Synthesis, Anti-Inflammatory Activity, and COX-1/2 Inhibition Profile of Some Novel Non-Acidic Polysubstituted Pyrazoles and Pyrano[2,3-c]pyrazoles

Article abstract of DOI:10.1002/ardp.201700025

The synthesis and evaluation of the anti-inflammatory activity of some structure hybrids comprising basically the 5-hydroxy-3-methyl-1-phenyl-4-substituted-1H-pyrazole scaffold directly linked to a variety of heterocycles and functionalities, or annulated

Full text of DOI:10.1002/ardp.201700025

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Full Paper
Synthesis, Anti-Inflammatory Activity, and COX-1/2 Inhibition
Profile of Some Novel Non-Acidic Polysubstituted Pyrazoles and
Pyrano[2,3-c]pyrazoles
2,3
Hassan M. Faidallah1 and Sherif A. F. Rostom
1
Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah,
2
Saudi Arabia
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria,
Egypt
The synthesis and evaluation of the anti-inflammatory activity of some structure hybrids comprising
basically the 5-hydroxy-3-methyl-1-phenyl-4-substituted-1H-pyrazole scaffold directly linked to a
variety of heterocycles and functionalities, or annulated as pyrano[2,3-c]pyrazoles, is described.
According to the in vivo results and a comprehensive structure–activity relationship study, five
analogs (5, 10, 17, 19, and 27) displayed remarkable anti-inflammatory profiles showing distinctive
% protection and ED50 values, especially 10 and 27 (ED50 35.7 and 38.7 mmol/kg, respectively) which
were nearly equiactive to celecoxib (ED50 32.1 mmol/kg). Compounds 10, 17, and 27 exhibited
distinctive COX-2 inhibition with a noticeable COX-2 selectivity (SI values 7.83, 6.87, and 7.16,
respectively), close to that of celecoxib (SI 8.68). Additionally, 5, 10, 17, 19, and 27 proved to be
gastrointestinal tract safe (0–20% ulceration) and non-toxic, being well tolerated by the experimental
animals up to 250 mg/kg orally and 80 mg/kg parenterally. Collectively, the in vivo ED50 values for the
most potent five derivatives agree with their in vitro COX-2 selectivity indices, suggesting their
usefulness as selective anti-inflammatory COX-2 inhibitors. The bipyrazole 10 and the pyranopyrazole
27 could be considered as the most active members in this study, being nearly equiactive to celecoxib,
besides their obvious selective COX-2 inhibition, high safety margin, and predicted pharmacokinetic
(ADME-T) suitability for oral use.
Keywords: Anti-inflammatory activity / COX-1 / COX-2 / Pyrazole / Selective COX-2 inhibitors
Received: January 22, 2017; Revised: March 1, 2017; Accepted: March 7, 2017
DOI 10.1002/ardp.201700025
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
processes (as in osteoarthritis), or different autoimmune
diseases, might be referred to as “inflammatory condi-
Introduction
The phenomenon of “inflammation” signalizes a reaction to
any mechanical damage or infection facing the human body.
Subsequently, cases, such as chronic infections, degenerative
tions” [1]. Non-steroidal anti-inflammatory drugs (NSAIDs)
constitute one of the most public classes of drugs that are
utilized for alleviation of pain and inflammatory conditions
connected with various pathophysiological conditions. Their
pharmacological action relies on the interference in the
biosynthesis of prostaglandins (PGs) through inhibition of
the cyclooxygenase (COX) enzyme [2]. PGs are known to be
implemented in the induction of inflammation, some
homeostatic processes as well as cytoprotection of the
gastrointestinal tract (GIT). Therefore, extensive and
Correspondence: Prof. Sherif A. F. Rostom, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz
University, PO Box 80260, Jeddah 21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: þ9662-6400000-22327
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inappropriate administration of NSAIDs mostly leads to GI
problems (e.g., induction of gastric ulcer), blood, kidney, and
liver serious harmful effects [3]. A new era in the anti-
inflammatory research area has been entered when it has
been assessed that COX is available as two isozymes, COX-1
and COX-2, which are controlled with different condi-
tions [4]. COX-1 is naturally occurring and is concerned with
the production of the protective PGs in the GI and
thromboxanes in blood platelets [5]. In contrast, COX-2 is
an inducible isozyme and is stimulated as a result of the
release of inflammatory mediators during the inflammation
process [6]. Subsequently, the peculiar distribution of the
two isozymes among different tissues has provoked the
search for novel NSAIDs with distinctive therapeutic activity
based on selective inhibition of COX-2 that would stop the
production of the inflammatory PGs, while sparing GI-
protective effects regulated by COX-1 [7]. As a result, many
selective inhibitors of COX-2 (coxibs) have been introduced
to the market with variable anti-inflammatory potentials
and safety margins. Unfortunately, prolonged use of some
coxibs (e.g., rofecoxib and valdecoxib) resulted in serious
cardiovascular disorders [8], which determined the dragging
of these coxibs [9] and clearly urged the need to find out new
scaffolds having COX-2 inhibitory activity.
celecoxib (Fig. 1) is a pyrazole derivative with distinctive anti-
inflammatory and GI safe effects [18]. During our continuous
search for novel pyrazole candidates endowed with potential
bioactivities [19–33], we have reported on the synthesis, in
vivo and in vitro anti-inflammatory activities of some lead
structures comprising mainly the 1-arylpyrazol(in)e counter-
part substituted with several functionalities and/or attached
to different heterocyclic rings through various linkages
[27–33]. In particular, potential anti-inflammatory activity
was ascribed to the pyrazolyl compounds A [30], B [31], and
C [32] (Fig. 1) and the bipyrazolyl derivatives D [33] (Fig. 1).
Inspired by these facts, the objective of this research is to
develop and investigate the in vivo as well as in vitro anti-
inflammatory potentials of some novel non-acidic polyfunc-
tional pyrazoles. The targeted compounds were rationalized
so as to comprise basically the 5-hydroxy-3-methyl-1-phenyl-4-
substituted-1H-pyrazole scaffold substituted at position-4
with a variety of heterocycles known for their relevant
biological activities including the isoxazole, pyrazole, pyrimi-
dine, tetrazolo[1,5-a]pyrimidine, triazolo[4,3-a]pyrimidine,
and pyrimido[2,1-c][1,2,4]triazine ring systems (E; Fig. 1). This
combination was suggested in an attempt to investigate the
influence of such hybridization and structure variation on the
anticipated anti-inflammatory potential and safety profile,
hoping to add some biological synergism to the target
molecules. Moreover, the substitution pattern of the target
compounds was aimed to include some pharmacophores that
are believed to be highly contributing to the anticipated
biological activity such as the sulfonamide, azomethine,
sulfonylureido, and sulfonylthioureido functionalities. Fur-
thermore, annulation of the main pyrazole ring into the
corresponding substituted pyrano[2,3-c]pyrazoles (F; Fig. 1)
was thought to be an interesting structure variation through
Among the family of biologically active heterocycles,
special interest has been given to pyrazole-containing
compounds as potential anti-inflammatory, antinociceptive,
and antipyretic agents [10–17] especially after the discovery of
antipyrine at 1884 as the first anti-inflammatory pyrazole
analog. As a consequence, a tremendous number of pyrazoles
has been prepared, biologically investigated, and some of
them have been introduced clinically. As
a prominent
example, the currently available selective COX-2 inhibitor
Figure 1. Structures of celecoxib, the lead compounds A–D, and the general formulae of the novel series of compounds E and F.
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affecting the electronic and/or steric environment of the
molecules that might have an impact on the targeted
biological activity. It should be noted that, in addition to
the targeted anti-inflammatory activity, the ulcerogenic and
acute toxicity profiles of the active compounds were also
evaluated. Besides, an in silico computation of the molecular
properties, physicochemical profile, drug score, and drug-
likeness of the biologically active compounds was performed
to predict their pharmacokinetic and toxicity properties
(ADME-T), and to assess their suitability as possible orally
active dug candidates.
chlorophenyl isothiocyanate and anhydrous K2CO3 in dry
acetone, afforded the corresponding substituted sulfonylur-
eido and sulfonylthioureido derivatives 12 and 13, respec-
tively. Their IR spectra exhibited two absorption bands at
1164–1169 cmꢀ1 and 1336–1368 cmꢀ1 due to the SO2N group
besides a ureido carbonyl band at 1652 cmꢀ1 (for 12) and a
thioureido absorption band at 1155 cmꢀ1 (for 13). In its turn,
cyclcondensation of the sulfonylthioureido derivative 13 with
ethyl bromoacetate or phenacyl bromide in the presence of
anhydrous Na acetate produced the corresponding 4-oxo-
thiazolidine 14 and the thiazoline 15. The IR spectrum of 14
was characterized by the sharp band at 1712 cmꢀ1 due to the
thiazolidine C4 carbonyl group, while its 1H NMR spectrum
showed a new singlet at d 3.42 ppm owing to the thiazolidine
C5 methylene protons. In contrast, the 1H NMR of the
thiazoline 15 exhibited a new singlet at d 5.63 ppm due to
the thiazoline C5 proton.
