Synthesis, molecular docking and evaluation of novel sulfonyl hydrazones as anticancer agents and COX-2 inhibitors

Article abstract of DOI:10.1007/s11030-019-09974-z

Abstract: In trying to develop new anticancer agents, a series of sulfonylhydrazones were synthesized. All synthesized compounds were checked for identity and purity using elemental analysis, TLC and HPLC and were characterized by their melting points, FT-IR and NMR spectral data. All synthesized compounds were evaluated for their cytotoxic activity against prostate cancer (PC3), breast cancer (MCF-7) and L929 mouse fibroblast cell lines. Among them, N′-[(2-chloro-3-methoxyphenyl)methylidene]-4-methylbenzenesulfonohydrazide (3k) showed the most potent anticancer activity against both cancer cells with good selectivity (IC₅₀ = 1.38?μM on PC3 with SI = 432.30 and IC₅₀ = 46.09?μM on MCF-7 with SI = 12.94). Further investigation confirmed that 3k displayed morphological alterations in PC3 and MCF-7 cells and promoted apoptosis through down-regulation of the Bcl-2 and upregulation of Bax expression. Additionally, compound 3k was identified as the most potent COX-2 inhibitor (91% inhibition) beside lower COX-1 inhibition. Molecular docking of the tested compounds represented important binding modes which may be responsible for their anticancer activity via inhibition of the COX-2 enzyme. Overall, the lead compound 3k deserves further development as a potential anticancer agent. Graphic abstract: Sulfonylhydrazones was synthesized and N′-[(2-chloro-3-methoxyphenyl)methylidene]-4-methylbenzenesulfonohydrazide (3k) was identified as the most potent anticancer agent and COX-2inhibitor. In addition, this compound docked inside the active site of COX-2 succesfully. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-019-09974-z

Molecular Diversity
https://doi.org/10.1007/s11030-019-09974-z
ORIGINAL ARTICLE
Synthesis, molecular docking and evaluation of novel sulfonyl
hydrazones as anticancer agents and COX‑2 inhibitors
Sevil Şenkardeş1 · M. İhsan Han2 · Necla Kulabaş1 · Mürüvvet Abbak3 · Özge Çevik4 · İlkay Küçükgüzel1
Ş. Güniz Küçükgüzel1
·
Received: 20 March 2019 / Accepted: 4 July 2019
© Springer Nature Switzerland AG 2019
Abstract
In trying to develop new anticancer agents, a series of sulfonylhydrazones were synthesized. All synthesized compounds
were checked for identity and purity using elemental analysis, TLC and HPLC and were characterized by their melting
points, FT-IR and NMR spectral data. All synthesized compounds were evaluated for their cytotoxic activity against prostate
cancer (PC3), breast cancer (MCF-7) and L929 mouse fbroblast cell lines. Among them, N′-[(2-chloro-3-methoxyphenyl)
methylidene]-4-methylbenzenesulfonohydrazide (3k) showed the most potent anticancer activity against both cancer cells
with good selectivity (IC50 =1.38 μM on PC3 with SI=432.30 and IC50 =46.09 μM on MCF-7 with SI=12.94). Further
investigation confrmed that 3k displayed morphological alterations in PC3 and MCF-7 cells and promoted apoptosis through
down-regulation of the Bcl-2 and upregulation of Bax expression. Additionally, compound 3k was identifed as the most
potent COX-2 inhibitor (91% inhibition) beside lower COX-1 inhibition. Molecular docking of the tested compounds repre-
sented important binding modes which may be responsible for their anticancer activity via inhibition of the COX-2 enzyme.
Overall, the lead compound 3k deserves further development as a potential anticancer agent.
The research was partly presented at the 6th International
Bahçeşehir University (BAU) Drug Design Congress on 13–15
December 2018, İstanbul, Turkey.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-09974-z) contains
supplementary material, which is available to authorized users.
* Sevil Şenkardeş
sevil.aydin@marmara.edu.tr
1
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Marmara University, Haydarpaşa,
34668 İstanbul, Turkey
2
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Erciyes University, Kayseri, Turkey
3
Scientifc Technology Research and Application Centre,
Adnan Menderes University, Aydın, Turkey
4
Department of Biochemistry, School of Medicine, Adnan
Menderes University, Aydın, Turkey
Vol.:(0123456789)
1 3

Molecular Diversity
Graphic abstract
Sulfonylhydrazones was synthesized and N′-[(2-chloro-3-methoxyphenyl)methylidene]-4-methylbenzenesulfonohydrazide
(3k) was identifed as the most potent anticancer agent and COX-2inhibitor. In addition, this compound docked inside the
active site of COX-2 succesfully.
Keywords Anticancer activity · Apoptosis · Cyclooxygenase · Molecular docking · Sulfonylhydrazones
anti-apoptotic Bcl-2 and proapoptotic Bax genes expression
are key regulators at the mitochondria. As long as a drug
alters the expression of apoptotic proteins, it might induce
the apoptosis in cancer cells [5].
Introduction
Cancer is a major leading cause of death all over the world
since the beginning of the twenty-frst century, and the eco-
nomic efect of this disease is important and increasing. Dis-
ease can develop various organs and systems of body at any
age with no certain etiopathology. Among the anticancer
therapies, chemotherapy receives much attention due to its
efects on tumor cells. However, many anticancer drugs dis-
play serious adverse efects and toxicity; there is an urgent
need to identify new compounds to fght against the fatal
disease [1].
Carcinogenesis mostly breaks through multistep pro-
cesses containing various somatic gene alterations. In
particular, prostate and breast cancers have been charac-
terized by alterations of genes and proteins involved in
proinfammatory pathways [6, 7]. Cyclooxygenase is a key
enzyme in prostanoid biosynthesis including prostaglan-
dins that are also involved in infammation. Cyclooxyge-
nase-2 isoform (COX-2), which is an important enzyme
in prostaglandin synthesis, is induced in many cells by
infammatory mediators and plays important roles in the
“infammogenesis of cancer” [8]. Several studies have
shown that COX-2 is overexpressed in solid malignan-
cies including breast [9], prostate [10] and colon [11]
cancers. Recently, studies have shown a notable correla-
tion between the expression of COX-2 and inhibition of
apoptosis, so COX-2 is an emerging drug target for cancer
treatment [12].
Apoptosis, or programmed cell death, is an important
process crucial for elimination of unnecessary or unwanted
cells. The feature of cancer is the ability of malignant cells
to escape from apoptosis [2]. Many compounds that have
been found to be a potential source of novel anticancer drugs
show their antitumor activity by inducing apoptosis. Apop-
tosis induction occurs via extrinsic and intrinsic pathways
[3]. The extrinsic pathway contains the binding of ligands to
cell surface “death receptors” [4]. In the intrinsic pathway,
1 3

Molecular Diversity
Fig. 1 Some reported anticancer
agents bearing sulfonylhydra-
zone
In drug design strategy, small organic molecules play
a critical role and act as extremely bioactive scaffolds.
Hydrazone group, a prominent moiety present richly in
medicinal molecules, has played a significant role in the
development of different therapeutic agents. On the other
hand, various biological [13] and chemotherapeutic activ-
ities of hydrazones [14–18] suggest them as an important
pharmacophoric character. In particular, sulfonylhydra-
zones have been recognized as potential antimicrobial
[19], antifungal [20], antiurease [21] and MAO inhibi-
tory [22] activities, and as illustrated in Fig. 1, numer-
ous sulfonylhydrazones have been recognized as poten-
tial anticancer agents such as compounds I and II which
showed a significant anticancer activity against melanoma
and liver cancer cell lines [23, 24], compounds III exhib-
ited displayed high anticancer activities against HCT116
colon cancer and A549 lung cancer cell lines [25], and
compound IV bearing benzotriazole ring was powerfully
effective against DAN-G pancreas cancer cell line [26]
(Fig. 1).
Results and discussion
Chemistry
The synthesis of sulfonylhydrazones (3a–o) followed the
general pathway outlined in Scheme 1. In the initial step,
p-toluenesulfonyl hydrazide (2) was synthesized via the
reaction of tosyl chloride (1) and hydrazine hydrate in THF.
The reaction of p-toluenesulfonyl hydrazide (2) with substi-
tuted aldehydes aforded 4-methyl-N′-(arylmethylidene)ben-
zenesulfonyl hydrazides (3a–o). The IR, 1H-NMR and 13C-
NMR data were in agreement with the proposed structures
of compounds. Among the newly synthesized compounds,
compounds 3i and 3n have been reported previously [27,
28] and compound 3g has got only a CAS number (CAS No:
461716-16-5) with no spectroscopic data. So, we presented
full structural characterization not reported in the above-
mentioned literature, including melting point, NMR data and
elemental analysis.
The HMBC spectra of 3g chosen as a prototype were
very useful for locating the tertiary and quaternary carbons
by identifying the various protons interacting with them
through two-bond, three-bond and occasionally four-bond
couplings as displayed in Fig. 2. Based on the HMBC spec-
trum, the correlations between H-1 (δH = 2.36) and C-2
(δC = 144.03), the correlations between H-3 (δH = 7.41)
and C-1, C-2, [δC=21.43 (C-1), 144.03 (C-2), the correla-
tions between H-4 (δH = 7.76) and C-2 (δC = 144.03), the
correlations between H-6 (δH = 7.92) and C-7, C-8, C-12
[δC = 126.78 (C-7), 123.56 (C-8), 132.61 (C-12)], the
On the basis of aforementioned fndings, herein some
new sulfonylhydrazones have been synthesized and evalu-
ated for their potential anticancer, apoptosis-inducing and
COX enzyme activities. Also, the designed compounds were
docked into COX-1 and COX-2 enzymes in order to exam-
ine the COX-1/2 selectivity and probable binding modes of
derivatives inside the active site.
1 3

