Molecular docking design and one-pot expeditious synthesis of novel 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2015.06.011

Abstract A series of novel 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines were designed as COX-2 inhibitors by molecular docking studies and their synthesis was accomplished via an expeditious one-pot reaction. Structures of the compounds were established by

Full text of DOI:10.1016/j.ejmech.2015.06.011

Accepted Manuscript
Molecular docking design and one-pot expeditious synthesis of novel 2,5-
diarylpyrazolo[1,5-a]pyrimidin-7-amines as anti-inflammatory agents
Ranjana Aggarwal, Gulshan Singh, Pawan Kaushik, Dhirender Kaushik, Deepika
Paliwal, Ajay Kumar
PII:
S0223-5234(15)30085-4
10.1016/j.ejmech.2015.06.011
EJMECH 7938
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2014
Revised Date: 2 June 2015
Accepted Date: 3 June 2015
Please cite this article as: R. Aggarwal, G. Singh, P. Kaushik, D. Kaushik, D. Paliwal,
A. Kumar, Molecular docking design and one-pot expeditious synthesis of novel 2,5-
diarylpyrazolo[1,5-a]pyrimidin-7-amines as anti-inflammatory agents, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.011.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Molecular docking design and one-pot expeditious synthesis of novel 2,5-diarylpyrazolo[1,5-
a]pyrimidin-7-amines as anti-inflammatory agents
Ranjana Aggarwala, Gulshan Singha, Pawan Kaushikb, Dhirender Kaushikb, Deepika
Paliwalb, Ajay Kumarb
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, Haryana, India
bDepartment of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, India
ae-mail: ranjanaaggarwal67@gmail.com, ranjana67in@yahoo.com.
NH2
O
N
Toluene / EtOH (9:1)
Reflux, 4h
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NH2NH2.H2O
+
Catalytic PTSA
R
N
1
R'/R
2a-e
4a-k, Yield 62-71%
R'
c
e
a
b
d
f
g
h
i
j
k
4
R
H
Br CH3 Br
CH3
H
H
CH3
H
CH3
CH3 Cl
Cl
CH3
Cl Br Cl Br
R'
F
Cl
Br
CH3

ACCEPTED MANUSCRIPT
Molecular docking design and one-pot expeditious synthesis of novel 2,5-
diarylpyrazolo[1,5-a]pyrimidin-7-amines as anti-inflammatory agents
Ranjana Aggarwala, Gulshan Singha, Pawan Kaushikb, Dhirender Kaushikb, Deepika
Paliwalb, Ajay Kumarb
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, Haryana, India
bDepartment of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, India
ae-mail: ranjanaaggarwal67@gmail.com, ranjana67in@yahoo.com.
Abstract
A series of novel 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines were designed as COX-2
inhibitors by molecular docking studies and their synthesis was accomplished via an
expeditious one-pot reaction. Structures of the compounds were established by NMR (1H-
13C), IR spectroscopy and high resolution mass spectrometry. All the eleven compounds have
been screened for their in vivo anti-inflammatory activity on rats by carrageenan-induced rat
paw edema assay. Correlation studies of calculated moldock score and observed percentage
inhibition have also been carried out which concluded that the synthesized 2,5-
diarylpyrazolo[1,5-a]pyrimidin-7-amines act as potent anti-inflammatory agents up to the 4th
hour of study.
Keywords 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines, hydrazine hydrate, 3-aryl-3-
oxopropanonitriles, p-toluenesulphonic acid, anti-inflammatory agents, molecular docking.
1

