Discovery of simplified N2-substituted pyrazolo[3,4-d]pyrimidine derivatives as novel adenosine receptor antagonists: Efficient synthetic approaches, biological evaluations and molecular docking studies

Article abstract of DOI:10.1016/j.bmc.2014.01.018

In the present study, a molecular simplification approach was employed to design novel bicyclic pyrazolo[3,4-d]pyrimidine (PP) derivatives from tricyclic pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines (PTP) as promising human A₃ adenosine receptor (hA₃AR) antagonists. All the target compounds were synthesized using novel and efficient synthetic schemes and the structure-activity relationship studies of these PPs were explored through the synthesis of a series of PTP analogues with various substituents. Substituents with different lipophilicity and steric hindrance (e.g., alkyl and aryl-alkyl) functions were introduced at N² position of the pyrazole ring, while acyl groups with different electronic properties were introduced at C ⁶ position of the bicyclic nucleus to probe both electronic and positional effects. Most of the synthesized derivatives of the PP series presented good affinity at the hA₃AR, as indicated by the low micromolar range of K_i values and among them, compound 63 with N ² neopentyl substituents showed most potent hA₃AR affinity with K_i value of 0.9 μM and high selectivity (hA ₁AR/hA₃AR = >111 & hA_2AAR/hA ₃AR = >111) towards other adenosine receptor subtypes. Interestingly, small isopropyl groups at N² position displayed high affinity at another receptor subtype (hA_2AAR, e.g., compound 55, with K_i hA_2AAR = 0.8 μM), while they were less favorable at the hA₃AR. Molecular docking analysis was also performed to predict the possible binding mode of target compounds inside the hA₃AR and hA_2AAR. Overall, PP derivatives represent promising starting points for new AR antagonists.

Full text of DOI:10.1016/j.bmc.2014.01.018

Bioorganic & Medicinal Chemistry 22 (2014) 1751–1765
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of simplified N2-substituted pyrazolo[3,4-d]pyrimidine
derivatives as novel adenosine receptor antagonists: Efficient
synthetic approaches, biological evaluations and molecular
docking studies
Gopalakrishnan Venkatesan a, Priyankar Paira d, Siew Lee Cheong a, Kosaraju Vamsikrishna a,
Stephanie Federico b, Karl-Norbert Klotz c, Giampiero Spalluto b, , Giorgia Pastorin a,
⇑
⇑
a Department of Pharmacy, Faculty of Science, National University of Singapore, 3 Science Drive 2, Singapore
b Department of Pharmaceutical Sciences, University of Trieste, Piazzale Europa 1, 34128 Trieste, Italy
c Institut für Pharmakologie und Toxikologie, Universität Würzburg, D-97078 Würzburg, Germany
d Pharmaceutical Chemistry Division, School of Advance Science, Vellore Institute of Technology University, Vellore 632014, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a molecular simplification approach was employed to design novel bicyclic pyraz-
olo[3,4-d]pyrimidine (PP) derivatives from tricyclic pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines
(PTP) as promising human A3 adenosine receptor (hA3AR) antagonists. All the target compounds were
synthesized using novel and efficient synthetic schemes and the structure–activity relationship studies
of these PPs were explored through the synthesis of a series of PTP analogues with various substituents.
Substituents with different lipophilicity and steric hindrance (e.g., alkyl and aryl–alkyl) functions were
introduced at N2 position of the pyrazole ring, while acyl groups with different electronic properties were
introduced at C6 position of the bicyclic nucleus to probe both electronic and positional effects. Most of
the synthesized derivatives of the PP series presented good affinity at the hA3AR, as indicated by the low
micromolar range of Ki values and among them, compound 63 with N2 neopentyl substituents showed
Received 11 July 2013
Revised 10 January 2014
Accepted 13 January 2014
Available online 31 January 2014
Keywords:
Pyrazolo[3,4-d]pyrimidine
Adenosine receptor antagonist
Homology modeling
Molecular docking
most potent hA3AR affinity with Ki value of 0.9
AR/hA3AR = >111) towards other adenosine receptor subtypes. Interestingly, small isopropyl groups at
2 position displayed high affinity at another receptor subtype (hA2AAR, e.g., compound 55, with Ki hA2A-
AR = 0.8 M), while they were less favorable at the hA3AR. Molecular docking analysis was also per-
lM and high selectivity (hA1AR/hA3AR = >111 & hA2A-
Structure affinity relationship
N
l
formed to predict the possible binding mode of target compounds inside the hA3AR and hA2AAR.
Overall, PP derivatives represent promising starting points for new AR antagonists.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
pathways involving phospholipases C and D, Ca2+ and mitogen-
activated protein kinases (MAPK) have also been described. In
Adenosine is an endogenous nucleoside that mediates a wide
range of physiological responses through interaction with specific
adenosine receptors (ARs), which are G protein-coupled receptors
(GPCRs) comprising the characteristic seven transmembrane do-
mains connected by three extracellular (EL) and three intracellular
(IL) loops. There are four basic types of ARs that have been cloned
and pharmacologically characterized, namely, A1, A2A, A2B and A3
ARs.1,2 Each of these ARs is associated with its own distinct
biochemical pathways. Typically, the activation of adenosine
receptors (ARs) can induce inhibition (A1 and A3) or activation
(A2A and A2B) of the adenylate cyclase. In addition, other signaling
particular, A3 adenosine receptor (A3AR) subtype is implicated in
pathological conditions such as cardiac and cerebral ischemia, neu-
rodegenerative diseases as well as chronic inflammatory diseases
including rheumatoid arthritis and asthma.3,4 Furthermore, A3AR
has been found to be over expressed in some tumor cell lines, such
as HL60 and K562 human leukemia,5 jurkat lymphoma,6 U937
monocytic macro phagichuman cell line,7 A375 human mela-
noma,8 human glioblastoma,9 and human prostatic cells,10 thus
rendering the A3AR an intriguing therapeutic target. Moreover,
A3ARs are involved in the tumor growth and in the regulation of
cell cycle and mediate both pro- and anti-apoptotic effects closely
associated with the level of receptor activation. It has been sug-
gested that the selective inhibition of A3ARs might result beneficial
in several human pathologies (i.e. glaucoma and asthma), but
⇑
Corresponding authors. Tel.: +65 6516 1876; fax: +65 6779 1554.
E-mail address: phapg@nus.edu.sg (G. Pastorin).
http://dx.doi.org/10.1016/j.bmc.2014.01.018
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

1752
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
antagonists could be also useful as diagnostic tools in diseases
where hA3ARs are over-expressed (i.e. cancer) thus stimulating
the search for new selective A3AR antagonists.
From the SAR studies of previously synthesized PTP deriva-
tives,22,25 we deduced certain features that could be vital for the
hA3AR antagonistic activity: (I) the pyrazolo[3,4-d]pyrimidine
(PP) nucleus has shown to represent the core domain in the inter-
action with AR’s binding sites, suggesting that its presence is crit-
ical in affecting the binding of this structure to the receptor of
interest. Therefore, we proposed a new series of hA3AR antagonists,
in which the core PP domain was kept intact (Chart 1). (II) The size
of the substituents at N2 position of the PP bicyclic nucleus (corre-
sponding to N8 position of the PTP scaffold) could play an impor-
tant role in determining the affinity and selectivity towards A3
receptors. Our previous studies showed that small alkyl groups
(e.g., methyl or ethyl) are well tolerated at the hA3AR, since there
In the past few years, there has been an intensive effort to
develop different heterocyclic scaffolds as hA3AR antagonists.
These scaffolds include pyridine and dihydropyridine analogues,11
flavonoid,12 isoquinoline,13 triazoloquinazolines,14 triazolo[4,3-
d]pyrimidine,15 pyrazolo-[3,4-c]or-[4,3-c]quinolines,16 2-phen-
ylpyrazolo[4,3-d]pyrimidine-7-one,17 pyrazolo[3,4-d]pyrimidine,18
pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines19 (Fig. 1). Re-
cently, it was reported a new series of 2-amino/2-oxoquinazo-
line-4-carboxamido derivatives, resulting from an in silico
molecular simplification approach of the 1,2,4-triazolo[4,3-a]-qui-
noxalin-1-one template, which was extensively investigated in the
search for new A3AR antagonists.20 Concurrently, an analogous
strategy, applied to the same tricyclic skeleton, was confirmed to
be effective for the identification of structurally simplified A3AR
antagonists with the advantage of less complex synthetic routes.21
This study promoted the 2-phenylphthalazin-1(2H)-one ring
system (compound 5 in Fig. 1) as a new versatile scaffold suitable
for further molecular manipulation. Moreover, a recent structure–
activity relationship (SAR) study on the 2-phenyl-pyrazolo[4,3-
d]pyrimidine-7-one (compound 6 in Fig. 1) and some simplified
structures were reported17, and these simplified analogues showed
potent activity in the nanomolar range with improved selectivity
towards the hA3AR as compared to the parent compound. Overall,
these studies proved the significance of simplified structures,
which can act as hA3AR antagonists to the same extent as more
complex structures.
On the other hand, our group has recently reported new potent
pyrazolo-triazolo-pyrimidines (PTPs) centered on the replacement
of the conventional furan ring at position C2 with a 2-(para substi-
tuted) phenyl ring and various substituents at N5 and N8 posi-
tions.22 These new modifications not only enhanced the
pharmacological profile of the antagonists significantly, but they
also improved selectivity against the other adenosine receptor sub-
types. However, PTP derivatives, and also most of the tricyclic com-
pounds, have a number of limitations such as poor aqueous
solubility due to high molecular weight and a complex nature of
the structure, which makes the synthetic preparation difficult
and time consuming. Moreover, these problems may affect the
drug-like properties of the analogues.23,24 Taking these drawbacks
into account, the synthesis of more simplified heterocyclic deriva-
tives has been scouted.17,20,21
is
a steric control in the receptor pocket surrounding that
position.19 Hence, we introduced various substituents with differ-
ent lipophilicity and steric hindrance such as alkyl (methyl 31, iso-
propyl 32, and neopentyl 33) and phenyl ethyl (34) functions at N2
position. III) Assuming that substituents at C6 position (correspon-
dent to position C5 of PTP scaffold) are essential for retaining the
selectivity and potency,26 various acyl groups (benzoyl, para
substituted benzoyl and phenyacetyl) were substituted at C6 posi-
tion in the new series of analogues. In fact, these moieties have gi-
ven rise to potent hA3AR antagonists when introduced at the C5
position of the PTP scaffold.
Differently from the previous triazole ring in the PTP series, two
types of substituents are introduced in our PP derivatives. First, a
small steric chloro functional group at the C4 position is chosen
to allow the PP scaffold to hopefully attain the same binding pose
as PTP due to restricted rotation of C4 position while enabling a fur-
ther point of functionalization. Second, a rotatable ethyl ester chain
with hydrogen bonding property is introduced, in order to explore
the effect of the different steric and rotatable bonds at the C4 posi-
tion. Thus, the development of a new class of compounds targeting
the hA3AR derives from a molecular simplification (Chart1) of the
tricyclic PTP structure into a bicyclic pyrazolo[3,4-d]pyrimidine
bearing key substituents, hoping that these new compounds could
preserve their pharmacological profile at the hA3AR while simplify-
ing the synthetic procedures.
2. Results and discussion
2.1. Chemistry
The pyrazolo[3,4-d]pyrimidine derivatives 31–63 were pre-
pared following four synthetic pathways, as depicted in Schemes
1–4.
Alkylation (or arylation) of 3-amino-4-pyrazole carbonitrile
with alkyl (aryl) iodide or bromide in dry DMF, using dry potas-
sium carbonate as a weak base, led to N1 substituted regioisomer
(7–10) as a major product. Among them, compounds 7 & 10 were
already reported by our group.22,27 In addition, compound 8 was
also reported in the literature and was prepared by different proce-
dure.28Subsequently,3-amino-N1-substituted-4-pyrazolocarbo-
nitriles (7–10) were hydrolyzed to corresponding carboxamides
(11–14) under acidic conditions at cold temperature for 30 min,
followed by stirring at ambient temperature for 5 h.
F
O
O
N
H
NH
N
O
N
N
N
NH
N
NH
N
N
N
N
N
N
O
N
O
N
H3C
F
Cl
2
1
3
Triazolo[4,5-d]pyrimidine
Triazoloquinazoline
Pyrazolo[3,4-
d]pyrimidine
O
Among the carboxamide derivatives (11–14), compound 11 was
already reported in the literature29 and compound 12 was commer-
cially available. Further, the carboxamide derivatives (11–14) were
refluxed with benzoylisothiocyanate in acetone for 12 h, to be
transformed into the benzoylthioureido derivatives (15–18). Treat-
ment of 15–18 with a small excess of methyl iodide in aqueous so-
dium hydroxide solution at ambient temperature for 3 h gave the
corresponding carbamidothioates 19–22. The resulting methyl
mercapto derivatives were heated in DMF containing 2% ammonia
at 120 °C for 3 h in a sealed tube to afford benzoylguanidino
O
N
N
O
NH
N
N
O
NH
NH
HN
N
N
N
O
N
N
H3CO
N
N
OCH3
4
6
5
Pyrazolo[4,3-e]-1,2,4-triazolo-
[1,5-c]pyrimidine
2-phenyl pyrazolo[4,3-
d]pyrimidin-7-one
2-phenylphthalazin-1(2H)
-one
Figure 1. Examples of human A3 adenosine receptor (hA3AR) antagonists.