Molecular Diversity
Scheme 1 The synthetic route
for compounds 3a–o
Fig. 2 HMBC spectrum and (H to C) interactions in compound 3g
correlations between H-8 (δH = 8.04) and C-6, C-9, C-10,
C-12 [δC = 145.19 (C-6), 139.72 (C-9), 153.45 (C-10),
132.61 (C-12)], the correlations between H-11 (δH=7.39)
and C-10 (δC = 153.45), the correlations between H-12
(δH = 7.85) and C-8, C-10 [δC = 123.56 (C-8), 153.45
(C-10)] and the correlations between H-13 (δH=3.94) and
C-10 (δC=153.45) indicated the structure of 3g should be
as shown in Fig. 2.
1 3

Molecular Diversity
Table 1 In vitro cytotoxic efect of compounds 3a–o against human
Table 2 The in vitro COX inhibition percentages and docking results
cancer cell lines and a normal cell line
of the active compounds
Compound IC50 (μM)
Compound COX-1
COX-2
PC3
MCF-7 L929
176.18 154.57 1.55
66.23 164.30 2.32
140.29 240.68 3.63
331.50 0.46
SI* (PC3) SI* (MCF-7)
Inhibition Binding afn- Inhibition Binding
% (50 μM) ity ∆G (kcal/ % (50 μM) afnity ∆G
2
99.20
70.65
66.20
0.87
2.48
1.71
11.09
ND
0.02
5.48
ND
0.025
1.78
1,11
12.94
5.47
ND
mol)
(kcal/mol)
3a
3b
3c
3d
3e
3f
2
26
13
29
49
76
43
56
69
82
28
64
48
63
56
16
32
n.t.
n.d.
25
41
20
75
14
11
80
19
13
82
73
91
64
88
22
64
100
n.t.
3a
3b
3c
3d
3e
3f
−7.3
−6.7
−7.6
−6.9
−7.1
−8.0
−6.7
−7.2
−8.0
−7.8
−8.6
−7.0
−8.4
−7.2
−7.0
−12.0
713.59 29.89
173.63 529.80 ND
43.77
341.61 28.31
188.05 360.43 ND
138.09 405.18 10.20
23.77
28.33
1.38
−6.2
−8.1
−8.5
−7.1
−7.9
−8.5
−8.7
−6.8
−8.1
−7.7
−8.0
−7.5
n.d.
ND
0.17
287.77 7.49
155.10 0.45
3g
3h
3i
3j
3k
3l
3m
3n
3o
ND
0.07
3g
3h
3i
3j
3k
3l
3m
3n
3o
181.34 324.60 13.66
283.25 314.90 11.12
46.09
596.58 432.30
143.17 167.95 920.03 6.42
189.06 204.69 ND
314.40 114.30 0.99
ND
0.003
ND
0.006
ND
4.08
35.39
104.09 ND
Celecoxib 150.00 128.01 523
Cisplatin 3.3* 22.2*
3.4
−7.4
−10.5
Celecoxib 92
ND not determined, *Lit: [31]
SI: IC50 for L929 cell line/IC50 for PC3 and MCF-7 cell line
n.d. not determined, n.t. not tested
3k has SI yielding 432.30 for PC3, and 12.94 for MCF-7
cell line.
Anticancer activity
Celecoxib was used as a positive control because its selec-
tive COX-2 inhibition correlated with induction of apopto-
sis in several cancer models, anticancer properties [29, 30],
and it is the bound ligand of COX-2 enzyme used in our
molecular modeling studies. Cisplatin has been one of the
most widely used chemotherapeutic drugs and has been used
in clinics for the treatment of various cancers. Altaf et al.
reported the cytotoxicity values of cisplatin against PC3
and MCF-7 cell lines with IC50 values of 3.3 and 22.2 μM,
respectively [31]. Compared with data, the lead compound
3k showed potent anticancer activity superior to those of
celecoxib and cisplatin.
The anticancer activity of the synthesized compounds was
evaluated on two human cancer cell lines, namely human
breast adenocarcinoma cell line (MCF-7), prostate cell line
(PC-3) and mouse normal fbroblasts (L929) using MTT
assay.
The four compounds 3i–k and 3o demonstrated potent
antiproliferative activity against PC3 cell line, and com-
pounds 3c, 3f and 3k demonstrated potent antiproliferative
activity against MCF-7 cell line. The starting compound
2 did not show promising anticancer activity against both
cancer cells.
Moreover, compound 3k showed the most promis-
ing efects for both tumor cell lines with lower IC50 val-
ues (IC50 =1.38 μM against PC3 cell and IC50 =46.09 μM
against MCF-7 cell). The ideal anticancer drug candidate
should be cytotoxic to cancer cells and be harmless for nor-
mal cells. The toxicity profles of compounds were deter-
mined against L929 cells indicating a considerable safety
profle. The IC50 and selectivity index (SI) results of the
compounds are presented in Table 1. Strikingly, the results
propose that active derivatives clearly are more cytotoxic to
cancer cells than the tested normal cell L929 according to
the selectivity index values. The most selective compound
In vitro COX‑1/2 inhibitory activities
It is well known that COX-2 plays a critical role in a num-
ber of solid tumors. Therefore, this enzyme is taken as a
target for anticancer drug development. The results of the
COX-1/2 inhibition studies of the compounds are listed
in Table 2. Celecoxib, which was used as a positive con-
trol, provided 100% inhibition at 50 μM. In the present
series, compounds 3c, 3f, 3i, 3j, 3k and 3m showed high
COX-2 inhibition (73–91%) at a concentration of 50 μM
1 3

