Synthesis and anti-inflammatory activity of novel pyridazine and pyridazinone derivatives as non-ulcerogenic agents

Article abstract of DOI:10.1007/s12272-012-1205-5

Herein, we report the synthesis and pharmacological properties of several series of pyridazine and pyridazinone derivatives. All the synthesized compounds were tested, in vivo, for their anti-inflammatory and ulcerogenic properties against indomethacin, a

Full text of DOI:10.1007/s12272-012-1205-5

Arch Pharm Res Vol 35, No 12, 2077-2092, 2012
DOI 10.1007/s12272-012-1205-5
Synthesis and Anti-inflammatory Activity of Novel Pyridazine and
Pyridazinone Derivatives as Non-ulcerogenic Agents
Makarem M. Saeed, Nadia A. Khalil, Eman M. Ahmed, and Kholoud I. Eissa
Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
(Received October 13, 2011/Revised November 27, 2011/Accepted December 6, 2011)
Herein, we report the synthesis and pharmacological properties of several series of pyridazine
and pyridazinone derivatives. All the synthesized compounds were tested, in vivo, for their
anti-inflammatory and ulcerogenic properties against indomethacin, as a reference compound.
Compounds 4a and 9d have shown a potent anti-inflammatory activity more than indometha-
cin with rapid onset of action and safe gastric profile. The latter compounds were then
selected for further investigation. In the MTT assay in vitro, both compounds were identified
as potent and selective COX-2 inhibitors.
Key words: Pyridazine, Pyridazinone, Anti-inflammatory activity, Ulcerogenicity
inflammatory efficacy as traditional NSAIDs, but with
improved gastrointestinal safety profile would be of
INTRODUCTION
Currently available non-steroidal anti-inflammatory therapeutic value. Diaryl heterocycles have become
drugs (NSAIDs), like ibuprofen and indomethacin, the major class of selective COX-2 inhibitors, such as
remain widely prescribed medications worldwide for celecoxib, rofecoxib, etoricoxib, and valdecoxib, which
the treatment of pain, fever, and swelling, which is display lower GI toxicity potential than compared to
associated with arthritis (Palomer et al., 2002). However, that of the traditional NSAIDs (Talley et al., 2000;
long-term use of these drugs result in gastrointestinal Stichtenoth and Frölich, 2003). However, concerns
(GI) side effects, which are inseparable from their phar- have been raised about the cardiovascular safety of
macological activities, such as ulceration, bleeding and selective COX-2 inhibitors, as some of them, like rofecoxib
renal toxicity (Sostres et al., 2010). The GI damage from and valdecoxib, have been withdrawn from the market
NSAIDs is generally attributed to the suppression of (Hsiao et al., 2009). Therefore, synthetic approaches
prostaglandin biosynthesis from arachidonic acid via based upon chemical modification of NSAIDs have
nonselective inhibition of the two isoforms of the cyclo- been taken with the aim of improving their safety
oxygenase (COX) enzyme (COX-1 and COX-2) (Warner profile.
et al., 1999; Habeeb et al., 2001). COX-1 is expressed
Pyridazines and pyridazinones are an important
in many organs and is responsible for homeostatic pro- class of heterocycles, which have been the subject of
cesses, such as platelet aggregation, gastric protection extensive research due to their broad-activities, such
and renal function, whereas the COX-2 enzyme is in- as anti-inflammatory (Gökçe et al., 2009; Asif, 2010),
ducible and expressed during inflammation, pain, and analgesic (Gökçe et al., 2009; Malinka et al., 2011),
oncogenesis. Due to this reason, the non-selective in- antihypertensive (Siddiqui et al., 2010, 2011), antican-
hibition of COX-1, by traditional NSAIDs, lead to GI cer (Al-Tel, 2010), anti-diabetic (Rathish et al., 2009),
complications (Almansa et al., 2003). On the other hand, anticonvulsant (Guan et al., 2010), anti-microbial
selective COX-2 inhibitors that achieve the same anti- (Kandile et al., 2009) and anti-fungal (Ting et al., 2011)
activities.
It has been reported that pyridazine and pyridazinone
moieties are an excellent templates for many selective
Correspondence to: Eman M. Ahmed, Department of Organic
Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Tel: 2-100-527-0276
E-mail: dr_eman2001@hotmail.com
I
(Beswick et al.,
COX-2 inhibitors, such as RS-57067
2004), Syntex compound II (Cesari et al., 2006) and
2077

2078
M. M. Saeed et al.
requirement for COX-2 selectivity. So, many N-sub-
stituted pyridazinone derivatives have been reported
to possess anti-inflammatory activity (Dogruer et al.,
2003; Asif, 2010; Rao and Knaus, 2008).
In this study, along with the continuous effort to
find distinctive structural template to selective COX-2
inhibitors, we utilized the pyridazinone moiety with a
suitable substitution at
N (2) and connecting the
pyridazinone ring at C (6) with an aryl group via an
ethenyl spacer, instead of methylene or ethyl spacer,
compounds 4a-i, 5a-d, 7a-c and 9a-d. It is assumed
that these modifications may provide additional sites
of interaction, thus, giving the desired selectivity, low
ulcerogenicity and increased potency. Furthermore,
the anti-inflammatory activity of certain fused pyrida-
zines had been reported (Abdel-Hakim, 2004). On this
Fig. 1. Representatives of pyridazinone lead compounds and
our pyridazinone scaffold.
the 4,5-dihydropyridazinone derivatives, IIIa
and Bekhit, 2008) (Fig. 1).
Structure activity relationship studies (SAR) employ- 12a
ing pyridazinone have shown that -substitution is a and ulcerogenic properties. Moreover, 6-arylethenyl-
,b
(Abouzid basis, we became interested in synthesizing certain
triazolopyridazines 11a and pyridazinoquinazolines
for the evaluation of their anti-inflammatory
-d
-d
N
Scheme 1. Reagents and conditions:
a
) morpholine, HOAc, dry benzene, 6 h,
b) N2H4H2O 99%, 3 h, c) anhyd.CuCl2/CH3CN/
60oC 3 h,
d) HCHO/appropriate secondary amine, 20 h,
e) N-bromomethylphthalimide/anhydrous K2CO3/DMF, 24 h.

Synthesis of Pyridazine as Anti-inflammatory Agent
2079
Scheme 2. Reagents and conditions:
a) 1-bromo-2-bromoethane or 1-bromo-3-chloropropane/K2CO3/DMF, 24 h,
b) N-(4-
methoxyphenyl)piperazine/KI/K2CO3/DMF, 20 h.
Scheme 3. Reagents and conditions:
a) INH, dioxan/absolute ethanol (1:1), 20 h, b) Anthranilic acid, dioxan/absolute
ethanol (1:1), 4 h, ) PABA, absolute ethanol/3 h.
c
pyridazine derivatives 13a-d were also synthesized (Sigma-Aldrich), and were used without further purifi-
and tested for anti-inflammatory activity and gastric cation. Reactions were monitored by TLC, performed
toxicity.
on silica gel glass plates that contained 60 GF-254, and
visualization on TLC was achieved, by UV light or iodine
indicator. IR spectra were determined on Shimadzu
IR 435 spectrophotometer (KBr, cm−1). 1H-NMR spectra
were carried out using a Mercury 300-BB 300 MHz,
MATERIALS AND METHODS
Chemistry
All chemicals and reagents were obtained from Aldrich using TMS as internal standard. Chemical shifts (δ)

