Synthesis of some new 2,6-disubstituted-3(2H)-pyridazinone derivatives and investigation of their analgesic, anti-inflammatory and antimicrobial activities

Article abstract of DOI:10.1007/s00044-012-0253-1

In this study, 12 new 3(2H)-pyridazinone derivatives carrying 4-substituted phenylpiperazinylethyl moiety on lactam nitrogen were synthesized and their chemical structures were confirmed by ¹H-NMR, mass, and elemental analysis. Analgesic and anti-inflammatory activities of the synthesized compounds were evaluated in mice. Among the synthesized compounds, compound 9c showed the best analgesic and anti-inflammatory activities without causing any gastric effect in stomachs of tested animals. In addition, the synthesized compounds were screened for their antibacterial and antifungal activities against some pathogenic strains.

Full text of DOI:10.1007/s00044-012-0253-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2553–2560
DOI 10.1007/s00044-012-0253-1
ORIGINAL RESEARCH
Synthesis of some new 2,6-disubstituted-3(2H)-pyridazinone
derivatives and investigation of their analgesic, anti-inflammatory
and antimicrobial activities
•
Didem Tiryaki Murat Sukuroglu Deniz S. Dogruer
•
•
•
Esra Akkol Selda Ozgen M. Fethi Sahin
•
Received: 29 June 2012 / Accepted: 20 September 2012 / Published online: 29 September 2012
ꢀ Springer Science+Business Media New York 2012
Abstract In this study, 12 new 3(2H)-pyridazinone
derivatives carrying 4-substituted phenylpiperazinylethyl
moiety on lactam nitrogen were synthesized and their
chemical structures were confirmed by 1H-NMR, mass, and
elemental analysis. Analgesic and anti-inflammatory activi-
ties of the synthesized compounds were evaluated in mice.
Among the synthesized compounds, compound 9c showed
the best analgesic and anti-inflammatory activities without
causing any gastric effect in stomachs of tested animals. In
addition, the synthesized compounds were screened for their
antibacterial and antifungal activities against some patho-
genic strains.
treatment of rheumatic disorders and other degenerative
inflammatory joint diseases. The main mechanism of action
of these drugs is the inhibition of cyclooxygenase enzyme
(COX) which is catalyzing the conversion of arachidonic acid
to the prostaglandins (PGs) mediating both inflammation
response and physiologic homeostasis. Accordingly, chronic
use of NSAIDs is often accompanied by side effects such
as gastrointestinal lesions, bleeding, and nephrotoxicity
(Williams and Lemke, 2008).
In the early 1990s, two isoforms of the COX enzyme,
known as COX-1 and COX-2, have been identified
(Masferrer et al., 1992). Subsequent studies showed that
COX-1 is a constitutive enzyme and synthesizes PGs
mediating normal homeostasis in the gastrointestinal tract,
kidneys, and platelets, whereas COX-2, mostly inducible,
is related to the production of PGs mediating inflammation,
pain, and fever (Dannhardt and Laufer, 2000). In the
meantime, classical NSAIDs were determined to inhibit
both isoforms (Dannhardt and Lauferm, 2000). These
findings have led to the development of selective COX-2
inhibitors to improve the therapeutic potency and to reduce
the classical side effects coupled with the use of NSAIDs
(Penning et al., 1997; Prasit et al., 1999). However,
selective COX-2 inhibitors have been reported to be
associated with serious cardiovascular side effects (Dogne’
et al., 2005). Therefore, the search for new analgesic and
anti-inflammatory agents devoid of side effects continues
to be an active area of research in medicinal chemistry.
On the other hand, despite many significant progresses
in antimicrobial therapy, the existing antimicrobial drugs
(antibacterial and antifungal) have become less effective or
ineffective due to the development of multidrug-resistant
pathogens (i.e., microbial isolates such as bacteria and
fungi), which is a serious health problem (Perea and
Patterson, 2002; Grare et al., 2007).
