Antitussive effects of azepino[2,1-b]quinazolones

Article abstract of DOI:10.1007/s00044-011-9641-1

A series of azepino[2,1-b]quinazolones (C-1-C-16) have been synthesized and evaluated for their antitussive activity using citric acid-induced cough model in Guinea pigs. The compounds C-1-C-16 caused notable decrease in cough frequency and increase in co

Full text of DOI:10.1007/s00044-011-9641-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1271–1277
DOI 10.1007/s00044-011-9641-1
ORIGINAL RESEARCH
Antitussive effects of azepino[2,1-b]quinazolones
•
Kunal Nepali Mukunda S. Bande Sameer Sapra Atul Garg
•
•
•
•
Sunil Kumar Punita Sharma Rohit Goyal Naresh Kumar Satti
•
•
•
•
Om Parkash Suri Kanaya Lal Dhar
Received: 4 December 2010 / Accepted: 31 March 2011 / Published online: 11 April 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of azepino[2,1-b]quinazolones (C-1–C-
16) have been synthesized and evaluated for their antitussive
activity using citric acid-induced cough model in Guinea
pigs. The compounds C-1–C-16 caused notable decrease in
cough frequency and increase in cough latency induced by
citric acid. C-3 [2,4-dibromo-7,8,9,10-tetrahydroazepi-
no[2,1-b]quinazolin-12(6H)-one] showed notable antitus-
sive effect as compared with codeine (10 mg/kg). Various
substitutions were made at Rings A and B, and all substitu-
ents like methoxy, hydroxyl, nitro, amino (Ring A) and alkyl,
acetyl, benzoyl (Ring B) other than bromine were found to
reduce the potential of the unsubstituted 7,8,9,10-tetra-
hydroazepino[2,1-b]quinazolin-12(6H)-one as antitussive.
2002). Although cough is not life-threatening, it causes
general liability, social embarrassment, and nuisance,
thereby deteriorating the quality of life (French et al.,
1998). The medication involves huge expenditure on pre-
scription and over the counter (OTC) cough remedies
(Dicpinigaitis, 2007). Currently used antitussives are
attributed with side effects along with limited efficacy
(Chung, 2005; Schroeder and Fahey, 2002; Eccles, 2002).
Keeping this in view, development of an effective and safer
antitussive agent is required.
Alkaloids having a long history of use as antitussives are
codeine (Freestone and Eccles, 1997) and dextromethor-
phen (Wong et al., 1988). Stemona alkaloids croomine (A),
stemoninine (B), neotuberostemonine (C), and tubero-
stemonine (D) have also been reported as antitussives
(Fig. 1) (Chung et al., 2003; Xu et al., 2006).
Keywords Antitussive Á Azepinoquinazolone Á
Codeine Á Cough
The azepine ring with nitrogen at the bridgehead position
appears to be an important structural feature among the
structures in Fig. 1. Antitussive potential of quinazolones
have earlier been proven (Dua et al., 1967). With this back-
ground, in this study, we have further explored 7,8,9,10-tet-
rahydroazepino[2,1-b]quinazolin-12(6H)-one(C-1) (Fig. 1),
synthetic analog of alkaloid vasicine (E) (Fig. 1), first syn-
thesized and reported as having a bronchodilatory effect 6–10
times more potent than aminophylline by Dhar et al. (Sharma
et al., 1979; Malhotra et al., 1988; Malhotra et al., 1989;
Zabeer et al., 2006) along with its several derivatives for their
antitussive effects using citric acid-induced cough model in
conscious guinea pigs.
