Design, synthesis and molecular modeling study of certain 4-Methylbenzenesulfonamides with CDK2 inhibitory activity as anticancer and radio-sensitizing agents

Article abstract of DOI:10.1016/j.bioorg.2018.06.010

Two series of 2-aminopyridine derivatives 6-17 and tyrphostin AG17 analogs 18-22 bearing 4-methylbenzenesulfonamide moiety were designed and synthesized as anticancer compounds. The synthesized compounds were biologically evaluated for their cytotoxic act

Full text of DOI:10.1016/j.bioorg.2018.06.010

Accepted Manuscript
Design, Synthesis and Molecular Modeling Study of certain 4-Methylbenzene-
sulfonamides with CDK2 inhibitory Activity as Anticancer and Radio-sensitiz-
ing agents
Mostafa M. Ghorab, Fatma A. Ragab, Helmy I. Heiba, Mohamed S.A. Elsayed,
Walid M. Ghorab
PII:
S0045-2068(18)30358-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.010
YBIOO 2390
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
13 April 2018
21 May 2018
3 June 2018
Please cite this article as: M.M. Ghorab, F.A. Ragab, H.I. Heiba, M.S.A. Elsayed, W.M. Ghorab, Design, Synthesis
and Molecular Modeling Study of certain 4-Methylbenzenesulfonamides with CDK2 inhibitory Activity as
Anticancer and Radio-sensitizing agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.06.010
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Design, Synthesis and Molecular Modeling Study of certain 4-
Methylbenzenesulfonamides with CDK2 inhibitory Activity as
Anticancer and Radio-sensitizing agents
Mostafa M. Ghoraba, Fatma A. Ragabb, Helmy I. Heibaa, Mohamed S. A.
Elsayedc, Walid M. Ghoraba
a
Department of Drug Radiation Research, National Centre for Radiation
Research and Technology (NCRRT), Egyptian Atomic Energy Authority
(EAEA), PO box 29, Nasr City, Cairo, Egypt.
b
Department of Pharmaceutical and Medicinal Chemistry, Faculty of
Pharmacy, Cairo University.
c
Department of Medicinal Chemistry and Molecular Pharmacology,
College of Pharmacy, and the Purdue Center for Cancer Research, Purdue
University, West Lafayette, Indiana, 47907, United States.
Correspondening author; E-Mail: mmsghorab@yahoo.com ;
Tel.: +20 2 2747413; fax: +20 2 2749298;
Abstract
Two series of 2-aminopyridine derivatives 6-17 and tyrphostin AG17
analogs 18-22 bearing 4-methylbenzenesulfonamide moiety were
designed and synthesized as anticancer compounds. The synthesized
compounds were biologically evaluated for their cytotoxic activity
against human breast cancer cell line MCF-7. From 2-aminopyridine and
tyrphostin AG17 series, compounds 14, 16 and 20 showed the best
activities with IC50 values of 20.4, 18.3 and 26.3 µM, respectively
compared to E7070 IC50 36.3 µM. Further biological evaluation of 14, 16
and 20 against cyclin dependent kinase-2 (CDK2) revealed good
inhibitory activity with IC50 of 2.53, 1.79 and 2.92 µM, respectively
compared to roscovitine IC50 0.43 µM. Additionally, capability of γ-
radiation to augment the cytotoxic activity of 14, 16 and 20 was studied
and showed a dramatic increase in the cell killing effect at lower
concentrations after irradiation. Docking was used to investigate the
possible binding modes of compounds 14, 16 and 20 inside the active site
of CDK2 enzyme.
1

Keywords: 2-Aminopyridine, Tyrphostin AG17 analogs, 4-
methylbenzenesulfonamide, CDK2, cytotoxic, γ-radiation.
1. Introduction
In the last few decades, a great attention has been paid to cyclin-
dependent kinases (CDKs). CDKs represent a class of serine/threonine
kinases that play a pivotal role in cell cycle regulation [1, 2]. One of the
main cell cycle deregulation causes is the abnormal CDK activity either
due to CDKs overexpression or mutations of their encoded genes [3-5].
CDKs inhibition is still under intense investigation as one of the most
important medicinal chemistry strategies in cancer management.
Consequently, the design of a wide array of many CDKs ATP
competitive inhibitors has been reported [5-9]. Remarkable growth
inhibition of tumor cells transformed with several oncogenes has been
obtained with selective CDK2 inhibitors [10]. Deans et al [11] has also
demonstrated that CDK2 inhibitors constitute a potential target in the
treatment of BRCA-deficient tumors. Recently, Galons et al. [12] and
santo et al. [13] suggested CDK2 inhibitors as promising antitumor
agents. On the other hand, it has been reported that the main reason for
Trastuzumab resistance is the overexpression of cyclin E in HER2+
breast tumor patients, and treatment of such patients with CDK2
inhibitors may be a significant strategy to conquer this resistance [14].
Furthermore, Yang et al. [15] showed that CDK2 inhibitors are inspiring
targets in ovarian carcinoma management.
Sulfonamide derivatives have been greatly studied for various biological
activities [16-18] including antitumor, antibacterial, antiparasitic and
diuretic activity. Several mechanisms have been suggested to explain the
antineoplastic activity of sulfonamides, such as cell cycle arrest in the G1
phase [19], binding to β-Tubulin, and disruption of microtubule assembly
[20]. Furthermore, certain sulfonamides act as inhibitors for several
enzymes as cyclin-dependent kinase (CDK) [21], carbonic anhydrase
[22], methionine amino peptidases [23], matrix metalloproteinase [24]
and nicotinamide adenine dinucleotide oxidase [25]. N-(3-Chloro-7-
indolyl)-1,4-benzenesulfonamide E7070 (Indisulam) (Fig. 1) is a
disulfonamide anticancer agent against different cancer types which
target the G1 phase of the cell cycle through CDK2 and cyclin E
inhibition. Besides, E7070 is indicated to be potent anti-breast cancer
candidate against MCF-7 cell line by in vitro and in vivo findings [26 a-
c]. Moreover, SLC-0111, is an urea derivative bearing one sulfonamide
moiety (Fig. 1) currently in phase I clinical trials for the treatment of solid
2

