Synthesis of 2-substituted-4-aryl-6-phenylpyridazin-3(2H)-ones as potential anti-inflammatory and analgesic agents with cardioprotective and ulcerogenic sparing effects

Article abstract of DOI:10.1007/s00044-016-1588-9

Several new 2-substituted-4-aryl-6-phenylpyridazin-3(2H)-ones were synthesized and evaluated for in vivo anti-inflammatory and analgesic activities and possible in vitro COX-2 selectivity. To critically evaluate the possibility of deleterious effects of n

Full text of DOI:10.1007/s00044-016-1588-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1588-9
ORIGINAL RESEARCH
Synthesis of 2-substituted-4-aryl-6-phenylpyridazin-3(2H)-ones
as potential anti-inflammatory and analgesic agents
with cardioprotective and ulcerogenic sparing effects
Deepika Sharma1 Ranju Bansal1
•
Received: 14 February 2016 / Accepted: 21 April 2016
Ó Springer Science+Business Media New York 2016
Abstract Several new 2-substituted-4-aryl-6-phenylpyri-
dazin-3(2H)-ones were synthesized and evaluated for
in vivo anti-inflammatory and analgesic activities and
possible in vitro COX-2 selectivity. To critically evaluate
the possibility of deleterious effects of newly synthesized
agents on normal hemostasis, anti-platelet activity and
whole blood clotting time were also assessed. The struc-
tures of the synthesized compounds were confirmed on the
basis of spectral data and elemental analysis. The present
study has led to the identification of 4-(4-methoxyphenyl)-
2-(2-(4-phenylpiperazin-1-yl)-2-oxoethyl)-6-phenylpyridazin-
3(2H)-one (17) as an ideal anti-inflammatory agent with good
affinity and remarkable selectivity for COX-2 enzyme with-
out any ulcerogenic and cardiovascular side effects. In addi-
tion, at lower doses all the pyridazinone derivatives do not
seem to affect normal hemostatic balance.
Introduction
Inflammation can originate from both infectious and non-
infectious processes of chronic injury or irritation (Gu
et al., 2007). The majority of currently known nonsteroidal
anti-inflammatory and analgesic drugs (NSAIDs) cause
serious gastrointestinal side effects. The GIT damage from
NSAIDs is because of non-selective inhibition of both
COX-1 and COX-2. Thus, the selective inhibition of COX-
2 over COX-1 is a useful strategy for development of new
agents for treating inflammation. However, increasing
selectivity for COX-2 also results in increased cardiovas-
cular toxicity (Dannhardt and Kiefer, 2001). Therefore,
search for more effective and less toxic drugs to treat
inflammation has become mandatory.
Pyridazines represent an important class of biologically
active compounds. Among pyridazine derivatives, 3(2H)-
pyridazinones form an important class of compounds due
to their diverse pharmacological properties. Easy func-
tionalization of various ring positions of pyridazinone core
structure makes it an attractive synthetic and therapeutic
target for designing and synthesis of new drugs (Bansal and
Thota, 2013). The phenomenal research efforts to develop
pyridazinone-based anti-inflammatory agents have led to
many interesting findings (Cesari et al., 2006). Emorfazone
(Fig. 1), a 3(2H)-pyridazinone derivative, has been laun-
ched in Japan as an analgesic drug without any gastric
complications (Takaya et al., 1979). This encouraged us to
synthesize and assess new pyridazinone-based chemical
entities for anti-inflammatory potential.
Keywords Pyridazin-3(2H)-one Á Anti-inflammatory Á
Analgesic Á Hemostasis Á Cyclooxygenase
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1588-9) contains supplementary
material, which is available to authorized users.
There has been much focus recently in respect to car-
diovascular risks associated with long-term usage of both
non-selective and selective NSAIDs (Chelucci et al.,
2014). In 2007, the American Heart Association published
a focused update discouraging the use of NSAIDs for
& Ranju Bansal
ranju29in@yahoo.co.in
1
University Institute of Pharmaceutical Sciences, Sector-14,
Panjab University, Chandigarh 160014, India
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Med Chem Res
Fig. 1 Pyridazinone-based
anti-inflammatory and
vasodilatory agents
N
N
C
N
NH
H
COC
3
HN
C
O
N
H
C2 5O
H
3C
H
3C
N
N
N
NH
NH
O
N
H
C
3
O
Levosimendan
O
SK&F-93741
Emorfazone
patients with established cardiovascular disease (Olsen
et al., 2012). Keeping in mind the good properties of
pyridazinone-based compounds (SK&F-93741 and levosi-
mendan, Fig. 1) toward controlling cardiovascular dis-
eases, particularly as vasodilatory and anti-platelet agents
(Sur et al., 2003), we envisaged new structural modifica-
tions of the pyridazinone nucleus to produce derivatives
with good anti-inflammatory potential without any car-
diovascular toxicity.
ring due to the presence of p-methoxy electron-donating
substituent led to bromination of aromatic ring by elec-
trophilic substitution along with unsaturation at desired
position to obtain compound 4-(3-bromo-4-methox-
yphenyl)-6-phenylpyridazin-3(2H)-one (4) as shown in
Scheme 1, the structure of which was confirmed by various
spectral analyses. Unsaturated 5–CH proton appeared
downfield at d 7.78 in the nuclear magnetic resonance
spectrum of compound 4 in comparison with its saturated
counterpart 3, where it appeared at 3.29 ppm. The presence
of bromine ortho to –OCH3 functionality was further
1
Results and discussion
Chemistry
confirmed by H NMR analysis by appearance of doublet
of ortho coupled aromatic protons at d 7.85 with
Jo = 7.38 Hz, while meta coupled aromatic proton ortho to
bromo group resonated downfield at 8.13 ppm (Jm
=
2-(4-Methoxyphenyl)-4-phenyl-4-oxobutyronitrile (1) was
synthesized according to the reported procedure (Ellis
et al., 1997). Hydrolysis of nitrile 1 in refluxing
hydrochloric acid (10 N) yielded 2-(4-methoxyphenyl)-4-
oxobutanoic acid (2). The formation of the acid was con-
2.16 Hz).
The presence of an acetamide side chain on lactam
nitrogen of pyridazinone ring has been reported to posi-
tively modulate the anti-inflammatory activity of the
compounds with less gastrointestinal complications
(Sahina et al., 2004; Banoglu et al., 2004; Su¨ku¨roglu et al.,
2005); therefore, introduction of an acetamide linker was
planned. Pyridazinone derivative 4 was treated with methyl
chloroacetate in ethyl methyl ketone to afford 4-(3-bromo-
4-methoxyphenyl)-2-(methoxycarbonylmethyl)-6-phenylpyri-
dazin-3(2H)-one (5) as depicted in Scheme 2. Additional
singlets for –NCH2 at d 5.04 and –COOCH3 at 3.95 ppm
appeared in the proton nuclear magnetic spectrum of
compound 5. The presence of a prominent [MH?2]? peak
almost equal in intensity to that of the molecular ion
peak [MH]? was also observed in the mass spectrum of
product 5, which is in agreement with the proposed
structure.
1
firmed using H NMR spectral analysis. For c-keto acid 2,
NMR signals at d 3.27 (dd, 1H, –COC(H)H–), 3.80 (s, 3H,
–OCH3), 3.87 (dd, 1H, –COC(H)H) and 4.25 ppm (dd, 1H,
–COCH2CHCOOH–) were observed. Subsequent cycliza-
tion reaction of c-keto acid 2 using hydrazine hydrate in
1-butanol afforded 4,5-dihydropyridazinone derivative 3.
Double doublets of germinal and vicinal protons of
4-CH and 5-CH2 of pyridazinone ring appeared separately
at d 3.19 (dd, 1H, 5–C(H)H, pyridazinone, Jgem
=
16.92 Hz, Jvic = 9.4 Hz), 3.29 (dd, 1H, 5–C(H)H, pyri-
dazinone, Jgem = 16.9 Hz, Jvic = 7.18 Hz) and 3.79 ppm
(m, 4H, –OCH3 and 4–CH, pyridazinone). A signal for
2-unsubstituted –NH of dihydropyridazinone ring was
observed at d 8.89 ppm. The formation of pyridazinone
nucleus was attempted by dehydrogenating the 4 and 5
positions of dihydropyridazinone 3 using bromine–acetic
acid (Coudert et al., 1988). However, activation of the aryl
Thermal fusion of 2-(methoxycarbonylmethyl)-substi-
tuted pyridazinone derivative 5 with heterocyclic amines
such as morpholine, N-methylpiperazine, piperidine and
pyrrolidine yielded desired 2-substituted-6-phenylpyri-
123

Med Chem Res
Scheme 1 Synthetic route to
the formation of 4-(3-bromo-4-
methoxyphenyl)-6-
Ha
Hc
C
O
C
O
C
C
COOH
CH2 CH CN
a
Hb
phenylpyridazin-3(2H)-one (4).
