Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols and their derivatives including natural products: Vidalol B

Article abstract of DOI:10.1016/j.ejmech.2012.05.025

A series of bisphenol, bromophenol, and methoxyphenol derivatives (2-24) including the natural bromophenols vidalol B, 3,4,6-tribromo-5-(2,5-dibromo-3,4- dihydroxybenzyl)benzene-1,2-diol (2) and 5,5′-methylenebis(3,4,6-tribromo- benzene-1,2-diol) (3) were

Full text of DOI:10.1016/j.ejmech.2012.05.025

European Journal of Medicinal Chemistry 54 (2012) 423e428
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and carbonic anhydrase inhibitory properties of novel bromophenols
and their derivatives including natural products: Vidalol B
,
,
a,
Halis T. Balaydın a b, Murat S¸ entürk c, Süleyman Göksu a , Abdullah Menzek
*
**
a Atatürk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey
b Artvin Çoruh University, Faculty of Education, 08000 Artvin, Turkey
c Agri Ibrahim Cecen University, Department of Chemistry, 04100 Agri, Turkey
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< A series of bisphenol, bromophenol,
and methoxyphenol derivatives
were prepared.
< The first and convenient synthesis
of vidalol B was also achieved.
< Inhibition of four human CA
isozymes, with these compounds
were investigated.
< The compounds were found to be
promising
inhibitors.
carbonic
anhydrase
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bisphenol, bromophenol, and methoxyphenol derivatives (2e24) including the natural bro-
mophenols vidalol B, 3,4,6-tribromo-5-(2,5-dibromo-3,4-dihydroxybenzyl)benzene-1,2-diol (2) and 5,50-
methylenebis(3,4,6-tribromo-benzene-1,2-diol) (3) were prepared. In the current study, inhibition of
four human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, I, II, IV, and VI, with these compounds 2e24
was investigated. The compounds 2e24 were found to be promising carbonic anhydrase inhibitors, some
of which showed interesting inhibitory activities. Some of the compounds investigated here showed
effective hCA 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, and osteoporosis.
Received 7 January 2012
Received in revised form
14 April 2012
Accepted 18 May 2012
Available online 27 May 2012
Keywords:
Phenol
Bromination
Bromophenols
Diphenylmethane
Carbonic anhydrase
Glaucoma
Ó 2012 Elsevier Masson SAS. All rights reserved.
Enzyme inhibition
Vidalol A
Vidalol B
1. Introduction
Natural bromophenols, frequently isolated from red algae of the
family Rhodomelaceae, have prominent biological activities [1e9].
From these natural compounds, 1 exhibits isocitrate lyase [2] and
microbial [3e5] activities, while 2 and 3 show significant aldose
reductase inhibitory activities (Fig. 1) [5]. Additionally, it was
reported that phenolic natural [6,7,10] and synthetic dopaminergic
* Corresponding author. Tel.: þ90 442 2314389; fax: þ90 442 2360948.
** Corresponding author. Tel.: þ90 442 2314423; fax: þ90 442 2360948.
E-mail addresses: sgoksu@atauni.edu.tr (S. Göksu), amenzek@atauni.edu.tr
(A. Menzek).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.025

424
H.T. Balaydın et al. / European Journal of Medicinal Chemistry 54 (2012) 423e428
Fig. 1. Some naturally occurring bromophenols.
[11] compounds are inhibitors of human carbonic anhydrases.
Antioxidant activities of and have also been reported
Our group and Supuran’s group recently investigated the
interaction of CA I and II isozymes with several types of phenols,
such as hydroxy-/methoxysubstituted benzoic acids as well as di-/
tri-methoxy benzenes, natural and unnatural bromophenols, and
several of its substituted derivatives, e.g., salicylates and some of
their derivatives [20e22]. In the current research, the first synthesis
of natural bromophenol vidalols A (25) and B (22) was studied. As
CA inhibitors are valuable molecules for therapeutic and pharma-
cological applications, we have evaluated these bromophenol
derivatives as novel carbonic anhydrase inhibitors.
