Synthesis and analgesic activity of new pyridine-based heterocyclic derivatives

Article abstract of DOI:10.1007/s00044-010-9489-9

A series of new heterocyclic derivatives having a pyridine nucleus were synthesized. 4-(5-(2-Chlorophenyl)- 4H-1,2,4-triazol-3-yl)pyridine (7c) and 4-(5-(2- Nitrophenyl)-4H-1,2,4-triazol-3-yl)pyridine (7d) presented the best analgesic profie of this series in hot-plate, tail-flick, and formalin-induced licking tests, which was partially prevented by pretreatment with mecamylamine, a nicotinic receptor antagonist. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9489-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:27–37
DOI 10.1007/s00044-010-9489-9
ORIGINAL RESEARCH
Synthesis and analgesic activity of new pyridine-based
heterocyclic derivatives
•
Ganesh Nigade Pradeep Chavan Meenakshi Deodhar
•
Received: 5 April 2010 / Accepted: 28 October 2010 / Published online: 21 November 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of new heterocyclic derivatives having
a pyridine nucleus were synthesized. 4-(5-(2-Chloro-
phenyl)-4H-1,2,4-triazol-3-yl)pyridine (7c) and 4-(5-(2-
Nitrophenyl)-4H-1,2,4-triazol-3-yl)pyridine (7d) presented
the best analgesic profie of this series in hot-plate, tail-flick,
and formalin-induced licking tests, which was partially
prevented by pretreatment with mecamylamine, a nicotinic
receptor antagonist.
Neuronal nicotinic receptors have been implicated in a
number of conditions like schizophrenia, autism, Alzhei-
mer’s disease, Parkinson’s disease, dementia with Lewy
bodies, and mood and anxiety disorders, besides pain
(Whitehouse et al., 1988; Court et al., 2000; Perry et al.,
2001; Lee et al., 2002; Pimlott et al., 2004; Bohr et al.,
2005; Marutle et al., 2001; Newhouse et al., 2004; Ripoll
et al., 2004). The literature reveals synthesis and analgesic
activity of nicotine and epibatidine analogs acting through
these receptors. Most of these analogs contain a pyridine
nucleus (Fig. 1) (Bai et al., 1996; Corey et al., 1993;
Holladay et al., 1998; Silva et al., 2002).
Keywords Pyridine ꢀ Nicotine ꢀ Epibatidine ꢀ
Mecamylamine ꢀ Analgesic activity
The reported analogs generally contain another hetero-
cyclic ring attached at the 3rd position of pyridine ring. In
this study, we report synthesis of some pyridine-based
analogs substituted with different heterocyclic rings
attached to the pyridine nucleus at the 3rd and 4th positions
directly (7–12) or through a carbonyl linkage (3–6).
Introduction
Pain is a typical sensory experience that may be described
as the unpleasant awareness of a noxious stimulus or bodily
harm. It is initiated by stimulation of nociceptors in the
peripheral nervous system, or by damage to or malfunction
of the peripheral or central nervous systems. The term
analgesic means a drug that selectively relives pain by
acting in the central nervous system or peripheral pain
mechanisms, without significantly altering consciousness
(Bennett and Villa, 2000). Though a host of popular
analgesics are available, they suffer from side effects like
dependence, tolerance, and gastrointestinal effects (Reisine
and Pasternak, 1996; Insel, 1996). Hence, there is a need to
explore new targets and agents acting on these targets.
Chemistry
The target compounds, 5-methyl-1-(pyridin-4-yl carbonyl)-
4-pyrazoline-3-(2H)-one (3) and 5-methyl-1-(pyridin-3-yl
carbonyl)-4-pyrazoline-3-(2H)-one (4), were synthesized by
reacting isoniazid and nicotinohydrazide, respectively, with
ethylacetoacetate in the presence of acetic acid (Scheme 1).
Similarly, 4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]pyr-
idine (5) and 3-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]
pyridine (6) were synthesized by reacting isoniazid and
nicotinohydrazide, respectively, with acetyl acetone in
the presence of acetic acid (Scheme 1). Compounds
4-(5-substituted-4H-1,2,4-triazol-3-yl)pyridine 7(a–d) and
3-(5-substituted-4H-1,2,4-triazol-3-yl)pyridine 8(a–d) were
synthesized by reacting isoniazid and nicotinohydrazide,
G. Nigade ꢀ P. Chavan ꢀ M. Deodhar (&)
Department of Pharmaceutical Chemistry, Seth Govind
Raghunath Sable College of Pharmacy, Saswad, Pune,
Maharashtra, India
e-mail: meenakshideodhar@yahoo.com
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Med Chem Res (2012) 21:27–37
Cl
H
base 9(a–d) and 10(a–d) were synthesized by the addition of
isoniazid and nicotinohydrazide, respectively, to aldehydes
(Scheme 1).
N
O
N
O
N
N
H
N
N
Epibatidine
ABT-594
2-(3-methyl-5-isoxazolyl) pyridine
Fig. 1 Epibatidine and epibatidine analogs contain
nucleus
a pyridine
Biology
Three acute pain models—tail-flick, hot-plate, and forma-
lin-induced licking tests—were used to evaluate the syn-
thesized compounds. The results are listed in Tables 1, 2,
and 3. In the tail-flick test of D’Amour and Smith (1941),
the tail is exposed to heat, and the amount of time taken for
the animal to move (flick) its tail away from the heat is
recorded. A control response (2–4 s) was determined for
each mouse before treatment, and test latency was deter-
mined after drug administration. For the hot-plate test
(Eddy and Leimbach, 1953; Atwell and Jacobson, 1978),
mice were placed into a 10-cm-wide glass cylinder on a hot
plate (Thermo just apparatus) maintained at 55.0°C. Two
control latencies with at least 10 min apart were deter-
mined for each mouse. The normal latency (reaction time)
was 8–10 s. The reaction time was scored when the animal
jumped or licked its paws. The mice were tested 30 min
after i.p. injections of the compounds (25 mg/kg) for the
dose–response determination.
respectively, with different aldyhydes in the presence of
ammonium acetate (Scheme 1).
