Synthesis and evaluation of anticancer activity of some novel 6-aryl-2-(p-sulfamylphenyl)-pyridazin-3(2H)-ones

Article abstract of DOI:10.1016/j.ejmech.2012.01.026

A series of novel pyridazinone derivatives bearing benzenesulfonamide moiety (2a-h) has been synthesized by the condensation of appropriate aroylacrylic acid and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol. Five derivatives (2a, 2b, 2d, 2g and 2h) were evaluated for their anticancer activity toward human cancer cell lines by the National Cancer Institute. The 2h showed remarkable activity against SR (leukemia) and NCI-H522 (non-small cell lung) with a GI₅₀ value of less than 0.1 μM. 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) and breast (MCF7) cancer cell lines with a GI ₅₀ less than 1.0 μM. The acute toxicity study of 2h indicated that it is well tolerated intra-peritoneally (400 mg/kg) by athymic nude mice. The 2h may possibly be used as lead compound for developing new anticancer agents.

Full text of DOI:10.1016/j.ejmech.2012.01.026

European Journal of Medicinal Chemistry 49 (2012) 304e309
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of anticancer activity of some novel
6-aryl-2-(p-sulfamylphenyl)-pyridazin-3(2H)-ones
,
a
a
a
b
I.G. Rathish a, Kalim Javed a , Shamim Ahmad , Sameena Bano , M.S. Alam , Mymoona Akhter ,
*
K.K. Pillai c, Syed Ovais a, Mohammed Samim a
a Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
b Drug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi, India
c Department of Pharmacology, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India
a r t i c l e i n f o
a b s r a c t
Article history:
A series of novel pyridazinone derivatives bearing benzenesulfonamide moiety (2aeh) has been
synthesized by the condensation of appropriate aroylacrylic acid and 4-hydrazinobenzenesulfonamide
hydrochloride in ethanol. Five derivatives (2a, 2b, 2d, 2g and 2h) were evaluated for their anticancer
activity toward human cancer cell lines by the National Cancer Institute. The 2h showed remarkable
Received 22 July 2011
Received in revised form
14 December 2011
Accepted 12 January 2012
Available online 20 January 2012
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), mela-
noma (MALME-3M, M14, MDA-MB-435 SK-MEL-5), ovarian (OVCAR-3, NCI/ADR-RES) and breast (MCF7)
cancer cell lines with a GI50 less than 1.0 mM. The acute toxicity study of 2h indicated that it is well
tolerated intra-peritoneally (400 mg/kg) by athymic nude mice. The 2h may possibly be used as lead
compound for developing new anticancer agents.
Keywords:
Pyridazinones
Benzenesulfonamides
Sulfonamides
Anticancer
Antiproliferative activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
anticancer drugs, devoid of the side effects of the presently avail-
able pharmacological agents [3e5].
Among all diseases that affect humanity, cancer ranks high as
a major killer [1,2]. Although major advances have been made in the
chemotherapeutic management of some patients, the continued
commitment to the arduous task of discovering new anticancer
agents remains critically important. A large number of structurally
novel sulfonamide derivatives have recently been reported to show
substantial antitumor activity, both in vitro and/or in vivo.
Although they have a common chemical motif of aromatic/
heterocyclic sulfonamide, there are a variety of mechanisms of their
antitumor action, most of them poorly understood at this moment.
It is believed that carbonic anhydrase inhibition, cell cycle arrest in
the G1 phase, disruption of microtubule assembly, functional
suppression of the transcriptional activator NF-Y, and angiogenesis
(matrix metalloproteinase, MMP) inhibition may be among such
relevant mechanisms of these anticancer compounds. Some of
these derivatives are currently being evaluated in clinical trials, and
there is much optimism that they may lead to novel alternative
Pyridazinone derivatives have been reported to exhibit a wide
range of pharmacological activities such as antidepressant [6],
antihypertensive [7,8], antithrombotic [9], anticonvulsant [10],
cardiotonic [11], antibacterial [12], diuretic [13], anti-HIV [14],
aldose reductase inhibitors [15], hepatoprotective agents [16] and
COX-2 inhibitors [17]. It has also been reported that pyridazinone
derivative have remarkable anticancer activity [18,19].
