Sulfonamides containing coumarin moieties selectively and potently inhibit carbonic anhydrases II and IX: Design, synthesis, inhibitory activity and 3D-QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2013.04.035

A series of sulfonamides containing coumarin moieties had been prepared that showed a very interesting profile for the inhibition of two human carbonic anhydrase inhibitors. These compounds were evaluated for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA II and the tumor-associated isozyme hCA IX. The most potent inhibitor against hCA II and IX were compounds 5d (IC₅₀ = 23 nM) and 5l (IC₅₀ = 24 nM), respectively. These sulfonamides containing coumarin moieties may prove interesting lead candidates to target tumor-associated CA isozymes, wherein the CA domain is located extracellularly. Eighteen compounds were scrutinized by CoMFA and CoMSIA techniques of 3D quantitative structure-activity relationship. Nine of the compounds were evaluated for cytotoxicity against human macrophage.

Full text of DOI:10.1016/j.ejmech.2013.04.035

European Journal of Medicinal Chemistry 66 (2013) 1e11
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Sulfonamides containing coumarin moieties selectively and potently
inhibit carbonic anhydrases II and IX: Design, synthesis, inhibitory
activity and 3D-QSAR analysis
1
1
1
Zhong-Chang Wang , Ya-Juan Qin , Peng-Fei Wang , Yong-An Yang, Qing Wen, Xin Zhang,
Han-Yue Qiu, Yong-Tao Duan, Yan-Ting Wang, Ya-Li Sang, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 February 2013
Received in revised form
A series of sulfonamides containing coumarin moieties had been prepared that showed a very interesting
profile for the inhibition of two human carbonic anhydrase inhibitors. These compounds were evaluated
for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA II and the
tumor-associated isozyme hCA IX. The most potent inhibitor against hCA II and IX were compounds 5d
13 April 2013
Accepted 15 April 2013
Available online 9 May 2013
(
IC50 ¼ 23 nM) and 5l (IC50 ¼ 24 nM), respectively. These sulfonamides containing coumarin moieties
may prove interesting lead candidates to target tumor-associated CA isozymes, wherein the CA domain is
located extracellularly. Eighteen compounds were scrutinized by CoMFA and CoMSIA techniques of 3D
quantitative structureeactivity relationship. Nine of the compounds were evaluated for cytotoxicity
against human macrophage.
Keywords:
Sulfonamide derivatives
Carbonic anhydrase
Antitumor activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
3D-QSAR
CCK-8 assay
1. Introduction
and proliferation which make for the extracellular acidification of
the solid tumor microenvironment. Tumor cells are prone to sur-
The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc
enzymes which are involved in pH buffering of extra- and
intracellular spaces by catalyzing the reversible hydration of
vive in hypoxic and acidic microenvironment owing to these en-
zymes [5]. Hypoxic tumor cells alter their metabolism that favors
cell survival and migration, when in a hypoxic conditions to sur-
vive, proliferate, and metastasize [6]. Hypoxia-inducible factor
(HIF), in addition to genetic and epigenetic changes, is largely
responsible for alterations in cell metabolism in hypoxic tumor
cells-one core aspect of which is the shift from aerobic to
fermentative glycolysis [7]. Fermentative glycolysis leads to
elevated lactic acid production; hence, a drop in the intracellular
carbon dioxide and water to bicarbonate and
a proton:
ꢀ
þ
CO
2
þ H
2
O 4 HCO
3
þ H [1]. This reaction is known to regulate a
broad range of physiological functions which has proven a reliable
treatment for a range of human disease states by the respiration
ꢀ
and transport of CO
2
/HCO
3
[2]. Carbonic anhydrases, specifically
isozymes IX (CA IX) is the most strongly overexpressed gene in
response to hypoxia in human cancer cells including breast, clear
cell renal, colorectal, head and neck, and nonsmall cell lung carci-
nomas [3]. This enzyme is a multidomain protein with the CA
pH (pH
is established [8]. Suggested by Svastova et al., the assumption that
pH in different tumor cell cultures recovers to an extent more
i
) of hypoxic tumor cells is expected unless pH homeostasis
e
subdomain situated outside the cell and possessing a very high CO
2
normal range along with a remarkable increased apoptosis of the
tumor cells, when CA IX is inhibited by potent and selective sul-
fonamide inhibitors is being widely accepted [9]. For example,
Pouyssegur’s group showed recently that in hypoxic LS174Tr tumor
cells expressing either one or both CA isoforms, in response to a
hydrase catalytic activity, which is also inhibited by the classical CA
inhibitors belonging to the sulfonamide, sulfamate, and sulfamide
classes of compounds [4]. There are evidences that CA IX contrib-
utes to creating a pH-regulating system suitable for cell viability
“
CO
2
load”, both enzymes contribute to extracellular acidification
), an
and to maintain a more alkaline resting intracellular pH (pH
i
action that preserves ATP levels and cell survival in a range of acidic
outside pH (6.0e6.8) and low bicarbonate medium. In vivo exper-
iments showed that silencing of CA IX alone leads to a 40%
*
Corresponding author. Tel./fax: þ86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These three authors equally contributed to this paper.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.035

2
Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
reduction in xenograft tumor volume, with up-regulation of the
second gene, that encoding for CA XII, whereas invalidation of both
CAIX and CA XII gives an impressive 85% reduction of tumor growth
2. Results and discussion
According to the analysis of the CA active site and the structure
of inhibitors [23], Thiry et al. [24] ever reported a general phar-
macophore (Fig. 2) for the compounds which acted as carbonic
anhydrase inhibitors. In order to make the pharmacophore interact
with CA inhibitors, it should include the structural elements that
are required to be present in the compounds. This pharmacophore
matches this requirement well. A sulfonamide moiety should be
included which coordinates with the zinc ion of the active site of
the CA and the sulfonamide is attached to a scaffold which is
usually a benzene ring. The side chain, which might interact with
the hydrophobic and hydrophilic parts of the CA active site, can
substitute an aromatic or heterocyclic sulfonamide scaffold.
Therefore, different hydrophobic side chains were incorporated in
the sulfonamide scaffold with an amide linker that can interact
with the hydrophilic part of the active site, and a hydrophobic
moiety which can interact with the hydrophobic part of the CA
active site.
[10]. Accompany with the fact that it has been shown earlier that
some CA inhibitors do demonstrate potential antitumor activity
in vivo [11], CA IX inhibition may compose a fascinating new
approach for the management of hypoxic tumors, which generally
do not respond to the classical radio- and chemotherapy [12].
