Anti-inflammatory drug approach: Synthesis and biological evaluation of novel pyrazolo[3,4-d]pyrimidine compounds

Article abstract of DOI:10.1016/j.bioorg.2019.02.014

In this study, the acid chlorides of pyrazolo[3,4-d]pyrimidine compounds were prepared and reacted with a number of nucleophiles. The novel compounds were experimentally tested via enzyme assay and they showed cyclooxygenase-2 inhibition activity in the middle micro molar range (4b had a COX-1 IC₅₀ of 26 μM and a COX-2 IC₅₀ of 34 μM, 3b had a COX-1 IC₅₀ of 19 μM and a COX-2 IC₅₀ of 31 μM, 3a had a COX-2 IC₅₀ of 42 μM). These compounds were analyzed via docking and were predicted to interact with some of the COX-2 key residues. Our best hit, 4d (COX-1 IC₅₀ of 28 μM, COX-2 IC₅₀ of 23 μM), appears to adopt similar binding modes to the standard COX-2 inhibitor, celecoxib, proposing room for possible selectivity. Additionally, the resultant novel compounds were tested in several in vivo assays. Four compounds 3a (COX-2 IC₅₀ of 42 μM), 3d, 4d and 4f were notable for their anti-inflammatory activity that was comparable to that of the clinically available COX-2 inhibitor celecoxib. Interestingly, they showed greater potency than the famous non-steroidal anti-inflammatory drug, Diclofenac sodium. In summary, these novel pyrazolo[3,4-d]pyrimidine analogues showed interesting anti-inflammatory activity and could act as a starting point for future drugs.

Full text of DOI:10.1016/j.bioorg.2019.02.014

Accepted Manuscript
Anti-inflammatory Drug Approach: Synthesis and Biological Evaluation of
Novel Pyrazolo[3,4-d]pyrimidine Compounds
Noor Atatreh, Amal M. Youssef, Mohammad A.Ghattas, Mohammad Al
Sorkhy, Sara Alrawashdeh, Khaled B. Al-Harbi, Ibrahim M. El-Ashmawy,
Tariq I. Almundarij, Amani A. Abdelghani, Alaa S. Abd-El-Aziz
PII:
S0045-2068(18)31491-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.014
YBIOO 2789
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
17 December 2018
3 February 2019
4 February 2019
Please cite this article as: N. Atatreh, A.M. Youssef, M. A.Ghattas, M. Al Sorkhy, S. Alrawashdeh, K.B. Al-Harbi,
I.M. El-Ashmawy, T.I. Almundarij, A.A. Abdelghani, A.S. Abd-El-Aziz, Anti-inflammatory Drug Approach:
Synthesis and Biological Evaluation of Novel Pyrazolo[3,4-d]pyrimidine Compounds, Bioorganic Chemistry
(
2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.014
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Anti-inflammatory Drug Approach: Synthesis and Biological
Evaluation of Novel Pyrazolo[3,4-d]pyrimidine Compounds
1
1,2
1
1
Noor Atatreh , Amal M. Youssef *, Mohammad A.Ghattas , Mohammad Al Sorkhy ,
1
3
3,4
Sara Alrawashdeh , Khaled B. Al-Harbi , Ibrahim M. El-Ashmawy , Tariq I.
Almundarij , Amani A. Abdelghani , Alaa S. Abd-El-Aziz
3
5
5
1
College of Pharmacy, Al Ain University of Science and Technology, Al Ain, United
Arab Emirates
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
2
Alexandria, Egypt
3
Department of Veterinary Medicine, College of Agricultural and Veterinary Medicine,
Al Qassim University, Kingdom of Saudi Arabia
4
Department of Pharmacology, Faculty of Veterinary Medicine, Alexandria University,
Alexandria, Egypt
5
Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince
Edward Island, Canada C1A 4P3
*
Correspondence: Prof. Amal Youssef, College of Pharmacy, Al Ain University of
Science and Technology, P.O. Box: 64141, Al Ain, United Arab Emirates. Tel: +971 3
024873; Fax: +9713 7024777. E-mail: ayoussef65@yahoo.com
7
Synthesis; Pyrazolo[3,4-d]pyrimidines; Anti-inflammatory drugs; Docking; COX
inhibitor.

Abstract
In this study, the acid chlorides of pyrazolo[3,4-d]pyrimidine compounds were prepared
and reacted with a number of nucleophiles. The novel compounds were experimentally
tested via enzyme assay and they showed cyclooxygenase-2 inhibition activity in the
middle micro molar range (4b had a COX-1 IC50 of 26 µM and a COX-2 IC50 of 34 µM,
3
b had a COX-1 IC50 of 19 µM and a COX-2 IC50 of 31 µM, 3a had a COX-2 IC50 of 42
µM). These compounds were analyzed via docking and were predicted to interact with
some of the COX-2 key residues. Our best hit, 4d (COX-1 IC50 of 28 µM, COX-2 IC50 of
2
3 µM), appears to adopt similar binding modes to the standard COX-2 inhibitor,
celecoxib, proposing room for possible selectivity. Additionally, the resultant novel
compounds were tested in several in vivo assays. Four compounds 3a (COX-2 IC50 of 42
µM), 3d, 4d and 4f were notable for their anti-inflammatory activity that was comparable
to that of the clinically available COX-2 inhibitor celecoxib. Interestingly, they showed
greater potency than the famous non-steroidal anti-inflammatory drug, Diclofenac
sodium. In summary, these novel pyrazolo[3,4-d]pyrimidine analogues showed
interesting anti-inflammatory activity and could act as a starting point for future drugs.

