Carbonic anhydrase inhibitors: Synthesis, characterization and inhibition activities of furan sulfonylhydrazones against carbonic anhydrase i (hCA I)

Article abstract of DOI:10.1016/j.molstruc.2015.10.054

The methane sulfonic acide hydrazide (1) was used to obtain furan sulfonylhydrazones; 2-acetylfuranmethanesulfonylhydrazone (2), 2-furaldehydemethanesulfonylhydrazone (3), 5-nitro-2-furaldehydemethanesulfonylhydrazone (4). The structures of furan sulfonylhydrazones were determined by using elemental analysis, FT-IR, ¹H NMR, ¹³C NMR and UV-vis methods. The structure of 5-nitro-2-furaldehydemethanesulfonylhydrazone (4) was also supported with X-ray difraction method and found that compound 4 was crystallized in triclinic, space group P1ˉ. In order to gain insight into the structure of the compounds, we performed computational studies by using 6-311G(d,p) basic set in which B3LYP correlation function was implemented. The geometry of the sulfonylhydrazones were optimized at DFT method with Gaussian 09 program package and the global reactivity descriptors were also calculated by this basic set. The enzyme inhibition activities of the sulfonylhydrazones were investigated on carbonic anhydrase I (hCA I) isoenzyme and their activity parameters (Km, IC₅₀ and Ki) were calculated by spectrophotometric method. And also, their inhibitor effects were also investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Inhibition results show that compound 4 containing electron withdrawing group (NO₂) has higher inhibition effect on hCA I isoenzyme than other's.

Full text of DOI:10.1016/j.molstruc.2015.10.054

Journal of Molecular Structure 1105 (2016) 332e340
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Carbonic anhydrase inhibitors: Synthesis, characterization and
inhibition activities of furan sulfonylhydrazones against carbonic
anhydrase I (hCA I)
a
,
*
a
a
Ayla Balaban Gündüzalp , G o€ khan Parlakgümü s¸ , Demet Uzun ,
€
€
a
€
b
c
d
Ümmuhan Ozdemir Ozmen , Neslihan Ozbek , Musa Sarı , Tuncay Tunç
Department of Chemistry, Faculty of Science, Gazi University, 06500 Ankara, Turkey
Department of Chemistry, Faculty of Education, Ahi Evran University, Kır s¸ ehir, Turkey
Faculty of Education, Department of Physics, Gazi University, 06500 Ankara, Turkey
a
b
c
d
Department of Science Education, Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 August 2015
Received in revised form
The methane sulfonic acide hydrazide (1) was used to obtain furan sulfonylhydrazones; 2-
acetylfuranmethanesulfonylhydrazone (2), 2-furaldehydemethanesulfonylhydrazone (3), 5-nitro-2-
furaldehydemethanesulfonylhydrazone (4). The structures of furan sulfonylhydrazones were deter-
mined by using elemental analysis, FT-IR, 1H NMR, C NMR and UVevis methods. The structure of 5-
nitro-2-furaldehydemethanesulfonylhydrazone (4) was also supported with X-ray difraction method
and found that compound 4 was crystallized in triclinic, space group P1. In order to gain insight into the
structure of the compounds, we performed computational studies by using 6e311G(d,p) basic set in
which B3LYP correlation function was implemented. The geometry of the sulfonylhydrazones were
optimized at DFT method with Gaussian 09 program package and the global reactivity descriptors were
also calculated by this basic set. The enzyme inhibition activities of the sulfonylhydrazones were
investigated on carbonic anhydrase I (hCA I) isoenzyme and their activity parameters (Km, IC50 and Ki)
were calculated by spectrophotometric method. And also, their inhibitor effects were also investigated by
cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Inhibition results show that
1
October 2015
13
Accepted 19 October 2015
Available online 23 October 2015
Keywords:
Carbonic anhydrase I (hCA I)
Enzyme inhibition
Furan sulfonylhydrazone
Cyclic voltammetry (CV)
Differantial pulse voltammetry (DPV)
compound 4 containing electron withdrawing group (NO
2
) has higher inhibition effect on hCA I isoen-
zyme than other's.
