Design, synthesis and molecular modeling of phenyl dihydropyridazinone derivatives as B-Raf inhibitors with anticancer activity

Article abstract of DOI:10.1016/j.bioorg.2020.104148

Three new series of phenyl dihydropyridazinone derivatives 4b–8i have been designed, synthesized and evaluated for their anticancer activity against different cancer cell lines. Nine compounds showed strong inhibitory activity, among which compound 8b exh

Full text of DOI:10.1016/j.bioorg.2020.104148

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Design, Synthesis and Molecular Modeling of Phenyl Dihydropyridazinone
Derivatives as B-Raf Inhibitors with Anticancer Activity
Mohamed G. Thabit, Amany S. Mostafa, Khalid B. Selim, Magda A. A.
Elsayed, Magda N. A. Nasr
PII:
S0045-2068(20)31445-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.104148
YBIOO 104148
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
26 November 2019
25 June 2020
26 July 2020
Please cite this article as: M.G. Thabit, A.S. Mostafa, K.B. Selim, M. A. A. Elsayed, M. N. A. Nasr, Design,
Synthesis and Molecular Modeling of Phenyl Dihydropyridazinone Derivatives as B-Raf Inhibitors with
Anticancer Activity, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104148
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Design, Synthesis and Molecular Modeling of Phenyl Dihydropyridazinone
Derivatives as B-Raf Inhibitors with Anticancer Activity
Mohamed G. Thabit,1 Amany S. Mostafa,1 Khalid B. Selim,1,* Magda A. A. Elsayed1,2 and Magda
N. A. Nasr1
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University, New Dammeitta,
Egypt
*Corresponding author: K. B. Selim (e-mail: khbselim@mans.edu.eg)
tel: +2050-2247496 fax: +2050-2247496
Abstract
Three new series of phenyl dihydropyridazinone derivatives 4b–8i have been designed, synthesized
and evaluated for their anticancer activity against different cancer cell lines. Nine compounds showed
strong inhibitory activity, among which compound 8b exhibited potent activity against PC-3 cell line
with IC50 value of 7.83 µM in comparison to sorafenib (IC50 11.53 µM). Compounds 6a, 6c, 7f-h and
8a-d were further screened for their B-Raf inhibitory activity where seven compounds 7f-h and 8a-d
showed high B-Raf inhibition with ranges of IC50 values 70.65-84.14 nM and 24.97-44.60 nM,
respectively when compared to sorafenib (IC50 44.05 nM). Among the tested compounds, 8b was the
most potent B-Raf inhibitor with IC50 value of 24.79 nM. Cell cycle analysis of MCF-7 cells treated
with 8b showed cell cycle arrest at G2-M phase with significant apoptotic effect. Molecular modeling
study was performed to understand the binding mode of the most active synthesized compounds with
B-Raf enzyme.
Keywords
Antitumor; B-Raf; Cell cycle analysis; Molecular modeling; Pyridazinone.
1. Introduction
Cancer ranks high as a major cause of mortality worldwide.1 Many Kinases are involved in signalling
transduction pathways inside the cancer cells so that, these kinases are considered as good therapeutic
targets for the discovery of new antitumor agents.2-5 B-Raf protein (Ser/Thr kinase) plays an important
role in activating RAS–RAF–MEK–ERK signalling pathway and promoting normal cell
development.6 B-Raf kinase is mutated in approximately 7% of all human cancer cells and the most
common oncogenic mutation is V600E which is found in 90% of all B-Raf mutated cancer cells and

involves a substitution of valine by glutamic acid at codon 600.7 The deregulation of normal B-Raf
signalling pathway leads to its over expression in different types of cancers; including colorectal
(15%),8-10 hepatocellular (14%),10,11 mammary gland (10%),12 melanoma (60%)10,13 and prostate
cancer (10%).8 Several Raf inhibitors have been approved by FDA as anticancer drugs such as
sorafenib,10 dabrafenib13 and vemurafenib14, while others are still under clinical trials such as
WO201106818715 (Figure 1).
NH2
N
O
CF3
N
O
S
O
F
F
O
Cl
O
N
H
N
N
S
H
N
N
F
N
H
H
Sorafenib
Dabrafenib
Cl
O
N
H
N
H
N
O
CF3
HN
O
O
O
F
F
H
O
S
N
O
N
N
HN
Vemurafenib
WO2011068187
Figure 1: Reported Raf inhibitors.
2. Rational design
The study of the molecular modeling analysis of the crystal structure of B-Raf kinase with sorafenib
(PDB code: 1UWJ)16 concluded common pharmacophoric features which are responsible for the good
activity of B-Raf inhibitors. These features can be summarized as i) hetero aromatic ring that binds to
the ATP pocket via hydrogen bonds which is necessary for high-binding affinity with the hinge region,
mostly with Cys 531, ii) cyclic or non-cyclic linker, responsible for extending the molecule within the
binding site, iii) a hydrogen bond network moiety which interacts with Asp 593 and/or Glu 500
residues at the DFG motif, iv) a lipophilic terminal which consists of a moiety that occupies the
allosteric hydrophobic pocket that is formed between the DFG motif and the catalytic loop17, 18 (Figure
2).

Figure 2: 2D interaction of the co-crystallized sorafenib inside the active site of B-Raf (PDB code:
1UWJ).16
The presence of nitrogenous heterocyclic moieties in the structure of the anticancer agents became
essential to have potent activity such as pyridine, pyrimidine and pyridazine rings which are present
in sorafenib, dabrafenib and vatalanib, respectively.19 While the presence of hydrogen bond network
moiety in the skeleton of various anticancer agents plays an important role in improving their inhibitory
activity against several types of kinases; this moiety may be urea as in sorafenib, heterocyclic ring as
in vatalanib and encorafenib14 or imino group as in azastilben isoster I20 (Figure 3).
H
N
N
N
N
N
N
F
N
H
O
H
N
Cl
N
N
N
O
O
O
HO
HO
HN
S
Cl
Vatalanib
Encorafenib
Figure 3: Reported antitumor agents.
I
Sorafenib was developed initially as an inhibitor for Raf kinase, but its efficacy in renal and
hepatocellular cancer was later attributed to inhibition of VEGFR2, PDGFR and other targets.21 Later
on, it faces a drug resistance,22 therefore we explored the structural modifications of sorafenib with the
goal of optimizing its B-Raf kinase inhibition and/or anti-cancer activity.

As shown in figure 4, our target compounds were designed based on the structure of the lead
compound; sorafenib; through computer-based design approach by incorporating different types of
hydrogen bond network linkers at the center with phenyl pyridazinone scaffold from one end, and
different hydrophobic moieties from the opposite end. The pyridazinone ring on the target series
mimics the distal pyridine-carboxamide moiety of sorafenib. Also, the imino, triazole and (thio) urea
moieties in the central core of the designed series mimic the urea moiety of sorafenib as hydrogen
bond network linkers, likewise, the hydrophobic terminal in the target compounds and sorafenib. This
optimization aims to give new derivatives which might be good B-Raf inhibitors with anticancer
activity against a broad panel of cancer cell.
N
R
N
X
O
N
N
R
N
Molecular
O
N
CF3
O
Cl
N
O
modification
R
H
N
N
N
N
H
N
H
H
H
Hinge-binding
moiety
H-bonding Lipophilic
Hinge-binding
moiety
H-bonding Lipophilic
moiety terminal
moiety
terminal
Sorafenib
lead compound
The newly designed series
Figure 4: Rational design of the newly synthesized compounds.
3. Results and discussion
3.1. Chemistry
The designed target compounds were prepared as outlined in schemes 1-4. The structures of the target
1
13
compounds were established based on elemental analysis, IR, H-NMR, C-NMR and MS data. As
shown in scheme 1, the preparation of pyridazinone derivatives 4a23 and 4b, which are the key
intermediates for the synthesis of our target compounds, was achieved via Friedel-Craft acylation24,25
of acetanilide (1) with succinic anhydride (2) in the presence of anhydrous AlCl3 as a Lewis acid in
DMF as a solvent, which was followed by acidic hydrolysis to give the corresponding γ-keto acid,26-
29
as free amino derivative 3. This intermediate was cyclized with hydrazine hydrate or phenyl
hydrazine to afford the required pyridazinone derivatives 4a and 4b.

