Aryl azide-sulfonamide hybrids induce cellular apoptosis: synthesis and preliminary screening of their cytotoxicity in human HCT116 and A549 cancer cell lines

Article abstract of DOI:10.1007/s00044-019-02438-x

Simple, small, drug-like molecules bearing aryl azide and aryl sulfonamide moieties were designed and synthesized. The cytotoxic activity of these compounds was measured on colon cancer HCT116, lung cancer A549, and normal fibroblast cells F180 cell lines. All the synthesized compounds showed a significant cytotoxic activity below 100 μM in both HCT116 and A549 cells. Compounds 10e and 10f exhibited the most potent activity with IC₅₀ values of 2.20 and 6.27 μM on A549 and HCT116 cells, respectively. Also, compounds 10e and 10f showed significant tumor selectivity on HCT116 and A549 cell lines when compared with the reference cytotoxic agent staurosporine. This indicated the promising safety of these compounds on normal cells. In addition, flow cytometry studies showed that HCT116 cell lines treated with the most active compound 10f were arrested in the G2/M phase of the cell cycle. 10f boosted both early and late apoptosis at HCT116 cells. A hypothetical pharmacophore model was built using 14 reported potent carbonic anhydrase I inhibitors. The pharmacophoric study revealed that the tested sulfonamide derivatives 10e and 10f showed significant fitting on the pharmacophore query with reasonable RMSD values. Molecular docking study showed a chelation reaction with the key Zn atom, in addition to different hydrogen bonding, and van der Waals interactions with several important amino acids inside the CA Ι active site. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-019-02438-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02438-x
ORIGINAL RESEARCH
Aryl azide-sulfonamide hybrids induce cellular apoptosis: synthesis
and preliminary screening of their cytotoxicity in human HCT116
and A549 cancer cell lines
Ahmed El-Kardocy1 Muhamad Mustafa Esam R. Ahmed Samy Mohamady Yaser A. Mostafa5
2 ●
3 ●
4 ●
●
Received: 17 May 2019 / Accepted: 4 September 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Simple, small, drug-like molecules bearing aryl azide and aryl sulfonamide moieties were designed and synthesized. The
cytotoxic activity of these compounds was measured on colon cancer HCT116, lung cancer A549, and normal fibroblast
cells F180 cell lines. All the synthesized compounds showed a significant cytotoxic activity below 100 µM in both HCT116
and A549 cells. Compounds 10e and 10f exhibited the most potent activity with IC50 values of 2.20 and 6.27 µM on A549
and HCT116 cells, respectively. Also, compounds 10e and 10f showed significant tumor selectivity on HCT116 and A549
cell lines when compared with the reference cytotoxic agent staurosporine. This indicated the promising safety of these
compounds on normal cells. In addition, flow cytometry studies showed that HCT116 cell lines treated with the most active
compound 10f were arrested in the G2/M phase of the cell cycle. 10f boosted both early and late apoptosis at HCT116 cells.
A hypothetical pharmacophore model was built using 14 reported potent carbonic anhydrase I inhibitors. The
pharmacophoric study revealed that the tested sulfonamide derivatives 10e and 10f showed significant fitting on the
pharmacophore query with reasonable RMSD values. Molecular docking study showed a chelation reaction with the key Zn
atom, in addition to different hydrogen bonding, and van der Waals interactions with several important amino acids inside
the CA Ι active site.
Graphical Abstract
●
●
●
●
Keywords Sulfonamides Cytotoxicity Carbonic anhydrase Molecular docking Pharmacophoric study
*
Yaser A. Mostafa
y3abdelh@uwaterloo.ca
3
4
VACSERA, Cairo, Egypt
Faculty of Pharmacy, The British University in Egypt, Cairo-Suez
Desert Road, Al-Sherouk 11837, Egypt
1
2
Student Research Center, Faculty of Pharmacy, Assiut University,
Assiut 71515, Egypt
5
Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Deraya University, Minya, Egypt

Medicinal Chemistry Research
Introduction
of an aromatic or heteroaromatic sulfonamide moiety
(Gokcen et al. 2016; Eldehna et al. 2017). Also, several
It is well known that cancer cells respond to cancer ther-
apeutics through three complexed processes including
induction of apoptosis, regulation of cell cycle, and DNA
damage-repair processes (Hoeijmakers 2009). Despite a
large number of protocols used for the treatment of cancer,
there are still few tumor cells able to resist cancer ther-
apeutics leading to therapy impairment or even tumor
rebound. Unfortunately, using high doses of cancer ther-
apeutics to overcome such impairment or rebound resulted
in emerging severe or even life-threatening side effects (El-
Awady et al. 2016). Carbonic anhydrases (CAs) are one of
the metalloenzymes that are ubiquitously expressed in the
human body. CAs are zinc enzymes that catalyze the
reversible hydration of carbon dioxide to bicarbonate and a
proton. This simple reaction is crucial for several physio-
logical mechanisms either desirable such as electrolyte
secretion, bone resorption, pH regulation, biosynthetic
sulfonamide containing compounds were found to possess
potent anticancer and CA inhibitory activity, compounds 1–
3 (Fig. 1) (Salmon et al. 2012; Arslan et al. 2002; Alaoui
et al. 2017; Bagheri et al. 2014; Jain et al. 2010; Prasanna
Kumar et al. 2014; El-Din et al. 2015). Moreover, the lit-
erature survey showed that aryl azides 4–6 were widely
used as the foremost scaffold to design and synthesize novel
compounds with antiproliferative activity (Fig. 2) (Tanabe
et al. 2012; Nicolaus et al. 2010; da Cruz et al. 2016;
Thamilarasan et al. 2015; Lipeeva et al. 2015; Legigan et al.
