Novel metronidazole-sulfonamide derivatives as potent and selective carbonic anhydrase inhibitors: Design, synthesis and biology analysis

Article abstract of DOI:10.1039/c4ra03819c

Metronidazole-sulfonamide derivatives 4a-4l, a new class of human carbonic anhydrase inhibitors (hCA), were designed, synthesized, isolated, and evaluated for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA II

Full text of DOI:10.1039/c4ra03819c

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Novel metronidazole-sulfonamide derivatives as
potent and selective carbonic anhydrase inhibitors:
design, synthesis and biology analysis
Cite this: RSC Adv., 2014, 4, 33029
Zhong-Chang Wang,†a Yong-Tao Duan,†a Han-Yue Qiu,†a Wan-Yun Huang,ab
a
a
ac
a
*
*
Peng-Fei Wang, Xiao-Qiang Yan, Shu-Feng Zhang
and Hai-Liang Zhu
Metronidazole–sulfonamide derivatives 4a–4l, a new class of human carbonic anhydrase inhibitors (hCA),
were designed, synthesized, isolated, and evaluated for their ability to inhibit the enzymatic activity of the
physiologically dominant isozymes hCA II and the tumor-associated isozyme hCA IX (h ¼ human). Many
of these compounds inhibited CA II and IX in the range of 16–137 and 38–169 nM, respectively. Among
all the compounds, the most potent inhibitor against hCA II and IX were compounds 4b (IC50 ¼ 16 nM)
and 4h (IC50 ¼ 38 nM). Conversely compounds 4e and 4d displayed the most potent growth inhibitory
activity against B16-F10 and MCF-7 cancer cell lines in vitro, respectively, with an IC50 value of 150 nM
for B16-F10 and 6.5 nM for MCF-7. These metronidazole–sulfonamide derivatives may prove interesting
lead candidates to target tumor-associated CA isozymes, wherein the CA domain is located
extracellularly. All the new compounds were evaluated for cytotoxicity against human macrophages by
MTT assay.
Received 2nd May 2014
Accepted 23rd June 2014
DOI: 10.1039/c4ra03819c
www.rsc.org/advances
very high CO2 hydrase catalytic activity, which is also inhibited
by the classical CA inhibitors belonging to the sulfonamide,
sulfamate, and sulfamide classes of compounds.6 CA IX may be
functionally linked to the regulation of the tumour pH, because
it contributes to extracellular acidication.7
Introduction
In many types of solid tumors, hypoxia is an advantageous
condition for the culture of pluripotent stem cells. It induces
the activation of a transcription factor, aptly named hypoxia-
inducible factor (HIFa).1 High levels of HIF regulate a signaling
cascade, which adapts cellular functions to allow solid tumor
cells to not only survive hypoxia but also to proliferate and
metastasize. Solid tumors are remarkably unregulated in the
functioning of some house-keeping enzymes, among which are
the carbonic anhydrases (CAs, EC 4.2.1.1) involved in pH
homeostasis, ion transport, and biosynthetic processes.2 CAs
are Zn(II) metalloenzymes involved in pH buffering of extra- and
intra-cellular spaces by catalyzing the reversible hydration of
carbon dioxide and water to bicarbonate and a proton: CO2 +
H2O 4 HCO3ꢀ + H+.3 This reaction is known to regulate a broad
range of physiological functions by respiration and the trans-
CA IX expression is strongly increased in many types of solid
tumors such as mesotheliomas, kidneys, lungs and breast.
Furthermore, such hypoxic tumors do not generally respond to
the classic chemo- and radio-therapy and the strong acidica-
tion produced by CA IX overexpression also triggers the devel-
opment of metastases.8 As suggested by Svastova et al., the
assumption that pHe in different tumor cell cultures recovers
more naturally, to an extent along with a remarkably increased
apoptosis of the tumor cells, when CA IX is inhibited by potent
and selective sulfonamide inhibitors, is being widely accepted.9
For example, Pouyssegur's group showed recently that in
hypoxic LS174Tr tumor cells either one or both CA isoforms
were expressed, in response to a “CO2 load,” and both enzymes
contributed to extracellular acidication and maintained a
more alkaline resting intracellular pH (pHi), which is an action
that preserves ATP levels and cell survival in a range of acidic
outside pH (6.0–6.8) and low bicarbonate medium. In vivo
experiments showed that silencing of CA IX alone leads to a
40% reduction in xenogra tumor volume, with up-regulation
of the second gene, encoding for CA XII, whereas invalidation of
both CAI X and CA XII gives an impressive 85% reduction of
tumor growth.10 Correlated with this fact, it has been shown
earlier that some CA inhibitors showed anticancer activity in
vivo.11 Moreover, CA IX inhibition may compose a fascinating
ꢀ
4
port of CO2/HCO3
.
In humans, the carbonic anhydrase
enzymes, specically isozymes IX (CAs IX), have recently been
shown to be druggable targets for imaging and treatment of
hypoxic tumors.5 This enzyme is a multidomain protein with
the CA subdomain situated outside the cell and possessing a
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, China. E-mail: zhuhl@nju.edu.cn; hxxyzsf@mail.tjnu.edu.cn; Fax: +86-25-
83592672; Tel: +86-25-83592672
bDepartment of Pharmacology, Guilin Medical University, Guilin 541004, China
cCollege of Chemistry, Tianjin Normal University, Tianjin, 300074, China
† These authors contributed equally to this work.
