Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; Design, synthesis and biological evaluation as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejps.2014.05.025

A novel set of 4-substituted-1-phenyl-pyrazolo[3,4-d]pyrimidine and 5-substituted-1-phenyl-pyrazolo[3,4-d]pyrimidin-4-one derivatives were synthesized and evaluated as potential anti-inflammatory agents. The newly prepared compounds were assessed through the examination of their in vitro inhibition of four targets; cyclooxygenases subtypes (COX-1 and COX-2), inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB). Compounds 8a, 10c and 13c were the most potent and selective ligands against COX-2 with inhibition percentages of 79.6%, 78.7% and 78.9% at a concentration of 2 μM respectively, while compound 13c significantly inhibited both COX subtypes. On the other hand, fourteen compounds showed high iNOS inhibitory activities with IC₅₀ values in the range of 0.22-8.5 μM where the urea derivative 11 was the most active compound with IC₅₀ value of 0.22 μM. Most of the tested compounds were found to be devoid of inhibitory activity against NF-kB. Moreover, almost all compounds were not cytotoxic, (up to 25 μg/ml), against a panel of normal and cancer cell lines. The in silico docking results were in agreement with the in vitro inhibitory activities against COXs and iNOS enzymes. The results of in vivo anti-inflammatory and antinociceptive studies were consistent with that of in vitro studies which confirmed that compounds 8a, 10c and 13c have significant anti-inflammatory and analgesic activities comparable to that of the control, ketorolac. Taken together, dual inhibition of COXs and iNOS with novel pyrazolopyrimidine derivatives is a valid strategy for the development of anti-inflammatory/ analgesic agents with the probability of fewer side effects.

Full text of DOI:10.1016/j.ejps.2014.05.025

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Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
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Novel pyrazolopyrimidine derivatives targeting COXs and iNOS
enzymes; design, synthesis and biological evaluation as potential
anti-inflammatory agents
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Ahmed H. Abdelazeem a, Shaimaa A. Abdelatef a, Mohammed T. El-Saadi a, Hany A. Omar b,
8 Q1
Shabana I. Khan c, Christopher R. McCurdy d, Samir M. El-Moghazy e,
⇑
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a Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
b Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
c National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, MS 38677, USA
d Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi, MS 38677, USA
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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A novel set of 4-substituted-1-phenyl-pyrazolo[3,4-d]pyrimidine and 5-substituted-1-phenyl-pyrazolo
[3,4-d]pyrimidin-4-one derivatives were synthesized and evaluated as potential anti-inflammatory agents.
The newly prepared compounds were assessed through the examination of their in vitro inhibition of four
targets; cyclooxygenases subtypes (COX-1 and COX-2), inducible nitric oxide synthase (iNOS) and nuclear
Received 25 March 2014
Received in revised form 7 May 2014
Accepted 27 May 2014
Available online xxxx
factor kappa B (NF-
COX-2 with inhibition percentages of 79.6%, 78.7% and 78.9% at a concentration of 2
compound 13c significantly inhibited both COX subtypes. On the other hand, fourteen compounds showed
high iNOS inhibitory activities with IC50 values in the range of 0.22–8.5 M where the urea derivative 11
was the most active compound with IC50 value of 0.22 M. Most of the tested compounds were found to
be devoid of inhibitory activity against NF-kB. Moreover, almost all compounds were not cytotoxic, (up
to 25 g/ml), against a panel of normal and cancer cell lines. The in silico docking results were in agreement
j
B). Compounds 8a, 10c and 13c were the most potent and selective ligands against
l
M respectively, while
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Keywords:
Anti-inflammatory
Analgesic
COX
iNOS
NF-jB
l
l
l
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Pyrazolopyrimidines
with the in vitro inhibitory activities against COXs and iNOS enzymes. The results of in vivo anti-
inflammatory and antinociceptive studies were consistent with that of in vitro studies which confirmed that
compounds 8a, 10c and 13c have significant anti-inflammatory and analgesic activities comparable to that
of the control, ketorolac. Taken together, dual inhibition of COXs and iNOS with novel pyrazolopyrimidine
derivatives is a valid strategy for the development of anti-inflammatory/analgesic agents with the
probability of fewer side effects.
Ó 2014 Published by Elsevier B.V.
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1. Introduction
(Ialenti et al., 1992; Ianaro et al., 1994; Salvemini et al., 1995;
Seibert et al., 1994). PGE2 is an important prostaglandin, which
plays a crucial role in regulation of vascular permeability, platelet
aggregation, and thrombus formation through the progression of
inflammation.
Currently, it is well documented that there are, at least two
designated COX isoforms; COX-1 and COX-2 (Dannhardt and
Kiefer, 2001; Gund and Shen, 1977). COX-1 is the constitutive
isoform which is widely expressed in most tissues and it is respon-
sible for the synthesis of cytoprotective PGs in GIT, maintenance of
normal renal function and the biosynthesis of pro-aggregatory
TXA2 in blood platelets (Almansa et al., 2003; Yoshimura et al.,
2011). The inducible COX-2 is the second isoform which is promi-
nent at sites of inflammation and it is rapidly induced in response
to mitogenic and proinflammatory stimuli. COX-2 significantly
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Over the past several decades, the quest to discover novel rem-
edies for inflammation has resulted in new chemical entities with
more improved therapeutic efficacy and lower toxicity than the
existing anti-inflammatory drugs. It is well-documented that the
inflammation process involves a sequential activation of signaling
molecules, proinflammatory mediators, such as prostaglandins
(PGs) and nitric oxide (NO) which are generated by cyclooxygenas-
es (COXs) and nitric oxide synthase (iNOS) respectively
⇑
Corresponding author. Address: Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Cairo University, Kasr-El-Eini Street, Cairo 11562, Egypt.
Tel.: +20 002 01223662720.
E-mail address: drsamirelmoghazy@yahoo.com (S.M. El-Moghazy).
http://dx.doi.org/10.1016/j.ejps.2014.05.025
0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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contributes in the production of inflammatory PGs (Peri et al.,
1995). Traditional non-steroidal anti-inflammatory drugs (NSAIDs)
which inhibit both COX subtypes are still the most commonly used
medications for inflammation and pain (Salgin-Goksen et al.,
2007). However, it is well known that NSAIDs have several serious
side effects such as gastric ulceration, renal injury and cardiotoxic-
ity (Allison et al., 1992; Wolfe et al., 1999). To limit the risk of GI
damage induced by NSAIDs, highly selective COX-2 inhibitors
(coxibs) have been developed where they exhibited equivalent
anti-inflammatory/analgesic activities to those of non-selective
COX inhibitors but with least GI toxicity (Micklewright et al.,
2003).
and comparable activity with celecoxib at a dose of 25 mg/kg,
Fig. 1 (Yewale et al., 2012). Moreover, the pyrazolopyrimidine
derivative 2 was reported to inhibit selectively and potently
COX-2 activity in human monocytes (IC50 = 0.9 nM for COX-2 vs.
IC50 = 59.6 nM for COX-1) with anti-angiogenic activity as well.(-
Devesa et al., 2004; Quintela et al., 2003) Raffa et al. prepared
and evaluated a set of 5-benzamido-1H-pyrazolo[3,4-d]pyrimi-
din-4-ones as anti-inflammatory agents. Compound 3 revealed a
better inhibitory profile against COX-2 than that of reference com-
pounds N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphona-
mide (NS398) and indomethacin (Raffa et al., 2009). Furthermore,
the piperazine ring system is a common core template in various
pharmacologically active compounds toward several receptors,
especially G-coupled receptors, such as 5-HT and sigma receptor
subtypes (Mesangeau et al., 2008; Seminerio et al., 2012). Also, it
was found that some compounds containing piperazine core such
as compound 4 display good anti-inflammatory and analgesic
activity, Fig. 1 (Gökhan et al., 1996; Viaud et al., 1995).
Based on the aforementioned studies and the substantial need
for superior anti-inflammatory compounds devoid of the classical
NSAIDs liabilities, we have undertaken the synthesis of novel pyr-
azolopyrimidine derivatives as COXs and iNOS dual inhibitors with
the hope of realizing compounds with improved anti-inflamma-
tory/analgesic activity and diminished side effects. Our strategy
to accomplish these goals was to tether the pyrazolopyrimidine
system to various piperazine derivatives or sulfonamides at
positions 4 and 5 utilizing a fragment-based drug design approach.
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Furthermore, the inducible transcription factor (NF-kB) regu-
lates the expression of several genes such as iNOS, COX-2, and
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TNF-a that are involved in inflammatory responses at the tran-
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scriptional level (Ghosh and Karin, 2002). Meanwhile, iNOS is
responsible for overproduction of the endogenous free radical
nitric oxide (NO) in the inflammation site. This free radical is an
important mediator in the process of vasodilation, nonspecific host
defense, and acute or chronic inflammation causing tissue injuries
(Cirino et al., 2006; Clancy et al., 1998; MacMicking et al., 1997).
Therefore, it has been proposed that a combined inhibition of
COX-2 and iNOS would be a useful and proper strategy for the
treatment of inflammatory diseases by suppression of the overpro-
duction of PGE2 and NO respectively (Lim et al., 2009; Ma et al.,
2011).
The pyrazolopyrimidine fused heterocyclic system is a fre-
quently found scaffold in a wide variety of bioactive molecules
including antivirals (Chern et al., 2004; El-Bendary and Badria,
2000), antimicrobials (Abunada et al., 2008; Bakavoli et al., 2010;
Bondock et al., 2008), anticancer (Celano et al., 2008; Spreafico
et al., 2008), antileukemic (Cottam et al., 1984), CNS agents
(Chen, 2000), tuberculostatic (Trivedi et al., 2010, 2012), antileish-
manials (Looker et al., 1986), anticardiovascular diseases
(Guccionea et al., 1996; Xia et al., 1997) and anti-inflammatory
agents (Rathod et al., 2005; Yewale et al., 2012). Recently, Yewale
et al. synthesized and evaluated a novel series of 3-substituted-1-
aryl-5-phenyl-6-anilinopyrazolo[3,4-d]pyrimidin-4-ones as poten-
tial anti-inflammatory agents. Compound 1 exhibited superior
anti-inflammatory activity in comparison with diclofenac sodium
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2. Results and discussion
2.1. Chemistry
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All the target compounds were prepared according to the
synthetic pathways outlined in Schemes 1–3. The synthesis of
the new pyrazolopyrimidine derivatives proceeded through the
general intermediate, 5-amino-1-phenyl-4-cyanopyrazole (5)
which was obtained through the reaction of phenylhydrazine with
commercially available ethoxymethylenemalononitrile in alcohol
with a good yield (Cheng and Robins, 1956). The preparation of
1-phenyl-pyrazolo[3,4-d]pyrimidin-4-one (6) was accomplished
by heating the starting material 5 with formic acid which, in turn
was N-alkylated with excess dibromoethane in the presence of
sodium hydride as a base to give intermediate 7. The coupling reac-
tion of the bromo intermediate 7 with various primary and second-
ary amines upon heating in DMF and potassium carbonate afforded
the final compounds 8a–f. The chloro derivative 8g was formed via
the treatment of compound 8f with excess thionyl chloride in
chloroform, as shown in Scheme 1.
On the other hand, heating of compound 5 with formamide in
ethanol gave the corresponding 1-phenyl-pyrazolo[3,4-d]pyrimi-
din-4-amine (9) which was subsequently treated with various
aldehydes through reductive amination using sodium triacetoxy-
borohydride in dichloroethane to afford the target compounds
10a–c. In addition, condensation of compound 9 with phenylisocy-
anate in DCM provided the desired urea derivative 11, in a high
yield. The treatment of 1-phenyl-pyrazolo[3,4-d]pyrimidin-4-one
(6) with phosphorus oxychloride in dimethyl formamide yielded
4-chloro-1-phenyl-pyrazolo[3,4-d]pyrimidine 12 which then was
utilized for the synthesis of pyrazolopyrimidine derivatives 13a-
m by nucleophilic displacement of the chlorine atom with various
piperazine derivatives and other amines, as shown in Scheme 2. It
should be mentioned that 13c was reported earlier as a Trp-p8
modulator and it had been synthesized by a different procedure
(Natarajan et al., 2005). Compound 13l was selected for further
structural modification to study the effect of incorporating new
Fig. 1. Chemical structures of some reported pyrazolo[3,4-d]pyrimidine and
piperazine derivatives with anti-inflammatory potential (Devesa et al., 2004;
Gökhan et al., 1996; Quintela et al., 2003; Raffa et al., 2009; Viaud et al., 1995;
Yewale et al., 2012).
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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Scheme 1. aReagents and reaction conditions: (a) ethanol, rt, overnight; (b) HCOOH, reflux, 6 h; (c) dibromoethane, NaH, DMF, 0 °C - rt; (d) appropriate amine, K2CO3, DMF,
60 °C; (e) SOCl2, CHCl3, reflux, 2 h.