R
O
O
N
O
N
OH
NH2NHR
O
i) AlCl3 /DMF
ii) HCl
O
+
Heat
75% Yield
O
N
H2N
H
NH2
O
4a,b
3
1
2
4a
4b
C6H5
Comp.
R
H
Scheme 1: Synthesis of the key intermediates 4a,b.
The azido derivatives 5a,b were obtained through diazotization of amino compounds 4a,b by sodium
nitrite and HCl, followed by reacting with sodium azide.30 The IR spectra of the compounds 5a,b
showed the disappearance of the absorption band at 3300-3400 cm-1 of NH2 group and the appearance
of a new absorption band at 2136-2143 cm-1 of N3 group. The triazolo derivatives 6a-d were obtained
through 1,3-dipolar cycloaddition reaction between 5a or 5b and different active methylene-containing
compounds such as ethyl cyanoacetate, ethyl acetoacetate and acetyl acetone.31 It is worthy to mention
that compound 6c was obtained as carboxylic acid derivatives due to the hydrolysis of the ester group
during the course of the reaction (Scheme 2).
R
N
R
R
O
O
O
N
O
N
N
N
N
X
X\
1) NaNO2\HCl
EtONa
N
stirring
2) NaN3
N
N3
N
NH2
60-65% Yield
60-65% Yield
R'
4a,b
COR''
5a,b
6a-d
6c
Comp. 5a 5b
6a
6b
6d
For synthesis
O
6a,b
: X = CN, X' = OC2H5
: X = COCH3, X' = OC2H5
: X = COCH3, X' = CH3
H
NH2
C6H5
NH2
H
CH3
H
CH3
CH3
R
R'
R''
H


C6H5


X
6c
6d
X'
OC2H5 OC2H5 OH
Scheme 2: Synthesis of the designed triazole derivatives 6a-d.
The Schiff base32 series 7a-h was afforded by reacting 4a or 4b with different aromatic aldehydes. The
1H-NMR spectra of compounds 7a-h showed a signal in the range of 8.55-8.88 ppm characteristic for
the (=Ar-H) proton.

R
R
O
N
O
N
N
N
O
Heat
R'
60-72% Yield
N
NH2
R'
4a, b
7a-h
7a 7b 7c
7d
7e
7f
7g 7h
Comp.
H
R
H
H
H
H
C6H5
H
H
R'
4-Br 4-F 2-Br 3-NO2 3-NO2 4-Cl OCH3 H
Scheme 3: Synthesis of the designed Schiff base derivatives 7a-h.
Reaction of aryl iso(thio)cyanate derivatives with compounds 4a,b afforded the diphenylurea (or
thiourea) derivatives 8a-i.33 The 1H-NMR spectra of compounds 8a-i showed two adjacent signals in
the range of 8.60-10.48 ppm indicating the presence of (NHCONH) and (NHCSNH) protons.
R
R
R
O
N
O
N
O
N
N
N
N
R\
R\
S
O
Ar N C S
Stirring
Ar N C O
Stirring
N
H
N
NH2
N
N
H
H
H
72-74% Yield
71-75% Yield
8a-d
4a,b
8e-i
8i
8a 8b
8c
8d
8e
8f
8g 8h
C6H5
Comp.
Comp.
R
H C6H5
H
C6H5
R
H
C6H5
H
H
R\
R\
4-NO2
4-CH3
4-Cl 4-Cl
4-NO2
H
H
4-NO2 4-NO2
Scheme 4: Synthesis of the designed (thio)urea derivatives 8a-i.
The 1H-NMR spectra of the all synthesized compounds showed a signal in the range of 10.79-10.98
ppm that have disappeared with the blocking of the amide nitrogen in the pyridazine ring indicating
the presence of (-CONH) proton, in addition to two triplet signals at the range of 2.41-2.74 and 2.91-
3.22 ppm characterizing the presence of (CH2-CH2) protons of the pyridazine ring.
3.2. Biological evaluation
3.2.1. In vitro cytotoxic evaluation
MTT procedure was used to evaluate the antitumor activity of the synthesized compounds 4b-8i
against five human tumor cell lines, using sorafenib as a reference drug. A pair of models was selected
based on B-Raf expression; where four cell lines represented over-expressed mutated B-Raf; including
colon carcinoma (HCT-116),8 hepatocellular carcinoma (HEPG-2),10 mammary gland (MCF-7)12 and
human prostate cancer (PC-3),8 while the fifth one; human cervical carcinoma (Hela); was selected as

a model which lacks an over-expression of mutated B-Raf.34 Biological data (Table1), revealed that
nine compounds 6a, 6c, 7f-h and 8a-d exhibited the highest antitumor activities with IC50 range of
7.50–19.07 µM. Compound 8b was the highest cytotoxic agent against all the tested cell lines and
showed potent activity against PC-3 cell line (IC50 7.83 µM) in comparison to sorafenib (IC50 11.53
µM). While, compound 7h showed high cytotoxicity against HEPG-2 cell line with IC50 equal to 8.71
µM when compared with sorafenib (IC50 9.18 µM). Compounds 7f-h were active against HEPG-2,
HCT-116 and MCF-7 rather than Hela and PC-3. Compound 6a was active against all the tested cell
lines, while compound 6c was active mainly against HCT-116 and Hela cell lines. Compounds 4b, 5a,
5b, 6b, 6d, 7a-c and 8e showed moderate cytotoxic activity, while compounds 7d, 8h and 8i were the
weakest. Unfortunately, compounds 7e, 8f and 8g were non cytotoxic.
Table 1. In vitro cytotoxicity of the synthesized compounds and sorafenib.
IC50 µM
Compound
HEPG2
MCF-7
PC3
HCT-116
Hela
4b
5a
74.08±3.80 39.04±2.60 59.76±3.70 81.52±4.30 60.64±3.40
31.11±2.20 19.68±1.90 40.81±2.70 25.37±2.70 38.43±2.50
24.83±1.90 28.94±2.00 49.60±3.10 20.78±1.80 41.86±2.70
5b
6a
10.57±1.00
9.87±0.80
12.83±1.20 7.50±0.60 11.63±1.00
6b
6c
29.11±2.00 54.02±3.50 42.05±3.00 32.41±2.20 40.57±2.60
18.79±1.50 14.56±1.30 25.61±1.70 11.19±1.10 13.06±1.20
6d
7a
43.16±2.70 36.23±2.40
63.94±3.8 52.38±3.40 27.18±1.90
69.55±3.50 45.12±2.70 62.04±3.80 76.82±4.00 64.80±3.80
53.81±3.10 73.08±3.90 90.18±5.10 48.17±3.40 82.75±4.70
50.97±2.90 34.81±2.30 54.86±3.50 43.79±3.20 58.13±3.00
83.10±4.00 59.16±3.30 77.23±4.00 88.14±4.50 75.13±3.90
7b
7c
7d
7e
68.73±3.60 93.72±5.20
>100
72.80±3.80
>100
7f
13.95±1.20 16.71±1.50 33.42±2.30 14.91±1.40 27.38±2.00
7g
9.78±1.00
8.71±0.60
36.02±2.40
9.46±0.80
18.52±1.80 29.85±2.30 13.78±1.20 32.84±2.30
15.83±1.50 28.03±1.80 10.36±0.90 23.91±1.90
7h
8a
7.59±0.40
8.26±0.60
8.96±0.70 26.38±2.50 12.60±1.10
7.83±0.50 11.35±1.10 15.02±1.30
8b
8c
17.86±1.50 13.25±1.30 27.74±1.40 19.07±1.60 21.78±1.50
29.50±2.10 12.13±1.10 11.78±0.90 22.36±1.90 17.31±1.60
42.34±2.60 30.40±2.20 51.20±3.20 37.56±2.90 46.78±2.80
8d
8e
8f
>100
>100
87.26±4.70
80.34±4.30
>100
>100
>100
91.42±5.30
8g
95.43±5.40 86.42±4.60
8h
8i
94.23±4.60 52.73±2.90 86.52±4.80 89.25±4.70 71.49±3.70
61.72±3.40 65.65±3.70 82.33±4.40 70.12±3.70 79.22±4.20
Sorafenib
9.18±0.60
7.26±0.30
11.53±0.90 5.47±0.30
8.04±0.50
3.2.2. B-Raf inhibition assay

