Synthesis of antipyrine/pyridazinone hybrids and investigation of their in vivo analgesic and anti-inflammatory activities

Article abstract of DOI:10.3906/kim-1111-29

Eleven antipyrine/pyridazinone hybrids were synthesized and evaluated for their in vivo analgesic and anti-inflammatory activities by p-benzoquinone- induced writhing test and carrageenan-induced paw edema model, respectively. The test results indicated t

Full text of DOI:10.3906/kim-1111-29

Turk J Chem
36 (2012) , 734 – 748.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1111-29
Synthesis of antipyrine/pyridazinone hybrids and
investigation of their in vivo analgesic and
anti-inflammatory activities
1
2
Sultan BAYTAS¸1,∗, Nazan INCELER , Khatereh Fattahpour MAVANEH ,
˙
2
2
1
˘
˘
¨
Mecit Orhan ULUDAG , Nurettin ABACIOGLU , Mehtap GOKC¸ E
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University,
06330, Ankara-TURKEY
e-mail: baytas@gazi.edu.tr
2Department of Pharmacology, Faculty of Pharmacy, Gazi University,
06330, Ankara-TURKEY
Received: 16.11.2011
Eleven antipyrine/pyridazinone hybrids were synthesized and evaluated for their in vivo analgesic and
anti-inflammatory activities by p-benzoquinone-induced writhing test and carrageenan-induced paw edema
model, respectively. The test results indicated that compounds 6a, 6c, and 6d were equally or more potent
analgesic and anti-inflammatory agents than aspirin and indomethacin, respectively. Side effects of the
compounds were examined on gastric mucosa. Most of the compounds were found to be non-ulcerogenic
under test conditions.
Key Words: Hybrid, antipyrine, pyridazinone, writhing, carrageenan
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are useful for the treatment of inflammation, pain, and fever.
The clinical effects of NSAIDs are based on the inhibition of the enzyme cyclooxygenase (COX), which catalyzes
the rate-limiting step in the metabolism arachidonic acid to prostaglandin H2 (PGH2). PGH2 is further
metabolized to prostanoids, prostaglandins (PGs), and thromboxane A2 (TxA2). Various physiological effects
of PGs include inflammatory reactions, blood pressure change, platelet aggregation, induction of labor, and
∗Corresponding author
734

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
intensification of pain and fever. Utility of NSAIDs in the treatment of inflammation and pain is often
limited by gastrointestinal side effects including ulceration and bleeding.1−5 Developing safer analgesic and
anti-inflammatory compounds with no such side effects has recently been the goal of many researchers.
Antipyrine was the first pyrazolin-5-one derivative used as an analgesic and antipyretic and anti-inflamma-
tory drug. Bioactive antipyrine derivatives have been synthesized and evaluated as potent anti-inflammatory,
analgesic, antipyretic, and antimicrobial agents.6−9 Furthermore, the synthesis of pyridazinone derivatives
possessing such diverse pharmacological properties as anti-inflammatory, analgesic, antimicrobial, and antiviral
activities has been attracting widespread attention.
In designing new bioactive agents, besides the development of completely new agents, there is another
approach involving the synthesis of hybrid molecules. Combination of different pharmacophores each with a
different mode of action in the same structure may lead to compounds having more efficiency in biological
activity.10 Diverse pharmacological properties of the compounds containing antipyrine and pyridazinone moi-
eties have prompted us to design and synthesize hybrid molecules incorporating those scaffolds in a single
molecule.
To identify new candidates that may be of value in designing new, potent, selective and less toxic analgesic
and anti-inflammatory agents, we report herein the synthesis of new antipyrine derivatives incorporating
pyridazinone pharmacophore as hybrid molecules possessing analgesic and anti-inflammatory activities. All
compounds were also tested for the irritative and ulcerogenic action on the gastric mucosa. Finally, these
derivatives were submitted to in silico oral biodisponibility screening to analyze their overall potential to qualify
as a drug.
Experimental
The chemicals were purchased from commercial vendors and were used without purification. Thin-layer chro-
matography (TLC) was performed on Merck 60F254 plates. The reactions were monitored by TLC on silica
gel, with detection by UV light (254 nm) or charring Dragendorff reagent.11 Melting points were determined
with an SMP-II Digital Melting Point Apparatus and are uncorrected (Schorpp Geaetetechnik, Germany). IR
spectra were obtained in-house using a Perkin Elmer Spectrum 400 FTIR/FTNIR spectrometer equipped with
a Universal ATR Sampling Accessory and only carbonyl stretching frequencies were given. 1 H-NMR spectra
were recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400 MHz High Performance Digital FT-NMR
spectrometer using tetramethylsilane as the internal standard at the NMR facility of the Faculty of Pharmacy,
Ankara University. All chemical shifts were recorded as δ (ppm). High resolution mass spectra data (HRMS)
were collected in-house using a Waters LCT Premier XE Mass Spectrometer (high sensitivity orthogonal accel-
eration time-of-flight instrument) operating in either ESI (+) or ESI (–) methods, also coupled to an AQUITY
Ultra Performance Liquid Chromatography system (Waters Corporation, Milford, MA, USA). Flash chromatog-
r
raphy was performed with a Combiflashꢀ Rf automated flash chromatography system with RediSep columns
(Teledyne-Isco, Lincoln, NE, USA) using hexane-ethyl acetate or dichloromethane-methanol solvent gradients.
735

