Synthesis and cytotoxic activities of some pyrazoline derivatives bearing phenyl pyridazine core as new apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2016.01.048

The cyclization of chalcones 3a-3u with 3-hydrazinyl-6-phenylpyridazine 7 under basic condition led to the formation of new pyrazoline derivatives 8a-8u. All final compounds were characterized by spectral and elemental analyses. They were screened for their antiproliferative activities against A549 (lung), HepG-2 (liver), CaCo-2 (intestinal) and MCF-7 (breast) cancer cell lines. Some of the synthesized compounds exhibited promising antiproliferative activities especially compound 8k with IC₅₀values of 8.33, 1.67 and 10?μM against HepG-2, MCF-7 and CaCo-2 cancer cell lines, respectively. Moreover, their antiproliferative activity was due to apoptosis rather than necrosis induction except compound 8h which exhibited equal apoptotic and necrotic properties. Compound 8k showed 5 fold increase in caspase-3 activity indicating that the apoptosis proceeds via caspase-3 activation.

Full text of DOI:10.1016/j.ejmech.2016.01.048

Accepted Manuscript
Synthesis and cytotoxic activities of some pyrazoline derivatives bearing phenyl
pyridazine core as new apoptosis inducers
Riham F. George, Marwa A. Fouad, Iman E.O. Gomaa
PII:
S0223-5234(16)30048-4
10.1016/j.ejmech.2016.01.048
EJMECH 8338
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 November 2015
Revised Date: 20 January 2016
Accepted Date: 25 January 2016
Please cite this article as: R.F. George, M.A. Fouad, I.E.O. Gomaa, Synthesis and cytotoxic activities
of some pyrazoline derivatives bearing phenyl pyridazine core as new apoptosis inducers, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.048.
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ACCEPTED MANUSCRIPT
Synthesis and cytotoxic activities of some pyrazoline derivatives bearing phenyl
pyridazine core as new apoptosis inducers
Riham F. Georgea*, Marwa A. Fouada, Iman E. O. Gomaab

ACCEPTED MANUSCRIPT
Synthesis and cytotoxic activities of some pyrazoline derivatives bearing phenyl
pyridazine core as new apoptosis inducers
Riham F. Georgea*, Marwa A. Fouada, Iman E. O. Gomaab
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-kasr Elaini Street,
Cairo 11562, Egypt
b Biotechnology sector, Faculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New
Cairo City, Egypt.
Abstract
The cyclization of chalcones 3a-3u with 3-hydrazinyl-6-phenylpyridazine 7 under
basic condition led to the formation of new pyrazoline derivatives 8a-8u. All final
compounds were characterized by spectral and elemental analyses. They were screened
for their antiproliferative activities against A549 (lung), HepG-2 (liver), CaCo-2
(intestinal) and MCF-7 (breast) cancer cell lines. Some of the synthesized compounds
exhibited promising antiproliferative activities especially compound 8k with IC50 values
of 8.33, 1.67 and 10 ꢀM against HepG-2, MCF-7 and CaCo-2 cancer cell lines,
respectively. Moreover, their antiproliferative activity was due to apoptosis rather than
necrosis induction except compound 8h which exhibited equal apoptotic and necrotic
properties. Compound 8k showed 5 fold increase in caspase-3 activity indicating that the
apoptosis proceeds via caspase-3 activation.
Keywords: Pyrazolylpyridazines, chalcones, MTT assay, apoptosis, caspase-3.
* Corresponding author.
Riham F. George, E-mail address: rihamfgeorge@yahoo.com
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1. Introduction
Cancer is a group of diseases characterized by uncontrolled growth and spread of
the abnormal cells [1]. Among all diseases that affect humanity, cancer ranks as the
second leading cause of death after cardiovascular diseases [2]. The worldwide cancer
burden is expected to increase by as much as 15 million new cancer cases per year by
2020, according to the World Health Organization, unless further preventive measures
are put into practice [3]. Generally, cancers of the breast, lung, colorectal, and prostate
are the most frequent types in developed countries while those of the stomach, liver, oral
cavity, and cervix, in developing countries, although this pattern seems to be evolving,
especially due to population aging and life style changes [4,5]. Strategies used for
treatment of cancer include surgery, radiation, chemotherapy, hormone therapy, immune
therapy, and targeted therapy [1]. Despite major advances in the chemotherapeutic
management of some patients, the use of available chemotherapeutics is often limited
mainly due to toxicities and emerging drug resistance [6]. Therefore, there is a continued
commitment to discover new anticancer agents.
Pyrazoline is an important scaffold of many bioactive agents that display different
biological activities. This can be represented by some pyrazolines that exhibit antiviral
[7], antitproliferative [8-15], antibacterial [16-18], anti-inflammatory [9, 16, 19, 20],
analgesic [16], and antihyperglycemic activities [21]. Recently, it was reported that 3,5-
diaryl pyrazolines I and II (Fig.1) exhibited potent cytotoxic activity against different
cancer cell lines [12, 14]. Furthermore, 6-phenylpyridazine can be considered as an
essential core responsible for various biological activities of different candidates. In
literature, some pyridazines act as antidepressant [22], antinociceptive [23], interleukin-
1β inhibitory [24], formyl peptide receptors agonists [25], antidiabetic [26], antimicrobial
[27] and antiproliferative agents [27-33]. 6-Arylpyridazines III and IV (Fig. 1) revealed
promising cytotoxic activity [29, 32].
From the molecular design point of view, the combination of two pharmacophores
into a single molecule represents one of the methods that can be adopted for the synthesis
of new anticancer molecules [34-40]. This approach was supported by compound V
which is a pyrazole substituted pyridazinone with potent antiproliferative and c-Met
inhibitory activities [41]. Therefore, based on the previous findings, this work deals with
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synthesis of a novel series 8a-u through hybridization of the pharmacophores, 3,5-
diarylpyrazoline and 6-phenylpyridazine (Fig. 2), hoping to obtain new promising
antiproliferative hits. Additionally, all the synthesized derivatives were screened against
diverse human tumor cell lines, including A549 “lung”, HEPG2 “liver”, MCF7 “breast”
and CaCo2 “intestinal” cancer cell lines. Moreover, the most active compounds were
further tested for their necrotic and/or apoptotic properties. Additionally, the apoptotic
mechanism of compound 8k having the most promising IC50 was investigated through
caspase-3 colorimetric assay.
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Fig. 1. Structures of some 3,5-diarylpyrazolines, 6-arylpyridazines and pyrazole-pyridazine hybrid with
antiproliferative activity
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Fig.2. General structure of target compounds
2. Results and discussion
2.1. Chemistry
The target compounds 8a–8u were prepared as outlined in Scheme 3. First, a
series of chalcones 3a-u were prepared according to scheme 1 by the Claisen–Schmidt
condensation between acetophenones 1a-c and different aromatic aldehydes 2a-g in
absolute ethanol in the presence of sodium hydroxide in 80–85% yield [42-53].
