Design, synthesis and evaluation of some pyrazolo[3,4-d]pyrimidines as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2018.03.030

New pyrazolo[3,4-d]pyrimidines substituted with various functionalities or attached to a substituted pyrazole ring through different linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory activity using in vitro COX-1/COX-2 inhibition assay and in vivo formalin induced paw edema and cotton pellet-induced granuloma assays. Results revealed that compounds 17b and 18 possessed COX-1/COX-2 selectivity indices higher than diclofenac sodium and celecoxib. However, compounds 16a,b exhibited selectivity indices higher than diclofenac sodium and nearly equivalent to celecoxib, whereas, 9b displayed selectivity index comparable to diclofenac sodium. In vivo anti-inflammatory data showed that compounds 9b, 16a, 18 displayed anti-inflammatory activity higher than both references in the formalin induced paw edema model. On the other hand, the pyrazolyl derivatives 9b, 16b and 17b displayed anti-inflammatory activity about 2–2.5-fold that of diclofenac sodium and nearly 8–10.5-fold that of celecoxib in the cotton pellet-induced granuloma assay. The ulcerogenic effect of the active compounds was also investigated and results revealed that compounds 16a, 17a,b and 18 showed good gastrointestinal safety profile. Based on this, compounds 16a and 18 were considered as safe and effective leads in managing acute inflammation, while, 17b was prominent in controlling chronic inflammation.

Full text of DOI:10.1016/j.bioorg.2018.03.030

Accepted Manuscript
Design, synthesis and evaluation of some pyrazolo[3,4-d]pyrimidines as anti-
inflammatory agents
Gina N. Tageldin, Salwa M. Fahmy, Hayam M. Ashour, Mounir A. Khalil,
Rasha A. Nassra, Ibrahim M. Labouta
PII:
S0045-2068(17)30874-X
https://doi.org/10.1016/j.bioorg.2018.03.030
YBIOO 2320
DOI:
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 November 2017
25 March 2018
31 March 2018
Please cite this article as: G.N. Tageldin, S.M. Fahmy, H.M. Ashour, M.A. Khalil, R.A. Nassra, I.M. Labouta,
Design, synthesis and evaluation of some pyrazolo[3,4-d]pyrimidines as anti-inflammatory agents, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.03.030
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Design, synthesis and evaluation of some
pyrazolo[3,4-d]pyrimidines as anti-inflammatory
agents
a*
a
a
a
Gina N. Tageldin , Salwa M. Fahmy , Hayam M. Ashour , Mounir A. Khalil , Rasha A.
b
a
Nassra , Ibrahim M. Labouta
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria,
Alexandria 21521, Egypt
b
Department of Medical Biochemistry, Faculty of Medicine, University of Alexandria,
Alexandria, Egypt
*
Author for correspondence:
Tel.: +20 34 871 317
Fax: +20 34 873 273
ginatageldin@yahoo.com
Abstract:
New pyrazolo[3,4-d]pyrimidines substituted with various functionalities or attached to a
substituted pyrazole ring through different linkages were synthesized. The synthesized
compounds were evaluated for their anti-inflammatory activity using in vitro COX-1/COX-2
inhibition assay and in vivo formalin induced paw edema and cotton pellet-induced granuloma
assays. Results revealed that compounds 17b and 18 possessed COX-1/COX-2 selectivity indices
higher than diclofenac sodium and celecoxib. However, compounds 16a,b exhibited selectivity
indices higher than diclofenac sodium and nearly equivalent to celecoxib, whereas, 9b displayed
selectivity index comparable to diclofenac sodium. In vivo anti-inflammatory data showed that
compounds 9b, 16a, 18 displayed anti-inflammatory activity higher than both references in the
formalin induced paw edema model. On the other hand, the pyrazolyl derivatives 9b, 16b and
1
7b displayed anti-inflammatory activity about 2-2.5 fold that of diclofenac sodium and nearly
8
-10.5 fold that of celecoxib in the cotton pellet-induced granuloma assay. The ulcerogenic
effect of the active compounds was also investigated and results revealed that compounds 16a,
7a,b and 18 showed good gastrointestinal safety profile. Based on this, compounds 16a and 18
1
were considered as safe and effective leads in managing acute inflammation, while, 17b was
prominent in controlling chronic inflammation.
Keywords:
Pyrazolo[3,4-d]pyrimidines, Pyrazoles, Anti-inflammatory activity, Ulcerogenic effect, COX-
1
/COX-2 selectivity index
1

1. Introduction:
Non-steroidal anti-inflammatory drugs (NSAIDs) are usually prescribed for treatment of
acute and chronic pain, inflammation and fever. Their clinical efficacy is greatly related to their
suppression of prostaglandin biosynthesis from arachidonic acid by inhibiting COX enzyme
[
1-5]
which exists in three distinct isoforms: COX-1, COX-2, and COX-3.
The constitutive COX-1
isoform provides cytoprotection in the gastrointestinal (GI) tract and controls renal function in
the kidneys, whereas the inducible COX-2 is upregulated by proinflammatory mediators such as
endotoxins, mitogens, or cytokines including tumor necrosis factor a (TNF-a) and interleukins
[6, 7]
(
ILs) such as IL-6 and IL-1.
Furthermore, COX-3 which is a variant of COX-1 present in
central nervous system and has been proposed to be another target for anti-inflammatory
[8]
agents. GI injury is one of the major hallmarks of NSAIDs and is contraindicated in patients
[9]
with history of GI bleeding or distress. Moreover, use of additional gastroprotective therapy in
conjunction with NSAIDs increases the risk of polypharmacy and drug-induced organ injury in
[10]
elderly and infants. Accordingly, research was directed towards the development of selective
COX-2 inhibitors such as celecoxib that should retain the therapeutic potency of NSAIDs with
less gastrointestinal adverse effects. However, significant cardiovascular side effects associated
with these drugs led to reconsideration of their clinical use. Therefore, development of novel
compounds having anti-inflammatory activity with an improved gastrointestinal safety profile is
a necessity.
Pyrazolo[3,4-d]pyrimidines either linked to or fused with other heterocyclic ring systems
constitute an important scaffold in several pharmacologically active compounds including anti-
[
11-16]
[17-20]
4
inflammatory,
(
and analgesic agents.
Literature survey revealed that DPP; (N -benzyl-1-
6
6
tert-butyl)-N ,N -dimethyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine) (A, Figure 1) possess
anti-inflammatory and analgesic activities in addition to higher selectivity for COX-2 over COX-
[21]
1
.
Moreover, some pyrazolo[3,4-d]pyrimidines comprising benzamide or substituted
benzamide moieties at position 4 or 5 showed remarkable anti-inflammatory activity and COX-2
[22, 23]
selectivity (B, C, Figure 1).
On the other hand, pyrazoles represent a key scaffold in heterocyclic chemistry and
occupy a significant position in medicinal chemistry because of their wide range of bioactivities.
[
24-26]
[27, 28]
They were found to display anti-inflammatory
COX-2 inhibitory activity.
and analgesic activities
Moreover, the pyrazole unit is one of the core structures in a
number of selective COX-2 inhibitors such as celecoxib (D, Figure 1) and SC-558 (E, Figure
in addition to
[29, 30]
[31-33]
1
).
inflammatory and antimicrobial agents.
to pyrazoline derivatives as a potent class of anti-inflammatory, analgesic, and antipyretic agents
Additionally, much research has been focused N-aryl pyrazole derivatives as dual anti-
[34-36]
On the other hand, great attention has been devoted
[25, 37, 38]
since the development of antipyrine, the first pyrazoline derivative used in the
management of pain, inflammation, and fever.
2

Figure 1. Chemical structures of compounds A-E
Motivated by these findings we deemed it interesting to synthesize some new pyrazolo
3,4-d]pyrimidines to be investigated for their anti-inflammatory activity. The synthesized
[
compounds were designed so as to comprise the pyrazolo[3,4-d]pyrimidine core substituted with
various functionalities or attached to a substituted pyrazole ring at position 5 or 6 through
different linkages (Figure 2). Such substituents were selected so as to confer different electronic,
lipophilic and steric environment to the molecules which would influence the targeted biological
activity. The target compounds were evaluated for their in vitro COX-1/COX-2 inhibition assay
and their in vivo anti-inflammatory activity in two inflammatory models and ulcerogenic liability
in addition to histopathological examination to confirm the degree of inflammatory reaction in
the gastric layers of treated rats’ stomachs.
3

Figure 2. Design of new COX-2 inhibitors
2
2
. Results and Discussion
.1. Chemistry
The synthetic strategies adopted for the synthesis of the intermediate and final compounds
[
20]
are depicted in Schemes 1 and 2. In Scheme 1, reaction of the oxazine 1
with hydrazine
[39]
hydrate following previously published reaction conditions gave rise to the 5-amino
pyrazolopyrimidine 2 which upon condensation with different pyrazole aldehydes
[40-42]
furnished the corresponding azomethines 3a-c. Moreover, fusion of the oxazine 1 with 4-
o
aminoantipyrine or 4-aminoacetanilide at 180 C resulted in formation of the antipyrinyl
pyrazolopyrimidine 4 and the acetamide derivative 5 respectively. On the other hand, reflux of 1
with 4-aminoacetophenone afforded the target 5-acetylphenylpyrazolopyrimidine derivative 6.
Finally, reaction of the latter compound with hydroxylamine hydrochloride in ethanol containing
sodium acetate furnished the oxime derivative 7.
[43, 44]
In Scheme 2, cyclocondensation of the hydrazine derivative 8
with acetyl acetone
or benzoyl acetone in ethanol afforded the corresponding pyrazolyl derivatives 9a, b. However,
heating the hydrazine 8 with ethyl acetoacetate in ethanol in an attempt to synthesize the
[
45]
pyrazolinyl derivative 10 gave the open chain ester 11 whereas, fusion with ethyl acetoacetate
gave unexpectedly the corresponding pyranopyrazole 12. The possible mechanism is clearly
[
46]
illustrated in Figure 3. On the other hand, reaction of 8 with antipyrylazoacetoacetate,
[46]
[47]
antipyryl azoacetylaetone
and antipyrylazobenzoylacetone
in boiling ethanol yielded the
corresponding antipyrylazopyrazolyl derivatives 13a-c. Moreover, refluxing the hydrazine 8 with
4

