Novel benzoyl thioureido benzene sulfonamides as highly potent and selective inhibitors of carbonic anhydrase IX: Optimization and bioactive studies

Article abstract of DOI:10.1039/c8md00392k

CA IX has attracted much attention as a promising target for the development of new anticancer agents. In this study, a series of sulfonamide derivatives were designed and synthesized as potential CA IX inhibitors from a lead compound (benzoyl thioureido

Full text of DOI:10.1039/c8md00392k

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RESEARCH ARTICLE
Novel benzoyl thioureido benzene sulfonamides
as highly potent and selective inhibitors of
carbonic anhydrase IX: optimization and bioactive
studies
Cite this: Med. Chem. Commun.,
2018, 9, 2100
Li Liu,a Wanqi Wang,a Jin Huang, b Zhenjiang Zhao,b
b
a
*
*
Honglin Li
and Yufang Xu
CA IX has attracted much attention as a promising target for the development of new anticancer agents. In
this study, a series of sulfonamide derivatives were designed and synthesized as potential CA IX inhibitors
from a lead compound (benzoyl thioureido benzene sulfonamide) discovered by virtual screening. The bio-
assay demonstrated that some of the synthesized compounds exhibited potent inhibitory activity against
CA IX in the subnanomolar range and high selectivity over isozymes CA I and CA II. Among them, com-
pound 6a displayed inhibitory activity against CA IX with an IC50 value of 0.48 nM and about 1500-fold se-
lectivity over CA II. The structure–activity relationship and CA IX selectivity of the new sulfonamide deriva-
tives were also analyzed by molecular docking.
Received 11th August 2018,
Accepted 5th November 2018
DOI: 10.1039/c8md00392k
rsc.li/medchemcomm
tive transcription factor.15 Then it binds to a hypoxia re-
sponse element (HRE) and induces the expression of CA IX.16
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are abundant zinc
metalloenzymes found in a diversity of organisms including
higher vertebrates, green plants, algae, bacteria, and ar-
chaea.1,2 These CAs catalyze a fundamental physiological reac-
tion, hydration of carbon dioxide to bicarbonate and a pro-
ton.3 So far, sixteen different CA isoforms (designated as h
CA) belonging to the α-CA class are presently known in
humans.4 They are involved in many biological processes,
such as pH homeostasis,5 ion transport,6 respiration, gluco-
neogenesis,7 glaucoma,8 bone resorption,9 renal acidifica-
tion,10 and formation of cerebrospinal fluid and gastric
acid.11
In addition, many reports have been published on the role
of CA IX in tumor physiology as well,12 such as the control of
tumor pH and its influence on other processes in the cell
microenvironment that promotes cell proliferation, invasion,
and metastasis.13,14 Hypoxia is an important characteristic of
the tumor microenvironment.13 Under hypoxia, hypoxia-
inducible factor 1α (HIF-1α) translocates to the nucleus and
dimerizes with the HIF-1β constitutive subunit to form an ac-
Thus, the level of CA IX is dramatically increased in a variety
of human tumors, while it has low expression in normal tis-
sues.17,18 Considering the abnormally high expression of CA
IX in many hypoxic tumors and its demonstrated role in tu-
mor acidification processes and oncogenesis, CA IX has be-
come a potential drug target for anticancer therapy.19–21
The sulfonamide group (R-SO2NH2) is one of the most im-
portant and largely used zinc-binding groups for the design
of CA inhibitors. Some classical sulfonamides/sulfamates and
their derivatives such as acetazolamide (AZA), methazolamide
(MZA), ethoxzolamide (EZA), dichlorphenamide (DCP), etc.
