Design, synthesis, molecular modeling, and biological evaluation of sulfanilamide-imines derivatives as potential anticancer agents

Article abstract of DOI:10.1007/s00210-013-0883-y

A series of sulfanilamide Schiff base derivatives (1 to 15) have been designed as potential antitubulin agents depending on the chemical structures of combretastatine A-4 and isoquinoline sulfamate (antimitotic agents under investigation). The designed co

Full text of DOI:10.1007/s00210-013-0883-y

Naunyn-Schmiedeberg's Arch Pharmacol
DOI 10.1007/s00210-013-0883-y
ORIGINAL ARTICLE
Design, synthesis, molecular modeling, and biological
evaluation of sulfanilamide-imines derivatives as potential
anticancer agents
Sofian S. Mohamed & Abdalkarem R. Tamer & Salah M. Bensaber &
Mousa I. Jaeda & Nouri B. Ermeli & Aemen Ali Allafi & Ibrahim A. Mrema &
Mabrouk Erhuma & Anton Hermann & Abdul M. Gbaj
Received: 1 March 2013 /Accepted: 14 May 2013
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract A series of sulfanilamide Schiff base derivatives
(1 to 15) have been designed as potential antitubulin agents
depending on the chemical structures of combretastatine A-
4 and isoquinoline sulfamate (antimitotic agents under in-
vestigation). The designed compounds were synthesized by
microwave chemical synthesis, their purity was confirmed
by melting point and HPLC and chemical structures were
potentially constitute an interesting novel class of anticancer
agents, which deserve further studies.
.
.
.
Keywords Schiff base Sulfanilamide Imines Antitumor
.
.
.
.
activities Cellcountingassay Microtubules Betatubulin
.
Microtubules targeting drugs Colchicine binding site
1
determined by FT-IR, UV, and H and 13C-NMR spectro-
scopic techniques. The synthesized compounds have been
docked in the colchicine binding site of β-tubulin using
molecular modeling programs and the antitumor activities
were screened on human breast and lung cancer cells by cell
counting assay. Some tested compounds showed potent and
selective activity against breast cancer (MCF-7) with IC50
range of 90 to 166 μM. With regarding broad-spectrum
activity, compounds 4, 8, and 13 have shown potent
antitumor activity against human breast and human lung
cells with IC50 range of 96 to 140 μM. The obtained results
suggest that the sulfanilamide Schiff base derivatives might
Introduction
The term “cancer” comprises a large group of diseases that can
affect almost any part of the body and is characterized by rapid
creation of abnormal cells that grow beyond their usual
boundaries (Amvrosieva et al. 2006; Al Rashood et al. 2006;
Eckhardt 2002; Medina et al. 1998). Cancer is the leading
cause of death worldwide, as it accounted for 7.6 million
deaths in 2008 and is projected to continue rising with an
estimated 11 million deaths in 2030 (Asai et al. 2012). Many
cancer patients have received chemotherapeutic agents during
the course of their disease, a treatment based on cell destruc-
tion by growth interruption. Antimitotic agents constitute an
important class of the current anticancer chemotherapeutic
agents that act by preventing polymerization or depolymeri-
zation of microtubules (motor of mitotic stage of cell cycle) by
binding to its tubulin subunits (Desai and Mitchison 1997;
Gadde and Heald 2004; Prota et al. 2013). The vital role of
tubulin in the cell division cycle together with the fact that
aberrant cell division is the feature of cancer has made tubulin
and microtubules prime targets for cancer therapy. Tubulin
contains three main sites for attacks, namely vinca alkaloids
binding sites, taxane binding sites, and the colchicine binding
site (Chang et al. 2004; Pfisterer et al. 2010). A number of
compounds react in the same manner like colchicine as
combretastatine A-4, indanocine, T138067, and isoquinoline
:
:
:
S. S. Mohamed A. A. Allafi M. Erhuma A. M. Gbaj
Department of Genetics, National Medical Research Centre,
Zawia Z16, Libya
N. B. Ermeli
Department of Natural Products, Faculty of pharmacy,
University of Tripoli, Tripoli M16, Libya
:
:
:
:
A. R. Tamer S. M. Bensaber M. I. Jaeda I. A. Mrema
A. M. Gbaj (*)
Department of Medicinal Chemistry, Faculty of pharmacy,
University of Tripoli, Tripoli M16, Libya
e-mail: abdulgbaj1@hotmail.com
A. Hermann
Department of Cell Biology, Division of Cellular and Molecular
Neurobiology, University of Salzburg, Salzburg 5020, Austria

Naunyn-Schmiedeberg's Arch Pharmacol
sulfamate. Some of these agents are now under study in
clinical trials (Dowlati et al. 2002; Cortes and Baselga 2007;
Thomas 2009; Thomas and Abbruzzese 2005).
removed from the surface of the protein and the non-polar
hydrogen atoms were removed as well. The protein in
AutoDock 4.2 is treated as a rigid molecule.
Owing to their high affinity to the colchicine binding
site, the natural product, combretastatin A-4, is regarded
as one of the most promising antineoplastic and cancer
vascular-targeting agents which are under investigation.
