Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: Novel sulfamoylcarbamates and sulfamides derived from acetophenones

Article abstract of DOI:10.1016/j.bmc.2015.04.019

Abstract In this study, several novel sulfamides were synthesized and evaluated for their acetylcholine esterase (AChE) and human carbonic anhydrase I, and II isoenzymes (hCA I and II) inhibition profiles. Reductive amination of methoxyacetophenones was u

Full text of DOI:10.1016/j.bmc.2015.04.019

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent carbonic anhydrase and acetylcholine esterase
inhibitors: Novel sulfamoylcarbamates and sulfamides derived from
acetophenones
Akın Akıncıog˘lu a,b, Hülya Akıncıog˘lu a,b, Ilhami Gülçin b,c, Serdar Durdagi d, Claudiu T. Supuran e,
_
Süleyman Göksu b,
⇑
a
_
Ag˘rı Ibrahim Çeçen University, Central Researching Laboratory, Ag˘rı, Turkey
b Atatürk University, Faculty of Science, Department of Chemistry, Erzurum, Turkey
c Fetal Programming of Diseases Research Chair, Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
d
_
Department of Biophysics, School of Medicine, Bahçeßsehir University, Istanbul, Turkey
e Università degli Studi di Firenze, Neurofarba dept., Via U Schiff 6, I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, several novel sulfamides were synthesized and evaluated for their acetylcholine esterase
(AChE) and human carbonic anhydrase I, and II isoenzymes (hCA I and II) inhibition profiles. Reductive
amination of methoxyacetophenones was used for the synthesis of amines. Amines were converted to
sulfamoylcarbamates with chlorosulfonyl isocyanate (CSI) in the presence of BnOH. Pd-C catalyzed
hydrogenolysis of sulfamoylcarbamates afforded sulfamides. These novel compounds were good inhibi-
tors of the cytosolic hCA I, and hCA II with Ki values in the range of 45.9 8.9–687.5 84.3 pM for hCA I,
and 48.80 8.2–672.2 71.9 pM for hCA II. The inhibitory effects of the synthesized novel compounds on
AChE were also investigated. The Ki values of these compounds were in the range of 4.52 0.61–
38.28 6.84 pM for AChE. These results show that hCA I, II, and AChE were effectively inhibited by the
novel sulfamoylcarbamates 17–21 and sulfamide derivatives 22–26. All investigated compounds were
docked within the active sites of the corresponding enzymes revealing the reasons of the effective inhi-
bitory activity.
Received 11 March 2015
Revised 7 April 2015
Accepted 8 April 2015
Available online xxxx
Keywords:
Acetylcholine esterase
Carbonic anhydrase
Enzyme inhibition
Hybrid molecules
Sulfamide
Sulfamoylcarbamate
Molecular docking
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
dementia of Alzheimer’s17 and Parkinson’s diseases18 (Fig. 1).
Rivastigmine (6) is cholinergic drug that inhibits AChE in an irre-
Sulfamides are frequently studied organic compounds and they
exhibit a wide range of biological activities for diverse targets.1 The
following drugs can be given as examples of sulfamide analogues:
Doripenem (1)2 an antibiotic; Quinagolide (2)3 an anti-hyperpro-
lactinemic agent; JNJ-26990990 (3)4 and JNJ-26489112 (4)5 anti-
convulsants. (Fig. 1) It has been reported that sulfamide analogue
5 shows multifunctional properties (i.e., LDL receptor agonist pro-
files,6 HCV replication inhibitory effect,7 hypoglycemic8 and
antibacterial9 activities) (Fig. 1). Sulfamide compounds are
reported in the literature for different therapeutic actions, such
as HIV-1 protease inhibition,10 b-secretase activity,11 protein tyro-
sine kinase inhibitory,12 endothelin receptor antagonists activity,13
anti-trypanosomal,14 AChE inhibitory15 and CA inhibitory proper-
ties.16 On the other hand, Rivastigmine (Exelon, 6) is a well known
drug used in the treatment of neurodegenerative disorders such as
versible manner. For this reason, Rivastigmine has central nervous
system selectivity over peripheral inhibition.19
CAs are metalloenzymes expressed in both prokaryotes and
eukaryotes that catalyze the reversible conversion of carbon diox-
ide (CO2) to bicarbonate ions (HCOꢀ3 ) and protons (H+).20–23 CO2 is
the end product of aerobic metabolism. In mammals, CO2 passes
into the blood by secretion after transported to the lungs. CO2
remains in erythrocytes for a while. During this time it reacts with
H2O. The product of this reaction is the carbonic acid (H2CO3).
H2CO3 then loses an H+ and it produces HCOꢀ3 .24,25
So far, six genetic families (
CAs were reported.24 The mammalian carbonic anhydrase enzymes
belong to the -CA family and consist of sixteen active members
that have different kinetic parameters, inhibitory properties, func-
tions, and localization. -CA family divides into sixteen isoforms
a-, b-, c-, d-, f-, and g-CAs) encoding
a
a
including cytosolic (CA I, II, III, VII, and XIII), membrane-bound
(CA IV, IX, XII, XIV, and XV), mitochondrial (CA VA and VB), and
secreted (CA VI) forms. Three cytosolic forms, the CA related
⇑
Corresponding author. Tel.: +90 442 2314389; fax: +90 442 2314109.
E-mail address: sgoksu@atauni.edu.tr (S. Göksu).
http://dx.doi.org/10.1016/j.bmc.2015.04.019
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
OH
O
N
O
O
S
O
H
N
H
N
S
NH2
O
S
O
O
S
O
H
N
N
NH2
H
H
N
H
HO
N
H
H
OH
S
1
3
2
O
HN
S
O
NH2
O
O
N
O
N
H
S NH2
O
N
O
Cl
O
4
5
6
Figure 1. Some selected drugs 1–4, 6 and biologically active sulfamides 5.
proteins (CARP) (CA VIII, X and XI) have also been identified, but
they are acatalytic.26–29 These CA isoenzymes are targets for many
diseases such as glaucoma, cancer, infertility, epilepsy, neurode-
generation, and obesity.
in cortical cholinergic activity and AD patients was found.37
Accordingly, the increasing of cholinergic neurotransmission
has been considered as one potential therapeutic approach
against AD. Although the pathogenesis of AD is obstructed and
involves numerous pathways, two major hypotheses are cur-
rently under consideration regarding the molecular mechanism:
the cholinergic hypothesis and the amyloid cascade hypothe-
sis.38,39 Thus, the main focus in this study is to find selective
AChE inhibition in order to relieve cholinergic deficits and
improve neurotransmission. Donepezil, Rivastigmine and
Galantamine are the most extensively studied AChE inhibitors
(AChEIs). It was demonstrated that these AChEIs significantly
improve cognitive function in AD.40 In addition to these drugs;
alternative and complementary drugs are also required to be
developed.
