Synthesis, molecular docking analysis and carbonic anhydrase I-II inhibitory evaluation of new sulfonamide derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103153

New sulfonamide-hydrazone derivatives (3a-3n) were synthesized to evaluate their inhibitory effects on purified human carbonic anhydrase (hCA) I and II. The inhibition profiles of the synthesized compounds on hCA I-II isoenzyme were investigated by comparing their IC₅₀ and K_i values. Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AZA) has also been used as a standard inhibitor. The compound 3e demonstrated the best hCA I inhibitory effect with a K_i value of 0.1676 ± 0.017 μM. Besides, the compound 3m showed the best hCA II inhibitory effect with a K_i value of 0.2880 ± 0.080 μM. Cytotoxicity of the compounds 3e and 3m toward NIH/3T3 mouse embryonic fibroblast cell line was observed and the compounds were found to be non-cytotoxic. Molecular docking studies were performed to investigate the interaction types between active compounds and hCA enzymes. Pharmacokinetic profiles of compounds were assessed by theoretical ADME predictions. As a result of this study a novel and potent class of CA inhibitors were identified with a good activity potential.

Full text of DOI:10.1016/j.bioorg.2019.103153

Accepted Manuscript
Synthesis, molecular docking analysis and carbonic anhydrase I-II inhibitory
evaluation of new sulfonamide derivatives
Begüm Nurpelin Sağlık, Ulviye Acar Çevik, Derya Osmaniye, Serkan Levent,
Betul Kaya Çavuşoğlu, Yeliz Demir, Sinem Ilgın, Yusuf Özkay, Ali Savaş
Koparal, Ş ükrü Beydemir, Zafer Asım Kaplancıklı
PII:
S0045-2068(18)31535-9
https://doi.org/10.1016/j.bioorg.2019.103153
103153
DOI:
Article Number:
Reference:
YBIOO 103153
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 December 2018
23 May 2019
24 July 2019
Please cite this article as: B. Nurpelin Sağlık, U. Acar Çevik, D. Osmaniye, S. Levent, B. Kaya Çavuşoğlu, Y. Demir,
S. Ilgın, Y. Özkay, A. Savaş Koparal, S. Beydemir, Z. Asım Kaplancıklı, Synthesis, molecular docking analysis
and carbonic anhydrase I-II inhibitory evaluation of new sulfonamide derivatives, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103153
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Synthesis, molecular docking analysis and carbonic anhydrase I-II inhibitory evaluation
of new sulfonamide derivatives
Begüm Nurpelin Sağlıka,b*, Ulviye Acar Çevika,b, Derya Osmaniyea,b, Serkan Leventa,b,
Betul Kaya Çavuşoğlua, Yeliz Demirc, Sinem Ilgınd, Yusuf Özkaya,b, Ali Savaş Koparale,
Şükrü Beydemirf, Zafer Asım Kaplancıklıa
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470
Eskişehir, Turkey
b
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu
University, 26470 Eskişehir, Turkey
c Department of Chemistry, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey
d
Department of Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
e Open Education Faculty, Anadolu University, 26470 Eskişehir, Turkey
f
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
* Corresponding author.
E-mail address: bnsaglik@anadolu.edu.tr (B.N. Sağlık).
Tel: +90-222-3350580/3774 Fax: +90-222-3350750.
Address: Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470,
Eskişehir, Turkey.
1

Abstract
New sulfonamide-hydrazone derivatives (3a-3n) were synthesized to evaluate their inhibitory
effects on purified human carbonic anhydrase (hCA) I and II. The inhibition profiles of the
synthesized compounds on hCA I-II isoenzyme were investigated by comparing their IC50 and
Ki values. Acetazolamide (5-acetamido-1,3,4- thiadiazole-2-sulfonamide, AZA) has also been
used as a standard inhibitor. The compound 3e demonstrated the best hCA I inhibitory effect
with a Ki value of 0.1676±0.017 µM. Besides, the compound 3m showed the best hCA II
inhibitory effect with a Ki value of 0.2880±0.080 µM. Cytotoxicity of the compounds 3e and
3m toward NIH/3T3 mouse embryonic fibroblast cell line was observed and the compounds
were found to be non-cytotoxic. Molecular docking studies were performed to investigate the
interaction types between active compounds and hCA enzymes. Pharmacokinetic profiles of
compounds were assessed by theoretical ADME predictions. As a result of this study a novel
and potent class of CA inhibitors were identified with a good activity potential.
Keywords: Carbonic anhydrase; Sulfonamide; Cytotoxicity; Molecular docking; ADME
2

1. Introduction
Carbonic anhydrases (CAs) are zinc enzymes strongly involved in regulating cell homeostasis,
intracellular pH, fluid secretion, ion transport and biosynthetic reactions by catalyzing the
-
reversible hydration of carbon dioxide (CO2) to bicarbonate ion (HCO3 ) and proton (H+) [1-6].
CAs are common in almost all organisms, from simple to complex [7,8].
To date, fifteen isoforms (CA I to CA XV) of CA enzymes have been reported in humans.
Among these isoforms, CA I-III, VII and XIII have been identified in cytosol, while CA IV,
IX, XII and XIV have been defined as membrane bound enzymes. The subtypes of CA VA and
CA VB are restricted to the mitochondrion, whereas CA VI is secreted in milk and saliva. These
enzymes can be classified in terms of affinity to various inhibitors, subcellular localization,
tissue distribution, and catalytic activity [9-13]. These processes include CA isoenzymes that
serve as therapeutic targets prone to be inhibited to treat various disorders such as glaucoma,
obesity, cancer, epilepsy, arthritis, neuropathic pain and osteoporosis [14-17]. CA I is found
together with CA II in erythrocytes at high concentrations. CA II is the most active isoenzyme
among CAs in human. hCA II is the most commonly found in the eye, kidney, central nervous
system (CNS), and inner ear [18]. This enzyme is a target for drugs such as diuretics,
antiglaucoma and anticonvulsants that are used clinically [19-22].
Sulfonamides (R-SO2NH2) represent an important class of chemicals possessing the potency to
inhibit CA isoenzymes, which are involved in different pathological and physiological
processes. These compounds bind to active site of CAs by an interaction between catalytic zinc
ion of enzyme and deprotonated nitrogen of the sulfonamide moiety and block the CA
enzymatic activity. Despite the supplementary interaction points offered from the sulfonamides
within the CA, such a structural feature per se does not lead to selective isoenzyme binding.
The CA isoenzyme selectivity of sulfonamide type CA inhibitors is related to molecular regions
of compounds that are able to interact with hydrophobic and/or hydrophilic residues of the CA
catalytic site [23-26].
Acetazolamide, dorzolamide, brinzolamide, methazolamide, ethoxzolamide, and pazopanib are
clinically important CA inhibitors that carry sulfonamide pharmacophore (Figure 1). CA
inhibitors have in vitro selectivity differences against the 13 active isozymes, however none of
them is convincingly selective in vivo or clinically [27]. Thus, there common side effects
including taste alterations, depression, weight loss, blurred vision, burning of the eye, and
3

