Synthesis, antiproliferative and anti-inflammatory activities of some novel 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi-3(2H)-ones

Article abstract of DOI:10.1016/j.ejmech.2013.06.050

Sixteen new 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi-3(2 H)-ones (2a-p) were synthesized and tested for in vitro anticancer and in vivo anti-inflammatory activities. Eleven (2b, 2d , 2-ej and 2m-p) of the obtained compounds were screened for their antiproliferative activity towards 60 human cancer cell lines by the National Cancer Institute (USA). Compound 2f showed remarkable activity with GI₅₀ less than 1 μM on 36 human tumor cell lines and has been referred to Biological Evaluation Committee (NCI) for advance study. Compound 2g also displayed promising antiproliferative activity against 20 different cell lines with GI50 less than 1 μM. Compounds 2k and 2n were found to have a comparable anti-inflammatory activity to that of standard drug etoricoxib in carrageenan-induced rat hind paw edema model at 5 h.

Full text of DOI:10.1016/j.ejmech.2013.06.050

European Journal of Medicinal Chemistry 67 (2013) 352e358
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative and anti-inflammatory activities of some
novel 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi-
3(2H)-ones
*
Syed Ovais, Kalim Javed , Shafiya Yaseen, Rafia Bashir, Pooja Rathore, Raed Yaseen,
Alhamzah D. Hameed, Mohammad Samim
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen new 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi-3(2H)-ones (2aep) were syn-
thesized and tested for in vitro anticancer and in vivo anti-inflammatory activities. Eleven (2b, 2d, 2eej
and 2mep) of the obtained compounds were screened for their antiproliferative activity towards 60
human cancer cell lines by the National Cancer Institute (USA). Compound 2f showed remarkable activity
Received 19 October 2012
Received in revised form
13 June 2013
Accepted 19 June 2013
Available online 5 July 2013
with GI50 less than 1
Committee (NCI) for advance study. Compound 2g also displayed promising antiproliferative activity
against 20 different cell lines with GI50 less than 1 M. Compounds 2k and 2n were found to have a
mM on 36 human tumor cell lines and has been referred to Biological Evaluation
m
Keywords:
Pyridazinone
(p-(Methanesulfonyl)phenyl)hydrazine
Antiproliferative activity
Anti-inflammatory activity
Ulcerogenic effect
comparable anti-inflammatory activity to that of standard drug etoricoxib in carrageenan-induced rat
hind paw edema model at 5 h.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
antihypertensive [7,8], antithrombotic [9], anticonvulsant [10],
cardiotonic [11], antibacterial, diuretic [12], anti-HIV [13], aldose
Cancer is not a single pathological state but a broad group of
diseases characterized by a high proliferative index and the spread
of aberrant cells from their site of origin [1]. It is the major cause of
death in the 21st century [2,3]. Despite major breakthroughs in
many areas of cancer therapies over the past few years, the suc-
cessful treatment of cancer remains a significant challenge in the
21st century. The therapeutic treatment of cancer is a combination
of surgery and/or radiotherapy with chemotherapy [4,5]. Current
chemotherapy consists of cytotoxic (cell-killing) agents and anti-
hormonal drugs, which reduce the proliferation of the tumors
[4,5]. However, therapeutic use of anticancer drugs is complicated
by systemic toxicity, usually observed in the bone narrow, the
gastrointestinal (GI) tract and hair, and by development of resis-
tance. Therefore, the design of new antitumor agents is one of the
most urgent research areas in medicinal chemistry.
reductase inhibitors [14], hepatoprotective agents [15], anti-
inflammatory [16e18] and COX-2 inhibitors [19]. Pyridazinone
derivatives have also been reported to have remarkable anticancer
activity [20,21]. Recently, our research group has reported the
interesting anticancer activity of 6-arylpyridazinones bearing
benzenesulfonamide moiety (16, 22). Some compounds of the se-
ries were found to have GI50 values in the low micromolar or
submicromolar concentration range against human cancer cell
lines and reaching, in the case of most active derivative bearing NSC
code 747558 (Fig. 1). This compound showed remarkable activity
against SR (leukemia) and NCI-H522 (non-small cell lung) with a
GI50 value of less than 0.1 mM. It also displayed good activity against
leukemia (CCRF-CEM, HL-60 (TB), K-562, MOLT-4, RPMI-8226),
non-small cell lung cancer (NCI-H460), colon (HCT-116, HCT-15,
HT29, KMI2, SW-620), CNS (SF-295), melanoma (MALME-3M,
M14, MDA-MB-435 SK-MEL-5), ovarian (OVCAR-3, NCI/ADR-RES)
Pyridazinone derivatives have been reported to exhibit wide
range of pharmacological activities such as antidepressant [6],
and breast (MCF7) cancer cell lines with a GI50 less than 1.0 mM.
The acute toxicity study of the compound (NSC 747558) indicated
that it is well tolerated intra-peritoneally (400 mg/kg) by athymic
nude mice [22].
In continuation of an ongoing program aiming at finding new
structure leads (based on pyridazinone skeleton) with potential
* Corresponding author. Tel.: þ91 9873463272.
E-mail addresses: kjaved@jamiahamdard.ac.in, Kjavedchem@yahoo.co.in
(K. Javed).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.050

S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
353
O
O
N
N
N
N
O
H3C
O
H3C
S
S
CH3
O
NH2
2f
NSC 747558
O
Fig. 1. Structure of the most potent antiproliferative agents.
chemotherapeutic activities, here, we report the synthesis and
evaluation for in vitro anticancer efficacy against human cancer cell
lines of a series of new 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-ones molecules (2aep). The data of these
synthesized compounds (structure, stereoisomerism, molecular
formula, molecular weight and melting point) were submitted to
National Cancer Institute (NCI), USA for antiproliferative activity. As
per the protocol of NCI, only eleven representative compounds 2b,
2d, 2eej and 2mep were selected and granted NSC codes Viz; NSC
762909, NSC 763763, NSC 762910, NSC 762908, NSC 762911, NSC
763765, NSC 762913, NSC 762915, NSC 763764, NSC 762912 and
NSC 762914 respectively and screened at NCI for antiproliferative
activity at a single high dose (10ꢀ5 M) in full 60 cell panel. Three
compounds namely 2f, 2g and 2p exhibited good activity at a single
dose and were selected for further evaluation at five dose level
screening. Among these three compounds 2f (NSC 762908) (Fig. 1)
showed remarkable antiproliferative activity at five dose level
screening. This compound has been referred to Biological Evalua-
tion Committee of NCI for advanced study.