In continuation of an ongoing program aiming at finding new
structure leads with potential chemotherapeutic activities [20e22]
it was thought worthwhile to synthesize and investigate the anti-
tumor activity of some novel pyridazinone derivatives comprising
the benzenesulfonamide moiety (2aeh). The data of these
synthesized compounds were submitted to National Cancer Insti-
tute (NCI), USA for antiproliferative activity. As per the protocol of
NCI, only five representative compounds 2a, 2b, 2d, 2g and 2h were
selected and granted NSC codes Viz; NSC 749115, NSC 749114, NSC
747557, NSC 749116 and NSC 747558 respectively and screened at
NCI for antiproliferative activity at a single high dose (10ꢀ5 M) in
full 60 cell panel. Compound (2h) NSC 747558 exhibited the best
result at a single dose and was selected for further evaluation at five
dose level screening. It showed remarkable antiproliferative
activity at five dose level screening and was referred to Biological
* 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 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.026

I.G. Rathish et al. / European Journal of Medicinal Chemistry 49 (2012) 304e309
305
Evaluation Committee of NCI for advanced study. This paper pres-
ents the synthesis of novel compounds (2aeh) and in vitro anti-
cancer efficacy against human cancer cell lines of 2a, 2b, 2d, 2g and
2h compounds.
sulforhodamine 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
[25e27]. The anticancer activity of tested compounds is given by
three parameters for each cell line: log GI50 value (GI50 ¼ molar
concentrationof thecompoundthatinhibits50%netcellgrowth), log
TGI value (TGI ¼ molar concentration of the compound leading to
totalinhibition)andlogLC50 value(LC50 ¼ molarconcentrationof 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
withrespect to one or morecell lines of the screen is characterized by
a high deviation (Δ) of the particular cell line parameter compared to
the MG_MID value.
2. Results and discussion
2.1. Chemistry
The synthetic route used to synthesize title compounds (2aeh)
is outlined in Scheme 1. The b-aroylacrylic acids (1aeh) required for
the synthesis of pyridazinones were obtained by a Friedel Craft’s
acylation through reported methods [23,24]. The cyclization to
pyridazinone derivatives bearing a benzenesulfonamide moiety
was afforded by the condensation of appropriate aroylacrylic acid
and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol in
9.1e16.2% yield. The purity of the compounds was checked by TLC.
The structures of 2aeh were determined on the basis of elemental
analysis and by various spectroscopic methods such as IR, 1H NMR,
13C NMR and MS. Elemental analysis (C, H, N & S) data were within
ꢁ0.4% of the theoretical values. Spectral data IR, 1H NMR, 13C NMR
and MS of compounds were found in full agreement with the
proposed structure. IR spectra showed prominent bands for NH2 at
3302e3299 cmꢀ1, and 3164e3158 cmꢀ1, for cyclic carbonyl at
1659e1651 cmꢀ1, for C]N at 1599e1579 cmꢀ1 and for SO2N < at
1343e1336 cmꢀ1 and 1166e1163 cmꢀ1. In 1H NMR spectra the
aromatic protons were observed at expected ppms. The signal for
SO2NH2 was observed as two-proton singlet or merged with the
signals of aromatic protonsin aromatic region.
The 2h inhibited the growth of 34 cells of the tested 59 tumor cell
lines (log GI50 ꢀ7.05 to ꢀ6.65). It 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) and breast (MCF7)
cancer cell lines with a GI50 less than 1.0 mM (Table 1).
2.2.2. Acute toxicity determination
Animal toxicity experiment was performed on the 2h. To
determine maximum tolerated dose (MTD) this compound was
intraperitoneal (ip) administrated to athymic nude mice at the
doses of 100, 200 and 400 mg/kg. The highest dose resulted in no
body weight loss or lethality, indicating that 2h was well tolerated.
2.2. Pharmacology
2.2.1. In vitro anticancer activity
Primary in vitro one-dose (10ꢀ5 M) anticancer assay was per-
formed using full panel of about 60 human tumor cell lines in
accordancewiththeprotocoloftheDrugEvaluationBranch, National
Cancer Institute (NCI), Bethesda, and described elsewhere [25e31].
The human tumor cell lines were derived from nine different cancer
types: Leukemia, melanoma, lung, colon, CNS, ovarian, renal, pros-
tate, and breast cancers. Out of the synthesized compounds (2aeh),
five compounds, namely 2a, 2b, 2d, 2g and 2h were selected by the
National Cancer Institute (NCI). The compounds 2a, 2b, 2d and 2g
displayed no sensitivity towards human tumor cell lines. The
compound 2h (NSC 747558) possessed considerable anti-
proliferative activity (Table S1, Supplementary data) and it was
selected for an advanced assayagainst a full panel (approximately 60
cell lines) at five concentrations at 10-fold dilution (100,10,1, 0.1 and
2.2.3. Hollow fiber assay
On the basis of these results, 2h was selected for a preliminary
in vivo testing. The hollow fiber assay, developed at the NCI, is
a screening tool for assessing the potential anticancer activity of
compounds against human tumor cells cultivated in hollow fibers
and implanted intra-peritoneally and subcutaneously in mice [28].
