Synthesis and molecular docking of hybrids ionic azole Schiff bases as novel CDK1 inhibitors and anti-breast cancer agents: In vitro and in vivo study

Article abstract of DOI:10.1016/j.molstruc.2021.131041

Three new hybrid ionic vanillyl-azole-Schiff bases (IVASBs) have synthesized and their antitumor performances were assessed in vitro against MCF-7 cells and in vivo against Ehrlich solid tumor (EST). The high binding energy score of IVASB3?CDK1 (?3.43 kca

Full text of DOI:10.1016/j.molstruc.2021.131041

Journal of Molecular Structure 1245 (2021) 131041
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis and molecular docking of hybrids ionic azole Schiff bases as
novel CDK1 inhibitors and anti-breast cancer agents: In vitro and in
vivo study
Waleed M. Seraga, Faten Zahranb, Yasmin M. Abdelghanya, Reda F.M. Elshaarawya,c,∗
,
Moustafa S. Abdelhamidb
a Chemistry department, Faculty of Science, Suez University, Suez 43533, Egypt
b Biochemistry Division, Chemistry Department, Faculty of Science, Zagazig University, Egypt
c Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new hybrid ionic vanillyl-azole-Schiff bases (IVASBs) have synthesized and their antitumor perfor-
mances were assessed in vitro against MCF-7 cells and in vivo against Ehrlich solid tumor (EST). The high
binding energy score of IVASB3–CDK1 (−3.43 kcal/mol) makes IVASB3 the most potent anti-breast can-
Received 7 June 2021
Accepted 3 July 2021
Available online 7 July 2021
cer candidate (IC50 3.73
0.2 μg/mL). Cell cycle analysis showed that IVASB3 was significantly increased
the percent of EST cells undergoing the sub G1 phase and decreased the percent of cells in the S phase.
In addition, the administration of IVASB1, IVASB2, and IVASB3 to tumorized mice caused a significant
reduction in cyclin-dependent kinase-1 (CDK1) expression by 41.27%, 53.31%, and 78.57%, respectively.
Moreover, concentrations of poly ADP-ribose polymerase (PARP) and vascular endothelial growth factor
(VEGF) significantly decreased in the serum of tumorized mice which received IVASBs. Consequently, the
antitumor effects of IVASBs could be attributed to their capacity to diminish CDK1, PAPR, and VEGF ex-
pressions.
Keywords:
Ionic vanillyl-azole-schiff bases
Molecular docking
In vitro and in vivo Cytotoxicity
Flow-cytometry
PAPR and VEGF
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
For the last few decades, an infinite number of researchers have
demonstrated great interest in the nitrogen- and sulfur-containing
Cancer is one of the most serious human health problems and
considered the second major reason for death all over the world
[1,2].Cancer is widely progressed in the modern era and is ex-
pected to hit ~25 million people in the next 20 years [3]. Breast
cancer is the most frequent type of cancer among women world-
wide; it is still a leading cause of global women’s death. It accounts
for about 450,000 deaths around the world yearly [4]. Current
treatment strategies are based on surgical removal of the tumor or
radiotherapy followed by chemotherapy [5]. Although chemother-
apy and radiotherapy are broadly being used to treat numerous
types of tumors, they suffer from many problems such as (i) non-
selectivity and destructive side effects on normal cells; (ii) narrow-
spectrum; and ii) chemotherapeutic agent resistance [6]. Therefore,
there is still an urgent need for the development of more safe and
effective anticancer agents for wiping out this disease.
heterocyclic compounds for the development of varieties of new
biomolecules to enrich their therapeutic potential in pharmacolog-
ical applications [7,8]. Amongst these compounds, 2-aminothiazole
(AT) and its derivatives (Fig. S1, ESI†), which found in many natu-
ral products, always offer a class of promising bioactive scaffolds
to design and develop diverse pharmacological agents with ex-
cellent biological activities [9]. In particular, Abafungin, Cefdinir,
Famotidine, and Riluzole (Fig. S1, ESI†) are well-known market-
ing drugs bearing the 2-aminothiazole moiety [10]. Moreover, 2-
aminothiazoles have proved to have various therapeutic potentials
including antibacterial [11,12], antifungal [13], antiviral [14], anti-
inflammatory [15], antioxidant [16], antidiabetic [17], anticonvul-
sant [18], and antitubercular [19]. In spite of the diverse biological
impacts of 2-aminothiazole derivatives, 2-aminothiazole is still cat-
egorized as a promising scaffold for the construction of new potent
anticancer agents due to their broad-spectrum antitumor activities
through acting on different biological targets such as tubulin pro-
tein, HAT/HDAC enzymes, PI3K, Src/Abl, EGFR, BRAF, and SphK ki-
nase [10,20]. Meanwhile, 1,3,4-thiadiazole ring is also considered
a versatile scaffold for the development of multifunctional phar-
∗
Corresponding author at: Chemistry Department, Faculty of Science, Suez Uni-
versity, Suez 43533, Egypt.
E-mail address: reda.elshaarawy@suezuniv.edu.eg (R.F.M. Elshaarawy).
https://doi.org/10.1016/j.molstruc.2021.131041
0022-2860/© 2021 Elsevier B.V. All rights reserved.

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
macological agents [21]. Where, the mesoionic nature of this ring
enables thiadiazole-based compounds to easily go through the cel-
lular membrane and strongly interact with biological targets [22].
Furthermore, the anticancer potentials of thiadiazole derivatives
are undeniable [22,23].
2.2. Synthesis of IVSBs
In general, a solution of SMIILs (5 mmol) in ethanol (25 mL)
was added dropwise under vigorous stirring to a solution of 2-
aminothiazole/ 2,5-diaminothiadiazole (0.25/ 0.28 g, 5/ 2.5 mmol)
in 10 mL of ethanol containing few drops of glacial acetic acid. The
reaction mixture was further stirred under reflux temperature for
4–6 h (thin-layer chromatography (TLC) was used to monitor reac-
tion progress). At the end of the reaction, the content was cooled
to ambient temperature and the formed solids were collected by
filtration, washed with cold ethanol (2 × 3 mL) followed by di-
ethyl ether (3 × 5 mL), dried, and then recrystallized from ethanol
to give the desired products.
Notable, Schiff bases also have attracted great interest of many
pharmaceutical researchers due to their biological performances in
many fields [24] including anticancer efficacy [25].
Interestingly, the exceptional and amazing physicochemical
properties of ionic liquids (ILs) makes them very attractive for
many researchers to design numerous smart materials, rendering
them inimitably suited for applications in many fields such as
synthesis [26], electrochemistry [27], analytics [28], active phar-
maceutical ingredients (API) [29], extraction [30], and so far. Re-
cently, ionic liquid-supported Schiff bases were effectively used as
chemical sensors [31,32] and to convert the toxic pollutants (pri-
mary amines and heavy metal ions) into pharmacological candi-
dates [33,34].