1
. Introduction
Pyrazolopyrimidines are fused heterocyclic compounds that have considerable chemical
and pharmacological importance as purine analogs. They are of interest as potential
bioactive molecules. They exhibit wide pharmacological activities like tuberculostatic [1]
antimicrobial [2], neuroleptic [3], antitumor [1], antihypertensive [4] and anti-leishmanial
activities [5]. They may act as tropomyosin receptor kinase (Trk) and c-Src inhibitors [6,
7
], C-C chemokine receptor type 1 and purine antagonists [8], HIV reverse transcriptase
inhibitors [9] as well as showing activity as antitumor/anti-leukemia [10-13]
andherbicidal/fungicidal agents [14].
Several compounds as xanthene attached amino acids [15], piperazine-1-carbothioamide
chitosan silver nanoparticles[16] , dihydrazones [17], coumarins [18], benzo[d]thiazoles
[
19] and sulfur containing motifs [20] have exhibited anti-inflammatory activities. The
anti-inflammatory properties of pyrazolo[3,4-d]pyrimidine compounds as potential
inhibitors of cyclooxygenase (COX) [21] and p38α isoenzymes [22, 23] are well known
and gaining more interest. Traditional non-steroidal anti-inflammatory drugs (NSAIDs)
are still the most commonly prescribed medications to alleviate acute and chronic
inflammation, pain and fever. The mechanism of action of NSAIDs is lowering the
production of inflammatory mediators such as prostaglandins (PGs). They inhibit the
cyclooxygenase enzyme (COX) that catalyzes the biosynthesis of prostaglandins (PGs)
and thromboxanes from arachidonic acid [24].
There are two COX isoforms COX-1 and COX-2 [25]. COX-1 is the constitutive isoform
which is mainly responsible for the synthesis of cytoprotective PGs in gastrointestinal

(
GI) tract, maintenance of renal function in the kidney [26]. The inducible isoform COX-
plays a major role in PG biosynthesis in inflammatory cells [27]. It is believed that
2
inhibition of COX-1 results in GI damage, ulceration, hematologic effects and
nephrotoxicity [28]. Therefore, highly selective COX-2 inhibitors (or “coxibs”) have
been developed to alleviate pain and inflammation without the untoward side effects
associated with non-selective NSAIDs. Among this class of compounds is celecoxib; 4-
[
5-p-tolyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide, which is a pyrazole
derivative that is known to inhibit COX-2 selectively [29]. However, its long-term use is
associated with cardiovascular adverse effects [30]. Therefore, there is a need for the
development of novel drugs having anti-inflammatory activity with better safety profile.
Methyl 4,5-dihydro-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-6-carboxylate 1 [31]
(
Fig. 1) which waspreviously prepared in our lab,was found to be toxic to monocytes at
0 μM and also showed a neuroprotective effect at 20 μM (2-fold difference). This
4
compound showed in vitro anti-neurotoxic and neuroprotective activity at concentrations
below its toxic range and partially inhibited both COX-1 and COX-2 enzymes.
As a continuation of our research program on the synthesis of novel anti-inflammatory
agents, the objectives of the present investigation have been directed toward the synthesis
of new derivatives of pyrazolo[3,4-d]pyrimidines. The new derivatives incorporate
various linear and branched chains in addition to morpholino group and aromatic
moieties. The different derivatives attached to pyrazolopyrmidine ring at position-2 have

been synthesized in order to investigate their impact on the anticipated anti-inflammatory
activity.
The new compounds were investigated in vitro and in vivo for their anti-inflammatory
activity. Docking was conducted for these compounds to suggest their binding mode and
correlate that with biological activities.
2
2
. Results and discussion
.1. Chemistry
Synthetic strategies adopted for the synthesis of the intermediate and target compounds
are depicted in Scheme 1.The general intermediate, 5-amino-4-cyano-1-phenylpyrazole
2
was obtained through the reaction of phenyl hydrazine with commercially available
ethoxymethylenemalononitrile in alcohol with a good yield [32].
Reaction of 2 with oxalyl chloride produced the acid chloride of pyrazolo[3,4-
d]pyrimidine which was immediately reacted with different nucleophiles such as alcohols
and amines to produce 3a-d and 4a-f, respectively.
The synthesized compounds were supported by spectroscopic data. IR spectra of 3a-d
-
and 4a-f lacked CN absorption bands and showed vibrational bands at 1735-1739cm
1
-1
(
CO ester) and 1686-1695 cm (CO amide) which support the conversion of pyrazole-4-
carbonitrile to the corresponding pyrazolo[3,4-d]pyrimidine esters and amides,
respectively.
1
H-NMR spectra of 3a-d were characterized by the presence of a D
-H, in addition to
isopropyl, propyl, butyl and sec-butyl protons at the expected range of δ–ppm scale.
2
O exchangeable
singlet for the NH of pyrazolopyrmidine ring, a singlet for pyrazole-C
5

DEPT 135 (Distortionless Enhancement of Polarization Transfer using a 135 degree
decoupler pulse) of 3a showed a carbon spectrum with methyl (CH ) and methyne (CH)
carbons appearing at 21.8 and 71.8 respectively, whereas DEPT 135 of 3b–d showed
3
methene (CH
2
) carbon, in addition to the methyl (CH ) and methyne (CH) carbons.
3
1
H-NMR spectra of 4a-f displayed the characteristic chemical signals corresponding to
the pyrazolopyrmidine ring in addition to other protons which were observed at their
expected chemical shifts, whereas DEPT 135 showed methyl (CH
ormethene (CH ) carbons for each compound at the expected range.
3
), methyne (CH)
2
2
.2 In vitro cyclooxygenase inhibition assay
The ability of our compounds to inhibit the conversion of Arachidonic acid (AA) to
Prostaglandin H2 (PGH2) by human recombinant COX-1 and COX-2 was determined
using a COX inhibitor screening assay kit Celecoxib served as positive control for COX-
2
and Diclofenac for COX-1. The results show significant COX-2 inhibition for
compounds 3b, 4b and 4d as shown in Table 1. Remarkably, 4d exhibited comparable
inhibition percentage to that of Celecoxib, with an IC50 of 23.8 µM. On the other hand,
compound 3b showed more specificity toward COX-1when compared to Diclofenac with
an IC50 of 19.45 µM and 86 % inhibition, putting it in close proximity with the classical
COX-1 inhibitor.
2
.3 In vivo assays

