Synthesis, molecular docking analysis, and carbonic anhydrase inhibitory evaluations of benzenesulfonamide derivatives containing thiazolidinone

Article abstract of DOI:10.3390/molecules24132418

To find novel human carbonic anhydrase (hCA) inhibitors, we synthesized thirteen compounds by combining thiazolidinone with benzenesulfonamide. The result of the X-ray singlecrystal diffraction experiment confirmed the configuration of this class of compounds. The enzyme inhibition assays against hCA II and IX showed desirable potency profiles, as effective as the positive controls. The docking studies revealed that compounds (2) and (7) efficiently bound in the active site cavity of hCA IX by forming sufficient interactions with active site residues. The fragment of thiazolidinone played an important role in the binding of the molecules to the active site.

Full text of DOI:10.3390/molecules24132418

molecules
Article
Synthesis, Molecular Docking Analysis, and Carbonic
Anhydrase Inhibitory Evaluations of
Benzenesulfonamide Derivatives
Containing Thiazolidinone
Zuo-Peng Zhang, Ze-Fa Yin, Jia-Yue Li, Zhi-Peng Wang, Qian-Jie Wu, Jian Wang, Yang Liu * and
Mao-Sheng Cheng *
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education,
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
* Correspondence: y.liu@syphu.edu.cn (Y.L.); mscheng@syphu.edu.cn (M.-S.C.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 May 2019; Accepted: 27 June 2019; Published: 30 June 2019
Abstract: To find novel human carbonic anhydrase (hCA) inhibitors, we synthesized thirteen
compounds by combining thiazolidinone with benzenesulfonamide. The result of the X-ray
single-crystal diffraction experiment confirmed the configuration of this class of compounds. The
enzyme inhibition assays against hCA II and IX showed desirable potency profiles, as effective as the
positive controls. The docking studies revealed that compounds (2) and (7) efficiently bound in the
active site cavity of hCA IX by forming sufficient interactions with active site residues. The fragment
of thiazolidinone played an important role in the binding of the molecules to the active site.
Keywords: human carbonic anhydrase IX; thiazolidinone; benzenesulfonamide; docking study
1. Introduction
Human carbonic anhydrases (hCAs, EC 4.2.1.1) are important metalloenzymes that can catalyze
the interconversion of CO2 and H2O with HCO3 and H+. These enzymes play a pivotal role in
−
many cellular and physiological processes, such as electrolyte secretion, pH homeostasis, respiration,
and biosynthetic pathways. There are at least 15 CA isoforms in humans, which differ in molecular
properties, subcellular localization, and tissue distribution [
in many normal tissues, but it is overexpressed in numerous solid tumors, assisting in tumor cell
survival, proliferation, and metastasis in the tumor microenvironment [ 11]. Therefore, CA IX has
1–4]. Uniquely, CA IX has low expression
5
–
been validated as a promising target for cancer diagnostics and treatments. Currently, there are a large
number of CA IX inhibitors reported in the literature, but only SLC-0111 (Figure 1) has completed
Phase I clinical trials [12–14].
Figure 1. The chemical structure of SLC-0111.
In the area of medicinal chemistry, five-membered heterocyclic compounds, especially heterocycles
having more than one hetero atom, have shown a diverse range of biological activities. Among them,
thiazolidinone, which bears a sulfur atom at 1-position, a nitrogen atom at 3-position and a carbonyl
group at 2-, 4-, or 5-positions, has received a great deal of attention over the years since it is a significant
and versatile moiety that has occupied a prominent position in medicinal chemistry [15–24]. Derivatives
Molecules 2019, 24, 2418; doi:10.3390/molecules24132418
www.mdpi.com/journal/molecules

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containing a thiazolidinone moiety have demonstrated a wide range of pharmacological properties,
including anti-HIV, anti-malarial, anti-inflammatory and hypoglycaemic activities [25 27]. In many
–
cases, the introduction of thiazolidinone into molecules can enhance the interaction between the whole
molecule and the biological target. In the field of anti-tumor drugs, thiazolidinone fragments are also
frequently chosen to modify lead compounds. A variety of thiazolidinone-based derivatives with
different substituents are being considered as potential anticancer agents [24,28]. In particular, previous
work by the Azam group pointed out that introducing a five-membered heterocyclic ring in some
compounds can help increase hCA IX inhibitory activity, achieving Ki values in the low micromolar
range [28]. Supuran and Akdemir’s work also drew the same conclusion [29].
