Synthesis and carbonic anhydrase isoenzymes inhibitory effects of brominated diphenylmethanone and its derivatives

Article abstract of DOI:10.1002/ardp.201300349

Known and novel derivatives including CO, Br, and OH (benzylic and phenolic), and the corresponding benzylic alcohols of (3,4-dimethoxyphenyl)(2,3, 4-dimethoxyphenyl)methanone were synthesized, and their inhibitory effects on the carbonic anhydrase (CA) i

Full text of DOI:10.1002/ardp.201300349

354
Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
Full Paper
Synthesis and Carbonic Anhydrase Isoenzymes Inhibitory
Effects of Brominated Diphenylmethanone and Its Derivatives
3
1
1
Yasin Çetinkaya1,2, Hülya Göçer , Ilhami Gülçin , and Abdullah Menzek *
_
1
Faculty of Science, Department of Chemistry, Atatürk University, Erzurum, Turkey
Department of Food Technology, Oltu Vocational School, Atatürk University, Oltu-Erzurum, Turkey
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey
2
3
Known and novel derivatives including CO, Br, and OH (benzylic and phenolic), and the corresponding
benzylic alcohols of (3,4-dimethoxyphenyl)(2,3,4-dimethoxyphenyl)methanone were synthesized, and
their inhibitory effects on the carbonic anhydrase (CA) isoenzymes I and II were investigated. CAs are
the metalloenzymes catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate
(HCO3ꢀ). The inhibitory effects of diphenylmethanone derivatives 5–18 were tested on human CA (hCA,
EC 4.2.1.1) isoenzymes (hCA I and hCA II) and they inhibited both isoenzymes at micromolar levels.
Compounds 5 and 10 were found to be the best inhibitors against both CA isoenzymes. According to our
data, compound 10 was the best inhibitor for isoenzyme hCA I (IC50 ¼ 3.48 mM, Ki ¼ 2.19 mM) whereas
compound 5 was found to be the best inhibitor for isoenzyme hCA II (IC50 ¼ 1.33 mM, Ki ¼ 2.09 mM).
Probably, stable conformations of 5 and 10 are more convenient for interaction with CA isoenzymes
than those of the other compounds.
Keywords: Benzylalcohol / Bromination / Carbonic anhydrase / Enzyme inhibition / Reduction
Received: September 15, 2013; Revised: November 27, 2013; Accepted: December 4, 2013
DOI 10.1002/ardp.201300349
Introduction
Carbonic anhydrase ꢁ Zn2þ ꢀ HCO3ꢀ þ H2O
, carbonic anhydrase ꢁ Zn2þ ꢀ H2O þ HCO3ꢀ
Diphenylmethanone derivatives, including Br and OH groups,
are both natural and biological active compounds (Fig. 1)
[1–5]. The naturally occurring bromophenols are frequently
Carbonic anhydrase ꢁ Zn2þ ꢀ H2O þ X
, carbonic anhydrase ꢁ Zn2þ ꢀ OHꢀ þ XHþ
isolated from red algae of the family Rhodomelaceae [1].
Compounds 1–4 are also natural products and have carbonic
anhydrase (CA) inhibitory properties [6–10].
Enzymes are naturally occurring biocatalysts in living
forms, CA IV, CA IX, CA XII, and CA XIV are membrane-
organisms that regulate the metabolic activities [11]. CAs (EC
4.2.1.1) are metalloenzymes in plant and animal kingdoms
In general, there are several cytosolic forms, including one
mitochondrial form, four membrane-bound isoenzymes, as
well as a secreted CA isoenzyme. CA I, III, and VII are cytosolic
bound, CA V is mitochondrial, and CA VI is a secreted CA
isoenzyme [13, 14]. CA isoenzymes have been purified and
and isoenzymes of CA catalyze one of the most important
characterized in numerous tissues. CA isoforms are found in a
reactions for life, the conversion of CO2 into bicarbonate
(HCO3ꢀ) with a metal hydroxide in nucleophilic conver-
variety of tissues where they participate in several important
biological processes such as acid–base balance, respiration,
CO2 and ion transport, bone resorption, ureagenesis,
gluconeogenesis, lipogenesis, and electrolyte secretion [15].
CA is very active in the kidney, gastric mucosa, eye lens,
sion [12]. This reaction can be summarized as follows
Carbonic anhydrase ꢁ Zn2þ ꢀ OHꢀ þ CO2
, carbonic anhydrase ꢁ Zn2þ ꢀ HCO3ꢀ
salivary glands, brain, nerve myelin sheath, pancreas,
_
Correspondence: Prof. Ilhami Gülçin, Faculty of Sciences, Department of
Chemistry, Ataturk University, 25240 Erzurum, Turkey.