Introduction
Cough, a reflex defence mechanism, can be considered not
only as idiopathic in nature but also as a common symptom
of various airway inflammatory diseases (Higenbottam,
K. Nepali (&) Á M. S. Bande Á S. Sapra Á A. Garg Á S. Kumar Á
O. P. Suri Á K. L. Dhar
Laboratory for Drug Design, I.S.F. College of Pharmacy,
Moga, Punjab, India
e-mail: Kunal_8855@rediffmail.com; kunalbvk3@gmail.com
P. Sharma Á N. K. Satti
Natural Product Chemistry Division, Indian Institute
of Integrative Medicine (IIIM), Jammu, India
Chemistry
R. Goyal
Department of Pharmacology, I.S.F. College of Pharmacy,
Moga, Punjab, India
Anthranilic acid was treated with thionyl chloride to yield
corresponding sulfonamide anhydride which was further
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Med Chem Res (2012) 21:1271–1277
Fig. 1 Structures having
azepine ring with nitrogen at the
bridgehead position
H
H
O
H
H
H
H
H
H
H
O
H
O
O
H
O
O
H
O
O
H
O
H
O
H
N
N
O
H
N
O
H
H
H
O
H
A
B
C
H
H
O
CH3
H
O
O
N
CH3
H
H
O
N
N
O
H
O
H
H
N
N
H
OH
D
E
C-1
condensed with caprolactam to yield the 7,8,9,10-tetra-
hydroazepino [2,1-b]quinazolin-12(6H)-one (C-1) (Fig. 2).
Various substitutions on Rings A and B of C-1 have been
made to synthesize a series of azepinoquinazolones as
shown in Figs. 3, 4, and 5. All products reported showed
1H NMR spectra that are in agreement with the assigned
structures. Reaction courses and product mixtures were
routinely monitored by TLC on silica gel (precoated F254
Merck plates). Dragondorff reagent was used as a spraying
reagent.
Among all the synthesized compounds, C-3 having dibromo
substitution proved to be the most effective antitussive.
Moreover, the unsubstituted C-1 was found to be better
active than all the other derivatives except C-3. C-2 formed
by the reduction of C=N bond at position 5 of C-1 resulted in
drastic decline of the antitussive effect as C-2 was found to
have only mild antitussive effect. Alkylation, acetylation,
benzylation, and benzoylation at position 5 of C-2 further
reduced the potential of the synthesized compounds as an-
titussives, and the decline in the antitussive effect was
notably higher with the bulkier groups such as benzyl and
benzoyl.
Results and discussion
Citric acid induction caused notable cough in Guinea pigs
as evidenced by decreased cough latency time and
increased cough frequencies. It has been demonstrated that
the guinea pigs and humans respond to similar concentra-
tions of citric acid and responses are well correlated with
concentration–response relationship (Laude, 1993). Pre-
treatment with the compounds C1–C16 (i.p) and Codiene as
standard showed notable increase in cough latency and
decrease in cough frequencies/10 min in comparison to
vehicle control (Figs. 6, 7). The compounds C-3 showed
notable antitussive effect at 10, 20 mg/kg as compared to
codeine (10 mg/kg). C-1 also showed notable antitussive
effect in comparison with codeine, but the effect of C-3 at
20 mg/kg was more pronounced than that of C-1 (20 mg/kg).
Conclusion
Antitussive potential of the synthesized compounds was
evaluated. Decrease in cough frequency and increase in
cough latency were observed in all the tested compounds as
compared with the vehicle. The overall weight of evidence
led us to conclude that the azepinoquinazolone skeleton
represents an active moiety as far as antitussive activity is
concerned, with the dibromo derivative (C-3) showing
better activity than even codeine (10 mg/kg). All the other
substituents except bromine were found to reduce the
potential of C-1 as an antitussive. Our study can thus give
new dimensions to the investigation of the molecules with
azepinoquinazolone skeleton as antitussive agents.