The anti-inflammatory activity of ten compounds was evaluated by two screening
protocols, namely the carrageenan-induced paw edema and turpentine oil–induced
granuloma pouch bioassays. The paw edema was employed as a model for acute and sub-
acute inflammation, while the turpentine oil–induced granuloma pouch assay was used as
another model for sub-acute inflammation. The ED50 valuesof the most potent
compounds were also determined. Both Diclofenac sodium and celecoxib (10 mg/kg)
were used as reference drugs. The data obtained were presented in Tables 2, 3 & 4 and
expressed as means ± SE. Statistical differences of control and tested groups were carried
out using the Analysis of Variance (ANOVA) followed by ‘Student–Newman–Keuls
Multiple Comparison Test’. They were performed using the Statistical Analysis System
(
SAS, 1987)computer package, SAS Incorporation Institute. The difference inresults was
considered significant when p< 0.05.
2
.3.1. Carrageenan-induced paw edema bioassay (Acute Inflammatory Model)
In this acute inflammatory model each test compound was dosed orally (po) at 10 mg/kg
body weight 1 h prior to induction of inflammation by carrageenan injection. Celecoxib
and dicofenac sodium were utilized as reference anti-inflammatory drugs at a dose of 10
mg/kg, po. The anti-inflammatory activity was then calculated at 1, 2, 3, and 4 hafter
induction. The data obtained are presented in (Table 2) as the mean paw volume (mL) in
addition to the percentage anti-inflammatory activity (AI %). All of the test compounds
significantly reduced paw edema volume 1, 2, 3, and 4 h after induction. However, a

comparison of the anti-inflammatory activity of the test compounds, relative to the
control, indicated distinctive pharmacokinetic profiles.
A comparative study of the AI activity of tested compounds relative to the reference
drugs at different time intervals indicated the following: after 1 h, compounds 3a, 4c, 4d
and 4f showed distinctive pharmacokinetic profile as revealed from its potent AI activity
(
53%, 66%, 64% and 55%, respectively) and rapid onset of action which was higher than
Diclofenac Na (51%). After 2 h interval, compounds 3a, 4b, 4d and 4f displayed
significant inhibition of paw edema (55%, 55%, 65% and 63%, respectively) equipotent
and higher than Diclofenac sodium(53%).Taking the AI activity after 4 h as acriterion for
comparison, the data indicated that compounds 3a, 3d, 4b, 4d and 4f displayed higher or
equipotent inhibition of pawedema with percentage activity of 57-66% when compared
with that of Diclofenac sodium (57%) but less than that of celecoxib (69%).
The most active compounds (3a, 3d, 4d and 4f) were further tested at 5, 10, 20, 40 and 50
mg/kg body weight in order to determine their ED50values. The results revealed that
compounds 3a, 3d, 4d and 4f (ED50= 8.3, 8.1, 7.6 and 7.4 mg/kg, respectively)
werefound to be nearly equipotent to celecoxib (ED50= 7.2 mg/kg).
2
.3.2. Carrageenan-induced paw edema bioassay (sub-acute inflammatory model)
In this sub-acute inflammatory model, inflammation was induced by carrageenan
injection on the first and third days, and test compounds were administered orally (at 10
mg/kg daily) for 7 days. Again, celecoxib and Diclofenac sodium were used as a
reference anti-inflammatory agents in this assay. The anti-inflammatory activity was

st
th
calculated at the 1 and 8 day after induction and the data presented in Table 3as the
mean paw volume and the percentage anti-inflammatory activity relative to a control.
st
The obtained data revealed that, according to the measurements performed on both the 1
th
and 8 day, all tested compounds displayed significant anti-inflammatory activity. At day
1
, compounds 3a, 3d, 4d and 4f showed significant AI activities (61-66%) when
compared to Diclofenac Na (57%); while compounds 3b, and 4bshowed AI activities
55% and 57%) similar to Diclofenac sodium. At day 8, compound 4f demonstrated
(
distinctive AI activity (66%) higher than Diclofenac sodium (57%) and similar to
celecoxib (65%);whereas, compounds 3a, 3dand 4d showed an inhibition of paw edema
(
56-60%) higher than that of Diclofenac sodium (57%)
2
.3.3. Turpentine oil-induced granuloma pouch bioassay
In this sub-acute inflammatory model, each test compound was administered orally (10
mg/kg) 1 h prior to turpentine oil injection and administration was continued for 7 days.
At the 8th day, the volume of exudates (mL) was measured and the percentage of
granuloma inhibition was calculated. Celecoxib and Diclofenac sodium were used as a
reference drugs. Table 4 illustrates that all of the studied compounds significantly
reduced the exudate volume when comparedto the control group of animals. Compounds
3
a, 4cand 4f appeared to be equipotent to celecoxib (54%) and higher than Diclofenac
sodium (51%); whereas Compounds 3d and 4d showed similar AI to Diclofenac sodium.
The in vivo experiments (Tables 1–3) demonstrated anti-inflammatory activity of
compounds 3a, 3d, 4d and 4f in carrageenan paw edema model of acute inflammation, as