[48]
ethyl acetoacetate coupled with azobenzoic acid in ethanol/ acetic acid mixture gave the open
chain ester 14 whereas, upon refluxing 8 with acetyl acetone or benzoyl acetone coupled with
[48]
azobenzoic acid under the same reaction conditions, the corresponding pyrazolyl derivatives
5b, c were obtained. However, cyclization of the open chain ester 14 to the corresponding
1
pyrazole derivative 15a was readily achieved by refluxing in glacial acetic acid. On the other
hand, Cyclocondensation of hydrazine derivative 8 with phenacyl cyanides in ethanol/glacial
acetic acid mixture afforded the corresponding aminopyrazoles 16a-e. Similarly, reaction of
hydrazine derivative 8 with (ethoxymethylidene) derivatives in ethanol/glacial acetic acid
mixture gave rise to the corresponding pyrazoles 17a,b. This procedure was successful when
ethyl 2-cyano-3-ethoxypropenoate (va) and (ethoxymethylidene)propanedinitrile (vb) were used.
However, applying the same method with diethyl (ethoxymethylidene)propanedioate (vc)
resulted in the formation of open chain counterpart 18 rather than pyrazole derivative 17c.
However, fusion of the hydrazine derivative 8 with diethyl 2-(ethoxymethylidene)propanedioate
(
vc) at 180 °C succeeded in giving the requisite pyrazole 17c.
Scheme 1
5

Scheme 2
6

Figure 3. The possible mechanism for pyranopyrazolyl formation
2
.2. Biological evaluation
2
.2.1. In vitro cyclooxygenase (COX) inhibition assay
All the synthesized compounds were tested for their in vitro inhibition of COX-1 and
COX-2 isoenzymes using Cayman colorimetric COX (ovine) inhibitor screening assay kit. The
Colorimetric COX Inhibitor Screening Assay utilizes the peroxidase component of
7

cyclooxygenase. The peroxidase activity was assayed colorimetrically by monitoring the
appearance of oxidized N,N,N ,N -tetramethyl-1,4-phenylenediamine (TMPD) which is produced
’
’
during the reduction of PGG2 to PGH2, at 590 nm. The concentration produced 50 % inhibition
of COX-1 and COX-2 isoenzymes (IC50 values) and the selectivity indices (SI = IC50 COX-1/
IC50 COX-2) of the test compounds were determined and results are recorded in Table 1.
In general, all the tested compounds showed relatively higher selectivity towards COX-2 than
COX-1. Compounds (3a, 4, 6, 9a, 9b, 12, 13a, 13c, 15a, 15c, 16a, 16b, 16d, 16e, 17a, 17b and 18)
showed COX-1 IC50 values (IC50 = 2.74-6.59 μM) lower than celecoxib and diclofenac sodium
(
IC50 = 5.64 and 6.74 μM, respectively). On the other hand, compounds 16a, 16b, 17b and 18
exhibited high COX-2 inhibitory activities (IC50 = 0.22-0.69 μM) which were lower than
celecoxib (IC50 = 0.78 μM). Further investigation revealed that compound 17b possesses
selectivity index (SI = 12.45) higher than both references diclofenac sodium (SI = 6.12) and
celecoxib (SI = 7.23). However, compounds 16a,b exhibited selectivity indices (SI= 7.20 and
6
.86 μM respectively) higher than diclofenac sodium and nearly equivalent to celecoxib,
whereas, 9b and 17a displayed selectivity indices (SI= 5.44 and 5.78 μM respectively)
comparable to diclofenac sodium. Interestingly, the open chain derivative 18, exhibited
selectivity index (SI= 8.52) higher than both references.
Table 1: In vitro COX-1 and COX-2 enzymes inhibitory activities, IC50 values and
selectivity indices (SI) of the tested compounds:
a
b
Comp. No.
IC50 (µM)
COX-1
IC50 (µM)
COX-2
SI
(COX-1/COX-2)
3
a
5.91
9.11
7.89
3.64
8.24
4.21
6.74
6.21
4.74
4.56
5.97
7.85
6.59
3.98
7.64
6.52
3.89
4.74
8.74
6.52
5.24
1.36
2.67
1.81
0.85
2.34
0.97
1.52
1.33
0.87
1.17
1.34
1.97
1.54
0.89
2.11
1.74
0.54
0.69
2.54
1.89
1.23
4.34
3.41
4.35
4.20
3.50
4.30
4.43
4.67
5.44
3.89
4.45
3.98
4.28
4.47
3.62
3.70
7.20
6.86
3.44
3.45
4.26
3
b
3
4
5
6
7
c
9
9
1
a
b
2
1
1
3a
3b
1
1
3c
5a
1
5b
1
1
5c
6a
1
1
1
1
6b
6c
6d
6e
8

1
1
1
7a
7b
8
5.61
2.74
5.88
5.46
6.74
0.97
0.22
0.69
0.78
1.10
5.78
12.45
8.52
7.23
6.12
Celecoxib
Diclofenac Sodium
a
Values are means of three determinations acquired using an ovine COX-1/COX-2 assay kit (catalog no.
760111, Cayman Chemicals, MI, USA) and the deviation from the mean is ±10 % of the mean value
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Based on the in vitro results, compounds showing selectivity indices higher or nearly
equivalent to celecoxib were selected for further evaluation of their in vivo anti-inflammatory
and analgesic activities.
2
.2.2. In vivo anti-inflammatory activity
2
.2.2.1 Formalin-induced paw edema bioassay
In this acute inflammatory model, each test compound (9b, 16a, 16b, 17a, 17b and 18) was
dosed orally at a dose of (5 mg/kg body weight) for seven days prior to induction of
[49, 50]
inflammation by formalin injection.
Celecoxib and diclofenac sodium were utilized as
reference drugs at a dose of (5 mg/kg, po). The anti-inflammatory activity was then calculated 4
3
h after induction of inflammation and presented in Table 2 as the mean paw volume (cm ) ± SD
and the percentage anti-inflammatory activity (AI %).
Table 2: In vivo anti-inflammatory activities of selected compounds in formalin-induced
rat paw edema bioassay (acute inflammation model)
3
Volume of edema (cm )
%
Inhibition
% AI)
Mean ± SD
(
Comp. No.
0
4 h
a
9
b
0.5±0.1
5.0±0.0
0.5±0.1
0.5±0.0
0.4±0.1
0.5±0.0
0.5±0.0
0.5±0.0
0.6 ±0.0
57.1
a
b,c
16a
16b
17a
17b
0.5 ±0.0
92.9
42.8
42.9
a
0.6 ±0.0
a
0.6 ±0.1
b
0.6±0.1
23.8
57.1
46.4
50.0
-
a
1
8
0.6 ±0.0
Celecoxib
Diclofenac sodium
Control
0.6±0.1
a
0.6 ±0.0
b
0.5 ±0.0
0.7±0.1
a
b
c
statistically significant difference in comparison with control group
statistically significant difference in comparison with diclofenac sodium treated group
statistically significant difference in comparison with celecoxib treated group
9

A comparative study of the anti-inflammatory activity of the test compounds relative to
the reference drug indicated that three compounds (9b, 16a and 18) showed outstanding anti-
inflammatory activity (% AI = 57.1- 92.2) that was superior to both diclofenac sodium and
celecoxib (% AI = 50, 46.4 respectively) (Figure 4). Interestingly, compound 16a displayed the
highest potency as it showed anti-inflammatory activity (% AI = 92.9) twice that of celecoxib
and diclofenac sodium (% AI = 46.4 and 50 % respectively). Meanwhile, the pyrazolyl
derivatives 16b and 17a displayed anti-inflammatory activity (% AI = 42.8, and 42.9 respectively)
equivalent to celecoxib and slightly lower than diclofenac sodium.
Figure 4: Anti-inflammatory activity of selected compounds in the formalin-induced
rat paw edema test (% inhibition)
2
.2.2.2 Cotton pellet-induced granuloma assay
In order to investigate the test compounds’ efficacy against the later proliferative phase of
inflammation caused by tissue degeneration and fibrosis, the cotton pellet induced granuloma
[51]
assay was used.
cotton in (mg) ± SD in animals pretreated with the reference drugs and test compounds (9b, 16a,
6b, 17a, 17b and 18) after 7 days from the insertion of the cotton pellet and induction of
The results listed in Table 3 represent the mean changes in weight of dry
1
inflammation, together with the percent inhibition of granuloma by the test compounds (% anti-
inflammatory activity).
The screening results revealed that the pyrazolyl derivatives 9b, 16b and 17b displayed
anti-inflammatory activity (% inhibition of granuloma = 71.2, 90.0 and 85.3 respectively) about
2
-2.5 fold that of diclofenac sodium and nearly 8-10.5 fold that of celecoxib. On the other hand,
compound 16a displayed anti-inflammatory activity (% inhibition of granuloma = 30.8)
10

comparable to diclofenac sodium. The other compounds showed weak to moderate activity
compared to the reference diclofenac sodium. (Figure 5)
Table 3: In vivo anti-inflammatory activities of selected compounds in Cotton pellet
induced granuloma test (Chronic inflammation model)
Dry weight of granuloma (mg)
% Granuloma inhibition
Comp. No.
Mean ± SD
ac
9
b
13.4 ±10.6
71.2
30.8
90.0
8.1
ac
16a
16b
17a
17b
32.2 ±5.4
abc
4.63 ±2.43
50.3±79.9
abc
6.86 ±3.46
85.3
18.1
8.6
c
1
8
38.1 ±10.5
Celecoxib
Diclofenac sodium
Control
50.5±11.7
a
29.7 ±13.3
36.1
-
46.5±6.2
a
b
c
statistically significant difference in comparison with control group
statistically significant difference in comparison with diclofenac sodium treated group
statistically significant difference in comparison with celecoxib treated group
Figure 5: Anti-inflammatory activity of selected compounds in the Cotton pellet-induced
granuloma test (% inhibition)
11