Inflammation is closely linked to cancer, and many anticancer
agents are also used to treat inflammatory diseases, such as rheu-
matoid arthritis. Moreover, chronic inflammation increases the risk
for various cancers, indicating that eliminating inflammation may
represent a valid strategy for cancer prevention and therapy [23].
Several studies have shown that the pyridazinone ring can serve as
excellent core template for designing anti-inflammatory agent
[16,17and18]. It prompted us to investigate the anti-inflammatory
potential of these synthesized compounds (2aep).
observed in the expected range C-5 (
d
21.98e22.93), C-4 (
d
28.47e
28.96), C-6 (d 153.01ed 155.21) and C-3 (d 165.10e166.87). All other
peaks (13C NMR spectra) were observed at expected ppms and are
provided as Supplementary data.
2.2. Pharmacology
2.2.1. In vitro antiproliferative activity
Primary in vitro one-dose (10ꢀ5 M) anticancer assay was per-
formed using full panel of about 59 or 60 human tumor cell lines in
accordance with the protocol of the Drug Evaluation Branch, Na-
tional Cancer Institute (NCI), Bethesda, and described elsewhere
[26e32]. The human tumor cell lines were derived from nine
different cancer types: Leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate, and breast cancers. Out of the synthesized
compounds (2aep), eleven compounds, namely 2b, 2dej, 2m, 2n
and 2p were selected by the National Cancer Institute (NCI). The
compounds 2b, 2d, 2e, 2hej, 2m and 2n displayed mild sensitivity
against few cell lines (Table S1 Supplementary data). Compounds
2f, 2g and 2p displayed considerable antitumor activity (Table S2
Supplementary data) and were therefore selected for an
advanced assay against a full panel (approximately 60 cell lines) at
five concentrations at 10-fold dilution (100, 10, 1, 0.1 and 0.01 mM).
A 48 h continuous drug exposure protocol was used and sulfo-
rhodamine B (SRB) protein assay was used to estimate cell growth.
Details of this system and the information which is encoded by the
activity pattern over all cell lines have been published [26e28]. The
anticancer activity of tested compounds is given by three param-
eters for each cell line: log GI50 value (GI50 ¼ molar concentration of
the compound that inhibits 50% net cell growth), log TGI value
(TGI ¼ molar concentration of the compound leading to total in-
hibition) and log LC50 value (LC50 ¼ molar concentration of the
compound leading to 50% net cell death). Furthermore, a mean
graph midpoint (MG_MID) is calculated for each of the mentioned
parameters, giving an averaged activity parameter over all cell
lines. For the calculation of the MG_MID, insensitive cell lines are
included with the highest concentration tested. Selectivity of the
compound with respect to one or more cell lines of the screen is
This paper describes the synthesis, in vitro anticancer and in vivo
anti-inflammatory activity of some new 6-aryl-2-(p-(meth-
anesulfonyl)phenyl)-4,5-dihydropyridazi-3(2H)-ones
(2aep).
molecules
2. Result and discussion
2.1. Chemistry
The synthetic route used to synthesize titled compounds (2aep)
is outlined in Scheme 1. The -aroylpropionic acids (1aep) required
b
characterized by a high deviation (D) of the particular cell line
for the synthesis of pyridazinones were obtained from Friedele
Crafts acylation through reported methods [24,25]. The cyclization
to the pyridazinone derivatives bearing p-(methanesulfonyl)phenyl
moiety was afforded by the condensation of appropriate aroyl-
propionic acid and (p-(methanesulfonyl)phenyl)hydrazine. The
purity of compounds was checked by TLC. The structures of com-
pounds (2aep) was determined on the basis of elemental analysis
and by various spectroscopic methods such as IR, 1H NMR, 13C NMR
and MS. IR spectra showed a band at 1648e1697 cmꢀ1 for cyclic
carbonyl, 1579e1594 cmꢀ1 for C]N and two bands for SO2C at
1311e1379 cmꢀ1 and 1137e1155 cmꢀ1. In the 1H NMR spectra ar-
omatic protons were observed at expected ppms. The signal for
SO2CH3 was observed as three-proton singlet in the range of
parameter compared to the MG_MID value.
Compounds 2f, 2g and 2p exhibited considerable broad spec-
trum antitumor activities and showed effective growth inhibition
GI50 (MG_MID) values of 0.93, 1.94 and 9.54
(Table 1), beside a cytostatic activity TGI (MG_MID) 45.7, 43.6, and
75.8 M, respectively (Supplementary Table S3). The compound 2f
displayed remarkable antiproliferative activity in 36 different cell
lines with GI50 less than 1 M and showed better activity than that
mM, respectively
m
m
of 5-fluorouracil (5-FU) a clinically used anticancer drug in 29
different cell lines. This compound 2f has been referred to Biolog-
ical Evaluation Committee (NCI) for in vivo anticancer activity.
Compound 2g displayed significant antiproliferative activity in
20 different cell lines with GI50 less than 1
2p displayed moderate activity against 4 cells of the tested 59 tu-
mor cell lines (GI50 less than 3 M) (Table 1). The cytotoxic effects
mM (Table 1). Compound
d
3.20e3.24. In the 13C NMR spectra of these compounds, the
chemical shift values of carbon atoms of pyridazinone ring were
m

354
S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
R3
R3
R4
O
R2
R1
R4
R2
R1
O
O
OH
anh. AlCl3
O
O
1a-p
NH
2
HN
R3
R4
/
/
3
5
4
2
/
3
R2
6
4
O
N N
O S O
R1
//
//
CH
6
2
3
//
//
3
5
EtOH; reflux; 10-12 h
O S O
CH
3
2a-p
1a, 2a: R1=R2=R3=R4=H
1b, 2b: R2= Cl, R1= R3=R4=H
1d, 2d: R2= F, R1= R3=R4=H
1f, 2f: R2= CH3 , R1= R3=R4=H
1h, 2h: R2= -C(Me)3, R1= R3=R4=H
1j, 2j: R2= OC6H5, R1= R3=R4=H
1l, 2l: R2=R3= Cl, R1= R4=H
1c, 2c: R2= Br, R1= R3=R4=H
1e, 2e: R2= OCH3, R1= R3=R4=H
1g, 2g: R2= C2H5, R1= R3=R4=H
1i, 2i: R2= C6H5, R1= R3=R4=H
1k, 2k: R2=R3= OCH3, R1= R4=H
1m, 2m: R2= OH, R3=Cl, R1= R4=H
1o, 2o: R2=OH, R3=CH3, R1=R4=H
1n, 2n: R4= OH, R1= CH3, R2=R3=H
1p, 2p: R2=OH, R4=CH3, R1=R3=H
Scheme 1. Synthesis of 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi-3(2H)-ones (2aep).