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
General procedures for the synthesis of compounds
Synthesis of 6-substituted-3(2H)-pyridazinone derivatives 2a-f
A solution of 0.05 mol of a 6-substituted-3-chloropyridazine 1 derivative in 30 mL of glacial acetic acid was
refluxed for 6 h. The acetic acid was removed under reduced pressure, and the residue was dissolved in water
and extracted with CHCl3 . The organic phase was dried over Na2 SO4 and evaporated under reduced pressure.
The new residue was purified by recrystallization from ethanol.
Synthesis of 6-substitutedphenyl-3(2H)-pyridazinone derivatives 3a,b
In an oil bath 0.05 mol (4.6 g) glyoxalic acid and 0.15 mol appropriate acetophenone derivative were heated at
100-105 ◦ C for 2 h. After the reaction mixture cooled down to 40 ◦ C, 20 mL of water and 5 mL of ammonium
hydroxide solution (25%) were added until the medium pH became 8. Then the reaction mixture was extracted
with CHCl2 (4 × 25 mL). To the aqueous layer was added 0.05 mol hydrazine hydrate (2.5 mL) and the
reaction mixture was refluxed for 2 h. After completion of the reaction, the reaction mixture cooled down to
room temperature. The resulting white precipitate was filtered and recrystallized from ethanol.
Synthesis of ethyl 6-substituted-3(2H)-pyridazinone-2-yl-acetate/propionate derivatives 4a-k
A mixture of required 6-substituted-3(2H )pyridazinones 2a-f or 3a,b (0.01 mol), ethyl bromoacetate/ethyl
bromopropionate (0.02 mol), and potassium carbonate (0.02 mol) in acetone (40 mL) was refluxed for 12 h.
After the mixture cooled, the organic salts were filtered off, the solvent evaporated, and the residue was purified
by recrystallization with appropriate alcohol to give the esters.
Synthesis of 6-substituted-3(2H)-pyridazinone-2-yl-acetic/propionic acid derivatives 5a-k
To 50 mL of HCl (37%) was added 0.01 mol ethyl 6-substituted-3(2H )-pyridazinone-2-yl-acetate/propionate
derivatives 4a-k and the reaction mixture was refluxed for 1 h. After completion of hydrolysis, the reaction
mixture was transferred into 300 mL of ice-cold water. The formed colorless crystals were collected by filtration.
Synthesis of 6-(4-substituted phenyl)-3(2H)-pyridazinone-2-ylacetamide and propionamide deriva-
tive 6a-k
To a solution of appropriate carboxylic acid derivative (1 mmol) and aminoantipyrine (1 mmol) in 10 mL of
dichloromethane (DCM) were added dimethylaminopyridine (DMAP) (0.2 mmol) and N-(3-dimethylaminopropyl)-
N’-ethylcarbodiimide hydrochloride (EDCI) (1.1 mmol) and the resulting solution was stirred overnight at room
temperature. The reaction mixture was then quenched with 1 N HCl and extracted with DCM. The organic
phase was washed with a 1% NaHCO3 solution and brine, dried over Na2 SO4 , and evaporated under vacuum.
r
The residue was purified by flash column chromatography (Combiflashꢀ Rf) using DCM-MeOH as eluents.
736

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-[6-oxo-3-(4-phenylpiperazin-1-yl)
pyridazin-1(6H)-yl]acetamide 6a
Elution with DCM-MeOH (0%-4%) yielded 6a as a white solid (yield 62%); mp 198-200 ◦ C; IR (FTIR/FTNIR-
ATR): 1709 cm−1 (C = O), 1668 cm−1 (C = O), 1645 cm−1 (C = O). 1 H-NMR (CDCl3)δ 7.97 (1H, s, NH),
7.41-7.45 (2H, m, J = 7.6 Hz, J = 8 Hz, phenyl H-2, 6), 7.35-7.38 (2H, d, J = 7.2 Hz, phenyl H-3, 5), 7.26-7.30
(3H, m, phenyl H-4, phenylpiperazin H-3, 5), 7.16-7.19 (1H, d, J = 10 Hz, pyridazine H-4), 6.91-6.93 (1H, d, J
= 10 Hz, pyridazine H-5), 6.89-6.96 (3H, m, phenylpiperazin H-2, 4, 6), 4.83 (2H, s, pyridazine-CH2), 3.43-3.48
(4H, m, J = 4.8 Hz, J = 5.2 Hz, piperazine H-2, 6), 3.24-3.27 (4H, m, J = 4.8 Hz, J = 5.2 Hz, piperazine H-3,
5), 3.05 (3H, s, pyrazole-1-CH3), 2.67 (3H, s, pyrazole-5-CH3). C27 H29 N7 O3 ESI-MS 500.2418 [M+H]+ .
2-[3-[4-(4-Chlorophenyl)piperazin-1-yl]-6-oxopyridazin-1(6H)-yl]-N -(1,5-dimethyl-3-oxo-2-phenyl-
2,3-dihydro-1H-pyrazol-4-yl)acetamide 6b
Elution with DCM-MeOH (0%-4%) yielded 6b as a white solid (yield 44%); mp 204-205 ◦ C; IR (FTIR/FTNIR-
ATR): 1703 cm−1 (C = O), 1646 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 7.99 (1H, s, NH), 7.27-7.45 (5H, m,
phenyl), 7.21 (1H, d, J = 5.2 Hz, pyridazine H-4), 7.16 (2H, d, J = 10.4 Hz, chlorophenyl H-2, 6), 6.92 (2H,
d, J = 10.4 Hz, chlorophenyl H-3, 5), 6.86 (1H, d, J = 4.8 Hz, pyridazine H-5), 4.83 (2H, s, pyridazine-CH2),
3.43 (4H, t, J = 5.2 Hz, piperazine H-2, 6), 3.21 (4H, m, J = 4.4 Hz, J = 5.6 Hz, piperazine H-3,5), 3.05
(3H, s, pyrazole-1-CH3), 2.26 (3H, s, pyrazole-5-CH3). C27 H28 ClN7 O3 ESI-MS 534.2010 [M+1]+ (100%),
536.4231 [M+1+2]+ (35%).
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-[3-[4-(4-fluorophenyl)piperazin-1-
yl]-6-oxopyridazin-1(6H)-yl]acetamide 6c
Elution with DCM-MeOH (0%-4%) yielded 6c as a white solid (yield 63%); mp 174-176 ◦ C; IR (FTIR/FTNIR-
ATR): 1711 cm−1 (C = O), 1665 cm−1 (C = O), 1642 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 7.93 (1H, s, NH),
7.42-7.45 (2H, m, J = 7.6 Hz, J = 8 Hz, phenyl H-2, 6), 7.35-7.37 (2H, d, J = 7.2 Hz, phenyl H-3, 5), 7.26-7.29
(1H, m, J = 7.6 Hz, phenyl H-4), 7.16-7.19 (1H, d, J = 10.4 Hz, pyridazine H-4), 6.93-6.95 (1H, d, J = 10.4
Hz, pyridazine H-5), 6.88-6.99 (4H, m, fluorophenyl H-2, 3, 5, 6), 4.83 (2H, s, pyridazine-CH2), 3.43-3.45 (4H,
m, J = 4.8 Hz, J = 5.6 Hz, piperazine H-2, 6), 3.16-3.18 (4H, m, J = 4.4 Hz, J = 5.2 Hz, piperazine H-3, 5),
3.05 (3H, s, pyrazole-1-CH3), 2.26 (3H, s, pyrazole-5-CH3). C27 H28 FN7 O3 ESI-MS 540.2147 [M+Na]+ .
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-[3-[4-(2,3-dimethylphenyl)pipe-
razin-1-yl]-6-oxopyridazin-1(6H)-yl]acetamide 6d
Elution with DCM-MeOH (0%-4%) yielded 6d as a white solid (yield 50%); mp 225-227 ◦ C; IR (FTIR/FTNIR-
ATR): 1705 cm−1 (C = O), 1660 cm−1 (C = O), 1643 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 7.82 (1H, s, NH),
7.42-7.45 (2H, m, J = 7.6 Hz, J = 8 Hz, phenyl H-2, 6), 7.36-7.38 (2H, d, J = 7.6 Hz, phenyl H-3, 5), 7.26-7.29
(1H, m, J = 7.2 Hz, phenyl H-4), 7.19-7.21 (1H, d, J = 10 Hz, pyridazine H-4), 7.06-7.10 (2H, m, J = 7.2 Hz,
J = 8 Hz, dimethyl phenyl H-5, 6), 6.90-6.93 (2H, m, J = 10.4 Hz, pyridazine H-5, dimethyl phenyl H-4), 4.83
(2H, s, pyridazine-CH2), 3.43-3.45 (4H, m, piperazine H-2, 6), 3.06 (3H, s, pyrazole-1-CH3), 2.95-2.97 (4H, m,
737