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new approach for the management of hypoxic tumors, which
In this paper, we report the synthesis of a series of metro-
generally do not respond to the classical radio- and chemo- nidazole–sulfonamide derivatives, which might exhibit syner-
therapy.12
gistic effect in anticancer activity, as well as the evaluation of
One approach aimed at improving the selectivity of tumor their CA inhibition, their anticancer activity and their interac-
cell killing by antitumor drugs is the use of less toxic prodrug tion with CA by docking studies.
forms that can be selectively activated in the tumor tissue
(tumor-activated prodrugs; TAP), utilizing some unique aspects
of tumor physiology such as selective enzyme expression or
Results and discussion
hypoxia.13 To characterize the potential effect of inhibition of
CA IX and XII in tumor hypoxia, there is an implied need to
develop small molecules, which are capable of targeting tumor
cells in the further research.14 Since the discovery of E-7010 in
1992,15 sulfonamides have been proven to be an important class
of anticancer agents, which interact with various cellular
targets. In fact, indisulam (E7070),16 HMN-214,17 T138067
(ref. 18) are in phase II clinical trials as an antitumor sulfon-
amide with a complex mechanism of action that also involves
CA inhibition of several isozymes participating in tumor genesis
(Fig. 1).
It is also known that sulfonamides are found to be an
important class of drugs possessing various types of biological
properties such as anti-cancer,19 and antibacterial.20 Many of
the structurally novel sulfonamide derivatives have recently
been reported to show substantial antitumor activity and low
toxicities, both in vitro and/or in vivo. Some of these derivatives
are currently being evaluated in clinical trials, and there is
much optimism that they may lead to novel alternative anti-
cancer drugs, which are devoid of the side effects of the pres-
ently available pharmacological agents.21
Thiry et al.24 reported that, from the analysis of the CA active site
and the structure of inhibitors described in the literature,25
a
general pharmacophore (Fig. 2) for the compounds that acted
as carbonic anhydrase inhibitors was identied. In order to
make this pharmacophore interact with CA inhibitors, it should
include the structural elements that are required to be present
in the compounds. A sulfonamide moiety should be included,
which coordinates with the zinc ion of the active site of the CA,
and it should be attached to a benzene ring scaffold. The side
chain, which might interact with the hydrophobic and hydro-
philic parts of the CA active site, can substitute an aromatic or
heterocyclic sulfonamide scaffold. Therefore, different hydro-
phobic side chains were incorporated in the indanesulfona-
mide scaffold with an amide linker that can interact with the
hydrophilic part of the active site, and a hydrophobic moiety
that can interact with the hydrophobic part of the CA active site.
Fig. 3 illustrates representative examples of the newly
synthesized compounds showing compliance with the above-
mentioned pharmacophore model. The new compounds were
synthesized according to Scheme 1.
Moreover, in a previous study, nitroimidazole derivatives
have attracted considerable attention as they showed a tendency
to penetrate and accumulate in regions of tumors,22 and can
undergo bioreduction to yield electrophilic substances which
can damage proteins and nucleic acids.23 All these ndings
encouraged us to explore the synthesis of metronidazole–
sulfonamide derivatives as potential CA inhibition agents.
Chemistry
All novel metronidazole–sulfanilamide derivatives CA inhibi-
tors (4a–4l) described herein were synthesized following the
Fig. 2 Structural elements of CA inhibitors in the CA enzymatic active
site.
Fig.
3 Representative examples of the synthesized compounds
Fig. 1 Known human CA inhibitors including some clinically used showing compliance to the general pharmacophore of sulfonamide
drugs.
compounds, acting as CA inhibitors.
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Table 1 In vitro anticancer activities (IC50, nM) against two human
tumor cell lines
IC50a (nM)
B16-F10b
IC50a (nM)
MCF-7b
Compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
270
310
200
190
220
220
230
150
300
160
270
180
72.3
3.6
87
6.8
9.5
6.5
7.2
120
93
47
58
30
7.8
90
Scheme 1 Reagents and conditions: (a) 30 equiv. tosyl chloride,
CH2Cl2, RT, 12 h, 75%; (b) 15.7 equiv. 3a, p-hydroxybenzaldehyde; 3b,
m-hydroxybenzaldehyde, K2CO3, DMF, 80 ꢁC, 20 h, 3a, 71.6%; 3b,
51.3%; (c) 1.0 equiv. p-RNHSO2PhNH2, CH3CH2OH, 50 ꢁC, 8 h, 50.0–
84.3%.
Doxorubicin
Semaxanib
65.3
3.1
a
Antiproliferation activity was measured using the MTT assay. Values
were the average of three independent experiments run in triplicate.
synthetic pathway depicted in Scheme 1. The starting MET-OTs
(2-(2-methyl-5-nitro-1H-imidazole-1-yl)-ethyl-4-methylbenzene
sulfonate, compound 2) has been previously synthesized from
metronidazole using excess of tosyl chloride in the presence of
triethylamine in dichloromethane.26 The synthesis of metroni-
dazole–benzaldehyde 3a–3b was performed by a nucleophilic
displacement reaction of the corresponding MET-OTs in the
presence of o- or p-hydroxybenzaldehyde and K2CO3 in DMF.
Subsequently, compounds 4a–4l were prepared from the reaction
of 3a, 3b with the corresponding sulfanilamide in ethanol at
room temperature. The reactions were monitored by thin layer
chromatography (TLC), and the crude products were puried by
recrystallization with ethanol, ethyl acetate and acetone
(1 : 1 : 0.05). All of the target compounds gave satisfactory
analytical and spectroscopic data, i.e. IR, 1H NMR, 13C NMR, ESI
MS, which are in accordance with their depicted structures.
b
Variation was generally 5–10%. Cancer cells kindly supplied by State
Key Laboratory of Pharmaceutical Biotechnology, Nanjing University.
inhibition to MCF-7 than to F10 with 1–10 folds. The more
potent antiproliferative activity for MCF-7 together with virtual
screening results both indicated that the over-expressed CA
might be a potential target with which these metronidazole–
sulfanilamide derivatives interacted.
