Facile synthesis and characterization of novel pyrazole-sulfonamides and their inhibition effects on human carbonic anhydrase isoenzymes

Article abstract of DOI:10.1016/j.bmc.2012.11.012

In the current study, a series of pyrazole-sulfonamide derivatives (2-14) were synthesized, characterized, and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these sulfonamides were confirmed by FT-IR, ¹H NMR, ¹³C NMR and LC-MS analysis. ¹H NMR and ¹³C NMR revealed the tautomeric structures. hCA I and hCA II isozymes were purified from human erythrocytes and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied. The K_i values of compounds were 0.062-1.278 μM for hCA I and 0.012-0.379 μM for hCA II. The inhibition effects of 7 for hCA I and 4 for hCA II isozymes were almost in nanomolar concentration range.

Full text of DOI:10.1016/j.bmc.2012.11.012

Bioorganic & Medicinal Chemistry 21 (2013) 21–27
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Facile synthesis and characterization of novel pyrazole-sulfonamides
and their inhibition effects on human carbonic anhydrase isoenzymes
a
b
a
b
b
c,d
_
Havva Balseven , M. Mustafa Ißsgör , Samet Mert , Zuhal Alım , Sßükrü Beydemir , Salim Ok
,
Rahmi Kasımog˘ulları a,
⇑
a Dumlupinar University, Art and Science Faculty, Department of Chemistry, 43100 Kütahya, Turkey
b Ataturk University, Faculty of Sciences, Department of Chemistry, Biochemistry Division, 25240 Erzurum, Turkey
c Institut für Chemie, Barbarastr. 7, Universität Osnabrück, 49069 Osnabrück, Germany
d King Fahd University of Petroleum and Minerals (KFUPM), Faculty of Natural Sciences, Chemistry Department, 31261-5048 Dhahran, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
In the current study, a series of pyrazole-sulfonamide derivatives (2–14) were synthesized, characterized,
and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were
investigated as in vitro. Structures of these sulfonamides were confirmed by FT-IR, 1H NMR, 13C NMR
and LC–MS analysis. 1H NMR and 13C NMR revealed the tautomeric structures. hCA I and hCA II isozymes
were purified from human erythrocytes and inhibitory effects of newly synthesized sulfonamides on
esterase activities of these isoenzymes have been studied. The Ki values of compounds were 0.062–
Received 11 September 2012
Revised 1 November 2012
Accepted 4 November 2012
Available online 27 November 2012
Keywords:
Pyrazole
Sulfonamide
Diazonium
Carbonic anhydrase
Inhibition
1.278
lM for hCA I and 0.012–0.379 lM for hCA II. The inhibition effects of 7 for hCA I and 4 for hCA
II isozymes were almost in nanomolar concentration range.
Published by Elsevier Ltd.
1. Introduction
neurological diseases.5 For this reason, there are several attempts
to illuminate the inhibition mechanism of CA and to synthesize
new compounds with higher inhibition potential. Novel CA inhib-
itors have been designed for pharmacological and medicinal ap-
proaches, and many inhibitors have been synthesized recently.6
Pyrazole derivatives have attracted great attention due to
widespread applications in pharmaceutical and agrochemical
industries.7–9 The presence of pyrazole as the central core in the
biologically active molecules has grown rapidly in the past
decade.7,10,11 Among various properties of pyrazole derivatives, for
example, anti-bacterial, anti-inflammatory, anti-obesity, anti-
depressant, anticoagulant, leishmanicidal and cytotoxic activities
have been reported.12–18
The present study was carried out in order to synthesize,
characterize and evaluate biological activity of a new series of pyr-
azole-sulfonamide derivatives. In this current study, our main aim
was to check whether the new derivatives may show higher
inhibitory effect on CA enzymes with respect to the compounds
previously investigated. The synthesized molecules have been
characterized by various techniques including NMR, FT-IR and
LC–MS. For in vitro inhibition studies human erythrocyte carbonic
anhydrase (CA I and CA II) isoenzymes were purified using Sephar-
Carbonic anhydrase (CA), a metalloenzyme (EC 4.2.1.1) contain-
ing Zn+2 ion in its actÀiv1e,2 region catalyses the hydration of CO2 and
dehydration of HCO3
Beside the hydration reaction of CO2, CA
catalyses the reaction of cyanamide to carbamic acid or urea to
cyanamide, hydration of aldehydes to geminal diol. It also hydro-
lyses carboxylic, sulfonic, and phosphoric acid esters.3
Sixteen CA isoenzymes isoenzymes have been identified till
now. It was shown that the vital functions of these isoenzymes
differ according to tissues and organs. Lung, kidney, gastric muco-
sa, eye lenses, salivary glands, muscles, nerve myelin sheath, pan-
creas, prostate and endometrial tissues come first among these
tissues. CA enzyme is characterized from most of these tissues
and functions of it are being tried to be determined.4
Carbonic anhydrase inhibitors (CAIs) block the function of
carbonic anhydrase enzyme. Primary sulfonamides, having the
molecular formula of RSO2NH2 are the most well-known CA
inhibitors. Sulfonamides, like acetazolamide, are useful antiglauco-
ma agents. However, the usages of sulfonamides are limited
because of numerous side effects. Sulfonamides, as well as the
usage as antiglaucoma agent, are used to treat cancer and some
ose-4B
L-tyrosine-sulfanilamide affinity chromatography. The
inhibitory effects of novel compounds on the activities of purified
human erythrocyte CA I and CA II isozymes were investigated.
⇑
Corresponding author.
E-mail address: rahmikasimoglu@hotmail.com (R. Kasımog˘ulları).
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.11.012

22
H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
and 13C NMR results showed that 5, 7 and 9–12 show tautomeric
2. Results and discussion
2.1. Chemistry
structures while 6 and 8 prefer keto-hydrazo form (See Fig. 1).
