Synthesis and evaluation of new 4-Chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated amino)pyridazin-3(2H)-one derivatives as anticancer, antiangiogenic, and antioxidant agents

Article abstract of DOI:10.1002/ardp.201400442

Pyridazinones are widely recognized as versatile scaffolds with a wide spectrum of biological activities. In the present work, a series of new 4-chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated amino)pyridazin-3(2H)-one derivatives 4a-i were synthesized and characterized by spectral techniques. The inhibitory effects of the synthesized compounds 4a-i on the viability of three human cancer cell lines, HEP3BPN 11 (liver), MDA 453 (breast), and HL 60 (leukemia), were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Among the compounds 4a-i screened, 4g and 4i exhibited inhibitory activity very close to the standard methotrexate; therefore, these lead compounds were further tested for their potential to inhibit the proangiogenic cytokines involved in tumor progression. Compound 4g was found to be a potent antiangiogenic agent against TNFα, VEGF, FGFb, and TGFβ, whereas 4i showed potent antiangiogenic activity against TNFα, VEGF, FGFb, and leptin. All the compounds 4a-i were screened for their antioxidant activities using 2,2-diphenyl-1-picryl hydrazine (DPPH), OH, and superoxide anion radicals. Compound 4f showed better OH radical scavenging activity than the standard ascorbic acid. A series of new 4-chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated amino)pyridazin-3(2H)-one derivatives (4a-i) were assessed for their anticancer potential by measuring their MTT-based cytotoxicity against selected cancer cell lines. The promising leads (4g and 4i) were further tested for their potential to inhibit the proangiogenic cytokines involved in tumor progression.

Full text of DOI:10.1002/ardp.201400442

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 338–346
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Full Paper
Synthesis and Evaluation of New 4-Chloro-2-(3-chloro-4-
fluorophenyl)-5-(aliphatic/cyclic saturated amino)pyridazin-3(2H)-
one Derivatives as Anticancer, Antiangiogenic, and Antioxidant
Agents
Vinod T. Kamble1, Ajay S. Sawant2, Sanjay S. Sawant2, Parshuram M. Pisal2, Rajesh N. Gacche3,
Sonali S. Kamble3, and Vilas A. Kamble4
1
Organic Chemistry Research Laboratory, Department of Chemistry, Institute of Science, Nagpur,
Maharashtra, India
2
School of Chemical Science, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra,
India
3
School of Life Science, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India
Department of Microbiology, Adarsha Mahavidyalaya, Amravati, Maharashtra, India
4
Pyridazinones are widely recognized as versatile scaffolds with a wide spectrum of biological activities.
In the present work, a series of new 4-chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated
amino)pyridazin-3(2H)-one derivatives 4a–i were synthesized and characterized by spectral techniques.
The inhibitory effects of the synthesized compounds 4a–i on the viability of three human cancer cell
lines, HEP3BPN 11 (liver), MDA 453 (breast), and HL 60 (leukemia), were assessed using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Among the com-
pounds 4a–i screened, 4g and 4i exhibited inhibitory activity very close to the standard methotrexate;
therefore, these lead compounds were further tested for their potential to inhibit the proangiogenic
cytokines involved in tumor progression. Compound 4g was found to be a potent antiangiogenic
agent against TNFa, VEGF, FGFb, and TGFb, whereas 4i showed potent antiangiogenic activity against TNFa,
VEGF, FGFb, and leptin. All the compounds 4a–i were screened for their antioxidant activities using
2,2-diphenyl-1-picryl hydrazine (DPPH), OH, and superoxide anion radicals. Compound 4f showed better OH
radical scavenging activity than the standard ascorbic acid.
Keywords: Antiangiogenic / Anticancer / Antioxidant activity / Pyridazinones
Received: December 10, 2014; Revised: February 9, 2015; Accepted: February 17, 2015
DOI 10.1002/ardp.201400442
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
for the treatment of cancer [1]. For the treatment of cancer,
Introduction
various anticancer drugs, i.e., 5-fluorouracil, cisplatin, pacli-
taxel, and docetaxel are available in the market. These drugs
cannot be used continuously for long time as they can cause
serious side effects such as neutropenia, venous thrombosis,
atrial arrhythmia, mucositis, and respiratory infection [2].
Thus, the search for novel anticancer agents and new
approaches to cancer treatment is a highly active research
field, stimulated by the discovery of new biological targets and
by the possibility of achieving newer anticancer agents with
high efficacy and selectivity, low toxicity, and minimum side
effects [3, 4].