2
.2.3. Structure activity relationship (SAR)
A deep insight in the structures of the tested compounds revealed that the 5-amino-3-
phenylpyrazolyl derivative 16a displayed nearly two fold the anti-inflammatory activity of
diclofenac sodium in the acute model although, it was slightly less active than the reference drug
in the chronic model. On the other hand, compound 16b which possesses the lipophilic 4-
bromophenyl group at position 3 of the pyrazole moiety showed a marked decrease in the anti-
inflammatory activity in the acute model, however, the activity in the chronic model was
markedly improved to exceed that of diclofenac sodium. In this respect, compound 16b exhibited
the highest activity among the tested compounds in the chronic model (% granuloma inhibition =
9
0.0) compared to diclofenac sodium (% granuloma inhibition = 36.1). Modification of structure
(
16) by replacing the lipophilic group (phenyl and p-bromophenyl at position 3) with another
lipophilic group as cyano while retaining the amino group as in compound 17b maintained the
anti-inflammatory activity in the chronic model but the activity in the acute model was
abolished. On the contrary, introduction of ester group (compound 17a) at the same position
reduced the anti-inflammatory activity in both models. In compound 16b, replacement of the
aromatic moiety at position 3 by methyl group and the 5-amino group by phenyl (compound 9b)
decreased the anti-inflammatory activity in the acute model, however, the activity in the chronic
model was highly increased to be twice that of diclofenac sodium.
2
.2.4. Gastric ulcerogenic activity
The tested compounds that exhibited in vitro COX-2 selectivity indices higher or nearly
[52, 53]
equivalent to reference drugs were further evaluated for their ulcerogenic potential in rats.
Gross observation of the isolated rats’ stomachs showed a normal stomach texture for the
tested compounds 16a, 17a, 17b, 18 as well as the reference celecoxib. While for compounds 9b
and 16b variable degrees of hyperemia were observed (Figure 6).
Figure 6a
Figure 6b
Figure 6: Gross appearance of gastric mucosa
6
a- Normal stomach texture for the tested compounds (16a, 17a, 17b, 18) and celecoxib
b- Hyperemia observed in case of compounds (9b, 16b) and diclofenac sodium
6
12

Further histopathological examination was performed to confirm the degree of
inflammatory reaction in the gastric layers of the treated rats’ stomachs. Histopathological
examination revealed that compounds 16a, 17a, 17b, 18 showed superior gastrointestinal safety
profile (free, normal gastric and esophageal mucosa) as well as celecoxib and diclofenac sodium
(
1
Figure 7a). On the other hand, histopathological examination indicated that the compound 9b,
6b revealed signs of gastro-esophageal inflammation (Figure 7b).
Figure 7: Histology of rat gastric mucosa (H&E stain)
Figure 7a: Free, normal gastro-esophageal junction, (H&Ex100)
Figure 7b: Gastro-esophageal inflammation, (H&Ex100)
3
. Conclusion
The aim of the present study was to synthesize and investigate the anti-inflammatory
activity of new pyrazolo[3,4-d]pyrimidines substituted with various functionalities or attached to
a substituted pyrazole ring through different linkages with the hope of discovering new lead
structures devoid of the GI side effects associated with conventional NSAIDs. The obtained
results from COX-1/COX-2 inhibition assay clearly revealed that compounds 17b and 18
possessed selectivity indices higher than diclofenac sodium and celecoxib. While, compounds
1
6a,b exhibited selectivity indices higher than diclofenac sodium and nearly equivalent to
celecoxib, whereas, 9b displayed selectivity index comparable to diclofenac sodium. In vivo anti-
inflammatory data showed that compounds 9b, 16a, 18 displayed anti-inflammatory activity
higher than both references in the formalin induced paw edema model, whereas, the pyrazolyl
13

derivatives 9b, 16b and 17b displayed anti-inflammatory activity about 2-2.5 fold that of
diclofenac sodium and nearly 8-10.5 fold that of celecoxib in the cotton pellet-induced
granuloma assay. Histopathological examination revealed that compounds 16a, 17a,b and 18
showed good gastrointestinal safety profile. Collectively, the promising anti-inflammatory
activity of 16a and 18 in acute model and 17b in chronic model as well as their reduced
ulcerogenic potential make them good lead candidates for further optimization and development
of potent and safe anti-inflammatory agents.
4
. Experimental
4
.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and were dried and
purified when necessary by standard techniques.
Melting points were determined in open glass capillaries using Griffin melting point
apparatus and are uncorrected. Infrared (IR) spectra were recorded on Perkine-Elmer RXIFT-IR
1
spectrophotometer using KBr discs. H-NMR spectra were scanned on were run on Jeol
spectrometer (500 MHz) at the Microanalytical Unit, Faculty of Science, Alexandria University,
on Bruker high performance digital FT-NMR spectrometer avance III (400 MHz) at Faculty of
Pharmacy, Cairo University and on Varian Mercury VX (300 MHz) spectrometer, Faculty of
1
3
Science, Cairo University. C-NMR proton decoupled spectra were run on Jeol spectrometer
125 MHz) at the Microanalytical Unit. Faculty of Science, Alexandria University and on Varian
Mercury VX (75 MHz) spectrometer, Faculty of Science, Cairo University, using deuterated
dimethylsulfoxide (DMSO-d ) as a solvent. The data were reported as chemical shifts or δ values
ppm) relative to tetramethylsilane (TMS) as internal standard. Mass spectra were run on a gas
(
6
(
chromatograph/mass spectrophotometer Shimadzu GCMS/QP-2010 plus (70 eV) at the faculty
of Science, Al-Azhar University. Relative intensity % corresponding to the most characteristic
fragments were recorded. Microanalyses were performed at the Microanalytical Unit, Faculty of
Science, Cairo University, Egypt and the found values were within ±0.4% of the theoretical
values. Follow up of the reactions and checking the purity of the compounds was made by thin
layer chromatography (TLC) on silica gel-precoated aluminum sheets (Type 60 GF254; Merck;
Germany) and the spots were detected by exposure to iodine vapour or UV lamp at λ 254 nm for
few seconds.
[20]
4
.1.1. 6-Methyl-1-phenylpyrazolo[3,4-d][1,3]oxazin-4(1H)-one (1) ; M.p. 94-95 °C as
[54]
reported
[39]
4
2
.1.2. 5-Amino-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (2) ; M.p. 220-
21 C, reported 215-217 C.
o
o
[39]
4
6
.1.3. General procedure for preparation of 5-{(Substituted pyrazol-4-yl)methylideneamino}-
-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (3a-c)
A mixture of the amino derivative 2 (0.24 g, 1 mmol) and the selected pyrazole aldehyde
(
1 mmol) in absolute ethanol (7 ml) was heated under reflux for 3 h. The separated product was
filtered, washed with ethanol, dried and recrystallized from dioxane. Physical and spectral data
for 3a-c are listed below.
14