1-(2-Substituted-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)yl)
ethanone 11(a–d) and 1-(2-substituted-5-(pyridin-3-yl)-1,3,
4-oxadiazol-3(2H)yl) ethanone 12(a–d) were synthesized by
reacting Schiff’s base of isoniazid 9(a–d) and nicotinohyd-
razide 10(a–d), respectively, with acetic anhydride. Schiff’s
H2N
NH
N
O
Isoniazid / Nicotinohydrazide
1/2
d
a
c
O
b
N
R
C
NHN
H
N
R
N
N
H
N
N
HN
In the formalin-induced licking test (Shibata et al.,
1989) mice injected (i.p.) with test compounds (25 mg/kg)
or vehicle (0.1% gum acacia solution). After 30 min, the
mice were injected with 0.025 ml of 5% formalin into the
dorsal portion of the front paw. Each individual mouse was
placed into a clear plastic cage for observation. Pain
responses were indicated by elevation or favoring of the
paw, or excessive licking and biting of the paw. A biphasic
response with an early (0–5 min) and a late (10–20 min)
phase with high licking activity were observed.
N
O
O
Schiff base
CH3
N
9 (a-d) & 10(a-d)
N
O
H3C
3/4
CH3
N
e
5/6
7 (a-d) &8(a-d)
O
C
CH3
N
O
N
N
R
11(a-d) & 12 (a-d)
Since several pyridine-based molecules have been found
to produce analgesia through their action on neuronal
nicotinic receptors, it was thought worthwhile to test the
more active synthesized compounds for nAChR inhibition
using mecamylamine antagonism test. Mecamylamine is a
nAChR antagonist and thus is able to block analgesic
action produced through activation of these receptors.
To evaluate whether the antinociceptive effects of syn-
thesized compounds involved neuronal nicotinic receptors,
separate groups of animals were pretreated with either
vehicle or mecamylamine (2 mg/kg) (Rui et al., 1996; Free
et al., 2006; Young and Shytle, 2001), and 20 min later,
were treated with a second injection of either saline or the
most effective synthesized compounds (25 mg/kg). After
an additional 30 min, all the animals were tested for tail-
flick test.
Compounds
R
7a, 8a, 9a, 10a, 11a, 12a
7b, 8b, 9b, 10b, 11b, 12b
-CH3
Cl
7c, 8c, 9c, 10c, 11c, 12c
7d, 8d, 9d, 10d, 11d, 12d
O2N
Scheme 1 Synthesis of pyridine analogs. Reagents and conditions:
(a) ethylacetoacetate, acetic acid, reflux—3 h; (b) acetylacetone,
acetic acid, reflux—3 h; (c) substituted aldehyde, ammonium acetate,
with stirring—24 h, and reflux—4 h; (d) substituted aldehyde, with
reflux—3 h; (e) acetic anhydride, with reflux—1 h
Comparisons between experimental groups were made
using one-way analysis of variance (ANOVA) followed by
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Med Chem Res (2012) 21:27–37
29
Table 1 Analgesic activity and mecamylamine antagonism of synthesized compounds in mice by tail-flick Test
Compound
(25 mg/kg)
Latency time (s)
mean ( SEM)
Latency time (s) mean ( SEM)
(mecamylamine antagonism)
Compound
(25 mg/kg)
Latency time (s)
mean ( SEM)
Latency time (s) mean ( SEM)
(mecamylamine antagonism)
Control
Pentazocinea
4.55 (0.09292)
12.84 (0.1877**)
5.7 (0.2309**)
5.26 (0.03)
4.55 (0.09292)
ND
8b
4.73 (0.09292)
5.53 (0.0881*)
6.15 (0.0851*)
11.03 (0.0656**)
6.03 (0.1764**)
6.03 (0.5457**)
4.76 (0.1054**)
8.95 (0.1906**)
8.44 (0.084**)
5.16 (0.066)
ND
8c
ND
3
ND
8d
ND
4
ND
11a
11b
11c
11d
12a
12b
12c
12d
5.14 (0.02186**)
5
6.40 (0.4064**)
7.69 (0.1906**)
7.62 (0.2709**)
5.33 (0.1426)
ND
ND
6
5.18 (0.0928**)
5.22 (0.05044**)
ND
ND
7a
7b
7c
7d
8a
ND
5.14 (0.04041**)
11.2 (0.3606**)
11.8 (0.2082**)
7.99 (0.1054**)
5.12 (0.01**)
5.23 (0.07424**)
5.17 (0.05364**)
5.13 (0.02333**)
ND
ND
4.93 (0.1202)
Results are expressed as means SEM and significant with * P \ 0.05, ** P \ 0.01. N = 6 mice per group
ND not done
a
17.5 mg/kg
Table 2 Analgesic activity of
synthesized compounds in mice
by hot-plate test
Compound
(25 mg/kg)
Latency time (s)
mean ( SEM)
Compound
(25 mg/kg)
Latency time (s)
mean ( SEM)
Control
Pentazocinea
7.26 (0.1457)
19.89 (0.1767**)
7.51 (0.3592)
8b
7.50 (0.4508)
8.55 (0.3044**)
8.47 (0.3716**)
12.93 (0.1079**)
7.78 (0.2718)
8c
3
8d
4
7.54 (0.3460)
11a
11b
11c
11d
12a
12b
12c
12d
5
8.11 (0.0152**)
8.30 (0.1650**)
9.49 (0.3755**)
7.81 (0.1682)
6
7.81 (0.1872)
7a
7b
7c
7d
8a
7.48 (0.1054)
10.03 (0.0680**)
10.03 (0.2203**)
7.77 (0.2868)
Results are expressed as
means SEM and significant
with * P \ 0.05, ** P \ 0.01.