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1753
hA3 Selectivity & Potency
Essential for activity
R1
Cl
4
5
3
4
N
3
9
8
N
5
N
N
6
Ar
N
R
2
2
6
N
N
R1
N
1
1
N
7
N
8
R
Essential for selectivity
Chart 1. Rationale for the design of 2-(alkyl and arylalkyl substituted)-2H-pyrazolo [3,4-d]pyrimidine derivatives (molecular simplification of PTP into PP).
O
C
O
H2N
HN
N
N
NH2
(c)
C
H2N
O
(a)
(b)
(d)
N
R
O
N
R
N
N
N
N
R
11-14
N
N
HN
H2N
H2N
R
N
S
15-18
7-10
O
N
H2N
H2N
O
N
C
C
N
Cl
O
N
O
N
(e)
(f)
HN
H2N
(g)
HN
HN
N
R
R
N
N
N
SCH3
N
N
R
R
H2N
N
NH2
23-26
27-30
19-22
31-34
Compd
R
31
32
33
34
CH3
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
Scheme 1. Reagents and conditions: (a) RI or RBr, K2CO3, DMF, 90 °C, 10 h; (b) conc H2SO4, 0 °C to rt, 5 h; (c) C6H5CONCS, acetone, reflux, 12 h; (d) CH3I, 0.1 N NaOH, rt, 3 h; (e)
2% aq NH3, DMF, 120 °C sealed tube, 3 h; (f) 1 N NaOH, reflux, 12 h; (g) POCl3, dimethylaniline, reflux, 24 h.
H2N
N
O
O
N
O
CN
Cl
N
(a)
N
N
(b)
(c)
N
N
N
N
N
N
N
N
H2N
N
35
H2N
N
H2N
H2N
N
37
36
33
Scheme 2. Reagents and conditions: (a) NaCN, p-toluene sulfinate sodium, DMF, 80 °C, 2 h; (b) K2CO3, 30% H2O2, rt, 1 h; (c) EtOH, concd H2SO4, reflux, 12 h.
derivatives 23–26. This nucleophilic substitution reaction was per-
formed with slight modification from the previously reported pro-
cedure applied for the conversion of methyl carbamidothioates to
guanidino derivatives.30 Interestingly, the synthesized compounds
23–26 were insoluble in any of the solvent. Hence, they were di-
rectly taken to the next reaction. Hydrolysis of the benzoyl guani-
dine derivatives under basic conditions led to the corresponding
free amines 27–30 with higher yields (from 68% to 81 %). In addi-
tion, compounds 27–30 are predominantly existed as tautomeric
keto form. The predominant keto form was confirmed by the pres-
ence of amide protons (NHAC@O) signals between 10.4 and
10.6 ppm in the series using 1H NMR spectroscopy. Conversely,
the enol form of the tautomer (N@CAOH) containing OH proton
was ruled out since it would have shown a signal at high down field
of about 12 ppm in 1H NMR spectroscopy. Subsequently, the 6-ami-
no-2-alkyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (27–30) were
further reacted with phosphoryl trichloride in the presence of
dimethylaniline under reflux condition to form the corresponding
chloro analogues (31–34). Among them, compound 31 was already
reported by Seela et al. in low yield using different procedures.31
The structural determination of 31 in the solid state was carried
out by using X-ray diffraction studies inorder to confirm the alkyl
substitution at N2 position. Single crystal of 31 was grown by vapor
diffusion of diethyl ether into a saturated solution of MeOH. The
detailed key crystallographic parameters can be found in the ESI
(Table S1 & S2). The X-ray crystallographic results confirmed the
successful substitution of the substituent (methyl group) at N2 po-
sition (Fig. 2).
Compound 35 was prepared by treating 33 with sodium
cyanide in the presence of p-toluene sulfinate sodium as catalyst
in dry DMF at 80 °C for 2 h. The nitrile groups were detected by
the presence of CN peak at 115 ppm at 13C NMR. The resulting ni-
trile compound was hydrolysed through the ‘Radziszewski reac-
tion’, using aqueous hydrogen peroxide and aqueous potassium
carbonate in cold or hot condition.32 However, the product was ob-
tained with low yield in these conditions. Hence, with a slight

1754
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
Cl
N
N
R
R1
N
N
N
Cl
N
38-57
)
a
(
O
N
N
R
R2
N
31-34
H2N
R
R1
R2
R
R1
R2
)
(b
31
32
33
CH3
-
-
-
-
-
-
48
CH2C(CH3)3
CH2C(CH3)3
CH2CH2C6H5
H
H
H
CF3
CH3
H
CH(CH3)2
49
50
CH2C(CH3)3
Cl
34
38
39
40
41
42
43
44
45
46
47
CH2CH2C6H5
CH3
-
-
H
51
52
53
54
55
56
57
58
59
60
61
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH3
H
F
CF3
CH3
H
H
H
H
-
N
O
N
R
H
H
H
H
H
H
H
H
H
H
H
N
HN
N
CH3
F
H
58-61
CH3
CF3
C6H5CO
CH3
CH3
H
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
CH3
C6H5CO
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2C(CH3)3
CH2C(CH3)3
C6H5CO
F
C6H5CO
CF3
CH3
H
-
-
-
-
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
-
-
F
-
Scheme 3. Reagents and conditions: (a) substituted benzoyl chloride, DIPEA, toluene, reflux, 24 h; (b) phenyacetyl chloride, DIPEA, toluene, reflux, 24 h.
O
N
O
O
N
O
and phenyl acetyl derivatives (58–61), respectively. Di-substituted
benzoyl derivatives (54–57) of analogues 31–34 were prepared un-
der this standard condition using 4 equiv of the benzoyl chloride
and DIPEA.
Similarly, acylation of compound 37 was carried out as reported
in Scheme 4. With a slight modification to the previous 4-chloro
substituted pyrazolopyrimidines (31–34), 3 equiv of substituted
benzoyl chlorides and diisopropylethylamine were used to obtain
monoacylated compounds 62–63.
O
(a)
N
N
N
N
N
H
N
H2N
N
37
62-63
R1
R1 = CF3(62), CH3(63)
Scheme 4. Reagents and conditions: (a) substituted benzoyl chloride, DIPEA,
toluene, reflux, 24 h.
2.2. Binding affinity and Structure–activity relationships
In this manuscript, we proposed to modify our previously syn-
thesized tricyclic pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]-pyrimi-
dines (PTPs)22 into
a bicyclic pyrazolo[3,4-d]pyrimidine (PP)
scaffold by considering the significance of simplified struc-
tures17,20,21 that can act as promising antagonists, hopefully with
similar or even improved affinity and selectivity at the hA3AR. To-
wards that purpose, a new series of 2-(ar)alkyl-2H-pyrazolo[3,4-
d]pyrimidines was successfully synthesized and characterized.
The receptor binding affinities of the synthesized PTP simplified
analogues (31–34 & 37) and their derivatives (38–61 & 62–63)
are summarized in Table 1.
Figure 2. X-ray crystal structure of compound 31 (CCDC-937929). All the atoms are
represented by spheres of arbitrary radii.
The binding affinity of antagonist was evaluated by measuring
the displacement of selective radioligands which were previously
bound to the receptor expressed (Chinese hamster ovary cells
(CHO) for hA1AR, hA2AAR and hA3AR) at the cellular surface. In this
assay, the displacement of (i) specific [3H]CCPA binding at hA1AR,
(ii) specific [3H]NECA binding at the hA2AAR and (iii) [3H]HEMADO
at the hA3AR were evaluated. Due to the lack of a suitable radioli-
gand for hA2BAR, the antagonists activity was determined in aden-
ylyl cyclase experiments in CHO cells expressing the hA2BAR.33,34 Ki
(dissociation constant) value of the data was calculated using
Cheng and Prusoff equation,35 with geometric means of at least
three experiments including 95% confidence intervals.
modification to the reported procedure, the reaction was carried
out at a room temperature to obtain carbamide (36) with good
yield. Subsequently, this intermediate compound (36) was esteri-
fied in the presence of concd H2SO4 with absolute ethanol under
reflux condition to obtain the corresponding carboxylate 37 as
solid.
Mono-and di-acylated derivatives of compounds 31–34 were
synthesized by using substituted benzoyl chlorides or phenylacetyl
chloride in different mole ratios (Scheme 2). Analogues 31–34
were treated with two equivalents of substituted benzoyl chloride
and phenylacetyl chloride in the presence of diisopropylethyl-
amine (DIPEA) to obtain the mono-benzoyl derivatives (38–53)
The binding assay results indicated that the reported molecular
simplification of the PTP structure, although with a remarkable