Cancer is responsible for many deaths every year. Thus, various
research groups worldwide are involved in search of safer
anticancer agents. Apart from the use of surgery and
radiotherapy, chemotherapy still remains an important option
Correspondence: Prof. Vinod T. Kamble, Organic Chemistry
Research Laboratory, Department of Chemistry, Institute of
Science, Nagpur 440001, Maharashtrra, India.
E-mail: vtkd@rediffmail.com
Fax: þ91 0712 2561148
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Pyridazin-3(2H)-one Derivatives as Anticancer Agents
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Angiogenesis: a process of neovascularization is a complex,
regulated, and multi-step physiological process, which initiates
with protease-mediated degradation of the basement mem-
brane, loss of endothelial cell adhesion, proliferation, and
migration of endothelial cells into the surrounding stroma,
and finally re-assembly of endothelial cells to form the lumen
of the new blood vessels [5]. This physiological process is
governed by numerous pro- and antiangiogenic factors,
including vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (FGFb), epidermal growth factor
(EGF), and transforming growth factor-beta (TGF-b). The
mechanism of action of each of these factors is different, as are
their origin and the stimuli for their production. The
angiogenic switch refers to the balance between pro- and
antiangiogenic factors. Therefore, profiling of these factors is
critical to understanding angiogenesis. The growth of
solid tumors is dependent on their capacity to induce
angiogenesis, to induce the growth of blood vessels to supply
them with oxygen and nutrients. Considering the side
effects associated with traditional chemotherapies and the
possibility of interrupting a tumor’s supply of oxygen and
nutrient, there has been great interest taken in the targeting
of tumor vasculature and much effort has been directed
toward the development of antiangiogenic agents that
could disrupt this angiogenesis. Normalization of tumor
vasculature by antiangiogenic drugs is an emerging concept
for improving the efficacy of cytotoxic chemotherapy and
radiotherapy [6–9].
Experimental investigations as well as clinical and epide-
miological findings attribute the role of reactive oxygen
species (ROS) or free radicals such as singlet oxygen O2,
superoxide anions (O2), hydrogen peroxide (H2O2), and
hydroxyl radicals in the etiology of cancer [10]. Human body
is equipped with certain enzymatic antioxidants especially
the radical scavenging enzymes such as superoxide dismu-
tase (SOD) and catalase (CAT), which can counteract the
deleterious actions of these ROS and protect against
oxidative damage [11]. Disturbances in the balance between
the free radicals and the endogenous antioxidants in the
human body may cause cellular damage and induce
carcinogenesis.
Pyridazinones, an important class of heterocyclic compounds
influence numerous cellular processes. Their synthesis has
attracted considerable attention from organic and medicinal
chemists for many years as members of this family have wide
applications in medicinal chemistry, being known as analge-
sic [12], anti-inflammatory [13], antimicrobial [14], antihyper-
tensive [15], antiasthmatic [16], antisecretory and antiulcer [17],
antianemic [18], antidepressant [19], antifungal [20], antinoci-
ceptive [21], cardiotonic [22], and anticancer [23].
Tempted by wide variety of biological activities exhibited
by pyridazinone derivatives and in continuation [24–26] of our
interest in nitrogen containing heterocycles, we have synthe-
sized new 4-chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic
saturated amino)pyridazin-3(2H)-one derivatives (Scheme 1) and
screened them for anticancer, antiangiogenic, and antioxidant
activities, which we wish to report in this paper.
Results and discussion
Chemistry
The compound [2-(3-chloro-4-fluorophenyl)]-4,5-dichloropyra-
dizin-3-one 3 required as intermediate was prepared from the
reaction of mucochloric acid 1 with 3-chloro-4-fluorophenyl
hydrazine 2 in the presence of methane sulfonic acid (MSA) in
EtOH under reflux temperature for 2 h. The earlier reported
approach [27] to 3 involved two step process: (a) diazotization
and reduction of 3-chloro-4-fluoro aniline to prepare 2 and (b)
subsequent reaction of 2 with mucochloric acid 1 in the
presence of acetic acid under reflux condition. Compared to
this, our approach involves simple route wherein we have
explored the potentiality of MSA; a well-known substitute for
mineral and Lewis acids [28–30], toward the synthesis of 3.
Further reaction of various amines with intermediate 3 in
acetonitrile using K2CO3 under reflux condition yielded new 4-
chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated
amino)pyridazin-3(2H)-one derivatives (Table 1, 4a–i). The main
aim of synthesizing new amino-pyridazinone derivatives is to
study anticancer, antiangiogenic, and antioxidant properties
by varying aliphatic/cyclic saturated amine moiety on pyrida-
zinone as these amines may enhance the biological activities.
