Synthesis and biological evaluation of 4-arylphthalazones bearing benzenesulfonamide as anti-inflammatory and anti-cancer agents

Article abstract of DOI:10.1002/ardp.201300056

Nine 4-arylphthalazones bearing benzenesulfonamide (2a-i) were synthesized by the condensation of the appropriate 2-aroylbenzoic acid (1a-i) and 4-hydrazinobenzenesulfonamide in ethanol. The structures of these compounds were elucidated by elemental analysis, IR, ¹H NMR, ¹³C NMR, and MS spectroscopy. Two compounds, 2b and 2i, showed significant anti-inflammatory activity comparable to that of the standard drug celecoxib in the carrageenan-induced rat paw edema model. These compounds (2b and 2i) had selective inhibitory activity towards the COX-2 enzyme. Compound 2b had a better selectivity ratio (COX-1/COX-2) compared to that of celecoxib and can be used as a novel template for the design of selective COX-2 inhibitors. Compounds 2d and 2i were screened for their antiproliferative activity toward 60 human cancer cell lines by the National Cancer Institute (USA). The compounds 2d and 2i displayed mild activity toward the renal cancer cell line UO-31. Nine 4-arylphthalazones bearing benzenesulfonamide (2a-i) were synthesized by the condensation of the appropriate 2-aroylbenzoic acid (1a-i) and 4-hydrazinobenzenesulfonamide in ethanol. Compounds 2b and 2i showed anti-inflammatory activity comparable to that of celecoxib in the carrageenan-induced rat paw edema model. Compounds 2d and 2i were screened for their antiproliferative activity towards 60 human cancer cell lines, displaying mild activity toward the renal cancer cell line UO-31. Copyright

Full text of DOI:10.1002/ardp.201300056

Arch. Pharm. Chem. Life Sci. 2013, 346, 491–498
491
Full Paper
Synthesis and Biological Evaluation of 4-Arylphthalazones
Bearing Benzenesulfonamide as Anti-Inflammatory and
Anti-Cancer Agents
Shafiya Yaseen1, Syed Ovais1, Rafia Bashir1, Pooja Rathore1, Mohammed Samim1,
Surender Singh2, Vinod Nair2, and Kalim Javed1
1 Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India
2 Department of Pharmacology, All India Institute of Medical Science, New Delhi, India
Nine 4-arylphthalazones bearing benzenesulfonamide (2a–i) were synthesized by the condensation of the
appropriate 2-aroylbenzoic acid (1a–i) and 4-hydrazinobenzenesulfonamide in ethanol. The structures of
these compounds were elucidated by elemental analysis, IR, 1H NMR, 13C NMR, and MS spectroscopy. Two
compounds, 2b and 2i, showed significant anti-inflammatory activity comparable to that of the standard
drug celecoxib in the carrageenan-induced rat paw edema model. These compounds (2b and 2i) had
selective inhibitory activity towards the COX-2 enzyme. Compound 2b had a better selectivity ratio (COX-
1/COX-2) compared to that of celecoxib and can be used as a novel template for the design of selective
COX-2 inhibitors. Compounds 2d and 2i were screened for their antiproliferative activity towards 60
human cancer cell lines by the National Cancer Institute (USA). The compounds 2d and 2i displayed mild
activity toward the renal cancer cell line UO-31.
Keywords: Anti-cancer / Anti-inflammatory / COX-2 inhibitor / Phthalazone / Sulfonamides
Received: February 11, 2013; Revised: March 21, 2013; Accepted: March 28, 2013
DOI 10.1002/ardp.201300056
Introduction
a rationale for developing anti-inflammatory and analgesic
agents that lack the GI liabilities. Unfortunately, some
NSAIDS including selective COX-2 inhibitors have been
shown to be associated with cardiovascular events, thus it
has become a challenge to explore novel NSAIDs with
reduced gastrointestinal tract and cardiovascular side effects.
In the last few years, attention was oriented towards the
synthesis and biological evaluation of phthalazine deriva-
tives as they exhibit a broad spectrum of biological activities
(Fig. 1). These derivatives have been reported to possess anti-
hypertensive [1, 2], anticonvulsant [3, 4], antidiabetic [5, 6],
antimicrobial [7], antitrypanosomal [8], anti-inflammatory
[9–11], and PDE4 inhibitory activities [12]. The phthalazinone
nucleus has been proven to be a versatile system in medicinal
chemistry. Some of the phthalazinone derivatives like hydra-
lazine [13], budralazine [14], azelastine [15], ponalrestat [16],
and zopolrestat [17] have found application in clinical
medicine. Recently, several groups have studied the
structure-activity relationship of novel series of 4-arylsubsti-
tuted phthalazinones and showed that the presence of 4-aryl
substituted on the phthalazinone nucleus contributes to
good anti-inflammatory and antinociceptive activities [18].
Inflammation is the first response of the body to infection,
irritation or other injuries. It is an essential process to
neutralize aggressor agents and to repair damaged tissues,
assuring this way the survival of the host. Unfortunately, the
available anti-inflammatory treatments are not always suffi-
ciently effective and frequently present numerous and severe
side effects especially in long-term use. Most of the current
non-steroidal anti-inflammatory drugs (NSAIDs) show serious
side effects including gastrointestinal disorders and kidney
damage.
