Synthesis of Some Novel 2,6-Disubstituted Pyridazin-3(2H)-one Derivatives as Analgesic, Anti-Inflammatory, and Non-Ulcerogenic Agents

Article abstract of DOI:10.1002/ardp.201700093

Some novel 2,6-disubstituted pyridazine-3(2H)-one derivatives were synthesized and evaluated for in vitro cyclooxygenase-2 (COX-2) inhibitory efficacy. Compounds 2-{[3-(2-methylphenoxy)-6-oxopyridazin-1(6H)-yl]methyl}-1H-isoindole-1,3(2H)-dione (5a), 2-pr

Full text of DOI:10.1002/ardp.201700093

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Full Paper
Synthesis of Some Novel 2,6-Disubstituted Pyridazin-3(2H)-one
Derivatives as Analgesic, Anti-Inflammatory, and Non-Ulcerogenic
Agents
Tamer H. Ibrahim1, Yasser M. Loksha
and Mohamed M. Said2
1, Hosam A. Elshihawy2, Dina M. Khodeer3,
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Industries, Sinai
University, Al-Arish, North Sinai, Egypt
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia,
Egypt
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia,
Egypt
2
3
Some novel 2,6-disubstituted pyridazine-3(2H)-one derivatives were synthesized and evaluated for in
vitro cyclooxygenase-2 (COX-2) inhibitory efficacy. Compounds 2-{[3-(2-methylphenoxy)-6-oxopyrida-
zin-1(6H)-yl]methyl}-1H-isoindole-1,3(2H)-dione (5a), 2-propyl-6-(o-tolyloxy)pyridazin-3(2H)-one (6a),
and 2-benzyl-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazin-3(2H)-one (16a) showed the most potent
COX-2 inhibitory activity with IC50 values of 0.19, 0.11, and 0.24 mM, respectively. The synthesized
compounds with the highest COX-2 selectivity indices were evaluated for their anti-inflammatory,
analgesic, and ulcerogenic activities. Compounds 6a and 16a demonstrated the most potent and
consistent anti-inflammatory activity over the synthesized compounds, which was significantly higher
than that of celecoxib in the carrageenin rat paw edema model and with milder ulcer scoring than
that of indomethacin in the ulcerogenicity screening.
Keywords: Analgesic activity / Anti-inflammatory / COX-1 / COX-2 / Pyridazinone
Received: March 19, 2017; Revised: June 29, 2017; Accepted: July 4, 2017
DOI 10.1002/ardp.201700093
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
side effects associated with long-term use of NSAIDs proved to
be major drawback restricting their clinical usefulness [1]. The
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) comprise an
important class of pharmacological agents with extensive
therapeutic applications in the treatment of fever, inflamma-
tion, and pain. Their use in the management of inflammatory
conditions such as rheumatoid arthritis (RA) and osteoarthritis
(OA) is widely established. However, the gastrointestinal (GI)
underlyingmechanismofNSAID-associatedGIadverseeffectsis
thesuppressionofprostaglandinbiosynthesis from arachidonic
acid by nonselective inhibition of the two isoforms of
cyclooxygenase (COX) enzyme (COX-1 and COX-2) [2]. COX-1
isconstitutivelyexpressedandwidelydistributedinmosttissues
providing prostaglandins that are required for homeostatic
functions, like cytoprotection of gastric mucosa, regulation of
renal hemodynamics, and platelet aggregation. COX-2 is
inducibly expressed in response to various stimuli, like pro-
inflammatory cytokines (IL-1b, TNFa), lipopolysaccharides, and
growth factors, resulting in the production of prostaglandins
implicated in inflammation and carcinogenesis [3, 4]. The
COX-1 and COX-2 isoforms share 60–65% sequence identity,
confirming the belief that the COX isoforms are structurally
Correspondence: Dr. Yasser M. Loksha, Department of Pharma-
ceutical Chemistry, Faculty of Pharmacy and Pharmaceutical
Industries, Sinai University, Al-Arish, North Sinai 31751, Egypt.
E-mail: yml@su.edu.eg
Fax: 0020 683336847
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homologous and quite superimposable. Subtle structural
differences within the binding sites of the COX isoforms
resulted in increased overall volume of the COX-2 active site by
almost 20% compared to that of COX-1, which provide broader
substrate flexibility and recognition in the COX-2 active site,
explaining the ability to detect drugs selective for COX-2 in
pharmacological screens [1, 2].
The tricyclic selective COX-2 inhibitors (coxibs) like cele-
coxib, rofecoxib, and valdecoxib (Fig. 1) showed remarkable
gastrointestinal safety profile with the same anti-inflamma-
tory efficacy as NSAIDs. However, concerns about the
cardiovascular safety of coxibs have been raised after
withdrawal of rofecoxib and valdecoxib from the market
[5, 6]. It was suggested that the presence of certain moieties
within the chemical structure of coxibs is responsible for the
increased cardiovascular risk [7]. Therefore, continuous
research on the development of novel anti-inflammatory
agents with increased COX-2 selectivity while moving away
from the classic coxib structures could provide agents with
improved cardiovascular and GI safety profiles [8].
3(2H)-Pyridazinone derivatives are important class of hetero-
cyclic compounds mainly due to their diverse pharmacological
activities such as antihypertensive, antidiabetic, and antiulcer
activities [9]. Pyridazinone skeleton was considered to be an
excellent template for novel selective COX-2 inhibitors such as
Syntex compound and RS-57067 (Fig. 1) [5, 6]. Furthermore,
various 3(2H)-pyridazinone derivatives have been reported as
analgesic and anti-inflammatory agents with low ulcerogenic
effect [5, 6, 10]. Structure activity relationship (SAR) studies
employing pyridazinone system revealed that N-substitution
was required for good in vitro COX-2 inhibitory activity [11].
This was confirmed further by several reports of N-substituted
pyridazinone derivatives that exhibited anti-inflammatory
activity with very low ulcerogenicity [12–15]. Additionally,
€ €
Sukuroglu et al. [16] reported that substitution at the
6-position of the pyridazinone ring participated in improving
the analgesic and anti-inflammatory activity.
We report the synthesis and in vitro evaluation of COX-1/
COX-2 inhibitory activity of some novel 2,6-disubstituted
pyridazinone derivatives. The analgesic, anti-inflammatory
activities in addition to gastric ulcerogenic effect were also
investigated for some of the title compounds with the highest
selectivity on COX-2 isoform.
Results and discussion
Chemistry
3-Aryloxy-6-chloropyridazines (2a–d) were synthesized by the
same procedure described by Loksha et al. [17] in which
nucleophilic aromatic substitution of one chlorine atom in
3,6-dichloropyridazine (1) with aryloxy group by treatment
with sodium phenoxide derivatives in dry dimethylforma-
mide. Compounds 2a–c were previously prepared by Tamura
and Jojima [18] through refluxing compound 1, phenol
derivatives, and sodium metal in dry benzene in the presence
of catalytic amount of copper powder for 3 h. Compound 2d
was prepared by Venkatraj et al. [19] by the same method
reported by Loksha et al. [17]. Hydrolysis of compounds 2a–d
by refluxing in acetic acid furnished 6-aryloxypyridazin-3(2H)-
ones (3a–d). This method was adopted by Venkatraj et al. [19]
for the synthesis of 3a,d and by Thuillier et al. [20] for the
preparation of 3c. N-Alkylation of 3a–d in dry dimethylfor-
mamide and anhydrous potassium carbonate was achieved by
coupling with benzyl bromide derivatives, N-bromomethylph-
thalimide, and/or n-propyl bromide to afford 2-arylmethyl-6-
aryloxypyridazin-3(2H)-ones (4a–h), 2-{[3-(aryloxy)-6-oxopyr-
idazin-1(6H)-yl]methyl}-1H-isoindole-1,3(2H)-diones (5a–d),
and 2-propyl-6-aryloxypyridazin-3(2H)-ones (6a–d), respec-
tively (Scheme 1).
3-Substituted amino-6-chloropyridazines 7a,b were pre-
pared in 55% and 83% yields, respectively, by fusing 1 with
the appropriate amines at 70°C. Yoneda et al. [21] reported
the synthesis of 7a,b by heating 1 with benzylamine for 4 h at
90°C to get 7a and by refluxing 1 with 4-chloroaniline in
ethanol for 3 h to obtain 7b. Hydrolysis of 7a by refluxing in
Figure 1. Some coxibs, pyridazinone-based selective COX-2
inhibitors, and compound 6a.
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potassium carbonate afforded 2-arylmethyl-6-arylaminopyr-
idazin-3(2H)-ones (9a–d) (Scheme 2).
Stirring of compound 1 in 40% aqueous solution of
N,N-dimethylamine at room temperature was performed
to furnish 6-chloro-N,N-dimethylpyridazin-3-amine (10).
6-(Dimethylamino)pyridazin-3(2H)-one (11) was prepared by
hydrolyzing 10 in refluxing acetic acid in the presence of
sodium acetate for 18 h. Coupling 11 with 4-bromobenzyl
bromide in dry dimethylformamide and anhydrous potassium
carbonate afforded 2-(4-bromobenzyl)-6-(dimethylamino)-
pyridazin-3(2H)-one (12) (Scheme 3).
3-Chloro-6-hydrazinylpyridazine (13) was synthesized
according to the reported procedure by Farina et al. [22].
