Anti-cancer activity of two novel heterocyclic compounds through modulation of VEGFR and miR-122 in mice bearing Ehrlich ascites carcinoma

Article abstract of DOI:10.1016/j.ejphar.2020.173747

Metastasis in breast cancer is a leading cause of mortality among women in many countries. This study investigated the anti-cancer role of benzoimidazoquinazoline and benzimidazotriazin; two novel compounds that were designed, synthesized, structurally elucidated, and biologically evaluated as potent anti-angiogenic agents that act through inhibition of vascular endothelial growth factor receptor-2 (VEGFR2). Breast cancer was induced by inoculation of Ehrlich Ascites Carcinoma (EAC) cells. Seventy swiss albino mice were randomly divided into 7 groups, 10 animals each: (1) normal, (2) control EAC group, (3) cisplatin treated group, (4&5) benzoimidazoquinazoline treated (5 mg/kg and 10 mg/kg), (6&7) benzimidazotriazin treated (5 mg/kg and 10 mg/kg). The expression of miR-122 was assessed in the tumor tissue by quantitative PCR, and the VEGF level was determined in serum by ELISA. VEGFR2 and cluster of differentiation (CD)34 were assessed by immunohistochemistry. Serum ALT, AST, creatinine, and urea were measured. Treatment with benzoimidazoquinazoline and benzimidazotriazin decreased tumor weight and serum levels of VEGF, and down-regulated expression of VEGFR2 and CD34 in the tumor tissue. miR-122 was upregulated, particularly in the benzimidazotriazin (10 mg/kg) group. Relative to cisplatin, the novel compounds were less toxic to kidneys. Benzoimidazoquinazoline and benzimidazotriazin are promising anti-cancer agents that act through inhibition of angiogenesis and thus provide a new strategy for advancement of chemotherapy.

Full text of DOI:10.1016/j.ejphar.2020.173747

European Journal of Pharmacology xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Anti-cancer activity of two novel heterocyclic compounds through
modulation of VEGFR and miR-122 in mice bearing Ehrlich
ascites carcinoma
Reem M. Hazema, Anhar A. Mohamedb, Nagat Gharebc, Eman T. Mehannab, Noha M. Mesbahb,
,e,*
Dina M. Abo-Elmattyb, Mohamed Saleh Elgawishd
a Department of Pharmacology, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt
b Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt
d Department of Medicinal Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt
e Center for Molecular Spectroscopy and Dynamic, Institute for Basic Science, Korea University, Seoul, 02841, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Metastasis in breast cancer is a leading cause of mortality among women in many countries. This study inves-
tigated the anti-cancer role of benzoimidazoquinazoline and benzimidazotriazin; two novel compounds that
were designed, synthesized, structurally elucidated, and biologically evaluated as potent anti-angiogenic agents
that act through inhibition of vascular endothelial growth factor receptor-2 (VEGFR2). Breast cancer was
induced by inoculation of Ehrlich Ascites Carcinoma (EAC) cells. Seventy swiss albino mice were randomly
divided into 7 groups, 10 animals each: (1) normal, (2) control EAC group, (3) cisplatin treated group, (4&5)
benzoimidazoquinazoline treated (5 mg/kg and 10 mg/kg), (6&7) benzimidazotriazin treated (5 mg/kg and 10
mg/kg). The expression of miR-122 was assessed in the tumor tissue by quantitative PCR, and the VEGF level was
determined in serum by ELISA. VEGFR2 and cluster of differentiation (CD)34 were assessed by immunohisto-
chemistry. Serum ALT, AST, creatinine, and urea were measured. Treatment with benzoimidazoquinazoline and
benzimidazotriazin decreased tumor weight and serum levels of VEGF, and down-regulated expression of
VEGFR2 and CD34 in the tumor tissue. miR-122 was upregulated, particularly in the benzimidazotriazin (10 mg/
kg) group. Relative to cisplatin, the novel compounds were less toxic to kidneys. Benzoimidazoquinazoline and
benzimidazotriazin are promising anti-cancer agents that act through inhibition of angiogenesis and thus provide
a new strategy for advancement of chemotherapy.
Breast cancer
Angiogenesis
VEGF
VEGFR-2
miR-122
Benzoimidazoquinazoline
Benzimidazotriazin
1. Introduction
occur through the proliferation, migration, and maturation of nearby
blood or lymph vessel endothelial cells (Lala et al., 2018). Angiogenesis
Breast cancer is the most widespread cancer among women around
the world, affecting more than 2 million new cases and resulting in
600,000 deaths in 2018 (Bray et al., 2018). Despite treatment with
surgery, radiotherapy or chemotherapeutics, 5–11% of patients exist
experience metastasis (Tao et al., 2016), and a large number of early
breast cancer patients have a micro-metastatic disease resistant to sys-
temic treatment. These patients eventually relapse. New treatment
strategies for breast cancer are required (Colozza et al., 2007).
is a basic biological process that is essential for development, repro-
duction, and wound repair. Angiogenesis is a key feature and distinctive
marker of cancer (Otrock et al., 2007). Since tumors cannot grow to 1–2
mm without an adequate blood supply, angiogenesis is fundamental for
uncontrolled growth of tumors to supply adequate oxygen and nutrients
(Lou et al., 2017). Similarly, lymphangiogenesis is involved in many
cancers, including breast cancer. The primary sites of metastasis in
breast cancer are often the regional lymph nodes, where lymphangio-
genesis can facilitate the migration of tumor cells to these sites (Ran
et al., 2010). In the last two decades, vascular endothelial growth factors
Angiogenesis and lymphangiogenesis stimulate the formation of new
blood and lymph vessels from pre-existing vasculature. These processes
* Corresponding author. Department of Medicinal Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt.,
E-mail addresses: mohamed_elgawish@pharm.suez.edu.eg, mohelgawish@gmail.com (M.S. Elgawish).
https://doi.org/10.1016/j.ejphar.2020.173747
Received 10 July 2020; Received in revised form 2 November 2020; Accepted 17 November 2020
Available online 21 November 2020
0014-2999/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Reem M. Hazem, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2020.173747

R.M. Hazem et al.