The most active compounds 6a, 6c, 7f-h and 8a-d were selected for further enzyme inhibition
screening against B-Raf (V600E).35, 36 Compounds 8a, 8b and 8d showed the highest affinity to B-Raf
with IC50 values range of 24.97-35.59 nM more than that of sorafenib (IC50 44.05 nM), whereas
compound 8c showed IC50 value of 44.60 nM; nearly similar to that of sorafenib. On the other hand,
compounds 7f-h showed lower affinity to B-Raf with IC50 value range of 70.65-84.14 nM, while
compounds 6a and 6c showed the weakest inhibitory activity (Table 2).
Table 2. B-Raf enzyme inhibitory activity for compounds 6a, 6c, 7f-h and 8a-d.
Compound
6a
6c
7f
7g
7h
B-Raf IC50 (nM) 110.23±0.04
120.92±0.04
74.84±0.03
70.65±0.01
84.14±0.03
Compound
Sorafenib
8a
8b
8c
8d
B-Raf IC50 (nM)
30.83±0.01
24.97±0.01
44.60±0.007
35.59±0.005
44.05±0.02
3.2.3. Cell cycle analysis
Results of the cytotoxicity and the B-Raf inhibitory activity encouraged us to further evaluate the effect
of compound 8b on cell cycle distribution in MCF-7 cancer cell line. MCF-7 cells were incubated with
compound 8b at a concentration of 3 µg/ml for 24 h, stained with propidium iodide (PI) and analyzed
by flow cytometry (FCM) using BD FACS caliber reader37 (Figure 5).
8b
Sorafenib
control
Figure 5: Effect of compound 8b and sorafenib on the cell cycle distribution of MCF-7 cell line.
Table 3. Effect of 8b and sorafenib on the cell cycle distribution of MCF-7 cell line.

Cell cycle distribution (%)
Compound
G0-G1
26.72
23.94
55.36
S
G2-M
43.62
48.25
5.39
Pre-G1
22.08
27.42
1.97
8b
Sorafenib
Control
29.66
27.81
39.25
Quantification of the results assessed that compound 8b not only arrested MCF-7 cells in G2-M phase
of the cell cycle at 24 h by 43.62% (Table 3); but it further caused induced block in G0-G1 phase by
26.72% as compared to that of sorafenib 23.94 % (Figure 6).
Figure 6: Effect of 8b and sorafenib on the cell phases of MCF-7 cell line.
3.3.4. Detection of apoptosis
By analysing the data from histograms shown in figure 7, it was concluded that compound 8b induced
an early apoptotic effect 5.33% in MCF-7 cells after 24 h, beside a late apoptotic induction 15.03%
nearly similar to sorafenib (15.78 %).

8b
Sorafenib
Control
Figure 7: Apoptosis induced by 8b and sorafenib on MCF-7 cell line.
The total apoptosis (Figure 8) was determined by measuring the percent of cells stalled in the pre-G1
peak. It was estimated after 24 h exposure to be 22.08 and 27.24% for 8b and sorafenib, respectively
compared to the untreated MCF-7 cells 1.97% (Table 4).

Table 4. Apoptosis induced by 8b and sorafenib on MCF-7.
Apoptosis
Compound
Necrosis
Total
22.08
27.24
1.97
Early
5.33
9.31
0.63
Late
15.03
15.78
0.46
8b
Sorafenib
Control
1.72
2.15
0.88
Figure 8. Early, late and total apoptosis induced by 8b in comparison to sorafenib.
3.3. Molecular modeling
In this modeling study, we carried out the docking simulation using molecular operating environment
software package (MOE 2013.08)38-40 in the binding site of B-Raf kinase41-46 crystal structure with the
active ligand sorafenib which was retrieved from the Protein Data Bank (PDB) at the Research
Collaboratory for Structural Bioinformatics (RCSB, http://www.rcsb.org) (PDB code: 1UWJ).16
Careful analysis revealed that the binding cavity is surrounded by the catalytic amino acids Ala 480,
Asp 593, Cys 531, Glu 500, Gly 595, His 573, Ile 512, Ile 571, Leu 504, Leu 513, Lys 482, Lys 566,
Phe 582, Phe 594, Ser 601, Thr 528, Trp 530 and Val 503. As shown in figure 2, sorafenib forms three
hydrogen bonds; one of them exists between the pyridyl nitrogen and Cys 531, while the other
hydrogen bonds are formed between the urea spacer and Glu 500 and Asp 593 amino acid residues
with energy of interaction –8.32 kcal/mol. It is observed that compound 8b (from thiourea series),
which gave the best B-Raf inhibitory activity, forms three hydrogen bonds; two of them are formed
between the thiourea nitrogens and the essential conserved amino acid residue Glu 500, while the third
bond is formed between the amide oxygen of pyridazine ring and Cys 531 with energy of interaction
–8.67 kcal/mol (Figure 9). Such binding mode is similar to that of sorafenib and well explains the high
inhibitory activity of 8b against B-Raf.

8b
Figure 9: 2D interaction of compound 8b with the binding site of B-Raf (PDB code: 1UWJ).
In a similar way, the synthesized compounds were found to form various interactions with the catalytic
amino acid residues in the active binding site with energy of interaction ranging from –6.60 to –8.55
kcal/mol. As shown in figure 10, compounds 6a (from triazole series) and 7g (from imine series)
retained the binding with the conserved amino acid residue Asp 593, while 7g lacks the binding with
Glu 500 in conrast to compound 6a. Both sompounds 6a and 7g interact with Thr 528 through the
nitrogen atom of the pyridazine ring, while the oxygen of the methoxy group in 7g binds to Ser 601
residue. The superior activity of 7g over 6a could be explained by the presence of the lipophilic phenyl
terminal in 7g, which lies in the hydrophobic pocket of the enzyme’s active site, and the presence of
the hydrophilic ester terminal in 6a instead of the essential aromatic moiety.

a
6a
b
7g
Figure 10: 2D interaction of compounds 6a and 7g within the binding site of B-Raf (PDB code:
1UWJ).
3.4. ADMET properties and Lipinski’s rule of five
Lipinski's rule of five47, 48 is a principle used to evaluate drug likeness. All calculated descriptors were
obtained using the molecular operating environment software package (MOE 2013.08)49. The obtained
results (Table 5) revealed that compounds 6a, 7g and 8b along with sorafenib fulfil Lipinski’s rule

with no violations. The calculated lipophilicity of 6a, 7g, 8b and sorafenib proposed a good
bioavailability with topological surface area lower than 140Å2.
Table 5. ADMET properties prediction parameters of 6a, 7g, 8b and sorafenib.
Compound
Mwt/g
328.33
307.35
400.50
464.83
TPSA/ Å2 LogP
nHBA
nHBD
nV
0
0
0
0
6a
7g
8b
124.49
63.05
88.82
92.35
1.31
2.59
3.83
2.91
6
4
2
7
2
1
2
3
Sorafenib
Mwt: molecular weight, TPSA: topological surface area, LogP: calculated lipophilicity,
nHBA: number of hydrogen bond acceptors, nHBD: number of hydrogen bond donors,
nV: number of violations of Lipinski's rules
3.5. SAR study
The results of the B-Raf inhibition assays (Table 2) revealed that compounds 6a and 6c form the
triazole series (R = H) showed the least inhibitory activity against B-Raf where compounds 6a and 6c
represented an IC50 value of about 3-folds than sorafenib. While, compounds 7f, 7g and 7h from the
imine series (R = H) showed better B-Raf inhibitory effect than triazole series with IC50 value of about
2-folds than sorafenib. These results indicate the importance of the lipophilic aromatic ring in the right
terminal of the linker (N=CHAr) in order to boost the activity. Interestingly, thiourea series (8a, 8b,
8c and 8d) exhibited the best inhibitory activity among other series indicating that the thiourea linker
is essential for B-Raf affinity. Comparison between compounds 8a, 8b, 8c and 8d revealed that the N-
phenyl substitution on the pyridazinone ring leads to improve the inhibitory activity rather than the
non substituted analogues. Moreover, p-nitro substitution on the phenyl ring (right lipophilic terminal)
lowers the activity in comparison to the unsubstituted analogue. Docking study assessed the biological
results, since it showed that the thiourea-containing derivatives form five hydrogen bonds with the
active site; with energies of interaction ranging from -8.85 to -10.99 kcal/mol. These interactions
increase the ligand-receptor affinity and stabilize the ligand-receptor complex, resulting in the
enhanced enzymatic activity of the whole series.
4. Conclusion
New series of phenyl dihydropyridazinone derivatives 4b–8i have been designed, synthesized and
evaluated for their in vitro cytotoxicity against different cell lines. Among the most promising
cytotoxic agents, compounds 6a, 6c, 7f-h and 8a-d, were selected to evaluate their B-Raf inhibitory
effect. Thiourea series including compounds 8a, 8b, and 8d showed the highest affinity to B-Raf with
IC50 values ranging from 24.97 to 35.59 nM when compared to sorafenib (IC50 44.05 nM). The results