1
3
[8,9]. Recently, we have achieved an alternative synthesis of 1 [8],
the first total synthesis of 2 and its derivative (3,4-dibromo-5-[2-
bromo-3,4-dihydroxy-6-(methoxymethyl)benzyl]benzene-1,2-
diol) [12,13]. Isolation of vidalols A [25, 2-bromo-4-(2,3-dibromo-
4,5-dihydroxybenzyl)benzene-1,3,5-triol] and B [22, 2-bromo-4,6-
bis(2,3-dibromo-4,5-dihydroxybenzyl)benzene-1,3,5-triol], anti-
inflammatory bromophenols, from the Caribbean marine red alga
Vidalia, was reported previously [14].
The carbonic anhydrases (CA; carbonate hydrolase, EC 4.2.1.1)
are a ubiquitous family of zinc-containing enzymes that classically
participate in the maintenance of pH homeostasis in the human
body, catalyzing the reversible hydration of carbon dioxide in
a two-step reaction to yield bicarbonate and protons [15e17]. The
enzyme plays an important role in physiological anion exchange
processes [15]. At least 16 CA isozymes have been described to date
in mammals, the most active ones as catalysts for carbon dioxide
hydration being CA II [17e19]. CA II is found primarily in red blood
cells but also in many other secretory tissues of the kidney, lung,
eye, etc [15e21]. Carbonic anhydrase VI (CA VI) is a secretory
enzyme that was initially described in the ovine parotid gland and,
saliva and normal human serum [18]. Other CA isoforms are found
in a variety of tissues where they participate in several important
biological processes such as acidebase balance, respiration, carbon
dioxide and ion transport, bone resorption, lipogenesis and elec-
trolyte secretion [15e22]. Many such CA isozymes involved in these
processes are important therapeutic targets with the potential to be
inhibited/activated for the treatment of a range of disorders such as
edema, glaucoma, obesity, cancer, epilepsy and osteoporosis
[15e17,19e22].
We have purified human CA I, II, IV, VI (hCA I, hCA II, hCA IV and
hCA VI) isoenzymes and examined the in vitro inhibition effects of
some phenolic, bisphenol, methoxy, and bromophenol compounds
mentioned above on these enzymes, using the esterase activity of
hCA I, hCA II, hCA IV, and hCA VI, with 4-nitrophenylacetate as
substrate.
2. Results and discussion
2.1. Chemistry
From the compounds shown in Fig. 2, 4e11 were synthesized by
the known method [7,8,12,13] and others 12e17 were bought
commercially.
To synthesize natural bromophenols vidalol A (25), B (22)
and their derivatives, we firstly prepared (2,3-dibromo-4,5-
dimethoxyphenyl)methanol (18) [12,13] from vanillin in four steps,
and mono- and dibromo-1,3,5-trimethoxybenzene (19 and 20) by
molecular bromination (1 and 2 eq., consecutively) of 1,3,5-
trimethoxybenzene in one step. The reaction of 18 (2 equivalent)
with 19 (1 equivalent) in polyphosphoric acid (PPA) by a known
R5
Br
Br
R4
Br
R1
R3
Br
Br
OMe
R
R
MeO
R1 R2
OMe
R3
R
R
R2
6
4. R1=R2=R4=Br, R3=OMe, R5=Br
5. R1=R2=R4=Br, R3=OMe, R5=H
. R: -OH
8. R1=R2=OMe, R3=CH2OH
9. R1=R2=OMe, R3=H
7. R: -OCH3
10
. R1=R2=OH, R3=H
11. R1=R2=OH, R3=Br
COOH
OH
N N
H2NO2S
NHAc
R
S
R
R
R
16
17
12
14
. R1=OH, R2=H
. R1=OH, R2=COOH
13. R1=OCH3, R2=H
15. R1=OCH3, R2=COOH
Fig. 2. From these compounds, 4e11 were synthesized and others 12e27 were bought as commercial.

