Novel thiazolidinone/thiazolo[3,2-a] benzimidazolone-isatin conjugates as apoptotic anti-proliferative agents towards breast cancer: One-pot synthesis and in vitro biological evaluation

Article abstract of DOI:10.3390/molecules23061420

In connection with our research program on the development of new isatin-based anticancer candidates, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates (4a–n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a–d), and in vitro evaluation of their antiproliferative activity towards two breast cancer cell lines; triple negative MDA-MB-231, and MCF-7. Compounds 4m and 7b emerged as the most active congeners against MDA-MB-231 cells (IC₅₀ = 7.6 ± 0.5 and 13.2 ± 1.1 μM, respectively). Compounds 4m and 7b were able to provoke apoptosis in MDA-MB-231 cells, evidenced by the up-regulation of Bax and down-regulation of Bcl-2, besides boosting caspase-3 levels. Hybrid 4m induced a fourfold increase in the percentage of cells at Sub-G₁, with concurrent arrest in G₂-M phase by 2.5-folds. Furthermore, hybrid 4m resulted in a sixfold increase in the percentage of annexin V-FITC positive apoptotic MDA-MB-231 cells as compared with the control. Moreover, the cytotoxic activities of the active conjugates were assessed towards two nontumorigenic cell lines (breast MCF-10A and lung WI-38) where both conjugates 4m and 7b displayed mean tumor selectivity index: 9.6 and 13.9, respectively. Finally, several ADME descriptors were predicted for the active conjugates via a theoretical kinetic study.

Full text of DOI:10.3390/molecules23061420

molecules
Article
Novel Thiazolidinone/Thiazolo[3,2-a]
Benzimidazolone-Isatin Conjugates as Apoptotic
Anti-Proliferative Agents Towards Breast Cancer:
One-Pot Synthesis and In Vitro Biological Evaluation
Mohamed El-Naggar 1, Wagdy M. Eldehna 2,*, Hadia Almahli 3,4, Amr Elgez 5
,
ID
Mohamed Fares 4,6, Mahmoud M. Elaasser 7 and Hatem A. Abdel-Aziz 8
ID
1
Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, UAE;
m5elnaggar@yahoo.com
2
3
4
5
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road,
OX1 3TA Oxford, UK; hadia.almahli@yahoo.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt; ph.fares@yahoo.com
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo 11829, Egypt; amro_elgez@yahoo.com
School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo 11759, Egypt;
mmelaasser@hotmail.com
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt;
hatem_741@yahoo.com
6
7
8
*
Correspondence: wagdy2000@gmail.com; Tel.: +20-106-883-7640
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 May 2018; Accepted: 8 June 2018; Published: 12 June 2018
Abstract: In connection with our research program on the development of new isatin-based anticancer
candidates, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates
(4a–n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a–d), and in vitro evaluation of
their antiproliferative activity towards two breast cancer cell lines; triple negative MDA-MB-231,
and MCF-7. Compounds 4m and 7b emerged as the most active congeners against MDA-MB-231
cells (IC50 = 7.6
±
0.5 and 13.2
±
1.1 µM, respectively). Compounds 4m and 7b were able to provoke
apoptosis in MDA-MB-231 cells, evidenced by the up-regulation of Bax and down-regulation of
Bcl-2, besides boosting caspase-3 levels. Hybrid 4m induced a fourfold increase in the percentage of
cells at Sub-G1, with concurrent arrest in G2-M phase by 2.5-folds. Furthermore, hybrid 4m resulted
in a sixfold increase in the percentage of annexin V-FITC positive apoptotic MDA-MB-231 cells as
compared with the control. Moreover, the cytotoxic activities of the active conjugates were assessed
towards two nontumorigenic cell lines (breast MCF-10A and lung WI-38) where both conjugates 4m
and 7b displayed mean tumor selectivity index: 9.6 and 13.9, respectively. Finally, several ADME
descriptors were predicted for the active conjugates via a theoretical kinetic study.
Keywords: triple-negative breast cancer; isatin-thiazolidinone hybrids; anticancer; apoptosis; QSAR
1. Introduction
Breast cancer has become the most frequently diagnosed malignancy among women, and the
second leading cause of cancer-related deaths in women [
1
–3]. The deficiency in the maintenance of
Molecules 2018, 23, 1420; doi:10.3390/molecules23061420
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1420
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genomic integrity, excessive exposure to estrogens and advanced age are considered as the leading
reasons for a high breast cancer risk [ ]. Breast cancer is considered as a diverse group of diseases
4,5
with several intrinsic tumor subtypes that have diverse treatment modalities and long-term survival
probabilities. The immunohistochemical expression of the estrogen receptor (ER), progesterone
receptor (PR), and human epidermal growth factor receptor-2 (HER-2) forms the platform of
characterization of clinically defined breast cancer subtypes [4]. Particularly, triple-negative breast
cancers (TNBCs) lacking the expression of the (ER), (PR) and (HER-2) represent the major cause of
breast cancer mortality due to their metastatic potential, invasiveness and the lack of suitable molecular
treatment targets [6–8].
Despite an early diagnosis and the diverse therapeutic regimens available for breast cancer
treatment, the development of chemo-resistance, the disease relapse, and the mortality rate are still
constantly on the rise [4]. This finding highlights the need for new drug leads as more effective and
affordable approaches for breast cancer therapies.
Pertaining to its wide presence endogenously in human and other mammalian tissues, isatin 1,
Figure 1, has emerged as a promising privileged scaffold that is endowed with diverse biological
activity [ 13], primarily anticancer activity [14]. During the last two decades, there is a growing
9–
interest regarding development of several isatin derivatives as promising drug candidates for the
treatment of different human malignancies. These research efforts have led to FDA approval of two
drugs and the discovery of many isatin derivatives with diverse cellular and enzymatic targets.
Figure 1. Structures of isatin-based approved anticancer drugs (II and III), some reported isatin-based
hybrids with potent antiproliferative activity against breast cancer (VII
(4a–n and 7a–d).
–IX), and the target conjugates
Sunitinib (Sutent®) II (Figure 1), an isatin-based multitarget tyrosine kinase inhibitor, was granted
its FDA approval in 2006 for the treatment of imatinib-resistant gastrointestinal stromal tumors
(GIST) and advanced metastatic renal cell carcinoma (RCC) [15]. By 2014, Nintedanib (III, Ofev®,
Figure 1), an orally available isatin-based triple-angiokinase inhibitor, was approved by the FDA for
the management of idiopathic pulmonary fibrosis [16]. One year later, the European Medicines Agency
approved Nintedanib, under a trade name Vargatef®, as a second-line treatment in combination