Combretastatin A-4 (Fig. 1), as potent inhibitors of tubulin
assembly and due to its success in phase II clinical trials
(Hsieh et al. 2005; Lu et al. 2012), has made it an attractive
lead molecule for the development of anticancer agents, and
promote synthesis of novel derivatives (Hori et al. 2010;
Mitrus et al. 2009; Cushman et al. 1991).
Preparation of the ligand
The ligand structure is initially drawn with ACD/ChemSketch
chemical structure drawing software and then transferred to
Discovery Studio Visualizer to obtain standard 3D structures.
The ligand chemical structure optimization was performed
using the AutoDock 4.2, Gasteiger charges optimization
and determination of all rotatable bonds of the ligand
was performed.
In the present study, we designed 15 compounds
with sulfanilamide-imine functionalities (Fig. 1) based
on structural similarity with combretastatin A-4, and we
hypothesized that sulfanilamide-imines series may fit
and interact at the colchicine active site and may be-
come a promising new tubulin-binding drug candidate
for treating various malignancies.
Definition of active site
The protein, using AutoGrid procedure of AutoDock 4.2, is
fixed in a three-dimensional grid and a probe atom is located
at each grid point. The active site determined with the grid
box and it must be large enough to accommodate all the
possible docked compounds. AutoGrid affinity grids are
calculated for each type of atom in the ligand, typically
carbon, oxygen, nitrogen, and hydrogen.
Experimental protocols
Molecular modeling tools
Docking
Two different molecular modeling tools have been used to
predict the interaction of the synthesized compounds at the
colchicine active site, namely Discovery Studio Visualizer and
AutoDock 4.2. The protein structure (tubulin) used in the
current study was downloaded from the protein data bank
(PDB) (Nogales et al. 1998) as bi units co-crystallized with
combretastatine A-4. The molecular modeling study was
performed in several steps using the above mentioned soft-
ware; these include protein preparation, ligand preparation,
and definition of active site, docking, and analysis of the
docking results. PDBQT file format, which is an extended
PDB format, is used in this part of our work.
This part was performed using AutoDock 4.2, where all
rotatable bonds of the ligand were randomized at the begin-
ning of the docking and treated as completely flexible dur-
ing the docking runs. Bond lengths, angles, and torsions
associated with non-rotatable bonds in the ligand were held.
For each independent Lamarckian genetic algorithm run, a
maximum number of 100,000 operations were performed.
The docking energy (Table 1) for the possible interaction
between the targeted microtubules with the compounds un-
der investigation was calculated and presented by AutoDock
4.2. Low value of the docking energy indicated the possible
high drug/target interaction, while the high docking energy
reflects the possible weak activity against the targeted mi-
crotubules. The Discovery Studio Visualizer is used for
viewing and analyzing the possible type on interaction as
it can easily detect the hydrogen bonds, lipophilic, and
dipolar interactions.
Preparation of the protein
The protein structure was prepared using Discovery Studio
Visualizer and AutoDock 4.2, where the water molecules
Chemical synthesis of the Schiff’s bases
Materials and solvents
All chemical materials and solvents used in the chemical
synthesis of Schiff bases were of highest purity and used
without further purification, purchased from Sigma/Aldrich
chemical company (UK) or Fluka analytical company, UK.
Fig. 1 Chemical structures of Combretastatine A-4 and Sulfanilamide-
imines derivatives

Naunyn-Schmiedeberg's Arch Pharmacol
Table 1 In vitro antitumor activity of the designed sulfanilamide Schiff base derivatives and binding free energies of docking studies, (−−)=no
effect
IC50 µM a
IC50 µM a
Breast Lung
carcinoma carcinoma
Compound
R group
Compounds
R group
Docking
energy
Breast
Lung
carcinoma
cell c
Docking
energy
carcinoma
cell b
cell b
cell c
1
9
--
--
4.65
166
--
6.47
2
3
10
11
105
90
--
--
--
--
--
--
5.40
5.66
5.11
4.85
4
12
110
120
120
--
5.87
5.65
5
6
13
14
101
130
--
--
99
--
140
--
5.45
7.02
6.11
4.81
7
8
15
105
96
--
--
--
6.05
6.23
5.23
130
a IC50, compound concentration required to inhibit tumor cell proliferation by 50% . Means were calculated from three
independent experiments. b Human breast cell line (MCF-7). c Human lung cell line (A549).

Naunyn-Schmiedeberg's Arch Pharmacol
Instruments
(s, 1H, HC=N), 8–7.40 (m, 9H, Ar–H). 13C-NMR
(400 MHz, DMSO-d6): δ [ppm] 162.77 (1C, *C=N),
154.35 (1C, C=*C–N), 153.56 (1C, C=*C–SO2NH2),
141.14 (1C, Ar), 132.65 (1C, Ar), 128.96 (2C, Ar), 128.90
(2C, Ar), 126.92 (2C, Ar) 121.24 (2C, Ar). lmax (nm): 298.
MP=191–192°C (literature=190°C) (Baluja et al. 2006),
% yield=83 %.