E-mail: igulcin@atauni.edu.tr
ꢂAdditional correspondence: Prof. Abdullah Menzek,
E-mail: amenzek@atauni.edu.tr,
Fax: þ90 4422360948
Fax: þ90 4422360948
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
Biological Activity of Diphenylmethanone Derivatives
355
may be important. Therefore, diphenylmethanone 5 and its
derivatives 6–13 with Br and OH were synthesized [23].
However, diphenylmethanol 15 and its derivatives 16–17 with
Br and OH were also synthesized. Furthermore, CA inhibitory
properties of the compounds 5–18 were investigated.
Et
O
Br
Br
Br
Br
Br
Br
H
O
H
O
H
O
B
r
H
O
H
O
H
O
H
O
H
O
1
2
Results and discussion
Br
O
Br
H
O
Br
Synthesis
Br
Br
Br
Diphenylmethanone 5 was obtained from the reaction of 3,4-
dimethoxybenzoic acid and 1,2,3-trimethoxybenzene in
polyphosphoric acid (PPA) and then its derivatives 6–13 and
14 with Br and OH were synthesized (Fig. 2) [23].
H
O
H
O
H
H
H
O
O
O
H
O
H
O
B
r
H
O
(Vidalol B)
4
3
To obtain corresponding benzyl alcohol derivatives from
diphenylmethanone 5 and its derivatives, reduction of CO
groups was aimed because they have benzylic OH in case of
CO. Reaction of the compound 5 with NaBH4 in MeOH for
1 day (d) gave benzylic alcohol in a yield of 95% (Scheme 1). A
mixture of compounds 15 and 16 was observed when HCl was
added in the last of this reaction step. Column chromatogra-
phy of the mixture allowed us to isolate compounds 15 and
16. The compound 16 is an ether compound forming in the
reaction condition.
In the same way, the reactions of the compounds 6 and 11
with NaBH4 in MeOH were treated. It was found that the
reaction rates with NaBH4 in MeOH are very low. For example,
the reactions of ketones 6 and 11 with NaBH4 approximately
were completed for 4 days. Therefore, their reduction to the
corresponding benzylic alcohols 17 and 18 were realized by
LiAlH4 in THF (Scheme 1).
Figure 1. Some natural compounds exhibiting carbonic anhydrase
inhibitory properties.
prostate, and uterus. Many studies have identified the
important role of this enzyme in chloroplasts, fish branchia
and secretory organs, some insects, and bacteria, and the
construction of shell and egg shell formation in some of
the animals, plants, and photosynthetic algae [16, 17]. In
physiological conditions, the activity of hCA from human
erythrocytes is quite changed, and these changes in hCA
activity are related with metabolic disorders, such as diabetes
and hypertension [18]. hCA enzyme inhibition studies can
give insights that will help us to understand the enzyme-
catalyzed reactions, and the vital functions and distribution
of enzymes in these tissues. Therefore, the synthesis of enzyme
inhibitors and activators of hCA are accelerated at present [19].
Specifically, CA I is located in many tissues, but some studies
showed that this enzyme is inclusive in retinal and cerebral
edema. Furthermore, CA II is involved in several diseases, such
as glaucoma, edema, and epilepsy [19–22].
CA isoenzymes purification and activity assay
In this study, hCA I and hCA II isoenzymes were purified
separately from human erythrocytes with Sepharose-4B-L-
thyrosine-sulfanilamide affinity column chromatography. In
the first part of our study, we have identified the inhibition
effects of brominated diphenylmethanone 5–18 on the
esterase activity of hCA I and hCA II. The inhibitions studies
were performed with esterase activity methods [24] previously
We synthesized the brominated diphenylmethanone deriv-
atives and investigated their CA inhibitory properties [6, 7]. To
compare CA inhibitory properties of diphenylmethanone,
diphenylmethanol, and their derivatives with Br and OH
R3
Me
O
O
R2
H
O
Br
O
R4
Me
Me
Me
O
R3
Me
Me
Me
Br
Br
Me
O
R1
O
O
R4
O
O
O
Me
O
R2
Me
O
R1
Me
O
=
=
=
=
5
6
7
8
9
R1 R2 R3 R4 H
=
=
=
=
=
11 R3 R4 H R R2 Br
14
,
1
=
=
=
=
H R2 Br
,
R1 R3 R4
=
=
=
12 R4 H R R2 R3 Br
,
1
1
=
=
=
= =
R2 R3 Br
= =
R1 R2 Br
R1 R4
H
,
H
,
=
=
= =
13 R3 H R
R2 R4 Br
,
=
R3 R4
Figure 2. Synthesized compounds without
benzylic OH or OMe.