H.T. Balaydın et al. / European Journal of Medicinal Chemistry 54 (2012) 423e428
425
method [8,12,13,23e25] gave 5,50-(5-bromo-2,4,6-trimethoxy-1,3-
phenylene)bis(methylene)bis(3,4-dibromo-1,2-dimethoxybenzene)
(21) as a sole product. We have achieved the first convenient
synthesis of the naturally occurring bromophenol vidalol B (22) from
the reaction of 21 with BBr3 in CH2Cl2 at 0e25 ꢀC (Scheme 1). Simi-
larly, 18 (1 equivalent) was subjected to reactions with 19 (1
equivalent) and 20 (1 equivalent), respectively, in PPA. From
these reactions, 2,3-dibromo-1-(3-bromo-2,4,6-trimethoxybenzyl)-
4,5-dimethoxybenzene(23),aprecursorofvidalolA(25),and24 were
isolated as the sole products (Scheme 1). On the other hand, the
demethylation reactions of 23 and 24 with BBr3 in CH2Cl2 at 0e25 ꢀC
for the synthesis of bromophenols vidalol A (25) and 26 failed. From
the synthesized compounds, vidalol B (22) is natural compound and
21 and 23 are new compounds.
have recently investigated the interactions of phenol and some of
its substituted derivatives with all mammalian carbonic anhydrase
enzymes [20e22], demonstrating some low micromolar/sub-
micromolar inhibitors as well as the possibility of designing
isozyme selective CAIs. The inhibition profile of various CA
isozymes with this class of agents is very variable, with inhibition
constants ranging from the millimolar to the submicromolar range
for many simple phenols [20e22]. Thus, it seemed reasonable to us
to extend the previous studies [7,8,10], including in this investiga-
tion phenolic, bisphenol, methoxy, and bromophenol compounds
with clinical and antioxidant applications as food additives, such as
compounds 2 and 3 and their derivatives [1e11]. Other structurally
related derivatives such as 4e24, were also included in our study
(Fig. 2, Table 1 and Scheme 1).
The purification of the CA isozymes was performed with
a simple one step method by Sepharose-4B-aniline-sulfanilamide
affinity column chromatography [10,11]. hCA I was purified 110-
fold with a specific activity of 941.4 EU mgꢁ1 and overall yield of
62.8%. hCA II was purified 781.3-fold with a specific activity of
7165 EU mgꢁ1 and overall yield of 69.4%. hCA IV was purified 86.5-
fold with a specific activity of 737.2 EU mgꢁ1 and overall yield of
27%. hCA VI was purified, 76.2-fold with a specific activity of
2.2. CA inhibition studies
Phenol 16 binds to CA in a different manner from the classical
inhibitors of the sulfonamide type, for example acetazolamide (17),
which coordinate to the Zn(II) ion from the enzyme active site by
substituting the fourth, non-protein ligand, a water molecule or
hydroxide ion (Fig. 2) [7,20e22]. Supuran’s group and our group
HO
Scheme 1.

426
H.T. Balaydın et al. / European Journal of Medicinal Chemistry 54 (2012) 423e428
422 EU mgꢁ1 and overall yield of 18.1% [10,26]. Inhibitory effects of
compounds 2, 3, 6, and 8e24 on enzyme activities were tested
under in vitro conditions; KI values were calculated from
LineweavereBurk graphs [27] and are given in Table 1.
We report here the first study of the inhibitory effects of these
compounds on the esterase activity of hCA I, II, IV, and VI. The
sulfonamide CAI acetazolamide AZA [15,16] has been used as
a negative control in our experiments, and for comparison reasons.
The previous reports by Senturk et al. [20] investigated other
antioxidant phenol derivatives (including salicylic acid and pro-
pofol) by esterase assay. The data in Table 1 show the following
regarding inhibition of hCA I, II, IV and VI, with these compounds,
by an esterase assay [28], with 4-nitrophenylacetate (4-NPA) as
substrate:
isozyme hCA II (Table 1). Five derivatives, i.e., 6, 12, 15, 16, and
22, showed moderate hCA II inhibitory activity with KI-s in the
range of 1.13e9.9 mM (Table 1). It must be stressed that KI-s
measured with the esterase method are always in the micro-
molar range because hCA I and II are weak esterases [30e32].
