Design and synthesis of 2-phenoxynicotinic acid hydrazides as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1002/ardp.200900294

A series of 2-phenoxynicotinic acid hydrazides were synthesized and evaluated for their analgesic and anti-inflammatory activities. Several compounds having an unsubstituted phenyl/4-pyridyl or C-4 methoxy substituent on the terminal phenyl ring showed moderate to high analgesic or antiinflammatory activity in comparison to mefenamic acid as the reference drug. The compounds with highest anti-inflammatory activity were subjected to in vitro COX-1/COX-2 inhibition assays and showed moderate to good COX-1 and weak COX-2 inhibition activities.

Full text of DOI:10.1002/ardp.200900294

Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
509
Full Paper
Design and Synthesis of 2-Phenoxynicotinic Acid Hydrazides
as Anti-inflammatory and Analgesic Agents
Alireza Moradi1, Latifeh Navidpour1, Mohsen Amini1, Hamid Sadeghian2, Hooman Shadnia3,
Omidreza Firouzi4, Ramin Miri4, Seyed Esmaeil Sadat Ebrahimi1, Mohammad Abdollahi5,
Mona Haddad Zahmatkesh1, and Abbas Shafiee1
1 Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
2 Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical
Sciences, Mashhad, Iran
3 Department of Chemistry, Carleton University, Ottawa, Ont., Canada
4 Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz,
Iran
5 Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran
A series of 2-phenoxynicotinic acid hydrazides were synthesized and evaluated for their analgesic and
anti-inflammatory activities. Several compounds having an unsubstituted phenyl/4-pyridyl or C-4
methoxy substituent on the terminal phenyl ring showed moderate to high analgesic or anti-
inflammatory activity in comparison to mefenamic acid as the reference drug. The compounds
with highest anti-inflammatory activity were subjected to in vitro COX-1/COX-2 inhibition assays
and showed moderate to good COX-1 and weak COX-2 inhibition activities.
Keywords: Analgesic / Anti-inflammatory activity / 2-Phenoxynicotinic acid hydrazide
Received: November 29, 2009; Accepted: February 9, 2010
DOI 10.1002/ardp.200900294
Introduction
homeostasis and gastroprotection, whereas COX-2 is induced
by pro-inflammatory stimuli at inflammatory sites [5]. Most of
the currently available NSAIDs show significant side effects
such as gastrointestinal injury, bleeding, and nephrotoxicity
in long term usage which causes some patients to abandon
NSAID therapy [6, 7]. Therefore, the discovery of new safer
anti-inflammatory drugs represents a challenging goal for
such a research area.
Non-steroidal anti-inflammatory drugs (NSAIDs) are useful
tools in the treatment of acute and chronic inflammation
and pain, particularly for different types of arthritis [1, 2].
The common mechanism of NSAIDs involves the nonselec-
tive inhibition of cyclooxygenases (COXs) thereby preventing
the biosynthesis of prostaglandins (PGs) which are the
important lipid mediators of inflammation as well as numer-
ous homeostatic physiological functions [3]. As it is now well
appreciated, COX exists in two isoforms, namely COX-1 and
COX-2 [4], while the existence of a third isoform (COX-3) is still
into debate. In general terms, COX-1 is the constitutive iso-
form providing normal production of PGs having a role in
Regio-isomeric pyridines have been explored previously as
replacement of the central core and were found to be useful
as analgesic as well as anti-inflammatory agents and for
treating Alzheimer’s disease [8, 9]. The pyridine nucleus is
present in niflumic acid 1 and flunixin 2, two traditional
NSAIDs belonging to the class of fenamates (Fig. 1). This class
includes derivatives of N-phenyl (or heteroaryl) anthranilic
acid that are employed for their analgesic, anti-inflamma-
tory, and anti-pyretic properties. Unlike most other NSAIDs,
the fenamates appear also to compete with prostaglandins
for binding at the prostaglandin receptor site and, thus,
potentially antagonize the physiopathological effects of pros-
taglandins that have already been formed. Fenamates are still
Correspondence: Abbas Shafiee, Department of Medicinal Chemistry,
Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran 14176, Iran.
E-mail: ashafiee@ams.ac.ir
Fax: þ98 21 664-61178.
Abbreviations: gastrointestinal (GI); N-acylhydrazones (NAH)
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

510
A. Moradi et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Figure 1. Representative examples of pyridine derivatives of
fenamates.
endowed with most of the adverse effects induced by NSAIDs,
particularly gastrointestinal (GI) bleeding, ulceration, and
perforation. Unlike aspirin and most NSAIDs, the fenamates
do not affect platelet aggregation and bleeding time [10].
GI damage from NSAIDs is generally attributed to two
factors; local irritation by the direct contact of the carboxylic
acid (–COOH) moiety of NSAIDs with GI mucosal cells (topical
effect) and decreased tissue prostaglandin production in
tissues which undermine the physiological role of cytopro-
tective prostaglandins in maintaining GI health and homeo-
stasis [11, 12].
Figure 2. Representative examples of amide or hydrazone deriva-
tives (3–8) and designed compounds 9.
Synthetic approaches based upon chemical modification of
NSAIDs have been taken with the aim of improving the safety
profile and, in turn, the therapeutic window of these NSAIDs.
Several studies have described the derivatization of the car-
boxylic function with amide or N-acylarylhydrazone having
less acidic amide hydrogen as well as the capacity of stabiliz-
ing free radicals [13–17]. Some evidences suggest that the
hydrazone moiety possesses a pharmacophoric character for
the inhibition of cyclooxygenase [14]. N-Acylhydrazones
(NAH) have been widely described as potent anti-inflamma-
tory, antinociceptive, and anti-platelet compounds, due to
their ability to mimic the bis-allylic moiety of unsaturated
fatty acids and amides, for example, arachidonic acid (AA),
precursor of the eicosanoid biosynthesis, involved in the
endocannabinoid system. This can be rationalized by the
relative acidity of the amide hydrogen of the NAH group
as well as its capacity of stabilizing free radicals [13].
In this respect, structure-activity relationship (SAR) studies
exhibited the potent anti-inflammatory activities for 2-ani-
Moreover, substitution of the carboxylic acid functionality
of several fenamates with heterocycles containing an acidic
functionality or hydrazone-type moieties provided dual
inhibitors of COX and 5-lipooxygenase [10]. Leukotrienes
produced through the 5-LO enzyme pathway, may also con-
tribute to both inflammation and NSAIDs-induced side-
effects. For these reason, compounds that are dual inhibitors
of both COX and 5-LO are being studied as potential analgesic
and anti-inflammatory agents with an improved safety pro-
file in comparison to NSAIDs [16].
Considering the above results and as a part of our ongoing
program to design analgesic and anti-inflammatory agents [17],
herein, we describe the design, synthesis, and biological evalu-
ation of a novel diverse group of N-acylarylhydrazone deriva-
tives of 2-phenoxynicotinic acid with different substituents on
the terminal phenyl (or heteroayl) ring (Fig. 2).
lino (or aryloxy) nicotinic acid amides 3, 4 (Fig. 2) [18]. Several Results and discussion
studies also showed that the presence of a N-heteroaromatic
ring having a lesser hydrophobic character in the N-acyl
group of these compounds 5–8 improves the analgesic
activity [15].
Chemistry
The synthetic reactions leading to the substituted arylidene-
2-phenoxynicotinic acid hydrazides 9a–u are outlined in
Scheme 1.
Although the exerted changes lead to nonionized deriva-
tives which resulted in compounds that selectively inhibit
COX-2 but not COX-1. The adverse reactions have been attrib-
uted, at least in part, to COX-1 inhibition occurring with long-
term exposure [19].
2-Phenoxynicotinic acid hydrazides 13 are key intermedi-
ates for the production of the title compounds 9. The starting
materials, 2-phenoxynicotinic acids 11, were prepared
according to the reported method in the literature [20] by
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Nicotinohydrazides: analgesic, anti-inflammatory agents
511
Scheme 1. Synthesis of designed compounds 9a–u.
the fusion of sodium 2-chloronicotinate and sodium phen-
oxide derivatives. Esterification of 11 using a general acid-
catalyzed method afforded the ethyl esters 12. Treatment of
12 with hydrazine hydrate in absolute ethanol generated the
required hydrazides 13 [21].
75.8 kcal/mol, while the energy found for the corresponding
(Z)-stereoisomer (D) was 82.2 kcal/mol (Fig. 3).