The relative integral of the enol and keto peaks of compounds 5,
7, 9, 10, 11, 12 are 1:0.95, 1:4.55, 1:6.69, 1:7.44, 1:11.90, 1:5.44,
respectively. Compound 5 has almost equally probable tautomeric
structures. Compounds 7 and 9–12 favor keto-hydrazo form with
respect to the enol form on the NMR time scale. As the –R moiety
is varied from the phenyl group to the alkyl groups, the novel mol-
ecules start showing tautomeric forms. In other words, the phenyl
group contributes to the stability of the molecule keeping only one
form rather than showing resonance structures.
On the other hand, the thermal decomposition of the compounds
upon heating did not allow the determination of melting tempera-
tures (see Fig. 2 and Table 1 showing the TGA results). Compound
12 exhibited the lowest degradation temperature while compound
2 had the highest value. Compounds 4, 13 and 14 started degrada-
tion right above 280 °C. Interestingly, 7, 8 and 9 began decomposi-
tion in the same temperature range of 215–225 °C. Such similar
thermal degradation temperatures are attributed to similar chemi-
cal structures. We think that the release of the –R moiety occurred at
relatively elevated temperatures. It was even observed that the TGA
In this study first, ethyl 1-(3-nitrophenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylate (2) was
synthesized by the reaction of ethyl 3-(chlorocarbonyl)-1-(3-nitro-
phenyl)-5-phenyl-1H-pyrazole-4-carboxylate (1) with 4-amino
benzenesulfonamide. Subsequent reduction of the nitro group of
2 by sodium polysulphur hydrogenation (Na2S/S/H2O) afforded 3.
The spectral analysis indicated the simultaneous hydrolysis of
the ester group of 2 with the reduction reaction. The hydrolysis
of the reactant (2) is explained by heating the mixture with HCl
solution. Compound 3 then underwent diazoniation and final reac-
tions with phenol, 2-naphthol and 1,3-dicarbonyl compounds to
yield novel pyrazole-sulfonamides (4–13). A nitrile derivative
(14) was also synthesized by interference of diazonium compound
with KCN. The synthesis of compounds is shown in Scheme 1.
Isotropic chemical shifts of Ar–OH and NH appear as downfield
shifts in 1H NMR spectra. The peaks of Ar–OH and NH are used to
distinguish between possible tautomeric structures. The 1H NMR
SO2NH2
SO2NH2
O
O
O
O
EtO
Ph
Cl
O
O
N
EtO
Ph
N
H
HO
Ph
N
H
H2N
THF, reflux, 5 h
N
SO2NH2
N
N
N
N
HO
NO2
NO2
N
N
OH
1
2
O
H2
/
l
C
H
4
,
x
t
S
E
a 2
O
N
SO2NH2
SO2NH2
R2
R1
-CH3
-C6H5
-CH3
-C6H5
O
O
5
-CH3
O
N
O
6
7
8
9
-C6H5
HO
Ph
N
HO
Ph
N
H
H
-C6H5
HCl, NaNO2, 0 °C
CH3-OH
-OEt
-OEt
-OEt
-OCH3
-OC(CH3)3
N
N
-CH3
N
-CH2CH2CH3
-CH(CH3)2
-CH3
10
11
12
Cl
N2
NH2
3
R1
OH
KCN
O
R2
CH
3
-CH
2
-OH
O
SO2NH2
SO2NH2
SO2NH2
O
O
O
O
N
O
O
N
HO
Ph
NH
N
HO
Ph
N
H
HO
Ph
NH
N
N
N
N
O
N
O
N
R1
N
H
OH
R2
CN
14
13
5-12
Scheme 1. Synthesis of target inhibitors 2–14.

H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
23
SO2NH2 H2NO2S
In the current study, isoenzymes of CA I and CA II were puri-
fied from human erythrocytes, intensive resource of CAs. For the
purification procedure Sepharose-4B L-tyrosine affinity chroma-
O
O
O
N
O
tography method was employed. Following the purification step,
the inhibitory effects of compounds from 2 to 14 on the activi-
ties of purified human erythrocyte CA I and CA II isozymes were
studied. It was not possible to investigate the inhibitory effect of
3. Ki values of the compounds are given in Table 2. Almost all of
the compounds inhibited the isoenzymes in a noncompetitive
manner, except compounds 4 and 7. Compound 7 inhibited CA
I and similarly compound 4 inhibited CA II uncompetitively.
Interestingly the compounds showing the uncompetitive inhibi-
tory effects on CA I and CA II had the smallest Ki values. The
strongest inhibitor on CA I was the compound 7 with a Ki value
HO
Ph
NH
N
HN
N
OH
Ph
N
N
N
O
O
R3
N
N
R1
H
H
R2
R4
O
O
Keto-Hydrazo
Enol-Azo
Figure 1. Only keto-hydrazo for (6,8) and tautomeric structures of keto-hydrazo
and enol-azo for (5,7, 9–12).
of 0.0623
lM. The compound 4 showed the strongest inhibition
on CA II with a Ki value of 0.0124
lM. Ki values of these two
compounds are almost in nanomolar concentration range.
According to decreasing inhibition power on CA I, compounds
can be ranked as 7, 2, 8, 6, 4, 12, 5, 10, 11, 14, 13 and 9. The
inhibition decreasing list of CA II is in the order of 4, 12, 10,
8, 6, 11, 2, 7, 9, 5, 13 and 14 (See Table 2). The compounds have
smaller Ki values for CA II in general. The smaller the Ki values
mean higher inhibitory effect.