tumors through the inhibition of carbonic anhydrase (CA, EC 4.2.2.1)
isoform IX [27].
On the other hand, the antiproliferative activity of several aminopyridines
has been examined against various cell lines and the observed cytotoxic
activity of this class is mainly attributed to inhibition of cyclin dependent
kinases (CDKs). The 2-aminopyridine moiety mimics the adenosine
triphosphate (ATP) 6-aminopurine core and considered a good candidate
for designing ATP-competitive kinase inhibitors. A series of aryl
aminopyridine was evaluated for cytotoxic activity and the most active
derivative was sp24 (Fig. 1). The mechanism of action involves the
disruption of signaling pathway and cell cycle through inhibition of CDK
and several other tyrosine kinases. It is also indicated that the presence of
a sulfonamide moiety with 2-aminopyridine contributed to selectivity and
potency, for example, compound I (Fig. 1) inhibits CDK1 and CDK2
while not interacting with other kinases [28]. The corresponding N-[(4-
hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide
E7010 (Fig. 1) is a sulfonamide derivative carrying 2-arylaminopyridine
substituent, showed a cytotoxic activity against different cell lines by
causing cell cycle arrest in the M phase due to inhibition of tubulin
polymerization [29]. All the synthesized derivatives have a nitrile
substituent attached whether to the aliphatic side chain or to the
aminopyridine ring, as this group acts as a hydrogen bond acceptor and
form nonspecific dipole interactions with protein side chain and thus
promote penetration with sterically occluded protein kinases. Bosutinib
(Fig. 1) is a known kinase inhibitor whose docking studies demonstrated
a crucial hydrogen bond between threonine and nitrile nitrogen which is a
common motif in these kinase inhibitors [30].
Tyrphostins are Low molecular weight compounds that act as general
tyrosine kinases inhibitors. AG17, a benzylidene malononitrile derivative
(Fig. 1) induces arrest at the G1 phase followed by apoptosis with a
general reduction of the intracellular level of tyrosine phosphorylated
proteins. Bcl-2 and cdk2 protein levels were not altered with AG17,
whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were
markedly reduced. These findings suggest that the target of AG17 is cdk2
inhibition [31]. The results offered by Burger et al. [32] reported that
AG17 is the most potent anti-breast cancer agent from tyrphostins series
investigated. Recently, Alafeefy et al. [33] studied antitumor synergism
from the coupling of certain quinazolines with tyrphostin AG99 (Fig. 1)
and discovered that ten of the synthesized candidates exhibited a
significant antitumor activity against three carcinoma cell lines.
3

Based on these studies, two novel series of 2-aminopyridines 6-17 and
tyrphostin AG17 analogs 18-22 (Fig. 1) bearing 4-
methylebenzenesulfonamide moiety were designed and synthesized. The
in vitro cytotoxic activity of the synthesized compounds 4-22 was
evaluated against breast carcinoma cell line MCF-7. In addition, the
ability of γ-radiation to synergize the cytotoxic activity of the most active
candidates from each series 14, 16 and 20 was also studied. Furthermore,
enzyme inhibition assay was carried out against CDK2 for the most
active compounds 14, 16 and 20. Finally, molecular modeling was
utilized to investigate the possible binding mode of the synthesized
compounds in CDK2.
O
N
N
O
N
Cl
O
H
N
Cl
NC
Bosutinib
NC
CN
H2NOC
CN
HO
HO
HO
AG17
AG99
4

Fig. 1
2. Results and Discussion
2.1. Chemistry
Schemes 1-3 show the synthetic routes adopted for the synthesis of target
derivatives 4-22. Ethylidenemalononitrile derivative 4, was obtained via
Knoevenagel condensation of the substituted acetophenone 3 [34] with
malononitrile in presence of anhydrous ammonium acetate. IR spectrum
of 4 revealed the absence of (C=O) band and the appearance of two
strong bands at 2191 and 2160 cm-1 (2C≡N). 1H-NMR displayed up-field
two singlets at 2.32, 2.45 ppm corresponding to CH3 (tolyl) and CH3
(aliph.), respectively and a downfield singlet at 10.78 ppm (SO2NH). 13C-
NMR exhibited a new signal at 86.3 ppm (C=(CN)2) and signal at 116.2
ppm (2C≡N). Reaction of 4 with dimethylformamide dimethylacetal
(DMF-DMA) in dry xylene afforded the novel enaminonitrile derivative
1
5. H-NMR displayed the absence of a singlet at 2.45 ppm (CH3) and
presence of an up-field singlet at 3.32 ppm (N-(CH3)2) and two doublets
13
at 7.05, 7.80 ppm (CH=CH). C-NMR exhibited absence of a signal at
25.7 ppm (CH3) and appearance of a new signal at 55.9 ppm (N-(CH3)2)
(Scheme 1). Reaction of 5 with certain aliphatic and aromatic amines or
4-aminobenzenesulfonamides in glacial acetic acid yielded the required
2-aminopyridine derivatives 6-17. 1H-NMR spectra showed the
disappearance of two doublets assigned to (CH=CH) and presence of
another downfield singlets at the range of 10.60-12.25 ppm (2NH) and 2
doublets at 6.60-8.20 ppm (2CH pyridine) in addition to the characteristic
peaks of each individual derivative (experimental section) (Scheme 2).
Reaction of arylethylidenemalononitrile 4 with certain aromatic
aldehydes in ethanol in presence of KOH afforded a new series of 1,1-
dicyano-4-(substituted phenyl)buta-1,3-dien derivatives 18-22 (Scheme
5

3). 1H-NMR spectra showed the appearance of two doublets at a range of
6.54-7.93 ppm (CH=CH) and a characteristic signals for each individual
compound. 13C-NMR spectra showed two new down-field signals
(CH=CH) (experimental section).
6

H
R
N
NC
NC
NH
N
R
NH2
N
N
Gl.AcOH
HN
HN
O
O
S
S
O
O
5
R
R
R
N
HN
N
H
NC
NC
NC
NH
N
N
N
HN
HN
HN
O
O
O
O
S
S
S
O
O
6-17
O
O
O
6, R=
9, R=
7, R=
8, R=
N
N
S
Cl
O
O
F
10, R=
13, R=
11, R=
N
HN
O
O
O
S
O
NH2
12, R=
S
NH
14, R=
S
NH
NH2
O
O
O
N
N
N
S
O
S
O
N
O
S
N
16, R=
15, R=
17, R=
NH
S
NH
NH
O
O
O
Scheme 2. Synthesis of 2-aminopyridine derivatives 6-17.
7

2.2. Biological evaluation
2.2.1. In-vitro anticancer evaluation against human tumor breast cancer
(MCF-7)
The results of the in vitro cytotoxic activity are shown in table 1. All the
tested compounds have good cytotoxic activity with varies degrees.
Thirteen compounds showed IC50 values lower than the reference
compound, E7070. From the aminopyridine series 6-17, compounds
bearing one sulfonamide moiety 6-8, 11 and all compounds with
disulfonamide moieties 12-17 showed a remarkable cytotoxic activity
with IC50 values between 18.3 and 35.7µM, compared to E7070 (IC50=
36.30 µM) [26c]. The substituents on the sulfonamide moiety attached to
8

the aminopyridine ring affected the cytotoxic activity, where the most
active derivative was 16 with (4-methylpyrimidin-2-yl) group and IC50
value of 18.3 µM followed by 14 that has 5-methyl-2-isoxazolyl moiety
with IC50 value of 20.4 µM then 17, 13 and 15 derivatives bearing 4,6-
dimethylpyrimidin-2-yl, diaminomethylene and thiazol-2-yl groups
revealed a significant IC50 values of 21.7, 28.8 and 30.5 µM respectively
compared to derivative 12 carrying free unsubstituted sulfonamide moiety
attached to 2-aminopyridine ring, displayed the lowest activity in the
disulfonamide series with IC50 33.1 µM. The most active derivative with
one sulfonamide moiety was 8 carrying pyrazolone ring which possessed
IC50 value of 26.5 µM followed by the thiophene derivative 7 with IC50
value of 26.7 µM then butyl derivative 6 with IC50 value of 28.7 µM and
ethylbenzoate derivative 11 with IC50 value of 34.3 µM. By contrast, the
substituted phenyl derivatives 9, 10 revealed lower cytotoxic activities
with IC50 values of 38.3 µM. Compound 12 showed comparable potency
to E7070. Regarding to tyrphostin AG17 analogs 18-22, the most
promising compound was the phenyl and 2-methoxy phenyl derivatives
18 and 20 with IC50 value of 35.7 and 26.3 µM. while the other
derivatives revealed lower cytotoxic activity compared to E7070 with
IC50 values ranging from 37.5-39.8 µM.
Table 1: In vitro antitumor screening of the targeted compounds 4-22
against human breast carcinoma cell line (MCF-7).
Compound No.
IC50 (µM)a
36.3 3.60
38.5 0.03
33.8 0.01
28.7 0.01
26.7 0.02
26.5 0.02
38.3 0.02
38.3 0.02
34.3 0.02
33.1 0.01
Compound No.
IC50
28.8 0.01
20.4 0.01
30.5 0.002
18.3 0.01
21.7 0.003
35.7 0.02
37.5 0.01
26.3 0.02
39.8 0.02
38.8 0.03
E7070
13
14
15
16
17
18
19
20
21
22
4
5
6
7
8
9
10
11
12
a Each value is the mean of three experiments standard error.
9