Treatment of compound 13 with acetylacetone afforded 3-
chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (14) as pre-
€ €
viously described by Sukuroglu et al. [16]. Hydrolysis of
compound 14 by refluxing in acetic acid in presence of sodium
acetate furnished6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine-3
(2H)-one (15) as reported by Banoglu et al. [23]. N-Alkylation of
15 with benzyl bromide, 4-bromobenzylbromide, and/or N-
bromomethylphthalimide in dry dimethylformamide in the
Scheme 1. Synthesis of compounds 2 to 6a–d. Reagents and
conditions: (a) NaH, DMF, RT; (b) AcOH, reflux 7 h; (c) anhyd.
K2CO3, DMF, RT.
acetic acid in the presence of sodium acetate furnished
6-(benzylamino)pyridazin-3(2H)-one (8a). Compound 7b was
hydrolyzed by refluxing in acetic acid to furnish 6-(4-
chlorophenylamino)pyridazin-3(2H)-one (8b). Compounds
8a,b were previously prepared by Yoneda et al. [21] via
acidic hydrolysis of 3-benzylamino-6-benzyloxypyridazine
and/or 3-(4-chloroanilino)-6-benzyloxypyridazine. Coupling
of 8a,b with benzyl bromide and/or 4-bromobenzyl bromide
in dry dimethylformamide in the presence of anhydrous
Scheme 2. Synthesis of compounds 9a–d. Reagents and
conditions: (a) Fusion, 70°C, 3.5 h (7a), 1.5 h (7b); (b) AcOH,
AcONa, reflux 18 h (8a), AcOH, reflux 10 h (8b); (c) anhyd. K2CO3,
DMF, RT.
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Scheme 3. Synthesis of compound 12. Reagents and conditions:
(a) Neat, RT, 3 h; (b) AcOH, AcONa, reflux 18 h; (c) 4-Br-C6H4-CH2-
Br, anhyd. K2CO3, DMF, RT, 48 h.
presence of anhydrous potassium carbonate furnished the
desired compounds 16a–c (Scheme 4).
Biological evaluation
In vitro COX-1 and COX-2 inhibition assay
All newly synthesized title compounds 4a–h, 5a–d, 6a–d, 9a–d,
12, and 16a–c were screened for in vitro COX-1/-2 inhibitory
potency adopting an enzyme immunoassay (EIA) kit (Cayman
Chemical Company, USA). IC50 values for the screened com-
poundsweredeterminedusingcelecoxib asthereference drug.
Furthermore, COX-2 selectivity index (COX-1 IC50/COX-2 IC50
)
was calculated for each compound. IC50 values for COX-1 and
COX-2 inhibition as well as the selectivity index are listed in
Table 1. From the assay results, it was obvious that all
compounds showed moderately potent inhibitory activity on
COX-2 enzyme with IC50 values that lower than those on COX-1
inrangeof0.11–1.12 mMforCOX-2and2.98–12.91 mMforCOX-
1. Among the tested compounds, 6a exhibited the highest
activity with IC50 value of 3.67 mM for COX-1 and 0.11 mM for
COX-2, demonstrating the highest COX-2 selectivity index of
33.3. Compounds 4b, 5a,c,d, 6a–d, 9c,d, and 16a,c with the
highest COX-2 selectivity indices were selected for further
pharmacological evaluation of in vivo analgesic, anti-inflam-
matory, and gastric ulcerogenic activities.
Scheme 4. Synthesis of compounds 16a–c. Reagent and
conditions: (a) 30% NH4OH, reflux 1.5 h; (b) absolute ethanol,
reflux 4h; (c) AcOH, AcONa, reflux 5 h; (d) anhydrous K2CO3,
DMF, RT, 24 h.
celecoxib and in good correlation with its anti-inflammatory
activity. Compounds 4b, 5a,d, 6a, 9d, and 16a displayed
moderate analgesic activity measured as % protection against
acetic acid-induced writhing ranging from 52.17 ꢀ 2.90 to
60.8 ꢀ 5.7.
Analgesic activity
The selected new compounds 4b, 5a,c,d, 6a–d, 9c,d, and 16a,c
were screened for analgesic activity at 10 mg/kg using acetic
acid induced writhing test as reported by Mishra et al. [24] and
compared with a reference drug celecoxib with the same dose
level (Table 2). Mouse abdominal constrictions were measured
as writhing response, and % protection was calculated. The
most potent analgesic activity was exhibited by compound 6d
with % protection 72.4 ꢀ 10.04, which was superior to
Anti-inflammatory activity
The anti-inflammatory activity of selected compounds 4b, 5a,
c,d, 6a–d, 9c,d, and 16a,c was evaluated by using carrageenin-
induced rat paw edema bioassay method reported by Winter
et al. [25]. Subplantar injection of 0.1 mL of 1% carrageenin
produced edema in rat paw which presented as increase in the
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Table 1. The results of in vitro COX-1 and COX-2 inhibition
IC50 and selectivity index (SI).
Table 2. Analgesic activity by acetic acid induced writhing
in mice.
COX-1
IC50
(mM)
COX-2
IC50
(mM)
COX-2
Number of
writhes in
30 min
Analgesic
activity
(% protection)
a)
a)
selectivity
Compound
indexb) (SI)
Compound
Celecoxib
14.8
3.9
0.05
0.8
296
4.8
Control
Celecoxib
4b
5a
5c
5d
6a
6b
6c
6d
9c
9d
16a
16c
69 ꢀ 4.94
40 ꢀ 2.08a)
31 ꢀ 3.47a)
33 ꢀ 2a)
0
Diclofenac
sodium
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
6a
6b
6c
6d
9a
9b
9c
9d
12
16a
16b
16c
42.04 ꢀ 3.01a)
55.07 ꢀ 5.02a)
52.17 ꢀ 2.90a)
40.5 ꢀ 2.89a)
60.8 ꢀ 5.7a)
4.51
3.98
4.67
7.11
10.21
8.53
6.54
8.42
4.86
9.45
8.65
4.98
3.67
2.98
4.29
5.14
7.24
5.24
11.42
12.91
6.33
5.22
6.24
4.75
0.61
0.22
0.54
0.87
1.12
0.78
0.83
0.98
0.19
0.96
0.69
0.33
0.11
0.16
0.26
0.38
0.86
0.52
1.04
0.86
0.74
0.24
0.68
0.34
7.3
18
8.6
8.17
9.1
10.93
7.8
8.5
25.5
9.84
12.5
15
33.3
18.6
16.5
13.5
8.4
10.07
10.98
15.01
8.5
41 ꢀ 2a)
27 ꢀ 4a)
31 ꢀ 3.47a)
49 ꢀ 0.58a)
48 ꢀ 1.16a)
19 ꢀ 6.94a),b)
51 ꢀ 2a)
55.07 ꢀ 5.02a)
28.9 ꢀ 0.83a)
30.4 ꢀ 1.67a)
72.4 ꢀ 10.04a),b)
26.08 ꢀ 2.9a)
55.07 ꢀ 5.02a)
56.5 ꢀ 0.83a)
31.8 ꢀ 2.90a)
31 ꢀ 3.47a)
30 ꢀ 0.58a)
47 ꢀ 2a)
Each value is the mean ꢀ SEM at p < 0.01 compared with
control. Data were analyzed by using one-way ANOVA
followed by Bonferroni test.
a)Significantly different from normal group.
b)Significantly different from celecoxib treated group.
and selectivity where 6a has the highest in vitro activity as
described earlier, and 16a gave COX-2 inhibition IC50 0.24 mM
and 21.7 selectivity index. Although less than celecoxib, the
N-phthalimidomethyl derivative 5d exhibited rapid onset
after 1 h and sustained anti-inflammatory effect after 3 h with
65.1% inhibition of edema. On the other hand, compounds
4b, 5a, 5c, 5d, 6b–d, 9c, 9d, and 16c showed less rapid anti-
inflammatory activity that began in 2 h and continued to after
3 h. Compound 5a was unexpectedly less active than 16a in
spite of having higher in vitro COX-2 selectivity and inhibitory
effect. This could be possibly due to less favorable pharmaco-
kinetic profile of 5a compared to 16a, leading to decreased
plasma concentration needed for optimal anti-inflammatory
activity.
21.7
9.1
13.9
a) The concentration of test compound producing 50%
inhibition of ovine COX-1 and COX-2 enzymes. The result
is the mean of two determinations obtained by assay of
enzyme kits obtained from Cayman Chemicals, Inc. (Ann
Arbor, MI, USA).
b) The in vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
paw volume in all the animals of various groups. Percentage
increase in left hind paw, in comparison to the uninjected
right hind paw, was calculated and expressed as the amount
of inflammation. The test compounds were orally adminis-
tered at equivalent doses of the reference drug celecoxib
(10 mg/kg, p.o.) after 1 h from induction of inflammation by
carrageenin. All the selected test compounds exhibited
significant (p < 0.01) reduction of rat paw edema, at 3 h,
when compared to that of the control group (Table 3). Among
all of the tested compounds, 6a and 16a demonstrated the
most potent and consistent anti-inflammatory activity with %
inhibition of edema 82.5 and 84, respectively that was
significantly (p < 0.01) higher than celecoxib with rapid onset
of action after 1 h, and sustained duration until the third hour
after the administration of the compound. The anti-inflam-
matory activity results of the previous compounds were in
good correlation with their in vitro COX-2 inhibitory potency
Gastric ulcerogenicity
Acute ulcerogenicity of the selected target compounds 4b, 5a,
c,d, 6a–d, 9c,d, and 16a,c was evaluated 6 h after their
administration to rats in a dose of 10 mg/kg. The ulcerative
effect was assessed by visual inspection of the resulting lesions
and compared to a known ulcerogenic drug, indomethacin.