Molecules 2018, 23, 1420
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with docetaxel for patients with advanced non-small cell lung cancer of adenocarcinoma who have
progressed to first-line chemotherapy [17].
Over the last few years, isatin-based hybrids have attracted considerable attention as promising
anticancer agents [18
approach for the development of diverse isatin-thiazolidine/thiazolidinone [25
IV, Figure 1) and isatin-benzimidazole (such as compound , Figure 1) [30] hybrids as potent
–
24]. In this context, numerous research groups adopted a hybridization
–
29] (such as compound
V
antiproliferative agents towards different breast cancer cell lines. Recently, our research group
has focused on the design and synthesis of novel and potent isatin-based hybrids as promising
anticancer agents [31–40]. In 2017, we developed two novel series of thiazolidinone-isatin hybrids as
effective anticancer agents targeting tumor-associated carbonic anhydrase isoform IX. Among these
hybrids, compound VI (Figure 1) displayed potent activity against MCF-7 breast cancer cell line
(
IC50 = 3.96 ± 0.21 µM) with induction of the intrinsic apoptotic mitochondrial pathway in MCF-7
cells [41].
Taking the above into account, herein we adopted the hybrid pharmacophore approach to design
and synthesize two novel series of thiazolidinone-isatin (4a ) and thiazolo[3,2-a]benzimidazolone-
isatin conjugates (7a ) (Figure 1), with the goal of developing potent antiproliferative agents toward
–n
–
d
TNBC MDA-MB-231, and MCF-7 breast cancer cell lines. Furthermore, the most potent antiproliferative
congeners were evaluated for their apoptosis induction potential in MDA-MB-231 cells, to gain insight
into the mechanism of the anticancer activity for the prepared hybrids. Finally, the cytotoxic activity
of the active conjugates was tested towards nontumorigenic human normal lung fibroblast cell line
(WI-38) to investigate the safety of the newly prepared conjugates.
2. Results
2.1. Chemistry
The target conjugates 4a
in Schemes 1 and 2. In Scheme 1, preparation of conjugates 4a
synthetic routes. The first route comprised a one-pot three-component synthesis, involving a
Knoevenagel condensation, followed by cyclization among isatins 1a , utilising an equimolar amount
of N,N0-diphenyl/p-tolyl thiourea 2a
and bromoacetic acid in refluxing glacial acetic acid in the
presence of sodium acetate to furnish conjugates 4a . This route proved successful to prepare
conjugates 4a
in high yields; 70–86% (Scheme 1). Alternatively, N,N0-diphenyl/p-tolyl thiourea
2a reacted with bromoacetic acid to furnish compounds 5a , which subsequently condensed
with isatins 1a in acetic acid in the presence of sodium acetate to afford the target conjugates 4a
with overall 55–68% yield over the two steps (Scheme 1).
–
n
and 7a
–d
were synthesized adopting the chemical pathways outlined
–
n
was accomplished via two
–
g
,b
3
–
n
–
n
,b
3
,b
–g
–n,
Scheme 1. Synthesis of target hybrids 4a–n; reagents and conditions: (i) glacial acetic acid/sodium
acetate/reflux 3–4 h.

Molecules 2018, 23, 1420
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Scheme 2. Synthesis of target hybrids 7a
acetate/reflux 2 h; (ii) ethanol/ KOH/reflux 1 h; (iii) acetic anhydride/pyridine 100 C 1 h.
–d; Reagents and conditions: (i) glacial acetic acid/sodium
◦
Similar to Scheme 1, target conjugates 7a
reaction of isatins 1c with equimolar amounts of 2-mercaptobenzimidazole
acid in refluxing glacial acetic acid in the presence of sodium acetate to produce conjugates
7a , in good yields; 80–88% (Scheme 2). The latter conjugates prepared through cyclization of
2-((benzimidazol-2-yl)thio)acetic acid into compound , with subsequent condensation with isatins
1c, e–g, with low overall yield; 45–61%.
–
d
were prepared in a one-pot three-component
,
e–
g
6
and bromoacetic
3
–
d
8
9
Postulated structures of the newly synthesized conjugates 4a–n and 7a–d were in full agreement
with their spectral and elemental analyses data.
2.2. Biological Evaluation
2.2.1. In Vitro Antiproliferative Activity
The in vitro antiproliferative activity of the newly synthesized conjugates 4a–n and 7a–d was
evaluated against breast cancer MDA-MB-231 and MCF-7 cell lines, following the Crystal Violet (CV)
cell cytotoxicity assay [41]. Doxorubicin was included in this assay as a positive control, and Sunitinib
was used as a reference drug. The results were expressed as median growth inhibitory concentration
(IC50) values that represent the compounds concentrations required to afford a 50% inhibition of cell
growth after 48 h of incubation, compared to untreated controls (Table 1).
Breast cancer is composed of multiple subtypes with distinct morphologies and clinical
implications. In this study, the sensitivity of two different breast cancer subtypes towards the
target conjugates was tested to examine if the target compounds possess selective growth inhibitory
activity toward certain subtype. MCF-7 is a Luminal A breast cancer cells which is hormone-receptor
positive (estrogen-receptor and/or progesterone-receptor positive), HER2 negative, and has low
levels of the protein Ki-67, which helps control how fast cancer cells grow. Luminal A cancers
are low-grade, tend to grow slowly and have the best prognosis. Whereas, MDA-MB-231 is a
triple-negative/basal-like breast cancer cell line, which is hormone-receptor negative (estrogen-receptor
and progesterone-receptor negative) and HER2 negative. This type of cancer is more common in
women with BRCA1 gene mutations.

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Table 1. In vitro antiproliferative activity of hybrids 4a–n and 7a–d against MDA-MB-231 and MCF-7
breast cancer cell lines.
IC50 (µM) a
Compound
R1
R
MDA-MB-231
MCF-7
NA b
NA b
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
7a
7b
7c
7d
H
5-F
5-Cl
H
H
H
H
H
108.3 ± 8.2
24.1 ± 2.1
15.7 ± 0.9
42.6 ± 3.4
60.9 ± 4.2
43.6 ± 3.1
51.7 ± 2.1
152.1 ± 11.5
50.8 ± 3.5
NA b
47.1 ± 4.1
7.6 ± 0.5
36.3 ± 2.9
47.2 ± 3.8
13.2 ± 1.7
40.3 ± 2.5
23.5 ± 1.0
4.7 ± 0.4
5.5 ± 0.5
58.5 ± 3.2
29.2 ± 2.5
17.1 ± 1.1
52.9 ± 5.0
22.8 ± 2.0
47.4 ± 3.9
56.0 ± 3.7
104.9 ± 7.5
NA b
5-Br
5-OCH3
5-CH3
5,7-(CH3)2
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
-
5-F
5-Cl
5-Br
NA b
5-OCH3
5-CH3
5,7-(CH3)2
5-Cl
5-OCH3
5-CH3
5,7-(CH3)2
53.5 ± 3.6
8.4 ± 0.5
23.7 ± 1.9
127.3 ± 9.9
21.7 ± 1.5
48.2 ± 2.7
27.6 ± 2.4
3.8 ± 0.4
3.4 ± 0.3
-
-
-
Dox.
Sunitinib
a
IC50 values are the mean
±
S.D. of three separate experiments; b NA: Compounds
having IC50 value > 200 µM.
From the obtained results, it was obvious that many of the target conjugates 4a–n and 7a–d have
excellent to modest growth inhibitory activity against the tested breast cancer cell lines; MDA-MB-231
and MCF-7. Also, it was noted that MDA-MB-231 cells is more sensitive to the influence of the target
conjugates than MCF-7 cells, especially for the thiazolobenzimidazole conjugates 7a–d.
Concerning activity against MDA-MB-231 cells, conjugate 4m was the most active one and
showed potent antiproliferative activity with IC50 7.6
doxorubicin (IC50 = 4.7 0.4 M). Furthermore, compounds 4c
against MDA-MB-231 cells with IC50 values of 24.1 2.1, 15.7
7a and 7c possessed moderate activity with an IC50
±
0.5
µ
M, in comparison to the standard drug
4d 7b and 7d displayed good activity
0.9, 13.2 1.1 and 23.5 1.0 M,
±
µ
,
,
±
±
±
±
µ
respectively. Moreover, conjugates 4e
range 36.3 ± 2.9–47.1 ± 4.1 µM.
, 4g, 4l, 4n,
On the other hand, examination of the antiproliferative activity in MCF-7 cells elucidated that
conjugate 4m had the best growth inhibitory activity (IC50 = 8.4
than doxorubicin (IC50 = 3.80 0.4 M). Furthermore, hybrid 4g showed good antiproliferative
activity against MCF-7 cancer cell line (IC50 = 17.1 1.1). Also, hybrids 4c 4f 4n 7b and 7d were
2.0, 23.7 1.9, 21.7 1.5
±
0.5), with 2.2-fold decreased activity
±
µ
±
,
,
,
moderately active towards MCF-7 cells with IC50 values of 29.2
±
2.5, 22.8
±
±
±
and 27.6 ± 2.4, respectively.

