Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols including natural products

Article abstract of DOI:10.3109/14756366.2011.574131

(2-Bromo-3,4-dimethoxyphenyl) (3,4-dimethoxyphenyl)methanone (10) and its derivatives with Br, one dibromide and isomeric three tribromides, were synthesized. Demethylation of these compounds afforded a series of new bromophenols. Inhibition of human cyto

Full text of DOI:10.3109/14756366.2011.574131

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 43–50
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.574131
RESEARCH ARTICLE
Synthesis and carbonic anhydrase inhibitory properties of
novel bromophenols including natural products
Halis Türker Balaydın1,2, Hakan Soyut1,3, Deniz Ekinci4, Süleyman Göksu1, Şükrü Beydemir1,5,
Abdullah Menzek1, and Ertan Şahin1
1Atatürk University, Faculty of Science, Department of Chemistry, Erzurum, Turkey, 2Artvin Çoruh University, Education
Faculty, Department of Elementary Science Education, Artvin, Turkey, 3Bayburt University, Education Faculty,
Department of Elementary Science Education, Bayburt, Turkey, 4Ondokuz Mayıs University, Faculty of Agriculture,
Department of Agricultural Biotechnology, Samsun, Turkey, and 5Atatürk University, Biotechnology Application and
Research Center, Erzurum, Turkey
Abstract
(2-Bromo-3,4-dimethoxyphenyl) (3,4-dimethoxyphenyl)methanone (10) and its derivatives with Br, one dibromide
and isomeric three tribromides, were synthesized. Demethylation of these compounds afforded a series of new
bromophenols. Inhibition of human cytosolic carbonic anhydrase II (hCA II) isozyme by these new bromophenols
and naturally occurring 3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)benzene-1,2-diol (3), and 5,5′-
methylenebis(3,4,6-tribromo-benzene-1,2-diol) (4) was investigated. The synthesized compounds showed carbonic
anhydrase inhibitory capacities with IC values in the range of 0.7–372 μM against hCA II. Some bromophenols
investigated here showed effective hC5A0 II inhibitory activity and might be used as leads for generating novel
carbonic anhydrase inhibitors which are valuable drug candidates for the treatment of glaucoma, epilepsy, gastric
and duodenal ulcers, neurological disorders, or osteoporosis.
Keywords: Bromophenols, diphenylmethane, carbonic anhydrase, glaucoma, enzyme inhibition
Introduction
been reported10,11. Recently, we have achieved an alter-
native synthesis of 110, first total synthesis of 2, 3, 412,13
Naturally occurring bromophenols, frequently iso-
lated from red algae of the family Rhodomelaceae,
have prominent biological activities1,2. Of these natural
compounds, 5,5′-methylenebis(3,4-dibromobenzene-
1,2-diol) (1) and 3,4-dibromo-5-(2-bromo-3,4-dihy-
droxy-6-(methoxymethyl)benzyl)benzene-1,2-diol (2)
exhibit enzyme inhibition, e.g. isocitrate lyase3 cytotoxic-
ity4, feeding deterrent5, and microbial6,7 activities, while
3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)
benzene-1,2-diol (3) and 5,5′-methylenebis(3,4,6-
tribromo-benzene-1,2-diol) (4) exhibit significant aldose
reductaseinhibitoryactivity8.Additionally,itwasreported
that bromophenol 1 is an inhibitor of protein tyrosine
phosphatase9. Antioxidant activities of 1 and 4 have also
,
and a series of diphenylmethanone like bromophenols
510. We have reported that compound 1 and a series of 5
show high antioxidant and radical scavenging activities10.
Compound 6 and its derivatives with different number of
bromines are also diphenylmethanone like compounds
which are similar to 5 (Figure 1).
e carbonic anhydrases (CA; Carbonate hydrolyase,
EC 4.2.1.1) are a ubiquitous family of zinc-containing
enzymes that classically participate in the maintenance
of pH homeostasis in human body, catalyzing the revers-
ible hydration of carbon dioxide in a two-step reaction to
yield bicarbonate and protons14. Sixteen isozymes have
been described so far, that differ in their subcellular
Address for Correspondence: Dr. Sukru Beydemir, Ataturk University, Chemistry, Ataturk University, Faculty of Sciences, Department
of Chemistry, Erzurum, 25240 Turkey. Tel.: +90 442 2314388. Fax: +90 442 2360948. E-mail: beydemir@atauni.edu.tr; Abdullah Menzek,
Ataturk University, Chemistry, Ataturk University, Faculty of Sciences, Department of Chemistry, Erzurum, 25240 Turkey.
Tel.: +90 442 2314423. Fax: +90 442 2360948. E-mail: amenzek@atauni.edu.tr
(Received 05 November 2010; revised 18 March 2011; accepted 18 March 2011)
43

44 H.T. Balaydın et al.
mesh, Merck, Darmstadt, Germany). Preparative thick
layer chromatography: 1 mm of SiO2 60 PF (Merck) on
glass plates. Mp: cap. melting-point apparatus (BUCHI
530: Flawil, Switzerland); uncorrected. IR Spectra: solns.
in 0.1 mm cells with a Mattson 1000 FT-IR spectropho-
1
tometer (Cambridge, England). H- and 13C- NMR spec-
tra: 200 (50) and 400 (100)-MHz Varian spectrometer
(Danbury, CT); δ in ppm; Me4Si as the internal standard.
