Design, synthesis and anticervical cancer activity of new benzofuran–pyrazol-hydrazono- thiazolidin-4-one hybrids as potential EGFR inhibitors and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bioorg.2019.103035

This study represents the synthetic approaches of a new set of 2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(aryl)thiazolidin-4-one derivatives 4–22 aiming to obtain new antiproliferative candidates against human cervix carcino

Full text of DOI:10.1016/j.bioorg.2019.103035

Accepted Manuscript
Design, synthesis and anticervical cancer activity of new benzofuran–pyrazol-
hydrazono- thiazolidin-4-one hybrids as potential EGFR inhibitors and apop-
tosis inducing agents
Hebat-Allah S. Abbas, Somaia S. Abd El-Karim
PII:
S0045-2068(19)30440-7
https://doi.org/10.1016/j.bioorg.2019.103035
103035
DOI:
Article Number:
Reference:
YBIOO 103035
Bioorganic Chemistry
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21 March 2019
23 May 2019
3 June 2019
Please cite this article as: H.S. Abbas, S.S. Abd El-Karim, Design, synthesis and anticervical cancer activity of new
benzofuran–pyrazol-hydrazono- thiazolidin-4-one hybrids as potential EGFR inhibitors and apoptosis inducing
agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103035
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1
Design, synthesis and anticervical cancer activity of new benzofuran–pyrazol-hydrazono-
thiazolidin-4-one hybrids as potential EGFR inhibitors and apoptosis inducing agents
Hebat-Allah S. Abbas1,2 , Somaia S. Abd El-Karim3, *
1Chemistry Department, College of Science, King Khalid University, Abha, Saudi Arabia.
2Photochemistry Department, National Research Centre, Dokki, Cairo, 12622,Egypt.
3Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo, 12622, Egypt
*Corresponding author: Somaia S. Abd El-Karim
E-mail address: somaia_elkarim@hotmail.com
Abstract
This study represents the synthetic approaches of a new set of 2-(((3-(benzofuran-2-yl)-1-
phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(aryl)thiazolidin-4-one derivatives 4-22 aiming
to obtain new antiproliferative candidates against human cervix carcinoma cells (Hela) of EGFR
PK inhibiting potency. The cancer cells represented promising sensitivity towards the
compounds 6, 7, 11, 13, 14, 16, 17 more than or equal to that against the reference drug
doxorubicin. In addition, the latter compounds were tested as EGFR protein kinase inhibitors.
The results revealed that compound 14 showed more significant EGFR PK inhibitory activity
than the reference drug erlotinib (IC50; 0.07, 0.08 µM, respectively). Moreover, cell cycle
analysis and apoptosis assay were performed for compound 14 proving its ability to cause G1/S
phase arrest and apoptosis in Hela cancer cells, in addition to its activation of the caspases-7 and
-3. In addition, derivative 14 increased the expression level of p53 and the ratio of Bax/Bcl-2
which confirmed its mode of action. Molecular docking study of 14 was performed to investigate
its binding mode of interaction with EGFR PK in the active site with the aim of rationalizing its
promising inhibitory activity. Accordingly, compound 14 might be considered as a promising
scaffold anticervical cancer chemotherapeutic and deserves further optimization and in-depth
biological studies.
Keywords: Benzofuran; Pyrazole; Thiazolidin-4-one; Cervical cancer; Apoptosis and EGFR PK
suppressing agents.

2
1. Introduction
Cancer disease is a life-threatening disease and its treatment is a significant challenge
over the past 100 years [1]. It has been documented that the organs; liver, prostate, lung, stomach
and colorectal are mostly affected by cancer disease in men, whereas among women, breast,
cervix, thyroid, colorectal and lung are the mostly affected by cancer [2], where the cervical
cancer is considered the fourth most frequent cancer among women with an estimated 570,000
new cases in 2018 representing 6.6% of all female cancers [3]. Approximately ninety percent of
deaths which occur as a result of cervical cancer appear in low- and middle-income countries [3].
Although the majority of women present with early-stage disease and can be treated successfully,
25% of women are present with advanced disease. Tremendous efforts are still needed to
improve the overall survival rate in patients with advanced-stage cervical cancer in addition to
the discovery of new drugs that have selectivity to inhibit the growth of the tumor cells or
completely kill them without producing toxic effects on the healthy cells [2,3].
Receptor protein tyrosine kinases (PTKs) play crucial roles in activating signal
transduction pathways in the cells, resulting in cell division, differentiation, and regulatory
mechanisms. Epidermal growth factor receptor (EGFR) kinase is a receptor tyrosine kinase
(RTK) of the ErbB family [4]. It regulates numerous biological pathways, such as; cell motility,
adhesion, and cell cycle regulation, as well as angiogenesis, apoptosis, and metastasis [5,6].
Over-expression of EGFR PK is also crucial for the evolution of malignant tumors, such as
colon, ovarian, breast and non-small cell lung cancer (NSCLC) [7].Figure 1 represents various
approved anticancer drugs targeting inhibition of EGFR kinase [8-12].
<Insert Fig. 1>
Fig.1. Drugs of EGFR PK inhibiting activity
It has been reported that, oxygen bearing heterocyclic rings reveal an eminent class of
compounds displaying interesting biological activities, due to their similarity to various natural
bioactive molecules [13]. Benzofurans have the ability to inhibit particular serine/threonine
kinases whose deregulation is correlated with cancer development, and reduce the proliferation
of different tumor cells such as cervical and liver cancer cells by stopping the cycle of the cell at
G2/M phase [13,14]. In addition, different compounds carrying the pyrazole skeleton were
reported as potent anticancer agents inducing strong apoptosis and cell cycle arrest in addition to

3
effective EGFR tyrosine kinase suppression of inhibiting IC50 values reaching single digit
nanomolar range [15]. Moreover, the hydrazone functional groups were reported to optimize the
anticancer potency of different compounds inducing anticancer activity [16]. Furthermore,
thiazole and thiazolidine pharmacophores occupy an important place in the field of anticancer
chemotherapy targeting different proteins [17,18]. Due to the appearance of intrinsic resistance
and an insufficient response towards many effective anticancer drugs [19] and the hope to
generate new more effective and selective anticancer agents, the scope of the design of the new
derivatives in this study was based on hybridization of benzofuran-pyrazole core with substituted
thiazolidinone moieties via a hydrazone linkage aiming to gain a new scaffold for the synthesis
of novel antiproliferative agents of synergistic activity. The impact of molecular orientation, ring
size variance and heteroatoms existence which could participate in the interaction with EGFR
PK binding pocket via hydrogen bonding were taken in consideration (Fig. 2).The newly
synthesized target compounds were evaluated against cancerous cervical cells in addition to
evaluating the activity of the most active candidates against the target EGFR tyrosine kinase.
Moreover, the cell cycle activity was detected for the most potent compound, to get an overview
about the possible stage at which the new derivatives could suppress the growth of cancer cells.
Also, up regulation ofp53, Bax/ Bcl-2ratio andcaspases -3 and -7 was also determined in this
study.
<Insert Fig. 2>
Fig. 2. Proposed hypothetic model for the new benzofuran-pyrazole-thiazolidinone compounds
2. Results and discussion
2.1. Chemistry
The target 2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-
(aryl)thiazolidin-4-ones (4-22) were prepared in good yields using the following synthetic
protocol. Firstly, the intermediate carbothioamide 2 [20] was allowed to react with ethyl
bromoacetate in absolute ethanol containing few drops of piperidine as a catalytic basic medium
to
afford
the
corresponding
2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-
yl)methylene)hydrazono)thiazolidin-4-one (3). The new hybrids 4–22 were obtained via
subjecting the key reactant thiazolidin-4-one 3 to Knoevenagel condensation reaction with a
series of aromatic and heterocyclic aldehydes in alcoholic sodium hydroxide with continuous