Scheme 2. aReagents and reaction conditions: (a) formamide, reflux, 4 h; (b) appropriate aldehyde, sodium triacetoxyborohydride, DCE, rt, overnight; (c) phenylisocyanate,
DCM, rt, overnight; (d) HCOOH, reflux, 6 h; (e) POCl5, DMF, reflux, 2 h; (f) appropriate amine, Ethanol, reflux, 2 h.
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groups such as gem-difluoro or increasing its size on the activity.
The introduction of the gem-difluoro substituent in 1-(1-phenyl-
pyrazolo[3,4-d]pyrimidin-4-yl) piperidin-4-one 13l was achieved
by treatment of 13l with deoxofluor in DCM at 0 °C to afford com-
pound 14 (Robichaud et al., 2008). Meanwhile, the condensation
reaction of 13l with o-hydroxyacetophenone proceeded in metha-
nol and was mediated by pyrrolidine afforded the spiro derivative
15. Subsequent reduction of 13l with sodium borohydride in meth-
anol and reductive amination (Abdel-Magid et al., 1996) with sul-
fanilamide using sodium triacetoxyborohydride in dichloroethane
gave the corresponding target compounds 17 and 16 respectively
depicted in Scheme 3.
their inhibitory activities on three targets; COXs, iNOS and NF-
B. The results of iNOS and NF- inhibitory assays were
expressed in terms of IC50 values (the concentration that caused
a 50% inhibition) while the inhibition of COX-1 and COX-2 was
expressed in terms of% inhibition at 2 lM as displayed in Table
1. In addition, the cytotoxicity of all the compounds was deter-
mined against a panel of normal and cancerous mammalian cell
lines. Based on the in vitro assays results, the most active eight
compounds were selected to be tested for their in vivo efficacy.
Three in vivo measures were utilized; the carrageenan-induced
paw hyperalgesia assay was used for testing the anti-hyperalgesia,
the carrageenan-induced paw edema assay was used for assess-
ment of the anti-inflammatory activity and finally, acetic acid-
induced writhing assay was used for evaluating the anti-nocicep-
tion potential.
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jB
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2.2. Pharmacology
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The newly synthesized pyrazolopyrimidine derivatives were
evaluated through in vitro and in vivo assays for their anti-
inflammatory and analgesic potentials. All compounds were ini-
tially underwent in vitro screening in cellular assays to examine
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2.2.1. Inhibition of COX subtypes activity
Thirteen representative compounds were selected to be tested
for their ability to inhibit COX-1 and COX-2 enzyme activity. The
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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Scheme 3. aReagents and reaction conditions: (a) deoxofluor, DCM, 0 - rt °C, overnight; (b) 2-hydroxyacetophenone, pyrrolidine, reflux, 6 h; (c) sulfanilamide, sodium
triacetoxyborohydride, DCE, rt, overnight; (d) NaBH4, MeOH, 0 °C, 2 h.
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results revealed that compounds 8a, 10c and 13c were the most
potent and the most selective ligands against COX-2 with inhibi-
tion percentages of 79.6%, 78.7% and 78.9%, at a concentration of
0.22
pound 10c, was well tolerated inside the extended iNOS binding
site and it exerted high activity with IC50 value of 0.3 M. In con-
trast, the less bulky compound 10a and the nitro derivative 10b
were inactive. For piperazine derivatives, it was observed that
the phenylpiperazine derivatives bearing an electron withdrawing
group were more active than those containing electron donating
moieties. Compounds 13c, 13e, 13h and 13j were active ligands
lM. The introduction of a bulky phenoxybenzyl, as in com-
l
2
lM respectively. On the other hand, compounds 8e, 13e, 13m,
15 and 16 showed moderate activity against COX-2 with inhibition
percentages of 42.9%, 24.9%, 38.9%, 43% and 33%, respectively. On
the contrary, compounds 8d and 13a exhibited a good selectivity
against COX-1 with moderate inhibition percentages of 28.7%
and 22.4%, respectively. Moreover, compound 13b was non-selec-
tive ligand that could inhibit both COX subtypes with moderate
inhibition percentages of 43.1% for COX-1 and 37.5% for COX-2
respectively. It could be suggested that the sulfonamide moiety
in compounds, 8a, 13n and 16 may largely contribute to their
COX-2 selectivity. However, compounds 8e, 10c, 13c, 15 and 13e
have COX-2 selectivity and they do not incorporate a sulfamoyl
(ASO2NH2) moiety in their structures. This finding could affirm
that the importance of sulfonamide as a structural basis for COX-
2 selectivity is still controversial. Interestingly, the 4-substituted
pyrazolopyrimidines (Scaffold B) were more active than the 5-
substituted derivatives (Scaffold A). The structure activity relation-
ship for the whole series needs further studies to be explained in
details. However, in silico docking studies confirmed the main
structural features required for activity and selectivity against both
COX subtypes.
with IC50 values of 0.4, 0.97, 2.8 and 2.1 lM respectively. On the
other hand, compounds 13b, 13d, 13f, 13i and 13k were less active
or devoid of any iNOS inhibitory activity. In addition, the piperidin-
4-one derivative 13l was more active than the piperidin-4-ol deriv-
ative 17 with intermediate IC50 values. Despite of the bulkiness of
the spiro derivative 15, it showed no activity which confirmed the
size of the ligand is crucial for activity. Finally, the gem-difluoro
derivative 14 possessed moderate activity with IC50 value of
3.8
l
M while sulfonamide compounds 13m and 16 were inactive.
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2.2.3. Inhibition of NF-
jB transcriptional activity
All compounds were devoid of any activity against NF-kB except
compounds 8e and 13d which exhibited moderate inhibition with
IC50 values of 5.3 and 2.8 lM respectively.
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2.2.4. Cytotoxicity
All novel pyrazolopyrimidine derivatives were evaluated for
cytotoxicity against a panel of four human solid tumor cell lines
(SK-MEL: malignant melanoma; KB: oral epidermal carcinoma;
BT-549: breast ductal carcinoma, and SK-OV-3: ovary carcinoma)
as well as and two normal kidney cell lines (Vero: monkey kidney
fibroblasts and LLC-PK1: pig kidney epithelial cells). In addition, all
compounds were tested against Mouse leukemic monocyte
macrophage cells (RAW 264.7) to determine whether their iNOS
inhibitory activity was related to a decrease in cell viability due
to the toxicity of tested compounds. Most of the compounds were
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2.2.2. Inhibition of iNOS activity
The iNOS inhibitory assay was performed in LPS-induced mouse
macrophages (RAW264.7) where the concentration of NO was
determined by measuring the level of nitrite in the cell culture
supernatant using Griess reagent. Parthenolide was used as positive
control. It was found that most of the newly synthesized pyrazol-
opyrimidines exerted good to moderate inhibitory activities with
IC50 values between 0.22 and 8.5 lM. In general, it was noticed
that the substitution at position 5 was more favorable for the iNOS
activity than the substitution at position 4 of the pyrazolopyrimi-
dine scaffold. Compounds 8a–8g, except compounds 8b and 8f,
showed remarkable activity with IC50 values between 0.24 and
not cytotoxic up to a concentration of 25 lg/mL and do not seem to
have any significant anti-cell proliferative activity toward cancer
cells or any observable cytotoxic effect toward the normal cells
except compound 13a which showed cytotoxicity in LLC-PK1 cells
0.71 lM. It was apparent that replacement of the azepine moiety
at high concentrations (IC50 values 42.35 and 37.83
tively). The results are shown in Table 2.
lM, respec-
with the smaller pyrrolidine, abolished the activity in compound
8b. On the other hand, the replacement of the electron withdraw-
ing group (chloride) in compound 8g with a hydroxyl group in
compound 8f abolished the activity as well. Regarding the substi-
tution at position 4 of the pyrazolopyrimidine nucleus, the urea
derivative 11 was the most active derivative with IC50 value of
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2.2.5. Carrageenan-induced rat paw edema assay
The assessment of anti-inflammatory activity of the selected
eight compounds was performed using carrageenan-induced rat
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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Table 1
Inhibition of COX-1, COX-2, iNOS and NF-kB by the newly synthesized pyrazolopyrimidine derivatives.
Compound
Scaffold
R
% Inhibition at 2
COX-1
lM
IC50 (lM)
COX-2
iNOS
NF-kB
7
8a
A
A
Br
ND
NA
ND
79.60
0.78
0.34
NA
NA
8b
8c
A
A
ND
ND
ND
ND
NA
NA
NA
0.27
8d
8e
A
A
28.70
NA
NA
0.71
0.38
NA
5.3
42.90
8g
A
B
ND
ND
ND
ND
0.24
ND
NA
ND
10a
10b
10c
B
B
ND
NA
ND
5.2
NA
NA
78.70
0.30
11
B
NA
NA
0.22
NA
13a
13b
B
B
22.40
43.10
2.30
NA
4.4
NA
NA
37.50
13c
13d
13e
13f
B
B
B
B
NA
ND
NA
ND
78.90
ND
0.40
NA
NA
2.8
NA
NA
24.90
ND
0.97
NA
13g
B
ND
ND
NA
NA
13h
13i
13j
B
B
B
ND
ND
NA
ND
ND
NA
2.8
NA
2.1
NA
NA
NA
(continued on next page)
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biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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Table 1 (continued)
Compound
Scaffold
R
% Inhibition at 2
lM
IC50 (lM)
COX-1
ND
COX-2
ND
iNOS
NA
NF-kB
NA
13k
13l
B
B
B
B
NA
NA
ND
NA
6.3
NA
3.8
NA
NA
NA
13m
14
38.9
ND
15
16
B
B
14.90
NA
43.00
33.00
NA
NA
NA
NA
17
B
ND
ND
8.5
–
NA
Indomethacin
Parthenolide
–
–
–
–
92.10
–
24.60
–
–
0.02
0.015
Inhibition of COX-1 and COX-2 is represented in terms of % inhibition at 2
l
M. Inhibition of iNOS and NF-kB is represented in terms of IC50 values obtained from dose response
curves. NA = No activity was observed up to 50
lM. ND = Not determined. Positive controls: indomethacin for COX inhibition and Parthenolide for iNOS and NF-jB
inhibition.
Table 2
In vitro cytotoxicity of newly synthesized pyrazolopyrimidine derivatives.
Compound
IC50 values
SK-MEL
lM
KB
BT-549
SK-OV-3
VERO
LLC-PK11
RAW 264.7
7
8a-g
10a-c
13a
13b-m
14
NC
NC
NC
NC
NC
NC
NC
NC
–
NC
NC
NC
NC
NC
NC
NC
NC
2.75
NC
NC
NC
NC
NC
NC
NC
NC
2.00
NC
NC
NC
NC
NC
NC
NC
NC
2.57
NC
NC
NC
NC
NC
NC
NC
NC
12.9
NC
NC
NC
37.83
NC
NC
NC
NC
2.20
NC
NC
NC
NC
NC
NC
NC
NC
ND
15
17
Doxorubicin
Cytotoxicity was determined in a panel of cell lines which included human solid tumor cell lines [SK-MEL (Melanoma), KB (epidermal carcinoma), BT-549 (breast carcinoma)
and SK-OV-3 (ovarian carcinoma)], normal kidney cell lines [Vero (Monkey kidney fibroblast) and LLC-PK1 (Pig kidney epithelial cells)] and Mouse leukemic monocyte
macrophage cells (RAW 264.7).
Doxorubicin for was used as positive control in cytotoxicity assay.
NC = No cytotoxicity.
299
300
301
302
303
304
305
306
307
308
309
310
311
paw edema model using ketorolac as a reference anti-inflamma-
tory drug. Mean changes in paw edema thickness of animals pre-
contrary, the least potent compounds were 8c and 10b which
showed no activity in vitro against COX subtypes although they
showed considerable inhibition of iNOS.
treated with the test compounds after 1,
3 and 6 h from
induction of inflammation is shown in Fig. 2. The results were in
accordance with the in vitro results of the compounds in both
COX and iNOS assays where compounds 8a, 10c and 13c showed
the most potent anti-inflammatory activities compared to ketoro-
lac. However, compounds 13b and 13m displayed moderate anti-
inflammatory activities and this could be explained by their weak
to moderate activities on both COXs and iNOS targets. On the
312
313
314
315
316
317
2.2.6. Carrageenan-induced thermal hyperalgesia assay
To evaluate the analgesic activity of eight compounds from the
newly synthesized pyrazolopyrimidine derivatives, carrageenan-
induced thermal hyperalgesia model was used. Ketorolac was used
as a reference analgesic drug. Fig. 3 shows the withdrawal latency
of the test compounds compared to vehicle-treated animals at the
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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7
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2.3. Docking studies
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
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381
382
383
To shed light into the required structural features for the activ-
ity and the proper binding mode of the newly synthesized pyrazol-
opyrimidine ligands, some of these compounds were docked into
the active sites of both COX subtypes and iNOS by using LIGANDFIT
embedded in Discovery Studio software (Accelrys Software Inc.,
2007). The protein crystal structures of COX-1, COX-2 and iNOS
complexed with their cocrystallized inhibitors (PDB codes: 1PGF,
1CX2 and 3E65, respectively) were selected for this study. It was
reported that the substitution of Ile523 in COX-1 with the less
bulky Val523 in COX-2 creates an additional polar side pocket
and increases the volume of the COX-2 active site that makes it
accommodate more bulky structures (Kurumbail et al., 1996).