©
2015 Elsevier B.V. All rights reserved.
1
. Introduction
Inhibitors of the carbonic anhydrases (CAs) have clinical usage of
major diseases such as glaucoma, epilepsy, gastroduodenal ulcers,
acid-base disequilibria and neurological disorders [4]. CA inhibition
by sulfonamides have been reported by Mann and Keilin [5], and
subsequently other inhibitors have been investigated for medical
applications. The synthesis of CA inhibitors from various classes of
sulfonamides (such as sulfonylhydrazide, sulfonylhydrazone, sul-
fonylurea etc.) have importance of specificity towards isoenzymes
[6] and affect biologic systems by interacting with enzyme active
sites. For this reason, new CA inhibitors interacting with CA iso-
enzymes have importance in bioinorganic and metallodrug chem-
istry. Many of metabolic processes are based on redox processes and
there are many similarities between electrochemical and biological
reactions relating with electron transfer mechanisms in systems [7].
In this study, methane sulfonic acide hydrazide (1) was syn-
thesized as reported by us [8,9] and used to obtain furan
The carbonic anhydrases (CAs) are the metalloenzymes con-
taining zinc center which classically participate in the maintenance
of pH homeostasis. CAs catalyze the reversible hydration of carbon
dioxide in two-step reaction to yield bicarbonate ion and proton.
Sixteen CA isozymes have been described and some of them are
cytosolic (CA I, CA II, CA III, CA VII and CA XIII), other CA isoensymes
are membrane bound (CA IV, CA IX, CA XII and CA XIV), two of CAs
are mitochondrial (CA VA and CA VB) and one of CAs is in saliva (CA
VI). The two important CA isozymes (CA I and CA II) are present at
higher concentrations in the cytosol in erythrocytes [1e3].
*
Corresponding author.
E-mail address: balaban@gazi.edu.tr (A.B. Gündüzalp).
http://dx.doi.org/10.1016/j.molstruc.2015.10.054
022-2860/© 2015 Elsevier B.V. All rights reserved.
0

A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
333
sulfonylhydrazones; 2-acetylfuranmethanesulfonylhydrazone (2),
-furaldehydemethanesulfonylhydrazone (3) and 5-nitro-2-
2.3. X-ray structure determination of compound 4
2
furaldehydemethanesulfonylhydrazone (4). The structures of
Crystallographic data of compound was recorded on a Bruker
Kappa APEX II CCD area-dedector X-ray diffractometer employing
1
compounds 2e4 were characterized by using elemental analysis, H
NMR, 13C NMR, FT-IR and UVevis methods. Gaussian 09 software
plane graphite monochromatized with MoK
radiation
a
was used to obtain the most stable conformation of the compounds
(
l
¼ 0.71073 Å), using
u
ꢀ 2
q
scan mode. The structures were
2
e4 based on DFT/B3LYP/6-311G(d,p) method and global reactivity
descriptors such as the highest occupied molecular orbital (εHOMO),
the lowest occupied molecular orbital (εLUMO), electronegativity ( ),
chemical potential ( ), global hardness ( ), global softness (S),
global electrophilicity index ( ) were calculated by this basic set.
solved by the direct methods and refined by full-matrix least-
squares techniques on F using the solution program SHELXS-97
2
c
and refined using SHELXL-97 [12]. The empirical absorption cor-
rections were applied by multi-scan via Bruker, SADABS software
[13]. The H atoms positions were calculated geometrically at dis-
tances of 0.95 Å (CH) from the parent C atoms; a riding model was
used during the refinement process. The molecular structure plots
were prepared using ORTEP-3 for Windows [14].
m
h
u
The inhibition effects of the heteroaromatic sulfonylhydrazones
on carbonic anhydrase I (h CAI) were determined by electronic
spectra. The activity parameters (Km, IC50 and Ki) were calculated
by LineweavereBurk graph, activity % graph and Cheng-Prusoff
equation. Inhibition activities of furan sulfonylhydrazones were
also evaluated by voltammetric techniques. The enzyme activities
were investigated by using enzymatic hydrolysis of substrate (p-
nitrophenylacetate, PNFA) to p-nitrophenolate, PNF via hCA I
isoenzyme. In the presence of sulfonylhydrazones, the enzyme
function was inhibited, resulting in a decrease in PNF formation in
other words the inhibition degree of hCA I was correlated to the
decrease in PNF reductive current. Biological activity results show
2.4. Computational section
Molecular geometry optimization and global reactivity de-
scriptors such as highest occupied molecular orbital (εHOMO), lowest
occupied molecular orbital (εLUMO), electronegativity (
potential ( ), global hardness ( ), global softness (S) and global
electrophilicity index ( ) were carried out by Gaussian 09 quantum
chemistry program-package examined with Becke's three-
parameter exchange functional in combination with the Lee-
eYangeParr correlation functional (B3LYP) in density functional
theory (DFT) method with 6-311G(d,p) basis set [15e18]. Global
c), chemical
m
h
u
that compound 4 containing electron withdrawing group NO
2
has
higher inhibition effect on hCA
sulfonylhydrazones.