O
O
O
COOH
SOCl2
O
O
C
N
HN
S
NH
NH2
N
N
O
H
C-1
Fig. 2 Mechanism showing the formation of C-1
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1273
Fig. 3 a NaBH4, Stirring, r.t.,
2 h. b Br2, CCl4, stirring, r.t.
c NaOMe, CuCl2, methanol, N2,
reflux, 12 h. d 48%HBr, reflux,
12 h
O
N
O
O
O
H3CO
c
Br
N
N
N
N
a
b
N
N
N
H
OCH3
Br
C-1
C-2
C-4
C-3
d
O
HO
N
N
OH
C-5
O
N
O
O
N
O
O2N
H2N
R-H2C-HN
N
N
a
b
N
c
N
N
N
C-1
C-7
C-6
C-(8-11)
OCH3
OCH3
NO2
CN
C-10=
C-11=
C-8=
C-9=
OCH3
OCH3
OCH3
Fig. 4 a Concentrated HNO3-H2SO4, stirring 0°C. b Sn-concentrated HCl, reflux, 2 h. c (i) R-CHO, methanol (ii) NaBH4, 0°C
Experimental
O
a
N
Chemistry
N
O
COCH3
All melting points (m.p.) were observed on a Veego
melting point apparatus and were uncorrected. IR spectra
were recorded as KBr pellets on Perkin-Elmer 882 spec-
trophotometer model. Proton (1H) nuclear magnetic reso-
nance spectroscopy was performed using Varian EM-360L
and Bruker AC-300F, 200 and 500 MHz spectrometer with
TMS as an internal standard. Plates for TLC were prepared
using silica gel G.
N
C-12
N
H
O
N
2
b
N
R
C-13 to C-16
Procedure for the synthesis of azepinoquinazolone
derivatives
C-15 = H2C
C-13 =
CH3
C-16 =
C
O
C-14 = C2H5
7,8,9,10-Tetrahydroazepino[2,1-b]quinazolin-12(6H)-one
(C-1) 13.7 g (0.1 mol) of anthranilic acid was dissolved
in dry benzene (200 ml) in a 500-ml round-bottomed flask,
and 29.5 ml of thionyl chloride (0.25 mol) was added to it.
Fig. 5 a Acetic anhydride, pyridine, stirring, r.t., 2 h. b R–X,
acetone, K2CO3, Reflux, 2 h
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Med Chem Res (2012) 21:1271–1277
Fig. 6 The effect of synthesized compounds and codiene (i.p.) on
cough frequency induced by citric acid. Results: Mean SEM.
a = P \ 0.05 vs VC; b = P \ 0.05 vs. Codiene 10 mg/kg;
c = P \ 0.05 vs. C1—20 mg/kg; [VC: vehicle control; COD:
codiene; and C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12,
C13, C14, C15, and C16: synthesized compounds in series]
Fig. 7 The effect of synthesized compounds and codeine (i.p.) on
cough latency induced by citric acid. Results: Mean SEM;
a = P \ 0.05 vs. VC; b = P \ 0.05 vs. codiene 10 mg/kg; and
c = P \ 0.05 vs. C1—20 mg/kg; [VC: vehicle control, COD:
Codiene and C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12,
C13, C14, C15 & C16: synthesized compounds in series]
The reaction mixture was refluxed for 3 h on a water bath
under anhydrous conditions. After completion of the
reaction, excess of thionyl chloride and benzene were
distilled off. Benzene (100 ml) was again added to remove
residual thionyl chloride. 11.3 g (0.1 mol) of caprolactam
was dissolved in benzene in a 100-ml round-bottomed
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flask. The solution was added slowly to the sulfinylanth-
raniloyl chloride under cold conditions and kept overnight.
Progress of the reaction was monitored on TLC. The
reaction mixture was basified with aqueous ammonia and
extracted with chloroform. The chloroform extract was
dried and distilled under reduced pressure, which yielded
crude product. The crude product was purified by column
chromatography on neutral alumina with increasing per-
centage of ethyl acetate in hexane (hexane:ethyl ace-
sodium methoxide and dried under reduced pressure. The
residue obtained was crystallized from ethanol to yield C-4
(0.55 g, 75.34%). m.p. 193°C. 1H NMR (CDCl3):d 7.15 (1H,
d, J = 2.62 Hz), 6.75 (1H, d, J = 2.62 Hz), 4.34 (2H, d,
J = 6.75 Hz), 3.93 (3H, s), 3.85 (3H, s), 3.07 (2H, d,
J = 6.45 Hz), 1.87 (6H, bs).
