Synthesis and Biological Evaluation of New Phthalazinone Derivatives as Anti-Inflammatory and Anti-Proliferative Agents

Article abstract of DOI:10.1002/ardp.201500336

The chemistry of phthalazine derivatives has been of increasing interest since many of these compounds have found many chemotherapeutic applications. So this study aims to synthesize a library of phthalazine derivatives and to investigate their anti-inflammatory and anti-proliferative activities. Sixteen new phthalazinone derivatives (2a-p) were synthesized and tested for their in vitro antiproliferative and in vivo anti-inflammatory activities. All the synthesized compounds were identified and characterized by IR, ¹H NMR, ¹³C NMR spectroscopy, and MS. Two compounds, 2b and 2i, showed significant anti-inflammatory activity comparable with that of the standard drug etoricoxib in the carrageenan-induced rat paw edema model at 3 and 5 h, respectively. Three compounds (2h, 2j, and 2g) showed moderate sensitivity toward the renal cancer cell line UO-31. A library of new phthalazone compounds (2a-p) was synthesized as dual inhibitors (COX-2/LOX-5) and evaluated for their anti-inflammatory, anticancer activities. Two compounds showed significant anti-inflammatory activity comparable with that of the standard drug etoricoxib, whereas three compounds showed moderate sensitivity toward the renal cancer cell line UO-31.

Full text of DOI:10.1002/ardp.201500336

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Arch. Pharm. Chem. Life Sci. 2016, 349, 150–159
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Full Paper
Synthesis and Biological Evaluation of New Phthalazinone
Derivatives as Anti-Inflammatory and Anti-Proliferative Agents
Alhamzah Dh. Hameed1, Syed Ovais1, Raed Yaseen1, Pooja Rathore1, Mohammed Samim1,
Surender Singh2, Kalicharan Sharma3, Mymona Akhtar3, and Kalim Javed1
1
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India
Department of Pharmacology, All India Institute of Medical Science, New Delhi, India
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Drug Design and Medicinal Chemistry
Laboratory, Jamia Hamdard (Hamdard University), New Delhi, India
2
3
The chemistry of phthalazine derivatives has been of increasing interest since many of these
compounds have found many chemotherapeutic applications. So this study aims to synthesize a
library of phthalazine derivatives and to investigate their anti-inflammatory and anti-proliferative
activities. Sixteen new phthalazinone derivatives (2a–p) were synthesized and tested for their in vitro
antiproliferative and in vivo anti-inflammatory activities. All the synthesized compounds were
1
identified and characterized by IR, H NMR, 13C NMR spectroscopy, and MS. Two compounds, 2b and
2i, showed significant anti-inflammatory activity comparable with that of the standard drug
etoricoxib in the carrageenan-induced rat paw edema model at 3 and 5 h, respectively. Three
compounds (2h, 2j, and 2g) showed moderate sensitivity toward the renal cancer cell line UO-31.
Keywords: Anti-inflammatory activity / Anti-proliferative activity / COX-2 / LOX-5 inhibitors /
Phthalazone
Received: September 29, 2015; Revised: December 1, 2015; Accepted: December 4, 2015
DOI 10.1002/ardp.201500336
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
mainly responsible for mucus formation in the gastrointesti-
nal (GI) tract, COX-1 inhibition is blamed for inducing GI
irritation, the main undesired side effect of such drugs [1].
Introduction
Cyclooxygenase (COX) and lipoxygenase (LOX) produce two
groups of arachidonic acid (ARA) metabolites, prostaglandins
Another side effect of selective COX-1 inhibitors, mild
bleeding diathesis, also results from the inhibition of the
COX-1 catalyzed synthesis of the platelet aggregation factor,
thromboxan (TXA2) [2].
(PGs) and leucotrienes (LTs), that play
a key role in
inflammation. Traditional non-steroidal anti-inflammatory
drugs (NSAIDs) act via the inhibition of the COX-1 isoenzyme
or the combined inhibition of COX-1 and COX-2 isoenzymes.
For example, acetylsalicylate (aspirin) is a COX-1 selective
inhibitor, whereas indomethacin (indocin) and naproxen
(naprosyn) are COX-1/COX-2 inhibitors. Because COX-1 is
Because COX-2 isoenzyme was found to be overexpressed
during inflammation, drug investigation was focused on
selective COX-2 inhibition, hoping to prevent inflammation
by sidestepping the undesired side effect of COX-1
inhibitors [3, 4]. Consequently, selective COX-2 inhibitors
(coxibs) based on a diarylheterocyclic ring template were
developed. These agents were characterized by the presence
of a para-sulfonamide (SO2NH2) or a para-methanesulfonyl
(SO2Me) pharmacophore present on one of the aryl rings.
Crystal structure studies supported the hypothesis that the
p-SO2NH2 or p-SO2Me pharmacophore was conferring COX-2
selectivity by orienting in a secondary pocket accessible only in
the COX-2 active site [5, 6]. However, these drugs (COX-2
Correspondence: Dr. Kalim Javed, Department of Chemistry,
Faculty of Science, Jamia Hamdard (Hamdard University), New
Delhi 110 062, India.
E-mail: kjaved@jamiahamdard.ac.in, kjavedchem@yahoo.co.in
Fax: þ91 011 26059663
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Phthalazinone Derivatives as Anti-Inflammatory and Anti-Proliferative Agents
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inhibitors) were associated with increased risk of myocardial
Results and discussion
Chemistry
infarction and cardiovascular thrombotic events. The throm-
botic effect of these drugs seems to be due to the decrease of
the level of the prostacyclin (PGI2), a molecule with vaso-
dilatory and antiaggregatory properties, synthesized via the
action of COX-2. In addition, COX-2 was found to exhibit a
protective role in asthma [7] and GI tract irritation.