kidney stones for non-spesific CA inhibitors [28-31]. This situation reveals that there is a need
to enhance the selectivity and inhibition potency of CA inhibitors towards isoenzymes
Hydrazones, characterized by the presence of a open chain -CO-NH-N=CH-group, are used as
building blocks for the synthesis of a variety of useful bioactive compounds due to their
flexibility and structural similarities to various natural substances of biological importance. The
extensive pharmacological activity of hydrazone analogs has led to considerable research. [32-
35]. Moreover, several examples of compounds including hydrazone moiety have been reported
as CA inhibitors [36-38].
Based on the above information, we focused on the synthesis of compounds that carry the
hydrazone and sulfonamide pharmacophores in the same chemical structure and investigated
their inhibitor effects on hCA I and II isoenzymes.
O
O
O
O
S
S
S
S
N
O
SO2NH2
H
SO2NH2
S
N
SO2NH2
N N
O
HN
HN
Brinzolamide
Dorzolamide
Acetazolamide
H
N
N
N
N
N
S
S
SO2NH2
N
N
SO2NH2
N
N N
Methazolamide
O
O
SO2NH2
Ethoxzolamide
Pazopanib
Figure 1. Chemical structures of some carbonic anhydrase inhibitors which are in clinical use.
2. Experimental
2.1. Chemistry
Chemicals used in the synthesis were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St.
Louis, MO, USA) or Merck (Merck KGaA, Darmstadt, Germany) chemical companies and
were used without further chemical purifications. Melting points (MP) were determined by an
automatic melting point apparatus (MP90, Mettler-Toledo, OH, USA). The NMR spectra (1H
and 13C) were recorded in DMSO-d6 by a Bruker digital FT-NMR spectrometer (Bruker
Bioscience, MA, USA) at 300 MHz and 75 MHz, respectively. The IR spectra were recorded
by an IRAffinity-1S Fourier transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan).
HRMS studies were performed on an LCMS-IT-TOF system (Shimadzu, Tokyo, Japan).
4

Chemical purities of the compounds were checked by classical TLC applications performed on
silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).
2.1.1. 2-Chloro-N-(4-sulfamoylphenyl)acetamide (1)
Chloroacetyl chloride (2.16 mL, 27.8 mmol) was added dropwise with stirring to a solution of
4-aminobenzenesulfonamide (4 g, 23.2 mmol) in DMF (20 mL). After the completion of
dropping, the mixture was stirred for 1h and then poured into the iced-water (100 mL).
Precipitated product was filtered, washed with water, dried, and recrystallized from ethanol.
2.1.2. 2-Hydrazinyl-N-(4-sulfamoylphenyl)acetamide (2)
Excess of hydrazine hydrate (5 mL) was added in portions into a solution of 2-chloro-N-(4-
sulfamoylphenyl)acetamide (1) (5.19 g, 20.9 mmol) in EtOH. The reaction mixture was stirred
for 15h at room temperature. The precipitated product was filtered and washed with cold EtOH
to remove the excess of the hydrazine hydrate. Raw product was dried, and recrystallized from
EtOH.
2.1.3. General procedure for the synthesis of target compounds (3a-3n)
A mixture of 2-hydrazinyl-N-(4-sulfamoylphenyl)acetamide (2) (0.3 g, 1.24 mmol), appropriate
aromatic benzaldehydes (1.24 mmol) and catalytic quantity of acetic acid was refluxed in EtOH
for 2h. The mixture was cooled down in an ice-bath, precipitated product was filtered, dried
and recrystallized from EtOH.
2.1.3.1. N-(4-Sulfamoylphenyl)-2-(2-(thiophen-2-ylmethylene)hydrazinyl)acetamide (3a)
Yield: 81 %, M.P. = 163-164 ℃, FTIR (ATR, cm-1): 3313 (N-H), 1707 (C=O), 1155 (C-N),
825. 1H-NMR (300 MHz, DMSO-d6): δ = 3.92 (2H, d, J=5.30 Hz, -CH2), 7.00 (1H, dd, J=5.04
Hz- 3.60 Hz, Thiophene CH), 7.09 (1H, d, J=3.60 Hz, Thiophene CH), 7.24 (2H, s, -NH2), 7.36
(1H, d, J=5.04 Hz, Thiophene CH), 7.53 (1H, t, J=5.30 Hz, -NH), 7.76 (2H, s, Disubstituted
Benzene -CH), 7.77 (2H, s, Disubstituted Benzene -CH), 7.90 (1H,s, -CH=N), 10.27 (1H, s, -
NH) 13C-NMR (75 MHz, DMSO-d6): δ = 52.67, 119.24, 125.53, 126.27, 127.17, 127.81,
132.36, 138.90, 141.77, 142.16, 169.99. HRMS (m/z): [M+H]+ calcd for C15H13N3O2S3:
364.0243; found: 364.0249
2.1.3.2. 2-(2-((3-Methylthiophen-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3b)
5