4
.1.3.1.
pyrazolo[3,4-d]pyrimidin-4(5H)-one (3a) Yield: 80 %; Mp: 226-227 C. IR (KBr, cm ): 3113,
046, 2926 (CH); 1706 (C=O); 1598 (C=N); 1563, 1539, 1504, 1455 (C=C). Anal. Calcd for
O (471.51): C, 71.32; H, 4.49; N, 20.79. Found: C, 71.53; H, 4.57; N, 21.06.
5-{(1,3-Diphenyl-1H-pyrazol-4-yl)methylideneamino}-6-methyl-1-phenyl-1H-
o
-1
3
C
28
H
21
N
7
4
.1.3.2. 5-{(5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylideneamino}-6-methyl-1-
o
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (3b) Yield: 87 %; Mp: 232-234 C. IR (KBr,
cm ): 3063, 3042, 2963, 2921 (CH); 1714 (C=O); 1606 (C=N); 1565, 1531, 1505, 1462 (C=C).
-1
+
•
+•
MS (m/z, %): 445.45 (M +2, 8); 443.72 (M , 24); 442 (13); 419 (9); 412 (15); 411 (11); 400
11); 338 (22); 333 (16); 318 (10); 302 (26); 266 (16); 211 (21); 197 (11); 195 (13); 192 (54);
(
1
5
58 (21); 150 (20); 141 (100); 137 (14); 128 (32); 108 (19); 106 (68); 93 (18); 79 (81); 73 (41);
7 (16); 47 (11); 44 (50). Anal. Calcd for C23 O (443.89): C, 62.23; H, 4.09; N, 22.09.
H18ClN
7
Found: C, 62.41; H, 4.15; N, 22.34.
4
1
3
.1.3.3. 5-{(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylideneamino}-6-methyl-1-phenyl-
o
-1
H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (3c) Yield: 86 %; Mp: 216-217 C. IR (KBr, cm ):
1
105, 3037, 2964, 2920 (CH); 1709 (C=O); 1607 (C=N); 1563, 1535, 1501, 1460 (C=C). H-
NMR (400 MHz, DMSO-d
H); 8.11 (d, J = 7.8 Hz, 2H, phenyl C2,6-H); 8.37 (s, 1H, pyrazolopyrimidine C
N=CH). Anal. Calcd for C24 O (423.47): C, 68.07; H, 5.00; N, 23.15. Found: C, 68.25; H,
6
) δ 2.47, 2.50, 2.56 (3s, each 3H, 3 CH
3
); 7.42-7.61 (m, 8H, phenyl-
3
-H); 8.77 (s, 1H,
H
21
N
7
4
.98; N, 23.34.
4
.1.4. 5-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-6-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (4)
A mixture of the oxazine derivative 1 (0.23 g, 1 mmol), 4-aminoantipyrine (0.20 g, 1
mmol) and a catalytic amount of p-toluene sulphonic acid was heated in an oil bath at 180 °C for
2
h. The reaction mixture was left to cool, then triturated with ethanol, filtered, washed with
o
-1
diethyl ether, dried and crystallized from ethanol. Yield: 66 %; Mp: 232-234 C. IR (KBr, cm ):
1
3
023, 2985, 2923 (CH); 1708, 1678 (C=O); 1597 (C=N); 1568, 1544, 1512, 1494 (C=C). H-
NMR (300 MHz, DMSO-d ) δ 2.20, 2.46 (2s, each 3H, 2 C-CH ); 3.28 (s, 1H, N-CH ); 7.38-
.61 (m, 8H, phenyl-H); 8.08 (d, J = 7.5 Hz, 2H, phenyl C2,6-H); 8.36 (s, 1H, pyrazolopyrimidine
6
3
3
7
+
•
+•
C
3
-H). MS (m/z, %): 413.20 (M +1, 32); 412.19 (M , 100); 397 (21); 291 (34); 210 (24); 143
(412.44): C, 66.98; H, 4.89; N, 20.38. Found:
(
58); 77 (80); 56 (99). Anal. Calcd for C23
H
20
N
6
O
2
C, 67.23; H, 4.94; N, 20.52.
4
.1.5. N-{4-(6-methyl-4-oxo-1-phenyl-1H,5H-pyrazolo[3,4-d]pyrimidin-5-
yl)phenyl}acetamide (5)
A mixture of oxazine derivative 1 (0.23 g, 1 mmol), 4-aminoacetanilide (0.15 g, 1 mmol)
and a catalytic amount of p-toluene sulphonic acid was heated in an oil bath at 180 °C for 2 h.
The reaction mixture was left to cool, triturated with ethanol. The separated solid was filtered,
o
washed with diethyl ether, dried and crystallized from ethanol. Yield: 67 %; Mp. 265-266 C. IR
-1
1
(
KBr, cm ): 3419, 3293 (NH); 3064 (CH); 1693 (C=O); 1601 (C=N); 1548, 1504 (C=C). H-
NMR (300 MHz, DMSO-d ) δ 2.09, 2.19 (2s, each 3H, 2 CH ); 7.31-7.76 (m, 7H, phenyl-H);
6
3
15

8
.09 (d, J = 7.5 Hz, 2H, phenyl C2,6-H); 8.34 (s, 1H, pyrazolopyrimidine C
3
-H); 10.15 (s, 1H,
1
3
NH, D
2
O exchangeable). C-NMR (125 MHz, DMSO-d
6
) δ 24.57 (CH
3
); 25.23 (CH ); 105.97
3
(
pyrazolopyrimidine C3a); 120.25, 122.22, 132.54 (phenyl C2-6); 127.66, 129.28, 129.85 (phenyl
C
2-6); 136.87 (phenyl C
1
); 138.78 (phenyl C
1
); 140.32 (pyrazolopyrimidine C
3
); 151.20
);
(359.38): C, 66.84; H, 4.77; N, 19.49. Found: C,
(
pyrazolopyrimidine C7a); 158.20 (pyrazolopyrimidine C6,); 159.90 (pyrazolopyrimidine C
4
1
6
69.22 (NHC=O). Anal. Calcd for C20
7.01; H, 4.81; N, 19.62.
H
17
N
5
O
2
4
.1.6. 5-(4-Acetylphenyl)-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (6)
A mixture of oxazine derivative 1 (2.27 g, 10 mmol) and 4-aminoacetophenone (1.35 gm,
1
0 mmol) in dry pyridine (30 ml) was heated under reflux for 6 h. The reaction mixture was
poured into ice-cold water and the reaction mixture was neutralized with diluted hydrochloric
acid. The separated product was filtered, washed with water, dried and crystallized from
o
-1
methanol. Yield: 55 %; Mp. 216-217 C. IR (KBr, cm ): 3050, 3014 (CH); 1706, 1688 (C=O);
1
1
581 (C=N); 1562, 1502 (C=C). H-NMR (300 MHz, DMSO-d
CH ); 7.43-7.64 (m, 5H, phenyl-H) 7.99 (d, J = 8.4 Hz, 2H, phenyl C2,6-H); 8.11 (d, J = 8.4 Hz,
H, phenyl C3,5-H); 8.37 (s, 1H, pyrazolopyrimidine C -H). Anal. Calcd for C20
344.37): C, 69.76; H, 4.68; N, 16.27. Found: C, 69.94; H, 4.73; N, 16.43.
6
) δ 2.19, 2.66 (2s, each 3H, 2
3
2
(
3
H
16
N
4
O
2
4
4
.1.7. 5-{4-(1-Hydroxyiminoethyl)phenyl}-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
(5H)-one (7)
A mixture of the acetophenone derivative 6 (0.34 g, 1 mmol), hydroxylamine hydrochloride
0.07 g, 1 mmol) and sodium acetate (0.08 g, 1 mmol) in ethanol (10 ml) was heated under reflux
for 4 h. The separated beage shiny crystals were filtered while hot, washed with ethanol, dried
(
o
-1
then recrystallized from dioxane. Yield: 78 %; Mp. 248-249 C. IR (KBr, cm ): 3281 (OH);
1
3
076, 2958, 2907(CH); 1685 (C=O); 1598 (C=N); 1561, 1543, 1501 (C=C). H-NMR (300 MHz,
DMSO-d ) δ 2.19, 2.22 (2s, each 3H, 2 CH ), 7.39-7.62 (m, 5H, phenyl-H); 7.83 (d, J = 8.7 Hz,
H, phenyl C2,6-H); 8.10 (d, J = 8.7 Hz, 2H, phenyl C3,5-H); 8.36 (s, 1H, pyrazolopyrimidine C
H); 11.37 (s, 1H, OH, D O exchangeable). Anal. Calcd for C20 (359.38): C, 66.84; H,
.77; N, 19.49. Found: C, 67.01; H, 4.81; N, 19.62.
6
3
2
3
-
2
H
17
N
5
O
2
4
[44]
4
2
.1.8. 6-Hydrazinyl-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (8)
70 C as reported.
Mp. 268-
o
[44]
4
.1.9. General procedure for preparation of 6-(3-Methyl-5-substituted-1H-pyrazol-1-yl)-1,5-
diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (9a,b)
A mixture of the hydrazine derivative 8 (0.32 g, 1mmol) and acetylacetone (ia) or
benzoylacetone (ib) (1 mmol) in absolute ethanol (10 ml) was heated under reflux for 3-6 h. The
reaction mixture was concentrated under reduced pressure, left overnight at room temperature
and the separated crystals were filtered, washed with diethyl ether, dried and recrystallized from
ethanol. Yield, physical properties and elemental microanalyses of compounds (9a,b) are listed
below.
4
.1.9.1. 6-(3,5-Dimethyl-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
o
-1
one (9a) Yield: 85 %; Mp: 184-186 C. IR (KBr, cm ): 3082, 3036, 2968, 2925 (CH); 1710
1
(
C=O); 1575(C=N); 1537, 1496, 1460 (C=C). H-NMR (500 MHz, DMSO-d ) δ 1.78, 2.37 (2s,
6
16

