J BIOCHEM MOLECULAR TOXICOLOGY

Volume 30, Number 6, 2016

Investigation of the Effect of Some Optically Active

Imine Compounds on the Enzyme Activities of hCA-I
and hCA-II under In Vitro Conditions: An Experimental
and Theoretical Study
Osman Tektas,1 Ebru Akkemik,2 and Haci Baykara1,3
1 Department of Chemistry, Faculty of Arts and Sciences, Siirt University 56100, Siirt, Turkey; E-mail: hacibaykara@gmail.com
2 Faculty of Engineering and Architecture, Food Engineering, Siirt University 56100, Siirt, Turkey; E-mail: eakkemik@siirt.edu.tr
3 Centerof Nanotechnology Research and Development (CIDNA), Facultad de Ingenierı́a Mecánica y Ciencias de la Producción, Escuela
Superior Politécnica del Litoral, ESPOL, Campus Gustavo Galindo Km 30.5 Vı́a Perimetral, Guayaquil, Ecuador

Received 10 November 2015; revised 6 December 2015; accepted 15 December 2015

ABSTRACT: Inhibitors of carbonic anhydrase (hCA; INTRODUCTION

EC 4.2.1.1) are used as medicines for many diseases.
Therefore, they are very important. In this study, a
known series of Schiff bases were synthesized and
their effects on the activities of hCA-I and hCA-II,
which are cytosolic isoenzymes of carbonic anhydrase,
were investigated under in vitro conditions. The syn-
thesized compounds (H1, H2, H3, and H4) were found
to cause inhibition on enzyme activities of hCA-1 and
hCA-II. IC50 values of H1, H2, H3, and H4 compounds
were 140, 88, 201, and 271 µM for hCA-I enzyme activ-
ity and 134, 251, 79, and 604 µM for hCA-II enzyme ac-
tivity, respectively. The synthesized Schiff bases were
characterized by several methods, including 1 H NMR,
FT-IR, elemental analysis, and polarimetric measure-
ments. Correlation coefficient square values (R2 ) of
comparison of the theoretical and experimental 1 H
NMR shifts for H1, H2, H3, and H4 compounds were
found as 0.9781, 0.9814, 0.9758, and 0.8635, respectively.
C 2016 Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol.
30:277–286, 2016; View this article online at wileyonlineli-
brary.com. DOI 10.1002/jbt.21788
KEYWORDS: Carbonic Anhydrase; DFT; Chirality; Inhi-
bition; Theoretical NMR
Correspondence to: Ebru Akkemik and Haci Baykara.
Contract Grant Sponsor: Scientific Research Projects Unit
of Siirt University.Contract Grant Number: 2013-SİÜFED-F6.
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.

C 2016 Wiley Periodicals, Inc.
Carbonic anhydrase (CA; EC 4.2.1.1) is a metal-
loenzyme with Zn2+ in its active site. In living or-
ganisms, it catalyzes interconversion of bicarbonate–
carbon dioxide as well as the hydration reaction of
cyanate to carbamic acid, urea to cyanide, and alde-
hyde to germinal diol [1, 2]. Carbonic anhydrases are
found in a large variety of living things including mam-
mals, bacteria, and archaea. In mammals, 16 isoen-
zymes of carbonic anhydrase have been identified.
They are located in different tissues and organs. CA-I
and CA-II are cytosolic isoenzymes. It was reported that
these enzymes can be used in treatment of glaucoma,
epilepsy, and diuretic therapy [2]. Each carbonic anhy-
drase isoenzyme plays a vital role in a variety of critical
processes such as respiration, arthritis, and acid bal-
ance [3–5]. Because they are diagnostic agents or pro-
teins directly causing a disease in many areas, including
antiglaucoma, antiobesity, antidiuretic, antitumor, etc.
[6–9]. Particularly, CA I, CA II, CA IV, and CA XII isoen-
zymes are involved in vision by producing aqueous
secretions rich in bicarbonate in the eye. Their dysfunc-
tion causes high intraocular pressure, hence glaucoma
[10, 11]. Sulfonamides have been known to be powerful
CA inhibitors since 1954. A derivative of sulfonamides
such as asetolazamid is used as an antiglaucoma agent.
However, the use of sulfonamides is limited because
it has various side effects [12, 13]. Therefore, many re-
searchers are working in this field to design and syn-
thesize new drugs.
Schiff bases are obtained as a result of a condensa-
tion reaction of primary amines with aldehydes and
ketones [14]. Chemical characteristics of the Schiff