2,4-Dihydroxy-7,8,9,10-tetrahydroazepino[2,1-b]quinazolin-
12(6H)-one (C-5) 300 mg of C-4 (0.0010 mol) was taken
in a 25-ml round-bottomed flask and 3 ml of 48% HBr was
added to it. The reaction mixture was refluxed for 12 h and
then dried under vacuum. 5 ml of distilled water was added
to it and the solution was basified with ammonia solution.
White precipitate were formed which were filtered and dried
under vacuum to yield C-5 (0.214 g, 79.55%). m.p. 259°C.
1H NMR (CDCl3): d 6.82(1H, d, J = 2.34 Hz), 6.66 (1H, d,
J = 2.34 Hz), 4.26(2H, d, J = 6.75 Hz), 2.98 (2H, d,
J = 6.43 Hz), 1.69 (6H, s).
1
tate::90:10) to yield C-1 (11.0 g, 51%). m.p. 97–99°C. H
NMR (CDCl3): d 8.25 (1H, d, J = 8.12 Hz), 7.10–7.90
(3H, m), 4.20 (2H, d, J = 6.75 Hz), 3.15 (2H, d,
J = 6.43 Hz), 1.86 (6H, bs).
5a,6,7,8,9,10-Hexahydroazepino[2,1-b]quinazoline-12(5H)-
one (C-2) 10 g (0.046 mol) of C-1 was dissolved in etha-
nol (75 ml) in a 250-ml round-bottomed flask containing a
magnetic stirring piece 20 g of sodium borohydride
(0.054 mol) was added in small proportions and the reaction
was monitored on TLC. After completion of the reaction, the
reaction mixture was basified with ammonia solution and
extracted with chloroform. The chloroform layer was dried,
and the solid residue was subjected to column chromatog-
raphy on neutral alumina with increasing percentage of ethyl
acetate in hexane (hexane:ethyl acetate::85:15) to yield C-2
(6.12 g, 60.71%). m.p. 168°C. 1H NMR (CDCl3):d 7.89 (1H,
dd, J = 1.32 and 7.76 Hz), 7.27 (1H, m) 6.82 (1H, m), 6.64
(1H, d, J = 7.79 Hz) 4.94 (2H, m), 4.42 (2H, m), 2.93 (2H,
m), 1.25–2.14 (6H, m).
2-Nitro-7,8,9,10-tetrahydroazepino[2,1-b]quinazolin-12(6H)-
one (C-6) 2 g of C-1 was taken in a 100-ml round-bot-
tomed flask which was kept in an ice bath, an ice cold
solution of nitrating mixture (4 ml; 2 ml each of conc. HNO3
and conc. H2SO4) with constant stirring. After 1 h, the
reaction mixture was kept at room temperature for 10 h. It
was then poured on to crushed ice and rendered alkaline with
aq. Ammonia. The yellow solid thus obtained was filtered,
washed with ice cold water, dried, and chromatographed
over silica gel column. Elution with hexane–ethyl acetate
(95:5) yielded the nitro compound which was recrystallized
from ethanol m.p. 170°C. 1H NMR (CDCl3) d 9.11 (1H, d,
J = 2.58 Hz), 8.49 (1H, dd, J = 2.58 and 8.96 Hz), 7.71
(1H, d, J = 8.97 Hz), 4.43 (2H, d, J = 5.51 Hz), 3.12 (2H,
bs) 1.89 (6H, bs).
2,4-Dibromo-7,8,9,10-tetrahydroazepino[2,1-b]quinaz-
olin-12(6H)-one (C-3) 2 g (0.009 mol) of C-2 was dis-
solved in 20 ml of carbon tetrachloride, and the solution was
kept in a 100-ml round-bottomed flask containing a magnetic
stirring piece. Bromine (2.99 g, 0.018 mol) was dissolved in
10 ml of carbon tetrachloride, and the solution was added to
the reaction mixture dropwise accompanied by constant
stirring. Completion of the reaction was monitored on TLC.