Reagents and reaction
conditions: a 10 N HCl, reflux;
b n-butanol, hydrazine hydrate,
reflux; c acetic acid, bromine,
heat
OCH3
OCH3
(1)
(2)
b
N
N
N
H
N
O
H
c
O
H3CO
Br
H3CO
(4)
(3)
Scheme 2 Synthetic route to
the formation of pyridazinones
5–9. Reagents and reaction
conditions: a methyl
chloroacetate, anhyd. K2CO3,
ethyl methyl ketone; b requisite
amine, heating
O
N
N
a
NCH2C R
NCH2COOCH3
b
4
O
O
H3CO
Br
H3CO
N
Br
(6-9)
(5)
N
(6) R=
(8) R=
(7) R=
(9) R=
N
N
CH3
N
O
dazin-3(2H)-ones (6–9). A characteristic amidic shift to a
lower wave number * 1649 was observed for carbonyl
absorption bands in infrared spectra of compounds 6–9 in
comparison with parent ester 5, where it appeared at a
higher frequency 1751 cm-1. Almost equal intensity
[MH?2]? and [MH]? peaks due to the presence of
molecular ions containing Br81 and Br79 isotopes were
prominently present in the mass spectra of all these
derivatives 6–9. This further confirmed the presence of
bromine in these compounds.
The observation that bromination of phenyl ring occurs
while introducing double bond at 4,5 positions of dihy-
dropyridazinone nucleus by using bromine–acetic acid led
us to adopt an alternative synthetic pathway (Sotelo et al.,
2002) for aromatization to prepare targeted pyridazinones
(Scheme 3). Copper (II) chloride was reported by Sotelo
et al. as an extremely efficient and versatile reagent capable
of carrying out successful aromatization of 4,5-dihy-
dropyridazin-3(2H)-ones (Sotelo and Ravina, 2000). Con-
sequently to serve the purpose, the intermediate 3 was
123

Med Chem Res
Scheme 3 Synthetic route to
the formation of pyridazinones
11–19. Reagents and reaction
conditions: a acetonitrile,
copper (II) chloride, reflux;
b methyl chloroacetate, ethyl
methyl ketone, anhyd. K2CO3; c
requisite amine, fusion
N
a
b
N
3
NH
NCH2COOCH3
O
O
(11)
H3CO
H3CO
(10)
c
O
N
NCH2C R
O
H3CO
(12-19)
N
N
N
N
(13) R =
(15) R =
(12) R =
(14) R =
(16) R =
N
CH3
O
N
H
N
(17) R =
(19) R =
N
N
OCH3
OCH3
N
NO2
N
N
F
(18) R =
Biological evaluation
reacted with copper (II) chloride in the presence of ace-
tonitrile to afford compound 10. A downfield singlet of
5-CH of pyridazinone (d 7.81 ppm) was observed in the
nuclear magnetic resonance spectrum of compound 10. As
mentioned earlier, substitution at N2 position to afford
pyridazinone methyl ester 11 and subsequent thermal
fusion with requisite amines afforded aimed derivatives
12–19.
Anti-inflammatory activity
The 2-substituted-4-aryl-6-phenylpyridazin-3(2H)-ones 5–
9 and 11–19 were evaluated for anti-inflammatory activity
using carrageenan-induced hind paw edema model
(Abouzid and Bekhit, 2008) in male Wistar rats.
The compounds at 20 and 40 mg/kg were given orally as
suspensions, and activity profile was compared with stan-
dard drugs indomethacin and celecoxib (20 mg/kg).
Additionally, all compounds were also investigated for
ulcerogenic side effects. The results of the studies are
summarized in Table 1.
A characteristic shift of vibrational frequency of car-
bonyl stretch of ester to lower wave number * 1660 cm-1
due to carbonyl stretch of tertiary amides was observed.
Two downfield signals at *d 160 and 166 ppm repre-
senting carbonyl carbons were observed in 13C NMR of all
the final products.
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Table 2 Analgesic activity of various pyridazinones and standard drugs
Compound no.
20 mg/kg
40 mg/kg
No. of writhes
% protection
No. of writhes
% protection
Control/VEHICLE
35.4 1.2
Indomethacin
13.3 1.3***
9.83 1.07***
31.67 2.23
28.83 1.30**
31.83 2.15
30.33 1.31*
32.50 1.54
33.17 1.96
26.83 2.01**
28.50 1.38**
32.33 1.02
25.50 1.38**
27.67 1.43**
29.17 2.86
30.83 3.05
30.33 1.61
62.3
72.16
10.37
18.4
9.9
Celecoxib
5
30.8 2.21
12.73
35.8
18.9
14.1
18.4
13.67
31.6
28.3
16.5
32.5
50.0
32.1
43.4
36.3
6
22.67 1.50***
28.67 1.89*
30.33 1.20*
28.83 2.15*
30.5 3.06
7
8
10.8
8.0
9
11
12
13
14
15
16
17
18
19
6.12
24.04
19.3
8.5
24.17 2.17**
25.33 1.56***
29.50 1.23**
23.83 2.23**
17.67 1.80***
24.00 2.08**
20.00 1.73***
22.50 2.51***
27.82
21.7
17.4
12.7
14.1
Data are represented as mean SEM (n = 5). Results were analyzed using one-way ANOVA followed by post hoc Dunnett’s test
* p \ 0.05, ** p \ 0.01 and *** p \ 0.001 as compared to control value
In general, most of the tested compounds (40 mg/kg)
displayed anti-inflammatory activity comparable to the
reference compound indomethacin (20 mg/kg). The com-
pounds produced dose-dependent inhibition of rat paw
edema, being more effective at 40 than 20 mg/kg. The
intermediate esters 5 and 11 were found to be less active
than their corresponding acetamide derivatives. All the
pyridazinone derivatives except compounds 5, 9 and 11
showed more than 50 % inhibition of edema at 40 mg/kg.
Homoveratrylamine-substituted derivative 16 exhibited
80.43 % inhibition of rat paw edema (40 mg/kg), which is
comparable to standard drugs indomethacin and celecoxib
(20 mg/kg). Also p-fluorophenylpiperazine (18) and p-ni-
trophenylpiperazine (19) analogs showed remarkably
potent anti-inflammatory activity. These results are in
accordance with the literature reports where the presence of
an arylpiperazine moiety at 2 position of pyridazinone ring
has been shown to increase anti-inflammatory activity of
pyridazinone derivatives (Dogruer et al., 2004). It was
interesting to note that bromo-unsubstituted derivatives
11–19 displayed better potency than bromo-substituted
counterparts 5–9.
were removed (Chan et al., 1995). At the maximum tested
dose (40 mg/kg), none of the synthesized derivative
showed any sign of gastric complications.
Analgesic activity
All the newly synthesized compounds were also evaluated
for analgesic activity at 20 and 40 mg/kg by acetic acid-
induced writhing test as reported (Ozkay et al., 2011).
Mouse abdominal constrictions were measured as writhing
response, and % protection was calculated. The obtained
results are summarized in Table 2.
Analgesic activity of the compounds was in good cor-
relation with their anti-inflammatory activity profile. In
accordance with literature reports, introduction of
arylpiperazine moieties on the amide side chain of pyri-
dazinones 17–19 seems to be contributing positively
toward anti-nociceptive activity, with p-fluorophenyl-sub-
stituted derivative 18 displaying maximum protection of
43.4 % at 40 mg/kg in 2-substituted-4-aryl-6-phenylpyri-
dazin-3(2H)-one series. Furthermore, it is also noticed that
the presence of bromine on the phenyl ring (except com-
pound 6) resulted in a remarkable decrease in analgesic
activity. The presence of N-methylpiperazine group as a
substituent in compounds 8 and 14 led to drastic decrease
in % protection against acetic acid-induced writhing in
mice at both the tested doses.
Gastric ulcerogenic activity
Rats were killed under deep ether anesthesia 24 h after the
anti-inflammatory activity experiment, and their stomachs
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In vitro COX inhibitory activity
assay are given in Table 3. It is evident from the in vitro
assay that many of the synthesized derivatives demon-
strated COX inhibitory potency and reasonable selectivity
for COX-2 isoform, but none showed activity superior to
that of standard drug celecoxib. Intermediate esters 5 and
11 displayed negligible affinity for either form of
cyclooxygenase enzyme, which is in strong agreement with
their poor in vivo anti-inflammatory activity. The com-
pound 17 carrying a phenylpiperazine substituent at 2-po-
sition of the pyridazinone ring showed good affinity and
remarkable selectivity for COX-2 enzyme being 6.37 times
more selective for COX-2 in comparison with COX-1
isoform. However, addition of a nitro group at para posi-
tion of phenylpiperazine as in compound 19 leads to
marginal reduction in the affinity as well as selectivity
toward COX-2 enzyme. COX-2 specificity was also
observed to a good extent in case of homoveratrylamine-
substituted (16) and pyrrolidine-derived (13) diarylpyri-
dazinones as they displayed preferential inhibition of
COX-2 enzyme with selectivity of the order of 3.89 and
3.63, respectively, versus COX-1 enzyme. In addition to
this, we noticed that compounds 9 and 15 having mor-
pholine ring in side chain inhibited both the enzymes with
similar potency. Only compound 7 showed more potency
and selectivity toward COX-1 enzyme.