Molecules 2018, 23, 1420
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It is worth highlighting that both target thiazolidinone-isatin conjugates (4a
–n) and thiazolo[3,2-a]
benzimidazolone-isatin conjugates (7a–d), didn’t display significant selectivity in their growth
inhibitory activities towards the tested cell line; TNBC MDA-MB-231 or MCF-7.
2.2.2. In Vitro Cytotoxic Activity towards Nontumorigenic Human WI-38 and MCF-10A Cells
The cytotoxic activity of the active conjugates towards MDA-MB-231 cells (4c
–e, 4g, 4l–n and
7a–d) was examined against nontumorigenic human lung fibroblast cell line (WI-38) and human breast
epithelial cell line (MCF-10A) to investigate the potential safety of the newly prepared conjugates
towards the normal cells. Cultures derived from human fibrocystic mammary tissue (MCF-10A)
are nontumorigenic and possess the features of primary cultures of breast tissue including dome
formation [42]. The results were expressed as IC50 values, and selectivity index was calculated
(Table 2). Most of the tested hybrids (4c
impact towards WI-38 and MCF-10A cells with IC50 range 49.1
9.8 M, respectively. Compounds 4m and 7a displayed good selectivity index range 6.0–13.9,
–
e,
4g
,
4l
–
n
and 7a
–
d) exhibited non-significant cytotoxic
±
1.9–353.0 17.1 and 73.1 2.7–250.3
±
±
±
µ
–d
thereby providing a good safety profile as anticancer agents.
Table 2. In vitro cytotoxic activity against nontumorigenic cell lines (WI-38 and MCF-10A), and
selectivity index (MCF-10A/ MDA-MB-231) for the active compounds.
IC50 (µM) a
Selectivity Index
Compound
Lung WI-38
Breast MCF-10A
MDA-MB-231
4c
4d
4e
4g
4l
4m
4n
7a
7b
7c
87.2 ± 5.2
65.5 ± 4.2
112.6 ± 5.9
80.7 ± 3.4
89.6 ± 5.1
141.7 ± 7.9
157.4 ± 6.8
73.1 ± 2.7
154.0 ± 5.2
233.8 ± 9.6
183.9 ± 8.0
242.2 ± 11.5
250.3 ± 9.8
24.1 ± 2.1
15.7 ± 0.9
42.6 ± 3.4
43.6 ± 3.1
47.2 ± 4.1
7.6 ± 0.5
4.7
5.1
2.1
3.25
3.3
9.6
4.2
5.0
13.9
6.0
10.7
193.1 ± 10.6
120.0 ± 7.8
176.2 ± 11.3
49.1 ± 1.9
218.3 ± 13.5
353.0 ± 17.1
121.1 ± 6.9
192.0 ± 10.8
234.0 ± 15.4
36.3 ± 2.9
47.2 ± 3.8
13.2 ± 1.1
40.3 ± 2.5
23.5 ± 1.00
7d
a
IC50 values are the mean ± S.D. of three separate experiments.
2.2.3. Apoptosis Induction in TNBC MDA-MB-231 Cells
In the current medical era, induction of apoptosis in cancer cells has emerged as one of the most
successful strategies for the development of cancer therapies [43 45]. As mentioned before, 4m and
–
7b emerged as the most active hybrids towards MDA-MB-231 cells. Consequently, we examined the
ability of conjugates 4m and 7b to provoke apoptosis in MDA-MB-231 cells to determine the principle
mechanism for their antiproliferative activity.
Effects on Mitochondrial Apoptosis Pathway Proteins Bcl-2 and Bax
Several members of the human Bcl-2 family of apoptosis-regulating proteins have been discovered,
including antiapoptotic proteins such as Bcl-2 protein, and structurally similar pro-apoptotic proteins
such as Bax protein, the first anti-death gene identified [44–46]. Bcl-2-family proteins regulate all major
types of cell death, including apoptosis, necrosis and autophagy, therefore operating as nodal points at
the convergence of diverse pathways with broad relevance to oncology.
In our study, we investigated the effect of conjugates 4m and 7b towards the level of the
antiapoptotic Bcl2 and the level of the pro-apoptotic Bax, (Table 3, Figure 2). As displayed in Table 2,
hybrid 4m induced the protein expression of Bax with 11.1 folds of the control while 7.3 folds were
evaluated for hybrid 7b. On the other hand, treatment of MDA-MB-231 cancer cells with conjugates

Molecules 2018, 23, 1420
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4m and 7b significantly downregulated the expression levels of the antiapoptotic protein Bcl-2 by 66.6
and 55.1%, respectively, compared to the control.
Table 3. Impact of hybrids 4m and 7b on the expression levels of Bcl-2 and Bax and on the active
caspase-3 level in MDA-MB-231 cancer cells treated with each hybrid at its IC50 concentration.
Bax (pg/mg of
Total Protein)
Bcl-2 (ng/mg of
Total Protein)
Caspase-3 (ng/mg
of Total Protein)
Compound
Bax/Bcl-2
4m
7b
Control
274.8
±
13.5 (11.1) *
2.1 ± 0.1 (0.33) *
2.8 ± 0.1 (0.45) *
6.3 ± 0.2
130.5
64.1
3.9
0.19 ± 0.01 (6.4) *
0.15 ± 0.01 (4.9) *
0.03 ± 0.01
180.0 ± 7.3 (7.3) *
24.7 ± 1.7
* Numbers given between parentheses are the numbers of folds of control.
Figure 2. Effect of compounds 4m and 7b on the protein levels of (
ratio in MDA-MB-231 cells treated with the compounds at their IC50 concentrations against control
(1% DMSO). Data are mean S.D. (n = 3). The experiment was done in triplicates. * Significantly
different from control at p < 0.05.
A) Bax; (B) Bcl-2; (C) Bax/Bcl2
±
The ratio between Bax (apoptosis inducer) and Bcl-2 (apoptosis suppressor) is an important
parameter and gave more profound insight into the apoptotic activity of the compounds as
a determining factor for cell fate regulation, and a key indicator of therapeutic response to
chemotherapy [47]. Calculating the values for Bax/Bcl2 ratio indicated that conjugates 4m and
7b boosted the Bax/Bcl-2 ratio by 33- and 16-folds, respectively, compared to the control. Conclusively,
the ability of conjugates 4m and 7b to up-regulate Bax level, down-regulate Bcl2 level while significantly
boosting the Bax/Bcl2 ratio proves their effectiveness as apoptosis inducers.
Effects on the Levels of Active Caspase-3 (Key Executor of Apoptosis)
The down-regulation of the antiapoptotic Bcl-2 leads to an increase in the levels of free
pro-apoptotic Bax which in turn accumulates at the inner mitochondrial membrane forming channels,
altering membrane permeability. Accordingly, the apoptotic factors can leak into the cytoplasm
resulting in the caspases cascade activation. As a key executioner protease, caspase-3 is activated by
upstream initiator caspases as caspase-9 [48]. Thence, the elevated Bax/Bcl-2 ratios obtained herein
triggered the examination of the protein expression levels of active caspases-3. Treatment of TNBC