Results and discussion
Chemistry
The synthetic strategies employed to obtain the intermediate
and target compounds are illustrated in Schemes 1–4. In
Scheme 1, condensation of the precursor dehydroacetic acid
(DHA) with the appropriate arylhydrazine afforded the
As shown in Scheme 3, cyclocondensation of the
pyrazolylbutane-1,3-dione 4 with thiourea or methylthiourea
produced the corresponding pyrimidine-2-thiones 16 and 17,
respectively. Their IR spectra exhibited characteristic absorption
bandsat1182–1192cmꢀ1,whiletheir13C NMR exhibitedpeeksat
d 180.40–185.02 ppm attributed to the thiocarbonyl functional-
ity. Cyclocondensation of the pyrimidinethione 16 with
hydrazine hydrate gave the key intermediate 2-hydrazino
derivative 18. In its turn, when a solution 18 in acetic acid was
reacted with sodium nitrite at room temperature, the tetrazolo-
[1,5-a]pyrimidine 19 was obtained. Moreover, refluxing 18 with
acetylacetone or ethyl bromoacetate yielded the targeted
substituted [1,2,4]triazolo[3,4-a]-pyrimidines 20 and 21,
respectively, following a reported procedure [34]. Whereas
reacting 18 with an excess of diethyl oxalate gave the
corresponding ethyl [1,2,4]triazolo[3,4-a]-pyrimidine-3-carbox-
ylate analog 22. Here, it should be noted that reaction of 18 with
an equivalent amount of diethyl oxalate in the presence of
sodium acetate yielded the pyrimido[2,1-c][1,2,4]triazine-3,4-
dione 23, where the 1H-NMR lacked the triplet and quartet
protons of the ethoxy group which proved the aminolysis of the
ester function. Meanwhile, the IR spectrum of 23 showed two
corresponding arylhydrazones
2 and 3. Heating these
arylhydrazones with acetic acid gave rise to the corresponding
substituted 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
butane-1,3-diones 4 and 5, which were employed as the
principle key intermediates in this study. Their IR spectra were
characterized by the appearance of two carbonyl absorption
bands at 1622–1635 cmꢀ1 and 1715–1724 cmꢀ1, respectively,
as well as an exchangeable OH band at 3328–3398 cmꢀ1
.
Reacting the pyrazoles 4 and 5 with 2-bromoaniline yielded
the target 2-bromophenylamino derivatives 6 and 7.
In Scheme 2, condensation of the pyrazole precursors 4
and 5 with hydroxyl amine gave rise to the corresponding
3-methylisoxazol-5-yl derivatives 8 and 9. Furthermore, cyclo-
condensation of the pyrazolylbenzenesulfonamide 5 with
phenylhydrazine yielded the corresponding bipyrazole 10
which was subjected to different condensation reactions. For
instance, condensing 10 with 4-chlorobenzaldehyde in acetic
acid afforded the expected azomethine derivative 11.
Meanwhile, nucleophilic addition reaction of the bipyrazole
10 across the N═C bond of the 4-chlorophenyl isocyanate or 4-
carbonyl absorptions at 1665 and 1678 cmꢀ1
.
Finally,
Scheme 1. Synthesis of compounds 2–7.
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Scheme 2. Synthesis of compounds 8–15.
cyclocondensation of the 18 with b-bromoketones in acetic acid
gave rise to the substituted pyrimido[2,1-c][1,2,4]triazines 24 and
25.
spectrum of the 5-chloropyranopyrazole 29 showed a
carbonyl absorption at 1665 cmꢀ1, while the analogs 32
and 33 exhibited two carbonyl bands at 1662–1668 cmꢀ1
and 1669–1672 cmꢀ1. The 13C NMR data showed a charac-
teristic carbonyl carbon at d186.56 ppm for 29, and two
At this stage, it was attempted to annulate the
pyrazolylbutane-1,3-diones 4 and 5 into the corresponding
pyrano[2,3-c]pyrazoles as illustrated in Scheme 4. In this
view, ring closure of 4 and 5 into the 3,6-dimethyl-1-
(4-substitutedphenyl)pyrano[2,3-c]pyrazol-4(1H)-ones 26
and 27 was easily accomplished via treatment of their
solutions in acetic acid with sulfuric acid at room tempera-
ture. Their IR spectra exhibited carbonyl absorption bands
at 1660–1664 cmꢀ1, whereas their 1H NMR showed two
carbonyl signals at
d 183.66–185.23 and d 196.20–
196.60 ppm for 32 and 33, respectively.
Biological evaluation
Carrageenan-induced paw edema
All the synthesized 27 target compounds (4–27, 29, 32, and 33)
were evaluated for their in vivo systemic anti-inflammatory
activity using the carrageenan-induced paw edema bioassay
in rats [35] as a model of acute inflammation. Indomethacin
and celecoxib were employed as standard anti-inflammatory
agents against COX-1 and COX-2 enzymes, respectively. The
data obtained are presented in Table 1 as the mean increase in
paw edema (mL) ꢁ SE. The results showed that 18 compounds
(4, 5, 7, 9–13, 16–20, 24, 26, 27, 29, and 33) were able to exhibit
variable degree of systemic anti-inflammatory potential.
methyl singlets at
d 2.04–2.10 and d 2.44–2.47 ppm.
Furthermore, treatment of the starting pyrazoles 4 and 5
with sulfuryl chloride or trifluoroacetic anhydride gave rise
to the corresponding 3-chloro 28 or the 3-trifluoroacetyl
derivatives 30 and 31, respectively, which were directly
cyclodehydrated (without purification) with concentrated
sulfuric acid at room temperature to the corresponding
4-oxo-1H-pyrano[2,3-c]pyrazoles 29, 32, and 33. The IR
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Scheme 3. Synthesis of compounds 16–25.
Compounds 5, 10, 17, 19, and 27 proved to be the most active
anti-inflammatory agents in the present study as evidenced
from their distinctive % protection values (68.5, 74.2, 69.7, 67.4,
and 70.8%, respectively), which are comparable or even better
than those of indomethacin and celecoxib (% protection 70.8
and75.3,respectively)underthesameexperimentalconditions.
Meanwhile, the analogs 4, 16, 20, and 24 displayed noticeable
anti-inflammatory potentials as seen from their % protection
values (60.7–65.2%), constituting 85.7–92.1% of the activity of
indomethacin and 80.6–86.6% of that of celecoxib. Moreover,
7,9,13,18,26,and33exhibitedmoderateactivity(%protection
range 50.6–59.6%) representing 71.5–84.2% of the activity of
indomethacin and 67.2–79.2% of that of celecoxib. Whereas,
11, 12, and 29 showed weak anti-inflammatory activity (%
protection 38.2–49.4%).
a considerable anti-inflammatory potential (ED50 values 68.2,
35.7, 53.1, 75.2, and 38.7 mmol/kg, respectively), when
compared with celecoxib (ED50 32.1 mmol/kg). In particular,
the bipyrazole 10 and pyranopyrazole 27 (ED50 35.7 and
38.7 mmol/kg, respectively) stemmed as the most potent
members being nearly equiactive to celecoxib (ED50
32.1 mmol/kg). The remaining three analogs showed moder-
ate anti-inflammatory potency with ED50 values ranging
between 53.1 and 75.2 mmol/kg.
Structure–activity correlation
A close examination of the structures of the active compounds
in relation to their % protection capabilities revealed that the
anti-inflammatory activity was greatly affected by the type of
substituent at C4 of the main pyrazole scaffold. For instance,
better anti-inflammatory activity was often linked with
analogs comprising the pyrazolyl, pyrimidinyl, tetrazolopyr-
imidinyl counterparts, in addition to the pyrano[2,3-c]-
pyrazole structure variant. In this view, in Scheme 1, among
For further assessment of the anti-inflammatory potential,
the ED50 values of the five prominently active compounds 5,
10, 17, 19, and 27 were determined [36]. The results presented
in Table 1 revealed that all the five tested compounds possess
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Scheme 4. Synthesis of compounds 26–33.
the parent 1-(5-hydroxy-3-methyl-1-(4-substituted phenyl)-
ledtoanoticeableloweringoftheanti-inflammatorypotential.
Subsequent rigidification of the sulfonylthioureido group into
either thiazolidinedione 14 or thiazolines 15 ring systems led to
complete abolishment of activity. Such dramatic collapse in the
bioactivity of the analogs 11–15 could be attributed to their
steric bulkiness which would probably affect bioavailability
and/or binding with COX enzymes. Variation of the substituent
type at C4 of the main pyrazole scaffold 4 was extended to
produce the substituted pyrazolylpyrimidines 16 and 17 which
showed acceptable anti-inflammatory capability, especially 17
(R¼ SO2NH2) with % protection of 69.7 (Scheme 3). Conversion
of the thione into the hydrazine function as in 18 led to a clear
drop in the activity, presumably due to increasing compound’s
polarity. Further cylization of 18 into the corresponding
tetrazolo[1,5-a]pyrimidine 19 has obviously improved the
activity probably due to the presence of multiple H-bond
forming centers that would assist binding with the COX
enzymes. However, bioisosteric replacement of the tetrazolo-
[1,5-a]pyrimidine moiety with substituted 1,2,4-triazolo[4,3-a]-
pyrimidine counterparts 20–22 furnished only one active
compound 20 with less anti-inflammatory potential. Whereas,
increasing ring size by incorporating the pyrimidine ring of 18
into a pyrimido[2,1-c][1,2,4]triazine ring system yielded three
compounds 23–25 among which the analog 24 was the only
1H-pyrazol-4-yl)butane-1,3-diones
4 and 5, compound 5
(R ¼ SO2NH2) showed remarkable anti-inflammatory activity
(% protection 68.5) comparable with indomethacin and
celecoxib (70.8 and 75.3%, respectively). Introduction of a
(2-bromophenyl)amino counterpart as in 7 (R ¼ SO2NH2)
resulted in a marked reduction in the activity (% protection
50.6). Similarly, incorporation of the butane-1,3-dione moiety
of the parent pyrazoles 4 and 5 into an isoxazole ring (9;
R ¼ SO2NH2) led to an obvious decrease in the activity
(Scheme 2). On the other hand, a significant improvement
in the anti-inflammatory potential was obtained via cycliza-
tion of the pyrazole 5 into the corresponding bipyrazole
structure 10 which was stemmed as the most active member in
this study (% protection 74.2), being more potent in vivo than
indomethacin and nearly equiactive to celecoxib. Here, it is
worth mentioning that the substitution pattern of the
bipyrazole 10 is structurally relevant to the vicinal diaryl
heterocycles template of COX-2 inhibitors represented by
celecoxib, in which the benzenesulfonamide moiety was
replaced with a sulfonamide-substituted arylpyrazole coun-
terpart (Fig. 2). However, further derivatization of the
sulfonamido group of 10 into the substituted benzylidene
11, sulfonylureido 12, and sulfonylthioureido 13 functionalities
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Table 1. In vivo anti-inflammatory activity (carrageenan-induced rat paw edema bioassay) and the ulcerogenic effect of
the active compounds.