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1. Introduction
Inflammation, an important natural and beneficial response imposed by the body
defence mechanism in order to cease infections, involves certain inflammatory reactions like
pain, swelling and mental discomfort. Thus relieving and reducing the severity of symptoms
stand as an important goal with the use of therapeutic agents. Use of non-steroidal anti-
inflammatory drugs (NSAIDs), disease modifying anti-rheumatic drugs (DMARDs),
corticosteroids and immunosuppressive agents is generally accepted stepwise
chemotherapeutic approach to treat the inflammatory disorder.
Pyrazole [1] and fused pyrazole [2] with six-membered ring nucleus are main skeletal feature
among different types of non-steroidal anti-inflammatory drugs. Among these, pyrazolo[1,5-
a]pyrimidine system, due to its purine analogy, represents an important class and is found to
display anti-inflammatory activity by acting as selective cyclooxygenase-2 (COX-2) inhibitor
[3]. Almansa et al have synthesized a series of pyrazolo[1,5-a]pyrimidines (I) which exhibit
in vitro inhibitory activitiy of COX-1 and COX-2 in a human whole blood (HWB ) assay
along with in vivo anti-inflammatory action studies in rat carrageenan-induced paw edema
assay [4]. Structure activity relationship (SAR) studies have proposed that the compounds
having substitution at the 5th, 6th and 7th position of pyrazolo[1,5-a]pyrimidine nucleus
display anti-inflammatory activity (Figure-1). The compound, 3-(4-fluorophenyl)-6,7-
dimethyl-2-(4-(methylsulphonyl)phenyl)pyrazolo[1,5-a]pyrimidine (II), was found to be the
most promising among the series of compounds listed as I where the position 6 and 7 was
occupied by methyl groups.
Along with the bio-active profile, synthesis of pyrazolo[1,5-a]pyrimidine by developing
simpler routes from readily available reagents and compounds stands as a challenging task in
organic synthesis. Multi-component condensation reactions (MCRs) and one-pot
condensation reactions provide a fair pathway to overcome this obstacle where the ease of
simplicity in procedures, high atom economy, high yields and avoidance of intermediate
isolation are observed [5]. Moreover, multi-component condensation reactions may also be a
useful synthetic strategy to bring out the regioselectivity in the reaction [6].
Thus keeping in mind the anti-inflammatory ability of pyrazolo[1,5-a]pyrimidine nucleus, it
was envisaged in the present study to design some new pyrazolo[1,5-a]pyrimidines by
applying few more structural modifications to the compound I (Figure-1) considering the
SAR studies of Almansa et al [4]. Pyrazolo[1,5-a]pyrimidines thus designed were
synthesized by one pot efficient and regioselective multicomponent reaction and were
evaluated for their anti-inflammatory activity.
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2. Molecular Docking Designing
In an effort to develop some novel pyrazolo[1,5-a]pyrimidines, the compounds 4a-k
(Figure-2) were taken into the consideration where methyl group at 7th position of the
pyrazolo[1,5-a]pyrimidine is replaced by amino group having steric profile comparable to
methyl group (Compound-II in Figure-1) but having additional advantage of ability to bind
with H-bonds in active site of responsible enzyme. Position-2 of the nucleus is occupied with
the aryl residue similar to compounds (I) but with a difference of substitution at the para
position. Variously para substituted aryl residues were shifted from position-3 to 5 of the
pyrazolo[1,5-a]pyrimidine nucleus so as to bring out structural deviation towards planarity in
the whole structure for better fitting compared to compound II (Figure-1).
The core domain of COX-2 with a standard ligand SC-558 was chosen for the interactive
studies of designed compounds 4a-k. In an effort to understand the binding between
pyrazolo[1,5-a]pyrimidines (4a-k) and COX-2, these compounds were docked to the core
domain of COX-2. Molegro Virtual Docker allows the flexible docking of ligands into its site
of action. It has the ability to use all the rotatable bonds of the ligands to give a number of
conformations from which the best mode could be achieved. All compounds were embedded
in the hydrophobic pocket formed by the amino acids. The results of docking studies with
respect to COX-2 inhibitor are summarised in Table 1.
Standard compound indomethacin showed three hydrogen bond interactions with OH and
C=O of carboxylic acid group, -O- of methoxy group, respectively. Compound II also
showed three interaction via hydrogen bonds with N of pyrimidine part and oxygen of SO2
region. Nitrogen of the sulphonamide group of internal ligand SC-558 of PDB (Protein Data
Bank) showed two hydrogen bond interactions with amino acids Ser353 & His90 with
hydrogen bond length 2.76Aº & 2.96Aº, respectively. Oxygen of sulphonamide group of SC-
558 showed interaction with Arg513 having distance 3.27Aº. Among these three,
indomethacin and compound II underwent a common hydrogen bond interaction with amino
acid residue Tyr355. Likewise, in most of the compounds (4), N of NH2 group showed
interaction with amino acid Tyr355 (2.55Aº -2.84Aº) and His90 (3.42Aº-3.52Aº) except
compound 4j and 4e, which showed interaction with Phe518 (2.54Aº-2.56Aº) and Gly519
(3.50Aº-3.57Aº). N of pyrazole ring showed strong interactions with amino acids Tyr355 and
Arg120 in compound 4d,. The secondary structure of PDB COX-2, compound SC-558 and
4k embedded in PDB were shown in Figure 3 and 4, respectively.
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Molecular docking results indicated that the designed compounds 4 may be potential COX-2
inhibitor, therefore a reterosynthetic strategy was taken in account so as to achieve this
scaffold from readily available starting materials such as hydrazine hydrate (1) and 3-aryl-3-
oxopropanonitriles (2a-e) (Figure-5).
3. Result and Discussion
In our earlier attempts, the reaction of hydrazine hydrate (1), 3-oxo-3-
phenylpropanonitriles (2a) and 3-oxo-3-p-tolylpropanonitrile (2b) was carried out in
refluxing ethanol with or without the presence of p-toluenesulphonic acid (PTSA). However,
it led to the formation of mixture of 5-aminopyrazoles corresponding to two 3-aryl-3-
oxopropanonitriles. Performing reaction at elevated temperature by refluxing in toluene
without PTSA again afforded 5-aminopyrazoles mixture. When the reaction of hydrazine
hydrate (1) with successive addition of 2a followed by 2b was performed with catalytic
PTSA in refluxing toluene, formation of the desired product 4a was observed but with poor
yield. Finally, on turning the reaction solvent from toluene to a mixture of toluene and
ethanol (9:1), the reaction led to desired product 2-phenyl-5-p-tolylpyrazolo[1,5-a]pyrimidin-
7-amine 4a in 65% yield within 4 hours (Scheme-1).
1
13
The structure elucidation of the product formed (4a) was carried out by TLC, H NMR, C
NMR, IR spectroscopy and high resolution mass spectrometry.
IR spectrum of 4a showed two characteristic absorption stretches at 3325 and 3350 cm-1
1
corresponding to the symmetric and asymmetric N-H stretches of 7-amino group. H NMR
spectrum of 4a showed two singlets of one proton intensity each at δ 6.59 ppm and 6.90 ppm
of protons 6-H and 3-H, respectively. A broad singlet exchangeable with D2O of two protons
intensity at δ 7.72 ppm was observed for amino group at position-7 of pyrazolo[1,5-
a]pyrimidine nucleus. A sharp singlet appeared at δ 2.38 ppm for the methyl group of p-tolyl
13
residue at position-5 besides the protons of aryl groups. In C NMR, two signals at δ 84.7
ppm and δ 91.5 ppm were observed corresponding to 6-C and 3-C. Signal for 7-C appeared at
δ 148.45 of pyrazolo[1,5-a]pyrimidine nucleus thus confirming the formation of 2-phenyl-5-
p-tolylpyrazolo[1,5-a]pyrimidin-7-amine isomer (4a). The spectral data is in conformity with
our recent report where in regioselective synthesis of 7-aminopyrazolo[1,5-a]pyrimidines
have been achieved by the reaction of 3(5)-amino5(3)-hydrazinopyrazole with 3-oxo-3-