In order to obtain additional evidence about the relative
configurations, this assignment was further confirmed by a
Differential-NOE experiment.
The title hydrazone compounds 9 were prepared in very
good yields by the condensation of hydrazides 13 with differ-
ent substituted aromatic aldehydes or acetophenones in
absolute ethanol.
The next step of this work was to determine the relative
configuration of the imine double bond of arylidenenicotinic
acid derivatives 9, in order to assure the stereoisomeric ratio
essential for the complete understanding of the biological
1
effect. The careful analysis of the H-NMR of 9 allowed us to
detect the presence of one singlet signal related to the imine
hydrogen at 8.2 to 8.5 ppm except for 9j and 9s which
appeared at 7.6 ppm.
According to the literature, the hydrazone may exist as Z/E
–
geometrical isomers about the C N double bond and cis/trans
–
amide conformers.
To investigate the possible influence of the conformational
restriction in the pharmacophoric N-acylhydrazone side chain,
promoted by a possible intramolecular H-bond formation,
conformational analysis studies using AM1 Hamiltonian were
carried out in MOE 2008.10 program using compound 9c. This
study indicated that the compound bearing a S-cis like con-
formation (A) has lower energy (75.8 kcal/mol) than the iso-
meric S-trans like conformation (B; 80.2 kcal/mol; Fig. 3).
Systematic conformational analysis was then carried out to
determine the heat of the formation value of the more stable
stereoisomeric form. For derivative 9c, the obtained value for
the more stable conformer of the (E)-stereoisomer (C) was
Figure 3. Different possible conformations for 9c and 9j.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

512
A. Moradi et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Table 1. Physical properties and analgesic activities of compounds 9a-u.
O
N
Ar
NH
R'
N
O
R
Compound
R
R0
Ar
Yield
(%)
M. p.
Constriction No§
Inhibition
(%)
Relative
activity#
(8C)
(mean ꢀ S.E.)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
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
phenyl
4-methylphenyl
4-chlorophenyl
4-dimethylaminophenyl
4-nitrophenyl
2,4-dimethoxyphenyl
2-thienyl
4-pyridyl
3-pyridyl
2-pyridyl
Phenyl
4-methylphenyl
4-chlorophenyl
4-methoxyphenyl
4-nitrophenyl
2-thienyl
4-pyridyl
3-pyridyl
2-pyridyl
4-chlorophenyl
4-methoxyphenyl
68
78
64
91
70
83
94
88
91
82
65
69
68
71
68
91
91
89
81
52
65
169–171
196–198
201–203
200–202
259–261
203–205
189–191
209–211
178–180
188–190
183–185
210–212
179–181
168–170
209–211
183–185
228–230
185–187
196–198
208–210
159–161
21.5 ꢀ 2.01
24.5 ꢀ 1.56
33.33 ꢀ 3.6
30.83 ꢀ 3.29
34 ꢀ 3.68
47.78
40.49
19.04
25.12
17.42
59.12
29.97
66.41
44.55
2.08
58.29
38.47
17.00
35.63
–3.64
31.58
48.19
43.72
9.37
0.94ꢁꢁ
0.79
0.37
0.49
0.34
16.83 ꢀ 2.02
28.83 ꢀ 1.96
13.83 ꢀ 2.27
22.83 ꢀ 3.39
40 ꢀ 1.88
1.16ꢁꢁꢁ
0.59
1.30ꢁꢁꢁ
0.87ꢁ
0.07
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
17.17 ꢀ 2.76
25.33 ꢀ 4.71
34.17 ꢀ 5.49
26.5 ꢀ 2.45
42.67 ꢀ 3.07
28.17 ꢀ 2.93
21.33 ꢀ 2.4
23.17 ꢀ 3.49
37.33 ꢀ 1.5
44.33 ꢀ 4.88
33.5 ꢀ 4.96
41.17 ꢀ 3.94
20.17 ꢀ 3.6
1.14ꢁꢁꢁ
0.75
0.33
0.70
–0.07
0.62
0.94ꢁꢁ
0.86ꢁ
0.34
9p
9q
9r
9s
9t
9u
H
H
CH3
CH3
ꢂ7.68
18.63
–
ꢂ0.15
H
0.37
–
Control
Mefenamic acid
51.01
1.00ꢁꢁ
The compounds were administered at the dose of 31 mM/kg; analgesic activity relative to mefenamic acid; , ꢁꢁ, and
from the control group P < 0.05, P < 0.01, and P < 0.001, respectively.
§
#
ꢁ
ꢁꢁꢁ
differed
More specifically, the assigned E-stereochemistry of com-
pound 9c is consistent with the observed enhancement of the
imine hydrogen when the ortho-hydrogens of the phenoxy
moiety (Ha) were irradiated and the absence of any enhance-
ment for the ortho-hydrogens of the 4-chlorophenyl group (Hb)
(C, Fig. 3). On the contrary, for compound 9j, irradiation of
the ortho-hydrogens of the phenoxy group (Hc) resulted in
enhancement of the pyridyl-hydrogen (Hd) and no enhance-
ment was observed for the imine hydrogen. Therefore, these
results are indicative of the Z-configuration (F) for this com-
pound. It seems that in 2-pyridyl derivative 9j the (Z)-confor-
mation (F) is favored over the (E)-conformation (E), since the
imino-2-pyridyl ring could be co-planar with the carbonyl
imino moiety by a possible additional intra-molecular hydro-
gen-bond formation with the acidic-amide hydrogen (Fig. 3).
This observation is in good agreement with the N-H chemical
shift of this compound which appeared more deshielded
(15.34 ppm) than the other E-analogs (10.69 to 11.06 ppm).
Pharmacology
Analgesic activity
The analgesic activities of the compounds were studied by
using the acetic-acid-induced abdominal constriction test
(writhing test) [22]. Qualitative structure-activity relationship
data, acquired using the analgesic assay, showed that most of
the arylidene-2-phenoxynicotinic acid hydrazides were able to
reduce the AcOH-induced constrictions with a moderate to
good activity range (17–66% inhibition) in comparison with
mefenamic acid as the reference drug (51% inhibition) (Table 1).
The analysis of these results allowed us to state that most of
the aldehyde-condensed title compounds (9a–s) revealed
respectable analgesic activities, while in acetophenone-
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Nicotinohydrazides: analgesic, anti-inflammatory agents
513
condensed compounds (9t–u), substitution of H with a
methyl group resulted in less to relatively inactive com-
pounds. It seems that there is a limited space available (steric
hindrance) for a methyl substituent attached to the imino
group of the hydrazone moiety on the target binding site.
Moreover, introduction of a small lipophilic substituent
such as chloro on the phenoxy moiety resulted in a compound
9k with a slightly improved analgesic activity (58% inhibition)
relative to the unsubstituted derivative 9a (47% inhibition).
The substituents of the terminal phenyl moiety, attached
to the imine function of the nicotinic acid hydrazides 9 were
rationally elected in order to acquire information about the
influence of electronic and physico-chemical parameters on
the pharmacological activity.
possible hydrogen-bond formation between the nitrogen of
the 4-pyridyl or C-4 methoxy substituent on the phenyl ring
with the amino acids of the target binding site.
Anti-inflammatory activity
In-vivo pharmacological evaluation of 9a–u was carried out to
assess their potential anti-inflammatory activity. Qualitative
structure-activity relationship data, acquired using the car-
rageenan-induced rat paw edema assay [23], showed that this
group of arylidene-2-phenoxynicotinic acid hydrazides
exhibit anti-inflammatory activity with a moderate to good
activity range (20–61% inhibition) in comparison with mefe-
namic acid as the reference drug (47% inhibition) at 2 h
(Table 2).
In general, most of the substituents resulted in less active
compounds (except for 9f and 9h), but among different para-
substituted ones (9b, 9c, and 9e), electron-donating substitu-
ents was slightly preferred over electron-withdrawing ones.
Finally, among substituted analogues, 2,4-dimethoxy-
phenyl 9f and 4-pyridyl derivatives 9h exhibited the most
potent analgesic activity with 59% and 66% inhibition,
respectively, relative to the reference drug mefenamic acid
with 51% inhibition. This phenomenon can be explained by a
As shown in Table 2, the presence of any substituent,
electron-donating or electron-withdrawing, or substitution
of the phenyl moiety with any heterocycles (thienyl or pyr-
idyl) produced compounds with a noticeable reduction in
activity. Moreover, a decrease in anti-inflammatory activity
was observed with introduction of a chloro substituent in the
ortho-position of the phenoxy moiety. Among these com-
pounds, benzylidene-2-phenoxynicotinic acid hydrazide 9a,
which was unsubstituted, exhibited the highest anti-
Table 2. Anti-inflammatory activities and IC50 values for COX-1 and COX-2 inhibition by 9a-u.