One approach aimed at improving the selectivity of tumor cell
killing by antitumor drugs mainly using prodrugs with less toxicity
which target the tumor cells by special physiology of tumor cells
such as selective enzyme expression or hypoxia and therefore are
highly specific active to tumor cells [13]. To characterize the po-
tential 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 to make for experimental advances [14]. Since
the discovery of E-7010 in 1992 [15], sulfonamides have emerged as
an important class of anticancer agents which interact with a wide
range of different cellular targets. Such that indisulam (E7070 [16],
HMN-214 [17], T138067 [18]) is in phase II clinical trials as an
antitumor sulfonamide with a complex mechanism of action also
involving CA inhibition of several isozymes participating in tumor
genesis (Fig. 1).
Fig. 3 illustrates representative examples of the newly synthe-
sized compounds showing compliance with the above-mentioned
pharmacophore model and the compounds which were synthe-
sized according to Scheme 1.
Sulfonamides constitute an important class of drugs, and they
possess various types of biological properties including anti-
carbonic anhydrase [19], antibacterial [20], Many of structurally
novel sulfonamide derivatives have recently been reported to show
substantial antitumor activity, both in vitro and in vivo. Some of
these derivatives are currently being evaluated in clinical trials.
Optimistically, these may lead to novel alternative anticancer drugs,
devoid of the side effects of the presently available pharmacological
agents [21].
2
.1. Chemistry
All novel sulfonamides containing coumarin moieties carbonic
anhydrase IX inhibitors (5ae5j) described herein were synthesized
following the synthetic pathway depicted in Scheme 1. The starting
diverse substituted malonic acid mono phenol esters were synthe-
sized by Meldrum’s acid and different substituted phenol under a
solvent-free condition in high yield, then cyclized by Eaton’s reagent
Moreover, Claudiu T. Supuran’s group reported a novel class of
inhibitors of the metalloenzyme carbonic anhydrase with couma-
rins [22]. All these findings encouraged us to explore the synthesis
of sulfonamides containing coumarin moieties and evaluate their
activities as antitumor agents.
In this paper we report the synthesis of a series of sulfonamides
containing coumarin moieties CAIs that possess high affinity for CA
IX, an acceptable selectivity profile for inhibiting the tumor-
associated isoforms CAs IX over the cytosolic ones CAs II, and
excellent in vivo inhibitory effects in a breast cancer xenograft
model.
(
phosphoric anhydride and methylsulfonic acid) in mild conditions
to give corresponding 4-hydroxycoumarin 3ae3c [25]. The different
substituted 3-formyl-4-chlorocoumarin 4ae4c were synthesized by
a VilsmeiereHaack reactions. Subsequently, compounds 5ae5r
were prepared from reaction of 4ae4c with the corresponding
different substituted sulfonamide in ethanol at room temperature.
The reactions were monitored by thin layer chromatography (TLC)
and the crude products were purified by recrystallization with
ethanol, ethyl acetate and acetone (1:1:0.05). The chemical struc-
tures of these sulfonamides containing coumarin moieties were
summarized in Table 1. All of the target compounds gave satisfactory
analytical and spectroscopic data, which in accordance with their
1
depicted structures by H NMR, ESI MS.
2
2
.2. Biological activity
.2.1. Antitumor assay and CCK-8 assays
In the present work, eighteen of the newly synthesized com-
pounds 5ae5r were evaluated on their in vitro growth inhibitory
Fig. 1. Known human CA inhibitors including some clinically used drugs.
Fig. 2. Structural elements of CA inhibitors in the CA enzymatic active site.

Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
3
prognosis for patients with breast cancer and it is significantly
associated with distant metastasis (Lou et al., unpublished results).
Therefore, we have chosen breast cancer as a malignancy model in
which to test, in vivo, the inhibitory activity of some of the CA IX
inhibitors described here [10]. So, the newly described sulfonamides
containing coumarin moieties 5ae5r were tested in an in vitro
carbonic anhydrase assay to evaluate their potential as antitumor
drugs. The results are summarized in Table 1 as IC50 values.
The following structureeactivity relationship (SAR) can be
observed from data of Table 1, CA activities of these compounds
were tested against the standard clinically used inhibitors AAZ and
SA. The physiologically dominant and highly active cytosolic iso-
form hCA II was inhibited by compounds 5ae5r with IC50 in the
Fig. 3. Representative examples of the synthesized compounds showing compliance to
the general pharmacophore of sulfonamide compounds acting as carbonic anhydrase
inhibitors.
activities against two cultured cell lines, which are mouse mela-
noma cells (B16-F10) and breast carcinoma cell lines (MCF-7) in
comparison to the known anticancer drugs: doxorubicin and
semaxanib as reference drugs. IC50 values were calculated using an
inhibitory model, with the sum of squares of the residuals mini-
mized by Origin 8.6 software. The values obtained for the eighteen
compounds against the B16-F10 and MCF-7 cell lines were shown
in Table 1. As shown in Table 1, it was observed that sulfonamides
containing coumarin moieties had been found to show the inhibi-
tion of growth of two tumor cell lines with moderate IC50 values.
For MCF-7 cell line which over-expressed CA that previously ref-
erences had reported, sulfonamide derivatives in particular dis-
played more potent anti-tumor activity than the other one tumor
cell lines. The IC50 values of most sulfonamides containing
range of 0.023e0.217
ing IC50 value of 0.023
active with IC50 value of 0.217
m
M. Compound 5d was the most active hav-
mol, whereas compound 5q was the least
mol. We simplified the situations by
m
m
only treating steric complexities as the single factor to deal with.
Most of these compounds with un-substituted moieties on the
benzene ring of the coumarin and the same substituted moieties on
the amino were more effective as hCA II inhibitors compared to
those with substituted moieties on the benzene ring of the
t
coumarin, for instance the active gradient is eH > eCH
3
> e Bu. But
t
for hCA IX it’s eCH
isoform hCA IX was inhibited by compounds 5ae5r with IC50 in the
range of 0.024e0.188 M (Table 1). Result indicated that the sul-
3
> e Bu > eH. Moreover, the tumor-associated
m
coumarin moieties ranged from 0.0088
Among hem, compound 5j displayed the most potent anti-tumor
activity with IC50 of 0.0088 M, compared to the positive control
doxorubicin (IC50 ¼ 0.072 M) and semaxanib (IC50 ¼ 0.012 M).
m
M to 0.16
m
M in MCF-7.
fonamides incorporating the coumarin moiety lead to highly
effective hCA IX inhibitors. In the series of reported compounds
with the same substituted moieties on the coumarin, the best in-
hibitors were incorporating methylpyrimidine. Compound 5l was
m
m
m
Nevertheless, IC50 values would have a 1e10 folds in B16-F10 on
average. More potent anti-tumor activity for MCF-7 together with
virtual screening results both indicated that the over-expressed CA
might be a potential target which these sulfonamides containing
coumarin moieties interacted with.
the most active having IC50 value of 0.024
mmol, whereas com-
pound 5c was the least active with IC50 value of 0.188
mmol. From
above, we can conclude compounds 5d and 5l had been identified
as the most potent inhibitors.