(Fig. 1) have been reported to be effective inhibitors of
CAs.22–25
There are so many CA isoforms in humans, so it is diffi-
cult to find the specific inhibitors of one isoform. In fact, un-
til now, there haven't been any clinically used CAIs that show
selectivity for a specific CA isoform.2,26 Therefore, it's of great
importance to develop novel isozyme-specific CAI drugs for
avoiding various undesired side effects. Through complex
X-ray crystallography studies, the selectivity to small mole-
cules of classical CAIs is low, which means that the interac-
tion of small molecules with the CA isoforms at active sites is
indiscriminate.27 To improve the CAIs' selectivity, a tail ap-
proach of designing effective sulfonamides is utilized, in
which some groups are introduced by special links and sub-
stituents to enhance the activity and selectivity efficiently due
to the favorable interactions with some specific amino acid
a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
Chemical Biology, East China University of Science and Technology, Shanghai
200237, China. E-mail: yfxu@ecust.edu.cn
b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, 130 Mei Long Road, Shanghai 200237,
China. E-mail: hlli@ecust.edu.cn; Tel: +86 21 64251399
2100 | Med. Chem. Commun., 2018, 9, 2100–2105
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Fig. 1 Structures of clinically used CA inhibitors.
residues of CA IX catalytic sites.28,29 In this way, benzene sul-
fonamides containing ureido, thioureido and acyl
selenoureido moieties show selective CA inhibitory properties
against different CA isozymes30 because the flexible linker
makes the inhibitors adopt suitable conformations and bind
to the most energetically favored hydrophobic region.
We previously discovered the lead compound 1a as a CA
IX inhibitor with a carbonyl thioureido linker by virtual
screening.31 It exhibits inhibition against CA IX with an IC50
value of 2.86 nM and some selectivity over CA I and CA II.
Based on previously reported CA inhibitors,30 tail approaches
and molecular docking, two new series of structurally opti-
mized derivatives from the lead compound 1a were designed
and synthesized to improve the bioactivity and selectivity to
CA IX, as shown in Fig. 2.
oxygen atom of the carbonyl thioureido moiety can form hy-
drogen bonds with the side chains of Asn62 and Gln67. Com-
pared with benzenesulfonamides containing an ureido or
thioureido moiety,30 compound 1a may form stronger hydro-
gen bond interactions in the hydrophilic region since the car-
bonyl thioureido moiety provides one more H-bond acceptor
from the carbonyl group which can form an additional hydro-
gen bond with Gln67. Meanwhile, in the hydrophobic region,
the benzene moiety can interact with Leu91, Val131, Leu135
and Leu141, and there is still some space around the ben-
zene ring for further substitution. These common factors
contribute to the high affinities with CA IX. Based on the
docking analysis, some substituents such as chloride and
methoxy were introduced to the benzene ring of the lead
compound 1a to improve its inhibitory potency against CA IX
and the selectivity over isozyme CA II. The structures of the
target compounds are shown in Fig. 2.
2. Results and discussion
2.1 Docking analysis and the target compound design
2.2 Chemistry
The discovery of the lead compound was conducted by virtual
screening in our previous work.31 To achieve structural opti-
mization efficiency, we first analyzed the potential binding
mode of the lead compound 1a shown in Fig. 3. According to
the binding mode, we found that the compound bound in
slightly twisted conformations with CA IX allowed for the for-
mation of stable intermolecular hydrogen bonds. The
deprotonated sulfonamide nitrogen coordinates to Zn2+ in
the same way as other benzene sulfonamide inhibitors. Be-
sides, the coordinated sulfonamide nitrogen atom also forms
a hydrogen bond with the hydroxyl group of Thr199, and one
of the oxygen atoms of the sulfonamide moiety forms a hy-
drogen bond with the backbone amide of Thr199 as well.
These directional hydrogen bond interactions are beneficial
for stabilizing the ligand in the active site. At the hydrophilic
part of the pocket (including residues Asn62, His64, Ser65,
Gln67, Thr69, Gln92 and His94), the polar sulfur atom and
The compounds were obtained from the coupling of the cor-
responding substituted benzoyl isothiocyanate and 3- and
4-aminobenzenesulfonamide (Scheme 1). The preparation
route of the target compounds was as follows.
2.3 Bioactivity and inhibition analysis
Based on the lead compound 1a, thirteen novel compounds
were synthesized (Scheme 1) and their bioactivities were eval-
uated (Table 1).
According to the bioassay results, the sulfonamide group
at the para-position (compounds 1a–7a) showed better inhibi-
tory activity than that at the meta-position (compounds
1b–7b) on the benzene ring. Based on docking analysis, as
shown in Fig. 4, we deduced that the main reason was that
the carbonyl thioureido moiety in compounds 1a–7a can
form more stable hydrogen bonds with residues Asn62 and
Fig. 2 Structures of the lead compound 1a and the target compounds.
