Synthesis and analgesic activity of 2-phenoxybenzoic acid and N-phenylanthranilic acid hydrazides

Article abstract of DOI:10.1248/bpb.29.1180

A series of 2-phenoxybenzoic acid and N-phenylanthranilic acid hydrazides were synthesized and evaluated for their analgesic activities. Several compounds were significantly more potent than mefenamic acid and diclofenac sodium in abdominal constriction a

Full text of DOI:10.1248/bpb.29.1180

1180
Biol. Pharm. Bull. 29(6) 1180—1185 (2006)
Vol. 29, No. 6
Synthesis and Analgesic Activity of 2-Phenoxybenzoic Acid and
N-Phenylanthranilic Acid Hydrazides
Ali ALMASIRAD,a Rohollah HOSSEINI,b Hassan JALALIZADEH,c Zohreh RAHIMI-MOGHADDAM,b
Nazila ABAEIAN,b Maryam JANAFROOZ,b Mohammad ABBASPOUR,c Vahid ZIAEE,c
Afshin DALVANDI,c and Abbas SHAFIEE*,c
a Department of Medicinal Chemistry, Faculty of Pharmacy, Islamic Azad University; Tehran, 19419 Iran: b Department of
Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences; and c Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Terhan
University of Medical Sciences; Tehran 14174, Iran. Received November 4, 2005; accepted February 28, 2006
A series of 2-phenoxybenzoic acid and N-phenylanthranilic acid hydrazides were synthesized and evaluated
for their analgesic activities. Several compounds were significantly more potent than mefenamic acid and di-
clofenac sodium in abdominal constriction and formalin tests.
Key words 2-phenoxybenzoic acid hydrazide; N-phenylanthranilic acid hydrazide; analgesic activity
Non steroidal anti-inflammatory drugs (NSAID’s) are first compounds 3—5).8—10) On the basis of these reports, we de-
line therapeutic agents for the treatment of arthritis. NSAID’s scribed previously the synthesis and analgesic profile of N-
reduce the pain and swelling associated with arthritis by arylhydrazone derivatives of mefenamic acid 6 (Fig. 3), a
blocking the metabolism of arachidonic acid (AA) through known NSAID drug with fenamate structure.11) Most of
the enzyme cyclooxygenase (COX) and thereby the produc- these synthesized compounds were effective as analgesic
tion of prostaglandins, e.g. PGE2, which sensitizes nocicep- agents in comparison to control and showed more analgesic
tors at nerve fiber terminals.1,2)
activity in comparison to mefenamic acid. In fact, compound
Additionally, the 5-lipoxygenase (5-LO) products such as 7 (Fig. 3) displayed 3.6-fold greater potency than mefenamic
leukotriene B4 (LTB4) also contributes to the hyperalgesia acid as an analgesic agent, in a model of abdominal constric-
seen during inflammation by decreasing the mechanical and tions induced by acetic acid. These results led us to design
thermal thresholds of C fiberes.3) Leukotrienes, especially new structurally related derivatives 10, 11. Target compounds
LTB4 together with prostaglandins are implicated in the acute 10, 11 were obtained according to Chart 1. The starting hy-
ulceration induced by NSAID’s.4) For these reasons, com- drazides 8 (XꢀO, RꢀH and XꢀNH, RꢀH) were prepared
pounds that achieve dual inhibition of enzymes COX and 5- by the known procedures and hydrazide 8 (XꢀO, RꢀCl) was
LO reduce side effects and improved efficacy in the combat synthesized as described previously.12—17) Compounds 10, 11
of pain in inflammatory diseases.5) Some evidences suggest were synthesized by acid-catalyzed condensation of hy-
that the hydrazone moiety present in phenylhydrazone deriv- drazides 8 with corresponding substituted benzaldehydes or
ative 1 (Fig. 1), possesses a pharmacophoric character for the acetophenones.10,11) The synthesis and antimicrobial activity
inhibition of COX and hydrazone type containing com-
pounds such as compound 2 (Fig. 1) were described as dual
COX/5-LO inhibitors.6,7) According to these evidences, there
are some reports about synthesis and pharmacological evalu-
ation of new bioactive compounds with N-aroylarylhydra-
zone structure acting at the AA cascade enzyme level (Fig. 2,
Fig. 3
Fig. 1
Chart 1
Fig. 2
∗ To whom correspondence should be addressed. e-mail: ashafiee@ams.ac.ir
© 2006 Pharmaceutical Society of Japan

June 2006
1181
of some of the target compounds 11 (11a—f) has been previ- ethanol (20 ml) was stirred at room temperature for 0.5 to
ously reported.15,18,19)
1 h, in the presence of hydrochloric acid (2 drops) as a cata-
lyst. The end of the reaction was observed by TLC, and the
hydrazones 10a—11j were isolated by concentration of the
reaction mixture uner reduced pressure, followed by neutral-
CHEMISTRY
Chemicals were purchased from Merck Chemical Com- ization with a 10% aqueous solution of sodium bicarbonate.
pany (Darmstadt, Germany). Melting points were taken on a The resulting precipitate was filtered, washed with water
Kofler hot stage apparatus (Reichert, Vienna, Austria) and (10 ml) and crystallized from a suitable solvent. Melting
are uncorrected. 1H-NMR spectra were obtained using a points, crystallization solvents and yields are reported in
Brucker FT-80 spectrometer (Brucker, Rheinstetten, Ger- Table 1.
many). Tetramethylsilane was used as an internal standard.