NSAIDs exert their side effects by inhibition of the COX-1
enzyme. Since the COX-1 isoform is the constitutive one that
is responsible for regulation of physiological processes and
the COX-2 isoform is discovered to be the enzyme induced by
inflammatory stimuli, selective inhibition of COX-2 provides
Correspondence: Kalim Javed, Department of Chemistry, Faculty of
Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
E-mail: kjaved@jamiahamdard.ac.in
Fax: þ91-11-26059666
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4-aryl substitution of phthalazone nucleus:
A structural requirement for potent PDE4 inhibition
(Van der Mey et al., 2001)
MY5445 classical PDE inhibitor
Hagiwara et al.,1984
R1
NH
O
N N
N N
H
Cl
R
Phthalazine based PDE4 inhibitor, Haack et al., 2005
O
O
_
Target compounds (2a i)
N N
Cl
R
N
Cl
N
SO2NH2
N
Vatalanib (PTK787): A VEGFR inhibitor currently in
phase III clinical trials for metastatic colorectal cancer,
Scott et al., 2007
O
N
N
H
N
F
N
O
O
NH
Olaparib:
A
potent
PARP-1
and
PARP-2
inhibitor
N N
currently undergoing clinical development for the treatment
of
BRCA1/2-defective
breast,
ovarian,
pancreatic
N
and colorectal tumors and melanoma. Vasiliou et al., 2009
Cl
Figure 1. Structures of some biologically active phthalazine pharmacophores and the target compounds [18, 22, 23, 46, 47].
Substitution with acetamide derivative on the 2 position
(the lactam nitrogen) in the 4-arylphthalazinone derivatives
resulted in compounds having considerably high antinoci-
ceptive and anti-inflammatory activities [9, 19]. A large num-
ber of phthalazine derivatives have been studied for their
antitumor activities [20–22]. Vatalanib (PTK-787), substituted
phthalazine, is a VEGFR (vascular endothelial growth factor
receptor) inhibitor. It is currently in phase III clinical trials
for metastatic colorectal cancer [23].
In view of the aforementioned facts, it was thought worth-
while to incorporate the benzenesulfonamide moiety in the
2 position of some 4-arylphthalazone derivatives in the hope to
yield safe and potent compounds with anti-inflammatory and
anti-cancer potential. This paper describes the synthesis, in vivo
anti-inflammatory and in vitro anticancer activities of some
new 4-arylphthalazone molecules (2a–i). Compounds showing
maximum anti-inflammatory activity were also evaluated for
in vitro COX activity and ulcerogenic potential in rats.
The sulfonamides constitute an important class of drugs,
with several types possessing a number of biological proper- Results and discussion
ties including antibacterial [24], anti-carbonic anhydrase [25,
26], diuretic [25, 27], hypoglycemic [28], antithyroid [29], and
antiprotons activities [30–32]. The SO2NH2 pharmacophore is
believed to induce COX-2 selectivity [33].
Chemistry
The synthesis of the title compounds (2a–i) was carried
out by the condensation of appropriate 2-aroylbenzoic acid
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Arch. Pharm. Chem. Life Sci. 2013, 346, 491–498
4-Arylphthalazones as Anti-Inflammatory Agents
493
R1
R2
a
Appropriate aromatic
hydrocarbon
O
O
+
O
O
O
OH
Phthalic anhydride
_
1a i
R2
5'
6
R1
4'
3'
6'
5
7
8
+
HN
.HCl
NH2
4
1'
2'
3
N
1
b
O
N
2
1''
2''
3''
6''
5''
O
S
O
4''
NH2
4-Hydrazinobenzenesulfonamide
hydrochloride
O
S
O
NH2
_
2a i
1a, 2a: R1 = R2 = H
1f, 2f: R1 = Phenyl, R2 = H
1g, 2g: R1 = Methoxy, R2 = H
1h, 2h: R1 = Phenoxy, R2 = H
1i, 2i: R1 = R2 = Methoxy
1b, 2b: R1 = Methyl, R2 = H
1c, 2c: R1 = Ethyl, R2 = H
1d, 2d: R1 = Chloro, R2 = H
1e, 2e: R1 = Bromo, R2 = H
Reagents and conditions: (a) anhydrous AlCl3, room temperature; (b) absolute alcohol,
reflux.
Scheme 1. Synthesis of 4-arylphthalazones bearing benzenesulfonamide, 2a–i.
(1a–i) with 4-hydrazinobenzenesulfonamide in ethanol
(Scheme 1). The intermediates (1a–i) were prepared by
Friedel–Crafts method wherein the appropriate aromatic
hydrocarbon was reacted with phthalic anhydride using
anhydrous AlCl3 as catalyst in nitrobenzene [34, 35]. A
literature survey revealed that 2a, 2g, and 2i were
registered with CAS numbers 847734-10-5, 735324-06-8,
and 852904-89-3, respectively. But no reference was
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available for work on its method of synthesis and bio-
logical activity.
to significant reduction in the activity (2a vs. 2c, 2a vs. 2e, 2a
vs. 2h, respectively). Introduction of methyl (at C-40) or two
methoxyl groups (at C-40 and C-30) significantly influenced the
anti-inflammatory activity (2a vs. 2b, 2a vs. 2i) at both study
times 3 and 5 h.