CA II and IX enzyme assay. Inhibition of two physiologically
relevant CA isoforms (hCAs II and hCAs IX) with compounds
4a–4l AAZ and SA, tow clinically used drugs were presented in
Table 2.27 This study has included hCAs II (cytosolic, widespread
enzymes) and hCAs IX (transmembrane, tumor-associated CAs)
Biological activity
Table 2 In vitro anticancer activities (IC50, nM) against two human
tumor cell lines
Antiproliferation assays and MTT assays. In the present
work, twelve of the newly synthesized compounds 4a–4l were
evaluated for their in vitro growth inhibitory activities against
two cell lines, which are mouse melanoma cells (B16-F10) and
breast carcinoma cell lines (MCF-7) in comparison to the known
anticancer drugs: doxorubicin and semaxanib as reference
drugs. The values obtained for the twelve compounds against
the B16-F10 and MCF-7 cell lines were shown in Table 1. As
shown in Table 1, it was observed that metronidazole–sulfa-
nilamide derivatives were found to inhibit the growth of two
tumor cell lines with moderate IC50 values. As previously shown,
for the MCF-7 cell line, which over-expressed CA, metronida-
zole–sulfanilamide derivatives, in particular, displayed more
potent antiproliferative action than in the other tumor cell
lines. The IC50 values of most metronidazole–sulfanilamide
derivatives ranged from 6.5 nM to 120 nM in MCF-7. Among
them, compound 4d displayed the most potent antiproliferative
activity with IC50 of 6.5 nM, comparable to the positive control
doxorubicin (IC50 ¼ 65.3 nM) and semaxanib (IC50 ¼ 3.1 nM).
Nevertheless, compounds 4a–4l displayed a more potent
IC50a (nM)
hCA IIb
IC50a (nM)
hCA IXb
Compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
AAZ
SA
137
16
70
48
22
33
53
89
114
81
169
93
135
110
81
112
42
38
69
61
126
56
28
86
52
16
256
298
a
Errors were in the range of 5–10% of the reported values from three
b
different assays. Two different human recombinant enzymes, by the
esterase assay (4-nitrophenylacetate as substrate).
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account of their relevance as targets/off targets when developing inhibitors compared to the p-substituted metronidazole resi-
CAIs. In fact, CA II is the drug target for developing anti- dues on the benzene ring. The opposite effect was noticed for
glaucoma CAIs.28
hCA IX; moreover, the tumor-associated isoform hCA IX was
CA IX is overexpressed in breast cancer, and studies have inhibited by compounds 4a–4l with IC50 in the range of 38–169
shown that CA IX and CA XII are variably expressed in breast nM (Table 2). The results indicated that the sulfonamides
cancer cell lines. Moreover, CA IX expression is associated with incorporating the metronidazole led to highly effective hCA IX
poor survival in an unselected cohort of patients with invasive inhibitors. Compound 4h showed the most potent inhibition to
breast carcinoma, and it is signicantly associated with distant hCA IX with an IC50 value of 38 nM, whereas compound 4a was
metastasis (Lou et al., unpublished results).10 Therefore, we the least active with an IC50 value of 169 nM among compounds
chose breast cancer as a malignancy model for in vivo testing, 4a–4l. Thus, we can conclude that compounds 4b and 4h have
and the antimetastatic prole of some of the CA IX inhibitors. been identied as the most potent inhibitors.
The newly described metronidazole–sulfanilamide derivatives
4a–4l were tested in an in vitro carbonic anhydrase assay to
evaluate their potential as antitumor drugs. The results are
Molecular modeling
To gain better understanding on the potency of the 12
compounds, we examined the interaction of these compounds
with hCA II by molecular docking, which was performed by
simulation of the 12 compounds into the ATP binding site in
hCA II. The protein structure of the hCA II was downloaded
from the PDB (3N4B.pdb)29 and was preprocessed using the
Schrodinger Protein Preparation Guide; moreover, hydrogen
atoms were added to the structure, and H-bonds within the
protein were optimized, and the protein was minimized to an
summarized in Table 2 as IC50 values.
The following structure–activity relationship (SAR) can be
observed from the data of Table 2. CA activities of these
compounds were tested against the standard clinically used
inhibitors AAZ and SA. The physiologically dominant and highly
active cytosolic isoform hCA II was inhibited by compounds 4a–
4l with IC50 in the range of 16–137 nM. Compound 4b was the
most active having an IC50 value of 16 nM, whereas compound
4a was the least active with IC50 value of 137 nM. We simplied
the situation by only treating steric complexities as the single
factor to deal with. Most of these compounds with p-substituted
metronidazole residues on the benzene ring and the same
sulfanilamide derivatives residues were more effective as hCA II
˚
˚
rmsd of 0.3 A. A 9.9 A sphere of water molecules was added
around the ligand and a short (3 ps) dynamics run was carried
out, followed by several cycles of minimization using Quanta/
CHARMm. The entire protein–ligand–water complex was
allowed to move during calculations.30
Fig. 4 Docking of compounds 4c and 4f in the ATP binding site of hCA II: (a) left: 2D model of the interaction between compounds 4c, 4f and the
ATP binding site. Right: 3D model of the interaction between compounds 4c, 4f and the ATP binding site, respectively.