well as in the carrageenan paw edema and turpentine oil-induced granuloma pouch assays
sub-acute inflammatory models). Therefore, these compounds might be effective in
managing acute inflammation, as well as in controlling chronic inflammatory conditions.
(
2
.4. Docking study
There are two main NSAIDs binding regions in the COX catalytic pocket: the top of the
active site comprising Ser530 and Tyr385 (e.g. involved in Diclofenac binding) or the
bottom of the active site comprising Arg120 and Tyr355 (e.g. involved in flurbiprofen
binding)[33]. The above interactions are mainly electrostatics and are primarily formed
by the NSAIDs' carboxylate. There is an additional binding region only accessed by
selective COX-2 inhibitors containing four main residues Gln192, Ser353, Arg 513 and
Val523.
In the present study, docking was employed to predict the binding modes of the tested
compounds inside the target pocket. Table 5 shows the binding energies of our ten
compounds docked into the COX-1 and COX-2 binding site. Docked compounds were
predicted to have favorable binding energies although celecoxib showed a superior
docking score compared to our hits. Interestingly, and in line with the in vitro and in
vivofindings, 4d was predicted to have the best docking score amongst all tested
compounds against the COX-2 enzyme.
4
d was predicted to have a docking score of -9.501 kcal/mol for COX-2 and 6.2 kcal/mol
for COX-1, which comes consistent to the enzymatic assays results where 4d possessed

slighter greater selectivity to COX-2 than COX-1;though selectivity prediction was not
consistent with experimental findings for other compounds. As shown in Fig.2, 4d forms
hydrogen bonds with the key amino acid Tyr355 and several π-hydrogen interactions
with the Leu352 and Val523 side chains along with multiple van der Waals contacts with
the surrounding hydrophobic residues. Although 4d does not possess a similar binding
mode to celecoxib in the COX-2 active site, its pyrazolo[3,4-d]pyrimidine system seems
to sit in the same site of celexocib’s pyrazole ring, as shown in Fig. 3.
Although docking was able to predict the experimental superiority of 4d over other tested
analogues, it was not quite successful to explain why the other enantiomer 4e showed
weaker COX-2 inhibition in vitro. Instead, 4e was predicted to have as low binding
energy as 4d; and looking at their docked poses aligned over each other showed that they
have very similar binding mode apart from the methyl group orientation that appears
sitting in opposite direction (Fig. 4).
When the in vitro activities and in silico findings are considered together, it can be
suggested that:
(
(
(
i)
Our best hit 4d was predicted to have its pyrazolo[3,4-d]pyrimidine scaffold
sitting in the same place of celecoxib’spyrazole ring [33].
ii)
iii)
4d was also predicted to interact with the COX-2 key residues such as Tyr355
and Ser530.
4d showed promising experimental activity but only slight selectivity towards
COX-2 which can be further enhance by optimizing the pyrazolopyrimidine

ring binding so that it reaches the selectivity region in the COX-2 active site
Fig. 3).
(
3
. Conclusion
Novel pyrazolo[3,4-d]pyrimidine compounds were prepared. Using enzymatic COX-1
and COX-2 inhibition assays the compounds were tested for their in vitro ability to
inhibit COX-1 and COX-2. The results show significant COX-2 inhibition for
compounds 3b, 4b and 4d. The synthesized compounds were also evaluated for their in
vivo anti-inflammatory activity using two different screening protocols, namely,
carrageenan–induced paw edema and the turpentine oil-induced granuloma pouch
bioassays. Among the tested analogs, compounds 3a, 3d, 4d and 4f were notable for their
comparable anti-inflammatory effect to the clinically available COX-2 inhibitor
celecoxib in both the acute inflammatory and sub-acute inflammatory models. The in
silico findings come in line with the in vivo and in vitro assays results and also suggest
that these compounds have the potential to be COX-2 selective inhibitors since they have
typical binding modes of the benchmark drug celecoxib. Structure activity relationship
showed that branched isomers 3a and 3d showed significant in vitro and in vivo COX-
2
inhibition. While linear isomer 3b showed more specificity toward COX-1 inhibition.
d the enantiomer containing the bulky aromatic group was identified as the best
4
compound adopting comparable activity to the clinically available COX-2 inhibitor
celecoxib.

Based on all previously mentioned results,pyrazolo[3,4-d] pyrimidine-6-carboxylate and
pyrazolo[3,4-d]pyrimidine-6-carboxamide derivatives could represent a fruitful matrix
for the future development of a new class of anti-inflammatory agents.
4
4
. Experimental
.1. Chemistry
All chemicals and reagents were obtained from Sigma-Aldrich and were used without
any further purification. All solvents were dried and stored over 3 Å molecular sieves
before being used. Melting points were determined in open glass capillaries on a Stuart
melting point apparatus and are uncorrected. The infrared (IR) spectra were recorded on a
1
FTIR Nicolet IR 200 spectrophotometer. A Bruker Avance NMR spectrometer ( H, 300
1
3
MHz and C, 75 MHz) was used to characterize all synthesized complexes in DMSO-
d
6
with the chemical signals referenced to solvent residual signal in ppm. Elemental
analyses for all the synthesized compounds 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 glass plates, and the spots were detected by exposure to
UV-lamp at  254.
4
.1.1.
Subsbstituted-4-oxo-l-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-
carboxylate (3)
To a stirred solution of oxalyl chloride (l.27 g, 0.01 mol) in THF (5 mL), a solution of 5-
amino-1 phenyl-1H-pyrazole-4-carbonitrile 2 (0.92 g, 0.005 mol) in THF (5 mL) was
added dropwise over a period of 2 h. The reaction mixture was further stirred for 24 h