100
2
3
4
90
5
6
7
80
70
60
50
The structure–activity relationship (SAR) mainly depends on
the tautomeric structures and the –R moiety. Compounds 8, 6
and 4 being stable only in one form depict higher inhibition effect
on both of the enzymes CA I and II. However, compounds 7, 10, 11
and 12 having resonance structures do not show consistent inhibi-
tion effect. For instance, compound 10 has relatively lower inhibi-
tion effect on CA I than CA II. Both of the compounds 13 and 14
exhibit the lowest inhibition on the enzymes. These results indi-
cate that varying the –R moiety has significant effect on the biolog-
ical activity of the sulfonamide derivatives. The compound 4
including 2-naphtol group is the most effective over CA II isoen-
zyme. We proposed that the OH group in this compound increase
the inhibition effect by interacting with other amino acids in CA II
enzyme. The values obtained for this compound are consistent
with one of our previous works.21 Also the coupling reactions of
3 with 1,3-dicarbonyl compounds gave eight novel sulfonamide
derivatives. Although inhibition effects of these compounds are
relatively weak compare with 4, the values are still quite good
8
9
10
11
12
13
14
0
100
200
Temp (°C)
300
400
500
Figure 2. TGA results of compounds 2–14.
Table 1
Thermal decomposition temperatures of the compounds
Compound
Thermal degradation temperature (°C)
2
3
4
5
6
7
8
9
300
210
280
265
187
210
218
225
195
183
120
280
280
(0.0623–1.278 lM for CA I and 0.0954–0.259 lM for CA II). Among
these compounds, compound 10 includes propyl and ethoxy
groups as R substituent. Also 12 includes methyl and t-butoxide.
It is estimated that these compounds have relatively less inhibition
effects on CA II because of the steric effect of whole or some part of
these R substituents. Ki values of 10 and 12 (0.09 and 0.111 lM for
10
11
12
13
14
CA II, respectively) are consistent with the Ki values of the
compounds, which have similar R substituents, in our previous
studies.22,23 Although the nitrile substituent in 14 is less bulky
according to others, this compound exhibited less inhibition effect
on CA II. Ki value of this compound (0.379
lM for CA II) is consis-
tent with the Ki value (0.410
lM for CA II) of nitrile derivative in
pans were enlarged at the end of the experiments, which is ex-
plained by the release of the –R moiety to the gas phase. The –R moi-
ety release did not allow to performing DSC experiments for
determining the melting temperatures of the molecules.
our previous study.21
Our inhibition type results (Table 2) in this study are consistent
with those of our previous ones.24 Almost all of the chemicals
inhibited the enzyme noncompetitively. However, Lineweaver–
Burk graphs of the compounds 7 and 4 depict uncompetitive inhi-
bition on CA I and CA II, respectively (Fig. 3). In an uncompetitive
inhibition, inhibitors bind to enzyme-substrate complex not to
the enzyme itself. The inhibitor prevents formation of the products
by binding to this complex. Small Ki values indicate the stability of
enzyme-substrate-inhibitor complex and production of fewer
amount of the products. Consequently, 4 and 7 are better inhibitors
than the other compounds on activity of CA isozymes.
2.2. Biological activity studies
CA inhibitors (CAIs) have been used in many pharmacological
applications including treatment of glaucoma, diuretics, anticon-
vulsants, antiobesity and/or antitumor agent/diagnostic tools.19
Since CA II plays an important role in the production of aqueous
humor in humans,20 CA II inhibitors are particularly used in the
treatment of glaucoma, epilepsy, and used as diuretics.

24
H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
Table 2
Ki values and inhibition types of chemicals for CA I and CA II
_
hCA I
hCA II
Inhibitor
Ki (lM)
Inhibition type
Ki (l
M)
Inhibition type
2
3
0.102 0.0503
—
Noncompetitive
—
0.144 0.0227
—
Noncompetitive
—
4
5
6
7
8
9
10
11
12
13
14
0.318 0.159
0.448 0.202
0.219 0.115
0.0623 0.0227
0.115 0.0266
1.278 0.046
0.485 0.0887
0.490 0.191
0.384 0.174
1.079 0.437
0.806 0.0202
Noncompetitive
Noncompetitive
Noncompetitive
Uncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
0.0124 0.0037
0.259 0.0713
0.128 0.0424
0.159 0.0298
0.126 0.0482
0.197 0.0503
0.111 0.0123
0.136 0.0743
0.0954 0.0398
0.349 0.0144
0.379 0.0764
Uncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Changes in CA activity can be related with diabetes and related
metabolic diseases such as hypertension.25 The inhibition studies
on CAs help in understanding mechanisms of enzyme catalysis.
Studies about the enzyme’s tissue distribution and understanding
the vital functions of the enzymes in the tissues gained signifi-
cance. Due to such important factors, syntheses of enzyme inhibi-
tors and activators attract more attention.3,23,26 Up to now,
inhibitory effects of various different anions, metal ions, drugs,
phenols and sulfonamides, functioning as specific inhibitors, have
been investigated on the activity of CA isozymes.22,27–30 In the cur-
rent contribution, the compounds showed strong inhibitory effects
on CA isozymes, especially on CA II. Among these compounds, 4
and 7 one are powerful inhibitors for CA and may be used in the
generation of potent CAIs.
use. All reactions were monitored by analytical thin-layer chroma-
tography (TLC) on 0.25 mm pre-coated Kieselgel 60F 254 plates
(Merck); compounds were visualized by Camag TLC devices UV
(254 and 366 nm).
3.2. Characterization
NMR: All the compounds were dissolved in DMSO-d for NMR
measurements. NMR experiments were performed on a JEOL 500
Lambda instrument (operating at 500 MHz for 1H, 125 MHz for
13C) with a multi-nuclei 5 mm solution probe (50TH5/FG2). The
1H NMR spectra were acquired with recycle delay time of 10 s,
and with 32 repetitions. The proton spectra were internally refer-
enced to the signal of the solvent at 2.50 ppm. The 13C NMR spectra
were acquired by cross-polarization pulse sequence including pro-
ton decoupling with 2 s recycle delay time and with 1024 repeti-
tions. The 13C NMR spectra were again internally referenced to
the solvent signal at 39.5 ppm.