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The predicted binding interaction energy was used as the
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Conclusion
criterion for ranking. The estimated interaction energy of other
synthesized compounds was ranging from ꢀ53.72 to ꢀ40.84
kcal molꢀ1. The selected compounds of 4c and 4f had an esti-
mated binding free energy of ꢀ53.72 kcal molꢀ1 and ꢀ42.04
kcal molꢀ1, respectively. The binding model of compounds 4c,
4f and hCA II was depicted in Fig. 4. The amino acid residues
that had interaction with hCA II were labeled. In the binding
mode, compound 4c was nicely bound to the ATP binding site of
hCA II by a hydrophobic interaction and binding was stabilized
by one hydrogen bond. The hydrogen atom of THR199 formed
one hydrogen bond interaction with the oxygen atom of N]O
bonds of compound 4c (angle N HD22 ¼ 172.3ꢁ, distance ¼ 2.42
In summary, CA is an emerging target for the development of
novel antitumour chemotherapeutics. We had designed and
synthesized novel series of metronidazole–sulfanilamide
derivatives, which were tested for their inhibitory activities
against B16-F10 and MCF7. These compounds showed a very
interesting prole for the inhibition of hCAs II (cytosolic, off-
target isoform) and hCAs IX (transmembrane, tumor-associated
enzyme). Most of them exhibited CA inhibitory activity and did
not show any toxicity. Docking simulation was performed to
decipher the probable binding models and poses. The results
indicate that metronidazole–sulfanilamide derivatives, which
acted as potent antibacterials and also as novel and potent
antitumour agents. Given the unforeseen structural differences
within the active site of some pathogenic enzymes, the key to
discovering inhibitors with broad-spectrum antitumour activity
lies in a detailed understanding of the CA active sites. Further
studies on the CA inhibition ability of these compounds, and
further modications of the current series, with the hope of
improving both enzymatic inhibition and physical properties,
are currently underway.
˚
A). Moreover, compound 4f was also nicely bound to the ATP
binding site of hCA II by a hydrophobic interaction and binding
was stabilized by four hydrogen bonds (angle TRP5:HE1
ꢁ
˚
4f:O29 ¼ 119.7 , distance ¼ 2.23 A; ASN67:HD22 4f:N17 ¼
ꢁ
ꢁ
˚
154.3 , distance ¼ 2.23 A; THR199:HN 4f:O32 ¼ 112.5 , distance
ꢁ
˚
¼ 2.42 A and 4f:H53 A:THR199:OG1 ¼ 145.9 , distance ¼
˚
1.97 A), and a p-cation interaction (4f A:HIS94). This molecular
docking result, along with the biological assay data, suggested
that compounds 4c and 4f were potential inhibitors of CA.
Experiments
Materials and measurements
Cytotoxicity
All compounds were evaluated for their toxicity against human
All chemicals used were purchased from Aldrich (USA). All
reagents used in the current study were of analytical grade. Thin
layer chromatography (TLC) was performed on silica gel plates
with uorescent indicator. All analytical samples were homo-
geneous on TLC in at least two different solvent systems.
Melting points (uncorrected) were determined on a X-4 MP
macrophages with the median cytotoxic concentration (CC50
)
data of tested compounds by the MTT assay, as shown in Table
3. These compounds were tested at multiple doses to study the
viability of macrophages.
As shown in Table 3, all compounds showed that they almost
did not exhibit cytotoxicity.
apparatus (Taike Corp, Beijing, China). All the 1H NMR and 13
C
NMR spectra were recorded on a Bruker DPX 300 model Spec-
trometer in DMSO-d6, and chemical shis (d) were reported as
parts per million (ppm). ESI-MS spectra were recorded on a
Mariner System 5304 Mass spectrometer. FT-IR spectra on KBr
pellets were recorded on a Thermo Nicolet NEXUS870 model
spectrometer.
Analysis of apoptosis by Annexin V-PE uorescence-activated
cell sorting (FACS)
To test whether the inhibition of cell growth of A549 was related
to cell apoptosis, A549 cell apoptosis induced by compound 4d
was determined using ow cytometry. The uptake of Annexin
V-PE was signicantly increased, and the uptake of normal cells
was signicantly decreased in a time-dependent manner.
Finally, the percentage of early apoptotic cells was markedly
elevated in a density-dependent manner from 6.28% to 19.6%
aer 48 h (Fig. 5).
General procedure for the synthesis of compounds 3a, 3b
Anhydrous K2CO3 (8.0 g, 58 mmol) was added to a stirred
solution of 2-(2-methyl-5-nitro-1H-imidazole-1-yl)-ethyl-4-
methylbenzenesulfonate (2) (4.0 g, 12.1 mmol) in anhydrous
DMF (150 mL) at 80 ꢁC. Subsequently, p- or m-hydroxy-
benzaldehyde (1.9 g, 15.7 mmol) was added to the reaction
mixture and stirring continued for 20 h. The mixture
was poured into water (200 mL) and extracted with EtOAc (3 ꢂ
250 mL). The combined organic extracts were washed with
saturated Na2CO3 solution, brine (200 mL), dried MgSO4, and
concentrated in vacuo. The crude product was puried by
column chromatography (silica gel, EtOAc/PE 1 : 4) to give
compounds 3a, 3b.
Table 3 The median cytotoxic concentration (CC50) data of tested
compounds
Compounds
CC50a, mmol
Compounds
CC50a, mmol
4a
4b
4c
4d
4e
4f
0.53
0.46
0.58
0.72
0.64
0.68
4g
4h
4i
4j
4k
4l
0.63
0.49
0.52
0.62
0.56
0.68
3a. Yellow solid, yield 71.6%, Rf ¼ 0.44 (EtOAc/PE 1 : 1);
m.p. 41–43 C; 1H NMR (300 MHz, DMSO-d6) d: 9.95 (s, 1H,
ꢁ
a
CHO), 7.91 (s, 1H, CH), 7.47–7.05 (m, 4H, ArH), 4.75 (t, J ¼ 4.7
Minimum cytotoxic concentration required to cause a microscopically
detectable alteration of normal cell morphology.
Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz, 2H, –CH2), 2.64 (s, 3H, CH3).
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Fig. 5 Compound 4d induced apoptosis in A549 cells with the density of 2.5, 10, 40, 160 mg mLꢀ1. A549 cells were treated for 48 h. Values
represent the mean, n ¼ 3. P < 0.05 versus control. The percentage of cells in each part was indicated.
13C NMR (75.4 MHz, DMSO-d6): d 190.46, 151.81, 142.24, CHO), 7.91 (s, 1H, CH), 7.96 (s, 1H, ArH), 7.47–7.05 (m, 3H,
138.63, 136.56, 133.23, 128.35, 127.38, 62.51, 44.96. ESI-MS: ArH), 4.75 (t, J ¼ 4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz, 2H, –CH2),
292.1 [M + H]+.
2.64 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): d 190.44,
3b. Faint yellow solid, yield 51.3%, Rf ¼ 0.43 (EtOAc/PE 1 : 1); 151.76, 142.24, 138.63, 136.56, 133.23, 127.38, 126.98, 62.51,
m.p. 68–70 ꢁC; 1H NMR (DMSO-d6, 300 MHz) d: 9.95 (s, 1H, 44.96. ESI-MS: 292.1 [M + H]+.
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General procedure for the synthesis of compounds 4a–4l
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (4e).
Yellow crystals, yield 63.2%. Rf ¼ 0.26 (EtOAc/PE 1 : 1); m.p.
207–209 ꢁC; 1H NMR (DMSO-d6, 300 MHz) d: 11.12 (s, 1H,
SO2NH), 9.02 (s, 1H, CH), 8.18 (s, 1H, CH), 8.04 (d, J ¼ 1.95 Hz,
1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (d, J ¼ 2.0 Hz, 1H, CH),
4.74 (t, J ¼ 4.9 Hz, 2H, CH2), 4.45 (t, J ¼ 4.9 Hz, 2H, –CH2), 2.53
(s, 3H, CH3), 2.28 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): d
190.06, 167.66, 157.00, 156.77, 151.96, 132.47, 131.40, 129.91,
125.64, 114.30, 114.18, 112.16, 66.49, 45.10, 23.32, 14.06. IR
(KBr, n, cmꢀ1): 3218 (NH), 1592 (C]N), 1576, 1365 (NO2), 1344,
1139 (SO2). ESI-MS: 522.4 [M + H]+.
To a solution of compound 3a or 3b (7.6 mmol) in ethanol (15
mL), the requisite substituted sulfonamide (7.6 mmol) and
acetic acid (0.5 mL) were added. The mixture was heated at 55
ꢁC for 3 h. A solid product was immediately formed, which was
ltered and washed with water. The crude products were puri-
ed by recrystallization with ethanol, ethyl acetate and acetone
(1 : 1 : 0.5), and then washed with ice-water (25 mL) three times
and dried to give the compounds 4a–4l as yellow solids.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)benzenesulfonamide (4a). Yellow crystals, yield
66.5%. Rf ¼ 0.25 (EtOAc/PE 1 : 1); m.p. 195–197 ꢁC; 1H NMR
(DMSO-d6, 300 MHz) d: 8.52 (s, 1H, CH), 8.03 (s, 1H, CH), 7.88–
7.03 (m, 8H, ArH), 7.04 (s, 1H, NH), 7.02 (s, 1H, NH), 4.75 (t, J ¼
4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz, 2H, –CH2), 2.48 (s, 3H,
CH3). 13C NMR (75.4 MHz, DMSO-d6): d 191.03, 157.82, 151.21,
137.54, 131.98, 129.76, 127.68, 123.58, 121.08, 113.23, 112.15,
66.31, 45.16, 30.31, 14.06. IR (KBr, n, cmꢀ1): 3334, 3222, 3200
(NH, NH2), 1596 (C]N), 1576, 1374 (NO2), 1343, 113 6 (SO2).
ESI-MS: 430.5 [M + H]+.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)-N-(thiazol-2-yl)benzenesulfonamide (4b). Yellow
crystals, yield 50.0%. Rf ¼ 0.32 (EtOAc/PE 1 : 1); m.p. 258–
259 ꢁC; 1H NMR (DMSO-d6, 300 MHz) d: 8.49 (s, 1H, CH), 8.02 (s,
1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J ¼ 5.0 Hz, 2H, ArH),
7.23 (d, J ¼ 2.7 Hz, 1H, CH), 7.02 (d, J ¼ 5.2 Hz, 2H, ArH), 6.81 (d,
J ¼ 2.7 Hz, 1H, CH), 4.75 (t, J ¼ 4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0
Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-
d6): d 168.36, 158.12, 152.01, 149.23, 131.36, 129.86, 126.46,
125.02, 121.38, 112.68, 107.36, 14.08. IR (KBr, n, cmꢀ1): 3220
(NH), 1596 (C]N), 1583, 1379 (NO2), 1348, 1139 (SO2). ESI-MS:
513.4 [M + H]+.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide
(4f).
Yellow crystals, yield 61.8%. Rf ¼ 0.30 (EtOAc/PE 1 : 1); m.p.