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
J = 4 Hz, J = 4.8 Hz, piperazine H-3, 5), 2.27 (3H, s, phenyl-3-CH3), 2.27 (3H, s, pyrazole-5-CH3), 2.23 (3H,
s, phenyl-2-CH3). C29 H33 N7 O3 ESI-MS 528.2740 [M+H]+ .
2-[3-(4-Benzylpiperidin-1-yl)-6-oxopyridazin-1(6H)-yl]-N -(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihyd-
ro-1H -pyrazol-4-yl)acetamide 6e
Elution with DCM-MeOH (0%-4%) yielded 6e as a white solid (yield 62%); mp 198-200 ◦ C; IR (FTIR/FTNIR-
ATR): 1664 cm−1 (C = O), 1642 cm−1 (C = O). 1 H-NMR (CDCl3) δ: 7.73 (1H, s, NH), 7.20-7.45 (10H,
m, phenyl, benzyl), 7.15 (1H, d, J = 9.2 Hz, pyridazine H-4), 6.86 (1H, d, J = 10 Hz, pyridazine H-5), 4.80
(2H, s, pyridazine-CH2), 3.82 (2H, d, J = 13.2 Hz, phenyl-CH2), 3.05 (3H, s, pyrazole-1-CH3), 2.26 (3H, s,
pyrazole-5-CH3), 2.55-2.72 (4H, m, piperidine H-2, 6), 1.66-1.71 (5H, m, piperidine H-3, 4, 5). C29 H32 N6 O3
ESI-MS 535.2410 [M+Na]+ .
3-[3-[4-(4-Chlorophenyl)piperazin-1-yl]-6-oxopyridazin-1(6H)-yl]-N -(1,5-dimethyl-3-oxo-2-phenyl-
2,3-dihydro-1H-pyrazol-4-yl)propanamide 6f
Elution with DCM-MeOH (0%-4%) obtained 6f as a white solid (yield 51%); mp 185-187 ◦ C; IR (FTIR/FTNIR-
ATR): 1681 cm−1 (C = O), 1655 cm−1 (C = O), 1623 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 7.88 (1H, s, NH),
7.39-7.43 (2H, m, J = 7.2 Hz, J = 8.4 Hz, phenyl H-2, 6), 7.34-7.36 (2H, d, J = 7.2 Hz, phenyl H-3, 5),
7.25-7.29 (1H, m, J = 7.2 Hz, phenyl H-4), 7.20-7.22 (2H, d, J = 4.8 Hz, chlorophenyl H-3, 5), 7.12-7.15 (1H,
d, J = 9.6 Hz, pyridazine H-4), 6.89-6.92 (1H, d, J = 9.6 Hz, pyridazine H-5), 6.82-6.84 (2H, d, J = 4.8
Hz, chlorophenyl H-2, 6), 4.39-4.42 (2H, t, J = 6.8 Hz, pyridazine-CH2 -CH2), 3.36-3.39 (4H, t, J = 5.2 Hz,
piperazine H-2, 6), 3.18-3.21 (4H, t, J = 5.2 Hz, piperazine H-3, 5), 3.05 (3H, s, pyrazole-1-CH3), 2.83-2.87
(2H, t, J = 6.8 Hz, pyridazine-CH2 -CH2), 2.22 (3H, s, pyrazole-5-CH3). C28 H30 ClN7 O3 ESI-MS 548.2176
[M+H]+ (100%), 550.3263 [M+1+2]+ (35%).
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-[3-[4-(4-fluorophenyl)piperazin-1-
yl]-6-oxopyridazin-1(6H)-yl]propanamide 6g
Elution with DCM-MeOH (0%-4%) obtained 6g as a white solid (yield 42%); mp 206-208 ◦ C; IR (FTIR/FTNIR-
ATR): 1662 cm−1 (C = O), 1628 cm−1 (C = O). 1 H-NMR (CDCl3) δ: 7.83 (1H, s, NH), 7.35-7.43 (4H, m,
phenyl H-2, 3, 5, 6), 7.28 (1H, m, phenyl H-4), 7.13-7.15 (1H, d, J = 9.6 Hz, pyridazine H-4), 6.95-6.97 (1H,
d, J = 8.8 Hz, pyridazine H-5), 6.86-6.91 (4H, m, fluorophenyl H-2, 3, 5, 6), 4.39-4.43 (2H, t, J = 6.4 Hz,
pyridazine-CH2 -CH2), 3.38-3.40 (4H, t, J = 5.2 Hz, piperazine H-2, 6), 3.14-3.16 (4H, t, J = 5.2 Hz, piperazine
H-3, 5), 3.05 (3H, s, pyrazole-1-CH3), 2.84-2.88 (2H, dd, J = 6.8 Hz, J = 7.2 Hz, pyridazine-CH2 -CH2), 2.23
(3H, s, pyrazole-5-CH3). C28 H30 FN7 O3 ESI-MS 532.2464 [M+H]+
.
3-[3-[4-(3-Chlorophenyl)piperazin-1-yl]-6-oxopyridazin-1(6H)-yl]-N -(1,5-dimethyl-3-oxo-2-phenyl-
2,3-dihydro-1H-pyrazol-4-yl)propanamide 6h
Elution with DCM-MeOH (0%-4%) yielded 6h as a white solid (yield 54%); mp 145-147 ◦ C; IR (FTIR/FTNIR-
ATR): 1650 cm−1 (C = O), 1620 cm−1 (C = O). 1 H-NMR (CDCl3) δ: 7.93 (1H, s, NH), 7.38-7.42 (2H, m,
J = 7.2 Hz, J = 7.6 Hz, phenyl H-2, 6), 7.34-7.36 (2H, d, J = 7.2 Hz, phenyl H-3, 5), 7.28 (1H, m, phenyl
738