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1755
Table 1
Binding affinity (Ki) of synthesized compounds at hA1AR, hA2AAR, and hA3ARa and selectivity against hA1AR and hA2AAR (R denotes N position, R1 denotes C position)
O
O
Cl
Cl
Cl
N
O
N
O
O
N
N
N
R
N
R
N
R
N
R
R1
N
N
N
N
N
HN
N
N
N
H
N
H2N
N
58-61
38-57
62-63
31-34
R1
R2
Compd
R
R1
R2
Ki,
lM (95% CI)
Selectivity
hA1/A3 hA2A/A3
b
c
d
e
hA1
hA2A
hA2B
hA3
31
32
33
34
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
CH3
—
—
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
—
—
—
H
F
CF3
CH3
H
29.7 (28.6–30.7)
9.0 (6.4–12.5)
12.0 (10.6–13.7)
4.3 (2.9–6.4)
>100
>100
>100
.>100
19.6 (19.3–20)
3.4 (3.41–3.47)
13.8 (9.3–20.3)
4.1 (2.3–7.4)
11.0 (5.7–20.0)
16.5 (6.8–39.8)
>100
11.1 (7.3–16.6)
3.8 (2.2–6.5)
2.7 (2.1–3.4)
>60
9.1 (4.4–19.1)
3.3 (1.5–7.2)
4.1 (2.2–7.6)
3.7 (3.4–4.1)
24.2 (7.7–76.5)
20.5 (12.7–33.2)
25.6 (16–41)
12.0 (6.6–21.8)
4.6 (1.8–11.8)
1.6 (0.85–3.29)
2.7 (1.8–4.1)
1.1 (0.66–2.0)
3.5 (2.9–4.3)
2.2 (1.6–3.1)
>100
>20
>10
>10
>20
>20
>20
>20
>20
>20
>20
>20
>20
P10
>10
>20
>10
>20
>20
>20
>20
>20
>20
>20
>20
>20
>10
>20
>20
>20
>20
>100
>100
>60
>0.29
>0.09
>0.2
>0.04
>4.2
>3.1
>2.9
7.4
1.5
>0.05
1.3
>0.04
0.3
2.7
>15
2.2
0.4
0.02
>21.4
7.2
>0.09
0.03
>0.6
>0.03
1.0
0.6
0.7
0.8
0.3
>0.02
0.2
0.01
0.1
0.3
>15
0.4
0.3
0.02
5.8
6.6
2.4
0.1
>15.3
2
4.5
0.17
2.1
0.7
>77
>111
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
CH3
CH3
CH3
>100
23.6 (15.8–35.4)
31.9 (25.7–39.6)
33.5 (28.2–39.7)
13.5 (5.8–31.2)
12.8 (6.9–23)
>60
CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2C(CH3)3
CH2C(CH3)3
CH2C(CH3)3
CH2C(CH3)3
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH2CH2C6H5
CH3
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
CH3
CH(CH3)2
CH2C(CH3)3
CH2CH2C6H5
CH2C(CH3)3
CH2C(CH3)3
F
CF3
CH3
H
10.3 (6.0–17.6)
>100
30.3 (17.4–52.7)
5.9 (3.0–11.4)
6.6 (3.6–12.1)
5.0 (4.6–5.4)
8.6 (3.3–21.9)
>100
2.8 (1.7–4.5)
1.5 (0.71–3.2)
7.1 (6.2–8.1)
6.7 (2.2–20.5)
6.5 (3.8–12.4)
2.9 (1.9–4.6)
6.6 (2.6–16.9)
27.1 (18.5–39.7)
10.9 (4.3–27.6)
11.4 (7.4–17.5)
1.3 (1.3–1.4)
0.9 (0.7–1.0)
F
CF3
CH3
H
2.1 (1.8–2.4)
3.1 (2.3–4.3)
2.06 (0.83–5.06)
16.4 (8.3–32.6)
9.9 (9.3–15.5)
17.5 (10.5–29.4)
0.8 (0.61–1.2)
ꢀ100
F
CF3
CH3
H
H
H
H
—
—
—
10.8 (9.5–12.1)
>60
7.9 (7.6–8.1)
>100
C6H5CO
C6H5CO
C6H5CO
C6H5CO
—
—
—
—
CF3
8.4
1.1
>15.3
>34.5
2.4
0.07
2.9
0.5
>77
>111
>100
5.4
16.3 (14–18.9)
1.9 (1.0–3.6)
31.9 (26.4–38.6)
6.5 (3.9–10.7)
>100
29.9 (13–69.2)
4.7 (3.7–5.9)
23.6 (9.5–58.2)
8.6 (5.0–14.9)
>100
—
—
—
CH3
>100
>100
a
b
c
Adenylyl cyclase activity of synthesized compounds at the hA2BAR.
Displacement of specific [3H]-CCPA binding at hA1AR expressed in Chinese hamster ovary (CHO) cells (n = 3–6).
Displacement of specific [3H]-5’-N-ethylcarboxamido adenosine (NECA) binding at hA2AAR expressed in CHO cells (n = 3–6).
Ki values for inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells (n = 3–6).
d
e
Displacement of specific [3H]-2-(1-hexynyl)-N6-methyl adenosine (HEMADO) binding at hA3AR expressed in CHO cells (n = 3–6).
drop in the Ki values, still enabled the binding at the hARs (Table 1).
This result seems very promising, considering that the PP repre-
sents a much smaller scaffold for the hA3AR. Indeed, most of the
pyrazolo[3,4-d]pyrimidines were able to maintain moderate
affinity at hA3 receptors as indicated by the low micromolar range
those of the other N2-substituted analogues (58 vs 59–61). In addi-
tion, in the N2 isopropyl series there are two inactive compounds at
the hA3AR (43, 45) and, in general, they presented a better hA2AAR
affinity profile (42–45, 55 and 59, with Ki hA2AAR values between
0.8 and 4.7 lM).
(0.9–33.5
l
M) of Ki values and a few compounds showed high
Interestingly, neopentyl substituted analogues (46–49 and 56)
of C4-chloro series and C4-carboxylate series exerted a relatively
more favourable effect at the hA3AR receptor. They also displayed
a 3–5 fold increased affinity in comparison with the N2 methyl
derivatives, but only when a substituted phenyl group was present
at the C6 position (47–49). In fact, the presence of an unsubstituted
phenyl moiety in compounds 60 and 46 lead to a lower affinity at
the hA3AR in respect to N2-methyl analogues 58 and 38 (i.e. 60, Ki
selectivity towards the other receptor subtypes.
2.2.1. N2 position
To examine the impact of the alkyl group substitutions at N2-
position on the pharmacological profile, we compared the binding
assay results of the analogues bearing various alkyl substitutions
(i.e. methyl, isopropyl and neopentyl) or a phenyl ethyl substitu-
tion at this position. First of all, all the synthesized compounds of
this series are inactive towards the hA2BAR subtype. In fact, aden-
hA3AR = 10.9 lM vs 58, Ki hA3AR = 6.6 l
M). Among all the C4-
chloro N2 alkyl substituted derivatives (38–49, 54–56 & 58–60),
compound 49 showed the best affinity at the hA3AR (Ki
ylate cyclase assays displayed Ki upper than 20 lM. The alkyl sub-
stitutents (methyl, isopropyl and neopentyl) at N2 position of the
pyrazole ring were found to modulate the affinity at hA3AR to a
considerable extent. Among them, N2 isopropyl and N2 methyl
substituted analogues showed the lowest binding affinity towards
hA3AR (38–45, 54, 55 & 59) with Ki values between 10.3 and
hA3AR = 5
lM). Moreover, derivatives 48 (Ki hA3AR = 6.6
lM) &
56 (Ki hA3AR = 6.5
lM) of the same series are comparable to com-
pound 49 in terms of affinity at the hA3AR but exhibited the high-
est selectivity towards other receptor subtypes (48 & 56; hA1AR/
hA3AR >15; hA2AAR/hA3AR >15). Similarly, few compounds with
C4 carboxylate group and N2 neopentyl substitution showed higher
affinity and selectivity as compared to the C4 chloro counterparts.
33.5
l
M, except for C6-phenylacetamido compound 58 which dis-
M towards hA3AR that is better than
played an affinity of 6.6
l

1756
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
Among them, compounds 62 & 63 displayed 4–6 fold improvement
in affinity and 5–55 fold improvement in selectivity.
different contributions of electron donating and electron with-
drawing groups at the para position of the benzoyl group at C6.
In the N2-phenylethyl series, para unsubstituted benzoyl (50,
Finally, in C4 chloro series the presence of a phenyl ethyl group
at N2 position generally resulted in a significant increase of hA3
affinity (50–53, 57 and 61, Ki hA3AR values between 1.5 and
with Ki hA3AR = 8.6
with Ki hA3AR = 11.4
l
l
M) and chain lengthened phenylacetyl (61,
M) at the C6 position, appeared to reduce
11.4
l
M) and better selectivity in comparison to the other N2 alkyl
moderately the affinity of the PP scaffold at the hA3AR in compar-
counterparts (38–49 and 54–56 & 58–60), except for compound
ison with the para substituted benzoyl derivatives (52 & 53). Sim-
60, which was unexpectedly inactive at the hA3AR (Ki hA3AR
ilarly, para-F group (51, with Ki hA3AR >ꢁ100
had a detrimental effect on hA3AR, however, it showed good affin-
ity towards hA2A receptor subtype (Ki hA2AAR = 2.06 M, hA3AR/
hA2AAR >50). On the other hand, analogue 53, with the electron
donating para-toluoyl substituent at C6 position, displayed sub-
stantial increase in the affinity towards the hA3 receptor (Ki hA3-
lM) in the acyl chain
>100 l
M). This result indicates that the phenylethyl group at N2
position played a more essential role in the binding at the hA3AR
in C4chloro series; in fact, it suggests that the steric hindrance far
from N2 position is more favourable for the bulky phenyl ethyl
group to orient itself inside the binding cavity, in comparison with
little groups in close proximity of N2 position (i.e. 52 vs 44). How-
ever, these findings are in contrast with the PTP scaffold, where
small substituents (e.g., CH3) at N8 position are preferable than lar-
ger alkyl and aryl substituents.19
l
AR = 1.5
lM) and it also imparted a 6–8 fold increment in affinity
in comparison with corresponding unsubstituted (50, Ki hA3-
AR = 8.6
AR = 5.0
l
M and 61, Ki hA3AR = 11.4
lM) and neopentyl (49, Ki hA3-
lM) compounds, respectively. Similarly, analogue 52,
with an electron withdrawing para-CF3 benzoyl group, also exhib-
ited comparable affinity at the hA3AR (Ki hA3AR = 2.8 M). Interest-
2.2.2. C6 position
l
Analogues bearing a free amino group at C6 (31–34) showed
ingly, a >36 fold increase in affinity was observed for the derivative
with para-CF3 substituent in comparison with the para-F analogue,
overall indicating that bulky, either electron-donating or electron-
acceptor functional groups, are preferred over less bulky groups for
a better affinity at the hA3AR.
affinity towards the hA2AAR (Ki hA2AAR = 3.3–9.1
AR (Ki hA1AR = 4.3–29.7 M) subtypes but not towards the hA3AR.
lM) and the hA1-
l
This was somehow expected, as it was already observed that PTPs
with a free amino group at C5 position are able to bind hA1AR and
hA2AARs better than hA3ARs.
In addition, a few compounds of the C4 carboxylate series with
In addition, analogue 54, bearing a dibenzoyl group at C6 posi-
tion, displayed an enhancement of affinity at the hA3AR subtype
C6-benzamide substitution and N2 neopentyl groups overall dis-
played the best affinity (Ki hA3AR = 0.9 lM) and a remarkable
(Ki hA3AR = 7.1
logue (38, with Ki hA3AR = 23.6
placed by a slightly longer phenyl acetyl group, affinity was
maintained (58, with Ki hA3AR = 6.6 M) and selectivity versus
l
M) in comparison with the mono-acylated ana-
improvement in selectivity towards other AR subtypes as com-
pared to the C4-chloro series. Also, when the para position of the
C4 carboxylates was substituted with the electron withdrawing
steric CF3 group, the activity and selectivity were increased by 5
l
M). When the C6 group was re-
l
the hA2AAR was increased (54, hA1AR/hA3AR = 2.4 vs 58, hA1AR/
hA3AR = 4.5). This indicates that the binding cavity is spacious en-
ough to accommodate the bulky diacyl group and longer phenyl-
acetyl group at C6 position. In contrast, shorter chains like single
benzoyl groups seemed less favourable.
fold (62, Ki hA3AR = 1.3 lM, hA1AR/hA3AR = >77 & hA2AAR/hA3-
AR = >77) as compared to its chloro counterpart (48). Similarly,
when electron donating –CH3 group was introduced at the para po-
sition of the benzamide chain, the affinity was further improved
(63, Ki hA3AR = 0.9 lM, hA1AR/hA3AR = >111 & hA2AAR/hA3-
As seen in Table 1, C6 acyl substituents of N2-isopropyl deriva-
tives (42–45, 55 and 59) impelled the affinity towards the hA2AAR;
in fact the best result was observed for the di-acylated analogue
AR = >111) up to 5 fold and the selectivity was improved up to
50 fold towards hA1AR & 278 fold towards hA2AAR as compared
to the corresponding chloro compound (49). Moreover, this com-
pound (63) showed the best pharmacological profile in the whole
series. These changes are observed due to the additional carboxyl-
ate substitution at C4 position. This strongly suggests that C4 ester
substituents, along with either electron withdrawing CF3 or elec-
tron donating CH3 groups in the para position of acyl chain, are
highly essential for enhancing the affinity at hA3AR and selectivity
towards the other receptor subtypes. Molecular modeling studies
further rationalized the reason for such an improvement.
In summary, structure–affinity relationship analysis showed
that substituents at N2, C4 and C6 positions play a key role in mod-
ulating the binding affinity and selectivity at the adenosine recep-
tors. Our results confirmed the importance of the contemporary
introduction in the PP system of (a) large lipophilic substituents
(e.g., neopentyl and phenyethyl) at N2 position to maintain the
affinity at hA3AR; (b) sterically bulky electron donating or electron
withdrawing para substituted acyl chains at C6 position to increase
the affinity towards hA3AR; (c) a slightly smaller chain (i.e. isopro-
pyl) at N2 to confer the affinity towards the hA2AAR, if further study
is intended on hA2A receptor; (d) an ester substituent at C4 position
to increase the selectivity towards other receptor subtypes.
Among the newly synthesized PP derivatives, analogue 63, with
a neopentyl substitution at N2 position and a para-methylbenza-
mide at C6 position, possessed the best hA3AR affinity profile (Ki
(55, with Ki hA2AAR = 0.8 lM). This derivative was also about
8–10 times more selective towards the hA1AR and hA3AR receptor
subtypes. Also, the presence of additional functional groups at the
para position of the C6-benzoyl substituent maintained the affinity
and remarkably increased the selectivity against the hA3AR sub-
type, independently of the electronic nature of substituent. In fact,
this behaviour was observed both for the electron donating methyl
group (45, Ki hA2AAR = 1.1
electron withdrawing group fluorine (43, with Ki hA2AAR = 1.6
l
M, hA3AR/hA2AAR = 91) and for the
M,
l
and hA3AR/hA2AAR >37). Moreover, it was noted that high affinity
towards the hA2A subtype was reflected in the whole N2 isopropyl
series.
Nevertheless, affinity switched towards hA3AR for the neopen-
tyl series. Although the free amino group of neopentyl prompted
a better affinity at the hA2AAR (33, Ki hA2AAR = 4.1
AR >60 M), corresponding acyl substitutions made a significant
improvement in the affinity at hA3ARs (e.g., 46, Ki hA2AAR = 3.5
and Ki hA3AR = 30
M). Notably, replacement of C6 benzoyl with
groups in para (e.g., F, CF3) slightly ameliorated the affinity towards
the hA3AR (e.g., 47, with Ki hA3AR = 5.9 M & 48, with Ki hA3-
AR = 6.6 M). Remarkably, while compound 49 (with an electron
lM and Ki hA3-
l
lM
l
l
l
donating para-toluoyl group) and 47 (with an electron withdraw-
ing para-F group) showed the best affinity at the hA3AR (49, Ki hA3-
AR = 5.0
l
M; 47, Ki hA3AR = 5.9
l
M) in the neopentyl series, its
hA3AR = 0.9 lM) and >100 fold selectivity towards the hA1 and
para-CF3 analogue 48 displayed the highest selectivity (hA1AR/hA3-
AR >15, hA2AAR/hA3AR >15) in the whole series. This indicates the
hA2AARs. Overall, compounds of the C4 chloro series showed
weaker affinity and poorer selectivity as compared to C4 ester