2080
M. M. Saeed et al.
are recorded in ppm on
δ
scale, Micro analytical Center, EIMS (% rel. abundance): 209 (100), 406 (M+, 0.46),
Cairo University, Egypt. 13C-NMR spectra were carried 408 (M+2, 8.73). Anal. Calcd for C23H23ClN4O: C,
out using a Mercury 300-BB 300 MHz that used TMS 67.89; H, 5.70; N, 13.77. Found C, 68.24; H, 5.63; N,
as an internal standard. Chemical shifts (
δ) are record- 13.71.
ed in ppm on scale, Micro analytical Center, Cairo
δ
University, Egypt. Mass spectra were recorded on 2-[4-(4-Chlorophenyl)piperazin-1-yl)methyl]-6-[2-
Shimadzu Qp-2010 plus, spectrometer, Micro analyti- (4-methoxyphenyl)ethenyl]-pyridazin-3(2H)-one
cal Center, Cairo University, Egypt. Elemental analyses (4b)
were carried out at the Micro analytical Center, Cairo Yield: 75%; mp 155-156oC; IR (cm−1): 3021 (C-H aro-
University, Egypt. Melting points were determined matic), 2920, 2850 (C-H aliphatic), 1658 (C=O), 1600
1
with Stuart apparatus and are uncorrected.
6-(4-Aryl)-4-oxo-hex-5-enoic acids (1a
Bekhit, 2008), 6-[2-(4-aryl)ethenyl]-4,5-dihydropyridazin- (s, 3H, -OCH3), 4.80 (s, 2H, CH2-N), 6.81 (d, 1H, =CH-
3(2H)-ones (2a ) (Abouzid et al., 2007), 6-[2-(4-aryl) olefinic), 6.89 (d, 1H, =CH-olefinic), 6.92 (d, 1H, pyri-
ethenyl]pyridazin-3(2H)-ones (3a ) (Abouzid et dazinone H-4, J = 8.7 Hz), 7.17-7.42 (m, 8H, Ar-H), 7.45
al., 2008) and 3-chloro-6-[2-(4-aryl)ethenyl]pyridazines (d, 1H, pyridazinone H-5, J = 8.7 Hz); EIMS (% rel.
10a
) (Abouzid et al., 2008) were synthesized abundance): 209 (100), 436 (M+, 0.6), 438 (M+2, 8.99).
(C=N); H-NMR (300 MHz, CDCl3): 2.81-2.90 (br m,
-d) (Abouzid and 4H, piperazine), 3.19-3.22 (br m, 4H, piperazine), 3.84
-d
,
b, d
(
, b, d
according to the reported procedures.
Anal. Calcd for C24H25ClN4O2: C, 65.97; H, 5.77; N,
12.82. Found: C, 65.93; H, 5.81; N, 12.76.
6-[2-(4-Bromophenyl)ethenyl]pyridazin-3(2
H)-one
(3c)
6-[2-(4-Methoxyphenyl)ethenyl]-2-[4-(phenyl)piper-
This compound was prepared according to the report- azin-1-yl)methyl]pyridazin-3(2H)-one (4c)
ed procedure (Abouzid et al., 2008). Yield: 65%; mp Yield: 68%; mp 157-158oC; IR (cm−1): 3063 (C-H aro-
249-250oC; IR (cm−1): 3441 (N-H), 3055 (C-H aromatic), matic), 2920, 2823 (C-H aliphatic), 1658 (C=O), 1604
1
1666 (C=O), 1593 (C=N); 1H-NMR (300 MHz, DMSO- (C=N); H-NMR (300 MHz, CDCl3): 2.87-2.89 (br m,
d6): 6.91 (d, 1H, =CH-olefinic, J = 10.2 Hz), 7.07 (d, 1H, 4H, piperazine), 3.17-3.21 (br m, 4H, piperazine), 3.84
=CH-olefinic, J = 10.2 Hz), 7.12 (d, 2H, ArH), 7.37 (d, (s, 3H, -OCH3), 4.79 (s, 2H, CH2-N), 6.82 (d, 1H, =CH-
2H, ArH), 7.55-7.58 (m, 2H, pyridazinone H-4 and H- olefinic), 6.89 (d, 1H, =CH-olefinic), 6.93 (d, 1H, pyri-
5), 13.08 (s, 1H, NH, D2O exchangeable); EIMS (% rel. dazinone H-4), 7.25-7.42 (m, 9H, ArH), 7.45 (d, 1H,
abundance): 128 (100), 276 (M+, 86.16), 278 (M+2, pyridazinone H-5); EIMS (% rel. abundance): 175 (100),
85.85). Anal. Calcd for C12H9BrN2O: C, 52.01; H, 3.27; 402 (M+, 0.40). Anal. Calcd for C24H26N4O2: C, 71.62;
N, 10.11. Found: C, 52.14; H, 3.28; N, 10.04.
H, 6.51; N, 13.92. Found: C, 71.73; H, 6.51; N, 13.95.
6-[2-(4-Substitutedphenyl)ethenyl]-2-[(substituted) 6-[2-(4-Bromophenyl)ethenyl]-2-[4-(4-chlorophenyl)
aminomethyl]pyridazin-3(2H)-ones (4a-i) piperazin-1-yl)aminomethyl]pyridazin-3(2H)-one
To a solution of an appropriate 3a-d (0.001 mol), in (4d)
absolute ethanol (10 mL), was added a mixture of Yield: 85%; mp 121-122oC; IR (cm−1): 3101 (C-H aro-
secondary amino compound (0.001 mol) and formalin matic), 2927, 2820 (C-H aliphatic), 1658 (C=O), 1604
1
37% (1.5 mL, 0.05 mol) in absolute ethanol (15 mL). (C=N); H-NMR (300 MHz, CDCl3): 2.82-2.91 (br m,
The reaction mixture was heated under reflux for 20 4H, piperazine), 3.18-3.20 (br m, 4H, piperazine), 4.81
h, filtered while hot and concentrated to half its (s, 2H, CH2-N), 6.79 (d, 1H, =CH-olefinic), 6.81 (d, 1H,
volume. After cooling, the separated solid was filtered =CH-olefinic), 6.88 (d, 1H, pyridazinone H-4), 7.18-7.49
and crystallized from ethanol.
(m, 8H, ArH), 7.52 (d, 1H, pyridazinone H-5); EIMS (%
rel. abundance): 209 (100), 486 (M+2, 4.20), 488 (M+4,
2-[4-(4-Chlorophenyl)piperazin-1-yl)methyl]-6-[2- 5.10). Anal. Calcd for C23H22BrClN4O: C, 56.86; H, 4.56;
(phenylethenyl)]pyridazin-3(2H)-one (4a)
N, 11.53; Found: C, 56.89; H, 4.62; N, 11.56.
Yield: 45%; mp 175-176oC; IR (cm−1): 3023 (C-H aro-
matic), 2951, 2831 (C-H aliphatic), 1670 (C=O), 1603 6-[2-(4-Bromophenyl)ethenyl]-2-[4-(phenyl)pipera-
1
(C=N); H-NMR (300 MHz, CDCl3): 2.85-2.88 (br m, zin-1-yl)aminomethyl]pyridazin-3(2H)-one (4e)
4H, piperazine), 3.12-3.15 (br m, 4H, piperazine), 4.78 Yield: 78%; mp 126-127oC; IR (cm−1): 3067 (C-H aro-
(s, 2H, CH2-N), 6.80 (d, 1H, =CH-olefinic), 6.83 (d, 1H, matic), 2939, 2823 (C-H aliphatic), 1658 (C=O), 1600
1
=CH-olefinic), 6.91 (d, 1H, pyridazinone H-4), 7.17- (C=N); H-NMR (300 MHz, CDCl3): 2.80-2.87 (br m,
7.48 (m, 9H, ArH), 7.51 (d, 1H, pyridazinone H-5); 4H, piperazine), 3.17-3.19 (br m, 4H, piperazine), 4.78

Synthesis of Pyridazine as Anti-inflammatory Agent
2081
(s, 2H, CH2-N), 6.83 (d, 1H, =CH-olefinic), 6.89 (d, 1H, C23H23ClN4O: C, 67.89; H, 5.70; N, 13.77. Found C,
=CH-olefinic), 6.93 (d, 1H, pyridazinone H-4, J = 8.7 68.21; H, 5.93; N, 13.84.
Hz), 7.22-7.48 (m, 9H, ArH), 7.51 (d, 1H, pyridazinone
H-5, J = 8.7 Hz); 13C-NMR (DMSO-d6):
δ 48.34 (2C of 6-[2-(4-Chlorophenyl)ethenyl]-2-[piperidin-1-yl)
piperazine), 49.92 (2C of piperazine), 68.61 (-CH2-N), methyl]pyridazin-3(2H)-one (4i)
118.42-151.12 (aromatic C), 166.54 (C=O); EIMS (% Yield: 72%; mp 96-97oC; IR (cm−1): 3107 (C-H aromatic),
rel. abundance): 175 (100), 450 (M+, 0.36), 452 (M+2, 2927, 2804 (C-H aliphatic), 1662 (C=O), 1600 (C=N);
25.73). Anal. Calcd for C23H23BrN4O: C, 61.20; H, 5.14; 1H-NMR (300 MHz, CDCl3): 1.57-1.61 (m, 2H, CH2 of
N, 12.41. Found: C, 61.26; H, 5.18; N, 12.52.
piperidine), 2.55-2.62 (m, 4H, 2CH2 of piperidine), 2.79
(t, 4H, 2CH2 of piperidine), 4.66 (s, 2H, CH2-N), 6.83 (d,
6-[2-(4-Bromophenyl)ethenyl]-2-[piperidin-1-yl) 1H, =CH-olefinic), 6.87 (d, 1H, =CH-olefinic), 6.88 (d, 1H,
methyl]pyridazin-3(2H)-one (4f)
pyridazinone H-4), 7.26-7.40 (m, 4H, ArH), 7.43 (d, 1H,
Yield: 58%; mp 128-129oC; IR (cm−1): 3054 (C-H aro- pyridazinone H-5); EIMS (% rel. abundance): 98 (100),
matic), 2939, 2830 (C-H aliphatic), 1660 (C=O), 1604 331 (M+2, 0.50). Anal. Calcd for C18H20ClN3O: C, 65.55;
1
(C=N); H-NMR (300 MHz, CDCl3): 1.64-1.68 (m, 2H, H, 6.11; N, 12.74. Found C, 65.84; H, 6.02; N, 12.60.
CH2 of piperidine), 2.56-2.61 (m, 4H, 2CH2 of piperi-
dine), 2.82 (t, 4H, 2CH2 of piperidine), 4.93 (s, 2H,
CH2-N), 6.86 (d, 1H, =CH-olefinic), 6.88 (d, 1H, =CH-
olefinic), 6.89 (d, 1H, pyridazinone H-4), 7.27-7.52 (m,
4H, ArH), 7.53 (d, 1H, pyridazinone H-5); 13C-NMR
2-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2yl)meth-
yl]-6-[2-(4-substitutedphenyl)ethenyl]pyridazin-
3(2H)-ones (5a-d)
A mixture of the respective 3a-d (0.001 mol), N-bro-
(DMSO-d6 ppm):
δ 48.34 (2C of piperazine), 49.92 (2C momethylphthalimide (0.24 g, 0.001 mol) and anhydrous
of piperazine), 68.61 (-CH2-N), 118.42-151.12 (15 aro- K2CO3 (0.41 g, 0.003 mol) in dry DMF (10 mL) was
matic C, 2 olefinic C), 166.54 (C=O); EIMS (% rel. stirred, at room temperature for 24 h. The reaction
abundance): 279 (100), 373 (M+, 0.06), 375 (M+2, 0.73). mixture was poured onto ice-cold water (25 mL) and
Anal. Calcd for C18H20BrN3O: C, 57.76; H, 5.39; N, the resulting precipitate was filtered, washed with
11.23. Found: C, 57.70; H, 5.43; N, 11.31.
water and crystallized from ethanol.
6-[2-(4-Chlorophenyl)ethenyl]-2-[4-(4-chlorophenyl) 2-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2yl)methyl]-
piperazin-1-yl)methyl] pyridazin-3(2H)-one (4g)
6-[2-(phenyl)ethenyl]pyridazin-3(2H)-one (5a)
Yield: 66%; mp 159-160oC; IR (cm−1): 3011 (C-H aro- Yield: 50%; mp >300oC; IR (cm−1): 3008 (C-H aromatic),
matic), 2947, 2831 (C-H aliphatic), 1674 (C=O), 1600 2950, 2835 (C-H aliphatic), 1728, 1639 (C=O), 1600
1
1
(C=N); H-NMR (300 MHz, CDCl3): 2.85-2.87 (br m, (C=N); H-NMR (300 MHz, CDCl3): 5.65 (s, 2H, CH2-
4H, piperazine), 3.13-3.16 (br m, 4H, piperazine), 4.78 N), 6.89 (d, 1H, =CH-olefinic), 6.92 (d, 1H, =CH-
(s, 2H, CH2-N), 6.79 (d, 1H, =CH-olefinic), 6.85 (d, 1H, olefinic), 7.26-7.89 (m, 11H: 9 ArH, 2H of pyridazinone
=CH-olefinic), 6.87 (d, 1H, pyridazinone H-4), 7.17-7.41 H-4 and H-5); EIMS (% rel. abundance): 55 (100), 357
(m, 8H, ArH), 7.44 (d, 1H, pyridazinone H-5); EIMS (% (M+, 0.23). Anal. Calcd for C21H15N3O3: C, 70.58; H,
rel. abundance): 70 (100), 440 (M+, 0.78), 442 (M+2, 4.23; N, 11.76. Found: C, 71.02; H, 4.51; N, 11.64.
6.13), 444 (M+4, 4.02). Anal. Calcd for C23H22Cl2N4O:
C, 62.59; H, 5.02; N, 12.69. Found: C, 62.64; H, 5.13; 2-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2yl)methyl]-
N, 12.73.
6-[2-(4-methoxyphenyl)ethenyl] pyridazin-3(2H)-
one (5b)
6-[2-(4-Chlorophenyl)ethenyl]-2-[4-(phenyl)piper- Yield: 58%; mp 151-152oC; IR (cm−1): 3035 (C-H aro-
azin-1-yl)methyl]pyridazin-3(2H)-one (4h)
matic), 2970, 2850 (C-H aliphatic), 1730, 1685 (C=O),
1
Yield: 76%; mp 128-129oC; IR (cm−1): 3043 (C-H aro- 1604 (C=N); H-NMR (300 MHz, CDCl3): 3.84 (s, 3H,
matic), 2951, 2881 (C-H aliphatic), 1662 (C=O), 1597 OCH3), 5.65 (s, 2H, CH2-N), 6.80 (d, 1H, =CH-olefinic),
1
(C=N); H-NMR (300 MHz, CDCl3): 2.83-2.89 (br m, 6.85 (d, 1H, =CH-olefinic), 6.92 (d, 1H, pyridazinone
4H, piperazine), 3.17-3.21 (br m, 4H, piperazine), 4.79 H-4, J = 8.7 Hz), 7.45 (d, 1H, pyridazinone H-5, J = 8.7
(s, 2H, CH2-N), 6.85 (d, 1H, =CH-olefinic, J = 8.1 Hz), Hz), 7.72-7.74 (m, 4H, ArH), 7.86-7.89 (m, 4H, ArH);
6.90 (d, 1H, =CH-olefinic, J = 8.1 Hz), 6.93 (d, 1H, EIMS (% rel. abundance): 229 (100), 387 (M+, 0.02).
pyridazinone H-4), 7.22-7.41 (m, 9H, ArH), 7.44 (d, Anal. Calcd for C22H17N3O4: C, 68.21; H, 4.42; N,
1H, pyridazinone H-5); EIMS (% rel. abundance): 175 10.85. Found: C, 68.24; H, 4.44; N, 10.81.
(100), 406 (M+, 0.48), 408 (M+2, 6.65). Anal. Calcd for