I isoenzyme than other
reactivity descriptors consist of electronegativity
(
c
)
¼ ꢀ1/
2
. Experimental
2(εLUMO þ εHOMO), chemical potential ( ) ¼ 1/2 (εLUMO þ εHOMO),
m
global hardness (
h
) ¼ 1/2(εLUMO ꢀ εHOMO), global softness (S) ¼ 1/
2
2.1. Physical measurements
2h
and electrophilicity index (
u
) ¼
m
/2
h
were highly successful in
predicting global reactivity trends. These parameters were related
not only to the spectral properties, but also to the reactivity prop-
erties [19].
The solvents used were purified and distilled according to routine
procedures. Methane sulfonyl chloride, hydrazine hydrate were
commercial products (purum). Elemental analyses were performed
according to standard micro analytical procedures by Leco CHNS-
2.5. Procedures for hCA I enzyme inhibitor activities
1
13
9
32, H and C NMR spectra of the compounds in dimethylsulf-
oxide-d (DMSO-d ) were registered on a Bruker WM-400 spec-
6
6
2.5.1. Spectrophotometric studies
trometer (400 MHz) using tetramethylsilane as internal standard.
The infrared spectra of the compounds as KBr-disks were recorded in
Carbonic anhydrase activities were assayed by the hydrolysis of
substrate (p-nitrophenylacetate, PNFA) [20] to p-nitrophenolate,
PNF via hCA I isoenzyme and activity parameters (Km, IC50 and Ki)
were calculated with LineweavereBurk graph, activity % - [inhibi-
tor] graph and Cheng-Prusoff equation. Acetazolamide ((5-
acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) clinically used
in hCA I inhibition was also been investigated as standard inhibitor.
ꢀ
1
the range of 4000e400 cm with a Mattson 1000 FT-IR spectrom-
eter. UVevis spectra were recorded on UNICAM-UV 2-100 spectro-
photometer. Melting points of furan sulfonylhydrazones were
determined with a Gallenkamp melting point apparatus. The crystal
structure of 5-nitro-2-furaldehydemethanesulfonylhydrazone (4)
was determined by using a on a Bruker Kappa APEX II CCD area-
detector. The inhibition activities of synthesized compounds on
carbonic anhydrase I (h CAI) were investigated by measuring ab-
sorbances at 400 nm on UVevis spectropohotometer. For electro-
chemical analysis, cyclic voltammetry (CV) and differential pulse
voltammetry (DPV) methods were studied by using CHI 660B
voltammeter.
In order to determine IC50 values, 100
mL of 3.0 mM p-nitro-
phenylacetate as substrate and four different concentrations
ꢀ2
ꢀ3
ꢀ4
ꢀ4
(3 ꢂ 10 ; 3 ꢂ 10 ; 5 ꢂ 10 ; 3 ꢂ 10 M) of inhibitors were used.
Reaction was started by adding of 170 L of 0.05 M tris-SO buffer
(pH: 7.4) and 0.1 L enzyme solution for total volume of 300 L. The
m
4
m
m
absorbance of the product (PNF) was determined at 400 nm after
6 min [21]. This study was repeated three times for each inhibitor.