European Journal of Pharmacology xxx (xxxx) xxx
(VEGFs) and their receptors (VEGFRs) have been identified as the key
drivers of lymphangiogenesis and angiogenesis in the vascular system.
The VEGF signaling pathway is a crucial regulator in many tissues and
plays a vital role in the pathogenesis of cancer, cardiovascular and
intraocular neovascular diseases (Shibuya, 2011).
breast, ovarian, bladder and colon cancers (Svoronos et al., 2016; Kian
et al., 2018). miRNAs can be used as biological markers for the diag-
nosis, prognosis and prediction of cancer. The diagnostic and prog-
nostic role of miR-122 was established in various tumors including
liver, breast, and colorectal cancers (Coulouarn et al., 2009; Ahsani
et al., 2017; Maierthaler et al., 2017). In breast cancer, miR-122 was
suggested to act as a tumor suppressor and to inhibit tumorigenesis
through targeting insulin-like growth factor 1 receptor (IGF1R) and
regulating PI3K/Akt/mTOR/p70S6K pathway (Wang et al., 2012).
Since VEGF/VEGFR signaling pathway and miRNAs are valuable
targets for cancer management, developing novel molecules with dual
activity is important. Inhibition of VEGFR occurs through two mecha-
nisms. The first mechanism is to block ligand binding to the extracellular
domain with monoclonal antibodies (e.g. bevacizumab). The other
mechanism relies on using small-molecule receptor tyrosine kinase (TK)
inhibitors that function at the intracellular domain. Gefitinib, Sorafenib,
pazopanib, and Sunitinib are 4-anilinoquinazoline derivatives which
competitively bind to the adenosine triphosphate (ATP) binding pocket
of intracellular VEGFR domain (Fig. 1), thereby inhibiting downstream
signaling essential for tumor survival and proliferation (Musumeci et al.,
2012; Peng et al., 2017). Despite high response rate of patients to these
agents, drug resistance limits the therapeutic benefits of these drugs.
Resistance develops because of secondary mutations. Therefore, devel-
opment of second-generation VEGFR tyrosine kinase inhibitors through
the discovery of a new scaffold can be beneficial for gefitinib-resisting
patients.
The VEGFRs play a vital role in vasculogenesis and angiogenesis in
embryonic development, wound repair, menstruation, and pregnancy.
VEGFR-2, a member of the VEGFR family, helps in the generation of new
blood vessels from existing tumor mass. Disruption of VEGFR-2
signaling results in inhibition of angiogenesis, thereby blocking oxy-
gen and nutrients from reaching tumor cells and decreasing clearance of
catabolic products (Ferrara et al., 2003). Inhibition of the VEGF/VEGFR
signaling cascade has emerged as an attractive therapeutic tactic for
inhibition of tumor angiogenesis and proliferation (Ferrara et al., 2003;
Olsson et al., 2006). CD34 is a specific biomarker of cellular vascular
endothelium. CD34 is especially sensitive to tumor angiogenesis, as it
can clearly identify the condition of neovascularization during tumor
growth (Zorgetto et al., 2013). In this manner, CD34 and VEGF are two
significant markers of tumor angiogenesis and the relationship between
the expression of CD34 and VEGF and pathological attributes of patients
with malignancy have been reported (Chen et al., 2015). Many mole-
cules have been implicated in angiogenesis regulation. Among them,
VEGF is the most pivotal angiogenic factor and their expression is
regulated by many factors including hypoxia-inducible factors (HIFs),
chemokines, and microRNAs (Hua et al., 2006; Olsson et al., 2006).
MicroRNAs (miRNAs) are small non-coding RNAs ~22 nucleotides
in length (Hu et al., 2017). MiRNAs play a major role in the regulation
of expression of 30–60% of human genes and are considered important
modulators of cell differentiation, proliferation, cell-cycle progression,
epithelial-mesenchymal transition, angiogenesis, stem cell renewal,
apoptosis and cell migration, invasion, and metastasis (Hamam et al.,
2017). Imbalance in miRNA expression is associated with cancer,
where they can act as promoters or suppressors of tumorigenesis
(Hamam et al., 2017; Song and Ajani, 2013). Many miRNAs have been
reported to be downregulated in malignant tumors including lung,
Modulations to the central core pyridine ring is a feature of the
medicinal chemistry strategy, and serve to look for potency, structure-
activity relationship (SAR), and reduce log p of TK inhibitors. A range
of heteroatoms have been reported to provide alternatives to the pyri-
dine and/or pyrimidine ring of Sorafenib and Pazopanib. Amongst these
rings, quinazoline and naphtamide derivatives, furo[2,3-d] pyrimidines,
pyridinyltriazines, and pyrimidinylindazoles have been reported as
VGEFR-2 inhibitors (Holmes et al., 2007). In this study, diverse structure
modifications in hit compounds were assessed to determine potency
Fig. 1. Chemical structures of US FDA approved drugs (VEGFR inhibitors) for cancer treatment and the designed compounds.
2

R.M. Hazem et al.
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against VEGFR. The effect of two novel compounds, benzoimidazoqui-
nazoline and benzimidazotriazin on breast cancer induced in mice was
studied. Their effect on the expression of miR-122, CD34, VEGFR, and
VEGF in the tumor tissues was measured.
disulfide in KOH producing a cyclized benzo[4,5]imidazo[1,2-c]
quinazoline-6-thiol. The free thiol was reacted with chloroacetyl-
chloride under alkaline conditions to release S-(benzo[4,5]imidazo[1,2-
c]quinazolin-6-yl) 2-chloroethanethioate in reasonable yield. Finally,
the chloroethanethioate derivative was condensed in alkaline media
with dichloroaniline producing the target compound (C1). Similarly, o-
phenylenediamine was condensed in acidic media with 2-mercaptoace-
tic acid releasing (1H-benzo[d]imidazole-2-yl) methanethiol in good
yield, which was then refluxed for 20 h with an excess concentration of
hydrazine hydrate in ethanol producing 2-(hydrazinylmethyl)-1H-benzo
[d]imidazole. The hydrazine derivative was cyclized producing imidazo
[1,2-d] [1,2,4] triazine after condensation with trimethoxymethane.
