Facile synthesis of bis(4,5-dihydro-1H-pyrazole-1-carboxamides) and their thio-analogues of potential PGE2 inhibitory properties

Article abstract of DOI:10.1016/j.ejmech.2008.10.020

A variety of bis(3-aryl-4,5-dihydro-1H-pyrazole-1-thiocarboxamides) 2a-h were prepared via reaction of bis(2-propen-1-ones) 1a-h with thiosemicarbazide in ethanolic KOH solution. Meanwhile, bis(3-aryl-4,5-dihydro-1H-pyrazole-1-carboxamides) 3a-d were obta

Full text of DOI:10.1016/j.ejmech.2008.10.020

European Journal of Medicinal Chemistry 44 (2009) 2172–2177
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Facile synthesis of bis(4,5-dihydro-1H-pyrazole-1-carboxamides)
and their thio-analogues of potential PGE2 inhibitory properties
,
Flora F. Barsoum a, Adel S. Girgis b
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
b Pesticide Chemistry Department, National Research Centre, El-Behoos St., Dokki, 12622 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of bis(3-aryl-4,5-dihydro-1H-pyrazole-1-thiocarboxamides) 2a–h were prepared via reaction of
bis(2-propen-1-ones) 1a–h with thiosemicarbazide in ethanolic KOH solution. Meanwhile, bis(3-aryl-
4,5-dihydro-1H-pyrazole-1-carboxamides) 3a–d were obtained through reaction of 1a–d with semi-
carbazide hydrochloride in refluxing with acetic acid. Anti-inflammatory activity screening of the
synthesized compounds 2a–f,h; 3a–d (at a dose of 50 mg/kg of body weight) utilizing in vivo acute
carrageenan-induced paw oedema standard method in rats exhibited that many of the tested
Received 16 June 2008
Received in revised form
9 October 2008
Accepted 20 October 2008
Available online 26 October 2008
compounds reveal considerable anti-inflammatory properties, especially 2e and
f which reveal
Keywords:
remarkable activities relative to indomethacin (which was used as a reference standard at a dose of
10 mg/kg of body weight). Ulcerogenic liability for the most promising prepared anti-inflammatory
active agents (2b,c,e and f) (at a dose of 50 mg/kg of body weight) using indomethacin as a reference
standard (at a dose of 10 mg/kg of body weight) indicated that compounds 2b and c exhibit lower ulcer
index values than the used reference standard itself. PGE2 inhibitory properties of the highly promising
synthesized anti-inflammatory active agents (2b,c,e and f) were determined by PGE2 assay kit technique,
which reveal remarkable activities coincide greatly with the observed anti-inflammatory properties.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Bis(2-propen-1-ones)
Bis(3-aryl-4,5-dihydro-1H-pyrazole-1-
carboxamides)
Bis(3-aryl-4,5-dihydro-1H-pyrazole-1-
thiocarboxamides)
Anti-inflammatory
PGE2
1. Introduction
antiamoebic properties than their corresponding ligands [3–5].
Invasive amoebiasis caused by E. histolytica is one of the world’s
2-Pyrazolines represent an important class of heterocycles due
to their highly pronounced biological and pharmacological activi-
ties. It has been reported that, 3,5-diaryl-4,5-dihydro-1-thio-
most prevalent and fatal infectious diseases in many Asian, South
American and tropical parts of Africa [3,8]. Recently, 1-thio-
carbamoyl-2-pyrazolines were exhibited anti-tubercular properties
against Mycobacterium tuberculosis H37Rv [9], beside antidepres-
sant and anticonvulsant activities [10–13].
In the present work, it is intended to investigate synthesis of
bis(2-pyrazoline-1-carboxamide) containing compounds as well as
their thio-analogues, i.e. construction of new heterocyclic deriva-
tives possessing two pharmacologically active unites, utilizing
easily accessible starting chemicals and facile synthetic approaches.
The anti-inflammatory properties of the newly prepared hetero-
cycles will be screened. In addition, the PGE2 inhibitory properties
for the most promising anti-inflammatory active agents will be
determined.
carbamoyl-1H-pyrazole
derivatives
possess
considerable
anti-inflammatory activity with no ulcerogenic effects [1]. Addi-
tionally, many analogues were found to be highly inhibitory active
agents against both monoamine oxidases A and B (MAO-A and
MAO-B) isoforms, which indicate their efficiency in treating
Alzheimer’s disease [1]. A recent approach in the treatment of many
serious diseases such as cancer, AIDS, cardiovascular diseases and
Alzheimer’s disease is the development of drugs with multiple
actions [1,2]. In this regard, compounds such as pyrazolines, which
possess different biochemical and pharmacological actions, may be
helpful in this respect.