The presence of such side pocket allows additional interactions
with amino acids such as Arg513, replaced by a His513 in COX-
1. The structure of traditional COX-2 inhibitors exploits binding
with Arg513 in the COX-2 side-pocket, often via sulfone or sulfon-
amide groups, to accomplish their selectivity.
In this regard, we examined the docking results of two of the
most COX-2 active newly synthesized pyrazolopyrimidines, 8a
and 10c, into COX-2 active sites. It was clear that the binding mode
and interactions of our compounds were similar with the cocrys-
tallized bromocelecoxib, SC-558 ligand. Clearly from Fig. 5(A),
positioning the sulfonamide moiety of 8a within the side pocket
of COX-2 and forming H-bond with NH of Arg513 residue was sim-
ilar to fitting of same moiety of SC-558 into the same side pocket in
addition to forming two H-bonds with NHs of Arg513 and His90
residues, Fig. 5(C). Interestingly, the side pocket of COX-2 could
accommodate the phenoxy ring of 10c which showed a similar
binding pattern and orientation within the entire COX-2 active site.
In addition, 10c phenoxy formed an H-bonding interaction with
Arg513 residue which is important for COX-2 selectivity,
Fig. 5(B). Furthermore, positioning the phenyl ring attached to
the pyrazolopyrimidine nucleus in both 8a and 10c within the
hydrophobic pocket of the side chains of Tyr348 and Trp387, cor-
relates well with the position of the bromophenyl fragment of
SC-558 into the same pocket via p-stacking interactions, Fig. 5.
On the other hand, docking of 8d and 13a revealed that both com-
pounds were forced to adopt another binding position within the
long COX-1 binding pocket due to presence of Ile523 which pre-
vented the formation of the side pocket and pushed the com-
pounds to the bottom of the active site. Fig. 6 shows the similar
analogy between the aromatic hydrophobic stacking of the phenyl
ring in 8d and 13a with the aromatic residues in Trp387 and
Tyr385 and the iodophenyl in the IMM cocrystallized ligand with
the same residues. We can also notice the H-bonding interaction
Fig. 2. Results of carrageenan-induced rat paw edema assay. Data are mean SD
(N = 6–8). ꢀP < 0.05, #P < 0.01 represents significant change in paw volume changes
compared to vehicle-treated group.
318
319
320
321
322
323
324
325
326
327
indicated time points. All the tested compounds, except 8c and
10b, showed variable analgesic activities. Compounds 8a, 10c
and 13c showed potent analgesic activities compared to ketorolac
while compounds 13b and 13m displayed moderate analgesic
activities. It was noteworthy that these results were consistent
with the in vitro inhibitory values of the same compounds on both
COX and iNOS where compounds 8a, 10c and 13c showed the high-
est COX-2 inhibition percentages of 79.60, 78.70 and 78.90%
respectively. Also, these three compounds have good iNOS inhibi-
tory IC50 values of 0.34, 0.30, 0.40 lM respectively.
328
329
330
331
332
333
334
335
336
337
2.2.7. Acetic acid-induced writhing test
Test compounds were further investigated for their analgesic
activity using the acetic acid-induced writhing test in mice. Ketor-
olac was used as a reference standard analgesic drug. Four com-
pounds (13b, 13c, 8a, 10c) exhibited relatively good analgesic
activity compared to the used standard analgesic, ketorolac. The
most active compound was 13c which also has a good activity
against both COX-2 and iNOS as mentioned previously. The effects
of the drug probes on the acetic acid abdominal writhing assay are
summarized in Fig. 4.
Fig. 3. Results of carrageenan-induced thermal hyperalgesia assay. Data are
mean SD (N = 6–8). ⁄P < 0.05, #P < 0.01 represents significant change in paw
withdrawal latency changes compared to vehicle-treated group.
Fig. 4. Results of acetic acid-induced writhing test. Data are mean SD (N = 6–8).
ꢀP < 0.05, #P < 0.01 represents significant change in number of writhes compared to
vehicle-treated group.
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

PHASCI 3032
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A.H. Abdelazeem et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
Fig. 5. (A) Docking and binding mode of compound 8a into COX-2 active site (PDB code: 1CX2). (B) Docking and binding mode of compound 10c into the same COX-2 binding
pocket. (C) X-ray crystallographic structure of bromocelecoxib analog, SC-558 co-crystallized within COX-2 active site (PDB code: 1CX2). (D) The superposition of the docked
poses 8a (green) and the co-crystallized SC-558 (blue) within active site of COX-2. Hydrogen bonds are represented by dashed green lines. All hydrogens are removed for the
purposes of clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
384
385
386
387
388
389
390
391
392
393
394
395
396
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400
401
402
403
404
405
of the oxygen in 13a with Arg120 which is essential for COX-1
affinity and selectivity.
Similarly, the active compounds 10c and 11 were docked into
the active site of the X-ray structure of iNOS complexed with the
selective inhibitor AR-C120011 (pdb code: 3E65). The two com-
pounds adopted a position and orientation similar to that of the
selective spiro inhibitor, AR-C120011, Fig. 8. The reported extend-
ing pocket exposed from rotations of Gln257, Arg260 and other
residues (Garcin et al., 2008) can explain the accommodation of
iNOS binding site for bulky phenoxy fragment of 10c and the phe-
nyl ring of 11 and their enhanced inhibitory activity. Apparently,
the two docked compounds showed a common critical hydropho-
bic stacking interaction between the phenyl ring linked to the
Interestingly, docking of compound 13b into both COX-1 and
COX-2 active sites explained the non-selectivity and affinity of this
compound for both COX subtypes where it could successfully bind
to both active sites, Fig. 7. As mentioned, the absence of the side
pocket in COX-1 forced the compound to have a longitudinal bind-
ing pattern similar to the former selective compounds 8d and 13a.
On the contrary, the COX-2 side corridor accommodated the bulky
hexylpiperazine fragment by forming an additional H-bond with
Tyr355 residue and an electrostatic interaction with Arg513.
Fig. 6. (A) Docking and binding mode of compound 8d into COX-1 active site (PDB code: 1PGF). (B) Docking and binding mode of compound 13a into the same COX-1 binding
pocket. (C) The superposition of the docked poses AH-6 (green) and the co-crystallized Iodoindomethacin, IMM (blue) within active site of COX-1. Hydrogen bonds are
represented by dashed green lines. All hydrogens are removed for the purposes of clarity. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

PHASCI 3032
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9
Fig. 7. (A) Docking and binding mode of compound 13b into COX-1 active site (PDB code: 1PGF). (B) Docking and binding mode of the same compound into COX-2 binding
pocket (PDB code: 1CX2). Hydrogen bonds are represented by dashed green lines. All hydrogens are removed for the purposes of clarity. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. (A) Docking and binding mode of compound 11 into iNOS active site (PDB code: 3E65). (B) Docking and binding mode of compound 10c into the same iNOS binding
pocket. (C) The superposition of the docked poses 10c (green) and the co-crystallized spiro ligand, AR-C120011 (blue) within the active site of iNOS. Hydrogen bonds are
represented by dashed green lines. All hydrogens are removed for the purposes of clarity. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
406
407
408
409
410
411
412
413
414
415
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
pyrazole moiety in 10c and phenyl ring attached to the urea group
in 11 and heme within the binding pocket of iNOS. An additional
hydrogen bond tethered the nitrogen of the pyrimidine heterocycle
in 11 to Gln257 amino acid, as shown in Fig. 8(A). It could also be
appreciated from Fig. 8(B) how compound 10c formed two hydro-
gen-bonding interactions with Glu371 and Tyr341 residue as well.
These results indicated that hydrophobic aromatic stacking, as well
as hydrogen bonds within the active sites of COXs and iNOS signif-
icantly contribute to the binding modes of these compounds and
their activity.
Meanwhile, most of the tested compounds were devoid of inhibitory
activity against NF-kB except compounds 8e and 13d which showed
moderate inhibition with IC50 values of 5.3 and 2.8
lM respectively.
In addition, most of the compounds were not cytotoxic, up to 25
lg/
ml, on a panel of normal and cancer cell lines as well. Interestingly,
the docking results were in agreement with that of the inhibitory
activity against the COX subtypes and iNOS enzymes where the
important structural features and proper orientations and binding
modes required for activity were determined. In vivo anti-
inflammatory and analgesic studies revealed that compounds 8a,
10c and 13c have significant anti-inflammatory and analgesic activ-
ities comparable to that of ketorolac. It was noticed that the results
of in vivo studies were consistent with that of in vitro COXs and iNOS
assays. In conclusion, compounds 8a, 10c and 13c showed a dual
inhibition for both COX-2 and iNOS which appears to be a valid
strategy for further development of novel anti-inflammatory drugs.
416
3. Conclusion
417
418
419
420
421
422
423
424
425
426
427
428
429
In this study, two sets of compounds from 4-substituted-1-
phenyl-pyrazolo[3,4-d]pyrimidine and 5-substituted-1-phenyl-
pyrazolo[3,4-d]pyrimidin-4-one were synthesized and evaluated
for their anti-inflammatory potential. The newly synthesized
compounds were screened in vitro against four anti-inflammatory
targets; COX subtypes (COX-1 and COX-2), iNOS and NF-kB. In COXs
inhibitory assays, three compounds 8a, 10c and 13c showed signif-
icant inhibition and good selectivity against COX-2. On the contrary,
compounds 8d and 13a exhibited good selectivity against COX-1
446
447
4. Experimental protocols
4.1. Chemistry
448
449
450
451
452
Reagents and starting materials were obtained from commer-
cial suppliers and were used without purification. Precoated silica
gel GF Uniplates from Analtech were used for thin-layer chroma-
tography (TLC). Column chromatography was done on silica gel
60 (Sorbent Technologies). Melting points (m.p.) were uncorrected
with moderate inhibition. Moreover, compound 13c was
a
non-selective ligand against both COXs. On the other hand, com-
pounds 8c, 8g, 10c and 11 were the most active ligands against iNOS
with IC50 values of 0.27, 0.24, 0.30 and 0.22 lM respectively.
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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513
514
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521
522
523
524
525
526
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531
532
533
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540
541
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and were carried out by open capillary tube method using IA
9100MK-Digital Melting Point Apparatus. 1H and 13C NMR spectra
were obtained on a Bruker APX400 at 400 and 100 MHz, respec-
tively. Chemical shifts were reported on the d scale and were
related to that of the solvent and J values are given in Hz. The mass
spectra (MS) were recorded on a Waters Acquity Ultra Performance
LC with ZQ detector in ESI mode. The infrared spectra IR were
obtained from PerkinElmer Spectrum 100FT-IR Spectrometer. Ele-
mental analyses (C, H, N) were recorded on an elemental analyzer,
Perkin-Elmer CHN/SO series II Analyzer. Chemical names were
generated using ChemDraw Ultra (Cambridge Soft, version 11.0.1).
5-Amino-1-phenyl-1H-pyrazole-4-carbonitrile (5). Ethoxymeth-
ylenemalononitrile (20.3 gm, 0.17 mol) was added slowly with
shaking to phenylhydrazine (18 gm, 0.17 mol) in 40 mL absolute
ethanol at room temperature. After the addition was completed,
the solution was carefully heated to boiling for about one hour.
The reaction mixture was then set aside overnight in the refriger-
ator. The product was filtered, washed with a little ether and fur-
ther purified by recrystallization from ethanol to give white
crystals, m.p. 140 °C. 1H NMR (400 MHz, DMSO-d6) d 3.52 (s, 2H,
NH2), 7.22–7.29 (m, 1H), 7.43–7.55 (m, 2H), 8.15–8.21 (d, J
= 8.47 Hz, 2H), 8.40 (s, 1H). 13C NMR (101 MHz, DMSO) d 158.77,
157.10, 153.71, 139.44, 129.48, 126.39, 120.96, 101.91. MS (EI)
m/z 185.18 (M++1).
1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (6). Compound
5 (3.44 g, 0.01 mol) was refluxed in formic acid (30 mL, 85%) for
6 h. The reaction mixture was cooled and poured into water. The
formed solid was filtered off, dried, and recrystallized from dioxane
to give compound 6 (75%) as a white solid, m.p. 265–267 °C. 1H
NMR (400 MHz, DMSO-d6) d 7.37 (t, J = 4.34 Hz, 1H), 7.53 (t, J
= 8.31 Hz, 2H), 8.02 (d, J = 7.85 Hz, 2H), 8.18 (s, 1H), 8.31 (s, 1H),
12.44 (s, 1H, NH). 13C NMR (101 MHz, DMSO) d 157.65 (C@O),
152.27, 149.20, 138.66, 136.41, 129.61, 127.52, 122.14, 108.06.
MS (EI) m/z 212.20 (M+).