277
278 TEKTAS ET AL.
bases, which came into the limelight as biologically ac-
tive compounds, are quite well known. Another reason
for the interest of researchers in Schiff bases is their elec-
tron donor properties arising from the double bonds
between their oxygen and nitrogen atoms [15]. Their
metal complexes are also known to stop tumor growth
[15–18]. The effects of Schiff bases on carbonic anhy-
drase isoenzymes have been investigated for the past
20 years [2, 15, 18–22].
Owing to their very flexible and variable struc-
tural characteristics, multiple Schiff bases and their
complexes have been synthesized and studied. It is
possible to use Schiff bases in many biological appli-
cations [14, 23–26]. They are considered as an impor-
tant class of organic compounds. Some Schiff bases
have been reported to possess antibacterial, antifungal,
anticarcinogenic, anticonvulsant, antituberculosis, an-
tioxidant, antimalarial, antiflammatory, anticorrosive,
anti-HIV, and antitumor activities [14, 23–30]. When
Schiff bases used as inhibitors were analyzed, their
inhibitory effects were observed to vary by the type
of functional groups attached to the benzene ring in
the structure, if these bases contained aryl substituents
[31, 32].
Theoretical NMR calculations and their compar-
isons with experimental NMR values are as impor-
tant as the experimental characterization methods and
constitute one of supportive methods of characteriza-
tion generally referred to by scientists in recent studies
[33–49]
In this study, four optically active Schiff bases,
which are well known [50–53] but no theoretical stud-
ies or enzyme inhibition/activation studies were con-
ducted, were synthesized and their inhibition and ac-
tivation effects on human CA I and II enzymes were
investigated. In addition, each compound was opti-
mized by the density functional theory (DFT) method
and B3LYP/6-311+G(2d,p) basis set using Gaussian 09
[54] software package, and theoretical 1 H NMR shift
values were calculated. Experimental and theoretical
1
H NMR values were compared, and it was demon-
strated that the compounds with proposed structures
were synthesized successfully.
MATERIALS AND METHODS
Chemicals and Instruments
R-(+)-α-Methylbenzylamine was obtained
from Fluka (Germany), and protein assay reagents,
Sepharose-4B, (S)-(–)1-(4-methoxyphenyl)ethan-1-
amine, (S)-(+)-1-cyclohexylethan-1-amine, (R)-(–)-3,3-
dimethylbutan-2-amine, acetic acid, and chemicals
for electrophoresis were obtained from Sigma-Aldrich
Volume 30, Number 6, 2016
Chemie (Taufkirchen, Germany). Para-aminobenzene
sulfonamide, absolute ethanol, salicylaldehyde, and
L-tyrosine were obtained from E. Merck (Darmstadt
Germany). All the other chemical substances used
were analytical grade and obtained from either
Sigma-Aldrich or E. Merck. The NMR analyses of
the compounds synthesized were recorded on both a
Bruker 300 MHz Ultrashield, and an Avance III HD
Ascend 600 ULH spectrometers, tetramethylsilane
was used as the internal standard. FT-IR analyses of
the compounds recorded on a Thermo Nicolet IS10
spectrometer, and the melting points were recorded on
a Stuart SMP40 apparatus. All the kinetic studies were
carried out using a Shimadzu UVmini-1240 UV–vis
spectrophotometer. Experimental mass spectra of the
compounds were obtained on a Thermo Scientific Velos
pro dual-pressure linear ion trap mass spectrometer.
Synthesis of Chiral Schiff Bases
Synthesis of Chiral Schiff Base (R)-(+)-2-(((1-
Phenylethyl)imino)methyl)phenol (H1)
A 6 g of 2-hydroxy benzaldehyde was transferred
to a flask and dissolved in ethyl alcohol. Then, 5.94 g
(d = 0.952; ࣙ98%) of R-(+)-α-methylbenzylamine, an
optically active chiral amine, was added to 2-hydroxy
benzaldehyde solution contained in the flask. A con-
denser was attached to the flask, and its content was
allowed to reflux for 3 h. After the reaction started,
one drop of concentrated CH3 COOH was added to the
flask and the resulting mixture was refluxed for another
4 h. After the completion of the reaction, the substance
was allowed to stand in the refrigerator for 24 h. Af-
ter filtration and removal of solvent, the product was
purified by crystallization in ethanol (Figure 1). The
purity of the synthesized compound was checked by
thin layer chromatography. The Schiff base was char-
acterized by elemental analysis, FT-IR, and 1 H NMR
techniques. The results of the experimental character-
ization of this compound, which are presented in the
literature [50], are as follows: melting point: 82–85°C,
yield (%): 906.. Found: mass spectrum (LC/MS-MS):
m/z 226 [M + 1]+ Calcd. For C15 ONH15 ; 225 g/mol. IR
(ATR, cm−1 ); vAr-H ; 3089–3061, vC=N ; 1623, vC=C ; 1577,
vC-N ; 1493, vC-O ; 1377. 1 H NMR (CDCl3 ); δ ppm, 13.54
(s, 1H, Ar-OH); 8.40 (s, 1H, -CH=N-); 7.37–6.85 (9H,
Ar-H); 1.63–1.62 (d, 3H, =N–CH–CH3 , J = 6); 4.56–4.52
ο
(q, 1H, –CH=N–CH–). [α]30 3
D : 108.2 (c: 0.66 g/100 cm ).
Synthesis of Chiral Schiff Base (S)-(+)-2-(((1-
Cyclohexylethyl)imino)methyl)phenol (H2)
A 4.20 g of 2-hydroxy benzaldehyde was trans-
ferred to a flask and dissolved in ethyl alcohol. Then,
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 30, Number 6, 2016