A variety of 1-thiocarbamoyl-2-pyrazoline derivatives and their
palladium II complexes were reported to be antiamoebic active
agents by screening their in vivo activity against Entameoba histo-
lytica [3–7] where, the palladium complexes show better
2. Results and discussion
2.1. Chemistry
Reaction of bis(2-propen-1-ones) 1a–h with thiosemicarbazide
in refluxing ethanolic KOH solution afforded directly bis(3-aryl-4,5-
dihydro-1H-pyrazole-1-thiocarboxamides) 2a–h (Scheme 1). The
* Corresponding author. Tel.: þ20 2 22352405; fax: þ20 2 33370931.
E-mail address: girgisas10@yahoo.com (A.S. Girgis).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.020

F.F. Barsoum, A.S. Girgis / European Journal of Medicinal Chemistry 44 (2009) 2172–2177
2173
S
O
A
NH2
NH2
HCl
H2N
N
H
H2N
N
H
R
R
KOH, EtOH
AcOH
O
O
(1)
A
A
R
R
R
R
N
N
N
N
NH2
N
N
H2N
N
N
NH2
S
H2N
S
O
O
(2)
(3)
1a, 2a, 3a; A = 2-O(CH2)2O-2', R = Ph
1b, 2b, 3b; A = 2-O(CH2)2O-2', R = 4-ClC6H4
1c, 2c, 3c; A = 2-O(CH2)2O-2', R = 4-FC6H4
1d, 2d, 3d; A = 2-O(CH2)2O-2', R = 4-H3CC6H4
1e, 2e; A = 2-O(CH2)2O-2', R = 2-thienyl
1f, 2f; A = 4-O(CH2)2O-4', R = Ph
1g, 2g; A = 4-O(CH2)2O-4', R = 4-ClC6H4
1h, 2h; A = 4-O(CH2)2O-4', R = 4-H3CC6H4
Scheme 1.
structure of 2a–h was established through spectroscopic (IR, 1H
NMR) as well as elemental analyses data. The IR spectra of 2a–h
reveal the presence of thiocarboxamide amino stretching vibration
Structure–activity relationships based on the observed results
indicated that, the type of aryl group substitution attached to the 3-
position of pyrazoline nucleus plays a controlling role for devel-
oping the exhibited pharmacological properties. It has been noticed
that, attachment of the phenyl group oriented at the 3-position of
pyrazoline heterocycle with a chlorine atom seems more favorable
for constructing an anti-inflammatory active agent than the case of
using a methyl residue as exhibited in pairs 2b,d (potency, 81.8,
54.5, respectively) and 3b, 3d (potency, 67.1, 33.6, respectively). It is
also obvious that, adopting a thienyl function (a bio-isostere of
phenyl group) at the 3-position of pyrazoline heterocycle seems
preferable for obtaining an effective pharmacological agent as
exhibited in pairs 2a, 2e (potency, 36.3, 100.0, respectively). Alter-
natively, no precise rule could be attained through the observed
results explaining the role of carbamoyl or thiocarbamoyl function
in affecting the pharmacological properties.
bands at
n
¼ 3482–3123 cmꢀ1
.
1H NMR spectra of 2a–h exhibit
three signal sets each appears as a double doublets due to the
presence of two symmetrical pyrazoline systems, assignable for
nonmagnetically equivalent pyrazoline H2C-4 (at
3.80–3.89) coupled with each other and in turn with the vicinal
methine proton HC-5 (
¼ 5.98–6.10, Jgem ¼ 17.7–18.0 Hz, Jvic ¼ 3.3–
3.6, 11.1–11.7 Hz), in addition to the two methylene ether linkage
which appears as a sharp singlet signal at
d
¼ 3.00–3.20,
d
d
¼ 4.26–4.38.
Similarly, reaction of 3,30-[1,2-ethanediylbis(oxy-2,1-phenyl-
ene)]bis(1-aryl-2-propen-1-ones) 1a–d with semicarbazide
hydrochloride in refluxing acetic acid gave directly bis(3-aryl-4,
5-dihydro-1H-pyrazole-1-carboxamides) 3a–d. The IR spectra of
3a–d exhibit the amidic carbonyl amino function (at
n
¼ 3489–
3132 cmꢀ1) beside the amidic carbonyl residue (at
n
¼ 1683–
1677 cmꢀ1). 1H NMR spectral features of 3a–d are closely similar to
those of 2a–h exhibiting the nonmagnetically equivalent methy-
lene H2C-4 as double doublet signals at
d
¼ 2.91–2.95, 3.63–3.70
Table 1
beside the double doublet pyrazoline HC-5 at
(Jgem ¼ 17.7–18.0 Hz, Jvic ¼ 5.1–5.4, 12.0–12.6 Hz).
d
¼ 5.54–5.59
Anti-inflammatory activity of the tested compounds using acute carrageenan-
induced paw oedema in rats at a dose of 50 mg/kg of body weight.