Yield: 75 %, M.P. = 150-151 ℃, FTIR (ATR, cm-1): 3329 (N-H), 1697 (C=O), 1151 (C-N),
1
827. H-NMR (300 MHz, DMSO-d6): δ = 2.40 (3H, s, CH3), 4.17 (2H, d, J=4.17 Hz, -CH2),
6.91 (1H, d, J=3.63 Hz, Thiophene CH), 7.25 (2H, s, -NH2), 7.39 (1H, d, J=3.60 Hz, Thiophene
CH), 7.41 (1H, t, J=5.25 Hz, -NH), 7.77 (4H, s, Disubstituted Benzene -CH), 7.88 (1H, s, -
CH=N), 10.43 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ = 14.19, 51.16, 119.38, 125.63,
127.23, 130.38, 131.77, 138.35, 139.06, 142.05, 142.53, 168.29. HRMS (m/z): [M+H]+ calcd
for C16H15N3O2S3: 378.0399; found: 378.0390.
2.1.3.3. 2-(2-((5-Methylthiophen-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3c)
Yield: 78 %, M.P. = 163-164 ℃. FTIR (ATR, cm-1): 3327 (N-H), 1703 (C=O), 1151 (C-N). 1H-
NMR (300 MHz, DMSO-d6): δ = 2.38 (3H, s, -CH3), 3.92 (2H, d, J=5.25 Hz, -CH2), 6.69 (1H,
d, J=3.48 Hz, Thiophene CH), 6.88 (1H, d, J=3.48 Hz, Thiophene CH), 7.25 (2H, s, -NH2),
7.41 (1H, t, J=5.25 Hz, -NH), 7.77 (2H, s, Disubstituted Benzene -CH), 7.78 (2H, s,
13
Disubstituted Benzene -CH), 7.89 (1H,s, -CH=N), 10.26 (1H, s, -NH). C-NMR (75 MHz,
DMSO-d6): δ = 15.64, 52.78, 119.22, 126.04, 126.47, 127.16, 127.24, 133.01, 138.89, 142.05,
142.16, 168.29. HRMS (m/z): [M+H]+ calcd for C16H15N3O2S3: 377.0399; found: 377.0392.
2.1.3.4. 2-(2-((5-Bromothiophen-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3d)
Yield: 84 %, M.P. = 188-189 ℃, FTIR (ATR, cm-1): 3336 (N-H), 1681 (C=O), 1155 (C-N),
1
825. H-NMR (300 MHz, DMSO-d6): δ 3.96 (2H, d, J=5.10 Hz, -CH2), 6.91 (1H, d, J=3.84
Hz, Thiophene CH), 7.10 (1H, d, J=3.81 Hz, Thiophene CH), 7.24 (2H, s, -NH2), 7.74 (1H, t,
J=5.67 Hz, -NH), 7.76 (4H, br. s, Disubstituted Benzene -CH), 7.78 (1H,s, -CH=N), 10.28 (1H,
s, -NH) 13C-NMR (75 MHz, DMSO-d6): δ = 52.38, 110.67, 119.25, 126.33, 127.17, 130.90,
131.14, 138.92, 142.14, 143.93, 169.66. HRMS (m/z): [M+H]+ calcd for C15H12N3O2S3Br:
441.9348; found: 440.9343.
2.1.3.5. 2-(2-((5-Nitrothiophen-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3e)
Yield: 92 %, M.P. = 177-178 ℃, FTIR (ATR, cm-1): 3329 (N-H), 1695 (C=O), 1151 (C-N),
1
829. H-NMR (300 MHz, DMSO-d6): δ 4.13 (2H, d, J=5.10 Hz, -CH2), 7.12 (1H, d, J=4.44
Hz, Thiophene CH), 7.25 (2H, s, -NH2), 7.75 (1H, s, -CH=N), 7.77 (4H, br. s, Disubstituted
Benzene -CH), 8.00 (1H, d, J=4.41 Hz, Thiophene CH), 8.68 (1H, t, J=4.90 Hz, -NH), 10.39
6

(1H, s, -NH) 13C-NMR (75 MHz, DMSO-d6): δ = 51.82, 119.30, 124.34, 127.16, 127.20,
131.66, 139.01, 142.08, 147.62, 151.66, 168.64. HRMS (m/z): [M+H]+ calcd for
C15H12N4O4S3: 409.0093; found: 408.0087.
2.1.3.6. 2-(2-((1H-pyrrol-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide (3f)
1
Yield: 76 %, M.P. = oil, FTIR (ATR, cm-1): 3257 (N-H), 1693 (C=O), 1149 (C-N), 831. H-
NMR (300 MHz, DMSO-d6): δ 4.08 (2H, d, J=5.00 Hz, -CH2), 6.22 (1H, m, Pyrrole -CH), 6.64
(1H, m, Pyrrole -CH), 7.08 (1H, m, Pyrrole -CH), 7.25 (2H, s, -NH2), 7.76 (1H, s, -CH=N),
7.77 (2H, s, Disubstituted Benzene -CH), 7.78 (2H, s, Disubstituted Benzene -CH), 8.08 (1H,
t, J=4.90 Hz, -NH), 10.41 (1H, s, -NH), 11.17 (1H, s, pyrrole -NH) 13C-NMR (75 MHz,
DMSO-d6): δ = 51.63, 104.67, 115.16, 119.72, 124.47, 126.38, 127.81, 133.94, 139.21, 142.42,
168.64. HRMS (m/z): [M+H]+ calcd for C15H14N4O2S2: 347.0631; found: 347.0623.
2.1.3.7.
2-(2-((1-Methyl-1H-pyrrol-2-yl)methylene)hydrazinyl)-N-(4-
sulfamoylphenyl)acetamide (3g)
1
Yield: 77 %, M.P. = oil, FTIR (ATR, cm-1): 3246 (N-H), 1660 (C=O), 1151 (C-N), 829. H-
NMR (300 MHz, DMSO-d6): δ 3.81 (3H, s, -OCH3), 3.87 (3H, s, –CH3), 4.59 (2H, d, J=4.17
Hz, -CH2), 6.09 (1H, dd, J=3.75 Hz- 2.61 Hz, Pyrrole CH), 6.47 (1H, dd, J=3.78 Hz- 1.74 Hz,
Pyrrole CH), 6.95-6.96 (1H, m, Pyrrole CH), 7.26 (2H, s, -NH2), 7.76 (1H, s, -CH=N), 7.77
(2H, s, Disubstituted Benzene -CH), 7.78 (2H, s, Disubstituted Benzene -CH), 8.08 (1H, t,
J=4.90 Hz, -NH), 10.39 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ = 35.74, 51.68,
104.71, 115.43, 119.72, 124.85, 126.38, 127.81, 133.94, 139.21, 142.42, 168.64. HRMS (m/z):
[M+H]+ calcd for C16H16N4O2S2: 361.0787; found: 361.0779.
2.1.3.8. 2-(2-(Furan-2-ylmethylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide (3h)
Yield: 79 %, M.P. = 146-147 ℃, FTIR (ATR, cm-1): 3267 (N-H), 1681 (C=O), 1151 (C-N). 1H-
NMR (300 MHz, DMSO-d6): δ 4.62 (2H, d, J=4.32 Hz, -CH2), 6.61-6.64 (1H, m, Furan CH),
6.92 (1H d, J=3.42 Hz, Furan CH), 7.26 (2H, s, -NH2), 7.76 (1H, s, -CH=N), 7.77 (2H, s,
Disubstituted Benzene -CH), 7.78 (2H, s, Disubstituted Benzene -CH), 7.82-7.83 (1H, m, Furan
CH), 7.95 (1H, t, J=4.50 Hz, -NH), 10.46 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =
51.99, 112.65, 114.43, 119.72, 127.81, 133.94, 139.21, 142.42, 143.86, 149.19, 168.10. HRMS
(m/z): [M+H]+ calcd for C15H13N3O3S2: 348.0471; found: 348.0466.
2.1.3.9.
2-(2-((5-Methylfuran-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3i)
7