4
1
13
stirring at room temperature overnight (Scheme 1). IR, H NMR, C NMR, Mass spectra and
1
the elemental analyses were used to confirm the new molecules. H NMR chemical shifts
allowed the characterization of the protons of the new derivatives that were presented at their
expected regions, such as a singlet signal appeared at δ 2.36, 3.03 ppm due to the three protons
of CH3 groups of compounds 5, 12, respectively. The signals of aromatic protons appeared as
multiplet signals in their characteristic regions. While the methoxy protons OCH3 of compounds
13
6-10 presented as singlets at the range δ 3.71 - 3.82 ppm. Also, C NMR spectral data showed
the typical signals for the aliphatic carbons in the expected regions, such as the signal for CH3
carbon of compounds 5, 12 which appeared at δ 21.46,33.92 ppm, respectively and the signals
due to OCH3 carbons of compounds 6-10 which are presented at the region δ 55.63 - 56.56 ppm.
<Insert Scheme 1>
Scheme 1: Synthesis of new 2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-
(aryl)thiazolidin-4-one derivatives. Reagents and conditions: (i) NH2CSNHNH2, EtOH, CH3COOH, reflux 2h; (ii)
BrCH2COOEt, EtOH, piperidine, reflux 3h; (iii) different aromatic aldehydes, alcoholic NaOH (10%), stirring
overnight.
2.2. Biological evaluation
2.2.1. In vitro anticancer activity
The aim of the present investigation is to synthesize a new series of benzofuran–pyrazol-
hydrazono-thiazolidin-4-ones (4-22) to be evaluated as anticancer candidates against the human
cervix epithelioid carcinoma cells. Accordingly, the anticancer activity of the new derivatives
was assessed against Hella cell line using MTT assay [21-22]. Doxorubicin served as a standard
drug.IC50 values for the tested compounds are reported in Table 1. It could be noted from the
obtained data that the significance of the potency depends of the type of the substituents of the
phenyl ring hybridized with the thiazolidinone ring. The most potent activity was gained by the
2,4-dichlorobenzylidene derivative 16 which was twice that obtained by the reference
doxorubicin (IC50; 0.60 µM, IC50 Doxorubicin; 1.10 µM). Also, the 4-bromo derivative 17
produced equipotent activity to that of doxorubicin. The presence of the two chlorine or the
bromine substituents on the phenyl ring yields an electron withdrawing effect. Even so, the
halogen atoms increased the Lipophilicity of the molecules, which is considered as one reason
for enhancement of the anticancer activity of the compounds 16, 17. On the other hand, the mono
halo substitution of the phenyl ring as 4-fluoro/3-chloro derivatives 13, 14 halved their activities

5
when compared to the reference drug (IC50; 2.52, 2.99 µM, respectively). Dramatic reduction in
the potency was observed by the 4-chloro derivative 15 (IC50; 22.68 µM). At the same time, the
3- and 4-methoxybenzylidene analogues 6, 7 represented significant cytotoxic activity of about
1.8 or 1.3 folds higher than that of doxorubicin (IC50; 0.63, 0.82 µM, respectively), a result
which could be explained due to the oxygen atom of the methoxy group that can function as H-
bond acceptor with the target protein kinase. A reverse result was obtained upon increasing the
number of the methoxy substituents on the phenyl ring. It was detected that 2,5-/3,4-/3,4,5-
dimethoxybenzylidene analogues 8, 9, 10 and 4-(dimethylamino)benzylidene derivative 12
exhibited 8-16 folds reduction in the potency with respect to doxorubicin (IC50; 8.64, 16.79
,9.01, 8.04 µM, respectively). The increase in the bulkiness of the substituents might negatively
affect the potency by changing the geometry of the molecules. Unfortunately, a detectable drop
in the activity was observed by the unsubstituted phenyl derivative 4 (IC50; 97.52 µM).
Furthermore, the replacement of the phenyl ring with two fused aromatic ring substituents such
as 1-naphthalenyl and 2-naphthalenyl analogues 18, 19 or with different heterocyclic rings such
as; 5-CH3-furyl, thienyl, pyridyl derivatives 20, 21, 22 was not in the favor the potency of the
anticancer activity since the effect decreased by 4.45-16 folds regarding to doxorubicin
2.2.2. EGFR PK inhibition assay
EGFR PK inhibition activity was evaluated for the compounds that represented the most potent
anticancer activity 6, 7, 11, 13, 14, 16, 17 using Hela cancer cells. The obtained results revealed
that some of the tested compounds are promising EGFR PK inhibiting candidates (Table 1).
Comparing to the potent EGFR PK inhibiting drug erlotinib, the 3-chlorobenzylidene compound
14 exhibited the most pronounced activity of IC50; 0.07 µM, more potent than the reference drug
of IC50; 0.08 µM. Although the rest of the examined derivatives produced potent cytotoxic
activity, their EGFR PK inhibitory activity was less than the reference drug by about 1.8-7.5
folds (IC50; 0.15-0.62 µM). In the light of the obtained results, although compound 14
represented the highest potency against EGFR PK, it showed the least anti-proliferative activity
among the tested derivatives (2.99 ±0.09 µM). Such discrepancies between enzyme potency and
the anticancer activity are frequently problematic issues in the field of the development of new
enzyme inhibiting agents. There are many factors that are related to such issue, such as improper
physicochemical properties leading to less accumulation in the cancer cells [24,25]. In addition,
there is no direct correlation between the anti-angiogenic effects of the tested compounds and

6
their anticancer activity against Hela cells, since the in vitro conditions do not simulate the in
vivo conditions since there is no blood supply or vascular endothelial cells [26]. So that, the
cytotoxic effect of the synthesized compounds is not necessarily related to the EGFR PK
inhibition, but mainly contribute to another mechanism of action
2.2.3. Effect of various representative target compounds against thenormal cervical cells
Damage to the normal cells is a very important obstacle that occurs due to the great
toxicity of antiproliferative drugs. Thus, the derivatives that showed the best activity against the
cervix cancer (Hela) cells (6, 7, 11, 13, 14, 16, 17) were selected as representative compounds to
be examined against the normal human cervical epithelium HCvEpC cell lines and their IC50
were determined via MTT assay [21-23]. It was detected that, the IC50 doses of all the tested
analogues against the healthy cells were much higher than their IC50 doses against the affected
cells (>20 folds of IC50; 17.2-32.12 µM) (Table 1), confirming the safety profile of the tested
compounds.
<Insert Table 1>
Table 1: In vitro anticancer activity against carcinoma Hela cell line, in vitro radiometric EGFR
PK assay and the effects against the normal human cervix cells of the different target compounds
2.2.4. Cell cycle analysis and apoptosis assay
The most effective EGFR PK inhibiting candidate 14 was subjected to cell cycle analysis
and apoptotic assay.
2.2.4.1. Cell cycle analysis and apoptosis detection
Typical neoplastic cells evade apoptosis since accumulation of mutations block apoptosis
pathways. Numerous anticancer researches confirmed the induction of apoptosis upon effective
cancer treatment. Autophagy is a metabolic process that degrades intracellular macromolecules
and endogenous substrate to maintain a stable internal environment. It is an important regulatory
mechanism in cell growth, maturation and death and is associated with a variety of human
diseases, including tumors. Chemotherapy agents may lead to an autophagic response, which is
one possible method of inducing apoptosis. There is a unique association between autophagy and
apoptosis [27].While chemotherapeutic drugs induce autophagy, cell cycle arrest and cell
senescence [21,22], it is imperative the induction of apoptosis and permanent removal of