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1757
substituents. This difference was purely attributable to the addi-
tional ester substituents at C4.
derivatives were docked into the orthosteric trans-membrane
(TM) binding cavities of both adenosine receptors.
From the docking simulation analysis, most of the newly syn-
thesized derivatives were seen to share a similar binding pose in
the TM region of the hA3AR. For these compounds, ligand-recogni-
tion occurred in the upper region of the TM bundle, and the pyraz-
olo[3,4-d]pyrimidine scaffold was surrounded by TMs 3, 5, 6, 7
with the acyl ring at the C6 position located towards the intracellu-
lar environment. Figure 3 (panel A) presents the hypothetical bind-
ing pose of compound 53 at the hA3AR, which possesses the
highest hA3AR affinity among all the newly synthesized derivatives
2.3. Molecular modeling studies
2.3.1. Homology modeling
The crystallographic structure of hA2AAR complexed with ZM-
241385 as high affinity antagonist (PDB code: 3EML),36 was used
to build up homology model of the hA3AR (Fig. S1). Although sev-
eral hA2AAR crystal structures are available, considering the high
resolution (2.6 A°) and accuracy of the structure, 3EML was se-
lected as a template. Moreover, the accuracy of 3EML has been
thoroughly investigated by various research groups,17,22 thus sub-
stantiating our approach. The homology modelling was performed
using programme Modeller 9.1137–40 and the quality of the model
was evaluated using the Ramachandran Plot. Subsequently, the
prediction ability of the constructed hA3AR homology model was
evaluated in the molecular docking experiments using MOE
programme.
(Ki hA3AR = 1.5
d]pyrimidine core was anchored within the binding cleft through
an aromatic stacking interaction with Phe168 (EL2). Moreover,
lM). It appeared that the bicyclic pyrazolo[3,4-
p–p
the carbonyl group of the exocyclic acyl chain at the bicyclic core
interacted with polar residue Asn250 (6.55), forming a lone stabi-
lizing H-bonding interaction. In addition, the phenyl ring of the
acyl chain was located deep within the ligand binding cavity and
formed hydrophobic interactions with the highly conserved
Trp243 (6.48), an important residue in receptor activation and
antagonist binding. The N2 phenyl ethyl chain of the ligand was di-
rected towards the TM2 and TM3. Compound 53 also formed
hydrophobic interaction with many residues in the binding cleft
including Thr87(3.29), Leu90(3.33), Leu91(3.34), Val169(EL2),
2.3.2. Molecular docking
A
molecular docking study was performed for the new
4-chloro-pyrazolo[3,4-d]pyrimidine (31–34 and 38–61)and pyraz-
olo[3,4-d]pyrimidine 4-carboxylate (62–63) derivatives on both
hA3AR and hA2AAR to identify the hypothetical binding mode at
the hA3AR and to rationalize the results obtained from the pharma-
cological evaluation. As reported in the experimental section, MOE
was used as a docking programme for the pose inspection of the
novel pyrazolo[3,4-d]pyrimidine derivatives in both hA2AAR and
hA3AR. All the newly synthesized pyrazolo[3,4-d]pyrimidine
Met177(5.38),
Trp243(6.48),
Leu246(6.51),
Tyr265(7.36),
Leu264(EL3) and Ile268(7.39).
In contrast, the binding pattern of compounds bearing non
bulky substituents, such as N2-methyl and N2-isopropyl deriva-
tives, displayed slightly different binding pose as compared to
53. The pyrazole ring of these compounds is directed towards the
Figure 3. (A) Hypothetical binding mode of compound 53 obtained after docking simulations inside the hA3AR binding site. (B) Hypothetical binding mode of compound 63
inside the hA3AR binding site. (C) Crystallographic binding mode of ZM-241385 inside the hA2AAR(PDB code 3EML) and (D) hypothetical binding mode of compound 55 inside
the hA2AAR binding site and poses are viewed from the membrane side facing TM6, TM7, and TM1. The view of TM7 is omitted. Side chains of some amino acids important for
ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.

1758
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
extracellular environment rather than towards TM2 and TM3, and
also the main nucleus pyrazolo[3,4-d]pyrimidine was rotated of
about 45° compared to the binding pose of compound 53. Because
of this different orientation of the molecule inside the binding cleft,
these derivatives (38–45 and 59) might have lost the H-bonding
interaction with Asn250 (6.55).The lack of these important interac-
tions resulting from different orientation seems to be responsible
for the lower affinity, as well as derivatives with less bulky groups
at N2 showed lower hA3AR affinities (data not shown). However, as
an exception, compound 54 and 58 in the N2 methyl series and
compound 55 in the isopropyl series showed a similar pattern as
compound 53, which in turn was reflected in the high binding
activity of these compounds. Interestingly, the pyrazolo[3,4-
d]pyrimidine derivatives bearing a lipophilic moieties at N2 posi-
tion, such as phenyl ethyl and neopentyl derivatives, shared similar
binding pattern which accounted for high binding affinity of the
compounds in these series (52,53 & 57). In fact, for all these deriv-
atives, the pyrazolo[3,4-d]pyrimidine scaffold is utterly aligned in-
side the TM region of hA3AR with the exception of compound 60 in
the neopentyl series and compound 61 in the phenyl ethyl series.
In particular, the aromatic stacking interaction with Phe168
(EL2), a lone H-bonding interaction with Asn250 (6.55) and the
hydrophobic interaction with Trp243 (6.48) are conserved among
these derivatives. As seen in Table 1, compounds 52 and 53 of
the N2 phenylethyl series and compounds 47, 48 and 49 of the
N2 neopentyl series showed close binding affinity values which
are reflected in the overlapping binding pattern with one another
inside the binding cleft. Notably, due to the flexible nature of the
neopentyl and phenylethyl substituents, which possess lengthy
and multiple rotatable bonds (2 for neopentyl and 3 for phenyl-
ethyl), enabled the scaffold to obtain a different conformation to
fit into the binding cleft. Although, compounds 46–48 of neopentyl
series showed comparable affinity towards hA3AR, the selectivity
against hA1AR and hA2AAR was improved for compound 48, be-
cause of the para-CF3 group. As seen from the docking studies, at
the hA2AAR, the hydrophobic side cleft delimited by TM2 and
TM3 could accommodate less steric para-substituted compounds
[i.e. 47 (F) and 49 (CH3)], but could hardly accommodate the
slightly bulkier para-CF3(48). This steric effect could be accounted
for the difference in affinity of compound 48 at hA2AAR versus 47
and 49. Therefore, it is deduced that the presence of sterically
bulky but lengthy chains at N2 position ensures high binding affin-
ity of the derivatives at the hA3AR.
chain enabled the hydrophobic interaction with Trp243, Leu91,
Ser181 and Ser247. The central PP scaffold was involved with an
essential
p–p stacking interaction with Phe168, which is consid-
ered to be crucial for the hA3 antagonistic activity.22 Notably, this
interaction was also present in the binding modes of 4-chloro PP
and PTPs. Data from literature suggest the importance of the bind-
ing of the Phe168 and the mutagenesis analyses conducted at the
A
2AAR demonstrated the importance of Phe168 for antagonist
binding.22
Among the isopropyl derivatives, compound 55 (Fig. 3 panel D),
with diacyl substitution at C6 position and with the highest hA2AAR
affinity (Ki = 0.8 M) among all the isopropyl derivatives, showed a
l
binding pattern close to ZM-241385. Notably, impeded free rota-
tion of isopropyl group due to ring constraints directed the ligand
to obtain a fixed conformation and this accounted for the shifting
of affinity towards the hA2AAR. The isopropyl substituents of the
main scaffold are located towards the TM2, one of the diacyl chains
pointing towards the EL2, whereas other acyl chains pointed to-
wards the TM6. This result indicates that the receptor pocket,
where the C6 appended moiety of these derivatives accommodated
is roomy enough to hold the rather bulky and broadened diacyl
chain. In addition, N1 of the pyrazole ring formed a H-bonding
interaction with Asn253 (6.55) and a stable interaction with
Glu169 (EL2), which is considered to be vital for ligand binding
at the hA2AAR.41–43 Similarly, compound 45, with a para-toluoyl
group of the C6-benzoyl substituent, bound to hA2AAR in a similar
fashion as compound 55, thus justifying an equally potent affinity
at this receptor subtype. The electrostatic contribution per residue
to the whole interaction energy for the ZM-241385 in hA2AAR
(Fig. S2-D) was compared to the one of compound 53 inside the
hA2AAR. It was noticed that the three main stabilizing residues
such as Glu169 (EL2), Asn253 (6.55), and Tyr271 (7.36) contributed
for the affinity of ZM-241385 in the hA2AAR. In contrast, compound
53 displayed slightly different binding mode, which in turn re-
flected in weak stabilizing interaction with these three residues
(Fig. S2-E).
Eventually, we compared the binding pose of the most potent
compounds of both chloro (53) and ester series (63) inside the hA3-
AR. It was observed that compound 63 was oriented in a different
pattern from compound 53 of the chloro series. It is speculated that
rotatable ester chain at C4 position caused the ligand to attain dif-
ferent conformation. However, compound 63 retained all essential
hydrophobic interactions with residues such as Met177, Leu246,
Leu264, Trp243, Val169 and Val259. As seen earlier, compound
63 gained an extra stabilizing hydrogen bonding interaction with
Asn250, whereas with compound 53, a single hydrogen bonding
interaction was observed (Fig. 3A). These findings substantiate
the experimental results of increased affinity and selectivity for
compound 63 over compound 53.
Interestingly, C4-carboxylate N2 neopentyl substituted benzam-
ide compounds (62 and 63) displayed a different binding pose in
comparison to compound 53. Among them, compound 63
(Fig. 3B), which was the most potent analogue of the C4-carboxyl-
ate series, oriented a little away from the centre of the binding
pocket, towards the TM5. In addition, the ester group was oriented
towards the TM2 and the phenyl ring of the benzamide pointed to-
wards the ECL2. Due to this reason, the additional ester group was
able to make a potential hydrogen bonding interaction with
Asn250. Surprisingly, three stabilizing hydrogen bonding interac-
tions were observed between ligand 63 and Asn250. Among them,
one of the hydrogen bonding was seen with NH of the amide chain,
and other two bondings were observed with the N-1 of the pyra-
zole and N-2 of pyrimidine, respectively. Similarly, another potent
and selective compound of the series, compound 62, also shared
with 63 a close resemblance of the binding pose and was also in-
volved in similar binding interactions. This in turn reflected the
higher binding affinity and selectivity of these compounds. In both
compounds (62 and 63), para-CF3 and para-CH3 made identical
hydrophobic interaction with residues such as Val169, Val259
and Leu264 and one of the residues, Val169, was also reported in
previous work on PTP analogues, where Val169 was deemed to
be essential for antagonist recognition.22 In addition, the neopentyl
Docking evaluation confirmed why newly synthesized PP-4-car-
boxylate derivatives are better than the 4-chloro-PP derivatives.
Apparently, compounds of the PP-4-carboxylate series made an
additional stabilizing hydrogen bonding interaction with the
Asn250, which was not found with previous PP-chloro series deriv-
atives. In addition, the most potent compounds of the series dis-
played some specific hydrophobic interactions with Val169 and
Val259. These hydrogen bonding and hydrophobic interactions have
remarkably contributed to the improved affinity and selectivity.
3. Conclusions
In summary, the bicyclic pyrazolo[3,4-d]pyrimidine scaffold
was designed based on the molecular simplification approach from
the tricyclic pyrazolo-triazolo-pyrimidine. In addition, all the
target compounds were synthesized using a novel and efficient
synthetic approaches. This study has led to the identification of a