The structures of the synthesized compounds were con-
1
firmed by elemental analysis and spectral data (IR, H NMR,
13C NMR and mass). Elemental analysis (C, H, N and S) data
were within ꢀ0.4% of the theoretical values. In the IR spectra
of 2a–i, six bands characteristic of the phthalazone moiety
out of which two bands for NH (3339–3162 cmꢁ1, 3173–
3052 cmꢁ1), one band for the carbonyl group of the phthal-
azone moiety at 1714–1635 cmꢁ1, one band for C¼N of
the phthalazone ring (1591–1520 cmꢁ1) and two bands
for SO2N (1380–1338 cmꢁ1 and 1191–1143 cmꢁ1) were
observed. In the 1H NMR spectra of 2a–i a two-proton singlet
for –SO2NH2 was observed at d 4.93–7.47. Phthalazinone,
4-aryl and N-phenyl ring protons appeared in the aromatic
region (d 7.10–8.63).
Two compounds (2b and 2i) showing maximum anti-
inflammatory activity were evaluated for their ulcerogenic
potential in rats according to Daidone et al. [37]. The com-
pounds (2b and 2i) did not show any ulceration or harmful
effects on the stomach at a dose of 0.25 mmol/kg po, when
administered twice at 2 h interval in fasted rats [32].
These two compounds (2b and 2i) were also evaluated for
their ability to inhibit COX-2 and COX-1 by in vitro colorimet-
ric COX (ovine) inhibitor assay method [38], which utilizes the
peroxidase component of cyclooxygenase. The peroxidase
activity is assayed colorimetrically by monitoring the appear-
ance of oxidized N,N,N0N0-tetramethyl-p-phenylene diamine
(TMPD) during the reduction of PGG2 to PGH2, at 590 nm.
The IC50 values for 2b and 2i were calculated using non-linear
regression analysis. The results of the in vitro COX enzyme
inhibition assay are summarized in Table 2. The results
showed that both compounds (2b and 2i) exhibited effective
inhibition against COX-2, compared to the inhibition of
COX-1. To assess the selectivity profiles of the tested com-
pounds, their approximate selectivity ratios (COX-1/COX-2)
were compared to that of the standard COX-2 selective inhibi-
tor, celecoxib. The compound 2b showed better selectivity
compared to that of celecoxib.
Biological results and discussion
The in vivo anti-inflammatory activities for all synthesized
compounds 2a–i (at 0.05 mmol/kg dose) were evaluated using
carrageenan-induced rat paw edema model by adopting the
reported method of Winter et al. [36]. These derivatives (2a–i)
showed mild to strong anti-inflammatory activity (inhibition
percentages are 20.0–85.8% and 36.4–85.8% at 3 and 5 h,
respectively). Among them, two compounds 2b and 2i have
significant activity, which is comparable to that of celecoxib
(Table 1). Introduction of chloro or phenyl group at para
position (C-40) of phenyl ring attached with C-4 of phthalazi-
none nucleus leads to significant increase in the activity at
3 h (2a vs. 2d and 2a vs. 2f, respectively). On the other hand,
introduction of bromo, ethyl or phenoxy group at C-40 leads
The structures of the synthesized compounds (2a–i) were
submitted to the National Cancer Institute (NCI), Bethesda,
Maryland, USA, for their anti-proliferative activity. Only two
compounds (2d and 2i) were selected by NCI on the basis of
degree of structural variation and computer modeling
techniques. These compounds (2d and 2i) were granted
NSC codes, viz. NSC 750151 and NSC 750152, respectively.
The selected compounds were subjected to in vitro anticancer
assay against tumor cells in a full panel of 60 cell lines taken
from nine different tissues (blood, lung, colon, CNS, skin,
ovary, kidney, prostate, and breast) at a concentration
Table 1. Effect of phthalazone derivatives (2a–i) at 0.05 mmol/kg
in carageenan-induced rat paw edema assay.
Treatment
Increase in paw volume (mL) after
carageenan administrationa)
3 h
5 h
Vehicle
Celecoxib
0.325 ꢀ 0.05
0.425 ꢀ 0.05
0.06 ꢀ 0.02 (81.5%)b)
0.13 ꢀ 0.03 (60.0%)
0.05 ꢀ 0.02 (84.6%)
0.18 ꢀ 0.05 (44.6%)
0.07 ꢀ 0.02 (78.4%)
0.20 ꢀ 0.06 (38.4%)
0.07 ꢀ 0.07 (78.4%)
0.12 ꢀ 0.06 (63.0%)
0.26 ꢀ 0.06 (20.0%)
0.046 ꢀ 0.025 (85.8%)
0.083 ꢀ 0.02 (80.4%)
0.16 ꢀ 0.03 (62.3%)
0.06 ꢀ 0.02 (85.8%)
0.18 ꢀ 0.05 (57.6%)
0.13 ꢀ 0.03 (69.4%)
0.27 ꢀ 0.07 (36.4%)
0.15 ꢀ 0.05 (64.7%)
0.17 ꢀ 0.05 (60.0%)
0.22 ꢀ 0.02 (48.2%)
0.08 ꢀ 0.04 (81.1%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
Table 2. In vitro COX enzyme inhibition data for compounds 2b
and 2i.