Leukotrienes (LTs) are synthesized via 5-LOX pathway and
are involved in the pathogenesis of bronchial asthma and
edema formation [8]. It is also believed that they play a role in
the damage of gastric mucosa [9–11]. The inhibition of this
metabolic pathway led to the development of new therapeu-
tic treatments for pathologies such as asthma, allergies, and
other inflammatory disorders.
The failure of COX-1 and COX-2 selective inhibitors evoked
the concept that inflammation should be considered as a
multifactorial process and all biochemical pathways should
be taken into account, including the LOX pathway. Com-
pounds that combine COX and LOX inhibition may present
multiple advantages because they synergistically block
metabolic pathways of arachidonic acid cascade and
thrombosis and possess a wide range of anti-inflammatory
activities [12].
The synthetic path which is used to synthesize the desired
compounds (2a–p) is outlined in Scheme 1. The b-aroylbenzoic
acids (1a–p) which are required for the synthesis of
phthalazinones derivatives were obtained from Friedel–Crafts
acylation through reported method wherein appropriate
aromatic hydrocarbon was mixed with phthalic anhydride
using anhydrous AlCl3 as catalyst in nitrobenzene. The
cyclization to the phthalazinone derivatives bearing
p-(methanesulfonyl)phenyl moiety was afforded by the
condensation of appropriate aroylbenzoic acid (1a–p) and
p-(methanesulfonyl)phenylhydrazine in absolute alcohol.
The structures of the synthesized compounds were deter-
mined on the basis of elemental analysis and by various
spectroscopic methods such as IR, 1H NMR, 13C NMR, and mass.
Elemental analysis (C, H, N, and S) data were within ꢀ0.4% of
the theoretical values. IR spectra of the compounds 2a–p
showed four bands characteristic of phthalazone moiety
–
out of which one band for C O of phthalazone moiety at
–
1718–1648 cmꢁ1, one band for C N of phthalazone ring
–
–
(1584–1496 cmꢁ1), and two bands for SO2C (1342–1310 and
1168–1142 cmꢁ1) were observed. In the 1H NMR spectra of
2a–p a three-proton singlet for –SO2CH3 was observed in a
range of d 3.20–3.24. Phthalazinone, 4-aryl, and N-phenyl ring
protons appeared in aromatic region (d 7.22–8.68).
In addition, both COX-2 and 5-LOX enzymes have been
involved in the development and progression of numerous
types of cancer [13–19]. So, the use of dual inhibitors opens up
new perspectives in the prophylactic treatment of this
dreadful disease.
Pharmacology
Phthalazine derivatives have been attracting increasing
attention over the years because of their broad spectrum
biological activities and strong efficacy. Phthalazines form the
structural profile for several biologically active compounds
and hence they are considered important key compounds
(Fig. 1). These derivatives have been reported to possess
antihypertensive [20, 21], anticonvulsant [22, 23], antidia-
betic [24, 25], antimicrobial [26], antitrypanosomal [27], anti-
inflammatory [28–30], and PDE4 inhibitory activities [31].
The phthalazinone nucleus has been proven to be a
versatile system in medicinal chemistry. Some of the
phthalazinone derivatives like hydralazine [32], budrala-
zine [33], azelastine [34], ponalrestat [35], and zopolrestat [36]
have found application in clinical medicine. Azelastine has
also been reported to have moderate 5-LOX inhibitory activity
in intact murine peritoneal cells and in chopped guinea pig
liver [37].
In view of the aforementioned facts, it was thought
worthwhile to incorporate the para-methanesulfonyl-
phenyl moiety at the 2 position of some 4-arylphthalazone
derivatives in the hope to yield safe and potent compounds
with anti-inflammatory and anticancer potential.
Therefore, this paper describes the synthesis, in vivo
anti-inflammatory and in vitro COX, 5-LOX, and anti-
proliferative activities of some new 4-arylphthalazone
compounds (2a–p). Compounds showing maximum anti-
inflammatory activity were also evaluated for ulcerogenic
potential in rats.
The in vivo anti-inflammatory activity of all synthesized
compounds 2a–p (0.05 mmol/kg) doses was evaluated by using
carrageenan-induced rat paw edema bioassay by the method
of Winter et al. [38]. Results were compared with etoricoxib as
it has some structural resemblance with the synthesized
compounds. Two compounds (2b and 2i) exhibited significant
anti-inflammatory activity and therefore, they were further
evaluated for their ulcerogenic potential in rats according to
Daidone et al. [39]. An in vitro anti-proliferative assay of 2h,
2j, and 2g was performed using a full panel of about 60
human tumor cell lines, with respect to the protocol of the
Drug Evaluation Branch, National Cancer Institute (NCI),
Bethesda [40–42]. Compounds 2b, 2i, 2j, and 2k were also
evaluated for their ability to inhibit COX-2 and 5-LOX by in
vitro an enzyme immuno-assay and an enzyme immune
according to previously reported methods [43, 44],
respectively.