Cl
O
Cl
Cl
F
F
Cl
Cl
F
O
HO
O
O
a
+
Cl
Cl
b
NH2
N
N
Cl
N
H
N
N
R
2
1
3
4
(Compounds 4a i)
−
R = Aliphatic/cyclic saturated amine
Scheme 1. Synthesis of 4-chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturated amino)pyridazin-3(2H)-one. Reagents and
conditions: (a) Methane sulfonic acid, EtOH, 2 h reflux; (b) aliphatic amine/cyclized saturated amine, K2CO3, acetonitrile, 1.5–2.5 h
reflux.
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Table 1. Synthesis of the [2-(3-chloro-4-flurophenyl)]-4-chloro-(5-substituted amino)-3(2H)-pyridazinone derivatives 4a–i.
Aliphatic amine/cyclized
Entry
Producta)
Cl
saturated amine
Time (h)
Yieldb) (%)
F
O
HN
Cl
N
4a
2.30
65
N
N
Cl
F
O
HN
N
Cl
4b
2.00
75
N
N
N
N
Cl
Cl
F
O
HN
N
Cl
N
4c
2.00
2.00
75
72
N
N
N
F
O
HN
N
Cl
4d
N
N
N
N
Cl
F
O
Cl
4e
4f
1.30
2.00
75
90
N
H2N
HN
N
N
H
Cl
F
O
Cl
N
N
N
Boc
N
N
Boc
Cl
F
O
Cl
N
4g
4h
2.30
2.30
85
85
N
N
HN
O
O
Cl
F
O
Cl
N
HN
N
N
(Continued)
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Arch. Pharm. Chem. Life Sci. 2015, 348, 338–346
Pyridazin-3(2H)-one Derivatives as Anticancer Agents
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Table 1. (Continued)
Aliphatic amine/cyclized
saturated amine
Entry
4i
Producta)
Time (h)
Yieldb) (%)
Cl
F
O
N
1.30
65
H2N
Cl
N
N
N
N
H
a)All the products were characterized by 1H NMR, LC–MS, IR, and 13C NMR.
b)Isolated yields.
Biological results
cell line, compound containing piperidine 4a at C-5 position
exhibited higher cytotoxicity than the rest of compounds in the
series. As far as HL 60 cell line is concerned, compound
containing N-ethyl piperazine 4c at C-5 position showed better
activity when compared to remaining compounds.
Cytotoxicity assay
MTT assay was used for the evaluation of cytotoxic properties of
the novel amino-pyridazinone derivatives 4a–i. The growth
inhibitory effect was assessed using three human cancer cell
lines, HEP3BPN 11 (liver), MDA 453 (breast), and HL 60
(leukemia) and the results are tabulated in Table 2. This table
indicates the percentage cytotoxic activity (dose dependent) of
the synthesized compounds at 0.01mM concentration. All these
compounds possess common [2-(3-chloro-4-fluorophenyl)]-4,5-
dichloropyradizin-3-one nucleus. The substitutions at C-5
position of the pyridazinone moiety play an important role
in determining the potency of the compounds 4a–i.
Through cytotoxicity assay, it was found that the amino-
pyridazinone derivatives 4a–i have moderate to good cytotoxic
activity against various human cell lines. With respect to the
HEP3BPN 11 cell line, compounds 4g and 4i were found to have
an activity very close to the standard. Although, the derivatives
4a–i are showing good activity against MDA 453 and HL60 cell
lines, their overall activity is moderate. The SAR study of amino-
pyridazinone derivatives 4a–i with respect to HEP3BPN 11 cell
line revealed that compounds containing morpholine 4g and
N,N-dimethyl ethylene diamine 4i at C-5 position have higher
cytotoxicity than piperidine 4a, substituted piperazines 4b–d
and 4f, morpholine 4g, and pyrrolidine 4h. In case of MDA 453
Inhibition of proangiogenic cytokines
The compounds 4g and 4i showing promising cytotoxicity
against selected cancer cell lines were further evaluated for
inhibition of selected cytokines such as TNFa, IGF1, VEGF, IL6,
FGFb, TGFb, EGF, and leptin. The selected cytokines are
described to be involved in progression of tumor growth
especially by recruiting massive vasculature for the promotion
of tumor growth [31]. Compound 4g is found to be potent
antiangiogenic agent against TNFa, VEGF, FGFb, and TGFb,
whereas compound 4i have shown potent TNFa, VEGF, FGFb,
and leptin inhibition. The results are summarized in Table 3.