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
H-4), 7.15-7.19 (1H, t, J = 8 Hz, chlorophenyl H-5), 7.12-7.15 (1H, d, J = 10.4 Hz, pyridazine H-4), 6.89-6.92
(1H, d, J = 9.6 Hz, pyridazine H-5), 6.77-6.88 (3H, m, chlorophenyl H-2, 4, 6), 4.38-4.41 (2H, t, J = 6.8 Hz,
pyridazine-CH2 -CH2), 3.35-3.38 (4H, t, J = 5.6 Hz, piperazine H-2, 6), 3.22-3.25 (4H, t, J = 5.6 Hz, piperazine
H-3, 5), 3.05 (3H, s, pyrazole-1-CH3), 2.82-2.85 (2H, dd, J = 6.8 Hz, J = 7.2 Hz, pyridazine-CH2 -CH2), 2.22
(3H, s, pyrazole-5-CH3). C28 H30 ClN7 O3 ESI-MS 548.2174 [M+H]+ (100%), 550.6259 [M+1+2]+ (35%).
3-[3-(4-Benzylpiperidin-1-yl)-6-oxopyridazin-1(6H)-yl]-N -(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihyd-
ro-1H -pyrazol-4-yl)propanamide 6i
Elution with DCM-MeOH (0%-4%) yielded 6i as a white solid (yield 51%); mp 173-175 ◦ C; IR (FTIR/FTNIR-
ATR): 1688 cm−1 (C = O), 1658 cm−1 (C = O), 1623 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 7.71 (1H, s, NH),
7.42-7.45 (2H, t, J = 7.6 Hz, phenyl H-2, 6), 7.37 (2H, d, J = 7.6 Hz, phenyl H-3, 5), 7.26-7.30 (3H, m, benzyl
H-3, 4, 5), 7.20 (1H, t, J = 7.6 Hz, phenyl H-4), 7.13-7.15 (2H, d, J = 7.6 Hz, benzyl H-2, 6), 7.08-7.11 (1H, d,
J = 9.6 Hz, pyridazine H-4), 6.83-6.85 (1H, d, J = 10 Hz, pyridazine H-5), 4.38-4.41 (2H, dd, J = 6.8 Hz, J
= 7.2 Hz, pyridazine-CH2 -CH2), 3.76-3.79 (2H, d, J = 13.2 Hz, phenyl-CH2), 3.04 (3H, s, pyrazole-1-CH3),
2.85-2.88 (2H, dd, J = 6.8 Hz, J = 7.2 Hz, pyridazine-CH2 -CH2), 2.54-2.69 (4H, m, piperidine H-2, 6), 2.23
(3H, s, pyrazole-5-CH3), 1.68-1.77 (5H, m, piperidine H-3, 4, 5). C30 H34 N6 O3 ESI-MS 527.2777 [M+H]+
.
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-(6-oxo-3-phenylpyridazin-1(6H)-
yl)propanamide 6j
Elution with DCM-MeOH (0%-4%) yielded 6j as a white solid (yield 47%); mp 188-190 ◦ C; IR (FTIR/FTNIR-
ATR): 1671 cm−1 (C = O), 1644 cm−1 (C = O). 1 H-NMR (CDCl3) δ: 8.10 (1H, s, NH), 7.77-7.79 (2H, d, J
= 8 Hz, phenylpyridazine H-2, 6), 7.67-7.69 (1H, d, J = 9.2 Hz, pyridazine H-5), 7.32-7.46 (7H, m, phenyl H-2,
3, 5, 6, phenylpyridazine H-3, 4, 5), 7.23-7.26 (1H, m, J = 7.2 Hz, phenyl H-4), 7.02-7.04 (1H, d, J = 9.6 Hz,
pyridazine H-4), 4.56-4.60 (2H, t, J = 6.4 Hz, pyridazine-CH2 -CH2), 3.01 (3H, s, pyrazole-1-CH3), 2.90-2.93
(2H, t, J = 6.4 Hz, pyridazine-CH2 -CH2), 2.10 (3H, s, pyrazole-5-CH3). C24 H23 N5 O3 ESI-MS 430.1868
[M+H]+
.
N -(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-[3-(4-methylphenyl)-6-oxopyri-
dazin-1(6H )-yl]propanamide 6k
Elution with DCM-MeOH (0%-4%) yielded 6k as a white solid (yield 60%); mp 233-235 ◦ C; IR (FTIR/FTNIR-
ATR): 1680 cm−1 (C = O), 1663 cm−1 (C = O), 1641 cm−1 (C = O). 1 H-NMR (CDCl3)δ: 8.01 (1H, s, NH),
7.65-7.68 (3H, m, methylphenyl H-2, 6, pyridazine H-5), 7.39-7.42 (2H, m, phenyl H-2, 6), 7.33-7.35 (1H, m,
phenyl H-3, 5), 7.22-7.26 (3H, m, methylphenyl H-3, 5, phenyl H-4), 7.00-7.02 (1H, d, J = 10 Hz, pyridazine H-
4), 4.56-4.59 (2H, dd, J = 6.8 Hz, J = 7.2 Hz, pyridazine-CH2 -CH2), 3.02 (3H, s, pyrazole-1-CH3), 2.90-2.93
(2H, dd, J = 6.8 Hz, J = 7.2 Hz, pyridazine-CH2 -CH2), 2.38 (3H, s, phenyl-CH3), 2.13 (3H, s, pyrazole-5-
CH3). C25 H25 N5 O3 ESI-MS 444.2026 [M+H]+
.
739