Anti-inflammatory activity
Phthalazinone derivatives 2a–p showed moderate to strong
anti-inflammatory activity (20.27–65.96 and 23.52–69.32% at
3 and 5 h, respectively). Among them, two compounds 2b
(66.97 and 68.16) and 2i (65.96 and 69.32) have higher anti-
inflammatory activity compared to that of standard drug
etoricoxib (64.69 and 67.14) (Table 1). With regard to
structural–activity relationship (SAR), it was observed that
introduction of chlorine atom at (C-40) position of
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Figure 1. Structures of some biologically active pharmacophores and the target compounds. See Refs. [37, 45, 46] for further
information on azelastine, vatalanib, and olaparib, respectively.
phthalazinone nucleus increases the activity whereas intro-
duction of bromine and fluorine atoms at the same position
decreases the activity at both 3 and 5 h (2f vs. 2g and 2f vs. 2h,
respectively). Introduction of biphenyl group showed better
activity when compared to the biphenyl ether (2b vs. 2j) and
introduction of hexyl group showed increase in activity
comparing to butyl, n-propyl, and tert-butyl groups (2m vs. 2p
and 2m vs. 2e and 2m vs. 2l, respectively). Tetralin-substituted
compound showed significant activity as compared to other
compounds at 3 and 5 h.
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Phthalazinone Derivatives as Anti-Inflammatory and Anti-Proliferative Agents
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Scheme 1. Synthesis of 4-arylphthalazones 2a–p. Reagents and conditions: (a) anhydrous AlCl3, room temperature; (b) absolute
alcohol, reflux.
Ulcerogenic activity
immune-assay according to previously reported method [43],
while the ability of these compounds to inhibit potato 5-LOX
were determined using an enzyme immune assay according to
previously reported method [44]. The IC50 values were deter-
mined following the instruction given in the kit manual (Table 2).
The compounds 2b and 2i were found safe as they do not
cause 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 [39].
In vitro cyclooxygenase/lipoxygenase inhibition studies
The ability of the test compounds to inhibit ovine COX-1 and
human recombinant COX-2 were determined using an enzyme
Anti-proliferative activity
The structures of the phthalazinone products (2a–p) were
submitted to National Cancer Institute (NCI), 20 Bethesda,
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Table 1. Effect of phthalazinone derivatives (2a–p) at 0.05 mmol/kg in carageenan-induced rats paw edema assay.
Increase in paw volume (mL) after carageenan administrationa)
Treatment
3 h
5 h
Vehicle
Etoricoxib
1.62 ꢀ 0.021
1.70 ꢀ 0.023
0.707 ꢀ 0.22 (64.69%)b)
1.36 ꢀ 0.019 (20.27%)
0.643 ꢀ 0.017 (64.97%)
0.92 ꢀ 0.012 (49.16%)
1.16 ꢀ 0.592 (34.52%)
1.22 ꢀ 0.019 (28.34%)
1.04 ꢀ 0.024 (55.62%)
1.036 ꢀ 0.021 (42.06%)
1.22 ꢀ 0.019 (28.34%)
0.661 ꢀ 0.019 (65.96%)
0.7145 ꢀ 0.019 (61.12%)
0.81 ꢀ 0.022 (56.92%)
1.18 ꢀ 0.6146 (33.70%)
0.98 ꢀ 0.18 (47.03%)
1.22 ꢀ 0.018 (28.13%)
1.29 ꢀ 0.018 (25.22%)
1.053 ꢀ 0.026 (44.03%)
0.657 ꢀ 0.019 (67.14%)
1.22 ꢀ 0.020 (23.52%)
0.59 ꢀ 0.013 (68.16%)
0.95 ꢀ 0.024 (48.09%)
1.11 ꢀ 0.0145 (41.22%)
1.12 ꢀ 0.011 (37.38%)
1.04 ꢀ 0.024 (42.69%)
0.27 ꢀ 0.07 (36.40%)
1.16 ꢀ 0.014 (33.34%)
0.572 ꢀ 0.012 (69.32%)
0.716 ꢀ 0.021 (58.19%)
0.79 ꢀ 0.012 (59.17%)
1.12 ꢀ 0.018 (39.25%)
1.04 ꢀ 0.024 (43.59%)
1.26 ꢀ 0.020 (27.52%)
1.31 ꢀ 0.020 (23.42%)
0.25 ꢀ 0.04 (37.63%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
All data are markedly different compared to respective control values, p < 0.05.
b)
a)Values are expressed as mean. Values in parenthesis (percent inhibitions).
Maryland, USA, and the three compounds 2h, 2j, and 2g were
selected on the basis of degree of structural variation and
computer modeling techniques for evaluation of their anti-
proliferative activity. These compounds (2h, 2j, and 2g) were
granted NSC codes, viz, NSC 776884, NSC 776885, and NSC
776886, respectively. The elected compounds were submitted
to in vitro anticancer assay against tumor cells in a full panel of
59 or 60 cell lines taken from different tissues (blood, lung,
colon, CNS, skin, ovary, kidney, prostate, and breast). The
compounds were tested at a single dose concentration of
10 mM, and the percentages of growth inhibition over the 60
tested cell lines were determined [40–42]. The compounds 2h,
2j, and 2g displayed mild sensitivity toward a renal cancer cell
line UO-31 (Table 3) which is comparable to 5-fluorouracil (5-
FU), a clinically used anticancer drug.
Conclusion
Condensation of p-(methanesulfonyl)phenylhydrazine with
aroyl benzoic acids yielded the target compounds 2a–p. The
structures of the synthesized compounds (2a–p) are well
supported by spectroscopic data (IR, 1H NMR, 13C NMR, and
mass). Two compounds, 2b and 2i, showed significant anti-
Table 2. In vitro COX-1, COX-2, and 5-LOX enzyme inhibition assay data for compounds 2b, 2j, 2i, and 2k.