FIGURE 1. Synthesis of Schiff bases.
4.40 g (d = 0.856; ࣙ98%) of (S)-(+)-1-cyclohexylethan-
1-amine, an optically active chiral amine, was added
to 2-hydroxy benzaldehyde solution contained in the
flask. Following the same procedure for the synthe-
sis of H1 Schiff base, H2 was obtained and purified
(Figure 1). The results of the experimental characteriza-
tion of this compound, which are presented in the liter-
ature [52], are as follows: melting point: 46°C, yield (%):
90.7. Found: mass spectrum (LC/MS-MS): m/z 232 [M +
1]+ Calcd. For C15 ONH17 : 231 g/mol. IR (ATR, cm−1 );
vAr-H ; 3058, vC=N ; 1628, vC=C ; 1580, vC-N ; 1496, vC-O ;
1379. 1 H NMR (CDCl3 ); δ ppm, 13.83 (s, 1H, Ar-OH);
8.27 (s, 1H, –CH=N–); 7.32–6.96 (4H, Ar-H); 1.26–1.24
(d, 3H, =N–CH–CH3 , J = 6); 3.13–3.04 (q, 1H, –CH=N–
ο
CH–); 1.77–1.13 (m, 11H, CH2 -cyclohexan). [α]30 D : 63
3
(c: 1.33 g/100 cm ).
Synthesis of Chiral Schiff Base (S)-(–)-2-(((1-(4-
Methoxyphenyl) ethyl) imino) methyl)phenol (H3)
A 4.88 g of 2-hydroxy benzaldehyde was trans-
ferred to a flask and dissolved in ethyl alcohol.
Then, 6.04 g (d = 1.024; ࣙ99%) of (S)-(–)1-(4-
methoxyphenyl)ethan-1-amine, an optically active
chiral amine, was added to 2-hydroxy benzaldehyde
solution contained in the flask. Following the same
procedure for the synthesis of H1 Schiff base, H3
was obtained and purified (Figure 1). The results of
the experimental characterization of this compound,
which are presented in the literature [51], are as
follows: melting point: 70—74°C, yield (%): 93.75.
J Biochem Molecular Toxicology DOI 10.1002/jbt
INHIBITION OF HCAs AND A DFT STUDY 279
Found: mass spectrum (LC/MS-MS): m/z 256 [M +
1]+ Calcd. For C16 O2 NH17 ; 255 g/mol. IR (ATR, cm−1 );
vAr-H ; 3087–3047, vC=N ; 1624, vC=C ; 1578–1607, vC-N ;
1510, vC-O ; 1375. 1 H NMR (CDCl3 ); δ ppm, 13.58 (s, 1H,
Ar-OH); 8.36 (s, 1H, –CH=N–); 7.30–6.84 (8H, Ar-H);
3.78 (s, 3H, Ar–OCH3 ); 1.59–1.60 (d, 3H, =N–CH–CH3 ,
ο
J = 6); 4.52–4.49 (q, 1H, –CH=N–CH–). [α]30 D : – 106.9
3
(c: 0.66 g/100 cm ).
Synthesis of Chiral Schiff Base (R)-(+)-2-(((3,3-
Dimethylbutan-2-yl)imino)methyl)phenol (H4)
A 5 g of 2-hydroxy benzaldehyde was transferred
to a flask and dissolved in ethyl alcohol. Then, 4.20
g (d = 0.762; ࣙ%99) of (R)-(–)-3,3-dimethylbutan-2-
amine, an optically active chiral amine, was added
to 2-hydroxy benzaldehyde solution contained in the
flask. Following the same procedure for the synthe-
sis of H1 Schiff base, H4 was obtained and purified
(Figure 1). The results of the experimental character-
ization of this compound, which are presented in the
literature [53], are as follows: It is liquid at ambient con-
ditions, yield (%): 92.8. Found: mass spectrum (LC/MS-
MS): m/z 206 [M + 1]+ Calcd. For C13 ONH19 ; 205 g/mol.
IR (ATR, cm−1 ); vAr-H ; 3061, vC=N ; 1630, vC=C ; 1583,
vC-N ; 1497, vC-O ; 1395. 1 H NMR (CDCl3 ); δ ppm, 13.82 (s,
1H, Ar-OH); 8.28 (s, 1H, –CH=N–); 7.31–6.83 (4H, Ar-
H); 1.20–1.18 (d, 3H, =N–CH–CH3 , J = 6); 3.03–2.96 (q,
ο
1H, –CH=N–CH–); 0.93 (s, 9H, (CH3 )). [α]30 D : – 97.7 (c:
3
0.66 g/100 cm ).
280 TEKTAS ET AL.
FIGURE 2. (A) The results of regression analysis obtained without neglecting deviated values. (B) The results of regression analysis obtained
after neglecting deviated values.
Theoretical Calculations by Gaussian 09 Program
Imine compounds were characterized by experi-
mental NMR [55–58] values; however, their theoretical
NMR values were calculated by Gaussian 09W soft-
ware package [54]. Both 1 H and 13 C NMR calculations
were performed only for the compound coded H1.
For this purpose, input files of the compounds were
prepared using GaussView 5.0.9 [59] program, and the
same program was used to view output files and 1 H
NMR results. Theoretical calculations (optimization
and theoretical 1 H NMR) were performed by the
DFT method [35–37] using RB3LYP/6-311+G(2d,p)
basis set. NMR calculations were performed according
to the Gauge-Independent Atomic Orbital (GIAO)
[55–58, 60] method. The fit between experimental
and theoretical NMR shifts was performed by linear
regression analysis (Figures 2a and 2b) according to
the equation y = ax + b. Regression analyses were
conducted in both cases where the values deviating
Volume 30, Number 6, 2016
between theoretical and experimental 1 H NMR val-
ues are either included or neglected, and necessary
descriptions were made according to the literature
[48, 61–67]. Optimized three-dimensional structures of
the compounds obtained using the said methods and
basis sets are shown in Figure 3.
Biochemical Studies
hCA-I and hCA-II isoenzymes were purified
in a single step using Sepharose-4B-L tyrosine-
sulfanilamide affinity column [68, 69]. The column ma-
terial, i.e., Sepharose 4B-L-tyrosine-sulfanilamide affin-
ity gel, was prepared according to previously described
methods [70]. Before hemolysate was packed into the
column, pH was adjusted to 8.7 using solid Tris. Then,
the column was washed with 25 mM of Tris HCl/22
mM Na2 SO4 buffer (pH 8.7). The washing process was
continued till to get an absorbance value of 0.05 at
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 30, Number 6, 2016 INHIBITION OF HCAs AND A DFT STUDY 281