2.2.2. In vitro Cyclin dependent kinase 2 (CDK2) inhibitory assay
In order to investigate the CDK2 inhibitory activity of the synthesized
compounds, the IC50 values of the most potent compounds from each
series 14, 16 and 20 were determined. From the results illustrated in
Table 2, Compound 16 was the most potent with IC50 value of 1.79 µM,
followed by compound 14 and 20 with IC50 value of 2.53 and 2.92 µM
respectively, compared to Roscovitine which exhibited IC50 value of 0.43
µM. The CDK2 inhibitory activity matched the cytotoxic activities of
compound 14 and 16 giving a good indication that the cytotoxic activity
is due to inhibition of this enzyme.
Table 2: IC50 values for the most active compounds 14, 16 and 20 against
CDK2 enzyme.
CDK2
IC50 (µM)a
Compound no.
14
16
2.53 0.01
1.79 0.01
2.92 0.01
0.43 0.01
20
Roscovitine
a Each value is the mean of three experiments standard error.
2.2.3. Radio-sensitizing evaluation
The capability of γ-irradiation to augment the cytotoxic activity of the
tested compounds was also evaluated. The most active derivatives among
the two series 14, 16 and 20 with IC50 20.4, 18.3 and 26.3 µM
respectively, were further evaluated against MCF-7 after exposure of the
cells to a single dose of ɤ-irradiation. A dramatic cytotoxic activity
enhancement was observed in potency for the tested compounds after
irradiation. The IC50 values decreased to 11.8, 9.1 and 12.2 µM,
respectively as shown in table 3. Thus γ-irradiation sensitize tumor cells
to the cytotoxic effect of the tested compounds which allows decreasing
the doses of the drug to avoid toxic effects. (Fig. 2. A, B and C)
10

Table 3: In vitro antitumor screening of compounds 14 and 16 against
human breast carcinoma cell line (MCF-7) in combination with γ-
radiation (8Gy).
Compound No.
IC50 (µM)a
11.8 3.6
14
16
20
9.10 0.03
12.20 0.04
a Each value is the mean of three experiments standard error.
1.2
A
Compound 14
Compound 14 + 8 Gy
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Concentrations (µm)
1.2
1
B
Compound 16
Compound 16 + 8 Gy
0.8
0.6
0.4
0.2
0
0
50
100
150
Concentrations (µm)
11

1.2
1
C
Compound 20
Compound 20 + 8 Gy
0.8
0.6
0.4
0.2
0
0
50
100
150
Concentrations (µm)
Fig. 2. Survival curve of MCF-7 for: (A) compound 14, (B) compound 16
and (C) compound 20 alone or in combination with γ-irradiation (8 Gy).
2.3. Molecular modeling and docking study
The hypothetical binding modes of the most active compounds 14, 16 and
20 were calculated using GOLD docking in the active site of the CDK2-
CA complex. Based on the structural similarity between the tested
compounds and type II kinase inhibitors, a crystal structure of the inactive
conformation of CDK2 (PDB: 2WXV) was chosen. This crystal structure
was also selected because of the similar structural motifs in the tested
compounds and the structure of the co-crystallized ligand, Pyrazolo[4,3-
h]quinazoline-3-carboxamide (Lead). First, the docking algorithm was
validated for its ability to reproduce the co-crystal binding mode of the
reference compound. The reference compound was extracted from the
complex and redocked in the active site using the same parameters that
will be used for the tested compounds. As expected the GOLD algorithm
could regenerate the co-crystal structure with RMSD of 0.5 Å giving high
confidence in the calculated binding mode of compounds (Fig. 3).
12

Fig. 3. RMSD, docking validation and binding mode of tthe lead
compound (cyan) in CD
K
K
2 pocket.
The calculated binding
mmodes of 14, 16 revealed very interestingg results
As shown in Fig. 4-6, the aminopyridine moiety in compound 166 showed
strong binding to the hi ge residue Leu83 with two hydrogen bbonding.
The nitrile group forced the sulfonamide side chain to the non-co-planar
position giving the pe fect orientation for hydrogen bondingg of the
sulfonamide group to two important residues Asp86 and Lys89. This
pattern of binding was r peated in compound 14 because it has aa similar
ins were directed to the solvent-expossed area
sidues and solvent molecules. In compoound 16,
n
rr
e
e
structure. The side ch
aa
which has some polar r
ee
the pyrimidine ring was directed to a solvent exposed area of thee enzyme
which was expected in the original design. Fig. 5 and 6 demonstrated the
binding mode of compoound 16 and its superimposition with thhe Lead
compound. The pyrimidine ring is very important as a solvent exposed
group as the hydrophiliic nature of this group will give the required
stabilization of the correct binding mode of this compoound. In
compounds 14 the methyl isoxazole ring also was directed to tthe same
area and showed very ggood binding interaction Fig. 4. Also, tthe most
active molecule 20 in t
active site Fig. 7. Com
nitrogen as the hinge
y
y
rphostin analogs 18-22, was docked intoo CDK2
ound 20, the nitrile group replaced the pyridine
inder and showed good binding to thhe hinge
hydrogen bonding, while, the tolyl gr up was
on with the tyrosyl residue. On the other hhand, the
p
p
bb
residue Leu83 with on
e
e
oo
involved in the interacti
o
methoxyphenyl group was directed to the solvent exposed area and the
13

polar amino acids in the binding site. The docking scores of 14, 16 and 20
are listed in table 4.
Table 4: Docking scores of promising compounds 14, 16 and 20 inside
CDK2 active site.
Compound GOLD Chemscore Amino
Interacting groups
N of quinazoline
Length (Å)
(Kcal/mol)
acids
-43
Leu 83
2.55
2.57
3.15
2.94
2.83
2.53
2.25
3.02
2.61
3.09
2.89
3.04
Lead
Leu 83 NH linked to quinazoline
Lys 89
Asp 86
Leu 83
Leu 83
Asp 86
Leu 83
Leu 83
Lys 89
Asp 86
SO2 of sulfonyl
SO2 of sulfonyl
-51
-52
N of pyridine
14
16
NH linked to pyridine
SO2 of sulfonamide
N of pyridine
NH linked to pyridine
SO2 of sulfonamide
SO2 of sulfonamide
Tyr 15 SO2 of tolyl sulfonamide
-45
Leu 83
CN of dinitrile
2.83
20
14

Fig. 4. Binding mode (stick view) of compound 14 (pink) in CDK2
pocket.
15

Fig. 5. Binding mode of compound 16 (orange) in CDK2 pocket and
surface view displaying the hydrophobic interaction between the tyrosine
residue and the p-tolyl group.
Fig. 6. Overlay of compound 16 (orange) with lead compound (green) in
CDK2 pocket.
16