After treatment by ip route, fiber cultures were collected and the
viable cell mass was determined. A scoring system was developed
to simplify evaluation of the results. For this, a value of 2 was
assigned for each compound dose which results in a 50% or greater
reduction (%T/C ꢂ 50) in viable cell mass. The intraperitoneal and
subcutaneous sample was scored separately. Compound with
a combined ip þ sc score ꢃ20, a sc score ꢃ8 or a net cell kill of one
or more cell lines is considered as active in this testing. The 2h
displayed no activity in this preliminary in vivo testing.
0.001
mM). A 48 h continuous drug exposure protocol was used and
NHNH .HCl
2
R
3'
2
R
R
1
5
2'
3
4
R
4'
2
R
2
O
R
R
1
3
3
R
R
1
5'
3
N
N
2
O
O
6'
1
SO NH
O
2
2
OH
2"
3''
6"
5''
Anhy. AlCl3
Absolute ethyl
alcohol/reflux
O
O
1a-h
SO NH
2
2
2a-h
(a) R1 = R2 = R3 = H
(c) R1 = R2 = H, R3 = Methyl
(e) R1 = H, R2 = Methyl, R3 = Chloro
(g) R1 = R2 = H, R3 = Biphenyl
(b) R1 = R2 = H, R3 = Chloro
(d) R1 = R2 = H, R3 = Methoxy
(f) R1 = Methyl, R2 = H, R3 = Methyl
(h) R1 = R2 = H, R3 = Ethyl
Scheme 1. Synthesis of 6-aryl-2-benzenesulfonamide-pyridazinones (2aeh).

306
I.G. Rathish et al. / European Journal of Medicinal Chemistry 49 (2012) 304e309
Table 1
The in vitro activitya and selectivity toward tumor cell lines for 2h (NSC: 747558).
Panel/Tumor
cell lines
Log GI50 (M)
Log TGI (M)
Log LC50 (M)
Selectivity toward tumor
GI50 (mM)
cell lines (d) for log GI50/log
TGI/log LC50 (M)b
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
ꢀ6.10
ꢀ6.42
ꢀ6.59
ꢀ6.24
ꢀ6.32
ꢀ7.05
> ꢀ4.00
ꢀ5.52
> ꢀ4.00
>ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
GI50 0.42
GI50 0.74/TGI 1.10
GI50 0.91
GI50 0.56
GI50 0.64/TGI 0.65
GI50 1.37/TGI 1.89
0.786
0.376
0.257
0.573
0.479
> ꢀ4.00
> ꢀ4.00
ꢀ5.07
ꢀ6.31
0.0887
(0.0238)*
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
ꢀ5.59
ꢀ5.55
ꢀ5.61
ꢀ4.76
ꢀ4.87
ꢀ4.77
ꢀ4.85
ꢀ6.36
> ꢀ4.00
> ꢀ4.00
ꢀ4.65
> ꢀ4.00
> ꢀ4.00
ꢀ4.15
e
2.58
2.79
2.48
17.3
13.5
16.9
14.3
0.438
e
TGI 0.23/LC50 0.14
e
ꢀ4.12
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
ꢀ4.20
> ꢀ4.00
> ꢀ4.00
ꢀ4.00
e
e
e
ꢀ5.35
GI50 0.68/TGI
0.93/LC50 0.19
GI50 1.34/TGI 1.78
NCI-H522
ꢀ7.02
ꢀ6.20
> ꢀ4.00
0.0966
(7.28)*
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
ꢀ6.01
ꢀ5.58
ꢀ6.27
ꢀ6.18
ꢀ6.36
ꢀ6.31
ꢀ6.34
ꢀ4.51
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
GI50 0.33/TGI 0.09
e
0.976
2.62
> ꢀ4.00
ꢀ4.72
GI50 0.59/TGI 0.30
GI50 0.50
GI50 0.68
GI50 0.63
GI50 0.66
0.535
0.661
0.436
0.486
0.454