phenylpropanonitrile (2a) and the position of substituents on pyrazolopyrimidine ring has
been established by the combined use of NMR (HMBC & HMQC) and DFT calculations [7].
High resolution mass spectrum of compound 4a showed a mass peak at m/z value of
301.1363 [M+1]+.
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With the optimized conditions in hand, further reaction of hydrazine hydrate (1) with two
different 3-aryl-3-oxopropanonitriles (2a-e) in refluxing toluene/ethanol (9:1) in presence of
catalytic p-toluenesulphonic acid (Scheme-2) resulted in a series of 2,5-diarylpyrazolo[1,5-
a]pyrimidin-7-amines (4b-k) in 60-70% yield.
4. Biological Activity
All the synthesized compounds (4a-k) have been tested for their anti-inflammatory
using Winter et al. carrageenan-induced rat paw edema assay protocol approved by the
Institutional Animal Ethics Committee (IAEC) [8]. 50 mg/kg body weight dose level of each
test compound was given orally to the test animals 30 min prior to induction of inflammation
by carrageenan injection. Indomethacin, reference anti-inflammatory drug was given as a
dose of 10 mg/kg, orally. The results of anti-inflammatory activity of the evaluated
compounds and the reference non-steroidal anti-inflammatory drug (indomethacin) are listed
in Table 2 and Table 3.
Examination of activity results from Table 1 and Table 2 showed that all the tested
compounds 4a-k were having moderate to comparable activity as comparable to the standard
drug indomethacin. Activity tables shows that the synthesized compounds 4c, 4f, 4g, 4h, 4j
and 4k start acting in first hour and also remain active up to the 4th hour of study. Except
compound 4d, all the remaining tested compounds show their potency as anti-inflammatory
agent in the 4th hour which can be observed in Figure-5 where hour wise % inhibition is
plotted of tested compounds and the standard drug.
On observing the dock results in Table-1 and Figure 6, this can be ascertained that
interaction of compounds 4a-k with amino acid Tyr355 in COX-2 active pocket is an
important interaction as observed by the compound II and standard drug as well. Whereas,
the excessive Tyr355 interaction in case of compound 4d might have caused the deformation
in the active site as it stands as the least active compound in activity comparison graph
(Figure-6). Interaction with amino acid His90 common in case of compounds 4c, 4g, 4k and
SC-558 also stands an important interaction for activity. Noteworthy interaction of amino
group nitrogen with amino acid residue Tyr355 in all compounds 4a-k makes this an
important SAR around pyrazolo[1,5-a]pyrimidine nucleus (Table-1) as this is the only
bonding that compounds 4a, b, f, h and i show in their docking results but still these
compounds are prominently active.
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Without specific tests, it is quite difficult to hypothesize the mechanism of action of active
compounds. Probable inhibition of the cyclooxygenase enzymes like other non-steroidal anti-
inflammatory agents might be the mode of action in case of active compounds.
After comparing the moldock score with data obtained from biological evaluation, an
interesting correlation was obtained between the two. We studied the correlation between
moldock score and percentage inhibition of these compounds as given in Figure-7. As the
anti-inflammatory effect of our compounds was evaluated further after fix interval of times
the r2 value between moldock score and percentage inhibition also increased proportionally.
But after 3hr the correlation value slightly decrease and then increases. This interesting
relation confirms that our compounds are potential anti-inflammatory agents which were
confirmed from the biological evaluation as well as molecular docking studies.
5. Conclusion
In conclusion, we have described an efficient, simple, expeditious one-pot protocol
for the preparation of novel 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines (4a-k) designed by
molecular docking. Compounds have been screened for their anti-inflammatory activity and
their activity results have been correlated with dock score. Good agreement in anti-
inflammatory potential of screened compounds (4a-k) and molecular docking studies make
these a new scaffold in the development of potent anti-inflammatory therapeutics.
6. Experimental
6.1 Materials and methods
Melting point were determined in open capillaries with an electrical apparatus and are
uncorrected, The IR spectra of the compounds were recorded on ABB MB3000 FT-IR
1
13
laboratory analyser combined with Horizon MBTM FTIR software (νmax in cm-1). H and C
NMR spectra were recorded on a Bruker instrument at 300 and 75 MHz, respectively.
Chemical shifts are expressed in δ-scale downfield from TMS as an internal standard. High
resolution mass spectra (HRMS) were measured in EI mode on a Kratos MS-50 spectrometer.
Hydrazine hydrate (1) was commercially available and 3-aryl-3-oxopropanonitriles (2a-e)
were prepared according to literature procedure [9].
6.2 General procedure for synthesis 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines (4a-
k):
To the 1 mol eq. of 3-aryl-3-oxopropanonitrile (2) was added 1 mol eq. of hydrazine hydrate
(1) in Toluene/EtOH (9:1) and the reaction mixture was refluxed for 30 min then another 1
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mol eq. of different 3-aryl-3-oxopropanonitrile (2) and catalytic amount of PTSA (0.01 mol
eq.) was added. Again reaction mixture was refluxed up to 4 hr. On completion of the
reaction, excess solvent was distilled off. The solid so obtained was filtered and washed with
cold ethanol. Solid was neutralised with aq. sodium bicarbonate solution and again filtered.
Compound thus obtained was air dried and recrystallized from ethanol.
Analytical and spectral data of synthesized compounds 4a-k:
6.2.1. 2-Phenyl-5-p-tolylpyrazolo[1,5-a]pyrimidin-7-amine (4a):
1
Yield 65%; Mp: 218-220°C; IR (cm-1): 3325, 3350 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.38 (s, 3H, 4"-CH3), 6.59 (s, 1H, 6-H), 6.90 (s, 1H, 3-H), 7.31-7.50 (m, 5H, Ar-H), 7.72
(bs, 2H, 7-NH2), 7.93-8.09 (m, 4H, Ar-H); 13C-NMR δ (DMSO-d6): 21.30, 84.70, 91.51,
126.61, 127.10, 129.15. 129.74, 133.49, 135.82, 139.77, 148.45, 150.77, 154.90, 154.97,
156.23; HRMS (EI) m/z for C19H16N4 calculated: 300.1375, found 301.1363 [M+1]+.
6.2.2. 2-Phenyl-5-p-chlorophenylpyrazolo[1,5-a]pyrimidin-7-amine (4b):
1
Yield 62%; Mp: 178-180°C; IR (cm-1): 3317, 3410 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 6.60 (s, 1H, 6-H), 6.93 (s, 1H, 3-H), 7.42-7.58 (m, 5H, Ar-H), 7.72 (bs, 2H, 7-NH2),
8.05-8.08 (m, 4H, Ar-H); 13C-NMR δ (DMSO-d6): 84.84, 92.07, 126.63, 128.96, 129.17,
129.29, 133.36, 134.88, 137.40, 148.57, 150.47, 154.94, 155.09; HRMS (EI) m/z for
C18H13ClN4 calculated: 320.0829, found: 321.0815 [M+1]+ 323.0816 [M+1+2]+(3:1).
,
6.2.3. 2-Phenyl-5-p-bromophenylpyrazolo[1,5-a]pyrimidin-7-amine (4c):
1
Yield 69%; Mp: 198-200°C; IR (cm-1): 3171, 3286 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 6.60 (s, 1H, 6-H), 6.93 (s, 1H, 3-H), 7.42-7.72 (m, 5H, Ar-H), 7.81 (bs, 2H, 7-NH2),
7.98-8.08 (m, 4H, Ar-H); 13C-NMR δ (DMSO-d6): 84.74, 92.10, 122.49, 123.63, 126.64,
128.59, 129.22, 132.12, 133.40, 137.83, 148.58, 150.68, 154.95, 155.08; HRMS (EI) m/z for
C18H13BrN4 calculated: 364.0324, found: 365.0314 [M+1]+ 366.0316 [M+1+2]+(1:1).
,
6.2.4. 2-p-Tolyl-5-phenylpyrazolo[1,5-a]pyrimidin-7-amine (4d):
1
Yield 63%; Mp: 236-238°C; IR (cm-1): 3325, 3350 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.37 (s, 3H, 4'-CH3), 6.60 (s, 1H, 6-H), 6.87 (s, 1H, 3-H), 7.29-7.32 (d, 2H, Ar-H), 7.49-
7.52 (m, 3H, Ar-H ), 7.72 (bs, 2H, 7-NH2), 7.95-7.98 (d, 2H, Ar-H), 8.03-8.05 (d, 2H, Ar-H);
13C-NMR δ (DMSO-d6): 21.38, 84.81, 91.69, 126.55, 127.15, 129.14, 129.72, 130.03,
130.75, 138.71, 148.47, 150.58, 155.02, 156.11; HRMS (EI) m/z for C19H16N4: calculated
300.1375, found: 301.1373 [M+1]+.
6.2.5. 2-p-Tolyl-5-p-fluorophenylpyrazolo[1,5-a]pyrimidin-7-amine (4e):
7