Synthesis of compounds was performed in a microwave-
closed system (milestone start E 2,450 MHz, Italy). Thin
layer chromatographic (TLC) analysis was performed on
pre-coated aluminum plates (silica gel 60778, Fluka analyt-
ical, UK). TLC spots were visualized with both short
(254 nm) and long (366 nm) UV light. Melting points were
measured in open capillary tubes on an Electrothermal
SMP30 melting point apparatus (Stuart, UK). The IR spec-
tra of samples were recorded in region 4,000–400 cm−1 by
Varian FT-IR spectrophotometer 660 (Varian, Australia),
UV-visible spectra were recorded by Cary 5000
UV\VIS\NIR spectrophotometer (Varian, Australia), using
4-(3-Phenyl-propylideneamino)-benzene sulfonamide (2)
FT-IR (cm−1): 3,176(C–H aromatic), 1,621 (HC=N), 1,575 (C=C
aromatic), 3,306 (N–H str), 1,150 (S=O asym), 1,330 (S=O sym).
1H-NMR (400 MHz, DMSO-d6): δ [ppm] 9.70 (d, 1H, HC=N),
8.45–7.51 (m, 9H, Ar–H), 7.10 (d, 1H, Ar–HC=C), 6.60 (d, 1H,
Ar–C=CH). 13C-NMR (400 MHz, DMSO-d6): δ [ppm] 164.02
(1C, *C=N), 154.30 (1C, C=*C–N), 145.62 (1C, C=*C–
SO2NH2), 140.92 (1C, Ar), 135.12 (1C, Ar–*C=C), 129.83 (2C,
Ar), 128.91 (2C, Ar), 127.95 (2C, Ar), 127.69 (1C, Ar), 126.84
(2C, Ar), 121.02 (1C, Ar–C=*C). lmax (nm)=231, 295. MP=
203°C, % yield=90 %.
1
1-cm quartz cuvettes. H-NMR spectra (400 MHz) recorded
in dimethyl sulfoxide (DMSO) by employing
tetramethylsilane as an internal standard on Bruker Avance
400 MHz NMR Spectrometer (Bruker, France).
General procedure for synthesis of Schiff bases (1a-15a)
by microwave synthesis
4-[(2-Hydroxy-benzylidene)-amino]-benzenesulfonamide (3)
Equimolar amounts (0.01 M) of sulfanilamide and substituted
aldehydes were triturated to form uniform mixture using clean
and dry Teflon vessels (Kundu et al. 2009; Venugopala and
Jayashree 2008). In case of solid aldehydes, minimum
amounts of ethanol (10–15 ml) were used to dissolve the
reaction mixture. This mixture was subjected to microwave
irradiation at 350–400 W power for about 0.5–1 min with
maximum heating of 60 °C. The optimum reaction time was
determined depending on the reaction competition using TLC
and the appropriate solvent system. The reaction mixture was
allowed to cool and the crude solid product was collected
through vacuum filtration and washed several times with
ethanol, and finally recrystallized from absolute ethanol. The
achieved crystals were dried and their melting points were
determined. The chemical purity where investigated by high
performance liquid chromatography (HPLC) (Waters, UK)
using chloroform/methanol (80:20) or acetonitrile/methanol
(90:10) as mobile phases and Luna C18 (2) as a column
(length 25 cm, inner diameter 4.6 mm, particle size 5 μm),
while the chemical structure for all the synthesized com-
pounds was elucidated using several spectroscopic techniques
FT-IR (cm−1): 1,614 (HC=N), 3,239 (N–H str), 1,151 (S=O
asym), 1,307 (S=O sym), 3,335 (O–H str), 1,570 (phenolic
OH). 1H-NMR (400 MHz, DMSO-d6): δ [ppm] 12.7 (s, 2H,
NH2), 9.67 (s, 1H, HC=N), 7.90–7.40 (m, 8H, Ar–H). 13C-
NMR (400 MHz, DMSO-d6): δ [ppm] 164.97 (1C, *C=N),
160.21(1C, *C–OH), 151.19 (1C, C=*C–N), 141.93 (1C,
C=*C–SO2NH2), 133.89 (1C, Ar), 132.51 (1C, Ar), 127.04
(2C, Ar), 121.79 (2C, Ar), 119.33 (1C, Ar), 119.27 (1C,
Ar), 116.68 (1C, Ar). lmax (nm): 350. MP=212°C (lit-
erature=212°C) (Bharat et al. 2007), % yield=88 %.
4-[(2,3-Dihydroxy-benzylidene)-amino]-
benzenesulfonamide (4)
FT-IR (cm−1): 3,248 (N–H str), 1,617 (HC=N), 1,572 (C=C
aromatic), 1,148 (S=O asym), 1,328 (S=O sym), 1,209 (C–O
1
phenolic), 1,273 (OH). H-NMR (400 MHz, DMSO-d6): δ
[ppm] 8.55 (s, 1H, HC=N), 7.95–7.1 (m, 7H, Ar–H). 13C-
NMR (400 MHz, DMSO-d6): δ [ppm] 165.4 (1C, *C=N),
150.54 (1C, C=*C–N), 149.1 (1C, *C–OH), 145.5 (1C, *C–
OH), 141.53 (1C, C=*C–SO2NH2), 127 (2C, Ar), 121.52
(2C, Ar), 119.30 (2C, Ar), 118.90 (2C, Ar). MP=
243°C, % yield=78 %.