To extend the applicability of thiazolidinone to CA inhibitors, in this work, we synthesized thirteen
novel compounds by combining sulfonamide with thiazolidinone. These compounds were then tested
for enzyme inhibition of hCA IX and off-target hCA II isoform (as a subtype of the CA family, CA II is
widely distributed in the body and participates in various physiological activities). The inhibitory
activities were determined to be in the low nanomolar region. To determine the binding mechanism
and rationalize the trend of the inhibition profiles, molecular docking studies of the designed hybrids
in the active site of hCA IX and II were carried out, respectively.
2. Results and Discussion
2.1. Chemistry
The general synthetic route for the synthesis of the target benzenesulfonamide-thiazolidinone
derivatives is shown in Scheme 1. Isothiocyanatobenzene reacted with mafenide hydrochloride to
afford benzenesulfonamide (1) as the intermediate, which continued in the next cyclization reaction to
form compound (2). Thereafter, compounds (3–14) were obtained via the Knoevenagel condensation
of compound (2) reacting with different substituted benzaldehydes.
Scheme 1. The general synthesis route of compounds 3–14. Reagents and conditions: (i) NaHCO3,
DMF 4 Å MS, r.t., 4 h (ii) chloroacetic acid, NaOAc, EtOH, reflux, 12 h (iii) substituted benzaldehydes,
piperidine, EtOH, reflux, 5 h.
2.2. hCA Enzyme Inhibition Studies
The synthesized compounds (2–14) were evaluated for inhibitory activity against hCA II and IX
and by monitoring the hydrolysis of 4-nitrophenyl acetate (4-NPA). The inhibition data are summarized
in Table 1.

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Table 1. Inhibitory activity of compounds (2–14) against hCAs and molecular docking binding scores.
Docking Scores
(Kcal/mol)
Compound
R
hCA II (IC50, nM)
hCA IX (IC50, nM)
2
3
4
5
6
7
8
9
10
11
12
13
14
/
H
3-F
3-Cl
3-Br
266.2
166.3
146.5
270.3
148.0
335.0
151.3
216.7
160.8
115.4
1725
692.1
121.3
8912
30.47
137.8
242.1
182.9
130.5
292.3
154.2
544.4
140.9
246.1
144.9
117.2
>10,000
1246
−5.16
−6.14
−6.68
−5.00
−6.18
−3.24
−6.90
−5.02
−6.30
−6.79
0.70
−2.15
−6.51
NA a
−5.46
−6.01
3-CF3
3-OH
4-F
4-OH
4-OCH3
4-OBn
4-i-Pr
3,4-2OCH3
146.5
>30,000
88.1
Mafenide hydrochloride
AcetazolaMide (AZM)
SLC-0111
180.7
a
Not available. Autodock is not suitable to score mafenide hydrochloride as a salt-type compound. The value of
mafenide’s docking score is −5.06.
According to the data in Table 1, the structure-activity relationships (SAR) of compounds (
are preliminarily summarized below.
2–14)
i. Compared to the lead compound mafenide hydrochloride, most synthesized compounds
showed a significant increase in the inhibitory activity against both hCA II and IX, which were
comparable to the positive control drugs AZM and SLC-0111, except for the compound (12) and (13).
