Synthesis and preliminary evaluation activity studies of novel 4-(aryl/heteroaryl-2-ylmethyl)-6-phenyl-2-[3-(4-substitutedpiperazine-1-yl) propyl]pyridazin-3(2H)-one derivatives as anticancer agents

Article abstract of DOI:10.1007/s00044-011-9851-6

A series of new 4-(aryl/heteroaryl-2-ylmethyl)- 6-phenyl-2-[3-(4- substituted piperazine-1-yl)propyl] pyridazin- 3(2H)-one derivatives were synthesized. The structures of the compounds were confirmed by IR, ¹H NMR, and mass spectral data. All the compounds were evaluated for their cytotoxicity toward five human cancer cell lines of different origins viz; HeLa (Cervical), SKBR3 (Breast), HCT116 (Colon), A375 (Skin) & H1299 (Lung) at different concentrations and the IC₅₀ values were determined. HCT116 and HeLa are the most sensitive against the compounds studied. One of them displayed moderate cytotoxicity against SKBR3. Majority of the compounds exhibited good to moderate activity.

Full text of DOI:10.1007/s00044-011-9851-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3161–3169
DOI 10.1007/s00044-011-9851-6
ORIGINAL RESEARCH
Synthesis and preliminary evaluation activity studies of novel
4-(aryl/heteroaryl-2-ylmethyl)-6-phenyl-2-[3-(4-substituted-
piperazine-1-yl)propyl]pyridazin-3(2H)-one derivatives
as anticancer agents
•
M. S. R. Murty B. Ramalingeswara Rao
•
•
•
•
Kesur R. Ram J. S. Yadav Jayesh Antony
Ruby John Anto
Received: 4 September 2011 / Accepted: 25 October 2011 / Published online: 11 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of new 4-(aryl/heteroaryl-2-ylmethyl)-
6-phenyl-2-[3-(4-substituted piperazine-1-yl)propyl] pyri-
dazin-3(2H)-one derivatives were synthesized. The struc-
Introduction
The discovery that several novel series of 3(2H)-pyridazi-
nones possess characteristic pharmacological and biologi-
cal activities stimulated great interest in pyridazine
chemistry, which continues to this day. Thus, the pyrida-
zine and its 3-oxo derivatives, i.e., the pyridazinones have
attracted a great deal of attention because of the wide
spectrum of their pharmaceutical and agrochemical activ-
ities. They are widely recognized as versatile scaffolds with
a diverse set of biological activities. Particularly pyridazi-
none derivatives were found to exhibit various pharma-
ceutical activities such as analgesic (Asif et al., 2011),
1
tures of the compounds were confirmed by IR, H NMR,
and mass spectral data. All the compounds were evaluated
for their cytotoxicity toward five human cancer cell lines of
different origins viz; HeLa (Cervical), SKBR3 (Breast),
HCT116 (Colon), A375 (Skin) & H1299 (Lung) at differ-
ent concentrations and the IC50 values were determined.
HCT116 and HeLa are the most sensitive against the
compounds studied. One of them displayed moderate
cytotoxicity against SKBR3. Majority of the compounds
exhibited good to moderate activity.
¨
anti-inflammatory (Gokc¸e et al., 2009, Sahin et al., 2004),
Keywords Pyridazinones Á Cyclic amines Á Hydrazide Á
antidepressant (Coelho et al., 2003), antihypertensive
(Demirayak et al., 2004, Anees Siddiqui et al., 2011),
antithrombotic (Monge et al., 1987), anticonvulsant (Rubat
et al., 1990), cardiotonic (Sircar et al., 1987), diuretics
(Akahane et al., 1999), and anti-HIV (Livermone et al.,
1993) activities. Certain pyridazinone derivatives contain-
ing the 2-phenyl-indolyl moiety have shown anti-tumor
activity (Ghaffar et al., 2011). Siddiqui et al. synthesized
and evaluated the antinociceptive (Anees Siddiqui et al.,
2010), activities of the compounds having 6-(substituted-
phenyl)-2-(substitutedmethyl)-4,5-dihydropyridazin-
3(2H)-one derivatives. 3(2H)-Pyridazinone derivatives
have been reported as analgesic and anti-inflammatory
agents without gastrointestinal side effect. Imazodan (I) is
reported to show ionotropic properties and Emorfazone A
(II) is an analgesic and anti-inflammatory compound
marketed as pentoil and nandron. Emorfazone E (III)
contains pyridazinone skeleton bearing piperazine moiety
which is also a pharmacologically active molecule and
the structures of the above biomolecules are illustrated
below.
Anticancer activity
M. S. R. Murty (&) Á B. R. Rao Á K. R. Ram Á J. S. Yadav
Organic Chemistry Division-I, Discovery Laboratory,
Indian Institute of Chemical Technology, Uppal Road,
Hyderabad 500607, India
e-mail: msrmurty@ymail.com
J. Antony Á R. J. Anto
Integrated Cancer Research Program, Division of Cancer
Research, Rajiv Gandhi Centre for Biotechnology, Thycaud PO,
Kerala, Thiruvananthapuram 695014, India
123

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Med Chem Res (2012) 21:3161–3169
O
O
O
NH
H2N
HO
O
N
N
N
N
N
N
N
Cl
N
N
O
(I)
(III)
(II)
N
A number of [(3-chlorophenyl) piperazinyl propyl] py-
ridazinone and the corresponding isoxazolo pyridazinones,
having the arylpiperazinyl substructure present in the
molecule are found to be very potent antinociceptive agents
(Giovannoni et al., 2003). Substituted 6-aryl-pyridazin-
3(2H)-ones were found to exhibit anti cancer activity
(Ahmad et al., 2010), against various cell lines. Various
piperazine derivatives were found to exhibit different
pharmaceutical activities. This motif was found in many
drug candidates displaying anticancer (Can-Cheng et al.,
2004), anti microbial (Chaudhary et al., 2006) activity.