Compound
Thickness variation (in mm)
AI activity3 (in %)
IC50 (in mM)&
at 2 h
at 4 h
Inhibition at 2 h#
Inhibition at 4 h
COX-1
COX-2
Control
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
2.45 ꢀ 0.19
0.95 ꢀ 0.15
1.54 ꢀ 0.21
1.55 ꢀ 0.30
1.54 ꢀ 0.12
1.27 ꢀ 0.18
2.21 ꢀ 0.21
1.56 ꢀ 0.16
1.43 ꢀ 0.17
1.94 ꢀ 0.15
1.81 ꢀ 0.23
1.65 ꢀ 0.36
1.73 ꢀ 0.29
1.42 ꢀ 0.17
1.24 ꢀ 0.18
1.68 ꢀ 0.20
1.82 ꢀ 0.17
1.12 ꢀ 0.20
1.54 ꢀ 0.33
1.77 ꢀ 0.19
1.54 ꢀ 0.09
1.18 ꢀ 0.21
1.30 ꢀ 0.04
n. t.
3.58 ꢀ 0.16
1.90 ꢀ 0.28
2.73 ꢀ 0.10
2.10 ꢀ 0.19
2.85 ꢀ 0.42
2.22 ꢀ 0.1
2.78 ꢀ 0.17
3.09 ꢀ 0.19
2.50 ꢀ 0.22
2.94 ꢀ 0.25
3.21 ꢀ 0.11
2.90 ꢀ 0.22
2.18 ꢀ 0.09
2.83 ꢀ 0.22
2.11 ꢀ 0.07
2.53 ꢀ 0.43
3.08 ꢀ 0.23
1.87 ꢀ 0.17
3.13 ꢀ 0.38
2.95 ꢀ 0.2
2.26 ꢀ 0.22
2.61 ꢀ 0.29
2.01 ꢀ 0.14
n. t.
0
0
–
29.8 ꢀ 15.8
n. t.
–
152.2 ꢀ 0.07
n. t.
61.27ꢁꢁꢁ
32.84
36.68
37.09
48.16ꢁ
9.80
46.93ꢁꢁꢁ
23.94
41.29ꢁꢁ
20.54
37.99ꢁ
22.43
13.84
30.13
18.08
10.33
19.20
39.23ꢁ
21.09
41.07ꢁꢁ
29.46
14.06
47.77ꢁꢁꢁ
12.72
17.60
37.05ꢁ
27.12
43.96ꢁꢁ
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
36.11
41.50
20.59
26.12
32.60
29.25
41.83
49.35ꢁ
31.37
25.49
54.25ꢁꢁ
37.01
27.75
37.25
51.96ꢁꢁ
47.03ꢁ
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
21.1 ꢀ 16.9
n. t.
82.0 ꢀ 55.5
n. t.
n. t.
n. t.
n. t.
9p
9q
9r
9s
9t
9u
68.5 ꢀ 46.7
>200
n. t.
n. t.
n. t.
n. t.
n. t.
n. t.
98.8 ꢀ 27.1
22.8 ꢀ 8.6
6.4 (2.0–9.9)$
12.4 ꢀ 1.8
Mefenamic acid
Celecoxib
–
2.7 ꢀ 0.5
§ Theꢁcꢁoꢁmpounds were administered at the dose of 31 mM/kg; # percentage of inhibition obtained by comparison with the vehicle; ꢁ, ꢁꢁ
,
&
and
different experiments; value was reported in [25]; n. t.: not tested.
differed from control group P < 0.05, P < 0.01 and P < 0.001, respectively; values represent the mean ꢀ S.D. of three
$
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

514
A. Moradi et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
inflammatory activity (61% inhibition) relative to mefenamic
acid.
further modification to achieve compounds with more
potent analgesic and anti-inflammatory activities.
Finally, among chlorophenoxy and ketone-condensed
analogues, possible hydrogen-bond formation between the Experimental
nitrogen of the 4-pyridyl or C-4 methoxy substituent on
the phenyl ring with the amino acids of the target binding
site explain the potent anti-inflammatory activity of 4-
methoxyphenyl 9n and 9u and 4-pyridyl derivatives 9q with
49, 51, and 54% inhibition of the activity relative to mefe-
namic acid (51% inhibition).
Chemistry
Melting points were determined with a Reichert-Jung hot-stage
microscope (Reichert-Jung, Germany) and are uncorrected.
Infrared spectra were recorded on a Nicolet Magna 550-FT spec-
trometer (Nicolet, Madison, WI, USA). 1H-NMR (500 MHz) spectra
were measured on a Brucker FT-500 MHz spectrometer (Bruker
Bioscience, USA) in CDCl3 or DMSO-d6 with TMS as the internal
standard, where J (coupling constant) values are estimated in
Hertz. Spin multiples are given as s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), m (multiplet), and
b (broad). Mass spectra were obtained with a Finnigan Mat TSQ-70
spectrometer (Thermo-Electron, Germany). Elemental micro-
analyses were carried out with a Perkin-Elmer 240-C apparatus
(Perkin-Elmer, Beaconsfield, UK) and were within ꢀ0.4% of the
theoretical values for C, H, and N. All solvents and reagents were
purchased from the Fluka, Aldrich (UK), or Merck Chemical
Company (Germany). Male NMRI mice and male Wistar rats,
used in the analgesic and anti-inflammatory screens, respect-
ively, were purchased from Pasteur Institute (Karaj, Iran), and
experiments were carried out using protocols approved by the
ethics committee of Tehran University of Medical Sciences.
In-vitro cyclooxygenase inhibition assay
Based on finding these preclinical results, in-vitro assays were
carried out to investigate the possible mechanism-of-action
by which these compounds exhibit potential analgesic and
anti-inflammatory activities. The compounds which exhib-
ited significant anti-inflammatory activities comparable to
that of the mefenamic acid were subjected to in-vitro COX-1/
COX-2 enzyme inhibition assay.
The ability of compounds 9a, 9n, 9q and 9u to inhibit COX-
1 and/or COX-2 was determined by in-vitro human whole-
blood assay (Table 2) [24, 25]. In-vitro enzyme inhibition stud-
ies for the latter exhibited weak COX-2 inhibition (82 mM to
higher than 200 mM range) with moderately weak to good
COX-1 inhibition in the 21.1 to 68.5 mM range. In this series,
General procedure for preparation of ethyl 2-
phenoxynicotinates 12a–b
4-methoxybenzylidene-2-(2-chlorophenoxy)nicotinic
acid
2-Phenoxynicotinic acid 11 (10 mmol) and 1 mL H2SO4 in
absolute ethanol (30 mL) were refluxed for 8 h. After cooling,
the reaction mixture was concentrated under reduced pressure.
Aqueous sodium carbonate solution (20 mL, 10%, w/v) was added
to the residue and the ester was extracted with chloroform
(2 ꢃ 15 mL). The solvent was dried over anhydrous Na2SO4, fil-
tered, and evaporated. The compound was further purified by
column chromatography using silica gel (60–120 mesh; EtOAc/
hexane, 2:8) to afford 12.
hydrazide 9n was the most potent COX-1 inhibitor (COX-1
IC50 ¼ 21.1 mM) relative to reference drug mefenamic acid
(COX-1 IC50 ¼ 6.4 mM) [24].