(iii) Compound 7 is a weak inhibitor of hCA IV, with a KI value of
145.12
mM. However, again compounds 8, 9, and 20e24 are
medium potency inhibitors (KI-s of 42.26e57.61
mM), and
natural bromophenol compound 22, AZA show a higher
affinity for this isozyme, with inhibition constant in the range
of 0.578 mM (Table 1).
(iv) Phenol 16 and some of its congeners such as 7e9,12 and 13 are
also weak inhibitors of the secreted isozyme hCA VI, with KI-s
of 158.592e919.182
derivatives 2e6, 10, 11, 14, 15, and 21e24 are medium potency
inhibitors (KI of 2.48e45.36 M (Table 1).
mM. However, again the remaining
m
(i) Against the slow cytosolic isozyme hCA I, compounds 7e9, 12
and 21 behave as weak inhibitors, with KI values in the range of
The inhibition effect of halogenated sulfonamide derivatives has
been investigated previously [35]. In a recent study it was reported
that derivatives of salicylic acid, some phenolic compounds, and
some benzoic acid derivatives [20e30], a simple compound lacking
the sulfonamide, sulfamate, or related functional groups that are
typically found in all known CA inhibitors, act as a CA I inhibitor,
and could represent the starting point for a new class of inhibitors
that may have advantages for patients with sulfonamide allergies
[31]. The sulfonamide zinc-binding group is thus superior to the
hydroxyl for generating CA inhibitors with a varied and sometimes
isozyme-selective inhibition profile against mammalian enzymes
[32e38]. However, it is critically important to explore further
classes of potent CAIs in order to detect compounds with a different
inhibition profile compared to the sulfonamides and their bio-
isosteres and to find novel applications for the inhibitors of these
widespread enzymes [37,38].
93.42e4003
I inhibitor (KI of 4003
and 24 showed better inhibitory activity as compared to the
previously mentioned compounds, with KI values of
57.87e61.14 mM (Table 1). Therefore, the nature of the groups in
m
M [22,29,30]. Catechol 12 was an ineffective hCA
mM). A second group of compounds 23
ortho-, para-, and meta- to the phenolic OH and OMe moiety
strongly influences hCA I inhibitory activity. It is also inter-
esting to note that the hydroxybenzoic acid derivatives 14 and
15 were much better hCA I inhibitors compared to the corre-
sponding phenolic compounds 12 and 13 from which they
were prepared. Kinetic investigations (LineweavereBurk plots,
data not shown) indicate that, similarly to sulfonamides and
inorganic anions [26,30e34], all the investigated compounds
act as noncompetitive inhibitors with 4-NPA as substrate, i.e.,
they bind in different regions of the active site cavity as
compared to the substrate. However, the binding site of 4-NPA
itself is unknown, but it is presumed to be in the same region as
that of CO2, the physiological substrate of this enzyme [29e31].
(ii) A better inhibitory activity has been observed with
compounds 13 and 14 for the inhibition of the rapid cytosolic
3. Conclusions
The first and convenient synthesis of the naturally occurring
bromophenol vidalol B (22) from the corresponding compounds
was achieved. Carbonic anhydrase inhibitory properties of a series
of bisphenol, bromophenol and methoxyphenol derivatives (2e24)
including natural bromophenols were investigated.
Table 1
hCA I, II, IV and VI inhibition data with the tested compounds (KI values, mM).
