Potential inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis and docking studies of new 1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1016/j.bmc.2017.05.005

In the last years, inhibition of carbonic anhydrase (CA) has emerged as a promising approach for pharmacologic intervention in a variety of disorders such as glaucoma, epilepsy, obesity, and cancer. As a consequence, the design of CA inhibitors (CAIs) is a highly dynamic field of medicinal chemistry. Due to the therapeutic potential of thiadiazoles as CAIs, new 1,3,4-thiadiazole derivatives were synthesized and investigated for their inhibitory effects on hCA I and hCA II. Although the tested compounds did not carry a sulfonamide group, an important pharmacophore for CA inhibitory activity, it was a remarkable finding that most of them were more effective on hCAs than acetazolamide (AAZ), the reference agent. Among these compounds, N′-((5-(4-chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (3) was found to be the most effective compound on hCA I with an IC₅₀ value of 0.14?nM, whereas N′-((5-(2-chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (1) was found to be the most potent compound on hCA II with an IC₅₀ value of 0.15?nM. According to molecular docking studies, all compounds exhibited high affinity and good amino acid interactions similar to AAZ on the both active sites of hCA I and hCA II enzymes.

Full text of DOI:10.1016/j.bmc.2017.05.005

Accepted Manuscript
Potential inhibitors of human carbonic anhydrase isozymes I and II: Design,
synthesis and docking studies of new 1,3,4-thiadiazole derivatives
Mehlika Dilek Altıntop, Belgin Sever, Ahmet Özdemir, Kaan Kucukoglu,
Hicran Onem, Hayrunnisa Nadaroglu, Zafer Asım Kaplancıklı
PII:
S0968-0896(17)30473-X
http://dx.doi.org/10.1016/j.bmc.2017.05.005
BMC 13723
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 March 2017
29 April 2017
3 May 2017
Please cite this article as: Altıntop, M.D., Sever, B., Özdemir, A., Kucukoglu, K., Onem, H., Nadaroglu, H.,
Kaplancıklı, Z.A., Potential inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis and docking
studies of new 1,3,4-thiadiazole derivatives, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.05.005
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Potential inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis
and docking studies of new 1,3,4-thiadiazole derivatives
Mehlika Dilek Altıntop1, Belgin Sever1, Ahmet Özdemir1, Kaan Kucukoglu2, Hicran Onem3,
Hayrunnisa Nadaroglu3,4, Zafer Asım Kaplancıklı1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470
Eskişehir, Turkey
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bülent Ecevit University,
Zonguldak, Turkey
3
Department of Nano-Science and Nano-Engineering, Faculty of Engineering, Atatürk
University, 25240 Erzurum, Turkey
4 Department of Food Technology, Erzurum Vocational Training School, Atatürk University,
25240 Erzurum, Turkey
ABSTRACT
In the last years, inhibition of carbonic anhydrase (CA) has emerged as a promising approach
for pharmacologic intervention in a variety of disorders such as glaucoma, epilepsy, obesity,
and cancer. As a consequence, the design of CA inhibitors (CAIs) is a highly dynamic field of
medicinal chemistry. Due to the therapeutic potential of thiadiazoles as CAIs, new 1,3,4-
thiadiazole derivatives were synthesized and investigated for their inhibitory effects on hCA I
and hCA II. Although the tested compounds did not carry a sulfonamide group, an important
pharmacophore for CA inhibitory activity, it was a remarkable finding that most of them were
more effective on hCAs than acetazolamide (AAZ), the reference agent. Among these
compounds,
N'-((5-(4-chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-
thiadiazol-2-yl)thio)acetohydrazide (3) was found to be the most effective compound on hCA
I with an IC50 value of 0.14 nM, whereas N'-((5-(2-chlorophenyl)furan-2-yl)methylene)-2-((5-
(phenylamino)-1,3,4-thiadiazol-2-yl)thio)acetohydrazide (1) was found to be the most potent
compound on hCA II with an IC50 value of 0.15 nM. According to molecular docking studies,
all compounds exhibited high affinity and good amino acid interactions similar to AAZ on the
both active sites of hCA I and hCA II enzymes.
Keywords: Carbonic anhydrase, Furan, Hydrazone, Thiadiazole, Molecular docking
1

1. Introduction
Carbonic anhydrases (CAs; EC 4.2.1.1) are ubiquitous zinc-containing
metalloenzymes involved in the catalysis of a simple but essential physiological reaction: the
reversible hydration of carbon dioxide to bicarbonate ion and proton.1,2 These enzymes play a
pivotal role in crucial physiological processes related to respiration and acid-base regulation,
electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions (e.g.
gluconeogenesis, lipogenesis and ureagenesis). Several studies have showed that abnormal
levels or activities of CAs have been often associated with different human diseases. As a
consequence, CA isozymes have been identified as potential drug targets for the design of
inhibitors or activators with clinical applications.1-10
Inhibition of CAs has emerged as a promising approach for the treatment of a variety
of disorders such as glaucoma, epilepsy, obesity, and cancer. Recent studies have also pointed
out the importance of CAs for the design of anti-infective agents with a novel mechanism of
action.1-10
Thiadiazole has attracted a great deal of interest as a privileged scaffold due to its
significant therapeutic potential.11-15 The sulfur atom of thiadiazole ring imparts improved
liposolubility and the mesoionic nature of 1,3,4-thiadiazoles also allows these compounds to
cross cellular membranes and interact with biological targets with distinct affinities.11,12
Thiadiazoles exhibit a broad spectrum of biological activities through the inhibition of CAs,
cyclooxygenases (COXs), neutral endopeptidase (NEP), aminopeptidase N (APN), matrix
metalloproteinases (MMPs), phosphodiesterases (PDEs) and c-Src/Abl tyrosine kinase.13 In
particular, medicinal chemists have focused on the inhibitory effects of 1,3,4-thiadiazoles on
CAs.11-24 Among thiadiazole derivatives, acetazolamide (AAZ) (N-(5-sulfamoyl-1,3,4-
thiadiazole-2-yl)acetamide), a potent carbonic anhydrase inhibitor, is used in the treatment of
glaucoma, acute mountain sickness, epileptic seizures.15,24 On the other hand, furan-based
hydrazone derivatives were also reported to exhibit notable inhibitory effects on hCA I.25
Prompted by the afore-mentioned findings and in the continuation of our ongoing
research in the field of design, synthesis and biological evaluation of thiadiazole derivatives
as hCA inhibitors23, herein we reported the synthesis and inhibitory effects of a new series of
N'-(5-arylfuran-2-yl)methylene-2-[(5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio]acetohydrazide derivatives on hCA I and hCA II. Ultimately, all compounds were
docked to the active sites of hCA I and hCA II with AAZ in order to speculate the possible
binding modes of these molecules in the active sites of these enzymes.
2. Results and Discussion
The synthesis of compounds 1-10 followed the general pathway outlined in Scheme 1.
Initially, 4-phenylthiosemicarbazide (A) was obtained by the reaction of phenyl
isothiocyanate with hydrazine hydrate. 5-(Phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B)
were synthesized via the ring closure reaction of 4-phenylthiosemicarbazide (A) with carbon
disulfide in the presence of potassium hydroxide. Ethyl 2-[[5-(phenylamino)-1,3,4-thiadiazol-
2-yl]thio]acetate (C) was obtained by the reaction of 5-(phenylamino)-1,3,4-thiadiazole-
2(3H)-thione (B) with ethyl chloroacetate in the presence of potassium carbonate. The
2