It is known that carrageenan-induced edema is a biphasic
event where histamine-, bradykinin- and serotonin-like
mediators are involved in the first stage of inflammation. On
the contrary, second stage of inflammation is inhibited by
NSAIDs indicating contribution of COX enzyme (Dogruer
et al., 2004). In the present study, the involvement of COX
enzyme toward anti-inflammatory activity is expected as all
the tested compounds were found to be effective even after
240 min of the oral administration in rat paw edema assay.
Moreover, structure activity relationship (SAR) studies
employing pyridazinones have shown N-substitution as a
structural requirement for COX-2 selectivity (Saeed et al.,
2012).These observations motivated us to study in vitro
COX enzyme inhibitory screening of the newly synthesized
compounds. The literature suggests the possibility of a weak
in vitro–in vivo correlation between biological activity and
enzyme selectivity (Chintakunta et al., 2002; Unsal-Tan
et al., 2010). Thus, compounds with maximum structural
diversity were selected for in vitro COX activity irrespec-
tive of their in vivo analgesic and anti-inflammatory profile.
This would further help in understanding the mechanism of
action of pyridazinones as anti-inflammatory agents. The
inhibitory activity of selected compounds on both isoforms
of cyclooxygenase enzymes, COX-1 and COX-2, was
assayed using the COX Inhibitor Screening Assay Kit
(Cayman No: 560101, Table 3) according to the protocol
recommended by the supplier.
Anti-platelet activity
All the synthesized compounds were also examined for
cardiovascular safety profile. A diversity of effects was
observed in tail transaction bleeding test (Table 4). In
general, treatment with test compounds at 20 mg/kg did not
affect the platelet function in comparison to control except
compounds 15, 17 and 19, which produced dose-dependent
reduction in the bleeding time. However, at 40 mg/kg, out
of 2-substituted-4-aryl-6-phenylpyridazin-3(2H)-ones 5–9
and 11–19, piperidinyl-substituted compound 12 signifi-
cantly increased the bleeding time. On the contrary, aryl
piperazine-substituted compounds 17 and 19 and
homoveratrylamine-based derivative 16, which emerged as
selective COX-2 inhibitors with potent analgesic and anti-
inflammatory activity, tend to shorten the bleeding time at
both the tested doses in comparison with control. Inter-
estingly, treatment with p-fluorophenylpiperazine deriva-
tive 18 brought no change in bleeding time at both the
tested doses. Marginal shifts in bleeding time were
observed after oral administration of rest of the compounds
of the series at 40 mg/kg. 4-(4-Methoxyphenyl)-2-(2-
pyrrolidin-1-yl-2-oxoethyl)-6-phenylpyridazin-3(2H)-one
(13) although depicted selective inhibition of COX-2
(3.89 times) versus COX-1, but displayed no change in
bleeding time with respect to control. Therefore,
Screening of the selected compounds and reference
drugs (indomethacin and celecoxib) was performed at
40 lM to determine the percent inhibition of the COX-1
and COX-2 isoforms. Results of COX inhibitor screening
Table 3 In vitro COX-1 and COX-2 enzyme inhibition data
Compound no.
% Inhibition (40 lM)a
COX-1
COX-2
5
1.88
66.84
17.33
16.54
26.25
76.24
23.38
12.44
21.91
79.99
6.44
8.21
39.60
26.25
9.07
7
9
11
13
95.45
64.89
91.02
79.83
59.50
97.40
98.18
15
16
17
19
Indomethacin
Celecoxib
a
The determination was performed in duplicate for two independent
experiments
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Table 4 Effect on bleeding time (sec) after oral administration of test
compounds
Table 5 Effect on clotting time (sec) after oral administration of test
compounds
Compound no.
Bleeding time (s)
20 mg/kg
Compound no.
Clotting time (s)
20 mg/kg
40 mg/kg
40 mg/kg
Control
220.0 13.0
290.0 11.8**
216.0 11.7
216.0 17.2
228.0 16.2
212.0 21.5
216.0 11.7
216.0 11.7
252.0 10.2
220.0 16.7
236.0 11.7
184.0 11.7
208.0 10.2
196.0 7.5
Control
158 7.8
192 9.6*
154 8.4
158 4.6
152 3.4
160 4.0
170 5.4
162 4.4
152 6.3
172 9.6
166 3.7
146 10.7
154 6.2
154 7.2
154 4.6
156 2.3
Aspirin (20 mg/kg)
Aspirin (20 mg/kg)
5
244.0 13.3
240.0 14.1
252.0 10.2
196.0 16.0
228.0 8.0
5
156 9.7
150 10.1
182 10.4
154 11.8
160 10.4
160.0 6.3
152 11.1
150 6.6
6
6
7
7
8
8
9
9
11
12
13
14
15
16
17
18
19
208.0 12.0
260.0 8.9*
208.0 16.2
248.0 10.2
176.0 7.5*
154.0 10.8**
180.0 14.1
204.0 11.7
172.0 10.2*
11
12
13
14
15
16
17
18
19
158 6.1
142 8.7
124 4.6**
162 4.7
204.0 14.1
192.0 10.2
150 5.6
180.0 11.5
Data are represented as mean SEM (n = 5). Results were analyzed
using one-way ANOVA followed by post hoc Dunnett’s test
Data are represented as mean SEM (n = 5). Results were analyzed
using one-way ANOVA followed by post hoc Dunnett’s test
* p \ 0.05 and ** p \ 0.01 as compared to control value
* p \ 0.01 and ** p \ 0.01 as compared to control value
Conclusions
compound 13 could be considered as a lead compound of
the series for further structural exploration for develop-
ment of therapeutically useful anti-inflammatory agents.
The preliminary structure activity relationship (SAR) data
designate pyridazinone ring as a suitable template to design
a new class of analgesic and anti-inflammatory compounds.
In addition, these compounds displayed excellent gas-
trointestinal safety profile. Further, most of the pyridazi-
none derivatives did not affect normal hemostatic balance.
However, these results were obtained following short-term
exposure with the drugs, other sub-chronic and chronic
level studies are required to confirm the results. It is further
interesting to mention that unlike anti-inflammatory and
analgesic activity, bleeding time and clotting time assays
did not show dose-dependent effects in majority of cases.
Whole blood clotting time assay
The discovery of cardioprotective NSAIDs remains a
challenge in the field of medicinal chemistry and may
prove beneficial in the treatment of chronic inflammatory
diseases. Thus, in addition to bleeding time studies, effect
on whole blood clotting time was also checked for all the
synthesized pyridazinone-based anti-inflammatory agents
to further evaluate their safety . The clotting time observed
in the mice, treated with various compounds at different
doses, was compared with aspirin and vehicle control. The
results obtained are summarized in Table 5.
Materials and methods
Experimental
Most of the tested pyridazinone derivatives did not
affect clotting time significantly except compound 2-(2-
(3,4-dimethoxyphenethylamine)-2-oxoethyl)-4-(4-methox-
Chemistry
yphenyl)-6-phenylpyridazin-3(2H)-one
(16),
which
showed reduction in clotting time in contrast to compounds
7, 17 and 19 which displayed increase in clotting time as
compared to control at 40 mg/kg. Most of the synthesized
compounds showed cardiovascular safety profile in both
anti-platelet and whole blood clotting time assays.
Melting points were determined on a Veego melting point
apparatus and are uncorrected. Infrared (IR) spectra were
recorded on a Perkin-Elmer 882 and Perkin-Elmer spec-
trum RX 1, FT-IR spectrophotometer models using potas-
sium bromide pellets (tmax in cm-1). Proton (1H) and
123

Med Chem Res
carbon (13C) nuclear magnetic resonance spectra were
recorded on a Bruker Avance II 400 MHz spectrometer
using deuterated chloroform (CDCl3) or deuterated
dimethylsulfoxide (DMSO-d6) as solvents containing
tetramethylsilane as internal standard (chemical shifts in d,
ppm). The spin multiplicities are indicated by symbols, s
(singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), p (pentet), m (multiplet) and br (broad). Mass
spectra were obtained on an Applied Biosystems API
2000TM mass spectrophotometer. The purity of compounds
was established by thin-layer chromatography (TLC) and
elemental analyses. Plates for TLC were prepared with
silica gel G according to Stahl’s method (E. Merck) using
ethyl acetate as solvent and activated at 110 °C for 30 min.
