759-767; b)
M. Elmastas, İ. Gülçin, Ö. Işıldak, Ö.İ. Küfrevioğlu, K. İbaoğlu, H.Y. Aboul-Enein, J. Iran.
Chem. Soc. 3 (2006) 258-266; c) M. Elmastas, İ. Türkekul, L. Öztürk, İ. Gülçin, Ö. Işıldak, H.Y.
Aboul-Enein, Comb. Chem. High Thrp. Scr. 9 (2006) 443-448.
61. a) İ. Gülçin, Int. J. Food Sci. Nut. 56 (2005) 491-499; b) İ. Gülçin, V. Mshvildadze, A.
Gepdiremen, R. Elias, Phytother. Res. 20 (2006) 130-134; c) İ. Gülçin, V. Mshvildadze, A.
Gepdiremen, R. Elias, Phytomedicine 13 (2006) 343-351.
62. G.L. Ellman, K.D. Courtney, V.Jr. Andres, R.M. Featherstone, Biochem. Pharmacol. 7 (1961) 88-
95.
21

TABLES, FIGURES AND SCHEMES
Table 1. Determination of reducing power of same concentration (20 µg/mL) of novel
bromophenols and their methylated precursors by FRAP methods, ferric ions (Fe3+) reducing
and cupric ions (Cu2+) reducing capacity by Cuprac method.
Fe3+-Fe2+reducing
Cu2+-Cu+ reducing
Fe3+-TPTZreducing
Antioxidants
BHA
λ700
R2
R2
λ450
R2
λ593
2.354 0.003
1.962 0.002
0.9999
1.869 0.001
0.9806
0.9575
1.405 0.003
0.927 0.008
0.893 0.002
0.136 0.003
0.135 0.001
0.145 0.004
0.156 0.013
0.182 0.002
0.152 0.002
0.252 0.002
2.094 0.004
2.123 0.011
1.947 0.003
2.563 0.003
1.163 0.002
1.407 0.004
0.854 0.003
1.005 0.002
0.117 0.005
0.083 0.002
0.225 0.003
0.074 0.003
0.057 0.003
0.086 0.002
0.077 0.002
1.602 0.001
2.014 0.004
1.966 0.002
2.018 0.005
0.655 0.003
0.9972
0.9998
0.9992
0.9438
0.9926
0.9748
0.9970
0.9913
0.9587
0.9896
0.9430
0.9984
0.9707
0.9997
0.9692
1.328 0.004
1.123 0.002
1.742 0.002
0.387 0.002
0.401 0.001
0.389 0.001
0.406 0.002
0.404 0.003
0.382 0.001
0.421 0.001
2.012 0.001
1.986 0.004
1.974 0.005
2.093 0.004
0.401 0.002
0.9739
0.9986
0.9997
0.9369
0.9510
0.9493
0.9427
0.9410
0.9373
0.9422
0.9591
0.9916
0.9546
0.9709
0.9404
BHT
0.9630
0.9819
α-Tocopherol
0.9930
0.9570
0.9589
0.9540
0.9976
0.9805
0.9990
0.9898
0.9711
0.9999
0.9757
0.9895
0.9700
Trolox
10
11
14
15
16
18
19
20
21
22
23
24
a expressed as absorbance values.
22

Table 2. Determination of half maximal concentrations (IC50) of novel bromophenols, their
methylated precursors and standards for DPPH· and ABTS•+ radical scavenging and AChE
inhibition assays
DPPH·
scavenging§ scavenging§
ABTS•+
Antioxidant
Compounds
AChE inhibition*
IC50 Ki
22.35
2.72
BHA
-
-
34.65
21.65
2.88
4.81
BHT
-
-
-
-
α-Tocopherol
16.11
2.86
Trolox
10
-
-
346.50
227.36
230.34
224.53
231.00
226.67
229.63
138.60
138.60
173.25
173.25
138.60
4.41
4.20
7.21
4.22
4.68
5.25
3.53
4.34
4.25
3.77
5.28
5.88
11
14
15
16
18
221.37
224.60
19
4.05
3.65
15.75
15.40
14.74
14.15
28.92
-
2.55
2.58
2.61
2.23
22.35
-
20
21
22
23
4.71
4.84
4.58
4.88
4.06
3.75
2.78
3.24
24
Tacrine♯
4.81
7.53
5.72
7.64
*: They were determined as nM
§: They were determined as µg/mL
♯: Tacrine was used as positive control for AChE inhibition
23