Molecular Diversity
while the least active compounds like 3d and 3h with weak
cyclooxygenase-2 inhibition potency. Conversely, 3d and
3h showed the highest COX-1 inhibitory activity (76% and
82%, respectively) with high selectivity and no anticancer
efect. All synthesized compounds were less potent COX
inhibitors than celecoxib.
displayed notable cytotoxicity against PC3 cell line with an
IC50 value of 28.33 μM.
Compound 3k inhibits cell proliferation and induced
morphological changes
Compound 3k bearing 2-chloro-3-methoxyphenyl moi-
ety is the most active derivative as COX-2 inhibitor by
91% inhibition (with a binding energy of -8.6 kcal/mol),
while compound 3e with 2-chloro-3-trifuoromethylphenyl
moiety is the least active compound (11% inhibition with
binding energy of -7.1 kcal/mol). These data indicated that
the methoxy substituent at the meta-position of the phenyl
rings is essential for their inhibitory activity. Moreover,
compound 3d bearing 4-fuoro-3-phenoxy showed 14%
COX-2 enzyme inhibition while replacing the phenoxy
group by methoxy on the phenyl ring increases the inhibi-
tion as seen in the compound 3f displayed 80% inhibition.
According to the acquired data, there was a generally
relationship between COX-2 enzyme inhibition and anti-
cancer activity results. Compounds 3k and 3i that showed
the highest COX-2 inhibition appeared as the most active
anticancer agents, while the weak inhibitors 3h and 3d
showed the lowest anticancer activity. Although celecoxib
was the strongest COX-2 inhibitor in this study, it showed
weak cytotoxicity against MCF-7 and PC-3 cell lines.
This is because the inhibitory efect of celecoxib on the
proliferation of breast and prostate cancer cells in vitro
was observed in a dose- and time-dependent manner [30,
32]. For celecoxib, 4 days was required to achieve 37%
cell growth inhibition against PC3 cell line at 5 μM [33].
The exact mechanisms for its anticancer activity are not
clear yet, and Grosch et al. showed that celecoxib showed
antiproliferative efect through COX-independent mecha-
nisms [34].
Herein, compound 3k was chosen for further studies on
the possible mechanism of its anticancer activity. MTT
assay was performed to assess the efect of 3k on PC3
and MCF-7 cell proliferation. When PC3 and MCF-7 cells
were incubated with 3k at IC50 values for 12, 24 and 48 h,
the collected data suggested that compound 3k decreased
the viability of PC3 and MCF-7 cells in a time-dependent
manner (Fig. 3). In addition, PC3 and MCF-7 cells treated
with 3k at IC50 value for 24 h were stained with crys-
tal violet and cell growth inhibition and morphological
changes were observed under inverted microscope. While
both cell lines clearly exhibited an increase in cell number
(Fig. 3, DMSO only control), treatment with compound 3k
gave rise to a marked decrease in cellular confuency such
as reduction of cell population, reduction of cell volume,
cell shrinkage and loss of cellular architecture.
Compound 3k induces apoptosis
through mitochondrial pathway
To reveal the possible mechanisms responsible for the
apoptotic efects of compound 3k, we evaluated its efects
on the expression level of a series of proteins related to
mitochondrial apoptosis. Therefore, PC3 and MCF-7 cells
were blotted for Bax and Bcl-2, following treatment with
compound 3k (Figs. 4a and 5a) and quantifed (Figs. 4b–d
and 5b–d). As expected, the proapoptotic Bax levels were
increased in PC3 cells, and the level of increase in Bax
expression was similar in response to MCF-7 cells. On the
other hand, the decrease in Bcl-2 levels varied between the
cells. While there was little to no change in Bcl-2 expres-
sion following compound 3k treatment in cell lines, Bax/
Bcl-2 ratio of cells treated with compound 3k, expres-
sion levels were signifcantly increased in PC3 and MCF-7
cells.
While anticancer efect of COX-2 inhibitors depends on
dose and time, this efect cannot be associated with COX-2
enzyme inhibition alone because COX-2 inhibitors show
anticancer efects via COX-2-dependent and COX-2-inde-
pendent pathways [35]. Among them, COX-2 inhibitors
trigger apoptosis in cancer cell as a result of a decreased
level of Bcl-2 protein. SC-58125, which is a derivative of
celecoxib, induced apoptosis by down-regulation of Bcl-2
like lead compound 3k [36], whereas celecoxib induced
apoptosis independent of Bcl-2 down-regulation in vari-
ous cancer cells [30]. The induction of apoptosis in can-
cer cells by COX-2 inhibitors is a unique process, and the
increased COX-1/COX-2 selectivity may not display a cor-
related anticancer efect [30, 37]. As shown in Tables 1 and
2, compound 3m, selective COX-2 inhibitor, did not show
any signifcant anticancer activity in both cancer cell lines.
Moreover, compound 3j, which is less selective than 3m,
In silico docking studies
Clinical studies have shown that COX-2 levels are increased
in a number of human cancers and COX-2 isoenzyme
released in cancer cells is an ideal drug target to inhibit car-
cinogenesis [38]. Computer-assisted molecular modeling
techniques have been used to estimate the possible inhibitory
activities and mechanism of binding to COX enzymes of the
synthesized compounds. Trans-isomers of compounds 3a-o
1 3

Molecular Diversity
Fig. 3 Efect of compound 3k on cell survival in human prostate
cancer cells PC3 and human breast cancer cells MCF-7. a Efect of
compound 3k (46 µM) on cell growth of MCF-7 cells for 24 h. Mor-
phological changes with bright light (up) and stained with crystal vio-
let (down). b Percentage of cell growth in MCF-7 cells treated with
3k (46 µM) for 12 h, 24 h, 48 h (++p<0.01, +++p<0.001 com-
pared to control in MCF-7 cells). c Efect of compound 3k (1.4 µM)
on cell growth of PC3 cells for 24 h. Morphological changes with
bright light (up) and stained with crystal violet (down). d Percentage
of cell growth in PC3 cells treated with 3k (1.4 µM) for 12 h, 24 h,
48 h (++p<0.01, +++p<0.001 compared to control in PC3 cells)
(+p<0.05, ++p<0.01, +++p<0.001 compared to control in PC3
cells)
were evaluated since they have more appropriate poses than
cis-isomers in COX active site. Binding energy obtained
from the docking studies of the compounds 3a–o as well
as celecoxib as reference ligand with COX-1 and COX-2 by
using Autodock Vina is presented in Table 2.
were compared with celecoxib in active site, and it was
detected that these compounds have not appropriate ori-
entation in binding pocket. The reason for the weak inhibi-
tion of these compounds was that they did not overlap with
the ligand as shown in Fig. 6b.
The in silico studies were in agreement with the in vitro
results where docking of hydrazone derivatives showed a
relative selectivity of these derivatives to COX-II active
site. Results of both in vitro enzyme inhibition assay and
molecular modeling studies have shown that compounds
3k and 3m are the best COX-2 inhibitors among the syn-
thesized compounds. When binding poses of both synthe-
sized compounds and celecoxib were compared in active
site, we detected highly similar orientations of compounds
3k, 3m and celecoxib (Fig. 6a). In addition, binding poses
of compounds 3b and 3g with minimum binding afnity
When the interactions of compound 3k with active
site of COX-2 enzyme were examined, hydrogen bonds
between sulfonyl group with side chains of both Tyr341
and Ser339 were detected. Moreover, we observed that
while sulfonylhydrazone group formed backbone hydrogen
bond with Val509, the methoxy group formed hydrogen
bond with the side chain of Ser516, too. Also, contribution
of pi-alkyl interaction of among methyl group and Val509
besides alkyl–alkyl weak interaction of between this group
and Ala502 was observed. Finally, pi-alkyl hydrophobic
interactions between phenyl ring which is substituted with
1 3

Molecular Diversity
Fig. 4 Efects of 3k on apoptotic proteins in breast cancer cells. a
Efects of compound 3k (46 µM) compound treatment at 24 h on the
Bcl-2 and Bax proteins in MCF-7 cells, representatively. b–d West-
ern blot densitometry analysis of expression levels of Bcl-2 and Bax
proteins c Ratio of Bax/Bcl-2 expression in MCF-7 cells (++p<0.01,
+++p<0.001 compared to control in MCF-7 cells). Each protein
band was normalized to the intensity of GAPDH used
sulfonyl group and the side chains of Ser339 and Val509
are thought to contribute to the activity (Fig. 7).
As a result of these interactions, by holding constant the
sulfonyl hydrazone core, substitution of electron-withdraw-
ing groups on both phenyl rings may increase the H-bond
interaction with Ser339, Leu338, Tyr341 and Ser516 as
similar to celecoxib.
(http://www.swissadme.ch/) online tools are given in
Table 3. Regarding in silico toxicity results for mutagenic-
ity, tumorigenicity, irritant and reproductive efects, almost
all synthesized compounds were predicted to be safe. The
clogP is an important data indicating the lipophilicity and
according to Lipinski’s rule of fve, the clogP value of ≤ 5
is necessity for a compound to be a drug candidate. Our
results showed that cLogP values for the target compounds
were well within the acceptable criteria. Polar surface area
(PSA) values of the drug candidates have been shown to
correlate very well with the human intestinal absorption,
Caco-2 monolayer’s permeability, and blood–brain bar-
rier penetration and values of 140 or more are expected
to exhibit poor intestinal absorption. The results show
that all the compounds have optimal TPSA values. The
aqueous solubility of a compound signifcantly afects its
absorption and distribution characteristics. Typically, a
low solubility means a bad absorption and suitable values
for logS > 4 were observed for the hydrazones that were
synthesized herein. The title compounds do not violate
the Lipinski rule, and they fall well in the range (Table 3).
Molecular modeling studies have shown that compounds
3d and 3h are the best COX-1 inhibitors among the synthe-
sized compounds as in the in vitro enzyme inhibition assay.
Besides, no interaction could be detected between COX-1
active site and compounds showing below 20% inhibition
during biological studies. Furthermore, for COX-2 selective
compounds 3c, 3f, 3i, 3k and 3m, in vitro enzyme inhibition
assay and molecular modeling studies results were found to
be correlated with each other.
In silico prediction of potential pharmacokinetic
and toxicological properties
The in silico toxicity risks obtained by OSIRIS Data War-
rior software (http://www.openmolecules.org/datawarrio
r/) and ADME properties calculated using SwissADME
1 3