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
Pharmacological screening
Animals
Male Swiss albino mice (25-30 g) were purchased from the animal breeding laboratories of Refik Saydam Central
Institute of Health (Ankara, Turkey). The animals were kept and housed in a controlled temperature (22
1
◦ C), humidity (55
10%), and photoperiod (12 h light and 12 h dark, lights on at 0800) room for at
least a week before they were used and throughout the experiments. The animals were allowed free access to
standard pellet diet (Korkuteli Yem Sanayii T.A.S., Ankara) and tap water. A minimum of 6 animals were
caged separately in each group. Throughout the experiments, the animals were treated under the audit of Gazi
University’s Commission of Animal Ethics according to the suggested international ethical guidelines for the
care of laboratory animals (Permission No: 10.094).
Drugs
All chemicals used in this study, including p-Benzoquinone (PBQ) and λ-carrageenan (type I) were purchased
from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).
Analgesic activity
Analgesic activity was assessed using the PBQ-induced writhing test (visceral pain model) in mice according
to Okun et al.12 Prior to the chemical induction of algesia, all test samples were subcutaneously injected (100
mg/kg body weight) with a 30 min latency time and concomitantly followed by the intraperitoneal injection
of 0.1 mL/10 g body weight of PBQ solution in saline (at a dose of 2.5 mg/kg) into mice. In the control
groups the animals were given an appropriate volume of dosing vehicle. After PBQ challenge, the mice were
housed individually in transparent glass cages for observation. The observers were blinded as to the agents the
mice received while assessing the time of onset (approximately 3 min after PBQ injection) and the total count
of writhing during the 15 min post-treatment period. The data represent the average of the total number of
writhing movements observed. Antinociceptive activity was then expressed as the percentage change from the
writhing controls.
Anti-inflammatory activity
Anti-inflammatory activity was assessed by the carrageenan-induced hind paw edema model.13 The control
and treated groups included a minimum of 7 animals, and 30 min after the subcutaneous administration of a
test sample (100 mg/kg body weight) or dosing vehicle each mouse was injected subplantarly in the right hind
paw with a freshly prepared suspension of carrageenan (0.5 mg/25 μL) in saline. Control injections (25 μL of
saline) were administered to paired contralateral left hind paws. Monitoring of carrageenan induced paw edema
was maintained in a timebase scale of 90, 180, 270, and 360 min. The difference in hind paw thicknesses was
measured by caliber compasses (Ozaki Co., Tokyo, Japan). Mean values of each treated group were compared
with the control group and analyzed by statistical methods.
740

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
Acute toxicity
Following the end of the anti-inflammatory activity experiments, the mice were observed for 48 h to any possible
recordings of morbidity or mortality.14
Gastric ulcerogenic effect
Following the end of the analgesic activity experiments, as described elsewhere15 the animals were sacrificed
with an overdose of diethyl ether after 270 min of administration of the compounds for the investigation of
ulcerogenicity. Each esophagus was then tied in a knot nearest the cardia by a surgical suture and the stomach
was injected duodenally with a formalin solution (1.5 mL of 10%). The distended stomach was immediately
tied to the pyloric sphincter using another surgical suture to avoid leakage of the formalin solution. After the
removal of the stomachs from the abdominal cavity the outer layers were fixed by the immersion of the same
formalin solution. Each stomach was then dissected along the greater curvature, rinsed with tap water to remove
the gastric contents and examined under a dissecting microscope (20 × 6.3) to assess the formation of ulcers.
The sum of the length (mm) of all lesions for each stomach was used as the ulcer index.
Statistical analysis
The data were expressed as means
SEM. The significance of differences between the treatment and the
control group of animals was determined by one-way ANOVA with Bartlett’s test following a post hoc Student-
Newman-Keuls multiple comparisons test for analgesic activity, and two-way ANOVA following a post hoc
Bonferroni test for anti-inflammatory activity. Values of P < 0.05 were considered statistically significant.
In silico oral biodisponibility
The theoretical study of oral biodisponibility (Lipinski rule-of-five) was performed in the molinspiration on-
line program (http://www.molinspiration.com). Lipinski et al. have described an approach to estimate drug
solubility and permeability in the human body. Lipinski’s rule-of-five is a method routinely used for identifying
leads and drug candidates. The rule-of-five predicts that an active compound shows poor absorption and
permeation if it presents more than 5 hydrogen-bond donors (HBD) and 10 hydrogen-bond acceptors (HDA),
and molecular weight (MW) and the calculated logP (clogP) are greater than 500 and 5, respectively.16
Results and discussion
Chemistry
New 6-(4-substituted piperazin/piperidin-1-yl)-3(2H )-pyridazinone-2-acetamide and propionamide derivatives
6a-i were synthesized according to Scheme 1. Initially, nucleophilic displacement reaction of commercial 3,6-
dichloropyridazine with arylpiperazines in ethanol afforded 3-chloro-6-substituted pyridazines 1. The physical
and spectral properties of 3-chloro-6-substitutedpyridazine 1 were in compliance with the literature.17,18 There-
fore, we carried out the next steps of the reaction without any further analysis. The synthesis procedures for
derivatives 2 and 3 were reported by us previously.19−21 Hydrolysis of 3-chloro-6-substituted pyridazines 1
741