COX-2 selectivity
index (COX-1
a)
a)
a)
Compound
COX-1 IC50 (mM)
COX-2 IC50 (mM)
IC50/COX-2 IC50
)
5-LOX IC50 (mM)
2b
2j
2i
2k
14.74
12.30
10.20
20.30
21.10
–
1.00
0.78
0.72
0.34
0.18
–
14.74
15.76
14.17
59.7
116.5
–
6.2
5.3
8.0
6.0
–
Etoricoxib
Caffeic acid
3.5
Values were obtained using an enzyme inhibition assay (EIA) kit [catalog no. (560131 and 760700), respectively, Cyman Chemical
Inc., Ann Arbor, MI].
a)IC50 are means of two determinations and the deviation from the mean is >10% of the mean value.
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Table 3. Anticancer screening data for 2h, 2j, and 2g at concentration of 10ꢁ5 M.
Most sensitive
Growth of most
sensitive cell line (%)
Compound
2h
Code
Mean growth (%)
cell line
NSC: 776884
NSC: 776885
NSC: 776886
NSC: 19893
97.40
93.38
95.79
43.22
UO-31 (renal cancer)
UO-31 (renal cancer)
UO-31 (renal cancer)
UO-31 (renal cancer)
67.89
61.40
65.44
41.3
2j
2g
5-Fluorouracil (5-FU)
inflammatory activity which is comparable to that of standard
drug etoricoxib. The data of these synthesized derivatives
were subjected to NCI, USA for antiproliferative activity and
three compounds, 2h, 2j, and 2g, were selected. These
compounds showed a moderate sensitivity toward renal
cancer cell line UO-31. Compounds 2b and 2i can act as lead
for the medicinal chemists to develop effective and safer anti-
inflammatory drugs.
General procedure for the preparation of 4-
arylphthalazone derivatives (2a–p)
A mixture of the appropriate aroylbenzoic acid (1a–p) (0.001
mole) and (p-(methanesulfonyl)phenylhydrazine) (0.001 mol)
in absolute alcohol (20–30 mL) was refluxed for 18–22 h. The
volume of the reaction mixture was reduced to one-third by
evaporating the solvent and left at room temperature when a
solid separated out. The crude product was filtered off and
was stirred with 5% sodium bicarbonate solution (25 mL). It
was filtered and washed with 5% HCl and then with water.
The product was dried and crystallized from methanol or
acetone to give pure compound (2a–p). The solvent system
used for TLC was toluene/ethyl acetate/formic acid (5:4:1).
Please see the Supporting Information for the InChI codes of
compounds 2a–p.
Experimental
Chemistry
Melting points were determined by open capillary tubes and
are uncorrected. All the Fourier transform infra-red (FTIR)
spectra were recorded on a Brukers Vector 22 spectrophotome-
ter in film; ymax values are given in cmꢁ1
.
1H NMR spectra
2-[4-(Methylsulfonyl)phenyl]-4-phthalazin-1(2H)-one (2a)
were recorded on a Bruker Spectrospin DPX 300/400 MHz
spectrometer using deuterated chloroform as solvent. Chemical
shifts are given ind (ppm) scaleand coupling constants (J-values)
are expressed in hertz. 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 spectrospin DPX 300 MHz using deuterated chloroform
as solvent. Purity of the compounds was checked on TLC plates
(silica gel G) which were visualized byexposing toiodine vapors.
Yield ¼ 58%, m.p. 217–219°C, Rf ¼ 0.74 (toluene/ethyl acetate/
ꢁ1
formic acid, 5:4:1). IR ymax (KBr): 1652 cmꢁ1 (C O), 1566 cm
–
–
ꢁ1
1
–
(C N), 1151 and 1325 cm (SO2C). H NMR (400 MHz, CDCl3,
–
d): 3.22 (3H, s, SO2CH3), 7.26–7.33 (2H, m, J ¼ 7.2 Hz, H-30, H-50),
7.60–7.71 (2H, m, J ¼ 7.8 Hz, H-20, H-60), 8.06–8.11 (4H, m, H-200,
H-300, H-500, H-600), 7.84–7.91 (2H, m, H-6, H-7), 8.53–8.66 (1H, m,
H-5), 7.75 (1H, m, H-8). ESI-MS (m/z): 377 [Mþ1]. Anal. calcd.
for C21H16N2O3S: C 67.00, H 4.28, N 7.44, S 8.52. Found: C 67.13,
H 4.25, N 7.41, S 8.55.
General procedure for the synthesis of aroyl benzoic acids
(1a–p)
4-(4-Biphenyl)-2-[4-(methylsulfonyl)phenyl]phthalazin-1
(2H)-one (2b)
To liquid aromatic hydrocarbon (30 mL), anhydrous aluminum
chloride (16.6 g, 0.125 mole) was added. It was stirred on
magnetic stirrer at room temperature for 30 min to get a
colored complex. To it, phthalic anhydride (5 g, 0.05 mole) was
added in five 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 precipitated solid was dissolved in 5%
sodium carbonate solution and was then filtered. The filtrate
so obtained was acidified with dilute HCl to give solid mass. It
was then filtered, washed with cold water, dried, and
crystallized from appropriate solvent to give 2-aroylbenzoic
acids.
Yield ¼ 53%, m.p. 260–264°C, Rf ¼ 0.68 (toluene/ethyl acetate/
ꢁ1
formic acid, 5:4:1). IR ymax (KBr): 1688 cmꢁ1 (C O), 1558 cm
–
–
ꢁ1
1
–
(C N), 1148 and 1316 cm (SO2C). H NMR (400 MHz, CDCl3,
–
d): 3.21 (3H, s, SO2CH3), 7.45–7.52 (5H, m, H-20, H-30, H-40, H-50,
H-60), 7.76–7.79 (3H, m, H-6, H-7,H-8), 8.09–8.11 (4H, m,
N-phenyl protons), 8.66 (1H, m, H-5). 13C NMR (300 MHz,
CDCl3, d): 44.67, 126.1, 127.1, 127.4, 127.9, 128.0, 128.7, 128.9,
129.4, 129.8, 132.4, 133.7, 138.8, 142.5, 146.3, 148.4, 158.9.