2082
M. M. Saeed et al.
2-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2yl)methyl]-
Yield: 74%; mp 119-120oC; IR (cm−1): 3055 (C-H aro-
6-[2-(4-bromophenyl)ethenyl]pyridazin-3(2H)-one matic), 2931, 2820 (C-H aliphatic), 1666 (C=O), 1600
(5c)
(C=N); 1H-NMR (300 MHz, DMSO-d6): 3.79 (t, 2H, -CH2-
Yield: 62%; mp 194-195oC; IR (cm−1): 3055 (C-H aro- CH2-), 3.99 (t, 2H, -CH2-CH2-), 6.94 (d, 1H, =CH-olefinic),
matic), 2931, 2850 (C-H aliphatic), 1728, 1670 (C=O), 6.99 (d, 1H, =CH-olefinic), 7.01 (d, 1H, H-4 of pyrida-
1
1612 (C=N); H-NMR (300 MHz, CDCl3): 5.65 (s, 2H, zinone), 7.10-7.58 (m, 5H: 4 ArH, 1H, H-5 of pyridazin-
CH2-N), 6.84 (d, 1H, =CH-olefinic), 6.85 (d, 1H, =CH- one); EIMS (% rel. abundance): 63 (100), 339 (M+H,
olefinic), 7.32 (d, 1H, pyridazinone H-4, J = 8.4 Hz), 9.24), 342 (M+4, 11.30). Anal. Calcd for C14H12BrClN2O:
7.51 (d, 1H, pyridazinone H-5, J = 8.4 Hz), 7.71-7.73 (m, C, 49.51; H, 3.56; N, 8.25. Found: C, 49.49; H, 3.56; N,
4H, ArH), 7.86-7.88 (m, 4H, ArH); 13C-NMR (DMSO- 8.25.
d6 ppm):
δ 72.24 (-CH2-N), 131.77-144.35 (15 aromatic
C, 2 olefinic C), 163.87, 166.98 (3 C=O); EIMS (% rel. 2-(2-Chloroethyl)-6-[2-(4-chlorophenyl)ethenyl]
abundance): 104 (100), 437 (M+2, 0.18). Anal. Calcd pyridazin-3(2H)-one (6c)
for C21H14BrN3O3: C, 57.82; H, 3.23; N, 9.63. Found: C, Yield: 68%; mp 186-187oC; IR (cm−1): 3059 (C-H aro-
57.89; H, 3.28; N, 9.68.
matic), 2931, 2827 (C-H aliphatic), 1666 (C=O), 1600
(C=N); H-NMR (300 MHz, DMSO-d6): 3.80 (t, 2H,
1
2-[(1,3-Dioxo-1,3-dihydro-2H-isoindol-2yl)methyl]- CH2-CH2-), 4.02 (t, 2H, -CH2-CH2-), 6.93 (d, 1H, =CH-
6-[2-(4-chlorophenyl)ethenyl]pyridazin-3(2H)-one olefinic), 7.02 (d, 1H, =CH-olefinic), 7.41-7.45 (m, 3H:
(5d)
2 Ar-H, 1H, H-4 of pyridazinone), 7.60-7.63 (m, 3H: 2
Yield: 68%; mp 199-200oC; IR (cm−1): 3055 (C-H aro- ArH, 1H, H-5 of pyridazinone); EIMS (% rel. abundance):
matic), 2939, 2854 (C-H aliphatic), 1728, 1666 (C=O), 247 (100), 294 (M+, 2.93), 296 (M+2, 97.58), 298 (M+4,
1597 (C=N); H-NMR (300 MHz, CDCl3): 5.64 (s, 2H, 66.30). Anal. Calcd for C14H12Cl2N2O: C, 56.97; H,
1
CH2-N), 6.85 (d, 1H, =CH-olefinic), 6.94 (d, 1H, =CH- 4.10; N, 9.49. Found: C, 56.97; H, 4.18; N, 9.52.
olefinic), 7.26-7.42 (m, 6H, 4Ar-H and 2H of pyrida-
zinone H-4 and H-5), 7.73 (d, 2H, ArH), 7.88 (d, 2H,
ArH); EIMS (% rel. abundance): 233 (100), 393 (M+2,
0.19). Anal. Calcd for C21H14ClN3O3: C, 64.37; H, 3.60;
N, 10.72. Found C, 64.48; H, 3.69; N, 10.74.
2-{2-[4-(4-Methoxyphenyl)piperazin-1yl]ethyl}-
6-[2-(4-substitutedphenyl)ethenyl]pyridazin-3
(2H)-ones (7a-c)
A mixture of an appropriate 6a-c (0.001 mol), an-
hydrous K2CO3 (0.69 g, 0.005 mol) and few specs of KI
in dry acetonitrile (20 mL) was heated under reflux
for 30 min. 4-Methoxyphenylpiperazine (0.58 g, 0.003
2-(2-Chloroethyl)-6-[2-(4-substitutedphenyl)
ethenyl]-pyridazin-3(2H)-ones (6a-c)
A mixture of an appropriate 3a-d (0.01 mol), 1-bromo- mol) was added to the hot reaction mixture, which
2-chloroethane (7.17 g, 4.16 mL, 0.05 mol) and anhydrous was heated under reflux for 20 h. After cooling, the
K2CO3 (6.9 g, 0.05 mol) in dry DMF (20 mL) was stirred, reaction mixture was poured onto ice-cold water (25
at room temperature for 24 h. The reaction mixture mL) with continuous stirring. The resulting solid was
was poured slowly with continuous stirring onto ice- filtered, dried then washed with dry ether.
cold water (50 mL). The formed solid was filtered and
washed with ethanol.
2-{2-[4-(4-Methoxyphenyl)piperazin-1yl]ethyl}-6-
[2-(4-methoxyphenyl)ethenyl]pyridazin-3(2H)-one
(7a)
2-(2-Chloroethyl)-6-[2-(4-methoxyphenyl)ethen-
yl]pyridazin-3(2H)-one (6a)
Yield: 58%; mp 110-111oC; IR (cm−1): 3028 (C-H aro-
Yield: 82%; mp 169-170oC; IR (cm−1): 3005 (C-H aro- matic), 2935, 2814 (C-H aliphatic), 1685 (C=O), 1604
matic), 2970, 2827 (C-H aliphatic), 1681 (C=O), 1604 (C=N); 1H-NMR (300 MHz, CDCl3): 2.56 (t, 2H, -CH2-
(C=N); 1H-NMR (300 MHz, DMSO-d6): 2.57 (t, 2H, -CH2- CH2-), 2.72-2.80 (br m, 4H, piperazine), 2.82 (t, 2H, -
CH2-), 2.82 (t, 2H, -CH2-CH2-), 3.84 (s, 3H, -OCH3), CH2-CH2-), 3.11-3.21 (br m, 4H, piperazine), 3.77 (s, 3H,
6.73 (d, 1H, =CH-olefinic), 6.84 (d, 1H, =CH-olefinic), -OCH3), 3.84 (s, 3H, -OCH3), 6.79 (d, 1H, =CH-olefinic),
7.27-7.44 (m, 6H: 4 ArH, 2H of pyridazinone); EIMS 6.86 (d, 1H, =CH-olefinic), 6.90-7.45 (m, 10H: 8 ArH,
(% rel. abundance): 149 (100), 294 (M+2+H, 16.84). 2H of pyridazinone); 13C-NMR (DMSO-d6 ppm):
Anal. Calcd for C15H15ClN2O2: C, 61.97; H, 5.20; N, 9.64. (-CH2-CH2-), 25.90 (-CH2-CH2-), 49.60 (2Cof piperazine),
δ
20.19
Found: C, 62.21; H, 5.24; N, 9.66.
51.10 (2C of piperazine), 55.17 (2-OCH3), 114.25-150.78
(aromatic C), 167.05 (C=O); EIMS (% rel. abundance):
205 (100), 219 (3.31), 227 (11.98), 446 (M+, 0.00). Anal.
Calcd for C26H30N4O3: C, 69.93; H, 6.77; N, 12.55.
2-(2-Chloroethyl)-6-[2-(4-bromophenyl)ethenyl]
pyridazin-3(2H)-one (6b)