Furthermore, the reaction of acetophenone 1a with glyoxilic acid 4 was followed by
treatment with hydrazine hydrate to afford 6-phenyl-3-pyridazinone 5 [54]. Moreover,
reflux of 5 in phosphorous oxychloride resulted in 3-chloropyridazine 6 that was reacted
with hydrazine hydrate in absolute ethanol to obtain 7 (Scheme 2) [55]. The target
pyrazoline derivatives 8a-u were prepared by reaction of 3-hydrazinyl-6-
phenylpyridazine 7 with the appropriate chalcones 3a-u in absolute ethanol in the
presence of sodium hydroxide (Scheme 3). All the final compounds 8a-u were
1
1
characterized by IR, H NMR and elemental analyses. H NMR spectra showed three
signals doublet of doublet at 3.05-3.32, 3.77-3.94 and 5.83-6.17 ppm corresponding to
the pyrazoline protons in addition to the signals of pyridazine and aromatic protons (c.f.
experimental part). On the other hand, 13C NMR of 8u as a representative example
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revealed signals at 43.4 and 62.1 of CH2 and CH of the pyrazoline ring, respectively, as
well as signals at 55.4, 56.1 and 60.7 attributed to four methoxy moieties.
Scheme 1. Preparation of chalcones 3a-u
O
H
O
O
N
NH
CH3
NH2NH2.H2O
+
O
OH
1a
4
5
POCl3
N N
NH2
NH
N N
NH2NH2.H2O
Cl
6
7
Scheme 2. Preparation of 3-hydrazinyl-6-phenylpyridazine 7
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Scheme 3. Preparation of target compounds 8a-u
2.2. Biological evaluation
2.2.1. Antiproliferative activity
In-vitro antiproliferative activity of the tested compounds was performed utilizing
MTT cell viability assay [56]. The target compounds 8a-u were screened against A549
“lung”, HEPG2 “liver”, MCF7 “breast” and CaCo2 “intestinal” cancer cell lines,
Doxorubicin was used as a reference standard. From the observed data (Table 1), it has
been noticed that many compounds exerted significant activity against HepG-2 “liver”
cell line compared with the other assayed cell lines. Considering the observed
antiproliferative screening data against A549 “lung” cancer cell line, only compounds 8d
and 8f revealed the highest activities with IC50 values 9.16 and 9.56 ꢀM, respectively.
However, compounds 8c and 8k resulted in 50% viability of CaCo-2 “intestinal” cancer
cell line at concentration of 10 ꢀM. Additionally, compound 8q exerted moderate activity
with IC50 value 18.07 ꢀM, while, compounds 8a, 8d and 8r revealed mild activity with
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IC50 values 40.86 - 65.00 ꢀM against the same cell line. Otherwise, compounds 8k, 8j, 8i
and 8h exhibited promising activity against MCF-7 “breast” cancer cell line with IC50
values 1.67, 3.75, 8.80 and 10 ꢀM, respectively. On the other hand, compounds 8a and
8q revealed moderate activity, meanwhile, compounds 8p and 8r showed mild activity.
Furthermore, compounds 8b-d, 8h-k and 8q resulted in promising activity against
HepG-2 “liver” cancer cell line with IC50 values 6.31-10 ꢀM. Structure activity
relationship study of the obtained antiproliferative activity of the target compounds 8a-u
against HepG-2 cancer cell line indicated that substitution with electron withdrawing
groups at one or both phenyl rings of the pyrazoline moiety, seemed more favorable for
activity than the case when electron donating groups were incorporated in either phenyl
rings. However, compound 8q is an exception (IC50= 9.16 ꢀM).
Moreover, the most active compounds 8b, 8c, 8d, 8f, 8h, 8i, 8j and 8k were
screened for their cytotoxicity against human embryonic kidney cells 293 as example of
normal cells at 10, 100, 200 and 300 ꢀM. The % viable cells and IC50 values were shown
in Table 2. All tested compounds were safe to the tested normal cells up to 300 ꢀM.
Therefore, these compounds were selective to the tested cancer cell lines.
2.2. 2. Evaluation of Cell Death Pathways
The most active compounds were further screened at 10 ꢀM for their cytotoxicity
mechanism either apoptosis or necrosis by applying the flow cytometry technique. The
obtained results were shown in the flow cytometry charts (Figures 3-6). The % apoptosis
or necrosis induction for each compound was calculated by taking the difference between
% apoptotic or necrotic cells of each compound and that of the negative control, and they
were summarized in Table 3. From the obtained data, it can be concluded that all tested
compounds can be considered as apoptotic inducers rather than necrotic inducers except
compound 8h which exhibited equal apoptotic and necrotic properties.
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Table 1: IC50 of the target compounds 8a-u against the tested cancer cell lines in MTT
assay
Compound
R
R`
IC50 (
µM) against
ID
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
A549
>100
100
89.2
9.16
HepG2 MCF7 CaCo2
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
4-Cl
4-F
10
23.75
100
100
100
100
>100
70
40.86
100
10
6.31
8.42
10
>100
100
100
8.75
10
4-CF3
50
4-OCH3
2,4-(OCH3)2
3,4,5-(OCH3)3
H
91.67
9.56
>100
>100
>100
100
>100
>100
10
>100
>100
>100
>100
>100
18.07
65
83.75
96.67
>100
>100
>100
100
>100
83.33
75.90
100
>100
>100
>100
100
>100
2.12
10
4-Cl
4-F
4-CF3
8.80
3.75
1.67
100
>100
100
>100
58
10
8.33
>100
>100
>100
>100
>100
9.16
>100
>100
>100
>100
3.82
4-OCH3
2,4-(OCH3)2
3,4,5-(OCH3)3
H
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
---------
4-Cl
4-F
4-CF3
8p
8q
8r
8s
8t
8u
20
46.8
100
100
100
2.78
4-OCH3
2,4-(OCH3)2
3,4,5-(OCH3)3
--------------
>100
>100
>100
3.00
Doxorubicin
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Table 2: % viable cells and IC50 of the most active compounds against normal human
embryonic kidney cells 293
Compd.
