Total synthesis and analgesic activity of 6-fluoroindan-1-carboxylic acid

Article abstract of DOI:10.1016/j.tet.2008.07.010

6-Fluoroindan-1-carboxylic acid (4) was conveniently synthesised from 3-fluorobenzaldehyde in six steps. The structure of this new compound and three other intermediates, 3-fluorophenylcyanoethylacrylate (1), 3-fluorophenyl succinic acid (2) and 6-fluoro-

Full text of DOI:10.1016/j.tet.2008.07.010

Tetrahedron 64 (2008) 8642–8645
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis and analgesic activity of 6-fluoroindan-1-carboxylic acid
Sharmistha Das a, Hasina Yasmin a, M. Mehedi Masud a, Suvas C. Roy b, Lutfun Nahar c,
,
M. Mukhlesur Rahman d, Simon Gibbons d, Sitesh C. Bachar e, Satyajit D. Sarker c
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh
b Department of Pharmacy, University of Science & Technology Chittagong, Foy’s Lake, Chittagong, Bangladesh
c School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Co. Londonderry, Northern Ireland, UK
d Centre for Pharmacognosy and Phytochemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
e Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Fluoroindan-1-carboxylic acid (4) was conveniently synthesised from 3-fluorobenzaldehyde in six steps.
The structure of this new compound and three other intermediates, 3-fluorophenylcyanoethylacrylate (1),
3-fluorophenyl succinic acid (2) and 6-fluoro-3-oxo-indan-1-carboxylic acid (3) was elucidated by
comprehensive spectral data analyses. The analgesic activity of compounds 3 and 4 was assessed by the
acetic acid induced writhing in Swiss albino mice.
Received 23 April 2008
Received in revised form 19 June 2008
Accepted 3 July 2008
Available online 8 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
3-Fluorophenylcyanoethylacrylate
3-Fluorophenyl succinic acid
6-Fluoro-3-oxo-indan-1-carboxylic acid
6-Fluoroindan-1-carboxylic acid
Analgesic activity
1. Introduction
2. Results and discussion
Compounds containing indan ring systems, especially with
a carboxylic acid functionality, possess anti-inflammatory activity.
Among indan derivatives 1H-indene-3-acetic acid-5-fluoro-2-
methyl-1-[4-(methylsulfinyl)-phenyl]methylene (Sulindac) and
indan-1,3-dione are well known anti-inflammatory agents.1 Juby
et al.2 observed significant anti-inflammatory activity among a se-
ries of substituted indan-1-carboxylic acids. Moreover, a number of
methoxy indan-1-alkanoic acids were synthesised with consider-
able anti-inflammatory properties.3 Indan derivatives, with a halo-
substituted indanyl group were found to possess analgesic and
anti-inflammatory activities.4–7 It was observed that aromatic
halogen substitution could be a reasonable means of increasing the
analgesic and anti-inflammatory activities and widening the mar-
gin of safety.7 As part of our quest for new and more effective an-
algesic and anti-inflammatory agents,7 we now report on the total
synthesis of 6-fluoroindan-1-carboxylic acid (4) from 3-fluo-
robenzaldehyde in six steps, and the assessment of analgesic ac-
tivity of the intermediate 6-fluoro-3-oxo-indan-1-carboxylic acid
(3) and the target compound 4 by the acetic acid induced writhing
in Swiss albino mice.
6-Fluoroindan-1-carboxylic acid (4) was conveniently syn-
thesised from 3-fluorobenzaldehyde (5) in six steps with an overall
24% yield (Scheme 1). The five intermediates were 3-fluo-
rophenylcyanoethylacrylate (1), 3-fluorophenyl-a,b-dicyanoethyl
propionate (6), 3-fluorophenyl succinic acid (2), 3-fluorophenyl
succinyl chloride (7) and 6-fluoro-3-oxo-indan-1-carboxylic acid
(3). Among these intermediates, compounds 1–3 were isolated in
pure form. The structures of compounds 1–4 were elucidated by
spectroscopic means, notably, UV, IR, HRMS and extensive 1D and
2D NMR analyses (1H and 13C NMR, 1H–1H COSY, 1H–13C HSQC and
1H–13C HMBC).