Elemental analyses: Leco CHNS-932 apparatus (MI,
USA). Antioxidant activities of samples were determined
in a spectrophotometer (UV-1208, Shimadzu, Japan).
Figure 1. Some naturally occurring bromophenols.
localization, catalytic activity and susceptibility to dif-
ferent classes of inhibitors. Some of these isozymes are
cytosolic (CA I, CA II, CA III, CA VII and CA XIII), oth-
ers are membrane-bound (CA IV, CA IX, CA XII and CA
XIV), two are mitochondrial (CA VA and CA VB), and
one is secreted in saliva (CA VI). It has been reported
that CA XV isoform is not expressed in humans or in
other primates, but it is abundant in rodents and other
higher vertebrates15–17. CAs are produced in a variety of
tissues where they participate in several important bio-
logical processes such as acid-base balance, respiration,
carbon dioxide and ion transport, bone resorption, ure-
agenesis, gluconeogenesis, lipogenesis and body fluid
generation15,18,19. e two major CA isozymes (CA I and
CA II) are present at high concentrations in the cytosol
in erythrocytes, and CA II has the highest turnover rate
among all CAs. Many of the CA isozymes involved in
these processes are important therapeutic targets with
the potential to be inhibited to treat a range of disorders
including oedema, glaucoma, obesity, cancer, epilepsy
Synthesis of (2-bromo-3,4-dimethoxyphenyl)(3,4-
dimethoxyphenyl)methanone (10)
Polyphosphoric acid (PPA), prepared from conc. H3PO4
(85%, 2.63 g) and P2O5 (4.72 g, 33.2 mmol), was heated to
80°C in a beaker (100 mL). To this mixture were added 8
(0.84 g, 4.6 mmol) and 936 (1.0 g, 4.6 mmol) quickly. e
mixture was stirred with a glass stick at 80°C for 45 min
and was then carefully poured onto 35 mL of ice/water.
e organic phase was extracted with EtOAc (2 × 125 mL).
e combined organic layers were dried over Na2SO4 and
the solvent was evaporated. Monobromide 10 (85%) was
the sole product and was crystallized from ethyl acetate
as white crystals. Mp 166–167°C; 1H-NMR (400 MHz,
CDCl3): δ 7.54 (d, J = 2.2 Hz, 1 H), 7.23 (dd, A part of
AB-system, J = 8.3 Hz, 2.2 Hz, 1 H), 7.07 (d, A part of
AB-system, J = 8.4 Hz, 1 H), 6.93 (d, B part of AB-system,
J = 8.4 Hz, 1 H), 6.82 (d, B part of AB-system, J = 8.4 Hz,
1 H), 3.93 (s, methoixde, 6 H), 3.92 (s, methoxide, 3 H),
3.88 (s, methoixde, 3 H); 13C-NMR (100 MHz, CDCl3): δ
194.24 (CO), 154.89 (C), 154.05 (C), 149.45 (C), 146.95
(C), 134.35 (C), 129.95 (C), 126.51 (CH), 124.78 (CH),
116.18 (C), 111.46 (CH), 111.18 (CH), 110.14 (CH), 60.84
(OCH3), 56.41 (OCH3), 56.31 (OCH3), 56.25 (OCH3); IR
(CH2Cl2, cm−1): 3003, 2938, 2839, 1657, 1586, 1512, 1487,
1463, 1417, 1394, 1341, 1294, 1274, 1240, 1217, 1171,
1135, 1032, 991, 904, 879, 813, 796, 759, 729, 636, 569;
Anal. Calcd for C17H17BrO5: C, 53.56; H 4.49. found: C,
53.52; H 4.40.
and osteoporosis18–20
.
Interaction of most CA isozymes with several types
of phenols, such as simple phenol and its substituted
derivatives, clioquinol, salicyclates and some of their
derivatives, has been recently investigated20–23. Here, we
extend these earlier investigations to a novel series of
bromophenols.
Chemicals are generally known to activate or inhibit
several enzymes in vivo and affect metabolic pathways.
Inhibitory effects of different anions, metal ions, drugs,
phenols and sulfonamides, which are specific inhibitors,
have been so far investigated against many CAs21,24–28. CA
II inhibitors are used for several purposes, in particular
for the treatment of glaucoma, epilepsy, and as diuretics
Bromination of compound 10
To a stirring solution of monobromide 10 (2.0 g, 5.2 mmol)
in CHCl3 (50 mL) was added a solution of bromine (5.0 g,
31.2 mmol, 6 eq.) in CHCl3 (30 mL) drop wise at room
temperature (RT) over 10 min. After the reaction mixture
was stirred at RT for 3 days, the solvent was evaporated.
Chromatography of the residue (2.71 g) on silica gel (SiO2,
100 g) with ethyl acetate/hexane (5:95) gave dibromide
11 (0.68 g, 28%), 14 (0.32 g, 12%), 13 (0.71 g, 26%) and 12
(0.76 g, 27%), respectively.
or antitumor agents/diagnostic tools18,19,29
.
Many chemical substances and synthesized drugs
affect metabolisms by changing enzyme activities30–33. As
CA II inhibitors are valuable molecules for therapeuti-
cal and pharmacological applications, we have synthe-
sized novel bormophenols in the current research and
evaluated their potency to be novel carbonic anhydrase
inhibitors.