5-(2-Bromoethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (7). A solution of compound 6 (3 g, 14.2 mmol) in dry DMF
(20 mL) was cooled to 0 °C. Sodium hydride (65% dispersion in
oil, 0.55 g, 16.25 mmol) was added, and the solution was stirred
for 30 min at 0 °C. 1,2 Dibromoethane (13.18 g, 70.5 mmol) was
added, the ice bath was removed, and the solution was stirred
for 4 h at room temperature. The mixture was poured onto H2O
and washed with ethyl acetate (5 ꢁ 40 mL). The organic layer
was washed with H2O and brine. The organic layer was then dried
over Na2SO4 and filtered, and the solvent was removed in vacuo to
give compound 7 as yellowish white solid (63%). mp 125–127 °C.
1H NMR (400 MHz, DMSO-d6) d 3.83 (t, J = 7.24 Hz, 2H), 4.42 (t, J
= 7.32 Hz, 2H), 7.37–7.41 (m, 1H), 7.55 (t, J = 7.79 Hz, 2H), 8.02
(d, J = 7.71 Hz, 2H), 8.37 (s, 1H), 8.52 (s, 1H). 13C NMR (101 MHz,
DMSO) d 156.67 (C@O), 151.77, 151.58, 138.46, 136.56, 129.67,
127.63, 122.06, 107.19, 47.39, 31.35. MS (EI) m/z 319.06 (M++1).
Anal. Calcd. for C13H11BrN4O: C, 48.92; H, 3.47.; N, 17.55. Found:
C, 49.27; H, 3.41.; N, 17.54.
General procedure A. Synthesis of compounds (8a–f). 4-((2-(4-oxo-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)ethyl)amino)benzene-
sulfonamide (8a). K2CO3 (0.74 g, 5.33 mmol) and 4-sulfanilamide
(0.31 g, 1.78 mmol) were added, while stirring, to a solution of com-
pound 7 (0.56 g, 1.78 mmol) in anhydrous DMF (15 mL). The
reaction mixture was heated at 60 °C for 2 h. After cooling, the mix-
ture was poured onto H2O (100 mL), extracted with ethyl acetate
(3 ꢁ 40 mL), washed with saturated aqueous NaCl and dried over
Na2SO4. The solvent was removed in vacuo, and the residue was
chromatographed on a silica gel column using methylene chloride/
methanol (9:1) as the eluent to afford 4-((2-(4-oxo-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-5(4H)-yl)ethyl)amino)benzenesulfon-
amide 8a (71%) as a white solid. mp 215–217 °C. 1H NMR (400 MHz,
DMSO-d6) d 3.67 (t, J = 4.18 Hz, 2H), 4.19 (t, J = 4.36 Hz, 2H), 4.41
(s, 1H, NH), 5.01 (s, 2H, NH2), 6.58–6.64 (m, 4H), 7.41–7.47 (m,
3H), 7.99 (d, J = 8.17 Hz, 2H), 8.53 (s, 1H), 8.77 (s, 1H). 13C NMR
(101 MHz, DMSO)
d 152.66, 152.33, 152.13, 140.18, 137.90,
130.41, 129.75, 128.03, 127.87, 122.78, 113.14, 112.98, 103.94,
57.61, 52.02. MS (EI) m/z 411.17 (M++1). Anal. Calcd. for C19H18N6
O3S: C, 55.60; H, 4.42; N, 20.48. Found: C, 55.67; H, 4.36; N, 20.08.
1-Phenyl-5-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimi-
din-4(5H)-one (8b). General Procedure A, yield 67%, Creamy white
solid, mp 153–155 °C. 1H NMR (400 MHz, DMSO-d6) d 1.63 (t, J
= 4.45 Hz, 4H), 2.47 (t, J = 8.17 Hz, 4H), 2.68 (t, J = 8.11 Hz, 2H),
4.08 (t, J = 4.05 Hz, 2H), 7.37 (t, J = 8.55 Hz, 1H), 7.54 (t, J
= 8.70 Hz, 2H), 8.02 (d, J = 4.24 Hz, 2H), 8.33 (s, 1H), 8.40 (s, 1H).
13C NMR (101 MHz, DMSO) d 156.72 (C@O), 151.95, 151.66,
138.57, 136.49, 129.66, 127.51, 121.97, 107.30, 54.54, 54.02,
44.67, 23.65. MS (EI) m/z 310.03 (M++1). Anal. Calcd. for
C17H19N5O: C, 66.00; H, 6.19; N, 22.64. Found: C, 66.37; H, 5.82;
N, 22.91.
5-(2-(Azepan-1-yl)ethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (8c). General Procedure A, yield 67%, white solid, mp
105–107 °C. 1H NMR (400 MHz, DMSO-d6) d 1.42 (s, 4H), 2.50–
264 (m, 4H), 2.72 (t, J = 4.77 Hz, 2H), 3.40 (t, J = 16.53 Hz, 4H),
4.04 (t, J = 8.81 Hz, 2H), 7.34–7.40 (m, 1H), 7.55 (t, J = 7.93 Hz,
2H), 8.04 (d, J = 4.04 Hz, 2H), 8.34 (s, 1H), 8.38 (s, 1H). 13C NMR
(101 MHz, DMSO) d 156.76, 152.07, 151.72, 138.61, 136.46,
129.66, 127.46, 121.89, 107.24, 56.28, 55.46, 44.11, 28.50, 26.87.
MS (EI) m/z 338.10 (M++1). Anal. Calcd. for C19H23N5O: C, 67.63;
H, 6.87; N, 20.76. Found: C, 68.31; H, 6.35; N, 20.71.
1-Phenyl-5-(2-(phenylthio)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (8d). General Procedure A, yield 67%, white solid, mp 108–110 °C. 1H
NMR (400 MHz, DMSO-d6) d 3.66 (t, J = 4.28 Hz, 2H), 4.08 (t, J = 8.28 Hz,
2H), 7.32–7.41 (m, 3H), 7.98–8.05 (m, 5H), 8.37 (d, J = 4.26 Hz, 2H), 8.43
(s, 1H), 8.52 (s, 1H). 13C NMR (101 MHz, DMSO) d 156.88 (C = O), 156.68,
152.24, 151.77, 139.36, 138.46, 134.30, 129.80, 129.68, 125.94, 122.07,
121.93, 107.19, 47.40, 31.35. MS (EI) m/z 349.39 (M++1). Anal. Calcd.
for C19H16N4OS: C, 65.50; H, 4.63; N, 16.08. Found: C, 65.54; H, 4.35;
N, 15.91.
3-(2-(4-oxo-1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)ethyl)
benzo[d]thiazol-2(3H)-one (8e). General Procedure A, yield 67%, white
solid, mp 211–213 °C. 1H NMR (400 MHz, DMSO-d6) d 3.63 (t, J
= 27.12 Hz, 2H), 4.31 (t, J = 17.15 Hz, 2H), 7.15 (t, J = 7.58 Hz, 1H),
7.27–7.40 (m, 3H), 7.50–7.60 (m, 3H), 7.94 (d, J = 8.20 Hz, 2H), 8.25
(s, 1H), 8.29 (s, 1H). 13C NMR (101 MHz, DMSO) d 169.76 (S-C@O),
157.06 (C@O), 151.46, 151.35, 138.34, 136.91, 136.49, 129.72,
127.69, 127.03, 123.76, 123.35, 122.01, 121.84, 111.45, 106.98,
43.79, 41.56. MS (EI) m/z 390.02 (M++1). Anal. Calcd. for C20H15N5O2S:
C, 61.68; H, 3.88; N, 17.98. Found: C, 61.41; H, 3.64; N, 17.91.
5-(2-(Bis(2-hydroxyethyl)amino)ethyl)-1-phenyl-1H-pyrazolo[3,4
-d]pyrimidin-4(5H)-one (8f). General Procedure A, yield 67%, light
brown solid, mp 121–123 °C. MS (EI) m/z 344.17 (M++1). It was
used directly for the preparation of compound 8g.
5-(2-(Bis(2-chloroethyl)amino)ethyl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (8g).
A mixture of compound 8f (0.4 g,
1.17 mmol) in thionyl chloride (3 mL) and dry benzene (10 mL)
was heated gently under reflux until a homogenous solution was
obtained, then for a further one hour. The solution was evaporated
to dryness under vacuum to remove excess thionyl chloride. The
residue was azeotroped with dry benzene (3 ꢁ 5 mL) where the last
traces of thionyl chloride were removed. The residue obtained was
purified by flash column chromatography (SiO2) using methylene
chloride/methanol (9.5:0.5) as eluent to give 0.25 g (62%) of com-
pound 8g as a yellowish white solid. 1H NMR (400 MHz, DMSO-
d6) d 3.41–3.57 (m, 6H), 3.97 (t, J = 8.15 Hz, 2H), 4.35–4.41 (m,
4H), 7.40 (t, J = 8.36 Hz, 1H), 7.56 (t, J = 7.84 Hz, 2H), 8.02 (d, J
= 8.23 Hz, 2H), 8.38 (s, 1H), 8.51 (s, 1H). 13C NMR (101 MHz, DMSO)
d 156.74 (C@O), 151.97, 151.60, 138.46, 136.56, 129.70, 127.67,
122.12, 107.19, 47.54, 47.48, 42.80, 42.75. MS (EI) m/z 380.12
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

PHASCI 3032
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No. of Pages 15, Model 5G
A.H. Abdelazeem et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
11
(M++1). Anal. Calcd. for C17H19Cl2N5O: C, 53.69; H, 5.04; N, 18.42.
Found: C, 53.48; H, 4.93; N, 18.14.
114.61, 100.47. MS (EI) m/z 331.24 (M++1). Anal. Calcd.. for
C18H14N6O: C, 65.44; H, 4.27; N, 25.44. Found: C, 65.51; H, 4.21; N,
25.49.
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1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9). 5-amino-1-
phenyl-1H-pyrazole-4-carbonitrile 5 (3.5 gm, 19 mmol) was added
to formamide (20 mL). The solution was refluxed for 4 h and
allowed to cool. The reaction mixture was poured onto water
and the product was filtered and recrystallized from dioxane to
4-Chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (12). 1-Phenyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 6 (10 g) was suspended
in phosphorus oxychloride (50 mL) containing few drops of DMF.
The mixture was refluxed for two hours. The excess phosphorus
oxychloride was removed from the clear yellow solution under
reduced pressure and the residue was then poured slowly with vig-
orous stirring onto ice water (250 mL). The mixture was allowed to
stand for 30 min, and the white suspension was extracted with
methylene chloride (3 ꢁ 60 mL.). The solvent was evaporated and
the residue obtained was dried and purified by flash column chro-
matography (SiO2) using CH2Cl2/CH3OH (9.5:0.5) as eluent to give
the chloro-derivative 12 as a yellow solid (92%), m.p. to 198–199.
1H NMR (400 MHz, DMSO-d6) d 7.37 (t, J = 8.42 Hz, 1H), 7.52 (t, J
= 7.30 Hz, 2H), 8.00 (d, J = 4.47 Hz, 2H), 8.32 (s, 1H), 8.55 (s, 1H).
13C NMR (101 MHz, DMSO) d 156.56, 151.70, 151.59, 138.49,
136.59, 129.60, 127.54, 122.05, 107.36. MS (EI) m/z 231.11 (M++1).
General procedure C. Synthesis of compounds (13a–m). 8-(1-phe-
nyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-1,4-dioxa-8-azaspiro[4.5]
decane (13a). A mixture of 4-chloro-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine 12 (1 g, 4.34 mmol) and 1,4-dioxa-8-azaspiro[4.5]
decane (0.62 g, 4.34 mmol) in 2-propanol (30 mL) or 95% ethanol
for other amines was refluxed for two hour. The white solid formed
in the hot solution was collected after cooling and further recrystal-
lized from ethanol yielded white crystals of compound 13a (67%).
mp 170–172 °C. 1H NMR (400 MHz, Chloroform-d) d 1.88 (t, J
= 7.99 Hz, 4H), 4.03 (s, 4H), 4.11 (t, J = 8.21 Hz, 4H), 7.31–7.35 (m,
1H), 7.50–7.54 (m, 2H), 8.11–8.14 (m, 3H), 8.45 (s, 1H). 13C NMR
(101 MHz, CDCl3) d 156.75, 155.63, 154.39, 138.87, 133.78, 129.08,
126.64, 122.05, 106.87, 101.67, 64.57, 43.75, 34.93. MS (EI) m/z
338.17 (M++1). Anal. Calcd. for C18H19N5O2: C, 64.08; H, 5.68; N,
20.76; Found: C, 64.12; H, 5.32; N, 20.47.
4-(4-Cyclohexylpiperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrim-
idine (13b). General Procedure C, yield 67%, white solid, mp 109–
111 °C. 1H NMR (400 MHz, Chloroform-d) d 1.09–1.23 (m, 5H),
1.54–1.94 (m, 5H), 2.22–2.41 (m, 1H), 2.72 (t, J = 8.04 Hz, 4H), 4.00
(t, J = 8.06 Hz, 4H), 7.26–7.33 (m, 1H), 7.47–7.52 (m, 2H), 8.10–8.12
(m, 3H), 8.43 (s, 1H). 13C NMR (101 MHz, CDCl3) d 156.99, 155.56,
154.34, 138.88, 133.88, 129.07, 126.61, 122.02, 101.69, 63.57, 48.74,
48.73, 28.90, 26.23, 25.78. MS (EI) m/z 363.21 (M++1). Anal. Calcd.
for C21H26N6: C, 69.58; H, 7.23; N, 23.19. Found: C, 69.81; H, 7.07;
N, 23.42.