Molecular Diversity
Fig. 5 Efects of 3k on apoptotic proteins in prostate cancer cells.
a Efects of compound 3k (1.4 µM) treatment at 24 h on the Bcl-2
and Bax proteins in PC3 cells, representatively. b-d Western blot
densitometry analysis of expression levels of Bcl-2 and Bax pro-
teins. c Ratio of Bax/Bcl-2 expression in PC3 cells (++p<0.01,
+++p<0.001 compared to control in PC3 cells). Each protein band
was normalized to the intensity of GAPDH used
Fig. 6 The comparison of a
orientations of most active
compounds 3k (cyan) and 3m
(green) b orientations of least
active compounds 3b (brown)
and 3g (black) with celecoxib
(red) in COX-2 active site.
(Color fgure online)
Estimated intestinal absorption (%ABS) was calculated
by: %ABS = 109 − [0.345 × topological polar surface area
(TPSA)] according to the method of Zhao et al. [39].
Swissadme server was also used to obtain compara-
tive information derived from poorly and highly absorbed
drugs to model human passive intestinal absorption. It
works by calculating the lipophilicity and polarity of the
molecules. The graphical prediction of intestinal absorp-
tion and blood–brain barrier permeation of the synthesized
compounds is shown in Fig. 8. Boiled egg plot denotes
bioavailable region of property space with respect to wlog
P and TPSA [yellow: well penetrated within the brain with
good intestinal absorption; white: intestinal absorption;
gray: poor intestinal absorption]. This provides a simple
visual cue for profling new compounds in terms of their
potential to be orally absorbed [40]. Inside yellow circle
depicts BBB—blood brain barrier; two of the compounds
are in this region. The white region which is outer to yel-
low depicts the human intestinal absorption. Almost all
compounds lie in this white area. None of the compounds
1 3

Molecular Diversity
Fig. 7 Interaction of compound
3k with COX-2 active site
are lying outside [gray area] which indicates poor intestinal
absorption. The blue dots indicate that compound 3g could
be a substrate for P-glycoprotein, reducing its absorption and
penetration within brain. Hence, these compounds may be
said to exhibit good bioavailability. In particular, compound
3k, the most promising agent, can be absorbed very easily by
the gastrointestinal tract with no potential BBB permeability.
lines besides COXs activities. Compound 3k was identi-
fed as the most potent anticancer agent in the series with
IC50 values of 1.38 μM and 46.09 μM on PC3 and MCF-7
cell lines, respectively, and showed the highest selectiv-
ity index values. Curiously, the potent anticancer com-
pounds showed high COX-2 inhibition percentages (up to
64%) at one dose comparable to celecoxib used as a posi-
tive control. After analysis of the results, we can deduce
that a remarkable relationship exists between MTT assay
results and COX-2 inhibitory activity of our compounds.
The docking study showed that compound 3k ftted well
into COX-2 enzyme active site with good selectivity. Com-
pound 3k also induced apoptosis via the mitochondrial
pathway accompanied by upregulation of Bax and down-
regulation of Bcl-2 protein expression. The morphological
alterations of PC3 and MCF-7 cells afected by 3k can be
directly recognized by inverted microscopy. Our data dem-
onstrate that compound 3k may be a potential anticancer
agent for further investigation in our future research. Other
pathways may a possible mechanism for our molecules
as anticancer agents beside COX-2 inhibition. Therefore,
Conclusions
In recent years, selective and nonselective COX inhibitors
have been evaluated for their efects on cancer treatment
and prevention, but COX-2 selective inhibitors have become
rather popular because of their lower side efects. Otherwise,
the combination of COX-2 inhibitors with radiation or drug
therapies may decrease their side efects in future cancer
prevention and treatment.
In the current work, we described the synthesis of new
sulfonylhydrazones which were investigated for their
cytotoxic and apoptotic efects on PC3 and MCF-7 cell
1 3

Molecular Diversity
Table 3 Predicted drug-likeness properties of all compounds according to OSIRIS property explorer tools
additional mechanistic studies are also needed to under-
stand the underlying.
the Electrothermal IA9300 apparatus and are uncorrected.
FT-IR spectra were recorded on a Schimadzu FT-IR-8400S
spectrometer. 1H and 13C-NMR spectra were recorded on
Bruker 300 MHz Ultrashield TM spectrometer. Elemental
analyses were determined by CHNS-932 (LECO). The liq-
uid chromatographic system consists of an Agilent Tech-
nologies 1100 series instrument equipped with a quater-
nary solvent delivery system and a model Agilent series
G1315 A photodiode array detector. The chromatographic
data were collected and processed using Agilent Chem-
station Plus software. Chromatographic separation was
performed at ambient temperature using a reverse phase
Experimental
Chemistry
General
All materials used were obtained from Sigma-Aldrich
(Saint Lewis, USA). Melting points were determined using
1 3

Molecular Diversity
Fig. 8 Graphical distribution of
compounds according to chemi-
cal classes using the boiled egg
predictive model
Zorbax C8 (4.0 × 250 mm) column. All experiments were
performed using acetonitrile–water (v/v, 70/30) mobile
phase with UV detection at 254 nm.
N′‑[(2,2‑difuoro‑1,3‑benzodioxol‑5‑yl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3a)
Yield 88%; m.p. 123–125 °C; HPLC tR (min.): 5.04;
FT-IR: ν/cm−1: 3203 (NH), 1639 (C=N), 1369, 1161
General procedure for the synthesis of p‑toluenesulfonyl
hydrazide (2)
1
(SO2); H NMR (300 MHz), (DMSO-d6/TMS) δ ppm:
2.36 (s, 3H, Ar-CH3); 7.41 (d, 4H, J = 9 Hz, ArH); 7.58
(s, 1H, Ar–H); 7.75 (d, 2H, J = 9 Hz, Ar–H); 7.91 (s,1H,
CH=N–); 11.54 (s,1H, SO2NH–N=); 13C NMR (75 MHz)
(DMSO-d6/TMS) δ ppm: 21.42 (CH3), 107.63 (CH),
110.81 (CH), 124.73 (CH), 125.96 (C), 127.72 (2CH),
128.54 (C), 130.14 (2CH), 131.24 (C), 131.56 (C), 136.46
(N=CH), 143.97 (C-F, J=20 Hz), 145.93 (C); Anal. calcd
for C15H12F2N2O4S.1/3H2O (360.331): C, 50.00; H, 3.54;
N, 7.77; S, 8.90 Found: C, 49.87; H, 3.37; N, 7.56; S, 9.14.
A mixture of p-toluenesulfonyl chloride (1) (26 mmol) and
hydrazine hydrate (66 mmol, 99%) was stirred at 0–5 °C
for 1–2 h in tetrahydrofuran (THF) (20 mL). The reaction
progress and completion were monitored using TLC. After
completion of the reaction, excess THF was distilled of.
The product was subsequently washed with water, dried and
recrystallized from ethanol [41, 42].
General procedure for the synthesis
4‑methyl‑N′‑(arylmethylidene)benzensulfonohydrazide
(3a‑o)
N′‑[(4‑bromothiophen‑2‑yl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3b)
A solution of p-toluenesulfonohydrazide (2) (25 mmol) was
mixed with a solution of substituted aldehydes (25 mmol)
in ethanol and refuxed for 3–4 h to obtain a precipitate of
hydrazone which was then fltered, dried and recrystallized
from ethanol [41, 42].
Yield 82%; m.p. 163 °C; HPLC tR (min.): 4.71; FT-IR:
1
ν/cm−1: 3203 (NH), 1597 (C=N), 1321, 1157 (SO2); H
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.37 (s, 3H,
Ar-CH3); 7.39–7.73 (m, 6H, Ar–H); 8.03 (s, 1H, CH=N–);
11.56 (s, 1H, SO2NH–N=); 13C NMR (75 MHz) (DMSO-
d6/TMS) δ ppm: 21.46 (CH3), 109.61 (C), 125.97 (CH),
126.41 (2CH), 127.64 (2CH), 132.59 (C), 136.32 (CH),
1 3