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1759
novel series of pyrazolo[3,4-d]pyrimidine derivatives as selective
hA3AR or hA2AAR antagonists. The present study has highlighted
that the pyrazolo-pyrimidine moiety is a promising scaffold for
obtaining novel human AR antagonists. Indeed, most of the synthe-
sized compounds showed affinity at the hA3AR in the low micro-
molar range, with the most potent antagonist bearing a C4
carboxylate and N2 neopentyl with a C6 para-toluoyl group (63,
carbonate (1.65 g, 0.012 mol, 1.2 equiv) was added at 0 °C and
the resulting mixture was stirred at the same temperature for
45 min. Appropriate alkyl/aryl iodide or bromide (0.012 mol,
1.2 equiv) was added slowly over 15 min and the reaction mixture
was heated at 90 °C for 10 h. Then the resulting reaction mixture
was cooled, poured over ice cold water and the aqueous phase
was extracted with EtOAc (3 ꢂ 10 mL), the combined organic lay-
ers were dried over Na2SO4, filtered, and the solvent was removed
under reduced pressure to give a oily residue that was purified via
column chromatography eluting with a mixture of hexane/ethyl-
acetate (1:1) to obtain the desired products (7–10) as solids.
Ki hA3AR = 0.9
lM, hA1AR/hA3AR = >111 & hA2AAR/hA3AR = >111)
and its fluorinated acyl derivative (62, Ki hA3AR = 1.3
lM, hA1AR/
hA3AR = >77 & hA2AAR/hA3AR = >77). In addition, most of the com-
pounds of the N2 isopropyl series exhibited affinity in very low
micromolar range towards hA2AAR, especially compound 55 (Ki
hA2AAR = 0.8
l
M), which displayed the most potent affinity at
4.2.1. 3-Amino-1-methyl-1H-pyrazole-4-carbonitrile (7)
Pale yellow solid: Yield (0.723 g, 67%); mp 125–127 °C.
hA2AAR, whereas compounds 45 and 43 displayed remarkable
selectivity towards the hA3AR subtype (45, hA3AR/hA2AAR >91;
43, hA3AR/hA2AAR >38).
4.2.2. 3-Amino-1-isopropyl-1H-pyrazole-4-carbonitrile (8)
Pale yellow solid: Yield (0.745 g, 69%); mp 120–122 °C.
To delineate the SAR of this pyrazolo[3,4-d]pyrimidine series as
hA3AR and hA2AAR antagonists, we have investigated different por-
tions of this structure, modifying the substituents at the C6, N2 and
C4 positions. It emerged that, within the pyrazolo[3,4-d]pyrimidine
nucleus, certain functionalities such as C4 carboxylate, the acyl
linkage at C6 position and chain-elongated lipophilic alkyl groups
or phenylethyl groups at N2 position are important for the activity
of these compounds at the hA3AR; conversely, an isopropyl group
at N2 position is essential for activity at hA2AAR. Through a molec-
ular modeling investigation, the experimental findings have been
rationalized by depicting the hypothetical binding mode between
these newly synthesized series and the specific amino acid resi-
dues within the binding site of hA3AR and hA2AAR. Generally, bind-
ing assay results exemplified that C4 ester substituted derivatives
(62–63) are more potent than that of C4 chloro substituted com-
pounds (38–61) due to additional stabilizing interactions at the
receptor site. Although the simplified analogues showed lower
affinity as compared to the parent PTP analogues, this could be
considered as the starting point for developing more potent hA3AR
antagonists. Further synthetic and biological studies on these C4
carboxylate series lead structures are on-going in our laboratories
and will be reported in due course.
4.2.3. 3-Amino-1-neopentyl-1H-pyrazole-4-carbonitrile (9)
Pale yellow solid: Yield (0.766 g, 71%); mp 123–125 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.88 (s, 9H, 3CH3), 3.67 (s, 2H, CH2), 5.49 (s,
2H, NH2), 8.04 (s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z) calcd
for C9H14N4 (178.23): found 179.2 [M+H]+.
4.2.4. 3-Amino-1-phenethyl-1H-pyrazole-4-carbonitrile (10)
Pale yellow solid: Yield (0.820 g, 76%); mp 132–134 °C.
4.3. General procedure for synthesis of 3-amino-1-(alkyl or aryl
alkyl)-1H-pyrazole-4-carboxamide (11–14)
Carbonitriles 7–10 (0.03 mol) was dissolved in 7 mL concd
H2SO4 at 0 °C and stirred at room temperature for 5 h. Then the
reaction mixture was poured into ice cold water and neutralized
to pH 7 with 28% NH3 solution. The white precipitate was formed
which was further washed with cold water and dried under
vacuum.
4.3.1. 3-Amino-1-methyl-1H-pyrazole-4-carboxamide (11)
White solid: Yield (3.0 g, 83%); mp 155–157 °C.
4. Experimental section
4.1. Chemistry
4.3.2. 3-Amino-1-isopropyl-1H-pyrazole-4-carboxamide (12)
White solid: Yield (3.82 g, 85%); mp 159–161 °C.
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) on silica gel plate (precoated 60 F254 Merck plate). Column
chromatographies were performed using silica gel 60 (Merck,70–
230 mesh). Melting points were determined on a Gallenkamp
instrument and were uncorrected. Compounds were dissolved in
HPLC (high performance liquid chromatography) grade methanol
for determination of mass to charge m/z on a LCQ Finnigan MAT
mass spectrometer (source of ionization: Electron spin ionization
(ESI) probe). Purity of the compounds were determined by HPLC
using HITACHI, version 3.1.8b. 1H and 13C NMR spectra were deter-
mined in the deuterated dimethylsulfoxide (DMSO-d6) solutions on
Bruker DPX ultrashield NMR (400 MHz) spectrometers, with chem-
ical shifts given in parts per million (d) downfield relative to tetra-
methylsilane (TMS) as internal standard, and J values (coupling
constants) given in hertz. The following abbreviations were used:
s, singlet; d, doublet; t, triplet; sep, septet; m, multiplet; br, broad.
4.3.3. 3-Amino-1-neopentyl-1H-pyrazole-4-carboxamide (13)
White solid: Yield (4.17 g, 78%); mp 161–163 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.90 (s, 9H, 3CH3), 3.64 (s, 2H, CH2), 5.32
(s, 2H, NH2), 6.72 (br s, 1H, NH), 7.21 (br, 1H, NH), 7.85 (s, 1H, pyr-
azole-H). LC–MS (ESI) analysis (m/z) calcd for C9H16N4O (196.25):
found 197.1 [M+H]+.
4.3.4. 3-Amino-1-phenethyl-1H-pyrazole-4-carboxamide (14)
White solid: Yield (5.73 g, 90%); mp 166–168 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.08 (t, J = 7.16 Hz, 2H, CH2), 4.12 (t,
J = 7.16 Hz, 2H, CH2), 5.44 (s, 2H, NH2), 6.24 (br s, 1H, NH), 6.74
(br s, 1H, NH), 7.19–7.35 (m, 5H, Ar-H), 7.80 (s, 1H, pyrazole-H).
LC–MS (ESI) analysis (m/z) calcd for C12H14N4O (230.27): found
231.0 [M+H]+, 254.2 [M+Na]+.
4.4. General procedure for synthesis of 3-(3-benzoylthioureido)-
1-(alkyl or aryl alkyl)-1H-pyrazole-4-carboxamide (15–18)
4.2. General procedure for the synthesis of 3-amino-1-alkyl or
arylalkyl-1H-pyrazole-4- carbonitriles (7–10)
To a solution of carboxamides 11–14 (0.015 mol) in dry acetone
(10 mL), benzoyl isothiocyante (0.0165 mol, 1.1 equiv) was added
and refluxed at 60 °C for 12 h. The reaction mixture was cooled.
The resulting yellow solid was filtered, washed with acetone and
To a solution of 3-amino-1H-pyrazole-4-carbonitrile (1.08 g,
0.01 mol) in anhydrous DMF (5 mL), anhydrous potassium

1760
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
dried under vacuum. The crude product was further recrystalized
from methanol to obtain a pure solids.
4.02 (s, 2H, CH2), 7.40–7.95 (m, 5H, CH), 8.02 (d, 2H, CONH2),
8.17 (s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z) calcd for C18-
H23N5O2S (373.47): found 374.1 [M+H]+, 396.2 [M+Na]+.
4.4.1. 3-(3-Benzoylthioureido)-1-methyl-1H-pyrazole-4-
carboxamide (15)
4.5.4. (E)-Methyl-N0-benzoyl-N-(4-carbamoyl-1-phenethyl-1H-
pyrazol-3-yl) carbamimidothioate (22)
Yellow solid: Yield (1.28 g, 61%); mp 218–220 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.61 (s, 3H, N–CH3), 7.17 (br s, 1H, NH),
7.46–7.60 (m, 5H, CH), 7.73 (d, 2H, CONH2), 7.78 (s, 1H, pyrazole-
H), 11.47 (br s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for
White solid: Yield: (0.743 g, 63%); mp 235–237 °C. 1H NMR
(400 MHz, DMSO-d6): d 2.41 (s, 3H, SCH3), 3.13 (t, J = 7.04 Hz, 2H,
CH2), 4.45 (t, J = 6.64 Hz, 2H, CH2), 7.11–7.23 (m, 5H, CH), 7.39–
7.87 (m, 5H, CH), 8.00 (d, 2H, CONH2), 8.12 (s, 1H, pyrazole-H),
13.10 (br s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for C21H21N5-
O2S (407.49): found 408.2 [M+H]+, 430.2 [M+Na]+.
C
13H13N5O2S (303.34): found 304.0 [M+H]+.
4.4.2. 3-(3-Benzoylthioureido)-1-isopropyl-1H-pyrazole-4-
carboxamide (16)
Yellow solid: Yield (1.58 g, 63%); mp 212–214 °C. 1H NMR
(400 MHz, DMSO-d6): 1.42 (d, J = 5.76 Hz, 6H, 2CH3), 4.44–4.50 (m,
1H, CH), 7.07 (br, 1H, NH), 7.57–7.68 (m, 5H, CH), 8.00 (d, 2H,
CONH2), 8.31 (s, 1H, pyrazole-H), 11.06 (br s, 1H, NH). LC–MS (ESI)
analysis (m/z) calcd for C15H17N5O2S (331.39): found 332.0 [M+H]+.
4.6. General procedure for synthesis of (Z)-3-(2-
benzoylguanidino)-1-(alkyl or aryl alkyl)-1H-pyrazole-4-
carboxamide (23–26)
Carbamimidothioate derivatives 19–22 (0.003 mol) were dis-
solved in dry DMF (5 mL) containing 2% ammonia (5 mL) and
heated to 120 °C for 3 h in a sealed tube. At the end of the reaction
the odour of methyl mercaptan was recognized, cooled and poured
over ice water mixture. The resulting white precipitate was
washed with water and dried under vacuum.
4.4.3. 3-(3-Benzoylthioureido)-1-neopentyl-1H-pyrazole-4-
carboxamide (17)
Yellow solid: Yield (1.76 g, 60%); mp 215–217 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.91 (s, 9H, 3CH3), 3.91 (s, 2H, CH2), 7.09
(br s, 1H, NHCO), 7.57–7.70 (m, 5H, CH), 8.00 (d, 2H, CONH2),
8.18 (br s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z) calcd for
4.6.1. (Z)-3-(2-Benzoylguanidino)-1-methyl-1H-pyrazole-4-
carboxamide (23)
C
17H21N5O2S (359.45): found 360.2 [M+H]+.
White solid: Yield (0.504 g, 53%); mp >300 °C. Insoluble.
4.4.4. 3-(3-Benzoylthioureido)-1-phenethyl-1H-pyrazole-4-
carboxamide (18)
4.6.2. (Z)-3-(2-Benzoylguanidino)-1-isopropyl-1H-pyrazole-4-
carboxamide (24)
Yellow solid: Yield (2.44 g, 71%); mp 208–210 °C. 1H NMR (400 MHz,
DMSO-d6): d 3.07 (br d, 2H, CH2), 4.34 (brs, 2H, CH2), 7.21–7.27 (m, 5H,
CH), 7.49–7.69 (m, 5H, CH), 7.99 (s, 2H, CONH2), 8.01 (s, 1H, pyrazole-H),
11.51 (br s, 1H, NHCO), 12.7 (br s, 1H, NH). LC–MS (ESI) analysis (m/z)
calcd for C20H19N5O2S (393.46): found 393.9 [M+H]+.
White solid: Yield (0.642 g, 62%); mp >300 °C. Insoluble.
4.6.3. (Z)-3-(2-Benzoylguanidino)-1-neopentyl-1H-pyrazole-4-
carboxamide (25)
White solid: Yield (0.627 g, 56%); white mp >300 °C. Insoluble.
4.5. General procedure for synthesis of (E)-methyl N0-benzoyl-N-
(4-carbamoyl-1-(alkyl or aryl alkyl)-1H-pyrazol-3-yl)
carbamimidothioate (19–22)
4.6.4. (Z)-3-(2-Benzoylguanidino)-1-phenethyl-1H-pyrazole-4-
carboxamide (26)
White solid: Yield (0.646 g, 53%); mp >300 °C. Insoluble.
To a solution of benzoylthioureidos 15–18 (0.003 mol) in 10 mL
of 0.1 N NaOH, methyl iodide (0.0036 mol, 1.2 equiv) was added at
room temperature and the resulting mixture was stirred for 3 h.
The white suspension was observed and it was adjusted to pH 6 with
glacial acetic acid. The white precipitate was obtained, which was
filtered and washed with cold water and finally dried under vacuum.
4.7. General procedure for synthesis of 6-amino-2-(alkyl or aryl
alkyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (27–30)
A
suspension of benzoylguanidino derivatives 23–26
(0.0035 mol) in 1 N NaOH (10 mL) was refluxed at 110 °C for 12 h.
The resulting milky white mixture was adjusted to pH 6 with acetic
acid. A mixture of benzoic acid and free amine derivatives were ob-
tained which was washed with water and dried. Finely divided
powder was suspended in hot ethanol with stirring to remove ben-
zoic acid and filtered hot. The resulting ethanolic portion was evap-
orated to dryness to obtain a pure desired product as white solid.
4.5.1. (E)-Methyl-N0-benzoyl-N-(4-carbamoyl-1-methyl-1H-
pyrazol-3-yl) carbamimidothioate (19)
White solid: Yield: (0.682 g, 75%); mp 196–198 °C. 1H NMR
(400 MHz, DMSO-d6): d 2.44 (s, 3H, SCH3), 3.57 (s, 3H, N–CH3),
7.00 (s, 1H, NH), 7.42–7.52 (m, 5H, CH), 7.70 (br s, 1H, pyrazole-
H), 7.78 (d, 2H, CONH2), 11.52 (br s, 1H, NH). LC–MS (ESI) analysis
(m/z) calcd for C14H15N5O2S (317.37): found 318.1 [M+H]+.
4.7.1. 6-Amino-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (27)
4.5.2. (E)-Methyl-N0-benzoyl-N-(4-carbamoyl-1-isopropyl-1H-
pyrazol-3-yl) carbamimidothioate (20)
White solid: Yield (0.721 g, 72%); mp >300 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.69 (s, 3H, N–CH3), 6.69 (s, 2H, NH2),
7.73 (s, 1H, pyrazole-H), 10.58 (s, 1H, NHCO). LC–MS (ESI) analysis
(m/z) calcd for C6H7N5O (165.16): found 166.1 [M+H]+.
White solid: Yield: (0.665 g, 67%); mp 223–225 °C. 1H NMR
(400 MHz, DMSO-d6): 1.45 (d, J = 6.52 Hz, 6H, 2CH3), 2.41 (s, 3H,
SCH3), 4.59 (br s, 1H, CH), 7.39–7.91(m, 5H, CH), 8.02 (d, 2H,
CONH2), 8.24 (s, 1H, pyrazole-H), 13.09 (br s, 1H,NH). LCMS (ESI)
analysis (m/z) calcd for C16H19N5O2S (345.42): found 346.0 [M+H]+.
4.7.2. 6-Amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (28)
White solid: Yield (0.825 g, 75%); mp >300 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.42 (d, J = 6.52 Hz, 6H, 2CH3), 4.50 (sep,
J = 6.52 Hz, 1H, CH), 6.90 (s, 2H, NH2), 8.11 (s, 1H, pyrazole-H),
10.61 (s, 1H, NHCO). LC–MS (ESI) analysis (m/z) calcd for
C8H11N5O (193.21): found 194.2 [M+H]+, 216.0 [M+Na]+.
4.5.3. (E)-Methyl-N0-benzoyl-N-(4-carbamoyl-1-neopentyl-1H-
pyrazol-3-yl) carbamimidothioate (21)
White solid: Yield: (0.600 g, 56%); mp 219–221 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.90 (s, 9H, 3CH3), 2.42 (s, 3H, SCH3),