Together with sulfonamides and their isoesters, there are many
other classes of CA inhibitors (CAIs) such as the dithiocarbamates,
xanthates, coumarins, phenolics and polyamines. Sulfonamide
derivatives are specific and potent CAIs and still attract much
interest due to their pharmacological properties, facility of prepa-
ration, stability and ease of administration.30–32 Recently, the inhi-
bition of physiologically dominant isoenzymes hCA I and II was
extensively studied.33–35 It is well known that CAIs have some mul-
ti-functional applications as diuretics, antiepileptics, tumor diag-
nostic tools, or for the treatment of several other neurological
minor disorders.27
Alzheimer’s disease (AD) is the most common neurodegener-
ative disorder and it is the most frequent and predominant cause
of dementia among the elderly people.36 Reduction of AChE
levels in the brain is the most considerable, biochemical change
in AD.15 Neuropathological argument has showed that choliner-
gic functions decrease in the basal forebrain and cortex in senile
dementia of the AD. A significant correlation between decrease
As sulfamides exhibit important biological activities, CA and
AChE inhibitory properties of hybrid molecules structurally related
to the parasympathomimetic drug Rivastigmine (6) might be use-
ful for further synthetic and biological applications. In that respect,
we report here in the CA and AChE inhibitory properties of a series
of sulfamides and sulfamoylcarbamates, which are hybrid mole-
cules of Rivastigmine (6).
R1
R4
R1
R4
R1
R2
R3
R2
R3
O
S
O
O
O
NH2
H
N
R2
R3
i
ii
N
H
O
Bn
R5
R5
R5
R4
7 R1=OMe, R2=R3=R4=R5= H
8 R3=OMe, R1=R2=R4=R5= H
12 R1=OMe, R2=R3=R4=R5= H 72%
13 R3=OMe, R1=R2=R4=R5= H 69%
17 R1=OMe, R2=R3=R4=R5= H 71%
18 R3=OMe, R1=R2=R4=R5= H 68%
9
14
19
R2=R3=OMe, R1=R4=R5= H 70%
R2=R3=OMe, R1=R4=R5= H
R2=R3=OMe, R1=R4=R5= H 75%
10 R1=R4=OMe, R2=R3=R5= H
11 R1=R5=OMe, R2=R3=R4= H
15 R1=R4=OMe, R2=R3=R5= H 75%
16 R1=R5=OMe, R2=R3=R4= H 67%
20 R1=R4=OMe, R2=R3=R5= H 67%
21 R1=R5=OMe, R2=R3=R4= H 72%
iii
R1
O
R2
N
H
S
O
NH2
R3
R5
R4
22 R1=OMe, R2=R3=R4=R5= H 95%
23 R3=OMe, R1=R2=R4=R5= H 96%
24
R2=R3=OMe, R1=R4=R5= H 96%
25 R1=R4=OMe, R2=R3=R5= H 92%
26 R1=R5=OMe, R2=R3=R4= H 90%
Scheme 1. Synthesis of sulfamides. Reagents and conditions: (i) NH4OAc/Zn/NaBCNH3/NH3(aq), EtOH, 25–80 °C 36 h; (ii) CSI/NEt3/BnOH, CH2Cl2 0–25 °C, 5 h; (iii) H2/Pd-C,
MeOH, 25 °C, 4 h.
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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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studied the inhibition effects of carbamates and sulfamoylcarba-
mate derivatives on hCA I, and II isoenzymes. The inhibition stud-
ies were performed with esterase activity method.58–60 On the
basis of this method, in general, the phenyl acetate is hydrolyzed
and the resulting product gives the absorbance at 348 nm.61
2. Results and discussion
2.1. Chemistry
Reductive amination of ketones is one of the most convenient
methods for the preparation of amines.41 The reductive amination
of aldehydes with NaCNBH3 in the presence of Zn and NH4OAc/NH3
has been reported by Dangerfield et al.42 This method was applied
to acetophenones 7–11 to give amines 12–16. For this purpose,
ketones 7–11 were converted to related imines with NH4OAc/
NH3 and followed by the in situ reduction of these imines with
NaCNBH3 in the presence of activated Zn in EtOH at 80 °C to give
amines 12,43 13,44 14,45 1546 and 1647 in moderate yields. In our
earlier studies, we have synthesized a series of sulfamoylcarba-
mates from the reactions of amines and benzyl alcohols (BnOH)/
tert-Butanol (tert-BuOH) with chlorosulfonyl isocyanate (CSI) in
the presence of Et3N. In these studies, we also addressed the syn-
thesis of sulfamides via Pd-C catalyzed hydrogenolysis of the ben-
zyl sulfamoylcarbamates48–50 or deprotection of Boc group of tert-
butyl sulfamoylcarbamates with CF3CO2H.51 We knew from these
studies that the deprotection of Boc group of tert-butyl sulfamoyl-
carbamates derived from benzyl amines led to elimination reac-
tions. Therefore, BnOH was chosen for the synthesis of
carbamates. The reaction of amines 12–16 and BnOH with CSI in
the presence of Et3N at 0–25 °C for 5 h furnished new benzyl sul-
famoylcarbamates 17–21 in good yields. Pd-C catalyzed
hydrogenolysis of benzyl carbamates gives amines.52,53 Hence, car-
bamates 17–21 were hydrogenated with H2 in the presence of Pd-C
catalyzed at room temperature for 4 h to yield a novel series of sul-
famides 22–26 in high yields (Scheme 1). The structures of all new
compounds were determined by spectroscopic techniques such as
1H, 13C NMR, IR, and elemental analysis.