The precipitate thus obtained was filtered and washed with
water. The residue obtained was crystallized from ethanol to
yield C-3 (1.74 g, 50.58%). m. p. 168°C. 1H NMR (CDCl3):
d 8.35 (1H, d, J = 2.21 Hz), 8.09 (1H, d, J = 2.21 Hz), 4.37
(2H, d, J = 6.36 Hz) 3.11 (2H, d, J = 6.21 Hz), 1.86 (6H,
bs).
General procedure for the synthesis of benzylamines C
(8–11)
For the synthesis of benzylamines, C-6 was reduced to
2-amino-7,8,9,10 -tetrahydroazepino-[2,1-b]quinazolin-12
(6H)-one (C-7). A solution of C-7 (0.4 g, 1.75 mmol) in
MeOH (8.0 ml) was added to a solution of aldehydes
(4.0 mmol) in MeOH (8.0 ml) with constant stirring. The
reaction mixture was allowed to stand overnight at room
temperature. Sodium borohydride (0.8 g, 21.1 mmol) was
added in small portions to the chilled and stirred reaction
mixture over a period of 2 h. The reaction mixture was
further stirred for 2 h. Crushed ice was added to the reac-
tion mixture and the precipitate was filtered, washed with
water, dried, and recrystallized to yield the required amines
C(8–11).
2,4-Dimethoxy-7,8,9,10-tetrahydroazepino[2,1-b]quinazolin-
12(6H)-one (C-4) 1 g of C-3 (0.0026 mol) was dissolved
in 10 ml methanol in a 100-ml round-bottomed flask. Freshly
prepared sodium methoxide (0.14 g, 0026 mol) and cupric
chloride (25 mg) was added to it. The reaction mixture was
refluxed for 12 h and the completion of the reaction was
monitored on TLC. Solvent was removed by distillation. The
solid residue obtained was washed with water to remove
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Med Chem Res (2012) 21:1271–1277
2-(3,4,5-Trimethoxybenzylamino)-7,8,9,10-tetrahydroazepino
[2,1-b]quinazolin-12(6H)-one (C-8) Yield: 36%, m.p.
170–172°C. 1H NMR (CDCl3): d 7.49 (1H, d, J = 7.45 Hz),
7.24 (1H, d, J = 7.23 Hz), 7.16 (1H, dd, J = 2.03 and
7.90 Hz), 6.69 (2H, s), 4.41 (1H, s), 4.31 (4H, m), 3.88 (9H,
s), 3.12 (2H, t, J = 6.79 Hz), 1.84 (6H, m).
5-Methyl-5a,6,7,8,9,10-hexahydroazepino[2,1-b]quinaz-
oline-12(5H)-one (C-13) m.p. -102°C. 1HNMR (CDCl3)
d 7.95 (1H, m), 7.31 (1H, m), 6.86 (1H, t, J = 7.46 Hz),
6.63 (1H, t, J = 8.55 Hz), 4.98 (1H, dd, J = 3.54 and
5.27 Hz), 4.53 (2H, m), 2.94 (3H, s), 1.25–2.14 (8H, m).
5-Ethyl-5a,6,7,8,9,10-hexahydroazepino[2,1-b]-quinazoline-
1
2-(3,4-Dimethoxybenzylamino)-7,8,9,10-tetrahydroazepino
[2,1-b]quinazolin-12(6H)-one (C-9) Yield: 67.43%, m.p.
12(5H)-one (C-14) m.p. semisolid. HNMR (CDCl3) d
7.93 (1H, m), 7.34 (1H, m), 6.83 (1H, t, J = 7.53 Hz), 6.66
(1H, t, J = 8.23 Hz), 4.93 (1H, dd, J = 3.63 and 5.34 Hz),
4.46 (2H, m), 3.40 (2H, q, J = 7.33 Hz), 1.25–2.14 (11H,
m).