Antioxidant activity
The 2,2-diphenyl-1-picryl hydrazine (DPPH) radicals scavenging
assay has been used for preliminary screening of the amino-
pyridazinone derivatives for their antioxidant activity. The
proton radical scavenging action is known as an important
mechanism of antioxidants. It is clear from the results that all
compound were found to interact with the stable free radical
Table 2. Cytotoxicity by MTT at 0.01 mM 4a–i.
% Cytotoxicity
Sr. no.
Compound
HEP 3B PN11
MDA 453
HL 60
1
2
3
4
5
6
7
8
9
10
4a
4b
4c
4d
4e
4f
4g
4h
NR
NR
69.13
60.64
60.76
63.17
66.65
59.71
65.97
66.46
61.93
88.73
64.73
58.80
67.42
62.87
64.12
62.18
62.13
61.06
60.30
82.77
64.83
69.23
63.92
51.32
83.02
69.01
82.11
84.64
4i
Methotrexate
NR, no reaction.
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DPPH, which indicates their potent radical scavenging ability.
Compound 4f was found to have an antioxidant activity
(83.29%) higher than standard ascorbic acid (AA; 76.45%),
whereas rest of the compounds showed moderate DPPH
radical scavenging activity. The overall range of DPPH radical
activity was found to be 49.21–83.29%. The OH radicals are
most hyperreactive among the relative oxygen species and
that affects every type of molecule found in living system.
Physiologically important bimolecules such as sugar, amino
acids, phospholipids, DNA bases, and organic acids may
undergo reaction with OH radicals and may change normal
physiological function of cells [32]. It was observed that
compounds 4d (65.87%) and 4g (63.01%) have shown OH
radical scavenging activities closer to standard sample AA
(69.37%), whereas rest of the compounds have moderate
activity. The profile of SOR scavenging activities indicates that
compound 4h (69.28%) exhibited SOR scavenging activity
closer to the standard AA (72.58%), while other test
compounds showed moderate SOR scavenging activity
(37.20–69.28%). The results are summarized in Table 4.
The SAR study revealed that compound 4f containing N-Boc
piperazine at C-5 position have maximum DPPH activity than
compounds containing piperidine 4a, substituted piperazines
4b–d, cyclopentylamine 4e, morpholine 4g, pyrrolidine 4h, and
aliphatic amine 4i at C-5 position. It is noteworthy to mention
that compounds containing N-methyl piperazine 4d, and
morpholine 4g at C-5 position exhibited similar OH radical
scavenging activity as the standard AA. Interestingly, com-
pound containing pyrrolidine 4h at C-5 position exhibited SOR
scavenging activity closer to standard AA when compared to
remaining compounds.
Conclusion
In conclusion, this work demonstrates the synthesis of new 4-
chloro-2-(3-chloro-4-fluorophenyl)-5-(aliphatic/cyclic saturat-
ed amino)pyridazin-3(2H)-one derivatives, characterization
and biological evaluation of cytotoxic, antiangiogenic, and
antioxidant activities. In case of cytotoxic study against three
Table 3. Effect of selected compounds on the tumor growth promoting cytokines (growth factors) at 0.01 mM.
% Inhibition of tumor growth promoting cytokines
Entry
Compound
TNFa
IGF1
VEGF
IL6
FGFb
TGFb
EGF
Leptin
1
2
3
4g
4i
Suramin
ꢀ77.81 ꢁ 0.10
ꢀ76.88 ꢁ 0.13
67.64 ꢁ 0.08
84.82 ꢁ 0.12
59.92 ꢁ 0.05 ꢀ78.71 ꢁ 0.09 ꢀ81.72 ꢁ 0.13
73.76 ꢁ 0.11
62.98 ꢁ 0.04
72.76 ꢁ 0.06 ꢀ87.34 ꢁ 0.09 ꢀ76.87 ꢁ 0.04 ꢀ87.55 ꢁ 0.03
73.72 ꢁ 0.04 ꢀ75.61 ꢁ 0.06 ꢀ78.24 ꢁ 0.15
ꢀ89.76 ꢁ 0.04 ꢀ87.89 ꢁ 0.13 ꢀ93.88 ꢁ 0.02 ꢀ89.90 ꢁ 0.11 ꢀ90.34 ꢁ 0.03 ꢀ91.25 ꢁ 0.10 ꢀ86.67 ꢁ 0.06 ꢀ85.37 ꢁ 0.03
The results summarized above are the mean values of n ¼ 3, ꢁSD.
ꢀp ꢂ 0.05 as compared to control.
Table 4. % DPPH, OH, and SOR activity of the synthesized compounds 4a–i.