Fig. 7. Binding mode (stick view) of compound 20 in CDK2 pocket.
3. Conclusion
In Conclusion, two series of 2-aminopyridines 6-17 and tyrphostin AG17
analogs 18-22 bearing 4-methylbenzenesulfonamide moiety have been
designed and synthesized as prospective cytotoxic agents and CDK2
inhibitors. The candidates were tested for their cytotoxic activity against
human breast carcinoma cell line (MCF-7). Compounds 6-8, 11-18 and
20 were found to be more potent than E7070. From SAR, we may deduce
that the presence of 4-methylbenzenesulfonamide scaffold bearing
another sulfonamide moiety or tyrphostin AG17 analog skeleton
incorporating 2-methoxyphenyl moiety is associated with promising
cytotoxic activity and gave the most active compounds 14, 16 and 20
(IC50= 20.4, 18.3 and 26.3 µM), respectively. On the other hand, the
enzyme inhibition assay of compounds 14, 16 and 20 against CDK2
enzyme demonstrated good inhibitory activity with IC50 values of 2.53,
1.79 and 2.92 µM, respectively compared to Roscovitine (IC50 = 0.43
µM). Molecular modeling study showed that compound 16 has
considerable docking score value (S= -52 Kcal mol-1) in the CDK2 active
site compared with the lead compound (S= -43 Kcal mol-1). Finally, γ-
irradiation sensitize tumor cells to the cytotoxic effect of the most active
compounds 14, 16 and 20 which allows to decrease the doses of drugs to
avoid toxic effects.
17

4. Experimental
4.1. Chemistry
Within an open capillary tube on a melting point Stuart equipment (Stuart
Scientific, Redhill, UK) melting points were obtained and were un
rectified. Molecules IR spectra were listed on FT-IR Shimadzu
spectrometer (Shimadzu, Tokyo, Japan). Mass spectra analyses were
completed on MS-5988 HP Model (Hewlett Packard, Palo Alto,
1
13
California, USA). H-NMR and C-NMR spectra were carried out using
Bruker NMR spectrometers running at 300 and 400 MHz, respectively.
Microanalyses were achieved on a Carlo Erba 1108 Elemental Analyzer
(Heraeus,
Hanau,
Germany),
and
all
readings
were
within 0.4 % of the hypothetical values. Substances purity was
confirmed through thin layer chromatography (TLC) with precoated
Aluminum plates SiO2 gel (HF254, 200 mesh) and were visualized via UV
lamp (Merck, Damstadt, Germany). Solvent system consumed was
chloroform/ethylacetate (8:2) and the spots were shown in UV chamber.
1
13
IR, H-NMR, C-NMR and elemental analyses were matched with the
determined structures. IR was accomplished at NCRRT-EAEA (National
Center for Radiation Research and Technology, Egyptian Atomic Energy
1
13
Authority). While H-NMR and C-NMR was carried out at both of
department of Chemical Warfare, Ministry of Defense and NCRRT,
EAEA. Elemental analyses were done at the Laboratories Microanalytical
chemistry of the Faculty of Science, Cairo University. P-toluenesulfonyl
chloride and 4- aminoacetophenon were bought from Sigma-Aldrich.
4.1.1.N-(4-(1,1-dicyanoprop-1-en-2-yl)phenyl)-4-
methylbenzenesulfonamide (4).
A mixture of N-(4-acetylphenyl)-4-methylbenzenesulfonamide 3 (3 g,
0.01 mol), malononitrile (0.66 g, 0.015 mole) and ammonium acetate
(0.77 g, 0.01 mole) was fused together in an oil bath at 100 °C for 4 hrs.
The resinous matter formed was dissolved in hot ethanol (15 ml) with
continuous stirring. The mixture was left overnight in the refrigerator, and
then poured onto ice water, the yellow solid obtained was dried and
º
crystallized from dioxan to give 4. Yield, 88 %; m.p. 120-122 C. IR
(KBr, cm-1): 3246 (NH), 3043 (CH arom.), 2926, 2871 (CH aliph.), 2191,
1
2160 (2C≡N), 1310, 1121 (SO2). H-NMR (DMSO-d6): 2.32 [s, 3H, CH3
tolyl], 2.45 [s, 3H, CH3], 7.21, 7.63, 7.72, 7.83 [4d, 8H, Ar-H AB system,
18

13
J= 8.7 Hz, J= 7.8 Hz], 10.78 [s, 1H, SO2NH]. C-NMR (DMSO-d6):
22.1, 24.7, 86.3, 116.2 (2), 119.3 (2), 124.4, 128.1 (2), 129.1 (2), 129.8
(2), 135.2, 136.3, 137.1, 174.1. MS, m/z (%): 337 [M+] (3.35), 274 (100).
Anal. Calcd. For C18H15N3O2S (337.40): C, 64.08; H, 4.48; N, 12.45.
Found: C, 63.90; H, 4.68; N, 12.20.
4.1.2.(E)-N-(4-(1,1-dicyano-4-(dimethylamino)buta-1,3-dien-2-
yl)phenyl)-4-methylbenzenesulfonamide (5)
A
mixture
of
N-(4-(1,1-dicyanoprop-1-en-2-yl)phenyl)-4-
methylbenzenesulfonamide 4 (3.3 g, 0.01 mol) and DMF/DMA (1.8 g,
0.015 mole) was refluxed for 24 hrs in dry xylene (20 ml). The mixture
was concentrated and left in the refrigerator for 24 hrs. The obtained solid
was crystallized from ethanol to give 5. Yield, 45%; m.p. 90-92 °C. IR
(KBr, cm-1): 3267 (NH), 3031 (CH arom.), 2926, 2868 (CH aliph.), 2192
1
(2C≡N), 1319, 1115 (SO2). H-NMR (DMSO-d6)δ: 2.32 [s, 3H, CH3
tolyl], 3.32 [s, 6H, N(CH3)2], 7.05, 7.80 [2d, 2H, CH=CH], 7.17- 7.70 [m,
13
8H, Ar-H], 10.20 [s, 1H, SO2NH]. C-NMR (DMSO-d6): 20.7, 55.9 (2),
67.3, 103.2, 113.2 (2), 119.4 (2), 122.0, 122.6 (2), 124.2, 126.2 (2), 127.2,
128.3, 134.8, 138.4, 155.5, 170.2. MS, m/z (%): 390 [M-2] (1.15), 91
(100). Anal. Calcd. For C21H20N4O2S (392.47): C, 64.27; H, 5.14; N,
14.28. Found: C, 64.47; H, 5.54; N, 13.97.
4.1.3.N-(4-(3-cyano-2-((4-(N-
(substituted)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamides(6-17)
A mixture of 5 (0.5 g, 0.0013 mol) and substituted amines (0.0013 mole)
namely, butan-1-amine, ethyl 2-amino-4,5-dimethylthiophene-3-
carboxylate, 4-amino-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one, 4-
fluoroaniline, 2-chloroaniline, ethyl-4-aminobenzoate, sulfanilamide,
sulfaguanidine,
sulfathiazole,
sulfamethoxazole,
sulfamerazine,
sulfamethazine, was refluxed for 20 hrs in glacial acetic acid (10 ml). The
mixture was cooled then poured onto ice water. The solid obtained was
crystallized from ethanol to give 6-17, respectively.
4.1.4.N-(4-(2-(butylamino)-3-cyanopyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (6)
19