206–210°C. 1H NMR (CDCl3):
d
7.49 (1H, d,
J = 8.75 Hz), 7.34 (1H, d, J = 3 Hz), 7.04 (1H, dd,
J = 2.73 and 8.95 Hz), 6.81 (2H, m), 6.79 (1H, d,
J = 6.23 Hz), 4.39 (5H, m), 3.79 (6H, s), 3.12 (2H, m),
1.85 (6H, m).
5-Benzyl-5a,6,7,8,9,10-hexahydroazepino[2,1-b]quinazoline-
1
12(5H)-one (C-15) m.p. semisolid. HNMR (CDCl3) d
2-(4-Nitrobenzylamino)-7,8,9,10-tetrahydroazepino[2,1-b]
quinazolin-12(6H)-one (C-10) Yield: 68.84%, m.p.
7.98 (1H, dd, J = 1.51 and 7.72 Hz), 7.26–7.34 (6H, m),
6.86 (1H, t, J = 7.23 Hz), 6.65 (1H, d, J = 8.22 Hz), 4.77
(2H, s), 4.50 (2H, m), 2.71 (1H, m), 1.20–2.13 (8H, m).
13CNMR (CDCl3) d 162.50, 146.27, 137.22, 133.24, 128.88,
128.79, 127.69, 127.31, 118.76, 118.22, 118.16, 114.03,
75.64, 53.47, 53.41, 44.80, 31.68, 27.59, 25.24, 25.03.
218–224°C. 1H NMR (CDCl3):
J = 8.23 Hz), 7.09–7.68(5H, m, aromatic), 5.53 (1H, s),
4.22–4.68 (4H, m), 3.07 (2H, s), 1.85 (6H, s).
d
8.17 (2H, d,
2-(4-Cyanobenzylamino)-7,8,9,10-tetrahydroazepino[2,1-b]
quinazolin-12(6H)-one (C-11) Yield: 77.33%, m.p.
5-Benzoyl-5a,6,7,8,9,10-hexahydroazepino[2,1-b]quinazo-
line-12(5H)-one (C-16) 1 g of C-2 (0.004 mol) was taken
in a 250-ml flask, and 20 ml of acetone was added into the
flask. Anhydrous potassium carbonate (2.5 g) and benzoyl
chloride (0.504 g, 0.004 mol) were added to this reaction
mixture. The reaction mixture was refluxed for 2 h. The
completion of the reaction was monitored with the help of
TLC. The work up of the reaction was done with water and
ethyl acetate. The ethyl acetate portion was dried under
reduced pressure and then subjected to column chroma-
tography to yield the required product (16) which was
eluted with hexane:ethyl acetate (95:5).
1
188–194°C. H NMR (CDCl3): d 7.10–7.93 (7H, m), 4.43
(5H, m), 3.02 (2H, s), 1.80 (6H, s).
5-Acetyl-5a,6,7,8,9,10-hexahydroazepino[2,1-b]quinazo-
line-12(5H)-one (C-12) 1 g of R-2(0.004 mol) was taken
in a 100-ml round-bottomed flask and dissolved in 10 ml
acetic anhydride. A few drops of pyridine were added to it.
The reaction mixture was refluxed for 6 h. under anhydrous
conditions. The completion of the reaction was monitored
with the help of TLC. The reaction mixture was poured on
to crushed ice. The precipitates were filtered and dried
under vacuum to yield C-12 1.09 g (90.83%). m.p.
1
m.p. -189°C. H NMR (CDCl3) d 8.08 (1H, dd, J =
2.35 and 9.46 Hz, H-1), 7.11–7.45 (7H, m), 6.50 (1H, d,
J = 7.60 Hz), 5.97 (1H, d, J = 3.92 and 10.40 Hz), 4.61
(1H, m), 3.02 (1H, m), 1.46–1.82 (8H, m). 13C (CDCl3) d
168.50, 161.41, 138.27, 134.08, 133.53, 131.57, 131.41,
130.19, 129.21, 128.47, 125.14, 124.26, 122.14, 70.14,
45.10, 32.81, 27.32, 25.31, 24.96.