Entry
Compound
4a
Conc. mM
% DPPH activity
% OH activity
% SOR activity
1
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
58.74
67.28
53.97
62.91
NR
NR
NR
35.16
51.20
35.94
62.59
34.35
62.91
33.60
49.32
82.00
35.94
39.85
59.11
18.75
41.00
46.10
69.28
18.75
37.20
72.58
2
4b
48.06
58.37
46.06
55.91
54.55
65.87
NR
NR
NR
NR
54.55
63.01
37.67
61.69
17.02
28.80
69.37
3
4c
NR
4
4d
57.15
60.89
41.27
59.02
70.00
83.29
50.80
67.89
47.62
58.95
13.00
49.21
76.45
5
4e
6
4f
7
4g
8
4h
100
50
100
100
9
4i
STD
Ascorbic acid
The results summarized are the mean value of two parallel experiments. NR, no reaction.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 338–346
Pyridazin-3(2H)-one Derivatives as Anticancer Agents
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human cancer cell lines by MTT assay, the results revealed that,
the compounds 4g and 4i were the most promising cytotoxic
agents. Therefore, these compounds were further evaluated
for inhibition of selected cytokines and found that 4g was
potent antiangiogenic agent against TNFa, VEGF, FGFb, and
TGFb, whereas 4i showed potent antiangiogenic activity
against TNFa, VEGF, FGFb, and leptin. Furthermore, com-
pound 4f showed better OH radical scavenging activity than
the standard AA. Finally, it is conceivable that further
derivatization of such compounds will be of interest with
hope to get more selective agents.
4H), 1.65–1.61 (m, 6H); 13C NMR (CDCl3, 300 MHz): d 158.48 (C-
Ar), 154.55 (CO), 147.64 (C-Ar), 136.53 (C-Ar), 132.39 (C-Ar),
125.46 (C-Ar), 123.98 (C-Ar), 121.42 (C-Ar), 128.37 (C-Ar),
106.31 (C-Ar), 45.70 (2, CH2), 25.74 (3, CH2); LC–MS: m/e 342
(Mþ1). Anal. calcd. for C15H14Cl2FN3O: C, 52.65; H, 4.12; N,
12.28. Found: C, 52.87; H, 4.31; N, 12.73%.
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-(4-methyl-1,4-
diazepan-1-yl)-pyridazin-3(2H)-one (4b)
Light brown solid; m.p.: 100–101°C; IR (KBr) cmꢄ1: 3335, 3075,
2942, 2765, 1645, 1493; 1H NMR (DMSO-d6, 400 MHz): d 8.06 (s,
1H), 7.81 (d, J ¼ 4.0 Hz, 1H), 7.54 (d, J ¼ 8.8 Hz, 2H), 3.76–3.73
(m, 4H), 2.70–2.66 (m, 4H), 2.28 (s, 3H), 1.91–1.90 (m, 2H);
13C NMR (CDCl3, 400 MHz): d 158.20 (C-Ar), 155.88 (CO), 146.71
(C-Ar), 137.88 (C-Ar), 130.84 (C-Ar), 127.52 (C-Ar), 124.90
(C-Ar), 120.80 (C-Ar), 116.22 (C-Ar), 110.51 (C-Ar), 58.52
(CH2), 57.37 (CH2), 51.33 (CH2), 51.16 (CH2), 46.43 (CH3),
28.34 (CH2); LC–MS: m/e 371 (Mþ1). Anal. calcd. for
Experimental
Chemistry
All the chemicals were purchased from Aldrich Chemical Co.
(USA). All the solvents were purchased from S. D. Fine
Chemicals. IR spectra (KBr disks) were recorded using a
Perkin-Elmer 237 spectrophotometer. 1H NMR spectra were
recorded on Bruker Advance (300 and 400 MHz) spectrometer
using DMSO-d6 and CDCl3 as solvent, with TMS as an internal
reference. Mass spectra were recorded on a Shimadzu LC–MS-
QP 1000 EX. Thin layer chromatography (TLC) was performed
on silica gel-coated plates for monitoring the reactions. The
spots could be visualized easily under UV light.
C
16H17Cl2FN4O: C, 51.77; H, 4.62; N, 15.09. Found: C, 51.43;
H, 4.83; N, 15.63%.