=
=
=
=
R4
H
,
R1 R2 R3 Br
=
=
=
=
10 R1 R2 R3 R4 Br
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

356
Y. Çetinkaya et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
OH OMe
O
OMe
OMe
OMe
OMe
OMe
a
MeO
MeO
OMe
OMe
15
5
b
OMe OMe
OH OMe
OMe
OMe
OMe
OMe
+
MeO
MeO
OMe
OMe
16
15
70%
27%
R2
O
R1
Br
R2 OH R1
OMe
OMe
c
OMe
OMe
MeO
MeO
OMe
OMe
Br
6
R1 = OMe, R2 = H
17 R1 = OMe, R2 = H, 65%
18 R1 = OH, R2 = Br, 58%
Scheme 1. Reagents and conditions: (a) NaBH4,
MeOH, RT, 1 d, 95%; (b) 1: NaBH4, MeOH, RT,
1 d, 2: HCl, 10 min; (c) LiAlH4, THF, RT, 20 min.
11 R1 = OH, R2 = Br
described by Gocer and Gülçin [25]. This method is based on
the destruction of CA ester bonds. In the process, the CA
enzyme hydrolyzes the phenyl acetate and the resulting
product is composed at 348 nm absorption. In our study, this
method was preferred due to its high sensitivity compared to
methods of Wilbur–Anderson. The isoenzyme purities were
controlled by SDS–PAGE and single band was observed for
each isoenzyme.
are presented in Table 1. It was reported that CA isoenzyme
inhibitors coordinate to the zinc ion from the active site of CA
isoenzyme [30]. Among the investigated brominated diphe-
nylmethanone compounds, the compounds coded as 5 and 10
were shown to be effective inhibitors of cytosolic isoenzymes
hCA I and hCA II (Table 1). According to the data obtained from
this study, it is clear that the brominated diphenylmethanone
CA isoenzymes inhibition effects
Table 1. Human carbonic anhydrase isoenzymes (hCA I and II)
inhibition values with some brominated diphenylmethanone
derivatives by an esterase assay with 4-nitrophenylacetate as
substrate.
As is known, there are many studies in the scientific world on
the interactions of various compounds and the CA isoen-
zymes. In recent years, the CA isoenzymes have become an
interesting target for the design of inhibitors or activators
with biomedical applications. The CA inhibitors have been
essentially used as diuretics, antiglaucoma and antiepileptic
agents, while novel compounds are subject to clinical
research as antiobesity and antitumor drugs/diagnostic
tools [19, 26–29].
For this purpose, we have investigated the inhibitory effects
of brominated diphenylmethanone compounds 5–18 on the
hCA I and hCA II in vitro. Initially inhibition effects of
investigated compounds on hCA I and hCA II were determined
by drawing Lineweaver–Burk charts. Then, IC50 and the
average of Ki values were calculated for 5–18 based on the
drawn charts (Table 1).
IC50 (mM)
Ki (mM)
hCA II
Compounds
hCA I
hCA II
hCA I
5
6
10.59
8.50
38.51
–
1.33
9.94
67.95
–
6.68
3.17
12.71
–
2.09
2.66
33.06
–
7
8a)
9
19.41
3.48
52.91
93.54
–
29.37
4.91
31.79
99.44
–
14.34
2.19
6.67
21.99
–
18.66
4.51
6.28
41.93
–
10
11
12
13a)
14
15
16
17
18a)
203.26
362.90
582.47
391.60
–
171.14
297.48
563.53
239.84
–
55.74
84.54
293.13
152.31
–
12.96
197.36
115.06
126.72
–
In the first step, we determined the inhibitory effects of the
synthesized brominated diphenylmethanone derivatives 5–18
on the esterase activity of the cytosolic isoform hCA I, and
physiologically dominant isoform II. The inhibition results
a)
Human carbonic anhydrase isoenzymes results belonging to
these compounds could not be determined.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
Biological Activity of Diphenylmethanone Derivatives
357
derivatives compound 10 showed the strongest inhibitory
activity on hCA I with Ki of 2.19 mM and compound 5 showed
the strongest inhibitory activity on hCA II with Ki of 2.09 mM
(Table 1). Furthermore, the other remaining brominated
diphenylmethanone derivatives have quite strong inhibition
effect on cytosolic isoenzyme hCA I with IC50, which ranged
from 3.48 to 582.47mM and on hCA II with IC50 in the range of
1.33–563.53 mM (Table 1). It is well known that the nature of
the groups in ortho-, para-, and meta-positions to the phenolic
OH and OMe moiety strongly influences hCA I and hCA II
inhibitory activity. The inhibition effect of halogenated
sulfonamide derivatives has been investigated previously [31,
32]. It was indicated that the phenols, biphenyl, or diphenols
bear bulky ortho-moieties in their molecules [32]. It was
previously reported that the inhibition effect of halogenated
sulfonamide derivatives has been investigated [33]. In another
study, it was demonstrated that halogenated derivatives of
sulfonamide such as bromosulfonamide are more effective as
compared with the corresponding sulfonamides [31, 32].