(2-Bromo-3,4-dimethoxyphenyl)(2-bromo-4,5-
dimethoxyphenyl)methanone (11)
Materials and methods
Mp 122–123°C as white crystals; 1H-NMR (400 MHz,
CDCl3) δ 7.21 (d, A part of AB-system, J = 8.4 Hz, 1 H),
7.06 (s, 1 H), 7.03 (s, 1 H) 6.88 (d, B part of AB-system,
J = 8.4 Hz, 1 H) 3.93 (s, methoxide, 3 H), 3.92 (s,
All chemicals and solvents are commercially available
and were used after distillation or treatment with drying
agents. Column chromatography (CC): silica gel (SiO2; 60
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and CAI’s inhibitory properties 45
methoxide, 3 H), 3.86 (s, methoxide, 3 H), 3.85 (s, meth-
oxide, 3 H); 13C-NMR (100 MHz, CDCl3) δ 194.19 (CO),
156.31 (C), 152.18 (C), 148.49 (C), 147.26 (C), 133.38
(C), 131.96 (C), 127.67 (CH), 117.97 (C), 116.56 (CH),
114.19 (CH), 113.61 (C), 110.83 (CH), 60.80 (OCH3),
56.56 (OCH3), 56.45 (OCH3), 56.37 (OCH3); IR (CH2Cl2,
cm−1): 3005, 2964, 2842, 2591, 1668, 1584, 1505, 1486,
1463, 1445, 1399, 1375, 1336, 1271, 1211, 1171, 1159,
1059, 1030, 994, 919, 867, 820, 785, 735, 702, 647, 584;
Anal. Calcd for C17H16Br2O5: C, 44.38, H 3.51 found: C,
44.38; H 3.52.
992, 931, 844, 815, 785, 756, 733, 701, 588; Anal. Calcd
for C17H16Br3O5: C, 37.88, H 2.80 found: C, 37.66; H 2.82.
Standard procedure for demethylation of compounds
with OMe by ether cleavage (2-bromo-3,4-
dihydroxyphenyl)(3,4-dihydroxyphenyl)methanone
(6)
A solution of monobromide 10 (0.43 g, 1.32 mmol)
in CH2Cl2 (15 mL) was cooled to 0°C and then a solu-
tion of BBr3 (0.9 mL) in CH2Cl2 (10.0 mL) was added
drop wise under N2(g) over 5 min. After the cold bath
was removed, the mixture was stirred at RT and under
N2 for 1 day. Methanol (35 mL) was slowly added over
15 min and then the solvent was evaporated. After water
(45 mL) and EtOAc (2 × 40 mL) were added, the mixture
was shaken. e organic phase was separated and the
water phase was extracted with EtOAc (2 × 30 mL). e
combined organic phases were dried over Na2SO4 and
the solvent was evaporated. Bromophenol 6 (0.40 g,
93%) was obtained as pale yellow amorphous. Mp
77–78°C; 1H-NMR (400 MHz, CD3COCD3) δ 9.06 (m,
1 OH), 8.77 (m, 1 OH), 8.43 (m, 1 OH) 8.28 (m, 1 OH),
7.32 (d, J = 2.2 Hz, 1 H), 7.17 (dd, A part of AB-system,
J = 8.1, 2.2 Hz, 1 H), 6.94 (d, A part of AB-system, J = 8.1
Hz, 1 H), 6.90 (d, B part of AB-system, J = 8.1 Hz, 1 H),
6.74 (d, B part of AB-system, J = 8.1 Hz, 1 H); 13C-NMR
(100 MHz, CD3COCD3) δ 193.59 (CO), 150.79 (C), 146.86
(C), 145.12 (C), 143.33 (C), 133.88 (C), 129.86 (C), 124.26
(CH), 120.21 (CH), 116.69 (CH), 115.08 (CH), 113.89
(CH), 107.53 (C); IR (CH2Cl2, cm−1): 3434, 2967, 2075,
1638, 1595, 1524, 1442, 1388, 1300, 1201, 1120, 1032,
1015, 943, 816, 782, 763; Anal. Calcd for C13H9BrO5: C, C,
48.03; H 2.79 found: C, 48.01; H 2.80.
(2-Bromo-3,4-dimethoxyphenyl)(2,3-dibromo-4,5-
dimethoxyphenyl)methanone (12)
Mp100–101°Caspaleyellowcrystals;1H-NMR(400 MHz,
CDCl3) δ 7.24 (d, A part of AB-system, J = 8.8 Hz, 1 H),
6.98 (s, 1 H), 6.86 (d, part of AB-system, J = 8.8 Hz, 1 H),
3.93 (s, methoxide, 3 H), 3.91 (s, methoxide, 3 H), 3.87
(s, methoxide, 3 H), 3.86 (s, methoxide, 3 H); 13C-NMR
(100 MHz, CDCl3) δ 193.60 (CO), 157.03 (C), 152.76 (C),
147.67 (C), 138.11 (C), 131.33 (C), 129.07 (CH), 126.18
(C), 123.27 (C), 118.75 (C), 114.70 (C), 113.20 (CH),
110.54, (CH), 60.93 (OCH3), 60.81 (OCH3), 56.62 (OCH3),
56.41 (OCH3); IR (CH2Cl2, cm−1): 3003, 2938, 1673, 1588,
1564, 1507, 1464, 1403, 1337, 280, 1262, 1217, 1166, 1141,
1070, 1032, 996, 924, 865, 837, 790, 733, 681, 609. Anal.
Calcd for C17H16Br3O5: C, 37.88, H 2.80 found: C, 37.93;
H 2.85.
