1724
I.M.I. Fakhr et al. / European Journal of Medicinal Chemistry 44 (2009) 1718e1725
absolute ethanol for a period of 4 h. After cooling, the mixture
was filtered off to provide the corresponding product (com-
pounds 7 and 8), which was crystallized from the appropriate
solvent.
approval from the Ethics Committee of the National Research
Centre and in accordance with the recommendations for the
proper care and use of laboratory animals (NIH Publication
No. 85-23, revised 1985). Equal groups of six rats or mice/
group were used in all experiments. Experiments were per-
formed between 9 am and 3 pm.
4
.1.6.1.
N-((1H-indol-3-yl)methylene)-5-nitrobenzo[b]thio-
phene-2-carbohydrazide (7). Yellow solid (0.25 g, 90%); mp
ꢂ
9
(
0e92 C (from EtOH/DMF). IR (KBr) n
2NH), 1677 (C]O), 1574 (C]N) cm
3220e3310
1
max
1
4.2.2. Tests of inflammation: carageenin-induced
paw oedema assay
ꢀ
;
H NMR
0
0
(DMSO-d ) d 7.17 (br s, 1H, NH), 7.21 (m, 2H, H-5 , H-6 ,
indole), 7.82 (d, 1H, H-7 , indole), 8.2 (s, 1H, H-2 , indole),
6
All tested compounds 2, 6e9 were screened for anti-
inflammatory activity using the carrageenan-induced paw
oedema assay in rats. Their activity was compared with the clas-
sic NSAID piroxicam. Paw oedema was induced by subplantar
injection of 100 mL of 1% sterile carrageenan lambda in saline
into the right hind paw [20]. Contra-lateral paw received an
equal volume of saline. Paw volume was determined immedi-
ately before carrageenan injection and at selected times thereaf-
ter using a plethysmometer (Ugo Basile, Milan, Italy). The
oedema component of inflammation was quantified by measur-
ing the increase in paw volume (mL) at before carrageenan in-
jection and at 1, 2, 3 and 4 h after carrageenan injection with
respect to the pre-injection value for each animal. Oedema
was expressed as a percentage of change from control (pre-
drug) values. The effect of systemic administration of each of
the test drugs at doses of 25 or 50 mg/kg (0.5 mL, s.c., n ¼ 6
per group) or piroxicam (10 mg/kg, s.c., 0.5 mL) given as
a 30 min pretreatment was studied. The control groups received
saline (0.5 mL, n ¼ 6 per group, s.c.) instead.
0
0
8
1
(
.25e8.35 (two dd, 2H, H-6, H-7, benzothiophene), 8.41 (s,
0
H, H-3, benzothiophene), 8.45 (d, 1H, H-4 , indole), 8.75
s, 1H, H-4, benzothiophene), 8.9 (s, 1H, CH]N), 12.01 (s,
þ
1
H, NH). MS: m/z (%) 364 (M , 7), 318 (15), 222 (13), 208
14), 156 (72), 110 (100), 77 (67). Anal. Calcd for
(
C H N O S: C, 59.33; H, 3.32; N, 15.38; O, 13.17; S,
1
8 12 4 3
8
.80. Found: C, 59.26; H, 3.28; N, 15.34; O, 13.26; S, 8.76.