Conclusion
Various substituted arylidene-2-phenoxynicotinic acid hydra-
zide derivatives were synthesized and screened for their
potential analgesic and anti-inflammatory activities. Most
compounds exhibited significant analgesic and anti-inflam-
matory activities. In acetic-acid-induced abdominal constric-
tion test, among substituted analogues, 2,4-dimethoxy-
phenyl 9k and 4-pyridyl derivatives 9h exhibited the most
potent analgesic activity. Moreover, compounds 9a and 9q
exhibited the most prominent and consistent anti-inflamma-
tory activity. The likely hydrogen-bond formation between
the nitrogen of the 4-pyridyl or C-4 methoxy substituent on
the phenyl ring with amino acids of the target-binding site
may explain the potent analgesic and anti-inflammatory
activity of these compounds. As shown in the results, the
most active anti-inflammatory compounds showed moderate
to good COX-1 inhibition. Since these compounds don’t have
an acidic moiety, they have a good framework as leads for
Ethyl 2-phenoxynicotinate 12a
Yield: 68%; m. p.: 48–508C; IR (KBr): 1731 (C O) cm
ꢂ1
;
1H-NMR
–
–
(CDCl3) d: 8.27–8.25 (m, 2H, H4,6, Pyr), 7.41 (t, J ¼ 8.0 Hz, 2H, H3,5
,
,
Ph-O), 7.21 (t, J ¼ 8.0 Hz, 1H, H4, Ph-O), 7.16 (d, J ¼ 8.0 Hz, 2H, H2,6
Ph-O), 7.05 (dd, J ¼ 7.5, 4.5 Hz, 1H, H5, Pyr), 4.40 (q, J ¼ 7.0 Hz, 2H,
CH2), 1.39 (t, J ¼ 7.0 Hz, 3H, CH3); MS m/z (%): 243 [Mþ] (29), 198 (30),
171 (100), 94 (21), 77 (17). Anal. calcd. for C14H13NO3: C, 69.12; H,
5.39; N, 5.76. Found: C, 69.28; H, 5.27; N, 5.58.
Ethyl 2-(2-chlorophenoxy)nicotinate 12b
Yield: 70%; m. p.: 58–598C; IR (KBr): 1710 (C O) cmꢂ1; 1H-NMR (CDCl3)
–
–
d: 8.29 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.23 (dd, J ¼ 5.0, 2.0 Hz,
1H, H6, Pyr), 7.47 (dd, J ¼ 8.0, 1.5 Hz, H3, Ph-O), 7.35–7.31 (m, 1H, H5,
Ph-O), 7.26 (dd, J ¼ 8.5, 2.0 Hz, 1H, H6, Ph-O), 7.21–7.18 (m, 1H, H4,
Ph-O), 7.07 (dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr), 4.43 (q, J ¼ 7.0 Hz, 2H,
CH2), 1.40 (t, J ¼ 7.0 Hz, 3H, CH3); MS m/z (%): 279 [Mþ þ 2] (5), 277
[Mþ] (15), 242 (100), 214 (100), 169 (45). Anal. calcd. for C14H12ClNO3:
C, 60.55; H, 4.36; N, 5.04. Found: C, 60.38; H, 4.47; N, 5.20.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Nicotinohydrazides: analgesic, anti-inflammatory agents
515
Ph-O), 7.35 (t, J ¼ 7.5 Hz, 1H, H4, Ph-O), 7.27–7.19 (m, 5H, H3,5, Ph &
Ph-O, H5, Pyr), 2.38 (s, 3H, CH3); MS m/z (%): 331 [Mþ] (10), 214 (40), 198
(100), 93 (88), 77 (36). Anal. calcd. for C20H17N3O2: C, 72.49; H, 5.17; N,
12.68. Found: C, 72.37; H, 5.31; N, 12.54.
General procedure for preparation of 2-phenoxynicotinic
acid hydrazides 13a–b
Ethyl 2-phenoxynicotinate 12 (1 mmol) and hydrazine hydrate
(5 mmol) were refluxed in ethanol for 2.5 h. Then, reaction
mixture was concentrated in vacuo and left overnight. The result-
ing crystals were filtered and washed with cooled ethanol to give
pure acid hydrazides 13.
(4-Chlorobenzylidene)-2-phenoxynicotinic acid hydrazide
9c
Yield: 64%; m. p.: 201–2038C (ethanol); IR (KBr): 3298 (NH), 1675
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 10.88 (bs, 1H, NH), 8.74 (dd,
–
2-Phenoxynicotinic acid hydrazide 13a
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.27 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6,
–
–
Yield: 92%; m. p.: 201–2038C; IR (KBr): 3320, 3272 (NH), 1642 (C O)
cmꢂ1; 1H-NMR (CDCl3) d: 9.03 (bs, 1H, NH), 8.62 (dd, J ¼ 7.5, 2.0 Hz,
1H, H4, Pyr), 8.22 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6, Pyr), 7.45 (t, J ¼ 7.5 Hz,
2H, H3,5, Ph-O), 7.30 (t, J ¼ 7.5 Hz, 1H, H4, Ph-O), 7.18–7.13 (m,
3H, H2,6, Ph-O & H5, Pyr), 4.23 (bs, 2H, NH2); MS m/z (%): 229 [Mþ]
(3), 150 (78), 122 (100), 94 (43). Anal. calcd. for C12H11N3O2: C, 62.87;
H, 4.84; N, 18.33. Found: C, 62.73; H, 4.71; N, 18.45.
–
–
Pyr), 8.22 (s, 1H, CH–), 7.73 (d, J ¼ 8.0 Hz, 2H, H2,6, Ph), 7.51 (t,
J ¼ 8.0 Hz, 2H, H3,5, Ph-O), 7.38 (d, J ¼ 8.0 Hz, 2H, H3,5, Ph), 7.35
(t, J ¼ 8.0 Hz, 1H, H4, Ph-O), 7.24–7.21 (m, 3H, H2,6, Ph-O & H5,
Pyr); MS m/z (%): 353 [Mþ þ 2] (10), 351 [Mþ] (30), 214 (92), 198
(100), 115 (98), 94 (97), 77 (87). Anal. calcd. for C19H14ClN3O2: C,
64.87; H, 4.01; N, 11.94. Found: C, 64.77; H, 4.15; N, 12.08.
(4-(Dimethylamino)benzylidene)-2-phenoxynicotinic acid
hydrazide 9d
2-(2-Chlorophenoxy)nicotinic acid hydrazide 13b
Yield: 94%; m. p.: 217–2198C; IR (KBr): 3307, 3190 (NH), 1646
–
2.0 Hz, 1H, H6, Pyr), 8.09 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 7.56
(dd, J ¼ 8.0, 1.5 Hz, 1H, H3, Ph-O), 7.44–7.38 (m, 2H, H5,6, Ph-O),
7.31–7.28 (m, 1H, H4, Ph-O), 7.23 (dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr),
4.63 (bs, 2H, NH2); MS m/z (%): 265 [Mþ þ 2] (13), 263 [Mþ] (40), 232
(100), 169 (30). Anal. calcd. for C12H10ClN3O2: C, 54.66; H, 3.82; N,
15.94. Found: C, 54.84; H, 3.70; N, 16.11.
(C O) cmꢂ1; 1H-NMR (CDCl3) d: 9.53 (bs, 1H, NH), 8.14 (dd, J ¼ 5.0,
–
Yield: 91%; m. p.: 200–2028C (ethanol); IR (KBr): 3308 (NH), 1659
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 10.69 (bs, 1H, NH), 8.75 (dd,
–
J ¼ 8.0, 2.0 Hz, 1H, H4, Pyr), 8.24 (dd, J ¼ 5.0, 2.0 Hz, 1H, H6,
Pyr), 8.06 (s, 1H, ¼CH–), 7.67 (d, J ¼ 8.0 Hz, 2H, H2,6, Ph), 7.51 (t,
J ¼ 7.5 Hz, 2H, H3,5, Ph-O), 7.35 (t, J ¼ 7.5 Hz, 1H, H4, Ph-O), 7.27–
7.24 (m, 2H, H2,6, Ph-O), 7.20 (dd, J ¼ 8.0, 5.0 Hz, 1H, H5, Pyr), 6.69
(d, J ¼ 8.0 Hz, 2H, H3,5, Ph), 3.03 (s, 6H, -N(CH3)2); MS m/z (%): 360
[Mþ] (17), 198 (12), 162 (7), 146 (100). Anal. calcd. for C21H20N4O2:
C, 69.98; H, 5.59; N, 15.55. Found: C, 69.89; H, 5.43; N, 15.67.
General procedure for synthesis of arylidene-2-
phenoxynicotinic acid hydrazides 9a–u
(4-Nitrobenzylidene)-2-phenoxynicotinic acid hydrazide 9e
Yield: 70%; m. p.: 259–2618C (ethanol); IR (KBr): 3328 (NH), 1712
The synthesis of final hydrazones 9a–u was carried out by the
reaction of hydrazide 13 and the corresponding aldehydes or
acetophenones in absolute ethanol. A mixture of hydrazide 13
(1.5 mmol) and the corresponding aldehyde or acetophenone
(1.5 mmol) in absolute ethanol (10 mL) was stirred at room
temperature or refluxed in case of acetophenones for 3 to
24 h in the presence of hydrochloric acid (2 drops) as the catalyst.