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
CH3
O
C
Cl
HO
C
H
O
O
+
N
a
R
N
R1
1
NH2NH2.H2O
a
O
NH
O
N
NH
R1
b
3a,b
R
N
2a-f
b
O
O
c
N
N
2a R= phenylpiperazine
R2
N
R2
N
(CH2)n
COOC2H5
(CH2)n
COOH
2b R= 4-Cl-phenylpiperazine
2c R= 4-F-phenylpiperazine
2d R= 3-Cl-phenylpiperazine
5a-k
4a-k
2e R= 3,4-dimethylphenylpiperazine
2f R= 4-benzylpiperidine
d
O
N
N
R2
(CH2)n
C
NH
O
CH3
CH3
O
6a-k
N
N
a. CH3COOH, reflux; b.ethyl bromoacetate/ethyl bromopropionate, K2CO3, acetone; c. conc. HCl;
d. 5, EDCI, DMAP, 4-aminoantipyrine, DCM
Scheme 1. Synthetic route for antipyrine/pyridazinone hybrids.
was carried out upon heating in glacial acetic acid to obtain 6-substituted-3(2H )-pyridazinone 2 derivatives.
6-(4-Substituted phenyl)-3(2H )-pyridazinone-2-ylacetamide and propionamide derivatives 6j and 6k were syn-
thesized according to Scheme 1. The starting reagents for the preparation of 6-phenyl-3(2H)-pyridazinone
3a and 6-(4-methylphenyl)-3(2H )-pyridazinone 3b were glyoxalic acid, appropriate acetophenone derivative,
and hydrazine hydrate. Condensation of these reagents provided an efficient route to 3a and 3b. Alkylation
of derivatives 2a-f and 3a,b was performed using ethyl bromoacetate or ethyl bromopropionate in the pres-
ence of potassium carbonate in acetone at reflux temperature and the resulting ester derivatives 4a-k were
prepared in good yields. Hydrolysis of these esters with concentrated HCl 37% resulted in carboxylic acid
742

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
5a-k derivatives. By treatment of these carboxylic acid derivatives with aminoantipyrine in the presence of
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) and dimethylaminopyridine (DMAP),
which was used as carboxylate activator, the resulting amide derivatives 6a-k were prepared in good yield.
Chemical structures of these compounds were elucidated by their elemental analysis, IR, HRMS, and 1 H-NMR
spectral data (Table 1).
Table 1. Synthesized compounds for analgesic and anti-inflammatory activity.
743

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
Pharmacology
Analgesic activities of the resultant compounds were investigated by p-benzoquinone-induced writhing test
in mice,12 which is a well-established method for testing the analgesic activity of compounds and sufficiently
sensitive to detect the effect of analgesics that are less active than aspirin.
As seen in Figures 1 and 2 and Table 2, compounds having phenylpiperazine 6a and 4-(2,3-dimethylphenyl)
piperazine 6d at the 6 position of the pyridazine ring show analgesic activities higher than aspirin, which serves
as the reference drug in the assays, although these results do not show statistical significance. Meanwhile,
compounds 6c, 6e, and 6h, as shown in Table 2, result in activities approximately equipotent to aspirin at the
same subcutaneous dose of 100 mg/kg.
Analgesic activities of derivatives 6a-k seem to be sensitive to electronic effects of the substituent at
the sixth position of the pyridazine ring. While the substituted piperazine or piperidine derivatives 6a-i show
potent analgesic activities (except for 6f), the derivatives possessing phenyl or methylphenyl groups (6j, 6k)
have diminished analgesic activities.
25
20
15
10
5
25
20
15
10
5
*
*
**
***
***
***
***
***
***
0
0
Figure 1. Writhing responses (means
SEM, n = 6-
Figure 2. Writhing responses (means
SEM, n = 6-9) of
9) of antipyrine/pyridazinone hybrids (6a, 6c, 6d, and
6e) and acetylsalicylic acid (ASA) compared to control
*Significantly different from the control (***P < 0.0001).
antipyrine/pyridazinone hybrids (6f, 6g, 6h, 6i, 6j, and
6k) and acetylsalicylic acid (ASA) compared to control
*Significantly different from the control (*P < 0.01, **P
< 0.001, ***P < 0.0001).
Recent studies have shown that preparation of amide derivatives of well-known NSAID drugs with
free carboxylic acid for developing new analgesic and anti-inflammatory drugs with reduced side effects re-
sulted in compounds with good analgesic and anti-inflammatory activity.22,23 In our study, to achieve hybrid
molecules, pyridazinone was linked to the antipyrine through an acetylenic or propionic amide bond. From
this perspective, the acetamides have generally been found more potent than propionamides. In the case of
4-fluorophenylpiperazine and benzylpiperidine derivatives, compounds 6c and 6e in which the pyridazinone ring
744