Molecular Diversity
140.18 (C), 141.08 (N=CH), 144.11 (C); Anal. calcd for
C12H11BrN2O2S2 (359.262): C, 40.12; H, 3.09; N, 7.80; S,
17.85 Found: C, 40.45; H, 3.08; N, 7.59; S, 17.76.
N′‑[(4‑fuoro‑3‑methoxyphenyl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3f)
Yield 77%; m.p. 123–125 °C; HPLC tR (min.): 4.25; FT-IR:
ν/cm−1: 3155 (NH), 1595 (C=N), 1361, 1155 (SO2); H
1
4‑methyl‑N′‑[(4‑phenylthiophen‑2‑yl)methylidene]
benzenesulfonohydrazide (3c)
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 3.85 (s, 3H, Ar-OCH3); 7.11–7.33 (m, 3H, Ar–H);
7.41 (d, 2H, J=9 Hz, Ar–H); 7.77 (d, 2H, J=9 Hz, Ar–H);
7.87 (s,1H, CH=N–); 11.46 (s,1H, SO2NH–N=); 13C NMR
(75 MHz) (DMSO-d6/TMS) δ ppm: 21.43 (CH3), 58.38
(OCH3), 111.80 (CH), 116.56 (CH), 120.31 (CH), 127.73
(2CH), 130.11 (2CH), 131.10 (C), 136.50 (C), 143.97
(C), 146.51 (N=CH), 147.77 (C), 151.35&154.64 (CF,
J=247 Hz); Anal. calcd for C15H15FN2O3S (322.354): C,
55.89; H, 4.69; N, 8.69; S, 9.95 Found: C, 55.64; H, 5.25;
N, 8.11; S, 9.28.
Yield 84%; m.p. 185–186 °C; HPLC tR (min.): 4.68; FT-IR:
ν/cm−1: 3186 (NH), 1610 (C=N), 1340, 1165 (SO2);
1H NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s,
3H, Ar-CH3); 7.27–7.92 (m, 11H, Ar–H); 8.12 (s, 1H,
CH=N–); 11.46 (s, 1H, SO2NH–N=); 13C NMR (75 MHz)
(DMSO-d6/TMS) δ ppm: 21.46 (CH3), 123.85 (CH), 126.32
(2CH), 127.67 (2CH), 127.93 (CH), 129.40 (2CH), 129.80
(CH), 130.14 (2CH), 134.83 (C), 136.44 (C), 139.51 (C),
142.08 (N=CH), 142.43 (C), 144.03 (C); Anal. calcd for
C18H16N2O2S2 (356.462): C, 60.65; H, 4.52; N, 7.86; S,
17.99 Found: C, 60.57; H, 4.51; N, 7.71; S, 17.96.
N′‑[(4‑methoxy‑3‑nitrophenyl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3g) (CAS
No: 461716‑16‑5)
N′‑[(4‑fuoro‑3‑phenoxyphenyl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3d)
Yield 87%; m.p. 172–173 °C; HPLC tR (min.): 3.97; FT-IR:
1
ν/cm−1: 3147 (NH), 1620 (C=N), 1359, 1157 (SO2); H
Yield 84%; m.p. 159–161 °C; HPLC tR (min.): 5.74; FT-IR:
1
ν/cm−1: 3240 (NH), 1610 (C=N), 1357, 1153 (SO2); H
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 3.94 (s, 3H, Ar-OCH3); 7.39 (d, 1H, J=8.7 Hz,
Ar–H); 7.41 (d, 2H, J = 8.1 Hz, Ar–H); 7.76 (d, 2H,
J = 8.1 Hz, Ar–H); 7.85 (dd, 1H, J1 = 8.7 Hz, J2 = 2.1 Hz,
Ar–H); 7.92 (s, 1H, CH=N–); 8.04 (d, 1H, J = 2.1 Hz,
Ar–H); 11.56 (s, 1H, SO2NH–N=); 13C NMR (75 MHz)
(DMSO-d6/TMS) δ ppm: 21.43 (CH3); 57.38 (OCH3);
115.25 (CH); 123.56 (CH); 126.78 (C); 127.67 (2CH);
130.16 (2CH); 132.61 (CH); 136.48 (C); 139.72 (CH);
144.03 (C); 145.19 (–N=CH); 153.45 (C); Anal. calcd for
C15H15N3O5S (349.362): C, 51.57; H, 4.33; N, 12.03; S, 9.18
Found: C, 51.17; H, 4.31; N, 11.62; S, 9.55.
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 7.03 (d, 2H, J=9 Hz, Ar–H); 7.18–7.45 (m, 8H,
Ar–H); 7.66 (d, 2H, J=9 Hz, Ar–H); 7.84 (s,1H, CH=N–);
11.48 (s, 1H, SO2NH–N=); 13C NMR (75 MHz) (DMSO-
d6/TMS) δ ppm: 21.45 (CH3), 118.04 (2CH), 118.23 (CH),
118.42 (CH), 119.12 (CH), 124.34 (C), 127.65 (2CH),
130.09 (2CH), 130.62 (2CH), 131.72 (CH), 136.36 (C),
141.00 (C), 144.17 (N=CH), 145.70 (C), 153.03&156.35
(CF, J=249 Hz), 156.69 (C); Anal. calcd for C20H17FN2O3S
(384.424): C, 62.49; H, 4.46; N, 7.29; S, 8.34 Found: C,
62.08; H, 4.49; N, 6.67; S, 8.11.
N′‑{[2‑chloro‑3‑(trifuoromethyl)phenyl]
methylidene}‑4‑methylbenzenesulfonohydrazide (3e)
4‑methyl‑N′‑[(3‑phenyl‑1H‑pyrazol‑4‑yl)methylidene]
benzenesulfonohydrazide (3h)
Yield 87%; m.p. 153–155 °C; HPLC tR (min.): 5.76; FT-IR:
Yield 85%; m.p. 90 °C; HPLC tR (min.): 3.67; FT-IR: ν/
cm−1: 3188 (NH), 1620 (C=N), 1321, 1157 (SO2); 1H NMR
(300 MHz), (DMSO-d6/TMS) δ ppm: 1.09 (t, CH3 ethanol);
2.36 (s, 3H, Ar-CH3); 2.51 (m, CH2 ethanol); 3.65 (bs, OH
ethanol); 7.37–7.52 (m, 8H, Ar–H, CH=N, pyrazole NH),
7.72 (d, 2H, J=9 Hz, Ar–H); 7.93 (d, 2H, J=9 Hz, Ar–H);
11.01 (s,1H, SO2NH–N=); 13C NMR (75 MHz) (DMSO-d6/
TMS) δ ppm: 21.24 (ethanol CH3), 21.45 (CH3), 65.39 (eth-
anol CH2), 113.71 (C), 125.97 (CH), 127.73 (2CH), 128.61
(2CH), 129.19 (C), 129.64 (2CH), 130.03 (2CH), 130.86
(CH), 136.65 (C), 141.96 (C), 143.74 (N=CH), 145.90 (C);
Anal. calcd for C17H16N4O2S. C2H5OH (386.467): C, 59.05;
H, 5.74; N, 14.50; S, 8.30 Found: C, 58.97; H, 5.44; N,
14.53; S, 8.81.
1
ν/cm−1: 3147 (NH), 1595 (C=N), 1361, 1157 (SO2); H
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.33 (s, 3H,
Ar-CH3); 7.40 (d, 2H, J = 8.1 Hz, Ar–H); 7.54 (t, 1H,
Ar–H), 7.77 (d, 2H, J = 8.1 Hz, Ar–H); 7.83 (dd, 1H,
J1 =7.8 Hz, J2 =0.9 Hz, Ar–H), 7.99 (dd, 1H, J1 =7.8 Hz,
J2 = 0.9 Hz, Ar–H), 8.33 (s, 1H, CH=N–); 11.96 (s, 1H,
SO2NH–N=); 13C NMR (75 MHz) (DMSO-d6/TMS) δ ppm:
21.42 (CH3), 124.92 (CF3, J=278 Hz), 125.95 (CH), 127.64
(2CH), 127.94 (CH), 129.43 (C), 130.27 (2CH), 130.63
(CH), 131.05 (C), 133.80 (C), 136.30 (C), 141.98 (N=CH),
144.26 (C); Anal. calcd for C15H12ClF3N2O2S.1/2C2H5OH
(399.811): C, 48.06; H, 3.78; N, 7.01; S, 8.02 Found: C,
48.34; H, 3.38; N, 7.34; S, 8.92.
1 3