All the test compounds demonstrated significantly less ulcer
scoring than exhibited by indomethacin, except for com-
pounds 6c and 6d that exhibited ulcer scores that were
equivalent to indomethacin (Table 4). Compounds 5c and 5d
displayed the best GI safety profile in rats with ulcer scoring
0.83 ꢀ 0.17 and 0.67 ꢀ 0.17, respectively.
All our target N-substituted pyridazinone derivatives
exhibited moderately potent in vitro inhibitory activity on
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Table 3. Percentage change and inhibition in rat paw edema after carrageenin subplantar injection.
Anti-inflammatory
activity (% inhibition
% Change in paw volume (mean ꢀ SEM)
of edema)
Compound
1 h
2 h
3 h
3 h
Control
Celecoxib
4b
5a
5c
5d
6a
6b
6c
6d
9c
9d
16a
16c
0.23 ꢀ 0.03
0.15 ꢀ 0.02
0.15 ꢀ 0.02
0.19 ꢀ 0.01
0.42 ꢀ 0.01
0.07 ꢀ 0.01a)
0.09
0.40 ꢀ 0.01
0.19 ꢀ 0.02
0.19 ꢀ 0.01
0.19
0.6 ꢀ 0.03
2 ꢀ 0.12
0.38 ꢀ 0.06a)
0.30 ꢀ 0.01a)
0.31 ꢀ 0.01a)
0.49 ꢀ 0.01a)
0.42 ꢀ 0.01a)
0.20 ꢀ 0.01a),b)
0.50 ꢀ 0.01b)
0.28 ꢀ 0.01a)
0.12 ꢀ 0.02a)
0.31 ꢀ 0.01a)
0.50 ꢀ 0.01b)
0.10a),b)
0.47 ꢀ 0.07a)
0.33 ꢀ 0.07a)
0.37 ꢀ 0.03a)
0.55 ꢀ 0.03a)
0.57 ꢀ 0.03a)
0.18 ꢀ 0.02a),b)
0.55 ꢀ 0.03a)
0.38 ꢀ 0.01a)
0.32 ꢀ 0.01a)
0.47 ꢀ 0.03a)
0.67 ꢀ 0.07a)
0.15 ꢀ 0.03a),b)
0.53 ꢀ 0.03a)
69.6
75.7
74.2
65.9
65.1
82.5b)
65.7
73.6
76.3
69.6
60.6
84.0b)
66.6
0.19 ꢀ 0.01
0.05 ꢀ 0.02a)
0.19 ꢀ 0.01
0.39 ꢀ 0.01a)
Each value is the mean ꢀ SEM at P < 0.01 compared with control.
Data were analyzed by using one-way ANOVA followed by Bonferroni test.
a)Significantly different from control group.
b)Significantly different from celecoxib treated group.
both COX-1 and COX-2 isoforms, emphasizing the importance
of N-substitution. It was clear that compounds 4b, 5a,c,d, and
6a–d containing aryloxy substituents in position 6 of the
pyridazinone ring generally demonstrated better COX-2
selectivity and anti-inflammatory activity than compounds
9c,d which contain arylamino substituents at the same
position. Moreover, the methyl group in ortho position of
the aryloxy moiety in compounds 5a and 6a demonstrated
higher COX-2 selectivity and marked anti-inflammatory
activities than the other derivatives with 3-methyl (in
compounds 4b,f, 5b, and 6b), 4-methyl group (in compounds
4c,g, 5c, and 6c) and/or 2,6-dimethyl group (in compounds 4d,
h, 5d, and 6d). Also, decreasing the bulkiness of the
substituent at position N2 of pyridazinone ring to propyl
group in compounds 6a–d generally improved the COX-2
potency compared with the other bulkier N-arylmethyl
derivatives 4a,c–g, 5c,d, 9a–c, 12, and 16b. Presence of directly
attached pyrazolyl substituent at position 6 in pyridazinone
ring of compound 16a enhanced the COX-2 selectivity and
inhibitory efficacy and resulted in displaying the highest anti-
inflammatory activity among all the screened compounds.
Our important goal of increasing COX-2 selectivity is to
decrease COX-1 related GI events that are normally associated
with non-selective agents. Ulcerogenicity evaluation results of
the selected compounds with the highest COX-2 selectivity
indices were in good correlation with this goal. All the tested
compounds exhibited significantly decreased ulcerogenicity
compared to indomethacin, except for compounds 6c and 6d
which showed equivalent ulcer scoring with indomethacin
that may result from increased local irritant effect of these
compounds.
Table 4. Gastric ulcerative effect of test compounds.
Compound
Ulcer scoring
Control
Indomethacin
0
5a)
4b
5a
5c
5d
6a
6b
6c
6d
9c
9d
16a
16c
2 ꢀ 0.59a),b)
1.33 ꢀ 0.34b)
0.83 ꢀ 0.17b)
0.67 ꢀ 0.17b)
2.67 ꢀ 0.34a),b)
2.33 ꢀ 0.34a),b)
4 ꢀ 0.593a)
5a)
1.67 ꢀ 0.34b)
1.67 ꢀ 0.34b)
2.67 ꢀ 0.34a),b)
2a),b)
Conclusion
Data were analyzed by using one-way ANOVA followed by
Bonferroni test.
a)Significantly different from control group.
b)Significantly different from indomethacin treated group.
In view of the biological results and structure–activity
relationship (SAR) study of our synthesized 2,6-disubstituted
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pyridazin-3(2H)-ones, we can conclude that the presence of
2-tolyloxy or pyrazolyl groups at position 6 in addition to
propyl or benzyl group, respectively, at position N2 of the
pyridazinone ring provided the best substituents required for
good in vitro COX-2 inhibitory and anti-inflammatory
activities in compounds 5a, 6a, and 16a. Further optimizations
on these derivatives are still required to enhance their COX-2
selectivity and anti-inflammatory activity while improving the
GI safety profile.
6-Aryloxypyridazin-3(2H)-ones 3a–d
Each compound of 2a–d (5 mmol) was refluxed in acetic acid
(15 mL) for 7 h. The reaction mixture was concentrated,
poured on ice-cold water (40 mL), and the solid product
formed was filtered off, washed with water, and recrystal-
lized from the appropriate solvent to give compounds 3a–d.
6-(o-Tolyloxy)pyridazin-3(2H)-one 3a
Yield 82%; recrystallized from water; m.p. 181–183°C. Lit. m.p.
187–188°C [26].
6-(m-Tolyloxy)pyridazin-3(2H)-one 3b
Experimental
Yield 91%; recrystallized from water; as white crystals; m.p.
127–129°C; 1H-NMR (400 MHz, CDCl3) d (ppm): 2.39 (s, 3H,
CH3), 6.91–6.99 (m, 2H, Harom), 7.03 (d, 1H, J ¼ 10.0 Hz, H5),
7.06 (s, 1H, Harom), 7.20 (d, 1H, J ¼ 10.0 Hz, H4), 7.29 (t, 1H,
J ¼ 7.7 Hz, Harom), 11.07 (brs., 1H, NH); 13C-NMR (100 MHz,
CDCl3) d (ppm): 21.33 (CH3), 117.69, 121.32, 126.39, 128.29
(Carom), 129.43 (C5), 133.32 (C4), 140.02, 152.95 (Carom), 153.74
(C6), 160.74 (C3); GC-MS (EI): m/z ¼ 91 (100%), 202 [Mþ] (89%).
Anal. calcd. for C11H10N2O2 (202): C, 65.34; H, 4.98; N, 13.85.
Found: C, 65.38; H, 4.95; N, 14.02.
Chemistry
General
NMR spectra were recorded on Bruker NMR spectrometer at
400 MHz for 1H and 100 MHz for 13C, with TMS as internal
standard and on Mercury-300BB NMR spectrometer at
300 MHz for 1H and 75 MHz for 13C, with TMS as internal
standard. Mass spectra were recorded on Shimadzu Qp-2010
plus spectrometer for compounds 4a–c,g,h and 5a–d, Thermo
Scientific Trace GC Ultra/ISQ spectrometer for compounds 4d,
e,f, 6a–c, 9a,b,d, and 16a,b, and on Agilent 6400 Series Triple
Quadrupole LC-MS-MS for compounds 9c, 12, and 16c.
Melting points were determined on a Mel Temp melting
point apparatus and are uncorrected. Elemental analyses
were performed at Micro Analytical Center, Cairo University,
Cairo, Egypt. Reactions were monitored by TLC, performed on
silica gel TLC plate 60 F254 that was purchased from Merck. The
starting material 3,6-dichloropyridazine (1) is commercially
available and was purchased from Sigma–Aldrich.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
6-(p-Tolyloxy)pyridazin-3(2H)-one 3c
Yield 76%; recrystallized from water; m.p. 168–170°C. Lit. m.p.
170°C [20].
6-(2,6-Dimethylphenoxy)pyridazin-3(2H)-one 3d
Yield 81%; recrystallized from water-glacial acetic acid; m.p.
242–244°C [19].