1
including IR, H NMR, 13C NMR, and UV spectra the fol-
lowing abbreviations for the NMR are used: singlet (s), dou-
blet (d), triplet (t), quartet (q), multiplet (m).
4-{[(2-
4-(Benzylidene-amino)-benzenesulfonamide (1)
Hydroxyphenyl)methylidene]amino}benzenesulfonamide
(5)
FT-IR (cm−1): 3,292 (N–H str), 1,619 (HC=N), 3,002 (C–H
aromatic), 1,574 (C=C aromatic), 1,150 (S=O asym), 1,331
FT-IR (cm−1): 3,246 (N–H str), 1,618 (HC=N), 1,156 (S=O
1
1
(S=O sym). H-NMR (400 MHz, DMSO-d6): δ [ppm] 8.65
asym), 1,275 (S=O sym). H-NMR (400 MHz, DMSO-d6):

Naunyn-Schmiedeberg's Arch Pharmacol
δ [ppm] 12 (s, 1H, OH), 8.95 (s, 1H, HC=N), 7.92–6.92 (m,
7H, Ar–H), 3.42 (s, 3H, OCH3). 13C-NMR (400 MHz,
DMSO-d6): δ [ppm] 164.26 (1C, *C=N), 154.29 (1C,
C=*C–N), 151.92 (1C, *C–OCH3), 151.56 (1C, *C–OH),
141.82 (1C, C=*C–SO2NH2), 127.06 (2C, Ar), 121.71 (2C,
Ar), 121.25 (1C, Ar), 119.21 (1C, Ar), 117.64 (1C, Ar),
114.54 (1C, Ar), 55.49 (1C, OCH3). lmax (nm): 286, 373.
MP=180°C, % yield=78 %.
4-[(4-Dimethylamino-benzylidene)-amino]-
benzenesulfonamide (9)
FT-IR (cm−1): 3,278 (N–H str), 1,569(HC=N), 1,518(C=C
aromatic), 1,143(S=O asym), 1,329 (S=O sym). H-NMR
1
(400 MHz, DMSO-d6): δ [ppm] 9.67(s, 2H, NH2), 8.43 (s,
1H, N=CH), 8.83–6.60 (m, 8H, Ar–H), 6.10 (s, 6H,
N(CH3)2). 13C-NMR (400 MHz, DMSO-d6): δ [ppm]
162.70 (1C, *C=N), 158.20 (1C, C=*C–N), 146.80 (1C,
*C–N(CH3)2), 137.00 (1C, C=*C–SO2NH2), 129.80 (2C,
Ar), 127.80 (2C, Ar), 123.00 (1C, Ar), 120.70 (2C, Ar),
113.20 (2C, Ar), 43.60 (2C, N(CH3)2). MP=204–205°C
(literature=202°C) (Santosh et al. 2010), % yield=75 %.
4-[(2-Hydroxy-naphthalen-1-ylmethylene)-amino]-
benzenesulfonamide (6)
FT-IR (cm−1): 3,023 (C–H aromatic), 3,165 (N–H str),
1,621 (HC=N), 1,587 (C=C aromatic), 1,310 (S=O sym),
1,292 (OH). 1H-NMR (400 MHz, DMSO-d6): δ [ppm] 15.5
(s, 2H, NH2), 10.82 (s, 1H, HC=N), 9.70–7.00 (m, 10H,
Ar–H). 13C-NMR (400 MHz, DMSO-d6): δ [ppm] 156.38
(1C, *C=N), 146.62 (1C, C=*C–N), 141.34 (1C, C=*C–
SO2NH2), 137.70 (1C, *C–OH), 133.07 (1C, Ar), 129.07
(1C, Ar), 128.28 (2C, Ar), 127.39 (1C, Ar), 127.16 (1C,
Ar), 126.74 (1C, Ar), 123.79 (1C, Ar), 122.17 (1C, Ar),
120.71 (1C, Ar), 120.50 (1C, Ar), 112.37 (1C, Ar), 108.74
(1C, Ar). lmax (nm): 302, 383, 441, 464. MP=274°C, %
yield=85 %.
4-[(4-Methoxy-benzylidene)-amino]-benzenesulfonamide (10)
FT-IR (cm−1): 3,272 (N–H str), 2,913 (C–H aromatic),
1,606 (HC=N), 1,569 (C=C aromatic), 1,147 (S=O asym),
1,315 (S=O sym), 1,265 (C–O phenolic). 1H-NMR
(400 MHz, DMSO-d6): δ [ppm] 8.60 (s, 1H, HC=N),
7.90–7.00 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3). 13C-NMR
(400 MHz, DMSO-d6): δ [ppm] 162.28 (1C, *C=N), 161.85
(1C, *C–OCH3), 154.67 (1C, C=*C–N), 140.72 (1C,
C=*C–SO2NH2), 130.85 (1C, Ar), 128.49 (2C, Ar),
126.89 (2C, Ar), 121.18 (2C, Ar), 114.33 (2C, Ar), 55.44
(OCH3). lmax (nm): 275. MP=205°C (literature=206°C)
(Baluja et al. 2006), % yield=83 %.