ESI-MS (m/z): 453 [Mþ1]. Anal. calcd. for C27H20N2O3S: C 71.66,
H 4.45, N 6.19, S 7.09. Found: C 71.63, H 4.37, N 6.22,
S 8.13.
4-(2,3-Dihydro-1H-inden-5-yl)-2-[4-(methylsulfonyl)-
phenyl]phthalazin-1(2H)-one (2c)
Yield ¼ 60%, m.p. 212–214°C, Rf ¼ 0.72 (toluene/ethyl acetate/
ꢁ1
formic acid, 5:4:1). IR ymax (KBr): 1662 cmꢁ1 (C O), 1496 cm
–
–
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ꢁ1
1
–
(C N), 1158 and 1310 cm (SO2C). H NMR (300 MHz, CDCl3,
4-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-
–
d): 3.22 (3H, s, SO2CH3), 2.94 (4H, t, H-40, H-60), 2.01–2.04 (2H, t,
H-50), 7.41 (2H, d, H-30, H-70), 7.50 (1H, s, H-20), 7.72–7.81
(3H, m, H-6, H-7, H-8), 8.10–8.13 (4H, m, N-phenyl protons),
8.61–8.73 (1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 25.50,
32.8, 44.68, 124.5, 125.3, 126.1, 127.4, 127.9, 128.7, 129.2,
131.9, 132.4, 133.6, 138.6, 145.2, 146.1, 146.4, 149.2, 159.0. ESI-
MS (m/z): 417 [Mþ1]. Anal. calcd. for C24H20N2O3S: C 69.26, H
4.65, N 6.59, S 7.05. Found: C 69.53, H 4.77, N 6.72, S 7.12.
phthalazin-1(2H)-one (2g)
Yield ¼ 58.7%, m.p. 244–245°C, Rf ¼ 0.76 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1656 cmꢁ1 (C O),
–
–
1562 cmꢁ1 (C N), 1155 and 1332 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 3.20 (3H, s, SO2CH3), 7.28–7.32 (2H, m,
J ¼ 7.2 Hz, H-30, H-50), 7.63–7.75 (2H, m, J ¼ 7.8 Hz, H-20, H-60),
8.08–8.10 (4H, m, H-200, H-300, H-500, H-600), 7.87–7.92 (2H, m, H-6,
H-7), 8.52–8.64 (1H, m, H-5), 7.76 (1H, m, H-8). 13C NMR
(300 MHz, CDCl3, d): 44.68, 124.2, 126.5, 126.7, 128.3, 128.6,
131.4, 132.2, 132.5, 133.7, 138.7, 146.2, 147.7, 157.4, 158.6. ESI-
MS (m/z): 395 [Mþ1]. Anal. calcd. for C21H15FN2O3S: C 63.95, H
3.83, N 7.10, S 8.13. Found: C 63.63, H 3.75, N 7.22, S 8.31.
4-(4-Isopropyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1(2H)-one (2d)
Yield ¼ 48%, m.p. 214–216°C, Rf ¼ 0.74 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBrꢁ):1 1655 cmꢁ1 (C O),
–
–
1536 cmꢁ1 (C N), 1167 and 1323 cm
(SO2C). 1H NMR
4-(4-Bromophenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1(2H)-one (2h)
–
–
(300 MHz, CDCl3, d):1.41 (6H, d, PhCH(CH3)2), 3.10 (1H, m,
PhCH(CH3)2), 3.21 (3H, s, SO2CH3), 7.37–7.40 (2H, d, J ¼ 8.1 Hz,
H-30, H-50), 7.59 (2H, d, J ¼ 8.1 Hz, H-20, H-60), 8.05 (2H, d,
J ¼ 9.0 Hz, H-300, H-500), 8.12 (2H, d, J ¼ 8.70 Hz, H-200, H-600),
7.82–7.91 (3H, m, H-6, H-7, H-8), 8.63 (1H, m, H-5). 13C NMR
(300 MHz, CDCl3, d): 25.21, 31.82, 44.65, 126.1, 127.3, 127.8,
128.0, 128.7, 128.3, 129.1, 129.3, 131.9, 132.0, 133.6, 138.7,
144.4, 146.3, 148.8, 158.9. ESI-MS (m/z): 417 [M–1]. Anal.
calcd. for C24H22N2O3S: C 68.88, H 5.30, N 6.69, S 7.66. Found:
C 68.57, H 5.71, N 6.94, S 7.32.
Yield ¼ 45.3%, m.p. 246–248°C, Rf ¼ 0.75 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1664 cmꢁ1 (C O),
–
–
1581 cmꢁ1 (C N), 1152 and 1341 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 3.23 (3H, s, SO2CH3), 7.58–7.62 (2H, d,
J ¼ 7.6 Hz, H-30, H-50), 7.68–7.74 (2H, d, J ¼ 7.7 Hz, H-20, H-60),
8.06–8.09 (4H, m, H-200, H-300, H-500, H-600), 7.83–7.92 (2H, m, H-6,
H-7), 8.64–8.68 (1H, m, H-5), 7.80 (1H, m, H-8). 13C NMR
(300 MHz, CDCl3, d): 44.66, 124.0, 126.0, 126.7, 128.0, 128.7,
131.0, 132.2, 133.5, 138.9, 146.1, 147.6, 157.2, 158.8. ESI-MS
(m/z): 457 [Mþ2]. Anal. calcd. for C21H15BrN2O3S: C 55.39, H
3.32, N 6.15, S 7.04. Found: C 55.63, H 3.51, N 6.47, S 7.24.