General procedure for the synthesis of 2-arylmethyl-
6-aryloxypyridazin-3(2H)-ones (4a–h), 2-{[3-(aryloxy)-
6-oxopyridazin-1(6H)-yl]methyl}-1H-isoindole-1,3(2H)-
diones (5a–d) and 2-propyl-6-aryloxypyridazin-3(2H)-one
(6a–d)
3-Aryloxy-6-chloropyridazines 2a–d
To a stirred solution of compound 1 (1.49 g, 10 mmol) and
phenol derivatives (10 mmol) in dry DMF (10 mL), sodium
hydride (0.48 g, 11 mmol, 55% susp. in paraffin oil) was
added portionwise. The reaction mixture was stirred for 3 h
at room temperature, poured onto ice-cold water (40 mL),
and the solid product formed was filtered off, washed with
water, and recrystallized from ethanol–water to give
compounds 2a–d
A mixture of 3a–d (1 mmol), substituted methyl bromide
(1.1 mmol), and anhydrous potassium carbonate (0.41 g,
3 mmol) in dry dimethylformamide (10 mL) was stirred at
room temperature for 24–48 h. The reaction mixture was
poured on ice-cold water (25 mL) with continuous stirring,
and the resulting solid product was filtered off, washed with
water, and recrystallized from the appropriate solvent to
afford compounds 4a–h, 5a–d, and 6a,c. In case of 6b,d, the
mixture was extracted with ether (3 ꢁ 15 mL). The combined
ethereal extracts were dried over sodium sulfate and the
solvent was removed under reduced pressure. For 6b, the
residual material was recrystallized from petroleum ether. For
6d, the residual material was chromatographed on a column
of silica gel using petroleum ether/ethyl acetate (4:1, v/v).
3-Chloro-6-(o-tolyloxy)pyridazine 2a
Yield 56%; m.p. 83–85°C. Lit. m.p. 85–85.5°C [18].
3-Chloro-6-(m-tolyloxy)pyridazine 2b
Yield 80%; m.p. 69–71°C. Lit. m.p. 71°C [18].
3-Chloro-6-(p-tolyloxy)pyridazine 2c
2-Benzyl-6-(o-tolyloxy)pyridazin-3(2H)-one 4a
Yield 84%; m.p. 105–107°C. Lit. m.p. 107–108°C [18].
The reactants were 3a and benzyl bromide, the reaction time
was 24 h; recrystallized from petroleum ether; yield 36%; m.p.
66–68°C; 1H-NMR (300 MHz, CDCl3) d (ppm): 2.12 (s, 3H, CH3),
5.06 (s, 2H, CH2), 6.96–7.02 (m, 2H, Harom, H5), 7.06 (d, 1H,
3-Chloro-6-(2,6-dimethylphenoxy)pyridazine 2d
Yield 66%; m.p. 89–91°C [19].
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T. H. Ibrahim et al.
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J ¼ 9.9 Hz, H4), 7.12–7.32 (m, 8H, Harom); 13C-NMR (100 MHz,
CDCl3) d (ppm): 16.11 (CH3), 54.24 (CH2), 120.88, 125.40,
125.98, 126.90, 127.78, 128.34, 129.01, 130.07 (Carom), 131.24
(C5), 133.57 (C4), 136.04, 151.55 (Carom), 151.86 (C6), 158.74
(C3); GC-MS (EI): m/z ¼ 106 (100%), 292 [Mþ] (59%). Anal.
calcd. for C18H16N2O2 (292): C, 73.95; H, 5.52; N, 9.58. Found:
C, 74.18; H, 5.03; N, 9.90.
127.97 (Carom), 130.09 (C5), 130.85, 131.31, 131.49 (Carom),
133.61 (C4), 134.95, 151.50 (Carom), 152.05 (C6), 158.69 (C3);
GC-MS (EI): m/z ¼ 370 [Mþ] (98%), 372 [Mþ2] (100%). Anal.
calcd. for C18H15BrN2O2 (370): C, 58.24; H, 4.07; N, 7.55. Found:
C, 58.60; H, 4.35; N, 7.23.
2-(4-Bromobenzyl)-6-(m-tolyloxy)pyridazin-3(2H)-one 4f
The reactants were 3b and 4-bromobenzyl bromide, the
reaction time was 24 h; recrystallized from petroleum ether;
yield 92%; as white crystals; m.p. 120–122°C; 1H-NMR
(300 MHz, CDCl3) d (ppm): 2.38 (s, 3H, CH3), 5.06 (s, 2H,
CH2), 6.99 (d, 1H, J ¼ 9.9 Hz, H5), 7.01–7.10 (m, 2H, Harom, H4),
7.14–7.27 (m, 5H, Harom), 7.39 (d, 2H, J ¼ 8.3 Hz, Harom); 13C-
NMR (100 MHz, CDCl3) d (ppm): 21.36 (CH3), 53.70 (CH2),
117.23, 120.85, 122.06, 125.88, 126.72 (Carom), 129.28 (C5),
130.87, 131.57 (Carom), 133.47 (C4), 135.05, 139.84, 152.09
(Carom), 153.34 (C6), 158.77 (C3); GC-MS (EI): m/z ¼ 370
[Mþ] (88%), 372 [Mþ2] (100%). Anal. calcd. for C18H15BrN2O2
(370): C, 58.24; H, 4.07; N, 7.55. Found: C, 58.01; H, 4.41;
N, 7.78.
2-Benzyl-6-(m-tolyloxy)pyridazin-3(2H)-one 4b
The reactants were 3b and benzyl bromide, the reaction time
was 36 h; recrystallized from petroleum ether; yield 81%; as
white crystals; m.p. 104–106°C; 1H-NMR (400 MHz, CDCl3) d
(ppm): 2.28 (s, 3H, CH3), 5.04 (s, 2H, CH2), 6.78–6.82 (m, 2H,
H
arom), 6.90 (d, 1H, J ¼ 9.8 Hz, H5), 6.94 (d, 1H, J ¼ 7.6 Hz,
Harom), 6.97 (d, 1H, J ¼ 9.8 Hz, H4), 7.14–7.31 (m, 6H, Harom);
13C-NMR (100 MHz, CDCl3) d (ppm): 21.35 (CH3), 54.42 (CH2),
117.16, 120.79, 125.74, 126.59, 127.88, 128.45, 129.06 (Carom),
129.25 (C5), 133.47 (C4), 136.17, 139.77, 151.88 (Carom), 153.45
(C6), 158.86 (C3); GC-MS (EI): m/z ¼ 106 (100%), 292
[Mþ] (61%). Anal. calcd. for C18H16N2O2 (292): C, 73.95;
H, 5.52; N, 9.58. Found: C, 74.16; H, 5.81; N, 9.32.
2-(4-Bromobenzyl)-6-(p-tolyloxy)pyridazin-3(2H)-one 4g
The reactants were 3c and 4-bromobenzyl bromide, the
reaction time was 24 h; recrystallized from petroleum ether;
yield 94%; as white crystals; m.p. 135–137°C; 1H-NMR
(400 MHz, CDCl3) d (ppm): 2.30 (s, 3H, CH3), 4.96 (s, 2H,
CH2), 6.88 (d, 2H, J ¼ 8.6 Hz, Harom), 6.89 (d, 1H, J ¼ 9.8 Hz, H5),
6.99 (d, 1H, J ¼ 9.8 Hz, H4), 7.10 (d, 2H, J ¼ 8.3 Hz, Harom),
7.14 (d, 2H, J ¼ 8.6 Hz, Harom), 7.34 (d, 2H, J ¼ 8.3 Hz, Harom);
13C-NMR (100 MHz, CDCl3) d (ppm): 20.82 (CH3), 53.79 (CH2),
120.21, 122.04, 126.63 (Carom), 130.05 (C5), 130.85, 131.57
(Carom), 133.45 (C4), 134.80, 135.04, 151.04 (Carom), 152.28
(C6), 158.77 (C3); GC-MS (EI): m/z ¼ 108 (100%), 370
[Mþ] (27%), 372 [Mþ2] (27%). Anal. calcd. for C18H15BrN2O2
(370): C, 58.24; H, 4.07; N, 7.55. Found: C, 58.16; H, 3.25;
N, 7.82.
2-Benzyl-6-(p-tolyloxy)pyridazin-3(2H)-one 4c
The reactants were 3c and benzyl bromide, the reaction time
was 36 h; recrystallized from petroleum ether; yield 90%; as
white crystals; m.p. 81–82°C; 1H-NMR (300 MHz, CDCl3) d
(ppm): 2.37 (s, 3H, CH3), 5.10 (s, 2H, CH2), 6.94–7.00 (m, 3H,
H
H
arom, H5), 7.05 (d, 1H, J ¼ 9.7 Hz, H4), 7.17 (d, 2H, J ¼ 8.3 Hz,
arom), 7.25–7.39 (m, 5H, Harom); 13C-NMR (75 MHz, CDCl3) d
(ppm): 20.73 (CH3), 54.40 (CH2), 120.07, 126.40, 127.77, 128.36,
128.93 (Carom), 129.95 (C5), 133.36 (C4), 134.52, 136.121,
151.12 (Carom), 151.96 (C6), 158.76 (C3); GC-MS (EI): m/z ¼ 106
(100%), 292 [Mþ] (55%). Anal. calcd. for C18H16N2O2 (292):
C, 73.95; H, 5.52; N, 9.58. Found: C, 73.52; H, 5.37; N, 10.04.