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
CNS Cancer
SF-268
SF-295
ꢀ5.37
ꢀ6.26
ꢀ4.32
ꢀ4.76
> ꢀ4.00
ꢀ4.12
ꢀ4.25
GI50 0.58/TGI
0.554
0.34/LC50 0.11
GI50 0.06/TGI 0.22
TGI 0.02
GI50 0.08/TGI 0.12
GI50 0.12/TGI 0.20
SF-539
SNB-19
SNB-75
U 251
ꢀ5.74
ꢀ5.36
ꢀ5.76
ꢀ5.80
ꢀ4.64
ꢀ4.44
ꢀ4.54
ꢀ4.62
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
1.84
4.41
1.76
1.58
Melanoma
LOX IMVI
MALME-3M
ꢀ5.28
ꢀ6.73
> ꢀ4.00
ꢀ4.79
> ꢀ4.00
ꢀ4.12
e
5.27
0.188
GI50 1.05/TGI
0.37/LC50 0.11
GI50 0.79/TGI 0.89
GI50 0.97/TGI 0.24
e
M14
ꢀ6.47
ꢀ6.65
ꢀ4.97
ꢀ5.86
ꢀ6.34
> ꢀ4.00
ꢀ6.14
ꢀ5.31
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
0.335
0.223
10.7
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ5.66
ꢀ4.14
> ꢀ4.00
ꢀ5.12
GI50 0.18
GI50 0.66/TGI 0.70
e
1.37
0.453
>100
0.724
> ꢀ4.00
> ꢀ4.00
GI50 0.46
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
ꢀ5.42
ꢀ6.37
> ꢀ4.00
ꢀ4.38
ꢀ4.96
ꢀ6.50
ꢀ5.40
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
ꢀ4.89
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
e
3.81
GI50 0.69
e
0.429
>100
41.4
11.0
0.314
4.02
e
e
GI50 0.82/TGI 0.47
e
> ꢀ4.00
Renal Cancer
786-0
A498
ꢀ4.99
ꢀ5.71
ꢀ498
ꢀ4.20
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
ꢀ4.11
e
10.2
1.97
10.5
2.73
3.51
5.84
10.8
>100
ꢀ4.42
GI50 0.03
e
ACHN
> ꢀ4.00
> ꢀ4.00
ꢀ4.55
CAKI-1
RXF 393
SN12C
TK-10
ꢀ5.56
ꢀ5.45
ꢀ5.23
ꢀ4.96
> ꢀ4.00
e
TGI 0.13
e
> ꢀ4.00
ꢀ4.54
TGI 0.12/LC50 0.10
e
UO-31
> ꢀ4.00
> ꢀ4.00

I.G. Rathish et al. / European Journal of Medicinal Chemistry 49 (2012) 304e309
307
Table 1 (continued )
Panel/Tumor
cell lines
Log GI50 (M)
Log TGI (M)
Log LC50 (M)
Selectivity toward tumor
GI50
13.8
(mM)
cell lines (d) for log GI50/log
TGI/log LC50 (M)b
Prostate Cancer
DU-145
ꢀ4.86
> ꢀ4.00
> ꢀ4.00
e
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
ꢀ6.21
ꢀ4.92
ꢀ5.99
ꢀ6.12
ꢀ4.72
> ꢀ4.00
ꢀ4.77
ꢀ4.75
> ꢀ4.00
> ꢀ4.00
> ꢀ4.00
ꢀ4.07
GI50 0.53/TGI 0.30
e
0.615
12.1
1.03
GI50 0.31/TGI 0.35
GI50 0.44/TGI
0.33/LC50 0.6
BT-549
0.756
T-47D
MDA-MB-468
MG_MID
ꢀ5.59
ꢀ5.50
L5.68
> ꢀ4.00
> ꢀ4.00
L4.42
> ꢀ4.00
> ꢀ4.00
L4.01
e
2.56
3.18
e
*The value in parenthesis represent the GI50 for the 5-FU (Fluorouracil) standard drug.
a
Data obtained from hte NCI’s in vitro disease-oriented human tumor cells.
The reported data represent the logarithmic difference between the parametric value referred to the most sensitive cell line and the same mean parameter, d is considered
b
low if <1, moderate >1 and <3, high if >3.
3. Conclusion
4.1.1.1. 6-Phenyl-2-benzenesulfonamidepyridazine-3(2H)-one (2a).