Compound
IC50 (mM)a)
Selectivity
index
COX-1/COX-2
COX-1
COX-2
2b
2i
Celecoxib
6.0
7.0
6.5
0.018
0.068
0.020
333.33
102.94
325.00
All data are significantly different compared to respective
control values, p < 0.05.
a)
Values are acquired using an ovine COXs assay kit (Catalog No.
760111, Cyman Chemical Inc., Ann Arbor, MI). Experiments were
carried out in duplicate and have <10% error.
a)
Values are expressed as mean ꢀ SEM and analyzed by ANOVA.
b)
Values in parenthesis (percent inhibition).
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4-Arylphthalazones as Anti-Inflammatory Agents
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Table 3. Anticancer screening data for 2d and 2i on 60 cell lines at a concentration of 10ꢁ5 M.
Compound
Code
Mean
growth (%)
Most sensitive
cell line
Growth of most
sensitive cell line (%)
2d
2i
NSC: 750151
NSC: 750152
97.72
102.57
UO-31 (renal cancer)
UO-31 (renal cancer)
52.56
58.88
of 10ꢁ5 M. These two compounds 2d and 2i displayed mild
portions with continuous stirring. Vigorous reaction started
with evolution of HCl gas. Stirring was further continued for
6 h at room temperature. It was left at room temperature for
48 h and then decomposed by adding ice-cold hydrochloric
acid (50%, 100 mL). The excess solvent was removed by steam
distillation. It was allowed to cool to room temperature. The
crude product was dissolved in 5% sodium carbonate solution
and was then filtered. The filtrate so obtained was acidified with
dilute HCl to give a precipitate. It was then filtered, washed
with cold water, dried and crystallized from ethyl alcohol to
give 2-aroylbenzoic acids (1a–i). The melting point of the
compounds (1a–i) was compared to those already mentioned
in the literature [34, 35].
sensitivity towards the renal cancer cell line UO-31 (Table 3).
Conclusion
Cyclocondensation
of 4-hydrazinobenzenesulfonamide
hydrochloride with aroyl benzoic acids yielded respective
phthalazone derivatives (2a–i). The structures proposed to
the synthesized compounds (2a–i) are well supported by
elemental analysis and spectroscopic data (IR, 1H NMR,
13C NMR and mass). Two compounds 2b and 2i showed
significant anti-inflammatory activity, which is comparable
to that of the standard drug celecoxib. These compounds
(2b and 2i) had selective inhibitory activity towards the
COX-2 enzyme. One of these derivatives (2b) had a better
selectivity ratio (COX-1/COX-2) compared to that of celecoxib
and can be used as a new template for the design of selective
COX-2 inhibitors. The compounds 2d and 2i displayed mild
sensitivity toward the renal cancer cell line UO-31.
General procedure for the preparation of 4-arylphthalazone
derivatives (2a–i)
On complete dissolution of 4-hydrazinobenzenesulfonamide
hydrochloride (0.001 mole) in absolute ethanol (25 mL), desired
2-aroylbenzoic acid was added (0.001 mole) and the mixture was
heated to reflux for 18–24 h. The volume was reduced to half by
evaporating the solvent and left at room temperature till the
solid product separated out. It was filtered and then converted
into fine powder, which was stirred with 5% sodium bicarbonate
solution (25 mL). It was filtered, washed with 2% HCl and then
with water. It was dried and crystallized with appropriate solvent
to give TLC pure compound.
Experimental
Chemistry
Melting points were determined by open capillary tubes and are
uncorrected. All the Fourier transform infrared (FTIR) spectra
were recorded on a Bruker Vector 22 spectrophotometer in KBr;
ymax values are given in cmꢁ1. 1H NMR spectra were recorded on
a Bruker Spectrospin DPX 300 MHz/400 MHz spectrometer using
deuterated DMSO or deuterated chloroform as solvent and tet-
ramethyl 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 ESI Bruker
Esquire 3000. The m/z values of the more intense peaks are
mentioned. 13C NMR spectra were recorded on Bruker spectro-
spin DPX 300 MHz using deuterated DMSO or deuterated chloro-
form as solvent and TMS as internal standard. 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).
4-(1-Oxo-4-phenylphthalazin-2(1H)-yl)-
benzenesulfonamide 2a
Yield ¼ 53%, m.p. 262–2638C, Rf ¼ 0.67 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3303 cmꢁ1 and 3127 cmꢁ1
(NH2), 1691 cmꢁ1 (C¼O), 1552 cmꢁ1 (C¼N), 1343 cmꢁ1 and
1173 cmꢁ1 (SO2N). 1H NMR (400 MHz, DMSO, d): 6.89 (2H,
s, SO2NH2), 7.46–7.56 (5H, m, H-20, H-30, H-40, H-50, H-60), 7.71–
7.79 (3H, m, H-6, H-7, H-8), 7.83–7.95 (4H, m, N-phenyl protons),
8.46–8.48 (1H, m, H-5). 13C NMR (300 MHz, DMSO, d): 126.1, 126.3,
126.9, 127.1, 128.0, 128.5, 128.6, 129.4, 129.5, 132.4, 134.2, 134.4,
142.7, 144.2, 147.4, 158.1. ESI-MS (m/z): 376 [Mꢁ1], 400 [MþNa].
Anal. calcd. for C20H15N3O3S: C 63.66, H 4.01, N 11.13, S 8.50.
Found: C 63.63, H 4.00, N 11.09, S 8.53.