Keywords 3(2H)-pyridazinone ꢀ Analgesic activity ꢀ
Anti-inflammatory activity ꢀ Antimicrobial activity
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are impor-
tant therapeutic agents for the treatment of pain, fever, and
inflammation associated with a number of pathological con-
ditions. Especially, NSAIDs are the first choice in the
D. Tiryaki ꢀ M. Sukuroglu ꢀ D. S. Dogruer (&) ꢀ M. F. Sahin
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Gazi University, 06330 Etiler, Ankara, Turkey
e-mail: denizdogruer2002@yahoo.com
E. Akkol
Department of Pharmacognosy, Faculty of Pharmacy, Gazi
University, 06330 Etiler, Ankara, Turkey
S. Ozgen
Department of Microbiology, Faculty of Pharmacy, Gazi
University, 06330 Etiler, Ankara, Turkey
123

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Med Chem Res (2013) 22:2553–2560
As a result, there is an increasing need to design new
Results and discussion
antibacterial and antifungal agents with better activity
profile and lower toxicity.
Chemistry
Many compounds carrying 3(2H)-pyridazinone have
been reported to be associated with analgesic and anti-
In this study, 12 new 2,6-disubstituted-3(2H)-pyridazinone
derivatives were synthesized as shown in the Scheme 1.
6-Phenyl-3(2H)-pyridazinone (1), 6-(4-methoxyphenyl)-
3(2H)-pyridazinone (2), and 6-(4-chlorophenyl)-3(2H)-
pyridazinone (3) used as starting compounds were
synthesized by the reaction of glyoxylic acid, appropriate
acetophenones and hydrazine hydrate. Subsequently, the
treatment of compounds 1–3 with 1-bromo-2-chloroethane
in the presence of potassium carbonate in anhydrous
DMF gave the compounds 2-(2-chloroethyl)-6-phenyl-3
(2H)-pyridazinone (4), 2-(2-chloroethyl)-6-(4-methoxyphenyl)
-3(2H)-pyridazinone (5), and 2-(2-chloroethyl)-6-(4-
chlorophenyl)-3(2H)-pyridazinone (6). The synthesis of
˘
inflammatory activities (Takaya et al., 1979; S¸u¨ku¨roglu
˘
¨
et al., 2005; S¸u¨ku¨roglu et al., 2006; Gokc¸e et al., 2009;
Abouzid et al., 2010), as well as antimicrobial activity
˘
(Sonmez et al., 2006; Dogruer et al., 2008). In addition, it
has been reported that compounds bearing arylpiperaziny
lalkyl moiety on lactam nitrogen of pyridazinone ring have
analgesic and anti-inflammatory activities (Rohet et al.,
1996; Dal Piaz et al., 2003; Giovanni et al., 2003).
Consequently, we synthesized 12 new pyridazinone
derivatives carrying 4-substituted phenylpiperazinylethyl
moiety at position 2 to investigate their analgesic, anti-
inflammatory, and antimicrobial activities.
Scheme 1 Synthetic route of
the title compounds
123

Med Chem Res (2013) 22:2553–2560
2555
compounds 1–3 (Coates and McKillop, 1993) and 4
(Yamada et al., 1983) was previously reported. However,
compounds 5 and 6 were prepared for the first time in this
study.
damage to gastric mucosa to some extent at 100 mg/kg
dose. The rest of the compounds were less active than
ASA.
On the other hand, anti-inflammatory activities of com-
pounds 8b, 9b, and 9c demonstrated parallel results with their
corresponding analgesic activities. However, except for
compounds 8b, 9b, and 9c other compounds did not have any
significant anti-inflammatory activity. When the chemical
structures of the active compounds are taken into consider-
ation, compounds 9b and 9c having p-chloro and p-fluoro
substituents on phenyl ring of phenylpiperazine moiety at the
side chain, respectively, are 6-(4-chlorophenyl)-3(2H)-pyrid-
azinone derivative. However, compounds 8b and 8c having
same substituents at same position are 6-(4-methoxyphenyl)-
3(2H)-pyridazinone’s derivatives.
Finally, new title compounds (7a–d, 8a–d, and 9a–d) were
prepared by the reaction of 2-(2-chloroethyl)-6-(4-non-
substituted/methoxy/chlorophenyl)-3(2H)-pyridazinones, NaI
with appropriate phenylpiperazine derivatives in the presence
of potassium carbonate in CH3CN. The chemical structures
of the newly synthesized compounds were elucidated by
1
1H-NMR, mass, and elemental analysis. The H-NMR, mass
spectra, and elemental analysis data of the compounds are in
agreement with the proposed structures.