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1761
4.7.3. 6-Amino-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (29)
(C–NH2), 161.7 (N–C–N–). LC–MS (ESI) analysis (m/z) calcd for C13-
H12ClN5 (273.72): found 272.0 [MꢁH]+. HPLC purity (254 nm);
99.3%, eluent: 60% ACN/H2O, tR = 5.5 min.
White solid: Yield (0.815 g, 68%); mp >300 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.96 (s, 9H, 3CH3), 3.70 (s, 2H, CH2), 6.24
(s, 2H, NH2), 7.67 (s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z)
calcd for C10H15N5O (221.26): found 222.0 [M+H]+, 244.0 [M+Na]+
4.9. General procedure for synthesis of 6-amino-2-neopentyl-
2H-pyrazolo[3,4-d]pyrimidine-4-carbonitrile (35)
4.7.4. 6-Amino-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (30)
A mixture of 4-chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimi-
din-6-amine 33 (0.00054 mol), sodium cyanide (0.0011 mol,
2 equiv), sodium p-toluene sulfinate (0.00016 mol, 0.3 equiv) in
5 mL of anhydrous DMF was heated on an oil bath at 80 °C for
2 h. The resultant mixture was cooled, poured into ice water mix-
ture and stirred for 10 min. The solid was collected by filtration,
washed with water and recrystallized from dimethylformamide
and methanol mixture to obtain the desired products (35) as solids.
Yield: 62%; pale yellow solid, mp 148–150 °C; 1H NMR
(400 MHz, DMSO-d6): d 0.98 (s, 9H, 3CH3), 4.06 (s, 2H, CH2), 7.48
(s, 2H, NH2), 8.27 (s, 1H, pyrazole-H).13C NMR (400 MHz, DMSO-
d6): d 28.1 (CH3), 33.7 (C), 57.2 (CH2), 108.5 (C), 115.1(CN), 131.8
(CH-pyrazole), 135.4 (C–CN), 156.6 (C–NH2), 162.3 (N–C–N–).
LC–MS (ESI) analysis (m/z) calcd for C11H14N6 (230.27): found
229.2 [MꢁH]+.
White solid: Yield (1.067 g, 81%); mp >300 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.10 (t, J = 6.64 Hz, 2H, CH2), 4.36 (t,
J = 6.76 Hz, 2H, CH2), 7.34 (s, 2H, NH2), 7.14–7.30 (m, 5H, CH),
7.89 (s, 1H, pyrazole-H), 10.62 (s, 1H, NHCO). LC–MS (ESI) analysis
(m/z) calcd for C13H13N5O (255.28): found 256.0 [M+H]+, 278.0
[M+Na]+.
4.8. General procedure for synthesis of 4-chloro-2-(alkyl or
arylalkyl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (31–34)
A mixture of 6-amino-2-(alkyl or arylalkyl)-2H-pyrazolo[3,4-
d]pyrimidin-4(5H)-ones (27–30) (0.005 mol), phosphoroyl trichlo-
ride (0.1 mol, 20 equiv) and N–N dimethylamine (0.005 mol,
1 equiv) was refluxed at 110 °C for 24 h. Then the reaction mixture
was cooled and the excess phosphoroyl chloride was removed un-
der reduced pressure. The resulting red oil was poured onto ice
mixture slowly, stirred for 10 min and the aqueous part was ex-
tracted with EtOAc (3 ꢂ 10 mL). The combined organic layers were
dried over Na2SO4, filtered, and the solvent was removed under re-
duced pressure to give a oily residue that was purified via column
chromatography eluting with a mixture of hexane/ethyl acetate
(5:5) to obtain pale yellow solids as desired products (31–34).
4.10. General procedure for synthesis of 6-amino-2-neopentyl-
2H-pyrazolo[3,4-d]pyrimidine-4-carboxamide (36)
To a stirred solution of 6-amino-2-neopentyl-2H-pyrazolo[3,4-
d]pyrimidine-4-carbonitrile 35 (0.00057 mol) in DMSO (3 mL) at
cold condition, 30% H2O2 (0.0023 mol, 4 equiv) and anhydrous
K2CO3 (0.0011 mol, 2 equiv) were added. The mixture was stirred
at room temperature for 1 h. Then the resultant mixture was
poured into ice water mixture and stirred for 10 min. The solid
was collected by filtration, washed with water and recrystallized
from methanol to obtain the desired products (36) as solids.
Yield: 79%; White solid, mp 174–176 °C; 1H NMR (DMSO-d6): d
0.99 (s, 9H, 3CH3), 4.07 (s, 2H, CH2), 7.03 (s, 2H, NH2), 7.89 (br s, 1H,
NH), 8.00 (br s, 1H, NH), 8.24 (s, 1H, pyrazole-H). LC–MS (ESI) anal-
ysis (m/z) calcd for C11H16N6O (248.28) : found 247.0 [MꢁH]+.
4.8.1. 4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-amine
(31)
Pale yellow solid: Yield (0.536 g, 65%); mp 247–249 °C. HPLC
purity (254 nm); 98.2%, eluent: 60% ACN/H2O, tR = 2.2 min.
4.8.2. 4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
amine (32)
Pale yellow solid: Yield (0.647 g, 67 %); mp 151–153 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.48 (d, J = 6.64 Hz, 6H, 2CH3), 4.53 (sep,
J = 6.52 Hz, 1H, CH), 6.18 (s, 2H, NH2), 8.30 (s, 1H, pyrazole-H).
13C NMR (400 MHz, DMSO-d6): d22.0 (CH3), 48.4 (CH-alkyl),
106.7 (C), 132.1 (CH-pyrazole), 153.7 (C–Cl), 155.1 (C–NH2),
166.6 (N–C–N–). LC–MS (ESI) analysis (m/z) calcd for C8H10ClN5
(211.65): found 212.1 [M+H]+, HPLC purity (254 nm); 96.8%, elu-
ent: 60% ACN/H2O, tR = 3.3 min.
4.11. General procedure for synthesis of ethyl-6-amino-2-
neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (37)
To a solution of 6-amino-2-(alkyl or aralkyl)-2H-pyrazolo[3,4-
d]pyrimidine-4-carboxamides 36 (0.00052 mol) in absolute etha-
nol (30 mL), conc. H2SO4 (3 mL) was added slowly and the resulting
mixture was refluxed at 80 °C for 12 h. The reaction mixture was
cooled and excess ethanol was evaporated and the resulting oily
residue was poured into ice water mixture, stirred for 10 min. The
solid was collected by filtration, washed with water and recrystal-
lized from methanol to obtain the desired products (37) as solids.
Yield: 84%; pale yellow solid, mp 120–122 °C; 1H NMR (DMSO-
d6): d 0.99 (s, 9H, 3CH3), 1.43 (t, J = 7.0 Hz, 3H, CH3), 4.07 (s, 2H,
CH2), 4.47 (q, J1 = 7.0 Hz, J2 = 14.18 Hz, 2H, COOCH2), 7.27 (s, 2H,
NH2), 8.18 (s, 1H, pyrazole-H). 13C NMR (400 MHz, DMSO-d6): d
14.5 (CH3), 28.2 (CH3)3, 33.8 (C), 57.0 (CH2), 62.3 (COOCH2),
106.0 (C), 133.7 (CH-pyrazole), 151.0 (C–COO), 157.7 (C–NH2),
162.7 (N–C–N–), 163.9 (COO). LC–MS (ESI) analysis (m/z) calcd
for C13H19N5O2 (277.32): found 278.3 [M+H]+, 300.2 [M+Na]+. HPLC
purity (254 nm); 97.9 %, eluent: 60% ACN/H2O, tR = 3.6 min.
4.8.3. 4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-6-
amine (33)
Pale yellow solid: Yield (0.815 g, 77%); mp 154–156 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.96 (s, 9H, 3CH3), 4.01 (s, 2H, CH2), 7.29 (s,
2H, NH2), 8.02 (s, 1H, pyrazole-H).13C NMR (400 MHz, DMSO-d6): d
28.1 (CH3), 33.7 (C-Alkyl), 57.3 (CH2), 106.2 (C), 132.2 (CH-pyra-
zole), 153.9 (C–Cl), 156.9 (C–NH2), 161.8 (N–C–N–). LC–MS (ESI)
analysis (m/z) calcd for C10H14ClN5 (239.70): found 240.0[M+H]+.
HPLC purity (254 nm); 100%, eluent: 60% ACN:H2O, tR = 6.0 min.
4.8.4. 4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-6-
amine (34)
Pale yellow solid: Yield (0.918 g, 72%); mp 146–148 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.10 (t, J = 6.95 Hz, 2H, CH2), 4.38 (t,
J = 7.55 Hz, 2H, CH2), 7.10–7.22 (m, 5H, Ar-H), 7.26 (s, 2H, NH2),
7.97 (s,1H, pyrazole-H). 13C NMR (400 MHz, DMSO-d6): d 35.0
(CH2), 47.8 (N–CH2), 106.5 (C), 126.8 (CH), 128.8 (CH), 129.0
(CH), 132.4 (CH), 138.6 (CH-pyrazole), 153.8 (C–Cl), 156.1
4.12. General procedure for synthesis of N-(4-chloro-2(alkyl or
arylalkyl)-2H-pyrazolo[3,4-d]pyrimidin-6-yl) benzamide (38–
41, 42–45, 46–49, 50–53)
To a suspension of 4-chloro-2-(alkyl or arylalkyl)-2H-pyrazol-
o[3,4-d]pyrimidin-6-amines 31–34 (0.001 mol) in toluene,