atroom temperature (rt). The solvent was evaporated under reduced pressure, and the
formed product was filtered, dried and refluxed in excess alcohol for 5 min. The beige
crystals were collected by suction filtration.
4
.1.1.1.
carboxylate (3a)
Yield 85%; mp 158-160 °C.IR (cm ): 1738 (CO ester), 1686 (CO amide); H NMR (
ppm): 12.84 (s, 1H, NH, D O exchangeable), 8.42 (s, IH, pyrazole-C -H), 8.05-7.42 (m,
Isopropyl 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-
-1
1
2
5
1
3
5
1
4
H, aromatic-H), 4.1-3.8 (m, 1H, CH), 1.36 (d, J=6.6 Hz, 6H, 2 CH ,); C NMR (ppm):
3
36.7, 129.8, 127.9, 122.4, 71.8, 21.8. Anal.Calcd for C15
.73; N, 18.78. Found: C, 60.75; H, 4.84; N, 18.39.
H
14
N
4
O
3
(298.3): C, 60.40; H,
4
.1.1.2.
Propyl 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d] pyrimidine-6-
carboxylate (3b)
-1
1
Yield 80%; mp 164-166 °C. IR (cm ): 1735 (CO ester), 1692 (CO amide); H NMR (
ppm): 12.87 (s, 1H, NH, D
2
O exchangeable), 8.43 (s, IH, pyrazole-C
H, aromatic-H), 4.3 (t, J=13.2 Hz, 2H, CH ), 1.80-1.69 (m, 2H, CH
); C NMR ( ppm): 136.7, 129.8, 127.9, 122.3, 68.6, 21.8, 10.6. Anal.Calcd
(298.3): C, 60.40; H, 4.73; N, 18.78. Found: C, 60.05; H, 4.93; N, 19.15.
5
-H), 8.06-7.41 (m,
5
2
2
), 0.98 (t, J=15.3
1
3
Hz,3H, CH
3
for C15
H
14
N
4
O
3

4
.1.1.3.
carboxylate (3c)
Yield 82% ; mp 167-169 °C.IR (cm ): 1739 (CO ester), 1695 (CO amide); H NMR (
Butyl 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d] pyrimidine-6-
-
1
1
ppm): 12.91 (s, 1H, NH, D
2
O exchangeable ), 8.43 (s, IH, pyrazole-C
), 1.77-1.67 (m, 2H, CH
); C NMR ( ppm): 136.7, 129.8, 127.9, 122.3,
(312.32): C, 61.53; H, 5.16; N, 17.94.
5
-H), 8.07-7.41 (m,
5
2
6
H, aromatic-H), 4.35 (t, J= 13.2 Hz, 2H, CH
2
2
), 1.50-1.38 (m,
1
3
H, CH
2
), 0.95 (t, J=14.7 Hz,, 3H, CH
3
7.0, 30.3, 19.1, 14.2. Anal.Calcd for C16
H
16
N
4
O
3
Found: C, 61.85; H, 4.95; N, 18.30.
4
.1.1.4. sec-Butyl 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d] pyrimidine-6-
carboxylate (3d)
Yield 80%; mp 162-164 °C.IR (cm ): 1737 (CO ester), 1686 (CO amide); H NMR
-1
1
(
ppm): 12.89 (s, 1H, NH, D
2
O exchangeable), 8.43 (s, IH, pyrazole-C
), 1.32 (d, J = 6.3 Hz,
); C NMR ( ppm): 136.7, 129.8, 127.9, 122.3,
6.1, 28.5, 19.4, 9.8. Anal.Calcd for C16 (312.32): C, 61.53; H, 5.16; N, 17.94.
5
-H), 8.08-7.41
(
m, 5H, aromatic-H), 4.5-4.2 (m, IH, CH), 1.79-1.63 (m, 2H, CH
2
1
3
3
7
H, CH
3
), 0.94 (t, J=15.9 Hz, 3H, CH
3
H
16
N
4
O
3
Found: C, 61.15; H, 5.34; N, 18.34.
4
.1.2. Substituted 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-
carboxamide (4a-f)
A mixture of carboxylate derivative 3a(0.3 g, 0.001 mol) and the appropriate amine
(
0.004 mol) in dry THF (10 mL) was heated under reflux for 10-12 h. The solvent was

evaporated under reduced pressure, diethyl ether was added and the formed product was
filtered, washed with diethyl ether, dried and crystallized from chloroform–hexane.
4
.1.2.1. 6-(morpholine-4-carbonyl)-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-4(5H)one
4a)
Yield 80%; mp 138–140 °C.IR (cm ): 1685, 1667 (CO amide); H NMR ( ppm): 12.21
s, 1H,NH, D O exchangeable), 8.38 (s, 1H, pyrazole-C -H), 7.99-7.39 (m, 5H, aromatic-
H), 3.68-3.53 (m, 8H, CH ); C NMR ( ppm): 136.6, 129.8, 127.7, 122.3, 66.6, 47.1.
Anal.Calcd for C16 (325.32): C, 59.07; H, 4.65; N, 21.53. Found: C, 59.37; H,
(
-1
1
(
2
5
13
2
H
15
N
5
O
3
4
.55; N, 21.88.
4
.1.2.2. 4-oxo-N-phenethyl-1-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d] pyrimidine-6-
carboxamide(4b)
Yield 85%; mp 170–172 °C.IR (cm ): 1686, 1664 (CO amide); H NMR ( ppm): 12.65
s, 1H,NH, D O exchangeable), 8.45 (s, 1H, pyrazole-C -H), 8.22-7.52 (m, 10H,
aromatic-H), 3.57-3.50 (m, 2H, CH
29.6, 129.1, 128.9, 126.9, 126.7, 121.4, 41.2, 35.3. Anal.Calcd for C20
C, 66.84; H, 4.77; N, 19.49. Found: C, 66.53; H, 4.57; N, 19.87.
-1
1
(
2
5
1
3
2
), 2.89-2.83 (m, 2H, CH
2
); C NMR ( ppm): 136.3,
1
H
17
N
5
O
2
(359.38):
4
.1.2.3.
carbohydrazide (4c)
Yield 78%; mp 177–179 °C.IR (cm ): 1692, 1664 (CO amide); H NMR ( ppm): 12.60
s, 1H, NHD O exchangeable), 10.87 (s, IH, NH, D O exchangeable), 8.43 (s, IH,
4-oxo-N',1-diphenyl-4,5-dihydro-1H-pyrazolo[3,4-d]
pyrimidine-6-
-1
1
(
2
2