3. Experimental protocols
3.1. Chemicals
FT-IR: The FT-IR transmission spectra of each compound were
recorded on Bruker Optics, Vertex 70 FT-IR spectrometer. For each
sample, 100 scans were taken at a resolution of 4 cmÀ1 with N2
exposure.
LC–MS: The LC–MS data were acquired by positive ionization on
a Bruker Esquire HCT connected with an electrospray ionization
unit.
Deuterated dimethyl sulfoxide (d-DMSO) with 99.98% purity,
ethyl benzoyl acetate, benzoyl acetone, CNBr-activated Sepharose
4B, protein assay reagents, and chemicals for electrophoresis were
purchased from Sigma-Aldrich. Acetyl acetone, 2-naphtol, sodium
azide, sodium nitrite, L-tyrosine, sodium acetate, and solvents such
as propanol, ethanol and tetrahydrofuran were purchased from
Merck. Dibenzoyl methane and 4-aminobenzene sulfonamide were
obtained from Fluka. Tetrahydrofuran was freshly distilled before
TGA: The thermal decomposition temperatures were measured
with a Thermogravimetric analyzer. TGA measurements were per-
Control
Control
Control
Control
14
14
[I1]=0,0158 µM
[I1]=0,0307
µ
M
[I1]=0,0158 µM
12
[I1]=0,0307 µM
12
[I2]=0,079 µM
[I2]=0,107
µ
M
M
[I2]=0,079 µM
10
[I2]=0,107 µM
10
[I3]=0,158 µM
[I3]=0,230
µ
[I3]=0,158 µM
8
6
4
2
0
[I3]=0,230 µM
8
6
4
2
0
-4
-2
0
2
4
6
8
-4
-2
0
2
4
6
8
1/[NPA]
1/[NPA]
1/[NPA]
1/[NPA]
4
7
Figure 3. Lineweaver–Burk graphs of the compound 4 on CA II and compound 7 on CA I.

H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
25
formed on Netzch STA 449 C (Selb, Germany) with the component
1732 (C@O, acid), 1683 (C@O, amide), 1591–1447 (C@N and
C@C), 1318 and 1151 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm): 15.47 (s, 1H, Ar–OH), 14.61 (br, s,
1H, CONH), 8.20–6.76 (m, 19 H, ArH), 7.30 (s, 2H, SO2NH2); 13C
NMR (125 MHz, DMSO-d6) d (ppm): 165.9 (C@O, acid), 160.9
(C@O, amide), 146.1, 145.8, and 119.9 (pyrazole C-3, C-5 and C-
4), 145.2 (@CÀOH), 142.9 (@C–N@N), 141.6, 140.7, 138.6, 132.9,
131.0, 130.8, 130.3, 129.9, 129.8, 129.5, 129.2, 128.3, 128.3,
127.2, 126.9, 124.7, 1221.0, 119.4, 114.7, 108.7; MS (ESI): m/z
633.1 [M+1] (calcd for C33H24N6O6S: 632.15).
of DSC (/TG) HIGH RG 4 unit, at a heating rate of 10 °C minÀ1 under
a helium purge of 65 ml minÀ1
.
3.3. Synthesis
Compound 1 was prepared as the starting material by following
the previous literature reported procedures.23
3.3.1. Ethyl 1-(3-nitrophenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylate (2)
Compound 1 (0.399 g, 1 mmol) was dissolved in 20 mL dry THF.
To this solution 4-aminobenzenesulfonamide (0.344 g, 2 mmol)
was added. Mixture was refluxed for 5 h and solvent was evapo-
rated. The crude product was washed with water and crystallized
3.3.5. 1-(3-[(2-Hydroxy-4-oxopent-2-en-3-yl)diazenyl]phenyl)-
5-phenyl-3-(4-sulfamoylphenyl carbamoyl)-1H-pyrazole-4-carb
oxylic acid (5)
Synthesized from
3 (0.477 g, 1 mmol) and acetylacetone
from ethanol. Yield: 96%; IR (m
, cmÀ1): 3353 and 3221 (NH), 3059
(0.103 ml, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 95%;
(ArCH), 2980 (Aliphatic CH), 1690 (C@O, ester), 1670 (C@O, amide),
1594–1427 (C@N and C@C), 1322 and 1131 (S@O, assym. and
sym.); 1H NMR (500 MHz, DMSO-d6) d (ppm): 11.02 (s, 1H, CONH),
8.19–7.32 (m, 13H, ArH), 7.30 (s, 2H, SO2NH2), 4.04 (q, J = 7.1 Hz,
2H, OCH2), 0.95 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (125 MHz,
DMSO-d6) d (ppm): 162.3 (C@O, ester), 160.9 (C@O, amide),
148.6 (@CÀNO2), 148.2, 146.2 and 120.1 (pyrazole C-3, C-5 and
C-4), 61.2 (OCH2), 14.1 (CH3), 141.9, 139.4, 132.2, 131.2, 130.7,
130.5, 129.0, 127.8, 127.3, 123.9, 120.9, 113.9; MS (ESI): m/z
536.1 [M+1] (calcd for C25H21N5O7S: 535.12).
IR (m
, cm–1): 3500–2500 (COOH), 3257 (NH), 3064 (ArCH), 2986
(Aliphatic CH), 1720 and 1671 (C@O), 1589–1430 (C@N and
C@C), 1313 and 1159 (S@O, assym. and sym.); 1H NMR
(125 MHz, DMSO-d6) d (ppm): 13.69 (s, 1H, C@CÀOH), 11.59 (br,
s, 1H, CONH), 7.86–7.08 (m, 13H, ArH), 7.29 (s, 2H, SO2NH2), 2.39
and 2.26 (s, 6H, 2CH3); 13C NMR (125 MHz, DMSO-d6) d (ppm):
197.8 and 197.3 (C@O, keton), 164.2 (C@O, acid), 161.4 (C@O,
amide), 147.1 (@C–N@N), 146.1, 145.9 and 120.3 (pyrazole C-3,
C-5 and C-4) 31.6 and 26.8 (CH3), 142.8, 141.9, 139.9, 139.3,
135.1, 134.6, 129.9, 128.2, 122.6, 121.8, 116.9, 114.9, 113.5; MS
(ESI): m/z 611.1 [M+Na] (calcd for C28H24N6O7S: 588.14).