Increase in
Compound
number
paw edema
AI % relative
to indom.b)
AI % relative
to celecoxibc)
ED50
(mmol/kg)
Ulcerationd)
(%)
(mL)a)
Protection (%)
Control
0.89 ꢁ 0.025
0.26 ꢁ 0.021
0.22 ꢁ 0.015
0.35 ꢁ 0.021
0.28 ꢁ 0.015
0.44 ꢁ 0.019
0.41 ꢁ 0.025
0.23 ꢁ 0.015
0.49 ꢁ 0.023
0.55 ꢁ 0.011
0.38 ꢁ 0.017
0.31 ꢁ 0.024
0.27 ꢁ 0.018
0.36 ꢁ 0.023
0.29 ꢁ 0.018
0.32 ꢁ 0.023
0.34 ꢁ 0.021
0.37 ꢁ 0.015
0.26 ꢁ 0.014
0.45 ꢁ 0.012
0.42 ꢁ 0.023
0.0
70.8
75.3
60.7
68.5
50.6
53.9
74.2
44.9
38.2
57.3
65.2
69.7
59.6
67.4
64.0
61.2
58.4
70.8
49.4
52.8
0.0
100
0.0
94
100
–
–
Indomethacinb)
NDe)
32.1
ND
100
40
ND
Celecoxibc)
4
5
7
106.4
85.7
96.8
71.5
76.1
104.8
63.4
53.9
80.9
92.1
98.4
84.2
95.2
90.4
86.4
82.5
100
80.6
91.0
67.2
71.5
98.5
59.6
50.7
76.1
86.6
92.6
79.2
89.5
85.0
81.3
77.6
94.1
65.6
70.1
68.2
ND
ND
35.7
ND
ND
ND
ND
53.1
ND
75.2
ND
ND
ND
38.7
ND
ND
20
ND
ND
20
9
10
11
12
13
16
17
18
19
20
24
26
27
29
33
ND
ND
ND
ND
0.0
ND
0.0
ND
ND
ND
0.0
ND
ND
69.8
74.6
a) Values were determined after 4 h and are expressed as mean (mL) ꢁ SE (no. of animals per experimental group N ¼ 5). All data
are significantly different from control (P < 0.05).
b,c) AI%: percentage anti-inflammatory activity relative to indomethacin and celecoxib; reference standard COX-1 and COX-2
inhibitors, respectively.
d) Five animals per experimental group.
e) ND: not determined.
active analog with moderate bioactivity. On the other hand,
annulation of the parent pyrazolyl butane-1,3-diones 4 and 5
into the corresponding bicyclic substituted 3,6-dimethyl-1-
phenylpyrano[2,3-c]pyrazole-4(1H)-ones (Scheme 4) furnished
four active compounds (26, 27, 29, and 33), which carry some
structurerelevancetothevicinaldiarylheterocyclestemplateof
COX-2 inhibitors represented by celecoxib (Fig. 2). The anti-
inflammatoryprofileofthesederivativeswasmodulatedbythe
type of substituent at position 5. For instance, the sulfonamido
derivative 27 (R ¼ SO2NH2) revealed remarkably improved anti-
Figure 2. Structures of celecoxib (selective COX-2 inhibitor) and the most active anti-inflammatory compounds 10 and 27.
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inflammatory potential as compared with the parent analog 5,
being almost equipotent with indomethacin and celecoxib.
Yet, introduction of Cl or COCF3 moieties at position-5 of the
pyrano[2,3-c]pyrazole ring system as in 29 and 33 resulted in an
obvious drop in the biological activity.
respectively) superior to that of celecoxib (IC50 0.84 mM),
whereas the activity of the analog 17 (IC50 0.91 mM).was nearly
equiactive to celecoxib. Furthermore, most of the investigated
compounds displayed considerable COX-2 selectivity index
values (SI) lying between 2.04 and 7.83, when compared with
celecoxib (SI value 8.68). Prominently, compounds 10, 17, and
27 were found to be obviously selective COX-2 inhibitors (SI
values 7.83, 6.87, and 7.16, respectively), while the analogs 5
and 19 revealed moderate selectivity towards the same
isozyme (SI values 6.15 and 5.27, respectively). The rest of the
tested compounds showed mild to weak selectivity indices
with SI values range lying between 2.04 and 3.93.
Collectively, the in vivo ED50 values for the most potent five
derivatives 5, 10, 17, 19, and 27 get along with their in vitro
COX-2 selectivity indices, suggesting their usefulness as
selective anti-inflammatory COX-2 inhibitors. The bipyrazole
10 and pyranopyrazole 27 (structurally relevant to celecoxib)
could be considered as the most active anti-inflammatory
members in this study, being more potent than indomethacin
and nearly equiactive to celecoxib, besides their obvious
selective COX-2 inhibitory activity.
In vitro COX-1 and COX-2 inhibitory assay
All the developed 27 target compounds (4–27, 29, 32, and 33)
were evaluated for their ability to in vitro inhibit COX-1 and
COX-2 isozymes employing an ovine COX-1/COX-2 inhibitor
screening assay kit (Catalog No. 760111, Cayman Chemicals
Inc., Ann Arbor, MI, USA) according to the manufacturer’s
instructions and a literature procedure [37, 38]. The method
relied on the peroxidase component of COX which is assayed
colorimetrically by monitoring the appearance of oxidized
N,N,N0,N0-tetramethyl-1,4-phenylenediamine (TMPD), that is
generated during the reduction of PGG2 to PGH2, at 590 nm.
The efficiencies of the investigated compounds were mea-
sured as the concentration causing 50% enzyme inhibition
(IC50; mM), the selectivity index (SI value) was calculated as IC50
(COX-1)/IC50 (COX-2).
The results recorded in Table 2 indicated that all the tested
compounds showed mild inhibitory effects on the activity of
COX-1 (IC50 range 5.64–12.2 mM) as compared with indometh-
acin (IC50 3.54 mM), while they proved to be moderate to good
COX-2 inhibitors (IC50 values range 0.72–2.74 mM), when
correlated to celecoxib (IC50 0.84 mM), except the analogs 11,
12, 26, 29, and 33 (IC50>3 mM). In particular, compounds 10
and 27 exerted COX-2 inhibition (IC50 0.72 and 0.76 mM,
Ulcerogenic effect
Compounds that showed remarkable in vivo and in vitro anti-
inflammatory potential (5, 10, 17, 19, and 27) were further
investigated for their ulcerogenic liability in rats [39]. The
results denoted that all the five tested compounds revealed
eminent gastrointestinal (GI) safety profiles (0–20% ulcera-
tion) in the population of the test animals at oral doses of
Table 2. In vitro COX-1/COX-2 enzymatic inhibition assay of the active compounds.a)
Compound number
COX-1 IC50 (mM)b)
COX-2 IC50 (mM)c)
COX-2 SId)
Indomethacin
Celecoxib
4
5
7
9
10
11
12
13
16
17
18
19
20
24
26
27
29
33
3.54
7.29
8.96
7.63
8.58
10.1
5.64
9.26
8.48
5.93
6.34
7.55
9.23
7.48
9.89
9.48
10.4
9.41
8.51
12.2
3.73
0.84
2.08
1.24
1.73
2.47
0.72
3.01
3.38
1.51
1.97
0.91
2.55
1.42
2.66
2.74
3.11
0.76
4.17
5.01
0.95
8.68
4.31
6.15
4.96
4.09
7.83
3.08
2.51
3.93
3.21
6.87
3.62
5.27
3.72
3.46
3.35
7.16
2.04
2.43
a) Values obtained using an ovine COX assay kit (Catalog No. 760111, Cayman Chemical, Inc., Ann Arbor, MI, USA).
b,c) Compounds’ concentrations causing 50% inhibition of COX-1 and COX-2, respectively. Results are the mean of two readings.
d) COX-2 selectivity index: (COX-1 IC50/COX-2 IC50).