4-[(4-Chloro-benzylidene)-amino]-benzene sulfonamide (7)
FT-IR (cm−1): 3,289 (N–H str), 3,001 (C–H aromatic),
1,620 (HC=N), 1,581 (C=C aromatic), 1,150 (S=O
asym), 1,331 (S=O sym). H-NMR (400 MHz, DMSO-
4-[(3-Methoxy-benzylidene)-amino]-benzene sulfonamide (11)
1
d6): δ [ppm] 8.67 (s, 1H, HC=N), 8.02–7.40 (m, 8H,
Ar–H). 13C-NMR (400 MHz, DMSO-d6): δ [ppm]
161.62 (1C, *C=N), 154 (1C, C=*C–N), 141.33 (1C,
C=*C–SO2NH2), 136.57 (1C, *C–Cl), 134.42 (1C, Ar),
130.57 (2C, Ar), 129.06 (2C, Ar), 126.92 (2C, Ar),
121.30 (2C, Ar). MP=181–182°C (literature=184°C)
(Santosh et al. 2010), % yield=80 %.
FT-IR (cm−1): 3,329 (N–H str), 3,175 (C–H aromatic),
1,618 (HC=N), 1,579 (C=C aromatic), 1,296 (C–O pheno-
lic), 1,150 (S=O asym), 1,329 (S=O sym). 1H-NMR
(400 MHz, DMSO-d6): δ [ppm] 8.62 (s, 1H, HC=N),
7.85–7.15 (m, 8H, Ar–H), 3.84 (s, 3H, OCH3). 13C-NMR
(400 MHz, DMSO-d6): δ [ppm] 162.67 (1C, *C=N), 159.53
(1C, *C–OCH3), 154.25 (1C, C=*C–N), 141.18 (1C,
C=*C–SO2NH2), 137 (1C, Ar), 130.02 (2C, Ar), 126.91
(1C, Ar), 121.96 (2C, Ar), 121.24 (1C, Ar), 118.30 (1C,
Ar), 112.67 (1C, Ar), 55.23 (1C, OCH3). lmax (nm): 278,
320. MP=161°C, % yield=77 %.
4-[(3-Ethoxy-4-hydroxy-benzylidene)-amino]-
benzenesulfonamide (8)
FT-IR (cm−1): 3,271 (N–H str), 1,598 (HC=N), 1,518 (C=C
aromatic), 1,145 (S=O asym), 1,330 (S=O sym), 1,286 (C–O
4-[(4-Nitro-benzylidene)-amino]-benzenesulfonamide (12)
1
phenolic), 1,192 (OH). H-NMR (400 MHz, DMSO-d6): δ
[ppm] 9.76 (s, 2H, SO2NH2), 8.46 (s, 1H, HC=N), 7.85–6.6
(m, 7H, Ar–H), 5.83 (q, 2H, OCH2*CH3), 1.96 (t, 3H,
OCH2CH3*). 13C-NMR (400 MHz, DMSO-d6): δ [ppm]
162.13 (1C, *C=N), 154.83 (1C, C=*C–N), 151.88 (1C,
*C–OH), 150.91 (1C, C*–OCH2CH3), 145.15 (1C, C=*C–
SO2NH2), 140.05 (1C, Ar), 129.93 (2C, Ar), 127.39 (1C, Ar),
124.55 (2C, Ar), 121.14 (1C, Ar), 115.42 (1C, Ar), 63.70 (1C,
O*CH2CH3), 16.46 (1C, OCH2*CH3). lmax (in nanometer):
205, 371. MP=185°C, % yield=90 %.
FT-IR (cm−1): 3,265 (N–H str), 3,105 (C–H aromatic), 1,591
(C=C aromatic), 1,628 (HC=N), 1,598 (NO2), 1,145 (S=O
1
asym), 1,341 (S=O sym), 1,277 (OH). H-NMR (400 MHz,
DMSO-d6): δ [ppm] 12.70 (s, 2H, NH2), 8.39 (s, 1H, HC=N),
7.50–8.22 (m, 8H, Ar–H). 13C-NMR (400 MHz, DMSO-d6):
δ [ppm] 162.7 (1C, *C=N), 156.4 (1C, C=*C–N), 150.7 (1C,
*C–NO2), 137.8 (1C, C=*C–SO2NH2), 137.3 (1C, Ar), 129.9
(2C, Ar), 126.8 (2C, Ar), 123.7 (2C, Ar), 122.3 (2C, Ar). lmax
(nm): 271. MP=193°C, % yield=92 %.