3.3.2. 1-(3-Aminophenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (3)
Compound 3 containing aromatic amine group was prepared as
described previously21 and crystallized from methanol. Yield: 75%;
3.3.6. 1-(3-[2-(1,3-Dioxo-1,3-diphenylpropan-2-ylidene)hydrazi
nyl]phenyl)-5-phenyl-3-(4-sulfamoylphenylcarbamoyl)-1H-pyr
azole-4-carboxylic acid (6)
IR (m
, cm–1): 3500–2500 (COOH), 3308 (NH), 3176 and 3053
(ArCH), 1706 (C@O, acid), 1630 (C@O, amide), 1587–1421 (C@N
and C@C), 1324 and 1151 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm): 11.03 (s, 1H, CONH), 7.83–6.26 (m,
13H, ArH), 7.29 (s, 2H, SO2NH2), 6.54 (m, 2H, Ar–NH2); 13C NMR
(125 MHz, DMSO-d6) d (ppm): 164.0 (C@O, acid), 161.7 (C@O,
amide), 149.7 (@CÀNH2), 146.3, 146.0, and 120.4 (pyrazole C-3,
C-5 and C-4), 141.9, 139.6, 139.4, 130.5, 129.7, 128.8, 128.6,
127.2115.2, 114.5, 113.8, 111.7; MS (ESI): m/z 478.0 [M+1] (calcd
for C23H19N5O5S: 477.11).
Synthesized from 3 (0.477 g, 1 mmol) and dibenzoylmethane
(0.224 g, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 79%;
IR (m
, cm–1): 3500–2500 (COOH), 3256 (NH), 3059 (ArCH), 1722
and 1625 (C@O), 1591–1447 (C@N and C@C), 1327 and 1156
(S@O, assym. and sym.); 1H NMR (500 MHz, DMSO-d6) d (ppm):
11.66 (s, 1H, Ar–NH –N@), 11.05 (s, 1H, CONH), 8.09–6.79 (m,
23H, ArH), 7.30 (s, 2H, SO2NH2); 13C NMR (125 MHz, DMSO-d6) d
(ppm): 194.2 and 191.1 (C@O, benzoyl), 163.8 (C@O, acid), 161.7
(C@O, amide), 145.9, 144.0 and 120.3 (pyrazole C-3, C-5 and C-
4), 141.9, 139.7, 139.4, 138.2, 137.1, 136.6, 136.3, 134.9, 133.6,
130.5, 130.4, 129.6, 129.4, 129.0, 128.9, 128.7, 128.6, 127.7,
127.3, 115.6, 114.4, 93.7; MS (ESI): m/z 735.2 [M+Na] (calcd for
3.3.3. General procedure for the syntheses of compounds 4–13
1-(3-aminophenyl)-5-phenyl-3-(4-sulfamoylphenylcarba-
moyl)-1H-pyrazole-4-carboxylic acid (3) (0.477 g, 1 mmol) was
dissolved in a mixture of methanol (30 ml) and concentrated
hydrochloric acid (2 ml). The solution was then cooled to 0–5 °C.
Sodium nitrite (0.104 g, 1.5 mmol) in water (10 ml) was then
added to this solution dropwise with vigorous stirring while keep-
ing at 0À5 °C. After dissolving an aromatic or b-dicarbonyl com-
pound (1 mmol) in a sufficient amount of ethanol, the solution
was cooled and added dropwise into the already prepared diazo-
nium salt solution. The pH of the coupling mixture, in each case,
was maintained at 7À8 through the coupling process by adding
aqueous sodium acetate. Stirring was continued for 1 h at 0À5 °C
and 2 h at room temperature. The precipitated products were fil-
tered off, washed with water several times, dried, and recrystal-
lized from an appropriate solvent.
C38H28N6O7S: 712.17).
3.3.7. 1-(3-[(3-Hydroxy-1-oxo-1-phenylbut-2-en-2-yl)diazenyl]
phenyl)-5-phenyl-3-(4-sulfamoylphenylcarbamoyl)-1H-
pyrazole-4-carboxylic acid (7)
Synthesized from 3 (0.477 g, 1 mmol) and benzoylacetone
(0.162 g, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 86%;
IR (m
, cm–1): 3500–2500 (COOH), 3258 (NH), 3060 (ArCH), 2970
(aliphatic CH), 1721 (C@O), 1591–1421 (C@N and C@C), 1323
and 1156 (S@O, assym. and sym.); 1H NMR (500 MHz, DMSO-d6)
d (ppm): 13.74 (s, 1H, C@CÀOH enol tautomer), 11.15 (s, 1H,
Ar–NH –N@ keto tautomer), 11.04 (s, 1H, CONH), 7.86–6.89 (m,
18H, ArH), 7.29 (s, 2H, SO2NH2), 2.38 (s, 3H, CH3); 13C NMR
(125 MHz, DMSO-d6) d (ppm): 196.9 (C@O, acetyl), 195.4 (C@O,
benzoyl), 163.9 (C@O, acid), 161.6 (C@O, amide), 147.4, 146.1,
120.3 (pyrazole C-3, C-5, C-4), 25.3 (CH3), 144.1, 141.8, 140.0,
139.7, 139.4, 135.8, 135.1, 130.6, 129.6, 129.2, 128.8, 128.7,
127.2, 126.4, 120.4, 115.4, 114.4, 112.3, 97.4; MS (ESI): m/z 651.2
[M+1] C33H26N6O7S: 650,16).