Synthesis of Pyridazine as Anti-inflammatory Agent
Found C, 70.25; H, 6.39; N, 12.43.
2083
65.53; H, 5.49; N, 10.32.
2-{2-[4-(4-Methoxyphenyl)piperazin-1yl]ethyl}-6-
[2-(4-bromophenyl)ethenyl]pyridazin-3(2H)-one
(7b)
2-(3-Chloropropyl)-6-[2-(4-methoxyphenyl)ethen-
yl]pyridazin-3(2H)-one (8b)
Yield: 72%; mp 79-80oC; IR (cm−1): 3055 (C-H aromatic),
Yield: 43%; mp 126-127oC; IR (cm−1): 3050 (C-H aro- 2924, 2854 (C-H aliphatic), 1666 (C=O), 1597 (C=N);
matic), 2947, 2823 (C-H aliphatic), 1665 (C=O), 1600 1H-NMR (300 MHz, DMSO-d6):
δ 2.15-2.19 (m, 2H,
(C=N); 1H-NMR (300 MHz, CDCl3): 2.63 (t, 2H, -CH2- -CH2-CH2-CH2-), 2.37 (t, 2H, -CH2-CH2-CH2-), 2.75 (t,
CH2-), 2.85 (t, 2H, -CH2-CH2-), 3.08-3.18 (br m, 4H, pi- 2H, -CH2-CH2-CH2-), 3.78 (s, 3H, OCH3), 6.78 (d, 1H,
perazine), 3.44-3.46 (br m, 4H, piperazine), 3.78 (s, =CH-olefinic), 6.80 (d, 1H, =CH-olefinic), 6.91-7.95 (m,
3H, -OCH3), 6.79 (d, 1H, =CH-olefinic), 6.86 (d, 1H, 6H, 4 ArH, 2H, H-4 and H-5 of pyridazinone). EIMS
=CH-olefinic), 6.90-7.10 (m, 5H, ArH), 7.26 (d, 1H, H- (% rel. abundance): 304 (M+, 100), 306 (M+2, 34.39).
5 of pyridazinone), 7.33 (d, 2H, ArH), 7.52 (d, 2H, Anal. Calcd for C16H17ClN2O2: C, 63.05; H, 5.62; N,
ArH); EIMS (% rel. abundance): 205 (1.94), 219 (2.88), 9.19. Found C, 62.84; H, 5.83; N, 9.26.
275 (4.72), 277 (62.45), 289 (0.82), 291 (100), 494 (M+,
0.00), 496 (M+2, 0.00). Anal. Calcd for C25H27BrN4O2: 2-(3-Chloropropyl)-6-[2-(4-bromophenyl)ethenyl]
C, 60.61; H, 5.49; N, 11.31. Found: C, 60.64; H, 5.53; pyridazin-3(2H)-one (8c)
N, 11.29.
Yield: 81%; mp 143-144oC; IR (cm−1): 3051 (C-H aro-
matic), 2954, 2858 (C-H aliphatic), 1654 (C=O), 1585
2-{2-[4-(4-Methoxyphenyl)piperazin-1yl]ethyl}-6-
[2-(4-chlorophenyl)ethenyl]pyridazin-3(2H)-one
(7c)
(C=N); 1H-NMR (300 MHz, DMSO-d6):
δ 2.17-2.20 (m,
2H, -CH2-CH2-CH2-), 3.70 (t, 2H, -CH2-CH2-CH2-), 4.19
(t, 2H, -CH2-CH2-CH2-), 6.99 (d, 1H, =CH-olefinic), 7.02
Yield: 46%; mp 130-131oC; IR (cm−1): 3047 (C-H aro- (d, 1H, =CH-olefinic), 7.14 (d, 2H, ArH), 7.35 (d, 2H,
matic), 2943, 2823 (C-H aliphatic), 1670 (C=O), 1585 ArH), 7.58 (d, 1H, H-4 of pyridazinone), 7.92 (d, 1H,
(C=N); 1H-NMR (300 MHz, CDCl3): 2.57 (t, 2H, -CH2- H-5 of pyridazinone). EIMS (% rel. abundance): 352
CH2), 2.63-2.78 (br m, 4H, piperazine), 2.82 (t, 2H, -CH2- (M+, 48.73), 354 (M+2, 65.55), 356 (M+4, 17.27). Anal.
CH2), 3.05-3.18 (br m, 4H, piperazine), 3.77 (s, 3H, Calcd for C15H14BrClN2O: C, 50.94; H, 3.99; N, 7.92.
-OCH3), 6.84 (d, 1H, =CH-olefinic), 6.98 (d, 1H, =CH- Found: C, 51.16; H, 4.13; N, 8.11.
olefinic), 7.27-7.43 (m, 10H: 8 ArH, 2H of pyridazinone);
EIMS (% rel. abundance): 205 (100), 219 (2.25), 450 (M+, 2-(3-Chloropropyl)-6-[2-(4-chlorophenyl)ethenyl]
13.29), 452 (M+2, 24.15). Anal. Calcd for C25H27ClN4O2: pyridazin-3(2H)-one (8d)
C, 66.58; H, 6.03; N, 12.42. Found: C, 66.29; H, 6.21; Yield: 75%; mp 193-194oC; IR (cm−1): 3047 (C-H aro-
N, 12.81.
matic), 2974, 2831 (C-H aliphatic), 1681 (C=O), 1604
(C=N); 1H-NMR (300 MHz, DMSO-d6):
δ
2.39-2.45 (m,
2H, -CH2-CH2-CH2-), 2.49 (t, 2H, -CH2-CH2-CH2-), 2.77
(t, 2H, -CH2-CH2-CH2-), 6.93 (d, 1H, =CH-olefinic), 7.02
2-(3-Chloropropyl)-6-[2-(4-substitutedphenyl)
ethenyl]-pyridazin-3(2H)-ones (8a-d)
The compounds were prepared from 3a-d and 1- (d, 1H, =CH-olefinic), 7.41-7.44 (m, 3H: 2ArH, 1H, H-
bromo-3-chloropropane (4.72 g, 2.96 mL, 0.03 mol), 4 of of pyridazinone), 7.60-7.63 (m, 3H: 2ArH, 1H, H-5
following the procedure described for the compounds of of pyridazinone). EIMS (% rel. abundance): 128
6a-d.
(100), 310 (M+2, 7.94), 312 (M+4, 4.81). Anal. Calcd
for C15H14Cl2N2O: C, 58.27; H, 4.56; N, 9.06. Found: C,
2-(3-Chloropropyl)-6-[2-(phenyl)ethenyl]pyridazin- 58.48; H, 4.71; N, 9.12.
3(2H)-one (8a)
Yield: 77%; mp >300oC; IR (cm−1): 3035 (C-H aromatic),
6-[2-(4-Aryl)ethenyl]-2-{3-[4-(4-Methoxyphenyl)
2924, 2835 (C-H aliphatic), 1662 (C=O), 1600 (C=N);
1H-NMR (300 MHz, DMSO-d6):
2.38-2.40 (m, 2H,
-CH2-CH2-CH2-), 2.48 (t, 2H, -CH2-CH2-CH2-), 2.77 (t,
2H, -CH2-CH2-CH2-), 6.84 (d, 1H, =CH-olefinic), 7.01 the procedure described for the compounds 7a
piperazin-1yl]propyl}-pyridazin-3(2H)-ones
(9a-d)
δ
The compounds were prepared from 8a-d, following
-c. The
(d, 1H, =CH-olefinic), 7.23-7.59 (m, 4H, 3 ArH, 1H, H- reaction mixture was cooled and poured slowly onto
4 of pyridazinone), 7.60-7.80 (m, 3H: 2 ArH, 1H, H-5 of ice-cold water (25 mL) with continuous stirring, then
pyridazinone); EIMS (% rel. abundance): 199 (100), extracted with methylene chloride (3
× 5 mL). The
274 (M+, 10.82), 276 (M+2, 10.26). Anal. Calcd for organic layer was washed with water and dried
C15H15ClN2O: C, 65.57; H, 5.50; N, 10.20. Found: C, (Na2SO4). After distillation off the solvent, the residue