were consistent with the docking simulation. Compound 8b, which exhibited the best cytotoxicity and
B-Raf affinity, was induced early and late apoptosis, and showed cell cycle arrest at G2-M phase. The
results of molecular modeling and biological screening reveal that the structural modification of
sorafenib as a lead structure affected the activity in a predictable manner.
5. Experimental
5.1. Chemistry
Melting points (°C) were recorded using Stuart SMP10 melting point apparatus and are uncorrected.
IR spectra were recorded on a Thermo Fisher Scientific, Nicolet Is10 FT-IR spectrometer (in cm-1)
using KBr disk at the Faculty of Pharmacy, Mansoura University, Egypt. NMR spectra were recorded
on Bruker Avance NMR spectrometer (400 MHz) in DMSO-d6 at Georgia State University, Atlanta,
GA (1H-NMR for compounds 4b, 5b and 7a-h); Bruker Avance NMR spectrometer (400 MHz) in
DMSO-d6 at Faculty of Pharmacy, Mansoura University, Egypt (1H and 13C-NMR for compounds 6a
and 6c-d) and on Jeol ECA NMR spectrometer (500 MHz) in DMSO-d6 at Faculty of Science
13
13
Mansoura University (1H and C-NMR for compounds 6b and 8a-i; C-NMR of compounds 7a-
h).The chemical shifts in ppm are expressed in  units, using TMS as an internal standard and coupling
constants in Hz. Mass spectrum analyses were performed on Thermo Scientific ISQ-LT quadrupole
GC-MS at the regional centre for mycology and biotechnology (RCMB) Al-Azhar University.
Elemental analysis was performed at micro analytical Unit, Cairo University and the results were
within the valid range. Reaction times were monitored on TLC plates (F254, Merck) using
chloroform/methanol (9:1) as elution solvent and the spots were visualized by U.V. (366, 245 nm).
Compound 4a was prepared according to the procedure described in the literature.50
5.1. 1. Synthesis of 6-(4-aminophenyl)-2-phenyl-4,5-dihydropyridazin-3(2H)-one (4b).
A mixture of 4-(4-aminophenyl)-4-oxobutanoic acid (3) (1.930 g, 10 mmol) and phenyl hydrazine
(1.080 g, 10 mmol) in absolute ethanol (10 ml) was heated under reflux for 8 h and then cooled. The
resultant precipitate was collected by filtration, dried, and crystallized from ethanol to give 4b:
Yield (1.99 g, 75%); m.p. 135-137 C; IR (KBr) cm-1, 3410, 3360 (NH2), 3234 (CH), 1668 (CO); 1H-
NMR (400MHz, DMSO-d6): δ 2.64 (t, J = 7.7 Hz, 2H, CH2), 3.01 (t, J = 7.7 Hz, 2H, CH2), 5.59 (s,
2H, NH2), 6.59 (d, J = 8.0 Hz, 2H, Ar-H), 7.25 (dd, J= 7.2, 7.2 Hz, 1H, Ar-H), 7.41 (dd, J= 7.5, 7.5
Hz, 2H, Ar-H), 7.54-7.56 (m, 4H, Ar-H).
5.1.2. General method for synthesis of 6-(4-azidophenyl)-4,5-dihydropyridazin-3(2H)-one
derivatives (5a,b).

Compound 4a or 4b (10 mmol) was dissolved in hydrochloric acid (17%, 20 mL) at room temperature.
The solution was cooled to 0 C using ice bath; and sodium nitrite (0.104 g, 15mmol) was added in
small portions with stirring, then sodium azide (0.098 g, 15 mmol) was slowly added portion wise.
The reaction mixture was then stirred for additional 2 h at room temperature. The separated solid was
filtered, dried, and crystallized from ethanol to give the titled azido compounds 5a, b.
6-(4-Azidophenyl)-4,5-dihydropyridazin-3(2H)-one (5a).
Yield (1.29 g, 60%); m.p. 96-98 C; IR (KBr) cm-1, 3448 (NH), 2918 (CH), 2143 (N3), 1678 (CO);
1H-NMR (400 MHz, DMSO-d ): 2.57 (t, J = 6.2 Hz, 2H, CH2), 3.22 (t, J = 6.2 Hz, 2H, CH2), 7.24
δ
6
(d, J = 8.5 Hz, 2H, Ar-H), 8.01 (d, J = 8.5 Hz, 2H, Ar-H), 12.16 (s, 1H, NH).
6-(4-Azidophenyl)-2-phenyl-4,5-dihydropyridazin-3(2H)-one (5b).
Yield (1.89 g, 65%); m.p. 89-91 C; IR (KBr) cm-1, 2998 (CH), 2136 (N3), 1676 (CO); 1H-NMR (400
MHz, DMSO-d ): 2.72 (t, J = 7.8 Hz, 2H, CH2), 3.13 (t, J = 7.8 Hz, 2H, CH2), 7.20 (d, J = 8.6 Hz,
δ
6
2H, Ar-H), 7.28 (dd, J = 7.3, 7.3 Hz, 1H, Ar-H), 7.43 (dd, J = 7.7, 7.7 Hz, 2H, Ar-H), 7.53 (d, J = 7.7
Hz, 2H, Ar-H), 7.88 (d, J = 8.6 Hz, 2H, Ar-H).
5.1.3. General method for synthesis of 6-(4-triazolophenyl)-4,5-dihydropyridazin-3(2H)-one
derivatives (6a-d).
The methylene-activated compound (ethyl cyanoacetate, ethyl acetoacetate or acetyl acetone) (15
mmol) was added to freshly prepared sodium ethoxide (15 mL) and stirred for 10 min at room
temperature. The reaction mixture was treated with the azide 5a or 5b (10 mmol) and stirred at room
temperature for 20 h. The obtained solid was filtered, dried, and recrystallized from ethanol to give the
titled compounds 6a-d.
Ethyl 5-amino-1-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]-1H-1,2,3-triazole-4-carboxylate (6a).
For ethyl cyanoacetate with 5a: Yield (2.07 g, 63%); m.p. 208-210 C; IR (KBr) cm-1, 3431, 3311
(NH ), 3243 (NH), 2976 (CH), 1683 (CO), 1628 (C=N). 1H-NMR(400 MHz, DMSO-d ): 1.30 (t, J
δ
2
6
= 7.1 Hz, 3H, CH3), 2.61 (t, J = 6.1 Hz, 2H, CH2), 3.30 (t, J = 6.1 Hz, 2H, CH2), 4.3 (q, J = 7.1 Hz,
2H, CH2), 6.72 (s, 2H, NH2), 7.75 (d, J = 8.6 Hz, 2H, Ar-H), 8.18 (d, J = 8.6 Hz, 2H, Ar-H), 12.23 (bs,
1H, NH). 13C-NMR (100 MHz, DMSO-d ): 14.88, 28.32, 33.79, 60.21, 119.74, (124.80, CH  2),
δ
6
(129.92, CH  2), 136.93, 138.58, 146.74, 162.13, 174.26, 198.31. EI-MS: (m/z, %): 328.15 (M+, 100),
329.10 (M++1, 18.95). Elemental analysis for C15H16N6O3 (328.33), calcd.: C, 54.87; H, 4.91; N,
25.60. Found: C, 54.91; H, 4.94; N, 25.61.