Compound
Mean swelling volume (ml) % Inhibition of oedema Potencyc
2.2. Anti-inflammatory activity
Control
Indomethacin
0.375 ꢁ 0.063b
0.122 ꢁ 0.068a
0.283 ꢁ 0.063b
0.168 ꢁ 0.099a
0.195 ꢁ 0.115a
0.237 ꢁ 0.112a
0.122 ꢁ 0.086a
0.062 ꢁ 0.023a
0.245 ꢁ 0.166a,b
0.213 ꢁ 0.087a
0.205 ꢁ 0.093a
0.297 ꢁ 0.172b
0.290 ꢁ 0.086b
00.0
67.5
24.5
55.2
48.0
36.8
67.5
83.5
34.7
43.2
45.3
20.8
22.7
–
100.0
36.3
81.8
71.1
54.5
100.0
123.7
51.4
64.0
67.1
30.8
33.6
Anti-inflammatory activity of the synthesized compounds 2a–
f,h; 3a–d (at a dose of 50 mg/kg of body weight) was determined
in vivo by the acute carrageenan-induced paw oedema standard
method in rats [14–17]. The anti-inflammatory properties were
recorded after 4 h of inflammation induction and compared with
that of indomethacin (at a dose of 10 mg/kg of body weight)
which was used as a reference standard. From the obtained results
(Table 1, Figs. 1–3), it has been noticed that many of the tested
compounds exhibit considerable anti-inflammatory properties,
especially 2e and f which reveal remarkable activities with
potency (% oedema inhibition of the tested compounds relative to
2a
2b
2c
2d
2e
2f
2h
3a
3b
3c
3d
a
Statistically significant from the control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
Potency was expressed as % oedema inhibition of the tested compounds relative
b
c
%
oedema inhibition of indomethacin) 100.0 and 123.7,
respectively.
to % oedema inhibition of indomethacin ‘‘reference standard’’.

2174
F.F. Barsoum, A.S. Girgis / European Journal of Medicinal Chemistry 44 (2009) 2172–2177
140
120
100
80
60
40
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
20
0
0.05
0
Tested Compounds
Tested Compounds
Fig. 3. Anti-inflammatory activity potency of the tested compounds relative to indo-
methacin which was used as a reference standard.
Fig. 1. Mean oedema volume (ml) of the tested compounds.
exhibits the highest promising anti-inflammatory properties also
reveals the highest inhibitory level of PGE2 (83.33 pg/ml) compa-
rable with indomethacin (98.33 pg/ml) which was used as a refer-
ence standard in this study. Similarly, compounds 2b,c and e reveal
considerable PGE2 inhibitory activities (110.83, 114.67, 96.50 pg/ml,
respectively).
The most promising anti-inflammatory active agents (2b,c,e and
f) were selected to be screened for their pharmacological properties
at lower doses (20, 10 mg/kg of body weight). From the obtained
results (Table 2), it has been noticed that, compound 2f reveals
highly distinguished activity at both 20 and 10 mg/kg of body
weight (% inhibition of oedema 79.5, 74.4, respectively) comparable
to indomethacin (% inhibition of oedema 67.3) which was used as
a reference standard (at a dose of 10 mg/kg of body weight). Based
on these observed pharmacological results, the ED50 values for the
most promising prepared anti-inflammatory active agents (2b,c,e
and f) were statistically calculated using the linear best fit tech-
nique giving values 34.59, 40.84, 28.82 and 23.05, respectively.
3. Experimental
Melting points are uncorrected and recorded on an Electro-
thermal 9100 digital melting point apparatus. IR spectra (KBr) were
recorded on a Bruker Vector 22 spectrophotometer. 1H NMR spectra
were recorded on a Varian MERCURY 300 (300 MHz) spectrometer.
The starting compounds 1a–h were prepared according to the
previously reported procedures [18].
2.3. Ulcerogenic liability
Ulcerogenic liability of the most promising prepared anti-
inflammatory active agents (2b,c,e and f) was determined using the
standard method [18,19] at a dose of 50 mg/kg of body weight using
indomethacin (at a dose of 10 mg/kg of body weight) as a reference
standard. From the obtained data (Table 3, Fig. 4) it has been
noticed that, compounds 2b and c reveal the lowest ulcer index
values (12.16, 11.51, respectively) and they are considered more
safer than indomethacin (reference standard) itself which reveals
ulcer index 13.49.