2.2.3. Cytotoxicity
2.2.2. Carbonic anhydrase inhibition
Nine compounds selected on the basis of their structural fea-
Inhibition of tow physiologically relevant CA isoforms with
tures were evaluated for their toxicity against human macrophage
with the median cytotoxic concentration (CC50) data of tested
compounds by the CCK-8 assay, as showed in Table 2. These com-
pounds were tested at multiple doses to study the viability of
macrophage.
As shown in Table 2, nine compounds showed that they almost
did not exhibit cytotoxic activity. So, we guess that all other com-
pounds were not exhibit cytotoxic activity in vitro against
macrophage.
compounds 5ae5r, AAZ and SA, tow clinically used drugs were
presented in Table 1 [26]. HCAs II (cytosolic, widespread enzymes)
and IX (transmembrane, tumor-associated CAs) have been included
in this study because of their relevance as targets/off targets when
developing CAIs [27].
CA IX is highly expressed in breast malignancies, and studies
have demonstrated that CA IX and CA XII are variably expressed in
breast cancer cell lines. Furthermore, CA IX is associated with a poor
Scheme 1.

4
Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
Table 1
Anticancer activities (IC50, mM) of sulfonamides containing coumarin moieties (5ae5r).
Compounds
R
1
R
2
R
3
IC50
(m
M)a
IC50
(
m
M)a
IC50
(
m
M)c
IC50 (m
M)c
B16-F10b
MCF-7b
hCAs IId
hCA IXd
5
5
a
b
H
H
H
H
H
0.30
0.30
0.12
0.16
0.033
0.058
0.091
0.138
5
5
c
H
H
H
H
0.18
0.17
0.058
0.020
0.085
0.023
0.188
0.124
d
5e
H
H
0.27
0.012
0.026
0.141
5
5
5
f
H
H
0.23
0.24
0.16
0.031
0.016
0.012
0.051
0.112
0.122
0.084
0.034
0.051
g
h
CH(CH
CH(CH
3
)
)
3
3
CH(CH
3
)
)
3
3
H
3
CH(CH
3
5
5
i
j
CH(CH
CH(CH
3
)
)
3
3
CH(CH
3
)
)
3
3
0.25
0.22
0.018
0.103
0.173
0.074
0.090
3
CH(CH
3
0.0088
5
k
CH(CH
3
)
)
3
3
CH(CH
3
)
)
3
3
0.24
0.064
0.173
0.043
5
5
5
l
CH(CH
3
CH(CH
3
0.19
0.36
0.28
0.032
0.27
0.063
0.088
0.121
0.024
0.085
0.087
m
n
H
H
CH
3
3
H
CH
0.033
5
5
o
p
H
H
CH
3
3
0.24
0.30
0.067
0.025
0.100
0.141
0.050
0.067
CH
5
5
q
r
H
H
CH
3
3
0.25
0.27
0.016
0.061
0.217
0.061
0.052
0.048
CH
Doxorubicin
Semaxanib
e
e
e
e
e
e
0.072
0.0036
0.065
0.0031
e
e
e
e

Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
5
Table 1 (continued )
Compounds
R
1
R
2
R
3
IC50
(
m
M)a
IC50
(
m
M)a
IC50
(m
M)c
IC50 (m
M)c
B16-F10b
MCF-7b
hCAs IId
hCA IXd
AAZ
SA
e
e
e
e
e
e
e
e
e
e
0.016
0.26
0.028
0.29
a
Antiproliferation activity was measured using the CCK-8 assay. Values are the average of three independent experiments run in triplicate. Variation was generally 5e10%.
Cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University.
Errors were in the range of 5e10% of the reported values, from three different assays.
b
c
d
Human recombinant enzymes, by the esterase assay (4-nitrophenylacetate as substrate).
ꢁ
ꢀ
2
.2.4. Analysis of apoptosis by Annexin V-PE fluorescence-activated
O HE21 ¼135.7 , distance ¼ 1.92 A), and oxygen atom of carbonyl of
ꢁ
ꢀ
cell sorting (FACS)
compound 5c (angle H HeN ¼ 153.0 , distance ¼ 2.23 A) each other.
And compound 5d was also nicely bound to the ATP binding site of
hCA II hydrophobic interaction and the binding was stabilized by
two hydrogen bonds, and a
sulfonyl formed two hydrogen bonds with the backbone NH of
To test whether the inhibition of cell growth of A549 was related
to cell apoptosis, A549 cell apoptosis induced by compound 5d was
determined using flow cytometry. The uptake of Annexin V-PE was
significantly increased, and the uptake of normal cells was signifi-
cantly decreased in a time-dependent manner. Finally the per-
centage of early apoptotic cells was markedly elevated in a density-
dependent manner from 6.28 to19.6% at 48 h (Fig. 4).
p-cation interaction. The oxygen of the
ꢁ
ꢀ
Asn67 (angle O/HeN ¼ 129.0 , distance ¼ 2.17 A), Gln92 (angle
ꢁ
ꢀ
NeH/O ¼ 137.7 , distance ¼ 2.11 A), respectively. In addition, a
benzene ring formed one interactions with Val135. This mo-
pep
lecular docking result, along with the biological assay data, sug-
gested that compounds 5a, 5c and 5d were potential inhibitors
of CA.
2
2
.3. Computational
.3.1. Molecular modeling
To gain better understanding on the potency of the 18 com-
2.3.2. 3D-QSAR model [30]
In order to acquire a systematic SAR profile on sulfonamides
pounds and guide further SAR studies, we proceeded to examine
the interaction of these compounds with hCA II by molecular
docking, which was performed by simulation of the 18 compounds
into the ATP binding site in hCA II. The protein structure of the hCA
II was downloaded from the PDB (3N4B.pdb) [28], then pre-
processed using the Schrodinger Protein Preparation Guide. Also
hydrogens were added to the structure, while H-bonds within the
protein were optimized, and the protein was minimized to a 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/CHARM. The entire
proteineligandewater complex was allowed to move during cal-
culations [29].
The predicted binding interaction energy was used as the cri-
terion for ranking. The estimated interaction energy of other syn-
thesized compounds was ranging from ꢀ54.42 to ꢀ37.0 kcal/mol.