Iodine was used to develop the TLC plates. Elemental
analyses were carried out on a Perkin-Elmer-2400 model
CHN analyzer. All solvents were distilled prior to use
according to standard procedures. Anhydrous sodium sul-
fate was used as drying agent.
pyridazinone),
3.19
(dd,
1H,
Jgem = 16.92 Hz,
Jvic = 9.4 Hz, 5-C(H)H, pyridazinone) ppm; Anal. Calcd.
for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99. Found: C,
72.51; H, 5.59; N, 9.88.
4-(3-Bromo-4-methoxyphenyl)-6-phenylpyridazin-3(2H)-
one (4) A vigorously stirred solution of 4-(4-methox-
yphenyl)-6-phenyl-4,5-dihydro-3(2H)-pyridazinone (3, 0.
5 g, 1.78 mmol) in acetic acid (9 ml) was heated to about
60–70 °C. Bromine (0.5 ml) was added drop wise for
15 min. The mixture was stirred for 3 h and then poured
into ice water. The solid that separated was filtered off,
washed with water, dried and recrystallized from ethanol
(95 %).
Yield 96.8 %; mp 220–224 °C; IR (KBr)tmax 3286 (N–
H), 3009 (C–H, aromatic), 2845 (C–H, aliphatic), 1645
(C=O), 1590 (C=C, aromatic), 1497 (C=N), 1018 (C–Br),
1
700 cm-1; H NMR (400 MHz, CDCl3): d = 12.4 (s, 1H,
–NH, pyridazinone), 8.13 (d, 1H, Jm = 2.16 Hz, ArH,
ortho to bromine), 7.94 (dd, 1H, ArH, Jo = 8.6 Hz,
Jm = 2.10 Hz, meta to methoxy), 7.85 (d, 2H,
Jo = 7.38 Hz, ArH), 7.78 (s, 1H, 5-CH, pyridazinone),
7.49–7.43 (m, 3H, ArH), 7.00 (d, 1H, Jo = 8.40 Hz, ArH,
ortho to methoxy), 3.96 (s, 3H, –OCH3) ppm; Anal. Calcd.
for C17H13N2O2Br: C, 57.16; H, 3.67; N, 7.84. Found: C,
57.41; H, 3.59; N, 7.58.
2-(4-Methoxyphenyl)-4-phenyl-4-oxobutanoic acid (2)
A
solution of 2-(4-methoxyphenyl)-4-phenyl-4-oxobutyroni-
trile (1, 1 g, 3.77 mmol) in hydrochloric acid (10 N, 23 ml)
was stirred at room temperature for 2 h and then heated on
a steam bath for 3 h. The reaction mixture was cooled, and
the product obtained was filtered, washed with water and
dried.
Yield = 37.38 %; mp 135–137 °C; 1H NMR
(400 MHz, CDCl3): d = 8.56 (d, 2H, Jo = 8.56 Hz, ArH),
7.56 (t, 1H, Jo = 6.8 Hz, ArH), 7.45 (t, 2H, Jo = 7.64 Hz,
ArH), 7.29 (d, 2H, Jo = 8.68 Hz, ArH), 6.88 (d, 2H,
Jo = 8.72 Hz, ArH), 4.25 (dd, 1H, Jac = 4.28 Hz,
Jbc = 10.00 Hz, –COCH2CHCOOH), 3.87 (dd, 1H,
Jbc = 9.98 Hz, Jab = 18.06 Hz, –COC(H)H–), 3.80 (s,
3H,–OCH3), 3.27 (dd, 1H, Jac = 4.32 Hz, Jab = 18.04 Hz,
–COC(H)H–) ppm; Anal. Calcd. For C17H16O4: C, 71.82;
H, 5.67. Found: C, 71.51; H, 5.79.
4-(3-Bromo-4-methoxyphenyl)-2-(methoxycarbonyl-
methyl)-6-phenylpyridazin-3(2H)-one
(5) Methyl
chloroacetate (1 ml, in excess) was added to a stirred and
refluxing suspension of 4-(3-bromo-4-methoxyphenyl)-6-
phenylpyridazin-3(2H)-one (4, 0.57 g, 1.38 mmol) and
anhydrous potassium carbonate (1.0 g) in ethyl methyl
ketone (40 ml). The reaction mixture was further refluxed
for 2 h with continuous stirring. The completion of the
reaction was monitored by TLC. On completion, the
reaction mixture was cooled and filtered and the solvent
was removed under reduced pressure. Ice cold water was
added to the reacting mixture. The solid thus separated was
filtered off, washed with water, dried and recrystallized
from methanol.
4-(4-Methoxyphenyl)-6-phenyl-4,5-dihydropyridazin-
3(2H)-one (3) Hydrazine hydrate (2 ml) was added to a
solution of 2-(4-methoxyphenyl)-4-phenyl-4-oxobutanoic
acid (2, 0.5 g, 1.76 mmol) in 1-butanol (9.5 ml). The
reaction mixture was refluxed for 6 h, and the reaction was
monitored by TLC for completion. Then, the mixture was
concentrated and cooled. The solid product separated was
filtered off, washed with water, dried and recrystallized
from ethanol (95 %).
Yield 70 %; mp 152–155 °C; 1H NMR (400 MHz,
CDCl3): d = 8.89 (s, 1H, –NH, pyridazinone), 7.69–7.74
(m, 2H, ArH), 7.42 (t, 3H, Jo = 7.16 Hz, ArH), 7.21 (d,
2H, Jo = 8.68 Hz, ArH), 6.88 (d, 2H, Jo = 8.76 Hz, ArH),
3.81–3.77 (m, 4H, –OCH3 and 4-CH, pyridazinone), 3.29
(dd, 1H, Jgem = 16.92 Hz, Jvic = 7.18 Hz, 5-C(H)H,
Yield 44.11 %; mp 120–122 °C; IR (KBr)tmax 3049 (C–
H, aromatic), 2954 (C–H, aliphatic), 1751 (C=O, ester),
1647 (C=O), 1599 (C=C, aromatic), 1439 (CH2 bending),
1232 (C–O–C), 1178 (C–N, amide), 1025 (C–Br, aryl
1
bromide), 1098 (C–O–C), 833 cm-1; H NMR (400 MHz,
CDCl3): d = 8.10 (d, 1H, Jm = 2.20 Hz, ArH, ortho to
bromine), 7.93 (dd, 1H, Jo = 8.68 Hz, Jm = 2.24 Hz,
ArH, meta to methoxy), 7.81 (d, 2H, Jo = 6.52 Hz, ArH),
7.78 (s, 1H, 5-CH, pyridazinone), 7.48–7.46 (m, 3H, ArH),
6.97 (d, 1H, ArH, ortho to methoxy, Jo = 8.72 Hz), 5.04
(s, 2H, –NCH2), 3.95 (s, 3H, –COOCH3), 3.81 (s, 3H,
–OCH3) ppm; 13C NMR (400 MHz, CDCl3): d = 168.1
123

Med Chem Res
(C=O), 160.9 (C=O), 159.5 (ArC), 145.4 (ArC), 139.3
(ArC), 135.0 (ArC), 130.3 (ArC), 129.4 (ArC), 128.9
(ArC), 128.8 (ArC), 128.7 (ArC), 126.1 (2xArC), 126.0
(ArC), 125.8 (ArC), 114.4 (ArC), 113.9 (ArC), 55.4
(OCH3), 54.4 (OCH3), 52.5 (CH2CO) ppm; Anal. Calcd.
for C20H17BrN2O4: C, 55.96; H, 3.99; N, 6.53. Found: C,
55.51; H, 3.89; N, 6.97. ESIMS m/z 429 [MH]?, 431
[MH?2]?.
1182 (C–N), 1054 (C–Br, aryl bromide), 1020 (C–O–C),
1
822, 698 cm-1; H NMR (400 MHz, CDCl3): d = 8.11(d,
1H, Jm = 2.20 Hz, ArH, ortho to bromine), 7.89 (dd, 1H,
Jo = 8.66 Hz, Jm = 2.20 Hz, ArH, meta to methoxy), 7.83
(dd, 2H, Jo = 8.32 Hz, Jm = 1.74 Hz, ArH), 7.77 (s, 1H,
5-CH, pyridazinone), 7.46–7.43 (m, 3H, ArH), 6.96 (d, 2H,
Jo = 8.72 Hz, ArH, ortho to methoxy), 5.04 (s, 2H,
–NCH2), 3.95 (s, 3H, –OCH3), 3.58–3.56 (m, 4H,
–N(CH2)2–, pyrrolidine), 2.04 (p, 2H, –CH2–, pyrrolidine),
1.89 (p, 2H, –CH2–, pyrrolidine) ppm; 13C NMR
(400 MHz, CDCl3): d = 164.40 (C=O), 159.39 (C=O),
156.87 (ArC), 145.09 (ArC), 137.67 (ArC), 135.12 (ArC),
133.55 (ArC), 129.49 (ArC), 129.31 (2xArC), 128.83
(ArC), 127.68 (ArC), 126.37 (2xArC), 126.21 (ArC), 111.
