Development of pyridazine derivatives as potential EGFR inhibitors and apoptosis inducers: Design, synthesis, anticancer evaluation, and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2020.104473

Novel hybrids of pyridazine-pyrazoline were synthesized aiming to develop new antiproliferative candidates. All compounds were submitted to the National Cancer Institute (NCI), USA, and many were proved to have significant antiproliferative activity. In addition, in vitro studies of the epidermal growth factor receptor (EGFR) inhibition showed that compounds IXn, IXg, IXb and IXl exhibited excellent inhibitory effect (IC₅₀ = 0.65, 0.75, 0.82 and 0.84 μM, respectively) compared to Erlotinib (IC₅₀ = 0.95 μM). The mechanistic effectiveness in cell cycle progression, apoptotic induction and gene regulation were assessed for the promising compounds IXg and IXn due to their significant EGFR inhibition. Flow cytometeric analysis indicated that compounds IXg and IXn result in increased cell numbers in phase G2/M, suggesting cell cycle arrest in phase G2/M in UO-31cells. Furthermore, real time PCR assay illustrated that compounds IXg and IXn elevated Bax/Bcl2 ratio which confirmed the mechanistic pathway of them. Moreover, the apoptotic induction of UO-31 renal cancer cells was enhanced effectively through activation of caspase-3 by compounds IXg and IXn. On the other hand, molecular docking study was performed to investigate binding mode of interaction of compounds with EGFR-PK in the active site with the aim of rationalizing its promising inhibitory activity. Finally, based on the aforementioned findings, compounds IXg and IXn could be considered as effective apoptosis modulators and promising leads for future development of new anti-renal cancer agents.

Full text of DOI:10.1016/j.bioorg.2020.104473

Journal Pre-proofs
Development of Pyridazine Derivatives as Potential EGFR inhibitors and
Apoptosis Inducers: Design, Synthesis, Anticancer Evaluation, and Molecular
Modeling Studies
Marwa F. Ahmed, Eman Y. Santali, Eman M. Mohi El-Deen, Ibrahim A.
Naguib, Radwan El-Haggar
PII:
S0045-2068(20)31771-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104473
YBIOO 104473
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 October 2020
10 November 2020
11 November 2020
Please cite this article as: M.F. Ahmed, E.Y. Santali, E.M. Mohi El-Deen, I.A. Naguib, R. El-Haggar,
Development of Pyridazine Derivatives as Potential EGFR inhibitors and Apoptosis Inducers: Design, Synthesis,
Anticancer Evaluation, and Molecular Modeling Studies, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.104473
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Inc. All rights reserved.

Development of Pyridazine Derivatives as Potential EGFR inhibitors
and Apoptosis Inducers: Design, Synthesis, Anticancer Evaluation,
and Molecular Modeling Studies
Marwa F. Ahmed 1,2*; Eman Y. Santali 1; Eman M. Mohi El-Deen 3; Ibrahim A.
Naguib 1,4; Radwan El-Haggar 2*
1 Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, P.O. Box
11099, Taif 21944, Saudi Arabia.
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo,
11795, Egypt.
3 Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo, 12622
Egypt.
4 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
University, Alshaheed Shehata Ahmad Hegazy St., 62514, Beni-Suef, Egypt
Key words: Pyridazine, Pyrazoline; Antiproliferation; epidermal growth factor
receptor, Apoptosis, Molecular docking.
* Corresponding authors:
Dr. Radwan El-Haggar, Email: radwan_elhaggar@pharm.helwan.edu.eg
Dr. Marwa F. Ahmed, Email: marwa.farg@tu.edu.sa
1

2

Highlights:
 Novel hybrids of pyridazine-pyrazoline were synthesized aiming to develop new
antiproliferative candidates
 All compounds were submitted to the National Cancer Institute (NCI), USA, and
many were proved to have significant antiproliferative activity.
 The enzyme inhibitory activity of compounds against epidermal growth factor
receptor (EGFR) was evaluated
 A cell cycle analysis and an annexin V-FITC apoptosis assay of the most active
compounds was tested.
 all synthesized compounds were subjected to molecular modeling studies using
MOE software on the EGFR 3D structure and using Erlotinib as a reference
compound.
3

Abstract
Novel hybrids of pyridazine-pyrazoline were synthesized aiming to develop new
antiproliferative candidates. All compounds were submitted to the National Cancer
Institute (NCI), USA, and many were proved to have significant antiproliferative
activity. In addition, in vitro studies of the epidermal growth factor receptor (EGFR)
inhibition showed that compounds IXn, IXg, IXb and IXl exhibited excellent
inhibitory effect (IC50=0.65, 0.75, 0.82 and 0.84 μM, respectively) compared to
Erlotinib (IC50=0.95 μM). The mechanistic effectiveness in cell cycle progression,
apoptotic induction and gene regulation were assessed for the promising compounds
IXg and IXn due to their significant EGFR inhibition. Flow cytometeric analysis
indicated that compounds IXg and IXn result in increased cell numbers in phase G2/M,
suggesting cell cycle arrest in phase G2/M in UO-31cells. Furthermore, real time PCR
assay illustrated that compounds IXg and IXn elevated Bax/Bcl2 ratio which confirmed
the mechanistic pathway of them. Moreover, the apoptotic induction of UO-31 renal
cancer cells was enhanced effectively through activation of caspase-3 by compounds
IXg and IXn. On the other hand, molecular docking study was performed to investigate
binding mode of interaction of compounds with EGFR-PK in the active site with the
aim of rationalizing its promising inhibitory activity. Finally, based on the
aforementioned findings, compounds IXg and IXn could be considered as effective
apoptosis modulators and promising leads for future development of new anti-renal
cancer agents.
1. Introduction
Despite the rapid advance in drug discovery, the lack of effectiveness, safety and
selectivity of anticancer drugs currently available remained as the most important
unsolved issue [1]. Targeted therapy, a special type of chemotherapy, is a useful method
4

for preventing the growth and spread of cancer cells by targeting specific genes or
proteins [2]. Advancing our knowledge of molecular and cellular biology enhances our
ability to target specific cancer cell functions [3]. The human epidermal growth factor
receptor (EGFR) is a tyrosine kinase (RTK) transmembrane receptor consisting of a
single polypeptide chain of 1186 amino acids [4, 5]. This receptor belongs to the
receptor family ErbB (human epidermal receptor, HER) which comprises four
structurally related members EGFR (ErbB1), ErbB2 (HER2), ErbB3 (HER3) and
ErbB4 (HER4) [6]. EGFR and members of its family play crucial roles in controlling
many cellular processes including cell migration, survival, and proliferation [7]. EGFR
may be triggered aberrantly by mutations or over-expression, leading to cell
proliferation, angiogenesis, metastasis, and anti-apoptosis. EGFR is over-expressed in
a variety of epidermal cancers including head and neck, chest, prostate, breast, cervical,
brain and pancreatic cancers [8-10]. Considering that EGFR is a rational target for
antitumor strategies, one of the most effective methods of inhibiting EGFR is blocking
tyrosine kinase by small-molecule inhibitors at the ATP-binding site in the cytoplasmic
domain [6].
In recent years, various nitrogen heterocyclic rings have remarkably complex
biological properties and belong to one of the most important classes of medicinal
chemistry compounds [11]. Pyrazole motif shows many applications in the field of
pharmaceutical and medicinal chemistry. They have anti-inflammatory [12], fungistatic
[13], analgesic [14], antibacterial [15], and potent antitumor activities [16-21]. A series
of novel compounds containing the pyrazole motifs were reported as potent anticancer
agents targeting EGFR tyrosine kinase [22-26]. Furthermore, pyrazoline nucleus
represent an important core for triaryl substituted derivatives such as compound 1 (Fig.
1) that had been reported to exhibit powerful cytotoxic activity against different cancer
cell lines [27]. In addition, the strategy of hybridization of pyrazoline core with different
heterocyclic nuclei such as thiophene (compound 2) [18] and pyrimidine (compound 3)
[17] proved effective in production of potent antiproliferative analogs. Therefore, the
pyrazoline ring is an advantageous choice for the synthesis of pharmaceutical
compounds with diverse biological activities and good safety profiles [28-30]. On the
other hand, pyridazine is considered a significant group of heterocyclic compounds
which contains two adjacent nitrogen atoms. In recent years, there has been an
5

increasing interest in the pyridazine ring system due its diverse biological activities and
other beneficial applications [31, 32]. Various pyridazine derivatives have been
reported to possess increasing attention in the synthesis and evaluation of their
biological properties. This indicates that various substituted pyridazine compounds
possess different types of pharmacological activities, such as antidiabetic [33],
antimicrobial [34], antihypertensive [35], anti-inflammatory [36], and antiproliferative
agents [37-40]. Pyridazine 4 (Fig. 1) revealed promising cytotoxic activity [41]. A
series of novel compounds containing the pyridazine moiety were reported as potent
anticancer agents targeting EGFR tyrosine kinase [42]. Finally, from the molecular
design point of view, the combination of two pharmacophores into one single molecule
represents one of the most important methods that can be used to synthesize new
anticancer molecules [43-45].
Cl
N
CH3
CN
NH
O
Cl
O
N
Pyrazoline-Heterocyclic
Core Hybridization
S
N
H3C
N
N
N
N
N
N
Cl
H3C
H3C
F
H3C
O
H3C
1
O
3
2
Pyrazoline Core
with triaryl substitution
Pyrazoline-Pyrimidine Hybrids
Core with triaryl substitution
Pyrazoline-Thiophene Hybrids
Core with triaryl substitution
R1
R2
N
2-Cl
H
HN
N
N
Pyrazoline-Pyridazine
Core Hybridization
3-OCH3
2-F
3-F
CN
N
N
4-F
R2
H3C
3-CH3
4-Cl
O
R1
4
Target
Compounds
Pyridazine Core
with triaryl substitution
3-OCH3 - 4-F
Fig. 1. Structures of reported antiproliferative compounds with heterocyclic core and triaryl
substitution and the structure of the target pyrazoline-pyridazines core hybridization.
This work therefore adopted the hybridization of pyrazoline and pyridazine core
with triaryl substitution for the synthesis of a novel pyrazoline/pyridazine hybrids IXa-
n, aiming to obtain new promising antiproliferative compounds (Fig. 1). The newly
synthesized target compounds were sent to the National Cancer Institute (NCI) for
assessment of their anticancer activity. Additionally, the most active candidates have
6