3-(Thiazol-2-ylimino)methyl)vanillyl-2-methylimidazolium chloride
(IVASB1); Obtained as yellowish-orange powder (96%). FTIR (KBr,
cm−1): 3421 (vs, br), 3098 (m, br), 3061 (m, br), 1621 (s, sh),
1573 (w, br), 1509 (vs, sh), 1459, 1385 (w, b), 1283 (m, sh), 1149
(m, sh), 1065 (w, br), 745 (m, br). 1H NMR (500 MHz, DMSO–
d6) δ (ppm): 11.13 (s, 1H), 9.12 (s, 1H), 8.89 (s, 1H), 8.77 (s, br,
1H), 7.67–7.61 (m, 1H), 7.65–7.55 (m, 1H), 7.28 (s, 1H), 7.17 (d,
J = 10.85 Hz, 1H), 7.10 (s, 1H), 5.25 (s, 2H), 3.86 (s, 3 H), 2.48
(s, 3H,). 13C NMR (125 MHz, DMSO–d6) δ (ppm): 161.95, 158.82,
157.09, 147.19, 144.65, 131.11, 125.71, 123.03, 121.28, 118.65, 106.38,
101.73, 55.88, 51.36, and 11.93. ESI-MS, m/z: 315.3 ([C15H15N4O2S]+,
M - Cl–). Anal. Calcd. for C15H15ClN4O2S (M = 350.82): C, 51.36; H,
4.31; N, 15.97; S, 9.14%. Found: C, 51.33; H, 4.32; N, 15.94; S, 9.07%.
There are a number of in vivo experimental models based on
laboratory animals including the Ehrlich solid tumor (EST), derived
from the mouse breast adenocarcinoma, which is an aggressive and
fast growing carcinoma able to develop both in the ascetic or the
solid form depending on whether inoculated intraperitoneally or
subcutaneously, respectively [35]. It is a neoplasm of epithelial ori-
gin and can be used to study the mechanisms of carcinogenesis
and evaluate the effect of new therapeutic approaches on tumors
[36].
2,5-Bis-(5-(2-methylimidazolium
chloride)−3-
The cell cycle is composed of four functional phases (Fig. S2,
ESI†) that lead to the duplication and division of cell: S phase in-
volves DNA duplication; M phase (mitosis) where DNA and cellular
components have divided into two daughter cells; G2 phase, where
cells prepare for mitosis; G1 where cells prepare itself for other
DNA and cellular divisions [37]. The transition from one phase to
another is regularly occurred by the action of key regulatory pro-
teins (Cyclin-dependent kinases (CDKs)) (Fig. S2, ESI†) [38]. Cyclin-
dependent kinase 1 (CDK1) serves an important role in the control
of the cell cycle by regulating the centrosome cycle, sponsoring
G2-M transition, moderating G1 phase progression, and involved
in the regulation of apoptosis, as well [39]. Noteworthy, CDK1 is
overexpressed in several tumors and contributes to their develop-
ment [40]. Therefore, studying the cell cycle through flow cytomet-
ric analysis could be very helpful in offering an insight concerning
the mode of action for a new anticancer drug.
methoxysalicylideneimino)−1,3,4-thiadiazole
(IVASB2):
Obtained
as orange powder (79%). FTIR (KBr, cm−1): 3429 (s, br), 2927 (m,
br), 1629 (m, br), 1568 (m, sh), 1449 (m, br), 1284 (s, sh), 1201
(w, br), 1167 (w, sh), 1043 (m, br), 720 (w, br). 1H NMR (500 MHz,
DMSO–d6) δ (ppm): 11.12 (s, 2H), 11.09 (s, 1H), 10.83 (s, 1H), 10.12
(s, 1H), 9.97 (s, 1H), 8.07 (d, J = 1.83 Hz, 2H), 7.85–7.49 (m, 4H),
7.43 (d, J = 1.71 Hz, 2 H), 7.16–6.91 (m, 2H), 5.38 (s, 4H), 3.79 (s,
3H), 3.77 (s, 3H), 2.66 (s, 3H), 2.63 (s, 3H). 13C NMR (125 MHz,
DMSO–d6) δ (ppm): 164.93, 161.28, 158.19, 136.31, 128.65, 125.73,
123.31, 121.35, 118.51, 118.08, 57.58, 39.42 and 10.63. ESI-MS, m/z:
582.00 ([C26H26ClN8O4S]+, M - Cl–), 273.2 ([C26H26N8O4S]2+, M -
2Cl–). Anal. Calcd. for C26H26Cl2N8O4S (M = 617.51): C, 50.57; H,
4.24; N, 18.15; S, 5.19%. Found: C, 50.58; H, 4.26; N, 18.11; S, 5.16%.
2,5-Bis-(5-(2,4-lutidinium
chloride)−3-
methoxysalicylideneimino)−1,3,4-thiadiazole
(IVASB3):
Obtained
as yellow powder (82%). FTIR (KBr, cm−1): 3423 (s, br), 2928 (w,
br), 1632 (vs, sh), 1551 (w, br), 1449 (s, sh), 1273 (s, sh), 1209 (w,
sh), 1155 (m, sh), 1039 (w, br), 710 (m, sh). 1H NMR (500 MHz,
DMSO–d6) δ (ppm): 11.13 (s, 2H), 10.28 (s, 1H), 10.03 (s, 1H),
8.79 (d, J = 4.3 Hz, 2H), 8.49 (s, 1H), 8.13–8.02 (m, 1H), 7.89 (d,
J = 8.2 Hz, 2H), 7.75 (s, 1H), 7.46–7.35 (m, 1H), 7.26 (d, J = 6.1 Hz,
2H), 7.18 (dd, J = 11.9, 8.4 Hz, 2H), 5.31 (s, 4H), 3.87 (s, 6H),
3.78 (s, 6H), 2.76 (s, 6H). 13C NMR (125 MHz, DMSO–d6) δ (ppm):
160.12, 155.89, 153.87, 147.11, 145.88, 133.15, 130.59, 128.90, 127.29,
126.31, 118.79, 117.81, 57.59, 22.13, and 21.21. ESI-MS, m/z: 646.10
([C33H34ClN6O4S]+, M – Cl–), 305.30 ([C33H34N6O4S]2+, M - 2Cl–).
Anal. Calcd. for C34H36Cl2N6O4S (M = 695.66): C, 58.70; H, 5.22;
N, 12.08; S, 4.61%. Found: C, 58.64; H, 5.26; N, 12.91; S, 4.42%.