The completion of the reaction was monitored by TLC. The
reaction mixture was concentrated under reduced pressure
and was neutralized with a 10% aqueous solution of sodium
bicarbonate. The resulting precipitate was filtered, washed with
water, and crystallized from a suitable solvent to give 9.
ꢂ1
1H-NMR (CDCl3) d: 11.06 (bs, 1H, NH), 8.74 (dd,
–
(C O) cm
–
;
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.41 (s, 1H, CH–), 8.29 (dd,
–
J ¼ 5.0, 2.0 Hz, 1H, H6, Pyr), 8.26 (d, J ¼ 8.5 Hz, 2H, H3,5, Ph),
7.95 (d, J ¼ 8.5 Hz, 2H, H2,6, Ph), 7.52 (t, J ¼ 7.5 Hz, 2H, H3,5, Ph-
O), 7.36 (t, J ¼ 7.5 Hz, 1H, H4, Ph-O), 7.25–7.22 (m, 3H, H2,6, Ph-O
& H5, Pyr); MS m/z (%): 362 [Mþ] (9), 213 (100), 170 (34), 77 (21).
Anal. calcd. for C19H14N4O4: C, 62.98; H, 3.89; N, 15.46. Found: C,
62.81; H, 3.97; N, 15.30.
(2,4-Dimethoxybenzylidene)-2-phenoxynicotinic acid
hydrazide 9f
Benzylidene-2-phenoxynicotinic acid hydrazide 9a
Yield: 68%; m. p.: 169–1718C (ethanol); IR (KBr): 3325 (NH), 1671
Yield: 83%; m. p.: 203–2058C (ethanol); IR (KBr): 3170 (NH), 1654
ꢂ1
1H-NMR (CDCl3) d: 10.70 (bs, 1H, NH), 8.75 (dd,
–
(C O) cm
–
;
ꢂ1
;
1H-NMR (CDCl3) d: 10.86 (bs, 1H, NH), 8.75 (dd,
–
(C O) cm
–
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.46 (s, 1H, CH–), 8.24 (dd,
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.26 (dd, J ¼ 5.0, 2.0 Hz, 1H, H6,
J ¼ 5.0, 2.0 Hz, 1H, H6, Pyr), 8.13 (d, J ¼ 8.5 Hz, 1H, H6, Ph),
7.51 (t, J ¼ 7.5 Hz, 2H, H3,5, Ph-O), 7.35 (t, J ¼ 7.5 Hz, 1H, H4,
Ph-O), 7.25 (t, J ¼ 7.5 Hz, 2H, H2,6, Ph-O), 7.19 (dd, J ¼ 7.5, 5 Hz,
1H, H5, Pyr), 6.55 (dd, J ¼ 8.5, 2.5 Hz, 1H, H5, Ph), 6.42 (d,
J ¼ 2.5 Hz, 1H, H3, Ph), 3.85 (s, 3H, OCH3), 3.83 (s, 3H, OCH3);
MS m/z (%): 377 [Mþ] (9), 198 (51), 163 (30), 93 (100). Anal. calcd.
for C21H19N3O4: C, 66.83; H, 5.07; N, 11.13. Found: C, 66.64; H,
5.17; N, 10.98.
–
Pyr), 8.24 (s, 1H, CH–), 7.82–7.79 (m, 2H, H2,6, Ph), 7.52 (t,
–
J ¼ 7.5 Hz, 2H, H3,5, Ph-O), 7.42–7.41 (m, 3H, H3,4,5, Ph), 7.35 (t,
J ¼ 7.5 Hz, 1H, H4, Ph-O), 7.24 (d, J ¼ 7.5 Hz, 2H, H2,6, Ph-O), 7.21
(dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr); MS m/z (%): 317 [Mþ] (9), 198 (65),
170 (100), 77 (14). Anal. calcd. for C19H15N3O2: C, 71.91; H, 4.76; N,
13.24. Found: C, 71.78; H, 4.90; N, 13.37.
(4-Methylbenzylidene)-2-phenoxynicotinic acid hydrazide
9b
2-Phenoxy-N0-(thiophen-2-ylmethylidene)nicotinic acid
hydrazide 9g
Yield: 78%; m. p.: 196–1988C (ethanol); IR (KBr): 3298 (NH), 1654
(C O) cmꢂ1; 1H-NMR (CDCl3) d: 10.82 (bs, 1H, NH), 8.74 (dd, J ¼ 7.5,
–
–
Yield: 94%; m. p.: 189–1918C (ethyl acetate); IR (KBr): 3317 (NH),
1
1689 (C O) cmꢂ1; H-NMR (CDCl3) d: 10.80 (bs, 1H, NH), 8.74 (s,
2.0 Hz, 1H, H4, Pyr), 8.25 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6, Pyr), 8.18 (s, 1H,
–
–
–
–
CH–), 7.69 (d, J ¼ 8.0 Hz, 2H, H2,6, Ph), 7.51 (t, J ¼ 7.5 Hz, 2H, H3,5
,
–
1H, CH–), 8.70 (dd, J ¼ 7.5, 2.0 Hz, 1H, H , Pyr), 8.25 (dd, J ¼ 5.0,
–
4
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

516
A. Moradi et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
2.0 Hz, 1H, H6, Pyr), 7.50 (t, J ¼ 8.0 Hz, 2H, H3,5, Ph-O), 7.42 (d,
J ¼ 5.0 Hz, 1H, H5, thiophene), 7.35–7.32 (m, 2H, H4, Ph-O & H3,
thiophene), 7.23–7.20 (m, 3H, H2,6, Ph-O & H5, Pyr), 7.06 (dd,
J ¼ 5.0, 3.5 Hz, 1H, H4, thiophene); MS m/z (%): 323 [Mþ] (6),
213 (100), 198 (34), 77 (11). Anal. calcd. for C17H13N3O2S: C,
63.14; H, 4.05; N, 12.99. Found: C, 63.01; H, 4.20; N, 13.18.
(4-Methylbenzylidene)-2-(2-chlorophenoxy)nicotinic acid
hydrazide 9l
Yield: 69%; m. p.: 210–2128C (ethanol); IR (KBr): 3320 (NH), 1685
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 10.80 (bs, 1H, NH), 8.72 (dd,
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.22 (dd, J ¼ 5.0, 2.0 Hz, 1H, H6,
–
Pyr), 8.19 (s, 1H, CH–), 7.70 (d, J ¼ 8.0 Hz, 2H, H2,6, Ph), 7.56 (dd,
–
J ¼ 8.0, 1.5 Hz, 1H, H3, Ph-O), 7.43–7.40 (m, 1H, H5, Ph-O), 7.36
(dd, J ¼ 8.0, 1.5 Hz, 1H, H6, Ph-O), 7.32–7.28 (m, 1H, H4, Ph-O),
7.24–7.21 (m, 3H, H3,5, Ph & H5, Pyr); MS m/z (%): 367 [Mþ þ 2] (6),
365 [Mþ] (18), 232 (63), 213 (100), 127 (25), 122 (23). Anal. calcd.
for C20H16ClN3O2: C, 65.67; H, 4.41; N, 11.49. Found: C, 65.55; H,
4.30; N, 11.64.
2-Phenoxy-N0-(pyridin-4-ylmethylidene)nicotinic acid
hydrazide 9h
Yield: 89%; m. p.: 209–2118C (ethanol/hexane); IR (KBr): 3308
ꢂ1
–
–
(NH), 1690 (C O) cm
;
1H-NMR (CDCl3) d: 11.04 (bs, 1H,
NH), 8.73 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.67 (d,
–
0
0
J ¼ 5.0 Hz, 2H, H2 ,6 , Pyr), 8.30 (s, 1H, CH–), 8.28 (dd,
–
(4-Chlorobenzylidene)-2-(2-chlorophenoxy)nicotinic acid
hydrazide 9m
J ¼ 5.0, 2.0 Hz, 1H, H6, Pyr), 7.64 (d, J ¼ 5.0 Hz, 2H, H3’,5’,
Pyr), 7.51 (t, J ¼ 8.0 Hz, 2H, H3,5, Ph-O), 7.36 (t, J ¼ 8.0 Hz,
1H, H4, Ph-O), 7.25–7.21 (m, 3H, H5, Pyr & H2,6, Ph-O); MS m/z
(%): 318 [Mþ] (5), 198 (100), 115 (18), 94 (28). Anal. calcd.
for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: C, 68.04;
H, 4.31; N, 17.71.