4-(4-(4-Fluorophenyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-
d] pyrimidine (13c) (Natarajan et al., 2005). General Procedure C,
yield 67%, white solid, mp 140–142 °C. 1H NMR (400 MHz,
DMSO-d6) d 3.24 (t, J = 4.21 Hz, 4H), 4.07 (t, J = 4.11 Hz, 4H),
6.93–6.97 (dd, J = 4.59, 9.02 Hz, 2H), 7.02–7.08 (m, 2H), 7.31–
7.35 (m, 1H), 7.52 (t, J = 7.85 Hz, 2H), 8.17 (d, J = 8.00 Hz, 2H),
8.37 (s, 1H), 8.54 (s, 1H). 13C NMR (101 MHz, DMSO) d 156.43,
give
a
light brown solid of compound
7.29 (t, = 7.43 Hz, 1H), 7.51 (t, J
9
(56%). 1H NMR
(400 MHz, DMSO-d6)
d
J
= 5.68 Hz, 2H), 7.98 (br. s, 2H, NH2), 8.20 (d, J = 7.27 Hz, 2H), 8.34
(s, 1H), 8.40 (s, 1H). 13C NMR (101 MHz, DMSO) d 158.89, 157.23,
153.83, 139.56, 134.62, 129.60, 126.51, 121.08, 102.03. MS (EI)
m/z 212.27 (M++1).
General procedure B. Synthesis of compounds (10a–c). N-(furan-3-
ylmethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10a). 1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 9 (1.0 g, 4.73 mmol)
and 3-furaldehyde (0.45 g, 4.73 mmol) were mixed in 1,2-dichloro-
ethane (20 mL) and then treated with sodium triacetoxyborohydride
(1.5 g, 6.62 mmol) and acetic acid (0.28 g, 4.73 mmol). The mixture
was stirred at room temperature for 24 h until the reactants were
consumed. The reaction mixture was quenched by 1 N NaOH, and
the product was extracted with CH2Cl2. The organic extract was
washed with brine and dried (Na2SO4). The solvent was evaporated
and the residue obtained was purified by flash column chromatogra-
phy (SiO2) using methylene chloride/methanol (9.5:0.5) as eluent to
give compound 10a as a white solid (82%). mp 143–145 °C. 1H NMR
(400 MHz, DMSO-d6) d 3.48 (s, 1H, NH), 3.84 (s, 2H, CH2), 6.74–6.77
(dd, J = 4.52, 7.46 Hz, 1H), 6.89 (d, J = 8.31 Hz, 1H), 7.43 (t, J
= 4.24 Hz, 1H), 7.61–7.68 (m, 3H), 8.25 (d, J = 4.35, 2H), 8.48 (s, 1H),
8.64 (s, 1H). 13C NMR (101 MHz, DMSO) d 158.64, 156.81, 155.63,
154.11, 147.88, 139.00, 135.37, 129.33, 126.55, 121.36, 113.29,
107.03, 101.52, 43.89. MS (EI) m/z 292.16 (M++1). Anal. Calcd. for
C16H13N5O: C, 65.97; H, 4.50; N, 24.04. Found: C, 65.84; H, 4.12; N,
23.83.
N-(4-Nitrobenzyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(10b). General Procedure B, yield 67%, white solid, mp 81–84 °C. 1H
NMR (400 MHz, DMSO-d6) d 4.02 (s, 2H, CH2), 4.76 (s, 1H, NH), 6.79
(d, J = 4.52, 2H), 6.97 (d, J = 4.31 Hz, 2H), 7.27 (t, J = 8.24 Hz, 1H), 7.46
(t, J = 8.21 Hz, 2H), 8.10 (d, J = 4.35, 2H), 8.31 (s, 1H), 8.51 (s, 1H). 13
C
NMR (101 MHz, DMSO) d 155.92, 154.58, 153.34, 148.48, 138.14,
134.09, 128.24, 127.99, 125.52, 121.86, 120.47, 115.83, 100.61,
46.65. MS (EI) m/z 347.22 (M++1). Anal. Calcd. for C18H14N6O2: C,
62.42; H, 4.07; N, 24.27. Found: C, 63.10; H, 3.98; N, 24.13.
N-(3-Phenoxybenzyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (10c). General Procedure B, yield 67%, white solid, mp
230–232 °C. 1H NMR (400 MHz, DMSO-d6) d 3.55 (s, 1H, NH),
4.85 (s, 2H), 6.95–7.00 (m, 3H), 7.09–7.13 (m, 2H), 7.34–7.39
(m, 6H), 8.10 (d, J = 4.35 Hz, 2H), 8.44 (s, 1H), 8.68 (s, 1H). 13C
NMR (101 MHz, DMSO)
d 157.27, 157.10, 156.82, 145.46,
135.42, 130.58, 130.46, 130.06, 129.70, 127.37, 123.97, 123.09,
121.80, 119.13, 118.28, 117.76, 117.25, 116.69, 102.13, 44.48.
MS (EI) m/z 394.19 (M++1). Anal. Calcd. for C24H19N5O: C,
73.27; H, 4.87; N, 17.80. Found: C, 73.25; H, 4.81; N, 17.96.
1-Phenyl-3-(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)urea (11).
155.34–155.29 (d,
131.19, 129.03, 126.27, 121.10, 117.30–117.22 (d,
J
= 5.05), 153.87, 147.41, 138.70, 134.90,
= 8.08),
J
115.49–115.27 (d, J = 22.22), 101.15, 48.61, 48.56. MS (EI) m/z
375.22 (M++1). Anal. Calcd. for C21H19FN6: C, 67.37; H, 5.11; N,
22.45. Found: C, 67.41; H, 4.94; N, 22.12.
A
mixture of 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 9
(1.0 g, 4.73 mmol) in methylene chloride (25 mL), phenylisocyanate
(0.56 g, 4.73 mmol.) and few drops of triethylamine were stirred
and left overnight. The reaction mixture was then evaporated and
the residue was dissolved in acetone, concentrated; set aside where
the solid obtained is collected, dried & recrystallized from ethanol-
acetone to give a white crystals from compound 11 (74%). mp 187–
189 °C. 1H NMR (400 MHz, DMSO-d6) d 5.52 (s, 2H, 2NH), 7.00–7.28
(m, 3H), 7.30 (t, J = 8.31 Hz, 1H), 7.47–7.57 (m, 3H), 7.72 (d, J
= 8.21 Hz, 1H), 8.14 (d, J = 8.35, 2H), 8.32 (s, 1H), 8.49 (s, 1H). 13C
NMR (101 MHz, DMSO) d 159.04 (C@O), 155.78, 154.67, 153.18,
149.75, 137.99, 134.30, 128.37, 128.34, 125.62, 120.43, 118.33,
1-Phenyl-4-(4-phenylpiperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimi-
dine (13d). General Procedure C, yield 67%, white solid, mp 118–
120 °C. 1H NMR (400 MHz, DMSO-d6) d 3.34 (t, J = 8.3 Hz, 4H),
4.11 (t, J = 4.2 Hz, 4H), 6.80 (t, J = 8.22 Hz, 1H), 6.90–7.01 (m,
2H), 7.22–7.36 (m, 3H), 7.50–7.57 (m, 2H), 8.18 (d, J = 8.2 Hz,
2H), 8.40 (s, 1H), 8.58 (s, 1H). 13C NMR (101 MHz, DMSO) d
156.93, 155.82, 154.33, 150.91, 139.14, 135.46, 129.52, 129.50,
126.77, 121.59, 119.48, 115.76, 101.62, 48.12, 48.08. MS (EI) m/
z 357.22 (M++1). Anal. Calcd. for C21H20N6: C, 70.77; H, 5.66; N,
23.58. Found: C, 70.55; H, 5.93; N, 23.61.
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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1-Phenyl-4-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)-1H-pyr-
azolo[3,4-d]pyrimidine (13e). General Procedure C, yield 67%, white
solid, mp 210–212 °C. 1H NMR (400 MHz, DMSO-d6) d 3.61 (t, J
= 4.21, 4H), 4.20 (t, J = 8.34, 4H), 7.04 (d, J = 8.54 Hz, 2H), 7.36–
7.41 (m, 1H), 7.52–7.58 (m, 4H), 8.09–8.12 (m, 2H), 8.45 (s, 1H),
8.66 (s, 1H). 13C NMR (101 MHz, DMSO) d 154.95, 153.46, 153.17,
152.56, 138.62, 136.26, 129.63, 127.35, 126.75–126.71 (d, J = 4.04),
124.12, 122.02, 118.23–117.91 (d, J = 32.3), 113.78, 101.66, 45.86,
45.84. MS (EI) m/z 425.20 (M++1). Anal. Calcd. for C22H19F3N6: C,
62.26; H, 4.51; N, 19.80. Found:. C, 62.51; H, 4.31; N, 20.14.
1-Phenyl-4-(4-(pyridin-2-yl)piperazin-1-yl)-1H-pyrazolo[3,4-d]
pyrimidine (13f). General Procedure C, yield 67%, white solid, mp
138–140 °C. 1H NMR (400 MHz, DMSO-d6) d 3.74 (t, J = 8.18 Hz,
4H), 4.08 (t, J = 4.44 Hz, 4H), 6.64–6.67 (dd, J = 4.84, 7.07 Hz,
1H), 6.79 (d, J = 8.56 Hz, 1H), 7.33 (t, J = 4.37 Hz, 1H), 7.51–7.58
(m, 3H), 8.13–8.18 (m, 3H), 8.38 (s, 1H), 8.54 (s, 1H). 13C NMR
(101 MHz, DMSO) d 158.80, 156.97, 155.79, 154.27, 148.04,
139.16, 138.06, 135.53, 129.49, 126.71, 121.52, 113.45, 107.19,
101.68, 44.05, 43.99. MS (EI) m/z 358.30 (M++1). Anal. Calcd.
for C20H19N7: C, 67.21; H, 5.36; N, 27.43. Found: C, 67.08; H,
5.70; N, 27.19.
4H), 7.33 (t, J = 7.89 Hz, 1H), 7.53 (t, J = 8.34 Hz, 2H), 8.18 (d, J
= 8.43 Hz, 2H), 8.38 (s, 1H), 8.55 (s, 1H). 13C NMR (101 MHz, DMSO)
d 156.89, 155.77, 154.34, 153.72, 145.37, 139.15, 135.36, 129.47,
126.71, 121.55, 118.22, 114.76, 101.59, 55.60, 49.95, 49.89. MS
(EI) m/z 387.15 (M++1). Anal. Calcd. for C22H22N6:O: C, 68.38; H,
5.74; N, 21.75. Found C, 68.65; H, 6.07; N, 21.71.
1-(1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-one (13l).
General Procedure C, yield 67%, white solid, mp 199–201 °C. 1H
NMR (400 MHz, DMSO-d6) d 2.62 (t, J = 6.20 Hz, 4H), 4.20 (t, J
= 8.21 Hz, 4H), 7.33 (t, J = 7.46 Hz, 1H), 7.53 (t, J = 8.47 Hz, 2H),
8.17 (d, J = 7.99 Hz, 2H), 8.37 (s, 1H), 8.48 (s, 1H). 13C NMR
(101 MHz, DMSO) d 207.93 (C@O), 156.78, 155.75, 154.19, 139.17,
135.58, 129.46, 126.66, 121.44, 101.83, 39.77, 39.71. MS (EI) m/z
393.20 (M+). Anal. Calcd. for C16H15N5O: C, 65.52; H, 5.15; N,
23.88. Found: C, 65.11; H, 5.42; N, 23.50.
4-(4-(Methylsulfonyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidine (13m). A mixture of 4-chloro-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine 12 (1 g, 4.34 mmol) and piperazine (1.1 g, 13 mmol)
in 2–95% ethanol (30 mL) was refluxed for two hours. The reaction
mixture was then concentrated and poured onto ice water
(100 mL). The crude product was extracted with ethyl acetate
(3 ꢁ 40 mL) and evaporated to give a white residue. Methyl sulfonyl
chloride (0.4 mL, 3.6 mmol) was added slowly over 5 min to a 0 °C
solution of the crude residue (1 g, 3.6 mmol) in CH2Cl2 (20 mL) con-
taining few drops of triethylamine. The reaction mixture was stirred
for30 min at0 °C thenthe ice bath was removed,andthe reaction was
stirred for 2 h at room temperature. The mixture was onto H2O and
the product was extracted with methylene chloride (3 ꢁ 40 mL).
Theorganic layer waswashed brine and dried overNa2SO4. The meth-
ylene chloride solution was concentrated and purified by flash col-
umn chromatography (SiO2) using hexane/acetone (6:4) as eluent
to give the methylsulfone derivative 13m as a white solid (55%). mp
220–222 °C.