2-Benzyl-6-(2,6-dimethylphenoxy)pyridazin-3(2H)-one 4d
The reactants were 3d and benzyl bromide, the reaction time
was 36 h; recrystallized from petroleum ether; yield 53%; as
white solid; m.p. 93–95°C; 1H-NMR (300 MHz, CDCl3) d (ppm):
2.07 (s, 6H, 2CH3), 5.00 (s, 2H, CH2), 6.99 (d, 1H, J ¼ 10.8 Hz, H5),
7.09 (s, 3H, Harom), 7.11 (d, 1H, J ¼ 10.8 Hz, H4), 7.22–7.26 (m,
5H, Harom); 13C-NMR (75 MHz, CDCl3) d (ppm): 16.17 (2CH3),
53.91 (CH2), 125.25, 125.46, 127.62, 128.16, 128.51, 128.85
(Carom), 130.36 (C5), 133.69 (C4), 136.00, 149.76 (Carom), 151.17
(C6), 158.57 (C3); EI-MS: m/z ¼ 306 [Mþ] (100%). Anal. calcd.
for C19H18N2O2 (306): C, 74.49; H, 5.92; N, 9.14. Found:
C, 74.49; H, 5.96; N, 9.15.
2-(4-Bromobenzyl)-6-(2,6-dimethylphenoxy)pyridazin-3
(2H)-one 4h
The reactants were 3d and 4-bromobenzyl bromide, the
reaction time was 24 h; recrystallized from hexane; yield 93%;
as white crystals; m.p. 148–149°C; 1H-NMR (300 MHz, CDCl3) d
(ppm): 2.06 (s, 6H, 2CH3), 4.94 (s, 2H, CH2), 6.98 (d, 1H,
J ¼ 9.8 Hz, H5), 7.05–7.10 (m, 5H, Harom), 7.13 (d, 1H, J ¼ 9.8 Hz,
H4), 7.37 (d, 2H, J ¼ 7.3 Hz, Harom); 13C-NMR (75 MHz, CDCl3) d
(ppm): 16.22 (2CH3), 53.91 (CH2), 121.87, 125.49, 125.58,
128.58 (Carom), 130.33 (C5), 130.73, 131.34 (Carom), 133.73 (C4),
134.91, 149.75 (Carom), 151.35 (C6), 158.55 (C3); GC-MS (EI):
m/z ¼ 122 (100%), 384 [Mþ] (45%), 386 [Mþ2] (45%). Anal.
calcd. for C19H17BrN2O2 (384): C, 59.23; H, 4.45; N, 7.27. Found:
C, 59.55; H, 4.14; N, 7.51.
2-(4-Bromobenzyl)-6-(o-tolyloxy)pyridazin-3(2H)-one 4e
The reactants were 3a and 4-bromobenzyl bromide, the
reaction time was 24 h; recrystallized from hexane; yield 91%;
as white crystals; m.p. 86–87°C; 1H NMR (300 MHz, CDCl3) d
(ppm): 2.12 (s, 3H, CH3), 4.99 (s, 2H, CH2), 6.95–7.02 (m, 2H,
2-{[3-(2-Methylphenoxy)-6-oxopyridazin-1(6H)-yl]methyl}-
1H-isoindole-1,3(2H)-dione 5a
The reactants were 3a and N-bromomethylphthalimide, the
H
arom, H5), 7.10 (d, 1H, J ¼ 9.9 Hz, H4), 7.12–7.30 (m, 5H, Harom),
7.39 (d, 2H, J ¼ 8.3 Hz, Harom); 13C-NMR (100 MHz, CDCl3) d
(ppm): 16.14 (CH3), 53.55 (CH2), 120.95, 122.01, 125.56, 126.16,
reaction time was 48 h; recrystallized from ether; yield 55%; as
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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700093
Novel 2,6-Disubstituted Pyridazin-3(2H)-one Derivatives
Archiv der Pharmazie
white crystals; m.p. 163–165°C; 1H-NMR (400 MHz, CDCl3) d
(ppm): 1.93 (s, 3H, CH3), 5.72 (s, 2H, CH2), 6.70–6.87 (m, 4H,
2-Propyl-6-(o-tolyloxy)pyridazin-3(2H)-one 6a
The reactants were 3a and n-propyl bromide, the reaction
time was 12 h; recrystallized from hexane; yield 75%; m.p.
84–85°C; as white crystals; 1H-NMR (400 MHz, CDCl3) d
(ppm): 0.88 (t, 3H, J ¼ 7.3 Hz, CH3CH2CH2), 1.70 (sextet, 2H,
J ¼ 7.3 Hz, CH3CH2CH2), 2.23 (s, 3H, CH3Ar), 3.91 (t, 2H,
J ¼ 7.3 Hz, N-CH2CH2), 6.99 (d, 1H, J ¼ 9.8 Hz, H5), 7.01–7.06
(m, 1H, Harom), 7.10 (d, 1H, J ¼ 9.8 Hz, H4), 7.13–7.36 (m,
H
arom), 7.04 (d, 1H, J ¼ 9.8 Hz, H5), 7.17 (d, 1H, J ¼ 9.8 Hz, H4),
7.74–7.78 (m, 4H, Harom); 13C-NMR (100 MHz, CDCl3) d (ppm):
15.82 (CH3), 50.60 (CH2), 121.07, 123.65, 125.43, 126.48,
129.91, 130.60 (Carom), 131.63 (C5), 133.54 (C4), 134.05, 134.35,
–
150.76 (Carom), 152.38 (C6), 158.51 (C3), 166.77 (2C O); GC-MS
–
(EI): m/z ¼ 160 (100%), 361 [Mþ] (6%); Anal. calcd. for
C
20H15N3O4 (361): C, 66.48; H, 4.18; N, 11.63. Found: C,
3H, H d (ppm): 10.90
arom); 13C-NMR (100 MHz, CDCl3)
66.81; H, 4.52; N, 11.91.
(CH3CH2CH2), 16.17 (CH3CH2CH2), 21.36 (CH3Ar), 52.44
(N-CH2), 120.60, 125.29, 125.70, 126.96, 129.92 (Carom),
131.31 (C5), 133.34 (C4), 151.78 (Carom), 151.78 (C6),
159.09 (C3); GC-MS (EI): m/z ¼ 244 [Mþ] (100%). Anal. calcd.
for C14H16N2O2 (244): C, 68.83; H, 6.60; N, 11.47. Found:
C, 68.41; H, 6.28; N, 11.72.
2-{[3-(3-Methylphenoxy)-6-oxopyridazin-1(6H)-yl]methyl}-
1H-isoindole-1,3(2H)-dione 5b
The reactants were 3b and N-bromomethylphthalimide, the
reaction time was 48 h; recrystallized from dichloromethane/
petroleum ether; yield 58%; as white crystals; m.p.
164–166°C; 1H-NMR (400 MHz, CDCl3) d (ppm): 2.17 (s, 3H,
CH3), 5.77(s, 2H, CH2), 6.63–6.71 (m, 2H, Harom), 6.73 (s, 1H,
2-Propyl-6-(m-tolyloxy)pyridazin-3(2H)-one 6b
The reactants were 3b and n-propyl bromide, the reaction
time was 12 h; yield 69%; m.p. 60–62°C; as white crystals;
1H-NMR (300 MHz, CDCl3) d (ppm): 0.89 (t, 3H, J ¼ 7.3 Hz,
CH3CH2CH2), 1.73 (sextet, 2H, J ¼ 7.3 Hz, CH3CH2CH2), 2.34 (s,
3H, CH3Ar), 3.93 (t, 2H, J ¼ 7.3 Hz, 2H, N-CH2CH2), 6.87–7.06 (m,
5H, Harom, H4 & H5), 7.24 (t, 1H, J ¼ 7.5, Harom); 13C-NMR
H
arom), 6.87 (t, 1H, J ¼ 7.8 Hz, Harom), 7.01 (d, 1H, J ¼ 9.7 Hz,
H5), 7.12 (d, 1H, J ¼ 9.7 Hz, H4), 7.68–7.85 (m, 4H, Harom); 13C-
NMR (100 MHz, CDCl3) d (ppm): 21.13 (CH3), 50.64 (CH2),
117.66, 121.23, 123.60, 125.77, 127.051 128.74 (Carom), 131.65
(C5), 133.34 (C4), 134.08, 139.32, 152.50 (Carom), 152.74 (C6),
158.55 (C3), 166.80 (2C ¼ O); GC-MS (EI): m/z ¼ 160 (100%),
361 [Mþ] (15%). Anal. calcd. for C20H15N3O4 (361): C, 66.48; H,
4.18; N, 11.63. Found: C, 66.72; H, 3.98; N, 11.23.
(75 MHz, CDCl3)
d
(ppm): 10.89 (CH3CH2CH2), 21.24
(CH3CH2CH2), 21.37 (CH3Ar), 52.52 (N-CH2), 116.79, 120.44,
125.52, 126.19 (Carom), 129.23 (C5), 133.41 (C4), 139.78, 151.60
(Carom), 153.73 (C6), 159.06 (C3); GC-MS (EI): m/z ¼ 244
[Mþ] (100%). Anal. calcd. for C14H16N2O2 (244): C, 68.83; H,
6.60; N, 11.47. Found: C, 69.20; H, 6.79; N, 11.33.