Recent studies reveals that certain triazine substituted
piperzines have shown anticancer activity (Patel et al.,
2011).
following sequence of the reactions. The starting material
3-aroyl propionic acid (1) was prepared by condensing ben-
zene with succinic anhydride in presence of anhydrous alu-
minum chloride by Friedel-Crafts acylation conditions. The
compounds 3-substituted-5-phenyl-2-yl-methylene-2-(3H)-
furanones (2a–d) were synthesized from (1) by reacting with
aromatic aldehydes in the presence of sodium acetate in
acetic anhydride. The structures of the furanones (2a–d) were
confirmed by IR spectrum which show an absorption band at
1765–1787 cm-1 for tC=O, characteristic of the lactone
carbonyl group. The 2-(3H)-furanones (2a–d) were reacted
with hydrazine hydrate in ethanol at 10–15°C to give the
corresponding 3-aroyl-2-(substituted-4-yl-methylene)-pro-
pionic acid hydrazide 3a–d. The presence of IR absorption
band at 3110–3340 cm-1 (broad band) characteristic of the
NH group, and bands at 1650–1665 cm-1 and 1678–
1700 cm-1, characteristic of the amide carbonyl and ketone
groups, respectively. The hydrazide derivatives 3a–d, after
cyclization with 1 N HCl in benzene furnished the corre-
sponding 4-alkyl-6-phenyl-3(2H)-pyridazinone 4a–d as
depicted in Scheme 1.
Results and discussion
Based on the above observations and continuing our efforts
toward the synthesis of new pharmacologically active
heterocyclic compounds with expected biological activi-
ties, prompted us to synthesize pyridazin-3 (2H)-one
derivatives bearing cyclic amine moiety i.e., morpholine or
substituted piperazines. The cyclic amine component will
be linked to the lactam nitrogen of the pyridazinone ring
with a three-carbon chain spacer. Thus, as a part of our
program aimed at developing simple and efficient synthe-
ses of pharmacologically useful pyridazinones, we have
synthesized a new series of 4-(aryl/heteroaryl-2-ylmethyl)-
6-phenyl-2-[3-(4-substituted piperazine-1-yl)propyl]pyri-
dazin-3(2H)-one derivatives to evaluate the new com-
pounds for anti cancer activity.
The structures of the compounds were confirmed by
1
Mass and H NMR spectral data. The infrared spectra of
compounds (4a–d) showed a broad absorption band at
3165–3310 cm-1, which is the characteristic of the NH
group and an absorption band at 1650–1660 cm-1 for
1
tC=O, characteristic of the amide carbonyl group. The H
NMR showed a singlet at d 3.98 ppm region, (for example
1H NMR signal for 4a observes at d = 3.97 ppm) which is
a characteristic peak for R–CH2 protons. Various N-
substituted cyclic amines were reacted with 1-bromo-3-
chloropropane and activated zinc under neutral conditions
to produce the 1-(3-chloropropyl)-4-substituted cyclic
amine derivatives (A) (Scheme 2) (Murty et al., 2003).
The target compounds 4-substituted-6-phenyl-2-[3-(4-
substituted-piperzin-1-yl)-propyl]-pyridazin-3(2H)-ones
5a–t were prepared by treating pyridazinones 4a–d with
the appropriate chloro alkyl substituted cyclic amines with
KF-Al2O3 in acetonitrile solvent as shown in Scheme 3.
The structures of all the synthesized compounds 5a–
t were confirmed by 1H NMR, IR, and Mass spectral
analysis. In the IR Spectrum of the products, the disap-
pearance of a band at 3165–3300 cm-1 confirmed that the
Chemistry
The main idea of synthesizing new piperazine containing
pyridazin-3(2H)-ones derivatives is to study the cytotoxicity
of the products by varying the substituents on pyridazin-
3(2H)-one moiety as well as on cyclic amine moiety as these
additional groups may enhance the biological activity. The
title compounds were synthesized from 3-aroyl propionic
acid via 2(3H)-furanones (2) as key intermediate by the
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Med Chem Res (2012) 21:3161–3169
3163
a
b
1
2a-d
3a-d
c
4a-d
Scheme 1 R = phenyl, 2-furyl, 2-pyridyl, 2-thienyl. Reagents and conditions: (a) R–CHO, Ac2O, NaOMe, 90°C, 2 h; (b) Hydrazine hydrate,
MeOH, rt; (c)1.0 N HCl, benzene, rt
Scheme 2 X = N-phenyl,
N-benzyl, N-ethyl, O,
N-3-chlorophenyl
X
Zn
X
Cl
N
+
Cl
Br
H N
THF, r.t.