4-(4-Propyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2e)
Yield ¼ 40.6%, m.p. 210–212°C, Rf ¼ 0.74 (toluene/ethyl ace-
2-(4-Methanesulfonyl-phenyl)-4-(5,6,7,8-tetrahydro-
naphthalen-2-yl)-phthalazin-1-one (2i)
tate/formic acid, 5:4:1). IR ymax (KBr): 1668 cmꢁ1 (C O),
–
–
1528 cmꢁ1 (C N), 1164 and 1318 cmꢁ1 (SO2C). 1H NMR
Yield ¼ 66.3%, m.p. 282–284°C, Rf ¼ 0.74 (toluene/ethyl ace-
–
–
tate/formic acid, 5:4:1). IR ymax (KBr): 1665 cmꢁ1 (C O),
–
–
(300 MHz, CDCl3, d): 1.1 (3H, t, PhCH2CH2CH3), 1.78 (2H, m,
PhCH2CH2CH3), 2.76 (2H, t, PhCH2CH2CH3), 3.24 (3H, s,
SO2CH3), 7.37–7.40 (2H, d, J ¼ 7.5 Hz, H-30, H-50), 7.60 (2H, d,
J ¼ 7.8 Hz, H-20, H-60), 8.06 (2H, d, J ¼ 8.4 Hz, H-300, H-500), 8.14
(2H, d, J ¼ 8.4.0 Hz, H-200, H-600), 7.86 (3H, m, H-6, H-7, H-8), 8.63
(1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 13.86, 24.46, 37.89,
44.69, 126.1, 127.3, 127.8, 128.0, 128.7, 128.4, 129.1, 129.3,
131.9, 132.0, 133.6, 138.7, 144.4, 146.3, 148.8, 158.9. ESI-MS
(m/z): 419 [Mþ1]. Anal. calcd. for C24H22N2O3S: C 68.88, H 5.30,
N 6.69, S 7.66. Found: C 68.67, H 5.61, N 6.74, S 7.42.
1584 cmꢁ1 (C N), 1147 and 1325 cmꢁ1 (SO2C). 1H NMR
–
–
(300 MHz, CDCl3, d): 3.12 (3H, s, SO2CH3), 1.94 (4H, t, H-50,
H-60), 2.85 (4H, t, H-40, H-70), 7.38 (2H, m, H-30, H-80), 7.25 (1H, d,
H-20), 7.82–7.85 (3H, m, H-6, H-7, H-8), 8.04 (2H, d, J ¼ 8.7 Hz, H-
300, H-500), 8.12 (2H, d, J ¼ 8.3 Hz, H-200, H-600), 8.45–8.47 (1H, m,
H-5). 13C NMR (300 MHz, CDCl3, d): 28.50, 37.84, 44.69, 113.1,
124.7, 125.5, 125.9, 126.1, 126.5, 127.6, 128.0, 129.8, 132.9,
133.6, 144.4, 146.6, 148.2, 154.9, 159.7. ESI-MS (m/z): 431
[Mþ1]. Anal. calcd. for C25H22N2O3S: C 69.75, H 5.15, N 6.51, S
7.45. Found: C 69.57, H 5.63, N 6.77, S 7.71.
4-(4-Chlorophenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1(2H)-one (2f)
4-(4-Methanesulfonyl-phenyl)-4-(4-phenoxy-phenyl)-
phthalazin-1-one (2j)
Yield ¼ 50.4%, m.p. 242–243°C, Rf ¼ 0.77 (toluene/ethyl
acetate/formic acid, 5:4:1). IR ymax (KBr): 1648 cmꢁ1 (C O),
Yield ¼ 30.1%, m.p. 284–286°C, Rf ¼ 0.75 (toluene/ethyl ace-
–
–
ꢁ1
1554 cmꢁ1 (C N), 1168 and 1322 cm
(SO2C). 1H NMR
tate/formic acid, 5:4:1). IR ymax (KBr): 1679 cmꢁ1 (C O),
–
–
–
–
1566 cmꢁ1 (C N), 1145 and 1315 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 3.12 (3H, s, SO2CH3), 7.56–7.60 (2H, m,
J ¼ 7.4 Hz, H-30, H-50), 7.66–7.70 (2H, m, J ¼ 7.4 Hz, H-20, H-60),
8.04–8.08 (4H, m, H-200, H-300, H-500, H-600), 7.85–7.91 (2H, m, H-
6, H-7), 8.51–8.63 (1H, m, H-5), 7.78 (1H, m, H-8). 13C NMR
(300 MHz, CDCl3, d): 44.65, 125.2, 126.5, 127.7, 128.3, 129.6,
131.4, 132.2, 132.5, 133.5, 138.6, 145.1, 149.2, 158.2, 158.6.
ESI-MS (m/z): 411 [Mþ1]. Anal. calcd. for C21H15ClN2O3S: C
61.39, H 3.68, N 6.82, S 7.80. Found: C 61.53, H 3.81, N 6.97,
S 7.51.
(400 MHz, CDCl3, d): 3.14 (3H, s, SO2CH3), 7.11–7.21 (5H, m,
H-3000, H-5000, H-2000, H-6000, and H-4000), 7.39–7.43 (2H, m, H-30, H-
50), 7.61 (2H, d, J ¼ 8.7 Hz, H-20, H-60), 7.85–7.91 (3H, m, H-6,
H-7, and H-8), 8.09–8.11 (4H, m, N-phenyl protons), 8.61–8.63
(1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 44.69, 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, 158.3.