Yield, 53.11%; m.p. 126-128 °C. IR (KBr, cm-1): 3248, 3199 (2NH),
3043 (CH arom.), 2922, 2872 (CH aliph.), 2189 (C≡N), 1331, 1149
1
(SO2). H-NMR (DMSO-d6)δ: 0.88 [t, 3H, CH3], 1.34 [m, 2H, CH2CH3],
1.56 [m, 2H, CH2CH2CH3], 2.32 [s, 3H, CH3 tolyl], 3.03 [t, 2H, NH-
CH2], 7.00-7.65 [m, 8H, Ar-H], 7.72, 7.83 [2d, 2H, 2CH pyridine, J = 7.2
13
Hz], 10.20, 10.78 [2s, 2H, SO2NH, NH]. C-NMR (DMSO-d6): 15.2,
20.9, 22.0, 26.1, 40.3, 84.2, 113.2, 115.8, 119.5 (2), 122.6 (2), 124.1 (2),
126.2, 127.2 (2), 128.2, 134.8, 138.2, 152.2, 155.5, 165.1. Anal. Calcd.
For C23H24 N4O2S (420.53): C, 65.69; H, 5.75; N, 13.32. Found: C, 65.41;
H, 5.52; N, 13.56.
4.1.5.Ethyl 2-((3-cyano-4-(4-(4-methylphenylsulfonamido)phenyl)pyridin-
2-yl)amino)-4,5-dimethylthiophene-3-carboxylate (7)
Yield, 54.9%; m.p. 108-110 °C. IR (KBr, cm-1): 3359, 3295 (2NH), 3044
(CH arom.), 2974, 2870 (CH aliph.), 2208 (C≡N), 1709.01 (C=O), 1360,
1
1156 (SO2). H-NMR (DMSO-d6)δ: 1.23 [t, 3H, CH2CH3], 2.06 [s, 3H,
CH3 tolyl], 2.32, 2.45 [2s, 6H, 2CH3 thiophene], 4.16 [q, 2H, CH2CH3],
6.91-7.54 [m, 8H, Ar-H], 7.74, 7.86 [2d, 2H, 2CH pyridine, J = 8.7 Hz],
13
10.34, 10.79 [2s, 2H, SO2NH, NH]. C-NMR (DMSO-d6): 9.5, 10.5,
21.1, 22.0, 67.0, 84.2, 113.2, 115.8, 118, 119.4 (2), 122.6 (2), 124.1 (2),
126.2, 127.2 (2), 128.2, 134.6, 138.4, 140.1, 145.1, 150.0, 152.2, 155.6,
165.1, 168.1. Anal. Calcd. For C28H26 N4O4S2 (546.66): C, 61.52; H, 4.79;
N, 10.25. Found: C, 61.30; H, 4.51; N, 10.56.
4.1.6.N-(4-(3-cyano-2-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)amino)pyridin-4-yl)phenyl)-4-methylbenzenesulfonamide (8)
Yield, 54.3%; m.p. 128-130°C. IR (KBr, cm-1): 3316, 3198 (2NH), 3047
(CH arom.), 2918, 2870 (CH aliph.), 2201 (C≡N), 1663 (C=O), 1334,
1
1157 (SO2). H-NMR (DMSO-d6)δ: 2.32 [s, 3H, CH3 pyrazol], 2.36 [s,
3H, CH3 tolyl], 2.55 [s, 3H, N-CH3], 7.05-7.66 [m, 13H, Ar-H], 7.83,
7.89 [2d, 2H, 2CH pyridine, J = 8.1 Hz], 10.13, 10.80 [2s, 2H, SO2NH,
13
NH]. C-NMR (DMSO-d6): 12.4, 22.4, 34.2, 84.2, 113.4, 115.8, 116.4,
119.5 (2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 130.1 (2), 131.0
(2) 132.0, 134.8, 136.0, 137.0, 138.2, 152.2, 155.6, 165.2, 170.2. Anal.
Calcd. For C30H26 N6O3S (550.63): C, 65.44; H, 4.76; N, 15.26. Found: C,
65.66; H, 4.54; N, 15.57.
4.1.7.N-(4-(3-cyano-2-((4-fluorophenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (9)
20

Yield, 50 %; m.p. 126-128 °C. IR (KBr, cm-1): 3328, 3260 (2NH), 3043
(CH arom.), 2927, 2870 (CH aliph.), 2191 (C≡N), 1352, 1186 (SO2). 1H-
NMR (DMSO-d6)δ: 2.37 [s, 3H, CH3 tolyl], 7.08-7.92 [m, 12H, Ar-H],
7.83, 7.92 [2d, 2H, 2CH pyridine, J = 8.7 Hz], 10.32, 10.80 [2s, 2H,
13
SO2NH, NH]. C-NMR (DMSO-d6): 20.9, 84.2, 113.2, 115.8, 116.2 (2),
119.5 (2), 120.2 (2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 134.8,
136.0, 138.2, 150.0, 152.2, 155.6, 165.2. Anal. Calcd. For C25H19 FN4O2S
(458.51): C, 65.49; H, 4.18; N, 12.22. Found: C, 65.79; H, 4.30; N, 12.56.
4.1.8.N-(4-(2-((2-chlorophenyl)amino)-3-cyanopyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (10)
Yield, 40 %; m.p. 136-138 °C. IR (KBr, cm-1): 3328, 3248 (2NH), 3046
(CH arom.), 2955, 2925 (CH aliph.), 2190 (C≡N), 1391, 1129 (SO2), 652
(C-Cl). 1H-NMR (DMSO-d6)δ: 2.32 [s, 3H, CH3], 6.78-7.42 [m, 12H, Ar-
H,], 7.80, 7.89 [2d, 2H, 2CH pyridine, J = 8.7 Hz], 10.49, 10.79 [2s, 2H,
13
SO2NH, NH]. C-NMR (DMSO-d6): 20.9, 88.2, 113.2, 115.8, 116.2 (2),
119.5 (2), 120.2 (2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 134.8,
136.1, 138.2, 152.2, 155.6, 157.5 165.2, Anal. Calcd. For C25H19 ClN4O2S
(474.96): C, 63.22; H, 4.03; N, 11.80. Found: C, 63.35; H, 4.28; N, 11.53.
4.1.9.Ethyl-4-((3-cyano-4-(4-(4-
methylphenylsulfonamido)phenyl)pyridin-2-yl)amino)benzoate (11)
Yield, 56.6 %; m.p. 144-146 °C. IR (KBr, cm-1): 3315, 3246 (2NH), 3049
(CH arom.), 2973, 2942 (CH aliph.), 2204 (C≡N), 1731 (C=O), 1363,
1
1142 (SO2). H-NMR (DMSO-d6)δ: 1.65 [t, 3H, CH2CH3], 2.33 [s, 3H,
CH3 tolyl], 4.16 [q, 2H, CH2CH3], 7.03-7.73 [m, 12H, Ar-H,], 7.80, 7.89
[2d, 2H, 2CH pyridine, J = 8.1 Hz], 10.54, 10.93 [2s, 2H, SO2NH, NH].
13C-NMR (DMSO-d6): 14.4, 20.9, 60.8, 84.2, 113.2, 115.8, 116.2 (2),
119.5 (2), 120.2 (2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 134.8,
136.3, 138.2, 150.4, 152.2, 155.6, 165.2, 172.2. Anal. Calcd. For C28H24
N4O4S (512.58): C, 65.61; H, 4.72; N, 10.93. Found: C, 65.36; H, 4.99;
N, 11.23.
4.1.10.N-(4-(3-cyano-2-((4-sulfamoylphenyl)amino)pyridin-4-yl)phenyl)-
4-methylbenzenesulfonamide (12)
Yield, 48 %; m.p. 138-140 °C. IR (KBr, cm-1): 3480, 3356, 3249, (NH2,
NH), 3039 (CH arom.), 2922, 2855 (CH aliph.), 2193 (C≡N), 1333, 1181
1
(2SO2). H-NMR (DMSO-d6)δ: 2.43 [s, 3H, CH3], 6.60, 7.80 [2d, 2H,
2CH pyridine, J= 8.1 Hz], 7.22-7.82 [m, 12H, Ar-H], 8.10 [s, 2H,
13
SO2NH2] 10.20, 10.70 [2s, 2H, SO2NH, NH]. C-NMR (DMSO-d6):
21