Phenolic, bisphenol, methoxy, and bromophenol compounds
influence the activity of hCA isozymes due to the presence of
different functional groups (OH, OMe, COOH, and Br) in their
aromatic scaffold. Our findings indicate thus another class of
possible CAIs of interest, in addition to the well-known sulfon-
amides/sulfamates/sulfamides, the phenols/biphenyl diphenols
bearing bulky ortho moieties in their molecules. Compounds 6e15
investigated here showed effective hCA I and II inhibitory activity
[22,29e32,37], in the low micromolar range, by the esterase
method, which usually gives KI-s an order of magnitude higher
compared to the CO2 hydrase assay [31]. These findings point out
that substituted phenolic, bisphenol, methoxy, and bromophenol
compounds may be used as leads for generating potent CAIs
eventually targeting other isoforms that have not been assayed yet
for their interactions with such agents.
Compound
hCA I
hCA II
hCA IV
hCA VI
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
21
22
23
24
34.41a
21.16a
21.47a
35.45
34.62
5.636b
59.107b
34.56
69.12
26.376b
29.820b
9.9c
27.48a
28.04a
32.72
30.78
7.64
145.12
46.35
48.73
13.12
17.48
10.9c
14.32
2.45
17.43a
17.83a
29.67
35.12a
43.51
41.36
28.17
18.356b
537.311b
193.24a
892.109a
25.341b
32.145b
4003c
13.692b
382.473b
158.592a
919.182a
11.143b
29.138b
606c
10.4d
0.50d
0.47d
6.18d
5.5c
234.7
4.72
1.08d
6.83d
6.48
24.42
10.2c
9.5c
208c
36.2e
0.37e
0.578e
57.61
1.84
0.34e
93.42
12.24
57.87
61.14
78.49
1.13
54.43
60.17
45.36
3.41
35.45
38.19
42.26
45.74
4. Experimental
Sepharose 4B, protein assay reagents, 4-nitrophenylacetate and
chemicals for electrophoresis were purchased from SigmaeAldrich
Co. All other chemicals and solvents are commercially available and
were used after distillation or treatment with drying agents.
Melting points were determined on a capillary melting apparatus
(BUCHI 530) and are uncorrected. IR spectra were obtained from
Mean from at least three determinations. Errors in the range of w1% of the reported
value (data not shown).
a
Ref. [6].
Ref. [29].
Ref. [22].
Ref. [30].
b
c
d
e
Ref. [35].

H.T. Balaydın et al. / European Journal of Medicinal Chemistry 54 (2012) 423e428
427
solutions in 0.1 mm cells with a PerkineElmer spectrophotometer.
(s, OCH3, 3H), 3.79 (s, OCH3, 3H), 3.71 (s, OCH3, 3H), 3.62 (s, OCH3,
3H), 13C NMR (100 MHz, CDCl3)
158.43(C), 157.31 (C), 156.47 (C),
The 1H and 13C NMR spectra were recorded on a 400 (100)-MHz
d
Varian spectrometer;
d
in ppm, Me4Si as the internal standard.
152.27 (C), 145.74 (C), 137.80 (C), 121.47 (C), 117.45 (C), 114.98 (C),
112.02 (CH), 98.21 (C), 9.59 (CH), 61.17 (OCH3), 60.46 (OCH3), 56.47
(OCH3), 56.08 (OCH3), 55.94 (OCH3), 31.84 (CH2), IR (CH2Cl2, cmꢁ1):
3000, 2937, 2840, 2593, 2427, 2284, 2120, 2017, 1963, 1913, 1592,
1549, 1463, 1435, 1422, 1394, 1372, 1337, 1321, 1282, 1254, 1199,
1164, 1107, 1057, 1008, 963, 946, 895, 857, 802, 757, 739, 656, 588,
491, 458, Anal. Calcd for (C18H19Br3O5): C 38.95, H 3.45; Found C
38.90, H 3.46.
Elemental analyses were performed on a Leco CHNS-932 apparatus.
All column chromatography was performed on silica gel (60-mesh,
Merck). PLC is preparative thick-layer chromatography: 1 mm of
silica gel 60 PF (Merck) on glass plates.
4.1. Synthesis
The compounds 4e11 were synthesized by the known method
[7,8,12,13].