3-Fluorobenzaldehyde (5) was condensed with ethyl-
cyanoacetate in the presence of pyridine in 1:1 molar ratio using dry
benzene as reaction solvent to produce 3-fluorophenyl-
cyanoethylacrylate (1) with an excellent yield of 86% (Scheme 1).
The reaction between compound 1 and sodium cyanide yielded
compound 3-fluorophenyl
a,b-dicyanoethylpropionate (6), which
was subjected to acid hydrolysis to obtain 3-fluorophenyl succinic
acid (2) (yield 66%) (Scheme 1).
The diacid (2) was cyclised by Friedel–Crafts acylation reaction
to obtain 6-fluoro-3-oxo-indan-1-carboxylic acid (3) via the for-
mation of 3-fluorophenyl succinyl chloride (7) as a highly moisture
sensitive liquid material (Scheme 1). The reaction was carried out in
two different solvents, nitrobenzene and CS2 to compare the yields.
* Corresponding author. Tel.: þ44 28 7032 4302; fax: þ44 28 7032 4965.
E-mail address: s.sarker@ulster.ac.uk (S.D. Sarker).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.07.010

S. Das et al. / Tetrahedron 64 (2008) 8642–8645
8643
CN
9
1.5 h, Δ
2
7
CN
F
CN
F
F
F
CHO
EtOH, NaCN
CNCH2COOEt, C6H5N
8
3
4
1
6
COOC2H5
COOC2H5
CH3COOH, C6H6, Δ, 20 h
10
6
5
1
5
Conc. HCl, 18 h, Δ
86%
COOH
COCl
COOH
AlCl3, CS2 or C6H5NO2
F
F
COOH
SOCl2, C6H6, Δ, 1.5 h
COCl
Stirring, 20 h, r.t.
7
2
O
3
10
Zn-Hg
66%
55%
COOH
7
8
F
1
2
Δ, 16 h
6
5
9
3
4
4
76%
Scheme 1. Synthesis of compounds 1–4.
It was found that the reaction carried out in CS2 gave a slightly
better yield (55% as opposed to 40%). The 3-oxo-derivative (3) was
subjected to reduction by Clemmensen reduction to obtain the
target compound 6-fluoroindan-1-carboxylic acid (4) with a yield
of 76% (Scheme 1).
fluoro substitution potentiates the analgesic activity of compounds
3 and 4, and the 6-fluoroindan-1-carboxylic acid and (4), which is
a reduced product of 3 possess slightly better analgesic activity
than the keto or 3-oxo compound.
The writhing reflex in mice induced by acetic acid is a sensitive
procedure to evaluate the potential analgesic activity of drugs. It
has been suggested that acetic acid acts by releasing endogenous
mediators, which stimulate the nociceptive neurons in mice.14
Acetic acid is sensitive to cyclooxygenase inhibitors and has been
used to evaluate the effect of analgesic agents, which primarily
inhibit the cyclooxygenase involved in prostaglandin synthesis.
Acetic acid is also sensitive to non-steroidal anti-inflammatory
drugs (NSAIDs)andto narcoticsand othercentrallyacting drugs.14–16
Recently it has been found that the nociceptive activity of acetic acid
The analgesic activity of compounds 3 and 4 was assessed by the
acetic acid induced writhing in Swiss albino mice.8 Since the anal-
gesic and the anti-inflammatory activities of various indan-1-acids
and tetrazoles were reported previously3,9–13 the 6-fluoro-3-oxo-
indan-1-carboxylic acid (3) and 6-fluoroindan-1-carboxylic acid (4)
were evaluated for their analgesic activity. The results showed that
the writhing induced by acetic acid was significantly reduced by the
test compounds in a dose dependent manner (Table 1). 6-Fluoro-3-
oxo-indan-1-carboxylic acid (3) showed a 19.5% (p<0.05) inhibition
at the dose of 25 mg/kg body weight and 30.54% (p<0.005) in-
hibition at the dose of 50 mg/kg body weight. 6-Fluoroindan-1-
carboxylic acid (4) showed 21.56% (p<0.005) inhibition at the dose
of 25 mg/kg body weight and 35.93% (p<0.0005) inhibition at the
dose of 50 mg/kg body weight. It is notable that the absence of the
ketonic carbonyl functionality at C-3 in 4, increased the cyclo-
pentane ring flexibility, and contributed to the slight increase in the
analgesic activity measured by % inhibition of induced writhing in
mice. The analgesic activity of 3 and 4 was comparable to those of
the positive controls, e.g., aminopyrine with 47.89% (p<0.0005)
inhibition at 30 mg/kg body weight, indomethacin with 48.5%
(p<0.0005) inhibition at 8 mg/kg body weight and diclofenac Na
with 62.88% (p<0.0005) inhibition at 10 mg/kg body weight. None
of the test compounds (3 and 4) displayed any significant side ef-
fects at test doses. However, the behavioural pattern of the mice
was slightly affected, e.g., reduced movement, head down and in-
creased respiration. The present study has demonstrated that the
may be due to the release of cytokines, such as TNF-a, interleukin-1b
and interleukin-8, by resident peritoneal macrophages and mast
cells.17 Based on this report, it can be assumed that in the present
study the antinociceptive action showed by compounds 3 and 4 in
theacetic acidinduced writhingtest might bedue to inhibitionof the
release of TNF-a, interleukin-1b and interleukin-8, by resident
peritoneal macrophages and mast cells.