4-[4-(4-Methylphenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2b
Yield ¼ 50%, m.p. 210–2118C, Rf ¼ 0.70 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3277 cmꢁ1 and 3052 cmꢁ1
(NH2), 1656 cmꢁ1 (C¼O), 1520 cmꢁ1 (C¼N), 1374 cmꢁ1 and
1143 cmꢁ1 (SO2N). 1H NMR (300 MHz, CDCl3, d): 2.31
(3H, s, CH3), 6.42 (2H, s, SO2NH2), 7.19 (2H, d, J ¼ 7.8 Hz, H-30,
H-50), 7.38 (2H, d, J ¼ 7.8 Hz, H-20, H-60), 7.67–7.71 (3H, m, H-6, H-7,
H-8), 7.79 (2H, d, J ¼ 8.7 Hz, H-300, H-500), 7.87 (2H, d, J ¼ 9.0 Hz,
H-2‘‘, H-600), 8.41–8.43 (1H, m, H-5). ESI-MS (m/z): 390 [Mꢁ1], 414
General procedure for the synthesis of aroyl benzoic acids
(1a–i)
To a slurry of anhydrous aluminum trichloride (13.2 g, 0.1 moles)
in nitrobenzene (30 mL), liquid aromatic hydrocarbon (0.05
moles) was added in portions while stirring on magnetic stirrer
at room temperature for 30 min to obtain a colored complex.
To it, phthalic anhydride (4.9 g, 0.05 moles) was added in five
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[MþNa]. Anal. calcd. for C21H17N3O3S: C 64.43, H 4.38, N 10.73,
4-[4-(4-Methoxyphenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2g
S 8.19. Found: C 64.32, H 4.42, N 10.89, S 8.12.
Yield ¼ 19.1%, m.p. 248–2498C, Rf ¼ 0.67 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3320 cmꢁ1 and 3130 cmꢁ1
(NH2), 1689 cmꢁ1 (C¼O), 1529 cmꢁ1 (C¼N), 1367 cmꢁ1 and
1191 cmꢁ1 (SO2N), 1046 cmꢁ1 (OCH3). 1H NMR (300 MHz,
DMSO, d): 3.85 (3H, s, OCH3), 7.13 (2H, d, J ¼ 8.4 Hz, H-30, H-50),
7.46 (2H, s, SO2NH2), 7.64 (2H, d, J ¼ 8.4 Hz, H-20, H-60), 7.79–7.99
(7H, m, H-200, H-300, H-500, H-600, H-6, H-7, H-8), 8.45–8.47 (1H, m, H-5).
13C NMR (300 MHz, DMSO, d): 54.4, 113.1, 124.7, 125.5, 125.9,
126.1, 126.5, 127.6, 128.0, 129.8, 130.9, 132.6, 141.4, 143.6, 146.9,
157.7, 159.4. ESI-MS (m/z): 406 [Mꢁ1], 430 [MþNa]. Anal. calcd.
for C21H17N3O4S: C 61.90, H 4.21, N 10.31, S 7.87. Found: C 61.82,
H 4.31, N 10.29, S 7.98.
4-[4-(4-Ethylphenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2c
Yield ¼ 30.5%, m.p. 214–2158C, Rf ¼ 0.78 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3217 cmꢁ1 and 3136 cmꢁ1
(NH2), 1635 cmꢁ1 (C¼O), 1583 cmꢁ1 (C¼N), 1338 cmꢁ1 and
1159 cmꢁ1 (SO2N). 1H NMR (300 MHz, CDCl3, d): 1.31 (3H, t,
CH3), 2.76 (2H, q, CH2), 5.05 (2H, s, SO2NH2), 7.37 (2H, d,
J ¼ 8.1 Hz, H-30, H-50), 7.56 (2H, d, J ¼ 8.1 Hz, H-20, H-60), 7.80
(3H, m, H-6, H-7, H-8), 7.98–8.02 (4H, m, N-phenyl protons), 8.59–
8.62 (1H, m, H-5). ESI-MS (m/z): 404 [Mꢁ1], 428 [MþNa]. Anal. calcd.
for C22H19N3O3S: C 65.17, H 4.72, N 10.36, S 7.91. Found: C 65.09,
H 4.81, N 10.42, S 7.89.
4-[4-(4-Phenoxyphenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2h
4-[4-(4-Chlorophenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2d
Yield ¼ 30.1%, m.p. 185–1868C, Rf ¼ 0.75 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3336 cmꢁ1 and 3127 cmꢁ1
(NH2), 1691 cmꢁ1 (C¼O), 1533 cmꢁ1 (C¼N), 1380 cmꢁ1 and
1180 cmꢁ1 (SO2N). 1H NMR (400 MHz, CDCl3, d): 4.93 (2H, s,
SO2NH2), 7.11–7.21 (5H, m, H-30, H-50, H-2‘‘0, H-5000 and H-4000),
7.39–7.43 (2H, m, H-3000, H-5000), 7.61 (2H, d, J ¼ 8.4 Hz, H-20,
H-60), 7.85–7.90 (3H, m, H-6, H-7 and H-8), 8.00–8.06 (4H, m,
N-phenyl protons), 8.61–8.63 (1H, m, H-5). 13C NMR (300 MHz,
DMSO, d): 117.6, 118.8, 123.4, 125.1, 125.9, 126.4, 127.0, 127.9,
128.2, 128.5, 129.3, 130.4, 131.3, 133.0, 141.6, 143.9, 147.0, 155.5,
157.9, 158.1. FAB-MS (m/z): 492 [MþNa], 300 [M–Ph–O–Ph]. Anal.
calcd. for C26H19N3O4S: C 66.51, H 4.08, N 8.95, S 6.83. Found: C
66.45, H 4.12, N 8.89, S 6.90.