2012; Pinney et al. 2000; Fu et al. 2017; Spletstoser et al.
2004).
The importance and the remarkable existence of CAs in
various sorts of tumors, and the induction of apoptosis by
aryl azides made them an excellent target for developing
new drug-like small molecules. Hence it is crucial to dis-
cover new successful candidates as therapeutics for the
treatment of cancer. In the present work, new azide-
sulfonamide hybrids (10a–f) were designed and synthe-
sized Scheme 1. The sulfonamide derivatives were pre-
liminarily tested and evaluated for their cytotoxic activity
against two different cancer cell lines; colon cancer
(HCT116) and non-small cell lung cancer (A549) cells,
which were deliberately preferred because CA isozymes
were found to be notably expressed in colon cancer and
lung cancer (Bekku et al. 2000; Wang et al. 2016). More-
over, flow cytometry and cell cycle analysis were imple-
mented on the most active derivatives. In addition, for
defining the potential mechanism of action, the most active
derivatives were chosen for fitting on the pharmacophoric
query and extensive docking studies.
reactions, and calcification which need HCO as a substrate
3
or undesirable such as tumorigenesis. The CA isozymes are
found to be interesting therapeutic targets where their
inhibition could be utilized to treat a variety of diseases
including cancer, oxidative stress, epilepsy, edema, and
osteoporosis (Salmon et al. 2012; Gulcin and Beydemir
2
2
013; Carradori et al. 2015; Huang et al. 2017; Bruno et al.
016). Human CAs I and II are cytosolic enzymes that are
prevalent throughout the human body (Arslan et al. 2002).
Moreover, CAs IX and XII are human-associated CA iso-
forms, which are having an extracellular active site, they
were found to be a marker for many hypoxic tumors
(
Barathova et al. 2008; Gondi et al. 2013). Interestingly,
literature survey revealed that the vast majority of CAs
inhibitors were characterized by being small molecules with
promising anticancer activities. Moreover, several studies
revealed that there are three major classes of carbonic
anhydrase inhibitors (CAIs): sulfonamides, coumarins, and
polyamines (Riafrecha et al. 2014). Since the discovery of
the sulfonamide inhibitors of CA isozymes, several pow-
erful inhibitors of CAs were designed and synthesized with
a common structural pharmacophore, which is the presence
Results and discussion
Chemistry
The synthesis of target compounds (10a–f) is outlined in
Scheme 1. The key intermediate 9 was obtained after four
Fig. 1 Chemical structures of carbonic anhydrase inhibitors containing sulfonamide moiety

Medicinal Chemistry Research
Fig. 2 Chemical structures of
aryl azide derivatives possessing
anti-proliferative activity
Scheme 1 Synthetic approach of
compounds (10a–f). I Na2CO3,
NaNO2, HCl, 0–5 °C; II (a)
NaN3; (b) SOCl2, DMF, heat; III
appropriate aniline, Pyridine, rt,
1
6 h
Table 1 IC50 (µM) values for the
tested compounds against colon
cancer cells (HCT116), lung
cancer cells (A594), normal
human fibroblast cells (F180),
and tumor selectivity (TS)
IC50 (μM)
Compound
R1
R2
HCT116 (A) A549 (B)
F180 (C)
TS (C)/(A) TS (C)/(B)
1
2
10a
H
H
13.48 ± 0.44 39.21 ± 1.18 17.22 ± 0.29 1.28
28.66 ± 0.94 7.31 ± 0.22 39.32 ± 1.18 1.37
11.48 ± 0.37 12.07 ± 0.42 15.26 ± 0.55 1.33
0.44
5.38
1.26
2.31
10b
10c
10d
10e
10f
H
Br
Cl
H
H
OCH3 34.96 ± 1.15 16.37 ± 0.52 37.85 ± 0.63 1.08
H
NO2
H
52.99 ± 1.74
2.20 ± 0.07 53.90 ± 0.18 1.02
24.5
NO2
6.27 ± 0.20 34.59 ± 0.93 54.52 ± 1.27 8.69
1.57
Positive control
Staurosporine
7.92 ± 0.26 10.55 ± 0.34
8.26 ± 0.16 1.04
0.78
IC50 resembles the concentration required to inhibit tumor growth by 50%
steps with a high yield (76%) without further purification.