ID
8b
8c
8d
8f
8h
8i
8j
% Viability of normal human embryonic kidney 293± SE at
100 200 300
IC50 (µM)
10
µM
µM
µM
µM
96.63 ± 3.13
93.68 ± 4.26
89.75 ± 1.96
85.16 ± 4.62
83.96 ± 2.33
85.60 ± 2.08
85.93 ± 4.30
92.70 ± 5.57
91.17 ± 2.58
91.88 ± 5.41
86.25 ± 1.79
82.87 ± 4.50
81.56 ± 1.64
81.67 ± 3.60
82.32 ± 1.79
87.67 ± 0.93
88.33 ± 1.26
86.31 ± 2.46
82.32 ± 1.61
81.12 ± 0.76
78.12 ± 2.13
79.81 ± 2.62
75.77 ± 2.02
84.62 ± 3.45
74.03 ± 1.64
78.61 ± 4.91
70.75 ± 5.24
74.19 ± 1.47
71.57 ± 2.46
76.42 ± 1.94
68.78 ± 4.45
79.10 ± 1.80
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
8k
Control
Compound 8d
Compound 8f
80
60
40
20
0
Necrosis
***
Figure 3. Effect of compound 8d, 8f on the induction of apoptosis/necrosis in A549 cells.
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Control
Compound 8b
Compound 8c
Compound 8h
Compound 8k
**
Apoptosis
Necrosis
***
80
60
40
20
0
***
***
***
***
NC
B
C
H
K
Figure 4. Effect of compound 8b, 8c, 8h and 8k on the induction of apoptosis/necrosis in HepG2 cells.
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Control
Compound 8i
Compound 8j
Compound 8k
*
80
Apoptosis
Necrosis
***
***
***
60
40
20
0
NC
I
J
K
Figure 5. Effect of compound 8i, 8j and 8k on the induction of apoptosis/necrosis in MCF-7 cells.
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Control
Compound 8k
40
Apoptosis
Necrosis
***
30
20
10
0
NC
K
Figure 6. Effect of compound 8k on the induction of apoptosis/necrosis in CaCo2 cells.
Table 3: % apoptotic and necrotic induction of the most active compounds at 10 ꢀM
A549
HepG-2
MCF7
CaCo2
Compd.
ID
%Apoptotic
induction
% necrotic %Apoptotic
% necrotic %Apoptotic % necrotic %Apoptotic % necrotic
induction
induction
44.40
30.50
NT
NT
22.70
NT
induction
4.40
8.84
NT
induction
induction
induction
induction
NT
NT
46.60
27.00
NT
NT
NT
NT
NT
NT
11.11
1.04
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
13.40
NT
NT
NT
8b
8c
8d
8f
8h
8i
NT
NT
NT
23.45
NT
NT
NT
NT
NT
NT
NT
1.87
39.00
32.10
27.20
0.10
1.98
1.26
NT
24.80
8j
8k
4.97
* NT: not tested
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2.2.3. Caspase-3 activation assay
Caspase-3 is a ubiquitously-expressed cysteine protease involved in programmed cell
death (apoptosis). In non-apoptotic cells, this enzyme exists as an inactive zymogen
known as procaspase-3. Intracellular procaspase-3 is activated when cells are committed
to die via apoptosis. The active enzyme cleaves a number of intracellular protein targets
important for cell survival. Therefore, monitoring caspase-3 enzyme activity is a
fundamental means to measure apoptosis in cells. In an attempt to find out whether the
induction of apoptosis of compounds 8b, 8c, 8d, 8f, 8i, 8j and 8k is dependent on caspase-3
activity or not, 8k was selected as a representative for testing caspase-3 activity in MCF-7
cell lines as it is the most active candidate with promising IC50 = 1.67
ꢀM. Caspase-3
colorimetric detection kit was used. The cleavage of the caspase 3-specific tetrapeptide
substrate (ac-Asp-Glu-Val-Asp-pNA) that is labeled with the chromophore p-nitroaniline
(pNA) by the activated caspase-3 leads to the release of the chromophore pNA. The free
pNA group has a yellow color which is visible in the wells and was quantitated
spectrophotometrically at 405 nm. A significant 5-fold increase was observed in the
induction of caspase-3 in MCF-7 cells treated with compound 8k relative to the control
(Fig. 7). Therefore, the apoptotic pathway may proceed through caspase-3 activation.
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control
8k
Figure 7. Effect of compound 8k on caspase-3 activity in MCF-7 cell lines compared to the negative
control (untreated cells).
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3. Conclusion
In summary, novel pyrazolyl pyridazine derivatives 8a-8u were synthesized by
the cyclization of chalcones 3a-3u with 3-hydrazinyl-6-phenylpyridazine 7 under basic
condition. All target compounds 8a-u were screened for their antiproliferative activities
against A549 (lung), HepG-2 (liver), CaCo-2 (intestinal) and MCF-7 (breast) cancer cell
lines using MTT cell viability assay. Many compounds exhibited moderate to good
activity against MCF-7 and HepG-2 cancer cell lines. On the other hand, only compounds
8d and 8f showed promising activity against A549 cancer cell lines, whereas, compounds
8c and 8k exhibited good activity against CaCo-2 cell lines. Moreover, these compounds
can be considered as apoptotic inducers except compound 8h which showed equal
apoptotic and necrotic properties. They may exhibit their apoptosis through caspase-3
activation as illustrated by compound 8k that resulted in 5 fold increase in caspase-3
activity in MCF-7 cell lines.
4. Experimental
4.1. Chemistry
Melting points were recorded on Stuart SMP10 digital melting point apparatus.
IR spectra (KBr disc) were recorded on a Shimadzu FT-IR 8400S infrared
spectrophotometer. NMR spectra were recorded on a Bruker Ascend 400/ R (1H: 400,
13C: 100 MHz) spectrometer. Elemental analyses were carried out at the Regional center
for mycology and biotechnology, Al-Azhar University, Egypt. Chalcones 3a-u [42-53]
and 6-phenylpyridazines 5-7 [54, 55] were prepared according to the reported procedures.
4.1.1. General procedure for preparation of 8a-u
A mixture of the appropriate chalcone 3a-u (1.5 mmol), the hydrazine derivative 7
(1.5 mmol) and sodium hydroxide (3.75 mmol) in absolute ethanol (5 ml) was heated
under reflux for 12 hours. The obtained precipitate on cooling was filtered and washed
with water and crystallized from ethanol.
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4.1.1.1. 3-(3,5-Diphenyl-4,5-dihydropyrazol-1-yl)-6-phenylpyridazine 8a
Obtained from the reaction of 3a with 7, mp 198-199˚C, yield 64% (0.36 g). IR: νmax/cm-1
3061, 3030, 2916, 1589, 1547, 1458, 1435. 1H NMR (CDCl3, 400 MHz): δ 3.23 (dd, 1H,
J = 5.26, 17.42 Hz, pyrazoline proton), 3.84 (dd, 1H, J = 12.20, 17.40 Hz, pyrazoline
proton), 5.94 (dd, 1H, J = 5.22, 12.14 Hz, pyrazoline proton), 7.11-7.23 (m, 3H, arom.
protons), 7.27-7.38 (m, 8H, arom. protons), 7.64 (d, 1H, J = 9.40 Hz, pyridazine proton),
7.71 (d, 2H, J = 6.68 Hz, arom. protons), 7.76 (d, 1H, J = 9.40 Hz, pyridazine proton),
7.89 (d, 2H, J = 7.44 Hz, arom. protons). 13C NMR (CDCl3, 100 MHz): δ 42.9
(pyrazoline CH2), 61.9 (pyrazoline CH), 115.2, 125.3, 126.0, 126.1, 126.2, 127.4, 128.7,
128.8, 129.6, 132.0, 136.8, 142.3, 151.5, 152.4, 155.6 (aromatic carbons).Anal.Calcd. for
C25H20N4 (376.46): C, 79.76; H, 5.35; N, 14.88. Found: C, 79.92; H, 5.41; N, 15.03.