N = 6 mice per group
13.12 (0.1320**)
15.10 (0.4394**)
9.65 (0.2203**)
7.29 (0.1266)
a
17.5 mg/kg
Table 3 Analgesic activity of synthesized compounds in mice by formalin-induced licking test
Compound
(25 mg/kg)
Number of paw licking
(0–5 min) mean ( SEM) (10–20 min) mean ( SEM) (25 mg/kg) (0–5 min) mean ( SEM) (10–20 min) mean ( SEM)
Number of paw licking
Compound
Number of paw licking
Number of paw licking
Control
Pentazocinea 4.33 (0.333**)
11.66 (0.8819)
11.33 (0.8819)
3.33 (0.333**)
4.33 (0.333**)
5.33 (0.333**)
4.66 (0.333**)
8 (1.00**)
8b
7.66 (0.333*)
8.33 (1.202)
6 (1.155**)
7 (0.5774**)
6.66 (0.333**)
3.66 (0.333**)
4.33 (0.881**)
5 (0.577**)
8c
3
10.33 (0.666)
6.33 (0.8819**)
7.66 (0.333*)
8.33 (0.666)
8d
9.33 (1.453)
4
11a
11b
11c
11d
12a
12b
12c
12d
5.66 (0.333**)
7.33 (0.8819**)
8.66 (0.8819)
10.66 (1.202)
6.33 (0.333**)
8.66 (1.453)
5
6
7a
7b
7c
7d
8a
6.66 (0.333**)
11 (0.5774)
4 (0.577**)
4.33 (0.666**)
5 (1.00**)
6 (0.5774**)
4.33 (0.333**)
4.33 (0.333**)
5.66 (0.666**)
5.33 (0.8819**)
5 (0.5774**)
6.66 (0.333**)
7.66 (0.333**)
7.33 (0.333**)
8 (0.5774**)
10.33 (0.8819)
9.66 (0.666)
Results are expressed as means SEM and significant with * P \ 0.05, ** P \ 0.01. N = 6 mice per group
a
17.5 mg/kg
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Med Chem Res (2012) 21:27–37
Dunnet’s multiple comparison test (dose–response and
time course studies). All the data were analyzed using
Graph Pad Instat. Statistical significance was accepted at a
value of P \ 0.05.
were recorded in DMSO-d6. The pyridine protons appeared
as doublets between 7.57–7.58 and 8.23–8.27 ppm. Pyrazole
4-H showed a peak at 5.81 ppm as singlet. Pyrazole 3-CH3
and 5-CH3 showed peaks at 2.47 and 2.35 ppm, respectively,
1
as singlet. H NMR spectra of triazole derivatives [7(a–
d) and 8(a–d)] were recorded in DMSO-d6. The pyridine
protons showed peaks between 7.79–7.82 and 8.44–8.74 ppm
as doublets in 7(a–d). While in compounds 8(a–d), the
pyridine protons appeared as triplets at 7.71–8.09 ppm, as
doublets at 8.14–8.75 and 8.24–9.24 ppm, and as singlet
at 8.82–9.24 ppm. The aromatic protons appeared at 7.68–
7.79 ppm as multiplet and at 8.14–8.24 ppm as doublet.
Triazole NH showed peaks between 10.92 and 12.27 ppm.
1H NMR spectra of all the oxadiazoline derivatives
[11(a–d) and 12(a–d)] were recorded in CDCl3. The pyridine
protons showed peaks between 8.10 and 8.20 ppm as triplets,
at 7.91–8.70 ppm as doublets, and at 9.10–9.46 ppm as
singlets. The aromatic protons displayed peaks at
7.49–7.89 ppm as multiplets. Oxadiazoline 5-H appeared
between 6.20 and 6.60 ppm.
Results and discussion
The desired compounds were synthesized using very simple
synthetic procedures outlined in Scheme 1. All the com-
1
pounds were characterized by IR and H NMR spectros-
copy. These compounds when evaluated for analgesic
action were found to elevate the latency time in the tail-flick
and hot-plate tests. Significant reduction in the number of
paw licking was observed in formalin-induced licking test.
IR (KBr) spectra of compounds 3–6 reveal a character-
istic aromatic stretch between 3,034 and 3,100 cm-1 and
sharp carbonyl stretching vibration between 1,681 and
1,690 cm-1. The stretching vibrations for C=N group of
pyrazolone are seen at 1,501–1,576 cm-1. IR (KBr) spectra
of compounds 7(a–d) and 8(a–d) reveal a characteristic NH
The analgesic activity of synthesized compounds was
evaluated using tail-flick test, hot-plate test, and formalin-
induced test. The data obtained are presented in Tables 1,
2, and 3, respectively, and expressed as mean SEM.
In tail-flick test, each group was injected with test drug
25 mg/kg (i.p.) or vehicle (0.1% gum acacia solution)
(i.p.). Pentazocine 17.5 mg/kg (i.p.) was used as reference.
As shown in Fig. 2, a single i.p. administration of the target
compounds 6, 7a, 7c, 7d, 8a, 11a, 12a, and 12b resulted in
a remarkable analgesic effect.
stretching vibration for the triazole at 3,400–3,500 cm-1
.
The IR spectra of compounds 11(a–d) and 12(a–d) show
stretching vibrations for C=O group between 1,681 and
1,700 cm-1. Also the disappearance of NH stretching
vibration for intermediate Schiff’s bases [9(a–d) and 10(a–
d)] at 3,400–3,500 cm-1, confirms the formation of 1,3,4-
oxadiazoline ring.
1
The H NMR spectra of 4-pyrazoline-3-(2H)-one deriv-
atives (3, 4) were recorded in DMSO-d6. The pyridine pro-
tons showed peaks between 7.57–7.58 and 8.23–8.74 ppm as
doublets. Pyrazoline NH appeared as singlet at 10.80 ppm.