The detailed synthesis procedures, as well as the char-
acterization data, are represented in the experimental part.
The target sulfonamides (10a–f) were synthesized in a
moderate to a high yield from the main intermediate 9
upon its reaction with different anilines in presence of
pyridine as a solvent at room temperature (Table 1). The
H, N, and S microanalyses of all synthesized compounds
were determined and were within ±0.4 of theoretical
values.
Biological investigation
In vitro cytotoxic activities
1
H NMR spectra of compounds (10a–f) revealed a com-
mon broad signal of sulfonamide group NH; around
In vitro cytotoxic activity of the synthesized sulfonamides
10a–f was tested using the MTT assay on the viability of
two different human cancer cells (colon cancer HCT116 and
non-small cell lung cancer A549 cell lines (Table 1).
Staurosporine served as a positive control. Because it is a
matter of concentration dependence, the percentages of
viable cells declined substantially upon increasing the
concentrations of the sulfonamide derivatives. After 48 h of
1
0.30 ± 0.2 ppm except for the two nitro derivatives,
Compounds 10e and 10f, which were downfield shifted to
0.91 ppm because of the strong electron-withdrawing
1
1
3
effect of the nitro group. In addition, the C NMR
spectra of the sulfonamides (10a–f) displayed common
signals around 128 and 121 ppm corresponding to CHs
of p-azido-benzene sulfonamide aromatic ring. The C,

Medicinal Chemistry Research
incubation, cell viability was analyzed at five different
concentrations of 100, 25, 6.25, 1.56, and 0.39 μM. The
HCT116 cell viability was ~73% at a concentration of
0
.39 μM when treated with the m-NO sulfonamide deri-
2
vative 10f (black), the viability percentages significantly
lessened upon increasing the concentration to reach 28% at
1
00 μM (Fig. 3). Compared with the synthesized sulfona-
mides, 10f showed the highest ability to inhibit HCT116
growth with an IC50 value of 6.26 μM, while the reference
staurosporine showed an IC50 value of 9.92 µM. While the
sulfonamide derivatives 10a (red) and 10c (green) exhibited
a remarkable drop in the viability percentages from about
Fig. 3 Effect of the sulfonamide derivatives (10a–f) on viability of
HCT116 colon cancer cells
7
4 and 81% at 0.39 μM to 35 and 30% at 100 μM,
respectively. 10a and 10c showed IC50 values of about
.7–1.5-folds lower in the inhibitory activity than the
1
reference staurosporine, respectively. Regarding A549 cell
lines, 10e (orange) effectively decreased cell viability from
6
4
2
1% at a concentration of 0.39 μM to 29% at 100 μM (Fig.
). The p-NO sulfonamide 10e exhibited an IC50 value of
2
.20 μM, which is markedly lower than that of Staur-
osporine (IC = 10.55 μM). Moreover, the p-Br sulfona-
5
0
mide derivative 10b significantly lessened cell viability
from 70% at a concentration of 0.39 μM to 35% at 100 μM.
Fig. 4 Effect of the sulfonamide derivatives (10a–f) on viability of
A549 lung cancer cells
1
0b showed an IC50 value of 7.31 μM which is also better
than that of staurosporine. Furthermore, compounds 10c
and 10d showed comparable inhibitory activity to staur-
osporine with IC50 values of 12.07 and 16.36 μM, respec-
tively. Compounds 10f and 10a were less active in
comparison to their congeners or even to the reference
compound on A549 cells with IC50 values of 34.59 and
than staurosporine. Also, the sulfonamide 10e was 31-fold
more selective towards A549 cancer cells than the reference
drug. These strong findings revealed that these two potent
sulfonamides have high selectivity towards cancer cell lines
which could be used as possible anticancer drug candidates.
3
9.21 μM, respectively.
In vitro cytotoxicity towards normal F180 cells
Cell cycle analysis by flow cytometry
Cytotoxic activities of the synthesized sulfonamides were
tested on the non-tumor normal fibroblast cells F180 (Table
Apoptosis induction represents one of the most successful
approaches for the development of cancer therapy (Hu and
Kavanagh 2003). In addition, several sulfonamide contain-
ing compounds were found to induce apoptosis and arrest
cell cycle at different phases (Sabt et al. 2018; Bagheri et al.
2014). Consequently, cell cycle analysis was performed on
HCT116 cells for 10f to determine whether the cytotoxic
activity resulted from arresting the cell cycle. The m-NO2
sulfonamide 10f which possessed the best IC50 concentra-
tion and showed the best TS on HCT116 cancer cells,
affected significantly the cell cycle distribution at its IC50
value (6.26 μM). The results showed that the cells treated
with 10f induced a significant growth arrest at G2/M phase
(Fig. 5). Compared with 6.13% for the untreated HCT116
cells, compound 10f arrested 26.45% of the cells (Table 2).