A
Scheme 3 R = phenyl, 2-furyl,
2-pyridyl, 2-thienyl; X =
N-phenyl, N-benzyl, N-ethyl,
O, N-3-Chlorophenyl. Reagents
and conditions: KF-Al2O3,
CH3CN, reflux, 4 h
O
R
N
N
d
N
X
4 + A
5a-t
piperazine moiety has substituted at N-2 position of the
pyridazinone. The results are summarized in Table 1.
Pyridazinone derivatives 5a–t can be divided from a
structural point of view in three principal parts that may be
responsible for pharmacological activity: (i) A pharmaco-
phoric portion constituted by a substituted 6-phenyl-pyri-
dazin-3(2H)-one, (ii) A terminal fragment constituted by a
cyclic amine moiety and (iii) A three carbon linker
between these two substructures.
cell lines using MTT assay as described earlier (Smitha
et al., 2005). The cytotoxicity studies were determined
against five human cancer cell lines, Cervical (HeLa),
Breast (SKBR3), Colon (HCT116), Skin (A375), and Lung
(H1299) cell lines and the results were presented in the
Tables 2 and 3. These two tables indicate the percentage
cytotoxic activity (dose dependent) of the synthesized
compounds at concentrations ranging from 50 to 100 lM.
Herein, in the Table 4, we represented the IC50 values of
the compounds 5a–t against the three cell lines (the cyto-
toxicity of the compounds against the other two cell lines
did not exhibit significant activity). All these com-
pounds possess common 6-phenyl-2H-pyridazin-3(2H)-one
nucleus. The substitutions at N-2 and C-4 positions of the
pyridazinone moiety play an important role in determining
the potency of the compounds 5a–t. Table 4 reveals that
Cytotoxic activity
The synthesized compounds, 5a–t were tested for in vitro
biological screening for their cytotoxicity toward cancer
123

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Med Chem Res (2012) 21:3161–3169
have same activity which is comparable with the standard
drug. This indicates the negligible effect because of C-4
substitution in the pyridazinone skeleton. Compounds 5i–
j and 5l exhibited good activity against cervical cancer cell
line (HeLa). Compound 5l (R = 2-thienyl and X =
N-C2H5) shows better activity against the same cell line
(HeLa). Here, the substitution at C-4 affects the cytotox-
icity of the compounds. As far as breast cancer cell line is
concerned 5a–e (R = phenyl or X = N-phenyl) showed
better cytotoxic activity when compared to other substitu-
ents. Hence, the effect because of pipezine moiety is
nominal against the breast cancer cell line (SKBR3).
Thus, the activity profile of these pyridazinone-piper-
zine compounds can be used as new lead molecules in the
development of effective anticancer agents.
Table 1 Substituted pyridazin-3-(2H)-one derivatives (5a–t)
O
R
N
N
N
X
5a-t
Compounda
R
X
Yieldb
5a
5b
5c
5d
5e
5f
Phenyl
–N–C6H5
–N–C6H5
–N–C6H5
–N–C6H5
80
71
68
74
82
75
66
68
72
70
70
80
74
77
75
80
75
78
72
68
2-Furyl
2-Pyridyl
2-Thienyl
Phenyl
–N–CH2–C6H5
–N–CH2–C6H5
–N–CH2–C6H5
–N–CH2–C6H5
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–O
2-Furyl
Conclusions
5g
5h
5i
2-pyridyl
2-Thienyl
Phenyl
As part of our continuous search for the potential anti-
cancer heterocyclic compounds, a series of new 4-substi-
tuted 6-phenyl pyridazin-3(2H)-one derivatives 5a–t were
synthesized and assessed for their anticancer activity. The
synthesis involves the Friedel-Crafts acylation of benzene
with succinic anhydride followed by various chemical
transformations. The N-alkylation of the pyridazin-3(2H)-
ones with chloropropyl piperazines was achieved by using
KF/Al2O3 in acetonitrile solvent. The products were
obtained in high purity with excellent yields. All the newly
synthesized pyrdiazin-3(2H)-one derivatives were screened
for anticancer activity on human cell lines. Hence, our
preliminary studies indicate that, the compounds 5q, 5s, 5t,
and 5l are the potent molecules possessing anti-prolifera-
tive activity. Compounds 5g, 5p, and 5r also may be
considered for further studies in more cell lines. In short,
our findings might be beneficial as leads for designing new
compounds with potential antitumoral activity. However,
further studies are necessary to evaluate the signal trans-
duction pathways induced by these compounds.
5j
2-Furyl
5k
51
5m
5n
5o
5p
5q
5r
5s
5t
2-Pyridyl
2-Thienyl
Phenyl
2-Furyl
–O
2-Pyridyl
2-Furyl
–O
–O
Phenyl
–N–3–Cl–C6H4
–N–3–Cl–C6H4
–N–3–Cl–C6H4
–N–3–Cl–C6H4
2-Furyl
2-Pyridyl
2-Thienyl
a
All the compounds were characterized by 1H NMR, IR and mass
spectroscopy
b
Isolated and optimized yields
compounds 5e, 5i, 5j, and 5t (IC50 \ 100) exhibited good
cytotoxicity against the cervical cell line, HeLa, where as
the compound 5l showed maximum cytotoxicity
(IC50 = 36) over the remaining compounds against HeLa
cell line in the series. Moderate activity was exhibited by
the compounds 5b, 5d, and 5e in SKBR3, 5o in A375 and
5e and 5o in H1299. The compounds 5g and 5p–t showed
good activity against colon cancer cell line (HCT116). The
compounds 5c and 5j show moderate activity against
HCT116 cell line. By stimulating these findings, we con-
cluded that in addition to the C-4 and N-2 substitution, the
type of cell line will also affect the cytotoxicity of the
compounds. In case of HCT116 (Colon cancer cell line)
substitution at N-2 will be more effective in determining
the potency of the compound. This was indicated by IC50
values of 5p–t. Among these compounds 5q, 5s, and 5t
Experimental
All the reagents were obtained from commercial sources.