associated with these compounds 2f, 2g and 2p were measured by
LC50 displayed in supplementary data (Table S4). The LC50 values for
compounds 2f and 2p are greater than 100 for all cell lines, while as
LC50 values for compounds 2g are greater than 100 except for two
cell lines (COLO 205 and SK-MEL-5). These LC50 values indicate low
toxicity of these compounds for normal human cell lines, as
required for development of potential antitumor agent.
activity at both 3 h and 5 h (2k vs 2e). Introduction of chlorine or
bromine atom at C-40 diminished the activity at both study time 3 h
and 5 h (2a vs 2b, 2a vs 2l, 2a vs 2c) while the introduction of
fluorine at same position increased the activity (2a vs 2d). In
compounds 2n, 2o and 2p hydroxyl group was found more effective
at ortho position (2n vs 2o and 2p) while as methyl group was
found more effective at meta position (2n and 2o vs 2p). Compound
2i with phenyl moiety at C-40 exhibited least activity in this study.
2.2.2. In vivo anti-inflammatory activity
The anti-inflammatory activity of target compounds (2aep) was
evaluated by applying carrageenan-induced hind paw edema
bioassay in rats [33]. Results were compared with etoricoxib as it
has some chemical structural resemblance with target compounds.
The results are summarized in Table 2. These compounds (2aep)
showed moderate to strong anti-inflammatory activity (6.6e66.6%
at 3 h and 21.0e86.4% at 5 h). The anti-inflammatory activity of 2k
and 2n at 5 h was almost comparable to that exhibited by standard
drug etoricoxib (83.3% at 3 h and 86.8% at 5 h).
2.2.3. Ulcerogenic effect
The ulcerogenic effect of the most potent anti-inflammatory
compounds 2k and 2n was studied at dose of 0.15 mmol/kg and
compared with that of reference drug etoricoxib (0.15 mmol/kg).
Like the etoricoxib, compounds 2k and 2n were found safe from the
point of view of ulcer induction.
3. Conclusion
With regard to structural activity relationship (SAR) it was
observed that introduction of alkyl group at C-40 position caused
elevation in the activity and it was further increased when the bulk
of alkyl was increased (2a vs 2feh) at both 3 h and 5 h. Introduction
of oxy group (hydroxyl, methoxyl and phenoxyl) in phenyl ring also
seems to be favorable for increasing the activity as compounds 2e,
2j, 2k, 2m, 2n, 2o and 2p showed better activity than that of 2a.
Introduction of second methoxyl group at C-30 increased the
Cyclocondensation of appropriate aroylpropionic acid and (p-
(methanesulfonyl)phenyl)hydrazine yielded pyridazinone de-
rivatives (2aep). The structures proposed for the synthesized
compounds are well supported by spectroscopic data and
elemental analysis. The data of these synthesized compounds were
submitted to National Cancer Institute (NCI), USA for anti-
proliferative activity. As per the protocol of NCI, only eleven
representative compounds 2b, 2d, 2eej and 2mep were selected.

S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
355
Table 1
Table 2
Growth inhibitory concentration (GI50
,
m
M) in tumor cell lines assay.a
Effect of pyridazinones derivatives on carrageenan induced hind paw edema in rats.
b
Cancer type
Leukemia
Cell
GI50
Group no. Treatment
Increase in paw volume ml ꢁ SEM
(0.05 mmol/kg) after carrageenan administration
5-FU 2f
2g
2.31
2p
3 h
5 h
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
SR
9.79
2.30
3.58
0.35
0.02
0.04
0.18
61.8
0.39
77.9
54.7
0.33
0.05
7.27
0.15
0.05
0.22
0.11
0.17
0.21
0.92
1.62
0.22
0.06
3.81
78.7
0.90
0.24
1.16
0.40
0.37
0.86
0.29
2.78
0.52
3.02
1.30
0.42
0.43
2.50
0.38
0.22
0.52
2.75
0.41
0.61
0.33
0.44
9.09
3.52
3.74
14.3
3.39
25.6
6.20
57.9
1.20
0.42
3.52
5.65
0.42
0.91
4.19
4.30
1.58
I
Vehicle
0.30 ꢁ 0.02
0.38 ꢁ 0.04
II
III
Etoricoxib
2a
0.05 ꢁ 0.02*** (83.3%) 0.05 ꢁ 0.02** (86.8%)
0.22 ꢁ 0.02* (26.6%)
0.22 ꢁ 0.03** (26.6%)
0.27 ꢁ 0.01* (10.0%)
0.12 ꢁ 0.02* (60.0%)
0.16 ꢁ 0.04** (46.7%)
0.16 ꢁ 0.02** (46.7%)
0.16 ꢁ 0.02* (46.7%)
0.15 ꢁ 0.02** (50.0%)
0.28 ꢁ 0.01** (6.6%)
0.15 ꢁ 0.02** (50.0%)
0.1 ꢁ 0.01*** (66.6%)
0.22 ꢁ 0.03* (26.6%)
0.17 ꢁ 0.01** (43.3%)
0.1 ꢁ 0.01*** (66.6%)
0.22 ꢁ 0.01* (26.6%)
0.2 ꢁ 0.03* (33.3%)
0.20 ꢁ 0.01** (47.3%)
0.24 ꢁ 0.02* (36.8%)
0.26 ꢁ 0.01* (31.5%)
0.10 ꢁ 0.01* (73.6%)
0.12 ꢁ 0.01** (68.4%)
0.15 ꢁ 0.02*** (60.5%)
0.13 ꢁ 0.02** (65.7%)
0.12 ꢁ 0.02* (68.4%)
0.30 ꢁ 0.01* (21.0%)
0.13 ꢁ 0.01** (65.7%)
0.06 ꢁ 0.03** (82.4%)
0.25 ꢁ 0.02* (34.2%)
0.17 ꢁ 0.02** (55.2%)
0.05 ꢁ 0.02** (86.8%)
0.15 ꢁ 0.02** (60.5%)
0.18 ꢁ 0.02** (52.6%)
IV
2b
RPMI-8226
V
2c
Non-small cell lung A549/ATCC
VI
2d
EKVX
VII
VIII
IX
2e
2f
2g
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
9.07
6.49
0.20 >100
X
XI
2h
2i
3.19
0.82
0.33
1.02
5.27
0.73
1.90
0.41
0.93
38.5
3.87
2.53
6.62
XII
XIII
XIV
XV
XVI
XVII
XVIII
2j
2k
2l
2m
2n
2o
Colon
46.7
3.78
7.91
3.70
4.22
3.32
40.0
2.87
13.2
50.4
5.53
4.30
7.25
5.37
3.85
1.28
3.48
9.56
5.49
5.65
4.00
7.13
3.41
2p
*P < 0.05, **p < 0.01, ***p < 0.001, compared to control (one-way ANOVA followed
by Dunnett’s test). Values are presented as mean ꢁ S.E.M. (n ¼ 6). Values in pa-
rentheses represent percent inhibitions.