Molecules 2018, 23, 1420
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MDA-MB-231 cells with conjugates 4m and 7b led to a significant increase in the active caspase-3
levels by 6.4 and 4.9 folds, respectively, compared to control (Table 3, Figure 3).
Figure 3. Effect of compounds 4m and 7b on the protein levels of caspase-3 in MDA-MB-231 cells
treated with the compounds at their IC50 concentrations against control (1% DMSO). Data are mean
±
S.D. (n = 3). The experiments were done in triplicate. * Significantly different from control at p < 0.05.
Cell Cycle Analysis
The effect of hybrid 4m on cell cycle progression was evaluated in TNBC MDA-MB-231 cells
after 24 h of treatment (Figure 4). Such impact was illustrated by DNA flow cytometric assay where
MDA-MB-231 cells were treated with conjugate 4m at its IC50 concentration.
Control
4m
Figure 4. Effect of compound 4m on the phases of cell cycle of MDA-MB-231 cells. * Significantly
different from control at p < 0.05. (Two-way ANOVA test).

Molecules 2018, 23, 1420
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As displayed in Figure 4, exposure of MDA-MB-231 cells to hybrid 4m resulted in a significant
rise in the percentage of cells at Sub-G1 by 4-folds, with concurrent significant arrest in the G2-M
phase by 2.5-folds compared to control. Arrest of G2-M phase and alteration of the Sub-G1 phase were
significant remarks for hybrid 4m to prompt apoptosis in TNBC MDA-MB-231 cells.
Annexin V-FITC Apoptosis Assay
Annexin V-based flow cytometry assay elucidates either that cell death is achieved via
programmed apoptosis or nonspecific necrosis [49]. The apoptotic impact of conjugate 4m was
further proved by Annexin V-FITC/PI (AV/PI) dual staining analysis to examine the occurrence
of phosphatidylserine externalization (Figure 5). Flow cytometric analysis revealed that treatment
of TNBC MDA-MB-231 cells with conjugate 4m resulted in a significant elevation in the percent of
annexin V-FITC-positive apoptotic cells, including both the early and late apoptotic phases (UR + LR),
from 1.92% to 11.43% which represents about 6-folds increase as compared with the control.
Figure 5. Effect of compound 4m on the percentage of annexin V-FITC-positive staining in MDA-MB-231
cells. The experiments were done in triplicates. The four quadrants identified as: LL, viable; LR
,
early apoptotic; UR, late apoptotic; UL, necrotic. * Significantly different from control at p < 0.05. (One-way
ANOVA test).
In conclusion, enhanced expression of the pro-apoptotic protein Bax in addition to the reduced
expression of the antiapoptotic protein Bcl-2, as well as the up-regulated active caspase-3 levels
together with a harmonized increase in the Bax/Bcl-2 ratio, suggests that the antiproliferative effect of
the target conjugates might be attributed, at least in part, to the induction of the intrinsic apoptotic
mitochondrial pathway.
Collectively, these data have highlighted conjugate 4m as an ideal lead compound for further
optimization and development as an effective anti-breast cancer therapy.

Molecules 2018, 23, 1420
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2.3. In Silico ADME Profiling
In order to examine drug-like physicochemical and pharmacokinetics properties of the target
conjugates, certain ADME descriptors for the prepared conjugates (4a and 7a ) were assessed
–
n
–d
through a computer-aided theoretical kinetic study using Discovery Studio software (Accelrys,
San Diego, CA, USA), [50,51] Table 4.
Table 4. Computer-aided ADME study for the synthesized conjugates 4a–m and 7a–d.
Compound AlogP98 a
Solubility c
Absorption Level e
PSA_2D b
Solubility Level d
CYP2D6 f
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
7a
7b
7c
4.476
4.682
5.141
5.225
4.46
4.962
5.449
5.449
5.654
6.113
6.197
5.432
5.935
6.421
3.581
2.9
62.087
62.087
62.087
62.087
71.017
62.087
62.087
62.087
62.087
62.087
62.087
71.017
62.087
62.087
64.021
72.951
64.021
64.021
−6.305
−6.605
−7.005
−7.079
−6.292
−6.765
−7.226
−7.197
−7.484
−7.885
−7.958
−7.159
−7.645
−8.093
−6.005
−5.327
−5.771
−6.263
c
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
2
2
1
0
0
0
0
0
0
0
0
1
1
1
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.403
3.889
7d
a
b
Lipophilicity descriptor. Polar surface area. Solubility parameter.(0:
−
2 = optimal,
−
2:
−
4 = good, −4:−6 = low
,
d
−
6:
−
8 = very low). Solubility level. (0 = extremely low, 1 = very low but possible, 2 = low, 3 = good, 4 = optimal).
e
f
Absorption level. (0 = good, 1 = moderate, 2 = low, 3 = very low). CYP2D6 inhibition. (0 = noninhibitor,
1 = inhibitor).
The tested conjugates exhibited low aqueous solubility levels, except compounds 7b and 7c
,
which showed better solubility levels. The tested conjugates displayed good levels of human intestinal
absorption, except compounds 4i 4m and 4n, which showed moderate intestinal absorption levels.
–k,
On the other hand, the investigated conjugates were predicted to be CYP2D6 non-inhibitors, Table 4.
3. Conclusions
In summary, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates
(
4a
evaluated for their antiproliferative activity towards breast cancer cell lines MDA-MB-231, and MCF-7.
Conjugates 4m and 7b were the most active members towards MDA-MB-231 (IC50 = 7.6 0.51 and
13.2 1.07 M, respectively). Conjugates 4d and 4m emerged as the most active derivatives against
MCF-7 cells with IC50 values of 17.1 1.14 and 8.4 0.47 M, respectively. Conjugates 4m and 7b
–n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a–d). All the prepared conjugates were
±
±
µ
±
±
µ
induced apoptosis in MDA-MB-231 cells, confirmed by the up-regulation of the Bax, downregulation
of the Bcl-2 and boosting caspase-3 levels. Compound 4m arrested the G2-M phase by 2.5-folds with
concurrent significant increase in the percentage of cells at Sub-G1 by 4-folds, compared to control.
Also, 4m showed significant increase in the percentage of annexin V-FITC positive apoptotic cells from
1.92% to 11.43%, approximately a 6-folds increase as compared with the control. Finally, the cytotoxic
activity of the active conjugates (4c
lung fibroblast WI-38 cell line and breast MCF-10A cell line, and compounds 4m and 7a
good selectivity index range 6.0–13.9, thereby providing a good safety profile as anticancer agents.
–e
,
4g
,
4l
–
n
and 7a
–d) were tested towards nontumorigenic human
–
d
displayed