been tested against target EGFR tyrosine kinase inhibition. Also, this study determined
annexin V-FITC apoptosis assay. Moreover, the cell cycle activity was detected for the
most potent compounds, to get an overview about the possible stage at which the new
derivatives could suppress the growth of cancer cells. Furthermore, levels of Bax, Bcl2
and caspase-3 was also determined in this study.
2. Results and discussion
2.1. Chemistry
Compound III was prepared via the reaction of acetophenone I with glyoxilic acid
II in the presence of hydrazine hydrate [46]. Furthermore, the reaction between
phosphorous oxychloride and compound III afford 3-chloropyridazine IV which has
been reacted with hydrazine hydrate to obtain V (Scheme 1) [47].
On the other hand, Chalcones derivatives VIIIa-n were prepared by the Claisene
Schmidt condensation between acetophenones VIa or VIb and appropriate aldehyde
derivatives VII [48-57]. Finally, the target compounds IXa-n were prepared by reaction
of V with the appropriate chalcones VIII a-n in absolute ethanol and in the presence of
sodium hydroxide (Scheme 1). All the final compounds IXa-n were characterized by
IR, MS, 1HNMR, 13CNMR and elemental analysis.
7

H
N
O
O
H
O
O
N
CH3
i
+
OH
III
N
I
II
ii
N
NHNH2
Cl
N
V
N
iii
IV
O
O
O
iv
CH3
H
R1
R2
R1
R2
+
VIII
VI
VII
IX R1
IX R1
R2
R2
a
b
c
d
e
f
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
h
i
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
H
H
2-F
3-F
4-F
3-CH3
4-Cl
2-F
N
N
v
j
3-F
V
+
VIII
k
l
4-F
N
N
R2
3-CH3
4-Cl
m
n
R1
g
3-OCH3 - 4-F
3-OCH3 - 4-F
IX
Scheme 1: Synthesis of compounds IXa-n; Reagents and conditions: (i) hydrazine hydrate, EtOH,
reflux, 4h; (ii) phosphorous oxychloride, reflux, 2h; (iii) hydrazine hydrate, EtOH, reflux,
10h.; (iv) ethanolic NaOH, r.t. 6h; (v) ethanolic NaOH, reflux, 15h.
2.2 Biological activity
2.2.1. Screening of anticancer activity and SAR discussion:
Data analysis showed that most synthesized compounds demonstrated different
level of growth inhibition against the nine panels of tumor cell lines (Table 1 and 2).
Furthermore, cell lines of ovarian cancer IGROV1, breast cancer T-47D, renal cancer
CAKI-1 and UO-31 were sensitive to almost all evaluated compounds. In this study,
we have two series of compounds, first series bearing Cl moiety at position 2 (lead
compound IXa), the other series bearing methoxy group at position 3 (lead compound
IXh). Structure–activity correlation was based on the obtained results and in
comparison, to the lead compounds, IXa and IXh by introducing different aryl moieties
at 5-position of pyrazole moiety.
8

The first series, lead compound IXa with phenyl moiety at position 5 displayed
cell growth inhibition for ovarian cancer IGROV1 (30.19%), renal cancer CAKI-1
(38.28%), renal cancer UO-31 (34.60%), and breast cancer T-47D (41.75%).
Substitution with flouro group at position 2 of the phenyl moiety IXb increase
selectivity against renal cancer CAKI-, renal cancer UO-31, and breast cancer T-47D
with growth inhibition (51.05%, 39.54%, 49.22%) respectively.
On the other hand substitution with flouro group at position 3 IXc increase
selectivity on renal cancer CAKI-1 and UO-31 with cell growth inhibition 40.10%,
46.04%, while substitution with flouro group at position 4 IXd shows marked increase
growth inhibition against ovarian cancer OVCAR-4 (46.44%), renal cancer CAKI-1
(62.98%), renal cancer UO-31 (51.58%), breast cancer T-47D (55.56%). Compound
IXe with 3 methyl group at position 3 show only increase in sensitivity toward renal
cancer UO-31 (cell growth inhibition: 46.54%). Compound IXf with Cl moiety at
position 4 decreased in sensitivity toward ovarian cancer IGROV1, breast cancer T-
47D, renal cancer CAKI-1, UO-31, cell lines. Furthermore compound IXg with flouro
group at position 4 and methoxy group at position 3 of the phenyl moiety show marked
increase in cell growth inhibition of ovarian cancer IGROV1 (47.17%), renal cancer
CAKI-1 (45.86%), renal cancer UO-31 (49.22%).
Table 1: In vitro antiproliferative activity results of compounds IXa - IXg against sixty
human tumor cell lines; results were provided as a percentage of cell growth
promotion.
Panel
Subpanel
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
IXa
IXb
93.16
103.32
88.71
81.35
86.37
81.35
96.50
89.85
IXc
IXd
IXe
IXf
IXg
95.17
98.84
81.65
81.48
87.78
77.36
90.54
90.78
98.76
96.07
87.85
88.11
92.61
87.07
92.13
82.98
81.77
92.04
65.68
56.77
64.11
75.76
77.62
77.68
98.56
91.22
81.57
87.58
85.27
82.51
88.47
84.38
98.99
108.66
95.98
94.93
94.73
85.40
98.96
93.69
107.00
95.37
88.97
82.30
92.04
83.78
88.83
79.42
Leukemia
A549/ATCC
EKVX
HOP-62
90.19
87.29
87.45
89.08
78.90
89.73
88.15
75.41
81.97
84.10
79.79
74.29
94.55
79.65
86.45
91.65
94.24
91.77
86.29
76.02
77.67
HOP-92
NCI-H226
NCI-H23
Non-small
cell lung
cancer
95.44
96.73
97.59
84.59
91.38
99.26
97.96
85.27
94.12
97.98
98.02
84.31
77.87
95.68
87.15
69.25
85.96
100.53
88.14
98.53
103.83
99.42
86.56
100.12
97.57
NCI-H322M
NCI-H460
NCI-H522
80.86
96.21
88.20
9

COLO 205
HCC-2998
100.76
107.11
105.93
91.34
81.80
104.77
97.59
102.88
81.35
92.20
103.70
80.60
90.15
106.01
85.92
100.53
99.42
57.66
81.24
94.09
69.17
91.25
98.82
93.94
106.80
102.10
80.33
98.59
HCT-116
HCT-15
HT29
Colon cancer
CNS cancer
80.69
94.17
97.48
92.58
101.96
93.55
94.08
88.73
89.79
91.78
92.66
100.91
101.65
98.77
85.36
105.77
81.50
69.81
100.31
80.47
103.28
93.50
92.60
90.39
92.86
78.45
99.31
61.72
98.54
92.49
103.62
65.40
76.29
105.04
80.57
91.78
99.92
94.98
87.69
99.04
92.61
90.07
68.90
87.90
92.61
91.42
100.74
102.71
101.52
90.05
100.44
74.53
73.86
97.84
81.84
96.41
91.45
87.92
96.86
97.68
82.57
94.76
48.95
88.25
91.46
111.44
60.46
76.87
102.57
82.85
85.12
96.77
91.23
91.12
100.12
94.46
92.93
86.82
89.45
92.19
97.55
101.44
107.21
104.43
89.89
101.79
78.60
66.17
98.08
83.29
98.22
94.93
94.04
87.85
100.17
90.80
95.97
59.90
98.64
91.61
131.33
53.96
76.75
106.17
84.78
69.48
78.34
85.94
74.16
86.94
83.32
83.90
76.41
77.22
86.84
76.90
96.33
93.70
95.62
78.17
100.23
72.37
58.39
72.38
53.56
98.63
80.87
81.18
78.50
89.38
79.34
83.88
37.02
88.06
79.67
130.89
48.42
56.88
91.83
66.25
58.71
92.58
89.23
83.15
101.02
96.72
88.94
66.06
84.36
88.58
87.96
98.06
97.30
101.89
84.12
98.22
76.15
68.46
92.77
79.22
100.68
83.56
86.84
83.71
89.35
84.79
95.03
60.37
93.25
92.87
108.60
53.46
71.43
99.00
80.42
101.87
97.67
98.75
92.14
101.28
98.98
100.18
65.63
92.82
95.15
96.19
101.20
114.51
106.58
90.08
103.78
89.63
85.40
97.26
97.44
101.42
97.83
101.30
97.73
105.45
103.84
101.48
64.66
109.71
94.68
146.06
67.36
91.59
109.86
95.27
81.63
92.94
89.65
87.11
99.90
95.30
94.82
68.24
90.24
94.52
94.32
98.94
102.37
102.97
79.05
95.92
82.50
52.83
80.63
80.94
99.38
89.39
89.19
83.00
94.91
88.26
90.37
54.14
96.45
97.66
151.25
50.78
71.05
96.85
82.33
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
Melanoma
Ovarian
cancer
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
Renal cancer
Prostate
cancer
MDA-MB-
231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
89.09
93.32
95.54
58.25
96.89
77.05
93.84
98.34
50.78
102.35
85.11
89.59
95.11
73.51
107.89
75.21
80.00
87.39
44.44
79.02
91.55
94.30
94.01
61.76
97.37
92.22
100.42
97.55
69.53
113.45
91.95
91.86
95.15
60.67
99.70
Breast cancer
On the other hand, the second series, lead compound IXh displayed cell growth
inhibition for ovarian cancer IGROV1, renal cancer CAKI-1, renal cancer UO-31 and
breast cancer T-47D (47.11%, 33.37%, 41.31%, 20.31%) respectively. Substitution
with flouro group at position 2 of the phenyl moiety IXi increase selectivity against
10