20.9, 84.2, 113.2, 115.8, 116.2 (2), 119.5 (2), 120.2 (2), 122.6 (2), 124.1
(2), 126.2, 127.2 (2), 128.2, 134.8, 136.4, 138.2, 150.3, 152.2, 155.6,
165.2. Anal. Calcd. For C25H21N5O4S2 (519.60): C, 57.79; H, 4.07; N,
13.48. Found: C, 57.99; H, 4.37; N, 13.18.
4.1.11.N-(4-(3-cyano-2-((4-(N-
(diaminomethylene)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (13)
Yield, 38.4 %; m.p. 148-50 °C. IR (KBr, cm-1): 3345, 3319, 3278 (NH2,
NH), 3044 (CH arom.), 2920, 2894 (CH aliph.), 2203 (C≡N), 1331, 1158
1
(2SO2). H-NMR (DMSO-d6)δ: 2.31 [s, 3H, CH3], 2.62 [s, 1H, NH
guanido], 6.05 [s, 2H, NH2], 6.85, 8.21 [2d, 2H, 2CH pyridine, J= 8.4
Hz], 7.20-7.90 [m, 12H, Ar-H], 10.21, 10.62, 10.93 [3s, 3H, 2SO2NH,
13
NH]. C-NMR (DMSO-d6): 21.4, 84.2, 113.2, 115.8, 116.2 (2), 119.5
(2), 120.2 (2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 134.8, 136.2,
138.2, 150.3, 152.2, 155.6, 158.2, 160.4. Anal. Calcd. For C26H23N7O4S2
(561.64): C, 59.03; H, 4.10; N, 11.87; Found: C, 59.28; H, 4.43; N, 12.05.
4.1.12.N-(4-(3-cyano-2-((4-(N-(5-methylisoxazol-3-
yl)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (14)
Yield, 44.9 %; m.p. 135-137 °C. IR (KBr, cm-1): 3319, 3217 (3NH), 3016
(CH arom.), 2972, 2873 (CH aliph.), 2202 (C≡N), 1324, 1116 (2SO2). 1H-
NMR (DMSO-d6)δ: 2.28 [s, 3H, CH3 tolyl], 2.32 [s, 3H, CH3 isoxazol],
6.07 [s, H, CH isoxazol], 6.56-7.47 [m, 12H, Ar-H], 7.72, 7.83 [2d, 2H,
CH pyridine, J= 8.4 Hz], 10.89 [s, 3H, 2SO2NH+NH]. 13C-NMR
(DMSO-d6): 37.8, 45.1, 88.1, 95.2, 112.5, 113.1, 115.8, 117.9 (2), 124.2
(2), 126.6 (2), 128.7, 129.6 (2), 129.8, 132 (2), 136.5, 137.1, 138.9, 142.2,
148.5, 151.9, 152.8, 153.2, 157.2, 169.8. Anal. Calcd. For C29H24N6O5S2
(600.12): C, 57.99; H, 4.03; N, 13.99. Found: C, 58.19; H, 4.20; N, 14.30.
4.1.13.N-(4-(3-cyano-2-((4-(N-(thiazol-2-
yl)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (15)
Yield, 31 %; m.p. 122-124 °C. IR (KBr, cm-1): 3270, 3216 (3NH), 3107
(CH arom.), 2967, 2926 (CH aliph.), 2191 (C≡N), 1335, 1157 (2SO2).
1H-NMR (DMSO-d6)δ: 2.34 [s, 3H, CH3], 5.40, 6.55 [2d, 2H, CH
thiazole, J= 8.1 Hz], 7.10-7.80 [m, 12H, Ar-H], 7.85, 7.95 [2d, 2H, CH
13
pyridine, J= 8.2 Hz], 10.60, 10.80 [2s, 3H, 2SO2NH, NH]. C-NMR
22

(DMSO-d6): 22.4, 84.2, 110.2, 113.2, 115.8, 116.2 (2), 119.5 (2), 120.2
(2), 122.6 (2), 124.1 (2), 126.2, 127.2 (2), 128.2, 130.3, 134.8, 136, 138.2,
150.3, 152.2, 155.6, 165.2, 172.1. Anal. Calcd. For C28H22N6O4S3
(602.71): C, 55.80; H, 3.68; N, 13.94. Found: C, 55.46; H, 3.88; N,
14.24.
4.1.14.N-(4-(3-cyano-2-((4-(N-(4-methylpyrimidin-2-
yl)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (16).
Yield, 35 %; m.p. 132-135 °C. IR (KBr, cm-1): 3316, 3260 (2NH), 3032
(CH arom.), 2919, 2873 (CH aliph.), 2195 (C≡N), 1335, 1150 (2SO2).
1H-NMR (DMSO-d6)δ: 2.33 [s, 3H, CH3 tolyl], 2.41 [s, 3H, CH3], 6.95,
7.84 [2d, 2H, 2CH pyrimidine, J= 6.9 Hz], 7.20-7.67 [m, 12H, Ar-H],
7.68, 7.73 [2d, 2H, CH pyridine, J= 10.2 Hz] 10.50, 10.90 [2s, 3H,
13
2SO2NH, NH]. C-NMR (DMSO-d6): 20.4, 25.3, 85.6, 110.5, 113.6,
113.9 (2), 115.2, 117.2 (2), 128.1 (2), 128.4 (2), 129.1, 129.4 (2), 129.8,
130.2 (2), 136.6, 137.6, 138.9, 144.2, 152.2, 152.8, 154.5, 155.4, 168.8,
170.1 Anal. Calcd. For C30H25N7O4S2 (611.69): C, 58.91; H, 4.12; N,
16.03. Found: C, 58.63; H, 4.42; N, 15.88.
4.1.15.N-(4-(3-cyano-2-((4-(N-(4,6-dimethylpyrimidin-2-
yl)sulfamoyl)phenyl)amino)pyridin-4-yl)phenyl)-4-
methylbenzenesulfonamide (17)
Yield, 43 %; m.p. 108-110 °C. IR (KBr, cm-1): 3363, 3236 (2NH), 3022
(CH arom.), 2924, 2844 (CH aliph.), 2204 (C≡N), 1311, 1129 (2SO2).
1H-NMR (DMSO-d6)δ: 2.24 [s, 6H, 2CH3 pyrimidine], 2.32 [s, 3H, CH3
tolyl], 6.53-7.64 [m, 12H, Ar-H], 6.74 [s, H, CH pyrimidine], 7.69, 7.83
[2d, 2H, CH pyridine, J= 8.7 Hz], 10.53, 10.78, 12.25 [3s, 3H, 2SO2NH,
13
NH]. C-NMR (DMSO-d6): 20.3, 24.1, 96.1, 110.5, 111.7, 113.9 (2),
115.2, 117.2 (2), 126.7 (2), 128.7 (2), 129.1, 129.4 (2), 129.8, 130.2 (2),
136.6, 137.6, 138.9, 144.2, 152.2, 152.8, 154.5, 155.4, 166.2 (2), 168.1.
Anal. Calcd. For C31H27N7O4S2 (625.72): C, 59.50; H, 4.35; N, 15.67.
Found: C, 59.22; H, 4.55; N, 15.82.
(E)-N-(4-(1,1-dicyano-4-(substitutedphenyl)buta-1,3-dien-2-yl)phenyl)-4-
methylbenzenesulfonamides (18-22)
General procedure
A mixture of 4 (0.5 g, 0.0015 mol), aromatic aldehydes (0.0015 mole)
namely, benzaldehyde, 4-methylbenzaldehyde, 2-methoxybenzaldehyde,
23