Mass spectra were obtained using a Finnigan Mat TSQ-70 lows:
spectrometer at 70 eV (Finnigan Mat, Bremen, Germany).
The spectral data of the synthesized compounds are as fol-
1
2-Phenoxybezoic Acid (Benzylidene)hydrazide 10a: H-
The IR spectra were obtained using a Nicolet FT-IR Magna NMR (CDCl3) d: 6.78—7.47 (m, 13H, aromatic), 8.30 (s,
550 spectrographs (KBr disks) (Nicolet, Madision, WI, 1H, CH), 8.67 (d, 1H, Jꢀ7.4 Hz, aromatic), 10.93 (bs, 1H,
U.S.A.). Elemental analyses were carried out with a Perkin- NH). IR (KBr) cmꢂ1: 3323 (NH), 1666 (CꢀO). MS m/z: 316
Elmer Model 240-c apparatus (Perkin Elmer, Norwalk, CT, (Mꢃ), 197, 92; Anal. Calcd for C20H16N2O2: C, 75.95; H,
U.S.A.). The results of the elemental analyses (C, H, N) were 5.06; N, 8.86. Found: C, 76.23; H, 4.84; N, 9.11.
within ꢁ0.4% of the calculated amounts.
2-Phenoxybenzoic Acid (4-Chlorobenzylidene)hydrazide
1
General Procedure for Synthesis of 2-Phenoxybenzoic 10b: H-NMR (CDCl3) d: 6.70—7.78 (m, 12H, aromatic),
Acid and N-Phenylanthranilic Acid Hydrazides (10a— 8.29 (s, 1H, CH), 7.41 (d, 1H, Jꢀ7.6 Hz, 1H, aromatic),
11j) A mixture of hydrazide 8 (2.2 mmol) and correspon- 10.72 (bs, 1H, NH). IR (KBr) cmꢂ1: 3304 (NH), 1660
ding aldehyde or acetophenone (2.2 mmol) in absolute (CꢀO). MS m/z: 350 (Mꢃ), 197, 141; Anal. Calcd for
Table 1. Physical Data of Synthesized Compounds
Comp.
X
R
Rꢄ
Ar
Recrystallization solvnet
mp (°C)
Yeild (%)
Molecular formulaa)
10a
10b
10c
10d
10e
10f
10g
10h
10i
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
Phenyl
EtOH
EtOH
EtOH
154—156
167—169
147—149
245—247
181—184
189—190
186—188
192—194
153—155
168—169
178—179
188—190
98—100
155—157
162—164
142—144
128—131
101—103
207—209
162—164
131—133
110—111
179—181
230—232
193—195
180—182
194—196
221—224
218—220
160—162
152—154
91—93
89
53
70
92
90
86
88
43
45
79
82
88
61
53
61
75
80
76
77
61
36
40
95
89
92
96
92
95
84
93
77
80
C20H16N2O2
C20H15ClN2O2
C21H17ClN2O2
C20H16N2O3
C21H18N2O3
C21H18N2O3
C21H18N2O3
C19H15N3O2
C19H15N3O2
C20H15FN2O2
C21H18N2O2
C22H21N3O2
C20H15ClN2O2
C20H14Cl2N2O2
C21H17ClN2O3
C21H17ClN2O3
C21H17ClN2O2
C20H14ClFN2O2
C20H15ClN2O3
C22H20ClN3O2
C19H14ClN3O2
C19H14ClN3O2
C20H17N3O
H
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Chlorophenyl
4-Chlorophenyl
4-Hydroxyphenyl
4-Hydroxyphenyl
2-Methyoxyphenyl
4-Methoxyphenyl
3-Pyridyl
i-PrOH
i-PrOH
EtOAc
EtOAc
MeOH
EtOAc
EtOH
EtOAc
EtOH
MeOH
EtOH
EtOAc
EtOAc
EtOAc
EtOH
i-PrOH
EtOH
EtOAc
EtOAc
i-PrOH
EtOAc
EtOAc
EtOH
4-Pyridyl
10j
10k
10l
4-Fluorophenyl
4-Methylphenyl
4-Dimethylaminophenyl
Phenyl
4-Chlorophenyl
2-Methoxyphenyl
4-Methyoxyphenyl
4-Methylphenyl
4-Fluorophenyl
4-Hydroxyphenyl
4-Dimethylaminophenyl
3-Pyridyl
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
11a
11b
11c
11d
11e
11f
4-Pyridyl
Phenyl
2-Methoxyphenyl
4-Methoxyphenyl
4-Chlorophenyl
4-Hydroxyphenyl
4-Dimethylaminophenyl
4-Methylphenyl
4-Fluorophenyl
3-Pyridyl
C21H19N3O2
C21H19N3O2
C20H16ClN3O
C20H17N3O2
C22H22N4O
C21H19N3O
C20H16FN3O
C19H16N4O
EtOH
i-PrOH
EtOAc
MeOH
EtOAc
EtOAc
11g
11h
11i
11j
4-Pyridyl
C19H16N4O
a) All compounds were analyzed for C, H, N. Analytical results obtained for these elements were within ꢁ0.4% of the calculated value for the formula shown.

1182
Vol. 29, No. 6
C20H15ClN2O2: C, 68.47; H, 4.28; N, 7.99. Found: C, 68.14; N, 8.48. Found: C, 76.61; H, 5.51; N, 8.71.
H, 4.14; N, 7.75.