Shiny off white crystals (m.p. 290e291 ꢄC). Yield 42.7%. Rf ¼ 0.67. 1H
Cyclocondensation of appropriate aroylacrylic acid and 4-
hydrazinobenzenesulfonamide hydrochloride yielded pyridazinone
derivatives bearing a benzenesulfonamide moiety (2aeh). The
structures proposed to the synthesized compounds are well sup-
ported by spectroscopic data and elemental analyses. One of these
derivatives (2h) had excellent in vitro anticancer activity and can be
used as lead compound for developing new anticancer agents.
NMR (DMSO-d6,
d
): 7.24 (1H, d, J ¼ 9.71 Hz, H-4), 7.53 (5H, m,
SO2NH2, H-30, H-40, H-50), 7.94 (6H, m, H-20, H-60 and N-phenyl
protons), 8.19 (1H, d, J ¼ 9.70 Hz, H-5). IR ymax (KBr): 3311 cmꢀ1 and
3161 cmꢀ1 (NH2), 1656 cmꢀ1 (C]O), 1587 cmꢀ1 (C]N), 1338 cmꢀ1
and 1166 cmꢀ1 (SO2N). FAB-MS (m/z): 327 [Mþ]. 13C NMR (75 MHz,
DMSO, d): 131.89 (C-5 pyridazinone), 134.48 (C-4 pyridazinone),
145.11 (C-6 pyridazinone), 159.07 (C-3 pyridazinone). Anal. Calcd for
C16H13N3O3S: C, 58.7; H, 4.00; N, 12.84; S, 9.80; Found: C, 58.5; H,
4.03; N, 13.02; S, 9.71.
4. Experimental
4.1.1.2. 6-(4-Chlorophenyl)-2-benzenesulfonamidepyridazine-3(2H)-
one (2b). Shiny off white crystals (m.p. 282e283 ꢄC). Yield 60.3%.
4.1. Chemistry
Rf ¼ 0.68. 1H NMR (DMSO-d6,
): 7.21 (1H, d, J ¼ 9.77 Hz, H-4), 7.32
d
(2H, d, J ¼ 7.81 Hz, H-30, H-50), 7.49 (2H, s, SO2NH2), 7.84 (2H, d,
J ¼ 7.86 Hz, H-20, H-60), 7.91 (2H, d, J ¼ 8.53 Hz, H-300, H-500) 7.97 (2H,
d, J ¼ 8.48 Hz, H-200, H-600), 8.15 (1H, d, J ¼ 9.79 Hz, H-5). IR ymax
(KBr): 3302 cmꢀ1 and 3158 cmꢀ1 (NH2), 1659 cmꢀ1 (C]O),
1596 cmꢀ1 (C]N), 1340 cmꢀ1 and 1163 cmꢀ1 (SO2N).FAB-MS (m/z):
361 [Mþ], 362 [Mþ1], 363 [Mþ2], 364 [Mþ3]. 13C NMR (75 MHz,
Melting points were determined by open capillary tubes and are
uncorrected. All the Fourier Transform Infra Red (FTIR) spectra were
recorded on a Bio-rad FTS-135 spectrophotometer using KBr
pellets; nmax values are given in cmꢀ1 1H NMR spectra were
.
recorded on a Bruker Spectrospin DPX 300-MHz spectrometer
using deuterated DMSO as solvent and tetramethyl silane (TMS) as
an internal standard. Chemical shifts are given in d (ppm) scale and
coupling constants (J values) are expressed in Hz. Mass spectra (MS)
were scanned by affecting FAB ionization JEOL-JMS-DX 303 system,
equipped with direct inlet probe system. The m/z values of the more
intense peaks are mentioned. Purity of the compounds was
checked on TLC plates (silica gel G) which were visualized by
exposing to iodine vapors. Elemental analysis was carried out on
CHNS Elementar (Vario EL III).
DMSO, d): 131.80 (C-5 pyridazinone), 133.35 (C-4 pyridazinone),
144.38 (C-6 pyridazinone), 159.00 (C-3 pyridazinone). Anal. Calcd
for C16H12ClN3O3S: C, 53.11; H, 3.34; N, 11.61; S, 8.86. Found: C,
53.02; H, 3.37; N, 11.52; S, 8.91.
4.1.1.3. 6-(4-Methylphenyl)-2-benzenesulfonamidepyridazine-3(2H)-
one (2c). Shiny off white crystals (m.p. 281e282 ꢄC).Yield 70.5%.