Herein, through our continuous efforts to explore new multi-
functional bioactive ionic liquids-supported Schiff bases (ILSSBs)
[41–43], three hybrid ionic vanillyl-azole-Schiff bases (IVASBs),
(3-(thiazol-2-ylimino)methyl)vanillyl-2-methylimidazolium
chlo-
ride (IVASB1), 2,5-Bis-(5-(2-methylimidazolium chloride)−3-
methoxysalicylideneimino)−1,3,4-thiadiazole (IVASB2), and 2,5-Bis-
(5-(2,4-lutidinium chloride)−3-methoxysalicylidene-imino)−1,3,4-
thiadiazole (IVASB3), have been successfully fabricated. The effect
of IVSBs against EST, induced experimentally in mice, was in-
vestigated in comparison to a reference chemotherapeutic drug,
5-fluorouracil, and examined their effect on CDK, PARP, and VEGF
as well.
2. Materials and methods
2.3. Docking study
2.1. Materials and instrumentation
AutoDock4 was employed for docking the tested compounds
(IVASBs) in the binding site of CDK1. The X-ray crystal structure of
CDK1 was obtained from RCSB Protein Data Bank (PDB ID: 4YC6)
Solvents and reagents coupled with their suppliers, methods
used for the preparation of vanillyl ionic liquids (VILs) (2a,b) and
2,5-diaminothiadiazole, and the instrumental techniques applied
for the full characterization of the as-prepared ionic liquids were
given in the online electronic supplementary information (ESI†).
˚
with a 2.6 A resolution. The protein targets were prepared for
molecular docking simulation by removing water molecules and
bound ligands. IVASBs were built and modeled using the MDL ISIS
Draw 2.5 software.
2

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
2.4. In vitro cytotoxicity experiment
ꢀ Group V: tumorized mice L3-treated group (EST + IVASB3): fif-
teen tumorized mice treated with L3 (1/10 LD50; 45 mg/Kg) ev-
ery other day for 13 days.
The in vitro cytotoxic impact of ionic vanillyl azole Schiff bases
(IVASBs) on human breast carcinoma cell lines (MCF-7) was evalu-
ated according the protocol adapted by the regional center for My-
cology and Biotechnology, Al-Azhar University, Egypt as described
in our previous work [44]. 5-Fluorouracil (5-Fu) was used as a ref-
erence drug. The cell viability and IC50 values were used as cyto-
toxicity markers for the tested compounds.
ꢀ Group VI: tumorized mice 5-fluorouracil (5-FU)-treated group
(EST
+
5-FU): fifteen tumorized mice treated with 5-FU
(20 mg/Kg) every other day for 13 days.
The volume of the solid tumor was evaluated using Vernier
caliper (after shaving the tumor-bearing thigh of each animal) and
was calculated using the formula of A × B × 0.5, where A and B
are the longest and the shortest diameter of tumor, respectively
[47]. Then, the tumor mass was removed, weighed, tabulated, and
preserved in buffered formalin and stored at −80 °C until further
use and processed for flow cytometry investigations of the tumor
marker cyclin-dependent kinase 1 (CDK1) and cell cycle analysis.
2.5. In vivo study
2.5.1. Determination of median lethal dose (LD50
)
The median lethal dose (LD50) of the test compounds was de-
termined according to the method described by Reed and Muench
[45]. Briefly, the method involves the intraperitoneal injection of
single dose of different dilutions for each test compound separately
to various groups, which has ten mice each. After that the injected
mice were observed for behavior and mortality for 24 h. The num-
ber of survival and dead mice was recorded and the percent of
mortality was calculated for each group and LD50 was calculated
using the following formula;
2.6. Hematological parameters
At the end of the in vivo experiment, the animals were sac-
rificed under slight ether anesthesia by cervical dislocation and
blood samples were taken in tubes containing EDTA for determi-
nation of hematological parameters. Hemoglobin (HGB), count of
white blood cells (WBCs), and red blood cells (RBCs) were analyzed
using standard automated procedures.
Log LD50 =log LD next below 50%
+
[log increasing fac-
tor × Proportional distance] where, proportional distance = [50 –
mortality at dilution next below] / [mortality next above – mor-
tality next below]. LD50 values of IVSB1, IVSB2 and IVSB3 were
recorded at 3.31 μg/Kg b.w., 55 and 450 mg/Kg b.w., respectively.
2.7. Biochemical parameters
Blood samples were taken in tubes without anticoagulant (for
serum biochemical analysis in accordance with the method of
Frankenberg [48]. Sera were separated and stored in aliquots at
−80 °C until using for analysis of biochemical parameters of as-
partate transaminase (AST), alanine transaminase (ALT), the con-
centration of albumin (Alb), total bilirubin (T bil), urea and crea-
tinine, and ELISA technique of Poly-ADP-ribose polymerase (PARP)
and vascular endothelial growth factor (VEGF).
2.5.2. Experimental animals, ethics and cancer cell lines
Swiss albino mice samples (20–25 g) were housed in plastic
cages under standard laboratory conditions (25
2 °C; 70–80 hu-
midity; 12 h light/darkness cycle) with standard pellet diet and
water ad libitum. The animals were handled in accordance with
current guidelines for the care of laboratory animals and ethical
guidelines for the investigation of experimental pain in conscious
animals. The first inoculum of Ehrlich ascites carcinoma (EAC) and
MCF-7 cell lines was purchased from the national cancer institute
(Cairo, Egypt). EAC cells were propagated in the peritoneal cavity
of mice through serial intraperitoneally (IP) transplantation of 106
cells (0.2 ml of PBS/animal) and transferred every 7 days to normal
animals.
2.7.1. Determination of poly ADP-ribose polymerase (PARP)
The concentration of PARP in the serum of mice was deter-
mined using a commercial ELISA kit applying the quantitative
sandwich enzyme immunoassay technique. The microtiter plate
has been pre-coated with a monoclonal antibody specific for PARP.
Standards or samples were added to the microtiter plate wells
and PARP will bind to the antibody pre-coated wells. In order to
quantitatively determine the amount of PARP present in the sam-
ple, a standardized preparation of horseradish peroxidase (HRP)-
conjugated polyclonal antibody, specific for PARP is added to each
well to “sandwich” the PARP immobilized on the plate. Then the
wells are thoroughly washed to remove all unbound components.
Next, substrate solutions are added to each well. The enzyme (HRP)
and substrate are allowed to react over a short incubation period.
Only those wells that contain PARP and enzyme-conjugated anti-
body will exhibit a change in color. A standard curve is plotted re-
lating the intensity of the color (OD) to the concentration of stan-
dards. The PARP concentration in each sample is interpolated from
this standard curve. The Optical Density (OD) was determined at
450 nm using a microplate reader.
