Design and synthesis of some novel 4-Chloro-N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide derivatives as anticancer and radiosensitizing agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.009

A novel series of sulfonamide derivatives 4e21 have been synthesized starting from the strategic starting material (E)-4-Chloro-N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide 4. Two series of hydrazone 5e9, and pyridone 10e21 derivat

Full text of DOI:10.1016/j.ejmech.2016.04.009

Accepted Manuscript
Design and synthesis of some novel 4-Chloro-N-(4-(1-(2-(2-
cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide derivatives as anticancer
and radiosensitizing agents
Mostafa M. Ghorab, Fatma A. Ragab, Helmy I. Heiba, Aiten M. Soliman
PII:
S0223-5234(16)30291-4
10.1016/j.ejmech.2016.04.009
EJMECH 8524
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 February 2016
Revised Date: 30 March 2016
Accepted Date: 4 April 2016
Please cite this article as: M.M. Ghorab, F.A. Ragab, H.I. Heiba, A.M. Soliman, Design and synthesis
of some novel 4-Chloro-N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide
derivatives as anticancer and radiosensitizing agents, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.04.009.
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Graphical Abstract
Title: Design and synthesis of some novel 4-Chloro-N-(4-(1-(2-(2-cyanoacetyl)
hydrazono)ethyl)phenyl)benzenesulfonamide derivatives as anticancer and
radiosensitizing agents.
Novel series of sulfonamide derivatives bearing a biologically active hydrazone or
pyridone moiety were synthesized. The most potent compounds in this study 4, 10
and 12 were evaluated for their radiosensitizing activity.

ACCEPTED MANUSCRIPT
Design and synthesis of some novel 4-Chloro-N-(4-(1-(2-(2-
cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide
derivatives as anticancer and radiosensitizing agents.
Mostafa M. Ghoraba,b*, Fatma A. Ragabc, Helmy I. Heibaa, Aiten M. Solimana.
a
Department of Drug Radiation Research, National Center for Radiation Research and Technology,
Nasr City, Cairo, Egypt.
b Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh
11451, Saudi Arabia.
c Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Cairo University.
*Correspondence author; E-Mail: mmsghorab@yahoo.com ; Tel.: +966-53-4292-860; Fax: +966-01-
4670-560.
Abstract
A novel series of sulfonamide derivatives 4-21 have been synthesized
starting from the strategic starting material (E)-4-Chloro-N-(4-(1-(2-(2-
cyanoacetyl)hydrazono)ethyl)phenyl) benzenesulfonamide 4. Two series of
hydrazone 5-9, and pyridone 10-21 derivatives bearing a sulfonamide moiety were obtained.
All the newly synthesized compounds were evaluated for their in vitro cytotoxic activity
against human liver cancer cell line (HepG2). Compounds 4-6, 8, 9, 10-14 and 16-18 showed
higher activity compared to doxorubicin as a positive control. The radiosensitizing ability of
the most promising compounds 4, 10 and 12 was studied which showed an increase in the cell
killing effect of γ-radiation after combination with these derivatives. The molecular design
was performed to predict the binding mode of the most promising compounds 4, 10 and 12
with the active site of hCA IX, that showed appropriate fitting with the relevant amino
acids in the binding pocket on the basis of standard bond lengths, angles, S score and
E conformation data.
Keywords: Sulfonamide, anticancer, radiosensitizers.
1. Introduction
Based on the World Health Organization (WHO) report, cancer is one of the
most prominent causes of morbidity and mortality worldwide [1]. The discovery of
novel small molecules with potential usefulness as potent and selective anticancer
agents is still a major challenge to medicinal chemistry researchers [2]. Current
antitumor chemotherapy suffers from two major limitations, the first being the lack of
selectivity of conventional chemotherapeutic agents for cancer tissues, causing
unwanted side effects. The second is the acquisition of multiple-drug resistance by
cancer cells, rendering them unresponsive to conventional chemotherapeutic agents
[3,4].
Sulfonamides were found to posses many types of biologically interesting
activities including anticancer activity [5-7]. There are a variety of mechanisms
describing the antitumor action of sulfonamides, such as cell cycle arrest in the G1
phase, disruption of microtubules and angiogenesis inhibition. The most prominent
mechanism is the inhibition of carbonic anhydrase isozymes (CAs) [8]. Aromatic or
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heteroaromatic sulfonamides have been shown to reverse the effect of tumor
acidification, consequently they inhibit the growth of cancer cells and suppress tumor
invasion mediated by carbonic anhydrases [9, 10]. CA inhibitors compromise the
survival of tumor cells due to perturbed pH control, or can be used in combination
with other conventional chemotherapeutics as doxorubicin to improve their uptake
and efficacy due to modulated pH gradient. [11-18].
On the other hand, Literature survey has revealed that hydrazone moiety has
significant biological activity as antibacterial, antimicrobial [19] antioxidant [20],
HIV-1 integrase inhibitor [21] and anticancer [22-25] due to their ability to inhibit
dihydrofolate reductase enzyme [26], also act as EGFR dimerization inhibitor [27]
and c-Met kinase inhibitor, with pronounced selectivity towards T-lymphoblastic
leukaemia cells, HT-29 colon cancer cell line and HepG2 Liver cancer cell line[28].
Also, pyridones have many biological activities as antifungal [29],
antibacterial [30], anti-inflammatory [31], antiviral [32], antimalarial [33], oxytocin
antagonist [34], Liver-targeting SCD1 inhibitor which is a crucial factor in lipid
metabolism and body weight control [35], and anticancer [36] through many
mechanisms as inhibition of FGFR and other members of the tyrosine kinase family,
including VEGFR, c-Kit and Flt3. Met and TAM family kinases inhibitor, through
Axl inhibition [37].
.
As a recent approach for designing new compounds is the development of
hybrid molecules through the combination of different pharmacophores that leads to
compounds with interesting biological profiles [38, 39]. Following this approach and
as a part of an ongoing effort to find alternate chemotherapeutic agents for
hepatocellular carcinoma [40-43], we herein, are reporting the design and synthesis of
some novel sulfonamide derivatives bearing biologically active hydrazone and
pyridone moeities for evaluation as anticancer and radiosensitizing agents on human
liver tumor cell line (HepG2).
2. Results and Discussion
2.1. Chemistry
Schemes 1 and 2 illustrated the synthetic strategies utilized for the synthesis of
the target compounds 4-21 from the starting material N-(4-acetylphenyl)-4-
chlorobenzenesulfonamide 3 [44]. Reaction of 3 with the nitrogen nucleophile 2-
cyanoacetohydrazide yielded the strategic starting material (E)-4-chloro-N-(4-(1-(2-
(2-cyanoacetyl)hydrazono)ethyl)phenyl)benzenesulfonamide 4 in a good yield
(scheme 1). IR spectrum of compound 4 revealed characteristic strong intensity bands
at 3282 and 3202 and 2185 cm-1 for the introduced 2NH and C≡N groups,
respectively, confirming the formation of cyanoacetyl hydrazono derivative. 1H-NMR
spectrum displayed an up-field singlet at 4.16 ppm for the introduced CH2 group and
a downfield shifted singlet appearing at 10.52 ppm due to the addition of NH group,
13
which is exchangeable with D2O. C-NMR exhibited a new up-field signal at 25.3
ppm for the CH2 group and a new signal at 119.9 ppm assigned to the C≡N group.