3.3.4. 1-(3-[(2-Hydroxynaphthalen-1-yl)diazenyl]phenyl)-5-
phenyl-3-(4-sulfamoylphenyl carbamoyl)-1H-pyrazole-4-
carboxylic acid (4)
Synthesized from 3 (0.477 g, 1 mmol) and b-naphtol (0.144 g,
1 mmol) according to the general procedure. The crude product
was purified by crystallization from ethanol. Yield: 88%; IR (
m,
cm–1): 3590 (OH), 3500–2500 (COOH), 3250 (NH), 3060 (ArCH),

26
H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
3.3.8. 1-(3-[2-(1-Ethoxy-1,3-dioxo-3-phenylpropan-2-
ylidene)hydrazinyl]phenyl)-5-phenyl-3-(4-
crude product was purified by crystallization from ethanol. Yield:
88%; IR (
, cm–1): 3500–2500 (COOH), 3259 (NH), 3059 (ArCH),
m
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (8)
Synthesized from 3 (0.477 g, 1 mmol) and ethylbenzoylacetate
(0.172 ml, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 82%;
2971 (Aliphatic CH), 1735 and 1683 (C@O), 1593–1436 (C@N and
C@C), 1312 and 1158 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm):13.65 (s, 1H, C@CÀOH enol tauto-
mer), 11.47 (s, 1H, Ar–NH–N@ keto tautomer), 11.04 (s, 1H, CONH),
7.88–7.02 (m, 13H, ArH), 7.30 (s, 2H, SO2NH2), 3.76 (s, 3H, OCH3),
3.38 (m, 1H, CH(CH3)2) 0.98 (d, J = 6.8 Hz, 6H, CH(CH3)2); 13C
NMR (125 MHz, DMSO-d6) d (ppm): 200.6 (C@O, keton), 163.9
(C@O, acid), 163.4 (C@O, ester), 161.5 (C@O, amide), 147.4, 145.9,
120.3 (pyrazole C-3, C-5, C-4), 52.8 (OCH3), 34.3 (CH), 19.3
(2CH3), 143.4, 141.8, 139.8, 139.4, 131.0, 130.8, 129.9, 128.8,
128.5, 127.2, 121.1, 120.4, 115.8, 114.5, 112.3; MS (ESI): m/z
633.2 [M+1] (calcd for C30H28N6O8S: 632.17).
IR (m
, cm–1): 3500–2500 (COOH), 3253 (NH), 3055 (ArCH), 2971
(aliphatic CH), 1726 (C@O), 1590–1422 (C@N and C@C), 1326
and 1157 (S@O, assym. and sym.); 1H NMR (500 MHz, DMSO-d6)
d
(ppm): 11.89 (s, 1H, Ar–NH –N@), 11.02 (s, 1H, CONH),
7.86–6.83 (m, 18H, ArH); 7.29 (s, 2H, SO2NH2), 4.26 (q, J = 7.1 Hz,
2H, OCH2), 1.20 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (125 MHz,
DMSO-d6) d (ppm): 189.6 (C@O, benzoyl), 163.8 (C@O, acid),
162.7 (C@O, ester) 161.5 (C@O, amide), 147.4, 146.1, 120.3 (pyra-
zole C-3, C-5, C-4), 62.0 (OCH2), 14.3 (CH3), 143.5, 141.9, 139.8,
139.4, 137.3, 133.3, 130.6, 130.3, 130.1, 129.9, 128.9, 128.8,
128.7, 128.4, 127.2, 120.9, 115.9, 114.4, 112.7; MS (ESI): m/z
681.2 [M+1] (calcd for C34H28N6O8S: 680.17).
3.3.12. 1-(3-[(1-tert-Butoxy-3-hydroxy-1-oxobut-2-en-2-
yl)diazenyl]phenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (12)
Synthesized from 3 (0.477 g, 1 mmol) and t-butyl acetoacetate
(0.163 ml, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 86%;
3.3.9. 1-(3-[(1-Ethoxy-3-hydroxy-1-oxobut-2-en-2-yl)diazenyl]
phenyl)-5-phenyl-3-(4-sulfamoylphenylcarbamoyl)-1H-pyrazo
le-4-carboxylic acid (9)
IR (m
, cm–1): 3500–2500 (COOH), 3227 (NH), 3060 (ArCH), 2971
Synthesized from 3 (0.477 g, 1 mmol) and ethyl acetoacetate
(0.126 ml, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 91%;
(Aliphatic CH), 1738 and 1674 (C@O), 1592–1435 (C@N and
C@C), 1327 and 1151 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm):13.69 (s, 1H, C@CÀOH enol tauto-
mer), 11.87 (s, 1H, Ar–NH–N@ keto tautomer), 11.51 (s, 1H, CONH),
7.88–6.96 (m, 13H, ArH), 7.30 (s, 2H, SO2NH2), 2.24 (s, 3H, COCH3),
1.46 (s, 9H, C(CH3)3); 13C NMR (125 MHz, DMSO-d6) d (ppm): 194.9
(C@O, acetyl), 164.4 (C@O, acid), 161.9 (C@O, ester), 161.4 (C@O,
amide), 146.9, 146.1, 120.2 (pyrazole C-3, C-5, C-4), 84.0
(OC(CH3)3), 26.4 and 25.8 (CH3), 143.3, 142.1, 139.9, 139.2, 132.2,
130.7, 130.6, 129.8, 128.9, 128.7, 127.2, 121.2, 115.9, 115.7,
IR (m
, cm–1): 3500–2500 (COOH), 3260 (NH), 3060 (ArCH), 2970
(Aliphatic CH), 1722 and 1683 (C@O), 1590–1402 (C@N and
C@C), 1313 and 1158 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm): 13.89 (s, 1H, C@CÀOH enol tauto-
mer), 11.52 (s, 1H, Ar–NH–N@ keto tautomer), 11.29 (s, 1H, CONH),
7.88–6.97 (m, 13H, ArH), 7.29 (s, 2H, SO2NH2), 4.24 (q, J = 7.1 Hz,
2H, OCH2), 2.42 (s, 3H, COCH3) 1.21 (t, J = 7.1 Hz, 3H, CH3); 13C
NMR (125 MHz, DMSO-d6) d (ppm): 194.8 (C@O, acetyl), 164.0
(C@O, acid), 162.8 (C@O, ester), 161.5 (C@O, amide), 147.2, 146.1,
120.3 (pyrazole C-3, C-5, C-4), 62.0 (OCH2), 25.9 and 14.3 (CH3),
143.4 141.9, 139.9, 139.3, 132.4, 130.7, 130.6, 129.9, 128.8, 128.7,
127.2, 121.1, 115.8, 114.8, 112.6; MS (ESI): m/z 641.2 [M+Na]
(calcd for C29H26N6O8S: 618.15).