Finally, the cyclized triazine was condensed with salicylaldehyde pro-
ducing compound C2 in good yield. The complete reaction steps, con-
ditions and yields are illustrated in Scheme 1. The purity of the
synthesized compounds was confirmed by elemental analysis and
chromatography (Supporting information) and found to be >95%
2. Materials and methods
2.1. Chemicals and reagents
Synthesis of benzoimidazoquinazoline and benzimidazotriazin is
described below. Complete physicochemical data are provided in sup-
porting information. Cisplatin was purchased from EIMC United Phar-
maceuticals, Badr City, Cairo, Egypt. Disodium EDTA was from Alpha
Chem (USA). Phosphate buffered saline (PBS) was purchased from Bio
Whittaker® Lonza (Switzerland). Monoclonal mouse antibodies for
CD34 and VEGFR-2 were from Santa Cruz Biotechnology (Santa Cruz,
CA, USA).
2.2. Synthesis of novel compounds
2.3. Tumor cells
Benzoimidazoquinazoline (C1) and benzimidazotriazine (C2) were
synthesized for testing as anticancer lead molecules that modulate the
activity of VEGFR, CD34, and microR-122. Full synthesis and charac-
terization details are given in the Supporting Information. o-Phenyl-
enediamine was refluxed with an equimolar concentration of anthranilic
acid producing 2-(1H-benzo[d]imidazole-2-yl) aniline in high yield. The
2-(1H-benzo[d]imidazole-2-yl) aniline was condensed with carbon
Human breast cancer cell line MCF-7 and Ehrlich Ascites Carcinoma
(EAC) cells were purchased from Tumor Biology Department, National
Cancer Institute, Cairo University. The cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% heat-
inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and
50
midified air atmosphere containing 5% CO2 and were sub-cultured two
μ
g/ml gentamycin. All cells were maintained at 37 ◦C in a fully hu-
Scheme 1. Synthetic pathways of the designed compounds
3

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European Journal of Pharmacology xxx (xxxx) xxx
times a week. EAC is a murine spontaneous breast cancer that served as
the original tumor from which an ascites variant was obtained. On
intraperitoneal inoculation, an ascitic fluid rich in tumor cells is pro-
duced. The tumor cell line was maintained in our laboratory by serial
intraperitoneal passages into Swiss female albino mice at 7–10 days
interval.
2.9. Sample collection
At the 21st day of the study, blood samples were collected from the
orbital sinus (retro-orbital plexus) under light ether anesthesia. A clean
sterile capillary tube was inserted at the inner canthus of the eye. The
blood was left to clot for 20 min, then was centrifuged at 1968 g for 15
min. Serum samples were separated, stored at (ꢀ 20 ◦C), and used for
measurement of liver enzymes, urea, creatinine, and vascular endothe-
lial growth factor (VEGF). After cervical dislocation, tumor discs were
separated, weighed, and fixed in 10% neutral buffered formalin for
immunohistochemical investigations, and a portion of the tumor tissue
was stored at ꢀ 80ᴼC for RNA isolation.
2.4. Experimental animals
Seventy Swiss albino mice weighing 25–30g were obtained from the
Egyptian Organization for Biological Products and Vaccines (Vacsera,
Egypt). Animals were housed in plastic cages with mesh floor and
hardwood bedding. They were kept under controlled laboratory condi-
tions with normal light/dark cycle at 25–30 ◦C. Food and water were
provided ad libitum during the study period. Mice were left to accli-
matize for 1 week before the experiments. All animal procedures and
experimental protocols were carried out in accordance with the Guide
for the Care and Use of Laboratory animals. The study was approved by
the ethical committee of Faculty of Pharmacy, Suez Canal University
(201704AM1).
2.10. Methods of analysis
2.10.1. Determination of liver and kidney functions
Serum activity of aspartate transaminase (AST) and alanine trans-
aminase (ALT) were measured by enzymatic colorimetric method (Bio-
diagnostic, Egypt). Serum urea and creatinine levels were measured
calorimetrically (Diamond, Egypt). All procedures were according to
manufacturer’s instructions.
2.5. In vitro study
2.10.2. Determination of vascular endothelial growth factor (VEGF)
Serum VEGF levels were determined using Quantikine® ELISA kit
(R&D systems, USA) according to the manufacturer’s protocol. Con-
centration was expressed as pg/g tissue.
Anticancer activity of benzoimidazoquinazoline and benzimidazo-
triazin was tested in the MCF-7 cell line with an HTScan® VEGF Re-
ceptor 2 Kinase Assay Kit. The kit provides a means of performing kinase
activity assays with recombinant human VEGFR-2 kinase. It includes
active VEGFR-2 kinase (supplied as a GST fusion protein), a biotinylated
peptide substrate and a phospho-tyrosine antibody for detection of the
phosphorylated form of the substrate peptide. Kit contents include
Phospho-Tyrosine Mouse mAb (P-Tyr-100), HTScan® Tyrosine Kinase
Buffer (4X), DTT (1000x, 1.25 M), ATP (10 mM), Gastrin Precursor
(Tyr87) Biotinylated Peptide, VEGF Receptor 2 Kinase (recombinant,
human).
2.10.3. Determination of miR-122 expression by quantitative real time
polymerase chain reaction (qPCR)
Total RNA, including small RNA, was isolated from the tumor tissue
using Qiagen miRNeasy Mini kit (Cat. No. 217004) (Qiagen, Germany)
following manufacturer protocol. RNA concentration was determined
with a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).
The expression of miR-122 was quantified by qRT-PCR. RNA was con-
verted to cDNA by TaqMan® microRNA reverse transcriptase kit (Cat.