In the media with or without inhibitor, the substrate concentrations
were 0.3, 0.6, 1.0, 3.0 mM. For this aim, inhibitor solutions were
used for the reaction medium in four different concentrations
2.2. General procedure for the synthesis of compounds
ꢀ2
ꢀ3
ꢀ4
ꢀ4
The nucleophilic substitution reaction of the hydrazine hydrate
(3 ꢂ 10 ; 3 ꢂ 10 ; 5 ꢂ 10 ; 3 ꢂ 10 M). Km values were
calculated from LineweavereBurk graphs, IC50 values were calcu-
lated from activity%-[inhibitor] graphs and Ki values were calcu-
lated according to Cheng Prusoff equation using Km and IC50
parameters [22,23].
with methane sulfonyl chloride was carried out methane sulfonic
acid hydrazide (1) as reported [8,10,11]. Compounds 2e4 were
synthesized according to the following general procedure: The
solution of methane sulfonic acid hydrazide (4.55 mmol) in 50 mL
of methanol was mixed with hot solution of 4.55 mmol of the
corresponding
furanaldehyde and 5-nitro-2-furanaldehyde, respectively) in
0 mL of methanol and stirred for 24 h. Obtained crystals were
carbonyl
compound
(2-acetylfuran,
2-
2.5.2. Electrochemical studies
Electrochemical measurements by using cyclic voltammetry
(CV) and differential pulse voltammetry (DPV) were conducted on a
CHI 660B electrochemical workstation (Shanghai, China). A con-
ventional three-electrode system was employed with a glassy
3
recrystallized with ethylacetate, then washed with ethylacetate/
ꢁ
ether and dried at 50 C in oven.

334
A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
carbon (GC) electrode (3 mm diameter) as working electrode, an
Ag/AgCl electrode as reference electrode, and a platinum wire as
counter electrode. For enzyme inhibition studies, 10 mM TRIS (pH
seen in Fig. 3.
3.2. FT-IR spectra
2 4
7.4) with 1 M H SO was used as supporting electrolyte. Stock so-
lutions of 10 mM inhibitors (compounds 1e4, AAZ) in acetonitrile
containing 0.1 M tetrabutylammonium tetrafluoroborate (TBATFB)
were prepared at room temperature. GC electrode was mechani-
cally polished first with fine wet emery papers (Buehler) with grain
The selected vibration frequencies of furan sulfonylhydrazones
are listed in Table 3. The assignment of the bands are made by
taking into consideration the literature data for compounds con-
taining appropriate structural fragments such as furan and sul-
fonamide derivatives [32e34]. NH vibrations in compounds 2e4
size of 4000 and then with 0.3
mm and 0.05 mm alumina slurry
ꢀ1
(
Baikowski Int. Corp.) on a microcloth pad (Buehler). Polished GC
are observed between 3220 and 3231 cm as strong bands. And
also ySO2 , ySO2 stretching vibrations are observed between
electrode was sonicated in ultrapure water and then with a mixture
of 1:1 (v/v) isopropyl alcohol/acetonitrile (IPA/MeCN) for 10 min for
removal of trace alumina from the surface. Then, GC electrode was
ðasÞ
ðsÞ
1
ꢀ
ꢀ1
1
333 and 1358 cm , 1031e1166 cm for compounds 2e4. Furan
rings have characteristic vibration bands ( CeOeC) in the range of
n
ꢀ1
rinsed with acetonitrile and dried under N
. Results and discussion
Analytical data and some physical properties of the furan sul-
2
stream [24e26].
1
162e1227 cm as seen in Fig. 4 [35].
3
3.3. Crystal structure of compound 4
The crystal and instrumental parameters used in the unit-cell
determination and data collection are summarized in Table 4, the
selected bond lengths and angles are listed in Table 5, respectively.
The 5-nitro-2-furaldehydemethanesulfonylhydrazone (4) was
crystallizes in the triclinic, P1 space group and the asymmetric unit
consists of two independent and similar molecules (C H N O S) as
fonylhydrazones are summarized in Table 1. The general synthetic
route used to prepare the compounds are illustrated in Fig. 1. The
exothermic nucleophilic substitution reaction of corresponding
methane sulfonyl chloride with hydrazine hydrate were employed
to form methane sulfonic acide hydrazide (1) which reacted with
aromatic ketone/aldehydes to form; 2-acetylfuranmethane
sulfonylhydrazone (2), 2-furaldehydemethanesulfonylhydrazone
6
7 3 5
seen in Fig. 5. The SeO and SeN bond distances lie within expected
range of 1.4239(18)e1.4265(19) Å and 1.647(2) Å, respectively. The
S1eC6 and S2eC12 bond distances are 1.748(3) and 1.749(3) Å. The
SeC bond distances still have an intermediate value between a
single (1.82 Å) and double (1.56 Å) [36]. This is an indication that
there is some electron delocalization in the methane sulfonic acide
hydrazide chain. All bond lengths and angles for compound 4 are
consistent with those found in related compound as seen in Table 5
(3), 5-nitro-2-furaldehydemethanesulfonylhydrazone (4). The ge-
ometry optimization was performed on DFT/B3LYP/6-311G(d,p)
methods with Gaussian 09 program. Optimized structure of the
sulfonylhydrazones are given in Fig. 2.