56 (ArC), 111.37 (ArC), 56.36 (OCH3), 55.15 (CH2CO),
46.14(CH2), 45.80 (CH2), 26.19 (CH2), 24.12 (CH2) ppm;
Anal. Calcd. for C23H22BrN3O3: C, 58.98; H, 4.73; N, 8.97.
Found: C, 58.51; H, 4.89; N, 8.47. ESIMS m/z 468.2
[MH]?, 470.2 [MH?2]?.
General procedure for the synthesis of 2-(2-amino-2-ox-
oethyl)-4-(3-bromo-4-methoxyphenyl)-6-phenylpyridazin-3(2H)-
one derivatives (6–9) A mixture of 4-(3-bromo-4-methox-
yphenyl)-2-(methoxycarbonylmethyl)-6-phenylpyridazin-3(2H)-
one (5, 0.2 g, 0.47 mmol) and requisite amine (1 ml, in
excess) was heated at 100 °C with continuous stirring. The
mixture was further stirred with heating for 6 h, reaction
being monitored by TLC. On completion, ice cold water was
added to the reaction mixture. The precipitated compound
was filtered, washed several times with distilled water to
remove any unreacted amine and recrystallized using
methanol to provide desired compounds 6–9.
4-(3-Bromo-4-methoxyphenyl)-2-(2-(4-methylpiperazin-1-
yl)-2-oxoethyl)-6-phenylpyridazin-3(2H)-one (8) Yield
43.29 %;mp 140–141 °C; IR (KBr)tmax 2953 (C–H, ali-
phatic), 1640 (C=O, amide), 1597 (C=C, aromatic), 1445
(CH2 bending), 1256 (C–O–C), 1165 (C–N), 1052 (C–Br,
4-(3-Bromo-4-methoxyphenyl)-2-(2-piperidin-1-yl-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (6) Yield 44.
49 %; mp 190–192 °C; IR (KBr)tmax 3014 (C–H, aro-
matic), 2937 (C–H, aliphatic), 1649 (C=O, amide), 1594
(C=C, aromatic), 1441 (CH2 bending), 1256 (C–O–C),
1174 (C–N, amide), 1049 (C–Br, aryl bromide), 1012 (C–
1
aryl bromide), 1017 (C–O–C), 768, 691 cm-1; H NMR
(400 MHz, CDCl3): d = 8.21 (m, 2H, ArH, ortho to bro-
mine), 7.92–7.85 (m, 3H, ArH), 7.71 (s, 1H, 5-CH, pyri-
dazinone), 7.48–7.43 (m, 3H, ArH), 7.02 (d, 2H, Jo = 8.
60 Hz, ArH, ortho to methoxy), 4.93 (s, 2H, –NCH2), 3.94
(s, 3H, –OCH3), 3.60 (s(br), 2H, –NCH2–, piperazine), 3.43
(s(br), 2H, –NCH2–, piperazine), 2.74 (t, 2H, J = 4.
00 Hz,–NCH2–, piperazine), 2.52 (t, 2H, J = 4.52 Hz,
–NCH2–, piperazine), 2.36 (s, 3H, N–CH3) ppm; Anal.
Calcd. for C24H25BrN4O3: C, 57.95; H, 5.07; N, 11.26.
Found: C, 57.58; H, 5.19; N, 11.57.
1
O–C), 889, 694 cm-1; H NMR (400 MHz, CDCl3): d =
8.11 (d, 1H, Jm = 2.20 Hz, ArH, ortho to bromine), 7.92
(dd, 1H, Jo = 8.66 Hz, Jm = 2.20 Hz, ArH, meta to
methoxy), 7.81 (dd, 2H, Jo = 6.48 Hz, Jm = 1.72 Hz,
ArH), 7.77 (s, 1H, 5-CH, pyridazinone), 7.47–7.42 (m, 3H,
CH, aromatic), 6.95 (d, 1H, Jo = 8.72 Hz, ArH, ortho to
methoxy), 5.13 (s, 2H, –NCH2), 3.95 (s, 3H, –OCH3), 3.60
(t, 2H, J = 5.42 Hz, –NCH2–, piperidine), 3.48 (s(br), 2H,
–NCH2–, piperidine), 1.68 (s(br), 4H, –CH2–, piperidine),
1.59 (s(br), 2H, –CH2–, piperidine) ppm; 13C NMR
(400 MHz, CDCl3): d = 164.15 (C=O), 159.45 (C=O),
156.88 (ArC), 145.11 (ArC), 137.68 (ArC), 135.16 (ArC),
133.56 (ArC), 129.52 (ArC), 129.31 (2xArC), 128.84
(ArC), 127.72 (ArC), 126.36 (ArC), 126.22 (2xArC), 111.
56 (ArC), 111.36 (ArC), 56.36 (OCH3), 54.28 (CH2CO),
45.94 (CH2), 43.29 (CH2), 26.25 (CH2), 25.33 (CH2), 24.
43 (CH2) ppm; Anal. Calcd. for C24H24BrN3O3: C, 59.76;
H, 5.01; N, 8.71 %. Found: C, 59.51; H, 5.39; N, 8.97.
ESIMS m/z 482.2 [MH]?, 484.2 [MH?2]?.
4-(3-Bromo-4-methoxyphenyl)-2-(2-morpholin-4-yl-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (9) Yield 22.22 %
; mp 180–186 °C; IR (KBr)tmax 3065 (C–H, aromatic),
2925 (C–H, aliphatic), 1646 (C=O, amide), 1591 (C=C,
aromatic), 1446 (CH2 bending), 1239 (C–O–C), 1178 (C–
N), 1053 (C–Br, aryl bromide), 1023 (C–O–C), 891,
1
692 cm-1; H NMR (400 MHz, CDCl3): d = 8.11 (d, 1H,
Jm = 2.20 Hz, ArH, ortho to bromine), 7.92 (dd, 1H, Jo =
8.68 Hz, Jm = 2.20 Hz, ArH, meta to methoxy), 7.81 (dd,
2H, Jo = 8.16 Hz, Jm = 1.72 Hz, ArH), 7.78 (s, 1H, 5-CH,
pyridazinone), 7.42–7.49 (m, 3H, ArH), 6.96 (d, 1H, Jo =
8.72 Hz, ArH, ortho to methoxy), 5.12 (s, 2H, –NCH2), 3.
95 (s, 3H, –OCH3), 3.78–3.72 (m, 4H, –O(CH2)2–, mor-
pholine), 3.67 (t, 2H, J = 4.92 Hz, –NCH2–, morpholine),
3.57 (t, 2H, J = 4.54 Hz, –NCH2–, morpholine) ppm; 13C
NMR (400 MHz, CDCl3): d = 164.75 (C=O), 159.41 (C=
4-(3-Bromo-4-methoxyphenyl)-2-(2-pyrrolidin-1-yl-2-
oxoethyl)-6-phenylpyridazin-3(2H)-one (7) Yield 45.
83 %; mp 150–151 °C; IR (KBr)tmax 3051 (C–H, aro-
matic), 2948 (C–H, aliphatic), 1643 (C=O, amide), 1593
(C=C, aromatic), 1444 (CH2 bending), 1255 (C–O–C),
123

Med Chem Res
O), 156.96 (ArC), 145.35 (ArC), 137.73 (ArC), 134.99
(ArC), 133.53 (ArC), 129.49 (ArC), 129.43 (2xArC), 128.
88 (ArC), 127.51 (ArC), 126.45 (ArC), 126.20 (2xArC),
111.61 (ArC), 111.38 (ArC), 66.75 (CH2), 66.35 (CH2),
56.36 (OCH3), 54.03 (CH2CO), 45.32 (CH2), 42.39 (CH2)
ppm; Anal. Calcd. For C23H22BrN3O4: C, 57.04; H, 4.58;
N, 8.68. Found: C, 57.41; H, 4.19; N, 8.59. ESIMS m/z 484.
2 [MH]?, 486.2 [MH?2]?.
128.9 (ArC), 127.3 (2xArC), 126.4 (ArC), 126.1 (ArC),
111.7 (ArC), 111.4 (ArC), 56.4(OCH3), 54.5 (OCH3), 52.6
(CH2CO) ppm; Anal. Calcd. for C20H18N2O3: C, 71.84; H,
5.43; N, 8.38. Found: C, 71.51; H, 5.59; N, 8.57.
General procedure for the synthesis of 2-(2-amino-2-ox-
oethyl)-4-(4-methoxyphenyl)-6-phenylpyridazin-3(2H)-one
derivatives (12–19) A mixture of 2-(methoxycarbonyl-
methyl)-4-(4-methoxyphenyl)-6-phenylpyridazin-3(2H)-one
(11, 0.2 g, 0.51 mmol) and requisite amine (1 ml, in excess)
was heated at 100 °C with continuous stirring. The mixture
was further stirred with heating for 6 h, reaction being
monitored by TLC. On completion, ice cold water was
added to the reaction mixture. The precipitated compound
was filtered, washed several times with distilled water to
remove any unreacted amine and recrystallized using ethyl
acetate to provide desired compounds 12–19.