treatment of the ester (C) with hydrazine hydrate gave the corresponding hydrazide (D).
Finally, the nucleophilic addition-elimination reaction of the hydrazide (D) with 5-
arylfurfurals afforded compounds 1-10.
Scheme 1. The synthetic route for the preparation of the thiadiazole derivatives (1-10).
Reagents and conditions: (a) NH2NH2.H2O, ethanol, rt, 5h; (b) (1) CS2/KOH, ethanol, reflux,
10h; (2) HCl, pH 4-5; (c) ClCH2COOEt, K2CO3, acetone, reflux, 10h; (d) NH2NH2.H2O,
ethanol, rt, 3h; (e) 5-arylfurfural, ethanol, reflux, 6h.
1
The H NMR and 13C NMR data were consistent with the assigned structures. In the
1H NMR spectra of compounds 1-10, the S-CH2 protons were observed at 4.03-4.44 ppm. The
N=CH proton appeared at 7.86-8.16 ppm. The NH-N= proton was observed at 11.69-11.82
1
ppm. The Ph-NH-Ar proton was observed at 10.32-10.40 ppm. In the H NMR spectra of
most compounds, S-CH2, N=CH and N-H protons gave rise to two singlet peaks in
3

accordance with the presence of the E and Z isomers.26 All other protons were observed
within the expected regions.
In their 13C NMR spectra, the signal due to the N=CH carbon was observed in the
region 146.26-150.28 ppm, whereas the signal due to the C=O carbon appeared at 165.13-
168.69 ppm. In the mass spectra of the compounds, [M+H]+ peaks were observed in
agreement with their molecular formula.
Compounds 1-10 were investigated for their in vitro inhibitory effects on hCA I and
hCA II and IC50 values were calculated for all derivatives (Table 1). AAZ was used as the
reference agent.
Table 1. The results obtained from regression analysis graphs for hCA I and hCA II in the
presence of the compounds
hCA I Inhibition
hCA II Inhibition
Ki (nM)
hCA I/
hCA II
Compound
Type of
Type of
IC50 (nM)
Ki (nM)
IC50 (nM)
inhibition
inhibition
1
0.77
0.57
0.14
0.21
0.56
0.29
4.21
0.29
0.45
0.28
5.8
1.12 0.11
0.77 1.12
0.12 0.16
0.12 .0.18
0.90 0.03
0.63 0.21
6.23 0.23
0.37 0.06
0.69 0.63
0.90 0.51
8.89 2.01
Competitive
Competitive
0.15
0.53
0.19
0.34
0.47
-
0.64 1.05
0.61 0.36
0.27 1.25
0.88 0.25
0.66 0.36
-
Competitive
Competitive
Competitive
Non-Competitive
Non-Competitive
-
5.25
1.07
0.76
0.28
1.19
-
2
3
Competitive
4
Non-Competitive
Non-Competitive
Non-Competitive
Competitive
5
6
7
0.41
0.39
-
5.12 0.86
0.66 0.39
-
Competitive
Competitive
-
10.29
0.76
-
8
Competitive
9
10
Competitive
Competitive
-
-
-
-
Acetazolamide
6.7
10.70 4.23
0.87
The compounds were more effective on hCA I than AAZ (IC50= 5.8 nM). The
compounds showed notable inhibitory effects on hCA I with IC50 values ranging between
0.14 and 4.21 nM. In particular, compounds 3 and 4 were found to be the most potent hCA I
inhibitors with IC50 values of 0.14 and 0.21 nM, respectively. The inhibitory effects of
compounds 6, 8 and 10 on hCA I were fairly close. The effects of the chloro and nitro
substituents on hCA I inhibitory activity revealed the following potency order: p-Cl > o-NO2
> 4-Cl-2-NO2 > p-NO2 > 2,4-diCl > 3,4-diCl > m-NO2 > m-Cl > o-Cl. Thus it can be
concluded that the position of chloro and nitro substituents is important for hCA I inhibitory
activity. Although bromo-substituted compound 7 was more effective than AAZ on hCA I,
the compound showed less inhibitory activity than other derivatives against hCA I. It is
noteworthy to indicate that p-bromo substituent significantly decreased hCA I inhibitory
4

activity when compared with p-chloro substituent. It can be attributed to the steric hindrance
caused by the bulky bromo substituent.
Compounds 6, 9 and 10 showed no inhibitory activity against hCA II, whereas the
other compounds were significantly more effective on hCA II than AAZ (IC50= 6.7 nM). The
compounds showed notable inhibitory effects on hCA II with IC50 values ranging between
0.15 and 0.53 nM. In particular, chloro-substituted compounds 1 and 3 showed notable
inhibitory effects on hCA II with the IC50 values of 0.15 and 0.19 nM, respectively. Although
compound 2 bearing m-chloro substituent was more effective than AAZ on hCA II, the
compound showed less inhibitory activity than other derivatives against hCA II. 2,4-Dichloro
substituted compound 8 exhibited inhibitory effect on hCA II with an IC50 value of 0.39 nM,
whereas 3,4-dichloro substituted compound 9 did not show any inhibitory activity against
hCA II. The results pointed out the importance of the position of chloro substituent for hCA II
inhibitory activity. On the other hand, nitro-substituted compounds 4 and 5 showed inhibitory
effects on hCA II with IC50 values of 0.34 and 0.47 nM, respectively. But compound 6
bearing p-nitro group did not show any inhibitory activity against hCA II. This outcome
pointed out the significance of the position of nitro group for hCA II inhibitory activity.
Ki constants were calculated between 0.12 and 6.23 nM for hCA I using Lineweaver-
Burk plots and are presented in Table 1. Most of the compounds showed competitive
inhibition, whilst the other compounds showed non-competitive inhibition. On the other hand,
AAZ, showed a Ki value of 8.89 2.01 nM against hCA I. The hCA I inhibitory effects of
compounds 1-10 were found to be greater than that of AAZ, which was a clinically standard
CA inhibitor.
Compounds 1-10 showed Ki values varying from 0.27 to 5.12 nM against hCA II, the
physiologically dominant isoform (Table 1). Thus, compound 1-10 were found to inhibit hCA
II isozyme at high levels. The synthesized compounds probably interact with the distinct
hydrophilic and hydrophobic halves of the active site of hCA II.
Molecular docking studies were performed for the potent hCA inhibitors on the active
sites of hCA I (PDB code: 2FW4) and hCA II (PDB code: 5SZ5) which were previously
defined27,28. AAZ, the potent carbonic anhydrase inhibitor, was chosen as a reference ligand
for docking studies due to its structural resemblance with the compounds. According to the
results, all compounds showed good affinity to the active site of both enzymes as indicated in
Figure 1. The docking score, glide gscore and glide emodel results of the compounds were
also detected similar to AAZ and each other as shown in Table 2. In particular, the residual
interactions of compound 3 on hCA I and compound 1 on hCA II were demonstrated in
Figures 2 and 3. Generally the thiadiazole and the furan ring systems and the phenylamino,
phenyl substitutions on these rings presented pi-pi stacking contacts with His64, His67, His
200 and Tyr204 residues on hCA I. However, the hydrazone group formed H-bonds with
Gln92 and Tyr20 residues similar to the sulfonamide group of AAZ. Moreover, the nitro
substituent in some compounds was engaged in salt-bridge formation with Asp72 on hCA I.
As regards to hCA II, the thiadiazole, the furan ring systems and the phenylamino, phenyl
substitutions on these rings presented pi-pi stacking and H-bonds with His4, His64, His94 and
Trp5 residues.
5