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
is incorporated to antipyrine via an acetylenic amide bond showed higher analgesic activity than derivatives 6g
and 6i possessing propionic amide bonds. Dogruer et al. reported that, in the case of [6-(4-methoxyphenyl)-
3(2H)-pyridazinone-2-yl]acetamide and propanamide derivatives, the highest analgesic activity was observed
with acetamide 4-fluorophenylpiperazine derivative in the amide portion of the compounds.24
In vivo systemic anti-inflammatory activities of the compounds were assessed by carrageenan-induced
hind paw edema model in mice at 100 mg/kg body weight doses.25 Carrageenan edema has been used in
the development of indomethacin. Carrageenan-induced paw edema is a traditional model to evaluate anti-
inflammatory drugs. This model is a non-specific acute inflammation resulting from a complex of diverse
mediators.26 The edema formed is a multi-mediated case divided into 2 phases. The first phase is mediated
by release of histamine and serotonin for 1 h followed by the kinin-mediated increased vascular permeability
up to 2.5, h whereas the second phase (3 and 4 h after carrageenan injection) is mainly mediated by release of
prostaglandins and prostaglandin-associated leukocytes into the site of edema.27−29 Subcutaneous injection of
carrageenan into the rat paw causes inflammation resulting from plasma extravasations, increased tissue water,
and plasma protein exudation along with neutrophil extravasations, all due to the metabolism of arachidonic
acid.30,31
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(B)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
DMSO
(A)
DMSO
INDO
6f
INDO
6a
6g
6b
6h
6c
6i
6j
6d
6e
6k
Time (min) after carrageenan
Time (min) after carrageenan
Figure 3. Timebase scale of carrageenan-induced paw edema in 90, 180, 270, and 360 min. Panel A: Control,
indomethacin (INDO), 6a, 6b, 6c, 6d, and 6e. Panel B: Control, INDO, 6f, 6g, 6h, 6i, 6j, and 6k.
Compound 6c showed remarkably potent anti-inflammatory activity, especially 270 min after the ad-
ministration of carrageenan to mice, indicating that cyclooxygenase inhibition may be related to the observed
anti-inflammatory activities. Analgesic activity result of the compound also showed good correlation with its
anti-inflammatory activity in that compound 6c strongly inhibited the peripheral pain response in the mice,
as the amount of writhing was found to be significantly diminished as compared to the control animals treated
with the vehicle. Compounds 6b, 6h, and 6j produced poor anti-inflammatory activity.
When the chemical structures of the active compounds are taken into consideration, para-fluoro substi-
tution in the phenyl ring of the phenylpiperazine moiety caused both analgesic and anti-inflammatory activities
to increase in antipyrine/pyridazinone hybrids possessing acetylenic amide bond.
745

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
Moreover, acute toxicity and gastric ulcerogenic effects of the title compounds were investigated in test
animals. As for the ulcerogenic effects of the synthesized compounds, compounds 6a, 6c, and 6h caused weak
damage to the gastric mucosa at 100 mg/kg dose. The other compounds had no ulcerogenic side effect (Table 2).
Table 2. Analgesic and anti-inflammatory effects of test compounds on p-benzoquinone (PBQ)-induced abdominal
constriction test and carrageenan (CG)-induced hind paw edema model in mice, respectively, and ratio of ulceration.
Swelling in paw thickness (× 10−2 mm)
Test
% Analgesic activity
SEMa
SEM (% inhibition of edema)
270th minute after CGb
Ratio of
ulcerationc
0/6
compounds
DMSO (Control)
—
41.67 2.37 (n = 19)
ASA
INDO
6a
78.21 7.22 (n = 14)
—
—
2/6
20.04 1.44 (51.92)*** (n = 16)
31.82 5.23 (23.64) (n = 11)
27.89 2.30 (33.05)*** (n = 19)
13.75 3.43 (67.00)*** (n = 12)
34.09 3.56 (18.18) (n = 11)
35.79 3.69 (14.11) (n = 19)
37.22 6.02 (10.67) (n = 9)
42.92 6.14 (–3.00) (n = 12)
27.50 2.79 (34.00)*** (n = 12)
32.00 2.80 (23.20)* (n = 20)
26.25 4.65 (37.00)** (n = 12)
50.56 5.43 (–21.33) (n = 9)
1/6
88.33 5.87 (n = 6)
—
1/6
6b
6c
0/6
73.33 13.02 (n = 6)
82.14 8.72 (n = 7)
77.14 10.34 (n = 7)
26.00 18.87 (n = 10)
64.29 19.01 (n = 7)
73.33 18.47 (n = 6)
62.22 9.93 (n = 9)
40.00 20.41 (n = 7)
30.71 14.03 (n = 7)
1/6
6d
6e
0/6
0/6
6f
0/6
6g
0/6
6h
6i
1/6
0/6
6j
0/6
6k
0/6
Data obtained from animal experiments were expressed as means
standard error of mean (SEM); Dimethylsulfoxide:
DMSO, Indomethacin: INDO, acetylsalicylic acid: ASA, carrageenan: CG. a,c Groups were employed for the PBQ-
induced writhing test, b Groups were employed for the CG-induced paw edema model. All test drugs were subcutaneously
administered to mice, INDO at the dose of 10 mg/kg and the other drugs at the doses of 100 mg/kg (body weight).
*Swelling in paw-thickness significantly different from the control (Unpaired t test, *P < 0.05, **P < 0.005, ***P <
0.0005).
In silico oral biodisponibility–molecular modeling approach
The synthesized compounds (6a–k) were submitted to an in silico evaluation using a molecular modeling
approach. Good absorption after oral administration is obligatory for anti-inflammatory and antiplatelet medical
use. Therefore, we analyzed these derivatives according to the rule-of-five developed by Lipinski et al. (Table
3).16 The rule-of-five theoretically characterizes molecular properties for orally active drugs. The rule states
that the 90% of orally active drugs present number of hydrogen bond acceptors ≤ 10 and donors ≤ 5, clogP
≤ 5 and molecular weight (MW) ≤ 500. Molecules violating more than one of these rules may have problems
with bioavailability. The results show that compounds 6a, 6j, and 6k fulfill the Lipinski rule-of-five (Table 3).
The rest of the compounds violated only the criterion of molecular weight.
746