Yield: 68%; m. p.: 179–1818C (ethanol); IR (KBr): 3324 (NH), 1693
(C O) cmꢂ1; 1H-NMR (CDCl3) d: 10.85 (bs, 1H, NH), 8.73 (dd, J ¼ 7.5,
–
–
2.0 Hz, 1H, H4, Pyr), 8.25 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6, Pyr), 8.24 (s,
–
1H, CH–), 7.76 (d, J ¼ 8.5 Hz, 2H, H3,5, Ph), 7.57 (dd, J ¼ 8.0,
–
1.5 Hz, 1H, H3, Ph-O), 7.43–7.37 (m, 4H, H5,6, Ph-O & H2,6, Ph),
7.34–7.31 (m, 1H, H4, Ph-O), 7.25 (dd, J ¼ 7.5, 4.5 Hz, 1H, H5, Pyr);
MS m/z (%): 389 [Mþ þ 4] (4), 387 [Mþ þ 2] (24), 385 [Mþ] (36), 232
(88), 213 (100), 169 (20), 127 (24). Anal. calcd. for C19H13Cl2N3O2: C,
59.08; H, 3.39; N, 10.88. Found: C, 59.27; H, 3.23; N, 11.14.
2-Phenoxy-N0-(pyridin-3-ylmethylidene)nicotinic acid
hydrazide 9i
Yield: 91%; m. p.: 178–1808C (ethanol/hexane); IR (KBr): 3201
ꢂ1
–
(NH), 1675 (C O) cm
;
1H-NMR (CDCl3) d: 10.98 (bs, 1H, NH),
–
8.83 (s, 1H, H2’, Pyr), 8.75 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.64 (d,
(4-Methoxybenzylidene)-2-(2-chlorophenoxy)nicotinic acid
hydrazide 9n
–
J ¼ 3.5 Hz, 1H, H ’, Pyr), 8.33 (s, 1H, CH–), 8.28–8.24 (m, 2H, H ,
–
6
6
Pyr & H4’, Pyr), 7.51 (t, J ¼ 8.0 Hz, 2H, H3,5, Ph-O), 7.37–7.34 (m,
2H, H4, Ph-O & H5’, Pyr), 7.25–7.21 (m, 3H, H2,6, Ph-O & H5, Pyr); MS
m/z (%): 318 [Mþ] (11), 213 (45), 198 (100), 94 (13). Anal. calcd.
for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: C, 67.83; H,
4.52; N, 17.48.
Yield: 71%; m. p.: 168–1708C (ethanol); IR (KBr): 3168 (NH), 1654
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 10.72 (bs, 1H, NH), 8.71 (dd,
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.21 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6,
–
Pyr), 8.16 (s, 1H, CH–), 7.75 (d, J ¼ 8.5 Hz, 2H, H2,6, Ph), 7.55 (d,
–
J ¼ 8.0 Hz, 1H, H3, Ph-O), 7.41 (t, J ¼ 8.0 Hz, 1H, H5, Ph-O), 7.36
(dd, J ¼ 8.0, 1.5 Hz, 1H, H6, Ph-O), 7.30 (t, J ¼ 8.0 Hz, 1H, H4, Ph-
O), 7.22 (dd, J ¼ 7.5, 4.5 Hz, 1H, H5, Pyr), 6.92 (d, J ¼ 8.5 Hz,
2H, H3,5, Ph), 3.84 (s, 3H, OCH3); MS m/z (%): 383 [Mþ þ 2] (5),
381 [Mþ] (15), 232 (55), 213 (100), 169 (20), 149 (24), 127 (30). Anal.
calcd. for C20H16ClN3O3: C, 62.91; H, 4.22; N, 11.01. Found: C,
63.17; H, 4.30; N, 11.16.
2-Phenoxy-N0-(pyridin-2-ylmethylidene)nicotinic acid
hydrazide 9j
Yield: 82%; m. p.: 188–1908C (ethanol/hexane); IR (KBr): 3320
ꢂ1
–
(NH), 1677 (C O) cm
;
1H-NMR (CDCl3) d: 15.34 (bs, 1H, NH),
–
8.78 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.26 (dd, J ¼ 4.5, 2.0 Hz,
–
1H, H , Pyr), 7.77–7.74 (m, 1H, H ’, Pyr), 7.59 (s, 1H, CH–), 7.54–
6
–
6
(4-Nitrobenzylidene)-2-(2-chlorophenoxy)nicotinic acid
hydrazide 9o
Yield: 68%; m. p.: 209–2118C (ethanol); IR (KBr): 3298 (NH), 1687
7.48 (m, 3H, H3,5, Ph-O & H4’, Pyr), 7.42 (d, J ¼ 7.5 Hz, 1H, H3’, Pyr),
7.35 (t, J ¼ 8.0 Hz, 1H, H4, Ph-O), 7.29–7.27 (m, 2H, H2,6, Ph-O),
0
7.17 (dd, J ¼ 7.5, 4.5 Hz, 1H, H5, Pyr), 7.07–7.05 (m, 1H, H5 , Pyr);
MS m/z (%): 318 [Mþ] (18), 227 (84), 120 (100), 92 (45), 65 (29). Anal.
calcd. for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: C, 67.70;
H, 4.31; N, 17.73.
ꢂ1
(C O) cm
;
1H-NMR (CDCl3) d: 11.02 (bs, 1H, NH), 8.71 (dd,
–
–
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.39 (s, 1H, CH–), 8.27–8.25 (m,
–
3H, H6, Pyr & H3,5, Ph), 7.96 (d, J ¼ 8.5 Hz, 2H, H2,6, Ph), 7.56 (d,
J ¼ 8.0 Hz, 1H, H3, Ph-O), 7.41 (t, J ¼ 8.0 Hz, 1H, H5, Ph-O), 7.37 (d,
J ¼ 8.0 Hz, 1H, H6, Ph-O), 7.32 (t, J ¼ 8.0 Hz, 1H, H4, Ph-O), 7.25
(dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr); MS m/z (%): 398 [Mþ þ 2] (4), 396
[Mþ] (12), 232 (100), 205 (11), 169 (21). Anal. calcd.
for C19H13ClN4O4: C, 57.51; H, 3.30; N, 14.12. Found: C, 57.62;
H, 3.21; N, 14.28.
Benzylidene-2-(2-chlorophenoxy)nicotinic acid hydrazide
9k
Yield: 65%; m. p.: 183–1858C (ethanol); IR (KBr): 3328 (NH), 1693
ꢂ1
(C O) cm
;
1H-NMR (CDCl3) d: 10.80 (bs, 1H, NH), 8.72 (dd,
–
–
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.24 (s, 1H, CH–), 8.23 (dd,
–
(Thiophen-2-ylmethylidene)-2-(2-chlorophenoxy)nicotinic
acid hydrazide 9p
J ¼ 5.0, 2.0 Hz, 1H, H6, Pyr), 7.82–7.80 (m, 2H, H2,6, Ph), 7.56
(dd, J ¼ 8.5, 1.5 Hz, 1H, H3, Ph-O), 7.44–7.41 (m, 4H, H3,4,5, Ph
& H5, Ph-O), 7.37 (dd, J ¼ 8.5, 2.0 Hz, 1H, H6, Ph-O), 7.32–7.29 (m,
1H, H4, Ph-O), 7.23 (dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr); MS m/z (%):
353 [Mþ þ 2] (5), 351 [Mþ] (15), 232 (78), 213 (100), 127 (23). Anal.
calcd. for C19H14ClN3O2: C, 64.87; H, 4.01; N, 11.94. Found: C,
64.72; H, 4.11; N, 11.83.
Yield: 91%; m. p.: 183–1858C (ethyl acetate); IR (KBr): 3278 (NH),
1685 (C O) cmꢂ1; 1H-NMR (CDCl3) d: 10.80 (bs, 1H, NH), 8.71 (dd,
–
–
–
–
J ¼ 7.5, 2.0 Hz, 1H, H , Pyr), 8.69 (s, 1H, CH–), 8.23 (dd, J ¼ 4.5,
4
2.0 Hz, 1H, H6, Pyr), 7.55 (dd, J ¼ 8.0, 1.5 Hz, 1H, H3, Ph-O), 7.45–
7.40 (m, 2H, H5, thiophene & H5, Ph-O), 7.37–7.34 (m, 2H, H3
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
Nicotinohydrazides: analgesic, anti-inflammatory agents
517
thiophene & H6, Ph-O), 7.32–7.29 (m, 1H, H4, Ph-O), 7.23 (dd,
J ¼ 7.5, 4.5 Hz, 1H, H5, Pyr), 7.07 (dd, J ¼ 5.0, 4.0 Hz, 1H, H4,
thiophene); MS m/z (%): 359 [Mþ þ 2] (7), 357 [Mþ] (21), 247 (30),
232 (100), 127(14), 77 (9). Anal. calcd. for C17H12ClN3O2S: C, 57.06;
H, 3.38; N, 11.74. Found: C, 56.89; H, 3.24; N, 11.66.