2.3. Inhibition studies
Sulfamide and its derivatives continue to attract attention, as
many enzyme inhibitors containing sulfamide derivatives exhibit
a wide spectrum of activities16 Recently the CA isoenzymes
attracted this type of interest for the design of activators or inhibi-
tors with biomedical and pharmaceutical applications. For this
purpose we measured effects of sulfamide and sulfamoylcarba-
mate derivatives against hCA I, and II. We used the esterase activity
method, for determining the inhibition parameters by the
Lineweaver–Burk method.62 CA activity (%) versus inhibitory con-
centration and 1/V versus 1/[S] graphs were drawn. Then IC50
and Ki values were calculated from these graphs (Table 1). Thus,
we report the inhibition effects of sulfamoylcarbamates 17–21
and sulfamide derivatives 22–26 on the esterase activity of hCA I,
II, and AChE under the in vitro conditions. The results were pre-
sented in Table 1.
The following results were observed for the inhibition of the CA
isoforms with sulfamoylcarbamates 17–21 and sulfamide deriva-
tives 22–26:
(i) The slow cytosolic isoform hCA I was inhibited by the inves-
tigated sulfamoylcarbamates 17–21 and sulfamide deriva-
tives 22–26 which with Ki values ranging between
45.9 8.9 and 672.2 71.9 pM. The standard drugs acetazo-
lamide and dorzolamide had Ki values of 61.1 0.02 nM and
1.68 0.35 lM, respectively. Thus, the investigated com-
pounds showed better inhibitory profiles compared to these
2.2. CA purification and activity assay
clinically used CA inhibitors.
(ii) The hCA II was also efficiently inhibited by sulfamoylcarba-
mates 17–21 and sulfamide derivatives 22–26 investigated
here. Compounds 17–21 and 22–26 appeared to strongly
inhibit hCA II with Ki values ranging from 48.8 8.2 to
156.1 73.0 pM and 136.8 28.6 to 672.2 71.9 pM, respec-
tively. These values are better than those of the clinically
used drugs Acetazolamide (Ki of 184.3 0.03 nM) and dorzo-
hCA I, and II isoenzymes were purified by sepharose-4B-L-thy-
rosine-sulfanilamide affinity column chromatography.54,55 The
purity of both isoenzymes being eluted from the column was
detected by sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS–PAGE).56,57 After completion of this step, we then
lamide (Ki of 0.025 0.01 lM). The investigated compounds
(17–26) demonstrated marked inhibition against hCA I, and
II, in the picomolar range.
Table 1
Acetylcholine esterase (AChE) enzyme and human carbonic anhydrase isoenzymes
(hCA I, and II) inhibition value with sulfamoylcarbamates (17–21) and sulfamide
derivatives (22–26)
(iii) AD can be treated by using of pharmaceuticals, which
restore the level of acetylcholine (ACh) through AChE inhibi-
tion.63 Recently, several cholinergic drugs have been initi-
ated in the market, including the synthetic compounds
such as Galantamine,64 Donepezil,65 Tacrine,66 and
Rivastigmine.67 It is well known that usage of these drugs
produced significant cognitive improvement.37 However,
these drugs have undesirable side effects such as gastroin-
testinal disturbances and hepatotoxicity.37,68,69 Some stud-
ies of the AChE inhibitor complexes by X-ray
crystallography techniques indicated that AChE had two
separate ligand binding sites including a peripheral anionic
and an acylation sites. Lately, some biochemical studies have
demonstrated that the peripheral anionic site is also impli-
cated in promoting aggregation of the b-amyloid peptide
responsible for the neurodegenerative effects in AD.70
Therefore, the designing of new inhibitors, which interacts
with the peripheral anionic site poses extremely important
for the treatment of AD.37 The inhibitory effects of the newly
synthesized sulfamoylcarbamates 17–21 and sulfamide
derivatives 22–26 on AChE are shown in Table 1. It was
Compounds
Ki (pM)
hCA II
hCA I
AChE
17*
127.0 48.0
127.1 25.0
48.3 9.0
139.7 15.7
45.9 8.9
274.7 81.7
479.4 73.5
657.1 82.8
564.5 79.3
687.5 84.3
61.1 0.018
1.686 0.35
—
60.3 10.0
156.1 73.0
88.8 9.3
5.6 0.44
4.5 0.61
5.1 1.04
22.9 9.72
10.5 1.96
38.2 6.84
6.3 1.68
4.9 0.56
5.6 0.58
8.1 3.22
—
18*
19*
20*
48.8 8.2
21*
105.1 14.9
192.7 32.5
672.2 71.9
453.9 43.0
383.8 39.2
136.8 28.6
184.3 0.03
0.025 0.011
—
22**
23**
24**
25**
26**
W
,h
AZA
DZA
W
,/
—
u
,/
GLT
0.25 0.21
*
Sulfamoylcarbamates (17–21).
Sulfamide derivatives (22–26).
Acetazolamide (AZA) and dorzolamide (DZA) were used as a standard inhibitor
**
W
for all CAs.
u
Galantamine (GLT) was used as a standard inhibitor for all AChE.
The results were expressed as nanomolar (nM).
The results were expressed as micromolar (lM).
h
/
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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
reported previously that the presence of heterocyclic frag-
ments lead more inhibition compared to the phenyl group.37
The AChE inhibition profile with the compounds investi-
gated here was quite interesting. Overall, the synthesized
sulfamoylcarbamates 17–21 and sulfamide derivatives 22–
26 showed excellent inhibitory activity with IC50 and Ki val-
ues ranging from 26.85 to 57.61 pM and from 4.52 0.61 to
38.28 6.84 pM. Galantamine (GLT) is a standard inhibitor
construct p–p stacking interactions, Tyr121 and water molecules
at the binding cavity form hydrogen bonds with the ligand.
Phe330, Trp84, Ile439, Phe290, Phe331, and Ile287 amino acid resi-
dues form main hydrophobic interactions with the compound 18.