FIGURE 3. Optimized three-dimensional (3D) structures of the synthesized compounds.

280 nm. Finally, hCA I and hCA II isoenzymes were RESULTS

eluted with 1.0 M NaCl/25 mM Na2 HPO4 (pH 6.3)
and 0.1 M CH3 COONa/0.5 M NaClO4 (pH 5.6), re- Synthesis of Chiral Schiff Bases (H1– H4)
spectively.
In view of FT-IR results of optically active chi-
Activity of hCA-I and hCA-II isoenzymes was per-
ral Schiff bases, it is clear that all compounds show
formed according to the Verpoorte method, which is
peaks near 1625 cm−1 [64, 66, 67], which is characteris-
based on hydrolysis of p-nitrophenylacetate, used as a
tic for CH=N stretching vibration. This indicates that
substrate, to p-nitrophenol or p-nitrophenolate, which
neither the peak (dual peak) near 3300 cm−1 for the
give absorption at 348 nm [71]. Enzyme purity was
amino groups in the starting compounds nor the peak
checked by sodium dodecyl sulfate–polyacrylamide-
near 2700 for aldehydes was observed and all amino
gel electrophoresis (SDS-PAGE). Accumulation and
(–NH2 ) and formyl (–COH) groups were transformed
separation gels at acrylamide concentrations of 3% and
into imine groups. Expected characteristic peaks for
10% were prepared according to the Laemmli proce-
each synthesized optically active chiral imine com-
dure [72]. SDS-PAGE was observed a single band for
pound were observed.
both isoenzymes. In the purification steps, amounts
Additionally, experimental mass analysis results
of proteins were performed according to the Bradford
confirmed theoretical m/z ratios for all synthesized
method, in which bovine serum albumin (BSA) is used
compounds. Namely, Found for H1: mass spec-
as a standard [73]. Enzyme activities were measured
trum (LC/MS-MS): m/z 226 [M + 1]+ Calcd. For
at constant substrate and different inhibitor concentra-
C15 ONH15 ; 225 g/mol, Found for H2: mass spec-
tions to find IC50 value. Stock solutions of inhibitors
trum (LC/MS-MS): m/z 232 [M + 1]+ Calcd. For
were prepared at a concentration of approximately
C15 ONH17 , 231 g/mol; Found for H3: mass spec-
5 mM (10 mg/10 mL in dimethyl sulfoxide (DMSO);
trum (LC/MS-MS): m/z 256 [M + 1]+ Calcd. For
w/v) and diluted with DMSO. At least seven different
C16 O2 NH17 , 255 g/mol; Found for H4: mass spectrum
inhibitor concentrations were used for measuring the
(LC/MS-MS): m/z 206 [M + 1]+ Calcd. For C13 ONH19 ,
inhibition constant. In preliminary experiments, DMSO
205 g/mol.
was found to have no significant inhibition effect on
The synthesized compounds were already re-
hCA I and hCA II isoenzymes. The tube not containing
ported in the literature [50–53] so only 1H NMR
inhibitor was used as control, and its activity was con-
results of these compounds were taken, and the re-
sidered as 100%. Each experiment was repeated three
sults obtained were consistent with the literature and
times. Activity–% [inhibitor] plots were drawn for in-
confirmed the structures of compounds. Experimental
hibitors. The Ki constants were calculated according to
NMR results of all compounds were provided as the
the Cheng–Prusoff equation [74].
Supporting Information.