1762
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
diisopropylethylamine (0.002 mol, 2 equiv) and benzoyl chloride
or substituted benzoyl chlorides (0.002 mol, 2 equiv) were added.
The mixture was heated under reflux at 120 °C for 24 h. The sol-
vent was removed under reduced pressure and the resulting resi-
due was purified by column chromatography (hexane/EtOAc, 5:5).
C16H13ClF3N5O (383.76): found 384.2 [M+H]+, 406.2 [M+Na]+. HPLC
purity (254 nm); 99.6%, eluent: 60% ACN/H2O, tR = 7.3 min.
4.12.8. N-(4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-methylbenzamide (45)
White solid: Yield (0.0719 g, 34%); mp 131–133 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.50 (d, , J = 6.64 Hz, 6H, 2CH3), 2.39 (s,
3H, CH3), 5.00 (sep, J = 6.64 Hz, 1H, CH), 7.32 (d, J = 8.04 Hz, 2H,
Ar-H), 7.91 (d, J = 8.12 Hz, 2H, Ar-H), 8.34 (s, 1H, pyrazole-H),
11.31 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for C16H16ClN5O
(329.78): found 330.2 [M+H]+, 352.1 [M+Na]+. HPLC purity
(254 nm); 95.7%, eluent: 60% ACN/H2O, tR = 5.2 min.
4.12.1. N-(4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)benzamide (38)
White solid: Yield (0.066 g, 36%); mp 165–167 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.99 (s, 3H, N–CH3), 7.51–7.64 (m, 3H,
Ar-H), 7.99 (d, J = 7.52 Hz, 2H, Ar-H), 8.34 (s, 1H, pyrazole-H),
11.42 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for C13H10ClN5O
(287.70): found 288.1 [M+H]+, 310.1 [M+Na]+. HPLC purity
(254 nm); 97.8%, eluent: 60% ACN:H2O, tR = 2.9 min.
4.12.9. N-(4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)benzamide (46)
4.12.2. N-(4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-fluorobenzamide (39)
White solid: Yield (0.105 g, 44%); mp 135–137 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.95 (s, 9H, 3CH3), 4.16 (s, 2H, CH2),
7.51–8.0 (m, 5H, Ar-H), 8.38 (s, 1H, pyrazole-H), 11.38 (s, 1H,
NH). LC–MS (ESI) analysis (m/z) calcd for C17H18ClN5O (343.81):
found 344.2 [M+H]+, 366.3 [M+Na]+. HPLC purity (254 nm);
97.8%, eluent: 60% ACN/H2O, tR = 7.2 min.
White solid: Yield (0.0786 g, 43%); mp 140–142 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.98 (s, 3H, N–CH3), 7.33–7.37 (m, 2H,
Ar-H), 8.05–8.09 (m, 2H, Ar-H), 8.35 (s, 1H, pyrazole-H), 11.46 (s,
1H, NH). LC–MS (ESI) analysis (m/z) calcd for
C21H18ClN5O
(391.85): found 392.0 [M+H]+. HPLC purity (254 nm); 99.4%, elu-
ent: 60% ACN/H2O, tR = 3.3 min.
4.12.10. N-(4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-4-fluorobenzamide (47)
4.12.3. N-(4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-(trifluoromethyl)benzamide (40)
White solid: Yield (0.129 g, 54%); mp 132–134 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.95 (s, 9H, 3CH3), 4.15 (s, 2H, CH2),
7.33–8.05 (m, 4H, Ar-H), 8.37 (s, 1H, pyrazole-H), 11.42 (s, 1H,
NH). LC–MS (ESI) analysis (m/z) calcd for C17H17ClFN5O (361.80):
found 362.2 [M+H]+, 384.2 [M+Na]+. HPLC purity (254 nm);
98.3%, eluent: 60% ACN/H2O, tR = 8.0 min.
White solid: Yield (0.104 g, 57%); mp 145–147 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.98 (s, 3H, N–CH3), 7.88-7.90 (m, 2H,
Ar-H), 8.16–8.17 (m, 2H, Ar-H), 8.35 (s, 1H, pyrazolo-H), 11.67 (s,
1H, NH). LC–MS (ESI) analysis (m/z) calcd for C14H9ClF3N5O
(355.70): found 356.2 [M+H]+, 378.2 [M+Na]+. HPLC purity
(254 nm); 99.8%, eluent: 60% ACN:H2O, tR = 5.7 min.
4.12.11. N-(4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-4-(trifluoromethyl)benzamide (48)
4.12.4. N-(4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-methylbenzamide (41)
White solid: Yield (0.0935 g, 39%); mp 129–131 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.92 (s, 9H, 3CH3), 4.10 (s, 2H, CH2),
7.87–8.13 (m, 4H, Ar-H), 8.38 (s, 1H, pyrazole-H), 11.63 (s, 1H,
White solid: Yield (0.0768 g, 42%); mp 149–151 °C. 1H NMR
(400 MHz, DMSO-d6): d 2.39 (s, 3H, CH3), 3.98 (s, 3H, N–CH3),
7.31–7.92 (m, 4H, Ar-H), 8.33 (s, 1H, pyrazole-H), 11.32 (s, 1H,
NH). LC–MS (ESI) analysis (m/z) calcd for C14H12ClN5O (301.73):
found 302.0 [M+H]+. HPLC purity (254 nm); 99.4%, eluent: 60%
ACN/H2O, tR = 3.8.
NH). LC–MS (ESI) analysis (m/z) calcd for
C18H17ClF3N5O
(411.81): found 412.3 [M+H]+, 434.3 [M+Na]+. HPLC purity
(254 nm); 95.4%, eluent: 60% ACN/H2O, tR = 10.9 min.
4.12.12. N-(4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-4-methylbenzamide (49)
4.12.5. N-(4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)benzamide (42)
White solid: Yield (0.0839 g, 35%); mp 134–136 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.94 (s, 9H, 3CH3), 2.36 (s, 3H, CH3), 4.15
(s, 2H, CH2), 7.28–7.89 (m, 4H, Ar-H), 8.34 (s, 1H, pyrazole-H),
11.28 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for C18H20ClN5O
(357.84): found 358.3 [M+H]+, 380.2 [M+Na]+. HPLC purity
(254 nm); 98.4%, eluent: 60% ACN/H2O, tR = 8.3 min.
White solid: Yield (0.059 g, 28%); mp 151–153 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.50 (d, J = 6.76 Hz, 6H, 2CH3), 5.01 (sep,
J = 6.52 Hz, 1H, CH), 7.50–7.99 (m, 5H, Ar-H), 8.35 (s, 1H, pyra-
zole-H), 11.39 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for
C
15H14ClN5O (315.76): found 316.2 [M+H]+, 338.3 [M+Na]+. HPLC
purity (254 nm); 100%, eluent: 60% ACN/H2O, tR = 4.2.
4.12.13. N-(4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl) benzamide (50)
4.12.6. N-(4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-fluorobenzamide (43)
White solid: Yield (0.123 g, 45%); mp 140–142 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.23 (t, J = 7.28 Hz, 2H, CH2), 4.60 (t,
J = 7.04 Hz, 2H, CH2), 7.17–7.26 (m, 5H, Ar-H), 7.51–8.00 (m, 5H,
acyl-H), 8.34 (s, 1H, pyrazole-H), 11.38 (s, 1H, NH). LC–MS (ESI)
White solid: Yield (0.0846 g, 40 %); mp 120–122 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.51 (d, J = 6.76 Hz, 6H, 2CH3), 5.02 (sep,
J = 6.76 Hz, 1H, CH), 7.46–7.85 (m, 4H, Ar-H), 8.36 (s, 1H, pyra-
zole-H), 11.49 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for
analysis (m/z) calcd for
C20H16ClN5O (377.83): found 378.3
[M+H]+, 400.2 [M+Na]+. HPLC purity (254 nm); 99.7%, eluent: 60%
C
15H13ClFN5O (333.75): found 334.2 [M+H]+, 356.3 [M+Na]+. HPLC
ACN/H2O, tR = 6.7 min.
purity (254 nm); 100%, eluent: 60% ACN/H2O, tR = 4.6 min.
4.12.14. N-(4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-4-fluorobenzamide (51)
4.12.7. N-(4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-4-(trifluoromethyl)benzamide (44)
White solid: Yield (0.101 g, 37%); mp 127–129 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.23 (t, J = 7.44 Hz, 2H, CH2), 4.61 (t,
J = 7.04 Hz, 2H, CH2), 7.15–7.19 (m, 5H, Ar-H), 7.22–7.26 (m, 2H,
CH), 8.05–8.08 (m, 2H, CH), 8.34 (s, 1H, pyrazole-H), 11.43 (s, 1H,
NH). LC–MS (ESI) analysis (m/z) calcd for C20H15ClFN5O (395.82):
White solid: Yield (0.0842 g, 38 %); mp 146–148 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.47 (d, J = 6.8 Hz, 6H, 2CH3), 4.96 (sep,
J = 6.64 Hz, 1H, CH), 7.86–8.15 (m, 4H, Ar-H), 8.35 (s, 1H, pyra-
zole-H), 11.65 (s, 1H, NH). LC–MS (ESI) analysis (m/z) calcd for