No. 4366596) and TaqMan assays hsa-miR-122 (5 × ) (Assay ID:
002245) and RNU6B (5 × ) (Assay ID: 001093) (Cat. No. 4427975)
(Applied Biosystems, Thermo Fisher Scientific, USA). Expression of miR-
122 was assessed against RNU6B as an endogenous control using the
same Taqman assays; hsa-miR-122 and RNU6B (both at concentration of
20x). ΔCT and fold change were calculated. The PCR reactions were
2.6. Preparation of Ehrlich Ascites Carcinoma (EAC) cells
Ascetic fluid was withdrawn under aseptic conditions from tumor-
bearing mice by needle aspiration from peritoneal cavity. Seven to ten
days after EAC cells implantation, EAC cells were tested for viability and
contamination using the Trypan blue dye exclusion technique (Lazarus
et al., 1966). Only EAC cells with at least 90% viability were used. EAC
cells were suspended in normal saline so that each 0.1 ml contains 2.5 ×
106 cells. Cells were counted under microscope with a hemocytometer.
performed in 20
Master Mix (Cat. No. 4440043) (Applied Biosystems, Thermo Fisher
Scientific, USA), 1 l from each assay, 4 l complementary DNA (cDNA),
and 5
μl total volume, including 10 μl TaqMan® Universal
μ
μ
μ
l RNase-free water. PCR conditions consisted of 1 min at 95 ◦C,
followed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for 1 min and a final
extension at 72 ◦C for 5 min. Real-time PCR was conducted using Ste-
pOnePlus Real-Time PCR thermal cycler (Applied Biosystems, Thermo
Fisher Scientific, USA).
2.7. Induction of solid tumors
Each mouse was inoculated intradermally at 2 sites bilaterally on the
lower ventral side (after shaving this area) with 100 μl EAC suspension
2.11. Immunohistochemistry
(2.5 × 106 cells) at each site.
Tumor discs for immunohistochemical examination were fixed in
10% neutral buffered formalin overnight and then embedded in
paraffin. When analyzed, all paraffin-embedded tissues were sectioned
2.8. Study design
Animals were randomly divided into 7 groups, 10 animals each.
Group (1) received PBS (phosphate buffer saline) and was considered as
normal group. Group (2) served as the EAC control untreated group.
Group (3) received e Cisplatin (i.p.) at a dose of 2 mg/kg (Williams et al.,
2001). Groups (4 & 5) received benzoimidazoquinazoline (i.p.) at doses
of 5 mg/kg and 10 mg/kg respectively. Groups (6 & 7) received benzi-
midazotriazin (i.p.) at doses of 5 mg/kg and 10 mg/kg respectively. The
synthetic compounds were dissolved in PBS. Cisplatin was available as
vials ready to use. All treatments started at the 7th day after inoculation
of EAC cells and were continued daily for 14 days. Cisplatin was given
every 3 days for two weeks.
at 4-μm, deparaffinized in xylene, and hydrated through graded ethyl
alcohol series in decreasing concentrations (100%, 90%, 80% and 70%).
Antigen retrieval was performed according to a Tris/EDTA buffer (pH =
9) antigen retrieval protocol. Tumor discs were stained applying the
EnVision™ FLEX HRP label, high pH method according to the manu-
facturer’s staining protocol (Dako). Endogenous peroxidase activity was
quenched by incubating the specimen for 10 min with phosphate buffer
containing 3% hydrogen peroxide. Sections were incubated with cor-
responding primary antibodies at 4 ◦C overnight. After conjugation with
dextran coupled with peroxidase molecules and goat secondary anti-
body molecules against rabbit immunoglobulins, sections were
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European Journal of Pharmacology xxx (xxxx) xxx
incubated in 3,3′-diaminobenzidine (DAB) substrate chromogen for 2
Table 1
Liver enzymes and kidney markers in the study groups.
min. Finally, Mayer’s hematoxylin was used for counter staining.
ALT
AST
Urea
Creatinine
(mg/dL)
(IU/L)
(IU/L)
(mg/dL)
2.12. Evaluation of antibody expression
Normal
Control
Cisplatin
43.50 ±
4.92
52.87 ±
5.13
16.25 ±
2.33
0.91 ± 0.07
1.82 ± 0.19a
ImageJ was used for the semi-quantitative analysis of immunohis-
tochemical reactions. The images were captured by an optical micro-
scope with a 40X objective (Optika B-352A, Italia) coupled to a camera
(HDCE30C), and quantified using the ImageJ MacBiophotonics (Na-
tional Institutes of Health, USA) software package developed by
McMaster University (Ontario, Canada). The expression of VEGFR-2 and
CD34 were assessed and percentage of stained area was measured.
51.69 ±
62.16 ±
29.99 ±
5.50*
7.11a
7.99a
55.20 ±
6.22a
67.34 ±
8.25a
44.15 ±
4.88 ±
6.42ab
1.11ab
Benzoimidazoquinazoline
(5 mg/kg)
50.32 ±
61.13 ±
30.23 ±
5.55ac
1.77 ± 0.22ac
5.20a
6.30a
Benzoimidazoquinazoline
(10 mg/kg)
52.13 ±
6.19a
63.50 ±
6.80a
34.67 ±
2.01 ± 0.28ac
2.10 ± 0.29ac
2.55 ± 0.78ac
5.78ac
Benzimidazotriazine
(5 mg/kg)
53.15 ±
65.33 ±
35.38 ±
5.60ac
6.20a
6.98a
2.13. Statistical analysis
Benzimidazotriazine
(10 mg/kg)
56.23 ±
7.09a
68.23 ±
8.11a
37.16 ±
6.00ac
All data are presented as mean ± standard deviation (S.D). Differ-
ences among groups was assessed using unpaired Student’s t-test and
one-way ANOVA. P value less than 0.05 was considered to be statisti-
cally significant. Calculations were performed with the SPSS 21.0 sta-
tistical software package (IBM, USA).
Values are expressed as mean ± S.D. Data were analyzed using ANOVA followed
a
by Bonferroni post hoc test. significantly different compared to the normal
group at p < 0.05, b significantly different compared to the control group at P <
0.05. c significantly different compared to the cisplatin group at P < 0.05.
3.3. Effect on serum VEGF
3. Results
Serum VEGF was measured in this study as a marker for angiogen-
esis. VEGF levels were significantly higher in the EAC control group
versus the normal mice (P < 0.001). The serum levels of VEGF decreased
significantly on treatment with cisplatin, the higher dose of benzoimi-
dazoquinazoline, and both doses of benzimidazotriazine (P < 0.05). The
lower dose of benzoimidazoquinazoline had no effect on VEGF (Fig. 3).