The selected pose of 5a, 5c and 5d had an estimated binding free
energy of ꢀ39.4 kcal/mol, ꢀ43.5 kcal/mol, ꢀ47.6 kcal/mol, respec-
tively. The binding model of compounds 5a, 5c, 5d and hCA II was
depicted in Fig. 5. The amino acid residues which had interaction
with hCA II were labeled. In the binding mode, compound 5a was
nicely bound to the ATP binding site of hCA II hydrophobic inter-
action and the binding was stabilized by three hydrogen bonds. The
hydrogens of Asn67 and Thr200 formed three hydrogen bonds
interaction with nitrogen atom of C]N bonds of compound 5a
containing coumarin moieties as antitumor agents and to explore
the more powerful and selective dual hCA II and IX inhibitors, 3D-
QSAR model was built to choose activity conformation of the
designed molecular and reasonably evaluated the designed mole-
cules by using the corresponding pIC50 values which were con-
verted from the obtained IC50 (mM) values of hCA II inhibition and
performed by built-in QSAR software of DS 3.5 (Discovery Studio
3.5, Accelrys, Co. Ltd). The way of this transformation was derived
from an online calculator developed from an Indian medicinal
chemistry lab (http://www.sanjeevslab.org/tools-IC50.html). The
training and test set was divided by the random diverse molecules
method of DS 3.5, in which the test set accounts for 83% while the
training set was set to 17%. The training set composes 15 agents and
ꢀ
ꢀ
3
agents were consisted of the relative test set. The success of this
model depended on docking study and the reliability of previous
study about activities data.
In default situation, the alignment conformation of each mole-
cule that possessed the lowest CDOCKER_INTERACTION_ENENGY
among the eighteen docked poses. The 3D-QSAR model generated
from DS 3.5, defined the critical regions (steric or electrostatic)
affecting the binding affinity. It was a PLS model set up 400 inde-
2
pendent variables (conventional R ¼ 0.861). The graphical rela-
tionship of observed and predicted values had been illustrated in
Fig. 6a, in which the plot of the observed IC50 versus the predicted
values showed that this model could be used in prediction of ac-
tivity for sulfonamides containing coumarin moieties.
ꢁ
ꢀ
(
angle N HD22 ¼ 148.8 , distance ¼ 2.30 A), nitrogen atom of amino
ꢁ
ꢀ
of compound 5a (angle N HeN ¼ 116.4 , distance ¼ 2.46 A) and
ꢁ
A contour plot of the electrostatic field region favorable (in blue)
or unfavorable (red) for anticancer activity based on hCA II protein
target were shown in Fig. 6b while the energy grids corresponding
to the favorable (in green) or unfavorable (yellow) steric effects for
the hCA II affinity were shown in Fig. 6c. It was widely acceptable
that a better inhibitor based on the 3D-QSAR model should have
strong Van der Waals attraction in the green areas and a polar
group in the blue electrostatic potential areas (which were domi-
nant close to the skeleton). As shown in these two pictures, this
promising model would provide a guideline to design and optimize
more effective hCA II inhibitors based on the sulfonamides con-
taining coumarin moieties and pave the way for us to further study
in future.
hydrogen atom of amino of compound 5a (angle H HeN ¼ 133.8 ,
ꢀ
distance ¼ 2.23 A) each other. Besides, the hydrogen of Thr199,
Gln92 and Trp5 formed three hydrogen bonds interaction with
ꢁ
nitrogen atom of amino of compound 5c (angle N HeN ¼ 133.9 ,
ꢀ
distance ¼ 2.10 A), oxygen atom of sulfonyl of compound 5c (angle
Table 2
The median cytotoxic concentration (CC50) data of tested compounds.
Compounds
5a
5c
5e
5g
5j
5l
5m 5g
5r
CC50
a
,
m
mola
0.58 0.62 0.42 0.58 0.73 0.36 1.2
0.84 0.56
Minimum cytotoxic concentration required to cause a microscopically detect-
able alteration of normal cell morphology.

6
Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
Fig. 4. Compound 5d induced apoptosis in A549 cells with the density of 2.5, 10, 40, 160
versus control. The percentage of cells in each part was indicated.
m
g/mL. A549 cells were treated with for 48 h. Values represent the mean, n ¼ 3. P < 0.05
3
. Conclusion
binding conformations to begin our work in this paper as well as
to provide a reliable tool for reasonable design of CA inhibitors
in future. The results indicate that sulfonamides containing
coumarin moieties which acted as potent antibacterial activity
and as such have the potential to be novel and potent antitumor
agents. Given the unforeseen structural differences within the
active site of some pathogenic enzymes, the key to discovering
inhibitors with broad-spectrum antitumor activity lies in a
detailed understanding of the CA active sites. Further studies on
the CA inhibition ability of this compound, new structural data
were guiding further modifications of the current series with the
hopes of improving both enzymatic inhibition and physical
properties.
In summary, CA is an emerging target for the development of
novel antitumor chemotherapeutics. We had designed and syn-
thesized novel series of sulfonamides containing coumarin
moieties which had been tested for their inhibitory activities
against B16-F10 and MCF-7. These compounds showed a very
interesting profile for the inhibition of hCAs II (cytosolic, off-
target isoform) and hCAs IX (transmembrane, tumor-associated
enzyme). Most of them exhibited CA inhibitory activities and
almost no toxicity toward morphological. Docking simulation
was performed to get the probable binding models and poses.
Finally, QSAR models were built with previous activity data and

Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
7
Fig. 5. Docking of compound 5a, 5c and 5d in the ATP binding site of hCA II: (a) Left: 2D model of the interaction between compound 5a, 5c, 5d and the ATP binding site. Right: 3D
model of the interaction between compound 5a, 5c, 5d and the ATP binding site, respectively.
4
. Experiments
2H-chromen-2-one (14.6 mmol) in DMF (10 mL) was then
gradually added to the mixture with constant stirring and
ꢁ
4.1. Materials and measurements
maintaining the temperature of the reaction mixture below 5 C.
The reaction mixture was then allowed to stand at room tem-
perature for 2 h and then heated on a steam bath for 1 h. The
reactions were monitored by thin layer chromatography (TLC).
After cooling, the reaction mixture was poured onto crushed ice
and neutralized with sodium carbonate. A solid product was
immediately formed which was filtered, washed with water. And
the crude products were purified by recrystallization with
ethanol, ethyl acetate and petroleum ether (1:1:4) washed by
ice-water (25 mL) for three times and dried to give a pure yellow
solid product.