0.34 43.1
CNS
2.94
0.40
1.02
1.25
0.54
0.45
0.74
6.99
0.93
2.35
0.13
1.32
1.91
3.50
6.80
0.63
0.22
0.90
1.84
1.48
3.30
0.78
1.48
0.93
0.01
8.51
3.25
0.40
2.44
5.13
0.14
8.59
1.00
2.04
4.81
8.31
5.23
2.76
5.66
3.13
3.46
0.83
0.96
4.40
3.75
1.94
respectively, beside a cytostatic activity TGI (MG_MID) 45.7, 43.6,
and 75.8
remarkable activity against 36 different cell lines with GI50 less
than 1 M. This compound (2f) can be used as lead compound for
mM, respectively. Particularly compound 2f displayed most
Melanoma
0.05
0.98
0.07
e
m
0.38
0.12
0.43
0.66
0.41
0.61
0.46
0.83
0.36
developing new anticancer agents. Two compounds (2k and 2n)
were found to have a comparable anti-inflammatory activity to that
of standard drug etoricoxib in carrageenan-induced hind rat paw
edema model at 5 h and were also found safe from the point of view
of ulcer induction.
56.7
1.03
0.46
3.55
0.52
1.22
0.01
4.43 >100
10.9 3.69
1.74
0.31
Ovarian
4. Experimental
76.9
53.8
12.3
3.26
9.77
92.5
2.78
37.8
4.1. Chemistry
0.74
0.27
1.15
5.78
0.14
4.02
0.29
0.63
21.8
Melting points were determined using open capillary tubes and
are uncorrected. All Fourier Transform Infrared (FTIR) spectra were
recorded on a bio-rad FTS-135 spectrophotometer using KBr pel-
lets; nmax values are given in cmꢀ1. 1H NMR spectra were recorded
Renal
0.72
0.35
0.29
0.07
2.61
0.49
1.12
1.42
2.36
0.36
0.07
6.60
9.77
27.9
12.8
7.18
on
a Bruker spectrospin DPX 300-MHz spectrometer using
e
deuterated dimethylsulfoxide (DMSO) as solvent and tetrame-
thylsilane (TMS) as internal standard. Chemical shifts are given in
(ppm) scale and coupling constants (J values) are expressed in Hz.
Mass spectra (MS) were recorded on ESI Q-TOF Water. 13C NMR
spectra were recorded on a Bruker spectrospin DPX at 75 MHz
using deuterated DMSO as a solvent and tetramethylsilane (TMS) as
internal standard. Purity of the compounds was checked on thin
layer chromatography (TLC) plates (silica gel G) which were visu-
alized by exposing to iodine vapors. Elemental analysis was carried
out on a CHNS elementar (Vario El III) system.
7.69
4.72
1.43
2.91
1.38
0.83
0.46
0.60
2.08
1.23
0.93
80.5
14.8
14.8
24.7
20.7
17.1
4.15
4.97
20.2
11.0
9.54
Prostate
Breast
10.6
8.12
e
MG_MID
a
Data was obtained from NCI’s in vitro disease-oriented human tumor cell lines
screen.
4.1.1. General procedure for the synthesis of aroylpropionic acids
(1aep)
b
GI50 was the drug concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in the control cells during the drug incu-
bation, determined at five concentration levels (100, 10, 1.0, 0.1 and 0.01 M).
To liquid aromatic hydrocarbon (30 ml), anhydrous aluminum
chloride (16.6 g, 0.125 mol) was added. Nitrobenzene (30 ml) was
used as solvent in case of solid aromatic hydrocarbons. The mixture
was stirred using a magnetic stirrer at room temperature for
30 min. To this, succinic anhydride (5 g, 0.05 mol) was added in five
portions with continuous stirring. Vigorous reaction started with
the evolution of HCl gas. Stirring was continued for another 6 h at
room temperature. The mixture was left at room temperature for
m
The in vitro antiproliferative activity showed that, among the eleven
compounds selected and evaluated by NCI, compounds 2f, 2g and
2p exhibited higher activity. Compounds 2f, 2g and 2p exhibited
considerable broad-spectrum antitumor activities with effective
growth inhibition GI50 (MG_MID) values of 0.93, 1.94 and 9.54 mM,

356
S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
48 h and then decomposed by adding ice-cold hydrochloric acid
(50%, 100 ml). The excess solvent was removed by steam distilla-
tion. The solid precipitated out was treated with aqueous saturated
sodium bicarbonate solution and filtered. Filtrate was acidified with
dilute HCl (4% v/v) to give a precipitate. It was filtered and residue
was washed with cold water, dried and crystallized from the
appropriate solvent to give 1aep [24,25].
346 [Mþ], 347 [Mþ þ 1], 348 [Mþ þ 2]. 13C NMR (75 MHz, DMSO,
d):
22.01 (C-5), 28.38 (C-4), 43.89 (SO2C), 153.21 (C-6), 165.76 (C-3).
Elemental analysis for: C17H15FN2O3S found (calculated) C: 58.98
(58.95), H 4.33 (4.36), N: 8.08 (8.09), S: 9.27 (9.26).
4.1.2.5. 2-(p-(Methanesulfonyl)phenyl)-6-(4-methoxy-phenyl)-4,5-
dihydropyridazi-3(2H)-one (2e). Light yellow crystals (m.p. 170e
171 ꢂC) from methanol. Yield ¼ 57%. Rf ¼ 0.39. IR nmax (KBr):
1693 cmꢀ1 (C]O), 1592 cmꢀ1 (C]N) 1144 cmꢀ1 & 1366 cmꢀ1
4.1.2. General procedure for the synthesis of pyridazinones (2aep)
A
mixture of the appropriate aroylpropionic acid (1aep)
(SO2C). 1H NMR (300 MHz, DMSO,
d
): 2.76 (2H, t, J ¼ 8.1 Hz, H-5),
(0.001 mol) and (p-(methanesulfonyl)phenylhydrazine) (0.001 mol)
in absolute ethanol (20e30 ml) was refluxed for 10e12 h. The re-
action mixture was concentrated to one-third of its volume and left
at room temperature, when a solid separated out. The crude product
was filtered off, washed with a small volume of alcohol, and stirred
with 5% sodium bicarbonate solution (25 ml). It was filtered, and
washed with 2% acetic acid and then with water. The product was
dried and recrystallized from the appropriate solvent to give pure
compounds 2aep. The solvent system used for TLC was tolue-
ne:ethyl acetate:formic acid (5: 4:1).