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Table 1 The inhibition assay against CA IX, CA I and CA II in vitro
IC50 (nM)
Selectivity
Selectivity
Compounds CA IX CA I
CA II
ratios (I/IX) ratios (II/IX)
AZA
EZA
1a
1b
2a
7.72
0.25
2.86
25.75
4.14
509.60
67.64
197.64
866.74 166.58 33.66
423.42 12.94 102.28
37.59 66.01
4.83 270.56
60.93 69.10
4.87
19.32
21.30
6.47
3.13
2b
3a
3b
4a
4b
5a
5b
6a
42.23 1541.18 239.58 36.50
5.67
6.68
14.58
54.42
35.61
6.97
0.38
1561
—
98.50
214.85
3.42
18.98
0.47
25.50
0.30
54.35
0.48
660.65
772.88 276.83 40.72
693.64 25.58 1475.83
664.71 908.13 26.07
22.86 193.17
266.56
44.38
2.09 888.53
20.75 0.82
503.74 749.28 1049.46
60.46 142.48
631.92 11.82 5266
1341.59 949.64 303.53
6b
7a
7b
>104
0.12
—
4.42
Fig. 3 Docking result of compound 1a against CA IX (PDB code: 3IAI).
The protein and the key residues are colored light pink and green,
respectively, and the docked molecule is colored cyan. The gray
sphere represents the zinc ion. The hydrogen bonds and coordinate
interactions are shown as red dashed lines.
locates in CA IX. As a consequence, CA IX has a larger room
at the active site than CA II. Therefore, the phenyl group of
compound 7a may form strong clashing interactions with
Phe131 of CA II, while compound 7a can interact well with
Val131 by hydrophobic interaction.
3. Conclusion
Gln67 than those formed by the same functional group in
1b–7b in the hydrophilic region, since the carbonyl thio-
ureido moiety of the latter deviated from these hydrophilic
residues. The linker moiety was kept, because its hydrophilic-
ity can be beneficial to the binding affinities. In the hydro-
phobic region, the bis-substituents at the 2,4-position on the
benzene ring could improve the activity compared to the
mono-substituted benzene at the 2-position (7a vs. 3a) or
3-position (7a vs. 2a) alone. It is presumed that the
substituted phenyl can strengthen the van der Waals interac-
tions with the hydrophobic residues around.
Meanwhile, the data in Table 1 clearly show that all the
compounds had relatively high selectivity ratios for CA IX
over CA I and CA II. Research has shown that CA II is the
most susceptible to be inhibited by sulfonamides,32 whereas
CA I generally has a lower affinity for this type of inhibitor
and much lower for inorganic anions, such as cyanide, cya-
nate, and thiocyanate.33 Therefore, we focused on the selec-
tivity for CA IX over CA II. An important difference between
these two isozymes is the amino acid in position 131 (Fig. 5).
The phenylalanine with its bulky side chain locates in CA II,
while the valine with its less sterically demanding side chain
In this paper, some potent CA IX inhibitors with high selec-
tivity over other CA isozymes (CA I and CA II) were designed
and synthesized based on the lead compound 1a. Compound
7a was the most potent CA IX inhibitor with an IC50 value of
0.12 nM and exhibited moderate selectivity over CA I and CA
II. To our satisfaction, compound 6a displayed inhibitory ac-
tivity against CA IX with an IC50 value of 0.48 nM and about
1500-fold selectivity over CA II. The structure–activity relation-
ship and CA IX selectivity of the newly synthesized com-
pounds were analyzed by docking analysis. The compounds
with the novel scaffold discovered in this work may serve as
new lead compounds for further development of therapeutics
to treat cancer.
4. Experimental
4.1 Chemistry: synthetic methods and experimental
All the starting materials and reagents were of analytical
grade and used without further purification. 1H NMR was
measured on a Bruker AV-500 spectrometer with chemical
shifts reported in ppm (in DMSO-d6/CDCl3, TMS as an
Scheme 1 The preparation route of compounds 1a–7a and 1b–7b.