285–287 ꢁC; 1H NMR (DMSO-d6, 300 MHz) d: 11.78 (s, 1H,
SO2NH), 9.02 (s, 1H, CH), 8.18 (s, 1H, CH), 8.97–7.44 (m, 8H,
ArH), 6.21 (s, 1H, CH), 4.74 (t, J ¼ 4.9 Hz, 2H, –CH2), 4.45 (t, J ¼
4.9 Hz, 2H, –CH2), 2.53 (s, 3H, CH3), 2.23 (s, 3H, CH3). 13C NMR
(75.4 MHz, DMSO-d6): d 169.69, 159.64, 157.06, 152.56, 151.37,
142.42, 137.68, 132.96, 130.37, 129.93, 129.11, 125.79, 125.03,
124.70, 119.00, 118.69, 116.94, 95.15, 12.02. IR (KBr, n, cmꢀ1):
3221 (NH), 1588 (C]N), 1585, 1376 (NO2), 1346, 1139 (SO2). ESI-
MS: 511.6 [M + H]+.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)benzenesulfonamide (4g). Yellow crystals, yield
64.3%. Rf ¼ 0.29 (EtOAc/PE 1 : 1); m.p. 193–195 ꢁC; 1H NMR
(DMSO-d6, 300 MHz) d: 8.52 (s, 1H, CH), 8.03 (s, 1H, CH), 7.93–
7.08 (m, 8H, ArH), 7.04 (s, 1H, NH), 7.02 (s, 1H, NH), 4.75 (t, J ¼
4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz, 2H, –CH2), 2.48 (s, 3H,
CH3). 13C NMR (75.4 MHz, DMSO-d6): d 191.17, 157.84, 151.16,
137.79, 132.52, 129.82, 127.43, 123.50, 120.78, 113.34, 112.33,
66.36, 45.21, 30.32, 14.06. IR (KBr, n, cmꢀ1): 3338, 3221, 3208,
3115 (NH, NH2), 1594 (C]N), 1577, 1374 (NO2), 1345, 1133
(SO2). ESI-MS: 430.3 [M + H]+.
N-Carbamimidoyl-4-(4-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)-
ethoxy)benzylideneamino)benzenesulfonamide (4c). Orange
crystals, yield 84.3%. Rf ¼ 0.28 (EtOAc/PE 1 : 1); m.p. 235–
1
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)-N-(thiazol-2-yl)benzenesulfonamide (4h). Yellow
crystals, yield 68.9%. Rf ¼ 0.23 (EtOAc/PE 1 : 1); m.p. 148–
ꢁ
237 C; H NMR (DMSO-d6, 300 MHz) d: 11.43 (s, 1H, SO2NH),
8.49 (s, 1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J ¼ 5.0 Hz, 2H,
ArH), 7.23 (d, J ¼ 2.7 Hz, 1H, CH), 7.02 (d, J ¼ 5.2 Hz, 2H, ArH),
6.80 (s, 3H, NH), 4.75 (t, J ¼ 4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz,
2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): d
190.23, 162.37, 160.52, 157.77, 154.03, 150.87, 141.15, 132.49,
131.44, 130.47, 127.01, 126.57, 120.39, 114.48, 114.32, 112.41,
66.61, 45.17, 14.06. IR (KBr, n, cmꢀ1): 3396, 3236, 3110 (NH,
NH2), 1588 (C]N), 1570, 1368 (NO2), 1341, 1144 (SO2).
1
ꢁ
150 C; H NMR (DMSO-d6, 300 MHz) d: 12.43 (s, 1H, SO2NH),
9.95 (s, 1H, CH), 8.04 (s, 1H, CH), 7.44–6.54 (m, 7.40, 4H, ArH),
7.19 (d, J ¼ 5.0 Hz, 2H, ArH), 7.17 (s, 1H, CH), 7.02 (d, J ¼ 5.2 Hz,
2H, ArH), 6.58–6.54 (m, 4H, ArH), 4.75 (t, J ¼ 4.7 Hz, 2H, –CH2),
4.39 (t, J ¼ 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4
MHz, DMSO-d6): d 167.98, 158.32, 151.96, 149.03, 130.78,
129.32, 127.46, 124.42, 120.98, 112.48, 108.96, 14.06. IR (KBr,
n, cmꢀ1): 3221 (NH), 1594 (C]N), 1585, 1378 (NO2), 1348, 1140
(SO2). ESI-MS: 513.5 [M + H]+.
N-(4,6-Dimethylpyrimidin-2-yl)-4-(4-(2-(2-methyl-5-nitro-1H-
imidazole-1-yl)ethoxy) benzylideneamino)benzenesulfonamide
(4d). Yellow crystals, yield 64.3%. Rf ¼ 0.32 (EtOAc/PE 1 : 1);
1
N-Carbamimidoyl-4-(3-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)-
ethoxy)benzylideneamino)benzenesulfonamide (4i). Light
yellow crystals, yield 65.9%. Rf ¼ 0.25 (EtOAc/PE 1 : 1); m.p.
229–231 ꢁC; 1H NMR (DMSO-d6, 300 MHz) d: 11.43 (s, 1H,
SO2NH), 8.49 (s, 1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J ¼ 5.0
Hz, 2H, ArH), 7.23 (s, 1H, CH), 7.02 (d, J ¼ 5.2 Hz, 2H, ArH), 6.80
(s, 3H, NH), 4.75 (t, J ¼ 4.7 Hz, 2H, –CH2), 4.39 (t, J ¼ 5.0 Hz, 2H,
–CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): d
189.26, 162.31, 160.42, 156.07, 153.95, 150.27, 141.11, 133.06,
ꢁ
m.p. 235–237 C; H NMR (DMSO-d6, 300 MHz) d: 9.02 (s, 1H,
CH), 8.04 (s, 1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (s, 1H, CH),
5.95 (s, 1H, SO2NH), 4.74 (t, J ¼ 4.9 Hz, 2H, –CH2), 4.45 (t, J ¼ 4.9
Hz, 2H, –CH2), 2.53 (s, 3H, CH3), 2.25 (s, 6H, CH3). 13C NMR
(75.4 MHz, DMSO-d6): d 171.28, 159.01, 156.78, 153.06, 151.68,
141.96, 137.48, 132.76, 129.86, 129.56, 125.86, 124.75, 119.36,
118.43, 116.54, 95.35, 14.08, 12.02. IR (KBr, n, cmꢀ1): 3187 (NH),
1591 (C]N), 1581, 1376 (NO2), 1345, 1140 (SO2). ESI-MS: 536.4
[M + H]+.