3. Experimental
3.1. General
The chemicals and solvents used in various reactions were
purchased from Merck (Germany), BDH (India) or SD Fine Chem-
icals (India), and used without purification. The melting points
were determined by using Adco Melting Point Apparatus and were
uncorrected. Thin-layer chromatography was performed using
Table 1
Analgesic activity of 6-fluoro-3-oxo-indan-1-carboxylic acid (3) and 6-fluoroindan-1-carboxylic acid (4)
Test compounds/controls
Group
Dose (mg/kg
body weight)
Total number of writhing
MeanꢀSD
% Inhibition
6-Fluoro-3-oxo-indan-1-carboxylic acid (3)
6-Fluoroindan-1-carboxylic acid (4)
A
B
C
D
E
F
G
H
25
50
25
50
30
8
31, 29, 16, 18, 24, 17
26, 24, 13, 15, 18, 20
22, 19, 20, 22, 27, 25
19, 15, 23, 18, 12, 20
17, 08, 19, 20, 14, 09
12, 14, 18, 12, 13, 20
07, 14, 14, 05, 10, 12
32, 30, 28, 25, 28, 24
22.5ꢀ5.90
19.33ꢀ4.61
21.83ꢀ2.54
17.83ꢀ3.53
14.50ꢀ4.64
14.33ꢀ3.68
10.33ꢀ3.39
27.83ꢀ2.73
19.15a
30.54b
21.56b
35.93c
47.89c
48.50c
62.88c
d
Aminopyrine
Indomethacin
Diclofenac Na
Saline
10
d
All values are means of six mice.
a
Probability values (calculated as compared to control using student’s t-test): <0.05.
Probability values (calculated as compared to control using student’s t-test): <0.005.
Probability values (calculated as compared to control using student’s t-test): <0.0005.
b
c

8644
S. Das et al. / Tetrahedron 64 (2008) 8642–8645
Kieselgel 60 F254 plates (Merck). The absorption maxima (lmax) of
all the newly synthesised compounds were determined in absolute
methanol by using Genesis-2 spectrophotometer. By using 8010M
FTIR spectrometer, the characteristic absorption bands (nmax) of the
newly synthesised compounds were recorded on KBr disk. NMR
spectra were recorded in CD3OD on a Bruker AVANCE 500 MHz
NMR spectrometer (500 MHz for 1H and 125 MHz for 13C) using the
residual solvent peaks as internal standard. MS analyses were
performed, on a Finnigan MAT95 spectrometer. HMBC spectra were
optimised for a long range JH–C of 9 Hz and NOESY experiment was
carried out with a mixing time of 0.4 s.
0.04 mol) was hydrolysed by refluxing with concd. HCl (11.65 M,
100 mL) for 18 h (Scheme 1). On completion of the reaction, the
reaction mixture was cooled and the precipitated white crystals
were filtered, washed with water (20ꢁ3¼60 mL) and recrystallised
from hot water to obtain a pure white crystalline solid in 65.5%
yield as 3-fluorophenyl succinic acid (2) (5.55 g).