Figure 1. Docking positions of all synthesized compounds on hCA I (PDB code: 2FW4) and
hCA II (PDB code: 5SZ5) together, respectively. Compounds 6, 9 and 10 were not docked to
hCA II. (Ligand custom carbons are colored given as in the figure and the zinc atom is
displayed in grey sphere).
Figure 2. Docking positions of compound 3 and AAZ on hCA I and interactions between
compound 3 and hCA I, respectively. (Ligand custom carbons are colored in aquamarine for
6

compound 3 and in dark green for AAZ; Green line: Pi-pi stacking and the zinc atom is
displayed in grey sphere).
Figure 3. Docking positions of compound 1 and AAZ on hCA II and interactions between
compound 1 and hCA II, respectively. (Ligand custom carbons are colored in cyan for
compound 1 and in dark green for AAZ; Green line: Pi-pi stacking and the zinc atom is
displayed in grey sphere).
Table 2. Docking score (kcal/mol), glide gscore (kcal/mol) and glide emodel (kcal/mol)
results of all compounds for hCA I and hCA II
hCA I
hCA II
Compound
R
Docking
score
Glide
Glide
Docking
score
Glide
Glide
gscore
emodel
gscore
emodel
1
2
3
4
5
6
7
8
2-Cl
3-Cl
-0.78
-1.22
-3.08
-2.94
-1.14
-1.17
-3.23
-3.21
-0.78
-1.22
-3.08
-2.94
-1.14
-1.17
-3.23
-3.21
-39.87
-37.21
-41.20
-43.26
-38.71
-37.50
-44.28
-45.15
-4.50
-3.84
-3.74
-3.81
-4.02
-
-4.50
-3.84
-3.74
-3.81
-4.02
-
-65.08
-60.96
-54.96
-60.38
-60.94
-
4-Cl
2-NO2
3-NO2
4-NO2
4-Br
-4.12
-3.87
-4.12
-3.87
-59.35
-58.07
2,4-diCl
7

9
3,4-diCl
-2.30
-2.93
-3.32
-2.30
-2.93
-5.02
-36.81
-41.40
-40.52
-
-
-
-
-
-
10
4-Cl-2-NO2
Acetazolamide
3. Conclusion
-6.88
-7.88
-72.92
In this paper, N'-(5-arylfuran-2-yl)methylene-2-[(5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio]acetohydrazide derivatives (1-10) were synthesized and evaluated for their ability to
inhibit human carbonic anhydrase isozymes (hCA I and hCA II). In order to speculate the
exact positions and the possible binding modes of these molecules, the potent hCA inhibitors
were docked to the active sites of hCA I and hCA II with AAZ. Although the synthesized
compounds do not carry a sulfonamide group, an important pharmacophore for hCA
inhibitory activity, most of them showed more significant inhibitory activity against hCAs
than AAZ. The inhibitory effects of four derivatives (1, 2, 5, 7) were more significant on hCA
II than hCA I, whereas the inhibitory effects of three derivatives (3, 4 and 8) were more
significant on hCA I than hCA II. Compounds 6, 9 and 10 showed notable inhibitory effects
on hCA I, whereas these compounds showed no inhibitory activity against hCA II. According
to docking studies, the potent hCA inhibitors were detected at the active sites of hCA I and
hCA II enzymes reasonably forming pi-pi stacking and H-bonds with proper residues. In
particular, compound 3 was identified as a potential hCA I inhibitor with an IC50 value of 0.14
nM when compared with AAZ (IC50= 5.8 nM), whilst compound 1 was found to be a
potential hCA II inhibitor with an IC50 value of 0.15 nM when compared with AAZ (IC50= 6.7
nM). These agents stand out as promising candidates for further in vivo studies.
4. Experimental
4.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. The melting points (M.p.) of the compounds were determined on an
Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and
are uncorrected. IR spectra were recorded on an IRPrestige-21 Fourier Transform Infrared
1
spectrophotometer (Shimadzu, Tokyo, Japan). H NMR and 13C NMR spectra were recorded
on a Bruker spectrometer (Bruker, Billerica, MA, USA). Mass spectra were recorded on a
Shimadzu LCMS-8040 (Shimadzu, Kyoto, Japan). Elemental analyses were performed on a
Perkin Elmer EAL 240 elemental analyser (Perkin-Elmer, Norwalk, CT, USA).
4.1.1. General procedure for the synthesis of the compounds
4.1.1.1. 4-Phenylthiosemicarbazide (A)
A mixture of phenyl isothiocyanate (0.1 mol) and hydrazine hydrate (0.2 mol) in
ethanol (30 mL) was stirred at room temperature for 5 hours and then filtered. The residue
was crystallized from ethanol.29
4.1.1.2. 5-(Phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B)
8