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30 mmol/kg per day, as compared with indomethacin (100%
ulceration) and celecoxib (40% ulceration), the reference anti-
inflammatory drugs utilized in the present study (Table 1). It is
to be noted that, although the analogs 10 and 27 displayed
equipotent or even higher anti-inflammatory profiles than
indomethacin, yet they kept their GI safety properties (20 and
0% ulceration, respectively). Gross observation of the isolated
rat stomachs showed a normal stomach texture for com-
pounds 17, 19, and 27 (0% ulceration), whereas the analogs 10
and 27 exhibited slight hyperemia (20% ulceration).
expected good permeability and oral bioavailability. Addi-
tionally, all the evaluated compounds showed TPSA range
2
2
̊
̊
55.88–132.36 A (<140 A ), indicating good permeability and
transport of the compounds in the cellular plasma membrane.
Furthermore, all the tested compounds exhibited a consider-
able % ABS range 63.3–89.7%, which is a designation of good
bioavailability upon oral administration. It is to be noted that
TPSA is inversely proportional to %ABS, for example, the
analog 17 possesses the maximum absorption (89.7%),
whereas its corresponding TPSA was the least among the
2
̊
series (55.88 A ).
Acute toxicity
On the other hand, the OSIRIS Property Explorer (2014:
Version 2) software [46] was utilized to calculate the aqueous
solubility (Log S) of the tested compounds (being significantly
affecting absorption as well as distribution characteristics),
where they gave moderate Log S values ranging between
ꢀ1.96 and ꢀ4.63 mol/L. Finally, the same OSIRIS software was
employed to determine the overall and drug score values
which is an expression representing integration of drug-
likeness, several physicochemical parameters, and toxicity
probabilities in one numerical value that can be utilized to
foresee compound’s ability to act as a drug candidate. A
positive value for drug-likeness and drug score indicates that
the compound contains fragments that are often present in
most of currently used drugs. The results revealed that
compounds 10, 17, 19, and 27 gave positive values for drug-
likeness lying between 4.48 and 6.79, while the analog 5
showed negative value of drug-likeness (ꢀ2.85). Additionally,
all the evaluated compounds (including those compounds
with negative drug-likeness scores) have displayed positive
values ranging from 0.14 to 0.66 in the drug score calculation
(Table 3). Finally, according to OSIRIS findings, prediction of
the expected toxicity risks pointed out that none of the five
investigated compounds would exert tumorigenic, muta-
genic, irritant, or reproductive toxicity.
The same biologically active derivatives 5, 10, 17, 19, and 27
were further investigated for their oral acute toxicity in male
mice according to the method described by Litchfield and
Wilcoxon [40]. The obtained acute lethal doses (ALD50
)
indicated that the test compounds proved to be nontoxic
and well tolerated by experimental animals up to 250 mg/kg.
Moreover, the same compounds were tested for their toxicity
through the parenteral route [41], where they proved to be
non-toxic up to 80 mg/kg.
In silico studies
In silico calculation of the pharmacokinetic properties
(ADME-T) and drug-likeness
Drug-likeness is a term that describes an integrated equilib-
rium between multiple molecular properties and structure
features that define whether a particular compound is
comparable to already known drugs. Among the common
principles applied to evaluate the drug-like properties of a
compound, stemmed prominently the Lipinski’s rule of 5
(RO5) [42] and Veber’s criteria [43]. These properties comprise
hydrophobicity, electronic distribution, hydrogen-bonding
capability, molecule size, and flexibility that would affect
molecule’s behavior in a living system including bioavailabil-
ity, transport properties, affinity to proteins, reactivity, and
toxicity.
In this context, a computational study for the most
biologically active compounds 5, 10, 17, 19, and 27 utilizing
the Molinspiration online property calculation toolkit [44]
was carried out to determine the Lipinski’s molecular
properties and the number of rotatable bonds (nROTB),
together with the topographical polar surface area (TPSA; a
sum of polar atoms’ surfaces: A descriptor for drug absorp-
tion, penetrability and bioavailability), the percentage of
absorption (ABS%) calculated as (ABS% ¼ 109 ꢀ 0.345 ꢂ
TPSA) [45] and the molecular volume (a determinant of the
transport characteristics).
The results presented in Table 3 revealed that all the tested
compounds comply with Lipinski’s rule of 5, where logP values
ranged between 0.17 and 2.03 (<5), MW range 307–409
(<500), HBA range 5–8 (ꢃ10), and HBD range 1–3 (<5),
suggesting that these compounds would not be expected to
cause problems with oral bioavailability. Moreover, all the
tested compounds showed nROTB values of 2–5 (<10),
indicating acceptable molecular flexibility with consequent
Conclusion
The objective of the present research work was to develop a
series of novel non-acidic polysubstituted pyrazoles to be
evaluated for their in vivo and in vitro anti-inflammatory
activities. Our aim has been verified by the synthesis of 27
structure hybrids comprising basically the 5-hydroxy-3-
methyl-1-phenyl-4-substituted-1H-pyrazole scaffold directly
attached to a variety of non-acidic heterocycles and function-
alities or annulated as pyrano[2,3-c]pyrazoles for synergistic
purpose. In general, obvious anti-inflammatory activity was
linked with analogs comprising a sulfonamido functionality
and/or the pyrazolyl, pyrimidinyl, and tetrazolo[1,5-a]pyrimi-
dinyl counterparts, in addition to the pyrano[2,3-c]pyrazole
structure variant. Eighteen compounds were able to exert
variable in vivo anti-inflammatory activities, among which the
analogs 5, 10, 17, 19, and 27 proved to be the most active
candidates as evidenced from their distinctive % protection
values which were comparable or even better than the
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Table 3. In silico ADME-T calculations, Lipinski’s parameters drug-likeness, and drug scorea,b)c)1 of the biologically active
compounds.
Compound
number
%
Logpc) MWd)
HBAe)
HBDf)
Viol.g) nROTBh) TPSAi)
ABSj)
Vol.k)
LogSl)
D.L.m)
D.S.n)
5
10
17
19
0.17
2.03
1.96
1.59
0.78
4.08
3.61
337.35
409.47
312.40
307.32
319.34
343.77
381.38
8
8
5
8
7
5
5
3
3
1
1
2
1
2
0
0
0
0
0
0
0
5
4
2
2
2
4
4
132.36 63.33
116.04 68.97
55.88 89.70
90.04 77.94
108.20 71.67
68.54 85.35
77.99 82.09
275.29 ꢀ2.72
344.55 ꢀ3.73
274.84 ꢀ1.96
261.94 ꢀ3.14
257.05 ꢀ4.63
286.68 ꢀ5.40
298.65 ꢀ4.17
ꢀ2.85
5.68
6.34
4.48
6.79
9.41
8.11
0.14
0.46
0.66
0.59
0.37
0.56
0.37
27
Ind.o)
Cel.p)
a) Molinspiration chemoinformatics property calculator (2014).
b) OSIRIS property explorer (2014).
c) Partition coefficient.
d) Molecular weight.
e) Number of H-Bond acceptors (O and N atoms).
f) Number of H-Bond donors (OH and NH groups).
g) Number of Rule of 5 violations.
h) Number of rotatable bonds.
i) Topological polar surface area.
j) Absorption %.
k) Molecular volume.
l) Aqueous solubility prediction.
m) Drug likeness.
n) Drug score.
o) Indomethacin.
p) Celecoxib.
reference standards. The ED50 values of the most active
analogs 5, 10, 17, 19, and 27 (35.7–75.2 mmol/kg) affirmed the
anti-inflammatory potential, especially the bipyrazole 10
(ED50 35.7 mmol/kg) which was nearly equiactive with
celecoxib (32.1 mmol/kg). Meanwhile, all the tested com-
pounds showed in vitro mild COX-1 inhibitory effects, while
they proved to be moderate to good COX-2 inhibitors. In
particular, 10, 17, and 27 exhibited distinctive COX-2
inhibition (IC50 0.72, 0.91 and 0.76 mM, respectively) as
compared with celecoxib (IC50 0.84 mM), meanwhile they
possessed noticeable COX-2 selectivity (SI values 7.83, 6.87,
and 7.16, respectively), close to that of celecoxib (SI 8.68).
Additionally, 5, 10, 17, 19, and 27 proved to be GI safe (0ꢀ20%
ulceration), and non-toxic being well tolerated the by
experimental animals up to 250 mg/kg orally and 80 mg/kg
parenterally. Collectively, the in vivo ED50 values for the most
potent five derivatives get along with their in vitro COX-2
selectivity indices, suggesting their usefulness as selective
anti-inflammatory COX-2 inhibitors. The bipyrazole 10 and
pyranopyrazole 27 (structurally relevant to celecoxib) could
be considered the most active anti-inflammatory members in
this study, being more potent than indomethacin and nearly
equiactive to celecoxib, besides their obvious selective COX-2
inhibitory activity, high safety margin, and predicted phar-
macokinetic (ADME-T) suitability for oral use.
of a new class of non-acidic anti-inflammatory agents that
deserves further investigation and derivatization.
Experimental
Chemistry
General
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. The infrared (IR) spectra
were recorded on Shimadzu FT-IR 8400S infrared spectropho-
tometer using the KBr pellet technique. 1H and 13C NMR
spectra were recorded on
a Bruker DPX-400 FT NMR
spectrometer using tetramethylsilane as the internal standard
and CDCl3 as a solvent (chemical shifts in d, ppm). Splitting
patterns were designated as follows: s: singlet; d: doublet; m:
multiplet; q: quartet. Elemental analyses were performed on a
2400 Perkin Elmer Series 2 analyzer and the found values were
within ꢁ0.4% of the theoretical values. Follow-up of the
reactions and checking the homogeneity of the compounds
were made by TLC on silica gel-protected aluminum sheets
(Type 60 F254, Merck) and the spots were detected by
exposure to UV-lamp at l 254 nm. Most of the starting
compounds and target products were synthesized according
to known literature procedures [47–49]. The synthesis of and
mechanism of formation of compound 20 was carried out
following the method described by Faidallah et al. [34].