7
neoplastic cells for effective cancer treatment. Studies on the effect of compound 14 on cell cycle
development and induction of apoptosis in cancer Hela cells was carried out. The cancer cells
were incubated with the compound 14 at its IC50 concentration 2.99 µM for 24 h. The cells were
stained with Annexin V/PI and analyzed by flow cytometry technique. Investigation of the
resultant data (Fig. 3) revealed a high percentage of cell accumulation of 27.11 % at pre G1
phase in Hela cells treated with the tested derivative after 24 h incubation vs 1.39 % of the
untreated Hela cells, indicating cell cycle arrest at G1/S phase.
<Insert Fig. 3>
Fig. 3. Cell Cycle analysis results of compound 14
2.2.4.2. Apoptosis assay
Cell cycle assay of Hela cancer cells treated with compound 14 showed the appearance of
pre-G1 peak which emphasized the induction of apoptosis. To prove the potency of compound14
to induce apoptosis, the cells were stained with Annexin V/PI, incubated for 24 h and analyzed.
It has been detected that the early and late apoptosis produced by the examined compound 14
certainly indicated its capability to induce significant levels of apoptosis with necrosis percent
1.99 (Fig. 4). Also, the percentage of late apoptosis (16.57 %) induced by compound 14 was
higher than that of the early apoptosis (8.55 %) which making recovery of apoptotic cells to be
healthy is more difficult (Fig. 5).
<Insert Fig. 4>
Fig. 4. Apoptosis induction analysis using Annexin V/PI for compound 14
<Insert Fig. 5>
Fig. 5. Cell cycle analysis and Apoptosis induction analysis of compound 14 on Hela cells

8
2.2.4.3.Effect of compound 14 on the level of p53/Bax/ Bcl-2
Apoptosis is triggered in a cell via two main pathways, which are; the extrinsic pathway
(the death receptor) or the intrinsic pathway (mitochondrial pathway) [28]. The cancer cells can
resist apoptosis by modulating the expression of Bcl-2 family proteins which in turn regulate the
mitochondrial apoptotic pathway via production anti-apoptotic proteins as Bcl-2 or down
regulating pro-apoptotic proteins, such as Bax [29]. Accordingly, the expression of Bcl-2 and
Bax genes is controlled by the tumor suppressor gene p53 [30]. Thus, this study also represented
the impact of 14 on p53 expression and the ratio of Bax/Bcl-2 (Table 2). Hela cells were
exposed to compound 14 at its IC50 concentration 2.99 µM for 24 h. The tested compound 14
represented overexpression of the tumor suppressor gene p53 and the pro-apoptotic protein Bax
by 7.07 and 5.7 folds, respectively with concurrent reduction in the expression levels ofthe anti-
apoptotic protein Bcl-2 by 0.45 folds (Table 2). Consequently, an observable increase in
Bax/Bcl-2 ratio was detected. These results suggested the participation of p53, Bax and Bcl-2
proteins in the apoptotic act of compound 14.
<Insert Table 2>
Table 2: Results of p53/Bax/BCL-2 analysis after treatment of Hela cells with compound 14 for
24 h.
2.2.4.4. Upregulation of caspases-3 and -7
Caspase cascade events mediate the induction of cell apoptosis via various intrinsic or
extrinsic pathways. Activated caspase-3 and caspase-7 can cleave multiple structural and
regulatory proteins, which are critical for cell survival and maintenance [31]. In this experiment,
the bioluminescent intensities of caspases -3 and -7 indicated that their activities were measured
in Hela cells treated with IC50 concentration of compound 14 for 24 hours of treatment. As
shown in (Table 3), a pronounced elevation in the caspases -3 and -7 levels by 16.21 and
7.60foldsrespectively comparing to the untreated Hela cells.
<Insert Table 3>
Table 3: Caspases-3, -7 concentrations in Hela cells after treatment with compound 14 for 24 h.

9
3. Molecular docking study
Molecular docking study was carried out to investigate the binding mode of interaction of
the most potent EGFR PK inhibiting compound 14 to show its interactions with the key amino
acids (hot spots) in the active site of the EGFR PK with the aim of explaining its promising
inhibitory activity. All the molecular modeling studies were carried out using Molecular
Operating Environment (MOE, 2010.10) software. All minimizations were performed with MOE
until an RMSD gradient of 0.05 kcal∙mol−1Å−1 with MMFF94x force field and the partial charges
were automatically calculated [32,33]. H-bonding with Met769, through water mediated H-
bonding with Thr766 and cation-π interaction with Lys721 (Fig.6 & 7). The validated setup was
then used in predicting the ligand-receptor interactions at the binding site for the compound of
interest.
<Insert Fig. 6>
Fig. 6. 2D interaction diagram showing erlotinib docking pose interactions with the key amino
acids in the EGFR PK binding site.
<Insert Fig. 7>
Fig.7. (a) 2D representation and (b) 3D representation of the superimposition of the co-
crystallized (red) and the docking pose (blue) of erlotinib in the EGFR PK binding site with
RMSD of 1.321Å. (Ligand Hydrogen atoms were removed for clarity).
The ability of compound 14 to interact with the key amino acids in the binding site
rationalizes its good activity as indicated by its docking pattern and docking score compared to
that of erlotinib. The general binding pattern of the tested compound 14 is that it interacts
through hydrogen bonding with the key amino acid Met769 by its N atom of the imine moiety,
through water mediated H-bonding with Thr766 by its S atom of the thiazole ring and through
cation-π interaction with Lys721 by its m-chlorophenyl moiety (Fig. 8 & Table 4).
<Insert Fig. 8>
Fig.8. (a) 2D diagram and (b) 3D representation of compound 14 in the EGFR PK binding site

10
<Insert Table 4>
Table 4: Docking energy scores (S) in kcal/mol for the reference drug and the tested compound
14
4. Conclusion
A
new
series
of
2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-
yl)methylene)hydrazono)-5-(aryl)thiazolidin-4-one derivatives have been designed and
synthesized as anticancer EGFR kinase inhibitors. All the new derivatives were subjected to
anticancer evaluation against Hela cell line by MTT technique using doxorubicin as a standard
drug. The compounds 6, 7, 11, 13, 14, 16, 17 exhibited the most potent activity of IC50 values
ranging from 0.60 to 2.99 µM comparing to 1.10 µM the IC50 value of doxorubicin.
Furthermore, the latter derivatives were evaluated for their EGFR kinase inhibitory activity. The
3-chlorobenzylidene compound 14 represented the highest inhibitory activity among all the
tested compounds more than the reference drug erlotinib of IC50; 0.07 µM vs IC50erlotinib; 0.08
µM. Also, compound 14 showed high cell accumulation at pre G1 phase in Hela cells confirming
its arresting cellcycle at G1/S phase. Compound 14 was certainly able to induce significant levels
of apoptosis percent 27.11 %. The apoptotic mechanistic pathway of 14 was also emphasized by
the enhanced expression of the tumor suppressor gene p53 and the increase of Bax/Bcl-2 ratio. In
addition, a pronounced enhancement in the levels of the active caspases -3 and -7 was also
investigated indicating the potent pro apoptotic activity of 14 by inducing the intrinsic apoptotic
pathway. Molecular docking study of compound 14 represented its good fitting and proper
interaction with the key amino residues in the binding site of EGFR kinase comparing to
erlotinib.
5. Experimental
5.1. Chemistry
All melting points are uncorrected and were taken in open capillary tubes using
Electrothermal apparatus 9100. Elemental microanalyses were carried out at Microanalytical
Unit, Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt, using Vario
Elementar and were found within ±0.4% of the theoretical values. Infrared spectra were recorded
on a Schimadzu FT-IR Affinity-1 Spectrometer, Infrared spectrometer at cm-1 scale using KBr
1
13
disc technique at Faculty of Pharmacy-Cairo University, Cairo, Egypt. H NMR and C NMR