renal cancer CAKI-1 (cell growth inhibition: 49.73%), while substitution with flouro
group at position 3 IXj increase cell growth inhibition of renal cancer UO-31 (47.27%).
Furthermore, compounds IXk (with flouro moiety at position 4) IXl (with methyl
moiety at position 3), and IXm (with Cl moiety at position 4) show decreased in
sensitivity toward ovarian cancer IGROV1, breast cancer T-47D, renal cancer CAKI-
1and UO-31, cell lines. Compound IXn with flouro group at position 4 and methoxy
group at position 3 of the phenyl moiety show increase in cell growth inhibition for
renal cancer UO-31 (35.44%), breast cancer T-47D (59.08%).
Table 2: In vitro antiproliferative activity results of compounds IXh - IXn against sixty
human tumor cell lines; results were provided as a percentage of cell growth
promotion.
Panel
Subpanel
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
IXh
99.59
101.11
92.60
90.64
97.99
95.27
93.99
NT
IXi
95.14
104.71
88.84
76.66
87.99
82.32
88.73
76.41
81.98
70.54
74.42
IXj
IXk
99.70
98.66
96.80
96.78
104.50
86.70
96.99
91.46
91.27
87.34
89.69
IXl
96.26
103.81
87.33
86.48
89.85
82.86
94.54
82.79
89.82
85.08
85.03
IXm
90.56
87.37
84.32
82.54
78.08
79.14
91.02
83.26
90.71
91.47
85.24
IXn
94.47
97.89
84.03
82.21
80.57
84.67
95.14
86.73
99.67
93.01
92.93
96.27
86.76
83.25
80.54
81.10
78.32
85.57
83.81
95.20
75.85
82.55
Leukemia
79.40
77.68
71.14
HOP-92
NCI-H226
Non-small cell
lung cancer
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
90.40
85.48
97.28
93.49
106.93
98.98
86.20
88.64
94.38
86.51
107.55
105.04
89.19
92.34
97.79
80.65
94.30
98.51
92.08
92.98
99.28
94.34
107.01
100.56
85.75
91.18
97.71
89.29
99.73
103.84
82.20
96.54
88.38
83.83
88.30
97.28
89.90
101.50
96.79
91.48
96.53
105.27
HCT-116
HCT-15
HT29
80.09
NT
80.57
94.95
93.14
87.20
89.46
88.64
95.42
94.57
87.70
78.85
90.61
93.28
91.33
82.45
85.39
83.66
92.78
90.80
85.33
101.51
91.44
82.17
82.99
86.21
88.39
88.74
100.80
100.15
102.06
98.68
100.04
93.75
101.06
94.45
91.94
83.87
97.04
97.34
102.05
81.78
88.59
91.00
97.31
92.62
90.92
95.61
94.79
87.85
98.18
92.25
95.19
88.90
68.22
85.74
67.28
89.32
82.00
87.19
100.22
95.74
87.68
78.34
84.53
93.94
85.99
68.24
91.02
82.86
93.00
92.26
91.28
100.24
96.31
92.84
103.49
90.87
100.96
94.59
Colon cancer
100.76
96.87
93.34
86.68
NT
93.49
87.63
91.83
91.13
84.38
87.93
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
CNS cancer
Melanoma
U251
MALME-3M
M14
MDA-MB-
435
101.22
102.59
100.95
98.46
99.98
83.75
102.04
105.21
100.83
97.46
97.92
94.64
100.87
98.06
SK-MEL-2
11

SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
99.68
88.62
96.56
75.70
52.89
91.33
93.48
95.02
94.23
105.33
91.86
98.35
73.07
61.59
91.72
82.61
98.16
93.71
98.38
89.43
96.58
77.23
57.14
89.90
84.39
92.50
87.66
103.04
98.36
108.24
94.59
89.38
106.96
99.68
99.58
99.54
101.89
98.22
99.92
75.52
72.10
99.90
85.46
90.35
93.71
98.85
94.52
98.01
79.65
72.62
96.92
89.54
96.34
89.08
106.00
84.86
104.54
86.96
85.75
100.73
76.78
100.07
93.38
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
Ovarian cancer
92.91
94.32
86.23
101.68
95.01
93.79
95.24
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
84.91
102.02
57.41
87.47
66.63
91.73
91.35
145.99
58.69
84.53
101.12
NT
89.75
106.26
78.32
87.51
50.27
102.26
90.47
170.13
62.38
79.65
102.87
83.96
85.32
86.19
66.28
90.98
68.52
85.00
89.07
131.49
52.73
73.51
100.08
79.82
96.28
99.25
79.22
98.65
79.54
96.96
93.20
125.03
85.86
100.02
102.87
93.95
92.99
100.25
76.01
97.95
65.27
92.08
88.15
143.16
75.67
85.93
98.84
82.71
79.34
89.56
75.27
95.81
71.06
98.11
98.59
137.87
67.82
71.20
99.93
76.22
100.10
98.43
76.08
101.58
73.33
110.69
101.32
109.39
64.56
Renal cancer
78.78
102.32
85.62
Prostate cancer
MDA-MB-
231/ATCC
HS 578T
BT-549
T-47D
74.15
78.02
86.48
86.96
77.70
85.54
97.06
94.46
88.41
79.69
81.30
91.37
74.00
88.48
93.30
77.44
95.79
96.65
99.94
95.78
94.15
85.94
85.05
91.63
72.82
94.14
97.45
40.92
Breast cancer
MDA-MB-
468
89.67
97.15
92.35
100.47
99.54
99.50
95.35
NT: Not tested.
2.2.2. IC50 determination of the synthesized compounds against EGFR
All new synthesized compounds IXa-n were further investigated for their
inhibitory effect on human EGFR using Erlotinib as a positive control to determine
their IC50 values as shown in (Table 3). The tested compounds showed significant
inhibitory effect (IC50=5.5–0.65 μM). It was found that compounds IXn, IXg, IXb and
IXl inhibition effect more than Erlotinib towards EGFR (IC50=0.65, 0.75, 0.82 and 0.84
μM, respectively) compared to Erlotinib (IC50=0.95 μM). Compound IXc had the same
potency as Erlotinib against EGFR.
Table 3: Inhibition activities of compounds IXh - IXn against EGFR.
Comp.
IXa
EGFR IC50 (µM)
2.1 ± 0.12
IXb
0.82 ± 0.06
IXc
0.95 ± 0.07
12

IXd
IXe
3.7 ± 0.26
3.9 ± 0.2
IXf
IXg
IXh
IXi
4.8 ± 0.25
0.75 ± 0.05
1.7 ± 0.14
5.5 ± 0.41
4.1 ± 0.3
IXj
IXk
IXl
IXm
IXn
Erlotinib
5.3 ± 0.43
0.84 ± 0.06
3.1 ± 0.23
0.65 ± 0.03
0.95 ± 0.08
2.2.3. Cell cycle analysis
Results in Tables 1 and 2 revealed that compounds IXg and IXn exhibited potent
cytotoxic activity towards renal cancer cell line UO-31. In this manner, the effect of
both compounds on the cell cycle progression of UO-31 cancer cells were performed
to carry out preliminary mechanistic studies of their anticancer activity. The cancer
cells were incubated for 24 h with compounds IXg and IXn at 10 μM. Results analysis
indicated that both compounds resulted in increased of cell numbers in phase G2/M
(50.11 % vs. 45.26 % and 12.39 %), suggesting that cell cycle was arrested in phase
G2/M (Table 4, Fig. 2).
Table 4: Effect of compounds IXg and IXn on the cell cycle progression of UO-31
cells.
Code
%G0/G1
24.45
%S
25.44
26.6
%G2/M
50.11
%Pre-G1
20.17
Comment
Cell growth arrest@G2/M
IXg/ UO-31
IXn/ UO-31
Cont. (UO-31)
Cell growth arrest@G2/M
28.14
45.26
15.36
51.28
36.33
12.39
1.85
13

Control
IXg
IXn
Fig. 2. Cell cycle analysis and apoptosis effect in UO-31 cell line when treated with compounds IXg and
IXn.
2.2.4. Annexin V-FITC apoptosis assay
Cell cycle assay of UO-31 cancer cells treated with compounds IXg and IXn
showed pre-G1 peak appearance which emphasized apoptosis induction. The cells were
stained with Annexin V / PI, incubated for 24 h and analyzed to confirm the efficacy of
these compounds to cause apoptosis. It has been found that the early and late apoptosis
induced by the compounds being studied definitely suggested their ability to cause
meaningful apoptosis rates with 1.5 and 2.7 % necrosis respectively (Table 5, Fig. 3).
In addition, the percentages of late apoptosis (10.29% and 7.25%) induced by IXg and
IXn were higher than those of early apoptosis (8.37% and 5.41%) which makes
recovery of apoptotic cells more difficult to be healthy.
Table 5. Induced apoptosis and necrosis on UO-31 cells by IXg and IXn.
Apoptosis
Necrosis
Total
20.17
15.36
1.85
Early
8.37
5.41
1.13
Late
10.29
7.25
IXg/UO-31
IXn/UO-31
Cont. (UO-31)
1.51
2.7
0.29
0.43
14