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1
Yield 69%; Mp: 202-204°C; IR (cm-1): 3250, 3290 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.37 (s, 3H, 4'-CH3), 6.57 (s, 1H, 6-H), 6.87 (s, 1H, 3-H), 7.32-7.41 (m, 4H, Ar-H), 7.75
13
(bs, 2H, 7-NH2), 7.95-7.98 (m, 2H, Ar-H), 8.08-8.11 (m, 2H, Ar-H); C-NMR δ (DMSO-
d6): 21.37, 84.79, 91.07, 126.52, 127.16, 129.18, 129.69, 130.10, 130.71, 138.75, 148.45,
150.52, 155.08, 156.16; HRMS (EI) m/z for C19H15FN4 calculated: 318.1281, found:
319.1278 [M+1]+.
6.2.6. 2-p-Chlorophenyl-5-p-tolylpyrazolo[1,5-a]pyrimidin-7-amine (4f):
1
Yield 70%; Mp: 264-266°C; IR (cm-1): 3190, 3260 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.38 (s, 3H, 4''-CH3), 6.60 (s, 1H, 6-H), 6.93 (s, 1H, 3-H), 7.31-7.38 (d, 2H, Ar-H), 7.55-
7.58 (d, 2H, Ar-H), 7.73 (bs, 2H, 7-NH2), 7.93-7.95 (d, 2H, Ar-H); 8.08-8.10 (d, 2H, Ar-H);
13C-NMR δ (DMSO-d6): 21.31, 84.80, 92.07, 126.63, 128.96, 129.17, 133.36, 134.88,
137.40, 148.57, 154.94, 155.09; HRMS (EI) m/z for C19H15ClN4 calculated: 334.0985, found:
335.0978 [M+1]+ 337.0976 [M+1+2]+(3:1).
,
6.2.7. 2-p-Tolyl-5-p-chlorophenylpyrazolo[1,5-a]pyrimidin-7-amine (4g):
1
Yield 65%; Mp: 240-242°C; IR (cm-1): 3240, 3279 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.37 (s, 3H, 4-CH3), 6.59 (s, 1H, 6-H), 6.88 (s, 1H, 3-H), 7.29-7.32 (d, 2H, Ar-H), 7.56-
7.58 (d, 2H, Ar-H), 7.77 (bs, 2H, 7-NH2), 7.95-7.98 (d, 2H, Ar-H); 8.05-8.08 (d, 2H, Ar-H);
13C-NMR δ (DMSO-d6): 21.38, 84.67, 91.80, 126.56, 128.92, 129.17, 129.72, 130.68,
134.80, 137.53, 138.68, 148.53, 150.68, 154.77, 155.13; HRMS (EI) m/z for C19H15ClN4
calculated : 334.0985, found: 335.1066 [M+1]+ 337.1047 [M+1+2]+(3:1).
,
6.2.8. 2-p-Chlorophenyl-5-p-bromophenylpyrazolo[1,5-a]pyrimidin-7-amine (4h):
1
Yield 69%; Mp: 252-254°C; IR (cm-1): 3240, 3286 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 6.61 (s, 1H, 6-H), 6.96 (s, 1H, 3-H), 7.56-7.59 (d, 2H, Ar-H), 7.70-7.72 (d, 2H, Ar-H),
7.84 (bs, 2H, 7-NH2), 7.98-8.01 (d, 2H, Ar-H); 8.08-8.11 (d, 2H, Ar-H); 13C-NMR δ
(DMSO-d6): 84.97, 92.25, 123.70, 128.31, 128.51, 128.59, 129.22, 132.10, 133.97, 137.73,
148.57, 150.72, 153.88, 155.15; HRMS (EI) m/z for C18H12BrClN4 calculated: 397.9934,
found: 398.9929 [M+1]+ 400.9928 [M+1+2]+, 402.9927 [M+1+4]+, (3:4:1).
,
6.2.9. 2-p-Bromophenyl-5-p-chlorophenylpyrazolo[1,5-a]pyrimidin-7-amine (4i):
1
Yield 62%; Mp: 262-264°C; IR (cm-1): 3210, 3250 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 6.61 (s, 1H, 6-H), 6.96 (s, 1H, 3-H), 7.55-7.58 (d, 2H, Ar-H), 7.69-7.72 (d, 2H, Ar-H),
7.83 (bs, 2H, 7-NH2), 8.01-8.07 (m, 4H, Ar-H); 13C-NMR δ (DMSO-d6): 84.91, 92.30,
123.66, 128.31, 129.22, 132.12, 137.79, 148.50, 150.76, 153.84; HRMS (EI) m/z for
C18H12BrClN4 calculated: 397.9934, found: 398.9987 [M+1]+ 400.0022 [M+1+2]+, 402.9937
,
[M+1+4]+ (3:4:1).
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6.2.10. 2-p-Tolyl-5-p-bromophenylpyrazolo[1,5-a]pyrimidin-7-amine (4j):
1
Yield 67%; Mp: 258-260°C; IR (cm-1): 3294, 3441 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.37 (s, 3H, 4'-CH3), 6.58 (s, 1H, 6-H), 6.88 (s, 1H, 3-H), 7.29-7.32 (d, 2H, Ar-H), 7.69-
7.72 (d, 2H, Ar-H); 7.78 (bs, 2H, 7-NH2), 7.95-8.00 (m, 4H, Ar-H); 13C-NMR δ (DMSO-d6):
21.38, 84.63, 91.82, 123.58, 126.56, 129.19, 129.72, 130.67, 132.10, 137.89, 138.68, 148.53,
150.68, 154.84, 155.14; HRMS (EI) m/z for C19H15BrN4 calculated: 378.0480, found:
379.0547 [M+1]+ 381.0526 [M+1+2]+(1:1).
,
6.2.11. 2-p-Bromophenyl-5-p-tolylpyrazolo[1,5-a]pyrimidin-7-amine (4k):
1
Yield 71%; Mp: 238-240°C; IR (cm-1): 3279, 3402 (-NH2); H-NMR δ (300 MHz, DMSO-
d6): 2.38 (s, 3H, 4''-CH3), 6.60 (s, 1H, 6-H), 6.93 (s, 1H, 3-H), 7.31-7.33 (d, 2H, Ar-H), 7.69-
7.72 (d, 2H, Ar-H); 7.59 (bs, 2H, 7-NH2), 7.93-7.95 (d, 2H, Ar-H), 8.01-8.04 (d, 2H, Ar-H);
13C-NMR δ (DMSO-d6): 21.32, 84.82, 92.01, 122.39, 127.17, 128.56, 129.75, 131.90,
132.12, 132.84, 135.84, 139.76, 150.88, 153.68, 156.31; HRMS (EI) m/z for C19H15BrN4
calculated: 378.0480, found: 379.0471 [M+1]+ 381.0469 [M+1+2]+(1:1).
,
7. Pharmacological assay
7.1 In vivo anti-inflammatory assay
Carrageenan-induced rat paw edema assay-
Male wistar albino rats weighing 200-250 g were used for the study. They were kept in the
animal house under standard conditions of light and temperature with free access to food and
water. Animals were deprived of the food 12 hr before and during experimental hours. Three
groups of five rats each were divided as control, standard drug and synthesized compounds
treated. Control group of five rats was given tween 80 (95%) only. Standard group received
indomethacin at a dose of 10 mg/kg body weight orally. Test group of animals was treated
with test compounds at a dose of 50 mg/kg body weight. A mark was made on the left hind
paw just beyond the tibiotarsal articulation for constant paw volume measurement. 0.1 ml
freshly prepared suspension of carrageenan (1% in 0.9% saline) was injected under the
planter region of the left hind paw of each rat. Test compounds and standard drug were
administered orally to the animals, respectively 30 min before the carrageenan injection. The
paw volume measurements of each rat were carried out at 0 hr (before carrageenan injection)
and after 1 hr, 2 hr, 3 hr and 4 hr of carrageenan treatment with the help of a Plethysmometer
(model 7140, Ugo Basile, Italy. The anti-inflammatory effect was calculated by the following
equation:
Anti-inflammatory activity (%) = (Vc-Vt/Vc) x 100
9