3.1. Effect on tumor weight
Tumor discs were removed and weighed on the 21st day of treat-
ment. All treatment groups showed a significant decrease in the tumor
weight compared to the EAC control group (P < 0.01) except for the
group treated with benzoimidazoquinazoline (5 mg/kg) where the
average tumor weight was significantly higher than that observed in the
cisplatin group (P < 0.01) (Fig. 2).
3.4. Effect on the expression of VEGFR2 and CD34
3.2. Effect on liver and kidney functions
Immunohistochemical analysis showed that EAC control group had
high expression of VEGFR2, the main responder to VEGF signal that
regulates endothelial migration and proliferation. Significant reduction
in expression was observed in the cisplatin group (P < 0.001). Treatment
with the test compounds in both doses decreased the expression
compared to control EAC (P < 0.001) but was still significantly higher
than the cisplatin treated group (Fig. 4). The highest expression of CD34
was observed in the control EAC group. All other treated groups showed
significantly lower expression compared to control (P < 0.001).
Expression of CD34 in the group treated with the higher dose of benzi-
midazotriazine (10 mg/kg) was not significantly different from that of
ALT and AST levels were increased in the EAC control mice and in all
treatment groups compared to normal group (Table 1). However, the
observed increase cannot be considered as an indication of liver toxicity.
Creatinine and urea levels were also increased in the control mice
compared to the normal group and were further increased after treat-
ment with cisplatin compared to the control group (P < 0.01). Treat-
ment with benzoimidazoquinazoline (C1) and benzimidazotriazine
(C2), in both doses (5 mg/kg and 10 mg/kg) resulted in a decrease in
creatinine and urea compared to the cisplatin group (P < 0.05).
Fig. 2. Effect of treatment on the tumor weight in EAC bearing mice. C1 = benzoimidazoquinazoline, C2 = benzimidazotriazine. Values are expressed as mean ± S.D.
All data were analyzed using ANOVA followed by Bonferroni post hoc test. # significantly different compared to the control group, $ significantly different compared
to the cisplatin group. Differences were considered significant at P < 0.05.
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Fig. 3. Effect of treatment on serum VEGF. C1= Benzoimidazoquinazoline, C2= Benzimidazotriazine. Values are expressed as mean ± S.D. All data were analyzed
using ANOVA followed by Bonferroni post hoc test. * significantly different compared to the normal group, # significantly different compared to the control group, $
significantly different compared to the cisplatin group. Differences were considered significant at P < 0.05.
Fig. 4. Expression of VEGFR2 and CD34 in tumor tissue. (A & B) Representative photomicrographs of (A) VEGFR2 and (B) CD34. (C & D) The percentage of positive
immunohistochemical reactions (brown stained area) analyzed by Image J software. C1 = benzoimidazoquinazoline, C2= Benzimidazotriazine. Values are expressed
as mean ± S.D. The. Data were analyzed using ANOVA followed by Bonferroni post hoc test. # significantly different compared to the control group, $ significantly
different compared to the cisplatin group. Differences were considered significant at P < 0.05.
the cisplatin treated group (Fig. 4).
treatment groups. Treatment with benzoimidazoquinazoline (10 mg/kg)
gave almost similar expression levels as the group treated with cisplatin.
The expression of miR-122 was significantly higher in both groups
treated with benzimidazotriazine compared to the cisplatin group (P <
0.05). Interestingly, expression levels upon treatment with the higher
dose of benzimidazotriazine (10 mg/kg) were not significantly different
from the normal group (Fig. 5).
3.5. Effect on levels of expression of miR-122 in tumor tissue
The expression of miR-122 was determined in the tumor tissue of
killed mice. The expression of miR-122 was significantly downregulated
in the mice with induced breast cancer (P < 0.001) compared to the
normal group. Its expression was significantly upregulated in all
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Fig. 5. Levels of expression of miRNA-122 in
tumor tissue determined by real time PCR. C1 =
benzoimidazoquinazoline, C2= Benzimidazo-
triazine. Fold change was calculated by the 2–ΔΔCt
method, where RNU6B was the endogenous con-
trol. Values are expressed as mean of log 2 fold
change vs the normal group ± S.D. The expression
of the normal group is normalized to 1 where log2
(1) = zero. Data were analyzed using ANOVA fol-
lowed by Bonferroni post hoc test. * significantly
different compared to the normal group, # signifi-
$
cantly different compared to the control group,
significantly different compared to the cisplatin
group. Differences were considered significant at P
< 0.05.
3.6. Molecular modeling
number of rotatable bonds (0–15) and polar surface area (7–200 Å). The
two compounds had a reasonable rotatable bond and polar surface area
compared to pazopanib (Table 2).
Computer-aided drug design simulation saves effort, time, and re-
sources required by traditional drug development methodologies. Mo-
lecular modeling was used to predict the potential interactions of the
target compounds with VEGFR. Modulation activity of pazopanib and
sorafenib derivatives towards VEGFR receptor was detected using glide
docking of the Schrodinger program. The docking study showed that the
docking profile of the target compounds were at the pazopanib binding
site (Fig. 6). Thus, they are expected to have a similar mode of action.
The binding of pazopanib to VEGFR in its inactive conformation is
believed to be similar to its interaction with its originally recognized
kinase target B-RAF, a component of the RAS signal transduction
network. Assuming so, the pyrimidine nitrogen in its un-ionized form
and the nearby aniline NH moiety form a H-bond with the amide group
of Cys919 in the hinge region (NH and carbonyl oxygen, respectively).