2.5.3. Ehrlich solid tumor (EST) model
A total of 90 male albino mice were randomly divided into six
groups (15 mice/ group). Group I was kept as control while the
solid tumor was induced in the other five groups. On the day of
induction, Ehrlich cells were collected from the ascitic fluid of a
female Swiss albino mouse bearing an 8-day-old ascetic tumor ob-
tained from the National Cancer Institute (Cairo, Egypt). The ascitic
fluid was diluted with normal saline (1: 10 v/v). Solid tumors were
induced by intramuscular inoculation of 0.2 mL of ascitic fluid,
containing approximately 2.5 × 106 EAC cells, in the right thigh
of the hind limb of each mouse [46]. After 2 weeks of tumor inoc-
ulation mice were treated intraperitoneally with test compounds
(IVASB1, IVASB2 and IVASB3) or 5-fluorouracil (5-FU) every other
day according to the following scheme:
ꢀ Group I: normal mice saline-control group (normal control): fif-
teen normal mice have injected IP with 0.2 mL physiological
saline (0.9 g/dL) every other day for 13 days.
2.7.2. Determination of vascular endothelial growth factor (VEGF)
The concentration of VEGF in serum of mice was determined
using ELISA commercial Kit (Invitrogen, USA) and the procedure
was followed according to the manufacturer’s instructions. Briefly,
a mouse monoclonal antibody specific for rat VEGF is coated on a
96-well plate. Standards and test samples are added to the wells
and VEGF present in a sample is bound by the immobilized an-
tibody. A biotinylated polyclonal antibody from goat specific for
VEGF is added subsequently. After washing away the unbound
ꢀ Group II: fifteen tumorized mice control group (Ehrlich control).
ꢀ Group III: tumorized mice L1-treated group (EST + IVASB1): fif-
teen tumorized mice treated with L1 (1/10 LD50; 0.331 μg/Kg)
every other day for 13 days.
ꢀ Group IV: tumorized mice treated with L2-treated group
(EST + IVASB2): fifteen tumorized mice treated with L2 (1/10
LD50; 5.5 mg/Kg) every other day for 13 days.
3

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
biotinylated antibody with PBS or TBS buffer, the avidin-Biotin-
Peroxidase complex is added to the wells. The wells were again
washed with PBS or TBS buffer to remove the unbound conju-
gates. HRP substrate TMB is used to visualize the HRP enzymatic
reaction. TMB is catalyzed by HRP to produce a blue product that
changes into yellow after adding the acidic solution. The density of
yellow color is proportional to the quantity of VEGF captured onto
the plate. The Optical Density (OD) was determined at 450 nm us-
ing a microplate reader.
3. Results and discussion
3.1. Chemistry
Different chemical processes including chloromethylation, quat-
ernization, heterocyclization, Schiff-base condensation were used
in our protocol to synthesize the target compounds (see
Scheme 1). Initially, chloromethylation of salicylaldehyde was per-
formed using a mixture of paraformaldehyde and hydrochloric acid
in presence of ZnCl2 to yield chloromethylsalicylaldehyde (1) that
used as alkylating agent for quaternization of 2-methylimidazole
(2-MeIm), 2,4-dimethylpyridine (2,4-lutidine, 2,4-Lut) to fabricate
salicylaldehyde ionic liquids (SILs, 2a,b).
2.8. Flow-cytometric analysis
On the other hand, 2,5-diamino-1,3,4-thiadiazole was obtained
by the heterocyclization of bis-thiourea by the action of hydrogen
peroxide. Eventually, the Schiff base condensation of SILs (2a,b)
with 2-AT or diaminothiadiazole produce the desired ionic vanil-
lyl azole Schiff bases (IVASBs) (Scheme 1). These ionic Schiff bases
were obtained with high to excellent yields and their structures
were elucidated based on the elemental and spectral (FTIR, 1H
NMR, 13C NMR, ESI-MS) analyses.
The flow-cytometric analysis was performed in the global cen-
ter for scientific research, Mansoura, Egypt using FACS (Flow ac-
tivated cell sorter) Caliber Flow Cytometer (Becton Dickinson,
Sunnyvale, CA, USA) equipped with a compact air-cooked low
power 15 mW argon ion laser beam (488 nm). Data analysis was
conducted using DNA analysis program MODFIT (verity software
house, Inc. Po Box 247, Topsham, ME 04,086 USA, version: 2.0
powers Mac with 131,072 KB Registration No: 42,000,960,827 Date
made: 16-Sep, 1996). This software measured cell cycle analysis,
which was calculated the coefficient of variation (CV) around the
G0/G1 peak and the percentage of cells in each phase (G0/G1, S,
and G2/M) of the DNA cell cycle for each sample. The analysis of
apoptotic cell death was performed by measuring DNA content.
3.2. Structural characterizations of IVASBs
3.2.1. Elemental analysis and mass spectrometry
The new ionic Schiff bases exhibited acceptable microanalyti-
cal data that in full consistent with the proposed structural for-
mula for them (see the Experimental part). Moreover, the positive-
mode electrospray ionization mass spectrometry (ESI-MS) of these
Schiff bases exhibited main peaks at m/z 315.3, 582.0, and 646.1
assigned to the singly-charged cation [M - Cl–] of IVASB1, IVASB2
and IVASB3, respectively. In addition, IVASB2 and IVASB3 display
another peak at m/z 273.2 and 305.3, respectively, assigned to the
doubly-charged cation [M - 2Cl–]. These findings offer further sup-
port for the authenticity of the suggested structures.
2.8.1. Cell cycle analysis
After washing the cells with PBS, EAC cells were fixed by ice-
cold absolute alcohol and then centrifuged and excessive alcohol
was removed. The cells were re-suspended in citrate buffer and
then were stained with PI. The DNA content was measured us-
ing an Accuri C6 flow cytometer (Becton Dickinson, Sunnyvale, CA,
USA).