Yield ¼ 50.1%, m.p. 242–2438C, Rf ¼ 0.77 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3315 cmꢁ1 and 3123 cmꢁ1
(NH2), 1695 cmꢁ1 (C¼O), 1591 cmꢁ1 (C¼N), 1372 cmꢁ1 and
1192 cmꢁ1 (SO2N). 1H NMR (300 MHz, DMSO, d): 7.46 (2H, s,
SO2NH2), 7.53–7.76 (4H, m, H-20, H-30, H-50, H-60), 7.89–7.99
(7H, m, N-phenyl protons, H-6, H-7, H-8), 8.47 (1H, m, H-5).
13C NMR (300 MHz, DMSO, d): 126.2, 126.3, 126.8, 127.1,
128.0, 128.3, 128.7, 131.4, 132.5, 133.3, 134.3, 142.8, 144.1,
146.4, 158.1. ESI-MS (m/z): 410 [Mꢁ1]. Anal. calcd. for
C20H14ClN3O3S: C 58.32, H 3.43, N 10.20, S 7.79. Found: C
58.23, H 3.49, N 10.14, S 7.73.
4-[4-(4-Bromophenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2e
4-[4-(3,4-Dimethoxyphenyl)-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2i
Yield ¼ 35.1%, m.p. 224–2258C, Rf ¼ 0.57 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3337 cmꢁ1 and 3123 cmꢁ1
(NH2), 1651 cmꢁ1 (C¼O), 1579 cmꢁ1 (C¼N), 1376 cmꢁ1 and
1161 cmꢁ1 (SO2N). 1H NMR (400 MHz, DMSO, d): 7.19
(2H, s, SO2NH2), 7.46–7.63 (4H, m, H-20, H-30, H-50 H-60), 7.81–
7.85 (5H, m, H-300, H-500, H-6, H-7, H-8), 7.91 (2H, d, J ¼ 8.4 Hz,
H-200, H-600), 8.42–8.44 (1H, m, H-5). 13C NMR (300 MHz,
DMSO, d): 123.0, 126.2, 126.3, 127.1, 128.0, 128.7, 129.5,
131.7, 132.5, 133.7, 134.4, 142.8, 144.2, 146.4, 147.5, 158.1.
ESI-MS (m/z): 453 [Mꢁ2], 478 [MþNa]. Anal. calcd. for
C20H14BrN3O3S: C 52.64, H 3.09, N 9.21, S 7.03. Found: C
52.59, H 3.13, N 9.19, S 7.10.
Yield ¼ 30.1%, m.p. 206–2078C, Rf ¼ 0.54 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3339 cmꢁ1 and 3173 cmꢁ1
(NH2), 1714 cmꢁ1 (C¼O), 1533 cmꢁ1 (C¼N), 1362 cmꢁ1
,
1157 cmꢁ1 (SO2N) and 1049 cmꢁ1 (OCH3). 1H NMR (300 MHz,
DMSO, d): 3.78 (3H, s, OCH3), 3.83 (3H, s, OCH3), 7.10–7.23
(3H, m, H-20, H-50, H-60), 7.47 (2H, s, SO2NH2), 7.81–
7.96 (7H, m, N-phenyl protons, H-6, H-7, H-8), 8.42–8.45
(1H, m, H-5). 13C NMR (300 MHz, DMSO, d): 55.6, 55.7, 111.6,
112.9, 122.2, 126.1, 126.3, 126.8, 127.0, 127.2, 127.9, 128.7,
132.3, 134.2, 142.7, 144.2, 147.4, 148.6, 149.7, 158.1. ESI-MS
(m/z): 436 [Mꢁ1]. Anal. calcd. for C22H19N3O5S: C 60.40, H 4.38,
N 9.61, S 7.33. Found: C 60.25, H 4.45, N 9.55, S 7.27.
4-[4-Biphenyl-1-oxophthalazin-2(1H)-yl]-
benzenesulfonamide 2f
Pharmacology
Yield ¼ 50.1%, m.p. 255–2568C, Rf ¼ 0.73 (toluene/ethyl acetate/
formic acid, 5:4:1). IR ymax (KBr): 3162 cmꢁ1 (NH2), 1652 cmꢁ1
(C¼O), 1583 cmꢁ1 (C¼N), 1341 cmꢁ1 and 1161 cmꢁ1 (SO2N).
1H NMR (400 MHz, DMSO, d): 7.04 (2H, s, SO2NH2), 7.28–
7.31 (1H, m, H-4000), 7.37–7.41 (2H, m, H-3000, H-5000), 7.58
(2H, d, J ¼ 7.6 Hz, H-2000, H-6000), 7.63–7.70 (4H, m, H-20, H-30,
H-50, H-60), 7.79–7.80 (3H, m, H-6, H-7, H-8), 7.84 (2H, d,
J ¼ 8.8 Hz, H-200, H-600), 7.92 (2H, d, J ¼ 8.4 Hz, H-300, H-500),
8.45–8.46 (1H, m, H-5). ESI-MS (m/z): 452 [Mꢁ1], 476 [MþNa].