11
spectra were determined by using a Bruker High Performance Digital FT-NMR Spectrometer
Avance III 400MHz, Faculty of Pharmacy-Cairo University, Cairo, Egypt. Chemical shifts were
expressed in δ (ppm) downfield from TMS as an internal standard. The mass spectrawere
measured with a Finnigan MATSSQ-7000 mass spectrometer at Central Services Laboratory,
National Research Centre, Dokki, Cairo, Egypt. Follow up of the reactions and checking the
purity of the compounds were made by TLC on silica gel-precoated aluminum sheets (Type 60,
F 254, Merck, Darmstadt, Germany) using chloroform/methanol (20:2, v/v) and the spots were
detected by exposure to UV lamp at 254 nanometer for few seconds and by iodine vapor. The
chemical names given for the prepared compounds are according to the IUPAC system. 3-
(Benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1), 2-((3-(benzofuran-2-yl)-1-phenyl-
1H-pyrazol-4-yl)methylene)hydrazine-1-carbothioamide (2) were prepared according to the
reported method [17].
5.1.1. 2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)thiazolidin-4-
one (3)
A mixture of 2-((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazine-1-
carbothioamide (2) (7.22 g, 0.02 mol) and ethyl bromoacetate (3.52 mL, 0.02 mol) was
dissolved in absolute ethanol (50ml) containing a few drops of piperidine was refluxed for 3 h.
The formed precipitate was filtered, dried and recrystallized from absolute ethanol to give the
title compound 3.
Yield 82%, mp 262-263ᴼC, light orange solid, IR (νmax/cm−1): 3135 (NH), 3051 (CH-aromatic),
1
1711 (C=O), 1631 (C=N), 1598 (C=C). H NMR (DMSO-d6): δ 3.98 (s, 2H, thiazolidinone
protons), 7.30-7.33 (m, 1H, Ar-H), 7.37-7.45 (m, 2H, Ar-H), 7.57-7.60 (s, 2H, Ar-H), 7.70-7.75
(s, 2H, Ar-H), 7.88 (s, 1H, Ar-H), 8.00 (d, 2H, Ar-H, J= 7.92 Hz), 8.73 (s, 1H, CH=N proton),
9.04 (s, 1H, pyrazole proton), 11.02 (br s, 1H, NH, D2O exchangeable);13C NMR (DMSO-d6): δ
55.68 (CH2), 107.39, 111.83, 114.73, 119.34, 121.79, 122.35, 123.76, 125.69, 127.73, 128.78,
130.18, 131.06, 132.31, 139.24, 141.72, 143.98, 149.84, 154.67, 159.36, 174.18
(C=O).MS,m/z(%): 401 [M+] (33), 77[C6H5] (100).Anal. For C21H15N5O2S (401.44): Calcd. C,
62.83; H, 3.77; N, 17.45; S, 7.99; Found: C, 62.56; H, 3.44; N, 17.27; S, 7.61.
5.1.2. General procedure for the synthesis of 2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-
yl)methylene)hydrazono)-5-(aryl)thiazolidin-4-one 4–22

12
A mixture of thiazolidin-4-one derivative 3 (0.41 gm, 0.001 mol) and the appropriate
aldehyde namely; benzaldehyde, 4-methylbenzaldehyde, 3-methoxybenzaldehyde, 4-
methoxybenzaldehyde, 2,5-dimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 3,4,5-
trimethoxybenzaldehyde,
4-nitrobenzaldehyde,
4-(dimethylamino)benzaldehyde,
4-
fluorobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 2,4-dichlorobenzaldehyde, 4-
bromobenzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 5-methylfuran-2-carbaldehyde,
thiophene-2-carbaldehyde and pyridine-4-carbaldehyde (0.0015 mol) was dissolved in alcoholic
sodium hydroxide (20 mL, 10%) was continuously stirred at room temperature overnight. The
formed precipitate wasfiltered, washed several times with water, dried and crystallized from
acetic acid to afford the title compounds 4–22 respectively.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-
(benzylidene)thiazolidin-4-one (4)
Yield 88%, mp 284-285ᴼC, yellow solid, IR (νmax/cm−1): 3132 (NH), 3059 (CH-aromatic), 1709
(C=O), 1637 (C=N), 1597 (C=C). 1H NMR (DMSO-d6): δ 7.26-7.75 (m, 11H, Ar-H), 7.84 (s,
1H, CH=C), 7.97-8.05 (m, 4H, Ar-H), 8.84 (s, 1H, CH=N proton), 9.17 (s, 1H, pyrazole proton),
12.68 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6): δ 107.30, 107.94, 111.72,
111.90,, 117.46, 119.50, 119.64, 121.91, 123.71, 123.95, 125.75, 125.84, 126.18, 127.93, 128.08,
128.49, 128.55, 128.76, 129.69, 130.19, 130.31, 131.30, 131.75, 134.10, 139.14, 149.42, 154.77,
174.01. MS, m/z (%): 489 [M+] (22), 77 [C6H4] (100).Anal. For C28H19N5O2S (489.55): Calcd.
C, 68.70; H, 3.91; N, 14.31; S, 6.55; Found: C, 68.56; H, 3.78; N, 14.47; S, 6.30.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
methylbenzylidene)thiazolidin-4-one (5)
Yield 78%, mp 223-225ᴼC, dark yellow solid,IR (νmax/cm−1): 3129 (NH), 3047 (CH-
aromatic),1704 (C=O), 1632 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 2.36 (s, 3H, CH3),
7.26-7.42 (m, 6H, Ar-H), 7.55-7.61 (m, 4H, Ar-H), 7.75-7.76 (m, 2H, Ar-H), 8.03 (d, 2H, Ar-H,
J = 7.22 Hz), 8.09 (s, 1H, CH=C), 8.73 (s, 1H, CH=N proton), 9.03 (s, 1H, pyrazole proton),
12.03 (br s, 1H, NH, D2O exchangeable);13C NMR (DMSO-d6): δ21.46 (CH3), 107.39, 111.83,
119.14, 119.38, 121.80. 122.49, 123.77, 125.64, 127.68, 128.82, 129.50, 129.88, 130.16, 131.84,
133.73, 137.89, 139.32, 141.75, 144.28, 149.93, 154.70, 179.64 (C=O). MS, m/z (%): 503 [M+]
(29), 91 [C7H7](100). Anal. ForC29H21N5O2S (503.58): Calcd. C, 69.17; H, 4.20; N, 13.91; S,
6.37; Found: C, 69.28; H, 4.35; N, 13.72; S, 6.18.

13
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(3-
methoxybenzylidene)thiazolidin-4-one (6)
Yield 77%, mp 259-260ᴼC, canarysolid,IR (νmax/cm−1): 3125 (NH), 3048 (CH-aromatic), 2947,
2836 (CH-aliphatic), 1716 (C=O), 1639 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 3.72 (s, 3H,
OCH3), 6.99 (d, 1H, Ar-H, J = 8.92 Hz), 7.19-7.17 (m, 2H, Ar-H), 7.25-7.39 (m, 4H, Ar-H),
7.50-7.55 (m, 3H, Ar-H), 7.65 (d, 2H, Ar-H, J = 8.00 Hz), 7.81 (s, 1H, CH=C), 7.96 (d, 2H, Ar-
H, J = 8.04 Hz), 8.74 (s, 1H, CH=N proton), 9.07 (s, 1H, pyrazole proton), 12.57 (br s, 1H,
NH,D2O exchangeable); 13C NMR (DMSO-d6): δ55.63 (OCH3), 107.52, 111.76, 115.29, 115.87,
117.84, 119.52, 121.75, 121.92, 123.89, 124.02, 125.86, 128.03, 128.52, 130.23, 130.64, 131.30,
139.08, 149.42, 154.66, 160.03, 174.28 (C=O).MS, m/z (%): 518 [M-1] (18), 76 [C6H4]
(100).Anal. ForC29H21N5O3S (519.58): Calcd. C, 67.04; H, 4.07; N, 13.48; S, 6.17; Found: C,
67.26; H, 4.30; N, 13.25; S, 6.02.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
methoxybenzylidene)thiazolidin-4-one (7)
Yield 84%, mp 297-298ᴼC, yellow solid,IR (νmax/cm−1): 3138 (NH), 3058 (CH-aromatic),1710
(C=O), 1629 (C=N), 1594 (C=C).1H NMR (DMSO-d6): δ 3.82 (s, 3H, OCH3), 7.00 (d, 2H, Ar-H,
J = 8.56 Hz), 7.22 (s, 1H, Ar-H), 7.35-7.44 (m, 3H, Ar-H), 7.57-7.62 (m, 4H, Ar-H), 7.74-7.77
(m, 2H, Ar-H), 8.03 (d, 2H, Ar-H, J = 7.92 Hz), 8.10 (s, 1H, CH=C), 8.69 (s, 1H, CH=N proton),
9.00 (s, 1H, pyrazole proton), 12.31 (br s, 1H, NH, D2O exchangeable);13C NMR (DMSO-d6): δ
56.56 (OCH3), 107.37, 111.69, 114.81, 117.65, 119.48, 122.04, 123.83, 125.75, 127.96, 128.05,
128.35, 128.51, 130.15, 130.83, 133.38, 139.06, 142.28, 143.61, 144.41, 149.38, 149.61, 154.66,
164.49, 165.31, 174.69 (C=O).MS, m/z (%): 519 [M+] (25), 77 [C6H5] (100).Anal.
ForC29H21N5O3S (519.58): Calcd. C, 67.04; H, 4.07; N, 13.48; S, 6.17; Found: C, 67.32; H, 4.29;
N, 13.19; S, 6.41.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(2,5-
dimethoxybenzylidene)thiazolidin-4-one (8)
Yield 76%, mp 283-285ᴼCcanarysolid,IR (νmax/cm−1): 3136 (NH), 3043 (CH-aromatic), 2939,
2831 (CH-aliphatic), 1712 (C=O), 1632 (C=N), 1597 (C=C). 1H NMR (DMSO-d6): δ 3.71 (s,
3H, OCH3), 3.83 (s, 3H, OCH3),7.08-7.10 (m, 3H, Ar-H), 7.27-7.46 (m, 3H, Ar-H), 7.57-7.61
(m, 2H, Ar-H), 7.67-7.72 (m, 2H, CH=C, Ar-H), 7.84 (d, 2H, Ar-H, J = 10.67 Hz), 8.03 (d, 2H,
Ar-H, J = 7.88 Hz), 8.82 (s, 1H, CH=N proton), 9.14 (s, 1H, pyrazole proton), 12.60 (br s, 1H,