4-(4-Methoxyphenyl)-6-phenylpyridazin-3(2H)-one (10) Cop-
per (II) chloride (0.30 g, 2.25 mmol) was added to a solution
of 4-(4-methoxyphenyl)-6-phenyl-4,5-dihydro-3(2H)-pyridazi-
none (3, 0.25 g, 0.89 mmol) in acetonitrile (10 ml). The
reaction mixture was refluxed for 3 h, and the reaction was
monitored by TLC. On completion of the reaction, the mix-
ture was added to crushed ice. The resulting solid was filtered
off, washed with water and recrystallized from ethanol.
Yield 60.43 %; mp 212–214 °C; IR (KBr)tmax 3290
(N–H), 3025 (C–H, aromatic), 2911 (C–H, aliphatic), 1650
(C=O), 1593, 1511, 1262 (C–O–C), 1178 (C–N), 1018 (C–
4.1.8.1.4-(4-Methoxyphenyl)-2-(2-piperidin-1-yl-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (12) Yield 34.
73 %, mp 160–161 °C; IR (KBr)tmax 3037 (C–H, aro-
matic), 2940 (C–H, aliphatic), 1643 (C=O, amide), 1604
(C=C, aromatic), 1449 (CH2 bending), 1252 (C–O–C),
O–C), 841, 603 cm-1 1H NMR (400 MHz, CDCl3):
;
d = 7.92 (d, 2H, Jo = 6.88 Hz, ArH, meta to methoxy),
7.84–7.82 (m, 2H, ArH), 7.81 (s, 1H, 5-CH, pyridazinone),
7.48–7.46 (m, 3H, ArH), 7.01 (d, 2H, Jo = 6.84 Hz, ArH,
ortho to methoxy), 3.87 (s, 3H, –OCH3), ppm; Anal. Calcd.
for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C,
73.61; H, 4.89; N, 10.39.
1188 (C–N), 1020 (C–O–C), 835, 698 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 7.87 (d, 2H, Jo = 8.80 Hz, ArH,
meta to methoxy), 7.82–7.80 (m, 2H, ArH), 7.76 (s, 1H,
5-CH, pyridazinone), 7.47–7.38 (m, 3H, ArH), 6.95 (d, 2H,
Jo = 8.80 Hz, ArH, ortho to methoxy), 5.12 (s, 2H, –
NCH2), 3.86 (s, 3H, –OCH3), 3.60 (t, 2H, J = 4.01 Hz, –
NCH2–, piperidine), 3.48 (s(br), 2H, –NCH2–, piperidine),
1.67 (s(br), 4H, –(CH2)2–, piperidine), 1.58 (s(br), 2H, –
CH2–, piperidine) ppm; 13C NMR (400 MHz, CDCl3):
d = 164.3 (C=O), 160.7 (C=O), 159.7 (ArC), 145.1 (ArC),
139.1 (ArC), 135.4 (ArC), 130.3 (2xArC), 129.1 (ArC),
128.8 (2xArC), 126.5 (ArC), 126.2 (2xArC), 125.9 (ArC),
113.7 (2xArC), 55.3 (OCH3), 54.1 (CH2CO), 45.9 (CH2),
43.2 (CH2), 26.2 (CH2), 25.3 (CH2), 24.4 (CH2) ppm;
Anal. Calcd. for C24H25N3O3: C, 71.44; H, 6.25; N, 10.41.
Found: C, 71.51; H, 6.59; N, 10.67. ESIMS m/z 404.3
[MH]?.
2-(Methoxycarbonylmethyl)-4-(4-methoxyphenyl)-6-phenylpyri-
dazin-3(2H)-one (11) Methyl chloroacetate (1 ml, in
excess) was added to a stirred and refluxing suspension of
4-(4-methoxyphenyl)-6-phenylpyridazin-3(2H)-one (10, 1.
0 g, 3.59 mmol) and anhydrous potassium carbonate (1.
0 g) in ethyl methyl ketone (40 ml). The reaction mixture
was further refluxed for 6 h with continuous stirring. The
completion of the reaction was monitored by TLC. On
completion, the reaction mixture was cooled and filtered
and the excess solvent was removed under reduced pres-
sure. Ice cold water was added to the reaction mixture. The
solid thus separated was filtered off, washed with water,
dried and recrystallized using methanol.
Yield 62.50 %; mp 108–110 °C; IR (KBr)tmax 3049 (C–
H, aromatic), 2951 (C–H, aliphatic), 1754 (C=O, ester),
1640 (C=O), 1594 (C=C, aromatic), 1441 (CH2 bending),
1224 (C–O–C), 1180 (C–N), 1032 (C–O–C), 829 cm-1; 1H
NMR (400 MHz, CDCl3): d = 7.88 (d, 2H, Jo = 8.88 Hz,
ArH, meta to methoxy), 7.83–7.80 (m, 2H, ArH), 7.78 (s,
1H, 5-CH, pyridazinone), 7.47–7.45 (m, 3H, ArH), 6.98 (d,
2H, Jo = 9.00 Hz, ArH, ortho to methoxy), 5.04 (s, 2H, –
NCH2), 3.86 (s, 3H, –COOCH3), 3.80 (s, 3H, –OCH3)
ppm; 13C NMR (400 MHz, CDCl3): d = 167.9 (C=O),
159.2 (C=O), 157.1 (ArC), 145.3 (ArC), 137.9 (ArC),
134.8 (ArC), 133.4 (ArC), 129.5 (ArC), 129.4 (ArC),
4.1.8.2.4-(4-Methoxyphenyl)-2-(2-pyrrolidin-1-yl-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (13) Yield 54.
05 %; mp 128–132 °C; IR (KBr)tmax 3051 (C–H, aro-
matic), 2945 (C–H, aliphatic), 1651 (C=O, amide), 1600
(C=C, aromatic), 1439 (CH2 bending), 1254 (C–O–C),
1
1182 (C–N, amide), 1024 (C–O–C), 830, 694 cm-1; H
NMR (400 MHz, CDCl3): d = 7.89 (d, 2H, Jo = 8.72 Hz,
ArH, meta to methoxy), 7.83 (dd, 2H, Jo = 7.80 Hz, Jm
=
1.26 Hz, ArH), 7.79 (s, 1H, 5-CH, pyridazinone), 7.49–7.
43 (m, 3H, ArH), 6.98 (d, 2H, Jo = 8.84 Hz, ArH, ortho to
methoxy), 5.06 (s, 2H, –NCH2), 3.87 (s, 3H, –OCH3), 3.59
(p, 4H, J = 6.84 Hz, –N(CH2)2–, pyrrolidine), 2.04 (p, 2H,
123

Med Chem Res
J = 6.76 Hz, –CH2–, pyrrolidine), 1.91 (p, 2H, J = 6.
80 Hz, –CH2–, pyrrolidine) ppm; 13C NMR (400 MHz,
CDCl3): d = 164.5 (C=O), 160.7 (C=O), 159.7 (ArC),
145.1 (ArC), 139.1 (ArC), 135.4 (ArC), 130.3 (2xArC),
129.2 (ArC), 128.8 (2xArC), 126.4 (ArC), 126.2 (2xArC),
126.0 (ArC), 113.8 (2xArC), 55.3 (OCH3), 55.1 (CH2CO),
46.1 (CH2), 45.8 (CH2), 26.2 (CH2), 24.1 (CH2) ppm;
Anal. Calcd. for C23H23N3O3: C, 70.93; H, 5.95; N, 10.79.
Found: C, 70.51; H, 5.89; N, 10.97. ESIMS m/z 390.4
[MH]?.
129.3 (ArC), 128.8 (2xArC), 126.3 (ArC), 126.2 (2xArC),
126.0 (ArC), 113.8 (2xArC), 66.7 (CH2), 66.4 (CH2), 55.3
(OCH3), 53.9 (CH2CO), 45.3 (CH2), 42.4 (CH2) ppm;
Anal. Calcd. for C23H23N3O4: C, 68.13; H, 5.72; N, 10.36.
Found: C, 68.51; H, 5.89; N, 9.97. ESIMS m/z 406.3
[MH]?.
2-(2-(3,4-Dimethoxyphenethylamine)-2-oxoethyl)-4-(4-meth-
oxyphenyl)-6-phenylpyridazin-3(2H)-one (16) Yield 35.