2084
M. M. Saeed et al.
was triturated with dry ether to give 9a
-
d
.
2931, 2831 (C-H aliphatic), 1666 (C=O), 1597 (C=N);
1H-NMR (300 MHz, CDCl3):
δ 2.39-2.42 (m, 2H, -CH2-
2-{3-[4-(4-Methoxyphenyl)piperazin-1yl]propyl}-
6-[2-(phenyl)ethenyl]pyridazin-3(2H)-one (9a)
CH2-CH2-), 2.51 (t, 2H, -CH2-CH2-CH2-), 2.75 (t, 2H,
-CH2-CH2-CH2-), 2.82-3.05 (br m, 4H, piperazine),
Yield: 66%; mp 144-145oC; IR (cm−1): 3050 (C-H aro- 3.25-3.59 (br m, 4H, piperazine), 3.67 (s, 3H, OCH3),
matic), 2947, 2831 (C-H aliphatic), 1670 (C=O), 1604 6.78 (d, 1H, =CH-olefinic), 6.85 (d, 1H, =CH-olefinic),
(C=N); 1H-NMR (300 MHz, CDCl3):
δ
2.59-2.61 (m, 2H, 7.01-7.63 (m, 6H: 4 Ar-H, 2H of pyridazinone), 7.41 (d,
-CH2-CH2-CH2-), 2.73 (t, 2H, -CH2-CH2-CH2-), 2.90 (t, 2H, Ar-H, = 8.4 Hz), 7.63 (d, 2H, Ar-H, = 8.4 Hz).
J
J
2H, -CH2-CH2-CH2), 3.10-3.16 (br m, 4H, piperazine), EIMS (% rel. abundance): 205 (8.77), 231 (7.97), 233
3.24-3.39 (br m, 4H, piperazine), 3.77 (s, 3H, - OCH3), (100), 261 (4.69), 464 (M+, 0.00). Anal. Calcd for
6.87 (d, 1H, =CH-olefinic), 6.90 (d, 1H, =CH-olefinic), C26H29ClN4O2: C, 67.16; H, 6.29; N, 12.05. Found: C,
7.09-7.50 (m, 11H: 9 Ar-H, 2H of pyridazinone). EIMS 67.31; H, 6.31; N, 12.11.
(% rel. abundance): 150 (100), 197 (5.40), 205 (24.97),
211 (1.19), 219 (1.40), 225 (0.53), 233 (0.62), 430 (M+,
3-Chloro-6-[2-(4-bromophenyl)ethenyl]pyrida-
1.02). Anal. Calcd for C26H30N4O2: C, 72.53; H, 7.02; N,
13.01. Found: C, 72.36; H, 7.18; N, 13.20.
zine (10c)
This compound was prepared according to reported
procedure (Abouzid et al., 2008). Yield: 67%; mp 184-
185oC; IR (cm−1): 3032 (C-H aromatic), 1595 (C=N).
2-{3-[4-(4-Methoxyphenyl)piperazin-1yl]propyl}-
6-[2-(4-methoxyphenyl)ethenyl]pyridazin-3(2H)-
one (9b)
1H-NMR (300 MHz, DMSO-d6):
δ 7.47 (d, 1H, =CH-
olefinic), 7.52 (d, 1H, =CH-olefinic), 7.61-7.82 (m, 2H
Yield: 42%; mp 134-135oC; IR (cm−1): 3045 (C-H aro- pyridazine H-4 and H-5), 7.94 (d, 2H, Ar-H), 8.10 (d,
matic), 2920, 2850 (C-H aliphatic), 1685 (C=O), 1604 2H, Ar-H); EIMS (% rel. abundance): 294 (M+, 21.60),
1
(C=N); H-NMR (300 MHz, CDCl3):
δ 2.34-2.39 (m, 295 (M+H, 100), 296 (M+2, 26.21), 298 (M+4, 6.56).
2H, -CH2-CH2-CH2-), 2.49 (t, 2H, -CH2-CH2-CH2-), 2.75 Anal. Calcd for C12H8BrClN2: C, 48.76; H, 2.73; N,
(t, 2H, -CH2-CH2-CH2-), 3.01-3.11 (br m, 4H, piperazine), 9.48. Found: C, 49.12; H, 2.66; N, 9.53.
3.25-3.45 (br m, 4H, piperazine), 3.68 (s, 3H, OCH3),
3.77 (s, 3H, OCH3), 6.71 (d, 1H, =CH-olefinic), 6.77 (d,
1H, =CH-olefinic), 6.85-7.54 (m, 10H: 8 ArH, 2H of
pyridazinone). EIMS (% rel. abundance): 227 (2.14),
3-(Pyridin-4-yl)-6-[2-(4-substitutedphenyl)ethe-
nyl]-1,2,4-triazolo[4,3-b]pyridazines (11a-d)
An equimolar mixture of a respective 10a-d and
229 (100), 233 (0.34), 460 (M+, 0.00). Anal. Calcd for isonicotinic acid hydrazide (0.002 mol each) in abso-
C27H32N4O3: C, 70.41; H, 7.00; N, 12.16. Found: C, lute ethanol/dry dioxan mixture (40 mL, 1:1) was
70.52; H, 7.08; N, 12.19.
heated under reflux for 20 h. The reaction mixture
was filtered while hot, the filtrate was concentrated
under reduced pressure to half its volume, then cooled.
The separated solid was filtered, washed with NaHCO3
solution, dried and crystallized from the benzene/
2-{3-[4-(4-Methoxyphenyl)piperazin-1yl]propyl}-
6-[2-(4-bromophenyl)ethenyl]pyridazin-3(2H)-one
(9c)
Yield: 63%; mp 174-175oC; IR (cm−1): 3055 (C-H aromatic), petroleum ether (60-80oC) mixture.
2939, 2850 (C-H aliphatic), 1666 (C=O), 1604 (C=N);
1H-NMR (300 MHz, CDCl3):
δ 1.94-2.00 (m, 2H, -CH2- 6-[2-(Phenyl)ethenyl]-3-(pyridin-4-yl)-1,2,4-triazolo
CH2-CH2-), 2.59 (t, 2H, -CH2-CH2-CH2-), 2.82 (t, 2H, [4,3-b]pyridazines (11a)
-CH2-CH2-CH2-), 3.00-3.08 (br m, 4H, piperazine), 3.20- Yield: 44%; mp >300oC; IR (cm−1): 3032 (C-H aromatic),
3.30 (br m, 4H, piperazine), 3.68 (s, 3H, OCH3), 6.83 1616 (C=N); 1H-NMR (300 MHz, DMSO-d6):
δ 7.04 (d,
(d, 1H, =CH-olefinic), 6.90 (d, 1H, =CH-olefinic), 7.26- 1H, =CH-olefinic), 7.21 (d, 1H, =CH-olefinic), 7.28-8.2
7.51 (m, 10H: 8 ArH, 2H of pyridazinone); EIMS (% (m, 11H, 9Ar-H and 2H pyridazine H-4 and H-5);
rel. abundance): 219 (0.71), 234 (2.88), 277 (91.85), 279 EIMS (% rel. abundance): 105 (100), 299 (M+, 1.31).
(100), 508 (M+,0.00), 510 (M+2, 0.00). Anal. Calcd for Anal. Calcd for C18H13N5: C, 72.23; H, 4.38; N, 23.40.
C26H29BrN4O2: C, 61.30; H, 5.74; N, 11.00. Found: C, Found: C, 72.20; H, 4.36; N, 23.49.
61.44; H, 5.82; N, 11.13.
3-(Pyridin-4-yl)-6-[2-(4-methoxyphenyl)ethenyl]-
2-{3-[4-(4-Methoxyphenyl)piperazin-1yl]propyl}- 1,2,4-triazolo[4,3-b]-pyridazines (11b)
6-[2-(4-chlorophenyl)ethenyl]pyridazin-3(2H)-one Yield: 62%; mp 174-175oC; IR (cm−1): 3059 (C-H aro-
1
(9d)
matic), 2920, 2850 (C-H aliphatic), 1604 (C=N); H-
Yield: 55%; mp 79-80oC; IR (cm−1): 3055 (C-H aromatic), NMR (300 MHz, DMSO-d6):
δ
3.80 (s, 3H, OCH3), 6.96