4.1.1.2. 3-[5-(4-Chlorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine 8b
Obtained from the reaction of 3b with 7, mp 225-227˚C, yield 50% (0.31 g). IR: νmax
/
cm-1 3048, 3028, 2924, 1591, 1553, 1468, 1439. 1H NMR (CDCl3, 400 MHz): δ 3.29 (dd,
1H, J = 5.48, 17.44 Hz, pyrazoline proton), 3.94 (dd, 1H, J = 12.20, 17.44 Hz, pyrazoline
proton), 6.00 (dd, 1H, J = 5.46 , 12.18 Hz, pyrazoline proton), 7.27-7.34 (m, 5H, arom.
protons), 7.41- 7.49 (m, 5H, arom. protons), 7.75 (d, 1H, J = 9.36 Hz, pyridazine proton),
7.80 (dd, 2H, J = 1.66, 7.82 Hz, arom. protons), 7.85 (d, 1H, J = 9.40 Hz, pyridazine
proton), 7.99 (d, 2H, J = 7.08 Hz, arom. protons). Anal.Calcd. for C25H19ClN4 (410.91):
C, 73.08; H, 4.66; N, 13.63. Found: C, 73.17; H, 4.69; N, 13.78.
4.1.1.3. 3-[5-(4-Fluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine 8c
Obtained from the reaction of 3c with 7, mp 166-168˚C, yield 41% (0.24 g). IR: νmax/cm-1
3061, 2935, 1591, 1553, 1506, 1470, 1439. 1H NMR (CDCl3, 400 MHz): δ 3.31 (dd, 1H,
J = 5.36, 17.44 Hz, pyrazoline proton), 3.94 (dd, 1H, J = 12.16, 17.44 Hz, pyrazoline
proton), 6.02 (dd, 1H, J = 5.32, 12.16 Hz, pyrazoline proton), 7.00 (t, 2H, J = 8.70 Hz,
arom. protons), 7.35- 7.49 (m, 8H, arom. protons), 7.75 (d, 1H, J = 9.40 Hz, pyridazine
proton), 7.81 (dd, 2H, J = 1.62, 7.90 Hz, arom. protons), 7.85 (d, 1H, J = 9.40 Hz,
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pyridazine proton), 7.99 (d, 2H, J = 7.04 Hz, arom. protons). Anal.Calcd. for C25H19FN4
(394.46): C, 76.12; H, 4.86; N, 14.20. Found: C, 76.28; H, 4.89; N, 14.37.
4.1.1.4. 3-Phenyl-6-{3-phenyl-5-[4-(trifluoromethyl)phenyl]-4,5-dihydropyrazol-1-
yl}pyridazine 8d
Obtained from the reaction of 3d with 7, mp 225-227˚C, yield 60% (0.40 g). IR: νmax
/
cm-1 3055, 2950, 2849, 1591, 1553, 1470, 1441. 1H NMR (CDCl3, 400 MHz): δ 3.20 (dd,
1H, J = 5.64, 17.44 Hz, pyrazoline proton), 3.88 (dd, 1H, J = 12.28, 17.44 Hz, pyrazoline
proton), 5.97 (dd, 1H, J = 5.62, 12.22 Hz, pyrazoline proton), 7.30-7.39 (m, 8H, arom.
protons), 7.49 (d, 2H, J = 8.20 Hz, arom. protons), 7.68 (d, 1H, J = 9.40 Hz, pyridazine
proton), 7.70 (dd, 2H, J = 1.80, 7.72 Hz, arom. protons), 7.78 (d, 1H, J = 9.40 Hz,
pyridazine proton), 7.89 (d, 2H, J = 7.08 Hz, arom. protons). 13C NMR (CDCl3, 100
MHz): δ 42.8 (pyrazoline CH2), 61.6 (pyrazoline CH), 115.3, 122.7, 125.4, 125.5, 125.8,
125.9, 126.4, 128.7, 128.8, 128.9, 130.3, 131.6, 136.6, 146.3, 151.4, 152.9, 155.5
(aromatic carbons). Anal.Calcd. for C26H19 F3N4 (444.45): C, 70.26; H, 4.31; N, 12.61.
Found: C, 70.44; H, 4.37; N, 12.80.
4.1.1.5. 3-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine
8e
Obtained from the reaction of 3e with 7, mp 173-175˚C, yield 66% (0.40 g). IR: νmax/cm-1
1
3049, 3011, 2955, 2930, 2835, 1587, 1549, 1508, 1470, 1456, 1435. H NMR (CDCl3,
400 MHz): δ 3.32 (dd, 1H, J = 5.18, 17.42 Hz, pyrazoline proton), 3.77 (s, 3H, OCH3),
3.94 (dd, 1H, J = 12.06, 17.42 Hz, pyrazoline proton), 6.00 (dd, 1H, J = 5.14, 12.06 Hz,
pyrazoline proton), 6.84 (d, 2H, J = 8.64 Hz, arom. protons), 7.33 (d, 2H, J = 8.68 Hz,
arom. protons), 7.38- 7.48 (m, 6H, arom. protons), 7.72 (d, 1H, J = 9.40 Hz, pyridazine
proton), 7.80-7.85 (m, 3H, 2H arom. protons + 1H pyridazine proton), 7.99 (d, 2H, J =
7.28 Hz, arom. protons). Anal.Calcd. for C26H22N4O (406.49): C, 76.83; H, 5.46; N,
13.78. Found: C, 76.98; H, 5.53; N, 13.96.
17

ACCEPTED MANUSCRIPT
4.1.1.6. 3-[5-(2,4-Dimethoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-6-phenyl-
pyridazine 8f
Obtained from the reaction of 3f with 7, mp 185-187˚C, yield 69% (0.45 g). IR: νmax/cm-1
3050, 2928, 2890, 1589, 1551, 1466, 1437. 1H NMR (CDCl3, 400 MHz): δ 3.09 (dd, 1H,
J = 5.00, 17.36 Hz, pyrazoline proton), 3.66 (s, 3H, OCH3), 3.74-3.81 (m, 4H, OCH3 +
pyrazoline proton), 6.10 (dd, 1H, J= 4.98, 11.98 Hz, pyrazoline proton), 6.24 (dd, 1H, J =
2.24, 8.40 Hz, arom. proton), 6.40 (d, 1H, J = 2.24 Hz, arom. proton), 6.90 (d, 1H, J =
8.44 Hz, arom. proton), 7.28-7.38 (m, 6H, arom. protons), 7.65 (d, 1H, J = 9.44 Hz,
pyridazine proton), 7.69 (d, 2H, J = 6.52 Hz, arom. protons), 7.77 (d, 1H, J = 9.40 Hz,
pyridazine proton), 7.91 (d, 2H, J = 7.24 Hz, arom. protons). Anal.Calcd. for C27H24N4O2
(436.51): C, 74.29; H, 5.54; N, 12.84. Found: C, 74.52; H, 5.57; N, 13.03.