Reaction of 4 with dimethylformamide dimethylacetal (DMF-DMA) in dry xylene
yielded the corresponding acrylohydrazide derivative 5 through the formation of
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1
carbanion (scheme 1). H-NMR spectrum of 5 displayed an up-field singlet at 3.19
ppm for the addition of N(CH3)2 group and a downfield shifted singlet appearing at
1
7.80 ppm due to the introduced CH group. Also, H-NMR spectrum showed the
disappearance of the singlet corresponding to the CH2 group of 4. 13C-NMR exhibited
a new up-field signal at 66.8 ppm for the introduced N(CH3)2 group and a down-field
signal at 154.0 ppm assigned to the CH group. Reaction of 4 with triethylorthoformate
in acetic anhydride yielded the corresponding acrylohydrazide derivative 6 (scheme
1). 1H-NMR spectrum of 6 displayed up-field triplet and quartet at 1.30 and 4.10 ppm
for the addition of CH3 and CH2 groups, respectively. A downfield shifted singlet
appearing at 8.37 ppm assigned to the CH group. Also, 1H-NMR spectrum showed the
disappearance of the singlet corresponding to the CH2 group of 4. 13C-NMR exhibited
two new up-field signals at 25.5 and 79.8 ppm for the introduced CH3 and CH2
groups, respectively. A down-field signal at 163.2 ppm for the addition of CH group.
Reaction of 4 with CS2 in N,N-dimethylformamide containing KOH followed by
acidification afforded the propanedithioic acid derivative 8 via the non-isolated
intermediate potassium dithiolate 7, while treatment of 7 with 2 moles of methyl
iodide afforded the acrylohydrazide derivative 9 (scheme 1). IR spectrum of 8
revealed two bands at 2300 and 1310 cm-1 corresponding to the SH and C꞊S groups,
1
respectively. H-NMR spectrum displayed an up-field singlet at 2.14 ppm for the SH
group and another singlet at 4.16 ppm assigned to the CH group. 13C-NMR exhibited
1
a new down-field signal at 196.9 ppm for the introduction of C꞊S group. H-NMR
spectrum of 9 displayed an up-field singlet at 2.49 ppm for the 2(S-CH3) groups.
Also, 1H-NMR spectrum showed the disappearance of the singlet corresponding to the
CH2 group of 4. 13C-NMR exhibited a new up-field signal at 28.3 for the 2(S-CH3)
groups and a down-field signal at 176.9 ppm for the addition of S-C-S group.
The synthesis of the target pyridone derivatives 10-21 involved reaction of 4
with different ethyl α-cyanocinnamate derivatives in ethanol through nucleophilic
substitution followed by intramolecular cyclization to yield the corresponding
pyridone derivatives 10-21 (scheme 2). IR spectra confirmed the assigned structures
by the presence of significant bands for NH2, C≡N, 2 C=O and SO2 at their specified
1
regions. H-NMR spectra of 10-21 showed downfield shifted singlet at (10.66-10.74)
ppm due to the introduction of NH2 group which is exchangeable with D2O. Also, 1H-
NMR spectra showed the disappearance of the singlet corresponding to the CH2 group
13
of 4. C-NMR spectra of 10-21 exhibited two new up-field signals for the CH3 and
CH2 groups, another C-C=N signal responsible for the ring closure and a downfield
signal assigned to the C=O ester group at their specified regions.
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2.2. In-vitro anticancer evaluation against human tumor liver cancer
(HepG2)
A closure look to the structure activity relationship (SAR) of the results in
Table 1, it was found that the cyanoacetohydrazone derivative 4 showed the most
potent activity (IC50 ꞊ 8 µM) i.e. four times higher in activity than doxorubicin. The
formation of hydrazone derivatives 5, 6, 8 and 9 (IC50 ꞊ 19.4, 16.8, 17.5 and 19.4 µM,
respectively) still gave potent derivatives compared to the reference drug. The most
active derivative was the ethoxy derivative 6 (IC50 ꞊ 16.8 µM).
Concerning the pyridone derivatives the 4- aryl substituent markedly affected
the cytotoxic activity. Where, compounds 10, 12, 13, 14 and 16 (IC50 ꞊ 10.3, 8.38,
10.7, 11.4 and 11.4 µM, respectively) showed better activity compared to the
reference drug. The most active derivative was 12 with p-tolyl substituent (IC50 ꞊ 8.38
µM). On the other hand compounds 19 and 20 (IC50 ꞊ 37 and 29.7 µM) showed
relatively equipotent activity compared to that of the reference drug, due to the
introduction of m-nitrophenyl and piperonyl substituents to the pyridone ring. While
compounds 15 and 21 with p-methoxyphenyl and p-nitrophenyl substituents showed
poor activity compared to that of doxorubicin.
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Table 1: In vitro anticancer screening of the synthesized compounds against human
liver cell line (HepG2).
Compound concentration (µM)
Cpd. No. 12.5 (µM)
25 (µM)
50 (µM)
100 (µM) IC 50 (µM)
Surviving fraction (mean ± SE) a
0.551 ± 0.026 0.480 ± 0.003 0.139 ± 0.005 0.130 ± 0.016
0.180 ± 0.056 0.159 ± 0.025 0.078 ± 0.008 0.064 ± 0.044
0.642 ± 0.041 0.386 ± 0.049 0.311 ± 0.017 0.281 ± 0.022
0.556 ± 0.020 0.406 ± 0.029 0.228 ±0.014 0.222 ± 0.021
0.542 ± 0.018 0.448 ± 0.044 0.387 ± 0.021 0.248 ± 0.008
0.579 ± 0.030 0.440 ± 0.014 0.329 ± 0.027 0.266 ± 0.010
0.413 ± 0.032 0.367 ± 0.014 0.370 ± 0.016 0.342 ± 0.006
0.617 ± 0.025 0.287 ± 0.008 0.212 ± 0.015 0.179 ± 0.007
0.264 ± 0.021 0.250 ± 0.022 0.214 ± 0.028 0.107 ± 0.015
0.414 ± 0.027 0.336 ± 0.032 0.275 ± 0.020 0.114 ± 0.009
0.489 ± 0.009 0.129 ± 0.006 0.105 ± 0.018 0.087 ± 0.011
0.651 ± 0.025 0.637 ± 0.098 0.613 ±0.003 0.147 ± 0.034
0.488± 0.023 0.545± 0.033 0.342 ± 0.010 0.192 ± 0.015
0.555 ± 0.032 0.463 ± 0.014 0.394 ± 0.016 0.314 ± 0.006
0.653 ± 0.015 0.432 ± 0.006 0.396 ± 0.010 0.196 ± 0.006
0.691 ± 0.023 0.606 ± 0.020 0.373 ± 0.018 0.134 ± 0.015
0.661 ± 0.027 0.524 ± 0.032 0.381 ± 0.020 0.298 ± 0.009
0.713 ± 0.009 0.586 ± 0.006 0.483 ± 0.018 0.212 ± 0.011
32
DOX.
4
8
19.4
16.8
17.5
19.4
10.3
16.8
8.38
10.7
11.4
61.7
11.4
20.2
21
5
6
8
9
10
11
12
13
14
15
16
17
18
19
20
21
37
29.7
41.1
a Each value is the mean of three experiments ± standard error.
2.3. Radiosensitizing evaluation
The ability of the most active compounds 4, 10 and 12 to enhance the cell
killing effect of ɤ-irradiation was studied. From the results obtained in table 2,
compound 4 showed in vitro cytotoxic activity with IC50 value of 8 µM, when the
cells were subjected to different concentrations of the compound alone. While when
the cells were subjected to the same concentrations of compound 4, and irradiated
with a single dose of ɤ-radiation at a dose level of 8 Gy, the IC50 value was
synergistically decreased to 6.5 µM. Similarly, compound 10 showed IC50 value of
10.3 µM when tested alone. The IC50 value was decreased to 8.8 µM, when the cells
were treated with compound 10 in combination with ɤ-radiation. Also, compound 12
showed IC50 value of 8.38 µM when used alone. The IC50 value was decreased to 7.3
µM, when the cells were treated with compound 12 in combination with ɤ-radiation.