4-(4,4-Difluoropiperidin-1-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrim-
idine (14). Deoxofluor (1 mL, 5.5 mmol) was added slowly over
5 min to a 0 °C solution of 1-(1-phenyl-1H-pyrazolo[3,4-d]pyrimi-
din-4-yl)piperidin-4-one, 13l (2.16 g, 5.5 mmol) in CH2Cl2 (20 mL).
The reaction mixture was stirred for 30 min at 0 °C then the ice
bath was removed, and the reaction was stirred for 24 h at room
temperature. The mixture was quenched with equivolume of H2O
and the product was extracted with methylene chloride
(3 ꢁ 40 mL). The organic layer was washed with H2O and brine
and dried over Na2SO4. The methylene chloride solution containing
the crude product was concentrated and purified by flash column
chromatography (SiO2) using hexane/ethyl acetate (8:2) as eluent
to give the final compound 14 as a white solid (45%). mp 203–
205 °C. 1H NMR (400 MHz, DMSO-d6) d 2.10–2.18 (m, 2H), 2.38–
2.43 (m, 2H), 4.06–4.18 (m, 4H), 7.34–7.39 (m, 1H), 7.53–7.57
(m, 2H), 8.15–8.17 (d, J = 8.19 Hz, 2H), 8.41 (s, 1H), 8.62 (s, 1H).
MS (EI) m/z 316.26 (M++1). Anal. Calcd. for C16H15F2N5: C, 60.94;
H, 4.79; N, 22.21. Found: C, 61.13; H, 5.09; N, 22.17.
4-(4-(4-Fluorobenzyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidine (13g). General Procedure C, yield 67%, white solid, mp
182–184 °C. 1H NMR (400 MHz, DMSO-d6) d 3.14 (t, J = 4.45 Hz,
4H), 3.91 (t, J = 4.32 Hz, 4H), 4.61 (s, 2H, CH2), 6.73–6.83 (m, 2H),
6.84–6.88 (m, 2H), 7.11–7.15 (m, 1H), 7.32 (t, J = 7.85 Hz, 2H),
7.97 (d, J = 8.00 Hz, 2H), 8.17 (s, 1H), 8.34 (s, 1H). 13C NMR
(101 MHz, DMSO) d 152.43, 151.34–151.29 (d, J = 5.05), 149.87,
149.41, 143.41, 134.70, 130.90, 125.03, 122.27, 117.10, 113.22–
113.30 (d, J = 8.1), 111.27–111.49 (d, J = 22.2), 97.15, 63.57, 48.12,
48.08. MS (EI) m/z 389.22 (M++1). Anal. Calcd. for C22H21FN6: C,
68.02; H, 5.45; N, 21.64. Found: C, 68.31; H, 5.20; N, 22.01.
4-(4-(4-Chlorophenyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine (13h). General Procedure C, yield 67%, white solid,
mp 148–150 °C. 1H NMR (400 MHz, DMSO-d6)
d 3.31 (t, J
= 8.31 Hz, 4H), 4.06 (t, J = 4.18 Hz, 4H), 7.90 (d, J = 8.32 Hz, 2H),
7.21–7.34 (m, 3H), 7.50–7.53 (m, 2H), 8.17 (d, J = 8.34 Hz, 2H),
8.37 (s, 1H), 8.51 (s, 1H). 13C NMR (101 MHz, DMSO) d 157.06,
155.72, 154.48, 149.62, 139.28, 135.23, 129.39, 129.14, 126.66,
123.01, 121.62, 116.98, 101.76, 47.79, 44.97. MS (EI) m/z 391.08
(M++1). Anal. Calcd. for C21H19CIN6: C, 64.53; H, 4.90; N, 21.50.
Found: C, 64.17; H, 5.45; N, 22.01
1-Phenyl-4-(4-(p-tolyl)piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimi-
dine (13i). General Procedure C, yield 67%, white solid, mp 152–
154 °C. 1H NMR (400 MHz, DMSO-d6) d 2.21 (s, 3H, CH3), 3.27 (t, J
= 4.31 Hz, 4H), 4.11 (t, J = 4.22 Hz, 4H), 6.88 (d, J = 8.28 Hz, 2H), 7.06
(d, J = 8.05 Hz, 2H), 7.36 (t, J = 8.40 Hz, 1H), 7.55 (t, J = 7.95 Hz, 2H),
8.18 (d, J = 8.13, 2H), 8.40 (s, 1H), 8.60 (s, 1H). 13C NMR (101 MHz,
DMSO) d 156.96, 155.85, 154.36, 148.93, 139.13, 135.48, 129.94,
129.54, 128.52, 126.81, 121.63, 116.28, 101.61, 48.86, 48.80, 20.50.
MS (EI) m/z 371.16 (M++1). Anal. Calcd. for C22H22N6: C, 71.33; H,
5.99; N, 22.69. Found: C, 70.96; H, 6.03; N, 22.94.
4-(4-(4-Nitrophenyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidine (13j). General Procedure C, yield 67%, yellow solid, mp
239–242 °C. 1H NMR (400 MHz, DMSO-d6) d 3.76 (t, J = 4.38 Hz,
4H), 4.18 (t, J = 4.21 Hz, 4H), 6.94 (d, J = 8.30 Hz, 2H), 7.34 (t, J
= 8.11 Hz, 1H), 7.53 (t, J = 7.98 Hz, 2H), 8.04–8.10 (m, 2H), 8.18
(d, J = 7.97 Hz, 2H), 8.40 (s, 1H), 8.48 (s, 1H). 13C NMR (101 MHz,
DMSO) d 155.77, 154.38, 139.25, 137.46, 135.40, 129.44, 126.73,
126.14, 125.95, 121.60, 113.84, 112.23, 101.85, 45.47, 44.15. MS
(EI) m/z 402.21 (M++1). Anal. Calcd. for C21H19N7O2: C, 62.83; H,
4.77; N, 24.42. Found: C, 62.44; H, 4.94; N, 24.16.
10-(1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)spiro[chroman-2,4’-
piperidin]-4-one (15). Pyrrolidine (0.1 mL, 12 mmol) was added drop-
wise at room temperature to a solution of 13l (1.82 g, 6 mmol) and
2-hydroxyacetophenone (0.85 g, 6 mmol) in anhydrous methanol
(30 mL). The reaction mixture was refluxed overnight and then con-
centrated under reduced pressure. Ethyl acetate (50 mL) was added
and the organic mixture was washed with 1 N HCl, then 1 N NaOH
and brine. The organic layer was dried over Na2SO4, concentrated
and purified by flash column chromatography (SiO2) using hexane/
ethyl acetate (8:2) as eluent to give the spiro derivative 15 as a white
solid (79%). mp 178–180 °C. 1H NMR (400 MHz, DMSO-d6) d 1.81 (t, J
= 12.85 Hz, 2H), 2.06 (t, J = 18.86 Hz, 2H), 2.87 (s, 2H), 3.56 (t, J
= 32.01 Hz, 2H), 4.49 (t, J = 24.26 Hz, 2H), 7.04–7.12 (m, 2H), 7.34 (t, J
= 7.99 Hz, 1H), 7.51–7.61 (m, 3H), 7.76 (d, J = 8.19 Hz, 1H), 8.18 (d, J =
7.98 Hz, 2H), 8.36 (s, 1H), 8.53 (s, 1H). 13C NMR (101 MHz, DMSO) d
4-(4-(4-Methoxyphenyl)piperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine (13k). General Procedure C, yield 67%, shiny white
solid, mp 143–145 °C. 1H NMR (400 MHz, DMSO-d6) d 3.15 (t, J
= 4.41 Hz, 4H), 3.67 (s, 3H), 4.07 (t, J = 4.02 Hz, 4H), 6.80–6.92 (m,
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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191.80 (C = O), 159.04, 156.68, 155.79, 154.37, 139.18, 136.93, 135.37,
129.47, 126.69, 126.34, 121.66, 121.54, 120.90, 118.88, 101.54, 78.55,
47.23, 33.56, 33.50. MS (EI) m/z 412.06 (M++1). Anal. Calcd. for
C24H21N5O2: C, 70.06; H, 5.14; N, 17.02. Found: C, 70.35; H, 5.11; N,
17.10.
4.2.2. Assay for iNOS inhibition
The assay was performed using mouse macrophages
(RAW264.7, obtained from ATCC). Cells were cultured in phenol
red free RPMI medium supplemented with 10% bovine calf serum
and 100 U/mL penicillin G sodium, and 100 lg/mL streptomycin
4-((1-(1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-yl)
amino)benzenesulfonamide (16). Sulfanilamide (1.0 g, 5.8 mmol)
and 1-(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-
one, 13l (1.7 g, 5.8 mmol) were mixed in 1,2-dichloroethane
(30 mL) and then treated with sodium triacetoxyborohydride
(1.8 g, 8.12 mmol) and acetic acid (0.6 g, 10 mmol). The mixture
was stirred at rt under a N2 atmosphere for 24 h. The reaction mix-
ture was quenched by adding 1 N NaOH, and the product was
extracted with ethyl acetate. The organic extract was washed with
brine and dried over MgSO4. The solvent was evaporated and the
residue was purified by flash column chromatography (SiO2) using
CH2Cl2/CH3OH (9.5:0.5) as eluent to give the target compound 16
as a white solid (59%). mp 157–159 °C. 1H NMR (400 MHz,
DMSO-d6) d 3.22–3.37 (m, 5H), 3.89–3.98 (m, 4H), 4.42 (s, 1H,
NH), 5.73 (br s, 2H, NH2), 7.26–7.34 (m, 2H), 7.56 (t, J = 7.89 Hz,
1H), 7.73–7.83 (m, 3H), 7.98 (d, J = 8.11 Hz, 1H), 8.40 (d, J =
11.98 Hz, 2H), 8.58 (s, 1H), 8.75 (s, 1H). 13C NMR (101 MHz, DMSO)
d 158.86, 155.62, 154.20, 139.00, 136.75, 135.20, 129.30, 126.51,
126.16, 121.36, 120.72, 118.70, 101.37, 57.36, 47.06, 33.38. MS
(EI) m/z 449.33 (M+). Anal. Calcd. for C22H23N7O2S: C, 58.78; H,
5.16; N, 21.81. Found: C, 59.01; H, 5.31; N, 22.16.
at 37 °C in an atmosphere of 5% CO2 and 95% humidity as above.
Cells were seeded in 96-well plates at 5 ꢁ 104 cells/well and incu-
bated for 24 h. Test compounds diluted in serum free medium
were added to the cells. After 30 min of incubation, LPS (5 lg/
mL) was added and the cells were further incubated for 24 h. The
concentration of nitric oxide (NO) was determined by measuring
the level of nitrite released in the cell culture supernatant by using
Griess reagent (Quang et al., 2006). Percent inhibition of nitrite pro-
duction by the test compound was calculated in comparison to the
vehicle control. IC50 values were obtained from dose curves.
Parthenolide was used as positive control.
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4.2.3. Assay for NF-jB inhibition
The assay was performed using human chondrosarcoma
(SW1353, obtained from ATCC) cells as described earlier (Ma
et al., 2007; Winter et al., 1962). Cells were cultured in 1:1 mixture
of DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin G
sodium, and 100 lg/mL streptomycin at 37 °C in an atmosphere
of 5% CO2 and 95% humidity. Cells (1.2 ꢁ 107) were washed once
in an antibiotic and FBS-free DMEM/F12, and then re-suspended
in 500
B luciferase plasmid construct was added to the cell suspension
at a concentration of 50 g/mL and incubated for 5 min at room
lL of antibiotic-free DMEM/F12 containing 2.5% FBS. NF-
1-(1-Phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-ol (17).
j
To
a
solution of 1-(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)
l
piperidin-4-one, 13l (1 g, 3.4 mmol) in MeOH (30 mL) was added
NaBH4 (0.25 g, 6.8 mmol) at 0 °C under argon. The reaction mixture
was warmed to room temperature and left to stir for 2 h. The reaction
mixture was quenched with water and extracted with ethyl acetate
(3 ꢁ 30 mL). The organic extract was washed with saturated aqueous
NaHCO3 and brine and dried over Na2SO4. The solvent was concen-
trated and purified by flash column chromatography (SiO2) using
hexane/acetone (6:4) as eluent to give the compound 17 as a white
solid (89%). mp 182–185 °C. 1H NMR (400 MHz, DMSO-d6) d 1.140–
1.52 (m, 2H), 1.78–1.89 (m, 1H), 3.28–3.64 (m, 4H), 3.83–3.87 (m,
1H), 4.29 (s, 1H, OH), 7.32–7.40 (m, 1H), 7.51–7.56 (m, 2H), 8.16 (d,
J = 8.10 Hz, 2H), 8.18 (s, 1H), 8.55 (s, 1H). 13C NMR (101 MHz, DMSO)
d 157.71, 152.28, 149.25, 138.67, 136.40, 129.61, 127.50, 122.13,
101.40, 65.66, 34.31, 34.25. MS (EI) m/z 296.26 (M++1). Anal. Calcd.
for C16H17N5O: C, 65.07; H, 5.80; N, 23.71. Found: C, 65.34; H, 6.22;
N, 23.53.
temperature. The cells were electroporated at 160 V and one 70-
ms pulse using BTX disposable cuvettes model 640 (4-mm gap)
in a BTX Electro Square Porator T 820 (BTX I, San Diego, CA). After
electroporation, cells were plated to the wells of 96-well plates at a
density of 1.25 ꢁ 105 cells per well. After 24 h, cells were treated
with different concentrations of test compounds for 30 min prior
to the addition of PMA (70 ng/mL) and incubated for 8 h. Luciferase
activity was measured using the Luciferase Assay kit (Promega).