Yield: 79 %, M.P. = 145-146 ℃, FTIR (ATR, cm-1): 3246 (N-H), 1701 (C=O), 1151 (C-N),
790.1H-NMR (300 MHz, DMSO-d6): δ 2.34 (3H, s, -CH3), 4.61 (2H, d, J=4.32 Hz, -CH2), 6.25-
6.26 (1H, m, Furan CH), 6.80 (1H d, J=3.24 Hz, Furan CH), 7.26 (2H, s, -NH2), 7.76 (1H, s, -
CH=N), 7.77 (2H, s, Disubstituted Benzene -CH), 7.78 (2H, s, Disubstituted Benzene -CH),
7.95 (1H, t, J=4.50 Hz, -NH), 10.46 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =14.01,
52.06, 105.00, 109.07, 114.19, 119.15, 127.68, 134.68, 137.33, 147.92, 155.10, 168.23. HRMS
(m/z): [M+H]+ calcd for C16H15N3O3S2: 362.0628; found: 362.0632.
2.1.3.10. 2-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide (3j)
Yield: 80 %, M.P. = 169-170 ℃, FTIR (ATR, cm-1): 3334 (N-H), 1701 (C=O), 1149 (C-N),
1
833. H-NMR (300 MHz, DMSO-d6): δ 4.17 (2H, d, J=4.41 Hz, -CH2), 6.79 (1H, d, J=3.99
Hz, Furan CH), 7.25 (2H, s, -NH2), 7.52 (1H, s, -CH=N), 7.71 (1H, d, J=3.96 Hz, Furan CH),
7.77 (4H, br. s, Disubstituted Benzene -CH), 8.80 (1H, t, J=4.44 Hz, -NH), 10.41 (1H, s, -NH)
13C-NMR (75 MHz, DMSO-d6): δ = 51.89, 109.88, 116.51, 119.27, 121.78, 127.22, 139.01,
142.07, 150.99, 155.77, 168.77. HRMS (m/z): [M+H]+ calcd for C15H12N4O5S2: 393.0322;
found: 393.0315.
2.1.3.11.
2-(2-((1H-imidazol-2-yl)methylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide
(3k)
Yield: 82 %, M.P. = 178-179 ℃, FTIR (ATR, cm-1): 3344 (N-H), 1695 (C=O), 1155 (C-N),
839. 1H-NMR (300 MHz, DMSO-d6): δ 4.69 (2H, d, J=4.32 Hz, -CH2-), 6.67 (2H, s, Imidazole
-CH), 7.26 (2H, s, -NH2), 7.51 (2H, s, Imidazole -NH), 7.72 (1H, s, -CH=N), 7.77 (2H, s,
Disubstituted Benzene -CH), 7.78 (2H, s, Disubstituted Benzene -CH), 7.82-7.83 (1H, m, Furan
13
CH), 7.95 (1H, t, J=4.50 Hz, -NH), 10.49 (1H, s, -NH). C-NMR (75 MHz, DMSO-d6): δ
=51.98, 119.25, 121.54, 126.73, 130.37, 133.94, 141.56, 144.02, 168.97. HRMS (m/z): [M+H]+
calcd for C16H14N4O2S2: 359.0631; found: 359.0635.
2.1.3.12.
2-(2-((1-Methyl-1H-imidazol-2-yl)methylene)hydrazinyl)-N-(4-
sulfamoylphenyl)acetamide (3l)
Yield: 82 %, M.P. = 160-161 ℃, FTIR (ATR, cm-1): 3342 (N-H), 1687 (C=O), 1153 (C-N),
839. 1H-NMR (300 MHz, DMSO-d6): δ 4.17 (2H, d, J=4.38 Hz, -CH2), 6.81 (1H, s, Imidazole
-CH), 7.09 (1H, s, Imidazole -CH), 7.25 (2H, s, -NH2), 7.65 (1H, s, -CH=N), 7.77 (4H, br. s,
Disubstituted Benzene -CH), 8.80 (1H, t, J=4.44 Hz, -NH), 10.36 (1H, s, -NH) 13C-NMR (75
8

MHz, DMSO-d6): δ = 13.82, 51.89, 119.27, 121.16, 126.83, 127.22, 139.01, 142.07, 144.42,
149.82, 168.77. HRMS (m/z): [M+H]+ calcd for C16H14N4O2S2: 359.0631; found: 359.0628.
2.1.3.13. 2-(2-(Pyridin-3-ylmethylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide (3m)
Yield: 82 %, M.P. = 162-163 ℃, FTIR (ATR, cm-1): 3350 (N-H), 1689 (C=O), 1151 (C-N),
1
896. H-NMR (300 MHz, DMSO-d6): δ 4.10 (2H, d, J=4.11 Hz, -CH2), 7.25 (2H, s, -NH2),
7.42 (1H, m, Pyridine CH), 7.59 (1H, s, -CH=N), 7.76 (2H, br. s, Disubstituted Benzene -CH),
7.78 (2H, br. s, Disubstituted Benzene -CH), 8.13 (1H, m Pyridine CH), 8.23 (1H, t, J=4.08
Hz, -NH), 8.78 (2H, m Pyridine CH), 10.39 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =
52.03, 119.24, 122.15, 127.14, 130.83, 131.72, 134.13, 138.90, 143.72, 148.02, 149.65, 168.11.
HRMS (m/z): [M+H]+ calcd for C16H14N4O2S2: 359.0631; found: 359.0628.
2.1.3.14. 2-(2-(Pyridin-4-ylmethylene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide (3n)
Yield: 82 %, M.P. = 157-158 ℃, FTIR (ATR, cm-1): 3350 (N-H), 1700 (C=O), 1151 (C-N),
1
840. H-NMR (300 MHz, DMSO-d6): δ 4.10 (2H, d, J=4.08 Hz, -CH2), 7.25 (2H, s, -NH2),
7.40 (2H, d, J=6.12 Hz, Pyridine CH), 7.57 (1H, s, -CH=N), 7.76 (2H, br. s, Disubstituted
Benzene -CH), 7.78 (2H, br. s, Disubstituted Benzene -CH), 8.29 (1H, t, J=4.08 Hz, -NH), 8.45
13
(2H, d, J=6.09 Hz, Pyridine CH), 10.53 (1H, s, -NH). C-NMR (75 MHz, DMSO-d6): δ =
52.19, 119.24, 122.96, 127.14, 131.99, 138.90, 142.22, 143.91, 150.23, 169.47. HRMS (m/z):
[M+H]+ calcd for C16H14N4O2S2: 359.0631; found: 359.0628.
2.2. Biological Activity
2.2.1. Purification of CA isozymes from human erythrocytes by affinity chromatography
Erythrocytes were purified from fresh human blood obtained from the Blood Center, Atatürk
University. The blood samples were centrifuged to separate erythrocytes at 1500 rpm for 15
min and plasma and buffy coat were carefully removed. Then, underlying erythrocytes washed
twice with 0.9 % NaCl and hemolysed with 1.5 volumes of ice-cold water. It was stirred for
half an hour at 4 °C. The hemolysate was centrifuged at 20,000 rpm for 30 min at 4 °C and cell
membranes were separated. pH was adjusted to 8.7 with solid Tris. The hemolysate was applied
to the prepared Sepharose 4B-L-tyrosine-sulfanylamide 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 CA isozymes (hCA-I and hCA-II) were eluted with 1 M
NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6), respectively.
All procedures were performed at 4 ◦C [39].
9