The benzimidazole moiety binds in a hydrophobic pocket, possibly
occupying the site normally reserved for Phe1047 in the “DFG-in”
(active) conformation. Compound 1 and Compound 2 docked well in the
pazopanib binding site and set up many hydrogen bonds, hydrophobic
and electrostatic interactions (Fig. 6). Cys919, Phe1046, Glu917,
Thr916, Glu885, Phe918, Leu840, Ile1044, Ile888, Ile892, Cys1045
were the main amino acids involved in interactions. Generally, quina-
zoline and triazine scaffolds occupy a deep pocket of the ATP binding
site and form a hydrogen bond with the main amino acid involved in
phosphorylation processes, Cys919. Additionally, the two compounds
can form extra-hydrogen bonds with certain amino acids in the hinge
region such as Glu917, Glu885, and Thr916. Hydroxy-phenyl and
dichloro-phenyl were occupied in the hydrophobic pocket in DFG-in
active conformation forming hydrophobic and van der Wall’s interac-
tion with Phe1046, Phe918, Leu840, Ile1044, IleE888, Ile892, and
Cys1045. The triazine containing compound had the highest docking
score of ꢀ 9.1 and glide emodel of ꢀ 62.6, while compound with qui-
nazoline moiety showed ꢀ 8.5 and ꢀ 87.4 docking score and glide
emodel, and pazopanib scored ꢀ 9.5, and ꢀ 93.6.
Intestinal absorption and permeation are important factors for drug
absorption. They were assessed by the predicted Caco-2 cell perme-
ability (QPPCaco), which is used as a model for the gut-blood barrier
by non-active transport. Caco-2 cell permeability of the proposed
compounds was 6.3-fold greater than that of pazopanib, indicating
excellent drug permeability. The oral absorption of the designed
compounds is superior to pazopanib. QPPMDCK descriptor was uti-
lized to predict permeability of the proposed compounds through the
blood brain barrier. MDCK cells are an excellent mimic of the blood-
brain barrier. Both compounds showed excellent BBB permeability
(QPPMDCK>25) in comparison to pazopanib. This factor should be
considered in future to diminish side effects. Human serum albumin
binding capability was predicted with the QPlogkhsa descriptor of
QikProp. The proposed compounds exhibited good binding capability
to plasma proteins. The other physicochemical properties of the com-
pounds are listed in Table 2.
4. Discussion
Despite the improved survival rates of breast cancer patients, met-
astatic breast cancer is still prevalent (Eroles et al., 2012). The current
study aimed to investigate the effect of two novel heterocyclic com-
pounds in the treatment of breast cancer. The rational in this study was
to design novel drug-like molecules that competitively bind to the ATP
binding pocket of VEGFR intracellular domain inhibiting its activity. To
achieve this, the central core adenine ring of ATP or 2-aminopyrimidine
of pazopanib was modulated. A range of heterocyclic systems have been
reported to be alternatives to adenine, including thienopyrimidines,
quinoline, imidazopyridazine, and the pyrido pyrimidine (Peng et al.,
2017; Holmes et al., 2007). Herein, imidazoquinazoline and imidazo-
triazine were selected to mimic adenine and pyrimidine in the hinge
region, presenting a hydrogen bond to the key amino acid cysteine.
Moreover, the current ring system formed extra hydrogen bonds to
amino acids in the hinge region allowing the designed compounds to
bind tightly to VEGFR. Pazopanib binds well to VEGFR, where the
indazole ring reaches out to the protein lipophilic pocket. In this study,
this feature was kept by modifying indazole to hydroxyphenyl and
dichlorophenyl, groups which occupy the same pose of indazole and
form van der Wall and hydrophobic interactions with key aromatic
amino acids. The hydroxyl group of benzimidazotriazine forms an extra
hydrogen bond with Thr916 in the hydrophobic pocket giving this
compound superiority. The novel compounds, therefore, can be
considered lead compounds which disrupt cell proliferation in solid
tumor mediated by aberrant VEGF/VEGFR signaling pathway.
3.7. Lipinski’s rule and in silico absorption, distribution, metabolism,
excretion (ADME) prediction of the designed compounds
The pharmacokinetic parameters of the designed molecules were
determined by ADME prediction using Schrodinger QikProp v4.3. Both
molecules were surveyed for primary parameters of Lipinski’s rule of 5
and other pharmacokinetic parameters. Orally dynamic compounds
should not violate the Lipinski rule multiple times. The designed com-
pounds in the current study did not reach maximum permissible viola-
tions, indicating they have medication similarity properties (Table 2).
Oral bioavailability of medication-like molecules is constrained by the
7

R.M. Hazem et al.
European Journal of Pharmacology xxx (xxxx) xxx
Fig. 6. (A) 3D binding mode and overlay of Pazopanib and the designed compounds in kinase domain of human VEGFR-2 (PDB 3EWH). (B) 2D of ligand receptor
interaction of Benzimidazotriazine, (C) Benzoimidazoquinazoline, and (D) Pazopanib.
The VEGF and its receptor VEGFR2 play a major role in generating
new blood vessels around the tumor mass. Binding of VEGF to its re-
ceptor induces endothelial cell proliferation and development. Inhi-
bition of VEGFR2 and tumor angiogenesis are necessary for treatment
of breast cancer (Cohen, 2001; Sebolt-Leopold and English, 2006).
VEGF is associated with poor prognosis in breast cancer as there is
increased synthesis in breast cancer tissues (Maae et al., 2012). VEGF
can stimulate proliferation and migration of naturally inert endothelial
cells during tumor angiogenesis and growth resulting in the formation
of new vessel structures. Hypoxia is considered as the main cause of
tumor angiogenesis, even though growth factors and a variety of
transcriptional regulators are able to stimulate VEGF production
through paracrine or autocrine mechanisms (Fox et al., 2007; Jia et al.,
2013). The higher dose of benzoimidazoquinazoline (C1) and both
doses of benzimidazotriazine (C2) caused a marked decrease in the
tumor weight and reduction in serum levels of the angiogenic factor
VEGF. All treatment doses significantly reduced the expression of
VEGFR2 and CD34 in the tumor tissue. VEGFR-2 represents a major
8

R.M. Hazem et al.
European Journal of Pharmacology xxx (xxxx) xxx
Table 2
Results of ADME study for the designed compounds in comparison with Pazopanib.
Comp.
No.