3.1. Synthesis of bis(3-aryl-4,5-dihydro-1H-pyrazole-1-
thiocarboxamides) 2a–h (general procedure)
A solution of 1a–h (2.5 mmol) and thiosemicarbazide (5 mmol)
in absolute ethanol (25 ml) containing KOH (6 mmol) was boiled
under reflux for the appropriate time. The separated solid while
refluxing was collected, washed with water and crystallized from
a suitable solvent affording 2a–e. In case of 1f–h, the clear reaction
mixture was stored at room temperature (25 ꢂC) overnight so, the
separated solid was collected, washed with water and crystallized
from a suitable solvent affording 2f–h.
2.4. PGE2 inhibitory properties
PGE2 inhibitory properties of the most promising prepared anti-
inflammatory active agents (2b,c,e and f) were determined using
the previously described 6-day-old air pouch standard method in
rats [20] via a commercial PGE2 assay kit (R&D Systems, Inc.,
Minneapolis, USA). From the obtained results (Table 4, Fig. 5), it has
been noticed that all the tested compounds effectively reduce PGE2
level. It has also been noticed that the PGE2 inhibitory properties of
the tested compounds coincide greatly with our described anti-
inflammatory properties. In other words, compound 2f, which
3.1.1. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis(4,5-dihydro-
3-phenyl-1H-pyrazole-1-thiocarboxamide) (2a)
Reaction time 1 h, pale yellow crystals from N,N-dime-
thylformamide – water as 1:1 v/v, mp 285–286 ꢂC, yield 52%. IR:
nmax/cmꢀ1 3482, 3360 (NH2), 1673, 1595, 1566, 1450 (C]N, C]C,
C]S). 1H NMR (DMSO-d6):
d 3.03 (dd, 2H, upfield H’s of pyr. H-4,
Table 2
Anti-inflammatory activity of the most promising prepared anti-inflammatory
active agents using acute carrageenan-induced paw oedema in rats at doses of 20
and 10 mg/kg of body weight.
90
80
70
60
50
40
30
20
10
0
Compound
Mean swelling volume (ml)
% Inhibition of oedema
Control
Indomethacin
0.352 ꢁ 0.054b
0.115 ꢁ 0.038a
0.163 ꢁ 0.024a
0.175 ꢁ 0.025a
0.200 ꢁ 0.025a
0.208 ꢁ 0.038a
0.135 ꢁ 0.020a
0.142 ꢁ 0.018a
0.072 ꢁ 0.027a
0.090 ꢁ 0.014a
00.0
67.3
53.7
50.3
43.2
40.9
61.6
59.7
79.5
74.4
2b
2c
2e
2f
20 mg/kg
10 mg/kg
20 mg/kg
10 mg/kg
20 mg/kg
10 mg/kg
20 mg/kg
10 mg/kg
-10
Tested Compounds
a
Statistically significant from the control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b
Fig. 2. % Inhibition of oedema for the tested compounds.

F.F. Barsoum, A.S. Girgis / European Journal of Medicinal Chemistry 44 (2009) 2172–2177
2175
IR: nmax/cmꢀ1 3438, 3239, 3130 (NH2), 1659, 1583, 1470 (C]N, C]C,
C]S). 1H NMR (DMSO-d6):
2.28 (s, 6H, 2ArCH3), 3.00 (dd, 2H,
upfield H’s of pyr. H-4, H-40, J ¼ 3.6, 17.7 Hz), 3.80 (dd, 2H, downfield
H’s of pyr. H-4, H-40, J ¼ 11.7, 18.0 Hz), 4.35 (s, 4H, 2OCH2), 6.07 (dd,
2H, pyr. H-5, H-50, J ¼ 3.6, 11.4 Hz), 6.87–7.68 (m, 16H, arom. H), 7.75
(br. s, 4H, 2NH2). Anal. Calcd. for C36H36N6O2S2 (648.82): C, 66.64;
H, 5.59; N, 12.95. Found: C, 66.50; H, 5.51; N, 12.89.