4
.1.6.2. N-((5-methylfuran-2-yl)methylene)-5-nitrobenzo[b]thi-
ophene-2-carbohydrazide (8). Yellow (0.23 g, 92%); mp
ꢂ
7
8e80 C (from EtOH/DMF). IR (KBr) n
ꢀ
3214 (NH),
max
H NMR (DMSO-d6)
1
1
1
d 2.7 (s, 3H, CH ), 6.1 (d, 1H, H-4, furan proton), 6.7 (d,
694 (C]O), 1590 (C]N) cm
;
3
1
H, H-3, furan proton), 7.2 (br s, 1H, NH), 8.24e8.38 (two
dd, 2H, H-6, H-7, benzothiophene), 8.40 (s, 1H, H-3, benzo-
thiophene), 8.74 (s, 1H, CH]N), 8.8 (s, 1H, H-4, benzothio-
þ
phene). MS: m/z (%) 329 (M , 24), 283 (41), 234 (69), 222
(
(
12), 208 (19), 167 (18), 136 (31), 103 (19), 92 (32), 77
100), 64 (52). Anal. Calcd for C H N O S: C, 54.71; H,
3.37; N, 12.76; O, 19.43; S, 9.74. Found: C, 54.63; H, 3.32;
N, 12.75; O, 19.48; S, 9.70.
In anti-inflammatory study, the percentage of oedema
inhibition was calculated from the mean effect in the control
and treated animals according to the following equation:
1
5 11 3 4
%
Oedema inhibition
f½ð% oedema formation of control group
% oedema formation of treated groupÞꢃ=
% oedema formation of control groupꢃ100g
4
2
.1.7. Synthesis of the 1-(5-nitrobenzo[b]thiophene-
-carbonyl)-4-phenylthiosemicarbazide (9)
A mixture of 5-nitrobenzo[b]thiophene-2-carbohydrazide 2
¼
ꢀ
½
(0.237 g, 1 mmol) and phenylisothiocyanate (0.135 g, 1 mmol)
was refluxed in absolute ethanol for 4 h. After cooling, the
mixture was filtered off to afford 1-(5-nitrobenzo[b]thiophene-
2
from EtOH/DMF gave analytically pure material; Yellow
-carbonyl)-4-phenyl-thiosemicarbazide (9). Recrystallization
4.2.3. Anti-nociceptive activity
This activity was determined by measuring the responses of
animals to the Koster test and hot-plate test.
ꢂ
(
(
0.34 g, 91%); mp 217e219 C. IR (KBr) n
3164e3325
max
ꢀ
1
3NH), 1687 (C]O) cm ; H NMR (DMSO-d ) d 3.3 (br
1
6
s, 2H, 2NH), 7.15e7.4 (m, 5H, Ph), 8.20e8.35 (two dd, 2H,
H-6, H-7), 8.45 (s, 1H, H-3), 9.0 (s, 1H, H-4), 11.1 (br s,
4.2.3.1. Hot-plate assay. The hot-plate test was performed on
rats by using an electronically controlled hot plate (Ugo
þ
ꢂ
ꢂ
1
2
H, NH, D O-exchangeable). EIMS: m/z (%) 373, M (5),
2
67 (25), 222 (8), 208 (12), 167 (10), 135 (40), 104 (19), 93
Basile, Italy) heated to 52 C (ꢁ0.1 C) [21]. The cut-off
time was 30 s. Groups of rats (n ¼ 6 per group) were given
different compounds 2, 6, 7, 8 or 9 at the dose of 25 mg/kg,
s.c., saline (control), or piroxicam at 10 mg/kg, s.c., 1 h mini-
mum prior to testing. The experimenter was blind to dose and
treatment. Latency to lick a hind paw or jump out of the appa-
ratus was recorded for the control and drug-treated groups.
Pain thresholds were measured sequentially before and at 1
and 2 h post-treatment.
(
42), 77 (100), 64 (22). Anal. Calcd for C H N O S : C,
16 12 4 3 2
5
5
1.60; H, 3.25; N, 15.04; O, 12.89; S, 17.22. Found: C,
1.51; H, 3.22; N, 15.01; O, 12.94; S, 17.19.
4
4
.2. Pharmacological assay
.2.1. Animals
SpragueeDawley strain rats weighing 120e130 g or Swiss
albino mice 20e25 g body weight (National Research Centre,
Cairo) was used. All animal procedures were performed after
4.2.3.2. Acetic acid-induced writhing (Koster test). Separate
groups of six mice each were administered vehicle and/or