2.2.2. Measurement of CA activity
2.2.2.1. Hydratase activity assay
CA activity was determined using the Wilbur-Anderson Method [40]. 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. CO2–Hydratase activity as an
enzyme unit (EU) was calculated by using the equation (to-tc/tc) where to and tc are the times
for pH change of the nonenzymatic and the enzymatic reactions, respectively.
2.2.2.2. Esterase activity assay
Esterase activity of human erythrocyte CA 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. [41].
2.2.3. Inhibition assays
The inhibitory effects of newly synthesized compounds on CA enzyme activity were tested in
triplicate at each concentration used. IC50 values were calculated for the compounds at different
concentrations by utilizing graphs of activity %. The activities of enzymes in the medium
without inhibitors were used as 100% activity. The Ki values of the compounds were calculated
by Lineweaver-Burk graphics.
2.3. Cytotoxicity Test
NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL 1658) was used for cytotoxicity
study. Cells were replicated into Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% fetal bovine serum (FBS), 1% 100 IU/mL penicillin / 100 mg/mL streptomycin, at 37 °C
in a humidified atmosphere of 95% air and 5% CO2. The proliferation of the NIH/3T3 cell line
was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) assay.
Briefly, cells were inoculated into 96-well culture plates at densities of 10,000 cells per well.
After 24 h, they were treated with synthesized compounds (concentrations of 200-0.39 µg/mL)
for 24 h. After the incubation period, MTT solution (5 mg/mL) was added to each well and
incubated for 3 h at 37 ºC. At the end of the incubation, the purple MTT-formazan crystals were
dissolved by adding 100 µg/ml of DMSO to each well. The plates were then read on a
Microplate reader at 540 nm wavelength [42,43]. The IC50 value was calculated from the plots
of cell proliferations against concentrations by applying regression analyses on GraphPad Prism
Version 5.
10

2.4. Prediction of ADME Parameters
Physicochemical parameters of synthesized compounds (3a-3n) were calculated by using
QikProp 4.8. [44].
2.5. Molecular Docking Studies
A Docking study was performed to seek binding modes of compounds 3e and 3m to CA I and
CA II enzyme active sites, respectively. The crystal structures of CA I (PDB ID: 1AZM) [45]
and CA II (PDB ID: 3HS4) [46] were retrieved from the Protein Data Bank server
(www.pdb.org).
The chemical structures of ligands were drawn using the Schrödinger Maestro [47] interface
and then were submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite
2016 Update 2 [48]. Then the ligands were prepared by the LigPrep 3.8 [49] to set to
protonation states at pH 7.4±1.0 and the atom types, correctly. Bond orders were assigned, and
hydrogen atoms were added to the structures. The grid generation was formed using Glide 7.1
[50]. Flexible docking runs were performed with single precision docking mode (SP).
3. Results and Discussion
3.1. Chemistry
The compounds 3a-3n were synthesized as summarized as Scheme 1. Initially, 2-Chloro-N-(4-
sulfamoylphenyl)acetamide (1) was prepared by means of the acetylation reaction using
chloroacetyl chloride. In the second step, 2-Chloro-N-(4-sulfamoylphenyl)acetamide (1) and
excess of hydrazine hydrate were stirred at room temperature to obtain 2-hydrazinyl-N-(4-
sulfamoylphenyl)acetamide (2). Finally, compound 2 and appropriate aromatic benzaldehydes
were reacted to gain the compounds 3a-3n.
Structure of the synthesized were elucidated by IR, 1H NMR, 13C NMR and mass spectroscopic
analyses. In the IR spectroscopy, N-H bond of hydrazide was observed over 3246 cm-1.
1
Carbonyl group (C=O) had a band about 1681-1707 cm-1. In H NMR spectrum, methoxy (-
OCH2) protons and methylene (-CH2-) protons between sulphur atom and carbonyl group gave
singlet peaks at 3.81-3.82 ppm and between 4.55-4.70 ppm, respectively. Benzimidazole H5
gave a doublet of doublet peak at 6.99 ppm with coupling constants values 8.8 Hz and 2.5 Hz.
Benzimidazole H4 and H6 protons were observed between 7.37-7.42 ppm and 7.86-7.87 ppm,
respectively. Methine (-CH=) proton of hydrazide group recorded as a singlet peak between
7.87-8.29 ppm, depending on the electronic nature of substituents. Proton of hydrazone nitrogen
11

(-CONHN=) had a singlet peak over 11.39 ppm. The aromatic peaks of variable heterocyclic
13
rings were observed between 6.09-8.88 ppm. In C NMR spectrum, aliphatic carbons were
recorded between 14.01-56.01 ppm, while aromatic carbons were observed between 104.96-
169.23 ppm. In the HRMS, all masses were matched well with the expected M+H values.
H
NH2
N
NH2NH2.H2O
EtOH/ Rt
ClCOCH2Cl
DMF/ 0°C
Cl
O
H2NO2S
H2NO2S
H
H
N
N
CH3COOH
NHN
NHNH2
CHO-Het
EtOH/
Het
O
O
H2NO2S
H2NO2S
Comp.
3a
Het
Thiophen-2-yl
3b
3c
3-Methylthiophen-2-yl
5-Methylthiopen -2-yl
5-Bromothiophen-2-yl
5-Nitrothiophen-2-yl
1H-pyrrol-2-yl
3d
3e
3f
3g
1-Methyl-1H-pyrrol-2-yl
Furan-2-yl
3h
3i
5-Methylfuran-2-yl
5-Nitrofuran-2-yl
1H-imidazol-2-yl
1-Methyl-1H-imidazol-2-yl
Pyridin-3-yl
3j
3k
3l
3m
3n
Pyridin-4-yl
Scheme 1. Synthesis of target compounds.
3.2. Biological activity
The newly prepared sulfonamides 3a-3n were evaluated for their ability to inhibit two
physiologically relevant hCA isoforms, hCA I and II. The inhibition data (IC50 and Ki) of the
prepared compounds and the standard inhibitor acetazolamide (AZA) against hCA isoforms are
summarized in Table 1.
12