4.1.4. Synthesis of 1,3-dibromo-5-(2,3-dibromo-4,5-dimethoxy
benzyl)-2,4,6-trimethoxybenzene (24)
The standard procedure described above (Section 4.1.1.) for 21
was applied, but, the ratio of reactants 18 and 20 was also 1. The
known [39] compound 24 (78%) was crystallized from ethyl
acetate/hexane (3/1) as colorless crystals. mp. 122e124 ꢀC, 1H NMR
4.1.1. Standard procedure: synthesis of 5,50-(5-bromo-2,4,6-
trimethoxy-1,3-phenylene)bis(methylene)bis(3,4-dibromo-1,2-
dimethoxybenzene) (21)
Polyphosphoric acid (PPA), prepared from conc. H3PO4 (85%,
2.32 g, 24 mmol) and P2O5 (4.15 g, 29 mmol), was heated to 80 ꢀC in
a beaker (50 mL) [12,13,23e25]. To this mixture were added 19
(0.5 g, 2.02 mmol) and 18 (1.32 g, 4.05 mmol) consecutively and
quickly. The mixture was stirred with a glass rod at 80 ꢀC for 1 h and
cooled to room temperature (RT). A mixture of water and ice
(50 mL) was carefully added to the cooled mixture. The organic
phase was separated and then the water phase was extracted with
EtOAc (2 ꢂ 40 mL). The combined organic layers were dried over
MgSO4 and the solvent was evaporated. The product 21 (1.01 g,
65%) was obtained and crystallized from ethyl acetate/hexane (3/1)
as pale yellow crystals. Mp: 136e137 ꢀC, 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
3.80 (s, OCH3, 3H), 3.73 (s, OCH3, 6H), 3.65 (s, OCH3, 3H), 13C NMR
(100 MHz, CDCl3) 156.55(C), 154.85 (C), 152.43 (C), 146.17 (C),
d 6.36 (s, 1H), 4.17 (s, CH2, 2H), 3.93 (s, OCH3, 3H),
d
136.81 (C), 125.66 (C), 121.77 (C), 117.40 (C), 112.35 (CH), 109.62 (C),
61.27 (2 OCH3), 60.75 (OCH3), 60.50 (OCH3), 56.21 (OCH3), 33.00
(CH2), IR (CH2Cl2, cmꢁ1): 3000, 2965, 2936, 2853, 2581, 2025, 1582,
1550, 156, 1422, 1397, 1382, 1322, 1284, 1264, 1198, 1164, 1085, 1058,
1003, 970, 947, 904, 868, 841, 810, 767, 713, 686, 672, 593, 497. Anal.
Calcd for (C18H18Br4O5): C 34.10, H 2.86; Found C 34.15, H 2.85.
4.2. Purification of carbonic anhydrase isoenzymes from human by
affinity chromatography
d
6.39 (s, 2H), 4.16 (s, CH2, 4H), 3.80 (s, OCH3, 6H), 3.76 (s, OCH3, 6H),
3.59 (s, OCH3, 6H), 3.57 (s, OCH3, 3H), 13C NMR (100 MHz, CDCl3)
157.97(C), 156.53 (C), 152.36 (C), 146.20 (C), 137.24 (2C), 124.65 (C),
d
Purification of hCA I and hCA II was previously described [10].