4.1.1.7. 3-Phenyl-6-[3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl]
pyridazine 8g
Obtained from the reaction of 3g with 7, mp 183-185˚C, yield 46% (0.32 g). IR: νmax/cm-1
3051, 2968, 2833, 1589, 1549, 1458, 1435. 1H NMR (CDCl3, 400 MHz): δ 3.21 (dd, 1H,
J = 5.56, 17.44 Hz, pyrazoline proton), 3.71 (s, 3H, OCH3), 3.72 (s, 6H, 2 OCH3), 3.82
(dd, 1H, J = 12.20, 17.44 Hz, pyrazoline proton), 5.86 (dd, 1H, J = 5.58, 12.14 Hz,
pyrazoline proton), 6.47 (s, 2H, arom. protons), 7.30-7.40 (m, 6H, arom. protons), 7.68
(d, 1H, J = 9.48 Hz, pyridazine proton), 7.71 (dd, 2H, J = 1.70, 7.90 Hz, arom. protons),
7.79 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.92 (d, 2H, J = 7.20 Hz, arom. protons).
Anal.Calcd. for C28H26N4O3 (466.53): C, 72.09; H, 5.62; N, 12.01. Found: C, 72.26; H,
5.69; N, 12.17.
4.1.1.8. 3-[3-(4-Chlorophenyl)-5-phenyl-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine 8h
Obtained from the reaction of 3h with 7, mp 215-217˚C, yield 42% (0.26 g). IR: νmax
/
cm-1 3075, 3057, 3028, 2918, 2849, 1593, 1545, 1493, 1462, 1439. 1H NMR (CDCl3, 400
MHz): δ 3.30 (dd, 1H, J= 5.32, 17.36 Hz, pyrazoline proton), 3.91 (dd, 1H, J = 12.26,
17.38 Hz, pyrazoline proton), 6.06 (dd, 1H, J = 5.32, 12.20 Hz, pyrazoline proton), 7.24-
7.48 (m, 10H, arom. protons), 7.73 (d, 2H, J = 8.44 Hz, arom. protons), 7.75 (d, 1H, J =
9.32 Hz, pyridazine proton), 7.84 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.99 (d, 2H, J
18

ACCEPTED MANUSCRIPT
= 7.16 Hz, arom. protons). Anal.Calcd. for C25H19ClN4 (410.91): C, 73.08; H, 4.66; N,
13.63. Found: C, 73.22; H, 4.71; N, 13.81.
4.1.1.9. 3-[3,5-bis(4-Chlorophenyl)-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine 8i
Obtained from the reaction of 3i with 7, mp 189-191˚C, yield 46% (0.31 g). IR: νmax/ cm-1
1
3084, 3032, 2922, 2853, 1591, 1551, 1464, 1441. H NMR (CDCl3, 400 MHz): δ 3.26
(dd, 1H, J= 5.56, 17.44 Hz, pyrazoline proton), 3.91 (dd, 1H, J = 12.26, 17.42 Hz,
pyrazoline proton), 6.01 (dd, 1H, J = 5.54, 12.22 Hz, pyrazoline proton), 7.27-7.33 (m,
3H, arom. protons), 7.39-7.49 (m, 6H, arom. protons), 7.72 (d, 2H, J = 8.56 Hz, arom.
Protons), 7.76 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.83 (d, 1H, J = 9.40 Hz,
pyridazine proton), 7.99 (d, 2H, J = 7.08 Hz, arom. protons). Anal.Calcd. for
C25H18Cl2N4 (445.36): C, 67.42; H, 4.07; N, 12.58. Found: C, 67.53; H, 4.10; N, 12.73.
4.1.1.10. 3-[3-(4-Chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydropyrazol-1-yl]-6-phenyl-
pyridazine 8j
Obtained from the reaction of 3j with 7, mp 203-205˚C, yield 63% (0.40 g). IR: νmax/cm-1
1
3075, 3053, 3036, 2922, 2853, 1603, 1541, 1514, 1460, 1441. H NMR (CDCl3, 400
MHz): δ 3.15 (dd, 1H, J = 5.42, 17.42 Hz, pyrazoline proton), 3.78 (dd, 1H, J = 12.24,
17.40 Hz, pyrazoline proton), 5.91 (dd, 1H, J = 5.38, 12.18 Hz, pyrazoline proton), 6.89
(t, 2H, J = 8.64 Hz, arom. protons), 7.24 (dd, 2H, J= 5.34, 8.50 Hz, arom. protons), 7.30–
7.37 (m, 5H, arom. protons), 7.60-7.65 (m, 3H, 2H arom. protons + pyridazine proton),
7.71 (d, 1H, J = 9.36 Hz, pyridazine proton), 7.88 (d, 2H, J = 7.24 Hz, arom. protons).
13C NMR (CDCl3, 100 MHz): δ 42.8 (pyrazoline CH2), 61.5 (pyrazoline CH), 115.2,
115.6, 115.8, 125.4, 126.2, 127.1, 127.4, 127.7,127.8, 128.7, 128.8, 128.9, 129.0, 130.2,
130.4, 135.6, 136.7, 137.9, 138.0, 150.3, 152.8, 155.5, 160.9, 163.3 (aromatic carbons).
Anal.Calcd. for C25H18ClFN4 (428.90): C, 70.01; H, 4.23; N, 13.06. Found: C, 70.19; H,
4.29; N, 13.22.