3.13 (2H, t, J ¼ 7.8 Hz, H-4), 3.23 (3H, s, SO2CH3), 3.81 (3H, s, OCH3),
7.03 (2H, d, J ¼ 9.0 Hz, H-30, H-50), 7.84 (2H, d, J ¼ 9.0 Hz, H-20, H-60),
7.89 (2H, d, J ¼ 8.7 Hz, H-300, H-500), 7.97 (2H, d, J ¼ 8.7 Hz, H-200, H-
600). ESI-MS (m/z): 358 [Mþ], 359 [Mþ þ 1]. 13C NMR (75 MHz,
DMSO, d): 22.81 (C-5), 28.92 (C-4), 44.14 (SO2C), 46.81 (OCH3),
153.26 (C-6), 166.46 (C-3). Elemental analysis for: C18H18N2O4S
found (calculated) C: 60.34 (60.32), H: 5.04 (5.06) N: 7.85 (7.82) S:
8.93 (8.95).
4.1.2.6. 2-(p-(Methanesulfonyl)phenyl)-6-p-tolyl-4,5-dihydropyridazi-
3(2H)-one (2f). Brown crystals (m.p. 169e170 ꢂC) from acetone.
Yield ¼ 54%, Rf ¼ 0.40 (toluene:ethyl acetate:formic acid, 5:4:1), IR
4.1.2.1. 2-(p-(Methanesulfonyl)phenyl)-6-phenyl-4,5-dihydropyridazi-
3(2H)-one (2a). Light yellow crystals (m.p. 167e168 ꢂC) from
acetoneemethanol. Yield ¼ 31%. Rf ¼ 0.44. IR nmax (KBr): 1690 cmꢀ1
(C]O), 1591 cmꢀ1 (C]N) 1137 cmꢀ1 & 1314 cmꢀ1 (SO2C). 1H NMR
nmax (KBr): 1692 cmꢀ1 (C]O), 1586 cmꢀ1 (C]N), 1145 cmꢀ1
&
1326 cmꢀ1 (SO2C). 1H NMR (300 MHz, DMSO,
d): 2.36 (3H, s, CH3 at
C-40), 2.77 (2H, t, J ¼ 8.1 Hz, H-5), 3.14 (2H, t, J ¼ 8.1 Hz, H-4), 3.23 (3H,
s, SO2CH3), 7.29 (2H, d, J ¼ 7.8 Hz, H-30, H-50), 7.78 (2H, d, J ¼ 7.8 Hz,
H-20, H-60), 7.89 (2H, d, J ¼ 8.7 Hz, H-300, H-500), 7.99 (2H, d, J ¼ 8.7 Hz,
H-200, H-600). ESI-MS (m/z): 342 [Mþ], 343 [Mþ þ 1]. 13C NMR
(300 MHz, DMSO,
d
): 2.79 (2H, t, J ¼ 8.1 Hz, H-5), 3.14 (2H, t,
J ¼ 8.1 Hz, H-4), 3.20 (3H, s, SO2CH3), 7.47e7.49 (3H, m, H-30, H-40, H-
50), 7.86e7.91 (4H, m, H-20, H-60, H-300, H-500), 7.98 (2H, d, J ¼ 9.0 Hz,
H-200, H-600). ESI-MS (m/z): 328 [Mþ], 327 [Mþ ꢀ 1]. 13C NMR
(75 MHz, DMSO, d): 21.39 (CH3), 22.62 (C-5), 28.03 (C-4), 44.15
(75 MHz, DMSO,
d): 22.81 (C-5), 28.47 (C-4), 44.61 (SO2C), 153.29 (C-
(SO2C), 153.75 (C-6), 166.36 (C-3). Elemental analysis for:
18H18N2O3S found (calculated) C: 63.17 (63.14), H: 5.31 (5.30), N:
6), 166.87 (C-3). Elemental analysis for: C17H16N2O3S found (calcu-
lated) C: 62.21 (62.18), H: 4.89 (4.91), N: 8.55 (8.53), S: 9.78 (9.76).
C
8.20 (8.18) S: 9.39 (9.36).
4.1.2.2. 6-(4-Chloro-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2b). White crystals (m.p. 157e159 ꢂC)
from methanol. Yield ¼ 56%. Rf ¼ 0.37. IR nmax (KBr): 1693 cmꢀ1 (C]
O), 1590 cmꢀ1 (C]N) 1150 cmꢀ1 & 1311 cmꢀ1 (SO2C). 1H NMR
4.1.2.7. 6-(4-Ethyl-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2g). Yellow crystals (m.p. 165e166 ꢂC)
from methanol. Yield ¼ 59%, Rf ¼ 0.58. IR nmax (KBr): 1691 cmꢀ1
(C]O), 1586 cmꢀ1 (C]N), 1155 cmꢀ1 & 1316 cmꢀ1 (SO2C). 1H NMR
(300 MHz, DMSO,
d
): 2.79 (2H, t, J ¼ 8.4 Hz, H-5), 3.17 (2H, t,
(300 MHz, DMSO,
d
): 1.19 (3H, t, J ¼ 7.5 Hz, eCH2eCH3), 2.66 (2H, q,
J ¼ 8.4 Hz, H-4), 3.24 (3H, s, SO2CH3), 7.55 (2H, d, J ¼ 8.7 Hz, H-300, H-
500), 7.87 (2H, d, J ¼ 6.3 Hz, H-30, H-50), 7.90 (2H, d, J ¼ 6.3 Hz, H-20,
H-60), 7.98 (2H, d, J ¼ 8.7 Hz, H-200, H-600). ESI-MS (m/z): 362, 364
J ¼ 7.5 Hz, eCH2eCH3), 2.77 (2H, t, J ¼ 8.1 Hz, H-5), 3.15 (2H, t,
J ¼ 7.8 Hz, H-4), 3.24 (3H, s, SO2CH3), 7.32 (2H, d, J ¼ 6.9 Hz, H-30, H-
50), 7.80 (2H, d, J ¼ 6.6 Hz, H-20, H-60), 7.89 (2H, d, J ¼ 8.7 Hz, H-300,
H-500), 7.97 (2H, d, J ¼ 8.7 Hz, H-200, H-600). ESI-MS (m/z): 357
[Mþ], 363 [Mþ þ 1], 359 [Mþ ꢀ 3]. 13C NMR (75 MHz, DMSO,
d):
21.98 (C-5), 28.01 (C-4), 44.22 (SO2C), 153.42 (C-6), 166.17 (C-3).