J Biochem Molecular Toxicology DOI 10.1002/jbt

282 TEKTAS ET AL.
TABLE 1. Inhibitory Effects on the Enzyme Activity (hCA-I, II) of the Schiff Bases
Code of the Compound hCA-I
H1 140
H2 88
H3 201
H4 271
Acetazolamidea [75, 76]
a
Acetazolamide (AZA) and dorzolamide (DZA) were used as standard inhibitors for all CAs.
FIGURE 4. The proposed inhibition mechanism of CA isoenzymes
with synthesized compounds [77, 78].
The compounds showed a turning angle in po-
larimeter, evidencing that they are optically active chi-
ral compounds.
Theoretical Calculations by Gaussian
09 Program
The results of regression analysis between theoret-
ical and experimental 1 H NMR shifts of the compounds
were very close to each other (Figure 2), and theoretical
data supported experimental data.
Biochemical Studies
hCA-I and hCA-II isoenzymes were purified
in a single step using Sepharose-4B-L tyrosine-
sulfanilamide affinity colon. Enzyme purity was
checked by SDS-PAGE. SDS-PAGE was observed a
single band for both isoenzymes (data not shown).
Enzyme activities were measured at constant sub-
strate and different inhibitor concentrations to find
IC50 value. The tube not containing inhibitor was used
as control, and its activity was considered as 100%.
Activity–% [inhibitor] plots were drawn for inhibitors.
From the resulting plots, IC50 values of each compound
Volume 30, Number 6, 2016
IC50 (μM) Ki (μM)
R2 hCA-II R2 hCA-I hCA-II
0.9200 134 0.9726 91.55 33.70
0.9005 251 0.9887 57.55 63.125
0.9219 79 0.9532 131.45 19.86
0.9734 604 0.9281 177.22 151.90
36.2 0.37
(H1, H2, H3, and H4) were 140, 88, 201, and 271 μM for
hCA-I and 134, 251, 79, and 604 μM for hCA-II, respec-
tively (Table 1). Ki constants were calculated according
to the Cheng–Prusoff equation [74]. From the resulting
plots, Ki values of each compound (H1, H2, H3, and
H4) were 91.55, 57.55, 131.45, and 177.22 μM for hCA-
I and 33.70, 63, 125, 19.86, and 151.90 μM for hCA-II,
respectively (Table 1).
DISCUSSION
In this study, four Schiff bases known in the lit-
erature were synthesized (Figures 1 and 3) and char-
acterized by various characterization techniques. The
effects of these Schiff bases on the activity of carbonic
anhydrase isoenzymes (I, II) were investigated under
in vitro conditions for the first time.
Regression analysis was performed to establish
whether theoretical and experimental 1 H NMR shifts
of the compounds fit each other or not. Accordingly, R2
values obtained from the regression analyses for H1,
H2, H3, and H4 were 0.3336, 0.6077, 0.3885, and 0.4996,
respectively. Calculated R2 values are very low. This
can be ascribed to the deviation between theoretical
1
H NMR values only found for –OH groups and ex-
perimental values. If these values (experimental and
theoretical NMR values of OH groups) are neglected,
R2 values for H1, H2, H3, and H4 will be 0.9781, 0.9814,
0.9758, and 0.8635, respectively. Such deviations arise
from acidic properties of phenolic OH groups, and
that theoretical calculations are made in the gas phase
by considering only one molecule. In that case, inter-
actions of molecules with solvent and among them-
selves are neglected. Moreover, differences between
experimental and theoretical NMR values for pro-
tons attached to electronegative atoms were previously
reported in the literature. Our results substantiate pre-
vious findings in the literature [61–67].
The synthesized compounds are believed to in-
hibit hCA-I and hCA-II enzymes by the interaction
of their hydroxyl groups with cofactor Zn2+ ions in
the structure of the enzymes [21, 77–79]. Previous
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 30, Number 6, 2016
theoretical studies demonstrated that Zn2+ incarbonic
anhydrase enzymes form chelates with hydroxyl, car-
boxyl groups of phenolic compounds and that these
groups (–OH and –COOH) form hydrogen bonds with
other parts of the enzyme, resulting in inhibition of en-
zymes [77, 80]. It is suggested that phenolic OH and
–CH=N groups in the structure of the compounds
synthesized in this study interacted with Zn2+ and
amino acids in the structure of human carbonic an-
hydrase enzymes and caused inhibition. The inhibition
mechanism/interaction between the compounds syn-
thesized in this study and the enzyme as thought to be
is shown in Figure 4, by also considering the literature
[21, 77, 78, 80].
According to %Activity/[Inhibitor] plots, IC50 val-
ues of each compound (H1, H2, H3, and H4) were 140,
88, 201, and 271 μM for hCA-I and 134, 251, 79, and
604 μM for hCA-II, respectively (Table 1). Ki constants
were calculated according to the Cheng–Prusoff equa-
tion [74]. From the resulting plots, Ki values of each
compound were 91.55, 57.55, 131.45, and 177.22 μM for
hCA-I and 33.70, 63.125, 19.86, and 151.90 μM for hCA-
II, respectively (Table 1). The compound H2 was found
to have the highest inhibitory effect on hCA-I enzyme
activity, whereas the compound H3 was found to have
the highest inhibitory effect on hCA-II enzyme activ-
ity. It is evident that the synthesized compounds have
different effects on CA isoenzymes. This is an expected
result because isoenzymes have different amino acid
sequence analyses but also catalyze the same biochem-
ical reactions. They also typically have different kinetic
parameters, such as IC50 values [78].
CA isoenzymes have become targets in drug de-
sign. So in the current study, we synthesized and char-
acterized by both experimental and theoretical appli-