Consequently, such type of hybrid structures would
represent the appropriate matrix for future development
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Selected NMR spectra as well as the InChI codes of the
investigated compounds together with some biological
activity data are provided as Supporting Information.
3). 1H NMR (d-ppm) of compound 2: 2.14 (s, 3H, CH3), 2.71 (s,
3H, CH3), 5.80 (s, 1H, H-5), 7.06–7.65 (m, 6H, ArH þ NH), 16.52
(exchangeable OH). 13C NMR (d-ppm) of compound 2: 16.95
(CH3), 20.27 (CH3), 95.58, 102.83, 115.45, 118.56, 129.32,
144.15, 146.47, 171.35 (Ar-C), 163.66 (CN), 179.53 (CO).
1H NMR (d-ppm) of compound 3: 2.46 (s, 3H, CH3), 2. 56 (s,
3H, CH3), 5.63 (s, 1H, H-5), 7.13–7.74 (m, 7H, ArH þ NH þ NH2),
16.48 (exchangeable OH). 13C NMR (d-ppm) of compound 3:
17.54 (CH3), 20.23 (CH3), 96.45, 103.64, 115.73, 126.28, 129.35,
141.41, 149.94, 174.23, 162 (ArC), 161.96 (CN), 178.86 (CO).
General procedure for the synthesis of 4-hydroxy-6-
methyl-3-[1-(substituted hydrazono)ethyl]pyran-2-ones 2
and 3
To a solution of dehydroacetic acid (DHA) 1 (1.68 g, 10 mmol)
in benzene (20 mL) was added the appropriate aryl hydrazine
(10 mmol). The mixture was refluxed for 15 min and allowed
to stand at room temperature for 2 h. After being cooled, the
hydrazone was collected by filtration and recrystallized from
ethanol. Physicochemical and analytical data are recorded in
Table 4 (see the Supporting Information for an extended
version of Table 4).
General procedure for the synthesis of 1-(5-hydroxy-3-
methyl-1-(4-substituted phenyl)-1H-pyrazol-4-yl)butane-
1,3-diones 4 and 5
A solution of the appropriate hydrazono derivative 2 or 3
(10 mmol) in acetic acid (20 mL) was refluxed for 1 h. The
reaction mixture was concentrated, cooled, and the formed
IR (cmꢀ1) of compounds 2 and 3: 1705–1712 (CO), 3298–
3323 (OH), 3638–3456 (NH), 1157, 1360 (SO2N for compound
Table 4. Physicochemical and analytical dataa) for compounds 2–33.
Mol. formula
(mol. weight)
Compound number
R
Yield (%)
m.p. (°C)
2
3
4
5
6
7
8
9
H
70
79
91
85
86
92
67
92
75
77
73
72
66
70
71
66
75
67
60
59
67
70
65
68
72
68
74
72
67
198–200
222–224
201–202
214–216
136–138
324–326
126–128
324–326
312–314
143–145
242–244
225–227
164–166
174–176
163–165
145–147
185–187
250–252
201–203
222–224
250–252
134–136
272–274
230–232
152–154
272–274
145–147
148–150
286–288
C14H14N2O3 (258.28)
C14H15N3O5S (337.35)
C14H14N2O3 (258.28)
C14H15N3O5S (337.35)
SO2NH2
H
SO2NH2
H
C
20H18BrN3O2 (412.28)
C20H19BrN4O4S (491.36)
C14H13N3O2 (255.27)
SO2NH2
H
SO2NH2
C14H14N4O4S (334.35)
C20H19N5O3S (409.46)
C27H22ClN5O3S (532.01)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
29
32
33
–
–
–
C27H23ClN6O4S (563.03)
C27H23ClN6O3S2 (579.09)
C29H23ClN6O4S2 (619.11)
C35H27ClN6O3S2 (679.21)
C15H14N4OS (298.36)
C16H16N4OS (312.39)
C15H16N6O (296.33)
C15H13N7 (307.31)
C17H16N6O (320.35)
C17H15BrN6O (399.24)
C19H18N6O3 (378.38)
C17H14N6O3 (350.33)
C18H18N6O (334.38)
C23H20N6O (396.44)
C14H12N2O2 (240.26)
C14H13N3O4S (319.34)
C14H11ClN2O2 (274.70)
C16H11F3N2O3 (336.27)
C16H12F3N3O5S (415.34)
–
–
–
H
CH3
–
–
–
–
–
–
CH3
C6H5
H
SO2NH2
–
H
SO2NH2
a)
The found values in the elementary microanalysis are within ꢁ0.4% of the calculated values.
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precipitate was filtered, washed with water, dried, and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 4.
compound 8: 13.96 (CH3), 19.55 (CH3), 96.42, 106.78, 112.59,
120.99, 127.31, 129.44, 137.08, 146.77, 153.57, 161.65 (ArC).
1H NMR (d-ppm) of compound 9: 2.61 (s, 3H, CH3), 2.98 (s, 3H,
CH3), 6.18 (s, 1H, isoxazole C4-H), 7.22–7.75 (m, ArH, 4H), 14.83
(exchangeable OH). 13C NMR (d-ppm) of compound 9: 13.44
(CH3), 19.23 (CH3), 96.67, 106.34, 112.95, 120.46, 127.22,
129.76, 137.16, 146.83, 153.36, 161.83 (ArC).
IR (cmꢀ1) of 4 and 5: 1622–1635 (CO), 1715–1724 (CO), 3328–
3398 (OH), 1149, 1355 (SO2N for compound 4). 1H NMR (d-
ppm) of compound 4: 2.11 (s, 3H, CH3), 2.34 (s, 3H, CH3), 3.88 (s,
2H, CH2), 7.26–7.80 (m, ArH, 5H), 14.85 (exchangeable OH).
13C NMR (d-ppm) of compound 4: 15.50 (CH3), 22.24 (CH3),
30.66 (CH2), 96.82, 100.40, 120.40, 121.37, 126.77, 126.83,
129.96, 129.06, 137.03, 147.12, 158.37 (ArC), 181.25 (CO),
188.58 (CO). 1HNMR (d-ppm) of compound 5: 2.56 (s, 3H, CH3),
2.43 (s, 3H, CH3), 3.97 (s, 2H, CH2), 7.64–7.84 (m, 6H,
ArH þ NH2), 15.28 (exchangeable OH). 13C NMR (d-ppm) of
compound 5: 16.0 (CH3), 22.45 (CH3), 30.18 (CH2), 96.64,
100.44, 120.98 121.57, 126.73, 126.38, 129.42, 129.97, 137.24,
147.71, 157.95 (ArC), 180.43 (CO), 188.25 (CO).
5-Hyroxy-3-methyl-1-(4-sulfamylphenyl)-4-(3-methyl-1-
phenyl-1H-pyrazol-5-yl)-1H-pyrazole 10
A solution of the starting pyrazole 5 (3.3 g, 10 mmol) in
ethanol (20 mL) was refluxed with phenylhydrazine (1.1 g,
10 mmol) for 2 h. The reaction mixture was cooled and the
formed precipitate was filtered, washed with ethanol, dried,
and recrystallized from ethanol. Physicochemical and analyti-
cal data are recorded in Table 4.
IR (cmꢀ1): 3342 (OH), 3405 (NH), 1153, 1376 (SO2N), 3349,
3355 (NH2). 1H NMR (d-ppm): 2.38 (s, 3H, CH3), 2.79 (s, 3H, CH3),
6.14 (s, 1H, pyrazole C4-H), 7.13–7.69 (m, 11H, ArH þ NH2),
13.54 (s, 1H, exchangeable OH). 13C NMR (d-ppm): 11.76 (CH3),
14.09 (CH3), 95.82, 101.38, 112.74, 120.28, 125.52, 128.84,
138.57, 140.39, 145.48, 146.58 (ArC).
General procedure for the synthesis of 3-((2-
bromophenyl)-amino)-1-(5-hydroxy-3-methyl-1-(4-
substituted phenyl)-1H-pyrazol-4-yl)but-2-en-1-ones 6 and
7
A solution of the appropriate pyrazole 4 or 5 (10 mmol) in
ethanol (20 mL) was refluxed with the 2-bromoaniline (1.7 g,
10 mmol) for 2 h. The reaction mixture was cooled and the
precipitated aryl aminopyrazole derivative was filtered,
washed with ethanol, and recrystallized from DMF. Physico-
chemical and analytical data are recorded in Table 4.
IR (cmꢀ1) of 6 and 7: 1662–1679 (CO), 3265–3320 (OH), 3395–
3405 (NH), 1156, 1372 (SO2N for compound 7). 1H NMR (d-
ppm) of compound 6: 2.45 (s, 3H, CH3), 2.18 (s, 3H, CH3), 5.46 (s,
5-Hyroxy-3-methyl-1-[4-(N-4-chlorobenzylidenesulfamyl)-
phenyl]-4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-1H-
pyrazole 11
A stirred solution of the bipyrazole 10 (4.1 g, 10 mmol) in
acetic acid (25 mL) was heated under reflux with 4-chlor-
obenzaldehyde (1.4 g, 10 mmol) for 8 h. The reaction mixture
was concentrated and the separated azomethine was filtered,
dried, and recrystallized from the ethanol. Physicochemical
and analytical data are recorded in Table 4.