(2-Bromo-4,5-dimethoxyphenyl)(2,6-dibromo-3,4-
dimethoxyphenyl)methanone (13)
Mp 115–117°C as pale yellow crystals; 1H-NMR
(400 MHz, CDCl3) δ 7.35 (s, 1 H), 7.11 (s, 1 H), 7.02
(s, 1 H) 3.94 (s, methoxide, 3 H), 3.91 (s, methoxide, 3
H), 3.87 (s, methoxide, 3 H), 3.84 (s, methoxide, 3 H);
13C-NMR (100 MHz, CDCl3) δ 192.88 (CO), 153.66 (C),
152.80 (C), 152.14 (C), 148.59 (C), 137.82 (C), 130.58
(C), 129.52 (CH), 117.00 (C), 116.81 (C), 116.74 (CH),
114.36 (CH), 114.23 (C), 61.39 (OCH3), 61.25 (OCH3),
56.62 (OCH3), 56.50 (OCH3); IR (CH2Cl2, cm−1): 2938,
2841, 1671, 1579, 1541, 1512, 1464, 1419, 1399, 1366,
1300, 1270, 1212, 1185, 1142, 1080, 1032, 1004, 918, 804,
775, 734, 665; Anal. Calcd for C17H16Br3O5: C, 37.88, H
2.80 found: C, 37.86; H 2.84.
Synthesis of bromophenols 15–18 from the corre-
sponding compounds 11–14, respectively.
e standard procedure10,12,13,35 described above for
the synthesis of 6 with BBr3 was applied. From these reac-
tions, bromophenols 15–18 were obtained.
(2-Bromo-3,4-dihydroxyphenyl)(2-bromo-4,5-
dihydroxyphenyl)methanone (15)
It was crystallized from ethyl acetate/hexane as pale
1
yellow crystals (0.382 g, 85%); mp 186–187°C; H-NMR
(400 MHz, CD3COCD3) δ 9.29 (s, 1 OH), 8.96 (s, 1 OH),
8.53 (s, 1 OH) 8.26 (s, 1 OH), 7.11 (s, 1H), 6.96 (s, 1H),
6.91 (d, A part of AB-system, J= 8.2 Hz, 1 H), 6.86 (d, B
part of AB-system, J= 8,2 Hz, 1 H); 13C-NMR (100 MHz,
CD3COCD3) δ 193.29 (CO), 149.15 (C), 148.58 (C), 144.52
(C), 143.70 (C), 132.57 (C), 131.49 (C), 123.31 (CH),
120.58 (CH), 118.66 (CH), 113.64 (CH), 111.01 (C), 108.98
(C); IR (CH2Cl2, cm−1): 3368, 2947, 2834, 2526, 2041, 1655,
1594, 1452, 1419, 1295, 1115, 1032, 668; Anal. Calcd for
C13H8Br2O5: C, 38.65; H 2.00. found: C, 38.64; H 2.01.
(2-Bromo-4,5-dimethoxyphenyl)(2,5-dibromo-3,4-
dimethoxyphenyl)methanone (14)
Mp 138–139°C as colourless crystals; 1H-NMR (400 MHz,
CDCl3) δ 7.42 (s, 1 H), 7.10 (s, 1 H), 7.09 (s, 1 H) 3.86 (s,
methoxide, 3 H), 3.85 (s, methoxide, 3 H), 3.85 (s, meth-
oxide, 3 H), 3.84 (s, methoxide, 3 H); 13C-NMR (100 MHz,
CDCl3) δ 191.38 (CO), 154.50 (C), 153.46 (C), 148.58 (C),
146.65 (C), 134.96 (C), 128.23 (C), 117.55 (CH), 116.61
(C), 116.29 (CH), 115.80 (C), 115.02 (CH), 114.60 (C),
60.97 (OCH3), 56.63 (2 OCH3), 56.43 (OCH3); IR (CH2Cl2,
cm−1): 3003, 2936, 2841, 1679, 1655, 1586, 1508, 1476,
1442, 1380, 1338, 1298, 1262, 1212, 1158, 1065, 1027,
(2-Bromo-3,4-dihydroxyphenyl)(2,3-dibromo-4,5-
dihydroxyphenyl)methanone (16)
Yellow amorphous (0.34g, 95%); mp 121–123°C; H-NMR
1
(400MHz, CD3COCD3) δ 6.96 (s, 1 H), 6.91 (s, 1 H), 6.90 (s, 1
© 2012 Informa UK, Ltd.

46 H.T. Balaydın et al.
H); 13C-NMR (100MHz, CD3COCD3), δ 193.05 (CO), 149.24
(C), 146.94 (C), 144.55 (C), 143.96 (C), 133.79 (C), 131.19
(C), 124.47 (CH), 116.19 (CH), 114.38 (C), 113.54 (CH),
113.37 (C), 109.43 (C); IR (CH2Cl2, cm−1): 3400, 2950, 2839,
2076, 1648, 1452, 1396, 1295, 1114, 1019, 667; Anal. Calcd
for C13H7Br3O5: C, 32.33; H 1.46. found: C, 32.11; H 1.45.
eluted with 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6). All
procedures were performed at 4°C.
Hydratase activity assay
Carbonic anhydrase activity was assayed by following the
hydration of CO2 according to our previous studies37,38
.