There was a certain relationship between the molecular docking binding scores with the value of IC50,
and the compound with better in vitro activity owned more negative score value.
ii. Compounds (3–14) were synthesized by introducing substituted aromatic groups into the
adjacent carbonyl group of thiazolidinone. In most cases, the introduction of an electron-withdrawing
group into the benzenesulfonamide-thiazolidinone scaffold caused a clear decrease in the inhibitory
potencies against hCA. Electron-donating groups provided better inhibition results, but when there
was more than one electron-donating group, the activity of the compound decreased slightly as in
compound (14).
iii. Among the halogenated benzene substituted derivatives (
position, the fluorine substituted derivative showed the highest inhibitory activity. Comparing
compounds ( ) and ( )–(10), we found that the compounds with substituents in the meta position
4–6) with the halogen at the meta
4
8
showed better inhibition activity than the ones with substituents in the para position.
iv. The weakest compound among the series was the benzyloxy substituted compound (12).
The isopropyl substituted compound (13) also exhibited poor inhibitory activity. Therefore, we can
conclude that increased steric hindrance of the substituents decreases the inhibition of hCA. However,
the effect of larger steric hindrance on the inhibitory activity of hCA II was not as great as that of
hCA IX.

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2.3. Docking Studies into the Active Site of hCAs
To further annotate the binding mode of the series compounds, compounds (
4), (
7
), (11) and
5FL4-ref were chosen for docking studies into the active site of hCA IX. The results were shown in
Figure 2.
Figure 2. The study of binding mode within CA IX. (A) 5FL4-ref (pink) (B) compound (4) (green) (C)
compound (7) (cyan) (D) compound (11) (khaki).
As shown in part A of Figure 2, compound 5FL4-ref interacted with the active site of CA IX
by one hydrogen bond between the Thr200 and the SO2 group. Additionally, the nitrogen atom
of the sulfonamide group formed a coordination bond with the zinc ion. The naphthyl moiety of
5FL4-ref formed interactions with Val130 and Leu134, and the heterocycle moiety of 5FL4-ref interacted
with Gln92 and Thr199 via hydrogen and hydrophobic bond, respectively. The residues with which
compounds (4), (7) and (11) interacted were essentially identical. Compounds (4), (7) and (11) also
formed interactions with the zinc cation by coordinating the nitrogen atom of the sulfonamide. However,
the interaction with the important residue Val130 was formed by the fragment of thiazolidinone in
compounds (
thiazolidinone fragments of compounds (
the rigidity of the heterocycle fragments in molecules, the substituted phenyl groups of compounds (
and (11) achieved the same torsion angle in the active site when bound to Gln92. Due to the presence
of trifluoromethyl, the conformation of compound ( ) was slightly different from that of other two
compounds and did not form interaction with residues Leu91 and Gln92, which may be the reason for
the weaker activity of compound ( ) than that of compounds ( ) and (11). In the enzyme inhibition
tests, the series of compounds also showed quite good inhibitory activity against hCA II. To illustrate
the reasons, compounds ( ), ( ) and (11) and 4IWZ-ref were chosen for docking studies into the active
site of hCA II. The results were shown in Figure 3.
4), (7) and (11) (part B, C and D of Figure 2). At the same time, carbonyl groups in the
4
) and (11) also formed hydrogen bonds with Gln92. Due to
4)
7
7
4
4
7

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Figure 3. The study of binding mode within CA II. (
A) 4IWZ-ref (yellow) (B) compound (4) (green) (C)
compound ( ) (cyan) ( ) compound (11) (khaki). As shown in part A of Figure 3, compound 4IWZ-ref
7
D
interacted with the active site of CA II by one hydrogen bond between the Thr199 and the SO2 group.
Additionally, the nitrogen atom of the sulfonamide group formed a coordination bond with the zinc ion.
The thiadiazole fragment of 4IWZ-ref formed interactions with Leu198, and the thiophene fragment of
4IWZ-ref interacted with some residues including Asn67, Ile91, Gln92 and Phe131. As shown in part B,
C and D of Figure 3, the residues interacting with compounds (4), (7) and (11) at the active site were
substantially identical. The different conformation and torsion angle of compounds in the active cavity
led to the difference of activity.
Although most of the compounds showed good inhibitory activity, some of them showed little
activity. To explain this phenomenon, the dominant conformations of compounds (
and (13) in CA IX were superimposed. As shown in Figure 4, compounds ( ) and ( ) with better
activity maintained almost the same conformation in the active site, whereas compounds (12) and (13
2), (3), (12)
2
3
)
underwent considerable torsion, which led to an increase in the distance between the nitrogen atom
of the sulfonamide group and the zinc ion. The increased distance may be the reason for the poor
activities of these two compounds.