2-hydroxybenzaldehyde and 4-dimethylaminobenzaldehyde in ethanolic
KOH (1.2 g, 20 ml) was stirred overnight while a precipitate was formed.
The obtained product was separated and crystallized from dioxane to give
18-22, respectively.
4.1.16.(E)-N-(4-(1,1-dicyano-4-phenylbuta-1,3-dien-2-yl)phenyl)-4-
methylbenzenesulfonamide (18)
Yield, 40 %; m.p. 140-142°C. IR (KBr, cm-1): 3203 (NH), 3083 (CH
1
arom.), 2993, 2920 (CH aliph.), 2203 (2C≡N), 1374, 1183 (SO2). H-
NMR (DMSO-d6)δ: 2.31 [s, 3H, CH3], 6.96-7.80 [m, 13H, Ar-H], 7.82,
13
7.92 [2d, 2H, CH=CH, J= 7.8 Hz], 9.34 [s, 1H, SO2NH]. C-NMR
(DMSO-d6): 20.8, 88.2, 114.2 (2), 118.6 (2), 122.2, 126.8, 127.8, 128.3
(2), 128.5 (2), 128.8 (2), 129.3 (2), 130.2 (2), 132.2, 135.2, 136.4, 136.9,
137.4, 162.1. Anal. Calcd. For C25H19N3O2S (425.5): C, 70.57; H, 4.50;
N, 9.88. Found: C, 70.77; H, 4.81; N, 9.68.
4.1.17.(E)-N-(4-(1,1-dicyano-4-(p-tolyl)buta-1,3-dien-2-yl)phenyl)-4-
methylbenzenesulfonamide (19)
Yield, 36.4 %; m.p. 240-242°C. IR (KBr, cm-1): 3218 (NH), 3033 (CH
1
arom.), 2916, 2871 (CH aliph.), 2217 (2C≡N), 1338, 1146 (SO2). H-
NMR (DMSO-d6)δ: 2.26 [s, 6H, 2CH3], 6.54, 6.61 [2d, 2H, CH=CH, J=
13
8.7 Hz], 6.76-7.57 [m, 12H, Ar-H], 12.82 [s, 1H, SO2NH]. C-NMR
(DMSO-d6): 20.7 (2), 55.9, 115.6 (2), 116.8 (2), 125.8, 126.4, 127.6,
128.3 (2), 128.6 (2), 128.8 (2), 129.4 (2), 130.2 (2), 132.2, 135.2, 136.4,
136.9, 137.4, 155.4. MS, m/z (%): 440.5 [M+] (5.58), 62.28 (100). Anal.
Calcd. For C26H21N3O2S (439.53): C, 71.05; H, 4.82; N, 9.56. Found: C,
71.34; H, 4.78; N, 9.71.
4.1.18.(E)-N-(4-(1,1-dicyano-4-(2-methoxyphenyl)buta-1,3-dien-2-
yl)phenyl)-4-methylbenzenesulfonamide (20)
Yield, 41 %; m.p. 236-238°C. IR (KBr, cm-1): 3360 (NH), 3089 (CH
arom.), 2973, 2868 (CH aliph.), 2188, 2160 (2C≡N), 1376, 1124 (SO2).
1H-NMR (DMSO-d6)δ: 2.26 [s, 3H, CH3], 3.91 [s, 3H, OCH3], 6.62, 6.64
[2d, 2H, CH=CH, J= 8.7 Hz], 7.05-7.57 [m, 12H, Ar-H], 10.36 [s, 1H,
13
SO2NH]. C-NMR (DMSO-d6): 20.7, 55.9, 68.1, 115.8 (2), 116.4 (2),
118.8, 120.6, 125.7, 125.9, 127.2, 128.4 (2), 128.8, 129.2 (2), 130.2 (2),
131.1, 135.2, 136.4, 136.8, 137.6, 159.2, 191.3. Anal. Calcd. For
C26H21N3O3S (455.53): C, 68.55; H, 4.65; N, 9.22. Found: C, 68.77; H,
4.88; N, 9.55.
24

4.1.19.(E)-N-(4-(1,1-dicyano-4-(2-hydroxyphenyl)buta-1,3-dien-2-
yl)phenyl)-4-methylbenzenesulfonamide (21)
Yield, 52.85 %; m.p. 218-220°C. IR (KBr, cm-1): 3585 (OH), 3388 (NH),
3032 (CH arom.), 2970, 2862 (CH aliph.), 2289, 2229 (2C≡N), 1384,
1
1164 (SO2). H-NMR (DMSO-d6)δ: 2.25 [s, 3H, CH3], 5.65 [s, H, OH],
6.56, 6.60 [2d, 2H, CH=CH, J= 8.7 Hz], 6.82-7.56 [m, 12H, Ar-H], 8.55
13
[s, 1H, SO2NH]. C-NMR (DMSO-d6): 20.7, 55.8, 114.8 (2), 117.4 (2),
119.8, 121.2, 122.4, 125.8, 126.6, 128.2 (2), 128.6, 129.1, 129.4 (2),
130.2 (2), 131.4, 136.2, 136.8, 137.4, 157.2, 166.4. Anal. Calcd. For
C25H19N3O3S (441.5): C, 68.01; H, 4.34; N, 9.52. Found: C, 68.24; H,
4.59; N, 9.19.
4.1.20.(E)-N-(4-(1,1-dicyano-4-(4-(dimethylamino)phenyl)buta-1,3-dien-
2-yl)phenyl)-4-methylbenzenesulfonamide (22)
Yield, 53 %; m.p. 116-118°C. IR (KBr, cm-1): 3393 (NH), 3042 (CH
arom.), 2966, 2865 (CH aliph.), 2189, 2155 (2C≡N), 1376, 1122 (SO2).
1H-NMR (DMSO-d6)δ: 2.32 [s, 3H, CH3], 3.04 [s, 6H, N(CH3)2], 6.74,
7.83 [2d, 2H, CH=CH, J= 8.7 Hz], 6.77-8.00 [m, 12H, Ar-H], 10.79 [s,
1H, SO2NH]. 13C-NMR (DMSO-d6): 20.8, 40.3 (2), 64.2, 110.6 (2), 115.6
(2), 119.4 (2), 124.2, 125.4, 126.8, 128.2 (2), 129.4 (2), 129.8 (2), 130.4
(2), 131.4, 136.4, 136.8, 143.2, 143.6, 163.2. Anal. Calcd. For
C27H24N4O2S (468.5): C, 69.21; H, 5.16; N, 11.96. Found: C, 68.96; H,
5.38; N, 11.68.
4.2. Biological evaluation
4.2.1. In-vitro cytotoxic evaluation against human tumor breast cancer
cell line (MCF-7)
The in-vitro cytotoxic activity was evaluated for nineteen novel
compounds on human breast cancer cell line (MCF-7) via the Sulfo-
Rhodamine-B stain (SRB) assay in the pharmacology unit at the National
Cancer Institute, Cairo University. SRB assay stays one of the most
broadly used techniques for in-vitro cytotoxic screening which was
extended in 1990 [35]. The assay based on the capability of SRB to join
to protein constituents of cellular materials that have been linked to
tissue-culture sheets by trichloroacetic acid (TCA). SRB is a luminous-
25