3.1. NMR spectra
[
37].
1He13C NMR spectra of compounds 2e4 were obtained in
DMSO-d
6
at room temperature using TMS as an internal Standard.
3.4. Global reactivity descriptors analysis
1
13
6
The experimental He C NMR assignments in DMSO-d are listed
in Table 2.
The highest occupied molecular orbital (εHOMO) and the lowest
unoccupied molecular orbital (εLUMO) are very important for
quantum chemistry [38e40]. HOMO's and LUMO's are the main
orbitals in chemical stability. The HOMO's as an electron donors
representing the ability to donate an electron and LUMO's as an
electron acceptors representing the ability to obtain an electron.
The HOMO's and LUMO's are the main orbital taking part in the
chemical reactions. The HOMO energy levels are directly related to
the ionization potential and LUMO energy levels are directly related
to the electron affinity. Frontier molecular orbitals were computed
at B3LYP/6-311G(d,p) level of the title molecules as shown in
3 2 3
CH protons bonded to SO group (C(6)H , three H intensities) of
compounds 2e4 are easily distinguishable as a singlet and they are
observed at 3.15 ppm, 3.22 ppm and 3.11 ppm, respectively. Acetyl
protons (CeCH
.27 ppm as singlet, the aromatic protons of furan ring systems are
observed between 6.53 and 7.77 ppm, 6.64e7.82 ppm,
.23e7.78 ppm, respectively. Azomethine protons (C(5)H]N, one H
intensity) of compounds 3 and 4 are observed at 7.87 ppm and
.93 ppm as singlet, respectively. In addition, the experimental
3
, three H intensities) of compound 2 are observed at
2
7
7
seconder NH protons of compounds 2e4 are observed in the range
of 10.22e11.84 ppm as seen in Fig. 3 [27e31].
Table 6. The electronegativity (
hardness ( ), global softness (S) and global electrophilicity index
) are calculated by frontier molecular orbitals (εHOMO, εLUMO
energies and listed in Table 6. Compound 4 containing electron-
withdrawing group (NO ) has the lowest LUMO energy level
c), chemical potential (m), global
The CH
compounds 2e4 are observed in the range of 38.38e39.79 ppm.
Acetyl carbon (CeCH ) of compound 2 is observed at 14.14 ppm.
The aromatic carbons of furan ring systems are observed between
11.87 and 151.56 ppm, 112.50e149.21 ppm and
3
carbons of methyl moiety bonded to SO
2
groups of
h
(u
)
3
2
1
which is the most important descriptor showing higher electro-
1
15.81e153.46 ppm, respectively. Azomethine carbons (C(5) ¼ N) of
philicity of the compound 4. The rising of electronic descriptors
compounds 3 and 4 are observed at 145.48 ppm and 135.56 ppm as
(c
¼ 0.1847 eV and
u
¼ 0.2389 eV) causes the strongest
Table 1
Analytical and physical data for furan sulfonylhydrazones.
ꢁ
Compound
Formula M
w
(g/mol)
m.p. ( C)
Yield (%)
l
(nm)
Found (calculated)
%
C
%H
%N
%S
2
3
4
C
7
C
6
C
6
H
H
H
10
N
2
O
3
S 202.23
S 188.21
S 233.20
138e140
105
180
69
64
60
370, 305, 245
370, 305, 245
400, 245
41.32 (41.60)
38.54 (38.30)
30.57 (30.90)
5.14 (4.95)
4.54 (4.25)
3.13 (3.00)
13.79 (13.86)
14.69 (14.89)
17.62 (18.03)
15.83 (15.84)
16.88 (17.02)
13.58 (13.73)
8
N
2
O
O
3
7
N
3
5

A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
335
Fig. 1. Preparation of compounds (1e4).