each 3H, 2 CH
H, phenyl C2,6-H); 8.50 (s, 1H, pyrazolopyrimidine C
C, 69.10; H, 4.74; N, 21.98. Found: C, 69.24; H, 4.81; N, 22.21.
3
); 5.83 (s, 1H, pyrazole C
4
-H); 7.22-7.59 (m, 8H, phenyl- H); 7.96 (d, J = 7.7 Hz,
2
3
-H). Anal. Calcd for C22 O (382.42):
H
18
N
6
4
4
.1.9.2. 6-(3-Methyl-5-phenyl-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-
o
-1
(5H)-one (9b) Yield: 84 %; Mp: 196-198 C. IR (KBr, cm ): 3033 (CH); 1715 (C=O); 1647
1
(
C=N); 1577, 1540, 1495 (C=C). H-NMR (300 MHz, DMSO-d
6
) δ 2.02 (s, 3H, CH
-H); 7.15-7.50 (m, 15H, phenyl-H); 8.45 (s, 1H, pyrazolopyrimidine C
) δ 13.00 (CH ); 105.94 (pyrazole C ); 107.95 (pyrazolopyrimidine
3a); 121.48, 128.53, 129.18 (phenyl C2-6); 127.31, 128.76, 129.10 (phenyl C2-6); 128.17, 128.63,
28.86 (phenyl C2-6); 135.15 (phenyl C ); 136.88; (phenyl C ); 137.34 (phenyl C ); 146.41
pyrazolopyrimidine C ); 147.43 (pyrazolopyrimidine C7a); 148.71 (pyrazole C ); 150.84
pyrazole C ); 156.98 (pyrazolopyrimidine C ); 162.00 (pyrazolopyrimidine C ). MS (m/z, %):
45 (M +1, 13); 444 (M , 41); 443 (15); 288 (19); 287 (100); 77 (24). Anal. Calcd for
O (444.49): C, 72.96; H, 4.54; N, 18.91. Found: C, 73.17; H, 4.61; N, 19.05.
3
); 6.37 (s,
1
3
1
H, pyrazole C
4
3
-H). C-
NMR (75 MHz, DMSO-d
6
3
4
C
1
(
(
4
C
1
1
1
3
3
5
6
4
+
•
+•
27
H
20
N
6
4
.1.10
Ethyl 3-{2-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)
hydrazinylidene}butanoate (11)
A mixture of the hydrazine derivative 8 (0.32 g, 1 mmol) and ethyl acetoacetate (ic) (0.13
g, 0.127 ml, 1 mmol) in absolute ethanol/glacial acetic acid (10 ml) (2:1) was heated under reflux
for 6 h. The reaction mixture was left to cool to room temperature and poured into ice-cold
water, the separated solid was filtered, washed with water, dried and crystallized from ethanol.
o
-1
Yield: 59 %; Mp. 152-153 C. IR (KBr, cm ): 3382 (NH); 3104, 3063, 2978 (CH); 1737, 1709
1
(
C=O); 1596 (C=N); 1567, 1547, 1494 (C=C); 1257, 1027 (C-O-C). H-NMR (400 MHz,
DMSO-d ) δ 1.19, 1.26 (2t, J = 7.0 Hz, each 3H, 2 CH -CH3, E and Z isomers); 2.11, 2.19 (2s,
each 3H, 2 CH , E and Z isomers); 2.44, 2.61 (2s, each 2H, 2 CH , E and Z isomers); 4.19, 4.45
2q, J = 7.0 Hz, each 2H, 2 CH -CH , E and Z isomers); 7.16-8.36 (m, 22H, phenyl-H and NH, E
and Z isomers); 8.46, 8.51 (2s, each 1H, pyrazolopyrimidine C -H, E and Z isomers). Anal.
Calcd for C23 (430.46): C, 64.17; H, 5.15; N, 19.52. Found: C, 64.34; H, 5.21; N, 19.75.
6
2
3
2
(
2
3
3
H
22
N
6
O
3
4
.1.11. 6-(3,4-Dimethyl-6-oxo-1H-pyrano[2,3-c]pyrazol-1-yl)1,5-diphenyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (12)
A mixture of the hydrazine derivative 8 (0.32 g, 1 mmol) and ethyl acetoacetate (ic) (1 ml)
was heated in an oil bath at 180 °C for 15 minutes. The reaction mixture was left to attain room
temperature, triturated with ethanol, then the solid product was filtered, washed with ethanol,
-1
dried and crystallized from dioxane. Yield: 88 %; Mp. > 300 °C. IR (KBr, cm ): 3060, 2968,
1
2
924 (CH); 1741, 1710 (C=O); 1616 (C=N); 1597, 1541, 1485 (C=C); 1230, 1048 (C-O-C). H-
NMR (400 MHz, DMSO-d ) δ 2.19, 2.34 (2s, each 3H, 2 CH ); 5.98 (s, 1H, pyranopyrazole C
H), 7.34-7.60 (m, 8H, phenyl-H); 8.08 (d, J = 8.0 Hz, 2H, phenyl C2,6-H); 8.56 (s, 1H,
6
3
5
-
1
3
pyrazolopyrimidine C
3
-H). C-NMR (125 MHz, DMSO-d
6
) δ 14.51 (CH
3
); 19.53 (CH
); 128.17,
29.11, 129.45 (phenyl C2-6); 128.99, 129.18, 129.98 (phenyl C2-6); 135.57 (pyranopyrazole C );
37.42 (pyrazolopyrimidine C ); 137.61 (phenyl C ); 138.21 (phenyl C ); 144.77
); 146.71 (pyrazolopyrimidine C7a); 149.42 (pyrazolopyrimidine C ); 154.54
pyrazolopyrimidine C ); 157.40 (pyranopyrazole C ); 158.87 (pyranopyrazole C7a). MS (m/z,
): 451 (M +1, 6); 450 (M , 19); 288 (19); 287 (100.00); 258 (5); 232 (4); 77 (24); 69 (4).
3
); 106.10
(
pyrazolopyrimidine C3a); 122.31 (pyranopyrazole C3a); 122.62 (pyranopyrazole C
5
1
1
3
3
1
1
(
pyranopyrazole C
4
6
(
%
4
6
+
•
+•
17

Anal. Calcd for C25
H
18
N
6
3
O (450.45): C, 66.66; H, 4.03; N, 18.66. Found: C, 66.78; H, 4.11; N,
1
8.78.
4
1
.1.12. General procedure for preparation of 6-{4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-
H-pyrazol-4-yldiazenyl)-5-substituted-3-methyl-1H-pyrazol-1-yl}-1,5-diphenyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-ones (13a-c)
An equimolar amount of the hydrazine derivative 8 and the selected antipyrylazo
derivative (iia-c) in ethanol/glacial acetic acid mixture (15 ml) (2:1) was heated under reflux for
1
8 h. The separated solid product was filtered while hot, washed with ethanol, dried and
recrystallized from dimethylformamide. Yield, physical properties and elemental microanalyses
of compounds (13a-c) are listed below.
4
.1.12.1.
6-{4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yldiazenyl)-5-
hydroxy-3-methyl-1H-pyrazol-1-yl}-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
o
-1
(
(
13a) Yield: 89 %; Mp: 228-229 C. IR (KBr, cm ): 3217 (OH); 3059, 2978, 2929 (CH); 1688
1
C=O); 1594 (C=N); 1539, 1501, 1467 (C=C). H-NMR (500 MHz, DMSO-d
); 3.26 (s, 3H, N-CH ); 7.17-8.24 (m, 15H, phenyl-H); 8.54 (s, 1H,
pyrazolopyrimidine C -H); 13.36 (s, 1H, OH, D
61 (7); 319 (6) 229 (5); 207 (5); 202 (12); 193 (4); 131 (14); 117 (12); 116 (26); 105 (50); 104
15); 103 (16); 102 (12); 91 (91); 90 (10); 89 (11); 78 (13); 77 (65); 76 (12); 65 (20); 55 (16); 43
100). Anal. Calcd for C32 (598.61): C, 64.21; H, 4.38; N, 23.40. Found: C, 64.37; H,
6
) δ 2.44, 2.65 (2s,
each 3H, 2 CH
3
3
+
•
3
2
O exchangeable). MS (m/z, %): 598.80 (M , 1);
3
(
(
H
26
N
10
O
3
4
.43; N, 23.73.
4
.1.12.2.
6-{4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yldiazenyl)-3,5-
dimethyl-1H-pyrazol-1-yl}-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(13b)
o
-1
Yield: 87 %; Mp: 267-272 C. IR (KBr, cm ): 3054, 2978, 2919 (CH); 1716, 1674 (C=O); 1570
1
(
3
C=N); 1535, 1494, 1451 (C=C). H-NMR (300 MHz, DMSO-d
H, 3 CH ); 3.31 (s, 1H, N-CH ); 7.33-8.04 (m, 15H, phenyl-H); 8.55 (s, 1H,
pyrazolopyrimidine C -H). Anal. Calcd for C33 (596.64): C, 66.43; H, 4.73; N, 23.48.
Found: C, 64.59; H, 4.82; N, 23.71.
6
) δ 2.04, 2.45, 2.66 (3s, each
3
3
3
H
28
N
10
O
2
4
.1.12.3. 6-{4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yldiazenyl)-3-
methyl-5-phenyl-1H-pyrazol-1-yl}-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
o
-1
one(13c) Yield: 84 %; Mp: 279-280 C. IR (KBr, cm ): 3062 (CH); 1698, 1680 (C=O); 1648
1
(
3
C=N); 1516, 1460 (C=C). H-NMR (300 MHz, DMSO-d
.27 (s, 1H, N-CH ); 7.20-8.04 (m, 20H, phenyl-H); 8.50 (s, 1H, pyrazolopyrimidine C
Anal. Calcd for C38 (658.71): C, 69.29; H, 4.59; N, 21.26. Found: C, 69.48; H, 4.63; N,
6
) δ 2.22, 2.45 (2s, each 3H, 2 CH
3
);
3
3
-H).
H
30
N
10
O
2
2
1.82.
4
.1.13.
4-{1-Ethoxy-1-oxo-3-(2-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)hydrazinylidene)butan-2-yl}diazenylbenzoic acid (14)
A mixture of the hydrazine derivative 8 (0.32 g, 1 mmol) and 4-(1-ethoxy-1,3-
dioxobutan-2-yl)diazenylbenzoic acid (iiia) (0.28 g, 1 mmol) in ethanol/glacial acetic acid
mixture (15 ml) (2:1) was heated under reflux for 18 h. The formed red precipitate was filtered
while hot, washed with ethanol, dried and recrystallized from dimethylformamide. Yield: 77 %;
o
-1
Mp. 228-229 C. IR (KBr, cm ): 3421-3100 (OH); 3281 (NH); 3042, 2978, 2927 (C=C); 1734,
18