Ethyl
5-amino-1-[4-(6-oxo-1-phenyl-4,5-dihydropyridazin-3-yl)phenyl]-1H-1,2,3-triazole-4
carboxylate (6b).
For ethyl cyanoacetate with 5b: Yield (2.47 g, 61%); m.p. 145-147 C; IR (KBr) cm-1, 3447 (NH2),
2981 (CH), 1632 (CO). 1H-NMR (500 MHz, DMSO-d ): 1.31 (t, J = 7.2 Hz, 3H, CH ), 2.78 (t, J =
δ
6
3
8.1 Hz, 2H, CH2), 3.21 (t, J = 8.1 Hz, 2H, CH2), 4.30 (q, J = 7.2 Hz, 2H, CH2), 6.64 (s, 2H, NH2), 7.28
(dd, J= 7.3, 7.3 Hz, 1H, Ar-H), 7.45 (dd, J= 7.5, 7.5 Hz, 2H, Ar-H), 7.55 (d, J = 7.5 Hz, 2H, Ar-H),
7.65 (d, J = 8.2 Hz, 2H, Ar-H), 8.02 (d, J = 8.2 Hz, 2H, Ar-H). 13C-NMR (125 MHz, DMSO- d ):
δ
6
14.42, 22.29, 27.28, 59.67, 119.18, (124.46, CH  2), (124.91, CH  2), 126.28, (127.46, CH  2),
(128.16, CH  2), 135.37, 136.06, 141.32, 146.21, 150.82, 161.68, 165.25. EI-MS: (m/z, %): 404.09
(M+, 100), 405.19 (M++1, 24.55). Elemental analysis for C21H20N6O3 (404.43), calcd.: C, 62.37; H,
4.98; N, 20.78. Found: C, 62.34; H, 4.94; N, 20.79.
5-Methyl-1-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]-1H-1,2,3-triazole-4-carboxylic acid (6c).
For ethyl acetoacetate with 5a: Yield (1.80 g, 60%); m.p. 181-183 C; IR (KBr) cm-1, 3592 (OH), 3490
(NH), 2929 (CH), 1691 (CO), 1601 (C=N). 1H-NMR (400 MHz, DMSO-d ): 2.55 (s, 3H, CH ), 2.61
δ
6
3
(t, J = 6.1 Hz, 2H, CH2), 3.32 (t, J = 6.1 Hz, 2H, CH2), 7.81 (d, J = 8.5 Hz, 2H, Ar-H), 8.21 (d, J = 8.5
Hz, 2H, Ar-H), 12.44 (brs, 1H, NH). 13C-NMR (100 MHz, DMSO-d ): 10.28, 28.31, 33.85, (126.04,
δ
6
CH  2), (129.79, CH  2), 137.28, 137.63, 139.14, 139.70, 162.98, 174.27, 198.35; EI-MS: (m/z, %):
299.15 (M+, 100), 300.19 (M++1, 16.55). Elemental analysis for C14H13N5O3 (299.29), calcd.: C,
56.18; H, 4.38; N, 23.40. Found: C, 66.15; H, 4.34; N, 23.37.
6-[4-(4-Acetyl-5-methyl-1H-1,2,3-triazol-1-yl)phenyl]-4,5-dihydropyridazin-3(2H)-one (6d).
For acetyl acetone with 5a: Yield (1.93 g, 65%); m.p. 155-157 C; IR (KBr) cm-1, 3449 (NH), 2911
(CH), 1686 (CO), 1604 (C=N). 1H-NMR (400 MHz, DMSO-d ): 2.56 (s, 3H, CH ), 2.62 (t, J = 6.4
δ
6
3
Hz, 2H, CH2), 2.65 (s, 3H, CH3), 3.34 (t, J = 6.4 Hz, 2H, CH2), 7.81 (d, J = 8.6 Hz, 2H, Ar-H), 8.21
(d, J = 8.6 Hz, 2H, Ar-H). 13C-NMR (100 MHz, DMSO-d ): 10.26, 28.18, 28.31, 33.86, (126.04, CH
δ
6
 2), (129.81, CH  2), 137.73, 138.42, 138.83, 143.48, 174.27, 193.87, 198.36. EI-MS: (m/z, %):
297.10 (M+, 100), 298.19 (M++1, 17.45). Elemental analysis for C15H15N5O2 (297.32), calcd.: C,
60.60; H, 5.09; N, 23.56. Found: C, 60.65; H, 5.13; N, 23.59.
5.1.4. General method for synthesis of 6-[4-(substituted benzylidene)amino)phenyl]pyridazin-
3(2H)-one (7a-h).
A mixture of compound 4a or 4b (10 mmol) and the appropriate aromatic aldehydes (10 mmol) was
dissolved in absolute ethanol (15 mL) with few drops of glacial acetic acid; then stirred under reflux

for 8 h. After cooling, the obtained solid was filtered, washed with water, dried, and crystallized from
ethanol to give the titled compounds 7a-h.
6-{4-[(4-Bromobenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7a).
Yield (2.53 g, 71%); m.p. 262-264 C; IR (KBr) cm-1, 3193 (NH), 2938 (CH), 1682 (CO), 1608 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.48 (t, J = 8.0 Hz, 2H, CH ), 2.97 (t, J = 8.0 Hz, 2H, CH ), 7.33
δ
6
2
2
(d, J = 7.9 Hz, 2H, Ar-H), 7.74 (d, J = 7.9 Hz, 2H, Ar-H), 7.80 (d, J = 7.9 Hz, 2H, Ar-H), 7.89 (d, J =
7.9 Hz, 2H, Ar-H), 8.68 (s, 1H, =CH-Ar), 10.95 (s, 1H, NH). 13C-NMR (125 MHz, DMSO-d ): 21.82,
δ
6
25.99, (121.28, CH  2), 125.23, (126.67, CH  2), (130.57, CH  2), (131.95, CH  2), 133.85, 135.09,
148.96, 151.64, 160.03, 166.99. EI-MS: (m/z, %): 355.05 (M+, 100), 357.03 (M++2, 96.45). Elemental
analysis for C17H14BrN3O (356.22), calcd.: C, 57.32; H, 3.96; N, 11.80. Found: C, 57.30; H, 3.92; N,
11.77.
6-{4-[(4-Fluorobenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7b).
Yield (1.87 g, 72%); m.p. 246-248 C; IR (KBr) cm-1, 3449 (NH), 2988 (CH), 1630 (CO), 1600 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.46 (t, J = 8.0 Hz, 2H, CH ), 2.97 (t, J = 8.0 Hz, 2H, CH ), 7.33
δ
6
2
2
(d, J = 8.2 Hz, 2H, Ar-H), 7.39 (dd, J = 8.6, 8.6 Hz, 2H, Ar-H), 7.81 (d, J = 8.2 Hz, 2H, Ar-H), 8.02
13
(dd, J = 5.9, 5.9 Hz, 2H, Ar-H), 8.67 (s, 1H, =CH-Ar), 10.95 (s, 1H, NH). C-NMR (100 MHz,
DMSO-d6): δ 22.30, 26.48, (121.77, CH  2), (127.15, CH  2), (129.51, CH  2), (130.88, CH  2),
134.32, 135.26, 136.70, 149.45, 152.14, 160.39, 167.48. EI-MS: (m/z, %): 295.15 (M+, 100), 296.10
(M++1, 19.45). Elemental analysis for C17H14FN3O (295.32), calcd.: C, 69.14; H, 4.78; N, 14.23.
Found: C, 69.18; H, 4.80; N, 14.21.
6-{4-[(2-Bromobenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7c).
Yield (2.32 g, 65%); m.p. 163-165 C; IR (KBr) cm-1, 3293 (NH), 2968 (CH), 1680 (CO), 1613 (C=N).
1H-NMR (400 MHz, DMSO-d6): δ 2.45 (t, J = 8.1 Hz, 2H, CH2), 2.98 (t, J = 8.1 Hz, 2H, CH2), 7.34
(d, J = 8.4 Hz, 2H, Ar-H), 7.52 (dd, J = 8.6, 8.6 Hz, 2H, Ar-H), 7.77 (d, J = 7.5 Hz, 1H, Ar-H), 7.82
(d, J = 8.4 Hz, 2H, Ar-H), 8.81 (d, J = 7.5 Hz, 1H, Ar-H), 8.82 (s, 1H, =CH-Ar), 10.98 (s, 1H, NH).
13C-NMR (100 MHz, DMSO-d6): δ 22.29, 26.47, (121.94, CH  2), 123.35, 126.28, (127.18, CH  2),
131.03, 134.75, 135.15, 137.93, 148.67, 149.36, 151.55, 159.69, 167.48. EI-MS: (m/z, %): 355.15
(M+, 100), 357.09 (M++2, 96.65). Elemental analysis for C17H14BrN3O (356.22), calcd.: C, 57.32; H,
3.96; N, 11.80. Found: C, 57.36; H, 4.00; N, 11.77.
6-{4-[(3-Nitrobenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7d).