Moreover, 10f induced pre-G1 apoptotic cell death with
13.54% compared with the negative control cells which
showed only 1.39%. These results showed that the
1
). Selective cytotoxicity is the main problem of anticancer
drugs in the market. Subsequently, if the compound has
selectivity towards cancer cells, that compound will inevi-
tably be evaluated as a possible anticancer drug candidate
for further experiments. To investigate whether the prepared
compounds have such selectivity or not, tumor selectivity
(
TS) values were calculated. First TS (TS ) calculations
1
were performed by dividing the IC50 values towards normal
cells F180 to the IC50 values towards HCT116 cancer cells
(
TS = column C/column A) (Table 1). Second TS (TS )
1
2
calculations were made by dividing the IC50 values towards
normal cells F180 to the IC50 values towards A549 cancer
cells (TS = column B/column A). When the calculated TS
2
values were compared with that of the reference drug, it was
obvious that the tested compounds were more selective
towards cancer cells than staurosporine. Compound 10f was
eight times more selective towards HCT116 cancer cells

Medicinal Chemistry Research
Fig. 5 Cell cycle analysis of HCT116 cells treated with Annexin PI; a Untreated cells, b cells treated with 10f
Table 2 Percentages of arresting cell cycle at different phases
2
017; Gul et al. 2016; Kocyigit et al. 2016, 2017; Gul
Compound
G0-G1
S
G2/M
Pre-G1
et al. 2017; Taslimi et al. 2016; Ceylan et al. 2017; Budak
et al. 2017; Mete et al. 2016; Boztas et al. 2014; Kucu-
koglu et al. 2016). The model included hydrophobic
domain (Hyd), aromatic center domain (Aro), hydrogen
bond acceptors domain (Acc), hydrogen bond donor
domain (Don), charged or ionizable groups (Cat & Ani),
and/or metal ligator domain (ML) united together with
geometrical constraints, such as distances, angles, and
dihedral angles. The most active compounds 10e and 10f
were selected for the pharmacophore study. The tested
compounds 10e and 10f showed good fitting in the phar-
macophoric query with remarkable RMSD values of 7.336
and 0.7307, respectively.
The initial pharmacophoric query was executed and it
was formed of five features, as given in Table 3 and
demonstrated graphically in Fig. 7a. The tested sulfona-
mide derivatives 10e and 10f showed complete fitting on
the query (Fig. 7b, c, respectively) compared with the
reference acetazolamide (Fig. 7d). This emphasized the
remarkable CAI inhibitory activity of the synthesized
compounds. Also, the pharmacophore model revealed the
ability of the azide group to interact with Zn atom inside
the CA active site.
Control
49.75
37.26
44.12
36.29
6.13
1.39
1
0f
26.45
13.54
sulfonamide derivative 10f was able to arrest cells at G2/M
phase and to induce apoptosis.
Induction of cellular apoptosis
The active compound 10f was tested for its potency to
induce apoptosis in HCT116 cell lines by staining with
Annexin V-FITC and propodium iodide. After treatment
with its IC50 concentration, the obtained data showed a
considerable ability of 10f to initiate cellular apoptosis and
to disperse cellular integrity (Fig. 6). 10f elevated the per-
centage of early apoptotic cells up to 6.49% compared with
0
.76% of the negative control (the lower right quadrant).
Furthermore, 10f dispersed cellular integrity by raising the
percentage of late apoptotic cells to 5.33%, while the
negative control possessed only 0.27% (the upper right
quadrant). These results implied that the sulfonamide deri-
vative exhibited most of its cytotoxicity by inducing cellular
apoptosis.
Molecular docking study
Pharmacophore modeling
To rationalize the significant effect of the synthesized
sulfonamides, molecular modeling was performed to
illustrate the interactions of the most active compounds
within the CA Ι binding site. The docked compounds
showed coordinate bonds with zinc atom Table 4. More-
over, several hydrogen-bonding interactions and pi-
interactions were observed with key amino acids. Strong
hydrophobic interactions were further observed with
A pharmacophoric model was created to affirm the CA Ι
inhibitory mechanism of the synthesized compounds. The
pharmacophore query was performed using a training set
of 14 reported active CA Ι inhibitors. The most active
derivative was selected from each series (Bulut et al. 2017;
Turkan et al. 2019; Yıldırım et al. 2015; Gulçin et al.