Table 3
Ulcerogenic liability of the most promising prepared anti-inflammatory active
agents
d
Compound
Number of animals % Incidence
Average of
Average Ulcer
with ulcer
divided by 10 ulcer number severity index
Control
Indomethacin 5/6
2b
2c
2e
2f
0/6
0.00
8.33
8.33
6.67
8.33
0.00
3.83
2.83
3.67
4.33
7.17
0.00
1.33
1.00
1.17
1.67
1.33
0.00
13.49
12.16
11.51
14.33
18.50
5/6
4/6
5/6
6/6
3.1.5. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[4,5-dihydro-
3-(2-thienyl)-1H-pyrazole-1-thiocarboxamide] (2e)
10
Reaction time 1 h, pale yellow crystals from N,N-dime-
thylformamide – water as 2:1 v/v, mp 269–270 ꢂC, yield 44%. IR:
nmax/cmꢀ1 3443, 3230, 3125 (NH2), 1660, 1580, 1472 (C]N, C]C,
H-40, J ¼ 3.6, 18.0 Hz), 3.87 (dd, 2H, downfield H’s of pyr. H-4, H-40,
J ¼ 11.7, 18.0 Hz), 4.36 (s, 4H, 2OCH2), 6.09 (dd, 2H, pyr. H-5, H-50,
J ¼ 3.3, 11.4 Hz), 6.87–7.95 (m, 22H, 18 arom. H þ 2NH2). Anal. Calcd.
for C34H32N6O2S2 (620.77): C, 65.78; H, 5.20; N, 13.54. Found: C,
65.99; H, 5.34; N, 13.65.
C]S). 1H NMR (DMSO-d6):
d 3.04 (dd, 2H, upfield H’s of pyr. H-4, H-
40, J ¼ 3.6, 18.0 Hz), 3.89 (dd, 2H, downfield H’s of pyr. H-4, H-40,
J ¼ 11.4, 17.7 Hz), 4.38 (s, 4H, 2OCH2), 6.10 (dd, 2H, pyr. H-5, H-50,
J ¼ 3.6, 11.4 Hz), 6.87–7.72 (m, 14H, arom. H), 7.94 (br. s, 4H, 2 NH2).
Anal. Calcd. for C30H28N6O2S4 (632.83): C, 56.93; H, 4.46; N, 13.28.
Found: C, 56.71; H, 4.33; N, 13.37.
3.1.2. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[3-(4-
chlorophenyl)-4,5-dihydro-1H-pyrazole-1-thiocarboxamide] (2b)
Reaction time 1.5 h, pale yellow crystals from N,N-dime-
thylformamide – water as 3:1 v/v, mp 267–269 ꢂC, yield 41%. IR:
nmax/cmꢀ1 3443, 3231, 3123 (NH2), 1671, 1581, 1462 (C]N, C]C,
3.1.6. 5,50-[1,2-Ethanediylbis(oxy-4,1-phenylene)]bis(4,5-dihydro-
3-phenyl-1H-pyrazole-1-thiocarboxamide] (2f)
C]S). 1H NMR (DMSO-d6):
d 3.03 (dd, 2H, upfield H’s of pyr. H-4, H-
Reaction time 12 h, yellow crystals from ethanol, mp 145–
147 ꢂC, yield 45%. IR: nmax/cmꢀ1 3434, 3263 (NH2), 1566, 1509, 1466
40, J ¼ 3.6, 18.0 Hz), 3.81 (dd, 2H, downfield H’s of pyr. H-4, H-40,
J ¼ 11.4, 18.0 Hz), 4.31 (s, 4H, 2OCH2), 6.05 (dd, 2H, pyr. H-5, H-50,
J ¼ 3.6, 11.4 Hz), 6.85–7.81 (m, 16H, arom. H), 7.95 (br. s, 2H, NH2),
8.00 (br. s, 2H, NH2). Anal. Calcd. for C34H30Cl2N6O2S2 (689.66): C,
59.21; H, 4.38; N, 12.19. Found: C, 59.40; H, 4.44; N, 12.42.
(C]N, C]C, C]S). 1H NMR (CDCl3):
d 3.20 (dd, 2H, upfield H’s of
pyr. H-4, H-40, J ¼ 3.6, 17.7 Hz), 3.82 (dd, 2H, downfield H’s of pyr. H-
4, H-40, J ¼ 11.4, 17.7 Hz), 4.26 (s, 4H, 2OCH2), 6.00 (dd, 2H, pyr. H-5,
H-50, J ¼ 3.6, 11.4 Hz), 6.87–7.76 (m, 22H, 18 arom. H þ 2NH2). Anal.
Calcd. for C34H32N6O2S2 (620.77): C, 65.78; H, 5.20; N, 13.54. Found:
C, 65.62; H, 5.09; N, 13.46.