25
20
15
10
5
0
Cont. (UO-
31)
IXg/UO-31
IXn/UO-31
Apoptosis Total
Apoptosis Early
Fig. 3. Effect of compounds IXg and IXn on the percentage of V-FITC-positive annexin staining in UO-
31 cells.
2.2.5. Effects on the levels of Bax, Bcl-2 and active caspase-3
Apoptosis is regulated by various factors including stimulation of the apoptotic
pathway through caspase-3, inducers (Bax protein) and suppressors (Bcl-2 protein)
[58]. The key members of apoptotic pathways are the cysteine protease caspase family
[59]. Caspase-3 is an apoptosis executioner and that its activation contributes to
apoptosis induction [58]. Many studies on human cancers have revealed a relationship
between increased Bcl-2 levels, decreased Bax levels and uncontrolled cell growth [59,
60]. Therefore, in this study, the levels of Bcl-2, Bax and caspase-3 were determined
after treatment with IXg and IXn in UO-31cells in order to examine the effect of both
compounds on these apoptotic biomarkers. The findings presented in (Table 6) showed
that IXg and IXn raised the Bax level by 7.3 and 6.6 folds, respectively, and decreased
the Bcl-2 level by 0.31 and 0.44 folds, respectively, compared with the control. Also
results showed that the active caspase-3 was significantly overexpressed by 7.1 and 5.9
folds relative to the control indicating the apoptosis induction potential of these
compounds.
15

Table 6. Bax, Bcl2 and Caspase-3 analysis results after treatment with compound IXg
and IXn.
Comp.
13
14
Cont.
Bax conc. pg FLD
Bcl-2 Conc. pg
FLD
0.317
0.44
1
Caspase-3 conc. pg FLD
392.17
356.25
53.49
7.33
6.6
1
1.84
2.56
5.79
419.25
347.11
58.31
7.19
5.95
1
2.3. Molecular docking
Molecular docking studies are an important method for studying molecular
interactions between the synthesized ligands and main amino acids at the respective
protein binding site [61]. Ligand – protein interaction activity at the binding active site
was measured on the basis of the docking score values determined using MOE 2015.10.
In the present research, all synthesized compounds were subjected to molecular
modeling studies using MOE software on the EGFR 3D structure and using Erlotinib
as a reference compound Table 7, in order to predict the behavior of the synthesized
compounds as an anticancer agents. The results of the docking studies showed that the
binding free energy of the compounds being examined exhibited favorable docking
complexes with the active target protein site. The compounds being studied displayed
interactions with the active EGFR sites with the same amino acid as did the reference
drug, Erlotinib. Although, all the tested compounds showed significant docking score
free energy compared to Erlotinib, compounds IXc, IXg, IXj, IXe, and IXn showed
the best docking score free energy (Table 7).
Table 7. Docking energy scores (kcal/mol) derived from the MOE for Compounds IXa-
n and Erlotinib.
Comp.
No.
IXa
IXb
IXc
IXd
IXe
IXf
rmsd_
refine
1.1158
1.1512
1.5914
1.2320
1.1562
2.3522
1.2496
2.0378
Score
E_conf
E_place E_score1 E_refine E_score2
-7.5032
-7.4828
-8.0715
-7.5669
-7.6098
-7.5063
-7.7437
-7.4173
52.5396 -67.4750 -13.0213 -43.1217 -7.5032
52.4754 -82.2369 -12.2609 -35.5909 -7.4828
46.3532 -53.7557 -11.9093 -42.3909 -8.0715
52.2871 -76.9072 -13.2147 -41.1207 -7.5669
50.0204 -99.3674 -11.9157 -40.4771 -7.6098
49.8316 -71.1179 -12.5182 -43.0492 -7.5063
70.0082 -93.1728 -12.3985 -37.4756 -7.7437
61.9342 -66.4482 -12.0435 -40.5859 -7.4173
IXg
IXh
16

IXi
IXj
IXk
IXl
IXm
IXn
-7.4513
-7.6264
-7.4602
-7.4967
-7.4797
-7.5941
1.1826
1.1773
1.9206
1.9904
1.3109
1.8792
2.9191
55.3965 -67.8661 -11.5407 -38.7661 -7.4513
62.3024 -58.6817 -12.0223 -41.9693 -7.6264
64.0970 -66.7235 -12.1795 -40.9835 -7.4602
62.7921 -66.7189 -11.4621 -39.5500 -7.4967
55.0005 -86.0764 -13.1680 -38.6300 -7.47977
78.2140 -68.6826 -12.3753 -40.3379 -7.5941
-34.3322 -71.5678 -12.4086 -42.4795 -7.3430
Erlotinib -7.3430
Score; lower scores are more favorable, rmsd_refine; the root mean square deviation of the pose from
the docking pose compared to the co-crystal ligand position, E_conf; free binding energy of the
conformer, E_place; free binding energy from the placement stage, E_score 1; free binding energy from
the first rescoring stage, E_refine; free binding energy from the refinement stage, E_score 2; free binding
energy from the second rescoring stage.
Compound IXg and IXn showed an excellent docking score (-7.7437 kcal/mol and
-7.5941 kcal/mol) compared to Erlotinib docking score (-7.3430 kcal/mol), and both
formed a direct interaction with most of the amino-acids that Erlotinib interacted with.
Where, Erlotinib had a direct interaction with amino-acids VAL702, LYS721,
CYS751 and THR766 in the active site (Fig. 4A), compound IXg and IXn showed
interactions with amino-acids LEU694, LYS721 and THR766 in the active site (Fig4
4B and 4C). Also, compounds IXg and IXn showed high degrees of superimposition
Erlotinib into EGFR active sites (Fig. 4B and 4C). These results were in a perfect
match with the EGFR enzyme inhibition assay where compounds IXg and IXn had
IC50s of (0.75 µM/mL and 0.65 µM/mL, respectively) compared to that of Erlotinib
(0.95 µM/mL) (Table 3).
A) Interaction of Erlotinib with amino-acids VAL702, LYS721, CYS751 and THR766.
17

B) Interaction of IXg with amino-acids LEU694, LYS721 and THR766 and superimposition of IXg
(shown as grey sticks) with Erlotinib (shown as cyan sticks).
C) Interaction of IXn with amino-acids LEU694, LYS721 and THR766 and superimposition of IXn
(shown as grey sticks) with Erlotinib (shown as cyan sticks).
Fig. 4. Docking of compounds IXg, IXn and Erlotinib into EGFR active sites.
3. Experimental
3.1. Chemistry
IR spectra had been measured on a Perkin Elmer-9712 spectrophotometer.
Elemental microanalysis was performed at the Micro-analytical Laboratory at Cairo
1
University, Egypt. H NMR spectrometer and 13C NMR spectra were measured on
Bruker 400 MHz NMR. For the evaluation of mass spectra, a Shimadzu Qp-2010 Plus
was used. 6-phenylpyridazines V [46, 47] and Chalcones VIII [48-57] have been
prepared according to the procedures indicated.
3.1.1. General procedure for preparation of IXa-n
In absolute ethanol (15 mL), a mixture of the corresponding chalcones VIIIa-n (1
mmol), hydrazine derivatives V (1 mmol) and sodium hydroxide (2.5 mmol) were
heated for 15 h under reflux. Filtered and washed with water and crystallized with
ethanol.
18