1
1
704, 1653 (C=O); 1599 (C=N); 1530, 1451 (C=C); 1268, 1084 (C-O-C). H-NMR (300 MHz,
DMSO-d ) δ 1.18, 1.28 (2t, J = 7.0 Hz, each 3H, 2 CH CH , E and Z isomers); 1.78, 1.88 (2s,
each 3H, 2 CH , E and Z isomers); 4.24, 4.36 (2q, J = 7.0 Hz, each 2H, 2 CH CH , E and Z
isomers); 6.84-8.51 (m, 30H, phenyl-H and pyrazolopyrimidine C -H, E and Z isomers); 10.66
s, 1H, NH, D O exchangeable, one isomer); 12.55 (s, 2H, NH and OH, D O exchangeable, one
isomer); 13.75 (s, 1H, OH, D O exchangeable, one isomer). Anal. Calcd for C30
578.58): C, 62.28; H, 4.53; N, 19.37. Found: C, 62.49; H, 4.62; N, 19.59.
6
2
3
3
2
3
3
(
2
2
2
H
26
N
8
O
5
(
4
.1.14.
4-{5-Hydroxy-3-methyl-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)-1H-pyrazol-4-yl}diazenylbenzoic acid (15a)
The intermediate 14 (0.58 g, 1 mmol) was heated under reflux in glacial acetic acid (4
ml) for 3 h. The reaction mixture was left to attain room temperature and the obtained yellow
crystals were filtered, washed with ethanol, dried and recrystallized from dioxane. Yield: 70 %;
o
-1
Mp. > 300 C. IR (KBr, cm ): 3398-3103 (OH); 3058, 2929, 2819 (CH); 1723, 1683 (C=O);
1
1
7
601 (C=N); 1538, 1494, 1427 (C=C). H-NMR (500 MHz, DMSO-d
6
) δ 2.02 (s, 3H, CH );
3
.28-8.05 (m, 14H, phenyl-H); 8.52 (s, 1H, pyrazolopyrimidine C3-H); 12.89 (s, 2H, 2 OH, D
2
O
1
3
exchangeable). C-NMR (125 MHz, DMSO-d
6
) δ 10.20 (CH
); 122.60, 126.55, 129.56 (phenyl C2-6); 128.01, 128.09, 129.14 (phenyl
2-6); 128.21, 129.96, 131.36 (phenyl C2-6); 135.69 (phenyl C ); 137.46 (phenyl C ); 138.26
); 145.30 (pyrazole C ); 146.47 (pyrazolopyrimidine C ); 149.94 (pyrazolopyrimidine
7a); 150.03 (pyrazolopyrimidine C ); 156.46 (pyrazolopyrimidine C ); 157.59 (pyrazole C );
(532.51): C, 63.15; H, 3.79; N, 21.04.
3
); 106.82 (pyrazolopyrimidine
C
C
3a); 116.86 (pyrazole C
4
1
1
(
phenyl C
1
3
3
C
1
6
4
5
67.22 (carboxylic C=O). Anal. Calcd for C28
H
20
N
8
O
4
Found: C, 63.24; H, 3.84; N, 21.31.
4
4
.1.15. General procedure for preparation of 4-{3-Methyl-5-sustituted-1-(4-oxo-1,5-diphenyl-
,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H-pyrazol-4-yl}diazenylbenzoic
acids
(
15b,c)
An equimolar amount of the hydrazine derivative 8 and the selected 4-substituted azo
benzoic acid derivative (iiib,c) in ethanol/glacial acetic acid mixture (15 ml) (2:1) was heated
under reflux for 18 h. The reaction mixture was concentrated, left to cool then poured into ice-
cold water, the separated solid was filtered, washed with water, dried and crystallized from
dioxane/ethanol. Yield, physical properties and elemental microanalyses of compounds (15b,c)
are listed below.
4
.1.15.1. 4-{3,5-Dimethyl-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
-yl)-1H-pyrazol-4-yl}diazenylbenzoic acid (15b) Yield: 85 %; Mp: 237-238 C. IR (KBr, cm
o
-
6
1
)
: 3392-3100 (OH); 3056, 2983, 2930, 2829 (CH); 1709, 1681 (C=O); 1633 (C=N); 1603, 1575
1
(
C=C). H-NMR (300 MHz, DMSO-d
phenyl-H); 8.34 (s, 1H, pyrazolopyrimidine C
Calcd for C29 (530.54): C, 65.65; H, 4.18; N, 21.12. Found: C, 65.81; H, 4.25; N, 21.37.
6
) δ 2.15, 2.81 (2s, each 3H, 2 CH
3
); 7.36-8.16 (m, 14H,
3
-H); 13.66 (s, 1H, OH, D O exchangeable). Anal.
2
H
22
N
8
O
3
4
.1.15.2. 4-{3-Methyl-5-phenyl-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-
o
d]pyrimidin-6-yl)-1H-pyrazol-4-yl}diazenylbenzoic acid (15c) Yield: 78 %; Mp: 251-253 C.
IR (KBr, cm ): 3361-3218 (OH); 3030, 2885 (CH); 1693 (C=O); 1648 (C=N); 1608, 1578, 1510
-
1
1
(
(
C=C). H-NMR (300 MHz, DMSO-d
s, 1H, pyrazolopyrimidine C3-H); 11.32 (s, 1H, OH, D
6
) δ 2.29 (s, 3H, CH
3
); 7.23-8.07 (m, 19H, phenyl-H); 8.51
1
3
2
O exchangeable). C-NMR (75 MHz,
19

DMSO-d
6
) δ 14.67 (CH
3
); 106.21 (pyrazolopyrimidine C3a); 114.08 (pyrazole C
4
); 121.56,
1
26.91, 130.03 (phenyl C2-6); 121.85, 127.46, 128.87 (phenyl C2-6); 128.44, 128.73, 129.18
(
1
1
phenyl C2-6); 128.99 , 130.47, 130.87 (phenyl C2-6); 132.15 (phenyl C
1
); 134.36 (phenyl C
1
3
);
);
34.81 (phenyl C
1
); 137.29 (phenyl C
1
); 142.47 (pyrazole C
3
); 146.78 (pyrazolopyrimidine C
48.38 (pyrazolopyrimidine C7a); 154.89 (pyrazole C
5
); 156.78 (pyrazolopyrimidine C ); 166.62
6
(
pyrazolopyrimidine C
4
); 166.90 (carboxylic C=O). Anal. Calcd for C34
H
24
N
8
3
O (592.61): C,
6
8.91; H, 4.08; N, 18.91. Found: C, 69.08; H, 4.14; N, 19.12.
4
.1.16. General procedure for preparation of 6-(5-Amino-3-aryl-1H-pyrazol-1-yl)-1,5-
diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (16a-e)
An equimolar amount of the hydrazine derivative 8 and the selected phenacyl cyanide
derivative (iva-e) in ethanol/glacial acetic acid mixture (2:1) (10 ml) was heated under reflux for
6
h. The reaction mixture was left to attain room temperature then the separated solid product
was filtered, washed with diethyl ether, dried and recrystallized from dioxane. Yield, physical
properties and elemental microanalyses of compounds (16a-e) are listed below.
4
4
.1.16.1. 6-(5-Amino-3-phenyl-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-
o
-1
(5H)-one (16a) Yield: 80 %; Mp: 226-227 C. IR (KBr, cm ): 3452, 3339 (NH); 3095, 3058
O (445.48): C,
(
CH); 1719 (C=O); 1610 (C=N); 1546, 1479 (C=C). Anal. Calcd for C26
H
19
N
7
7
0.10; H, 4.30; N, 22.01. Found: C, 70.36; H, 4.38; N, 22.19.
4
.1.16.2. 6-(5-Amino-3-(4-bromophenyl)-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-
o
-1
d]pyrimidin-4(5H)-one (16b) Yield: 80 %; Mp: 285-286 C. IR (KBr, cm ): 3457, 3316 (NH);
1
3
111, 3063 (CH); 1700 (C=O); 1616 (C=N); 1572, 1542, 1484 (C=C). H-NMR (500 MHz,
DMSO-d ) δ 5.52 (s, 1H, pyrazole C -H); 6.11 (s, 2H, NH2, O exchangeable); 7.23-7.59 (m,
2H, phenyl-H); 8.03 (d, J = 8.5 Hz, 2H, phenyl C2,6-H); 8.51 (s, 1H, pyrazolopyrimidine C -H).
); 106.81 (pyrazolopyrimidine C3a);
21.74, 128.86, 131.85 (phenyl C2-6); 122.52, 127.85, 129.95 (phenyl C2-6); 128.11, 128.72,
28.99 (phenyl C2-6); 132.41 (phenyl C ); 136.56 (phenyl C ); 137.42 (phenyl C ); 138.38
); 147.59 (pyrazolopyrimidine C7a); 150.17 (pyrazole C ); 151.25
); 151.41 (pyrazole C ); 158.03 (pyrazolopyrimidine C ). MS (m/z, %):
26 (M +2, 48); 524 (M , 53); 315 (7); 313 (7); 287 (100); 261 (5); 258 (7); 232 (5); 205 (4);
43 (5); 102 (8); 77 (22). Anal. Calcd for C26 O (524.37): C, 59.55; H, 3.46; N, 18.70.
6
4
D
2
1
3
1
3
C-NMR (125 MHz, DMSO-d
6
) δ 84.45 (pyrazole C
4
1
1
1
1
1
(
(
pyrazolopyrimidine C
pyrazolopyrimidine C
3
5
6
3
4
+
•
+•
5
1
H18BrN
7
Found: C, 59.79; H, 3.49; N, 18.94.
4
.1.16.3.
d]pyrimidin-4(5H)-one (16c) Yield: 70 %; Mp: > 300 C. IR (KBr, cm ): 3458, 3315 (NH);
110, 3065 (CH); 1702 (C=O); 1618 (C=N); 1573, 1543, 1486 (C=C). Anal. Calcd for
O (479.92): C, 65.07; H, 3.78; N, 20.43. Found: C, 65.31; H, 3.75; N, 20.82.
6-(5-Amino-3-(4-chlorophenyl)-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-
o
-1
3
C
26
H18ClN
7
4
.1.16.4. 6-(5-Amino-3-(4-methylphenyl)-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-
o
-1
d]pyrimidin-4(5H)-one (16d) Yield: 65 %; Mp: 237-238 C. IR (KBr, cm ): 3457, 3305 (NH);
1
4
705 (C=O); 1612 (C=N); 1540, 1478 (C=C). Anal. Calcd for C27
.61; N, 21.34. Found: C, 70.74; H, 4.67; N, 21.58.
H
21
7
N O (459.50): C, 70.57; H,
20