The IC50 values of compounds against hCA I were calculated between 0.176 µM and 2.398 µM,
whereas IC50 values against hCA II were in the range of 0.526 µM to 2.484 µM. The compounds
3e and 3m showed notable inhibitory effects against hCA I and hCA II, respectively. Compound
3e (IC50=0.176 µM) showed the strongest and selective inhibition toward hCA I enzyme, while
3m was found to be the most active (IC50=0.526 µM) and selective derivative against hCA II.
Table 1 gives some important information about structure activity relationships. Compounds
3e (IC50=0.176 µM) and 3j (IC50=0.264 µM), bearing 5-nitrothiophene and 5-nitrofuran
substructures, have higher inhibition potency against hCA I when compared with other
derivatives. Due to nitro substituent in both compounds, it can be suggested that electron
withdrawing group has very important contribution to enhance inhibition potency against hCA
I. Furthermore, higher inhibition potencies of thiophene based compounds 3a, 3c and 3e than
those of furan bearing derivatives 3h, 3i and 3j indicate that thiophene ring has more impact to
enhance hCA I inhibition when compared with furan ring. When the CA II inhibition potentials
of the compounds are examined, it is seen that the compounds 3m and 3n exhibit higher activity
in the series. The compounds 3a-3l include five membered thiophene, furan, pyyrole, or
imidazole rings. On the other hand, there is a pyridine ring in the structure of compounds 3n
and 3m. Thus, it can be proposed that unlike other compounds (3a-3l), the six-membered
pyridine ring in the structures of 3n and 3m may be the reason for their higher activity.
The enzyme kinetic studies of all synthesized compounds 3a-3n on both hCA I and hCA II
enzymes were carried out in order to determine the inhibition type and calculate Ki values. The
inhibition types are divided as reversible and irreversible inhibition. The uncompetitive,
competitive, non-competitive and mixed type inhibitions constitute the subtypes of reversible
inhibitors. All these subtypes of inhibition can be determined with the help of Lineweaver-Burk
plots according to the style of graphics [51]. Uncompetitive type inhibition has parallel lines of
inhibitor and control without any cross in the graphic. If there is an intersection in any area of
the graph except for the x and y axes, the mixed type inhibition is concerned. The graph formed
by the plots intersecting on y-axis describes the competitive inhibition type and this graphic has
different slopes and intercepts on x-axis. Non-competitive inhibitors have the same intercept
on x-axis and diverse slopes and intercepts on y-axis in the graphics. Lineweaver-Burk plots of
the most active compounds 3e and 3m are presented in Figures 2 and 3. Besides, Lineweaver-
Burk plots of the other compounds are presented as supporting information (Supplementary
Material Figures S1-26). These graphs intersect at the x-axis as in the non-competitive
inhibition type described above. Hence, enzyme kinetic studies reveal that all synthesized
13

compounds 3a-3n are reversible and non-competitive inhibitors against hCA I and hCA II
enzymes. Ki values were calculated by using the data gained from Lineweaver-Burk graphics.
Ki values of the synthesized compounds were considered in the ranges of 0.1676-3.1280 µM
and 0.2880-2.4822 µM on hCA I and hCA II, respectively. In addition to lowest IC50
compounds 3e and 3m also showed the lowest Ki values towards hCA I and hCA II enzyme
(Table 1).
The Ki and IC50 of a drug to cause the inhibition of an enzyme is related to the concentration
required to reduce the activity of this enzyme by half. More specifically, the Ki reflects the
enzyme binding affinity of a drug, while the IC50 more exhibits the inhibitory potency of a drug
on the enzyme. With lower Ki, the binding affinity required to inhibit the activity of the enzyme
increases, while the amount of inhibitor decreases. Obtained results indicated that compounds
3e and 3m possess lower IC50 and Ki values against hCA I and hCA II than those of AZA.
Besides, selectivity of compounds towards hCA I and hCA II are significantly different as seen
in Table 1. These findings improve the biological importance of compounds 3e and 3m as
selective inhibitors of hCA I and hCA II, respectively.
Table 1. Human CA isoenzymes (hCA I and II) inhibition value of the compounds (3a-3n).
Compounds
IC50(µM)
Ki(µM)
hCA I
0.732
1.586
0.692
0.772
0.176
2.003
1.627
1.205
1.963
0.264
1.098
2.063
1.462
2.398
0.263
r2
hCA II
1.754
1.378
1.853
1.893
1.974
1.634
2.094
2.044
2.484
2.415
1.456
1.791
0.526
0.637
0.108
r2
hCA I
hCA II
3a
3b
3c
0.9347
0.9512
0.9399
0.9568
0.9504
0.9505
0.9547
0.9856
0.9914
0.9445
0.9713
0.9716
0.9576
0.9643
0.9567
0.9668
0.9517
0.9903
0.9479
0.9637
0.9510
0.9825
0.9443
0.9809
0.9852
0.9561
0.9852
0.9430
0.9897
0.9496
0.3110±0.103 1.3594±0.354
0.8190±0.205 1.2442±0.342
0.3445±0.112 1.0974±0.259
0.2653±0.047 1.5036±0.451
0.1676±0.017 1.5154±0.375
1.1014±0.396 0.9596±0.215
1.1364±0.238 1.2963±0.357
0.6454±0.115 2.4180±0.415
1.7470±0.355 2.4822±0.490
0.2383±0.057 1.8232±0.474
2.0480±0.314 1.6259±0.408
2.2538±0.338 1.1614±0.422
1.8580±0.409 0.2880±0.080
3.1280±0.403 0.5722±0.108
0.2892±0.010 0.1355±0.004
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
AZA
14