2-Phenoxybenzoic Acid (4-Dimethylaminobenzylidene)-
2-Phenoxybenzoic Acid (Methyl-4-chlorobenzylidene)hy- hydrazide 10l: 1H-NMR (CDCl3) d: 3.00 (s, 6H, CH3),
1
drazide 10c: H-NMR (CDCl3) d: 2.17 (s, 3H, CH3), 6.88— 6.64—7.70 (m, 12H, aromatic), 7.99 (s, 1H, CH), 8.24 (dd,
7.79 (m, 12H, aromatic), 8.43 (d, 1H, Jꢀ7.6 Hz, aromatic), Jꢀ7.6, 2.3 Hz, 1H, aromatic), 10.48 (bs, 1H, NH). IR (KBr)
10.86 (bs, 1H, NH). IR (KBr) cmꢂ1: 3329 (NH), 1680 cmꢂ1: 3304 (NH), 1665 (CꢀO). MS m/z: 359 (Mꢃ), 197,
(CꢀO). MS m/z: 364 (Mꢃ), 350, 279, 231; Anal. Calcd for 146, 77; Anal. Calcd for C22H21N3O2: C, 73.54; H, 5.85; N,
C21H17ClN2O2: C, 69.13; H, 4.66; N, 7.68. Found: C, 68.84; 11.70. Found: C, 73.25; H, 6.03; N, 11.78.
H, 4.33; N. 7.84.
2-(2-Chlorophenoxy)benzoic Acid (Benzylidene)hydrazide
1
2-Phenoxybenzoic Acid (4-Hydroxybenzylidene)hydrazide 10m: H-NMR (CDCl3) d: 6.74—7.76 (m, 12H, aromatic),
10d: 1H-NMR (DMSO-d6) d: 6.76—7.92 (m, 13H, aro- 8.19 (s, 1H, CH), 8.33 (dd, Jꢀ7.3, 2.0 Hz, 1H, aromatic),
matic), 8.18 (s, 1H, CH), 9.91 (bs, 1H, NH), 10.77 (bs, 1H, 10.58 (bs, 1H, NH). IR (KBr) cmꢂ1: 3285 (NH), 1668
OH). MS m/z: 332 (Mꢃ), 197, 174, 92; Anal. Calcd for (CꢀO). MS m/z: 350 (Mꢃ), 231, 212, 127; Anal. Calcd for
C20H16N2O3: C, 72.29; H, 4.82; N, 8.43. Found: C, 72.64; H, C20H15ClN2O2: C, 68.47; H, 4.28; N, 7.99. Found: C, 68.60;
4.94; N, 8.35.
H, 4.49; N, 8.18.
2-Phenoxybenzoic Acid (Methyl-4-hydroxybenzylidene)-
2-(2-Chlorophenoxy)benzoic Acid (4-Chlorobenzylidene)-
1
hydrazide 10e: H-NMR (DMSO-d6) d: 2.14 (s, 3H, CH3), hydrazide 10n: 1H-NMR (DMSO-d6) d: 6.74—8.01 (m, 12H,
6.78—7.75 (m, 12H, aromatic), 8.38 (dd, Jꢀ7.6, 2.4 Hz, 1H, aromatic), 8.30 (s, 1H, CH), 11.83 (bs, 1H, NH). IR (KBr)
aromatic), 9.04 (bs, 1H, NH), 10.73 (bs, 1H, OH). IR (KBr) cmꢂ1: 3199 (NH), 1673 (CꢀO). MS m/z: 385 (Mꢃ), 231,
cmꢂ1: 3333 (OH), 3161 (NH), 1652 (CꢀO). MS m/z: 346 212, 168, 127; Anal. Calcd for C20H14Cl2N2O2: C, 62.34; H,
(Mꢃ), 330, 252, 226, 197; Anal. Calcd for C21H18N2O3: C, 3.64; N, 7.28. Found: C, 61.99; H, 3.93; N, 6.97.
72.83; H, 5.20; N, 8.09. Found: C, 72.94; H, 5.29; N, 8.21.
2-(2-Chlorophenoxy)benzoic Acid (2-Methoxybenzyli-
2-Phenoxybenzoic Acid (2-Methoxybenzylidene)hydrazide dene)hydrazide 10o: 1H-NMR (CDCl3) d: 3.85 (s, 3H,
1
10f: H-NMR (CDCl3) d: 3.82 (s, 3H, OCH3), 6.86—7.29 OCH3), 6.72—7.55 (m, 10H, aromatic), 8.13 (dd, Jꢀ7.7,
(m, 11H, aromatic), 8.12 (d, Jꢀ7.2 Hz, 1H, aromatic), 8.32 2.4 Hz, 1H, aromatic), 8.32 (dd, Jꢀ7.3, 2.6 Hz, 1H, aro-
(d, Jꢀ7.8 Hz, 1H, aromatic), 8.52 (s, 1H, CH), 10.64 (bs, 1H, matic), 8.56 (s, 1H, CH), 10.54 (bs, 1H, NH). IR (KBr)
NH). IR (KBr) cmꢂ1: 3172 (NH), 1678 (CꢀO). MS m/z: 346 cmꢂ1: 3167 (NH), 1668 (CꢀO). MS m/z: 380 (Mꢃ), 231,
(Mꢃ), 213, 197, 93; Anal. Calcd for C21H18N2O3: C, 72.83; 212, 168, 127; Anal. Calcd for C21H17ClN2O3: C, 66.23; H,
H, 5.20; N, 8.09. Found: C, 72.56; H, 5.08; N, 8.28.