4-Phenylthiosemicarbazide (A) was dissolved in a solution of potassium hydroxide in
ethanol. Carbon disulfide was then added while stirring and the reaction mixture was heated
under reflux for 10 h. The solution was cooled and acidified to pH 4-5 with hydrochloric acid
solution and crystallized from ethanol.29
4.1.1.3. Ethyl 2-[[5-(phenylamino)-1,3,4-thiadiazol-2-yl]thio]acetate (C)
A mixture of 5-(phenylamino)-1,3,4-thiadiazole-2(3H)-thione (B) (0.05 mol) and ethyl
chloroacetate (0.05 mol) in the presence of potassium carbonate (0.05 mol) in acetone was
refluxed for 10 h. The reaction mixture was cooled, filtered and the crude product was solved
in water and then extracted with ether.29
4.1.1.4. 2-[[5-(Phenylamino)-1,3,4-thiadiazol-2-yl]thio]acetohydrazide (D)
A mixture of the ester (C) (0.05 mol) and hydrazine hydrate (0.1 mol) in ethanol was
stirred at room temperature for 3 h and then filtered.29
4.1.1.5.
N'-(5-Arylfuran-2-yl)methylene-2-[(5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio]acetohydrazide derivatives (1-10)
A mixture of the hydrazide (D) (0.01 mol) and 5-arylfurfural (0.01 mol) was refluxed
in ethanol for 6 h, filtered and crystallized from ethanol.
N'-((5-(2-Chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (1)
Vanilla colored solid. Yield: 80%; m.p. 207.1 °C. IR νmax (cm−1): 3178.69 (N-H stretching),
3043.67 (Aromatic C-H stretching), 2927.94, 2902.87, 2858.51, 2825.72, 2785.21 (Aliphatic
C-H stretching), 1678.07 (C=O stretching), 1600.92, 1570.06, 1531.48, 1496.76, 1462.04,
1435.04 (N-H bending, C=N and C=C stretching), 1411.89, 1379.10, 1332.81, 1294.24 (C-H
bending), 1251.80, 1226.73, 1197.79, 1165.00, 1138.00, 1082.07, 1041.56, 1022.27 (C-N, C-
O stretching and aromatic C-H in plane bending), 927.76, 883.40, 869.90, 813.96, 798.53,
785.03, 746.45, 729.09, 713.66, 688.59, 675.09, 653.87, 592.15 (aromatic C-H out of plane
bending and C-S stretching). 1H NMR (400 MHz, DMSO-d6): 4.03 + 4.42 (2H, s + s, S-CH2),
6.96-7.11 (2H, m, Ar-H), 7.24-7.60 (8H, m, Ar-H), 7.89-7.91 (1H, m, Ar-H), 7.95 + 8.16 (1H,
s + s, CH=N), 10.35 + 10.39 (1H, s + s, NH), 11.74 + 11.78 (1H, s + s, NH). 13C NMR (100
MHz, DMSO-d6): 35.50 and 36.47 (CH2), 113.11 (CH, d, J= 8.9 Hz), 114.93 (CH), 115.86
(CH), 117.38 (2CH, d, J= 3.2 Hz), 121.96 (CH, d, J= 6.4 Hz), 127.65 (2CH), 128.13 (CH, d,
J= 16.7 Hz), 129.02 (CH, d, J= 5.1 Hz), 129.43 (CH, t, J= 8.0, 14.1, 22.4 Hz), 130.81 (C),
133.31 (C), 136.68 (C), 140.33 (C), 148.88 (CH, d, J= 5.7 Hz), 150.57 and 150.88 (C), 151.78
and 152.27 (C), 163.47 and 165.03 (C), 165.17 and 168.55 (C). MS (ESI) (m/z): 470 [M+H]+,
471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2 Calculated: C, 53.67; H, 3.43; N, 14.90.
Found: C, 53.63; H, 3.47; N, 14.91.
N'-((5-(3-Chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (2)
White solid. Yield: 82%; m.p. 211.6 °C. IR νmax (cm-1): 3251.98, 3196.05 (N-H stretching),
3138.18, 3082.25, 3010.88 (Aromatic C-H stretching), 2870.08 (Aliphatic C-H stretching),
1672.28 (C=O stretching), 1614.42, 1602.85, 1560.41, 1489.05, 1458.18, 1446.61 (N-H
9

bending, C=N and C=C stretching), 1429.25, 1404.18, 1338.60, 1313.52 (C-H bending),
1217.08, 1130.29, 1093.64, 1078.21, 1026.13, 1002.98 (C-N, C-O stretching and aromatic C-
H in plane bending), 948.98, 933.55, 871.82, 779.24, 746.45, 711.73, 686.66, 673.16
1
(aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz, DMSO-d6):
4.03 + 4.44 (2H, s + s, S-CH2), 6.96-7.06 (2H, m, Ar-H), 7.22-7.58 (7H, m, Ar-H), 7.71-7.81
(2H, m, Ar-H), 7.93 + 8.13 (1H, s + s, CH=N), 10.35 + 10.40 (1H, s + s, NH), 11.74 + 11.77
(1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.56 and 36.47 (CH2), 109.66 (CH, d, J=
8.4 Hz), 115.52 (CH), 116.41 (CH), 117.38 (2CH, d, J= 4.4 Hz), 121.99 (CH, d, J= 7.7 Hz),
122.40 (CH, d, J= 12.1 Hz), 123.41 (CH), 127.92 (CH, d, J= 10.9 Hz), 129.07 (2CH, d, J= 6.4
Hz), 130.94 and 131.41 (C), 133.40 and 133.87 (C), 136.70 (C), 140.35 (C), 149.28 (CH, d,
J= 7.1 Hz), 151.84 and 152.35 (C), 152.83 and 153.18 (C), 163.49 and 165.03 (C), 165.17 and
168.58 (C). MS (ESI) (m/z): 470 [M+H]+, 471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2
Calculated: C, 53.67; H, 3.43; N, 14.90. Found: C, 53.63; H, 3.47; N, 14.89.
N'-((5-(4-Chlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (3)
White solid. Yield: 86%; m.p. 234.8 °C. IR νmax (cm−1): 3277.06, 3192.05 (N-H stretching),
3138.18, 3086.11, 3041.74 (Aromatic C-H stretching), 2978.09, 2914.44 (Aliphatic C-H
stretching), 1670.35 (C=O stretching), 1651.07, 1616.35, 1600.92, 1552.70, 1485.19,
1475.54, 1446.61 (N-H bending, C=N and C=C stretching), 1409.96, 1396.46, 1328.95,
1305.81 (C-H bending), 1251.80, 1215.15, 1149.57, 1089.78, 1043.49, 1024.20, 1001.06 (C-
N, C-O stretching and aromatic C-H in plane bending), 925.83, 831.32, 794.67, 750.31,
1
729.09, 692.44, 648.08 (aromatic C-H out of plane bending and C-S stretching). H NMR
(400 MHz, DMSO-d6): 4.03 + 4.44 (2H, s + s, S-CH2), 6.96-7.05 (2H, m, Ar-H), 7.14 + 7.17
(1H, d + d, J= 4 Hz + J= 3.2 Hz, Ar-H), 7.29-7.35 (2H, m, Ar-H), 7.49-7.58 (4H, m, Ar-H),
7.76-7.79 (2H, m, Ar-H), 7.92 + 8.12 (1H, s + s, CH=N), 10.35 + 10.39 (1H, s + s, NH),
11.70 + 11.75 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.49 and 36.47 (CH2),
108.95 (CH, d, J= 9.0 Hz), 115.73 (CH), 116.53 (CH), 117.37 (2CH, d, J= 3.9 Hz), 121.98
(CH, d, J= 6.4 Hz), 125.59 (2CH, d, J= 12.2 Hz), 128.28 (CH, d, J= 3.8 Hz), 129.07 (2CH, d,
J= 4.5 Hz), 132.61 and 132.70 (C), 133.43 (C), 136.72 (C), 140.34 (C), 148.99 (CH, d, J= 9.6
Hz), 151.82 and 152.36 (C), 153.34 and 153.70 (C), 163.43 and 165.00 (C), 165.17 and
168.50 (C). MS (ESI) (m/z): 470 [M+H]+, 471 [M+H]++, 472 [M+H]+++. For C21H16ClN5O2S2
Calculated: C, 53.67; H, 3.43; N, 14.90. Found: C, 53.64; H, 3.45; N, 14.90.
N'-((5-(2-Nitrophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (4)
Yellow solid. Yield: 83%; m.p. 195.7 °C. IR νmax (cm−1): 3325.28, 3273.20 (N-H stretching),
3118.90, 3074.53 (Aromatic C-H stretching), 2935.66, 2897.08 (Aliphatic C-H stretching),
1666.50 (C=O stretching), 1624.06, 1597.06, 1548.84, 1521.84, 1496.76, 1440.83 (N-H
bending, C=N and C=C stretching), 1415.75, 1396.46, 1369.46, 1344.38, 1301.95 (C-H
bending), 1230.58, 1192.01, 1072.42, 1028.06 (C-N, C-O stretching and aromatic C-H in
plane bending), 999.13, 964.41, 947.05, 921.97, 893.04, 848.68, 802.39, 746.45, 686.66,
646.15, 626.87 (aromatic C-H out of plane bending and C-S stretching). 1H NMR (400 MHz,
DMSO-d6): 4.03 + 4.38 (2H, s + s, S-CH2), 6.96-7.08 (3H, m, Ar-H), 7.29-7.35 (2H, m, Ar-
10