The light output was detected on a SpectraMax plate reader. Per-
cent inhibition of luciferase activity was calculated as compared
to vehicle control and IC50 values were obtained from dose curves.
Parthenolide was used as positive control. Sp-1 was used as a con-
trol transcription factor which is unresponsive to inflammatory
mediators such as PMA. This is useful in detecting agents that
nonspecifically inhibit luciferase expression due to cytotoxicity or
inhibition of luciferase enzyme activity.
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4.2.4. Assay for in vitro cytotoxicity assay
893
4.2. Pharmacology
Cytotoxicity was determined against four human tumor cell
lines [SK-MEL (malignant melanoma); KB (epidermal carcinoma,
oral); BT-549 (ductal carcinoma, breast); SK-OV-3 (ovary carci-
noma)] and two non-cancerous kidney cell lines [Vero cells (Afri-
can green monkey kidney fibroblasts) and LLC-PK11 (pig kidney
epithelial cells)] as described earlier in addition to Mouse leukemic
monocyte macrophage cells (RAW 264.7) as described earlier
(Mustafa et al., 2004). All the cell lines were obtained from ATCC
and cultured in RPMI-1640 medium supplemented with bovine
calf serum (10%) and amikacin (60 mg/L), at 37 °C, 95% humidity,
5% CO2. Cells were seeded at a density of 25,000 cells/well and
grown for 24 h. Test compounds, diluted in serum free medium,
were added to the cells and incubated for 48 h. The number of via-
ble cells was determined by Neutral Red assay (Borenfreund et al.,
1990). Briefly, after treatment, cells were washed with saline and
incubated for 3 h with the medium containing Neutral Red
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4.2.1. Assay for cyclooxygenases (COXs) inhibition
Inhibition for Cox-1 and Cox-2 activity was determined by a
colorimetric method using a Cox inhibitor screening assay kit (Cay-
man Chemical Ann Arbor, Michigan) in a total volume of 220 lL
according to the directions provided with the kit manufacturer
(Kulmacz and Lands, 1983). The inhibitory activities of the tested
compounds were measured by monitoring the production of oxi-
dized N, N, N0, N0-tetramethyl-p-phenylenediamine (TMPD) at
590 nm followed by incubation of either ovine COX-1 or COX-2
with arachidonic acid. The enzymes were preincubated for 5 min
at 25 °C with the test compounds prior to addition of arachidonic
acid (final concentration 1.1 mM) and TMPD and incubation for
5 min at 25 °C. Indomethacin was used as positive control. The
COX-inhibiting activity was calculated according to the equation:
907 Q3
908
910
ð% inhibitionÞ ¼ ½1 ꢂ ðA2 ꢂ A0Þ=ðA1 ꢂ A0Þꢃ ꢁ 100
where A0 is the absorbance of blank, A1 was the absorbance of the
vehicle control and A2 is the absorbance in the presence of the test
compound.
(166 lg/mL). The cells were washed to remove extracellular dye.
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913
A solution of acidified ethanol (0.33% HCl) was then added to lyse
the cells; as a result, the incorporated dye was liberated from the
viable cells. The absorbance was measured at 450 nm. IC50 was
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

PHASCI 3032
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calculated from the dose curves generated by plotting percent
growth vs. the test concentration on a logarithmic scale. Doxorubi-
cin was used as positive control. All assays were performed in trip-
licate and the mean values are given in Table 1.
and iNOS protein structures (PDB codes: 1PGF, 1CX2 and 3E65),
respectively. Selected active pyrazolopyrimidine derivatives were
energy minimized using CHARMm ForceField and then docked into
the former prepared proteins active sites using LIGANDFIT imbed-
ded into Discovery Studio Software with the following docking
protocol: (i) number of Monte Carlo search trials = 30000, search
step for torsions with polar hydrogens = 30°. (ii) The Root Mean
Square Difference (RMS) threshold for ligand-to-binding site shape
match was set to 2.0 employing a maximum of 1.0 binding site par-
titions and 1.0 site partition seed. (iii) The interaction energies
were assessed employing Consistent Force Field (CFF) force field
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4.2.5. In vivo assays
Wistar rats (150–175 g) obtained from the National Research
Center, Cairo, Egypt) were used throughout these studies and were
kept at controlled conditions (temperature 23 2 °C, humidity
60 10%) and a 12/12 h light/dark cycle. All procedures relating
to animal care and treatments were conducted in accordance with
protocols approved by the Research Ethical Committee of Faculty
of Pharmacy Beni-Suef University, Beni-Suef, Egypt (REC/BSU/
P017B-2013).
with
a non-bonded cutoff distance of 10.0 Å and distance-
dependent dielectric. An energy grid extending 3.0 Å from the
binding site was implemented. (iv) Rigid body ligand minimization
parameters were: 10 iterations of steepest descend (SD) minimiza-
tion followed by 20 Broyden–Fletcher–Goldfarb–Shanno (BFGS)
iterations applied to every successful orientation of the docked
ligand. (v) A maximum of 10 diverse docked conformations/poses
of optimal interaction energies were saved. (vi) The saved con-
formers/poses were further energy-minimized within the binding
site for a maximum of 1000 rigid-body iterations.
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4.2.5.1. Carrageenan-induced thermal hyperalgesia. Animals under
light anesthesia received 100 lL of vehicle or carrageenan (1% in
993
saline) s.c. on the plantar surface of the left hindpaw. The rats
received orally vehicle, test compounds (10 mg/kg) or ketorolac
(10 mg/kg) 2 h after carrageenan administration and were evalu-
ated for paw hyperalgesia. Hyperalgesic responses to heat were
measured at the indicated time points as described before
(Hargreaves et al., 1988). A cutoff latency of 30 s was used to pre-
vent heat-induced tissue damage. Rats were individually placed
into plexiglass chambers. A high-intensity projector bulb (8-V,
50-watt) was used to deliver a focused thermal stimulus directly
to individual hindpaws from beneath the chamber. The withdrawal
latency period of paws was determined to the nearest 0.1 s. Each
point represented the change (s) in withdrawal latency compared
to vehicle-treated animals at each time point. Results are
expressed as Paw-withdrawal latency change (s).
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Acknowledgments
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We deeply thank Dr. Min Hye Yang and Ms. Katherine Martin
for their excellent technical help and support in carrying out the
in vitro bioassays.
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References
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1103
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1105
1106
1107
1108
Abdel-Magid, A.F., Carson, K.G., Harris, B.D., Maryanoff, C.A., Shah, R.D., 1996.
Reductive
amination
of
aldehydes
and
ketones
with
sodium
triacetoxyborohydride. Studies on direct and indirect reductive amination
procedures(1). J. Org. Chem. 61, 3849–3862.
Abunada, N.M., Hassaneen, H.M., Kandile, N.G., Miqdad, O.A., 2008. Synthesis and
antimicrobial activity of some new pyrazole, fused pyrazolo[3,4-d]-pyrimidine
and pyrazolo[4,3-e][1,2,4]-triazolo[1,5-c]pyrimidine derivatives. Molecules 13,
1501–1517.
Accelrys Software Inc., 2007. Discovery Studio Modeling Environment, Release 2.5.
Accelrys Software Inc., San Diego.
Allison, M.C., Howatson, A.G., Torrance, C.J., Lee, F.D., Russell, R.I., 1992.
Gastrointestinal damage associated with the use of nonsteroidal
antiinflammatory drugs. N. Engl. J. Med. 327, 749–754.
Almansa, C., Alfon, J., de Arriba, A.F., Cavalcanti, F.L., Escamilla, I., Gomez, L.A.,
Miralles, A., Soliva, R., Bartroli, J., Carceller, E., Merlos, M., Garcia-Rafanell, J.,
2003. Synthesis and structure–activity relationship of a new series of COX-2
selective inhibitors: 1,5-diarylimidazoles. J. Med. Chem. 46, 3463–3475.
Arrigoni-Martelli, E., 1979. Screening and assessment of antiinflammatory drugs.
Methods Find. Exp. Clin. Pharmacol. 1, 157–177.
Bakavoli, M., Bagherzadeh, G., Vaseghifar, M., Shiri, A., Pordel, M., Mashreghi, M.,
Pordeli, P., Araghi, M., 2010. Molecular iodine promoted synthesis of new
pyrazolo[3,4-d]pyrimidine derivatives as potential antibacterial agents. Eur. J.
Med. Chem. 45, 647–650.
Bondock, S., Rabie, R., Etman, H.A., Fadda, A.A., 2008. Synthesis and antimicrobial
activity of some new heterocycles incorporating antipyrine moiety. Eur. J. Med.
Chem. 43, 2122–2129.
Borenfreund, E., Babich, H., Martin-Alguacil, N., 1990. Rapid chemosensitivity assay
with human normal and tumor cells in vitro. In Vitro Cell. Dev. Biol. 26, 1030–
1034.
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4.2.5.2. Carrageenan-induced rat paw edema assay. Rats were
administered orally vehicle, test compounds (10 mg/kg) or ketoro-
lac (10 mg/kg). Immediately thereafter, the rats received 100 lL of
vehicle or carrageenan (1% in saline) s.c. on the plantar surface of
the left hindpaw under light anesthesia, essentially, as reported
before (Salvemini et al., 1996). The development of paw edema
was assessed by measuring paw-volume changes at 1, 3 and 6 h
after carrageenan injection using plethysmometer. The right hind-
paw served as a reference of non-inflamed paw for comparison.
Results are expressed as paw-volume change (mL).
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4.2.5.3. Acetic acid-induced writhing assay. The writhing tests were
carried as described before (Arrigoni-Martelli, 1979). Briefly, mice
were administered orally vehicle, test compounds (10 mg/kg) or
ketorolac (10 mg/kg) 30 minutes prior to intraperitoneal adminis-
tration of 0.7% v/v acetic acid solution at 10 mL/kg body weight.
The number of writhes (i.e., abdominal constriction followed by
dorsiflexion and extension) occurring during a 20 min period
beginning 5 min after acetic acid injection was measured. The
results are expressed as the number of writhes per 20-min period.
Celano, M., Schenone, S., Cosco, D., Navarra, M., Puxeddu, E., Racanicchi, L., Brullo, C.,
Varano, E., Alcaro, S., Ferretti, E., Botta, G., Filetti, S., Fresta, M., Botta, M., Russo,
D., 2008. Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped
in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors
in vivo. Endocr. Relat. Cancer 15, 499–510.
Chen, Y.L., 2000. Pyrazolopyrimidines for Treatment of CNS Disorders. United States
Patent 6051578.
Cheng, C.C., Robins, R.K., 1956. Potential purine antagonists. VI. Synthesis of 1-
Alkyl- and 1-Aryl-4-substituted pyrazolo[3,4-d]pyrimidines. J. Org. Chem. 21,
1240–1256.
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4.3. Statistical analysis
1027
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1031
1032
Statistical analyses were performed by SPSS 9.0 software (SPSS
Inc., Chicago, IL, USA). All results are presented as mean standard
deviation (SD) values. Statistical differences between means were
evaluated by one-way analysis of variance (ANOVA) followed by
the Neuman–Keults test for multiple comparisons. Differences
were considered significant at P < 0.05.
Chern, J.H., Shia, K.S., Hsu, T.A., Tai, C.L., Lee, C.C., Lee, Y.C., Chang, C.S., Tseng, S.N.,
Shih, S.R., 2004. Design, synthesis, and structure–activity relationships of
pyrazolo[3,4-d]pyrimidines:
a novel class of potent enterovirus inhibitors.
1033
Bioorg. Med. Chem. Lett. 14, 2519–2525.
4.4. Docking study
Cirino, G., Distrutti, E., Wallace, J.L., 2006. Nitric oxide and inflammation. Inflamm.
Allergy Drug Targets 5, 115–119.
Clancy, R.M., Amin, A.R., Abramson, S.B., 1998. The role of nitric oxide in
inflammation and immunity. Arthritis Rheum. 41, 1141–1151.
1034 Q4
The binding sites were generated from the co-crystallized
ligands (IMM, SC-558 and AR-C120011) within COX-1, COX-2
1035
Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025

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Cottam, H.B., Petrie, C.R., McKernan, P.A., Goebel, R.J., Dalley, N.K., Davidson, R.B.,
Robins, R.K., Revankar, G.R., 1984. Synthesis and biological activity of certain
3,4-disubstituted pyrazolo[3,4-d]pyrimidine nucleosides. J. Med. Chem. 27,
1119–1127.