Pyrazoline 4-H displayed a peak at 5.81 ppm as singlet.
Methyl protons of pyrazoline exhibited a singlet at
In hot-plate test, each group was injected with test drug
25 mg/kg (i.p.) or vehicle (0.1% gum acacia solution)
(i.p.). Pentazocine 17.5 mg/kg (i.p.) was used as reference.
As evident in Fig. 3, compounds 6, 7a, 7c, 7d, 8a, 11a, and
12a have displayed remarkable analgesic effects.
1
2.35 ppm. H NMR spectra of pyrazole derivatives (5, 6)
Fig. 2 Effect of i.p.
administration of synthesized
derivatives (25 mg/kg) during
the course of the latency times
in the tail-flick test in mice.
Results are expressed as
means SEM and significant
with * P \ 0.05, ** P \ 0.01.
N = 6 mice per group
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Med Chem Res (2012) 21:27–37
31
Fig. 3 Effect of i.p.
administration of synthesized
derivatives (25 mg/kg) during
the course of the latency times
in the hot-plate test in mice.
Results are expressed as
means SEM and significant
with * P \ 0.05, ** P \ 0.01.
N = 6 mice per group
In formalin-induced test. each group was injected with
test drug 25 mg/kg (i.p.) or vehicle (0.1% gum acacia
solution) (i.p.). Pentazocine 17.5 mg/kg (i.p.) was used as
reference. After 30 min, each group was injected with
0.025 ml of 5% formalin into the dorsal portion of the front
paw. Pain responses were indicated by elevation or favor-
ing of the paw or excessive licking and biting of the paw. A
biphasic response with an early (0–5 min) and a late
(10–20 min) phase with high licking activity was observed.
A single i.p. administration of the compounds 6, 7a, 7c, 7d,
8a, 11a, 12a, and 12b resulted in a remarkable analgesic
effect (Fig. 4).
Mecamylamine is a nAChR antagonist and thus is able
to block analgesic action produced through activation of
these receptors.
To evaluate whether the analgesic effects of synthesized
compounds involved neuronal nicotinic receptors, separate
groups of animals were pretreated with either vehicle or
mecamylamine (2 mg/kg) (Rui et al., 1996; Free et al.,
2006), and 20 min later, they were treated with a second
injection of either saline or synthesized compounds 6, 7a,
7c, 7d, 8a, 11a, 12a, and 12b (25 mg/kg). After a 30 min,
all the animals were tested for tail-flick test. As shown in
Fig. 5, the analgesic effects of synthesized compounds 6,
7a, 7c, 7d, 8a, 11a, 12a, and 12b were reduced by
mecamylamine.
Fig. 4 Effect of i.p. administration of synthesized derivatives
(25 mg/kg) on the course of the latency times in the formalin test
in mice (a) 0–5 min (b) 10–20 min. Results are expressed as
means SEM and significant with * P \ 0.05, ** P \ 0.01. N = 6
mice per group
Based on the activity data (Tables 1, 2, 3), the following
observations were made:
substituted at ortho position of phenyl ring in triazole
derivatives (7c, 7d) increased the activity.
(1) Compounds having a 5-membered heterocyclic ring
containing three heteroatoms (7a–d, 8a–d, 11a–d,
and 12a–d) were more active than those containing
ring with only two heteroatoms (3–6).
(3) Similarly, 2-substituted 1,3,4-oxadiazoline at the 4th
position of pyridine ring (11a) possessed better
analgesic activity as compared to when it is attached
to the 3rd position of pyridine ring (12a).
(2) 5-Substituted 1, 2, 4-triazol-3-yl at the 4th position of
pyridine ring (7a–d) was found to enhance the
analgesic activity as compared to when it is attached
to the 3rd position of pyridine ring. Electron-
withdrawing groups, like chloro and nitro, when
(4) Methyl substitution on oxadiazoline ring (11a, 12a)
favors activity as compared with the substituted and
unsubstituted phenyl rings (11b, 11c, 11d, 12b, 12c,
and 12d).
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Med Chem Res (2012) 21:27–37
5-Methyl-1-(pyridin-4-yl carbonyl)-4-pyrazoline-3-
(2H)-one (3) and 5-methyl-1-(pyridin-3-yl carbonyl)-4-
pyrazoline-3-(2H)-one (4)
Isoniazid (1) or nicotinohydrazide (2) 2.74 g (0.02 mol)
and 2.60 ml (0.02 mol) ethylacetoacetate were dissolved in
30 ml ethanol taken in a 100-ml round-bottomed flask and
refluxed for 3 h. To this solution was added 1–2 ml of
acetic acid and refluxed again for 1 h. It was then poured
into ice water. The separated solid was filtered, washed
with water, and dried in air. The crude product was re-
crystallized from ethanol to yield pure product.
Fig. 5 Effect of mecamylamine (2 mg/kg) on synthesized deriva-
tive’s elicited analgesia in mice. Results are expressed as mean-
s SEM and significant with * P \ 0.05, ** P \ 0.01. N = 6 mice
per group. C ? M compound ? mecamylamine, C compound
5-Methyl-1-(pyridin-4-yl carbonyl)-4-pyrazoline-3-
(2H)-one (3)
The compound 6, 7a, 7c, 7d, 8a, 11a, 12a, and 12b
(25 mg/kg, i.p.) were found to be the most active in all the
tests. Moreover, the analgesic effect of these compounds
was reduced by mecamylamine (Fig. 5) suggesting the
possible involvement of neuronal AChRs in producing
analgesic effects.
Yield: 83%; orange, solid; mp 256–258°C.
IR (KBr, cm-1): 1570.11 (C=N), 1593.25 (C=C),
1681.98 (C=O), 2980.12 (Me-CH), 3034.16, 3066.35 (Ar–
CH), 3462.31 (NH).