2.8.2. Flowcytometric evaluation of cyclin dependent-kinase 1(CDK1)
in Ehrlich cells
3.2.2. FTIR
Usually, the IR analysis provides a shred of preliminary evidence
for the success of the preparation of a new compound [49]. For this
purpose, the comprehensive comparison between the FTIR spectra
of SIL1 (2a), 2-AT, and, IVASB1 (Fig. 1A) revealed the disappearance
of the stretching vibration bands distinctive for the amine group of
AT (centered at 3412 and 3295 cm–1 in the 2-AT spectrum) and
the aldehydic carbonyl group of SIL1 (centered at 1674 cm–1 in the
SIL1 spectrum) in the spectrum of IVASB1. Besides, new stretches
have emerged in the IVASB1 spectrum at 3411, 1614, and 1279
To prepare the cells, they were fixed in
a solution of 1%
formaldehyde for 15 min at 4 °C, then washed with phosphate-
buffered saline (PBS) containing 1% fetal calf serum (FCS, Sigma,
St. Lois, USA), after that the cells were fixed in 80% ethanol with
vortexing to prevent the aggregation of cells. The fixed cells were
kept in 80% ethanol at –20 °C until use. Before the assay, the cells
were washed with wash buffer (WB) at 400 g for 10 min, so as to
remove the ethanol, and then incubated with WB containing Triton
X-100 for 5 min at 4 °C in order to perforate the cells.
cm–1 characteristic for the phenolic O H, azomethine, and Aryl–O
–
The expression of CDK1 was performed using the FITC conju-
gates of the antibodies specific to CDK1 (Pharmingen, USA). The
percent of cells expressed CDK1 was assessed using fluorescence
threshold by FACS Caliber Flow Cytometer (Becton Dickinson, Sun-
nyvale, CA, USA).
groups, respectively, of salicylidene Schiff base segment. Further-
more, the main peaks distinctive for the imidazolium moiety can
be noticed 1450 and 852 cm–1 in the IVASB1 spectrum [50].
In the same context, comparison of the spectra of ionic bis-
(salicylideneimino)-thiadiazoles (IVASB2 and IVASB3) with those of
SIL1,2 (2a,b), and 2,5-diamino-1,3,4-thiadiazole (DAT) (Fig. 1B) ev-
ident missing of the stretches of amine group of DAT (3490 and
3420 cm–1 in DAT spectrum) and the aldehydic carbonyl groups
2.9. Statistical analysis
of SIL1,2 (1680
7 cm–1 in the SIL1,2 spectra) in the spectrum of
All the data were expressed as a mean
standard error of the
IVASB2,3. Instead, a new set of vibration bands have emerged in
the spectra IVASB2,3 round 3425, 1610, 1289, 1423, and 845 cm–1
mean (SEM) to compare between EAC control and treated groups.
The statistical significance was evaluated by one-way analysis of
variance using SPSS, 16.0 software (SPSS Corp, Chicago, IL), and in-
dividual comparisons were done by Duncan’s multiple range test.
Values were considered statistically significant when p < 0.05, high
statistically significant when p < 0.01 and very high statistically
significant when p < 0.001.
–
assignable for the vibration of phenolic O H, azomethine (H–C=N),
Aryl–O groups, and imidazolium or pyridinium ring, respectively, of
ionic salicylidene Schiff base segments [51].
These findings confirm the successful formation of IVASBs by
Schiff base condensation of the amine group(s) of 2-AT or DAT
with carbonyl group of SILs.
4

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Scheme 1. Stepwise protocol used for preparation of salicylaldehyde ionic liquids (SILs, 2a,b), 2,5-diaminothiadiazole, and ionic vanillyl azole Schiff bases (IVASBs).
Fig. 1. Collective FTIR spectra of 2-AT, DAT, SILs, and ionic vanillyl azole Schiff bases (IVASBs) for comparison of their respective spectral signatures, particularly, the azome-
–
thine (H C=N) and aryl–O vibration bands.
3.2.3. NMR spectroscopy
spectrum of IVASB2 (Fig. 2) at the chemical shift ranges 11.13 –
The common spectral features of 1H NMR spectra for new
Schiff bases (IVASBs) are the disappearance of the low-field sin-
glets distinctive for the aldehydic and amine protons of native SILs
(~10.30 ppm) and AT or DAT (~6.20 ppm), respectively. Instead,
new singlet peaks have observed around 8.48 ppm assigned to
10.0, 7.62 – 6.91, and 5.51 – 2.95 ppm assignable to the resonance
–
–
of imidazolium N H, phenolic O H, aromatic Ar-H, methylene CH2,
and methyl CH3 protons of the imidazolium salicylidene fragment
[52].
13C NMR spectra of IVASBs offers additional evidences empha-
sizing their successful preparation as evident from notice of two
down-field peaks around δ 161 ppm and 147 ppm corresponding
to carbon atoms bonded to azomethine nitrogen and phenolic oxy-
gen, respectively.
–
the azomethinic proton (H C=N), confirming the success of Schiff
base condensation between the amine group(s) of 2-AT or DAT and
aldehydic group of SILs to form IVASBs. Furthermore, compared to
the spectrum of DAT, three new sets signals have emerged in the
5

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Fig. 2. 1H NMR spectra of 2,5-diamino-1,3,4-thiadiazole (DAT) and IVASB2 in d6-DMSO at 300 MHz.
3.3. Docking study
IVASB1 (−3.52 kcal/mol) and IVASB3 (−3.43 kcal/mol) makes them
the most potent anti-breast cancer candidates.
CDKs play a vital role in the regulation of cell cycle and prolifer-
ation and their functions qualify them to be convenient targets for
anticancer agents [53,54]. CDK1 in combination with cyclin B pro-
motes entry into mitosis and regulates several processes of mitotic
progression [53,55]. An increase in the expression of CDK1 was ob-
served in human cancer [56], therefore CDK1 inhibition represents
a great benefit in cancer treatment. The molecular docking study
was applied to investigate the binding modes and affinities of the
newly synthesized Schiff bases (IVASBs) with the active sites of
CDK1. The calculated docking scores for IVASBs and the interacted
amino acid residues of CDK1 with new ionic Schiff bases were tab-
ulated in Table S1 (ESI†). As evident in Table S1, all Schiff bases
exhibit good binding affinities toward CDK1 with binding energy
(BE) values ranged from −2.30 to −3.52 kcal/mol, indicating that
the CDK1 inhibition is a reasonable mechanism for interpreting the
anticancer effect of these compounds.
3.4. Cytotoxicity experiment
The MTT assay was applied to in vitro assess the cytotoxic
effects of new ionic Schiff bases (IVASBs) toward human breast
carcinoma cell lines (MCF-7) in comparison to a clinical drug 5-
Fluorouracil (5-Fu). The MCF-7 cell viability and IC50 values (Fig. 4)
were used as biomarkers for cytotoxic performance. In general, the
ionic liquids-supported Schiff bases (IVASB1 and IVASB3) are more
cytotoxic than 5-Fu, whereas, IVASB2 is less than the clinical drug
as revealed from the survival MCF-7 cells after each treatment
(Fig. 4A). Just to name a few, the surviving MCF-7 cells were found
to be 42.19, 83.53, 26.96, and 46.53 remediation of these cells with
16.60 μg/mL of IVASB1, IVASB2, IVASB3, and 5-Fu, respectively. Fur-
thermore, the IC50 values of IVASB1, IVASB2, IVASB3, and 5-Fu were
13.5 1.14, 102 6.3, 3.73 0.2, and 28.12 2.25 μg/mL, respec-
tively (Fig. 4B). According to our results, IVASB1 and IVASB3 are
the most cytotoxic against MCF-7 human breast cancer cell lines
and could offer promising anti- breast cancer candidates. Our find-
ings agreed with the molecular docking results.