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-(4-ethylpiperazin-
1-yl)-pyridazin-3(2H)-one (4c)
Brown solid; m.p.: 110–111°C; IR (KBr) cmꢄ1: 3055, 2941, 2750,
2710, 2261, 1643; 1H NMR (DMSO-d6, 400 MHz): d 8.10 (s, 1H),
7.81 (d, J ¼ 5.6 Hz, 1H), 7.55 (d, J ¼ 6.0 Hz, 2H), 3.50–3.48 (m,
4H), 2.50–2.46 (m, 4H), 2.38 (q, J ¼ 7.2 Hz, 2H), 1.02 (t, 3H); 13
C
Synthesis of [2-(3-chloro-4-fluorophenyl)]-4,5-
dichloropyridazin-3-one (3)
NMR (CDCl3, 400 MHz): d 158.52 (C-Ar), 156.03 (CO), 147.11 (C-
Ar), 137.82 (C-Ar), 131.88 (C-Ar), 127.61 (C-Ar), 125.10 (C-Ar),
121.94 (C-Ar), 120.85 (C-Ar), 116.26 (C-Ar), 52.61 (2, CH2), 52.24
(2, CH2), 48.85 (CH2), 11.88 (CH3); LC–MS: m/e 371 (Mþ1). Anal.
calcd. for C16H17Cl2FN4O: C, 51.77; H, 4.62; N, 15.09. Found: C,
51.46; H, 4.33; N, 15.57%.
To a solution of mucochloric acid 1 (14.99 g, 88.75 mmol) in
EtOH (150 mL) was added 3-chloro-4-fluorophenyl hydrazine 2
(14.25 g, 88.75 mmol), and MSA (20 mL) at 25°C. The solution
was refluxed for 2 h. After completion of reaction as indicated
by TLC, the suspension was filtered, washed with water, and
dried under high vacuum to afford crude solid compound 3.
Yield: 90%; 1H NMR (DMSO-d6, 400 MHz): d 8.36 (s, 1H), 7.86 (d,
J ¼ 6.4 Hz, 1H), 7.61 (d, J ¼ 6.8 Hz, 2H); LC–MS: m/e 293 (Mþ1).
5-Chloro-1-(3-chloro-4-fluorophenyl)-1,6-dihydro-4-(4-
methylpiperazin-1-yl)-pyridazine (4d)
White solid; m.p.: 130–131°C; IR (KBr) cmꢄ1: 3150, 2941, 2835,
2810, 2371, 1661; 1H NMR (DMSO-d6, 400 MHz): d 8.11 (s, 1H),
7.81 (d, J ¼ 5.6 Hz, 1H), 7.56 (d, J ¼ 6.0 Hz, 2H), 3.50–3.48 (m,
4H), 2.46–2.41 (m, 4H), 2.22 (s, 3H); 13C NMR (CDCl3, 300 MHz): d
158.78 (C-Ar), 155.45 (CO), 146.99 (C-Ar), 137.61 (C-Ar), 131.89
(C-Ar), 127.46 (C-Ar), 124.98 (C-Ar), 124.88 (C-Ar), 120.90 (C-Ar),
116.31 (C-Ar), 54.70 (2, CH2), 48.74 (2, CH2), 45.92 (CH3); LC–MS:
m/e 357 (Mþ1). Anal. calcd. for C15H15Cl2FN4O: C, 50.44; H,
4.23; N, 15.68. Found: C, 50.91; H, 4.02; N, 15.98%.
Synthesis of 4-chloro-2-(3-chloro-4-fluorophenyl)-5-
(aliphatic/cyclic saturated amino)pyridazin-3(2H)-one (4a–i)
To the solution of crude [2-(3-chloro-4-fluorophenyl)]-4,5-
dichloropyridazin-3-one 3 (2.30 g, 7.84 mmol) in acetonitrile
(20 mL) was added amine (10 mmol) and anhydrous potassium
carbonate (1.08 g, 7.84 mmol) at 25°C. The reaction mixture
was then refluxed for 1.5–2.5 h. After completion of reaction
(TLC), reaction mixture was poured over crushed ice, extracted
with ethyl acetate (30 mL ꢃ 3), washed with water (30 mL ꢃ 2),
brine (30 mL), dried (anhydrous Na2SO4), and concentrated
under reduced pressure. The crude product was purified by
recrystallization with diethyl ether.