cations the Schiff bases which are already available in
the literature, but the effects on CAs of which have not
been investigated yet. We investigated the effects of
the resulting compounds on hCA-I and hCA-II enzyme
activities.
REFERENCES
1. Supuran CT, Scozzafava A. Carbonic anhydrase in-
hibitors. Curr Med Chem Endocr Metab Agents
2001;1:61–97.
2. Nasr G, Cristian A, Barboiu M, Vullo D, Winum JY,
Supuran CT. Carbonic anhydrase inhibitors. Inhibition
of human cytosolic isoforms i and II with (reduced)
Schiff’s bases incorporating sulfonamide, carboxylate
and carboxymethyl moieties. Bioorganic Med Chem
2014;22:2867–2874.
3. Supuran CT. Carbonic anhydrases: from biomedical ap-
plications of the inhibitors and activators to biotechno-
logical use for CO2 capture. J Enzyme Inhib Med Chem
2013;28:229–230.
J Biochem Molecular Toxicology DOI 10.1002/jbt
INHIBITION OF HCAs AND A DFT STUDY 283
4. Supuran CT. Structure-based drug discovery of car-
bonic anhydrase inhibitors. J Enzyme Inhib Med Chem
2012;27:759–772.
5. Alterio V, Di Fiore A, D’Ambrosio K, Supuran CT, De
Simone G. Multiple binding modes of inhibitors to car-
bonic anhydrases: how to design specific drugs targeting
15 different isoforms?. Chem Rev 2012;112:4421–4468.
6. Thiry A, Dogné JM, Masereel B, Supuran CT. Targeting
tumor-associated carbonic anhydrase IX in cancer ther-
apy. Trends Pharmacol Sci 2006;27:566–573.
7. Carta F, Supuran CT. Diuretics with carbonic anhydrase
inhibitory action: a patent and literature review (2005–
2013). Expert Opin Ther Pat 2013;23:681–691.
8. Masini E, Carta F, Scozzafava A, Supuran CT. Antiglau-
coma carbonic anhydrase inhibitors: a patent review. Ex-
pert Opin Ther Pat 2013;23:705–716.
9. Scozzafava A, Supuran CT, Carta F. Antiobesity carbonic
anhydrase inhibitors: a literature and patent review. Ex-
pert Opin Ther Pat 2013;23:725–735.
10. Eroglu E. Some QSAR studies for a group of sulfonamide
schiff base as carbonic anhydrase CA II inhibitors. Int J
Mol Sci 2008;9:181–197.
11. Supuran CT, Scozzafava A. Carbonic anhydrases as
targets for medicinal chemistry. Bioorg Med Chem
2007;15:4336–4350.
12. Balseven H, İşgör M, Mert S, Alım Z, Beydemir S, Ok S,
Kasımoğullarıa R. Facile synthesis and characterization
of novel pyrazole-sulfonamides and their inhibition ef-
fects on human carbonic anhydrase isoenzymes. Bioorg
Med Chem 2013;21:21–27.
13. Becker B. The mechanism of the fall in intraocular pres-
sure induced by the carbonic anhydrase inhibitor. Di-
amox*. Am J Ophthalmol 1955;39:177–184.
14. Ganguly A, Chakraborty P, Banerjee K, Choudhuri SK.
The role of a Schiff base scaffold, N-(2-hydroxy acetophe-
none) glycinate-in overcoming multidrug resistance in
cancer. Eur J Pharm Sci 2014;51:96–109.
15. Durgun M, Turkmen H, Ceruso M, Supuran CT. Syn-
thesis of Schiff base derivatives of 4-(2-aminoethyl)-
benzenesulfonamide with inhibitory activity against car-
bonic anhydrase isoforms I, II, IX and XII. Bioorg Med
Chem Lett 2015;25:2377–2381.
16. Gust R, Ott I, Posselt D, Sommer K. Development of
cobalt(3,4-diarylsalen) complexes as tumor therapeutics
J Med Chem. 2004;47:5837–5846.
17. Lv J, Liu T, Cai S, Wang X, Liu L, Wang Y. Synthesis,
structure and biological activity of cobalt(II) and cop-
per(II) complexes of valine-derived schiff bases J Inorg
Biochem. 2006;100:1888–1896.
18. Puccetti L, Fasolis G, Vullo D, Chohan ZH, Scozzafava
A, Supuran CT. Carbonic anhydrase inhibitors. In-
hibition of cytosolic/tumor-associated carbonic anhy-
drase isozymes I, II, IX, and XII with Schiff’s bases in-
corporating chromone and aromatic sulfonamide moi-
eties, and their zinc complexes. Bioorg Med Chem Lett
2005;15:3096–3101.
19. Nasr G, Petit E, Supuran CT, Winum JY, Barboiu M. Car-
bonic anhydrase II-induced selection of inhibitors from
a dynamic combinatorial library of Schiff’s bases. Bioorg
Med Chem Lett 2009;19:6014–6017.
20. Supuran CT, Nicolae A, Popescu A. Carbonic anhy-
drase inhibitors. Part 35: Synthesis of Schiff bases de-
rived from sulfanilamide and aromatic aldehydes: the
first inhibitors with equally high affinity towards cytoso-
lic and membrane-bound isozymes. Eur J Med Chem
1996;31:431–438.
284 TEKTAS ET AL.
21. Supuran CT, Scozzafava A, Popescu A, Bobes-Tureac R,
Banciu A, Creanga A, Bobes-Tureac G, Banciu MD. Car-
bonic anhydrase inhibitors. Part 43: Schiff bases derived
from aromatic sulfonamides: towards more specific in-
hibitors for membrane-bound versus cytosolic isozymes.
Eur J Med Chem 1997;32:445–452.
22. Supuran CT, Clare BW. Carbonic anhydrase inhibitors,
Part 47: Quantum chemical quantitative structure-
activity relationships for a group of sulfanilamide Schiff
base inhibitors of carbonic anhydrase. Eur J Med Chem
1998;33:489–500.
23. Li L, Guo Q, Dong J, Xu T, Li J. DNA binding,
DNA cleavage and BSA interaction of a mixed-ligand
copper(II) complex with taurine Schiff base and 1,10-
phenanthroline. J Photochem Photobiol B 2013;125:56–62.
24. Anitha C, Sheela CD, Tharmaraj P, Johnson Raja S. Syn-
thesis and characterization of VO(II), Co(II), Ni(II), Cu(II)
and Zn(II) complexes of chromone based azo-linked
Schiff base ligand. Spectrochim Acta A Mol Biomol Spec-
trosc 2012;98:35–42.
25. Kursunlu AN, Guler E, Sevgi F, Ozkalp B. Synthesis,
spectroscopic characterization and antimicrobial studies
of Co(II), Ni(II), Cu(II) and Zn(II) complexes with Schiff
bases derived from 5-bromo-salicylaldehyde. J Mol Struct
2013;1048:476–481.
26. Cui Y, Dong X, Li Y, Li Z, Chen W. Synthesis, structures