Fresh citrated human whole blood was obtained from the Blood
Center of the Research Hospital at Atatürk University. Cells were
washed three times by centrifugation at 1000 ꢂ g at 4 ꢃ 6 ꢀC, for
20 min in four volumes of 25 mM Na2HPO4 (pH ¼ 7.4) buffer. The
supernatant and fluffy coat were removed. The erythrocytes were
lysed in 10 volumes of 5 mM Na2HPO4 (pH ¼ 7.4) buffer, containing
1 mM EDTA. After 20 min the hemolysate was centrifuged at
10.000 ꢂ g for 60 min. The particulate fraction was washed four
times in the same buffer. The membranes were centrifuged down at
15.000 ꢂ g for 60 min. pH was adjusted to 8.3 with solid Tris. A
sepharose-4B-aniline-sulfanilamide affinity column was equili-
brated with 25 mM TriseHCl/0.1 M Na2SO4 (pH 8.3). The affinity gel
was washed with 25 mM TriseHCl/25 mM Na2PO4 (pH 8.3). Finally,
human carbonic anhydrase IV (hCA IV) isozyme was eluted with
25 mM TriseHCl/0.5 M NaClO4 (pH 7.4). Fresh non-citrated human
whole blood was obtained from the Blood Center of the Research
Hospital at Atatürk University. The blood samples were centrifuged
at 5000 rpm for 15 min and the precipitant was removed. The
serum was isolated. The pH was adjusted to 8.7 with solid Tris. A
sepharose-4B-aniline-sulfanilamide affinity column was equili-
brated with 25 mM TriseHCl/0.1 M Na2SO4 (pH 8.7). The affinity gel
was washed with 25 mM TriseHCl/22 mM Na2SO4 (pH 8.7). The
human carbonic anhydrase (hCA VI) isozyme was eluted with
0.25 M H2NSO3H/25 mM Na2HPO4 (pH ¼ 6.7) [18].
121.79 (C), 117.54 (C), 112.68 (CH), 61.84 (OCH3), 61.28 (OCH3), 60.50
(OCH3), 56.23 (OCH3), 32.72 (CH2). IR (CH2Cl2, cmꢁ1): 2999, 2937,
2868, 2840, 2591, 2283, 2013, 1582, 1549, 1466, 1422, 1404, 1372,
1321, 1283, 1255, 1221, 1195, 1161, 1105, 1082, 1058, 1005, 966, 950,
903, 881, 843, 819, 804, 775, 737, 704, 662, 591, 510, 487. Anal. Calcd
for (C27H27Br5O7): C 37.58, H 3.15; Found C 37.65, H 3.16.
4.1.2. Synthesis of 2-bromo-4,6-bis(2,3-dibromo-4,5-dihydroxy
benzyl)benzene-1,3,5-triol (vidalol B, 22)
A solution of 21 (0.500 g, 0.58 mmol) in CH2Cl2 (10 mL) was
cooled to 0 ꢀC and then a solution of BBr3 (0.65 mL) in CH2Cl2 (7 mL)
was added dropwise under N2 (g) over 5 min. After the cold bath
was removed, the mixture was stirred at RT and under N2 for 1 day.
Methanol (15 mL) was slowly added over 15 min at 0 ꢀC and then
the solvent was evaporated. After water (40 mL) and EtOAc (50 mL)
were added, the mixture was shaken. The organic phase was
separated and the water phase was extracted with EtOAc
(2 ꢂ 30 mL). The combined organic phases were dried over MgSO4
and the solvent was evaporated. Natural product 22 (0.412 g, 93%)
was obtained as brownish solid. Mp: 188.5e189 ꢀC, 1H NMR
(400 MHz, CD3COCD3)
7.77 (s, 2OH), 6.38 (s, 2H), 4.04 (s, CH2, 4H), 13C NMR (100 MHz,
CDCl3) 154.76 (C), 151.88 (C), 144.70 (C), 142.59 (C), 132.46 (2C),
d 8.59 (s, 2OH), 8.07 (s, 2OH), 7.83 (s, OH),
d
115.96 (C), 114.20 (CH), 112.93 (C), 106.67 (C), 31.68 (CH2), IR
(CH2Cl2, cmꢁ1): 2975, 2863, 2112, 1646, 1468, 1401, 1340, 1273,
1220, 1172, 1105, 1055, 1033, 1014, 915, 856, 666, 551, 434, 408, 400.
4.3. CA inhibition
4.1.3. Synthesis of 2,3-dibromo-1-(3-bromo-2,4,6-trimethoxy
benzyl)-4,5-dimethoxybenzene (23)
Carbonic anhydrase activity was assayed by following 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 spec-
trophotometer according to the method described by Verpoorte
et al. [28]. The enzymatic reaction, in a total volume of 3.0 mL,
contained 1.4 mL of 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL of 3 mM
4-nitrophenylacetate, 0.5 mL of H2O and 0.1 mL of enzyme solution.