3. Conclusion
In conclusion, starting from acetophenones five novel benzyl
sulfamoylcarbamates and their sulfamide analogues were synthe-
sized in good yields. In our study, we have combined the synthesis,
in silico studies and biological evaluation of newly synthesized sul-
famoylcarbamates 17–21 and sulfamide derivatives 22–26 as hCA
I, II, and AChE inhibitors. The in vitro results of these compounds
both on CAs and AChE targets showed very strong inhibition (pico-
molar level) profiles. Thus these new compounds investigated here
may be considered in generating potent hCA I, and II inhibitors for
the treatment of glaucoma, epilepsy, idiopathic intracranial hyper-
tension, and altitude sickness. Also, these compounds possessed
excellent AChE inhibition, which is the mainly used target for
treatment of AD.
for AChE has Ki value of 0.253 0.21 lM. Some novel sul-
famides and sulfonamides tested on AChE at the literature
with Ki values in the range of 56.04 0.02 to
537.3 49.1 l
M.15 Similarly, five monoterpene indole alka-
loids were evaluated for their inhibition of AChE activities,
and showed moderate activities, with IC50 values in the
range of 4.1–10.3
tuted thiosemicarbazides and thiadiazoles demonstrated Ki
values in the range of 0.32–24.75 M. As can be seen in
l
M.71 In a recent study, some disubsti-
l
these results, the inhibition of AChE of the synthesized sul-
famoylcarbamates 17–21 and sulfamide derivatives 22–26
is much better than that of standard drug and newly synthe-
sized or purified compounds.
4. Experimental section
4.1. General information
2.4. In silico docking studies
All investigated molecules are docked at the binding sites of
hCA I, II, and AChE targets. Maestro’s Induced Fit Docking (IFD)
module is used for all docking simulations. IFD is a novel method
for fast and accurate prediction of conformational changes of small
molecules at the active site of the protein. Table 2 shows top-dock-
ing scores of each ligand at CA and AChE targets. Docking scores
showed a good agreement with in vitro results. Figures 2–4 show
top-docking poses and corresponding 2D ligand interaction dia-
grams of potent compounds at the screened enzymes. Figure 2
shows top-docking pose of compound 21 at the hCA I isoenzyme.
While His67, His94 and His119 form polar interactions; Pro202,
Ala135, Ala132, Leu131, Phe91, Leu198, Ala121, Val207, Val143
and Trp209 residues construct hydrophobic interactions with the
ligand. Gln92 and a water molecule near ligand form hydrogen
bond interactions with the compound 21. Figure 3 shows top-
docking pose of compound 20 at the hCA II target. While polar
interactions are formed by Thr199, Thr200, His119, His94 and
Gln92, hydrophobic interactions are mainly formed by Leu198,
Val143, Val121, Leu141, Trp209, Ala142, Val207, Pro202, Ile91,
Phe131 and Val135 amino acid residues. A few hydrogen bonds
are formed between compound 20 and water molecules at the
binding cavity. In addition, Gln92 forms hydrogen-bonding inter-
actions. Figure 4 shows top-docking pose of compound 18 at the
binding site of the AChE. While Phe330 and Trp84 residues
All chemicals and solvents are commercially available and were
used without purification or after distillation and treatment with
drying agents. Melting points are uncorrected and they were deter-
mined on a capillary melting apparatus (BUCHI 530). IR spectra
were obtained from solutions in 0.1 mm cells with a Perkin–
Elmer spectrophotometer. The 1H and 13C NMR spectra were
recorded on a 400 (100)-MHz Varian and 400 (100)-MHz Bruker
spectrometer;
d in ppm, Me4Si as the internal standard.
Elemental analyses were performed on a Leco CHNS-932 appara-
tus. 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. All biologically
evaluated compounds were demonstrated to exist in >95% purity
by elemental analysis.
4.2. Chemistry
4.2.1. General procedure for the synthesis of amines via
reductive amination of acetophenones
To a saturated solution of NH4OAc in EtOH (40 mL) were
added activated Zn (5 equiv), acetophenone (1 equiv), NaBCNH3
(3 equiv) and 30% aq NH3 (10 mL) respectively. The mixture was
stirred at 80 °C for 36 h. The reaction mixture was cooled to rt
and concentrated under reduced pressure. The residue was dis-
solved in CH2Cl2 and made basic using 1 M NaOH (50 mL). The
organic phase was separated and aqua phase was extracted with
(2x25 mL) CH2Cl2. The organic phases were combined and acidi-
fied with HCl (pH: 2.0). The organic layer was separated and
H2O layer was extracted with CH2Cl2 (2 ꢁ 25 mL). The H2O layer
was made basic with NaOH (pH: 10.0), The organic layer was
extracted with CH2Cl2 (3 ꢁ 25 mL). Combined organic layers were
dried over Na2SO4 and evaporation of the solvent afforded the
desired amines.
Table 2
Induced-Fit Docking (IFD) is performed for sulfamoylcarbamates (17–21) and
sulfamide derivatives (22–26)
Compounds Docking score for
hCA I (kcal/mol)
Docking score for
hCA II (kcal/mol)
Docking score for
AChE (kcal/mol)
17
18
19
20
21
22
23
24
25
26
ꢀ8.91
ꢀ8.18
ꢀ8.80
ꢀ7.68
ꢀ8.15
ꢀ6.50
ꢀ5.44
ꢀ5.91
ꢀ5.84
ꢀ6.02
ꢀ9.09
ꢀ9.93
ꢀ9.72
ꢀ8.43
ꢀ8.48
ꢀ8.42
ꢀ7.65
ꢀ8.74
ꢀ7.89
ꢀ7.54
ꢀ11.12
ꢀ11.43
ꢀ12.04
ꢀ10.43
ꢀ10.16
ꢀ7.93
1H and 13C NMR data of amines 12,43 13,44 14,45 1546 and 1647
are in agreement with data given in the literature.
ꢀ8.80
4.2.2. General procedure for the synthesis of
sulfamoylcarbamates
CSI (1.2 equiv) in CH2Cl2 (10 mL) was reacted with benzylalco-
hol (1.2 equiv) at 0 °C under N2 atmosphere. Then the mixture
ꢀ7.26
ꢀ7.21
ꢀ7.99
Table shows XP Glide docking scores (in kcal/mol) for hCA I, II, and AChE targets.