pink aminoxanthene pigment with double sulfonic groups that attach to
basic amino-acid residues under moderate acidic conditions, and detach
under basic conditions. The quantity of pigment extracted from stained
cellular materials is directly comparative to the cellular material mass
[36]. Cellular materials were plated in 96-multiwell plate (104 cells/ well)
for 24 hrs before conduction with the molecules to permit binding of the
Cellular materials to plate wall. Test molecules were disoluted in
dimethylsulfoxide (DMSO) and diluted with saline to the appropriate
volume. Four different concentrations of the compounds under test (12.5,
25, 50 and 100 µM) were inserted to the cell monolayer. For each
individual dose, Triplicate wells were prepared. Monolayer cells were
incubated with the compounds for 48 hrs at 37ºC and in atmosphere of
5% CO2. After 48 hrs, cells were fixed, washed and stained for 30 min.
with 0.4% (wt/ vol) SRB dissolved in 1% acetic acid. Acetic acid 1%
conc. was used to remove unbounded pigment via four washes, and
bounded stain was recovered with Tris-EDTA buffer. The intensity of
colour was estimated through an enzyme linked immunosorbent assay
(ELISA) reader. The relation between drug concentration and surviving
fraction is plotted to get the survival curve of each carcinoma cell line
after the definite time. The concentration demanded for 50% mortality of
viable cell (IC50) was calculated and compared with E7070 which is
presented to be effective anti-breast cancer agent by in vitro and in vivo
findings [26 b,c] and the results are displayed in table 1.
4.2.2. In vitro Cyclin dependent kinase 2 (CDK2) inhibitory assay
In vitro enzymatic inhibitory assay was completed in the Egyptian
Company for Production of Vaccines, Sera and Drugs (VACSERA),
Dokki, Egyptian. Sandwich enzyme immunoassay is a kit used for in
vitro quantitative evaluation of CDK2 in human tissue homogenates and
other biological fluids (Clound-Clone.Corp.). The microtiter plate has
been pre-coated with an antibody specific to CDK2. Standards or samples
are further added to the proper microtiter plate wells with a biotin-
conjugated antibody specific to CDK2. Then, Avidin conjugated to
Horseradish Peroxidase (HRP) is added to each individual microplate
well and incubated. After addition of TMB substrate solution, only those
well that include CDK2, enzyme conjugated Avidin and biotin
conjugated antibody will exhibit alteration in color. The enzyme-substrate
reaction is finished by the sulphuric acid solution addition and the
variation in color is determined spectrophotometrically at 450 nm
26

±ꢀ10ꢀnm wavelength. CDK2 concentration in the samples is further
determined by comparing the optical density (O.D.) of the samples to the
standard curve. Prepare all reagents, samples and standards; add 100 µL
standard or sample to each well. Incubate 2 hrs at 37 ºC; aspirate and add
100 µL prepared Detection Reagent A. Incubate 1 hr at 37 ºC. Aspirate
and wash 3 times; add 100 µL prepared Detection Reagent B. Incubate 30
minutes at 37 ºC. Aspirate and wash 5 times; add 90 µL substrate
solution. Incubate 15-25 minutes at 37 ºC; add 50 µL Stop Solution. Read
at 450 nm instantly. The results are expressed as IC50 and presented in
Table 2.
4.2.3. Radio-sensitizing evaluation
Irradiation was completed in the National Center for Radiation Research
and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA),
using Gamma cell-40 (137Cs) source. The impressive derivatives 14, 16
and 20 were further elected to be re-evaluated for their in-vitro cytotoxic
activity after combination with γ-irradiation. Cells were plated in 96-
multiwell plate (104cells/well) for one day before γ-irradiation with one
single dose of 8 Gy. Cells were incubated for two days at 37 ˚C in
atmosphere of 5% CO2. After two days, cells were fixed, washed and
pigmented with 0.4% (wt/vol) SRB dissolved in 1% acetic acid, for 30
minutes. Extra unbound dye was separated via four washes with 1%
acetic acid and attached stain was recovered with Tris–EDTA buffer. The
intensity of color was measured in an ELISA reader at 570 nm
wavelength. In another multiwell plate, cells were incubated with the
compounds 14, 16 and 20 in molar concentrations of 12.5, 25, 50 and
100 µM. After 2 h., cells were subjected to a single dose of γ-radiation at
a dose level of 8 Gy with a dose rate of 0.758 rad/sec for 17.73 min, and
then the cytotoxicity was measured two days after irradiation. The
surviving fractions were measured using the above stated techniques by
ELISA reader. The surviving fractions were expressed as mean values
standard error. The results were analyzed using 1-way ANOVA test and
given in Table 3.
27

4.4. Molecular modeling and docking
For docking calculations the x-ray crystal structure of CDK2-CA protein
was downloaded from the protein data bank website (code: 2WXV, res
2.6 Å) [37]. The protein structure was organized for docking via
Accelry’s Discovery Studio 2.5.5 according to the following criteria; the
unwanted monomers were deleted leaving only one kinase and cyclin
complex, the amino acid residues were corrected for errors and the
missing ones were added via loop builder, the pH for ionization was
adjusted at 7.4 and dielectric constant of 10 and protein was protonated
accordingly, the hydrogen atoms were minimized using CHARMm force
fields to relieve bumping and orient hydrogen atoms in the right direction,
finally, all of the water molecules were deleted and only the water
molecules involved in bridging the protein structure or those present in
the binding site of ligand, were retained for accuracy. The ligands for
docking were constructed using the drawing tools in Accelry’s discovery
studio and then they were prepared for docking by adding hydrogens and
minimization using smart minimizer for 1000 steps. The docking
calculations were performed using genetic optimization of ligand docking
method in GOLD 4.1 software. The chemscore_kinase template was
loaded and the protein and ligands structures were saved by GOLD. The
binding site atoms were defined as all the atoms within 8 Å around the
co-crystalized ligand. The genetic algorithm parameters were used
without change except that the number of iterations was increased to
300000 to obtain the maximum accuracy for the binding poses. The early
termination option was disabled and the top 10 poses were saved for each
ligand. Chemscore scoring function was the primary scoring function
used by the chemscore-kinase template and the ligand poses were
rescored using GOLDscore. At last, all the figures were produced using
Pymol 0.99.
Acknowledgments
The authors appreciate the staff members of gamma irradiation unit at the
National Center for Radiation Research and Technology (NCRRT) for
carrying the irradiation process.
28

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