Fig. 2. Optimized geometries of furan sulfonylhydrazones (2e4).
Table 2
interaction of compounds with enzyme active sites. Acetazolamide
5-acetamido-1,3,4-thiadiazole-2-sulfonamide), AAZ) has also been
The experimental 1H NMR data of furan sulfonylhydrazones.
(
Assign.
1H NMR (
d, ppm)
13C NMR (
d, ppm)
investigated as standard inhibitor which clinically used against hCA
I. One of the most important findings of this study is that compound
2
3
4
2
3
4
4
2
containig NO group has the nearest activity to AAZ.
NH (s,1H)
10.22
7.77
6.53
6.94
e
10.76
6.90
6.64
7.82
e
11.84
e
7.78a
7.23
e
e
C(1)H (d,1H)
C(2)H (dd,1H)
C(3)H (d,1H)
C(4)
145.80
144.99
112.26
151.56
e
145.48
112.50
114.15
149.21
137.21
37.72
e
153.46
117.86
115.81
151.98
135.56
39.79
e
3.5.2. Electrochemical method
Redox behaviors of methane sulfonic acid hydrazide (1) and
C(5)H]N (s,1H)
e
7.87
3.22
e
7.93
3.11
e
furan sulfonylhydrazones (2e4) were investigated by CV and DPV
techniques [42,43]. Two oxidation peaks belong to methane sul-
fonic acid hydrazide (1) were observed in the positive potential
ranges in CVs. Furan sulfonylhydrazones (2e4) show redox
behavior compared with free methane sulfonic acid hydrazide (1),
but there are some changes including negative and/or positive
shifts of redox waves and appearance/disappearance of some redox
signals. In CVs of sulfonylhydrazones (2e4), the reduction peaks
which belong to azomethine groups (C]N / CHeNH) were
observed at ꢀ0.803 V, ꢀ0.772 V and ꢀ0.753 V, respectively. Two
reduction peaks of nitro group belong to compound 4 were
observed at about 1.06 V and ꢀ1.42 V, respectively. The first peak
SO
2
eC(6)H
3
(s,3H)
3.15
2.27
38.38
14.14
C(5)-CH
3
(s,3H)
a
C(2)H (d,1H).
electrophilicty, which plays dominant role on predicting global
chemical reactivity trend.
3
3
.5. Determination of hCA I inhibitory effects
.5.1. Spectrophotometric method
The inhibitory effects of the furan sulfonylhydrazones were
can be attributed to reduction of NO
reduction of NHOH / NH
2
/ NHOH and the second to
investigated by spectrophotometric method (Fig. 6). The enzyme
activity of compounds 2e4 were evaluated by using K (Michaelis
constant), IC50 (IC50 represents the molarity of inhibition as 50%
decrease of enzyme activity), and K (inhibitoreenzyme dissocia-
tion constant) values that are three of the most appropriate pa-
rameters of the inhibitons (Table 7) [41].
2
.
m
hCA I inhibitor activities of methane sulfonic acid hydrazide (1)
and furan sulfonylhydrazones (2e4) were investigated by cyclic
voltammetry (CV) and also differential-pulse voltammetry (DPV).
The inhibition effects of the compounds are concluded by
measuring the reduction peaks of the product (PNF) produced from
the enzymatic hydrolysis of substrate (PNFA) by hCA I. PNFA
exhibited two peaks occurring at approximately ꢀ0.782 V as
reduction peak and 0.025 V as oxidation peak. Notably, PNF for-
mation is also defined by a single, non-overlapping cathodic peak at
approximately ꢀ0.2 V (vs Ag/AgCl). In the presence of sulfonylhy-
drazones, the enzymatic function is inhibited resulting in a
i
As seen in Table 7, furan sulfonylhydrazones behave as good
inhibitors against hCA I isoenzyme. Compound 4 containing NO
2
ꢀ
3
group shows enhanged inhibition effect (IC50: 1.02 ꢂ 10 , Ki:
ꢀ
6
5
.88 ꢂ 10 M) on hCA I than other compounds. This is probably
due to the further effect of the electron withdrawing group such as
NO . The presence of electronegative group may increase the
2

336
A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
Fig. 3. 1H-NMR and 13C-NMR spectrum (aef) of furan sulfonylhydrazones.