Figure 4. Compounds superposed in CA IX active site: compound (
compound (12) (green), compound (13) (yellow).
2) (pink), compound (3) (turquoise),

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3. Materials and Methods
3.1. Chemistry
All the reagents (Energy Chemical, Shanghai, China) were used without further purification
unless otherwise specified. Solvents were dried and redistilled prior to use in the usual manner.
Analytical TLC was per-formed using silica gel HF254 (Qingdao Haiyang Chemical, Qingdao, China).
Preparative column chromatography was performed with silica gel H. Melting points were obtained on
1
a Büchi melting point B-540 apparatus. H and 13C NMR spectra (details of raw data for compounds,
see Figures S1–S26) were recorded on a Bruker ARX 600 MHz or 400 MHz spectrometer (Bruker,
Zurich, Switzerland) HRMS were obtained on an Agilent ESI-QTOF instrument (Agilent, Santa Clara,
CA, USA).
3.1.1. 4-((3-phenylthioureido)methyl)benzenesulfonamide (1)
Compound (1) was prepared according to the literature procedures [28]. To a solution of mafenide
hydrochloride (1.0 g, 4.5 mmol) and isothiocyanatobenzene (0.54 mL, 4.5 mmol) in DMF (10 mL)
sodium bicarbonate (0.42 g, 5.0 mmol) and molecular sieves (4 Å, 1.0 g) were added. The mixture was
stirred under room temperature for 4 h. After the molecular sieves were filtered off, 15 mL of water
was added. The mixture was stirred vigorously to form white solids, which were filtered off to afford
compound (1) (0.99 g, 68%).
3.1.2. (Z)-4-((4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (2)
To a mixture of compound (1) (0.55 g, 1.7 mmol) and chloroacetic acid (0.24 g, 2.6 mmol) in EtOH
(20 mL), sodium acetate anhydrous (0.28 g, 3.4 mmol) was added. The mixture was stirred at 78 ◦C for
12 h. The solvent was removed under reduced pressure, and the resulting residue was dissolved in
ethyl acetate and washed by water. Then, ethyl acetate was removed under reduced pressure, affording
light yellow solid, which was recrystallized from ethanol to give compound (
45%). m.p. 179.2–180.3 ◦C; 1H-NMR (DMSO-d6, 600 MHz)
7.81 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.2 Hz,
2H), 7.42–7.29 (m, 4H), 7.13 (t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.4 Hz, 2H), 4.99 (s, 2H), 4.13 (s, 2H); 13
NMR (DMSO-d6, 151 MHz) 172.49, 155.47, 148.28, 143.66, 140.48, 129.79, 128.49, 126.32, 124.88, 121.33,
45.45, 33.10; HRMS (ESI): Calcd. for [M + Na]+ C16H15N3O3S2: 361.0555, Found 361.0548.
2) as white powder (0.28 g,
δ
C
δ
3.1.3. General Procedure of Synthesis of 4-(((Z)-5-((Z)-benzylidene)-4-oxo-2-(phenylimino)thiazolidin-
3-yl)methyl)benzenesulfonamide hybrids (3–14)
A mixture of compound (2) (0.14 g, 0.39 mmol), different aldehydes (0.39 mmol), piperidine
(0.43 mmol, 0.040 mL), and ethanol (25 mL) were refluxed for 5 h. The reaction mixture was cooled
to room temperature. The solvent was removed under reduced pressure, and the resulting residue
was purified by column chromatography (150:1, dichloromethane-methanol) to give compounds
(3–14), respectively.