4
.1.16.5. 6-(5-Amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)-1,5-diphenyl-1H-pyrazolo[3,4-
o
-1
d]pyrimidin-4(5H)-one (16e) Yield: 70 %; Mp: 267-268 C. IR (KBr, cm ): 3447, 3320 (NH);
3
1
112, 3068, 2951, 2925, 2827 (CH); 1703 (C=O); 1616 (C=N); 1580, 1544, 1482 (C=C); 1243,
1
028 (C-O-C). H-NMR (400 MHz, DMSO-d
, D O exchangeable); 6.85 (d, 2H, J = 8.8 Hz, methoxy phenyl C3,5-H);
.28-7.62 (m, 10H, phenyl-H); 8.07 (d, J = 7.7 Hz, 2H, phenyl C2,6-H); 8.52 (s, 1H,
pyrazolopyrimidine C -H). Anal. Calcd for C27 (475.50): C, 68.20; H, 4.45; N, 20.62.
Found: C, 68.43; H, 4.51; N, 20.80.
6
) δ 3.74 (s, 3H, OCH
3
); 5.47 (s, 1H, pyrazole C
4
-
H); 6.06 (s, 2H, NH
7
2
2
3
H
21
N
7
O
2
4
1
1
.1.17. General method for preparation of Ethyl 5-amino-1-(4-oxo-1,5-diphenyl-4,5-dihydro-
H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H-pyrazole-4-carboxylate (17a) and 5-Amino-1-(4-oxo-
,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-1H-pyrazole-4-carbonitrile
(
17b)
An equimolar amount of the hydrazine derivative 8 and ethyl 2-cyano-3-
ethoxypropenoate (va) or (ethoxymethylidene)propanedinitrile (vb) in ethanol/glacial acetic acid
mixture (4:1) (10 ml) was heated under reflux for 6-8 h. The reaction mixture was concentrated
under reduced pressure and allowed to cool. The separated crystals were filtered, washed with
water, dried and recrystallized from ethanol. Yield, physical properties and elemental
microanalyses of compounds (17a,b) are listed below.
4
.1.17.1. Ethyl 5-amino-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-
o
-1
yl)-1H-pyrazole-4-carboxylate (17a) Yield: 65 %; Mp: 234-235 C. IR (KBr, cm ): 3402, 3291
(
(
2
NH); 2977, 2932 (CH); 1735, 1694 (C=O); 1633 (C=N); 1576, 1526, 1497 (C=C); 1253, 1058
1
C-O-C). H-NMR (300 MHz, DMSO-d
H, CH ); 6.97 (s, 2H, NH , D O exchangeable); 7.29-7.63 (m, 9H, phenyl-H and pyrazole C
H); 8.01 (d, J = 7.2 Hz, 2H, phenyl C2,6-H); 8.55 (s, 1H, pyrazolopyrimidine C
6
) δ 1.21 (t, J = 7.0 Hz, 3H, CH ); 4.12 (q, J = 7.0 Hz,
3
2
2
2
3
-
1
3
3
-H). C-NMR
); 92.90 (pyrazole C ); 107.36
pyrazolopyrimidine C3a); 122.58, 128.25, 130.02 (phenyl C2-6); 128.82, 129.16, 129.32 (phenyl
(
(
125 MHz, DMSO-d
6
) δ 14.93 (CH
3
); 59.55 (OCH
2
4
C
2-6); 135.80 (phenyl C
1
); 137.42 (phenyl C
); 152.32 (pyrazolopyrimidine C6); 157.89 (pyrazolopyrimidine C4)
(441.15): C, 62.58; H, 4.34; N, 22.21. Found:
1
); 138.24 (pyrazole C
3
); 146.79 (pyrazolopyrimidine
C7a); 150.03 (pyrazole C
5
;
1
63.25 (ester C=O). Anal. Calcd for C23
H
19
N
7
O
3
C, 62.80; H, 4.38; N, 22.48.
4
1
.1.17.2.
5-Amino-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-
o
-1
H-pyrazole-4-carbonitrile (17b) Yield: 60 %; Mp: 284-285 C. IR (KBr, cm ): 3317, 3246
1
(
NH); 2971 (CH); 2224 (CN); 1704 (C=O); 1646 (C=N); 1592, 1524, (C=C). H-NMR (300
MHz, DMSO-d ) δ 7.27 (dd, J = 7.8, 1.8 Hz, 2H, phenyl-H); 7.39-7.64 (m, 9H, phenyl-H, NH
and pyrazole C -H); 8.00 (dd, J = 8.7, 1.2 Hz, 2H, phenyl-H); 8.56 (s, 1H, pyrazolopyrimidine
-H). Anal. Calcd for C21 O (394.13): C, 63.95; H, 3.58; N, 28.41. Found: C, 64.14; H,
.62; N, 28.67.
6
2
3
C
3
3
H
14
N
8
4
6
.1.17.3. Ethyl 5-hydroxy-1-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
-yl)-1H-pyrazole-4-carboxylate (17c)
21

A
(
°
mixture of the hydrazine derivative
ethoxymethylidene)propanedioate (vc) (0.43 g, 0.4 ml, 2 mmol) was heated in an oil bath at 180
C for 15 min. The reaction mixture was allowed to attain room temperature, triturated with
ethanol, then the solid product was filtered, washed with diethyl ether, dried and crystallized
8
(0.32 g,
1
mmol) and diethyl
o
-1
from ethanol. Yield: 52 %; Mp. > 300 C. IR (KBr, cm ): 3391, 3363 (OH); 3102, 3066, 2982
1
(
(
(
CH); 1719 (C=O); 1646 (C=N); 1568, 1541, 1519, 1497 (C=C); 1250, 1025 (C-O-C). H-NMR
400 MHz, DMSO-d ) δ 1.22 (t, J = 6.9 Hz, 3H, CH ); 4.15 (q, J = 6.9 Hz, 2H, CH ); 7.10-8.15
m, 11H, phenyl-H and pyrazole C -H); 8.33 (s, 1H, pyrazolopyrimidine C -H); 9.91 (s, 1H, OH,
O exchangeable). Anal. Calcd for C23 (442.43): C, 62.44; H, 4.10; N, 19.00. Found:
C, 63.67; H, 4.16; N, 19.23.
6
3
2
3
3
D
2
H
18
N
6
O
4
4
.1.18.
Diethyl
2-{(2-(4-oxo-1,5-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)hydrazin-1-yl)methylidene}propanedioate (18)
A mixture of the hydrazine derivative 8 (0.32 g, 1 mmol), diethyl 2-
(
ethoxymethylidene)propanedioate (vc) (0.21 g, 0.2 ml, 1 mmol) and anhydrous potassium
carbonate (0.14 g, 1 mmol) in absolute ethanol (10 ml) was heated under reflux for 12 h. The
reaction mixture was allowed to cool, acidified with (1 N) hydrochloric acid (until pH = 5) in an
ice-water bath. The precipitated product was filtered, washed with water, dried and crystallized
o
-1
from ethanol; Yield 70 %; Mp. 215-217 C. IR (KBr, cm ): 3272 (NH); 3070, 2976, 2902 (CH);
1
1
(
718, 1698 (C=O); 1657 (C=N); 1612, 1551, 1492, 1419 (C=C); 1255, 1026 (C-O-C). H-NMR
400 MHz, DMSO-d ) δ 1.17, 1.26 (2t, J = 8.0 Hz, each 3H, 2 CH CH ); 4.07, 4.22 (2q, J = 8.0
CH ); 7.29-7.67 (m, 8H, phenyl-H); 7.97 (d, J = 12 Hz, 2H, phenyl C2,6-H);
-H and =CH); 9.31 (s, 1H, NH, D O exchangeable);
6
2
3
Hz, each 2H, 2 CH
2
3
8
1
2
7
.20-8.26 (m, 2H, pyrazolopyrimidine C
0.27 (d, J = 12 Hz, 1H, NH, D O exchangeable). MS (m/z, %): 488.45 (M , 9); 309.22 26.12);
60.24 (22.36); 203.07 (24.24); 122.42 (20.02); 122.15 (25.53); 97.22 (23.52); 73.29 (30.20);
1.19 (22.97); 65.28 (100.00); 63.00 (24.96); 60.04 (24.27); 46.20 (29.95); 44.05 (94.71). Anal.
3
2
+
•
2
Calcd for C25
H
24
N
6
O
5
(488.50): C, 61.47; H, 4.95; N, 17.20. Found: C, 61.72; H, 5.02; N, 17.49.
4
4
.2. Biological evaluation
.2.1. In vitro cyclooxygenase inhibition assay
The ability of the tested compounds to inhibit both COX-1 and COX-2 isozymes at three
concentrations (25, 50 and 100 μM) was carried out using Cayman colorimetric COX (ovine)
[55]
inhibitor screening assay kit (Catalog No. 760111)
supplied by Cayman chemicals, Ann
Arbor, MI, USA. The Colorimetric COX Inhibitor Screening Assay utilizes the peroxidase
component of cyclooxygenase. The peroxidase activity was assayed colorimetrically by
’
’
monitoring the appearance of oxidized N,N,N ,N -tetramethyl-1,4-phenylenediamine (TMPD)
[
56]
which is produced during the reduction of PGG2 to PGH2, at 590 nm.
The preparation of
[55]
reagent was carried out following the manufacturer instructions (Catalog No. 760111).
Briefly, aliquots contain 160 μl of assay buffer (0.1N Tris–HCl, pH 8.0), 10 μl of heme, 10 μl of
enzyme (either ovine COX-1 or COX-2), and 10 μl of tested compounds were placed in a 96-
well plate. Different concentrations of celecoxib, diclofenac sodium or tested compounds were
22