decrease in PNF formation. The inhibition degree of hCA I is
therefore correlated by the decrease in PNF reductive current which
form was applied from ꢀ1.4 V to 0.70 V. Fig. 7 shows that hCA I
activity is related with the decrease of reduction peak current of
substrate (PNFA) and the increase of reduction peak current of
product (PNF). In other approach, inhibition degree of inhibitor can
be correlated to the decrease in PNF reductive current which de-
pends on the increasing concentration of the inhibitor in the
sample.
depends
on
the
increasing
concentration
of
the
sulfonylhydrazones.
To determine the enzyme inhibition activity of substrate (PNFA),
CVs were taken first in tris buffer media (pH 7.4) containing 3 mM
substrate (PNFA) and after adding 5
mL carbonic anhydrase I
enzyme (hCA I) into the cell, respectively. The CV potential wave
The effects of different concentration (0.1e100 mM) of inhibitors

A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
337
Table 3
Selected FT-IR vibration bands of furan sulfonylhydrazones (cmꢀ1).
Compound
y
(NH)
n
CH(ar)
n
CH(alph)
C
n ]
N
n
SO2ðasÞ
n
CeOeC
n
SO2ðsÞ
d
NH
d
SO2
d
CH(ar)
2
3231
3224
3220
3018
3028
3021
2929
852
2937
853
2930
860
1622
1634
1333
1346
1358
1227
1164
1162
1166
1031
1067
637
639
626
510
541
564
757
755
779
2
3
4
2
1642
1623
2
Fig. 4. FT-IR spectrum of furan sulfonylhydrazones (2e4).
Table 4
Crystal data and structure refinement details for compound 4.
Chemical formula
6 7 3 5
C H N O S
Formula weight
Temperature (K)
233.22
293(2)
Wavelength (Å)
0.71073
Crystal system, space group
Triclinic, P1
Unit cell dimensions:(Å)
a
b
c
a
b
g
7.6005(15)
8.2485(17)
16.226(3)
77.718(5)
86.492(5)
78.812(5)
974.9(3)
4
3
Volume (Å )
Z
Absorbtion coefficient (mmꢀ1)
Calculated density (Mg m 3)
F(000)
0.339
1.589
480
ꢀ
Crystal size (mm)
Theta range for data collection( )
0.25 ꢂ 0.17 ꢂ 0.11
ꢁ
2.57e28.49
Limiting indices
Reflections collected
ꢀ9 ꢃ h ꢃ 10, ꢀ10 ꢃ k ꢃ 11, ꢀ21 ꢃ l ꢃ 21
11,455
Independent reflections
Number of reflections used
Number of parameters
Max. and min. transmission
Refinement method
4952
3617
273
0.933, 0.963
Ful-matrix least-squares on F2
Final R indices [I ꢄ 2
s
(I)]
R
R
1
¼ 0.0514, wR
2
¼ 0.1552
R indices (all data)
1
¼ 0.0660, wR
2
¼ 0.1720
2
Goodness -of- fit (GOF) on F
1.092
0.488 and ꢀ0.571
Largest difference in peak and hole (e Åꢀ3)

338
A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
Table 5
Selected bond distances [Å] and angles [ ] of compound 4.
Table 6
ꢁ
Calculated global reactivity descriptors in eV.
S1eO4
S1eO5
S1eN3
N2eN3
1.4239 (18)
1.4265 (19)
1.647 (2)
1.385 (3)
1.748 (3)
S2eO9
S2eO10
S2eN6
N5eN6
1.4326 (18)
1.4162 (18)
1.649 (2)
1.385(2)
1.749 (3)
Compound
ε
LUMO
ε
HOMO
c
m
h
S
u
2
3
4
ꢀ0.0556 ꢀ0.2165 0.1361 ꢀ0.1361 0.0805 0.0403 0.1151
ꢀ0.1000 ꢀ0.2234 0.1617 ꢀ0.1617 0.0617 0.0309 0.2119
ꢀ0.1133 ꢀ0.2560 0.1847 ꢀ0.1847 0.0714 0.0357 0.2389
S1eC6
S2eC12
O4eS1eO5
O4eS1eN3
N2eN3eS1
N3eS1eC6
119.75 (12)
107.14 (11)
115.20 (15)
106.32 (13)
O9eS2eO10
O10eS2eN6
N5eN6eS2
N6eS2eC12
119.38 (12)
107.22 (11)
115.50 (7)
109.27 (7)
on the reduction peak of product (PNF) formed by the interaction of
the enzyme with the substrate were investigated by DPV method.