1H NMR (DMSO-d6, d ppm): 2.35 (s, 3H, CH3), 5.81 (s,
1H, CH), 7.56 (d, 2H, J = 5.6, Py), 8.25 (d, 2H, J = 5.7,
Py), 10.80 (s, 1H, NH).
Conclusion
5-Methyl-1-(pyridin-3-yl carbonyl)-4-pyrazoline-3-
(2H)-one (4)
To conclude, results of this study suggest that the synthe-
sized compounds 6, 7a, 7c, 7d, 8a, 11a, 12a, and 12b
displayed the best analgesic profile of this series. More-
over, the analgesic effects of these compounds were greatly
reduced by mecamylamine, the noncompetitive neuronal
nicotinic acetylcholine receptor antagonist, suggesting the
involvement of neuronal AChRs in producing analgesic
effects besides any other mechanism.
Yield: 73%; orange, solid; mp: 236–238°C.
IR (KBr, cm-1): 1572.04 (C=N), 1597.11 (C=C),
1681.98 (C=O), 2956.97 (Me–CH), 3005.20, 3194.23 (Ar–
CH), 3387.11 (NH).
1H NMR (DMSO-d6, d ppm): 2.35 (s, 3H, CH3), 5.81 (s,
1H, CH), 7.56 (t, 1H, J = 7.4, Py), 8.24 (d, 1H, J = 5.6, Py),
8.77 (d, 1H, J = 5.6, Py), 9.0 (s, 1H, Py), 10.80 (s, 1H, NH).
4-[(3,5-dimethyl-1H-pyrazol-1yl)carbonyl]pyridine (5)
and 3-[(3,5-dimethyl-1H-pyrazol-1-
yl)carbonyl]pyridine (6)
Experimental
All the chemicals used in the synthesis were of laboratory
grade. The melting points were determined in open capil-
lary on Veego (model:-VMP-D) electronic apparatus and
are uncorrected. The IR spectra of the synthesized com-
pounds were recorded on Shimadzu 8400-S FT-IR Spec-
trophotometer using potassium bromide. The 1H NMR
were recorded in CDCl3 and DMSO-d6 using NMR Varian-
Mercury 300 MHz spectrometer and chemical shifts are
given in parts per millions, downfield from tetramethyl-
silane (TMS) as an internal standard. The reactions were
monitored by pre-coated aluminum silica gel 60 F254 thin
layer plates procured from Merck (Germany), using tolu-
ene–methanol, chloroform–ethyl acetate, and dioxane as
the solvent systems, and the spots were visualized by
exposure to iodine vapors.
Isoniazid (1) or nicotinohydrazide (2) 2.74 g (0.02 mol)
and 2.60 ml (0.02 mol) acetyl acetone were dissolved in
30 ml ethanol taken in a 100-ml round-bottomed flask and
refluxed for 3 h. To this solution was added 1–2 ml of
acetic acid and refluxed again for 1 h. The resulting solu-
tion was poured into ice water. The separated solid was
filtered, washed with water, and dried in air. The crude
product was recrystallized from ethanol to yield pure
product.
4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]pyridine (5)
Yield: 82%; white, solid; mp 234–236°C.
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Med Chem Res (2012) 21:27–37
33
IR (KBr, cm-1): 1570.11 (C=N), 1593.25 (C=C), 1698.43
(C=O), 2856.63 (Me–CH), 3034.13, 3074.63 (Ar–CH).
1H NMR (DMSO-d6, d ppm): 2.36 (s, 3H, CH3), 2.47 (s,
3H, CH3), 5.82 (s, 1H, CH), 7.56 (d, 2H, J = 5.6, Py), 8.24
(d, 2H, J = 5.6, Py)
1H NMR (DMSO-d6, d ppm): 7.48 (d, 2H, J = 7.5, Ar),
7.72–7.78 (m, 2H, Ar), 8.40 (d, 2H, J = 5.4, Py), 8.73 (d,
2H, J = 5.6, Py),12.08 (s, 1H, NH).
4-(5-(2-Nitrophenyl)-4H-1,2,4-triazol-3-yl)
pyridine (7d)
3-[(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl]
pyridine (6)
Yield: 66%; yellow, solid; mp 225–227°C.
IR (KBr, cm-1): 1354.07, 1525.74 (C–NO2), 1560.82
(C=N), 1600.57 (C=C), 3059.20, 3062.35 (Ar–CH),
3460.31 (NH).
1H NMR (DMSO-d6, d ppm): 7.68–7.79 (m, 2H, Ar),
8.14 (d, 1H, J = 7.4, Ar), 8.24 (d, 1H, J = 7.6, Ar), 8.51
(d, 2H, J = 5.4, Py), 8.74 (d, 2H, J = 5.5, Py), 12.27 (s,
1H, NH).
Yield: 72%; brown, solid; mp 230–232°C.
IR (KBr, cm-1): 1588.54 (C=N), 1593.25 (C=C), 1674.27
(C=O), 2846.53 (Me–CH), 3059.20, 3186.51 (Ar–CH).
1H NMR (DMSO-d6, d ppm): 2.35 (s, 3H, CH3), 2.47 (s,
3H, CH3), 5.89 (s, 1H, CH), 7.56 (t, 1H, J = 7.3, Py), 8.25 (d,
1H, J = 5.4, Py), 8.77 (d, 1H, J = 5.6, Py), 9.05 (s, 1H, Py).
General procedure for synthesis of 4-(5-substituted-4H-
1,2,4-triazol-3-yl)pyridine 7(a–d) and 3-(5-substituted-
4H-1,2,4-triazol-3-yl)pyridine 8(a–d)
3-(5-Methyl-4H-1,2,4-triazol-3-yl)pyridine (8a)
Yield: 60%; brown, solid; mp 152–154°C.
IR (KBr, cm-1): 1554.56 (C=N), 1591.33 (C=C),
2872.18 (Me–CH), 3057.27–3070.78 (Ar–CH), 3392.50
(NH).