H), 7.54-7.65 (3H, m, Ar-H), 7.71-7.78 (1H, m, Ar-H), 7.86-8.14 (3H, m, Ar-H and CH=N),
10.36 + 10.39 (1H, s + s, NH), 11.72 + 11.76 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-
d6): 35.40 and 36.47 (CH2), 112.02 (CH, d, J= 19.3 Hz), 114.66 (CH), 115.44 (CH), 117.39
(2CH, d, J= 3.2 Hz), 121.97 (CH, d, J= 8.4 Hz), 124.06 (CH, d, J= 26.3 Hz), 128.93 (2CH, d,
J= 32 Hz), 129.34 (CH), 129.76 (CH, d, J= 23.1 Hz), 132.39 (C), 132.86 (C), 136.67 and
140.37 (C), 147.02 (C), 149.21 and 149.64 (C), 150.28 (CH, d, J= 7.7 Hz), 151.85 and 152.34
(C), 163.53 and 165.08 (C), 165.15 and 168.62 (C). MS (ESI) (m/z): 481 [M+H]+. For
C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49. Found: C, 52.45; H, 3.40; N, 17.50.
N'-((5-(3-Nitrophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (5)
Pale yellow solid. Yield: 85%; m.p. 218.1 °C. IR νmax (cm−1): 3253.91, 3196.05 (N-H
stretching), 3138.18, 3086.11 (Aromatic C-H stretching), 2991.59, 2862.36 (Aliphatic C-H
stretching), 1670.35 (C=O stretching), 1610.56, 1562.34, 1525.69, 1489.05, 1446.61 (N-H
bending, C=N and C=C stretching), 1348.24, 1309.67 (C-H bending), 1217.08, 1130.29,
1091.71, 1024.20, 1002.98 (C-N, C-O stretching and aromatic C-H in plane bending), 945.12,
891.11, 862.18, 792.74, 754.17, 736.81, 723.31, 711.73, 690.52, 680.87, 667.37 (aromatic C-
H out of plane bending and C-S stretching). 1H NMR (400 MHz, DMSO-d6): 4.05 + 4.43 (2H,
s + s, S-CH2), 6.93-7.08 (2H, m, Ar-H), 7.26-7.37 (3H, m, Ar-H), 7.51 (1H, d, J= 8.0 Hz),
7.56 (1H, d, J= 8.8 Hz), 7.67-7.72 (1H, m, Ar-H), 7.93-8.16 (3H, m, Ar-H and CH=N), 8.43-
8.46 (1H, m, Ar-H), 10.32 + 10.39 (1H, s + s, NH), 11.75 + 11.81 (1H, s + s, NH). 13C NMR
(100 MHz, DMSO-d6): 35.56 and 36.49 (CH2), 110.56 (CH, d, J= 7.7 Hz), 115.41 (CH),
116.47 (CH), 117.37 (2CH, d, J= 6.4 Hz), 118.01 (CH), 121.96 (CH, d, J= 10.3 Hz), 122.41
(CH, d, J= 14.8 Hz), 129.04 (2CH, d, J= 9.0 Hz), 129.87 (CH, d, J= 18.6 Hz), 130.60 and
130.87 (C), 133.31 and 136.61 (C), 140.33 (C), 148.36 (C), 149.72 (CH, d, J= 7.1 Hz), 151.83
and 152.03 (C), 152.22 and 152.38 (C), 163.60 and 165.15 (C, d, J= 6.4 Hz), 168.67 (C). MS
(ESI) (m/z): 481 [M+H]+. For C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49. Found:
C, 52.46; H, 3.39; N, 17.49.
N'-((5-(4-Nitrophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (6)
Yellow solid. Yield: 90%; m.p. 237.4 °C. IR νmax (cm−1): 3253.91, 3194.12 (N-H stretching),
3053.32 (Aromatic C-H stretching), 2914.44, 2821.86 (Aliphatic C-H stretching), 1672.28
(C=O stretching), 1622.13, 1595.13, 1573.91, 1508.33, 1498.69, 1462.04 (N-H bending, C=N
and C=C stretching), 1417.68, 1390.68, 1325.10 (C-H bending), 1159.22, 1103.28, 1041.56,
1029.99 (C-N, C-O stretching and aromatic C-H in plane bending), 977.91, 923.90, 852.54,
800.46, 742.59, 694.37, 682.80, 655.80 (aromatic C-H out of plane bending and C-S
1
stretching). H NMR (400 MHz, DMSO-d6): 4.04 + 4.43 (2H, s + s, S-CH2), 6.95-7.01 (1H,
m, Ar-H), 7.10 + 7.13 (1H, d + d, J= 3.6 Hz + J= 3.6 Hz, Ar-H), 7.28-7.35 (2H, m, Ar-H),
7.42 + 7.45 (1H, d + d, J= 4.0 Hz + J= 3.2 Hz, Ar-H), 7.52 (1H, d, J= 8.0 Hz), 7.56 (1H, d, J=
8.0 Hz), 7.95-8.16 (3H, m, Ar-H and CH=N), 8.28-8.31 (2H, m, Ar-H), 10.33 + 10.39 (1H, s
+ s, NH), 11.78 + 11.82 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.48 and 36.47
(CH2), 112.31 (CH), 115.74 (CH), 116.60 (CH), 117.36 (2CH, d, J= 7.1 Hz), 121.97 (CH, d,
J= 9.0 Hz), 124.43 (CH), 124.54 (2CH, d, J= 13.5 Hz), 129.05 (2CH, d, J= 7.0 Hz), 133.13
11