FIGURES LEGENDS
Figure 1. Some selected natural products 1-6
Figure 2. Determination of the half maximal inhibitory concentration (IC50) value (A) and
inhibition constant (Ki) value (B) of novel bromophenol 22 for acetylcholinesterase
enzyme (AChE) by using a Lineweaver-Burk graph
Br
OH
Br
Br
Br
OH
Br
Br
Br
Br
HO
HO
OH
Br
HO
HO
OH
OH
OH
OH
OH
1 Vidalol A
2 Vidalol B
Br
Br
Br
CH2OR
Br
Br
Br
Br
Br
Br
HO
OH HO
Br
OH HO
OH
Br
Br
OH
OH
OH
OH
OH
OH
6
3
4 R=Me
5
R=Et
Figure 1
24

100
80
100
80
60
40
20
0
60
40
20
0
-3
0
1
2
3
4
5
6
-18
12
27
42
1/[S] (mM)-1
Concentration (nM)
A
B
Figure 2
25

SCHEMES
R1
O
Br
R1
O
Br
R2
R3
R2
R3
PPA
OH
80 oC
+
OMe
OMe
1.5 h
7 R1 = H, R2 = R3 = OMe
8
9
10 R1 = H, R2 = R3 = OMe, 60%
11
R1 = R2 = OMe, R3 = H
R1 = R2 = OMe, R3 = H, 55%
OMe
OMe
O
O
OMe
PPA
OH
+
o
80 C
Br
R
R
Br
1.5 h
OMe
12
R = OMe
13 R = H
15
R = OMe, 76%
16 R = H, 50%
14
Scheme 1. The synthesis of compounds 10, 11, 15 and 16
26

OMe
O
O
OMe
PPA
OH +
80 oC
1.5 h
73%
OMe
MeO
MeO
OMe
18
17
12
25 oC
Br2
CHCl3
12 h
62%
O
OMe
MeO
OMe
Br
19
Scheme 2. The synthesis of compound 19
27

R1
O
R4
R1
O
R4
R2
R3
R2
R3
1. BBr3, 0-25 oC
CH2Cl2
24 h
R5
R5
R6
R6
2. MeOH or H2O
0 oC
10
R1=R6=H, R4=Br, R2=R3 =R5=OMe
11R3=R6=H, R4=Br, R1=R2=R5=OMe
20
R1=R6=H, R4=Br, R2=R3 =R5=OH, 91%
21 R3=R6=H, R4=Br, R1=R2=R5=OH, 80%
22
15
R1=R2= H, R5=Br, R3=R4=R6=OMe
16 R1=R2=R3=H, R5=Br, R4=R6=OMe
19 R1=R2=H, R6=Br, R3=R4=R5=OMe
R1=R2= H, R5=Br, R3=R4=R6=OH, 92%
23 R1=R2=R3=H, R5=Br, R4=R6=OH, 93%
24 R1=R2=H, R6=Br, R3=R4=R5=OH, 83%
Scheme 3. The synthesis of new bromophenols 20-24
28

HIGHLIHTS
•
•
•
•
•
Series of novel bromophenol derivatives were synthesized.
Ferric ions (Fe3+) and cupric ions (Cu2+) reducing capacities were investigated.
The synthesized compounds showed DPPH· and ABTS•+ scavenging activities.
The bromophenols were found to be the most potent antioxidants.
All compounds demonstrated low nanomolar inhibition against acetylcholinesterase.
29