16
12
8
4
0
0
1
2
3
4
5
6
1/[S] (mM)-1
Figure 2. Lineweaver–Burk graphs of the compound 3e on CA I.
3
2
1
0
-1
0
1
2
3
4
5
6
1/[S] (mM)-1
Figure 3. Lineweaver–Burk graphs of the compound 3m on CA II.
3.3. Cytotoxicity Test
The simultaneous optimization of the desired drug properties and potential adverse effects are
aimed in the early stages of new drug development studies. This approach allows scientists to
select drug candidates with the highest probability of success for preclinical safety trials. Thus,
some in vitro models are preferred for evaluating toxicity should be to predict human specific
toxicity due to their high sensitivity in the determination of toxic agents [52]. In the present
study MTT assay, a classical instance for such in vitro toxicity models, was performed using
healthy NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658), recommended cell
type for general cytotoxicity screening by ISO (10993-5, 2009) [53]. As presented in the Table
2, the IC50 values of compounds 3e and 3m against NIH/3T3 cells were found to be higher than
15

1000 µM, which is significantly greater than the IC50 values of compounds against hCA I and
II. Thus, it may be declared that both compounds are not toxic at their effective concentrations
against hCA I and II.
Table 2. IC50 (μM) values of the compounds 3a, 3c, 3d, 3e, 3j, 3m and 3n derivatives against NIH/3T3 cell line.
Compound
IC50 (µM)
3a
3c
3d
3e
3j
3m
3n
>1000
>1000
>1000
>1000
>1000
>1000
>1000
3.4. Theoretical Calculation of ADME Parameters
Evaluation of the pharmacological properties of a drug candidate such as Absorption,
Distribution, Metabolism and Excretion (ADME) has critical importance for initial selection.
Thus, criteria for assessment of synthesized compounds during parent compound optimization
can be established. Therefore, in the present study ADME properties were calculated using
QikProp 4.8. [44] software and presented in the Table 3. QikProp also provides acceptable
ranges for comparing the predicted properties of compounds with those of 95% of known drugs.
Furthermore, the drug-likeness properties were estimated with the help of this software. The
drug-likeness of all compounds was assessed according to Lipinski’s Rule of Five [54] and
Jorgensen's Rule of Three [55]. Lipinski’s Rule of Five considers molecular weight (<500 Da),
number of hydrogen bond acceptors (≤10), number of hydrogen bond donors (≤5), and
octanol/water partition coefficient (≤5), whereas Jorgensen's Rule of Three regards logS (>-
5.7), PCaco (>22 nm/s), primary metabolites (PM) (<7). The violations of these rules are
essential for the optimization of a biologically active compounds.
Table 3 contains the following parameters: Molecular weight (MW), number of rotatable bonds
(RB), dipole moment (DM), molecular volume (MV), number of hydrogen donors (DHB),
number of hydrogen acceptors (AHB), polar surface area (PSA), octanol/water partition
coefficient (log P), aqueous solubility (log S), apparent Caco-2 cell permeability (PCaco),
brain/blood partition coefficient (logBB), apparent MDCK cell permeability (PMDCK),
number of likely primer metabolic reactions (PM), and percent of human oral absorption
(%HOA), the violations of rules of three (VRT) and five (VRF). According to Lipinski's rule
of five and Jorgensen's rule of three all compounds 3a-3n are in accordance with the rule by
causing no more than one violation. Also, all ADME parameters of synthesized compounds 3a-
3n are in the recommended ranges. As a consequence of ADME predictions, it can be suggested
that the final compounds 3a-3n may have a good pharmacokinetic profile.
16

Table 3. Calculated ADME parameters.
Comp.
MW
RB
DM
MV
DHB AHB
PSA
logP
logS
PCaco
logBB PMDCK PM %HOA
VRF VRT
3a
3b
3c
3d
3e
3f
338.399
352.425
352.425
417.295
383.396
321.353
335.380
322.338
336.365
367.336
322.341
336.368
333.364
333.364
8
8
8
8
9
8
8
8
8
9
8
8
8
8
10.531 1029.269
4
4
4
4
4
5
4
4
4
4
5
4
4
4
9.5
9.5
9.5
9.5
10.5
9.5
9.5
10
126.840
120.894
123.862
126.824
173.861
140.313
126.734
135.655
132.802
183.310
151.938
140.156
136.385
139.254
0.55
-3.398
-3.213
-3.463
-4.170
-3.276
-3.037
-2.997
-2.848
-2.977
-2.880
-2.519
-2.662
-2.747
-2.993
85.135
125.023
99.369
85.246
9.439
-2.130
-1.776
-1.983
-2.005
-3.202
-2.495
-2.027
-2.295
-2.079
-3.401
-2.589
-2.274
-2.354
-2.514
61.661
96.890
68.771
161.973
5.214
3
4
4
3
3
2
2
3
4
3
2
2
4
4
64.709
69.145
67.198
67.830
43.218
57.115
65.473
59.974
64.058
25.715
49.215
56.834
56.782
56.087
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
7.996
8.382
9.961
1053.538
1073.871
1083.843
0.797
0.77
1.081
-0.201
-0.031
0.524
-0.018
0.273
-0.717
-0.807
-0.248
-0.222
-0.198
10.175 1099.002
9.341
8.977
10.422
8.443
9.888
9.528
1008.847
1047.045
993.846
49.625
95.728
71.020
96.424
7.759
19.577
39.826
28.841
40.139
2.635
3g
3h
3i
1041.415
1071.889
984.063
10
3j
11
3k
3l
11
32.237
56.374
54.918
49.299
12.281
22.470
21.843
19.438
10.156 1036.825
10.786 1031.598
10.245 1044.408
11
3m
3n
11
11
MW: Molecular weight RB: Number of rotatable bonds DM: Computed dipole moment MV: Total solvent-accessible volume DHB: Estimated number of hydrogen bond
donors AHB: Estimated number of hydrogen bond acceptors PSA: Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms logP: Predicted
octanol/water partition coefficient logS: Predicted aqueous solubility PCaco: Predicted apparent Caco-2 cell permeability logBB: Predicted brain/blood partition coefficient
PMDCK: Predicted apparent MDCK cell permeability PM: Number of likely metabolic reactions %HOA: Predicted human oral absorption percent VRF: Number of violations
of Lipinski’s rule of five. The rules are: MW < 500, logP < 5, DHB ≤ 5, AHB≤ 10, Positive PSA value. VRT: Number of violations of Jorgensen’s rule of three. The three rules
are: logS > -5.7, PCaco > 22 nm/s, PM < 7.
1