(C), 135.10 (C, d, J= 4.4 Hz), 136.45 (C), 140.31 (C), 146.26 (CH, d, J= 7.7 Hz), 150.58 and
151.79 (C, d, J= 8.3 Hz), 152.19 and 152.52 (C), 163.59 and 165.13 (C, d, J= 9.0 Hz), 168.67
(C). MS (ESI) (m/z): 481 [M+H]+. For C21H16N6O4S2 Calculated: C, 52.49; H, 3.36; N, 17.49.
Found: C, 52.45; H, 3.40; N, 17.50.
N'-((5-(4-Bromophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (7)
Beige solid. Yield: 85%; m.p. 234.0 °C. IR νmax (cm−1): 3277.06, 3194.12 (N-H stretching),
3138.18, 3086.11, 3039.81 (Aromatic C-H stretching), 2978.09, 2914.44 (Aliphatic C-H
stretching), 1670.35 (C=O stretching), 1649.14, 1616.35, 1600.92, 1550.77, 1485.19,
1473.62, 1444.68 (N-H bending, C=N and C=C stretching), 1406.11, 1305.81 (C-H bending),
1251.80, 1213.23, 1147.65, 1087.85, 1070.49, 1043.49, 1024.20, 1002.98 (C-N, C-O
stretching and aromatic C-H in plane bending), 921.97, 894.97, 829.39, 796.60, 748.38,
690.52 (aromatic C-H out of plane bending and C-S stretching). 1H NMR (400 MHz, DMSO-
d6): 4.04 + 4.44 (2H, s + s, S-CH2), 6.96-7.04 (2H, m, Ar-H), 7.13 + 7.15 (1H, d + d, J= 3.6
Hz + J= 3.6 Hz, Ar-H), 7.29-7.34 (2H, m, Ar-H), 7.54-7.71 (6H, m, Ar-H), 7.93 + 8.13 (1H, s
+ s, CH=N), 10.34 + 10.38 (1H, s + s, NH), 11.69 + 11.74 (1H, s + s, NH). 13C NMR (100
MHz, DMSO-d6): 35.50 and 36.49 (CH2), 108.95 (CH, d, J= 8.3 Hz), 115.62 (CH), 116.46
(CH), 117.37 (2CH, d, J= 4.5 Hz), 121.24 (CH, d, J= 9.7 Hz), 121.95 (CH, d, J= 7.1 Hz),
125.78 (CH, d, J= 12.2 Hz), 128.57 (CH, d, J= 3.8 Hz), 129.02 (2CH, d, J= 4.5 Hz), 131.89
(C), 133.43 (C), 136.74 (C), 140.33 (C), 149.01 (CH, d, J= 8.3 Hz), 151.77 and 152.30 (C),
153.36 and 153.72 (C), 163.42 and 165.01 (C), 165.18 and 168.48 (C). MS (ESI) (m/z): 514
[M+H]-, 516 [M+H]+. For C21H16BrN5O2S2 Calculated: C, 49.03; H, 3.14; N, 13.61. Found: C,
49.05; H, 3.13; N, 13.60.
N'-((5-(2,4-Dichlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (8)
White solid. Yield: 83%; m.p. 231.7 °C. IR νmax (cm−1): 3219.19, 3184.48 (N-H stretching),
3136.25, 3066.82, 3028.24 (Aromatic C-H stretching), 2956.87, 2825.72, 2748.56 (Aliphatic
C-H stretching), 1662.64 (C=O stretching), 1598.99, 1560.41, 1496.76, 1456.26, 1435.04 (N-
H bending, C=N and C=C stretching), 1400.32, 1386.82, 1357.89, 1323.17, 1301.95 (C-H
bending), 1253.73, 1240.23, 1213.23, 1141.86, 1112.93, 1097.50, 1033.85 (C-N, C-O
stretching and aromatic C-H in plane bending), 989.48, 912.33, 864.11, 819.75, 806.25,
792.74, 756.10, 738.74, 688.59, 671.23, 644.22, 601.79 (aromatic C-H out of plane bending
1
and C-S stretching). H NMR (400 MHz, DMSO-d6): 4.03 + 4.42 (2H, s + s, S-CH2), 6.95 -
7.01 (1H, m, Ar-H), 7.04 + 7.08 (1H, d + d, J= 3.2 Hz + J= 3.6 Hz, Ar-H), 7.25- 7.34 (3H, m,
Ar-H), 7.51 -7.57 (3H, m, Ar-H), 7.69 + 7.72 (1H, d + d, J= 2.0 Hz + J= 2.0 Hz, Ar-H), 7.87
(1H, d, J= 8.4 Hz), 7.94 + 8.16 (1H, s + s, CH=N), 10.33 + 10.38 (1H, s + s, NH), 11.75 (1H,
br s, NH). 13C NMR (100 MHz, DMSO-d6): 35.48 and 36.46 (CH2), 113.52 (CH, d, J= 9.6
Hz), 114.98 (CH), 115.89 (CH), 117.35 (2CH, d, J= 5.8 Hz), 121.94 (CH, d, J= 7.6 Hz),
126.60 (CH), 127.89 (CH), 129.01 (2CH, d, J= 5.8 Hz), 129.22 (C), 130.05 and 130.17 (C, d,
J= 4.5 Hz), 132.93 and 133.10 (C, d, J= 7.7 Hz), 136.53 (C), 140.30 (C), 149.12 (CH, d, J=
7.7 Hz), 149.58 and 149.87 (C), 151.75 and 152.18 (C), 163.49 and 165.05 (C), 165.15 and
12