Mp 170–172 ꢂC. UV (MeOH): 278 nm. IR (KBr): 1650 (COOH) and
1120 (C–F) cmꢃ1 1H NMR (500 MHz, CD3OD) and 13C NMR
.
(125 MHz, CD3OD): Table 2. ESI-MS m/z: [MꢃH]þ 211. HR-ESIMS
m/z: [MꢃH]þ 211.0406 calcd 211.0406 for C10H8FO4.
3.2.3. Synthesis of 6-fluoro-3-oxo-indan-1-carboxylic acid (3)
3-Fluorophenyl succinic acid (2) was cyclised to 6-fluoro-3-oxo-
indan-1-carboxylic acid (3) by Friedel–Crafts acylation reaction
using two different methods, one using nitrobenzene and the other
using CS2 as the solvent, to compare the percent yield of 6-fluoro-3-
oxo-indan-1-carboxylic acid (3). In the Friedel–Crafts acylation re-
action using nitrobenzene as a solvent, compound 2 (2.33 g,
0.011 mol) was first converted to acylchloride (7) by refluxing it
with thionyl chloride (specific gravity 1.631, 2.83 g; 0.024 mol) for
1.5 h in benzene (dry, 25 mL) (Scheme 1). The benzene and excess
thionyl chloride were removed in vacuo to obtain 3-fluorophenyl
succinyl chloride (7) as a liquid. Anhydrous aluminium chloride
(8.25 g, 0.06 mol) was poured into the solution of 7 in nitrobenzene
(30 mL), and stirred thoroughly for 20 h at room temperature
(Scheme 1). The reaction mixture was poured into ice–water mix-
ture (100 mL), and the solid mass thus formed was isolated by
evaporation of nitrobenzene with steam and recrystallisation from
alcohol–water (30 mL) to give compound 6-fluoro-3-oxo-indan-1-
carboxylic acid (3) (0.85 g, 40%).
3.2. Synthesis of compounds 1–4
3.2.1. Synthesis of 3-fluorophenylcyanoethyl acrylate (1)
3-Fluorophenylcyanoethylacrylate (1) was prepared from 3-
fluorobenzaldehyde (5) (7.45 g, 0.06 mol) by the reaction with
ethylcyanoacetate (6.78 g, 0.06 mol) through refluxing in benzene
(dry, 60 mL) in presence of pyridine (2 mL) and glacial acetic acid
(2 mL) for 20 h (Scheme 1). On completion of the reaction, benzene
was removed in vacuo to obtain a solid residue. The residue was
treated with concd. HCl (5ꢁ2¼10 mL) to remove pyridine as salt
present in the reaction mixture. Then the reaction mixture was
washed repeatedly with water (50ꢁ3¼150 mL) to remove all water
soluble impurities, filtered and recrystallised in acetone–water to
obtain compound 1 as a white crystalline solid (yield 11.38 g,
86.64%).
Mp 86–88 ꢂC. UV (MeOH): 280 nm. IR (KBr): 2235 (CN), 1690
(–COOC2H5 ester) and 1120 (C–F) cmꢃ1. 1H NMR (500 MHz, DMSO-
d6) and 13C NMR (125 MHz, DMSO-d6): Table 2. CIMS m/z:
[MþNH3]þ 237. HR-EIMS m/z: [M]þ 219.0692 calcd 219.0695 for
In the Friedel–Crafts acylation reaction using CS2 as the solvent,
compound 2 (2.33 g, 0.011 mol) was converted to acylchloride by
refluxing it with thionyl chloride (specific gravity 1.631, 2.830 g;
0.024 mol) for 1.5 h in benzene (dry, 30 mL). The benzene and ex-
cess thionyl chloride were removed in vacuo to obtain 3-fluo-
rophenyl succinyl chloride (7) as a liquid. Anhydrous aluminium
chloride (8.25 g, 0.06 mol) was poured into the solution of 7 in CS2
(30 mL), and stirred thoroughly for 20 h at room temperature
(Scheme 1). The reaction mixture was poured into ice–water mix-
ture (100 mL). The solvent CS2 was evaporated in a hot water bath
(50 ꢂC). After cooling the mixture a precipitate was formed, filtered,
washed thoroughly with water and recrystallised from alcohol–
water to afford compound 6-fluoro-3-oxo-indan-1-carboxylic acid
(3) (1.18 g, 55.3%).