CO2-hydratase activity as an enzyme unit was calculated
by using the equation (t0-tc/tc) where t0 and tc are the
times for pH change of the non-enzymatic and the enzy-
matic reactions, respectively.
(2-Bromo-4,5-dihydroxyphenyl)(2,6-dibromo-3,4-
dimethoxyphenyl)methanone (17)
Red amorphous; (0.47 g, 87%); mp 240–242°C′(its color
was changed at ≥ 180°C); 1H-NMR (400 MHz, CD3COCD3)
δ 7.20 (s, 1 H), 7.15 (s, 2 H); 13C-NMR (100 MHz,
CD3COCD3) δ 190.55 (CO), 150.60 (C), 147.04 (C), 144.49
(C), 143.42 (C), 133.90 (C), 122.01 (CH), 120.16 (CH),
118.75 (C), 118.29 (CH), 113.62 (C), 112.90 (C), 108.33
(C); IR (CH2Cl2, cm−1): 3681, 2973, 2863, 2071, 1654, 1587,
1495, 1476, 1454, 1286, 1213, 1144, 1054, 1033; Anal. Calcd
for C13H7Br3O5: C, 32.33; H 1.46. found: C, 32.33; H 1.45.
Protein determination
Protein quantity was determined spectrophotometrically
at 595 nm according to the Bradford method during the
purification steps, using bovine serum albumin as the
standard39.
SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed
after purification of the enzymes. It was carried out in 10
and 3% acrylamide for the running and the stacking gel,
respectively, containing 0.1% SDS according to Laemmli
procedure. A 20-μg sample was applied to the electro-
phoresis medium. Gels were stained for 1.5h 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 dye40.
(2-Bromo-4,5-dihydroxyphenyl)(2,5-dibromo-3,4-
dihydroxyphenyl)methanone (18)
It was crystallized from ethyl acetate/hexane as pale
yellow crystals (0.382 g, 85%); mp 186–188°C (its color
1
changed at ≥ 160°C); H-NMR (400 MHz, CD3COCD3),
9.09 (bs, 1 OH), 9.05 (bs, 1 OH), 8.80 (bs, 1 OH) 8.58 (bs,
1 OH), δ 7.16 (s, 1 H), 7.13 (s, 1 H), 7.02 (s, 1 H); 13C-NMR
(100 MHz, CD3COCD3), δ 192.02 (CO), 149.66 (C), 146.28
(C), 144.65 (C), 144.30 (C), 133.35 (C), 130.49 (C), 125.70
(CH), 120.78 (CH), 118.94 (CH), 111.33 (C), 108.45 (C),
108.15 (C); IR (CH2Cl2, cm−1): 3436, 3225, 2076, 1638,
1285, 1033, 720; Anal. Calcd for C13H7Br3O5: C, 32.33; H
1.46. found: C, 32.33; H 1.49.
Crystal structure determination
For the crystal structure determination, the single-crys-
tal of 13 and 14 were used for data collection on a four-
circle Rigaku R-AXIS RAPID-S diffractometer (equipped
with a two-dimensional area IP detector). e graphite-
monochromatized Mo Kα radiation (λ= 0.71073 Å) and
oscillation scans technique with Δω= 5° for one image
wereusedfordatacollection.elatticeparameterswere
determined by the least-squares method on the basis of
all reflections with F2 > 2σ (F2). Integration of the inten-
sities, correction for Lorentz and polarization effects
and cell refinement were performed using CrystalClear
(Rigaku/MSC Inc., 2005) software41. e structures
were solved by direct methods using SHELXS-9742 and
refined by a full-matrix least-squares procedure using
the program SHELXL-97. H atoms were positioned geo-
metrically and refined using a riding model. e final
difference Fourier maps showed no peaks of chemical
significance. Crystal data for 13: C17H15O5Br3, crystal
system, space group: triclinic, P-1; (no:2); unit cell
dimensions: a = 8.2823(2), b = 10.2487(2), c = 12.7924(3)
Å, α= 72.529(5), β= 68.923(5), γ= 87.664(7)°; volume:
963.71(6) Å3; Z = 2; calculated density: 1.86 mg/m3;
absorption coefficient: 6.302 mm−1; F(000): 524; θ range
for data collection 2.6–30.5°; refinement method: full-
matrix least-square on F2; data/parameters: 3936/228;
goodness-of-fit on F2: 1.267; final R indices [I > 2σ(I)]:
R1 = 0.089, wR2 = 0.103; R indices (all data): R1 = 0.137,
wR2 = 0.115; largest diff. peak and hole: 0.391 and −0.556
e Å−3; CCDC: 774839. Crystal data for 14: C17H15O5Br3,
CA purification assay
e purification of the CA II isozyme was performed in
a simple single-step method by means of Sepharose-4B-
aniline-sulfanilamideaffinitycolumnchromatoghrapy36.
hCA II was purified 311-fold with a specific activity of
2500 EU mg−1 and an overall yield of 16%. Erythrocytes
were purified from fresh human blood obtained from
the Blood Centre of the Research Hospital at Atatürk
University. e blood samples were centrifuged at
1500 rpm for 15 min and the plasma and buffy coat were
removed. e red cells were isolated and washed twice
with 0.9% NaCl and hemolyzed with 1.5 volumes of ice-
cold water. e ghost and intact cells were removed by
centrifugation at 20,000 rpm for 30 min at 4°C. e pH of
the hemolysate was adjusted to 8.7 with solid Tris. Firstly,
Sepharose-4B was oxidized by KMnO4 and subsequently
activated by SOCl2. Subsequently, aniline was attached
to the activated gel as a spacer arm and finally diazotized
sulfanilamide was clamped to the para position of ani-
line molecule as ligand. e hemolysate was applied to
the prepared Sepharose 4B-aniline-sulfanylamide affin-
ity column which had been equilibrated with 25 mM
Tris-HCl/0.1 M Na2SO4 (pH 8.7). e affinity gel was
washed with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7).