All chemicals used were purchased from Aldrich (USA). All
reagents used in current study were of analytical grade. Thin
layer chromatography (TLC) was performed on silica gel plates
with fluorescent indicator. All analytical samples were homoge-
neous on TLC in at least two different solvent systems. Melting
points (uncorrected) were determined on a X-4 MP apparatus
1
(
Taike Corp, Beijing, China). All the H NMR spectra were recor-
ded on a Bruker DPX 300 model Spectrometer in DMSO-d
6
and
chemical shifts ( ) were reported as parts per million (ppm). ESI-
d
MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN-O-
Rapid instrument and were within ꢂ0.4% of the theoretical
values.
4.2.1. 4-Chloro-2-oxo-2H-chromene-3-carbaldehyde (4a) [31]
ꢁ
1
Yellow solid, yield 80%, m.p. 118e120 C; H NMR (DMSO-d
300 MHz) : 10.35 (s, 1H, COH), 8.44e8.25 (m, 2H, ArH).
6
,
d
4.2. General procedure for the synthesis of compounds 4ae4c
4.2.2. 4-Chloro-6-methyl-2-oxo-2H-chromene-3-carbaldehyde
(4b) [32]
Phosphorus oxychloride (10 mL) was added dropwise to a
Yellow solid, yield 70%, m.p. 126e128 C; 1H NMR (DMSO-d
ꢁ
6
,
solution of dimethylformamide (DMF, 20 mL) keeping the tem-
perature below 5 C. Solution of different substituted 4-hydroxy-
300 MHz) d: 10.35 (s, 1H, COH), 8.44e8.25 (m, 2H, ArH), 1.35 (s,
3H, CH ).
3
ꢁ

8
Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
Fig. 6. (a) Using linear fitting curve to compare the predicted pIC50 value with that of experiment. (b) Isosurface of the 3D-QSAR model coefficients on electrostatic potential grids.
The blue triangle mesh represents positive electrostatic potential and the red area represents negative electrostatic potential. (c) Isosurface of the 3D-QSAR model coefficients on
Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates negative coefficients. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
þ
4
(
.2.3. 6, 8-Ditertbutyl-4-chloro-2-oxo-2H-chromene-3-carbaldehyde
4c)
Yellow solid, yield 73%, m.p. 134e136 C; 1H NMR (DMSO-d
00 MHz) : 10.35 (s, 1H, COH), 8.44e8.25 (m, 2H, ArH), 1.54 (s, 9H,
), 1.42 (s, 9H, C(CH ).
8H, ArH), 3.80 (s, 2H, NH). ESI-MS: 363.61 [M þ H] . Anal. Calcd for
16 2 4
C H11ClN O S: C, 52.97; H, 3.06; Cl, 9.77; N, 7.72; O, 17.64; S, 8.84;
Found: C, 52.93; H, 3.07; Cl, 9.79; N, 7.71; O, 17.65; S, 8.85.
ꢁ
6
,
3
d
C(CH
3
)
3
3
)
3
4.3.2. 4-((4-Chloro-2-oxo-2H-chromen-3-yl) methyleneamino)-N-
(
thiazol-2-yl) benzenesulfonamide (5b)
Yellow crystals, yield 73%, m.p. 276e278 C; H NMR (DMSO-
, 300 MHz) : 11.68 (s, 1H, SO NH), 9.61 (s, 1H, eCH), 8.89e
ꢁ
1
4.3. General procedure for the synthesis of compounds 5ae5r
d
6
d
2
To a solution of compounds 4ae4c (7.6 mmol) in ethanol
15 mL) was added different substituted sulfonamide (7.6 mmol)
and acetic acid (0.5 mL). The mixture was heated at room tem-
perature for 3 h. A solid product was immediately formed which
was filtered, washed with water. And the crude products were
purified by recrystallization with ethanol, ethyl acetate and acetone
7.69 (m, 8H, ArH), 7.30 (s, J ¼ 2.7 Hz, 1H, CH), 6.89 (s, J ¼ 2.7 Hz,
þ
(
1H, eCH), 6.90 (s, 1H, NH). ESI-MS: 446.8.11 [M þ H] . Anal.
3 4 2
Calcd for C19H12ClN O S : C, 51.18; H, 2.71; Cl, 7.95; N, 9.42; O,
14.35; S, 14.38; Found: C, 51.10; H, 2.73; Cl, 7.97; N, 9.43; O,
14.36; S, 14.40.
(
1:1:0.5) washed by ice-water (25 mL) for three times and dried to
4.3.3. N-Carbamimidoyl-4-((4-chloro-2-oxo-2H-chromen-3-yl)
give a yellow solid product 5ae5r.
methyleneamino) benzenesulfonamide (5c)
ꢁ
1
Orange crystals, yield 66%, m.p. 261e262 C; H NMR (DMSO-d
300 MHz) : 11.70 (s, 1H, SO NH), 10.01 (s, 1H, CH), 7.82e7.06 (m,
8H, ArH), 6.76 (s, 4H, NH). ESI-MS: 405.30 [M þ H] . Anal. Calcd for
13ClN S: C, 50.44; H, 3.24; Cl, 8.76; N, 13.84; O, 15.81; S, 7.92;
Found: 50.40; H, 3.26; Cl, 8.78; N, 13.81; O, 15.82; S, 7.95.
6
,
4
.3.1. 4-((4-Chloro-2-oxo-2H-chromen-3-yl) methyleneamino)
benzenesulfonamide (5a)
Yellow crystals, yield 79%, m.p. 271e273 C; H NMR (DMSO-d
00 MHz) : 11.78 (s, 1H, SO NH), 10.05 (s, 1H, CH), 7.89e7.07 (m,
d
2
þ
ꢁ
1
6
,
C
17
H
4 4
O
3
d
2

Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
9
4
.3.4. 4-((4-Chloro-2-oxo-2H-chromen-3-yl) methyleneamino)-N-
4.3.11. 4-((6,8-Di-tert-butyl-4-chloro-2-oxo-2H-chromen-3-yl)
methyleneamino)-N-(5-methylisoxazol-3-yl) benzenesulfonamide
(5k)
(
4, 6-dimethyl-pyrimidin-2-yl) benzenesulfonamide (5d)
ꢁ
1
Yellow crystals, yield 62%, m.p. 245e247 C; H NMR (DMSO-d6,
3
00 MHz)
d
: 11.65 (s, 1H, SO
2
NH), 9.63 (s, 1H, CH), 9.01 (s, 1H, CH),
Light yellow crystals, yield 63%. m.p. 217e219 C; 1H NMR
ꢁ
þ
8
.94e7.33 (m, 8H, ArH), 2.24 (s, 6H, CH
17ClN
3.65; S, 6.84; Found: C, 56.30; H, 3.63; Cl, 7.59; N, 11.97; O, 13.66; S,
3
). ESI-MS: 469.90 [M þ H] .