Synthesis of Pyridazine as Anti-inflammatory Agent
2085
(d, 1H, =CH-olefinic), 6.99 (d, 1H, =CH-olefinic), 7.25 matic), 2920, 2850 (C-H aliphatic), 1670 (C=O), 1604
(d, 1H, pyridazinone H-4, J = 6.9 Hz 3.72 (s, 3H,
, 7.27-8.06 (m, (C=N); 1H-NMR (300 MHz, DMSO-d6):
)
δ
8H, Ar-H), 7.88 (d, 1H, pyridazinone H-5, J = 6.9 Hz); -OCH3), 6.89 (d, 1H, =CH-olefinic), 6.95 (d, 1H, =CH-
EIMS (% rel. abundance): 57 (100), 329 (M+, 4.01). olefinic), 7.20-7.66 (m, 10H, 8Ar-H and 2H pyridazine
Anal. Calcd for C19H15N5O: C, 69.29; H, 4.59; N, 21.26. H-4 and H-5); EIMS (% rel. abundance): 45 (100), 329
Found: C, 69.34; H, 4.55; N, 21.19.
(M+, 6.76). Anal. Calcd for C20H15N3O2: C, 72.94; H,
4.59; N, 12.76. Found: C, 72.96; H, 4.63; N, 12.70.
3-(Pyridin-4-yl)-6-[2-(4-bromophenyl)ethenyl]-1,2,
4-triazolo[4,3-b]pyridazines (11c)
6-[2-(4-Bromophenyl)ethenyl]-10H-pyridazino[6,
Yield: 65%; mp 184-185oC; IR (cm−1): 3035 (C-H aro- 1-b]quinazolin-10-ones (12c)
1
matic), 1600 (C=N); H-NMR (300 MHz, DMSO-d6):
δ
Yield: 43%; mp 194-195oC; IR (cm−1): 3032 (C-H aro-
1
6.85 (d, 1H, =CH-olefinic), 6.90 (d, 1H, =CH-olefinic), matic), 1631 (C=O), 1604 (C=N); H-NMR (300 MHz,
7.47-7.93 (m, 6H, ArH), 8.06 (d, 2H, Ar-H,
J = 9 Hz), DMSO-d6): δ 6.88 (d, 1H, =CH-olefinic), 6.96 (d, 1H,
8.09 (d, 2H, Ar-H,
J = 9 Hz); EIMS (% rel. abundance): =CH-olefinic), 7.46-7.81 (m, 6H, 4ArH and 2H pyridazine
128 (100), 377 (M+, 66.33), 379 (M+2, 56.05). Anal. H-4 and H-5), 7.89 (d, 2H, Ar-H, J = 8.7 Hz), 8.05 (d,
Calcd for C18H12BrN5: C, 57.16; H, 3.20; N, 18.52. 2H, Ar-H, J = 8.7 Hz); EIMS (% rel. abundance): 295
Found: C, 56.94; H, 3.31; N, 18.64.
(100), 377 (M+, 4.12), 379 (M+2, 4.10). Anal. Calcd for
C19H12BrN3O: C, 60.34; H, 3.20; N, 11.11. Found: C,
6-[2-(4-Chlorophenyl)ethenyl]-3-(pyridin-4-yl)-1,2, 60.34; H, 3.29; N, 11.08.
4-triazolo[4,3-b]pyridazines (11d)
Yield: 71%; mp 193-194oC; IR (cm−1): 3047 (C-H aro- 6-[2-(4-Chlorophenyl)ethenyl]-10H-pyridazino[6,
1
matic), 1600 (C=N); H-NMR (300 MHz, DMSO-d6):
δ
1-b]quinazolin-10-ones (12d)
6.87 (d, 1H, =CH-olefinic,
=CH-olefinic,
2H pyridazine H-4 and H-5), 8.06 (d, 2H, Ar-H,
Hz), 8.09 (d, 2H, Ar-H, = 9 Hz); EIMS (% rel. olefinic), 7.41-7.84 (m, 6H, 4Ar-H and 2H pyridazine H-4
J
= 18 Hz), 6.92 (d, 1H, Yield: 68%; mp 187-188oC; IR (cm−1): 3035 (C-H aro-
1
J
= 18 Hz), 7.36-7.93 (m, 6H, 4ArH and matic), 1670 (C=O), 1604 (C=N); H-NMR (300 MHz,
J
= 9 DMSO-d6): δ 6.92 (d, 1H, =CH-olefinic), 7.01 (d, 1H, =CH-
J
abundance): 233 (100), 334 (M+H, 0.20). Anal. Calcd and H-5), 7.90 (d, 2H, Ar-H, J = 9 Hz), 8.06 (d, 2H,
for C18H12ClN5: C, 64.77; H, 3.62; N, 20.98. Found: C, ArHs, J = 9 Hz); EIMS (% rel. abundance): 111 (100),
64.73; H, 3.62; N, 20.93.
333 (M+, 56.45). Anal. Calcd for C19H12ClN3O: C, 68.37;
H, 3.62; N, 12.59. Found: C, 68.28; H, 3.57; N, 12.63.
6-[2-(4-Substitutedphenyl)ethenyl]-10H-pyridazi-
no[6,1-b]quinazolin-10-ones (12a-d)
4-{[6-(2-(4-Substitutedphenyl)ethenyl)pyridazin-
3-yl]amino}benzoic acids (13a-d)
An equimolar mixture of an appropriate 10a
-d and
anthranilic acid (0.001 mol each) in absolute ethanol/
An equimolar mixture of an appropriate 10a-d and
dry dioxan mixture (40 mL, 1:1) was heated under 4-aminobenzoic acid (0.001 mol each) was heated
reflux for 4 h. After cooling, the precipitated solid was under reflux for 3 h. The reaction mixture was filtered
filtered and crystallized from absolute ethanol.
while hot and the filtrate was concentrated under
reduced pressure to half its volume, then cooled. The
separated solid was filtered and crystallized from
isopropanol.
6-[2-(Phenyl)ethenyl]-10H-pyridazino[6,1-b]qui-
nazolin-10-ones (12a)
Yield: 45%; mp >300oC; IR (cm−1): 3055 (C-H aromatic),
1635 (C=O), 1605 (C=N); 1H-NMR (300 MHz, DMSO- 4-{[6-(2-(Phenyl)ethenyl)pyridazin-3-yl]amino}ben-
d6):
olefinic), 7.35-8.35 (m, 11H, 9Ar-H and 2H pyridazine Yield: 35%; mp >300oC; IR (cm−1): 3140 (N-H), 3248
H-4 and H-5); 13C-NMR (DMSO-d6 ppm):
133.41- (O-H), 3057 (C-H aromatic), 1688 (C=O), 1608 (C=N);
145.17 (16 aromatic C, 2 olefinic C), 167.17 (C=O); 1H-NMR (300 MHz, DMSO-d6):
6.74 (d, 1H, =CH-
δ 6.80 (d, 1H, =CH-olefinic), 6.95 (d, 1H, =CH- zoic acid (13a)
δ
δ
EIMS (% rel. abundance): 119 (100), 300 (M+H, 1.32). olefinic), 6.80 (d, 1H, =CH-olefinic), 7.38-8.20 (m, 11H,
Anal. Calcd for C19H13N3O: C, 76.24; H, 4.38; N, 14.04. 9Ar-H and 2H of pyridazine H-4 and H-5), 11.90 (s,
Found: C, 76.29; H, 4.35; N, 14.12.
1H, D2O exchangeable). EIMS (% rel. abundance): 120
(100), 317 (M+, 8.63). Anal. Calcd for C19H15N3O2: C,
71.91; H, 4.76; N, 13.24. Found: C, 71.99; H, 4.78; N,
13.28.
6-[2-(4-Methoxyphenyl)ethenyl]-10H-pyridazino
[6,1-b]quinazolin-10-ones (12b)
Yield: 52%; mp 162-163oC; IR (cm−1): 3035 (C-H aro-

2086
M. M. Saeed et al.
4-{[6-(2-(4-Methoxyphenyl)ethenyl)pyridazin-3-
yl]amino}benzoic acid (13b)
dispensed in 10% Tween-80 solution in the distilled
water. Animals’ treatment protocol was approved by the
Yield: 32%; mp 129-130oC; IR (cm−1): 3213 (N-H), 3444 National Research Center Animal Rights Committee.
(O-H), 3012 (C-H aromatic), 2966, 2839 (C-H aliphatic),
1681 (C=O), 1604 (C=N); 1H-NMR (300 MHz, DMSO-
Assessment of anti-inflammatory activity
d6):
δ
3.80 (s, 3H, -OCH3), 6.53 (d, 1H, =CH-olefinic),
Anti-inflammatory activity of the compounds was
6.77 (d, 1H, =CH-olefinic), 7.01 (d, 1H, pyridazine H- assessed using carrageenan-induced paw edema. Rats
4), 7.32-7.84 (m, 4H, Ar-H), 7.87 (d, 1H, pyridazine H- were divided into groups of six rats each. One hour
5), 8.02 (d, 2H, Ar-H), 8.05 (d, 2H, Ar-H), 10.77 (s, 1H, after oral administration of test compounds (10 mg/
D2O exchangeable); EIMS (% rel. abundance): 231 kg), 0.1 mL of 1% carragenan solution was injected,
(100), 349 (M+2H, 8.17). Anal. Calcd for C20H17N3O3: sub-planter to the left hind paw of each animal. Paw
C, 69.15; H, 4.93; N, 12.10. Found: C, 69.21; H, 4.95; volumes were measured using standard fluid displace-
N, 12.19.
ment procedures (7140-plesthysmometer Ugo Basile)
by dipping the hind left paw in 0.45% saline solution,
4-{[6-(2-(4-Bromophenyl)ethenyl)pyridazin-3-yl] at 1, 2, 3, and 4 h, post carrageenan injection. The
amino}benzoic acid (13c)
percent change in paw volume relative to that of the
Yield: 56%; mp 169-170oC; IR (cm−1): 3217 (N-H), 3429 base line measurement was taken as the criteria of
(O-H), 3032 (C-H aromatic), 1681 (C=O), 1608 (C=N); comparison. Indomethacin (10 mg/kg) was used as an
1H-NMR (300 MHz, DMSO-d6):
δ
6.52 (d, 1H, =CH- internal standard anti-inflammatory agent. Significant
olefinic), 6.70 (d, 1H, =CH-olefinic), 7.44 (d, 1H, pyrida- differences between the control and the treated groups
zine H-4), 7.49-7.89 (m, 8H, Ar-H), 8.04 (d, 1H, pyrida- were obtained, using Student,s
-test and values. The
zine H-5), 10.90 (s, 1H, D2O exchangeable); EIMS (% differences in the results were considered significant
t
p
rel. abundance): 295 (100), 394 (M-H, 0.04). Anal. at
Calcd for C19H14BrN3O2: C, 57.59; H, 3.56; N, 10.60.
Found: C, 57.58; H, 3.56; N, 10.71.
p < 0.05 (Table I).
Gastric ulcerative effect
Ulcerative effect (gastric hemorrhagic gross lesions)
4-{[6-(2-(4-Chlorophenyl)ethenyl)pyridazin-3-yl] of test compounds, which showed anti-inflammatory
amino}benzoic acids (13d)
action, was assessed after intragastric administration
Yield: 68%; mp 179-180oC; IR (cm−1): 3305 (N-H), 3217 of 10 mg/kg as previously described. Six hours after
(O-H), 3032 (C-H aromatic), 1685 (C=O), 1606 (C=N); drug administration, rats were euthanized by cervical
1H-NMR (300 MHz, DMSO-d6):
δ 6.49 (d, 1H, =CH- dislocation and the stomachs were removed, inflated
olefinic), 6.75 (d, 1H, =CH-olefinic), 7.48-8.07 (m, 10H, and opened along the greater curvature. The hemor-
8ArHs and 2H pyridazine H-4 and H-5), 10.91 (s, 1H, rhagic lesions were stretched out and scored from 0 to
D2O exchangeable); EIMS (% rel. abundance): 249 5, according to the method of Clementi et al. Gastric
(100), 351 (M+, 0.13). Anal. Calcd for C19H14ClN3O2: C, tissue samples were fixed in neutral buffered formalin
64.87; H, 4.01; N, 11.94. Found: C, 64.89; H, 4.12; N, for 24 h. indomethacin was used as positive control
11.82.
drug, (Table II, Fig. 2).
Statistical analysis
Pharmacology
Chemicals and drugs
Data are presented as mean ± S.E.M. Analysis of
Carrageenan and indomethacin were purchased from variance (ANOVA) with Dunnet's post hoc test was
Sigma-Aldrich.
used for testing the significance of data, using SPSS®
for windows, version 17.0.
off value for significance.
p < 0.05 was taken as a cut
Animals
Male Sprague Dawley rats (120-130 g weight) were
purchased from the animal house facility of the Na-
tional Research Center. Animals were acclimatized in
the animal house unit of the Pharmacology Dept.,
In vitro studies
Cell culture
Raw murine macrophage (RAW 264.7) was purchased
National Research Center of Egypt for at least one from the American Type Culture collections. Cells were
week prior to the experiments. Animals were kept at routinely cultured in RPMI-1640. Media were supple-
22 ± 3oC and 55 ± 5% relative humidity, during the mented with 10% fetal bovine serum (FBS), 2 mM L-
whole experiment. Standard food pellets and water glutamine, containing 100 U/mL penicillin G sodium,
were supplied ad labium. All test compounds were 100 U/mL streptomycin sulphate, and 250 mg/mL