The results proved the ability of the synthesized compounds to sensitize cancer cells
to the lethal effect of ionizing radiation in order to decrease the dose of the drug and
decrease its toxicity. The change in IC50 (µM) for compounds 4, 10 and 12 against
HepG2 after irradiation is illustrated in table 2.
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Table 2: In vitro anticancer screening of compounds 4, 10 and 12 against human liver
cell line (HepG2) in combination with radiation.
Compound concentration (µM) +
irradiation (8 Gy)
Surviving fraction (mean ± SE) a
Cpd.
No.
Irradiated
(8 Gy)
IC 50
(µM)
Control
12.5
0.26 ±
0.08*
0.32 ±
0.01*
0.18 ±
0.02*
25
50
100
0.25±
0.01*
0.22±
0.02*
0.15 ±
0.01*
0.12 ±
0.01*
0.10 ±
0.01*
0.12±
0.01*
0.11±
0.01*
0.10 ±
0.01*
0.11 ±
0.01*
1.000
1.000
1.000
0.927 ±0.02*
0.927 ±0.02*
0.927 ±0.02*
6.5
8.8
7.3
4
10
12
a: Each value is the mean of three values ± Standard Error
*: Significant difference from control group at p<0.001
2.4. Molecular modeling and docking study
Carbonic anhydrases (CA, EC 4.2.1.1) represent a family of Zn based
metalloenzymes that catalyzes the interconversion between carbon dioxide and
bicarbonate with generation of protons. Thus inhibition of which has proven
beneficial in the field of selective cancer therapy [11].
Crystallographic data revealed that CA IX, which is a membrane-associated α-
CA, is comprised of a dimer each monomer of which consists of 10-stranded
antiparallel sheets forming the core of the molecule with an intramolecular disulfide
bond between Cys23 and Cys203 [45, 46]. CA IX active site contains a Zn(II) in
coordination with His 94, 96, 119 and a water molecule. The essential binding pocket
amino acids are the proton shuttle residues involved in the binding with inhibitors [44,
45]. Thus, Leu91, Val121, Val131, Leu135, Leu141, Val143, Leu198 and Pro202
define the hydrophobic region of the active site, whereas Arg58, Arg60, Asn62,
His64, Ser65, Gln67, Thr69 and Gln92 identify the hydrophilic one.
In an attempt to rationalize the cytotoxic activity profile exhibited by the
synthesized compounds, a molecular modeling study was carried out. A
conformational search using an implicit solvent model was accomplished for the
prepared compounds; this was followed by refinement of the geometry of local
minima through a quantum-mechanical (QM) method. Subsequently, flexible docking
of the compounds was performed in the crystallographic structure of the CA IX co-
crystallized with a sulfonamide native ligand obtained from the Protein DataBank
(PDB ID: 4BCW) [46] to assess the plausible ability of the new sulfonamide
derivatives to bind in the active pocket of CA IX as a potential molecular target. The
poses obtained for our sulfonamides were found to bind in a co-crystallized ligand-
like fashion with CA IX (Figures 1–5).
Initially, docking validation was performed to assert the ability of the docking
protocol to recognize the active site and reproduce the docking results. Figure 1
displays the superposition of the co-crystallized ligand and the re-docked ligand
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which was found to bind at the same position and with the same manner (S= -14.04
Kcal mol-1).
Figure 1. Docking validation for the re-docked ligand (red) compared to the co-
crystallized ligand (blue) in the active site of CA IX (S=-14.04 Kcal mol-1).
Docking of all the synthesized compounds was performed and herein the
findings obtained for the three most active compounds in this study, sulfonamide
derivatives 4 (Figures 2 and 3) and 10 (Figures 4), 12 (Figure 5) were presented. 3D
ligand interaction of the sulfonamide derivative 4 (Figure 2) shows that the compound
binds in the same site as the co-crystallized ligand with a binding energy reported by
an S score of -10.92 Kcal mol-1. On the other hand, the 2D ligand interaction (Figure
3) demonstrates that the compound binds with the amino acids of the active site
Thr200, Thr199, His119, His96 and His94 as well as the Zn atom through a network
of hydrogen bonds (2.03–3.08Å). Regarding the docking results of compound 10, the
2D ligand interaction simulation (Figures 4) showed that 10 binds in the same fashion
to the co-crystallized ligand displaying a set of hydrogen bonds with the active pocket
amino acids Thr200, Thr199, His119, His96, His94 and Gln92 as well as water and
Zn interactions leading to an overall binding energy of =-10.49 Kcal mol-1. Regarding
the docking results of compound 12, the 2D ligand interaction simulation (Figures 5)
showed that 12 binds in the same fashion to the co-crystallized ligand displaying a set
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of hydrogen bonds with the active pocket amino acids Thr199, His119 and His94 as
well as Zn interactions leading to an overall binding energy of =-10.99 Kcal mol-1.
These observed favorable interactions between CA IX and the new
sulfonamide derivatives might, at least in part, explain the observed cytotoxic activity
of this series of compounds. Further investigations to explore more the plausible
mechanism of action of these derivatives are underway.
Figure 2. 3D Docking of sulfonamide derivative 4 (red) (S=-10.92 Kcal mol-1)
compared to the co-crystallized ligand (blue) in the active site of CA IX.
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Figure 3. 2D ligand interaction of sulfonamide derivative 4 with active site amino
acids of CA IX.
Figure 4. 2D ligand interaction of sulfonamide derivative 10 with active site amino
acids of CA IX.
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Figure 5. 2D ligand interaction of sulfonamide derivative 12 with active site amino
acids of CA IX.
3. Conclusion
In summary, a novel series of sulfonamide derivatives were synthesized. The
prepared compounds were designed and synthesized as potential carbonic anhydrase
inhibitors (CAIs) and evaluated for their potential anticancer activity against human
liver cancer cell line (HepG2). Compounds 4-6, 8-14, 16 and 18 were found to be
more potent than doxorubicin. From the SAR, we may conclude that the introduction
of cyanoacetohydrazone derivative is associated with enhanced anticancer activity
and gave the most potent compound in this study 4 (IC50= 8.0 µM). Combination of
sulfonamide with pyridone moiety with different substitution in compound 12 and 10
also showed potent activity (IC50= 8.38 and 10.3 µM). This study may provide
valuable information for further investigations as anticancer agents. Moreover, the
most active compounds showed interesting radiosensitizing activity when evaluated
for their in-vitro anticancer activity in combination with γ-radiation.
4. Experimental
4.1. Chemistry
Melting points were uncorrected and were taken in an open capillary tube on a
Stuart melting point apparatus (Stuart Scientific, Redhill, UK). The IR spectra of the
compounds were recorded on ABB Bomem FT-IR spectrometer MB 104 with KBr
1
13
pellets. H-NMR and C-NMR spectra were recorded using a Bruker 300 and 400
NMR spectrometers operating at 300 and 400 MHz, respectively. Mass spectra were
run on Shimadzu. GCMS/ QP 5050 mass spectrometer (Shimadzu, Tokyo, Japan).
Microanalyses were obtained with an Elemental analyses system GmbH VarioEL
V300 element analyzer which were found within the limit of 0.4 % of theoretical
values for all the synthesized compounds. The purity of the compounds was checked
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by thin layer chromatography (TLC) with precoated Aluminum sheets Silica gel
Merck 60 F254 and were visualized by UV lamp (Merck, Damstadt, Germany). The
developing solvent system was chloroform/methanol (7:3) and the spots were
1
visualized in UV chamber. IR, H-NMR, 13C-NMR, mass spectra and elemental
analysis were consistent with the assigned structures. IR spectra were performed at
National Center for Radiation Research and Technology, Atomic Energy Authority.