Yield (2.26 g, 70%); m.p. 220-222 C; IR (KBr) cm-1, 3211 (NH), 2945 (CH), 1679 (CO), 1610 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.46 (t, J = 8.2 Hz, 2H, CH ), 2.99 (t, J = 8.2 Hz, 2H, CH ), 7.38
δ
6
2
2
(d, J = 8.4 Hz, 2H, Ar-H), 7.83–7.85 (m, 3H, Ar-H), 8.37 (dd, J = 6.5, 6.5 Hz, 2H, Ar-H), 8.75 (s, 1H,
13
Ar-H), 8.68 (s, 1H, =CH-Ar), 10.79 (s, 1H, NH). C-NMR (125 MHz, DMSO-d ): 21.83, 25.99,
δ
6
(121.47, CH  2), 122.98, 125.83, (126.72, CH  2), 130.58, 134.30, 134.68, 137.48, 148.28, 148.91,
151.10, 159.27, 167.01. EI-MS: (m/z, %): 322.21 (M+, 100), 323.23 (M++1, 22.91). Elemental analysis
for C17H14N4O3 (322.32), calcd.: C, 63.35; H, 4.38; N, 17.38. Found: C, 63.31; H, 4.42; N, 17.37.
6-{4-[(3-Nitrobenzylidene)amino]phenyl}-2-phenyl-4,5-dihydropyridazin-3(2H)-one (7e).
Yield (2.39 g, 60%); m.p. 211-213 C; IR (KBr) cm-1, 3063 (CH), 1679 (CO), 1630 (C=N). 1H-NMR
(400 MHz, DMSO-d ): 2.75 (t, J = 7.9 Hz, 2H, CH ), 3.17 (t, J = 7.9 Hz, 2H, CH ), 7.27 (dd, J =
δ
6
2
2
7.2, 7.2 Hz, 1H, Ar-H), 7.41–7.46 (m, 4H, Ar-H), 7.56 (d, J = 7.9 Hz, 2H, Ar-H), 7.83 (dd, J = 7.9, 7.9
Hz, 1H, Ar-H), 7.92 (d, J = 8.0 Hz, 2H, Ar-H), 8.38 (d, J = 7.5 Hz, 2H, Ar-H) 8.76 (s, 1H, Ar-H), 8.87
(s, 1H, =CH-Ar). 13C-NMR (125 MHz, DMSO-d6): δ 21.11, 25.83, (121.31, CH  2), (121.02, CH 
2), 123.01, 125.95, (125.99, CH  2), (126.02, CH  2), 130.49, 131.49, 132.38, 134.02, 134.28,
136.33, 146.75, 148.99, 150.85, 158.31, 167.81. EI-MS: (m/z, %): 398.10 (M+, 100), 399.19 (M++1,
26.65). Elemental analysis for C23H18N4O3 (398.42), calcd.: C, 69.34; H, 4.55; N, 14.06. Found: C,
69.30; H, 4.51; N, 14.07.
6-{4-[(4-Chlorobenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7f).
Yield (2.18 g, 71%); m.p. 271-273 C; IR (KBr) cm-1, 3195 (NH), 2940 (CH), 1684 (CO), 1609 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.48 (t, J = 8.1 Hz, 2H, CH ), 2.97 (t, J = 8.1 Hz, 2H, CH ), 7.33
δ
6
2
2
(d, J = 8.1 Hz, 2H, Ar-H), 7.60 (d, J = 8.0 Hz, 2H, Ar-H), 7.81 (d, J = 8.1 Hz, 2H, Ar-H), 7.97 (d, J =
13
8.0 Hz, 2H, Ar-H), 8.69 (s, 1H, =CH-Ar), 10.96 (s, 1H, NH). C-NMR (125 MHz, DMSO-d ):
δ
6
21.82, 25.99, (121.28, CH  2), (126.66, CH  2), (129.02, CH  2), (130.40, CH  2), 133.83, 134.78,
136.23, 148.96, 151.65, 159.89, 166.99. EI-MS: (m/z, %): 311.09 (M+, 100), 313.19 (M++2, 33.65).
Elemental analysis for C17H14ClN3O (311.77), calcd.: C, 65.49; H, 4.53; N, 13.48. Found: C, 65.55;
H, 4.57; N, 13.50.
6-{4-[(4-Methoxybenzylidene)amino]phenyl}-4,5-dihydropyridazin-3(2H)-one (7g).
Yield (1.91 g, 62%); m.p. 211-213 C; IR (KBr) cm-1, 3208 (NH), 2931 (CH), 1679 (CO), 1622 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.45 (t, J = 8.1 Hz, 2H, CH ), 2.97 (t, J = 8.1 Hz, 2H, CH ), 3.85
δ
6
2
2
(s, 3H, -OCH3), 7.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.28 (d, J = 8.2 Hz, 2H, Ar-H), 7.78 (d, J = 8.2 Hz,

2H, Ar-H), 7.90 (d, J = 8.4 Hz, 2H, Ar-H), 8.58 (s, 1H, =CH-Ar), 10.93 (s, 1H, NH). 13C-NMR (100
MHz, DMSO-d6): δ 22.29, 26.50, 55.91, (114.79, CH  2), (121.61, CH  2), (127.11, CH  2), 129.30,
(131.12, CH  2), 133.65, 149.55, 152.86, 160.74, 162.54, 167.49. EI-MS: (m/z, %): 307.15 (M+, 100),
308.19 (M++1, 20.65). Elemental analysis for C18H17N3O2 (307.35), calcd.: C, 70.34; H, 5.58; N,
13.67. Found: C, 70.30; H, 5.57; N, 13.65.
6-[4-(Benzylideneamino)phenyl]-4,5-dihydropyridazin-3(2H)-one (7h).
Yield (1.66 g, 60%); m.p. 248-250 C; IR (KBr) cm-1, 3218 (NH), 2991 (CH), 1670 (CO), 1620 (C=N).
1H-NMR (400 MHz, DMSO-d ): 2.48 (t, J = 7.9 Hz, 2H, CH ), 2.98 (t, J = 7.9 Hz, 2H, CH ), 7.32
δ
6
2
2
(d, J = 8.0 Hz, 2H, Ar-H), 7.46 – 7.55 (m, 3H, Ar-H), 7.44 (d, J = 8.1 Hz, 2H, Ar-H), 7.81 (d, J = 8.0
Hz, 2H, Ar-H), 8.68 (s, 1H, =CH-Ar), 10.98 (s, 1H, NH). 13C-NMR (125 MHz, DMSO-d6): δ 21.22,
25.19, (121.87, CH  2), (125.96, CH  2), (128.82, CH  2), (129.81, CH  2), 133.12, 134.15, 131.11,
148.56, 151.53, 160.01, 166.69. EI-MS: (m/z, %): 277.15 (M+, 100), 278.19 (M++1, 20.15). Elemental
analysis for C17H15N3O (277.33), calcd.: C, 73.63; H, 5.45; N, 15.15. Found: C, 73.69; H, 5.50; N,
15.12.
5.1.5. General method for synthesis of1-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]-3-phenylurea
(or thiourea) derivatives (8a-i).
A mixture of compound 4a or 4b (10 mmol) and the appropriate aryl iso(thio)cyanate derivative (10
mmol) was dissolved in THF (10 mL) and stirred at room temperature for 4h. The resultant precipitate
was collected by filtration, dried, and recrystallized from THF to give the titled compounds 8a-i.
1-[4-(6-Oxo-4,5-dihydropyridazin-3-yl)phenyl]-3-phenylthiourea (8a).
Yield (2.37 g, 73%); m.p. 220-222 C; IR (KBr) cm-1, 3227 (NH), 3058 (NH), 2977 (CH); 1673 (CO).
1H-NMR (500 MHz, DMSO-d ): 2.43 (t, J = 8.2 Hz, 2H, CH ), 2.93 (t, J = 8.2 Hz, 2H, CH ), 7.13
δ
6
2
2
(dd, J = 7.2, 7.2 Hz, 1H, Ar-H), 7.32 (dd, J = 7.8, 7.8 Hz, 2H, Ar-H), 7.47 (d, J = 7.8 Hz, 2H, Ar-H),
7.56 (d, J = 8.8 Hz, 2H, Ar-H), 7.70 (d, J = 8.8 Hz, 2H, Ar-H), 9.90 (s, 1H, NH), 9.95 (s, 1H, NH),
10.89 (s, 1H, NH). 13C-NMR (125 MHz, DMSO-d ): 22.17, 27.11, (122.29, CH  2), (124.01, CH 
δ
6
2), 124.38, (124.49, CH  2), 125.97, (126.26, CH  2), 140.23, 141.10, 150.29, 164.59, 179.29. EI-
MS: (m/z, %): 324.12 (M+, 100), 325.19 (M++1, 19.19). Elemental analysis for C17H16N4OS (324.40),
calcd.: C, 62.94; H, 4.97; N, 17.27. Found: C, 62.98; H, 5.00; N, 17.25.
1-[4-(6-Oxo-1-phenyl-4,5-dihydropyridazin-3-yl)phenyl]-3-phenylthiourea (8b).