Synthesis of Pyridazine as Anti-inflammatory Agent
2087
Table I. Percent change in rat paw edema after carrageenan sub-planter injection
% Change in paw volume (mean ± S.E.M)a
Compound No.
1 h
2 h
3 h
4 h
Control
Indomethacin
4 a
89.7 ± 19.6
62.7 ± 16.4
47.7* ± 10.6
49.6* ± 10.3
68.9 ± 12.2
39.3* ± 13.0
43.0* ± 8.4
65.0 ± 8.8
51.0* ± 11.6
41.6* ± 5.5
53.8* ± 6.0
54.1 ± 8.2
54.6* ± 6.2
53.3* ± 6.8
47.9* ± 4.6
90.7 ± 4.4
66.8 ± 16.2
76.0 ± 13.6
60.5 ± 7.5
79.4 ± 9.9
50.8 ± 17.3
42.0* ± 12.9
50.4* ± 3.8
70.7 ± 8.5
89.2 ± 18.0
82.1 ± 7.0
62.4 ± 19.8
136.5 ± 24.8
69.1 ± 6.7
51.8* ± 4.1
67.0 ± 21.0
77.4 ± 20.6
81.9 ± 10.8
66.9 ± 10.1
67.7 ± 12.2
74.7 ± 8.5
118.2 ± 26.2
48.8* ± 16.4
33.5* ± 8.8
48.5* ± 11.8
88.2 ± 18.5
104.8 ± 25.9
100.7 ± 19.0
52.6* ± 6.8
94.3 ± 10.2
112.6 ± 24.1
48.5* ± 2.1
56.5* ± 13.0
47.1* ± 11.0
73.0 ± 12.1
35.3* ± 6.5
56.0* ± 13.7
89.2 ± 19.5
59.9* ± 11.8
95.5 ± 15.1
43.1* ± 1.2
99.7 ± 17.8
31.9* ± 13.1
63.2* ± 12.9
69.0 ± 10.6
65.1* ± 15.9
57.2* ± 12.7
47.9* ± 15.3
76.7 ± 8.2
104.8 ± 13
40.3* ± 14.8
42.6* ± 6.0
43.8* ± 6.2
73.2 ± 22.1
70.8* ± 13.9
87.4 ± 4.0
51.7* ± 12.0
78.0 ± 17.2
72.6 ± 12.1
55.6* ± 7.0
32.9* ± 7.2
40.1* ± 5.0
65.0 ± 21.6
37.2* ± 7.6
65.2* ± 16.2
77.4 ± 21.1
44.9* ± 7.6
76.0* ± 8.7
73.2 ± 17.3
84.3 ± 7.6
31.0* ± 14.7
59.3 ± 17.7
51.9* ± 10.3
36.3* ± 4.2
68.3 ± 21.2
62.7* ± 4.4
85.3 ± 10.9
79.8 ± 19.6
78.4 ± 17.5
82.0 ± 21.1
59.0* ± 14.1
64.3* ± 12.1
98.9 ± 19.5
4 b
4 c
4 d
4 e
4 f
4 g
4 h
4 i
5 a
5 b
5 c
5 d
7 a
7 b
7 c
9 a
84.6 ± 6.0
86.6 ± 18.4
72.1 ± 12.6
90.8 ± 12.9
33.1* ± 10.0
48.2* ± 5.3
48.7* ± 5.1
90.8 ± 22.0
75.0 ± 16.5
75.8 ± 11.7
90.9 ± 18.9
104.6 ± 13.4
64.2 ± 20.1
74.2 ± 10.0
93.1 ± 25.2
70.9 ± 14.1
120.1 ± 11.5
9 b
9 c
9 d
47.0* ± 8.9
74.9 ± 12.1
23.8* ± 5.6
37.2* ± 4.5
42.7* ± 4.0
68.7 ± 7.7
11 a
11 b
11 c
11 d
12 a
12 b
12 c
12 d
13 a
13 b
13 c
13 d
42.2* ± 10.2
66.9 ± 8.5
42.5* ± 12.7
54.6 ± 14.3
65.8 ± 17.9
38.9* ± 4.9
66.8 ± 16.0
61.3 ± 10.4
51.1 ± 17.9
115.8 ± 17.0
85.8 ± 12.9
78.6 ± 14.1
102.9 ± 10.5
85.1 ± 12.8
86.0 ± 14.7
Significantly different from corresponding control value at *p < 0.05; aThe data are the result of six rats/experiment.
amphotericin B. Cells were maintained in humidified via the addition of 180 µL acidified isopropanol/well
air, which contains 5% CO2 at 37oC. RAW 264.7 cells and plate was shacked at room temperature, followed
were collected by scraping. All experiments were re- by photometric determination of the absorbance at
peated four times, unless mentioned, and the data was 570 nm using 96 wells microplate ELISA reader. Tri-
represented as (mean ± S.D.). All cell culture material plicate repeats were performed for each concentration
was obtained from Cambrex, BioScience.
and the average was calculated. Data was expressed
Cells 0.5 × 105 cells/well in serum-free media were as the percentage of relative viability compared with
plated in a flat bottom 96-well microplate, and treated that of the untreated control cells, as the relative fold
with 20
compound for 24 h at 37oC, in a humidified 5% CO2
atmosphere. After incubation, media were removed and culated, using the following equation: Absorbance of
40 L MTT solution/well were added and incubated treated cells/Absorbance of control untreated cells.
for an additional 4 h. MTT crystals were solubilized,
µ
L of different concentrations of each tested of increase in the cell viability (Fig. 3).
The fold of increase in the relative viability was cal-
µ

2088
M. M. Saeed et al.
Table II. Gastric ulcerative effect of test compounds
compared to indomethacin
Ulcer severity
Compound No.
Ulcer numbera
(score)a
Control
Indomethacin
4 a
0.0 ± 0.0
2.6 ± 0.4
0.0 ± 0.0
2.6 ± 0.5
3.0 ± 1.0
2.3 ± 1.2
0.0 ± 0.0
1.3 ± 0.7
3.6 ± 0.5
5.0 ± 0.5
1.2 ± 0.6
4.6 ± 1.0
4.0 ± 1.3
3.6 ± 0.5
1.6 ± 1.1
4.0 ± 1.3
1.6 ± 1.0
0.6 ± 0.5
1.2 ± 0.6
2.6 ± 1.2
0.0 ± 0.0
7.0 ± 1.8
2.6 ± 1.0
2.6 ± 0.5
1.3 ± 0.6
5.3 ± 2.2
0.0 ± 0.0
4.5 ± 2.2
0.0 ± 0.0
0.0 ± 0.0
6.2 ± 0.7
0.0 ± 0.0
3.0 ± 0.8
3.0 ± 1.0
2.6 ± 1.5
0.0 ± 0.0
1.3 ± 0.7
6.0 ± 2.1
5.3 ± 0.3
1.6 ± 0.8
9.3 ± 0.5
5.0 ± 1.7
6.0 ± 2.1
1.6 ± 1.1
5.0 ± 1.2
2 ± 1.2
0.6 ± 0.5
2.2 ± 1.3
4.3 ± 2.4
0.0 ± 0.0
9.3 ± 3.2
4.3 ± 2.4
6.3 ± 0.5
1.6 ± 0.8
6.3 ± 1.6
0.0 ± 0.0
6.3 ± 3.1
0.0 ± 0.0
4 b
4 d
4 e
4 f
4 g
4 h
4 i
5 a
5 b
5 c
5 d
7 a
7 c
9 a
9 b
9 c
Fig. 3. Cell viability of RAW 264.7 cells after the treatment
with different concentrations of the test compounds for 48 h
as measured by MTT assay. The data are presented as fold
of increase compared to control untreated cells.
specific against murine COX-2. Commercially available
matched paired antibodies were used (R&D Systems
Inc.).
9 d
11 a
11 b
11 c
11d
12 a
12 b
13 a
13 b
13 c
Cells were treated for 24 h, with or without bacterial
lipopolysaccharide (25
duction in the cells, and then cells were treated with
samples (100 g/mL) for another 24 h. The cell lysate
was coated onto 96-well flat bottom microtiter plate
µg/mL), to stimulate COX-2 pro-
µ
(Griener Labortechnik) in diluent, 50 µL/well and in-
cubated 1 h at 37oC, which was then placed in the
humidified chamber overnight, at 4oC. Plates were
washed three times with washing buffer and blocked
aData is expressed as mean ± S.E.M.; The data are the result
of six rats/experiment.
with 200
µL/well blocking buffer, which was then
incubated, at 37oC for 1.5 h. The plates were washed
three times with washing buffer and incubated with
the diluted primary antibody. At the end of the incu-
bation period, the plates were washed three times with
washing buffer and diluted second biotin labeled anti-
body was added for 1 h incubation, at 37oC. After wash-
ing away any unbound substances, the peroxidase-
conjugated streptavidin (Jackson Immunsearch Lab)
diluted 1:1000 was added to as 50 µL/well, then the
plates were incubated for 1 h, at 37oC. After an inten-
sive washing, the enzyme reaction was carried out by
Fig. 2.
(A) Non-ulcerative stomach (Negative control); (B)
adding a 50
µL/well of substrate solution. Color devel-
Ulcerative stomach (indomethacin).
opment was stopped by addition of 50
µL/well of stopping
Estimation of COX-2 in the macrophages lysate buffer (1M HCl) (Surechern Products, Needham Marker).
The level of COX-2 was measured in the cells by The intensity of the developed color was measured by
ELISA. The assay uses the quantitative indirect im- reading optical absorbance at 450 nm, using a microplate
munoassay technique that uses polyclonal antibody reader FLUOstar OPTIMA, BMG LABTECH GmbH)
and biotin-linked polyclonal antibody, both of which are (Hansen et al., 1989) (Fig. 4).