4.1.1.11. 3-{3-[4-Chlorophenyl]-5-[4-(trifluoromethyl)phenyl]-4,5-dihydropyrazol-1-yl}-
6-phenylpyridazine 8k
19

ACCEPTED MANUSCRIPT
Obtained from the reaction of 3k with 7, mp 201-203˚C, yield 49% (0.35 g). IR: νmax
/
1
cm-1 3067, 3024, 2995, 2849, 1589, 1555, 1464, 1443. H NMR (CDCl3, 400 MHz): δ
3.17 (dd, 1H, J= 5.76, 17.44 Hz, pyrazoline proton), 3.86 (dd, 1H, J = 12.34, 17.46 Hz,
pyrazoline proton), 5.98 (dd, 1H, J = 5.74, 12.26 Hz, pyrazoline proton), 7.30-7.40 (m,
7H, arom. protons), 7.49 (d, 2H, J = 8.20 Hz, arom. protons), 7.63 (d, 2H, J = 8.52 Hz,
arom. protons), 7.68 (d, 1H, J = 9.36 Hz, pyridazine proton), 7.77 (d, 1H, J = 9.40 Hz,
pyridazine proton), 7.89 (d, 2H, J = 7.12 Hz, arom. protons). Anal.Calcd. for C26H18Cl
F3N4 (478.90): C, 65.21; H, 3.79; N, 11.70. Found: C, 65.34; H, 3.76; N, 11.82.
4.1.1.12. 3-[3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-phenyl
pyridazine 8l
Obtained from the reaction of 3l with 7, mp 173-175˚C, yield 70% (0.46 g). IR: νmax/ cm-1
1
3055, 3038, 2984, 2957, 2918, 2837, 1589, 1574, 1549, 1464, 1439. H NMR (CDCl3,
400 MHz): δ 3.18 (dd, 1H, J = 5.14, 17.42 Hz, pyrazoline proton), 3.66 (s, 3H, OCH3),
3.77 (dd, 1H, J = 12.14, 17.42 Hz, pyrazoline proton), 5.93 (dd, 1H, J = 4.94, 11.98 Hz,
pyrazoline proton), 6.74 (d, 2H, J = 8.60 Hz, arom. protons), 7.21 (d, 2H, J = 8.60 Hz,
arom. protons), 7.30-7.38 (m, 5H, arom. protons), 7.63 (d, 3H, J = 8.80 Hz, arom. protons
+ pyridazine proton), 7.71 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.88 (d, 2H, J = 7.44
Hz, arom. protons). 13C NMR (CDCl3, 100 MHz): δ 42.7 (pyrazoline CH2), 55.3 (OCH3),
61.6 (pyrazoline CH), 114.2, 115.2, 125.3, 126.1, 127.4, 128.7, 128.8, 129.0, 130.6,
134.3, 135.4, 136.8, 150.3, 152.6, 155.5, 158.9 (aromatic carbons). Anal.Calcd. for
C26H21ClN4O (440.92): C, 70.82; H, 4.80; N, 12.71. Found: C, 70.94; H, 4.85; N, 12.83.
4.1.1.13. 3-[3-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-
phenylpyridazine 8m
Obtained from the reaction of 3m with 7, mp 195-197˚C, yield 57% (0.40 g). IR: νmax
/
cm-1 3057, 3003, 2951, 2934, 2832, 1589, 1551, 1508, 1462, 1437. 1H NMR (CDCl3, 400
MHz): δ 3.05 (dd, 1H, J = 4.96, 17.32 Hz, pyrazoline proton), 3.66 (s, 3H, OCH3), 3.70-
3.77 (m, 4H, OCH3 + pyrazoline proton), 6.10 (dd, 1H, J = 4.92, 11.96 Hz, pyrazoline
proton), 6.25 (d, 1H, J = 8.36 Hz, arom. protons), 6.40 (s, 1H, arom. proton), 6.89 (d, 1H,
J = 8.40 Hz, arom. proton), 7.29-7.38 (m, 5H, arom. protons), 7.62 (d, 2H, J = 8.32 Hz,
20

ACCEPTED MANUSCRIPT
arom. protons), 7.65 (d, 1H, J = 9.48 Hz, pyridazine proton), 7.74 (d, 1H, J = 9.36 Hz,
pyridazine proton), 7.91 (d, 2H, J = 7.60 Hz, arom. protons). 13C NMR (CDCl3, 100
MHz): δ 41.7 (pyrazoline CH2), 55.4 (OCH3), 55.5 (OCH3), 57.8 (pyrazoline CH), 99.1,
103.8, 114.9, 121.8, 125.2, 126.1, 126.9, 127.4, 128.7, 128.9, 130.9, 135.2, 136.8, 151.4,
152.2, 155.7, 157.4, 160.2 (aromatic carbons). Anal.Calcd. for C27H23ClN4O2 (470.95):
C, 68.86; H, 4.92; N, 11.90. Found: C, 69.03; H, 4.98; N, 12.04.
4.1.1.14. 3-[3-(4-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-
phenylpyridazine 8n
Obtained from the reaction of 3n with 7, mp 196-198˚C, yield 50% (0.38 g). IR: νmax
/
1
cm-1 3032, 2967, 2916, 2887, 1587, 1576, 1547, 1458, 1439. H NMR (CDCl3, 400
MHz): δ 3.17 (dd, 1H, J = 5.50, 17.42 Hz, pyrazoline proton), 3.71 (s, 3H, OCH3), 3.72
(s, 6H, 2OCH3), 3.79 (dd, 1H, J = 12.24, 17.36 Hz, pyrazoline proton), 5.87 (dd, 1H, J =
5.48, 12.12 Hz, pyrazoline proton), 6.45 (s, 2H, arom. protons), 7.32-7.40 (m, 5H, arom.
protons), 7.63 (d, 2H, J = 8.40 Hz, arom. protons), 7.68 (d, 1H, J = 9.40 Hz, pyridazine
proton), 7.76 (d, 1H, J = 9.36 Hz, pyridazine proton), 7.91 (d, 2H, J = 7.48 Hz, arom.
protons). Anal.Calcd. for C28H25ClN4O3 (500.98): C, 67.13; H, 5.03; N, 11.18. Found: C,
67.24; H, 5.08; N, 11.27.
4.1.1.15. 3-[3-(4-methoxyphenyl)-5-phenyl-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine
8o
Obtained from the reaction of 3o with 7, mp 201-202˚C, yield 48% (0.29 g). IR: νmax/cm-1
1
3082, 3024, 2916, 2849, 1609, 1591, 1547, 1516, 1456, 1437. H NMR (CDCl3, 400
MHz): δ 3.30 (dd, 1H, J = 5.22, 17.30 Hz, pyrazoline proton), 3.87-3.95 (m, 4H, OCH3 +
pyrazoline proton), 6.02 (dd, 1H, J = 5.22, 12.06 Hz, pyrazoline proton), 6.98 (d, 2H, J =
8.84 Hz, arom. protons), 7.21-7.33 (m, 5H, arom. protons), 7.37-7.47 (m, 5H, arom.
protons), 7.73 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.74 (d, 2H, J = 8.76 Hz, arom.
protons), 7.83 (d, 1H, J = 9.40 Hz, pyridazine proton), 7.99 (d, 2H, J = 7.20 Hz, arom.
protons). 13C NMR (CDCl3, 100 MHz): δ 43.1 (pyrazoline CH2), 55.4 (OCH3), 61.8
(pyrazoline CH), 114.2, 115.0, 124.7, 125.2, 126.1, 127.3, 127.8, 128.6, 128.7, 128.8,
21

ACCEPTED MANUSCRIPT
136.9, 142.5, 151.4, 152.1, 155.7, 160.8 (aromatic carbons).Anal.Calcd. for C26H22N4O
(406.49): C, 76.83; H, 5.46; N, 13.78. Found: C, 76.97; H, 5.52; N, 13.89.