4.47; N, 7.35. Found: C, 66.37; H, 4.39; N, 7.21.
2-Phenoxybenzoic Acid (4-Methoxybenzylidene)hydrazide
2-(2-Chlorophenoxy)benzoic Acid (4-Methoxybenzyli-
1
10g: H-NMR (CDCl3) d: 3.82 (s, 3H, OCH3), 6.77—7.76 dene)hydrazide 10p: 1H-NMR (CDCl3) d: 3.82 (s, 3H,
(m, 12H, aromatic), 8.09 (s, 1H, CH), 8.35 (dd, Jꢀ7.5, OCH3), 6.73—7.78 (m, 11H, aromatic), 8.12 (s, 1H, CH),
2.3 Hz, 1H, aromatic), 10.58 (bs, 1H, NH). IR (KBr) cmꢂ1: 8.32 (dd, Jꢀ7.3, 2.0 Hz, 1H, aromatic), 10.51 (bs, 1H, NH).
3234 (NH), 1652 (CꢀO). MS m/z: 346 (Mꢃ), 212, 197, 93; IR (KBr) cmꢂ1: 3201 (NH), 1665 (CꢀO). MS m/z: 380
Anal. Calcd for C21H18N2O3: C, 72.83; H, 5.20; N, 8.09. (Mꢃ), 231, 212, 168, 127; Anal. Calcd for C21H17ClN2O3: C,
Found: C, 72.71; H, 5.05; N. 8.33.
2-Phenoxybenzoic Acid (3-Pyridinylidene)hydrazide 10h:
66.23; H, 4.47; N, 7.35. Found: C, 66.41; H, 4.35; N, 7.42.
2-(2-Chlorophenoxy)benzoic Acid (4-Methylbenzylidene)-
1H-NMR (CDCl3) d: 6.79—7.83 (m, 12H, aromatic), 8.15 (s, hydrazide 10q: 1H-NMR (CDCl3) d: 2.37 (s, 3H, CH3),
1H, CH), 8.37 (dd, Jꢀ7.6, 1.9 Hz, 1H, aromatic), 10.71 (bs, 6.74—7.73 (m, 11H, aromatic), 8.13 (s, 1H, CH), 8.34 (dd,
1H, NH). IR (KBr) cmꢂ1: 3188 (NH), 1678 (CꢀO). MS m/z: Jꢀ7.4, 2.5 Hz, 1H, aromatic), 10.53 (bs, 1H, NH). IR (KBr)
317 (Mꢃ), 212, 197, 140, 93; Anal. Calcd for C19H15N3O2: C, cmꢂ1: 3244 (NH), 1668 (CꢀO). MS m/z: 364 (Mꢃ), 233,
71.92; H, 4.73; N, 13.25. Found: C, 72.1; H, 4.58; N, 12.96.
2-Phenoxybenzoic Acid (4-Pyridinylidene)hydrazide 10i: N, 7.68. Found: C, 69.29; H, 4.37; N. 7.34.
1H-NMR (CDCl3) d: 6.80—7.67 (m, 11H, aromatic), 8.26 (s,
2-(2-Chlorophenoxy)benzoic Acid (4-Fluorobenzylidene)-
231, 212. Anal. Calcd for C21H17ClN2O2: C, 69.13; H, 4.66;
1
1H, CH), 8.65 (d, Jꢀ5.6 Hz, 2H, aromatic), 10.91 (bs, 1H, hydrazide 10r: H-NMR (CDCl3) d: 6.73—7.84 (m, 11H,
NH). IR (KBr) cmꢂ1: 3247 (NH), 1660 (CꢀO). MS m/z: 317 aromatic), 8.19 (s, 1H, CH), 8.33 (dd, Jꢀ7.9, 2.5 Hz, 1H,
(Mꢃ), 197, 168; Anal. Calcd for C19H15N3O2: C, 71.92; H, aromatic), 10.55 (bs, 1H, NH). IR (KBr) cmꢂ1: 3206 (NH),
4.73; N, 13.25. Found: C, 72.17; H, 4.89; N, 13.06.
1686 (CꢀO). MS m/z: 380 (Mꢃ), 212, 168, 127; Anal. Calcd
2-Phenoxybenzoic Acid (4-Flourobenzylidene)hydrazide for C20H14ClFN2O2: C, 65.13; H, 3.80; N, 7.60. Found: C,
1
10j: H-NMR (CDCl3) d: 6.78—7.84 (m, 12H, aromatic), 64.79; H, 4.03; N, 7.31.
8.17 (s, 1H, CH), 8.34 (dd, Jꢀ7.4, 2.1 Hz, 1H, aromatic),
2-(2-Chlorophenoxy)benzoic Acid (4-Hydroxybenzyli-
10.67 (bs, 1H, NH). IR (KBr) cmꢂ1: 3242 (NH), 1651 dene)hydrazide 10s: 1H-NMR (DMSO-d6) d: 6.80—8.02 (m,
(CꢀO). MS m/z: 334 (Mꢃ), 197, 168; Anal. Calcd for 12H, aromatic), 8.22 (s, 1H, CH), 10.85 (bs, 1H, NH). IR
C20H15FN2O2: C, 71.85; H, 4.49; N, 8.38. Found: C, 71.94; (KBr) cmꢂ1: 3323 (OH), 3167 (NH), 1652 (CꢀO): MS m/z:
H, 4.42; N, 8.51.