–
1H, CH ), 7.18–7.86 (m, 11H, ArH þ NH), 12.26 (exchangeable
–
OH). 13C NMR (d-ppm) of compound 6: 15.43 (CH3), 20.44
–
(CH ), 94.28 (CH ) 100.42, 117.32, 120.28, 126.13, 129.53,
IR (cmꢀ1): 3352 (OH), 1150, 1345 (SO2N). 1H NMR (d-ppm):
2.12 (s, 3H, CH3), 2.58 (s, 3H, CH3), 6.36 (s, 1H, pyrazole, C4-H),
–
3
129.46, 131.66, 136.75, 137.90, 146.86, 159.74, 161.44 (ArC),
184.86 (CO). 1H NMR (d-ppm) of compound 7: 2.33 (s, 3H, CH3),
7.21–7.55 (m, 13H, ArH), 8.21 (s, 1H, CH N), 15.24 (s, 1H,
–
–
–
2.43 (s, 3H, CH ), 5.65 (s, 1H, CH ), 7.09–7.79 (m, 11H,
exchangeable OH). 13C NMR (d-ppm): 15.61(CH3), 20.44 (CH3),
94.89, 100.74, 113.32, 117.52, 120.15, 122.65, 126.60, 127.58,
128.82, 129.25, 129.17, 130.31, 137.81, 139.66, 146.52, 147.23,
–
3
ArH þ NH þ NH2), 12.56 (exchangeable OH). 13C NMR (d-ppm)
–
–
of compound 7: 15.84 (CH ), 20.82 (CH ), 94.38 (CH ) 100.76,
3
3
–
117.34, 120.48, 126.59, 129.43, 129.56, 131.23, 136.94, 137.93,
146.55, 160.47, 161.32 (ArC), 184.52 (CO).
158.69, 160.32 (ArC), 161.24 (CH N).
–
N1-[4-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-5-hydroxy-5-
methyl-1H-pyrazol-1-yl]benzenesulfonyl-N3-(4-
chlorophenyl)urea 12
General procedure for the synthesis of 5-hydroxy-3-
methyl-4-(3-methylisoxazol-5-yl)-1-(4-substituted phenyl)-
1H-pyrazoles 8 and 9
A stirred mixture of the bipyrazole 10 (4.1 g, 10 mmol),
anhydrous K2CO3 (1.4 g, 10 mmol), and 4-chlorophenyl
isocyanate (1.5 g, 10 mmol) in dry acetone (25 mL) was heated
under reflux for 18 h. The solvent was removed under reduced
pressure and the remaining solid residue was dissolved in
water (30 mL). After neutralization of the resulting solution
with drops of 2 N HCl, the precipitated crude product was
filtered, washed with water, dried, and recrystallized from
ethanol. Physicochemical and analytical data are recorded in
Table 4.
A solution of the appropriate pyrazole 4 or 5 (10 mmol) and
hydroxyl amine hydrochloride (0.69 g, 10 mmol) in a mixture
of ethanol and acetic acid (1:1, 20 mL) was refluxed for 2 h.
The reaction mixture was allowed to attain room temperature
then allowed to stand in the refrigerator for further 12 h. The
solid thus precipitated was collected by filtration and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 4.
IR (cmꢀ1) of 8 and 9: 3345–3378 (OH), 1152, 1368 (SO2N for
compound 9). 1H NMR (d-ppm) of compound 8: 2.09 (s, 3H,
CH3), 2.47 (s, 3H, CH3), 6.33 (s, 1H, isoxazole C4-H), 7.04–7.88
(m, ArH, 5H), 14.62 (exchangeable OH). 13C NMR (d-ppm) of
IR (cmꢀ1): 1652 (CO), 1336, 1164 (SO2N), 3343 (OH), 3402
(NH). 1H NMR (d-ppm): 2.11 (s, 3H, CH3), 2.48 (s, 3H, CH3), 6.57
(s, 1H, pyrazole C4-H), 7.11–7.86 (m, 15H, ArH þ 2NH), 12.06
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(s, 1H, exchangeable OH). 13C NMR (d-ppm): 15.79 (CH3), 20.28
(CH3), 97.16, 101.49, 101.65, 114.49, 123.61, 123.87, 130.22,
130.94, 138.67, 143.18, 147.81, 160.37, 161.29, 162.14, 165.19,
169.10 (ArC), 173.75 (CO).
IR (cmꢀ1): 1336, 1166 (SO2N), 3328 (OH). 1H NMR (d-ppm): 2.15
(s, 3H, CH3), 2.42 (s, 3H, CH3), 5.63 (s, 1H, thiazoline C5-H), 6.42
(s, 1H, pyrazole C4-H), 6.68–7.46 (m, 18H, ArH), 14.44 (s, 1H,
exchangeable OH). 13C NMR (d-ppm): 15.71 (CH3), 20.89 (CH3),
97.78, 110.87, 114.24, 120.02, 121.65, 126.04, 126.92, 128.54,
129.12, 129.29, 129.44, 129.94, 136.37, 136.62, 139.85, 142.40,
N1-[4-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-5-hydroxy-5-
methyl-1H-pyrazol-1-yl]benzenesulfonyl-N3-(4-
–
153.64, 155.76 (ArC), 169.32 (N C).
–
chlorophenyl)thiourea 13
A solution of 4-chlorophenyl isothiocyanate (1.7 g, 10 mmol)
in dry acetone (5 mL) was added to a stirred mixture of the
bipyrazole 10 (4.1 g, 10 mmol) and anhydrous K2CO3 (1.4 g,
10 mmol) in dry acetone (25 mL), and the mixture was heated
under reflux for 10 h. Working up of the reaction mixture was
done as mentioned above for compound 12. The crude
product was recrystallized from DMF. Physicochemical and
analytical data are recorded in Table 4.
6-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-4-
methyl-1-substituted-1H-pyrimidine-2-thiones 16 and 17
A solution of the pyrazole 4 (2.58 g, 10 mmol) in absolute
ethanol (20 mL) was refluxed with the appropriate thiourea
(10 mmol) for 6 h. The reaction mixture was concentrated and
the separated solid product was filtered, washed with ethanol
and recrystallized from ethanol/DMF (5:1). Physicochemical
and analytical data are recorded in Table 4.
IR (cmꢀ1): 1155 (CS), 1181, 1368, 1169 (SO2N), 3358 (OH),
3402 (NH). 1H NMR (d-ppm): 2.09 (s, 3H, CH3), 2.47 (s, 3H, CH3),
6.59 (s, 1H, pyrazole C4-H), 7.04–7.88 (m, 15H, ArH þ 2NH),
11.86 (s, 1H, exchangeable OH). 13C NMR (d-ppm): 15.92 (CH3),
20.08 (CH3), 94.87, 100.92, 120.53, 120.98, 122.73, 123.29,
126.03, 160.02, 160.99 (ArC), 184.80 (CS).
IR (cmꢀ1): of compounds 16 and 17: 3322–3356 (OH), 1182–
1192 (CS), 3375 (NH for compound 16). 1H NMR (d-ppm) of
compound 16: 2.16 (s, 3H, CH3), 2.58 (s, 3H, CH3), 5.76 (s, 1H,
exchangeable NH), 7.11–7.43 (m, 6H, Ar H þ pyrimidine C5-H),
15.85 (s, 1H, exchangeable OH). 13C NMR (d-ppm) of
compound 16: 19.99 (CH3), 20.31 (CH3), 97.58, 107.10,
126.94, 129.86, 134.02, 134.93, 163.32, 163.73, 165.03 (ArC),
185.02 (C S). 1H NMR (d-ppm) of compound 17: 1.72 (s, 3H,
–
–
2-{[4-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-5-hydroxy-5-
methyl-1H-pyrazol-1-yl]benzenesulfonylimino}-3-(4-
chlorophenyl)thiazolidin-4-one 14
CH3), 2.45 (s, 3H, CH3), 2.76 (s, 3H, CH3), 6.89–7.49 (m, 6H,
Ar H þ pyrimidine C5-H), 15.12 (s, 1H, exchangeable OH). 13 MR
(d-ppm) of compound 17: 19.62 (CH3), 20.63 (CH3), 37.22 (CH3),
99.8, 108.2, 118.7, 126.0, 129.1, 129.3, 139.8, 152.4, 157.9,
To a stirred solution of the sulfonylthioureido derivative 13
(3.0 g, 5 mmol) in absolute ethanol (20 mL) was added ethyl
bromoacetate (1.84 g, 11 mmol) and anhydrous sodium
acetate (1.64 g, 0.02 mol) and the mixture was heated under
reflux for 2 h. The reaction mixture was left for 1 h to attain
room temperature, then poured onto ice water (30 mL). The
solid product thus formed was filtered, washed with water,
dried, and recrystallized from ethanol/benzene mixture (1:1).
Physicochemical and analytical data are recorded in Table 4.