o
incubated with the enzymes for a period of 5 min at 25 C. After the incubation period an
addition of 20 μl of the colorimetric substrate (TMPD) and 20 μl of arachidonic acid was done
then the plate was carefully shaken for a few seconds and then incubated for 5 min at 25 °C. The
absorbance was measured at 590 nm using plate reader. The average absorbance of samples was
determined; the absorbance of background wells was subtracted from absorbances of the 100%
initial activity and the inhibitor wells; each inhibitor sample was subtracted from the 100% initial
activity sample then divided by the 100% initial activity sample and multiplied by 100 to give
the percent inhibition. Percent inhibition was graphed by the inhibitor concentration to determine
the IC50 values.
4
.2.2. In vivo anti-inflammatory activity
Adult female wistar rats weighing 150-250 g were used (procured from the Experimental
Animal Centre in Alexandria University). All animals accessed to food and water ad libitum and
o
were housed in 12 h dark/light cycle in a controlled condition at 23-25 C. They were allowed to
acclimatize for 1 week prior to experimentation. Procedures involving animals and their care
were conducted in conformity with the Guide for the Care and Use of Laboratory Animals
published by US National Institute of Health (NIH publication No. 83-23, revised 1996) and
following the ethical guidelines of Alexandria University on laboratory animals. In all tests,
adequate considerations were adopted to reduce pain or discomfort of animals.
Celecoxib and diclofenac sodium (European Egyptian Pharmaceutical industries,
Alexandria, Egypt), formalin 5 % made from formaldehyde 37 % and saline (Merck, Germany)
were used as reference compounds. The novel compounds were synthesized based on the
previously described methods.
Inflammatory models:
Compounds that showed in vitro selectivity indices higher or nearly equivalent to reference
drugs towards COX 2 enzyme (9b, 16a, 16b, 17a, 17b and 18) were further evaluated for their in
vivo anti-inflammatory activity applying the formalin-induced paw edema screening protocol as
[
49, 50]
an acute inflammation model
inflammation model.
and cotton pellet-induced granuloma protocol as a chronic
[51]
Celecoxib (5 mg/kg) and diclofenac sodium (5 mg/kg) were used as
reference drugs. Animals were divided randomly into six groups of six rats each, treated with the
different test compounds. Groups treated with celecoxib and diclofenac sodium served as
reference and rats which were given the vehicle (DMSO) served as control. Same groups of rats
were employed in both inflammation models and in ulcerogenic examination.
4
.2.2.1. Formalin-induced paw edema test (acute inflammation model):
A solution of freshly prepared 5 % formalin was used as a phlogistic agent. A mark was
made on the lateral malleolus of the rats’ paws delineating the injection sites. The novel test
compounds (5 mg/kg body weight), celecoxib (5 mg/kg body weight), diclofenac sodium (5
mg/kg body weight), were dissolved in DMSO and administered orally using gastric gavage once
daily for 7 consecutive days, while DMSO was given to the control group. After the last dose, on
th
the 8 day, the initial volume of paw was measured by means of digital calibrated Vernier
caliper then 40 μl formalin were injected subcutaneously into the sub-plantar tissue of the right
hind paw of all groups under light ether anesthesia.
23

The volume of the paw was measured in different treatment groups, 4 h following the
formalin injection and the amount of increase in paw volume (edema volume) was calculated by
subtracting the volumes before and 4 h after the injection of formalin.
Edema was expressed as an increase in the volume of the paw, and the percentage of
edema inhibition (or percent protection against inflammation) for each rat and each group was
calculated according to the following equation:
Where Vt is the mean volume of edema after 4 h at specific time interval (4 h) and Vo is the
mean volume of edema at zero time interval.
Statistical differences between control and test groups were estimated using the F test
[
57]
(
ANOVA), and pairwise comparison was done using Post Hoc Test (Tukey). Data were fed to
[
58]
the computer and analyzed using IBM SPSS software package version 20.0.
4
.2.2.2. Cotton pellet-induced granuloma assay (chronic inflammation model):
Adsorbent cotton wool was cut into pieces weighing 20 ± 1 mg and made into pellets.
o
The pellets were then sterilized in a hot air oven at 120 C for 2 h. The abdomen of the rat was
shaved cleanly, swabbed with 70 % ethanol and two sterilized cotton pellets were implanted
subcutaneously, one on each side of the abdomen of the animal under Ketamine/xylazine
anesthesia (50 mg/kg and 9 mg/kg respectively intraperitoneal). Gentamycin injection (4 mg/kg
intramuscular) was used for three days following the procedure to avoid post-operative infection.
Test compounds, celecoxib (5 mg/kg), diclofenac sodium (5 mg/kg) or vehicle (DMSO) were
administered orally using gastric gavage once daily throughout the experimental period of 7
th
consecutive days. On the 8 day after implantation, rats were anaesthetized under light ether
o
anesthesia. The pellets were dissected, dried at 60 C for 24 h and weighed after cooling. The
mean weight of the dried cotton pellets of the control group, the test groups as well as of
reference groups was calculated. Percentage of granuloma inhibition of the test compounds and
reference compounds was calculated relative to control.
The net dry weights were determined as follows:
Dry granuloma weight = weight of the cotton pellet of test, reference and control groups (dry) -
weight of the cotton pellet before the experiment.
Statistical differences between control and test groups were estimated using the F test
[
57]
(
ANOVA), and pairwise comparison was done using Post Hoc Test (Tukey). Data were fed to
[
58]
the computer and analyzed using IBM SPSS software package version 20.0.
4
.2.3 Ulcerogenic activity:
Compounds were also evaluated for chronic gastric ulcerogenic effect
[52, 53]
on the same
th
groups of rats. On the 8 day and after scarification and removal of cotton pellets, rats’ stomachs
were also removed and opened through the greater curvature, washed under running water and
fixed in saline solution.
24

Gross examination was performed for any evidence of hyperemia, hemorrhage, definite
hemorrhagic erosion or ulcer. In addition, histopathological examination was performed to
confirm the degree of inflammatory reaction in the gastric mucosal layers of the treated rats’
stomachs.
5
. Conflict of Interest
The authors have declared no conflict of interest.
Acknowledgments
The authors would like to express their thanks to Dr.Waleed Ali Mohamed, Assistant
professor of Medicinal Biochemistry, Faculty of Medicine, Cairo University, for carrying out the
in vitro anti-inflammatory screening.
Highlights

Synthesis of new pyrazolo[3,4-d] pyrimidines linked to different functionalities at 5 and 6
positions




In vitro COX-1/ COX-2 inhibitory assay
In vivo formalin paw edema and cotton pellets induced granuloma assays
Compounds 16a and 18 were highly effective in formalin paw edema assay (acute model)
Compound 17b showed promising activity in cotton-pellets granuloma assay (chronic
model)

Compounds 16a, 17b and 18 showed safe gastrointestinal margin
References
1
.
Dannhardt G, Kiefer W. Cyclooxygenase inhibitors–current status and future prospects.
European journal of medicinal chemistry 36(2), 109-126 (2001).
2
.
Bertolini A, Ottani A, Sandrini M. Selective COX-2 inhibitors and dual acting anti-
inflammatory drugs: critical remarks. Current medicinal chemistry 9(10), 1033-1043
(
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