1
3
pyrazole-C
5
-H), 8.29-6.74 (m, 10H, aromatic-H); C NMR (ppm): 136.8, 129.8, 129.2,
27.6, 122.1, 119.5, 113.0. Anal.Calcd for C18 (346.34): C, 62.42; H, 4.07; N,
4.27. Found: C, 62.72; H, 4.25; N, 24.59.
1
2
H
14
N
6
O
2
4
.1.2.4.
S-(-)-4-oxo-1-phenyl-N-(1-phenylethyl)-4,5-dihydro-1H-pyrazolo[3,4-d]
pyrimidine-6-carboxamide (4d)
Yield 83%; mp 160-162 °C.IR (cm- ): 1690, 1664 (CO amide); H NMR ( ppm): 12.37
s, 1H, NH, D O exchangeable), 9.19 (s, IH, NH, D O exchangeable), 8.39 (s, IH,
pyrazole-C -H ), 8.19-7.24 (m, 10H, aromatic-H), 5.16 (q, J= 7.2 Hz,1H, CH), 1.54 (d, J
7.2 Hz, 3H, CH
1
1
(
2
2
5
13
=
3
); C NMR ( ppm): 136.7, 129.8, 128.8, 127.7, 127.5, 126.6, 122.2,
4
6
9.4, 22.3. Anal.Calcd for C20
6.49; H, 4.58; N, 19.17.
H
17
N
5
2
O (359.38): C, 66.84; H, 4.77; N, 19.49. Found: C,
4
.1.2.5.
R-(+)-4-oxo-1-phenyl-N-(1-phenylethyl)-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidine-6-carboxamide (4e)
1
1
Yield 82%; mp 160-162 °C. IR (cm- ): 1690, 1664 (CO amide); H NMR ( ppm): 12.37
s, 1H, NH, D O exchangeable), 9.19 (s, IH, NH, D O exchangeable), 8.39 (s, IH,
pyrazole-C -H ), 8.19-7.24 (m, 10H, aromatic-H), 5.16 (q, J= 7.2 Hz,1H, CH), 1.54 (d, J
7.2 Hz, 3H, CH
(
2
2
5
13
=
3
); C NMR ( ppm): 136.7, 129.8, 128.8, 127.7, 127.5, 126.6, 122.2,
4
6
9.4, 22.3. Anal.Calcd for C20
7.18; H, 5.01; N, 19.85.
H
17
N
5
2
O (359.38): C, 66.84; H, 4.77; N, 19.49. Found: C,

4
.1.2.6.
R/S-(±)-4-oxo-1-phenyl-N-(1-phenylethyl)-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidine-6-carboxamide (4f)
1
1
Yield 85%; mp 160-162 °C.IR (cm- ): 1690, 1664 (CO amide); H NMR ( ppm): 12.37
s, 1H, NH, D O exchangeable), 9.19 (s, IH, NH, D O exchangeable), 8.39 (s, IH,
pyrazole-C -H ), 8.19-7.24 (m, 10H, aromatic-H), 5.16 (q, J= 7.2 Hz,1H, CH), 1.54 (d, J
7.2 Hz, 3H, CH
(
2
2
5
13
=
3
); C NMR ( ppm): 136.7, 129.8, 128.8, 127.7, 127.5, 126.6, 122.2,
4
6
9.4, 22.3. Anal.Calcd for C20
6.45; H, 4.55; N, 19.82.
H
17
N
5
2
O (359.38): C, 66.84; H, 4.77; N, 19.49. Found: C,
4
.2 In vitro cyclooxygenase inhibition assay
The inhibition of the conversion of AA toPGH2 by human recombinant COX-1 and
COX-2 was determined using a COX inhibitor screening assay kit (No. 701230; Cayman
Chemical, USA). The COX human inhibitor screening assay directly measures
Prostaglandin F2alpha (PGF2α) by stannous chloride (SnCl ) reduction of COX-derived
2
PGH2 produced in the COX reaction. The prostanoid product is quantified via enzyme-
linked immunosorbent assay (ELISA) using a broadly specific antiserum that binds to all
the major PG compounds.
Cyclooxygenase inhibitory activity of 3a-d and 4a-f towards COX-1 and COX-2 activity
was performed in accordance to the one recommended by the supplier of the kit.
Celecoxib (Sigma, Germany)served as positive control for COX-2 and Diclofenac
(
Sigma, Germany) for COX-1. The test compounds were dissolved in DMSO for a final