Melting points were determined on a Buchi capillary melting
1
point apparatus. The H NMR (200 and 300 MHz) and
spectra was recorded on Varian Gemini and Bruker Avance
spectrometers. Chemical shifts are expressed in ppm down
field from internal tetramethyl silane (TMS). The mass
spectra were recorded on a VG Auto Spec mass spectrome-
ter. Elemental analyses were performed on Elemental
VARIO EL elemental analyzer. IR spectra were recorded on
Perkin-Elmer Infrared-683 Infrared spectrometer.
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Med Chem Res (2012) 21:3161–3169
3165
Table 2 The percentage cell viability induced by the compounds 5a–t in five different cancer cell lines
Compound
Percentage viability of the cells compared to untreated control
Cell lines
HeLa
50
SKBR3
50
HCT116
50
A375
50
H1299
50
Concentration in lM
100
100
82.0
100
100
100
5a
5b
5c
5d
5e
5f
79.6
75.0
78.2
81.6
67.9
72.5
71.9
80.4
65.1
68.4
70.5
45.2
81.9
70.3
73.1
78.7
72.1
70.1
75.3
67.0
77.7
61.3
69.9
77.2
42.7
61.7
63.8
80.0
45.8
42.8
61.9
37.1
61.8
64.6
73.0
67.1
56.6
67.8
72.8
37.8
100.0
69.9
72.9
67.1
70.6
80.9
83.6
87.2
88.3
82.8
100.0
96.3
100.0
93.4
96.0
100.0
83.3
80.6
90.2
78.6
100.0
77.8
66.3
78.6
73.4
86.6
54.9
78.4
77.2
72.2
100.0
78.8
94.9
83.6
81.0
49.7
40.3
63.1
43.5
44.0
97.0
73.5
56.2
78.5
70.9
86.1
34.3
68.2
75.7
59.5
98.3
66.8
91.3
63.5
78.5
42.3
34.8
42.3
37.0
43.8
100.0
98.7
94.2
96.0
91.1
100.0
90.8
98.5
93.0
92.0
93.6
86.8
89.5
90.1
83.8
93.2
83.2
100.0
85.7
84.8
100.0
96.5
90.8
93.6
80.1
97.6
85.8
97.0
82.6
90.3
91.0
86.0
89.3
86.3
81.6
93.0
77.1
92.0
81.8
76.5
100.0
78.9
88.0
86.2
78.5
95.7
92.4
96.1
96.0
96.4
100.0
98.8
95.2
100.0
82.4
96.1
94.1
94.1
97.3
93.2
100.0
78.5
77.6
81.5
71.4
93.0
90.2
95.5
94.7
95.4
97.2
96.0
90.5
90.5
74.3
95.5
92.8
90.1
94.2
92.8
45.6
60.5
56.8
59.0
79.9
80.0
75.6
85.1
80.9
92.2
96.2
100.0
90.5
96.2
87.1
75.1
78.5
86.5
72.6
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
Table 3 The percentage cell viability induced by Curcumin (positive
control) in five different cancer cell lines
Table 4 Cytotoxic activity (IC50, lM) of compounds 5a–t against
three human cancer cell lines
Cell Line
Concentration in lM
Compound
R
X
HeLa SKBR3 HCT116
5
10
25
5a
Phenyl
2-Furyl
–N–C6H5
–N–C6H5
150
150
82
150
150
114
150
150
150
55
5b
5c
129
HeLa
78.3
53.7
61.6
92.0
86.1
53.7
74.0
49.6
72.3
62.4
28.0
56.5
22.4
50.2
44.7
2-Pyridyl –N–C6H5
2-Thienyl –N–C6H5
150
126
115
121
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
27
SKBR3
HCT116
A375
5d
5e
150
78
Phenyl
2-Furyl
–N–CH2–C6H5
5f
–N–CH2–C6H5 131
H1299
5g
2-Pyridyl –N–CH2–C6H5 138
2-Thienyl –N–CH2–C6H5 150
5h
5i
150
150
123
150
150
150
150
150
49
Chemistry
Phenyl
2-Furyl
–CH2CH3
–CH2CH3
71
79
5j
General procedure for the synthesis of 3-substituted-5-
phenyl-2-yl-methylene-2-(3H)-furanones (2a–d)
5k
5l
2-Pyridyl –CH2CH3
2-Thienyl –CH2CH3
131
36
5m
5n
5o
Phenyl
2-Furyl
–O
–O
131
141
150
150
A solution of 3-(aroyl) propionic acid (3 mmol) and aro-
matic aldehyde (equimolar, 3 mmol) in acetic anhydride
(15 mL) with anhydrous sodium acetate (4.5 mmol) was
refluxed for 4 h under anhydrous conditions. After com-
pletion of reaction, the contents were poured into crushed
ice in small portions while stirring. A solid mass separated
out, which was filtered, washed with water and crystal-
lized from a mixture of methanol/chloroform (1:1) to give
(2a–d).