Molecules 2018, 23, 1420
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4. Experimental
4.1. Chemistry
4.1.1. General
Using a Stuart melting point apparatus, melting points were measured and uncorrected.
Using Schimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan), Infrared (IR) Spectra were
recorded as KBr disks. NMR Spectra were recorded on a Bruker spectrophotometer (Bruker, Karlsruhe,
1
Germany). H spectrum was run at 400 MHz and 13C spectrum was run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d6). All coupling constant (J) values are given in hertz. The abbreviations
used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were done at the Regional
Center for Microbiology and Biotechnology, Al-Azhar University, Egypt. Mass spectral data were
given by a GCMS-QP1000 EX spectrometer (Shimadzu, Kyoto, Japan) at 70 e.V. High-resolution mass
spectra were obtained using a Bruker MicroTOF spectrometer (Bruker Daltonics, Bremen, Germany).
Compounds 5a,b [52], 8 [53], and 9 [54] were previously prepared.
4.1.2. General Procedure for the Preparation of Target Compounds 4a–n
Route A:
0
A mixture of isatins 1a
–
g
(1 mmoL) with an equimolar amount of N,N -diphenyl/(p-tolyl)thiourea
2a
,b
(1 mmoL) and bromoacetic acid
3
(0.14 g, 1 mmoL) in glacial acetic acid (10 mL) in the presence
of sodium acetate (0.16 gm, 2 mmoL), was heated under reflux for 3 h. The formed solid was filtered
off while hot, washed with hot ethanol, dried and recrystallized from DMF to furnish the target
hybrids 4a–n.
Route B:
To a hot solution of N,N0-diphenyl/(p-tolyl)thiourea 2a
2 mmoL) in glacial acetic acid (10 mL), bromoacetic acid (0.14 gm, 1 mmoL) was added. The reaction
mixture was refluxed for 4 h then allowed to cool to room temperature. The obtained solid was filtered
off, washed with water and dried to give intermediates 3a . The later intermediates were added,
without further purification, to a stirred solution of the appropriate isatin derivative 1a (1 mmoL)
,b (1 mmoL) and sodium acetate (0.16 gm,
3
,b
–g
and sodium acetate (0.16 gm, 2 mmoL) in glacial acetic acid (5 mL) then the reaction mixture was
refluxed for 3 h. The formed solid was filtered off, washed with ethanol and recrystallized from DMF
to give compounds 4a–n.
5-(2-Oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4a)
Red powder (yield 83%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3251 (NH) and 1687 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 6.95 (d, J = 7.7 Hz, 1H, Ar-H), 6.99 (d, J = 7.3 Hz, 2H, Ar-H), 7.04 (t,
δ
J = 7.3 Hz, 1H, Ar-H), 7.17 (t, J = 7.4 Hz, 1H, Ar-H), 7.35–7.43 (m, 3H, Ar-H), 7.49–7.54 (m, 1H, Ar-H),
7.58 (d, J = 7.7 Hz, 4H, Ar-H), 8.77 (d, J = 7.7 Hz, 1H, Ar-H), 11.18 (s, 1H, NH isatin, D2O exchangeable);
MS, m/z [%]: 397.0 [M+, 100]; Anal. Calcd. for C23H15N3O2S: C, 69.51; H, 3.80; N, 10.57; found C, 69.34;
H, 3.91; N, 10.83; HRMS m/z 398.09583 M+ + 1, calcd for C23H15N3O2S: 398.09577.
5-(5-Fluoro-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4b)
◦
Orange powder (yield 75%), m.p. > 300 C; IR (KBr,
(DMSO-d6, 400 MHz) ppm: 6.81–6.87 (m, 3H, Ar-H), 7.22 (d, J = 7.8 Hz, 2H, Ar-H), 7.42–7.60 (m,
ν
cm−1): 3343 (NH) and 1683 (C=O); 1H-NMR
δ
7H, Ar-H), 8.57 (d, J = 7.2 Hz, 1H, Ar-H), 11.25 (s, 1H, NH isatin, D2O exchangeable); Anal. Calcd.
for C23H14FN3O2S: C, 66.50; H, 3.40; N, 10.11; found C, 66.78; H, 3.26; N, 10.40.