3.1.1.1. 3-(3-(2-chlorophenyl)-4,5-dihydro-5-phenylpyrazol-1-yl)-6-phenyl-
pyridazine (IXa)
White powder (yield 64%); m.p. 107-109 oC; IR (υmax./cm-1) 3031 (C-H aromatic),
1649 (C=N) and at 1588 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.08 (d, J = 9.3
Hz, 1H, Ar-H), 8.00 (d, J = 7.6 Hz, 2H, Ar-H), 7.81-7.85 (m, 1H, Ar-H), 7.78 (d, J =
9.5 Hz, 1H, Ar-H), 7.56-7.60 (m, 1H, Ar-H), 7.41-7.50 (m, 6H, Ar-H), 7.30-7.36 (m,
4H, Ar-H), 5.94 (dd, J = 5.1, 12.0 Hz, 1H, pyrazoline-CH), 4.16 (dd, J = 12.0, 17.9 Hz,
1H, one proton of pyrazoline-CH2), 3.31 (d, J = 5.4 Hz, 1H, one proton of pyrazoline-
CH2); 13CNMR (100 MHz, DMSO-d6) δ ppm: 155.9, 152.4, 151.1, 142.9, 136.8, 131.9,
131.4, 131.2, 131.1, 130.7, 129.4, 129.3, 129.2, 127.9, 127.7, 126.7, 126.3, 126.2,
115.4, 62.1, 45.7; MS (m/z (R.I. %)); [M]+ 410 (12.7), [M+2]+ 412 (4.7), 272 (100),
170 (17.4); Anal. Calcd. for C25H19ClN4 (410.13); % C, 73.08; H, 4.66; N, 13.64,
Found: % C, 73.21; H, 4.75; N, 13.71.
3.1.1.2. 3-(3-(2-chlorophenyl)-5-(2-fluorophenyl)-4,5-dihydropyrazol-1-yl)-6-
phenyl-pyridazine (IXb)
White powder (yield 60%); m.p. Over 280 oC; IR (υmax./cm-1)3054 (C-H aromatic),
1649 (C=N) and at 1589 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.10 (d, J = 9.5
Hz, 1H, Ar-H), 8.00 (d, J = 7.6 Hz, 2H, Ar-H), 7.82-7.86 (m, 1H, Ar-H), 7.79 (d, J =
9.3 Hz, 1H, Ar-H), 7.55-7.61 (m, 1H, Ar-H), 7.42-7.50 (m, 5H, Ar-H), 7.29-7.34 (m,
1H, Ar-H), 7.20-7.28 (m, 2H, Ar-H), 7.13 (t, J = 7.3 Hz, 1H, Ar-H), 6.09 (dd, J = 5.6,
12.2 Hz, 1H, pyrazoline-CH), 4.19 (dd, J = 12.4, 17.7 Hz, 1H, one proton of pyrazoline-
CH2), 3.30 (m, 1H, one proton of pyrazoline-CH2); MS (m/z (R.I. %)); [M]+ 428 (25.5),
[M+2]+ 430 (9.7), 290 (79.5), 272 (100), 170 (31.2); Anal. Calcd. For C25H18ClFN4
(428.12); % C, 70.01; H, 4.23; N, 13.06, Found: % C, 70.09; H, 4.35; N, 13.15.
3.1.1.3. 3-(3-(2-chlorophenyl)-5-(3-fluorophenyl)-4,5-dihydropyrazol-1-yl)-6-
phenylpyridazine (IXc)
o
Yellowish powder (yield 59%); m.p. 209-211 C; IR (υmax./cm-1) 3064 (C-H
aromatic), 1647 (C=N) and at 1590 (C=C); 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.10
(d, J = 8.8 Hz, 1H, Ar-H), 7.97-8.04 (m, 2H, Ar-H), 7.77-7.87 (m, 2H, Ar-H), 7.57 (br.
s, 1H, Ar-H), 7.44-748 (m, 6H), 7.16 (br. s, 2H, Ar-H), 7.06-7.13 (m, 1H, Ar-H), 5.87-
6.03 (m, 1H, pyrazoline-CH), 4.05-4.24 (m, 1H, one proton of pyrazoline-CH2), 3.32
19

(m, 1H, one proton of pyrazoline-CH2); MS (m/z (R.I. %)); [M-F]+ 409 (31.5), 386
(20.7), 306 (43.1), 55 (100); Anal. Calcd. for C25H18ClFN4 (428.12); % C, 70.01; H,
4.23; N, 13.06, Found: % C, 70.12; H, 4.19; N, 12.98.
3.1.1.4. 3-(3-(2-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydropyrazol-1-yl)-6-
phenyl-pyridazine (IXd)
o
Yellowish powder (yield 63%); m.p. 183-185 C; IR (υmax./cm-1) 3038 (C-H
aromatic), 1649 (C=N) and at 1588 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.07
(d, J = 9.5 Hz, 1H, Ar-H), 8.00 (d, J = 7.3 Hz, 2H, Ar-H), 7.80-7.86 (m, 1H, Ar-H),
7.77 (d, J = 9.5 Hz, 1H, Ar-H), 7.54-7.60 (m, 1H, Ar-H), 7.41-7.50 (m, 5H, Ar-H), 7.36
(dd, J = 5.5, 8.0 Hz, 2H, Ar-H), 7.16 (t, J = 8.8 Hz, 2H, Ar-H), 5.94 (dd, J = 5.4, 12.0
Hz, 1H, pyrazoline-CH), 4.14 (dd, J = 12.2, 17.9 Hz, 1H, one proton of pyrazoline-
CH2), 3.31-3.37 (m, 1H, one proton of pyrazoline-CH2); 13CNMR (100 MHz, DMSO-
d6) δ ppm: 155.9, 152.5, 151.1, 136.7, 131.9, 131.4, 131.3, 131.1, 130.6, 129.4, 129.3,
128.4, 128.3, 128.0, 126.3, 126.2, 116.1, 115.8, 115.5, 61.5, 45.6; MS (m/z (R.I. %));
[M]+ 428 (12.9), [M+2]+ 430 (4.7), 290 (100), 170 (15.5), 115 (35.3); Anal. Calcd. for
C25H18ClFN4 (428.12); % C, 70.01; H, 4.23; N, 13.06, Found: % C, 70.09; H, 4.27; N,
13.01.
3.1.1.5. 3-(3-(2-chlorophenyl)-4,5-dihydro-5-m-tolylpyrazol-1-yl)-6-phenyl-
pyridazine (IXe)
Buff powder (yield 55%); m.p. Over 280 oC; IR (υmax./cm-1) 3050 (C-H aromatic),
1648 (C=N) and at 1590 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.08 (d, J = 9.3
Hz, 1H, Ar-H), 8.00 (d, J = 6.9 Hz, 2H, Ar-H), 7.83 (br. s, 1H, Ar-H), 7.78 (d, J = 9.3
Hz, 1H, Ar-H), 7.54-7.61 (m, 1H, Ar-H), 7.41-7.51 (m, 5H, Ar-H), 7.18-7.25 (m, 1H,
Ar-H), 7.15 (br. s, 1H, Ar-H), 7.07 (t, J = 8.2 Hz, 2H, Ar-H), 5.83-5.95 (m, 1H,
pyrazoline-CH), 4.15 (dd, J = 12.4, 17.2 Hz, 1H, one proton of pyrazoline-CH2), 3.30
(br. s, 1H, one proton of pyrazoline-CH2), 2.28 (s, 3H, CH3); 13CNMR (100 MHz,
DMSO-d6) δ ppm: 155.9, 152.4, 151.1, 143.0, 138.4, 136.7, 131.9, 131.4, 131.2, 131.1,
130.7, 129.3, 129.2, 128.4, 128.0, 126.7, 126.3, 126.2, 123.1, 115.4, 62.1, 45.8, 21.5;
MS (m/z (R.I. %)); [M]+ 424 (9.7), [M+2]+ 426 (3.6), 286 (100), 170 (14.6), 115 (37.3)
); Anal. Calcd. for C26H21ClN4 (424.15); % C, 73.49; H, 4.98; N, 13.19, Found: % C,
73.55; H, 4.87; N, 13.25.
20

3.1.1.6. 3-(3-(2-chlorophenyl)-5-(4-chlorophenyl)-4,5-dihydropyrazol-1-yl)-6-
phenylpyridazine (IXf)
o
Yellowish powder (yield 60%); m.p. Over 280 C; IR (υmax./cm-1) 3059 (C-H
aromatic), 1677 (C=N) and at 1587 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.10
(d, J = 9.5 Hz, 1H, Ar-H), 8.00 (d, J = 7.6 Hz, 2H, Ar-H), 7.81-7.86 (m, 1H, Ar-H),
7.78 (d, J = 9.3 Hz, 1H, Ar-H), 7.35-7.42 (m, 6H, Ar-H), 7.11 (d, J = 8.1 Hz, 2H, Ar-
H), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 5.94 (dd, J = 5.5, 11.9 Hz, 1H, pyrazoline-CH), 4.16
(dd, J = 12.2, 17.6 Hz, 1H, one proton of pyrazoline-CH2), 3.33 (d, J = 5.6 Hz, 1H, one
proton of pyrazoline-CH2); 13CNMR (100 MHz, DMSO-d6) δ ppm: 155.9, 152.5, 151.2,
141.9, 138.6, 136.7, 136.3, 132.8, 131.9, 131.4, 131.2, 130.0, 129.3, 129.2, 128.3,
127.9, 126.4, 126.2, 115.4, 61.6, 45.5; MS (m/z (R.I. %)); [M]+ 444 (9.8), [M+2]+ 446
(6.7), 306 (100), 308 (37), 115 (50.2); Anal. Calcd. for C25H18Cl2N4 (444.09); % C,
67.42; H, 4.07; N, 12.58, Found: % C, 67.40; H, 3.93; N, 12.65.
3.1.1.7. 3-(3-(2-chlorophenyl)-5-(4-fluoro-3-methoxyphenyl)-4,5-dihydropyrazol-1-
yl)-6-phenylpyridazine (IXg)
Buff powder (yield 64%); m.p. 166-168 oC; IR (υmax./cm-1) 3059 (C-H aromatic),
1648 (C=N) and at 1589 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.09 (d, J = 9.5
Hz, 1H, Ar-H), 7.99 (d, J = 7.6 Hz, 2H, Ar-H), 7.81-7.86 (m, 1H, Ar-H), 7.73-7.76 (m,
2H, Ar-H), 7.33-7.37 (m, 4H, Ar-H), 7.06-7-09 (m, 2H, Ar-H), 6.94 (d, J = 8.8 Hz, 2H,
Ar-H), 5.92 (dd, J = 5.6, 12.2 Hz, 1H, pyrazoline-CH), 4.14 (dd, J = 12.2, 17.6 Hz, 1H,
one proton of pyrazoline-CH2), 3.84 (s, 3H, OCH3), 3.33 (d, J = 5.6 Hz, 1H, one proton
of pyrazoline-CH2); MS (m/z (R.I. %)): [M]+ 458 (13.5), [M+2]+ 460 (5.4), 320 (100),
197 (15.2), 115 (27); Anal. Calcd. for C26H20ClFN4O (458.13); % C, 68.05; H, 4.39; N,
12.21, Found: % C, 67.96; H, 4.42; N, 12.16.
3.1.1.8. 3-(4,5-dihydro-3-(3-methoxyphenyl)-5-phenylpyrazol-1-yl)-6-phenyl-
pyridazine (IXh)
Buff powder (yield 60%); m.p. 162-164 oC; IR (υmax./cm-1) 2960 (C-H aromatic),
1668 (C=N) and at 1597 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.06 (d, J = 8.8
Hz, 1H, Ar-H), 8.01 (d, J = 7.6 Hz, 2H, Ar-H), 7.80-7.84 (m, 2H, Ar-H), 7.62 (br. s,
1H, Ar-H), 7.29-7.44 (m, 6H, Ar-H), 7.16 (br. s, 2H, Ar-H), 7.01-7.8 (m, 2H, Ar-H),
21