As seen in Fig. 7, the reduction peak (at about ꢀ0.2 V) belongs to
PNF is observed by negative scan in DPV and so, the decreasing rate
of PNF reduction peak is employed to assay the hCA I inhibition
activity of compound 4. Accordingly, analysis over a wider range of
inhibitor concentrations (0.1e100
mM) show that the gradual
decrease in PNF peak current intensity with increasing inhibitor
concentration reveals the inhibition effects of the inhibitors against
hCA I isoenzyme [44]. When the inhibitory activities of the com-
pounds (1e4) compared with the standard (acetazolamide, AAZ),
2
compound 4 having NO group is found to has good inhibitory
properties than others. Sulfonylhydrazone containing acetyl CH
3
group has lower enzyme inhibition activity than other furan sul-
fonylhydrazones. Methane sulfonic acid hydrazide has poor activity
which resulting of absence of active sites such as imine, furan, nitro
etc. [45].
4
. Conclusion
In this study, the synthesis of new furan sulfonylhydrazones
from methane sulfonic acide hydrazide is reported. The structural
characterization of the synthesized compounds (2e4) was made by
using the elemental analyses and spectroscopic methods. Based on
physicochemical evidence, the proposed structure of compounds
2
e4 are exhibited in Fig. 2. The structure of 5-nitro-2-fur-
aldehydemethanesulfonylhydrazone is also supported by X-ray
crystal diffraction. The enzyme inhibition effects of the compounds
Fig. 6. LineweavereBurk (a) and Activity % (b) graphs of compound 4.
are evaluated using activity parameters (K
m i
, IC50 and K ) found by
spectrophotometric method. The remarkable inhibition efficiency
Fig. 5. Crystal structure of compound 4 (ortep diagram (a) and package model (b)).

A.B. Gündüzalp et al. / Journal of Molecular Structure 1105 (2016) 332e340
339
Table 7
Enzyme inhibition results of furan sulfonylhydrazones (2e4) and AAZ.
Compound
Inhibitor
Km (M)
IC50 (M)
Ki (M)
Inhibition type
ꢀ
ꢀ
ꢀ
5
5
5
5
1.12 ꢂ 10ꢀ3
2.55 ꢂ 10ꢀ5
2
24.21 ꢂ 10
Competitive
2
1
1
0.78 ꢂ 10
7.51 ꢂ 10
6.24 ꢂ 10ꢀ
ꢀ
ꢀ
ꢀ
5
5
5
5
1.10 ꢂ 10ꢀ3
1.28 ꢂ 10ꢀ5
3
24.74 ꢂ 10
Competitive
2
1
1
1.30 ꢂ 10
8.04 ꢂ 10
6.23 ꢂ 10ꢀ
ꢀ
ꢀ
ꢀ
5
5
5
5
1.02 ꢂ 10ꢀ4
5.47 ꢂ 10ꢀ5
5.88 ꢂ 10ꢀ6
7.03 ꢂ 10ꢀ6
4
25.30 ꢂ 10
Competitive
Competitive
2
1
1
0.80 ꢂ 10
7.60 ꢂ 10
5.80 ꢂ 10ꢀ
ꢀ
ꢀ
ꢀ
5
5
5
5
AAZ
64.94 ꢂ 10
5
4
3
5.96 ꢂ 10
6.97 ꢂ 10
9.64 ꢂ 10ꢀ
Science and Technology Application and Research Center, for the
use of the Bruker SMART BREEZE CCD diffractometer (purchased
under grant No. 2010K120480 of the State of Planning Organiza-
tion). I'm thankful to my mother (Gül s¸ ahin Balaban, died in July 13,
2014) for her support in my life.
Appendix A. Supplementary data
CCDC 1409649 contain the supplementary crystallographic data
for complex. This data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/data_request/cif, (or from the Cambridge
Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: þ44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or
www://www.ccdc.cam.ac.uk).
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