06 %; mp 162–164 °C; IR (KBr)tmax 3287 (N–H), 3095
(C–H, aromatic), 2935 (C–H, aliphatic), 1654 (C=O,
amide), 1599 (C=C, aromatic), 1513, 1454 (CH2 bending),
1266 (C–O–C), 1178 (C–N), 1023 (C–O–C), 841,
4-(4-Methoxyphenyl)-2-(2-(4-methylpiperazin-1-yl)-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (14) Yield 60.69 %
; mp 166–168 °C; IR (KBr)tmax 2937 (C–H, aliphatic),
1649 (C=O, amide), 1598 (C=C, aromatic), 1458 (CH2
bending), 1244 (C–O–C), 1175 (C–N), 1028 (C–O–C),
833, 698 cm-1; 1H NMR (400 MHz, CDCl3): d = 7.79 (d,
2H, Jo = 8.27 Hz, ArH, meta to methoxy), 7.73 (d, 2H,
Jo = 7.36 Hz, ArH), 7.69 (s, 1H, 5-CH, pyridazinone),
7.39–7.33 (m, 3H, ArH), 6.88 (d, 2H, Jo = 8.76 Hz,
ArH, ortho to methoxy), 5.05 (s, 2H, –NCH2), 3.77 (s,
3H, –OCH3), 3.61 (t, 2H, J = 4.60 Hz, –NCH2–,
piperazine), 3.50 (t, 2H, J = 4.28 Hz, –NCH2–, piper-
azine), 2.42 (t, 2H, J = 4.36 Hz, –NCH2–, piperazine),
2.36 (t, 2H, J = 4.84 Hz, –NCH2–, piperazine), 2.35 (s,
3H, N–CH3) ppm; 13C NMR (400 MHz, CDCl3): d =
164.5 (C=O), 160.8 (C=O), 159.7 (ArC), 145.3 (ArC),
139.1 (ArC), 135.3 (ArC), 130.3 (ArC), 130.1 (ArC),
129.2 (ArC), 128.9 (ArC), 128.8 (ArC), 126.3 (ArC),
126.2 (ArC), 126.0 (ArC), 126.0 (ArC), 114.0 (ArC),
113.8 (ArC), 55.3 (OCH3), 54.7 (CH2CO), 54.4 (CH2),
54.0 (CH2), 46.0 (CH2), 44.7 (CH2), 42.0 (CH3) ppm;
Anal. Calcd. for C24H26N4O3: C, 68.88; H, 6.26; N, 13.
39. Found: C, 68.59; H, 5.89; N, 12.97. ESIMS m/
z 419.4 [MH]?.
1
690 cm-1; H NMR (400 MHz, CDCl3): d = 7.75 (d, 4H,
Jo = 8.60 Hz, ArH), 7.69 (s, 1H, 5-CH, pyridazinone), 7.
42–7.37 (m, 3H, ArH), 6.92 (d, 2H, Jo = 8.68 Hz, ArH,
ortho to methoxy), 6.57–6.51 (m, 3H, ArH, homoveratry-
lamine), 6.41 (s, 1H, –NH), 4.85 (s, 2H, –NCH2), 3.79 (s,
3H, –OCH3), 3.68 (s, 3H, –OCH3), 3.63 (s, 3H, –OCH3), 3.
43 (q, 2H, J = 6.76 Hz, –NHCH2CH2), 2.67 (t, 2H, J = 6.
90 Hz, –NHCH2CH2,) ppm; 13C NMR (400 MHz, CDCl3):
d = 167.1 (C=O), 161.0 (C=O), 159.9 (ArC), 148.9 (ArC),
147.5 (ArC), 145.6 (ArC), 139.3 (ArC), 134.7 (ArC), 131.
1 (ArC), 130.2 (2xArC), 129.6 (ArC), 128.9 (2xArC), 126.
1 (2xArC), 125.9 (ArC), 125.8 (ArC), 120.5 (ArC), 113.9
(2xArC), 111.8 (ArC), 111.1 (ArC), 57.3 (OCH3), 55.7
(OCH3), 55.7 (OCH3), 55.4 (CH2CO), 40.7 (CH2), 34.9
(CH2) ppm; Anal. Calcd. for C29H29N3O5: C, 69.72; H, 5.
85; N, 8.41. Found: C, 69.59; H, 5.59; N, 8.80. ESIMS m/
z 500.3 [MH]?.
4-(4-Methoxyphenyl)-2-(2-(4-phenylpiperazin-1-yl)-2-ox-
oethyl)-6-phenylpyridazin-3(2H)-one (17) Yield 36.45 %
; mp 142–144 °C; IR (KBr)tmax 3049 (C–H, aromatic),
2955 (C–H, aliphatic), 1643 (C=O, amide), 1595 (C=C,
aromatic), 1449 (CH2 bending), 1234 (C–O–C), 1184 (C–
4-(4-Methoxyphenyl)-2-(2-morpholin-4-yl-2-oxoethyl)-6-
phenylpyridazin-3(2H)-one (15) Yield 43.30 %; mp
152–156 °C; IR (KBr)tmax 3040 (C–H, aromatic), 2957
(C–H, aliphatic), 1648 (C=O, amide), 1600 (C=C, aro-
matic), 1450 (CH2 bending), 1240 (C–O–C), 1176 (C–N),
N, amide), 1025 (C–O–C), 845, 690 cm-1 1H NMR
;
(400 MHz, CDCl3): d = 7.89 (d, 2H, Jo = 8.92 Hz, ArH,
meta to methoxy), 7.83 (dd, 2H, Jo = 8.24 Hz, Jm = 1.
72 Hz, ArH), 7.80 (s, 1H, 5-CH, pyridazinone), 7.49–7.43
(m, 3H, ArH), 7.31 (t, 2H, ArH, phenylpiperazine), 7.00–6.
92 (m, 5H, ArH, phenylpiperazine and aromatic ortho to
methoxy), 5.20 (s, 2H, –NCH2), 3.87 (s, 3H, –OCH3), 3.85
(t, 2H, J = 4.84 Hz, –NCH2–, piperazine), 3.75 (t, 2H,
J = 5.04 Hz, –NCH2–, piperazine), 3.30 (t, 2H, J = 4.
76 Hz, –NCH2–, piperazine), 3.23 (t, 2H, J = 4.87 Hz, –
NCH2–, piperazine) ppm; 13C NMR (400 MHz, CDCl3):
d = 164.7 (C=O), 160.8 (C=O), 159.7 (ArC), 145.4 (ArC),
139.1 (ArC), 135.2 (ArC), 130.3 (2xArC), 129.3 (2xArC),
128.8 (2xArC), 126.3 (ArC), 126.2 (2xArC), 126.0 (ArC),
1028 (C–O–C), 835, 698 cm-1 1H NMR (400 MHz,
;
CDCl3): d = 7.79 (d, 2H, Jo = 6.80 Hz, ArH), 7.75–7.72
(m, 2H, ArH), 7.70 (s, 1H, 5-CH, pyridazinone), 7.40–7.34
(m, 3H, ArH), 6.90 (d, 2H, Jo = 6.84 Hz, ArH), 5.05 (s,
2H, –NCH2), 3.78 (s, 3H, –OCH3), 3.69 (t(br), 2H, –
OCH2–, morpholine), 3.64 (t(br), 2H, –OCH2–, morpho-
line), 3.60 (t(br), 2H, –NCH2–, morpholine), 3.50 (t(br),
2H, –NCH2–, morpholine) ppm; 13C NMR (400 MHz,
CDCl3): d = 164.9 (C=O), 160.8 (C=O), 159.7 (ArC),
145.4 (ArC), 139.2 (ArC), 135.2 (ArC), 130.3 (2xArC),
123

Med Chem Res
Biological activity
116.8 (ArC), 113.8 (2xArC), 55.4 (OCH3), 54.0 (CH2CO),
49.6 (CH2), 49.3 (CH2), 44.9 (CH2), 42.0 (CH2) ppm;
Anal. Calcd. for C29H28N4O3: C, 72.48; H, 5.87; N, 11.66.
Found: C, 72.59; H, 5.59; N, 11.97. ESIMS m/z 481.4
[MH]?.
Anti-inflammatory activity
All the synthesized derivatives were evaluated for anti-
inflammatory activity using carrageenan-induced hind paw
edema model (Abouzid and Bekhit, 2008) in male Wistar
rats (120–130 g). Animals were provided with regular
rodent pellet diet (Ashirwad Industries, Chandigarh) and
purified water ad libitum. The food was withdrawn 1 day
before the experiment, but allowed free access to water.
The experimental study protocol was duly approved by
Institutional Animal Ethics Committee (IAEC), Panjab
University, and strictly carried out in accordance with the
guidelines of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA),
Government of India. Acute edema in the hind paws of the
rats was induced by injecting 0.1 ml of freshly prepared
1 % solution of carrageenan in distilled water under the
plantar aponeurosis of right hind paw. The suspensions of
20 and 40 mg/kg dose of the respective compounds, uni-
formly dispersed in distilled water by adding 0.1 ml of
Tween 80, were given to test animals orally an hour prior
to the administration of carrageenan. The control group
received the same experimental handling as test group
except that equivalent doses of vehicle alone were
administered by the same route in place of test compounds.