As shown in Figs. 3 (A, D) and S3 (ESI†), IVASB1 exhibited
an interesting binding model with CDK1 where it has interacted
through two types of bindings: (i) Three H-bonds (HB), between
nitrogen atoms of azomethine and thiazole moieties hydroxyl
groups of amino acid residues (AAR) (Thr25, Arg47, and Tyr26).
(ii) One hydrophobic binding (HPOB), between the phenyl ring and
carbon chain of IVASB1 and Glu32, respectively. Also, IVASB2 has
bound to CDK1 by H-bonding and HPOB but with different bind-
ing models as evident in Figs. 3 (B, E) and S4 (ESI†) with lower
BE values as compared to IVASB1. Interestingly, the IVASB3–CDK1
binding involves three types of interactions Figs. 3 (C, F) and S5
(ESI†); HB, between N-atom of DAT and OH group of Gly24; π-
stacking, between phenyl rings of DAT and Tyr26; HPOB, between
the phenyl ring and carbon chain of IVASB3 and Glu32, Thr25,
Lys138, respectively. In addition to the wonderful IVASB1–CDK1
and IVASB3–CDK1 binding models, the binding energy scores of
3.5. Influence of tested compounds on hematology of
Ehrlich-tumorized mice
The data in Table 1 showed that treatment with thiazole re-
sulted in significant EAC-induced alteration of hematological pa-
rameters compared to untreated Ehrlich-tumorized mice. White
blood cells (WBCs) level was significantly reduced by the admin-
istration of IVASB1, IVASB2, and IVASB3 (P ≤ 0.001, P ≤ 0.01, and
P ≤ 0.01, respectively) compared to normal control group. Red
blood cells count (RBCs) was increased in IVASB1-, IVASB2-, and
IVASB3-treated mice by (8.7, 12.3, and 20.5%, respectively) com-
6

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Fig. 3. (A–C) 2D molecular docking modeling of IVASB1, IVASB2, IVASB3 (green stick molecule) with amino acid residues (AAR) of CDK1; showing hydrogen bonding (HB)
(dotted lines), hydrophobic binding (HPOB), and π-stacking interactions (curved lines). (D,E) 3D model for the electrostatic map and interaction of IVASB1, IVASB2, and
IVASB3 with the CDK1 binding sites (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
Fig. 4. (A) The cytotoxicity performance of new ionic Schiff bases (IVASBs) toward human breast carcinoma cell lines (MCF-7) in comparison to a clinical drug 5-Fluorouracil
(5-Fu). (B) The IC50 values of the tested compounds against MCF-7.
Table 1
†
Effect of the tested compounds on hematological parameters
.
Parameter
Normal control M SE
EAC control M SE
EAC + SB1
M
SE
EAC + SB2
M
SE
EAC + SB3
M
SE
EAC +5-Fu M SE
WBCs
RBCs
HGB
HCT
10.3∗∗∗ 0.2
8.78∗∗ 0.15
11.8∗∗∗ 0.2
40.3∗∗∗ 0.4
46.9∗∗ 0.9
14.9∗∗ 0.3
32.02∗∗∗ 0.3
474∗∗ 22
13.2
7.6
0.38
0.2
0.4
1.9
7.73∗∗∗ 0.16
8.33∗ 0.03
9.65∗ 0.2
35.1∗∗ 0.7
46.9∗∗ 0.7
16.2∗∗∗ 0.2
29.3∗ 0.3
11.1∗2∗∗∗ 0.11
12.8∗∗∗ 0.3
46.3∗∗∗ 0.8
50∗∗∗ 0.7
15.8∗∗ 0.2
31.7∗∗∗ 0.5
473.1∗∗ 1.5
11.2∗1∗∗∗∗ 0.2
11.38∗∗ 0.5
8.64∗∗ 0.2
9.9∗ 0.2
35.7∗∗ 1.04
52.2∗∗∗ 0.7
15.8∗∗ 0.4
33∗∗∗ 0.3
8.67
0.3
9.56
0.02
8.6
12.8∗∗∗ 0.2
43.9∗∗∗ 0.6
45.9∗ 0.8
14.6∗ 0.3
32.6∗∗∗ 0.9
324.6∗ 5.8
27.9
42.7
13.2
27.5
MCV
MCH
MHC
PLT
0.7
0.5
0.7
381 22.4
541.2∗∗∗ 1.07
451.8∗ 19
†
∗
Values are represented as mean SE (n = 6), Where = Significant difference between test groups and Ehrlich control group,∗P ≤ 0.05,
∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. RBCs: red blood cell count; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; PLT: platelet count;
WBC: white blood cell count. SB1 = IVASB1, SB2 = IVASB2, and SB3 = IVASB3.
7

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Table 2
†
The effect of the tested compounds on biochemical parameters
.
Groups
ALT (U/L)
AST (U/L)
267.25
185.15∗∗∗ 2.73
170.25∗∗∗ 2.54
154.05∗∗∗ 2.12
149.91∗∗∗ 4.42
186.23∗∗∗ 4.19
Albumin (g/L)
2.14 0.005
2.396∗∗∗ 0.004
2.37∗∗∗ 0.023
2.34∗∗∗ 0.027
2.301∗∗∗ 0.007
2.46∗∗∗ 0.012
TBil (mg/dL)
Urea (mg/dL)
40.5 0.76
36.5∗∗ 0.43
35.16∗ 1.92
29.33∗∗∗ 0.88
35∗∗∗ 0.816
29.33∗∗∗ 0.88
Creatinine (mg/dL)
0.59 0.014
0.48∗∗ 0.03
0.45∗∗∗ 0.016
0.37∗∗∗ 0.008
0.53∗ 0.025
0.35∗∗∗ 0.009
EAC control
EAC + SB1
EAC + SB2
EAC + SB3
EAC +5-FU
Control
80.61
1.34
3.89
0.56 0.012
0.478∗∗ 0.015
47.86∗∗∗ 3.69
59.32∗∗∗ 1.88
59.93∗∗∗ 1.02
60.84∗∗∗ 0.199
56.98∗∗∗ 1.68
0.50
0.03
0.503∗∗ 0.11
0.49∗ 0.02
0.44∗∗∗ 0.017
†
∗
Values are represented as mean SE (n
group,∗P ≤ 0.05, ∗∗P ≤ 0.01 and ∗∗∗P ≤ 0.001.