14
NH,D2O exchangeable); 13C NMR (DMSO-d6): δ 55.89 (OCH3), 56.05 (OCH3), 107.54, 111.61,
112.24, 114.05, 115.28, 118.08, 119.18, 119.32, 121.91, 122.54, 122.94, 123.82, 125.61, 127.68,
128.43, 130.22, 133.31, 139.25, 142.19, 143.27, 147.86, 149.01, 149.62, 150.11, 154.58, 173.43
(C=O).MS, m/z (%): 548 [M-1] (21), 91 [C7H7] (100).Anal. ForC30H23N5O4S (549.61): Calcd. C,
65.56; H, 4.22; N, 12.74; S, 5.83; Found: C, 65.40; H, 4.36; N, 12.58; S, 5.65.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(3,4-
dimethoxybenzylidene)thiazolidin-4-one (9)
Yield 78%, mp 249-250ᴼC, light yellow,IR (νmax/cm−1): 3124 (NH), 3059 (CH-aromatic), 2951,
2831 (CH-aliphatic), 1701 (C=O), 1635 (C=N), 1593 (C=C).1H NMR (DMSO-d6): δ 3.76 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 7.04 (d, 1H, Ar-H, J = 8.16 Hz), 7.23-7.25 (m, 2H, Ar-H), 7.31-7.35
(m, 1H, Ar-H), 7.39-7.45 (m, 2H, Ar-H), 7.51 (s, 1H, Ar-H), 7.58-7.61 (m, 2H, Ar-H), 7.71-7.75
(m, 2H, Ar-H), 7.89 (s, 1H, s, 1H, CH=C), 8.01 (d, 2H, Ar-H, J = 7.92 Hz), 8.78 (s, 1H, CH=N
proton), 9.09 (s, 1H, pyrazole proton), 12.46 (br s, 1H, NH, D2O exchangeable);13C NMR
(DMSO-d6): δ 55.87 (OCH3), 55.97 (OCH3), 107.33, 111.76, 112.13, 114.23, 115.22, 118.14,
119.39, 119.45, 121.83, 122.41, 123.75, 125.72, 127.87, 128.59, 130.13, 133.11, 139.10, 142.11,
143.39, 147.98, 149.22, 149.55, 150.06, 154.65, 172.83 (C=O).MS, m/z (%): 550 [M+1] (29), 77
[C6H5] (100).Anal. ForC30H23N5O4S (549.61): Calcd. C, 65.56; H, 4.22; N, 12.74; S, 5.83;
Found: C, 65.72; H, 4.38; N, 12.50; S, 5.54.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(3,4,5-
trimethoxybenzylidene)thiazolidin-4-one (10)
Yield 71%, mp 206-207ᴼC,canarysolid,IR (νmax/cm−1): 3163 (NH), 3048 (CH-aromatic), 2936,
2835 (CH-aliphatic), 1701 (C=O), 1631 (C=N), 1585 (C=C).1H NMR (DMSO-d6): δ 3.71 (s, 3H,
OCH3), 3.72 (s, 6H, OCH3), 6.95 (s, 2H, Ar-H), 7.20 (s, 1H, Ar-H), 7.24-7.45 (m, 2H, Ar-H),
7.58-7.75 (m, 3H, Ar-H), 7.92-8.01 (m, 2H, Ar-H), 8.32 (s, 1H, CH=C), 8.64 (d, 2H, Ar-H, J =
7.28 Hz), 8.84 (s, 1H, CH=N proton), 9.27 (s, 1H, pyrazole proton), 11.50 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6): δ 56.18 (2-OCH3), 60.64 (OCH3), 106.89, 107.03, 111.67,
118.45, 119.25, 119.40, 121.77, 122.05, 124.05, 125.68, 127.99, 128.05, 128.43, 128.64, 130.22,
130.25, 132.41, 135.45, 137.74, 139.21, 139.28, 141.74, 142.57, 149.52, 149.79, 153.41, 154.61,
178.11 (C=O).MS, m/z (%): 579 [M+] (17), 90 [C7H6] (100).Anal. For C31H25N5O5S (579.63):
Calcd. C, 64.24; H, 4.35; N, 12.08; S, 5.53; Found: C, 64.06; H, 4.18; N, 12.24; S, 5.21.