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-
(cyclopentylamino)-pyridazin-3(2H)-one (4e)
Light brown solid; m.p.: 106–107°C; IR (KBr) cmꢄ1: 2935, 2770,
2830, 2345, 1549, 1525; 1H NMR (DMSO-d6, 400 MHz): d 8.13 (s,
1H), 7.81 (d, J ¼ 6.0 Hz, 1H), 7.53 (d, J ¼ 8.40 Hz, 2H), 6.52 (d,
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-(piperidin-1-yl)-
pyridazin-3(2H)-one (4a)
1H), 4.19–4.17 (m, 1H), 1.98–1.96 (m, 2H), 1.71–1.55 (m, 6H) 13
C
NMR (CDCl3, 400 MHz): d 158.46 (C-Ar), 156.00 (CO), 155.87 (C-
Ar), 143.43 (C-Ar), 137.83 (C-Ar), 127.55 (C-Ar), 125.14 (C-Ar),
120.78 (C-Ar), 116.22 (C-Ar), 107.25 (C-Ar), 54.84 (CH), 34.22 (2,
CH2), 23.81 (2, CH2); LC–MS: m/e 342 (Mþ1). Anal. calcd. for
White solid; m.p.: 140–141°C; IR (KBr) cmꢄ1: 2950, 2740, 2830,
2350, 1649, 1475; 1H NMR (DMSO-d6, 400 MHz): d 8.08 (s, 1H),
7.81 (d, J ¼ 5.6 Hz, 1H), 7.55 (d, J ¼ 8.0 Hz, 2H), 3.46–3.42 (m,
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C15H14Cl2FN3O: C, 52.65; H, 4.12; N, 12.28. Found: C, 52.97; H,
4.47; N, 12.83%.
India. 1,1-Diphenyl-2-picryl hydrazyl (DPPH) and 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were
procured from Sigma–Aldrich Co. (St. Louis MO, USA). Other
chemicals like rifampicin (Himedia Laboratories Pvt. Ltd.,
Mumbai). AA, solvents, reagents used were of AR grade and
were obtained from commercial sources.
tert-Butyl-4-(5-chloro-1-(3-chloro-4-fluorophenyl)-1,6-
dihydro-6-oxopyridazin-4-yl)-piperazine-1-carboxylate (4f)
White solid; m.p.: 170–171°C; IR (KBr) cmꢄ1: 3250, 3035, 2950,
2735, 2355, 1645, 1493; 1H NMR (DMSO-d6, 400 MHz): d 8.10 (s,
1H), 7.82 (d, J ¼ 5.6 Hz, 1H), 7.56 (d, J ¼ 6.4 Hz, 2H), 3.40–3.32
(m, 8H), 1.42 (s, 9H); 13C NMR (CDCl3, 300 MHz): d 157.66 (C-Ar),
155.58 (CO), 154.40 (COO), 147.11 (C-Ar), 137.54 (C-Ar), 131.74
(C-Ar), 127.50 (C-Ar), 125.00 (C-Ar), 121.03 (C-Ar), 117.40 (C-Ar),
116.42 (C-Ar), 80.40 (CO), 48.74 (4, CH2), 28.28 (3, CH3); LC–MS:
m/e 443 (Mþ1). Anal. calcd. for C19H22Cl2FN4O3: C, 51.48; H,
4.77; N, 12.64. Found: C, 51.87; H, 4.32; N, 12.95%.
Cytotoxic activity
The cytotoxic potential of the selected compounds against
selected cancer cell lines was determined by using the MTT
assay [33–36]. The assay uses a tetrazolium salt (MTT), to assess
cellular metabolism and hence viability. Cells were harvested
in the exponential phase and seeded into sterile 96-well plates
in 100 mL of medium and allowed to adhere overnight. Then
20 mL of the appropriate compound (0.01 mM) added. Cells
were incubated with the compound for a period of 24 h.
Following the required incubation period, 10 mL of MTT was
added to each well and the plates were incubated at 37°C in a
humid atmosphere with 5% CO2 and 95% air for 4 h. The
media was then gently aspirated, and 100 mL DMSO was added
to dissolve the formazan crystals. The amount of formazan
product generated was measured spectrophotometrically at
570 nm using Thermo Make Automatic Ex-Microplate Reader
(M 51118170).
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-
morpholinopyridazin-3(2H)-one (4g)
White solid; m.p.: 159–160°C; IR cmꢄ1 (KBr): 3091, 2913, 2840,
2360, 1645, 1585; 1H NMR (DMSO-d6, 400 MHz): d 8.10 (s, 1H),
7.82 (d, J ¼ 5.6 Hz, 1H), 7.55 (d, J ¼ 6.4 Hz, 2H), 3.78–3.74 (m,
4H), 3.50–3.46 (m, 4H); LC–MS: m/e 344 (Mþ1); 13C NMR (CDCl3,
300 MHz): d 158.75 (C-Ar), 156.45 (CO), 145.99 (C-Ar), 138.31
(C-Ar), 133.70 (C-Ar), 128.61 (C-Ar), 123.98 (C-Ar), 121.88 (C-Ar),
118.90 (C-Ar), 106.31 (C-Ar), 64.72 (2, CH2), 48.74 (2, CH2). Anal.
calcd. for C14H12Cl2FN3O2: C, 48.86; H, 3.51; N, 12.21. Found: C,
48.52; H, 3.19; N, 12.63%.