Rf ¼ 0.75. 1H NMR (DMSO-d6,
): 8.15 (1H, d, J ¼ 9.82 Hz, H-5), 7.96
d
(2H, d, J ¼ 8.50 Hz, H-200, H-600), 7.91 (2H, d, J ¼ 8.68 Hz, H-300, H-500),
7.84 (2H, d, J ¼ 7.96 Hz, H-20, H-60), 7.50 (2H, s, SO2NH2), 7.32 (2H, d,
J ¼ 7.87 Hz, H-30, H-50), 7.21 (1H, d, J ¼ 9.78 Hz, H-4), 2.36 (3H, s,
CH3). IR ymax (KBr): 3310 cmꢀ1 and 3160 cmꢀ1 (NH2), 1655 cmꢀ1
(C]O), 1584 cmꢀ1 (C]N), 1337 cmꢀ1 and 1165 cmꢀ1 (SO2N). FAB-
4.1.1. General procedure for the synthesis of 6-aryl-2-
benzenesulfonamide-pyridazinones (2aeh)
A mixture of appropriate b-aroylacrylic acid (1aeh) (0.001 mol)
and 4-hydrazinobenzenesulfonamide hydrochloride (0.001 mol) in
absolute ethanol (20e30 mL) was refluxed for 48 h. The solvent
ethyl alcohol was removed by distillation method. The solid residue
thus obtained was converted into fine powder, which was stirred
with 5% sodium bicarbonate solution (25 mL). It was filtered,
washed with 2% acetic acid and then with water. It was dried and
crystallized from methanol (2aec, 2eeh) or acetone (2d). The
purity of the compounds was checked on TLC plate (Silica gel G) in
the solvent system Toluene : Ethyl acetate : Formic acid (5 : 4 : 1).
MS (m/z): 341 [Mþ]. 13C NMR (75 MHz, DMSO,
d): 21.32 (CH3 at
C-40), 131.70 (C-5 pyridazinone), 131.81 (C-4 pyridazinone), 145.09
(C-6 pyridazinone), 159.04 (C-3 pyridazinone). Anal. Calcd for
C17H15N3O3S: C, 59.81; H, 4.43; N, 12.31; S, 9.39. Found: C, 59.69; H,
4.37; N, 12.22; S, 9.31.
4.1.1.4. 6-(4-Methoxyphenyl)-2-benzenesulfonamidepyridazine-
3(2H)one (2d). Shiny off white crystals (m.p. 290e291 ꢄC).Yield
48.6%. Rf ¼ 0.62. 1H NMR (DMSO-d6,
): 8.15 (1H, d, J ¼ 9.80 Hz,
d
The intermediates,
synthesis of pyridazinones were prepared through reported
procedures [23,24].
b-aroylacrylic acids (1aeh) required for the
H-5), 7.88e7.94 (6H, m, N-phenyl protons, H-20, H-60), 7.50 (2H, s,
SO2NH2), 7.19 (1H, d, J ¼ 9.78 Hz, H-4), 7.06 (2H, d, J ¼ 8.70 Hz, H-30,

308
I.G. Rathish et al. / European Journal of Medicinal Chemistry 49 (2012) 304e309
H-50), 3.82 (3H, s, OCH3). IR ymax (KBr): 3317 cmꢀ1 and 3294 cmꢀ1
(NH2), 1657 cmꢀ1 (C]O), 1599 cmꢀ1 (C]N), 1334 cmꢀ1 and
4.2. Pharmacology
1161 cmꢀ1 (SO2N), 1033 cmꢀ1 (OCH3). FAB-MS (m/z): 357 [Mþ]. 13
C
4.2.1. In vitro anticancer activity
NMR (75 MHz, DMSO,
d
): 55.77 (OCH3 at C-40), 131.62 (C-5 pyr-
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
idazinone), 131.95 (C-4 pyridazinone), 144.92 (C-6 pyridazinone),
158.94 (C-3 pyridazinone). Anal. Calcd for C17H15N3O4S: C, 57.13; H,
4.23; N, 11.76; S, 8.97. Found: C 57.09; H, 4.31; N, 11.62; S, 8.89.
serum and 2 mM L-glutamine. The tumor cells are inoculated over
4.1.1.5. 6-(4-Chloro-3-methylphenyl)-2-benzenesulfonamidepyrida-
a series of standard 96-well microtrite plates in 100 mL of medium
[29,30]. Density of inoculum depends on the type of tumor cell and
from its growth characteristics [27]. These cells are then pre-
incubated on the microtrite plate for 24 h before adding the
compounds. These were tested in DMSO solution at five different
concentrations (10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7 and 10ꢀ8 M). After an incu-
bation of the chemical agent for 48 h with the tumor cell lines
a sulforhodamine 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 [31].
zine-3(2H)-one (2e). Shiny off white crystals (m.p. 289e290ꢄꢄC).