Medicinal Chemistry Research
Fig. 6 Contour diagram of Annexin V/PI flow cytometry: a Untreated cells, b cells treated with 10f
Table 3 Pharmacophoric and structure features of the training set
Conclusion
Pharmacophoric features
Structural features
In conclusion, we reported the cytotoxic effects of certain
new azidosulfonamide derivatives in colon cancer cells
F1: Aro/Hyd
Substituted phenyl group
Substituted phenyl group
SO2NH
F2: Aro/Hyd
(HCT116), lung cancer cells (A549), and normal fibroblast
F3: (Acc/Don/ML)
F4: (Acc/ML)
F5: (Acc/ML)
cells (F180). Compounds 10e and 10f showed significant
cytotoxic effects in both A549 and HCT116 (IC = 2.20
Azide group
5
0
Azide group
and 6.26 μM, respectively), with powerful TS values. Pre-
liminary mode of action studies revealed that compound 10f
halted cell cycle progression at G2/M phase and induced
apoptosis in HCT116 cells. Pharmacophoric features were
recognized and the prepared compounds revealed a
remarkable fitting on the created model. Molecular docking
study showed a higher fitting of 10e and 10f on CAΙ active
site. This would warrants additional testing in the in vivo
cancer models.
Leu131, Asn69, His67, His200, phe91, Gln92, and
Leu198. The binding mode of 10e (cyan) which exhibited
the best inhibitory activity on A549 cells, showed a
coordinate bond between the azide nitrogen and zinc atom
with interaction energy of −0.5 kcal/mol (Fig. 8). Also,
the azide group showed a hydrogen bond with Thr199
(
−1.2 kcal/mol). The sulfonamide oxygen showed a
hydrogen bond with His67 (−1.4 kcal/mol). The role of
the p-niro group was observed by forming a hydrogen
bond with Ala132 with interaction energy of −0.3 kcal/
mol. Moreover, several hydrophobic interactions were
observed with Phe91, His94, Pro202, Ala135, Lue198,
and Leu131 amino acids. Compound 10f (magenta) which
possessed the best cytotoxic activity on HCT116 cells,
showed a coordinate bond between zinc atom and the
azide group with interaction energy of −0.3 kcal/mol (Fig.
Materials and methods
Chemistry
All starting materials and reagents were obtained from
Alpha Aesar Chemical Company and Sigma-Aldrich Che-
micals Company. Pyridine was distilled from KOH pellets,
thionyl chloride was freshly distilled under reduced pres-
sure, and toluene was distilled over CaH under reduced
pressure. Silica gel chromatography was performed using
9
). Also, the azide group made additional interactions
showing two hydrogen bonds with Thr199 and Trp209
amino acids. The nitro group served as a hydrogen bond
acceptor with His67 and Asn69 (−0.2 and −1.4 kcal/mol,
respectively). H–pi interaction was observed between the
phenyl sulfonamide ring and Leu198. 10f showed several
hydrophobic interactions with Gln92, His200, Asn69,
Leu198, and Phe91 amino acids.
TM
1
13
silica gel obtained from Fluka . Most of H and
spectra were recorded on a JEOL ECA-500 II spectrometer.
C
For NMR spectra obtained using DMSO-d as the solvent,
6
1
chemical shifts (δ) for H NMR spectra are reported relative
13
to internal Me Si (δ 0.0 ppm), and chemical shifts for
C
4
spectra are relative to the residual solvent peak (δ 39.5 ppm,

Medicinal Chemistry Research
Fig. 7 a Pharmacophoric query showing pharmacophoric features, b fitting of 10e to pharmacophore query, c fitting of 10f to pharmacophore
query, and d fitting of the reference compounds acetazolamide
Table 4 Energy scores (kcal/mol) and binding features for the top
potent compounds 10e and 10f with carbonic anhydrase Ι binding site
Zn
Compound Energy score Ligand-receptor interactions
(
S) (kcal/mol)
Residue Type
Length (Å)
1
0e
0f
−4.57
Zn Coordinate bond 3.13
Thr199 Hydrogen bond 3.91
Ala132 Hydrogen bond 3.68
His67
Zn
Hydrogen bond 3.28
Coordinate bond 3.15
1
−4.15
Thr199 Hydrogen bond 3.29
Trp209 Hydrogen bond 3.75
Asn69 Hydrogen bond 3.26
Fig. 9 Docking pose of 10f (magenta) with CAΙ active site (PDB
code: 4WR7)
His67
Hydrogen bond 3.75
3.70
Leu198 H–Pi
central peak). Microanalyses, determined for C, H, and
other elements, were within ± 0.4 of theoretical values.
Melting points were determined on a Fisher-Johns melting
point apparatus and are uncorrected.