4-(((Z)-5-((Z)-benzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (
yellow powder, m.p. 227.0–229.3 ◦C, 42% yield; 1H-NMR (CD3CN, 600 MHz)
7.89 (d, J = 8.4 Hz,
2H), 7.79 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.48 (dd, J = 10.2, 4.8 Hz, 2H), 7.43
t, J = 7.9 Hz, 3H), 7.24 (t, J = 7.5 Hz, 1H), 7.05–7.00 (m, 2H), 5.68 (s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN,
3): Light
δ
(
151 MHz) δ 166.87, 150.88, 148.55, 143.13, 141.45, 134.21, 131.29, 130.60, 130.42, 130.07, 129.74, 129.17,
126.86, 125.59, 122.15, 121.60, 46.16; HRMS (ESI): Calcd. for [M + Na]+ C23H19N3O3S2: 449.0868, Found
449.0872.
4-(((Z)-5-((Z)-3-fluorobenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (
Light yellow powder, m.p. 226.9–227.6 ◦C, 45% yield; 1H-NMR (CD3CN, 600 MHz)
7.89
d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.70–7.64 (m, 3H), 7.59 (dd, J = 8.0, 0.9 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H),
7.44 (t, J = 7.9 Hz, 2H), 7.40 (t, J = 7.9 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 7.4 Hz, 2H), 5.68
4):
δ
(

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(s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN, 151 MHz)
δ
166.76, 150.52, 148.57, 143.33, 141.46, 136.65, 133.33,
131.68, 130.26, 129.62, 129.39, 128.72, 127.01, 125.85, 124.11, 123.26, 121.70, 46.43; HRMS (ESI): Calcd.
for [M + Na]+ C23H18FN3O3S2: 467.0774, Found 467.0779.
4-(((Z)-5-((Z)-3-chlorobenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (
Light yellow powder, m.p. 218.9–220.9 ◦C, 41% yield; 1H-NMR (CD3CN, 600 MHz)
7.89 (d, J = 8.4 Hz
2H), 7.72 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.52 (s, 1H), 7.47–7.41 (m, 5H), 7.25 (t, J = 7.5 Hz, 1H), 7.02
dd, J = 8.3, 0.9 Hz, 2H), 5.68 (s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN, 151 MHz)
165.23, 149.00, 147.03,
5):
δ
,
(
δ
141.78, 139.92, 134.86, 133.65, 129.94, 128.83, 128.81, 128.15, 127.84, 126.84, 125.47, 124.30, 122.59, 120.15,
44.89; HRMS (ESI): Calcd. for [M + Na]+ C23H18ClN3O3S2: 483.0478, Found 483.0492.
4-(((Z)-5-((Z)-3-bromobenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (
Light yellow powder, m.p. 217.7–220.3 ◦C, 48% yield; 1H-NMR (CD3CN, 600 MHz)
7.89
d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.70–7.64 (m, 3H), 7.59 (dd, J = 8.0, 0.9 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H),
7.44 (t, J = 7.9 Hz, 2H), 7.40 (t, J = 7.9 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 7.4 Hz, 2H), 5.68
(s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN, 151 MHz)
166.76, 150.52, 148.57, 143.33, 141.46, 136.65, 133.33,
6):
δ
(
δ
131.68, 130.26, 129.62, 129.39, 128.72, 127.01, 125.85, 124.11, 123.26, 121.70, 46.43; HRMS (ESI): Calcd.
for [M + Na]+ C23H18BrN3O3S2: 526.9973, Found 526.9967.
4-(((Z)-4-oxo-2-(phenylimino)-5-((Z)-3-(trifluoromethyl)benzylidene)thiazolidin-3-yl)methyl)benzenesulfonami
◦
1
de (
d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.52 (s, 1H), 7.47–7.41 (m, 5H), 7.25 (t, J = 7.5
Hz, 1H), 7.02 (dd, J = 8.3, 0.9 Hz, 2H), 5.68 (s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN, 151 MHz)
165.23,
7): Light yellow powder, m.p. 226.6-227.4 C, 44% yield; H-NMR (CD3CN, 600 MHz) δ 7.89
(
δ
149.00, 147.03, 141.78, 139.92, 134.86, 133.65, 129.94, 128.92–128.62, 128.15, 127.84, 126.84, 125.47, 124.30,
122.59, 120.15, 44.89; HRMS (ESI): Calcd. for [M + Na]+ C24H18F3N3O3S2: 517.0742, Found 517.0760.