1H NMR (DMSO-d6, d ppm): 2.49 (s, 3H, CH3), 7.75 (t,
1H, J = 7.2, Py), 8.14 (d, 1H, J = 5.6, Py), 8.24 (d, 1H,
J = 5.6, Py), 8.82 (s, 1H, Py), 12.27 (s, 1H, NH).
Equimolar quantities (0.01 mol) of isoniazid (1) or nicot-
inohydrazide (2), ammonium acetate, and the appropriate
aldehyde were added to 15 ml acetic acid in 100-ml iodine
flask and stirred for 24 h. The solution was then neutralized
with liquid ammonia, and the resulting mixture was
refluxed for 4 h. The solid separated was filtered, washed
with water, and dried in air. The crude product was re-
crystallized from ethanol to yield pure product.
3-(5-Phenyl-4H-1,2,4-triazol-3-yl)pyridine (8b)
Yield: 68%; yellow, solid; mp 130–132°C.
IR (KBr, cm-1): 1566.02 (C=N), 1596.25 (C=C),
3026.11 (Ar–CH), 3450.10 (NH).
4-(5-Methyl-4H-1,2,4-triazol-3-yl)pyridine (7a)
Yield: 70%; brown, solid; mp 245–247°C.
1H NMR (DMSO-d6, d ppm): 7.29–7.70 (m, 3H, Ar),
7.89 (t, 1H, J = 7.2, Py), 8.57 (d, 2H, J = 7.2, Ar), 8.75 (d,
1H, J = 5.4, Py), 9.24 (s, 1H, Py), 9.95 (d, 1H, J = 5.6,
Py), 11.25 (s, 1H, NH).
IR (KBr, cm-1): 1558.54 (C=N), 1606.76 (C=C),
2924.18 (Me–CH), 3064.58 (Ar–CH), 3431.48 (NH).
1H NMR (DMSO-d6, d ppm): 2.49 (s, 3H, CH3), 7.78 (d,
2H, J = 5.3, Py), 8.77(d, 2H, J = 5.6, Py), 10.92(s, 1H, NH).
3-(5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl)
4-(5-Phenyl-4H-1,2,4-triazol-3-yl)pyridine (7b)
pyridine (8c)
Yield: 68%; yellow, solid; mp 241–242°C.
IR (KBr, cm-1): 1566.25 (C=N), 1588.02 (C=C),
3026.11 (Ar–CH), 3450.10 (NH).
1H NMR (DMSO-d6, d ppm): 7.46 (d, 2H, J = 7.2, Ar),
7.79 (t, 2H, J = 8.6, Ar), 7.81 (d, 2H, J = 5.5, Py), 8.44 (d,
1H, J = 7.4, Ar), 8.77 (d, 2H, J = 5.6, Py), 12.05 (s, 1H,
NH).
Yield: 73%; yellow, solid; mp 138–140°C.
IR (KBr, cm-1): 761.91 (C–Cl), 1553.24 (C=N),
1593.25 (C=C), 3099.20, 3186.51 (Ar–CH), 3512.31 (NH).
1H NMR (DMSO-d6, d ppm): 7.23–7.63 (m, 4H, Ar),
7.99 (t, 1H, J = 7.1, Py), 8.46 (d, 1H, J = 5.6, Py), 8.72 (d,
1H, J = 5.6, Py), 9.04 (s, 1H, Py), 12.17 (s, 1H, NH).
4-(5-(2-Nitrophenyl)-4H-1,2,4-triazol-3-yl)
4-(5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl)
pyridine (8d)
pyridine (7c)
Yield: 76%; yellow, solid; mp 145–147°C.
IR (KBr, cm-1): 1355.43, 1524.28 (C–NO2), 1554.64
(C=N), 1593.25 (C=C), 3099.20, 3186.51 (Ar–CH),
3512.31 (NH).
Yield: 71%; buff, solid; mp 215–217°C.
IR (KBr, cm-1): 750.33 (C–Cl), 1570.11 (C=N),
3034.16, 3066.35 (Ar–CH), 3462.31 (NH).
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Med Chem Res (2012) 21:27–37
1H NMR (DMSO-d6, d ppm): 7.56–7.73 (m, 4H, Ar),
N0-[ethylidene] pyridine-3-carbohydrazide (10a)
8.05 (t, 1H, J = 7.2, Py), 8.23 (d, 1H, J = 5.6, Py), 8.78 (d,
1H, J = 5.6, Py), 9.00 (s, 1H, Py), 12.40 (s, 1H, NH).
Yield: 73%; yellow, solid; mp 138–140°C.
IR (KBr, cm-1): 1556.51 (C=N), 1596.61 (C=C),
1681.58 (C=O), 2963.43 (Me–CH), 3064.99, 3188.03 (Ar–
CH), 3431.48 (NH).
Synthesis of target compounds
N0-[phenylmethylidine] pyridine-3-carbohydrazide (10b)
1-(2-substituted-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)yl)-ethanone 11(a–d) and 1-(2-substituted-5-
(pyridin-3-yl)-1,3,4-oxadiazol-3(2H)yl) ethanone
12(a–d)
Yield: 73%; yellow, solid; mp 130–132°C.
IR (KBr, cm-1): 1581.37 (C=N), 1602.13 (C=C),
1688.79 (C=O), 2903.13, 3001.31 (Ar–CH), 3460.41 (NH).
General procedure for synthesis of intermediate
(Schiff’s base) 9(a–d) and 10(a–d)
N0-[(2-chlorophenyl) methylidine] pyridine-3-
carbohydrazide (10c)
Isoniazid (1) or nicotinohydrazide (2) 1.37 g (0.01 mol)
and (0.01 mol) of aldehyde were dissolved in 20 ml ethanol
and refluxed for 3 h in a 100-ml round bottomed flask. The
mixture was poured on to crushed ice, and the solid sepa-
rated was washed with ethanol and dried in air. The crude
product was recrystallized from ethanol to yield pure
product.