366 (Mꢃ), 248, 231, 212, 127, 120; Anal. Calcd for
2-Phenoxybenzoic Acid (4-Methylbenzylidene)hydrazide C20H15ClN2O3: C, 65.48; H, 4.09; N, 7.64. Found: C, 65.72;
10k: 1H-NMR (CDCl3) d: 2.35 (s, 3H, CH3), 6.78—7.70 (m, H, 3.98; N, 7.85.
12H, aromatic), 8.11 (s, 1H, CH), 8.33 (dd, Jꢀ7.5, 2.1 Hz,
2-(2-Chlorophenoxy)benzoic Acid (4-Dimethylaminoben-
1
1H, aromatic), 10.61 (bs, 1H, NH). IR (KBr) cmꢂ1: 3312 zylidene)hydrazide 10t: H-NMR (CDCl3) d: 3.00 (s, 6H,
(NH), 1673 (CꢀO). MS m/z: 330 (Mꢃ), 213, 197, 168, 141, CH3), 6.64—7.71 (m, 11H, aromatic), 8.03 (s, 1H, CH), 8.30
115, 93, 77; Anal. Calcd for C21H18N2O2: C, 76.36; H, 5.45; (dd, Jꢀ7.1, 2.2 Hz), 10.39 (bs, 1H, NH). IR (KBr) cmꢂ1:

June 2006
1183
1
3293 (NH), 1650 (CꢀO). MS m/z: 393 (Mꢃ), 230, 145. Anal. drazide 11g: H-NMR (CDCl3) d: 2.35 (s, 3H, CH3), 6.81—
Calcd for C22H20ClN3O2: C, 67.09; H, 5.08; N, 10.67. Found: 8.33 (m, 13H, aromatic), 8.46 (s, 1H, CH), 10.23 (bs, 1H,
C, 66.97; H,4.99; N, 10.95.
NH), 11.44 (bs, 1H, NH). IR (KBr) cmꢂ1: 3371 (NH), 3210
2-(2-Chlorophenoxy)benzoic Acid (3-Pyridinylidene)hy- (NH), 1646 (CꢀO). MS m/z: 329 (Mꢃ), 195, 167, 139; Anal.
1
drazide 10u: H-NMR (CDCl3) d: 6.83—7.59 (m, 9H, aro- Calcd for C21H19N3O: C, 76.59; H, 5.77; N, 12.77. Found: C,
matic), 8.19 (s, 1H, CH), 8.27 (d, Jꢀ7.2 Hz, 1H, aromatic), 76.41; H, 5.92; N, 12.53.
8.62 (d, Jꢀ6.2 Hz, 1H, aromatic), 8.95 (s, 1H, aromatic),
2-Phenylaminobenzoic Acid (4-Fluorobenzylidene)hy-
1
10.52 (bs, 1H, NH). IR (KBr) cmꢂ1: 3261 (NH), 1652 drazide 11h: H-NMR (CDCl3) d: 6.91—8.15 (m, 13H, aro-
(CꢀO). MS m/z: 351 (Mꢃ), 231, 168; Anal. Calcd for matic), 8.52 (s, 1H, CH), 9.20 (bs, 1H, NH), 10.59 (bs, 1H,
C19H14ClN3O2: C, 64.86; H, 3.98; N, 11.95. Found: C, 64.75; NH). IR (KBr) cmꢂ1: 3352 (NH), 3186 (NH), 1634 (CꢀO).
H, 3.94; N, 11.78.
MS m/z: 333 (Mꢃ), 196, 167; Anal. Calcd for C20H16FN3O:
2-(2-Chlorophenoxy)benzoic Acid (4-Pyridinylidene)hy- C, 72.07; H, 4.08; N, 12.61. Found: C, 71.73; H, 4.04; N,
1
drazide 10v: H-NMR (CDCl3) d: 6.80—7.63 (m, 9H, aro- 12.80.
matic), 8.22 (s, 1H, CH), 8.29 (d, Jꢀ7.6 Hz, 1H, aromatic),
2-Phenylaminobenzoic Acid (3-Pyridinylidene)hydrazide
1
8.64 (bs, 2H, aromatic), 10.89 (bs, 1H, NH). IR (KBr) cmꢂ1: 11i: H-NMR (CDCl3) d: 6.54—7.72 (m, 12H, aromatic),
3272 (NH), 1657 (CꢀO). MS m/z: 351 (Mꢃ), 231, 212, 168, 8.25 (s, 1H, CH), 8.62 (dd, Jꢀ7.6, 1.9 Hz, 1H, aromatic),
139; Anal. Calcd for C19H14ClN3O2: C, 64.86; H, 3.98; N, 9.15 (bs, 1H, NH), 9.62 (bs, 1H, NH). IR (KBr) cmꢂ1: 3310
11.95. Found: C, 65.01; H, 4.09; N, 12.20.
(NH), 3237 (NH), 1662 (CꢀO). MS m/z: 316 (Mꢃ), 196,
2-Phenylaminobenzoic Acid (Benzylidene)hydrazide 11a: 167, 139. Anal. Calcd for C19H16N4O: C, 72.15; H, 5.06; N,
1H-NMR (CDCl3) d: 6.82—7.83 (m, 14H, aromatic), 8.14 (s, 17.72. Found: C, 72.02; H, 4.95; N, 17.91.