Zn
Synthesis of 4-azidobenezenesulfonyl chloride 9
To a solution of sodium carbonate (5.8 g) in H O (200 ml),
2
sulfanilic acid 7 (250 g) was added portion-wise with vig-
orous stirring. To the resultant solution, sodium nitrite
(8.4 g) was added and stirring was continued till complete
dissolution of sodium nitrite. This step was followed by a
portion-wise addition of a solution of concentrated HCl
(
14 mL) in H O (30 ml) with vigorous stirring in ice bath to
Fig. 8 Docking pose of 10e (cyan) with CAΙ active site (PDB
code: 4WR7)
2
maintain reaction between 0 and 5 °C. A white precipitate

Medicinal Chemistry Research
was obtained 8, separated by suction and washed with about
0 ml of cold water. The precipitate solid was then dis-
persed in 40 ml of cold water, whereupon a solution of
sodium azide (8 g in 20 ml water) was added portion-wise
116.3; Anal. Calcd. for C H BrN O S (351.963): C, 40.81;
H, 2.57; N, 15.86; S, 9.08; Found: C, 41.90; H, 2.84; N,
15.73; and S, 9.21.
1
2
9
4
2
8
till evolution of N ceased. After that sodium chloride (30 g)
4-Azido-4-chlorophenyl-benzenesulfonamide 10c Puri®-
cation was achieved using flash chromatography (ethyl
2
was added to salt out. The resulting precipitate was filtered
off and dried in vacuum to yield a yellowish-white pre-
cipitate. 5 g of the resultant azido derivative was dissolved
in thionyl chloride (20 ml) and three drops of DMF and
heated till boiling for 30 min. After completion of the
reaction, the excess thionyl chloride was removed under
vacuum and residue was extracted with diethyl ether
acetate/hexane, 4:6) which provided 10c as a buff solid
1
(71%). mp 122–124 °C; H NMR (DMSO-d , 500 MHz), δ
6
10.42 (s, 1H, –SO NH), 7.73 (app. ddd, J = 2, 3 and 5 Hz,
2
2H, Ar–H), 7.30–7.24 (m, 4H, Ar–H), 7.08 (m, 2H, Ar–H);
1
3
C NMR (DMSO-d , 125 MHz) δ 144.3, 136.6, 135.1,
6
129.2 (2CH ), 128.7 (2CH ), 128.3, 121.7 (2CH ), 119.9
Ar
Ar
Ar
(
100 ml). The etherial extract was evaporated under vacuum
(2CH ); Anal. Calcd. for C H ClN O S (308.0135): C,
Ar 12 9 4 2
to afford 4-azidobenzene sulfonyl chloride 9 as a yellowish-
white solid (4.1 g, 76%). mp 59–61 °C; H NMR (DMSO-
46.68; H, 2.94; N, 18.15; S, 10.39 Found: C, 47.04; H,
3.17; N, 18.41; and S, 10.20.
1
d , 500 MHz) δ 7.04–7.07 (m, 2H, Ar–H), 7.60–7.63 (m,
6
1
3
2
1
H, Ar–H); C NMR(DMSO-d , 125 MHz) δ 144.9, 139.5,
4-Azido-4-methoxyphenyl-benzenesulfonamide 10d Puri-
fication was achieved using flash chromatography (ethyl
acetate/hexane, 3:7) which provided 10d as a white solid
6
27.5 (2CH ), 118.4 (2CH ).
Ar
Ar
1
General procedure for synthesis of 4-azido-(un)substituted-
(63%). mp 74–75 °C (Mirian et al. 2011); H NMR
phenyl-benzenesulfonamides, compounds 10a–f
(DMSO-d , 500 MHz), δ 10.15 (s, 1H, SO NH), 7.77 (app.
6
2
dd, J = 2 & 4 Hz, 2H, Ar–H), 7.68–7.65 (m, 2H, Ar–H),
6.99 (m, 2H, Ar–H), 6.75 (m, 2H, Ar–H), 3.80 (s, 3H,
To a solution of 9 (0.01 mmol), appropriate anilines were
added (0.01 mmol) in dry pyridine (1 ml) at 0 °C. After
addition, the reaction was stirred for 16 h at room tem-
perature, and then pyridine was azeotropically removed
with toluene under vacuum. The residue was dissolved in
ethyl acetate, washed with water and brine, and then dried
with Na SO , filtered, and concentrated under vacuum.
1
3
OCH3); C NMR (DMSO-d , 125 MHz) δ 149.9, 144.3,
6
136.6, 135.1, 129.2 (2CH ), 128 (2CH ), 121.7 (2 CH ),
Ar
Ar
Ar
115.9 (2CH ); Anal. Calcd. for C H N O S (304.063): C,
Ar
13 12
4
3
51.31; H, 3.97; N, 18.41; S, 10.54; Found: C, 51.09; H,
3.89; N, 18.63; and S, 10.48.