=
6), Where
=
Significant difference between Test groups and EAC control
Table 3
The effect of tested compounds on the cell cycle of Ehrlich cells†.
Phases
EAC control M SE
EAC + SB1
M
SE
EAC + SB2
M
SE
EAC + SB3
M
SE
EAC +5-FU M SE
Sub G1
G0/1
S
8.5
1.4
0.8
0.7
0.6
70.5∗∗∗ 1.3
13.2∗∗∗ 1.2
11.6∗∗ 1.8
1.9∗∗ 0.3
41.8∗∗∗ 1.2
40.3∗ 1.4
15.1∗∗∗ 0.1
86.03∗∗∗ 2.7
10.1∗∗∗ 1.4
0.5∗∗∗ 0.3
37.2∗∗∗ 2.2
38.7∗ 1.9
21.03∗ 3.9
46.9
34.8
5.1
G2/M
6.2
0.5
0.03∗∗∗ 0.03
5.5
1.1
†
∗
Values are represented as mean SE (n = 6), Where = Significant difference between test groups and Ehrlich
control group,∗P ≤ 0.05.
∗∗
∗∗∗
P ≤ 0.01.
P ≤ 0.001.
pared to Ehrlich control group, while hemoglobin (HGB) concentra-
which confirmed that IVASB3 arrested Ehrlich cells at G0/G1 check
point. In the same way, the percent of cells undergoing apopto-
sis in Ehrlich-tumerized mice treated with compound IVASB2 in-
creased by 4.92-fold compared to control group (P ≤ 0.001) and
the percent of cells in S phase significantly decreased by 2.30-fold
compared to control group (P ≤ 0.001). Likewise, The administra-
tion of the compound IVASB1 to tumorized mice led to a signifi-
cant increase 8.29-fold in the percent of cells undergoing apoptosis
compared to control group (P ≤ 0.001) and a significant decrease
3-fold in the percent of cells in S phase compared to tumor con-
trol group (P ≤ 0.01). Moreover, It was observed that the adminis-
tration of IVASB1 and IVASB3 significantly decreases the percent of
cells undergoing mitosis by 2.68 and 170-fold (P ≤ 0.01), respec-
tively compared to Ehrlich control.
tion was increased by (10.8, 32.8, and 32.8%, respectively) as com-
pared to Ehrlich control group. Similarly, mean corpuscular vol-
ume (MCV) of red blood cells (RBCs) was significantly increased
by the administration of IVASB1 (8.9%), IVASB2 (14.6%), and IVASB3
(6.9%) compared to untreated Ehrlich control group. Only IVASB1
and IVASB2 can be induced significant elevation of platelet count
(PLT) by (29.6% and 19.4%, respectively) as compared to untreated
Ehrlich control group.
3.6. Effect of tested compounds on biochemical parameters of
Ehrlich-tumorized mice
The data in Table
2 showed that treatment with IVASBs
Based on the analysis of cell cycle by flowcytometry in
the present study, all three test compounds increased the per-
centage of cells in sub G1 phase which indicates that these
compounds exert their anticancer effect by inducing apopto-
sis. Previous report revealed that p-chlorophenethylamino and p-
methoxyphenethylamino thiazole derivatives induced apoptosis in
human U-937 leukemia cells in a time and concentration depen-
dent manner [57]. In addition, the cell cycle analysis confirmed the
G2/M-phase arrest and induction of apoptosis by thiazole deriva-
tives in HeLa cells [58].
caused significant Ehrlich-induced alteration of biochemical pa-
rameters compared to untreated Ehrlich control group. Results of
the present study demonstrated that the liver activities of AST
and ALT were increased in Ehrlich-tumorized mice when compared
to normal control group. It was found that Ehrlich led to some
liver dysfunction. The level of liver enzymes increased in serum
of Ehrlich-tumorized mice indicating general toxicity found that
Ehrlich mice exhibited raised activities of liver enzymes such as
AST, ALT, due to hepatocellular damages. There was a significant
decrease in serum ALT and AST of Ehrlich tumorized mice received
compounds (IVASBs) (P ≤ 0.001 at all).
3.7. Flowcytometric analysis of Ehrlich solid tumor cell cycle phase
distribution
3.8. Effect of tested compounds on the expression of CDK-1 in Ehrlich
cells
One of the most important strategies for cancer chemotherapy
to give an insight regarding the performance and mode of action
for a new chemotherapeutic agent is the flowcytometric analysis.
The cell cycle analysis of control and different treated mice groups
were performed and the obtained results were collected in Fig. 5
and Table 3. Noteworthy, treating of tumorized mice with IVASB3
has significantly reduced the percent of cells in the G1 phase by
46.9% (4.64-times lower than that Ehrlich control group (10.1%))
(P ≤ 0.001). Furthermore, the percent of cells undergoing apop-
tosis in the tumorized mice, IVASB3-remediated, has increased by
~10-fold compared to the control group (P ≤ 0.001). In addition,
the compound IVASB3 significantly reduced the percent of cells
in S phase by 69.6-fold compared to control group (P ≤ 0.001)
As shown in Fig. 6, the expression of CDK1 in Ehrlich cells
collected from mice which received the compound IVASB1 sig-
nificantly decreased by 41.27% when compared to tumor control
group (P ≤ 0.001) and the administration of compound IVASB2 led
to a decrease in the expression of CDK1 by 53.31% compared to
Ehrlich control group (P ≤ 0.001), while the compound IVASB3 was
the most effective one causing a reduction of CDK1 expression by
78.57% compared to tumor control group (P ≤ 0.001).
Based on flow-cytometric analysis in our study, all three tested
compounds were able to inhibit the expression of CDK1 by the per-
cent of change ranging from 41.27% to 78.57% when compared to
tumor control group and the most potent inhibitory effect was ob-
served at IVASB3. Moreover, IVASB1 and IVASB3 caused cell cycle
8

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Fig. 5. Flow cytometric histogram showing; (A) Ehrlich control and the effect of (B) 5-Fu, (C) IVASB1, (D) IVASB2, and (E) IVASB3 on the cell cycle in Ehrlich tumorized mice.
Fig. 6. Flow cytometric histogram showing; (A) Ehrlich control and the effect of (B) 5 –Fu, (C) IVASB1, (D) IVASB2, and (E) IVASB3 on the expression of CDK1 in Ehrlich
tumorized mice. (F) Effect of the tested compounds on CDK1 expression in Ehrlich cells.
arrest at G2/M-phase with respect to Ehrlich control which sug-
gested that the two test compounds inhibit CDK1 expression.