Elemental analysis for: C17H15ClN2O3S found (calculated) C: 56.25
(56.27), H: 4.15 (4.17), N: 7.74 (7.72), S: 8.86 (8.84).
[Mþ þ 1], 358 [Mþ þ 2]. 13C NMR (75 MHz, DMSO,
d): 15.91 (CH3e
CH2e), 22.68 (CH3eCH2e), 24.03 (C-5), 28.47 (C-4), 44.14 (SO2C),
153.80 (C-6), 166.36 (C-3). Elemental analysis for: C19H20N2O3S
found (calculated) C: 64.00 (64.02), H: 5.63 (5.66), N: 7.88 (7.86), S:
8.88 (9.00).
4.1.2.3. 6-(4-Bromo-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2c). White crystals (m.p. 161e162 ꢂC)
from methanol. Yield ¼ 42%. Rf ¼ 0.34. IR nmax (KBr): 1692 cmꢀ1
(C]O), 1588 cmꢀ1 (C]N), 1140 cmꢀ1 & 1379 cmꢀ1 (SO2C). 1H NMR
4.1.2.8. 6-(4-tert-Butyl-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2h). Yellow crystals (m.p. 179e180 ꢂC)
from methanol. Yield ¼ 53%, Rf ¼ 0.53. IR nmax (KBr): 1693 cmꢀ1
(C]O), 1579 cmꢀ1 (C]N), 1145 cmꢀ1 & 1352 cmꢀ1 (SO2C). 1H NMR
(300 MHz, DMSO,
d
): 2.79 (2H, t, J ¼ 7.5 Hz, H-5), 3.15 (2H, t,
J ¼ 7.8 Hz, H-4), 3.24 (3H, s, SO2CH3), 7.48e7.50 (2H, m, H-30, H-50),
7.89e7.92 (4H, m, H-20, H-60, H-300, H-500), 7.98 (2H, d, J ¼ 8.7 Hz, H-
(300 MHz, DMSO,
d
): 1.29 [9H, s, eC(CH3)3], 2.77 (2H, t, J ¼ 8.1 Hz,
200, H-600). ESI-MS (m/z): 406, 408 [Mþ]. 13C NMR (75 MHz, DMSO,
d
):
H-5), 3.14 (2H, t, J ¼ 8.7 Hz, H-4), 3.23 (3H, s, SO2CH3), 7.49 (2H, d,
J ¼ 8.4 Hz, H-30, H-50), 7.80 (2H, d, J ¼ 8.4 Hz H-20, H-60), 7.89 (2H, d,
J ¼ 8.7 Hz, H-300, H-500), 7.98 (2H, d, J ¼ 8.7 Hz, H-200, H-600). ESI-MS
22.52 (C-5), 28.41 (C-4), 44.44 (SO2C), 153.45 (C-6), 166.82 (C-3).
Elemental analysis for: C17H15BrN2O3S found (calculated) C: 50.16
(50.13), H: 3.73 (3.71), N: 6.91 (6.88) S: 7.89 (7.87).
(m/z): 384 [Mþ], 385 [Mþ þ 1]. 13C NMR (75 MHz, DMSO,
d): 21.80
(C-5), 28.61 (C-4), 31.5 [C(CH3)3], 34.2 [ C(CH3)3], 44.80 (SO2C),
153.21 (C-6), 165.89 (C-3). Elemental analysis for: C21H24N2O3S
found (calculated) C: 65.62 (65.60) H 6.27 (6.29) N: 7.28 (7.29) S:
8.37 (8.34).
4.1.2.4. 6-(4-Fluoro-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2d). White crystals (m.p. 68e69 ꢂC)
from methanol. Yield ¼ 59%. Rf ¼ 0.47. IR nmax (KBr): 1691 cmꢀ1 (C]
O), 1591 cmꢀ1 (C]N), 1143 cmꢀ1 & 1356 cmꢀ1 (SO2C). 1H NMR
(300 MHz, DMSO,
d
): 2.78 (2H, t, J ¼ 8.1 Hz, H-5), 3.15 (2H, t,
4.1.2.9. 6-Biphenyl-4-yl-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2i). Brown crystals (m.p. 147e148 ꢂC)
from chloroform. Yield ¼ 59%, Rf ¼ 0.62. IR nmax (KBr): 1693 cmꢀ1
J ¼ 9.0 Hz, H-4), 3.23 (3H, s, SO2CH3), 7.28e7.34 (2H, m, H-30, H-50),
7.86e7.98 (6H, m, H-20, H-60, H-300, H-500, H-200, H-600). ESI-MS (m/z):

S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
357
(C]O), 1586 cmꢀ1 (C]N), 1141 cmꢀ1 & 1317 cmꢀ1 (SO2C). 1H NMR
(300 MHz, DMSO,
): 2.81 (2H, t, J ¼ 8.1 Hz, H-5), 3.21 (2H, t,
SO2CH3), 6.85 (1H, d, J ¼ 8.4 Hz, H-30), 7.56 (1H, orthoemeta coupled
double doublet, J ¼ 2.0 Hz, J ¼ 8.0 Hz, H-40), 7.63 (1H, d, J ¼ 1.2 Hz,
meta coupled H-60), 7.89 (2H, d, J ¼ 8.8 Hz, H-300, H-500), 7.97 (2H, d,
J ¼ 8.8 Hz, H-200, H-600), 9.88 (1H, s, OH). ESI-MS (m/z): 358 [Mþ], 359
d
J ¼ 8.4 Hz, H-4), 3.25 (3H, s, SO2CH3), all aromatic protons appeared
between
(75 MHz, DMSO,
d
7.82e8.01. ESI-MS (m/z): 404 [Mþ], 405 [Mþ þ 1]. 13C NMR
d
): 22.14 (C-5), 28.71 (C-4), 44.09 (SO2C), 153.20 (C-
[Mþ þ 1], 360 [Mþ þ 2]. 13C NMR (75 MHz, DMSO,
d): 21.9 (CH3),
6), 165.74 (C-3). Elemental analysis for: C23H20N2O3S found (calcu-
lated) C: 68.28 (68.30), H: 4.96 (4.98), N: 6.95 (6.93) S: 7.95 (7.93).