e human carbonic anhydrase II (hCA II) isozyme was
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and CAI’s inhibitory properties 47
crystal system, space group: triclinic, P-1; (no:2); unit cell
dimensions: a = 8.3610(5), b = 8.3972(5), c = 14.7542(7)
Å, α= 98.097(2), β= 95.915(3), γ= 107.200(2)°; volume:
968.2(2) Å3; Z = 2; calculated density: 1.85 mg/m3;
absorption coefficient: 6.273 mm−1; F(000): 524; θ range
for data collection 2.6–30.5°; refinement method: full-
matrix least-square on F2; data/parameters: 4293/230;
goodness-of-fit on F2: 1.333; final R indices [I > 2σ(I)]:
R1 = 0.086, wR2 = 0.135; R indices (all data): R1 = 0.139,
wR2 = 0.146; largest diff. peak and hole: 0.367 and
−0.662 e Å−3; crystallographic data were deposited in
CSD under CCDC registration number 774807.
erefore, the exact structures of them were determined
by X-ray diffraction analysis (Figure 2).
Bromophenols derived from compounds 10–14 may
be potential biologically active compounds, because
they are similar to 5 with high antioxidant and radical
scavenging activities10. erefore, bromophenols 6 and
15–18 were synthesized from compounds 10–14 by ether
cleavage reaction with BBr3 in high yields (Figure 3).
Spectroscopic data of 6 and 15–18 are consistent with the
proposed structures.
Inhibitory effects of the compounds on CA II catalytic
activity were tested under in vitro conditions; IC50 values
were calculated and are given in Table 1.
We report here the first study on the inhibitory effects
of the bromophenols derivatives 3–6 and 10–18 on the
hydratase activity of hCA II. e data in Table 1 show
the following regarding the inhibition of hCA II by bro-
mophenol derivatives.
e strongest inhibitory activity has been observed
with compounds 11, 15–18, (Table 1). ree derivatives,
3, 10, 13, showed weak hCA II inhibitory activity with
IC50-s in the range of 86.4–372 μM, (Table 1), whereas
the remaining four derivatives were quite effective hCA
II inhibitors, with IC50-s in the range of 26.4–58 µM,
(Table 1). e best hCA II inhibitor in this series of deriv-
atives was the bulky, (2-Bromo-3,4-dihyroxyphenyl)(2,3-
dibromo-4,5-dihyroxyphenyl)methanone (16), with a
IC50 of 0.7 µM.
Results and discussion
We have synthesized natural product bromophenols
3 and 4 from corresponding materials by the known
method as shown in Scheme 113. Reactions of compound
7 with 1,4-dibromo-2,3-dimethoxybenzene and 1,2,5-
tribromo-3,4-dimethoxybenzene in the presence of PPA
at 80°C gave methylether substituted diarylmethanes in
high yields as sole product. e ether cleavage reaction
of these diarylmethanes with BBr3 under mild conditions
afforded naturally occurring bromophenols 3 and 413.
(2-Bromo-3,4-dimethoxyphenyl)(3,4-dimethoxyphe-
nyl)methanone (10) was synthesized from the reaction
of 3,4-dimethoxybenzoic acid (8) and 3-bromoveratrole
(9) with PPA in 85% yield as sole product (Scheme 1).
Bromination of monobromide 10 (in CHCl3) with Br2 (6
eq.) at RT for 3 days followed by CC allowed us to isolate
four products 11–14 (Scheme 1). e NMR analysis of 13
and 14 did not allow determination of their structures.
As revealed by a comparison of the inhibition ranges
of molecules, 13 has a higher IC50 value than those of its
isomers 12 and 14, and, likewise, 17 has a higher IC50
than those of 16 and 18.
Scheme 1. (A) 1,4-dibromo-2,3-dimethoxybenzene, PPA/80°C, (B) BBr3/CH2Cl2, 0–25°C, (C) 1,2,5-tribromo-3,4-dimethoxybenzene,
PPA/80°C.
© 2012 Informa UK, Ltd.

48 H.T. Balaydın et al.
Figure 2. (A) e molecular structure of tribromide 13 showing the atom numbering scheme. (B) Packing diagram for 13. (C) e molecular
structure of tribromide 14 showing the atom numbering scheme. (D) Packing diagram for 14.
Figure 3. e new synthesized bromophenols.
e phenolic compounds have been investigated
as CA inhibitors (CAIs) in this study. e rationale of
investigating these compounds as CAIs lies in the fact
that phenol has been shown to be the only competitive
inhibitor with CO2 as the substrate for the main isoform
of CA, i.e., human CA II (hCA II)20. In a very sound study,
Christianson and colleagues reported on the X-ray crystal
structure for the adduct of hCA II with phenol20, showing
this compound to bind to CA by anchoring its OH moiety
to the zinc-bound water/hydroxide ion of the enzyme
active site through a hydrogen bond as well as to the NH
amide of r199, an amino acid conserved in all α-CAs
and critically important for the catalytic cycle of these
enzymes18,19
.