15
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
nitrobenzylidene)thiazolidin-4-one (11)
Yield 60%, mp 286-287ᴼC, dark red solid,IR (νmax/cm−1): 3132 (NH), 3062 (CH-aromatic), 1713
(C=O), 1635 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 7.32-7.44 (m, 4H, Ar-H), 7.58-7.74
(m, 6H, Ar-H), 7.96 (s, 1H, CH=C), 7.87 (d, 1H, Ar-H, J = 8.60 Hz), 8.03 (d, 2H, Ar-H, J = 7.60
Hz), 8.30 (d, 1H, Ar-H, J = 8.68 Hz), 8.81 (s, 1H, CH=N proton), 9.09 (s, 1H, pyrazole proton),
12.13 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6): δ 107.15, 112.56, 117.92,
119.37, 120.38, 121.69, 122.23, 124.24, 125.74, 127.68, 128.21, 128.56, 129.07, 130.30, 132.51,
137.74, 138.43, 139.39, 141.62, 142.71, 149.77, 149.91, 152.81, 155.40, 177.66 (C=O).MS, m/z
(%): 534 [M+] (18), 77 [C6H5] (100). Anal. ForC28H18N6O4S (534.55): Calcd. C, 62.91; H, 3.39;
N, 15.72; S, 6.00; Found: C, 62.75; H, 3.51; N, 15.62; S, 6.18.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
(dimethylamino)benzylidene)thiazolidin-4-one (12)
Yield 61%, mp 259-260ᴼC,light orange solid,IR (νmax/cm−1): 3117 (NH), 3048 (CH-aromatic),
1732 (C=O), 1636 (C=N), 1582 (C=C).1H NMR (DMSO-d6): δ 3.03 (s, 6H, N(CH3)2), 6.78 (d,
1H, Ar-H, J = 8.72 Hz), 7.30-7.45 (m, 4H, Ar-H), 7.48-7.52 (m, 1H, Ar-H),7.57- 7.61 (m, 3H,
Ar-H), 7.70-7.75 (m, 3H, Ar-H), 7.89 (s, 1H, CH=C), 8.00 (d, 2H, Ar-H, J = 8.00 Hz), 8.73 (s,
1H, CH=N proton), 9.04 (s, 1H, pyrazole proton), 11.94 (br s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6): δ 33.92 (N(CH3)2), 107.34, 107.48, 111.78, 112.42, 117.78, 119.55, 121.89,
122.05, 123.86, 125.75, 127.96, 128.58, 130.18, 130.89, 132.12, 139.17, 142.31, 149.33, 149.48,
149.56, 151.31, 154.71, 154.76, 174.96 (C=O). MS, m/z (%): 533 [M+1] (23), 77 [C6H5]
(100).Anal. For C30H24N6O2S (532.62): Calcd. C, 67.65; H, 4.54; N, 15.78; S, 6.02; Found: C,
67.49; H, 4.27; N, 15.60; S, 6.15.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
fluorobenzylidene)thiazolidin-4-one (13)
Yield 84%, mp 284-286ᴼC, yellow solid,IR (νmax/cm−1): 3132 (NH), 3063 (CH-aromatic),1715
(C=O), 1620 (C=N), 1589 (C=C).1H NMR (DMSO-d6): δ 7.23-7.28 (m, 2H, Ar-H), 7.33-7.44
(m, 4H, Ar-H), 7.57-7.61 (m, 2H, Ar-H), 7.67-7.77(m, 4H, Ar-H), 7.99 (s, 1H,CH=C),8.03 (d,
2H, Ar-H, J = 7.84 Hz),8.69 (s, 1H, CH=N proton), 8.99 (s, 1H, pyrazole proton), 11.87 (br s,
13
1H, NH, D2O exchangeable); C NMR (DMSO-d6): δ 107.34, 112.04, 116.21, 117.64, 118.71,
119.58, 121.47, 122.11, 123.51, 123.90, 125.63, 128.70, 129.08, 129.71, 130.02, 130.75, 132.54,

16
140.48, 142.87, 149.28, 149.72, 151.46, 153.95, 156.62, 174.96 (C=O).MS, m/z (%): 506 [M-1]
(16), 77 [C6H5] (100).Anal. ForC28H18FN5O2S (507.54): Calcd. C, 66.26; H, 3.57; N, 13.80; S,
6.32; Found: C, 66.42; H, 3.29; N, 13.92; S, 6.16.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(3-
chlorobenzylidene)thiazolidin-4-one (14)
Yield 72%, mp 243-245ᴼC, yellow solid,IR (νmax/cm−1): 3125 (NH), 3059 (CH-aromatic), 1713
(C=O), 1639 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 7.30-7.45 (m, 3H, Ar-H), 7.52-7.61
(m, 5H, Ar-H), 7.70-7.75 (m, 3H, Ar-H), 7.80 (s, 1H,CH=C),7.88-7.96(m, 1H, Ar-H),8.01 (d,
2H, Ar-H, J = 8.16 Hz),8.82 (s, 1H, CH=N proton), 9.14 (s, 1H, pyrazole proton), 12.64 (br s,
1H, NH, D2O exchangeable);13C NMR (DMSO-d6): δ 106.84, 111.36, 117.69, 119.90,
120.43,121.56, 122.51, 123.22, 123.85, 124.80, 125.21, 125.68, 128.25, 128.96, 129.11, 129.62,
129.97, 131.06, 134.62, 139.04, 142.87, 148.20, 149.01, 151.13, 154.55, 174.82 (C=O). MS, m/z
(%): 525, 523 [M+] (23, 8), 77 [C6H5] (100).Anal. ForC28H18ClN5O2S (524.00): Calcd. C, 64.18;
H, 3.46; N, 13.37; S, 6.12; Found: C, 64.27; H, 3.51; N, 13.14; S, 6.30.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
chlorobenzylidene)thiazolidin-4-one (15)
Yield 82%, mp > 300ᴼC, yellow solid,IR (νmax/cm−1): 3124 (NH), 3048 (CH-aromatic),1713
(C=O), 1636 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 7.29-7.33 (m, 1H, Ar-H), 7.38-7.45
(m, 3H, Ar-H), 7.52-7.65 (m, 5H, Ar-H), 7.68-7.72 (m, 2H, Ar-H), 7.76 (s, 1H, CH=C), 7.95 (d,
1H, Ar-H, J = 8.28 Hz), 8.02 (d, 2H, Ar-H, J = 7.88 Hz), 8.80 (s, 1H, CH=N proton), 9.09 (s,
1H, pyrazole proton), 12.66 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6): δ107.21,
111.43, 117.50, 119.82, 120.11, 121.48, 122.33, 123.76, 124.65, 125.52, 127.87, 128.09, 128.37,
128.60, 129.44, 130.46, 134.41, 139.22, 141.63, 148.45, 149.33, 151.27, 154.61, 175.73
(C=O).MS, m/z (%): 526, 524 [M+1](16, 5), 76 [C6H4] (100). Anal. ForC28H18ClN5O2S (524.00):
Calcd. C, 64.18; H, 3.46; N, 13.37; S, 6.12; Found: C, 64.26; H, 3.31; N, 13.19; S, 6.28.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(2,4-
dichlorobenzylidene)thiazolidin-4-one (16)
Yield 77%, mp 229-230ᴼC, orange solid,IR (νmax/cm−1): 3128 (NH), 3047 (CH-aromatic),1709
(C=O), 1632 (C=N), 1595 (C=C).1H NMR (DMSO-d6): δ 7.31-7.32 (m, 1H, Ar-H), 7.38-7.47
(m, 4H, Ar-H), 7.57-7.61 (m, 2H, Ar-H), 7.65 (d, 1H, Ar-H, J = 7.36 Hz), 7.75 (d, 2H, Ar-H, J =
9.16 Hz), 7.81 (d, 1H, Ar-H, J = 8.44 Hz), 7.92 (s, 1H, CH=C proton), 8.04 (d, 2H, Ar-H, J =