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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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Figure 2. (top) Top-docking pose of compound 21 at the binding pocket of hCA-I. (bottom) 2D ligand interaction diagram of compound 21.
was allowed to warm to rt and it was stirred for 1 h at this temp. A
solution of amine (1 equiv) in CH2Cl2 and NEt3 (1.4 equiv) were
added to the solution of CSI drop wise and stirred at rt for 4 h.
The reaction mixture was added to a solution of 0.1 N HCl
(50 mL). Organic layer was separated and H2O layer was extracted
with CH2Cl2 (2 ꢁ 30 mL). Combined organic layers were dried over
Na2SO4 and the solvent was evaporated under reduced pressure.
The residue was purified by column chromatography.
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A. Akıncıog˘lu et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 3. (top) Top-docking pose of compound 20 at the binding pocket of hCA-II. (bottom) 2D ligand interaction diagram of compound 20.
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Figure 4. (top) Top-docking pose of compound 18 at the binding pocket of AChE. (bottom) 2D ligand interaction diagram of compound 18.
4.2.3. Benzyl N-(1-(2-methoxyphenyl)ethyl)sulfamoyl-
carbamate (17)
General procedure described at Section 4.2.2 was applied to
amine 12 to give 17. Purified by silica gel column chromatography
(EtOAc/hexane, 3:10). White solid, 71% yield. Mp: 138–140 °C. 1H
NMR (400 MHz, CDCl3): d 7.37–7.26 (m, 4H, 3Ph-H and NH),
7.25–7.13 (m, 4H, 2Ph-H and 2Ar-H), 6.88 (dt, 1H, Ar-H, J = 0.8
and 7.5 Hz), 6.81 (d, 1H, Ar-H, J = 8.3 Hz), 6.35 (d, 1H, NH,
J = 9.6 Hz), 4.84 (A part of AB, d, 1H, CH, J = 12.1 Hz), 4.69 (B part
of AB, d, 1H, CH, J = 12.1 Hz), 4.65–4.59 (m, 1H, CH), 3.80 (s, 3H,
OCH3), 1.54 (d, 3H, CH3, J = 7.1 Hz). 13C NMR (100 MHz, CDCl3):
156.8 (C), 150.7 (CO), 134.6 (C), 129.1 (CH), 128.8 (C), 128.6
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(3CH), 128.4 (CH), 128.3 (2CH), 120.8 (CH), 110.9 (CH), 68.0
(OCH2), 55.3 (OCH3), 53.5 (CH), 21.8 (CH3). IR (CH2Cl2, cmꢀ1):
3276, 3068, 3034, 2976, 2932, 2839, 1707, 1601, 1588, 1494,
1436, 1373, 1356, 1293, 1242, 1199, 1158, 1133, 1083, 1054,
1023. Anal. Calcd for (C17H20N2O5S): C, 56.03; H, 5.53; N, 7.69; S,
8.80. Found: C, 56.01; H, 5.70; N, 7.74; S, 8.77.
4.2.7. Benzyl N-(1-(2,6-dimethoxyphenyl)ethyl)sulfamoyl-
carbamate (21)
General procedure described at Section 4.2.2 was applied to
amine 16 to give 21. Purified by silica gel column chromatography
(EtOAc/hexane, 3:10). White solid, 72% yield. Mp: 115–117 °C. 1H
NMR (400 MHz, CDCl3): d 7.39–7.29 (m, 4H, 3Ph-H and NH),
7.26–7.19 (m, 2H, Ph-H), 7.16 (t, 1H, Ar-H, J = 8.4 Hz), 6.64 (d, 1H,
NH, J = 10.6 Hz), 6.50 (d, 2H, Ar-H, J = 8.4 Hz), 5.22 (dq, 1H, CH,
J = 7.0 and 10.6 Hz), 4.76 (A part of AB, d, 1H, CH, J = 12.2 Hz),
4.57 (B part of AB, d, 1H, CH, J = 12.2 Hz), 3.78 (s, 6H, 2OCH3),
1.49 (d, 3H, CH3, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): 157.6
(2C), 150.5 (CO), 134.7 (C), 128.9 (CH), 128.6 (2CH), 128.5 (CH),
128.0 (2CH), 116.9 (C), 104.0 (2CH), 67.8 (OCH2), 55.8 (2OCH3),
45.8 (CH), 20.9 (CH3). IR (CH2Cl2, cmꢀ1): 3305, 3260, 2979, 2936,
2840, 1742, 1596, 1478, 1439, 1416, 1355, 1310, 1287, 1275,
1242, 1222, 1197, 1182, 1153, 1117, 1075, 1018. Anal. Calcd for
(C18H22N2O6S): C, 54.81; H, 5.62; N, 7.10; S, 8.13. Found: C,
54.86; H, 5.58; N, 7.15; S, 8.10.
4.2.4. Benzyl N-(1-(4-methoxyphenyl)ethyl)sulfamoyl-
carbamate (18)
General procedure described at Section 4.2.2 was applied to
amine 13 to give 18. Purified by silica gel column chromatography
(EtOAc/hexane, 2:10). White solid, 68% yield. Mp: 111–113 °C. 1H
NMR (400 MHz, CDCl3): d 7.46 (bs, 1H, NH), 7.39–7.32 (m, 3H,
Ph-H), 7.27–7.21 (m, 2H, Ph-H), 7.19 (d, 2H, Ar-H, J = 8.7 Hz),
6.80 (d, 1H, Ar-H, J = 8.7 Hz), 5.71 (d, 1H, NH, J = 7.7 Hz), 4.99 (A
part of AB, d, 1H, CH, J = 11.9 Hz), 4.89 (B part of AB, d, 1H, CH,
J = 11.9 Hz), 4.55–4.48 (m, 1H, CH), 3.73 (s, 3H, OCH3), 1.48 (d,
3H, CH3, J = 6.9 Hz). 13C NMR (100 MHz, CDCl3): 159.1 (C), 151.3
(CO), 134.6 (C), 133.6 (C), 128.7 (CH), 128.6 (2CH), 128.4 (2CH),
127.6 (2CH), 114.0 (2CH), 68.3 (OCH2), 55.2 (OCH3), 54.1 (CH),
22.9 (CH3). IR (CH2Cl2, cmꢀ1): 3302, 3221, 2969, 2932, 2833,
1708, 1616, 1585, 1516, 1499, 1468, 1446, 1428, 1378, 1354,
1306, 1288, 1239, 1206, 1178, 1156, 1122, 1082, 1068, 1031.