3.1.3. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[4,5-dihydro-
3-(4-fluorophenyl)-1H-pyrazole-1-thiocarboxamide] (2c)
3.1.7. 5,50-[1,2-Ethanediylbis(oxy-4,1-phenylene)]bis[3-(4-
Reaction time 1 h, colourless crystals from N,N-dime-
thylformamide – water as 2:1 v/v, mp 264–266 ꢂC, yield 43%. IR:
nmax/cmꢀ1 3446, 3232, 3124 (NH2), 1673, 1581, 1465 (C]N, C]C,
chlorophenyl)-4,5-dihydro-1H-pyrazole-1-thiocarboxamide] (2g)
Reaction time 24 h, yellow crystals from ethanol, mp 142–
144 ꢂC, yield 29%. IR: nmax/cmꢀ1 3429, 3260, 3139 (NH2), 1576, 1509,
C]S). 1H NMR (DMSO-d6):
d 3.04 (dd, 2H, upfield H’s of pyr. H-4, H-
1460 (C]N, C]C, C]S). 1H NMR (CDCl3):
d 3.16 (dd, 2H, upfield H’s
40, J ¼ 3.3, 18.0 Hz), 3.84 (dd, 2H, downfield H’s of pyr. H-4, H-40,
J ¼ 11.4, 18.0 Hz), 4.34 (s, 4H, 2OCH2), 6.07 (dd, 2H, pyr. H-5, H-50,
J ¼ 3.3, 11.1 Hz), 6.87–7.87 (m, 16H, arom. H), 7.91 (br. s, 2H, NH2),
7.97 (br. s, 2H, NH2). Anal. Calcd. for C34H30F2N6O2S2 (656.75): C,
62.18; H, 4.60; N, 12.80. Found: C, 62.08; H, 4.46; N, 12.97.
of pyr. H-4, H-40, J ¼ 3.6, 17.7 Hz), 3.80 (dd, 2H, downfield H’s of pyr.
H-4, H-40, J ¼ 11.4, 17.7 Hz), 4.27 (s, 4H, 2OCH2), 6.00 (dd, 2H, pyr. H-
5, H-50, J ¼ 3.6, 11.4 Hz), 6.87–7.68 (m, 20H, 16 arom. H þ 2NH2).
Anal. Calcd. for C34H30Cl2N6O2S2 (689.66): C, 59.21; H, 4.38; N,
12.19. Found: C, 59.37; H, 4.49; N, 12.05.
3.1.4. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[4,5-dihydro-
3-(4-methylphenyl)-1H-pyrazole-1-thiocarboxamide] (2d)
Reaction time 1 h, colourless crystals from N,N-dime-
thylformamide – methanol as 1:3 v/v, mp 255–257 ꢂC, yield 56%.
3.1.8. 5,50-[1,2-Ethanediylbis(oxy-4,1-phenylene)]bis[4,5-dihydro-
3-(4-methylphenyl)-1H-pyrazole-1-thiocarboxamide] (2h)
Reaction time 24 h, yellow crystals from ethanol, mp 128–
130 ꢂC, yield 25%. IR: nmax/cmꢀ1 3431, 3368, 3261, 3137 (NH2), 1609,
1509, 1463 (C]N, C]C, C]S). 1H NMR (CDCl3):
d 2.41 (s, 6H, 2
ArCH3), 3.18 (dd, 2H, upfield H’s of pyr. H-4, H-40, J ¼ 3.6, 17.7 Hz),
3.80 (dd, 2H, downfield H’s of pyr. H-4, H-40, J ¼ 11.4, 17.7 Hz), 4.26
(s, 4H, 2OCH2), 5.98 (dd, 2H, pyr. H-5, H-50, J ¼ 3.6, 11.4 Hz), 6.87–
7.64 (m, 20H, 16 arom. H þ 2NH2). Anal. Calcd. for C36H36N6O2S2
20
18
16
14
12
10
8
Table 4
PGE2 inhibitory activity of the most promising prepared anti-inflammatory active
agents.
6
4
Compound
Concentration of PGE2 (pg/ml)
533.33 ꢁ 19.26b
98.33 ꢁ 3.33a
110.83 ꢁ 5.90a
114.67 ꢁ 1.84a
96.50 ꢁ 4.47a
83.33 ꢁ 4.84a
2
Control
Indomethacin
2b
2c
2e
2f
0
2c
2b
2e
2f
Control
Indomethacin
Tested compounds
Fig. 4. Ulcer index for the most promising prepared anti-inflammatory active agents.
Results are means of 6 experiments ꢁ standard error.
a
Statistically significant from the control at p < 0.05.