168.60 (C). MS (ESI) (m/z): 504 [M+H]+, 506 [M+H]+++. For C21H15Cl2N5O2S2 Calculated: C,
50.01; H, 3.00; N, 13.88. Found: C, 50.02; H, 3.00; N, 13.87.
N'-((5-(3,4-Dichlorophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (9)
Ivory solid. Yield: 84%; m.p. 208.1 °C. IR νmax (cm−1): 3259.70, 3194.12 (N-H stretching),
3051.39 (Aromatic C-H stretching), 2937.59, 2823.79 (Aliphatic C-H stretching), 1672.28
(C=O stretching), 1624.06, 1600.92, 1573.91, 1502.55, 1462.04, 1440.83 (N-H bending, C=N
and C=C stretching), 1417.68, 1402.25, 1392.61, 1381.03, 1357.89, 1317.38, 1280.73 (C-H
bending), 1226.73, 1159.22, 1134.14, 1099.43, 1043.49, 1026.13 (C-N, C-O stretching and
aromatic C-H in plane bending), 977.91, 929.69, 883.40, 808.17, 788.89, 740.67, 669.30,
655.80 (aromatic C-H out of plane bending and C-S stretching). 1H NMR (400 MHz, DMSO-
d6): 4.04 + 4.43 (2H, s + s, S-CH2), 6.95-7.04 (2H, m, Ar-H), 7.21-7.34 (3H, m, Ar-H), 7.52-
7.71 (4H, m, Ar-H), 7.91-8.14 (2H, m, Ar-H and CH=N), 10.32 + 10.37 (1H, s + s, NH),
11.72 + 11.77 (1H, s + s, NH). 13C NMR (100 MHz, DMSO-d6): 35.56 and 36.49 (CH2),
110.09 (CH, d, J= 9.6 Hz), 115.35 (CH), 116.26 (CH), 117.36 (2CH, d, J= 6.5 Hz), 121.93
(CH, d, J= 8.4 Hz), 123.73 (CH, d, J= 16.0 Hz), 125.33 (CH, d, J= 3.2 Hz), 129.49 (2CH, d,
J= 7.1 Hz), 129.85 (C, d, J= 3.8 Hz), 130.27 and 130.39 (C), 131.11 and 131.88 (C, d, J= 2.5
Hz), 133.26 and 136.64 (C), 140.32 (C), 149.48 (CH, d, J= 7.7 Hz), 151.81 (C, d, J= 11.5
Hz), 152.21 (C, d, J= 2.6 Hz), 163.49 and 165.06 (C), 165.17 and 168.57 (C). MS (ESI) (m/z):
504 [M+H]+, 506 [M+H]+++. For C21H15Cl2N5O2S2 Calculated: C, 50.01; H, 3.00; N, 13.88.
Found: C, 50.01; H, 3.01; N, 13.86.
N'-((5-(4-Chloro-2-nitrophenyl)furan-2-yl)methylene)-2-((5-(phenylamino)-1,3,4-
thiadiazol-2-yl)thio)acetohydrazide (10)
Yellow solid. Yield: 82%; m.p. 222.4 °C. IR νmax (cm−1): 3194.12 (N-H stretching), 3086.11,
3047.53 (Aromatic C-H stretching), 2954.95, 2920.23, 2873.94 (Aliphatic C-H stretching),
1668.43 (C=O stretching), 1620.21, 1600.92, 1573.91, 1562.34, 1531.48, 1500.62, 1463.97
(N-H bending, C=N and C=C stretching), 1417.68, 1363.67, 1342.46, 1317.38, 1261.45 (C-H
bending), 1220.94, 1199.72, 1174.65, 1085.92, 1053.13, 1031.92 (C-N, C-O stretching and
aromatic C-H in plane bending), 972.12, 933.55, 883.40, 875.68, 837.11, 796.60, 767.67,
746.45, 725.23, 686.66, 663.51, 653.87, 642.30 (aromatic C-H out of plane bending and C-S
1
stretching). H NMR (400 MHz, DMSO-d6): 4.03 + 4.38 (2H, s + s, S-CH2), 6.96-7.08 (3H,
m, Ar-H), 7.29-7.34 (2H, m, Ar-H), 7.53-7.58 (3H, m, Ar-H), 7.77-7.83 (1H, m, Ar-H), 7.87-
8.14 (2H, m, Ar-H and CH=N), 10.35 + 10.38 (1H, s + s, NH), 11.73 + 11.78 (1H, s + s, NH).
13C NMR (100 MHz, DMSO-d6): 35.38 and 36.47 (CH2), 112.60 (CH, d, J= 19.9 Hz), 114.71
(CH), 115.37 (CH), 117.38 (2CH, d, J= 3.8 Hz), 120.52 and 120.94 (CH), 121.97 (CH, d, J=
6.4 Hz), 123.83 and 124.07 (CH), 129.22 (2CH, d, J= 3.8 Hz), 132.20 and 132.60 (C, d, J=
9.6 Hz), 133.38 and 133.63 (C), 136.55 (C), 140.35 (C), 147.05 (CH, d, J= 4.5 Hz), 148.07
and 148.43 (C), 150.49 and 150.61 (C), 151.82 and 152.27 (C), 163.58 and 165.12 (C),
165.15 and 168.69 (C). MS (ESI) (m/z): 515 [M+H]+, 517 [M+H]+++. For C21H15ClN6O4S2
Calculated: C, 48.98; H, 2.94; N, 16.32. Found: C, 48.96; H, 2.95; N, 16.34.
4.2. Biochemistry
13

4.2.1. Purification of human carbonic anhydrase isozymes (hCA I and hCA II) from
human erythrocytes by affinity chromatography
Fresh human blood was obtained from the blood center, Atatürk University, it was
stored at 4oC used within 2-3 days at most. The blood samples were centrifuged to separate
erythrocytes at 2500 rpm for 15 min and plasma and buffy coat were carefully removed.
Then, underlying erythrocytes were washed with 0.9% NaCl solution twice and upper
portions were also discarded. The erythrocytes were hemolyzed with distilled water at 0oC, it
was stirred for half an hour at 4oC. The hemolysate was centrifuged at 20 000 rpm for 30 min
and cell membranes were separated. pH was adjusted to 8.7 with solid Tris. So, the
hemolysate was recovered to be applied to the column.30,31
The affinity gel was prepared on Sepharose-4B matrix. After Sepharose-4B was
activated with CNBr, L-tyrosine was covalently fitted. Then sulfanilamide was coupled to
tyrosine with diazotization reaction as a ligand. The hemolysate was applied to the prepared
Sepharose-4B-L-tyrosine-sulfanilamide affinity column equilibrated with 25 mM Tris-
HCl/0.1 M Na2SO4 (pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM
Na2SO4 (pH 8.7). The human carbonic anhydrase isozymes (hCA I and hCA II) were eluted
with 1 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6),
respectively. All procedures were performed at 4oC.32
4.2.2. Determination of CA activity
4.2.2.1. Hydratase Activity
Carbonic anhydrase activity was determined using the Wilbur-Anderson Method
which was modified by Rickli et al.33,34 This method, as a result hydration of CO2 is released
H+ ions and the pH changes were determined by means of bromine thymol blue indicator,
based on measuring the elapsed time. Enzyme Unit (EU) was calculated using the equation
(to-tc/tc) where to and tc are the times for pH change of the nonenzymatic and the enzymatic
reactions, respectively.
4.2.2.2. Inhibition assays
The inhibitory effects of compounds 1-10 and AAZ on the hydratase activity of hCA I
and hCA II enzymes were investigated. IC50 values were calculated for the compounds at
different concentrations while maintaining a constant substrate concentration. The activities of
enzymes in the medium without inhibitors were used as 100% activity. The activity % values
of enzymes were calculated by measuring the hydratase activity in the presence of different
concentrations of inhibitors. The IC50 value was calculated by utilizing graphs of activity%-[I]
for each inhibitor. The Ki values and inhibition types of the compounds were determined by
Lineweaver-Burk graphics. 33-35
4.2.2.3. Docking Studies
Compounds 1-10 were docked to the active site of hCA I and hCA II. Ligands were
set to the physiological pH (pH = 7.4) at the protonation step and crystal structures of hCA I
and hCA II were retrieved from Protein Data Bank server (PDB codes: 2FW4 for hCA I;
5SZ5 for hCA II). The structures of compounds 1-10 were submitted in protein preparation
14