Although we do not know in depth the mechanism of
interaction between the synthesized compounds and both
isoenzymes, we can speculate that the stable conformations of
5 and 10 are more appropriate for interaction with CA enzymes
than those of the others. Considering all this information, the
compounds 5 and 10 showing the highest inhibitory effects are
inevitable. Also, the physiologically dominant isoenzyme hCA I
was effectively inhibited by a range of compounds 5–18, with Ki
ranging from 2.19 to 293.13 mM, and isoenzyme hCA II by
compounds 5–18, with Ki ranging from 2.09 to 197.36 mM.
Reconnaissance of new CA inhibitors is of great importance for
pharmacological and medicinal approaches. Many inhibitors
have been designed and synthesized in the literature. However,
it is critically important to explore further classes of potent
CA I and CA II in order to detect compounds with a different
inhibition profile when compared to methanone derivatives,
and to find novel applications for the inhibitors of these
widespread enzymes [34, 35].
were obtained in the reactions of 6 and 11 with LiAlH4 in THF.
In addition, CA inhibitory properties of 5–18 have also been
evaluated. These known novel compounds may be important
synthons for further synthetic and biological purposes.
Our data suggest that the brominated diphenylmethanone
derivatives can be used as potential compounds in generating
convenient CA isoenzyme inhibitors. Specifically, brominated
diphenylmethanone derivatives 5–18 may be used in
generating potent hCA I and hCA II inhibitors, which
eventually target other isoforms that have not been assayed
yet for their interactions with such agents. Also, a net
distinction in the inhibition profile of the two cytosolic (hCA I
and II) has not been found. Phenolic and bromophenolic
compounds influence the activity of both hCA isozymes due to
the presence of different functional groups such as OH, OMe,
and Br in their aromatic scaffold.
Experimental section
General information
All chemicals and solvents are commercially available. All
solvents were distilled and dried according to standard
procedures. Silica gel (SiO2, 60 mesh; Merck, Darmstadt,
Germany) was used for column chromatography (CC). Melting
point of all compounds was determined by cap. melting-point
apparatus (BUCHI 530; Flawil, Switzerland) and are uncorrected.
IR spectra were recorded as solns. in 0.1 mm cells with a Mattson
1000 FT-IR spectrophotometer (Cambridge, England). 1H and 13
C
NMR spectra were recorded by 400 (100)-MHz Varian spectrome-
ter (Danbury, CT) in deuterated solvents (CDCl3 and D2O) with
tetramethylsilane (TMS, SiMe4) as an internal standard for
protons, and with solvent signals as an internal standard for
carbon spectra. Chemical shift values were mentioned as d in
ppm. Elemental analyses were recorded by Leco CHNS-932
apparatus (MI). CA inhibitory properties of samples were
determined by a spectrophotometer (UV-1208, Shimadzu, Japan).