In vivo analgesic and anti-inflammatory activities
These results will give some idea about further research
on these molecules.
Analgesic and anti-inflammatory activities of the title
compounds were determined by p-benzoquinone-induced
writhing test and carrageenan-induced hind paw edema
test, respectively (Okun et al., 1963; Kasahara et al., 1985).
In addition, all the synthesized compounds were also
evaluated in terms of their gastric effects. As shown in
Table 1, among the synthesized compounds, 9b and 9c
were found more active than ASA. The best activity was
obtained with compound 9c. Moreover, compounds 8b and
8c exhibited analgesic activity almost equal to that of
acetylsalicylic acid (ASA). In addition, while compounds
9b and 9c did not cause any gastric lesion and bleeding in
stomachs of tested animals, compounds 8b and 8c caused
Antimicrobial activity
The synthesized compounds were tested for their antibac-
terial activity against 4 gram-positive bacteria (Staphylo-
coccus aureus, methicillin-resistant S. aureus (MRSA,
isolate), Enterococcus faecalis, and E. faecalis isolate), 4
gram-negative bacteria (Escherichia coli, E. coli producing
extended spectrum b-lactamase, Pseudomonas aeruginosa,
and P. aeruginosa isolate), and for their antifungal activity
against Candida albicans and Candida krusei by microdi-
lution method (Clinical and Laboratory Standards Insti-
Table 1 Analgesic and anti-inflammatory activities of the synthesized compounds
Compound
Dose
(mg/kg)
Analgesic
activity
(inhibition of
writhing %)
Gastric
ulcerogenic
effect
Anti-inflammatory activity (inhibition of edema %)
90 min
180 min
270 min
360 min
7a
100
100
100
100
100
100
100
100
100
100
100
100
100
10
10.7
0/6
0/6
0/6
0/6
0/6
1/6
2/6
0/6
0/6
0/6
0/6
0/6
4/6
–
–
–
6.4
3.1
7b
23.3
16.2
10.6
–
14.9
26.3**
–
14.8
29.9**
–
12.1
19.5
–
7c
16.8
7d
8.6
8a
18.4
11.9
16.9
16.2
–
12.6
19.8
15.5
–
–
–
8b
44.4***
47.6***
5.6
27.5**
22.9*
13.6
7.4
26.2**
18.3
7.9
8c
8d
9a
20.1
–
1.4
–
9b
53.2***
57.5***
30.2**
45.9***
–
20.6
29.5**
4.7
29.7***
28.3**
12.3
–
31.0***
35.3***
18.5
–
28.2**
28.8**
15.5
–
9c
9d
ASA
–
Indomethacin
25.3**
36.9***
37.6***
39.4***
* p \ 0.05, ** p \ 0.01, *** p \ 0.001 significant from control
SEM standard error mean
123

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Med Chem Res (2013) 22:2553–2560
tute, 2006, 2008). Ampicillin, ofloxacin, fluconazole, and
amphotericin B were used as references.
employing a Varian Mercury 400 mHz FT spectrometer
(Varian Inc., Palo Alto, CA, USA), in DMSO-d6 at Faculty
of Pharmacy, Ankara University, Ankara, Turkey. The
mass spectra were recorded on a Micromass LCT Premier
XE (Waters, Milford, MA, USA) LC–MS spectrometer
using an positive electrospray ion source (ESI?) at Faculty
of Pharmacy, Gazi University, Ankara, Turkey. Elemental
analysis was performed on a Leco 932 CHNS instrument
(St. Joseph, MI, USA) at Faculty of Pharmacy, Ankara
University, Ankara, Turkey, and the results were within
0.4 % of the theoretic values.
As shown in the Table 2, the synthesized compounds
showed antimicrobial activity with MIC values in the range
of 32–128 lg/mL. Regarding the antibacterial activity, all
the compounds were found active at 64 lg/mL concen-
trations against all the tested gram bacteria except S. aur-
eus isolate (MIC 128 lg/mL). However, the antifungal
activity of the compounds was observed against both
C. albicans and C. krusei at 32 lg/mL concentration.
According to the antimicrobial activity results, fungi can be
said to be more sensitive to title compounds rather than
gram bacteria.
The synthesis of compounds 1–3 (Coates and McKillop,
1993) and 4 (Yamada et al., 1983) was previously reported.