Yield (2.96 g, 74%); m.p. 208-210 C; IR (KBr) cm-1, 3230 (NH), 3056 (NH), 2971 (CH); 1671 (CO).
1H-NMR (500 MHz, DMSO-d ): 2.71 (t, J = 8.1 Hz, 2H, CH ), 3.11 (t, J = 8.1 Hz, 2H, CH ), 7.13
δ
6
2
2
(dd, J = 7.3, 7.3 Hz, 1H, Ar-H), 7.27 (dd, J = 7.4, 7.4 Hz, 1H, Ar-H), 7.33 (dd, J = 7.9, 7.9 Hz, 2H,
Ar-H), 7.42 (dd, J = 7.9, 7.9 Hz, 2H, Ar-H), 7.47 (d, J = 7.7 Hz, 2H, Ar-H), 7.54 (d, J = 7.7, 2H, Ar-
H), 7.61 (d, J = 8.4 Hz, 2H, Ar-H), 7.78 (d, J = 8.4 Hz, 2H, Ar-H), 9.92 (s, 1H, NH), 9.99 (s, 1H, NH).
13C-NMR (125 MHz, DMSO-d ): 22.12, 27.43, (122.70, CH  2), (123.63, CH  2), 124.57, (124.82,
δ
6
CH  2), 126.07, (126.38, CH  2), (128.29, CH  2), (128.50, CH  2), 130.87, 139.30, 141.06,
141.44, 151.60, 165.30, 179.32. EI-MS: (m/z, %): 400.12 (M+, 100), 404.19 (M++1, 26.89). Elemental
analysis for C23H20N4OS (400.50), calcd.: C, 68.98; H, 5.03; N, 13.99. Found: C, 68.95; H, 5.00; N,
13.95.
1-(4-Nitrophenyl)-3-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]thiourea (8c).
Yield (2.66 g, 72%); m.p. 200-202 C; IR (KBr) cm-1, 3447 (NH), 3212 (NH), 3100 (NH), 2950 (CH),
1645 (CO). 1H-NMR (500 MHz, DMSO-d ): 2.43 (t, J = 8.3 Hz, 2H, CH ), 2.93 (t, J = 8.3 Hz, 2H,
δ
6
2
CH2), 7.57 (d, J = 8.7 Hz, 2H, Ar-H), 7.73 (d, J = 8.7 Hz, 2H, Ar-H), 7.81 (d, J = 9.1 Hz, 2H, Ar-H),
8.19 (d, J = 9.1 Hz, 2H, Ar-H), 10.41 (s, 1H, NH), 10.46 (s, 1H, NH), 10.91 (s, 1H, NH). 13C-NMR
(100 MHz, DMSO-d ): 22.17, 26.53, (118.24, CH  2), (120.28, CH  2), 125.96, (126.89, CH  2),
δ
6
(129.13, CH  2), 129.91, 139.02, 141.09, 149.64, 152.72, 167.46. EI-MS: (m/z, %): 369.15 (M+, 100),
370.19 (M++1, 20.85). Elemental analysis for C17H15N5O3S (369.40), calcd.: C,55.28; H, 4.09; N,
18.96. Found: C, 55.33; H, 4.14; N, 18.95.
1-(4-Nitrophenyl)-3-[4-(6-oxo-1-phenyl-4,5-dihydropyridazin-3-yl)phenyl]thiourea (8d).
Yield (3.30 g, 74%); m.p. 213-215 C; IR (KBr) cm-1, 3333 (NH), 3207 (NH), 2987 (CH), 1631 (CO).
1H-NMR (500 MHz, DMSO-d ): 2.72 (t, J = 8.1 Hz, 2H, CH ), 3.12 (t, J = 8.1 Hz, 2H, CH ), 7.26
δ
6
2
2
(dd, J = 7.4, 7.4 Hz, 1H, Ar-H), 7.42 (dd, J = 7.9, 7.9 Hz, 2H, Ar-H), 7.53 (d, J = 8.8 Hz, 2H, Ar-H),
7.61 (d, J = 8.8 Hz, 2H, Ar-H), 7.81–7.84 (m, 4H, Ar-H), 8.19 (d, J = 9.2 Hz, 2H, Ar-H), 10.45 (s, 1H,
NH), 10.48 (s, 1H, NH). 13C-NMR (125 MHz, DMSO-d ): 22.17, 26.85, (119.98, CH  2), (119.25,
δ
6
CH  2), 124.49, (127.02, CH  2), 126.92, (128.10, CH  2), (128.21, CH  2), (128.48, CH  2),
131.25, 139.82, 141.98, 142.01, 148.60, 162.95, 178.98. EI-MS: (m/z, %): 445.18 (M+, 100), 446.19
(M++1, 27.81). Elemental analysis for C23H19N5O3S (445.50), calcd.: C,62.01; H, 4.30; N, 15.72.
Found: C, 62.05; H, 4.34; N, 15.75.
1-(4-Nitrophenyl)-3-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]urea (8e).

Yield (2.65 g, 75%); m.p. 294-296 C; IR (KBr) cm-1, 3364 (NH), 3164 (NH), 2970 (CH), 1655 (CO);
1H-NMR (500 MHz, DMSO-d ): 2.42 (t, J = 8.2 Hz, 2H, CH2), 2.91 (t, J = 8.2 Hz, 2H, CH2), 7.53
δ
6
(d, J = 8.7 Hz, 2H, Ar-H), 7.68 (d, J = 8.7 Hz, 2H, Ar-H),7.70 (d, J = 8.9 Hz, 2H, Ar-H), 8.19 (d, J =
8.9 Hz, 2H, Ar-H), 9.11 (s, 1H, NH), 9.48 (s, 1H, NH), 10.85 (s, 1H, NH). 13C-NMR (125 MHz,
DMSO-d ): 21.52, 26.22, (117.22, CH  2), (118.02, CH  2), 125.99, (126.11, CH  2), (128.01,
δ
6
CH  2), 132.86, 140.88, 144.73, 145.81, 152.95, 165.92. EI-MS: (m/z, %): 353.18 (M+, 100), 354.16
(M++1, 19.83). Elemental analysis for C17H15N5O4 (353.34), calcd.: C, 57.79; H, 4.28; N, 19.82.
Found: C, 57.75; H, 4.23; N, 19.85.
1-(4-Nitrophenyl)-3-[4-(6-oxo-1-phenyl-4,5-dihydropyridazin-3-yl)phenyl]urea (8f).
Yield (3.22 g, 75%); m.p. 296-298 C; IR (KBr) cm-1, 3353 (NH), 3158 (NH), 3102 (CH), 1648 (CO).
1H-NMR (500 MHz, DMSO-d ): 2.47 (t, J = 8.1 Hz, 2H, CH ), 3.10 (t, J = 8.1 Hz, 2H, CH ), 7.27
δ
6
2
2
(dd, J = 7.3, 7.3 Hz, 1H, Ar-H), 7.41 (dd, J = 8.1, 8.1 Hz, 2H, Ar-H), 7.52–7.57 (m, 4H, Ar-H), 7.69
(d, J = 8.1 Hz, 2H, Ar-H), 7.80 (d, J = 8.7 Hz, 2H, Ar-H), 8.19 (d, J = 8.7 Hz, 2H, Ar-H), 9.18 (s, 1H),
9.48 (s, 1H, NH). 13C-NMR (125 MHz, DMSO-d ): 22.68, 27.38, (119.50, CH  2), (120.03, CH 
δ
6
2), 121.87, (122.02, CH  2), 125.97, (125.98, CH  2), (128.11, CH  2), (128.00, CH  2), 129.17,
139.30, 139.85, 140.13, 150.21, 152.09, 167.32. EI-MS: (m/z, %): 429.18 (M+, 100), 430.29 (M++1,
26.77). Elemental analysis for C23H19N5O4 (429.44), calcd.: C, 64.33; H, 4.46; N, 16.31. Found: C,
64.36; H, 4.50; N, 16.30.
1-(4-Chlorophenyl)-3-[4-(6-oxo-4,5-dihydropyridazin-3-yl)phenyl]urea (8g).
Yield (3.12 g, 72%); m.p. > 300 C; IR (KBr) cm-1, 3385 (NH), 3216 (NH), 3104 (NH), 2950 (CH),
1668 (CO). 1H-NMR (500 MHz, DMSO-d ): 2.41 (t, J = 8.2 Hz, 2H, CH ), 2.90 (t, J = 8.2 Hz, 2H,
δ
6
2
CH2), 7.31 (d, J = 8.4 Hz, 2H, Ar-H), 7.47 (d, J = 8.4 Hz, 2H, Ar-H), 7.49 (d, J = 8.8 Hz, 2H, Ar-H),
7.67 (d, J = 8.8 Hz, 2H, Ar-H), 8.87 (s, 1H, NH), 8.90 (s, 1H), 10.83 (s, 1H, NH). 13C-NMR (125 MHz,
DMSO-d ): 21.70, 26.05, (117.77, CH  2), (119.81, CH  2),125.49, (126.41, CH  2), (128.66,
δ
6
CH  2), 129.45, 138.57, 140.61, 142.09, 152.43, 166.98. EI-MS: (m/z, %): 342.23 (M+, 100), 344.21
(M++2, 33.78). Elemental analysis for C17H15ClN4O2 (342.78), calcd.: C, 59.57; H, 4.41; N, 16.35.
Found: C, 59.62; H, 4.48; N, 16.31.
1-(4-Chlorophenyl)-3-[4-(6-oxo-1-phenyl-4,5-dihydropyridazin-3-yl)phenyl]urea (8h).
Yield (3.14 g, 75%); m.p. 273-275 C; IR (KBr) cm-1, 3357 (NH), 3297 (NH), 3130 (CH), 1651 (CO).
1H-NMR (500 MHz, DMSO-d ): 2.70 (t, J = 8.1 Hz, 2H, CH ), 3.10 (t, J = 8.1 Hz, 2H, CH ), 7.27
δ
6
2
2