1H, CH), 9.33 (bs, 1H, NH), 10.53 (bs, 1H, NH). IR (KBr)
2-Phenylaminobenzoic Acid (4-Pyridinylidene)hydrazide
1
cmꢂ1: 3252 (NH), 3175 (NH), 1628 (CꢀO). MS m/z: 315 11j: H-NMR (CDCl3) d: 6.75—7.63 (m, 11H, aromatic),
(Mꢃ), 196, 167; Anal. Calcd for C20H17N3O: C, 76.19; H, 8.21 (s, 1H, CH), 8.65 (d, Jꢀ6.2 Hz, 2H, aromatic), 9.05 (bs,
5.40; N, 13.33. Found: C, 75.91; H, 5.29; N, 13.59.
1H, NH), 9.73 (bs, 1H, NH). IR (KBr) cmꢂ1: 3368 (NH),
2-Phenylaminobenzoic Acid (2-Methoxybenzylidene)hy- 3277 (NH), 1644 (CꢀO). MS m/z: 316 (Mꢃ), 196, 167;
drazide 11b: 1H-NMR (CDCl3) d: 3.87 (s, 3H, OCH3), Anal. Calcd for C19H16N4O: C, 72.15; H, 5.06; N, 17.72.
6.82—7.35 (m, 11H, aromatic), 7.72 (d, Jꢀ7.3 Hz, 1H, aro- Found: C, 72.43; H, 4.87; N, 17.84.
matic), 8.13 (d, Jꢀ7.4 Hz, 1H, aromatic), 8.77 (s, 1H, CH),
9.67 (bs, 1H, NH), 10.21 (bs, 1H, NH). IR (KBr) cmꢂ1: 3357 PHARMACOLOGY
(NH), 3164 (NH), 1623 (CꢀO). MS m/z: 345 (Mꢃ), 196,
167; Anal. Calcd for C21H19N3O2: C, 73.04; H, 5.51; N,
12.17. Found: C, 73.21; H, 5.72; N, 12.25.
Male NMRI weighing 20—25 g (Razi Institute of Iran)
were used for the abdominal constriction test (writhing test).
2-Phenylaminobenzoic Acid (4-Methoxybenzylidene)hy- The animals were housed in colony cages and conditions of
drazide 11c: 1H-NMR (CDCl3) d: 3.82 (s, 3H, OCH3), constant temperature (22ꢁ2 °C) and a 12 h light/dark sched-
6.81—8.17 (m, 13H, aromatic), 8.47 (s, 1H, CH), 9.95 (bs, ule and allowed free access to standard diet and tap water ex-
1H, NH), 10.86 (bs, 1H, NH). IR (KBr) cmꢂ1: 3368 (NH), cept during the experiment. The animals were allowed to ha-
3212 (NH), 1634 (CꢀO). MS m/z: 345 (Mꢃ), 196, 167; bituate to the laboratory environment for 2 h before the ex-
Anal. Calcd for C21H19N3O2: C, 73.04; H, 5.51; N, 12.17. periments were initiated. All ethical manners for use of labo-
Found: C, 72.73; H, 5.43; N, 12.41.
ratory animals were considered carefully and the protocol of
2-Phenylaminobenzoic Acid (4-Chlorobenzylidene)hy- study was approved by TUMS ethical committee. The com-
1
drazide 11d: H-NMR (CDCl3) d: 6.76—8.31 (m, 13H, aro- pounds were administered intraperitoneally (IP) (0.2 ml/20 g)
matic), 8.72 (s, 1H, CH), 9.76 (bs, 1H, NH), 11.19 (bs, 1H, as suspension in saline and tween 80 (2% w/v). In-
NH). IR (KBr) cmꢂ1: 3371 (NH), 3210 (NH), 1646 (CꢀO). domethacin,20) diclofenac sodium,21) mefenamic acid22)
MS m/z: 349 (Mꢃ), 195, 167; Anal. Calcd for C20H16ClN3O: (Hakim Pharmaceutical Co.) were used as standard drugs
C, 60.08; H, 4.57; N, 12.02. Found: C, 60.37; H, 4.51; N, under the same conditions. The control group received vehi-
12.16.
2-Phenylaminobenzoic Acid (4-Hydroxybenzylidene)hy-
cle (0.2 ml/20 g, IP) alone.
Abdominal Constrication Test The Analgesic activity
1
drazide 11e: H-NMR (CDCl3) d: 6.79—7.66 (m, 13H, aro- was determined in vivo by the abdominal constriction test in-
matic), 8.27 (s, 1H, CH), 9.81 (bs, 1H, NH), 11.06 (bs, 1H, duced by acetic acid (0.6%; 0.1 ml/10 g) in mice.23) An acetic
OH). IR (KBr) cmꢂ1: 3392 (NH), 3241 (OH), 3155 (NH), acid solution was administered IP 30 min after administration
1630 (CꢀO). MS m/z: 331 (Mꢃ), 196, 167, 76. Anal. Calcd of the compounds. Antinociception was recorded by count-
for C20H17N3O2: C, 72.51; H, 5.13; N, 12.69. Found: C, ing the number of writhings immediately after injection of
72.37; H, 5.22; N, 12.88.