(3,4-Dimethoxyphenyl)(2,3,4-trimethoxyphenyl)methanol (15)
Ketone 5 (500 mg, 1.5 mmol) dissolved in MeOH (40 mL) was added
to NaBH4 (340 mg, 8.9 mmol) at 0°C. The reaction solution was
stirred for 1 day. After it was controlled with TLC, the solvent was
evaporated. It was added to water (10 mL) and HCl (10 mL, 1%). The
mixture was extracted with CH2Cl2 (3 ꢃ 50 mL). Combined
organic layers were watched and dried over Na2SO4. Diphenyl-
methanol 15 (478 mg, 1.43 mmol, 95%) was recrystallized from
EtOAc/hexane. White crystals m.p. 104–106°C. 1H NMR (400 MHz,
CDCl3) d 6.96 (bs, aromatic, 1H), 6.93 (d, A part of AB-system, 1H,
J ¼ 8.6 Hz), 6.82 (dd, A part of AB-system, J ¼ 1.6 Hz, J ¼ 8.3 Hz, 1H),
6.79 (d, B part of AB-system, J ¼ 8.3 Hz, 1H), 6.63 (d, B part of AB-
system, J ¼ 8.6 Hz, 1H), 5.88 (d, J ¼ 5.4 Hz, 1H), 2.98 (d, OH,
J ¼ 5.6 Hz, 1H), 3.844 (s, OCH3, 3H), 3.841 (s, OCH3, 3H), 3.836 (s,
OCH3, 6H), 3.68 (s, OCH3, 3H). 13C NMR (400 MHz, CDCl3) d 153.58
(C), 151.49 (C), 149.05 (C), 148.35 (C), 136.88 (C), 130.20 (C), 122.33
(CH), 118.88 (CH), 111.04 (CH), 110.06 (CH), 109.95 (C), 107.18 (CH),
72.29 (COH), 61.05 (OCH3), 60.90 (OCH3), 56.19 (OCH3), 56.11
(OCH3), 56.06 (OCH3). IR (CH2Cl2, cmꢀ1): 3513, 3001, 2936, 2839,
2032, 1905, 1838, 1600, 1512, 1352, 1287, 1138, 1100, 1027, 954,
The inhibition effect of bromophenol derivatives on
cytosolic isoenzymes hCA I and II was previously repoted [15,
32]. Recently, the interaction of CA I and CA II isoenzymes
with several types of phenols, such as hydroxyl benzoic acids,
methoxy benzoic acids, natural and unnatural bromophe-
nols, and their substituted derivatives, was investigated. These
derivatives demonstrated low micromolar or submicromolar
inhibition as well as the possibility of designing isozyme
selective CAIs [32, 36–38].
Conclusion
According to literature [11], known compounds 5–14 were
synthesized. From novel compounds, 15 and 16 were obtained
in the reactions of 5 with NaBH4 in MeOH while 17 and 18
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

358
Y. Çetinkaya et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
933 Anal. calcd. for (C18H22O6): C 64.64; H 6.63. Found C 64.96;
H 6.71; MS (CI) m/z 317.1 (Mþ).
111.26 (CH), 107.38 (C), 71.11 (CH), 61.44 (OCH3), 60.98 (OCH3),
56.40 (OCH3), 56.30 (OCH3). IR (CH2Cl2, cmꢀ1): 3433, 2930, 2852,
1736, 1631, 1603, 1548, 1505, 1462, 1432, 1415, 1343, 1265, 1206,
1158, 1074, 1041.
1-((3,4-Dimethoxyphenyl)(methoxy)methyl)-2,3,4-
trimethoxybenzene (16)
Biochemistry
Ketone 5 (400 mg, 1,2 mmol) dissolved in MeOH (40 mL) was added
to NaBH4 (320 mg, 8.4 mmol) at 0°C. The reaction solution was
stirred for 1 day and controlled with TLC. After the reaction was
completed, HCl (20 mL, 1%) was added and stirred for 10 min. The
solvent was evaporated and the residue was extracted with
CH2Cl2 (3 ꢃ 50 mL). After drying the organic layer over Na2SO4
and evaporation of the solvent, chromatography of the residue on
silica gel (SiO2, 60 g) with ethyl acetate/hexane (1:9) was utilized to
obtain diphenyl methoxymethyl 16 (295 mg, 0.847 mmol, 70%) as
pale yellow diphenylmethanol 15 (110 mg, 0.328 mmol, 27%).
M.p. 51–54°C for compound 16. 1H NMR (400 MHz, CDCl3) d 7.03
(d, A part of AB-system, J ¼ 8.7 Hz, 1H), 6.92 (d, J ¼ 1.9 Hz, 1H), 6.85
(dd, A part of AB-system, J ¼ 1.9 Hz, J ¼ 8.2 Hz, 1H), 6.79 (d, A part
of AB-system, J ¼ 8.2 Hz, 1H), 6.65 (d, A part of AB-system,
J ¼ 8.7 Hz, 1H), 5.49 (s, CH, 1H), 3.838 (s, OCH3, 3H), 3.835 (s, OCH3,
3H), 3.82 (s, OCH3, 3H), 3.81 (s, OCH3, 3H), 3.74 (s, OCH3, 3H), 3.35
(s, OCH3, 3H). 13C NMR (400 MHz, CDCl3) d 153.28 (C), 151.71 (C),
149.14 (C), 148.46 (C), 142.30 (C), 135.00 (C), 128.58 (C), 121.75
(CH), 119.68 (CH), 111.20 (CH), 110.59 (CH), 107.61 (CH), 79.37
(CH), 61.02 (OCH3), 60.85 (OCH3), 57.13 (OCH3), 56.16 (OCH3),
56.08 (OCH3), 56.05 (OCH3). IR (CH2Cl2, cmꢀ1): 2935, 2835, 1598,
1514, 1494, 1463, 1416, 1286, 1261,1234, 1139, 1095, 1029. Anal.
calcd. for (C18H24O6): C 65.50; H 6.94. Found C 65.56; H 7.21; MS
(CI) m/z 348.1 (Mþ).