Anal. Calcd for (C17H20N2O5S): C, 56.03; H, 5.53; N, 7.69; S, 8.80.
Found: C, 56.08; H, 5.57; N, 7.63; S, 8.78.
4.2.8. Standard procedure for the synthesis of sulfamides
Pd-C (50 mg) was added to a stirred solution of sulfamoylcarba-
mate in MeOH (50 mL). A balloon filled with H2 gas (3 L) was fitted
to the flask containing the reaction mixture. After the mixture was
deoxygenated by flushing with H2, it was hydrogenated at rt for
4 h. the catalyst was removed by filtration. Recrystallization of
the residue from EtOAc–hexane gave sulfamides.
4.2.5. Benzyl N-(1-(3,4-dimethoxyphenyl)ethyl)sulfamoyl-
carbamate (19)
4.2.9. N-(1-(2-Methoxyphenyl)ethyl)sulfamide (22)
General procedure described at Section 4.2.8 was applied to sul-
famoylcarbamate 17 to give 22. White solid, 95% yield. Mp: 114–
116 °C. 1H NMR (400 MHz, CDCl3): d 7.25–7.21 (m, 2H, Ar-H),
6.92–6.86 (m, 2H, Ar-H), 5.57 (d, 1H, NH, J = 9.0 Hz), 4.75–4.68
(m, 1H, CH), 4.61 (bs, 2H, NH2), 3.82 (s, 3H, OCH3), 1.51 (d, 3H,
CH3, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): 156.6 (C), 130.4 (C),
128.7 (CH), 127.9 (CH), 121.0 (CH), 111.2 (CH), 55.5 (OCH3), 51.7
(CH), 22.1 (CH3). IR (CH2Cl2, cmꢀ1): 3340, 3267, 3002, 2974,
2942, 2839, 1601, 1588, 1493, 1464, 1437, 1431, 1372, 1341,
1307, 1290, 1276, 1242, 1200, 1178, 1167, 1158, 1133, 1085,
1053, 1037. Anal. Calcd for (C9H14N2O3S): C, 46.94; H, 6.13; N,
12.16; S, 13.92. Found: C, 46.88; H, 6.03; N, 12.13; S, 13.98.
General procedure described at Section 4.2.2 was applied to
amine 14 to give 19. Purified by silica gel column chromatography
(EtOAc/hexane, 3:10). White solid, 70% yield. Mp: 108–110 °C. 1H
NMR (400 MHz, CDCl3): d 7.76 (bs, 1H, NH), 7.30–7.28 (m, 3H,
Ph-H), 7.20–7.18 (m, 2H, Ph-H), 6.86 (s, 1H, Ar-H), 6.82 (dd, 1H,
Ar-H, J = 1.8 and 8.2 Hz), 6.73 (d, 1H, Ar-H, J = 8.2 Hz), 5.96 (d,
1H, NH, J = 7.8 Hz), 4.95 (A part of AB, d, 1H, CH, J = 11.9 Hz), 4.80
(B part of AB, d, 1H, CH, J = 11.9 Hz), 4.54–4.50 (m, 1H, CH), 3.85
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 1.49 (d, 3H, CH3, J = 6.9 Hz). 13C
NMR (100 MHz, CDCl3): 151.4 (CO), 149.3 (C), 148.9 (C), 134.6
(C), 134.3 (C), 128.9 (CH), 128.8 (2CH), 128.6 (2CH), 118.8 (CH),
111.2 (CH), 109.6 (CH), 68.6 (OCH2), 56.1 (OCH3), 56.0 (OCH3),
54.7 (CH), 23.2 (CH3). IR (CH2Cl2, cmꢀ1): 3273, 3216, 2963, 2842,
1707, 1595, 1515, 1467, 1452, 1374, 1349, 1318, 1289, 1255,
1240, 1146, 1113, 1077, 1025. Anal. Calcd for (C18H22N2O6S): C,
54.81; H, 5.62; N, 7.10; S, 8.13. Found: C, 54.77; H, 5.58; N, 7.10;
S, 8.15.
4.2.10. N-(1-(4-Methoxyphenyl)ethyl)sulfamide (23)
General procedure described at Section 4.2.8 was applied to sul-
famoylcarbamate 18 to give 23. Colorless liquid, 96% yield. 1H NMR
(400 MHz, CDCl3): d 7.28 (d, 2H, Ar-H, J = 8.7 Hz), 6.90 (d, 2H, Ar-H,
J = 8.7 Hz), 4.70 (d, 1H, NH, J = 6.3 Hz), 4.60–4.54 (m, 1H, CH), 4.22
(bs, 2H, NH2), 3.80 (s, 3H, OCH3), 1.53 (d, 3H, CH3, J = 6.8 Hz). 13C
NMR (100 MHz, CDCl3): 159.1 (C), 134.6 (C), 127.6 (2CH), 114.2
(2CH), 55.3 (OCH3), 53.6 (CH), 23.2 (CH3). IR (CH2Cl2, cmꢀ1):
3294, 2991, 2965, 2839, 1610, 1586, 1546, 1512, 1460, 1429,
1416, 1379, 1328, 1322, 1307, 1277, 1243, 1200, 1191, 1179,
1155, 1122, 1101, 1074, 1034, 1011. Anal. Calcd for
(C9H14N2O3S): C, 46.94; H, 6.13; N, 12.16; S, 13.92. Found: C,
46.91; H, 6.17; N, 12.24; S, 13.88.