acetic acid during 30 min. The analgesic activity was ex-
2-Phenylaminobenzoic Acid (4-Dimethylaminobenzyli- pressed as the percentage of inhibition of constrictions when
1
dene)hydrazide 11f: H-NMR (CDCl3) d: 5.96 (s, 6H, CH3), compared with the vehicle control group. The data were sta-
6.83—7.68 (m, 13H, aromatic), 8.56 (s, 1H, CH), 9.35 (bs, tistically analyzed by one way analysis of Variance (ANOVA)
1H, NH), 10.91 (bs, 1H, NH). IR (KBr) cmꢂ1: 3349 (NH), followed by Newman-Keuls multi comparison test, differ-
3201 (NH), 1635 (CꢀO). MS m/z: 358 (Mꢃ), 196, 163; ences with pꢅ0.05 between experimental groups were con-
Anal. Calcd for C22H22N4O: C, 73.74; H, 6.14; N, 15.64. sidered statistically significant. The ED50 values (the dose of
Found: C, 73.43; H, 6.02; N, 15.45.
compound that reduced responses by 50% in relation to con-
2-Phenylaminobenzoic Acid (4-Methylbenzylidene)hy- trol values) were estimated by graphical interpolation from

1184
Vol. 29, No. 6
Table 2. Effects of Compounds (10a—v and 11a—j), Mefenamic Acid, Indomethacin and Diclofenac Sodium in the Abdominal Constrictions Induced by
Acetic Acid in Mice
a)
Compound
ED50
M.I.b) (%)
Studied doses (mmol/kg)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
196.8 (168.8—223.1)c)
21.25d) (11.89—28.63)
299.5 (210.3—379.0)
18.79d) (11.34—24.90)
205.8 (145.1—250.2)
40.69 (27.33—51.17)
27.88d) (14.33—37.94)
65.89 (27.09—92.30)
50.80 (36.74—62.60)
110.67 (95.45—124.1)
58.78 (44.46—70.43)
322.50 (263.0—381.5)
76.36 (64.2—87.9)
151.4 (128.9—174.1)
113.5 (105.7—121.0)
95.04 (70.2—113.2)
145.07 (100.2—187.8)
100.6 (76.5—118.8)
529.7 (375.6—674.8)
114.7 (91.3—136.8)
211.3 (170.0—244.9)
97.9 (67.9—119.1)
26.37 (20.82—32.77)
22.61d) (16.73—28.52)
26.59d) (17.45—33.57)
10.21d,e) (5.23—14.12)
23.96d) (19.45—28.68)
45.78 (25.13—58.29)
16.22d,e) (12.88—19.20)
42.41 (29.24—51.51)
23.12d) (17.80—28.55)
29.68d) (16.37—39.32)
43.89 (34.12—52.68)
15.69 (12.75—18.92)
30.45 (25.48—35.56)
92.78ꢁ5.2
95.50ꢁ3.8
96.70ꢁ5.2
82.30ꢁ3.5
96.68ꢁ4.7
89.30ꢁ3.2
95.56ꢁ4.5
92.30ꢁ5.2
95.40ꢁ4.3
88.4ꢁ4.6
75.92ꢁ4.8
91.80ꢁ4.4
96.8ꢁ5.2
96.2ꢁ5.4
94.2ꢁ4.7
97.6ꢁ4.4
94.9ꢁ4.7
99.7ꢁ5.6
92.3ꢁ4.3
93.1ꢁ4.8
94.2ꢁ4.6
94.7ꢁ5.2
86.20ꢁ3.8
85.2ꢁ4.3
98.4ꢁ4.4
98.30ꢁ4.7
91.20ꢁ3.1
95.4ꢁ4.8
91.20ꢁ3.2
91.30ꢁ5.1
84.1ꢁ2.3
91.7ꢁ2.8
96.30ꢁ5.2
94.20ꢁ4.1
97.80ꢁ5.3
100, 150, 200, 400
12.5, 25, 50, 100
100, 200, 400, 600
12.5, 25, 50, 75
100, 200, 300, 400
25, 50, 100, 150
12.5, 25, 50, 100
25, 50, 100, 150
25, 50, 100, 200
50, 100, 150, 200
25, 50, 100, 150
100, 200, 400, 600
25, 50, 100, 150
50, 100, 150, 300
50, 100, 150, 200
50, 100, 150, 200
50, 100, 150, 300
50, 100, 150, 200
150, 300, 600, 1200
50, 75, 150, 200
100, 200, 300, 400
50, 100, 150, 200
5, 10, 25, 50
5, 10, 25, 50
10, 25, 50, 75
5, 10, 25, 50
5, 10, 25, 50
25, 50, 75, 100
5, 10, 25, 50
25, 50, 75, 100
10, 25, 50, 100
10, 25, 50, 75
15, 30, 60, 99
3.75, 7.5, 15, 30
7.5, 15, 35, 60
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
Mefenamic acid
Indomethacin
Diclofenac sodium
a) The number of animals in each group was 10 animals; b) maximal inhibition; c) 95% confidence interval; d) significant as compared to mefenamic acid group (pꢅ0.05); e)
significant as compared to diclofenac sodium group (pꢅ0.05).
individual experiments. ED50’s are presented as mean values RESULT AND DISCUSSION
and 95% confidence interval.