5.92 (dd, J = 5.6, 12.2 Hz, 1H, pyrazoline-CH), 3.99 (dd, J = 12.2, 17.9 Hz, 1H, one
proton of pyrazoline-CH2), 3.82 (s, 3H, OCH3), 3.31 (dd, J = 5.6, 17.9 Hz, 1H, one
proton of pyrazoline-CH2); MS (m/z (R.I. %)): [M]+ 406 (22.5), 272 (100), 170 (14.3),
115 (26); Anal. Calcd. for C26H22N4O (406.18); % C, 76.83; H, 5.46; N, 13.78, Found:
% C, 76.75; H, 5.42; N, 13.69.
3.1.1.9. 3-(5-(2-fluorophenyl)-4,5-dihydro-3-(3-methoxyphenyl)pyrazol-1-yl)-6-
phenylpyridazine (IXi)
o
Yellowish powder (yield 56%); m.p. 123-125 C; IR (υmax./cm-1) 3060 (C-H
aromatic), 1647 (C=N) and at 1596 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.08
(d, J = 9.3 Hz, 1H, Ar-H), 8.00 (d, J = 7.8 Hz, 2H, Ar-H), 7.86 (d, J = 9.3 Hz, 1H, Ar-
H), 7.77-7.81 (m, 1H, Ar-H), 7.63 (br. s, 1H, Ar-H), 7.31-7.44 (m, 5H, Ar-H), 7.17-24
(m, 3H, Ar-H), 7.03-7.6 (m, 1H, Ar-H), 6.03 (dd, J = 5.5, 12.1 Hz, 1H, pyrazoline-CH),
4.04 (dd, J = 12.2, 17.9 Hz, 1H, one proton of pyrazoline-CH2), 3.84 (s, 3H, OCH3),
3.28 (dd, J = 5.6, 17.9 Hz, 1H, one proton of pyrazoline-CH2); MS (m/z (R.I. %)): [M]+
424 (35.3), 290 (80.4), 272 (100), 170 (30), 115 (44.2); Anal. Calcd. for C26H21FN4O
(424.17); % C, 73.57; H, 4.99; N, 13.20, Found: % C, 73.49; H, 5.12; N, 13.13.
3.1.1.10. 3-(5-(3-fluorophenyl)-4,5-dihydro-3-(3-methoxyphenyl)pyrazol-1-yl)-6-
phenylpyridazine (IXj)
o
Yellowish powder (yield 55%); m.p. 177-179 C; IR (υmax./cm-1) 3082 (C-H
aromatic), 1649 (C=N) and at 1593 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.08
(d, J = 9.5 Hz, 1H, Ar-H), 8.01 (d, J = 7.6 Hz, 2H, Ar-H), 7.86 (d, J = 9.3 Hz, 1H, Ar-
H), 7.36-7.51 (m, 7H, Ar-H), 7.01-7.16 (m, 4H, Ar-H), 5.94 (dd, J = 5.5, 12.1 Hz, 1H,
pyrazoline-CH), 4.01 (dd, J = 12.2, 17.9 Hz, 1H, one proton of pyrazoline-CH2), 3.83
(s, 3H, OCH3), 3.30 (dd, J = 5.6, 17.9 Hz, 1H, one proton of pyrazoline-CH2); 13CNMR
(100 MHz, DMSO-d6) δ ppm: 161.6, 159.9, 155.9, 152.6, 152.3, 146.1, 136.8, 133.2,
131.2, 130.4, 129.3, 126.2, 122.2, 119.4, 116.3, 115.4, 114.5, 114.3, 113.3, 111.7, 61.8,
55.7, 43.1; MS (m/z (R.I. %)): [M]+ 424 (21.9), 290 (100), 170 (16.2), 115 (33.7); Anal.
Calcd. for C26H21FN4O (424.17); % C, 73.57; H, 4.99; N, 13.20, Found: % C, 73.62;
H, 4.94; N, 13.29.
22

3.1.1.11. 3-(5-(4-fluorophenyl)-4,5-dihydro-3-(3-methoxyphenyl)pyrazol-1-yl)-6-
phenylpyridazine (IXk).
White powder (yield 63%); m.p. 185-187 oC; IR (υmax./cm-1) 3056 (C-H aromatic),
1649 (C=N) and at 1598 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.06 (d, J = 9.3
Hz, 1H, Ar-H), 7.99 (d, J = 7.6 Hz, 2H, Ar-H), 7.83 (d, J = 9.3 Hz, 1H, Ar-H), 7.68 (d,
J = 7.6 Hz, 2H, Ar-H),7.33-7.45 (m, 5H, Ar-H), 7.21-7.26 (m, 2H, Ar-H),7.03-7.09 (m,
2H, Ar-H), 5.92 (dd, J = 5.5, 12.2 Hz, 1H, pyrazoline-CH), 3.98 (dd, J = 12.2, 17.9 Hz,
1H, one proton of pyrazoline-CH2), 3.82 (s, 3H, OCH3), 3.31 (dd, J = 5.5, 17.9 Hz, 1H,
one proton of pyrazoline-CH2); MS (m/z (R.I. %)): [M]+ 424 (2), 421 (23.1), 135 (56.1),
57 (100); Anal. Calcd. for C26H21FN4O (424.17); % C, 73.57; H, 4.99; N, 13.20, Found:
% C, 73.65; H, 4.89; N, 13.31.
3.1.1.12. 3-(4,5-dihydro-3-(3-methoxyphenyl)-5-m-tolylpyrazol-1-yl)-6-phenyl-
pyridazine (IXl).
Yellow powder (yield 66%); m.p. 195-197 oC; IR (υmax./cm-1)3057 (C-H aromatic),
1647 (C=N) and at 1598 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.06 (d, J = 8.8
Hz, 1H, Ar-H), 7.97-8.01 (m, 2H, Ar-H), 7.84 (d, J = 9.3 Hz, 1H, Ar-H), 7.33-7.49 (m,
6H, Ar-H), 6.97-7.10 (m, 5H, Ar-H), 5.92 (dd, J = 5.5, 12.1 Hz, 1H, pyrazoline-CH),
3.98 (dd, J = 12.2, 17.8 Hz, 1H, one proton of pyrazoline-CH2), 3.82 (s, 3H, OCH3),
3.28 (dd, J = 5.6, 17.8 Hz, 1H, one proton of pyrazoline-CH2), 2.26 (s, 3H, CH3);
13CNMR (100 MHz, DMSO-d6) δ ppm: 164.4, 159.1, 155.0, 152.7, 144.7, 136.4, 130.2,
129.9, 129.4, 128.6, 128.3, 128.0, 127.6, 126.7, 126.1, 122.0, 121.8, 118.2, 116.8,
115.3, 114.5, 63.3, 55.3, 46.7, 21.5; MS (m/z (R.I. %)): [M]+ 420 (5.4), 135 (78.1), 57
(100); Anal. Calcd. for C27H24N4O (420.20); % C, 77.12; H, 5.75; N, 13.32, Found: %
C, 77.17; H, 5.69; N, 13.21.
3.1.1.13. 3-(5-(4-chlorophenyl)-4,5-dihydro-3-(3-methoxyphenyl)pyrazol-1-yl)-6-
phenylpyridazine (IXm).
Buff powder (yield 67%); m.p. 144-146 oC; IR (υmax./cm-1) 3036 (C-H aromatic),
1695 1649 (C=N) and at 1592 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.07 (d,
J = 9.3 Hz, 1H, Ar-H), 8.00 (d, J = 7.3 Hz, 2H, Ar-H), 7.85 (d, J = 9.5 Hz, 1H, Ar-H),
7.45-7.51 (m, 2H, Ar-H), 7.37-7.44 (m, 6H, Ar-H), 7.30-7.34 (m, 2H, Ar-H), 7.03 (d,
J = 6.9 Hz, 1H, Ar-H), 5.93 (dd, J = 5.5, 12.10 Hz, 1H, pyrazoline-CH), 4.01 (dd, J =
23