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Fig. 4 Docking result of compounds 4a (A) and 4b (B) against CA IX (PDB code: 3IAI). The protein and the key residues are colored light pink and
green respectively, and the docked molecules are colored cyan. The gray sphere represents the zinc ion. The hydrogen bonds and coordinate
interactions are shown as red dashed lines.
internal standard). The mass spectra were measured on an
HP 1100 LC-MS spectrometer. The melting points were deter-
mined using an X-6 micro-melting point apparatus and
uncorrected.
added. After reflux overnight, the solution was evaporated un-
der reduced pressure. The product was purified by recrystalli-
zation or silica gel column chromatography, and the struc-
1
tures were identified by H-NMR, 13C-NMR and MS.
All the reactions were monitored by thin-layer chromatog-
raphy (TLC) using UV light (254 nm). Silica gel (300–400
mesh) was used for column chromatography.
General procedure for target compounds 1a–7a and
1b–7b. Substituted benzoyl isothiocyanate (5.0 mmol) was
dissolved in 20 mL acetone in a 50 mL round-bottom flask.
Then 3- and 4-aminobenzenesulfonamide (4.0 mmol) were
4.1.1 N-((4-Sulfamoylphenyl)carbamothioyl)benzamide (1a).
White crystal; yield: 35%; m.p: 204.7–205.4 °C; H NMR (400
1
MHz, DMSO-d6) δ (ppm): 12.72 (s, 1H), 11.69 (s, 1H), 8.01 (d,
J = 7.6 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.87–7.85 (m, 2H),
7.68 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 7.40 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δ: 180.00, 168.69, 141.80,
141.59, 134.00, 132.54, 129.20, 128.95, 128.67, 127.91, 126.76,
124.84; HRMS (ESI) calcd. for C14H13N3O3S2 [M − H+]:
334.0320, found: 334.0320.
4.1.2 N-((3-Sulfamoylphenyl)carbamothioyl)benzamide (1b).
1
White crystal; yield: 30%; m.p: 189.1–190.0 °C; H NMR (400
MHz, DMSO-d6) δ (ppm): 12.68 (s, 1H), 11.68 (s, 1H), 8.21 (s,
1H), 8.01 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 7.6 Hz, 1H), 7.74–
7.72 (m, 1H), 7.70–7.61 (m, 2H), 7.58 (t, J = 7.6 Hz, 2H), 7.46
(s, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 180.04, 168.66,
145.00, 139.00, 133.68, 132.50, 129.85, 129.20, 128.95, 128.17,
123.79, 122.00; HRMS (ESI) calcd. for C14H13N3O3S2 [M − H+]:
334.0320, found: 334.0321.
4.1.3 4-Chloro-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (2a). White crystal; yield: 32%; m.p: 204.9–206.7
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.62 (s, 1H),
11.79 (s, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H),
7.86 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.40 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δ: 179.78, 167.62, 141.84,
141.35, 138.55, 131.42, 131.18, 129.02, 126.76, 124.83; HRMS
(ESI) calcd. for C14H12ClN3O3S2 [M − H+]: 367.9930, found:
367.9929.
4.1.4 4-Chloro-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (2b). White crystal; yield: 28%; m.p: 192.7–194.2
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.57 (s, 1H),
11.78 (s, 1H), 8.20 (s, 1H), 8.01 (d, J = 8.0 Hz, 2H), 7.90 (d, J =
7.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 3H),
7.46 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 179.93, 167.58,
Fig. 5 Superposition of the crystal structures of CA II and CA IX. The
binding poses of compound 7a (cyan stick) were predicted by
molecular docking. The pink structure is CA IX (PDB code: 3IAI); the
green structure is CA II (PDB code: 3R16); the gray spheres represents
the zinc ion.
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145.00, 138.94, 138.53, 131.45, 131.19, 129.86, 129.03, 128.18,
123.82, 122.01; HRMS (ESI) calcd. for C14H12ClN3O3S2 [M −
H+]: 367.9930, found: 367.9931.
Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), 4.02 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ: 178.94, 165.85, 158.02, 145.04, 138.76,
135.57, 131.58, 129.92, 128.10, 123.92, 121.91, 121.78, 120.07,
131.32, 57.17; HRMS (ESI) calcd. for C15H15N3O4S2 [M − H+]:
364.0426, found: 364.0425.