Molecules 2018, 23, 1420
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5-(5-Chloro-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4c)
Light brown powder (yield 80%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3257 (NH) and 1687 (C=O);
1H-NMR (DMSO-d6, 400 MHz)
δ
ppm: 6.89 (d, J = 8.4 Hz, 1H, Ar-H), 6.97 (d, J = 7.6 Hz, 2H, Ar-H),
7.24–7.33 (m, 3H, Ar-H), 7.51–7.63 (m, 6H, Ar-H), 8.71 (d, J = 2.0 Hz, 1H, Ar-H), 11.29 (s, 1H, NH isatin,
D2O exchangeable); Anal. Calcd. for C23H14ClN3O2S: C, 63.96; H, 3.27; N, 9.73; found C, 64.23; H, 3.41;
N, 10.01.
5-(5-Bromo-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4d)
Red powder (yield 85%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3308 (NH) and 1689 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 6.93 (d, J = 8.3 Hz, 1H, Ar-H), 6.99 (d, J = 7.3 Hz, 2H, Ar-H), 7.20 (t, J = 7.4
δ
Hz, 1H, Ar-H), 7.42 (t, J = 7.8 Hz, 2H, Ar-H), 7.65–7.49 (m, 6H, Ar-H), 8.96 (d, J = 2.0 Hz, 1H, Ar-H),
11.37 (s, 1H, NH isatin, D2O exchangeable); Anal. Calcd. for C23H14BrN3O2S: C, 57.99; H, 2.96; N, 8.82;
found C, C, 58.17; H, 3.08; N, 9.04; HRMS m/z 476.00635 M+ + 1, calcd for C23H14BrN3O2S: 476.00629.
5-(5-Methoxy-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4e)
Red powder (yield 70%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3340 (NH) and 1692 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 3.72 (s, 3H, -OCH3), 6.86 (d, J = 8.5 Hz, 1H, Ar-H), 7.05–6.94 (m, 3H,
δ
Ar-H), 7.19 (t, J = 7.4 Hz, 1H, Ar-H), 7.41 (t, J = 7.8 Hz, 2H, Ar-H), 7.57–7.46 (m, 1H, Ar-H), 7.59
(d, J = 4.3 Hz, 4H, Ar-H), 8.48 (d, J = 2.6 Hz, 1H, Ar-H), 11.02 (s, 1H, NH isatin, D2O exchangeable);
13C-NMR (DMSO-d6, 100 MHz)
δ ppm: 56.10, 111.14, 113.90, 118.24, 121.09, 121.31 (2C), 125.03, 125.50,
129.18 (2C), 129.56 (2C), 129.85 (2C), 132.14, 135.05, 137.54, 137.57, 147.76, 153.77, 154.83, 166.10, 169.05;
MS, m/z [%]: 427 [M+, 100]; Anal. Calcd. for C24H17N3O3S: C, 67.43; H, 4.01; N, 9.83; found C, 67.70; H,
4.19; N, 10.02; HRMS m/z 428.10632 M+ + 1, calcd for C24H17N3O3S: 428.10634.
5-(5-Methyl-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4f)
Red powder (yield 75%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3285 (NH) and 1684 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.28 (s, 3H, -CH3), 6.83 (d, J = 8.0 Hz, 1H, Ar-H), 6.98 (d, J = 7.2 Hz,
δ
2H, Ar-H), 7.18 (d, J = 7.2 Hz, 2H, Ar-H), 7.39 (t, J = 7.6 Hz, 2H, Ar-H), 7.52–7.59 (m, 5H, Ar-H),
8.64 (s, 1H, Ar-H), 11.10 (s, 1H, NH isatin, D2O exchangeable); MS, m/z [%]: 411 [M+, 100]; Anal. Calcd.
for C24H17N3O2S: C, 70.06; H, 4.16; N, 10.21; found C, 69.84; H, 4.38; N, 10.38.
5-(5,7-Dimethyl-2-oxoindolin-3-ylidene)-3-phenyl-2-(phenylimino)thiazolidin-4-one (4g)
◦
Brown powder (yield 73%), m.p. > 300 C; IR (KBr,
(DMSO-d6, 400 MHz) ppm: 2.17 (s, 3H, -CH3), 2.23 (s, 3H, -CH3), 6.84 (s, 1H, Ar-H), 6.97 (d, J = 7.6 Hz
ν
cm−1): 3328 (NH) and 1682 (C=O); 1H-NMR
δ
,
2H, Ar-H), 7.19 (d, J = 7.6 Hz, 2H, Ar-H), 7.41 (t, J = 7.6 Hz, 2H, Ar-H), 7.49–7.58 (m, 4H, Ar-H),
8.43 (s, 1H, Ar-H), 11.07 (s, 1H, NH isatin, D2O exchangeable); MS, m/z [%]: 425 [M+, 100]; Anal. Calcd.
for C25H19N3O2S: C, 70.57; H, 4.50; N, 9.88; found C, 70.79; H, 4.61; N, 10.15.
5-(2-Oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4h)
Red powder (yield 70%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3271 (NH) and 1687 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.33 (s, 3H, -CH3), 2.41 (s, 3H, -CH3), 6.93 (d, J = 7.6 Hz, 1H, Ar-H), 6.98 (d,
δ
J = 7.2 Hz, 2H, Ar-H), 7.06 (t, J = 7.3 Hz, 1H, Ar-H), 7.21 (d, J = 8.0 Hz, 2H, Ar-H), 7.32–76 (m, 3H, Ar-H),
7.42 (d, J = 8.0 Hz, 2H, Ar-H), 8.75 (d, J = 7.6 Hz, 1H, Ar-H), 11.27 (s, 1H, NH isatin, D2O exchangeable);
Anal. Calcd. for C25H19N3O2S: C, 70.57; H, 4.50; N, 9.88; found C, 70.64; H, 4.64; N, 10.06.
5-(5-Fluoro-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4i)
◦
Orange powder (yield 75%), m.p. > 300 C; IR (KBr,
(DMSO-d6, 400 MHz) ppm: 2.32 (s, 3H, -CH3), 2.41 (s, 3H, -CH3), 6.87 (d, J = 8.2 Hz, 2H, Ar-H),
ν
cm−1): 3290 (NH) and 1686 (C=O); 1H-NMR
δ
6.92–6.95 (m, 1H, Ar-H), 7.26–7.15 (m, 3H, Ar-H), 7.38 (d, J = 8.3 Hz, 2H, Ar-H), 7.44 (d, J = 8.3 Hz, 2H,
Ar-H), 8.57 (dd, J = 10.4, 2.7 Hz, 1H, Ar-H), 11.23 (s, 1H, NH isatin, D2O exchangeable); 13C-NMR

Molecules 2018, 23, 1420
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(DMSO-d6, 100 MHz)
δ
ppm: 31.16 (2C), 118.67, 121.16 (2C), 121.30, 124.30, 128.77 (2C), 130.00 (2C),
130.33 (2C), 131.84, 132.32, 134.11, 134.28, 138.76, 140.06, 145.54, 153.37, 155.40, 156.73, 165.92, 168.63;
MS, m/z [%]: 443 [M+, 100]; Anal. Calcd. for C25H18FN3O2S: C, 67.71; H, 4.09; N, 9.47; found C, 68.03;
H, 4.35; N, 9.29; HRMS m/z 444.11771 M+ + 1, calcd for C25H18FN3O2S: 444.11765.
5-(5-Chloro-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4j)
◦
Orange powder (yield 73%), m.p. > 300 C; IR (KBr,
(DMSO-d6, 400 MHz) ppm: 2.32 (s, 3H, -CH3), 2.41 (s, 3H, -CH3), 6.87 (d, J = 8.2 Hz, 2H, Ar-H), 6.96 (d,
ν
cm−1): 3223 (NH) and 1691 (C=O); 1H-NMR
δ
J = 8.4 Hz, 1H, Ar-H), 7.21 (d, J = 8.1 Hz, 2H, Ar-H), 7.50–7.35 (m, 5H, Ar-H), 8.82 (d, J = 2.2 Hz, 1H,
Ar-H), 11.34 (s, 1H, NH isatin, D2O exchangeable); Anal. Calcd. for C25H18ClN3O2S: C, 65.28; H, 3.94;
N, 9.14; found C, 65.41; H, 4.12; N, 9.32; HRMS m/z 460.08807 M+ + 1, Calcd. for C25H18ClN3O2S:
460.08810.
5-(5-Bromo-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4k)
Red powder (yield 86%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3296 (NH) and 1690 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.33 (s, 3H, -CH3), 2.43 (s, 3H, -CH3), 6.91 (d, J = 8.0 Hz, 2H, Ar-H), 7.01 (d,
δ
J = 8.0 Hz, 1H, Ar-H), 7.25 (d, J = 7.6 Hz, 2H, Ar-H), 7.39–7.51 (m, 5H, Ar-H), 9.03 (s, 1H, Ar-H), 11.41 (s,
1H, NH isatin, D2O exchangeable); Anal. Calcd. for C25H18BrN3O2S: C, 59.53; H, 3.60; N, 8.33; found
C, 59.80; H, 3.78; N, 8.59.
5-(5-Methoxy-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4l)
Red powder (yield 75%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3256 (NH) and 1689 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.32 (s, 3H, -CH3), 2.40 (s, 3H, -CH3), 3.71 (s, 3H, -OCH3), 6.83–6.88 (m,
δ
3H, Ar-H), 6.95 (d, J = 7.8 Hz, 1H, Ar-H), 7.19 (d, J = 8.0 Hz, 2H, Ar-H), 7.37 (d, J = 8.0 Hz, 2H, Ar-H),
7.42 (d, J = 8.0 Hz, 2H, Ar-H), 8.47 (s, 1H, Ar-H), 11.01 (s, 1H, NH isatin, D2O exchangeable); 13C-NMR
(DMSO-d6, 100 MHz) δ ppm: 20.97, 21.27, 55.99, 111.14, 113.89, 118.27, 121.18, 121.22, 125.59, 128.62,
128.86, 129.00, 130.02 (2C), 130.30 (2C), 132.35, 132.56, 134.29, 137.53, 138.92, 145.49, 153.83, 155.02,
166.06, 169.03; MS, m/z [%]: 455 [M+, 100]; Anal. Calcd. for C26H21N3O3S: C, 68.55; H, 4.65; N, 9.22;
found C, 68.47; H, 4.60; N, 9.53; HRMS m/z 456.13754 M+ + 1, calcd for C26H21N3O3S: 456.13764.
5-(5-Methyl-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4m)
Red powder (yield 80%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3358 (NH) and 1693 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.26 (s, 3H, -CH3), 2.32 (s, 3H, -CH3), 2.41 (s, 3H, -CH3), 6.82 (d, J = 8.0 Hz,
δ
1H, Ar-H), 6.91 (d, J = 7.2 Hz, 2H, Ar-H), 7.19–7.23 (m, 3H, Ar-H), 7.35 (d, J = 7.6 Hz, 2H, Ar-H), 7.43 (d,
J = 7.6 Hz, 2H, Ar-H), 8.54 (s, 1H, Ar-H), 11.19 (s, 1H, NH isatin, D2O exchangeable); MS, m/z [%]:
439 [M+, 100]; Anal. Calcd. for C26H21N3O2S: C, 71.05; H, 4.82; N, 9.56; found C, 70.89; H, 4.96; N, 9.74.
5-(5,7-Dimethyl-2-oxoindolin-3-ylidene)-3-(p-tolyl)-2-(p-tolylimino)thiazolidin-4-one (4n)
◦
Brown powder (yield 71%), m.p. > 300 C; IR (KBr,
(DMSO-d6, 400 MHz) ppm: 2.19 (s, 3H, -CH3), 2.24 (s, 3H, -CH3), 2.32 (s, 3H, -CH3), 2.41 (s, 3H, -CH3),
ν
cm−1): 3319 (NH) and 1685 (C=O); 1H-NMR
δ
6.87 (d, J = 8.2 Hz, 2H, Ar-H), 7.02 (s, 1H, Ar-H), 7.21 (d, J = 8.1 Hz, 2H, Ar-H), 7.37 (d, J = 8.3 Hz, 2H,
Ar-H), 7.43 (d, J = 8.3 Hz, 2H, Ar-H), 8.49 (s, 1H, Ar-H), 11.11 (s, 1H, NH isatin, D2O exchangeable);
13C-NMR (DMSO-d6, 100 MHz)
δ ppm: 18.90, 21.35, 28.18 (2C), 119.54, 120.90 (2C), 126.31, 128.86 (2C),
129.87 (2C), 130.30 (2C), 130.93, 131.46, 132.59, 134.14, 138.97, 139.89, 143.02, 145.42, 153.76, 157.33,
163.30, 165.80, 169.33; MS, m/z [%]: 453 [M+, 100]; Anal. Calcd. for C27H23N3O2S: C, 71.50; H, 5.11; N,
9.26; found C, 71.67; H, 5.04; N, 9.51; HRMS m/z 454.15833 M+ + 1, calcd for C27H23N3O2S: 454.15837.