concentration in the reaction tubes of 40 μMin the screening run and 5 different
concentrations which were 10, 20, 40, 80 and 100 μM in theIC50 run.
4
.2.1 AChE Competitive ELISA
This assay is based on the competition between PGs and a PG-acetylcholinesterase
AChE) conjugate (PG tracer) for a limited amount of PG antiserum. Because the
(
concentration of the PG tracer is held constant while the concentration of PG varies, the
amount of PG tracer that is able to bind to the PG antiserum will be inversely
proportional to the concentration of PG in the well. This rabbit antiserum-PG (either free
or tracer) complex binds to a mouse monoclonal anti-rabbit antibody that has been
previously attached to the well. The plate is washed to remove any unbound reagents and
then Ellman’s Reagent (which contains the substrate to AChE) is added to the well. The
yellow product of this enzymatic reaction is determined spectrophotometrically in a
Microplate Reader (Multiskan™ GO Microplate Spectrophotometer) at 415 nm. The
intensity of this color is proportional to the amount of PG tracer bound to the well, which
is inversely proportional to the amount of free PG present in the well during the
incubation; or
Absorbance
∝ [Bound PG Tracer] ∝ 1/[PG].
4
.3. In vivo anti-inflammatory activity
Male Wistar strain albino rats weighing 180–200 g were used throughout the assay. They
were acquired from a closed randombred colony at the College of Agricultural and
Veterinary Medicine, Al Qassim University, Kingdom of Saudi Arabia. Rats were given
ad libitum access to food and water and housed in groups of four in isolated cages under

standard conditions of light and temperature. The animals were acclimatized for 2 weeks
prior to experiments. The investigation conformed to the guide for the Care and Use of
Laboratory Animals published by US National Institute of Health (NIH Publication
No.83-23, revised 1996). The Local Ethics Committee approved this study.
4
.3.1. The carrageenan-induced rat paw edema model (acute inflammatory model)
The methods of Winter et al. [34] and Adeyemi et al. [35] were adopted. A total of
seventeen groups of rats having 5 animals each were used for the study. In the control
group, only the vehicle, 2 % DMSO, was administered. Positive control groups were
treated with diclofenac sodium or celecoxib. All drugs and compounds were given orally
at the dose of 10 mg/kg. After 1 h, the rats were challenged with subcutaneous injection
of 0.1 ml of freshly prepared solution of 1% of carrageenan (Sigma Aldrich, St. Louis,
MO, USA) in sterile 0.9% normal saline was injected into the sub plantar region of the
left hind paw under light ether anesthesia. An equal volume of saline was injected into
the right hind paw and served as internal control for the degree of inflammation in the left
hind paw. The paw edema was measured Plethysmographically (UgoBasile 7150, Varese,
Italy Plethysmograph) and re-measured again 1, 2 and 4 h after injection of carrageenan.
Edema was expressed as an increase in the volume of 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:
%
Inhibition = (Vt-Vo) control-(Vt-Vo) test compound
Vt-Vo) control
x
100
(

Where Vt is the mean volume of edema at specific time interval and Vo is the mean
volume of edema at zero time intervals.
4
.3.2. Determination of effective dose 50 (ED50)
The selected compounds were further tested at 5, 10, 20, 40, and 50 mg/kg body weight
and the ED50 was determined by measuring the inhibition of edema volume 3 h after
carrageenan injection.
4
.3.3.The carrageenan-induced rat paw edema model (sub-acute inflammatory
model)
Rats in the first experiment were given the same test compounds daily for 7 successive
days. A solution of carrageenan (1%, 0.1 ml) was injected into the sub planter region of
the left hind paw under light ether anesthesia 1 h after oral administration of the test
material. A second injection of carrageenan (1%, 0.1 ml) was given on the third day. The
changes in the volume of paw were measured Plethymographically at the first and eighth
days [34, 35].
4
.3.4. Turpentine oil–induced granuloma pouch bioassay
This chronic inflammatory model was performed as previously described [36] and
modified using turpentine oil as irritant [37]. In ether-anaesthetized rats subcutaneously
dorsal granuloma pouch was made by injecting 2 mL of air, followed by injecting 0.5 mL
of turpentine oil into it. As mentioned previously, drugs and compounds were given

orally at the dose of 10 mg/kg, one hour before turpentine oil injection and continued for
seven consecutive days. On the eighth day, the pouch was opened under light ether
anesthesia and the exudates were collected by a syringe. The volume (mL) of the
exudates was measured and the percentage inhibition of inflammation relative to the
control was determined as follows:
%
Inhibition = (V control - V treated)
V control
x
100
4
4
.4. Computational Methods
.4.1. Proteins and ligands preparation
Crystal structures for COX-I and COX-II were downloaded from the protein data bank
(
(
PDB, ID: 3N8Z [38] and 3LN1 [39]) both of which were solved by X-ray diffraction
XRD).Then the co-crystallized ligand and all water molecules were removed from the
protein structure. The protein structure was prepared by MOE using the protein
preparation wizard [40]. This wizard prepares the crystal structure by checking the
protein for any missing atoms, residues or loop; consequently, it performs all the required
corrections. The structure was then processed by the Maestro protein preparation module
to set up partial charges on each atom and protonation states on each ionizable group
[
41]. The co-crystallized ligand from the crystal structures was used to define the binding
site of the COX enzymes then a grid box was created using the Receptor Grid Generation
module in Glide [42]. The ten synthesized compounds were built using the build panel in