17
7.92), 8.72 (s, 1H, CH=N proton), 8.99 (s, 1H, pyrazole proton)12.52 (br s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6): δ107.45, 112.26, 117.62, 119.71, 120.24, 121.53, 121.91,
122.84, 124.14, 125.69, 127.91, 128.36, 128.74, 128.91, 129.56, 131.26, 134.68, 138.90, 141.15,
148.62, 149.14, 151.30, 154.82, 176.09 (C=O). MS, m/z (%): 557, 558, 559 [M+] (19, 4, 7), 77
[C6H5] (100). Anal. ForC28H17Cl2N5O2S (558.44): Calcd. C, 60.22; H, 3.07; N, 12.54; S, 5.74;
Found: C, 60.38; H, 3.26; N, 12.33; S, 5.61.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(4-
bromobenzylidene)thiazolidin-4-one (17)
Yield 93%, mp 249-251ᴼC, orange solid,IR (νmax/cm−1): 3132 (NH), 3063 (CH-aromatic), 1701
(C=O), 1628 (C=N), 1589 (C=C).1H NMR (DMSO-d6): 7.22 (s, 1H, Ar-H), 7.31-7.49 (m, 3H,
Ar-H), 7.57-7.61 (m, 4H, Ar-H), 7.70-7.76 (m, 3H, Ar-H), 7.83-7.84 (m, 1H, Ar-H), 7.95-8.05
(m, 3H, CH=C, Ar-H), 8.71 (s, 1H, CH=N proton), 8.99 (s, 1H, pyrazole proton), 9.91 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6): δ106.97, 112.55, 117.02, 118.93, 119.69,
121.41, 121.97, 122.23, 122.82, 124.09, 125.42, 127.67, 128.40, 128.66, 128.97, 129.21, 131.5,
134.31, 141.15, 148.62, 149.14, 151.30, 154.82, 175.41 (C=O).MS, m/z (%): 568, 566 [M-1]
(100, 34).Anal. ForC28H18BrN5O2S (568.45): Calcd. C, 59.16; H, 3.19; N, 12.32; S, 5.64; Found:
C, 59.25; H, 3.31; N, 12.08; S, 5.42.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(naphthalen-1-
ylmethylene)thiazolidin-4-one (18)
Yield 82%, mp 283-284ᴼC, dark yellow solid,IR (νmax/cm−1): 3152 (NH), 3044 (CH-aromatic),
1705 (C=O), 1636 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 7.29-7.44 (m, 3H, Ar-H), 7.54-
7.72 (m, 7H, Ar-H), 7.84-7.89 (m, 2H, Ar-H), 8.00-8.07 (m, 4H, Ar-H), 8.15-8.19 (m, 2H, Ar-H,
CH=C proton), 8.78 (s, 1H, CH=N proton), 9.08 (s, 1H, pyrazole proton), 11.18 (br s, 1H, NH,
13
D2O exchangeable), C NMR (DMSO-d6): δ 106.86, 112.35, 118.33, 118.82, 119.22, 119.36,
119.84, 121.54, 121.95, 122.11, 122.72, 123.61, 124.14, 125.23, 127.21, 128.11, 128.56, 128.84,
129.54, 130.34, 132.51, 133.67, 134.27, 135.07, 139.41, 141.62, 142.40, 149.33, 149.91, 154.44,
177.41 (C=O). MS, m/z (%): 539 [M+] (45), 92 [C7H8] (100).Anal. ForC32H21N5O2S (539.61):
Calcd. C, 71.23; H, 3.92; N, 12.98; S, 5.94; Found: C, 71.42; H, 3.71; N, 12.68; S, 5.80.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(naphthalen-2-
ylmethylene)thiazolidin-4-one (19)

18
Yield 91%, mp 197-198ᴼC, yellow solid,IR (νmax/cm−1): 3159 (NH), 3051 (CH-aromatic), 1705
(C=O), 1628 (C=N), 1597 (C=C).1H NMR (DMSO-d6): δ 7.28-7.45 (m, 4H, Ar-H), 7.53-7.67
(m, 4H, Ar-H), 7.72-7.98 (m, 6H, Ar-H), 8.05 (d, 2H, Ar-H, J = 8.52 Hz), 8.64 (s, 1H, Ar-H),
8.72 (s, 1H, CH=C proton), 9.05 (s, 1H, CH=N proton), 9.27 (s, 1H, pyrazole proton), 11.50 (s,
1H, NH, D2O exchangeable);13C NMR (DMSO-d6): δ 107.32, 111.68, 118.45, 119.01, 119.25,
119.42, 121.84, 122.05, 122.69, 123.88, 124.05, 125.69, 127.09, 128.05, 128.43, 128.72, 130.21,
130.26, 132.69, 133.50, 134.10, 135.46, 139.21, 141.84, 142.58, 149.52, 149.78, 154.65, 178.11
(C=O).MS, m/z (%): 538 [M-1] (29), 77 [C6H5] (100). Anal. ForC32H21N5O2S (539.61): Calcd. C,
71.23; H, 3.92; N, 12.98; S, 5.94; Found: C, 71.38; H, 3.64; N, 12.81; S, 5.76.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-((5-methylfuran-
2-yl)methylene)thiazolidin-4-one (20)
Yield 77%, mp 245-247ᴼC, yellow solid,IR (νmax/cm−1): 3125 (NH), 3059 (CH-aromatic), 1701
(C=O), 1632 (C=N), 1595 (C=C).1H NMR (DMSO-d6): δ 2.33 (s, 3H, CH3), 6.35 (d, 1H, Ar-H, J
= 2.48 Hz), 6.78 (d, 1H, Ar-H, J = 2.80 Hz), 7.22 (s, 1H, Ar-H), 7.31-7.45 (m, 3H, Ar-H), 7.58-
7.62 (m, 2H, Ar-H), 7.72-7.75 (m, 2H, Ar-H), 7.90 (s, 1H, CH=C proton), 8.05 (d, 2H, Ar-H, J =
7.92), 8.76 (s, 1H, CH=N proton), 9.07 (s, 1H, pyrazole proton), 10.81 (br s, 1H, NH, D2O
exchangeable);13C NMR (DMSO-d6): δ 14.13, 107.17, 110.11, 111.77, 114.13, 117.05, 118.12,
119.51, 121.96, 123.86, 125.79, 127.96, 128.56, 130.21, 130.59, 139.14, 142.24, 148.06, 149.52,
149.65, 154.68, 155.75, 174.22 (C=O).MS, m/z (%): 493 [M+] (21), 77 [C6H5] (100). Anal. For
C27H19N5O3S (493.54): Calcd. C, 65.71; H, 3.88; N, 14.19; S, 6.50; Found: C, 65.54; H, 3.62; N,
14.37; S, 6.29.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(thiophen-2-
ylmethylene)thiazolidin-4-one (21)
Yield 65%, mp 240-242ᴼC, dark yellow solid,IR (νmax/cm−1): 3125 (NH), 3066 (CH-aromatic),
1700 (C=O), 1632 (C=N), 1591 (C=C). 1H NMR (DMSO-d6): δ 7.12 (s, 1H, Ar-H), 7.27-7.37
(m, 4H, Ar-H), 7.42 (s, 1H, Ar-H), 7.51-7.53(m, 2H, Ar-H), 7.64-7.74 (m, 3H, Ar-H), 7.95-7.98
(m, 3H, CH=C, Ar-H),8.65 (s, 1H, CH=N proton), 8.93 (s, 1H, pyrazole proton), 11.12 (br s, 1H,
NH, D2O exchangeable);13C NMR (DMSO-d6): δ 107.16, 111.78, 115.42, 119.15, 119.43,
122.06, 123.76, 125.61, 127.72, 128.41, 128.66, 128.77, 129.57, 130.05, 130.17, 132.21, 139.33,
141.56, 141.82, 144.20, 149.90, 154.70, 179.73 (C=O).MS, m/z (%): 494 [M-1] (26), 76 [C6H4]

19
(100).Anal. For C26H17N5O2S2 (495.58): Calcd. C, 63.01; H, 3.46; N, 14.13; S, 12.94; Found: C,
63.25; H, 3.31; N, 14.29; S, 12.68.
2-(((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-5-(pyridin-4-
ylmethylene)thiazolidin-4-one (22)
Yield 61%, mp 248-250ᴼC, dark orange solid,IR (νmax/cm−1): 3125 (NH), 3048 (CH-
aromatic),1710 (C=O), 1620 (C=N), 1598 (C=C).1H NMR (DMSO-d6): δ 7.20 (s, 1H, Ar-H),
7.33-7.44(m, 3H, Ar-H), 7.56-7.61 (m, 4H, Ar-H), 7.73-7.77 (m, 2H, Ar-H), 7.96 (s, 1H, CH=C),
8.05 (d, 2H, Ar-H, J = 7.88 Hz), 8.60 (d, 2H, Ar-H, J = 7.32 Hz), 8.75 (s, 1H, CH=N proton),
9.03 (s, 1H, pyrazole proton), 12.48 (br s, 1H, NH, D2O exchangeable), 13C NMR (DMSO-d6): δ
107.24, 112.28, 116.07, 118.68, 119.22, 121.53, 122.84,122.51, 123.54, 124.92, 125.04, 127.43,
128.55, 129.64, 130.26, 139.41, 141.73, 144.36, 146.28, 148.20, 149.67, 155.06, 178.49 (C=O).
MS, m/z (%): 491 [M+1] (19), 64 [C5H4] (100).Anal. ForC27H18N6O2S (490.54): Calcd. C, 66.11;
H, 3.70; N, 17.13; S, 6.54; Found: C, 66.24; H, 3.53; N, 17.24; S, 6.40.
5.2. Biological evaluation
5.2.1. In vitro anticancer activity
MTT assay was used to evaluate the in vitro cytotoxicity of the new compounds against
cervix cancer cell lines Hela [21-23]. MTT assay depends on the reduction of the soluble 3-(4,5-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into a blue purple formazan
product, mainly by mitochondrial reductase activity inside the living cells. The cells used in
cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10% fetal calf
serum. Cells suspended in the medium (2˟104 cells/mL) were plated in 96-well culture plates and
ο
incubated at 37 C in a 5% CO2incubator. After 12 h, the test sample (2 μL) was added to the
cells (2 ˟104) in 96-well plates and cultured at 37◦C for 3 days. The cultured cells were mixed
ο
with 20 μL of MTT solution and incubated for 4 h at 37 C. The supernatant was carefully
removed from each well and 100 μL of DMSO were added to each well to dissolve the formazan
crystals which were formed by the cellular reduction of MTT. After mixing with a mechanical
plate mixer, the absorbance of each well was measured by a microplate reader using a test
wavelength of 570 nm. The results were expressed as the IC50 (μM), which inducing a50%
inhibition of cell growth of the treated cells when compared to the growth of control cells. Each
experiment was performed at least 3 times. There was a good reproducibility between the
replicate wells with standard errors.