(1-(4-Methoxyphenyl)ethylidene)-2-phenoxynicotinic acid
hydrazide 9u
Yield: 65%; m. p.: 159–1618C (ethanol); IR (KBr): 3244 (NH), 1689
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 10.94 (bs, 1H, NH), 8.79 (dd,
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.26 (dd, J ¼ 5.0, 2.0 Hz, 1H, H6,
Pyr), 7.84 (d, J ¼ 9.0 Hz, 2H, H2,6, Ph), 7.49 (t, J ¼ 8.0 Hz, 2H, H3,5
,
Ph-O), 7.33 (t, J ¼ 8.0 Hz, 1H, H4, Ph-O), 7.24–7.21 (m, 3H, H2,6, Ph-
O & H5, Pyr), 6.91 (d, J ¼ 9.0 Hz, 2H, H3,5, Ph), 3.84 (s, 3H, OCH3),
2.25 (s, 3H, CH3); MS m/z (%): 361 [Mþ] (5), 346 (10), 198 (100), 77
(11). Anal. calcd. for C21H19N3O3: C, 69.79; H, 5.30; N, 11.63.
Found: C, 69.60; H, 5.39; N, 11.87.
(Pyridin-4-ylmethylidene)-2-(2-chlorophenoxy)nicotinic
acid hydrazide 9q
Yield: 91%; m. p.: 22ꢂ81–2308C (ethanol/hexane); IR (KBr): 3320
–
(NH), 1689 (C O) cm
;
1H-NMR (CDCl3) d: 11.0 (bs, 1H, NH),
–
8.73 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.69 (dd, J ¼ 4.5, 1.5 Hz,
–
0
0
2H, H2 ,6 , Pyr), 8.30 (s, 1H, CH–), 8.26 (dd, J ¼ 5.0, 2.0 Hz, 1H, H ,
–
6
Pyr), 7.67 (dd, J ¼ 4.5, 1.5 Hz, 2H, H3’,5’, Pyr), 7.57 (dd, J ¼ 8.0,
1.5 Hz, 1H, H3, Ph-O), 7.46–7.42 (m, 1H, H5, Ph-O), 7.38 (dd,
J ¼ 8.0, 1.5 Hz, 1H, H6, Ph-O), 7.35–7.32 (m, 1H, H4, Ph-O), 7.27
(dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr); MS m/z (%): 354 [Mþ þ 2] (6), 532
[Mþ] (18), 232 (100), 213 (95), 169 (22), 127 (17). Anal. calcd.
for C18H13ClN4O2: C, 61.28; H, 3.71; N, 15.88. Found: C, 61.19;
H, 3.60; N, 16.05.
Pharmacology
Analgesic activity
The analgesic activity of all compounds was determined in vivo by
acetic-acid-induced abdominal constriction test (writhing test) in
mice [22]. An aqueous acetic acid solution (0.6%; 0.1 mL/10 g) was
administered i. p. 30 min after administration of the compounds.
The number of writhes was counted for 30 min after acetic acid
injection. The analgesic activity was expressed as the percentage
of inhibition of constrictions when compared with the vehicle
control group and was calculated using the following formula:
(Pyridin-3-ylmethylidene)-2-(2-chlorophenoxy)nicotinic
acid hydrazide 9r
Yield: 89%; m. p.: 185–1878C (ethanol/hexane); IR (KBr) 3316 (NH),
1
1697 (C O) cmꢂ1; H-NMR (CDCl3) d: 10.94 (bs, 1H, NH), 8.83 (d,
–
% Inhibition ¼ ½ðcontrol mean ꢂ test meanÞ=control
–
(1)
0
J ¼ 1.5 Hz, 1H, H2 , Pyr), 8.71 (dd, J ¼ 7.5, 1.5 Hz, 1H, H4, Pyr),
meanꢄ ꢃ 100
–
8.62 (dd, J ¼ 4.5, 1.5 Hz, 1H, H6’, Pyr), 8.32 (s, 1H, CH–), 8.29–
–
0
8.27 (m, 1H, H4 , Pyr), 8.24 (dd, J ¼ 5.0, 1.5 Hz, 1H, H6, Pyr), 7.55
(dd, J ¼ 8.0, 1.5 Hz, 1H, H3, Ph-O), 7.42–7.40 (m, 1H, H5, Ph-O),
7.38–7.34 (m, 2H, H6, Ph-O & H5’, Pyr), 7.32–7.28 (m, 1H, H4, Ph-O),
7.24 (dd, J ¼ 7.5, 5.0 Hz, 1H, H5, Pyr); MS m/z (%): 354 [Mþ þ 2]
(10), 532 [Mþ] (30), 232 (87), 213 (100), 169 (20), 127 (20), 122 (18).
Anal. calcd. for C18H13ClN4O2: C, 61.28; H, 3.71; N, 15.88. Found:
C, 61.03; H, 3.59; N, 15.74.
Anti-inflammatory activity
The anti-inflammatory activity was determined in vivo using the
carrageenan-induced rat paw edema test. Edema was induced in
the right hind paw of all rats by subcutaneous injection of 0.1 mL
of 1% (w/v) carrageenan (Sigma-Aldrich, Dorset, UK) in saline into
their footpads 1 h after i. p. administration of compounds [23].
The paw thickness was measured from the ventral to the
dorsal surfaces using a dial caliper immediately before and then
two and four hours after carrageenan injection. The edema was
calculated as the thickness variation between thickness of paw
before and after carrageenan injection. Anti-inflammatory
activity was expressed as the percentage of inhibition of the
edema when compared with the control group and was calcu-
lated using the following formula:
(Pyridin-2-ylmethylidene)-2-(2-chlorophenoxy)nicotinic
acid hydrazide 9s
Yield: 81%; m. p.: 19ꢂ61–1988C (ethanol/hexane); IR (KBr): 3332
–
(NH), 1693 (C O) cm
;
1H-NMR (CDCl3) d: 15.4 (bs, 1H, NH),
–
8.81 (dd, J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.23 (dd, J ¼ 5.0, 2.0 Hz,
1H, H6, Pyr), 7.78–7.75 (m, 1H, H6’, Pyr), 7.63 (m, 1H, H4’, Pyr), 7.61
–
(s, 1H, CH–), 7.57 (dd, J ¼ 8.0, 2.0 Hz, 1H, H , Ph-O), 7.44–7.42
–
3
(m, 2H, H5, Ph-O & H3’, Pyr), 7.38 (dd, J ¼ 8.0, 2.0 Hz, 1H, H6, Ph-
O), 7.35–7.32 (m, 1H, H4, Ph-O), 7.20 (dd, J ¼ 7.5, 5.0 Hz, 1H, H5,
% Inhibition ¼ ð1 ꢂ Tt=TcÞ ꢃ 100 (2)
where Tt and Tc are defined as the thickness variation of test
group and control group, respectively.
þ
0
Pyr), 7.09–7.07 (m, 1H, H5 , Pyr); MS m/z (%): 354 [M þ 2] (5), 532
[Mþ] (15), 232 (43), 169 (24), 120 (100), 92 (47). Anal. calcd.
for C18H13ClN4O2: C, 61.28; H, 3.71; N, 15.88. Found: C, 61.45;
H, 3.63; N, 15.74.
Statistics
(1-(4-Chlorophenyl)ethylidene)-2-phenoxynicotinic acid
hydrazide 9t
The results are expressed as the mean ꢀ SEM of n animals per
group. The data were statistically analyzed by one way analysis of
Variance (ANOVA) followed by Tukey multicomparison test.
Differences with P < 0.05 between experimental groups were
considered statistically significant.