MOE then were energy minimized via the Maestro program [42] and the OPLS force
field [43].
4
.4.2. Glide docking
Next, ligands were docked into the previously identified binding site using the Glide
docking tool [42], where the extra-precision (XP) Algorithm [44] was used for
conformational sampling. Next, the resulting poses were given scores via the Glide-XP
scoring function which includes terms for van der Waals, hydrogen bond, electrostatic
interactions, desolvation penalty and intra-ligand contact penalty [44].
5
. Conflict of interest
The authors report no conflicts of interest. The authors alone are responsible for the
contents and writing the paper.
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Table 1. Data obtained from enzyme assay and expressed as means ± SE.
inhibition (40µM)
%
IC50)µM(
Comp. No.
COX-1
12.73
ꢁꢂ.91
2ꢁ.32
ꢂ1.9ꢂ
31.ꢃ9
ꢁ3.ꢀꢃ
7ꢂ.9ꢀ
21.1ꢂ
30.92
3ꢁ.ꢂꢂ
-
COX-2
54.15
79.61
47.76
37.63
19.65
67.69
14.91
91.25
23.50
41.60
96.61
-
COX-1
COX-2
3
3
3
3
4
4
4
4
4
a
b
c
d
a
b
c
d
e
-
ꢀ7.3±9.70
19.45±0.07
13.ꢀ±9.37
-
-
-
-
-
-
26.04±0.36
1ꢀ.ꢀ±9.39
-
-
28.39±0.03
23.8±9.32
-
-
4
f
-
-
Celecoxib
Diclofenac
-
33.2±9.71
0ꢀ.ꢁ1
8.72±0.28
-

Table 2. Anti-inflammatory activity of compounds in the carrageenan-induced rat paw
edema model (acute inflammatory model).
Treatment
Volume of edema
2h
ED50
0
1h
4h
a
Control
1.03±0.01
1.06±0.02
0.90±0.01
1.05±0.02
0.98±0.01
1.05±0.01
1.06±0.01
1.07±0.02
1.05±0.01
1.08±0.01
1.16±0.01
1.21±0.02
2.30±0,02
2.90±0.03
3.75±0.02
b
3
3
3
3
4
4
4
4
4
4
a
b
c
d
a
b
c
d
e
f
1.65±0,01* (53)
1.66±0,01* (40)
1.89±0,02* (33)
1.68±0,01* (44)
2.01±0,01* (24)
1.85±0,01* (37)
1.50±0,01* (66)
1.50±0,01* (64)
1.85±0,01* (39)
1.73±0,02* (55)
1.56±0,01* (72)
1.68±0,01* (51)
1.90±0.01* (55) 2.10±0.02* (61) 8.3
1.80±0.01* (51) 2.11±0.03* (55)
1.95±0.01* (51) 2.32±0.02* (53)
1.88±0.02* (51) 2.03±0.02* (61) 8.1
2.37±0.01* (29) 2.70±0.02* (39)
1.90±0.02* (55) 2.21±0.02* (57)
2.13±0.02* (43) 2.36±0.01* (52)
1.69±0.01* (65) 2.01±0.01* (64) 7.6
2.17±0.01* (41) 2.61±0.02* (43)
1.85±0.01* (63) 2.08±0.01* (66) 7.4
1.79±0.02* (68) 2.03±0.01* (69) 7.2
1.93±0.01* (53) 2.21±0.01* (57)
Celecoxib
Declofenac Na. 1.06±0.01
*
Significantly different compared to corresponding control p ≤ 0.05.
a
b
Number of animals = 5. Values are expressed as Mean ±SEM. Values in parentheses
are percent anti-inflammatory activity. All compounds and drugs are given at the dose of
1
0 mg/kg orally.

Table 3. Anti-inflammatory activity of compounds in the carrageenan-induced rat paw
edema model (sub-acute inflammatory model).
Treatment
Volume of edema
st
th
0
1 day
8 day
a
Control
1.03±0.01
1.06±0.02
0.90±0.01
1.05±0.02
0.98±0.01
1.05±0.01
1.06±0.01
1.07±0.02
1.05±0.01
1.08±0.01
1.16±0.01
1.21±0.02
1.06±0.01
3.75±0.02
4.06±0.03
b
3
3
3
3
4
4
4
4
4
4
a
b
c
d
a
b
c
d
e
f
2.10±0.02* (61)
2.11±0.03* (55)
2.32±0.02* (53)
2.03±0.02* (61)
2.70±0.02* (39)
2.21±0.02* (57)
2.36±0.01* (52)
2.01±0.01* (64)
2.61±0.02* (43)
2.08±0.01* (66)
2.03±0.01* (69)
2.21±0.01* (57)
2.38±0.02* (56)
2.55±0.02* (45)
2.74±0.03* (44)
2.31±0.02* (56)
2.68±0.01* (46)
2.73±0.02* (44)
2.45±0.01* (54)
2.26±0.01* (60)
2.88±0.02* (40)
2.16±0.02* (66)
2.25±0.01* (65)
2.36±0.01* (57)
Celecoxib
Declofenac Na.
*
Significantly different compared to corresponding control p ≤ 0.05.
a
b
Number of animals = 5. Values are expressed as Mean ±SEM. Values in parentheses
are percent of anti-inflammatory activity. All compounds and drugs aregiven at the dose
of 10 mg/kg orally.

Table 4. Anti-inflammatory activity of compoundsin the turpentine oil–induced
granuloma pouch model.
Treatment
Volume of
exudates
% of inhibition
a
Control
2.24±0.02
----------
54
3
3
3
3
4
4
4
4
4
4
a
b
c
d
a
b
c
d
e
f
1.02±0.01*
1.42±0.02*
1.39±0.02*
1.08±0.01*
1.63±0.02*
1.49±0.02*
1.03±0.01*
1.09±0.01*
1.66±0.01*
1.03±0.01*
1.02±0.01*
1.09±0.01*
36
37
51
27
33
54
51
25
54
Celecoxib
54
Declofenac Na.
51
*Significantly different compared to control p ≤ 0.05.
a
Number of animals = 5. Values are expressed as Mean ±SEM.
All compounds and drugs are given at the dose of 10 mg/kg orally.