2-Pyridyl –O
5p
5q
5r
2-Furyl
Phenyl
2-Furyl
–O
–N–3–Cl–C6H4 115
–N–3–Cl–C6H4 150
29
67
5s
2-Pyridyl –N–3–Cl–C6H4 150
2-Thienyl –N–3–Cl–C6H4 76
29
5t
28
Curcumin
–
17
10
–
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Med Chem Res (2012) 21:3161–3169
General procedure for the synthesis of 3-aroyl-2-
(substituted-4-yl-methylene)-propionic
acid hydrazide (3a–d)
J = 6.8 Hz, 2H). IR (KBr): 2945 (C–H), 1649 (C=O),
1602 (C = N) cm-1; ESI–MS: m/z 465(M ? H)?.
4-furan-2-ylmethyl-6-phenyl-2-[3-(phenyl-piperzin-1-yl)-
propyl]-2H-pyridazin-3-one (5b) gummy
To a solution of the furanones (2a–d) (1 mmol) in ethanol
(10 mL), hydrazine hydrate (1.3 mmol) was added and
maintain the temperature at 10–15°C. The reaction mixture
allowed to stand for about 2 h. Evaporate the solvent and
pour the reaction mixture into crushed ice. A solid mass is
separated. Filter off the solid and washed with water then
recrystallized form ethanol.
1H NMR (CDCl3, 300 MHz) d :7.68–7.64 (m, 2H),
7.44–7.19 (m, 10H), 6.34 (m, 1H) 6.22 (d, J = 3.2 Hz,
1H), 4.28 (t, J = 7.2 Hz, 2H), 3.97 (s, 2H), 2.76–2.50 (m,
8H), 2.34 (t, J = 8.1 Hz, 2H), 2.12 (q, J = 7.2 Hz, 2H). IR
(KBr): 2947 (C–H), 1651 (C=O), 1603 (C=N) cm-1; ESI–
MS: m/z 455 (M ? H)?.
General procedure for the synthesis of 4-substituted-6-
phenyl-3-(2H)-pyrdidazinone (4a–d)
6-phenyl-2-[3-(phenyl-piperzin-1-yl)-propyl]-4-pyridin-2-
ylmethyl-2H-pyridazin-3-one(5c) gummy
1.0 N HCl (5 mL) was added drop wise with stirring at
25–35°C over 30–60 min to a solution of the 3-aroyl-2-
(substituted-4-yl-methylene)-propionic acid hydrazides
(4a–d) (3 mmol) in benzene (20 mL), after completion of
the addition, the solid was continuously stirred for 1 h, then
filtered off and washed thoroughly with water. The product
obtained was recrystallized from ethanol.
1H NMR (CDCl3, 300 MHz) d: 8.41 (d, J = 4.5 Hz, 1H),
7.69–7.48 (m, 4H), 7.38–7.24 (m, 4H), 7.15–7.02 (m, 3H),
6.76 (d, J = 8.7 Hz, 2H), 6.70 (t, J = 7.2 Hz, 1H), 4.22 (t,
J = 7.2 Hz, 2H), 4.02(s, 2H), 3.10–3.00 (m, 4H),
2.55–2.49 (m, 4H), 2.43 (t, J = 6.8 Hz, 2H) 2.00 (q,
J = 7.0 Hz, 2H). IR (KBr): 2927 (C–H), 1649 (C=O),
1598 (C=N) cm-1; ESI–MS: m/z 466 (M ? H)?.
4-benzyl-6-phenyl-2H-pyridazin-3-one (4a) Solid.
1
mp.113–115°C; Yield 85%, H NMR (CDCl3, 300 MHz)
d: 7.62 (d, J = 6.8 Hz, 2H), 7.39–7.;16 (m, 2H), 6.85 (d,
J = 8.7 Hz, 2H), 6.79 (t, J = 7.8 Hz, 1H), 4.33 (t, J =
6.8 Hz, 2H), 3.94 (s, 2H), 3.16–3.07 (m, 4H), 2.62–2.56
(m, 4H), 2.53 (t, J = 6.8 Hz, 2H), 2.10 (q, J = 6.8 Hz,
2H). IR (KBr): 1649 (C=O), 1602 (C=N) cm-1; ESI–MS:
m/z 465(M ? H)?.
6-phenyl-2-[3-(phenyl-piperzin-1-yl)-propyl]-4-thiophen-
2-ylmethyl-2H-pyridazin-3-one (5d) gummy
1H NMR (CDCl3, 400 MHz) d: 7.72–7.61 (m, 1H),
7.50–7.14, (m, 8H), 6.92 (t, J = 5.2 Hz, 1H), 7.02–6.75
(m, 5H), 4.33 (t, J = 7.2 Hz, 2H), 4.15 (s, 2H), 3.41 (s,
2H), 3.24–3.10 (m, 4H), 2.70–7.51 (m, 6H), 2.54 (t,
J = 6.6 Hz, 2H), 2.11 (q, J = 7.2 Hz, 2H). IR (KBr): 2923
(C–H), 1649 (C=O), 1600 (C=N) cm-1; ESI–MS: m/z 471
(M ? H)?.