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Where Vt and Vc are the volume of edema in test compound/standard drug and control group,
respectively.
8. Molecular docking material and method
The 3D structures of the inhibitors were constructed using standard geometric parameters of
the molecular modelling software package Marvin Sketch [10]. All the structures were
minimized and optimized with the Merck Molecular Force Field (MMFF) method taking the
root mean square gradient (RMS) of 0.01 kcal/molA° and the iteration limit to 10,000. All the
structures were ionized at neutral pH 7. Conformers for each structure were generated using
Monte Carlo by applying MMMF force field method and least energy conformer was
selected for further study [11]. Molecular docking and scoring protocols as implemented in
Molegro Virtual Docker [12] were used to investigate the possible binding conformations of
the ligands within the COX-2 binding pocket. The X-ray crystallographic data for COX-2
determined at 2.05Aº (PDB ID 1COX-2) [13] used in the docking simulations were retrieved
from the Protein Data Bank (PDB) and the A-chain residues of 1COX-2 were remained to
dock. Before docking the protein is prepared by using the protein preparation wizard,
removing the water molecule and cofactors from the proteins, optimizing hydrogen bonding
and deleting the ligand present in crystal structure. Solvent molecules were deleted and bond
order for crystal ligand and protein were adjusted and minimized up to 0.30 A° RMS
distance.
Acknowledgement
We are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi,
India for providing financial assistance (JRF) to Gulshan Singh and the Mass Spectrometry
Facility, University of California, San Francisco, USA for running the mass spectra.
References:
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[2- hydroxy-3-(4-phenyl-1- piperazinyl)propyl]-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-5-carboxylic acid, Farmaco, 58 (2003) 25-32.
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protein kinase 2 (MAPKAP-K2) as an antiinflammatory target: discovery and in vivo
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García-Rafanell, J. Forn, Synthesis and SAR of a new series of COX-2-selective
inhibitors: pyrazolo[1,5-a]pyrimidines, J. Med. Chem. 44 (2001) 350-361.
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convenient one-pot synthesis of 2-(trifluoromethyl)-3,4,7,8-tetrahydro-2H-chromen-
5(6H)-one derivatives and their further transformations, J. Fluorine Chem., 133 (2012)
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synthesis of 3-aryl-4-unsubstituted-6-CF3-pyridin-2-ones via aryl migration reaction in
the presence of PhI(OAc)2/NaOH, Chem. Commun., 46 (2010) 6941–6943.(c) P.
Wang, L. Song, H. Yi, M. Zhang, S. Zhu, H. Deng, M. Shao, Convenient one-pot
synthesis of fluorinated DHPs derivatives and their further transformations,
Tetrahedron Lett., 51 (2010) 3975–3977.(d) S. Song, L. Song, B. Dai, H. Yi, G. Jin, S.
Zhu, M. Shao, A convenient one-pot synthesis of 2-(trifluoromethyl)-3,4,7,8-
tetrahydro-2H-chromen-5(6H)-one derivatives and their further transformations,
Tetrahedron, 64 (2008) 5728–5735.
11