IR (cmꢀ1): 1329, 1156 (SO2N), 1712 (CO), 3397 (OH). 1H NMR
(d-ppm): 2.51 (s, 3H, CH3), 2.42 (s, 3H, CH3), 3.42 (s, 2H,
thiazolidine CH2), 5.48 (s, 1H, pyrazole C4-H), 7.32–7.76 (m,
13H, ArH), 14.44 (s, 1H, exchangeable OH). 13C NMR (d-ppm):
17.53 (CH3), 20.04 (CH3), 37.32 (CH2), 96.95, 107.37, 115.29,
115.50, 121.58, 121.38, 124.84, 126.24, 127.72, 127.32, 128.38,
140.61, 140.63, 140.92, 161.25, 163.74, 163.32, 163.73 (ArC),
–
164.8 (ArC), 180.40 (C S).
–
6-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
hydrazino-4-methylpyrimidine 18
A solution of the pyrimidine-2-thione 16 (1.5 g, 5 mmol) and
98% hydrazine hydrate (1 g) in absolute ethanol (50 mL) was
stirred at room temperature for 2 h. The solid which was
separated during stirring was filtered and recrystallized from
ethanol. Physicochemical and analytical data are recorded in
Table 4.
IR (cmꢀ1): 3192–3342 (NHNH2), 3367 (OH). 1H NMR (d-ppm):
1.72 (s, 3H, CH3), 2.38 (s, 3H, CH3), 5.92 (s, 2H, exchangeable
NH2), 6.14 (s, 1H, exchangeable NH), 6.69–7.38 (m, 6H,
ArH þ H-5), 14.79 (s, 1H, exchangeable OH). 13C NMR (d-
ppm): 10.03 (CH3), 18.2 (CH3), 99.8, 108.2, 118.7, 126.0, 129.1,
129.3, 139.8, 152.4, 157.9, 164.8 (ArC).
–
172.47 (C N), 184.77 (CO).
–
2-{[4-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-5-hydroxy-5-
methyl-1H-pyrazol-1-yl]benzenesulfonylimino}-4-phenyl-
3-(4-chlorophenyl)thiazoline 15
5-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-
methyl-[1,2,3,4]tetrazolo[1,5-a]pyrimidine 19
A stirred solution of the hydrazino derivative 18 (2.9 g,
10 mmol) in glacial acetic acid (10 mL) was gradually treated
with 25% aqueous NaNO2 (15 mL) at room temperature. The
tetrazolo derivative which separated as white solid was
filtered, washed with water, and recrystallized from DMF.
Physicochemical and analytical data are recorded in Table 4.
IR (cmꢀ1): 3356 (OH). 1H NMR (d-ppm): 2.15 (CH3), 2.57 (CH3),
6.95–7.26 (m, 6H, ArH þ H-5), 15.58 (s, 1H, exchangeable OH).
13C NMR (d-ppm): 19.95 (CH3), 20.26 (CH3), 97.26, 107.27,
A solution of the sulfonylthioureido 13 (3.0 g,10 mmol) in
absolute ethanol (20 mL) was refluxed with phenacyl bromide
(2.2 g, 11 mmol) and anhydrous sodium acetate (1.64 g,
20 mmol) for 2 h, during which a solid product was partially
crystallized out. The mixture was left to attain room
temperature, filtered, washed with cold ethanol, dried, and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 4.
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114.76, 126.74, 129.07, 139.27, 147.53, 159.19, 163.28, 163.60,
166.56 (ArC).
pyrimidine H-5 þ NH), 14.85 (s, 1H, exchangeable OH).
13C NMR (d-ppm): 15.97 (CH3), 20.78 (CH3), 93.44, 100.51,
120.43, 125.06, 125.83, 128.99, 129.86, 135.66, 136.25, 138.03,
146.40 (ArC), 162.51 (CO), 167.78 (CO).
5-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3,7-
dimethyl[1,2,4]triazolo[3,4-a]pyrimidine 20
A mixture of the hydrazino derivative 18 (2.9 g, 10 mmol) and
acetylacetone (1.0 g, 10 mmol) in ethanol (20 mL) was refluxed
with stirring for 8 h. The reaction mixture was concentrated
and the separated product was filtered, washed, dried, and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 4.
General procedure for the synthesis of 6-(5-hydroxy-3-
methyl-1-phenyl-1H-pyrazol-4-yl)-8-methyl-3-substituted-
2H-pyrimido-[2,1-c][1,2,4]triazines 24 and 25
A solution of the hydrazino derivative 18 (2.9 g, 10 mmol) in
glacial acetic acid (15 mL) was refluxed with the appropriate
b-bromoketone (10 mmol) for 2 h. After being cooled to room
temperature, the mixture was poured onto crushed ice and
the separated solid was filtered, washed with water, and
recrystallized from DMF. Physicochemical and analytical data
are recorded in Table 4.
IR (cmꢀ1): 3375 (OH). 1H NMR (d-ppm): 2.10, (s, 3H, CH3), 2.36
(s, 3H, CH3), 2.74 (s, 3H,CH3), 7.04–7.88 (m, 5H, ArH), 5.33 (s,
1H, C6-H), 11.86 (s,1H, exchangeable OH). 13C NMR (d-ppm):
15.94 (CH3), 20.75 (CH3), 20.97 (CH3), 93.41, 100.49, 120.40,
125.03, 125.80, 128.96, 129.83, 135.64, 136.22, 138.01, 146.37,
160.49, 162.41 (ArC).
IR (cmꢀ1) of compounds 24 and 25: 3287–3298 (OH), 3358–
3373 (NH). 1H NMR (d-ppm) of compound 24: 2.18 (CH3), 2.39
(CH3), 2.52 (CH3), 5.75 (s, 1H, C4-H), 6.87–7.35 (m, 7H,
ArH þ NH þ H-7), 15.62 (s, 1H, exchangeable OH). 13C NMR
(d-ppm) of compound 24: 17.98 (CH3), 20.04 (CH3), 21.3 (CH3),
89.56, 98.76, 109.18, 118.78, 123.65, 126.20, 129.07, 129.14,
139.67, 146.32, 153.84, 155.82, 164.65 (ArC). 1H NMR (d-ppm)
of compound 25: 2.02 (CH3), 2.39 (CH3), 6.69–7.52 (m, 12H,
ArH þNH þ H-7), 15.25 (s, 1H, exchangeable OH). 13C NMR (d-
ppm) of compound 25: 19.97 (CH3), 22.47 (CH3), 93.52, 96.45,
107.43, 108.53, 114.55, 118.09, 121.23, 121.51, 124.91, 126.37,
128.64, 137.06, 140.50, 145.39, 146.61, 163.27, 163.79 (ArC).
3-Bromomethyl-5-(5-hydroxy-3-methyl-1-phenyl-1H-
pyrazol-4-yl)-7-methyl[1,2,4]triazolo[3,4-a]pyrimidine 21
A solution of the hydrazino derivative 18 (2.9 g, 10 mmol) in
ethanol (20 mL) was refluxed with ethyl bromoacetate
(0.167 g, 10 mmol) for 2 h. The reaction mixture was then
concentrated to yield a white solid which was filtered, washed
with ethanol, and recrystallized from ethanol. Physicochemi-
cal and analytical data are recorded in Table 4.
IR (cmꢀ1): 3356 (OH). 1H NMR (d-ppm): 2.37 (s, 3H, CH3), 2.44
(s, 3H, CH3), 4.76 (s, 2H, CH2), 7.18–7.82 (m, 6H, ArH þ H-6),
14.43 (s, 1H, exchangeable OH). 13C NMR (d-ppm): 15.62 (CH3),
22.47 (CH3), 30.79 (CH2), 96.82, 100.53, 120.99, 121.02, 126.99,
129.10, 129.15, 137.24, 147.12, 158.70, 163.56, 166.96 (ArC).
General procedure for the synthesis of 3,6-dimethyl-1-
substituted pyrano[2,3-c]pyrazole-4(1H)-ones 26 and 27
To a solution of the appropriate pyrazole diketone 4 or 5
(10 mmol) in acetic acid (20 mL) was added dropwise
concentrated sulfuric acid (1 mL). The mixture was allowed
to stand at room temperature for 2 h, then poured carefully
onto crushed ice. The formed precipitate was filtered, washed
several times with cold water, dried, and recrystallized from
ethanol/DMF (4:1). Physicochemical and analytical data are
recorded in Table 4.
Ethyl 5-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-
methyl[1,2,4]triazolo[3,4-a]pyrimidine-3-carboxylate 22
A mixture of 18 (2.9 g, 10 mmol) and an excess of diethyl
oxalate (7.3 g, 50 mmol) was refluxed for 3 h. The reaction was
allowed to attain room temperature and the separated solid
was collected by filtration and recrystallized from DMF.
Physicochemical and analytical data are recorded in Table 4.
IR (cmꢀ1): 1702 (CO), 3356 (OH). 1H NMR (d-ppm): 1.35 (t,
J ¼ 8 Hz, 3H, CH3), 2.39 (s, 3H, CH3), 2.79, (s, 3H, CH3), 4.30 (q,
J ¼ 8 Hz, 2H, CH2), 7.42–7.55 (m, 6H, ArH þ H-5), 15.13 (s, 1H,
exchangeable OH). 13C NMR (d-ppm): 8.6 (CH3), 13.5 (CH3),
21.2 (CH3), 59.2 (CH2), 107.8, 118.7, 119.63, 126.2, 129.0, 129.2,
139.6, 151.8, 152.2, 152.4, 164.2, 166.4, 167.3 (ArC), 167.3 (CO).