1
-125°C. H NMR (CDCl3) d 8.06 (1H, dd, J = 1.55 and
7.68 Hz), 7.26–7.63 (3H, m), 4.48 (1H, dd, J = 6.31 and
13.46 Hz), 2.95 (2H, m) 2.21 (3H, s) 1.25–2.04 (8H, m).
General procedure for the synthesis of C- (13–15)
2 g of C-2 (0.009 mol) was taken in a 250-ml round-bot-
tomed flask and dissolved in 20 ml of acetone. Anhydrous
potassium carbonate (2.5 g) and alkyl or aryl iodide
(0.009 mol) was added to this reaction mixture. The reac-
tion mixture was refluxed for 2 h. The completion of the
reaction was monitored with the help of TLC. The work up
of the reaction was done with water and ethyl acetate. The
ethyl acetate portion was dried under reduced pressure and
subjected to column chromatography. The required product
C-13 to C-15 was eluted with increasing percentage of
ethyl acetate in hexane.
Pharmacological assessment
Albino guinea pigs weighing 400–600 g of either sex were
employed in different groups (n = 6) selected. Each group
included six animals. Animals were maintained in the
animal house and exposed to normal day–night cycles
under standard conditions with the temperature at
25 2°C and the relative humidity of 55–65%. The
protocol of this study was approved by the Institutional
Animal Ethics Committee (IAEC). All the compounds
C-1–C-16 were evaluated for their antitussive effect using
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Acknowledgment The authors thank Mr. Parveen Garg, Chairman
of the ISF College of Pharmacy Moga, Punjab for providing the
support and facilities.
citric acid-induced cough model in guinea pig (Laude
et al., 1994). Animals underwent a screening procedure
before pretreatment with drugs. On the first day, after a
3 min acclimatization period, the animals were first
exposed to normal saline and 5 min later to aerosolized
7.5% citric acid for a period of 10 min. Animal selection
criterion was made either on the basis of number of coughs
(\7 or[15) on exposure to aerosolized 7.5% citric acid or
their tendency to cough on exposure to normal saline.
Animal producing cough outside the stated limit of citric
acid or on exposure to aerosolized saline was excluded as
this was taken as an indication of infection or hyper-reac-
tivity. Cough challenge was given at the same time of day
for each animal and a minimum of 24-h interval was
allowed between challenges to eliminate any short-term
prophylaxis. Animals were allowed free access to food and
water up to the time of testing. Each animal was placed in a
Perspex chamber, dimensions 30 cm 9 20 cm 9 20 cm,
and exposed to an aerosolized aqueous solution of 7.5%
w/v citric acid for a period of 10 min. The output of the
aerosolizer (INCO Laboratories, Ambala, India) was
0.25 0.02 ml per minute, and same aerosolizer was used
throughout the experiment. The animals were watched
continuously by the trained observer, and number of
coughs and latency time to initial cough response were
noted. Coughs could easily be distinguished from sneeze
since there is a clear difference in sound as well as in the
behavior of the animals (fox, 1996). All the drugs were
dissolved in 4% HCl and then diluted in saline. Their pH
values were adjusted to 6.5–7.0 with 1% Na2CO3. The
samples were intraperitoneally administered at 30 min.
before the second challenge of aerosolized citric acid
(7.5% w/v) solution for 10 min. The above protocol was
performed 30 min after the intraperitoneal administration
of following solutions for a period of 10 min. The syn-
thesized compounds C-1–C-16 were administered at doses
5, 10, and 15 mg/kg (dissolved in normal saline, i.p.).
Codeine 10 mg/kg was administered as standard. The
protocol of this study was approved by the Institutional
Animal Ethics Committee (IAEC).
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Statistical analysis
All the values were expressed as Mean SEM and were
statistically analyzed using one way ANOVA followed by
Tukey’s multiple comparison test. The p \ 0.05 was con-
sidered to be statistically significant.
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