Yield 57.8%. Rf ¼ 0.77. 1H NMR (DMSO-d6,
d): 8.19 (1H, d,
J ¼ 9.60 Hz, H-5), 7.81e7.98 (5H, m, N-phenyl protons and H-20), 7.80
(1H, d, J ¼ 8.33 Hz, H-60), 7.54 (3H, SO2NH2 and H-50), 7.25 (1H, d,
J ¼ 9.75 Hz, H-4), 2.40 (3H, s, CH3). IR ymax (KBr): 3302 cmꢀ1 and
3164 cmꢀ1 (NH2), 1651 cmꢀ1 (C]O), 1584 cmꢀ1 (C]N), 1343 cmꢀ1
and 1164 cmꢀ1 (SO2N). FAB-MS (m/z): 375 [Mþ], 376 [Mþ1], 377
[Mþ2], 378 [Mþ3]. 13C NMR (75 MHz, DMSO,
d
): 20.04 (CH3 at C-40),
132.40 (C-5 pyridazinone), 133.86 (C-4 pyridazinone), 154.35 (C-6
pyridazinone), 159.86 (C-3 pyridazinone). Anal. Calcd for
C17H14ClN3O3S: C, 54.33; H, 3.75; N, 11.18; S ¼ 8.53. Found: C, 54.19;
H, 3.61; N, 11.10; S, 8.41.
4.2.2. Acute toxicity determination
The determination of maximum tolerated dose (MTD) is per-
formed in a way that conserves compound and minimizes the
number of animals sacrificed. Thus, a single mouse is given
a single injection (IP) of 400 mg/kg; a second mouse receives
a dose of.
200 mg/kg and a third mouse receive a single dose of 100 mg/kg.
The mice are observed for a period of 2 weeks. They are sacrificed if
they lose more than 20% of their body weight or if there are other
signs of significant toxicity. If all 3 mice must be sacrificed, the next
3 dose levels (50, 35 and 12.5 mg/kg) are tested in a similar manner.
This process is repeated until a tolerated dose is found. This dose is
then designated the MTD and is used to calculate the amount of
material administered to mice during antitumor testing. The mice
are allowed ad libitum feed and water. Injections are administered
IP. The compounds are solubilized in DMSO and dose volumes were
4.1.1.6. 6-(2,4-dimethylphenyl)-2-benzenesulfonamidepyridazine-
3(2H)-one (2f). Shiny off white crystals (m.p. 230e231 ꢄC). Yield
40.2%. Rf ¼ 0.73. 1H NMR (DMSO-d6,
): 7.95 (2H, d, J ¼ 7.86 Hz, H-
d
200, H-600), 7.85 (2H, d, J ¼ 7.95 Hz, H-300, H-500), 7.76 (1H, d,
J ¼ 9.56 Hz, H-5), 7.49 (2H, s, SO2NH2), 7.38 (1H, d, J ¼ 9.56 Hz, H-
4), 7.14e7.15 (3H, m, H-30, H-50, H-60), 2.32 (3H, s, CH3), 2.34 (3H,
s, CH3). IR ymax (KBr): 3311 cmꢀ1 and 3161 cmꢀ1 (NH2), 1654 cmꢀ1
(C]O), 1580 cmꢀ1 (C]N), 1343 cmꢀ1 and 1166 cmꢀ1 (SO2N).FAB-
MS (m/z): 355 [Mþ]. 13C NMR (75 MHz, DMSO,
d): 20.81 (CH3 at C-
20), 21.42 (CH3 at C-40), 133.46 (C-4 pyridazinone), 134.20 (C-5
pyridazinone), 151.40 (C-6 pyridazinone), 157.78 (C-3 pyr-
idazinone). Anal. Calcd for C18H17N3O3S: C, 60.83; H, 4.82; N,
11.82; S ¼ 9.02. Found: C, 60.75; H, 4.77; N, 11.75; S, 9.10.
4.1.1.7. 6-Biphenyl-2-benzenesulfonamidepyridazine-3(2H)-one (2g).
Shiny off white crystals (m.p. 272e273 ꢄC). Yield 45.7%. Rf ¼ 0.55.
5 mL, 2.5 mL and 1.25 mL/g of body weight.