However, 2-(2-chloroethyl)-6-(4-methoxy)-3(2H)-pyridaz-
inone (5) and 2-(2-chloroethyl)-6-(4-chlorophenyl)-3(2H)-
pyridazinone (6) were prepared for the first time in this
study.
As a result, these compounds might be relatively good
candidates to develop better antifungal agents against
C. albicans and C. krusei.
Experimental
Synthesis of 6-(4-nonsubstituted/methoxy/
chlorophenyl)-3(2H)-pyridazinones (1, 2, 3)
All the chemicals used for the synthesis of the compounds
were purchased from Aldrich (Steinheim, Germany) and
Merck (Darmstadt, Germany). Melting points of the com-
pounds were recorded on an Electrothermal-9200 digital
melting points apparatus (Southent, Great Britain) and the
values are uncorrected. Thin-layer chromatography (TLC)
was performed on Merck 60F254 plates. Reactions were
monitored by TLC on silica gel with detection by UV light
Glyoxylic acid (0.05 mol) and (0.15 mol) acetophenone
derivative (nonsubstituted/4-methoxy/4-chloroacetophenone
derivatives) were heated at 100–105 ꢁC for 2 h. At the end
of this period, the reaction mixture was cooled to 40 ꢁC and
then, 20 mL water and 5 mL ammonium hydroxide solution
(25 %) were added to the reaction mixture until the medium
pH became 8. Then, reaction mixture was extracted with
dichloromethane (4 9 25 mL), then hydrazine hydrate
1
(254 nm). The H-NMR spectra (400 mHz) was recorded
Table 2 Antimicrobial activity of the synthesized compounds (MICs, lg/mL)
Compounds
S. aureus S. aureus
ATCC
29213
E. faecalis E.
ATCC
29212
E. coli
faecalis ATCC
E. coli
isolate
(ESBL)
P.
aeruginosa
ATCC 27853 isolate
P.
aeruginosa ATCC
C. albicans C. krusei
ATCC
6258
isolate
(MRSA)
isolate
25922
10231
7a
64
64
64
64
64
64
64
64
64
64
64
64
2
128
128
128
128
128
128
128
128
128
128
128
128
32
64
64
64
64
64
64
64
64
64
64
64
64
2
64
64
64
64
64
64
64
64
64
64
64
64
4
64
64
64
64
64
64
64
64
64
64
64
64
8
64
64
64
64
64
64
64
64
64
64
64
64
64
16
–
64
64
64
64
64
64
64
64
64
64
64
64
–
64
64
64
64
64
64
64
64
64
64
64
64
–
32
32
32
32
32
32
32
32
32
32
32
32
–
32
32
32
32
32
32
32
32
32
32
32
32
–
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
Ampicillin
Ofloxacin
Fluconazole
0.5
–
2
1
2
0.12
–
1
32
–
–
–
–
–
–
–
1
64
0.5
Amphotericin
B
–
–
–
–
–
–
–
–
\0.25
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Med Chem Res (2013) 22:2553–2560
2557
2-[2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl]-6-
phenylpyridazin-3(2H)-one (7c)
(0.05 mol) was added to the separated aqueous layer and the
reaction mixture was refluxed for 2 h. After the completion
of the reaction, reaction mixture was cooled to room tem-
perature. The resulting precipitate was filtered to give
compounds 1, 2, and 3.
Yield 53 %, m.p.: 102 ꢁC. Recrystallized from acetone/n-
hexane. 1H-NMR (DMSO-d6) d: 8.00 (d, J = 9.6 Hz, 1H),
7.86 (d, 2H), 7.48–7.41 (m, 3H), 7.02–6.85 (m, 5H), 4.27
(t, 2H), 2.98 (t, 4H), 2.74 (t, 2H), 2.57 (t, 4H). MS ESI(?)
m/e 379 (M?H, 100). Anal. (C22H23FN4O): C, H, N calc.
69.82, 6.13, 14.80 found 69.62, 6.24, 14.76.