Purification of CA isoenzymes from human erythrocytes by
affinity chromatography
CA isoenzymes were purified via a simple single-step process with
Sepharose-4B-L-tyrosine-sulfanilamide affinity gel chromatogra-
phy, as defined previously [39, 40]. The fresh human erythrocyte
samples were centrifuged at 10,000g for 30 min and the
precipitate was removed from serum. The pH was adjusted to
8.7 with solid Tris. Sepharose-4B-tirozyne-sulfanylamide affinity
column was equilibrated with 25 mM Tris-solution. The affinity
gel was washed with 25 mM Tris–HCl/22 mM Na2SO4 (pH 8.7). The
human carbonic anhydrase (hCA VI) isoenzyme was eluted with
0.25 M H2NSO3H/25 mM Na2HPO4 (pH 6.7). All procedures were
performed at 4°C [18, 41, 42].
Esterase activity assay
CA activity was assayed by the change in absorbance at 348 nm of
4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period
of 3 min at 25°C using a spectrophotometer (Shimadzu, UVmini-
1240 UV–VIS spectrophotometer) according to the method
described by Verpoorte [43]. The enzymatic reaction contained
1.4 mL of Tris–SO4 buffer (0.05 M; pH: 7.4), 1 mL of 4-nitro-
phenylacetate (3 mM), 0.5 mL H2O, and 0.1 mL of enzyme solution
(total volume, 3.0 mL). A reference measurement was obtained
by preparing the mixture without the enzyme solution. All
measurements were recorded in triplicate. The Ki values were
determined from a series of experiments using three different
brominated diphenylmethanone compounds 5–18 and 4-nitro-
phenylacetate as the substrate at five different concentrations to
construct Lineweaver–Burk curves [44].
General procedure for synthesis of benzyl alcohols 17, 18
(5-Bromo-2,3,4-trimethoxyphenyl)(3,4-dimethoxyphenyl)-
methanol (17)
Ketone 6 (100 mg, 0.24 mmol) dissolved in THF (25 mL) was added
to LiAlH4 (60 mg, 1.58 mmol) at 0°C. The reaction was stirred for
20 min and then the solvent was evaporated. The residues were
added to H2O (30 mL) and EtOAc (40 mL). The water layer was
extracted with EtOAc (2 ꢃ 40 mL). Combined organic layers were
dried over Na2SO4. Evaporation of the solvent and chromatogra-
phy of the residue on silica gel (SiO2, 70 g) with ethyl acetate/
hexane (1:9) afforded benzyl alcohol 17 (66 mg, 65%). 1H NMR
(400 MHz, CDCl3) d 7.27 (s, 1H), 6.96 (s, 1H), 6.83 (s, 2H), 5.90 (s, 1H),
3.88 (s, 6H, 2OCH3), 3.88 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.67 (s,
1H, –OH). 13C NMR (100 MHz, CDCl3) d 150.94 (C), 150.85 (C),
149.14 (C), 148.63 (C), 147.56 (C), 135.93 (C), 134.41 (C), 125.46
(CH), 119.00 (CH), 111.76 (C), 111.06 (CH), 109.94 (CH), 71.68 (CH),
61.23 (OCH3), 61.16 (OCH3), 61.09 (OCH3), 56.16 (OCH3), 56.11
(OCH3). IR (CH2Cl2, cmꢀ1): 3496, 2931, 2852, 1734, 1515, 1461,
1415, 1404, 1263, 1234, 1139, 1083, 1027, 1004.
Protein determination
The yield of protein during the purification steps was determined
spectrophotometrically at 595 nm according to the Bradford
method [45]. Bovine serum albumin was used as standard in this
study [46, 47].
SDS–polyacrylamide gel electrophoresis
The purity of the enzymes was confirmed using SDS–polyacryl-
amide gel electrophoresis. The running and stacking gels
contained 10 and 3% acrylamide, respectively, and 0.1% SDS,
according to the Laemmli procedure [48] and as described
previously [49, 50].
A 20 mg sample was applied to the
electrophoresis medium [50]. Gels were stained for 1.5 h in
0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10%
acetic acid, then destained with several changes of the same
solvent without the dye [51–53].