FAB-MS (m/z): 491 [MþNa], 300 [M–Ph–O–Ph]. Anal. calcd. for
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Phthalazinone Derivatives as Anti-Inflammatory and Anti-Proliferative Agents
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C27H20N2O4S: C 69.22, H 4.30, N 5.98, S 6.84. Found: C 69.58, H
4.611, N 5.79, S 6.59.
H-60, H-70), 7.91–8.11 (3H, m, H-6, H-7, H-8), 8.12 (2H, d,
J ¼ 9.0 Hz, H-300, H-500), 8.13 (2H, d, J ¼ 8.7 Hz, H-200, H-600), 8.62–
8.68 (1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 33.45, 44.67,
126.5, 127.4, 128.4, 128.9, 129.0, 129.7, 129.9, 130.4, 131.8,
132.4, 135.33, 136.23, 135.7, 138.8, 144.5, 145.55, 147.21,
147.3, 149.4, 159.9. ESI-MS (m/z): 463 [M–1]. Anal. calcd. for
4-(3,4-Difluorophenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1(2H)-one (2k)
Yield ¼ 52.2%, m.p. 246–247°C, Rf ¼ 0.56 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1684 cmꢁ1 (C O),
C28H20N2O3S: C 72.39, H 4.34, N 6.03, S 6.90. Found: C 72.65, H
4.72, N 6.28, S 6.81.
–
–
1568 cmꢁ1 (C N), 1142 and 1320 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 3.23 (3H, s, SO2CH3), 7.37–7.42 (2H, m,
H-30, H-50), 7.62 (2H, d, J ¼ 8.6 Hz, H-20, H-60), 7.86–7.92 (3H, m,
H-6, H-7, and H-8), 8.12–8.15 (4H, m, N-phenyl protons), 8.60–
8.64 (1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 44.62, 117.6,
117.9, 118.6, 118.9, 125.9, 126.0, 126.5, 128.1, 128.5, 131.4,
132.4, 133.9, 139.0, 146.0, 146.5, 158.7. FAB-MS (m/z): 411
[M–1]. Anal. calcd. for C21H14F2N2O3S: C 61.16, H 3.42, N 6.69, S
7.78. Found: C 61.42, H 3.71, N 6.88, S 7.66.
4-(4-Ethyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2o)
Yield ¼ 42.6%, m.p. 202–204°C, Rf ¼ 0.78 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1658 cmꢁ1 (C O),
–
–
1536 cmꢁ1 (C N), 1161 and 1323 cmꢁ1 (SO2C). 1H NMR
–
–
(300 MHz, CDCl3, d): 1.3 (3H, t, PhCH2CH3), 2.76 (2H, t,
PhCH2CH3), 3.23 (3H, s, SO2CH3), 7.33–7.38 (2H, d, J ¼ 7.5 Hz,
H-30, H-50), 7.62 (2H, d, J ¼ 7.8Hz, H-20, H-60), 8.05 (2H, d,
J ¼ 8.4 Hz, H-300, H-500), 8.11 (2H, d, J ¼ 8.4.0 Hz, H-200, H-600), 7.85
(3H, m, H-6, H-7, H-8), 8.60 (1H, m, H-5). 13C NMR (300 MHz,
CDCl3, d): 24.46, 37.89, 44.69, 126.1, 127.3, 127.8, 128.0, 128.7,
128.4, 129.1, 129.3, 131.9, 132.0, 133.6, 138.7, 144.2, 146.3,
148.8, 158.9. ESI-MS (m/z): 405 [Mþ1]. Anal. calcd. for
4-(4-tert-Butyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2l)
Yield ¼ 60.2%, m.p. 232–234°C, Rf ¼ 0.74 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1656 cmꢁ1 (C O),
–
–
1547 cmꢁ1 (C N), 1130 and 1342 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 1.4 (9H, s, Ph(CH3)3), 3.15 (3H, s, SO2CH3),
7.58–7.62 (4H, m, J ¼ 7.4 Hz, H-30, H-50, H-20, H-60), 7.50–7.61
(2H, d, J ¼ 7.3, H-20, H-60), 8.12 (2H, d, J ¼ 9.0 Hz, H-300, H-500),
8.21 (2H, d, J ¼ 8.7 Hz, H-200, H-600), 7.82–7.91 (3H, m, H-6, H-7,
H-8), 8.60–8.72 (1H, m, H-5). 13C NMR (300 MHz, CDCl3, d):
31.37, 34.86, 44.69, 125.7, 126.1, 127.3, 127.8, 128.0, 128.7,
129.1, 131.6, 132.0, 133.6, 138.7, 146.4, 148.7, 152.8, 159.0. ESI-
MS (m/z): 431 [M–1]. Anal. calcd. for C25H24N2O3S: C 69.42, H
5.59, N 6.48, S 7.41. Found: C 69.73, H 5.83, N 6.78, S 7.71.
C24H22N2O3S: C 68.55, H 5.75, N 6.66, S 7.63. Found: C
68.67, H 5.82, N 6.81, S 7.72.