Naunyn-Schmiedeberg's Arch Pharmacol
4-{[(2-methoxy-4-
nitrophenyl)methylidene]amino}benzenesulfonamide(13)
penicillin–G, kanamycin, amphotericin B). All cell culture
work was carried out in a sterile tissue culture laboratory under
a laminar flow hood. The frozen vials of the cells were held at
room temperature for 1 min before being placed in
37 °C incubator for a further minute until thawed. The
outside of the ampoule was sprayed with 70 % ethanol
before the cells were poured into a flask containing
trypsin-EDTA.
FT-IR (cm−1): 3,289 (N–H str), 3,029 (C–H aromatic),
1,620 (HC=N), 1,579 (C=C aromatic), 1,258 (C–O pheno-
lic), 1,334 (S=O sym), 1,153 (S=O asym), 1,378 (NO2). 1H-
NMR (400 MHz, DMSO-d6): δ [ppm] 8.90 (s, 1H, HC=N),
8.25–7.25 (m, 7H, Ar–H), 4.05 (s, 3H, OCH3). 13C-NMR
(400 MHz, DMSO-d6): δ [ppm] 159.41 (1C, *C=N),
156.02 (1C, *C–OCH3), 153.86 (1C, C=*C–N), 150.33
(1C, *C–NO2), 141.73 (1C, C=*C–SO2NH2), 141.03 (1C,
Ar), 128.98 (1C, Ar), 128.16 (2C, Ar), 127.30 (1C, Ar),
126.93 (1C, Ar), 121.32 (2C, Ar), 115.59 (1C, Ar), 107.15
(1C, Ar), 56.61 (1C, OCH3). MP=224°C, % yield=92 %.
Cell culturing
The cells were passaged every 4 to 7 days using trypsin-
EDTA and fed three times a week. Cells were split once
confluence was reached and 1×105 cells were placed into
25-ml tissue culture flasks. To split the cells, the medium
was removed, the cells washed with Hank’s Buffered Salt
Solution and gently harvested with 1-ml detachment trypsin
neutralizing solution. Then cells were poured into a sterile
falcon tube and centrifuged at 1,300 rpm for 5 min. The
supernatant was removed and the pellet gently resuspended
in an appropriate volume of medium. This mixture was then
placed into fresh culture flasks at 1:2 split ratios. Cells (5×
106 cells/ml) were plated in 1-ml cell suspension concentra-
tion in RPMI1640 supplement. The cells were allowed to
attach to the bottom for 24 h and finally harvested. After
48 h, the cells were treated with aseptic samples which were
sterilized by passing through 0.22-μm Millipore filter and
cisplatin was used as positive control. Untreated cells were
used as negative controls. Each plate was incubated for
another 72 h at 37 °C in a humidified incubator with 5 %
CO2. The duplicate plates were then used for screening the
chemical synthesized antitumor agents.
4-[(Furan-2-ylmethylene)-amino]-benzene sulfonamide (14)
FT-IR (cm−1): 2,978 (C–H aromatic), 3,284 (N–H str),
1,623 (HC=N), 1,578 (C=C aromatic), 1,147 (S=O asym),
1,325 (S=O sym). 1H-NMR (400 MHz, DMSO-d6): δ [ppm]
8.46 (s, 1H, HC=N), 8.10–6.75 (m, 7H, Ar–H). 13C-NMR
(400 MHz, DMSO-d6): δ [ppm] 154.00 (1C, *C=N), 151.55
(1C, C=*C–N), 150.15 (1C, O–*C–C=N), 147.08 (1C,
O–C*=C), 141.10 (1C, C=*C–SO2NH2), 126.93 (2C, Ar),
121.24 (2C, Ar), 118.42 (1C, Ar), 112.74 (1C, Ar). lmax (nm):
302. MP=191°C, % yield=70 %.
4-[(Thiophen-2-ylmethylene)-amino]-benzenesulfonamide (15)
FT-IR (cm−1): 1,605 (HC=N), 3,285 (N–H str), 1,149 (S=O
asym), 1,321 (S=O sym). H-NMR (400 MHz, DMSO-d6):
1
δ [ppm] 8.82 (d, 1H, HC=N), 7.90–6.60 (m, 7H, Ar–H).
13C-NMR (400 MHz, DMSO-d6): δ [ppm] 155.79 (1C,
*C=N), 153.54 (1C, C=*C–N), 141.85 (1C, S–*C–C=N),
141.03 (1C, C=*C–SO2NH2), 134.62 (1C, S–C*=C),
132.04 (1C, Ar), 128.35 (2C, Ar), 127.30 (1C, Ar), 126.85
(1C, Ar), 121.27 (2C, Ar). MP=208°C, % yield=89 %.
Evaluation of cytotoxicity
All synthesized compounds were tested for their cytotoxic-
ity on lung and breast carcinoma in our cell culture lab at the
National Medical Research Center, Zawia, Libya. The com-
pounds were dissolved in DMSO (1 ml) and tested on cells
at a concentration of 200 μg/ml for 72 h. DMSO was used
as negative control, after this period the cell culture was
examined under phase contrast inverted microscope.