20
5.2.2. In vitro radiometric EGFR kinase assay
Microliter plates were coated with 50 μL/well of the EGFR substrate ATF-2 (10 μg/mL
in TBS) for 1.5 h at 37οC. After washing three times with bidi stilled water, the remaining open
binding sites were blocked with blocking buffer (BB; 0.05% Tween20 (Bio-Rad), 0.25% BSA,
0.02% NaN3 in TBS) for 30 min at room temperature. Plates were washed again, 50 μL of the
ο
respective test solution was filled into the wells, and the plates were incubated for1 h at 37 C.
Test solutions containing 12 ng/well p38 a MAPK were diluted in kinase buffer (50 mM Tris-
HCl, pH 7.5, 10 mM MgCl2, 10mM-Glycerophosphate, 100 μg/mL BSA, 1 μM Dithiothreitol,
0.1mM Na3VO4, 100 μM rATP) with or without test substance (10-4–10-8 M). Test substances
were dissolved in DMSO to form stock solutions of 10–2 M; all further dilution steps were
carried out in kinase buffer. After subsequent washing, plates were blocked again with BB for 15
min followed by a fourth washing step. Wells were filled with 50 μL of the primary AB;
Phospho-ATF-2 (Thr69/71)-Antibody (1:500 in BB) and incubated for 1 h at 37οC of the
secondary AB; Antirabbit IgG-AP-Antibody (alkaline phosphataseconjugated) (1:4000 in BB).
Then 100 μL of 4-NPP (Nitrophenylphosphate)was pipetted in each well after a final washing
step, and the color development was measured 1.5–2 h later with an enzyme-linked
immunosorbent assay reader linked equipped with SOFT max PRO software at 405 nm [34].
5.2.3. Cell cycle analysis and apoptosis detection
Cell cycle analysis and apoptosis investigation were carried out by flow cytometry [35].
MCF-7 cells were seeded at 8 ˟ 104 and incubated at 37οC, 5% CO2 overnight. After treatment
with thetested compound, for 24 h, cell pellets were collected and centrifuged (300g, 5 min). For
cell cycle analysis, cell pellets were fixed with 70% ethanol on ice for 15 min and collected
again. The collected pellets were incubated with propidium iodide (PI) staining solution (50
mg/mL PI, 0.1 mg/mL RNaseA, 0.05% Triton X-100) at room temperature for 1 hand analyzed
by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Apoptosis detection was
performed by FITC Annexin V/PI commercial kit (Becton Dickenson,Franklin Lakes, NJ, USA)
following the manufacture protocol. The samples were analyzed by fluorescence-activated cell
sorting(FACS) with a Gallios flow cytometer (Beckman Coulter, Brea, CA,USA) within 1 h after
staining. Data were analyzed using Kaluzav 1.2 (Beckman Coulter).
5.2.4. Measurement of the effect of compound 14 on the level of p53, Baxand BCL-2

21
The levels of the tumor suppressor gene p53, anti-apoptotic markerBCL-2 as well as the
apoptotic marker Bax were assessed using BIO RAD iScript TM One-Step RT-PCR kit with
SYBR® Green. The procedure of the used kit was done according to the manufacturer’s
instructions [36].
5.2.5. Caspases-3, -7 assays
Activities of caspases-3, -7 were measured using DRG Caspase-3 (human) ELISA (EIA-
4860) kit (DRG International Inc., USA), Invitrogen. Caspase-7 (Active) (human) ELISA kit,
Catalog # KHO1091 (96 tests)(Invitrogen Corporation, USA) according to the manufacturer
instructions.
5.3. Molecular docking study
The X-ray crystallographic structure of Epidermal Growth Factor Receptor (EGFR) co-
crystallized with the 4-anilinoquinazoline derivative erlotinib (PDB ID: 1M17) was downloaded
from the protein data bank [32,33]. Water molecules and ligands that are not involved in binding
were removed. Then, the protein was prepared for docking study using Protonate 3D protocol in
MOE with default options. The co-crystalized ligand was used to define the binding site for
docking. The co-crystalized ligand was used to define the binding site for docking. Triangle
Matcher placement method and London dG scoring function were used for docking. Docking
setup was first validated by re-docking of the co-crystallized ligand (erlotinib) in the vicinity of
the binding site of the enzyme with energy score (S) = −11.08 kcal/mol and RMSD of 1.321 Å
and with the ability to reproduce all the key interactions accomplished by the co-crystallized
ligand with the key amino acids in the binding site. The validated docking protocol was then
used to study the ligand-target interactions in the active site for the most potent compound 14 to
predict its binding mode to rationalize its promising activity.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University for funding this work through General Research Project under Grant number (G.R.P
258-39) and thanking to Dr. Essam Rashwan, head of confirmatory diagnostic unit, Vacsera-
Egypt, for helping in performing the biological screening.

22
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26
Highlights
 A new set of 2-(((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-
5-(aryl)thiazolidin-4-one derivatives were designed and synthesized.

In vitro cytotoxic evaluation was carried out against human cervix carcinoma (Hela)
cells and the most active derivatives were examined against the normal human cervical
epithelium HCvEpC cell lines

The most active compounds were tested against the EGFR PK protein kinase.
 Cell cycle analysis, apoptosis assay and upregulation of p53 gene expression, Bax/Bcl-2
ratio and caspases-3 and -7 were performed for compound 14 as a representative
example.
 Molecular docking study of compound 14 was performed to investigate its binding mode
of interaction with EGFR PK.

27
O
O
O
N
O
O
N
O
O
O
N
N
N
N
O
N
HN
HN
Cl
HN
Erlotinib
(Tarceva)
N
HN
Cl
F
Gefitinib
(Iressa)
O
F
Afatinib (Gilotrif)
Dual HER2/EGFR
inhibitor
O
O
O
O
N
O
H
O
S
N
N
N
N
HN
O
O
HN
Icotinib
N
N
N
HN
N
Lapatinib (Tykerb)
Dual HER2/EGFR
inhibitor
F
(Conmana)
HN
O
N
O
Cl
(Tagrisso)
Osimertinib
Fig. 1. Drugs of EGFR PK inhibiting activity
g
n
i
d
n
O
o
b
-
H
HN
Ar
S
hydrophobic
interaction
N
N
N
O
N
hydrophobic
interaction
Van der waals
interaction
H
-
b
o
n
d
i
n
g
Fig. 2. Proposed hypothetic model for the new benzofuran-pyrazole-thiazolidinone compounds

28
14/Hela
untreated Hela
Fig. 3. Cell Cycle analysis results of compound 14
14/Hela
Hela
Fig. 4. Apoptosis induction analysis using Annexin V/PI for compound 14

29
14/Hela
14/Hela
Fig. 5. Cell cycle analysis and apoptosis induction analysis of compound 14 on Hela cells