and urease inhibition studies of Schiff base metal com-
plexes derived from 3,5-dibromosalicylaldehyde. Eur J
Med Chem 2012;58:323–331.
27. Ray A, Maity D, Pramanik A, Das KK, Nandi M, Bhaumik
A, Nethaji M, Mondal S, Mukherjee M, Ali M. Two highly
unsymmetrical tetradentate (N3 O) Schiff base copper(II)
complexes: template synthesis, structural characteriza-
tion, magnetic and computational studies. Polyhedron
2009;28:3659–3666.
28. Keypour H, Shayesteh M, Rezaeivala M, Chalabian F, El-
erman Y, Buyukgungor O. Synthesis, spectral characteri-
zation, structural investigation and antimicrobial studies
of mononuclear Cu(II), Ni(II), Co(II), Zn(II) and Cd(II)
complexes of a new potentially hexadentate N2 O4 Schiff
base ligand derived from salicylaldehyde. J Mol Struct
2013;1032:62–68.
29. Jayaseelan P, Akila E, Usha Rani M, Rajavel R. Syn-
thesis, spectral characterization, electrochemical, an-
timicrobial, DNA binding and cleavage studies of
new binuclear Schiff base metal(II) complexes derived
from o-hydroxyacetophenone. J Saudi Chem Soc DOI:
10.1016/j.jscs.2013.07.001 (In Press).
30. Taha Z, Ajlouni AM, Al Momani W, Al-Ghzawi A. Syn-
theses, characterization, biological activities and pho-
tophysical properties of lanthanides complexes with a
tetradentate Schiff base ligand. Spectrochim Acta A Mol
Biomol Spectrosc 2011;81:570–577.
31. Agrawal YK, Talati JD, Shah MD, Desai MN, Shah NK.
Schiff bases of ethylene diamine as corrosion inhibitors
of zinc in sulphuric acid. Corros Sci 2004;46:633–651.
32. Desai MN, Desai MB, Shah CB, Desai SM. Schiff bases
as corrosion inhibitors for mild steel in hydrochloric acid
solutions. Corros Sci 1986;26:827–837.
33. Sarkar S, Biswas S, Liao MS, Kar T, Aydogdu Y, Dagde-
len F, Mostafa G, Chattopadhyay AP, Yap GPA, Xie
RH, Khan AT, Dey K. An attempt towards coordination
supramolecularity from Mn(II), Ni(II) and Cd(II) with a
new hexadentate [N4 O2 ] symmetrical Schiff base ligand:
syntheses, crystal structures, electrical conductivity and
optical properties. Polyhedron 2008;27:3359–3370.
Volume 30, Number 6, 2016
34. Yıldız M, Karpuz Ö, Zeyrek CT, Boyacıoğlu B, Dal H,
Demir N, Yıldırım N, Ünver H. Synthesis, biological ac-
tivity, DNA binding and anion sensors, molecular struc-
ture and quantum chemical studies of a novel bidentate
Schiff base derived from 3,5-bis(triflouromethyl)aniline
and salicylaldehyde. J Mol Struct 2015;1094:148–160.
35. Kryachko ES, Ludeña EV. Density functional theory:
foundations reviewed. Phys Rep 2014;544:123–239.
36. Kohn W, Sham LJ. Self-consistent equations including ex-
change and correlation effects. Phys Rev 1965;140:A1133–
A1138.
37. Hohenberg P. Inhomogeneous electron gas. Phys Rev
1964;136:B864–B871.
38. Lee C, Yang W, Parr RG. Development of the Colle-
Salvetti correlation-energy formula into a functional of
the electron density. Phys Rev B 1988;37:785–789.
39. Miehlich B, Savin A, Stoll H, Preuss H. Results obtained
with the correlation energy density functionals of becke
and Lee, Yang and Parr. Chem Phys Lett 1989;157:200–
206.
40. Stephens PJ, Devlin FJ, Chabalowski CF, Frisch MJ. Ab
initio calculation of vibrational absorption and circular
dichroism spectra using density functional force fields. J
Phys Chem 1994;98:11623–11627.
41. Becke AD. Density-functional thermochemistry, III: The
role of exact exchange. J Chem Phys 1993;98:5648.
42. Tan X-J, Hao X-Q, Zhao Q-Z, Cheng S-S, Xie W-L, Xing
D-X, Liu Y, Song LZ. Mono-Schiff-base or di-Schiff-base?
Synthesis, spectroscopic, X-ray structural and DFT study
of a series of Schiff-bases derived from benzil dihydra-
zone. J Mol Struct 2015;1099:373–387.
43. de Toledo TA, Pizani PS, da Silva LE, Teixeira AMR,
Freire PTC. Spectroscopy studies on Schiff base N,N -
bis(salicylidene)-1,2-phenylenediamine by NMR, in-
frared, Raman and DFT calculations. J Mol Struct
2015;1097:106–111.
44. Tyagi P, Chandra S, Saraswat BS, Yadav D. Design, spec-
tral characterization, thermal, DFT studies and anticancer
cell line activities of Co(II), Ni(II) and Cu(II) complexes
of Schiff bases derived from 4-amino-5-(pyridin-4-yl)-
4H-1,2,4-triazole-3-thiol. Spectrochim Acta A Mol Biomol
Spectrosc 2015;145:155–164.
45. Balakrishnan C, Subha L, Neelakantan MA, Mariappan
SS. Synthesis, spectroscopy, X-ray crystallography, DFT
calculations, DNA binding and molecular docking of a
propargyl arms containing Schiff base. Spectrochim Acta
A Mol Biomol Spectrosc 2015;150:671–681.
46. Anbuselvan C, Jayabharathi J, Thanikachalam V, Tamil-
selvi G. Physico-chemical studies on some fluorescence
sensors: DFT based ESIPT process. Spectrochim Acta A
Mol Biomol Spectrosc 2012;97:125–130.
47. İskeleli NO, Alpaslan YB, Direkel Ş, Ertürk AG,
Süleymanoğlu N, Ustabaş R. The new Schiff base
4-[(4-hydroxy-3-fluoro-5-methoxy-benzylidene)amino]-
1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one:
experimental, DFT calculational studies and in vitro
antimicrobial activity. Spectrochim Acta A Mol Biomol
Spectrosc 2015;139:356–366.
48. Jin RY, Sun XH, Liu YF, Long W, Lu WT, Ma HX.
Synthesis, crystal structure, IR, 1H NMR and theoreti-
cal calculations of 1,2,4-triazole Schiff base. J Mol Struct
2014;1062:13–20.
49. Nagapandiselvi P, Baby C, Gopalakrishnan R. A
new Schiff base, (E)-4-((4-chlorophenylimino)methyl)-
2-methoxyphenol: crystal structure, thermal behavior,
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 30, Number 6, 2016 INHIBITION OF HCAs AND A DFT STUDY 285