Molecular Diversity
(N=CH), 156.32 (C); Anal. calcd for C15H15ClN2O3S
(338.809): C, 53.17; H, 4.46; N, 8.27; S, 9.46 Found: C,
53.28; H, 4.48; N, 8.09; S, 9.80.
N′‑[(5‑bromo‑2‑methoxyphenyl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3i) (CAS
No: 200625‑38‑3)
Yield 78%; m.p. 164–166 °C; HPLC tR (min.): 5.27;
FT-IR: ν/cm−1: 3159 (NH), 1595 (C=N), 1354, 1165
N′‑[(2‑chloro‑6‑methylphenyl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3l)
1
(SO2); H NMR (300 MHz), (DMSO-d6/TMS) δ ppm:
2.36 (s, 3H, Ar-CH3); 3.79 (s, 3H, Ar-OCH3); 7.05 (d,
1H, J = 9 Hz, Ar–H); 7.42 (d, 2H, J = 9 Hz, Ar–H); 7.52
(d, 1H, J = 2.7 Hz, Ar–H); 7.61 (dd, 1H, J1 = 8.7 Hz,
J2 = 2.7 Hz, Ar–H); 7.73 (d, 2H, J = 9 Hz, Ar–H); 8.12
(s, 1H, CH=N–); 11.56 (s,1H, SO2NH–N=); 13C NMR
(75 MHz) (DMSO-d6/TMS) δ ppm: 21.45 (CH3), 56.51
(O-CH3), 112.81 (C), 114.83 (CH), 124.20 (C), 127.50
(CH), 127.59 (2CH), 130.21 (2CH), 134.21 (CH), 136.52
(C), 141.22 (N=CH), 144.05 (C), 157.01 (C); Anal. calcd
for C15H15BrN2O3S (383.260): C, 47.01; H, 3.94; N, 7.31;
S, 8.37 Found: C, 47.25; H, 4.08; N, 7.04; S, 8.45.
Yield 76%; m.p. 175–176 °C; HPLC tR (min.): 5.42; FT-IR:
1
ν/cm−1: 3201 (NH), 1589 (C=N), 1373, 1161 (SO2); H
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.18 (s, 3H,
Ar-CH3); 2.37 (s, 3H, Ar-CH3); 7.17–7.33 (m, 3H, Ar–H);
7.41 (d, 2H, J=9 Hz, Ar–H); 7.75 (d, 2H, J=9 Hz, Ar–H);
8.25 (s, 1H, CH=N–); 11.70 (s, 1H, SO2NH–N=); 13C NMR
(75 MHz) (DMSO-d6/TMS) δ ppm: 21.46 (CH3), 21.86
(CH3), 127.76 (CH), 127.80 (2CH), 128.53 (CH), 129.93
(CH), 130.14 (2CH), 130.64 (C), 131.23 (C), 136.47 (C),
139.63 (C), 144.07 (C), 145.13 (N=CH); Anal. calcd for
C15H15ClN2O2S (322.809): C, 55.81; H, 4.68; N, 8.68; S,
9.93 Found: C, 55.25; H, 4.79; N, 8.35; S, 9.28.
N′‑{4‑fuoro‑2‑(trifuoromethyl)phenyl]
methylidene}‑4‑methylbenzenesulfonohydrazide (3j)
N′‑[(6‑bromopyridin‑2‑yl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3m)
Yield 82%; m.p. 165–166 °C; HPLC tR (min.): 6.14;
FT-IR: ν/cm−1: 3211 (NH), 1597 (C=N), 1319, 1155
1
Yield 88%; m.p. 117–119 °C; HPLC tR (min.): 4.17; FT-IR:
(SO2); H NMR (300 MHz), (DMSO-d6/TMS) δ ppm:
ν/cm−1: 3203 (NH), 1597 (C=N), 1361, 1159 (SO2); H
1
2.37 (s, 3H, Ar-CH3); 7.42 (d, 2H, J = 9 Hz, Ar–H); 7.59
(t, 1H, Ar–H); 7.67 (dd, 1H, J1 =9 Hz, J2 =2.7 Hz, Ar–H);
7.77 (d, 2H, J = 9 Hz, Ar–H); 7.97 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz, Ar–H); 8.16 (s, 1H, CH=N–); 11.88 (s,1H,
SO2NH–N=); 13C NMR (75 MHz) (DMSO-d6/TMS) δ
ppm: 21.38 (CH3), 144.24 (CH), 120.52&121.58 (CH,
J=21 Hz), 123.4 (CF3, J = 273 Hz), 127.55 (2CH), 128.17
(C), 129.01 (C), 129.78 (CH), 130.22 (2CH), 136.33
(C), 141.18 (C), 144.14 (N=CH), 160.90 &164.22 (C-F,
J = 249 Hz); Anal. calcd for C15H12F4N2O2S (360.326): C,
50.00; H, 3.36; N, 7.77; S, 8.90 Found: C, 49.74; H, 3.67;
N, 7.70; S, 9.23.
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 7.10–7.80 (m, 7H, Ar–H); 7.82 (s, 1H, CH=N–);
11.98 (s, 1H, SO2NH–N=); 13C NMR (75 MHz) (DMSO-
d6/TMS) δ ppm: 21.47 (CH3), 119.46 (CH), 127.63 (2CH),
129.05 (CH), 130.29 (2CH), 136.34 (CH), 140.69 (C),
141.40 (C), 144.24 (N=CH), 145.145 (C), 154.04 (C); Anal.
calcd for C13H12BrN3O2S (354.222): C, 44.08; H, 3.41; N,
10.86; S, 9.05 Found: C, 44.40; H, 3.57; N, 10.73; S, 8.34.
N′‑[(1‑methyl‑1H‑pyrrol‑2‑yl)
methylidene]‑4‑methylbenzenesulfonohydrazide (3n) (CAS
No: 113334‑34‑2)
N′‑[(2‑chloro‑3‑methoxyphenyl)
Yield 68%; m.p. 124 °C; HPLC tR (min.): 3.95; FT-IR:
methylidene]‑4‑methylbenzenesulfonohydrazide (3k)
1
ν/cm−1: 3157 (NH), 1618 (C=N), 1350, 1165 (SO2); H
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 3.64 (s, 3H, N-CH3); 6.00–6.87 (m, 3H, Ar–H);
7.40 (d, 2H, J = 8.1 Hz, Ar–H); 7.72(d, 2H, J = 8.4 Hz,
Ar–H); 7.78 (s, 1H, CH=N–); 10.92 (s, 1H, SO2NH–N=);
13C NMR (75 MHz) (DMSO-d6/TMS) δ ppm: 21.40 (CH3),
36.58 (CH3), 108.48 (CH), 115.60 (CH), 126.71 (CH),
127.76 (2CH), 128.56 (C), 129.35 (2CH), 136.40 (C),
141.37 (N=CH), 143.74 (C); Anal. calcd for C13H15N3O2S
(277.342): C, 56.30; H, 5.45; N, 15.15; S, 11.56 Found: C,
56.40; H, 5.18; N, 14.68; S, 11.36
Yield 80%; m.p. 161–163 °C; HPLC tR (min.): 4.68;
FT-IR: ν/cm−1: 3157 (NH), 1597 (C=N), 1354, 1157
1
(SO2); H NMR (300 MHz), (DMSO-d6/TMS) δ ppm:
2.97 (s, 3H, Ar-CH3); 3.85 (s, 3H, Ar-OCH3); 7.149–7.345
(m, 3H, Ar–H); 7.42 (d, 2H, J = 9 Hz, Ar–H); 7.75 (d,
2H, J = 9 Hz, Ar–H); 8.27 (s,1H, CH=N–); 11.75 (s, 1H,
SO2NH–N=); 13C NMR (75 MHz) (DMSO-d6/TMS) δ
ppm: 21.45 (CH3), 58.76 (OCH3), 114.08 (CH), 118.34
(CH), 121.73 (CH), 127.64 (2CH), 128.34 (CH), 130.23
(2CH), 132.43 (C), 138.45 (C), 143.37 (C), 144.11
1 3