2-{[3-(4-Methylphenoxy)-6-oxopyridazin-1(6H)-yl]methyl}-
1H-isoindole-1,3(2H)-dione 5c
The reactants were 3c and N-bromomethylphthalimide, the
reaction time was 48 h; recrystallized from dichloromethane/
petroleum ether; yield 49%; as white crystals; m.p. 188–190°C;
1H-NMR (400 MHz, CDCl3) d (ppm): 2.18 (s, 3H, CH3), 5.76 (s, 2H,
CH2), 6.68–6.82 (m, 4H, Harom), 7.01 (d, 1H, J ¼ 9.8 Hz, H5), 7.12
(d, 1H, J ¼ 9.8 Hz, H4), 7.71–7.85 (m, 4H, Harom); 13C-NMR
(100 MHz, CDCl3) d (ppm): 20.86 (CH3), 50.67 (CH2), 120.47,
123.55, 126.96, 129.52 (Carom), 131.74 (C5), 133.31 (C4), 134.00,
134.28, 150.19 (Carom), 152.76 (C6), 158.55 (C3), 166.82
(2C ¼ O); GC-MS (EI): m/z ¼ 160 (100%), 361 [Mþ] (11%). Anal.
calcd. for C20H15N3O4 (361): C, 66.48; H, 4.18; N, 11.63. Found:
C, 66.31; H, 4.51; N, 11.24.
2-Propyl-6-(p-tolyloxy)pyridazin-3(2H)-one 6c
The reactants were 3c and n-propyl bromide, the reaction
time was 36 h; recrystallized from hexane; yield 81%; m.p. 91–
92°C; as white crystals; 1H-NMR (400 MHz, CDCl3) d (ppm): 0.83
(t, 3H, J ¼ 7.3 Hz, CH3CH2CH2), 1.66 (sextet, 2H, J ¼ 7.3 Hz,
CH3CH2CH2), 2.29 (s, 3H, CH3Ar), 3.86 (t, 2H, J ¼ 7.3 Hz,
N-CH2CH2), 6.89 (d, 1H, J ¼ 9.8 Hz, H5), 6.90–6.95 (m, 2H,
H
H
arom), 6.98 (d, 1H, J ¼ 9.8 Hz, H4), 7.10 (d, 2H, J ¼ 8.3 Hz,
arom); 13C-NMR (100 MHz, CDCl3) d (ppm): 10.97 (CH3CH2CH2),
20.78 (CH3CH2CH2), 21.47 (CH3Ar), 52.61 (N-CH2), 119.91,
126.16 (Carom), 130.10 (C5), 133.19 (C4), 134.53, 151.40 (Carom),
151.96 (C6), 159.15 (C3); GC-MS (EI): m/z ¼ 244 [Mþ] (100%).
Anal. calcd. for C14H16N2O2 (244): C, 68.83; H, 6.60; N, 11.47.
Found: C, 68.52; H, 6.91; N, 11.13.
2-{[3-(2,6-Dimethylphenoxy)-6-oxopyridazin-1(6H)-yl]-
methyl}-1H-isoindole-1,3(2H)-dione 5d
The reactants were 3d and N-bromomethylphthalimide, the
reaction time was 48 h; crystallized from ethanol/water; yield
60%; as white crystals; m.p. 198–200°C; 1H-NMR (400 MHz,
CDCl3) d (ppm): 1.89 (s, 6H, 2CH3), 5.68 (s, 2H, CH2), 6.57 (s, 3H,
6-(2,6-Dimethylphenoxy)-2-propylpyridazin-3(2H)-one 6d
The reactants were 3d and n-propyl bromide, the reaction
time was 12 h; yield 63%; as an oil; 1H-NMR (400 MHz, CDCl3) d
(ppm): 0.79 (t, 3H, J ¼ 7.3 Hz, CH3CH2CH2), 1.60 (sextet, 2H,
J ¼ 7.3 Hz, CH3CH2CH2), 2.12 (s, 3H, CH3Ar), 3.81 (t, 2H,
J ¼ 7.3 Hz, N-CH2CH2), 6.96 (d, 1H, J ¼ 9.7 Hz, H5), 7.00–7.06
(m, 3H, Harom), 7.09 (d, 1H, J ¼ 9.7 Hz, H4); 13C-NMR (100 MHz,
CDCl3) d (ppm): 10.62 (CH3CH2CH2), 16.15 (2CH3), 21.07
(CH3CH2CH2), 52.01 (N-CH2), 124.84, 125.32, 128.45 (Carom),
130.22 (C5), 133.36 (C4), 149.65 (Carom), 151.12 (C6), 158.85
(C3); GC-MS (EI): m/z ¼ 258 [Mþ] (100%). Anal. calcd. for
H
arom), 7.05 (d, 1H, J ¼ 9.8 Hz, H5), 7.20 (d, 1H, J ¼ 9.8 Hz, H4),
7.65–7.81 (m, 4H, Harom); 13C-NMR (100 MHz, CDCl3) d (ppm):
15.89 (2CH3), 50.61 (CH2), 123.57, 125.27, 126.06, 127.92,
129.77 (Carom), 131.54 (C5), 133.70 (C4), 133.98, 149.17
(Carom), 151.47 (C6), 158.51 (C3), 166.70 (2C ¼ O); EI-MS:
m/z ¼ 215 (100%), 375 [Mþ] (4%). Anal. calcd. for C21H17N3O4
(375): C, 67.19; H, 4.56; N, 11.19. Found: C, 67.43; H, 4.22; N,
11.47.
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C15H18N2O2 (258): C, 69.74; H, 7.02; N, 10.84. Found: C, 69.43;
H, 7.32; N, 10.52.
CDCl3) d (ppm): 46.14 (CH2), 54.33 (CH2-NH), 126.10, 127.31,
127.45, 127.88, 128.31, 128.52, 128.66 (C5), 131.44 (C4),
136.84, 138.70 (Carom), 146.61 (C6) 158.10 (C3); GC-MS (EI): m/
z ¼ 91 (100%), 291 [Mþ] (39%). Anal. calcd. for
N-Benzyl-6-chloropyridazin-3-amine 7a
A mixture of compound 1 (1.49 g, 10 mmol) and benzylamine
(10 mmol) was heated at 70°C for 3.5 h. The reaction mixture
was left to reach room temperature, then ethanol (10 mL) was
added and the mixture was stirred for 15 min. The reaction
mixture was poured on ice-cold water (100 mL) and the
crystallized product formed was collected by filtration and
dried to give compound 7a. Yield 55%; m.p. 160–162°C.
Lit. m.p. 162–163°C [21].
C18H17N3O (291): C, 74.20; H, 5.88; N, 14.42. Found: C,
74.58; H, 5.66; N, 14.81.
6-(Benzylamino)-2-(4-bromobenzyl)pyridazin-3(2H)-one
9b
Yield 67%; purified by column chromatography of silica gel
using petroleum ether/ethyl acetate (1:1, v/v) as an eluent; as
yellow crystals; m.p. 113–115°C; 1H-NMR (300 MHz, DMSO-d6)
d (ppm): 4.26 (d, 2H, J ¼ 5.5 Hz, CH2-NH), 4.96 (s, 2H, CH2), 6.77
(d, 1H, J ¼ 9.7 Hz, H5), 6.98 (t, 1H, J ¼ 5.5 Hz, NH), 7.03 (d, 1H,
J ¼ 9.7 Hz, H4), 7.15 (d, 2H, J ¼ 8.1 Hz, Harom), 7.21–7.35 (m, 5H,
6-Chloro-N-(4-chlorophenyl)pyridazin-3-amine 7b
Compound
1 (1.49 g, 10 mmol) with 4-chloroaniline
(1.27 g, 10 mmol) was fused together at 70°C for 1.5 h.
The reaction mixture was left to reach room temperature,
then ethanol (10 mL) was added and the mixture was
stirred for 15 min. The reaction mixture was poured on ice-
cold water (100 mL) and the resulting solid product was
filtered off, washed with water, and recrystallized from
methanol to give compound 7b. Yield 83%; m.p.
204–206°C. Lit. m.p. 201–203°C [21].
H
arom), 7.44 (d, 2H, J ¼ 8.1 Hz, Harom); 13C-NMR (75 MHz,
DMSO-d6) d (ppm): 44.70 (CH2), 52.54 (CH2-NH), 120.32,
126.65, 127.35, 128.10 (Carom), 130.15 (C5), 130.65, 131.06
(Carom), 133.21 (C4), 136.75, 139.32 (Carom), 146.95 (C6), 157.08
(C3); GC-MS (EI): m/z ¼ 369 [Mþ] (100%), 371 [Mþ2] (95%).
Anal. calcd. for C18H16 BrN3O (369): C, 58.39; H, 4.36; N, 11.35.
Found: C, 58.72; H, 4.61; N, 11.64.
2-Benzyl-6-(4-chlorophenylamino)pyridazin-3(2H)-one 9c
Yield 75%; recrystallized from ethyl acetate; as yellow
crystals; m.p. 252–254°C; 1H-NMR (300 MHz, DMSO-d6) d
(ppm): 5.14 (s, 2H, CH2), 6.93 (d, 1H, J ¼ 9.6 Hz, H5), 7.19 (d,
1H, J ¼ 9.6 Hz, H4), 7.22–7.36 (m, 7H, Harom), 7.50 (d, 2H,
J ¼ 8.7 Hz, Harom), 9.21 (s, 1H, NH); 13C-NMR (75 MHz, DMSO-
d6) d (ppm): 53.25 (CH2), 118.71, 124.03, 127.35, 127.95,
128.23, 128.37 (Carom), 128.39 (C5), 131.62 (C4), 137.23,
139.63 (Carom), 143.76 (C6), 157.05 (C3); LC-MS (ESI): 310
[M–H] (100%), 311 [Mþ] (18%). Anal. calcd. for C17H14
ClN3O (311): C, 65.49; H, 4.53; N, 13.48. Found: C, 65.73; H,
4.84; N, 13.71.
6-(Benzylamino)pyridazin-3(2H)-one 8a
A solution of compound 7a (1.00 g, 5 mmol) and sodium
acetate (0.70 g, 10 mmol) in acetic acid (15 mL) was refluxed
for 18 h. The reaction mixture was concentrated, allowed to
reach room temperature, and then poured on ice-cold water.