Synthesis of 2-(2-chloroethyl)-6-(4-nonsubstituted/
methoxy/chlorophenyl)-3(2H)-pyridazinones (4, 5, 6)
Appropriate 3(2H)-pyridazinone derivative (0.003 mol),
anhydrous potassium carbonate (0.009 mol), and 1-bromo-
2-chloroethane (0.009 mol) in 7 mL of anhydrous DMF
were stirred at room temperature for 2.5 h. At the end of
this period, the reaction mixture was poured into ice water
and the resulting precipitate was filtered to give compounds
4, 5, and 6. These compounds were used to synthesize title
compounds without further purification.
2-[2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethyl]-6-
phenylpyridazin-3(2H)-one (7d)
Yield 67 %, m.p.: 117 ꢁC. Recrystallized from acetone/
n-hexane. 1H-NMR (DMSO-d6) d: 8.01 (d, J = 9.6 Hz,
1H), 7.86 (d, 2H), 7.48–7.41 (m, 3H), 7.01 (d, J = 9.6 Hz,
1H), 6.82 (d, 2H), 6.75 (d, 2H), 4.27 (t, 2H), 3.63 (s, 3H),
2.92 (t, 4H), 2.74 (t, 2H), 2.57 (t, 4H). MS ESI(?) m/e 391
(M?H, 100). Anal. (C23H26N4O2): C, H, N calc. 70.75,
6.71, 14.35 found 70.48, 6.57, 14.25.
General procedure for the synthesis of title compounds
(7a–d, 8a–d and 9a–d)
2-[2-(4-Phenylpiperazin-1-yl)ethyl]-6-(4-
methoxyphenyl)pyridazin-3(2H)-one (8a)
2-(2-Chloroethyl)-6-(4-nonsubstituted/methoxy/chlorophenyl)
-3(2H)-pyridazinone derivative (1.7 mmol) and NaI
(1.8 mmol) in 30 mL of CH3CN were refluxed for 30 min
and cooled room temperature; then, anhydrous potassium
carbonate (3.4 mmol) and appropriate phenylpiperazine
derivatives (3.4 mmol) were added. The resulting mixture
was refluxed for 2.5 h. At the end of this period, the reaction
mixture was evaporated to dryness and treated with cold
water; then, the forming precipitate was crystallized from an
appropriate solvent.
Yield 87 %, m.p.: 98 ꢁC. Recrystallized from n-hexane.
1H-NMR (DMSO-d6) d: 7.95 (d, J = 9.6 Hz, 1H), 7.80 (d,
2H), 7.15 (t, 2H), 7.00 (d, 2H), 6.97 (d, J = 9.6 Hz, 1H),
6.85 (d, 2H), 6.71 (t, 1H), 4.24 (t, 2H), 3.76 (s, 3H), 3.03 (t,
4H), 2.73 (t, 2H), 2.56 (t, 4H). MS ESI(?) m/e 391 (M?H,
100). Anal. (C23H26N4O2): C, H, N calc. 70.75, 6.71, 14.35
found 70.69, 6.64, 14.28.
2-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-6-(4-
methoxyphenyl)pyridazin-3(2H)-one (8b)
2-[2-(4-Phenylpiperazin-1-yl)ethyl]-6-phenylpyridazin-
3(2H)-one (7a)
Yield 60 %, m.p.: 118 ꢁC. Recrystallized from ethanol/
water. H-NMR (DMSO-d6) d: 7.93 (d, J = 9.6 Hz, 1H),
Yield 68 %, m.p.: 139 ꢁC. Recrystallized from n-hexane.
1H-NMR (DMSO-d6) d: 8.00 (d, J = 9.6 Hz, 1H), 7.86 (d,
2H), 7.48–7.41 (m, 3H), 7.15 (t, 2H), 7.01 (d, J = 9.6 Hz,
1H), 6.86 (d, 2H), 6.71 (t, 1H), 4.27 (t, 2H), 3.03 (t, 4H),
2.75 (t, 2H), 2.57 (t, 4H). MS ESI(?) m/e 361 (M?H, 100).
Anal. (C22H24N4O): C, H, N calc. 73.31, 6.71, 15.54 found
73.24, 6.72, 15.52.
1
7.78 (d, 2H), 7.14 (d, 2H), 6.98 (d, 2H), 6.96 (d,
J = 9.6 Hz, 1H), 6.84 (d, 2H), 4.22 (t, 2H), 3.75 (s, 3H),
3.01 (t, 4H), 2.71 (t, 2H), 2.54 (t, 4H). MS ESI(?) m/e 425
(M?H, 100). Anal. (C23H25ClN4O2): C, H, N calc. 65.01,
5.93, 13.19 found 64.81, 6.01, 13.09.