Yield: 52%; m. p.: 208–2108C (ethanol); IR (KBr): 3328 (NH), 1673
ꢂ1
–
(C O) cm
;
1H-NMR (CDCl3) d: 11.01 (bs, 1H, NH), 8.78 (dd,
–
J ¼ 7.5, 2.0 Hz, 1H, H4, Pyr), 8.27 (dd, J ¼ 4.5, 2.0 Hz, 1H, H6,
Pyr), 7.81 (d, J ¼ 8.5 Hz, 2H, H2,6, Ph), 7.50 (t, J ¼ 8.5 Hz, 2H, H3,5
,
Ph-O), 7.37–7.32 (m, 3H, H3,5, Ph & H4, Ph-O), 7.25–7.22 (m,
3H, H2,6, Ph-O & H5, Pyr), 2.25 (s, 3H, CH3); MS m/z (%): 367
[Mþ] (9), 365 [Mþ] (27), 350 (12), 198 (100), 149 (11), 115 (14).
Anal. calcd. for C20H16ClN3O2: C, 65.67; H, 4.41; N, 11.49. Found:
C, 65.55; H, 4.30; N, 11.61.
COX-1/COX-2 activity
Inhibition of COX-1 activity in human whole blood
COX-1 activity was measured by determination of the production
of thromboxane B2, during the spontaneous clotting of human
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

518
A. Moradi et al.
Arch. Pharm. Chem. Life Sci. 2010, 9, 509–518
whole blood [24, 25]. Fresh human venous blood from three
healthy donors was collected in glass tubes without anticoagu-
lant, one volunteer each day. The volunteers had not taken any
nonsteroidal anti-inflammatory drugs for the last two weeks.
Aliquots of blood (0.5 mL) were immediately transferred to tubes
containing 2 mL of inhibitor or vehicle (DMSO). Samples were
vortex-mixed and incubated at 378C for 1 h. Then, the tubes were
centrifuged at 5000 ꢃ g for 10 min and 120 mL of serum was
mixed with 480 mL of methanol to precipitate the proteins.
Samples were centrifuged again and the supernatants were
taken for measurements of thromboxane B2 by a commercially
available EIA kit (Cayman chemicals Europe). Samples were
tested at different concentrations and IC50 values were calcu-
lated with the software CurveExpert version 1.34 for Windows.
[7] M. C. Allison, G. Howatson, C. J. Torrance, F. D. Lee, R. I. G.
Russell, N. Engl. J. Med. 1992, 327, 749–754.
[8] S. M. Sondhi, N. Singhal, M. Johar, B. S. N. Reddy, J. W. Lown,
Curr. Med. Chem. 2002, 9, 1045–1074.
[9] R. C. Effland, J. T. Klein, Eur. Pat. Appl. EP 453, 222. 1991.
[10] M. T. Cocco, C. Congiu, V. Onnis, M. Morelli, V. Felipo, O.
Cauli, Bioorg. Med. Chem. 2004, 12, 4169–4177.
[11] C. J. Smith, Y. Zhang, C. M. Koboldt, J. Muhammad, B. S.
Zweifel, A. Shaffer, J. J. Talley, J. L. Masferrer, K. Seibert, P. C.
Isakson, Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 13313–
13318.
[12] C. Hawkey, L. Laine, T. Simon, A. A. Beaulieu, J. Maldonado-
Cocco, E. Acevedo, A. Shahane, H. Quan, J. Bolognese, E.
Mortensen, Arthritis Rheum. 2000, 43, 370–377.
[13] C. D. Duarte, J. L. M. Tributino, D. Lacerda, M. V. Martins, A.-
M. S. Moreira, F. Dutra, E. J. H. Bechara, F. S. Paula, M. O. F.
Goulart, J. Ferreira, J. B. Calixto, M. P. Nunes, A. L. Bertho,
A. L. P. Miranda, E. Barreiro, C. A. M. Fraga, Bioorg. Med. Chem.
2007, 15, 2421–2433.
Inhibition of COX-2 activity in human whole blood
COX-2 activity was assessed by measuring the formation of PGE2,
after incubation of blood samples with lipopolysacharide (LPS
from Escherichia coli serotype 0111-B4; Sigma-Aldrich) according
to a method described previously [24, 25]. Fresh venous blood
from healthy volunteers was collected in tubes containing hep-
arin (15 IU/mL). 0.5 mL of blood was transferred to tubes con-
taining 2 mL of inhibitor or DMSO and incubated at 378C
for 15 min. Then, 16 mL of LPS solution (final concentration
30 mg/mL) was added to each tube and the samples were further
incubated at 378C for 18 h. After the separation of plasma,
proteins were precipitated as described above and levels of
PGE2 were measured by a commercially available EIA kit
(Cayman chemicals). Samples were tested at different concen-
trations and IC50 values were calculated with the software
CurveExpert version 1.34 for Windows.
[14] P. C. Lima, L. M. Lima, K. C. M. da Silva, P. H. O. Leda, A. L. P.
de Miranda, C. A. M. Fraga, E. J. Barreiro, Eur. J. Med. Chem.
2000, 35, 187–203.
[15] J. M. Figueiredo, C. A. Camara, E. G. Amarante, A. L. P.
Miranda, F. M. Santos, C. R. Radrigues, C. A. M. Fraga,
E. J. Barreiro, Bioorg. Med. Chem. 2000, 8, 2243–2248.
[16] A. Almasirad, M. Tajik, D. Bakhtiari, A. Shafiee, M.
Abdollahi, M. J. Zamani, R. Khorasani, H. Esmaily,
J. Pharm. Pharm. Sci. 2005, 8, 419–425.
[17] A. Almasirad, R. Hosseini, H. Jalalizadeh, Z. Rahimi-
Moghaddam, N. Abaeian, M. Janafrooz, M. Abbaspour, V.
Ziaee, A. Dalvandi, A. Shafiee, Biol. Pharm. Bull. 2006, 29,
1180–1185.
This work was supported by grants from the INSF (Iran National Science
Foundation) and research council of Tehran University of Medical Sciences.
[18] M. V. Pavlova, A. I. Mikhalev, M. E. Kon’shin, M. Y. Vasil’eva,
L. G. Mardanova, T. F. Odegova, M. I. Vakhrin, Pharm. Chem. J.
2001, 35, 664–666.
The authors have declared no conflict of interest.
¨
[19] Y. Du¨ndar, S. Unlu¨, E. Banog˘lu, A. Entrena, G. Costantino, M.
References
Nunez, F. Ledo, M. F. ahin, N. Noyanalpan, Eur. J. Med. Chem.
2009, 44, 1830–1837.
[1] A. Palomer, F. Cabre, J. Pascual, J. Campos, M. A. Trugillo, A.
Entrena, M. A. Gallo, L. Garcia, D. Macleon, A. Espinosa,
J. Med. Chem. 2002, 45, 1402–1411.
[20] M. M. Perez, R. M. Docampo, J. M. Puigmarti Codina, J.
Repolles-Moliner, J. S. Sola, U.S. Pat. Appl. US 4623648 1986.
[21] A. Kamal, M. N. A. Khan, K. S. Reddy, K. Rohini, Bioorg. Med.
Chem. 2007, 15, 1004–1013.
[2] J. J. Talley, D. L. Brown, J. S. Carter, M. J. Graneto, C. M.
Koboldt, J. L. Masferrer, W. E. Perkins, R. S. Rogers, A. F.
Shaffer, Y. Y. Zhang, B. S. Zweifel, K. Seibert, J. Med. Chem.
2000, 43, 775–777.
[22] B. A. Whittle, Br. J. Pharmacol. Chemother. 1964, 22, 246–
253.
[3] J. R. Vane, R. M. Botting, Am. J. Med. 1998, 104 (3A), 2S–8S,
discussion 21S–22S.
[23] H. A. Al-Haboubi, I. J. Zeitlin, Eur. J. Pharmacol. 1983, 88, 169–
176.
[4] J. R. Vane, Y. S. Bakhle, R. M. Botting, Annu. Rev. Pharmacol.
Toxicol. 1998, 38, 97–120.
[24] J. K. Gierse, Y. Zhang, W. F. Hood, M. C. Walker, J. S. Trigg,
T. J. Maziasz, C. M. Koboldt, J. L. Muhammad, B. S. Zweifel,
P. C. Isakson, K. Seibert, J. Pharmacol. Exp. Ther. 2005, 312,
1206–1212.
[5] D. L. Simmons, R. M. Botting, T. Hla, Pharmacol. Rev. 2004, 56,
387–437.
[25] C. Brideau, S. Kargman, S. Liu, A. L. Dallob, E. W. Ehrich, I. W.
Rodger, C. C. Chan, Inflamm. Res. 1996, 45, 68–74.
[6] L. M. Jackson, C. J. Hawkey, Exp. Opin. Invest. Drugs 1999, 8,
963–971.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com