The solid product formed was filtered off, washed with water,
and recrystallized from ethyl acetate to give compound 8a.
Yield 86%; m.p. 175–177°C. Lit. m.p. 173°C [21].
6-(4-Chlorophenylamino)pyridazin-3(2H)-one 8b
A solution of compound 7b (1.20 g, 5 mmol) in 15-mL acetic
acid was refluxed for 10 h. The reaction mixture was poured
on water (20 mL), concentrated, cooled, and the resulting
solid product was filtered off, washed with petroleum ether,
and dried to give compound 8b. Yield 63%; m.p. 258–260°C.
Lit. m.p. 254°C [21].
2-(4-Bromobenzyl)-6-(4-chlorophenylamino)pyridazin-
3(2H)-one 9d
Yield 84%; recrystallized from ethyl acetate; as yellow
crystals; m.p. 242–244°C; 1H-NMR (300 MHz, DMSO-d6) d
(ppm): 5.02 (s, 2H, CH2), 6.91 (d, 1H, J ¼ 9.3 Hz, H5), 7.19 (d,
1H, J ¼ 9.3 Hz, H4), 7.24–7.54 (m, 8H, Harom), 9.22 (s, 1H, NH);
13C-NMR (75 MHz, DMSO-d6) d (ppm): 52.67 (CH2), 118.74,
120.61, 124.11, 128.08 (Carom), 128.39 (C5), 130.48, 131.22
(Carom), 131.29 (C4), 136.58, 139.57 (Carom), 143.84 (C6), 157.04
(C3); GC-MS (EI): m/z ¼ 91 (100%), 390 [Mþ] (18%). Anal. calcd.
for C17H13BrClN3O (390): C, 52.27; H, 3.35; N, 10.76. Found: C,
52.78; H, 3.18; N, 10.77.
2-Arylmethyl-6-arylaminopyridazin-3(2H)-ones 9a–d
A mixture of 8a,b (0.20 g, 1 mmol), benzyl bromide and/or
4-bromobenzyl bromide (2 mmol), and anhydrous potassium
carbonate (0.41 g, 3 mmol) in dry dimethylformamide (10 mL)
was stirred at room temperature for 48 h. The reaction
mixture was poured on ice-cold water (25 mL) and stirred for
1 h, and the resulting solid purified to give 9a–d.
2-Benzyl-6-(benzylamino)pyridazin-3(2H)-one 9a
6-Chloro-N,N-dimethylpyridazin-3-amine 10
Yield 62%; purified by column chromatography of silica gel
using petroleum ether/ethyl acetate (1:1, v/v) as an eluent; as
white crystals; m.p. 148–150°C; 1H-NMR (400 MHz, CDCl3) d
(ppm): 4.38 (d, 2H, J ¼ 5.4 Hz, CH2-NH), 4.62 (t, 1H, J ¼ 5.4 Hz,
NH), 5.16 (s, 2H, CH2), 6.71 (d, 1H, J ¼ 9.8 Hz, H5), 6.76 (d, 1H,
J ¼ 9.8 Hz, H4), 7.25–7.40 (m, 10H, Harom); 13C-NMR (100 MHz,
Compound 1 (1.49 g, 10 mmol) was stirred in 40% aqueous N,
N-dimethylamine (10 mL) at room temperature for 3 h.
Petroleum ether (10 mL) was added on the reaction mixture,
and the crystalline product formed was filtered off, washed
with petroleum ether, and dried to give 10. Yield 67%; m.p.
101–103°C. Lit. m.p. 103–104°C [27].
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6-(Dimethylamino)pyridazin-3(2H)-one 11
reaction mixture was concentrated, allowed to reach room
temperature, poured on ice-cold water (30 mL), and the solid
product formed was filtered off, washed with water, and
dried to give 15 as white solid. Yield 95%; m.p. 256–258°C. Lit.
258°C [23].
A solution of compound 10 (1.00 g, 6 mmol) and sodium
acetate (0.90 g, 12 mmol) in acetic acid (15 mL) was refluxed
for 18 h. The reaction mixture was concentrated, left to reach
room temperature, poured on water (30 mL), and extracted
with ethyl acetate (3 ꢁ 30 mL). The combined organic
extracts were dried over sodium sulfate and the solvent
was reduced to minimal amount under reduced pressure,
petroleum ether was added, and the solid product formed
was collected by filtration and dried to afford 0.6 g (68%
yield) of 11 as yellow solid. M.p. 190–192°C. Lit. m.p.
192–194°C [28].
2-Benzyl-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazin-3(2H)-
one 16a
A mixture of compound 15 (0.19 g, 1 mmol), benzyl bromide
(1.5 mmol), and anhydrous potassium carbonate (0.41 g,
3 mmol) in dry dimethylformamide (10 mL) was stirred, at
room temperature for 24 h. The reaction mixture was poured
on ice-cold water (25 mL) and extracted with ether
(3 ꢁ 15 mL). The combined ethereal extracts were dried over
sodium sulfate and the solvent was removed under reduced
pressure. The residual material was chromatographed on a
column of silica gel using petroleum ether/ethyl acetate (4:1,
v/v) as an eluent to afford 240 mg (85% yield) of 16a as
colorless oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 2.25 (s, 3H,
5-CH3 of pyrazole), 2.40 (s, 3H, 3-CH3 of pyrazole), 5.30 (s, 2H,
CH2), 5.97 (s, 1H, H4 of pyrazole), 7.05 (d, 1H, J ¼ 10.0 Hz, H4 of
pyridazinone), 7.28–7.37 (m, 3H, Harom), 7.41–7.46 (m, 2H,
2-(4-Bromobenzyl)-6-(dimethylamino)pyridazin-3(2H)-
one 12
A suspension of compound 11 (0.20 g, 1 mmol), 4-bromoben-
zyl bromide (0.70 g, 2 mmol), and anhydrous potassium
carbonate (0.41 g, 3 mmol) in dry dimethylformamide
(10 mL) was stirred, at room temperature for 48 h. The
reaction mixture was poured on ice-cold water (25 mL), and
the resulting solid product was filtered off, washed with
water, and purified by column chromatography on silica gel
using petroleum ether/ethyl acetate (1:1, v/v) as an eluent to
afford 0.28 g (72%) of compound 12 as yellow crystals. M.p.
138–140°C; 1H-NMR (300 MHz, DMSO-d6) d (ppm): 2.85 (s, 6H,
(CH3)2N), 5.03 (s, 2H, CH2), 6.83 (d, 1H, J ¼ 10.1 Hz, H5), 7.25 (d,
2H, J ¼ 8.3 Hz, Harom), 7.41 (d, 1H, J ¼ 10.1 Hz, H4), 7.50 (d, 2H,
J ¼ 8.3 Hz, Harom); 13C-NMR (75 MHz, DMSO-d6) d (ppm): 38.16
(CH3)2N), 52.79 (CH2), 120.40, 124.98, 130.10 (Carom), 130.51
(C5), 131.16 (C4), 136.66 (Carom), 148.84 (C6), 156.75 (C3); LC-
MS (ESI): 308 [Mþ] (100%), 310 [Mþ2] (98%). Anal. calcd. for
H
arom), 7.95 (d, 1H, J ¼ 10.0 Hz, H5 of pyridazinone); 13C-NMR
(100 MHz, CDCl3) d (ppm): 13.41 (5-CH3 of pyrazole), 13.90
(3-CH3 of pyrazole), 54.76 (CH2), 109.30 (C4 of pyrazole),
128.93, 128.15, 128.51 (Carom), 128.86 (C5 of pyridazinone),
131.81 (C4 of pyridazinone), 136.98 (Carom), 140.93 (C5 of
pyrazole), 142.29 (C3 of pyrazole), 150.16 (C6 of pyridazi-
none), 158.80 (C3 of pyridazinone); GC-MS (EI): m/z ¼ 280
[Mþ] (100%). Anal. calcd. for C16H16N4O (280): C, 68.55; H,
5.75; N, 19.99. Found: C, 68.83; H, 5.42; N, 19.63.
C
13H14BrN3O (308): C, 50.67; H, 4.58; N, 13.64. Found: C, 50.61;
H, 4.13; N, 14.13.
2-(4-Bromobenzyl)-6-(3,5-dimethyl-1H-pyrazol-1-yl)-
pyridazin-3(2H)-one 16b
3-Chloro-6-hydrazinylpyridazine 13
A mixture of compound 15 (0.19 g, 1 mmol), 4-bromobenzyl
bromide (0.27 g, 1.1 mmol), and anhydrous potassium
carbonate (0.41 g, 3 mmol) in dry dimethylformamide
(10 mL) was stirred at room temperature for 24 h. The
reaction mixture was poured on ice-cold water (25 mL), and
the resulting solid product was filtered off, washed with
water, and recrystallized from petroleum ether to afford
0.22 g (62% yield) of 16b as white crystals. M.p. 108–109°C;
1H-NMR (300 MHz, CDCl3) d (ppm): 2.24 (s, 3H, 5-CH3 of
pyrazole), 2.39 (s, 3H, 3-CH3 of pyrazole), 5.22 (s, 2H, CH2),
5.96 (s, 1H, H4 of pyrazole), 7.02 (d, 1H, J ¼ 9.6 Hz, H4 of
pyridazinone), 7.29 (d, 2H, J ¼ 7.8 Hz, Harom), 7.44 (d, 2H,
J ¼ 7.8 Hz, Harom), 7.95 (d, 1H, J ¼ 9.6 Hz, H5 of pyridazinone);
13C-NMR (75 MHz, CDCl3) d (ppm): 13.40 (5-CH3 of pyrazole),
13.93 (3-CH3 of pyrazole), 54.20 (CH2), 109.45 (C4 of
pyrazole), 122.10 (Carom), 128.38 (C5 of pyridazinone),
130.59, 131.69 (Carom), 131.86 (C4 of pyridazinone), 134.96
(Carom), 140.86 (C5 of pyrazole), 142.48 (C3 of pyrazole),
150.30 (C6 of pyridazinone), 158.72 (C3 of pyridazinone);
GC-MS (EI): m/z ¼ 357.9 [Mþ] (100%), 359.9 [Mþ2] (92%).