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131.61, 130.53, 127.31, 125.97, 120.29, 114.08, 114.23, 112.36, were determined in the same manner and subtracted from the
66.58, 45.22, 14.08. IR (KBr, n, cmꢀ1): 3390, 3233, 3111 (NH, total observed rates. Stock solutions of inhibitor (1 mM) were
NH2), 1593 (C]N), 1573, 1370 (NO2), 1343, 1138 (SO2). ESI-MS: prepared in distilled-deionized water with 10–20% (v/v) DMSO
472.6 [M + H]+.
(which is not inhibitory at these concentrations), and dilutions
N-(4,6-Dimethylpyrimidin-2-yl)-4-(3-(2-(2-methyl-5-nitro-1H- up to 0.01 nM were performed thereaer with distilled-deion-
imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide ized water. Inhibitor and enzyme solutions were preincubated
(4j). Yellow crystals, yield 71.2%. Rf ¼ 0.26 (EtOAc/PE 1 : 1); m.p. together for 15 min at room temperature prior to the assay in
1
ꢁ
226–228 C; H NMR (DMSO-d6, 300 MHz) d: 9.02 (s, 1H, CH), order to allow for the formation of the E–I complex. The inhi-
8.04 (s, 1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (s, 1H, CH), 5.95 bition constants were obtained by nonlinear least-squares
(s, 1H, SO2NH), 4.74 (t, J ¼ 4.9 Hz, 2H, CH2), 4.45 (t, J ¼ 4.9 Hz, methods using PRISM3. The curve-tting algorithm allowed us
2H, CH2), 2.53 (s, 3H, CH3), 2.25 (s, 6H, CH3). 13C NMR (75.4 to obtain the IC50 values, working at the lowest concentration of
MHz, DMSO-d6): d 171.18, 159.86, 157.08, 152.86, 151.28, substrate of 1.7 mM, from which Ki values were calculated by
141.78, 138.02, 132.56, 130.14, 129.58, 125.46, 124.70, 119.36, using the Cheng–Prusoff equation. The catalytic activity (in the
118.23, 116.54, 95.35, 14.08, 12.02. IR (KBr, n, cmꢀ1): 3228 (NH), absence of inhibitors) of these enzymes were calculated from
1587 (C]N), 1585, 1368 (NO2), 1346, 1139 (SO2). ESI-MS: 536.7 Lineweaver–Burk plots, as reported earlier, and represent the
[M + H]+.
mean from at least three different determinations. 38–42
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide- enzyme concentrations in the assay system were 7.3 nM for hCA
neamino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (4k). II and 8.5 nM for hCA IX. Enzymes used here were recombinant
Light yellow crystals, yield 62%. Rf ¼ 0.28 (EtOAc/PE 1 : 1); m.p. ones, and they were prepared and puried as described
1
128–130 C; H NMR (DMSO-d6, 300 MHz) d: 9.95 (s, 1H, CH), earlier.32
ꢁ
8.31 (d, J ¼ 5.1 Hz, 2H, ArH), 8.03 (s, 1H, CH), 7.66–7.51 (m, 6H,
ArH), 7.34 (s, 1H, SO2NH), 6.95 (s, 1H, CH), 6.88 (s, 1H, CH), 4.74 Cell proliferation assay
(t, J ¼ 4.9 Hz, 2H, CH2), 4.45 (t, J ¼ 4.9 Hz, 2H, –CH2), 2.55 (s, 3H,
MTT is much more convenient and helpful than MTT for
CH3), 2.31 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): d
191.37, 157.03, 156.81, 152.08, 132.44, 129.89, 129.58, 129.15,
123.13, 120.80, 120.10, 114.20, 112.80, 112.12, 66.44, 45.18,
23.32, 14.08. IR (KBr, n, cmꢀ1): 3220 (NH), 1592 (C]N), 1585,
1380 (NO2), 1347, 1139 (SO2). ESI-MS: 522.5 [M + H]+.
analyzing cell proliferation, because it can be reduced to soluble
formazan by dehydrogenase in mitochondria and has little
toxicity to cells. Cell proliferation was determined using MTT
dye (BeyotimeInst Biotech, China) according to manufacturer's
instructions. Briey, 1–5 ꢂ 103 cells per well were seeded in a
96-well plate, grown at 37 ꢁC for 12 h. Subsequently, cells were
treated with the target compounds at increasing concentrations
in the presence of 10% FBS for 24 or 48 h. Aer 10 mL MTT dye
was added to each well, cells were incubated at 37 ꢁC for 1–2 h
and plates were read in a Victor-V multilabel counter (Perkin-
Elmer) using the default europium detection protocol. Percent
inhibition or IC50 values of compounds were calculated by
comparison with DMSO-treated control wells.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylide-
neamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide
(4l).