solid-state fluorescence, DFT calculations and FT NMR new 3-alkyl-4-(p-methoxybenzoylamino)-4,5-dihydro-

spectral analysis. J Mol Struct 2014;1056(1057):110–120. 1H-1,2,4-triazol-5-ones. J Mol Struct 2008;873:142–148.
50. Lu W, Du G, Liu K, Jiang L, Ling J, Shen Z. Chiroptical 63. Ilhan S, Baykara H, Seyitoglu MS, Levent A, Özdemir S,
inversion induced by rotation of a carbon-carbon sin- Dündar A, Öztomsuk A, Cornejo MH. Preparation, spec-
gle bond: an experimental and theoretical study. J Phys tral studies, theoretical, electrochemical and antibacterial
Chem A 2014;118:283–292. investigation of a new Schiff base and its some metal
51. Chamayou A-C, Lüdeke S, Brecht V, Freedman TB, Nafie complexes. J Mol Struct 2014;1075:32–42.
LA, Janiak C. Chirality and diastereoselection of /- 64. Baykara H, Ilhan S, Levent A, Seyitoglu MS, Özdemir S,
configured tetrahedral zinc complexes through enan-
Okumuş V, Öztomsuk A, Cornejo M. Synthesis, charac-
tiopure Schiff base complexes: combined vibrational cir-
terization and experimental, theoretical, electrochemical,
cular dichroism, density functional theory, 1 H NMR,
antioxidant and antibacterial study of a new Schiff base
and X-ray structural studies. Inorg Chem 2011;50:11363–
and its complexes. Spectrochim Acta A Mol Biomol Spec-
11374.
trosc 2014;130:270–279.
52. Brunner H, Zwack T, Zabel M, Beck W, Böhm A.
65. Yüksek H, Cakmak I, Sadi S, Alkan M, Baykara H.
Optically active transition metal complexes, 130.1 Syn-
Synthesis and GIAO NMR calculations for some novel 4-
thesis, crystal structures, and catalytic properties of
heteroarylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-one
chiral-at-metal (η 6 -arene)ruthenium(ii) and (η 6 -
derivatives: comparison of theoretical and experimental
arene)osmium(ii) half-sandwich complexes. Crystal- 1
H and 13 C- chemical shifts. Int J Mol Sci 2005;6:219–229.
lization of pure diastereomers versus diastereomer.
66. Baykara H, Ilhan S, Oztomsuk A, Seyitoglu MS, Levent
Organometallics 2003;22:1741–1750.
A, Okumus V, Dündar A. Synthesis and characterization
53. Smith HE, Ensley HE. Optically active amines, Part XIII:
of a new difunctional ligand and its metal complexes: an
Circular dichroism of the N-salicylidene derivatives of
experimental, theoretical, cyclic voltammetric, and an-
alkylamines. Can J Chem 1971;49:2902–2906.
timicrobial study. Synth React Inorg, Met Nano-Metal
54. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E.,
Chem 2015;45(12):1795–1807.
Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone V,
67. Ilhan S, Baykara H, Oztomsuk A, Okumus V, Levent A,
Mennucci B, Petersson GA, Nakatsuji H, Caricato M,
Seyitoglu MS, Ozdemir S. Synthesis and characteriza-
Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G,
tion of 1,2-bis(2-(5-bromo-2-hydroxybenzilidenamino)-
Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R,
4-chlorophenoxy)ethane and its metal complexes: an ex-
Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O,
perimental, theoretical, electrochemical, antioxidant and
Nakai H, Vreven T, Montgomery JA, Jr, Peralta JE, Ogliaro
antibacterial study. Spectrochim Acta A Mol Biomol
F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov
Spectrosc 2014;118:632–42.
VN, Kobayashi R, Normand J, Raghavachari K, Rendell
68. Coban TA, Beydemir S, Gülçin I, Ekinci D. The effect of
A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Mil-
ethanol on erythrocyte carbonic anhydrase isoenzymes
lam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo
activity: an in vitro and in vivo study. J Enzyme Inhib
C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O.,
Med Chem 2008;23:266–270.
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin
69. Beydemir S, Gülçin I. Effects of melatonin on carbonic
RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P,
anhydrase from human erythrocytes in vitro and from
Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö, Fores-
rat erythrocytes in vivo. J Enzyme Inhib Med Chem
man JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09W,
2004;19:193–197.
Revision C.01, Wallingford, CT: Gaussian, Inc.; 2010.
70. Hisar O, Hisar Ş̧; Küfrevioǧlu Ö̈I, Yanik T. Inhibitory ef-
55. McWeeny R. Perturbation theory for the fock-dirac den-
fects of some antibiotics on activity of carbonic anhydrase
sity matrix. Phys Rev 1962;126:1028–1034.
from rainbow trout erythrocytes in vitro and in vivo. J
56. Wolinski K, Hinton JF, Pulay P. Efficient implementa-
Aquat Anim Health 2003;15:221–228.
tion of the gauge-independent atomic orbital method
71. Verpoorte JA, Mehta S, Edsall JT. Esterase activities
for NMR chemical shift calculations. J Am Chem Soc
of human carbonic anhydrases B and C. J Biol Chem
1990;112:8251–8260.
1967;242:4221–4229.
57. Ditchfield R. Self-consistent perturbation theory of dia-
72. Laemmli UK. Cleavage of structural proteins during
magnetism, I: A gauge-invariant LCAO method for
the assembly of the head of bacteriophage T4. Nature
N.M.R. chemical shifts. Mol Phys 1974;27:789–807.
1970;227:680–685.
58. Cheeseman JR, Trucks GW, Keith TA, Frisch MJ. A com-
73. Bradford MM. A rapid and sensitive method for the
parison of models for calculating nuclear magnetic reso-
quantitation of microgram quantities of protein utiliz-
nance shielding tensors. J Chem Phys 1996;104:5497.
ing the principle of protein-dye binding. Anal Biochem
59. Frisch Æ, Hratchian H.P, Dennington R.D II, T.A. Keith
1976;72:248–54.
JM. GaussView 5 reference. Wallingford, CT: Gaussian;
74. Cheng Y, Prusoff WH. Relationship between the inhi-
2009.
bition constant (K1 ) and the concentration of inhibitor
60. London F. Théorie quantique des courants inter-
which causes 50 per cent inhibition (I50 ) of an enzymatic
atomiques dans les combinaisons aromatiques. J Phys
reaction. Biochem Pharmacol 1973;22:3099–108.
Radium 1937;8:397–409.
75. Güney M, Çavdar H, Şentürk M, Ekinci D: Synthesis and
61. Cakmak I. GIAO calculations of chemical shifts in enan-
carbonic anhydrase inhibitory properties of novel uracil
tiometrically pure 1-trifluoromethyl tetrahydroisoquino-
derivatives. Bioorg Med Chem Lett 2015;25:3261–3263.
line alkaloids. J Mol Struct THEOCHEM. 2005;716:143–
76. Keller LBS and EB. Labile Acetylated uracil derivatives.
148.
J Biol Chem 1958;232:185–192.
62. Yüksek H, Alkan M, Bahçeci Ş, Cakmak I, Ocak Z,
77. Durdagi S, Senturk M, Ekinci D, Balaydin HT, Göksu S,
Baykara H, Aktaş O, Ağyel E. Synthesis, determination
of pKa values and GIAO NMR calculations of some Küfreviolu ÖI, Innocenti A, Scozzafava A, Supuran CT.
Kinetic and docking studies of phenol-based inhibitors of

J Biochem Molecular Toxicology DOI 10.1002/jbt

286 TEKTAS ET AL.
carbonic anhydrase isoforms I, II, IX and XII evidence a
new binding mode within the enzyme active site. Bioorg
Med Chem 2011;19:1381–1389.
78. Yıldırım A, Atmaca U, Keskin A, Topal M, Çelik M,
Gülçin İ, Supuran CT. N-Acylsulfonamides strongly in-
hibit human carbonic anhydrase isoenzymes I and II.
Bioorg Med Chem 2015;23:2598–605.
Volume 30, Number 6, 2016
79. Preisig PA, Toto RD, Alpern RJ. Carbonic anhydrase in-
hibitors. Ren Physiol 1987;10:136–159.
80. Huang H, Pan X, Ji C, Zeng G, Jiang L, Fu X, Liu
J, Hao X, Zhang Y, Tan N. Screening and dock-
ing studies of natural phenolic inhibitors of carbonic
anhydrase II. Sci China, Ser B Chem 2009;52:332–
337.
J Biochem Molecular Toxicology DOI 10.1002/jbt