Mol-MWa
DonorHBb
AcceptHBc
QPlogPo/wd
#rotore
PSAf
QPlogSg
QPlogHERGh
QPPCacoi
QPP
QPlogKhsak
% Human
Oral
MDCKj
Absorptionl
Compound-1
Compound-2
Pazopanib
453.3
276.2
437.5
1
2
3
5.5
4.7
8.5
5.5
2.4
2.7
5
3
6
63.4
62.8
115.1
ꢀ 7.6
ꢀ 4.1
ꢀ 5.88
ꢀ 7.1
ꢀ 5.9
ꢀ 6.2
2325
916.1
124.7
10000
450
0.714
ꢀ 0.03
0.265
100
94.11
80.6
53.3
Acceptable Ranges.
a
<500amu; b < 5; c < 10; d < 5;e 0-15; f 7–200; g < 0.5; h < - 5;i <25 poor, >500 great; j <25 poor, >500 great; k ꢀ 1.5 – 1.5; l >80% is high, <25% is poor.
target within the angiogenesis-related kinases, hence considered the
most important transducer of VEGF-dependent angiogenesis. Thus,
inhibition of VEGF/VEGFR signaling pathway is regarded as an
attractive therapeutic target for inhibition of tumor angiogenesis and
subsequent tumor growth (Holmes et al., 2007). Therefore, the
designed compound might inhibit angiogenesis.
diagnostic biomarker in cancer (Vo et al., 2014). Up-regulation of
miR-122 was reported in breast cancer cells. Functional analysis showed
that miR-122 plays dual function as a tumor suppressor and onco-mirR
dependent on the tumor cells phenotype by diminishing endurance and
˜
advancing radiosensitivity (Perez-Anorve et al., 2019). Many studies
have targeted miR-122 for disease management (Van Meter et al., 2020).
Benzoisoquinoline derivatives activated miR-122 in hepatocarcinoma
and treat hepatitis C virus infection by inducing apoptosis and reducing
cell viability via caspase activation. These compounds are considered
promising lead molecules (Young et al., 2010). Expression of miR-122 in
breast cancer tissue was significantly upregulated on treatment with
both compounds, especially with benzimidazotriazine. It has been pro-
posed that small molecules can act as novel anti-cancer therapeutic
agents via regulating miRNA expression either pre-transcriptionally, at
the transcriptional stage, or at the post-transcriptional stage (Deiters,
2010). The expression of miR-122 was reported to be enhanced by a
small molecule activator (benzoisoquinoline derivative) that acts at the
transcriptional level through upregulation of pri-miR-122 (Young et al.,
2010). Benzoisoquinoline was replaced in the current study by benzoi-
midazoquinazoline and benzimidazotriazin in an attempt to investigate
the role of heterocyclic systems in miR-122 modulation. Both systems
kept the modulation activity towards miR-122 activation. As a result,
they may be a valuable tool for exploring the regulation of miR-122 in
breast cancer and can possibly provide new targets and lead structures
for the advancement of new chemotherapeutics.
Assessment of tumoral micro-vessel density using immunohisto-
chemical endothelial markers like factors CD31, CD34, and the activated
endothelial cell marker CD105 is a commonly used procedure for
measuring tumor angiogenesis in breast cancer (Uzzan et al., 2004).
CD34 is a transmembrane anti-adhesive sialomucin that is expressed in
the luminal surface of endothelial cells (Nielsen and MCNagy, 2009).
The CD34 antigen has been widely used in studying tumor angiogenesis
(Ancuta et al., 2010). A role for CD34 in tumor angiogenesis was pro-
posed by the observation that CD34 deletion in mice impaired early
tumor growth due to delay in angiogenesis (Maltby et al., 2011). High
expression of CD34 on endothelial tip cells and their filopodia suggests
its role in angiogenesis (Siemerink et al., 2013).
Benzimidazotriazine showed promising activity in suppressing
CD34 expression in mice bearing EAC. It has been proposed that tumor
progression and metastasis in breast cancer is angiogenesis dependent
(Pyakurel et al., 2014). The relation between the VEGF and CD34
expression has been reported in various malignant tumors. Thus,
measuring their expression is vital for deciding the prognosis of tumor
patients.
Reports showed that levels of miR-122 variate in several cancer types
and stages (Akuta et al., 2016; Ahsani et al., 2017). In hepatocellular
carcinoma (HCC), overexpression of miR-122 stimulates cell cycle arrest
and apoptosis by inhibiting Cyclin G1 and Bcl-2 like expression (Ma
et al., 2010). On the other hand, overexpression of miR-122 in non-small
cell lung cancers (NSCLC) may result in reducing the number of invasion
and migration cells (Qin et al., 2015). These data confirm the hypothesis
that miR-122 acts as a tumor suppressor in various types of cancer. The
roles of specific miRNAs in regulating tumor-associated angiogenesis
and lymphangiogenesis have been reported. miRNAs represent a
fundamental regulatory tool for regulation of gene expression and
maintenance of cellular functions necessary for adequate angiogenic
A large number of drug-like molecules cure infectious diseases and
treat cancer but have adverse effects on the host. In the current study,
groups treated with benzoimidazoquinazoline and benzimidazotriazin
showed lower serum urea and creatinine levels compared to the cisplatin
group, indicating a reduced nephrotoxic effect.
Physicochemical properties also play an important role in deciding if
a compound is suitable for therapeutic use. Solubility directly affects
absorption; any therapeutic compound has to dissolve in biological
fluids to effectively pass through a biological membrane. The designed
compounds had the appropriate solubility as indicated by the partition
coefficient (QplogPO/W). Orally active medications must be absorbed in
the GIT and should penetrate the cellular membrane to reach their
proposed targets. Benzoimidazoquinazoline and benzimidazotriazine
had excellent permeability and oral absorptivity. Additionally, these
drugs showed an inhibitory activity against multidrug drug resistance
and P-glycoprotein which play a role in drug efflux to reduce the drugs
therapeutic effect (data not shown). Moreover, the novel molecules
exhibited promising binding to albumin and can penetrate the BBB,
criteria essential in drug-like molecules. The physiochemical properties
of the compounds determined in silico along with their anti-cancer ef-
fects in vivo in EAC animals give the compounds potential for use in
treatment of breast cancer and warrant further clinical study.