4-Bromo-6-((2-bromo-4,5-dimethoxyphenyl)(hydroxy)-
methyl)-2,3-dimethoxyphenol (18)
General procedure described for the synthesis of 17 was applied
to synthesize compound 11 (180 mg, 0.38 mmol) and LiAlH4
(0.8 g, 2.11 mmol) was used to yield diphenylmethanol 18
(115 mg, 58%) as oily. 1H NMR (400 MHz, CDCl3) d 7.08 (s, 1H),
7.02 (s, 1H), 6.76 (s, 1H), 6.59 (s, 1H), 6.28 (s, 1H), 3.96 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR
(100 MHz, CDCl3) d 149.60 (C), 149.32 (C), 148.87 (C), 147.54 (C),
141.12 (C), 132.46, 125.88 (CH), 125.39 (C), 115.47 (CH), 113.04 (C),
We thank to Ataturk University for the financial supports of this work
(BAP, Project Numbers: 2009/84 and 2009/251).
The authors have declared no conflict of interest.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 354–359
Biological Activity of Diphenylmethanone Derivatives
359
References
[27] C. T. Supuran, Curr. Top. Med. Chem. 2007, 7, 825–833.
[28] C. T. Supuran, Nat. Rev. Drug Disc. 2008, 7, 168–181.
[1] G. W. Gribble, Chem. Soc. Rev. 1999, 28, 335–346.
[29] G. De Simone, A. Di Fiore, C. T. Supuran, Curr. Pharm. Des.
2008, 14, 655–660.
[2] H. T. Balaydın, Y. Akbaba, A. Menzek, E. S¸ahin, S. Göksu,
Arkivoc 2009, XIV, 75–87.
[30] C. T. Supuran, Bioorg. Med. Chem. 2013, 21, 1377–1378.
_
[3] H. T. Balaydın, I. Gülçin, A. Menzek, S. Göksu, E. S¸ahin, J.
[31] D. Ekinci, M. Senturk, O. I. Kufrevioglu, Expert Opin. Ther. Pat.
2011, 21, 1831–1841.
Enzyme Inhib. Med. Chem. 2010, 25, 685–695.
_
[4] Y. Çetinkaya, H. Göçer, A. Menzek, I. Gülçin, Arch. Pharm.
[32] H. T. Balaydın, M. Senturk, S. Goksu, A. Menzek, Eur. J. Med.
Chem. 2012, 54, 423–428.
2012, 345, 323–334.
[5] N. Xu, X. Fan, X. Yan, X. Li, R. Niu, C. K. Tseng, Phytochemistry
2003, 62, 1221–1224.
[33] D. Ekinci, H. Cavdar, S. Durdagi, O. Talaz, M. Senturk,
C. T. Supuran, Eur. J. Med. Chem. 2012, 49, 68–73.
[6] Y. Akbaba, H. T. Balaydın, A. Menzek, S. Göksu, E. S¸ahin,
D. Ekinci, Arch. Pharm. 2013, 346, 447–454.
_
[34] A. Innocenti, S. B. Öztürk Sarıkaya, I. Gülçin, C. T. Supuran,
Bioorg. Med. Chem. 2010, 18, 2159–2164.
[7] H. T. Balaydın, S. Durdagi, D. Ekinci, M. Senturk, S. Göksu,
A. Menzek, J. Enzyme Inhib. Med. Chem. 2012, 27, 467–475.
[35] S. Pastorekova, D. Vullo, I. Nishimori, A. Scozzafava,
J. Pastoreka, C. T. Supuran, Bioorg. Med. Chem. 2008, 16,
3530–3536.
_
[8] M. Nar, Y. Çetinkaya, I. Gülçin, A. Menzek, J. Enzyme Inhib.
Med. Chem. 2013, 28, 402–406.
[36] C. T. Supuran, A. Scozzafava, Expert Opin. Ther. Pat. 2002, 12,
217–242.
[9] H. T. Balaydın, M. S¸entürk, A. Menzek, Bioorg. Med. Chem. Lett.
2012, 22, 1352–1357.
[37] C. T. Supuran, A. Scozzafava, Bioorg. Med. Chem. 2007, 15,
4336–4350.
_
ꢀ
[10] A. Akıncıoglu, M. Topal, I. Gülçin, S. Göksu, Arch. Pharm. 2014,
347, 68–76.
[38] M. Senturk, I. Gulcin, A. Dastan, O. I. Kufrevioglu, C. T.
[11] D. Shahwar, M. A. Raza, T. Khan, Turk. J. Chem. 2012, 36, 257–
Supuran, Bioorg. Med. Chem. 2009, 17, 3207–3211.
266.