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solvent-free versus stepwise solvent mediated reactions: Regiospecific formation of 6-
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Sanz, J. Elguero, An NMR and Computational Study of Azolo[a]pyrimidines with
Special Emphasis on Pyrazolo[1,5-a]pyrimidines, J. Het. Chem. 52 (2015) 336-345.
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[10] Marvin sketch, (2012) Version 5.11.0.
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of State Calculations by Fast Computing Machines, E. J. Chem. Phys. 21(1953) 1087-
1092.
[12] Molegro Virtual Docker 2008.3.2. software.
[13] http://www.rcsb.org/pdb/explore.do?structureId=1cx2
12

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Table 1: Docking results of pyrazolo[1,5-a]pyrimidines (4a-k) by Molegro Virtual
Docker
Compound
Mole Dock No of H-
Atoms of
Ligand/Compounds
N of NH2
Amino acid Distance
(Aº)
No.
4a
Score
Bonds
-125.811
-115.933
-124.808
1
1
2
Tyr355
Tyr355
Tyr355
His90
2.62
2.60
2.63
3.52
2.59
2.69
2.48
3.41
2.56
3.57
2.84
2.61
3.50
2.81
2.78
2.54
3.50
2.67
3.42
3.27
2.76
2.96
3.12
2.88
2.79
2.75
2.51
2.30
N of NH2
4b
4c
N of NH2
N of NH2(weak)
N of NH2
-102.883
4
Tyr355
Tyr355
Tyr355
Arg120
Phe518
Gly519
Tyr355
Tyr355
His90
4d
N1 of Pyrazole Ring
N7a of Pyrazole Ring
N7a of Pyrazole Ring
N of NH2
-119.854
2
4e
N of NH2(very weak)
N of NH2
-133.205
-129.829
1
2
4f
N of NH2
4g
N of NH2(weak)
N of NH2
-131.838
-131.662
-118.342
1
1
2
Tyr355
Tyr355
Phe518
Gly519
Tyr355
His90
4h
4i
N of NH2
N of NH2
4j
N of NH2(very weak)
N of NH2
-132.439
-156.097
2
3
4k
N of NH2
O of SO2NH2
N of SO2NH2
N of SO2NH2
OH of COOH
C=O of COOH
O of OCH3
Arg513
Ser353
His90
SC-558
-138.644
-135.815
3
3
Leu352
Tyr355
Arg120
Tyr355
Tyr285
Ser530
Indomethacin
Compound II
N4 of Pyrimidine
O of SO2CH3
O of SO2CH3