General procedure for the synthesis of 4-substituted-6-
phenyl-2-[3-(4-substituted-piperzin-1-yl)-propyl]-
pyridazin-3-(2H)-ones (5a–t)
A mixture of 6-phenyl-3-(2H)-pyridazinone (3.0 mmol)
and KF-Al2O3 (4.5 mmol) in dry acetonitrile (15 mL) was
stirred for 20 min under N2 atmosphere. 1-(3-chloro-pro-
pyl)-4-substituted-piperzin (3.2 mmol) was added to the
above mixture and stirred for 5 h. After the completion of
reaction (confirmed by TLC), the solvent was evaporated
and cold water was added to the reaction mixture and
stirred for 30 min. Extract the organic compound with
ethyl acetate. The ethyl acetate layer is dried over anhy-
drous sodium sulfate. The compound was purified by col-
umn chromatography on silica gel eluting with EtOAc-
n-hexane.
4-benzyl-2-[3-(4-benzyl-piperzin-1yl)-propyl]-6-phenyl-
2H-pyridazin-3-one (5e) gummy
1H NMR (CDCl3, 300 MHz) d : 7.67 (d, J = 7.4 Hz, 2H),
7.51–7.42 (m, 5H), 7.39–7.16 (m, 6H), 6.85 (d,
J = 8.7 Hz, 2H), 6.79 (t, J = 7.8 Hz, 1H), 4.28 (t,
J = 7.4 Hz, 2H), 3.93 (s, 2H), 3.47 (s, 2H), 2.54–2.41(m,
8H), 2.35(t, J = 8.1 Hz, 2H), 2.04 (q, J = 8.1 Hz, 2H IR
(KBr): 2937 (C–H), 1650 (C=O), 1604 (C=N) cm-1; ESI–
MS: m/z 479 (M ? H)?.
2-[3-(4-benzyl-piperzin-1yl)-propyl]-4-furan-2-ylmethyl-6-
phenyl-2H-pyridazin-3-one (5f) gummy
4-Benzyl-6-phenyl-2-[3-(phenyl-piperzin-1-yl)-propyl]-
1
2H-pyridazin-3-one (5a) solid. mp. 113–115°C; H NMR
(CDCl3, 300 MHz) d: 7.62 (d, J = 6.8 Hz, 2H), 7.39–7.16,
(m, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.79 (t, J = 7.8 Hz,
1H), 4.33 (t, J = 6.8 Hz, 2H), 3.94 (s, 2H), 3.16–3.07 (m,
4H), 2.62–2.56 (m, 4H), 2.53 (t, J = 6.8 Hz, 2H), 2.10 (q,
1H NMR (CDCl3, 300 MHz) d: 7.69–7.64 (m, 2H),
7.43–7.18 (m, 9H), 7.16 (s, 1H), 6.34 (m, 1H), 6.22 (d,
J = 3.2 Hz, 1H), 4.28 (t, J = 7.4 Hz, 2H), 3.97 (s, 2H),
3.50 (s, 2H), 2.54–2.41 (m, 8H), 2.35 (t, J = 8.1 Hz, 2H),
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Med Chem Res (2012) 21:3161–3169
3167
2-[3-(4-ethyl-piperzin-1yl)-propyl]-4-thiophen-2-ylmethyl-
6-phenyl-2H-pyridazin-3-one (5l) gummy
2.04 (q, J = 8.1 Hz, 2H). IR (KBr): 2924 (C–H), 1650
(C=O), 1602 (C=N) cm-1; ESI–MS: m/z 469 (M ? 1).
1H NMR (CDCl3, 300 MHz) d :7.83–7.59 (m, 2H),
7.46–7.18 (m, 5H), 6.98 (t, J = 5.0 Hz, 1H), 6.34 (m, 1H),
6.94 (s, 1H), 4.29 (t, J = 7.0 Hz, 2H), 4.14 (s, 2H), 3.50 (s,
2H), 2.74–2.58 (m, 8H), 2.54 (q, J = 7.4 Hz, 2H), 2.06 (q,
J = 8.1 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H). IR (KBr): 2941
(C–H), 1649 (C=O), 1604 (C=N) cm-1; ESI–MS: m/z
413.2 (M ? H)?.
2-[3-(4-benzyl-piperzin-1yl)-propyl]-6-phenyl-4-pyridn-2-
ylmethyl-2H-pyridazin-3-one (5g) gummy
1H NMR (CDCl3, 300 MHz) d: 8.51 (d, J = 4.9 Hz, 1H),
7.77–7.54 (m, 4H), 7.45–7.09 (m, 10H), 4.26 (t,
J = 7.2 Hz, 2H), 4.25 (s, 2H), 4.09 (s, 2H), 3.47 (s, 2H),
2.61–2.25 (m, 10H) 2.03 (q, J = 7.2 Hz, 2H). IR (KBr):
2939 (C–H), 1650 (C=O), 1601 (C=N) cm-1; ESI–MS: m/z
480 (M ? H)?.
4-Benzyl-2-(3-morpholin-4-ylpropyl)- 6-phenyl-2H-
pyridazin-3-one (5m) gummy
2-[3-(4-benzyl-piperzin-1yl)-propyl]-6-phenyl-4-thiophen-
2-ylmethyl-2H-pyridazin-3-one (5h) gummy
1H NMR (CDCl3, 300 MHz) d: 7.66–7.46 (m, 2H),
7.41–7.21 (m, 7H), 7.19 (s, 1H), 4.30 (t, J = 7.4 Hz, 2H),
3.94 (s, 2H), 3.68–3.58 (m, 4H), 2.52–2.39 (m, 4H) 2.35 (t,
J = 7.0 Hz, 2H), 2.05 (q, J = 7.4 Hz, 2H). IR (KBr): 2925
(C–H), 1649 (C=O), 1603 (C=N) cm-1; ESI–MS: m/z 390
(M ? H)?.