Statistically significant from indomethacin at p < 0.05.
b

2176
F.F. Barsoum, A.S. Girgis / European Journal of Medicinal Chemistry 44 (2009) 2172–2177
3.2.4. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[4,5-dihydro-
3-(4-methylphenyl)-1H-pyrazole-1-carboxamide] (3d)
600
500
400
300
200
100
0
Reaction time 4 h, colourless crystals from N,N-dime-
thylformamide – methanol as 2:3 v/v, mp 278–280 ꢂC, yield 52%. IR:
nmax/cmꢀ1 3480, 3269, 3196, 3140 (NH2), 1683 (C]O), 1588, 1448
(C]N, C]C). 1H NMR (DMSO-d6):
d 2.28 (s, 6H, 2ArCH3), 2.91 (dd,
2H, upfield H’s of pyr. H-4, H-40, J ¼ 5.1, 17.7 Hz), 3.64 (dd, 2H,
downfield H’s of pyr. H-4, H-40, J ¼ 12.0, 18.0 Hz), 4.31 (s, 4H,
2OCH2), 5.54 (dd, 2H, pyr. H-5, H-50, J ¼ 5.1, 12.0 Hz), 6.40 (s, 4H,
2NH2), 6.86–7.58 (m, 16H, arom. H). Anal. Calcd. for C36H36N6O4
(616.70): C, 70.12; H, 5.88; N, 13.63. Found: C, 70.20; H, 5.99; N,
13.82.
3.3. Anti-inflammatory activity screening
Tested compounds
Anti-inflammatory activity screening for the prepared
compounds was determined in vivo by the acute carrageenan-
induced paw oedema standard method in rats [14–17]. Wister
albino rats of either sex (pregnant female animals were excluded)
weighing 160–190 g were divided into 13 groups of 6 animals each.
Administration of indomethacin (reference standard at a dose of
10 mg/kg of body weight) and the tested compounds (2a–f,h and
3a–d) dissolved in DMSO, at a dose of 50 mg/kg (body weight) was
given intraperitoneally 1 h before induction of inflammation. The
control group was given DMSO only. Carrageenan paw oedema was
induced by subcutaneous injection of 1% solution of carrageenan in
saline (0.1 ml/rat) into the right hind paw of rats. Paw volumes were
measured volumetrically after 4 h of inflammation induction with
plethysmometer 7140 (UGO BASILE, Italy) and compared with the
initial hind paw volume of each rat for determining the oedema
volume. Data were collected, checked, revised and analyzed.
Quantitative variables from normal distribution were expressed as
means ꢁ SE ‘‘standard error’’. The significant difference between
groups was tested by using one-way ANOVA followed by post hoc
test and the chosen level of significance was p < 0.05.
Fig. 5. PGE2 inhibitory activity of the most promising prepared anti-inflammatory
active agents.
(648.82): C, 66.64; H, 5.59; N, 12.95. Found: C, 66.86; H, 5.79; N,
12.87.
3.2. Synthesis of bis(3-aryl-4,5-dihydro-1H-pyrazole-1-
carboxamides) 3a–d (general procedure)
A solution of 1a–d (2.5 mmol) and semicarbazide hydrochloride
(5 mmol) in glacial acetic acid (25 ml) was boiled under reflux for
the appropriate time. The separated solid upon storing the reaction
mixture to cool at room temperature (25 ꢂC) was collected, washed
with water and crystallized from a suitable solvent affording 3a–d.
3.2.1. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis(4,5-dihydro-
3-phenyl-1H-pyrazole-1-carboxamide) (3a)
Reaction time 3 h, colourless crystals from N,N-dime-
thylformamide – methanol as 3:1 v/v, mp 282–284 ꢂC, yield 48%. IR:
nmax/cmꢀ1 3481, 3263, 3177, 3135 (NH2), 1677 (C]O), 1588, 1449
The anti-inflammatory activity was expressed as percentage
inhibition of oedema volume in treated animals in comparison with
the control group (Table 1, Figs. 1–3):
(C]N, C]C). 1H NMR (DMSO-d6):
d 2.94 (dd, 2H, upfield H’s of pyr.
H-4, H-40, J ¼ 5.1, 17.7 Hz), 3.70 (dd, 2H, downfield H’s of pyr. H-4, H-
40, J ¼ 12.3, 17.7 Hz), 4.33 (s, 4H, 2OCH2), 5.59 (dd, 2H, pyr. H-5, H-50,
J ¼ 5.1, 12.3 Hz), 6.45 (s, 4H, 2NH2), 6.86–7.70 (m, 18H, arom. H).
Anal. Calcd. for C34H32N6O4 (588.65): C, 69.37; H, 5.48; N, 14.28.
Found: C, 69.54; H, 5.61; N, 14.49.
Vc ꢀ Vt
%Inhibition of oedema ¼
ꢃ 100
Vc
where, Vc and Vt are the volumes of oedema for the control and
drug-treated animal groups, respectively.
Potency of the tested compounds was calculated relative to
indomethacin ‘‘reference standard’’ treated group according to the
following equation.