G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
1763
found 396.3 [M+H]+, 418.3 [M+Na]+. HPLC purity (254 nm); 99.6%,
eluent: 60% ACN/H2O, tR = 7.3 min.
(s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z) calcd for C27H20-
ClN5O2 (481.93): found 482.1[M+H]+, 504.1[M+Na]+. HPLC purity
(254 nm); 98.6%, eluent: 60% ACN/H2O, tR = 12.3 min.
4.12.15. N-(4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin
-6-yl)-4-(trifluoromethyl)benzamide (52)
4.14. General procedure for synthesis of N-(4-chloro-2-(alkyl or
arylalkyl)-2H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-phenyl
acetamide (58–61)
White solid: Yield (0.139 g, 51%); mp 137–139 °C. 1H NMR
(400 MHz, DMSO-d6): d 3.20 (t, J = 7.28 Hz, 2H, CH2), 4.59 (t,
J = 7.16 Hz, 2H, CH2), 7.16–7.25 (m, 5H, Ar-H), 7.86–8.15 (m, 5H,
acyl-H), 8.34 (s, 1H, pyrazole-H), 11.63 (s, 1H, NH). LC–MS (ESI)
analysis (m/z) calcd for C21H15ClF3N5O (445.82): found 446.3
[M+H]+, 468.2 [M+Na]+. HPLC purity (254 nm); 98.7%, eluent: 60%
ACN:H2O, tR = 9.2 min.
To a suspension of 4-chloro-2-(alkyl or arylalkyl)-2H-pyrazol-
o[3,4-d]pyrimidin-6-amines 31–34 (0.001 mol) in toluene, diiso-
propylethylamine (0.002 mol, 2 equiv) and phenyl acetyl chloride
(0.002 mol, 2 equiv) were added. The mixture was heated under re-
flux for 12 h. Subsequently, another 0.001 mol equivalent of diiso-
propylethylamine and phenyl acetyl chloride were added and
refluxing continued for 12 h again. The solvent was then removed
under reduced pressure and the resulting residue was purified by
column chromatography (hexane/EtOAc, 5:5).
4.12.16. N-(4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-4-methylbenzamide (53)
White solid: Yield (0.109 g, 40 %); mp 131–133 °C. 1H NMR
(400 MHz, DMSO-d6): d 2.39 (s, 3H, CH3), 3.22 (t, J = 7.16 Hz, 2H,
CH2), 4.60 (t, J = 7.04 Hz, 2H, CH2), 7.17–7.25 (m, 5H, Ar-H), 7.25–
7.91 (m, 5H, acyl-H), 8.33 (s, 1H, pyrazole-H), 11.29 (s, 1H, NH).
LC–MS (ESI) analysis (m/z) calcd for C21H18ClN5O (391.85): found
392.0 [M+H]+. HPLC purity (254 nm); 97.6%, eluent: 60% ACN:H2O,
tR = 6.9 min.
4.14.1. N-(4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-2-phenylacetamide (58)
White solid: Yield (0.0771 g, 42%); mp 147–149 °C. 1H NMR
(DMSO-d6): d 3.83 (s, 2H, CH2), 3.94 (s, 3H, N–CH3), 7.24–7.34
(m, 5H, Ar-H), 8.30 (s, 1H, pyrazole-H), 11.23 (s, 1H, NH). LC–MS
(ESI) analysis (m/z) calcd for C14H12ClN5O (301.73): found 302.0
[M+H]+. HPLC purity (254 nm); 96.2%, eluent: 60% ACN/H2O,
tR = 4.2 min.
4.13. General procedure for synthesis of N-benzoyl-N-(4-chloro-
2-(alkyl or arylalkyl)-2H-pyrazolo[3,4-d]pyrimidin-6-yl)
benzamide (54–57)
To a suspension of 4-chloro-2-(alkyl or arylalkyl)-2H-pyrazol-
o[3,4-d]pyrimidin-6-amines 31–34 (0.001 mol) in toluene, diiso-
propylethylamine (0.004 mol, 4 equiv) and benzoyl chloride
(0.004 mol, 4 equiv) were added. The mixture was heated under re-
flux for 18 h.The solvent was removed under reduced pressure and
the resulting residue was purified by column chromatography
(hexane/EtOAc, 5:5).
4.14.2. N-(4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-
yl)-2-phenylacetamide (59)
White solid: Yield (0.0910 g, 43%); mp 146–148 °C. 1H NMR
(DMSO-d6): d 1.47 (d, J = 6.76 Hz, 6H, 2CH3), 3.83 (s, 2H, CH2),
4.94–4.98 (sep, J = 6.64 Hz, 1H, CH), 7.24–7.35 (m, 1H, CH), 8.30
(s, 1H, pyrazole-H), 11.21 (s, 1H, NH). LC–MS (ESI) analysis (m/z)
calcd for
C
16H16ClN5O (329.78): found 330.0 [M+H]+, 352.3
[M+Na]+. HPLC purity (254 nm); 99.5%, eluent: 60% ACN:H2O,
4.13.1. N-Benzoyl-N-(4-chloro-2-methyl-2H-pyrazolo[3,4-
d]pyrimidin-6-yl)benzamide (54)
tR = 5.3 min.
White solid: Yield (0.112 g, 61%); mp 163–165 °C. 1H NMR
(DMSO-d6): d 3.88 (s, 3H, N–CH3), 7.53-7.87 (m, 10H, Ar-H), 8.53
(s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z) calcd for C20H14-
ClN5O2 (391.81): found 392.1 [M+H]+. HPLC purity (254 nm);
99.9%, eluent: 60% ACN/H2O, tR = 4.5 min.
4.14.3. N-(4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-2-phenylacetamide (60)
White solid: Yield (0.1078 g, 45%); mp 155–157 °C. 1H NMR
(DMSO-d6): d 0.93 (s, 9H, 3CH3), 3.85 (s, 2H, CH2 Ar), 4.12 (s, 2H,
CH2), 7.24–7.34 (m, 5H, Ar-H), 8.33 (s, 1H, pyrazole-H), 11.19 (s,
1H, NH). LC–MS (ESI) analysis (m/z) calcd for C18H20ClN5O
(357.84): found 358.1 [M+H]+, 380.5 [M+Na]+. HPLC purity
(254 nm); 98.3%, eluent: 60% ACN/H2O, tR = 10.0 min.
4.13.2. N-Benzoyl-N-(4-chloro-2-isopropyl-2H-pyrazolo[3,4-
d]pyrimidin-6-yl)benzamide (55)
White solid: Yield (0.1206 g, 57%); mp 153–155 °C. 1H NMR
(DMSO-d6): d 1.21–1.22 (d, J = 6.8 Hz, 6H, 2CH3), 4.67–4.74 (sep,
1H, CH), 7.47–7.82 (m, 10H, Ar-H), 8.45 (s, 1H, pyrazole-H). LC–
MS (ESI) analysis (m/z) calcd for C22H18ClN5O (419.86): found
421.3 [M+H]+, 442.3[M+Na]+. HPLC purity (254 nm); 99.4%, eluent:
60% ACN/H2O, tR = 7.2 min.
4.14.4. N-(4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-
6-yl)-2-phenylacetamide (61)
White solid: Yield (0.1067 g, 39 %); mp 141–143 °C. 1H NMR
(DMSO-d6): d 3.20 (t, J = 7.16 Hz, 2H, CH2), 3.85 (s, 2H, CH2), 4.57
(t, J = 6.88 Hz, 2H, CH2), 7.12–7.21 (m, 5H, Ar-H), 7.25–7.34 (m,
5H, Ar-H), 8.30 (s,1H, pyrazole-H), 11.17 (s, 1H, NH). LC–MS (ESI)
4.13.3. N-Benzoyl-N-(4-chloro-2-neopentyl-2H-pyrazolo[3,4-
d]pyrimidin-6-yl)benzamide (56)
analysis (m/z) calcd for C21H18ClN5O (391.85): found 392.3
[M+H]+, 414.0 [M+Na]+. HPLC purity (254 nm); 99.5%, eluent: 60%
Yield: 52%; White solid, mp 161–162 °C; 1H NMR (400 MHz,
DMSO-d6): d 0.61 (s, 9H, 3CH3), 4.01 (s, 2H, CH2), 7.48–7.83 (m,
10H, Ar-H), 8.52 (s, 1H, pyrazole-H). LC–MS (ESI) analysis (m/z)
ACN/H2O, tR = 7.9 min.
4.15. General procedure for synthesis of ethyl 6-benzamido-
neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (62 &
63)
calcd
for
C24H22ClN5O2
(447.92):
found
448.1[M+H]+,
470.1[M+Na]+. HPLC purity (254 nm); 100.0%, eluent: 60% ACN/
H2O, tR = 11.6 min.
To a suspension of ethyl 6-amino-2-(neopentyl)-2H-pyrazol-
o[3,4-d]pyrimidine-4-carboxylates 37 (0.00045 mol) in anhydrous
toluene (5 mL), diisopropylethylamine (0.00135 mol, 3 equiv) and
substituted benzoyl chlorides (0.00135 mol, 3 equiv) were added.
The mixture was heated under reflux for 24 h. The solvent was
removed under reduced pressure and the resulting residue was
4.13.4. N-Benzoyl-N-(4-chloro-2-phenethyl-2H-pyrazolo[3,4-
d]pyrimidin-6-yl)benzamide (57)
Yield: 46%; White solid, mp 134–135 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.79 (t, 7.0 Hz, 3H, CH3), 4.45 (t, J = 7.04 Hz, 2H,
CH2), 6.86–7.20 (m, 5H, Ar-H), 7.49–7.82 (m, 10H, acyl-H), 8.41

1764
G. Venkatesan et al. / Bioorg. Med. Chem. 22 (2014) 1751–1765
purified by column chromatography (hexane/EtOAc, 4:6) to obtain
4.18. Computational methodologies
the desired products (62 & 63) as solids.
All modeling studies were carried out on a Intel(R) Core[TM] 2
Quad CPU Q9550, 2.83 GHz, 3.25 GB RAM, DELL system. Docking
simulation, energy calculation and the analyses of docking poses
were performed using the Molecular Operating Environment
(MOE, version 2010.10) suite.49 The software package MOPAC (ver-
sion 7),50 implemented in the MOE suite, was utilized for all quan-
tum mechanical calculations. PyMOL molecular graphics system
was used for graphical visualizations and manipulations.51
4.15.1. Ethyl-2-neopentyl-6-(4-(trifluoromethyl)benzamido)-
2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (62)
Yield: 61%; pale yellow solid, mp 116–118 °C; 1H NMR
(400 MHz, DMSO-d6): d 0.99 (s, 9H, 3CH3), 1.47 (t, J = 7.0 Hz, 3H,
CH3), 4.20 (s, 2H, CH2), 4.55 (q, J1 = 7.02 Hz, J2 = 14.18 Hz, 2H,
CH2), 7.93–8.20 (m, 4H, Ar-H), 8.53 (s, 1H, pyrazole-H), 11.76 (s,
1H, NH). LC–MS (ESI) analysis (m/z) calcd for C21H22F3N5O3
(449.43) : found 450.4 [M+H]+, 472.2 [M+Na]+. HPLC purity
(254 nm); 100%, eluent: 60% ACN:H2O, tR = 7.9 min.
4.18.1. Homology modeling
The crystallographic structure of human A2A adenosine receptor
complexed with ZM-241385 as high affinity antagonist (PDB code:
3EML),36 was employed to build up a homology model of the hA3-
AR by using software Modeller version 9.11.37–40 The tip of the sec-
ond extracellular loop (Gln148 to Ser156) is absent in the crystal
structure of A2AAR (PDB No. 3EML) and it was not modelled owing
to weak experimental electron density. Such missing tip of the loop
is spatially distinct from the ligand-binding site and most probably
does not directly interact with the binding cavity, as reported in
Jaakola et.al.36 The refined model was validated using PROCHECK
program in Protein Structure Validation Suite52 (PSVS). The num-
bering of the amino acids follows the arbitrary scheme as proposed
by Ballesteros and Weinstein. According to this scheme, each ami-
no acid identifier starts with the helix number, followed by the po-
sition relative to a reference residue among the most conserved
amino acid in that helix. The number 50 is arbitrarily assigned to
the reference residue.53
4.15.2. Ethyl-6-(4-methylbenzamido)-2-neopentyl-2H-
pyrazolo[3,4-d]pyrimidine-4-carboxylate (63)
Yield: 48%; pale yellow solid, mp 97–99 °C; 1H NMR (400 MHz,
DMSO-d6): d 1.01 (s, 9H, 3CH3), 1.48 (t, J = 7.16 Hz, 3H, CH3), 2.45
(s, 3H, CH3), 4.25 (s, 2H, CH2), 4.55 (q, J1 = 7.16 Hz, J2 = 14.16 Hz,
2H, CH2), 7.36–7.98 (m, 4H, Ar-H), 8.51 (s,1H, pyrazole-H), 11.39
(s, 1H, NH). 13C NMR (400 MHz, DMSO-d6): d 14.5 (CH3), 21.5
(CH3), 28.1 (CH3)3, 33.8 (C-alkyl), 57.8 (CH2), 62.7 (COOCH2),
109.4 (C), 128.9 (CH), 129.3 (CH), 129.9 (C), 131.7 (C), 133.9 (CH-
pyrazole), 142.8 (N–C–N), 150.9 (C–COO), 156.3 (C–NH), 163.5
(CO), 166.3 (COO). LC–MS (ESI) analysis (m/z) calcd for
C
21H25N5O3 (395.45): found 396.3 [M+H]+, 418.3 [M+Na]+. HPLC
purity (254 nm); 100%, eluent: 60% ACN/H2O, tR = 7.1 min.
4.16. X-ray diffraction studies
X-ray data were collected with a Bruker AXSSMART APEX dif-
fractometer using MoKa radiation at 223(2) K with the SMART
suite of Programs.44 Data were processed and corrected for Lorentz
and polarization effects by means of SAINT software,45 and for
absorption effects using the SADABS software.46 Structural solution
and refinement were carried out by using the SHELXTL suite of pro-
grams.47 The structure was solved by using Direct Methods. Non-
hydrogen atoms were located by using difference maps and were
given anisotropic displacement parameters in the final refinement.
All the H atoms were put at calculated positions by means of the
riding model. CCDC-937929 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
4.18.2. Molecular docking simulations
Ligand structures were built using MOE-builder tool,49 and
were subjected to MMFF94x energy minimization until the rms
of conjugate gradient was <0.05 kcal molꢁ1 Åꢁ1. Partial charges
for the ligands were calculated using PM3/ESP methodology.
All ligands were docked into the hypothetical TM binding site of
the hA3AR model and that of the hA2AAR crystal structure by using
the docking tool of the MOE suite.49 In the MOE-Dock, the Alpha
PMI placement method, followed by force field refinement and lon-
don dG scoring, were used for the docking runs. MOE-Dock per-
formed 100 independent docking runs (100 for our specific case)
and wrote the resulting conformations and their energies in a
molecular database file. The resulting docked complexes were sub-
jected to MMFF94x energy minimization until the rms of conjugate
gradient was <0.1 kcal molꢁ1 Åꢁ1. Prediction of antagonist–recep-
tor complex stability (in terms of corresponding pKi value) and
the quantitative analysis for non bonded intermolecular interac-
tions (H-bonds, hydrophobic, electrostatic) were calculated and
visualized using tools implemented in MOE suite.49 Electrostatic
contributions to the binding energy of individual amino acids have
been calculated as implemented in MOE suite.49
4.17. Binding assay and adenylyl cyclase activity assay
Binding experiments with respective tritiated radioligands at
hA1AR, hA2AAR and hA3AR were carried out for 3 h at room temper-
ature as described previously.48 Nonspecific binding was deter-
mined in the presence of 1 mM theophylline for hA1AR and
100 l
M (R)-N6-phenyliso-propyladenosine (R-PIA) for both hA2A-
AR and hA3AR. Bound and free radioactivity was separated by fil-
tering the assay mixture through Whatman GF/B glass-fiber
filters using a Micro-Mate 196-cell harvester (Packard Instrument
Company). The filter bound radioactivity was counted on Top
Count (efficiency of 57%) with Micro-Scint 20. The Ki values were
calculated from competition curves by nonlinear curve fitting with
the program SCTFIT.48 In the case of hA2BAR, adenylyl cyclase
experiments were carried out as described previously with minor
modifications.33,34 Membranes transfected with hA2B receptor
were incubated with 100 nM NECA, as well as 150,000 cpm of
Acknowledgements
This work was supported by the National University of Singa-
pore, and Singapore Ministry of Education (MOE2009-T2-2-011
and R-398-000-068-112; NUS-FRC R-148-000-129-112 and
A⁄STAR-SERC TSRPiNPB 102 152 0016). The authors would like
to thank Sonja Kachler for her assistance and expertise in the phar-
macological evaluation of the compounds.
[a-
32P]ATP and tested compounds in different concentrations for
Supplementary data
20 min in the incubation mixture without EGTA (ethylene glycol-
tetraacetic acid) and NaCl. The Ki values for concentration-depen-
dent inhibition of NECA-mediated adenylyl cyclase activity
caused by tested antagonists were calculated accordingly.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.018.

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