12.1, 17.7 Hz, 1H, one proton of pyrazoline-CH2), 3.83 (s, 3H, OCH3), 3.28 (dd, J =
13
5.5, 18.0 Hz, 1H, one proton of pyrazoline-CH2); CNMR (100 MHz, DMSO-d6) δ
ppm: 159.9, 155.8, 152.6, 152.2, 142.1, 136.8, 133.2, 132.1, 130.4, 129.3, 129.2, 128.2,
127.5, 126.2, 119.4, 116.3, 115.3, 111.7, 61.6, 55.7, 43.0; MS (m/z (R.I. %)): [M]+ 440
(25.8), [M+2]+ 442 (9.5), 306 (100), 308 (36.9); Anal. Calcd. for C26H21ClN4O
(440.14); % C, 70.82; H, 4.80; N, 12.71, Found: % C, 70.73; H, 4.89; N, 12.65.
3.1.1.14. 3-(5-(4-fluoro-3-methoxyphenyl)-4,5-dihydro-3-(3-methoxyphenyl)-
pyrazol-1-yl)-6-phenylpyridazine (IXn)
Yellow powder (yield 61%); m.p. 175-177 oC; IR (υmax./cm-1)3056 (C-H aromatic),
1650 (C=N) and at 1598 (C=C); 1HNMR (400 MHz, DMSO-d6) δ ppm: 8.07 (d, J = 9.3
Hz, 1H, Ar-H), 8.00 (d, J = 7.3 Hz, 2H, Ar-H), 7.86 (d, J = 9.5 Hz, 1H, Ar-H), 7.34-
7.53 (m, 6H, Ar-H), 7.07-7.20 (m, 2H, Ar-H), 7.03 (d, J = 6.6 Hz, 1H, Ar-H), 6.76 (br.
s, 1H, Ar-H), 5.91 (dd, J = 5.5, 11.9 Hz, 1H, pyrazoline-CH), 4.00 (dd, J = 12.2, 17.9
Hz, 1H, one proton of pyrazoline-CH2), 3.80-3.86 (m, 6H, 2-OCH3), 3.29 (dd, J = 5.5,
17.9 Hz, 1H, one proton of pyrazoline-CH2); 13CNMR (101 MHz, DMSO-d6) δ ppm:
159.9, 156.0, 152.6, 152.2, 149.8, 147.7, 139.9, 136.8, 133.3, 130.3, 129.3, 126.2,
126.1, 119.3, 117.9, 117.8, 116.5, 116.2, 115.4, 112.0, 111.7, 61.9, 56.4, 55.7, 43.2;
MS (m/z (R.I. %)): [M]+ 454 (25.8), 320 (100), 170 (13.5), 115 (25.5); Anal. Calcd. for
C27H23FN4O2 (454.18); % C, 71.35; H, 5.10; N, 12.33, Found: % C, 71.42; H, 4.98; N,
12.41.
3.2. In vitro cytotoxicity
In vitro cytotoxicity is made according to reported method[62]. The detailed procedures
were supplied in Supplementary data.
3.3. EGFR inhibition assay:
HTRF (homogeneous time-resolved fluorescence) assay method was used to
conduct inhibitory action of all newly synthesized compounds against EGFR [63].
EGFR kinase (BioAssay Systems - Kinase Assay Kit (EKIN-400)–(CA 94545, USA)
and its substrate were incubated for 5 min in a buffer solution with newly synthesized
compounds/Erlotinib to initiate the enzymatic reaction, then ATP was added to the
reaction mixture. Hold under 30 min at room temperature. The reaction was stopped by
24

adding detection reagents containing EDTA for 1 h then the IC50 values have been
determined.
3.4. Cell cycle analysis
UO-31 cells were seeded for 24 h at density of 1 x 105 cells per well and into six
well plates. Added fetal bovine serum (FBS, 10 percent), after cells incubated at 37 °C
and 5 percent CO2. The medium was replaced with (DMSO 0.1% v/v) containing the
10 μM of compounds IXn and IXg , then incubated for 48 h, washed with cold
phosphate buffered saline (PBS), fixed with 70% ethanol, rinsed with PBS then stained
with the DNA fuorochrome PI, kept for 15 min. at 37 °C. A FACS Caliber flow
cytometer was then used to test samples [64].
3.5. Annexin V-FITC apoptosis assay
Cells were incubated with the tested compounds IXn and IXg for 24. Then,
trypsinized and washed with cold phosphate-buffer saline (PBS), then stained with
Annexin V-FITC and PI in binding buffer for 15 min. at room temperature in the dark.
The samples were analyzed using the flow cytometer [65].
3.6. In vitro ElISA immunoassay for cell death modulators
The levels of anti-apoptotic marker Bcl2 as well as the apoptotic marker Bax were
measured in UO-31cells treated with the molecules IXg, IXn and PG for 24 h using
ELISA colorimetric kits. All the procedures were performed according to the
manufacturer’s instructions [66]. On the other hand, the activation of caspase 3 was
measured using DRG Caspase-3 (human) ELISA (EIA-4860) kit (DRG International
Inc., USA), according to the manufacturer instructions.
3.7. Molecular docking study
The crystal structures of EGFR domain co-crystallized with Erlotinib (PDB ID:
1M17) were retrieved from the RCSB Protein Data Bank [67, 68]. The bound water
and ligands not active in binding have been eliminated from the protein and added polar
hydrogen. Then, the protein was prepared in MOE with default options for docking
study using Protonate 3D protocol. The entire EGFR complex had been defined as a
docking receptor binding site. Docking was done using Triangle Matcher placement
25

method and London dG scoring function. The docking setup was initially validated by
redocking the co-crystallized ligand (Erlotinib) in the vicinity of the enzyme binding
site, then removing the Erlotinib molecule and inserting the target compound during
the molecular docking process. Types of docked protein interactions with ligand were
analyzed after molecular docking ended.
4. Conclusion
New series of pyridazine hybridized with pyrazoline derivatives were designed and
synthesized based on targeting EGFR. Preliminarily screening results at NCI, USA
showed the sensitivity of Ovarian cancer IGROV1, Breast cancer T-47D, Renal cancer
CAKI-1 and UO-31 cell lines to almost all screened compounds. EGFR inhibition for
the promising cytotoxic compounds showed that compounds IXn, IXg, IXb and IXl
exhibited inhibition effect more than Erlotinib. Also, compounds IXg and IXn showed
high cell accumulation at phase G2/M phase in UO-31cells confirming its arresting cell
cycle at G2/M phase. Furthermore, compounds IXg and IXn elevated Bax/Bcl2 ratio
and show a pronounced enhancement in the levels of the active caspases -3 which
confirmed the mechanistic pathway of them. Molecular docking study of compounds
IXg and IXn showed high degrees of superimposition Erlotinib into EGFR active sites.
These results were in a perfect match with the EGFR enzyme inhibition assay.
Acknowledgement
The authors would like to extend their sincere appreciation to Taif University
Researchers Supporting Project number (TURSP-2020/56), Taif University, Taif,
Saudi Arabia. Authors are thankful to the Developmental Therapeutics Program of the
United States, National Cancer Institute for performing the anticancer testing.
Supplementary data.
Supplementary materials associated with this research including NCI cytotoxicity
results and elemental and spectroscopic analyses are available.
References
26

[1] A. Ayati, S. Emami, A. Asadipour, A. Shafiee, A. Foroumadi, Recent applications
of 1,3-thiazole core structure in the identification of new lead compounds and drug
discovery, Eur J Med Chem 97 (2015) 699-718.
[2] R.M. Aly, R.A.T. Serya, A.M. El-Motwally, A. Esmat, S. Abbas, D.A. Abou El
Ella, Novel quinoline-3-carboxamides (Part 2): Design, optimization and synthesis
of quinoline based scaffold as EGFR inhibitors with potent anticancer activity,
Bioorg Chem 75 (2017) 368-392.
[3] G. Mountzios, Lung cancer: biology and technology foster therapeutic innovation,
Ann Transl Med 6 (2018) 137.
[4] S. Bishayee, Role of conformational alteration in the epidermal growth factor
receptor (EGFR) function, Biochem Pharmacol 60 (2000) 1217-23.
[5] H. Ogiso, R. Ishitani, O. Nureki, S. Fukai, M. Yamanaka, J.H. Kim, K. Saito, A.
Sakamoto, M. Inoue, M. Shirouzu, S. Yokoyama, Crystal structure of the complex
of human epidermal growth factor and receptor extracellular domains, Cell 110
(2002) 775-87.
[6] T. Holbro, G. Civenni, N.E. Hynes, The ErbB receptors and their role in cancer
progression, Exp Cell Res 284 (2003) 99-110.
[7] N.E. Hynes, K. Horsch, M.A. Olayioye, A. Badache, The ErbB receptor tyrosine
family as signal integrators, Endocr Relat Cancer 8 (2001) 151-9.
[8] J. Capdevila, E. Elez, T. Macarulla, F.J. Ramos, M. Ruiz-Echarri, J. Tabernero,
Anti-epidermal growth factor receptor monoclonal antibodies in cancer treatment,
Cancer Treat Rev 35 (2009) 354-63.
[9] D.A. Reardon, P.Y. Wen, I.K. Mellinghoff, Targeted molecular therapies against
epidermal growth factor receptor: past experiences and challenges, Neuro Oncol
16 Suppl 8 (2014) viii7-13.
[10]P. Seshacharyulu, M.P. Ponnusamy, D. Haridas, M. Jain, A.K. Ganti, S.K. Batra,
Targeting the EGFR signaling pathway in cancer therapy, Expert Opin Ther
Targets 16 (2012) 15-31.
[11]T.K. Olszewski, B. Boduszek, Synthesis of new thiazole-2,-4, and-5-yl-(amino)
methylphosphonates and phosphinates: unprecedented cleavage of thiazole-2
derivatives under acidic conditions., Tetrahedron 66 (2010) 8661-8666.
[12]T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter,
M.J. Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier,
S.S. Yu, AndersonGd, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt,
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, Synthesis
and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2
inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl]benze nesulfonamide (SC-58635, celecoxib), J Med Chem 40 (1997)
1347-65.
[13]R. Sridhar, P.T. Perumal, S. Etti, G. Shanmugam, M.N. Ponnuswamy, V.R.
Prabavathy, N. Mathivanan, Design, synthesis and anti-microbial activity of 1H-
pyrazole carboxylates, Bioorg Med Chem Lett 14 (2004) 6035-40.
[14]G. Menozzi, L. Mosti, P. Fossa, F. Mattioli, M. Ghia, ω-Dialkylaminoalkyl ethers
of phenyl-(5-substituted-1-phenyl-1H-pyrazol-4-yl)methanols with analgesic and
anti-inflammatory activity., J. Heterocycl. Chem. 34 (1997) 963-968.
[15]H.A. Abdel-Aziz, A.A. Mekawey, K.M. Dawood, Convenient synthesis and
antimicrobial evaluation of some novel 2-substituted-3-methylbenzofuran
derivatives, Eur J Med Chem 44 (2009) 3637-44.
27