Molecular Diversity
both docking studies. The molecular docking protocol
was validated by re-docking of the co-crystallized inhibi-
tor celecoxib back into respective enzymes COX-1 and
COX-2 with 0.8 Å and 0.7 Å root mean square deviation
(RMSD) values. During study of validations as well as
our docking of ligands was used fexible ligand in rigid
protein. The results fles were analyzed using Accelrys
Discovery Studio Visualizer 4.0 program and PyMOL
Molecular Graphics System.
N′‑{[2‑(trifuoromethoxy)phenyl]
methylidene}‑4‑methylbenzenesulfonohydrazide (3o)
Yield 74%; m.p. 139–140 °C; HPLC tR (min.): 5.41; FT-IR:
ν/cm−1: 3142 (NH), 1606 (C=N), 1361, 1157 (SO2); H
1
NMR (300 MHz), (DMSO-d6/TMS) δ ppm: 2.36 (s, 3H,
Ar-CH3); 7.41–7.81 (m, 8H, Ar–H); 8.12 (s, 1H, CH=N–);
11.74 (s, 1H, SO2NH–N=); 13C NMR (75 MHz) (DMSO-
d6/TMS) δ ppm: 21.43 (CH3), 118.73 (CH), 122.19 (CH),
125.96 (CH), 126.90 (C), 127.04 (CH), 127.62 (2CH),
128.51 (C), 130.21 (2CH), 132.21 (C), 136.44 (N=CH),
142.30 (CF3), 146.85 (C); Anal. calcd for C15H13N2O3S
(358.335): C, 50.28; H, 3.66; N, 7.82; S, 8.95 Found: C,
50.02; H, 3.67; N, 7.52; S, 9.21
Biological assays
Cell culture and cell viability assay
Cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Human breast
cancer cell line MCF-7, human prostate cancer cell line
PC3, and mouse fbroblast cell line L929 cells were main-
tained in Dulbecco’s modifed Eagle’s medium (DMEM,
Invitrogen) or RPMI-1640 supplemented with 10% FBS,
2 mM l‐glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin and kept in a humidifed atmosphere at 37 °C
incubator with 5% CO2 in air. To determine the cell viabil-
ity, cells were plated onto 96‐well plates (1 × 104 cells/
well). The cells were treated with diferent concentrations
(0, 0.1, 1, 10, 100 and 1000μM) of synthesized compound
(3a–o) incubated for 24 h. After the incubation, cells were
washed with PBS and added to 100 μL DMEM/RPMI-
1640. A total of 10 μL of the MTT (3‐(4,5‐dimethylthi-
azol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) (Vybrant,
Invitrogen) labeling reagent was added to each well and
incubated for 4 h in humidifed atmosphere at 37 °C incu-
bator with 5% CO2 in air. After the incubation, 100 μL of
the SDS bufer was added into each well for solubilization
of formazan precipitate. Then, absorbance was measured
by microplate reader at 570 nm and was carried out in trip-
licate of each assay [47].
Molecular modeling studies
Preparation of ligands
The synthesized compounds 3a–o and reference ligand
(celecoxib) were drawn with the Spartan 04 software
(SPARTAN 04, Wavefunction, Inc., Irvine, USA) and opti-
mized for each compound by using the semi-empirical PM3
method. For each compound, the most stable conformation
was utilized in docking calculation and The AutoDock Tools
program was used to generate the docking input fles [43].
Preparation of COXs structures for docking studies
The structures of COX-1 (PDB code: 3KK6, resolution:
2.75 Å) COX-2 (PDB code: 3LN1, resolution: 2.4 Å) were
obtained from the protein databank [44, 45]. Water mol-
ecules and co-crystallized inhibitor celecoxib were deleted
together with N-acetyl-D-glucosamine and β-octylglucoside
in the structure; at the same time, the charge of the Fe atom
was set to +2 manually. This structure was protonated using
the AutoDock Tools program, and thereafter the obtained
structure was energy-minimized.
Docking studies
Cell growth assays
AutoDock Vina software was used for the synthesized
compounds 3a–o into the COX-1 and COX-2 structures
docking calculations [46]. The size of binding pocket
was detected 40 × 40 × 40 points for both enzymes. For
COX-2 enzyme, x (30.518), y (− 21.298) and z (− 16.69)
dimensions were built, while x (− 31.867), y (42.146)
and z (− 5.089) dimensions were used in COX-1 enzyme
docking studies, appropriate to the position of celecoxib in
these structures. The Vina parameter “exhaustiveness” was
set to the value of 10; besides a grid spacing of 0.375 Å
was employed for the calculation of the energetic map for
In order to investigate the efects of compound 3k on cell
survival of PC3 and MCF-7 cancer cell lines, cells were
seeded in 24-well plates and incubated with compounds at
a fnal concentration of IC50 in growth medium (without
antibiotics) for 12 h, 24 h and 48 h. After the incubation,
methylene blue staining was used for cell survivals. The cells
were extracted with 1% SDS in PBS solution and stained
with 0.01% methylene blue solutions. The absorbance was
measured at 600 nm with microplate reader. Each experi-
ment was repeated three times.
1 3

Molecular Diversity
2. Hunter AM, Lacasse ÆEC, Korneluk ÆRG (2007) The inhibi-
tors of apoptosis (IAPs) as cancer targets. Apoptosis 12:1543–
1568. https://doi.org/10.1007/s10495-007-0087-3
Western blot analysis for protein expression
Cells were treated with fnal concentration of IC50 of syn-
thesized compound 3k in 6 well plates and incubated for
24 h. Cells were lysed using RIPA cell lysis bufer (89900,
Thermo), and the protein concentrations were determined
by the BCA protein assay (Thermo Scientifc). Samples
were denaturated with Laemli bufer, and 25 μg protein
was loaded to each well. The samples were resolved by
4–12% sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis and were transferred to polyvinylidene fuoride
membrane, which was then blocked with 3% BSA (Cap-
ricorn Scientifc, BSA-1T) in Tris bufered saline (TBS).
The membrane was washed twice in TBS containing
Tween-20 (0.1%) and incubated overnight with primary
antibodies (1:500 dilution anti-Bax sc-7480, anti-Bcl2
sc-492, anti-GAPDH sc-25778, Santa Cruz Biotechnol-
ogy, Heidelberg, Germany) and washed with TBS con-
taining Tween-20 (0.1%). The membrane was washed and
then incubated with horseradish peroxidase conjugated
secondary antibody (anti-mouse; sc-2060 or anti-rabbit;
sc-2004, Santa Cruz Biotechnology) for 2 h. The blots
were developed with chemiluminescence reagents (sc2048,
Santa Cruz Biotechnology, Texas, USA) and analyzed with
chemiluminescent imaging systems (Syngene, Cambridge,
UK). Data were analyzed using “Image J Programme Opti-
cal Density Analysis Software” (NIH) by measuring each
band three times for quantifcation. Signals were normal-
ized with respect to GAPDH.
3. Ashe PC, Berry MD (2003) Apoptotic signaling cascades. Prog
Neuropsychopharmacol Biol Psychiatry 308:199–214. https://doi.
org/10.1016/S0278-5846(03)00016-2
4. Kumar R, Herbert PE, Warrens AN (2005) An introduction to
death receptors in apoptosis. Int J Surg 3:268–277. https://doi.
org/10.1016/j.ijsu.2005.05.002
5. Elumalai P, Gunadharini DN, Senthilkumar K et al (2012) Induc-
tion of apoptosis in human breast cancer cells by nimbolide
through extrinsic and intrinsic pathway. Toxicol Lett 215:131–
142. https://doi.org/10.1016/j.toxlet.2012.10.008
6. Sfanos KS, De Marzo AM (2014) Prostate cancer and infamma-
tion: the evidence. Histopathology 60:199–215. https://doi.org/1
0.1111/j.1365-2559.2011.04033.x
7. Morrow RJ, Etemadi N, Yeo B, Ernst M (2017) Challenging a
misnomer? The role of infammatory pathways in infammatory
breast cancer. Mediators Infamm 2017:4754827. https://doi.
org/10.1155/2017/4754827
8. Liu B, Qu L, Yan S (2015) Cyclooxygenase-2 promotes tumor
growth and suppresses tumor immunity. Cancer Cell Int 15:106.
https://doi.org/10.1186/s12935-015-0260-7
9. Harris RE, Casto BC, Harris ZM (2014) Cyclooxygenase-2 and
the infammogenesis of breast cancer. World J Clin Oncol 5:677–
692. https://doi.org/10.5306/wjco.v5.i4.677
10. Gupta S, Srivastava M, Ahmad N et al (2000) Over-expression
of cyclooxygenase-2 in human prostate adenocarcinoma. Pros-
tate 42:73–78. https://doi.org/10.1002/(SICI)1097-0045(20000
101)42:1%3c73:AID-PROS9%3e3.0.CO;2-G
11. Maekawa M, Sugan K, San H et al (1998) Increased expression of
cyclooxygenase-2 to -1 in human colorectal cancers and adeno-
mas, but not in hyperplastic polyps. Jpn J Clin Oncol 28:421–426.
https://doi.org/10.1093/jjco/28.7.421
12. Meric J-B, Rottey S, Olaussen K et al (2006) Cyclooxygenase-2 as
a target for anticancer drug development. Crit Rev Oncol Hematol
59:51–64. https://doi.org/10.1016/j.critrevonc.2006.01.003
13. Rollas S, Küçükgüzel SG (2007) Biological activities of
hydrazone derivatives. Molecules 12:1910–1939. https://doi.
org/10.3390/12081910
In vitro COX inhibition assay
14. Aydın S, Kaushik-basu N, Arora P et al (2013) Microwave assisted
synthesis of some novel Flurbiprofen hydrazide- hydrazones as
anti-HCV NS5B and anticancer agents. Marmara Pharm J 17:26–
34. https://doi.org/10.12991/201317389
In order to determine the COX activity, inhibitors were dis-
solved in DMSO. Using a commercial COX (ovine/human)
inhibitor screening assay kit (Cayman 560131, USA) and
following the manufacturer’s instructions, compounds was
used for measuring COX inhibition assay. Celecoxib was
used as the reference drug for comparison of activity.
15. Çıkla P, Özsavcı D, Bingöl-Özakpınar Ö et al (2013) Synthesis,
cytotoxicity, and pro-apoptosis activity of etodolac hydrazide
derivatives as anticancer agents. Arch Pharm (Weinheim)
346:367–379. https://doi.org/10.1002/ardp.201200449
16. Küçükgüzel ŞG, Koç D, Çıkla-Süzgün P et al (2015) Synthe-
sis of tolmetin hydrazide-hydrazones and discovery of a potent
apoptosis inducer in colon cancer cells. Arch Pharm (Weinheim)
348:730–742. https://doi.org/10.1002/ardp.201500178
17. Şenkardeş S, Kaushik-basu N, Durmaz İ et al (2016) Synthesis
of novel difunisal hydrazide hydrazones as anti-hepatitis C virus
agents and hepatocellular carcinoma inhibitors. Eur J Med Chem
108:301–308. https://doi.org/10.1016/j.ejmech.2015.10.041
18. Tatar E, Şenkardeş S, Sellitepe E et al (2016) Synthesis, and pre-
diction of molecular properties and antimicrobial activity of some
acylhydrazones derived from N-(arylsulfonyl) methionine. Turk J
Chem 40:510–534. https://doi.org/10.3906/kim-1509-21
19. Siemann S, Evanof DP, Marrone L et al (2002) N-Arylsulfonyl
hydrazones as inhibitors of IMP-1 metallo-β-lactamase. Antimi-
crob Agents Chemother 46:2450–2457. https://doi.org/10.1128/
aac.46.8.2450-2457.2002
Acknowledgements This work was supported by the Research Fund of
Marmara University, Project Number: SAG-K-120917-0495.
Compliance with ethical standards
Conflict of interest The authors have declared no confict of interest.
References
1. Carmichael J (1994) Cancer chemotherapy: identifying novel
anticancer drugs. Br Med J 308:1288–1290. https://doi.
org/10.1136/bmj.308.6939.1288
20. Gao Z, Lv M, Li Q, Xu H (2015) Synthesis of heterocycle-attached
methylidenebenzenesulfonohydrazones as antifungal agents.
1 3