(dd, J = 8.4, 8.4 Hz,1H, Ar-H), 7.42 (d, J = 8.5 Hz, 2H, Ar-H), 7.54-7.60 (m, 4H, Ar-H), 7.68 (d, J =
8.5 Hz, 2H, Ar-H), 7.79 (d, J = 8.9 Hz, 2H, Ar-H), 8.18 (d, J = 8.9 Hz, 2H, Ar-H), 9.18 (s, 1H, NH),
9.48 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d ): 22.49, 27.93, (118.24, CH  2), (120.32, CH 
δ
6
2), (125.34, CH  2), 126.01, 126.52, (127.44, CH  2), (128.76, CH  2), (129.14, CH  2), 129.22,
138.98, 141.64, 141.97, 152.18, 152.69, 165.80. EI-MS: (m/z, %): 418.23 (M+, 100), 420.91 (M++2,
33.38). Elemental analysis for C23H19ClN4O2 (418.88), calcd.: C, 65.95; H, 4.57; N, 13.38. Found: C,
65.90; H, 4.60; N, 13.41.
1-[4-(6-Oxo-4,5-dihydropyridazin-3-yl)phenyl]-3-(p-tolyl)urea (8i).
Yield (2.30 g, 71%); m.p. > 300 C; IR (KBr) cm-1, 3348 (NH), 3196 (NH), 3100 (NH), 2918 (CH),
1673 (CO). 1H-NMR (500 MHz, DMSO-d ): 2.23 (s, 3H, CH ), 2.41 (t, J = 8.2 Hz, 2H, CH ), 2.90
δ
6
3
2
(t, J = 8.2 Hz, 2H, CH2), 7.08 (d, J = 8.3 Hz, 2H, Ar-H), 7.32 (d, J = 8.3 Hz, 2H, Ar-H), 7.48 (d, J =
8.70 Hz, 2H, Ar-H), 7.67 (d, J = 8.7 Hz, 2H, Ar-H), 8.59 (s, 1H, NH), 8.80 (s, 1H, NH), 10.82 (s, 1H,
13
NH). C-NMR (125 MHz, DMSO-d ): 20.36, 21.70, 26.07, (117.85, CH  2), (118.37, CH  2),
δ
6
(126.40, CH  2), (129.20, CH  2), 130.82, 136.93, 140.90, 142.09, 149.21, 152.37, 166.70. EI-MS:
(m/z, %): 322.33 (M+, 100), 323.85 (M++1, 20.28). Elemental analysis for C18H18N4O2 (322.37),
calcd.: C, 67.07; H, 5.63; N, 17.38. Found: C, 67.11; H, 5.68; N, 17.35.
5.2. Biological methodology
Five human tumor cell lines HCT-116, Hela, HEPG-2, MCF-7 and PC-3; were obtained from
American Type Culture Collection (ATCC)51 via Holding company for biological products and
vaccines (VACSERA), Cairo, Egypt. These cell lines were used to determine the inhibitory effects of
compounds 4a–8i on cell growth against the standard antitumor drug sorafenib.
5.2.1. MTT assay for cytotoxicity
MTT methodology52-55 is a colorimetric assay based on the conversion of the yellow tetrazolium
bromide to a purple formazan56 derivative by mitochondrial succinate dehydrogenase.57 Cell lines were
cultured in RPMI-1640 medium with 10% fetal bovine serum. 100 IU/mL Penicillin and 100 µg/mL
streptomycin were added at 37 ºC in a 5% carbon dioxide incubator for 48 h. The cell lines were then
seeded in a 96-well plate at a density of 104 cells/well. After incubation, the cells were treated with
different concentrations of the designed compounds 4b–8i and incubated for additional 24 h. Then,
MTT (5 mg/mL, 20 µl) solution was added and incubated for another 4 h. Dimethyl sulfoxide (100 µl)
was added into each well to dissolve the formed purple formazan. The colorimetric assay was

performed and results were measured and recorded at absorbance of 570 nm using a plate reader (EXL
800, USA). The relative cell viability was calculated as a percentage of (A570 of treated samples/A570
of untreated sample). The cytotoxic activity was expressed as IC50 mean ± SEM compared with the
growth of untreated cells.
5.2.2. B-Raf inhibition assay
The detecting reagent Kinase-Glo® MAX was used to measure B-Raf kinase activity. The B-Raf kinase
assay kit was added to 96-well plates with purified recombinant B-Raf (V600E)44, 45 enzyme, B-Raf
substrate, ATP and kinase assay buffer. After preparation of the positive control, test inhibitor and
blank wells, diluted Raf (V600E) enzyme was added to both the positive control and test inhibitor with
incubation for 45 minutes at 30 ºC. Then, Kinase-Glo® MAX reagent was added to each well and
incubated at room temperature for15 minutes.58 Measurement of the produced luminescence was
performed via a micro-plate reader. All samples and controls were tested in duplicate.59
5.2.3. Cell cycle analysis
The MCF-7 cell lines were treated with compound 8b for 24 h. After that, the cells were suspended in
0.5 mL of phosphate buffer saline (PBS), collected by centrifugation, fixed with ice-cold ethanol (70%
v/v), re-suspended with RNase (0.1 mg/mL), stained with propidium iodide (PI) (50 µg/mL) and
incubated at 37 ºC in a 5% carbon dioxide incubator for 55 min. The PI fluorescence intensity was
measured by flow cytometry using FACS caliber (Becton Dickinson).60, 61
5.2.4. Detection of apoptosis
The MCF-7 cell lines in complete growing medium were treated with compound 8b and incubated for
24 h. Then, 1-5 X 105 cells were harvested and suspended in 500 mL of 1X binding buffer after that,
5 mL of Annexin V-FITC and 5 mL of PI were added. The reaction media were then reincubated in
dark for further 5 min. Analysis of Annexin-V-FITC binding was performed using FACS caliber flow
cytometer.
5.3. Molecular docking methodology
Molecular docking was carried out using MOE software. The three-dimensional structures of the
ligand compounds 4b-8i were generated using Cambridge software program chem. office. The
3Dcrystal structure of the target protein 1UWJ was retrieved from the Protein Data Bank (PDB) at the

Research Collaboratory for Structural Bioinformatics (RCSB, http://www.rcsb.org) and was imported
in the MOE program.
MOE program recognized the binding cavity of the pre-existed ligand sorafenib and all the tested
ligands were docked in this cavity. The number of runs of ligands was set to 30 and the lowest energy
aligned conformations were identified. In a similar way the poses of the tested ligands 4b-8i were
selected and compared.
5. 4. ADMET properties and Lipinski’s rule of five
The physicochemical properties of compounds 6a, 7g, 8b and sorafenib were calculated using MOE
software by creating a new data base followed by importing the compounds in the form of mol2 files
then, the selected descriptors were computed.
6. Acknowledgments
The authors gratefully acknowledge Mansoura University for the continues support as well as the
Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt, for performing
the biological screening.
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