4.1.1.16.
3-[5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-
phenylpyridazine 8p
Obtained from the reaction of 3p with 7, mp 203-205˚C, yield 73% (0.48 g). IR: νmax/ cm-
1 3046, 3026, 2953, 2928, 2835, 1609, 1593, 1551, 1516, 1468, 1441. H NMR (CDCl3,
1
400 MHz): δ 3.26 (dd, 1H, J = 5.42, 17.34 Hz, pyrazoline proton), 3.87-3.94 (m, 4H,
OCH3 + pyrazoline proton), 5.96 (dd, 1H, J = 5.40, 12.08 Hz, pyrazoline proton), 6.98 (d,
2H, J = 8.84 Hz, arom. protons), 7.27-7.34 (m, 4H, arom. protons), 7.40-7.48 (m, 3H,
arom. protons), 7.73-7.75 (m, 3H, 2H arom. protons + pyridazine proton), 7.82 (d, 1H, J
=9.40 Hz, pyridazine proton), 7.98 (d, 2H, J = 7.08 Hz, arom. protons). Anal.Calcd. for
C26H21ClN4O (440.94): C, 70.82; H, 4.80; N, 12.71. Found: C, 70.95; H, 4.84; N, 12.86.
4.1.1.17. 3-[5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-phenyl
pyridazine 8q
Obtained from the reaction of 3q with 7, mp 168-170˚C, yield 52% (0.33 g). IR: νmax
/
cm-1 3038, 2959, 2920, 2837, 1609, 1593, 1549, 1506, 1468, 1439. 1H NMR (CDCl3, 400
MHz): δ 3.26 (dd, 1H, J = 5.30, 17.34 Hz, pyrazoline proton), 3.86- 3.93 (m, 4H, OCH3 +
pyrazoline proton), 5.98 (dd, 1H, J = 5.28, 12.04 Hz, pyrazoline proton), 6.97-7.02 (m,
4H, arom. protons), 7.34-7.48 (m, 5H, arom. protons), 7.71- 7.75 (m, 3H, 2H arom.
protons + pyridazine proton), 7.82 (d, 1H, J= 9.40 Hz, pyridazine proton), 7.99 (d, 2H, J
= 7.08 Hz, arom. protons). Anal.Calcd. for C26H21FN4O (424.48): C, 73.57; H, 4.99; N,
13.20. Found: C, 73.73; H, 5.03; N, 13.37.
4.1.1.18. 3-[3-(4-Methoxyphenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydropyrazol-1-
yl]-6-phenylpyridazine 8r
Obtained from the reaction of 3r with 7, mp 220-222˚C, yield 40% (0.28 g). IR: νmax/ cm-
1
1 3060, 2928, 2839, 1593, 1514, 1464, 1441. H NMR (CDCl3, 400 MHz): δ 3.17 (dd,
1H, J = 5.60, 17.36 Hz, pyrazoline proton), 3.79 (s, 3H, OCH3), 3.85 (dd, 1H, J = 12.20,
17.36 Hz, pyrazoline proton), 5.94 (dd, 1H, J = 5.60, 12.16 Hz, pyrazoline proton), 6.89
22

ACCEPTED MANUSCRIPT
(d, 2H, J = 8.80 Hz, arom. protons), 7.29-7.41 (m, 5H, arom. protons), 7.48 (d, 2H, J =
8.24 Hz, arom. protons), 7.63-7.67 (m, 3H, 2 arom. protons+ pyridazine proton), 7.75 (d,
1H, J = 9.40 Hz, pyridazine proton), 7.89 (d, 2H, J = 7.20 Hz, arom. protons).
Anal.Calcd. for C27H21F3N4O (474.49): C, 68.35; H, 4.46; N, 11.81. Found: C, 68.44; H,
4.52; N, 11.96.
4.1.1.19. 3-[3,5-bis(4-Methoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-phenylpyridazine 8s
Obtained from the reaction of 3s with 7, mp 195-196˚C, yield 64% (0.42 g). IR: νmax/cm-1
1
3042, 3009, 2949, 2934, 2832, 1595, 1549, 1514, 1466, 1439. H NMR (CDCl3, 400
MHz): δ 3.29 (dd, 1H, J = 5.08, 17.32 Hz, pyrazoline proton), 3.77 (s, 3H, OCH3), 3.84-
3.91 (m, 4H, OCH3 + pyrazoline proton), 5.97 (dd, 1H, J = 5.06, 11.98 Hz, pyrazoline
proton), 6.84 (d, 2H, J= 8.64 Hz, arom. protons), 6.98 (d, 2H, J = 8.80 Hz, arom.
protons), 7.32 (d, 2H, J = 8.68 Hz, arom. protons), 7.37-7.48 (m, 3H, arom. protons), 7.71
(d, 1H, J = 9.44 Hz, pyridazine proton), 7.75 (d, 2H, J = 8.80 Hz, arom. protons), 7.80 (d,
1H, J = 9.40 Hz, pyridazine proton), 7.99 (d, 2H, J = 7.24 Hz, arom. protons).
Anal.Calcd. for C27H24N4O2 (436.52): C, 74.29; H, 5.54; N, 12.83. Found: C, 74.53; H,
5.59; N, 13.01.
4.1.1.20. 3-[5-(2,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-6-
phenylpyridazine 8t
Obtained from the reaction of 3t with 7, mp 183-185˚C, yield 59% (0.41 g). IR: νmax/cm-1
1
3051, 2916, 2833, 1607, 1589, 1551, 1508, 1464, 1437. H NMR (CDCl3, 400 MHz): δ
3.15 (dd, 1H, J = 4.94, 17.26 Hz, pyrazoline proton), 3.76 (s, 3H, OCH3), 3.81-3.88 (m,
7H, 2 OCH3 + pyrazoline proton ), 6.17 (dd, 1H, J = 4.92, 11.84 Hz, pyrazoline proton),
6.34 (dd, 1H, J = 2.14, 8.38 Hz, arom. protons), 6.50 (d, 1H, J = 2.12 Hz, arom. protons),
6.94-7.00 (m, 3H, arom. protons), 7.37-7.48 (m, 3H, arom. protons + pyridazine proton),
7.73 (d, 2H, J = 8.72 Hz, arom. protons), 7.83 (d, 1H, J = 9.40 Hz, pyridazine proton),
8.01 (d, 2H, J = 7.40 Hz, arom. Protons). Anal.Calcd. for C28H26N4O3 (466.54): C, 72.09;
H, 5.62; N, 12.01. Found: C, 72.24; H, 5.64; N, 12.13.