2
4
4-Azido-4-nitrophenyl-benzenesulfonamide 10e Puri®ca-
4
-Azido-phenyl-benzenesulfonamide 10a Purification was
tion was achieved using flash chromatography (ethyl acet-
achieved using flash chromatography (ethyl acetate/hexane,
ate/hexane, 1:9) which provided 10e as a yellow solid
1
3
9
5
:7) which provided 10a as a faint buff solid (78%). mp
4–96 °C (Mirian et al. 2011); H NMR (DMSO-d6,
(81%). mp 104–105 °C; H NMR (DMSO-d , 500 MHz), δ
6
1
10.93 (s, 1H, SO NH), 8.12–8.10 (m, 2H, Ar–H), 7.93–7.84
2
00 MHz), δ 10.36 (brs, 1H, SO NH), 8.14 (app. dd, 2H,
(m, 2H, Ar–H), 7.27 (m, 2H, Ar–H), 6.58 (app. dd, J = 2
2
1
3
J = 2.5 and 2 Hz, Ar–H), 7.91 (app. t, 2H, J = 2 and 7 Hz,
Ar–H), 7.22 (app. dd, J = 5 and 7 Hz, 2H, Ar–H);
and 5.5 Hz, 2H, Ar–H); C NMR (DMSO-d , 125 MHz) δ
6
155.8, 144.9, 142.2, 142.7, 128.9 (2CH ), 126.5 (2CH ),
Ar
Ar
1
3
7
.17–7.04 (m, 2H, Ar–H), 6.59–6.57 (m, 1H, Ar–H);
C
120.3 (2CHAr), 112.5 (2CHAr). Anal. Calcd. for
C H N O S (319.0375): C, 45.14; H, 2.84; N, 21.93; S,
NMR (DMSO-d , 125 MHz) δ 145.1, 143.8, 138, 128.9
6
12
9
5
4
(
2CH ), 126.5 (2CH ), 120.1 (2CH ), 118.3 (CH ),
10.04; Found: C, 45.39; H, 2.97; N, 22.23; and S, 9.89.
Ar
Ar
Ar
Ar
1
16.0 (2CH ). Anal. Calcd. for C H N O S (274.0524):
Ar 12 10 4 2
C, 52.54; H, 3.67; N, 20.43; S, 11.69; Found: C, 52.31; H,
4-Azido-3-nitrophenyl-benzenesulfonamide 10f Puri®ca-
tion was achieved using flash chromatography (ethyl acet-
3
.90; N, 20.65; and S, 11.47.
ate/hexane, 1:9) which provided 10f as a yellow solid
1
4
-Azido-4-bromophenyl-benzenesulfonamide 10b Puri®-
(76%). mp 98–100 °C; H NMR (DMSO-d , 500 MHz), δ
6
cation was achieved using flash chromatography (ethyl
10.93 (s, 1H, SO NH), 7.92 (app. t, J = 2.5 Hz, 1H, Ar–H),
2
acetate/hexane, 4:6) which provided 10b as a brown solid
7.88–7.86 (m, 1H, Ar–H), 7.80–7.78 (m, 1H, Ar–H),
1
13
(
66%). mp 112–114 °C; H NMR (DMSO-d , 500 MHz), δ
7.55–7.49 (m, 2H, Ar–H), 7.28–7.23 (m, 3H, Ar–H);
C
6
1
2
0.44 (s, 1H, SO NH), 7.73 (app. dd, J = 2.5 and 8.5 Hz,
NMR (DMSO-d , 125 MHz) δ 150.1, 148.2, 144.8, 139,
2
6
H, Ar–H), 7.41 (m, 2H, Ar–H), 7.25 (m, 2H, Ar–H), 7.03
130.9, 128.8 (2CH ), 125.5, 120.1 (2CH ), 109.9, 107.1
Ar
Ar
13
(
(
(
apparent ddd, J = 2, 2.5 and 4.5 Hz, 2H, Ar-H); C NMR
DMSO-d , 125 MHz) δ 144.3, 137.0, 135.1, 132.1
2CH ), 128.7 (2CH ), 121.9 (2CH ), 119.9 (2CH ),
(C ); Anal. Calcd. for C H N O S (319.0375): C, 45.14;
Ar 12 9 5 4
H, 2.84; N, 21.93; S, 10.04; Found: C, 45.43; H, 3.06; N,
22.07; and S, 9.93.
6
Ar
Ar
Ar
Ar

Medicinal Chemistry Research
Biology
iodide (PI) Flow Cytometry Kit for cell cycle analysis
®
(Annexin V Apoptosis Detection Kit ) according to the
Cell culture
manufacturer’s instructions. Data analysis and nuclear
morphology of apoptotic cells were examined using BD
FACS Calibur. PI histograms of normal and treated cells
with the cell count on the y-axis and the PI fluorescence
intensity on the x-axis were recorded. Also, the cell cycle
arrest caused by compounds (10a–f) in a concentration-
dependent manner in HCT116 cells was examined.