Molecules 2018, 23, 1420
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4.1.3. General Procedure for the Preparation of Target Compounds 7a–d
Route A:
A mixture of isatins 1c
(0.15 gm, 1 mmoL) and bromoacetic acid
,
e
–
g
(1 mmoL) with an equimolar amount of 2-mercaptobenzimidazole
(0.14 g, 1 mmoL) in glacial acetic acid (10 mL) in the
6
3
presence of sodium acetate (0.16 g, 2 mmoL), was heated under reflux for 2 h. The obtained solid was
filtered off while hot, washed with hot ethanol, dried and recrystallized from DMF to afford the target
hybrids 7a–d.
Route B:
To a stirred solution of 2-mercaptobenzimidazole
1.1 mmoL) in ethanol (10 mL), bromoacetic acid (0.14 gm, 1 mmoL) was added, and then the reaction
mixture was refluxed for 1 h. The formed solid was filtered off, washed with ethanol to give compound
, which cyclized into intermediate
via heating with acetic anhydride in pyridine medium at 100 ◦C.
Intermediate (0.19 g, 1 mmoL) was added to a stirred solution of the appropriate isatin derivative
1c (1 mmoL) and sodium acetate (0.16 g, 2 mmoL) in glacial acetic acid (5 mL), then the reaction
6 (0.15 gm, 1 mmoL) and KOH (0.06 gm,
3
8
9
9
, e–g
mixture was refluxed for 2 h. The formed solid was filtered off, washed with ethanol and recrystallized
from DMF to furnish compounds 7a–d.
2-(5-Chloro-2-oxoindolin-3-ylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (7a)
Red powder (yield 80%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3325 (NH) and 1689 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 7.04 (d, J = 8.3 Hz, 1H), 7.36–7.48 (m, 1H), 7.52 (dd, J = 8.4, 2.0 Hz, 1H),
δ
7.69 (d, J = 6.6 Hz, 1H), 8.03 (d, J = 6.5 Hz, 1H), 8.93 (d, J = 2.1 Hz, 1H), 11. 51 (s, 1H, NH isatin,
D2O exchangeable); Anal. Calcd. for C17H8ClN3O2S: C, 57.72; H, 2.28; N, 11.88; found C, 58.06; H,
2.41; N, 12.16.
2-(5-Methoxy-2-oxoindolin-3-ylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (7b)
Red powder (yield 85%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3296 (NH) and 1692 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 3.83 (s, 3H, -OCH3), 6.93 (d, J = 8.5 Hz, 1H, Ar-H), 7.08 (dd, J = 8.5, 2.6 Hz,
δ
1H, Ar-H), 7.42 (m, 2H, Ar-H), 7.69 (d, J = 7.0 Hz, 1H, Ar-H), 8.01 (d, J = 6.6 Hz, 1H, Ar-H), 8.59 (d,
J = 2.6 Hz, 1H, Ar-H), 11.18 (s, 1H, NH isatin, D2O exchangeable); MS, m/z [%]: 349 [M+, 21.03]; Anal.
Calcd. for C18H11N3O3S: C, 61.88; H, 3.17; N, 12.03; found C, 62.14; H, 3.42; N, 12.31.
2-(5-Methyl-2-oxoindolin-3-ylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (7c)
Red powder (yield 87%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3405 (NH) and 1693 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.37 (s, 3H, -CH3), 6.90 (d, J = 7.6 Hz, 1H, Ar-H), 7.27 (d, J = 8.5, 1H,
δ
Ar-H), 7.33–7.43 (m, 2H, Ar-H), 7.68 (d, J = 7.2 Hz, 1H, Ar-H), 8.01 (d, J = 7.6 Hz, 1H, Ar-H), 8.75 (s,
1H, Ar-H), 11.26 (s, 1H, NH isatin, D2O exchangeable); MS, m/z [%]: 333 [M+, 23.48]; Anal. Calcd.
for C18H11N3O2S: C, 64.85; H, 3.33; N, 12.61; found C, 64.97; H, 3.50; N, 12.89.
2-(5,7-Dimethyl-2-oxoindolin-3-ylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (7d)
Red powder (yield 88%), m.p. > 300 ◦C; IR (KBr,
ν
cm−1): 3382 (NH) and 1689 (C=O); 1H-NMR
(DMSO-d6, 400 MHz) ppm: 2.20 (s, 3H, -CH3), 2.29 (s, 3H, -CH3), 7.09 (s, 1H, Ar-H), 7.34–7.48 (m,
δ
2H, Ar-H), 7.68 (d, J = 7.0 Hz, 1H), 8.02 (d, J = 7.0 Hz, 1H), 8.62 (s, 1H, Ar-H), 11.28 (s, 1H, NH isatin,
D2O exchangeable); MS, m/z [%]: 347 [M+, 100]; Anal. Calcd. for C19H13N3O2S: C, 65.69; H, 3.77; N,
12.10; found C, 65.91; H, 3.86; N, 11.97.