CAIs are a class of pharmaceuticals used as antiglau-
coma agents, diuretics, antiepileptics, in the manage-
ment of mountain sickness, gastric and duodenal ulcers,
neurological disorders, or osteoporosis. us, discovery
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and CAI’s inhibitory properties 49
Table 1. IC50 values (concentration that causes 50% inhibition of
the enzyme activity) for the molecules.
References
1. Gribble GW. Naturally Occurring Organohalogen Compounds-A
Survey. J Nat Prod 1992;55:1353–1395.
2. Gribble GW. e diversity of naturally occurring organobromine
compounds. Chem Soc Rev 1999;28:335–346.
Inhibitor
IC50 (µM)
86.4
38.29
26.4
372
3
4
3. Lee HS, Lee TH, Lee JH, Chae CS, Chung SC, Shin DS, Shin J,
Oho KB. Inhibition of the pathogenicity of Magnaporthe grisea
by bromophenols, isocitrate lyase inhibitors, from the red alga
Odonthalia corymbifera. J Agric Food Chem 2007;55:6923–6928.
4. Xu X, Song F, Wang S, Li S, Xiao F, Zhao J , Yang Y, Shang S, Yang L, Shi
J. Dibenzyl bromophenols with diverse dimerization patterns from
the brown alga Leathesia nana. J Nat Prod 2004;67:1661–1666.
5. Kurata K, Taniguchii K, Takashima K, Hayashi I, Suzuki M.
Feeding-deterrent bromophenols from Odonthalia corymbifera.
Phytochemistry 1997;45:485–487.
6
10
11
12
13
14
15
16
17
18
11.7
49.5
126
58
1.65
0.7
9.23
1.36
6. Oh KB, Lee JH, Chung SC, Shin J, Shin HJ, Kim HK, Lee HS.
Antimicrobial activities of the bromophenols from the red alga
Odonthalia corymbifera and some synthetic derivatives. Bioorg
Med Chem Lett 2008;18:104–108.
of novel CAIs is of great importance for pharmacological
and medicinal approaches, and many inhibitors have
been designed and synthesized in the literature. However,
it is critically important to explore further classes of
potent CAIs in order to detect compounds with a differ-
ent inhibition profile when compared to sulfonamides
and their bioisosteres, and to find novel applications for
the inhibitors of these widespread enzymes.
7. Xu N, Fan X, Yan X, Li X, Niu R, Tseng CK. Antibacterial
bromophenols from the marine red alga Rhodomela confervoides.
Phytochemistry 2003;62:1221–1224.
8. Wang W, Okada Y, Shi H, Wang Y, Okuyama T. Structures and
aldose reductase inhibitory effects of bromophenols from the red
alga Symphyocladia latiuscula. J Nat Prod 2005;68:620–622.
9. Fan X, Ma C, Han L, Shi D, Liu Q. Extraction of bromophenol
compounds from red algae and their uses in the treatment of
diabetes and obesity. CN Patent 1,853,618 2006; Chem Abstr
2007;146:13018v.
10. Balaydin HT, Gülçin Í, Menzek A, Göksu S, Sahin E. Synthesis
and antioxidant properties of diphenylmethane derivative
bromophenols including a natural product. J Enzyme Inhib Med
Chem 2010;25:685–695.
11. Duan XJ, Li XM, Wang BG. Highly brominated mono- and bis-
phenols from the marine red alga Symphyocladia latiuscula with
radical-scavenging activity. J Nat Prod 2007;70:1210–1213.
12. Akbaba Y, Balaydın HT, Göksu S, Şahin E, Menzek A. Total Synthesis
of the biologically active, naturally occurring 3,4-dibromo-5-[2-
bromo-3,4-dihydroxy-6-(methoxymethyl)benzyl]benzene-1,2-
diol and regioselective O-demethylation of aryl methyl ethers.
Helv Chim Acta 2010;93:1127–1135.
13. Balaydin HT, Akbaba Y, Menzek A, Sahin E, Goksu S. First and short
syntheses of biologically active, naturally occurring brominated
mono- and dibenzyl phenols. Arkivoc 2009;XIV:75–87.
14. Ekinci D, Beydemir S, Alim Z. Some drugs inhibit in vitro hydratase
and esterase activities of human carbonic anhydrase-I and II.
Pharmacol Rep 2007;59:580–587.
15. Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic
anhydrases: Current state of the art, therapeutic applications and
future prospects. J Enzyme Inhib Med Chem 2004;19:199–229.
16. Cankaya M, Hernandez AM, Ciftci M, Beydemir S, Ozdemir H,
Budak H et al. An analysis of expression patterns of genes encoding
proteins with catalytic activities. BMC Genomics 2007;8:232.
17. Hilvo M, Tolvanen M, Clark A, Shen B, Shah GN, Waheed A et al.
Characterization of CA XV, a new GPI-anchored form of carbonic
anhydrase. Biochem J 2005;392:83–92.
Conclusions
Diphenylmethanone derivative 10 was obtained and its
bromination gave dibromide 11 and tribromides 12–14.