1
While H-NMR and 13C-NMR spectra were performed at Chemical Warfare
department, Ministry of Defense. Mass spectra were done at the Microanalytical
Laboratories of Al-Azhar University. Elemental analysis were performed at the
Microanalytical Laboratories of the Faculty of Science, Cairo University. 4-
Clorobenzene sulfonyl chloride and 4- aminoacetophenon were purchased from
Sigma-Aldrich.
4.1.1.(E)-4-Chloro-N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl)
benzenesulfonamide (4).
A mixture of 2–cyanoacetohydrazide (0.99 g, 0.01 mol) and N-(4-acetylphenyl)-4-
chlorobenzenesulfonamide 3 (3.09 g, 0.01 mol) [44] was refluxed in 1,4-dioxane (15
mL) for 3h. The reaction mixture was poured onto ice/ water and the solid obtained
was crystallized from ethanol to give 4. Yield, 64 %; m.p. 210-212 ºC. IR(KBr,cm-1):
3282, 3202 (2NH), 3052 (CH arom.), 2922, 2830 (CH aliph.), 2185 (C≡N), 1720
1
(C=O), 1610 (C=N), 1347, 1162 (SO2), 754 (C-Cl). H-NMR (DMSO-d6): 2.15 [s,
3H, CH3], 4.16 [s, 2H, CH2], 7.10, 7.80 [2d, 4H, Ar-H AB system, J= 6.8 Hz], 7.61 [s,
1H, SO2NH exchangeable with D2O], 7.64, 7.76 [2d, 4H, Ar-H AB system, J= 6.8
13
Hz], 10.52 [s, 1H, NH exchangeable with D2O]. C-NMR (DMSO-d6): 22.0 (CH3),
25.3 (CH2), 119.9 (C≡N), 127.7, 127.7, 127.8, 129.0, 129.0, 129.9, 129.9, 130.0,
130.0, 134.5, 138.4, 138.7 (C-phenyl), 154.0 (C=N), 159.0 (C꞊O). MS m/z (%): 390.8
(M+) (1.4), 392.5 (0.4), 90.7 (100). Anal. Calcd. For C17H15ClN4O3S (390.84): C,
52.24; H, 3.87; N, 14.33 Found: C, 52.38; H, 3.90; N, 14.50.
4.1.2.4-Chloro-N-(4-((E)-1-(2-((E)-2-cyano-3-(dimethylamino)acryloyl)
hydrazono)ethyl)phenyl)benzenesulfonamide (5)
A mixture of 4 (3.9 g, 0.01 mol) and dimethylformamide-dimethylacetal (DMF-
DMA) (1.19 g, 0.01 mol), in dry xylene (15 ml) was refluxed for 5h. The reaction
mixture was cooled, and the solid obtained was crystallized from ethanol to give 5.
º
Yield, 56%; m.p. 240-242 C. IR (KBr, cm-1): 3385, 3321 (2NH), 3097 (CH arom.),
2922, 2859 (CH aliph.), 2185 (C≡N), 1710 (C=O), 1602 (C=N), 1373, 1180 (SO2),
1
755 (C-Cl). H-NMR (DMSO-d6)δ: 2.13 [s, 3H, CH3], 3.19 [s, 6H, N(CH3)2], 7.07-
7.76 [m, 8H, Ar-H], 7.60 [s, 1H, SO2NH exchangeable with D2O], 7.80 [s, 1H, CH],
13
10.51 [s, 1H, NH exchangeable with D2O]. C-NMR (DMSO-d6): 23.5 (CH3), 66.8,
66.8 (N(CH3)2), 119.9 (C-CN), 120.0 (C≡N), 127.7, 127.7, 129.0, 129.0, 129.0, 129.9,
129.9, 134.5 ,134.5, 138.3, 138.3, 138.5 (C-phenyl), 138.7 (C=N), 154.0 (CH), 159.0
(C꞊O). MS m/z (%): 445.8 (M+) (15.4), 447.7 (5.8), 63.6 (100). Anal. Calcd. For
C20H20ClN5O3S (445.92): C, 53.87; H, 4.52; N, 15.71; Found: C, 54.01; H, 4.73; N,
15.82.
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4.1.3.4-Chloro-N-(4-((E)-1-(2-((E)-2-cyano-3-ethoxyacryloyl)hydrazono)ethyl)
phenyl) benzenesulfonamide (6)
A mixture of 4 (3.9 g, 0.01 mol) and triethyl orthoformate (5 ml), in acetic anhydride
(5 ml) was refluxed for 8h. Acetic anhydride was evaporated, and the reaction mixture
was triturated with ethanol and filtered. The solid obtained was crystallized from
º
ethanol to give 6. Yield, 49%; m.p. 140-142 C. IR (KBr, cm-1): 3359, 3325 (2NH),
3098 (CH arom.), 2919, 2923 (CH aliph.), 1664 (C=O), 1605 (C=N), 1367, 1184
1
(SO2), 748 (C-Cl). H-NMR (DMSO-d6)δ: 1.30 [t, 3H, CH3CH2], 1.81 [s, 3H, CH3],
4.1 [q, 2H, CH2], 7.08-8.08 [m, 8H, Ar-H], 7.69 [s, 1H, SO2NH exchangeable with
D2O], 8.37 [s, 1H, CH], 10.63 [s, 1H, NH exchangeable with D2O]. 13C-NMR
(DMSO-d6): 25.5 (CH2-CH3), 26.8 (CH3), 79.8 (CH2), 107.9 (C-CN), 113.5 (C≡N),
118.6, 118.6, 119.8, 129.0, 129.0, 130.0, 130.0, 130.2, 130.2, 131.0, 132.6, 138.4 (C-
phenyl), 142.3 (C=N), 144.2 (C꞊O), 163.2 (CH). MS m/z (%): 446.6 (M+) (6.4), 448.5
(2.1), 90 (100). Anal. Calcd. For C20H19ClN4O4S (446.91): C, 53.75; H, 4.29; N,
12.54; Found: C, 54.11; H, 4.34; N, 12.69.
4.1.4.(E)-3-(2-(1-(4-(4-Chlorophenylsulfonamido)phenyl)ethylidene)hydrazinyl)-2-
cyano-3-oxopropanedithioic acid (8) and (E)-4-Chloro-N-(4-(1-(2-(2-cyano-3,3-
bis(methylthio)acryloyl)hydrazono)ethyl) phenyl)benzenesulfonamide (9)
Carbon disulfide (0.76 g, 0.01 mol) was added gradually to a cold solution of 4 (3.9 g,
0.01 mol) in N,N-dimethylformamide (15mL) containing finely grounded potassium
hydroxide (1.0 g). The reaction mixture was stirred at room temperature for 24h, then
triturated with cold water (50 mL) and neutralized with 1N HCl. The resulting
precipitated solid was collected, filtered and crystallized from dioxane to give 8.
While compound 9 was obtained by the same experimental procedure described
above for the synthesis of 8 except that two moles of methyl iodide (2.82 g, 0.02 mol)
were added before the reaction was left at room temperature.