Synthesis of Pyridazine as Anti-inflammatory Agent
2089
were prepared by alkylation of 3a
-d
with, either 1-
bromo-2-chloroethane or 1-bromo-3-chloropropane, re-
spectively. Nucleophilic displacement of chlorine in
6a
afforded the desired target compounds 7a
respectively in 42-66% yields. The structure of the
target compounds 7a and 9a was substantiated by
elemental analyses, as well as spectroscopic data.
1H-NMR spectra of compounds 7a
showed the
appearance of two broad signals of piperazine ring, in
the range of 2.63-3.18 and 3.05-3.46 ppm. Moreover,
these compounds displayed a singlet signal in the
range of 3.76-3.78 ppm assignable to 3H of OCH3.
13C-NMR spectrum of compound 7a showed the appear-
-c
and 8a-d
with 4-(4-methoxyphenyl) piperazine
-c
and 9a-d,
-c
-d
-c
δ
δ
ance of 4C of piperazine ring at
δ 49.60 ppm and 51.10
1
ppm. All the other signals in H-NMR and 13C-NMR
Fig. 4. The level of COX-2 protein in RAW 264.7 cells lysate
were observed in the expected regions. The mass spectra
of 7a-c showed two fragments at m/z = 219 and m/z =
after the treatment with the samples (100 µg/mL) for 48 h
compared LPS treated cells, as measured by ELISA assay.
The data are presented as absorbance (mean ± S.E).
205, corresponding to the characteristic fragmentation
pattern of these compounds. 1H-NMR spectrum of com-
pounds 9a
istic broad signals of piperazine ring, in the range of
2.92-3.16 and 3.25-3.50 ppm. Moreover, the spectra
-d showed the appearance of two character-
δ
RESULTS AND DISCUSSION
The synthetic pathways leading to the target com- showed the appearance of a singlet signal in the range
pounds 4a and 5a are presented in Scheme 1. The of 3.67-3.78 ppm assignable to 3H of OCH3 group.
intermediate 6-(4-substitutedphenyl)-4-oxo-hex-5-enoic The mass spectra of compounds 9a displayed two
acids (1a ) (Abouzid et al., 2007), 6-[2-(4-substituted- fragments at m/z = 233 and m/z = 219, which explained
phenyl)ethenyl]-4,5-dihydropyridazin-3(2H)-ones (2a the fragmentation pattern of these compounds.
) (Abouzid and Bekhit, 2008) and 6-[2-(4-substitut- Scheme 3 involves the chlorination of 3a with phos-
edphenyl)ethenyl] pyridazin-3(2H)-ones (3a 3b 3d phorous oxychloride to give the corresponding chloro
(Abouzid et al., 2008), were prepared following the pre- derivatives 10a . The physical and spectral data of
viously reported procedures. The Mannich bases 4a 10a were in accordance with the literature (Abouzid
were synthesized by condensation of 3a with for- et al., 2008). Compound 10c was obtained in 67% yield
-i
-d
δ
-d
-d
-
d
-d
,
,
)
-d
-i
,b,d
-d
maline and a secondary amino function of amino com- and was fully characterized by microanalysis and spec-
pounds, which follows the standard reaction conditions. tral data. The IR spectra revealed the disappearance
On the other hand, the preparation of the target com- of a band corresponding to NH. Cyclization of 10a
pounds 5a was achieved by alkylation of 3a-d with was carried out with either, isonicotinic acid hydrazide
N-bromomethylphthalimide. or anthranilic acid in ethanol/dioxan mixture (1:1) to
The final targets 4a and 5a were obtained in 45- give the target compounds 11a and 12a , respect-
85% and 50-68% yields respectively, and were struc- ively in 43-71% yieds. IR spectra of 12a showed the
-d
-d
-i
-d
-d
-d
-d
turally elucidated on the basis of microanalyses and appearance of a strong band in the range 1631-1670
spectral data. The IR spectra revealed the disappearance cm−1, which indicates the presence of C=O stretching
of a band corresponding to NH. 1H-NMR of 4a
d
-
i
and 5a
proved the presence of a singlet peak, which corres-
ponds to methylene group flanked by the two nitrogen 13a
atoms in the range of 4.72-4.93 and 5.64-5.65 ppm the appearance of a broad band in the range of 3240-
respectively. In addition, 1H-NMR spectrum of com- 3444, indicating the presence of NH and COOH. More-
pounds 4a
showed the appearance of two char- over, the 1H-NMR spectra displayed D2O exchangeable
acteristic broad multiplets of piperazine ring, in the signals in the range 10.77-11.90 ppm assignable to
range 2.80-2.91 ppm and 3.13-3.21 ppm. All the other NH protons.
-
band of amide. On the other hand, reaction of 10a
-aminobenzoic acid afforded the target compounds
in 32-68% yieds. The IR spectra of 13a showed
-d with
p
-d
-d
δ
-e, g, h
δ
δ
aromatic and aliphatic protons were observed in the
expected regions.
In the Scheme 2, the intermediates 6a-c and 8a-d
Pharmacology
The synthesized new final compounds were subjected

2090
M. M. Saeed et al.
to the evaluation of anti-inflammatory activity and displayed any significant ulcerogenicity (Table II).
acute ulcerogenicity studies. Indomethacin was used
as reference standard.
In vitro assays
The compounds that showed the most potent anti-
inflammatory activity 4a and 9d were selected to
Anti-inflammatory activity
Anti-inflammatory activity was determined using further investigation for COX-2 selectivity.
carrageenan-induced rat paw edema model (Winter et
al., 1962). Carrageenan (1% w/v) was used to produce
paw edema. Edema was presented as percentage in-
Cell viability assay
The effect of test compounds 4a and 9d, on the cell
crease in left hind paw, in comparison to the uninjected viability of RAW 264.7 cells, was measured using the
right hind paw. Percentage change in paw volume was MTT cell viability assay.
calculated and expressed as the amount of inflamma-
3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
tion. For anti-inflammatory activity, the test compounds bromide (MTT) assay is based on the ability of active
were orally administered, at molar equivalent doses of mitochondrial dehydrogenase enzyme of living cells to
indomethacin (10 mg/kg, p.o.). Sub-planter injection of cleave the tetrazolium rings of the yellow MTT, which
0.1 mL of 1% carrageenan in rat paw increased the paw forms a dark blue insoluble formazan crystals, which
volume (edema) in all the animals of various groups. is largely impermeable to cell membranes, resulting in
The onset of action was evident during 1 h in various its accumulation within healthy cell. Solubilization of
test groups. The significant (
p < 0.01) reduction of rat the cells results in the liberation of crystals, which are
paw edema was observed for most of the test compounds, then solubilized. The number of viable cells is directly
at 4 h, when compared to that of the control group and proportional to the level of soluble formazan dark blue
indomethacin (Table I).
color. The extent of the reduction of MTT was quanti-
Among all of the tested compounds, 4a and 9d exhibit- fied by measuring the absorbance at 570 nm (Hansen
ed the most prominent and consistent anti-inflamma- et al., 1989) (Fig. 3).
tory activity, which was higher than indomethacin with
rapid onset of action and sustained duration until the
fourth hour after the administration of the drug. Also,
Estimation of COX-2 in the macrophages lysate
The level of COX-2 was measured in the cells by
compounds 4b and 4i gave a high rapid and sustained ELISA. The assay uses the quantitative indirect immu-
anti-inflammatory effect for 4 h comparable to indo- noassay technique that uses polyclonal antibody and
methacin. On the other hand, compounds 4c
,
7b
,
9c
,
biotin-linked polyclonal antibody, both of which are
12c 12d and 13d did not show any significant change specific against murine COX-2. Commercially available
,
in paw volume, when compared to that of the untreat- matched paired antibodies were used (R&D Systems
ed group. It is also obvious that the triazolopyridazine Inc.).
derivatives 11a
more potent anti-inflammatory activity than the pyri- duction up to 2.34 fold of the control and that the com-
dazinoquinazoline derivatives 12a pounds 4a and 9d (100 g/mL) possessed a high in-
Moreover, it has been shown that compounds 4e hibitory activity of COX-2 to the extent that is nearly
4g 4h 5c 12b and 13a displayed a rapid short anti- to the control level. Treatment with different concen-
inflammatory effect at the first hour, only after drug trations of the test compounds revealed that IC50 of 4a
administration, whereas the compounds 9a 13b and and 9d is 48.4 g/mL and 56.4 g/mL, respectively
13c revealed a mild anti-inflammatory effect of delayed (Fig. 4).
-d
seems to exhibit rapid onset and
The results indicated that LPS induced COX-2 pro-
-
d
.
µ
,
,
,
,
,
µ
µ
onset at the fourth hour, only compared to indomethacin.
In summary, most of the synthesized compounds ex-
hibited anti-inflammatory activity and superior gas-
trointestinal safety profile in the animal study. The
Gastric ulcerative effect
Compounds with significant anti-inflammatory profile results showed that among the Mannich bases, 4a-i,
were tested for GI-ulcerogenicity potential. The ulcer- the compounds 4a and 4b containing 4-chlorophenyl-
ative effect of test compounds has been inspected visual- piperazine in their structure, displayed potent anti-
ly, relative to the known ulcerogenic drug, indomethacin, inflammatory effect, with a safe gastric profile. Also,
(Fig. 2). After gross visual inspection, it has been obvious in a series of compounds 9a
that compounds 4a 4f 9a 13a and 13c showed no ylpyridazinone pharmacophore is linked to the 4-meth-
ulcer formation, whereas compounds 4h 4i 5b 5d oxyphenylpiperazine via three carbon spacer, the com-
11b 11d 12b 13b and indomethacin showed signi- pound 9d, in which there is a chlorine atom in the aryl-
ficant ulcerogenic effect. Other test compounds haven’t ethenyl moiety that showed a superior action higher
-d, in which the 6-arylethen-
,
,
,
,
,
,
,
,
,
,

Synthesis of Pyridazine as Anti-inflammatory Agent
2091
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than indomethacin with very low ulcerogenicity. Also,
among the benzoic acid derivatives 13a-d, the unsub-
stituted and 4-bromo substituted derivatives, 13a and
13c have shown high anti-inflammatory activity, without
ulcer formation. The results of in vitro studies reveal-
ed that compounds 4a and 9d exhibited a high COX-2
inhibition effect.
Therefore, it can be concluded that the rational, on
which these compounds were designed, has been proven
to show promising results, which follow the SAR studies
previously discussed that revealed the importance of
N-substitution on pyridazine ring as a requirement for
COX-2 selectivity.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Wafaa El-Eraky, As-
sociate Professor of Pharmacology and Toxicology De-
partment, National Research Center, Cairo. Egypt for
carrying out the biological testing.
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