C
12H10O2FN.
3.2.2. Synthesis of 3-fluorophenyl succinic acid (2)
Compound 1 (10.95 g, 0.05 mol) was allowed to react with NaCN
(2.45 g, 0.05 mol) dissolved in water (10 mL) in presence of 50%
alcohol (100 mL) under reflux for 1.5 h (Scheme 1). The reaction
mixture was cooled, poured into water (200 mL), concd. HCl
(100 mL) was added to it and kept overnight. The biphasic mixture
produced was separated and the aqueous part was extracted with
chloroform (50ꢁ3¼150 mL) and added to the biphasic mixture
part. The combined chloroform extract was washed with water
(50 mL), dried over sodium sulfate and finally concentrated under
reduced pressure. 3-Fluorophenyl-a,b-dicyanoethyl propionate (6)
was obtained as a semisolid mass (10.30 g, yield 84%). Compound 6
was used without further purification or spectral analyses for the
synthesis of 3-fluorophenyl succinic acid (2). Compound 6 (9.84 g,
White crystalline solid. Mp 162–164 ꢂC. UV (MeOH): 281 nm. IR
(KBr): 1680 (pC]O), 1650 (COOH) and 1120 (C–F) cmꢃ1 1H NMR
.
(500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6): Table 2.
Table 2
1H and 13C NMR data of compounds 1–4
Position Chemical shifts
d in ppm
1H NMR (coupling constant J in Hz)
13C NMR
1a
2b
3a
4b
1a
2b
3a
46.4
37.2
4b
48.4
23.7
29.3
1
2
3
4
5
6
7
8
9
10
d
d
3.94 dd (12.5, 6.5)
2.96 dd (21.5, 12.5), 2.58 dd (21.5, 6.5) 2.54 m, 2.22 m
3.52 dd (12.0, 8.0) 133.8
141.7
115.2
161.2 196.8
7.81c
d
7.12 m
d
115.0
161.1
d
2.74 m, 2.33 m
7.36 br dt
7.57 m
7.79c
8.32 s
d
7.12 m
7.36 m
7.12 m
2.56 dd (5.0, 17.0)
7.13 d (8.0)
7.37 dd (8.0, 2.2)
d
7.11 d (8.0)
7.33 dd (8.0, 2.1)
d
120.9
132.4
128.3
154.5
115.2
107.8
162.1
14.4, 63.4
114.3 123.9 124.0
130.2 112.1 110.9
124.4 163.4 163.1
46.5 114.9 112.9
37.2 141.4 138.4
173.6 130.4 132.3
170.3 173.6 174.0
7.09 d (2.2)
7.04 d (2.1)
3.94 dd (5.0, 10.0), 2.96 dd (10.0, 17.0)
d
d
d
d
d
d
d
d
d
12.44 br s
d
12.23 br s
d
10-C2H5 1.38 t (9.0), 4.37 q (9.0)
d
d
d
a
Spectrum obtained in DMSO-d6.
Spectrum obtained in CD3OD.
Overlapped peaks.
b
c

S. Das et al. / Tetrahedron 64 (2008) 8642–8645
8645
CIMS m/z: [MþNH3]þ 212. HR-ESIMS m/z: [MþH]þ 195.0458 calcd
3.3.3. Protocol
195.0457 for C10H8FO3.
The mice were randomly divided into eight groups, which
consisted of six mice in each group. Groups A and B received the
test compound 3, and groups C and D received the test com-
pound 4. All test compounds were administered orally with
a help of a feeding needle at doses of 25 and 50 mg/kg body
weight in the groups, respectively. Mice groups E, F and G re-
ceived positive controls, aminopyrine 30 mg/kg body weight,
diclofenac Na 10 mg/kg body weight and indomethacin 8 mg/kg
body weight, respectively. Group H was kept as negative control
giving saline solution only. A 40 min interval was allowed to
ensure proper absorption of the administered compounds. Then
the writhing inducing chemical, acetic acid solution (0.7%, 0.1 mL/
10 g), was administered intraperitoneally (ip) to each of the an-
imals of a group. After an interval of 10 min, numbers of writhing
were counted for another 10 min. The average percent decrease
in writhing was calculated and compared against the control
(saline treated) group. Percent inhibition was calculated using the
following formula.