Anal. calcd. for C16H15BrN4O (358): C, 53.50; H, 4.21; N, 15.60.
Found: C, 53.79; H, 4.60; N, 15.31.
To a stirred solution of 25% hydrazine hydrate (2.8 mL,
14 mmol) and 30% aqueous ammonia (5 mL, 126 mmol) in
water (5 mL), compound 1 (2.00 g, 13 mmol) was added, and
the mixture was refluxed for 1.5 h. The solution obtained was
cooled to 0°C using ice bath and stirring was continued at this
temperature for 3 h. The solid product formed was collected
by filtration and dried to give 13 as a white solid. Yield
61%; m.p. 140–142°C. Lit. 140–142°C [22].
3-Chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine 14
A
solution of 3-chloro-6-hydrazinopyridazine 13 (1.00 g,
7 mmol) and acetylacetone (7 mmol) in 10 mL absolute
ethanol was heated to reflux for 4 h. After cooling, the
reaction mixture was poured on ice-cold water (30 mL) and
the resulting solid product was filtered off, washed with
water, and recrystallized from ethanol to obtain 14 as white
crystals. Yield 75%; m.p. 113–115°C. Lit. 115°C [16].
6-(3,5-Dimethyl-1H-pyrazol-1-yl)pyridazin-3(2H)-one 15
A solution of 14 (1.00 g, 5 mmol) and sodium acetate (0.41 g,
5 mmol) in acetic acid (10 mL) was refluxed for 5 h. The
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2-{[3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-oxopyridazin-
1(6H)-yl]methyl}-1H-isoindole-1,3(2H)-dione 16c
recorded as writhing episodes for 30 min. The % protection
against acetic acid induced writhing was calculated according
to the following formula:
% Analgesic activity ¼ fðn ꢂ n0Þ=ng ꢁ 100
A mixture of compound 15 (0.19 g, 1 mmol), N-bromome-
thylphthalimide (0.36 g, 1.5 mmol), and anhydrous potassium
carbonate (0.41 g, 3 mmol) in dry dimethylformamide
(10 mL) was stirred, at room temperature for 24 h. The
reaction mixture was poured on ice-cold water (25 mL), and
the resulting solid product was filtered off, washed with
water, and recrystallized from methanol/water to afford
190 mg (55% yield) of 16c as white crystals. M.p. 152–154°C;
1H-NMR (300 MHz, CDCl3) d (ppm): 2.18 (s, 3H, 5-CH3 of
pyrazole), 2.23 (s, 3H, 3-CH3 of pyrazole), 5.87 (s, 1H, H4 of
pyrazole), 5.97 (s, 2H, CH2), 7.01 (d, 1H, J ¼ 10.2 Hz, H4 of
pyridazinone), 7.72–7.90 (m, 4H, Harom), 7.97 (d, 1H, J ¼ 10.2
Hz, H5 of pyridazinone); 13C-NMR (100 MHz, CDCl3) d (ppm):
13.41 (5-CH3 of pyrazole), 13.82 (3-CH3 of pyrazole), 50.55
(CH2), 109.53 (C4 of pyrazole), 123.76 (Carom), 128.82 (C5 of
pyridazinone), 131.75 (C4 of pyridazinone), 131.94, 134.52
(Carom), 140.84 (C5 of pyrazole), 141.27 (C3 of pyrazole),
150.39 (C6 of pyridazinone), 158.48 (C3 of pyridazinone),
where n is the mean number of writhes of the control group
and n0 is the mean number of writhes of the test group.
Anti-inflammatory activity
Carrageenin-induced rat paw edema method [25] was
adopted for the assessment of anti-inflammatory activity of
the test compounds using Wistar albino rats (120–130 g). The
rats were marked and divided into 14 experimental groups of
six rats each. After 1 h of oral administration of test
compounds (10 mg/kg), a subplantar injection of 0.1 mL of
1% carrageenin solution to the left hind paw of each animal
was performed. Rat paw volumes were measured using
standard fluid displacement procedures (7140-plesthysmom-
eter Ugo Basile) by immersing the hind left paw in 0.45%
saline solution, at 1, 2, and 3 h, post carrageenin injection. The
percentage change in paw volume relative to the base line
measurement was taken as the criterion of comparison.
Celecoxib (10 mg/kg) was used as an internal standard anti-
inflammatory agent. Significant differences between the
mean values of the control and the treated groups were
obtained, using one-way ANOVA test and p values. The
differences in the obtained results were considered significant
at p < 0.01. The anti-inflammatory activity of the test
compounds after 3 h was expressed as percentage inhibition
of edema that was calculated by applying the following
formula:
–
166.82 (2C O); LC-MS (ESI): 350 [Mþ1] (87.7%). Anal. calcd.
–
for C18H15N5O3 (349): C, 61.89; H, 4.33; N, 20.05. Found: C,
61.54; H, 4.59; N, 20.31.
Biological activity
Chemicals and drugs
Celecoxib and carrageenin were purchased from Sigma–
Aldrich. Glacial acetic acid was purchased from ADWIC
Company (Cairo, Egypt).
Animals
Male Swiss albino mice and Wistar albino rats were used for
evaluation of analgesic and anti-inflammatory activities,
respectively. Animals were purchased from the Modern
Veterinary Office for Laboratory Animals (Cairo, Egypt) and
were acclimatized for 1 week before experimentation and
then divided into different experimental groups, each with six
animals with food and water ad libitum. All performed
experimental protocols were approved by the Research Ethics
Committee at the Faculty of Pharmacy, Suez Canal University,
Ismailia, Egypt.
Anti-inflammatory activityð%inhibitionÞ ¼ fðVc ꢂ VtÞ=Vcg ꢁ 100
where Vc is the edema volume in the control group and Vt is
the edema volume in groups treated with the test
compounds.
Gastric ulcerogenicity
Ulcerogenic effect of the test compounds was assessed after
p.o. administration of the test compounds to rats at the dose
of 10 mg/kg in comparison to a known ulcerogenic drug,
indomethacin, in equal dose. Six hours after the administra-
tion of the reference and test compounds, rats were
euthanized by cervical dislocation and the stomachs were
removed, inflated, and opened along the greater curvature.
The gastric lesions were stretched out and scored from 0 to 5,
according to the method of Clementi et al. [29]. Gastric tissue
samples were fixed in buffered neutral formalin for 24 h.
Analgesic activity
Evaluation of analgesic activity of the test compounds was
performed using acetic acid induced writhing method [24].
The Swiss albino mice weighing between 25 g and 33 g used
for the experiment were divided into 14 different groups each
containing six animals, and were marked individually. Food
was withdrawn 12 h before oral administration of the test
compounds (10 mg/kg, p.o.) and compared to an equal dose
of celecoxib as the reference drug and saline as a negative
control. After 60 min, writhing was provoked by intraper-
itonial injection of 1% acetic acid in a volume of 0.1 mL/10 g
body weight. Total number of stretching movements like
arching of the back, extension of hind limbs, twisting of the
trunk, and elongation of the body were counted and
In vitro COX-1 and COX-2 inhibition assay
The ability of the test compounds to inhibit ovine COX-1 and
human recombinant COX-2 was evaluated using COX Inhibi-
tor Screening Assay kit (catalog no. 560131, Cayman Chemical,
Ann Arbor, MI, USA) according to the instructions recom-
mended by the supplier.
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Statistical analysis
[13] D. S. Dogruer, M. F. Sahin, Turk. J. Chem. 2003, 27,
727–738.
Data were collected, tabulated, and presented as mean ꢀ
SEM. One-way analysis of variance (ANOVA) followed by
Bonferroni’s post hoc test for examining significance of data
using the Statistical Package for Social Sciences, version 17
(SPSS, Inc., Chicago, IL, USA). p < 0.01 was considered to be
statistically significant.
€
€
€
[14] M. GokcSe, Y. Dundar, E. Kupeli, FABAD J. Pharm. Sci.
2010, 35, 1–11.
[15] D. Tiryaki, M. Sukuroglu, D. S. Dogruer, E. Akkol,
S. Ozgen, M. F. Sahin, Med. Chem. Res. 2013, 22,
2553–2560.
€
€ €
€
€
€
[16] M. Sukuroglu, B.CS . Ergun, S. Unlu, M. F. Sahin, E. Kupeli,
E. Yesilada, E. Banoglu, Arch. Pharm. Res. 2005, 28,
509–517.
The authors have declared no conflict of interest.
[17] Y. M. Loksha, E. B. Pedersen, P. La Colla, R. Loddo, J.
Heterocycl. Chem. 2007, 44, 1351–1356.
[18] S. Tamura, T. Jojima, Agr. Biol. Chem. 1963, 27, 653–658.
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