From these compounds, potential biological active bro-
mophenols 6 and 15–18 were synthesized in high yields.
e structures of the products were determined and
characterized by spectroscopic methods. Bromophenol
derivatives 1–13 used in this study affect the activity of
CA II isozyme due to the presence of the different func-
tional groups (OH, OCH3) in their aromatic scaffold. It
has been determined in our study that compounds 15,
16 and 18 are effective inhibitors for CA II when com-
pared to Acetazolamide, which is used as the reference
inhibitor for carbonic anhydrase. Our findings here indi-
cate thus another class of possible CAIs of interest, in
addition to the well-known sulfonamides/sulfamates/
sulfamides. ese findings point out that substituted
phenolic compounds may be used for generation of
potent CAIs.
18. Supuran CT. Carbonic anhydrases: Novel therapeutic applications
for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–181.
19. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for
medicinal chemistry. Bioorg Med Chem 2007;15:4336–4350.
20. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors: Interactions of phenols with the 12
catalytically active mammalian isoforms (CA I-XIV). Bioorg Med
Chem Lett 2008;18:1583–1587.
Acknowledgment
e authors are indebted to the Department of Chemistry
(Atatürk University) for research conditions.
Declaration of interest
21. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors: Inhibition of mammalian isoforms I-XIV
with a series of substituted phenols including paracetamol and
salicylic acid. Bioorg Med Chem 2008;16:7424–7428.
is research was financed by grants from of TÜBİTAK
(e Scientific and Technological Research Council of
Turkey) (Project no: TBAG-107T348).
© 2012 Informa UK, Ltd.

50 H.T. Balaydın et al.
22. BayramE,SenturkM,KufreviogluOI,SupuranCT.Invitroinhibition
of salicylic acid derivatives on human cytosolic carbonic anhydrase
isozymes I and II. Bioorg Med Chem 2008;16:9101–9105.
23. Sentürk M, Gülçin I, Dastan A, Küfrevioglu OI, Supuran CT.
Carbonic anhydrase inhibitors. Inhibition of human erythrocyte
isozymes I and II with a series of antioxidant phenols. Bioorg Med
Chem 2009;17:3207–3211.
24. AbdülkadirCobanT,BeydemirS,GülcinI,GücinI,EkinciD,Innocenti
A et al. Sildenafil is a strong activator of mammalian carbonic
anhydrase isoforms I-XIV. Bioorg Med Chem 2009;17:5791–5795.
25. Supuran CT. Diuretics: From classical carbonic anhydrase
inhibitors to novel applications of the sulfonamides. Curr Pharm
Des 2008;14:641–648.
32. Ekinci D, Beydemir S. Evaluation of the impacts of antibiotic drugs
on PON 1; a major bioscavenger against cardiovascular diseases.
Eur J Pharmacol 2009;617:84–89.
33. Ekinci D, Beydemir S. Effect of some analgesics on paraoxonase-1
purified from human serum.
2009;24:1034–1039.
J Enzyme Inhib Med Chem
34. Stevens RV, Bisacchi GS. An efficient and remarkably regioselective
synthesis of benzocyclobutenones from benzynes and 1,1-
dimethoxyethylene. J Org Chem 1982;47:2393–2396.
35. Vickery EH, Pahler LF, Eisenbraun EJ. Selective O-demethylation
of catechol ethers. Comparison of boron tribromide and
iodotrimethylsilane. J Org Chem 1979;44:4444–4446.
36. Ekinci D, Beydemir S. Risk assessment of pesticides and fungicides
for acid–base regulation and salt transport in rainbow trout tissues.
Pestic Biochem Phys 2010;97:66–70.
37. Soyut H, Beydemir S. Purification and some kinetic properties of
carbonic anhydrase from rainbow trout (Oncorhynchus mykiss)
liver and metal inhibition. Protein Pept Lett 2008;15:528–535.
38. Soyut H, Beydemir S, Hisar O. Effects of some metals on carbonic
anhydrase from brains of rainbow trout. Biol Trace Elem Res
2008;123:179–190.
39. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal Biochem 1976;72:248–254.
40. Laemmli DK. Cleavage of structural proteins during in assembly of
the head of Bacteriophage T4. Nature 1970;227:680–685.
41. Rigaku/MSC, Inc., 9009 new Trails Drive, e Woodlands, TX
77381, USA.
26. Ekinci D, Beydemir S, Küfrevioglu OI. In vitro inhibitory effects of
some heavy metals on human erythrocyte carbonic anhydrases.
J Enzyme Inhib Med Chem 2007;22:745–750.
27. Coban TA, Beydemir S, Gülçin I, Ekinci D. e effect of ethanol on
erythrocyte carbonic anhydrase isoenzymes activity: An in vitro
and in vivo study. J Enzyme Inhib Med Chem 2008;23:266–270.
28. Ekinci D, Cavdar H, Talaz O, Sentürk M, Supuran CT. NO-releasing
esters show carbonic anhydrase inhibitory action against human
isoforms I and II. Bioorg Med Chem 2010;18:3559–3563.
29. Sly WS, Hu PY. Human carbonic anhydrases and carbonic
anhydrase deficiencies. Annu Rev Biochem 1995;64:375–401.
30. Alici HA, Ekinci D, Beydemir S. Intravenous anesthetics inhibit
human paraoxonase-1 (PON1) activity in vitro and in vivo. Clin
Biochem 2008;41:1384–1390.
31. Ekinci D, Beydemir S. Purification of PON1 from human serum
and assessment of enzyme kinetics against metal toxicity. Biol
Trace Elem Res 2010;135:112–120.
42. Sheldrick GM. SHELXS97 and SHELXL97. Crystal structure
solution and refinement programs. 1997.
Journal of Enzyme Inhibition and Medicinal Chemistry