112.8; MS (ESI): m/z 669.2 [M+Na] (calcd for
C31H30N6O8S:
646.18).
3.3.13. 1-(3-[(4-Hydroxyphenyl)diazenyl]phenyl)-5-phenyl-3-
(4-sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid
(13)
Synthesized from 3 (0.477 g, 1 mmol) and phenol (0.094 g,
1 mmol) according to the general procedure. The crude product
was purified by crystallization from ethanol. Yield: 75%; IR (m,
3.3.10. 1-(3-[(1-Ethoxy-3-hydroxy-1-oxohex-2-en-2-
yl)diazenyl]phenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (10)
Synthesized from 3 (0.477 g, 1 mmol) and ethyl butyrylacetate
(0.160 ml, 1 mmol) according to the general procedure. The crude
product was purified by crystallization from ethanol. Yield: 86%;
cm–1): 3385 (OH), 3500–2500 (COOH), 3260 (NH), 3064 (ArCH),
1736 (C@O, acid), 1624 (C@O, amide), 1590–1457 (C@N and
C@C), 1312 and 1156 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm): 11.07 (s, 1H, CONH), 7.89–6.90 (m,
18H, ArH and ArOH), 7.30 (s, 2H, SO2NH2); 13C NMR (125 MHz,
DMSO-d6) d (ppm): 163.8 (C@O, acid), 161.9 (C@O, amide), 152.8
(@C–OH), 147.4, 146.4, and 120.4 (pyrazole C-3, C-5 and C-4),
145.5, 141.8, 139.7, 139.4, 130.7, 130.7, 130.6, 130.6, 130.5,
130.1, 128.8, 128.5, 127.2, 125.7, 123.8, 122.6, 118.5, 116.6,
114.6; MS (ESI): m/z 583.1 [M+1] (calcd for C29H22N6O6S: 582.13).
IR (m
, cm–1): 3500–2500 (COOH), 3259 (NH), 3064 (ArCH), 2970
(Aliphatic CH), 1735 and 1684 (C@O), 1592–1458 (C@N and
C@C), 1335 and 1157 (S@O, assym. and sym.); 1H NMR
(500 MHz, DMSO-d6) d (ppm):13.81 (s, 1H, C@CÀOH enol tauto-
mer), 11.46 (s, 1H, Ar–NH–N@ keto tautomer), 11.23 (s, 1H, CONH),
7.88–6.96 (m, 13H, ArH), 7.29 (s, 2H, SO2NH2), 4.24 (q, J = 7.1 Hz,
2H, OCH2), 2.66 (t, J = 7.2 Hz, 2H, COCH2CH2) 1.51 (hextet,
J = 7.3 Hz, 3H, CH2CH2CH3), 1.21 (t, J = 7.1 Hz, 3H, OCH2CH3), 0.85
(t, J = 7.3 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) d (ppm):
197.0 (C@O, keton), 164.0 (C@O, acid), 162.9 (C@O, ester), 161.5
(C@O, amide), 147.3, 146.0, 120.3 (pyrazole C-3, C-5, C-4), 61.4
(OCH2), 18.0 and 14.5 (CH2), 14.3 and 14.1 (CH3), 143.5, 141.9,
139.9, 139.3, 132.3, 130.7, 130.6, 129.9, 128.7, 128.6, 127.2,
120.9, 115.7, 114.9, 112.4; MS (ESI): m/z 647.2 [M+1] (calcd for
3.3.14. 1-(3-Cyanophenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (14)
The diazonium salt solution of 3 (0.477 g, 1 mmol) was pre-
pared according to the general procedure. After dissolving potas-
sium cyanide (2 mmol) in 2 ml water, the solution was cooled
and added dropwise into the already prepared diazonium salt
solution. Following the addition, the cold mixture is allowed to
warm up to room temperature. When the temperature reached
about 15 °C, the formation of nitrogen gas began. Then the solution
was placed on a steam bath and heated at 60 °C for 45 min to com-
plete the decomposition. Finally the resulting precipitate was fil-
tered under vacuum and dried. The residue was purified by
C31H30N6O8S: 646.18).
3.3.11. 1-(3-[(3-Hydroxy-1-methoxy-4-methyl-1-oxopent-2-en-
2-yl)diazenyl]phenyl)-5-phenyl-3-(4-
sulfamoylphenylcarbamoyl)-1H-pyrazole-4-carboxylic acid (11)
Synthesized from 3 (0.477 g, 1 mmol) and methyl isobutyrylac-
etate (0.158 ml, 1 mmol) according to the general procedure. The
crystallization from ethanol. Yield: 87%; IR (
(COOH), 3263 (NH), 3032 (ArCH), 2110 (CN), 1719 and 1624
m
, cm–1): 3500–2500

H. Balseven et al. / Bioorg. Med. Chem. 21 (2013) 21–27
27
(C@O), 1589–1424 (C@N and C@C), 1329 and 1155 (S@O, assym.