The paw volumes were measured using plethysmometer
(UGO BASILE) before and after 30, 60, 90, 120, 180 and
240 min of injecting carrageenan. Indomethacin and cele-
coxib were used as the standard anti-inflammatory drugs.
The percent inhibition of inflammation was calculated
using following formula:
2-(2-(4-(4-Fluorophenyl)piperazin-1-yl)-2-oxoethyl)-4-(4-
methoxyphenyl)-6-phenylpyridazin-3(2H)-one (18) Yield
50.30 %; mp 158–160 °C; IR (KBr)tmax 2955 (C–H, ali-
phatic), 1642 (C=O, amide), 1591 (C=C, aromatic), 1509,
1457 (CH2 bending), 1386 (C–F), 1247 (C–O–C), 1176
1
(C–N, amide), 1035 (C–O–C), 870, 697 cm-1; H NMR
(400 MHz, CDCl3): d = 7.80–7.78 (m, 4H, ArH and ArH,
meta to methoxy), 7.69 (s, 1H, 5-CH, pyridazinone), 7.
45–7.41 (m, 3H, ArH), 6.94–6.87 (m, 4H, ArH,
phenylpiperazine and ArH, ortho to methoxy), 6.73–6.70
(m, 2H, ArH, phenylpiperazine), 4.90 (s, 2H, –NCH2), 3.80
(s, 3H, –OCH3), 3.16 (t, 4H, J = 3.30 Hz, –N(CH2)2–,
piperazine), 3.09 (t, 4H, J = 3.32 Hz, –N(CH2)2–, piper-
azine) ppm; 13C NMR (400 MHz, CDCl3): d = 185.6 (C=
O), 160.6 (C=O), 147.2 (ArC), 144.5 (ArC), 138.8 (ArC),
135.2 (ArC), 130.2 (2xArC), 129.2 (2xArC), 128.8
(2xArC), 126.4 (ArC), 126.0 (2xArC), 125.8 (ArC), 118.7
(ArC), 118.7 (ArC), 115.7 (ArC), 115.5 (ArC), 113.7
(2xArC), 55.3 (CH2), 47.7 (CH2), 43.3 (CH2) ppm; Anal.
Calcd. for C29H27N4O3F: C, 69.86; H, 5.46; N, 11.24.
Found: C, 69.99; H, 5.59; N, 10.97. ESIMS m/z 499.3
[MH]?.
2-(2-(4-(4-Nitrophenyl)piperazin-1-yl)-2-oxoethyl)-4-(4-
methoxyphenyl)-6-phenylpyridazin-3(2H)-one (19) Yield
38.17 %; mp 176–178 °C; IR (KBr)tmax 3049 (C–H, aro-
matic), 2841 (C–H, aliphatic), 1636 (C=O, amide), 1592
(C=C, aromatic), 1457 (CH2 bending), 1383, 1332 (NO2),
1256 (C–O–C), 1171 (C–N, amide), 1033 (C–O–C), 834,
ꢀ
ꢁ
a À x
b À y
% Inhibition of Inflammation ¼ 100 1 À
1
695 cm-1; H NMR (400 MHz, CDCl3): d = 8.06 (d, 2H,
where x and a are the mean foot volumes of the rats before
and after the administration of carrageenan injection,
respectively, treated with test compounds or standard drug,
whereas y and b are the mean foot volumes of the rats
before and after the administration of carrageenan,
respectively, in the control group. Animals were also
observed for 24 h, and the mortality rate was recorded for
each group at the end of the observation period.
Jo = 9.40 Hz, ArH, meta to methoxy), 7.91–7.88 (m, 4H,
ArH), 7.86 (s, 1H, 5-CH, pyridazinone), 7.47–7.41 (m, 3H,
ArH), 6.95 (d, 2H, Jo = 8.84 Hz, ArH, phenylpiperazine,
meta to nitro), 6.88 (d, 2H, Jo = 9.44 Hz, ArH, ortho to
methoxy), 4.85 (s, 2H, –NCH2), 3.84 (s, 3H, –OCH3), 3.50
(t, 4H, J = 4.84 Hz, –N(CH2)2–, piperazine), 3.07 (t, 4H,
J = 5.04 Hz, –N(CH2)2–, piperazine) ppm; 13C NMR
(100 MHz, CDCl3): d = 170.5 (C=O), 164.1 (C=O), 160.1
(ArC), 158.7 (ArC), 154.3 (ArC), 143.5 (ArC), 138.0
(ArC), 134.8 (ArC), 129.9 (2xArC), 128.6 (ArC), 128.3
(2xArC), 126.1 (ArC), 125.6 (2xArC), 125.3 (2xArC),
125.1 (ArC), 113.2 (2xArC), 112.4 (2xArC), 55.5 (OCH3),
54.8 (CH2CO), 45.8 (CH2), 44.8 (CH2), 43.6 (2xCH2)
ppm; Anal. Calcd. for C29H27N5O5: C, 66.27; H, 5.18; N,
13.32. Found: C, 66.59; H, 5.29; N, 12.97. ESIMS m/z 526.
2 [MH]?.
Gastric ulcerogenic effect
Rats were killed under deep ether anesthesia 24 h after the
anti-inflammatory experiment, and their stomachs were
removed. The abdomen of each rat was opened through
great curvature and examined for lesions or bleedings using
a hand lens. For each stomach, the mucosal damage was
assessed according to the following scoring system: 0.5:
123

Med Chem Res
Anti-platelet activity
redness; 1.0: spot ulcers; 1.5: hemorrhagic streaks; 2.0:
ulcers [3 but B5; 3.0: ulcers [5. The mean score of each
treated group minus the mean score of the control group
was regarded as severity index of the gastric mucosal
damage (Chan et al., 1995).
Anti-platelet activity was determined by using the tail
transaction bleeding test as reported (Wang et al., 2004;
Tanaka et al., 1998). Bleeding time of male mice (Laca
strain) weighing 20–24 g was determined by administering
compounds and aspirin (standard drug) suspended in car-
boxy methyl cellulose orally at 20 and 40 mg/kg or vehicle
control. After 1 h, 3 mm of the tails of the mice under light
diethyl ether anesthesia was transected and blood was
dripped on filter paper. The duration of bleeding was
recorded.
Analgesic activity
Anti-nociceptive activity of compounds against noxious
chemical stimuli was evaluated by acetic acid-induced
writhing test in Laca mice (20–25 g) as described previ-
ously (Ozkay et al., 2011). Sixty min after the p.o.
administration of saline or compounds, mice were treated
with an aqueous solution of acetic acid (0.6 % v/v, i.p.) at a
dose of 10 ml/kg to induce contractions. After 5 min, the
number of abdominal constrictions and stretches during the
following 10 min was recorded. After treatments, a sig-
nificant reduction in the number of writhes was considered
as a positive anti-nociceptive response. The percentage
protection against writhing was calculated according to the
following equation:
Whole blood clotting time assay
The whole blood clotting time test was essentially per-
formed as described by Mackenzie et al. (Mackenzie et al.,
1971). After the tail bleeding test was terminated, the tail
was wiped dry. One of the tail veins was sliced with a razor
blade, and the first drop of blood was allowed to fill a non-
siliconized glass capillary tube. The tube was regularly
tilted and checked for fluidity of the blood. When the blood
ceased to flow by tilting or when fragmented blood coagula
were observed to adhere to the inner walls, the tube was
broken at 10-s intervals until a fibrin strand was seen
between the broken ends. The whole blood clotting time
was measured from the moment of filling the tube to the
appearance of the first fibrin strands.
ðControl mean À Treated meanÞ
Protection % ¼
 100
Control mean
In vitro COX Inhibitory activity
The inhibitory activities of compounds for COX-1 and
COX-2 enzymes were assayed using the COX Inhibitor
Screening Assay Kit (Cayman No: 560101) according to
the protocol recommended by the supplier. This assay is
based on the competition between PGs and a PG-acetyl-
cholinesterase (AChE) conjugate (PG tracer) for a limited
amount of PG antiserum. Because the concentration of the
PG tracer is held constant while the concentration of PG
varies, the amount of PG tracer that is able to bind to the
PG antiserum will be inversely proportional to the con-
centration of PG in the well. This rabbit antiserum-PG
(either free or tracer) complex binds to a mouse mono-
clonal anti-rabbit antibody that has been previously
attached to the well. The plate is washed to remove any
unbound reagents, and then Ellman’s reagent (which con-
tains the substrate to AChE) is added to the well. The
product of this enzymatic reaction has a distinct yellow
color and absorbs strongly at 412 nm. The intensity of this
color, determined spectrophotometrically, is proportional
to the amount of PG tracer bound to the well, which is
inversely proportional to the amount of free PG present in
the well during the incubation.
Acknowledgments The authors are thankful to University Grants
Commission, India, and Council of Scientific and Industrial Research,
India, for providing financial support.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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