3.2.4. Synthesis of 6-fluoroindan-1-carboxylic acid (4)
6-Fluoroindan-1-carboxylic acid (4) was obtained from 6-flu-
oro-3-oxo-indan-1-carboxylic acid (3) by Clemmensen reduction
(Scheme 1). Compound 3 (1.94 g, 0.01 mol) was treated with
amalgamated zinc (10 g), water (10 mL), concd. HCl (20 mL,
11.65 M) and benzene (30 mL) and refluxed for 16 h. The layers
were separated and the aqueous layer was extracted with benzene
(20ꢁ3¼60 mL). The combined benzene layers were washed with
water and dried over anhydrous sodium sulfate. After removal of
the solvent under reduced pressure, the resulting solid mass was
recrystallised from alcohol–water to give 6-fluoroindan-1-carbo-
xylic acid (4) (1.36 g, 76%).
White crystalline solid. Mp 128–130 ꢂC. UV (MeOH): 280 nm. IR
(KBr): 1650 (COOH) and 1120 (C–F) cmꢃ1 1H NMR (500 MHz,
.
DMSO-d6) and 13C NMR (125 MHz, DMSO-d6): Table 2. CIMS m/z:
[MþNH3]þ 198. HR-ESIMS m/z: [MþH]þ 181.0665 calcd 181.0665
for C10H10FO2.
% Inhibition ¼ ½ðWc ꢃ WtÞ=Wcꢄ ꢁ 100
where, Wc¼average writhing counted for control group;
Wt¼average writhing calculated for individual test group.
3.3. Assessment of analgesic activity
The analgesic activity of compounds 3 and 4, and the positive
controls, aminopyrine (BDH, Germany), indomethacin (BDH, India)
and diclofenac Na (BDH, Germany) was studied by acetic acid in-
duced writhing test as described by Vogel and Vogel8 with little
modification.
Acknowledgements
One of the authors (S.C.B.) is grateful to the Ministry of Science,
Information, Communication and Technology, Government of
Bangladesh, Dhaka for a grant (No. BTAJOPROMA/Sha-9/B.:ANU.:
PRO.:/2007-2008/BS-74/118) to perform some parts of the project.
The mass spectral analyses were carried out in the EPSRC National
Mass Spectrometry Service Centre, Swansea, and the NMR spec-
troscopy was performed in the School of Pharmacy, University of
London.
3.3.1. Animals
Young Swiss albino mice aged 4–5 weeks weighed 20–25 g of
either sex were used for the assessment of analgesic activity. They
were collected from the animal house of the International Center
for Diarrheal Diseases and Research’ Bangladesh (ICDDR’B),
Mohakhali, Dhaka. The mice were kept in groups of six in plastic
polyvinyl cages (BIK industries, India) having dimensions of
(28ꢁ22ꢁ13) cm3. The animals were given standard mice feed de-
livered by ICDDR’B and water ad libitum. They were kept in the
laboratory environment for 7 days maintaining light and dark;
were fasted overnight and weighed before the experiment.
References and notes
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3.3.2. Test compounds and positive controls
6-Fluoro-3-oxo-indan-1-carboxlic acid (3) and 6-fluoroindan-1-
carboxlic acid (4) were weighed in 20 mg each and taken into
separate graduated test tubes. Compounds were then dissolved in
2 mL of saline solution and a few drops of 0.1 N NaOH in saline. The
pH of the solution was adjusted to 7.4ꢀ0.2 by drop wise addition of
0.1 N HCl in saline. Then the final volumes of the solutions were
adjusted to 10 mL with saline water.
The solutions of the positive controls aminopyrine (BDH, Ger-
many), indomethacin (BDH, India) and diclofenac Na (BDH, Ger-
many) were prepared as follows. Each of these drugs (5 mg) was
dissolved separately in 2 mL of saline solution and 2–3 drops of
0.1 N NaOH in saline. The pH of the solution was adjusted to 7.4ꢀ0.2
by drop wise addition of 0.1 N HCl in saline. Finally, the volume was
adjusted to 6 mL with saline.