Antiangiogenesis assay
The profiling of the inhibition of cytokines promoting tumor
growth such as TNFa, IGF1, VEGF, IL6, FGFb, TGFb, EGF, and
leptin was carried out as per the manufacturers manual
instructions (Signosis, Inc., Sunnyvale, CA, USA). In brief, the
sealing film over the plate was removed and 100 mL of
standard, control, or sample was added in each well and
incubated for 1 h at room temperature with gentle shaking.
The contents were aspirated from each well followed by
washing the well by adding 200 mL of 1ꢃ assay wash buffer.
The process of washing was repeated for three times. After the
last wash, the remaining liquid was removed by inverting the
plate against clean paper towels. Diluted biotin-labeled
antibody mixture (100 mL) was added to each well and
incubated for 1 h at room temperature with gentle shaking.
The contents were washed as described above. To each well a
100 mL of diluted streptavidin–horseradish peroxidase (HRP)
conjugate was added and incubated for 45 min at room
temperature with gentle shaking. Again the contents were
washed as described above. A substrate (tetramethylbenzine
(TMB); 100 mL) was added to each well and incubated for
25 min followed by addition of stop solution (50 mL). The
change in the color of the mixture from blue to yellow
indicates the occurrence of reaction. The optical density of
each well was recorded using microplate reader at 450 nm
within 30 min.
4-Chloro-[2-(3-chloro-4-fluorophenyl)]-5-(pyrrolidin-1-yl)-
pyridazin-3(2H)-one (4h)
White solid; m.p.: 154–155°C; IR (KBr) cmꢄ1: 3108, 2886, 2361,
1749, 1645, 1515; 1H NMR (DMSO-d6, 400 MHz): d 7.96 (s, 1H),
7.80 (d, J ¼ 5.2 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 2H), 3.75–3.71 (m,
4H), 1.90–1.87 (m, 4H); 13C NMR (CDCl3, 400 MHz): d 158.29
(C-Ar), 155.78 (CO), 143.55 (C-Ar), 138.02 (C-Ar), 130.23 (C-Ar),
127.48 (C-Ar), 125.02 (C-Ar), 120.81 (C-Ar), 116.25 (C-Ar),
106.65 (C-Ar), 59.63 (2, CH2), 25.53 (2, CH2); LC–MS: m/e 328
(Mþ1). Anal. calcd. for C14H12Cl2FN3O: C, 51.24; H, 3.69; N,
12.80. Found: C, 51.67; H, 3.28; N, 12.38%.
5-(2-(Dimethylamino)ethylamino)-4-chloro-[2-(3-chloro-4-
fluorophenyl)]-pyridazin-3(2H)-one (4i)
White solid; m.p.: 128–129°C; IR (KBr) cmꢄ1: 3040, 2950, 2765,
1640, 1615, 1501; 1H NMR (DMSO-d6, 400 MHz): d 8.09 (s, 1H),
7.81 (d, J ¼ 6.4 Hz, 1H), 7.53 (d, J ¼ 8.0 Hz, 2H), 6.26 (bs, 1H), 3.48
(t, J ¼ 4.80 Hz, 2H), 2.45 (t, 2H), 2.19 (s, 6H); 13C NMR (CDCl3,
400 MHz): d 158.20 (C-Ar), 156.55 (CO), 147.33 (C-Ar), 140.43
(C-Ar), 129.83 (C-Ar), 123.55 (C-Ar), 121.14 (C-Ar), 120.78 (C-Ar),
117.22 (C-Ar), 107.25 (C-Ar), 56.84 (CH2), 38.29 (2, CH2),
45.71 (2, CH3); LC–MS: m/e 345 (Mþ1). Anal. calcd. for
C
14H15Cl2FN4O: C, 48.71; H, 4.8; N, 16.23. Found: C, 48.39; H,
4.05; N, 16.71%.
Biological assays
Human cell lines were requested from National Centre for Cell
Science (NCCS: a National Cell Line Facility), Pune (MS), India.
ELISA kit was purchased from Unique Technology, Pune (MS),
Antioxidant activity
The involvement of ROS and the free radical-mediated
oxidative damage of cell membranes, DNA, and proteins in
degenerative process related to ageing, cancer, inflammation,
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atherosclerosis, is a cause of concern. Therefore, there is
increasing interest in the protective and preventative func-
tions of foods and their constituents against oxidative
damage caused by ROS and free radicals [37]. In the present
study, we investigated the antioxidant properties of amino-
pyridazinone derivatives using DPPH [38], OH [39], and
SOR (superoxide anion) radical scavenging activity [40],
with respect to the standard AA and using a spectrometric
assay.
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