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Table 2: Anti-inflammatory activity of test compounds (carrageenan-induced paw edema test
in rats)
Sr. No.
Compounds
Mean value of edema volume
1hr
2hr
3hr
4hr
0.37±0.09
0.41±0.012
0.18±0.04
0.38±0.07
0.37±0.15
0.22±0.08
0.21±0.06
0.21±0.07
0.45±0.07
0.15±0.05*
0.17±0.05*
0.14±0.05*
0.52±0.17
0.50±0.08
0.56±0.13
0.38±0.08**
0.41±0.10**
0.28±0.04**
0.55±0.05*
0.30±0.11**
0.16±0.05**
0.27±0.05**
0.20±0.04**
0.29±0.07**
0.28±0.05**
0.30±0.09**
0.13±0.05**
0.84±0.03
1
2
4a
0.69±0.04
0.59±0.05
4b
0.29±0.06**
0.66±0.03
0.36±0.05*
0.54±0.06
3
4c
4
4d
0.28±0.10**
0.20±0.08**
0.26±0.03**
0.32±0.11**
0.22±0.06**
0.35±0.07**
0.19±0.11**
0.16±0.06**
0.80±0.05
0.60±0.18
5
4e
0.25±0.11**
0.29±0.04**
0.24±0.07**
0.36±0.08*
0.36±0.07*
0.22±0.10**
0.14±0.02**
0.82±0.13
6
4f
7
4g
8
4h
9
4i
10
11
12
13
4j
4k
Indomethacin
Control (DMSO)
All values are expressed as mean ±SEM of five rats in each group. Statistically significant **p<0.01,*p<0.05
compared to control.