1H NMR (CDCl3, 400 MHz) d: 7.62–7.56 (m, 2H),
7.40–7.12 (m, 10H), 6.92 (t, J = 5.2 Hz, 1H), 6.88 (s, 1H),
4.23 (t, J = 8.3 Hz, 2H), 4.08 (s, 2H), 3.41 (s, 2H),
2.49–2.28 (m, 10H) 1.99 (q, J = 6.8 Hz, 2H). IR (KBr):
2924 (C–H), 1648 (C=O), 1603 (C=N) cm-1; ESI–MS: m/z
485(M ? H)?.
4-furan-2-ylmethyl -2-(3-morpholin-4-ylpropyl)-6-phenyl-
2H-pyridazin-3-one (5n) gummy
4-Benzyl-2-[3-(phenyl-piperzin-1-yl)-propyl]-6-phenyl-2H-
pyridazin-3-one (5i) gummy
1H NMR (CDCl3, 300 MHz) d: 7.70–7.64 (m, 2H),
7.45–7.29 (m, 5H), 6.34(m, 1H) 6.22 (d, J = 3.2 Hz, 1H),
4.30 (t, J = 7.4 Hz, 2H), 3.97 (s, 2H), 3.67–3.60 (m, 4H),
2.52–2.39 (m, 6H), 2.53 (t, J = 6.8 Hz), 2.03 (q,
J = 7.4 Hz, 2H). IR (KBr): 2926 (C–H), 1655 (C=O),
1606 (C=N) cm-1; ESI–MS: m/z 380(M ? H)?.
1H NMR (CDCl3, 500 MHz) d :7.66–7.60 (m, 2H),
7.45–7.22 (m, 8H), 7.18 (s, 1H), 4.29 (t, J = 6.8 Hz, 2H),
3.94 (s, 2H), 2.60–2.42 (m, 10H), 2.40 (q, J = 6.8 Hz, 2H),
2.05 (q, J = 7.8 Hz, 2H), 1.07 (t, J = 6.8 Hz, 3H). IR
(KBr): 2960 (C–H), 1647 (C=O), 1602 (C=N) cm-1; ESI–
MS: m/z 417 (M ? H)?.
2-[3-morpholin-4-ylpropyl)-6-phenyl-4-pyridin-2-
ylmethyl-2H-pyridazin-3-one (5o) gummy
2-[3-(4-ethyl-piperzin-1yl)-propyl]-4-furan-2-ylmethyl-6-
phenyl-2H-pyridazin-3-one (5j) gummy
1H NMR (CDCl3, 300 MHz) d :8.60 (d, J = 6.0 Hz, 1H),
8.10–8.04 (m,1H), 7.96–7.74 (m, 4H), 7.53–7.36 (m, 4H),
4.33 (t, J = 6.8 Hz, 2H), 3.75–3.63 (m, 4H), 2.63–2.43 (m,
6H), 2.10 (q, J = 7.2 Hz, 2H). IR (KBr): 2925 (C–H),
1654 (C=O), 1596 (C=N) cm-1; ESI–MS: m/z 390
(M ? H)?.
1H NMR (CDCl3, 300 MHz) d: 7.70–7.65 (m, 2H),
7.45–7.30 (m, 5H), 6.37–6.33 (m, 1H), 6.24–6.21 (m, 1H),
4.29 (t, J = 7.6 Hz, 2H), 3.97 (s, 2H), 3.08–3.00 (m, 4H),
2.44–2.70 (m, 12H), 2.05 (q, J = 6.8 Hz, 2H), 1.11 (t,
J = 7.6 Hz, 3H). IR (KBr): 2953 (C–H), 1648 (C=O),
1604 (C=N) cm-1; ESI–MS: m/z 407 (M ? H)?.
2-[3-morpholin-4-ylpropyl)6-phenyl-4-thiophen-2-
ylmethyl-2H-pyridazin-3-one (5p) gummy
2-[3-(4-ethyl-piperzin-1yl)-propyl]-6-phenyl-4-pyridin-2-
ylmethyl-2H-pyridazin-3-one (5k) gummy
1H NMR (CDCl3, 300 MHz) d: 8.41 (d, J = 4.5 Hz, 1H),
7.69–7.48 (m, 4H), 7.38–7.24 (m, 4H), 7.15–7.02 (m, 3H),
6.76 (d, J = 8.7 Hz, 2H), 6.70 (t, J = 7.2 Hz, 1H), 4.30 (t,
J = 7.0 Hz, 2H), 4.14 (s, 2H), 3.68–3.59 (m, 4H),
2.50–2.35 (m, 6H), 2.05 (q, J = 7.2 Hz, 2H). IR (KBr):
2953 (C–H), 1651 (C=O), 1606 (C=N) cm-1; ESI–MS: m/z
396 (M ? H)?.