Molecules 2018, 23, 1420
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4.2. Biological Evaluation
4.2.1. Antiproliferative Activity Against Breast Cancer Cell Lines
Breast cancer cell lines MDA-MB-231 and MCF-7 cells were obtained from VACSERA Tissue
Culture Unit. The cells were propagated in DMEM supplemented with 10% heat-inactivated fetal
bovine serum, 1% L-glutamine (2.5 mM), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
◦
buffer (10 mM) and 50
µ
g/mL gentamycin. All cells were maintained at 37 C in a humidified
atmosphere with 5% CO2 and were sub-cultured two times a week. Cytotoxicity was determined
following the Crystal Violet (CV) cell cytotoxicity assay [41].
Test samples were dissolved in DMSO and kept at a stock concentration of 100 mM. Exponentially
growing cells were collected using 0.25% Trypsin-EDTA and seeding was done at a density of 3000
cells/well in 96-well plates. After 24 h, the monolayer cultured cells were washed with sterile
phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 mL from
10 different dilutions of the test compounds as well as Doxorubicin was used as a standard compound
◦
in fresh maintenance medium and incubated at 37 C for 24 h. Control wells were exposed to the same
concentration of vehicle, DMSO (1%) used in the highest concentration of the test samples. Cell viability
was >99% compared to untreated cells. The number of the surviving cells was determined by staining
the cells with crystal violet followed by cell lysing using 33% glacial acetic acid. The absorbance was
measured at 490 nm using ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA) after gentle
shaking. The absorbance values from untreated cells were considered as 100% proliferation.
The number of viable cells was determined using ELISA reader as previously mentioned and
the percentage of viability was calculated as [1
−
(ODt/ODc)]
×
100% where ODt is the mean optical
density of wells treated with the test sample and ODc is the mean optical density of untreated cells.
The 50% inhibitory concentration (IC50) was estimated from graphic plots of the dose response curve
for each conc. using GraphPad Prism software, Version 5 (San Diego, CA, USA). The data presented
are the mean of at least three separate experiments.
4.2.2. In Vitro Cytotoxic Activity towards Nontumorigenic Human WI-38 and MCF-10A Cells
The cytotoxic activity of the active conjugates towards nontumorigenic human lung fibroblast cell
line (WI-38) and human breast epithelial cell line (MCF-10A) was determined using Crystal violet (CV)
cell cytotoxicity assay according to the previously published procedures [42,55].
4.2.3. ELISA Immunoassay
The levels of the apoptotic markers (Bax, caspase-3) as well as the antiapoptotic marker Bcl-2
were determined using ELISA colorimetric kits per the manufacturer’s instructions, as reported
earlier [41]. MDA-MB-231 cells were cultured as a monolayer in T-25 flasks and were seeded to attain
30% confluency prior to treatment. Cells were then treated separately with compounds 4m and 7b
at their IC50 concentrations for 48 h. At the end of treatment, cells were collected via trypsinization
and centrifuged at 10,000 rpm. The pellet was then rinsed with PBS and lysed in RIPA lysis buffer at
4 ◦C for 45 min, then centrifuged at 14,000 rpm for 20 min to remove the cellular debris. Lysates were
then collected and stored at
Kit (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer’s recommendations.
The cell lysate was diluted 10 times, and 100 L (50 mg protein) was added to the wells of four
−
80 ◦C for later protein determination using Pierce BCA Protein Assay
µ
separate microtiter plates for the four ELISA kits that were pre-coated with primary antibodies specific
to Bax, Bcl-2 and caspase-3 proteins, respectively. A secondary biotin-linked antibody specific to the
protein captured by the primary antibody was further added to bind the captured protein, forming a
“sandwich” of specific antibodies around the desired protein in the cell lysate. The streptavidin-HRP
complex was then used to bind the biotin-linked secondary antibody through its streptavidin portion.
The HRP domain reacted with the added TMB substrate to form a colored product that measured at

Molecules 2018, 23, 1420
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450 nm by a plate reader (ChroMate-4300, Orlando, FL, USA) after the reaction was terminated via the
addition of stop solution.
4.2.4. Cell Cycle Analysis
TNBC MDA-MB-231 cells were treated with compound 4m for 24 h (at its IC50 concentration),
and then cells were washed twice with ice-cold phosphate buffered saline (PBS). Subsequently,
the treated cells were collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS,
re-suspended with 100 µg/mL RNase, stained with 40 µg/mL PI, and analyzed by flow cytometry
using FACS Calibur (Becton Dickinson, BD, Franklin Lakes, NJ, USA). The cell cycle distributions were
calculated using CellQuest software 5.1 (Becton Dickinson) [35,49].
4.2.5. Annexin V-FITC Apoptosis Assay
Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis detection
kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions, as reported
earlier [35
its IC50 concentration as described earlier. Cells were then harvested via trypsinization, and rinsed
twice in PBS followed by binding buffer. Moreover, cells were re-suspended in 100 L of binding
buffer with the addition of 1
L of FITC-Annexin V (Becton Dickinson BD PharmingenTM, Heidelberg,
Germany) followed by an incubation period of 30 min at 4 C. Cells were then rinsed in binding
buffer and resuspended in 150 L of binding buffer with the addition of 1 L of DAPI (1 g/ L in
,49]. MDA-MB-231 cells were cultured to a monolayer then treated with conjugate 4m at
µ
µ
◦
µ
µ
µ
µ
PBS) (Invitrogen, Life Technologies, Darmstadt, Germany). Cells were then analyzed using the flow
cytometer BD FACS Canto II (BD Biosciences, San Jose, CA, USA) and the results were interpreted
with FlowJo7.6.4 software (Tree Star, Ashland, OR, USA).
4.2.6. Statistical Analysis
Data are presented as means
±
S.D. Individual groups were compared using the two-tailed
independent Student’s t-test. Multiple group comparisons were carried out using one-way analysis of
variance (ANOVA) followed by the Tukey–Kramer test for post-hoc analysis. Statistical significance
was accepted at a level of p < 0.05. All statistical analyses were performed using GraphPad InStat
software, version 3.05 (GraphPad Software, Inc., La Jolla, CA, USA). Graphs were sketched using
GraphPad Prism software, version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA).
Author Contributions: Wagdy M. Eldehna and Hatem A. Abdel-Aziz have formulated the research idea;
Wagdy M. Eldehna, Mohamed H. El-Naggar, Hadia Almahli and Mohaned Fares carried out the experimental,
interpreted the data and discussion, and prepared the manuscript; Mahmoud M. Elaasser performed the
cytotoxicity assay; Amr Elgez carried out the biological work and discussed its results. All the authors reviewed
and approved the manuscript.
Funding: This research received no external funding.
Acknowledgments: Patrick Maccosker, University of Wollongong, Australia, is highly appreciated for his efforts
in language editing of the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).