º
8; Yield, 30.1%; m.p. 130-132 C. IR (KBr, cm-1): 3386, 3351 (2NH), 3091 (CH
arom.), 2916, 2869 (CH aliph.), 2300 (SH), 2182 (C≡N), 1664 (C=O), 1597 (C=N),
1
1355, 1156 (SO2), 1310 (C=S), 762 (C-Cl). H-NMR (DMSO-d6)δ: 2.14 [s, 1H, SH
exchangeable with D2O], 2.24 [s, 3H, CH3], 4.16 [s, 1H, CH], 7.08-7.83 [m, 8H, Ar-
H], 7.66 [s, 1H, SO2NH exchangeable with D2O], 10.58 [s, H, NH exchangeable with
13
D2O]. C-NMR (DMSO-d6): 26.8 (CH3), 112.7 (C-CN), 112.7 (C≡N), 118.7, 118.7,
119.8, 129.0, 129.0, 130.0, 130.0, 130.3, 130.3, 132.7, 138.3, 138.5 (C-phenyl),
142.3 (C=N), 162.7 (C꞊O), 196.9 (C=S). MS m/z (%): 466.7 (M+) (1.0), 468.5 (0.3),
90.8 (100). Anal. Calcd. For C18H15ClN4O3S3 (466.98): C, 46.30; H, 3.24; N, 12.00;
Found: C, 46.06; H, 3.10; N, 11.89.
9; Yield, 48 %; m.p. 90-92 ºC. IR (KBr, cm-1): 3365, 3234 (2NH), 3043 (CH arom.),
2925, 2873 (CH aliph.), 2177 (C≡N), 1675 (C=O), 1600 (C=N), 1356, 1138 (SO2),
1
744 (C-Cl). H-NMR (DMSO-d6)δ: 2.08 [s, 3H, CH3], 2.49 [s, 6H, 2CH3], 6.72-7.64
[m, 8H, Ar-H], 7.35 [s, 1H, SO2NH exchangeable with D2O], 10.56 [s, H, NH
13
exchangeable with D2O]. C-NMR (DMSO-d6): 26.8 (CH3), 28.3, 28.3 (2(S-CH3)),
112.7 (C-CN), 112.7 (C≡N), 118.7, 118.7, 119.8, 129.0, 129.0, 130.0, 130.0, 130.3,
13

ACCEPTED MANUSCRIPT
130.3, 132.7, 138.3, 138.4 (C-phenyl), 142.3 (C=N), 162.7 (C꞊O), 176.9 (S-C-S). MS
m/z (%): 494.7 (M+) (11.4), 496.8 (3.2), 52.2 (100). Anal. Calcd. For C20H19ClN4O3S3
(495.04): C, 48.52; H, 3.87; N, 11.32; Found: C, 48.37; H, 3.44; N, 11.03.
4.1.5. General procedure for the preparation of compounds 10-21:
A mixture of 4 (3.9 g, 0.01 mol) and ethyl α-cyanocinnamate derivatives (0.01 mol)
namely, (E)-ethyl 2-cyano-3-(furan-2-yl)acrylate, (Z)-ethyl 2-cyano-3-phenylacrylate,
(Z)-ethyl 2-cyano-3-(p-tolyl)acrylate, (2Z,4E)-ethyl 2-cyano-5-phenylpenta-2,4-
dienoate, (Z)-ethyl 2-cyano-3-(2-methoxyphenyl)acrylate, (Z)-ethyl 2-cyano-3-(4-
methoxyphenyl)acrylate, (Z)-ethyl 3-(2-chlorophenyl)-2-cyanoacrylate, (Z)-ethyl 3-
(4-chlorophenyl)-2-cyanoacrylate,
(dimethylamino)phenyl)acrylate,
(Z)-ethyl
2-cyano-3-(4-
(Z)-ethyl
3-(benzo[d][1,3]dioxol-5-yl)-2-
cyanoacrylate, (Z)-ethyl 2-cyano-3-(3-nitrophenyl)acrylate, (Z)-ethyl 2-cyano-3-(4-
nitrophenyl)acrylate in 1,4-dioxane (15 mL) containing piperidine (0.5 mL) was
refluxed for 5 h. The reaction mixture was poured onto ice/water and the formed solid
was crystallized from dioxane to give 10-21, respectively.
4.1.5.1.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-4-(furan-2-yl)-6-oxo-1,6-
dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (10)
Yield, 76.2 %; m.p. >300 ºC. IR (KBr, cm-1): 3364, 3243, 3225 (NH2,NH), 3094 (CH
arom.), 2941, 2859 (CH aliph.), 2200 (C≡N), 1703, 1656 (2 C=O), 1599 (C=N), 1330,
1189 (SO2), 758 (C-Cl). 1H-NMR (DMSO-d6)δ: 0.82 [t, 3H, CH3 ester], 2.17 [s, 3H,
CH3], 3.63 [q, 2H, CH2], 7.06-7.80 [m, 8H, Ar-H], 7.07 [t, 1H, CH-CH-CH furan],
7.56 [s, 1H, SO2NH exchangeable with D2O], 7.60 [m, 1H, C-CH furan], 7.72 [m, 1H,
CH-O furan] 10.73 [s, 2H, NH2 exchangeable with D2O]. 13C-NMR (DMSO-d6): 22.6
(CH3 ester), 26.9 (CH3), 44.2 (CH2), 83.7 (C-C=O), 118.7, 118.7 (CH furan), 119.7
(C≡N), 128.0, 128.0 (C-phenyl), 129.0 (C-C≡N), 129.1, 130.0, 130.0, 130.1, 130.1,
130.2, 130.2, 130.3, 130.5, 134.0 (C-phenyl), 138.4, 138.7 (C-O furan), 139.2 (C-
NH2), 143.5 (C꞊O), 157.5 (C=N), 165.2 (C=O ester), 174.8 (C-pyridine). MS m/z
(%): 580 (M+) (13.4), 581.8 (4.1), 194.0 (100). Anal. Calcd. For C27H22ClN5O6S
(580.01): C, 55.91; H, 3.82; N, 12.07; Found: C, 55.58; H, 3.53; N, 12.01.
4.1.5.2.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-6-oxo-4-phenyl-1,6-
dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (11)
º
Yield, 28.3 %; m.p. 255-257 C. IR (KBr, cm-1): 3328, 3247, 3213 (NH2,NH), 3094
(CH arom.), 2941, 2808 (CH aliph.), 2195 (C≡N), 1670, 1667 (2 C=O), 1601 (C=N),
1345, 1161 (SO2), 746 (C-Cl). 1H-NMR (DMSO-d6)δ: 0.81 [t, 3H, CH3 ester], 2.14 [s,
3H, CH3], 3.65 [q, 2H, CH2], 7.3-7.70 [m, 13H, Ar-H], 7.46 [s, 1H, SO2NH
exchangeable with D2O], 10.74 [s, 2H, NH2 exchangeable with D2O]. 13C-NMR
(DMSO-d6): 22.6 (CH3 ester), 28.9 (CH3), 44.2 (CH2), 83.8 (C-C=O), 120.6 (C-C≡N),
120.6 (C≡N), 127.6, 127.6, 127.6, 127.6, 129.1, 129.6, 129.6, 129.6, 130.0, 130.0,
130.1, 130.1, 130.2, 130.2, 130.3, 130.5, 134.0, 134.6 (C-phenyl), 134.7 (C-NH2),
147.8 (C꞊O), 157.0 (C=N), 163.7 (C=O ester), 174.8 (C-pyridine). MS m/z (%): 590.0
14

ACCEPTED MANUSCRIPT
(M+) (1.8), 592.1 (0.6), 104 (100). Anal. Calcd. For C29H24ClN5O5S (590.05): C,
59.03; H, 4.10; N, 11.87; Found: C, 59.28; H, 4.43; N, 12.05.