Anal. calcd. for C26H19N3O3S: C 68.86, H 4.22, N 9.27, S 7.07.
Found: C 68.95, H 4.13, N 9.19, S 7.15.
Anti-inflammatory activity
In the present work, rats of the Wistar strain (either sex)
obtained from the central animal house of the university
were used. 1% Tween-10 in distilled water was used as vehicle
for dosing (10 mL/kg). All the treatments suspended in
vehicle were given orally.
The experiment was conducted in accordance with the
guidelines for the care and use of laboratory animals laid
down by the Committee for the Purpose of Control and
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 491–498
4-Arylphthalazones as Anti-Inflammatory Agents
497
USA, for carrying out the in vitro anticancer screening of the newly
synthesized compounds.
Supervision of Experiments on Animals (CPCSEA), Ministry
of Social Justice and Empowerment, Govt. of India.
The experiment was performed by the method of Winter
et al. [36]. Overnight fasted rats (150–200 g) were taken and
divided into groups of six animals each. Animals of group-I
were administered orally with vehicle (1% Tween-10, 10 mL/
kg) and served as control. Group II animals were fed with
celecoxib (0.05 mmol/kg b.w.) and served as standard. Test
groups were administered orally with respective test drugs
(2a–i) in the dose of 0.05 mmol/kg b.w. After 30 min, all
animals were injected with 0.1 mL of 1% carageenan solution
(prepared in normal saline) in the sub plantar aponeurosis of
left hind paw and the paw volume was measured by using a
plethysmometer at 0, 3, and 5 h post-carageenan treatment.
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:
The authors have declared no conflict of interest.
References
[1] S. Demirayak, A. Karaburun, R. Beis, Eur. J. Med. Chem. 2004,
39, 1089–1095.
[2] N. Watanabe, Y. Kabasawa, Y. Takase, M. Matsukura,
K. Miyazaki, H. Ishihara, J. Med. Chem. 1998, 41, 3367–3372.
[3] M. Kornet, G. Shackleford, J. Het. Chem. 1999, 36, 1095–1096.
[4] O. Nassar, Ind. J. Het. Chem. 1997, 7, 105–108.
[5] R. Madhavan, R. Chakrabarti, K. Kumar, P. Misra, N. Mamidi,
V. Balraju, Eur. J. Med. Chem. 2001, 36, 627–637.
[6] M. Lenz, D. Wilson, B. Wright, A. Partidge, T. Roddgers,
R. Haycock, J. Pharm. Biomed. Anal. 2002, 28, 31–43.
[7] C. Cardia, E. Distinto, E. Maccioni, A. Delogu, J. Het. Chem.
2003, 40, 1011.
ꢀ
ꢁ
Vt
Vc
Percent inhibition ¼ 1 ꢁ
ꢂ 100
[8] D. Olmo, G. Armas, J. Lopez-perez, G. Ruiz, F. Vargas,
A. Gimenez, Bioorg. Med. Chem. Lett. 2001, 11, 2755–2757.
where Vt is the mean paw volume of the test drug treated
rats and Vc is the mean paw volume of the control.
[9] S. Dogruer, E. Kupeli, E. Yesilada, F. Sahin, Arch. Pharm. 2004,
337, 303–310.
[10] M. Napoletano, G. Norcini, F. Pellacini, F. Marchini, G.
Moraazzoni, P. Ferlenga, Bioorg. Med. Chem. Lett. 2000, 10,
2235–2238.
Ulcerogenic activity
Two groups of six rats each weighing 180–200 g and fasted
for 24 h were used. The compounds 2a and 2i were given
orally at a dose of 0.25 mmol/ kg po, and administered twice
at 2 h interval. Rats were sacrificed by ether inhalation 6 h
after the first dose. Their stomachs were removed, opened
along the greater curvature and examined for the presence of
gastric ulcers or hyperemia [37].
[11] K. Chakraborti, B. Gopalakrishnan, E. Sobhia, A. Malde,
Bioorg. Med. Chem. Lett. 2003, 13, 2473–2479.
[12] K. Kagayama, T. Morinmoto, S. Nagata, F. Katoh, X. Zhang,
N. Inoue, Bioorg. Med. Chem. 2009, 17, 6959–6970.
[13] A. Kleemann, J. Engel, Pharmazeutische Wirkstoffe
Synthesen Patente Anwendungen, Thieme Stuttgart,
1982, Vol. 5.
[14] K. Ueno, R. Moroi, M. Kitagawa, K. Asano, S. Miyazaki, Chem.
Pharm. Bull. 1976, 24, 1068.
In vitro cylcooxygenase inhibition studies
[15] G. Scheffler, J. Engel, B. Kutscher, S. Sheldrick, P. Bell, Arch.
Pharm. 1998, 321, 205.
Two compounds (2b and 2i) were tested for their ability to
inhibit in vitro COX-1 and COX-2 using a colorimetric COX
(ovine) inhibitor screening kit (Catalog No. 760 111, Cayman
Chemicals Inc Ann Arbor, MI, USA) using the previously
established method [38].