2-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-6-
phenylpyridazin-3(2H)-one (7b)
2-[2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl]-6-
(4-methoxyphenyl)pyridazin-3(2H)-one (8c)
Yield 72 %, m.p.: 126 ꢁC. Recrystallized from ethanol/
water. H-NMR (DMSO-d6) d: 8.00 (d, J = 9.6 Hz, 1H),
1
Yield 33 %, m.p.: 100 ꢁC. Recrystallized from acetone/
n-hexane. 1H-NMR (DMSO-d6) d: 7.99 (d, J = 9.6 Hz, 1H),
7.85 (d, 2H), 7.05–6.89 (m, 7H), 4.29 (t, 2H), 3.81 (s, 3H),
3.02 (t, 4H), 2.77 (t, 2H), 2.61 (t, 4H). MS ESI(?) m/e 409
(M?H, 100). Anal. (C23H25FN4O2): C, H, N calc. 67.63,
6.17, 13.72 found 67.78, 6.25, 13.83.
7.86 (d, 2H), 7.50–7.38 (m, 3H), 7.16 (d, 2H), 7.01 (d,
J = 9.6 Hz, 1H), 6.87 (d, 2H), 4.27 (t, 2H), 3.03 (t, 4H),
2.74 (t, 2H), 2.56 (t, 4H). MS ESI(?) m/e 395 (M?H, 100).
Anal. (C23H24ClN3O): C, H, N calc. 66.91, 5.87, 14.19
found 66.88, 6.01, 14.23.
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Med Chem Res (2013) 22:2553–2560
2-[2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethyl]-6-
(4-methoxyphenyl)pyridazin-3(2H)-one (8d)
Biologic assays
Preparation of test mice for analgesic and anti-
inflammatory assays
Yield 86 %, m.p.: 123,5 ꢁC. Recrystallized from n-hexane.
1H-NMR (DMSO-d6) d: 7.99 (d, J = 9.6 Hz, 1H), 7.84 (d,
2H), 7.05–7.0 (m, 3H), 6.85 (d, 2H), 6.79 (d, 2H), 4.28 (t,
2H), 3.81 (s, 3H), 3.67 (s, 3H), 2.95 (t, 4H), 2.77 (t, 2H),
2.60 (t, 4H). MS ESI(?) m/e 421 (M?H, 100). Anal.
(C24H28N4O3): C, H, N calc. 68.55, 6.71, 13.32 found
68.70, 6.62, 13.37
Male Swiss albino mice (20–25 g) were purchased from
the animal breeding laboratories of Refik Saydam Central
Institute of Health (Ankara, Turkey). The animals, left for
2 days for acclimatization to animal room conditions, were
maintained on Standard pellet diet and water ad libitum.
The food was withdrawn 1 day before the experiment, but
free Access to water was allowed. Throughout the experi-
ments, animals were processed according to the suggested
ethical guidelines for the care of laboratory animals. The
experiments were confirmed by the Gazi University Fac-
ulty of Medicine Laboratory Animals Ethics Committee.
The mice were randomly assigned into the groups and six
animals were used in each group.
2-[2-(4-Phenylpiperazin-1-yl)ethyl]-6-
(4-chlorophenyl)pyridazin-3(2H)-one (9a)
Yield 40 %, m.p.: 121 ꢁC. Recrystallized from ethyl acetate/
1
n-hexane. H-NMR (DMSO-d6) d: 8.05 (d, J = 9.6 Hz, 1H),
7.93 (d, 2H), 7.56 (d, 2H), 7.19 (t, 2H), 7.06 (d, J = 9.6 Hz,
1H), 6.90 (d, 2H), 6.75 (t, 1H), 4.31 (t, 2H), 3.07 (t, 4H), 2.78
(t, 2H), 2.61 (t, 4H). MS ESI(?) m/e 395 (M?H, 100). Anal.
(C22H23ClN4O): C, H, N calc. 66.91, 5.87, 14.19 found
67.05, 5.732, 14.32.