4.1.5.3.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-6-oxo-4-(p-tolyl)-1,6-
dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (12)
º
Yield, 32.5 %; m.p. 263-265 C. IR (KBr, cm-1): 3327, 3228, 3213 (NH2,NH), 3086
(CH arom.), 2945, 2860 (CH aliph.), 2203 (C≡N), 1673, 1667 (2 C=O), 1607 (C=N),
1340, 1160 (SO2), 740 (C-Cl). 1H-NMR (DMSO-d6)δ: 0.82 [t, 3H, CH3 ester], 1.26 [s,
1H, CH3 tolyl], 1.56 [s, 3H, CH3], 3.68 [q, 2H, CH2], 6.91-7.77 [m, 12H, Ar-H], 7.48
[s, 1H, SO2NH exchangeable with D2O], 10.69 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 21.4 (CH3 ester), 22.5 (CH3), 29.1 (CH3 p-tolyl), 44.2 (CH2),
83.6 (C-C=O), 120.8 (C-C≡N), 120.8 (C≡N), 128.2, 128.2, 128.7, 129.2, 129.2, 129.6,
129.6, 129.6, 129.6, 130.0, 130.0, 130,0, 130.0, 130.1, 130.3, 130.5 131.7, 134.6 (C-
phenyl), 138.5 (C-NH2), 147.8 (C꞊O), 157.1 (C=N), 163.7 (C=O ester), 175.0 (C-
pyridine). MS m/z (%): 603.8 (M+) (9.4), 605.7 (3.1), 92.2 (100). Anal. Calcd. For
C30H26ClN5O5S (604.08): C, 59.65; H, 4.34; N, 11.59; Found: C, 59.28; H, 4.23; N,
11.55.
4.1.5.4.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-6-oxo-4-((E)-styryl)-1,6-
dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (13)
º
Yield, 53.8 %; m.p. 145-147 C. IR (KBr, cm-1): 3320, 3287, 3233 (NH2,NH), 3054
(CH arom.), 2939, 2856 (CH aliph.), 2334 (C≡N), 1738, 1687 (2 C=O), 1602 (C=N),
1348, 1169 (SO2), 756 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.05 [t, 3H, CH3 ester], 2.16 [s,
3H, CH3], 3.74 [q, 2H, CH2], 7.02, 7.04 [2d, 2H, CH=CH], 7.30-7.77 [m, 13H, Ar-H],
7.43 [s, 1H, SO2NH exchangeable with D2O], 10.69 [s, 2H, NH2 exchangeable with
D2O]. 13C-NMR (DMSO-d6): 22.6 (CH3 ester), 28.9 (CH3), 44.2 (CH2), 83.8 (C-
C=O), 120.6 (C-C≡N), 125.4 (C≡N), 127.6, 127.6 (C-phenyl), 127.8 (CH2 enol),
127.9, 128.0, 128.5, 128.5, 129.1, 129.1, 129.6, 129.6, 130.0, 130.0, 130.1, 130.1 (C-
phenyl), 130.2 (CH2 enol), 130.3, 130.5, 134.0, 134.6 (C-phenyl), 134.7 (C-NH2),
147.8 (C꞊O), 157.0 (C=N), 163.7 (C=O ester), 174.8 (C-pyridine). MS m/z (%): 615.5
(M+) (13.4), 617.8 (4.9), 97.7 (100). Anal. Calcd. For C31H26ClN5O5S (616.09): C,
60.43; H, 4.25; N, 11.37; Found: C, 60.28; H, 4.23; N, 11.15.
4.1.5.5.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-4-(2-methoxyphenyl)-6-
oxo-1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (14)
º
Yield, 29.1 %; m.p. 161-163 C. IR (KBr, cm-1): 3381, 3234, 3219 (NH2,NH), 3094
(CH arom.), 2942, 2856 (CH aliph.), 2216 (C≡N), 1732, 1657 (2C=O), 1600 (C=N),
1345, 1132 (SO2), 758 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.03 [t, 3H, CH3 ester], 2.06 [s,
3H, CH3], 3.60 [s, 3H, OCH3], 3.66 [q, 2H, CH2], 7.02-7.74 [m, 12H, Ar-H], 7.58 [s,
1H, SO2NH exchangeable with D2O], 10.69 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.6 (CH3 ester), 25.9 (CH3), 44.8 (OCH3), 48.2 (CH2), 83.8
(C-C=O), 112.9 (C-phenyl), 113.7 (C-C≡N), 118.8 (C≡N), 127.6, 127.6, 128.0, 128.5,
129.1, 129.6, 129.6, 129.8, 130.0, 130.0, 130.1, 130.1, 130.2, 130.3, 130.5, 134.6 (C-
phenyl), 134.7 (C-NH2), 140.0 (C- phenyl), 147.8 (C꞊O), 157.0 (C=N), 163.7 (C=O
15

ACCEPTED MANUSCRIPT
ester), 174.8 (C-pyridine). MS m/z (%): 619.8 (M+) (8.2), 621.5 (2.8), 90 (100). Anal.
Calcd. For C30H26ClN5O6S (620.08): C, 58.11; H, 4.23; N, 11.29; Found: C, 58.38; H,
4.45; N, 11.54.
4.1.5.6.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-4-(4-methoxyphenyl)-6-
oxo-1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (15)
º
Yield, 83 %; m.p. 161-163 C. IR (KBr, cm-1): 3328, 3294, 3203 (NH2,NH), 3094
(CH arom.), 2934, 2854 (CH aliph.), 2203 (C≡N), 1792, 1677 (2 C=O), 1602 (C=N),
1345, 1161 (SO2), 753 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.03 [t, 3H, CH3 ester], 2.14 [s,
3H, CH3], 3.52 [s, 3H, OCH3], 3.66 [q, 2H, CH2], 6.91-8.12 [m, 13H, Ar-H], 7.44 [s,
1H, SO2NH exchangeable with D2O], 10.69 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.6 (CH3 ester), 25.3 (CH3), 44.6 (OCH3), 48.0 (CH2), 83.5
(C-C=O), 112.9, 112.9 (C-phenyl), 113.7 (C-C≡N), 118.8 (C≡N), 126.5, 126.5, 126.7,
127.8, 128.8, 128.8, 131.2, 131.2, 132.1, 132.1, 132.6, 132.6, 133.3, 133.5, 134.6 (C-
phenyl), 134.7 (C-NH2), 140.0 (C- phenyl), 147.9 (C꞊O), 157.0 (C=N), 162.3 (C=O
ester), 163.8 (C-pyridine). MS m/z (%): 619.5 (M+) (3.4), 621.6 (1.0), 90.7 (100).
Anal. Calcd. For C30H26ClN5O6S (620.08): C, 58.11; H, 4.23; N, 11.29; Found: C,
58.28; H, 4.29; N, 11.37.
4.1.5.7.(E)-N-(4-(1-((2-amino-4-(2-chlorophenyl)-5-cyano-3-ethylcarboxylate-6-oxo-
1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (16)
º
Yield, 19.4 %; m.p. 110-112 C. IR (KBr, cm-1): 3315, 3257, 3223 (NH2,NH), 3086
(CH arom.), 2934, 2855 (CH aliph.), 2206 (C≡N), 1794, 1667 (2 C=O), 1597 (C=N),
1331, 1172 (SO2), 745 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.39 [t, 3H, CH3 ester], 2.15 [s,
3H, CH3], 3.60 [q, 2H, CH2], 6.90-8.02 [m, 12H, Ar-H], 7.44 [s, 1H, SO2NH
exchangeable with D2O], 10.73 [s, 2H, NH2 exchangeable with D2O]. 13C-NMR
(DMSO-d6): 21.5 (CH3 ester), 22.1 (CH3), 43.6 (CH2), 71.5 (C-C=O), 115.3 (C-C≡N),
115.8 (C≡N), 116.4, 116.4, 126.7, 127.5, 127.9, 128.7, 128.7, 129.1, 129.1, 129.3,
129.9, 130.0, 130.0, 135.3, 135.8, 137.5, 137.8, 140.0 (C-phenyl), 152.3 (C-NH2),
160.0 (C꞊O), 163.6 (C=N), 165.3 (C=O ester), 169.8 (C-pyridine). MS m/z (%): 624.5
(M+) (5.2), 626.5 (1.9), 185.2 (100). Anal. Calcd. For C29H23Cl2N5O5S (624.49): C,
55.77; H, 3.71; N, 11.21; Found: C, 55.88; H, 3.94; N, 11.37.