[16]H.A. Abdel-Aziz, H.S. El-Zahabi, K.M. Dawood, Microwave-assisted synthesis
and in-vitro anti-tumor activity of 1,3,4-triaryl-5-N-arylpyrazole-carboxamides,
Eur J Med Chem 45 (2010) 2427-32.
[17]F.M. Awadallah, G.A. Piazza, B.D. Gary, A.B. Keeton, J.C. Canzoneri, Synthesis
of some dihydropyrimidine-based compounds bearing pyrazoline moiety and
evaluation of their antiproliferative activity, Eur J Med Chem 70 (2013) 273-9.
[18]C.N. Cavasotto, M.A. Ortiz, R.A. Abagyan, F.J. Piedrafita, In silico identification
of novel EGFR inhibitors with antiproliferative activity against cancer cells,
Bioorg Med Chem Lett 16 (2006) 1969-74.
[19]Y.R. Liu, J.Z. Luo, P.P. Duan, J. Shao, B.X. Zhao, J.Y. Miao, Synthesis of pyrazole
peptidomimetics and their inhibition against A549 lung cancer cells, Bioorg Med
Chem Lett 22 (2012) 6882-7.
[20]P.C. Lv, D.D. Li, Q.S. Li, X. Lu, Z.P. Xiao, H.L. Zhu, Synthesis, molecular
docking and evaluation of thiazolyl-pyrazoline derivatives as EGFR TK inhibitors
and potential anticancer agents, Bioorg Med Chem Lett 21 (2011) 5374-7.
[21]I. Vujasinovic, A. Paravic-Radicevic, K. Mlinaric-Majerski, K. Brajsa, B. Bertosa,
Synthesis and biological validation of novel pyrazole derivatives with anticancer
activity guided by 3D-QSAR analysis, Bioorg Med Chem 20 (2012) 2101-10.
[22]H.S. Abbas, S.S. Abd El-Karim, Design, synthesis and anticervical cancer activity
of new benzofuran-pyrazol-hydrazono- thiazolidin-4-one hybrids as potential
EGFR inhibitors and apoptosis inducing agents, Bioorg Chem 89 (2019) 103035.
[23]K.M. Qiu, H.H. Wang, L.M. Wang, Y. Luo, X.H. Yang, X.M. Wang, H.L. Zhu,
Design, synthesis and biological evaluation of pyrazolyl-thiazolinone derivatives
as potential EGFR and HER-2 kinase inhibitors, Bioorg Med Chem 20 (2012)
2010-8.
[24]X.X. Tao, Y.T. Duan, L.W. Chen, D.J. Tang, M.R. Yang, P.F. Wang, C. Xu, H.L.
Zhu, Design, synthesis and biological evaluation of pyrazolyl-nitroimidazole
derivatives as potential EGFR/HER-2 kinase inhibitors, Bioorg Med Chem Lett 26
(2016) 677-683.
[25]H.H. Wang, K.M. Qiu, H.E. Cui, Y.S. Yang, L. Yin, M. Xing, X.Y. Qiu, L.F. Bai,
H.L. Zhu, Synthesis, molecular docking and evaluation of thiazolyl-pyrazoline
derivatives containing benzodioxole as potential anticancer agents, Bioorg Med
Chem 21 (2013) 448-55.
[26]W.M. Zhang, M. Xing, T.T. Zhao, Y.J. Ren, X.H. Yang, Y.S. Yang, P.C. Lv, H.L.
Zhu, Synthesis, molecular modeling and biological evaluation of cinnamic acid
derivatives with pyrazole moieties as novel anticancer agents., RSC advances 4
(2014) 37197-37207.
[27]M.V. Reddy, C.R. Su, W.F. Chiou, Y.N. Liu, R.Y. Chen, K.F. Bastow, K.H. Lee,
T.S. Wu, Design, synthesis, and biological evaluation of Mannich bases of
heterocyclic chalcone analogs as cytotoxic agents, Bioorg Med Chem 16 (2008)
7358-7370.
[28]J.V. Faria, P.F. Vegi, A.G.C. Miguita, M.S. Dos Santos, N. Boechat, A.M.R.
Bernardino, Recently reported biological activities of pyrazole compounds, Bioorg
Med Chem 25 (2017) 5891-5903.
[29]B. Insuasty, A. Tigreros, F. Orozco, J. Quiroga, R. Abonia, M. Nogueras, A.
Sanchez, J. Cobo, Synthesis of novel pyrazolic analogues of chalcones and their 3-
aryl-4-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole derivatives as
potential antitumor agents, Bioorg Med Chem 18 (2010) 4965-74.
28

[30]M. Labbozzetta, R. Baruchello, P. Marchetti, M.C. Gueli, P. Poma, M.
Notarbartolo, D. Simoni, N. D'Alessandro, Lack of nucleophilic addition in the
isoxazole and pyrazole diketone modified analogs of curcumin; implications for
their antitumor and chemosensitizing activities, Chem Biol Interact 181 (2009) 29-
36.
[31]M. Asif, The biological potentials of substituted 1, 2-diazines: a review on versatile
pyridazine derivatives., Journal of Chinese Pharmaceutical Sciences 25 (2016)
707-725.
[32]M. Asif, A Review on Pyridazinone Ring Containing Various Cardioactive
Agents., Journal of Chemical Reviews 1 (2019) 66-77.
[33]M.J. Kim, J. Lee, S.Y. Kang, S.H. Lee, E.J. Son, M.E. Jung, K.S. Song, M. Lee,
H.K. Han, J. Kim, Novel C-aryl glucoside SGLT2 inhibitors as potential
antidiabetic agents: Pyridazinylmethylphenyl glucoside congeners, Bioorg Med
Chem Lett 20 (2010) 3420-5.
[34]H.M. Faidallah, K.A. Khan, M.S. Makki, Synthesis and Biological Evaluation of
New Fused Isoxazolo [4, 5‐d] Pyridazine Derivatives., Journal of the Chinese
Chemical Society 58 (2011) 191-198.
[35]A.A. Siddiqui, R. Mishra, M. Shaharyar, Synthesis, characterization and
antihypertensive activity of pyridazinone derivatives, Eur J Med Chem 45 (2010)
2283-90.
[36]S. Ahmad, I.G. Rathish, S. Bano, M.S. Alam, K. Javed, Synthesis and biological
evaluation of some novel 6-aryl-2-(p-sulfamylphenyl)-4,5-dihydropyridazin-
3(2H)-ones as anti-cancer, antimicrobial, and anti-inflammatory agents, J Enzyme
Inhib Med Chem 25 (2010) 266-71.
[37]N.A. El-Ghaffar, M.K. Mohamed, M.S. Kadah, A.M. Radwan, G.H. Said, S.N.
Abd, Synthesis and anti-tumor activities of some new pyridazinones containing the
2-phenyl-1H-indolyl moiety., Journal of Chemical and Pharmaceutical Research 3
(2011) 248-259.
[38]E.F. Ewies, M.F. El-Shehry, L.S. Boulos, Synthesis of some novel pyridazine
derivatives of expected antitumor activity, Int. J. Chem. Tech. Res. 7 (2015) 2506–
2513.
[39]E. Lattmann, W.O. Ayuko, D. Kinchinaton, C.A. Langley, H. Singh, L. Karimi,
M.J. Tisdale, Synthesis and evaluation of 5-arylated 2(5H)-furanones and 2-
arylated pyridazin-3(2H)-ones as anti-cancer agents, J Pharm Pharmacol 55 (2003)
1259-65.
[40]M.S.R. Murty, B.R. Rao, K.R. Ram, J.S. Yadav, J. Antony, R.J. Anto, Synthesis
and preliminary evaluation activity studies of novel 4-(aryl/heteroaryl-2-
ylmethyl)-6-phenyl-2-[3-(4-substituted-piperazine-1-yl) propyl] pyridazin-3 (2H)-
one derivatives as anticancer agents., Medicinal chemistry research 21 (2012)
3161-3169.
[41]M.F. Brana, M. Cacho, M.L. Garcia, E.P. Mayoral, B. Lopez, B. de Pascual-Teresa,
A. Ramos, N. Acero, F. Llinares, D. Munoz-Mingarro, O. Lozach, L. Meijer,
Pyrazolo[3,4-c]pyridazines as novel and selective inhibitors of cyclin-dependent
kinases, J Med Chem 48 (2005) 6843-54.
[42]H. Djaballah, H.E. Varmus, D. Shum, R. Somwar, A. Chucholowski, M.S.
Thiruvazhi, Substituted pyridazines as EGFR and/or KRAS inhibitors, U.S. Patent
(2017) 9,562,019.
29