3.2.2. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[3-(4-
chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide] (3b)
Reaction time 4 h, colourless crystals from N,N-dime-
thylformamide, mp 291–293 ꢂC, yield 43%. IR: nmax/cmꢀ1 3487,
3266, 3184, 3132 (NH2), 1678 (C]O), 1588, 1451 (C]N, C]C). 1H
Potency ¼ ð%Oedema inhibition of tested compound
treated groupÞ=ð%Oedema inhibition of indomethacin
treated groupÞ
NMR (DMSO-d6):
d
2.95 (dd, 2H, upfield H’s of pyr. H-4, H-40, J ¼ 5.4,
17.7 Hz), 3.63 (dd, 2H, downfield H’s of pyr. H-4, H-40, J ¼ 12.6,
18.0 Hz), 4.27 (s, 4H, 2OCH2), 5.55 (dd, 2H, pyr. H-5, H-50, J ¼ 5.4,
12.3 Hz), 6.48 (s, 4H, 2NH2), 6.86–7.69 (m, 16H, arom. H). Anal.
Calcd. for C34H30Cl2N6O4 (657.54): C, 62.10; H, 4.60; N,12.78. Found:
C, 61.86; H, 4.49; N, 12.94.
Additionally, the most promising prepared anti-inflammatory
active agents (2b,c,e and f) were screened for their pharmacological
properties at lower doses (20, 10 mg/kg body weight) using the
same described experimental technique (Table 2).
3.2.3. 5,50-[1,2-Ethanediylbis(oxy-2,1-phenylene)]bis[4,5-dihydro-
3-(4-fluorophenyl)-1H-pyrazole-1-carboxamide] (3c)
Reaction time 4 h, colourless crystals from N,N-dime-
thylformamide – water as 4:1 v/v, mp 271–273 ꢂC, yield 45%. IR:
nmax/cmꢀ1 3489, 3267, 3187, 3135 (NH2), 1682 (C]O), 1584, 1449
3.4. Ulcerogenic liability
(C]N, C]C). 1H NMR (DMSO-d6):
d 2.94 (dd, 2H, upfield H’s of pyr.
H-4, H-40, J ¼ 5.1, 17.7 Hz), 3.65 (dd, 2H, downfield H’s of pyr. H-4, H-
40, J ¼ 12.0, 17.7 Hz), 4.30 (s, 4H, 2OCH2), 5.56 (dd, 2H, pyr. H-5, H-50,
J ¼ 5.1, 12.0 Hz), 6.47 (s, 4H, 2NH2), 6.86–7.74 (m, 16H, arom. H).
Anal. Calcd. for C34H30F2N6O4 (624.63): C, 65.37; H, 4.84; N, 13.46.
Found: C, 65.56; H, 4.92; N, 13.70.
The ulcerogenic liability was determined in albino rats following
the previously reported standard method [18,19]. Rats of either sex
(pregnant female rats were excluded) weighing 130–150 g were
divided into 6 groups of 6 animals each. The animals were fasted
18 h before drug administration. Indomethacin (reference standard

F.F. Barsoum, A.S. Girgis / European Journal of Medicinal Chemistry 44 (2009) 2172–2177
2177
at a dose of 10 mg/kg of body weight) and the tested compounds (at
a dose of 50 mg/kg of body weight), were suspended in saline
solution by the aid of few drops of Tween 80 and were adminis-
tered orally for three successive days to fasted rats. The control
group animals were given saline with few drops of Tween 80. One
hour following the last dose, the animals were sacrificed by cervical
dislocation and the stomach was removed, opened along the
greater curvature and rinsed with saline. The gastric mucosa was
examined with a magnifying lens (10ꢃ) for the presence of lesions
and erosions. The ulcer index was calculated (Table 3, Fig. 4) and the
degree of ulcerogenic effect was expressed in terms of:
and stored at ꢀ20 ꢂC until required for PGE2 assay. PGE2 was
measured by an ELISA (Beckman BiomekÔ 1000 automated labo-
ratory workstation apparatus) technique using PGE2 assay kit
supplied by R&D Systems Inc., Minneapolis, USA, according to the
manufacturer’s specifications.
Acknowledgment
This work is sponsored by the U.S.–Egypt Science and Tech-
nology Joint Fund under Project No. MAN10-007-002. Thanks are
also to Prof. M.T. Khayyal (Faculty of Pharmacy, Cairo University)
and Dr. Ebtehal El-Demerdash (Faculty of Pharmacy, Ain-Shams
University) for facilities allowed and valuable discussion.
1. percentage incidence of ulcer divided by 10;
2. average number of ulcers per stomach;
3. average severity of ulcers.
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