23

ACCEPTED MANUSCRIPT
4.1.1.21. 3-[3-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl]-
6-phenylpyridazine 8u
Obtained from the reaction of 3u with 7, mp 193-195˚C, yield 41% (0.31 g). IR: νmax
/
1
cm-1 3050, 2980, 2963, 2837, 1607, 1591, 1508, 1462, 1439. H NMR (CDCl3, 400
MHz): δ 3.18 (dd, 1H, J = 5.52, 17.32 Hz, pyrazoline proton), 3.71 (s, 3H, OCH3), 3.72
(s, 6H, 2 OCH3), 3.75-3.83 (m, 4H, OCH3 + pyrazoline proton), 5.83 (dd, 1H, J = 5.48,
12.04 Hz, pyrazoline proton), 6.47 (s, 2H, arom. protons), 6.88 (d, 2H, J = 8.84 Hz, arom.
protons), 7.29-7.39 (m, 3H, arom. protons), 7.65 (d, 2H, J = 8.76 Hz, arom. protons),
7.66 (d, 1H, J = 9.36 Hz, pyridazine proton), 7.76 (d, 1H, J = 9.40 Hz, pyridazine
13
proton), 7.92 (d, 2H, J = 7.24 Hz, arom. protons). C NMR (CDCl3, 100 MHz): δ 43.4
(pyrazoline CH2), 55.4 (OCH3), 56.1 (2OCH3), 60.7 (OCH3), 62.1 (pyrazoline CH),
102.5, 114.2, 115.2, 124.6, 125.2, 126.1, 127.8, 128.8, 136.8, 137.1, 138.4, 151.6, 152.3,
153.6, 155.9, 160.9 (aromatic carbons). Anal.Calcd. for C29H28N4O4 (496.56): C, 70.15;
H, 5.68; N, 11.28. Found: C, 70.29; H, 5.72; N, 11.44.
4.2. In-vitro MTT assay
Human cancer cell lines of different origins purchased from ATCC were used for
evaluation of the anticancer effect of the target pyrazolines. MCF7 (ATCC; HTB-22),
CaCo-2 (ATCC; HTB-37), HepG2 (ATCC; HB-8065) & A549 (ATCC; CRM-CCL-185).
Cells were cultured and maintained as monolayer in either Dulbecco’s modified Eagle’s
medium (DMEM) for A549 and HepG2, or Rose Well Park Memorial Institute medium
(RPMI) for MCF-7 and CaCo-2 cell lines. Each medium was supplemented with 10% of
fetal calf serum (Lonza), antibiotics: 100 IU/mL of penicillin (Lonza) and 100 mg/mL of
streptomycin (Lonza). All cells were cultured in 10 cm diameter culture plates at 370C in
100% humidity atmosphere and 5% of CO2 [56]. Only viable cells were used in the
assay. Stock solutions of 1M from the tested compounds were prepared in serum free
culture medium containing 1% (v/v) dimethylsulfoxide (DMSO). Cells were seeded
overnight in 96 multiwells plate (103 cells/well) before treatment with the prepared
compounds to allow the attachment of cells to the wall of the plate. A further dilution of
the stock solution from each compound in complete medium (10% serum) was added to
the seeded cells at concentrations of 0, 5, 10, 50, and 100 µM. Each cell line was
24

ACCEPTED MANUSCRIPT
incubated with the compounds for 24 h at standard culture conditions. The potential
cytotoxicity of the tested compounds was evaluated at 24 h post incubation with the
respective compounds using MTT colorimetric assay (WST-1 premix cell proliferation
assay – Takara, Germany), measured at a wavelength of 450 nm. Compounds mediated
cellular toxicity was calculated using the following formula (specified by the kit
protocol); % Cell viability = [(Absorbance of treated cells – absorbance of blank) /
(Absorbance of control cells – Absorbance of blank)] x 100.
After 24 h incubation with the cells, control (untreated cells) did not exhibit
significant changes compared to the 1% DMSO vehicle. The IC50, mean value and
standard error were determined, and data were expressed as the mean ± standard error of
the mean (SEM). Statistical analysis was done using one-way ANOVA Tukey’s Multiple
Comparison Test via GraphPad Prism 5.0 software.
4.3. Evaluation of Cell Death Pathways
Flow cytometry was applied in order to determine the mechanism of cancer cell
death for each cell line. Fluorescent markers of fluorescein isothiocyanate (FITC)-
Annexin V and Ethidium Homodimer III (EthD-III) from (Biotium, USA) for detection
of apoptotic and necrotic cell deaths were used respectively. A number of 2-3 x 105
cells/ml treated with the LC50 – PDT conditions were re-suspended in 1 ml of 1X binding
buffer diluted in distilled water, then 5 µl of FITC-Annexin V, together with 5 µl of
EthD-III were added to 100 µl of cell suspension and incubated at room temperature for
15 min in dark. 400 µl of 1X binding buffer was added to the cell suspension and
fluorescence was measured within 1 h of staining by fluorescence associated cell sorter
(FACS) analysis.
4.4. Caspase-3 activation assay
The activity of caspase-3 was determined at Vacsera, Dokki, Cairo, Egypt, using
MaxDiscovery™caspase-3 colorimetric detection kit, Bioo Scientific Corporation
(BIOO), USA. Briefly, MCF-7 cells (5×105 cells/well) in 6-well plate were treated with
IC50 concentrations of compound 8k for 24 h. The cell culture medium was gently
removed from the control and treated culture wells and then the cells were lysed by
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adding cell lysis buffer (1000 µL) to each culture well. The plate was gently shaken for
10 minutes to facilitate cell lysis and sample homogenization. Caspase-3 Substrate (100
µL) was diluted into 10 mL of Reaction Buffer. In the reaction microplate, 100 µL
Caspase-3 Reaction Buffer was added to each well, followed by 100 µL of cell lysate.
The absorbance was measured using a plate reader for the increase in absorbance at 405
nm in 30 minutes. Caspase-3 activity was expressed as the change of the activity in
treated cancer cells compared to the untreated controls [57].
Acknowledgement:
The authors would like to thanks Miss. Hadil Hamdy El-Sammak, Faculty of Pharmacy
and Biotechnology –GermanUniversity in Cairo for her technical assistance at the MTT
assay and the FACS analysis.
The antiproliferative study and the FACS analysis of this work has been partially funded
by a national grant from (Centre for Special Studies and Programs / CSSP)- Bibliotheca
Alexandrina – Project No. 145.
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