Yellow crystals, yield 65.7%. Rf ¼ 0.24 (EtOAc/PE 1 : 1); m.p. 88–
1
ꢁ
90 C; H NMR (DMSO-d6, 300 MHz) d: 9.31 (s, 1H, CH), 8.04–
7.83 (m, 4H, ArH), 7.63 (s, 1H, CH), 7.33–6.74 (m, 4H, ArH), 6.55
(s, 1H, CH), 5.95 (s, 1H, SO2NH), 4.74 (t, J ¼ 4.92 Hz, 2H, –CH2),
4.45 (t, J ¼ 4.92 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.23 (s, 3H,
CH3). 13C NMR (75.4 MHz, DMSO-d6): d 168.96, 157.88, 157.69,
151.60, 137.30, 132.46, 129.84, 128.50, 127.87, 125.83, 123.40,
120.78, 113.02, 112.46, 94.91, 66.38, 45.21, 14.08, 11.97. IR (KBr,
n, cmꢀ1): 3220 (NH), 1593 (C]N), 1582, 1379 (NO2), 1350, 1139
(SO2). ESI-MS: 511.6 [M + H]+.
Flow cytometry
Cells (1.3 ꢂ 105 cells per mL) were cultured in the presence or
not of novobiocin analogues at 200 mM. Nvb at the same
concentration served as reference inhibitor. Aer treatment for
48 and 72 h, cells were washed and xed in PBS/ethanol (30/70).
For cytouorometric examination, cells (104 cells per mL) were
incubated for 30 min in PBS/Triton X-100, 0.2% EDTA (1 mM),
and propidium iodide (PI) (50 mg mLꢀ1) in PBS supplemented
by RNase (0.5 mg mLꢀ1). The number of cells in the different
phases of the cell cycle was determined, and the percentage of
apoptotic cells was quantied. Analyses were performed with a
FACS Calibur (Becton Dickinson, Le Pont de Claix, France). Cell
Quest soware was used for data acquisition and analysis.33
CA inhibition assay
An Applied Photophysics stopped-ow instrument has been
used for assaying the CA-catalyzed CO2 hydration activity.31
Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH ¼ 7.5) as buffer and 0.1 M Na2SO4 (for
ꢁ
maintaining constant the ionic strength) at 25 C, followed by
the CA-catalyzed CO2 hydration reaction for a period of 10–100 s
(the uncatalyzed reaction needs around 60–100 s in the assay
conditions, whereas the catalyzed reactions need around 6–10
s). The CO2 concentrations ranged from 1.7 to 17 mM for the
Experimental protocol of docking study
determination of kinetic parameters. For each inhibitor, tested Molecular docking of compound 4c and 4f into the three-
in the concentration range between 0.01 nM and 100 mM, at dimensional X-ray structure of human hCA II (PDB code: 3N4B)
least six traces of the initial 5–10% of the reaction have been was carried out using the Discovery Studio (version 3.5) as
used for determining the initial velocity. The uncatalyzed rates implemented through the graphical user interface DS-CDOCKER
33036 | RSC Adv., 2014, 4, 33029–33038
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Paper
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protocol. The three-dimensional structures of the aforementioned 10 F. Pacchiano, F. Carta, P. C. McDonald, Y. M. Lou, D. Vullo,
compounds were constructed using Chem. 3D ultra 12.0 soware
[Chemical Structure Drawing Standard; Cambridge So corpora-
A. Scozzafava, S. Dedhar and C. T. Supuran, J. Med. Chem.,
2011, 54, 1896–1902.
tion, USA (2010)], and then they were energetically minimized by 11 C. T. Supuran, F. Briganti, S. Tilli, W. R. Chegwidden and
using MMFF94 with 5000 iterations and minimum RMS gradient A. Scozzafava, Bioorg. Med. Chem., 2001, 9, 703–714.
of 0.10. The crystal structures of protein complex was retrieved 12 (a) C. C. Wykoff, N. J. Beasley, P. H. Watson, K. J. Turner,
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/
home/home.do). All bound water molecules and ligands were
eliminated from the protein, and the polar hydrogen was added to
the proteins. Molecular docking of all 20 compounds was then
carried out using the Discovery Studio (version 3.5) as imple-
J. Pastorek, A. Sibtain, G. D. Wilson, H. Turley, K. L. Talks,
P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe and A. L. Harris,
Cancer Res., 2000, 60, 7075–7083; (b) C. P. S. Potter and
A. L. Harris, J. Cancer, 2003, 89, 2–7; (c) R. K. Jain, Science,
2005, 307, 58–62.
mented through the graphical user interface CDOCKER protocol. 13 W. A. Denny, Cancer Invest., 2004, 22, 604–619.
´
CDOCKER is an implementation of a CHARMm based molecular 14 J. Chiche, M. Brahimi-Horn and J. Pouyssegur, J. Cell. Mol.
docking tool using a rigid receptor.
Med., 2010, 14, 771–794.
15 Y. Yoshino, N. Ueda, J. Niijima, H. Sugumi, Y. Kotake,
N. Koyanagi, K. Yoshimatsu, M. Asada, T. Watanabe,
T. Nagasu, K. Tsukahara, A. Iijima and K. Kitoh, J. Med.
Chem., 1992, 35, 2496–2497.
Acknowledgements
The work was nanced by a grant from Major Projects on
Control and Rectication of Water Body Pollution (no. 16 A. Casini, A. Scozzafava, A. Mastrolorenzo and C. T. Supuran,
2011ZX07204–001–004), and supported by “PCSIRT” (IRT1020).
Curr. Cancer Drug Targets, 2002, 2, 55–75.
17 B. Shan, J. C. Medina, E. Santha, W. P. Frankmoelle,
T.-C. Chou, R. M. Learned, M. R. Narbut, D. Stott, P. Wu,
J. C. Jaen, T. Rosen, P. B. M. W. M. Timmermans and
H. Beckmann, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 5686–5691.
18 H. Tanaka, N. Ohshima, M. Ikenoya, K. Komori, F. Katoh
and H. Hidaka, Cancer Res., 2003, 63, 6942–6947.
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