[16] C. Laudadio, F. Sima, J. Diab. Comp. 1998, 12, 121–127.
[17] A. Beyer, J. Diecke, K. Mistry, E. Cruze, Pharmacology 1997, 54,
76–83.
[18] M. Van der Mey, A. Hatzelmann, P. Van Klink, J. Van der
Lann, J. Sterk, U. Thibaut, J. Med. Chem. 2001, 44, 2523–2535.
In vitro anticancer activity
[19] M. Abd alla, M. Hegab, N. Abo Taleb, M. Hasabelnaby,
A. Goudah, Eur. J. Med. Chem. 2010, 45, 1267–1277.
Primary in vitro one-dose (10ꢁ5 M) anticancer assay was per-
formed using full panel of about 60 human tumor cell lines
in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute (NCI), Bethesda, and
described elsewhere [39–45]. The human tumor cell lines
were derived from nine different cancer types: leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate, and
breast cancers.
[20] K. A. Menear, C. Adcock, F. C. Alonso, Bioorg. Med. Chem. Lett.
2008, 18, 3942–3945.
[21] X. Cockcroft, K. J. Dillon, L. Dixon, Bioorg. Med. Chem. Lett.
2006, 16, 1040–1044.
[22] S. Vasiliou, R. Castaner, J. Bolos, Drugs Future 2009, 34, 101–
105.
[23] E. N. Scott, G. Meinhardt, C. Jacques, Expert. Opin. Investig.
Drugs 2007, 16, 367–379.
[24] J. Drew, Science 2000, 287, 1960.
One of the authors (S.Y.) is thankful to DST for an INSPIRE fellowship. The
authors are very grateful to the staff members of Department of Health and
Human Services, National Cancer Institute (NCI), Bethesda, Maryland,
[25] T. Supuran, A. Scozzafava, Exp. Opin. Ther. Patents 2000, 10,
575–600.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

498
S. Yaseen et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 491–498
[26] T. Supuran, A. Scozzafava, Curr. Med. Chem. Immunol. Endoc.
Metab. Agents 2001, 1, 61–97.
[39] M. R. Boyd, Am. Assoc. Cancer Res. 1989, 30, 652–663.
[40] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull,
D. Vistica, C. Hose, J. Langley, P. Cronise, A. V. Wolff, M. G.
Goodrich, H. Campbell, J. Mayo, M. J. Boyd, Natl. Cancer Inst.
1991, 83, 757–766.
[27] H. Maren, Ann. Rev. Pharmacol. Toxicol. 1976, 16, 309.
[28] E. Boyd, Diabetes 1988, 37, 847–850.
[29] C. Thornber, Chem. Soc. Rev. 1979, 8, 563–580.
[41] J. N. Weinstein, T. G. Myers, P. M. O. Connor, S. H. Friend, A. J.
Fornance, Jr, K. W. Kohn, T. Fojo, S. E. Bates, L. V. Rubinstein,
N. L. Anderson, J. K. Buolamwini, W. W. V. Osdol, A. P.
Monks, D. A. Scudiero, E. A. Sausville, D. W. Zaharevitz,
B. Bunow, V. N. Viswanadhan, G. S. Johnson, R. E. Wittes,
K. D. Paull, Science 1997, 275, 343–349.
[30] C. Ogden, W. Flexner, Protease Inhibitors in AIDS Therapy,
Marcel Dekker, New York, Basel 2001.
[31] T. Supuran, A. Scozzafava, A. Mastrolorenzo, Exp. Opin. Ther.
Patents 2001, 11, 221–259.
[32] A. Scozzafava, T. Supuran, J. Med. Chem. 2000, 43, 3677–
3687.
[42] M. G. Hollingshead, M. C. Alley, R. F. Camalier, B. J. Abbott,
J. G. Mayo, L. Malspeis, M. R. Grever, Life Sci. 1995, 57, 131–
141.
[33] G. Amgad, N. Habeeb, R. Parveen, K. Edward, J. Med. Chem.
2001, 44, 3039–3042.
[43] M. R. Grever, S. A. Schepartz, B. A. Chabner, Semin. Oncol.
1992, 19, 622–638.
[34] A. F. M. Fahmy, S. A. Essawy, Egypt. J. Chem. 1976, 19 (6), 937–
944.
[44] M. R. Boyd, K. D. Paull, Drug Dev. Res. 1995, 34, 91–109.
[45] M. R. Boyd, Princ. Pract. Oncol. 1989, 3, 1–12.
[35] E. P. William, K. B. Charles, J. E. Kevin, J. Org. Chem. 1981, 46,
1057–1061.
[36] C. Winter, A. Risley, G. Nuss, Proc. Soc. Exp. Biol. Med. 1962, 111,
544–547.
[46] M. Hagiwara, T. Endo, T. Kanayama, H. Hidaka, J. Pharmacol.
Exp. Ther. 1984, 228, 467–471.
[37] G. Daidone, B. Maggio, D. Raffa, Eur. J. Med. Chem. 1994, 29,
707–711.
[47] T. Haack, R. Fattori, M. Napoletano, F. Pellacini, G. Fronza,
G. Raffaini, F. Ganazzoli, Bioorg. Med. Chem. 2005, 13, 4425–
4433.
[38] R. Kulmacz, M. Lands, Prostaglandins 1983, 25, 531–540.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com