Molecular Diversity
cancer cells by the selective COX-2 inhibitor celecoxib. FASEB
J 15:2742–2744. https://doi.org/10.1096/f.01-0299fe
35. Wang Z, Chen J, Liu J (2014) COX-2 Inhibitors and gas-
tric cancer. Gastroent Res Pract 2014:132320. https://doi.
org/10.1155/2014/132320
Bioorg Med Chem Lett 25:5092–5096. https://doi.org/10.1016/j.
bmcl.2015.10.017
21. Begum F, Almandil NB, Lodhi MA et al (2019) Synthesis and
urease inhibitory potential of benzophenone sulfonamide hybrid
in vitro and in silico. Bioorg Med Chem 27:1009–1022. https://
doi.org/10.1016/j.bmc.2019.01.043
36. Morrow D, Daniel R, Dubois N (1998) Modulation of apoptosis
and Bcl-2 expression by prostaglandin E2 in human colon cancer
cells. Cancer Res 58:362–366
22. Mobasher S, Abid A, Amna H et al (2017) Sulfonyl hydrazones
derived from 3-formylchromone as non-selective inhibitors of
MAO-A and MAO-B:Synthesis, molecular modelling and in
silico ADME evaluation. Bioorg Chem 75:291–302. https://doi.
org/10.1016/j.bioorg.2017.10.001
37. Abdelazeem AH, Gouda AM, Omar HA, Tolba MF (2014)
Design, synthesis and biological evaluation of novel diphenylth-
iazole-based cyclooxygenase inhibitors as potential anticancer
agents. Bioorg Chem 57:132–141. https://doi.org/10.1016/j.bioor
g.2014.10.001
23. Hayakawa M, Kawaguchi K, Kaizawa H et al (2007) Synthe-
sis and biological evaluation of sulfonylhydrazone- substituted
imidazo [1,2-a] pyridines as novel PI3 kinase p110 a inhibitors.
Bioorg Med Chem 15:5837–5844. https://doi.org/10.1016/j.
bmc.2007.05.070
38. Murakami M (2011) Lipid mediators in life science. Exp Anim
60:7–20. https://doi.org/10.1538/expanim.60.7
39. Zhao YH, Abraham MH, Le J et al (2015) Rate-limited steps of
human oral absorption and QSAR studies. Pharm Res 19:1446–
1457. https://doi.org/10.1023/A:1020444330011
24. Wei D, Pan Y, Wang H et al (2018) Synthesis of substituted aro-
matic heterocyclic sulfonyl hydrazone compounds and in vitro
anti-hepatoma activity: preliminary results. Eur Rev Med Phar-
macol Sci 22:4720–4729. https://doi.org/10.26355/eurrev_20180
7_15532
40. Daina A, Zoete V (2016) A BOILED-Egg to predict gastroin-
testinal absorption and brain penetration of small molecules.
ChemMedChem 11:1117–1121. https://doi.org/10.1002/
cmdc.201600182
25. George RF (2018) Facile synthesis of simple 2-oxindole-based
compounds with promising antiproliferative activity. Future Med
Chem 10:269–282. https://doi.org/10.4155/fmc-2017-0148
26. Korcz M, Franciszek S, Bednarski PJ, Kornicka A (2018) Cyto-
toxic properties of novel quinoline-3-carbaldehyde hydrazones
bearing a 1,2,4-triazole or benzotriazole moiety. Molecules
23:1497. https://doi.org/10.3390/molecules23061497
41. Neumann DM, Cammarata A, Backes G et al (2015) Synthesi-
sand anti fungal activity of substituted 2,4,6-pyrimidinetrione
carbalde hydehydrazones. Bioorg Med Chem 22:813–826. https
://doi.org/10.1016/j.bmc.2013.12.010
42. Murtaza S, Shamim S, Kousar N et al (2015) Synthesis, biological
investigation, calf thymus DNAbinding and docking studies of the
sulfonyl hydrazides and their derivatives. J Mol Struct 1107:99–
108. https://doi.org/10.1016/j.molstruc.2015.11.046
43. Morris GM, Huey R, Lindstrom W et al (2009) AutoDock4 and
AutoDockTools4: automated docking with selective receptor fex-
ibility. J Comput Chem 30:2785–2791. https://doi.org/10.1002/
jcc.21256
27. Yu Y, Huang W, Chen Y et al (2016) Calcium carbide as the
acetylide source: transition-metal-free synthesis of substituted
pyrazoles via [1,5]-sigmatropic rearrangements. Green Chem
18:6445–6449. https://doi.org/10.1039/c6gc02776h
28. Katsuhiro S, Hiraku I (1987) Thermolysis of sodium salts of tosyl-
hydrazones of some heterocyclic aldehydes in the presence of
silver chromate: 1,3 N→C migration of tosyl group. Heterocycles
26:2–9. https://doi.org/10.3987/R-1987-07-1891
44. Rimon G, Sidhu RS, Lauver DA et al (2010) Coxibs interfere
with the action of aspirin by binding tightly to one monomer of
cyclooxygenase-1. PNAS 107:28–33. https://doi.org/10.1073/
pnas.0909765106
29. Kısmet K, Akay MT, Abbasoğlu O, Ercan A (2004) Celecoxib:
a potent cyclooxygenase-2 inhibitor in cancer prevention.
Cancer Detect Prev 28:127–142. https://doi.org/10.1016/j.
cdp.2003.12.005
45. Wang JL, Limburg D, Graneto MJ et al (2010) The novel ben-
zopyran class of selective cyclooxygenase-2 inhibitors. Part 2:
the second clinical candidate having a shorter and favorable
human half-life. Bioorg Med ChemLett 20:7159–7163. https://
doi.org/10.1016/j.bmcl.2010.07.054
30. Hsu A, Ching T, Wang D et al (2000) The Cyclooxygenase-2
inhibitor celecoxib induces apoptosis by blocking Akt activation in
human prostate cancer cells independently of Bcl-2*. J Biol Chem
275:11397–11403. https://doi.org/10.1074/jbc.275.15.11397
31. Altaf M, Casagrande N, Mariotto E et al (2019) Potent in vitro
and in vivo anticancer activity of new bipyridine and bipyrimidine
gold (III) dithiocarbamate derivatives. Cancers 11:1–14. https://
doi.org/10.3390/cancers11040474
46. Trott O, Olson AJ (2009) AutoDock Vina: improving the speed
and accuracy of docking with a new scoring function, efcient
optimization, and multithreading. J Comput Chem 31:455–461.
https://doi.org/10.1002/jcc.21334
47. Çoruh I, Çevik Ö, Yelekçi K et al (2018) Synthesis, anticancer
activity, and molecular modeling of etodolac-thioether deriva-
tives as potentmethionine aminopeptidase (type II) inhibitors.
Arch Pharm (Weinheim) 351:e1700195. https://doi.org/10.1002/
ardp.201700195
32. Dai Z, Ma X, Kang H et al (2012) Antitumor activity of the
selective cyclooxygenase-2 inhibitor, celecoxib, on breast can-
cer in vitro and in vivo. Cancer Cell Int 12:1–8. https://doi.
org/10.1186/1475-2867-12-53
33. Patel MI, Subbaramaiah K, Du B et al (2007) Celecoxib inhibits
prostate cancer growth: evidence of a cyclooxygenase-2-inde-
pendent mechanism. Clin Cancer Res 11:1999–2007. https://doi.
org/10.1158/1078-0432.CCR-04-1877
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
34. Grösch S, Tegeder I, Niederberger E et al (2001) COX-2 inde-
pendent induction of cell cycle arrest and apoptosis in colon
1 3