4.2.6. Benzyl N-(1-(2,5-dimethoxyphenyl)ethyl)sulfamoyl-
carbamate (20)
General procedure described at Section 4.2.2 was applied to
amine 15 to give 20. Purified by silica gel column chromatography
(EtOAc/hexane, 2:10). White solid, 67% yield. Mp: 126–128 °C. 1H
NMR (400 MHz, CDCl3): d 8.00 (bs, 1H, NH), 7.35–7.30 (m, 3H,
Ph-H), 7.21–7.17 (m, 2H, Ph-H), 6.79 (d, 1H, Ar-H, J = 1.6 Hz),
6.77–6.69 (m, 2H, Ar-H), 6.52 (d, 1H, NH, J = 9.6 Hz), 4.84 (A part
of AB, d, 1H, CH, J = 12.1 Hz), 4.69 (B part of AB, d, 1H, CH,
J = 12.1 Hz), 4.64–4.54 (m, 1H, CH), 3.74 (s, 3H, OCH3), 3.68 (s,
3H, OCH3), 1.51 (d, 3H, CH3, J = 7.0 Hz). 13C NMR (100 MHz,
CDCl3): 153.4 (C), 150.9 (CO), 150.8 (C), 134.6 (C), 129.9 (C),
128.6 (2CH), 128.5 (CH), 128.2 (2CH), 114.6 (CH), 113.1 (CH),
111.7 (CH), 68.0 (OCH2), 55.7 (2OCH3), 53.1 (CH), 21.8 (CH3). IR
(CH2Cl2, cmꢀ1): 3276, 3208, 3013, 2938, 2832, 1707, 1595, 1496,
1474, 1454, 1433, 1423, 1375, 1356, 1308, 1268, 1232, 1194,
1183, 1161, 1152, 1102, 1080, 1046, 1021. Anal. Calcd for
(C18H22N2O6S): C, 54.81; H, 5.62; N, 7.10; S, 8.13. Found: C,
54.88; H, 5.53; N, 7.15; S, 8.04.
4.2.11. N-(1-(3,4-Dimethoxyphenyl)ethyl)sulfamide (24)
General procedure described at Section 4.2.8 was applied to sul-
famoylcarbamate 19 to give 24. White solid, 96% yield. Mp: 98–
100 °C. 1H NMR (400 MHz, CDCl3): d 6.90 (s, 1H, Ar-H), 6.88 (d,
1H, Ar-H, J = 8.3 Hz), 6.79 (d, 1H, Ar-H, J = 8.3 Hz), 5.50 (d, 1H,
NH, J = 7.2 Hz), 4.86 (bs, 1H, NH2), 4.49–4.45 (m, 1H, CH), 3.83 (s,
3H, OCH3), 3.80 (s, 3H, OCH3), 1.48 (d, 3H, CH3, J = 6.9 Hz). 13C
NMR (100 MHz, CDCl3): 149.2 (C), 148.5 (C), 135.3 (C), 118.5
(CH), 111.2 (CH), 109.6 (CH), 56.0 (OCH3), 55.9 (OCH3), 53.9 (CH),
23.3 (CH3). IR (CH2Cl2, cmꢀ1): 3297, 3242, 2993, 2981, 2957,
2935, 2841, 1678, 1597, 1569, 1519, 1465, 1447, 1434, 1418,
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1374, 1345, 1328, 1302, 1265, 1229, 1198, 1157, 1139, 1101, 1084,
4.5. Esterase activity and inhibition assays
1041, 1024. Anal. Calcd for (C10H16N2O4S): C, 46.14; H, 6.20; N,
10.76; S, 12.32. Found: C, 46.11; H, 6.15; N, 10.68; S, 12.36.
Esterase activity was determined according to the procedure
developed by Verpoorte.83 Both CA isoenzymes activities were
determined by following the change in absorbance at 348 nm of
4-nitrophenylacetate (NPA) to 4-nitrophenylate ions using a spec-
trophotometer (Shimadzu, UVmini-1240 UV–vis spectrophotome-
ter).48 The Ki values were calculated from experiments using
three different sulfamoylcarbamates 17–21 and sulfamide deriva-
tives 22–26 concentrations. PNF was used as substrate at five dif-
ferent concentrations for determination of Lineweaver–Burk
curves62 reported previously.61
4.2.12. N-(1-(2,5-Dimethoxyphenyl)ethyl)sulfamide (25)
General procedure described at Section 4.2.8 was applied to
sulfamoylcarbamate 20 to give 25. Yellow oil, 92% yield. 1H
NMR (400 MHz, CDCl3): d 6.86 (d, 1H, Ar-H, J = 3.0 Hz), 6.80 (d,
1H, Ar-H, J = 8.9 Hz), 6.74 (dd, 1H, Ar-H, J = 3.0 and 8.9 Hz), 6.67
(d, 1H, NH, J = 8.9 Hz), 4.80 (bs, 1H, NH2), 4.73–4.66 (m, 1H,
CH), 3.78 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 1.48 (d, 3H, CH3,
J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): 153.7 (C), 150.6 (C),
131.7 (C), 114.0 (CH), 112.8 (CH), 112.2 (CH), 56.0 (OCH3), 55.7
(OCH3), 51.2 (CH), 22.2 (CH3). IR (CH2Cl2, cmꢀ1): 3273, 2938,
2836, 1610, 1589, 1558, 1496, 1463, 1427, 1301, 1281, 1217,
1156, 1079, 1022. Anal. Calcd for (C10H16N2O4S): C, 46.14; H,
6.20; N, 10.76; S, 12.32. Found: C, 46.10; H, 6.26; N, 10.55; S,
12.38.