module of Schrodinger’s Maestro molecular modeling package. In molecular docking
simulations: Glide/XP docking protocols were applied for the prediction of topologies of
compounds 1-10 at the active sites of target structures.36-37
Acknowledgements
This study was supported by Anadolu University Scientific Research Projects Commission
under the grant no: 1605S318.
References
1. Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.
2. Supuran, C. T.; Winum, J.-Y. Future Med. Chem. 2015, 7, 1407.
3. Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168.
4. Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3233.
5. Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, C.T.; De Simone, G. Chem. Rev. 2012,
112, 4421.
6. Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759.
7. Gieling, R. G.; Williams K. J. Bioorg. Med. Chem. 2013, 21, 1470.
8. Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2016, 31, 345.
9. Yang,Y.; Hu, X.-Q.; Li, Q.-S.; Zhang, X.-X.; Ruan, B.-F.; Xu, J.; Liao C. Curr. Top. Med.
Chem. 2016, 16, 384.
10. Supuran, C. T. Expert Opin. Drug Discov. 2017, 12, 61-88.
11. Li, Y.; Geng, J.; Liu, Y.; Yu, S.; Zhao, G. Chem. Med. Chem. 2013, 8, 27.
12. Haider, S.; Alam, M. S.; Hamid H. Eur. J. Med. Chem. 2015, 92, 156.
13. Matysiak, J. Mini-Rev. Med. Chem. 2015, 15, 762.
14. Jain, A.K.; Sharma, S.; Vaidya, A.; Ravichandran, V.; Agrawal, R.K. Chem. Biol. Drug
Des. 2013, 81, 557.
15. Hu, Y.; Li, C.-Y.; Wang, X.-M.; Yang, Y.-H.; Zhu, H.-L. Chem. Rev. 2014, 114, 5572.
16. Almajan, G. L.; Innocenti, A.; Puccetti, L.; Manole, G.; Barbuceanu, S.; Saramet I.;
Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 2347.
17. Abdel-Hamid, M.K.; Abdel-Hafez, A.A.; El-Koussi, N.A.; Mahfouz, N.M.; Innocenti, A.;
Supuran, C.T. Bioorg. Med. Chem. 2007, 15, 6975.
18. Kasımoğulları, R.; Bülbül, M.; Günhan, H.; Güleryüz, H. Bioorg. Med. Chem. 2009, 17,
3295.
19. Kasımoğulları, R.; Bülbül, M.; Mert, S.; Güleryüz, H. J. Enzyme Inhib. Med. Chem. 2011,
26, 231.
20. Biswas, S.; McKenna, R.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 5646.
15

21. Mert, S.; Alım, Z.; İşgör, M. M.; Beydemir, Ş.; Kasımoğulları R. Bioorg. Chem. 2016, 68,
64.
22. Diaz, J. R. A.; Camí, G. E.; Liu-González, M.; Vega, D. R.; Vullo, D.; Juárez, A.;
Pedregosa, J. C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2016, 31, 1102.
23. Altintop, M.D.; Ozdemir, A.; Kucukoglu, K.; Turan-Zitouni, G.; Nadaroglu, H.;
Kaplancikli, Z.A. J. Enzyme Inhib. Med. Chem. 2015, 30, 32.
24. Xu, J.; Peng, Z.; Li, R.; Dou, T.; Xu, W.; Gu, G.; Liu, Y.; Kang, Z.; Tao, H.; Zhang, J. H.;
Ostrowski, R. P.; Lu, J.; Sun X. Neurosci. Lett. 2009, 451, 274.
25. Balaban Gündüzalp, A.; Parlakgümüs, G.; Uzun, D.; Özdemir Özmen, Ü.; Özbek, N.;
Sarı, M.; Tunç, T. J. Mol. Struct. 2016, 1105, 332.
26. Wyrzykiewicz, E.; Prukała, D. J. Heterocyclic Chem. 1998, 35, 381.
27. Temperini, C.; Scozzafava, A.; Supuran C.T. Bioorg. Med. Chem. Lett. 2006, 16, 5152.
28. Bhatt, A.; Mahon, B.P.; Cruzeiro, V.W.; Cornelio, B.; Laronze-Cochard, M.; Ceruso, M.;
Sapi, J.; Rance, G.A.; Khlobystov, A.N.; Fontana, A.; Roitberg, A.; Supuran, C.T.; McKenna,
R. Chembiochem. 2017, 18, 213.
29. Altintop, M.D.; Özdemir, A.; Mohsen, U.A.; Temel, H.E.; Akalın Çiftçi, G.; Kaplancikli,
Z.A. Lett. Drug Des. Discov. 2014, 11, 1062.
30. Nalbantoglu, B.; Demir, N.; Ozdemir, H.; Kufrevioglu, O.I. Turk. J. Med. Sci. 1996, 26,
163.
31. Demir, Y.; Demir, N.; Nadaroglu, H.; Bakan, E. Prep. Biochem. Biotechnol. 2000, 30, 49.
32. Demir, Y.; Demir, N.; Yildirim, S.; Nadaroglu, H.; Karaosmanoglu, M.; Bakan, E. Prep.
Biochem Biotechnol. 2001, 31, 291.
33. Wilbur, K.M.; Anderson, N.G. J. Biol. Chem. 1948, 176, 147.
34. Rickli, E.E.; Ghazanfar, S.A.; Gibbons, B.H.; Edsall, J.T. J. Biol. Chem. 1964, 239, 1065.
35. Altıntop, M.D.; Sever, B.; Akalın Çiftçi, G.; Kucukoglu, K.; Özdemir, A.; Soleimani,
S.S.; Nadaroglu, H.; Kaplancıklı, Z.A. Eur. J. Med. Chem. 2017, 125, 190.
36. Sever, B.; Altıntop, M.D.; Kuş G, Özkurt, M.; Özdemir, A.; Kaplancıklı, Z.A. Eur. J.
Med. Chem. 2016, 113, 179.
37. Altıntop, M.D.; Temel, H.E.; Sever, B.; Akalın Çiftçi, G.; Kaplancıklı, Z.A. Molecules.
2016, 21, 1598.
16

Highlights
► The efficient synthesis of new 1,3,4-thiadiazole derivatives was carried out.
► New thiadiazole derivatives were investigated for their hCA inhibitory effects.
► Generally, most of them were more effective on CAs than acetazolamide.
► Compound 3 was found to be the most effective compound on hCA-I.
► Compound 1 was found to be the most potent agent on hCA-II.
17

Graphical Abstract
Docking positions of all synthesized compounds on hCA I (PDB code: 2FW4) and hCA II
(PDB code: 5SZ5) together, respectively.
18