IR (cmꢀ1) of compounds 26 and 27: 1660–1664 (CO), 1152,
1375 (SO2N for compound 17). 1H NMR (d-ppm) of compound
26: 2.10 (s, 3H, CH3), 2.44 (s, 3H, CH3), 5.41 (s, 1H, pyrane C3-H),
7.04–7.32 (m, 5H, ArH). 13C NMR (d-ppm) of compound 26:
14.89 (CH3), 21.22 (CH3), 98.24, 112.54, 118.98, 126.02, 129.21,
129.43, 139.56, 150.43, 164.73 (ArC), 185.89 (CO). 1H NMR (d-
ppm) for compound 27: 2.04 (s, 3H, CH3), 2.47 (s, 3H, CH3), 5.32
(s, 1H, pyrane C3-H), 6.89–7.43 (m, 6H, ArH þ NH2). 13C NMR (d-
ppm) for compound 27: 15.94 (CH3), 20.66 (CH3), 93.10,
114.40, 120.37, 126.83, 128.96, 131.06, 138.03, 146.36, 162.96
(ArC), 183.66 (CO).
6-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-8-
methyl-2H-pyrimido-[2,1-c][1,2,4]triazin-3,4-dione 23
A mixture of 18 (2.9 g, 10 mmol), sodium acetate (1.0 g), and
diethyl oxalate (1.6 g, 11 mmol) in ethanol (30 mL) was
refluxed for 8 h. The reaction mixture was poured into ice
cold water and the precipitated solid product was filtered,
washed with water, recrystallized from DMF. Physicochemical
and analytical data are recorded in Table 4.
5-Chloro-3,6-dimethyl-1-phenylpyrano[2,3-c]pyrazole-4
(1H)-one 29
To a solution of the pyrazole 4 (2.5 g, 10 mmol) in dry
methylene chloride (20 mL) was added dropwise sulfuryl
chloride (1.35 g, 10 mmol). The reaction mixture was allowed
to stand at room temperature for 2 h, then poured into a
IR (cmꢀ1): 1662, 1667 (2 CO), 3356 (OH), 3386 (NH). 1H NMR
(d-ppm): 2.12 (CH3), 2.48 (CH3), 7.10–7.87 (m, 7H, ArH þ
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stirred solution of 10% aqueous K2CO3 (50 mL) over 5 min. The
aqueous layer was acidified with 10% hydrochloric acid and
extracted with chloroform (3 ꢂ 10 mL). The combined organic
extracts were washed with water and dried over anhydrous
sodium sulfate, then the solvent was removed under reduced
pressure. The left crude compound 28 was directly treated
with concentrated sulfuric acid (10 mL) and the mixture was
left at room temperature for 4 h then poured carefully onto
crushed ice. The product formed was extracted with chloro-
form (3 ꢂ 10 mL), the chloroformic extract was washed with
5% aqueous K2CO3 solution, dried over anhydrous sodium
sulfate, and evaporated in vacuo to dryness to give the
product 29, which was recrystallized from acetonitrile.
Physicochemical and analytical data are recorded in Table 4.
IR (cmꢀ1): 1665 (CO). 1H NMR (d-ppm): 2.20 (s, 3H, CH3), 2.49
(s, 3H, CH3), 7.21–7.67 (m, 5H, ArH). 13C NMR (d-ppm): 11.32
(CH3), 18.59 (CH3), 97.96, 101.51, 119.68, 125.04, 128.97,
137.74, 139.76, 141.75, 145.90, 164.82 (ArC), 186.56 (CO).
prior to carrageenan injection. Control group was injected
with DMSO only. The volume of paw edema (mL) was
determined by means of plethysmometer immediately after
injection of carrageenan and 4 h later. The increase in paw
volume between time 0 h and 4 h was measured and the
percentage protection against inflammation was calculated
according to the following equation:
Protection against inflammation ð%Þ ¼ðVc ꢀ VdÞ=Vc ꢂ 100
where Vc is the increase in paw volume in the absence of test
compound (control) and Vd is the increase in paw volume
after injection of the test compound. Data were expressed as
the mean ꢁ SE. Statistical differences of control and test
groups were carried out using the analysis of variance
(ANOVA) followed by “Student–Newman–Keuls Multiple
Comparison Test”. They were performed using computer
package of the Statistical Analysis System (SAS, 1987), SAS
Incorporation Institute. The difference in results was consid-
ered significant when P < 0.05. The obtained data are
recorded in Table 1. Additionally, the ED50 values for the
most potent derivatives 5, 10, 17, 19, and 27 were determined
using three different doses, and the paw thickness of each rat
was measured after 4 h of carrageenan injection according to
the literature procedure [36].
General procedure for the synthesis of 5-trifluoroacetyl-
3,6-dimethyl-1-substituted-pyrano[2,3-c]pyrazole-4(1H)-
ones 32 and 33
A solution of the appropriate pyrazole 4 or 5 (10 mmol) in THF
(20 mL) was refluxed with trifluoroacetic anhydride for 4 h.
Removal of the solvent in vacuo afforded compounds 30 and
31 in crude state. Working up of the reaction mixture was
carried out as described under compound 29. The products
were recrystallized from DMF. Physicochemical and analytical
data are recorded in Table 4.
In vitro COX-1 and COX-2 inhibitory assay
All the developed 27 target compounds (4–27, 29, 32, and 33)
were evaluated for their ability to inhibit COX-1 and COX-2
isozymes employing an ovine COX-1/COX-2 inhibitor screen-
ing assay kit (Catalog No. 760111, Cayman Chemicals, Inc.,
Ann Arbor, MI, USA) according to the manufacturer’s
instructions and literature procedure [37, 38]. The method
relied on the peroxidase component of COX which is assayed
colorimetrically by monitoring the appearance of oxidized
N,N,N0,N0-tetramethyl-1,4-phenylenediamine (TMPD), that is
generated during the reduction of PGG2 to PGH2, at 590 nm.
The efficiencies of the investigated compounds were mea-
sured as the concentration causing 50% enzyme inhibition
(IC50; mM), the selectivity index (SI value) was calculated as IC50
(COX-1)/IC50 (COX-2). Indomethacin and celecoxib were used
as reference standard in the study.
IR (cmꢀ1) of compounds 32 and 33: 1662–1668, 1669–1672 (2
CO), 1142, 1369 (SO2N for compound 33). 1H NMR (d-ppm) of
compound 32: 2.09 (s, 3H, CH3), 2.42 (s, 3H, CH3), 7.06–7.35 (m,
5H, ArH). 13C NMR (d-ppm) of compound 32: 16.80 (CH3), 20.77
(CH3),128.70(CF3),93.01,100.38,114.08,120.12,125.83,126.58,
126.83, 131.48, 138.49, 146.50, 158.60, 160.62, 162.16 (ArC),
183.66 (CO), 196.60 (COCF3). 1H NMR (d-ppm) of compound 33:
2.07 (s, 3H, CH3), 2.48 (s, 3H, CH3), 6.73–7.99 (m, 6H, ArHþ NH2).
13CNMR (d-ppm) of compound 33: 15.46 (CH3), 20.84 (CH3),
128.98 (CF3), 108.38, 112.86, 115.41, 119.62, 129.10, 129.32,
144.32, 152.42, 164.25 (ArC), 185.23 (CO), 196.20 (COCF3).
Biological evaluation
Carrageenan-induced paw edema bioassay
Ulcerogenic effects
All the synthesized 27 compounds (4–27, 29, 32, and 33) were
evaluated for their in vivo systemic anti-inflammatory activity
using the carrageenan-induced paw edema bioassay in rats as
a model of acute inflammation [35]. Male albino rats
weighing 120–150 g were used throughout the work. They
were kept in the animal house under standard conditions of
light and temperature with free access to food and water. The
animals were randomly divided into groups of five rats each.
The paw edema was induced by subplantar injection of 50 mL
of 2% carrageenan solution in saline (0.9%). Indomethacin,
celecoxib (reference anti-inflammatory drugs), and the test
compounds were dissolved in DMSO and were injected
subcutaneously in a dose of 10 mmol/kg body weight, 1 h
Compounds 5, 10, 17, 19, and 27 were evaluated for their
ulcerogenic potential in rats according to
a reported
procedure [39]. Indomethacin and celecoxib were used as
reference standard anti-inflammatory drugs. Male albino rats
(100–120 g) were fasted for 12 h prior to the administration of
the compounds. Water was given ad libitum. The animals
were divided into groups, each of five animals. Control group
received 1% gum acacia orally. Other groups received
indomethacin, celecoxib or the test compounds orally in
two equal doses at 0 and 12 h for three successive days at a
dose of 30 mmol/kg per day. Animals were sacrificed under
ether anesthesia 6 h after the last dose and the stomach was
removed. An opening at the greater curvature was made and
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the stomach was cleaned by washing with cooled saline and
inspected with a 3ꢂ magnifying lens for any evidence of
hyperemia, hemorrhage, definite hemorrhagic erosion, or
ulcer. An arbitrary scale was used to calculate the ulcer index
which indicates the severity of the stomach lesions, and the
percentage of ulceration for each group was calculated and
recorded in Table 1 as follows:
K. Chan, D. J. Schrier, A. Guglietta, D. A. Bornemeier,
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J-M. Jimenez, L. Soca, I. Cardelus, A. Heredia,
M. Miralpeix, J. Puig, J. Beleta, J. M. Huerta,
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Number of animals bearing ulcer in a group
Total number of animals in the same group
%Ulceration ¼
ꢂ 100
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