4.6. Determination of anticholinesterase activity
The inhibition effects of sulfamoylcarbamates 17–21 and sul-
famide derivatives 22–26 on AChE activities were measured by
of Ellman’s method.84 AChI and 5,50-dithio-bis(2-nitro-ben-
zoic)acid (DTNB; product no: D8130-1G, Sigma–Aldrich) were used
as substrate for this enzymatic reaction. To this end, 100 mL of
Tris/HCl buffer (1 M, pH 8.0) and 10 mL of sample solution at dif-
ferent concentrations and 50 mL AChE (5.32 ꢁ 10ꢀ3 U) solution
were mixed and incubated for 10 min at 25 °C. Then 50 mL of
DTNB (0.5 mM) was transferred. Then the reaction was initiated
by the addition of 50 mL of AChI (10 mM, product no: 01480-1G,
Sigma–Aldrich). The hydrolysis of AChI was recorded spectropho-
tometrically by the formation of yellow 5-thio-2-nitrobenzoate
anion as the result of the reaction of DTNB with thiocholine at a
wavelength of 412 nm.15,61
4.2.13. N-(1-(2,6-Dimethoxyphenyl)ethyl)sulfamide (26)
General procedure described at Section 4.2.8 was applied to
sulfamoylcarbamate 21 to give 26. White solid, 90% yield. Mp:
110–112 °C. 1H NMR (400 MHz, acetone-d6): d 7.21 (t, 1H, Ar-
H, J = 8.4 Hz), 6.68 (d, 2H, Ar-H, J = 8.4 Hz), 5.90 (d, 1H, NH,
J = 10.5 Hz), 5.26 (dq, 1H, CH, J = 7.0 and 10.5 Hz), 3.86 (s,
6H, 2OCH3), 1.47 (d, 3H, CH3, J = 7.0 Hz). 13C NMR
(100 MHz): 158.6 (2C), 129.4 (CH), 120.1 (C), 105.5 (2CH),
56.4 (2OCH3), 45.9 (CH), 21.8 (CH3). IR (CH2Cl2, cmꢀ1): 3342,
3309, 3256, 2999, 2983, 2968, 2945, 2845, 1594, 1551,
1476, 1459, 1445, 1438, 1423, 1350, 1337, 1317, 1288,
1269, 1243, 1199, 1185, 1168, 1152, 1137 1115, 1101, 1085,
1074, 1024. Anal. Calcd for (C10H16N2O4S): C, 46.14; H, 6.20;
N, 10.76; S, 12.32. Found: C, 46.07; H, 6.26; N, 10.84; S,
12.28.
4.7. Determination of acetylcholine esterase inhibition
For determination of effects of some sulfamoylcarbamates 17–
21 and sulfamide derivatives 22–26 on AChE, different some sul-
famoylcarbamates 17–21 and sulfamide derivatives 22–26 concen-
trations were added into the reaction flask. AChE activity was
measured, and the reaction medium in the absence of synthesized
compounds was used as control sample.60 The IC50 values were cal-
culated from activity (%) versus sulfamoylcarbamates 17–21 and
sulfamide derivatives 22–26 concentration plots. Also, to deter-
mine the inhibition constants (Ki) in the media with sulfamoylcar-
bamates 17–21 and sulfamide derivatives 22–26 as inhibitor, the
different substrate (ACh) concentrations were used. For this pur-
pose, the inhibitor solution was transferred to the reaction med-
ium, resulting in three different fixed concentrations of each
sulfamoylcarbamates 17–21 and sulfamide derivatives 22–26 as
inhibitor. Lineweaver–Burk graphs67 were drawn using 1/V versus
1/[S] values. Ki values were obtained from Lineweaver–Burk
graphs. Galantamine was used as a reference compound.
4.3. Purification of CA isoenzymes by affinity chromatography
The human erythrocytes were centrifuged at 10.000ꢁg for
30 min. Then precipitate and the serum were separated. The serum
pH was adjusted with solid Tris (pH: 8.7).20,72 Sepharose-4B-tiro-
zyne-sulfanylamide affinity column balanced with Tris–HCl/
Na2SO4 (25 mM/0.1 M, pH 8.7). Adjusted human erythrocyte sam-
ple was applied to the Sepharose-4B-tirozyne-sulfanylamide affin-
ity column. The affinity gel was washed with Tris–HCl/Na2SO4
(25 mM/22 mM, pH: 8.7) and hCA I, II isoenzyme were eluted with
H2NSO3H/Na2HPO4 (25 mM/25 mM, pH: 6.7). All of enzyme studies
were performed at 4 °C.73,74
4.4. SDS–PAGE and protein identification
4.8. In silico docking studies
The protein content during the purification steps was identified
using by Bradford method at 595 nm75 with slight modification.76
In this study, bovine serum albumin was used as standard
protein.77,78
Crystal structures of CAs in complex with inhibitors (PDB
Codes: 2FW4 for hCA I; 4PQ7 for hCA II; and 4TVK for AChE) were
used for docking simulations. Explicit water molecules and metal
ions from the X-ray structure were kept for all calculations.
Before the docking simulations, the complexes were submitted to
the Protein Preparation module of the Maestro Molecular
Modeling Suite.85 3D structures of investigated small molecules
were constructed using the Macromodel module of Maestro.
Geometry optimization calculations of studied ligands were per-
formed using Polak–Ribiere conjugate gradient (PRCG) minimiza-
tion method with 0.0001 kJ Åꢀ1 molꢀ1 convergence criteria.
Protonation states of ligands and residues were tested using
LigPrep and Protein Preparation (using PROPKA method at
pH = 7.0) modules under Schrodinger Molecular Modeling package.
Also, the purity of both hCA isoenzymes was controlled
using SDS–PAGE. This study was performed according to the
Laemmli’s metod79 and also been described our previous stud-
ies.80 The running and separating gels contained SDS (0.1%)
and 3% and 10% acrylamide respectively.81 Before loading into
the gel, samples were added 1% SDS and 10% by 2-mercap-
toethanol and then were waited in boiling water bath for 5
minutes. The gel was painted in colouring solution after bands
were detected. This colouring solution are prepared as1.5 h in
Coomassie Brilliant Blue (R-250, 0.1%) in (10%) acetic acid and
methanol (50%).82
Please cite this article in press as: Akıncıog˘lu, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.019

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Docking (IFD) have been used for the docking calculations. IFD uses
the Glide docking program to account for the ligand flexibility and
Prime algorithm to account for refinement of docking poses.86,87
Default parameters of IFD were used throughout the simulations:
(i) constrained minimization of the target with and RMSD cutoff
of 0.18 Å; (ii) initial Glide docking of each ligand using a softened
potential; (iii) side chain prediction for each protein/ligand com-
plex on residues within 5.0 Å from ligand using Prime module;
(iv) minimization of complex structures (protein/ligand poses);
(v) re-docking of each protein/ligand complex structure.
25. Çoban, T. A.; Beydemir, Sß.; Gülçin, I.; Ekinci, D. J. Enzyme Inhib. Med. Chem. 2008,
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Acknowledgments
_
_
We are greatly indebted to Ataturk University and Ag˘rı Ibrahim
2010, 20, 5050.
Çeçen University for their research and financial support. Also, I.G.
would like to extend his sincere appreciation to the Research
Chairs Program at King Saud University for funding this research.
S.D. acknowledges support from Turkish Academy of Science under
the BAGEP program.
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