Preparation of test samples for analgesic and anti-
inflammatory assays
2-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-6-
(4-chlorophenyl)pyridazin-3(2H)-one (9b)
Test samples were suspended in a mixture of distilled water
and 0.5 % sodium carboxymethylcellulose (CMC) and were
given orally to the test animals. The animals of the control
group received the same experimental handling except that
the drug treatment was replaced with appropriate volumes of
the vehicle. ASA (100 mg/kg) and indomethacin (10 mg/kg)
in 0.5 % CMC were used as reference drugs.
Yield 37 %, m.p.: 116 ꢁC. Recrystallized from ethyl acetate/
n-hexane. 1H-NMR (DMSO-d6) d: 8.05 (d, J = 9.6 Hz, 1H),
7.93 (d, 2H,), 7.56 (d, 2H), 7.20 (d, 2H), 7.06 (d, J = 9.6 Hz,
1H), 6.91 (d, 2H), 4.31 (t, 2H), 3.07 (t, 4H), 2.78 (t, 2H), 2.56 (t,
4H). MS ESI(?) m/e 430 (M?H, 100). Anal. (C22H22Cl2N4O):
C, H, N calc. 61.54, 5.16, 13.05 found 61.28, 5.158, 13.21.
Analgesic assay (p-benzoquinone-induced writhing
test)
2-[2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl]-6-(4-
chlorophenyl)pyridazin-3(2H)-one (9c)
Sixty minutes after the oral administration of the test
samples and ASA, the mice were intraperitoneally injected
with 0.1 mL/10 g body weight of 2.5 % (vlv) p-benzo-
quinone (PBQ, Merck, Darmstadt, Germany) solution in
distilled water. Control animals received an appropriate
volume of dosing vehicle. The mice were then kept indi-
vidually for observation and the total number of abdominal
contraction (writhing movements) was counted for the next
15 min, starting on the 5th min after the PBQ injection.
The data represent an average of the total number of
writhing movements observed. The analgesic activity was
expressed as the percentage change compared to writing
controls (Okun et al., 1963)
Yield 23 %, m.p.: 113 ꢁC. Recrystallized from acetone/
n-hexane. 1H-NMR (DMSO-d6) d: 8.05 (d, J = 9.6 Hz,
1H), 7.93 (d, 2H), 7.56 (d, 2H), 7.07–6.89 (m, 5H), 6.75 (t,
1H), 4.31 (t, 2H), 3.02 (t, 4H), 2.78 (t, 2H), 2.61 (t, 4H).
MS ESI(?) m/e 413 (M?H, 100). Anal. (C22H22ClFN4O):
C, H, N calc. 64.00, 5.37, 13.57 found 63.93, 5.51, 13.65.
2-[2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethyl]-6-(4-
chlorophenyl)pyridazin-3(2H)-one (9d)
Yield 59 %, m.p.: 129 ꢁC. Recrystallized from n-hexane.
1H-NMR (DMSO-d6) d: 8.05 (d, J = 9.6 Hz, 1H), 7.93
(d, 2H), 7.56 (d, 2H), 7.06 (d, J = 9.6 Hz, 1H), 6.85
(d, 2H), 6.79 (d, 2H), 4.30 (t, 2H), 3.67 (s, 3H), 2.95
(t, 4H), 2.78 (t, 2H), 2.60 (t, 4H). MS ESI(?) m/e 425
(M?H, 100). Anal. (C23H25ClN4O2): C, H, N calc. 65.01,
5.93, 13.19 found 64.76, 5.79, 13.31.
n ꢁ n0
Analgesic activity ðwrithing inhibition % ) ¼
ꢂ 100
n
n ¼ The mean writhing count of control group
n0 ¼ The mean writhing counts of test groups
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Med Chem Res (2013) 22:2553–2560
2559
Stock solutions of the tested compounds were dissolved
in DMSO. Stock solutions of the tested compounds and
reference drugs were diluted twofold in microplate wells.
All solvents and diluents, pure microorganisms, and pure
media were used in control wells.
Anti-inflammatory assay (Carrageenan-induced hind
paw edema test)
Sixty minutes after the oral adminstration of either test
sample or dosing vehicle, each mouse was injected with
freshly prepared (0.5 mg/25 lL) suspension of carrageenan
(Sigma, St. Louis, Missouri, USA) in physiologic saline
(154 nM NaCl) into subplantar tissue of the right hind paw.