22.66 (C-5), 28.42 (C-4), 44.61 (SO2C), 153.10 (C-6), 166.18 (C-3).
Elemental analysis for: C18H18N2O4S found (calculated) C: 60.33
(60.32), H: 5.08 (5.06) N: 7.81 (7.82) S: 8.93 (8.95).
4.1.2.10. 2-(p-(Methanesulfonyl)phenyl)-6-(4-phenoxy-phenyl)-4,5-
dihydropyridazi-3(2H)-one (2j). Yellow crystals (m.p. 168e169 ꢂC)
from acetone-methanol. Yield ¼ 51%, Rf ¼ 0.58. IR nmax (KBr):
1693 cmꢀ1 (C]O), 1586 cmꢀ1 (C]N), 1147 cmꢀ1 & 1319 cmꢀ1
4.1.2.15. 6-(4-Hydroxy-3-methyl-phenyl)-2-(p-(methanesulfonyl)
phenyl)-4,5-dihydropyridazi-3(2H)-one (2o). Cream white crystals
(m.p. 243e244 ꢂC) from methanol. Yield ¼ 41%. Rf ¼ 0.33. IR nmax
(SO2C). 1H NMR (300 MHz, DMSO,
d
): 2.78 (2H, t, J ¼ 8.1 Hz, H-5),
(KBr): 1648 cmꢀ1 (C]O), 1592 cmꢀ1 (C]N), 1148 cmꢀ1
&
3.16 (2H, t, J ¼ 8.4 Hz, H-4), 3.24 (3H, s, SO2CH3), all aromatic pro-
1340 cmꢀ1 (SO2C). 1H NMR (300 MHz, DMSO,
d): 2.50 (3H, s, CH3),
tons appeared between
d
7.07e7.98. ESI-MS (m/z): 420 [Mþ], 421
2.73 (2H, t, J ¼ 8.4 Hz, H-5), 3.09 (2H, t, J ¼ 7.5 Hz, H-4), 3.23 (3H, s,
SO2CH3), 6.85 (1H, d, J ¼ 8.4 Hz, H-50), 7.55 (1H, orthoemeta coupled
double doublet, J ¼ 2.5 Hz, J ¼ 8.7 Hz, H-60), 7.63 (1H, d, J ¼ 2.1 Hz,
meta coupled H-20), 7.89 (2H, d, J ¼ 9.0 Hz, H-300, H-500), 7.96 (2H, d,
J ¼ 9.0 Hz, H-200, H-600), 9.86 (1H, s, OH). ESI-MS (m/z): 358 [Mþ], 359
[Mþ þ 1]. 13C NMR (75 MHz, DMSO,
d): 22.48 (C-5), 28.61 (C-4),
43.14 (SO2C), 153.44 (C-6), 165.10 (C-3). Elemental analysis for:
C
23H20N2O4S found (calculated) C: 62.97 (63.00) H: 5.03 (5.06) N:
6.41 (6.39) S: 7.33 (7.31).
[Mþ þ 1]. 13C NMR (75 MHz, DMSO,
d
): 12.70 (CH3 at C-30), 22.61 (C-
5), 28.44 (C-4), 44.10 (SO2C), 154.67 (C-6), 166.75 (C-3). Elemental
analysis for: C18H18N2O4S found (calculated) C: 60.30 (60.32), H:
5.07 (5.06) N: 7.84 (7.82) S: 8.92 (8.95).
4.1.2.11. 6-(4-Dimethoxy-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2k). Off-white crystals (m.p.173e174 ꢂC)
acetoneemethanol. Yield ¼ 57%. Rf ¼ 0.48. IR nmax (KBr): 1694 cmꢀ1
(C]O), 1588 cmꢀ1 (C]N), 1148 cmꢀ1 & 1348 cmꢀ1 (SO2C). 1H NMR
(300 MHz, DMSO,
d
): 2.76 (2H, t, J ¼ 8.1 Hz, H-5), 3.15 (2H, t,
4.1.2.16. 6-(4-Hydroxy-2-methyl-phenyl)-2-(p-(methanesulfonyl)
phenyl)-4,5-dihydropyridazi-3(2H)-one (2p). Light yellow crystals
(m.p. 248e250 ꢂC) from methanol. Yield ¼ 39%. Rf ¼ 0.47. IR nmax
J ¼ 7.8 Hz, H-4), 3.23 (3H, s, SO2CH3), 3.81 (6H, s, OCH3 ꢃ2), 7.04 (1H,
d, J ¼ 8.4 Hz, H-60), 7.41e7.46 (2H, m, H-20, H-50), 7.90 (2H, d,
J ¼ 9.0 Hz, H-300, H-500), 7.98 (2H, d, J ¼ 9.0 Hz, H-200, H-600). ESI-MS (m/
(KBr): 1648 cmꢀ1 (C]O), 1594 cmꢀ1 (C]N), 1141 cmꢀ1
&
z): 388 [Mþ], 389 [Mþ þ 1]. 13C NMR (75 MHz, DMSO,
d): 22.79 (C-5),
1340 cmꢀ1 (SO2C). 1H NMR (300 MHz, DMSO,
d): 2.28 (3H, s, CH3),
28.65 (C-4), 44.04 (SO2C), 56.55 [(OCH3)2], 153.58 (C-6), 166.25 (C-3).
Elemental analysis for: C19H20N2O5S found (calculated) C: 58.78
(58.75), H: 5.23 (5.19), N: 7.25 (7.21), S: 8.27 (8.25).
2.77 (2H, t, J ¼ 7.8 Hz, H-5), 3.14 (2H, t, J ¼ 8.4 Hz, H-4), 3.25 (3H, s,
SO2CH3), 6.75 (1H, s, H-30), 6.77 (1H, d, J ¼ 7.2 Hz, H-50), 7.51 (1H, d,
J ¼ 8.4 Hz, H-60), 7.80 (2H, d, J ¼ 8.7 Hz, H-300, H-500), 8.00 (2H, d,
J ¼ 8.7 Hz, H-200, H-600), 11.04 (1H, s, OH). ESI-MS (m/z): 358 [Mþ],
4.1.2.12. 6-(3,4-Dichloro-phenyl)-2-(p-(methanesulfonyl)phenyl)-4,5-
dihydropyridazi-3(2H)-one (2l). Light yellow crystals (m.p. 206e
208 ꢂC) from methanol. Yield ¼ 42%. Rf ¼ 0.41. IR nmax (KBr):
1694 cmꢀ1 (C]O),1589 cmꢀ1 (C]O),1150 cmꢀ1 & 1313 cmꢀ1 (SO2C).