Dannhardt, G., Kiefer, W., 2001. Cyclooxygenase inhibitors – current status and
future prospects. Eur. J. Med. Chem. 36, 109–126.
Peri, K.G., Hardy, P., Li, D.Y., Varma, D.R., Chemtob, S., 1995. Prostaglandin G/H
synthase-2 is a major contributor of brain prostaglandins in the newborn. J. Biol.
Chem. 270, 24615–24620.
Quang, D.N., Harinantenaina, L., Nishizawa, T., Hashimoto, T., Kohchi, C., Soma, G.,
Asakawa, Y., 2006. Inhibition of nitric oxide production in RAW 264.7 cells by
azaphilones from xylariaceous fungi. Biol. Pharm. Bull. 29, 34–37.
Quintela, J.M., Peinador, C., Gonzalez, L., Devesa, I., Ferrandiz, M.L., Alcaraz, M.J.,
Riguera, R., 2003. 6-Dimethylamino 1H-pyrazolo[3,4-d]pyrimidine derivatives
as new inhibitors of inflammatory mediators in intact cells. Bioorg. Med. Chem.
11, 863–868.
Raffa, D., Maggio, B., Plescia, F., Cascioferro, S., Raimondi, M.V., Plescia, S., Cusimano,
M.G., 2009. Pyrazolo[3,4-d]pyrimidine derivatives as COX-2 selective inhibitors:
synthesis and molecular modelling studies. Arch. Pharm. (Weinheim) 342, 321–
326.
Rathod, I.S., Baheti, K.G., Shirsath, V.S., 2005. Synthesis and analgesic activity of 2-
(un) substituted-3-amino-5-aryl-6-phenyl pyrazolo(3,4-d)pyrimidine-4-(5H)-
ones. Ind. J. Pharm. Sci. 67, 593–597.
Devesa, I., Alcaraz, M.J., Riguera, R., Ferrandiz, M.L., 2004.
A new pyrazolo
pyrimidine derivative inhibitor of cyclooxygenase-2 with anti-angiogenic
activity. Eur. J. Pharmacol. 488, 225–230.
el-Bendary, E.R., Badria, F.A., 2000. Synthesis, DNA-binding, and antiviral activity of
certain pyrazolo[3,4-d]pyrimidine derivatives. Arch. Pharm. (Weinheim) 333,
99–103.
Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson, G.,
Andrews, G., Hamley, P.J., Mallinder, P.R., Nicholls, D.J., St-Gallay, S.A., Tinker,
A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuehr, D.J., Aberg,
A., Wallace, A.V., Tainer, J.A., Getzoff, E.D., 2008. Anchored plasticity opens doors
for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol. 4, 700–
707.
Robichaud, J., Black, W.C., Therien, M., Paquet, J., Oballa, R.M., Bayly, C.I., McKay, D.J.,
Wang, Q., Isabel, E., Leger, S., Mellon, C., Kimmel, D.B., Wesolowski, G., Percival,
M.D., Masse, F., Desmarais, S., Falgueyret, J.P., Crane, S.N., 2008. Identification of
Ghosh, S., Karin, M., 2002. Missing pieces in the NF-kappaB puzzle. Cell 109 (Suppl.),
S81–S96.
Gökhan, N., Erdogan, H., Tel, B., Demirdamar, R., 1996. Analgesic and
antiinflammatory activity screening of 6-acyl-3-piperazinomethyl-2-
benzoxazolinone derivatives. Eur. J. Med. Chem. 31, 625–628.
Guccionea, S., Modicaa, M., Longmoreb, J., Shawb, D., Barrettac, G., Santagatia, A.,
Santagatia, M., Russo, F., 1996. Synthesis and NK-2 antagonist effect of 1,6-
diphenyl-pyrazolo[3,4-d]-thiazolo[3,2-a]4H-pyrimidin-4-one. Bioorg. Med.
Chem. Lett. 6, 59–64.
a nonbasic, nitrile-containing cathepsin K inhibitor (MK-1256) that is
efficacious in a monkey model of osteoporosis. J. Med. Chem. 51, 6410–6420.
Salgin-Goksen, U., Gokhan-Kelekci, N., Goktas, O., Koysal, Y., Kilic, E., Isik, S., Aktay,
G., Ozalp, M., 2007. 1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones,
1,3,4-thiadiazoles and hydrazones containing 5-methyl-2-benzoxazolinones:
synthesis, analgesic-anti-inflammatory and antimicrobial activities. Bioorg.
Med. Chem. 15, 5738–5751.
Salvemini, D., Manning, P.T., Zweifel, B.S., Seibert, K., Connor, J., Currie, M.G.,
Needleman, P., Masferrer, J.L., 1995. Dual inhibition of nitric oxide and
prostaglandin production contributes to the antiinflammatory properties of
nitric oxide synthase inhibitors. J. Clin. Invest. 96, 301–308.
Salvemini, D., Wang, Z.Q., Wyatt, P.S., Bourdon, D.M., Marino, M.H., Manning, P.T.,
Currie, M.G., 1996. Nitric oxide: a key mediator in the early and late phase of
carrageenan-induced rat paw inflammation. Br. J. Pharmacol. 118, 829–838.
Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins, W., Lee, L., Isakson,
P., 1994. Pharmacological and biochemical demonstration of the role of
cyclooxygenase 2 in inflammation and pain. Proc. Natl. Acad. Sci. USA 91,
12013–12017.
Gund, P., Shen, T.Y., 1977.
A
model for the prostaglandin synthetase
cyclooxygenation site and its inhibition by antiinflammatory arylacetic acids.
J. Med. Chem. 20, 1146–1152.
Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive
method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32,
77–88.
Ialenti, A., Ianaro, A., Moncada, S., Di Rosa, M., 1992. Modulation of acute
inflammation by endogenous nitric oxide. Eur. J. Pharmacol. 211, 177–182.
Ianaro, A., O’Donnell, C.A., Di Rosa, M., Liew, F.Y., 1994. A nitric oxide synthase
inhibitor reduces inflammation, down-regulates inflammatory cytokines and
enhances interleukin-10 production in carrageenin-induced oedema in mice.
Immunology 82, 370–375.
Seminerio, M.J., Robson, M.J., Abdelazeem, A.H., Mesangeau, C., Jamalapuram, S.,
Avery, B.A., McCurdy, C.R., Matsumoto, R.R., 2012. Synthesis and
Kulmacz, R.J., Lands, W.E., 1983. Requirements for hydroperoxide by the
cyclooxygenase and peroxidase activities of prostaglandin
Prostaglandins 25, 531–540.
H
synthase.
pharmacological characterization of
improved metabolic stability
methamphetamine. AAPS J. 14, 43–51.
a
and
novel sigma receptor ligand with
antagonistic effects against
Kurumbail, R.G., Stevens, A.M., Gierse, J.K., McDonald, J.J., Stegeman, R.A., Pak, J.Y.,
Gildehaus, D., Miyashiro, J.M., Penning, T.D., Seibert, K., Isakson, P.C., Stallings,
W.C., 1996. Structural basis for selective inhibition of cyclooxygenase-2 by anti-
inflammatory agents. Nature 384, 644–648.
Lim, K.M., Lee, J.Y., Lee, S.M., Bae, O.N., Noh, J.Y., Kim, E.J., Chung, S.M., Chung, J.H.,
2009. Potent anti-inflammatory effects of two quinolinedione compounds, OQ1
and OQ21, mediated by dual inhibition of inducible NO synthase and
cyclooxygenase-2. Br. J. Pharmacol. 156, 328–337.
Looker, D.L., Marr, J.J., Berens, R.L., 1986. Mechanisms of action of
pyrazolopyrimidines in Leishmania donovani. J. Biol. Chem. 261, 9412–9415.
Ma, G., Khan, S.I., Benavides, G., Schuhly, W., Fischer, N.H., Khan, I.A., Pasco, D.S.,
2007. Inhibition of NF-kappaB-mediated transcription and induction of
apoptosis by melampolides and repandolides. Cancer Chemother. Pharmacol.
60, 35–43.
Ma, L., Xie, C., Ma, Y., Liu, J., Xiang, M., Ye, X., Zheng, H., Chen, Z., Xu, Q., Chen, T.,
Chen, J., Yang, J., Qiu, N., Wang, G., Liang, X., Peng, A., Yang, S., Wei, Y., Chen, L.,
2011. Synthesis and biological evaluation of novel 5-benzylidenethiazolidine-
2,4-dione derivatives for the treatment of inflammatory diseases. J. Med. Chem.
54, 2060–2068.
Spreafico, A., Schenone, S., Serchi, T., Orlandini, M., Angelucci, A., Magrini, D.,
Bernardini, G., Collodel, G., Di Stefano, A., Tintori, C., Bologna, M., Manetti, F.,
Botta, M., Santucci, A., 2008. Antiproliferative and proapoptotic activities of new
pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human
osteosarcoma cells. FASEB J. 22, 1560–1571.
Trivedi, A., Vaghasiya, S., Dholariya, B., Dodiya, D., Shah, V., 2010. Synthesis and
antimycobacterial evaluation of various 6-substituted pyrazolo[3,4-
d]pyrimidine derivatives. J. Enzyme Inhib. Med. Chem. 25, 893–899.
Trivedi, A.R., Dholariya, B.H., Vakhariya, C.P., Dodiya, D.K., Ram, H.K., Kataria, V.B.,
Siddiqui, A.B., Shah, V.H, 2012. Synthesis and anti-tubercular evaluation of some
novel pyrazolo[3,4-d]pyrimidine derivatives. Med. Chem. Res. 8, 1887–1891.
Viaud, M.C., Jamoneau, P., Flouzat, C., Bizot-Espiard, J.G., Pfeiffer, B., Renard, P.,
Caignard, D.H., Adam, G., Guillaumet, G., 1995. N-substituted oxazolo[5,4-
b]pyridin-2(1H)-ones: a new class of non-opiate antinociceptive agents. J. Med.
Chem. 38, 1278–1286.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind paw
of the rat as an assay for antiiflammatory drugs. Proc. Soc. Exp. Biol. Med. 111,
544–547.
Wolfe, M.M., Lichtenstein, D.R., Singh, G., 1999. Gastrointestinal toxicity of
nonsteroidal antiinflammatory drugs. N. Engl. J. Med. 340, 1888–1899.
Xia, Y., Chackalamannil, S., Czarniecki, M., Tsai, H., Vaccaro, H., Cleven, R., Cook, J.,
Fawzi, A., Watkins, R., Zhang, H., 1997. Synthesis and evaluation of polycyclic
pyrazolo[3,4-d]pyrimidines as PDE1 and PDE5 cGMP phosphodiesterase
inhibitors. J. Med. Chem. 40, 4372–4377.
MacMicking, J., Xie, Q.W., Nathan, C., 1997. Nitric oxide and macrophage function.
Annu. Rev. Immunol. 15, 323–350.
Mesangeau, C., Narayanan, S., Green, A.M., Shaikh, J., Kaushal, N., Viard, E., Xu, Y.T.,
Fishback, J.A., Poupaert, J.H., Matsumoto, R.R., McCurdy, C.R., 2008. Conversion
of a highly selective sigma-1 receptor-ligand to sigma-2 receptor preferring
ligands with anticocaine activity. J. Med. Chem. 51, 1482–1486.
Micklewright, R., Lane, S., Linley, W., McQuade, C., Thompson, F., Maskrey, N., 2003.
Review article: NSAIDs, gastroprotection and cyclo-oxygenase-II-selective
inhibitors. Aliment. Pharmacol. Ther. 17, 321–332.
Mustafa, J., Khan, S.I., Ma, G., Walker, L.A., Khan, I.A., 2004. Synthesis and anticancer
activities of fatty acid analogs of podophyllotoxin. Lipids 39, 167–172.
Natarajan, S.K., Moreno, O., Graddis, T.J., Duncan, D., Laus, R., Chen, F., 2005.
Compositions and methods using small mol. Trp-p8 Modulators for the
Treatment of Diseases Associated with Trp-p8 Expression. Patent WO
2005020897.
Yewale, S.B., Ganorkar, S.B., Baheti, K.G., Shelke, R.U., 2012. Novel 3-substituted-1-
aryl-5-phenyl-6-anilinopyrazolo[3,4-d]pyrimidin-4-ones: docking, synthesis
and pharmacological evaluation as
a potential anti-inflammatory agents.
Bioorg. Med. Chem. Lett. 22, 6616–6620.
Yoshimura, H., Sekine, S., Adachi, H., Uematsu, Y., Mitani, A., Futaki, N., Shimizu, N.,
2011. High levels of human recombinant cyclooxygenase-1 expression in
mammalian cells using a novel gene amplification method. Protein Expr. Purif.
80, 41–46.
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Please cite this article in press as: Abdelazeem, A.H., et al. Novel pyrazolopyrimidine derivatives targeting COXs and iNOS enzymes; design, synthesis and
biological evaluation as potential anti-inflammatory agents. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.025