Biological activities testing
Antitumor activity
Cell lines and culturing
Statistical analysis
Human breast (MCF7, Cell Line human breast adeno-
carcinoma) and lung carcinoma (A549, Cell Line human
lung carcinoma) were purchased from the European
Collection of Cell Culture, UK. The cells were stored in
a liquid nitrogen storage facility at the National Medical
Research Centre (Zawia/Libya). The cells were grown in
Dulbecco’s Minimum essential medium supplemented with
10 % fetal bovine serum containing antibiotics (streptomycin,
The experimental data were expressed as mean SEM. The
significance of differences among the various treated groups
and control group were analyzed by means of one–way
ANOVA. The level of significance was set at p <0.05.
IC50 (inhibitory concentration which caused 50 % inhibi-
tion) were estimated using linear regression method of the
percent of cell viability against the concentration of the
tested compounds using Microsoft Excel software program.

Naunyn-Schmiedeberg's Arch Pharmacol
Results and discussion
lower docking energy, which is in total agreement with the
biological results achieved for the same compounds. The
sulfonamide group in the tested compounds occupies the same
part used by the same group of isoquinoline sulfamate. They
further showed greater reactivity at the active site and a high
ability to form four different hydrogen bonds with amino acids
of the colchicine active site, namely histidine 94, histidine
119, threonine 199 and threonine 200 (Fig. 3a). The hydro-
phobic pocket of the active site, which accommodates the
lipophilic moieties of the tested compounds, has large size.
This can be concluded based on the comparison of naphthyl
side chain of compound 6 and colchicine phenyl group, as
they represented in different positions at the hydrophobic
pocket. Additionally, increasing the lipophilicity of the sub-
stituents at this position leads to decreasing of docking energy
and possibly increased the interaction with the targeted
microtubules.
Design of sulfanilamide-imine scaffold
A great variety of compounds have been reported in the
literature (Desai and Mitchison, 1997; Gadde and Heald
2004) as inhibitors for polymerization or depolymerization
of microtubules by binding to the colchicine binding
site of tubulin β-subunit. Combretastatine A-4 (Fig. 2a)
and isoquinoline sulfamate (Fig. 2b) are examples of the
most interesting compounds under investigation as anti-
microtubules which are currently in clinical trials. They
were used to design structurally related sulfanilamide-imine
derivatives (Fig. 2c) as possible antitubulin agents. The main
structural features used in the current study (red in Fig. 2)
are two phenyl rings separated by azomethene group rather
than the alkene moiety in combretastatine A-4. In addition,
sulfonamide functionality of isoquinoline sulfamate is used
instead the methoxy group of combretastatine A-4. Fifteen
sulfanilamide-imine derivatives have been designed and
then synthesized by varying the R group(s) on the second
benzene ring.
Chemical synthesis
The sulfanilamide-imines (1–15) were synthesized by con-
densation of sulfanilamide with the starting aldehydes in
equimolar amounts using microwave method for chemical
synthesis (Scheme 1). The optimum microwave power (in
Watt) and the corresponding reaction time were determined
depending on the completion of the reaction which is moni-
tored by TLC using the appropriate solvent system. Highly
purified crystals of all compounds were obtained via succes-
sive recrystallization using absolute ethanol and their purity
was determined by HPLC using either chloroform/methanol
(80:20) or acetonitrile/methanol (90:10) as a mobile phase.
The percentage of the achieved dried yield of the synthetic
compounds was high (70–93 %) for most of the synthesized
compounds.
Molecular modeling study
All the targeted compounds were docked to the single β-
tubulin colchicine active site (Fig. 3a, e.g., compound 6 and
colchicine) and the docking energy was calculated. At the
same time, the possible interaction of the designed compounds
with the colchicine active site of tubulin unit was studied. The
docking results showed that most of the synthesized
sulfanilamide-imines theoretically fit and interact with the
colchicine active site in the same manner as combretastatine
A-4 with extra binding to the active site via the sulfonamide
moiety. Interestingly, the docking results showed that most of
the high score compounds are planar and occupy the same part
of the active site (Fig. 3b). These flexible molecules are of
Chemical structures of the synthesized compounds were
determined on the basis of their FT-IR, UV, 1H NMR, and 13
C
NMR spectroscopic analysis. The synthesized compounds
Fig. 2 Chemical structures of
Combretastatine A-4,
Isoquinoline sulfamate and
Sulfanilamide-imines
derivatives

Naunyn-Schmiedeberg's Arch Pharmacol
Compounds with binding free energy bigger than −5.23
were ineffective against human breast cancer cells. The lung
human cell line proved to be sensitive toward compounds 4,
8, and 13 with a IC50 concentration range of 120–140 μM
compared to Combretastatine A4 (IC50 2.0 μM) and colchi-
cine (IC50 3.2 μM)(De Martino et al. 2004) with binding
free energy ranging from −5.87 to −6.23. Whereas, com-
pounds 1, 10, 11, 14, and 15 proved to be ineffective against
breast cancer cells while compounds 1, 2, 3, 5, 6, 7, 9, 10,
11, 12, 14, and 15 also proved to be ineffective against lung
cancer cells. These results indicate that cytotoxicity is gen-
erally correlated with binding affinity for the colchicine site.