4.1.5.8.(E)-N-(4-(1-((2-amino-4-(4-chlorophenyl)-5-cyano-3-ethylcarboxylate-6-oxo-
1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (17)
º
Yield, 30.8 %; m.p. 148-150 C. IR (KBr, cm-1): 3346, 3250, 3220 (NH2,NH), 3076
(CH arom.), 2941, 2865 (CH aliph.), 2217 (C≡N), 1792, 1636 (2 C=O), 1598 (C=N),
1334, 1169 (SO2), 740 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.23 [t, 3H, CH3 ester], 2.14 [s,
3H, CH3], 3.60 [q, 2H, CH2], 7.12-7.97 [m, 12H, Ar-H], 7.49 [s, 1H, SO2NH
exchangeable with D2O], 10.67 [s, 2H, NH2 exchangeable with D2O]. 13C-NMR
(DMSO-d6): 21.5 (CH3 ester), 22.1 (CH3), 43.6 (CH2), 71.5 (C-C=O), 115.3 (C-C≡N),
115.8 (C≡N), 116.4, 116.4, 126.7, 127.5, 127.5, 127.9, 127.9, 128.7, 128.7, 129.1,
129.1, 129.1, 129.1, 129.3, 129.9, 135.3, 135.8, 137.5 (C-phenyl), 152.3 (C-NH2),
160.0 (C꞊O), 163.6 (C=N), 165.3 (C=O ester), 169.8 (C-pyridine). MS m/z (%): 624.4
(M+) (6.4), 626.5 (2.2), 185.2 (100). Anal. Calcd. For C29H23Cl2N5O5S (624.49): C,
55.77; H, 3.71; N, 11.21; Found: C, 55.82; H, 3.91; N, 11.39.
16

ACCEPTED MANUSCRIPT
4.1.5.9.(E)-N-(4-(1-((2-amino-5-cyano-4-(4-(dimethylamino)phenyl)-3-
ethylcarboxylate-6-oxo-1,6-dihydropyridin)imino)ethyl)phenyl)-4-
chlorobenzenesulfonamide (18)
º
Yield, 38 %; m.p. 258-260 C. IR (KBr, cm-1): 3300, 3206, 3169 (NH2,NH), 3090
(CH arom.), 2934, 2858 (CH aliph.), 2207 (C≡N), 1708, 1687 (2 C=O), 1595 (C=N),
1324, 1188 (SO2), 753 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.50 [t, 3H, CH3 ester], 2.22 [s,
3H, CH3], 3.06 [s, 6H, N(CH3)2], 3.63 [q, 2H, CH2], 6.50-8.00 [m, 12H, Ar-H], 7.99
[s, 1H, SO2NH exchangeable with D2O], 10.66 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.6 (CH3 ester), 30.1 (CH3), 42.7, 42.7 (N(CH3)2), 44.2
(CH2), 83.8 (C-C=O), 116.4, 116.4 (C-phenyl), 119.2 (C-C≡N), 119.2 (C≡N), 125.1,
125.1, 127.6, 128.3, 128.3, 128.3, 128.6, 128.6, 129.6, 129.6, 130.2, 130.2, 130.3,
133.0, 134.0 (C-phenyl), 134.3 (C-N(CH3)2), 134.7 (C-NH2), 147.1 (C꞊O), 157.0
(C=N), 163.7 (C=O ester), 174.8 (C-pyridine). MS m/z (%): 632.8 (M+) (3.1), 634.5
(1.2), 90 (100). Anal. Calcd. For C31H29ClN6O5S (633.12): C, 58.81; H, 4.62; N,
13.27; Found: C, 58.99; H, 4.79; N, 13.39.
4.1.5.10.(E)-N-(4-(1-((2-amino-4-(benzo[d][1,3]dioxol-5-yl)-5-cyano-3-
ethylcarboxylate-6-oxo-1,6-dihydropyridin)imino)ethyl)phenyl)-4-
chlorobenzenesulfonamide (19)
Yield, 42.9 %; m.p. >300 ºC. IR (KBr, cm-1): 3340, 3258, 3210 (NH2,NH), 3088 (CH
arom.), 2958, 2895 (CH aliph.), 2202 (C≡N), 1798, 1648 (2 C=O), 1556 (C=N), 1364,
1154 (SO2), 748 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.51 [t, 3H, CH3 ester], 2.21 [s, 3H,
CH3], 3.67 [q, 2H, CH2 ester], 6.09 [s, 2H, CH2], 6.80-7.70 [m, 13H, Ar-H], 7.66 [s,
1H, SO2NH exchangeable with D2O], 10.67 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.0 (CH3 ester), 22.7 (CH3), 44.1 (CH2 ester), 83.8 (C-C=O),
93.8 (CH2), 101.5, 101.5 (C-phenyl), 107.7 (C-C≡N), 110.5 (C≡N), 120.7, 120.7,
120.7, 123.6, 123.6, 123.6, 123.6, 128.2, 128.2, 128.2, 128.2, 130.0, 130.2, 130.3,
130.5, 134.0 (C-phenyl), 146.6 (C-NH2), 147.8 (C꞊O), 156.7 (C=N), 163.7 (C=O
ester), 174.7 (C-pyridine). MS m/z (%): 633.9 (M+) (11.4), 635.5 (3.2), 90.7 (100).
Anal. Calcd. For C30H24ClN5O7S (634.06): C, 56.83; H, 3.82; N, 11.05; Found: C,
56.96; H, 4.00; N, 11.30.
4.1.5.11.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-4-(3-nitrophenyl)-6-oxo-
1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (20)
º
Yield, 34.4 %; m.p. 130-132 C. IR (KBr, cm-1): 3372, 3259, 3233 (NH2,NH), 3086
(CH arom.), 2932, 2852 (CH aliph.), 2200 (C≡N), 1790, 1657 (2 C=O), 1607 (C=N),
1507, 1310 (NO2), 1341, 1152 (SO2), 742 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.23 [t, 3H,
CH3 ester], 2.16 [s, 3H, CH3], 3.61 [q, 2H, CH2], 6.92-8.30 [m, 12H, Ar-H], 7.54 [s,
1H, SO2NH exchangeable with D2O], 10.69 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.6 (CH3 ester), 26.7 (CH3), 44.2 (CH2), 83.8 (C-C=O),
110.6 (C-C≡N), 110.6 (C≡N), 124.0, 124.0, 127.6, 127.8, 128.1, 128.9, 128.9, 129.0,
129.0, 129.9, 130.0, 130.0, 130.1, 130.2, 130.3, 130.5, 134.0 (C-phenyl), 134.2 (C-
NO2), 134.7 (C-NH2), 147.8 (C꞊O), 157.0 (C=N), 163.7 (C=O ester), 174.8 (C-
pyridine). MS m/z (%): 634.5 (M+) (5.9), 636.7 (1.8), 120.2 (100). Anal. Calcd. For
C29H23ClN6O7S (635.05): C, 54.85; H, 3.65; N, 13.23; Found: C, 54.99; H, 3.82; N,
13.41.