1H NMR (DMSO-d6,
d
): 7.33 (1H, d, J ¼ 9.72 Hz, H-4), 7.46e7.61 (5H,
4.2.3. Hollow fiber assay
m, SO2NH2, H-90, H-100, H-110), 7.82 (2H, d, J ¼ 7.83 Hz, H-80, H-120),
7.92 (2H, d, J ¼ 8.13 Hz, H-30, H-50), 8.02 (2H, d, J ¼ 8.66 Hz, H-300, H-
500), 8.06 (1H, d, J ¼ 8.47 Hz, H-200, H-600). 8.13 (2H, d, J ¼ 8.08 Hz, H-
20, H-60). IR ymax (KBr): 3311 cmꢀ1 and 3161 cmꢀ1 (NH2), 1654 cmꢀ1
(C]O), 1579 cmꢀ1 (C]N), 1336 cmꢀ1 and 1165 cmꢀ1 (SO2N). IR
ymax (KBr): 3311 cmꢀ1 and 3161 cmꢀ1 (NH2), 1654 cmꢀ1 (C]O),
1579 cmꢀ1 (C]N), 1336 cmꢀ1 and 1165 cmꢀ1 (SO2N). FAB-MS (m/
Human tumor cells were cultivated in polyvinylidene fluoride
hollow fibers, and a sample of each cell line was implanted into
each of two physiologic compartments (intraperitoneal and
subcutaneous) in mice. After treatment with 2h at two test doses
using a QD X 4 schedule, fiber cultures were collected and the viable
cell mass was determined using a formazan dye conversion assay. A
scoring system was developed to simplify evaluation of the results.
The cell lines used were: MDA-MB-231 and MDA-MB-435 (breast
cancer), NCI-H23 and NCI-H522 (lung cancer), OVCAR-3 and
OVCAR-5 (ovarian cancer), SF-295 and U-251 (CNS cancer), LOX
IMVI and UACC-62 (melanoma), COLO 205 and SW-620 (colon
cancer).
z): 403 [Mþ], 404 [Mþ1]. 13C NMR (75 MHz, DMSO,
d): 131.80 (C-5
pyridazinone), 133.84 (C-4 pyridazinone), 153.80 (C-6 pyr-
idazinone), 159.4 (C-3 pyridazinone). Anal. Calcd for C22H17N3O3S:
C, 65.49; H, 4.25; N, 10.42; S ¼ 7.95. Found: C, 65.39; H, 4.17; N,
10.22; S, 7.80.
4.1.1.8. 6-(4-Ethylphenyl)-2-benzenesulfonamidepyridazine-3(2H)-
Acknowledgment
one (2h). Off white crystals (m.p. 275e276 ꢄC). Yield 72.8%. Rf ¼ 0.7.
1H NMR (DMSO-d6,
d
): 8.15 (1H, d, J ¼ 9.76 Hz, H-5), 7.84e7.98 (6H,
This work was supported by Grant No. 32-228/2006 (SR) from
the University Grants Commission, New Delhi, India. One of the
authors, I. G. Rathish is thankful to UGC for fellowship. We thank
the Antitumor Evaluation Branch of the National Cancer Institute
for performing biological evaluations.
m, N-phenyl protons, H-20, H-60), 7.50 (2H, s, SO2NH2), 7.34 (2H, d,
J ¼ 7.73 Hz, H-30, H-50), 7.21 (1H, d, J ¼ 9.76 Hz, H-4), 2.65 (2H, q,
CH2), 1.20 (3H, t, CH3). IR ymax (KBr): 3313 cmꢀ1 and 3299 cmꢀ1
(NH2), 1654 cmꢀ1 (C]O), 1596 cmꢀ1 (C]N), 1336 cmꢀ1 and
1163 cmꢀ1 (SO2N). FAB-MS (m/z): 355 [Mþ], 356 [Mþ1]. 13C NMR
(75 MHz, DMSO, d
): 16.00 (CH3eCH2e at C-40), 21.42 (CH3eCH2e at
C-40), 131.86 (C-5 pyridazinone), 132.01 (C-4 pyridazinone), 146.19
(C-6 pyridazinone), 159.04 (C-3 pyridazinone). Anal. Calcd for
C18H17N3O3S: C, 60.83; H, 4.82; N, 11.82; S, 9.02. Found: C, 60.53; H,
4.77; N, 11.72; S, 8.92.
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2012.01.026.

I.G. Rathish et al. / European Journal of Medicinal Chemistry 49 (2012) 304e309
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