However, some results were not fully explained in terms of
binding affinity, suggesting that others factors, such as spe-
cific effects of the compound on the tubulin molecule, may
play a role in the cytotoxic activities of these derivatives.
The activity of the tested compounds could be correlated
to structure variations and modifications. By studying the
variation in selectivity of the tested compounds over the two
cell lines, 11 compounds showed significant inhibition of
the breast cancer cells (MCF-7) and only 3 compounds
affected lung cells. The agreement between the inhibition
of breast and lung cancer cells could be correlated to a
similar inhibitory mechanism related to the common struc-
tural features, while the variation in selectivity for breast
compared to lung cells is probably caused by the differences
in the substitution pattern in sulfanilamide Schiff base de-
rivatives. These variations could be further correlated to the
lipophilic substitution pattern on sulfanilamide Schiff base.
The high activity of compounds 4, 8, and 13 in breast cancer
over lung cancer cells may be attributed to the occurrence of
OH, OCH3, and NO2 moieties which may be important for
hydrogen bonding at the receptor site as described in the
docking section.
Fig. 3 a Interaction of compound 6 (red) and colchicine (green) at the
colchicine active site. b Planarity of compound 6 (red) and colchicine
(green) at the colchicine active site
were in agreement with those found in the literature
(Zevzikoviene et al. 2012; Kaya et al. 2012; Yang et al.
2010; Scozzafava et al. 2000; Popescu et al. 1999). The IR
spectra of the synthesized compounds confirmed the presence
of C=N stretching bands at 1,500–1,690 cm−1 and the absence
of C=O at 1,700 cm−1 for the starting aldehyde. The bands due
to symmetric stretching SO2 groups that are shifted to lower
frequencies, while NH disappeared or was hidden under the
broad band at 3,450–3,300 cm−1 of the Schiff’s base. 1H NMR
spectra of the synthesized compounds were recorded in
DMSO-d6, and the chemical shifts (δ), expressed in parts per
million (ppm) downfield from tetramethylsilane. The signals
for the methene protons of the azomethine group, −N=C (H)
were observed between 9.6 and 10.82 ppm.
In vitro antitumor evaluation
The antitumor activity of all compounds against breast and
lung cell lines was determined using cell counting assay and
cisplatin as a reference drug control. Each cell line was
incubated with four concentrations of each compound. The
response parameter (IC50) was calculated for each cell line
(Table 1). The predicted binding free energy as shown in
Table 1 is proportional to the in vitro cytotoxicity results.
The antitumor drug discovery screen has been designed to
distinguish between broad-spectrum antitumor compounds
and tumor selective agents. The active analogs showed a
characteristic potential model of selectivity as well as broad-
spectrum antitumor activity. With regard to selectivity
against individual cell lines, compounds 2, 3, 4, 5, 6, 7, 8,
9, 12, and 13 had a positive impact against cell line human
breast cancer MCF-7 with IC50 values range of 90–166 μM
compared to Combretastatine A4 (IC50 2.0 μM) and colchi-
cine (IC50 3.2 μM)(De Martino et al. 2004) (Table 1).
When the chemical structure of the synthesized com-
pounds was correlated with their anticancer activity, a num-
ber of structure activity relationships could be derived.
Table 1 shows that no anticancer activity is achieved either
against lung or breast carcinoma when the R substituent is
unsubstituted phenyl group (compound 1). However, the
introduction of an extra double bond between the R substit-
uent and the azomethine moiety leads to strong anti-breast
cancer activity of compound 2. Introduction of a hydrogen
bond donor group, such as the hydroxyl group (−OH) in the
ortho position of the phenyl group of the inactive compound
Scheme 1 Chemical synthesis
of sulfanilamide-imines (1–15)

Naunyn-Schmiedeberg's Arch Pharmacol
Open Access This article is distributed under the terms of the Creative
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source are credited.
1, leads to strong activity against breast cancer cells as
found for compound 3 (Table 1). Introduction of a methoxy
group (−OCH3) at the same phenyl group leads to a small
decrease of activity as in compound 5, while an additional
hydroxyl group promotes anticancer activity against both
breast and lung cancer cells, as found for compound 4.
Increasing the lipophilicity of compound 3 by substitution
of the phenyl with a naphthyl group leads to a small de-
crease in the activity as in compound 6. The para position of
phenyl group is important for activity as the introduction of
chloro-, hydroxyl-, dimethylamino- or nitro- substituents
improves activity as in compounds 7, 8, 9, and 12, respec-
tively. The methoxy group at the meta or para position
showed no activity as in compounds 10 and 11. The
presence of methoxy- or ethoxy- groups, however, appears
to be important for the activity on both types of cancer
cells as in compounds 8 and 13. This may be attributed to
the effect of both groups on the pharmacokinetics com-
pared to mono-substituted compounds. Finally, the
isosteric substitution of the phenyl group of compound 1
with thiophenyl or furfuryl rings showed no activity as in
compounds 14 and 15.
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