Formalin-Induced Test The most active compounds
As shown in Table 2, several synthesized derivatives ex-
were also analysed in the formalin-induced pain test.24) hibited potent antinociceptive effect when assessed by the
Twenty microliters of 5% (0.92% formaldehyde) made up writhing test in mice. In the first series of derivatives (10a—
PBS (phosphate buffered solution, containing: NaCl 137 mM; l), compounds 10b, 10d and 10g were the most potent com-
KCl 2.7 mM and phosphate buffer 10 mM) was injected s.c. pounds and their activity were comparable to indomethacin,
under the plantar surface of the left hindpaw with a Hamilton diclofenac sodium and mefenamic acid. In fact they are 2.1,
syringe. Animnals were acclimatized to the laboratory for at 2.3 and 1.5 fold more potent than mefenamic acid respec-
least 24 h before the experiments. Two mice (control and tively.
treated) were simultaneously observed from 0 up to 30 min
Comparison of compounds 10b and 10d with 10c and 10e,
following formalin injection. The initial nociceptive scores respectively, proves that the introduction of a methyl group in
normally peaked after 5 min (first phase, representing the the NꢀC double bond of the hydrazone moiety reduces the
neurogenic pain), and 15—30 min after formalin injection analgesic activity. These results may indicate that the methyl
(second phase, representing the inflammatory pain). The ani- group in 10c and 10e blocks the imine framework of the
mals were treated with saline 0.9% (10 ml/kg, i.p.) or with aroyl arylhydrazone moiety in the bioreceptor recognition,
test compounds 30 min before formalin injection. After intra- contributing to reduction of the analgesic activity.9) In
plantar irritant application, the animals were immediately NSAID’s with fenamate structure, substituents on N-aryl ring
placed into a glass cylinder (20 cm diameter). The time spent which force this ring to be non-coplanar with the anthranilic
by animals licking or biting the injected paw was measured acid ring can enhance binding to the receptor.25) Because of
with a chronometer and was considered indicative of pain.
this fact we synthesized the second series of derivatives
(10m—v) which possess ortho-chlorine on 2-phenoxy moi-
ety in order to have more potent compounds. However, this
strategy not only didn’t increase their analgesic effect but

June 2006
1185
Table 3. Effect of Compounds (10b, 10d, 10f—g and 11c—j) against the Second Phase (15—30 min) in the Formalin Test
a)
Compound
ED50
M.I.b) (%)
Studied doses (mmol/kg)
10b
10d
10f
10g
11a
11b
11c
11d
11e
11f
11g
11h
11i
8.40c,d) (4.64—11.47)e)
21.67 (10.83—29.21)
44.48 (33.89—54.61)
32.15 (19.74—41.91)
24.45 (20.94—28.13)
28.28 (27.14—29.47)
42.09 (37.59—46.72)
5.49c,d) (2.04—8.38)
17.84c) (7.98—24.91)
61.54 (56.19—66.83)
10.74c,d) (7.24—13.69)
57.41 (47.19—66.47)
85.43 (65.26—107.3)
37.63 (32.07—42.80)
32.18 (25.26—37.91)
13.53 (10.90—16.09)
24.96 (15.62—32.83)
86.80ꢁ3.7
92.30ꢁ5.3
94.30ꢁ4.8
96.40ꢁ4.7
84.90ꢁ3.7
85.70ꢁ3.2
81.30ꢁ4.1
97.30ꢁ4.7
97.80ꢁ4.2
82.70ꢁ3.6
92.2ꢁ3.8
82.7ꢁ5.2
96.3ꢁ5.3
98.2ꢁ5.6
98.10ꢁ4.1
86.80ꢁ3.6
91.3ꢁ4.6
6.25, 12.5, 25, 50
12.5, 25, 50, 75
12.5, 25, 50, 100
12.5, 25, 50, 100
5, 10, 25, 50
5, 10, 25, 50
10, 25, 50, 75
5, 10, 25, 50
5, 10, 25, 50
25, 50, 75, 100
5, 10, 25, 50
25, 50, 75, 100
10, 25, 50, 100, 200
10, 25, 50, 75
15, 30, 60, 90
3.75, 7.5, 15, 30
7.5, 15, 35, 60
11j
Mefenamic
Indomethacin
Diclofenac sodium
a) The number of animals in each group was 10 animals; b) maximal inhibition; c) significant as compared to mefenamic acid group (pꢅ0.05); d) significant as compared to
diclofenac sodium group (pꢅ0.05); e) 95% confidence interval.
also decreased the activity in comparison to the first series of
compounds (Table 2). The third series of compounds having
N-phenylbenzoly moiety in their structure were more potent
than compounds 10a—v bearing 2-phenoxybenzoyl moiety
in their structure. In addition, all of them (except 11f and
11h) were more potent than mefenamic acid (pꢅ0.05) and
compounds 11d and 11g showed significant analgesic effect
in comparison to diclofenec sodium (pꢅ0.05). The most po-
tent compound in this group (11d) was 4.3-fold and 2.9-fold
more potent than mefenamic acid and diclofenac sodium re-
spectively. As shown in Table 3, the most active compounds
were also analysed in the formalin-induced pain test. They
inhibited the second phase (inflammatory pain, pꢅ0.05) but
not the first phase (neurogenic pain, pꢆ0.05) in comparison
to control. Compounds 10b, 11d and 11g were the most po-
tent compounds in this test and they showed 3.8, 5.9 and 2.9-
fold greater potency than mefenamic acid and 2.9, 4.5 and
2.3-fold greater potency than diclofenac sodium respectively
(pꢅ0.05). We can deduce from these results that the fena-
mate structure plays an important role in the analgesic prop-
erties of synthesized compounds and bioisosteric replace-
ment of NH with oxygen, has a deleterious effect on the
analgesic potency. In conclusion, several synthesized com-
pounds showed comparable or better analgesic activity than
the reference drugs.
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Acknowledgments This work was supported by grants
from Iran National Science Foundation (INSF) and National
Research Center for Medical Sciences of I. R. Iran
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