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Table 3: Anti-inflammatory activity of test compounds showing percent inhibition
(carrageenan-induced paw edema test in rats)
Sr. No.
Compounds
Percent inhibition
1hr
2hr
3hr
4hr
28.84
21.15
65.38
26.92
28.84
57.69
59.61
59.61
13.46
71.15
67.30
73.07
37.50
13.75
63.75
17.50
65.00
75.00
67.50
60.00
72.50
56.25
76.25
80.00
31.70
28.04
56.09
34.14
26.82
69.51
64.63
70.73
56.09
56.09
73.17
82.92
54.76
51.16
66.66
34.52
64.28
80.95
67.85
76.19
65.47
66.66
64.28
84.52
1
2
4a
4b
3
4c
4
4d
5
4e
6
4f
7
4g
8
4h
9
4i
10
11
12
4j
4k
Indomethacin

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CH3
7
R3
1
N
1
N
7
H3C
7a
N
6
R2
R1
7a
N
6
2
2
5
SO2CH3
5
SO2CH3
3a
3
N
4
3a
3
N
4
F
II
I
F
R1 = H, CH3, CF3, COOH, COOC2H5
R3 = CH3, CF3, Cl, OH, OCH3, COOH, COOC2H5, C6H5
R2 = H, CH3, CH3CH2, CF3, Br, COOH, COOC2H5
Figure 1: Structure of pyrazolo[1,5-a]pyrimidines (I and II) showing variation at 5th, 6th and
7th position of nucleus

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NH2
1
N
7
7a
N
6
2
5
R
3a
N
4
3
R'
4a-k
c
e
a
b
d
f
g
h
i
j
k
4
R
H
Br CH3 Br
CH3
H
H
CH3
H
CH3
Cl
CH3 Cl
CH3
Cl
Br Cl
Br
R'
F
Cl
Br
CH3
Figure 2: Pyrazolo[1,5-a]pyrimidines (4a-k
)

ACCEPTED MANUSCRIPT
Figure 3: Secondary Structure of COX-2 with active binding pocket

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Figure 4: Binding mode of compound SC-558 and 4k into COX-2 pocket

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NH2
NH2NH2.H2O
1
+
N
N
O
R
CN
N
R'
R'/R
4a-k
2a-e
Figure 5: Disconnection of 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amine

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90
80
70
60
50
40
30
20
10
0
Series1
Series2
Series3
Series4
Compounds
Figure 6: Comparison graph between tested compounds and standard drug

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Time (hr)
Correlation Value
1
2
3
4
0.137
0.631
0.516
0.706
80
70
60
50
40
30
20
10
0
100
80
60
40
20
0
R² = 0.1374
R² = 0.6314
Mole Dock Score
Mol Dock score
-150
-100
-50
0
-150
-100
-50
0
Mole dock score v/s % inhibition after 1 Mole dock score v/s % inhibition after 2 hr
hr
80
60
40
20
0
100
80
60
40
20
0
R² = 0.706
R² = 0.5162
Mole Dock Score
Mole Dock Score
-150
-100
-50
0
-150
-100
-50
0
Mole dock score v/s % inhibition after 3 Mole dock score v/s % inhibition after 4 hr
hr
Figure 7: Correlation value at different time interval

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HN
N
N
HN
(i) / (ii) / (iii)
NH2NH2.H2O
H2N
H2N
+
1
CH3
+
O
Mixture of 5-aminopyrazoles
CN
CN
2a
NH2
+
O
N
N
N
2b
(iv) / (v)
4a
H3C
H3C
Reaction Conditions: (i) Ethanol, Reflux; (ii) Ethanol, PTSA, Reflux;(iii) Toluene, Reflux; (iv)Toluene,
PTSA, Reflux, Yield 30%; (v) Toluene / EtOH (9:1), Catalytic PTSA, Reflux,
4h,Yield 65 %.
Scheme 1: Reaction profile of hydrazine hydrate (
1) and 3-aryl-3-oxopropanonitriles (2) in
different conditions

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O
3'
2'
H2N
1
4'
R
CN
6"
N
7a
N
1'
7
2
O
6
5'
2a-e
6'
CN
3
3a
R'
N
1"
5
NH2NH2.H2O
4
+
5"
4"
Toluene / EtOH (9:1)
Catalytic PTSA
Reflux, 4h
2a-d
4a-k
1
2"
e
R
3"
R'
c
a
b
d
f
g
h
i
j
k
4
c
a
b
d
e
2
R
H
Br CH3 Br
CH3
H
H
CH3
H
CH3
Cl
CH3 Cl
CH3
Cl
R, R'
H
CH3
Cl
Br
F
Br Cl
Br
R'
F
Cl
Br
CH3
Scheme 2: General Scheme for synthesizing 2,5-diarylpyrazolo[1,5-a]pyrimidin-7-amines
4a-k
(
)

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Research highlights
‹ Molecular docking design and synthesis of some novel 2,5-diarylpyrazolo[1,5-
a]pyrimidin-7-amines.
‹ Efficient, simple, expeditious one pot protocol for the preparation.
‹
‹
‹
In vivo anti-inflammatory activity on rats by carrageenan-induced rat paw edema assay.
Correlation of activity results with dock score.
Potency as anti-inflammatory agents up to the 4th hour of study