4.1.5 2-Chloro-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (3a). White crystal; yield: 40%; m.p: 206.3–206.9
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.46 (s, 1H),
12.12 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H),
7.64 (d, J = 5.6 Hz, 1H), 7.59–7.54 (m, 2H), 7.50–7.46 (m, 1H),
7.40 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 179.40, 172.34,
168.10, 141.90, 141.21, 134.66, 132.70, 130.46, 130.18, 130.09,
129.82, 127.65, 126.76, 125.03; HRMS (ESI) calcd. for
C14H12ClN3O3S2 [M − H+]: 367.9930, found: 367.9930.
4.1.11 4-Nitro-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (6a). Faint yellow; yield: 32%; m.p: 202.0–202.3 °C;
1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.51 (s, 1H), 12.07 (s,
1H), 8.36 (d, J = 9.2 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H), 7.92 (d,
J = 8.8 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 7.42 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 179.59, 167.09, 150.34, 141.92,
141.32, 138.49, 130.76, 126.79, 124.88, 123.85; HRMS (ESI)
calcd. for C14H12N4O5S2 [M − H+]: 379.0717, found: 379.0710.
4.1.12 4-Nitro-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (6b). Faint yellow; yield: 30%; m.p: 207.0–207.8 °C;
1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.47 (s, 1H), 12.07 (s,
1H), 8.36 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 8.8 Hz, 3H), 7.91 (d,
J = 7.2 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz,
1H), 7.49 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 179.73,
167.05, 150.32, 145.03, 138.91, 138.52, 130.77, 129.90, 128.22,
123.92, 123.87, 122.05; HRMS (ESI) calcd. for C14H12N4O5S2
[M + Na+]: 403.0147, found: 403.0149.
4.1.6 2-Chloro-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (3b). White crystal; yield: 34%; m.p: 162.0–162.5
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.41 (s, 1H),
12.10 (s, 1H), 8.19 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.73 (d, J =
7.2 Hz, 1H), 7.64 (dd, J1 = 6.8 Hz, J2 = 2.4 Hz, 2H), 7.56 (d, J =
5.6 Hz, 2H), 7.46 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
179.58, 168.08, 145.00, 138.81, 134.73, 132.66, 130.47, 130.09,
129.87, 129.81, 128.39, 127.63, 123.91, 122.22; HRMS (ESI)
calcd. for C14H12ClN3O3S2 [M
391.9896.
+
Na+]: 391.9906, found:
4.1.7 4-Methoxy-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (4a). White crystal; yield: 38%; m.p: 206.0–206.9
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.85 (s, 1H),
11.51 (s, 1H), 8.04 (d, J = 9.2 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H),
7.87–7.85 (m, 2H), 7.40 (s, 2H), 7.08 (d, J = 8.0 Hz, 2H), 3.87
(s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 180.00, 167.78,
163.81, 141.71, 141.42, 131.55, 126.75, 124.78, 124.20, 114.31,
4.1.13
2,4-Dichloro-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (7a). White crystal; yield: 32%; m.p: 206.0–206.6
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.38 (s, 1H),
12.14 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H),
7.79 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.59–7.57 (m,
1H), 7.41 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 179.28,
167.16, 141.97, 141.17, 136.51, 133.85, 131.26, 129.69, 127.88,
125.04; HRMS (ESI) calcd. for C14H10Cl2N3O3S2 [M − H+]:
401.9541, found: 401.9539.
56.09; HRMS (ESI) calcd. for C15H15N3O4S2 [M
364.0426, found: 364.0420.
−
H+]:
4.1.8 4-Methoxy-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (4b). White crystal; yield: 35%; m.p: 223.0–223.8
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.65 (s, 1H),
11.31 (s, 1H), 8.23 (s, 1H), 7.93 (s, 2H), 7.74 (d, J = 6.4 Hz,
1H), 7.65 (d, J = 7.2 Hz, 2H), 7.47 (s, 2H), 7.30 (d, J = 6.4 Hz,
1H), 7.18 (s, 1H), 4.02 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ: 178.94, 165.84, 158.02, 145.03, 138.76, 135.59, 131.60,
129.91, 128.10, 123.93, 121.91, 121.79, 120.03, 113.31, 57.17;
HRMS (ESI) calcd. for C15H15N3O4S2 [M − H+]: 364.0426,
found: 364.0424.