4-(((Z)-5-((Z)-3-hydroxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (8):
Yellow powder, m.p. 268.2–269.3 ◦C, 30.30% yield; 1H-NMR (DMSO-d6, 400 MHz) δ 9.83 (s, 1H), 7.83
(d, J = 8.3 Hz, 2H), 7.70 (s, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.48 – 7.24 (m, 5H), 7.20 (t, J = 7.4 Hz, 1H),
7.00 (t, J = 7.8 Hz, 3H), 6.92 (t, J = 1.8 Hz, 1H), 6.84 (dd, J = 8.1, 1.7 Hz, 1H), 5.17 (s, 2H); 13C-NMR
(DMSO-d6, 101 MHz)
δ 166.24, 158.35, 150.14, 147.99, 143.77, 140.29, 134.81, 131.58, 130.82, 130.02,
128.46, 126.46, 125.47, 121.84, 121.46, 121.11, 118.05, 115.96, 45.87; HRMS (ESI): Calcd. for [M + Na]+
C23H19N3O4S2: 465.0817, Found 465.0814.
4-(((Z)-5-((Z)-4-fluorobenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (
9
):
7.92–7.83
(m, 2H), 7.77 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.60 – 7.52 (m, 2H), 7.47–7.39 (m, 2H), 7.27–7.18 (m, 3H),
7.07–6.98 (m, 2H), 5.69 (s, 2H), 5.22 (s, 2H); 13C-NMR (CD3CN, 151 MHz)
166.82, 164.57, 162.91,
◦
1
Light yellow powder, m.p. 209.7–211.0 C, 33% yield; H-NMR (CD3CN, 600 MHz)
δ
δ
150.70, 148.54, 143.15, 141.42, 132.73, 130.75, 130.09, 129.20, 126.87, 125.62, 121.92, 121.60, 116.87, 116.72,
46.20; HRMS (ESI): Calcd. for [M + Na]+ C23H18FN3O3S2: 467.0774, Found 467.0771.
4-(((Z)-5-((Z)-4-hydroxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (10):
Yellow powder, m.p. 176.3–178.2 ◦C, 30% yield; 1H-NMR (DMSO-d6, 400 MHz)
δ
7.83 (d, J = 8.3 Hz, 2H),
7.68 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.19 (t, J = 7.4 Hz, 1H),
7.00 (d, J = 7.4 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 5.15 (s, 2H); 13C-NMR (DMSO-d6, 101 MHz)
166.52,
δ
162.30, 150.49, 148.20, 143.72, 140.50, 132.79, 132.24, 129.98, 128.37, 126.45, 125.27, 123.24, 121.51, 117.31,
115.24, 46.47; HRMS (ESI): Calcd. for [M + Na]+ C23H19N3O4S2: 465.0817, Found 465.0824.
4-(((Z)-5-((Z)-4-methoxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (11):
Light yellow powder, m.p. 210.6–211.8 ◦C, 39% yield; 1H-NMR (CD3CN, 600 MHz)
(
δ
7.88
d, J = 8.4 Hz, 2H), 7.74 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H),
7.14–7.08 (m, 2H), 7.03 (dd, J = 7.8, 2.9 Hz, 3H), 5.68 (s, 1H), 5.22 (s, 2H), 3.85 (s, 3H), 3.81 (s, 3H);
13C-NMR (CD3CN, 151 MHz)
167.04, 161.70, 151.08, 148.70, 143.09, 141.57, 132.43, 131.25, 130.05,
δ

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129.13, 126.85, 126.70, 125.50, 121.63, 119.00, 115.24, 55.79, 46.08; HRMS (ESI): Calcd. for [M + Na]+
C24H21N3O4S2: 479.0973, Found 479.0981.