and sym.); 1H NMR (500 MHz, DMSO-d6) d (ppm): 11.06 (CONH),
7.88–6.99 (m, 13H, ArH), 7.30 (s, 2H, SO2NH2); 13C NMR
(125 MHz, DMSO-d6) d (ppm): 163.8 (C@O, acid), 161.7 (C@O,
amide), 147.5, 146.3 and 120.4 (pyrazole C-3, C-5, C-4), 116.9
(CN), 141.8, 140.8, 139.9, 139.4, 131.1, 130.7, 128.8, 128.5, 127.2,
126.4, 125.6, 122.6, 121.7, 116.1; MS (ESI): m/z 510.1 [M+Na]
(calcd for C24H17N5O5S: 487.10).
project (Grant No. 106T180). The facility of King Fahd University of
Petroleum and Minerals (KFUPM) is used, and KFUPM is gratefully
acknowledged for the support of this project. The authors are
thankful Stefan Walter and Kerstin Ruecker at Chemistry/Biology
Branch of Osnabrück University for LC/MS and TGA measurements,
respectively. S.O. is also grateful to German Science Foundation
(DFG) for the support of summer research stay at Osnabrück
University/Germany with Grant number STE 1127/15-1.
3.4. Biological activity evaluation
References and notes
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3.4.1. Hemolysate preparation
Fresh human blood obtained from the University Hospital Blood
center and erythrocytes were purified. After low-speed centrifuga-
tion (1.500 rpm for 15 min) and removal of plasma and buffy coat,
the red blood cells were isolated, washed twice with 0.9% NaCl, and
hemolyzed with 1.5 volumes of ice-cold water. Then, ghost and in-
tact cells were removed by high-speed centrifugation (20.000 rpm
for 30 min) at 4 °C and the pH of the hemolysate adjusted to 8.7
with solid Tris.
7. Machado, P.; Lima, G. R.; Rotta, M.; Bonacorso, H. G.; Zanatta, N.; Martins, M. A.
P. Ultrason. Sonochem. 2011, 18, 293.
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9. Sherman, T. D.; Duke, M. V.; Clark, R. D.; Sanders, E. F.; Matsumoto, H.; Duke, S.
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erythrocytes by affinity chromatography
Sepharose-4B L-tyrosine affinity chromatography column was
prepared according to our previous studies.21–24,31 During the
purification procedures of hCA I and hCA II, the absorbance was
measured at 280 nm to monitor protein elution by affinity chroma-
tog raphy. CO2-hydratase activity was determined in eluted frac-
tions and the active fractions were collected.32,33
10. Wei, F.; Zhao, B. X.; Huang, B.; Zhang, L.; Sun, C. H.; Dong, W. L.; Shin, D. S.;
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41, 80.
3.4.3. Hydratase activity assay
Carbonic anhydrase hydratase activity was assayed by follow-
ing the hydration of CO2 according to the method described by
Wilbur and Anderson.32
3.4.4. Esterase activity assay
18. Choi, W.-K.; El-Gamal, M. I.; Choi, H. S.; Baek, D.; Oh, C.-H. Eur. J. Med. Chem.
2011, 46, 5754.
19. Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 825.
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21. Kasımog˘ulları, R.; Bülbül, M.; Günhan, H.; Güleryüz, H. Bioorg. Med. Chem.
Esterase activity of human erythrocyte carbonic anhydrase was
assayed by following the change in absorbance at 348 nm of 4-
nitrophenyl acetate to 4-nitrophenolate ion over a period of
3 min at 25 °C using a spectrophotometer according to the method
described by Verpoorte et al.34
2009, 17, 3295.
22. Kasımogulları, R.; Bülbül, M.; Mert, S.; Güleryüz, H. J. Enzyme Inhib. Med. Chem.
˘
2011, 26, 231.
23. Kasımog˘ulları, R.; Bülbül, M.; Arslan, B. S.; Gökçe, B. Eur. J. Med. Chem. 2010, 45,
4769.
3.4.5. Quantitative protein determination
_
24. Sßen, E.; Alım, Z.; Duran, H.; Isßgör, M. M.; Beydemir, Sß.; Kasımog˘ulları, R.; Ok, S. J.
Quantitative protein determination was done by measuring the
absorbance at 595 nm according to Bradford, using bovine serum
albumin as a standard.35
Enzyme Inhib. Med Chem 2011, 651456. http://dx.doi.org/10.3109/14756366.
25. Gülcin, I.; Beydemir, S.; Büyükokurog˘lu, M. E. Biol. Pharm. Bull. 2004, 27, 613.
26. Beydemir, S.; Gülcin, I. J. Enzyme Inhib. Med. Chem. 2004, 19, 193.
27. Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 1583.
3.4.6. SDS–polyacrylamide gel electrophoresis
28. Coban, T. A.; Beydemir, S.; Gulcin, I.; Ekinci, D.; Innocenti, A.; Vullo, D.;
Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 5791.
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30. Ekinci, D.; Beydemir, S.; Kufrevioglu, O. I. J. Enzyme Inhib. Med. Chem. 2007, 22,
745.
The control of enzyme purity was carried out by using
Laemmli’s procedure in two different acrylamide concentrations,
3% and 8% for running and stacking gel, respectively.24,36
31. Beydemir, S.; Ciftci, M.; Ozmen, I.; Buyukokuroglu, M. E.; Ozdemir, H.;
Küfrevioglu, O. I. Pharmacol. Res. 2000, 42, 187.
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35. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
36. Laemmli, U. K. Nature 1970, 227, 680.
3.4.7. In vitro inhibition studies
The inhibitory effects of some synthesized sulfonamide deriva-
tives on carbonic anhydrase enzyme activity purified from human
erythrocyte were tested in triplicate at each concentration by using
esterase activity assay. CA activities were measured in the pres-
ence of different substrate concentrations as previously described.
Lineweaver–Burk curves were used for determination of Ki and
inhibitor type.24,37
37. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 57, 658.
Acknowledgements
The authors are very grateful to The Scientific and Research
Council of Turkey (TUBITAK) for providing financial support for this