6 2
(DMSO-d , 300 MHz) d: 11.68 (s,1H, SO NH), 9.65 (s,1H, eCH), 8.96e
Anal. Calcd for C22
1
H
4
O
4
S: C, 56.35; H, 3.65; Cl, 7.56; N,11.95; O,
7.44 (m, 8H, ArH), 6.23 (s, J ¼ 0.8 Hz,1H, CH), 2.29 (s, 6H, CH
3
).1.53 (s,
þ
9H, C(CH
3
)
3
), 1.41 (s, 9H, C(CH
30ClN
)
3 3
). ESI-MS: 557.01 [M þ H] . Anal.
6
.85.
Calcd for C28
H
3 5
O S: C, 60.48; H, 5.44; Cl, 6.38; N, 7.56; O,14.39;
S, 5.77; Found: C, 60.40; H, 5.45; Cl, 6.39; N, 7.58; O, 14.42; S, 5.78.
4.3.5. 4-((4-Chloro-2-oxo-2H-chromen-3-yl) methyleneamino)-N-
(
5-methylisoxazol-3-yl) benzenesulfonamide (5e)
Yellow crystals, yield 63%, m.p. 245e247 C; H NMR (DMSO-d
4.3.12. 4-((6,8-Di-tert-butyl-4-chloro-2-oxo-2H-chromen-3-yl)
methyleneamino)-N-(4-methylpyrimidin-2-yl) benzenesulfonamide
ꢁ
1
6
,
3
00 MHz) : 11.73 (s, 1H, SO NH), 9.61 (s,1H, CH), 8.96e7.44 (m, 8H,
d
2
(5l)
ꢁ
1
ArH), 6.2 (s, J ¼ 0.8 Hz, 1H, CH), 2.29 (s, 6H, CH
3
). ESI-MS: 445.56
S: C, 54.12; H, 3.18; Cl, 7.99;
Yellow crystals, yield 88%. m.p. 245e247 C; H NMR (DMSO-d
300 MHz) : 11.66 (s, 1H, SO
6
,
þ
[
M þ H] . Anal. Calcd for C20
H14ClN
3
O
5
d
2
NH), 9.64 (s, 1H, CH), 9.04 (s, J ¼ 5.2 Hz,
N, 9.47; O,18.02; S, 7.22; Found: C, 54.11; H, 3.19; Cl, 7.82; N, 9.45; O,
8.03; S, 7.20.
1H, CH), 8.68e7.47 (m, 8H, ArH), 6.86 (d, J ¼ 5.2 Hz, 1H, CH), 2.31 (s,
1
3H, CH
3
), 1.53 (s, 9H, C(CH
3
)
3
), 1.41 (s, 9H, C(CH
3 3
) ). ESI-MS: 568.40
31ClN S: C, 61.42; H, 5.51; Cl, 6.25;
þ
[
M þ H] . Anal. Calcd for C29
H
4 4
O
4.3.6. 4-((4-Chloro-2-oxo-2H-chromen-3-yl) methyleneamino)-N-
N, 9.88; O, 11.29; S, 5.65; Found: C, 61.49; H, 5.50; Cl, 6.23; N, 9.92;
O, 11.30; S, 5.66.
(
4-methylpyri-midin-2-yl) benzenesulfonamide (5f)
ꢁ
1
Yellow crystals, yield 88%, m.p. 245e247 C; H NMR (DMSO-d
6
,
3
1
00 MHz) : 11.68 (s, 1H, SO
d
2
NH), 9.64 (s, 1H, CH), 9.04 (s, J ¼ 5.2 Hz,
4.3.13. 4-((4-Chloro-6-methyl-2-oxo-2H-chromen-3-yl) methylen-
H, CH), 8.68e7.47 (m, 8H, ArH), 6.86 (d, J ¼ 5.2 Hz, 1H, eCH), 2.31
eamino) benzene-sulfonamide (5m)
þ
Yellow crystals, yield 57%. m.p. >300 C; 1H NMR (DMSO-d
ꢁ
(
s, 3H, CH
3
). ESI-MS: 455.9 [M þ H] . Anal. Calcd for C21
H15ClN
4
O
4
S:
6
,
C, 55.45; H, 3.32; Cl, 7.79; N,12.32; O,14.07; S, 7.05; Found: C, 55.38;
H, 3.31; Cl, 7.80; N, 12.36; O, 14.09; S, 7.06.
300 MHz)
ArH), 2.45 (s, 3H, CH
13ClN S: C, 54.19; H, 3.48; Cl, 9.41; N, 7.43; O, 16.98; S, 8.51;
d
: 11.71 (s, 1H, SO
2
NH), 9.54 (s, 1H, CH), 8.80e7.29 (m, 7H,
þ
3
). ESI-MS: 377.59 [M þ H] . Anal. Calcd for
C
17
H
2 4
O
4.3.7. 4-((6, 8-Di-tert-butyl-4-chloro-2-oxo-2H-chromen-3-yl)
Found: C, 54.10; H, 3.50; Cl, 9.44; N, 7.42; O, 16.99; S, 8.52.
methyleneamino) benzenesulfonamide (5g)
Yellow crystals, yield 45%, m.p. >300 C; 1H NMR (DMSO-d
00 MHz) : 9.61 (s, 1H, CH), 7.64 (s, 2H, NH), 8.81e7.66 (m, 6H,
ꢁ
,
4.3.14. 4-((4-Chloro-6-methyl-2-oxo-2H-chromen-3-yl) methylen-
eamino)-N-(thiazol-2-yl) benzenesulfonamide (5n)
6
3
d
ꢁ
1
ArH), 1.52 (s, 9H, C(CH
3
)
3
), 1.40 (s, 9H, C(CH
3
)
3
). ESI-MS: 476.2
Yellow crystals, yield 58%. m.p. 225e227 C; H NMR (DMSO-d
6
,
þ
[
M þ H] . Anal. Calcd for C24
H
27ClN S: C, 60.69; H, 5.73; Cl, 7.46;
2
O
4
300 MHz) : 11.62 (s, 1H, SO NH), 9.56 (s, 1H, eCH), 8.86e7. 45 (m,
d
2
N, 5.90; O, 13.47; S, 6.75; Found: C, 60.65; H, 5.75; Cl, 7.45; N, 5.92;
O, 13.51; S, 7.08.