Yield: 75%; yellow, solid; mp 148–150°C.
IR (KBr, cm-1): 750.12 (C–Cl), 1570.10 (C=N),
1593.25 (C=C), 1678.39 (C=O), 3023.65, 3034.13 (Ar–
CH), 3466.20 (NH).
1H NMR (DMSO-d6, d ppm): 7.53–7.75 (m, 4H, Ar),
8.05 (t, 1H, J = 7.1, Py), 8.23 (d, 1H, J = 5.5, Py), 8.44 (s,
1H, CH), 8.78 (d, 1H, J = 5.6, Py), 9.00 (s, 1H, Py), 12.40
(s, 1H, NH).
N0-[ethylidene] pyridine-4-carbohydrazide (9a)
N0-[(2-nitrophenyl) methylidine] pyridine-3-
carbohydrazide (10d)
Yield: 81%; yellow, solid; mp 200–202°C.
IR (KBr, cm-1): 1550.42 (C=N), 1604.23 (C=C),
1684.31 (C=O), 2903.13, 2935.43 (CH3–CH), 3063.06,
3182.65 (Ar–CH), 3432.40 (NH).
Yield: 77%; yellow, solid; mp 142–144°C.
IR (KBr, cm-1): 1342.50, 1519.96 (C–NO2), 1552.75
(C=N), 1600.06 (C=C), 1681.12 (C=O), 3037.99, 3074.63
(Ar–CH), 3432.19 (NH).
N0-[phenylmethylidine] pyridine-4-carbohydrazide (9b)
Yield: 83%; yellow, solid; mp 203–205°C.
IR (KBr, cm-1): 1581.93 (C=N), 1602.09 (C=C),
1696.49 (C=O), 2903.13, 3001.31 (Ar–CH), 3460.41 (NH).
General procedure for synthesis of target compound
11(a–d) and 12(a–d)
N0-[(2-chlorophenyl) methylidine] pyridine-
4-carbohydrazide (9c)
Appropriate Schiff base (0.002 mol) was dissolved in
0.1 mol of acetic anhydride, and the mixture was refluxed
for 1 h in a 100-ml round-bottomed flask. The reaction
mixture was poured on to crushed ice, and the solid sepa-
rated was dried in air. The crude product was recrystallized
from ethanol to yield pure product.
Yield: 82%; yellow, solid; mp 202–204°C.
IR (KBr, cm-1): 761.91 (C–Cl), 1570.10 (C=N),
1593.25 (C=C), 3099.20, 3186.51 (Ar–CH), 3512.31 (NH).
1H NMR (DMSO-d6, d ppm): 7.50–7.74 (m, 4H, Ar),
7.78 (d, 2H, J = 5.5, Py), 8.74 (d, 2H, J = 5.6, Py), 8.40
(s, 1H, CH), 12.08 (s, 1H, NH).
1-(2-Methyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)yl)
ethanone (11a)
N0-[(2-nitrophenyl) methylidine] pyridine-4-
carbohydrazide (9d)
Yield: 67%; gray, solid; mp 236–238°C.
IR (KBr, cm-1): 1288.49 (C–O), 1585.96 (C=N),
1604.83 (C=C), 1695.49 (C=O), 2928.04 (Me–CH),
3010.10 (Ar–CH).
Yield: 82%; yellow, solid; mp 203–205°C.
IR (KBr, cm-1): 1350.07, 1522.58 (C–NO2), 1569.33
(C=N), 1591.68 (C=C), 1684.19 (C=O), 3037.99, 3060.07
(Ar–CH), 3439.19 (NH).
1H NMR (CDCl3, d ppm): 1.34 (d, 3H, CH3), 2.40 (s,
3H, CH3), 6.74–6.84 (q, 1H, CH), 8.30 (d, 2H, J = 5.3,
Py), 8.76 (d, 2H, J = 5.6, Py).
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Med Chem Res (2012) 21:27–37
35
IR (KBr, cm-1): 1282.71 (C–O), 1581.91 (C=N),
1602.54 (C=C), 1696.48 (C=O), 2901.04 (Me–CH),
3001.34 (Ar–CH).
1-(2-Phenyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)
ethanone (11b)
1H NMR (CDCl3, d ppm): 2.40 (s, 3H, CH3), 6.52 (s,
1H, CH), 7.49–7.89 (m, 5H, Ar), 7.91 (d, 1H, J = 5.4, Py),
8.20 (t, 1H, J = 7.2, Py), 8.70 (d, 1H, J = 5.6, Py), 9.06 (s,
1H, Py).
Yield: 69%; brown, solid; mp 247–249°C.
IR (KBr, cm-1): 1237.71 (C–O), 1581.68 (C=N),
1602.34 (C=C), 1688.48 (C=O), 2901.04 (Me–CH),
3010.10 (Ar–CH).
1H NMR (CDCl3, d ppm): 2.27 (s, 3H, CH3), 7.25 (s,
1H, CH), 7.62 (t, 2H, J = 8.7, Ar), 7.71 (t, 1H, J = 8.8,
Ar), 7.83 (d, 2H, J = 7.3, Ar), 8.30 (d, 2H, J = 5.4, Py),
8.76 (d, 2H, J = 5.6, Py).
1-(2-(2-Chlorophenyl)-5-(pyridin-3-yl)-1,3,4-
oxadiazol-3(2H)yl) ethanone (12c)
Yield: 65%; brown, solid; mp 123–125°C.
IR (KBr, cm-1): 750.33 (C–Cl), 1271.13 (C–O),
1582.54 (C=N), 1607.61 (C=C), 1691.98 (C=O), 3007.13
(Me–CH), 3068.85 (Ar–CH).