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
Table 3. Parameters for the Lipinski rule-of-five for synthesized compounds predicted using a molecular modeling
approach.
Theoretical oral biodisponibility (Lipinski rule-of-five)a)
Compound
HBA HBD
MW
clogP
2.144
2.822
2.308
2.945
3.132
3.093
2.579
3.069
3.403
2.273
2.721
6a
6b
6c
6d
6e
6f
10
10
10
10
9
1
1
1
1
1
1
1
1
1
1
1
499.56
533.19
517.55
526.61
512.25
547.20
531.58
547.20
526.26
429.47
443.49
10
10
10
9
6g
6h
6i
6j
8
6k
8
a For good theoretical oral biodisponibility – number of hydrogen bond acceptors (HBA) ≤ 10 and donors (HBD) ≤ 5,
clog P ≤ 5 and molecular weight ≤ 500.
In conclusion, we report herein a simple and convenient route for the synthesis of antipyrine/pyridazinone
hybrids for analgesic and anti-inflammatory evaluation. According to the results of in vivo studies conducted
in the present study, the analgesic and anti-inflammatory activities of 6c are comparable to those of known
drugs, i.e. aspirin and indomethacin, without inducing any visible gastric damage. Therefore, our initial
screening results demonstrate that the presence of certain arylpiperazine substituents at the pyridazine ring
in antipyrine/pyridazinone hybrids may contribute to their analgesic and anti-inflammatory activities. Further
detailed studies are under way in order to investigate the effect of the synthesized hybrid molecules on chronic
inflammatory test models and COX-2 selective inhibitory effects.
Acknowledgment
This investigation was supported by a Scientific Research Grant 02/2010-35, awarded by Gazi University BAP.
References
1. Hashash, J. G.; Atweh L. A; Saliba, T.; Chakhachiro, Z. Clinical Therapeutics 2007, 29, 2448-2452.
2. Ray, W. A.; Chung, C. P; Stein, C. M.; Smalley, W. E.; Hall, K.; Arbogast, P. G.; Griffin, M. R. Gastroenterology
2007, 133, 790-798.
3. Marco, J. L.; Amariles, P.; Bosca, B.; Castello A. Curr. Ther. Res. 2007, 68, 107-119.
4. Funatsu, T.; Chono, K.; Hirata, T.; Keto, Y.; Kimoto, A.; Sasamata, M. Eur. J. Pharmacol. 2007, 554, 53-59.
747

Synthesis of antipyrine/pyridazinone hybrids and investigation of..., S. BAYTAS¸, et al.
5. Odabasoglu, F.; Cakır, A.; Suleyman, H.; Aslan, A.; Bayir, Y.; Halici, M.; Kazaz, C. Journal of Ethnopharmacology
2006, 103, 59-65.
6. Rostom, S. A .F.; El-Ashmawy, I. M.; Abd El Razik, H. A.; Badr, M. H.; Ashour, H. M. A. Bioorg. Med. Chem.
2009, 17, 882-895.
7. Bekhit, A. A.; Abdel-Azneim, T. Bioorg. Med. Chem. 2004, 12, 1935-1945.
8. Jain, S. C. Sinha, J.; Bhagat, S.; Errington, W.; Olsen, C. E. Synth. Commun. 2003, 33, 563-577.
9. Bondock, S.; Rabie, R.; Etman, H. A.; Fadda, A. A. Eur. J. Med. Chem. 2008, 43, 2122-2129.
10. Meunier, B. Accounts Chem. Res. 2008, 41, 69-77.
11. Stahl, E. Thin-layer Chromatography, Springer, New York, 1969.
12. Okun, R. Liddon, S. C.; Lasagnal, L. J. Pharmacol. Exp. Ther. 1963, 139, 107-114.
13. Kupeli, E.; Tatlı, I., Akdemir, Z. S.; Yesilada, E. J. Ethnopharmacol. 2007, 114, 234-240.
14. Cioli, V.; Putzolu, S.; Rossi, V., Barcellona, P. S.; Corradino, C. Toxicol. Appl. Pharm. 1979, 50, 283-289.
15. Yesilada, E.; Gurbuz, I.; J. Ethnopharmacol. 2010, 131, 17-21.
16. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev. 2001, 46, 3-26
17. Boissier, J. R.; Ratouis, R.; Dumont, C. J. Med. Chem. 1963, 6, 541-544.
18. Elvio, B.; Paravicini, F.; Emilio, T. Farmaco Ed. Sci. 1969, 24 919-929.
19. Gokce, M.; Dogruer, D.; Sahin, M. F. Il Farmaco, 2001, 56, 233-237.
20. Gokce, M.; Sahin, M. F.; Kupeli, E.; Yesilada, E. Arzneim. Forsch. Drug Res. 2004 54, 396-401.
21. Dundar, Y.; Gokce, M.; Kupeli, E.; Sahin, M. F. Arzneim. Forsch. Drug Res. 2007, 57, 777-781.
22. Kalgutkar, A. S.; Marnett, A. B.; Crews, B. C.; Remel, R. P.; Marnett, L. J. J. Med. Chem. 2000, 43, 2860-2870.
23. Kalgutkar, A. S.; Rowlinson, S. W.; Crews, B. C.; Marnett, L. J. Bioorg. Med. Chem. Lett. 2002, 12, 521-524.
24. Dogruer, D. S.; Sahin, M. F.; Unlu, S.; Shigeru, I. Arch. Pharm. Pharm. Med. Chem. 2000, 333, 79-86.
25. Kasahara, Y.; Hikino, H.; Tsurufuji, S.; Watanabe, M., Ohuchi, K. Planta Medica 1985, 51, 325-331.
26. Shen, T. Y. Non-Steroidal Anti-Inflammatory Agents. Burger’s Medicinal Chemistry, ed. M.E. Wolf. 1980, New
York: John Wiley and Sons. 1217-1219.
27. Vinegar, R.; Truax, J. F.; Selph, J. L. Fed. Proc. 1976, 35, 2447-2456.
28. Castro, J.; Sasame, H.; Sussman, H.; Buttett, P. Life. Sci. 1968, 7, 129-136.
29. Di Rosa, M.; Giroud, J. P.; Willoughby, D. A. J. Pathol. 1971, 104, 15-29.
30. Chatpaliwar, V. A.; Johrapurkar A. A.; Wanjari, M. M.; Chakraborty, R. R.; Kharkar, V. T. Indian Drugs, 2002,
39, 543-545.
31. Di Rosa, M. J. Pharm. Pharmacol. 1972, 24, 89-102.
748

Copyright of Turkish Journal of Chemistry is the property of Scientific and Technical
Research Council of Turkey and its content may not be copied or emailed to multiple sites or
posted to a listserv without the copyright holder's express written permission. However, users
may print, download, or email articles for individual use.