6H, ArH), 7.32 (s, J ¼ 5.0 Hz, 1H, eCH), 7.29 (s, 1H, ArH), 6.90 (s,
J ¼ 5.0 Hz, 1H, eCH), 6.90 (s, 1H, NH), 2.43 (s, 3H, CH
3
). ESI-MS:
þ
4
60.68 [M þ H] . Anal. Calcd for C20
3 4 2
H14ClN O S : C, 52.23; H,
4.3.8. 4-((6, 8-Di-tert-butyl-4-chloro-2-oxo-2H-chromen-3-yl)
3.07; Cl, 7.71; N, 9.14; O, 13.91; S, 13.94; Found: C, 52.18; H, 3.08; Cl,
7.70; N, 9.13; O, 13.94; S, 13.98.
methyleneamino)-N-(thiazol-2-yl) benzenesulfonamide (5h)
ꢁ
1
Yellow crystals, yield 88%, m.p. >300 C; H NMR (DMSO-d
00 MHz) : 11.74 (s, 1H, SO NH), 9.61 (s, 1H, eCH), 8.86e7. 65 (m,
H, ArH), 7.28 (s, J ¼ 2.7 Hz, 1H, CH), 6.88 (s, J ¼ 2.7 Hz, 1H, CH), 6.90
), 1.39 (s, 9H, C(CH ). ESI-MS:
28ClN : C, 58.10; H,
6
,
3
6
d
2
4.3.15. N-Carbamimidoyl-4-((4-chloro-6-methyl-2-oxo-2H-
chromen-3-yl) methylene-amino) benzenesulfonamide (5o)
(
s, 1H, NH), 1.50 (s, 9H, C(CH
3
)
3
3
)
3
Light yellow crystals, yield 50%. m.p. 250e252 C; 1H NMR
ꢁ
þ
5
5
6
59.30 [M þ H] . Anal. Calcd for C27
H
3
O
4
S
2
6 2
(DMSO-d , 300 MHz) d: 11.59 (s, 1H, SO NH), 9.52 (s, 1H, CH), 7.83e
7.23 (m, 6H, ArH), 6.77 (s, 3H, NH), 2.39 (s, 3H, CH
H15ClN O S: C, 51.62; H, 3.61; Cl, 8.46;
4 4
N,13.38; O,15.28; S, 7.66; Found: C, 51.56; H, 3.64; Cl, 8.43; N,13.42;
O, 15.31; S, 7.65.
.06; Cl, 6.35; N, 7.53; O, 11.47; S, 11.49; Found: C, 58.05; H, 5.05; Cl,
.34; N, 7.57; O, 11.49; S, 11.50.
3
). ESI-MS: 419.91
þ
[M þ H] . Anal. Calcd for C18
4.3.9. N-Carbamimidoyl-4-((6,8-di-tert-butyl-4-chloro-2-oxo-2H-
chromen-3-yl) methyleneamino) benzenesulfonamide (5i)
Light yellow crystals, yield 70%, m.p. 286e288 C; 1H NMR
DMSO-d , 300 MHz) : 11.77 (s, 1H, SO NH), 9.52 (s, 1H, CH), 8.77e
), 1.39 (s, 9H,
29ClN S:
ꢁ
4.3.16. 4-((4-Chloro-6-methyl-2-oxo-2H-chromen-3-yl) methylen-
(
7
6
d
2
eamino)-N-(4,6-dimethylpyrimidin-2-yl) benzenesulfonamide (5p)
ꢁ
1
.66 (m, 6H, ArH), 6.80 (s, 3H, NH), 1.51 (s, 9H, C(CH
3
)
3
Orange crystals, yield 51%. m.p. 170e172 C; H NMR (DMSO-d
6
,
þ
C(CH
3
)
3
). ESI-MS: 518.08 [M þ H] . Anal. Calcd for C25
H
4
O
4
300 MHz) : 11.59 (s,1H, SO NH), 9.58 (s,1H, CH), 8.98e7.28 (m, 8H,
d
2
C, 58.07; H, 5.65; Cl, 6.86; N,10.84; O,12.38; S, 6.20; Found: C, 58.13;
H, 5.62; Cl, 6.83; N, 10.83; O, 12.36; S, 6.22.
ArH), 6.70 (s, 1H, CH), 2.42 (s, 3H, CH
3
), 2.23 (s, 6H, CH
3
). ESI-MS:
þ
483.81 [M þ H] . Anal. Calcd for C23
H
19ClN S: C, 57.20; H, 3.97;
4 4
O
Cl, 7.34; N, 11.60; O, 13.25; S, 6.64; Found: C, 57.10; H, 3.98; Cl, 7.36;
N, 11.62; O, 13.27; S, 6.65.
4
.3.10. 4-((6,8-Di-tert-butyl-4-chloro-2-oxo-2H-chromen-3-yl)
methyleneamino)-N-(4, 6-dimethylpyrimidin-2-yl)
benzenesulfonamide (5j)
4.3.17. 4-((4-Chloro-6-methyl-2-oxo-2H-chromen-3-yl) methylen-
Yellow crystals, yield 51%, m.p. 140e142 C; 1H NMR (DMSO-
ꢁ
eamino)-N-(5-methylisoxazol-3-yl) benzenesulfonamide (5q)
ꢁ
1
d
6
, 300 MHz)
CH), 8.94e7.33 (m, 8H, ArH), 2.24 (s, 6H, CH
C(CH ), 1.39 (s, 9H, C(CH
Calcd for C30 33ClN
1.01; S, 5.52; Found: C, 62.18; H, 5.68; Cl, 6.06; N, 9.60; O, 9.97; S,
.50.
d
: 11.76 (s, 1H, SO
2
NH), 9.63 (s, 1H, CH), 9.01 (s, 1H,
Orange crystals, yield 55%. m.p. 240e242 C; H NMR (DMSO-d
6
,
3
). 1.51 (s, 9H,
300 MHz) : 11.78 (s, 1H, SO NH), 9.51 (s, 1H, CH), 8.88e7.33 (m, 7H,
d
2
þ
3
)
3
3
)
3
). ESI-MS: 582.3 [M þ H] . Anal.
ArH), 6.23 (s, J ¼ 0.9 Hz, 1H, CH), 2.36 (s, 3H, CH
3
). 2.22 (s, 3H, CH
3
).
þ
H
4
O
4
S: C, 62.00; H, 5.72; Cl, 6.10; N, 9.64; O,
ESI-MS: 458.92 [M þ H] . Anal. Calcd for C21
H
16ClN S: C, 55.08;
3 5
O
1
5
H, 3.52; Cl, 7.74; N, 9.18; O, 17.47; S, 7.00; Found: C, 55.00; H, 3.54;
Cl, 7.76; N, 9.22; O, 17.50; S, 6.99.