359 [Mþ þ 1]. 13C NMR (75 MHz, DMSO,
d
): 16.50 (CH3 at C-20),
22.50 (C-5), 28.44 (C-4), 44.14 (SO2C), 154.20 (C-6), 166.46 (C-3).
Elemental analysis for: C18H18N2O4S found (calculated) C: 60.33
(60.32), H: 5.08 (5.06), N: 7.81 (7.82) S: 8.93 (8.95).
1H NMR (300 MHz, DMSO,
d): 2.81 (2H, t, J ¼ 8.1 Hz, H-5), 3.17 (2H, t,
J ¼ 7.8 Hz, H-4), 3.24 (3H, s, SO2CH3), 7.75 (1H, d, J ¼ 8.7 Hz, H-60),
7.84e7.88 (3H, m, H-200, H-600, H-500), 7.98 (2H, d, J ¼ 8.7 Hz, H-300, H-
500), 8.08 (1H, d, J ¼ 2.1 Hz, meta coupled H-20). ESI-MS (m/z): 396,
4.2. Pharmacology
4.2.1. In vitro anticancer activity
398, 400 [Mþ]. 13C NMR (75 MHz, DMSO,
d): 22.46 (C-5), 28.24 (C-4),
A total of 60 human tumor cell lines, derived from nine cancer
types (leukemia, lung, colon, brain, melanoma, ovarian, renal,
prostate and breast) formed the basis of this test. The tumor cells
were cultured in PMI1640 medium supplemented with 5% fetal calf
44.54 (SO2C), 153.80 (C-6), 166.47 (C-3). Elemental analysis for:
17H14Cl2N2O3S found (calculated) C: 51.43 (51.40), H: 3.54 (3.55), N:
C
7.08 (7.05), S: 8.09 (8.07).
serum and 2 mM L-glutamine. The tumor cells are inoculated over a
4.1.2.13. 6-(3-Chloro-4-hydroxy-phenyl)-2-(p-(methanesulfonyl)
phenyl)-4,5-dihydropyridazi-3(2H)-one (2m). Yellow crystals (m.p.
230e231 ꢂC) from methanol. Yield ¼ 48%. Rf ¼ 0.61. IR nmax (KBr):
1697 cmꢀ1 (C]O), 1588 cmꢀ1 (C]N), 1155 cmꢀ1 & 1352 cmꢀ1
series of standard 96-well microtiter plates in 100 ml of medium
[30,31]. Density of inoculum depends on the type of tumor cell and
from its growth characteristics [28]. These cells are then pre-
incubated on the microtiter plate for 24 h before adding the com-
pounds. These were tested in DMSO solution at five different con-
centrations (10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7 and 10ꢀ8 M). After an incubation
of the chemical agent for 48 h with the tumor cell lines a sulfo-
rhodamine B (SRB) protein assay was used to estimate cell viability
or growth. The cytotoxic effects are evaluated and the assay results
and doseeresponse parameters were calculated as previously
described [32].
(SO2C). 1H NMR (300 MHz, DMSO,
d): 2.75 (2H, t, J ¼ 8.1 Hz, H-5), 3.11
(2H, t, J ¼ 7.8 Hz, H-4), 3.24 (3H, s, SO2CH3), 7.04 (1H, d, J ¼ 8.7 Hz, H-
60), 7.69 (1H, d, J ¼ 8.7 Hz, H-50), 7.84e7.89 (3H, m, H-20 H-300, H-500),
7.98 (2H, d, J ¼ 8.7 Hz, H-200, H-600), 10.77 (1H, s, OH). ESI-MS (m/z):
378, 380 [Mþ]. 13C NMR (75 MHz, DMSO,
d): 22.67 (C-5), 28.44 (C-4),
44.34 (SO2C), 153.01 (C-6), 166.81 (C-3). Elemental analysis for:
17H15ClN2O4S found (calculated) C: 53.91 (53.90), H: 3.97 (3.99), N:
C
7.38 (7.39), S: 8.49 (8.46).
4.2.2. Anti-inflammatory activity
4.1.2.14. 6-(2-Hydroxy-5-methyl-phenyl)-2-(p-(methanesulfonyl)
phenyl)-4,5-dihydropyridazi-3(2H)-one (2n). Yellow crystals (m.p.
248e249 ꢂC) from methanol. Yield ¼ 37%. Rf ¼ 0.47. IR nmax (KBr):
1649 cmꢀ1 (C]O), 1592 cmꢀ1 (C]N), 1148 cmꢀ1 & 1342 cmꢀ1
(SO2C). 1H NMR (300 MHz, DMSO,
(2H, t, J ¼ 8.4 Hz, H-5), 3.09 (2H, t, J ¼ 8.0 Hz, H-4), 3.24 (3H, s,
Carrageenan induced hind paw edema method was used for
evaluating anti-inflammatory activity [33]. Wistar rats (either sex)
weighing 150e175 g were procured from Central Animal House
facility of Jamia Hamdard, New Delhi (Registration no.173/CPCSEA).
The experiments were performed in accordance with the guide-
lines for the care and use of laboratory animals, laid down by the
d
) 2.17 (3H, s, CH3 at C-50), 2.73

358
S. Ovais et al. / European Journal of Medicinal Chemistry 67 (2013) 352e358
Committee for the Purpose of Control and Supervision of Experi-
ments in Animals (CPCSEA), Ministry of Social Justice and
Empowerment, Govt. of India, Jan. 2000. Overnight fasted rats
(16 h) were divided into 18 groups of 6 animals each. One group of
rats, which served as control was given vehicle (1% CMC in water in
a volume of 10 ml/kg) only. Test compounds (0.05 mmol/kg b.w),
etricoxib (0.05 mmol/kg b.w) suspended in vehicle (10 ml/kg) were
administered orally to respective groups. After 30 min, all animals
were injected with 0.1 ml of 1% carrageenan solution (prepared in
normal saline) in the subplantar aponeurosis of left hind paw to
induce inflammation and the volume of injected paw was
measured by using plethysmometer immediately (at 0 h). The paw
volume was again measured after 3 h and 5 h. The average paw
volume in a group of treated rats was compared with vehicle
(control group) and the percentage inhibition of edema was
calculated by using the formula:
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Acknowledgments
The authors are very grateful to the staff members of Depart-
ment of Health and Human Services, National Cancer Institute
(NCI), Bethesda, Maryland, USA, for carrying out in vitro anticancer
screening of the newly synthesized compounds.
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.050.