4-(4-Butyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2p)
Yield ¼ 44.4%, m.p. 225–227°C, Rf ¼ 0.75 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1662 cmꢁ1 (C O),
–
–
1552 cmꢁ1 (C N), 1124 and 1335 cmꢁ1 (SO2C). 1H NMR
–
–
(400 MHz, CDCl3, d): 0.98 (3H, t, PhCH2CH2CH2CH3), 1.8
(4H, m, PhCH2CH2CH2CH3), 2.71 (2H, t, PhCH2CH2CH2CH3),
3.16 (3H, s, SO2CH3), 7.38–7.43 (2H, d, J ¼ 7.1 Hz, H-30, H-50),
7.52–7.63 (2H, d, J ¼ 7.3, H-20, H-60), 8.13 (2H, d, J ¼ 8.7 Hz,
H-300, H-500), 8.32 (2H, d, J ¼ 8.7 Hz, H-200, H-600), 7.78–7.83
(3H, m, H-6, H-7, H-8), 8.63–8.74 (1H, m, H-5). ESI-MS (m/z): 433
[Mþ1]. Anal. calcd. for C25H24N2O3S: C 69.42, H 5.59, N 6.48, S
7.41. Found: C 69.45, H 5.56, N 6.44, S 7.45.
4-(4-Hexyl-phenyl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2m)
Yield ¼ 54.4%, m.p. 220–222°C, Rf ¼ 0.76 (toluene/ethyl acetate/
ꢁ1
formicacid,5:4:1).IRymax (KBr):1669 cmꢁ1(C O),1558 cm (C N),
–
–
–
–
1122and1334 cmꢁ1(SO2C).1HNMR(400 MHz,CDCl3,d):0.99(3H,t,
PhCH2CH2CH2CH2CH2CH3), 1.3 (4H, m, PhCH2CH2CH2CH2CH2CH3),
1.6 (2H, m, PhCH2CH2CH2CH2CH2CH3), 1.72 (2H, m,
PhCH2CH2CH2CH2CH2CH3), 2.77 (2H, t, PhCH2CH2CH2CH2CH2CH3),
3.17 (3H, s, SO2CH3), 7.36–7.41 (2H, d, J¼ 7.1 Hz, H-30, H-50), 7.50–
7.61 (2H, d, J¼ 7.3, H-20, H-60), 8.1 (2H, d, J¼ 8.7 Hz, H-300, H-500), 8.2
(2H, d, J¼ 8.7 Hz, H-200,H-600), 7.78–7.83 (3H, m, H-6, H-7, H-8), 8.61–
8.72 (1H, m, H-5). 13C NMR (300 MHz, CDCl3, d): 14.22, 24.11, 29.78,
32.66, 34.41, 36.89, 44.69, 126.1, 127.3, 127.8, 128.0, 128.7, 128.2,
129.1,129.3,131.9,132.0,133.6,138.7,144.4,146.3,148.8,158.ESI-
MS (m/z): 461 [Mþ1]. Anal. calcd. for C27H28N2O3S: C 70.41, H 6.13,
N 6.08, S 6.96. Found: C 70.75, H 6.43, N 6.38, S 6.78.
Pharmacology
Anti-inflammatory activity
Carrageenan-induced hind paw edema method was sued for
evaluating anti-inflammatory activity of the synthesized
compounds [38]. In the current study, rats of Wistar progeny
(either sex) obtained from Central Animal House facility of
Jamia Hamdard, New Delhi (registration no. 173/CPCSEA)
were used. The experiments were performed in accordance
with the guidelines for the care and use of laboratory animals,
laid down by the Committee for the Purpose of Control and
Supervision of Experiments in Animals (CPCSEA), Ministry of
Social Justice and Empowerment, Govt. of India, Jan 2000.
CMC 1%, w/v in distilled water was used as vehicle for dosing
(10 mL/kg). All the treatments suspended in vehicle were
given orally. Overnight fasted rats (150–200 g) were taken and
divided into 15 groups of six animals each. Group I was
administered orally with vehicle (1% Cween-10, 10 mL/kg) and
4-(9H-Fluoren-2-yl)-2-[4-(methylsulfonyl)phenyl]-
phthalazin-1-one (2n)
Yield ¼ 40.6%, m.p. 278–280°C, Rf ¼ 0.72 (toluene/ethyl ace-
tate/formic acid, 5:4:1). IR ymax (KBr): 1718 cmꢁ1 (C O),
–
–
1576 cmꢁ1 (C N), 1148 and 1339 cmꢁ1 (SO2C). 1H NMR
–
–
(300 MHz, CDCl3, d): 3.12 (3H, s, SO2CH3), 4.04 (2H, s, H-90),
7.38–7.49 (3H, m, H-20, H-30, H-80), 7.61–7.72 (4H, m, H-40, H-50,
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Archiv der Pharmazie
served as control. Group II was treated with etoricoxib
(0.05 mmol/kg b.w.) served as standards. Test groups were
administered orally with respective test drugs (2a–p) 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 volume of paw was measured by using plethys-
mometer at 0, 3, and 5 h postcarageenan 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 following
formula:
24 h before adding the compounds. These were tested in
DMSO solution at 10ꢁ5 M concentrations. After an incubation
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 dose–response parameters were
calculated as previously described [42].
The authors are very thankful to the staff members of
Department of Health and Human Services, National Cancer
Institute (NCI), Bethesda, Maryland, USA, for carrying out in
vitro anticancer screening of the newly synthesized
compounds.
% Inhibition ¼ ð1 ꢁ Vt=VcÞ ꢂ 100
where Vt is the mean paw volume of the test drug-treated rats
and Vc is the mean paw volume of the control.
The authors have declared no conflicts of interest.
Ulcerogenic activity
References
Acute gastric ulcerogenic effect of the compounds 2b and 2i
were evaluated in Wistar rats [39]. Albino rats of Wistar strain
(150–200 g) fasted over 24 h were randomly allotted into four
groups of six animals each. The animals of first group were
given vehicle 10 mL/kg (CMC 1% w/v in distilled water) orally.
Group II served as standard and was administered orally
etoricoxib (0.15 mmol/kg) suspended in vehicle. Groups III and
IV were administered orally compounds 2b and 2i at a dose of
0.25 mmol/kg po 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.
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