1-(2-(2-Chlorophenyl)-5-(pyridin-4-yl)-1,3,4-
oxadiazol-3(2H)yl) ethanone (11c)
1H NMR (CDCl3, d ppm): 2.40 (s, 3H, CH3), 6.60 (s,
1H, CH), 7.49–7.89 (m, 4H, Ar), 7.91 (d, 1H, J = 5.6, Py),
8.20 (t, 1H, J = 7.1, Py), 8.70 (d, 1H, J = 5.6, Py), 9.46 (s,
1H, Py).
Yield: 65%; brown, solid; mp 250–252°C.
IR (KBr, cm-1): 744.55 (C–Cl), 1303.92 (C–O),
1556.81 (C=N), 1596.61 (C=C), 1681.98 (C=O), 2983.98
(CH3–CH), 3064.99 (Ar–CH).
1H NMR (CDCl3, d ppm): 1.80 (s, 3H, CH3), 7.24 (s,
1H, CH), 7.64 (t, 1H, J = 8.7, Ar), 7.71 (t, 1H, J = 8.5,
Ar), 7.72 (d, 1H, J = 7.4, Ar), 7.82 (d, 1H, J = 7.8, Ar),
8.29 (d, 2H, J = 5.6, Py), 8.74 (d, 2H, J = 5.8, Py).
1-(2-(2-Nitrophenyl)-5-(pyridin-3-yl)-1,3,4-oxadiazol-
3(2H)yl) ethanone (12d)
Yield: 69%; buff, solid; mp 123–125°C.
IR (KBr, cm-1): 1305.85 (C–O), 1341.55, 1543.10 (C–
NO2), 1572.04 (C=N), 1597.61 (C=C), 1681.98 (C=O),
2985.91 (Me–CH), 3194.23 (Ar–CH).
1-(2-(2-Nitrophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)yl) ethanone (11d)
1H NMR (CDCl3, d ppm): 2.32 (s, 3H, CH3), 7.25 (s,
1H, CH), 7.48–7.75 (m, 4H, Ar), 7.91 (d, 1H, J = 5.6, Py),
8.10 (t, 1H, J = 7.2, Py), 8.30 (d, 1H, J = 5.6, Py), 9.10 (s,
1H, Py).
Yield: 68%; buff, solid; mp 257–259°C.
IR (KBr, cm-1): 1271.13 (C–O), 1344.35, 1529.60 (C–
NO2), 1551.59 (C=N), 1600.61 (C=C), 1664.34 (C=O),
2945.98 (Me–CH), 3060.70 (Ar–CH).
1H NMR (CDCl3, d ppm): 2.14 (s, 3H, CH3), 7.24 (s,
1H, CH), 7.61 (t, 1H, J = 8.7, Ar), 7.70 (t, 1H, J = 8.7,
Ar), 7.72 (d, 1H, J = 7.8, Ar), 7.84 (d, 1H, J = 7.6, Ar),
8.30 (d, 2H, J = 5.4, Py), 8.75 (d, 2H, J = 5.6, Py).
Biologic activity
The synthesized compounds were evaluated for analgesic
activity by the following tests:
1-(2-Methyl-5-(pyridin-3-yl)-1,3,4-oxadiazol-3(2H)yl)
ethanone (12a)
Yield: 67%; gray, solid; mp 121–123°C.
IR (KBr, cm-1): 1282.71 (C–O), 1581.96 (C=N),
1602.23 (C=C), 1688.49 (C=O), 2901.04 (Me–CH),
3001.34 (Ar–CH).
1H NMR (CDCl3, d ppm): 2.12 (d, 3H, CH3), 2.32 (s,
3H, CH3), 6.19–6.31 (q, 1H, CH), 8.10 (t, 1H, J = 7.1, Py),
8.29 (d, 1H, J = 5.4, Py), 8.71 (s, 1H, Py), 9.1 (d, 1H,
J = 5.6, Py).
Tail-flick test
Albino mice of either sex weighing 25–30 g were used for
the test. They were divided into three groups each con-
taining six mice. Each group was injected with test drug
25 mg/kg (i.p.) or vehicle (0.1% gum acacia solution)
(i.p.). Pentazocine 17.5 mg/kg (i.p.) was used as reference.
Mice were placed into cages leaving the tail exposed. Heat
to the proximal third of the tail was applied. Within a few
seconds the animal flicks the tail aside or tries to escape.
The time until this reaction occurs was recorded by a stop-
watch. The latency was recorded before and after 30 min
(Fig. 2).
1-(2-Phenyl-5-(pyridin-3-yl)-1,3,4-oxadiazol-3(2H)-yl)
ethanone (12b)
Yield: 69%; brown, solid; mp 122–124°C.
123

36
Med Chem Res (2012) 21:27–37
Hot-plate test
of less than 0.05 was considered statistically significant
(Tables 1, 2, 3; Figs. 2, 3, 4, 5).
Three groups containing six albino mice of either sex each
weighing 25–30 g were used for the test. Each group was
injected with test drug 25 mg/kg (i.p.) or vehicle (0.1%
gum acacia solution) (i.p.). Pentazocine 17.5 mg/kg (i.p.)
was used as reference. A hot plate was maintained at 55° to
56°C. The animals were placed on the hot plate and the
time until either licking or jumping occurs was recorded by
a stop-watch. The latency was recorded before and after
30 min (Fig. 3).
Acknowledgments One of the authors (MND) is thankful to the
University of Pune for the grant for this project, and Dr. Ashok
Bhosale for providing the necessary facilities.
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The albino mice of either sex each weighing 25–30 g were
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Statistical analysis
Results are expressed as mean SEM; n represents the
number of animals. Data obtained from pharmacological
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test (dose–response and time course studies) and used to
evaluate the results, employing Graph Pad Instat. A P value
Perry EK, Lee ML, Martin-Ruiz CM, Court JA, Volsen SG, Merrit J,
Folly E, Iversen PE, Bauman ML, Perry RH, Wenk GL (2001)
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