10
Z.-C. Wang et al. / European Journal of Medicinal Chemistry 66 (2013) 1e11
4.3.18. 4-((4-Chloro-6-methyl-2-oxo-2H-chromen-3-yl)
served as reference inhibitor. After treatment for 48 and 72 h, cells
methyleneamino)-N-(4-methylpyrimidin-2-yl) benzenesulfonamide
were washed and fixed in PBS/ethanol (30/70). For cyto-
fluorometric examination, cells (10 cells/mL) were incubated for
30 min in PBS/Triton X100, 0.2%/EDTA 1 mM, and propidium iodide
(PI) (50 mg/mL) in PBS supplemented by RNase (0.5 mg/mL). The
number of cells in the different phases of the cell cycle was deter-
mined, and the percentage of apoptotic cells was quantified. Ana-
lyses were performed with a FACS Calibur (Becton Dickinson, Le
Pont de Claix, France). Cell Quest software was used for data
acquisition and analysis [35].
4
(
5r)
Yellow crystals, yield 74%. m.p. 273e275 C; H NMR (DMSO-d
00 MHz) : 11.76 (s, 1H, SO
ꢁ
1
6
,
3
1
(
d
2
NH), 9.64 (s,1H, CH), 9.97 (d, J ¼ 2.0 Hz,
H, CH), 8.42e7.32 (m, 7H, ArH), 6.78 (d, J ¼ 5.2 Hz, 1H, eCH), 2.44
þ
s, 3H, CH
3
), 2.27 (s, 3H, CH
3
). ESI-MS: 469.83 [M þ H] . Anal. Calcd
4 4
for C22H17ClN O S: C, 56.35; H, 3.65; Cl, 7.56; N, 11.95; O, 13.65; S,
6
.84; Found: C, 56.30; H, 3.63; Cl, 7.58; N, 11.97; O, 13.67; S, 6.85.
4.4. CA inhibition assay
4.7. Experimental protocol of docking study
An Applied Photophysics stopped-flow instrument has been
2
used for assaying the CA-catalyzed CO hydration activity [33].
Phenol red (at a concentration of 0.2 mM) has been used as indi-
cator, working at the absorbance maximum of 557 nm, with 10 mM
Molecular docking of compound 5a, 5c and 5d into the three
dimensional X-ray structure of human hCA II (PDB code: 3N4B) was
carried out using the Discovery Studio (version 3.5) as implemented
through the graphical user interface DS-CDOCKER protocol. The
three-dimensional structures of the aforementioned compounds
were constructed using Chem. 3D ultra 12.0 software [Chemical
Structure Drawing Standard; Cambridge Soft corporation, USA
Hepes (pH ¼ 7.5) as buffer, 0.1 M Na
2
SO
4
(for maintaining constant
hy-
ꢁ
the ionic strength), at 25 C, following the CA-catalyzed CO
2
dration reaction for a period of 10e100 s (the uncatalyzed reaction
needs around 60e100 s in the assay conditions, whereas the cata-
lyzed ones are of around 6e10 s). The CO concentrations ranged
2
from 1.7 to 17 mM for the determination of kinetic parameters. For
each inhibitor, tested in the concentration range between 0.01 nM
and 100 mM, at least six traces of the initial 5e10% of the reaction
have been used for determining the initial velocity. The uncatalyzed
rates were determined in the same manner and subtracted from the
total observed rates. Stock solutions of inhibitor (1 mM) were pre-
pared in distillededeionized water with 10e20% (v/v) DMSO (which
is not inhibitory at these concentrations), and dilutions up to
(
2010)], then they were energetically minimized by using MMFF94
with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of protein complex was retrieved from the RCSB Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound
waters and ligands were eliminated from the protein and the polar
hydrogen was added to the proteins. Molecular docking of all twenty
compounds was then carried out using the Discovery Stutio (version
.5) as implemented through the graphical user interface CDOCKER
protocol. CDOCKER is an implementation of a CHARMm based
molecular docking tool using a rigid receptor.
3
0
.01 nM were done thereafter with distillededeionized water. In-
hibitor and enzyme solutions were preincubated together for
5 min at room temperature prior to assay, in order to allow for the
formation of the EeI complex. The inhibition constants were ob-
tained by nonlinear least-squares methods using PRISM . The curve-
fitting algorithm allowed us to obtain the IC50 values, working at the
lowest concentration of substrate of 1.7 mM, from which K values
1
4
.8. 3D-QSAR
3
Ligand-based 3D-QSAR approach was performed by QSAR
software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The
training sets were composed of inhibitors with the corresponding
i
were calculated by using the ChengePrusoff equation. The catalytic
activity (in the absence of inhibitors) of these enzymes was calcu-
lated from LineweavereBurk plots, as reported earlier, and repre-
sents the mean from at least three different determinations. 38-42
Enzyme concentrations in the assay system were 7.3 nM for hCA II,
and 8.5 nM for hCA IX. Enzymes used here were recombinant ones,
prepared and purified as described earlier [34].
pIC50 values which were converted from the obtained IC50
(mM),
and test sets comprised compounds of data sets. All the definition
of the descriptors can be seen in the “Help” of DS 3.5 software and
they were calculated by QSAR protocol of DS 3.5. The alignment
conformation of each molecule was the one with lowest interaction
energy in the docked results of CDOCKER. The predictive ability of
3
D-QSAR modeling can be evaluated based on the cross-validated
correlation coefficient, which qualifies the predictive ability of the
models. Scrambled test (Y scrambling) was performed to investi-
gate the risk of chance correlations. The inhibitory potencies of
compounds were randomly reordered for 30 times and subject to
leave-one-out validation test, respectively. The models were also
validated by test sets, in which the compounds are not included in
the training sets. Usually, one can believe that the modeling is
4
.5. Cell proliferation assay
CCK-8 is much more convenient and helpful than MTT for
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 CCK-8 dye (Beyo-
timeInst Biotech, China) according to manufacture’s instructions.
2
reliable, when the R for test sets is larger than 0.6.
3
Briefly, 1e5 ꢃ 10 cells per well were seeded in a 96-well plate,
ꢁ
grown at 37 C for 12 h. Subsequently, cells were treated with the
Acknowledgments
target compounds at increasing concentrations in the presence of
1
0% FBS for 24 or 48 h. After 10 mL CCK-8 dye was added to each
ꢁ
The work was financed by a grant (No. J1103512) from National
well, cells were incubated at 37 C for 1e2 h and Plates were read in
a Victor-V multilabel counter (PerkineElmer) using the default
europium detection protocol. Percent inhibition or IC50 values of
compounds were calculated by comparison with DMSO-treated
control wells.
Natural Science Foundation of China and
a
grant (No.
CXLX12_0047) from Jiangsu Ordinary University Graduate Students
Scientific Research Innovation Plan of China.
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