46.67% respectively, compared to Ehrlich control group (P ≤ 0.01).
In the same way, the administration of IVASB2 and IVASB3 to tu-
morized mice decreased the concentration of VEGF in serum by
34.99% and 28.49% respectively, compared to Ehrlich control group
(P ≤ 0.001).
3.9. Effect of tested compounds on PARP and VEGF
PARP is considered the principal regulator for repairing dam-
aged DNA [59], so that the inhibition of PARP in tumor cells de-
As shown in Table 4, the concentration of PARP in serum of tu-
morized mice received IVASB2 and IVASB3 decreased by 60% and
9

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
Table 4
Effect of the tested compounds on PARP and VEGF in Ehrlich cells†.
Biomarkers
EAC control M SE
Control M SE
EAC + SB1
M
SE
EAC + SB2
M
SE
EAC + SB3
M
SE
EAC +5-Fu M SE
PARP (ng/mg)
VEGF (ng/mg)
1.5
0.2
2.3
0.5∗∗∗ 0.02
71.3∗∗∗ 1.2
1.07∗ 0.01
74.8∗∗ 2.5
0.6∗∗ 0.2
55∗∗∗ 1.5
0.8∗∗ 0.02
60.5∗∗∗ 0.9
1.16∗ 0.02
58.5∗∗∗ 1.1
84.6
†
∗
Values are represented as mean SE (n = 6), Where = Significant difference between test groups and Ehrlich control group,∗P ≤ 0.05.
∗∗
∗∗∗
P ≤ 0.01.
P ≤ 0.001.
Fig. 7. Histopathological examination of Ehrlich solid tumor. Representative sections were obtained from (A) untreated Ehrlich control (Tumor mass showing wide prolifera-
tion of Ehrlich neoplastic cells of high mitotic figures (arrows), (B–D) Tumor mass of tumorized mice treated with IVASB1, IVASB2, and IVASB3, respectively, showing increase
the degenerative and necrotic lesions with decrease the neoplastic cells (arrow) (E) Tumor mass of tumorized mice treated with 5-Fu showing increase the necrotic lesions
(arrow) with decrease the neoplastic cells (arrowhead) Sections were stained with HE dyes (scale bar = 100 mM).
creases chemotherapy resistance and enhances treatment response
[60]. In our investigation, all three test compounds inhibit PARP
activity and the most potent inhibitor was found IVASB2. Further-
more, G0/G1 cell cycle arrest caused by IVASB1 and IVASB3 in-
dicates to the accumulation of DNA double-strand breaks, hence
apoptosis induction.
4. Conclusion
In the present work, three novel hybrid thiazole-vanillyl and
thiadiazole-vanillyl Schiff bases bearing imidazolium or lutidinium
ionic liquids terminals (IVASBs) have successfully synthesized
and structurally characterized based on their respective elemen-
tal and spectral (FTIR, NMR, and MS) analyses. They are coded
VEGFs stimulate cancerous cells angiogenesis which means
promoting the formation of new blood vessels to supply tu-
mor mass with nutrients and oxygen. Therefore, the inhibition of
VEGF is considered an efficient strategy in cancer treatment [61].
Based on our results, the test compounds significantly decreased
the concentration of VEGF in the serum of Ehrlich-tumorized
mice compared to Ehrlich control group. Several previous studies
proved that thiazole Schiff base derivatives act as VEGFR-1 and 2
inhibitors [62].
as
3-(thiazol-2-ylimino)methyl)vanillyl-2-methylimidazolium
chloride (IVASB1), 2,5-Bis-(5-(2-methylimidazolium chloride)−3-
methoxysalicylideneimino)−1,3,4-thiadiazole (IVASB2), and 2,5-Bis-
(5-(2,4-lutidinium chloride)−3-methoxysalicylidene- imino)−1,3,4-
thiadiazole (IVASB3). The in vitro anti-breast cancer study re-
vealed that IVASB1 (IC50 13.5
1.14 μg/mL) and IVASB3 (IC50
3.73 0.2 μg/mL) exhibited the highest ability to inhibit the pro-
liferation of MCF-7 cells. These findings agreed with the molecular
docking results. Flow-cytometric analysis showed that IVASBs
significantly increased the percent of Ehrlich tumor cells undergo-
ing sub G1 phase and decreased the percent of cells in S phase,
where IVASB3 was the most effective. In addition, the adminis-
tration of IVASB1, IVASB2, and IVASB3 to tumorized mice caused
a significant reduction in CDK1 expression by 41.27%, 53.31% and
78.57%, respectively compared to Ehrlich control group. Moreover,
the concentrations of PARP and VEGF significantly decreased in
serum of tumorized mice remediated by these compounds. Based
on our results, IVASB3 could act as a starting point for promis-
ing anti-breast cancer candidate, warrant further research and
development.
3.10. Histopathological examination
The inoculation of Ehrlich tumor cells into the control mice in-
duced intramuscular tumors at the point of inoculation. These tu-
mors were prominent and revealed fast growth with mixed in-
flammatory reaction predominantly lymphocytes, white conglom-
erated mass and sometimes infiltrated into the muscle fibers of
the animals. Examination of untreated tumorized mice presented
that Ehrlich cells penetrated and mostly replaced the subcuta-
neous tissue with necrosis of the remaining skeletal muscles. Mice
treated with 5-Fu revealed minimal tumor cell infiltrations, exten-
sive necrosis, and apoptosis at the edge of the tumor with de-
structed blood vessels and depletion. Mice treated with IVASB1,
IVASB2, and IVASB3 (3.31 μg/kg, 55 mg/Kg and 450 mg/Kg, re-
spectively) showed most necrosis with a decrease of the neoplastic
cells (Fig. 7 (B–D)). There was a marked reduction in tumor size af-
ter treatment with the test compounds, the tumor was found to be
intermittent and appeared growing slow and split. This indicates a
partial prevention of the effects of EAC cells by test compounds.
Declaration of Competing Interest
none
CRediT authorship contribution statement
Waleed M. Serag: Supervision, Methodology, Data curation,
Software, Validation, Writing – original draft, Writing – review
10

W.M. Serag, F. Zahran, Y.M. Abdelghany et al.
Journal of Molecular Structure 1245 (2021) 131041
& editing. Faten Zahran: Supervision, Data curation, Validation,
Visualization, Writing – original draft. Yasmin M. Abdelghany:
agents; Base-acid-base type architectures, J. Mol. Struct. 1200 (2020) 127126,
doi:10.1016/j.molstruc.2019.127126.
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Methodology, Data curation, Software, Visualization, Writing
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sion, Methodology, Data curation, Validation, Writing – original
draft, Writing – review & editing. Moustafa S. Abdelhamid: Super-
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