17

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4.1.5.12.(E)-N-(4-(1-((2-amino-5-cyano-3-ethylcarboxylate-4-(4-nitrophenyl)-6-oxo-
1,6-dihydropyridin)imino)ethyl)phenyl)-4-chlorobenzenesulfonamide (21)
º
Yield, 94.5 %; m.p. 141-143 C. IR (KBr, cm-1): 3360, 3234, 3213 (NH2,NH), 3096
(CH arom.), 2934, 2858 (CH aliph.), 2211 (C≡N), 1780, 1650 (2 C=O), 1600 (C=N),
1500, 1318 (NO2), 1349, 1142 (SO2), 740 (C-Cl). 1H-NMR (DMSO-d6)δ: 1.21 [t, 3H,
CH3 ester], 2.10 [s, 3H, CH3], 3.84 [q, 2H, CH2], 6.94-8.32 [m, 12H, Ar-H], 7.77 [s,
1H, SO2NH exchangeable with D2O], 10.68 [s, 2H, NH2 exchangeable with D2O].
13C-NMR (DMSO-d6): 22.6 (CH3 ester), 26.7 (CH3), 44.2 (CH2), 83.8 (C-C=O),
110.6 (C-C≡N), 110.6 (C≡N), 124.0, 124.0, 127.6, 127.6, 128.1, 128.9, 128.9, 129.0,
129.0, 129.9, 129.9, 130.2, 130.2, 130.3, 130.5, 134.0, 134.0 (C-phenyl), 134.2 (C-
NO2), 134.7 (C-NH2), 147.8 (C꞊O), 157.0 (C=N), 163.7 (C=O ester), 174.8 (C-
pyridine). MS m/z (%): 634.6 (M+) (7.2), 636.5 (2.4), 58.4 (100). Anal. Calcd. For
C29H23ClN6O7S (635.05): C, 54.85; H, 3.65; N, 13.23; Found: C, 55.01; H, 3.71; N,
13.47.
4.2. In-vitro anticancer evaluation against human tumor liver cancer
(HepG2)
The in-vitro anticancer activity was measured for seventeen new compounds
on liver human tumor cell line (HepG2) using the Sulfo-Rhodamine-B stain (SRB)
assay. The in-vitro anticancer screening was done by the pharmacology unit at the
National Cancer Institute, Cairo University.
The sulfo-rhodamine B (SRB) assay which was developed in 1990 [47], remains one
of the most widely used methods for in-vitro cytotoxic screening. The assay relies on the
ability of SRB to bind to protein components of cells that have been fixed to tissue-culture
plates by trichloroacetic acid (TCA). SRB is a bright-pink aminoxanthene dye with two
sulfonic groups that bind to basic amino-acid residues under mild acidic conditions, and
dissociate under basic conditions. The amount of dye extracted from stained cells is directly
proportional to the cell mass [48]. Cells were plated in 96-multiwell plate (104 cells/ well) for
24 hrs before treatment with the compounds to allow attachment of the cells to the wall of the
plate. Test compounds were dissolved in dimethylsulfoxide (DMSO) and diluted with saline
to the appropriate volume. Different concentrations of the compounds under test (12.5, 25, 50
and 100 ꢀM) were added to the cell monolayer. Triplicate wells were prepared for each
individual dose. 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. Unbounded dye was removed by four
washes with 1% acetic acid, and attached stain was recovered with Tris-EDTA buffer. Color
intensity was measured in an enzyme linked immunosorbent assay (ELISA) reader. The
relation between surviving fraction and drug concentration is plotted to get the survival curve
of each tumor cell line after the specified time. The concentration required for 50%
inhibition of cell viability (IC50) was calculated and compared with the reference drug
d
oxorubicin and the results are given in table
1.
Doxorubicin (CAS, 25316-40-9), the reference drug used in this study is a
drug used in cancer chemotherapy. It is an anthracycline antibiotic, it works by
18

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intercalating DNA and inhibition of macromolecular biosynthesis. It is commonly
used in the treatment of a wide range of cancers as in acute leukemia’s, Hodgkin’s
disease, and other lymphomas [49].
4.3. Radisensitizing evaluation
Irradiation was performed at the National Center for Radiation Research and
Technology, Atomic Energy Authority, using Gamma cell-40 (137Cs) source. The
most active compounds 4, 10 and 12 were selected to be re-evaluated for their in-vitro
cytotoxic activity in combination with γ-irradiation. Cells were plated in 96-multiwell
plate (104cells/well) for 24 h. before γ-irradiation with a single dose of 8 Gy. Cells
were incubated for 48 h. at 37 ˚C in atmosphere of 5% CO2. After 48 h., cells were
fixed, washed and stained with 0.4% (wt/vol) SRB dissolved in 1% acetic acid, for 30
minutes. Excess unbound dye was removed by four washes with 1% acetic acid and
attached stain was recovered with Tris–EDTA buffer. Color intensity was measured
by an ELISA reader at a wave length of 570 nm. In another multiwell plate, cells were
incubated with compounds 4, 10 and 12 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, then the cytotoxicity was
measured 48h. after irradiation. The surviving fractions were measured using the
above mentioned procedures 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 2 .
4.4. Molecular modeling and docking
The molecular model of the new sulfonamide derivatives was built using
standard bond lengths and angles, with the MOE software suite 2007.09. Following
geometry optimization, a systematic conformational search was carried out to an RMS
gradient of 0.01Å with energy minimization of the resultant conformations employing
the ConfSearch module implemented in MOE. All molecular mechanics computations
were performed with the Merck Force Field (MMFF94s). The experimental
crystallographic structures of CA IX complex was retrieved from the Protein
DataBank (PDB ID: 4BCW). Missing hydrogens were added to the enzyme and
partial charges were calculated. After removing the co-crystallized inhibitor,
validation followed by docking of the compounds were carried out using MOE
software suite 2007.09. The target protein was kept rigid, while the ligands were left
free to explore the conformational space inside the enzyme cavity; 100 separate
docking simulations were run using default parameters and the conformations were
chosen based on the combination of S score data, E conformation and appropriate
fitting with the relevant amino acids in the binding pocket.
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List of captions:
Scheme 1. Synthesis of the hydrazone derivatives 4-9
Scheme 2. Synthesis of the pyridone derivatives 10-21
Table 1: In vitro anticancer screening of the synthesized compounds against human
liver cell line (HepG2).
Table 2: In vitro anticancer screening of compounds 4, 10 and 12 against human liver
cell line (HepG2) in combination with radiation.
Figure 1. Docking validation for the re-docked ligand (red) compared to the co-
crystallized ligand (blue) in the active site of CA IX (S=-14.04 Kcal mol-1).
Figure 2. 3D Docking of sulfonamide derivative 4 (red) (S=-10.92 Kcal mol-1)
compared to the co-crystallized ligand (blue) in the active site of CA IX.
Figure 3. 2D ligand interaction of sulfonamide derivative 4 with active site amino
acids of CA IX.
Figure 4. 2D ligand interaction of sulfonamide derivative 10 with active site amino
acids of CA IX.
Figure 5. 2D ligand interaction of sulfonamide derivative 12 with active site amino
acids of CA IX.
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Research Highlights
•
•
•
•
Novel sulfonamide derivatives bearing a hydrazone moiety were synthesized.
Novel sulfonamide derivatives bearing a pyridone moiety were synthesized.
In-vitro anticancer evaluation on HepG2 cell line.
Radiosensitizer evaluation for the most potent compounds.