®
The pure cultures of A549 (ATCC CCL-185) (Lung cancer
cell line) and HCT116 (ATCC CCL-247) (Colon cancer cell
line) were obtained from (ATCC ) American Type Culture
Collection, F180 (normal fibroblast cells) was obtained from
®
®
VACSERA. The cells were grown and maintained in DMEM
®
(Dulbecco’s Modified Eagle’s Medium -ATCC 30-2002),
TM
supplemented with 10% FBS (HyClone ), 10 µg/ml Insulin
Pharmacophore study
®
(
Sigma ), and 1% penicillin-streptomycin to prevent the
bacterial contamination and incubated at 37 °C in a humidi-
Pharmacophore modeling and visualization processes were
performed using Molecular Operating Environment (MOE)
2014.09 software (Chemical Computing Group, Montreal,
QC, Canada). The database of the most active CA І inhi-
bitors was collected. The energy of the structures was
minimized using MMF94FX forcefield with gradient RMS
of 0.001 kcal/mol. Structures were aligned. The energy
cutoff was 10 kcal/mol, while the conformational search
was stochastic. The radius of the created pharmacophoric
features were from 1.2–1.5 Å. The lowest RMSD values
were selected for every compound in the pharmacophoric
database
fied atmosphere having 5% CO for 24 h. The entire plate was
2
observed after 24 h of treatment in an inverted phase-contrast
tissue culture microscope, and after treatment of cells with
serial concentrations of the compound to be tested in DMSO
was incubated for 48 h at 37°C, and then plates were exam-
ined and processed for the MTT assay.
MTT assay for in vitro cytotoxicity
The MTT method of monitoring in vitro cytotoxicity is well
suited for use with multiwell plates. The sample content in
the wells was removed after 48 h of the incubation period
and reconstitutes each vial of MTT (M-5655) to be used
with 3 ml of medium or balanced salt solution without
phenol red and serum. Add reconstituted MTT in an amount
equal to 10% of the culture medium volume. Return cul-
tures to the incubator for 2–4 h depending on cell type and
maximum cell density. An incubation period of 2 h is
generally adequate but may be lengthened for low cell
densities or cells with lower metabolic activity. The
supernatant was then removed and an equal amount of MTT
solubilization solution (M-8910; DMSO), Sigma-Aldrich,
USA) was added, and then the wells were mixed (in a
gyratory shaker) in order to enhance solubilization of the
insoluble formazan crystals. The cells were subjected to
different treatments after 24 h of seeding. The cells were
then incubated for 48 h with staurosporine as a positive
control, test drugs or vehicle (DMSO). Spectro-
photometrically measurement of absorbance at a wave-
length of 570 nm using Microplate reader, and the
percentage viability was calculated by using the formula: %
Cell viability = [(OD of treated)/(OD of control)] × 100, the
plot of % cell viability versus log concentrations was used
to calculate the concentration of compounds in micromoles
which reduce the viable cell number of by 50% (IC ).
Docking study
Molecular modeling and visualization processes were per-
formed within the CA
Ι active site using MOE
2014.09 software (Chemical Computing Group, Montreal,
QC, Canada). The co-crystal structure was retrieved from
the RCSB Protein Data Bank (PDB code 4WR7). The
compounds were prepared with the standard protocol
designated in MOE 2014.09. The energy of the docked
structures was minimized using MMF94FX forcefield with
gradient RMS of 0.001 kcal/mol. then the protein structure
was prepared by using the MOE LigX protocol. To validate
the docking study at the CA Ι active site, the native ligand
acetazolamide was re-docked into the binding site using the
same set of parameters as described above. The RMSD of
the best-docked pose was 0.6243 Å, thus validating the
docking using MOE. The ligands were then docked in the
binding site using the triangle matcher placement method.
Refinement was carried out using Forcefield and scored
using the Affinity dG scoring system. The resulting docking
poses were visually inspected, and the pose of the lowest
binding free energy value was considered.
5
0
Acknowledgements The authors extend their appreciation to faculty of
Pharmacy, the British University in Egypt and the Pharmaceutical
Organic Chemistry Department, Faculty of Pharmacy, Assiut University
for their support to synthesis these azide-sulfonamides and perform the
preliminary cytotoxic studies. Special thanks to Assoc. Prof. Ahmed S.
Aboraia for helping in performing the pharmacophoric study.
Cell cycle analysis (assessment of apoptosis)
The apoptotic effect of synthesized azido sulfonamides,
Compounds (10a–f), was analyzed by ab139418 Propidium

Medicinal Chemistry Research
Compliance with ethical standards
redox centres, and their mechanistic insights. Eur J Med Chem
1
22:1–16
El-Awady RA, Semreen MH, Saber-Ayad MM, Cyprian F, Menon V,
Al-Tel TH (2016) Modulation of DNA damage response and
induction of apoptosis mediates synergism between doxorubicin
and a new imidazopyridine derivative in breast and lung cancer
cells. DNA Repair 37:1–11
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
El-Din MMG, El-Gamal MI, Abdel-Maksoud MS, Yoo KH, Oh C-H
(2015) Synthesis and in vitro antiproliferative activity of new
1
,3,4-oxadiazole derivatives possessing sulfonamide moiety. Eur
J Med Chem 90:45–52
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