The standard procedure described above (Section 4.1.1.) for 21
was applied, but the ratio of reactants 18 and 19 was 1. Compound
23 (57%) was crystallized from ethyl acetate/hexane (3/1) as
colorless crystals. mp: 123e125 ꢀC, 1H NMR (400 MHz, CDCl3)
d
6.38
(s, 1H), 6.33 (s, 1H), 4.06 (s, CH2, 2H), 3.94 (s, OCH3, 3H), 3.80

428
H.T. Balaydın et al. / European Journal of Medicinal Chemistry 54 (2012) 423e428
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itors and activators, Nat. Rev. Drug Discov. 7 (2008) 168e181.
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J. Valjakka, S. Pastoreková, J. Pastorek, M.S. Kulomaa, H.R. Nordlund,
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A reference measurement was obtained by preparing the same
cuvette without enzyme solution. The inhibitory effects of
compounds 2e24 were examined. All compounds were tested in
triplicate at each concentration used. Different inhibitor concen-
trations were used. Control cuvette activity in the absence of
inhibitor was taken as 100%. For each inhibitor an Activity
%-[Inhibitor] graph was drawn. The curve-fitting algorithm
allowed us to obtain the IC50 values, working at the lowest
concentration of substrate of 0.15 mM, from which KI values were
calculated by using the ChengePrusoff equation [36]. The catalytic
activity (in the absence of inhibitors) of these enzymes was calcu-
lated from LineweavereBurk plots, as reported earlier, and represent
the mean from at least three different determinations. The enzymes
used here were purified from human blood as described earlier.
[21] S. Pastorekova, S. Parkkila, J. Pastorek, C.T. Supuran, Carbonic anhydrases:
current state of the art, therapeutic applications and future prospects,
J. Enzym. Inhib. Med. Chem. 19 (2004) 199e229.
Acknowledgments
[22] A. Innocenti, D. Vullo, A. Scozzafava, C.T. Supuran, Carbonic anhydrase
inhibitors. Interactions of phenols with the 12 catalytically active mammalian
isoforms (CA I-XIV), Bioorg. Med. Chem. Lett. 18 (2008) 1583e1587.
[23] M. Harig, B. Neumann, H.G. Stammler, D. Kuck, 2,3,6,7,10,11-
Hexamethoxytribenzo triquinacene: synthesis, solid-state structure and
functionalization of a rigid analogue of cyclotriveratrylene, Eur. J. Org. Chem.
11 (2004) 2381e2397.
[24] E. Sahin, H.T. Balaydin, S. Goksu, A. Menzek, 2,3-Dibromo-1-[4-(2,3-dibromo-
4,5-di-meth-oxy-benz-yl)-2,5-dimeth-oxy-benz-yl]-4,5-dimeth-oxy-
benzene, Acta Cryst. E66 (2010) o3029.
We are grateful to Prof. Dr. Claudiu T. Supuran, University of
Florence, for various valuable studies on CAIs. This study was
financed by TÜBITAK (The Scientific and Technological Research
Council of Turkey) (Project no: TBAG-107T348) for (HTB and SG)
and Ataturk University.
_
Appendix A. Supplementary data
[25] Y. Cetinkaya, A. Menzek, E. Sahin, H.T. Balaydın, Selective O-demethylation
during bromination of (3,4-dimethoxyphenyl)(2,3,4-trimethoxyphenyl)
methanone, Tetrahedron 67 (2011) 3483e3489.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.05.025.
[26] D. Ekinci, S.B. Ceyhun, M. Senturk, D. Erdem, O.I. Kufrevioglu, C.T. Supuran,
Characterization and anions inhibition studies of an
a-carbonic anhydrase
from the teleost fish Dicentrarchus labrax, Bioorg. Med. Chem. 19 (2011)
744e748.
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