As the control, 25 lL saline solution was injected into that
of the left hind paw. Paw edema was measured in every
90 min during 6 h after the induction of inflammation. The
difference in footpad thickness between the right and left
foot was measured with a pair of dial thickness gauge
callipers (Ozaki Co., Tokyo, Japan). Mean values of treated
groups were compared with mean values of a control group
and analyzed by statistical methods (Kasahara et al., 1985).
Bacteria were subcultured in Mueller–Hinton Agar
(MHA) (Merck) plates and incubated overnight at 37 ꢁC
and Candida was subcultured in Sabouraud Dextrose Agar
(SDA; Merck) plates at 35 ꢁC for 24–48 h.
Bacterial susceptibility testing was performed according
to the guidelines of CLSI M100-S18 (Clinical and Labora-
tory Standards Institute, 2008). Mueller–Hinton Broth
(MHB; Merck) was added to each microplate well. Then, the
tested compounds and reference drugs were added to this
medium in wells by twofold serial dilution to obtain the
required concentrations of 512, 256,128…0.5 lg/mL. The
bacterial suspensions used for inoculation were prepared at
105 CFU/mL by diluting fresh cultures at McFarland 0.5
density (107 CFU/mL). Suspensions of the bacteria at
105 CFU/mL concentrations were inoculated with a two-
folddiluted solution of the compounds. A 10 lL bacteria
inoculum was added to each microplate well. There were
104 CFU/mL bacteria in the wells after inoculation.
Ulcerogenic effect
After p-benzoquinone-induced writhing test, the surviving
mice were killed under deep ether anesthesia and stomachs
were removed. Then, each stomach was opened through
great curvature and examined under dissecting microscope
for lesions or bleedings.
Fungal susceptibility testing was performed according to
the guidelines of CLSI M27-A (Clinical and Laboratory
Standards Institute, 2006). Roswell Park Memorial Institute
(RPMI)-1640 medium with L-glutamine (Sigma) buffered to
pH 7 with 3-(N-morpholino)propanesulfonic acid (MOPS)
(Sigma) was added each microplate well. Then, the tested
compounds and reference drugs were added to this medium
in wells by twofold serial dilution to obtain the required
concentrations of 512, 256,128…0.125 lg/mL. The yeast
suspensions used for inoculation were prepared at 104 CFU/
mL by diluting fresh cultures at McFarland 0.5 density
(106 CFU/mL). Suspensions of the yeast at 104 CFU/mL
concentrations were inoculated to the twofold-diluted solu-
tion of the compounds. A 10 lL yeast inoculum was added
to each well of the microplates. There were 103 CFU/mL
yeast in the wells after inoculations.
Statistical analysis
Data obtained from animal experiments were expressed as
mean standard error ( SEM). Statistical differences between
the treatments and the control were tested byANOVA test and
Student–Newman–Keuls post hoc test. A value of p \ 0.05
was considered to be significant.
Antimicrobial assay
The in vitro minimum inhibitory concentrations (MICs) of the
synthesized compounds were determined by the two-fold serial
dilution technique in 96-well microtest plates according to the
methods recommended by the Clinical Laboratory Standards
Institute (CLSI). Standard and the isolated strains of the bac-
teria: S. aureus, ATCC 29213, methicillin-resistant S. aureus,
E. faecalis, ATCC 29212, E. faecalis isolate, E. coli ATCC
35218, E. coli, producing extended spectrum b-lactamase
(ESbL), P. aeruginosa, ATCC 27853 and its isolate were used
to determine antibacterial activity. As for antifungal activity,
C. albicans ATCC 10231 and C. krusei ATCC 6258 were used
to determine antifungal activity. Clinical isolates were obtained
from Gazi University Hospital, Microbiology Laboratory.
Standard powders of ampicillin, ofloxacin, fluconazole,
and amphotericin B were dissolved in appropriate solvents
recommended by CLSI guidelines.
Microplates were incubated at 37 ꢁC for 24 h for antibacterial
activity and at 37 ꢁC for 48 h for antifungal activity. After incu-
bation, the lowest concentration of the compounds that com-
pletely inhibits macroscopic growth was determined and reported
as MICs. All the experiments were done in 3 parallel series.
Acknowledgments This study was supported financially with grant from
Research Foundation of Gazi University (Project Number: 02/2011-07).
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