4.1.14
2,4-Dichloro-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (7b). White to pale yellow crystal; yield: 28%; m.p:
186.8–187.7 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 12.35
(s, 1H), 12.13 (s, 1H), 7.92 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 7.6
Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 9.6 Hz, 1H), 7.58
(d, J = 8.0 Hz, 1H), 7.47 (s, 3H); HRMS (ESI) calcd. for
C14H10Cl2N3O3S2 [M + Na+]: 425.9517, found: 425.9511.
1
4.2 Biological assay
4.1.9 2-Methoxy-N-((4-sulfamoylphenyl)carbamothioyl)-
benzamide (5a). White crystal; yield: 42%; m.p: 198.0–198.5
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.70 (s, 1H),
11.32 (s, 1H), 7.94 (d, J = 7.6 Hz, 3H), 7.88 (d, J = 7.6 Hz, 2H),
7.67 (t, J = 7.2 Hz, 1H), 7.42 (s, 2H), 7.30 (d, J = 8.0 Hz, 1H),
7.18 (t, J = 7.2 Hz, 1H), 4.02 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ: 178.79, 165.88, 158.02, 141.94, 141.15, 135.59,
131.57, 126.78, 124.77, 121.78, 120.04, 113.31, 57.16; HRMS
(ESI) calcd. for C15H15N3O4S2 [M − H+]: 364.0426, found:
364.0423.
4.1.10 2-Methoxy-N-((3-sulfamoylphenyl)carbamothioyl)-
benzamide (5b). White crystal; yield: 28%; m.p: 222.2–223.2
°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.65 (s, 1H),
11.32 (s, 1H), 8.23 (s, 1H), 7.93 (d, J = 6.8 Hz, 2H), 7.74 (d, J =
7.6 Hz, 1H), 7.69–7.61 (m, 2H), 7.48 (s, 2H), 7.30 (d, J = 8.4
The purification of recombinant protein h CA IX was
performed as described previously,28 and CA I and CA II were
both purchased from Sigma. The stock solutions of the inhibi-
tors (50 mM) were prepared and diluted to different concentra-
tions with DMSO. An Applied Photophysics SX 20 stopped-flow
instrument was used to test the CA-catalyzed CO2 hydration ac-
tivity, as described by I. Nishimori et al.34 The initial rates of
4-nitrophenyl acetate hydrolysis catalyzed were monitored
using a Synergy 2 Biotek Microplate reader, as described by E.
Truppo et al.35 The inhibitor and enzyme solutions were pre-
incubated together for 15 min prior to the assay study. The en-
zyme concentrations were 1 ng μL−1 for CA II, 50 ng μL−1 for CA
I and 3 ng μL−1 for CA IX. Triplicate experiments were done for
each inhibitor concentration, and the values reported through-
out the paper are the mean of such results.
2104 | Med. Chem. Commun., 2018, 9, 2100–2105
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4.3 Molecular modeling
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For better understanding, compounds 1a, 4a, 4b and 7a were
studied by molecular docking. First, the four compounds were
prepared by using the Builder tool and then optimized with the
Ligprep module in Maestro 9.0 (Schrödinger Inc.). The protein
structures of CA II (PDB ID: 3R16) and CA IX (PDB ID: 3IAI)
were derived from the Protein Data Bank. Then we utilized the
Glide module in Maestro to optimize these structures and all
the water molecules were removed. When defining the active
site, the grid-enclosing box was generated based on the ligand
in the complex structure, and other parameters were set up by
default. All of the compounds were docked into the protein
using Glide with the standard precision (SP) approach, and a
scaling factor of 1.0 was set to the van der Waals (VDW) radii
of protein atoms with partial atomic charges less than 0.25.
The best 20 docking poses of each compound ranked by
GlideScore were kept for further analysis.
Conflicts of interest
The authors declare no competing interests, no conflicts to
declare.
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Acknowledgements
This research is supported in part by the National Key Re-
search and Development Program (Grant 2016YFA0502304)
and the Shanghai Committee of Science and Technology
(Grant 14431902100).
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