4-(((Z)-5-((Z)-4-(benzyloxy)benzylidene)-4-oxo-2-(◦phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide
1
(12): Light yellow powder, m.p. 219.6–221.3 C, 48% yield; H-NMR (CD3CN, 600 MHz)
δ 7.88
(
d, J = 8.4 Hz, 2H), 7.74 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H),
7.14–7.08 (m, 2H), 7.03 (dd, J = 7.8, 2.9 Hz, 3H), 5.68 (s, 1H), 5.22 (s, 2H), 3.85 (s, 3H), 3.81 (s, 3H);
13C-NMR (CD3CN, 151 MHz)
167.00, 151.56, 151.00, 149.88, 148.60, 143.09, 141.59, 131.54, 130.04,
δ
129.13, 126.91, 125.51, 123.57, 121.65, 119.34, 114.33, 112.38, 57.51, 56.01, 46.09; HRMS (ESI): Calcd. for
[M + Na]+ C30H25N3O4S2: 555.1286, Found 555.1279.
4-(((Z)-5-((Z)-4-isopropylbenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide (13):
Light yellow powder, m.p. 200.7–201.6 ◦C, 49% yield; 1H NMR (CD3CN, 600 MHz)
δ 7.92–7.84 (m, 2H),
7.76 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.48–7.40 (m, 4H), 7.36 (d, J = 8.3 Hz, 2H), 7.26–7.20 (m, 1H),
7.06–6.98 (m, 2H), 5.68 (s, 2H), 5.21 (s, 2H), 2.95 (hept, J = 6.9 Hz, 1H), 1.23 (d, J = 6.9 Hz, 6H); 13C-NMR
(CD3CN, 151 MHz)
δ 166.94, 152.13, 150.99, 148.60, 143.11, 141.49, 131.83, 131.38, 130.63, 130.05,
+
129.17, 127.83, 126.85, 125.54, 121.61, 121.01, 46.11, 34.38, 23.46; HRMS (ESI): Calcd. for [M + Na
]
C26H25N3O3S2: 491.1337, Found 493.1343.
4-(((Z)-5-((Z)-3,4-dimethoxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)methyl)benzenesulfonamide
◦
1
(
(
14): Light yellow powder, m.p. 249.8–250.9 C, 41% yield; H-NMR (CD3CN, 600 MHz)
d, J = 8.4 Hz, 2H), 7.74 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H),
7.14–7.08 (m, 2H), 7.03 (dd, J = 7.8, 2.9 Hz, 3H), 5.68 (s, 1H), 5.22 (s, 2H), 3.85 (s, 3H), 3.81 (s, 3H);
13C-NMR (CD3CN, 151 MHz)
167.00, 151.56, 151.00, 149.88, 148.60, 143.09, 141.59, 131.54, 130.04,
δ 7.88
δ
129.13, 126.91, 125.51, 123.57, 121.65, 119.34, 114.33, 112.38, 57.51, 56.01, 46.09; HRMS (ESI): Calcd. for
[M + Na]+ C25H23N3O5S2: 509.1079, Found 509.1092.
3.2. hCA Enzyme Inhibition Assays
Carbonic anhydrases are able to catalyze the conversion 4-nitrophenyl acetate (4-NPA) to
4-nitrophenol. According to the method described previously by Verpoorte, the rate of this
reaction is monitored spectrophotometrically, at 405 nm, with a Perkin Elmer Envision 2104
plate reader [30,31] (Perkin Elmer, Waltham, MA, USA). 1X assay buffer consisted of 15 mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH = 7.40) 0.01% tetraethylene glycol
monododecyl ether (Brij) and 100 mmol/L NaCl. Recombinant human carbonic anhydrase II and IX
was commercially available (Sino Biological Inc, Beijing, China) and prepared in 1X assay buffer with
a concentration of 11.10 ng/µL. Then, 18 µL of enzyme solution was transferred into 384-well assay
plates in triplicate. Stock solutions of the inhibitor (10.00 mmol/L) were prepared using DMSO as
solvent, and then diluted 1:3 with DMSO. Ten different inhibitor concentrations were used: 600.00
µ
mol/L, 200.00
0.091 mol/L, 0.03
assay solution. All compounds were allowed to incubate with the enzyme for 15 min at 25 ◦C to form
µ
mol/L, 66.67
µ
mol/L, 22.22
µ
mol/L, 7.41
µ
mol/L, 2.47
µmol/L, 0.82 µmol/L, 0.27 µmol/L,
µ
µmol/L, and 0
µmol/L. A volume of 2.00
µL of each inhibitor was added into the
the Enzyme-Inhibitor (E-I) complex. After that, substrate 4-NPA (1.00 mmol/L, 20.00 µL, Sigma-Aldrich,
St. Louis, MI, USA) was added into the E-I complex solution and incubated for 90 min at 25 ◦C. The
absorbance of each compound was measured with Envision 2104 plate reader. The inhibitor AZM and
SLC-0111 were used as standards to investigate the inhibitory activity of these compounds.