1H NMR (CDCl3, 200 MHz) d: 7.66 (d, J = 7.9 Hz, 2H),
7.51–7.15 (m, 8H), 7.02–6.76 (m, 5H), 4.29 (t, J = 7.2 Hz,
2H), 4.15 (s, 2H), 3.25–3.11 (m, 4H), 2.75–2.57 (m, 10H),
2.54 (q, J = 7.3 Hz, 2H), 2.05 (q, J = 7.3 Hz, 2H), 1.14 (t,
J = 7.2 Hz, 3H). IR (KBr): 2961 (C–H), 1653 (C=O),
1598 (C=N) cm-1; ESI–MS: m/z 429.8 (M ? H)?.
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Med Chem Res (2012) 21:3161–3169
4-benzyl-2-{3-[4-(3-chlorophenyl)-piperzin-1-yl]-propyl}-
6-phenyl-2H-pyridazin-3-one (5q) gummy
DMEM containing 10% FBS with antibiotics and antimy-
cotics at 37°C in a 5% CO2 atmosphere.
1H NMR (CDCl3, 200 MHz) d: 7.67-7.57 (m, 2H),
7.41–7.20 (m, 6H), 7.18 (s, 1H), 7.10 (t, J = 8.1 Hz, 1H),
6.83–6.67 (m, 3H), 4.32 (t, J = 7.2 Hz, 2H), 3.93 (s, 2H),
3.19–3.08 (m, 4H), 2.61–2.48 (m, 6H), 2.09 (q,
J = 7.2 Hz, 2H). IR (KBr): 2925 (C–H), 1649 (C=O),
1598 (C=N) cm-1; ESI–MS: m/z 499 (M ? H)?.
MTT assay
The cytotoxic activity of the compounds was determined
by MTT assay as described earlier. This assay measures the
percentage viability of the cells in response to different
concentrations of the compounds. Active mitochondrial
dehydrogenases of living cells convert the water soluble
yellow tetrazolium salt to an insoluble purple formazan.
The intensity of color developed is an indicator of per-
centage of viable cells present.
2-{3-[4-(3-chlorophenyl)-piperzin-1-yl]-propyl}-4-furan-
2ylmethyl-6-phenyl-2H-pyridazin-3-one (5r) gummy
1H NMR (CDCl3, 200 MHz) d: 7.66–7.59 (m, 2H),
7.42–7.21 (m, 6H), 7.19 (s, 1H), 7.10 (t, J = 8.1 Hz, 1H),
6.83–6.66 (m, 3H), 4.33 (t, J = 7.2 Hz, 3.95 (s, 2H),
3.20–3.08 (m, 4H), 2.62–2.48 (m, 6H), 2.10 (q,
J = 7.2 Hz, 2H). IR (KBr): 2922 (C–H), 1650 (C=O),
1597 (C=N), cm-1; ESI–MS: m/z 488 (M ? H)?.
In brief, cells (3000/well) were plated in 96-well plates
and kept overnight at 37°C after which, the cells were
incubated with and without various concentrations of the
compounds (25, 50, 100, and 250 lM). Curcumin was used
as the positive control. At the end of the incubation,
medium was removed and fresh medium containing 20%
MTT solution (2 mg/mL in PBS) was added to each well.
After 2 h, 0.1 ml of the extraction buffer (20% SDS and
50% DMF) was added, and the optical density was mea-
sured at 570 nm using a plate reader (Bio-Rad) after 1 h
and compared with that of the untreated control. The per-
centage of inhibition of cell viability was determined with
reference to the untreated control. The data were subjected
to linear regression analysis and the regression lines were
plotted for the best straight-line fit. The IC50 concentrations
were calculated using the respective regression analysis.
2-{3-[4-(3-chlorophenyl)-piperzin-1-yl]-propyl}-6-phenyl-
4-pyridin-2ylmethyl-2H-pyridazin-3-one (5s) gummy
1H NMR (CDCl3, 300 MHz) d: 8.41 (d, J = 4.5 Hz, 1H),
7.69–7.48 (m, 4H), 7.38–7.24 (m, 4H), 7.15–7.02 (m, 3H),
6.76 (d, J = 8.7 Hz, 2H), 6.70 (t, J = 7.2 Hz, 1H), 4.35 (t,
J = 7.2 Hz, 2H), 3.97 (s, 2H), 3.28–3.14 (m, 4H),
2.53–2.48 (m, 6H), 2.11 (q, J = 7.0 Hz, 2H). IR (KBr):
2925 (C–H), 1654 (C=O), 1594 (C=N) cm-1; ESI–MS: m/z
413.2 (M ? H)?.
Acknowledgments BRR is thankful to University Grants Com-
mission (U.G.C.). KRR and JA are thankful to the Council of Sci-
entific & Industrial Research (CSIR), New Delhi, India for the award
of fellowships.
2-{3-[4-(3-chlorophenyl)-piperzin-1-yl]-propyl}-6-phenyl-
4-thiophen-2ylmethyl-2H-pyridazin-3-one (5t) gummy
1H NMR (CDCl3, 300 MHz) d: 7.68–7.52 (m, 2H),
7.40–7.12 (m, 9H), 6.88 (t, J = 4.6 Hz, 1H), 6.80 (s, 1H),
4.33 (t, J = 7.4 Hz, 2H), 4.15 (s, 2H), 3.47 (s, 2H),
3.21–3.06 (m, 4H), 2.62–2.48 (m, 4H), 2.44 (t, J = 7.4 Hz,
2H), 2.09 (q, J = 7.2 Hz, 2H). IR (KBr): 2924 (C–H),
1651 (C=O), 1597 (C=N) cm-1; ESI–MS: m/z 505
(M ? H)?.
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