3.5. Molecular Docking Studies
The compounds 3e and 3m were found to be the most active derivatives against CAI and CA
II enzymes as mentioned in the enzyme inhibition assay. Docking studies were performed in
order to gain more insight into the binding modes of compounds 3e and 3m to evaluate the
effects of structural modifications on the inhibitory activity against CAI and CA II. X-ray
crystal structures of CA I (PDB ID: 1AZM) [45] and CA II (PDB ID: 3HS4) [46] were obtained
from Protein Data Bank server (www.pdb.org). The docking poses of compounds 3e and 3m
are presented in Figures 4,5 and Supplementary Material Figures S27-36.
In the Supplementary Material Figures S27 and S 32, it can be seen that sulfonamide groups
of compounds 3e and 3m are in N-deprotonated form, which serves as a zinc-binding site [56].
As expected, compounds 3e and 3m bind to enzyme active site by their sulfonamide groups
coordinated to the zinc ion. (Supplementary Material Figures S28, S29, S33 and S34). The
deprotonated sulfonamide amino groups bind to the Zn+2 ion by salt bridge. Also, one of oxygen
atom of deprotonated sulfonamide group forms a metal coordination bond with Zn+2 ion. The
other oxygen atom of deprotonated sulfonamide group creates a hydrogen bond with amino of
Thr199 for compounds 3e and 3m. Both these interactions are typical for all sulfonamide-CA
complexes investigated so far [56-62].
When docking poses of compound 3e on CA I (Figure 4 and Supplementary Material Figures
S27-31) were analyzed, it can be seen that this compound has three interactions with enzyme
active region, consist of a hydrogen bond, a π-π interaction and a salt bridge, except for
mentioned above related to zinc ion. The hydrogen bond is seen between carbonyl of the
structure and amino of Gln92. The phenyl ring in the structure is in an interaction with
imidazole ring of Hid200 by doing π-π interaction. Furthermore, oxygen atom of nitro group is
essential for strengthening binding to enzyme active site. This oxygen atom forms a salt bridge
with quaternized amino of Lys57. Also, it can be seen from Supplementary Material Figures
S30 and 31 that van der Waals and electrostatic interactions provide stronger binding to enzyme
active site of CA I for compound 3e. This compound has favourable van der Waals interactions
with Asn69, Hid67, Phe91, Gln92, Hid94, Hid96, Hid119, Ala121, Leu131, Leu141, Leu198,
Thr199, Hid200 and Trp209, displayed with pink and red colours as described in the user guide
of Glide [50]. Especially, it is thought that thiophene ring, hydrazine and amide moieties
enhance van der Waals interaction with enzyme active region. Also, this compound shows
promising electrostatic contributions with Hid94, Hid96, Glu106 and Hid119.
1

As mentioned in the enzyme inhibition assay, compound 3e is the most active derivative against
CA I with an IC50 value of 0.176 µM, while 3j is the second active compound with an IC50
value of 0.264 µM. The structurally common feature of these compounds is the nitro group at
C-5 position of both thiophene and furan rings. In this context, the docking studies supports the
enzyme inhibition results. It is thought that the interactions related to nitro group at C-5 position
of thiophene or furan is important in terms of explaining their inhibitory activities. It is seen
that the presence of an electron withdrawing group such as nitro at this position is a positive
contribution to the activity.
According to the docking poses of compound 3m on CA II enzyme (Figure 5 and
Supplementary Material Figures S32-36), there are three hydrogen bonds apart from
sulfonamide group interactions. The carbonyl of amide moiety of compound 3m forms two
hydrogen bonds with amino of Asn62 and Asn67. The nitrogen atom of pyridine ring in the
structure has an additional interaction different from the interactions of the common chemical
structure. This nitrogen atom is in interaction with nitrogen atom of indole ring of Trp5 by
forming a hydrogen bond. Besides, van der Waals and electrostatic interactions of compound
3m are presented in Supplementary Material Figures S35 and 36. It can be seen that
piperidine ring, hydrazine and amide moieties support binding to residues of enzyme active
region of CA II by van der Waals interactions. This compound forms van der Waals interactions
with Trp5, Asn62, Hid64, Gln92, Hid94, Hid96, Hid119, Val121, Val143, Leu198, Thr199,
Thr200, Pro201, Pro202 and Trp209, which were shown as pink and red colours [50].
Electrostatic interactions between this compound and Hid96, Glu106 and Hid119 are also
observed.
As stated in the enzyme activity results, compounds 3m and 3n are the most active derivatives
against CA II enzyme with IC50 values of 0.526 and 0.637 µM, respectively. These compounds
bear pyridine-3-yl and -4-yl rings different from common chemical structure. Docking studies
provide to explain their inhibitory activities. The nitrogen atoms of pyridine rings have ability
to form a hydrogen bond by acting as a hydrogen acceptor because their lone pair is ready for
donation. Thus, pyridine nitrogen can interact with donor sites of amino acid residues in an
enzyme active site. Consequently, this difference influences and explains binding capability of
the compounds 3m and 3n to enzyme active site and enzyme inhibition potency.
2

Figure 4. Three-dimensional mode of compound 3e in the enzyme active site of CA I (CA I PDB Code: 1AZM).
3

Figure 5. Three-dimensional mode of compound 3m in the enzyme active site of CA II (CA II PDB Code: 3HS4).
4. Conclusion
In conclusion, we have reported the synthesis, characterization and evaluation of biological
activity of a series of sulfonamide bearing hydrazone compounds for the inhibition of the
physiologically relevant CA isozymes. According to Ki values, nitrothiophen compound 3e
toward hCA I and pyridin-3-yl compound 3m toward hCA II can be considered as leader
compounds for further studies. Enzyme inhibition is a very important issue for drug design and
biochemical applications. Hence, the results put forward that new sulfonamide derivatives
inhibit the hCA I and II enzyme activity, which will probably be enhanced in the further studies.
4

5. Acknowledgment
This study was financially supported by Anadolu University Scientific Projects Fund, Project
No: 1803S050.
6. Declaration of interest
The authors declare no conflicts of interest.
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8

Highlights
 New sulfonamide-hydrazone derivatives (3a-3n) were synthesized
 Synthesized compounds were evaluated for their inhibitory effects on purified human
carbonic anhydrase (hCA) I and II.
 The compound 3e demonstrated the best hCA I inhibitory effect with a Ki value of
0.1676±0.017 µM.
 The compound 3m showed the best hCA II inhibitory effect with a Ki value of
0.2880±0.080 µM.
 Molecular docking studies were performed to investigate the interaction types between
active compounds and hCA enzymes.
9

Synthesis, molecular docking analysis and carbonic anhydrase I-II inhibitory evaluation
of new sulfonamide derivatives
Three-dimensional binding mode of compounds 3e and 3m in the enzyme active site of CA
I (CA I PDB Code: 1AZM) and CA II (CA II PDB Code: 3HS4), respectively.
10