3.3. Single-Crystal Structure
The configuration of the target compound was determined by the single-crystal X-ray diffraction
(SuperNova, Rigaku, Tokyo, Japan) of compound (13). The compound crystallized in a centrosymmetric
space groups as a triclinic system. The resulting structure is shown in Figure 5 (CCDC Number:
1888237). The experiment showed that the double bonds between N2-C10 and C9-C17 are both in the
Z configuration.

Molecules 2019, 24, 2418
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Figure 5. The molecular structure (A) and single-crystal structure (B) of compound (13).
3.4. Preparation of Compound 2–14 for Docking Studies
Molecular docking calculations using the ligand molecules with CA IX (PDB code: 5FL4) [32
]
and CA II (PDB: 4IWZ) [33] were conducted using the modified version of Autodock 4(Zn) [34
,
35].
A grid box with a grid spacing of 0.375 Å was generated to define the binding pocket. AutoGrid
4 was used as the auxiliary program to generate affinity grid fields with the modified forcefield to
generate maps files. Compound structures were built and minimized with the Accelrys Discovery
Studio 3.0 software package [36] with flexible torsions assigned, and all dihedral angles were able
to freely rotate. We applied the Lamarckian genetic algorithm to determine the appropriate binding
positions, orientations, and conformations of the ligands. The optimized parameters were as follows:
The maximum number of energy evaluations was increased to 25,000,000 per run, the iterations of
Solis and Wets local search were 3000, the number of individuals in the population was 300, and the
number of generations was 25. The results differing by <2 Å in a positional root mean square deviation
were clustered together. In each group, the lowest binding energy configuration with the highest
percentage frequency was selected as the group representative. All other parameters were maintained
as default parameters.
4. Conclusions
A series of benzenesulfonamide-thiazolidinone derivatives was synthesized and evaluated for
the ability to inhibit hCA II and IX in vitro. Although the target compounds showed less selectivity
between CA II and IX, the inhibitory activities of most derivatives were equivalent to AZM but
were much higher than that of the precursor compound. The conclusion of SARs indicates that the
introduction of heterocyclic rings in the molecules can increase CA inhibitory activity and is worthy
of being further explored. In addition, the molecular docking results of hCA II and IX indicated that
the benzenesulfonamide derivatives containing thiazolidinone were structurally rigid and extended
into the active sites of CAs with relatively fixed conformations. Such a rigid and relatively fixed
conformation could interact with the key residue Val130 in the active site of CA IX, and without
interacting with Phe130 in CA II. Further exploration around this point of view is expected to help
improve the CA IX/II selectivity of this class of structure.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/13/2418/s1,
1
Table S1. Crystal data and structure refinement for compound 13; Figure S1
–
Figure S26. The H-NMR and
13C-NMR of compound 2-14.
Author Contributions: M.-S.C. and Y.L. supervised the whole experiment and provided technical guidance.
Z.-P.Z. designed and synthesized all of novel compounds. Z.-F.Y., J.-Y.L., Z.-P.W. and Q.-J.W. assisted in the
synthetic and biology research. J.W. supervised and carried out molecular modeling experiments.
Funding: We gratefully acknowledge the financial support from National Natural Science Foundation of
China (81473087, 81872752). Dr. Yang Liu wishes to express his thanks for the support by the Program for

Molecules 2019, 24, 2418
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Liaoning Innovative Talents in University (LR2017043) and the Career Development Support Plan for Young and
Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2016005).
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).