´
response (Suarez and Sessa, 2009). Interestingly, miR-122 was reported
to act as a tumor suppressor and to inhibit angiogenesis in bladder
cancer through down-regulating VEGFC expression (Wang et al., 2016).
In HCC, miR-122 was shown to induce apoptosis via targeting toll-like
receptor 4 (TLR4) and inhibiting the expression of inflammatory
markers including VEGF (Wei et al., 2019). miR-20b is implicated in the
regulation of VEGF in breast cancer cells by targeting HIF-1; this verified
its role as an angiogenic regulator (Casico et al., 2010). miR-128 acts as a
tumor suppressor through reducing the expression of both VEGFA and
VEGFC (Hu et al., 2014). The inhibitory effect of some miRNAs on tumor
angiogenesis has been attributed to their ability to target the VEGF re-
ceptors in endothelial cells and block their downstream signaling (Wang
et al., 2018). Examples include downregulation of VEGFR-2 and
VEGFR-3 by miR-128 in NSCLC (Hu et al., 2014), and targeting VEGFR2
by miR-497 to inhibit tumor angiogenesis (Tu et al., 2016).
5. Conclusion
Modulation of the activity of VEGF/VEGFR and miR-122 is prom-
ising for the management of cancer. Two heterocyclic compounds,
benzoimidazoquinazoline and benzimidazotriazine, exhibited anti-
cancer properties through reducing the angiogenesis effect and
The role of miR-122 was documented in various cancers where it
functions as an oncogene/tumor suppressor. Thus, it can serve as a
9

R.M. Hazem et al.
European Journal of Pharmacology xxx (xxxx) xxx
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CRediT authorship contribution statement
Fox, S.B., Generali, D.G., Harris, A.L., 2007. Breast tumour angiogenesis. Breast Cancer
Res. 9, 216–223.
Reem M. Hazem: Methodology, Development or design of meth-
odology, creation of models, Application of statistical, Verification of the
overall results. Preparation, creation and/or presentation of the pub-
lished work. Anhar A. Mohamed: Methodology, Development or design
of methodology, creation of models. Provision of study materials, re-
agents. Preparation, creation and/or presentation of the published work.
Nagat Ghareb: Methodology, Development or design of methodology,
creation of models, Application of statistical, Verification of the overall
results. Eman T. Mehanna: Methodology, Development or design of
methodology, creation of models. Noha M. Mesbah: Ideas; formulation
or evolution of overarching research goals and aims. Conducting a
research and investigation process. Oversight and leadership re-
sponsibility for the research activity. Dina M. Abo-Elmatty: Ideas;
formulation or evolution of overarching research goals and aims. Con-
ducting a research and investigation process. Oversight and leadership
responsibility for the research activity. Mohamed Saleh Elgawish:
Methodology, Ideas; formulation or evolution of overarching research
goals and aims. Development or design of methodology. Designing
computer programs. Conducting a research and investigation process,
Verification of the overall results. Preparation, creation and/or presen-
tation of the published work.
Hamam, R., Hamam, D., Alsaleh, K.A., Kassem, M., Zaher, W., Alfayez, M.,
Aldahmash, A., Alajez, N.M., 2017. Circulating microRNAs in breast cancer: novel
diagnostic and prognostic biomarkers. Cell Death Dis. 8, e3045.
Holmes, K., Roberts, O.L., Thomas, A.M., Cross, M.J., 2007. Vascular endothelial growth
factor receptor-2: structure, function, intracellular signalling and therapeutic
inhibition. Cell. Signal. 19, 2003–2012.
Hu, F., Xu, P., Sun, B., Xiao, Z., 2017. Differences in the MicroRNA profiles of
subcutaneous adipose-derived stem cells and omental adipose-derived stem cells.
Gene 625, 55–63.
Hu, J., Cheng, Y., Li, Y., Jin, Z., Pan, Y., Liu, G., Fu, S., Zhang, Y., Feng, K., Feng, Y.,
2014. microRNA-128 plays a critical role in human non-small cell lung cancer
tumorigenesis, angiogenesis and lymph angiogenesis by directly targeting vascular
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under hypoxia. PloS One 1, e116.
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Siemann, D.W., Zurakowski, D., Rodig, S.J., Moses, M.A., 2013. Transcriptional
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carcinogenesis. Exp. Oncol. 40, 2–9.
Lala, P.K., Nandi, P., Majumder, M., 2018. Roles of prostaglandins in tumor associated
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Declaration of competing interest
Lou, W., Liu, J., Gao, Y., Zhong, G., Chen, D., Shen, J., Bao, C., Xu, L., Pan, J., Cheng, J.,
Ding, B., Fan, W., 2017. MicroRNAs in cancer metastasis and angiogenesis.
Oncotarget 8, 115787–115802.
The authors declare no competing financial interest.
Ma, L., Liu, J., Shen, J., Liu, L., Wu, J., Li, W., Luo, J., Chen, Q., Qian, C., 2010.
Expression of miR-122 mediated by adenoviral vector induces apoptosis and cell
cycle arrest of cancer cells. Canc. Biol. Ther. 9, 554–561.
Acknowledgment
Maae, E., Olsen, D.A., Steffensen, K.D., Jakobsen, E.H., Brandslund, I., Sørensen, F.B.,
Jakobsen, A., 2012. Prognostic impact of placenta growth factor and vascular
endothelial growth factor A in patients with breast cancer. Breast Canc. Res. Treat.
133, 257–265.
The authors are thankful to staff members of Pharmacology
Department, Faculty of Pharmacy, Suez Canal University for help and
support.
Maierthaler, M., Benner, A., Hoffmeister, M., Surowy, H., Jansen, L., Knebel, P., Chang-
Claude, J., Brenner, H., Burwinkel, B., 2017. Plasma miR-122 and miR-200 family
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Maltby, S., Freeman, S., Gold, M.J., Baker, J.H., Minchinton, A.I., Gold, M.R.,
Roskelley, C.D., McNagny, K.M., 2011. Opposing roles for CD34 in B16 melanoma
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2020.173747.
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