_
[39] S. ArasHisar, O. Hisar, S¸. Beydemir, I. Gülçin, T. Yanik, Acta
_
ꢀ
[12] A. Akıncıoglu, Y. Akbaba, H. Göçer, S. Göksu, I. Gülçin, C. T.
Supuran, Bioorg. Med. Chem. 2013, 21, 1379–1385.
Vet. Hung. 2004, 52, 413–422.
_
[40] S. B. Öztürk Sarikaya, I. Gülçin, C. T. Supuran, Chem. Biol. Drug
[13] C. T. Supuran, A. Scozzafava, Exp. Opin. Ther. Pat. 2002, 12,
217–242.
Des. 2010, 75, 515–520.
_
_
ꢀ
[41] M. S¸entürk, I. Gülçin, S¸. Beydemir, Ö. I. Küfrevioglu,
C. T. Supuran, Chem. Biol. Drug Des. 2011, 77, 494–499.
[14] C. T. Supuran, A. Scozzafava, Exp. Opin. Ther. Pat. 2000, 10,
575–600.
_
[42] S¸. Beydemir, I. Gülçin, J. Enzyme Inhib. Med. Chem. 2004, 19,
_
[15] I. Gülçin, S. Beydemir, Mini Rev. Med. Chem. 2013, 13, 408–430.
193–197.
[16] T. H. Maren, Physiol. Rev. 1967, 47, 595–781.
[43] J. A. Verpoorte, S. Metha, J. T. Edsall, Biol. Chem. 1967, 242,
4221–4229.
[17] A. Fersht, Enzyme Structure and Mechanism, 2nd edn., W.H.
Freeman and Company, 1985, pp. 436–439.
[44] H. Lineweaver, D. Burk, Am. Chem. Soc. 1934, 57, 685–693.
[45] M. Bradford, Anal. Biochem. 1976, 72, 248–254.
_
ꢀ
[18] I. Gülçin, S¸. Beydemir, M. E. Büyükokuroglu, Biol. Pharm. Bull.
2004, 27, 613–616.
_
_
ꢀ
[46] I. Gülçin, Ö. I. Küfrevioglu, M. Oktay, J. Enzyme Inhib. Med.
Chem. 2005, 20, 297–302.
[19] N. Hen, M. Bialer, B. Yagen, A. Maresca, M. Aggarwal, A. H.
Robbins, R. McKenna, A. Scozzafava, C. T. Supuran, J. Med.
Chem. 2011, 54, 3977–3981.
[47] D. P. Iga, R. Schmidt, S. Iga, C. L. Hotoleanu, F. Duica,
A. Nicolescu, S. S. Gitman, Turk. J. Chem. 2013, 37, 299–307.
[20] F. Mincione, A. Scozzafava, C. T. Supuran, Curr. Pharm. Des.
2008, 14, 649–654.
[48] D. K. Laemmli, Nature 1970, 227, 680–685.
_
[49] T. A. Çoban, S¸. Beydemir, I. Gülçin, D. Ekinci, Biol. Pharm. Bull.
[21] E. R. Swenson, L. J. Teppema, J. Appl. Physiol. 2007, 102, 1305–
2007, 30, 2257–2261.
1307.
[50] T. A. Çoban, S. Beydemir, I. Gulcin, D. Ekinci, J. Enzyme Inhib.
Med. Chem. 2008, 23, 266–270.
[22] A. Scozzafava, C. T. Supuran, Med. Res. Rev. 2003, 23, 146–189.
[23] Y. Çetinkaya, A. Menzek, E. Sahin, H. T. Balaydın, Tetrahedron
2011, 67, 3483–3489.
_
_
ꢀ
[51] A. Atasaver, H. Özdemir, I. Gülçin, Ö. I. Küfrevioglu, Food
Chem. 2013, 136, 864–870.
[24] J. M. Armstrong, D. V. Myers, J. A. Verpoorte, J. T. Edsall, J. Biol.
Chem. 1966, 214, 5137–5149.
_
ꢀ
[52] M. S¸isSecioglu, I. Gülçin, M. Çankaya, H. Özdemir, Int. J. Food
Propert. 2012, 15, 1182–1189.
_
[25] H. Gocer, I. Gülçin, Int. J. Acad. Res. 2013, 5, 150–155.
_
[53] K. Aksu, M. Nar, M. Tanç, D. Vullo, I. Gülçin, S. Göksu,
[26] J. Y. Winum, A. Scozzafava, J. L. Montero, C. T. Supuran, Mini
Rev. Med. Chem. 2006, 6, 921–936.
F. Tümer, Bioorg. Med. Chem. 2013, 21, 2925–2931.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com